From b8973147248eef8e1e6aa31002ba5377ccbdee5d Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Wed, 22 Nov 2023 11:50:42 -0500 Subject: [PATCH 01/24] Init CRAFT eval --- src/ontogpt/evaluation/craft/__init__.py | 0 .../evaluation/craft/eval_craft_ner.py | 31 +++++++++++++++++++ src/ontogpt/evaluation/craft/eval_craft_re.py | 15 +++++++++ 3 files changed, 46 insertions(+) create mode 100644 src/ontogpt/evaluation/craft/__init__.py create mode 100644 src/ontogpt/evaluation/craft/eval_craft_ner.py create mode 100644 src/ontogpt/evaluation/craft/eval_craft_re.py diff --git a/src/ontogpt/evaluation/craft/__init__.py b/src/ontogpt/evaluation/craft/__init__.py new file mode 100644 index 000000000..e69de29bb diff --git a/src/ontogpt/evaluation/craft/eval_craft_ner.py b/src/ontogpt/evaluation/craft/eval_craft_ner.py new file mode 100644 index 000000000..5ee35c66d --- /dev/null +++ b/src/ontogpt/evaluation/craft/eval_craft_ner.py @@ -0,0 +1,31 @@ +""" +Evaluation on the CRAFT corpus. + +This is an NER evaluation, or specifically +concept recognition. + +It uses a specific template (craft_concepts) + +The corpus is retrieved from: +https://github.com/UCDenver-ccp/CRAFT + +This evaluation use the v5.0.2 release. + +Funk et al. 2014 BMC Bioinformatics +(https://doi.org/10.1186/1471-2105-15-59) +reported that the ConceptMapper dictionary +lookup tool generally performed the best +across most ontologies, +with F-scores on these: +Cell Type Ontology (CL) 0.83 +Gene Ontology (GO) Cellular Component 0.77 +Gene Ontology (GO) Biological Process 0.37 +Gene Ontology (GO) Molecular Function* 0.48 +Sequence Ontology (SO) 0.56 +Protein Ontology (PR) 0.57 +NCBI Taxonomy (NCBITaxon) 0.69 +CHEBI 0.56 + +* after adding synonyms without the word "activity" + +""" \ No newline at end of file diff --git a/src/ontogpt/evaluation/craft/eval_craft_re.py b/src/ontogpt/evaluation/craft/eval_craft_re.py new file mode 100644 index 000000000..fd9814a4f --- /dev/null +++ b/src/ontogpt/evaluation/craft/eval_craft_re.py @@ -0,0 +1,15 @@ +""" +Evaluation on the CRAFT corpus. + +This is an evaluation of relation extraction +on CRAFT - relations are not annotated +in the corpus beyond coreferences and +structural relationships, so this is +not a scored evaluation. + +The corpus is retrieved from: +https://github.com/UCDenver-ccp/CRAFT + +This evaluation use the v5.0.2 release. + +""" \ No newline at end of file From 11b91b4a4a82f71abc2d9e12dbb0803e7b929d36 Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Wed, 22 Nov 2023 12:51:17 -0500 Subject: [PATCH 02/24] Add placeholder test data dir --- src/ontogpt/evaluation/craft/database/README.md | 3 +++ 1 file changed, 3 insertions(+) create mode 100644 src/ontogpt/evaluation/craft/database/README.md diff --git a/src/ontogpt/evaluation/craft/database/README.md b/src/ontogpt/evaluation/craft/database/README.md new file mode 100644 index 000000000..2cd823be0 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/README.md @@ -0,0 +1,3 @@ +# CRAFT corpus + +The CRAFT corpus is retrieved from its own repository, at . From cb1ac0f1e62a041338fccb2aac57af0d36d22be0 Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Wed, 22 Nov 2023 13:17:35 -0500 Subject: [PATCH 03/24] Expand on eval --- .../evaluation/craft/eval_craft_ner.py | 301 +++++++++++++++++- 1 file changed, 299 insertions(+), 2 deletions(-) diff --git a/src/ontogpt/evaluation/craft/eval_craft_ner.py b/src/ontogpt/evaluation/craft/eval_craft_ner.py index 5ee35c66d..418ae815a 100644 --- a/src/ontogpt/evaluation/craft/eval_craft_ner.py +++ b/src/ontogpt/evaluation/craft/eval_craft_ner.py @@ -25,7 +25,304 @@ Protein Ontology (PR) 0.57 NCBI Taxonomy (NCBITaxon) 0.69 CHEBI 0.56 - * after adding synonyms without the word "activity" -""" \ No newline at end of file +Annotations in CRAFT also include: +MONDO Disease Ontology +Molecular Process Ontology (MOP) +UBERON Ontology + +""" + + +import gzip +import logging +from collections import defaultdict +from dataclasses import dataclass +from pathlib import Path +from random import shuffle +from typing import Any, Dict, Iterable, List, Optional, Set, Tuple + +import yaml +from oaklib import BasicOntologyInterface, get_adapter +from pydantic import BaseModel + +from ontogpt.engines.knowledge_engine import chunk_text +from ontogpt.engines.spires_engine import SPIRESEngine +from ontogpt.evaluation.evaluation_engine import SimilarityScore, SPIRESEvaluationEngine +from ontogpt.templates.craft_concept import ( + Document, + TextWithEntity, +) + +THIS_DIR = Path(__file__).parent +DATABASE_DIR = Path(__file__).parent / "database" + +# These are the entity types involved in this dataset. +TARGET_TYPES = [ + "AnatomicalElement", + "Biological Process", + "CellType", + "CellularComponent", + "Chemical", + "Disease", + "Molecular Function", + "MolecularProcess", + "Protein", + "SequenceEntity", + "Taxon", +] + +RESULT_TYPES = [ + "true_positives", + "num_true_positives", + "false_positives", + "num_false_positives", + "false_negatives", + "num_false_negatives", +] + +logger = logging.getLogger(__name__) + + +class PredictionNER(BaseModel): + """A prediction for a named entity recognition task.""" + + test_object: Optional[TextWithEntity] = None + """Source of truth to evaluate against.""" + + # results stores all the TP, FP, FN, and TP entities + # with entity name from TARGET_TYPES as key + results = Dict[Dict[List[Tuple]]] + for target in TARGET_TYPES: + results[target] = {} + for result_type in RESULT_TYPES: + results[target[result_type]] = [] + + scores: Optional[Dict[str, SimilarityScore]] = None + predicted_object: Optional[TextWithEntity] = None + named_entities: Optional[List[Any]] = None + +class EvaluationObjectSetNER(BaseModel): + """A result of performing named entity recognition.""" + + precision_ce: float = 0 + recall_ce: float = 0 + f1_ce: float = 0 + + precision_de: float = 0 + recall_de: float = 0 + f1_de: float = 0 + + training: Optional[List[TextWithEntity]] = None + predictions: Optional[List[PredictionNER]] = None + test: Optional[List[TextWithEntity]] = None + + +@dataclass +class EvalCRAFTConcepts(SPIRESEvaluationEngine): + + # TODO: nope, not these + subject_prefix = "MESH" + object_prefix = "MESH" + + def __post_init__(self): + self.extractor = SPIRESEngine( + template="craft_concept.Document", model=self.model + ) + + def load_test_cases(self) -> Iterable[Document]: + return self.load_cases(DATABASE_DIR) + + def load_cases(self, path: Path) -> Iterable[Document]: + logger.info(f"Loading {path}") + + # Retrieve corpus if not present + # Preprocess corpus + # Load documents + # Validate documents + + # with gzip.open(str(path), "rb") as f: + # collection = biocxml.load(f) + # chemicals_by_text = defaultdict(list) + # diseases_by_text = defaultdict(list) + # for document in collection.documents: + # these_annotations = [] + # doc = {} + # for p in document.passages: + # doc[p.infons["type"]] = p.text + # for a in p.annotations: + # if a.infons["type"] in TARGET_TYPES: + # these_annotations.append(a) + # title = doc["title"] + # abstract = doc["abstract"] + # logger.debug(f"Title: {title} Abstract: {abstract}") + # for a in these_annotations: + # i = a.infons + # if i["type"] == "Chemical": + # e = Chemical.model_validate( + # { + # "id": f"{self.subject_prefix}:{i[self.subject_prefix]}", + # } + # ) + # chemicals_by_text[(title, abstract)].append(e.id) + # elif i["type"] == "Disease": + # e = Disease.model_validate( + # { + # "id": f"{self.subject_prefix}:{i[self.subject_prefix]}", + # } + # ) + # diseases_by_text[(title, abstract)].append(e.id) + + # all_entities_by_text = chemicals_by_text | diseases_by_text + + # i = 0 + # for (title, abstract), _entities in all_entities_by_text.items(): + # i = i + 1 + # pub = Publication.model_validate( + # { + # "id": str(i), + # "title": title, + # "abstract": abstract, + # } + # ) + # chemical_entities = chemicals_by_text[(title, abstract)] + # disease_entities = diseases_by_text[(title, abstract)] + # logger.debug( + # f"Chemicals: {len(chemical_entities)} for Title: {title} Abstract: {abstract}" + # ) + # logger.debug( + # f"Diseases: {len(disease_entities)} for Title: {title} Abstract: {abstract}" + # ) + # yield ChemicalToDiseaseDocument.model_validate( + # { + # "publication": pub, + # "chemicals": chemical_entities, + # "diseases": disease_entities, + # } + # ) + + + # def eval(self) -> EvaluationObjectSetNER: + # """Evaluate the ability to extract chemical and disease named entities.""" + + # # TODO: need other labelers + # labeler = get_adapter("sqlite:obo:mesh") + # if self.num_tests and isinstance(self.num_tests, int): + # num_test = self.num_tests + # else: + # num_test = 1 + # ke = self.extractor + # docs = list(self.load_test_cases()) + # shuffle(docs) + # eos = EvaluationObjectSetNER( + # test=docs[:num_test], + # training=[], + # predictions=[], + # ) + # n = 1 + # for doc in eos.test: + # logger.info(f"Iteration {n} of {num_test}") + # n += 1 + # logger.info(doc) + # text = f"Title: {doc.publication.title} Abstract: {doc.publication.abstract}" + # pub = Publication.model_validate( + # { + # "id": str(doc.publication.id), + # "title": doc.publication.title, + # "abstract": doc.publication.abstract, + # } + # ) + # predicted_obj = Document.model_validate( + # { + # "publication": pub, + # "chemicals": [], + # "diseases": [], + # } + # ) + # named_entities: List[str] = [] # This stores the NEs for the whole document + # ke.named_entities = [] # This stores the NEs the extractor knows about + + # if self.chunking: + # text_list = chunk_text(text) + # else: + # text_list = iter([text]) + + # for chunked_text in text_list: + # extraction = ke.extract_from_text(chunked_text) + # if extraction.extracted_object is not None: + # logger.info( + # f"{len(extraction.extracted_object.chemicals)}\ + # chemical entities from window: {chunked_text}" + # ) + # logger.info( + # f"{len(extraction.extracted_object.diseases)}\ + # disease entities from window: {chunked_text}" + # ) + # if not predicted_obj and extraction.extracted_object is not None: + # predicted_obj = extraction.extracted_object + # else: + # if predicted_obj is not None and extraction.extracted_object is not None: + # predicted_obj.chemicals.extend(extraction.extracted_object.chemicals) + # predicted_obj.diseases.extend(extraction.extracted_object.diseases) + # logger.info( + # f"{len(predicted_obj.chemicals)} chemical entities, after concatenation" + # ) + # logger.info( + # f"{len(predicted_obj.diseases)} disease entities, after concatenation" + # ) + # logger.debug(f"concatenated chemical entities: {predicted_obj.chemicals}") + # logger.debug(f"concatenated disease entities: {predicted_obj.diseases}") + # if extraction.named_entities is not None: + # for entity in extraction.named_entities: + # if entity not in named_entities: + # named_entities.append(entity) + + # def included(t: str): + # if t.startswith("MESH:"): + # return t + + # predicted_obj.chemicals = [t for t in predicted_obj.chemicals if included(t)] + # predicted_obj.diseases = [t for t in predicted_obj.diseases if included(t)] + + # logger.info(f"{len(predicted_obj.chemicals)} filtered chemical entities (MESH only)") + # logger.info(f"{len(predicted_obj.diseases)} filtered disease entities (MESH only)") + # pred = PredictionNER( + # predicted_object=predicted_obj, test_object=doc, named_entities=named_entities + # ) + # named_entities.clear() + # logger.info("PRED") + # logger.info(yaml.dump(data=pred.model_dump())) + # logger.info("Calc scores") + # pred.calculate_scores(labelers=[labeler]) + # logger.info(yaml.dump(data=pred.model_dump())) + # eos.predictions.append(pred) + # self.calc_stats(eos) + # return eos + + # def calc_stats(self, eos: EvaluationObjectSetNER): + # num_true_positives_ce = sum(p.num_true_positives_ce for p in eos.predictions) + # num_false_positives_ce = sum(p.num_false_positives_ce for p in eos.predictions) + # num_false_negatives_ce = sum(p.num_false_negatives_ce for p in eos.predictions) + # if num_true_positives_ce + num_false_positives_ce == 0: + # logger.warning("No true positives or false positives for chemical entities.") + # return + # eos.precision_ce = num_true_positives_ce / (num_true_positives_ce + num_false_positives_ce) + # eos.recall_ce = num_true_positives_ce / (num_true_positives_ce + num_false_negatives_ce) + # if eos.precision_ce + eos.recall_ce == 0: + # logger.warning("No precision or recall for chemical entities.") + # return + # eos.f1_ce = 2 * (eos.precision_ce * eos.recall_ce) / (eos.precision_ce + eos.recall_ce) + + # num_true_positives_de = sum(p.num_true_positives_de for p in eos.predictions) + # num_false_positives_de = sum(p.num_false_positives_de for p in eos.predictions) + # num_false_negatives_de = sum(p.num_false_negatives_de for p in eos.predictions) + # if num_true_positives_de + num_false_positives_de == 0: + # logger.warning("No true positives or false positives for disease entities.") + # return + # eos.precision_de = num_true_positives_de / (num_true_positives_de + num_false_positives_de) + # eos.recall_de = num_true_positives_de / (num_true_positives_de + num_false_negatives_de) + # if eos.precision_de + eos.recall_de == 0: + # logger.warning("No precision or recall for disease entities.") + # return + # eos.f1_de = 2 * (eos.precision_de * eos.recall_de) / (eos.precision_de + eos.recall_de) From 607a48a4fd9dc5108623759bdbf3467629d55273 Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Wed, 22 Nov 2023 13:30:09 -0500 Subject: [PATCH 04/24] Add craft_concept template, first version --- src/ontogpt/templates/craft_concept.py | 155 +++++++++++++++++++++++ src/ontogpt/templates/craft_concept.yaml | 94 ++++++++++++++ 2 files changed, 249 insertions(+) create mode 100644 src/ontogpt/templates/craft_concept.py create mode 100644 src/ontogpt/templates/craft_concept.yaml diff --git a/src/ontogpt/templates/craft_concept.py b/src/ontogpt/templates/craft_concept.py new file mode 100644 index 000000000..447bae420 --- /dev/null +++ b/src/ontogpt/templates/craft_concept.py @@ -0,0 +1,155 @@ +from __future__ import annotations +from datetime import datetime, date +from enum import Enum +from typing import List, Dict, Optional, Any, Union +from pydantic import BaseModel as BaseModel, ConfigDict, Field +import sys +if sys.version_info >= (3, 8): + from typing import Literal +else: + from typing_extensions import Literal + + +metamodel_version = "None" +version = "None" + +class ConfiguredBaseModel(BaseModel): + model_config = ConfigDict( + validate_assignment=True, + validate_default=True, + extra='forbid', + arbitrary_types_allowed=True, + use_enum_values = True) + + +class NullDataOptions(str, Enum): + + + UNSPECIFIED_METHOD_OF_ADMINISTRATION = "UNSPECIFIED_METHOD_OF_ADMINISTRATION" + + NOT_APPLICABLE = "NOT_APPLICABLE" + + NOT_MENTIONED = "NOT_MENTIONED" + + + +class ExtractionResult(ConfiguredBaseModel): + """ + A result of extracting knowledge on text + """ + input_id: Optional[str] = Field(None) + input_title: Optional[str] = Field(None) + input_text: Optional[str] = Field(None) + raw_completion_output: Optional[str] = Field(None) + prompt: Optional[str] = Field(None) + extracted_object: Optional[Any] = Field(None, description="""The complex objects extracted from the text""") + named_entities: Optional[List[Any]] = Field(default_factory=list, description="""Named entities extracted from the text""") + + +class NamedEntity(ConfiguredBaseModel): + + id: str = Field(..., description="""A unique identifier for the named entity""") + label: Optional[str] = Field(None, description="""The label (name) of the named thing""") + + +class AnatomicalElement(NamedEntity): + + id: str = Field(..., description="""A unique identifier for the named entity""") + label: Optional[str] = Field(None, description="""The label (name) of the named thing""") + + +class Chemical(NamedEntity): + + id: str = Field(..., description="""A unique identifier for the named entity""") + label: Optional[str] = Field(None, description="""The label (name) of the named thing""") + + +class Disease(NamedEntity): + + id: str = Field(..., description="""A unique identifier for the named entity""") + label: Optional[str] = Field(None, description="""The label (name) of the named thing""") + + +class CompoundExpression(ConfiguredBaseModel): + + None + + +class Triple(CompoundExpression): + """ + Abstract parent for Relation Extraction tasks + """ + subject: Optional[str] = Field(None) + predicate: Optional[str] = Field(None) + object: Optional[str] = Field(None) + qualifier: Optional[str] = Field(None, description="""A qualifier for the statements, e.g. \"NOT\" for negation""") + subject_qualifier: Optional[str] = Field(None, description="""An optional qualifier or modifier for the subject of the statement, e.g. \"high dose\" or \"intravenously administered\"""") + object_qualifier: Optional[str] = Field(None, description="""An optional qualifier or modifier for the object of the statement, e.g. \"severe\" or \"with additional complications\"""") + + +class TextWithTriples(ConfiguredBaseModel): + """ + A text containing one or more relations of the Triple type. + """ + publication: Optional[Publication] = Field(None) + triples: Optional[List[Triple]] = Field(default_factory=list) + + +class TextWithEntity(ConfiguredBaseModel): + """ + A text containing one or more instances of a single type of entity. + """ + publication: Optional[Publication] = Field(None) + entities: Optional[List[str]] = Field(default_factory=list) + + +class Document(TextWithEntity): + """ + A document that contains biological and biomedical concepts. + """ + anatomicalelements: Optional[List[str]] = Field(default_factory=list, description="""One or more parts of the body or anatomy.""") + chemicals: Optional[List[str]] = Field(default_factory=list, description="""One or more chemical substances, drugs, or small molecules.""") + diseases: Optional[List[str]] = Field(default_factory=list, description="""One or more diseases or conditions.""") + publication: Optional[Publication] = Field(None) + entities: Optional[List[str]] = Field(default_factory=list) + + +class RelationshipType(NamedEntity): + + id: str = Field(..., description="""A unique identifier for the named entity""") + label: Optional[str] = Field(None, description="""The label (name) of the named thing""") + + +class Publication(ConfiguredBaseModel): + + id: Optional[str] = Field(None, description="""The publication identifier""") + title: Optional[str] = Field(None, description="""The title of the publication""") + abstract: Optional[str] = Field(None, description="""The abstract of the publication""") + combined_text: Optional[str] = Field(None) + full_text: Optional[str] = Field(None, description="""The full text of the publication""") + + +class AnnotatorResult(ConfiguredBaseModel): + + subject_text: Optional[str] = Field(None) + object_id: Optional[str] = Field(None) + object_text: Optional[str] = Field(None) + + + +# Model rebuild +# see https://pydantic-docs.helpmanual.io/usage/models/#rebuilding-a-model +ExtractionResult.model_rebuild() +NamedEntity.model_rebuild() +AnatomicalElement.model_rebuild() +Chemical.model_rebuild() +Disease.model_rebuild() +CompoundExpression.model_rebuild() +Triple.model_rebuild() +TextWithTriples.model_rebuild() +TextWithEntity.model_rebuild() +Document.model_rebuild() +RelationshipType.model_rebuild() +Publication.model_rebuild() +AnnotatorResult.model_rebuild() + diff --git a/src/ontogpt/templates/craft_concept.yaml b/src/ontogpt/templates/craft_concept.yaml new file mode 100644 index 000000000..61748eb7b --- /dev/null +++ b/src/ontogpt/templates/craft_concept.yaml @@ -0,0 +1,94 @@ +id: http://w3id.org/ontogpt/craft_concept +name: craft_concept +title: Concept Recognition in the CRAFT Corpus +description: >- + A template for Concept Recognition in the CRAFT Corpus. + + CRAFT includes annotation for these entity types. + CHEBI + Cell Type Ontology (CL) + Gene Ontology (GO) Biological Process + Gene Ontology (GO) Cellular Component + Gene Ontology (GO) Molecular Function + MONDO Disease Ontology + Molecular Process Ontology (MOP) + NCBI Taxonomy (NCBITaxon) + Protein Ontology (PR) + Sequence Ontology (SO) + UBERON Ontology + + Accordingly, the corresponding entity types are + represented within this schema. +see_also: + - https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-15-59 + - https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471-2105-13-161 +source: >- + https://github.com/UCDenver-ccp/CRAFT +license: https://creativecommons.org/publicdomain/zero/1.0/ +prefixes: + linkml: https://w3id.org/linkml/ + craft_concept: http://w3id.org/ontogpt/craft_concept + +default_prefix: craft_concept +default_range: string + +imports: + - linkml:types + - core + +classes: + + Document: + description: A document that contains biological and biomedical concepts. + is_a: TextWithEntity + attributes: + anatomicalelements: + range: AnatomicalElement + multivalued: true + description: One or more parts of the body or anatomy. + annotations: + prompt: >- + A semi-colon separated list of anatomy names or regions, for + example: conjunctival space; anterior surface of kidney; + medial surface of mandible; exoskeleton; choroidal gland + chemicals: + range: Chemical + multivalued: true + description: One or more chemical substances, drugs, or small molecules. + annotations: + prompt: >- + A semi-colon separated list of chemical names, for example: + Lidocaine; Hydroxychloroquine; Methyldopa; Monosodium Glutamate; + Imatinib + diseases: + range: Disease + multivalued: true + description: One or more diseases or conditions. + annotations: + prompt: >- + A semi-colon separated list of disease names, for example: cardiac + asystole; COVID-19; Hypotension; Headache; cancer + + AnatomicalElement: + is_a: NamedEntity + annotations: + annotators: "sqlite:obo:mesh, sqlite:obo:uberon, sqlite:obo:ncit, gilda:" + prompt.examples: conjunctival space, anterior surface of kidney, medial surface of mandible, exoskeleton, choroidal gland + id_prefixes: + - UBERON + + Chemical: + is_a: NamedEntity + annotations: + annotators: "sqlite:obo:mesh, sqlite:obo:chebi, sqlite:obo:ncit, bioportal:mdm, sqlite:obo:drugbank, gilda:" + prompt.examples: Lidocaine, Hydroxychloroquine, Methyldopa, Imatinib + id_prefixes: + - CHEBI + + Disease: + is_a: NamedEntity + annotations: + annotators: "sqlite:obo:mesh, sqlite:obo:mondo, sqlite:obo:hp, sqlite:obo:ncit, sqlite:obo:doid, bioportal:meddra" + prompt.examples: cardiac asystole, COVID-19, Headache, cancer + id_prefixes: + - MONDO From ae2c08a9a1e847c63194338343b1d9cb025ed7d2 Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Wed, 22 Nov 2023 13:44:47 -0500 Subject: [PATCH 05/24] Change some entity names --- src/ontogpt/evaluation/craft/eval_craft_ner.py | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/src/ontogpt/evaluation/craft/eval_craft_ner.py b/src/ontogpt/evaluation/craft/eval_craft_ner.py index 418ae815a..ddd8df919 100644 --- a/src/ontogpt/evaluation/craft/eval_craft_ner.py +++ b/src/ontogpt/evaluation/craft/eval_craft_ner.py @@ -61,12 +61,12 @@ # These are the entity types involved in this dataset. TARGET_TYPES = [ "AnatomicalElement", - "Biological Process", + "BiologicalProcess", "CellType", "CellularComponent", "Chemical", "Disease", - "Molecular Function", + "MolecularFunction", "MolecularProcess", "Protein", "SequenceEntity", From 9f430e18fe9a63b2540349225654cba3620cd5a5 Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Wed, 22 Nov 2023 13:49:25 -0500 Subject: [PATCH 06/24] Expand template --- src/ontogpt/templates/craft_concept.py | 42 ++++++++++ src/ontogpt/templates/craft_concept.yaml | 102 ++++++++++++++++++++++- 2 files changed, 142 insertions(+), 2 deletions(-) diff --git a/src/ontogpt/templates/craft_concept.py b/src/ontogpt/templates/craft_concept.py index 447bae420..705ccd716 100644 --- a/src/ontogpt/templates/craft_concept.py +++ b/src/ontogpt/templates/craft_concept.py @@ -22,6 +22,24 @@ class ConfiguredBaseModel(BaseModel): use_enum_values = True) +class GOBiologicalProcessType(str): + + + dummy = "dummy" + + +class GOCellComponentType(str): + + + dummy = "dummy" + + +class GOMolecularFunctionType(str): + + + dummy = "dummy" + + class NullDataOptions(str, Enum): @@ -58,6 +76,18 @@ class AnatomicalElement(NamedEntity): label: Optional[str] = Field(None, description="""The label (name) of the named thing""") +class BiologicalProcess(NamedEntity): + + id: str = Field(..., description="""A unique identifier for the named entity""") + label: Optional[str] = Field(None, description="""The label (name) of the named thing""") + + +class CellularComponent(NamedEntity): + + id: str = Field(..., description="""A unique identifier for the named entity""") + label: Optional[str] = Field(None, description="""The label (name) of the named thing""") + + class Chemical(NamedEntity): id: str = Field(..., description="""A unique identifier for the named entity""") @@ -70,6 +100,12 @@ class Disease(NamedEntity): label: Optional[str] = Field(None, description="""The label (name) of the named thing""") +class MolecularFunction(NamedEntity): + + id: str = Field(..., description="""A unique identifier for the named entity""") + label: Optional[str] = Field(None, description="""The label (name) of the named thing""") + + class CompoundExpression(ConfiguredBaseModel): None @@ -108,8 +144,11 @@ class Document(TextWithEntity): A document that contains biological and biomedical concepts. """ anatomicalelements: Optional[List[str]] = Field(default_factory=list, description="""One or more parts of the body or anatomy.""") + biologicalprocesses: Optional[List[str]] = Field(default_factory=list, description="""One or more biological processes, as defined by the Gene Ontology.""") + cellularcomponents: Optional[List[str]] = Field(default_factory=list, description="""One or more cellular components, as defined by the Gene Ontology.""") chemicals: Optional[List[str]] = Field(default_factory=list, description="""One or more chemical substances, drugs, or small molecules.""") diseases: Optional[List[str]] = Field(default_factory=list, description="""One or more diseases or conditions.""") + molecularfunctions: Optional[List[str]] = Field(default_factory=list, description="""One or more molecular functions, as defined by the Gene Ontology.""") publication: Optional[Publication] = Field(None) entities: Optional[List[str]] = Field(default_factory=list) @@ -142,8 +181,11 @@ class AnnotatorResult(ConfiguredBaseModel): ExtractionResult.model_rebuild() NamedEntity.model_rebuild() AnatomicalElement.model_rebuild() +BiologicalProcess.model_rebuild() +CellularComponent.model_rebuild() Chemical.model_rebuild() Disease.model_rebuild() +MolecularFunction.model_rebuild() CompoundExpression.model_rebuild() Triple.model_rebuild() TextWithTriples.model_rebuild() diff --git a/src/ontogpt/templates/craft_concept.yaml b/src/ontogpt/templates/craft_concept.yaml index 61748eb7b..4659ce767 100644 --- a/src/ontogpt/templates/craft_concept.yaml +++ b/src/ontogpt/templates/craft_concept.yaml @@ -37,7 +37,6 @@ imports: - core classes: - Document: description: A document that contains biological and biomedical concepts. is_a: TextWithEntity @@ -51,6 +50,28 @@ classes: A semi-colon separated list of anatomy names or regions, for example: conjunctival space; anterior surface of kidney; medial surface of mandible; exoskeleton; choroidal gland + biologicalprocesses: + range: BiologicalProcess + multivalued: true + description: >- + One or more biological processes, as defined by the Gene Ontology. + annotations: + prompt: >- + A semi-colon separated list of biological processes, for + example: nuclear axial expansion; intracellular transport; + medial surface of mandible; ribosomal subunit export from nucleus; + pole cell development + biologicalprocesses: + cellularcomponents: + range: CellularComponent + multivalued: true + description: >- + One or more cellular components, as defined by the Gene Ontology. + annotations: + prompt: >- + A semi-colon separated list of cellular components and structures, + for example: tubulin complex; proteasome complex; + cytoplasm; keratohyalin granule; nucleus chemicals: range: Chemical multivalued: true @@ -68,15 +89,57 @@ classes: prompt: >- A semi-colon separated list of disease names, for example: cardiac asystole; COVID-19; Hypotension; Headache; cancer + molecularfunctions: + range: MolecularFunction + multivalued: true + description: >- + One or more molecular functions, as defined by the Gene Ontology. + annotations: + prompt: >- + A semi-colon separated list of molecular functions, + for example: catalytic activity; amine binding; + peptide receptor activity; oxygen carrier activity; + structural constituent of cytoskeleton AnatomicalElement: is_a: NamedEntity annotations: annotators: "sqlite:obo:mesh, sqlite:obo:uberon, sqlite:obo:ncit, gilda:" - prompt.examples: conjunctival space, anterior surface of kidney, medial surface of mandible, exoskeleton, choroidal gland + prompt.examples: >- + conjunctival space, anterior surface of kidney, + medial surface of mandible, exoskeleton, choroidal gland id_prefixes: - UBERON + BiologicalProcess: + is_a: NamedEntity + annotations: + annotators: "sqlite:obo:go" + prompt.examples: >- + nuclear axial expansion, intracellular transport, + medial surface of mandible, ribosomal subunit export from nucleus, + pole cell development + id_prefixes: + - GO + slot_usage: + id: + values_from: + - GOBiologicalProcessType + + CellularComponent: + is_a: NamedEntity + annotations: + annotators: "sqlite:obo:go" + prompt.examples: >- + tubulin complex, proteasome complex, cytoplasm, keratohyalin granule, + nucleus + id_prefixes: + - GO + slot_usage: + id: + values_from: + - GOCellComponentType + Chemical: is_a: NamedEntity annotations: @@ -92,3 +155,38 @@ classes: prompt.examples: cardiac asystole, COVID-19, Headache, cancer id_prefixes: - MONDO + + MolecularFunction: + is_a: NamedEntity + annotations: + annotators: "sqlite:obo:go" + prompt.examples: >- + catalytic activity, amine binding, + peptide receptor activity, oxygen carrier activity, + structural constituent of cytoskeleton + id_prefixes: + - GO + slot_usage: + id: + values_from: + - GOMolecularFunctionType + +enums: + + GOBiologicalProcessType: + reachable_from: + source_ontology: obo:go + source_nodes: + - GO:0008150 # biological_process + + GOCellComponentType: + reachable_from: + source_ontology: obo:go + source_nodes: + - GO:0005575 # cellular_component + + GOMolecularFunctionType: + reachable_from: + source_ontology: obo:go + source_nodes: + - GO:0003674 # molecular_function From 3bcef788f6960ec704d4579b4e5533f552de4e5d Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Wed, 22 Nov 2023 14:04:23 -0500 Subject: [PATCH 07/24] Change SequenceEntity to SequenceFeature --- src/ontogpt/evaluation/craft/eval_craft_ner.py | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/src/ontogpt/evaluation/craft/eval_craft_ner.py b/src/ontogpt/evaluation/craft/eval_craft_ner.py index ddd8df919..fb62b3526 100644 --- a/src/ontogpt/evaluation/craft/eval_craft_ner.py +++ b/src/ontogpt/evaluation/craft/eval_craft_ner.py @@ -69,7 +69,7 @@ "MolecularFunction", "MolecularProcess", "Protein", - "SequenceEntity", + "SequenceFeature", "Taxon", ] From 30f7f135efb880e2ef34ca18b665e3f698dcb14d Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Wed, 22 Nov 2023 14:12:55 -0500 Subject: [PATCH 08/24] Expand template some more --- src/ontogpt/templates/craft_concept.py | 32 +++++++++ src/ontogpt/templates/craft_concept.yaml | 84 ++++++++++++++++++++++++ 2 files changed, 116 insertions(+) diff --git a/src/ontogpt/templates/craft_concept.py b/src/ontogpt/templates/craft_concept.py index 705ccd716..42076e3f7 100644 --- a/src/ontogpt/templates/craft_concept.py +++ b/src/ontogpt/templates/craft_concept.py @@ -106,6 +106,30 @@ class MolecularFunction(NamedEntity): label: Optional[str] = Field(None, description="""The label (name) of the named thing""") +class MolecularProcess(NamedEntity): + + id: str = Field(..., description="""A unique identifier for the named entity""") + label: Optional[str] = Field(None, description="""The label (name) of the named thing""") + + +class Protein(NamedEntity): + + id: str = Field(..., description="""A unique identifier for the named entity""") + label: Optional[str] = Field(None, description="""The label (name) of the named thing""") + + +class SequenceFeature(NamedEntity): + + id: str = Field(..., description="""A unique identifier for the named entity""") + label: Optional[str] = Field(None, description="""The label (name) of the named thing""") + + +class Taxon(NamedEntity): + + id: str = Field(..., description="""A unique identifier for the named entity""") + label: Optional[str] = Field(None, description="""The label (name) of the named thing""") + + class CompoundExpression(ConfiguredBaseModel): None @@ -149,6 +173,10 @@ class Document(TextWithEntity): chemicals: Optional[List[str]] = Field(default_factory=list, description="""One or more chemical substances, drugs, or small molecules.""") diseases: Optional[List[str]] = Field(default_factory=list, description="""One or more diseases or conditions.""") molecularfunctions: Optional[List[str]] = Field(default_factory=list, description="""One or more molecular functions, as defined by the Gene Ontology.""") + molecularprocesses: Optional[List[str]] = Field(default_factory=list, description="""One or more molecular processes, or named processes in chemical reactions.""") + proteins: Optional[List[str]] = Field(default_factory=list, description="""One or more protein names.""") + sequencefeatures: Optional[List[str]] = Field(default_factory=list, description="""One or more terms describing features and attributes of a biological sequence.""") + taxa: Optional[List[str]] = Field(default_factory=list, description="""One or more names of species or biological taxonomic groups.""") publication: Optional[Publication] = Field(None) entities: Optional[List[str]] = Field(default_factory=list) @@ -186,6 +214,10 @@ class AnnotatorResult(ConfiguredBaseModel): Chemical.model_rebuild() Disease.model_rebuild() MolecularFunction.model_rebuild() +MolecularProcess.model_rebuild() +Protein.model_rebuild() +SequenceFeature.model_rebuild() +Taxon.model_rebuild() CompoundExpression.model_rebuild() Triple.model_rebuild() TextWithTriples.model_rebuild() diff --git a/src/ontogpt/templates/craft_concept.yaml b/src/ontogpt/templates/craft_concept.yaml index 4659ce767..45b737225 100644 --- a/src/ontogpt/templates/craft_concept.yaml +++ b/src/ontogpt/templates/craft_concept.yaml @@ -100,6 +100,48 @@ classes: for example: catalytic activity; amine binding; peptide receptor activity; oxygen carrier activity; structural constituent of cytoskeleton + molecularprocesses: + range: MolecularProcess + multivalued: true + description: >- + One or more molecular processes, or named processes in + chemical reactions. + annotations: + prompt: >- + A semi-colon separated list of molecular processes, + for example: cyclization; exothermic reaction; + acid catalysis; dehydrogenation; N-thioacylation + proteins: + range: Protein + multivalued: true + description: >- + One or more protein names. + annotations: + prompt: >- + A semi-colon separated list of protein names, + for example: annexin A; flotillin; + lymphocyte antigen 6L; oncomodulin-1; serine protease 46 + sequencefeatures: + range: SequenceFeature + multivalued: true + description: >- + One or more terms describing features and attributes of a + biological sequence. + annotations: + prompt: >- + A semi-colon separated list of biological feature terms, + for example: junction; three prime UTR; + CpG island; pseudogene; syntenic region + taxa: + range: Taxon + multivalued: true + description: >- + One or more names of species or biological taxonomic groups. + annotations: + prompt: >- + A semi-colon separated list of species names or taxonomic + terms, for example: shark; Carcharodon carcharias; + elephant seal; lion; ungulates AnatomicalElement: is_a: NamedEntity @@ -171,6 +213,48 @@ classes: values_from: - GOMolecularFunctionType + MolecularProcess: + is_a: NamedEntity + annotations: + annotators: "sqlite:obo:mop, sqlite:obo:ncit" + prompt.examples: >- + cyclization, exothermic reaction, + acid catalysis, dehydrogenation, N-thioacylation + id_prefixes: + - MOP + + Protein: + is_a: NamedEntity + annotations: + annotators: "sqlite:obo:pr" + prompt.examples: >- + annexin A, flotillin, lymphocyte antigen 6L, oncomodulin-1, + serine protease 46 + id_prefixes: + - PR + + SequenceFeature: + is_a: NamedEntity + annotations: + annotators: "sqlite:obo:so, sqlite:obo:ncit" + prompt.examples: >- + junction, three prime UTR, CpG island, pseudogene, syntenic region + id_prefixes: + - SO + + Taxon: + is_a: NamedEntity + id_prefixes: + - NCBITaxon + - SNOMEDCT + annotations: + annotators: "sqlite:obo:ncbitaxon, bioportal:SNOMEDCT" + prompt.examples: >- + shark, Carcharodon carcharias, elephant seal, lion, + ungulates + + + enums: GOBiologicalProcessType: From c5a180c9531a923fc3c6bf6bbfbe8ab168155aad Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Wed, 22 Nov 2023 14:32:12 -0500 Subject: [PATCH 09/24] A couple more annotators --- src/ontogpt/templates/craft_concept.yaml | 2 +- 1 file changed, 1 insertion(+), 1 deletion(-) diff --git a/src/ontogpt/templates/craft_concept.yaml b/src/ontogpt/templates/craft_concept.yaml index 45b737225..a419e0240 100644 --- a/src/ontogpt/templates/craft_concept.yaml +++ b/src/ontogpt/templates/craft_concept.yaml @@ -193,7 +193,7 @@ classes: Disease: is_a: NamedEntity annotations: - annotators: "sqlite:obo:mesh, sqlite:obo:mondo, sqlite:obo:hp, sqlite:obo:ncit, sqlite:obo:doid, bioportal:meddra" + annotators: "sqlite:obo:mesh, sqlite:obo:mondo, sqlite:obo:hp, sqlite:obo:ncit, sqlite:obo:doid, sqlite:obo:nbo, sqlite:obo:efo" prompt.examples: cardiac asystole, COVID-19, Headache, cancer id_prefixes: - MONDO From 9ca3774e44a16170b71d382267bba15bf2e6a57b Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Wed, 29 Nov 2023 10:36:22 -0500 Subject: [PATCH 10/24] Add description of annotation file prep --- .../evaluation/craft/database/README.md | 77 ++++++++++++++++++- 1 file changed, 76 insertions(+), 1 deletion(-) diff --git a/src/ontogpt/evaluation/craft/database/README.md b/src/ontogpt/evaluation/craft/database/README.md index 2cd823be0..9ecfe177d 100644 --- a/src/ontogpt/evaluation/craft/database/README.md +++ b/src/ontogpt/evaluation/craft/database/README.md @@ -1,3 +1,78 @@ # CRAFT corpus -The CRAFT corpus is retrieved from its own repository, at . +The CRAFT corpus is available at its own repository, at . + +The version used in this evaluation is v5.0.2. + +To prepare the version used here, do the following. This process ensures the compatible Java version is available. + +## Get Clojure Boot in a Docker container + +```bash +$ docker pull clojure:openjdk-8-boot-2.8.1 +... +$ docker run clojure:boot +SLF4J: Failed to load class "org.slf4j.impl.StaticLoggerBinder". +SLF4J: Defaulting to no-operation (NOP) logger implementation +SLF4J: See http://www.slf4j.org/codes.html#StaticLoggerBinder for further details. +Retrieving tools.nrepl-0.2.13.pom from https://repo1.maven.org/maven2/ (3k) +Retrieving pom.contrib-0.2.2.pom from https://repo1.maven.org/maven2/ (7k) +Retrieving tools.nrepl-0.2.13.jar from https://repo1.maven.org/maven2/ (40k) +nREPL server started on port 42403 on host 127.0.0.1 - nrepl://127.0.0.1:42403 +REPL-y 0.3.7, nREPL 0.2.13 +Clojure 1.8.0 +OpenJDK 64-Bit Server VM 1.8.0_181-8u181-b13-2~deb9u1-b13 + Exit: Control+D or (exit) or (quit) + Commands: (user/help) + Docs: (doc function-name-here) + (find-doc "part-of-name-here") +Find by Name: (find-name "part-of-name-here") + Source: (source function-name-here) + Javadoc: (javadoc java-object-or-class-here) + Examples from clojuredocs.org: [clojuredocs or cdoc] + (user/clojuredocs name-here) + (user/clojuredocs "ns-here" "name-here") +boot.user=> Bye for now! +$ docker ps -a +CONTAINER ID IMAGE COMMAND CREATED STATUS PORTS NAMES +54d94e154884 clojure:openjdk-8-boot-2.8.1 "boot repl" 28 seconds ago Exited (0) 18 seconds ago crazy_stonebraker +``` + +## Enter the containter and retrieve the CRAFT corpus + +```bash +$ docker run -ti clojure:boot /bin/bash +root@cdd32ade99bd:/tmp# +root@cdd32ade99bd:/tmp# curl -OL https://github.com/UCDenver-ccp/CRAFT/archive/refs/tags/v5.0.2.tar.gz + % Total % Received % Xferd Average Speed Time Time Time Current + Dload Upload Total Spent Left Speed + 0 0 0 0 0 0 0 0 --:--:-- --:--:-- --:--:-- 0 +100 165M 0 165M 0 0 16.7M 0 --:--:-- 0:00:09 --:--:-- 10.6M +root@cdd32ade99bd:/tmp# ls -lh +total 166M +drwxr-xr-x 1 root root 4.0K Nov 17 2018 hsperfdata_root +-rw-r--r-- 1 root root 166M Nov 28 22:53 v5.0.2.tar.gz +root@cdd32ade99bd:/tmp# tar -xvzf v5.0.2.tar.gz +... decompress everything ... +root@cdd32ade99bd:/tmp# cd CRAFT-5.0.2/ +root@cdd32ade99bd:/tmp/CRAFT-5.0.2# export BOOT_JVM_OPTIONS='-Xmx5g -client' +``` + +## Convert to BRAT standoff format + +This format is most compatible with OntoGPT evaluations because it includes full input text (in each txt file) and one entity annotation per line (in each ann file). + +Note this command will not include the extension classes - this is intentional. + +```bash +root@cdd32ade99bd:/tmp/CRAFT-5.0.2# boot all-concepts convert -o ./converted/all --brat +``` + +## Copy over the converted files + +Back on the host machine, copy the full directory of converted annotations to the `/ontogpt/src/ontogpt/evaluation/craft/database/` directory. + +```bash +$ docker cp stoic_allen:/tmp/CRAFT-5.0.2/converted/all . +Successfully copied 8.58MB to /home/harry/ontogpt/src/ontogpt/evaluation/craft/database/. +``` From 44199dda439c31093b709a38c21c6f087855cf14 Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Wed, 29 Nov 2023 10:37:50 -0500 Subject: [PATCH 11/24] Add CRAFT annotations in BRAT standoff format --- .../craft/database/all/11319941.ann | 542 ++ .../craft/database/all/11319941.txt | 169 + .../craft/database/all/11532192.ann | 529 ++ .../craft/database/all/11532192.txt | 229 + .../craft/database/all/11597317.ann | 298 + .../craft/database/all/11597317.txt | 49 + .../craft/database/all/11604102.ann | 648 +++ .../craft/database/all/11604102.txt | 121 + .../craft/database/all/11897010.ann | 463 ++ .../craft/database/all/11897010.txt | 97 + .../craft/database/all/12079497.ann | 333 ++ .../craft/database/all/12079497.txt | 91 + .../craft/database/all/12546709.ann | 521 ++ .../craft/database/all/12546709.txt | 119 + .../craft/database/all/12585968.ann | 898 +++ .../craft/database/all/12585968.txt | 179 + .../craft/database/all/12925238.ann | 931 ++++ .../craft/database/all/12925238.txt | 145 + .../craft/database/all/14609438.ann | 331 ++ .../craft/database/all/14609438.txt | 113 + .../craft/database/all/14611657.ann | 1239 +++++ .../craft/database/all/14611657.txt | 181 + .../craft/database/all/14624252.ann | 919 ++++ .../craft/database/all/14624252.txt | 241 + .../craft/database/all/14675480.ann | 1287 +++++ .../craft/database/all/14675480.txt | 165 + .../craft/database/all/14691534.ann | 1352 +++++ .../craft/database/all/14691534.txt | 249 + .../craft/database/all/14723793.ann | 556 ++ .../craft/database/all/14723793.txt | 129 + .../craft/database/all/14737183.ann | 1454 +++++ .../craft/database/all/14737183.txt | 295 + .../craft/database/all/15005800.ann | 1276 +++++ .../craft/database/all/15005800.txt | 183 + .../craft/database/all/15018652.ann | 433 ++ .../craft/database/all/15018652.txt | 91 + .../craft/database/all/15040800.ann | 1733 ++++++ .../craft/database/all/15040800.txt | 229 + .../craft/database/all/15061865.ann | 530 ++ .../craft/database/all/15061865.txt | 125 + .../craft/database/all/15070402.ann | 1126 ++++ .../craft/database/all/15070402.txt | 131 + .../craft/database/all/15207008.ann | 1246 +++++ .../craft/database/all/15207008.txt | 163 + .../craft/database/all/15238161.ann | 1094 ++++ .../craft/database/all/15238161.txt | 169 + .../craft/database/all/15314655.ann | 1276 +++++ .../craft/database/all/15314655.txt | 237 + .../craft/database/all/15314659.ann | 683 +++ .../craft/database/all/15314659.txt | 223 + .../craft/database/all/15320950.ann | 940 ++++ .../craft/database/all/15320950.txt | 137 + .../craft/database/all/15328533.ann | 539 ++ .../craft/database/all/15328533.txt | 179 + .../craft/database/all/15328538.ann | 682 +++ .../craft/database/all/15328538.txt | 266 + .../craft/database/all/15345036.ann | 1920 +++++++ .../craft/database/all/15345036.txt | 221 + .../craft/database/all/15492776.ann | 1668 ++++++ .../craft/database/all/15492776.txt | 243 + .../craft/database/all/15550985.ann | 1097 ++++ .../craft/database/all/15550985.txt | 255 + .../craft/database/all/15560850.ann | 475 ++ .../craft/database/all/15560850.txt | 101 + .../craft/database/all/15588329.ann | 889 +++ .../craft/database/all/15588329.txt | 153 + .../craft/database/all/15615595.ann | 954 ++++ .../craft/database/all/15615595.txt | 131 + .../craft/database/all/15619330.ann | 595 ++ .../craft/database/all/15619330.txt | 183 + .../craft/database/all/15630473.ann | 1669 ++++++ .../craft/database/all/15630473.txt | 309 ++ .../craft/database/all/15676071.ann | 999 ++++ .../craft/database/all/15676071.txt | 129 + .../craft/database/all/15760270.ann | 1909 +++++++ .../craft/database/all/15760270.txt | 391 ++ .../craft/database/all/15784609.ann | 720 +++ .../craft/database/all/15784609.txt | 107 + .../craft/database/all/15819996.ann | 1105 ++++ .../craft/database/all/15819996.txt | 145 + .../craft/database/all/15836427.ann | 1670 ++++++ .../craft/database/all/15836427.txt | 303 ++ .../craft/database/all/15850489.ann | 834 +++ .../craft/database/all/15850489.txt | 147 + .../craft/database/all/15876356.ann | 748 +++ .../craft/database/all/15876356.txt | 149 + .../craft/database/all/15882093.ann | 1779 ++++++ .../craft/database/all/15882093.txt | 325 ++ .../craft/database/all/15917436.ann | 875 +++ .../craft/database/all/15917436.txt | 161 + .../craft/database/all/15921521.ann | 732 +++ .../craft/database/all/15921521.txt | 137 + .../craft/database/all/15938754.ann | 591 ++ .../craft/database/all/15938754.txt | 151 + .../craft/database/all/16026622.ann | 597 ++ .../craft/database/all/16026622.txt | 167 + .../craft/database/all/16027110.ann | 795 +++ .../craft/database/all/16027110.txt | 143 + .../craft/database/all/16098226.ann | 730 +++ .../craft/database/all/16098226.txt | 151 + .../craft/database/all/16103912.ann | 839 +++ .../craft/database/all/16103912.txt | 199 + .../craft/database/all/16109169.ann | 1154 ++++ .../craft/database/all/16109169.txt | 129 + .../craft/database/all/16110338.ann | 1565 ++++++ .../craft/database/all/16110338.txt | 461 ++ .../craft/database/all/16121255.ann | 1060 ++++ .../craft/database/all/16121255.txt | 213 + .../craft/database/all/16121256.ann | 684 +++ .../craft/database/all/16121256.txt | 203 + .../craft/database/all/16216087.ann | 1470 +++++ .../craft/database/all/16216087.txt | 335 ++ .../craft/database/all/16221973.ann | 1322 +++++ .../craft/database/all/16221973.txt | 201 + .../craft/database/all/16255782.ann | 640 +++ .../craft/database/all/16255782.txt | 157 + .../craft/database/all/16279840.ann | 1371 +++++ .../craft/database/all/16279840.txt | 345 ++ .../craft/database/all/16362077.ann | 1209 ++++ .../craft/database/all/16362077.txt | 269 + .../craft/database/all/16410827.ann | 1429 +++++ .../craft/database/all/16410827.txt | 227 + .../craft/database/all/16433929.ann | 840 +++ .../craft/database/all/16433929.txt | 153 + .../craft/database/all/16462940.ann | 632 +++ .../craft/database/all/16462940.txt | 273 + 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src/ontogpt/evaluation/craft/database/all/17677002.txt create mode 100644 src/ontogpt/evaluation/craft/database/all/17696610.ann create mode 100644 src/ontogpt/evaluation/craft/database/all/17696610.txt create mode 100644 src/ontogpt/evaluation/craft/database/all/annotation.conf diff --git a/src/ontogpt/evaluation/craft/database/all/11319941.ann b/src/ontogpt/evaluation/craft/database/all/11319941.ann new file mode 100644 index 000000000..8915c4bcf --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/11319941.ann @@ -0,0 +1,542 @@ +T1 NCBITaxon:10088 30 35 mouse +T2 UBERON:0002435 36 44 striatum +T3 SO:0000771 58 62 QTLs +T4 GO:0065007 63 71 modulate +T5 CL:0000540 83 89 neuron +T6 UBERON:0002435 124 132 striatum +T7 GO:0065007 157 167 modulating +T8 GO:0050890 194 203 cognitive +T9 UBERON:0002435 239 247 striatal +T10 CL:0002613 239 247;259 265 striatal ... neuron +T11 NCBITaxon:10088 276 280 mice +T12 SO:0000704 344 349 genes +T13 GO:0065007 355 363 modulate +T14 UBERON:0010011 385 398 basal ganglia +T15 UBERON:0000955 410 415 Brain +T16 UBERON:0000955 424 429 brain +T17 UBERON:0002435 434 442 striatal +T18 CL:0002613 434 442;451 457 striatal ... neuron +T19 UBERON:0002435 661 669 Striatal +T20 CL:0000540 703 709 Neuron +T21 CL:0000540 770 777 neurons +T22 UBERON:0002435 791 799 striatal +T23 CL:0002613 791 806 striatal neuron +T24 NCBITaxon:33208 857 864 animals +T25 UBERON:0000955 877 883 brains +T26 UBERON:0002435 895 902 striata +T27 CL:0000540 913 919 neuron +T28 CL:0000540 975 981 neuron +T29 CL:0000540 1067 1073 neuron +T30 SO:0000771 1121 1144 quantitative trait loci +T31 SO:0000771 1146 1150 QTLs +T32 UBERON:0002435 1178 1186 striatal +T33 UBERON:0002435 1301 1309 striatal +T34 UBERON:0000955 1341 1346 brain +T35 CL:0000540 1462 1469 neurons +T36 NCBITaxon:33208 1476 1483 animals +T37 NCBITaxon:10088 1570 1575 mouse +T38 UBERON:0002435 1576 1584 striatum +T39 SO:0000771 1619 1623 QTLs +T40 GO:0065007 1629 1637 modulate +T41 UBERON:0001062 1650 1658 anatomic +T42 UBERON:0005382 1688 1703 dorsal striatum +T43 UBERON:0000125 1717 1724 nucleus +T44 UBERON:0002743 1732 1747 basal forebrain +T45 GO:0065007 1777 1787 modulating +T46 GO:0050890 1814 1823 cognitive +T47 CL:0000540 1859 1866 neurons +T48 UBERON:0002435 1874 1882 striatum +T49 NCBITaxon:10088 1907 1911 mice +T50 NCBITaxon:9606 1941 1947 humans +T51 GO:0042995 1997 2007 projection +T52 CL:1001474 2028 2048 medium spiny neurons +T53 UBERON:0002435 2056 2064 Striatal +T54 CL:0002613 2056 2072 Striatal neurons +T55 SO:0000704 2164 2168 gene +T56 GO:0010467 2164 2179 gene expression +T57 GO:0098793 2220 2224;2233 2241 pre- ... synaptic +T58 GO:0098794 2229 2241 postsynaptic +T59 CL:1001474 2274 2294 medium spiny neurons +T60 UBERON:0002435 2333 2341 striatal +T61 CL:0000099 2342 2354 interneurons +T62 GO:0050890 2426 2435 cognition +T63 http://purl.obolibrary.org/obo/MONDO_0007739 2452 2470 Huntington disease +T64 CL:1001474 2518 2538 medium spiny neurons +T65 http://purl.obolibrary.org/obo/MONDO_0005395 2557 2574 movement disorder +T66 NCBITaxon:9606 2583 2589 humans +T67 NCBITaxon:10088 2605 2610 mouse +T68 SO:0000704 2641 2646 genes +T69 GO:0065007 2661 2668 control +T70 GO:0008283 2673 2686 proliferation +T71 UBERON:0002435 2721 2729 striatal +T72 CL:0002613 2721 2737 striatal neurons +T73 GO:0051867 2799 2816 adaptive behavior +T74 SO:0000704 2862 2869 genetic +T75 SO:0000704 2967 2972 genes +T76 GO:0065007 2991 2999 modulate +T77 UBERON:0002435 3031 3039 striatal +T78 CL:0002613 3031 3047 striatal neurons +T79 GO:0050767 3069 3089 neurogenetic control +T80 UBERON:0002435 3093 3101 striatal +T81 SO:0000704 3258 3265 genetic +T82 SO:0000704 3337 3342 genic +T83 NCBITaxon:10088 3564 3569 mouse +T84 UBERON:0002435 3570 3578 striatum +T85 NCBITaxon:40674 3669 3678 mammalian +T86 UBERON:0001017 3679 3682 CNS +T87 NCBITaxon:10088 3795 3800 mouse +T88 UBERON:0000955 3801 3806 brain +T89 UBERON:0005382 3820 3835 dorsal striatum +T90 SO:0000771 3924 3947 quantitative trait loci +T91 SO:0000771 3949 3953 QTLs +T92 UBERON:0002435 3967 3975 striatal +T93 CL:0002613 3967 3982 striatal neuron +T94 GO:0065007 4036 4043 control +T95 UBERON:0001893 4047 4060 telencephalic +T96 GO:0021537 4047 4072 telencephalic development +T97 SO:0000704 4102 4107 genes +T98 CL:0000540 4123 4129 neuron +T99 GO:0008283 4130 4143 proliferation +T100 UBERON:0002435 4171 4179 striatum +T101 UBERON:0001890 4202 4211 forebrain +T102 SO:0000704 4275 4282 genetic +T103 UBERON:0002435 4326 4334 striatum +T104 CL:0000540 4353 4360 neurons +T105 GO:0065007 4385 4394 modulated +T106 CL:0000540 4520 4528 neuronal +T107 NCBITaxon:10088 4544 4549 mouse +T108 UBERON:0002435 4550 4558 striatum +T109 SO:0000704 4597 4604 genetic +T110 SO:0000771 4620 4623 QTL +T111 CL:0000540 4662 4670 neuronal +T112 NCBITaxon:10088 4689 4694 mouse +T113 UBERON:0002435 4695 4703 striatum +T114 UBERON:0000955 4738 4743 Brain +T115 UBERON:0002435 4755 4763 Striatal +T116 UBERON:0000955 4829 4834 brain +T117 UBERON:0000955 4853 4858 Brain +T118 UBERON:0000955 5029 5034 brain +T119 UBERON:0002435 5192 5200 striatal +T120 UBERON:0000955 5244 5249 brain +T121 UBERON:0002435 5273 5280 striata +T122 NCBITaxon:10088 5335 5339 mice +T123 NCBITaxon:10088 5545 5549 mice +T124 UBERON:0000955 5555 5560 Brain +T125 UBERON:0002435 5724 5732 Striatal +T126 UBERON:0002435 5778 5786 striatal +T127 UBERON:0000955 5810 5815 brain +T128 UBERON:0000955 5858 5863 brain +T129 NCBITaxon:10088 5905 5909 mice +T130 UBERON:0000955 5944 5949 brain +T131 CL:0000540 6067 6073 Neuron +T132 UBERON:0002435 6097 6105 striatum +T133 UBERON:0000955 6119 6125 brains +T134 UBERON:0002435 6139 6146 striata +T135 CL:0000540 6160 6166 neuron +T136 CL:0000540 6285 6291 Neuron +T137 UBERON:0002435 6306 6314 striatum +T138 UBERON:0002435 6355 6363 striatal +T139 CL:0002613 6355 6372 striatal neuronal +T140 UBERON:0002435 6430 6438 Striatal +T141 CL:0002613 6430 6445 Striatal Neuron +T142 UBERON:0002435 6537 6545 striatal +T143 CL:0002613 6537 6553 striatal neurons +T144 CL:0000540 6632 6639 neurons +T145 CL:0000540 6696 6703 neurons +T146 CL:0000540 6734 6741 neurons +T147 CL:0000540 6772 6779 neurons +T148 NCBITaxon:10088 6917 6921 mice +T149 UBERON:0002435 6980 6988 striatal +T150 CL:0000540 7009 7017 neuronal +T151 UBERON:0002435 7047 7055 Striatal +T152 CL:0002613 7047 7062 Striatal Neuron +T153 UBERON:0002435 7176 7184 striatal +T154 CL:0002613 7176 7191 striatal neuron +T155 UBERON:0002435 7229 7237 striatal +T156 CL:0002613 7229 7244 striatal neuron +T157 SO:0000704 7617 7628 genetically +T158 NCBITaxon:33208 7659 7666 animals +T159 NCBITaxon:33208 7885 7892 animals +T160 UBERON:0000955 7893 7898 brain +T161 UBERON:0002435 7936 7944 striatal +T162 UBERON:0002435 7990 7998 Striatal +T163 CL:0000540 8036 8042 neuron +T164 UBERON:0002435 8136 8144 striatal +T165 CL:0000540 8167 8173 neuron +T166 UBERON:0002435 8251 8259 striatal +T167 CL:0002613 8251 8267 striatal neurons +T168 UBERON:0002435 8295 8303 striatal +T169 SO:0000704 8413 8424 genetically +T170 NCBITaxon:33208 8442 8449 animals +T171 UBERON:0000955 8566 8571 Brain +T172 UBERON:0002435 8607 8615 striatal +T173 UBERON:0002435 8651 8659 striatal +T174 CL:0002613 8651 8666 striatal neuron +T175 UBERON:0002435 8714 8722 striatal +T176 CL:0002613 8714 8729 striatal neuron +T177 UBERON:0002435 8970 8978 striatal +T178 UBERON:0000955 8991 8997 brains +T179 UBERON:0002435 9010 9018 striatal +T180 CL:0002613 9010 9025 striatal neuron +T181 UBERON:0002435 9051 9059 striatal +T182 CL:0002613 9051 9066 striatal neuron +T183 UBERON:0002435 9131 9139 striatal +T184 UBERON:0000955 9156 9161 brain +T185 CL:0000540 9173 9179 neuron +T186 UBERON:0002435 9188 9196 Striatal +T187 CL:0000540 9231 9237 neuron +T188 SO:0000771 9297 9300 QTL +T189 UBERON:0002435 9378 9386 striatal +T190 UBERON:0002435 9430 9438 striatal +T191 CL:0002613 9430 9445 striatal neuron +T192 UBERON:0000955 9576 9581 brain +T193 SO:0000771 9673 9676 QTL +T194 UBERON:0000955 9703 9708 brain +T195 UBERON:0002435 9724 9732 striatal +T196 UBERON:0000955 9733 9738 brain +T197 UBERON:0002435 9830 9838 striatal +T198 UBERON:0000955 9906 9911 brain +T199 UBERON:0002435 9946 9954 Striatal +T200 CL:0002613 9946 9954;9966 9972 Striatal ... Neuron +T201 SO:0001023 9984 9991 Alleles +T202 SO:0000771 10161 10164 QTL +T203 UBERON:0000955 10169 10174 brain +T204 NCBITaxon:10088 10314 10318 mice +T205 SO:0000704 10718 10725 genetic +T206 SO:0001026 10864 10870 genome +T207 UBERON:0002435 10970 10978 striatal +T208 SO:0001026 11297 11303 genome +T209 UBERON:0002435 11460 11468 striatal +T210 SO:0001023 11534 11541 Alleles +T211 UBERON:0002435 11652 11660 striatum +T212 SO:0001023 11669 11676 alleles +T213 SO:0001023 11986 11992 allele +T214 SO:0001023 12330 12336 allele +T215 UBERON:0002435 12378 12386 striatal +T216 UBERON:0002435 12503 12511 Striatal +T217 UBERON:0002435 12585 12593 striatal +T218 CL:0002613 12585 12600 striatal neuron +T219 UBERON:0000955 12712 12717 brain +T220 UBERON:0000955 12736 12741 brain +T221 UBERON:0002435 12767 12775 striatum +T222 SO:0001023 12871 12877 allele +T223 UBERON:0000955 12901 12906 brain +T224 UBERON:0001017 12971 12974 CNS +T225 UBERON:0002435 12998 13006 striatum +T226 UBERON:0000955 13140 13145 Brain +T227 GO:0065007 13151 13158 control +T228 UBERON:0002435 13186 13194 striatal +T229 UBERON:0002435 13256 13264 striatal +T230 UBERON:0000955 13305 13310 brain +T231 SO:0001023 13346 13352 allele +T232 GO:0065007 13608 13616 modulate +T233 UBERON:0002435 13617 13625 striatal +T234 CL:0002613 13617 13632 striatal neuron +T235 CL:0000540 13756 13762 neuron +T236 CL:0000540 13952 13959 neurons +T237 UBERON:0002435 13963 13971 striatum +T238 SO:0001023 13989 13995 allele +T239 CL:0000540 14226 14233 neurons +T240 CL:0000540 14294 14301 neurons +T241 UBERON:0002435 14345 14353 striatal +T242 CL:0002613 14345 14361 striatal neurons +T243 SO:0001026 14432 14438 genome +T244 SO:0000771 14506 14509 QTL +T245 UBERON:0002435 14899 14907 Striatal +T246 CL:0002613 14899 14914 Striatal Neuron +T247 SO:0001023 14937 14944 Allelic +T248 UBERON:0002435 15021 15029 striatal +T249 CL:0002613 15021 15036 striatal neuron +T250 SO:0000704 15142 15149 genetic +T251 CL:0000540 15169 15175 neuron +T252 CL:0000540 15216 15222 neuron +T253 UBERON:0002435 15336 15344 striatum +T254 UBERON:0000955 15391 15396 brain +T255 UBERON:0002435 15565 15573 striatal +T256 GO:0065007 15664 15675 controlling +T257 UBERON:0002435 15676 15684 striatal +T258 CL:0002613 15676 15684;15695 15701 striatal ... neuron +T259 UBERON:0002435 15723 15731 Striatal +T260 UBERON:0000955 15762 15767 brain +T261 UBERON:0002435 15837 15845 striatal +T262 CL:0002613 15837 15845;15857 15863 striatal ... neuron +T263 NCBITaxon:10088 15895 15899 mice +T264 UBERON:0000955 15937 15942 brain +T265 SO:0000704 16065 16070 genes +T266 UBERON:0005382 16127 16142 dorsal striatum +T267 UBERON:0000955 16154 16159 brain +T268 SO:0000771 16202 16205 QTL +T269 UBERON:0002435 16262 16270 striatum +T270 UBERON:0000955 16292 16297 brain +T271 SO:0000771 16359 16362 QTL +T272 CL:0000540 16399 16406 neurons +T273 UBERON:0002435 16414 16422 striatum +T274 UBERON:0001017 16513 16516 CNS +T275 UBERON:0002435 16588 16596 striatum +T276 UBERON:0001017 16630 16633 CNS +T277 UBERON:0002435 16649 16657 striatal +T278 CL:0002613 16649 16657;16670 16677 striatal ... neurons +T279 CHEBI:15355 16658 16669 cholinergic +T280 CL:0000108 16658 16677 cholinergic neurons +T281 CHEBI:5613 16806 16817 haloperidol +T282 CL:0000120 16855 16867 granule cell +T283 UBERON:0005381 16855 16876;16881 16894 granule cell layer of ... dentate gyrus +T284 NCBITaxon:10088 16954 16958 mice +T285 CL:0000540 17065 17071 neuron +T286 CL:0000598 17097 17106;17119 17123 pyramidal ... cell +T287 UBERON:0002313 17097 17106;17119 17133;17138 17149 pyramidal ... cell layers of ... hippocampus +T288 UBERON:0002304 17111 17133;17138 17149 dentate cell layers of ... hippocampus +T289 CL:0000120 17156 17168 Granule cell +T290 NCBITaxon:10088 17234 17238 mice +T291 NCBITaxon:10088 17343 17347 mice +T292 UBERON:0001016 17426 17440 nervous system +T293 UBERON:0009050 17455 17465;17470 17484 nucleus of ... solitary tract +T294 UBERON:0018545 17495 17512;17517 17532 spinal nucleus of ... bulbocavernosus +T295 UBERON:0001792 17543 17559 retinal ganglion +T296 CL:0000740 17543 17565 retinal ganglion cells +T297 CL:0000540 17646 17652 neuron +T298 UBERON:0001017 17667 17670 CNS +T299 NCBITaxon:10088 17674 17678 mice +T300 UBERON:0002435 17752 17760 striatum +T301 CL:0000540 17974 17980 neuron +T302 UBERON:0002435 18045 18053 striatal +T303 CL:0002613 18045 18053;18065 18071 striatal ... neuron +T304 CL:0000540 18121 18127 neuron +T305 CL:0000540 18153 18159 neuron +T306 CL:0000540 18305 18313 neuronal +T307 UBERON:0002304 18337 18365;18370 18381 dentate gyrus cell layers of ... hippocampus +T308 NCBITaxon:10088 18398 18403 mouse +T309 CL:0000598 18476 18490 pyramidal cell +T310 UBERON:0000119 18486 18497 cell layers +T311 CL:0000120 18543 18555 granule cell +T312 NCBITaxon:10088 18696 18701 mouse +T313 GO:0065007 18752 18758 govern +T314 CL:0000540 18784 18792 neuronal +T315 CL:0000540 18804 18810 neuron +T316 UBERON:0002435 18850 18858 striatum +T317 UBERON:0001017 18873 18876 CNS +T318 UBERON:0002435 18890 18898 striatum +T319 CL:1001474 18952 18972 medium spiny neurons +T320 GO:0065007 18989 18998 regulated +T321 CL:0000540 19004 19010 neuron +T322 CL:0000540 19087 19095 neuronal +T323 UBERON:0002435 19287 19295 striatal +T324 NCBITaxon:10088 19423 19428 Mouse +T325 UBERON:0000955 19429 19434 Brain +T326 SO:0000771 19462 19465 QTL +T327 UBERON:0002435 19512 19520 striatal +T328 CL:0002613 19512 19528 striatal neurons +T329 UBERON:0000955 19550 19555 brain +T330 NCBITaxon:10088 19652 19657 mouse +T331 UBERON:0001017 19658 19661 CNS +T332 SO:0000704 19691 19695 gene +T333 SO:0000704 19851 19858 genetic +T334 GO:0065007 19936 19945 modulates +T335 UBERON:0002435 19946 19954 striatal +T336 SO:0001026 19969 19975 genome +T337 UBERON:0002435 20085 20093 striatal +T338 CL:0002613 20085 20101 striatal neurons +T339 SO:0000704 20229 20236 genetic +T340 UBERON:0002435 20257 20265 striatal +T341 CL:0002613 20257 20265;20277 20283 striatal ... neuron +T342 SO:0000704 20318 20325 genetic +T343 UBERON:0002435 20344 20352 striatum +T344 NCBITaxon:10088 20545 20550 Mouse +T345 UBERON:0000955 20551 20556 Brain +T346 SO:0000771 20731 20734 QTL +T347 SO:0000704 20841 20845 gene +T348 SO:0001023 20890 20896 allele +T349 SO:0000771 20925 20928 QTL +T350 SO:0000704 21050 21055 genes +T351 SO:0000771 21092 21096 QTLs +T352 NCBITaxon:10088 21168 21173 mouse +T353 NCBITaxon:9606 21178 21183 human +T354 SO:0000704 21232 21236 gene +T355 GO:0010467 21232 21247 gene expression +T356 UBERON:0000955 21266 21271 brain +T357 UBERON:0002435 21276 21284 striatum +T358 SO:0000771 21325 21329 QTLs +T359 SO:0000771 21409 21412 QTL +T360 SO:0000018 21435 21458 cluster of linked genes +T361 NCBITaxon:33208 21538 21545 animals +T362 SO:0000771 21609 21612 QTL +T363 SO:0000704 21687 21692 genes +T364 UBERON:0000955 21709 21714 brain +T365 GO:0007420 21709 21726 brain development +T366 PR:000008241 21744 21748 Grk2 +T367 SO:0000704 21806 21811 genes +T368 GO:0065007 21846 21856 modulating +T369 http://purl.obolibrary.org/obo/MONDO_0007739 21857 21875 Huntington disease +T370 NCBITaxon:10088 21889 21894 mouse +T371 GO:0045202 21958 21966 synaptic +T372 UBERON:0002435 21983 21991 striatum +T373 SO:0000704 22006 22010 gene +T374 PR:000010184 22050 22054 Macs +T375 SO:0000704 22060 22064 gene +T376 MOP:0000124 22078 22091 myristoylated +T377 PR:000010184 22078 22123 myristoylated alanine-rich C kinase substrate +T378 PR:000010184 22125 22131 MARCKS +T379 CHEBI:36357 22147 22155 molecule +T380 UBERON:0001893 22172 22180 cerebral +T381 GO:0021537 22172 22192 cerebral development +T382 PR:000010184 22194 22200 MARCKS +T383 NCBITaxon:10088 22211 22215 mice +T384 http://purl.obolibrary.org/obo/MONDO_0009022 22254 22265;22270 22285 agenesis of corpus callosum +T385 UBERON:0002336 22270 22285 corpus callosum +T386 UBERON:0001890 22311 22320 forebrain +T387 UBERON:0001950 22352 22363 neocortical +T388 PR:000010185 22387 22409 MARCKS-related protein +T389 SO:0000704 22410 22414 gene +T390 GO:0010467 22418 22427 expressed +T391 UBERON:0002435 22435 22443 striatum +T392 UBERON:0000955 22457 22462 brain +T393 GO:0007420 22457 22474 brain development +T394 NCBITaxon:10114 22482 22485 rat +T395 SO:0000771 22513 22516 QTL +T396 GO:0065007 22517 22527 modulating +T397 UBERON:0002435 22528 22536 striatal +T398 CL:0002613 22528 22543 striatal neuron +T399 SO:0000704 22625 22630 genes +T400 UBERON:0001893 22693 22706 telencephalic +T401 GO:0021537 22693 22718 telencephalic development +T402 PR:000017268 22733 22737 Vax1 +T403 PR:000017268 22739 22743 Vax1 +T404 SO:0000704 22769 22773 gene +T405 PR:000011336 22813 22816 Not +T406 SO:0000704 22817 22822 genes +T407 PR:000017268 22824 22828 Vax1 +T408 UBERON:0001890 22885 22894 forebrain +T409 GO:0010467 22903 22912 expressed +T410 UBERON:0002435 22920 22928 striatum +T411 GO:0009790 22936 22949 embryogenesis +T412 CHEBI:36357 22961 22969 molecule +T413 GO:0030424 23000 23004 axon +T414 GO:0007411 23000 23013 axon guidance +T415 UBERON:0002336 23048 23063 corpus callosum +T416 UBERON:0000959 23072 23084 optic chiasm +T417 PR:000017268 23112 23116 Vax1 +T418 NCBITaxon:10088 23126 23130 mice +T419 PR:000017268 23150 23154 Vax1 +T420 CHEBI:36357 23178 23187 molecules +T421 PR:000014841 23198 23212 sonic hedgehog +T422 PR:000012315 23213 23217 Pax2 +T423 PR:000012318 23219 23223 Pax6 +T424 PR:000013771 23229 23231 Rx +T425 GO:0030900 23270 23284;23295 23304 development of ... forebrain +T426 UBERON:0002743 23289 23304 basal forebrain +T427 UBERON:0000955 23316 23321 Brain +T428 CL:0000540 23333 23339 neuron +T429 UBERON:0000955 23397 23402 brain +T430 UBERON:0000955 23436 23441 brain +T431 UBERON:0001017 23480 23483 CNS +T432 UBERON:0000955 23523 23528 brain +T433 NCBITaxon:10088 23611 23615 mice +T434 NCBITaxon:9606 23623 23629 humans +T435 UBERON:0000955 23707 23712 brain +T436 CL:0000540 23724 23730 neuron +T437 CL:0000540 23805 23811 neuron +T438 UBERON:0002435 23883 23891 striatal +T439 CL:0000540 23913 23919 neuron +T440 UBERON:0000125 23935 23942 nucleus +T441 UBERON:0002435 24011 24018 striata +T442 CL:0000540 24058 24065 neurons +T443 UBERON:0002435 24086 24093 striata +T444 UBERON:0002435 24149 24157 striatum +T445 CL:0000540 24197 24203 neuron +T446 SO:0000704 24293 24300 genetic +T447 SO:0000771 24337 24341 QTLs +T448 CL:0000540 24456 24462 neuron +T449 UBERON:0001893 24477 24485 cerebrum +T450 UBERON:0000955 24506 24511 brain +T451 NCBITaxon:10088 24651 24655 mice +T452 UBERON:0000955 24745 24750 brain +T453 UBERON:0002435 24780 24788 striatal +T454 UBERON:0000955 24802 24807 brain +T455 UBERON:0000955 24877 24882 brain +T456 UBERON:0002435 25104 25112 striatal +T457 CL:0002613 25104 25119 striatal neuron +T458 SO:0000771 25179 25183 QTLs +T459 GO:0065007 25189 25197 modulate +T460 UBERON:0001017 25211 25214 CNS +T461 UBERON:0000955 25292 25297 brain +T462 UBERON:0000955 25334 25339 brain +T463 NCBITaxon:33208 25531 25538 animals +T464 UBERON:0000955 25615 25620 brain +T465 UBERON:0000955 25691 25696 brain +T466 SO:0000771 25772 25776 QTLs +T467 GO:0065007 25782 25790 modulate +T468 UBERON:0000955 25797 25802 brain +T469 UBERON:0002037 25819 25829 cerebellar +T470 NCBITaxon:10088 25995 25999 mice +T471 SO:0001026 26056 26063 genomes +T472 SO:0001023 26156 26163 alleles +T473 NCBITaxon:10088 26200 26204 mice +T474 NCBITaxon:10088 26254 26258 mice +T475 NCBITaxon:10088 26382 26386 mice +T476 UBERON:0000955 26577 26583 brains +T477 NCBITaxon:10088 26623 26628 Mouse +T478 UBERON:0000955 26629 26634 Brain +T479 NCBITaxon:10088 26778 26782 Mice +T480 CHEBI:30879 26871 26878 alcohol +T481 CHEBI:15377 26882 26887 water +T482 UBERON:0002084 26929 26943 left ventricle +T483 CHEBI:37586 26954 26970 sodium phosphate +T484 CHEBI:64276 27019 27033 glutaraldehyde +T485 CHEBI:50913 27146 27154 fixative +T486 CHEBI:64276 27161 27175 glutaraldehyde +T487 UBERON:0000033 27258 27262 head +T488 UBERON:0000955 27268 27273 brain +T489 CHEBI:50913 27306 27314 fixative +T490 UBERON:0000955 27378 27384 brains +T491 UBERON:0000955 27411 27417 Brains +T492 CHEBI:16842 27517 27525 formalin +T493 UBERON:0000955 27590 27596 Brains +T494 CHEBI:52815 27719 27736 cresylechtviolett +T495 UBERON:0000955 27874 27879 Brain +T496 UBERON:0000955 27943 27948 brain +T497 UBERON:0000955 28003 28008 brain +T498 UBERON:0000955 28062 28067 brain +T499 UBERON:0000955 28076 28081 Brain +T500 NCBITaxon:10088 28247 28252 Mouse +T501 UBERON:0000955 28253 28258 Brain +T502 UBERON:0002435 28585 28593 striatal +T503 CL:0002613 28585 28593;28605 28611 striatal ... neuron +T504 UBERON:0002435 28743 28751 Striatal +T505 UBERON:0002435 28774 28782 striatum +T506 UBERON:0002435 29094 29102 Striatal +T507 CL:0002613 29094 29109 Striatal Neuron +T508 CL:0002613 29094 29102;29130 29136 Striatal ... Neuron +T509 CL:0000540 29145 29152 Neurons +T510 UBERON:0002435 29359 29367 striatum +T511 CL:0000540 29379 29386 neurons +T512 GO:0005730 29391 29399 nucleoli +T513 UBERON:0002435 29602 29610 striatal +T514 CL:0000540 29696 29702 Neuron +T515 UBERON:0002435 29814 29822 striatum +T516 CL:0000540 29889 29896 neurons +T517 UBERON:0000125 29905 29912 nucleus +T518 UBERON:0002435 30007 30015 striatal +T519 UBERON:0000955 30041 30047 brains +T520 UBERON:0002435 30107 30115 striatal +T521 CL:0000540 30488 30496 neuronal +T522 CL:0000540 30547 30553 neuron +T523 UBERON:0000955 30576 30582 brains +T524 CL:0000540 30723 30729 neuron +T525 SO:0000771 30763 30766 QTL +T526 SO:0001026 30776 30783 Genomic +T527 UBERON:0002106 30807 30814 spleens +T528 NCBITaxon:33208 30821 30828 animals +T529 CHEBI:24866 30842 30846 salt +T530 GO:0030849 30918 30927 autosomes +T531 GO:0000805 30936 30948 X chromosome +T532 NCBITaxon:33208 30978 30985 animals +T533 SO:0001026 31317 31323 genome +T534 SO:0000771 31884 31887 QTL +T535 UBERON:0002435 32034 32042 striatal +T536 UBERON:0000955 32110 32115 brain +T537 SO:0000771 32174 32178 QTLs +T538 UBERON:0002435 32218 32226 striatum +T539 UBERON:0000955 32335 32340 brain +T540 UBERON:0000955 32448 32453 brain +T541 UBERON:0001062 32955 32963 anatomic +T542 NCBITaxon:10088 33526 33530 mice diff --git a/src/ontogpt/evaluation/craft/database/all/11319941.txt b/src/ontogpt/evaluation/craft/database/all/11319941.txt new file mode 100644 index 000000000..954185451 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/11319941.txt @@ -0,0 +1,169 @@ +Complex trait analysis of the mouse striatum: independent QTLs modulate volume and neuron number + +Abstract + +Background + +The striatum plays a pivotal role in modulating motor activity and higher cognitive function. We analyzed variation in striatal volume and neuron number in mice and initiated a complex trait analysis to discover polymorphic genes that modulate the structure of the basal ganglia. + +Results + +Brain weight, brain and striatal volume, neuron-packing density and number were estimated bilaterally using unbiased stereological procedures in five inbred strains (A/J, C57BL/6J, DBA/2J, BALB/cJ, and BXD5) and an F2 intercross between A/J and BXD5. Striatal volume ranged from 20 to 37 mm3. Neuron-packing density ranged from approximately 50,000 to 100,000 neurons/mm3, and the striatal neuron population ranged from 1.4 to 2.5 million. Inbred animals with larger brains had larger striata but lower neuron-packing density resulting in a narrow range of average neuron populations. In contrast, there was a strong positive correlation between volume and neuron number among intercross progeny. We mapped two quantitative trait loci (QTLs) with selective effects on striatal architecture. Bsc10a maps to the central region of Chr 10 (LRS of 17.5 near D10Mit186) and has intense effects on striatal volume and moderate effects on brain volume. Stnn19a maps to distal Chr 19 (LRS of 15 at D19Mit123) and is associated with differences of up to 400,000 neurons among animals. + +Conclusion + +We have discovered remarkable numerical and volumetric variation in the mouse striatum, and we have been able to map two QTLs that modulate independent anatomic parameters. + +Background + +The dorsal striatum is a massive nucleus in the basal forebrain that plays a pivotal role in modulating motor activity and higher cognitive function. Approximately 90% of all neurons in the striatum - 1.5 to 2.5 million in mice [1,2] and 110-200 million in humans [3,4] - belong to an unusual type 'of inhibitory projection cell referred to as medium spiny neurons [5-8]. Striatal neurons are divided into two major subpopulations (patch and matrix), that have somewhat different gene expression profiles and have different patterns of pre- and postsynaptic connections [9-13]. + +Numbers of medium spiny neurons and ratios of these and less numerous striatal interneurons are critical variables that influence motor performance and aspects of cognition. In the case of Huntington disease the loss of 15-30% of the normal complement of medium spiny neurons leads to distinct movement disorder in both humans and transgenic mouse models [14-17]. The subset of genes that normally control the proliferation, differentiation, and survival of striatal neurons [18-20] are therefore of considerable importance in ensuring adaptive behavior at maturity. + +In this study we use a forward genetic approach [21, 22] to begin to map and characterize members of the subset of normally polymorphic genes that specifically modulate the production and survival of striatal neurons. Our analysis of the neurogenetic control of striatal cell populations relies on the combination of two complementary quantitative approaches. The first of these, complex trait analysis, is a comparatively new genetic method that makes it possible to map individual loci that underlie polygenic traits [22,23]. The second consists of a set of unbiased stereological procedures that can be used to obtain cell counts accurately and efficiently from large numbers of cases [24,25]. + +From a technical point of view the mouse striatum has several advantages that make it an excellent target for complex trait analysis of the mammalian CNS. First, it is a large, cytoarchitectonically distinct region comprising approximately 5-6% of the volume of the mouse brain. Second, the dorsal striatum has a comparatively homogenous cellular composition, potentially reducing the number of quantitative trait loci (QTLs) that affect striatal neuron number. Finally, recent experiments on the molecular control of telencephalic development have highlighted a number of genes that influence neuron proliferation and differentiation of the striatum and other neighboring forebrain structures [18][26-30]. + +We report here both neuroanatomic and genetic quantitative evidence that the size of the striatum and the number of neurons contained within it are modulated independently. + +Results + +The results are divided in two sections. The first is a biometric analysis of variation of size and neuronal populations in mouse striatum. The second section is a quantitative genetic dissection and QTL analysis of variation in the size and neuronal population of the mouse striatum. + +Phenotypes + +Strain differences + +Brain Weight and Striatal Volume. Five strains were chosen to represent low, mid, and high brain weights (Fig 1A). Brain weights of the two high strains, BXD5 (540 ± 9 mg SEM) and BALB/cJ (527 ± 13 mg), are significantly greater (P < .001) than that of C57BL/6J (476 ± 5 mg). Similarly, the brain weights of A/J (395 ± 5 mg) and DBA/2J (403 ± 5 mg) are significantly lower than those of the other three strains (P < .001). As anticipated, differences in striatal volume correspond well with differences in brain weight and volume. The striata of BXD5 (31.0 ± 0.9 mm3) and BALB/cJ (32.5 ± 1.6 mm3) mice are significantly larger (P < .001) than those of C57BL/6J (23.6 ± 0.6 mm3), A/J (21.7 ± 1.0 mm3), and DBA/2J (22.8 ± 0.6 mm3) strains. + +Figure 1 + +Histograms of histologic phenotypes for inbred strains of mice.  A. Brain weight differences are apparent between the three categories (F4,28 = 82.1, P < .001). Asterisk indicates significant differences on post-hoc tests (P < .005). B. Striatal volume. There are significant differences in striatal volume between the low brain weight strains (A/J, DBA.2J) and the high brain weight strains (BALB/cJ, BXD5). C57BL6/J mice differ from the high, but not low brain weight strains (F4,28 = 28.9, P < .001). Asterisk indicates significant differences on post-hoc tests (P < .005). C. Neuron-packing density in the striatum. In general, brains with smaller striata have greater neuron-packing density (F4,28 = 17.6, P < .001). Asterisks indicate significant differences on post-hoc tests (P < .005). D. Neuron number in the striatum. There are no significant difference in striatal neuronal number among the five inbred strains (F4,28 = 2.0. ns). + +Striatal Neuron-Packing Density. There is a significant difference among strains in the packing density of striatal neurons (P < .001 for all comparisons). A/J has a higher mean density (84,800 ± 3,500 neurons/mm3) than all strains other than DBA/2J (80,400 ± 2,700 neurons/mm3). BALB/cJ (57,700 ± 2,500 neurons/mm3) and BXD5 (62,700 ± 2,600 neurons/mm3) do not differ significantly from each other, but do differ from all other strains. C57BL/6J (73,100 ± 1,700) differs from all other mice with the exception of DBA/2J. Inbred strains with smaller striatal volumes have higher neuronal packing densities (Fig 1C). + +Striatal Neuron Number. As a result of the reciprocal relation between volume and density, there is no significant difference in striatal neuron number among the five strains. Total striatal neuron numbers ranges over a very modest range - from a low of 1.72 ± .015 million in C57BL/6J to a high of 1.93 ± .035 million in BXD5. + +Correlational Statistics + +Our comparisons are based on five strains, and one consequence of this modest sample size is that sampling errors and intraclass correlations may bias the results [31]. We therefore also analyzed a larger sample of genetically heterogeneous ABF2 intercross animals (an F2 intercross between A/J and BXD5). We first determined that the distribution of all four dependent measures were normally distributed (Fig 2), before subjecting the data to correlational analysis. In this set of animals brain weight correlates significantly with striatal volume (r = .82, df = 42, P < .001, Fig 3A). Striatal volume is negatively correlated with neuron-packing density overall (r = -0.32, df = 42, P < .05), again indicating that the greater the striatal volume, the lower the neuron-packing density (Fig 3B). Despite this relationship, the total population of striatal neurons correlates positively with striatal volume in this larger sample (r = .60, df = 42, P < .001; Fig 3C). In this crucial respect, results from the genetically heterogeneous F2 animals differ from those of inbred strains. + +Figure 2 + +Histograms of distribution of dependent measures in ABDF2 subjects. Brain weight (A: Χ2 = 2.91, df = 2, ns), striatal volume (B: Χ2 = 1.13, df = 2, ns), striatal neuron-packing density C: Χ2 = 1.64, df = 2, ns), and striatal neuron number (D: Χ2 = 0.73, df = 2, ns) are all normally distributed (Kolmogorov-Smirnov Normality Test). + +Figure 3 + +Scatterplots of subjects from an F2 intercross between a BXD5 and A/J inbred strains (ABF2, N = 44) illustrating correlations of striatal volume with brains weight (A), striatal neuron-packing density (B), and striatal neuron number (C). There are significant positive correlations between striatal volume and both brain weight and neuron number. Striatal volume negatively correlates with neuron-packing density in these subjects. ***P < .001; *P < .05. + +QTL Analysis + +The analysis in this section is focused principally on two traits: striatal volume (absolute and residual values), and striatal neuron number (also absolute and residual values). Residuals for these traits were computed for both traits to minimize the influence of brain weight. Two additional traits were mapped to assess specificity of the effects of putative QTL-bearing intervals, namely brain weight and non-striatal brain weight (Table 1). This last parameter was estimated by subtracting the estimated bilateral striatal weight (assuming a specific gravity of 1.0) from that of the whole brain. + +Table 1 + +Linkage Statistics for Striatal Volume and Neuron Number + +** Alleles inherited from BXD5 that increase a value are defined as positive additive effects. † LRS values can be converted to LOD scores by dividing by 4.6. Previously described QTL for brain weight [56]. Column headings:Trait, the phenotype used in linkage analysis; Marker, the symbol of the microsatellite loci used to genotype mice; Chr, the chromosome on which the marker is located; LRS is the likelihood ratio statistic (4.6 x the LOD score); %Var is the percentage of the total phenotypic variance apparently accounted for by differences in genotype in the an interval defined by the marker; P, the point-wise probability that the linkage is a false positive. Add and Dom are estimates of the additive and dominance effects of genetic variation. Units are the same as those of the traits (volume in mm3 or numbers of cells). The two bold loci marked with asterisks achieve genome-wide significance in this sample population. + +The strongest linkage was found between variation in striatal volume and an interval on chromosome 10 between the markers D10Mit194 at 29 cM and D10Mit209 at 49 cM. The likelihood ratio statistic - a value that can be read as a chi-square - peaks in this 20 cM interval at 17.5 and is associated with a P value of 0.00016 (Table 1), equivalent to a LOD score of 3.8 (Fig 4A). The genome-wide probability of achieving a linkage of this strength by chance is <0.05. This locus accounts for as much as a third of the total phenotypic variance in striatal volume and as much as 50% of the genotypic variance (h2 = 0.39). Alleles in this Chr 10 interval that are inherited from the BXD5 parental strain contribute to a significantly larger striatum than do alleles inherited from A/J. Mean bilateral volume corrected for shrinkage for the AA genotype at D10Mit186 (n = 11) is 25.3 ± 1.3 mm3 whereas that for AB and BB genotypes are 29.1 ± 0.7 mm3 and 30.0 ± 0.5 mm3 (n = 14 and 11), respectively. The insignificant difference between BB and AB genotypes suggests that the B allele is dominant. BXD5 is a recombinant inbred strain initially generated by crossing strains C57BL/6J and DBA/2J. Most of the proximal part of Chr 10 in BXD5 is derived from DBA/2J, but a short interval between 40 and 50 cM is derived exclusively from C57BL/6J. This C57BL/6J region corresponds to the peak LRS score. We estimate a single B allele inherited from the BXD5 parent increases striatal volume by approximately 2.0-2.5 mm3. + +Figure 4 + +Plots of LOD scores and LRS for each of the two traits. A. Plot for Striatal volume on Chr. 10. Peak values for the LRS are around 30 cM. B. Plot for striatal neuron number on Chr. 19. Peak values for the LRS are around 50 cM. + +The Chr 10 interval has an appreciable effect on brain size. Variance in brain weight minus that of the striatum is associated with an LRS of 14.2 in the same location between D10Mit106 and D10Mit186. Each B allele adds 20-30 mg to total brain weight. The Chr 10 locus clearly has pleiotropic effects on the CNS, but its effect on the striatum is more intense. Nonetheless, until we know more about the scope of effects, we have opted to give this Chr 10 locus a generic name, Brain size control 10a (Bsc10a). The specific striatal component of the Bsc10a was analyzed by mapping the residual striatal values that corrects for differences in brain volume. The specific effect of a B allele is reduced from 2.5 mm3 to 0.5-1.0 mm3, and the LRS is reduced to 6.9, a value which still has a point-wise probability of only 0.03, indicating a significant independent effect. + +We identified a second strong candidate interval on distal Chr 19 that may modulate striatal neuron number. The LRS peaks at 15.0 (a LOD of 3.26) at one of our more distal markers (D19Mit123, 51 cM, p = .00055). In mapping neuron number we actually used the residual cell population as a trait, and we are therefore confident that this interval has a selective, although not necessarily exclusive, effect on numbers of neurons in striatum (Fig 4B). Each B allele increases the population by approximately 200,000 cells. The AA genotype has an average residual population that is 116,000 less than the mean (i.e., a residual of -116,000; n = 12), the AB heterozygotes have an average of -8,000 neurons (n = 18), and the BB homozygotes have an average of 290,000 neurons (n = 6). Corresponding absolute numbers of striatal neurons for the three genotypes are 1.8, 1.9, and 2.2 million. The two-tailed genome-wide probability of this locus is at the threshold for declaring a QTL (p = 0.035 ± 0.01 two-tailed for an additive model and 0.08 ± 0.2 for a free model). No other chromosomal interval has an LRS score remotely as high as distal Chr 19. The next highest LRS value is only 7.2 on Chr 1 near D1Mit65 and has a point-wise probability that is 50 times higher than the Chr 19 interval. Given these findings we have given the distal chromosome 19 interval the name Striatal Neuron Number 19a (Stnn19a). Allelic differences in this interval account for up to 30% of the total variance in striatal neuron number. As the heritability of this trait is 0.64, this trait can be said to account for over 80% of the genetic variance. Residual neuron counts have a higher LRS than the total neuron counts (LRS of 15.0 vs. 11.9). This indicates that the Chr 19 interval is likely to have selective effect on the striatum. Consistent with this hypothesis, the LRS for brain weight on distal Chr 19 is under 1.0, and weights of all three genotypes average 480 ± 5 mg. Linkage on Chr 19 is not affected at all by remapping with control for the striatal volume locus on Chr 10. Thus, Chr 10 and Chr 19 intervals do not interact or cooperate in controlling striatal volume or neuron number. + +Discussion + +Striatal volume correlates strongly to brain weight. Nonetheless, a significant fraction of the variation in both striatal volume and neuron number among inbred strains of mice can not be predicted on the basis of brain weight or volume. This non-predictable variation is of particular interest to us because it is generated in large part by genes that have more intense or even selective effects on the dorsal striatum than other brain regions. We have succeeded in mapping one QTL with somewhat more intense effects on the volume of the striatum than the rest of the brain to the proximal half of chromosome 10. We have also mapped a QTL with selective effects on number of neurons in the striatum to the distal end of chromosome 19. + +Between-strain variability + +Variation in the size of CNS regions and cell populations is already known to be substantial in the striatum and in many other regions of the CNS. The number of striatal cholinergic neurons, for example, varies 50% among 26 BXD RI lines [32]. Interestingly, this variation appears to be unrelated to susceptibility to haloperidol-induced catalepsy. The volume of the granule cell layer of the dentate gyrus varies as much as 40-80% among different inbred strains of mice [33-35]. More recent experiments using stereologic techniques have reported substantial variation in both neuron number and volume of the pyramidal and dentate cell layers of the hippocampus [36]. Granule cell numbers in NZB/BINJ and DBA/2 were 37-118% greater than C57BL/6J mice, and differences in volume were even larger (up to 150% larger in the DBA/2 as compared to the C57BL/6J mice). There is also substantial among-strain variation in other structures in the nervous system including the nucleus of the solitary tract [37], the spinal nucleus of the bulbocavernosus [38], and retinal ganglion cells [39, 40]. Taken together, these results point to a high level of variability in neuron number in the CNS of mice. + +Based on these findings, we anticipated significant differences in the striatum of inbred strains. We did find large strain differences in volume. What was surprising was that in our set of five highly divergent strains the differences in volume were not matched by significant differences in neuron number. There was in fact a strong inverse relationship between striatal volume and neuron-packing density that led to a remarkably stabile neuron number. The variation in neuron-packing density with volume contrasts somewhat with the report of Abusaad and colleagues [36], who found no significant differences (P = .06) in neuronal packing density in the dentate gyrus cell layers of the hippocampus among the three mouse strains that they examined, but did see a significant difference in the pyramidal cell layers. A recent report demonstrates a 25% range of granule cell packing density among 35 BXD recombinant inbred lines [41], a finding supporting the notion that packing density varies significantly among mouse strains. + +These data suggest that principles that govern the relationship between neuronal volume and neuron-packing density may differ between the striatum and the other CNS regions. The striatum may be a special case - a region in which numbers of medium spiny neurons is more tightly regulated than neuron populations in some other regions. It could also be that measuring specific neuronal subtypes would demonstrate a greater amount of variance than we currently report [32]. Given the relatively small number of strains that we have sampled, our hypothesis of lower variation in striatal cell populations requires a more extensive test, a problem which we are now pursuing using the large numbers of strains in the Mouse Brain Library . + +Verification of QTL Results + +We have quantified the population of striatal neurons on both sides of the brain in 77 cases total. This is a large sample from the perspective of stereological analysis of the mouse CNS, but from the perspective of gene mapping and quantitative genetics this is, of course, a modest-sized sample size and one that will need to be treated as a starting point for more refined genetic analysis. Nonetheless, we have succeeded in mapping one locus, Bsc10a, which modulates striatal volume with a genome-wide significance of P < 0.05. We have also discovered linkage on Chr 19 to variation in the total number of striatal neurons. These mapping data are concordant with our strain comparison and collectively suggest that there is apparently no significant genetic correlation between striatal volume and neuron number. To confirm and refine our genetic dissection of the striatum we plan to analyze the AXB and BXA recombinant inbred (RI) strains generated by crossing A/J with C57BL/6J. This large RI set has already been processed and regenotyped and is now part of the Mouse Brain Library (see and ). An analysis of RI strains can be quickly extended by generating F1 intercrosses between A/J and the subset of RI strains that have recombinations in the QTL intervals on Chrs 10 and 19. Isogenic sets of RI-backcross progeny can be used to test specific models of gene action, for example, the dominance of the B allele at Bsc10a. + +A major goal of QTL mapping is to define loci that affect critical phenotypes with sufficient precision to generate short lists of candidate genes. Generating lists of candidates for QTLs will soon be greatly facilitated by more complete and better annotated mouse and human sequence databases combined with information on gene expression profiles of whole brain and striatum [42]. Once chromosomal positions of the QTLs have been determined to a precision of 1-3 cM, reducing the probability that a QTL actually represents a cluster of linked genes, it will become appropriate to assess strengths of candidates using transgenic animals and by sequence comparisons [43]. + +Interval mapping places the QTL for Bsc10a in the central portion of Chr 10 in proximity with a number of genes known to affect brain development. One of these is Grk2, a member of the family of ionotropic glutamate receptor genes that is thought to play a role in modulating Huntington disease [44]. In the mouse, members of this receptor type act to indirectly down-regulate synaptic activity in the striatum [45]. Another gene that falls into the Bsc10a interval is Macs, the gene encoding the myristoylated alanine-rich C kinase substrate (MARCKS protein). This molecule is important in cerebral development. MARCKS-deficient mice have a high incidence of exencephaly, agenesis of the corpus callosum, and abnormalities other forebrain structure including widespread neocortical ectopias [46, 47]. The MARCKS-related protein gene is expressed in the striatum during early brain development in the rat [48]. + +The location of the QTL modulating striatal neuron number to the distal part of chromosome 19 places it in proximity to a number of genes that have been recently been shown to be important factors in telencephalic development, particularly Vax1. Vax1 is a homeobox-containing gene and is a close relative of the Emx and Not genes. Vax1 is localized during development to the anterior ventral forebrain, and is expressed in the striatum during embryogenesis [28]. This molecule also has an important role in axon guidance: both the anterior portion of the corpus callosum and the optic chiasm are malformed or absent in Vax1 knockout mice [49]. In addition, Vax1 interacts with several molecules including sonic hedgehog,Pax2, Pax6, and Rx that are known to be important during development of the basal forebrain [27, 50]. + +Brain volume and neuron number + +It has previously been shown that differences in brain weight are proportional to total brain DNA content and consequently to total CNS cell number [51, 52]. For this reason, brain weight has been suggested to be a good surrogate measure for total cell number in mice, as in humans [53]. Moreover, previous work has demonstrated a tight link between regional brain volume and neuron number [54, 55], which implies that volumetric measures reliably estimate neuron number. With this literature in mind, we expected that our measures of striatal volume would predict neuron number in this nucleus. With the inbred strains, however, we found that strains with small striata (A/J) had virtually the same number of neurons as those with large striata (BALB/cJ). This result indicates that at least for the striatum, volume is not a reliable indicator of neuron number, and that they may be two independent traits. This conclusion is bolstered at the genetic level by our report of two distinct QTLs for these two morphologic phenotypes. Taken together with previous reports [51-53], we speculate that while total neuron number in the cerebrum may relate to total brain weight, the relationship of these two variables is flexible at the regional level. + +Materials and Methods + +Subjects + +Thirty-four of the 78 mice that we analyzed were common inbred strains that were selected to sample a wide range of brain weights, and by expectation, striatal volumes. Low brain weight strains included A/J (n = 5) and DBA/2J (n = 8). Mid and high brain weight strains include C57BL/6J (n = 10), BALB/cJ (n = 5), and BXD5 (n = 6, formally this recombinant inbred strain is known as BXD-5/Ty). One of the ten C57BL/6J subjects was removed from the analysis because values for striatal neuron number were anomalous with Z scores more than 2.5. + +To map QTLs that modulate variation in CNS size and cell populations we used an F2 intercross between a strain with low brain weight (A/J) and a strain with high brain weight (BXD5). A total of 518 of these ABDF2 progeny were generated, but for this study we selected a subset of 44 cases, of which 36 were fully genotyped (see below). The sample included 20 animals in the lowest and highest quartiles, and 24 cases within 0.5 SD of the mean brain weight. We therefore measured subjects representing the full range of brain weights (see Fig 2A). The ABDF2 intercross has been used previously to map QTLs that modulate total brain weight [56] and cerebellar volume [57]. The BXD5 strain used as the paternal strain in this intercross is a recombinant inbred strain that was derived by crossing C57BL/6J and DBA/2J lines of mice [58]. As a result, ABDF2 progeny are a mixture of three genomes (50% A/J, 25% C57BL/6J, and 25% DBA/2J). However, at any given locus there will be only two alleles, A and B, or A and D. All stocks of mice were obtained from the Jackson Laboratory . ABF2 mice were generated at the University of Tennessee by Dr. Richelle Strom [56] using Jackson Laboratory foundation stock. The F2 mice ranged in age from 35 to 143 days. The standard inbred strains ranged in age from 51 to 365 days. We studied approximately equal numbers of males and females. + +Histological Preparation + +All brains analyzed in this study are part of the Mouse Brain Library (MBL). The MBL is both a physical and Internet resource. High-resolution digital images of sections from all cases are available at . + +Mice were anesthetized deeply with Avertin (1.25% 2,2,2-tribromoethanol and 0.8% tert-pentyl alcohol in water, 0.5-1.0 ml ip) and perfused through the left ventricle with 0.9% sodium phosphate buffered (PB) saline (pH 7.4) followed by 1.25% glutaraldehyde/1.0% paraformaldehyde in 0.1 M PB (pH 7.40) over a period of 2 to 4 min. An additional 10-ml of double-strength fixative (2.5% glutaraldehyde/2.0% paraformaldehyde) was injected for 1 to 2 min at an increased flow rate. The head with brain was placed a vial with the last fixative and stored at 4°C until dissection. + +Following dissection, the brains were weighed immediately. Brains were subsequently shipped to Beth Israel Deaconess Medical Center. They were immersed in fresh 10% formalin for at least one week before being embedding in celloidin [59]. Brains were cut on a sliding microtome at 30 μm in either horizontal or coronal planes. Free-floating sections were stained with cresylechtviolett and four series of every tenth section were mounted on slides and coverslipped (see for further details). + +Histologic Phenotypes + +Total Brain Volume + +To accurately estimate histological shrinkage for each brain in the sample, we determined the volume of the entire brain and took a ratio of this value to the original fixed brain weight. Brain volumes were determined from serial sections using point counting and Cavalieri's rule. High-resolution (4.5 μm/pixel) images of entire sections were taken from the Mouse Brain Library, and point counting was performed on these images using NIH Image 1.55 and an Apple Macintosh computer . If the criteria for using the Cavalieri's estimator were not met (due to missing or damaged sections), a measurement method involving piecewise parabolic integration was employed [60]. Subsequent measurements of striatal volume and neuron packing density were corrected for volumetric shrinkage. The average shrinkage was 62.2 ± 0.4% (a mean residual volume of 37.8%). + +Striatal Volume + +Volume of the striatum was also determined from serial section analysis using point counting and Cavalieri's rule. Images from the sections were captured at 12.5 x and were projected onto a video monitor. Point counting was performed as above. Volume was computed separately for the right and left sides and corrected for shrinkage. + +Striatal Neuron-Packing Density and Neuron Number + +Neurons were counted using the 3-dimensional counting software of Williams and Rakic [24]. A series of six contiguous counting boxes (each 40 x 65 x 20 μm) aligned in a 3 x 2 matrix were placed randomly within the striatum, and those neurons the nucleoli of which were in focus were counted as described previously [61, 62]. This large functional counting box (80 x 195 x 20 μm) was chosen to minimize sampling variance by ensuring an equitable sampling of striatal patch and matrix. Two of these large fields were counted in each of the hemispheres. Neuron-packing density was computed as the number of cells/mm3 corrected for shrinkage. Multiplying the volume of the striatum by its cell-packing density permitted estimation of the number of neurons in that nucleus. + +Reliability + +We determined test-retest reliability by having an observer blindly re-measure striatal volume on a subset of 10 brains from the collection. The observer not only re-measured the striatal volume from the same series of sections as the original measure, but also estimated volume from a second series of 1 in 10 sections offset by 5 sections from the previous series. The correlations among the three estimations ranged from .95 to .99 (P < .05), indicating a high degree of reliability for this dependent variable. + +We assessed reliability of our estimates of neuronal numbers by having an observer blindly re-estimate neuron number in the same 10 brains above. The intra-observer correlation for this measure was .81 (P < .05), which is similar to the reliability seen in previous estimates of neuron number [39, 40]. + +Genotyping and QTL Mapping + +Genomic DNA was extracted from spleens of F2 animals using a high-salt procedure [63]. A set of 82 microsatellite loci distributed across all autosomes and the X chromosome were typed in a set of ABDF2 animals using a standard PCR protocol [64, 65] as detailed in Zhou and Williams [66]. F2 genotypes were entered into a spreadsheet program and transferred to Map Manager QTb28 for mapping and permutation analysis [67]. Map Manager implements both simple and composite interval mapping methods described by Haley and Knott [68]. Two-tailed genome-wide significance levels were estimated by comparing the highest likelihood ratio statistic (LRS) of correctly ordered data sets with LRSs computed for 10,000 permutations of those same data sets [69]. LRS scores can be converted to LOD scores by dividing by 4.6. The 2-LOD support interval of linkage was estimated directly from interval maps. The approximate 95% support interval was estimated by application of equations in Darvasi and Soller [70]. With a modest sample size such as we have been able to examine using unbiased stereological methods, even a QTL responsible for 30 to 50% of the variance. is associated with a 95% interval of 20 to 30 cM. + +Regression Analysis of Trait Values + +The unadjusted striatal estimates vary to a large extent as a result of variation in total brain weight. However, one of our goals in this study is to map QTLs with relatively intense effects on the striatum. For this reason we also have corrected all of the parameters used in the mapping analysis for variation in brain weight using linear regression analysis. We have mapped data with and without compensation for variance in brain weight. The corrected values are referred to as residuals. + +Analysis + +All data were analyzed using regression, correlation, and ANOVA statistical tests (see StrAnatData.xls for original data used to perform this analysis). A Bonferroni/Dunn correction was used for post hoc examination of significant main effects in the ANOVA. This post-hoc test is functionally identical to a Fisher PLSD, but the alpha level is more conservative (.005). + +Supplementary Material + +StrAnatData.xls + +This file contains anatomic data for each of the subjects used in the current experiment. + +Click here to download StrAnatData.xls + +StrMap.qtx + +This file contains the genotyping data from the 82 markers used in the current experiment, in MapManager QTX format. + +Click here to download StrMap.qtx + +Acknowledgements + +This work was supported, in part, by grants HD20806 and NS35485 from the Public Health Service of the USA. The authors wish to thank Dr. Jing Gu, Aaron Levine, Anna Ohlis, and Stefany Palmieri for technical assistance. We thank Richelle Strom for generating the F2 intercross mice. diff --git a/src/ontogpt/evaluation/craft/database/all/11532192.ann b/src/ontogpt/evaluation/craft/database/all/11532192.ann new file mode 100644 index 000000000..a9508ce46 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/11532192.ann @@ -0,0 +1,529 @@ +T1 UBERON:0000970 5 11 ocular +T2 SO:0000704 24 35 genetically +T3 NCBITaxon:10088 45 49 mice +T4 SO:0000704 124 131 genetic +T5 UBERON:0000970 155 161 ocular +T6 NCBITaxon:10088 180 184 mice +T7 NCBITaxon:40674 195 202 mammals +T8 SO:0000704 259 270 genetically +T9 NCBITaxon:10088 280 285 mouse +T10 SO:0000704 445 449 gene +T11 NCBITaxon:10088 503 508 mouse +T12 GO:0007568 612 617 aging +T13 GO:0007623 692 706 diurnal rhythm +T14 SO:0001023 793 799 allele +T15 SO:0000704 829 833 gene +T16 SO:0000704 918 922 gene +T17 http://purl.obolibrary.org/obo/MONDO_0011122 944 951 obesity +T18 http://purl.obolibrary.org/obo/MONDO_0005015 956 964 diabetes +T19 http://purl.obolibrary.org/obo/MONDO_0043209 991 997 Albino +T20 NCBITaxon:10088 1007 1011 mice +T21 CHEBI:26130 1087 1096 pigmented +T22 SO:0000704 1125 1136 Genetically +T23 NCBITaxon:10088 1146 1151 mouse +T24 SO:0000704 1271 1278 genetic +T25 GO:0065007 1340 1350 regulation +T26 http://purl.obolibrary.org/obo/MONDO_0011122 1377 1384 obesity +T27 http://purl.obolibrary.org/obo/MONDO_0005015 1389 1397 diabetes +T28 NCBITaxon:10088 1414 1419 mouse +T29 NCBITaxon:9606 1444 1450 humans +T30 NCBITaxon:species 1461 1468 species +T31 SO:0000704 1513 1518 genes +T32 NCBITaxon:10088 1590 1594 mice +T33 GO:0065007 1673 1683 regulation +T34 http://purl.obolibrary.org/obo/MONDO_0000001 1688 1695 disease +T35 http://purl.obolibrary.org/obo/MONDO_0005041 1753 1761 Glaucoma +T36 http://purl.obolibrary.org/obo/MONDO_0001941 1784 1793 blindness +T37 http://purl.obolibrary.org/obo/MONDO_0005041 1843 1851 Glaucoma +T38 UBERON:0001792 1861 1877 retinal ganglion +T39 CL:0000740 1861 1882 retinal ganglion cell +T40 GO:0008219 1878 1888 cell death +T41 UBERON:0000970 1893 1898 optic +T42 UBERON:0000970 1956 1962 ocular +T43 http://purl.obolibrary.org/obo/MONDO_0005041 2039 2047 glaucoma +T44 SO:0000704 2055 2062 genetic +T45 SO:0000704 2156 2161 genes +T46 http://purl.obolibrary.org/obo/MONDO_0005041 2181 2189 glaucoma +T47 NCBITaxon:1 2241 2252 individuals +T48 http://purl.obolibrary.org/obo/MONDO_0005041 2290 2298 glaucoma +T49 SO:0000704 2317 2322 genes +T50 http://purl.obolibrary.org/obo/MONDO_0005041 2356 2364 glaucoma +T51 NCBITaxon:33208 2402 2408 animal +T52 NCBITaxon:40674 2540 2549 mammalian +T53 NCBITaxon:10088 2587 2592 mouse +T54 NCBITaxon:10088 2603 2607 mice +T55 http://purl.obolibrary.org/obo/MONDO_0000001 2695 2702 disease +T56 SO:0000704 2712 2717 genes +T57 NCBITaxon:9606 2756 2761 human +T58 SO:0001026 2762 2768 genome +T59 NCBITaxon:10088 2809 2814 mouse +T60 http://purl.obolibrary.org/obo/MONDO_0005041 2834 2842 glaucoma +T61 NCBITaxon:10088 2860 2864 Mice +T62 SO:0000704 2920 2925 genes +T63 UBERON:0000970 2986 2991 optic +T64 UBERON:0000966 3002 3008 retina +T65 http://purl.obolibrary.org/obo/MONDO_0005041 3012 3024 glaucomatous +T66 NCBITaxon:10088 3098 3103 mouse +T67 NCBITaxon:10088 3134 3138 mice +T68 NCBITaxon:10088 3219 3223 mice +T69 NCBITaxon:10088 3274 3279 mouse +T70 UBERON:0001766 3373 3389 anterior chamber +T71 UBERON:0002398 3440 3445 hands +T72 UBERON:0000970 3454 3460 ocular +T73 UBERON:0000970 3592 3598 ocular +T74 NCBITaxon:10088 3628 3633 mouse +T75 NCBITaxon:10088 3915 3920 mouse +T76 SO:0000704 4330 4341 genetically +T77 NCBITaxon:10088 4351 4356 mouse +T78 NCBITaxon:10088 4420 4424 mice +T79 NCBITaxon:10088 4499 4503 mice +T80 NCBITaxon:10088 4506 4510 Mice +T81 NCBITaxon:10088 4671 4675 mice +T82 NCBITaxon:10088 4728 4732 mice +T83 UBERON:0000970 4875 4879 eyes +T84 UBERON:0001062 4934 4942 anatomic +T85 UBERON:0006206 5030 5048 iridocorneal angle +T86 CHEBI:15377 5053 5060 aqueous +T87 UBERON:0003956 5053 5086 aqueous humor drainage structures +T88 UBERON:0001766 5103 5119 anterior chamber +T89 NCBITaxon:10088 5175 5179 mice +T90 NCBITaxon:10088 5217 5221 mice +T91 http://purl.obolibrary.org/obo/MONDO_0005041 5273 5281 glaucoma +T92 NCBITaxon:9606 5285 5291 humans +T93 NCBITaxon:10088 5324 5328 mice +T94 UBERON:0000970 5382 5387 optic +T95 UBERON:0000966 5399 5406 retinas +T96 http://purl.obolibrary.org/obo/MONDO_0005041 5432 5440 glaucoma +T97 UBERON:0006206 5460 5479 iridocorneal angles +T98 UBERON:0006206 5518 5536 iridocorneal angle +T99 CHEBI:15377 5555 5562 aqueous +T100 UBERON:0003956 5555 5588 aqueous humor drainage structures +T101 UBERON:0004029 5590 5605 Schlemm's canal +T102 UBERON:0004029 5607 5609 SC +T103 UBERON:0005969 5615 5634 trabecular meshwork +T104 UBERON:0005969 5636 5638 TM +T105 CHEBI:26130 5742 5749 Pigment +T106 CL:0000235 5757 5768 macrophages +T107 NCBITaxon:9606 5783 5788 human +T108 NCBITaxon:10088 5844 5848 mice +T109 UBERON:0000964 6024 6030 cornea +T110 UBERON:0000964 6032 6033 C +T111 UBERON:0001769 6039 6043 iris +T112 UBERON:0001769 6050 6051 I +T113 CHEBI:15377 6080 6087 Aqueous +T114 UBERON:0001796 6080 6093 Aqueous humor +T115 UBERON:0000970 6201 6207 Ocular +T116 NCBITaxon:10088 6387 6391 mice +T117 UBERON:0000970 6395 6399 eyes +T118 NCBITaxon:10088 6470 6474 mice +T119 UBERON:0002506 6685 6700 iris epithelium +T120 UBERON:0000964 6751 6758 corneal +T121 http://purl.obolibrary.org/obo/MONDO_0005594 6759 6767 scarring +T122 UBERON:0000964 6831 6837 cornea +T123 UBERON:0000966 6860 6867 retinal +T124 http://purl.obolibrary.org/obo/MONDO_0004580 6860 6880 retinal degeneration +T125 PR:000012479 6919 6924 Pde6b +T126 SO:0000704 7330 7337 genetic +T127 NCBITaxon:10088 7580 7584 mice +T128 NCBITaxon:10088 8288 8292 mice +T129 NCBITaxon:10088 8590 8594 mice +T130 NCBITaxon:10088 8885 8889 mice +T131 NCBITaxon:33208 9151 9158 animals +T132 NCBITaxon:10088 9199 9204 mouse +T133 NCBITaxon:10088 9436 9440 mice +T134 NCBITaxon:10088 10175 10179 mice +T135 NCBITaxon:10088 10222 10226 mice +T136 NCBITaxon:10088 10336 10340 mice +T137 NCBITaxon:10088 10561 10565 mice +T138 NCBITaxon:10088 11003 11007 mice +T139 NCBITaxon:10088 11089 11093 mice +T140 NCBITaxon:10088 11358 11362 mice +T141 NCBITaxon:10088 11514 11518 mice +T142 http://purl.obolibrary.org/obo/MONDO_0005041 11749 11761 glaucomatous +T143 NCBITaxon:10088 12651 12655 mice +T144 CHEBI:6121 12891 12899 ketamine +T145 CHEBI:6121 13009 13017 ketamine +T146 NCBITaxon:10088 13160 13165 mouse +T147 SO:0000704 13282 13293 genetically +T148 NCBITaxon:10088 13307 13311 mice +T149 NCBITaxon:10088 13778 13782 mice +T150 NCBITaxon:10088 14194 14198 mice +T151 NCBITaxon:10088 14724 14728 mice +T152 CHEBI:38867 14790 14800 anesthetic +T153 CHEBI:6121 14836 14844 ketamine +T154 NCBITaxon:10088 15054 15058 mice +T155 NCBITaxon:10088 15231 15236 mouse +T156 NCBITaxon:10088 15459 15463 mice +T157 NCBITaxon:10088 15696 15700 mice +T158 NCBITaxon:10088 15788 15792 mice +T159 NCBITaxon:10088 15918 15922 mice +T160 UBERON:0000178 16120 16125 Blood +T161 UBERON:0000178 16209 16214 blood +T162 NCBITaxon:10088 16235 16239 mice +T163 UBERON:0000178 16330 16335 blood +T164 UBERON:0000178 16415 16420 blood +T165 NCBITaxon:10088 16496 16500 mice +T166 NCBITaxon:10088 16642 16646 mice +T167 PR:000010873 16742 16746 Myoc +T168 SO:0001023 16747 16754 alleles +T169 PR:000010873 16816 16824 myocilin +T170 SO:0000704 16825 16829 gene +T171 PR:000010873 16831 16835 MYOC +T172 NCBITaxon:9606 16843 16848 human +T173 http://purl.obolibrary.org/obo/MONDO_0005041 16849 16857 glaucoma +T174 SO:0001023 16875 16882 allelic +T175 NCBITaxon:10088 16900 16905 mouse +T176 PR:000010873 16906 16910 Myoc +T177 SO:0000704 16911 16915 gene +T178 NCBITaxon:10088 16939 16944 mouse +T179 SO:0000704 16970 16974 gene +T180 SO:0001023 17028 17035 alleles +T181 SO:0001023 17066 17073 alleles +T182 SO:0000167 17111 17119 promoter +T183 SO:0001023 17249 17255 allele +T184 SO:0001023 17267 17273 allele +T185 SO:0001023 17334 17340 allele +T186 SO:0000147 17408 17412 exon +T187 SO:0000167 17460 17468 promoter +T188 SO:0001023 17514 17520 allele +T189 PR:000010873 17621 17625 Myoc +T190 SO:0001023 17626 17633 alleles +T191 SO:0001023 17688 17695 alleles +T192 PR:000010873 17699 17703 Myoc +T193 NCBITaxon:10088 17713 17718 mouse +T194 NCBITaxon:10088 17739 17744 Mouse +T195 SO:0001023 17771 17777 allele +T196 SO:0000028 17790 17792 bp +T197 SO:0000167 17810 17818 promoter +T198 SO:0001023 17880 17886 allele +T199 SO:0000704 17975 17982 Genetic +T200 GO:0000806 18004 18016 Y Chromosome +T201 GO:0000806 18022 18034 Y chromosome +T202 UBERON:0000178 18074 18079 blood +T203 NCBITaxon:10114 18104 18108 rats +T204 GO:0000806 18133 18145 Y chromosome +T205 GO:0000806 18292 18304 Y chromosome +T206 NCBITaxon:10088 18399 18403 mice +T207 SO:0000704 18447 18454 genetic +T208 GO:0000806 18611 18623 Y chromosome +T209 GO:0000806 18625 18630 Y Chr +T210 GO:0000806 18641 18646 Y Chr +T211 SO:0001023 18802 18809 alleles +T212 http://purl.obolibrary.org/obo/MONDO_0005015 18919 18927 diabetes +T213 http://purl.obolibrary.org/obo/MONDO_0011122 18932 18939 obesity +T214 UBERON:0000178 19180 19185 Blood +T215 http://purl.obolibrary.org/obo/MONDO_0005015 19252 19260 diabetic +T216 NCBITaxon:10088 19610 19614 mice +T217 SO:0000704 19644 19655 genetically +T218 SO:0001023 19690 19697 alleles +T219 SO:0000704 19710 19714 gene +T220 NCBITaxon:10088 19781 19785 mice +T221 SO:0000704 19825 19832 genetic +T222 http://purl.obolibrary.org/obo/MONDO_0011122 19858 19865 obesity +T223 http://purl.obolibrary.org/obo/MONDO_0005015 19870 19878 diabetes +T224 NCBITaxon:10088 19906 19910 mice +T225 SO:0000704 19921 19932 genetically +T226 http://purl.obolibrary.org/obo/MONDO_0011122 20036 20043 obesity +T227 http://purl.obolibrary.org/obo/MONDO_0005015 20048 20056 diabetes +T228 http://purl.obolibrary.org/obo/MONDO_0011122 20173 20178 obese +T229 http://purl.obolibrary.org/obo/MONDO_0005015 20180 20188 diabetic +T230 http://purl.obolibrary.org/obo/MONDO_0011122 20242 20247 obese +T231 http://purl.obolibrary.org/obo/MONDO_0005015 20253 20261 diabetic +T232 http://purl.obolibrary.org/obo/MONDO_0043209 20337 20345 albinism +T233 NCBITaxon:10088 20373 20377 mice +T234 CHEBI:26130 20395 20404 pigmented +T235 http://purl.obolibrary.org/obo/MONDO_0043209 20408 20414 albino +T236 http://purl.obolibrary.org/obo/MONDO_0043209 20420 20426 albino +T237 NCBITaxon:10088 20427 20431 mice +T238 SO:0001023 20476 20482 allele +T239 CHEBI:26130 20535 20544 pigmented +T240 NCBITaxon:10088 20575 20579 mice +T241 CHEBI:26130 20673 20682 pigmented +T242 NCBITaxon:10088 20683 20687 mice +T243 NCBITaxon:10088 20764 20768 mice +T244 CHEBI:26130 20854 20863 pigmented +T245 NCBITaxon:10088 20867 20871 mice +T246 http://purl.obolibrary.org/obo/MONDO_0043209 20900 20906 albino +T247 NCBITaxon:10088 20910 20914 mice +T248 NCBITaxon:10088 21135 21139 mice +T249 NCBITaxon:10088 21304 21308 mice +T250 NCBITaxon:10088 21406 21410 mice +T251 NCBITaxon:10088 21558 21562 mice +T252 SO:0000704 21704 21711 genetic +T253 SO:0000704 21752 21763 genetically +T254 NCBITaxon:10088 21774 21779 mouse +T255 SO:0000704 21965 21972 genetic +T256 SO:0000704 21996 22001 genes +T257 NCBITaxon:10088 22103 22107 mice +T258 NCBITaxon:10088 22172 22176 mice +T259 SO:0000771 22181 22205 quantitative trait locus +T260 SO:0000771 22207 22210 QTL +T261 SO:0000704 22575 22580 genes +T262 http://purl.obolibrary.org/obo/MONDO_0005041 22594 22602 glaucoma +T263 CHEBI:15377 22667 22674 aqueous +T264 UBERON:0001796 22667 22680 aqueous humor +T265 http://purl.obolibrary.org/obo/MONDO_0005041 22736 22744 glaucoma +T266 CHEBI:38867 22842 22859 anesthetic agents +T267 CHEBI:6121 22890 22898 Ketamine +T268 CHEBI:6121 22965 22973 ketamine +T269 NCBITaxon:species 23045 23052 species +T270 NCBITaxon:10088 23205 23209 mice +T271 UBERON:0001179 23267 23277 peritoneal +T272 CHEBI:6121 23300 23308 ketamine +T273 SO:0000704 23390 23401 genetically +T274 NCBITaxon:10114 23847 23850 rat +T275 UBERON:0004535 23902 23916 cardiovascular +T276 UBERON:0001179 23943 23953 peritoneal +T277 CHEBI:6121 23972 23980 ketamine +T278 NCBITaxon:10114 23997 24001 rats +T279 UBERON:0000178 24051 24056 blood +T280 http://purl.obolibrary.org/obo/MONDO_0005468 24127 24138 hypotensive +T281 UBERON:0001179 24233 24245 peritoneally +T282 CHEBI:6121 24259 24267 ketamine +T283 NCBITaxon:10114 24329 24332 rat +T284 NCBITaxon:10114 24832 24835 rat +T285 NCBITaxon:10114 24921 24924 rat +T286 NCBITaxon:10088 24929 24934 mouse +T287 NCBITaxon:10114 25095 25099 rats +T288 NCBITaxon:10088 25123 25127 mice +T289 NCBITaxon:species 25246 25253 species +T290 CHEBI:6121 25313 25321 ketamine +T291 NCBITaxon:10114 25371 25374 rat +T292 NCBITaxon:10088 25379 25384 mouse +T293 NCBITaxon:10088 25401 25405 mice +T294 CHEBI:23888 25770 25774 drug +T295 GO:0017144 25770 25785 drug metabolism +T296 NCBITaxon:10114 25818 25822 rats +T297 NCBITaxon:10088 25942 25946 mice +T298 CHEBI:35186 25950 25958 terpenes +T299 CHEBI:35186 25960 25967 Terpene +T300 CHEBI:35186 26012 26020 terpenes +T301 CHEBI:23888 26045 26049 drug +T302 CHEBI:38867 26089 26106 anesthetic agents +T303 NCBITaxon:10114 26115 26119 rats +T304 NCBITaxon:10088 26124 26128 mice +T305 http://purl.obolibrary.org/obo/MONDO_0005015 26226 26234 diabetes +T306 http://purl.obolibrary.org/obo/MONDO_0005044 26236 26257 vascular hypertension +T307 UBERON:0001637 26259 26267 arterial +T308 http://purl.obolibrary.org/obo/MONDO_0005468 26268 26279 hypotension +T309 GO:1903522 26305 26329 regulation of blood flow +T310 UBERON:0000178 26319 26324 blood +T311 http://purl.obolibrary.org/obo/MONDO_0005041 26341 26349 glaucoma +T312 http://purl.obolibrary.org/obo/MONDO_0005041 26411 26419 glaucoma +T313 UBERON:0000178 26496 26501 blood +T314 http://purl.obolibrary.org/obo/MONDO_0011122 26512 26519 obesity +T315 http://purl.obolibrary.org/obo/MONDO_0005015 26524 26532 diabetes +T316 SO:0000704 26652 26663 genetically +T317 NCBITaxon:10088 26676 26681 mouse +T318 NCBITaxon:10088 26784 26788 mice +T319 NCBITaxon:10088 26804 26808 mice +T320 NCBITaxon:10088 26912 26916 mice +T321 NCBITaxon:10088 26976 26980 mice +T322 NCBITaxon:10088 27053 27057 mice +T323 NCBITaxon:10088 27103 27107 mice +T324 NCBITaxon:9606 27234 27239 human +T325 NCBITaxon:10088 27291 27295 mice +T326 NCBITaxon:10088 27396 27401 mouse +T327 http://purl.obolibrary.org/obo/MONDO_0005041 27534 27546 glaucomatous +T328 NCBITaxon:9606 27839 27844 human +T329 NCBITaxon:10088 28143 28147 mice +T330 UBERON:0000178 28241 28246 Blood +T331 NCBITaxon:9606 28274 28279 human +T332 UBERON:0000178 28341 28346 blood +T333 UBERON:0000178 28408 28413 blood +T334 UBERON:0007023 28441 28446 adult +T335 NCBITaxon:10088 28454 28458 mice +T336 NCBITaxon:10088 28472 28477 mouse +T337 UBERON:0000178 28493 28498 blood +T338 UBERON:0000178 28615 28620 blood +T339 UBERON:0000178 28702 28707 blood +T340 NCBITaxon:10088 28736 28741 mouse +T341 http://purl.obolibrary.org/obo/MONDO_0011122 28761 28768 Obesity +T342 http://purl.obolibrary.org/obo/MONDO_0005015 28773 28781 diabetes +T343 http://purl.obolibrary.org/obo/MONDO_0011122 28783 28790 Obesity +T344 http://purl.obolibrary.org/obo/MONDO_0005041 28906 28914 glaucoma +T345 http://purl.obolibrary.org/obo/MONDO_0005015 28938 28946 diabetes +T346 http://purl.obolibrary.org/obo/MONDO_0005015 28969 28977 diabetes +T347 http://purl.obolibrary.org/obo/MONDO_0011122 28982 28989 obesity +T348 http://purl.obolibrary.org/obo/MONDO_0005041 29042 29050 glaucoma +T349 SO:0000704 29077 29084 genetic +T350 http://purl.obolibrary.org/obo/MONDO_0011122 29110 29117 obesity +T351 http://purl.obolibrary.org/obo/MONDO_0005015 29122 29130 diabetes +T352 NCBITaxon:10088 29168 29172 mice +T353 SO:0000704 29183 29194 genetically +T354 http://purl.obolibrary.org/obo/MONDO_0011122 29323 29330 obesity +T355 http://purl.obolibrary.org/obo/MONDO_0005015 29335 29343 diabetes +T356 http://purl.obolibrary.org/obo/MONDO_0011122 29349 29354 obese +T357 http://purl.obolibrary.org/obo/MONDO_0005015 29356 29364 diabetic +T358 NCBITaxon:10088 29365 29369 mice +T359 http://purl.obolibrary.org/obo/MONDO_0005015 29407 29415 diabetic +T360 http://purl.obolibrary.org/obo/MONDO_0011122 29490 29497 obesity +T361 http://purl.obolibrary.org/obo/MONDO_0005015 29501 29509 diabetes +T362 http://purl.obolibrary.org/obo/MONDO_0011122 29602 29607 obese +T363 http://purl.obolibrary.org/obo/MONDO_0005015 29612 29620 diabetic +T364 http://purl.obolibrary.org/obo/MONDO_0005015 29624 29632 diabetic +T365 http://purl.obolibrary.org/obo/MONDO_0011122 29637 29642 obese +T366 NCBITaxon:10088 29643 29647 mice +T367 NCBITaxon:9606 29809 29815 humans +T368 NCBITaxon:33208 29831 29838 animals +T369 GO:0007623 29885 29899 diurnal rhythm +T370 CHEBI:15377 29930 29937 aqueous +T371 UBERON:0001796 29930 29943 aqueous humor +T372 NCBITaxon:9986 30013 30020 rabbits +T373 NCBITaxon:9606 30025 30031 humans +T374 CHEBI:15377 30076 30083 aqueous +T375 UBERON:0001796 30076 30089 aqueous humor +T376 NCBITaxon:10088 30236 30240 mice +T377 NCBITaxon:10114 30429 30433 rats +T378 NCBITaxon:10114 30444 30448 rats +T379 NCBITaxon:9986 30453 30460 rabbits +T380 NCBITaxon:10088 30585 30589 mice +T381 CL:0000604 30862 30865;30875 30889 rod ... photoreceptors +T382 CL:0000573 30870 30889 cone photoreceptors +T383 NCBITaxon:10088 30928 30932 mice +T384 PR:000012479 30961 30966 Pde6b +T385 GO:0042752 31012 31032 circadian regulation +T386 NCBITaxon:10088 31086 31090 mice +T387 CL:0000604 31099 31103 rods +T388 CL:0000573 31108 31113 cones +T389 GO:0007623 31323 31337 diurnal rhythm +T390 UBERON:0000970 31346 31352 ocular +T391 NCBITaxon:10088 31365 31369 mice +T392 NCBITaxon:10088 31473 31478 mouse +T393 GO:0065007 31542 31553 controlling +T394 GO:0007623 31554 31569 diurnal rhythms +T395 http://purl.obolibrary.org/obo/MONDO_0005041 31605 31613 glaucoma +T396 CHEBI:17544 31652 31663 Bicarbonate +T397 CHEBI:15377 31691 31698 aqueous +T398 UBERON:0001796 31691 31704 aqueous humor +T399 UBERON:0010427 31724 31741 ciliary processes +T400 SO:0001060 31842 31849 isoform +T401 UBERON:0010427 31896 31913 ciliary processes +T402 SO:0000704 31951 31958 genetic +T403 NCBITaxon:10088 31981 31985 mice +T404 SO:0000704 32024 32028 gene +T405 PR:000004920 32049 32053 CAIV +T406 UBERON:0010427 32096 32113 ciliary processes +T407 PR:000004920 32175 32179 CAIV +T408 CHEBI:15377 32200 32207 aqueous +T409 UBERON:0001796 32200 32213 aqueous humor +T410 CHEBI:15377 32284 32291 aqueous +T411 UBERON:0001796 32284 32297 aqueous humor +T412 NCBITaxon:10088 32328 32333 mouse +T413 PR:000004920 32334 32338 CAIV +T414 NCBITaxon:10088 32415 32419 mice +T415 CHEBI:15377 32537 32544 aqueous +T416 CHEBI:26130 32651 32658 pigment +T417 GO:0046148 32651 32669 pigment production +T418 http://purl.obolibrary.org/obo/MONDO_0043209 32708 32716 albinism +T419 UBERON:0000970 32733 32739 ocular +T420 UBERON:0000966 32821 32828 retinal +T421 GO:0030424 32829 32834 axons +T422 UBERON:0001769 32890 32894 iris +T423 NCBITaxon:40674 32947 32956 mammalian +T424 NCBITaxon:10088 32993 32997 mice +T425 SO:0000704 33048 33059 genetically +T426 CHEBI:26130 33070 33079 pigmented +T427 NCBITaxon:10088 33083 33087 mice +T428 CHEBI:26130 33185 33194 pigmented +T429 http://purl.obolibrary.org/obo/MONDO_0043209 33207 33213 albino +T430 NCBITaxon:10088 33217 33221 mice +T431 http://purl.obolibrary.org/obo/MONDO_0043209 33229 33237 albinism +T432 NCBITaxon:10088 33316 33320 mice +T433 http://purl.obolibrary.org/obo/MONDO_0043209 33326 33332 albino +T434 UBERON:0000970 33333 33337 eyes +T435 UBERON:0000970 33381 33384 eye +T436 GO:0007623 33417 33432 diurnal rhythms +T437 CHEBI:26130 33445 33454 pigmented +T438 NCBITaxon:10088 33455 33459 mice +T439 http://purl.obolibrary.org/obo/MONDO_0043209 33466 33474 Albinism +T440 GO:0007623 33523 33537 diurnal rhythm +T441 SO:0000704 33591 33598 genetic +T442 http://purl.obolibrary.org/obo/MONDO_0043209 33630 33636 albino +T443 GO:0007623 33700 33715 Diurnal rhythms +T444 GO:0007601 33739 33745 visual +T445 NCBITaxon:10114 33799 33803 rats +T446 GO:0007623 33968 33982 diurnal rhythm +T447 http://purl.obolibrary.org/obo/MONDO_0043209 33997 34003 albino +T448 NCBITaxon:10088 34007 34011 mice +T449 NCBITaxon:10088 34161 34166 mouse +T450 GO:0007623 34181 34195 diurnal rhythm +T451 NCBITaxon:9606 34309 34315 humans +T452 SO:0000704 34317 34328 Genetically +T453 NCBITaxon:10088 34338 34342 mice +T454 SO:0000704 34510 34517 genetic +T455 NCBITaxon:10088 34554 34558 Mice +T456 NCBITaxon:33208 34672 34678 Animal +T457 NCBITaxon:33208 34770 34777 animals +T458 GO:0007601 34796 34802 vision +T459 NCBITaxon:10088 34817 34821 mice +T460 NCBITaxon:10088 34874 34878 Mice +T461 NCBITaxon:3337 34917 34921 pine +T462 NCBITaxon:10088 34992 34996 mice +T463 GO:0007631 35002 35005 fed +T464 CHEBI:33290 35022 35026 chow +T465 CHEBI:15377 35049 35054 water +T466 NCBITaxon:10088 35090 35094 mice +T467 http://purl.obolibrary.org/obo/MONDO_0005015 35116 35124 diabetes +T468 http://purl.obolibrary.org/obo/MONDO_0005015 35225 35233 diabetes +T469 NCBITaxon:10088 35242 35246 mice +T470 GO:0007568 35254 35259 aging +T471 GO:0007631 35276 35279 fed +T472 CHEBI:33290 35295 35299 chow +T473 NCBITaxon:10088 35348 35352 mice +T474 NCBITaxon:10088 35432 35436 mice +T475 NCBITaxon:10088 35560 35564 mice +T476 UBERON:0000970 35811 35817 ocular +T477 UBERON:0000970 35833 35839 ocular +T478 NCBITaxon:10088 35900 35904 mice +T479 NCBITaxon:10088 36115 36119 mice +T480 NCBITaxon:33208 36244 36251 animals +T481 NCBITaxon:10088 36288 36292 mice +T482 NCBITaxon:33208 36490 36497 animals +T483 GO:0007568 36584 36589 aging +T484 NCBITaxon:10088 36717 36721 mice +T485 NCBITaxon:10088 36908 36912 mice +T486 NCBITaxon:10088 36972 36977 mouse +T487 CHEBI:38867 37024 37041 anesthetic agents +T488 NCBITaxon:10088 37127 37132 mouse +T489 UBERON:0000970 37274 37280 ocular +T490 NCBITaxon:10088 37529 37533 mice +T491 NCBITaxon:10088 37603 37608 mouse +T492 UBERON:0000178 37624 37629 Blood +T493 UBERON:0000178 37644 37649 blood +T494 NCBITaxon:10088 37673 37677 mice +T495 UBERON:0002415 37743 37747 tail +T496 NCBITaxon:10088 37824 37828 mice +T497 UBERON:0001766 37944 37961 anterior chambers +T498 NCBITaxon:10088 38024 38028 mice +T499 UBERON:0001766 38091 38108 anterior chambers +T500 NCBITaxon:10088 38122 38126 mice +T501 UBERON:0000970 38233 38237 Eyes +T502 NCBITaxon:10088 38254 38258 mice +T503 CHEBI:37527 38325 38329 acid +T504 CHEBI:30879 38330 38337 alcohol +T505 CHEBI:16842 38338 38346 formalin +T506 CHEBI:50913 38347 38355 fixative +T507 UBERON:0000970 38581 38585 eyes +T508 NCBITaxon:10088 38609 38613 mice +T509 UBERON:0001771 38786 38791 pupil +T510 UBERON:0000970 38796 38801 optic +T511 UBERON:0000970 38857 38863 ocular +T512 PR:000010873 38889 38893 Myoc +T513 SO:0000147 38895 38900 Exons +T514 SO:0001668 38909 38926 proximal promoter +T515 NCBITaxon:10088 38934 38939 mouse +T516 PR:000010873 38940 38944 Myoc +T517 SO:0000704 38945 38949 gene +T518 NCBITaxon:10088 38970 38975 mouse +T519 SO:0001026 38976 38983 genomic +T520 SO:0000112 39024 39031 primers +T521 SO:0000147 39034 39038 Exon +T522 SO:0000147 39169 39173 Exon +T523 SO:0000147 39241 39245 Exon +T524 SO:0000167 39377 39385 Promoter +T525 SO:0000006 39686 39698 PCR products +T526 UBERON:0000970 39765 39771 ocular +T527 NCBITaxon:10088 40056 40060 mice +T528 PR:000010873 40108 40112 Myoc +T529 SO:0001023 40113 40120 alleles diff --git a/src/ontogpt/evaluation/craft/database/all/11532192.txt b/src/ontogpt/evaluation/craft/database/all/11532192.txt new file mode 100644 index 000000000..a1828a9bb --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/11532192.txt @@ -0,0 +1,229 @@ +Intraocular pressure in genetically distinct mice: an update and strain survey + +Abstract + +Background + +Little is known about genetic factors affecting intraocular pressure (IOP) in mice and other mammals. The purpose of this study was to determine the IOPs of genetically distinct mouse strains, assess the effects of factors such as age, sex and time of day on IOP in specific strain backgrounds, and to assess the effects of specific candidate gene mutations on IOP. + +Results + +Based on over 30 studied mouse strains, average IOP ranges from approximately 10 to 20 mmHg. Gender does not typically affect IOP and aging results in an IOP decrease in some strains. Most tested strains exhibit a diurnal rhythm with IOP being the highest during the dark period of the day. Homozygosity for a null allele of the carbonic anhydrase II gene (Car2n) does not alter IOP while homozygosity for a mutation in the leptin receptor gene (Leprdb) that causes obesity and diabetes results in increased IOP. Albino C57BL/6J mice homozygous for a tyrosinase mutation (Tyrc-2J) have higher IOPs than their pigmented counterparts. + +Conclusions + +Genetically distinct mouse strains housed in the same environment have a broad range of IOPs. These IOP differences are likely due to interstrain genetic differences that create a powerful resource for studying the regulation of IOP. Age, time of day, obesity and diabetes have effects on mouse IOP similar to those in humans and other species. Mutations in two of the assessed candidate genes (Lepr and Tyr) result in increased IOP. These studies demonstrate that mice are a practical and powerful experimental system to study the genetics of IOP regulation and disease processes that raise IOP to harmful levels. + +Background + +Glaucoma is a leading cause of blindness but its molecular etiology is poorly understood. Glaucoma involves retinal ganglion cell death and optic nerve damage that is often associated with elevated intraocular pressure (IOP) [1-5]. + +It is becoming increasingly clear that many forms of glaucoma have a genetic component [6,7], and much current research is focused on identifying chromosomal regions and genes that contribute to glaucoma [8-10]. Identifying such loci allows screening for individuals with an increased risk of developing glaucoma [11]. Identifying genes contributing to elevated IOP and glaucoma is only the first step, however, and animal models will provide systems for subsequent hypothesis testing and experimental dissection of pathogenesis. + +Due to conservation in mammalian physiology and the powerful tools of mouse genetics, mice are a very important experimental system for probing the functions (both in health and disease) of many genes recently identified by sequencing the human genome [12]. We have focused on developing the mouse system for IOP and glaucoma studies [13-19]. Mice are expected to be extremely helpful in characterizing genes and mechanisms that affect IOP or the susceptibility of the optic nerve and retina to glaucomatous damage [20]. + +Very little is known about the magnitude of IOP of various mouse strains or IOP fluctuation in mice with time or other factors. Previously, we developed a method to measure IOP in mice and reported initial findings on the magnitude of mouse IOP [13]. The procedure involves direct measurement of pressure following cannulation of the anterior chamber. The initial experiments demonstrated that in our hands careful ocular cannulation has a very minor effect on IOP (average of -0.3 mmHg, mode -0.5 mmHg) and demonstrated significant differences in intraocular pressure levels between four mouse strains. Here, we provide an update, including an extensive strain survey, and show that the methodology is reliable and produces reproducible data over extended periods of time. + +Results + +A broad range of IOPs between strains + +Figure 1 shows the average IOP of a number of inbred mouse strains that were housed in the same environmental conditions. There is a wide range of IOP with strain BALB/cJ having one of the lowest average IOPs (11.1 ± 0.5 mmHg) and strain CBA/CaJ one of the highest IOPs (19.3 ± 0.3 mmHg). Significant differences exist among various strains (P < 0.0001 for all groups, ANOVA comparing strains within each sex group). + +Figure 1 + +An almost two-fold range of IOP between genetically distinct mouse strains. Mean IOP ± SEM is shown for each strain. Twelve to 25 mice were analyzed for each strain (typically 18 to 20), except for RIIIS/J (7 mice). Mice of most strains were 6 to 12 months old, except for SEC/1ReJ and SB/Le that were 2 to 3 months old. Approximately half of the CE/J and one third of the BUB/BnJ mice were 16 months old and were pooled with the younger mice, as the IOPs of both ages were the same. Strain name CBA/CaHN-Btkxid/J is abbreviated to CBA/CaHN. + +Clinical and histological analysis of the eyes of all studied strains (see Table 1) did not identify anatomic or pathologic features that might account for the differences in IOP. For example, the iridocorneal angle and aqueous humor drainage structures are open to the anterior chamber and have normal morphology in both BALB/cJ and CBA/CaJ mice (Figure 2). More than 20% of CBA/CaJ mice had IOPs of over 21 mmHg, which increases risk for glaucoma in humans. We aged a small group of these mice (n = 4) to 2 years and histologically analyzed their optic nerves and retinas but they did not develop glaucoma. + +Figure 2 + +Normal iridocorneal angles in strains with high and low IOP. The iridocorneal angle that contains the aqueous humor drainage structures (Schlemm's canal (SC) and trabecular meshwork (TM) had a normal morphology in strains CBA/CaJ (A, high IOP), BALB/cJ (B, low IOP) and all other strains. Pigment filled macrophages that resemble human clump cells were often located in the angle of CBA/CaJ mice but were never sufficient to block drainage. We have observed similar quantities of these cells at this location in other strains with lower IOP. The angle recess between the cornea (C) and iris root (I) was open and not occluded. Aqueous humor passes through this recess before entering the drainage structures. Original Magnification 630X. + +Table 1 + +Ocular abnormalities in various strains + +All strains shown in Figure 1 were evaluated but only strains with abnormalities are shown. For incidence, the numerator indicates the number of mice or eyes affected and the denominator indicates the total number analyzed. The mice were 6 to 12 months old at the time of analysis and were not studied at other ages unless indicated. Histological analysis confirmed these observations and also identified calcified cyst like structures in the iris epithelium of strains SB/Le and SEC/1ReJ. A low incidence of corneal scarring was noted in some strains and likely resulted from a scratched cornea. Previously described retinal degeneration [[81]] caused by homozygosity for the Pde6brd1mutation was noted in strains SB/Le, ST/bJ, BUB/BnJ, CBA/J, C3H/HeJ, SJL/J and SWR/J. + +Strain differences are reproducible + +To assess the consistency of IOP in specific strains, we measured IOP in different cohorts of each strain maintained under similar conditions at different times. We purposefully included strains at each end of the IOP spectrum, and strain C57BL/6J (B6) that is commonly used for genetic experiments (Figure 3). The average IOP of different cohorts of strains CBA/CaJ, CBA/CaHN (both high end of spectrum) and B6 were consistent over time. This was true of most strains assessed on multiple occasions. Average IOP for age matched mice of the same strain assessed at different times typically differed by no more than 1.5 mmHg, and the differences were usually smaller. Strain 129P3/J was the most variable strain with the average IOP fluctuating by up to 2.5 mmHg. + +Figure 3 + +The IOPs of different strains are stable and reproducible over time. Mean IOP ± SEM is shown for the indicated strain at different times of measurement. The measurement times within a single year were separated by a few months. The IOPs of strains that were analyzed multiple times in the same year are listed in the chronological order that they were obtained with the earliest measurement on left. The sex [Male (M) or Female (F)], age (months), and number of mice (n) analyzed for the CBA strains are listed starting with the earliest measurement and ending with the latest: CBA/CaJ, F, n = 22, age 6–7; M, n = 11, age 2; CBA/CaHN, F, n = 16, age 3; M, n = 8, age 3. For BALB/cJ and C57BL/6J, all groups were male and primarily 3 to 4 months old. The number of mice analyzed at each time follows: BALB/cJ 8, 10, 11, 15, 18 and 14, C57BL/6J 10, 14, 21,40,40 and 42. Strain name CBA/CaHN-Btkxid/J is abbreviated to CBA/CaHN. + +Despite the general consistency of IOP, the average IOPs of some strains have changed in a reproducible manner. The IOPs of BALB/cJ mice (low end of spectrum) were very similar for the past several years, around 11 mmHg. Between early 1996 and 1997, however, the IOP of this strain did jump from approximately 7.7 mmHg to approximately 11 mmHg (Figure 3). During this period, the room in which our animals were housed and the manufacturer of the mouse diet were changed. Over the same period, the IOP of A/J also increased dramatically, from 9.4 ± 0.5 mmHg (n = 11) in 1996 to 14.2 ± 0.4 mmHg (n = 19) in 1997. The increased IOP in A/J also was reproducible, with the average IOP of mice assessed in the year 2000 being 14.5 ± 0.4 mmHg (n = 16). Importantly, the IOPs of strains B6 and C3H/HeJ did not change during this time (B6, 12.3. ± 0.5 mmHg in 1996 and 12.4 ± 0.3 mmHg in 1997, n= 10 and 14; C3H/HeJ, 13.7 ± 0.8 mmHg in 1996 and 13.6 ± 0.2 mmHg in 1997, n = 9 and 19). + +Effect of age on IOP + +We focused on the commonly used B6, 129P3/J and C3H/HeJ strains to determine the effects of age on IOP (Figure 4). In B6, age had a significant effect on IOP (P < 0.001). IOP was slightly decreased at both 12 months (12.2 ± 0.2 mmHg) and 19 months (12.2 ± 0.3 mmHg) compared to 3 months (13.1 ± 0.3 mmHg) and 7 months (13.3 ± 0.3 mmHg). Although the decrease was of a similar level to the variation observed in 3 month old mice (see Figure 3), the IOPs of control young mice (see Methods) analyzed at the same times as the various B6 age groups did not decrease. For example, control mice analyzed at the same time as the 3 month, 12 month and 18 month B6 age groups had IOPs of 13.0 ± 0.2 mmHg (n = 14), 13.3 ± 0.2 mmHg (n = 14), 13.7 ± 0.4 mmHg (n = 12), respectively. IOP was even lower in the 24 month B6 mice (10.8 ± 0.4 mmHg), and again the average IOP of young controls measured at the same time was not changed (13.5 ± 0.2, n = 12). + +Figure 4 + +IOP changes with increasing Age. Mean IOP ± SEM is shown for the indicated strain at different ages. IOP decreased significantly in strains C57BL/6J and 129P3/J but not C3H/HeJ. The C57BL/6J and 129P3/J groups consisted of approximately equal numbers of males and females. The 3 months old C3H/HeJ mice were male while the other ages were composed of males and females. The number of mice analyzed at each age, listed from youngest to oldest, follow: C57BL/6J, 18, 18, 18, 18, 13; 129P3/J, 18, 15, 16, 14, 21; C3H/HeJ, 15, 23 17 and 16. + +In strain 129P3/J, IOP did not differ significantly with age between 3 and 14 months but was lower in 18 month old mice (P < 0.001 compared to all younger ages, Figure 4). Despite a 1 mmHg dip in IOP at 8 months, there were no significant IOP differences between C3H/HeJ mice at each age tested (P = 0.2 for age). Although the effect of age has not been thoroughly assessed in other strains, no obvious age-related differences have been identified in other strains analyzed at multiple ages except for the glaucomatous DBA/2J and AKXD-28/Ty strains [14,19]. + +Effect of sex on IOP + +Although we have not rigorously assessed the effect of sex on IOP in many strains, sex specific differences have not been detected in the majority of strains for which both sexes have been analyzed, and have proven inconsistent even within an individual strain analyzed multiple times. Strains B6 and 129P3/J have been extensively evaluated at multiple ages between 3 and 24 months of age. Sex differences were always absent in strain 129P3/J and typically absent in strain B6. In strain B6, however, males infrequently had significantly higher IOP than females. For example, in one experiment, B6 males had an average IOP of 14.2 ± 0.3 mmHg (n = 12) whereas the average IOP of females was 13.1 ± 0.3 mmHg (n = 12, P = 0.01). If real, this sporadic sex difference was not dependent on age, sometimes occurring in a group of B6 mice at a particular age and sometimes not occurring in a separate group of the same age. + +Anesthesia protocol avoids IOP alteration and allows detection of diurnal differences + +All IOPs were assessed using an anesthetic regime of 99 mg/kg ketamine and 9 mg/kg xylazine (defined as 1X). Initial experiments suggested that an almost identical dose (100 mg/kg ketamine and 9 mg/kg xylazine) of anesthesia had no effect on IOP during the experimental period with IOP being measured as soon as possible after the mouse was unconscious, typically minutes. [13]. To further assess the effects of anesthesia, we measured IOP in groups of genetically identical B6 mice subjected to different doses (1X, 1.5X and 2X) at 5 and 25 minutes after administration (Figure 5). For all doses, IOP decreased by 25 minutes (P = 0.005 for time). The greater the dose the greater the decrease in IOP. At the 5 minute measurement, however, IOP was the same using all doses suggesting that the anesthetic effect on IOP had not yet occurred. To identify any early window when it may be possible to assess IOP without an obvious anesthetic effect, 195 mice of strain B6 were anesthetized with the 1X dose and IOP was measured at 1 minute time points between 4 and 12 minutes after administration (Figure 5). The mean IOP of groups analyzed at each time point did not differ (P = 0.9) indicating that the IOP depressing effect of anesthesia occurs later than 12 minutes after administration. Similar results were obtained using 161 strain 129P3/J and 145 strain DBA/2J mice with the 1X dose (129P3/J, P = 0.1; DBA/2J, P = 0.2). In support of a later effect of anesthesia (since general anesthesia is reported to mask diurnal variation in IOP [21]), we identified increased IOP during the dark compared to the light period of the day in several tested strains (Figure 6). In these experiments, IOP measurements were made between 5 and 12 minutes after administration of anesthesia. + +Figure 5 + +No effect of 1X anesthetic dose within 12 minutes of administration. A Mean IOP ± SD is shown for C57BL/6J mice at 5 and 25 minutes after administration of various doses of anesthetic. The 1X dose consisted of 99 mg/kg ketamine and 9 mg/kg xylazine. All doses decreased IOP by 25 minutes. At all doses the IOP at 5 minutes was the same suggesting that the effect of anesthesia had not yet occurred. Approximately thirty 3 to 4 month old mice were analyzed at each dose and time B-D. Scatter-plots demonstrating no change in IOP over the 12 minutes following anesthetic administration in three relatively unrelated mouse strains. All strains consisted of males and females that ranged from 3 to 6 months of age. The sexes and ages were equally represented at each time point. The scatter-plots include 195 C57BL/6J, 161 129P3/J and 145 DBA/2J mice. + +Figure 6 + +IOP is increased during the dark compared to light periods in several strains. Mean IOP ± SEM is shown for the indicated strains at measurement during the light (open bars) or dark (filled bars) periods of the day. All mice were 2 to 4 months old except for CBA/CaJ that were 5 to 6 months old. 129B6 refers to mice of a mixed 129X1/SvJ and C57BL/6J background. IOP was increased during the dark in all strains except CBA/CaJ. The number of mice successfully analyzed during the light (L) or dark (D) periods follows, DBA/2J 10L,10D; C57BL/6J 32L, 22D; C57BR/cdJ 12L,10D; SWR/J 16L, 16D; BALB/cByJ 15L, 16D; 129B6 10L, 12D; CBA/CaJ 22L, 16D. + +Blood pressure does not correlate with IOP + +An initial study of the relationship between blood pressure and IOP in mice did not detect a good correlation (R2 = 0.1, Figure 7). + +Figure 7 + +No correlation between blood pressure and IOP. There is no strong correlation between the average systolic blood pressure of each strain and the average IOP of that strain (R2 = 0.1). All mice were female and 2 to 4 months old, with the exception that the female CBA/CaJ used for IOP assessment were 6 to 7 months old. If the CBA/CaJ mice are excluded, R2 drops to 0.01 of ARK/J, C57BL/6J and SJL/J, strain ARK/J had the lowest IOP. + +Myoc alleles do not associate with the magnitude of IOP + +Mutations in the myocilin gene (MYOC) cause human glaucoma. To determine if allelic variation in the mouse Myoc gene associated with IOP in mouse strains, we analyzed the gene in an assortment of strains with different IOPs. Two alleles were identified. One of these alleles had a 12 nucleotide insertion in the promoter region (ccagagcagggt, between positions -340 and -341) compared to the previously published sequence and is called the insertion allele. The other allele was identical to the published sequence [22]. The insertion allele also had a previously reported substitution (A to G, Thr164Ala) in exon 1 and several other single base changes in the promoter region [22]. The presence or absence of this allele does not associate with IOP as it is present in strains with a range of IOPs (Figure 8). + +Figure 8 + +Myoc alleles do not associate with IOP. Sequencing identified two alleles of Myoc in these mouse strains (see text). Mouse strains homozygous for an allele having a 12 bp insertion in the promoter region are shown as open bars and strains homozygous for the allele without this insertion have filled bars. The IOP data is the same as that in Figure 1. + +Genetic alterations and IOP + +Y Chromosome + +The Y chromosome has been implicated in strain specific blood pressure differences in rats [23,24]. To test if the Y chromosome of strain 129/Ola alters IOP in relation to that of strain B6, we compared the IOPs of pure B6 males and consomic B6 males that had the 129P2/Ola Y chromosome (backcrossed for 11 generations). No differences in IOP were detected between these groups of mice (Figure 9, P = 0.1). + +Figure 9 + +Effects of genetic alterations. Mean IOP ± SEM is shown. There is no difference in IOP between 3 to 4 month old males with either the 129P2/Ola (n = 18) or C57BL/6J (n = 20) Y chromosome (Y Chr). The 129 Y Chr had been backcrossed to strain C57BL/6J for 11 generations. Similarly, 6 to 9 month old males and females that are homozygous for normal (+) or mutant (n) alleles of Car2 have similar IOPs (n = 18 and 14, respectively). However, 4 months old males homozygous for the Lepr diabetes and obesity causing mutation (db) had significantly higher IOPs than their heterozygous age and sex matched littermates. The average body weight of the homozygotes and heterozygotes was 59 g (range 50 to 64 g) and 35 g (range 28 to 41 g) respectively. Blood sugar levels in db /db males of this age are almost always in the diabetic range whereas those of heterozygotes are not [[27]]. This was recently confirmed in cohorts of approximately 25 males of each genotype (mean ± SD; db/db 422 ± 110 mg/dl ; db/+ 147 ± 19 mg/dl; Anthony Nicholson, The Jackson Laboratory, personal communication) + +Car2 + +To test if deficiency of carbonic anhydrase II leads to decreased IOP, we analyzed mice of a B6 background that were genetically similar but with normal or mutant alleles of the Car2 gene [25,26]. There was no difference in IOP between normal and mutant mice (Figure 9, P = 0.5). + +Lepr + +To test if genetic perturbations that cause obesity and diabetes can alter IOP, we compared mice that were genetically similar but were either homozygous or heterozygous for a leptin receptor mutation (db) that results in obesity and diabetes before 4 months of age on the C57BLKS/J strain background used [27]. IOP was modestly but significantly elevated in obese, diabetic homozygous mutants (14.7 ± 0.3 mmHg) compared to non-obese, non-diabetic heterozygotes (13.4 ± 0.4 mmHg, P < 0.01, Figure 9). + +Tyr + +To determine if albinism alters IOP, we analyzed B6 mice that were either pigmented or albino. The albino mice were homozygous and coisogenic for a mutant allele of tyrosinase (Tyrc-2J) that arose on the otherwise pigmented B6 background. In 2 month old mice, homozygosity for Tyrc-2J resulted in increased IOP (14.2 ± 0.4 mmHg) compared to wild type, pigmented mice (12.4 ± 0.3 mmHg, P < 0.0001). The same was true for independent cohorts of mice of different ages that were analyzed at different times (Figure 10A). In contrast to pigmented B6 mice (Figure 6), the IOPs of the albino B6 mice were not increased at measurement during the dark compared to light period of the day (P = 0.6, Figure 10B). + +Figure 10 + +Tyr mutation results in increased IOP and altered diurnal changes A. Different cohorts of C57BL/6J mice that are homozygous for the spontaneous Tyrc-2J mutation have higher IOP than their normal counterparts at all tested ages. All groups consisted of male and female mice. B. IOP is not increased during the dark period of the day in Tyrc-2J homozygotes. The number of mice analyzed during the light period were: 2 month, 28 +/+, 29 c-2J/c-2J, 4 month, 20 +/+ and 20 c-2J/c-2J, 8 month, 15 +/+ and 15 c-2J/c-2J. Fourteen mice of each genotype were successfully analyzed during the dark period of the day. + +Discussion + +IOP differences are reproducible and amenable to genetic analyses + +We report the IOPs of over 30 genetically different mouse strains that were housed in the same environmental conditions. The magnitude of IOP differences between strains and the good reproducibility of readings over time will allow the use of genetic approaches to identify genes that underlie these differences. Using our method, a trained investigator can measure the IOPs of 10 mice in an hour. Thus it is feasible to assess sufficient numbers of mice for quantitative trait locus (QTL) analysis methods and to use these methods to identify chromosomal regions contributing to strain differences in IOP. The strain survey we report provides valuable information for designing these experiments. The throughput and reproducibility also is sufficient for mutagenesis screens [28-30]. These are important approaches as they may allow the association of genes with IOP and glaucoma whose currently known functions do not suggest that they affect aqueous humor dynamics or do not immediately identify them as likely glaucoma candidates. + +No effect of anesthetic protocol on IOP during a 12 minute measurement window + +Many anesthetic agents including xylazine lower IOP. Ketamine usually appears to increase IOP [31-33], but there are reports of ketamine having no effect on IOP or even reducing IOP [31,34]. Different doses, species used, routes of administration or environments may contribute to these differences. The relationship between the IOPs we measure and those in conscious mice depends upon the effect of our anesthetic protocol (intraperitoneal injection of 99 mg/kg ketamine and 9 mg/kg xylazine). An anesthetic effect could potentially alter IOP and mask genetically determined differences in IOP. Therefore, it is very important to understand this effect. Here, we show that, despite a depressant effect on IOP by 25 minutes, our anesthetic protocol has no detectable effect on IOP during the first 12 minutes after administration. Thus, to avoid effects of anesthesia on IOP, all measurements should be made within a window of up to 12 minutes after anesthetic administration. + +Similarities and differences to rat studies + +Our results agree with the time course of cardiovascular depression caused by intraperitoneal administration of ketamine and xylazine in rats. In that study, anesthesia had a minor effect on blood pressure during the first 15 minutes following injection but a strong hypotensive effect between 15 and 30 minutes that continued for more than an hour [35]. In contrast, intraperitoneally administered ketamine (100 mg/kg) was shown to rapidly decrease IOP in a different rat study [36]. IOP decreased significantly between a conscious measurement and the first possible measurement under anesthesia (defined as time 0). IOP decreased further by the next reading (at 5 minutes) after which it remained stable for the duration of the experiment (20 minutes)[36]. The time 0 measurement in that study was at a very similar state of anesthesia as our 4 and 5 minute time points (starting as soon as possible and within 20 to 60 seconds of adequate anesthesia), and the 5 minute rat time point was similar to our 9 and 10 minute measurement times. Thus, although both rat and mouse studies show that anesthesia decreases IOP, the studies do not agree on the timing of the effect. The IOP depression occurred very soon after anesthesia in the rats but was delayed in the mice. + +Environment may influence the effect of anesthesia + +Factors that may influence the effect of anesthesia include the species, strain and environment used. Essentially the same dose of ketamine and route of administration was used in both the rat and mouse IOP studies. In mice, the strain does not appear to be important as no early effect of anesthesia was present in the three distantly related laboratory strains [37] we studied in detail, and we have not observed any obvious effect during this period in any analyzed strain. Environmental differences may be important. Cage cleanliness, changing frequency and housing density can alter drug metabolism and the effect of anesthesia in rats [38,39]. The type of bedding used also may be important. We use wood shavings for bedding and wood shavings expose the mice to terpenes. Terpene administration or environmental exposure to terpenes in wood shavings alters drug resistance and decreases the effect of anesthetic agents in both rats and mice [40-45]. + +Risk factors for increased IOP + +Some epidemiological studies implicate factors such as diabetes, vascular hypertension, arterial hypotension, vasospasm, aberrant autoregulation of blood flow and sex in glaucoma. Other studies find no association between these factors and glaucoma [2,46-48]. Similarly, the effects of various factors including age, gender, blood pressure, obesity and diabetes have been variably associated with elevated IOP. We evaluated the relationship between these latter factors and IOP in genetically homogeneous mouse strains in a controlled environment. + +Age + +Although within the range of variability observed in young mice, the IOP of B6 mice modestly decreased around 1 year of age. This decrease was statistically significant compared to young mice measured at the same time. At 2 years of age the IOP of B6 mice had decreased further, though an effect of anesthesia in these very old mice cannot be ruled out [49]. The IOP of 129P3/J mice also decreased with age but only at the oldest age examined (18 months). Decreasing IOP correlates with increasing age in the human Japanese population [50,51]. Further studies of B6 mice may allow experimental investigation of this effect. Additional studies may also identify " normal" mouse strains that develop increased IOP with age as generally occurs in Western populations [52,53]. We previously demonstrated that the glaucomatous strains DBA/2J and AKXD-28/Ty develop elevated IOP with age [14,19]. + +Gender + +In the examined strains, we found no consistent differences in IOP between males and females. This was true at all ages for the 3 strains that were aged to 18 months or older. This is in agreement with a number of human studies, which show that IOP is equal between the sexes [52,53]. However, some studies have found sex-specific differences (typically with higher IOP in females and the magnitude of the difference increasing after 40 years of age) [53,54]. Of possible relevance, we previously reported that female mice of strains DBA/2J and AKXD-28/Ty develop elevated IOP at an earlier age than males [14,19]. + +Blood pressure + +Some but not all human studies have reported a positive association between IOP and blood pressure [48,52]. Our comparison of the relationship between blood pressure and IOP in young, adult female mice of different mouse strains, whose blood pressures differed up to 36 mmHg, did not reveal a positive correlation. Further studies are needed to determine if blood pressure correlates with IOP in males, and to determine the relationship between blood pressure and IOP in various mouse strains with age. + +Obesity and diabetes + +Obesity or higher body mass index have been implicated by some but not other studies as risk factors for increased IOP and glaucoma [51,55-57]. Similarly, diabetes or the combination of diabetes and obesity have been variably associated with elevated IOP and glaucoma [46,52,58-62]. To test if genetic perturbations that cause obesity and diabetes can alter IOP, we compared groups of mice that were genetically similar except that they were either homozygous or heterozygous for a leptin receptor mutation (db) that results in early onset obesity and diabetes. The obese, diabetic mice had higher IOPs than their lean, non-diabetic littermates. Thus, epidemiological associations between increased IOP and obesity or diabetes are supported by this work and appear to be functionally relevant. Further experiments with obese non-diabetic or diabetic non-obese mice will help to characterize the separate effects of these risk factors. + +IOP is increased during the dark period of the day + +Diurnal variation in IOP is common in humans and laboratory animals [53]. The molecular mechanisms underlying the diurnal rhythm are not defined but increased aqueous humor production or flow occurs during the period of increased IOP in both rabbits and humans [63-65]. Small changes in the resistance to aqueous humor drainage may also contribute to diurnal differences in IOP [66,67]. We first suspected IOP changes with time of day when the IOP of a group of B6 mice measured in the hour prior to onset of the dark period appeared to be higher than at other times of day. Previously, a rise of IOP was reported to occur before onset of the dark period in rats [21], and rats and rabbits were shown to have higher IOP during the dark period compared to the light period [21,68,69]. Thus, we compared the IOPs of mice at different times of day and identified several strains with significantly higher IOP during the dark compared to the light period. The magnitude of the difference varies with strain, and was greatest in SWR/J. The IOP increase in the dark is not dependent on functional rod and cone photoreceptors since these cells degenerate in SWR/J mice due to homozygosity for the Pde6brd1 mutation. In agreement with this finding, circadian regulation of wheel running by light was previously reported in mice lacking rods and cones [70]. Interestingly, the IOP of strain CBA/CaJ, which has one of the highest daytime IOPs does not appear to increase in the dark. Further more detailed studies are needed to define the characteristics of the diurnal rhythm of intraocular pressure in mice, and to determine whether it is lacking or has a different timing in strain CBA/CaJ. Analysis of these mouse strains may increase understanding of the molecular mechanisms controlling diurnal rhythms of IOP and that may be relevant to glaucoma. + +Car2 deficiency does not alter IOP + +Bicarbonate formation is important for aqueous humor secretion from the ciliary processes and carbonic anhydrase (CA) facilitates this secretion. There are multiple forms of CA and the CAII isoform is reported to be the predominant form in the ciliary processes [71,72]. Our experiments show that a genetic deficiency of CAII in mice homozygous for a mutation in the Car2 gene does not alter IOP. CAIV activity was recently demonstrated in the ciliary processes [73] and so our data may support a more substantial role for CAIV compared to CAII in aqueous humor secretion. It also is possible that CAII substantially contributes to aqueous humor secretion but that functional mouse CAIV is sufficient to prevent an effect of CAII deficiency on IOP in Car2 mutant mice. In support of a role for both enzymes, a greater than 90% inhibition of CA is required for significant reduction of aqueous secretion [71,72]. + +Tyrosinase deficiency results in increased IOP + +Tyrosinase is the first enzyme of the pigment production pathway. Tyrosinase deficiency causes albinism and has various ocular consequences. These include alteration of the number of ipsilaterally projecting retinal axons and substantially increased light penetration past the iris [74]. It is not known if these abnormalities affect mammalian IOP. Here, we show increased IOP in mice lacking tyrosinase activity compared to otherwise genetically identical pigmented B6 mice. Additionally, IOP differences between the light and dark period of the day were detected in the pigmented but not the albino B6 mice. Thus, albinism can affect the diurnal pattern of IOP changes. In agreement with this result, mice with albino eyes that are homozygous for tyrosinase or pink eye dilution mutations have altered diurnal rhythms compared to pigmented mice [75]. Albinism by itself is either not sufficient to alter the diurnal rhythm of IOP or alters it in different ways depending upon genetic background, however, since the albino strains BALB/cByJ and SWR/J had increased IOP during the dark. Diurnal rhythms are known to depend on visual pathways and to respond to light intensity. Exposing rats to 24 hours of low light abrogates the diurnal fluctuation of IOP and results in constantly elevated IOP [36]. Further experiments will determine the nature of the diurnal rhythm of IOP in the albino B6 mice and if its alteration or other mechanisms result in IOP elevation. + +Conclusions + +A broad range of reproducible IOP differences exists between inbred mouse strains and a diurnal rhythm of IOP exists in different strains. Various factors have been variably associated with risk for increased IOP in humans. Genetically uniform, mice can be used to study the effects of these risk factors on IOP. In trained hands, our measurement procedure is reliable, accurate and rapid enough to allow large scale genetic studies of factors determining IOP. Mice have great potential for helping to characterize the molecular mechanisms affecting IOP. + +Materials and Methods + +Animal husbandry + +All experiments were performed in compliance with the ARVO statement for use of animals in ophthalmic and vision research. All mice were bred and maintained at The Jackson Laboratory. Mice were housed in cages containing white pine bedding and covered with polyester filters. For most experiments, the mice were fed NIH31 (6 % fat) chow ad libitum, and their water was acidified to pH 2.8 to 3.2. B6 mice develop diet induced diabetes when maintained on a high fat diet [76]. To ensure that we were analyzing the effect of age and not diabetes, the B6 mice in the aging experiment were fed NIH31 (4% fat) chow. We have found no differences in IOP between B6 mice housed on the 4% fat and 6% fat versions of this otherwise identical diet. The mice were group housed and the cages were changed one time per week. If any cage appeared soiled between scheduled changes, the mice were placed in a clean cage. The environment was kept at 21°C with a 14 hour light: 10 hour dark cycle. The colony was monitored for specific pathogens by The Jackson Laboratory's routine surveillance program (see for specific pathogens). + +Intraocular pressure + +Intraocular pressures were measured as described elsewhere [13,77]. The mice were typically acclimatized to the procedure room for at least 2 weeks prior to measurement, but sometimes between 1 and 2 weeks. Although it was not possible to include all strains in each measurement period, mice of different strains were intermixed. As demonstrated here, the IOPs of C57BL/6J are very consistent over time and so these animals were interspersed with experimental mice during all experiments to ensure that calibration had not drifted and that the system was functioning optimally. Whenever possible, the investigator measuring IOP did not know the genotypes of the animals. It was not possible to analyze all age groups of each strain at the same time in the aging experiments. Therefore, to control for potential IOP changes due to measurement time and not age, approximately 3 month old B6 mice were assessed at the same time as each age group. All dark period measurements were made between 1 and 3 hours after the lights turned off. The room was equipped with dim red lights and mice were protected from all light exposure during set up. Each mouse was briefly exposed to the red light when the anesthetic agents were administered. When adequate anesthesia was achieved (after 3 to 4 minutes), the mouse was placed on the measurement platform and the white light of the microscope was turned on (for approximately 1 and a half minutes) to allow ocular cannulation, IOP measurement and post-measurement tests [13] that guard against artifactual data. The white light was used at very low intensity and was dim but we cannot rule out the possibility that this brief exposure altered the IOP. All other mice were protected from light exposure throughout the time an individual mouse was analyzed. + +Blood pressure + +The blood pressures of conscious mice of each strain (6 to 12 females per strain) were measured with a tail-cuff system as reported [78], except that 5 days of training were used. The mice were analyzed in the same procedure room as was used for IOP measurment. + +Clinical examinations + +For most strains, anterior chambers were examined with a slit lamp biomicroscope [16]. At least 8 mice of each strain shown in figure 1 were evaluated. However, the anterior chambers of C57BLKS/J mice were only evaluated under a dissection microscope at the time of IOP measurement. + +Histological analysis + +Eyes from at least 2 mice of the listed strains were fixed (4% paraformaldehyde or Fekete's acid-alcohol-formalin fixative) processed, paraffin embedded and sectioned as previously reported [15,79], except that the paraformaldehyde was buffered with 0.1 M phosphate buffer. All strains other than CBA/CaHN and C57BLKS/J strains were evaluated. The eyes of CBA/CaJ and BALB/cJ mice were also fixed and processed for plastic embedding (Historesin, Leica, Heidelberg, Germany), and sectioned as previously reported [14,79]. Saggital sections including the pupil and optic nerve were collected and analyzed as they contain most ocular structures. + +Analysis of Myoc + +Exons and the proximal promoter of the mouse Myoc gene were amplified from mouse genomic DNA using the following combinations of primers: + +Exon 1: 5'-cttgcaggagaactttccagaa-3' and 5'-atctcgaaggagattgttatagg-3' + +5'-gaccagctggagacccaaaccag-3' and 5'-gctcagatccactgacctaaa-3' + +Exon 2: 5'-tgaagccatactttaccaaccat-3' and 5'-caaaagggagaagtctaacttc-3' + +Exon 3: 5'-agtcaaggctcacagagctaa-3' and 5'-aagagtagctgctcaccgtgtacaag-3' + +5'-agacattgacttagctgtggat-3' and 5'-cggaacttcaccttttctggc-3' + +Promoter: 5'-taggagaagtctcattatactgc-3' and 5'-ttcactggaccagcataagga-3' + +5'-tctgaggatgttcacaggtttat-3' and 5'-tcttctggaaagttctcctgca-3' + +Samples underwent 30 cycles of amplification with Perkin-Elmer Taq polymerase in a PTC Thermal Cycler (MJ Research, MA) (94° for 40 sec, 57° for 1 min, 72° for 2 min). The PCR products we purified and sequenced as described [22]. + +Abbreviations + +Intraocular pressure (IOP), C57BL/6J (B6), chromosome (Chr), polymerase chain reaction (PCR), carbonic anhydrase (CA) + +Acknowledgments + +We thank Norma Buckley, Felicia Farley and Jennifer Smith for their assistance with data entry, references, and figures. We also thank Jane Barker for the Car2 mice, Marianna Mertts for her help with analysis of Myoc alleles, and members of the John laboratory, Tatyana Golovkina, Edward Leiter and Timothy O'Brien for critical reading of the manuscript. Supported in part by AHAF 97437 and G1999023, NIH EY11721 and HL55001. Core services were subsidized by grant CA34196. Major funding was provided by the Howard Hughes Medical Institute. SWMJ is an Assistant Investigator of The Howard Hughes Medical Institute. diff --git a/src/ontogpt/evaluation/craft/database/all/11597317.ann b/src/ontogpt/evaluation/craft/database/all/11597317.ann new file mode 100644 index 000000000..c2e55a091 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/11597317.ann @@ -0,0 +1,298 @@ +T1 PR:000004804 0 5 BRCA2 +T2 UBERON:0000310 113 119 breast +T3 http://purl.obolibrary.org/obo/MONDO_0007254 113 126 breast cancer +T4 SO:0000704 142 146 gene +T5 PR:000004804 147 152 BRCA2 +T6 GO:0006281 156 166 DNA repair +T7 SO:0000704 191 198 genetic +T8 PR:000004804 233 238 BRCA2 +T9 GO:0000724 242 260;279 313 homology-dependent ... repair of DNA double-strand breaks +T10 GO:0000724 262 277;279 313 recombinational ... repair of DNA double-strand breaks +T11 PR:000004804 396 401 BRCA2 +T12 GO:0000725 428 450 recombinational repair +T13 PR:000004804 452 457 BRCA2 +T14 NCBITaxon:2759 527 537 eukaryotic +T15 PR:000013672 550 555 RAD51 +T16 PR:000004804 733 738 BRCA2 +T17 PR:000013672 742 747 RAD51 +T18 GO:0006281 757 763 repair +T19 UBERON:0000310 776 782 breast +T20 http://purl.obolibrary.org/obo/MONDO_0007254 776 789 breast cancer +T21 PR:000004804 817 822 BRCA2 +T22 PR:000004804 824 829 BRCA2 +T23 UBERON:0000310 845 851 breast +T24 http://purl.obolibrary.org/obo/MONDO_0007254 845 858 breast cancer +T25 SO:0000704 874 878 gene +T26 UBERON:0000310 981 987 breast +T27 http://purl.obolibrary.org/obo/MONDO_0007254 981 994 breast cancer +T28 PR:000004804 1018 1023 BRCA2 +T29 http://purl.obolibrary.org/obo/MONDO_0004992 1047 1053 cancer +T30 SO:0000704 1061 1072 genetically +T31 SO:0000657 1257 1274 repeated segments +T32 NCBITaxon:40674 1357 1366 mammalian +T33 SO:0000855 1367 1376 orthologs +T34 UBERON:0000310 1385 1391 Breast +T35 http://purl.obolibrary.org/obo/MONDO_0007254 1385 1398 Breast cancer +T36 SO:0000704 1414 1419 genes +T37 GO:0006281 1424 1434 DNA repair +T38 PR:000013672 1517 1522 RAD51 +T39 GO:0005634 1541 1548 nuclear +T40 PR:000004803 1560 1565 BRCA1 +T41 PR:000004803 1635 1640 BRCA1 +T42 PR:000004804 1645 1650 BRCA2 +T43 GO:0000725 1667 1703 recombinational repair of DNA damage +T44 PR:000004803 1705 1710 BRCA1 +T45 PR:000004804 1715 1720 BRCA2 +T46 GO:0005634 1735 1742 nuclear +T47 GO:0051320 1759 1766 S phase +T48 CHEBI:50902 1789 1808 DNA damaging agents +T49 GO:0006281 1853 1862;1887 1897 repair of ... DNA damage +T50 PR:000004803 1941 1946 BRCA1 +T51 PR:000004804 1950 1955 BRCA2 +T52 GO:0000725 2065 2087 recombinational repair +T53 PR:000004804 2108 2113 BRCA2 +T54 PR:000013672 2133 2138 RAD51 +T55 PR:000004803 2255 2260 BRCA1 +T56 PR:000013672 2265 2270 RAD51 +T57 PR:000004804 2356 2361 BRCA2 +T58 PR:000013672 2389 2394 RAD51 +T59 PR:000004803 2422 2427 BRCA1 +T60 GO:0005634 2461 2468 nuclear +T61 PR:000013672 2469 2474 RAD51 +T62 GO:0065003 2556 2568;2581 2590 formation of ... complexes +T63 GO:0032991 2581 2590 complexes +T64 SO:0000704 2636 2643 genetic +T65 PR:000004803 2700 2705 Brca1 +T66 UBERON:0000922 2709 2718 embryonic +T67 CL:0002322 2709 2723;2729 2734 embryonic stem ... cells +T68 CL:0002322 2725 2727;2729 2734 ES ... cells +T69 GO:0000725 2752 2767;2772 2778 recombinational ... repair +T70 GO:0006302 2768 2778 DSB repair +T71 PR:000004803 2838 2843 BRCA1 +T72 PR:000004804 2848 2853 BRCA2 +T73 PR:000004803 2943 2948 BRCA1 +T74 PR:000004804 2953 2958 BRCA2 +T75 GO:0006281 2962 2968 repair +T76 PR:000004804 3014 3019 BRCA2 +T77 PR:000004803 3026 3031 BRCA1 +T78 GO:0000725 3059 3081 recombinational repair +T79 PR:000004804 3214 3219 BRCA2 +T80 SO:0000704 3422 3427 genes +T81 SO:0000061 3513 3529 restriction site +T82 PR:P03882 3534 3540 I-SceI +T83 SO:0000188 3550 3556 intron +T84 SO:0000028 3589 3598 base pair +T85 SO:0000409 3599 3615 recognition site +T86 GO:0009294 3627 3639 transfection +T87 PR:P03882 3646 3652 I-SceI +T88 GO:0010467 3653 3663 expression +T89 SO:0000440 3664 3670 vector +T90 GO:0042148 3781 3790 invade(s) +T91 GO:0006302 3859 3868;3873 3876 repair of ... DSB +T92 SO:0000704 3901 3905 gene +T93 GO:0035822 3901 3916 gene conversion +T94 GO:0006281 3994 4000 repair +T95 GO:0010467 4156 4166 expression +T96 PR:000004804 4282 4287 BRCA2 +T97 GO:0006281 4330 4336 repair +T98 PR:P03882 4341 4347 I-SceI +T99 GO:0010467 4394 4404 Expression +T100 PR:P03882 4408 4414 I-SceI +T101 NCBITaxon:9606 4497 4502 human +T102 UBERON:0001264 4503 4513 pancreatic +T103 http://purl.obolibrary.org/obo/MONDO_0021040 4503 4519 pancreatic tumor +T104 PR:000004804 4573 4578 BRCA2 +T105 PR:P03882 4708 4714 I-SceI +T106 PR:000004804 4798 4803 BRCA2 +T107 NCBITaxon:9606 4806 4811 human +T108 http://purl.obolibrary.org/obo/MONDO_0005070 4812 4817 tumor +T109 SO:0000704 4895 4906 genetically +T110 PR:000004804 4942 4947 BRCA2 +T111 GO:0000725 5064 5086 recombinational repair +T112 PR:000004804 5143 5148 BRCA2 +T113 GO:0000725 5276 5298 recombinational repair +T114 PR:000004804 5370 5375 BRCA2 +T115 NCBITaxon:10088 5427 5432 mouse +T116 CL:0002322 5433 5440 ES cell +T117 GO:0010467 5451 5460 expresses +T118 PR:000004804 5476 5481 BRCA2 +T119 GO:0010467 5583 5593 expressing +T120 PR:000004804 5606 5611 BRCA2 +T121 GO:0010467 5787 5794 express +T122 PR:000004804 5807 5812 BRCA2 +T123 GO:0010467 5901 5911 expressing +T124 PR:000004804 5924 5929 BRCA2 +T125 GO:0010467 5978 5988 expressing +T126 PR:000004804 6004 6009 BRCA2 +T127 GO:0000725 6273 6295 recombinational repair +T128 NCBITaxon:9606 6331 6336 human +T129 PR:000004804 6365 6370 BRCA2 +T130 PR:000004804 6473 6478 BRCA2 +T131 NCBITaxon:9606 6621 6626 human +T132 PR:000004804 6697 6702 Brca2 +T133 PR:000004804 6721 6726 Brca2 +T134 CL:0002322 6730 6738 ES cells +T135 PR:000004804 6786 6791 Brca2 +T136 SO:0001023 6815 6821 allele +T137 PR:000004804 6888 6893 BRCA2 +T138 PR:000004804 6972 6977 BRCA2 +T139 GO:0000725 7016 7038 recombinational repair +T140 NCBITaxon:9606 7047 7052 human +T141 NCBITaxon:10088 7057 7062 mouse +T142 PR:000004804 7081 7086 BRCA2 +T143 PR:000013672 7087 7092 RAD51 +T144 PR:000013672 7242 7247 RAD51 +T145 NCBITaxon:9606 7310 7315 human +T146 PR:000004804 7316 7321 BRCA2 +T147 GO:0010467 7323 7333 Expression +T148 CHEBI:35222 7406 7415 inhibitor +T149 GO:0006281 7419 7429 DNA repair +T150 GO:0010467 7482 7492 expression +T151 GO:0000086 7608 7612 G2/M +T152 GO:0009294 7665 7676 transfected +T153 GO:0005634 7698 7705 nuclear +T154 PR:000013672 7706 7711 RAD51 +T155 GO:0010467 7844 7854 expression +T156 CHEBI:10545 7902 7910 electron +T157 PR:000013672 7999 8004 RAD51 +T158 PR:000013672 8099 8104 RAD51 +T159 PR:000004804 8183 8188 BRCA2 +T160 PR:000013672 8248 8253 RAD51 +T161 PR:000004804 8410 8415 BRCA2 +T162 PR:000013672 8425 8430 RAD51 +T163 PR:000013672 8476 8481 RAD51 +T164 NCBITaxon:9606 8522 8527 human +T165 http://purl.obolibrary.org/obo/MONDO_0005070 8528 8533 tumor +T166 PR:000004804 8636 8641 BRCA2 +T167 GO:0010467 8650 8659 expressed +T168 GO:0005737 8701 8710 cytoplasm +T169 GO:0005634 8773 8780 nuclear +T170 SO:0001528 8773 8800 nuclear localization signal +T171 GO:0005634 8860 8867 nuclear +T172 PR:000013672 8884 8889 RAD51 +T173 PR:000013672 8920 8925 RAD51 +T174 GO:0005634 8953 8960 nucleus +T175 PR:000004804 8972 8977 BRCA2 +T176 PR:000013672 8999 9004 RAD51 +T177 PR:000004804 9037 9042 BRCA2 +T178 PR:000004804 9089 9094 BRCA2 +T179 GO:0005737 9102 9111 cytoplasm +T180 GO:0051235 9116 9125 sequester +T181 PR:000013672 9126 9131 RAD51 +T182 PR:000013672 9236 9241 RAD51 +T183 PR:000004804 9276 9281 BRCA2 +T184 PR:000004804 9327 9332 BRCA2 +T185 PR:000013672 9382 9387 RAD51 +T186 PR:000004804 9388 9393 BRCA2 +T187 PR:000004804 9456 9461 BRCA2 +T188 GO:0005634 9515 9522 nuclear +T189 PR:000013672 9538 9543 RAD51 +T190 PR:000013672 9629 9634 RAD51 +T191 PR:000004804 9635 9640 BRCA2 +T192 PR:000013672 9665 9670 RAD51 +T193 PR:000004804 9766 9771 BRCA2 +T194 PR:000013672 9773 9778 RAD51 +T195 GO:0006281 9858 9864 repair +T196 GO:1990391 9858 9872 repair complex +T197 PR:000013672 9938 9943 RAD51 +T198 GO:0065007 10073 10083 regulatory +T199 PR:000004804 10101 10106 BRCA2 +T200 PR:000013672 10133 10138 RAD51 +T201 GO:0051235 10169 10182 sequestration +T202 PR:000013672 10186 10191 RAD51 +T203 PR:000013672 10263 10268 RAD51 +T204 PR:000013672 10321 10326 RAD51 +T205 GO:0006260 10388 10403 DNA replication +T206 PR:000004804 10405 10410 BRCA2 +T207 PR:000013672 10428 10433 RAD51 +T208 GO:0065003 10434 10447;10471 10480 assembly into ... complexes +T209 GO:0000725 10448 10470 recombinational repair +T210 GO:1990391 10464 10480 repair complexes +T211 PR:000013672 10535 10540 RAD51 +T212 PR:000004804 10541 10546 BRCA2 +T213 PR:000013672 10635 10640 RAD51 +T214 PR:000013672 10742 10747 RAD51 +T215 PR:000004804 10748 10753 BRCA2 +T216 PR:000013672 10834 10839 RAD51 +T217 PR:000004804 10840 10845 BRCA2 +T218 PR:000004804 10892 10897 BRCA2 +T219 PR:000013672 10911 10916 RAD51 +T220 GO:0000725 10927 10949 recombinational repair +T221 PR:000013672 10984 10989 RAD51 +T222 PR:000013672 11054 11059 RAD51 +T223 PR:000013672 11097 11102 RAD51 +T224 PR:000013672 11146 11151 RAD51 +T225 SO:0000854 11152 11160 paralogs +T226 PR:000017505 11186 11191 XRCC2 +T227 PR:000017506 11193 11198 XRCC3 +T228 PR:000013676 11200 11206 RAD51B +T229 PR:000013675 11208 11214 RAD51C +T230 PR:000013677 11219 11225 RAD51D +T231 GO:0032991 11245 11252 complex +T232 PR:000013672 11276 11281 RAD51 +T233 PR:000004804 11297 11302 BRCA2 +T234 PR:000013672 11324 11329 RAD51 +T235 SO:0000854 11330 11338 paralogs +T236 PR:000013672 11352 11357 RAD51 +T237 PR:000004804 11438 11443 BRCA2 +T238 GO:0005634 11475 11482 nuclear +T239 PR:000013672 11493 11498 RAD51 +T240 PR:000004804 11542 11547 BRCA2 +T241 GO:0065003 11573 11584;11603 11610 assembly of ... complex +T242 GO:0032991 11603 11610 complex +T243 PR:000004804 11695 11700 BRCA2 +T244 PR:000013672 11701 11706 RAD51 +T245 PR:000004804 11765 11770 BRCA2 +T246 PR:000013672 11799 11804 RAD51 +T247 PR:000013672 11862 11867 RAD51 +T248 PR:000004804 12008 12013 BRCA2 +T249 PR:000013672 12018 12023 RAD51 +T250 GO:0000725 12031 12053 recombinational repair +T251 PR:000004804 12128 12133 BRCA2 +T252 PR:000013672 12161 12166 RAD51 +T253 GO:0005634 12174 12181 nucleus +T254 GO:0005634 12220 12227 nuclear +T255 SO:0001528 12220 12247 nuclear localization signal +T256 PR:000013672 12251 12256 RAD51 +T257 PR:000013672 12277 12282 RAD51 +T258 PR:000004804 12321 12326 BRCA2 +T259 PR:000004804 12414 12419 BRCA2 +T260 PR:000013672 12439 12444 RAD51 +T261 PR:000004804 12445 12450 BRCA2 +T262 PR:000004804 12520 12525 BRCA2 +T263 GO:0000725 12529 12551 recombinational repair +T264 PR:000004804 12619 12624 BRCA2 +T265 PR:000004804 12674 12679 BRCA2 +T266 UBERON:0000922 12775 12784 embryonic +T267 GO:0006281 12943 12949 repair +T268 PR:P03882 12966 12972 I-SceI +T269 GO:0006281 13054 13062 repaired +T270 SO:0000704 13082 13086 gene +T271 GO:0035822 13082 13097 gene conversion +T272 SO:0000704 13221 13225 gene +T273 SO:0000704 13251 13255 gene +T274 GO:0035822 13251 13266 gene conversion +T275 GO:0097617 13302 13311 annealing +T276 PR:000004804 13353 13358 BRCA2 +T277 PR:000013672 13384 13389 Rad51 +T278 SO:0000954 13414 13429 Chromosomal DNA +T279 PR:000013672 13467 13472 Rad51 +T280 PR:000004804 13494 13499 BRCA2 +T281 PR:000004804 13564 13569 BRCA2 +T282 PR:000013672 13570 13575 Rad51 +T283 PR:000013672 13612 13617 RAD51 +T284 PR:000004804 13618 13623 BRCA2 +T285 PR:000013672 13686 13691 Rad51 +T286 MOP:0000629 13731 13742 polymerizes +T287 PR:000004804 13788 13793 BRCA2 +T288 GO:0065003 13814 13825;13837 13846 assembly of ... complexes +T289 GO:0032991 13837 13846 complexes +T290 PR:000013672 13907 13912 Rad51 +T291 GO:0051235 13924 13935 sequestered +T292 PR:000004804 13966 13971 BRCA2 +T293 PR:000013672 13995 14000 Rad51 +T294 PR:000004804 14129 14134 BRCA2 +T295 PR:000013672 14166 14171 Rad51 +T296 GO:0000725 14200 14222 recombinational repair +T297 GO:1990391 14216 14232 repair complexes +T298 PR:000013672 14278 14283 Rad51 diff --git a/src/ontogpt/evaluation/craft/database/all/11597317.txt b/src/ontogpt/evaluation/craft/database/all/11597317.txt new file mode 100644 index 000000000..c432f7802 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/11597317.txt @@ -0,0 +1,49 @@ +BRCA2 and homologous recombination + +Abstract + +Two recent papers provide new evidence relevant to the role of the breast cancer susceptibility gene BRCA2 in DNA repair. Moynahan et al provide genetic data indicating a requirement for BRCA2 in homology-dependent (recombinational) repair of DNA double-strand breaks. The second paper, by Davies et al, begins to address the mechanism through which BRCA2 makes its contribution to recombinational repair. BRCA2 appears to function in recombination via interactions with the major eukaryotic recombinase RAD51 [1,2,3]. We briefly review the context in which the two studies were carried out, we comment on the results presented, and we discuss models designed to account for the role of BRCA2 in RAD51–mediated repair. + +Keywords: breast cancer, homologous recombination + +BRCA2 + +BRCA2 was the second breast cancer susceptibility gene to be discovered, and was isolated through positional cloning using data from families with inherited breast cancer [4]. Cells with mutant BRCA2 protein are, like many cancer cells, genetically unstable and accumulate gross chromosomal rearrangements [5,6]. The sequence of this large protein (3418 amino acids) offers very little clue to its function, although there are eight repeated segments (termed BRC repeats) in the middle of the protein that are highly conserved among mammalian orthologs [7,8]. + +Breast cancer susceptibility genes and DNA repair + +Following the landmark discovery by Scully et al that the homologous recombinase RAD51 colocalizes at subnuclear sites with BRCA1 [9], a number of additional results have provided evidence that both BRCA1 and BRCA2 are involved in recombinational repair of DNA damage. BRCA1 and BRCA2 form discrete nuclear foci during the S phase and after exposure to DNA damaging agents [9,10,11]. These foci are probably sites of repair of spontaneous and induced DNA damage [12,13,14]. Cell lines defective in either BRCA1 or BRCA2 are sensitive to damaging agents that form double-strand breaks (DSBs), as are other cell lines defective in recombinational repair (reviewed in [15]). BRCA2 interacts with the RAD51 recombinase via direct protein-protein contacts [16,17,18,19]. Biochemical analysis also showed interaction between BRCA1 and RAD51, although these detected interactions may have been indirect [9]. The BRC repeats of BRCA2 are responsible for direct RAD51 interaction. Cells lacking BRCA1/2 fail to form damage-induced subnuclear RAD51 foci with normal efficiency, suggesting that these proteins are required for the formation of recombinase complexes at the sites of DNA damage [20,21]. Finally, genetic measurements of recombination frequency have shown that Brca1-/- embryonic stem (ES) cells are deficient in recombinational DSB repair [22,23]. The similarity between phenotypes associated with BRCA1 and BRCA2 deficiency, together with data showing a similar effect of DNA damage on distribution of BRCA1 and BRCA2 in repair-proficient cells, led to the hypothesis that BRCA2, like BRCA1, is required for efficient recombinational repair. The paper by Moynahan et al [24] provides important support for this hypothesis. + +Measuring DSB-induced recombination frequency in BRCA2-defective cells + +Pierce et al have designed a set of recombination substrates for measuring the level of homologous recombination in vivo (Fig. 1) [25]. The DNA substrate contains a pair of mutated GFP genes (GFP encodes the easily detected green fluorescent protein), one of which contains a restriction site for I-SceI, a yeast intron encoded endonuclease with an 18 base pair recognition site. Transient transfection of an I-SceI expression vector results in the production of a DSB in the first mutated copy of GFP. One or both DNA ends formed by the break invade(s) the homologous sequence in the second mutant GFP copy, resulting in repair of the DSB via a homology-mediated gene conversion event. The configuration of the GFP construct is such that homology-mediated repair often results in the formation of a functional copy of GFP. Such events can be detected by fluorescence-activated cell sorting analysis by virtue of their expression of GFP. + +Moynahan et al [24] have used such a GFP recombination substrate to demonstrate that cells with defective BRCA2 protein are deficient in their ability to repair the I-SceI-induced DSB through homologous recombination. Expression of I-SceI resulted in 1 out of 1400 cells producing GFP via homologous recombination in the human pancreatic tumor cell line CAPAN-1. CAPAN-1 cells carry a deletion of BRCA2 on one homolog and codes for a protein truncated at amino acid 1981 on the other homolog. The authors indicate that the level of I-SceI-induced recombination in CAPAN-1 is over 100-fold less than that seen using other (BRCA2+) human tumor cell lines. The different lines examined, however, are very likely to differ genetically from CAPAN-1 cells not only at the BRCA2 locus, but also at a very large number of additional loci. This raises the possibility that some or even all of the recombinational repair defect seen in CAPAN-1 could be due to mutations at non-BRCA2 loci. + +While Moynahan et al were careful to point out this problem, two sets of results argue against the possibility that the recombinational repair deficiency of CAPAN-1 cells is completely independent of its defect in BRCA2. First, in the same study [24], recombination in a mouse ES cell line that expresses only truncated BRCA2 protein was measured. This line was found to have lower recombination efficiency than isogenic cells expressing full-length BRCA2 (the defect observed was about fivefold to sixfold). Second, in an independent study, Powell was able to compare derivatives of CAPAN-1 that differed only in their ability to express full-length BRCA2 protein (S. Powell, personal communication, 2001). In these experiments, the derivative expressing full-length BRCA2 yielded 10-fold more recombinants than controls expressing only truncated BRCA2 (S. Powell, personal communication, 2001). While the experimental design of these experiments was somewhat different from that of Moyna-han et al [24], the results raise the possibility that CAPAN-1 cells have more than one mutation that lowers the efficiency of recombinational repair relative to that observed in other human cell lines. Conversely, the BRCA2 defect in CAPAN-1 could be fully responsible for the 100-fold defect in recombination if the level of BRCA2 complementation observed by Powell was incomplete. Furthermore, the discrepancy between the 100-fold difference between CAPAN-1 and the other human lines as compared with the fivefold to sixfold difference between the Brca2lex1/lex2cells and Brca2+/+ ES cells might simply be accounted for by the fact that Brca2lex1/lex2 is not a null allele. Additional studies are likely to shed light on the efficiency of BRCA2-independent recombination pathways. Taken together, the results indicate that BRCA2 is indeed required for high levels of recombinational repair in both human and mouse. + +The function of BRCA2-RAD51 interaction + +The second paper providing evidence, by Davies et al [26], is a biochemical study of the interaction between the homologous recombinase RAD51 and a peptide consisting of one of the eight BRC repeats from human BRCA2. Expression of a single BRC repeat (BRC4) had previously been reported to act as an inhibitor of DNA repair by Chen et al [27]. These investigators showed that expression of constructs containing the BRC4 repeat in MCF-7 cells enhances the radiosensitivity of cells and blocks both the G2/M delay associated with damage and the ability of the transfected cells to assembly subnuclear RAD51 foci. In the paper by Davies et al, data provide evidence for a plausible mechanism of the dominant negative effect associated with expression of a single BRC repeat. DNA binding assays and electron microscopy methods were used to show that a BRC3 peptide interferes with the ability of RAD51 to assemble into oligomeric filaments on DNA. A BRC4 peptide was also reported to inhibit the RAD51–DNA interaction. These experiments raise the possibility that the full-length BRCA2 protein could act as a negative regulator of inappropriate RAD51–DNA interaction. + +The final experiment reported by Davies et al [26] is particularly important with respect to understanding the mechanism(s) through which BRCA2 promotes RAD51-dependent recombination. The localization of RAD51 was examined in CAPAN-1 cells, the same human tumor line examined by Moynahan et al [24]. It had been shown previously that the small amount of truncated BRCA2 protein expressed by CAPAN-1 cells was mislocalized to the cytoplasm, consistent with the fact that the truncation protein lacks a nuclear localization signal [28]. Davies et al showed CAPAN-1 cells to be defective in nuclear localization of RAD51, raising the possibility that RAD51 is normally carried to the nucleus by binding BRCA2. It is possible that RAD51 may alternatively be capable of BRCA2-independent transport, but mislocalization of BRCA2 to the cytoplasm may sequester RAD51 and block its normal mode of transport. Support for the hypothesis that the observed mislocalization of RAD51 is an indirect consequence of the BRCA2 defect requires reintroduction of functional BRCA2 into CAPAN-1. + +Given the elaborate nature of the RAD51–BRCA2 interaction, it seems somewhat unlikely that the only role of BRCA2 in promoting recombination is to serve as a specific nuclear transporter of RAD51. An attractive alternative, favored by Davies et al [26], is that the association of RAD51–BRCA2 is required to maintain RAD51 in a form that is in a state of 'readiness' (Fig. 2a). In the absence of this interaction with BRCA2, RAD51 might exist in a form that is not capable of being recruited into a functional repair complex when damage occurs. For example, previous studies indicated that RAD51 shows little binding preference for single-strand DNA (ssDNA) over double-strand DNA (dsDNA) [3,29]. This result could mean that regulatory factors, such as BRCA2, are required to suppress RAD51–dsDNA interactions to prevent sequestration of RAD51 in a nonfunctional form. Alternatively, or in addition, suppression of RAD51–ssDNA interaction might be important for preventing RAD51 from binding to the regions of ssDNA that form during normal DNA replication. BRCA2 may thus promote RAD51 assembly into recombinational repair complexes via a negative regulatory mechanism (i.e. by blocking RAD51–BRCA2 interaction until damage has occurred and factors required for 'productive' assembly of RAD51 at damaged sites are in place). As mentioned previously, the ability of a BRC peptide to repress the RAD51–BRCA2 interaction could reflect a role of full-length protein in suppressing unwanted RAD51–BRCA2 interactions. + +Yet another potential role for BRCA2 in promoting RAD51-dependent recombinational repair is a positive role in assembly of RAD51 at damaged sites (Fig. 2b). Several proteins that interact with RAD51 are thought to 'mediate' assembly of RAD51 at sites of DNA damage (reviewed in [30]). RAD51 paralogs in particular, including XRCC2, XRCC3, RAD51B, RAD51C and RAD51D, may function as a complex that actively promotes RAD51 assembly [31]. BRCA2 could cooperate with RAD51 paralogs in promoting RAD51 assembly, or even provide an alternative assembly pathway. The observation that BRCA2 localizes to damage-induced subnuclear foci with RAD51 seems consistent with the possibility that BRCA2 plays a positive role in assembly of the recombination complex [11]. + +Finally, it is important to note that the two models for the function of the BRCA2–RAD51 interaction shown in Figure 2 are not mutually exclusive. BRCA2 could prevent inappropriate RAD51 assembly in the absence of damage and promote functional RAD51 assembly at DNA lesions during the damage response. Further experiments are clearly required to clarify the functional interactions between BRCA2 and RAD51 during recombinational repair. It should be relatively straightforward to determine whether the role of BRCA2 is limited to transporting RAD51 to the nucleus. If this were the case, addition of a nuclear localization signal to RAD51 should suppress the RAD51-mediated phenotypes associated with a BRCA2 mutation. However, biochemical experiments are needed to determine whether full-length BRCA2 blocks or promotes RAD51–BRCA2 interaction. Such experiments could also reveal additional roles for BRCA2 in recombinational repair. However, several technical obstacles, including the large size of BRCA2, promise to make biochemical characterization of BRCA2 function difficult. + +Abbreviations + +DSB = double-strand break; dsDNA = double-strand DNA; ES = embryonic stem; GFP = green fluorescent protein; ssDNA = single-strand DNA. + +Figures and Tables + +Figure 1 + +Recombination substrates used for assaying homology-directed repair. Cutting at the I-SceI site within the mutant GFP (SceGFP) results in a double-strand break that can be repaired through homologous gene conversion using a 3'-truncated copy of GFP as sequence donor. The mechanism results in the formation of a functional copy of the GFP gene. The model shown assumes gene conversion occurs via the synthesis-dependent annealing mechanism. + +Figure 2 + +Potential roles of BRCA2 in promoting assembly of Rad51 at sites of DNA damage. Chromosomal DNA is shown as pairs of straight lines, Rad51 as open circles, and BRCA2 as grey bars. (a) Prevention of nonproductive DNA interactions. BRCA2–Rad51 interaction is proposed to suppress RAD51–BRCA2 interactions until DNA damage is present. When damage occurs, Rad51 is recruited to damaged sites where is polymerizes into nucleoprotein filaments. In this model, BRCA2 is not required for assembly of functional complexes at damaged sites, only to prevent a substantial fraction of Rad51 from being sequestered in a nonfunctional form. In a BRCA2-defective cell, mutant Rad51 becomes associated with DNA at random sites and is therefore not readily recruited to sites of damage. (b) Positive regulation. BRCA2 is proposed to be required for Rad51 to assemble into functional recombinational repair complexes at sites of damage. In BRCA-defective cells, Rad51 fails to associate with sites of damage due to lack of an assembly factor. diff --git a/src/ontogpt/evaluation/craft/database/all/11604102.ann b/src/ontogpt/evaluation/craft/database/all/11604102.ann new file mode 100644 index 000000000..1a28130ea --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/11604102.ann @@ -0,0 +1,648 @@ +T1 NCBITaxon:10088 24 29 mouse +T2 PR:000006288 30 36 Dazap1 +T3 SO:0000704 37 41 gene +T4 http://purl.obolibrary.org/obo/MONDO_0005047 94 105 infertility +T5 PR:000006284 114 117 DAZ +T6 PR:000006290 122 126 DAZL +T7 PR:000006288 150 156 DAZAP1 +T8 PR:000006288 158 182 DAZ Associated Protein 1 +T9 http://purl.obolibrary.org/obo/MONDO_0005372 279 295 male infertility +T10 PR:000006284 304 307 DAZ +T11 PR:000006284 309 331 Deleted in Azoospermia +T12 http://purl.obolibrary.org/obo/MONDO_0004983 320 331 Azoospermia +T13 PR:000006288 344 350 DAZAP1 +T14 GO:0000806 375 387 Y chromosome +T15 PR:000006284 396 399 DAZ +T16 GO:0030849 407 415 autosome +T17 PR:000006284 424 427 DAZ +T18 PR:000006290 442 446 DAZL +T19 PR:000006288 448 454 DAZAP1 +T20 SO:0000417 480 487 domains +T21 GO:0010467 541 550 expressed +T22 UBERON:0000473 574 580 testis +T23 PR:000006288 623 629 DAZAP1 +T24 PR:000006284 675 678 DAZ +T25 PR:000006290 683 687 DAZL +T26 NCBITaxon:10088 723 728 mouse +T27 PR:000006288 729 735 Dazap1 +T28 SO:0000704 736 740 gene +T29 GO:0010467 758 768 expression +T30 NCBITaxon:9606 840 845 human +T31 NCBITaxon:10088 850 855 mouse +T32 SO:0000704 856 861 genes +T33 SO:0001026 875 882 genomic +T34 SO:0005858 905 913;926 933 syntenic ... regions +T35 NCBITaxon:10088 939 944 mouse +T36 NCBITaxon:9606 949 954 human +T37 PR:000006288 955 961 DAZAP1 +T38 NCBITaxon:8353 1036 1043 Xenopus +T39 SO:0000855 1044 1054 orthologue +T40 PR:000006288 1055 1059 Prrp +T41 PR:000006288 1085 1091 Dazap1 +T42 GO:0010467 1095 1104 expressed +T43 UBERON:0000473 1116 1122 testis +T44 GO:0008584 1116 1134 testis development +T45 PR:000006288 1172 1178 DAZAP1 +T46 UBERON:0000473 1216 1222 testis +T47 PR:000006288 1247 1253 DAZAP1 +T48 GO:0005737 1272 1283 cytoplasmic +T49 GO:0005844 1323 1336 polyribosomes +T50 PR:000006288 1352 1358 DAZAP1 +T51 GO:0010467 1411 1421 expression +T52 UBERON:0000473 1425 1431 testes +T53 GO:0007283 1451 1466 spermatogenesis +T54 GO:0006412 1548 1559 translation +T55 GO:0007283 1574 1589 Spermatogenesis +T56 CL:0000015 1634 1649 male germ cells +T57 GO:0007067 1667 1674 mitotic +T58 GO:0008283 1675 1688 proliferation +T59 GO:0007126 1690 1706 meiotic division +T60 CL:0000019 1757 1762 sperm +T61 NCBITaxon:species 1811 1818 species +T62 SO:0001026 1856 1862 genome +T63 NCBITaxon:1 1869 1877 organism +T64 SO:0000704 1987 1992 genes +T65 NCBITaxon:7215 1996 2006 Drosophila +T66 http://purl.obolibrary.org/obo/MONDO_0005372 2021 2035 male sterility +T67 NCBITaxon:9606 2071 2077 humans +T68 http://purl.obolibrary.org/obo/MONDO_0005372 2135 2153 infertility in men +T69 SO:0000704 2169 2174 genes +T70 http://purl.obolibrary.org/obo/MONDO_0005372 2191 2207 male infertility +T71 PR:000006284 2215 2218 DAZ +T72 PR:000006284 2220 2242 Deleted in Azoospermia +T73 http://purl.obolibrary.org/obo/MONDO_0004983 2231 2242 Azoospermia +T74 SO:0000704 2244 2248 gene +T75 GO:0000806 2281 2282 Y +T76 PR:000006284 2290 2293 DAZ +T77 SO:0000704 2294 2299 genes +T78 NCBITaxon:9604 2325 2335 great apes +T79 NCBITaxon:9527 2340 2357 old world monkeys +T80 GO:0030849 2371 2380 autosomal +T81 PR:000006290 2381 2386 DAZL1 +T82 PR:000006290 2388 2398 DAZ-like 1 +T83 PR:000004789 2404 2409 BOULE +T84 SO:0000704 2410 2415 genes +T85 NCBITaxon:40674 2429 2436 mammals +T86 PR:000006284 2454 2457 DAZ +T87 SO:0000704 2458 2463 genes +T88 http://purl.obolibrary.org/obo/MONDO_0005047 2489 2498 infertile +T89 http://purl.obolibrary.org/obo/MONDO_0004983 2521 2532 azoospermia +T90 PR:000006290 2556 2561 Dazl1 +T91 http://purl.obolibrary.org/obo/MONDO_0005047 2569 2580 infertility +T92 NCBITaxon:10088 2605 2609 mice +T93 PR:000006284 2632 2635 DAZ +T94 NCBITaxon:7215 2654 2664 Drosophila +T95 NCBITaxon:6239 2669 2679 C. elegans +T96 NCBITaxon:8353 2688 2695 Xenopus +T97 SO:0000704 2775 2779 gene +T98 GO:0010467 2825 2834 expressed +T99 CL:0000586 2851 2861 germ cells +T100 PR:000006284 2863 2866 DAZ +T101 PR:000006290 2871 2875 DAZL +T102 GO:0010467 2880 2889 expressed +T103 GO:0005634 2897 2904 nucleus +T104 GO:0005737 2909 2918 cytoplasm +T105 CL:0000670 2922 2943 primordial germ cells +T106 CL:0000020 2948 2961 spermatogonia +T107 GO:0005737 2974 2983 cytoplasm +T108 GO:0007126 2987 2994 meiotic +T109 CL:0000017 2995 3008 spermatocytes +T110 PR:000004789 3017 3022 BOULE +T111 GO:0010467 3026 3035 expressed +T112 GO:0005737 3050 3059 cytoplasm +T113 GO:0000239 3063 3072 pachytene +T114 CL:0000017 3073 3086 spermatocytes +T115 SO:0000704 3092 3099 Genetic +T116 PR:000006284 3147 3150 DAZ +T117 GO:0006417 3165 3178;3184 3195 regulation of ... translation +T118 NCBITaxon:7215 3197 3207 Drosophila +T119 PR:000004789 3208 3213 Boule +T120 GO:0006412 3243 3254 translation +T121 GO:0007126 3262 3269 meiosis +T122 PR:000027506 3279 3284 CDC25 +T123 SO:0000853 3285 3294 homologue +T124 PR:Q03019 3296 3301 Twine +T125 PR:000006290 3312 3316 DAZL +T126 GO:0005844 3349 3362 polyribosomes +T127 NCBITaxon:10088 3366 3371 mouse +T128 UBERON:0000473 3372 3378 testes +T129 PR:000006290 3400 3404 DAZL +T130 SO:0000837 3559 3569;3577 3580 segment in ... UTR +T131 SO:0000204 3574 3580 5' UTR +T132 NCBITaxon:10088 3584 3589 mouse +T133 PR:000005193 3590 3596 Cdc25C +T134 PR:000006284 3656 3659 DAZ +T135 SO:0000704 3660 3664 gene +T136 NCBITaxon:9606 3772 3777 human +T137 SO:0000704 3778 3783 genes +T138 PR:000006284 3793 3796 DAZ +T139 PR:000006288 3845 3851 DAZAP1 +T140 GO:0010467 3856 3865 expressed +T141 UBERON:0000473 3883 3889 testes +T142 SO:0000417 3933 3940 domains +T143 PR:000006288 3984 3990 DAZAP1 +T144 PR:000006284 4020 4023 DAZ +T145 PR:000006290 4028 4032 DAZL +T146 NCBITaxon:10088 4120 4125 mouse +T147 PR:000006288 4126 4132 Dazap1 +T148 SO:0000704 4133 4137 gene +T149 PR:000006288 4195 4201 DAZAP1 +T150 GO:0006412 4252 4263 translation +T151 NCBITaxon:10088 4299 4304 mouse +T152 PR:000006288 4305 4311 Dazap1 +T153 NCBITaxon:10088 4318 4323 Mouse +T154 PR:000006288 4324 4330 Dazap1 +T155 SO:0000317 4331 4342 cDNA clones +T156 SO:0000028 4479 4481 bp +T157 SO:0000204 4482 4504 5' untranslated region +T158 SO:0000204 4482 4484;4506 4509 5' ... UTR +T159 GO:0006412 4487 4497 translated +T160 SO:0000236 4515 4533 open reading frame +T161 SO:0000028 4586 4588 bp +T162 SO:0000205 4589 4595 3' UTR +T163 NCBITaxon:9606 4687 4692 human +T164 SO:0000855 4693 4703 orthologue +T165 SO:0000205 4709 4715 3' UTR +T166 SO:0000028 4783 4785 bp +T167 SO:0000028 4791 4793 bp +T168 SO:0000028 4801 4803 bp +T169 SO:0000837 4849 4860;4874 4877 segments in ... UTR +T170 NCBITaxon:9606 4865 4870 human +T171 SO:0000205 4871 4877 3' UTR +T172 GO:0065007 4925 4935 regulatory +T173 SO:0005836 4925 4944 regulatory elements +T174 PR:000006288 4951 4957 DAZAP1 +T175 SO:0000417 4991 4998 domains +T176 NCBITaxon:10088 5110 5115 mouse +T177 NCBITaxon:9606 5124 5129 human +T178 NCBITaxon:40674 5224 5233 mammalian +T179 NCBITaxon:8353 5287 5294 Xenopus +T180 PR:000006288 5295 5299 Prrp +T181 PR:000006288 5305 5337 proline-rich RNA binding protein +T182 PR:000006288 5473 5479 DAZAP1 +T183 PR:000006288 5484 5488 Prrp +T184 PR:000006288 5599 5605 DAZAP1 +T185 PR:000006288 5610 5614 Prrp +T186 SO:0000028 5699 5701 bp +T187 PR:000006288 5713 5717 Prrp +T188 SO:0000717 5747 5760 reading frame +T189 PR:000006288 5786 5790 Prrp +T190 PR:000006288 5877 5883 DAZAP1 +T191 NCBITaxon:9606 5926 5931 human +T192 NCBITaxon:10088 5936 5941 mouse +T193 PR:000006288 5942 5949 DAZAP1s +T194 NCBITaxon:8353 5958 5965 Xenopus +T195 PR:000006288 5966 5970 Prrp +T196 SO:0000417 6005 6012 domains +T197 NCBITaxon:9606 6048 6053 human +T198 NCBITaxon:10088 6062 6067 mouse +T199 NCBITaxon:10088 6095 6100 mouse +T200 NCBITaxon:8353 6105 6112 Xenopus +T201 SO:0001026 6165 6172 Genomic +T202 PR:000006288 6186 6192 Dazap1 +T203 NCBITaxon:10710 6238 6244 lambda +T204 NCBITaxon:10088 6263 6268 mouse +T205 PR:000006288 6269 6275 Dazap1 +T206 SO:0001026 6276 6283 genomic +T207 SO:0000147 6326 6331 exons +T208 SO:0000147 6376 6380 exon +T209 SO:0000188 6381 6387 intron +T210 SO:0000147 6442 6446 exon +T211 SO:0001026 6477 6484 genomic +T212 NCBITaxon:9606 6502 6507 human +T213 PR:000006288 6508 6514 DAZAP1 +T214 SO:0000704 6515 6519 gene +T215 NCBITaxon:9606 6556 6561 human +T216 SO:0001026 6562 6568 genome +T217 NCBITaxon:9606 6636 6641 human +T218 PR:000006288 6642 6648 DAZAP1 +T219 NCBITaxon:10088 6668 6673 mouse +T220 NCBITaxon:9606 6682 6687 human +T221 SO:0000704 6688 6693 genes +T222 SO:0000147 6741 6746 exons +T223 SO:0000188 6773 6779 intron +T224 SO:0000366 6780 6795 insertion sites +T225 SO:0000147 6849 6854 exons +T226 SO:0000147 6892 6896 Exon +T227 SO:0000188 6897 6903 intron +T228 NCBITaxon:10088 6924 6929 Mouse +T229 NCBITaxon:9606 6934 6939 Human +T230 PR:000006288 6940 6946 DAZAP1 +T231 SO:0000704 6947 6952 genes +T232 SO:0000188 6957 6964 Introns +T233 PR:000006288 7022 7028 DAZAP1 +T234 NCBITaxon:10088 7075 7080 mouse +T235 NCBITaxon:9606 7085 7090 human +T236 SO:0000112 7153 7160 primers +T237 PR:000006288 7179 7185 Dazap1 +T238 SO:0000188 7186 7194 intronic +T239 NCBITaxon:10088 7220 7225 mouse +T240 NCBITaxon:10026 7234 7241 hamster +T241 SO:0001026 7242 7249 genomic +T242 NCBITaxon:10088 7278 7283 mouse +T243 NCBITaxon:10026 7284 7291 hamster +T244 NCBITaxon:10088 7315 7320 mouse +T245 PR:000006288 7321 7327 Dazap1 +T246 SO:0000704 7328 7332 gene +T247 SO:0000860 7437 7445 syntenic +T248 NCBITaxon:9606 7449 7454 human +T249 NCBITaxon:9606 7473 7478 human +T250 PR:000006288 7479 7485 DAZAP1 +T251 SO:0000704 7486 7490 gene +T252 SO:0001023 7539 7546 alleles +T253 http://purl.obolibrary.org/obo/MONDO_0005047 7572 7583 infertility +T254 GO:0010467 7586 7596 Expression +T255 PR:000006288 7600 7606 Dazap1 +T256 UBERON:0007023 7629 7634 adult +T257 NCBITaxon:10088 7635 7640 mouse +T258 UBERON:0000479 7641 7648 tissues +T259 PR:000006288 7676 7682 Dazap1 +T260 SO:0000673 7683 7694 transcripts +T261 SO:0000673 7760 7770 transcript +T262 SO:0000317 7792 7803 cDNA clones +T263 PR:000006288 7805 7811 Dazap1 +T264 GO:0010467 7816 7825 expressed +T265 UBERON:0000473 7849 7855 testis +T266 UBERON:0002107 7870 7875 liver +T267 UBERON:0000948 7877 7882 heart +T268 UBERON:0000955 7887 7892 brain +T269 UBERON:0000479 7917 7924 tissues +T270 GO:0010467 7942 7952 expression +T271 NCBITaxon:9606 7979 7984 human +T272 PR:000006288 7985 7991 DAZAP1 +T273 PR:000006288 8026 8032 Dazap1 +T274 UBERON:0000473 8067 8073 testes +T275 UBERON:0000922 8077 8086 embryonic +T276 PR:000006290 8106 8111 Dazl1 +T277 GO:0010467 8133 8143 expression +T278 PR:000006290 8152 8157 Dazl1 +T279 PR:000006288 8162 8168 Dazap1 +T280 UBERON:0000473 8190 8196 testes +T281 GO:0008584 8190 8208 testes development +T282 GO:0007567 8225 8230 natal +T283 GO:0007567 8239 8244 natal +T284 PR:000006290 8254 8259 Dazl1 +T285 PR:000006288 8264 8270 Dazap1 +T286 SO:0000673 8271 8282 transcripts +T287 UBERON:0000473 8308 8314 testes +T288 PR:000002065 8318 8319 W +T289 PR:000002065 8321 8322 W +T290 NCBITaxon:10088 8331 8335 mice +T291 CL:0000586 8373 8383 germ cells +T292 PR:000006288 8404 8410 Dazap1 +T293 GO:0010467 8415 8424 expressed +T294 NCBITaxon:10088 8430 8435 mouse +T295 CL:0000586 8436 8445 germ cell +T296 CL:0000216 8470 8482 Sertoli cell +T297 PR:000006288 8518 8524 Dazap1 +T298 GO:0010467 8528 8537 expressed +T299 CL:0002371 8546 8553;8563 8568 somatic ... cells +T300 CL:0000586 8558 8568 germ cells +T301 UBERON:0000473 8576 8582 testis +T302 GO:0010467 8595 8605 Expression +T303 PR:000006288 8609 8615 Dazap1 +T304 UBERON:0007023 8619 8624 adult +T305 NCBITaxon:10088 8625 8630 mouse +T306 UBERON:0000479 8631 8638 tissues +T307 NCBITaxon:10088 8642 8647 mouse +T308 UBERON:0000479 8657 8663 tissue +T309 GO:0097617 8682 8692 hybridized +T310 PR:000006288 8700 8706 Dazap1 +T311 GO:0097617 8735 8745 hybridized +T312 PR:000003676 8753 8760 β-actin +T313 PR:000006288 8768 8774 Dazap1 +T314 GO:0010467 8778 8787 expressed +T315 UBERON:0000473 8811 8817 testis +T316 GO:0010467 8844 8854 expression +T317 PR:000006288 8858 8864 Dazap1 +T318 PR:000006290 8869 8873 Dazl +T319 NCBITaxon:10088 8877 8882 mouse +T320 UBERON:0000473 8883 8889 testes +T321 UBERON:0000473 8921 8931 testicular +T322 UBERON:0000922 8983 8990 embryos +T323 GO:0007567 8996 9000 born +T324 NCBITaxon:10088 9001 9005 mice +T325 NCBITaxon:10088 9019 9023 mice +T326 GO:0007567 9046 9051 birth +T327 PR:000002065 9053 9054 W +T328 PR:000002065 9056 9057 W +T329 UBERON:0000473 9059 9065 testes +T330 CL:0000586 9085 9094 germ cell +T331 PR:000002065 9123 9124 W +T332 PR:000002065 9126 9139 White spotted +T333 SO:0000704 9141 9145 gene +T334 NCBITaxon:10088 9163 9168 mouse +T335 CL:0000586 9169 9178 germ cell +T336 CL:0000216 9183 9195 Sertoli cell +T337 NCBITaxon:10088 9229 9234 mouse +T338 SO:0001026 9235 9242 genomic +T339 SO:0000112 9256 9263 primers +T340 SO:0000188 9274 9281 introns +T341 SO:0001026 9330 9337 genomic +T342 GO:0010467 9357 9367 expression +T343 PR:000006288 9375 9381 DAZAP1 +T344 GO:0042571 9395 9405 antibodies +T345 NCBITaxon:10088 9414 9419 mouse +T346 PR:000006288 9420 9426 DAZAP1 +T347 PR:000006288 9452 9458 DAZAP1 +T348 GO:0042571 9461 9469 antibody +T349 PR:000006288 9572 9578 DAZAP1 +T350 GO:0042571 9581 9589 antibody +T351 CHEBI:25676 9612 9624 oligopeptide +T352 GO:0042571 9691 9701 antibodies +T353 PR:000006288 9734 9740 DAZAP1 +T354 NCBITaxon:10088 9811 9816 mouse +T355 UBERON:0000479 9817 9823 tissue +T356 UBERON:0000473 9898 9904 testis +T357 UBERON:0002106 9932 9938 spleen +T358 UBERON:0002107 9940 9945 liver +T359 UBERON:0002048 9947 9951 lung +T360 UBERON:0000955 9956 9961 brain +T361 UBERON:0000992 10010 10015 ovary +T362 GO:0010467 10021 10031 expression +T363 PR:000006288 10035 10041 DAZAP1 +T364 CL:0000586 10049 10058 germ cell +T365 GO:0007281 10049 10070 germ cell development +T366 PR:000006290 10090 10094 DAZL +T367 UBERON:0000473 10143 10149 testes +T368 NCBITaxon:10088 10164 10168 mice +T369 CL:0000020 10207 10220 spermatogonia +T370 GO:0010467 10226 10236 expression +T371 PR:000006288 10240 10246 DAZAP1 +T372 UBERON:0000473 10275 10281 testes +T373 GO:0008283 10313 10326 proliferating +T374 GO:0007126 10331 10338 meiotic +T375 CL:0000586 10339 10349 germ cells +T376 GO:0010467 10362 10372 Expression +T377 PR:000006288 10380 10386 DAZAP1 +T378 UBERON:0007023 10398 10403 adult +T379 NCBITaxon:10088 10404 10409 mouse +T380 UBERON:0000479 10410 10417 tissues +T381 UBERON:0000479 10463 10469 tissue +T382 CHEBI:8984 10501 10504 SDS +T383 PR:000006288 10558 10564 DAZAP1 +T384 GO:0042571 10567 10575 antibody +T385 GO:0010467 10617 10627 expression +T386 PR:000006288 10631 10637 DAZAP1 +T387 PR:000006290 10642 10646 DAZL +T388 NCBITaxon:10088 10650 10655 mouse +T389 UBERON:0000473 10656 10662 testes +T390 GO:0007567 10674 10679 natal +T391 PR:000006288 10722 10728 DAZAP1 +T392 NCBITaxon:10088 10760 10765 mouse +T393 UBERON:0000473 10766 10772 testis +T394 PR:000006290 10799 10803 DAZL +T395 GO:0005739 10829 10842 mitochondrial +T396 GO:0005844 10891 10904 polyribosomes +T397 PR:000006288 10954 10960 DAZAP1 +T398 UBERON:0007023 10964 10969 adult +T399 NCBITaxon:10088 10970 10975 mouse +T400 UBERON:0000473 10976 10982 testes +T401 GO:0005737 11007 11018 cytoplasmic +T402 CHEBI:17992 11055 11062 sucrose +T403 GO:0005739 11094 11106 mitochondria +T404 PR:000006290 11136 11140 DAZL +T405 PR:000006288 11142 11148 DAZAP1 +T406 GO:0005844 11174 11187 polyribosomes +T407 CHEBI:17992 11211 11218 Sucrose +T408 PR:000006288 11248 11254 DAZAP1 +T409 GO:0005844 11278 11291 polyribosomes +T410 GO:0005739 11302 11315 mitochondrial +T411 NCBITaxon:10088 11331 11336 mouse +T412 UBERON:0000473 11337 11343 testis +T413 CHEBI:17992 11378 11385 sucrose +T414 PR:000006288 11450 11456 DAZAP1 +T415 PR:000006290 11461 11465 DAZL +T416 SO:0000932 11640 11648 pre-mRNA +T417 GO:0051028 11661 11675 mRNA transport +T418 GO:0006412 11691 11702 translation +T419 PR:000006284 11738 11741 DAZ +T420 GO:0006417 11756 11769;11775 11786 regulation of ... translation +T421 PR:000006290 11866 11870 DAZL +T422 GO:0005844 11876 11889 polyribosomes +T423 PR:000006288 11911 11917 DAZAP1 +T424 GO:0005844 11923 11936 polyribosomes +T425 GO:0006412 11983 12000 protein synthesis +T426 PR:000003390 12059 12064 FXR1P +T427 PR:000003391 12069 12074 FXR2P +T428 GO:0005844 12136 12145 polysomal +T429 http://purl.obolibrary.org/obo/MONDO_0010383 12182 12210 fragile X mental retardation +T430 PR:000003389 12182 12218 fragile X mental retardation protein +T431 PR:000003390 12230 12235 FXR1P +T432 PR:000003391 12240 12245 FXR2P +T433 GO:0005844 12270 12283 polyribosomes +T434 PR:000006288 12335 12341 DAZAP1 +T435 PR:000006290 12346 12350 DAZL +T436 PR:000006284 12351 12354 DAZ +T437 GO:0010467 12428 12438 expression +T438 SO:0000704 12451 12456 genes +T439 CL:0000586 12460 12470 germ cells +T440 PR:000006288 12485 12491 DAZAP1 +T441 GO:0051028 12517 12529;12534 12539 transport of ... mRNAs +T442 SO:0000704 12554 12559 genes +T443 PR:000006290 12563 12567 DAZL +T444 PR:000006290 12584 12588 DAZL +T445 PR:000006288 12593 12599 DAZAP1 +T446 GO:0065007 12628 12636 regulate +T447 GO:0010467 12665 12675 expression +T448 CL:0000540 12777 12783 neuron +T449 GO:0005634 12793 12800 nuclear +T450 PR:000011339 12822 12828 Nova-1 +T451 PR:000011339 12830 12836 Nova-1 +T452 GO:0065007 12837 12846 regulates +T453 GO:0000380 12851 12874;12879 12888 alternative splicing of ... pre-mRNAs +T454 SO:0000932 12879 12888 pre-mRNAs +T455 CL:0000540 12898 12906 neuronal +T456 PR:000008043 12918 12937 glycine receptor α2 +T457 PR:000008043 12939 12946 GlyR α2 +T458 PR:000011339 12969 12975 Nova-1 +T459 SO:0000147 12988 12992 exon +T460 CL:0000540 13006 13013 neurons +T461 PR:000013411 13062 13067 brPTB +T462 UBERON:0000955 13069 13074 brain +T463 PR:000013411 13069 13120 brain-enriched polypyrimidine tract-binding protein +T464 SO:0000612 13084 13104 polypyrimidine tract +T465 PR:000011339 13144 13150 Nova-1 +T466 PR:000006288 13183 13189 DAZAP1 +T467 PR:000006290 13239 13243 DAZL +T468 PR:000006290 13301 13306 Dazl1 +T469 PR:000006288 13311 13317 Dazap1 +T470 NCBITaxon:10088 13346 13350 mice +T471 PR:000006290 13416 13421 Dazl1 +T472 NCBITaxon:10088 13432 13436 mice +T473 GO:0007283 13486 13499 spermatogenic +T474 GO:0007567 13558 13564 partum +T475 CL:0000586 13574 13584 germ cells +T476 GO:0007126 13603 13610 meiosis +T477 SO:0001026 13651 13658 genomic +T478 PR:000006288 13672 13678 Dazap1 +T479 PR:000006288 13733 13739 Dazap1 +T480 PR:000006288 13756 13762 DAZAP1 +T481 NCBITaxon:8353 13912 13919 Xenopus +T482 SO:0000855 13920 13930 orthologue +T483 PR:000006288 13934 13940 DAZAP1 +T484 PR:000006288 13942 13946 Prrp +T485 PR:000006288 13987 13991 Prrp +T486 SO:0000205 14026 14032 3' UTR +T487 NCBITaxon:8353 14036 14043 Xenopus +T488 GO:0051028 14112 14121;14137 14139;14144 14148 migration ... of ... mRNA +T489 GO:0005938 14164 14170 cortex +T490 CL:0000023 14181 14187 oocyte +T491 PR:000006288 14189 14193 Prrp +T492 SO:0000417 14234 14240 domain +T493 PR:000007072 14297 14301 Mena +T494 GO:0005938 14365 14371 cortex +T495 PR:000000046 14409 14437 transforming growth factor-β +T496 UBERON:0000926 14484 14492 mesoderm +T497 UBERON:0000108 14500 14514 blastula stage +T498 NCBITaxon:8353 14518 14525 Xenopus +T499 GO:0009790 14526 14539 embryogenesis +T500 PR:000006288 14588 14594 DAZAP1 +T501 PR:000006288 14599 14603 Prrp +T502 NCBITaxon:40674 14758 14767 mammalian +T503 SO:0000857 14795 14803 homology +T504 PR:000006288 14863 14867 Prrp +T505 PR:000006288 14882 14888 DAZAP1 +T506 PR:000006288 14937 14943 DAZAP1 +T507 PR:000006288 14959 14965 DAZAP1 +T508 UBERON:0000479 15059 15066 tissues +T509 GO:0010467 15084 15094 expression +T510 UBERON:0000473 15098 15104 testes +T511 GO:0007283 15124 15139 spermatogenesis +T512 NCBITaxon:10088 15144 15149 mouse +T513 UBERON:0000473 15150 15156 testes +T514 PR:000006288 15158 15164 DAZAP1 +T515 GO:0005634 15187 15193 nuclei +T516 GO:0005737 15205 15214 cytoplasm +T517 GO:0005844 15233 15246 polyribosomes +T518 GO:0006412 15298 15309 translation +T519 NCBITaxon:10088 15348 15353 mouse +T520 PR:000006288 15354 15360 Dazap1 +T521 SO:0000317 15361 15372 cDNA clones +T522 PR:000006288 15374 15380 Dazap1 +T523 SO:0000317 15381 15392 cDNA clones +T524 NCBITaxon:10088 15414 15419 mouse +T525 UBERON:0000473 15420 15426 testis +T526 NCBITaxon:9606 15484 15489 human +T527 PR:000006288 15490 15496 DAZAP1 +T528 SO:0000112 15619 15625 primer +T529 NCBITaxon:10088 15650 15655 mouse +T530 UBERON:0000473 15656 15662 testis +T531 SO:0000112 15725 15731 primer +T532 SO:0000112 15739 15746 primers +T533 SO:0000006 15774 15786 PCR products +T534 SO:0000440 15817 15823 vector +T535 PR:000006288 15863 15869 Dazap1 +T536 GO:0097617 15903 15916 hybridization +T537 SO:0000204 15967 15973 5' UTR +T538 SO:0000317 15989 15999 cDNA clone +T539 GO:0006266 16009 16016 ligated +T540 SO:0000317 16076 16086 cDNA clone +T541 PR:000006288 16088 16094 Dazap1 +T542 SO:0000667 16122 16128 insert +T543 PR:000006288 16154 16160 Dazap1 +T544 PR:000006288 16162 16168 Dazap1 +T545 SO:0000040 16169 16183 genomic clones +T546 NCBITaxon:10088 16205 16210 mouse +T547 SO:0001026 16217 16224 genomic +T548 SO:0000357 16270 16278 flanking +T549 SO:0000147 16284 16289 exons +T550 SO:0000112 16311 16318 primers +T551 SO:0000188 16404 16412 intronic +T552 SO:0000357 16423 16431 flanking +T553 PR:000006288 16432 16438 Dazap1 +T554 SO:0000147 16439 16443 exon +T555 SO:0000112 16453 16460 primers +T556 SO:0000028 16477 16479 bp +T557 NCBITaxon:10088 16494 16499 mouse +T558 NCBITaxon:10026 16508 16515 hamster +T559 SO:0001026 16516 16523 genomic +T560 NCBITaxon:10088 16588 16593 mouse +T561 NCBITaxon:10026 16620 16627 hamster +T562 NCBITaxon:10088 16711 16716 mouse +T563 PR:000006288 16717 16723 Dazap1 +T564 SO:0000704 16857 16861 gene +T565 GO:0010467 16864 16874 Expression +T566 PR:000006288 16878 16884 Dazap1 +T567 SO:0000673 16885 16896 transcripts +T568 GO:0097617 16907 16920 hybridization +T569 NCBITaxon:10088 16983 16988 mouse +T570 UBERON:0000479 16998 17004 Tissue +T571 GO:0097617 17071 17081 hybridized +T572 PR:000006288 17100 17106 DAZAP1 +T573 PR:000003676 17111 17118 β-actin +T574 GO:0001171 17180 17201 Reverse transcription +T575 GO:0097617 17286 17295 annealing +T576 SO:0000112 17326 17333 primers +T577 PR:000006288 17409 17415 Dazap1 +T578 SO:0000028 17449 17451 bp +T579 PR:000006290 17527 17532 Dazl1 +T580 SO:0000028 17566 17568 bp +T581 SO:0000112 17575 17581 primer +T582 GO:0097617 17588 17596 annealed +T583 SO:0000188 17604 17610 intron +T584 SO:0000366 17611 17626 insertion sites +T585 PR:000006288 17648 17654 DAZAP1 +T586 GO:0042571 17655 17665 antibodies +T587 GO:0042571 17667 17677 Antibodies +T588 NCBITaxon:562 17740 17747 E. coli +T589 CHEBI:25676 17755 17767 oligopeptide +T590 SO:0000913 17794 17803;17813 17823 insert of ... cDNA clone +T591 PR:000006288 17806 17812 Dazap1 +T592 PR:000006288 17869 17875 DAZAP1 +T593 SO:0001817 17912 17920 in-frame +T594 GO:0010467 17953 17963 expression +T595 SO:0000440 17964 17970 vector +T596 PR:000006288 18116 18122 DAZAP1 +T597 CHEBI:60809 18247 18255 adjuvant +T598 NCBITaxon:9986 18274 18281 rabbits +T599 PR:000006288 18303 18309 DAZAPl +T600 GO:0042571 18312 18320 antibody +T601 CHEBI:25676 18325 18337 oligopeptide +T602 NCBITaxon:10088 18387 18392 mouse +T603 PR:000006288 18393 18399 DAZAP1 +T604 MOP:0000779 18501 18511 conjugated +T605 NCBITaxon:9925 18550 18554 goat +T606 PR:000006288 18565 18571 DAZAP1 +T607 GO:0042571 18574 18582 antibody +T608 CHEBI:25676 18647 18659 oligopeptide +T609 CHEBI:59132 18660 18667 antigen +T610 NCBITaxon:10088 18688 18693 Mouse +T611 UBERON:0000479 18694 18701 tissues +T612 GO:0019835 18731 18736 lysis +T613 CHEBI:26710 18752 18756 NaCl +T614 CHEBI:63016 18763 18768 NP-40 +T615 CHEBI:8984 18785 18788 SDS +T616 CHEBI:9754 18796 18800 Tris +T617 UBERON:0000479 18838 18844 tissue +T618 UBERON:0000479 18987 18993 tissue +T619 UBERON:0000479 19120 19126 tissue +T620 CHEBI:8984 19158 19161 SDS +T621 PR:000006288 19215 19221 DAZAP1 +T622 GO:0042571 19224 19232 antibody +T623 PR:000006288 19269 19275 DAZAP1 +T624 GO:0042571 19278 19286 antibody +T625 NCBITaxon:3704 19334 19345 horseradish +T626 MOP:0000779 19357 19367 conjugated +T627 GO:0042571 19378 19388 antibodies +T628 GO:0042571 19405 19415 antibodies +T629 NCBITaxon:10088 19531 19536 mouse +T630 UBERON:0000473 19537 19547 testicular +T631 UBERON:0007023 19558 19563 Adult +T632 NCBITaxon:10088 19564 19569 mouse +T633 UBERON:0000473 19570 19576 testes +T634 CHEBI:9754 19623 19627 Tris +T635 CHEBI:32588 19644 19647 KCl +T636 CHEBI:6636 19654 19659 MgCl2 +T637 CHEBI:63016 19666 19671 NP-40 +T638 CHEBI:35222 19703 19712 inhibitor +T639 CHEBI:60004 19743 19750 mixture +T640 GO:0005634 19950 19956 nuclei +T641 GO:0005739 20036 20048 mitochondria +T642 CHEBI:17992 20101 20108 sucrose +T643 CHEBI:9754 20128 20132 Tris +T644 CHEBI:32588 20141 20144 KCl +T645 CHEBI:6636 20154 20159 MgCl2 +T646 PR:000006288 20411 20417 Dazap1 +T647 GO:0010467 20418 20428 expression +T648 SO:0000440 20429 20436 vectors diff --git a/src/ontogpt/evaluation/craft/database/all/11604102.txt b/src/ontogpt/evaluation/craft/database/all/11604102.txt new file mode 100644 index 000000000..b3598b376 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/11604102.txt @@ -0,0 +1,121 @@ +Characterization of the mouse Dazap1 gene encoding an RNA-binding protein that interacts with infertility factors DAZ and DAZL + +Abstract + +Background + +DAZAP1 (DAZ Associated Protein 1) was originally identified by a yeast two-hybrid system through its interaction with a putative male infertility factor, DAZ (Deleted in Azoospermia). In vitro, DAZAP1 interacts with both the Y chromosome-encoded DAZ and an autosome-encoded DAZ-like protein, DAZL. DAZAP1 contains two RNA-binding domains (RBDs) and a proline-rich C-terminal portion, and is expressed most abundantly in the testis. To understand the biological function of DAZAP1 and the significance of its interaction with DAZ and DAZL, we isolated and characterized the mouse Dazap1 gene, and studied its expression and the subcellular localization of its protein product. + +Results + +The human and mouse genes have similar genomic structures and map to syntenic chromosomal regions. The mouse and human DAZAP1 proteins share 98% identity and their sequences are highly similar to the Xenopus orthologue Prrp, especially in the RBDs. Dazap1 is expressed throughout testis development. Western blot detects a single 45 kD DAZAP1 protein that is most abundant in the testis. Although a majority of DAZAP1 is present in the cytoplasmic fraction, they are not associated with polyribosomes. + +Conclusions + +DAZAP1 is evolutionarily highly conserved. Its predominant expression in testes suggests a role in spermatogenesis. Its subcellular localization indicates that it is not directly involved in mRNA translation. + +Background + +Spermatogenesis is a complex developmental process in which male germ cells progress through mitotic proliferation, meiotic division and dramatic morphological changes to form mature sperm. This process is vital for the propagation of a species, and involves a large portion of the genome of an organism to ensure the quality and quantity of the final products. It is estimated that mutations in up to 11% of all genes in Drosophila might lead to male sterility [1]. This is likely to be true for humans also, considering the extremely high incidence (4–5%) of infertility in men [2]. Among the genes associated with male infertility is the DAZ (Deleted in Azoospermia) gene family. The family includes the Y-linked DAZ genes that are present only in great apes and old world monkeys [3], and the autosomal DAZL1 (DAZ-like 1) and BOULE genes [4,5] in all mammals. Deletion of the DAZ genes is found in about 10% of infertile males with idiopathic azoospermia [2], and disruption of Dazl1 causes infertility in both male and female mice [6]. Mutations in the DAZ family members of Drosophila[7], C. elegans[8], and Xenopus[9] also affect the fertility in either males, females, or both sexes. + +The DAZ gene family encodes RNA binding proteins that are expressed specifically in germ cells. DAZ and DAZL are expressed in the nucleus and cytoplasm of primordial germ cells and spermatogonia, and in the cytoplasm of meiotic spermatocytes [6,10]. BOULE is expressed later, in the cytoplasm of pachytene spermatocytes [5]. Genetic and biochemical studies suggest a role for the DAZ family in the regulation of mRNA translation. Drosophila Boule mutants was defective in the translation of the meiosis-specific CDC25 homologue, Twine [11], and DAZL was found to be associated with polyribosomes in mouse testes [12]. More recently, DAZL was shown both in vitro and in a yeast three-hybrid system to bind specifically to oligo(U) stretches interspersed by G or C residues, including a U-rich segment in the 5' UTR of mouse Cdc25C mRNA [13]. + +In an attempt to elucidate the function of the DAZ gene family and to understanding the mechanisms of its action, we used a yeast two-hybrid system to isolate two human genes encoding DAZ associated proteins (DAZAPs) [14]. One of them, DAZAP1, is expressed predominantly in testes. It encodes a protein with two RNA binding domains and a proline rich C-terminal portion. The DAZAP1 protein interacted with both DAZ and DAZL in vitro. It also bound to RNA homopolymers. We now report our characterization of the mouse Dazap1 gene and its protein product. The subcellular localization of DAZAP1 suggests that it is not involved directly in mRNA translation. + +Results + +Characterization of the mouse Dazap1 cDNA + +Mouse Dazap1 cDNA clones were isolated by library screening, and the 5' end of the cDNA was isolated by 5' RACE [15]. The near fall length cDNA consists of a 53 bp 5' untranslated region (UTR), an open reading frame for a protein of 405 amino acid residues, and a 362 bp 3' UTR (GenBank Accession No: AF225910). The coding region shares 89% similarity with that of the human orthologue. The 3' UTR sequence is remarkably conserved. It contains three segments of 35 bp, 133 bp and 90 bp that share 85%, 90%, and 97% similarity with segments in the human 3' UTR, respectively. These segments probably contain regulatory elements. + +The DAZAP1 protein contains two RNA-binding domains (RBDS) and a C-terminal portion that is rich in proline (Figure 1). It is highly conserved evolutionarily. The mouse and the human proteins differ in 9 amino acids only, with 7 substitutions and two deletions/insertions. The mammalian proteins shares 89% similarity and 81% identity with Xenopus Prrp (for proline-rich RNA binding protein) [16]. The two RBDs are especially highly conserved. They share 98% and 97% similarity and 97% and 92% identity, respectively, between DAZAP1 and Prrp. These proteins may therefore have a similar RNA binding specificity. The C-terminal proline-rich portions of DAZAP1 and Prrp are less conserved (81% similarity and 71% identity). There is an insertion of a 58 bp segment in Prrp cDNA that causes a change of reading frame and results in a shorter Prrp with a different C-terminal end sequence. + +Figure 1 + +Evolutionary conservation of the DAZAP1 proteins. The amino acid sequences of the human and mouse DAZAP1s and the Xenopus Prrp are compared. The two RNA binding domains are boxed. Differences between the human and the mouse sequences, and between the mouse and Xenopus sequences are marked by #'s and *'s, respectively. + +Genomic structure of Dazap1 and chromosomal mapping + +Several overlapping lambda clones containing mouse Dazap1 genomic sequences were isolated. The locations of exons were determined by PCR amplification across exon-intron boundaries following by sequencing. All but the first exon were isolated and mapped. The genomic structure of the human DAZAP1 gene was also determined by blasting the human genome sequence at National Center for Biotechnology Information with the human DAZAP1 cDNA sequence. The mouse and the human genes have very similar structures, consisting of 12 exons spanning about 28 kb. All intron insertion sites are conserved (Table 1). The two RBDs are encoded by exons 1–4 and 5–8, respectively. + +Table 1 + +Exon-intron Organization of the Mouse and Human DAZAP1 genes + +a: Introns are inserted after the indicated nucleotide positions of DAZAP1 cDNA sequences. GenBank accession numbers for mouse and human cDNAs are AF225910 and AF181719, respectively. + +A pair of PCR primers was designed from Dazap1 intronic sequences that amplified mouse but not hamster genomic sequences. Using a panel of mouse-hamster radiation hybrids, the mouse Dazap1 gene was mapped to chromosome 10 placed 27.84 cR from D10Mit260 (lod > 3.0) (data not shown). This region is syntenic to human 19p13.3 where the human DAZAP1 gene is located [14,17]. It contains no known mutant alleles that are associated with infertility. + +Expression of Dazap1 + +Northern analyses of adult mouse tissues showed the presence of two Dazap1 transcripts of 1.75 kb and 2.4 kb, respectively (Figure 2). Only the shorter transcript has been isolated in cDNA clones. Dazap1 was expressed most abundantly in the testis, much less in liver, heart and brain, and even less in other tissues. This pattern of expression is similar to that of the human DAZAP1 (14). RT-PCR analyses showed that Dazap1 mRNA was already present in fetal testes at embryonic day 15, similar to Dazl1 mRNA (Figure 3). The expression of both Dazl1 and Dazap1 persisted throughout testes development, in both the prenatal and postnatal periods. Dazl1 and Dazap1 transcripts were also present in the testes of Wv/Wv mutant mice which contained diminished number of germ cells [18]. However, only Dazap1 was expressed in a mouse germ cell line GCl-spg [19] and a Sertoli cell line MT4. The results suggest that Dazap1 is expressed in both somatic and germ cells in the testis. + +Figure 2 + +Expression of Dazap1 in adult mouse tissues. A mouse multiple-tissue Northern blot was hybridized with a Dazap1 cDNA probe, stripped, and rehybridized with a β-actin probe. Dazap1 is expressed most abundantly in the testis. + +Figure 3 + +Developmental expression of Dazap1 and Dazl in mouse testes. RT-PCR was performed on total testicular RNAs isolated from day 15 (El 5) and day 17 (El 7) embryos, new born mice (Day 0), and mice at various days after birth. Wv/Wv testes contain diminished germ cell population due to a mutated W (White spotted) gene. GC1 and MT4 are mouse germ cell and Sertoli cell lines, respectively, and gDNA is mouse genomic DNA. The PCR primers span over introns and produce much larger (if any) fragments from genomic DNA. + +To study the expression of the DAZAP1 protein, two antibodies against mouse DAZAP1 were generated. The anti DAZAP1-C antibody was raised against a recombinant protein containing the C-terminal proline-rich portion, and the anti DAZAP1-P antibody was raised against an oligopeptide containing the last 19 amino acid residue at the C-terminus. Both antibodies recognized in vitro synthesized DAZAP1 in an immunoprecipitation assay (data not shown). Western blotting of mouse tissue extracts detected a 45 kD protein that was present most abundantly in the testis, and to a lesser degree in spleen, liver, lung and brain (Figure 4). The protein was also present in the ovary. The expression of DAZAP1 during germ cell development paralleled that of DAZL (Figure 5). It is present at a low level in the testes of 6 days old mice which contained only primitive type A spermatogonia. The expression of DAZAP1 increased afterward, as the testes contained increasing number of proliferating and meiotic germ cells. + +Figure 4 + +Expression of the DAZAP1 protein in adult mouse tissues. Equal amounts of total protein from various tissue extracts were applied to a 10% SDS-polyacrylamide gel and western blotted with the anti-DAZAP1-P antibody. + +Figure 5 + +Western blot analyses of the expression of DAZAP1 and DAZL in mouse testes during postnatal development. + +Subcellular localization of DAZAP1 + +Our previous fractionation of mouse testis extracts showed that most DAZL were present in the post mitochondrial fraction, and some of them were associated with polyribosomes [12]. Similar analyses showed that a majority of DAZAP1 in adult mouse testes was also present in the cytoplasmic fraction (data not shown). However, sucrose gradient analyses of the post- mitochondria fraction showed that, unlike DAZL, DAZAP1 did not co-sediment with polyribosomes (Figure 6). + +Figure 6 + +Sucrose gradient analyses shows that DAZAP1 is not associated with polyribosomes. The post-mitochondrial supernatant of mouse testis extracts was analyzed on a 15–45% sucrose gradient. Sedimentation was from left to right. The presence of DAZAP1 and DAZL in each fractions was analyzed by Western blotting. + +Discussion + +RNA-binding proteins have been found to participate in many cellular functions, including RNA transcription, pre-mRNA processing, mRNA transport, localization, translation and stability [20]. A role for the DAZ family in the regulation of mRNA translation is supported by lines of circumstantial evidence, including the association of DAZL with polyribosomes [12]. The absence of DAZAP1 from polyribosomes indicates that it is not directly involved in protein synthesis. This finding is different from two RNA-binding proteins, FXR1P and FXR2P, that were identified through their interaction with another polysomal-associated RNA-binding protein, the fragile X mental retardation protein [21]. Both FXR1P and FXR2P are associated with the polyribosomes [22]. + +The significance of the interaction between DAZAP1 and DAZL/DAZ remains to be defined. These proteins may act together to facilitate the expression of a set of genes in germ cells. For example, DAZAP1 could be involved in the transport of the mRNAs of the target genes of DAZL. Alternatively, DAZL and DAZAP1 may act antagonistically to regulate the timing and the level of expression. Such an antagonistic interaction between two interacting RNA-binding proteins is exemplified by the neuron-specific nuclear RNA-binding protein, Nova-1. Nova-1 regulates the alternative splicing of the pre-mRNAs encoding neuronal inhibitory glycine receptor α2 (GlyR α2) [23]. The ability of Nova-1 to activate exon selection in neurons is antagonized by a second RNA-binding protein, brPTB (brain-enriched polypyrimidine tract-binding protein), which interacts with Nova-1 and inhibits its function [24]. DAZAP1 could function in a similar manner by binding to DAZL and inhibiting its function. Comparing the phenotypes of Dazl1 and Dazap1 single and double knock-out mice may provide some clues to the significance of their interaction. Dazl1 knock-out mice have already been generated and studied [6]. The spermatogenic defect in the male becomes apparent only after day 7 post partum when the germ cells are committing to meiosis (H. Cooke, personal communication). The genomic structure of Dazap1, delineated here, should facilitate the generating of Dazap1 null mutation. + +DAZAP1 was shown to bind RNA homopolymers in vitro, with a preference for poly U and poly G. Its natural substrates have not been identified. Recently, the Xenopus orthologue of DAZAP1, Prrp, was identified and characterized [16]. Prrp binds to a 340 nt sequence in the 3' UTR of Xenopus Vg1 mRNA. This Vg1 localization element (VLE) is sufficient for the migration and clustering of Vg1 mRNA to the vegetal cortex of mature oocyte. Prrp also interacts through its proline-rich domain with two microfilament-associated proteins profilin and Mena, which may facilitate the anchoring of Vg1 mRNA to the vegetal cortex. The Vg1 RNA encodes a member of the transforming growth factor-β family that is required for generating dorsal mesoderm at the blastula stage of Xenopus embryogenesis [25]. Sequence conservation between the RBDs of DAZAP1 and Prrp suggests that these proteins may bind to similar RNA sequences. However, a BLAST search of the GenBank for the 340 nt VLE sequence failed to identify any mammalian sequences with significant homology. Further mapping of the RNA sequence within VLE that binds Prrp, and possibly DAZAP1, may help to identify the natural substrates of DAZAP1. + +Conclusions + +DAZAP1 is an evolutionarily conserved RNA-binding protein. It is present at variable levels in many tissues. Its predominant expression in testes suggests a role in spermatogenesis. In mouse testes, DAZAP1 was found both in the nuclei and in the cytoplasm. Its absence from polyribosomes indicates that it is not directly involved in mRNA translation. + +Materials and methods + +Isolation of mouse Dazap1 cDNA clones + +Dazap1 cDNA clones were isolated from a mouse testis cDNA library (#937308, Stratagene, La Jolla, CA) using a human DAZAP1 cDNA as a probe. The 5' end of the cDNA was isolated by 5' RACE [15], using prdap11 (ttgcgggccatatccttg, #749–732) as the primer for cDNA synthesis from mouse testis RNA, and prdap37 (ttgttgccacgtgggcg, #734–718) and an adaptor primer as the primers for PCR amplification. The PCR products were cloned into a TA cloning vector pCR2.1-TOPO (Invitrogen, Carlsbad CA). Dazap1 clones were identified by colony hybridization and sequenced. The 5' RACE clone with the longest 5' UTR region and the cDNA clone P21 were ligated together through a unique PmlI site at # 722 to generate a cDNA clone (Dazap1-C) with a near full-length insert. + +Chromosomal mapping of Dazap1 + +Dazap1 genomic clones were isolated from a mouse 129SV genomic library (#946305, Stragagene), and sequences flanking each exons were determined. PCR primers (prdap25: cacctccaggatgtgttagc and prdazp26:gtcaccaagggtgtctgaag) were designed from intronic sequences flanking Dazap1 exon 8. These primers amplified a 271 bp fragment from mouse but not hamster genomic DNA. DNA samples of a panel of 100 radiation hybrids containing mouse chromosome fragments in a hamster background were purchased from Research Genetics (Huntsville, AL). The presence of mouse Dazap1 in the radiation hybrids was determined by PCR and the results were sent to the MIT server for computerized physical mapping of the gene. + +Expression of Dazap1 transcripts + +Northern hybridization was carried out according to standard procedures [26] using a mouse Multiple Tissue Northern Blot #7762–1 from Clontech (Palo Alto, CA). The blot was hybridized sequentially with DAZAP1 and β-actin cDNA probes, with stripping of the bound probes in between. + +Reverse transcription-polymerase chain reaction (RT-PCR) was carried out as preciously described using an annealing temperature of 54°C [27]. The primers were prdap35: agctcagggagtacttcaaga and prdap24 :ggagcttgattcttgctgtcc for Dazap1 which generated a product of 211 bp, and prdaz71: atcgaactggtgtgtcgaagg and prdaz72: ggaggctgcatgtaagtctca for Dazl1 which generated a product of 245 bp. Both primer pairs annealed across intron insertion sites. + +Generation of anti-DAZAP1 antibodies + +Antibodies were generated against both a recombinant protein produced in E. coli and an oligopeptide synthesized in vitro. The insert of a Dazap1 cDNA clone P21, which encoded the C-terminal portion of DAZAP1 (starting from aa #197), was cloned in-frame into the EcoRI/XhoI sites of an expression vector pET32b (Novagen, Madison, WI). Sequences at the junctions were verified by DNA sequencing. Milligrams of fusion proteins between thioredoxin and DAZAP1 were prepared and purified on His-Bind metal chelation resins (Novagen, Madison, WI). The proteins were mixed with Freund's adjuvant and injected into rabbits to generate the anti-DAZAPl-C antibody. An oligopeptide containing the last 19 ammo acid residues of the mouse DAZAP1 was synthesized in vitro using the services of Bethyl Laboratories (Montgomery, TX). The peptide was conjugated to KLH as carrier and injected into a goat. The anti-DAZAP1-P antibody thus produced was purified on an affinity column containing the oligopeptide antigen. + +Western blotting + +Mouse tissues were homogenized in the RIPA lysis buffer (150 mM NaCl, 1.0% NP-40, 0.5% DOC, 0.1% SDS, 50 mM Tris, pH 8.0) at a concentration of 0.2 g tissue per ml of buffer. The homogenized samples were cleared of debris by centrifugation at 10,000 × g for 10 minutes. Protein concentration of the tissue extracts was determined by the Bradford method using the Bio-Rad Protein Assay system (Bio-Rad, Hercules, CA). About 50 μg of tissue extracts were separated on 10% SDS-polyacrylamide gels and blotted with either the anti-DAZAP1-C antibody (at a 1/2,000 dilution) or the anti-DAZAP1-P antibody (at a 1/5,000 dilution). After incubation with horseradish peroxidase-conjugated secondary antibodies, the binding of antibodies was detected using the ECL Western Blotting System (Amersham Pharmacia Biotech, Piscataway, NJ). + +Fractionation of mouse testicular extracts + +Adult mouse testes were homogenized in a buffer containing 20 mM Tris, pH 7.5, 100 mM KCl, 5 mM MgCl2, 0.3% NP-40, 40 U/ml of Rnasin ribonuclase inhibitor (Promega, Madison, WI), and a mixture of 10 protease inhibitors provided in the Protease Inhibitors Set (Roche Molecular Biochemicals, Indianapolis, IN). Homogenates were centrifuged at 1,000 × g for 10 minutes to pellet cell debris and nuclei. After an additional centrifugation at 10,000 × g for 10 minutes to pellet the mitochondria, aliquots of the supernatant were applied to 15–45% sucrose gradients in 20 mM Tris, 100 mM KCl and 5 mM MgCl2 and centrifuged in a Beckman SW41 rotor at 39,000 rpm for 2 hours at 4°C. Fractions of 0.5 ml were collected from the bottom of the tubes and analyzed by western blotting. + +Acknowledgments + +We thank Gary Kuo for his involvement in the construction of Dazap1 expression vectors, and Ron Swerdloff's group for helpful discussion. The work was supported by grants from the National Institutes of Health (HD28009 and HD36347). Y. Vera was supported by an NIH grant (GM56902) on Initiative for Minority Student Development. diff --git a/src/ontogpt/evaluation/craft/database/all/11897010.ann b/src/ontogpt/evaluation/craft/database/all/11897010.ann new file mode 100644 index 000000000..d6100e0d2 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/11897010.ann @@ -0,0 +1,463 @@ +T1 NCBITaxon:10088 36 41 mouse +T2 PR:000010252 42 48 Mcoln1 +T3 SO:0000704 49 53 gene +T4 GO:0000380 65 86 alternatively spliced +T5 SO:1001187 65 97 alternatively spliced transcript +T6 NCBITaxon:9606 110 116 humans +T7 http://purl.obolibrary.org/obo/MONDO_0009653 140 161 Mucolipidosis type IV +T8 http://purl.obolibrary.org/obo/MONDO_0009653 163 167 MLIV +T9 GO:0030849 175 184 autosomal +T10 http://purl.obolibrary.org/obo/MONDO_0006025 175 194;213 221 autosomal recessive disorder +T11 GO:0005764 195 204 lysosomal +T12 http://purl.obolibrary.org/obo/MONDO_0002561 195 221 lysosomal storage disorder +T13 UBERON:0001016 246 256 neurologic +T14 UBERON:0000970 261 275 ophthalmologic +T15 http://purl.obolibrary.org/obo/MONDO_0009653 304 308 MLIV +T16 SO:0000704 309 313 gene +T17 PR:000010252 315 321 MCOLN1 +T18 PR:000000681 366 394;401 415 transient receptor potential ... cation channel +T19 PR:000000681 396 399;401 415 TRP ... cation channel +T20 CHEBI:36916 401 407 cation +T21 NCBITaxon:10088 484 489 mouse +T22 SO:0000853 490 499 homologue +T23 PR:000010252 501 507 Mcoln1 +T24 GO:0008380 528 534 splice +T25 NCBITaxon:9606 563 569 humans +T26 NCBITaxon:9606 585 590 human +T27 NCBITaxon:10088 595 600 mouse +T28 SO:0000704 601 606 genes +T29 SO:0000860 632 639 synteny +T30 PR:000010252 641 647 Mcoln1 +T31 PR:000010252 700 706 MCOLN1 +T32 PR:000010252 714 720 Mcoln1 +T33 SO:0000236 758 776 open reading frame +T34 SO:0000673 804 814 transcript +T35 SO:0000147 858 863 exons +T36 NCBITaxon:9606 880 885 human +T37 SO:0000673 903 913 transcript +T38 NCBITaxon:39107 932 938 murine +T39 GO:0000380 948 968 alternative splicing +T40 PR:000010252 1048 1054 Mcoln1 +T41 PR:000010252 1076 1082 MCOLN1 +T42 GO:0016020 1107 1115 membrane +T43 SO:0000417 1116 1123 domains +T44 CHEBI:24870 1128 1131 ion +T45 GO:0005770 1155 1169 late endosomal +T46 SO:0001529 1160 1169;1180 1196 endosomal ... targeting signal +T47 GO:0005764 1170 1179 lysosomal +T48 SO:0001530 1170 1196 lysosomal targeting signal +T49 GO:0012506 1276 1293 vesicle membranes +T50 NCBITaxon:species 1342 1349 species +T51 GO:0008380 1359 1365 splice +T52 PR:000010252 1400 1406 Mcoln1 +T53 NCBITaxon:10088 1440 1445 mouse +T54 http://purl.obolibrary.org/obo/MONDO_0009653 1457 1461 MLIV +T55 SO:0000673 1491 1502 transcripts +T56 NCBITaxon:10088 1506 1510 mice +T57 SO:0000704 1563 1567 gene +T58 http://purl.obolibrary.org/obo/MONDO_0009653 1624 1645 Mucolipidosis type IV +T59 http://purl.obolibrary.org/obo/MONDO_0009653 1647 1651 MLIV +T60 GO:0030849 1671 1680 autosomal +T61 http://purl.obolibrary.org/obo/MONDO_0006025 1671 1690;1709 1717 autosomal recessive disorder +T62 GO:0005764 1691 1700 lysosomal +T63 http://purl.obolibrary.org/obo/MONDO_0002561 1691 1717 lysosomal storage disorder +T64 UBERON:0000964 1743 1750 corneal +T65 GO:0036343 1769 1780 psychomotor +T66 UBERON:0000104 1864 1868 life +T67 http://purl.obolibrary.org/obo/MONDO_0043465 1954 1966 achlorhydric +T68 http://purl.obolibrary.org/obo/MONDO_0009653 2017 2021 MLIV +T69 CHEBI:37395 2034 2052 mucopolysaccharide +T70 GO:0007588 2053 2062 excretion +T71 UBERON:0004288 2064 2072 skeletal +T72 http://purl.obolibrary.org/obo/MONDO_0019248 2113 2126 mucolipidoses +T73 GO:0005764 2137 2146 lysosomal +T74 GO:0000322 2147 2161 storage bodies +T75 GO:0005773 2172 2180 vacuoles +T76 UBERON:0001811 2209 2220 conjuctival +T77 CHEBI:10545 2236 2244 electron +T78 SO:0000704 2270 2274 gene +T79 http://purl.obolibrary.org/obo/MONDO_0009653 2392 2396 MLIV +T80 NCBITaxon:9606 2524 2529 human +T81 SO:0000704 2530 2534 gene +T82 PR:000010252 2535 2541 MCOLN1 +T83 PR:000000681 2646 2674;2681 2695 transient receptor potential ... cation channel +T84 PR:000000681 2676 2679;2681 2695 TRP ... cation channel +T85 CHEBI:36916 2681 2687 cation +T86 SO:0000704 2696 2700 gene +T87 GO:0015031 2721 2740 Protein trafficking +T88 http://purl.obolibrary.org/obo/MONDO_0009653 2762 2766 MLIV +T89 GO:0006897 2805 2814 endocytic +T90 http://purl.obolibrary.org/obo/MONDO_0019248 2846 2859 mucolipidoses +T91 GO:0005764 2900 2909 lysosomal +T92 NCBITaxon:6239 2945 2967 Caenorhabditis elegans +T93 PR:000010252 3026 3032 MCOLN1 +T94 NCBITaxon:6239 3033 3043 C. elegans +T95 SO:0000853 3044 3053 homologue +T96 PR:000010252 3055 3060 cup-5 +T97 GO:0006897 3093 3104 endocytosis +T98 GO:0005773 3128 3136 vacuoles +T99 GO:0006897 3162 3173 endocytosed +T100 GO:0030163 3174 3191 protein breakdown +T101 GO:0010467 3204 3214 expression +T102 SO:0000704 3223 3227 gene +T103 NCBITaxon:10088 3293 3298 mouse +T104 SO:0000853 3299 3308 homologue +T105 PR:000010252 3312 3318 MCOLN1 +T106 NCBITaxon:10088 3353 3358 mouse +T107 http://purl.obolibrary.org/obo/MONDO_0009653 3369 3373 MLIV +T108 http://purl.obolibrary.org/obo/MONDO_0000001 3396 3404 disorder +T109 NCBITaxon:10088 3458 3463 mouse +T110 SO:0000853 3464 3473 homologue +T111 PR:000010252 3474 3480 Mcoln1 +T112 NCBITaxon:10088 3504 3509 mouse +T113 SO:0000853 3510 3519 homologue +T114 PR:000010252 3523 3529 MCOLN1 +T115 NCBITaxon:9606 3535 3540 human +T116 SO:0001026 3597 3604 genomic +T117 NCBITaxon:10088 3666 3671 mouse +T118 SO:0000153 3672 3675 BAC +T119 SO:0001026 3747 3753 Genome +T120 NCBITaxon:9606 3818 3823 Human +T121 SO:0001026 3824 3830 Genome +T122 SO:0000153 3869 3872 BAC +T123 NCBITaxon:10088 3876 3881 mouse +T124 SO:0001249 3925 3937 physical map +T125 SO:0000153 3973 3976 BAC +T126 NCBITaxon:10088 4011 4016 mouse +T127 SO:0000345 4017 4020 EST +T128 SO:0000345 4057 4061 ESTs +T129 SO:0001249 4132 4140;4156 4159 Physical ... map +T130 SO:0000673 4145 4155 transcript +T131 PR:000010252 4167 4173 Mcoln1 +T132 SO:0000704 4174 4178 gene +T133 SO:0001249 4191 4203 Physical map +T134 NCBITaxon:10088 4232 4237 mouse +T135 SO:0000860 4251 4260 syntenous +T136 NCBITaxon:9606 4264 4269 human +T137 SO:0000704 4301 4306 genes +T138 PR:000012946 4316 4319 Nte +T139 SO:0000153 4352 4356 BACs +T140 SO:0000673 4449 4459 transcript +T141 PR:000010252 4467 4473 Mcoln1 +T142 SO:0000147 4483 4488 exons +T143 SO:0000147 4590 4594 exon +T144 GO:0000380 4605 4626 alternatively spliced +T145 SO:1001187 4605 4637 alternatively spliced transcript +T146 SO:0000853 4684 4693 homologue +T147 NCBITaxon:9606 4701 4706 human +T148 SO:0000704 4719 4723 gene +T149 SO:0000673 4777 4787 transcript +T150 SO:0000153 4831 4834 BAC +T151 SO:0000673 4985 4995 transcript +T152 SO:0000236 5004 5022 open reading frame +T153 PR:000010252 5053 5059 Mcoln1 +T154 SO:0000153 5118 5121 BAC +T155 SO:0000704 5144 5148 gene +T156 SO:0000147 5164 5169 exons +T157 SO:0000673 5189 5199 transcript +T158 PR:000010252 5211 5217 Mcoln1 +T159 NCBITaxon:10088 5228 5233 mouse +T160 SO:0000853 5314 5323 homologue +T161 NCBITaxon:9606 5331 5336 human +T162 SO:0000704 5349 5353 gene +T163 PR:000010252 5429 5435 Mcoln1 +T164 PR:000012946 5441 5444 Nte +T165 NCBITaxon:10088 5450 5455 mouse +T166 SO:0000853 5456 5465 homologue +T167 NCBITaxon:9606 5473 5478 human +T168 http://purl.obolibrary.org/obo/MONDO_0005244 5479 5489 neuropathy +T169 PR:000012946 5479 5505 neuropathy target esterase +T170 SO:0000704 5506 5510 gene +T171 PR:000012946 5512 5515 NTE +T172 SO:0000028 5531 5541 base pairs +T173 GO:0043631 5552 5567 polyadenylation +T174 SO:0000551 5552 5574 polyadenylation signal +T175 PR:000010252 5579 5585 Mcoln1 +T176 SO:0000673 5591 5601 transcript +T177 PR:000010252 5632 5638 Mcoln1 +T178 SO:0000853 5685 5695;5709 5715 homologous ... region +T179 NCBITaxon:9606 5696 5701 human +T180 PR:000010252 5702 5708 MCOLN1 +T181 SO:0000704 5758 5762 gene +T182 PR:000012946 5767 5770 Nte +T183 SO:0005858 5796 5813 region of synteny +T184 NCBITaxon:9606 5822 5827 human +T185 NCBITaxon:10088 5846 5851 mouse +T186 PR:000010252 5887 5893 Mcoln1 +T187 NCBITaxon:10088 5913 5918 mouse +T188 NCBITaxon:9606 5923 5928 human +T189 NCBITaxon:6239 5981 5991 C. elegans +T190 SO:0000853 5992 6001 homologue +T191 PR:000010252 6002 6007 cup-5 +T192 PR:000010252 6032 6038 Mcoln1 +T193 PR:000010252 6062 6068 Mcoln1 +T194 NCBITaxon:7227 6091 6114 Drosophila melanogaster +T195 SO:0000853 6115 6124 homologue +T196 PR:000010252 6178 6184 MCOLN1 +T197 http://purl.obolibrary.org/obo/MONDO_0009653 6225 6229 MLIV +T198 PR:000010252 6336 6342 MCOLN1 +T199 GO:0016020 6362 6370 membrane +T200 SO:0000417 6371 6378 domains +T201 GO:0005737 6421 6430 cytoplasm +T202 PR:000010252 6487 6493 Mcoln1 +T203 NCBITaxon:9606 6497 6502 human +T204 NCBITaxon:7227 6504 6519 D. melanogaster +T205 NCBITaxon:6239 6525 6535 C. elegans +T206 PR:000010252 6537 6542 cup-5 +T207 SO:0000853 6544 6554 homologues +T208 GO:0016020 6581 6589 membrane +T209 SO:0000417 6590 6597 domains +T210 GO:0005770 6686 6700 late endosomal +T211 SO:0001529 6691 6700;6711 6727 endosomal ... targeting signal +T212 GO:0005764 6701 6710 lysosomal +T213 SO:0001530 6701 6727 lysosomal targeting signal +T214 GO:0010467 6730 6740 Expression +T215 PR:000010252 6753 6759 Mcoln1 +T216 NCBITaxon:10088 6761 6766 Mouse +T217 UBERON:0007023 6767 6772 adult +T218 UBERON:0000479 6782 6788 tissue +T219 UBERON:0000922 6793 6802 embryonic +T220 GO:0097617 6823 6833 hybridized +T221 NCBITaxon:10088 6863 6868 mouse +T222 SO:0000147 6869 6873 exon +T223 SO:0001060 6937 6944 isoform +T224 SO:0001060 7010 7017 isoform +T225 GO:0010467 7083 7093 expression +T226 GO:0010467 7112 7122 expression +T227 UBERON:0000955 7126 7131 brain +T228 UBERON:0002107 7133 7138 liver +T229 UBERON:0002113 7143 7149 kidney +T230 CHEBI:33695 7211 7218 message +T231 GO:0007565 7235 7246 gestational +T232 NCBITaxon:9606 7262 7267 human +T233 PR:000010252 7268 7274 MCOLN1 +T234 SO:0000704 7275 7279 gene +T235 SO:0000673 7297 7307 transcript +T236 GO:0097617 7341 7355 hybridizations +T237 SO:0000851 7391 7401;7406 7421 regions of ... coding sequence +T238 SO:0000205 7426 7432 3' UTR +T239 SO:0000673 7473 7484 transcripts +T240 NCBITaxon:10088 7492 7497 mouse +T241 NCBITaxon:10088 7560 7565 mouse +T242 GO:0097617 7576 7586 hybridized +T243 NCBITaxon:10088 7589 7594 mouse +T244 SO:0000147 7618 7622 exon +T245 PR:000010252 7802 7808 Mcoln1 +T246 NCBITaxon:10088 7814 7819 Mouse +T247 UBERON:0005291 7820 7832 fetal tissue +T248 NCBITaxon:10088 7850 7855 mouse +T249 UBERON:0000479 7865 7871 tissue +T250 GO:0097617 7898 7908 hybridized +T251 SO:0000147 7937 7941 exon +T252 GO:0097617 7968 7981 Hybridization +T253 UBERON:0000479 7998 8004 tissue +T254 GO:0000380 8032 8053 alternatively spliced +T255 SO:0000852 8054 8069 segment of exon +T256 GO:0097617 8109 8122 hybridization +T257 PR:000010252 8174 8180 Mcoln1 +T258 GO:0000380 8181 8199 alternative splice +T259 SO:0000673 8266 8276 transcript +T260 NCBITaxon:10088 8294 8299 mouse +T261 SO:0000345 8300 8303 EST +T262 SO:0000188 8324 8330 intron +T263 SO:0001026 8346 8353 genomic +T264 SO:0000357 8363 8371 flanking +T265 PR:000010252 8376 8382 Mcoln1 +T266 SO:0000704 8383 8387 gene +T267 SO:0000345 8393 8397 ESTs +T268 SO:0000188 8443 8449 intron +T269 SO:0000345 8544 8548 ESTs +T270 SO:0000147 8612 8616 exon +T271 SO:0000147 8646 8650 exon +T272 GO:0008380 8655 8662 splices +T273 SO:0000147 8676 8680 exon +T274 SO:0000345 8700 8703 EST +T275 SO:0000028 8732 8734 bp +T276 SO:0000147 8742 8746 exon +T277 GO:0008380 8759 8766 splices +T278 SO:0000147 8780 8784 exon +T279 NCBITaxon:10088 8791 8796 mouse +T280 UBERON:0000479 8806 8812 tissue +T281 GO:0097617 8826 8836 hybridized +T282 SO:0000188 8879 8885 intron +T283 SO:0000673 9028 9038 transcript +T284 SO:0000112 9053 9060 primers +T285 SO:0000147 9064 9069 exons +T286 SO:0000112 9094 9100 primer +T287 SO:0000188 9104 9110 intron +T288 NCBITaxon:10088 9141 9146 mouse +T289 UBERON:0000955 9147 9152 brain +T290 SO:0000673 9237 9247 transcript +T291 GO:0000380 9261 9279 alternative splice +T292 SO:0000147 9314 9318 exon +T293 SO:0000147 9337 9341 exon +T294 GO:0008380 9345 9352 splices +T295 SO:0000028 9356 9358 bp +T296 SO:0000188 9366 9372 intron +T297 SO:0000028 9399 9401 bp +T298 SO:0000147 9402 9406 exon +T299 GO:0008380 9415 9422 splices +T300 SO:0000147 9436 9440 exon +T301 SO:0000236 9449 9467 open reading frame +T302 GO:0000380 9476 9497 alternatively spliced +T303 SO:1001187 9476 9508 alternatively spliced transcript +T304 CHEBI:33695 9560 9567 message +T305 SO:0000673 9590 9600 transcript +T306 SO:0001060 9633 9640 isoform +T307 PR:000010252 9687 9693 Mcoln1 +T308 GO:0016020 9713 9721 membrane +T309 SO:0000417 9722 9729 domains +T310 GO:0005737 9836 9847 cytoplasmic +T311 SO:0000673 9875 9885 transcript +T312 NCBITaxon:39107 9953 9959 murine +T313 SO:0000673 9976 9986 transcript +T314 NCBITaxon:10088 10006 10011 Mouse +T315 GO:0097617 10034 10044 hybridized +T316 SO:0000147 10054 10058 exon +T317 SO:0000188 10065 10071 intron +T318 SO:0000673 10138 10149 transcripts +T319 GO:0010467 10172 10182 expression +T320 UBERON:0000479 10248 10255 tissues +T321 GO:0000380 10336 10357 alternatively spliced +T322 PR:000010252 10358 10364 Mcoln1 +T323 GO:0005737 10424 10435 cytoplasmic +T324 GO:0016020 10476 10484 membrane +T325 SO:0000417 10485 10492 domains +T326 GO:0000380 10568 10589 alternatively spliced +T327 SO:1001187 10568 10589;10596 10606 alternatively spliced ... transcript +T328 NCBITaxon:10088 10590 10595 mouse +T329 NCBITaxon:9606 10621 10626 human +T330 PR:000010252 10627 10633 MCOLN1 +T331 SO:0001026 10634 10641 genomic +T332 NCBITaxon:9606 10743 10748 human +T333 PR:000010252 10749 10755 MCOLN1 +T334 SO:0000673 10784 10794 transcript +T335 GO:0097617 10812 10822 hybridized +T336 NCBITaxon:9606 10825 10830 human +T337 UBERON:0000479 10840 10846 tissue +T338 NCBITaxon:9606 10860 10865 human +T339 NCBITaxon:9606 10891 10896 human +T340 SO:0000188 10897 10903 intron +T341 SO:0000147 10927 10931 exon +T342 SO:0000860 10972 10980 syntenic +T343 SO:1001187 11007 11016;11023 11033 alternate ... transcript +T344 NCBITaxon:10088 11017 11022 mouse +T345 SO:1001187 11154 11176 alternative transcript +T346 NCBITaxon:39107 11192 11198 murine +T347 PR:000010252 11199 11205 Mcoln1 +T348 SO:1001187 11237 11246;11254 11264 alternate ... transcript +T349 PR:000010252 11247 11253 Mcoln1 +T350 PR:000010252 11562 11568 Mcoln1 +T351 SO:0000673 11604 11615 transcripts +T352 PR:000043184 11645 11661 Mcoln1 isoform 1 +T353 SO:0001060 11652 11659 isoform +T354 NCBITaxon:9606 11669 11674 human +T355 SO:0000853 11675 11684 homologue +T356 GO:0016020 11776 11784 membrane +T357 SO:0000417 11785 11792 domains +T358 CHEBI:36916 11818 11824 cation +T359 GO:0005770 11933 11947 late endosomal +T360 SO:0001529 11938 11947;11958 11974 endosomal ... targeting signal +T361 GO:0005764 11948 11957 lysosomal +T362 SO:0001530 11948 11974 lysosomal targeting signal +T363 PR:000010252 12042 12047 cup-5 +T364 NCBITaxon:6239 12057 12067 c. elegans +T365 SO:0000853 12068 12077 homologue +T366 PR:000010252 12081 12087 MCOLN1 +T367 GO:0005770 12166 12180 late endosomes +T368 GO:0005764 12188 12197 lysosomes +T369 NCBITaxon:10088 12204 12209 mouse +T370 PR:000010252 12210 12216 Mcoln1 +T371 SO:0000704 12217 12221 gene +T372 GO:0000380 12230 12251 alternatively spliced +T373 SO:0001060 12267 12274 isoform +T374 GO:0005737 12307 12318 cytoplasmic +T375 GO:0005764 12376 12385 lysosomal +T376 SO:0001530 12376 12402 lysosomal targeting signal +T377 SO:0000417 12438 12445 domains +T378 SO:0001060 12636 12643 isoform +T379 GO:0042571 12653 12663 antibodies +T380 PR:000010252 12735 12741 Mcoln1 +T381 NCBITaxon:10088 12752 12756 mice +T382 NCBITaxon:10088 12830 12835 mouse +T383 SO:0000704 12836 12840 gene +T384 GO:0010467 12836 12851 gene expression +T385 PR:000010252 12894 12900 Mcoln1 +T386 SO:0001060 12901 12908 isoform +T387 NCBITaxon:9606 12924 12930 humans +T388 GO:0000380 12955 12976 alternatively spliced +T389 NCBITaxon:9606 13009 13012 man +T390 NCBITaxon:10088 13017 13022 mouse +T391 SO:0000704 13042 13047 genes +T392 NCBITaxon:species 13077 13084 species +T393 GO:0000380 13094 13112 alternative splice +T394 PR:000010514 13123 13126 MOG +T395 UBERON:0000345 13128 13134 myelin +T396 PR:000010514 13128 13163 myelin/oligodendrocyte glycoprotein +T397 CL:0000128 13135 13150 oligodendrocyte +T398 CHEBI:17089 13151 13163 glycoprotein +T399 GO:0008380 13184 13190 splice +T400 NCBITaxon:9606 13203 13209 humans +T401 NCBITaxon:10088 13232 13236 mice +T402 PR:000031229 13243 13249 ATP11B +T403 NCBITaxon:9986 13274 13280 rabbit +T404 GO:0008380 13290 13296 splice +T405 GO:0016020 13325 13333 membrane +T406 SO:0000417 13334 13340 domain +T407 NCBITaxon:9606 13430 13435 human +T408 SO:0001026 13436 13442 genome +T409 SO:0000704 13488 13493 genes +T410 GO:0010467 13595 13604 expressed +T411 SO:0000345 13595 13618 expressed sequence tags +T412 SO:0000345 13620 13624 ESTs +T413 GO:0000380 13701 13721 alternative splicing +T414 SO:0000704 13734 13741 genetic +T415 GO:0043484 13783 13802 splicing regulation +T416 NCBITaxon:species 13877 13884 species +T417 GO:0008380 13894 13900 splice +T418 GO:0000380 13956 13977 alternatively spliced +T419 SO:1001187 13956 13989 alternatively spliced transcripts +T420 NCBITaxon:9606 14096 14101 Human +T421 SO:0001026 14102 14108 Genome +T422 PR:000010252 14168 14174 Mcoln1 +T423 NCBITaxon:10088 14576 14581 mouse +T424 UBERON:0000955 14582 14587 brain +T425 SO:0000112 14787 14793 primer +T426 GO:0000380 14834 14855 alternatively spliced +T427 SO:0000112 14867 14874 primers +T428 SO:0001030 14903 14910 forward +T429 SO:0001031 14943 14950 reverse +T430 GO:0097617 14978 14987 annealing +T431 NCBITaxon:10088 15040 15045 mouse +T432 SO:0001026 15046 15053 genomic +T433 PR:P23940 15084 15089 BamHI +T434 CHEBI:2511 15148 15155 agarose +T435 CHEBI:37972 15276 15279 32P +T436 SO:0001384 15297 15308;15320 15333 fragment of ... coding region +T437 PR:000010252 15313 15319 Mcoln1 +T438 SO:0000147 15351 15355 exon +T439 SO:0000112 15379 15386 primers +T440 SO:0001030 15414 15421 forward +T441 SO:0001031 15454 15461 reverse +T442 GO:0097617 15471 15480 annealing +T443 GO:0097617 15503 15516 Hybridization +T444 NCBITaxon:10088 15599 15604 Mouse +T445 UBERON:0000922 15605 15611 embryo +T446 UBERON:0000479 15621 15627 tissue +T447 NCBITaxon:10088 15646 15651 mouse +T448 UBERON:0007023 15652 15657 adult +T449 UBERON:0000479 15667 15673 tissue +T450 GO:0097617 15772 15782 hybridized +T451 CHEBI:37972 15792 15795 32P +T452 SO:0001384 15813 15824;15832 15845 fragment of ... coding region +T453 PR:000010252 15825 15831 Mcoln1 +T454 SO:0000147 15863 15867 exon +T455 SO:1001187 15903 15913 transcript +T456 SO:0000147 15959 15964 exons +T457 SO:0000112 15980 15987 primers +T458 SO:0001030 16014 16021 forward +T459 SO:0001031 16055 16062 reverse +T460 GO:0097617 16072 16081 annealing +T461 GO:0097617 16125 16135 hybridized +T462 PR:000003676 16141 16148 β-actin +T463 GO:0097617 16157 16171 Hybridizations diff --git a/src/ontogpt/evaluation/craft/database/all/11897010.txt b/src/ontogpt/evaluation/craft/database/all/11897010.txt new file mode 100644 index 000000000..7b9523496 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/11897010.txt @@ -0,0 +1,97 @@ +Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans + +Abstract + +Background + +Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder characterized by severe neurologic and ophthalmologic abnormalities. Recently the MLIV gene, MCOLN1, has been identified as a new member of the transient receptor potential (TRP) cation channel superfamily. Here we report the cloning and characterization of the mouse homologue, Mcoln1, and report a novel splice variant that is not seen in humans. + +Results + +The human and mouse genes display a high degree of synteny. Mcoln1 shows 91% amino acid and 86% nucleotide identity to MCOLN1. Also, Mcoln1 maps to chromosome 8 and contains an open reading frame of 580 amino acids, with a transcript length of approximately 2 kb encoded by 14 exons, similar to its human counterpart. The transcript that results from murine specific alternative splicing encodes a 611 amino acid protein that differs at the c-terminus. + +Conclusions + +Mcoln1 is highly similar to MCOLN1, especially in the transmembrane domains and ion pore region. Also, the late endosomal/lysosomal targeting signal is conserved, supporting the hypothesis that the protein is localized to these vesicle membranes. To date, there are very few reports describing species-specific splice variants. While identification of Mcoln1 is crucial to the development of mouse models for MLIV, the fact that there are two transcripts in mice suggests an additional or alternate function of the gene that may complicate phenotypic assessment. + +Background + +Mucolipidosis type IV (MLIV; MIM 252650) is an autosomal recessive lysosomal storage disorder that is characterized by corneal clouding, delayed psychomotor development, and mental retardation that usually presents during the first year of life [1]. Another interesting clinical characteristic is that patients are constitutively achlorhydric with associated hypergastremia [2]. Patients with MLIV do not show mucopolysaccharide excretion, skeletal changes, or organomegaly like the other mucolipidoses. Abnormal lysosomal storage bodies and large vacuoles have been found in skin and conjuctival biopsies using electron-microscopy and, prior to gene identification, served as the only means of diagnosis [3-5]. A recent report estimates that the carrier frequency of MLIV in the Ashkenazi Jewish population is 1 in 100, and mutations have been reported in Jewish and non-Jewish families [6-9]. + +The human gene MCOLN1 (GenBank #AF287270) maps to chromosome 19p13.2-13.3 and encodes a novel protein that is a member of the transient receptor potential (TRP) cation channel gene superfamily [7-10]. Protein trafficking studies suggest that MLIV is the result of a defect in the late endocytic pathway, contrary to the other mucolipidoses which are typically caused by defective lysosomal hydrolases [11,12]. Recent work in Caenorhabditis elegans supports this hypothesis. Loss of function mutants of the MCOLN1 C. elegans homologue, cup-5, result in an increased rate of endocytosis, accumulation of large vacuoles, and a decreased rate of endocytosed protein breakdown; while over-expression of this gene reverses the phenotype [13]. Cloning and characterization of the mouse homologue of MCOLN1 is crucial for the development of mouse models of MLIV to further study this disorder. + +Results and discussion + +Cloning and mapping of the mouse homologue Mcoln1 + +In order to clone the mouse homologue of MCOLN1, the human amino acid sequence was compared to the high throughput genomic sequence (HTGS) database using TBLASTN, which identified the mouse BAC clone RPCI-23_387H4 (GB No. AC079544.1). Correspondence with the Joint Genome Institute and the Lawrence Livermore National Laboratory (LLNL) Human Genome Center confirmed the location of this BAC to mouse chromosome 8 and allowed us to construct a physical map of this region [14] (Fig. 1A). The BAC sequence was then compared to the mouse EST database using BLASTN, and multiple ESTs and their corresponding I.M.A.G.E. clones were identified. + +Figure 1 + +Physical and transcript map of the Mcoln1 gene region. (A) Physical map showing a 590 kb segment of mouse chromosome 8 syntenous to human chromosome 19, anchored by the genes Insr and Nte. The region is covered by three BACs: RPCI-23_334D24 (AC087153), RPCI-23_312B8 (AC087150), and RPCI-23_387H4 (AC079544). (B) The transcript map of Mcoln1 shows 14 exons, and the locations of the probes used in this study are illustrated. The map also shows the expanded exon 13 in the alternatively spliced transcript (hashed box). UniGene cluster Mm.39099 is the homologue of the human zinc finger gene (AC001252). It should be noted that the scale of the transcript map is in reference to the 201 kb scale of BAC RPCI-23_387H4. + +Three clones (ID Nos. 604971, 1228665, 1247566) from the UniGene cluster Mm. 8356 were obtained, sequenced, and assembled into a 2 kb transcript with an open reading frame of 580 amino acids designated Mcoln1 (GB No. AF302010). Comparison of the cDNA sequence to the BAC clone showed that the gene consists of 14 exons. We then created a transcript map of the Mcoln1 region of mouse chromosome 8 (Fig. 1B), noting the presence of the UniGene Cluster Mm. 39099, a homologue of the human zinc finger gene (GB No. AC001252) that terminates approximately 1.8 kb before the start of Mcoln1; and Nte, the mouse homologue of the human neuropathy target esterase gene (NTE) beginning 130 base pairs after the polyadenylation signal for Mcoln1. The transcript map of the region surrounding Mcoln1 is similar to the corresponding region of the homologous human MCOLN1 region [15], and the presence of the zinc finger gene and Nte confirms and extends the region of synteny between human chromosome 19 and mouse chromosome 8. + +Characterization of Mcoln1 + +Comparison of the mouse and human peptide sequences showed 91% identity (Fig. 2). The C. elegans homologue cup-5 shows 34% identity with Mcoln1 and BLASTP analysis of Mcoln1 identified a putative Drosophila melanogaster homologue that shows 38% identity (Fig. 2). Interestingly, two MCOLN1 amino acid substitutions that result in MLIV occur at conserved amino acids. TMPred analysis predicts a protein structure that is nearly identical to MCOLN1, containing 6 transmembrane domains with the N- and C-termini residing in the cytoplasm (Fig. 2) [9]. + +Figure 2 + +Peptide sequence comparison of Mcoln1 to human, D. melanogaster, and C. elegans (cup-5) homologues. Blue lines indicate transmembrane domains, the red box surrounds the putative channel pore, the orange box surrounds the putative late endosomal/lysosomal targeting signal. + +Expression analysis of Mcoln1 + +Mouse adult multiple tissue and embryonic Northern blots were hybridized using a probe generated from mouse exon 2 (probe 1, Fig. 1B), yielding a band of approximately 2.4 kb (isoform 1), as expected, and a less abundant and unexpected 4.4 kb band (isoform 2) (Figs. 3A &3B). The 2.4 kb band shows ubiquitous but variable expression, with the highest expression in brain, liver and kidney. The fetal tissue blot shows decreasing levels of the 2.4 kb message with increasing gestational age. Since the human MCOLN1 gene encodes a single transcript [7-9], we carried out additional hybridizations with probes generated from various regions of the coding sequence and 3' UTR, and all probes identified the same two transcripts in the mouse (data not shown). In order to verify the presence of a single mouse locus, we hybridized a mouse Southern blot with the exon 2 probe. Four different restriction enzymes were used, and only the expected size bands for the chromosome 8 locus were detected (data not shown). + +Figure 3 + +Northern analysis of Mcoln1. (A) Mouse fetal tissue Northern and (B) mouse multiple tissue Northern blots (Clontech) hybridized with a probe generated from exon 2 (probe 1, Fig. 1B). (C) Hybridization of the multiple tissue blot with a probe from the alternatively spliced segment of exon 13 (probe 2, Fig. 1B). β-Actin control hybridization is shown below each blot. + +Characterization of the Mcoln1 alternative splice variant + +In order to determine the coding sequence for the larger transcript, we searched the mouse EST database using each intron as well as the genomic sequence flanking the Mcoln1 gene. Two ESTs were identified that contained sequence from intron 12 (GB No. AI430291 and AA874645), and the corresponding clones were sequenced. Clone 408619 (ESTs: GB No. AI430291, AI429558) begins approximately 1.1 kb before exon 13 and continues through the exon and splices correctly to exon 14. Clone 1281641 (EST:GB No. AA874645) begins 175 bp before exon 13 and also splices correctly to exon 14. A mouse multiple tissue Northern was hybridized using a probe generated from the putative intron sequence in clone 408619 (Probe 2, Fig. 1B), which detected only the 4.4 kb band (Fig. 3C). + +In order to determine the sequence of the entire transcript, RT-PCR using primers in exons 10 and 11 paired with a primer in intron 12 was performed using BALB/c mouse brain total RNA and the resulting products sequenced. These products show that the larger transcript is due to an alternative splice event that results in an expanded exon 13. Specifically, exon 12 splices at bp 436 of intron 12, creating a large 1614 bp exon 13 that splices correctly to exon 14. The open reading frame of this alternatively spliced transcript is 611 amino acids, 28 amino acids longer than the message encoded by the 2.4 kb transcript. + +TMPred analysis predicts that isoform 2 encodes a protein identical in structure to Mcoln1, possessing 6 transmembrane domains and a channel pore, however the protein sequences diverge at amino acid 526. The 55 amino acid C-terminal cytoplasmic tail encoded by the 2.4 kb transcript is completely different from the 86 amino acid tail encoded by the murine specific 4.4 kb transcript (Fig. 4). Clontech Mouse RNA Master Blots were hybridized with the exon 2 and intron 12 probes mentioned above in an attempt to determine if these two transcripts showed differences in expression patterns, however, there was no significant difference in the 22 tissues represented (data not shown). + +Figure 4 + +Peptide sequence comparison of the two alternatively spliced Mcoln1 isoforms. The green box surrounds the divergent c-terminal cytoplasmic tails. The blue lines indicate the transmembrane domains. + +Next, we directly compared the nucleotide and amino acid sequence of the alternatively spliced mouse transcript to the entire human MCOLN1 genomic sequence and found no significant similarity. As mentioned previously, Northern blots performed with human MCOLN1 probes show only one 2.4 kb transcript. In addition, we hybridized a human multiple tissue Northern and human Southern with a probe in human intron 12 that is adjacent to exon 13. The probe was located in the region syntenic to that which encodes the alternate mouse transcript. Only the expected bands were detected on the Southern and no bands were detected on the Northern, confirming that this alternative transcript is specific to murine Mcoln1. Recent BLASTP analysis of the alternate Mcoln1 transcript yields a match to a putative 145 amino acid anonymous protein (GB No. BAB25862) predicted from a RIKEN clone. It is obvious from our results, however, that the identification of this sequence as a full-length protein is incorrect since probes unique to the clone, as well as probes containing the Mcoln1 coding sequence, identify the same transcripts. + +Conclusions + +Comparison of Mcoln1 isoform 1 to its human homologue shows striking similarity at both the amino acid and nucleotide level. All six of the transmembrane domains, as well as the putative cation channel are highly conserved. The putative di-leucine (L-L-X-X) motif at the C-terminus, which may act as a late endosomal/lysosomal targeting signal, is also conserved [9]. This speculation is supported by work with cup-5[13], the c. elegans homologue of MCOLN1, since cellular localization studies suggest that the protein is found in the late endosomes and/or lysosomes. + +The mouse Mcoln1 gene has two alternatively spliced isoforms, with isoform 2 having a different c-terminal cytoplasmic tail. The unique 86 amino acid c-terminal tail lacks the lysosomal targeting signal and does not contain any conserved domains when compared against the current profile databases. We speculate that this protein may have similar channel function but an alternate subcellular localization, but this must be proven once isoform-specific antibodies are raised. However, our results suggest that phenotypic assessment of Mcoln1 knock-out mice may be complicated and that care must be taken when interpreting data on mouse gene expression and phenotype. + +Interestingly, the second Mcoln1 isoform is not seen in humans and the sequence of the alternatively spliced region is not conserved between man and mouse. To date, very few genes have been reported that show species specific alternative splice variants. MOG, myelin/oligodendrocyte glycoprotein, has many different splice variants in humans that are not found in mice [17]. ATP11B, a P-type ATPase, has a rabbit-specific splice variant that deletes a transmembrane domain and therefore likely alters the putative function of the protein [18]. Sequencing of the human genome has led to estimates of approximately 32,000 genes, a total surprise given the previous significantly higher estimates that were based on the number of expressed sequence tags (ESTs) in the public databases. This apparent disparity suggests a major role for alternative splicing in creating genetic complexity, and has brought the study of splicing regulation to the forefront of molecular genetics. It is likely that an abundance of species-specific splice variants will be identified as the characterization of alternatively spliced transcripts progresses. + +Materials and methods + +Bioinformatics + +We conducted database searches using BLAST and Draft Human Genome Browser . Sequences from UniGene were used to confirm the Mcoln1 sequence. We performed motif searches using ProfileScan and TMPred and alignment of protein sequences using Pileup (GCG) and Boxshade . + +DNA Sequencing + +I.M.A.G.E. Clones were purchased from Research Genetics (Huntsville, AL). Sequencing was performed using the AmpliCycle sequencing kit from Applied Biosystems (Foster City, CA) on a Genomyx LR programmable DNA sequencer. + +RT-PCR Reaction + +BALB/c mouse brain total RNA was purchased from Clontech Laboratories (Palo Alto, CA) and made to a 1 μg / ml concentration. This RNA was used as a template to create cDNA via RT-PCR using random hexamers and oligo dT primer. The RT product was used to confirm the alternatively spliced form using primers: 5'-CATCTACCTGGGCTATTGC-3' (forward) and 5'-GCTCTCAGGTGGTGGACAC-3' (reverse) in a PCR reaction with an annealing temperature of 61°C. + +Southern Blot Analysis + +Total mouse genomic DNA was digested using EcoRI, BamHI, PstI, and XbaI. The digests were electrophoresed on a 1% agarose gel at 60V overnight and were transferred onto a Hybond N+ membrane from Amersham Pharmacia Biotech (Piscataway, NJ). A 32P-dATP labeled PCR fragment of the Mcoln1 coding region corresponding to exon 2 was used as a probe (primers 5'-CCCCACAGAAGAGGAAGAC-3' (forward) and 5'-AGATCTTGACCACCTGCAG-3' (reverse) with an annealing temperature of 59°C). Hybridization and washes were carried out in standard conditions [16]. + +Northern Blot Analysis + +Mouse embryo multiple-tissue northern blot and mouse adult multiple-tissue northern blot filters were purchased from Clontech Laboratories (Palo Alto, CA). The filters were hybridized with the 32P-dATP labeled DNA fragment of Mcoln1 coding region corresponding to exon 2 (see above). For the alternative transcript, a probe was generated in the region between exons 12 and 13 with primers 5'-GTGTCCACCACCTGAGAG-3' (forward) and 5'-GAAGTAGCATTCCTGCAGGC-3' (reverse) with an annealing temperature of 62°C. The filters were then hybridized with β-actin probes. Hybridizations and washes were carried out in standard conditions, with the stripping of previously bound probes in between [16]. + +Acknowledgements + +This work was supported by grant NS39995. diff --git a/src/ontogpt/evaluation/craft/database/all/12079497.ann b/src/ontogpt/evaluation/craft/database/all/12079497.ann new file mode 100644 index 000000000..8649c0819 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/12079497.ann @@ -0,0 +1,333 @@ +T1 UBERON:0000922 0 9 Embryonic +T2 CL:0002322 0 20 Embryonic stem cells +T3 NCBITaxon:10088 25 29 mice +T4 GO:0010467 30 40 expressing +T5 NCBITaxon:33208 121 127 animal +T6 NCBITaxon:1 252 261 organisms +T7 GO:0008218 320 334 bioluminescent +T8 NCBITaxon:6142 335 344 jellyfish +T9 NCBITaxon:6100 345 362 Aequoria Victoria +T10 NCBITaxon:10088 388 393 mouse +T11 SO:0000704 409 413 gene +T12 SO:0000704 484 488 gene +T13 PR:000033987 514 518 lacZ +T14 CHEBI:75050 590 601 chromogenic +T15 UBERON:0000922 762 771 embryonic +T16 CL:0002322 762 776;782 787 embryonic stem ... cells +T17 CL:0002322 778 780;782 787 ES ... cells +T18 NCBITaxon:10088 792 796 mice +T19 UBERON:0000922 839 848 embryonic +T20 CL:0002322 839 853;859 863 embryonic stem ... cell +T21 CL:0002322 855 857;859 863 ES ... cell +T22 NCBITaxon:86599 1156 1166 Discostoma +T23 SO:0000857 1190 1198 homology +T24 CL:0002322 1249 1257 ES cells +T25 NCBITaxon:10088 1291 1295 mice +T26 GO:0010467 1314 1324 expression +T27 SO:0000902 1498 1507 transgene +T28 GO:0010467 1508 1518 expression +T29 CL:0002322 1679 1687 ES cells +T30 UBERON:0000922 1695 1702 embryos +T31 GO:0010467 1742 1752 expressing +T32 NCBITaxon:33208 1815 1821 animal +T33 GO:0010467 1959 1969 expressing +T34 CL:0002322 1981 1989 ES cells +T35 NCBITaxon:10088 1994 1998 mice +T36 UBERON:0000479 2051 2058 tissues +T37 SO:0001026 2191 2197 genome +T38 NCBITaxon:10088 2223 2228 mouse +T39 NCBITaxon:1 2250 2258 organism +T40 NCBITaxon:40674 2263 2272 mammalian +T41 SO:0000704 2273 2280 genetic +T42 SO:0000704 2386 2390 gene +T43 CL:0002322 2404 2412 ES cells +T44 SO:0000704 2439 2450 Genetically +T45 NCBITaxon:10088 2463 2467 mice +T46 SO:0000704 2489 2493 gene +T47 NCBITaxon:40674 2561 2570 mammalian +T48 http://purl.obolibrary.org/obo/MONDO_0000001 2587 2594 disease +T49 SO:0000704 2688 2692 gene +T50 NCBITaxon:2 2721 2730 bacterial +T51 PR:000033987 2731 2735 lacZ +T52 NCBITaxon:9606 2739 2744 human +T53 UBERON:0001987 2745 2754 placental +T54 PR:000003969 2745 2775 placental alkaline phosphatase +T55 CHEBI:75050 2850 2861 chromogenic +T56 GO:0008218 3118 3132 bioluminescent +T57 NCBITaxon:6142 3133 3142 jellyfish +T58 NCBITaxon:6100 3143 3160 Aequorea Victoria +T59 NCBITaxon:10088 3383 3387 mice +T60 UBERON:0001017 3837 3859 central nervous system +T61 UBERON:0001017 3861 3864 CNS +T62 NCBITaxon:10088 3869 3873 mice +T63 NCBITaxon:10239 3900 3905 viral +T64 http://purl.obolibrary.org/obo/MONDO_0005550 3906 3915 infection +T65 NCBITaxon:10088 4037 4041 mice +T66 NCBITaxon:10088 4132 4136 mice +T67 GO:0010467 4153 4163 expression +T68 GO:0010467 4322 4332 expression +T69 CL:0002322 4337 4344 ES cell +T70 GO:0006412 4798 4809 translation +T71 NCBITaxon:6101 4940 4949 anthozoan +T72 NCBITaxon:44295 5124 5135 sea anemone +T73 NCBITaxon:86599 5136 5145 Discosoma +T74 NCBITaxon:species 5146 5148 sp +T75 GO:0010467 5361 5371 expression +T76 NCBITaxon:40674 5375 5384 mammalian +T77 SO:0000028 5425 5434 base pair +T78 NCBITaxon:9606 5460 5465 human +T79 SO:0000360 5466 5471 codon +T80 CL:0002322 5560 5568 ES cells +T81 NCBITaxon:10088 5573 5577 mice +T82 SO:0000155 5580 5588 Plasmids +T83 GO:0010467 5629 5639 expression +T84 SO:0000704 5655 5659 gene +T85 CL:0002322 5720 5727 ES cell +T86 CL:0002322 5789 5797 ES cells +T87 GO:0010467 5816 5826 expression +T88 GO:0010467 5925 5935 expressing +T89 GO:0010467 6112 6122 expressing +T90 GO:0010467 6267 6277 expressing +T91 CL:0002322 6350 6357 ES cell +T92 GO:0048471 6686 6708;6713 6722 perinuclear regions of ... cytoplasm +T93 NCBITaxon:11034 6812 6825 sindbis virus +T94 GO:0030425 6846 6855 dendrites +T95 GO:0030424 6860 6865 axons +T96 UBERON:0001017 6877 6880 CNS +T97 UBERON:0007023 6884 6889 adult +T98 NCBITaxon:10088 6890 6894 mice +T99 CL:0002322 6969 6977 ES cells +T100 NCBITaxon:10088 6982 6986 mice +T101 GO:0005634 7041 7048 nuclear +T102 CL:0002322 7132 7140 ES cells +T103 NCBITaxon:10088 7144 7148 mice +T104 CL:0002322 7313 7321 ES cells +T105 CL:0002322 7380 7388 ES cells +T106 CL:0002322 7752 7760 ES cells +T107 CL:0002322 7932 7939 ES cell +T108 UBERON:0000085 7944 7950 morula +T109 UBERON:0000922 7957 7963 embryo +T110 CL:0002322 7991 7998 ES cell +T111 NCBITaxon:10088 8110 8114 mice +T112 GO:0010467 8147 8157 expression +T113 GO:0010467 8243 8253 expression +T114 NCBITaxon:10088 8264 8268 mice +T115 GO:0010467 8287 8297 expression +T116 GO:0007566 8331 8343 implantation +T117 GO:0007566 8378 8390 implantation +T118 GO:0009790 8391 8404 embryogenesis +T119 UBERON:0000113 8408 8417 adulthood +T120 GO:0010467 8437 8447 expressing +T121 NCBITaxon:33208 8552 8559 animals +T122 SO:0000704 8592 8596 gene +T123 GO:0010467 8592 8607 gene expression +T124 SO:0000366 8616 8623;8634 8645 site of ... integration +T125 SO:0000902 8624 8633 transgene +T126 UBERON:0000922 8731 8738 embryos +T127 UBERON:0007023 8743 8748 adult +T128 NCBITaxon:10088 8749 8753 mice +T129 NCBITaxon:33208 8766 8773 animals +T130 SO:0000902 8825 8835 transgenes +T131 UBERON:0000358 8843 8853 blastocyst +T132 UBERON:0000922 8867 8874 embryos +T133 UBERON:0000922 8936 8943 embryos +T134 SO:0000902 9043 9053 transgenes +T135 UBERON:0000922 9084 9091 embryos +T136 UBERON:0000922 9457 9464 embryos +T137 UBERON:0005631 9489 9513 extraembryonic membranes +T138 NCBITaxon:10088 9919 9923 mice +T139 GO:0010467 9941 9951 expressing +T140 NCBITaxon:10088 10138 10143 mouse +T141 NCBITaxon:10088 10179 10184 mouse +T142 NCBITaxon:10088 10224 10229 mouse +T143 NCBITaxon:10088 10263 10268 mouse +T144 UBERON:0002415 10314 10319 tails +T145 NCBITaxon:10088 10327 10331 mice +T146 CL:0000365 10345 10352 Zygotic +T147 GO:0010467 10353 10363 expression +T148 SO:0000902 10374 10384 transgenes +T149 UBERON:0000358 10411 10421 blastocyst +T150 UBERON:0000922 10524 10531 embryos +T151 UBERON:0000922 10571 10578 embryos +T152 UBERON:0000922 10619 10626 embryos +T153 GO:0007566 10635 10647 implantation +T154 GO:0007566 10681 10693 implantation +T155 UBERON:0007023 10734 10739 adult +T156 NCBITaxon:10088 10740 10744 mice +T157 CL:0002322 10868 10875 ES cell +T158 GO:0010467 10916 10926 expression +T159 GO:0010467 11001 11011 expression +T160 GO:0010467 11106 11116 expression +T161 NCBITaxon:33208 11120 11127 animals +T162 GO:0010467 11197 11207 expression +T163 CL:0002322 11256 11263 ES cell +T164 CL:0002322 11290 11297 ES cell +T165 UBERON:0010166 11332 11336 coat +T166 SO:0000902 11366 11375 transgene +T167 GO:0010467 11402 11412 expression +T168 UBERON:0000062 11462 11467 organ +T169 UBERON:0007023 11471 11476 adult +T170 NCBITaxon:10088 11477 11481 mice +T171 UBERON:0000922 11489 11496 embryos +T172 GO:0010467 11589 11599 expression +T173 SO:0000167 11615 11623 promoter +T174 SO:0000165 11624 11632 enhancer +T175 NCBITaxon:10088 11759 11763 mice +T176 NCBITaxon:33208 11898 11904 animal +T177 UBERON:0000922 11988 11995 embryos +T178 CL:0002322 12005 12013 ES cells +T179 UBERON:0000922 12018 12025 embryos +T180 GO:0010467 12119 12129 expression +T181 UBERON:0000922 12196 12203 embryos +T182 UBERON:0000922 12223 12229 embryo +T183 UBERON:0000922 12282 12291 embryonic +T184 UBERON:0007023 12399 12404 adult +T185 UBERON:0000922 12518 12525 embryos +T186 UBERON:0000922 12539 12546 embryos +T187 CL:0002322 12551 12559 ES cells +T188 UBERON:0000062 12646 12651 organ +T189 UBERON:0007023 12735 12740 adult +T190 UBERON:0000062 12741 12747 organs +T191 UBERON:0000922 13069 13076 embryos +T192 UBERON:0000922 13333 13340 embryos +T193 CL:0000353 13355 13366 blastomeres +T194 UBERON:0000085 13527 13534 morulae +T195 CL:0002322 13538 13546 ES cells +T196 UBERON:0002450 13633 13641 deciduum +T197 CL:0002497 13664 13683 primary giant cells +T198 UBERON:0008800 13688 13705 parietal endoderm +T199 UBERON:0002532 13716 13724 epiblast +T200 UBERON:0000922 13740 13746 embryo +T201 UBERON:0004366 13790 13813 extraembryonic ectoderm +T202 UBERON:0004877 13819 13836 visceral endoderm +T203 UBERON:0000088 13841 13852 trophoblast +T204 UBERON:0000922 13878 13884 embryo +T205 UBERON:0005631 14158 14182 extraembryonic membranes +T206 UBERON:0001040 14211 14219 yolk sac +T207 UBERON:0000922 14238 14244 embryo +T208 UBERON:0001040 14273 14281 yolk sac +T209 UBERON:0000926 14310 14318 mesoderm +T210 UBERON:0000925 14323 14331 endoderm +T211 UBERON:0009571 14401 14419;14424 14430 ventral midline of ... embryo +T212 UBERON:0004877 14562 14579 visceral endoderm +T213 CL:0000223 14571 14585 endoderm cells +T214 UBERON:0000925 14630 14638 endoderm +T215 UBERON:0000922 14719 14725 embryo +T216 UBERON:0001987 14759 14767 placenta +T217 UBERON:0000922 14788 14794 embryo +T218 UBERON:0000088 14837 14854 trophoblast layer +T219 UBERON:0001987 14862 14870 placenta +T220 UBERON:0000062 14968 14974 organs +T221 UBERON:0007023 14978 14983 adult +T222 UBERON:0000948 15204 15209 heart +T223 UBERON:0001264 15214 15222 pancreas +T224 UBERON:0007023 15261 15266 adult +T225 UBERON:0007023 15307 15312 adult +T226 UBERON:0000948 15313 15318 heart +T227 UBERON:0002082 15349 15361;15373 15378 ventricle of ... heart +T228 UBERON:0000085 15419 15426 morulae +T229 SO:0000902 15468 15477 transgene +T230 SO:0000902 15528 15537 transgene +T231 UBERON:0002082 15719 15728 ventricle +T232 UBERON:0001264 15743 15751 pancreas +T233 UBERON:0000085 15782 15788 morula +T234 SO:0000902 15817 15826 transgene +T235 CL:0002322 15841 15849 ES cells +T236 UBERON:0001264 15942 15950 pancreas +T237 UBERON:0002107 15956 15961 liver +T238 UBERON:0007023 16002 16007 adult +T239 UBERON:0000085 16035 16041 morula +T240 CL:0002322 16076 16084 ES cells +T241 SO:0000902 16112 16121 transgene +T242 SO:0000902 16166 16175 transgene +T243 UBERON:0000055 16194 16200 vessel +T244 GO:0008283 16214 16226 proliferated +T245 UBERON:0001113 16247 16257 liver lobe +T246 UBERON:0000948 16285 16290 heart +T247 UBERON:0001264 16295 16303 pancreas +T248 UBERON:0002107 16396 16401 liver +T249 GO:0048513 16461 16471;16477 16482 genesis of ... organ +T250 UBERON:0000062 16477 16482 organ +T251 NCBITaxon:10088 16633 16637 mice +T252 NCBITaxon:10088 16698 16702 mice +T253 GO:0010467 16724 16734 expression +T254 CL:0002322 16851 16859 ES cells +T255 UBERON:0000922 16861 16868 embryos +T256 NCBITaxon:10088 16872 16876 mice +T257 GO:0010467 16893 16903 expression +T258 GO:0010467 16996 17006 expression +T259 SO:0000704 17105 17109 gene +T260 UBERON:0000922 17398 17404 embryo +T261 UBERON:0007023 17408 17413 adult +T262 UBERON:0000062 17414 17419 organ +T263 GO:0010467 17492 17502 expressing +T264 UBERON:0000922 17540 17547 embryos +T265 UBERON:0007023 17551 17556 adult +T266 UBERON:0000062 17557 17563 organs +T267 NCBITaxon:33208 18124 18130 animal +T268 UBERON:0000479 18311 18317 tissue +T269 NCBITaxon:10088 18467 18472 mouse +T270 SO:0001026 18473 18479 genome +T271 SO:0000440 18551 18557 Vector +T272 SO:0000155 18628 18636 plasmids +T273 SO:0000112 18747 18754 primers +T274 SO:0000112 18869 18876 primers +T275 SO:0000112 18996 19003 primers +T276 SO:0000994 19020 19029;19053 19057 consensus ... site +T277 GO:0006413 19030 19052 translation initiation +T278 SO:0000323 19030 19057 translation initiation site +T279 GO:0006412 19092 19103 translation +T280 NCBITaxon:2759 19118 19128 eukaryotic +T281 CL:0000255 19118 19134 eukaryotic cells +T282 SO:0000440 19154 19161 vectors +T283 SO:0000902 19181 19190 transgene +T284 GO:0010467 19191 19201 expression +T285 CL:0002322 19205 19213 ES cells +T286 NCBITaxon:10088 19218 19222 mice +T287 NCBITaxon:9031 19388 19395 chicken +T288 PR:000003676 19396 19406 beta-actin +T289 SO:0000167 19407 19415 promoter +T290 SO:0000190 19420 19432 first intron +T291 MOP:0000779 19433 19440 coupled +T292 NCBITaxon:10358 19448 19451 CMV +T293 SO:0000165 19468 19476 enhancer +T294 NCBITaxon:9986 19482 19488 rabbit +T295 PR:000008457 19489 19500 beta-globin +T296 CHEBI:5386 19494 19500 globin +T297 GO:0043631 19501 19516 polyadenylation +T298 SO:0000551 19501 19523 polyadenylation signal +T299 SO:0000167 19530 19538 promoter +T300 SO:0000165 19539 19547 enhancer +T301 SO:0000902 19617 19626 transgene +T302 GO:0010467 19627 19637 expression +T303 CL:0002322 19641 19649 ES cells +T304 UBERON:0000922 19651 19658 embryos +T305 UBERON:0007023 19663 19668 adult +T306 NCBITaxon:10088 19669 19673 mice +T307 UBERON:0000479 19684 19690 Tissue +T308 CL:0002322 19703 19711 ES cells +T309 PR:000009799 19786 19789 LIF +T310 SO:0000988 19936 19944 circular +T311 CHEBI:23888 19984 19988 drug +T312 SO:0000902 20377 20386 transgene +T313 GO:0010467 20387 20397 expression +T314 CHEBI:23888 20431 20435 drug +T315 CL:0002322 20494 20501 ES cell +T316 SO:0000902 20526 20535 transgene +T317 GO:0010467 20536 20546 expression +T318 SO:0000902 20611 20620 transgene +T319 GO:0010467 20621 20631 expression +T320 UBERON:0000922 20732 20739 embryos +T321 CL:0002322 20768 20775 ES cell +T322 UBERON:0000922 20784 20791 embryos +T323 UBERON:0000922 20918 20925 embryos +T324 UBERON:0000922 21066 21073 embryos +T325 SO:0000902 21135 21145 transgenes +T326 SO:0000902 21173 21183 Transgenes +T327 SO:0000704 21241 21248 genetic +T328 UBERON:0007023 21680 21685 Adult +T329 NCBITaxon:10088 21686 21690 mice +T330 GO:0010467 22163 22173 expressing +T331 NCBITaxon:10088 22174 22178 mice +T332 SO:0000155 22472 22479 plasmid +T333 http://purl.obolibrary.org/obo/MONDO_0004992 22655 22661 Cancer diff --git a/src/ontogpt/evaluation/craft/database/all/12079497.txt b/src/ontogpt/evaluation/craft/database/all/12079497.txt new file mode 100644 index 000000000..9668ac453 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/12079497.txt @@ -0,0 +1,91 @@ +Embryonic stem cells and mice expressing different GFP variants for multiple non-invasive reporter usage within a single animal + +Abstract + +Background + +Non-invasive autofluorescent reporters have revolutionized lineage labeling in an array of different organisms. In recent years green fluorescent protein (GFP) from the bioluminescent jellyfish Aequoria Victoria has gained popularity in mouse transgenic and gene targeting regimes [1]. It offers several advantages over conventional gene-based reporters, such as lacZ and alkaline phosphatase, in that its visualization does not require a chromogenic substrate and can be realized in vivo. We have previously demonstrated the utility and developmental neutrality of enhanced green fluorescent protein (EGFP) in embryonic stem (ES) cells and mice [2]. + +Results + +In this study we have used embryonic stem (ES) cell-mediated transgenesis to test the enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP), two mutant and spectrally distinct color variants of wild type (wt) GFP. We have also tested DsRed1, the novel red fluorescent protein reporter recently cloned from the Discostoma coral by virtue of its homology to GFP. To this end, we have established lines of ES cells together with viable and fertile mice having widespread expression of either the ECFP or EYFP GFP-variant reporters. However, we were unable to generate equivalent DsRed1 lines, suggesting that DsRed1 is not developmentally neutral or that transgene expression cannot be sustained constitutively. Balanced (diploid <-> diploid) and polarized (tetraploid <-> diploid) chimeras comprising combinations of the ECFP and EYFP ES cells and/or embryos, demonstrate that populations of cells expressing each individual reporter can be distinguished within a single animal. + +Conclusions + +GFP variant reporters are unique in allowing non-invasive multi-spectral visualization in live samples. The ECFP and EYFP-expressing transgenic ES cells and mice that we have generated provide sources of cells and tissues for combinatorial, double-tagged recombination experiments, chimeras or transplantations. + +Background + +The ease of manipulating its genome has helped establish the mouse as the premier model organism for mammalian genetic studies. Both directed and random mutagenesis approaches, including the technologies of transgenesis and gene targeting in ES cells, have become commonplace. Genetically manipulated mice, often incorporating gene-based reporters, are frequently being used to model and understand mammalian development and disease processes. Fluorescent protein reporters currently represent a superior alternative to other gene-based reporters such as the bacterial lacZ or human placental alkaline phosphatase in that their visualization is non-invasive, and as such does not require chromogenic substrates. Fluorescence can be monitored in real-time in vivo and in situ, and has the added advantage in that it can be quantified. The prototype fluorescent protein reporter is green fluorescent protein (GFP, reviewed in [3]), which is derived from the bioluminescent jellyfish Aequorea Victoria [1]. + +Green fluorescent variants of wild type GFP (wtGFP) with improved thermostability and fluorescence emission, including enhanced green fluorescent protein (EGFP, [4]), and mMGFP [5], have gained popularity for use in mice. Recently however, several additional mutants of wtGFP, with altered excitation and emission spectral profiles (i.e. fluoresce in colors other than green), as well as improved thermostability and fluorescence, have been described [4,6]. + +Dual color imaging of several GFP variant proteins is a very attractive prospect and has become a standard procedure in cell biology [7,8]. It has also recently been demonstrated in a subset of cells within the central nervous system (CNS) of mice using both transgenic and viral infection-based approaches [9,10]. We therefore wished to investigate the feasibility of non-invasive multiple reporter imaging in mice, and as a first step we wanted to ascertain whether we could establish viable and fertile mice with widespread expression of GFP variant reporters. + +Results and discussion + +We tested several of the available wtGFP variants for developmental neutrality and stability of widespread expression. An ES cell-mediated transgenic approach was used to evaluate the Enhanced Cyan Fluorescent Protein (ECFP) and Enhanced Yellow Fluorescent Protein (EYFP) wtGFP spectral variants [4,11]. ECFP harbors six amino substitutions (Y66W, F64L, S65T, N146I, M153T, V163A) as compared to GFP, whereas EYFP harbors four (S65G, V68L, S72A and T203Y). In addition to increasing the quantum yield and solubility of the proteins these mutations shift the spectral profiles of the translation products [4,11]. + +A breakthrough in the availability of spectrally distinct autofluorescent proteins came with the cloning of six anthozoan fluorescent proteins all having 20–30% identity to wtGFP [12]. Of these, DsRed1 (referred to as drFP585 by Matz et al., [12]), a 28 kDa protein isolated from the IndoPacific sea anemone Discosoma sp, posseses the longest excitation (558 nm) and emission (583 nm) wavelength maxima known for a wild-type autofluorescent protein. The sequence of DsRed1 has recently been modified and optimized for high levels of expression in mammalian cells by the introduction of 144 silent base pair changes corresponding to human codon usage preferences. We therefore also tested this novel red fluorescent protein (RFP) in ES cells and mice. + +Plasmids were constructed for driving widespread expression of each of the gene-based fluorescent protein (FP) reporters and were tested in ES cell-mediated transgenics as described previously [2]. Transgenic ES cells exhibiting robust expression of ECFP or EYFP were obtained at the same frequency as we had previously obtained for EGFP. Cells expressing one or other of the two GFP variant reporters were clearly distinguishable within a mixed population (Fig. 1a,1b,1c,1d). Interestingly however we were unable to recover clones expressing DsRed1 at the same frequency as for the GFP variant reporters after electroporation and selection under identical conditions. Indeed the DsRed1 expressing clones that we were able to recover did not retain their characteristic ES cell morphology, usually appearing smaller and more spherical (Fig. 1e and 1f), or occasionally spindle shaped (data not shown). Unlike the fluorescence produced by the GFP variants which was evenly distributed throughout the cells, the DsRed signal was never homogenous. Red fluorescence was often aggregated in what appeared to be perinuclear regions of the cytoplasm. Similar observations have been made by Furuta and colleagues when they used recombinant sindbis virus for the labeling of dendrites and axons within the CNS of adult mice [10]. In order to generate a DsRed variant that may be amenable to use in ES cells and mice, we recently introduced various sequences that direct nuclear localization to the DsRed coding region. However, again we were unable to generate ES cells or mice exhibiting robust widespread reporter activity (A.-K.H. and V.E. Papaioannou, unpublished observations). + +Figure 1 + +Non-invasive multiple reporter visualization in ES cells. (a-d) a mixture of CK6/ECFP (ECFP+) and YC5/EYFP (EYFP+) ES cells at low density. (a) image taken under bright field with no epifluorescence. (b) dark field image taken through an EYFP filter. (c) dark field image taken through an ECFP filter. (d) dark field double exposure image acquired by consecutively using ECFP and EYFP filters. ECFP+ cells can clearly be discerned from EYFP+ cells. (e-f) culture of mixed populations of ES cells comprising ECFP+, EYFP+ and RFP+ cells. Scale bars represent 100 μm. + +Two ECFP+ and two EYFP+ clones exhibiting robust fluorescence were used to generate chimeras through ES cell <-> morula stage embryo aggregation [13]. All four ES cell lines transmitted through the germline, leading to the establishment of, CK4/ECFP and CK6/ECFP, two strains of mice having cyan fluorescent protein expression and, YC5/EYFP and 2A4/EYFP, two strains having widespread yellow fluorescent protein expression. In these mice the respective FP expression is constituitive, starting at preimplantation stages and continuing through postimplantation embryogenesis to adulthood (Fig. 2). One line expressing each color was chosen for further analysis and breeding to homozygosity. The availability of homozygous animals demonstrated that both reporter gene expression and the site of transgene integration were developmentally neutral. + +Figure 2 + +Dual non-invasive reporter visualization in embryos and adult mice. Transgenic animals are hemizygous for either the CK6/ECFP or YC5/EYFP transgenes. (a-d) blastocyst stage (E3.5) embryos, that are either fluorescent or non-fluorescent. Fluorescent embryos are either blue/cyan or yellow/green, thereby being hemizygous for either the CK6/ECFP or YC5/EYFP transgenes respectively. Non-fluorescent embryos (marked with an asterix on panel a) are of the wild type outbred ICR strain. (a) image taken under bright field with no epifluorescence. (b) dark field image taken through an EYFP filter. (c) dark field image taken through an ECFP filter. (d) dark field double exposure image acquired by consecutively using ECFP and EYFP filters. (e-h) two transgenic (TG/+) E13.5 embryos dissected free of their extraembryonic membranes, one is blue/cyan fluorescent (CK6/ECFP transgenic, left) and the other is yellow/green fluorescent (7YC5/EYFP transgenic, right). (e) image taken under bright field with no epifluorescence. (f) dark field image taken through an EYFP filter. (g), dark field image taken through an ECFP filter. (h) double exposure acquired by consecutively using ECFP and EYFP filters. (i-k) two three week old transgenic mice each exclusively expressing either the ECFP or EYFP reporter. All images shown are double exposures taken by consecutively using ECFP and then the EYFP filters under epifluorescence. (i) CK6/ECFP (TG/+) transgenic mouse bottom, YC5/EYFP (TG/+) transgenic mouse bottom. (j) CK6/ECFP (TG/+) transgenic mouse left, YC5/EYFP (TG/+) transgenic mouse right. (k) higher maginification view of the tails of the mice in i and j. + +Zygotic expression of the FP transgenes is clearly evident by the blastocyst stage (Fig. 2a,2b,2c,2d) and remains widespread thereafter (Fig. 2). Figure 2 demonstrates that ECFP+ embryos can easily be distinguished from EYFP+ embryos and from non-transgenic non-fluorescent embryos from preimplantation stages (Fig. 2a,2b,2c,2d) to postimplantation stages (Fig. 2e,2f,2g,2h). Additionally adult mice can also be discriminated on the basis of the color of their fluorescence (Fig. 2i,2j,2k). + +Even though our RFP transgenic ES cell lines failed to maintain strong uniform expression with in vitro passage, we selected three lines exhibiting the most robust expression for making chimeras, in order to ascertain whether we could achieve, or possibly recover, RFP expression in animals. Cells were flow sorted prior to aggregation so as to enrich for RFP expression. Even though they had lost their characteristic ES cell morphology all three RFP+ ES cell lines went gemline as detected by coat color and PCR for the DsRed1 transgene (data not shown). However expression of the RFP reporter could not be detected in any organ of adult mice nor in embryos (data not shown). This suggests that the DsRed1 RFP reporter was unable to recapitulate the expression profile of the promoter/enhancer combination employed. Therefore it appears that GFP variant reporters are more versatile with respect to their application in mice. + +We produced double FP-tagged chimeras in order to assess whether the ECFP and EYFP reporters could be co-visualized within the same animal. The first series of chimeras were generated through the aggregation of tetraploid embryos with FP+ ES cells (or embryos) of the complementary color. The procedure is schematized in Fig. 3a. It is evident that the expression of the two FPs can easily be discerned in these double transgenic embryos where the diploid, embryo-proper compartment is ECFP+ and the tetraploid extraembryonic compartment is EYFP+ (Fig. 3b,3c,3d,3e,3f). Secondly we investigated reporter co-visualization in balanced adult chimeras comprised of both ECFP+ and EYFP+ compartments. These were generated through the aggregation of diploid embryos with diploid embryos (or ES cells) of the complementary color. The aggregated components of the chimera from which each organ originated is schematized in the top right of each panel in Fig. 4. These chimeric adult organs demonstrate that the ECFP+ and EYFP+ cells comprising these chimeras can easily be discerned (Fig. 4). It can therefore be envisaged that two different FP colors could be used in the construction of chimeras so as to tag both mutant and wild type lineages. + +Figure 3 + +Dual non-invasive reporter visualization in chimeric embryos generated by tetraploid <-> diploid aggregation. In all panels the tetraploid compartment is yellow/green fluorescent (EYFP+) and the diploid is blue/cyan fluorescent (ECFP+). (a) schematic representation of the tetraploid procedure. Electrofusion of E1.5 embryos to render the blastomeres tetraploid, in vitro culture, and subsequent aggregation schemes using double-tagged compartments. Sandwich aggregations were made using either ECFP+ (diploid) morulae or ES cells. (b-d) anterior view of an E7.5 double-tagged polarized chimera dissected free of its deciduum and therefore missing primary giant cells and parietal endoderm. Here the epiblast (lower half of embryo) and its derivatives are ECFP+ whereas the extraembryonic ectoderm, the visceral endoderm and trophoblast are EYFP+ (upper half or embryo). (b) dark field image taken through an EYFP filter. (c) dark field image taken through an ECFP filter. (d) dark field double exposure image acquired by consecutively using ECFP and EYFP filters. (e) dark field double filter exposure of an E9 chimera dissected free of all extraembryonic membranes except for a small piece of yolk sac (lower left). The embryo itself is ECFP+ whereas the yolk sac both ECFP+ and EYFP+ in the mesoderm and endoderm respectively. A small number of EYFP+ cells are also observed in the ventral midline of the embryo (arrowhead). This observation has been noted previously in tetraploid chimeras, and is presumed to represent a small population of visceral endoderm cells that fail to be displaced by the definitive endoderm at earlier stages. (f) dark field double filter exposure of an E10 chimera. The embryo is exclusively ECFP+ whereas its placenta (to the left of the embryo) is predominantly EYFP+. The labyrinthine trophoblast layer of the placenta comprises both ECFP+ and EYFP+ cells. + +Figure 4 + +Dual non-invasive reporter visualization in the organs of adult chimeras. All images depict dark field double exposures acquired by consecutively using ECFP and EYFP filters under dark field epifluorescence. Details of aggregates are schematized at the top right of each panel. (a-c) heart and pancreas from double-tagged double-compartment adult chimeras. (a) view of the surface of an adult heart. (b) close-up surface view of ventricle of a chimeric heart generated by aggregation of two diploid morulae, one hemizygous for the CK6/ECFP (ECFP+) transgene and the other hemizygous for the YC5/EYFP (EYFP+) transgene. The regions of yellow/green and cyan fluorescence are clearly mutually exclusive. The striations in the fields of fluorescence represent the proliferative zones present within the ventricle. (c) chimeric pancreas generated by aggregation of a morula hemizygous for the YC5/EYFP transgene with CK6/ECFP ES cells. The restricted fields of fluorescence represent the proliferative zones present within the pancreas. (d) liver from a double-tagged triple-compartment adult chimera. A non-fluorescent morula was aggregated with two clumps of ES cells, one carrying the CK6/ECFP transgene (ECFP+) and the other carrying the YC5/EYFP transgene (EYFP+). An ECFP+ vessel has clonally proliferated and infiltrated the liver lobe (top). Note that unlike in heart and pancreas there appears to be more interspersed blue/cyan vs green/yellow fluorescence present in the liver, possibly reflecting greater cell intermingling during the genesis of this organ. + +Conclusions + +From these observations we conclude that the blue-shifted ECFP, and the red-shifted EYFP variants of wtGFP are both amenable to use in mice. Also since we have established lines of viable and fertile mice having widespread FP expression we have demonstrated that ECFP and EYFP represent developmentally neutral reporters. Since we were unable to obtain ES cells, embryos or mice with widespread expression of a RFP (DsRed1) we conclude that this reporter is not developmentally neutral or that its expression cannot be sustained at levels high enough for detection, and as such is not as versatile as other gene-based fluorescent reporters. + +ECFP and EYFP can be distinguished on the basis of their unique non-overlapping spectral profiles, and chimeras comprised of ECFP and EYFP tagged compartments demonstrate that the two reporters can be simultaneously non-invasively visualized within the same embryo or adult organ. Therefore it should be possible to use flow cytometry to isolate cells expressing either or both of the reporters from embryos or adult organs [14]. + +These two reporters can also be used to create pairs of donors and acceptors for in vivo fluorescence energy transfer (FRET) [15]. Since the emission profiles of ECFP and EYFP are the least overlapping of all the usable GFP variants, these ECFP and EYFP currently represent the best combination for co-visualization and for use in multi-spectral double-tagged transgenic, chimeric and/or in vivo FRET experiments (reviewed in [16]). + +These experiments have demonstrated the feasibility of monitoring multiple FP reporters non-invasively within a single animal, and have shown the developmental neutrality of the FP reporters employed. This should pave the way for novel strategies designed to incorporate the use of compound transgenics or tissue recombination or chimeric analyses where several compartments are to be tagged. This technology should provide new possibilities in manipulating the mouse genome, and non-invasively studying the consequences. + +Materials and methods + +Vector construction + +cDNAs encoding ECFP, EYFP and DsRed were amplified from plasmids pECFP, pEYFP and pDsRed1-N1 (Clontech, Inc.) by PCR using HiFi Taq Polymerase (Invitrogen). For ECFP and EYFP primers used were GFP-L (5'-TTG AAT TCG CCA CCA TGG TGA GC) and GFP-R (5'-TTG AAT TCT TAC TTG TAC AGC TCG TCC), for dsRed primers were red-L (5'-TTG AAT TCG CCA CCA TGG TGC GCT C) and red-R (5'-TTG AAT TCT TAC GCT ACA GCT ACA GGA ACA GGT G). All 5' primers contain a Kozak consensus translation initiation site upstream of the ATG for increased translation efficiency in eukaryotic cells [17]. To construct vectors driving ubiquitous transgene expression in ES cells and mice, the PCR amplified cDNAs were digested with EcoRI and inserted into the EcoRI sites of pCAGGS [18] to generate pCX-ECFP, pCX-EYFP and pCX-DsRed. pCAGGS contains the chicken beta-actin promoter and first intron coupled to the CMV immediate early enhancer, and rabbit beta-globin polyadenylation signal. This promoter/enhancer combination has previously been shown to drive strong and widespread transgene expression in ES cells, embryos and adult mice [18,19]. + +Tissue culture + +R1 ES cells [20] were maintained under standard culture conditions in the presence of LIF, as described previously [21]. 20 μg of SalI linearized pCX-ECFP, pCX-EYFP or pCX-DsRed1 was co-electroporated into 2 × 107 cells with 5–10 μg of circular pPGKPuro [22]. This provided transient drug resistance which could be used as a selection for obtaining individual colonies. Selection was applied 24 hours after electroporation and maintained for 5 days, thereafter cells were propagated in in the absence of selection. Fluorescent colonies were picked into 96-well plates, expanded and archived as described previously ([2]. Clones were screened for strong, stable, and homogenous transgene expression during culture in the absence of drug selection. + +Chimera production and germline transmission + +ES cell lines exhibiting robust transgene expression in vitro were assessed in vivo for the spatiotemporal extent of transgene expression and their developmental potential in chimeras produced by aggregation with wild type tetraploid ICR embryos so as to produce completely ES cell-derived embryos, as described previously [20,23]. A detailed protocol can be found on the world wide web at . Clones giving fluorescent E12.5 embryos having no readily identifiable malformations were used to generate germline transmitting chimeras by aggregation with diploid wild type ICR embryos as described previously [21,23]. After germline transmission transgenes were bred to homozygosity. Transgenes were maintained on both mixedbred ICR and inbred 129/SvJ genetic backgrounds [24]. + +Imaging + +Photographs of live samples, freshly dissected in PBS ([24] were taken on a Leica MZFLIII stereo dissecting microscope equipped with epifluorescence optics. Filter sets used were: excitation 436/20 nm, dichroic mirror 455 nm, barrier 480/40 nm for ECFP, excitation 500/20 nm, dichroic mirror 515 nm, barrier 535/30 nm for EYFP and excitation 540/25 nm, dichroic mirror 565 nm, barrier 605/55 for DsRed. Adult mice were anaesthetized with avertin. They were then placed on the stage of a stereo dissecting microscope (Leica MZFLIII) from which the objective had been removed, such that they were illuminated using the epifluorescence optics (lamp and excitation filters). Photographs were taken using a Nikon FE10 35 mm SLR camera fitted with individual emission filters. Images were processed using Photoshop software (Adobe Systems). + +Footnote + +The cyan and yellow fluorescent protein-expressing mice described here have been acquired by, and are available from, the Jackson Laboratory Induced Mutant Resource . They are referred to therein as 003373 Stock TgN(ActbECFP) 1Nagy (CK6/ECFP) and 003772 Stock TgN(ActbEYFP)1Nagy (YC5/EYFP). + +Acknowledgements + +We thank J.-I. Miyazaki for the pCAGGS plasmid. A.-K.H. is particularly indebted to V. E. Papaioannou for helpful discussion and constructive comments on the manuscript. This work was supported by grants from the National Cancer Institute of Canada. diff --git a/src/ontogpt/evaluation/craft/database/all/12546709.ann b/src/ontogpt/evaluation/craft/database/all/12546709.ann new file mode 100644 index 000000000..b65165a33 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/12546709.ann @@ -0,0 +1,521 @@ +T1 PR:000005907 38 45;57 67 AlphaA- ... crystallin +T2 PR:000005908 50 67 AlphaB-crystallin +T3 NCBITaxon:10088 84 89 mouse +T4 NCBITaxon:33208 209 215 animal +T5 SO:0000704 265 269 gene +T6 NCBITaxon:10088 341 345 mice +T7 PR:000005907 497 503 alphaA +T8 PR:000005908 508 514 alphaB +T9 GO:0010467 528 537 expressed +T10 NCBITaxon:10088 638 642 mice +T11 NCBITaxon:10088 647 651 mice +T12 PR:000005907 684 691;703 713 alphaA- ... crystallin +T13 PR:000005908 696 713 alphaB-crystallin +T14 SO:0000704 714 719 genes +T15 PR:000005907 721 727 alphaA +T16 NCBITaxon:10088 767 771 mice +T17 PR:000005907 776 782 alphaA +T18 NCBITaxon:10088 787 791 mice +T19 CHEBI:10545 827 835 electron +T20 PR:000005907 953 959 alphaA +T21 NCBITaxon:10088 964 968 mice +T22 UBERON:4200215 1044 1051 sutures +T23 UBERON:0001804 1094 1104;1109 1113 capsule of ... lens +T24 PR:000005907 1128 1134 alphaA +T25 GO:0005634 1157 1164 nucleic +T26 UBERON:0003893 1212 1220 capsular +T27 UBERON:0003893 1252 1260 capsular +T28 UBERON:0001851 1261 1267 cortex +T29 PR:000005907 1277 1283 alphaA +T30 PR:000005907 1416 1422 alphaA +T31 NCBITaxon:10088 1427 1431 mice +T32 NCBITaxon:10088 1452 1456 mice +T33 PR:000005907 1505 1512;1524 1534 alphaA- ... crystallin +T34 PR:000005908 1517 1534 alphaB-crystallin +T35 GO:0048468 1566 1580 cell formation +T36 http://purl.obolibrary.org/obo/MONDO_0005129 1635 1643 cataract +T37 PR:000005907 1719 1736 alphaA-crystallin +T38 PR:000005907 1738 1744 alphaA +T39 PR:000005908 1750 1767 alphaB-crystallin +T40 PR:000005908 1769 1775 alphaB +T41 SO:0000857 1814 1822 homology +T42 CL:0011004 1867 1883 lens fiber cells +T43 GO:0010467 2004 2014 expression +T44 UBERON:0001803 2044 2059 lens epithelium +T45 UBERON:0000483 2130 2140 epithelium +T46 PR:000005908 2142 2148 AlphaB +T47 GO:0010467 2149 2159 expression +T48 UBERON:0000483 2183 2193 epithelium +T49 UBERON:0000483 2252 2262 epithelial +T50 CL:0000066 2252 2268 epithelial cells +T51 PR:000005907 2301 2307 AlphaA +T52 UBERON:0000483 2349 2359 epithelium +T53 PR:000005907 2388 2394 alphaA +T54 PR:000005908 2399 2405 alphaB +T55 PR:000005907 2598 2604 alphaA +T56 PR:000005908 2609 2615 alphaB +T57 CHEBI:75958 2836 2844 solution +T58 GO:0030164 3085 3097;3114 3116;3122 3130 denaturation ... of ... proteins +T59 PR:000005907 3132 3138 AlphaA +T60 PR:000005908 3143 3149 alphaB +T61 SO:0000857 3183 3191 homology +T62 PR:000025349 3244 3268 small heat shock protein +T63 GO:0010467 3297 3307 expression +T64 PR:000005908 3342 3348 alphaB +T65 PR:000005908 3394 3400 AlphaB +T66 GO:0010467 3417 3426 expressed +T67 UBERON:0000479 3479 3486 tissues +T68 PR:000005907 3494 3500 alphaA +T69 UBERON:0000479 3556 3563 tissues +T70 UBERON:0000966 3572 3578 retina +T71 UBERON:0002106 3580 3586 spleen +T72 UBERON:0002370 3591 3597 thymus +T73 PR:000005907 3973 3979 alphaA +T74 PR:000005908 3984 3990 alphaB +T75 GO:0005884 4064 4078 actin filament +T76 GO:0030154 4124 4142;4159 4164 differentiation of ... cells +T77 UBERON:0001803 4143 4158 lens epithelial +T78 CL:0002224 4143 4164 lens epithelial cells +T79 GO:0045098 4317 4347 type III intermediate filament +T80 PR:000004734 4390 4394 CP49 +T81 PR:000004733 4399 4404 CP115 +T82 UBERON:0001803 4576 4591 lens epithelium +T83 GO:0070307 4645 4667 lens fiber development +T84 GO:0009294 4750 4761 transfected +T85 PR:000005908 4773 4779 alphaB +T86 GO:0051325 4827 4837 interphase +T87 GO:0005634 4838 4844 nuclei +T88 GO:0005634 4888 4895 nucleus +T89 GO:0005634 4904 4911 nuclear +T90 PR:000005908 4921 4927 alphaB +T91 UBERON:0001803 4987 5002 lens epithelial +T92 CL:0002224 4987 5008 lens epithelial cells +T93 PR:000005908 5022 5028 alphaB +T94 NCBITaxon:10088 5038 5042 mice +T95 GO:0008283 5061 5074 proliferation +T96 SO:0001026 5079 5086 genomic +T97 NCBITaxon:27592 5178 5184 bovine +T98 UBERON:0001803 5185 5200 lens epithelial +T99 CL:0002224 5185 5205 lens epithelial cell +T100 NCBITaxon:33208 5513 5519 animal +T101 SO:0000704 5569 5573 gene +T102 NCBITaxon:10088 5667 5672 mouse +T103 PR:000005907 5673 5679 alphaA +T104 SO:0000704 5680 5684 gene +T105 PR:000005908 5802 5808 alphaB +T106 NCBITaxon:10088 6145 6150 mouse +T107 PR:000005908 6151 6157 alphaB +T108 SO:0000704 6158 6162 gene +T109 http://purl.obolibrary.org/obo/MONDO_0005129 6243 6251 cataract +T110 PR:000005908 6282 6288 alphaB +T111 NCBITaxon:33208 6312 6319 animals +T112 CL:0000187 6325 6336 muscle cell +T113 UBERON:0000104 6422 6432 life spans +T114 NCBITaxon:10088 6508 6512 mice +T115 PR:000005907 6664 6670 alphaA +T116 PR:000005908 6675 6681 alphaB +T117 GO:0010467 6695 6704 expressed +T118 NCBITaxon:10088 6828 6833 mouse +T119 PR:000005907 6878 6884 alphaA +T120 PR:000005908 6889 6895 alphaB +T121 SO:0000704 6896 6901 genes +T122 CHEBI:10545 6965 6973 electron +T123 GO:0048468 7158 7172 cell formation +T124 NCBITaxon:10088 7238 7243 mouse +T125 NCBITaxon:10088 7289 7294 mouse +T126 NCBITaxon:10088 7372 7376 mice +T127 PR:000005907 7379 7385 AlphaA +T128 NCBITaxon:10088 7457 7461 mice +T129 NCBITaxon:10088 7502 7506 mice +T130 PR:000008816 7521 7526 HSPB2 +T131 SO:0000704 7527 7531 gene +T132 NCBITaxon:10088 7614 7618 mice +T133 NCBITaxon:33208 7624 7631 animals +T134 GO:0007601 7696 7702 Vision +T135 NCBITaxon:33208 7746 7753 Animals +T136 UBERON:0000970 7757 7767 Ophthalmic +T137 GO:0007601 7772 7778 Vision +T138 NCBITaxon:33208 7820 7827 animals +T139 UBERON:0000970 7829 7833 eyes +T140 UBERON:0001766 7915 7931 anterior chamber +T141 UBERON:0000970 7963 7967 eyes +T142 NCBITaxon:33208 7984 7991 animals +T143 CHEBI:50913 8014 8022 fixative +T144 CHEBI:64276 8057 8071 glutaraldehyde +T145 CHEBI:62956 8087 8104 sodium cacodylate +T146 UBERON:0000970 8122 8126 Eyes +T147 CHEBI:64276 8710 8724 glutaraldehyde +T148 CHEBI:62956 8740 8757 sodium cacodylate +T149 CHEBI:9754 8818 8822 Tris +T150 CHEBI:9754 8846 8850 Tris +T151 CHEBI:26710 8859 8863 NaCl +T152 CHEBI:18059 8887 8892 lipid +T153 GO:0016020 8893 8902 membranes +T154 CHEBI:52029 8931 8997 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate +T155 CHEBI:52029 9000 9003 DiI +T156 CHEBI:52029 9005 9011 DiIC18 +T157 CHEBI:9754 9106 9110 Tris +T158 GO:0005634 9132 9139 nucleic +T159 CHEBI:52029 9625 9628 DiI +T160 CHEBI:10545 9730 9738 electron +T161 CHEBI:64276 9847 9861 glutaraldehyde +T162 CHEBI:62956 9877 9894 sodium cacodylate +T163 CHEBI:15377 9981 9986 water +T164 CHEBI:15377 10013 10020 aqueous +T165 CHEBI:16236 10098 10105 ethanol +T166 CHEBI:16236 10158 10165 ethanol +T167 CHEBI:16526 10201 10215 carbon dioxide +T168 CHEBI:10545 10457 10465 electron +T169 PR:000005907 10779 10785 AlphaA +T170 UBERON:0000104 10944 10948 life +T171 PR:000005907 10968 10974 alphaA +T172 PR:000005907 11147 11153 alphaA +T173 PR:000005907 11246 11252 AlphaA +T174 UBERON:0003893 11403 11411 capsular +T175 PR:000005907 11466 11472 alphaA +T176 UBERON:0001851 11500 11508 cortical +T177 http://purl.obolibrary.org/obo/MONDO_0045051 11500 11508;11521 11530 cortical cataracts +T178 UBERON:0000390 11513 11520 nuclear +T179 http://purl.obolibrary.org/obo/MONDO_0045050 11513 11530 nuclear cataracts +T180 UBERON:4200215 11557 11564 sutures +T181 PR:000005907 11593 11599 alphaA +T182 UBERON:0000970 11681 11684 eye +T183 UBERON:4200215 11738 11745 sutures +T184 UBERON:0000970 11807 11811 eyes +T185 PR:000005907 11815 11821 alphaA +T186 NCBITaxon:10088 11852 11856 mice +T187 UBERON:0006983 12090 12095 point +T188 UBERON:0016459 12166 12183;12188 12192 posterior pole of ... lens +T189 PR:000005907 12247 12253 alphaA +T190 NCBITaxon:10088 12258 12262 mice +T191 UBERON:0003893 12320 12328 capsular +T192 PR:000005907 12381 12387 alphaA +T193 NCBITaxon:10088 12392 12397 mouse +T194 http://purl.obolibrary.org/obo/MONDO_0005129 12428 12436 cataract +T195 NCBITaxon:10088 12522 12526 mice +T196 PR:000005907 12607 12613 alphaA +T197 UBERON:0003893 12708 12715 capsule +T198 PR:000005907 12741 12747 alphaA +T199 CHEBI:52029 12870 12873 DiI +T200 GO:0005634 12942 12949 nucleic +T201 PR:000005907 12972 12978 alphaA +T202 PR:000005907 13040 13046 alphaA +T203 GO:0005634 13079 13085 nuclei +T204 UBERON:0000483 13110 13120 epithelium +T205 UBERON:0003893 13155 13163 capsular +T206 GO:0005634 13185 13196 Cell nuclei +T207 GO:0005615 13300 13320 extracellular spaces +T208 PR:000005907 13376 13382 alphaA +T209 PR:000005907 13413 13419 alphaA +T210 GO:0005634 13461 13468 nucleic +T211 UBERON:0003893 13494 13502 capsular +T212 UBERON:0003893 13543 13551 capsular +T213 GO:0005634 13576 13583 nucleic +T214 UBERON:0001804 13779 13789;13794 13798 capsule of ... lens +T215 UBERON:0000922 13880 13889 embryonic +T216 UBERON:0000390 13896 13903 nucleus +T217 PR:000005907 14040 14046 alphaA +T218 UBERON:0000922 14062 14071 embryonic +T219 UBERON:0000390 14078 14085 nucleus +T220 CL:0002228 14087 14106 primary lens fibers +T221 UBERON:0003893 14182 14189 capsule +T222 UBERON:0003893 14306 14314 capsular +T223 GO:0005634 14315 14322 nucleic +T224 UBERON:0000922 14345 14354 embryonic +T225 UBERON:0000390 14361 14368 nucleus +T226 UBERON:0003893 14426 14433 capsule +T227 PR:000005907 14530 14536 alphaA +T228 CHEBI:52029 14684 14687 DiI +T229 GO:0016020 14698 14706 membrane +T230 PR:000005907 14807 14813 alphaA +T231 UBERON:0000483 14965 14975 epithelial +T232 GO:0005634 14976 14982 nuclei +T233 PR:000005907 15066 15072 alphaA +T234 CHEBI:52029 15220 15223 DiI +T235 GO:0016020 15234 15242 membrane +T236 PR:000005907 15326 15332 alphaA +T237 PR:000005907 15366 15372 alphaA +T238 UBERON:0001851 15500 15506 cortex +T239 UBERON:0000483 15511 15521 epithelium +T240 UBERON:0000922 15588 15597 embryonic +T241 UBERON:0000390 15604 15611 nucleus +T242 UBERON:0001851 15648 15654 cortex +T243 UBERON:0003893 15690 15698 capsular +T244 UBERON:4200215 15792 15798 suture +T245 GO:0005634 15839 15845 nuclei +T246 CHEBI:52029 15970 15973 DiI +T247 PR:000005907 16035 16041 alphaA +T248 UBERON:0000483 16095 16105 epithelial +T249 PR:000005907 16127 16133 alphaA +T250 GO:0005634 16221 16228 nuclear +T251 PR:000005907 16295 16301 alphaA +T252 UBERON:0000483 16363 16373 epithelium +T253 UBERON:0000483 16514 16524 epithelial +T254 GO:0005634 16525 16532 nuclear +T255 GO:0005634 16543 16550 nuclear +T256 UBERON:0000483 16561 16571 epithelial +T257 UBERON:0001851 16628 16636 cortical +T258 PR:000005907 16676 16682 alphaA +T259 PR:000005907 16778 16784 alphaA +T260 UBERON:0001851 16827 16835 cortical +T261 CHEBI:18059 16857 16862 lipid +T262 GO:0016020 16863 16872 membranes +T263 PR:000005907 16979 16985 alphaA +T264 GO:0016020 17033 17042 membranes +T265 UBERON:0001851 17072 17078 cortex +T266 GO:0005634 17088 17095 nucleic +T267 PR:000005907 17137 17143 alphaA +T268 GO:0016020 17238 17246 membrane +T269 GO:0005773 17308 17316 vacuoles +T270 GO:0016020 17436 17445 membranes +T271 GO:0005634 17507 17514 nucleic +T272 PR:000005907 17857 17863 alphaA +T273 PR:000005907 17991 17997 alphaA +T274 GO:0005634 18085 18091 nuclei +T275 UBERON:0003893 18232 18239 capsule +T276 UBERON:0000483 18256 18266 epithelium +T277 PR:000005907 18360 18366 alphaA +T278 UBERON:0000922 18473 18482 embryonic +T279 UBERON:0000390 18493 18500 nucleus +T280 PR:000005907 18510 18516 alphaA +T281 PR:000005907 18865 18871 alphaA +T282 UBERON:0001804 19055 19065;19082 19088 capsule of ... lenses +T283 PR:000005907 19071 19077 alphaA +T284 GO:0005634 19111 19118 nucleic +T285 GO:0016020 19178 19186 membrane +T286 GO:0042995 19187 19198 projections +T287 PR:000005907 19244 19250 alphaA +T288 PR:000005907 19271 19277 alphaA +T289 UBERON:0003893 19312 19319 capsule +T290 UBERON:0000922 19377 19386 embryonic +T291 UBERON:0000390 19393 19400 nucleus +T292 UBERON:0000922 19484 19493 embryonic +T293 UBERON:0000390 19500 19507 nuclear +T294 UBERON:0003893 19542 19549 capsule +T295 PR:000005907 19559 19565 alphaA +T296 PR:000005907 19610 19616 alphaA +T297 UBERON:4200215 19642 19649 sutures +T298 UBERON:4200215 19731 19738 sutures +T299 UBERON:0003893 19858 19865 capsule +T300 PR:000005907 20208 20214 alphaA +T301 UBERON:0001851 20439 20447 cortical +T302 PR:000005907 20463 20469 alphaA +T303 GO:0009986 20520 20532 cell surface +T304 GO:0042995 20533 20544 projections +T305 UBERON:0001851 20921 20929 cortical +T306 UBERON:0003893 21040 21048 capsular +T307 PR:000005907 21099 21105 alphaA +T308 GO:0009986 21173 21185 cell surface +T309 GO:0042995 21186 21197 projections +T310 UBERON:0000922 21212 21221 embryonic +T311 UBERON:0000390 21222 21229 nucleus +T312 GO:0042995 21285 21296 projections +T313 GO:0016020 21310 21319 membranes +T314 UBERON:0001804 21412 21422;21434 21440 capsule of ... lenses +T315 PR:000005907 21423 21429 alphaA +T316 UBERON:0003893 21613 21621 capsular +T317 UBERON:0003893 21759 21766 capsule +T318 UBERON:0000483 21830 21840 epithelium +T319 CHEBI:10545 21877 21885 electron +T320 PR:000005907 21901 21907 alphaA +T321 PR:000005907 21984 21990 alphaA +T322 PR:000005907 22042 22048 alphaA +T323 UBERON:0001851 22240 22246 cortex +T324 UBERON:0003893 22278 22286 capsular +T325 NCBITaxon:33208 22399 22405 animal +T326 SO:0000704 22455 22459 gene +T327 NCBITaxon:10088 22553 22558 mouse +T328 PR:000005907 22559 22565 alphaA +T329 SO:0000704 22566 22570 gene +T330 PR:000005908 22688 22694 alphaB +T331 NCBITaxon:10088 22955 22960 mouse +T332 PR:000005908 22961 22967 alphaB +T333 SO:0000704 22968 22972 gene +T334 http://purl.obolibrary.org/obo/MONDO_0005129 23057 23066 cataracts +T335 PR:000005907 23102 23108 alphaA +T336 PR:000005908 23182 23188 alphaB +T337 NCBITaxon:10088 23230 23234 mice +T338 PR:000005907 23386 23392 alphaA +T339 PR:000005908 23397 23403 alphaB +T340 GO:0010467 23417 23426 expressed +T341 UBERON:4200215 23530 23537 sutures +T342 UBERON:0001804 23580 23590;23595 23599 capsule of ... lens +T343 GO:0005634 23622 23629 nucleic +T344 UBERON:0003893 23656 23664 capsular +T345 UBERON:0003893 23696 23704 capsular +T346 UBERON:0001851 23705 23711 cortex +T347 PR:000005907 23823 23829 alphaA +T348 NCBITaxon:10088 23834 23838 mice +T349 NCBITaxon:10088 23859 23863 mice +T350 PR:000005907 23938 23944 alphaA +T351 PR:000005908 23948 23954 alphaB +T352 NCBITaxon:10088 23964 23968 mice +T353 PR:000005907 24006 24012 alphaA +T354 NCBITaxon:10088 24017 24021 mice +T355 PR:000008816 24036 24041 HSPB2 +T356 SO:0000704 24042 24046 gene +T357 PR:000008816 24219 24224 HSPB2 +T358 GO:0030154 24348 24363;24375 24377;24389 24394 differentiation ... of ... cells +T359 CL:0011004 24378 24394 lens fiber cells +T360 GO:0030154 24403 24421;24433 24438 differentiation of ... cells +T361 CL:0011004 24422 24438 lens fiber cells +T362 UBERON:0010077 24479 24498 cuboidal epithelial +T363 CL:0000066 24488 24503 epithelial cell +T364 GO:0005634 24518 24525 nucleus +T365 GO:0043226 24551 24561 organelles +T366 UBERON:0000119 24579 24587;24609 24614 layer of ... cells +T367 GO:0005634 24626 24632 nuclei +T368 GO:0043226 24637 24647 organelles +T369 GO:0030154 24649 24667;24679 24684 Differentiation of ... cells +T370 UBERON:0000483 24668 24678 epithelial +T371 CL:0000066 24668 24684 epithelial cells +T372 UBERON:0005614 24728 24732 lens +T373 UBERON:0000483 24740 24750 epithelial +T374 CL:0000066 24740 24756 epithelial cells +T375 UBERON:0000483 24901 24911 epithelium +T376 UBERON:0003893 24929 24936 capsule +T377 PR:000005907 25011 25017 alphaA +T378 PR:000005908 25022 25028 alphaB +T379 PR:000005907 25132 25138 alphaA +T380 PR:000005908 25143 25149 alphaB +T381 NCBITaxon:4679 25245 25250 onion +T382 UBERON:0000483 25310 25320 epithelium +T383 UBERON:0003893 25338 25345 capsule +T384 PR:000005907 25382 25388 alphaA +T385 GO:0005634 25428 25439 cell nuclei +T386 UBERON:0001851 25450 25458 cortical +T387 GO:0005634 25480 25491 cell nuclei +T388 UBERON:0001851 25546 25552 cortex +T389 GO:0005634 25570 25581 cell nuclei +T390 UBERON:0003893 25622 25629 capsule +T391 UBERON:0001803 25734 25749 lens epithelial +T392 CL:0002224 25734 25755 lens epithelial cells +T393 PR:000005907 25816 25822 alphaA +T394 NCBITaxon:10088 25827 25832 mouse +T395 PR:000005907 25982 25988 alphaA +T396 PR:000005908 25993 25999 alphaB +T397 UBERON:0000970 26042 26045 eye +T398 GO:0007567 26085 26090 birth +T399 NCBITaxon:10088 26105 26109 mice +T400 UBERON:0000970 26120 26124 eyes +T401 NCBITaxon:33208 26230 26237 animals +T402 NCBITaxon:33208 26263 26269 animal +T403 GO:0007601 26517 26523 visual +T404 UBERON:0000483 26567 26577 epithelium +T405 UBERON:0003893 26585 26593 capsular +T406 UBERON:0001851 26594 26600 cortex +T407 UBERON:0003893 26742 26750 capsular +T408 UBERON:0003893 26796 26804 capsular +T409 GO:0005634 26805 26812 nucleic +T410 UBERON:4200215 26849 26856 sutures +T411 UBERON:0000178 27002 27007 blood +T412 CHEBI:15377 27008 27015 aqueous +T413 GO:0005634 27118 27125 nucleic +T414 UBERON:0003893 27160 27168 capsular +T415 PR:000005907 27189 27195 alphaA +T416 GO:0005634 27208 27215 nucleic +T417 UBERON:0003893 27250 27258 capsular +T418 UBERON:0000483 27301 27311 epithelial +T419 CL:0000066 27301 27317 epithelial cells +T420 UBERON:0016459 27346 27360 posterior pole +T421 CL:0002228 27365 27384 primary fiber cells +T422 GO:0005634 27651 27658 nucleic +T423 UBERON:0003893 27693 27701 capsular +T424 PR:000005907 27760 27766 alphaA +T425 GO:0005634 27839 27846 nucleic +T426 UBERON:0003893 27886 27894 capsular +T427 GO:0009653 28048 28059;28076 28100 development ... of morphological changes +T428 PR:000005907 28173 28179 alphaA +T429 GO:0030154 28320 28335;28347 28349;28361 28366 differentiation ... of ... cells +T430 CL:0011004 28350 28366 lens fiber cells +T431 GO:0008150 28473 28489 biological event +T432 UBERON:0001803 28495 28510 lens epithelial +T433 CL:0002224 28495 28515 lens epithelial cell +T434 UBERON:0000483 28553 28563 epithelial +T435 CL:0000066 28553 28568 epithelial cell +T436 UBERON:0001851 28706 28712 cortex +T437 GO:0043226 28749 28759 organelles +T438 UBERON:4200215 28765 28771 suture +T439 UBERON:0000483 28842 28852 epithelial +T440 CL:0000066 28842 28857 epithelial cell +T441 GO:0010467 28948 28958 expression +T442 UBERON:0001803 29311 29326 lens epithelial +T443 CL:0002224 29311 29331 lens epithelial cell +T444 GO:0030154 29327 29347 cell differentiation +T445 PR:000005907 29412 29418 AlphaA +T446 PR:000005908 29423 29429 alphaB +T447 PR:000025349 29449 29453 shsp +T448 PR:000005907 29789 29795 alphaA +T449 GO:0010467 29909 29918 expressed +T450 GO:0010467 30008 30017 expressed +T451 GO:0030154 30029 30047;30096 30101 differentiation of ... cells +T452 NCBITaxon:40674 30048 30057 mammalian +T453 CL:0000062 30058 30069 osteoblasts +T454 CL:0000836 30074 30086 promelocytic +T455 http://purl.obolibrary.org/obo/MONDO_0005059 30087 30095 leukemia +T456 GO:0010467 30125 30135 expression +T457 NCBITaxon:9606 30181 30186 human +T458 CL:0000236 30187 30200 B lymphocytes +T459 CL:0000235 30210 30220 macrophage +T460 GO:0030154 30221 30239;30246 30251 differentiation of ... cells +T461 PR:000005908 30300 30306 alphaB +T462 PR:000010877 30363 30371 myogenin +T463 NCBITaxon:27592 30472 30478 bovine +T464 UBERON:0001803 30479 30494 lens epithelium +T465 GO:0005634 30788 30800 cell nucleus +T466 GO:0065007 30824 30834 regulating +T467 GO:0007049 30839 30849 cell cycle +T468 GO:0005634 30898 30909 cell nuclei +T469 PR:000005908 31013 31019 AlphaB +T470 GO:0010467 31021 31030 expressed +T471 GO:0009294 31034 31045 transfected +T472 GO:0051325 31099 31109 interphase +T473 GO:0005634 31110 31116 nuclei +T474 GO:0065007 31131 31141 regulatory +T475 GO:0005634 31171 31178 nucleus +T476 UBERON:0001803 31210 31225 lens epithelial +T477 CL:0002224 31210 31231 lens epithelial cells +T478 NCBITaxon:10088 31246 31250 mice +T479 GO:0008283 31275 31286 proliferate +T480 PR:000005908 31308 31314 alphaB +T481 SO:0001026 31347 31354 genomic +T482 UBERON:0001803 31369 31384 lens epithelium +T483 PR:000005907 31513 31519 alphaA +T484 GO:0065007 31523 31533 regulating +T485 GO:0007049 31538 31548 cell cycle +T486 GO:0005634 31647 31654 nucleus +T487 GO:0051726 31662 31683 cell cycle regulation +T488 GO:0005634 31775 31781 nuclei +T489 PR:000005907 31824 31830 alphaA +T490 GO:0005634 31847 31854 nucleic +T491 UBERON:0001851 31907 31913 cortex +T492 PR:000005907 31923 31929 alphaA +T493 GO:0005634 31996 32003 nucleus +T494 GO:0005856 32112 32124 cytoskeletal +T495 PR:000005907 32144 32150 alphaA +T496 PR:000005908 32155 32161 alphaB +T497 GO:0005884 32231 32245 Actin filament +T498 GO:0030154 32295 32313;32330 32335 differentiation of ... cells +T499 UBERON:0001803 32314 32329 lens epithelial +T500 CL:0002224 32314 32335 lens epithelial cells +T501 GO:0032991 32487 32494 complex +T502 GO:0045098 32500 32530 type III intermediate filament +T503 PR:000004734 32587 32591 CP49 +T504 PR:000004733 32596 32601 CP115 +T505 UBERON:0001803 32724 32739 lens epithelial +T506 CL:0002224 32724 32745 lens epithelial cells +T507 CL:0011004 32800 32816 lens fiber cells +T508 GO:0044249 32863 32872;32893 32895;32907 32912 synthesis ... in ... cells +T509 UBERON:0000483 32896 32906 epithelial +T510 CL:0000066 32896 32912 epithelial cells +T511 GO:0005856 32985 32997 cytoskeleton +T512 GO:0016477 33035 33039;33050 33059 cell ... migration +T513 PR:000005907 33153 33159 alphaA +T514 GO:0005856 33244 33256 cytoskeletal +T515 PR:000005907 33277 33283 alphaA +T516 CHEBI:10545 33455 33463 electron +T517 PR:000005907 33564 33569 Alpha +T518 NCBITaxon:10088 33574 33578 mice +T519 PR:000005907 33618 33624 AlphaA +T520 NCBITaxon:10088 33629 33633 mice +T521 GO:0007601 33908 33914 Vision diff --git a/src/ontogpt/evaluation/craft/database/all/12546709.txt b/src/ontogpt/evaluation/craft/database/all/12546709.txt new file mode 100644 index 000000000..91d1f3e8e --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/12546709.txt @@ -0,0 +1,119 @@ +Morphological characterization of the AlphaA- and AlphaB-crystallin double knockout mouse lens + +Abstract + +Background + +One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. The purpose of this study was to characterize gross lenticular morphology in normal mice and mice with the targeted disruption of alphaA- and alphaB-crystallin genes (alphaA/BKO). + +Methods + +Lenses from 129SvEvTac mice and alphaA/BKO mice were examined by standard scanning electron microscopy and confocal microscopy methodologies. + +Results + +Equatorial and axial (sagittal) dimensions of lenses for alphaA/BKO mice were significantly smaller than age-matched wild type lenses. No posterior sutures or fiber cells extending to the posterior capsule of the lens were found in alphaA/BKO lenses. Ectopical nucleic acid staining was observed in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk alphaA/BKO lenses. Gross morphological differences were also observed in the equatorial/bow, posterior and anterior regions of lenses from alphaA/BKO mice as compared to wild mice. + +Conclusion + +These results indicated that both alphaA- and alphaB-crystallin are necessary for proper fiber cell formation, and that the absence of alpha-crystallin can lead to cataract formation. + +Background + +Alpha-Crystallin is comprised of two polypeptides, alphaA-crystallin (alphaA) and alphaB-crystallin (alphaB), which share 55% amino acid sequence homology [1]. They are the most abundant proteins in lens fiber cells [2,3] and exist as heteroaggregates of approximately 800 kDa that can undergo inter-aggregate subunit exchange [4]. The expression of these two proteins in the lens epithelium, however, is not uniform throughout different regions of the anterior epithelium. AlphaB expression is detected in central epithelium and increases from the central to elongation zones, where epithelial cells differentiate into fiber cells. AlphaA, however, is not detected in the central epithelium. The relative proportion of alphaA and alphaB changes from a molar ratio of 1:3 in the pericentral and germative zones to a molar ratio of 3:1 in the elongation zone and fiber cells [2,3]. These differences in the relative proportions of alphaA and alphaB within the lens suggest different functions for the two subunits in the developing lens. + +Prior to the 1990's, alpha-crystallin was thought to be a structural protein whose major cellular function was to produce a dense solution necessary for the refraction of light in the lens. During the early 1990's, alpha-crystallin was shown to act as a molecular chaperone, binding to partially denatured proteins, both in vitro [5] and probably in vivo [6], to inhibit further denaturation and aggregation of lens proteins. AlphaA and alphaB were also shown to have sequence homology with several other proteins that are members of the small heat shock protein (hsp) family [7]. Moreover, expression of alpha-crystallin, particularly alphaB, was shown to not be restricted to the lens. AlphaB was found to be expressed at significant levels in a variety of nonlenticular tissues, while alphaA has only been detected in small amounts in a few other tissues such as retina, spleen and thymus [8-12]. Collectively, these findings have challenged the dogma that alpha-crystallin is purely a structural protein necessary for light refraction, and have led to the realization that alpha-crystallin may have a variety of biological functions in the lens. + +A much broader scope of cellular functions of alpha-crystallin in lens is inferred from in vitro observations. Both alphaA and alphaB can bind specifically to actin, in vitro [13] and in vivo [14]. Although actin filament formation has been shown to be necessary for differentiation of lens epithelial cells [15], the significance of alpha-crystallin's interaction with actin in differentiation is not known. In the lens, alpha-crystallin also associates with type III intermediate filament proteins and the beaded filament proteins CP49 and CP115, and correct beaded filament assembly has been shown depend on the presence of alpha-crystallin [16]. Beaded filament mRNA levels are greatly increased in differentiating lens epithelium and have been suggested as a pan-specific marker for lens fiber development [17]. Alpha-crystallin has also been shown to interact directly with DNA [18]. In transfected CHO cells, alphaB has also been shown to ectopically localize to interphase nuclei, suggesting a role for this protein in the nucleus [19]. A nuclear role for alphaB in the lens was supported by the findings that a subset of lens epithelial cells derived from alphaB knockout mice demonstrated hyperproliferation and genomic instability [20]. In addition, the administration of exogenous alpha-crystallin to primary bovine lens epithelial cell cultures resulted in the formation of lentoid bodies, consistent with a role for these proteins in lens differentiation [21]. These findings indicate that alpha-crystallin may have a multitude of in vivo functions. + +One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. Brady et al. [22] demonstrated, by targeted disruption of the mouse alphaA gene, that this protein was essential for the maintenance of lens transparency, possibly by maintaining the solubility of alphaB, or associated proteins, in the lens. These lenses were also reported to be smaller in equatorial and light axial dimensions than age matched wild type lens. It was not possible using the techniques employed in the study to determine if the smaller lenses were due to reduced volume or number of fiber cells. Targeted disruption of the mouse alphaB gene resulted in lenses similar in size to aged-matched wild type lens. Moreover, no cataract formation was observed in the alphaB knockout lenses. These animals have muscle cell abnormalities, severe postural anomalies, selective muscle degeneration, and shorter life spans compared to normal controls [23]. + +In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. The objectives of the current report were to characterize gross morphology of young (5 wk) and old (54 wk) mouse lenses with targeted disruption of both the alphaA and alphaB genes, in comparison to age matched wild type lenses, using scanning electron microscopy (SEM) and confocal microscopy, to elucidate the possible functions of alpha-crystallin in the lens. The results indicate that alpha-crystallin is necessary for proper fiber cell formation and resulting lens transparency. + +Methods + +Lenses + +The wild type mouse strain used in this study was the 129SvEvTac mouse. Lenses examined were from 5(8 lenses), 46(8 lenses) and 72(6 lenses) wk old mice. + +AlphaA/BKO was generated by cross breeding alphaAKO-127 [22] and alphaBKO-168 mice [23], also in a 129Sv background. These mice also lack the HSPB2 gene product [23]. Lenses examined were from 5 wk (6 lenses) and 54 wk (16 lenses) old mice. All animals were treated in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. + +Immediately after euthanizing animals, eyes were enucleated. A scalpel blade was then used to make a small incision into the anterior chamber near the equator and then both eyes from individual animals were immersed in 5 ml fixative (2% (w/v) paraformaldehyde and 2% glutaraldehyde (v/v) in 0.1 M sodium cacodylate buffer, pH 7.2). Eyes were fixed for at least 24 hr at room temperature (RT) prior to dissection of the lenses. At this time, equatorial and axial dimensions of lenses and gross lenticular appearance were recorded. Statistical analyses on data consisted of Student's t-test, means and standard deviation, using the statistical software package StatMost (Dataxiom Software Inc., Los Angeles, CA). + +Confocal microscopy + +Lenses were vibratome sectioned into 100–200 μm thick sections along the optical (anterior to posterior) or equatorial axes. Thick sections were fixed in 2% (w/v) paraformaldehyde and 2% glutaraldehyde (v/v) in 0.1 M sodium cacodylate buffer pH 7.2 at RT for 24 hrs then washed several times in Tris buffered saline (0.5 M Tris, 150 mM NaCl, pH 7.4). To visualize lipid membranes, sections were stained with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate ('DiI';DiIC18; Molecular Probes) as previously described [24]. Sections were then washed several times with Tris buffered saline, and nucleic acids were stained by incubating sections in TBS containing 1 μM SYTOX Green (Molecular Probes) for 10 min at RT followed by several washes with TBS prior to confocal imaging. + +Lens sections were viewed on a Zeiss laser scanning confocal microscope model LSM 410 equipped with an Axiovert 100 inverted microscope, an Argon-Krypton 488/568/647 laser, a KP line selection filter, a FT 488/568 Dichroic beam splitter, a FT 560 Dichroic beam splitter, a LP 590 emission filter for viewing DiI, a BP 515–540 emission filter for viewing SYTOX green, and the software package LSM 3.993. + +Scanning electron microscopy + +Lenses bisected along the optical axes were immersion fixed in 2% (w/v) paraformaldehyde and 2% glutaraldehyde (v/v) in 0.1 M sodium cacodylate buffer pH 7.2 at RT for 24 hrs. Samples were then washed several times with distilled water and postfixed in 1% (v/v) aqueous osmium tetroxide at RT for 1 hr. Lens halves were dehydrated in an ascending ethanol series from 50–100 %. Once lens halves were in 100% ethanol, they were critical point dried in carbon dioxide in a Samdri-790B (Tousimis Research Corp., Rockville, MD). Critical point dried lens halves were secured on aluminum stubs with double sided tape. Mounted specimens were then sputter coated with gold and viewed on a Hitachi S-3500N scanning electron microscope (Tokyo, Japan) at 1–5 kV. This microscope is equipped with Hitachi's patented "Dual-Bias" which allows extremely high emission currents at acceleration voltages of 5 kV and lower. + +Results + +Equatorial and axial (sagittal) dimensions of lenses, and gross lenticular appearance, were recorded (Table 1). AlphaA/BKO lenses at 5 wks of age were significantly smaller (p < 0.05), both equatorially and axially, than 5 wk old wild type lenses and remain smaller throughout life. Fifty-four wk old alphaA/BKO lenses were much smaller than similarly aged normal lenses, and were similar in size to 5 wk old wild type lenses. Both the 5 wk old wild type lenses and the 54 wk old alphaA/BKO lenses were significantly smaller (p < 0.01) than the 46 or 72 wk old wild type lenses. AlphaA/BKO lenses at 5 wks of age exhibited vacuoles at the equatorial region (Figure 1A see arrows) and an area of slight light scatter at the posterior subcapsular regions (Figure 1B see arrowheads). By 54 wks of age, alphaA/BKO lenses exhibited dense cortical and nuclear cataracts (Figure 1C). No posterior sutures were observed in any of the alphaA/BKO lenses examined. All wild type lenses were transparent upon removal from the eye (Figure 1D,1E and 1F) and had well defined posterior sutures. + +Figure 1 + +Light micrographs of lenses dissected from fixed eyes of alphaA/BKO (A-C) and wild type (D-F) mice. All micrographs were generated using a 4 × /0.10 objective on a Zeiss LSM 410 using transmitted light mode. Images in (A&B) were enlarged compared to images (C-F) (see scale bars). For all micrographs except (B) the objective focal point was set at the equatorial plane of the lens, while (B) was set at the posterior pole of the lens. (A&B) Representative micrographs of lenses from 5 wk alphaA/BKO mice, showing changes in the equatorial (A) and posterior sub capsular (B) regions. (C) Representative micrograph of 54 wk alphaA/BKO mouse lens showing dense whole lens cataract. (D-F) Representative clear lenses from 5 wk (D), 46 wk (E), and wk 72 (F) wild type mice. Arrows (A) indicate vacuoles in the equatorial region of a representative 5 wk alphaA/BKO lens. The arrowheads (B) indicate an area of minor light scattering deep to the posterior capsule in a representative 5 wk alphaA/BKO lens. + +Table 1 + +Dimensions and gross lenticular appearance of lenses. + +Examination of mid-axial sections stained with DiI and SYTOX green revealed unique differences in gross morphology and nucleic acid staining between alphaA/BKO lenses and wild type lenses (Figure 2). In five week old alphaA/BKO lenses, Sytox green stained nuclei (green) in the anterior epithelium, equatorial/bow, and posterior subcapsular regions (Figure 2A). Cell nuclei localized to the equatorial/bow region were disorganized as compared to wild type. There were enlarged extracellular spaces between cells in the equatorial/bow region in 5 wk old alphaA/BKO lenses. Fifty-four wk old alphaA/BKO lenses (Figure 2B) did not stain for nucleic acid in the posterior subcapsular region, however, the entire anterior subcapsular lens region stained for nucleic acid (refer to Figure 3I for high magnification/resolution image of this area). A defined equatorial/bow region was not observed in these older lenses, and fiber cells extending to the posterior capsule of the lens were not found. Disorganized cellular material capped the anterior region of the embryonic/fetal nucleus, and a distinctive indentation of the lens mass at the equatorial region was apparent in mid-axial sections as well as whole lenses. In alphaA/BKO lenses the embryonic/fetal nucleus (primary lens fibers) had migrated posteriorly and appeared to be in contact with the posterior capsule. In 5 wk, 46 wk or 72 wk old wild type lenses (Figure 2C,2D and 2E), however, there was no posterior or anterior subcapsular nucleic acid staining and the embryonic-fetal nucleus was centrally located, not in contact with the posterior capsule. + +Figure 2 + +Representative confocal optical sections taken from mid-axial vibratome sections of alphaA/BKO (A&B) and wild type (C-E) lenses. All optical sections were generated using a 4 × /0.10 objective on a Zeiss LSM 410. The red color represents DiI staining (membrane), while the green color represents SYTOX green staining (DNA). (A&B) were taken from 5 wk and 54 wk alphaA/BKO lenses, respectively. (C-E) were taken from 5 wk, 46 wk, and 72 wk wild type lenses, respectively. The arrowheads indicate representative anterior epithelial nuclei stained with SYTOX green. + +Figure 3 + +Representative confocal optical sections from alphaA/BKO (A-J) and wild type (K-T) lenses. All optical sections were generated using a 63 × /1.4 objective on a Zeiss LSM 410. The red color represents DiI staining (membrane), while the green color represents SYTOX green staining (DNA). (A-E) are from 5 wk alphaA/BKO lenses. (F-J) are from 54 wk alphaA/BKO lenses. (K-O) are from 5 wk wild type lenses. (P-T) are from 46 wk wild type lenses. (A, F, K, and P) are anterior central cortex and epithelium. (B, G, L, and Q) are equatorial bow region. (C, H, M, and R) are embryonic/fetal nucleus. (D, I, N, and S) are deep anterior cortex. (E, J, O, and T) are posterior subcapsular region. (*) indicates areas devoid of cellular material (B and G). Arrow indicates posterior suture (O). Arrowheads indicate representative nuclei stained with SYTOX green (A, B, E, F, G, I, K, L, P, and Q). + +At higher magnification, examination of sections stained with DiI and SYTOX green revealed ultrastructural differences between alphaA/BKO lenses and wild type lenses (Figure 3). Anterior epithelial staining in 5 wk old alphaA/BKO (Figure 3A) and 5 wk old wild type lenses were similar (Figure 3K) with respect to nuclear staining with SYTOX green. However, some differences in 54 wk old alphaA/BKO lenses (Figure 3F) were observed in the central anterior epithelium compared to 5 wk (Figure 3K), 46 wk (Figure 3P), and 72 wk (data not shown) wild type lenses. These differences included changes in central epithelial nuclear staining, nuclear shape and epithelial thickness and continuity. Superficial and deep anterior cortical staining was grossly different between alphaA/BKO (Figure 3A,3D,3F and 3I) and wild type lenses (Figure 3K,3N,3P and 3S). In 5 and 54 wk old alphaA/BKO lenses, superficial and deep anterior cortical regions, stained for lipid membranes, did not reveal any patterns typical of fiber cells cut in cross-section or longitudinal section. In 5 wk alphaA/BKO lenses there was a high density of stained membranes per unit area throughout the cortex, with no nucleic acid staining in these regions. In 54 wk alphaA/BKO lenses, large, irregularly shaped cells were observed, interspersed among regions of high membrane staining density per unit area. These large objects were not vacuoles, because examination of the interior of these structures by transmitted and reflected light microscopy showed that the membranes encompassed cellular material (data not shown). In addition, nucleic acid staining was observed (Figure 3F and 3I) within many of these cells exhibiting large cross-sectional profiles. In the wild type lenses, typical cross-sectional patterns of fiber cells organized in radial columns were observed (Figure 3K,3N,3P and 3S). + +Ultrastructural differences at the equatorial/bow region were also observed between alphaA/BKO lenses (Figure 3B and 3G) and wild type lenses (Figure 3L and 3Q). In contrast to wild type lenses (Figure 3L and 3Q), the alphaA/BKO lenses (Figure 3B and 3G) contained large areas devoid of cellular material, their nuclei were not limited to a well defined equatorial/bow region, and ordered radial columns of elongating fiber cells extending from the posterior capsule to the anterior epithelium were not observed. In addition to the equatorial region, ultrastructural differences between alphaA/BKO lenses (Figure 3C and 3H) and wild type lenses (Figure 3M and 3R) were observed in fiber cells of the embryonic and fetal nucleus. In 5 wk alphaA/BKO lenses (Figure 3C), a greater variation in cross sectional diameter was observed, with cell diameters ranging from less than 2 microns up to approximately 25 microns, compared to the 5 wk and 46 wk wild type lenses, whose diameters ranged from less than 2 microns up to approximately 10 microns (Figure 3M and 3R). At 54 wk, these cells in the alphaA/BKO lenses were larger in cross sectional diameter than wild type, and exhibited a much greater variation in the cross sectional size and cell shape. + +Cells adjacent to the posterior capsule of 5 wk alphaA/BKO lenses (Figure 3E) exhibited nucleic acid staining, were small and non-elongated, with numerous membrane projections., These were, however, not observed in 54 wk alphaA/BKO lenses. In 5 wk alphaA/BKO lenses, deep to the posterior capsule, larger diameter cells, similar to those observed in the embryonic/fetal nucleus were observed (data not shown). Fiber cells of similar dimension and appearance to embryonic/fetal nuclear fibers were seen at the posterior capsule in 54 wk alphaA/BKO lenses (Figure 3J). In both 5 and 54 wk alphaA/BKO lenses, no posterior sutures could be found in any axial or equatorial vibratome sections examined. Posterior sutures were readily found in vibratome sections of wild type lenses (Figure 3O, arrow). Fiber cells attached to the posterior capsule and extending anteriorly were observed in 5 wk (Figure 3O), 46 wk (Figure 3T) and 72 wk (data not shown) wild type lenses. + +SEM confirmed many of the observations made by confocal microscopy. In the equatorial/bow region, a disorganized array of relatively small irregular shaped cells were observed in 5 wk (Figure 4A) and 54 wk (Figure 4D) alphaA/BKO lenses, while elongated fiber cells organized into radial columns, with ball and socket interdigitations, were observed in 5 wk (Figure 4G), 46 wk (Figure 4J) and 72 wk (data not shown) wild type lenses. In the anterior cortical region of 5 wk alphaA/BKO lenses, radial columns of cells with numerous cell surface projections were observed (Figure 4B), but these radial columns were no longer present at 54 weeks of age (Figure 4E). In these older lenses, irregularly shaped, convoluted cells, with no recognizable pattern of organization, were present. In contrast, radial columns of elongated fiber cells of uniform size and shape, with ball and socket interdigitations, were present in the anterior cortical region of 5 wk (Figure 4H), 46 wk (Figure 4K) and 72 week (data not shown) wild type lenses. The posterior subcapsular regions of 5 wk (Figure 4C) and 54 wk (Figure 4F) alphaA/BKO lenses were filled with irregularly shaped cells with numerous cell surface projections. In the fetal/embryonic nucleus, elongated fiber cells with numerous long, finger-like projections and furrowed membranes were evident (not shown). No elongated cells extending from the bow region to the posterior capsule of alphaA/BKO lenses were observed. In contrast, radial columns of elongated fiber cells of uniform size and shape containing ball and socket interdigitations were observed in the posterior subcapsular region in 5 wk (Figure 4I), 46 wk (Figure 4L) and 72 wk (data not shown) wild type lenses, and fiber cells in contact with the posterior capsule could be traced back to the equatorial bow region and anterior epithelium. + +Figure 4 + +Representative scanning electron micrographs of alphaA/BKO (A-F) and wild type (G-L) lenses. (A-C) Representative images from 5 wk alphaA/BKO lenses. (D-F) Representative images from 54 wk alphaA/BKO lenses. (G-I) Representative images from 5 wk wild type lenses. (J-L) Representative images from 46 wk wild type lenses. (A, D, G, and J) Equatorial/bow region. (B, E, H, and K) Anterior cortex. C, (F, I, and L) Posterior subcapsular region. + +Discussion + +One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. Brady et al. [22] demonstrated, by targeted disruption of the mouse alphaA gene, that this protein was essential for the maintenance of lens transparency, possibly by maintaining the solubility of alphaB, or associated proteins, in the lens. These lenses were also reported to be smaller in equatorial and axial dimensions than age matched wild type lens, which was very similar to that which was observed with the double knockout lens. Targeted disruption of the mouse alphaB gene, however, resulted in lenses similar in size to aged-matched wild type lens with no cataracts reported [23]. This indicates that alphaA may play a greater role in maintaining the transparency of the lens then alphaB. In the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. This was supported by the morphological observation made in this study of no posterior sutures or fiber cells extending to the posterior capsule of the lens, ectopically staining nucleic acids in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk, gross morphological differences in the equatorial/bow, posterior and anterior regions of lenses from alphaA/BKO mice as compared to wild mice. None of these morphological differences have been reported in the single alphaA or alphaB knockout mice. It must be noted, however, that the alphaA/BKO mice also lack the HSPB2 gene product [23] and the contribution of this protein to normal lens morphology and functions should not be overlooked. Future studies should address the possible functions of HSPB2 in normal lens. + +The results of the current study support the hypothesis that alpha-crystallin plays an active role in the differentiation and growth of lens fiber cells. Normal differentiation of lens fiber cells consists of a progression from a simple cuboidal epithelial cell, containing a nucleus and a minimal numbers of organelles, to a stratified layer of elongated fiber-like cells, devoid of nuclei and organelles. Differentiation of epithelial cells occurs in the equatorial/bow region of the lens, where epithelial cells begin to elongate and differentiate into fiber cells of uniform cellular shape, arranged in radial columns of cells extending from the anterior epithelium to the posterior capsule. This process did not appear to have proceeded normally in lenses lacking alphaA and alphaB. The morphological observations presented in this study demonstrate that fiber cells in lenses lacking alphaA and alphaB fail to elongate symmetrically from the bow region and therefore do not establish the typical "onion skin" conformation in which cells extend from the anterior epithelium to the posterior capsule. Additionally, in lenses from 54 wk alphaA/BKO lenses, there was a persistence of cell nuclei in deeper cortical regions, and ectopic cell nuclei were present in large numbers in the anterior central cortex. At 5 wks of age cell nuclei were present, adjacent to the posterior capsule. These morphological observations are consistent with a defect in the normal differentiation pathway of lens epithelial cells into fiber cells. + +It is unlikely that these alterations in alphaA/BKO mouse lenses result from increased susceptibility of these lenses to light-induced damage in the absence of the molecular chaperone protection afforded by alphaA and alphaB in normal lenses. With the normal time of eye opening at approximately 14 days after birth, the 5 wk old mice had their eyes open and lenses exposed to light for only about 3 weeks prior to morphological analysis. Moreover, these animals had been exposed to only animal facility fluorescent lighting and were protected from UV light by plastic cages. If lack of protection from light-induced damage was the major factor affecting the changes in these lenses, then the bulk of the damage should have resided along the visual axis, particularly in the central anterior epithelium and subcapsular cortex in the 5 wk lenses, but this was not the case. In these lenses, gross morphological changes were apparent in the equatorial and posterior subcapsular regions. These changes included posterior subcapsular nucleic acid staining, absence of posterior sutures, and small irregularly shaped cells, not arranged in any discernable pattern, in the equatorial/bow region. Systemic stress factors crossing the blood/aqueous barrier might explain some morphological changes at the equatorial region, but this would not explain nucleic acid staining in the posterior subcapsular region. In the 5 wk alphaA/BKO lenses, nucleic acid staining in the posterior subcapsular region is consistent with either anterior epithelial cells migrating aberrantly to the posterior pole, or primary fiber cells failing to fully differentiate by 5 wks of age. These two possibilities could not be differentiated in the methods employed, and were beyond the objectives of the current study. Future studies are being designed to address which of these two processes might explain nucleic acid staining in the posterior subcapsular region. Staining in this region was not observed in older alphaA/BKO lenses, suggesting that this pattern was transient. The fate of the nucleic acid-containing cells in the posterior capsular region of younger lenses is not known at this time, nor is the morphology of earlier stage lenses. Future studies with defined objectives to address the development and progression of morphological changes seen in this study are being designed. The morphological differences in alphaA/BKO lenses, compared to age matched wild type lenses, were consistent with the hypothesis that alpha-crystallin plays an active role in the differentiation and growth of lens fiber cells. In addition, it was clearly evident that alpha-crystallin is necessary for lens transparency. + +The final biological event in a lens epithelial cell's life is the transformation from an epithelial cell into a fiber cell, which occurs at the equatorial/bow region of the lens. The newly forming fiber cell continues to differentiate in the cortex until a mature fiber cell devoid of organelles with suture formation at the ends of the cell is formed. This entire process from epithelial cell to mature fiber cell is defined as lens differentiation. The precise spatial and temporal expression of the crystallin proteins in the developing lens may not be simply a consequence of the differentiation process, but instead may play an important, if not essential, role in the differentiation process itself. The results of the current study support this hypothesis. + +The exact in vivo molecular mechanisms, by which alpha-crystallin might influence lens epithelial cell differentiation, and maintenance of lens transparency, remain to be determined. AlphaA and alphaB are members of the shsp family [7]. Previous studies have shown that the alpha-crystallin possesses molecular chaperone activity, binding to partially denatured proteins, both in vitro [5] and probably in vivo [6], to inhibit further denaturation. Although this property may be a major contributor to the maintenance of lens clarity, the early changes in the alphaA/BKO lenses indicate a much broader cellular function for alpha-crystallin. Stress proteins have been shown to be expressed in non-stressed cells during development and differentiation [25]. Hsps were shown to be expressed during the differentiation of mammalian osteoblasts and promelocytic leukemia cells [26]. In addition, hsp expression has been shown to accompany growth arrest in human B lymphocytes [27] and macrophage differentiation of HL 60 cells [28]. During myogenic differentiation, mRNA for alphaB increases in conjunction with the induction of mRNA for myogenin, the earliest known event in myogenesis [29]. The addition of exogenous alpha-crystallin to primary bovine lens epithelium was shown to induce rapid changes in cell shape, leading to the formation of lentoid bodies [21]. These studies strongly suggest that the hsp family of proteins has other functions in addition to protecting proteins and cells during stress. + +Alpha-crystallin may play a functional role in the cell nucleus and may have a role in regulating the cell cycle. Several heat shock proteins have been found in cell nuclei in the absence of stress [30], and alpha-crystallin has been shown to interact directly with DNA [18]. AlphaB, expressed in transfected CHO cells, has been shown to ectopically localize to interphase nuclei, suggesting a regulatory role for this protein in the nucleus [19]. A subset of immortalized lens epithelial cells from alphaBKO mice have been shown to hyperproliferate [20] suggesting that alphaB may be important in maintaining genomic stability. In lens epithelium derived from alphaAKO lenses, cell growth rates were reported to be 50% lower compared to wild type [31], suggesting a role for alphaA in regulating the cell cycle. All of these findings raise many questions as to the possible role(s) of alpha-crystallin in the nucleus and in cell cycle regulation during differentiation. In the current study, the observation of a disorganized pattern of nuclei localized to the equatorial bow region of alphaA/BKO lenses, and nucleic acid staining of structures throughout the anterior cortex of 54 wk alphaA/BKO lenses, is consistent with a role for alpha-crystallin in the nucleus. + +There is extensive evidence from previous studies demonstrating that alpha-crystallin plays a role in the cytoskeletal organization. Both alphaA and alphaB can bind specifically to actin, both in vitro [13] and in vivo [14]. Actin filament formation has been shown to be necessary for the differentiation of lens epithelial cells [15], however, the significance of alpha-crystallin interaction with actin in differentiation is not known. In the lens, alpha-crystallin also forms a complex with type III intermediate filament proteins and the lens-specific beaded filament proteins CP49 and CP115, which may be critical for proper filament assembly [16]. Beaded filament mRNA levels increase greatly in differentiating lens epithelial cells, and have been suggested as a pan-specific marker for lens fiber cells [17]. It is therefore possible that increased synthesis of alpha-crystallin in epithelial cells early in the differentiation process may have profound effects upon the cytoskeleton, which in turn may profoundly affect cell shape and migration. The lack of cellar organization and uniform cell shape at the equatorial region observed in alphaA/BKO lenses supports this hypothesis. Studies are currently underway to characterize cytoskeletal organization in the alphaA/BKO lens. + +Competing interests + +None declared + +Authors' contributions + +DLB conceived and designed the study, carried out sample preparation, confocal microscopy, scanning electron microscopy, statistical analysis, and drafted the manuscript. JPB assisted in the production of the Alpha/BKO mice. EFW assisted in the production of the AlphaA/BKO mice, experimental design, and initial fixation of samples. All authors read and approved the final manuscript. + +Pre-publication history + +The pre-publication history for this paper can be accessed here: + + + +Acknowledgements + +This research was supported in part by a NIH Grant for Vision Research EY02932 awarded to LT. diff --git a/src/ontogpt/evaluation/craft/database/all/12585968.ann b/src/ontogpt/evaluation/craft/database/all/12585968.ann new file mode 100644 index 000000000..be1e9a9db --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/12585968.ann @@ -0,0 +1,898 @@ +T1 PR:000013043 0 5 Brn3c +T2 NCBITaxon:10088 18 22 mice +T3 UBERON:0001846 77 86 inner ear +T4 UBERON:0001690 122 126 Ears +T5 PR:000013043 130 135 Brn3c +T6 CL:0000855 166 176 hair cells +T7 GO:0006915 238 247 apoptosis +T8 GO:0048468 274 288;294 299 development of ... cells +T9 CL:0000855 289 299 hair cells +T10 PR:000021998 321 333 neurotrophin +T11 GO:0010467 334 344 expression +T12 CL:0000101 356 371 sensory neurons +T13 UBERON:0000922 380 389 embryonic +T14 GO:0009790 380 401 embryonic development +T15 GO:0010467 486 496 expression +T16 PR:000021998 506 519 neurotrophins +T17 GO:0007567 534 539 birth +T18 GO:0010467 559 569 expression +T19 PR:000004716 573 577 BDNF +T20 PR:000011459 582 586 NT-3 +T21 UBERON:0006934 601 618 sensory epithelia +T22 CHEBI:52029 623 626 DiI +T23 UBERON:0006798 679 688 efferents +T24 PR:000021998 704 716 neurotrophin +T25 GO:0007567 740 745 natal +T26 PR:000010869 839 850 myosin VIIa +T27 CL:0000855 869 879 hair cells +T28 UBERON:0001844 906 913 cochlea +T29 CL:0000855 964 974 hair cells +T30 UBERON:0006932 995 1023 vestibular sensory epithelia +T31 CL:0000855 1053 1063 hair cells +T32 UBERON:0001844 1090 1097 cochlea +T33 CL:0000101 1116 1131 sensory neurons +T34 UBERON:0006932 1159 1179 vestibular epithelia +T35 NCBITaxon:10088 1206 1210 mice +T36 UBERON:0006932 1235 1263 vestibular sensory epithelia +T37 UBERON:0002819 1285 1298 cochlear apex +T38 PR:000010869 1311 1318 MyoVIIa +T39 CL:0000855 1328 1338 hair cells +T40 UBERON:0001844 1372 1379 cochlea +T41 GO:0042995 1380 1391 projections +T42 CL:0000855 1444 1454 hair cells +T43 GO:0042995 1490 1501 projections +T44 GO:0010467 1528 1538 expression +T45 PR:000021998 1542 1555 neurotrophins +T46 UBERON:0001844 1563 1570 cochlea +T47 PR:000013043 1574 1579 Brn3c +T48 NCBITaxon:10088 1585 1589 mice +T49 CL:0000101 1615 1630 sensory neurons +T50 UBERON:0001844 1652 1659 cochlea +T51 GO:0007567 1667 1672 birth +T52 CL:0011113 1731 1745 spiral neurons +T53 UBERON:0001690 1774 1777 ear +T54 GO:0043583 1774 1789 ear development +T55 CL:0000855 1807 1817 hair cells +T56 UBERON:0001690 1828 1831 ear +T57 UBERON:0006798 1845 1853 efferent +T58 UBERON:0001690 1854 1857 ear +T59 PR:000013043 1883 1888 Brn3c +T60 SO:0000417 1898 1904 domain +T61 UBERON:0001846 1932 1941 inner ear +T62 CL:0000202 1932 1951 inner ear hair cell +T63 GO:0060119 1932 1963 inner ear hair cell development +T64 PR:000013043 1979 1984 Brn3c +T65 CL:0000855 2008 2018 hair cells +T66 CL:0000855 2078 2088 hair cells +T67 PR:000013043 2097 2102 Brn3c +T68 GO:0010467 2120 2127 express +T69 PR:000010868 2153 2162 Myosin VI +T70 PR:000010869 2153 2159;2167 2171 Myosin ... VIIa +T71 PR:000004968 2173 2183 calretinin +T72 CL:0000855 2233 2243 hair cells +T73 PR:000013043 2247 2252 Brn3c +T74 GO:0006915 2274 2283 apoptosis +T75 CL:0000855 2347 2357 hair cells +T76 UBERON:0000395 2411 2426 spiral ganglion +T77 CL:0011113 2411 2432 spiral ganglion cells +T78 GO:0007567 2449 2454 natal +T79 CL:0000540 2570 2577 neurons +T80 CL:0011113 2594 2608 spiral neurons +T81 UBERON:0007023 2678 2684 adults +T82 PR:000013043 2804 2809 Brn3c +T83 GO:0048468 2837 2848;2877 2879;2885 2890 development ... of ... cells +T84 GO:0030154 2861 2879;2885 2890 differentiation of ... cells +T85 CL:0000855 2880 2890 hair cells +T86 PR:000013043 2894 2899 Brn3c +T87 PR:000021998 2936 2948 neurotrophin +T88 GO:0010467 2949 2959 expression +T89 CL:0000101 2986 3001 sensory neurons +T90 UBERON:0000922 3010 3019 embryonic +T91 GO:0009790 3010 3031 embryonic development +T92 PR:000021998 3080 3092 neurotrophin +T93 PR:000021998 3145 3157 neurotrophin +T94 CL:0000101 3200 3215 sensory neurons +T95 UBERON:0000922 3223 3232 embryonic +T96 UBERON:0001690 3233 3236 ear +T97 PR:000013043 3267 3272 Brn3c +T98 PR:000021998 3335 3347 neurotrophin +T99 CL:0000855 3360 3370 hair cells +T100 UBERON:0000922 3408 3415 embryos +T101 PR:000011459 3416 3420 NT-3 +T102 GO:0010467 3434 3443 expressed +T103 CL:0000630 3447 3463 supporting cells +T104 GO:0007567 3484 3489 birth +T105 CL:0000855 3495 3505 hair cells +T106 UBERON:0001844 3570 3578 cochlear +T107 CL:0000101 3579 3594 sensory neurons +T108 PR:000011459 3631 3635 NT-3 +T109 GO:0010467 3636 3646 expression +T110 PR:000013043 3676 3681 Brn3c +T111 PR:000004716 3700 3704 BDNF +T112 GO:0030154 3742 3760;3772 3777 differentiation of ... cells +T113 CL:0000630 3761 3777 supporting cells +T114 UBERON:0001690 3798 3801 ear +T115 PR:000021998 3832 3845 neurotrophins +T116 GO:0007567 3885 3890 natal +T117 NCBITaxon:40674 3891 3898 mammals +T118 PR:000013043 4010 4015 Brn3c +T119 GO:0010467 4040 4050 expression +T120 PR:000011459 4054 4058 NT-3 +T121 PR:000004716 4063 4067 BDNF +T122 CL:0000101 4136 4150 sensory neuron +T123 PR:000021998 4182 4194 neurotrophin +T124 CL:0000855 4218 4228 hair cells +T125 GO:0007567 4232 4237 birth +T126 NCBITaxon:33208 4251 4258 animals +T127 NCBITaxon:9606 4330 4335 human +T128 PR:000013043 4360 4366 Pou4f3 +T129 SO:0000704 4367 4371 gene +T130 PR:000013043 4373 4379 DFNA15 +T131 UBERON:0001844 4425 4433 cochlear +T132 CL:0000101 4434 4449 sensory neurons +T133 UBERON:0002819 4494 4507 cochlear apex +T134 PR:000013043 4512 4517 Brn3c +T135 NCBITaxon:10088 4530 4534 mice +T136 UBERON:0006798 4568 4577 efferents +T137 CL:0000855 4594 4603 hair cell +T138 GO:0030154 4599 4619 cell differentiation +T139 CL:0000855 4641 4651 hair cells +T140 GO:0007567 4681 4686 natal +T141 UBERON:0001844 4746 4753 cochlea +T142 CL:0011113 4780 4794 spiral neurons +T143 PR:000021998 4824 4837 neurotrophins +T144 GO:0010467 4877 4887 expression +T145 NCBITaxon:9989 4900 4907 rodents +T146 PR:000013043 4956 4961 Brn3c +T147 UBERON:0001846 4998 5007 inner ear +T148 PR:000004716 5061 5065 BDNF +T149 PR:000011459 5070 5074 NT-3 +T150 GO:0007567 5093 5098 birth +T151 PR:000004716 5126 5130 BDNF +T152 PR:000011470 5147 5151 trkB +T153 UBERON:0001840 5180 5199 semicircular canals +T154 UBERON:0001853 5238 5245 utricle +T155 UBERON:0001854 5247 5254 saccule +T156 UBERON:0001844 5278 5285 cochlea +T157 PR:000011459 5325 5329 NT-3 +T158 PR:000011471 5346 5350 trkC +T159 CL:0011113 5369 5383 spiral neurons +T160 PR:000013043 5533 5538 Brn3c +T161 NCBITaxon:10088 5544 5548 mice +T162 PR:000021998 5593 5606 neurotrophins +T163 UBERON:0006134 5657 5668 nerve fiber +T164 PR:000013043 5676 5681 Brn3c +T165 GO:0007567 5698 5703 birth +T166 UBERON:0002824 5710 5728 Vestibular ganglia +T167 PR:000013043 5744 5749 Brn3c +T168 PR:000004716 5831 5835 BDNF +T169 PR:000011470 5839 5843 trkB +T170 UBERON:0002824 5920 5938 vestibular ganglia +T171 CL:0000101 6057 6072 sensory neurons +T172 PR:000004716 6076 6080 BDNF +T173 PR:000011470 6085 6089 trkB +T174 CL:0000101 6133 6148 sensory neurons +T175 PR:000013043 6155 6160 Brn3c +T176 UBERON:0002824 6211 6238 vestibular sensory ganglion +T177 PR:000004716 6274 6278 BDNF +T178 CL:0000855 6306 6316 hair cells +T179 UBERON:0002824 6348 6366 vestibular ganglia +T180 CHEBI:52029 6414 6417 DiI +T181 NCBITaxon:33208 6430 6437 animals +T182 UBERON:0002824 6443 6462 vestibular ganglion +T183 PR:000013043 6493 6498 Brn3c +T184 UBERON:2000468 6595 6597 AC +T185 UBERON:2000468 6599 6614 anterior crista +T186 UBERON:0000045 6616 6619 ggl +T187 UBERON:0000045 6621 6629 ganglion +T188 UBERON:0001982 6631 6632 c +T189 UBERON:0001982 6641 6650 capillary +T190 UBERON:0001700 6652 6663 Genic. ggl. +T191 UBERON:0001700 6665 6684 geniculate ganglion +T192 UBERON:0000483 6699 6709 epithelial +T193 UBERON:0004721 6732 6738 crista +T194 CL:0000855 6740 6743 HaC +T195 CL:0000609 6745 6765 vestibular hair cell +T196 CL:0000589 6767 6770 IHC +T197 CL:0000589 6772 6787 inner hair cell +T198 CL:0000855 6803 6812 hair cell +T199 UBERON:0000045 6825 6835 ganglionic +T200 CL:0000601 6851 6854 OHC +T201 CL:0000601 6856 6871 outer hair cell +T202 UBERON:0004721 6887 6893 crista +T203 UBERON:0001854 6895 6896 S +T204 UBERON:0001854 6898 6905 saccule +T205 UBERON:0000395 6907 6909 SG +T206 UBERON:0000395 6911 6926 spiral ganglion +T207 UBERON:0002233 6928 6930 TM +T208 UBERON:0002233 6932 6950 tectorial membrane +T209 UBERON:0001853 6952 6953 U +T210 UBERON:0001853 6955 6962 utricle +T211 UBERON:0002828 6964 6967 VCN +T212 UBERON:0002828 6969 6993 ventral cochlear nucleus +T213 UBERON:0002824 6995 6997 VG +T214 UBERON:0002824 6999 7018 vestibular ganglion +T215 PR:000013043 7057 7062 Brn3c +T216 UBERON:0006798 7120 7128 efferent +T217 UBERON:0006932 7143 7171 vestibular sensory epithelia +T218 UBERON:0006798 7219 7227 efferent +T219 UBERON:0000025 7247 7252 canal +T220 UBERON:0004721 7253 7259 crista +T221 PR:000004716 7290 7294 BDNF +T222 PR:000011470 7299 7303 trkB +T223 UBERON:0006934 7407 7425 sensory epithelium +T224 UBERON:0004721 7435 7441 crista +T225 UBERON:0001853 7518 7525 utricle +T226 UBERON:0001854 7530 7537 saccule +T227 PR:000004716 7588 7592 BDNF +T228 NCBITaxon:10088 7605 7609 mice +T229 UBERON:0001854 7630 7637 saccule +T230 UBERON:0001853 7641 7648 utricle +T231 PR:000013043 7652 7657 Brn3c +T232 NCBITaxon:10088 7663 7667 mice +T233 MOP:0000030 7720 7730 acteylated +T234 PR:000028799 7731 7738 tubulin +T235 PR:000013043 7796 7801 Brn3c +T236 UBERON:0001690 7819 7823 ears +T237 NCBITaxon:10088 7846 7850 mice +T238 UBERON:0006585 7895 7914 vestibular endorgan +T239 CHEBI:52029 7933 7936 DiI +T240 MOP:0000030 7953 7963 acetylated +T241 PR:000028799 7964 7971 tubulin +T242 UBERON:0001844 8079 8086 cochlea +T243 PR:000013043 8090 8095 Brn3c +T244 CL:0000601 8213 8229 outer hair cells +T245 UBERON:0006798 8238 8246 Efferent +T246 UBERON:0001690 8261 8264 ear +T247 UBERON:0000045 8292 8302 ganglionic +T248 CL:0011113 8474 8480;8489 8496 spiral ... neurons +T249 CL:0000101 8481 8496 sensory neurons +T250 UBERON:0001844 8573 8580 cochlea +T251 PR:000013043 8587 8592 Brn3c +T252 CL:0011113 8641 8655 spiral neurons +T253 PR:000013043 8671 8676 Brn3c +T254 PR:000011459 8729 8733 NT-3 +T255 PR:000011471 8737 8741 trkC +T256 NCBITaxon:33208 8891 8898 animals +T257 PR:000004716 8936 8940 BDNF +T258 GO:0010467 8950 8959 expressed +T259 UBERON:0006798 9005 9013 efferent +T260 PR:000021998 9140 9152 neurotrophin +T261 CL:0011113 9200 9206;9215 9222 spiral ... neurons +T262 CL:0000101 9207 9222 sensory neurons +T263 UBERON:0006798 9312 9321 efferents +T264 PR:000004716 9423 9427 BDNF +T265 PR:000021998 9524 9536 neurotrophin +T266 CL:0000601 9584 9600 outer hair cells +T267 UBERON:0006798 9684 9692 efferent +T268 CL:0000601 9722 9738 outer hair cells +T269 PR:000013043 9742 9747 Brn3c +T270 CL:0000601 9893 9908 outer hair cell +T271 CL:0000601 9967 9982 outer hair cell +T272 PR:000013043 10013 10018 Brn3c +T273 PR:000013043 10033 10038 Brn3c +T274 UBERON:0006798 10172 10181 efferents +T275 UBERON:0001853 10209 10216 utricle +T276 UBERON:0001854 10234 10241 saccule +T277 UBERON:0000025 10296 10301 canal +T278 UBERON:0004721 10302 10309 cristae +T279 UBERON:0001843 10372 10388 horizontal canal +T280 UBERON:0006798 10426 10434 efferent +T281 PR:000013043 10450 10455 Brn3c +T282 UBERON:0001690 10513 10516 ear +T283 NCBITaxon:10088 10539 10543 mice +T284 UBERON:0006932 10555 10583 vestibular sensory epithelia +T285 UBERON:0006798 10613 10621 efferent +T286 UBERON:0006798 10694 10702 efferent +T287 UBERON:0001853 10718 10725 utricle +T288 UBERON:0000025 10751 10757 canals +T289 UBERON:0001844 10766 10773 cochlea +T290 CL:0000601 10925 10941 outer hair cells +T291 CL:0000601 11050 11066 outer hair cells +T292 CL:0000855 11140 11150 hair cells +T293 CHEBI:52029 11186 11189 DiI +T294 MOP:0000030 11231 11241 acetylated +T295 PR:000028799 11242 11249 tubulin +T296 UBERON:0001844 11322 11329 cochlea +T297 CL:0000601 11441 11457 outer hair cells +T298 UBERON:0006798 11488 11496 efferent +T299 CL:0000601 11588 11603 outer hair cell +T300 PR:000013043 11614 11619 Brn3c +T301 NCBITaxon:33208 11662 11669 animals +T302 CL:0000589 11700 11716 inner hair cells +T303 PR:000013043 11726 11731 Brn3c +T304 UBERON:0006798 11937 11946 efferents +T305 CL:0000601 11971 11986 outer hair cell +T306 UBERON:0006798 12073 12082 efferents +T307 NCBITaxon:33208 12195 12202 animals +T308 UBERON:0006798 12204 12212 efferent +T309 PR:000013043 12338 12343 Brn3c +T310 PR:000013043 12407 12412 Brn3c +T311 CL:0000855 12531 12541 hair cells +T312 UBERON:0001844 12549 12556 cochlea +T313 CL:0000855 12627 12636 hair cell +T314 UBERON:0002819 12650 12657;12662 12669 apex of ... cochlea +T315 CL:0000855 12698 12708 hair cells +T316 CL:0000855 12750 12760 hair cells +T317 UBERON:0006585 12768 12788 vestibular endorgans +T318 UBERON:0006585 12813 12834 vestibular end organs +T319 CL:0000855 12920 12930 hair cells +T320 UBERON:0006134 12932 12944 Nerve fibers +T321 UBERON:0006934 12968 12986 sensory epithelium +T322 PR:000013043 13000 13005 Brn3c +T323 CL:0011113 13081 13087;13096 13103 spiral ... neurons +T324 CL:0000101 13088 13103 sensory neurons +T325 UBERON:0006106 13107 13124 Rosenthal's canal +T326 PR:000013043 13143 13148 Brn3c +T327 UBERON:0001844 13180 13187 cochlea +T328 UBERON:0001982 13241 13250 capillary +T329 UBERON:0002227 13279 13293 organ of Corti +T330 CL:0000855 13357 13367 hair cells +T331 GO:0048468 13393 13405;13413 13418 formation of ... cells +T332 CL:1000191 13406 13418 pillar cells +T333 UBERON:0001844 13459 13466 cochlea +T334 CL:0000635 13480 13494 Deiters' cells +T335 CL:0000855 13614 13624 hair cells +T336 CL:0000630 13629 13645 supporting cells +T337 CL:0000855 13686 13696 hair cells +T338 UBERON:0001844 13780 13787 cochlea +T339 GO:0030154 13816 13843 cytological differentiation +T340 PR:000013043 13950 13955 Brn3c +T341 UBERON:0001844 13963 13971 cochleae +T342 GO:0042571 14019 14027 antibody +T343 CL:0000601 14049 14065 outer hair cells +T344 CL:0000601 14067 14070 OHC +T345 CL:0000589 14094 14110 inner hair cells +T346 CL:0000589 14112 14115 IHC +T347 CL:0000855 14171 14181 hair cells +T348 CL:0000855 14350 14360 hair cells +T349 GO:0030154 14408 14426;14432 14437 differentiation of ... cells +T350 CL:0000855 14427 14437 hair cells +T351 GO:0060384 14448 14459 innervating +T352 PR:000028799 14480 14487 tubulin +T353 UBERON:0001982 14552 14561 capillary +T354 UBERON:0001982 14563 14564 C +T355 UBERON:0005972 14576 14591 tunnel of Corti +T356 CL:0000589 14607 14612;14623 14633 inner ... hair cells +T357 CL:0000601 14617 14633 outer hair cells +T358 UBERON:0002227 14715 14729 organ of Corti +T359 UBERON:0005972 14754 14769 tunnel of Corti +T360 UBERON:0002233 14789 14807 tectorial membrane +T361 UBERON:0002233 14815 14817 TM +T362 GO:0030154 14849 14877 cyotological differentiation +T363 PR:000028799 14889 14896 tubulin +T364 CL:0000855 15017 15027 hair cells +T365 CL:0000101 15032 15047 sensory neurons +T366 PR:000013043 15067 15072 Brn3c +T367 CL:0000855 15197 15207 hair cells +T368 UBERON:0001844 15230 15238 cochlear +T369 CL:0000609 15284 15294;15308 15323 vestibular ... sensory neurons +T370 UBERON:0001844 15299 15307 cochlear +T371 CL:0000202 15299 15323 cochlear sensory neurons +T372 CL:0000855 15401 15411 hair cells +T373 PR:000010869 15429 15436 MyoVIIa +T374 UBERON:0014626 15468 15483;15488 15495 basal region of ... cochlea +T375 UBERON:0001844 15523 15531 cochlear +T376 CL:0000202 15523 15547 cochlear sensory neurons +T377 CL:0000855 15643 15653 hair cells +T378 PR:000010869 15659 15666 MyoVIIa +T379 CL:0000855 15756 15766 hair cells +T380 CL:0000101 15834 15849 sensory neurons +T381 CL:0000855 15863 15873 hair cells +T382 PR:000013043 15914 15919 Brn3c +T383 UBERON:0001844 15983 15991 cochlear +T384 CL:0000855 16001 16011 hair cells +T385 CL:0011113 16029 16035;16044 16051 spiral ... neurons +T386 CL:0000101 16036 16051 sensory neurons +T387 CL:0000855 16114 16124 hair cells +T388 UBERON:0006932 16148 16176 vestibular sensory epithelia +T389 UBERON:0001854 16178 16185 saccule +T390 CL:0000101 16255 16270 sensory neurons +T391 CHEBI:52029 16366 16369 DiI +T392 CHEBI:52024 16374 16377 DiA +T393 UBERON:0001720 16399 16414 cochlear nuclei +T394 UBERON:0004727 16561 16575 cochlear nerve +T395 UBERON:0002828 16594 16618 ventral cochlear nucleus +T396 UBERON:0002828 16620 16623 VCN +T397 GO:0042995 16667 16678 projections +T398 GO:0042995 16767 16777 projection +T399 UBERON:0001720 16781 16796 cochlear nuclei +T400 NCBITaxon:10088 16821 16825 mice +T401 GO:0007605 16854 16862 auditory +T402 CL:0000855 16884 16893 hair cell +T403 PR:000013043 17029 17034 Brn3c +T404 NCBITaxon:10088 17047 17052 mouse +T405 CHEBI:52029 17054 17057 DiI +T406 CHEBI:52024 17098 17101 DiA +T407 UBERON:0004727 17201 17215 cochlear nerve +T408 UBERON:0002211 17255 17259 root +T409 UBERON:2000280 17284 17290;17303 17312 medial ... divisions +T410 UBERON:2000532 17295 17312 lateral divisions +T411 UBERON:0002828 17320 17344 ventral cochlear nucleus +T412 GO:0042995 17395 17405 projection +T413 CL:0000855 17464 17474 hair cells +T414 UBERON:0001844 17482 17489 cochlea +T415 UBERON:0006798 17511 17519 efferent +T416 UBERON:0001018 17559 17564 tract +T417 PR:000013043 17598 17603 Brn3c +T418 UBERON:0006798 17653 17661 efferent +T419 UBERON:0006932 17670 17690 vestibular epithelia +T420 PR:000013043 17787 17792 Brn3c +T421 CL:0000101 17890 17905 sensory neurons +T422 UBERON:0000025 17913 17918 canal +T423 UBERON:0000483 17919 17928 epithelia +T424 UBERON:0001843 17948 17964 horizontal canal +T425 UBERON:0001853 18014 18021 utricle +T426 UBERON:0001854 18026 18033 saccule +T427 UBERON:0006798 18056 18065 efferents +T428 UBERON:0000483 18075 18084 epithelia +T429 NCBITaxon:10088 18159 18163 mice +T430 CHEBI:52029 18207 18210 DiI +T431 PR:000013043 18271 18276 Brn3c +T432 UBERON:0001853 18289 18296 utricle +T433 CL:0000855 18373 18383 hair cells +T434 UBERON:0006934 18441 18458 sensory epithelia +T435 PR:000013043 18483 18488 Brn3c +T436 NCBITaxon:10088 18501 18505 mice +T437 NCBITaxon:33208 18522 18529 animals +T438 UBERON:0006932 18579 18607 vestibular sensory epithelia +T439 UBERON:0006934 18641 18658 sensory epithelia +T440 CHEBI:52029 18694 18697 DiI +T441 CL:0000855 18756 18766 hair cells +T442 UBERON:0001844 18772 18779 cochlea +T443 CL:0011113 18861 18867;18876 18883 spiral ... neurons +T444 CL:0000101 18868 18883 sensory neurons +T445 UBERON:0006106 18899 18916 Rosenthal's canal +T446 UBERON:0002227 18948 18962 organ of Corti +T447 UBERON:0001018 18992 18998 tracts +T448 UBERON:0000483 19015 19025 epithelium +T449 UBERON:0001844 19054 19061 cochlea +T450 CL:0000601 19322 19338 outer hair cells +T451 CL:0011113 19413 19427 spiral neurons +T452 CL:0000855 19514 19524 hair cells +T453 UBERON:0002819 19628 19635;19640 19647 apex of ... cochlea +T454 PR:000021998 19749 19761 neurotrophin +T455 PR:000021998 19766 19778 neurotrophin +T456 PR:000013043 19819 19824 Brn3c +T457 NCBITaxon:10088 19832 19836 mice +T458 GO:0007567 19843 19848 birth +T459 GO:0097617 19859 19872 hybridization +T460 PR:000021998 19891 19903 neurotrophin +T461 GO:0010467 19904 19914 expression +T462 UBERON:0001690 19934 19938 ears +T463 PR:000013043 19952 19957 Brn3c +T464 GO:0010467 19991 20001 expression +T465 PR:000004716 20005 20009 BDNF +T466 PR:000011459 20014 20018 NT-3 +T467 PR:000021998 20085 20097 neurotrophin +T468 GO:0010467 20098 20108 expression +T469 NCBITaxon:33208 20124 20131 animals +T470 PR:000021998 20135 20148 neurotrophins +T471 GO:0010467 20191 20201 expression +T472 NCBITaxon:33208 20230 20237 animals +T473 UBERON:0000025 20250 20255 canal +T474 UBERON:0004721 20256 20263 cristae +T475 PR:000004716 20295 20299 BDNF +T476 CL:0000855 20341 20351 hair cells +T477 NCBITaxon:33208 20370 20377 animals +T478 PR:000013043 20442 20447 Brn3c +T479 CL:0000855 20553 20563 hair cells +T480 PR:000004716 20569 20573 BDNF +T481 UBERON:0001853 20588 20595 utricle +T482 CL:0000855 20624 20634 hair cells +T483 NCBITaxon:33208 20650 20656 animal +T484 PR:000013043 20716 20721 Brn3c +T485 GO:0010467 20768 20778 expression +T486 PR:000004716 20782 20786 BDNF +T487 PR:000011459 20791 20795 NT-3 +T488 UBERON:0006932 20820 20848 vestibular sensory epithelia +T489 UBERON:0000025 20954 20959 Canal +T490 UBERON:0004721 20960 20967 cristae +T491 PR:000004716 20981 20985 BDNF +T492 UBERON:0001854 21028 21035 saccule +T493 UBERON:0001853 21040 21047 utricle +T494 PR:000004716 21062 21066 BDNF +T495 PR:000011459 21071 21075 NT-3 +T496 PR:000004716 21102 21106 BDNF +T497 GO:0010467 21107 21117 expression +T498 CL:0000855 21125 21134 hair cell +T499 UBERON:0000025 21159 21164 canal +T500 UBERON:0001854 21173 21180 saccule +T501 GO:0010467 21203 21213 expression +T502 PR:000013043 21221 21226 Brn3c +T503 NCBITaxon:10088 21232 21236 mice +T504 CL:0000855 21257 21267 hair cells +T505 PR:000011459 21279 21283 NT-3 +T506 GO:0010467 21284 21294 expression +T507 PR:000013043 21336 21341 Brn3c +T508 UBERON:0001854 21392 21399 saccule +T509 PR:000004716 21426 21430 BDNF +T510 PR:000011459 21448 21452 NT-3 +T511 PR:000013043 21480 21485 Brn3c +T512 PR:000004716 21558 21562 BDNF +T513 CL:0000855 21589 21599 hair cells +T514 NCBITaxon:33208 21611 21618 animals +T515 CL:0000855 21646 21655 hair cell +T516 PR:000004716 21670 21674 BDNF +T517 PR:000013043 21689 21694 Brn3c +T518 PR:000011459 21713 21717 NT-3 +T519 PR:000013043 21770 21775 Brn3c +T520 UBERON:0006934 21861 21879 sensory epithelium +T521 PR:000013043 21892 21897 Brn3c +T522 UBERON:0001844 21919 21926 cochlea +T523 PR:000004716 21932 21936 BDNF +T524 PR:000013043 22008 22013 Brn3c +T525 PR:000011459 22083 22087 NT-3 +T526 UBERON:0002227 22104 22118 organ of Corti +T527 PR:000013043 22134 22139 Brn3c +T528 PR:000011459 22247 22251 NT-3 +T529 UBERON:0001844 22278 22285 cochlea +T530 CL:0000589 22310 22315 IHC's +T531 PR:000013043 22359 22364 Brn3c +T532 NCBITaxon:10088 22370 22374 mice +T533 PR:000004716 22400 22404 BDNF +T534 PR:000011459 22409 22413 NT-3 +T535 GO:0010467 22419 22429 expression +T536 UBERON:0001844 22446 22453 cochlea +T537 PR:000013043 22479 22484 Brn3c +T538 GO:0010467 22532 22542 expression +T539 PR:000004716 22546 22550 BDNF +T540 GO:0010467 22637 22647 expression +T541 PR:000011459 22651 22655 NT-3 +T542 CL:0000589 22691 22707 inner hair cells +T543 PR:000011459 22756 22760 NT-3 +T544 GO:0010467 22761 22771 expression +T545 PR:000013043 22814 22819 Brn3c +T546 NCBITaxon:10088 22825 22829 mice +T547 CL:0000589 22876 22892 inner hair cells +T548 NCBITaxon:33208 22904 22911 animals +T549 GO:0010467 23026 23036 expression +T550 PR:000021998 23040 23053 neurotrophins +T551 CL:0000530 23090 23105 primary neurons +T552 CL:0000855 23148 23158 hair cells +T553 PR:000021998 23176 23189 neurotrophins +T554 CL:0011113 23253 23267 spiral neurons +T555 CL:0000855 23299 23309 hair cells +T556 UBERON:0006798 23331 23339 efferent +T557 NCBITaxon:33208 23402 23409 animals +T558 GO:0007567 23426 23430 born +T559 GO:0007605 23445 23452 hearing +T560 http://purl.obolibrary.org/obo/MONDO_0005365 23445 23457 hearing loss +T561 CL:0000855 23469 23479 hair cells +T562 GO:0010467 23492 23502 expression +T563 PR:000021998 23506 23519 neurotrophins +T564 PR:000021998 23561 23574 neurotrophins +T565 GO:0008219 23638 23648 cell death +T566 GO:0065007 23677 23687 regulation +T567 CL:0000540 23691 23699 neuronal +T568 PR:000021998 23777 23790 neurotrophins +T569 UBERON:0000479 23818 23824 tissue +T570 CL:0000101 23860 23875 sensory neurons +T571 PR:000021998 23926 23939 neurotrophins +T572 PR:000021998 23993 24006 neurotrophins +T573 PR:000004716 24100 24104 BDNF +T574 NCBITaxon:33208 24119 24126 animals +T575 CL:0000101 24170 24185 sensory neurons +T576 UBERON:0001690 24193 24196 ear +T577 PR:000021998 24276 24288 neurotrophin +T578 PR:000021998 24289 24301 neurotrophin +T579 GO:0010467 24350 24360 expression +T580 UBERON:0000955 24399 24404 brain +T581 UBERON:0000922 24454 24461 embryos +T582 PR:000013043 24476 24481 Brn3c +T583 GO:0010467 24492 24501 expressed +T584 GO:0009790 24509 24522 embryogenesis +T585 UBERON:0001017 24530 24533 CNS +T586 UBERON:0001690 24573 24576 ear +T587 CL:0000530 24577 24592 primary neurons +T588 GO:0010467 24654 24664 expression +T589 PR:000021998 24668 24681 neurotrophins +T590 CL:0000530 24727 24742 primary neurons +T591 GO:0007567 24794 24799 natal +T592 PR:000013043 24800 24805 Brn3c +T593 NCBITaxon:10088 24811 24815 mice +T594 GO:0010467 24896 24906 expression +T595 PR:000004716 24910 24914 BDNF +T596 PR:000021998 25009 25022 neurotrophins +T597 GO:0010467 25038 25047 expressed +T598 UBERON:0006934 25055 25072 sensory epithelia +T599 UBERON:0000922 25076 25085 embryonic +T600 PR:000013043 25086 25091 Brn3c +T601 PR:000021998 25147 25160 neurotrophins +T602 UBERON:0006932 25168 25196 vestibular sensory epithelia +T603 PR:000011459 25300 25304 NT-3 +T604 GO:0010467 25305 25315 expression +T605 PR:000013043 25319 25324 Brn3c +T606 UBERON:0001844 25337 25344 cochlea +T607 PR:000021998 25377 25389 neurotrophin +T608 CL:0011113 25418 25424;25433 25440 spiral ... neurons +T609 CL:0000101 25425 25440 sensory neurons +T610 UBERON:0001844 25498 25506 cochlear +T611 PR:000013043 25530 25535 Brn3c +T612 PR:000004716 25557 25561 BDNF +T613 CL:0000855 25600 25610 hair cells +T614 GO:0030154 25642 25660;25666 25671 differentiation of ... cells +T615 CL:0000855 25661 25671 hair cells +T616 GO:0010467 25714 25724 expression +T617 PR:000004716 25728 25732 BDNF +T618 UBERON:0000025 25756 25761 canal +T619 UBERON:0004721 25762 25769 cristae +T620 PR:000004716 25812 25816 BDNF +T621 PR:000011470 25821 25825 trkB +T622 UBERON:0000025 25859 25864 canal +T623 UBERON:0004721 25865 25872 cristae +T624 GO:0007567 25888 25893 birth +T625 GO:0010467 25932 25942 expression +T626 UBERON:0000025 25991 25996 canal +T627 UBERON:0006934 25997 26014 sensory epithelia +T628 UBERON:0006798 26060 26069 efferents +T629 GO:0007567 26073 26078 birth +T630 UBERON:0001853 26124 26131 utricle +T631 UBERON:0001854 26133 26140 saccule +T632 UBERON:0001844 26145 26152 cochlea +T633 PR:000004716 26175 26179 BDNF +T634 PR:000011459 26184 26188 NT-3 +T635 GO:0010467 26193 26202 expressed +T636 PR:000011459 26249 26253 NT-3 +T637 GO:0010467 26254 26264 expression +T638 UBERON:0002819 26369 26376;26381 26388 apex of ... cochlea +T639 UBERON:0001844 26406 26413 cochlea +T640 UBERON:0000922 26417 26424 embryos +T641 UBERON:0001854 26456 26463 saccule +T642 UBERON:0001853 26468 26475 utricle +T643 UBERON:0000922 26479 26486 embryos +T644 PR:000011459 26488 26492 NT-3 +T645 GO:0010467 26510 26519 expressed +T646 CL:0000630 26523 26539 supporting cells +T647 GO:0010467 26550 26560 expression +T648 PR:000011459 26564 26568 NT-3 +T649 CL:0000630 26572 26588 supporting cells +T650 UBERON:0000922 26592 26599 embryos +T651 PR:000013043 26634 26639 Brn3c +T652 CL:0011113 26666 26672;26681 26688 spiral ... neurons +T653 CL:0000101 26673 26688 sensory neurons +T654 NCBITaxon:33208 26714 26721 animals +T655 CL:0000101 26766 26781 sensory neurons +T656 CL:0000855 26813 26823 hair cells +T657 PR:000021998 26853 26866 neurotrophins +T658 PR:000004716 26893 26897 BDNF +T659 PR:000011459 26902 26906 NT-3 +T660 GO:0010467 26932 26942 expression +T661 CL:0000589 26948 26964 inner hair cells +T662 GO:0007567 26972 26977 birth +T663 UBERON:0001690 26997 27000 ear +T664 UBERON:0001016 27024 27038 nervous system +T665 PR:000021998 27086 27099 neurotrophins +T666 UBERON:0001690 27126 27129 ear +T667 PR:000013043 27157 27162 Brn3c +T668 CL:0011113 27203 27216 spiral neuron +T669 GO:0048468 27248 27260;27273 27278 formation of ... cells +T670 CL:0000855 27268 27278 hair cells +T671 CL:0000855 27376 27386 hair cells +T672 GO:0010467 27441 27451 expression +T673 PR:000021998 27455 27468 neurotrophins +T674 UBERON:0000483 27507 27516 epithelia +T675 PR:000021998 27533 27546 neurotrophins +T676 UBERON:0007023 27573 27578 adult +T677 UBERON:0001844 27579 27587 cochleae +T678 PR:000021998 27619 27631 neurotrophin +T679 GO:0010467 27632 27642 expression +T680 GO:0007567 27673 27678 natal +T681 NCBITaxon:33208 27679 27686 animals +T682 UBERON:0001017 27694 27697 CNS +T683 GO:0010467 27709 27719 expression +T684 UBERON:0001017 27727 27730 CNS +T685 PR:000021998 27791 27802 neutrophins +T686 CL:0000530 27880 27895 primary neurons +T687 GO:0007567 27939 27944 birth +T688 UBERON:0001690 27967 27970 ear +T689 PR:000013043 28061 28066 Brn3c +T690 UBERON:0001017 28196 28199 CNS +T691 CL:0011113 28242 28248;28257 28264 spiral ... neurons +T692 CL:0000101 28249 28264 sensory neurons +T693 NCBITaxon:9685 28298 28301 cat +T694 NCBITaxon:9606 28335 28341 humans +T695 http://purl.obolibrary.org/obo/MONDO_0021140 28347 28357 congenital +T696 UBERON:0001844 28393 28400 cochlea +T697 CL:0000855 28425 28435 hair cells +T698 GO:0043005 28486 28498;28506 28513 processes of ... neurons +T699 CL:0011113 28499 28513 spiral neurons +T700 UBERON:0002227 28521 28535 organ of Corti +T701 UBERON:0002819 28575 28582;28587 28594 apex of ... cochlea +T702 CL:0000855 28632 28642 hair cells +T703 PR:000010869 28659 28667 Myo VIIa +T704 CL:0000855 28722 28731 hair cell +T705 GO:0016265 28794 28798 died +T706 GO:0007567 28806 28811 birth +T707 CL:0000601 28842 28858 outer hair cells +T708 PR:000013043 28879 28884 Brn3c +T709 UBERON:0001844 28897 28905 cochleae +T710 UBERON:0005972 28949 28964 tunnel of Corti +T711 CL:0000601 28968 28984 outer hair cells +T712 GO:0030154 29115 29133;29141 29146 differentiation of ... cells +T713 CL:1000191 29134 29146 pillar cells +T714 PR:000001450 29172 29177 Fgfr3 +T715 PR:000007499 29202 29206 Fgf8 +T716 CL:0000589 29251 29267 inner hair cells +T717 GO:0048468 29281 29293;29305 29310 formation of ... cells +T718 CL:0000630 29294 29310 supporting cells +T719 GO:0010467 29348 29358 expression +T720 PR:000008515 29391 29396 Hes 1 +T721 PR:000008519 29391 29394;29401 29402 Hes ... 5 +T722 GO:0065007 29422 29431 regulated +T723 GO:0007219 29439 29462 Notch signaling pathway +T724 CL:0000601 29544 29560 outer hair cells +T725 GO:0030154 29587 29605;29617 29622 differentiation of ... cells +T726 CL:0000630 29606 29622 supporting cells +T727 CL:0000855 29648 29658 hair cells +T728 GO:0010467 29683 29693 expression +T729 CL:0000630 29697 29712 supporting cell +T730 PR:000013043 29733 29738 Brn3c +T731 UBERON:0006798 29838 29846 efferent +T732 UBERON:0001456 29868 29874 facial +T733 CL:0000100 29885 29896 motoneurons +T734 UBERON:0006798 30069 30077 efferent +T735 UBERON:0006798 30261 30269 efferent +T736 UBERON:0006798 30410 30418 efferent +T737 UBERON:0001690 30433 30436 ear +T738 GO:0042995 30527 30538 projections +T739 NCBITaxon:40674 30588 30595 mammals +T740 GO:0042995 30641 30651 projection +T741 GO:0007605 30677 30684 hearing +T742 GO:0042995 30729 30739 projection +T743 UBERON:0004727 30747 30761 cochlear nerve +T744 UBERON:0002610 30770 30785 cochlear nuclei +T745 PR:000013043 30803 30809 Brn 3c +T746 GO:0042995 30907 30917 projection +T747 PR:000011459 30921 30925 NT-3 +T748 UBERON:0001844 31036 31043 cochlea +T749 PR:000013043 31082 31087 Brn3c +T750 NCBITaxon:10088 31198 31203 mouse +T751 PR:000004716 31213 31217 BDNF +T752 GO:0010467 31221 31230 expressed +T753 PR:000011459 31237 31241 NT-3 +T754 SO:0000167 31242 31250 promoter +T755 GO:0065007 31251 31258 control +T756 PR:000013043 31323 31328 Brn3c +T757 CL:0000855 31404 31413 hair cell +T758 NCBITaxon:9606 31507 31512 human +T759 SO:0001023 31513 31519 allele +T760 PR:000013043 31523 31529 Pou4f3 +T761 PR:000013043 31531 31536 Brn3c +T762 GO:0007605 31580 31587 hearing +T763 http://purl.obolibrary.org/obo/MONDO_0005365 31580 31592 hearing loss +T764 GO:0007605 31658 31665 hearing +T765 http://purl.obolibrary.org/obo/MONDO_0005365 31658 31670 hearing loss +T766 UBERON:0001844 31705 31712 cochlea +T767 PR:000013043 31743 31748 Brn3c +T768 NCBITaxon:10088 31763 31767 mice +T769 GO:0007605 31799 31806 hearing +T770 http://purl.obolibrary.org/obo/MONDO_0005365 31799 31811 hearing loss +T771 NCBITaxon:10088 31847 31851 mice +T772 PR:000013043 31870 31875 Brn3c +T773 NCBITaxon:10088 31881 31886 mouse +T774 GO:0007605 31959 31966 hearing +T775 http://purl.obolibrary.org/obo/MONDO_0005365 31959 31971 hearing loss +T776 NCBITaxon:9606 31975 31981 humans +T777 CL:0011113 32043 32056 spiral neuron +T778 UBERON:0000113 32071 32080 adulthood +T779 NCBITaxon:9606 32103 32108 human +T780 PR:000013043 32109 32115 Pou4f3 +T781 UBERON:0001844 32116 32124 cochleae +T782 CL:0011113 32150 32163 spiral neuron +T783 GO:0007605 32231 32238 hearing +T784 http://purl.obolibrary.org/obo/MONDO_0005365 32231 32248 hearing-deficient +T785 http://purl.obolibrary.org/obo/MONDO_0021140 32298 32310 congenitally +T786 NCBITaxon:9685 32352 32355 cat +T787 NCBITaxon:10088 32408 32412 mice +T788 CL:0000855 32442 32452 hair cells +T789 CL:0000601 32459 32475 outer hair cells +T790 CL:0000855 32548 32558 hair cells +T791 UBERON:0006798 32599 32607 efferent +T792 NCBITaxon:10088 32635 32639 mice +T793 UBERON:0006798 32691 32699 efferent +T794 PR:000013043 32715 32720 Brn3c +T795 PR:000004716 32826 32830 BDNF +T796 PR:000011459 32834 32838 NT-3 +T797 UBERON:0001690 32875 32878 ear +T798 PR:000021998 32888 32901 neurotrophins +T799 CL:0000101 32929 32944 sensory neurons +T800 GO:0007567 32952 32957 birth +T801 UBERON:0006798 33020 33028 efferent +T802 CL:0000101 33085 33099 sensory neuron +T803 PR:000021998 33136 33148 neurotrophin +T804 GO:0038179 33136 33158 neurotrophin signaling +T805 NCBITaxon:33208 33202 33209 animals +T806 PR:000007928 33234 33238 GDNF +T807 GO:0010467 33280 33290 expression +T808 UBERON:0002819 33377 33390 cochlear apex +T809 UBERON:0002819 33437 33450 cochlear apex +T810 GO:0030154 33482 33500;33506 33511 differentiation of ... cells +T811 CL:0000855 33501 33511 hair cells +T812 UBERON:0001844 33529 33537 cochlear +T813 GO:0007567 33555 33559 born +T814 NCBITaxon:9685 33643 33646 cat +T815 UBERON:0000922 33703 33712 embryonic +T816 CL:0002321 33703 33712;33731 33735 embryonic ... cell +T817 CL:0000855 33726 33735 hair cell +T818 PR:000013043 33785 33790 Brn3c +T819 NCBITaxon:10088 33791 33795 mice +T820 NCBITaxon:10088 33836 33840 Mice +T821 PR:000013043 33860 33865 Brn3c +T822 NCBITaxon:33208 33969 33976 animals +T823 CHEBI:7983 34007 34020 pentobarbital +T824 UBERON:0000948 34030 34039 cardially +T825 UBERON:0001690 34110 34114 Ears +T826 CHEBI:50913 34142 34150 fixative +T827 UBERON:0006134 34205 34217 nerve fibers +T828 CHEBI:52029 34219 34222 DiI +T829 UBERON:0002298 34240 34249 brainstem +T830 CHEBI:52029 34337 34340 DiI +T831 UBERON:0006798 34365 34373 efferent +T832 UBERON:0003079 34396 34407 floor plate +T833 CHEBI:37958 34518 34521 dye +T834 UBERON:0001690 34527 34531 ears +T835 UBERON:0001690 34754 34758 Ears +T836 MOP:0000030 34789 34799 acetylated +T837 PR:000028799 34800 34807 tubulin +T838 CHEBI:52029 34864 34867 DiI +T839 UBERON:0001690 34952 34956 ears +T840 MOP:0000030 34988 34998 acetylated +T841 PR:000028799 34999 35006 tubulin +T842 GO:0042571 35007 35017 antibodies +T843 GO:0042571 35059 35069 antibodies +T844 MOP:0000779 35070 35080 conjugated +T845 UBERON:0001690 35105 35109 ears +T846 CHEBI:16240 35136 35140 H2O2 +T847 UBERON:0001690 35245 35249 ears +T848 UBERON:0006934 35341 35358 sensory epithelia +T849 CHEBI:52029 35427 35430 DiI +T850 CHEBI:52024 35470 35473 DiA +T851 PR:000013043 35533 35538 Brn3c +T852 UBERON:0000955 35641 35647 brains +T853 UBERON:0004727 35666 35680 cochlear nerve +T854 CHEBI:5291 35698 35705 gelatin +T855 GO:0042995 35938 35948 projection +T856 GO:0097617 35993 36006 hybridization +T857 UBERON:0001690 36013 36017 ears +T858 PR:000013043 36026 36031 Brn3c +T859 PR:000004716 36149 36153 BDNF +T860 PR:000011459 36158 36162 NT-3 +T861 CL:0000855 36438 36448 hair cells +T862 CL:0000855 36471 36481 hair cells +T863 CL:0000540 36486 36493 neurons +T864 UBERON:0001844 36516 36524 cochlear +T865 CL:0000855 36548 36557 hair cell +T866 PR:000010869 36579 36586 MyoVIIa +T867 GO:0042571 36588 36596 antibody +T868 UBERON:0002227 36658 36673 organs of Corti +T869 CL:0000855 36769 36779 hair cells +T870 CL:0000855 36840 36850 hair cells +T871 CL:0000540 36854 36861 neurons +T872 UBERON:0006585 36865 36885 vestibular endorgans +T873 UBERON:0001800 36889 36904 sensory ganglia +T874 CHEBI:52815 36940 36953 Cresyl Violet +T875 UBERON:0006934 37041 37059 sensory epithelium +T876 UBERON:0000045 37104 37112 ganglion +T877 CL:0000540 37119 37126 neurons +T878 GO:0005634 37149 37156 nucleus +T879 GO:0005730 37161 37169 nucleoli +T880 UBERON:0000483 37221 37231 epithelium +T881 UBERON:0000045 37236 37244 ganglion +T882 NCBITaxon:10088 37412 37416 mice +T883 UBERON:0001018 37534 37539 tract +T884 UBERON:0001844 37559 37567 cochlear +T885 GO:0097617 37677 37690 hybridization +T886 PR:000021998 37695 37708 neurotrophins +T887 CL:0000540 37799 37807 neuronal +T888 MOP:0000030 37844 37854 acetylated +T889 PR:000028799 37855 37862 tubulin +T890 UBERON:0000970 37983 37986 Eye +T891 GO:0007567 38031 38036 Birth +T892 http://purl.obolibrary.org/obo/MONDO_0000839 38031 38044 Birth Defects +R1 NULL^SLOT Arg1:T201 Arg2:T200 +R2 NULL^SLOT Arg1:T196 Arg2:T197 +R3 NULL^SLOT Arg1:T343 Arg2:T344 +R4 NULL^SLOT Arg1:T200 Arg2:T201 +R5 NULL^SLOT Arg1:T197 Arg2:T196 +R6 NULL^SLOT Arg1:T344 Arg2:T343 diff --git a/src/ontogpt/evaluation/craft/database/all/12585968.txt b/src/ontogpt/evaluation/craft/database/all/12585968.txt new file mode 100644 index 000000000..1e9a821ae --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/12585968.txt @@ -0,0 +1,179 @@ +Brn3c null mutant mice show long-term, incomplete retention of some afferent inner ear innervation + +Abstract + +Background + +Ears of Brn3c null mutants develop immature hair cells, identifiable only by certain molecular markers, and undergo apoptosis in neonates. This partial development of hair cells could lead to enough neurotrophin expression to sustain sensory neurons through embryonic development. We have therefore investigated in these mutants the patterns of innervation and of expression of known neurotrophins. + +Results + +At birth there is a limited expression of BDNF and NT-3 in the mutant sensory epithelia and DiI tracing shows no specific reduction of afferents or efferents that resembles neurotrophin null mutations. At postnatal day 7/8 (P7/8), innervation is severely reduced both qualitatively and quantitatively. 1% of myosin VIIa-positive immature hair cells are present in the mutant cochlea, concentrated in the base. Around 20% of immature hair cells exist in the mutant vestibular sensory epithelia. Despite more severe loss of hair cells (1% compared to 20%), the cochlea retains many more sensory neurons (46% compared to 15%) than vestibular epithelia. Even 6 months old mutant mice have some fibers to all vestibular sensory epithelia and many more to the cochlear apex which lacks MyoVIIa positive hair cells. Topologically organized central cochlea projections exist at least until P8, suggesting that functional hair cells are not required to establish such projections. + +Conclusion + +The limited expression of neurotrophins in the cochlea of Brn3c null mice suffices to support many sensory neurons, particularly in the cochlea, until birth. The molecular nature of the long term survival of apical spiral neurons remains unclear. + +Keywords: ear development, POU factors and hair cells, afferent ear innervation, efferent ear innervation + +Background + +Brn3c is a POU domain factor that is crucial for inner ear hair cell development. Targeted null Brn3c mutants have no mature hair cells [1,2]. Close examination has revealed that some 'immature' hair cells form in Brn3c null mutants and express cellular markers such as Myosin VI and VIIa, calretinin and parvalbumin [3]. Furthermore, these immature hair cells of Brn3c null mutants undergo apoptosis in neonates [3]. Consistent with an apparent absence of mature hair cells, initial work suggested that all vestibular and most spiral ganglion cells are lost by postnatal day 14 (P14; [1]). However, more detailed quantification by others [3] reported that at P4 about 77% of vestibular neurons and only 29% of spiral neurons are lost. It was suggested that there is possibly a complete loss in adults [3]. Other than these preliminary statements, no data exists concerning the detailed pattern of loss of innervation in Brn3c null mutants. + +The initial development and partial differentiation of hair cells in Brn3c mutants could possibly lead to some neurotrophin expression in these cells to sustain sensory neurons through embryonic development and beyond. Data on various single and compound neurotrophin null mutants have shown that the loss of a specific neurotrophin leads to topologically restricted loss of sensory neurons in the embryonic ear [4,5]. Such selective loss in Brn3c null mutants would therefore indicate reduction of a specific neurotrophin in immature hair cells. Moreover, recent work shows that in embryos NT-3 is primarily expressed in supporting cells, moving only around birth into hair cells [6,7]. In fact, the selective loss of vestibular as compared to cochlear sensory neurons (77% versus 29%; [3]) suggests that NT-3 expression may be less downregulated in Brn3c null mutants than BDNF [6,8,9], provided that at least some differentiation of supporting cells takes place. In the ear [4] as well as elsewhere [10] neurotrophins are progressively downregulated in postnatal mammals and possibly replaced by other factors [11]. + +We have investigated in detail the pattern of innervation in the Brn3c mutants, as well as the expression of NT-3 and BDNF. We want to evaluate a possible correlation between the topology of sensory neuron loss and absence of a specific neurotrophin or topological loss of hair cells at birth and in older animals. This information could be important for an in-depth evaluation of the human deafness related to the Pou4f3 gene, DFNA15 [12]. + +We report here long term retention of cochlear sensory neurons for at least 6 months, in particular in the cochlear apex, in Brn3c null mutant mice. This retention of afferents and efferents is unrelated to hair cell differentiation as not even immature hair cells can be detected at early postnatal stages with MyoVII immunocytochemistry in this part of the cochlea. This retention of apical spiral neurons is also largely unrelated to neurotrophins which are known to be reduced in their expression in neonatal rodents [4]. + +Results + +To appreciate the effects of the Brn3c null mutation on the pattern of the inner ear innervation, we first want to present the effects of BDNF and NT-3 null mutations at birth [6,13,14]. Null mutants of BDNF or its receptor trkB lose all innervation to the semicircular canals and have a reduced innervation to the utricle, saccule and apical turn of the cochlea. In contrast, null mutations of either NT-3 or its receptor trkC result in loss of spiral neurons in the basal turn with formation of an inner spiral bundle of afferents extending to the basal tip. Our null hypothesis for this study would be that Brn3c null mice show severe compromised production of these neurotrophins and should therefore show a comparable pattern of nerve fiber loss. + +Brn3c null mutants at birth (P0) + +Vestibular ganglia are smaller in Brn3c null mutants (Fig. 1b) than in control littermates (Fig. 1a), but larger than in BDNF or trkB null mutants of the same age [13,15]. The reduction in apparent size of the vestibular ganglia is in agreement with quantitative data published previously [3,13]. These data suggest a loss of 80–85% of vestibular sensory neurons in BDNF and trkB null mutants [13] and of 77% of vestibular sensory neurons in P4 Brn3c null mutants [3]. Thus, the size reduction in the vestibular sensory ganglion could be compatible with a loss of BDNF production in the immature hair cells. + +Figure 1 + +Size variations of vestibular ganglia in control and mutant littermates labeled with DiI. In newborn animals, the vestibular ganglion shows a dramatic reduction in Brn3c null mutants (b) compared to control littermates (a). Abbreviations for this and other figures: AC, anterior crista; ggl, ganglion; c, spiral capillary; Genic. ggl., geniculate ganglion; GER, greater epithelial ridge; HC, horizontal crista; HaC, vestibular hair cell; IHC, inner hair cell; iHC, immature hair cell; IGSB, intraganglionic spiral bundle; OHC, outer hair cell; PC, posterior crista; S, saccule; SG, spiral ganglion; TM, tectorial membrane; U, utricle; VCN, ventral cochlear nucleus; VG, vestibular ganglion. Bar indicates 1000 μm. + +However, the Brn3c null mutants show only a reduced density of afferent and efferent fibers to all vestibular sensory epithelia. There is no specific loss of all afferent and efferent innervation to the canal crista (Fig. 2a), a hallmark of both BDNF and trkB null mutations [13,15]. In fact, the reduction of fibers seems to be rather uniform throughout a given sensory epithelium with the crista innervation being qualitatively no more reduced than the innervation of the utricle and saccule. No loss in specific areas, comparable to that in BDNF null mutant mice, is apparent in the saccule or utricle of Brn3c null mice. Similar patterns of innervation are obtained using acteylated tubulin immunocytochemistry (Fig. 2b). + +Figure 2 + +Innervation of Brn3c null and control ears are shown for newborn mice. There is no specific loss of fibers to any vestibular endorgan, as visualized by DiI labeling (a) or acetylated tubulin immunoreactivity (b). No major differences in pattern of projection through radial fibers are found in the cochlea of Brn3c null mutants (d,f) as compared to control littermates (c). Note, however, the lack of orderly fiber outgrowth to the outer hair cells (c, d). Efferent fibers to the ear show a well developed intraganglionic spiral bundle (IGSB) with no detectable differences compared to controls (e). Bar indicates 100 μm. + +Consistent with the finding of Xiang et al. [3] of only a 29% loss of spiral sensory neurons at P4, our data show little difference in the pattern of innervation of the cochlea in P0 Brn3c null mutants (Fig. 2c,2d). No selective loss of spiral neurons is observed in Brn3c null mutants in the basal turn, a feature of either NT-3 or trkC loss [6,9,14]. Likewise, the innervation of the apex (Fig. 2f) shows no detectable abnormality in overall pattern of innervation compared to control animals (data not shown), an indication that BDNF could be expressed in the apex [6]. In addition, the pattern of efferent innervation shows no deviation from normal either (Fig. 2e), whereas they show the same pattern of loss as afferent fibers in neurotrophin null mutants [16]. These data suggest that the spiral sensory neurons develop qualitatively normal at least until P0 and therefore allow normal pathfinding of efferents. Most interestingly, there is no increase in radial fiber spacing in the apex, a specific problem of BDNF null mutants [6,13]. + +However, there is one qualitative difference not recognized in any single neurotrophin null mutant. Afferents reach all three rows of outer hair cells in the basal turn of control wildtype littermates (Fig. 2c), but both afferent and efferent outgrowth is disorganized to outer hair cells in Brn3c null mutants (Fig. 2d) and does not show any clear organization into three distinct longitudinal fiber bundles paralleling the three rows in the outer hair cell region. These data suggest that fiber organization in the outer hair cell region is partly disrupted in Brn3c null mutant. + +Brn3c null mutants at P7/8 + +Reduction of vestibular innervation is more pronounced than at P0. Loss of afferents and, even more clearly of efferents, is most pronounced in the utricle (Fig. 3a,3b) and saccule (data not shown). In contrast, the innervation to the canal cristae, while reduced, has changed little from P0. In particular the horizontal canal receives a rather dense afferent and efferent innervation in Brn3c null mutants. + +Figure 3 + +Reduction of innervation of the ear is shown in 8-day old mice. While all vestibular sensory epithelia retain some afferent (a) and efferent (b) innervation, there is a more pronounced reduction, in particular of efferent fibers, to the utricle (a,b) as compared to the canals. In the cochlea there is an increased spacing between radial fiber bundles in the basal turn (c,e). No afferents can be traced to the three outer spiral bundles along outer hair cells of mutant littermates (c, e). The apex shows little change, except for the absence of fibers to the area of outer hair cells and the formation of inner spiral bundles passing along the few immature hair cells (d,f,g). The pattern obtained with DiI tracing is identical to that obtained by acetylated tubulin immunoreactivity (f,g). Bar indicates 1000 μm in a, 100 μm in b-g. + +The cochlea at P8 shows more qualitative deviations from the normal pattern of innervation. For example, all three rows of outer hair cells now receive both afferent and efferent innervation in the base of control littermates (Fig. 3c). However, no fibers extend to the outer hair cell region in Brn3c null mutants (Figs. 3d,3e,3f). In control animals there is dense innervation of inner hair cells, whereas Brn3c null mutants show a curious aggregation of fibers near focal spots around the habenula perforata. The apex shows less pronounced deviations from normal (Fig. 3f,3g). However, as in the base, afferents and efferents extend sparingly to the outer hair cell region. There is formation of an inner spiral bundle communicating both afferents and efferents between the focal points where fibers appear to pass through the habenula perforata (Fig. 3f,3g). As in newborn animals, efferent fibers closely follow the pattern of innervation displayed by afferents (data not shown). + +The limited qualitative effect of Brn3c null mutations on fiber loss is particularly obvious in the P8 Brn3c null mutant apical turn (Fig. 3g). Immunocytochemical and histochemcial data show the presence of only a few immature hair cells in the cochlea (Fig. 4), which amounted to about 1% (Table 1). Not a single immature hair cell forms in the apex of the cochlea. In contrast, more immature hair cells are found in the appropriate position of hair cells in the vestibular endorgans (Table 1). In fact, the vestibular end organs show no overt morphological defect other than the reduced presence of these immature hair cells. Nerve fibers can be traced into the sensory epithelium of P0 and P8 Brn3c null mutants past the habenula perforata and there is a normal location of spiral sensory neurons in Rosenthal's canal in the apex of P8 Brn3c null mutants (Fig. 3d,3g). The cochlea shows other defects as well. For example, the spiral capillary is not found underneath the organ of Corti but in the spiral limbus nearby (Fig. 4). The lack of immature hair cells seems to affect also the formation of pillar cells (undifferentiated almost throughout the cochlea; Fig. 4) and Deiters' cells (not recognizable based on their morphology), a suggestion consistent with recent molecular data on the interaction of hair cells and supporting cells [17,18]. + +Figure 4 + +The distribution of hair cells as identified with MyoVII immunocytochemistry is shown for 7-day old whole mounted cochlea (a-d) and the appearance of cytological differentiation is shown as revealed with epoxy resin sections in 7-day old (e-g) and 6-month old (h) control (a,c,e) and Brn3c mutant cochleae (b,d,f-h). Note the uniform staining with this antibody of all three rows of outer hair cells (OHC) and the single row of inner hair cells (IHC) in the wildtype (a,c). In contrast, only few immature hair cells (iHC) are found in the basal turn of the mutant littermate (b) and only an occasional cell is MyoVII immunopositive in the middle turn (d). No MyoVII positive immature hair cells exist in the apex. Sections show the degree of differentiation of hair cells and their innervating fibers stained with tubulin immunocytochemistry (arrow; e-h). Note the presence of a spiral capillary (C) under the tunnel of Corti separating the inner and outer hair cells in the P7 wildtype (e). In contrast, no such capillary is present underneath the organ of Corti in mutants (f-h) and no tunnel of Corti is found either. A tectorial membrane forms (TM) but otherwise there is little cyotological differentiation. Note that tubulin-positive fibers can be traced to the immature cells until 6 months (h). Bar in c indicates 100 μm. + +Table 1 + +Numbers of hair cells and sensory neurons in P7 wildtype and Brn3c mutant littermates + +Differences between wildtype and mutants are all highly significant. + +Comparison of numbers of immature hair cells in the vestibular and cochlear areas (Fig. 4) with the numbers of surviving vestibular and cochlear sensory neurons does not show any correlation (Table 1; Fig. 5). Only a few undifferentiated hair cells were detected by MyoVIIa immunostaining and only in the basal region of the cochlea. And yet only about 50% of cochlear sensory neurons are lost overall with a less severe loss in the apex in which we could not detect any immature hair cells with MyoVIIa immunocytochemistry. This does not exclude the possibility that even less differentiated hair cells may form in the apex. + +Figure 5 + +This graph shows the reduction in sensory neurons (top) and in hair cells (bottom) between 7-day old wildtype and Brn3c null mutant littermates. Note the sharp reduction to 1% in the cochlear immature hair cells (bottom) whereas spiral sensory neurons (SG) are only reduced by 50% (top). In contrast, 20% immature hair cells could be identified in vestibular sensory epithelia (saccule has been selected; bottom) whereas only around 15% of the vestibular sensory neurons survive to this age (VG; top). For details on the data see Table 1. + +The central projection of DiI and DiA traces fibers to the cochlear nuclei (Fig. 6) where they show a clear segregation of apical turn afferents (Fig. 6, green) and basal turn afferents (Fig. 6, red) in both the entering cochlear nerve (Fig. 6a) and the ventral cochlear nucleus (VCN) (Fig. 6b). Qualitative comparable central projections are found in wildtype control littermates (data not shown) and suggest that the central projection to cochlear nuclei is at least until P8 in mice not critically dependent on auditory information or other hair cell-mediated influences, in agreement with recent suggestions [5,19]. + +Figure 6 + +These confocal images show the projection pattern of a P8 Brn3c null mutant mouse. DiI (red) was inserted into the basal turn, DiA (green) was inserted into the apical turn. Note that the fibers run in discrete bundles inside the cochlear nerve (a), spiral around each other near the root and project to distinct medial and lateral divisions of the ventral cochlear nucleus (b). These data suggest that a crude cochleotopic projection can form independently of any signaling from the immature hair cells of the cochlea. Arrow indicates the efferent bundle as it leaves the afferent fiber tract. Bar indicates 100 μm. + +6 months Brn3c null mutants + +Very few fibers (both afferent and efferent) to the vestibular epithelia remain (Fig. 7a), thus displaying a further reduction of the pattern of innervation found in P8 Brn3c null mutants. Notably, there are still a number of fibers extending from the very few vestibular sensory neurons to the canal epithelia, in particular the horizontal canal (Fig. 7a,7b). Only very few fibers extend to the utricle and saccule with the reduction of efferents to these epithelia being almost complete (Fig. 7a and data not shown). Comparable to control mice, there is some transcellular labeling with DiI to cells apparently contacted by the afferent fibers in the Brn3c null mutant utricle (Fig. 7b). However, these cells could not be clearly identified as immature hair cells based on their morphology. + +Figure 7 + +The innervation of sensory epithelia is shown in 6-month old Brn3c null mutant mice. Compared to P8 animals there is a further reduction of fiber density to vestibular sensory epithelia, but fibers can be traced to all sensory epithelia (a,b). Transcellular labeling with DiI (b) suggest contacts between these afferents and immature hair cells. The cochlea shows a much reduced density of radial bundles, even in the apex (c,d). However, spiral sensory neurons are present in Rosenthal's canal (c,d) and fibers extend to the organ of Corti where they form longitudinal tracts in the immature epithelium. Bar indicates 100 μm. + +The cochlea, most prominently the apex, receives many afferent fibers, which form an interrupted inner spiral bundle (Fig. 7c). Some of the radial fiber bundles are myelinated (Fig. 7d). Only an occasional fiber extends beyond the inner spiral bundle into the area of the outer hair cells (Fig. 7d). Even at this late stage in maturation there are still numerous spiral neurons present in the apex (Fig. 7c,7d). + +In summary, these tracing data suggest that mature hair cells are not necessary to direct fiber outgrowth and to maintain some fibers at least up to 6 months in the apex of the cochlea. Importantly, the spatio-temporal loss of innervation does not follow the pattern of loss known from neurotrophin and neurotrophin receptor null mutations as it starts in Brn3c mutant mice after birth. + +In situ hybridization confirms neonatal neurotrophin expression + +We processed four ears of P0 and P8 Brn3c null and control littermates for expression of BDNF and NT-3 using the riboprobes previously described [7]. We did not analyze neurotrophin expression in 6-month old animals as neurotrophins are downregulated in neonates [20] and no expression can be found even in normal animals [4]. In the canal cristae we expected, and found, only a BDNF signal. This signal was very strong over hair cells of the P0 control animals (Fig. 8b). In contrast, only a faint signal was detected in the Brn3c null mutant (Fig. 8a) and the few silver grains were located predominantly over the area of the immature hair cells. The BDNF signal in the utricle was clearly centered on the hair cells in the control animal but some aggregation of silver grains appeared also in the Brn3c null mutants (data not shown). + +Figure 8 + +The expression of BDNF and NT-3 mRNA is shown in the P0 vestibular sensory epithelia. Silver grains are false colored as red to enhance visibility against the grey Nissl-stained background. Canal cristae contain only BDNF (a,b), but gravistatic endorgans, such as saccule and utricle, contain both BDNF and NT-3 (c-f). Note the prominent BDNF expression in the hair cell region (HaC) of control canal (b) and saccule (d) and low levels of expression in the Brn3c null mice (a,c) over immature hair cells (iHC). The NT-3 expression is much more diffuse in both control and Brn3c mutant littermates. Bar indicates 100 μm. + +In the saccule, we directly compared the BDNF (Fig. 8c,8d) and NT-3 signal (Fig. 8e,8f) of the Brn3c null mutant (Fig. 8c,8e) and control littermates (Fig. 8d,8f). A strong BDNF signal was found over the hair cells of control animals and an occasional immature hair cell also showed a BDNF signal in the Brn3c null mutants. The NT-3 signal was more diffuse in both the control and the Brn3c null littermates but showed some slightly above background signal in the area of the sensory epithelium even in the Brn3c null mutant. + +In the cochlea, the BDNF signal was very weak in the base in both the control (Fig. 9b) and the Brn3c null littermates (Fig. 9a). However, there was a surprisingly strong NT-3 signal over the organ of Corti in the apex of Brn3c null littermates (Fig. 9c) that closely matched the signal found in the control littermates (Fig. 9d). The NT-3 signal in the area of the cochlea that corresponds to the IHC's persisted at least until P8 in the apex of Brn3c null mice (Fig. 9e,9f). + +Figure 9 + +BDNF and NT-3 mRNA expression is shown in the cochlea of P0 (a-d) and P8 (e,f) Brn3c null and control littermates. Note the limited expression of BDNF in the basal turn of both wildtype and mutant littermates (a,b) and the much stronger expression of NT-3 in the apex over the region of the inner hair cells in both wildtype and control littermates (c,d). NT-3 expression persists at least until P8 in the apex of Brn3c null mice in an area that topologically compares to the inner hair cells of control animals (e,f, arrows). Bar indicates 100 μm. + +Discussion + +We will explore five points in this discussion: How the limited expression of neurotrophins relates to the apparent survival of primary neurons until P8; how the known absence of apical hair cells and of classical neurotrophins can be related to the presence of large numbers of apical turn spiral neurons; how absence of differentiated hair cells affects afferent and efferent targeting; and how these data possibly relate to other mutant animals and to children born with profound hearing loss. + +Immature hair cells and limited expression of neurotrophins rescue afferent projection until P8 + +The neurotrophins have long been implicated as the mediators for target mediated cell death. In essence, this theory of regulation of neuronal connections via neurotrophic support implies that only limited quantities of neurotrophins are released by the target tissue to support only properly connected sensory neurons by providing only these with a critical amount of neurotrophins [21]. Past experiments, which eliminated one or more neurotrophins entirely, only partially tested the basic assumption of this theory in vivo. For example, in BDNF heterozygotic animals there is a small decrease in the number of sensory neurons in the ear [13] but no change in the overall pattern of innervation. Moreover, all of the neurotrophin/neurotrophin receptor null mutants studied to date eliminate expression in both the peripheral target and the brain simultaneously and achieve their effects in late embryos. In contrast, Brn3c is barely expressed during embryogenesis in the CNS [22] thus arguing that the survival of ear primary neurons depends crucially on the periphery. + +We suggest that limited expression of neurotrophins in the immature target leads to retention of primary neurons and their afferent fibers in newborn and early postnatal Brn3c null mice. In agreement with this suggestion, our in situ data show a severe reduction in expression of BDNF (Figs. 8, 9). Despite this reduction, some, apparently biologically significant low levels of neurotrophins are apparently expressed in all sensory epithelia in embryonic Brn3c null mutants. It appears that even these low levels of neurotrophins in the vestibular sensory epithelia can sustain normal fiber outgrowth and a limited maintenance until P0. The comparatively high level of NT-3 expression in Brn3c null mutant cochlea, which has been shown to be the neurotrophin most prominently supporting spiral sensory neurons [6,9,14], is in agreement with the normal development of cochlear innervation in newborn Brn3c null mutants. + +Since BDNF appears to be exclusively produced by hair cells [6,7,20], the lack of terminal differentiation of hair cells appears to be accompanied by only limited expression of BDNF, as is the case in the canal cristae (Fig. 8). Past research has shown that in BDNF and trkB null mutants this innervation to canal cristae is lost before birth [6,13]. That even these low levels of expression are significant is clear from the fact that all canal sensory epithelia receive an innervation by both afferents and efferents at birth and later (Figs. 2, 3). The situation in the utricle, saccule and cochlea is less clear as both BDNF and NT-3 are expressed [6,7]. In fact, it appears that the levels of NT-3 expression hardly differ from those in control littermates (Figs. 8, 9). The latter is particularly obvious in the apex of the cochlea. In fact, in the cochlea of embryos and neonates as well as in the saccule and utricle of embryos, NT-3 is predominantly expressed in supporting cells [6]. This expression of NT-3 in supporting cells in embryos and neonates may even preserve in Brn3c null mutants the numerous spiral sensory neurons in the apex of 8-day old animals (Figs. 3, 4). + +Long term survival of apical sensory neurons is unrelated to differentiated hair cells and likely is independent of neurotrophins + +Past work has shown that BDNF and NT-3 appear to shift in their expression into inner hair cells around birth or are lost in the ear and other parts of the nervous system [6,10,20,23]. It has been suggested that other neurotrophins may come into play in the ear [11]. However, the apex of Brn3c null mutants, which retains most of the spiral neuron afferents at 6-month, shows no formation of mature hair cells at any time surveyed here (P0, P8, P10). Thus the factor(s) cannot be released by differentiated hair cells but there is an unexplored possibility of generalized expression of neurotrophins at low levels in the undifferentiated epithelia. However, known neurotrophins are largely absent in the adult cochleae [4] and significant amounts of neurotrophin expression appear to develop only in postnatal animals in the CNS [24]. Such expression in the CNS could possibly offset to some extent the peripheral loss of neutrophins in targeted null mutants. This would work only in cases in which the loss of primary neurons would not already be completed long before birth as is the case in the ear [6,13]. + +It therefore remains unclear what supports the many afferents in the apex of the Brn3c null mutants until 6 months of age or longer, unknown peripheral neurotrophic substances or neurotrophic support provided by the CNS. Comparable long term retention of apical spiral sensory neurons was described for the deaf white cat [25] and may also be the case in humans with congenital deafness. + +Growth of fibers to the cochlea does not require mature hair cells + +Formation of radial fibers that bring peripheral processes of spiral neurons to the organ of Corti seems to be rather normal, even in the apex of the cochlea which does not even develop immature hair cells recognizable by Myo VIIa. This does not preclude that even less differentiated hair cell precursors may form in the apex or that those precursors have died before birth. Interestingly, the growth to outer hair cells is most affected in Brn3c null mutant cochleae. Instead of extending radially through the tunnel of Corti to outer hair cells, afferents appear to stall and extend in longitudinal directions as inner spiral bundles (Figs. 3, 7). It has been shown that the differentiation of pillar cells depends on activation of Fgfr3 [26] by FGF's, probably Fgf8, a factor produced by developing and mature inner hair cells [18,27]. The formation of supporting cells also appears to depend on the proper expression of various bHLH factors such as Hes 1 and 5 which appear to be regulated by the Notch signaling pathway [17,28,29]. It is possible that the apparent inability of fibers to extend along outer hair cells is related to the lack of differentiation of supporting cells in the absence of mature hair cells. Probing for the proper expression of supporting cell specific markers in Brn3c null mutants is necessary to further evaluate these suggestions. + +Previous work has suggested that efferent fibers, derived from facial branchial motoneurons, follow afferents and for that reason reflect in detail all connectional deviations of afferents [16,30-32]. Our data agree with this scenario but extend it. Specifically, efferent fibers may suffer the same loss of their molecularly unknown neurotrophic support as do afferents and therefore show the same spatio-temporal profile of loss (Fig. 3). Alternatively, efferent fibers can sustain established connections only as long as afferent fibers are present. More information on the actual molecular support of efferent fibers in the ear is needed before those two possibilities can be distinguished. + +The onset of cochleotopic projections has been investigated in detail in only very few mammals [19]. These data suggest that a cochleotopic projection develops before onset of hearing. Our data show that at least a cochleotopic projection in the cochlear nerve and the cochlear nuclei develops even in Brn 3c null mutants (Fig. 6). Those data are also consistent with the formation of a crude cochleotopic projection in NT-3 null mutants [6,14]. Those mutants have a much more severely reduced density of innervation in the developing cochlea [6,14] than the reduction we found in Brn3c null mutants at P0. In this context, it would be interesting to see whether the recently generated transgenic mouse in which BDNF is expressed under NT-3 promoter control [33] could actually perform an even more pronounced rescue in a Brn3c null mutant background. + +Relevance of our findings to other mutations with hair cell loss + +Vahava et al. [12] and Frydman et al. [34] have demonstrated that mutation in a single human allele of Pou4f3 (Brn3c) results in a late onset of high frequency hearing loss. Thus far no data are available that correlate the late onset of hearing loss with morphological defects in the cochlea. In fact, an investigation of Brn3c heterozygotic mice showed no additional effect on hearing loss due to haploinsufficiency in these mice [35]. Our data on Brn3c null mouse mutants nevertheless suggest that the high to low frequency progressive hearing loss in humans may correlate with the unknown factor(s) that mediate apical spiral neuron survival into adulthood. Quantitative data on human Pou4f3 cochleae is needed to verify that spiral neuron loss is more pronounced in the basal, high frequency area of these hearing-deficient patients. Similar data are also needed for other congenitally deaf children [36] and in the deaf white cat [25]. Further work on the recently available mutant mice with specific absence of all hair cells [37], outer hair cells in the base [38] or apex [39] could further help to clarify the role of hair cells in forming and maintaining afferent and efferent innervation in these model mice. + +Conclusion + +The progressive loss of afferent and efferent innervation in Brn3c null mutants shows neither in spatial nor in temporal pattern a resemblance to losses reported in simple BDNF or NT-3 null mutations [6]. Null mutants of ear specific neurotrophins have completed the loss of sensory neurons before birth. In contrast, our results suggest a slow loss of afferent and efferent innervation between P0 and at least 6 months. This late sensory neuron loss is likely not related to known neurotrophin signaling which becomes reduced in neonatal wildtype animals. Other factors, such as GDNF [11,40] need to be investigated in their expression in these mutants and their functional role needs to be assessed, in particular in the cochlear apex. + +The long term retention of afferents in the cochlear apex in the absence of any apparent differentiation of hair cells raises hopes for cochlear implants in deaf-born children. Similar long term retention of apical afferents exists in the deaf white cat [25] and should be explored in other model systems with embryonic and neonatal hair cell loss [37,38]. + +Methods + +Breeding and genotyping + +Brn3c mice were bred as previously described [22]. Mice were genotyped and Brn3c null mutants were raised to a specific age (P0, N = 8; P7, N = 6; P8, N = 6; 6 months old, N = 2). The animals were deeply anesthetized with pentobarbital and transcardially perfused with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Ears were postfixed in the same fixative for at least one day prior to dissection. + +Tracing of nerve fibers + +DiI tracing from the brainstem was performed as previously described [41]. Small filter stripes soaked with saturated DiI were implanted into the efferent fiber bundle near the floor plate or into the ascending or descending afferents in the alar plate [42]. After appropriate diffusion time of the dye, the ears were dissected and mounted flat for visualization in an epifluorescent microscope. Images were taken on black and white film or acquired with a cooled CCD camera and processed using ImagePro software (Media Cybernetics). + +Ears were subsequently reacted for acetylated tubulin to reveal the pattern of innervation in addition to the DiI tracing with a different technique as previously described [14]. Briefly, dissected ears were incubated with 1:500 anti-acetylated tubulin antibodies (Sigma, St. Louis) followed by secondary antibodies conjugated to HRP. Dissected inner ears were reacted with DAB and H2O2 for HRP distribution and subsequently viewed as whole mounts. + +After photographing as whole mounts, the ears were embedded in epoxy resin and sectioned to reveal the distribution of fibers inside the sensory epithelia in more detail. + +Cochleotopic projection was evaluated by inserting DiI soaked filter strips into the base and DiA soaked filter strips into the apical turn of 2 P0 and 4 P8 Brn3c null mutants and a similar number of wildtype littermates. After appropriate diffusion time [43], the brains with the attached cochlear nerve were embedded in gelatin, hardened in 4% PFA over night, sectioned coronally on a vibratome (100 μm thickness) and viewed with a Biorad Radiance 2000 confocal system attached to a Nikon Eclipse 800 microscope. Image stacks were collapsed to view the entire projection in one section in one focal plane. + +In situ hybridization + +Four ears each of Brn3c null mutants (P0 and P8) and control littermates were embedded in paraffin, sectioned and probed for the presence of BDNF and NT-3 mRNA using the in situ technique previously described [7]. Sections were lightly counterstained and viewed with bright and dark field microscopy. Images were acquired with a CCD camera and displayed as false color images as previously described [18]. + +Immunocytochemistry of hair cells and quantification of hair cells and neurons + +Immunostaining of P7 cochlear whole mounts using the hair cell specific-myosin VII (MyoVIIa) antibody was performed as previously described [2]. Images of labeled organs of Corti were then acquired using a SPOT digital camera (Diagnostic Instruments Inc.) and the number of hair cells was scored from acquired images. To determine the number of hair cells or neurons in vestibular endorgans or sensory ganglia, serial sections were stained with Cresyl Violet and images were similarly acquired and scored. Every other section was scored for each sensory epithelium and every forth section was scored for each ganglion. Only neurons or cells with a clear nucleus and nucleoli were counted and 4–6 samples were counted for each epithelium and ganglion. All data were tested for significance using two-sample Student's t-test with unequal variances. + +Authors' contributions + +MX generated the null mutation, bred all the mice used for this study and generated the statistical data and the immunocytochemical data with MyoVII, AM conducted the tract tracing studies of cochlear afferents and helped with the histological analysis and assembly of image plates, UP carried out the in situ hybridization for neurotrophins and assembled the plates for these data, BF wrote the manuscript, carried out most of the neuronal tracing and immunocytochemistry for acetylated tubulin and data presentation. All authors read and approved the final manuscript. + +Acknowledgements + +Supported by the National Eye Institute (EY12020, MX), the March of Dimes Birth Defects Foundation (MX), the Egyptian Government (AM), the Juselius Foundation (UP), the NIDCD (2 P01 DC00215, BF; DC04594, M.X.), the Taub foundation (BF) and NASA (01-OBPR-06; BF). diff --git a/src/ontogpt/evaluation/craft/database/all/12925238.ann b/src/ontogpt/evaluation/craft/database/all/12925238.ann new file mode 100644 index 000000000..cc67abed1 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/12925238.ann @@ -0,0 +1,931 @@ +T1 GO:0010467 10 20 expression +T2 PR:000004080 41 51 annexin A7 +T3 CL:0000233 55 64 platelets +T4 CL:0000232 69 84 red blood cells +T5 UBERON:0000178 73 78 blood +T6 PR:000004080 111 121 annexin A7 +T7 NCBITaxon:10088 129 134 mouse +T8 PR:000004080 158 168 Annexin A7 +T9 CHEBI:29108 174 178 Ca2+ +T10 CHEBI:16247 184 196 phospholipid +T11 GO:0010467 213 222 expressed +T12 SO:0001060 242 249 isoform +T13 GO:0016020 286 294 membrane +T14 GO:0061025 286 301 membrane fusion +T15 SO:0001060 333 340 isoform +T16 CL:0000232 364 376 erythrocytes +T17 CHEBI:29108 443 447 Ca2+ +T18 GO:0017156 443 473 Ca2+-dependent vesicle release +T19 GO:0031982 458 465 vesicle +T20 CL:0000232 496 511 red blood cells +T21 UBERON:0000178 500 505 blood +T22 PR:000004080 615 625 annexin A7 +T23 CL:0000232 629 644 red blood cells +T24 UBERON:0000178 633 638 blood +T25 CL:0000232 662 674 erythrocytes +T26 PR:000004080 680 685 anxA7 +T27 NCBITaxon:10088 689 693 mice +T28 CHEBI:29108 714 718 Ca2+ +T29 GO:0006900 728 740 vesiculation +T30 GO:0016020 777 785 membrane +T31 PR:000004080 895 905 annexin A7 +T32 CL:0000232 971 986 red blood cells +T33 UBERON:0000178 975 980 blood +T34 PR:000004080 988 998 Annexin A7 +T35 CL:0000233 1022 1031 platelets +T36 CL:0000233 1159 1168 platelets +T37 PR:000004080 1222 1232 annexin A7 +T38 GO:0016020 1236 1244 membrane +T39 GO:0061025 1236 1251 membrane fusion +T40 CL:0000232 1275 1290 red blood cells +T41 UBERON:0000178 1279 1284 blood +T42 GO:0016020 1357 1365 membrane +T43 GO:0005856 1366 1378 cytoskeleton +T44 CL:0000232 1380 1395 Red blood cells +T45 UBERON:0000178 1384 1389 blood +T46 PR:000004080 1452 1462 annexin A7 +T47 GO:0009987 1466 1484 cellular processes +T48 PR:000004080 1541 1551 annexin A7 +T49 SO:0001060 1552 1560 isoforms +T50 CL:0000232 1564 1579 red blood cells +T51 UBERON:0000178 1568 1573 blood +T52 SO:0001060 1610 1617 isoform +T53 CL:0000233 1621 1630 platelets +T54 PR:000004080 1663 1673 annexin A7 +T55 PR:000004080 1774 1784 annexin A7 +T56 GO:0016020 1788 1796 membrane +T57 GO:0061025 1788 1803 membrane fusion +T58 PR:000004080 1848 1858 Annexin A7 +T59 CHEBI:29108 1864 1868 Ca2+ +T60 CHEBI:16247 1874 1886 phospholipid +T61 GO:0042583 1965 1984 chromaffin granules +T62 GO:0061025 1989 1998;2012 2021 fusion of ... membranes +T63 CHEBI:16247 1999 2011 phospholipid +T64 GO:0016020 2012 2021 membranes +T65 CHEBI:29108 2041 2045 Ca2+ +T66 PR:000004080 2084 2094 annexin A7 +T67 GO:0006887 2116 2126 exocytotic +T68 CHEBI:33567 2140 2154 catecholamines +T69 SO:0000417 2308 2314 domain +T70 SO:0000417 2331 2337 domain +T71 CHEBI:29108 2361 2365 Ca2+ +T72 CHEBI:16247 2371 2383 phospholipid +T73 SO:0000417 2408 2414 domain +T74 CHEBI:62643 2476 2508 negatively charged phospholipids +T75 CHEBI:29108 2538 2547 Ca2+ ions +T76 PR:000004080 2550 2560 Annexin A7 +T77 GO:0000380 2677 2697 Alternative splicing +T78 SO:0001060 2716 2724 isoforms +T79 SO:0001060 2748 2756 isoforms +T80 SO:0000147 2790 2794 exon +T81 SO:0000417 2840 2846 domain +T82 UBERON:0000479 2853 2860 tissues +T83 SO:0001060 2884 2891 isoform +T84 UBERON:0000955 2917 2922 brain +T85 UBERON:0000948 2927 2932 heart +T86 SO:0001060 2950 2957 isoform +T87 GO:0010467 2973 2982 expressed +T88 UBERON:0004288 2993 3001 skeletal +T89 PR:000004080 3033 3043 annexin A7 +T90 GO:0005829 3067 3074 cytosol +T91 GO:0005886 3083 3098 plasma membrane +T92 GO:0005634 3111 3118 nucleus +T93 GO:0031982 3123 3132 vesicular +T94 UBERON:0002369 3154 3161 adrenal +T95 GO:0042583 3162 3181 chromaffin granules +T96 GO:0030315 3194 3202 t-tubule +T97 PR:000004080 3217 3227 Annexin A7 +T98 GO:0016020 3244 3253 membranes +T99 CHEBI:29108 3259 3263 Ca2+ +T100 GO:0005622 3292 3305 intracellular +T101 CHEBI:29108 3306 3310 Ca2+ +T102 GO:0005886 3358 3364;3381 3389 plasma ... membrane +T103 GO:0031965 3373 3389 nuclear membrane +T104 GO:0005622 3404 3417 intracellular +T105 GO:0031982 3418 3426 vesicles +T106 PR:000004080 3441 3451 annexin A7 +T107 CHEBI:18059 3468 3473 lipid +T108 GO:0045121 3468 3479 lipid rafts +T109 CHEBI:18059 3485 3490 Lipid +T110 GO:0045121 3485 3496 Lipid rafts +T111 GO:0016020 3516 3524 membrane +T112 GO:0006900 3516 3532 membrane budding +T113 GO:0006900 3540 3552 vesiculation +T114 GO:0006887 3581 3591 exocytosis +T115 PR:000004080 3625 3635 annexin A7 +T116 PR:000015619 3658 3664 sorcin +T117 PR:000009771 3669 3679 galectin-3 +T118 PR:000015619 3689 3695 Sorcin +T119 CHEBI:29108 3701 3705 Ca2+ +T120 PR:000004080 3781 3791 annexin A7 +T121 GO:0016020 3804 3813 membranes +T122 CHEBI:29108 3819 3823 Ca2+ +T123 PR:000015619 3842 3848 Sorcin +T124 CHEBI:8925 3903 3912 ryanodine +T125 GO:0065007 3938 3946 modulate +T126 PR:000015619 3987 3993 sorcin +T127 PR:000004080 3994 4004 annexin A7 +T128 CHEBI:8925 4024 4033 ryanodine +T129 PR:000015619 4055 4061 Sorcin +T130 PR:000004080 4066 4076 annexin A7 +T131 GO:0010467 4083 4092 expressed +T132 UBERON:0000479 4100 4107 tissues +T133 PR:000004080 4145 4155 annexin A7 +T134 PR:000015619 4160 4166 sorcin +T135 CHEBI:29108 4170 4174 Ca2+ +T136 CHEBI:29108 4210 4214 Ca2+ +T137 SO:0000409 4235 4248 binding sites +T138 PR:000015619 4315 4321 sorcin +T139 PR:000004080 4369 4379 annexin A7 +T140 PR:000015619 4441 4447 sorcin +T141 CHEBI:36357 4448 4457 molecules +T142 PR:000004080 4462 4472 annexin A7 +T143 CHEBI:36357 4473 4481 molecule +T144 PR:000009771 4489 4499 Galectin-3 +T145 GO:0005615 4584 4603 extracellular space +T146 GO:0005634 4612 4619 nucleus +T147 GO:0005737 4624 4633 cytoplasm +T148 GO:0005739 4641 4653 mitochondria +T149 GO:0005737 4655 4666 Cytoplasmic +T150 PR:000009771 4667 4677 galectin-3 +T151 http://purl.obolibrary.org/obo/MONDO_0005070 4694 4699 tumor +T152 GO:0005739 4725 4738 mitochondrial +T153 PR:000004080 4769 4779 annexin A7 +T154 PR:000009771 4789 4799 galectin-3 +T155 GO:0005640 4821 4841 perinuclear membrane +T156 PR:000009771 4856 4866 galectin-3 +T157 PR:000004080 4882 4892 annexin A7 +T158 PR:000009771 4904 4914 galectin-3 +T159 GO:0042981 4928 4948 apoptosis regulation +T160 GO:0005739 4954 4967 mitochondrial +T161 PR:000004080 5019 5029 annexin A7 +T162 GO:0065007 5071 5079 regulate +T163 GO:0044425 5094 5110 membrane domains +T164 CHEBI:29108 5133 5137 Ca2+ +T165 GO:0042592 5138 5149 homeostasis +T166 CHEBI:29108 5154 5158 Ca2+ +T167 GO:0019722 5154 5187 Ca2+-dependent signaling pathways +T168 PR:000004080 5253 5263 annexin A7 +T169 NCBITaxon:10088 5274 5279 mouse +T170 PR:000004080 5293 5303 annexin A7 +T171 NCBITaxon:10088 5315 5319 mice +T172 NCBITaxon:10088 5443 5447 mice +T173 PR:000045358 5459 5466 insulin +T174 http://purl.obolibrary.org/obo/MONDO_0005070 5488 5493 tumor +T175 PR:000045358 5584 5591 insulin +T176 CL:0000746 5656 5670 cardiomyocytes +T177 CL:0000232 5758 5773 red blood cells +T178 UBERON:0000178 5762 5767 blood +T179 CL:0000233 5778 5787 platelets +T180 PR:000004080 5795 5800 anxA7 +T181 CL:0000232 5823 5838 red blood cells +T182 UBERON:0000178 5827 5832 blood +T183 CL:0000233 5843 5852 platelets +T184 PR:000004080 5881 5891 annexin A7 +T185 SO:0001060 5921 5928 isoform +T186 CL:0000232 5963 5978 red blood cells +T187 UBERON:0000178 5967 5972 blood +T188 CL:0000232 5985 6000 Red blood cells +T189 UBERON:0000178 5989 5994 blood +T190 CHEBI:29108 6072 6076 Ca2+ +T191 GO:0006887 6125 6135;6158 6169 release of ... exovesicles +T192 GO:0070382 6158 6169 exovesicles +T193 CL:0000232 6280 6294 red blood cell +T194 UBERON:0000178 6284 6289 blood +T195 CL:0000232 6351 6366 Red blood cells +T196 UBERON:0000178 6355 6360 blood +T197 GO:0006900 6393 6403 vesiculate +T198 http://purl.obolibrary.org/obo/MONDO_0000001 6412 6419 disease +T199 CL:0000232 6425 6439 red blood cell +T200 UBERON:0000178 6429 6434 blood +T201 GO:0008219 6435 6451 cell destruction +T202 http://purl.obolibrary.org/obo/MONDO_0003656 6456 6471 haemoglobinuria +T203 GO:0031982 6503 6511 vesicles +T204 GO:1990742 6513 6519;6528 6536 micro- ... vesicles +T205 CHEBI:16113 6605 6616 cholesterol +T206 CHEBI:26739 6621 6633 sphingolipid +T207 CHEBI:18059 6639 6644 lipid +T208 GO:0045121 6639 6657 lipid raft domains +T209 GO:0009986 6719 6731 cell surface +T210 CHEBI:18059 6782 6787 lipid +T211 GO:0045121 6782 6792 lipid raft +T212 PR:000015763 6802 6810 stomatin +T213 GO:0005856 6844 6856 cytoskeletal +T214 PR:000015619 6896 6902 sorcin +T215 PR:000004080 6907 6917 annexin A7 +T216 CHEBI:18059 6934 6939 lipid +T217 GO:0045121 6934 6945 lipid rafts +T218 GO:0031982 6986 6994 vesicles +T219 GO:0031982 7000 7007 vesicle +T220 GO:0006900 7000 7017 vesicle formation +T221 CL:0000232 7063 7077 red blood cell +T222 UBERON:0000178 7067 7072 blood +T223 GO:0005856 7083 7095 cytoskeletal +T224 CHEBI:16247 7130 7142 phospholipid +T225 GO:0005886 7162 7179 cellular membrane +T226 PR:000004080 7191 7201 Annexin A7 +T227 GO:0010467 7202 7212 expression +T228 CL:0000232 7216 7231 red blood cells +T229 UBERON:0000178 7220 7225 blood +T230 CL:0000232 7233 7247 red blood cell +T231 UBERON:0000178 7237 7242 blood +T232 GO:0070382 7256 7267 exovesicles +T233 CL:0000233 7275 7284 platelets +T234 SO:0001060 7345 7352 isoform +T235 PR:000004080 7356 7366 annexin A7 +T236 PR:000015619 7383 7389 sorcin +T237 GO:1990742 7393 7399;7408 7416 micro- ... vesicles +T238 CL:0000232 7430 7445 red blood cells +T239 UBERON:0000178 7434 7439 blood +T240 GO:0045121 7507 7521 membrane rafts +T241 SO:0001060 7593 7600 isoform +T242 NCBITaxon:9606 7641 7646 human +T243 CL:0000232 7647 7662 red blood cells +T244 UBERON:0000178 7651 7656 blood +T245 SO:0001060 7681 7689 isoforms +T246 SO:0001060 7743 7750 isoform +T247 PR:000004080 7809 7819 annexin A7 +T248 SO:0001060 7890 7897 isoform +T249 GO:0010467 7945 7955 Expression +T250 PR:000004080 7959 7969 annexin A7 +T251 NCBITaxon:9606 7973 7978 human +T252 CL:0000232 7979 7994 red blood cells +T253 UBERON:0000178 7983 7988 blood +T254 NCBITaxon:9606 8003 8008 human +T255 CL:0000233 8009 8018 platelets +T256 CHEBI:8984 8062 8065 SDS +T257 CHEBI:28619 8077 8087 acrylamide +T258 MOP:0000779 8190 8197 coupled +T259 GO:0042571 8198 8206 antibody +T260 SO:0001060 8229 8237 isoforms +T261 CL:0000232 8254 8269 red blood cells +T262 UBERON:0000178 8258 8263 blood +T263 CL:0000233 8274 8283 platelets +T264 SO:0001060 8299 8306 isoform +T265 GO:0031982 8336 8344 vesicles +T266 GO:0045121 8385 8397 raft domains +T267 PR:000004080 8399 8409 annexin A7 +T268 GO:0016020 8426 8434 membrane +T269 GO:0006900 8456 8468 vesiculation +T270 CL:0000232 8522 8537 red blood cells +T271 UBERON:0000178 8526 8531 blood +T272 PR:000004080 8555 8565 annexin A7 +T273 NCBITaxon:10088 8576 8580 mice +T274 PR:000004080 8582 8587 anxA7 +T275 GO:0070382 8600 8611 exovesicles +T276 NCBITaxon:9606 8736 8741 human +T277 UBERON:0000178 8742 8747 blood +T278 PR:000004080 8780 8790 annexin A7 +T279 GO:0070382 8805 8816 exovesicles +T280 GO:1990742 8818 8824;8833 8841 micro- ... vesicles +T281 CHEBI:29108 8863 8867 Ca2+ +T282 CHEBI:24869 8868 8877 ionophore +T283 CHEBI:15355 8905 8918 acetylcholine +T284 PR:000004080 8966 8976 Annexin A7 +T285 GO:0031982 8996 9003 vesicle +T286 GO:0031982 9043 9051 vesicles +T287 SO:0001060 9073 9080 isoform +T288 GO:0031982 9107 9115 vesicles +T289 GO:0031982 9138 9145 vesicle +T290 CL:0000232 9199 9213 red blood cell +T291 UBERON:0000178 9203 9208 blood +T292 GO:0031982 9214 9222 vesicles +T293 GO:0031982 9266 9274 vesicles +T294 GO:1990742 9288 9301 microvesicles +T295 PR:000004080 9347 9357 annexin A7 +T296 GO:0016020 9398 9407 membranes +T297 CL:0000232 9467 9481 red blood cell +T298 UBERON:0000178 9471 9476 blood +T299 GO:0070382 9482 9493 exovesicles +T300 PR:000015619 9530 9536 sorcin +T301 GO:0031982 9561 9569 vesicles +T302 PR:000015619 9588 9594 sorcin +T303 GO:0031982 9633 9641 vesicles +T304 PR:000004080 9677 9687 annexin A7 +T305 PR:000015619 9692 9698 sorcin +T306 GO:0070382 9702 9713 exovesicles +T307 CL:0000232 9727 9742 red blood cells +T308 UBERON:0000178 9731 9736 blood +T309 GO:0031982 9744 9752 Vesicles +T310 PR:000004080 9777 9782 anxA7 +T311 CHEBI:29108 9818 9822 Ca2+ +T312 CHEBI:24869 9823 9832 ionophore +T313 PR:000015619 9938 9944 sorcin +T314 GO:0042571 9956 9964 antibody +T315 PR:000004080 9978 9988 annexin A7 +T316 PR:000015619 9993 9999 sorcin +T317 GO:0031982 10025 10033 vesicles +T318 SO:0001060 10046 10053 isoform +T319 PR:000004080 10057 10067 annexin A7 +T320 GO:0031982 10101 10109 vesicles +T321 GO:0031982 10127 10135 vesicles +T322 CHEBI:15355 10177 10190 acetylcholine +T323 NCBITaxon:9606 10210 10215 Human +T324 CL:0000232 10216 10231 red blood cells +T325 UBERON:0000178 10220 10225 blood +T326 PR:000004080 10319 10329 annexin A7 +T327 CL:0000232 10333 10348 red blood cells +T328 UBERON:0000178 10337 10342 blood +T329 CHEBI:31705 10370 10379 iodixanol +T330 SO:0001060 10404 10412 isoforms +T331 GO:0016020 10532 10541 membranes +T332 GO:0016020 10549 10558 membranes +T333 GO:0005886 10575 10591 plasma membranes +T334 CL:0000232 10595 10610 red blood cells +T335 UBERON:0000178 10599 10604 blood +T336 GO:0043226 10627 10637 organelles +T337 PR:000004080 10743 10753 annexin A7 +T338 GO:0044425 10757 10776 membrane subdomains +T339 CHEBI:18059 10797 10802 lipid +T340 CHEBI:18059 10806 10811 lipid +T341 PR:000015619 10877 10883 sorcin +T342 GO:0031982 10974 10982 vesicles +T343 PR:000004080 11013 11023 annexin A7 +T344 CL:0000233 11049 11058 platelets +T345 PR:000004080 11063 11073 annexin A7 +T346 GO:0010467 11074 11084 expression +T347 SO:0001060 11109 11116 isoform +T348 PR:000004080 11153 11163 annexin A7 +T349 PR:000015619 11168 11174 sorcin +T350 CL:0000232 11189 11203 red blood cell +T351 UBERON:0000178 11193 11198 blood +T352 GO:0005886 11204 11219 plasma membrane +T353 GO:0019835 11231 11236 Lysed +T354 NCBITaxon:9606 11237 11242 human +T355 CL:0000232 11243 11258 red blood cells +T356 UBERON:0000178 11247 11252 blood +T357 CHEBI:31705 11288 11297 iodixanol +T358 PR:000015619 11466 11472 sorcin +T359 GO:0042571 11484 11492 antibody +T360 PR:000004080 11507 11517 annexin A7 +T361 CL:0000232 11526 11540 red blood cell +T362 UBERON:0000178 11530 11535 blood +T363 PR:000004080 11579 11589 annexin A7 +T364 CL:0000232 11608 11622 red blood cell +T365 UBERON:0000178 11612 11617 blood +T366 GO:0031982 11623 11631 vesicles +T367 PR:000004080 11694 11699 anxA7 +T368 NCBITaxon:10088 11702 11707 mouse +T369 CL:0000232 11712 11726 red blood cell +T370 UBERON:0000178 11716 11721 blood +T371 UBERON:0000178 11777 11782 blood +T372 PR:000004080 11910 11915 anxA7 +T373 CL:0000232 11918 11933 red blood cells +T374 UBERON:0000178 11922 11927 blood +T375 CL:0000232 12280 12295 red blood cells +T376 UBERON:0000178 12284 12289 blood +T377 PR:000004080 12301 12306 anxA7 +T378 NCBITaxon:10088 12335 12339 mice +T379 CL:0000232 12513 12527 red blood cell +T380 UBERON:0000178 12517 12522 blood +T381 UBERON:0001982 12567 12576 capillary +T382 GO:0016020 12657 12665 membrane +T383 CHEBI:18059 12666 12671 lipid +T384 GO:0016020 12695 12705 membranous +T385 GO:0005856 12706 12718 cytoskeleton +T386 PR:000004080 12730 12740 annexin A7 +T387 GO:0005576 12831 12844 extracellular +T388 CHEBI:24870 12845 12850 ionic +T389 CL:0000232 12903 12917 red blood cell +T390 UBERON:0000178 12907 12912 blood +T391 CHEBI:26710 12951 12966 sodium chloride +T392 GO:0019835 12990 13004 cellular lysis +T393 GO:0019835 13050 13055;13077 13079;13084 13089 lysis ... of ... cells +T394 GO:0009986 13149 13156 surface +T395 GO:0044237 13185 13204 cellular metabolism +T396 GO:0005622 13221 13234 intracellular +T397 CHEBI:24870 13235 13238 ion +T398 CL:0000232 13252 13267 red blood cells +T399 UBERON:0000178 13256 13261 blood +T400 CL:0000232 13360 13375 red blood cells +T401 UBERON:0000178 13364 13369 blood +T402 GO:0016020 13389 13397 membrane +T403 PR:000004080 13438 13448 annexin A7 +T404 CL:0000232 13459 13474 red blood cells +T405 UBERON:0000178 13463 13468 blood +T406 CHEBI:26710 13603 13607 NaCl +T407 CHEBI:75958 13608 13616 solution +T408 CHEBI:26710 13691 13695 NaCl +T409 CHEBI:75958 13696 13704 solution +T410 CHEBI:26710 13734 13738 NaCl +T411 CHEBI:26710 13890 13894 NaCl +T412 CHEBI:75958 13895 13903 solution +T413 CL:0000232 13951 13965 red blood cell +T414 UBERON:0000178 13955 13960 blood +T415 GO:0005886 13961 13974 cell membrane +T416 PR:000004080 14033 14043 annexin A7 +T417 GO:0016020 14051 14059 membrane +T418 CL:0000232 14157 14172 red blood cells +T419 UBERON:0000178 14161 14166 blood +T420 GO:0016020 14279 14287 membrane +T421 GO:0019835 14301 14306 lysis +T422 CL:0000232 14546 14561 red blood cells +T423 UBERON:0000178 14550 14555 blood +T424 CL:0000232 14603 14618 red blood cells +T425 UBERON:0000178 14607 14612 blood +T426 PR:000004080 14638 14643 anxA7 +T427 NCBITaxon:10088 14654 14658 mice +T428 GO:0005576 14706 14719 extracellular +T429 CHEBI:24870 14720 14725 ionic +T430 CL:0000232 14777 14791 red blood cell +T431 UBERON:0000178 14781 14786 blood +T432 CHEBI:26710 14823 14838 sodium chloride +T433 GO:0019835 14862 14876 cellular lysis +T434 GO:0019835 14922 14927;14949 14951;14964 14968 lysis ... of ... cell +T435 CL:0000232 14954 14968 red blood cell +T436 UBERON:0000178 14958 14963 blood +T437 PR:000004080 14996 15006 annexin A7 +T438 CL:0000232 15017 15032 red blood cells +T439 UBERON:0000178 15021 15026 blood +T440 PR:000004080 15140 15150 annexin A7 +T441 CL:0000232 15179 15194 red blood cells +T442 UBERON:0000178 15183 15188 blood +T443 PR:000004080 15260 15270 annexin A7 +T444 GO:0031941 15274 15281 F-actin +T445 GO:0016020 15342 15350 membrane +T446 CHEBI:24870 15384 15387 ion +T447 GO:0050801 15384 15399 ion homeostasis +T448 PR:000004080 15414 15424 annexin A7 +T449 GO:0007599 15441 15452 haemostasis +T450 NCBITaxon:39107 15605 15611 murine +T451 UBERON:0000178 15612 15617 blood +T452 CL:0000738 15638 15648 leukocytes +T453 CL:0000232 15650 15665 red blood cells +T454 UBERON:0000178 15654 15659 blood +T455 CL:0000233 15791 15800 platelets +T456 CL:0000775 15802 15814 neutrophiles +T457 CL:0000542 15816 15827 lymphocytes +T458 CL:0000576 15829 15838 monocytes +T459 CL:0000771 15840 15852 eosinophiles +T460 CL:0000767 15857 15867 basophiles +T461 UBERON:0000178 15893 15898 blood +T462 CL:0000233 15929 15937 platelet +T463 UBERON:0000178 15965 15979 haematological +T464 CL:0000233 16019 16027 platelet +T465 PR:000004080 16054 16059 anxA7 +T466 NCBITaxon:10088 16070 16074 mice +T467 CL:0000233 16142 16150 platelet +T468 NCBITaxon:10088 16171 16175 mice +T469 CL:0000233 16266 16274 platelet +T470 NCBITaxon:9606 16316 16322 humans +T471 CL:0000233 16324 16332 platelet +T472 http://purl.obolibrary.org/obo/MONDO_0024518 16381 16404 reactive thrombocytosis +T473 http://purl.obolibrary.org/obo/MONDO_0023370 16468 16487 neoplastic diseases +T474 PR:000004080 16506 16511 anxA7 +T475 CL:0000233 16567 16575 platelet +T476 CL:0000233 16630 16638 platelet +T477 UBERON:0001969 16644 16650 plasma +T478 CL:0000233 16652 16660 Platelet +T479 GO:0030168 16652 16671 Platelet activation +T480 GO:0031143 16775 16785 pseudopods +T481 GO:0005856 16824 16836 cytoskeletal +T482 CHEBI:75958 16933 16941 solution +T483 CL:0000233 17039 17047 platelet +T484 GO:0030168 17039 17058 platelet activation +T485 CHEBI:85129 17062 17072 ristocetin +T486 CHEBI:23357 17098 17106 cofactor +T487 CL:0000233 17196 17204 platelet +T488 GO:0030168 17196 17215 platelet initiation +T489 PR:000004080 17274 17284 Annexin A7 +T490 CL:0000233 17295 17304 platelets +T491 NCBITaxon:9606 17396 17401 human +T492 CL:0000233 17402 17411 platelets +T493 NCBITaxon:1 17464 17474 individual +T494 PR:000017364 17482 17503 von Willebrand factor +T495 NCBITaxon:10088 17625 17630 mouse +T496 CL:0000233 17631 17640 platelets +T497 PR:000004080 17645 17650 anxA7 +T498 CL:0000233 17653 17662 platelets +T499 NCBITaxon:39107 17690 17696 murine +T500 CL:0000233 17697 17706 platelets +T501 NCBITaxon:9606 17764 17769 human +T502 CL:0000233 17770 17779 platelets +T503 CL:0000233 17814 17822 Platelet +T504 GO:0070527 17814 17834 Platelet aggregation +T505 CL:0000233 17847 17855 platelet +T506 UBERON:0001969 17861 17867 plasma +T507 PR:000004080 17871 17881 annexin A7 +T508 NCBITaxon:10088 17906 17910 mice +T509 CHEBI:85129 18015 18025 ristocetin +T510 CL:0000233 18072 18080 platelet +T511 NCBITaxon:39107 18264 18270 murine +T512 CL:0000233 18338 18346 platelet +T513 GO:0030168 18338 18357 platelet initiation +T514 NCBITaxon:39107 18635 18641 Murine +T515 CL:0000233 18642 18651 platelets +T516 NCBITaxon:9606 18735 18740 human +T517 CL:0000233 18741 18750 platelets +T518 CL:0000233 18787 18795 platelet +T519 NCBITaxon:9606 18825 18830 human +T520 CL:0000233 18831 18840 platelets +T521 PR:000004080 18899 18909 Annexin A7 +T522 GO:0031982 18939 18946 vesicle +T523 GO:0006906 18939 18953 vesicle fusion +T524 GO:0065007 18961 18971 regulating +T525 GO:0044425 18988 19004 membrane domains +T526 GO:0005886 19048 19063 plasma membrane +T527 GO:0045121 19074 19089 raft subdomains +T528 GO:0005829 19124 19131 cytosol +T529 GO:0031982 19165 19172 vesicle +T530 UBERON:0000479 19241 19247 tissue +T531 GO:0010467 19322 19332 expression +T532 SO:0001060 19386 19394 isoforms +T533 GO:0010467 19404 19413 expressed +T534 CL:0000232 19426 19441 Red blood cells +T535 UBERON:0000178 19430 19435 blood +T536 PR:000004080 19478 19488 annexin A7 +T537 SO:0001060 19531 19538 isoform +T538 PR:000015619 19573 19579 sorcin +T539 SO:0001060 19622 19629 isoform +T540 CL:0000232 19646 19661 red blood cells +T541 UBERON:0000178 19650 19655 blood +T542 SO:0001060 19700 19707 isoform +T543 PR:000004080 19765 19775 annexin A7 +T544 CL:0000232 19779 19794 red blood cells +T545 UBERON:0000178 19783 19788 blood +T546 PR:000004080 19818 19828 annexin A7 +T547 PR:000015619 19833 19839 sorcin +T548 GO:0045121 19857 19878 membrane raft domains +T549 GO:0031982 19886 19894 vesicles +T550 CL:0000232 19907 19922 red blood cells +T551 UBERON:0000178 19911 19916 blood +T552 GO:0045121 19933 19945 Raft domains +T553 GO:0016020 19987 19995 membrane +T554 GO:0006900 20030 20042 vesiculation +T555 CHEBI:26739 20087 20100 sphingolipids +T556 CHEBI:16113 20105 20116 cholesterol +T557 GO:0097478 20130 20140;20145 20153 leaflet of ... membrane +T558 CHEBI:16247 20167 20180 phospholipids +T559 CHEBI:16113 20185 20196 cholesterol +T560 GO:0097478 20210 20217 leaflet +T561 GO:0045121 20270 20275 rafts +T562 GO:0016020 20291 20300 membranes +T563 CHEBI:9750 20342 20354 Triton X-100 +T564 CHEBI:17992 20394 20401 sucrose +T565 PR:000004080 20473 20483 annexin A7 +T566 GO:0016020 20493 20501 membrane +T567 PR:000004080 20534 20544 annexin A7 +T568 GO:0006900 20578 20590 vesiculation +T569 GO:0006900 20646 20658 vesiculation +T570 PR:000004080 20692 20702 annexin A7 +T571 CHEBI:35195 20778 20788 surfactant +T572 PR:000004080 20792 20802 annexin A7 +T573 PR:000004080 20860 20870 annexin A7 +T574 CHEBI:35195 20874 20884 surfactant +T575 PR:000004080 20917 20927 annexin A7 +T576 GO:0016020 20937 20945 membrane +T577 GO:0061025 20937 20952 membrane fusion +T578 PR:000004080 21022 21032 annexin A7 +T579 NCBITaxon:10088 21042 21047 mouse +T580 PR:000004080 21077 21087 annexin A7 +T581 GO:0006900 21091 21101;21117 21125 budding of ... vesicles +T582 CL:0000232 21102 21116 red blood cell +T583 UBERON:0000178 21106 21111 blood +T584 GO:0031982 21117 21125 vesicles +T585 GO:1990742 21167 21173;21182 21189 micro- ... vesicle +T586 CL:0000232 21255 21270 red blood cells +T587 UBERON:0000178 21259 21264 blood +T588 NCBITaxon:44689 21282 21306 Dictyostelium discoideum +T589 SO:0000704 21342 21346 gene +T590 GO:0016020 21405 21413 membrane +T591 GO:0061025 21405 21420 membrane fusion +T592 CHEBI:16247 21496 21508 phospholipid +T593 PR:000004080 21519 21529 annexin A7 +T594 CHEBI:29108 21554 21558 Ca2+ +T595 PR:000004080 21646 21656 annexin A7 +T596 GO:1990742 21660 21666;21675 21683 micro- ... vesicles +T597 CL:0000232 21739 21753 red blood cell +T598 UBERON:0000178 21743 21748 blood +T599 GO:0005886 21749 21762 cell membrane +T600 PR:000004080 21775 21785 annexin A7 +T601 CHEBI:29108 21806 21810 Ca2+ +T602 GO:0070382 21867 21878 exovesicles +T603 PR:000004080 21918 21928 annexin A7 +T604 SO:0001060 21929 21937 isoforms +T605 CL:0000232 21954 21969 red blood cells +T606 UBERON:0000178 21958 21963 blood +T607 GO:0016020 22033 22042 membranes +T608 GO:0016020 22046 22054 membrane +T609 PR:000004075 22073 22083 annexin A2 +T610 GO:0005768 22111 22120 endosomes +T611 GO:0016020 22147 22155 membrane +T612 GO:0061025 22147 22162 membrane fusion +T613 GO:0005768 22171 22180 endosomal +T614 PR:000004080 22190 22200 annexin A7 +T615 GO:0044853 22218 22246 plasma membrane raft domains +T616 GO:0031982 22299 22307 vesicles +T617 CL:0000232 22311 22326 red blood cells +T618 UBERON:0000178 22315 22320 blood +T619 PR:000004080 22335 22345 annexin A7 +T620 PR:000015619 22360 22366 sorcin +T621 PR:000004080 22439 22449 annexin A7 +T622 GO:0010467 22457 22467 expression +T623 CL:0000232 22475 22489 red blood cell +T624 GO:0048821 22475 22501 red blood cell development +T625 UBERON:0000178 22479 22484 blood +T626 GO:0031982 22549 22556 vesicle +T627 GO:0006900 22549 22566 vesicle formation +T628 GO:0016020 22571 22579 membrane +T629 GO:0061025 22571 22586 membrane fusion +T630 PR:000004080 22588 22598 annexin A7 +T631 CHEBI:29108 22628 22632 Ca2+ +T632 PR:000004080 22681 22691 annexin A7 +T633 GO:0016020 22720 22728 membrane +T634 PR:000004080 22788 22798 annexin A7 +T635 CHEBI:29108 22836 22840 Ca2+ +T636 GO:0042592 22841 22852 homeostasis +T637 GO:0016020 22870 22878 membrane +T638 PR:000004080 23112 23122 annexin A7 +T639 CL:0000232 23147 23162 red blood cells +T640 UBERON:0000178 23151 23156 blood +T641 CL:0000232 23206 23221 red blood cells +T642 UBERON:0000178 23210 23215 blood +T643 GO:0005886 23279 23294 plasma membrane +T644 GO:0016020 23303 23311 membrane +T645 GO:0044430 23323 23330;23335 23347 part of ... cytoskeleton +T646 GO:0016020 23383 23391 membrane +T647 GO:0005856 23392 23400 skeleton +T648 GO:0005884 23449 23464 actin filaments +T649 GO:0009898 23512 23534;23549 23562 cytoplasmic surface of ... cell membrane +T650 CL:0000232 23539 23553 red blood cell +T651 UBERON:0000178 23543 23548 blood +T652 GO:0016020 23569 23577 Membrane +T653 CHEBI:16113 23578 23589 cholesterol +T654 CL:0000232 23601 23615 red blood cell +T655 UBERON:0000178 23605 23610 blood +T656 GO:0005579 23634 23652 complement complex +T657 GO:0045121 23675 23679 raft +T658 CHEBI:16113 23680 23691 cholesterol +T659 GO:0045121 23721 23725 raft +T660 GO:0005886 23790 23803 cell membrane +T661 PR:000004080 23832 23842 Annexin A7 +T662 NCBITaxon:10088 23853 23858 mouse +T663 CL:0000232 23859 23874 red blood cells +T664 UBERON:0000178 23863 23868 blood +T665 PR:000004080 23952 23962 annexin A7 +T666 GO:0016020 23977 23985 membrane +T667 GO:0016020 24040 24048 membrane +T668 PR:000004080 24104 24114 Annexin A7 +T669 GO:0005886 24155 24170 plasma membrane +T670 CHEBI:29108 24244 24248 Ca2+ +T671 GO:0016020 24253 24261 membrane +T672 GO:0005856 24262 24270 skeletal +T673 CL:0000232 24315 24329 red blood cell +T674 UBERON:0000178 24319 24324 blood +T675 GO:0005886 24325 24338 cell membrane +T676 GO:0031941 24413 24420 F-actin +T677 PR:000004080 24468 24478 annexin A7 +T678 GO:0016020 24567 24575 membrane +T679 GO:0005856 24576 24588 cytoskeleton +T680 GO:0065007 24590 24601 controlling +T681 GO:0045121 24602 24606 raft +T682 CHEBI:24870 24647 24652 ionic +T683 CL:0000232 24665 24680 red blood cells +T684 UBERON:0000178 24669 24674 blood +T685 PR:000004080 24761 24771 annexin A7 +T686 GO:0005737 24797 24808 cytoplasmic +T687 CHEBI:29108 24809 24813 Ca2+ +T688 GO:0042592 24814 24825 homeostasis +T689 GO:0005622 24883 24896 intracellular +T690 CHEBI:29108 24897 24901 Ca2+ +T691 GO:0016020 24947 24955 membrane +T692 GO:0065007 24972 24980 regulate +T693 CL:0000232 24981 24995 red blood cell +T694 UBERON:0000178 24985 24990 blood +T695 GO:0005622 25036 25049 intracellular +T696 CHEBI:29108 25050 25054 Ca2+ +T697 GO:0065007 25068 25076 regulate +T698 GO:0016020 25081 25089 membrane +T699 GO:0065007 25108 25118 modulation +T700 GO:0005856 25122 25134 cytoskeletal +T701 CHEBI:29108 25162 25166 Ca2+ +T702 CHEBI:16247 25195 25207 phospholipid +T703 CL:0000232 25250 25265 red blood cells +T704 UBERON:0000178 25254 25259 blood +T705 CHEBI:29108 25285 25294 Ca2+ ions +T706 GO:0016020 25324 25332 membrane +T707 GO:0005856 25363 25375 cytoskeleton +T708 CL:0000233 25417 25425 platelet +T709 PR:000004080 25442 25452 annexin A7 +T710 CL:0000233 25468 25476 platelet +T711 GO:0070527 25468 25488 platelet aggregation +T712 CL:0000233 25575 25584 platelets +T713 PR:000004080 25593 25603 annexin A7 +T714 NCBITaxon:39107 25706 25712 murine +T715 CL:0000233 25713 25722 platelets +T716 NCBITaxon:9606 25752 25757 human +T717 NCBITaxon:10088 25832 25837 mouse +T718 NCBITaxon:10088 25873 25878 mouse +T719 CL:0000233 25879 25888 platelets +T720 CHEBI:9574 25979 25987 thrombin +T721 NCBITaxon:9606 26032 26037 human +T722 PR:000017364 26038 26041 vWF +T723 http://purl.obolibrary.org/obo/MONDO_0024574 26038 26050 vWF-syndrome +T724 GO:0007599 26060 26071 haemostatic +T725 http://purl.obolibrary.org/obo/MONDO_0003159 26060 26080 haemostatic diseases +T726 CL:0000233 26288 26296 platelet +T727 CL:0000233 26317 26325 platelet +T728 CHEBI:59132 26326 26333 antigen +T729 PR:000004080 26446 26456 annexin A7 +T730 SO:0001060 26457 26465 isoforms +T731 CL:0000232 26469 26484 red blood cells +T732 UBERON:0000178 26473 26478 blood +T733 GO:0031982 26516 26524 vesicles +T734 CHEBI:29108 26540 26544 Ca2+ +T735 GO:0006900 26601 26613 vesiculation +T736 GO:0044425 26681 26697 membrane domains +T737 CHEBI:18059 26706 26711 lipid +T738 GO:0045121 26706 26717 lipid rafts +T739 PR:000004080 26752 26762 annexin A7 +T740 GO:0016020 26831 26840 membranes +T741 CL:0000232 26854 26869 red blood cells +T742 UBERON:0000178 26858 26863 blood +T743 PR:000004080 26873 26878 anxA7 +T744 NCBITaxon:10088 26881 26885 mice +T745 CL:0000232 26921 26933 erythrocytes +T746 PR:000004080 26942 26952 annexin A7 +T747 CL:0000232 26988 26999 erythrocyte +T748 GO:0005856 27000 27012 cytoskeleton +T749 GO:0031941 27043 27050 F-actin +T750 CHEBI:29108 27109 27113 Ca2+ +T751 NCBITaxon:39107 27157 27163 murine +T752 NCBITaxon:9606 27168 27173 human +T753 UBERON:0000178 27174 27179 blood +T754 UBERON:0000178 27189 27194 Blood +T755 PR:000004080 27220 27230 annexin A7 +T756 NCBITaxon:10088 27241 27245 mice +T757 PR:000004080 27247 27252 anxA7 +T758 GO:0007631 27297 27300 fed +T759 CHEBI:33290 27314 27318 food +T760 UBERON:0002080 27389 27412 right cardiac ventricle +T761 UBERON:0005434 27455 27463 cervical +T762 GO:0050817 27477 27488 Coagulation +T763 NCBITaxon:10088 27591 27595 mice +T764 UBERON:0005434 27611 27619 cervical +T765 CHEBI:38867 27664 27685 anaesthetic chemicals +T766 UBERON:0000178 27689 27694 blood +T767 NCBITaxon:10088 27732 27736 mice +T768 UBERON:0000178 27802 27807 blood +T769 NCBITaxon:39107 27909 27915 murine +T770 UBERON:0000178 27916 27921 blood +T771 NCBITaxon:9606 28004 28009 Human +T772 UBERON:0000178 28010 28015 blood +T773 UBERON:0001638 28059 28063 vein +T774 UBERON:0000178 28207 28212 blood +T775 UBERON:0002415 28248 28252 tail +T776 UBERON:0000948 28257 28262 heart +T777 UBERON:0000178 28344 28349 blood +T778 SO:0000704 28383 28390 genetic +T779 NCBITaxon:10088 28438 28442 mice +T780 NCBITaxon:10088 28514 28518 mice +T781 SO:0000704 28557 28564 genetic +T782 NCBITaxon:10088 28593 28597 mice +T783 CL:0000233 28641 28649 platelet +T784 GO:0070527 28641 28661 platelet aggregation +T785 NCBITaxon:10088 28683 28687 mice +T786 CHEBI:31705 28747 28756 iodixanol +T787 NCBITaxon:9606 28778 28783 human +T788 CL:0000232 28784 28799 red blood cells +T789 UBERON:0000178 28788 28793 blood +T790 PR:000004080 28833 28843 annexin A7 +T791 CL:0000232 28877 28892 Red blood cells +T792 UBERON:0000178 28881 28886 blood +T793 GO:0019835 28979 28984 lysis +T794 CHEBI:46756 28999 29004 Hepes +T795 CHEBI:3312 29021 29026 CaCl2 +T796 CHEBI:17992 29035 29042 sucrose +T797 CHEBI:31705 29190 29199 iodixanol +T798 CHEBI:75958 29200 29208 solution +T799 CHEBI:75958 29244 29252 solution +T800 CHEBI:31705 29259 29268 iodixanol +T801 CHEBI:46756 29297 29302 Hepes +T802 CHEBI:3312 29319 29324 CaCl2 +T803 CHEBI:17992 29333 29340 sucrose +T804 CHEBI:31705 29368 29377 iodixanol +T805 CHEBI:75958 29378 29386 solution +T806 CL:0000232 29425 29439 red blood cell +T807 UBERON:0000178 29429 29434 blood +T808 CHEBI:75958 29440 29448 solution +T809 CHEBI:75958 29472 29480 solution +T810 CHEBI:31705 29499 29508 iodixanol +T811 CHEBI:60004 29514 29521 mixture +T812 CHEBI:31705 29596 29605 iodixanol +T813 GO:0070382 29942 29952 Exovesicle +T814 NCBITaxon:9606 29969 29974 human +T815 NCBITaxon:39107 29979 29985 murine +T816 CL:0000232 29986 30001 red blood cells +T817 UBERON:0000178 29990 29995 blood +T818 CL:0000232 30003 30018 Red blood cells +T819 UBERON:0000178 30007 30012 blood +T820 GO:0070382 30060 30071 exovesicles +T821 GO:0031982 30088 30095 vesicle +T822 GO:0006900 30088 30105 vesicle formation +T823 CHEBI:29108 30109 30113 Ca2+ +T824 CHEBI:28304 30149 30156 heparin +T825 GO:0050817 30161 30171 coagulated +T826 UBERON:0000178 30172 30177 blood +T827 CL:0000232 30241 30253 erythrocytes +T828 CHEBI:46756 30288 30293 Hepes +T829 CHEBI:26710 30314 30318 NaCl +T830 CL:0000232 30363 30378 red blood cells +T831 UBERON:0000178 30367 30372 blood +T832 GO:0006900 30406 30418 vesiculation +T833 CHEBI:46756 30433 30438 Hepes +T834 CHEBI:26710 30455 30459 NaCl +T835 CHEBI:3312 30466 30471 CaCl2 +T836 CHEBI:29108 30489 30493 Ca2+ +T837 CHEBI:24869 30494 30503 ionophore +T838 CHEBI:75958 30589 30597 solution +T839 CL:0000232 30892 30906 red blood cell +T840 UBERON:0000178 30896 30901 blood +T841 GO:1990742 30907 30920 microvesicles +T842 GO:0031982 31164 31172 vesicles +T843 GO:0031982 31198 31206 vesicles +T844 CHEBI:15355 31224 31237 acetylcholine +T845 CHEBI:15355 31288 31301 Acetylcholine +T846 CL:0000232 31336 31350 red blood cell +T847 UBERON:0000178 31340 31345 blood +T848 GO:0031982 31351 31359 vesicles +T849 CHEBI:46756 31408 31413 Hepes +T850 CHEBI:26710 31430 31434 NaCl +T851 CHEBI:9750 31442 31454 Triton X-100 +T852 CHEBI:37586 31523 31539 sodium phosphate +T853 CHEBI:86228 31578 31582 DTNB +T854 CHEBI:86228 31584 31619 5,5'-dithiobis-(2-nitrobenzoic acid +T855 CHEBI:37586 31631 31647 sodium phosphate +T856 CHEBI:15377 31706 31709 H2O +T857 CL:0000232 31909 31924 red blood cells +T858 UBERON:0000178 31913 31918 blood +T859 CHEBI:34683 31928 31935 Na2HPO4 +T860 CHEBI:37585 31936 31943 NaH2PO4 +T861 CHEBI:26710 31953 31957 NaCl +T862 CHEBI:26710 32003 32007 NaCl +T863 CHEBI:75958 32149 32157 solution +T864 UBERON:0000178 32168 32173 blood +T865 CL:0000233 32477 32485 Platelet +T866 GO:0070527 32477 32497 Platelet aggregation +T867 NCBITaxon:39107 32529 32535 murine +T868 UBERON:0000178 32536 32541 blood +T869 CL:0000233 32655 32663 platelet +T870 UBERON:0001969 32669 32675 plasma +T871 CL:0000233 32687 32695 platelet +T872 GO:0070527 32687 32707 platelet aggregation +T873 CHEBI:46756 32940 32945 Hepes +T874 CHEBI:26710 32954 32958 NaCl +T875 CL:0000233 32979 32988 platelets +T876 CL:0000233 33004 33012 platelet +T877 NCBITaxon:39107 33092 33098 murine +T878 NCBITaxon:9606 33103 33108 human +T879 CL:0000233 33134 33143 platelets +T880 CHEBI:85129 33272 33282 ristocetin +T881 CHEBI:75958 33283 33291 solution +T882 CHEBI:33893 33370 33377 reagent +T883 NCBITaxon:10088 33418 33423 mouse +T884 NCBITaxon:9606 33428 33433 human +T885 CL:0000233 33434 33443 platelets +T886 NCBITaxon:39107 33450 33456 murine +T887 NCBITaxon:33208 33486 33492 animal +T888 NCBITaxon:9606 33506 33511 human +T889 UBERON:0000178 33512 33517 blood +T890 NCBITaxon:9606 33630 33646 human individual +T891 CL:0000233 33898 33906 platelet +T892 UBERON:0000178 33975 33980 blood +T893 GO:0030097 34026 34039 haematopoetic +T894 NCBITaxon:39107 34072 34078 murine +T895 NCBITaxon:9606 34083 34088 human +T896 UBERON:0000178 34089 34094 blood +T897 UBERON:0000178 34119 34124 blood +T898 CL:0000232 34210 34225 red blood cells +T899 UBERON:0000178 34214 34219 blood +T900 CL:0000233 34230 34239 platelets +T901 CL:0000232 34342 34357 red blood cells +T902 UBERON:0000178 34346 34351 blood +T903 CL:0000233 34362 34371 platelets +T904 CL:0000738 34477 34487 leucocytes +T905 CL:0000232 34489 34504 red blood cells +T906 UBERON:0000178 34493 34498 blood +T907 CL:0000233 34622 34630 platelet +T908 CL:0000775 34639 34651 neutrophiles +T909 CL:0000542 34653 34664 lymphocytes +T910 CL:0000576 34666 34675 monocytes +T911 CL:0000771 34677 34689 eosinophiles +T912 CL:0000767 34691 34701 basophiles +T913 PR:000004080 34809 34819 annexin A7 +T914 GO:0031941 34852 34859 F-actin +T915 PR:000004080 34883 34893 annexin A7 +T916 CHEBI:8984 34922 34925 SDS +T917 GO:0042571 35051 35061 Antibodies +T918 NCBITaxon:10088 35105 35110 mouse +T919 PR:000004080 35111 35121 annexin A7 +T920 SO:0000417 35127 35133 domain +T921 NCBITaxon:9986 35155 35161 rabbit +T922 PR:000015619 35167 35173 sorcin +T923 GO:0042571 35174 35182 antibody +T924 CL:0000232 35707 35722 red blood cells +T925 UBERON:0000178 35711 35716 blood +T926 CL:0000233 35727 35736 platelets +T927 CL:0000233 35818 35827 platelets +T928 CL:0000233 35959 35967 platelet +T929 GO:0070527 35959 35979 platelet aggregation +T930 PR:000015619 36047 36053 sorcin +T931 GO:0005856 36078 36090 cytoskeletal diff --git a/src/ontogpt/evaluation/craft/database/all/12925238.txt b/src/ontogpt/evaluation/craft/database/all/12925238.txt new file mode 100644 index 000000000..e7ae85c3b --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/12925238.txt @@ -0,0 +1,145 @@ +Function, expression and localization of annexin A7 in platelets and red blood cells: Insights derived from an annexin A7 mutant mouse + +Abstract + +Background + +Annexin A7 is a Ca2+- and phospholipid-binding protein expressed as a 47 and 51 kDa isoform, which is thought to be involved in membrane fusion processes. Recently the 47 kDa isoform has been identified in erythrocytes where it was proposed to be a key component in the process of the Ca2+-dependent vesicle release, a process with which red blood cells might protect themselves against an attack by for example complement components. + +Results + +The role of annexin A7 in red blood cells was addressed in erythrocytes from anxA7-/- mice. Interestingly, the Ca2+-mediated vesiculation process was not impaired. Also, the membrane organization appeared not to be disturbed as assessed using gradient fractionation studies. Instead, lack of annexin A7 led to an altered cell shape and increased osmotic resistance of red blood cells. Annexin A7 was also identified in platelets. In these cells its loss led to a slightly slower aggregation velocity which seems to be compensated by an increased number of platelets. The results appear to rule out an important role of annexin A7 in membrane fusion processes occurring in red blood cells. Instead the protein might be involved in the organization of the membrane cytoskeleton. Red blood cells may represent an appropriate model to study the role of annexin A7 in cellular processes. + +Conclusion + +We have demonstrated the presence of both annexin A7 isoforms in red blood cells and the presence of the small isoform in platelets. In both cell types the loss of annexin A7 impairs cellular functions. The defects observed are however not compatible with a crucial role for annexin A7 in membrane fusion processes in these cell types. + +Background + +Annexin A7 is a Ca2+- and phospholipid-binding protein, which was isolated as the agent that mediated aggregation of chromaffin granules and fusion of phospholipid membranes in the presence of Ca2+. This activity led to the proposal of annexin A7's involvement in the exocytotic secretion of catecholamines [1]. The protein belongs to a family of evolutionarily conserved proteins of a bipartite structure with a variable N-terminal and a conserved C-terminal domain. The C-terminal domain is responsible for the Ca2+- and phospholipid-binding, the N-terminal domain appears to confer functional diversity [2-4]. The binding to negatively charged phospholipids is thought to be mediated by Ca2+ ions. + +Annexin A7 is unique in that it carries an extraordinarily long and hydrophobic amino terminus with more than 100 amino acids. Alternative splicing gives rise to two isoforms of 47 and 51 kDa. Both isoforms differ by an additional cassette exon located in the first third of the N-terminal domain. Most tissues harbor only the 47 kDa isoform, both forms are found in brain and heart, while the large isoform is exclusively expressed in mature skeletal muscle. + +At the cellular level annexin A7 can be detected in the cytosol, at the plasma membrane, around the nucleus, at vesicular structures including adrenal chromaffin granules, and at the t-tubule system [5,6]. Annexin A7 translocates to membranes in a Ca2+-dependent fashion and, when intracellular Ca2+ levels rise, sequentially redistributes to the plasma and the nuclear membrane as well as to intracellular vesicles. Furthermore, annexin A7 associates with lipid rafts [7]. Lipid rafts play a key role in membrane budding and in vesiculation processes such as endo- and exocytosis [8-10]. + +Two binding partners of annexin A7 have been identified, sorcin and galectin-3 [11-13]. Sorcin is a Ca2+-binding protein and belongs to the penta EF-hand protein family [14]. Like annexin A7 it binds to membranes in a Ca2+-dependent manner. Sorcin also has been described as interaction partner of the ryanodine receptor, and appears to modulate its function [15]. The influence of the sorcin/annexin A7 interaction on the ryanodine receptor is unknown. Sorcin and annexin A7 are coexpressed in all tissues examined so far [11]. The binding of annexin A7 and sorcin is Ca2+-dependent and occurs at micromolar Ca2+ concentrations. The binding sites have been localised to the amino terminal GGYY and GYGG motifs in sorcin and to the GYPP motif in the amino terminus of annexin A7. The proteins bind to each other with a stoichiometry of two sorcin molecules per annexin A7 molecule [12]. + +Galectin-3 is a multifunctional oncogenic protein with an anti-apoptotic activity found in the extracellular space, in the nucleus and cytoplasm and in mitochondria. Cytoplasmic galectin-3 correlates with tumor progression and protects mitochondrial integrity. Down regulation of annexin A7 prevents galectin-3 translocation to the perinuclear membrane and increases galectin-3 secretion. For annexin A7 a role for galectin-3 trafficking, apoptosis regulation, and mitochondrial integrity was proposed [13]. + +The cellular role of annexin A7 is not well understood. It is thought to regulate and stabilize membrane domains and to have a role in Ca2+ homeostasis and Ca2+-dependent signaling pathways. These proposals are supported by data obtained from analysis of annexin A7 deficient mouse mutants. Two annexin A7 129Sv null mice strains were generated independently using a different strategy. The one of Srivastava et al. [16] is lethal. Heterozygous mice exhibit an insulin secretion defect and tumor phenotypes. The null strain reported by Herr et al. [17] is viable, healthy, and shows no insulin secretion defect or other obvious defects. However, in isolated cardiomyocytes the frequency-induced shortening is disturbed. Here we have focused on the analysis of red blood cells and platelets of the anxA7-/- mutant. Generally, red blood cells and platelets were thought not to contain annexin A7 and only recently the 47 kDa isoform has been reported as component of red blood cells [7]. + +Red blood cells undergo various biochemical or morphological changes that appear to be Ca2+-dependent processes [18,19]. One of them is the release of hemoglobin-containing exovesicles occurring in vivo as well as in vitro [7,20]. This process is thought to represent a protective method of the red blood cell against an attack by for example complement components. Red blood cells which lack the ability to vesiculate cause a disease with red blood cell destruction and haemoglobinuria [21]. There exist two types of vesicles, micro- and nanovesicles with a size of 180 nm and 60 nm, respectively. They are enriched in cholesterol and sphingolipid rich lipid raft domains that are associated with proteins like acetylcholinesterase, cell surface proteins including a complement receptor, and the lipid raft proteins stomatin and flotillin, but they lack any cytoskeletal protein [7]. Furthermore, they contain sorcin and annexin A7 attached to the lipid rafts. Both proteins are more abundant in nanovesicles. The vesicle formation goes along with several other changes in the red blood cell like cytoskeletal rearrangements and changes in the phospholipid orientation in the cellular membrane. + +Results + +Annexin A7 expression in red blood cells, red blood cell derived exovesicles and in platelets + +Salzer et al. recently reported the presence of the 47 kDa isoform of annexin A7 and its partner sorcin in micro- and nanovesicles derived from red blood cells where they are located in the lumen and are also enriched in membrane rafts [7]. We have now extended these findings and show here that the 51 kDa isoform is present as well. In western blots of human red blood cells the 47 and 51 kDa isoforms were detected using mAb 203–217 (Fig. 1). The 47 kDa isoform was detected in silver stained gels and its identity with annexin A7 confirmed by peptide mass fingerprinting (data not shown). The 51 kDa isoform was only detected in western blots. + +Figure 1 + +Expression of annexin A7 in human red blood cells (1) and human platelets (2). Protein homogenates were separated by SDS-PAGE (12 % acrylamide). The resulting western blot was probed with mAb 203–217, visualization was by a secondary peroxidase coupled antibody followed by ECL. Both isoforms are detected in red blood cells, in platelets only the small isoform is present. + +Its presence in vesicles led to the suggestion, that, along with raft domains, annexin A7 plays a role in membrane organization and the vesiculation process. To examine this, we analysed the ability of red blood cells derived from the annexin A7 knock out mice (anxA7-/-) to form exovesicles. Because of better accessibility and larger available amounts that led to clear results, we included our data obtained with human blood. Independent of the presence of annexin A7 both types of exovesicles, micro- and nanovesicles, were released after Ca2+/ionophore treatment as determined by acetylcholine esterase activity and microscopic examination. Annexin A7 is present in both vesicle types where it is more enriched in nanovesicles (Fig. 2). The 51 kDa isoform is only detectable in nanovesicles. The quantity of both vesicle types did not differ between wild type and knock out red blood cell vesicles as determined by the AChE-values (A405 nanovesicles: ~0.18; A405 microvesicles: ~0.9; both, for wt and ko). It appears that annexin A7, although it has been described to fuse membranes, is not a key component in the process of the formation of red blood cell exovesicles. When we probed for the presence of sorcin in wild type and mutant vesicles we found that the sorcin levels were reduced in the mutant nanovesicles (Fig. 2). + +Figure 2 + +Enrichment of annexin A7 and sorcin in exovesicles derived from red blood cells. Vesicles from wild type (wt) and anxA7-/- mutant (ko) were generated with Ca2+/ionophore treatment, isolated by differential centrifugation and analysed by immunoblotting with mAb 203–217 and a sorcin polyclonal antibody. In general, annexin A7 and sorcin are more abundant in nanovesicles. The 51 kDa isoform of annexin A7 (arrows) is only observed in nanovesicles. Samples of both vesicles types were normalized according to their acetylcholine esterase activity. Human red blood cells (co) were used for control and normalized independently. + +To study the distribution of annexin A7 in red blood cells we used self forming iodixanol density gradients. Both isoforms were present in the soluble fraction as well as in the gradient fractions where they are assumed to be associated with membranes. These membranes are exclusively plasma membranes as red blood cells are free of any organelles. The distribution was however not homogeneous throughout the gradient. This could reflect the binding of annexin A7 to membrane subdomains that have different lipid or lipid/protein composition as discussed by Salzer et al. [7]. Likewise, sorcin does not exhibit a homogeneous distribution in the gradient. Moreover, it segregates into vesicles which are not associated with annexin A7 (Fig. 3). We also tested platelets for annexin A7 expression and detected the 47 kDa isoform (Fig. 1). + +Figure 3 + +Association of annexin A7 and sorcin with distinct red blood cell plasma membrane fractions. Lysed human red blood cells were added to a self forming iodixanol density gradient. The density of the gradient increases from left (1.06 g/ml) to right (1.20 g/ml). Fractions were analysed by immunoblotting using mAb 203–217 and the sorcin polyclonal antibody. + +The lack of annexin A7 changes red blood cell morphology and osmotic resistance + +As annexin A7 is a component of red blood cell vesicles we studied the consequences of the loss of the protein in the anxA7-/-mouse for red blood cell morphology and osmotic resistance. In a standard 'blood smear' we did not note a deformation of the cells, however dark field microscopy revealed changes in shape and diameter of the anxA7-/-red blood cells. They had a statistically significant larger cell diameter of 6.0 μm compared to wild type with 5.7 μm (p = 0.015, n = 40), a remarkably lower emphasized central impression and a more flat shape (Fig. 4). No significant change in the mean corpuscular volume (MCV) was measured with the ADVIA 120 cell counter. + +Figure 4 + +Dark field microscopy of red blood cells from anxA7-/- mutant (B) and wild type mice (A). The mean cellular diameters are 6.0 μm and 5.7 μm, respectively. The size differences are statistically significant (p = 0.015; n = 40). Bar, 3 μm. + +Shape and MCV of a red blood cell essentially influence its function and capillary passage. The typical biconcave form depends on various influences including the membrane lipid composition and the submembranous cytoskeleton [32] where annexin A7 might play a role. The osmotic resistance, which is the resistance towards changes in the extracellular ionic strength, is a convenient assay for analysis of the red blood cell integrity. It is measured as the sodium chloride concentration at which cellular lysis starts (minimal resistance) up to a complete lysis (maximal resistance) of the cells [33]. Osmotic resistance increases with higher MCV, larger surface area and a higher degree of cellular metabolism stabilizing the intracellular ion levels. Aged red blood cells show a lowered MCV and lower osmotic resistance [33]. Similar properties can be observed in red blood cells with lowered membrane permeability. The osmotic resistance of annexin A7 deficient red blood cells is significantly increased compared to wild type cells. The minimal resistance value of knock out cells is observed in a 0.60 % NaCl solution versus 0.65 % for wild type cells. 50 % haemolysis is achieved at 0.516 % NaCl solution for knock out versus 0.564 % NaCl for wild type cells (mean values, p = 0.00066, n = 8; Fig. 5). The resistance width of the knock out cells is slightly lower (0.20 % instead of 0.25 % NaCl solution). We also performed direct measurements of the red blood cell membrane deformability, to further characterize a possible role of annexin A7 on the membrane stability. With micropipette experiments we tried to correlate mechanical characteristics of the red blood cells with their morphology. We could not observe any statistically significant difference of values describing membrane rigidity and lysis force in these experiments (M. Heil, B. Hoffmann and R. Merkel, unpublished results). However, the distinction observed in the osmotic resistance experiments was highly significant and reflects a mean value over a high number of different red blood cells. + +Figure 5 + +Osmotic resistance curves of red blood cells from wild type and anxA7-/- mutant mice. The osmotic resistance towards changes in the extracellular ionic strength is a convenient assay for analysis of the red blood cell integrity. It is measured as a sodium chloride concentration in which cellular lysis starts (minimal resistance) up to a complete lysis (maximal resistance) of a red blood cell. The osmotic resistance of annexin A7 deficient red blood cells is significantly increased compared to the one of wild type (p = 0.00066; n = 8). + +These data suggest that annexin A7 contributes to the shape of red blood cells and the osmotic resistance. As we have not observed a binding of annexin A7 to F-actin (data not shown) it could do so either by alteration of the membrane rigidity and/or by affecting the ion homeostasis. + +The lack of annexin A7 affects primary haemostasis ex vivo + +The use of an advanced electronic cell counter/flow cytometer (ADVIA 120) allowed us to screen other parameters as well despite of the limited murine blood volume. We analysed leukocytes, red blood cells, haemoglobin content, haematocrit, mean cellular volume, mean cellular haemoglobin, mean cellular haemoglobin concentration, platelets, neutrophiles, lymphocytes, monocytes, eosinophiles and basophiles. In these tests of whole blood we found only a change in the platelet numbers, whereas all other haematological parameters were not affected. The mean platelet counts from wild type and anxA7-/- mutant mice are determined as 674 × 103/μl and 774 × 103/μl, respectively. The platelet counts in knock out mice are significantly higher (p = 0.0275; n knock out = 11, n wild type = 14). An increase in platelet counts is a rather uncommon disorder. In humans, platelet counts normally increase only transiently as in reactive thrombocytosis and under postoperative conditions or are largely increased in neoplastic diseases. By contrast, the anxA7-/-mice are healthy and viable. Therefore we tested the platelet function and performed aggregometry measurements with platelet rich plasma. Platelet activation is observed as a morphological change from the resting discoid state to activated spherical cells with pseudopods. The morphological changes are due to cytoskeletal rearrangements. In vitro the activated cells form aggregates recruiting additional cells in the solution thereby reducing its cloudiness. Analysis of the transmission values of aggregation curves after platelet activation by ristocetin addition (von Willebrand cofactor) showed that both curves differed significantly at the time point of seven seconds after platelet initiation (p = 0.0287, n knock out = 16, n wild type = 15; Fig. 6). Annexin A7 deficient platelets showed a slightly lowered aggregation velocity. When we compared the aggregation curves of human platelets from a healthy donor with the ones obtained from an individual with a von Willebrand factor type 1 defect, we found that the difference in the curves was much more pronounced as observed in our studies of healthy mouse platelets and anxA7-/-platelets. Furthermore we found that murine platelets responded immediately to the initiating chemical whereas human platelets have a lag phase [23]. + +Figure 6 + +Platelet aggregation curves from platelet rich plasma of annexin A7 knock out and wild type mice. The transmission values were measured with an APACT photometer and aggregation was initiated by adding ristocetin. The first thirty seconds are given. Constant platelet counts were used throughout all experiments. The aggregation curve data were analysed for slope values, the maximal aggregation amplitude of every single curve was set to 100 %. Both murine curves differ significantly at a time point of seven seconds after platelet initiation, the mutant shows a slightly lower aggregation velocity (p = 0.0287, n knock out = 16, n wild type = 15, mean knock out = 67.7 % transmission, mean wild type = 77.1 % transmission, standard error knock out = 8.3 % transmission, standard error wild type = 13.8 % transmission). Murine platelets react immediately to the initiating chemical and show no lag phase like the normal human platelets. For comparison of the range of the platelet impairment slightly affected human platelets are shown as well (von Willebrand syndrome). + +Discussion + +Annexin A7 is thought to have a role in vesicle fusion and in regulating and stabilizing membrane domains [1]. The protein has been localized to the plasma membrane including raft subdomains, furthermore it is present in the cytosol where it is found on subcellular vesicle-like structures. This distribution appears to be independent of the tissue or cell type analysed. In specialized or terminally differentiated cells, expression levels increase and frequently the larger of the two isoforms is being expressed [6,34,35]. + +Red blood cells, which were thought to be devoid of annexin A7, were recently shown to harbor the 47 kDa isoform together with its binding partner sorcin [7]. We showed the presence of the 51 kDa isoform as well, adding red blood cells to the list of cells that harbor this isoform. Salzer et al. [7] not only demonstrated the presence of annexin A7 in red blood cells, they also showed that annexin A7 and sorcin were enriched in membrane raft domains of nanovesicles formed from red blood cells in vitro. Raft domains in general are considered key players in membrane organization and in mediating the vesiculation process. They result from the clustering of sphingolipids and cholesterol in the outer leaflet of the membrane connected to phospholipids and cholesterol in the inner leaflet and are enriched in special proteins. Biochemically rafts are defined as membranes that are resistant to extraction by cold Triton X-100 and can be floated to low densities in sucrose gradient centrifugation [36]. + +The findings of Caohuy et al. [37] that annexin A7 mediates membrane aggregation also suggested that annexin A7 is an essential component during vesiculation. In addition they reported a further activation of the vesiculation process by GTP and proposed that annexin A7 acts as a GTPase. Similarly, the presence of GTP enhanced the secretion of surfactant by annexin A7. These results were thought to support a direct role for annexin A7 in surfactant secretion, but in these studies annexin A7 mediated membrane fusion was separated from a second GTP-dependent mechanism [38]. + +Having an annexin A7 knockout mouse we tested the involvement of annexin A7 in budding of red blood cell vesicles. Our data suggest that the efficiency of micro- and nanovesicle formation is not significantly different in wild type and mutant red blood cells. Likewise, Dictyostelium discoideum cells, in which the single annexin gene was inactivated, were not impaired in processes requiring membrane fusion [[39]; and data unpublished]. It might well be that the observed fusion of phospholipid layers by annexin A7 in the presence of high Ca2+ concentrations may turn out to be an in vitro effect. Similarly, the high abundance of annexin A7 in micro- and nanovesicles might be fortuitous as a significant percentage of the red blood cell membrane, with which annexin A7 associates when the Ca2+ levels rise, appears to be involved in the formation of exovesicles. Furthermore, the distribution of both annexin A7 isoforms in gradients of red blood cells indicates a function which differs from organizing subcellular membranes or membrane pathways. Whereas annexin A2 is tightly associated with endosomes and may have functions in membrane fusion and the endosomal pathway, annexin A7 distributes with plasma membrane raft domains of different types and densities. Interestingly, in vesicles of red blood cells lacking annexin A7 the amount of sorcin is lowered, indicating that its localisation is affected by the loss of annexin A7 or its expression during red blood cell development was reduced. + +Having excluded a direct role in vesicle formation and membrane fusion, annexin A7 might act by its property as Ca2+-binding protein [17]. Previous results in which annexin A7 was shown to play a role in membrane aggregation might be explained by a supportive function of annexin A7 in that it interferes with the local Ca2+ homeostasis thus influencing membrane organization. As the annexins belong to a wide spread and evolutionarily conserved protein family, redundant, but not identical functions are expected. However, so far no other members of the annexin family, which may substitute for annexin A7, have been described in red blood cells. + +The structural basis for the elasticy of red blood cells in circulation are long range molecular functions of the plasma membrane and the membrane associated part of the cytoskeleton [40-43]. Major constituents of the membrane skeleton are spectrin tetramers linked together by short actin filaments and several other proteins covering the entire cytoplasmic surface of the red blood cell membrane [44]. Membrane cholesterol diminishes red blood cell haemolysis by the complement complex, whereas depletion of raft cholesterol abrogates association of all raft proteins with no significant effect on areas in the rest of the cell membrane and deformability [45,46]. + +Annexin A7 knock out mouse red blood cells show a more flat shape and have a higher osmotic resistance. The presence of annexin A7 may alter the membrane flexibility in that it supports the typical biconcave membrane shape and leads to a lower minimal osmotic resistance. Annexin A7 does not appear to have an influence on plasma membrane integrity itself as the maximal osmotic resistance value stays constant. Ca2+ and membrane skeletal proteins are known to play key roles on the red blood cell membrane shape and stability [47]. Annexins have been reported to bind directly to F-actin, we could however exclude such an activity for annexin A7. Nevertheless it might interact with other components and have a role in organizing the membrane cytoskeleton, controlling raft protein associations or influencing the ionic strength of red blood cells on its own or by interfering with other signaling pathways. + +As discussed above annexin A7 could be involved in the cytoplasmic Ca2+ homeostasis. It has been demonstrated that micromolar changes of the intracellular Ca2+ concentration exert a profound effect on the membrane properties that regulate red blood cell deformability [48,49]. Furthermore, the intracellular Ca2+ was shown to regulate the membrane stability through modulation of cytoskeletal protein interactions [50]. Ca2+ also induces a transbilayer phospholipid redistribution inducing a shape change of red blood cells [51]. In addition, Ca2+ ions can drive spiculation of the membrane bilayer without involving the cytoskeleton [52]. + +We have also observed a defect in platelet function in the annexin A7 mutant. In the platelet aggregation experiments the initial aggregation velocity was slightly, but significantly lower in platelets lacking annexin A7. These data are not reflected by a change in the in vivo bleeding time. However the reaction speed of murine platelets is much higher than those of human and due to the technical setup we might have missed larger changes in the mouse samples. The immediate reaction of mouse platelets and the absence of a lag phase after triggering may be caused by an immediately initiated thrombin generation [22]. As known from the frequent human vWF-syndrome or other haemostatic diseases, small defects often are clinically silent and do not result in physiological changes under normal conditions. Only in situations with severe physical injury, distinct environmental influences or additional platelet attributes like the platelet antigen polymorphism [53] differences might become apparent. + +Conclusions + +In this paper we report the presence of both annexin A7 isoforms in red blood cells where they are abundant in nanovesicles that form upon Ca2+ addition. The proteins are however not required for the vesiculation process. They are also not essential for the formation of specific membrane domains such as lipid rafts, as exogenously added recombinant annexin A7 redistributed to similar positions in a density gradient containing membranes derived from red blood cells of anxA7-/-mice. The observed cell shape change of erythrocytes lacking annexin A7 might be due to alterations in the erythrocyte cytoskeleton. As a direct interaction with F-actin was ruled out, this effect might be mediated by annexin's Ca2+-binding activity. + +Methods + +Acquisition of murine and human blood samples + +Blood from 129Sv wild type and annexin A7 knock out mice (anxA7-/-) kept under pathogen-free conditions and fed with regular food, was collected in 1 ml syringes with a 22 gauge needle punctating the right cardiac ventricle immediately after they had been killed by cervical dislocation. Coagulation generally was inhibited with 1/10 volume of 10 mM NaEDTA, pH 7.5, already present in the syringe. The mice were killed by cervical dislocation in order to avoid any effect of anaesthetic chemicals on blood parameters. The wild type and mutant mice were matched regarding their age, weight and sex. The daytime of blood collection, sampling site and collection method were kept constant during all experiments. A typical murine blood sample volume consisted of 550 μl, with variations ranging from 200 μl to 900 μl. Human blood samples from the authors were collected by vein puncture using common EDTA-monovettes (Sarstedt). + +We performed our studies using well standardized sampling conditions, as differences in the blood sampling site, for example between tail and heart, show large changes in all cell type counts. First and second sample draw affect blood measurements, too [22]. Also the genetic background in common laboratory and transgenic mice affects the phenotype. A change from one strain to another may protect mice from effects of the primarily induced genetic defect. For example, BALB/c mice have a significantly increased velocity of platelet aggregation as compared to 129Sv mice, which were used throughout this study [23]. + +Self forming iodixanol density gradients of human red blood cells + +The subcellular distribution of annexin A7 was addressed according to [24]. Red blood cells were centrifuged and washed in isotonic buffered salt solution, collected in ice cold lysis buffer (20 mM Hepes, pH 7.4, 200 μM CaCl2, 0.25 M sucrose, and proteinase inhibitors (Roche)), homogenized by a loosely fitting dounce homogenizer and additional passages through a 22 gauge needle. A 50 % iodixanol solution was established by mixing Optiprep solution (60 % iodixanol; Sigma) with buffer (120 mM Hepes, pH 7.4, 1.2 mM CaCl2, 0.25 M sucrose). Seven parts of this 50 % iodixanol solution were mixed with thirteen parts of the red blood cell solution, generating a gradient solution containing 17.5 % iodixanol. The mixture was filled into a centrifuge tube underlayed by cushions of 30 % and 35 % iodixanol and centrifuged for 3 hours at 270,000 × g in a SW41 Ti swing out rotor (Beckman) to achieve a nearly linear density gradient (approximately 1.06 g/ml to 1.20 g/ml) [24]. Fractions of 0.6 ml were collected from the top of the gradient by a 1 ml syringe without using a needle and immediately frozen in liquid nitrogen for further use. + +Exovesicle generation from human and murine red blood cells + +Red blood cells naturally can form hemoglobin containing exovesicles. In vitro these vesicle formation is Ca2+ induced. Freshly collected lithium-heparin anticoagulated blood samples were centrifuged for 5 minutes at 400 × g and 4°C. The erythrocytes were washed five times with 20 mM Hepes, pH 7.5, and 150 mM NaCl (washing buffer). A volume of 500 μl washed red blood cells were combined with 3 ml of vesiculation buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM CaCl2 adjusted to 5 μM Ca2+ ionophore A23187) and incubated for 30 minutes at 37°C. After incubation EDTA was added to the solution to a final concentration of 5 mM and centrifuged for 5 minutes at 400 × g and 4°C. The supernatant was collected and centrifuged for 20 minutes at 15,000 × g and 4°C. The resulting pellet was resuspended in washing buffer and centrifuged at 400 × g again to collect a supernatant enriched with red blood cell microvesicles. The supernatant of the 15,000 × g step was further centrifuged for 60 minutes at 100,000 × g and 4°C. The final pellet of this step was resuspended in washing buffer and centrifuged at 15,000 × g again to purify a supernatant enriched in nanovesicles according to [7,20]. The vesicles were assayed for acetylcholine esterase activity and used for further analysis. + +Acetylcholine esterase assay + +A 30 μl sample of red blood cell vesicles was mixed with an equal volume of buffer (20 mM Hepes, pH 7.6, 150 mM NaCl, 0.5 % Triton X-100), vortexed and incubated for 5 minutes at 37°C. Subsequently 640 μl sodium phosphate buffer (100 mM Na2PO4, pH 7.6), 50 μl DTNB (5,5'-dithiobis-(2-nitrobenzoic acid), 10 mM in sodium phosphate buffer), and 50 μl acetylthiocholine chloride (12,5 mM in H2O) were added. The reaction took place at room temperature and was measured photometrically at a wavelength of 405 nm according to the Ellmann' method [25]. + +Determination of the osmotic resistance of red blood cells + +A Na2HPO4/NaH2PO4 buffered NaCl gradient (pH 7.4) comprised of the following NaCl steps was used for this assay: 0.90%, 0.65%, 0.60%, 0.55%, 0.50%, 0.45%, 0.40%, 0.35%, 0.30%, 0.10%. For each step 5 ml of the gradient step solution and 50 μl blood sample were carefully inverted four times and incubated for 30 minutes in the dark. Samples were carefully inverted again and centrifuged at 1,500 × g for 10 minutes. From 1 ml supernatant of each step the extinction at 546 nm wavelength was measured. The experiment was performed at room temperature. + +Platelet aggregation assay + +Approximately 500 μl of murine blood were centrifuged at 200 × g for 15 minutes. The resulting supernatants were transferred to new tubes and used as platelet rich plasma (PRP). The platelet aggregation measurements were done according to the method developed by Born and Cross [26] using an APACT photometer (Labor, Ahrendsburg, FRG) and the APACT software (APACT 1.4). Subsequently samples of the PRP were adjusted with buffer (5 mM Hepes, 150 mM NaCl, pH 7.3) to 250,000 platelets/μl. A constant platelet count was used throughout allowing comparisons between wild type and knock out murine and human samples. 250 μl adjusted platelets were prewarmed to 37°C and used in the assay, and the aggregation process was started by adding 25 μl of a prewarmed 16.5 mg/ml ristocetin solution (DiaMed Diagnostica, Bensheim, FRG; concentration in the assay: 1.5 mg/ml), a reagent which worked reliably in our hands with mouse and human platelets. Each murine assay represents a different animal. Analyses of human blood samples were performed as described in the manufacturer's protocol. Several repeated measurements were done per human individual to control the reproducibility. The acquired aggregation curve data, measured as ascending degree of transmission, were analysed for maximal slope values, while the maximal aggregation amplitude of every single curve was set to 100 %. In general, all platelet procedures were done at 22°C within four hours after collecting the blood sample. + +Miscellaneous methods + +For standard haematopoetic measurements and cell counts on murine and human blood samples about 300 μl of blood were analysed with an ADVIA 120 electronic cell counter (Bayer). With this equipment red blood cells and platelets are "transformed" into spherical bodies without changing their volume. Therefore number and volume of red blood cells and platelets are not calculated values, but directly measured values. The following data were statistically analysed: leucocytes, red blood cells, haemoglobin, haematocrit, mean cellular volume, mean cellular haemoglobin, mean cellular haemoglobin concentration, platelet number, neutrophiles, lymphocytes, monocytes, eosinophiles, basophiles. + +Peptide mass fingerprinting was performed on a Bruker Reflex IV MALDI-TOF mass spectrometer. Recombinant annexin A7 was purified according to [27], F-actin-binding of recombinant annexin A7 was done as described [28]. SDS-PAGE and western blotting were done as described [29,30]. Detection in immunoblots was with enhanced chemiluminescence [17]. Antibodies employed were mab 203–217 directed against mouse annexin A7 core domain [5] and a polyclonal rabbit anti-sorcin antibody [31]. + +For statistical analyses the following tests were performed: David et al. range test to ensure samples are from a normal distribution, F-test looking for a samples' homoscedascity, Student's T-test to judge if samples of different groups have a different mean value and are parts from different normal distributions. The exact probability values and the significance of an analysis are indicated when the experiments are described. + +Authors' contributions + +CH and CSC planned and carried out the experiments with the red blood cells and platelets and the fractionation studies and drafted the manuscript, GL was responsible for platelets, RK gave support on the ADVIA 120 cell counter, evaluated and discussed the results, SMP and BSG gave support on the APACT system, platelet aggregation experiments, and evaluated the results, CZ was responsible for the sorcin studies, MS studied the cytoskeletal activities of the protein, AAN conceived of the studies and participated in its design. All authors read and approved the manuscript. + +Acknowledgements + +We would like to thank M. Heil, B. Hoffmann and R. Merkel for the biophysical experiments, L. Eichinger for discussion, B. Gassen for expert technical assistance and R. Müller for protein purification. Mass spectrometric analysis was performed in the central service facilities of the ZMMK. This worked is supported by a grant from the ZMMK (Center of Molecular Medicine Cologne). diff --git a/src/ontogpt/evaluation/craft/database/all/14609438.ann b/src/ontogpt/evaluation/craft/database/all/14609438.ann new file mode 100644 index 000000000..1999b7d2a --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/14609438.ann @@ -0,0 +1,331 @@ +T1 PR:000016585 11 34 tripeptidyl-peptidase I +T2 PR:000016585 36 40 CLN2 +T3 PR:000016585 161 184 Tripeptidyl-peptidase I +T4 PR:000016585 200 204 CLN2 +T5 NCBITaxon:9606 279 285 humans +T6 GO:0010467 298 308 expression +T7 http://purl.obolibrary.org/obo/MONDO_0005559 340 365 neurodegenerative disease +T8 http://purl.obolibrary.org/obo/MONDO_0015674 377 422 late-infantile neuronal ceroid lipofuscinosis +T9 CL:0000540 392 400 neuronal +T10 SO:0001026 463 470 genomic +T11 NCBITaxon:species 492 499 species +T12 SO:0000855 663 672 orthologs +T13 NCBITaxon:9606 676 681 human +T14 PR:000016585 682 686 CLN2 +T15 SO:0001026 694 701 genomic +T16 NCBITaxon:2759 727 737 eukaryotic +T17 NCBITaxon:species 738 745 species +T18 SO:0001026 819 826 genomes +T19 NCBITaxon:9539 830 837 macaque +T20 NCBITaxon:10088 839 844 mouse +T21 NCBITaxon:10114 846 849 rat +T22 NCBITaxon:9615 851 854 dog +T23 NCBITaxon:9913 860 863 cow +T24 NCBITaxon:31032 938 942 fugu +T25 NCBITaxon:7955 947 952 zebra +T26 NCBITaxon:8342 969 974 frogs +T27 NCBITaxon:8364 976 994 Xenopus tropicalis +T28 NCBITaxon:9606 1026 1031 human +T29 PR:000016585 1032 1036 CLN2 +T30 SO:0000857 1067 1075 homology +T31 NCBITaxon:286 1081 1092 Pseudomonas +T32 NCBITaxon:species 1093 1096 sp. +T33 PR:000016585 1125 1129 CLN2 +T34 NCBITaxon:1 1191 1200 organisms +T35 UBERON:0000104 1241 1252 life cycles +T36 SO:0100019 1316 1327 active site +T37 PR:000016585 1380 1384 CLN2 +T38 CHEBI:35222 1658 1668 inhibitors +T39 PR:000016585 1672 1676 CLN2 +T40 PR:000016585 1717 1740 tripeptidyl-peptidase I +T41 PR:000016585 1742 1747 TPP-I +T42 PR:000016585 1940 1945 TPP-I +T43 PR:000016585 2006 2010 CLN2 +T44 PR:000016585 2104 2108 CLN2 +T45 http://purl.obolibrary.org/obo/MONDO_0024237 2142 2177 inherited neurodegenerative disease +T46 http://purl.obolibrary.org/obo/MONDO_0015674 2189 2234 late-infantile neuronal ceroid lipofuscinosis +T47 CL:0000540 2204 2212 neuronal +T48 PR:000016585 2305 2309 CLN2 +T49 SO:0100019 2411 2425 catalytic site +T50 PR:000016585 2454 2458 CLN2 +T51 NCBITaxon:2 2578 2587 bacterial +T52 PR:000016585 2634 2638 CLN2 +T53 CHEBI:35406 2927 2935 oxyanion +T54 NCBITaxon:2157 3059 3066 archaea +T55 NCBITaxon:2 3068 3076 bacteria +T56 NCBITaxon:4751 3078 3083 fungi +T57 NCBITaxon:5774 3088 3095 amoebae +T58 NCBITaxon:1 3116 3125 organisms +T59 PR:000016585 3159 3163 CLN2 +T60 NCBITaxon:1 3219 3228 organisms +T61 PR:000016585 3303 3307 CLN2 +T62 SO:0001026 3360 3367 genomic +T63 CHEBI:35222 3770 3780 inhibitors +T64 NCBITaxon:11118 3919 3930 coronavirus +T65 http://purl.obolibrary.org/obo/MONDO_0005091 3947 3980 severe acute respiratory syndrome +T66 UBERON:0001004 3960 3971 respiratory +T67 SO:0000857 4044 4052 homology +T68 PR:000016585 4091 4095 CLN2 +T69 CHEBI:35222 4226 4236 inhibitors +T70 PR:000016585 4291 4295 CLN2 +T71 NCBITaxon:40674 4310 4319 Mammalian +T72 SO:0000857 4328 4338 homologous +T73 NCBITaxon:9606 4342 4347 human +T74 PR:000016585 4348 4352 CLN2 +T75 GO:0010467 4452 4461 expressed +T76 SO:0001062 4469 4479 prosegment +T77 MOP:0000780 4515 4522 cleaved +T78 SO:0000417 4576 4582 domain +T79 PR:000016585 4621 4625 CLN2 +T80 NCBITaxon:species 4635 4642 species +T81 NCBITaxon:9606 4678 4683 human +T82 NCBITaxon:9539 4685 4692 macaque +T83 NCBITaxon:9615 4694 4697 dog +T84 NCBITaxon:10088 4699 4704 mouse +T85 NCBITaxon:10114 4706 4709 rat +T86 NCBITaxon:9913 4715 4718 cow +T87 SO:0001062 4831 4841 prosegment +T88 SO:0000417 4860 4866 domain +T89 NCBITaxon:10088 4890 4895 mouse +T90 PR:000016585 4896 4900 CLN2 +T91 SO:0001062 4935 4945 prosegment +T92 NCBITaxon:9606 5063 5068 human +T93 NCBITaxon:10088 5073 5078 mouse +T94 NCBITaxon:10088 5206 5211 mouse +T95 NCBITaxon:9606 5212 5217 human +T96 SO:0000855 5218 5227 orthologs +T97 NCBITaxon:40674 5240 5249 mammalian +T98 PR:000016585 5250 5254 CLN2 +T99 NCBITaxon:40674 5331 5340 mammalian +T100 PR:000016585 5341 5345 CLN2 +T101 SO:0001062 5425 5435 prosegment +T102 SO:0100019 5458 5469 active site +T103 MOP:0000780 5764 5772 cleavage +T104 NCBITaxon:40674 5878 5885 mammals +T105 PR:000016585 5887 5891 CLN2 +T106 NCBITaxon:31032 5923 5927 fugu +T107 NCBITaxon:31031 5929 5940 puffer fish +T108 NCBITaxon:31032 5993 5997 fugu +T109 NCBITaxon:7955 6023 6032 zebrafish +T110 SO:0000149 6034 6040 contig +T111 NCBITaxon:7955 6057 6066 zebrafish +T112 SO:0000345 6067 6070 EST +T113 NCBITaxon:7955 6224 6233 zebrafish +T114 NCBITaxon:31032 6280 6284 fugu +T115 PR:000016585 6285 6289 CLN2 +T116 SO:0000028 6414 6430 nucleotide pairs +T117 SO:0000704 6690 6694 gene +T118 NCBITaxon:7955 6711 6720 zebrafish +T119 PR:000016585 6721 6725 CLN2 +T120 NCBITaxon:31032 6832 6836 fugu +T121 NCBITaxon:31032 6954 6958 fugu +T122 PR:000016585 6959 6963 CLN2 +T123 NCBITaxon:40674 7055 7064 mammalian +T124 SO:0000855 7065 7074 orthologs +T125 GO:0006412 7120 7130 translated +T126 NCBITaxon:40674 7167 7176 mammalian +T127 NCBITaxon:7955 7181 7190 zebrafish +T128 SO:0001062 7231 7241 propeptide +T129 SO:0000717 7417 7429 coding frame +T130 SO:0001026 7515 7522 genomic +T131 NCBITaxon:31032 7551 7555 fugu +T132 SO:0000417 7618 7625 domains +T133 PR:000016585 7629 7633 CLN2 +T134 PR:000016585 7668 7672 CLN2 +T135 NCBITaxon:9606 7678 7683 human +T136 NCBITaxon:31032 7685 7689 fugu +T137 NCBITaxon:7955 7695 7704 zebrafish +T138 PR:000016585 7753 7757 CLN2 +T139 NCBITaxon:8364 7761 7779 Xenopus tropicalis +T140 SO:0001104 7877 7897 Active site residues +T141 PR:000016585 7926 7930 CLN2 +T142 NCBITaxon:8292 7972 7982 amphibians +T143 NCBITaxon:8364 8001 8019 Xenopus tropicalis +T144 NCBITaxon:species 8023 8030 species +T145 NCBITaxon:8342 8034 8038 frog +T146 SO:0000345 8105 8108 EST +T147 SO:0000417 8158 8164 domain +T148 SO:0100019 8199 8210 active site +T149 NCBITaxon:31032 8388 8392 fugu +T150 NCBITaxon:9606 8430 8435 human +T151 PR:000016585 8436 8440 CLN2 +T152 NCBITaxon:2 8476 8485 bacterial +T153 NCBITaxon:4751 8489 8495 fungal +T154 NCBITaxon:8342 8558 8563 frogs +T155 PR:000016585 8614 8618 CLN2 +T156 PR:000016585 8665 8669 CLN2 +T157 NCBITaxon:40674 8695 8702 mammals +T158 NCBITaxon:species 8724 8731 species +T159 NCBITaxon:8342 8743 8748 frogs +T160 NCBITaxon:7742 8815 8826 vertebrates +T161 NCBITaxon:9606 8872 8878 humans +T162 NCBITaxon:10088 8887 8891 mice +T163 NCBITaxon:9606 8960 8965 human +T164 PR:000016585 8966 8970 CLN2 +T165 PR:000016585 8998 9002 CLN2 +T166 GO:0016020 9127 9135 membrane +T167 GO:0016020 9252 9260 membrane +T168 GO:0031225 9261 9267 anchor +T169 CHEBI:15377 9344 9349 water +T170 PR:000016585 9447 9451 CLN2 +T171 SO:0000857 9589 9597 homology +T172 NCBITaxon:9606 9615 9620 human +T173 PR:000016585 9621 9625 CLN2 +T174 SO:0000857 9652 9660 homology +T175 GO:0032991 9690 9697 complex +T176 MOP:0000779 9718 9734 covalently-bound +T177 CHEBI:35222 9735 9744 inhibitor +T178 CHEBI:36357 9819 9827 compound +T179 NCBITaxon:9606 9840 9845 human +T180 PR:000016585 9846 9850 CLN2 +T181 CHEBI:17478 9883 9891 aldehyde +T182 CHEBI:35222 10057 10066 inhibitor +T183 PR:000016585 10082 10086 CLN2 +T184 CHEBI:35222 10137 10146 inhibitor +T185 GO:0032991 10281 10288 complex +T186 PR:000016585 10297 10301 CLN2 +T187 CHEBI:35222 10335 10344 inhibitor +T188 NCBITaxon:2 10467 10476 bacterial +T189 NCBITaxon:40674 10481 10490 mammalian +T190 NCBITaxon:9606 10558 10563 human +T191 PR:000016585 10564 10568 CLN2 +T192 PR:000016585 10754 10758 CLN2 +T193 CHEBI:29826 11020 11034 disulfide bond +T194 NCBITaxon:33208 11224 11230 animal +T195 NCBITaxon:species 11231 11238 species +T196 PR:000016585 11242 11246 CLN2 +T197 NCBITaxon:2 11317 11326 bacterial +T198 CHEBI:29826 11353 11362 disulfide +T199 PR:000016585 11401 11405 CLN2 +T200 SO:0000857 11478 11486 homology +T201 NCBITaxon:9606 11504 11509 human +T202 PR:000016585 11510 11514 CLN2 +T203 PR:000016585 11550 11554 CLN2 +T204 CHEBI:50325 11698 11709 Side chains +T205 SO:0000704 11763 11768 genes +T206 http://purl.obolibrary.org/obo/MONDO_0015674 11798 11843 late-infantile neuronal ceroid lipofuscinosis +T207 CL:0000540 11813 11821 neuronal +T208 PR:000016585 11928 11932 CLN2 +T209 PR:000016585 11986 11990 CLN2 +T210 CHEBI:35222 12185 12194 inhibitor +T211 GO:0032991 12287 12296 complexes +T212 CHEBI:35222 12337 12347 inhibitors +T213 NCBITaxon:9606 12719 12724 human +T214 PR:000016585 12725 12729 CLN2 +T215 CHEBI:50325 12824 12834 side chain +T216 CHEBI:35406 13037 13045 oxyanion +T217 CHEBI:50325 13139 13150 side chains +T218 CHEBI:46787 13266 13273 solvent +T219 SO:0000357 13356 13361 flank +T220 SO:0100019 13521 13532 active site +T221 NCBITaxon:9606 13536 13541 human +T222 PR:000016585 13542 13546 CLN2 +T223 GO:0032991 13571 13578 complex +T224 CHEBI:35222 13614 13623 inhibitor +T225 CHEBI:35222 13799 13808 inhibitor +T226 CHEBI:50325 14127 14138 side chains +T227 CHEBI:35222 14249 14258 inhibitor +T228 PR:000016585 14299 14303 CLN2 +T229 PR:000016585 14576 14580 CLN2 +T230 PR:000016585 14770 14774 CLN2 +T231 CHEBI:50325 14882 14892 side chain +T232 PR:000016585 14955 14959 CLN2 +T233 PR:000016585 15277 15281 CLN2 +T234 CHEBI:50325 15403 15414 side chains +T235 PR:000016585 15473 15477 CLN2 +T236 CHEBI:46787 15515 15522 solvent +T237 PR:000016585 15850 15854 CLN2 +T238 CHEBI:33250 15989 15994 atoms +T239 PR:000016585 16063 16067 CLN2 +T240 CHEBI:50325 16205 16215 side chain +T241 CHEBI:46882 16332 16337 amine +T242 PR:000016585 16378 16382 CLN2 +T243 CHEBI:24433 16576 16582 groups +T244 PR:000016585 16916 16920 CLN2 +T245 http://purl.obolibrary.org/obo/MONDO_0000001 16972 16979 disease +T246 SO:0000704 17019 17026 genetic +T247 http://purl.obolibrary.org/obo/MONDO_0015674 17057 17102 late-infantile neuronal ceroid lipofuscinosis +T248 CL:0000540 17072 17080 neuronal +T249 GO:0010467 17178 17188 expression +T250 SO:0000188 17232 17238 intron +T251 SO:0000147 17239 17243 exon +T252 GO:0008380 17244 17251 splices +T253 PR:000016585 17556 17560 CLN2 +T254 CHEBI:50325 17568 17578 side chain +T255 PR:000016585 17780 17784 CLN2 +T256 PR:000016585 18076 18080 CLN2 +T257 http://purl.obolibrary.org/obo/MONDO_0000001 18160 18167 disease +T258 CHEBI:35222 18185 18195 inhibitors +T259 PR:000016585 18199 18203 CLN2 +T260 PR:000016585 18276 18280 CLN2 +T261 PR:000016585 18352 18356 CLN2 +T262 SO:0000704 18380 18385 genes +T263 http://purl.obolibrary.org/obo/MONDO_0015674 18423 18468 late-infantile neuronal ceroid lipofuscinosis +T264 CL:0000540 18438 18446 neuronal +T265 http://purl.obolibrary.org/obo/MONDO_0000001 18502 18509 disease +T266 GO:0005764 18584 18593 lysosomal +T267 GO:0000322 18594 18608 storage bodies +T268 UBERON:0001016 18649 18663 nervous system +T269 PR:000022190 18725 18764 subunit c of mitochondrial ATP synthase +T270 GO:0005739 18738 18751 mitochondrial +T271 PR:000016585 18881 18885 CLN2 +T272 PR:000016585 19032 19036 CLN2 +T273 MOP:0000780 19142 19150 cleavage +T274 PR:000016585 19335 19339 CLN2 +T275 SO:0000409 19422 19434 binding site +T276 MOP:0000030 19570 19580 acetylated +T277 CHEBI:46882 19682 19693 amino group +T278 CHEBI:50325 19703 19713 side chain +T279 PR:000016585 19799 19803 CLN2 +T280 SO:0000409 20007 20019 binding site +T281 PR:000016585 20027 20031 CLN2 +T282 PR:000005110 20098 20113 cholecystokinin +T283 PR:000016585 20180 20184 CLN2 +T284 MOP:0000780 20212 20218 cleave +T285 NCBITaxon:192387 20445 20473 Alicyclobacillus sendaiensis +T286 NCBITaxon:192387 20487 20489 As +T287 MOP:0000780 20507 20513 cleave +T288 PR:000003263 20566 20581 type I collagen +T289 SO:0000409 20607 20619 binding site +T290 PR:000016585 20623 20627 CLN2 +T291 PR:000016585 20780 20784 CLN2 +T292 PR:000016585 20869 20873 CLN2 +T293 CHEBI:35222 21065 21075 inhibitors +T294 PR:000016585 21089 21093 CLN2 +T295 CHEBI:17478 21217 21225 aldehyde +T296 CHEBI:17087 21342 21348 ketone +T297 CHEBI:35222 21385 21395 inhibitors +T298 CHEBI:24433 21513 21519 groups +T299 CHEBI:35222 21561 21571 inhibitors +T300 PR:000016585 21799 21803 CLN2 +T301 CHEBI:24433 21932 21937 group +T302 CHEBI:35222 22047 22057 inhibitors +T303 PR:000016585 22114 22118 CLN2 +T304 SO:0000857 22153 22161 Homology +T305 CHEBI:33250 22191 22197 atomic +T306 PR:000016585 22214 22218 CLN2 +T307 CHEBI:33250 22361 22366 atoms +T308 SO:0000857 22961 22971 homologous +T309 NCBITaxon:9606 23207 23212 human +T310 PR:000016585 23213 23217 CLN2 +T311 NCBITaxon:9606 24031 24036 human +T312 PR:000016585 24037 24041 CLN2 +T313 GO:0032991 24085 24094 complexed +T314 CHEBI:35222 24104 24113 inhibitor +T315 CHEBI:35222 24257 24266 inhibitor +T316 PR:000016585 24322 24326 CLN2 +T317 CHEBI:33250 24393 24399 atomic +T318 SO:0000857 24491 24499 homology +T319 SO:0100019 24605 24616 active site +T320 CHEBI:50325 24754 24765 side chains +T321 CHEBI:50325 25228 25238 side chain +T322 SO:0000857 25407 25415 homology +T323 SO:0100019 25494 25505 active site +T324 CHEBI:33250 26031 26037 atomic +T325 CHEBI:24866 26058 26062 salt +T326 CHEBI:33250 26159 26165 atomic +T327 SO:0001026 26553 26560 genomic +T328 PR:000016585 26668 26672 CLN2 +T329 CHEBI:35222 26871 26881 inhibitors +T330 http://purl.obolibrary.org/obo/MONDO_0004992 27506 27512 Cancer +T331 NCBITaxon:9606 27703 27708 Human diff --git a/src/ontogpt/evaluation/craft/database/all/14609438.txt b/src/ontogpt/evaluation/craft/database/all/14609438.txt new file mode 100644 index 000000000..9ee59e502 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/14609438.txt @@ -0,0 +1,113 @@ +A model of tripeptidyl-peptidase I (CLN2), a ubiquitous and highly conserved member of the sedolisin family of serine-carboxyl peptidases + +Abstract + +Background + +Tripeptidyl-peptidase I, also known as CLN2, is a member of the family of sedolisins (serine-carboxyl peptidases). In humans, defects in expression of this enzyme lead to a fatal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis. Similar enzymes have been found in the genomic sequences of several species, but neither systematic analyses of their distribution nor modeling of their structures have been previously attempted. + +Results + +We have analyzed the presence of orthologs of human CLN2 in the genomic sequences of a number of eukaryotic species. Enzymes with sequences sharing over 80% identity have been found in the genomes of macaque, mouse, rat, dog, and cow. Closely related, although clearly distinct, enzymes are present in fish (fugu and zebra), as well as in frogs (Xenopus tropicalis). A three-dimensional model of human CLN2 was built based mainly on the homology with Pseudomonas sp. 101 sedolisin. + +Conclusion + +CLN2 is very highly conserved and widely distributed among higher organisms and may play an important role in their life cycles. The model presented here indicates a very open and accessible active site that is almost completely conserved among all known CLN2 enzymes. This result is somehow surprising for a tripeptidase where the presence of a more constrained binding pocket was anticipated. This structural model should be useful in the search for the physiological substrates of these enzymes and in the design of more specific inhibitors of CLN2. + +Background + +Although the existence of tripeptidyl-peptidase I (TPP-I) was first noted over 40 years ago [1], the structural and mechanistic basis of its activity has been largely misunderstood until quite recently. The situation changed after it was shown that TPP-I is identical to an independently characterized enzyme named CLN2. It was also demonstrated that mutations leading to abolishment of the enzymatic activity of CLN2 were the direct cause of a fatal inherited neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis [2]. This important observation was followed by the identification of CLN2 as a serine peptidase [3,4], without, however, specifying its structural fold and the details of the catalytic site. More accurate placement of CLN2 within the context of a family of related enzymes became possible only after high-resolution crystal structures of two bacterial enzymes with a limited sequence similarity to CLN2, sedolisin and kumamolisin, became available [5-7]. These structures defined a novel family of enzymes, now called sedolisins or serine-carboxyl peptidases, that is characterized by the utilization of a fully conserved catalytic triad (Ser, Glu, Asp) and by the presence of an Asp in the oxyanion hole [8]. Sedolisin and its several variants (e.g., kumamolisin, aorsin [9], and physarolisin [10]) have now been found in archaea, bacteria, fungi and amoebae, whereas the higher organisms seem to contain only variants of CLN2 [8]. The physiological role of sedolisins in the lower organisms has not yet been elucidated. + +Despite the potential medical importance of CLN2 and related enzymes, no systematic studies of their genomic distribution have been published to date. There are also no published reports of the crystallization of this enzyme. In the absence of an experimental structure obtained by crystallography or NMR it is sometimes necessary to resort to molecular modeling in order to provide a structural basis for the explanation of the biological properties of an enzyme, and, in particular, to initiate design of its inhibitors. Examples of such very successful and useful modeling efforts are provided by HIV protease [11], or very recently by the peptidase from a coronavirus involved in the severe acute respiratory syndrome [12], among others. We have now applied the tools of molecular homology modeling to predicting a structure of CLN2 that could be used as a basis for a search for the biological substrates of this family of enzymes and for the design of specific inhibitors. + +Results and discussion + +Sequence comparisons of the CLN2-like enzymes + +Mammalian enzymes homologous to human CLN2 [2,4] form a subfamily of sedolisins with highly conserved sequences (Figure 1). These enzymes are expressed with a prosegment consisting of 195 residues that is cleaved off during maturation, yielding the active catalytic domain. Complete sequences are available for CLN2 from six species in which it has been found so far (human, macaque, dog, mouse, rat, and cow). The full-length enzymes consist of 563 amino acids arranged in a single polypeptide chain containing both the prosegment and the catalytic domain, with the exception of mouse CLN2 that has a single deletion in the prosegment. The overall sequence identity for these enzymes is 81%, whereas the similarity is 92%. A pairwise comparison of the human and mouse enzymes yields 88% identity and 94% similarity, considerably higher than the median 78.5% identity reported for all identified mouse-human orthologs [13]. Thus, mammalian CLN2 appears to be a highly conserved enzyme. + +Figure 1 + +Sequence comparisons of mammalian CLN2-like enzymes.These sequences correspond to the complete enzymes, including the prosegment. Residues forming the active site are shown in yellow on red background, other conserved residues identified as important for the stability of the enzyme are marked with yellow background, residues identical in at least 5 of the structures are green, and residues similar in their character are shown in magenta. The maturation cleavage point generating the N terminus of the active enzyme is marked with black triangles. + +In addition to the mammals, CLN2-like enzymes are also found in fugu (puffer fish – unannotated record SINFRUP00000077297 in the NCBI fugu sequence database, ) and zebrafish (contig wz4596.2 in the zebrafish EST database, ). Only a fragment of the sequence of the latter enzyme agreed with the former. However, a few comparatively minor modifications can bring the zebrafish sequence into good agreement with that of the fugu CLN2. These modifications include a deletion of a single nucleotide from a run of three, as well as three insertions of repeated nucleotide pairs (Figure 2). It must be stressed that these modifications are speculative and may lead to prediction of several incorrect amino acids; however, they bring the two sequences into good global agreement (69% identities and 83% similarities). + +Figure 2 + +Corrected gene sequence of the zebrafish CLN2.This putative sequence shows the manual corrections that bring it into alignment with the sequence of the fugu enzyme. Inserted nucleotides are marked in green and a deleted one in red. + +The available amino acid sequence of the fugu CLN2 analog, named by us sedolisin-TPP [8], is also in good agreement with the sequences of the mammalian orthologs (Figure 3). The only major difference in the translated amino acid sequence compared to the mammalian and zebrafish enzymes is in the amino terminus of the propeptide region that is shorter by 30 amino acids (not shown). It is very likely that this represents a fault in the assembled sequence rather than a real variation, since the current coding frame is not initiated with a methionine, and a few extra residues are present in the full genomic sequence available from the fugu sequencing consortium . + +Figure 3 + +Sequences of the catalytic domains of CLN2. Complete sequences are shown for CLN2 from human, fugu, and zebrafish, together with the partial sequence of putative CLN2 in Xenopus tropicalis. Residues identical in all four enzymes are colored green and those similar are colored magenta. Active site residues are marked as in Figure 1. + +CLN2 is present not only in fish, but also in amphibians, in particular in Xenopus tropicalis (a species of frog). A partial sequence of its sedolisin-TPP (AL594774) found in the EST database spans the middle part of the catalytic domain, without reaching the part of the active site closer to the N terminus that contains the aspartic and glutamic acids that belong to the catalytic triad. However, the sequenced part of the enzyme shows 75% identity with the fugu sedolisin-TPP, and 69% identity with human CLN2 (Figure 3). Sequence similarity to bacterial or fungal sedolisins is much lower, indicating that the enzyme found in frogs might also share the functional properties of the CLN2 subfamily. + +The discovery of highly conserved CLN2-like enzymes not only in mammals but also in two fish species and one of frogs may indicate that these peptidases are universally present in the vertebrates, and that their important role identified in humans [2] and mice [14] might be a more general feature. + +Modeling of the structure of human CLN2 + +The medical importance of CLN2 and the lack of a crystal structure inspired attempts at protein modeling. The first such model assumed that this enzyme is membrane-bound, with the sequence 271–294 (numbering corresponding to the mature enzyme, Figure 1) forming the putative transmembrane anchor [15]. However, in view of the subsequently obtained structures of the fully water-soluble sedolisins, this model was clearly incorrect. Exploiting the sequence similarity between CLN2, sedolisin, and kumamolisin (Figure 4), we have now used the experimentally obtained structures of the latter two enzymes to form a new, homology-derived model of human CLN2. The primary basis of the homology model was the structure of a complex of sedolisin with a covalently-bound inhibitor, pseudo-iodotyrostatin. Although it has not been directly shown that this compound can inhibit human CLN2, other similar peptides with an aldehyde functionality on their "C-termini" are weak, but detectable, inhibitors of this enzyme (Oda, unpublished). It is thus likely that pseudo-iodotyrostatin or a similar inhibitor might work for CLN2 as well, although the actual contacts between the inhibitor and the enzyme that are seen in the model have to be treated with caution. Another reason for the modeling of a pseudo-iodotyrostatin complex is that CLN2 is a tripeptidase, and that this inhibitor it represents the only experimental structure of a tripeptide analog bound to sedolisin. + +Figure 4 + +Sequence alignment of bacterial and mammalian enzymes. Alignment of the sequences of sedolisin, kumamolisin, and human CLN2 used in the construction of the model of the latter enzyme. The colors scheme is the same as in Figure 2. + +The r.m.s. deviation between the corresponding Cα coordinates of the model of CLN2 (Figure 5) and the experimental structure of sedolisin is about 1.75 Å, not much larger than the experimental difference between sedolisin and kumamolisin. Interestingly, the Cys327 and Cys342 residues in the model were found to be ideally positioned to form a disulfide bond even though this was not part of the design strategy. That this bond likely occurs in the real protein is suggested by the fact that these two cysteines are strictly conserved in all known animal species of CLN2 (Figures 1 and 3), although they are absent in all known sequences of bacterial sedolisins. Thus, if this disulfide were experimentally found to exist in CLN2 it would provide support for the correctness of the model. + +Figure 5 + +A homology-derived model of human CLN2. Ribbon diagram of the Cα trace of CLN2, with the segments that were modeled based on the highly conserved core of sedolisin and kumamolisin (r.m.s. deviation of 1 Å) colored in red. Side chains of the residues that were found to be mutated in the genes of families of patients with late-infantile neuronal ceroid lipofuscinosis [17] are marked in ball-and-stick. + +Comparisons of the substrate binding pockets of CLN2 and sedolisin + +Since the principal known activity of CLN2 is that of a tripeptidase, it is expected that three substrate-binding pockets, S1 through S3 (using the nomenclature of Schechter and Berger [16]), should be discernible. Residues P1-P3 of the inhibitor that should occupy these pockets are shown in Figure 6. All the available structures of the complexes of either sedolisin or kumamolisin with inhibitors contain either a tyrosine or a phenylalanine occupying the S1 pocket. Parts of this pocket are fully conserved among different sedolisins, whereas other parts of it differ. The right-hand side of the pocket (in the view used in Figure 6) is made of the main chain including residues 164–165 (unless otherwise indicated, the numbering refers to the sequence of the mature human CLN2). This part of the main chain is held in place through a hydrogen-bonded interaction with the side chain of Thr279, part of the signature sequence SGTSAS surrounding the catalytic Ser280 and its equivalents in the other enzymes. Asp165 itself is also conserved since that residue provides the lining of the oxyanion hole, so it can be safely assumed that this part of the S1 pocket is virtually identical. No side chains point into the pocket from there, though, so its importance is limited to providing a steric barrier and excluding solvent. Another wall of the pocket is made up of the main chain of residues 130–132 that flank the conserved Gly131. Again, this part of the chain does not provide any specific interactions with the P1 residue of the substrate. + +Figure 6 + +A model of the active site of human CLN2. The enzyme is shown in complex with pseudo-iodotyrostatin, a good inhibitor of the sedolisin family of peptidases. Only selected residues of the enzyme are explicitly shown on the background consisting of the molecular surface. The stick model of the inhibitor is colored gold and the P1-P3 residues are labeled in black. Similar views have been previously published for the experimentally-determined structures of sedolisin and kumamolisin [8]. The figure was prepared using the program DINO . + +Considerable differences are seen, however, at the bottom of the pocket, where the side chains of Asp179 in kumamolisin and the equivalent Ser190 in sedolisin make hydrogen bonds to the P1 tyrosine of the inhibitor (if present). The equivalent residue in CLN2 is Thr182, but it is very unlikely that it can assume an orientation that would allow it to make a hydrogen bond to a P1 tyrosine. Other polar residues in the vicinity are Glu175 in sedolisin and the corresponding Asp169 in kumamolisin. However, the residue found here in CLN2 is Cys170, much less polar than its counterparts. There is also no equivalent to the polar interaction between Glu175 and Glu171 in sedolisin, since the equivalent of the latter residue in CLN2 is Ser166, much smaller and pointing away. + +The side of the S1 pocket that is created by the very flexible side chain of Arg179 in sedolisin contains only a much smaller Ser174 in CLN2, and thus is much more open in the latter protein. This part of the pocket, with the main chain of the protein quite distant from the substrate, is indeed not well conserved in these proteins, with kumamolisin missing it entirely due to a deletion in the corresponding sequence position. In summary, the S1 pocket in CLN2 has less polar character than the equivalent pocket in the related proteins, and is lacking direct polar anchors for any side chains that might be present in the substrate. + +The S2 pocket in CLN2 is also quite open and accessible to solvent. It is most likely larger than the equivalent pockets in either sedolisin or kumamolisin, since these are limited by Trp81 in the former and Trp129 in the latter (these residues originate from different parts of the backbone in the two enzymes and are not topologically related). Tyr130, an equivalent of the latter residue in CLN2, is unlikely to come into direct contact with the P2 residue of the substrate due to its greater distance (almost 4 Å for the closest atoms). + +An important remaining puzzle is that the predicted structure of CLN2 does not show any clear limitations of the S3 pocket that could explain the tripeptidase activity of this enzyme. The location of the P3 side chain of the substrate is ambiguous, since it could point in either one of two directions by exchange with the N-terminal amine. The only negatively charged residue of CLN2 that is found in this vicinity is Asp132. Although in the current model the distance between its carboxylate and the nitrogen of the N-terminus of the modeled substrate is about 6 Å, these two groups could be brought into hydrogen-bonding range by some allowed changes in the torsion angles of the protein. Such a conformational change would involve breaking of the hydrogen bond between Asp132 and Ser139. However, this latter interaction is not likely to be structurally crucial since the serine is not absolutely conserved in all CLN2-like enzymes. + +Location of mutations implicated in disease + +Location of mutations found through a genetic survey of families of classic late-infantile neuronal ceroid lipofuscinosis patients has been described previously [17]. Most such mutations result in expression of either truncated enzyme or in incorrect intron-exon splices. However, some of the mutations lead to single amino acid substitutions in the mature enzyme. Such mutations include I92N, E148K, C170R and C170Y, V190D, G194E, Q227H, R252H, A259E, and S280L (Figure 5). Only the role of the latter mutation is completely clear, since it replaces the catalytic serine of CLN2 with a side chain that cannot support its enzymatic activity. No other residues appear to be located in the immediate vicinity of the substrate. Residues Val190, Gly194, and Arg252 are very highly conserved not only in CLN2 but also in other sedolisins and must play an important structural role. The reasons why the remaining mutations would lead to the loss of enzymatic activity are much less clear, but the wide distribution of these mutations in the structure supports the conclusion that any modifications to CLN2 that would abolish or impair its function could lead to the development of the disease. + +Substrates and inhibitors of CLN2 + +Little is known at this time about biologically-relevant substrates of CLN2. Various defects that include truncations and single-site mutations in CLN2 have been found in the genes of patients that display symptoms of late-infantile neuronal ceroid lipofuscinosis [17]. One of the symptoms of the disease is the accumulation of an autofluorescent material, ceroid-lipofuscin, in lysosomal storage bodies in various cell types, primarily in the nervous system. Since a major component of such bodies appears to be intact subunit c of mitochondrial ATP synthase, this protein has been implicated as a potential biological target of the protease. It has been shown recently that CLN2 can indeed degrade this subunit on its N terminus [18], but the unambiguous proof that this is indeed the most important target is still lacking. CLN2 is capable of processing a number of different angiotensin-derived peptides [19], with the efficiency of cleavage dependent on the length of such peptides. The most efficiently processed peptide consisted of 14 amino acids, with the tripeptide Asp-Arg-Val removed from its N terminus. The model of CLN2 presented here can easily accommodate this peptide on the P side of the substrate-binding site, although the exact mode of binding of the long P' portion of the substrate remains obscure. The observation that an analogous peptide acetylated on its N terminus cannot be processed supports the postulate that the interactions of the N-terminal amino group with the side chain of Asp132 may be the most important feature defining the tripeptidase specificity of CLN2. A number of different tripeptides can be serially processed from glucagon, with their sequences varying widely [20]. Again, however, all of these tripeptides can be easily accommodated in the substrate-binding site of the CLN2 model. Other potentially biologically relevant substrates include cholecystokinin and possibly other neuropeptides [21]. + +An intriguing property of CLN2 is its reported ability to cleave collagen-related peptides [22]. The tripeptides resulting from such processing include Gly-Pro-Met, Gly-Pro-Arg, and Gly-Pro-Ala. It has been recently reported that kumamolisin, and particularly a closely related protein from Alicyclobacillus sendaiensis (kumamolisin-As) can efficiently cleave not only collagen-related peptides, but also native type I collagen [23]. With the substrate-binding site of CLN2 resembling that of kumamolisin more than sedolisin (the latter enzyme has low, if any, collagenase activity), the potential collagen-processing role of CLN2 might warrant further investigation. + +Conclusions + +Since the catalytic machinery of CLN2 matches closely that of sedolisin, kumamolisin, and other members of the family of serine-carboxyl peptidases, the enzymatic mechanism of all these enzymes is most likely the same. Design of inhibitors specific for CLN2 should incorporate the features that have been proven to be important for the related enzymes, such as the placement of an aldehyde functionality capable of making covalent interactions with the catalytic serine, or the utilization of chloromethyl ketone for the same purpose. Since the few inhibitors that have been successfully used in the studies of sedolisins are either longer than tripeptides or contain blocking groups on their N termini, new tripeptide-based inhibitors with free N termini are now being synthesized (Oda, unpublished). It will be necessary to test the binding properties of different substrates in order to determine the most promising peptide sequences. Analysis of the model of CLN2 suggests that the size of the S1 subsite is much larger than in either sedolisin or kumamolisin, and thus the use of a large P1 group might be indicated. Of course, the availability of an experimental crystal structure will make the design of inhibitors easier and we are continuing our efforts to crystallize CLN2 from different sources. + +Methods + +Homology modeling + +Three-dimensional, atomic-scale models of CLN2 were developed by exploiting the sequence similarity to the sedolisin and kumamolisin proteins (r.m.s. deviation of 1.0 Å for 273 pairs of Cα atoms in the core of the enzymes). Presently, these two enzymes are the only members of the newly-defined sedolisin/serine-carboxyl peptidase family [8] for which the crystal structures have been published [5-7]. The actual Protein Data Bank [24]: ) entries used in the modeling were 1GA4.pdb and 1GT9.pdb for sedolisin and kumamolisin, respectively. + +The first step was to form a global, multiple sequence alignment between all known members of the sedolisin family. Studies have shown that incorporating the specific patterns of amino acid residue-type variation and conservation among a family of homologous proteins provides superior results over simple, pair-wise sequence alignment [25]. Sequence files representing the different subfamilies were extracted from the non-redundant GenBank database [26] using sedolisin, kumamolisin, and the human CLN2 sequences as queries to the web-based version of the BLAST program [27]: . Initial multiple sequence alignments were formed with the ClustalX computer program [28]. As is expected for a family of proteins, highly-conserved segments were found aligned to the crystal structure-identified core regions of the sedolisin and kumamolisin sequences. Subsequently, the sequences were divided into two groups: those closer to sedolisin than kumamolisin and vise-a-versa. The alignment of these two groups was then manually set by the observed structural alignment of the sedolisin and kumamolisin proteins. Finally, some additional adjustment was required to correct the few places where highly conserved residues of the core regions were slightly out of alignment among different subfamilies of sequences. + +The model of human CLN2 was built using the structure of sedolisin complexed with the inhibitor pseudo-tyrostatin [5,6] as a template. The reason for this choice is that while different protein models were generally comparable, the chosen inhibitor was most compatible with the tripeptidase character of CLN2. With the correspondence of residues specified in the alignments, atomic coordinates were transferred to the target sequence by a variety of methods, including the homology modeling modules of the Look/GeneMine [29] and DeepView [30] computer program packages. For the core and active site of the protein, coordinates for identical residues were simply transferred unchanged; whereas, special care was required to position the side chains of residues differing from the template. This was first accomplished automatically by the two computer packages, then manually adjusted in the Quanta molecular modeling package (Accelrys, Inc.) to better mimic the templates and optimize the interactions with surrounding residues. A similar two-step approach was used to manifest the insertions and deletions in the variable, loop regions of the protein, where it was necessary to create new backbone as well as side chain coordinates for the models. It should be noted that, for obvious reasons, the conformation of poorly conserved loop regions is generally the least accurate aspect of a homology model. Fortunately, these problematic loops will not significantly affect the active site of the model, since only two of them impinge on the boundary of this highly conserved, functional region. + +Refinement and analysis of the model + +The model was finished by performing energy minimization in vacuo with the computer program CHARMM [31]. This refined the structure by bringing the covalent geometry and non-bonded interactions into agreement with experimentally observed and calculated values. Such optimizations included adjusting bond lengths, 3-point angles and 4-point dihedral angles, as well as eliminating atomic overlap and forming salt-bridges and hydrogen bonds. Since presently the potential energy functions used to describe the atomic-scale models are not sufficiently comprehensive and accurate, the final energy of the model was not used as an indicator of the realistic quality of the structure. The final quality of the structure was analyzed with the computer program PROCHECK [32]. The structure was deposited at the PDB under accession code 1R60. + +Authors' contributions + +AW initiated this project and analyzed the genomic distribution of this family of enzymes. SRD contributed the modeling of the three-dimensional structure of CLN2. ML analyzed the model and compared it to the crystal structures of sedolisins. HO, KO and BMD contributed their experience gained from studies of serine-carboxyl peptidases and the design of their inhibitors, aimed at analysis of substrate-enzyme interactions and enzyme specificity. All authors read and approved the final manuscript. + +Acknowledgements + +Extensive discussions with Dr. A. Barrett (Wellcome Trust Sanger Institute, Hinxton, UK) are gratefully acknowledged. Modeling efforts were facilitated by Dr. Robert Jernigan (Iowa State University). This work was supported in part by a Grant-in-Aid for Scientific Research (B), 15380072, from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to K.O.); by NIH grants DK18865 and AI39211 (to B.M.D.); and in part with Federal funds from the National Cancer Institute, National Institutes of Health, under contract No. NO1-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products or organizations imply endorsement by the U. S. Government. diff --git a/src/ontogpt/evaluation/craft/database/all/14611657.ann b/src/ontogpt/evaluation/craft/database/all/14611657.ann new file mode 100644 index 000000000..808b23a73 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/14611657.ann @@ -0,0 +1,1239 @@ +T1 GO:0010467 17 26 expressed +T2 SO:0000345 17 40 expressed sequence tags +T3 GO:0007608 53 62 olfactory +T4 GO:0010467 63 73 expression +T5 SO:0000704 86 91 genes +T6 GO:0000380 103 121 alternate splicing +T7 GO:0010467 134 144 expression +T8 NCBITaxon:10088 221 226 mouse +T9 GO:0007608 227 236 olfactory +T10 GO:0007608 285 294 olfactory +T11 NCBITaxon:10088 343 348 mouse +T12 GO:0007608 349 358 olfactory +T13 UBERON:0001997 349 369 olfactory epithelium +T14 GO:0007608 406 415 olfactory +T15 GO:0010467 425 434 expressed +T16 SO:0000345 425 448 expressed sequence tags +T17 GO:0007608 472 481 olfactory +T18 GO:0007608 511 520 olfactory +T19 SO:0000704 555 559 gene +T20 SO:0000167 583 591 promoter +T21 GO:0007608 628 637 olfactory +T22 SO:0000704 647 651 gene +T23 NCBITaxon:40674 688 697 mammalian +T24 SO:0001026 698 704 genome +T25 NCBITaxon:10088 774 779 mouse +T26 GO:0007608 780 789 olfactory +T27 GO:0007608 838 847 olfactory +T28 GO:0007608 883 892 olfactory +T29 NCBITaxon:10088 928 933 mouse +T30 GO:0007608 934 943 olfactory +T31 UBERON:0001997 934 954 olfactory epithelium +T32 GO:0007608 978 987 olfactory +T33 GO:0010467 997 1006 expressed +T34 SO:0000345 997 1020 expressed sequence tags +T35 GO:0007608 1044 1053 olfactory +T36 GO:0007608 1083 1092 olfactory +T37 SO:0000704 1127 1131 gene +T38 SO:0000167 1155 1163 promoter +T39 SO:0000704 1263 1268 genes +T40 GO:0010467 1301 1311 expression +T41 SO:0000673 1368 1378 transcript +T42 GO:0007608 1393 1402 olfactory +T43 UBERON:0001997 1393 1413 olfactory epithelium +T44 GO:0007608 1433 1442 olfactory +T45 SO:0000704 1492 1496 gene +T46 GO:0010467 1548 1558 expressing +T47 SO:0000673 1577 1587 transcript +T48 GO:0010467 1599 1609 expressing +T49 GO:0007608 1639 1648 olfactory +T50 SO:0001060 1719 1727 isoforms +T51 SO:0000204 1736 1738;1746 1766 5' ... untranslated regions +T52 SO:0000205 1743 1766 3' untranslated regions +T53 GO:0006412 1748 1758 translated +T54 SO:0000673 1773 1784 transcripts +T55 GO:0008380 1798 1804 splice +T56 SO:0000162 1798 1810 splice sites +T57 SO:0000851 1805 1817;1822 1835 sites within ... coding region +T58 GO:0007608 1865 1874 olfactory +T59 SO:0000704 1884 1888 gene +T60 SO:0000673 1914 1925 transcripts +T61 GO:0007608 1947 1956 olfactory +T62 GO:0007608 2061 2070 olfactory +T63 NCBITaxon:10088 2112 2117 mouse +T64 GO:0007608 2118 2127 olfactory +T65 SO:0000704 2153 2158 genes +T66 GO:0007608 2188 2197 olfactory +T67 GO:0007608 2272 2281 olfactory +T68 GO:0010467 2307 2317 expression +T69 CL:0000540 2353 2359 neuron +T70 SO:0000704 2365 2369 gene +T71 GO:0010467 2370 2380 expression +T72 GO:0007608 2391 2400 olfactory +T73 SO:0000204 2423 2445 5' untranslated region +T74 GO:0006412 2428 2438 translated +T75 SO:0000167 2470 2478 promoter +T76 GO:0007608 2505 2514 olfactory +T77 GO:0065007 2563 2573 regulatory +T78 GO:0007608 2607 2616 olfactory +T79 GO:0007608 2701 2710 olfactory +T80 GO:0007608 2832 2851 perception of smell +T81 GO:0007608 2857 2866 olfactory +T82 SO:0000704 2876 2880 gene +T83 NCBITaxon:40674 2917 2926 mammalian +T84 SO:0001026 2927 2933 genome +T85 NCBITaxon:10088 2973 2978 mouse +T86 SO:0001026 2979 2985 genome +T87 GO:0007608 2993 3002 Olfactory +T88 GO:0016020 3119 3127 membrane +T89 SO:0000417 3128 3134 domain +T90 SO:0000704 3172 3176 gene +T91 GO:0010467 3190 3200 expression +T92 GO:0007608 3222 3231 olfactory +T93 UBERON:0001997 3222 3242 olfactory epithelium +T94 GO:0010467 3375 3384 expressed +T95 NCBITaxon:9606 3501 3506 human +T96 NCBITaxon:10088 3517 3522 mouse +T97 GO:0007608 3529 3538 olfactory +T98 SO:0000704 3548 3552 gene +T99 SO:0000704 3604 3609 genes +T100 GO:0007608 3613 3622 olfactory +T101 NCBITaxon:10088 3727 3732 mouse +T102 GO:0007608 3733 3742 olfactory +T103 SO:0000704 3783 3787 gene +T104 GO:0007608 3819 3828 olfactory +T105 SO:0001026 3883 3889 genome +T106 SO:0000704 3938 3943 genes +T107 SO:0000704 4011 4016 genes +T108 GO:0010467 4078 4087 expressed +T109 UBERON:0000479 4101 4108 tissues +T110 SO:0000704 4145 4149 gene +T111 GO:0007608 4182 4191 olfactory +T112 GO:0010467 4216 4225 expressed +T113 GO:0007608 4233 4242 olfactory +T114 UBERON:0000479 4243 4250 tissues +T115 UBERON:0000473 4268 4274 testis +T116 GO:0050909 4289 4294 taste +T117 UBERON:0000479 4295 4302 tissues +T118 UBERON:0002367 4308 4316 prostate +T119 CL:0000764 4323 4338 erythroid cells +T120 UBERON:0002328 4345 4354 notochord +T121 UBERON:0000479 4378 4385 tissues +T122 GO:0010467 4387 4397 Expression +T123 UBERON:0000473 4405 4411 testis +T124 GO:0007608 4467 4476 olfactory +T125 CL:0000018 4503 4512 spermatid +T126 NCBITaxon:9606 4555 4560 human +T127 UBERON:0000473 4561 4567 testis +T128 GO:0010467 4568 4577 expressed +T129 GO:0007608 4578 4587 olfactory +T130 CL:0000019 4638 4643 sperm +T131 GO:0006935 4644 4654 chemotaxis +T132 GO:0007608 4712 4721 olfactory +T133 SO:0000704 4739 4744 genes +T134 GO:0007608 4784 4793 olfactory +T135 GO:0007608 4819 4828 olfactory +T136 GO:0010467 4838 4847 expressed +T137 SO:0000345 4838 4860 expressed sequence tag +T138 SO:0000345 4862 4865 EST +T139 GO:0007608 4886 4895 olfactory +T140 UBERON:0001997 4886 4906 olfactory epithelial +T141 GO:0010467 4907 4917 expression +T142 NCBITaxon:10088 4933 4938 mouse +T143 SO:0000704 4956 4961 genes +T144 GO:0007608 4975 4984 olfactory +T145 UBERON:0001997 4975 4995 olfactory epithelium +T146 GO:0007608 5008 5017 olfactory +T147 SO:0000704 5027 5032 genes +T148 GO:0010467 5052 5062 expression +T149 GO:0007608 5077 5086 olfactory +T150 GO:0010467 5099 5108 expressed +T151 UBERON:0000483 5158 5168 epithelium +T152 GO:0007608 5196 5205 olfactory +T153 CL:0000207 5196 5212 olfactory neuron +T154 GO:0010467 5213 5222 expresses +T155 SO:0001023 5232 5238 allele +T156 GO:0007608 5256 5265 olfactory +T157 SO:0000704 5275 5279 gene +T158 SO:0000704 5327 5332 genes +T159 GO:0010467 5403 5413 expression +T160 GO:0016444 5487 5512 somatic DNA recombination +T161 GO:0007608 5529 5538 olfactory +T162 SO:0000704 5548 5552 gene +T163 SO:0001026 5585 5592 genomic +T164 GO:0007618 5634 5640 mating +T165 SO:0001789 5634 5651 mating type locus +T166 NCBITaxon:40674 5665 5674 mammalian +T167 SO:0000704 5690 5695 genes +T168 SO:0000235 5764 5798 transcription factor binding sites +T169 SO:0000704 5814 5818 gene +T170 GO:0007608 5855 5864 olfactory +T171 SO:0000902 5874 5884 transgenes +T172 GO:0010467 5921 5930 expressed +T173 CL:0000540 5944 5951 neurons +T174 SO:0000167 6133 6141 promoter +T175 GO:0007608 6154 6163 olfactory +T176 SO:0000704 6173 6177 gene +T177 GO:0010467 6194 6204 expression +T178 NCBITaxon:5690 6231 6243 trypanosomes +T179 GO:0010467 6263 6273 expression +T180 GO:0009986 6301 6308 surface +T181 CHEBI:17089 6309 6321 glycoprotein +T182 SO:0000704 6322 6327 genes +T183 GO:0007608 6416 6425 olfactory +T184 SO:0001023 6435 6441 allele +T185 GO:0007608 6504 6513 olfactory +T186 GO:0010467 6568 6578 expression +T187 GO:0007608 6620 6629 olfactory +T188 SO:0000704 6639 6644 genes +T189 GO:0007608 6718 6727 olfactory +T190 SO:0000704 6737 6742 genes +T191 SO:0001532 6764 6784 recombination signal +T192 SO:0000315 6909 6935 transcriptional start site +T193 GO:0007608 6957 6966 olfactory +T194 SO:0000704 6976 6981 genes +T195 GO:0007608 6987 6996 olfactory +T196 SO:0000345 7006 7009 EST +T197 SO:0000204 7030 7052 5' untranslated region +T198 SO:0000204 7030 7031;7054 7057 5 ... UTR +T199 GO:0006412 7035 7045 translated +T200 SO:0000704 7078 7083 genes +T201 SO:0000167 7129 7137 promoter +T202 GO:0007608 7148 7157 Olfactory +T203 SO:0000704 7167 7172 genes +T204 SO:0000188 7181 7187 intron +T205 GO:0007608 7255 7264 olfactory +T206 GO:0007608 7312 7321 olfactory +T207 SO:0000704 7333 7337 gene +T208 SO:0000445 7380 7401 5' untranslated exons +T209 GO:0006412 7385 7395 translated +T210 GO:0000380 7447 7468 alternatively spliced +T211 SO:0000205 7485 7507 3' untranslated region +T212 GO:0006412 7490 7500 translated +T213 SO:0000188 7521 7527 intron +T214 NCBITaxon:9606 7603 7608 human +T215 GO:0007608 7609 7618 olfactory +T216 SO:0000704 7728 7732 gene +T217 SO:0000704 7756 7761 genes +T218 SO:0000147 7855 7860 exons +T219 SO:0000205 7883 7889 3' UTR +T220 SO:0001026 7895 7902 genomic +T221 GO:0008380 7959 7965 splice +T222 SO:0000162 7959 7970 splice site +T223 GO:0007608 8007 8016 olfactory +T224 SO:0000147 8072 8076 exon +T225 SO:0000704 8101 8106 genes +T226 GO:0007608 8224 8233 olfactory +T227 SO:0000704 8254 8258 gene +T228 SO:0000704 8422 8427 genes +T229 NCBITaxon:10088 8443 8448 mouse +T230 GO:0007608 8449 8458 olfactory +T231 UBERON:0001997 8449 8469 olfactory epithelium +T232 GO:0007608 8494 8503 olfactory +T233 SO:0000345 8551 8555 ESTs +T234 GO:0007608 8599 8608 olfactory +T235 GO:0010467 8609 8619 expression +T236 GO:0007608 8632 8641 olfactory +T237 SO:0000704 8667 8671 gene +T238 GO:0007608 8708 8717 olfactory +T239 GO:0010467 8732 8741 expressed +T240 GO:0007608 8779 8788 olfactory +T241 SO:0000704 8798 8803 genes +T242 SO:0001060 8834 8842 isoforms +T243 NCBITaxon:10088 8867 8872 mouse +T244 GO:0007608 8873 8882 olfactory +T245 SO:0000704 8892 8897 genes +T246 GO:0010467 8902 8911 expressed +T247 GO:0007608 8919 8928 olfactory +T248 UBERON:0001997 8919 8939 olfactory epithelium +T249 GO:0007608 8964 8973 olfactory +T250 SO:0000317 8983 8994 cDNA clones +T251 GO:0007608 9023 9032 olfactory +T252 UBERON:0001997 9023 9043 olfactory epithelial +T253 GO:0010467 9044 9054 expression +T254 GO:0007608 9072 9081 olfactory +T255 SO:0000704 9091 9096 genes +T256 GO:0097617 9121 9134 hybridization +T257 GO:0007608 9151 9160 olfactory +T258 UBERON:0007023 9241 9246 adult +T259 NCBITaxon:10088 9247 9252 mouse +T260 GO:0007608 9253 9262 olfactory +T261 UBERON:0001997 9253 9273 olfactory epithelium +T262 UBERON:0000922 9316 9325 embryonic +T263 GO:0007608 9326 9335 olfactory +T264 UBERON:0001997 9326 9346 olfactory epithelium +T265 GO:0097617 9389 9402 hybridization +T266 GO:0007608 9503 9512 olfactory +T267 GO:0097617 9613 9626 hybridization +T268 GO:0007608 9725 9734 olfactory +T269 GO:0007608 9812 9821 olfactory +T270 GO:0007608 9875 9884 olfactory +T271 SO:0000857 10132 10140 homology +T272 SO:0001031 10169 10176 reverse +T273 SO:0001026 10195 10202 genomic +T274 GO:0007608 10238 10247 olfactory +T275 SO:0000204 10289 10295 5' UTR +T276 SO:0000028 10303 10305 bp +T277 SO:0001026 10332 10339 genomic +T278 GO:0008380 10353 10360 spliced +T279 SO:0000188 10381 10387 intron +T280 SO:0001026 10457 10464 genomic +T281 GO:0007608 10627 10636 olfactory +T282 SO:0000704 10664 10668 gene +T283 SO:0000318 10671 10682 start codon +T284 SO:0000204 10701 10707 5' UTR +T285 SO:0001026 10752 10759 genomic +T286 NCBITaxon:10088 10806 10811 mouse +T287 GO:0007608 10812 10821 olfactory +T288 SO:0000704 10831 10836 genes +T289 SO:0001026 10871 10877 genome +T290 NCBITaxon:10088 10930 10935 mouse +T291 GO:0007608 10936 10945 olfactory +T292 SO:0001248 11048 11056 scaffold +T293 SO:0000730 11093 11097 gaps +T294 GO:0007608 11190 11199 olfactory +T295 GO:0007608 11263 11272 olfactory +T296 SO:0000704 11282 11287 genes +T297 GO:0007608 11312 11321 olfactory +T298 SO:0001026 11481 11488 genomic +T299 GO:0007608 11513 11522 olfactory +T300 SO:0000357 11548 11556 flanking +T301 GO:0007608 11623 11632 olfactory +T302 SO:0001026 11636 11642 genome +T303 SO:0000704 11705 11710 genes +T304 GO:0007608 11743 11752 olfactory +T305 GO:0007608 11841 11850 olfactory +T306 GO:0007608 11896 11905 olfactory +T307 SO:0000704 11915 11920 genes +T308 GO:0007608 11960 11969 olfactory +T309 GO:0007608 12075 12084 olfactory +T310 SO:0000704 12094 12098 gene +T311 GO:0007608 12130 12139 olfactory +T312 SO:0000112 12160 12166 primer +T313 GO:0007608 12215 12224 olfactory +T314 SO:0000704 12234 12239 genes +T315 NCBITaxon:40674 12266 12275 mammalian +T316 GO:0007608 12276 12285 olfactory +T317 GO:0007608 12370 12379 olfactory +T318 GO:0007608 12416 12425 olfactory +T319 GO:0007608 12461 12470 olfactory +T320 GO:0010467 12608 12618 expression +T321 GO:0007608 12635 12644 olfactory +T322 SO:0000704 12654 12658 gene +T323 GO:0007608 12695 12704 olfactory +T324 SO:0000704 12714 12719 genes +T325 GO:0007608 12768 12777 olfactory +T326 SO:0000704 12787 12792 genes +T327 SO:0001026 12811 12818 genomic +T328 GO:0007608 12936 12945 olfactory +T329 GO:0007608 13051 13060 olfactory +T330 GO:0097617 13130 13143 hybridization +T331 UBERON:0007023 13196 13201 adult +T332 UBERON:0000922 13226 13235 embryonic +T333 SO:0000112 13449 13456 primers +T334 GO:0097617 13471 13484 hybridization +T335 SO:0000704 13515 13520 genes +T336 GO:0010467 13530 13539 expressed +T337 GO:0007608 13571 13580 olfactory +T338 UBERON:0001997 13571 13591 olfactory epithelium +T339 GO:0007608 13631 13640 olfactory +T340 GO:0010467 13655 13664 expressed +T341 GO:0007608 13672 13681 olfactory +T342 UBERON:0001997 13672 13692 olfactory epithelium +T343 SO:0000132 13708 13722;13734 13740 reverse-strand ... primer +T344 GO:0016020 13770 13778 membrane +T345 SO:0000417 13779 13785 domain +T346 GO:0007608 13836 13845 olfactory +T347 GO:0007608 13904 13913 olfactory +T348 SO:0000112 13963 13969 primer +T349 SO:0000704 14028 14033 genes +T350 SO:0000704 14086 14091 genes +T351 GO:0007608 14120 14129 olfactory +T352 SO:0000112 14150 14156 primer +T353 SO:0000112 14173 14179 primer +T354 GO:0007608 14213 14222 olfactory +T355 NCBITaxon:10088 14238 14243 mouse +T356 SO:0001026 14244 14251 genomic +T357 SO:0000006 14281 14293 PCR products +T358 GO:0007608 14334 14343 olfactory +T359 GO:0007608 14396 14405 olfactory +T360 GO:0007608 14456 14465 olfactory +T361 GO:0001171 14534 14553 reverse-transcribed +T362 GO:0007608 14554 14563 olfactory +T363 UBERON:0001997 14554 14574 olfactory epithelium +T364 GO:0007608 14614 14623 olfactory +T365 GO:0010467 14638 14647 expressed +T366 SO:0000704 14684 14688 gene +T367 UBERON:0007023 14764 14769 adult +T368 GO:0007608 14783 14792 olfactory +T369 UBERON:0001997 14783 14803 olfactory epithelium +T370 SO:0000112 14837 14843 primer +T371 GO:0007608 14910 14919 olfactory +T372 GO:0010467 14949 14959 expression +T373 GO:0007608 14979 14988 olfactory +T374 SO:0000704 15126 15130 gene +T375 SO:0000112 15140 15146 primer +T376 GO:0001171 15180 15199 reverse-transcribed +T377 SO:0000704 15239 15244 genes +T378 GO:0010467 15264 15273 expressed +T379 SO:0000673 15298 15308 transcript +T380 GO:0010467 15355 15365 Expression +T381 SO:0000704 15408 15413 genes +T382 GO:0007608 15484 15493 olfactory +T383 GO:0007608 15537 15546 olfactory +T384 UBERON:0001997 15537 15557 olfactory epithelium +T385 GO:0097617 15662 15675 hybridization +T386 GO:0007608 15690 15699 olfactory +T387 SO:0000704 15709 15714 genes +T388 GO:0010467 15719 15728 expressed +T389 GO:0007608 15794 15803 olfactory +T390 SO:0000704 15813 15818 genes +T391 GO:0010467 15823 15832 expressed +T392 GO:0007608 15870 15879 olfactory +T393 SO:0000704 15889 15894 genes +T394 GO:0010467 15900 15909 expressed +T395 GO:0007608 16002 16011 olfactory +T396 GO:0007608 16065 16074 olfactory +T397 GO:0007608 16186 16195 olfactory +T398 GO:0007608 16280 16289 olfactory +T399 SO:0000336 16304 16315 pseudogenes +T400 SO:0000704 16427 16431 gene +T401 GO:0007608 16515 16524 olfactory +T402 GO:0007608 16561 16570 olfactory +T403 GO:0010467 16649 16658 expressed +T404 GO:0007608 16687 16696 olfactory +T405 SO:0000704 16706 16711 genes +T406 SO:0000704 16732 16737 genes +T407 SO:0001026 16808 16819 genomically +T408 GO:0007608 16853 16862 olfactory +T409 CL:0000084 16892 16898 T-cell +T410 GO:0042101 16892 16907 T-cell receptor +T411 GO:0007608 16955 16964 olfactory +T412 GO:0010467 16989 16999 expression +T413 SO:0000704 17043 17048 genes +T414 GO:0007608 17098 17107 olfactory +T415 UBERON:0001997 17098 17118 olfactory epithelium +T416 SO:0000673 17119 17129 transcript +T417 GO:0007608 17144 17153 olfactory +T418 SO:0000704 17163 17168 genes +T419 GO:0005840 17177 17186 ribosomal +T420 PR:000014258 17177 17190 ribosomal S16 +T421 SO:0000704 17191 17195 gene +T422 NCBITaxon:10088 17205 17209 mice +T423 SO:0000704 17254 17259 genes +T424 GO:0007608 17272 17281 olfactory +T425 GO:0007608 17377 17386 olfactory +T426 SO:0000673 17449 17459 transcript +T427 SO:0001026 17470 17477 genomic +T428 SO:0000704 17490 17494 gene +T429 SO:0000704 17519 17523 gene +T430 SO:0000112 17533 17539 primer +T431 GO:0001171 17555 17574 reverse-transcribed +T432 GO:0006277 17612 17628;17643 17646 amplification of ... DNA +T433 NCBITaxon:10088 17629 17634 mouse +T434 SO:0001026 17635 17642 genomic +T435 GO:0010467 17661 17671 expression +T436 SO:0000704 17715 17720 genes +T437 SO:0000704 17735 17740 genes +T438 GO:0010467 17811 17821 expression +T439 SO:0000112 17910 17917 primers +T440 SO:0000704 17960 17965 genes +T441 SO:0000704 18029 18034 genes +T442 SO:0000205 18048 18055 3' UTRs +T443 GO:0010467 18088 18098 expression +T444 NCBITaxon:10088 18124 18128 mice +T445 SO:0000704 18141 18146 genes +T446 SO:0000704 18194 18199 genes +T447 GO:0010467 18244 18254 expression +T448 SO:0000704 18274 18279 genes +T449 SO:0000704 18333 18338 genes +T450 GO:0010467 18359 18369 Expression +T451 NCBITaxon:10088 18417 18421 mice +T452 GO:0010467 18439 18449 expression +T453 GO:0007608 18476 18485 olfactory +T454 SO:0000704 18495 18500 genes +T455 NCBITaxon:10088 18514 18518 mice +T456 SO:0000704 18564 18569 genes +T457 NCBITaxon:10088 18597 18601 mice +T458 GO:0010467 18635 18645 expression +T459 SO:0000704 18660 18665 genes +T460 NCBITaxon:10088 18674 18678 mice +T461 SO:0000704 18693 18697 gene +T462 GO:0097617 18721 18734 hybridization +T463 GO:0010467 18778 18788 expressing +T464 SO:0000673 18847 18857 transcript +T465 SO:0000704 18884 18889 genes +T466 SO:0000704 18915 18920 genes +T467 GO:0097617 18946 18956 hybridized +T468 GO:0007608 19010 19019 olfactory +T469 UBERON:0001997 19010 19030 olfactory epithelium +T470 NCBITaxon:10088 19042 19047 mouse +T471 SO:0000704 19069 19073 gene +T472 SO:0000704 19080 19084 gene +T473 GO:0097617 19157 19166 hybridize +T474 SO:0000704 19192 19197 genes +T475 SO:0000704 19225 19229 Gene +T476 GO:0010467 19235 19244 expressed +T477 UBERON:0000483 19262 19272 epithelium +T478 GO:0010467 19344 19354 expression +T479 SO:0000704 19366 19370 gene +T480 GO:0007608 19424 19433 olfactory +T481 UBERON:0001997 19424 19444 olfactory epithelial +T482 GO:0010467 19564 19574 expression +T483 GO:0007608 19631 19640 olfactory +T484 UBERON:0000483 19756 19766 epithelium +T485 SO:0000704 19781 19785 gene +T486 GO:0010467 19791 19800 expressed +T487 SO:0000704 19848 19852 gene +T488 GO:0010467 19865 19874 expressed +T489 GO:0010467 19938 19948 expressing +T490 SO:0000673 20066 20076 transcript +T491 GO:0010467 20087 20097 expressing +T492 SO:0000704 20107 20111 gene +T493 SO:0000673 20144 20154 transcript +T494 GO:0010467 20169 20179 expressing +T495 SO:0000704 20189 20193 gene +T496 GO:0097617 20210 20223 hybridization +T497 CL:0000540 20249 20255 neuron +T498 SO:0000704 20276 20280 gene +T499 SO:0000704 20288 20292 gene +T500 SO:0000673 20361 20371 transcript +T501 GO:0010467 20422 20432 expression +T502 GO:0007608 20525 20534 olfactory +T503 UBERON:0001997 20525 20545 olfactory epithelial +T504 SO:0000704 20560 20564 gene +T505 SO:0000704 20579 20583 gene +T506 GO:0007608 20604 20613 olfactory +T507 SO:0000704 20623 20628 genes +T508 SO:0001060 20658 20666 isoforms +T509 GO:0007608 20728 20737 olfactory +T510 GO:0000380 20761 20781 alternative splicing +T511 SO:0000445 20791 20812 5' untranslated exons +T512 GO:0006412 20796 20806 translated +T513 SO:0001026 20875 20882 genomic +T514 GO:0008380 20925 20931 splice +T515 SO:0000704 20962 20967 genes +T516 GO:0000380 21018 21036 alternate splicing +T517 GO:0008380 21096 21103 spliced +T518 SO:0000704 21129 21134 genes +T519 GO:0000380 21215 21233 alternate splicing +T520 GO:0000380 21253 21271 alternative splice +T521 SO:0000204 21312 21318 5' UTR +T522 SO:0000147 21331 21335 exon +T523 GO:0000380 21349 21365 alternate splice +T524 GO:0007608 21413 21422 olfactory +T525 GO:0043631 21490 21505 polyadenylation +T526 SO:0000553 21490 21510 polyadenylation site +T527 SO:0000205 21537 21543 3' UTR +T528 SO:0001060 21544 21552 isoforms +T529 SO:0000205 21580 21586 3' UTR +T530 SO:0000317 21602 21613 cDNA clones +T531 SO:0000667 21639 21645 insert +T532 SO:0000205 21700 21706 3' UTR +T533 SO:0001060 21707 21714 isoform +T534 SO:0000704 21751 21756 genes +T535 SO:0000205 21802 21808 3' UTR +T536 GO:0043631 21856 21871 polyadenylation +T537 SO:0001060 21872 21880 isoforms +T538 SO:0000704 21910 21915 genes +T539 SO:0000317 21947 21958 cDNA clones +T540 SO:0000610 22012 22024 poly(A) tail +T541 SO:0000028 22038 22040 bp +T542 SO:0000319 22052 22062 stop codon +T543 SO:0000188 22085 22091 intron +T544 GO:0008380 22096 22103 spliced +T545 SO:0000205 22115 22121 3' UTR +T546 GO:0007608 22166 22175 olfactory +T547 SO:0000704 22185 22189 gene +T548 GO:0007608 22214 22223 olfactory +T549 GO:0008380 22248 22256 splicing +T550 GO:0007608 22292 22301 olfactory +T551 GO:0007608 22334 22343 olfactory +T552 GO:0007608 22362 22371 olfactory +T553 SO:0000336 22381 22391 pseudogene +T554 GO:0008380 22400 22406 splice +T555 SO:0000162 22400 22411 splice site +T556 SO:0000851 22407 22418;22431 22444 site within ... coding region +T557 SO:0000010 22423 22437 protein-coding +T558 SO:0000704 22462 22467 genes +T559 GO:0007608 22541 22550 olfactory +T560 SO:0000147 22655 22659 exon +T561 SO:0000704 22671 22675 gene +T562 NCBITaxon:9606 22717 22722 human +T563 GO:0007608 22723 22732 olfactory +T564 NCBITaxon:10088 22765 22770 mouse +T565 SO:0000704 22771 22776 genes +T566 SO:0000318 22784 22795 start codon +T567 SO:0000195 22825 22836 coding exon +T568 GO:0008380 22850 22858 splicing +T569 SO:0000704 22912 22916 gene +T570 SO:0000673 22966 22976 transcript +T571 GO:0008380 23003 23009 splice +T572 SO:0000673 23021 23031 transcript +T573 GO:0008380 23091 23097 splice +T574 SO:0000147 23220 23224 exon +T575 SO:0000317 23238 23248 cDNA clone +T576 SO:0001026 23288 23295 genomic +T577 SO:0000673 23454 23465 transcripts +T578 GO:0008380 23533 23541 splicing +T579 SO:0001026 23554 23561 genomic +T580 GO:0007608 23616 23625 olfactory +T581 SO:0000673 23635 23645 transcript +T582 SO:0000704 23675 23680 genes +T583 GO:0008380 23701 23708 spliced +T584 SO:0001060 23769 23776 isoform +T585 GO:0008380 23795 23801 splice +T586 SO:0000162 23795 23807 splice sites +T587 SO:0000851 23802 23814;23819 23832 sites within ... coding region +T588 SO:0000704 23866 23871 genes +T589 GO:0008380 23884 23892 splicing +T590 SO:0001060 23965 23973 isoforms +T591 GO:0008380 24035 24043 splicing +T592 NCBITaxon:9606 24047 24052 human +T593 GO:0007608 24053 24062 olfactory +T594 GO:0042611 24082 24114 major histocompatibility complex +T595 GO:0042611 24116 24119 MHC +T596 SO:0000704 24144 24149 genes +T597 GO:0008380 24150 24156 splice +T598 SO:0000147 24198 24202 exon +T599 GO:0007608 24232 24241 olfactory +T600 GO:0065007 24267 24274 control +T601 SO:0000673 24355 24366 transcripts +T602 GO:0007608 24470 24479 olfactory +T603 SO:0000345 24489 24492 EST +T604 SO:0000704 24519 24523 gene +T605 SO:0001026 24526 24533 genomic +T606 SO:0000704 24605 24609 gene +T607 SO:0000704 24628 24632 gene +T608 SO:0000319 24635 24645 stop codon +T609 SO:0000188 24672 24679 introns +T610 GO:0007608 24698 24707 olfactory +T611 SO:0000704 24717 24722 genes +T612 GO:0007608 24740 24749 olfactory +T613 NCBITaxon:9606 24767 24772 human +T614 GO:0042611 24773 24776 MHC +T615 SO:0000704 24835 24839 gene +T616 GO:0008380 24963 24969 splice +T617 SO:0000162 24963 24975 splice sites +T618 SO:0000837 24970 24982;24990 24993 sites within ... UTR +T619 SO:0000205 24987 24993 3' UTR +T620 SO:0000319 25060 25070 stop codon +T621 GO:0008380 25105 25111 splice +T622 SO:0000851 25118 25130;25135 25148 sites within ... coding region +T623 GO:0007608 25170 25179 olfactory +T624 SO:0000188 25230 25236 intron +T625 GO:0008380 25240 25247 spliced +T626 SO:0000205 25259 25265 3' UTR +T627 GO:0007608 25305 25314 olfactory +T628 GO:0065007 25340 25347 control +T629 SO:0000673 25404 25414 transcript +T630 SO:0000167 25443 25451 promoter +T631 SO:0000673 25481 25491 transcript +T632 SO:0000236 25515 25518 ORF +T633 SO:0000317 25603 25614 cDNA clones +T634 SO:0000028 25649 25651 bp +T635 SO:0001026 25681 25688 genomic +T636 SO:0000730 25749 25753 gaps +T637 SO:0001026 25761 25768 genomic +T638 GO:0007608 25903 25912 olfactory +T639 SO:0001026 25994 26000 genome +T640 NCBITaxon:10088 26029 26034 mouse +T641 SO:0001026 26035 26041 genome +T642 SO:0000810 26206 26226 chimeric cDNA clones +T643 GO:0007608 26362 26371 olfactory +T644 GO:0065007 26397 26407 regulation +T645 SO:0000167 26451 26459 promoter +T646 SO:0000833 26477 26484;26489 26499 part of ... transcript +T647 GO:0007608 26519 26528 olfactory +T648 GO:0007608 26543 26552 olfactory +T649 SO:0000336 26562 26573 pseudogenes +T650 GO:0010467 26581 26590 expressed +T651 GO:0007608 26622 26631 olfactory +T652 SO:0001026 26678 26684 genome +T653 SO:0001026 26733 26740 genomic +T654 GO:0007608 26757 26766 olfactory +T655 SO:0000704 26877 26881 gene +T656 GO:0007608 26906 26915 olfactory +T657 GO:0007608 26950 26959 olfactory +T658 GO:0007608 26986 26995 olfactory +T659 SO:0000336 27035 27046 pseudogenes +T660 GO:0007608 27056 27065 olfactory +T661 GO:0007608 27178 27187 olfactory +T662 GO:0007608 27276 27285 olfactory +T663 GO:0007608 27368 27377 olfactory +T664 GO:0010467 27387 27397 expression +T665 GO:0010467 27433 27442 expressed +T666 SO:0000704 27453 27458 genes +T667 GO:0007608 27482 27491 olfactory +T668 GO:0007608 27526 27535 olfactory +T669 GO:0007608 27621 27630 olfactory +T670 SO:0000336 27640 27651 pseudogenes +T671 GO:0010467 27656 27665 expressed +T672 GO:0007608 27711 27720 olfactory +T673 GO:0007608 27754 27763 olfactory +T674 SO:0000704 27773 27777 gene +T675 SO:0000336 27867 27878 pseudogenes +T676 GO:0007608 27961 27970 olfactory +T677 GO:0010467 28010 28019 expressed +T678 SO:0000336 28020 28031 pseudogenes +T679 SO:0000704 28044 28049 genes +T680 NCBITaxon:10088 28064 28069 mouse +T681 SO:0001026 28070 28076 genome +T682 SO:0000704 28128 28133 genes +T683 NCBITaxon:10088 28207 28212 mouse +T684 GO:0010467 28259 28268 expressed +T685 SO:0000336 28269 28280 pseudogenes +T686 SO:0000336 28292 28303 pseudogenes +T687 SO:0000343 28315 28331 sequence matches +T688 GO:0010467 28342 28351 expressed +T689 SO:0000336 28352 28362 pseudogene +T690 GO:0007608 28475 28484 olfactory +T691 SO:0000704 28494 28499 genes +T692 GO:0010467 28518 28528 expression +T693 GO:0007608 28536 28545 olfactory +T694 UBERON:0001997 28536 28556 olfactory epithelium +T695 GO:0007608 28645 28654 olfactory +T696 SO:0000704 28664 28669 genes +T697 NCBITaxon:10088 28687 28692 mouse +T698 SO:0001026 28693 28699 genome +T699 GO:0007608 28797 28806 olfactory +T700 GO:0007608 28862 28871 olfactory +T701 SO:0000704 28881 28886 genes +T702 GO:0007608 29009 29018 olfactory +T703 SO:0000112 29088 29095 primers +T704 GO:0007608 29115 29124 olfactory +T705 GO:0010467 29125 29135 expression +T706 GO:0007608 29162 29171 olfactory +T707 GO:0007608 29212 29221 olfactory +T708 SO:0000704 29231 29236 genes +T709 GO:0007608 29367 29376 olfactory +T710 GO:0007608 29443 29452 olfactory +T711 GO:0010467 29467 29476 expressed +T712 GO:0010467 29559 29569 expression +T713 GO:0007608 29614 29623 olfactory +T714 GO:0010467 29633 29642 expressed +T715 GO:0010467 29692 29702 expression +T716 SO:0000673 29736 29746 transcript +T717 GO:0007608 29790 29799 olfactory +T718 CL:0000207 29790 29807 olfactory neurons +T719 SO:0000704 29825 29830 genes +T720 SO:0000704 29848 29853 genes +T721 GO:0010467 29865 29875 expression +T722 SO:0000704 29983 29988 genes +T723 GO:0010467 30021 30031 expressing +T724 SO:0000673 30042 30052 transcript +T725 GO:0007608 30085 30094 olfactory +T726 GO:0007608 30176 30185 olfactory +T727 SO:0000704 30195 30200 genes +T728 GO:0007608 30229 30238 olfactory +T729 SO:0000902 30248 30257 transgene +T730 SO:0001026 30271 30278 genomic +T731 GO:0010467 30293 30302 expressed +T732 SO:0000673 30377 30387 transcript +T733 SO:0000704 30423 30428 genes +T734 GO:0010467 30480 30489 expressed +T735 GO:0007608 30542 30551 olfactory +T736 CL:0000207 30542 30558 olfactory neuron +T737 SO:0001023 30568 30574 allele +T738 GO:0010467 30575 30585 expression +T739 GO:0010467 30621 30631 expression +T740 GO:0007608 30686 30695 olfactory +T741 GO:0010467 30708 30717 expressed +T742 GO:0007608 30746 30755 olfactory +T743 UBERON:0001997 30746 30766 olfactory epithelium +T744 GO:0007608 30812 30821 olfactory +T745 GO:0010467 30855 30862 express +T746 GO:0007608 30868 30877 olfactory +T747 GO:0010467 30955 30964 expressed +T748 GO:0007608 30965 30974 olfactory +T749 SO:0000704 30986 30990 gene +T750 GO:0010467 31036 31045 expressed +T751 GO:0007608 31063 31072 olfactory +T752 UBERON:0001997 31063 31083 olfactory epithelium +T753 CL:0000540 31109 31117 neuronal +T754 GO:0007608 31181 31190 olfactory +T755 GO:0007608 31263 31272 olfactory +T756 SO:0000167 31297 31305 promoter +T757 GO:0007608 31326 31335 olfactory +T758 SO:0000673 31394 31404 transcript +T759 GO:0010467 31416 31426 expressing +T760 GO:0007608 31449 31458 olfactory +T761 SO:0001747 31477 31491 open chromatin +T762 GO:0000785 31482 31491 chromatin +T763 SO:0001026 31524 31531 genomic +T764 GO:0007608 31581 31590 olfactory +T765 SO:0000704 31600 31605 genes +T766 GO:0007608 31645 31654 olfactory +T767 SO:0000704 31664 31668 gene +T768 SO:0001026 31721 31728 genomic +T769 GO:0007608 31747 31756 olfactory +T770 GO:0007608 31825 31834 olfactory +T771 SO:0000902 31844 31854 transgenes +T772 GO:0010467 31862 31871 expressed +T773 GO:0007608 31913 31922 olfactory +T774 SO:0000336 31965 31975 pseudogene +T775 GO:0007608 31998 32007 olfactory +T776 SO:0000704 32017 32022 genes +T777 GO:0007608 32054 32063 olfactory +T778 SO:0000704 32073 32078 genes +T779 SO:0001026 32102 32109 genomic +T780 GO:0007608 32138 32147 olfactory +T781 SO:0000704 32157 32161 gene +T782 SO:0000336 32293 32303 pseudogene +T783 GO:0007608 32325 32334 olfactory +T784 SO:0000704 32347 32351 gene +T785 GO:0035822 32347 32362 gene conversion +T786 GO:0007608 32383 32392 olfactory +T787 SO:0000704 32452 32457 genes +T788 GO:0007608 32508 32517 olfactory +T789 SO:0000704 32527 32531 gene +T790 GO:0010467 32588 32598 expression +T791 SO:0000704 32628 32633 genes +T792 GO:0007608 32652 32661 olfactory +T793 SO:0000704 32671 32675 gene +T794 SO:0000673 32749 32759 transcript +T795 GO:0007608 32783 32792 olfactory +T796 SO:0000704 32802 32807 genes +T797 SO:0000167 32866 32874 promoter +T798 GO:0065007 33000 33010 regulation +T799 GO:0007608 33014 33023 olfactory +T800 SO:0000167 33135 33143 promoter +T801 SO:0000204 33157 33163 5' UTR +T802 GO:0097617 33263 33277 hybridizations +T803 GO:0007608 33301 33310 olfactory +T804 SO:0000704 33432 33436 gene +T805 GO:0007608 33466 33475 olfactory +T806 SO:0000704 33532 33537 genes +T807 GO:0007608 33561 33570 olfactory +T808 SO:0000203 33618 33622 UTRs +T809 SO:0000010 33632 33646 protein-coding +T810 SO:0000203 33692 33696 UTRs +T811 GO:0007608 33822 33831 olfactory +T812 SO:0000704 33841 33846 genes +T813 SO:0001026 33878 33885 genomic +T814 SO:0000204 33941 33947 5' UTR +T815 SO:0000704 33965 33970 genes +T816 SO:0000205 33991 33997 3' UTR +T817 GO:0007608 34013 34022 olfactory +T818 SO:0000704 34032 34037 genes +T819 SO:0001060 34103 34111 isoforms +T820 GO:0007608 34130 34139 olfactory +T821 SO:0001060 34271 34279 isoforms +T822 SO:0000147 34294 34299 exons +T823 SO:0001060 34329 34336 isoform +T824 GO:0007608 34383 34392 olfactory +T825 GO:0007608 34418 34427 olfactory +T826 SO:0000704 34437 34442 genes +T827 SO:0001060 34480 34488 isoforms +T828 GO:0000380 34500 34520 alternative splicing +T829 SO:0000445 34524 34545 5' untranslated exons +T830 GO:0006412 34529 34539 translated +T831 GO:0043631 34560 34575 polyadenylation +T832 SO:0000553 34560 34580 polyadenylation-site +T833 GO:0008380 34599 34607 splicing +T834 GO:0006406 34646 34662;34667 34674 mRNA export from ... nucleus +T835 GO:0005634 34667 34674 nucleus +T836 MOP:0000779 34686 34692 couple +T837 GO:0007608 34693 34702 olfactory +T838 SO:0001026 34732 34743 genomically +T839 SO:0000167 34752 34761 promoters +T840 GO:0008380 34787 34794 spliced +T841 SO:0000673 34795 34805 transcript +T842 SO:0001060 34846 34854 isoforms +T843 GO:0008380 34908 34914 splice +T844 SO:0000162 34908 34920 splice sites +T845 SO:0001060 34987 34995 isoforms +T846 SO:0000203 35035 35039 UTRs +T847 GO:0065007 35060 35071 controlling +T848 GO:0006402 35072 35076;35104 35115 mRNA ... degradation +T849 GO:0007608 35159 35168 olfactory +T850 SO:0000673 35178 35189 transcripts +T851 GO:0007608 35223 35232 olfactory +T852 SO:0000704 35242 35246 gene +T853 SO:0000188 35275 35281 intron +T854 GO:0008380 35285 35292 spliced +T855 SO:0000205 35304 35326 3' untranslated region +T856 GO:0006412 35309 35319 translated +T857 GO:0008380 35350 35356 splice +T858 SO:0000162 35350 35362 splice sites +T859 GO:0007608 35374 35383 olfactory +T860 SO:0000236 35395 35398 ORF +T861 SO:0001026 35484 35491 genomic +T862 SO:0000673 35531 35541 transcript +T863 GO:0007608 35568 35577 olfactory +T864 SO:0000147 35668 35672 exon +T865 NCBITaxon:9606 35725 35730 human +T866 GO:0007608 35731 35740 olfactory +T867 SO:0000704 35750 35754 gene +T868 SO:0000236 35773 35776 ORF +T869 GO:0008380 35777 35785 splicing +T870 SO:0000010 35801 35815 protein-coding +T871 SO:0000704 35863 35868 genes +T872 GO:0008380 35879 35887 splicing +T873 GO:0008380 35981 35987 splice +T874 SO:0000673 36037 36048 transcripts +T875 SO:0000318 36086 36097 start codon +T876 GO:0007608 36152 36161 olfactory +T877 SO:0000673 36199 36210 transcripts +T878 GO:0008380 36252 36260 splicing +T879 GO:0065007 36312 36324 surveillance +T880 CL:0000540 36346 36353 neurons +T881 GO:0010467 36354 36364 expressing +T882 SO:0000673 36380 36391 transcripts +T883 SO:0000673 36415 36426 transcripts +T884 SO:0000704 36440 36445 genes +T885 GO:0007608 36476 36485 olfactory +T886 GO:0065007 36539 36549 regulation +T887 GO:0007608 36553 36562 olfactory +T888 GO:0008380 36600 36606 splice +T889 GO:0010467 36618 36627 expressed +T890 GO:0010467 36729 36739 expressing +T891 SO:0001060 36755 36763 isoforms +T892 SO:0000673 36802 36813 transcripts +T893 GO:0007608 36837 36846 olfactory +T894 SO:0000336 36856 36867 pseudogenes +T895 NCBITaxon:9606 36903 36908 human +T896 GO:0007608 36909 36918 olfactory +T897 SO:0000336 36928 36939 pseudogenes +T898 SO:0000336 36969 36980 pseudogenes +T899 SO:0000704 36993 36998 genes +T900 CL:0000540 37045 37052 neurons +T901 GO:0007608 37060 37069 olfactory +T902 UBERON:0001997 37060 37080 olfactory epithelium +T903 GO:0010467 37091 37098 express +T904 GO:0007608 37109 37118 olfactory +T905 GO:0060384 37193 37202 innervate +T906 GO:0007608 37207 37216 olfactory +T907 UBERON:0002264 37207 37221 olfactory bulb +T908 GO:0007608 37289 37298 olfactory +T909 SO:0000704 37308 37312 gene +T910 GO:0007608 37377 37386 olfactory +T911 CL:0000207 37377 37393 olfactory neuron +T912 SO:0000336 37404 37414 pseudogene +T913 GO:0010467 37415 37425 expressing +T914 CL:0000540 37426 37433 neurons +T915 UBERON:0001047 37456 37466 glomerulus +T916 GO:0007608 37474 37483 olfactory +T917 UBERON:0002264 37474 37488 olfactory bulb +T918 GO:0007608 37514 37523 olfactory +T919 SO:0000336 37578 37588 pseudogene +T920 GO:0010467 37589 37599 expressing +T921 CL:0000540 37600 37607 neurons +T922 GO:0016265 37608 37611 die +T923 GO:0010467 37625 37632 express +T924 GO:0007608 37645 37654 olfactory +T925 SO:0000704 37664 37668 gene +T926 SO:0000336 37696 37706 pseudogene +T927 GO:0010467 37707 37717 expressing +T928 CL:0000540 37718 37725 neurons +T929 UBERON:0007023 37729 37734 adult +T930 NCBITaxon:10088 37735 37739 mice +T931 CL:0000540 37783 37790 neurons +T932 GO:0010467 37791 37801 expressing +T933 GO:0007608 37809 37818 olfactory +T934 GO:0007608 37870 37879 olfactory +T935 GO:0007608 37938 37947 olfactory +T936 SO:0000704 37957 37961 gene +T937 GO:0007608 38014 38023 olfactory +T938 SO:0000704 38033 38038 genes +T939 GO:0007608 38049 38058 olfactory +T940 GO:0007608 38133 38142 olfactory +T941 SO:0000704 38152 38156 gene +T942 SO:0001060 38193 38201 isoforms +T943 GO:0007608 38208 38217 olfactory +T944 SO:0000673 38227 38238 transcripts +T945 GO:0007608 38322 38331 olfactory +T946 SO:0000704 38341 38346 genes +T947 GO:0010467 38373 38383 expression +T948 CL:0000540 38442 38448 neuron +T949 SO:0000704 38453 38457 gene +T950 GO:0007608 38488 38497 olfactory +T951 UBERON:0001997 38488 38508 olfactory epithelium +T952 GO:0007608 38623 38632 olfactory +T953 SO:0000704 38642 38646 gene +T954 GO:0010467 38647 38657 expression +T955 GO:0007608 38701 38710 olfactory +T956 UBERON:0007023 38730 38735 adult +T957 NCBITaxon:10088 38736 38741 mouse +T958 GO:0007608 38769 38778 olfactory +T959 UBERON:0001997 38769 38789 olfactory epithelium +T960 NCBITaxon:33208 38802 38808 animal +T961 UBERON:0000922 38893 38902 embryonic +T962 GO:0007608 38926 38935 olfactory +T963 UBERON:0001997 38926 38945 olfactory epithelia +T964 UBERON:0000922 38969 38976 embryos +T965 SO:0000440 39134 39140 vector +T966 UBERON:0007023 39178 39183 adult +T967 UBERON:0000922 39246 39255 embryonic +T968 UBERON:0007023 39355 39360 adult +T969 UBERON:0000922 39382 39391 embryonic +T970 GO:0097617 39394 39407 Hybridization +T971 NCBITaxon:10088 39446 39451 mouse +T972 SO:0001026 39452 39459 genomic +T973 SO:0000112 39526 39532 primer +T974 GO:0097617 39543 39552 annealing +T975 GO:0097617 39599 39612 hybridization +T976 SO:0000440 39776 39782 vector +T977 SO:0000112 39783 39790 primers +T978 CHEBI:2511 39884 39891 agarose +T979 SO:0000667 39917 39924 inserts +T980 SO:0000112 39971 39977 primer +T981 CHEBI:37958 39986 39989 dye +T982 SO:0000155 40156 40163 plasmid +T983 SO:0000155 40246 40253 Plasmid +T984 SO:0000006 40291 40303 PCR products +T985 SO:0000610 40365 40378 poly(A) tract +T986 GO:0007608 40498 40507 olfactory +T987 GO:0007608 40544 40553 olfactory +T988 SO:0000704 40563 40567 gene +T989 SO:0000704 40664 40669 genes +T990 SO:0000440 40837 40843 vector +T991 SO:0000028 40921 40923 bp +T992 SO:0000205 40955 40961 3' UTR +T993 SO:0000667 41010 41016 insert +T994 SO:0000205 41158 41164 3' UTR +T995 SO:0000704 41202 41206 gene +T996 GO:0043631 41230 41245 polyadenylation +T997 SO:0000553 41230 41250 polyadenylation site +T998 SO:0000205 41260 41266 3' UTR +T999 SO:0000028 41307 41309 bp +T1000 GO:0007608 41450 41459 olfactory +T1001 SO:0000704 41469 41474 genes +T1002 SO:0001026 41495 41502 genomic +T1003 SO:0000704 41524 41528 gene +T1004 SO:0000318 41608 41619 start codon +T1005 SO:0000704 41647 41651 gene +T1006 SO:0000319 41743 41753 stop codon +T1007 SO:0001026 41793 41800 genomic +T1008 SO:0000610 41853 41864 polyA tails +T1009 SO:0000147 41916 41921 exons +T1010 GO:0008380 41948 41954 splice +T1011 SO:0000162 41948 41959 splice-site +T1012 SO:0000993 41960 41969 consensus +T1013 SO:0001026 42120 42127 genomic +T1014 SO:0001026 42144 42151 genomic +T1015 SO:0000704 42382 42386 gene +T1016 SO:0000704 42420 42424 gene +T1017 SO:0000147 42534 42539 exons +T1018 SO:0001026 42641 42648 genomic +T1019 SO:0000704 42664 42668 gene +T1020 GO:0008380 42758 42766 splicing +T1021 SO:0000205 42778 42784 3' UTR +T1022 SO:0000667 42938 42944 insert +T1023 SO:0001031 42974 42993 reverse orientation +T1024 SO:0000704 43042 43046 gene +T1025 SO:0000147 43101 43106 exons +T1026 SO:0000147 43150 43155 exons +T1027 SO:0000147 43375 43380 exons +T1028 SO:0000345 43417 43420 EST +T1029 SO:0000028 43542 43544 bp +T1030 SO:0000147 43566 43571 exons +T1031 SO:0000704 43802 43806 gene +T1032 SO:0000704 43855 43859 gene +T1033 GO:0007608 43897 43906 olfactory +T1034 SO:0000704 43916 43921 genes +T1035 GO:0007608 43961 43970 olfactory +T1036 GO:0010467 44002 44011 expressed +T1037 SO:0000336 44012 44023 pseudogenes +T1038 GO:0007608 44040 44049 olfactory +T1039 SO:0000336 44059 44070 pseudogenes +T1040 SO:0001026 44099 44106 genomic +T1041 NCBITaxon:10088 44191 44196 mouse +T1042 GO:0007608 44197 44206 olfactory +T1043 SO:0001060 44657 44665 isoforms +T1044 SO:0000704 44675 44679 gene +T1045 SO:0000147 44766 44771 exons +T1046 GO:0008380 44807 44813 splice +T1047 SO:0000147 44872 44876 exon +T1048 SO:0001060 44913 44921 isoforms +T1049 SO:0001060 45053 45061 isoforms +T1050 GO:0007608 45111 45120 olfactory +T1051 UBERON:0001997 45111 45130 olfactory epithelia +T1052 UBERON:0007023 45157 45162 adult +T1053 NCBITaxon:10088 45178 45182 mice +T1054 UBERON:0000479 45194 45201 tissues +T1055 UBERON:0003129 45218 45223 skull +T1056 UBERON:0003037 45228 45234 septum +T1057 GO:0007608 45708 45717 olfactory +T1058 SO:0000112 45759 45766 primers +T1059 GO:0097617 45824 45833 annealing +T1060 SO:0000112 45959 45966 primers +T1061 GO:0010467 45983 45993 expression +T1062 GO:0007608 46008 46017 olfactory +T1063 SO:0000704 46027 46032 genes +T1064 SO:0000006 46075 46086 PCR product +T1065 SO:0000704 46130 46134 gene +T1066 SO:0001026 46315 46322 genomic +T1067 SO:0000673 46338 46348 transcript +T1068 SO:0000112 46465 46471 primer +T1069 CHEBI:39442 46481 46498 fluorescent probe +T1070 SO:0000112 46661 46667 primer +T1071 NCBITaxon:10088 46701 46706 mouse +T1072 SO:0001026 46707 46714 genomic +T1073 SO:0001026 46808 46814 genome +T1074 GO:0010467 46826 46836 expression +T1075 SO:0000704 46851 46855 gene +T1076 GO:0001171 46999 47018 reverse-transcribed +T1077 SO:0000112 47067 47074 primers +T1078 SO:0000704 47114 47118 gene +T1079 GO:0005840 47120 47129 ribosomal +T1080 PR:000014258 47120 47133 ribosomal S16 +T1081 GO:0010467 47322 47332 Expression +T1082 SO:0000704 47359 47364 genes +T1083 NCBITaxon:10088 47390 47395 mouse +T1084 PR:000014258 47404 47407 S16 +T1085 GO:0097617 47459 47472 hybridization +T1086 GO:0007608 47509 47518 olfactory +T1087 UBERON:0001997 47509 47528 olfactory epithelia +T1088 UBERON:0007023 47535 47540 adult +T1089 NCBITaxon:10088 47541 47546 mouse +T1090 NCBITaxon:10088 47583 47588 mouse +T1091 GO:0097617 47602 47615 hybridization +T1092 CHEBI:42098 47669 47680 digoxigenin +T1093 SO:0000077 47689 47698 antisense +T1094 SO:0000205 47727 47734 3' UTRs +T1095 SO:0000704 47738 47743 genes +T1096 SO:0000112 47833 47839 primer +T1097 SO:0000112 47923 47929 primer +T1098 GO:0097617 48019 48032 Hybridization +T1099 CHEBI:16397 48056 48065 formamide +T1100 GO:0097617 48152 48161 hybridize +T1101 GO:0007608 48243 48252 olfactory +T1102 SO:0000704 48262 48266 gene +T1103 NCBITaxon:10088 48334 48339 mouse +T1104 SO:0001026 48340 48346 genome +T1105 SO:0001026 48427 48434 genomic +T1106 GO:0097617 48464 48475 hybridizing +T1107 GO:0007608 48514 48523 olfactory +T1108 GO:0007608 48597 48606 olfactory +T1109 SO:0000673 48616 48626 transcript +T1110 SO:0000112 48769 48776 primers +T1111 GO:0010467 48797 48807 expression +T1112 GO:0007608 48811 48820 olfactory +T1113 SO:0000704 48830 48835 genes +T1114 SO:0000112 49076 49083 primers +T1115 GO:0010467 49104 49114 expression +T1116 GO:0007608 49118 49127 olfactory +T1117 SO:0000704 49137 49142 genes +T1118 http://purl.obolibrary.org/obo/MONDO_0004992 49484 49490 Cancer +T1119 PR:000014258 49701 49704 S16 +T1120 SO:0000112 49705 49712 primers +T1121 GO:0007608 49927 49936 Olfactory +T1122 SO:0000704 49946 49951 genes +T1123 GO:0010467 49958 49968 expression +T1124 NCBITaxon:10088 49976 49981 mouse +T1125 GO:0007608 49982 49991 olfactory +T1126 UBERON:0001997 49982 50002 olfactory epithelium +T1127 SO:0000704 50032 50037 Genes +T1128 GO:0010467 50044 50054 expression +T1129 NCBITaxon:10088 50149 50154 mouse +T1130 GO:0007608 50155 50164 olfactory +T1131 SO:0000704 50176 50181 Genes +T1132 GO:0010467 50188 50198 expression +T1133 SO:0000704 50245 50250 genes +T1134 GO:0001171 50338 50357 reverse-transcribed +T1135 SO:0000704 50367 50372 genes +T1136 SO:0000112 50412 50418 primer +T1137 GO:0007608 50524 50533 olfactory +T1138 GO:0007608 50613 50622 olfactory +T1139 GO:0007608 50666 50675 olfactory +T1140 GO:0010467 50746 50756 expression +T1141 GO:0007608 50778 50787 olfactory +T1142 GO:0007608 50882 50891 olfactory +T1143 SO:0000704 50901 50905 gene +T1144 GO:0007608 50918 50927 olfactory +T1145 GO:0010467 51012 51022 expression +T1146 GO:0007608 51040 51049 olfactory +T1147 SO:0000704 51059 51064 genes +T1148 GO:0010467 51101 51111 Expression +T1149 GO:0007608 51122 51131 olfactory +T1150 SO:0000704 51141 51146 genes +T1151 SO:0000704 51189 51194 genes +T1152 GO:0010467 51214 51224 expression +T1153 GO:0007608 51248 51257 olfactory +T1154 SO:0000704 51267 51272 genes +T1155 GO:0007608 51375 51384 olfactory +T1156 UBERON:0001997 51375 51395 olfactory epithelium +T1157 NCBITaxon:10088 51419 51423 mice +T1158 GO:0010467 51425 51435 Expression +T1159 SO:0000704 51452 51456 gene +T1160 NCBITaxon:10088 51519 51524 mouse +T1161 SO:0001026 51525 51532 genomic +T1162 NCBITaxon:10088 51573 51578 mouse +T1163 SO:0000704 51618 51622 gene +T1164 GO:0005840 51624 51633 ribosomal +T1165 PR:000014258 51624 51637 ribosomal S16 +T1166 SO:0000704 51753 51758 Genes +T1167 GO:0007608 51803 51812 olfactory +T1168 SO:0000704 51885 51889 gene +T1169 GO:0010467 51900 51910 Expression +T1170 SO:0000704 51926 51930 gene +T1171 NCBITaxon:10088 51981 51985 mice +T1172 NCBITaxon:10088 52046 52050 mice +T1173 GO:0010467 52074 52084 expression +T1174 NCBITaxon:10088 52117 52122 mouse +T1175 GO:0007608 52190 52199 Olfactory +T1176 GO:0010467 52228 52238 expression +T1177 GO:0010467 52257 52267 expression +T1178 GO:0007608 52290 52299 olfactory +T1179 GO:0010467 52341 52351 expressing +T1180 SO:0000673 52372 52382 transcript +T1181 GO:0097617 52412 52425 hybridization +T1182 CHEBI:42098 52431 52442 digoxigenin +T1183 SO:0000704 52466 52470 gene +T1184 SO:0000704 52492 52496 gene +T1185 GO:0007608 52537 52546 olfactory +T1186 UBERON:0001762 52537 52557 olfactory turbinates +T1187 UBERON:0007023 52564 52569 adult +T1188 NCBITaxon:10088 52570 52575 mouse +T1189 GO:0007608 52727 52736 olfactory +T1190 SO:0000704 52746 52751 genes +T1191 GO:0000380 52757 52775 alternate splicing +T1192 SO:0001060 52823 52831 isoforms +T1193 GO:0007608 52852 52861 olfactory +T1194 GO:0007608 52943 52952 olfactory +T1195 GO:0008380 52972 52978 splice +T1196 SO:0000162 52972 52984 splice sites +T1197 SO:0000851 52979 52991;52996 53009 sites within ... coding region +T1198 GO:0007608 53061 53070 olfactory +T1199 GO:0016020 53100 53108 membrane +T1200 GO:0005622 53138 53151 intracellular +T1201 GO:0005622 53153 53155 IC +T1202 GO:0005576 53161 53174 extracellular +T1203 GO:0005576 53176 53178 EC +T1204 GO:0007608 53350 53359 olfactory +T1205 GO:0008380 53391 53399 splicing +T1206 SO:0000993 53448 53457 consensus +T1207 SO:0001060 53581 53588 isoform +T1208 SO:0001060 53656 53664 isoforms +T1209 SO:0000704 53679 53683 gene +T1210 SO:0000188 53792 53800 intronic +T1211 GO:0007608 53915 53924 olfactory +T1212 SO:0000440 54001 54007 vector +T1213 SO:0001031 54015 54034 reverse orientation +T1214 SO:0000188 54036 54043 Introns +T1215 GO:0007608 54088 54097 olfactory +T1216 SO:0000994 54107 54116 consensus +T1217 SO:0001817 54161 54169 in-frame +T1218 SO:0000319 54230 54240 stop codon +T1219 SO:0000610 54461 54473 poly(A) tail +T1220 SO:0000147 54513 54517 exon +T1221 SO:0001026 54543 54550 genomic +T1222 SO:0000704 54581 54585 gene +T1223 NCBITaxon:10088 54615 54620 mouse +T1224 SO:0001026 54621 54627 genome +T1225 SO:0000147 54678 54682 exon +T1226 SO:0001031 54703 54710 reverse +T1227 GO:0016020 54741 54749 membrane +T1228 SO:0000417 54750 54756 domain +T1229 GO:0007608 54780 54789 olfactory +T1230 SO:0000704 54799 54803 gene +T1231 SO:0000147 54834 54838 exon +T1232 SO:0000317 54852 54862 cDNA clone +T1233 SO:0001026 54902 54909 genomic +T1234 SO:0000188 54949 54955 intron +T1235 GO:0007608 54983 54992 olfactory +T1236 NCBITaxon:10088 55051 55056 mouse +T1237 SO:0001026 55057 55063 genome +T1238 SO:0000112 55179 55186 primers +T1239 GO:0097617 55195 55204 annealing diff --git a/src/ontogpt/evaluation/craft/database/all/14611657.txt b/src/ontogpt/evaluation/craft/database/all/14611657.txt new file mode 100644 index 000000000..30dfae17e --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/14611657.txt @@ -0,0 +1,181 @@ +Odorant receptor expressed sequence tags demonstrate olfactory expression of over 400 genes, extensive alternate splicing and unequal expression levels + +Previous computational analyses have identified approximately 1,500 mouse olfactory receptors, but experimental evidence confirming olfactory function is available for very few receptors. A mouse olfactory epithelium cDNA library was screened to obtain olfactory receptor expressed sequence tags, providing evidence of olfactory function for many additional olfactory receptors, as well as identifying gene structure and putative promoter regions. + +Abstract + +Background + +The olfactory receptor gene family is one of the largest in the mammalian genome. Previous computational analyses have identified approximately 1,500 mouse olfactory receptors, but experimental evidence confirming olfactory function is available for very few olfactory receptors. We therefore screened a mouse olfactory epithelium cDNA library to obtain olfactory receptor expressed sequence tags, providing evidence of olfactory function for many additional olfactory receptors, as well as identifying gene structure and putative promoter regions. + +Results + +We identified more than 1,200 odorant receptor cDNAs representing more than 400 genes. Using real-time PCR to confirm expression level differences suggested by our screen, we find that transcript levels in the olfactory epithelium can differ between olfactory receptors by up to 300-fold. Differences for one gene pair are apparently due to both unequal numbers of expressing cells and unequal transcript levels per expressing cell. At least two-thirds of olfactory receptors exhibit multiple transcriptional variants, with alternative isoforms of both 5' and 3' untranslated regions. Some transcripts (5%) utilize splice sites within the coding region, contrary to the stereotyped olfactory receptor gene structure. Most atypical transcripts encode nonfunctional olfactory receptors, but can occasionally increase receptor diversity. + +Conclusions + +Our cDNA collection confirms olfactory function of over one-third of the intact mouse olfactory receptors. Most of these genes were previously annotated as olfactory receptors based solely on sequence similarity. Our finding that different olfactory receptors have different expression levels is intriguing given the one-neuron, one-gene expression regime of olfactory receptors. We provide 5' untranslated region sequences and candidate promoter regions for more than 300 olfactory receptors, valuable resources for computational regulatory motif searches and for designing olfactory receptor microarrays and other experimental probes. + +Background + +The interaction of olfactory (or odorant) receptors with their odorant ligands is the first step in a signal transduction pathway that results in the perception of smell. The olfactory receptor gene family is one of the largest in the mammalian genome, comprising about 1,500 members in the mouse genome [1,2]. Olfactory receptors were originally identified in an elegant experiment based on the hypothesis that they would be seven-transmembrane-domain proteins encoded by a large, diverse gene family whose expression is restricted to the olfactory epithelium [3]. Subsequent studies have shown that some of these receptors do indeed respond to odorants and can confer that responsivity when expressed in heterologous cell types (for example [4]). Recent computational investigations have provided the almost complete human [5,6] and mouse [1,2] olfactory receptor-gene catalogs. However, the assignment of most of these genes as olfactory receptors is based solely on similarity to one of a relatively small number of experimentally confirmed mouse olfactory receptors or, worse, on similarity to a gene that in turn was defined as an olfactory receptor solely by similarity. While similarity-based genome annotation is a good initial method to identify genes and predict their function, in some cases it can be misleading, as genes of similar sequence can carry out different functions and be expressed in different tissues (for example, the sugar transporter gene family [7]). + +A small subset of olfactory receptors appears to be expressed in non-olfactory tissues, principally the testis [8], but also taste tissues [9], prostate [10], erythroid cells [11], notochord [12] and perhaps other tissues. Expression in the testis has led some investigators to suggest that a subset of olfactory receptors may function as spermatid chemoreceptors [8]. Recent studies of one human testis-expressed olfactory receptor indicate that it does indeed function in sperm chemotaxis [13]. Due to the paucity of experimental evidence of the olfactory function of most genes in the family and suggestions of extra-olfactory roles, we embarked on an olfactory receptor expressed sequence tag (EST) project to confirm olfactory epithelial expression of hundreds of mouse odorant receptor genes. + +Within the olfactory epithelium, individual olfactory receptor genes show an intriguing expression pattern. Each olfactory receptor is expressed in a subset of cells in one of four zones of the epithelium [14,15]. Furthermore, each olfactory neuron expresses only one allele [16] of a single olfactory receptor gene [17,18], and the remaining approximately 1,499 genes are transcriptionally inactive. While the mechanism ensuring singular expression is unknown, many hypotheses have been proposed [14,16,19]. In one model, somatic DNA recombination would bring one olfactory receptor gene into a transcriptionally active genomic configuration, as observed for the yeast mating type locus [20] and the mammalian immunoglobulin genes [21]. Alternatively, a second model invokes a combinatorial code of transcription factor binding sites unique to each gene. This is unlikely, however, as even olfactory receptor transgenes with identical upstream regions are expressed in different neurons [18]. In a third model, there would be a limiting quantity of transcription factors - the cell might contain a single transcriptional 'machine' that is capable of accommodating the promoter of only one olfactory receptor gene, similar to the expression site body used by African trypanosomes to ensure singular expression of only one set of variant surface glycoprotein genes [22]. Finally, in a fourth model, transcriptional activity at one stochastically chosen olfactory receptor allele might send negative feedback to repress activity of all other olfactory receptors and/or positive feedback to enhance its own expression. In the latter three models, some or all olfactory receptor genes might share transcription factor binding motifs, and in the first model, olfactory receptor genes might share a common recombination signal. In order to perform computational and experimental searches for such signals, it is important to have a better idea of the transcriptional start site of a large number of olfactory receptor genes. Our olfactory receptor EST collection provides 5' untranslated region (UTR) sequences for many genes and, therefore, a large dataset of candidate promoter regions. + +Olfactory receptor genes have an intronless coding region, simplifying both computational and experimental olfactory receptor identification. For a small number of olfactory receptors, gene structure has been determined. Additional 5' untranslated exons lie upstream of the coding region and can be alternatively spliced [19,23-26]. The 3' untranslated region is typically intronless. Exceptions to this stereotyped structure have been described for some human olfactory receptors, but are thought to be rare [25-27]. cDNA identification and RACE data have been used to determine gene structure for about 30 genes, see, for example, [19,23]. However, computational prediction of the location of 5' upstream exons and the extent of the 3' UTR from genomic sequence has been extremely difficult. A combination of splice site predictions and similarity to other olfactory receptors has allowed some investigators to predict 5' exon locations for around 15 genes [25,28]. Experimental validation shows that some, but not all, predictions are accurate [24,25]. The total number of olfactory receptors for which gene structure is known is vastly increased by our study. + +In this report, we describe the isolation and analysis of over 1,200 cDNAs representing 419 odorant receptor genes. We screened a mouse olfactory epithelium library with degenerate olfactory receptor probes and obtained 5' end sequences (ESTs) from purified cDNAs. These clones confirm olfactory expression of over 400 olfactory receptors, provide their gene structure, demonstrate that not all olfactory receptors are expressed at the same level and show that most olfactory receptor genes have multiple transcriptional isoforms. + +Results + +At least 419 mouse olfactory receptor genes are expressed in the olfactory epithelium + +We have isolated 1,264 olfactory receptor cDNA clones, which together confirm the olfactory epithelial expression of 419 annotated olfactory receptor genes. We used low-stringency hybridization with degenerate olfactory receptor DNA probes to screen around 4,100,000 plaque-forming units (pfu) of an adult mouse olfactory epithelium cDNA library and around 640,000 pfu of an embryonic olfactory epithelium library. We obtained sequences from 1,715 hybridization-positive cDNAs following secondary screens to isolate single clones. Of these clones, 1,264 yielded olfactory receptor-containing sequences. The 26% false-positive rate is a consequence of using low-stringency hybridization to obtain maximal sensitivity. Continuing the screen would have resulted in cDNAs from additional olfactory receptors, but we reached a point of limiting returns: our final batch of 45 olfactory receptor-positive sequences represented 33 different olfactory receptors, of which only four had not been encountered previously in our screen. + +Sequence analysis shows that the libraries are of high quality. Firstly, directional cloning was successful: only eight out of 1,430 cDNA sequences with any protein homology matched that protein on the reverse strand. Secondly, genomic contamination is rare: when the 83 olfactory receptor-containing sequences that had a 5' UTR of 400 bp or longer were aligned to genomic sequence, 80 spliced across at least one intron, leaving a maximum of three clones (3.6%) that potentially represent genomic contamination of the libraries. Thirdly, most clones are of a reasonable length: although we did not determine whether clones are full-length, 881 of 1,264 (70%) olfactory receptor cDNAs contain the gene's start codon and at least some 5' UTR sequence. + +In order to match cDNAs to their genomic counterparts, first we updated our catalog of mouse olfactory receptor genes [1] based on Celera's most recent genome assembly (Release 13) [29]. Previous reports of the mouse olfactory receptor repertoire [1,2] were based on the Release 12 assembly. Release 13 consists of fewer, longer scaffold sequences containing fewer, smaller gaps than Release 12. Using the BLAST-based methods detailed previously [1], we identified 1,490 olfactory receptor sequences in the new assembly, including 1,107 intact olfactory receptor genes (compared to 866 intact olfactory receptors in the old assembly) reflecting the reduced sequence error rate and increased coverage of the new assembly (Table 1). We created a local database of genomic sequences including all olfactory receptor loci and 0.5 Mb flanking sequences (if available) and compared each cDNA sequence to this 'olfactory subgenome' database using sim4 [30]. + +cDNAs were assigned to individual genes based on their best match to an olfactory receptor coding region or its upstream region (see Materials and methods). Of the 1,264 olfactory receptor cDNAs, 1,176 matched a total of 419 olfactory receptor genes; the remaining cDNAs either matched an olfactory receptor below our 96% nucleotide identity threshold or had ambiguous matches encompassing more than one olfactory receptor gene region (see below). + +A class I olfactory receptor degenerate primer broadens phylogenetic distribution of confirmed olfactory receptor genes + +Previous analyses of the mammalian olfactory receptor family define two major phylogenetic clades, referred to as class I and II olfactory receptors, and suggest that class I olfactory receptors are more similar to fish olfactory receptors than are class IIs [5]. Figure 1 illustrates the phylogenetic diversity of our cDNA collection, showing that we have confirmed expression of at least one olfactory receptor gene in each major clade of the class II olfactory receptor genes, or 391 out of 983 (40%) of all intact class II olfactory receptor genes where full-length genomic sequence data are available (blue branches). The screen thus appears relatively unbiased in its coverage of class II olfactory receptors. However, our random screen provided cDNAs for only two out of 124 intact, full-length class I olfactory receptors. In an attempt to broaden the phylogenetic coverage of our hybridization screen, we used additional degenerate probes on the adult library and screened an embryonic library (Table 2). These experiments did not increase the diversity of clones identified (not shown). + +This severe class I underrepresentation could be due to experimental bias - a consequence of using degenerate primers to create our hybridization probe. Alternatively, class I genes might be expressed at extremely low levels in the olfactory epithelium. In order to determine whether class I olfactory receptors are expressed in the olfactory epithelium, we designed a reverse-strand degenerate primer to recognize a motif in transmembrane domain 7 (PP{V/M/A/T}{F/L/I/M}NP) enriched among class I olfactory receptor sequences. Most of the motif is shared among all olfactory receptors, but the first proline residue (at the primer's 3' end) is found in 121 out of 124 (98%) intact class I genes compared to only 37 out of 983 (4%) intact class II genes. When combined with another olfactory receptor degenerate primer, P26 [17], this primer preferentially amplifies class I olfactory receptors from mouse genomic DNA: of 33 sequenced, cloned PCR products, 17 represented seven different class I olfactory receptors, six represented three different class II olfactory receptors, and ten represented five different non-olfactory receptor contaminants. Degenerate PCR, cloning, and sequencing from reverse-transcribed olfactory epithelium RNA showed that at least seven class I olfactory receptors are expressed, as well as one additional class II gene (colored red in Figure 1). However, no products could be obtained from the adult or the fetal olfactory epithelium cDNA libraries using the class I primer, suggesting that the libraries contain very low levels of class I olfactory receptors. We also confirmed expression of nine additional olfactory receptors (three class I and six class II, colored red in Figure 1) from subclades that were poorly represented in our cDNA screen using gene-specific primer pairs to amplify cDNA library or reverse-transcribed RNA templates. + +For two of the class I genes we had shown to be expressed, we determined relative transcript levels using quantitative RT-PCR (see below). Expression levels were similar to those observed for genes that were represented in our cDNA collection, suggesting that class I olfactory receptors are not under-represented in the olfactory epithelium, and that the dearth of class I cDNAs in our screen is likely to be due to bias in the libraries and/or hybridization probes. + +Some olfactory receptor genes are expressed at higher levels than others + +Our cDNA screen suggests that some olfactory receptor genes are expressed at higher levels than others. If all olfactory receptor genes were expressed at equal levels, and our screen and library were unbiased in their coverage of the class II olfactory receptors, the number of cDNAs detected per class II olfactory receptor should follow a Poisson distribution, calculated based on the assumption that all 983 intact class II olfactory receptors have an equal chance of being represented in the screen, but that class I olfactory receptors and pseudogenes cannot be found (Figure 2). We calculate a low probability (approximately one in 28) that we would observe any gene with at least eight matching cDNAs in the set of 1,176 cDNAs we assigned to single olfactory receptor sequences. However, for 17 olfactory receptors, we found ten or more matching cDNAs, suggesting that they might be expressed at higher levels than other olfactory receptor genes (Figure 2). The two genes for which we found most cDNAs (AY318726/MOR28 and AY318727/MOR10) are genomically adjacent and in the well-studied olfactory receptor cluster next to the T-cell receptor α/δ locus [18,31]. + +Quantitative RT-PCR of six olfactory receptors confirms that expression levels do indeed vary considerably between genes. We used quantitative (real-time) PCR to measure olfactory epithelium transcript levels of six olfactory receptor genes and the ribosomal S16 gene in three mice of the same inbred strain (Figure 3). These genes include two olfactory receptors with more than 20 matching cDNAs, two with one or two matching cDNAs and two class I olfactory receptors with no matching cDNAs. In these assays, we measure transcript level per genomic copy of the gene by comparing how well a gene-specific primer pair amplifies reverse-transcribed RNA, relative to a standard curve of amplification of mouse genomic DNA. We find that expression levels can vary by almost 300-fold between genes (for example, genes A and D, Figure 3). However, cDNA numbers are not a good indicator of expression level, a discrepancy that is likely to be due to bias in the screen (we used degenerate primers to make the probes, which will favor some genes over others) and in the libraries (oligo-dT priming will favor genes with shorter 3' UTRs). For example, we observe large expression differences in all three mice between two genes for which similar numbers of cDNAs were found (genes A and B, Figure 3), and conversely, similar expression levels between two genes with a ten-fold difference in number of cDNAs found (genes B and C, Figure 3). Expression levels are mostly consistent between different mice: we find similar expression-level differences between olfactory receptor genes in all three mice examined (that is, the rank order of the six genes is similar among the three mice), although there is variation in expression level of some genes between mice (for example, gene E, Figure 3). + +In situ hybridization (Figure 4) shows that increased numbers of expressing cells account for some, but not all, of the difference in transcript levels between two of the genes tested by real-time PCR (genes A and D in Figure 3). We hybridized alternate coronal serial sections spanning an entire olfactory epithelium of a young mouse (P6) with probes for gene A and gene D. Southern blot and BLAST analyses show that both probes are likely to hybridize to their intended target genes and no others (not shown). Gene A is expressed in zone 4 of the epithelium according to the nomenclature of Sullivan et al. [32] (Figure 4a). The expression pattern of gene D does not correspond to any of the four 'classical' olfactory epithelial zones [14,15,32]: positive cells are found in regions of endoturbinates II and III and ectoturbinate 3, resembling the expression pattern seen previously for the OR37 subfamily and ORZ6 olfactory receptors [33,34] (Figure 4b). Counting the total number of positive cells in alternate sections across the entire epithelium, we find that gene A is expressed in 2,905 cells, about 12 times more cells than gene D, which is expressed in a total of 249 cells. This 12-fold difference in numbers of expressing cells does not account for the almost 300-fold difference in RNA levels observed by real-time PCR, implying that the transcript level per expressing cell for gene A is about 25 times higher than transcript level in each expressing cell for gene D. We note that hybridization intensities per positive neuron appear stronger for gene A than gene D after comparable exposure times, in accordance with the idea that transcript levels are higher per cell. Thus, we suggest that expression in more cells and in higher levels per cell together account for the almost 300-fold higher olfactory epithelial RNA levels of gene A relative to gene D (Figure 3). + +Most olfactory receptor genes have several transcriptional isoforms + +Our cDNA collection reveals that at least two thirds of the olfactory receptors sampled show alternative splicing of their 5' untranslated exons. Using a custom script to process sim4 alignments of cDNA and genomic sequences, we find two to eight different splice forms for 85 (45%) of the 191 genes for which we have had some opportunity to observe alternate splicing (that is, a minimum of two cDNAs, at least one of which is spliced), and 55 (67%) of the 82 genes for which we have four or more cDNAs (and thus a higher chance of observing any alternate splicing) (Figure 5). These alternative splice events are almost all restricted to the 5' UTR and include exon skipping and alternate splice-donor and -acceptor use. + +At least half of the olfactory receptors represented in our cDNA collection utilize more than one polyadenylation site, resulting in alternative 3' UTR isoforms. We have crudely estimated 3' UTR size for 1,169 cDNA clones by combining approximate insert size information with 5' sequence data. More than one 3' UTR isoform is predicted for 43 of the 77 (56%) genes for which there are at least four cDNAs with 3' UTR size information. We confirmed the alternative polyadenylation isoforms of four out of five selected genes by sequencing the 3' end of 14 cDNA clones. These 14 sequences also revealed one cDNA where the poly(A) tail was added 27 bp before the stop codon, and another where an intron was spliced out of the 3' UTR, contrary to the conventional stereotype of olfactory receptor gene structure. + +A subset of olfactory receptors shows unusual splicing + +We identified 62 cDNAs (5% of all olfactory receptor clones) from 38 intact olfactory receptors and one olfactory receptor pseudogene where a splice site within the protein-coding region is used. For two genes (top two cDNAs, Figure 6), the predicted protein appears to be an intact olfactory receptor with three or ten amino acids, including the initiating methionine, contributed by an upstream exon. A similar gene structure was described previously for a human olfactory receptor [25]. One of these two mouse genes has no start codon in its otherwise intact main coding exon. The unusual splicing thus rescues what would otherwise be a dysfunctional gene. In most cases (60 out of 62 cDNAs), the unusual transcript appears to be an aberrant splice form - the transcript would probably not encode a functional protein because the splice introduces a frameshift or removes conserved functional residues (Figure 6). For two clones (bottom two cDNAs, Figure 6), exon order in the cDNA clone is inconsistent with the corresponding genomic sequence. It is difficult to imagine what kind of cloning artefact resulted in these severely scrambled cDNAs: we suggest that they derive from real but rare transcripts. However, their low frequency in our cDNA collection suggests that splicing contrary to genomic organization does not contribute significantly to the olfactory receptor transcript repertoire. For 21 of the 26 genes for which unusually spliced cDNAs were found, we also observe an alternative ('normal') isoform that does not use splice sites within the coding region. (For the remaining 13 of the 3' genes showing odd splicing, we have identified only one cDNA so have not determined whether normal isoforms are present.) + +We were intrigued both by previous reports of splicing of human olfactory receptors near the major histocompatibility complex (MHC) cluster, where several genes splice over long distances to a common upstream exon [26,27] and by the idea that olfactory receptor transcriptional control could be achieved by DNA recombination mechanisms, perhaps with the result that transcripts would contain some sequence from another locus. We therefore verified that the entire sequence of each olfactory receptor EST matches the corresponding gene's genomic 'territory' (defined for this purpose as from 1 kb after the preceding gene to 1 kb after the gene's stop codon). We found no cDNAs where introns encompassed other olfactory receptor genes, as reported for olfactory receptors in the human MHC region [26,27]. Six cDNAs do extend further than a single gene's 'territory' and appear not to be artifacts of the sequencing or analysis process. In each of these cases, the clones use splice sites within the 3' UTR and thus extend further than the arbitrary 1 kb downstream of the stop codon. Five of these six cDNAs also use splice-donor sites within the coding region and encode disrupted olfactory receptors (Figure 6). In the sixth cDNA, a 2.6-kb intron is spliced out of the 3' UTR, leaving the coding region intact. + +If olfactory receptor transcriptional control is achieved by DNA recombination, the beginning of each transcript might derive from a donated promoter region, with the rest of the transcript coming from the native ORF-containing locus. In order to examine the recombination hypothesis, we analyzed 115 cDNA clones for which sim4 failed to align 20 bp or more to the corresponding genomic locus. In most cases, the missing sequence was explained by gaps in the genomic sequence or by matches that fell below our percent identity-based cutoff for reporting matches. For three cDNAs (from three different olfactory receptors), we found that the missing piece of sequence matched elsewhere in the genome. Comparison with the public mouse genome assembly confirmed the distant matches. With such a small number of cDNAs exhibiting a possible sign of DNA recombination (a sign that could also be interpreted as chimeric cDNA clones), we conclude that such rearrangement is unlikely to occur. However, the possibility remains that DNA recombination is responsible for olfactory receptor transcriptional regulation, with the donated region contributing only promoter sequences but no part of the transcript. + +Both unclustered olfactory receptors and olfactory receptor pseudogenes can be expressed + +We were interested in whether olfactory receptors need to be part of a cluster in the genome in order to be transcribed, or if the clustered genomic organization of olfactory receptors is simply a consequence of the fact that local duplication is the major mechanism for expanding the gene family [1]. 'Singleton' olfactory receptors (defined as full-length olfactory receptors without another olfactory receptor within 0.5 Mb) are more often pseudogenes than are olfactory receptors in clusters (8 out of 16 versus 271 out of 1,358; χ2 = 8.8, P < 0.005). Of the eight intact singleton olfactory receptors, two have matching cDNAs in our collection, a similar proportion as found for olfactory receptors in clusters, showing that clustering is not an absolute requirement for olfactory receptor expression. However, it is possible these two expressed singleton genes are part of 'extended' olfactory receptor clusters - their nearest olfactory receptor neighbors are 1.7 Mb and 2.6 Mb away, respectively. + +We also find that some olfactory receptor pseudogenes are expressed, albeit with a lower probability than intact olfactory receptors. Considering the 1,392 olfactory receptor gene sequences for which reliable full-length data are available, 15 out of 285 (5%) apparent pseudogenes are represented in our cDNA collection, compared to 393 out of 1,107 (36%) intact olfactory receptors. However, three of these 15 'expressed pseudogenes' are intact genes in the public mouse genome sequence. The defects in Celera's version of these genes may be due to sequencing errors or true polymorphism. Publicly available mouse sequence confirms that 11 of the 12 remaining expressed pseudogenes are indeed pseudogenes. No public sequence matches the 12th 'expressed pseudogene' with 99% identity or more. + +Discussion + +We have identified and sequenced 1,264 odorant receptor cDNAs from 419 olfactory receptor genes, confirming their expression in the olfactory epithelium. We have thus validated the similarity-based prediction of over one-third of the intact olfactory receptor genes annotated in the mouse genome [1,2], thereby vastly increasing the proportion of the family for which experimental evidence of olfactory function is available. We have not found cDNAs for all olfactory receptor genes or an even phylogenetic distribution of cDNAs, probably because the libraries and/or our screen are biased toward certain olfactory receptor subfamilies. Using RT-PCR with both degenerate and specific primers, we have confirmed olfactory expression of a number of additional olfactory receptors, bringing the total number of olfactory receptor genes verified in this study to 436, and ensuring that almost all phylogenetic clades have at least one representative with evidence of olfactory function. + +Results of our cDNA library screen suggested that some olfactory receptors are expressed at significantly higher levels than others. We used quantitative PCR to show that expression levels are indeed highly variable, with one olfactory receptor expressed at almost 300 times the level of another. Higher expression levels could be due to increased transcript number per cell and/or a greater number of olfactory neurons 'choosing' those genes. For one pair of genes we tested, expression level differences appear to be due to both factors. It would be interesting to collect data for additional genes to determine how the numbers of expressing cells and transcript levels per cell vary across the olfactory receptor family. Data from a number of previous studies also show that different olfactory receptor genes, or even copies of the same olfactory receptor transgene in different genomic locations are expressed in different numbers of cells [14,18,35], but do not address the issue of transcript level per cell. The fact that some genes are chosen more frequently, and when chosen may be expressed at higher levels per cell, is intriguing given each olfactory neuron's single-allele expression regime. The observation of unequal expression leads to a number of questions. It is known that each olfactory receptor is expressed in one of four zones of the olfactory epithelium [14,15]; do some zones choose from a smaller olfactory receptor sub-repertoire and thus express each olfactory receptor in a larger number of cells? We note that several apparently highly expressed olfactory receptors (gene A, this study, and MOR10 and MOR28 [36]) are expressed in zone 4 of the olfactory epithelium. Does activity-dependent neuronal competition [37] contribute to increased representation of the olfactory receptors that respond to common environmental odorants? Do the favored olfactory receptors have stronger promoter sequences? Are some olfactory receptor mRNAs more stable than others, leading to higher transcript levels per expressing cell? Are the favored olfactory receptors in more open chromatin conformation or more accessible genomic locations? Transcription of apparent 'singleton' olfactory receptor genes (0.5 Mb or more from the nearest other olfactory receptor gene) suggests that there is no absolute requirement for genomic clustering for an olfactory receptor to be transcribed, consistent with observations that small olfactory receptor transgenes can be expressed correctly when integrated outside native olfactory receptor clusters [35]. However, the high pseudogene count among singleton olfactory receptor genes (50%, versus 20% for clustered olfactory receptor genes) suggests that not all genomic locations are favorable for olfactory receptor gene survival, perhaps due to transcriptional constraints. It is also possible that evolutionary factors may be responsible for reduced pseudogene content of clustered olfactory receptors - gene conversion between neighboring olfactory receptors could rescue inactivating mutations in clustered genes, but not singletons. Before these questions about olfactory receptor gene choice can be answered, it will be important to measure expression levels of a larger number of genes, perhaps using an olfactory receptor gene microarray. + +Our study provides at least partial data about the upstream transcript structures of over 300 olfactory receptor genes. These data provide tentative locations of a large set of promoter regions, allowing computational searches for shared sequence motifs that might be involved in the intriguing transcriptional regulation of olfactory receptors. However, given that not all cDNAs are full-length clones, some of these candidates will not be true promoter regions. The 5' UTR sequences we obtained will also aid in the design of experimental probes, for example, for in situ hybridizations or to immobilize on an olfactory receptor microarray. One of the challenges of such an array will be to design unique probes with which to represent each gene. Often, the coding region of olfactory receptors is highly similar between recently duplicated genes. Many pairs of similar olfactory receptors show more sequence divergence in the UTRs than the protein-coding region (J.Y., unpublished observations). The UTRs would therefore make a better choice of sequence from which to design unique oligonucleotides to distinguish closely related olfactory receptor genes. Locations of these regions in genomic sequence are difficult to predict - our study provides 5' UTR sequences of 343 genes and the approximate 3' UTR length for 399 olfactory receptor genes. Probe design must also account for the multiple transcriptional isoforms observed for many olfactory receptors - depending on the question being asked, probes could be designed in shared sequence to determine the total level of all isoforms, or in unique exons to measure the level of each isoform separately. + +We find that the majority of the olfactory receptors, like most non-olfactory receptor genes [38,39], are transcribed as multiple isoforms, involving alternative splicing of 5' untranslated exons and alternate polyadenylation-site usage. The act of splicing itself may be important for efficient mRNA export from the nucleus [40] or to couple olfactory receptor coding regions with genomically distant promoters. The exact nature of the spliced transcript might be unimportant, such that several isoforms might be produced simply because multiple functional splice sites are available. Alternatively, the multiplicity of transcriptional isoforms might have functional significance, as UTRs may contain signals controlling mRNA stability, localization or degradation [41,42]. + +Our study shows that about 5% of olfactory receptor transcripts do not fit the current notion of olfactory receptor gene structure. Occasionally, an intron is spliced out of the 3' untranslated region. A number of cDNAs use splice sites within the olfactory receptor's ORF, meaning that their protein product is different from that predicted on the basis of genomic sequence alone. In two such cases, the transcript would encode a functional olfactory receptor, with the initiating methionine and first few amino acids encoded by an upstream exon, as has been observed previously for a subtelomeric human olfactory receptor gene [25]. Such within-ORF splicing might increase protein-coding diversity, although, given the small number of genes involved, splicing is unlikely to significantly affect the functional receptor repertoire. Most of the atypical splice forms we observe appear to encode non-functional transcripts, containing frameshifts or lacking a start codon or other functional residues conserved throughout the olfactory receptor family. These nonfunctional transcripts are probably aberrant by-products of the splicing system [43] that have not yet been degraded by RNA surveillance systems [40,41]. The neurons expressing these aberrant transcripts might also make normal transcripts for the same genes and thus produce a functional olfactory receptor. Alternatively, the unusual transcriptional regulation of olfactory receptors might ensure that only one splice isoform is expressed per cell (unlikely, but possible if an RNA-based feedback mechanism operates), thus condemning cells expressing these aberrant isoforms to be dysfunctional. + +We also observe transcripts from a small number of olfactory receptor pseudogenes, as previously described for three human olfactory receptor pseudogenes [26,44]. Although many fewer pseudogenes than intact genes were represented in our cDNA collection, some neurons in the olfactory epithelium evidently express disrupted olfactory receptors and thus might be unable to respond to odorants or to correctly innervate the olfactory bulb. Wang, Axel and coworkers have shown that an artificial transgenic olfactory receptor gene containing two nonsense mutations can support development of an olfactory neuron, but that pseudogene-expressing neurons fail to converge on a glomerulus in the olfactory bulb [45]. By analogy with an olfactory receptor deletion mutant [45], it is likely that most pseudogene-expressing neurons die or switch to express a different olfactory receptor gene, leaving a small number of pseudogene-expressing neurons in adult mice, but at greatly reduced levels compared to neurons expressing intact olfactory receptors. + +Conclusions + +Our study has provided an olfactory receptor cDNA resource representing over one-third of the olfactory receptor gene family. We have thus established over 400 annotated olfactory receptor genes as having olfactory function. The sequences we generated demonstrate that the majority of the olfactory receptor gene family has multiple transcriptional isoforms. Most olfactory receptor transcripts encode functional receptor proteins, with rare exceptions. We show that individual olfactory receptor genes can have vastly different expression levels, an intriguing finding in light of the unusual one-neuron one-gene transcriptional regime of the olfactory epithelium. Our results and the sequences we provide will facilitate future global studies of the mechanisms and dynamics of olfactory receptor gene expression. + +Materials and methods + +Identification of olfactory receptor cDNAs + +An adult mouse cDNA library made from the olfactory epithelium of a single animal was provided by Leslie Vosshall (Rockefeller University, New York, NY, USA), and an embryonic library (made from the olfactory epithelia of several E16.5-E18.5 embryos) was provided by Tyler Cutforth (Columbia University, New York, NY, USA). Both libraries were oligo-dT primed and directionally cloned into the lambdaZAP-XR vector (Stratagene, La Jolla, CA, USA). The adult library has a complexity of 6.5 × 106 primary clones, and the embryonic library has a complexity of 1.65 × 106. Libraries were amplified to give titers of 5 × 109 pfu/ml (adult) or 2 × 1010 pfu/ml (embryonic). Hybridization probes were made by degenerate PCR of mouse genomic DNA, in a fashion similar to those described previously [1], with primer pairs and annealing temperatures given in Table 2. Low-stringency hybridization conditions were as described [1]. Clonally-pure plaques were obtained through secondary screens using the same probe as the corresponding primary screen. PCR with vector primers (M13F/R) was performed to prepare sequencing templates. cDNA size estimates were obtained by agarose gel electrophoresis, and inserts were sequenced from the 5' end using the M13R primer and big-dye terminator chemistry according to ABI's protocols (Applied Biosystems, Foster City, CA, USA). In order to obtain 3' sequence, selected phage clones were converted to plasmid stocks following a scaled-down version of Stratagene's in vivo excision protocol. Plasmid DNA gave better 3'-end sequence than PCR products, which often suffered from polymerase stuttering through the poly(A) tract. + +cDNA sequences and associated information are available through dbEST (Genbank accessions CB172832-CB174569) and our olfactory receptor database [46]. The updated olfactory receptor gene catalog is available through Genbank (accessions AY317244-AY318733). Throughout the manuscript, genes are referred to by their Genbank accession numbers. + +Sequence analysis + +cDNA sequences were base-called and quality-trimmed using phred (trim_cutoff = 0.05) [47], and vector sequences were removed using cross_match [48]. Any sequences of less than 50 bp after trimming were discarded. 3' UTR lengths were estimated by combining approximate insert sizes determined by PCR with 5' sequence data where possible (if the 5' sequence did not extend into the coding region we could not estimate 3' UTR size). We counted cDNAs from a given gene as showing alternative polyadenylation site usage if 3' UTR length estimates varied by at least 400 bp - smaller variation could be real, but may not be distinguishable from error in our size estimates. + +To assign cDNAs to their corresponding olfactory receptor genes, we first defined a genomic 'territory' for each gene, with the following attributes: strand, start position (100 kb upstream of the start codon or 1 kb after the previous gene upstream on the same strand, whichever is closer) and end position (1 kb downstream of the stop codon). Trimmed sequences were compared with genomic sequences using sim4 [30] (settings P = 1 to remove polyA tails and N = 1 to perform an intensive search for small exons). The sim4 algorithm uses splice-site consensus sequences to refine alignments. Only matches of 96% or greater nucleotide identity were considered. RepeatMasked sequences [49] were also compared to genomic sequences; cDNA:genomic sequence pairings not found in both masked and unmasked alignments were rejected. Coordinates from the unmasked alignment were used for further analysis. Any cDNA sequence matching entirely within a territory was assigned to that gene. If a cDNA matched more than one gene territory, the best match was chosen (that is, the one with highest 'score', where score is the total of all exons' lengths multiplied by their respective percent identities). We found 27 cDNAs that spanned a larger genomic range than one gene territory and flagged them for more careful analysis. Of these, six cDNAs showed unusual splicing within the 3' UTR, but the remaining 'territory violators' were found to be artifacts of the analysis process which fell into three types. These included: cDNAs where the insert appeared to be cloned in the reverse orientation (six cDNAs); sequences from recently duplicated gene pairs, where sim4 assigned coding region and upstream exons to different members of the pair, although exons could equally well have been aligned closer to one another (six cDNAs); and artifacts due to use of sim4's N = 1 parameter (nine cDNAs). This parameter instructs the program to make extra effort to match small upstream exons, allowing a greater total length of EST sequence to be matched. However, occasionally the N = 1 parameter caused the program to assign very small sequences (1-4 bp) to distant upstream exons, when they probably match nearer to the corresponding coding sequence. + +The expected distribution shown in Figure 2 was calculated using the equation P(x) = e-μμx/x!, where P(x) is the Poisson probability of observing x cDNAs per gene, and μ is the mean number of cDNAs observed per gene (μ = 1,176/983: 1,176 cDNAs matching olfactory receptor genes in our dataset and 983 intact class II olfactory receptors). In our analysis of expressed pseudogenes, we ignored two olfactory receptor pseudogenes found very near the ends of genomic sequences and thus likely to be error-prone. A protein sequence alignment of intact mouse olfactory receptors was generated using CLUSTALW [50], edited by hand, and used to produce the phylogenetic tree shown in Figure 1 using PAUP's neighbor-joining algorithm (v4.0b6 Version 4, Sinauer Associates, Sunderland, MA). The tree was colored using a custom script. Information content (the measure of sequence conservation shown in Figure 6) was calculated for each position in the alignment using alpro [51]. + +To determine the number of transcriptional isoforms for each gene, we examined the sim4 output for every matching cDNA in decreasing order of number of exons. The first cDNA was counted as one splice form, and for each subsequent cDNA, we determined whether exon structure was mutually exclusive to isoforms already counted. We were conservative in our definition of mutually exclusive, and thus our count represents the minimum number of isoforms represented in the cDNA collection. + +RT-PCR + +The olfactory epithelia were dissected from three adult female C57BL/6 mice, including tissues attached to the skull and septum. RNA was isolated using the Qiagen RNeasy midi kit (Qiagen, Valencia, CA, USA), including a DNase treatment step. First-strand cDNA was produced from 2.5 μg of RNA in a volume of 50 μl using random hexamers and Invitrogen's Superscript II reverse transcriptase (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's recommendations. One-twenty-fifth of the resulting cDNA was used as template in subsequent PCR reactions. PCR amplification biased towards class I olfactory receptors was performed using degenerate primers P26 [17] and classI_R1 (5'-GGRTTIADIRYIGGNGG-3') with an annealing temperature of 44°C. The product was cloned (TA cloning kit, Invitrogen), and individual clones were sequenced. Specific PCR primers used to confirm expression of individual olfactory receptor genes are given in Additional data file 1. Each PCR product was sequenced to confirm that the expected gene and no others had been amplified. Control reactions on a template made by omitting reverse transcriptase gave no product, indicating that the RNA preparation was uncontaminated by genomic DNA. + +Relative transcript levels were estimated using real-time PCR according to Applied Biosystems' protocols, with magnesium concentration, primer pair and fluorescent probe given in Additional data file 2. The increase in fluorescence during thermocycling was measured on an ABI PRISM 7900HT. Standard curves were constructed for each primer pair using triplicate samples of mouse genomic DNA of nine known concentrations (range 0.01-100 ng, or about 3-30,000 copies of the haploid genome). Relative expression level of each gene was determined by comparing the mean Ct (cycle where fluorescence reaches an arbitrary threshold value) obtained with six replicate samples of reverse-transcribed RNA to the standard curve for the corresponding primers. Relative RNA levels of a housekeeping gene, ribosomal S16, were measured as previously described [52]. Control reactions on template prepared by omitting reverse transcriptase amplified at a relative level of 0.03 ± 0.01 ng or less in each case. Expression measurements of the seven genes were normalized for each mouse so that S16 levels were equal to 1 (arbitrary units). + +In situ hybridization + +Coronal sections were cut from the olfactory epithelia of an adult mouse (Figure 4) and a young (P6) C57BL/6 mouse. RNA in situ hybridization was carried out as described previously [15,53] with digoxigenin-labeled antisense riboprobes specific for the 3' UTRs of genes AY318555 (0.5 kb) and AY317365 (0.5 kb). Riboprobe sequences were generated by PCR using primer pairs 5'-TCTTCCAAACCTGGACCCCCC-3' and 5'-ATCTCTCCAGCACCTTACTTG-3' for AY318555 and primer pairs 5'-TAAGATGTAAGTGATAATTTAGATTACAGG-3' and 5'-TTTCTGCCTCAGCTATGACAG-3' for AY317365. Hybridization was carried out in 50% formamide at 65°C, and slides were washed at high stringency (65°C, 0.2 × SSC). The probes each hybridize to only one band on a Southern blot, indicating that each probe only detects one olfactory receptor gene. BLAST analyses show that the AY318555 probe is unique in Celera's mouse genome assembly (Release 13), and that the AY317365 probe is similar to only one other genomic region. This potential cross-hybridizing region is over 10 Mb from the nearest olfactory receptor coding region and is thus highly unlikely to be included in any olfactory receptor transcript. Low-power images are composed of three overlapping micrographs (40×) assembled in Adobe Photoshop 7.0. + +Additional data files + +A list of the primers used to confirm the expression of olfactory receptor genes by RT-PCR and PCR from cDNA library templates can be found in Additional data file 1. The experimental conditions used for real-time PCR can be found in Additional data file 2. + +Supplementary Material + +Additional data file 1 + +A list of the primers used to confirm the expression of olfactory receptor genes by RT-PCR and PCR from cDNA library templates + +Click here for additional data file + +Additional data file 2 + +The experimental conditions used for real-time PCR + +Click here for additional data file + +Acknowledgements + +We thank Leslie Vosshall and Tyler Cutforth for providing cDNA libraries, staff of the core facilities at the Fred Hutchinson Cancer Research Center and the University of Washington's former Department of Molecular Biotechnology for sequencing assistance, Linda Buck and Michael Schlador for comments on the manuscript and Colin Pritchard for S16 primers. The data in this paper were analyzed in part through use of the Celera Discovery System™ and Celera Genomics' associated databases. This work was supported by NIH grant R01 DC04209. + +Figures and Tables + +Figure 1 + +Olfactory receptor genes whose expression in the mouse olfactory epithelium was confirmed in this study. Genes whose expression has been confirmed by our cDNA screen are colored blue on a phylogenetic tree of 1,107 intact mouse olfactory receptors. Genes whose expression was confirmed by PCR methods are colored red (genes listed in Additional data file 1 were confirmed by specific PCR of the cDNA library or reverse-transcribed RNA, and genes confirmed using the class I degenerate primer for RT-PCR are AY317681, AY317698, AY317700, AY317767, AY317773, AY317774, AY317797 and AY317923). Other olfactory receptors are colored gray, and a chemokine outgroup is colored black. Class I olfactory receptors are bracketed, and the remaining olfactory receptors are class II. + +Figure 2 + +The cDNA screen suggests different expression levels for different olfactory receptors. Distribution of number of cDNAs observed (dots) and expected (triangles, line) per olfactory receptor gene among 1,176 olfactory receptor cDNAs identified, based on a Poisson distribution. + +Figure 3 + +Differential expression levels among six olfactory receptor genes determined by quantitative PCR. (a) Expression levels of olfactory receptor genes can vary by almost 300-fold (for example, genes A and D). Relative expression levels of six selected olfactory receptor genes (A, AY318555; B, AY318107; C, AY318644; D, AY317365; E, AY317773; and F, AY317797) were determined in olfactory epithelium RNA samples from three mice. Expression levels for each gene were first determined relative to a standard curve made using mouse genomic DNA templates, and then values for each mouse were normalized so that a housekeeping gene, ribosomal S16, had a value of 1 (arbitrary units) (not shown). Error bars show one standard deviation (six replicate reactions). Genes E and F (AY317773 and AY317797) are class I olfactory receptors. Numbers of cDNAs observed in our screen are shown under each gene name. (b) Expression levels of each gene are similar, with some variation, among the three mice sampled. Graphs show pairwise comparisons between the three mice sampled, with relative expression levels (arbitrary units) in one mouse plotted along the x-axis and in a second on the y-axis. + +Figure 4 + +Olfactory receptors showing different expression levels. Different expression levels of one pair of olfactory receptors is due to different numbers of expressing cells and different transcript levels per cell. RNA in situ hybridization with digoxigenin-labeled probes for (a) gene A (AY318555) and (b) gene D (AY317365) on coronal sections of the olfactory turbinates of an adult mouse, shown at low magnification and inset (boxed) at high magnification. Endoturbinates II and III and ectoturbinate 3 are labeled in (b). + +Figure 5 + +Many olfactory receptor genes show alternate splicing. Distribution of the number of transcriptional isoforms observed for the 82 olfactory receptors for which we have identified at least four cDNAs. + +Figure 6 + +Sixty-two olfactory receptor cDNAs use splice sites within the coding region. The bar at the top represents an alignment of all olfactory receptor proteins, with transmembrane (TM) regions shaded gray and intracellular (IC) and extracellular (EC) loops in white. Above the bar, the jagged line plots information content [51] for each alignment position, with higher values representing residues conserved across more olfactory receptors. cDNAs with atypical splicing are plotted below, aligned appropriately to the consensus representation. Genbank accessions for each cDNA are shown on the right, and where more than one clone represents the same isoform, both names are given, but a composite sequence is drawn. Multiple isoforms from the same gene are grouped by gray background shading. Thick black lines represent cDNA sequence, and thin lines represent intronic sequence (with diagonal slash marks if not drawn to scale). The uppermost two cDNAs encode potentially functional olfactory receptors. A single cDNA drawn as white boxes (CB173065) is cloned into the vector in the reverse orientation. Introns that result in a frameshift relative to the olfactory receptor consensus are drawn as single dashed lines. The first in-frame methionine in the cDNA is marked with an 'M', and the first stop codon 5' to this methionine (if any) is marked with *. Most sequences are incomplete at the 3' end, as represented by paired dotted lines, although two sequences (CB174400 and CB174364), marked with '(A)n', contain the cDNA's poly(A) tail. The 'X' on sequence CB173500 marks an exon that does not align with genomic sequence near the rest of the gene or anywhere else in Celera's mouse genome sequence, and 'TM4' on sequence CB172879 notes an exon that matches to the reverse-complement of the fourth transmembrane domain of the next downstream olfactory receptor gene. For the two lowermost cDNAs, exon order in the cDNA clone is inconsistent with the corresponding genomic sequence, as represented by the curved intron lines. + +Table 1 + +Number of olfactory receptors in old (Release 12) and new (Release 13) Celera mouse genome assemblies + +Table 2 + +Summary of cDNA screen for each library and probe. + +Probe names comprise the names of the two primers and the annealing temperature used during PCR to generate the probes, separated by underscores diff --git a/src/ontogpt/evaluation/craft/database/all/14624252.ann b/src/ontogpt/evaluation/craft/database/all/14624252.ann new file mode 100644 index 000000000..607cb6885 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/14624252.ann @@ -0,0 +1,919 @@ +T1 GO:1990265 20 24 PDGF +T2 GO:0035791 20 54 PDGF Receptor β Signaling Pathways +T3 UBERON:0004237 58 80 Vascular Smooth Muscle +T4 CL:0000359 58 85 Vascular Smooth Muscle Cell +T5 GO:0097084 58 97 Vascular Smooth Muscle Cell Development +T6 CL:0000233 113 121 platelet +T7 GO:1990265 113 143 platelet-derived growth factor +T8 CHEBI:36357 190 199 molecules +T9 GO:0007169 260 294 receptor tyrosine kinase signaling +T10 SO:0001023 435 442 allelic +T11 UBERON:0004237 586 608 vascular smooth muscle +T12 CL:0000359 586 614 vascular smooth muscle cells +T13 CL:0000669 615 624 pericytes +T14 CL:0000359 626 627 v +T15 CL:0000669 628 629 p +T16 GO:0010467 706 715 expressed +T17 GO:0010467 804 814 expression +T18 CL:0000359 913 914 v +T19 CL:0000669 915 916 p +T20 PR:000013743 938 944 RasGAP +T21 CHEBI:35224 1042 1050 effector +T22 GO:0035791 1186 1212 PDGFRβ signal transduction +T23 CL:0000359 1252 1253;1256 1261 v ... cells +T24 CL:0000669 1254 1261 p cells +T25 GO:0007169 1287 1335 signal transduction by receptor tyrosine kinases +T26 GO:0007169 1287 1309;1337 1341 signal transduction by ... RTKs +T27 GO:0007169 2169 2182 RTK signaling +T28 GO:0007169 2236 2239;2248 2257 RTK ... signaling +T29 GO:0005737 2302 2313 cytoplasmic +T30 SO:0000417 2314 2321 domains +T31 NCBITaxon:10088 2342 2346 mice +T32 CL:0000233 2744 2752 platelet +T33 GO:1990265 2744 2774 platelet-derived growth factor +T34 GO:0035790 2744 2785;2795 2804 platelet-derived growth factor receptor α ... signaling +T35 GO:0035790 2787 2793;2795 2804 PDGFRα ... signaling +T36 CHEBI:28874 2836 2856 phosphatidylinositol +T37 GO:0014065 2836 2866;2902 2930 phosphatidylinositol 3′-kinase ... signal transduction pathways +T38 GO:0014065 2868 2872;2902 2930 PI3K ... signal transduction pathways +T39 PR:000002997 2878 2895 Src family kinase +T40 PR:000002997 2897 2900 SFK +T41 CL:0000128 2945 2960 oligodendrocyte +T42 GO:0014003 2945 2972 oligodendrocyte development +T43 CL:0000233 3183 3191 platelet +T44 GO:1990265 3183 3213 platelet-derived growth factor +T45 GO:0046983 3372 3381 dimerizes +T46 GO:0046777 3389 3407 autophosphorylated +T47 GO:0005737 3425 3436 cytoplasmic +T48 SO:0000409 3494 3507 binding sites +T49 SO:0000417 3516 3522 domain +T50 GO:0065007 3691 3698 control +T51 GO:0008283 3738 3751 proliferation +T52 GO:0031012 3764 3770 matrix +T53 SO:0000704 3803 3807 gene +T54 SO:0000417 3909 3915 domain +T55 CHEBI:36357 4031 4040 molecules +T56 PR:000002997 4049 4052 SFK +T57 PR:000003177 4160 4163 Shc +T58 PR:000013743 4186 4192 RasGAP +T59 PR:000001933 4239 4289 signal transducers and activators of transcription +T60 PR:000001933 4291 4296 STATs +T61 PR:000008224 4321 4325 Grb2 +T62 PR:000008225 4351 4355 Grb7 +T63 PR:000013449 4378 4420 SH2-containing phosphotyrosine phosphatase +T64 PR:000013449 4422 4427 SHP-2 +T65 PR:000013449 4443 4450 SH-PTP2 +T66 PR:000011033 4580 4583 Nck +T67 GO:0035791 4827 4849;4854 4860 signal transduction by ... PDGFRβ +T68 SO:0001023 4892 4898 allele +T69 GO:0035791 4938 4964 PDGFRβ signal transduction +T70 UBERON:0004237 4993 5015 vascular smooth muscle +T71 CL:0000359 4993 5021 vascular smooth muscle cells +T72 CL:0000669 5026 5035 pericytes +T73 CL:0000359 5037 5038 v +T74 CL:0000669 5039 5040 p +T75 UBERON:0003104 5098 5109 mesenchymal +T76 CL:0000630 5110 5123 support cells +T77 UBERON:0001981 5138 5151 blood vessels +T78 UBERON:0000955 5194 5199 Brain +T79 CL:0000669 5200 5209 pericytes +T80 UBERON:0002113 5211 5217 kidney +T81 CL:0000650 5211 5233 kidney mesangial cells +T82 UBERON:0002319 5218 5227 mesangial +T83 UBERON:0000966 5235 5242 retinal +T84 CL:0009004 5235 5242;5249 5254 retinal ... cells +T85 UBERON:0002101 5260 5264 limb +T86 CL:0000669 5274 5283 pericytes +T87 GO:0008283 5481 5494 proliferation +T88 GO:1990265 5603 5607 PDGF +T89 GO:0048008 5603 5627 PDGF signal transduction +T90 CL:0000359 5658 5659;5662 5666 v ... cell +T91 CL:0000669 5660 5666 p cell +T92 UBERON:0012101 5693 5702 perinatal +T93 GO:0007567 5697 5702 natal +T94 UBERON:0000055 5720 5726 vessel +T95 SO:0000409 5864 5877 binding sites +T96 GO:0005737 5896 5907 cytoplasmic +T97 SO:0000417 5908 5914 domain +T98 NCBITaxon:33208 6104 6111 animals +T99 SO:0001023 6237 6244 allelic +T100 NCBITaxon:10088 6255 6259 mice +T101 CL:0000359 6399 6400;6403 6407 v ... cell +T102 CL:0000669 6401 6407 p cell +T103 GO:0035791 6432 6458 PDGFRβ signal transduction +T104 GO:0010467 6506 6515 expressed +T105 CL:0000359 6604 6605;6608 6613 v ... cells +T106 CL:0000669 6606 6613 p cells +T107 GO:0007169 6661 6684 RTK signal transduction +T108 GO:0065007 6700 6711 controlling +T109 GO:0048468 6712 6732 cellular development +T110 SO:0001023 6762 6769 Allelic +T111 CL:0000359 6862 6863 v +T112 GO:0097084 6862 6863;6866 6877 v ... development +T113 CL:0000669 6864 6865 p +T114 SO:0001023 7141 7148 allelic +T115 PR:000013743 7439 7445 RasGAP +T116 SO:0000409 7446 7458 binding site +T117 SO:0001587 7601 7621 premature stop codon +T118 PR:000013743 7640 7646 RasGAP +T119 SO:0000409 7647 7659 binding site +T120 SO:0001644 7879 7895 targeting vector +T121 UBERON:0000922 7972 7981 embryonic +T122 CL:0002322 7972 7986;7992 7997 embryonic stem ... cells +T123 CL:0002322 7988 7990;7992 7997 ES ... cells +T124 SO:0001023 8085 8091 allele +T125 NCBITaxon:10088 8175 8179 mice +T126 SO:0001023 8241 8247 allele +T127 SO:0000417 8295 8301 domain +T128 PR:000013449 8310 8315 SHP-2 +T129 SO:0000409 8326 8339 binding sites +T130 GO:0071897 8459 8472 DNA synthesis +T131 UBERON:0000922 8566 8573 Embryos +T132 SO:0001023 8596 8602 allele +T133 GO:0016265 8603 8606 die +T134 UBERON:0012101 8607 8618 perinatally +T135 GO:0007567 8611 8618 natally +T136 UBERON:0000922 8673 8680 embryos +T137 UBERON:0000922 8686 8693 embryos +T138 UBERON:0000479 8737 8744 tissues +T139 UBERON:0002113 8760 8766 kidney +T140 UBERON:0000955 8768 8773 brain +T141 CL:0000359 8868 8869 v +T142 GO:0097084 8868 8869;8872 8883 v ... development +T143 CL:0000669 8870 8871 p +T144 CL:0000071 8977 9002 vascular endothelial cell +T145 PR:000007563 8977 9027 vascular endothelial cell growth factor receptor 1 +T146 UBERON:0001986 8986 8997 endothelial +T147 CL:0000359 9072 9073;9076 9081 V ... Cells +T148 CL:0000669 9074 9081 P Cells +T149 UBERON:0001981 9099 9112 blood vessels +T150 NCBITaxon:10088 9136 9140 mice +T151 CL:0000359 9247 9248;9251 9255 v ... cell +T152 CL:0000669 9249 9255 p cell +T153 NCBITaxon:10088 9337 9341 mice +T154 NCBITaxon:10088 9365 9370 mouse +T155 GO:0010467 9371 9380 expresses +T156 GO:0005634 9381 9388 nuclear +T157 GO:0008283 9450 9463 proliferating +T158 CL:0000359 9464 9465;9468 9473 v ... cells +T159 CL:0000669 9466 9473 p cells +T160 UBERON:0000922 9481 9487 embryo +T161 UBERON:0007023 9496 9501 adult +T162 UBERON:0007023 9565 9570 adult +T163 NCBITaxon:33208 9571 9578 animals +T164 NCBITaxon:10088 9628 9632 mice +T165 UBERON:0000479 9640 9647 tissues +T166 UBERON:0000970 9655 9659 eyes +T167 UBERON:0000948 9661 9667 hearts +T168 UBERON:0000955 9673 9679 brains +T169 SO:0001023 9773 9780 alleles +T170 UBERON:0000479 9806 9813 tissues +T171 GO:0035791 9831 9857 PDGFRβ signal transduction +T172 CL:0000359 9989 9990 v +T173 CL:0000669 9991 9992 p +T174 CL:0000359 10078 10079;10082 10087 v ... cells +T175 CL:0000669 10080 10087 p cells +T176 CL:0000359 10089 10090;10093 10097 V ... cell +T177 CL:0000669 10091 10097 p cell +T178 NCBITaxon:10088 10151 10155 mice +T179 UBERON:0002113 10168 10174 kidney +T180 CL:0000650 10168 10190 kidney mesangial cells +T181 UBERON:0002319 10175 10184 mesangial +T182 CL:0000669 10195 10204 pericytes +T183 UBERON:0004288 10221 10229 skeletal +T184 GO:0005634 10307 10313 nuclei +T185 UBERON:0000074 10340 10356 kidney glomeruli +T186 http://purl.obolibrary.org/obo/MONDO_0000490 10380 10398 glomerulosclerosis +T187 UBERON:0000479 10493 10500 tissues +T188 CL:0000359 10539 10540;10543 10548 v ... cells +T189 CL:0000669 10541 10548 p cells +T190 NCBITaxon:10088 10600 10604 mice +T191 GO:0010467 10631 10640 expressed +T192 UBERON:0000479 10655 10662 tissues +T193 UBERON:0000479 10678 10685 tissues +T194 CL:0000359 10728 10729;10732 10736 v ... cell +T195 CL:0000669 10730 10736 p cell +T196 CL:0000359 10775 10776;10779 10784 v ... cells +T197 CL:0000669 10777 10784 p cells +T198 GO:0035791 10816 10842 PDGFRβ signal transduction +T199 GO:0010467 10895 10904 expressed +T200 CL:0000359 10926 10927 v +T201 CL:0000669 10928 10929 p +T202 UBERON:0003104 10993 11004 mesenchymal +T203 CL:0000134 10993 11009 mesenchymal cell +T204 CL:0000359 11165 11166 v +T205 CL:0000669 11167 11168 p +T206 GO:0010467 11181 11188 express +T207 NCBITaxon:10088 11256 11260 mice +T208 NCBITaxon:10088 11295 11300 mouse +T209 GO:0010467 11308 11317 expresses +T210 GO:0005634 11320 11327 nuclear +T211 GO:0065007 11380 11387 control +T212 SO:0000167 11402 11410 promoter +T213 NCBITaxon:10088 11454 11458 mice +T214 GO:0010467 11480 11490 expression +T215 CL:0000359 11517 11518;11521 11526 v ... cells +T216 CL:0000669 11519 11526 p cells +T217 UBERON:0002113 11534 11540 kidney +T218 UBERON:0000970 11542 11545 eye +T219 UBERON:0000955 11551 11556 brain +T220 CL:0000359 11637 11638;11641 11646 v ... cells +T221 CL:0000669 11639 11646 p cells +T222 UBERON:0001135 11648 11661 smooth muscle +T223 PR:000003675 11648 11669 smooth muscle actin α +T224 PR:000003675 11671 11675 αSMA +T225 PR:000006427 11678 11684 desmin +T226 SO:0000902 11701 11710 transgene +T227 GO:0010467 11728 11738 expressing +T228 UBERON:0000479 11767 11774 tissues +T229 CL:0000359 11778 11779;11782 11786 v ... cell +T230 CL:0000669 11780 11786 p cell +T231 GO:0010467 11820 11830 expression +T232 CL:0000359 11854 11855;11858 11862 v ... cell +T233 CL:0000669 11856 11862 p cell +T234 UBERON:0001637 11878 11886 arteries +T235 UBERON:0001638 11890 11895 veins +T236 UBERON:0000970 11918 11921 eye +T237 UBERON:0000955 11926 11931 brain +T238 GO:0010467 11940 11950 expressing +T239 UBERON:0002113 12004 12010 kidney +T240 UBERON:0000074 12066 12083 kidney glomerulus +T241 UBERON:0002113 12117 12123 kidney +T242 CL:0000650 12117 12139 kidney mesangial cells +T243 UBERON:0002319 12124 12133 mesangial +T244 UBERON:0005742 12152 12163 adventitial +T245 CL:0000057 12164 12175 fibroblasts +T246 GO:0010467 12225 12232 express +T247 GO:0010467 12306 12315 expressed +T248 UBERON:0000479 12359 12366 tissues +T249 NCBITaxon:10088 12381 12385 mice +T250 UBERON:0000970 12391 12394 eye +T251 UBERON:0000955 12403 12408 brain +T252 CL:0000359 12479 12480 V +T253 GO:0097084 12479 12480;12483 12494 V ... Development +T254 CL:0000669 12481 12482 P +T255 CL:0000359 12534 12535 v +T256 CL:0000669 12536 12537 p +T257 CL:0000669 12606 12614 pericyte +T258 UBERON:0000922 12651 12658 embryos +T259 NCBITaxon:10088 12671 12676 mouse +T260 CL:0000359 12717 12718;12721 12725 v ... cell +T261 CL:0000669 12719 12725 p cell +T262 CL:0000669 12777 12786 pericytes +T263 CL:0000359 12822 12823 v +T264 CL:0000669 12824 12825 p +T265 NCBITaxon:33208 12852 12859 animals +T266 UBERON:0000479 12871 12878 tissues +T267 UBERON:0002240 12905 12916 spinal cord +T268 UBERON:0012198 12921 12932 intercostal +T269 UBERON:0002049 12933 12944 vasculature +T270 CL:0000359 13001 13002;13005 13009 v ... cell +T271 CL:0000669 13003 13009 p cell +T272 UBERON:0000922 13041 13048 embryos +T273 UBERON:0000922 13085 13091 embryo +T274 UBERON:0000922 13151 13158 embryos +T275 UBERON:0000922 13251 13258 embryos +T276 UBERON:0000922 13297 13304 embryos +T277 UBERON:0001981 13331 13344 blood vessels +T278 CL:0000359 13358 13359 v +T279 CL:0000669 13360 13361 p +T280 UBERON:0002240 13454 13465 spinal cord +T281 CL:0000669 13466 13474 pericyte +T282 UBERON:0000922 13560 13566 embryo +T283 UBERON:0002240 13670 13681 spinal cord +T284 UBERON:0002049 13825 13836 vasculature +T285 UBERON:0000922 13873 13879 embryo +T286 CL:0000669 13889 13898 pericytes +T287 NCBITaxon:10088 13988 13992 mice +T288 CL:0000669 14006 14014 pericyte +T289 CL:0000669 14182 14191 pericytes +T290 UBERON:0000922 14223 14230 embryos +T291 CL:0000669 14249 14257 pericyte +T292 UBERON:0002113 14398 14404 kidney +T293 UBERON:0000922 14424 14431 embryos +T294 UBERON:0000922 14445 14452 embryos +T295 CL:0000669 14503 14512 pericytes +T296 UBERON:0000948 14520 14525 heart +T297 UBERON:0002113 14570 14576 kidney +T298 CL:0000669 14657 14665 pericyte +T299 GO:0042592 14710 14721 homeostasis +T300 UBERON:0000479 14729 14735 tissue +T301 SO:0001023 14790 14796 allele +T302 UBERON:0000948 14845 14850 heart +T303 UBERON:0002113 14855 14861 kidney +T304 CL:0000359 14904 14905 v +T305 GO:0097084 14904 14905;14908 14917 v ... formation +T306 CL:0000669 14906 14907 p +T307 CL:0000669 14919 14927 Pericyte +T308 UBERON:0002113 14984 14990 kidney +T309 UBERON:0000922 15000 15007 embryos +T310 UBERON:0000948 15041 15046 heart +T311 CL:0000669 15058 15066 pericyte +T312 CL:0000359 15126 15127 v +T313 CL:0000669 15128 15129 p +T314 UBERON:0000922 15182 15189 embryos +T315 UBERON:0001135 15307 15320 smooth muscle +T316 CL:0000192 15307 15325 smooth muscle cell +T317 CL:0000359 15391 15392 v +T318 GO:0097084 15391 15392;15395 15408 v ... developmental +T319 CL:0000669 15393 15394 p +T320 CL:0000359 15495 15496;15499 15504 v ... cells +T321 CL:0000669 15497 15504 p cells +T322 NCBITaxon:33208 15531 15538 animals +T323 SO:0001023 15563 15569 allele +T324 CL:0000669 15653 15661 pericyte +T325 CL:0000669 15705 15714 pericytes +T326 UBERON:0000922 15742 15749 embryos +T327 UBERON:0000922 15785 15792 embryos +T328 CL:0000669 15915 15924 pericytes +T329 CL:0000669 15999 16008 pericytes +T330 UBERON:0000922 16067 16074 embryos +T331 GO:0048468 16086 16102 cell development +T332 NCBITaxon:33208 16154 16161 animals +T333 CHEBI:36357 16293 16302 molecules +T334 SO:0001023 16395 16402 alleles +T335 CL:0000359 16429 16430;16433 16438 v ... cells +T336 CL:0000669 16431 16438 p cells +T337 SO:0001023 16447 16453 allele +T338 UBERON:0002240 16489 16500 spinal cord +T339 CL:0000669 16501 16510 pericytes +T340 UBERON:0000922 16676 16683 embryos +T341 UBERON:0000922 16698 16705 embryos +T342 CL:0000669 16729 16738 pericytes +T343 UBERON:0000922 16751 16758 embryos +T344 PR:000013743 16863 16869 RasGAP +T345 CL:0000359 17001 17002 v +T346 CL:0000669 17003 17004 p +T347 GO:0065007 17032 17042 controlled +T348 GO:0035791 17052 17068 PDGFRβ signaling +T349 CL:0000359 17155 17156 v +T350 CL:0000669 17157 17158 p +T351 CL:0000359 17212 17213 v +T352 CL:0000669 17214 17215 p +T353 UBERON:0000966 17234 17240 retina +T354 PR:000012491 17276 17281 PDGFB +T355 GO:0035791 17276 17302 PDGFB and PDGFRβ signaling +T356 GO:0065007 17303 17311 controls +T357 CL:0000669 17312 17320 pericyte +T358 UBERON:0000970 17340 17343 eye +T359 UBERON:0007023 17411 17416 Adult +T360 NCBITaxon:10088 17417 17421 mice +T361 SO:0001023 17453 17459 allele +T362 SO:0001023 17477 17483 allele +T363 UBERON:0000970 17501 17504 eye +T364 UBERON:0000970 17605 17608 eye +T365 GO:0035791 17650 17676 PDGFRβ and PDGFB signaling +T366 PR:000012491 17661 17666 PDGFB +T367 NCBITaxon:10088 17762 17766 mice +T368 UBERON:0001981 17797 17810 blood vessels +T369 UBERON:0000966 17829 17836 retinal +T370 CL:0009004 17829 17842 retinal cells +T371 GO:0007567 17981 17986 birth +T372 http://purl.obolibrary.org/obo/MONDO_0000001 18006 18028 pathological condition +T373 SO:0001023 18057 18064 alleles +T374 SO:0001023 18147 18154 alleles +T375 UBERON:0000966 18177 18184 retinal +T376 CL:0000669 18185 18194 pericytes +T377 GO:0035791 18221 18246 PDGFRβ signaling pathways +T378 UBERON:0000966 18287 18294 retinal +T379 CL:0000669 18295 18304 pericytes +T380 NCBITaxon:10088 18343 18347 mice +T381 SO:0000902 18367 18376 transgene +T382 UBERON:0004864 18397 18416 retinal vasculature +T383 CHEBI:26130 18465 18474 pigmented +T384 UBERON:0000483 18475 18485 epithelium +T385 SO:0001023 18573 18580 alleles +T386 UBERON:0000970 18618 18622 eyes +T387 UBERON:0000970 18649 18653 eyes +T388 CL:0000669 18681 18690 pericytes +T389 UBERON:0000966 18818 18825 retinas +T390 CL:0000359 18846 18847 v +T391 CL:0000669 18848 18849 p +T392 UBERON:0000970 18877 18881 eyes +T393 GO:0035791 18924 18959 PDGFRβ signal transduction pathways +T394 CL:0000359 18963 18964 v +T395 GO:0097084 18963 18964;18967 18978 v ... development +T396 CL:0000669 18965 18966 p +T397 UBERON:0000479 18989 18995 tissue +T398 CL:0000359 19019 19020 v +T399 GO:0097084 19019 19020;19023 19032 v ... formation +T400 CL:0000669 19021 19022 p +T401 UBERON:0000948 19040 19045 heart +T402 UBERON:0000948 19147 19153 hearts +T403 UBERON:0000970 19209 19212 eye +T404 UBERON:0001016 19221 19235 nervous system +T405 SO:0001023 19258 19265 alleles +T406 UBERON:0004797 19295 19311 vascular coating +T407 UBERON:0001621 19321 19338 coronary arteries +T408 UBERON:0004148 19321 19329;19343 19348 coronary ... veins +T409 NCBITaxon:10088 19391 19395 mice +T410 UBERON:0000948 19425 19430 heart +T411 UBERON:0002082 19465 19475 ventricles +T412 UBERON:0000948 19487 19492 heart +T413 UBERON:0002081 19518 19523 atria +T414 NCBITaxon:10088 19578 19582 mice +T415 CL:0000359 19615 19616 v +T416 CL:0000669 19617 19618 p +T417 UBERON:0001621 19636 19653 coronary arteries +T418 UBERON:0001016 19702 19716 nervous system +T419 UBERON:0000970 19725 19728 eye +T420 SO:0001023 19751 19758 alleles +T421 CL:0000359 19791 19792;19795 19800 v ... cells +T422 CL:0000669 19793 19800 p cells +T423 GO:0035791 19876 19902 PDGFRβ signal transduction +T424 CL:0000669 19925 19934 pericytes +T425 CL:0000669 20052 20061 pericytes +T426 SO:0000417 20147 20153 domain +T427 CL:0000359 20181 20182 v +T428 CL:0000669 20183 20184 p +T429 CL:0000359 20353 20354 v +T430 GO:0097084 20353 20354;20357 20368 v ... development +T431 CL:0000669 20355 20356 p +T432 SO:0001023 20390 20396 allele +T433 SO:0000417 20472 20478 domain +T434 SO:0000410 20490 20511 protein-binding sites +T435 CL:0000359 20528 20529 v +T436 GO:0097084 20528 20529;20532 20543 v ... development +T437 CL:0000669 20530 20531 p +T438 NCBITaxon:10088 20965 20970 mouse +T439 UBERON:0000922 20971 20977 embryo +T440 CL:0000057 20978 20989 fibroblasts +T441 GO:0010467 21053 21062 expressed +T442 GO:0009986 21215 21222 surface +T443 GO:0010467 21223 21233 expression +T444 GO:0005737 21805 21816 cytoplasmic +T445 PR:000027234 21943 21946 Src +T446 SO:0000409 21947 21960 binding sites +T447 PR:000027234 21987 21990 Src +T448 PR:000027234 22108 22111 Src +T449 PR:000027234 22303 22306 Src +T450 PR:000027234 22474 22477 Src +T451 GO:0005576 22661 22674 extracellular +T452 PR:000000104 22661 22699 extracellular signal-related kinases 1 +T453 PR:000000103 22661 22697;22704 22705 extracellular signal-related kinases ... 2 +T454 PR:000000104 22707 22711 ERK1 +T455 PR:000029189 22719 22722 AKT +T456 PR:000000104 22777 22781 ERK1 +T457 PR:000029189 22788 22791 AKT +T458 GO:0010467 22878 22888 expressing +T459 CHEBI:36357 22992 23001 molecules +T460 PR:000000104 23249 23253 ERK1 +T461 PR:000013743 23338 23344 RasGAP +T462 SO:0000409 23345 23357 binding site +T463 CL:0000359 23461 23462 v +T464 CL:0000669 23463 23464 p +T465 GO:0007169 23669 23692 RTK signal transduction +T466 GO:0048468 23802 23822 cellular development +T467 CL:0000359 23902 23903 v +T468 CL:0000669 23904 23905 p +T469 CL:0000359 24029 24030 v +T470 CL:0000669 24031 24032 p +T471 CL:0000084 24187 24193 T cell +T472 GO:0048468 24189 24205 cell development +T473 UBERON:0002405 24213 24226 immune system +T474 GO:0010467 24490 24499 expressed +T475 GO:0009986 24507 24519 cell surface +T476 CL:0000359 24521 24522 V +T477 CL:0000669 24523 24524 p +T478 UBERON:0000922 24570 24577 embryos +T479 GO:0035791 24816 24842 PDGFRβ signal transmission +T480 SO:0000417 24878 24884 domain +T481 CL:0000359 24960 24961 v +T482 CL:0000669 24962 24963 p +T483 CL:0000359 25051 25052 v +T484 CL:0000669 25053 25054 p +T485 SO:0001023 25124 25131 alleles +T486 CL:0000359 25267 25268 v +T487 CL:0000669 25269 25270 p +T488 SO:0001023 25352 25359 alleles +T489 CL:0000359 25490 25491 v +T490 CL:0000669 25492 25493 p +T491 CHEBI:35224 25565 25573 effector +T492 GO:0035791 25678 25704 PDGFRβ signal transduction +T493 GO:0065007 25708 25717 regulated +T494 CHEBI:36357 25768 25777 molecules +T495 GO:0010467 25800 25810 expression +T496 GO:0035791 25959 25989 transmission of PDGFRβ signals +T497 SO:0001023 26011 26017 allele +T498 GO:0005576 26231 26244 extracellular +T499 SO:0000417 26245 26251 domain +T500 GO:0005622 26274 26287 intracellular +T501 SO:0000417 26288 26294 domain +T502 SO:0001023 26354 26360 allele +T503 PR:000013743 26493 26499 RasGAP +T504 NCBITaxon:10088 26577 26581 mice +T505 GO:0010467 26627 26637 expression +T506 CL:0000359 26747 26748 v +T507 CL:0000669 26749 26750 p +T508 CL:0000359 26891 26892 v +T509 CL:0000669 26893 26894 p +T510 CL:0000359 27108 27109 v +T511 CL:0000669 27110 27111 p +T512 PR:000013449 27187 27192 SHP-2 +T513 PR:000013743 27197 27203 RasGAP +T514 CHEBI:36357 27249 27258 molecules +T515 GO:0035791 27440 27466 PDGFRβ signal transduction +T516 CL:0000359 27626 27627 v +T517 CL:0000669 27628 27629 p +T518 PR:000002997 27739 27742 SFK +T519 GO:0008283 27782 27795 proliferation +T520 CL:0000359 28020 28021 v +T521 CL:0000669 28022 28023 p +T522 SO:0000704 28282 28286 gene +T523 GO:0010467 28282 28297 gene expression +T524 NCBITaxon:10088 28683 28687 mice +T525 CL:0000359 28720 28721 v +T526 CL:0000669 28722 28723 p +T527 NCBITaxon:10088 28784 28788 mice +T528 CL:0000359 28851 28852 v +T529 CL:0000669 28853 28854 p +T530 NCBITaxon:10088 28962 28967 mouse +T531 UBERON:0000922 28968 28975 embryos +T532 UBERON:0000922 29043 29050 embryos +T533 GO:0010467 29051 29061 expressing +T534 SO:0001023 29074 29080 allele +T535 SO:0001023 29109 29115 allele +T536 SO:0001023 29191 29198 alleles +T537 PR:000013449 29247 29252 SHP-2 +T538 PR:000002997 29254 29257 SFK +T539 PR:000008224 29259 29263 Grb2 +T540 PR:000013743 29269 29275 RasGAP +T541 CL:0000669 29288 29296 pericyte +T542 SO:0001023 29389 29396 alleles +T543 PR:000013449 29525 29530 SHP-2 +T544 PR:000013743 29535 29541 RasGAP +T545 GO:0035791 29609 29625 PDGFRβ signaling +T546 PR:000013743 29694 29700 RasGAP +T547 GO:0035791 29734 29750 PDGFRβ signaling +T548 PR:000013743 29798 29804 RasGAP +T549 PR:000013449 29878 29883 SHP-2 +T550 GO:0065007 29919 29929 modulating +T551 CL:0000359 30051 30052 v +T552 GO:0097084 30051 30052;30055 30064 v ... formation +T553 CL:0000669 30053 30054 p +T554 NCBITaxon:10088 30098 30102 mice +T555 NCBITaxon:33208 30129 30136 animals +T556 SO:0001023 30145 30151 allele +T557 CHEBI:62488 30282 30304 signal relay molecules +T558 PR:000002997 30380 30384 SFKs +T559 PR:000001933 30386 30391 STATs +T560 PR:000002997 30481 30484 SFK +T561 PR:000002997 30603 30607 SFKs +T562 PR:000001933 30700 30704 STAT +T563 PR:000008224 30709 30713 Grb2 +T564 CHEBI:35224 30807 30815 effector +T565 CHEBI:36357 30816 30825 molecules +T566 GO:1990265 30829 30833 PDGF +T567 GO:0036120 30829 30860 PDGF-induced cellular responses +T568 SO:0000417 30970 30976 domain +T569 SO:0000417 31118 31124 domain +T570 PR:000013449 31133 31138 SHP-2 +T571 SO:0000409 31149 31162 binding sites +T572 CHEBI:36357 31206 31215 molecules +T573 CL:0000359 31415 31416;31419 31423 v ... cell +T574 CL:0000669 31417 31423 p cell +T575 GO:0010467 31454 31463 expressed +T576 CHEBI:36357 31481 31490 molecules +T577 PR:000025844 31527 31534 ephrins +T578 PR:000001817 31544 31592 low-density lipoprotein receptor-related protein +T579 PR:000002058 31593 31596 LRP +T580 SO:0001023 31893 31899 allele +T581 GO:0035791 32024 32050 PDGFRβ signal transduction +T582 PR:000013743 32211 32217 RasGAP +T583 PR:000013743 32369 32375 RasGAP +T584 SO:0000409 32376 32388 binding site +T585 SO:0000704 32409 32413 gene +T586 SO:0000704 32454 32458 gene +T587 PR:000013743 32511 32517 RasGAP +T588 GO:0035791 32575 32601 PDGFRβ signal transduction +T589 NCBITaxon:10088 32626 32630 mice +T590 GO:0007169 32660 32683 RTK signal transduction +T591 CL:0000359 32753 32754;32757 32761 v ... cell +T592 GO:0097084 32753 32754;32757 32773 v ... cell development +T593 CL:0000669 32755 32761 p cell +T594 CL:0000359 32797 32798;32801 32805 v ... cell +T595 GO:0097084 32797 32798;32801 32817 v ... cell development +T596 CL:0000669 32799 32805 p cell +T597 GO:0035791 32835 32861 PDGFRβ signal transduction +T598 CL:0000359 32983 32984;32987 32991 v ... cell +T599 GO:0097084 32983 32984;32987 33003 v ... cell development +T600 CL:0000669 32985 32991 p cell +T601 UBERON:0000922 33094 33101 embryos +T602 CL:0000359 33103 33104;33107 33112 v ... cells +T603 CL:0000669 33105 33112 p cells +T604 CL:0000359 33202 33203;33206 33211 v ... cells +T605 CL:0000669 33204 33211 p cells +T606 NCBITaxon:33208 33299 33306 animals +T607 CL:0000669 33312 33320 pericyte +T608 CL:0000359 33447 33448;33451 33456 v ... cells +T609 CL:0000669 33449 33456 p cells +T610 CL:0000359 33539 33540 v +T611 CL:0000669 33541 33542 p +T612 NCBITaxon:10088 33601 33605 mice +T613 CL:0000359 33669 33670 v +T614 CL:0000669 33671 33672 p +T615 GO:0008283 33759 33772 proliferation +T616 UBERON:0007023 33780 33785 adult +T617 NCBITaxon:33208 33796 33803 animals +T618 GO:0006915 33843 33852 apoptosis +T619 NCBITaxon:10088 33876 33880 mice +T620 CL:0000359 33981 33982;33985 33990 v ... cells +T621 CL:0000669 33983 33990 p cells +T622 CL:0000359 34027 34028;34031 34035 v ... cell +T623 CL:0000669 34029 34035 p cell +T624 UBERON:0001986 34105 34116 endothelial +T625 CL:0000115 34105 34122 endothelial cells +T626 GO:0046903 34126 34133 secrete +T627 GO:1990265 34138 34142 PDGF +T628 UBERON:0003915 34225 34242 endothelial tubes +T629 CL:0000359 34254 34255;34258 34263 v ... cells +T630 CL:0000669 34256 34263 p cells +T631 CL:0000359 34346 34347;34350 34355 v ... cells +T632 CL:0000669 34348 34355 p cells +T633 UBERON:0002203 34396 34411 eye vasculature +T634 UBERON:0000055 34451 34457 vessel +T635 UBERON:0000922 34538 34544 embryo +T636 CL:0000359 34667 34668;34671 34676 v ... cells +T637 CL:0000669 34669 34676 p cells +T638 GO:0065007 34691 34700 modulated +T639 GO:0010467 34736 34745 expressed +T640 GO:0009986 34753 34765 cell surface +T641 GO:0035791 34895 34921 PDGFRβ signal transduction +T642 CL:0000359 34925 34926;34929 34934 v ... cells +T643 CL:0000669 34927 34934 p cells +T644 NCBITaxon:10088 35036 35040 Mice +T645 SO:0000061 35274 35290 restriction site +T646 NCBITaxon:10088 35351 35356 Mouse +T647 SO:0001644 35456 35472 targeting vector +T648 SO:0000440 35551 35557 vector +T649 SO:0000147 35563 35568 exons +T650 SO:0001644 35647 35663 targeting vector +T651 SO:0001026 35740 35747 genomic +T652 NCBITaxon:10088 35775 35779 mice +T653 SO:0001644 35825 35841 targeting vector +T654 SO:0001644 35881 35897 targeting vector +T655 SO:0001026 35906 35913 genomic +T656 SO:0000147 35942 35947 exons +T657 PR:000027234 35963 35966 Src +T658 PR:000008224 35972 35976 Grb2 +T659 SO:0000409 35977 35990 binding sites +T660 PR:000027234 36055 36058 Src +T661 PR:000008224 36063 36067 Grb2 +T662 SO:0001644 36096 36112 targeting vector +T663 GO:0009294 36117 36128 transfected +T664 CL:0002322 36150 36158 ES cells +T665 SO:0001587 36283 36303 premature stop codon +T666 PR:000013743 36359 36365 RasGAP +T667 SO:0000409 36366 36378 binding site +T668 CL:0002322 36380 36387 ES cell +T669 SO:0005853 36562 36571 cassettes +T670 NCBITaxon:10088 36597 36601 mice +T671 NCBITaxon:10088 36823 36828 mouse +T672 NCBITaxon:10088 36953 36957 mice +T673 CL:0000669 37042 37050 Pericyte +T674 UBERON:0000922 37065 37072 Embryos +T675 UBERON:0000479 37077 37084 tissues +T676 UBERON:0002049 37185 37196 vasculature +T677 UBERON:0000479 37221 37228 tissues +T678 NCBITaxon:33208 37252 37259 animals +T679 UBERON:0000479 37267 37274 tissues +T680 UBERON:0002048 37292 37296 lung +T681 UBERON:0001348 37298 37318 brown adipose tissue +T682 UBERON:0002369 37328 37341 adrenal gland +T683 UBERON:0002113 37366 37373 Kidneys +T684 UBERON:0000970 37534 37537 eye +T685 CHEBI:26130 37564 37573 pigmented +T686 UBERON:0000483 37574 37584 epithelium +T687 NCBITaxon:10088 37606 37611 mouse +T688 UBERON:0000966 37612 37619 retinas +T689 UBERON:0000966 37665 37672 Retinas +T690 UBERON:0002113 37677 37683 kidney +T691 CL:0000359 37765 37766 v +T692 CL:0000669 37767 37768 p +T693 GO:0042571 37777 37787 Antibodies +T694 PR:000003675 37865 37869 αSMA +T695 PR:000006427 37924 37930 desmin +T696 CL:0000669 38095 38103 Pericyte +T697 UBERON:0000922 38124 38131 embryos +T698 UBERON:0000033 38184 38188 head +T699 UBERON:0000974 38189 38193 neck +T700 UBERON:0000974 38195 38199 neck +T701 UBERON:0002107 38200 38205 liver +T702 UBERON:0002107 38207 38212 liver +T703 UBERON:0002113 38213 38219 kidney +T704 UBERON:0002113 38225 38231 kidney +T705 UBERON:0002415 38232 38236 tail +T706 CHEBI:16842 38295 38307 formaldehyde +T707 CHEBI:64276 38314 38328 glutaraldehyde +T708 CHEBI:75055 38395 38400 X-Gal +T709 CHEBI:16842 38454 38462 formalin +T710 CHEBI:75055 38532 38537 X-Gal +T711 GO:0005634 38547 38553 nuclei +T712 UBERON:0001049 38573 38584 neural tube +T713 UBERON:0000948 38605 38610 heart +T714 UBERON:0002113 38619 38625 kidney +T715 CL:0000669 38728 38737 Pericytes +T716 UBERON:0001049 38770 38781 neural tube +T717 GO:0005634 38822 38828 nuclei +T718 UBERON:0000966 38932 38939 Retinas +T719 CHEBI:26130 38979 38988 pigmented +T720 UBERON:0000483 38989 38999 epithelium +T721 UBERON:0000966 39120 39126 retina +T722 UBERON:0000922 39327 39334 embryos +T723 UBERON:0000922 39336 39343 Embryos +T724 UBERON:0000479 39403 39409 tissue +T725 PR:000027795 39432 39439 trypsin +T726 GO:0019835 39860 39865 lysed +T727 GO:0042571 39972 39982 Antibodies +T728 PR:000029189 40185 40188 Akt +T729 CHEBI:32958 40263 40270 Phospho +T730 PR:000029189 40271 40274 AKT +T731 PR:000013743 40310 40316 RasGAP +T732 PR:000008224 40350 40354 Grb2 +T733 GO:0009293 40367 40379 Transduction +T734 PR:000000104 40432 40436 ERK1 +T735 PR:000027234 40472 40477 c-Src +T736 CHEBI:32958 40522 40529 Phospho +T737 PR:000027234 40530 40533 Src +T738 CHEBI:32958 40612 40619 Phospho +T739 PR:000000104 40620 40624 ERK1 +T740 PR:000013449 40700 40705 SHP-2 +T741 SO:0000667 40821 40827 insert +T742 SO:0000417 40828 40834 domain +T743 GO:0042571 41122 41132 antibodies +T744 http://purl.obolibrary.org/obo/MONDO_0004992 41394 41400 Cancer +T745 UBERON:0000948 41502 41507 Heart +T746 GO:0007567 41563 41568 Birth +T747 http://purl.obolibrary.org/obo/MONDO_0000839 41563 41576 Birth Defects +T748 NCBITaxon:9606 41677 41682 Human +T749 GO:0005576 41724 41737 extracellular +T750 UBERON:0000922 41766 41775 embryonic +T751 CHEBI:52290 41828 41835 mitogen +T752 NCBITaxon:10088 41868 41873 mouse +T753 UBERON:0000922 41874 41883 embryonic +T754 CL:0000057 41884 41894 fibroblast +T755 GO:1990265 41896 41900 PDGF +T756 CL:0000233 41903 41911 platelet +T757 GO:1990265 41903 41933 platelet-derived growth factor +T758 CL:0000233 41943 41951 platelet +T759 GO:1990265 41943 41973 platelet-derived growth factor +T760 CHEBI:28874 41991 42011 phosphatidylinositol +T761 PR:000002997 42080 42083 SFK +T762 PR:000002997 42086 42103 Src family kinase +T763 SO:0000417 42109 42115 domain +T764 PR:000027234 42118 42121 Src +T765 SO:0000857 42122 42130 homology +T766 SO:0000417 42131 42137 domain +T767 PR:000013449 42141 42146 SHP-2 +T768 PR:000013449 42149 42191 SH2-containing phosphotyrosine phosphatase +T769 PR:000003675 42193 42197 αSMA +T770 UBERON:0001135 42200 42213 smooth muscle +T771 PR:000003675 42200 42221 smooth muscle actin α +T772 PR:000001933 42223 42227 STAT +T773 PR:000001933 42230 42278 signal transducer and activator of transcription +T774 CL:0000359 42280 42281 v +T775 CL:0000669 42282 42283 p +T776 UBERON:0004237 42286 42308 vascular smooth muscle +T777 CL:0000359 42286 42313 vascular smooth muscle cell +T778 CL:0000669 42314 42322 pericyte +T779 CL:0000359 42324 42328 VSMC +T780 UBERON:0004237 42331 42353 vascular smooth muscle +T781 CL:0000359 42331 42358 vascular smooth muscle cell +T782 SO:0001023 42397 42404 Allelic +T783 SO:0001023 42444 42451 alleles +T784 NCBITaxon:10088 42469 42474 mouse +T785 SO:0001026 42482 42489 genomic +T786 SO:0000409 42537 42552 binding site(s) +T787 CHEBI:36357 42590 42598 molecule +T788 SO:0001023 42607 42613 allele +T789 PR:000002997 42643 42646 SFK +T790 SO:0000409 42647 42659 binding site +T791 SO:0001023 42760 42766 allele +T792 SO:0000319 42840 42850 stop codon +T793 PR:000013743 42875 42881 RasGAP +T794 SO:0000409 42882 42894 binding site +T795 SO:0001644 42949 42965 Targeting vector +T796 SO:0001023 42991 42997 allele +T797 SO:0000147 43003 43008 exons +T798 SO:0001644 43050 43066 Targeting vector +T799 SO:0000147 43094 43099 exons +T800 SO:0001023 43131 43137 allele +T801 SO:0001023 43154 43160 allele +T802 SO:0001023 43176 43182 allele +T803 PR:P43870 43280 43281 H +T804 PR:P43870 43283 43290 HindIII +T805 CL:0002322 43386 43394 ES cells +T806 SO:0000147 43425 43429 exon +T807 SO:0000147 43524 43529 exons +T808 SO:0000147 43550 43555 exons +T809 SO:0000350 43734 43743 FRT sites +T810 SO:0000346 43762 43772 loxP sites +T811 CL:0002322 43814 43821 ES cell +T812 SO:0001023 43934 43940 allele +T813 SO:0001023 43976 43982 allele +T814 CL:0002322 44018 44026 ES cells +T815 CL:0002322 44090 44097 ES cell +T816 UBERON:0000970 44116 44119 Eye +T817 NCBITaxon:10088 44136 44140 Mice +T818 UBERON:0000970 44146 44150 Eyes +T819 NCBITaxon:10088 44166 44171 mouse +T820 UBERON:0000970 44255 44259 eyes +T821 NCBITaxon:10088 44291 44295 mice +T822 UBERON:0000970 44347 44350 eye +T823 UBERON:0000970 44408 44411 eye +T824 UBERON:0000966 44422 44423 R +T825 UBERON:0000966 44425 44431 retina +T826 CL:0000359 44444 44445 V +T827 CL:0000669 44446 44447 P +T828 UBERON:0000966 44467 44474 Retinas +T829 UBERON:0000966 44488 44495 retinal +T830 UBERON:0000970 44535 44539 eyes +T831 CHEBI:26130 44541 44550 Pigmented +T832 UBERON:0000483 44551 44561 epithelium +T833 UBERON:0000966 44664 44671 retinal +T834 UBERON:0000966 44724 44730 retina +T835 UBERON:0001637 44807 44813 artery +T836 UBERON:0001638 44818 44822 vein +T837 UBERON:0000970 44843 44847 eyes +T838 UBERON:0004237 44860 44882 Vascular Smooth Muscle +T839 CL:0002592 44860 44891;44896 44913 Vascular Smooth Muscle Cells of ... Coronary Arteries +T840 UBERON:0001621 44896 44913 Coronary Arteries +T841 UBERON:0000948 44946 44952 hearts +T842 SO:0001023 44980 44987 alleles +T843 NCBITaxon:10088 44998 45002 mice +T844 UBERON:0000948 45004 45010 Hearts +T845 UBERON:0000948 45085 45090 heart +T846 UBERON:0000948 45172 45178 hearts +T847 UBERON:0000948 45214 45220 Hearts +T848 UBERON:0000479 45297 45303 Tissue +T849 CL:0000359 45320 45321;45324 45328 V ... Cell +T850 CL:0000669 45322 45328 P Cell +T851 GO:0010467 45348 45358 Expression +T852 UBERON:0000479 45360 45366 Tissue +T853 NCBITaxon:10088 45420 45425 mouse +T854 PR:000003675 45465 45469 αSMA +T855 PR:000006427 45481 45487 desmin +T856 GO:0010467 45522 45532 expression +T857 UBERON:0002113 45555 45561 Kidney +T858 UBERON:0000074 45610 45620 glomerulus +T859 UBERON:0001980 45648 45657 arteriole +T860 UBERON:0000966 45665 45671 Retina +T861 GO:0005634 45744 45750 nuclei +T862 CL:0000359 45776 45777;45780 45785 V ... Cells +T863 CL:0000669 45778 45785 P Cells +T864 UBERON:0000915 45793 45808 Thoracic Region +T865 UBERON:0000922 45818 45825 Embryos +T866 NCBITaxon:10088 45896 45901 mouse +T867 GO:0005634 45946 45952 nuclei +T868 CL:0000359 45963 45964;45967 45972 v ... cells +T869 CL:0000669 45965 45972 p cells +T870 UBERON:0002370 45974 45976 Th +T871 UBERON:0002370 45978 45984 thymus +T872 CL:0000669 45997 46006 Pericytes +T873 UBERON:0001016 46024 46038 Nervous System +T874 UBERON:0000922 46058 46065 Embryos +T875 UBERON:0001049 46103 46115 neural tubes +T876 UBERON:0000922 46119 46126 embryos +T877 SO:0001023 46149 46156 allelic +T878 UBERON:0000922 46196 46203 embryos +T879 SO:0001023 46224 46231 allelic +T880 SO:0001023 46282 46288 allele +T881 UBERON:0000948 46339 46344 heart +T882 UBERON:0002113 46349 46355 kidney +T883 CL:0000669 46357 46366 Pericytes +T884 GO:0005634 46385 46392 nuclear +T885 CL:0000359 46454 46455 v +T886 CL:0000669 46456 46457 p +T887 CL:0000669 46513 46522 Pericytes +T888 UBERON:0001016 46526 46540 Nervous System +T889 GO:0005634 46567 46573 nuclei +T890 UBERON:0001049 46598 46609 neural tube +T891 UBERON:0000922 46683 46689 embryo +T892 UBERON:0000922 46740 46746 embryo +T893 GO:0010467 46871 46880 expressed +T894 UBERON:0002240 47024 47035 spinal cord +T895 CL:0000669 47036 47045 perictyes +T896 NCBITaxon:10088 47322 47326 Mice +T897 CHEBI:8984 47515 47518 SDS +T898 GO:0042571 47588 47596 antibody +T899 CHEBI:8984 47749 47752 SDS +T900 PR:000027234 47803 47806 Src +T901 GO:0042571 47815 47823 antibody +T902 CHEBI:8984 47938 47941 SDS +T903 GO:0042571 48024 48034 antibodies +T904 GO:0042571 48073 48083 antibodies +T905 GO:0042571 48344 48354 antibodies +R1 NULL^SLOT Arg1:T774 Arg2:T777 +R2 NULL^SLOT Arg1:T71 Arg2:T73 +R3 NULL^SLOT Arg1:T13 Arg2:T15 +R4 NULL^SLOT Arg1:T777 Arg2:T774 +R5 NULL^SLOT Arg1:T73 Arg2:T71 +R6 NULL^SLOT Arg1:T14 Arg2:T12 +R7 NULL^SLOT Arg1:T12 Arg2:T14 +R8 NULL^SLOT Arg1:T72 Arg2:T74 +R9 NULL^SLOT Arg1:T74 Arg2:T72 +R10 NULL^SLOT Arg1:T775 Arg2:T778 +R11 NULL^SLOT Arg1:T15 Arg2:T13 +R12 NULL^SLOT Arg1:T779 Arg2:T781 +R13 NULL^SLOT Arg1:T778 Arg2:T775 +R14 NULL^SLOT Arg1:T781 Arg2:T779 diff --git a/src/ontogpt/evaluation/craft/database/all/14624252.txt b/src/ontogpt/evaluation/craft/database/all/14624252.txt new file mode 100644 index 000000000..7b53220f3 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/14624252.txt @@ -0,0 +1,241 @@ +Additive Effects of PDGF Receptor β Signaling Pathways in Vascular Smooth Muscle Cell Development + +Abstract + +The platelet-derived growth factor β receptor (PDGFRβ) is known to activate many molecules involved in signal transduction and has been a paradigm for receptor tyrosine kinase signaling for many years. We have sought to determine the role of individual signaling components downstream of this receptor in vivo by analyzing an allelic series of tyrosine–phenylalanine mutations that prevent binding of specific signal transduction components. Here we show that the incidence of vascular smooth muscle cells/pericytes (v/p), a PDGFRβ-dependent cell type, can be correlated to the amount of receptor expressed and the number of activated signal transduction pathways. A decrease in either receptor expression levels or disruption of multiple downstream signaling pathways lead to a significant reduction in v/p. Conversely, loss of RasGAP binding leads to an increase in this same cell population, implicating a potential role for this effector in attenuating the PDGFRβ signal. The combined in vivo and biochemical data suggest that the summation of pathways associated with the PDGFRβ signal transduction determines the expansion of developing v/p cells. + +Introduction + +Although signal transduction by receptor tyrosine kinases (RTKs) has been studied extensively, the roles of individual signaling proteins downstream of these receptors are a matter of debate. Some studies have shown that disruption of particular pathways leads to loss of specific cellular functions (Valius and Kazlauskas 1993). Others have suggested that it is the sum of the signals that results in the unique cellular outcomes directed by each receptor (Fambrough et al. 1999). Yet others have demonstrated that the interpretation of receptor signals is determined by the distinct cellular history (Flores et al. 2000; Halfon et al. 2000; Xu et al. 2000). Because many of these conclusions have been reached in diverse cell types and through the analysis of different RTKs, it is difficult to determine whether results from one receptor system can be used to generalize the functions of RTK signaling. + +Recently, several labs have dissected the roles of RTK modular signaling components by generating point mutations in cytoplasmic domains of the receptors in mice (Partanen et al. 1998; Heuchel et al. 1999; Blume-Jensen et al. 2000; Kissel et al. 2000; Tallquist et al. 2000; Klinghoffer et al. 2001, 2002; Maina et al. 2001). These studies have revealed a unique requirement for individual signaling components in specific cell types (Partanen et al. 1998; Blume-Jensen et al. 2000; Kissel et al. 2000; Maina et al. 2001). In contrast, similar experiments on platelet-derived growth factor receptor α (PDGFRα) signaling mutants have demonstrated that phosphatidylinositol 3′-kinase (PI3K) and Src family kinase (SFK) signal transduction pathways play roles in oligodendrocyte development (Klinghoffer et al. 2002). These experiments suggest that requirements for signal transduction vary not only by the receptor under consideration, but also by the cell lineage that is receiving the signal. + +The platelet-derived growth factor receptor β (PDGFRβ) has not only been studied physiologically, but also has been the focus of intensive biochemical analysis. Upon ligand binding, the PDGFRβ dimerizes and is autophosphorylated on as many as 13 cytoplasmic tyrosine residues. These phosphorylated tyrosines become binding sites for SH2 domain-containing proteins that initiate a number of signal transduction pathways (reviewed by Claesson-Welsh 1994; Heldin et al. 1998). The pathways downstream of the PDGFRβ control multiple cellular functions, including proliferation, migration, matrix deposition, and immediate early gene induction (reviewed by Heldin and Westermark 1999; Betsholtz et al. 2001). At least ten distinct SH2 domain-containing proteins can bind the phosphorylated PDGFRβ and activate downstream signal transduction cascades. These molecules include SFK (Kypta et al. 1990), PI3K (Kazlauskas and Cooper 1990; Kundra et al. 1994; Wennstrom et al. 1994a, 1994b), Shc (Yokote et al. 1994), RasGAP (Kaplan et al. 1990; Kazlauskas et al. 1990), signal transducers and activators of transcription (STATs) (Vignais et al. 1996), Grb2 (Arvidsson et al. 1994), Grb7 (Yokote et al. 1996), SH2-containing phosphotyrosine phosphatase (SHP-2, also known as SH-PTP2) (Kazlauskas et al. 1993; Lechleider et al. 1993), phospholipase Cγ (PLCγ) (Meisenhelder et al. 1989; Morrison et al. 1990), and Nck (Nishimura et al. 1993). While multiple downstream effects have been attributed to activation of these pathways, their relative importance downstream of the PDGFRβ has not been determined in vivo. + +We have concentrated our present analyses on signal transduction by the PDGFRβ. Previous studies using a null allele of the receptor have demonstrated that PDGFRβ signal transduction is required for a subset of vascular smooth muscle cells and pericytes (v/p) (Levéen et al. 1994; Soriano 1994). These cells are the mesenchymal support cells that surround blood vessels (reviewed by Hungerford and Little 1999). Brain pericytes, kidney mesangial cells, retinal mural cells, and limb and skin pericytes have all been recognized as PDGFRβ-dependent cells (Lindahl et al. 1997a, 1998; Hellström et al. 1999; Enge et al. 2002). Studies have indicated that the PDGFRβ is likely to play a key role in the proliferation, migration, or both of a progenitor population (Hellström et al. 1999). These results explain why defective PDGF signal transduction results in a reduction of the v/p cell lineage and ultimately in perinatal lethality due to vessel instability (Hellström et al. 2001). + +To examine the roles of PI3K and PLCγ downstream of the PDGFRβ, we have previously disrupted their binding sites in the receptor's cytoplasmic domain (Heuchel et al. 1999; Tallquist et al. 2000). Surprisingly, no overt phenotypes were detected in homozygous mutants lacking these two pathways, and deficiencies were observed only when the animals were challenged physiologically. To assess the roles of the remaining signal transduction pathways, we have created a PDGFRβ allelic series in mice (Figure 1). We refer to this series as the F series because it contains Y–F mutations at the known phosphorylated tyrosine residues. Using v/p cell number as a readout for PDGFRβ signal transduction, we have determined that the level of receptor expressed as well as the sum of signaling pathways induced by the PDGFRβ determines the number of v/p cells that form. These results provide an example of RTK signal transduction quantitatively controlling cellular development. + +Results + +Generation of the Allelic Series + +Previous studies of the PDGFRβ have revealed an essential role for this receptor in v/p development, but attempts to identify essential biochemical signals thus far have demonstrated that loss of certain signaling pathways only diminishes PDGFRβ-driven responses (Heuchel et al. 1999; Tallquist et al. 2000). To study key signaling pathways, we have generated an allelic series of PDGFRβ mutants. Figure 1 illustrates the mutations that we have generated in the PDGFRβ locus and the signaling pathways that are disrupted by these mutations. Each mutant will be referred to by the number of tyrosines (Y) that have been mutated. For example, the mutation in the RasGAP-binding site is the PDGFRβF1/F1 or F1/F1 mutant. The truncation mutation of the PDGFRβ (βT) was created by the introduction of a frameshift and subsequent premature stop codon downstream of the RasGAP-binding site. Figure 2 illustrates the targeting events that were used to generate the series of mutants. The F1-, F2-, F3-, F5-, and βT-targeted mutations were generated by engineering Y–F, Y–I, or frameshift mutations in the same targeting vector (Figure 2A). The F7 mutation was generated by targeting the F5 heterozygous embryonic stem (ES) cells (Figure 2B; see Materials and Methods). Cells that contained all mutations on the same allele, as determined by Southern blotting, were used to generate the F7 line. All mutant mice were viable and fertile as homozygotes except the truncation allele, βT, which lacks the second half of the kinase domain and the SHP-2- and PLCγ-binding sites. Based on a similar mutation in the PDGFRβ, we assume that this receptor is kinase deficient and incapable of inducing DNA synthesis, but it still should bind ligand and undergo receptor downregulation (Escobedo et al. 1988). Embryos homozygous for the βT allele die perinatally with a phenotype identical to that of the PDGFRβ null embryos. E18 embryos exhibit edema and hemorrhaging in multiple tissues, including the kidney, brain, and skin (data not shown). These results suggest that PDGFRβ kinase activity is required for v/p development and that the receptor cannot function in the absence of kinase activity, unlike another RTK, vascular endothelial cell growth factor receptor 1 (Hiratsuka et al. 1998). + +Identification of V/P Cells + +We examined the blood vessels of F series homozygous mice by histology and detected no gross abnormalities (data not shown). To obtain a more global perspective of v/p cell populations, we introduced the XlacZ4 transgenic marker into our F series mutant mice. The XlacZ4 transgenic mouse expresses nuclear β-galactosidase in certain populations of differentiated, nonproliferating v/p cells in the embryo and the adult (Tidhar et al. 2001). As described below, using this marker in adult animals, we identified vascular defects in the F5 and F7 mice in the tissues of the eyes, hearts, and brains (see Figures 7, 8, and 9; data not shown). This observation suggests that both the F5 and F7 alleles function suboptimally in tissues known to require PDGFRβ signal transduction (Lindahl et al. 1997a; Hellström et al. 1999; Enge et al. 2002). Although both of these mutations cause notable phenotypes in some v/p populations, we have not observed pathologies in all populations of PDGFRβ-dependent v/p cells. V/p cell populations with no overt phenotype in the F5 and F7 mice include the kidney mesangial cells and pericytes in the skin and skeletal muscle (data not shown). We have observed a modest decrease in the number of nuclei present in F5/F5 and F5/− kidney glomeruli, but have not detected glomerulosclerosis with Masson trichrome stain (data not shown). The lack of any pathological phenotype in these tissues suggests either that the reduction in v/p cells is less severe than in the case of the PDGFRβ null mice, that the PDGFRα may be coexpressed in these same tissues, or that these tissues can function adequately even with reduced v/p cell numbers. + +Because some populations of v/p cells appear to be more dependent on PDGFRβ signal transduction than others, we reasoned that the PDGFRα might be coexpressed in the less-affected v/p populations. Although PDGFRα has been reported in a variety of mesenchymal cell lineages (Schatteman et al. 1992; Lindahl et al. 1997b; Takakura et al. 1997; Zhang et al. 1998; Karlsson et al. 2000), we wanted to determine whether any v/p populations express the PDGFRα or whether it may be upregulated in any of the F series mice. We crossed the PDGFRαGFP line of mouse, which expresses a nuclear-localized green fluorescent protein (GFP) under the control of the PDGFRα promoter (Hamilton et al. 2003), with the F5 mutant mice and compared the GFP expression pattern to the pattern of v/p cells in the kidney, eye, and brain (Figure 3; data not shown). We have used three independent markers to designate v/p cells: smooth muscle actin α (αSMA), desmin, and the XlacZ4 transgene. Although PDGFRα-expressing cells are found in the same tissues as v/p cell markers, there is no overlapping expression of GFP with any of the v/p cell markers in the arteries or veins in the vessels of the eye and brain. PDGFRα-expressing cells are also absent from the larger vessels of the kidney, but a population of GFP+ cells is detected within the kidney glomerulus (Figure 3A). These may be either kidney mesangial cells or vascular adventitial fibroblasts. Both are populations of cells that are known to express the PDGFRα (Seifert et al. 1998). These data indicate that PDGFRα is not expressed or upregulated in two of the most affected tissues of the mutant mice, the eye and the brain, but could be functioning as a surrogate coreceptor with the PDGFRβ. + +V/P Development + +To determine whether the reduction in v/p was caused by a gradual loss or a developmental defect, we examined pericyte populations in wild-type and mutant embryos. The XlacZ4 mouse marker can be used to identify specific v/p cell populations as early as E12.5. We chose to observe pericytes at E14.5 because at this timepoint v/p are abundant in wild-type animals in several tissues, including the developing spinal cord and intercostal vasculature. Figure 4 demonstrates whole-mount visualization of the v/p cell populations in E14.5 wild-type embryos and the most severe F series mutant embryo (F7/−). After examining several litters of F series mutant embryos bearing the XlacZ4 marker, it was clear that the entire panel of F series homozygous mutant embryos could be distinguished from wild-type embryos simply by the degree that blood vessels had acquired v/p (data not shown). + +To obtain a quantitative view of these results, we chose to focus on the spinal cord pericyte population. These cells begin to form at E10.5 in a rostral-to-caudal fashion in the embryo and require PDGFRβ signals for development (Levéen et al. 1994). Cross-sections through the developing spinal cord (neural tube) provide a relatively uniform area for quantitation. We can consistently identify a particular maturation stage of the developing vasculature based on its axial level within the embryo, and the pericytes can often be found as isolated cells (Figure 5). Using the entire panel of PDGFRβ mutant mice, we compared pericyte numbers between the different F series mutants (Figures 5 and 6). In all mutants examined, with the exception of the F1 mutation, we observed a decreased incidence of pericytes when compared to the wild-type embryos. The reduction in pericyte numbers ranged from 42% to 77%. This reduction was present at the more mature axial level of the heart as well as at the axial level of the kidney. + +The F7/F7 mutant embryos are the only embryos that exhibited a difference between the number of pericytes at the heart level versus the number at the level of the kidney. All other mutants demonstrated similar numbers at both levels, indicating that pericyte development is disrupted and does not reach homeostasis as the tissue matures. Because the F7 is the most severely affected allele, it is possible that the difference between the heart and kidney levels is due to a developmental delay in v/p formation. Pericyte development may still be proceeding at the level of the kidney in these embryos. At the more mature level of the heart, the F7/F7 pericyte populations have reached a steady-state level and resemble v/p numbers more similar to those observed in the F5/F5 embryos. + +Previously, chimeric analysis had demonstrated that PDGFRβ heterozygous cells do not contribute extensively to the smooth muscle cell compartment, suggesting that heterozygous cells may have reduced v/p developmental potential (Crosby et al. 1998). To find out whether receptor levels had any impact on v/p cells in our system, we crossed animals bearing the PDGFRβ null allele to our mutant series (Figures 5B and 6B). We observed an even further reduction in pericyte levels, resulting in a 70%–92% decrease in pericytes when compared to wild-type embryos. Interestingly, even the PDGFRβ+/− embryos, which exhibit a approximately 50% reduction in PDGFRβ messenger RNA (Soriano 1994), demonstrate a nearly 40% decrease in pericytes. This result suggests that the quantity of receptor impacts the number of pericytes. Another observation from this data is that even the F7/− embryos can induce cell development at levels greater than the null. In fact, the F7/− animals survive, whereas the PDGFRβ nulls do not. This is a rather surprising result given that most of the downstream signal transduction molecules that directly interact with the receptor have been dissociated. + +While most of the F series alleles demonstrate a decrease in v/p cells, the F1 allele results in an apparent increase in spinal cord pericytes. Although the increase is most pronounced when we compare the F1 hemizygote to the PDGFRβ heterozygote (Figure 6), an increase is also observed when comparing F1/F1 embryos and wild-type embryos. In fact, the level of pericytes in the F1/− embryos is very similar to those in the wild-type. These data demonstrate two interesting findings. One is that RasGAP may play a role in PDGFRβ signal attenuation and that loss of this pathway results in increased PDGFRβ signals. The second is that v/p numbers may not be tightly controlled and that PDGFRβ signaling can result in more cells. + +To determine whether the signaling pathways affected other v/p populations in the same manner, we have examined the v/p population in the retina. It has been shown previously that PDGFB and PDGFRβ signaling controls pericyte development in the eye (Benjamin et al. 1998; Klinghoffer et al. 2001; Enge et al. 2002). Adult mice transheterozygous for one null allele and one F5 or F7 allele exhibited severe eye defects. These defects were first observed as an opacity and sometimes as visible hemorrhage in the eye (Figure 7A), as previously described for PDGFRβ and PDGFB signaling mutants (Klinghoffer et al. 2001; Enge et al. 2002). The F5 and F7 hemizygous mutant mice possessed fewer discontinuous blood vessels and overgrowth of retinal cells. This phenotype occurred with 100% penetrance, but with variable severity (Figure 7C), and was detectable sometimes as early as 4 d after birth. The presence of a pathological condition suggests that the F5 and F7 alleles have compromised receptor function when compared to the wild-type, F1, F2, and F3 alleles and demonstrates that retinal pericytes are also dependent on the PDGFRβ signaling pathways that we have disrupted. + +To examine the retinal pericytes in the entire F series, we again used mice bearing the XlacZ4 transgene. At 4 wk of age the retinal vasculature is mature and can be isolated from the lens and pigmented epithelium for visualization. Figure 8 illustrates that homozygotes for the F1, F2, and F3 mutant alleles are indistinguishable from wild-type eyes; however, F5/F5 and F7/F7 eyes exhibit reduced numbers of pericytes. Even without the ability to quantitate these differences, it is clear that the PDGFRβ+/−, F5/F5, F7/F7, F5/−, and F7/− mutant retinas have a reduction in v/p when compared to wild-type eyes, reinforcing the requirement for multiple PDGFRβ signal transduction pathways in v/p development. + +A final tissue where we have examined v/p formation is the heart. F2 and F3 homozygotes and transheterozygotes were indistinguishable from wild-type and heterozygous hearts, respectively. Consistent with our observations in the eye and the nervous system, the F5 and F7 mutant alleles display abnormalities in the vascular coating of their coronary arteries and veins (Figure 9; data not shown). F5/− and F7/− mice often exhibited a variety of heart abnormalities, including enlarged ventricles, increased heart:body mass ratio, dilated atria, and fibrosis (data not shown). In contrast, the F1/+ mice appeared to have more extensive v/p coating on their coronary arteries (Figure 9). In agreement with the data from the nervous system and the eye, the F5 and F7 mutant alleles have a significant reduction in v/p cells. + +Taken together, these results demonstrate several important findings for PDGFRβ signal transduction. First, the number of pericytes formed directly correlates with the number of signaling pathways transducing PDGFRβ activity. Second, a reduction in pericytes is observed even when only the amount of receptor is affected. Finally, although SH2 domain-containing proteins impact v/p numbers, the intrinsic kinase activity of the receptor may play a role in transmitting the PDGFRβ signal because the truncation mutation does not exhibit any rescue of v/p development, while the F7 mutant allele that transmits primarily through kinase activity (owing to loss of the SH2 domain-containing protein-binding sites) still supports v/p development sufficient for viability. + +Downstream Signal Transduction + +Because F2, F3, and F5 mutant receptors have been previously studied biochemically (Valius and Kazlauskas 1993; Heuchel et al. 1999; Tallquist et al. 2000), we have focused our biochemical analysis on the F1 and F7 mutant receptors' signal transduction to verify the effects of these particular mutations on downstream signal transduction cascades. We have used mouse embryo fibroblasts (MEFs) for these analyses. All lines of MEFs that we generated expressed the PDGFRβ at similar levels (Figure 10D) as well as the PDGFRα (data not shown). To avoid stimulation of the PDGFRα by PDGFBB, we downregulated PDGFRα surface expression by pretreatment with PDGFAA 2 h before PDGFBB stimulation. In all cell lines examined, we observed an increase in tyrosine phosphorylation in response to ligand (Figure 10A). The most evident phosphorylated bands are around 200 kd, which are likely to be the PDGFRα and PDGFRβ. Although we have mutated seven of the 13 tyrosines, a significant amount of phosphorylation is observed in all cell lines, albeit at lower levels in the F7/− cell line (Figure 10A and 10B). In the whole-cell lysate phosphotyrosine blot, the phosphorylated protein detected at 200 kd is likely cytoplasmic PDGFRα, as it is reduced in F7 cells after downregulation of the PDGFRα. + +Because we have disrupted only one of the potential Src-binding sites, we examined the level of Src activation downstream of our F7 cell line (Figure 10B). Upon PDGFBB addition, there was an increase in the amount of Src phosphorylated on tyrosine 418 (a site whose phosphorylation is required for full catalytic activity; Johnson et al. 1996). In contrast, in the F7/F7 MEFs, we did not observe any increase in Src activation. These results are in agreement with other reports that demonstrate that a mutation at amino acid 578 of the PDGFRβ is sufficient for reducing the level of Src binding and activation (Mori et al. 1993; Twamley et al. 1993; Vaillancourt et al. 1995; Fanger et al. 1997). + +Two potential downstream targets of PDGFRβ activation are activation of extracellular signal-related kinases 1 and 2 (ERK1/2) and AKT (Franke et al. 1995). As expected, phosphorylation of ERK1/2 and AKT is reduced or absent in the F7 homozygous and hemizygous mutant cell lines, but cells expressing at least one copy of the wild-type receptor are capable of inducing the activation of these downstream molecules (Figure 10C). These data demonstrate that loss of seven tyrosine residues on the PDGFRβ results in a severe loss of downstream signal transduction. In contrast, cells bearing even just one copy of the F1 receptor show increased phosphorylation of ERK1/2. These data are in concordance with the in vivo data, which show that lack of the RasGAP-binding site on the receptor results in an increase in the downstream signaling events and a subsequent increase in v/p. Therefore, the F1 mutant receptor has increased activity while the F7 receptors have decreased activation of these same pathways, despite having apparently normal levels of kinase activity. + +Discussion + +RTK signal transduction plays an important role in directing many cellular activities. We have used an in vivo system to analyze how cellular development relates to the signaling pathways downstream of the PDGFRβ. Examination of the v/p population demonstrates a quantitative relationship between the extent that signals are being transduced and the number of v/p that form. Several other studies have demonstrated that combinations of signal transduction pathways may dictate cellular outcomes. Examples of these are T cell development in the immune system and gradients of morphogens in developmental systems (Heemskerk and DiNardo 1994; Nellen et al. 1996; Zecca et al. 1996; Ferrell and Machleder 1998; Gong et al. 2001). + +In our system, the signal can be affected in two ways. The first is by the amount of receptor expressed at the cell surface. V/p numbers are significantly lower in PDGFRβ+/− embryos when compared to wild-type controls. In this situation there is a decrease in overall signal, but no specific, directly associated pathway is disrupted. This demonstrates a quantitative role for receptor activity. The second influence on PDGFRβ signal transmission is by the number of associated SH2 domain-containing proteins. Loss of even a single pathway results in reduction of v/p, and as the number of disrupted pathways increases, there is a concomitant decrease in v/p. There is no significant difference between the F2 and the F3 mutant alleles, but there is a noticeable difference when the number of mutations is further increased. These signaling differences as illustrated by v/p number and the presence of vascular pathologies can be categorized in the mutant alleles by the following hierarchy: F1 > wild-type > F2 = F3 > F5 > F7 > null. In addition, hemizygotes show an even further reduction in v/p when compared to the F series homozygotes. This suggests that specific effector pathways may play more of a role in fine-tuning PDGFRβ signals. + +In total, our results demonstrate that PDGFRβ signal transduction is regulated not only by direct binding of signal transduction molecules, but also by receptor expression levels, possibly reflecting inherent kinase activity. In addition, no particular signaling pathway that we have analyzed is absolutely required for transmission of PDGFRβ signals, because even the F7 allele has a phenotype less severe than the null. In support of the observation that receptor levels and kinase activity may have a direct role in signal transduction, we have observed that a chimeric PDGFR that has the extracellular domain of the PDGFRβ but the intracellular domain of the PDGFRα exhibits a more severe phenotype than the F5 allele (Klinghoffer et al. 2001). This chimeric receptor can signal through all of the same downstream components as the PDGFRβ except for RasGAP, suggesting that the more severe vascular defects in these chimeric receptor mice may be due to reduced kinase activity and/or expression levels of the chimeric receptor. + +The PDGFRβ's signaling pathways appears to dictate the absolute numbers of v/p that form, but how the individual pathways contribute to this phenotype remains to be tested. In fact, very little difference in numbers of v/p is observed between the F2 and the F3 mutants, suggesting that PLCγ signals may be somehow redundant with or dependent on the PI3K pathway. In contrast, loss of additional pathways leads to an incremental loss of v/p. The difference between the F3 and the F5 mutations is the ability to bind SHP-2 and RasGAP, and it has been proposed that both of these molecules play roles in downregulating the PDGFRβ signal (Klinghoffer and Kazlauskas 1995; Ekman et al. 1999). Our results demonstrate that loss of these signaling pathways is detrimental to PDGFRβ signal transduction and that both may have positive and negative influences on receptor activity. + +There are several potential ways that loss of these signaling pathways leads to v/p reduction. One mechanism would be that each pathway contributes to a specific cellular outcome. For example, SFK's predominant role could be to promote proliferation (Roche et al. 1995; Hansen et al. 1996), whereas PI3K activity could be more important for migration (Kundra et al. 1994; Wennstrom et al. 1994b). Therefore, the combined loss of these pathways results in a net reduction in v/p, albeit for entirely different cellular reasons. A second scenario would be that all pathways lead to a single or few specific cellular conclusions. Thus, loss of any one pathway only reduces the outcome but does not ablate it. Evidence from immediate-early gene expression analysis suggests that this mechanism may occur (Fambrough et al. 1999), although this possibility does not require that all pathways contribute equally. Last, some pathways may play a primary role downstream of the receptor, while others may be more secondary. + +Our data suggest that PI3K may be a principal pathway, while the other pathways may be less significant. The F2/F2 mutant mice have a significant reduction in v/p numbers when compared to the wild-type and the heterozygous mice, and additional mutations have less of an effect than PI3K on v/p numbers. A similar situation has been observed with the PDGFRα (Klinghoffer et al. 2002): the phenotype of mouse embryos with loss of the PDGFRα–PI3K pathway was just as severe as that of embryos expressing a PDGFRα F7 allele (which is similar to the F7 allele of the PDGFRβ). The pathological difference observed between the F5 and F7 alleles versus all of the other mutations suggests that SHP-2, SFK, Grb2, and RasGAP also impact pericyte development. Assessing the importance of these pathways would require generating additional alleles with different combinations of mutant sites. The only signaling differences between the F3 mutation and the F5 mutation are the SHP-2 and RasGAP pathways. This suggests that one or both of these pathways promote PDGFRβ signaling, in contrast to the F1 mutation, which demonstrates that absence of RasGAP leads to a potential increase of PDGFRβ signaling. This apparent contradiction may indicate that RasGAP plays both a positive and a negative role in signal transduction or that SHP-2 may have mainly a positive role in modulating this response. + +Although we find that overall loss of downstream pathways attenuates receptor actions as demonstrated by v/p formation, it is surprising that the F7/F7 mice do not phenocopy the null animals. The F7 allele possesses disruptions at seven of the 13 known phosphorylated tyrosine residues. These mutations should disrupt a majority of the signal relay molecules downstream of the receptor. The remaining tyrosines are capable of binding SFKs, STATs, and Grbs. Based on several previous reports, disruption of Y578 affects the majority of SFK binding (Mori et al. 1993; Twamley et al. 1993; Vaillancourt et al. 1995; Fanger et al. 1997), and we have shown that SFKs do not become activated after stimulation of the F7 receptor. As for the signaling roles of STAT and Grb2 downstream of the receptor, little direct function has been demonstrated for these remaining effector molecules in PDGF-induced cellular responses (Heldin et al. 1998). Therefore, F7 signal transmission must use some other means than direct binding by SH2 domain-containing proteins. The receptor should still have full kinase activity, unlike the lethal βT mutation, which is lacking half of the kinase domain and the SHP-2- and PLCγ-binding sites. Possibly, the receptor is phosphorylating molecules that are only transiently associated. Another possibility is that other receptors may function as surrogates. The most likely surrogate is the PDGFRα, but we have demonstrated that in several of the v/p cell populations the PDGFRα is not expressed. Other candidate molecules for such a mechanism are integrins, ephrins, and the low-density lipoprotein receptor-related protein LRP (Miyamoto et al. 1996; Schneller et al. 1997; Woodard et al. 1998; Boucher et al. 2002; Loukinova et al. 2002). Although these proteins are known to cross-talk with the PDGFRβ, it is unclear whether they have the capability to substitute for the PDGFRβ's own signaling components. + +The F1 mutant allele is an interesting corollary to the F mutant series. While all of the other mutations appear to have a detrimental effect on PDGFRβ signal transduction, the F1 mutation results in an apparent increase in PDGFRβ activity as determined by v/p incidence. These data are in agreement with previous observations that RasGAP function decreases the Ras/MAP kinase pathway activity and migration (Kundra et al. 1994; Ekman et al. 1999). In addition, an add-back mutation of the RasGAP-binding site induced a different gene profile from the PDGFRβ immediate-early gene profile (Fambrough et al. 1999). This suggests that RasGAP may have different signaling capabilities from the other PDGFRβ signal transduction components. + +The mutant mice not only uncover the role of RTK signal transduction in vivo, but they also reveal some interesting information regarding v/p cell development. For example, although v/p cell development is impaired when PDGFRβ signal transduction is disrupted, a basal level of cells forms, in agreement with previous observations that propagation, not initiation, of v/p cell development is directed by the PDGFRβ (Lindahl et al. 1997a; Hellström et al. 1999). Even in the null embryos, v/p cells can be found. It has been proposed that PDGFRβ signals are required for the expansion of v/p cells (Lindahl et al. 1998). While this may be the case, it is curious that in the F5 and F7 animals, the pericyte numbers never reach wild-type levels, resulting in vascular pathologies. + +There are two explanations for the observation that v/p cells never reach wild-type levels. The first is that there is constant turnover in the v/p population and that the rate of replacement in the mutant mice is below the rate of loss, resulting in a net reduction in the v/p population. Evidence against this mechanism is the failure to observe any significant proliferation in the adult wild-type animals under normal conditions or significant apoptosis in the mutant panel of mice (data not shown). The second possibility is that there is a specific window during development when v/p cells can expand. After a specified time, v/p cell number expansion could be limited, perhaps related to the ability of endothelial cells to secrete the PDGF ligand (Benjamin et al. 1998). Support for this model is the inability of nascent endothelial tubes to recruit v/p cells in tumors (Abramsson et al. 2002). The inability to develop sufficient numbers of v/p cells also appears to be recapitulated in the eye vasculature, suggesting that the maturation of the vessel is more dependent on the local environment than on the chronological age of the embryo. + +Our findings demonstrate that the combination of signaling pathways downstream of PDGFRβ determines the total number of v/p cells. These can be modulated not only by the amount of receptor expressed at the cell surface, but also by the number of specific downstream signaling pathways activated by the receptor. Whether these results are unique to PDGFRβ signal transduction in v/p cells or whether they can be extrapolated to other RTK remains to be demonstrated. + +Materials and Methods + +Mice + +Point mutations that disrupt the designated signal transduction pathways were generated by changing the tyrosine residue to phenylalanine. The exception was Y1020, which was mutated to encode an isoleucine, thus generating a unique restriction site that facilitated identification of homologous recombinants. Mouse mutants F2 and F3 have been previously described (Heuchel et al. 1999; Tallquist et al. 2000). The targeting vector for the F1, F5, and βT mutations utilized the same arms of homology as the F3 vector. The exons containing the point mutations were introduced in the arms of homology of the targeting vector by site-directed mutagenesis and verified by sequence data of PCR-amplified genomic DNA from homozygous mutant mice. The F7 mutation was generated by creating a targeting vector that incorporated the 5′ arm of the F5 targeting vector with 5′ genomic sequences that included the exons containing the Src- and Grb2-binding sites. Tyrosines 578 and 715 were mutated to phenylalanine to disrupt Src and Grb2 binding, respectively. This targeting vector was transfected into F5 heterozygous ES cells and screened for homologous recombination. The truncation mutation possesses a frameshift at amino acid 780, resulting in a premature stop codon after amino acid 801, 11 amino acids downstream of the RasGAP-binding site. ES cell colonies were screened initially by PCR, and positive clones were further verified by Southern blot analysis for the correct recombination at the 5′ and 3′ arms. The PGK–neo cassettes were removed by crossing mice to Meox2Cre (Tallquist and Soriano 2000) and ROSA26FlpeR (Farley et al. 2000) deleters. + +The majority of analyses have been carried out on a mixed 129S4 × C57BL/6 background, except where indicated. The XlacZ4 transgenic mouse (Tidhar et al. 2001) was kindly provided by Moshe Shani and crossed into the F series. We also crossed the F5 and wild-type mice to the PDGFRαGFP line (Hamilton et al. 2003). + +Histology, Immunohistochemistry, and Pericyte Quantitation + +Embryos and tissues were processed and embedded for sectioning according to standard protocol. We have not examined the vasculature of all PDGFRβ-dependent tissues in the F series mutant animals. Those tissues not examined are lung, brown adipose tissue, and the adrenal gland. + +Immunohistochemistry + +Kidneys were removed and fixed for 20 min in 4% paraformaldehyde, 200 μm sections were then obtained by vibratome sectioning, and immunofluorescence was performed. For eye immunohistochemistry, the pigmented epithelium was removed from the mouse retinas and fixed for 10 min in 4% paraformaldehyde. Retinas and kidney slices were then blocked and subjected to immunohistochemistry for the indicated v/p marker. Antibodies were β-galactosidase (55976; Cappel, Costa Mesa, California, United States), αSMA (1A4; Sigma, St. Louis, Missouri, United States), and desmin (D33; Dako Cytomation, Glostrup, Denmark). Photographs were obtained on a Zeiss Axiophot microscope (Carl Zeiss MicroImaging, Thornwood, New York, United States). + +Pericyte quantitation + +E14.5 embryos were divided into quarters at the following levels: head–neck, neck–liver, liver–kidney, and kidney–tail. Quarters were rinsed with PBS and fixed for 20 min in 2% formaldehyde, 0.2% glutaraldehyde. They were then washed three times in PBS, stained overnight with X-Gal, transferred to PBS, photographed, post-fixed in 10% formalin, and then processed and embedded. Sections (7 μm) were generated and X-Gal-positive nuclei quantitated in the neural tube at the level of the heart and the kidney. Seven to ten samples were counted for each level; the mean of these data is represented in Figure 6. Pericytes surrounding the exterior of the neural tube were excluded from the sample. Positive nuclei were counted at 20× magnification and photographed at 10× magnification on Zeiss Axiophot microscopes. Retinas were prepared in a similar manner. The pigmented epithelium was removed prior to the initial fixation step, and the lens was not removed until after the final fixation to maintain retina shape. Images were obtained on a Nikon SMZ1000 with a Coolpix 900 camera (Nikon Corporation, Tokyo, Japan). + +Immunoprecipitation and Western Blotting + +MEFs were generated from E9-d-old or E14.5-d-old embryos. Embryos were isolated, decapitated, and eviscerated. The remaining tissue was then treated with trypsin and plated. Cells were frozen down at passages 2 and 3. Most experiments were completed on cells at passages 3–6, except for the wild-type line, which was spontaneously immortalized. + +Cells were plated at 1 × 105 to 3 × 105 cells per well and starved for 48 h. Receptor downregulation was achieved by treating starved cells for 2 h with 100 ng/ml PDGFAA (R&D). Cells were then stimulated with PDGFBB (R&D) for 5 min and lysed. + +Immunoprecipitation and Western blotting were executed as previously described (Tallquist et al. 2000). Antibodies were obtained from the following sources: PDGFRβ (06-498; Upstate Biotechnology, Lake Placid, New York, United States); PDGFRα (sc-338; Santa Cruz Biotechnology, Santa Cruz, California, United States); Akt (9272; Cell Signaling Technology, Beverly, Massachusetts, United States); Phospho-AKT (9271; Cell Signaling Technology); RasGAP (05-178; Upstate Biotechnology); Grb2 (610111; BD Transduction Laboratories, San Jose, California, United States); ERK1/2 (06-182; Upstate Biotechnology); c-Src (SRC2 and sc-18; Santa Cruz Biotechnology); Phospho-Src Y418 (44-660; Biosource International, Camarillo, California, United States); Phospho-ERK1/2 (9101; Cell Signaling Technology); PLCγ (05-163; Upstate Biotechnology); SHP-2 (sc-280; Santa Cruz Biotechnology); and phosphotyrosine (4G10) (05-321; Upstate Biotechnology). PDGFRβ 97A (kinase insert domain) was a kind gift from Andrius Kazlauskas. + +Supporting Information + +Accession Numbers + +The LocusLink ID numbers discussed in this paper are PDGFRα (Locus ID 18595) and PDGFRβ (Locus ID 18596). + +Acknowledgements + +We thank Moshe Shani for the XlacZ4 transgenic line; Andrius Kazlauskas for antibodies; Elizabeth Behler, Philip Corrin, Triston Dougall, Jason Frazier, and Tara Swamy for excellent technical support; and our laboratory colleagues for discussions and critical comments on the manuscript. MDT was supported in part by a fellowship from the American Cancer Society (PF-98–149-01). This work was supported in part by research grant 0330351N from the American Heart Association and grant 5-FY03–14 from the March of Dime Birth Defects Foundation to MDT and by grants HD24875 and HD25326 from the National Institute of Child Health and Human Development to PS. + +Abbreviations + +ERK - extracellular signal-related kinase + +ES - embryonic stem + +GFP - green fluorescent protein + +MAP kinase - mitogen-activated protein kinase + +MEF - mouse embryonic fibroblast + +PDGF - platelet-derived growth factor + +PDGFR - platelet-derived growth factor receptor + +PI3K - phosphatidylinositol 3′-kinase + +PLCγ - phospholipase Cγ + +RTK - receptor tyrosine kinase + +SFK - Src family kinase + +SH2 domain - Src homology domain 2 + +SHP-2 - SH2-containing phosphotyrosine phosphatase + +αSMA - smooth muscle actin α + +STAT - signal transducer and activator of transcription + +v/p - vascular smooth muscle cell/pericyte + +VSMC - vascular smooth muscle cell + +Figures and Tables + +Figure 1 + +PDGFRβ Allelic Series + +This figure depicts the mutant alleles generated in the mouse PDGFRβ genomic locus. X represents a mutation in the tyrosine-binding site(s) for a particular signal transduction molecule. The F7 allele contains a disruption in one SFK-binding site because loss of both sites results in diminished kinase activity (Mori et al. 1993). The truncation allele (βT) was created by deletion and subsequent frameshift that results in a stop codon 32 amino acids past the RasGAP-binding site. + +Figure 2 + +Targeting Strategy and Southern Blot + +(A) Targeting vector used to create F5 mutant allele. Two exons contain all five mutated tyrosines. + +(B) Targeting vector containing mutations in 5′ exons used to generate the F7 mutant allele. + +(C) Wild-type allele. + +(D) Targeted allele with PGK–neo removal. Restriction enzyme abbreviations: Sp, SpeI; A, Asp718; S, SacI; RV, EcoRV; H, HindIII; X, XhoI; and RI, EcoRI. Green boxes indicate probes used in Southern blotting for F7-targeted ES cells. The blue arrow indicates the exon where point mutation causes frameshift in truncation mutation. Black boxes indicate wild-type exons. Red boxes indicate exons containing targeted mutations. Restriction enzymes in red indicate sites introduced by mutagenesis to verify proper homologous recombination by Southern blotting. Circles denote FRT sites. Triangles denote loxP sites. + +(E) Southern blot results from various ES cell lines. SpeI digest using P1 probe. Blot with P2 probe gave expected results (data not shown). Lanes 1 and 3, F5 allele targeted. Lanes 2 and 4, wild-type allele targeted. Lanes 5 and 7, F5 mutant ES cells after and before Cre activity, respectively. Lane 6, wild-type ES cell clone. + +Figure 7 + +Eye Defects in F5/− Mice + +(A) Eyes from a P4 F5/− mouse demonstrating severe hemorrhaging. (B and C) H/E-stained sagittal sections through eyes of wild-type and F5/− 3-mo-old mice, respectively. The absence of the lens of the F5/− eye is a histological defect and not a phenotype of the F5/− eye. L, lens; R, retina. + +Figure 8 + +V/P Populations in P28 Retinas + +Whole-mount retinal preparations from wild-type and mutant eyes. Pigmented epithelium was removed for visualization of β-galactosidase. Note F7/F7 and F7/− had extensive thickening of the retinal layers that resulted in a contraction of the entire retina and apparent reduction in size. The far right panel shows a close-up of the artery and vein of three homozygous eyes. + +Figure 9 + +Vascular Smooth Muscle Cells of the Coronary Arteries + +(Top) Whole-mount views of P21 hearts from littermates of the F5 alleles of mutant mice. Hearts were sliced coronally, and the ventral surface was photographed. The F5/− heart was sliced disproportionately and therefore appears to be smaller. + +(Bottom) P28 hearts from wild-type and F1 littermates. Hearts were sliced sagittally. Both the left and right views are shown. + +Figure 3 + +Tissue Localization of V/P Cell Markers and PDGFRα Expression + +Tissue preparations from P21 PDGFRαGFP/+;PDGFRβF5/F5 mutant mouse. Immunofluorescence was used to detect αSMA (A and B), desmin (C), β-galactosidase (D), and GFP expression for PDGFRα (A–D). (A) Kidney (200 μm vibratome section). The arrow indicates glomerulus. The asterisk indicates an arteriole. (B–D) Retina (whole-mount preparation). Arrowheads point to β-galactosidase-positive nuclei. + +Figure 4 + +Reduction in V/P Cells in the Thoracic Region of E14.5 Embryos + +Ventral view of E14.5 wild-type and F7/− littermates with the XlacZ4 mouse marker background. β-Galactosidase-positive nuclei represent v/p cells. Th, thymus. + +Figure 5 + +Pericytes within the E14.5 Nervous System of F Series Mutant Embryos + +(A) Representative sections through neural tubes of embryos from the homozygous F allelic series. (B) Representative sections of embryos from the hemizygous allelic series (F series mutant with one copy of the null allele). Sections are from the rostral level between the heart and kidney. Pericytes are visualized by nuclear-localized β-galactosidase staining in cells committed to the v/p lineage. Sections are 7 μm. + +Figure 6 + +Quantitation of Pericytes in Nervous System + +β-Galactosidase-positive nuclei were counted within the neural tube. Each datapoint represents a mean of seven to ten sections from a single embryo. Data were gathered at two rostral levels in each embryo. The genotypes are ordered by the predicted strength of the signal, depending on the number of copies of the receptor being expressed and the signal transduction pathways remaining downstream of the receptor. A two-tailed Student's t-test was performed comparing the number of spinal cord perictyes in mutants to those in wild-type, and all homozygous mutants and hemizygous mutant values were statistically different (p < 0.005) from wild-type values. The F1/− was also statistically different from the PDGFRβ+/− (p < 0.001). + +Figure 10 + +Biochemistry of MEFs from F7 and F1 Mice + +(A) Whole-cell lysates were generated from MEFs that were unstimulated or stimulated with PDGFAA and/or PDGFBB alone, 100 ng/ml and 30 ng/ml, respectively. Lysates were then subjected to SDS–PAGE and Western blotting accomplished with the anti-phosphotyrosine antibody (4G10). + +(B) Immunoprecipitation of tyrosine-phosphorylated proteins from wild-type and the F7 series of mutant MEFs. The precipitates were then run on SDS–PAGE, and a Western blot was performed using anti-Src [pY418] antibody and anti-PDGFRβ 97A. + +(C) Whole-cell lysates from unstimulated or stimulated MEFs. Lysates were then subjected to SDS–PAGE and Western blotting accomplished with the indicated phosphorylated-specific antibodies. Blots were stripped and blotted with antibodies to the corresponding unphosphorylated proteins to demonstrate protein loading. Data are representative of multiple experiments from at least two independently derived cell lines. + +(D) Western blots of whole-cell lysates from 5 ×104 cells per lane blotted with antibodies to PDGFRβ (06-498) and ERK as a loading control. + +Footnotes + +Conflicts of Interest. The authors have declared that no conflicts of interest exist. + +Author Contributions. MDT and PS conceived and designed the experiments. MDT and WJF performed the experiments. MDT analyzed the data. MDT wrote the paper. + +Academic Editor: Roel Nusse, Stanford University School of Medicine diff --git a/src/ontogpt/evaluation/craft/database/all/14675480.ann b/src/ontogpt/evaluation/craft/database/all/14675480.ann new file mode 100644 index 000000000..b07348622 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/14675480.ann @@ -0,0 +1,1287 @@ +T1 PR:000010159 12 16 ERK5 +T2 UBERON:0001987 44 53 placental +T3 GO:0001890 44 53;68 79 placental ... development +T4 UBERON:0000922 58 67 embryonic +T5 GO:0009790 58 79 embryonic development +T6 PR:000010159 102 106 ERK5 +T7 CHEBI:52290 126 133 mitogen +T8 CHEBI:52290 187 196 mitogenic +T9 PR:000010159 321 325 ERK5 +T10 SO:0000704 339 343 gene +T11 SO:0000704 373 377 gene +T12 NCBITaxon:10088 381 385 mice +T13 PR:000010159 407 411 ERK5 +T14 NCBITaxon:10088 421 425 mice +T15 GO:0016265 426 429 die +T16 GO:0097617 462 475 hybridisation +T17 PR:000010159 480 484 ERK5 +T18 PR:000010127 513 517 MKK5 +T19 GO:0010467 533 543 expression +T20 UBERON:0008816 551 555;566 568;573 579 head ... of ... embryo +T21 UBERON:0002100 560 565 trunk +T22 PR:000010159 682 686 ERK5 +T23 UBERON:0000922 690 697 embryos +T24 GO:0006915 724 733 apoptosis +T25 UBERON:0005253 741 767 cephalic mesenchyme tissue +T26 UBERON:0001046 790 798 hind gut +T27 UBERON:0000948 845 852 cardiac +T28 GO:0007507 845 864 cardiac development +T29 UBERON:0001987 869 878 placental +T30 PR:000010159 901 905 Erk5 +T31 UBERON:0019248 923 938 early embryonic +T32 GO:0009790 929 950 embryonic development +T33 UBERON:0007798 998 1013 vascular system +T34 CHEBI:52290 1046 1053 Mitogen +T35 GO:0000165 1046 1078;1086 1094 Mitogen activated protein kinase ... cascades +T36 GO:0000165 1080 1084;1086 1094 MAPK ... cascades +T37 GO:0009987 1124 1142 cellular processes +T38 GO:0008283 1153 1171 cell proliferation +T39 GO:0030154 1153 1157;1173 1188 cell ... differentiation +T40 GO:0006915 1153 1157;1203 1212 cell ... apoptosis +T41 UBERON:0000467 1282 1289 systems +T42 UBERON:0002405 1319 1325;1339 1346 immune ... systems +T43 CL:0000540 1330 1338 neuronal +T44 SO:0001060 1360 1368 isoforms +T45 NCBITaxon:40674 1387 1396 mammalian +T46 PR:000000104 1472 1476 ERK1 +T47 PR:000000103 1481 1485 ERK2 +T48 PR:000002206 1514 1519 SAPK1 +T49 PR:000003106 1522 1526 p38s +T50 PR:000010152 1548 1553 SAPK2 +T51 PR:000010153 1555 1560 SAPK3 +T52 PR:000010154 1565 1570 SAPK4 +T53 PR:000010158 1599 1603 ERK3 +T54 PR:000010159 1605 1609 ERK5 +T55 PR:000010155 1614 1618 ERK8 +T56 PR:000010158 1642 1646 ERK3 +T57 GO:0005576 1862 1875 extracellular +T58 PR:000010159 1886 1890 ERK5 +T59 CHEBI:52290 1981 1989 mitogens +T60 PR:000006928 1998 2001 EGF +T61 MOP:0000568 2036 2045 oxidative +T62 PR:000010136 2137 2142 MEKK3 +T63 PR:000010135 2146 2151 MEKK2 +T64 PR:000010127 2161 2165 MKK5 +T65 PR:000010159 2191 2195 ERK5 +T66 PR:000010159 2219 2223 ERK5 +T67 PR:000010159 2283 2287 ERK5 +T68 PR:000010127 2291 2295 MKK5 +T69 CHEBI:35222 2380 2390 inhibitors +T70 CHEBI:35222 2409 2419 inhibitors +T71 GO:0000165 2437 2449 MAPK cascade +T72 PR:000010159 2552 2556 ERK5 +T73 PR:000000104 2648 2652 ERK1 +T74 PR:000000103 2657 2661 ERK2 +T75 PR:000010159 2691 2695 ERK5 +T76 PR:000000104 2700 2704 ERK1 +T77 PR:000000103 2705 2709 ERK2 +T78 PR:000010159 2789 2793 ERK5 +T79 GO:0010467 2825 2835 expression +T80 PR:000010127 2863 2867 MKK5 +T81 NCBITaxon:10088 2871 2875 mice +T82 UBERON:0000948 2887 2894 cardiac +T83 GO:0016265 2911 2916 death +T84 NCBITaxon:10088 2924 2928 mice +T85 PR:000010159 2984 2988 ERK5 +T86 UBERON:0000948 2996 3001 heart +T87 UBERON:0000948 3023 3030 cardiac +T88 GO:0007507 3023 3042 cardiac development +T89 PR:000010159 3044 3048 ERK5 +T90 GO:0048745 3081 3109 development of smooth muscle +T91 UBERON:0001135 3096 3109 smooth muscle +T92 PR:000010159 3114 3118 ERK5 +T93 SO:0000077 3119 3128 antisense +T94 PR:000010159 3171 3175 ERK5 +T95 GO:0030154 3224 3242;3257 3262 differentiation of ... cells +T96 UBERON:0001135 3243 3256 smooth muscle +T97 CL:0000192 3243 3262 smooth muscle cells +T98 PR:000010159 3339 3343 ERK5 +T99 PR:000008373 3400 3411 connexin 43 +T100 PR:000010311 3446 3451 MEF2C +T101 NCBITaxon:10088 3461 3466 Mouse +T102 PR:000010311 3480 3485 MEF2C +T103 UBERON:0000922 3490 3499 embryonic +T104 PR:000010311 3512 3517 MEF2C +T105 UBERON:0000922 3521 3528 embryos +T106 GO:0016265 3529 3532 die +T107 UBERON:0000948 3568 3573 heart +T108 GO:0001947 3592 3599 looping +T109 PR:000010136 3625 3630 MEKK3 +T110 UBERON:0000922 3647 3656 embryonic +T111 PR:000010136 3675 3680 MEKK3 +T112 UBERON:0000922 3684 3691 embryos +T113 UBERON:0002349 3711 3721 myocardium +T114 GO:0048738 3711 3731 myocardium formation +T115 GO:0001525 3733 3745 angiogenesis +T116 UBERON:0001987 3750 3759 placental +T117 GO:0001890 3750 3769 placental formation +T118 PR:000010159 3823 3827 ERK5 +T119 PR:000010136 3839 3844 MEKK3 +T120 PR:000010311 3859 3864 MEF2C +T121 UBERON:0000948 3872 3879 cardiac +T122 GO:0007507 3872 3891 cardiac development +T123 PR:000010136 3917 3922 MEKK3 +T124 SO:0001060 3947 3955 isoforms +T125 PR:000003107 3970 3974 p38α +T126 PR:000003107 3996 4002 SAPK2A +T127 PR:000003107 4025 4029 p38α +T128 PR:000003107 4071 4075 p38α +T129 UBERON:0000922 4079 4086 embryos +T130 UBERON:0000948 4131 4138 cardiac +T131 GO:0007507 4131 4150 cardiac development +T132 GO:0001525 4152 4164 angiogenesis +T133 UBERON:0001987 4169 4178 placental +T134 GO:0001890 4169 4188 placental formation +T135 PR:000010159 4249 4253 ERK5 +T136 GO:0010467 4269 4279 expression +T137 SO:0000704 4284 4288 gene +T138 NCBITaxon:10088 4310 4314 mice +T139 PR:000010159 4316 4320 ERK5 +T140 GO:0009790 4360 4373 embryogenesis +T141 UBERON:0000922 4439 4446 embryos +T142 PR:000010159 4534 4538 ERK5 +T143 PR:000010159 4585 4589 ERK5 +T144 PR:000010159 4658 4662 ERK5 +T145 NCBITaxon:10088 4672 4676 mice +T146 NCBITaxon:10088 4696 4701 mouse +T147 PR:000010159 4702 4706 ERK5 +T148 SO:0000704 4707 4711 gene +T149 SO:0000147 4742 4747 exons +T150 SO:0001026 4767 4774 genomic +T151 SO:0000147 4795 4800 exons +T152 PR:000010159 4832 4836 ERK5 +T153 SO:0000204 4848 4870 5' untranslated region +T154 GO:0006412 4853 4863 translated +T155 SO:0000147 4886 4891 exons +T156 SO:0000205 4909 4931 3' untranslated region +T157 GO:0006412 4914 4924 translated +T158 SO:0000852 4925 4939 region in exon +T159 SO:0001644 4968 4984 targeting vector +T160 SO:0000147 5008 5013 exons +T161 PR:000010159 5025 5029 ERK5 +T162 CL:0002322 5033 5041 ES cells +T163 SO:0000440 5081 5087 vector +T164 CL:0002322 5127 5135 ES cells +T165 PR:000010159 5218 5222 ERK5 +T166 CL:0002322 5223 5225 ES +T167 PR:000010159 5242 5246 ERK5 +T168 NCBITaxon:10088 5250 5254 mice +T169 PR:000010159 5329 5333 ERK5 +T170 NCBITaxon:10088 5337 5341 mice +T171 PR:000010159 5385 5389 ERK5 +T172 NCBITaxon:10088 5393 5397 mice +T173 PR:000010159 5410 5414 ERK5 +T174 NCBITaxon:10088 5418 5422 mice +T175 PR:000010159 5458 5462 ERK5 +T176 GO:0009790 5489 5502 embryogenesis +T177 UBERON:0000922 5541 5548 embryos +T178 GO:0007618 5613 5620 matings +T179 NCBITaxon:10088 5676 5680 mice +T180 UBERON:0000922 5746 5753 embryos +T181 GO:0060047 5787 5794 beating +T182 UBERON:0000948 5795 5800 heart +T183 PR:000010159 5839 5843 ERK5 +T184 UBERON:0000922 5853 5860 embryos +T185 GO:0016265 5871 5875 died +T186 PR:000010159 5960 5964 ERK5 +T187 CL:0002322 6042 6049 ES cell +T188 PR:000010159 6083 6087 ERK5 +T189 SO:0000704 6088 6092 gene +T190 CHEBI:7507 6168 6176 neomycin +T191 SO:0005853 6188 6196 cassette +T192 GO:0043631 6210 6225 polyadenylation +T193 SO:0000610 6210 6234 polyadenylation sequence +T194 PR:000010159 6244 6248 ERK5 +T195 SO:0000704 6249 6253 gene +T196 SO:0000147 6261 6266 exons +T197 SO:0000704 6297 6301 gene +T198 CHEBI:7507 6320 6328 neomycin +T199 SO:0005853 6329 6337 cassette +T200 GO:0008380 6399 6407 splicing +T201 SO:0000147 6414 6418 exon +T202 SO:0000147 6429 6433 exon +T203 GO:0008380 6447 6453 splice +T204 SO:0000147 6459 6463 exon +T205 SO:0000704 6482 6486 gene +T206 GO:0006412 6574 6587;6593 6600 production of ... protein +T207 PR:000010159 6588 6592 ERK5 +T208 UBERON:0000922 6621 6628 embryos +T209 GO:0042571 6680 6688 antibody +T210 PR:000010159 6714 6718 ERK5 +T211 PR:000010159 6728 6732 ERK5 +T212 UBERON:0000922 6759 6766 embryos +T213 PR:000010159 6774 6778 ERK5 +T214 UBERON:0000922 6782 6789 embryos +T215 PR:000010159 6813 6817 ERK5 +T216 PR:000010159 6842 6846 ERK5 +T217 UBERON:0000922 6850 6857 embryos +T218 UBERON:0000922 6880 6887 embryos +T219 PR:000010159 6925 6929 ERK5 +T220 PR:000010159 6937 6941 ERK5 +T221 UBERON:0000922 6945 6952 embryos +T222 PR:000010159 7007 7011 ERK5 +T223 PR:000010159 7019 7023 ERK5 +T224 UBERON:0000922 7027 7034 embryos +T225 GO:0010467 7086 7096 Expression +T226 PR:000010127 7100 7104 MKK5 +T227 PR:000000104 7129 7133 ERK1 +T228 PR:000000103 7135 7139 ERK2 +T229 PR:000003106 7144 7147 p38 +T230 PR:000010159 7184 7188 ERK5 +T231 PR:000010159 7224 7228 ERK5 +T232 NCBITaxon:10088 7238 7242 mice +T233 PR:000010159 7247 7251 ERK5 +T234 NCBITaxon:10088 7261 7265 mice +T235 SO:0001644 7284 7300 targeting vector +T236 SO:0000147 7311 7316 exons +T237 NCBITaxon:39107 7332 7338 murine +T238 PR:000010159 7339 7343 ERK5 +T239 SO:0000704 7344 7348 gene +T240 CHEBI:7507 7375 7383 neomycin +T241 SO:0005853 7394 7402 cassette +T242 SO:0005853 7423 7431 cassette +T243 CL:0002322 7475 7482 ES cell +T244 CL:0002322 7497 7504 ES cell +T245 PR:P43870 7532 7540 Hind III +T246 SO:0001644 7606 7622 targeting vector +T247 UBERON:0000922 7744 7751 embryos +T248 PR:P43870 7770 7778 Hind III +T249 UBERON:0000922 7903 7910 embryos +T250 CHEBI:28619 7928 7938 acrylamide +T251 GO:0042571 7987 7997 antibodies +T252 PR:000010159 8015 8019 ERK5 +T253 PR:000010127 8021 8025 MKK5 +T254 PR:000000104 8027 8031 ERK1 +T255 PR:000003106 8037 8040 p38 +T256 PR:000010159 8062 8066 ERK5 +T257 UBERON:0007023 8067 8073 adults +T258 UBERON:0000922 8078 8085 embryos +T259 UBERON:0000922 8111 8118 embryos +T260 UBERON:0000922 8164 8171 embryos +T261 PR:000010159 8235 8239 ERK5 +T262 UBERON:0000922 8249 8256 embryos +T263 UBERON:0000922 8366 8373 embryos +T264 UBERON:0000922 8388 8395 embryos +T265 UBERON:0000922 8451 8458 embryos +T266 PR:000010159 8576 8580 ERK5 +T267 UBERON:0000922 8590 8597 embryos +T268 UBERON:0001987 8614 8623 placental +T269 GO:0001890 8614 8623;8641 8652 placental ... development +T270 UBERON:0001981 8628 8640 blood vessel +T271 GO:0001568 8628 8652 blood vessel development +T272 UBERON:0000922 8731 8738 embryos +T273 UBERON:0000033 8867 8871 head +T274 UBERON:0002355 8876 8894 lower trunk region +T275 UBERON:0000922 8920 8927 embryos +T276 UBERON:0000922 8967 8974 embryos +T277 GO:0060322 8984 8998;9003 9007 development of ... head +T278 UBERON:0000033 9003 9007 head +T279 UBERON:0002355 9012 9023 lower trunk +T280 UBERON:0004362 9057 9062;9074 9090 first ... branchial arches +T281 UBERON:0003066 9067 9090 second branchial arches +T282 UBERON:0000922 9112 9119 embryos +T283 UBERON:0000033 9142 9146 head +T284 GO:0060485 9167 9181;9195 9205 development of ... mesenchyme +T285 UBERON:0005253 9186 9205 cephalic mesenchyme +T286 PR:000010159 9254 9258 ERK5 +T287 PR:000010159 9329 9333 ERK5 +T288 UBERON:0000922 9345 9352 embryos +T289 UBERON:0000922 9354 9361 Embryos +T290 GO:0007618 9387 9394 matings +T291 PR:000010159 9398 9402 ERK5 +T292 NCBITaxon:10088 9406 9410 mice +T293 UBERON:0001040 9457 9465 yolk sac +T294 PR:000010159 9537 9541 ERK5 +T295 PR:000010159 9616 9620 ERK5 +T296 UBERON:0000922 9624 9631 embryos +T297 UBERON:0000033 9639 9643 head +T298 UBERON:0005253 9673 9692 cephalic mesenchyme +T299 UBERON:0000922 9705 9712 embryos +T300 PR:000010159 9744 9748 ERK5 +T301 UBERON:0000922 9752 9759 embryos +T302 UBERON:0000922 9803 9810 embryos +T303 PR:000010159 9812 9816 ERK5 +T304 UBERON:0000922 9820 9827 embryos +T305 UBERON:0000033 9847 9851 head +T306 UBERON:0000922 9881 9888 embryos +T307 PR:000010159 9919 9923 ERK5 +T308 UBERON:0000922 9927 9934 embryos +T309 UBERON:0005253 9975 9994 cephalic mesenchyme +T310 UBERON:0004362 9999 10002;10011 10027 1st ... branchial arches +T311 UBERON:0003066 10007 10027 2nd branchial arches +T312 GO:0060174 10045 10059;10069 10078 Development of ... limb buds +T313 UBERON:0005418 10064 10078 hind limb buds +T314 UBERON:0002355 10090 10101 lower trunk +T315 PR:000010159 10127 10131 ERK5 +T316 UBERON:0000922 10135 10142 embryos +T317 GO:0010467 10145 10155 Expression +T318 PR:000010159 10159 10163 ERK5 +T319 PR:000010127 10168 10172 MKK5 +T320 GO:0009790 10180 10193 embryogenesis +T321 GO:0010467 10211 10221 expression +T322 PR:000010159 10225 10229 ERK5 +T323 PR:000010127 10255 10259 MKK5 +T324 GO:0097617 10284 10297 hybridisation +T325 SO:0000644 10304 10317 antisense RNA +T326 GO:0010467 10341 10351 expression +T327 GO:0065007 10385 10394 regulated +T328 UBERON:0000922 10402 10411 embryonic +T329 GO:0009790 10402 10423 embryonic development +T330 PR:000010159 10454 10458 ERK5 +T331 GO:0010467 10459 10469 expression +T332 UBERON:0009748 10505 10525 cephalic neural fold +T333 UBERON:0007026 10530 10543 primitive gut +T334 PR:000010159 10553 10557 ERK5 +T335 GO:0010467 10558 10568 expression +T336 UBERON:0004362 10585 10590;10602 10616 first ... branchial arch +T337 UBERON:0003066 10595 10616 second branchial arch +T338 UBERON:0000033 10618 10626 cephalic +T339 UBERON:0002329 10635 10642 somites +T340 UBERON:0000309 10671 10680 body wall +T341 PR:000010159 10700 10704 ERK5 +T342 GO:0010467 10705 10715 expression +T343 UBERON:0004347 10748 10757 limb buds +T344 PR:000010159 10810 10814 ERK5 +T345 GO:0010467 10820 10830 expression +T346 PR:000010127 10842 10846 MKK5 +T347 PR:000010159 10882 10886 ERK5 +T348 PR:000010159 10922 10926 ERK5 +T349 GO:0010467 10954 10963 expressed +T350 UBERON:0000948 10982 10987 heart +T351 PR:000010159 11068 11072 ERK5 +T352 GO:0097617 11093 11107 hybridisations +T353 PR:000010159 11147 11151 ERK5 +T354 GO:0010467 11152 11162 expression +T355 UBERON:0002539 11179 11193 branchial arch +T356 UBERON:0005253 11195 11214 cephalic mesenchyme +T357 UBERON:0002101 11276 11281 limbs +T358 UBERON:0001046 11283 11291 hind gut +T359 UBERON:0004161 11293 11311 septum transversum +T360 UBERON:0000044 11313 11333 dorsal root ganglion +T361 UBERON:0002329 11335 11342 somites +T362 UBERON:0002415 11347 11351 tail +T363 GO:0010467 11363 11373 expression +T364 PR:000010159 11377 11381 ERK5 +T365 UBERON:0000948 11398 11403 heart +T366 GO:0010467 11463 11473 expression +T367 PR:000010159 11477 11481 ERK5 +T368 UBERON:0002081 11503 11520;11525 11530 atrial chamber of ... heart +T369 GO:0010467 11556 11566 expression +T370 PR:000010159 11570 11574 ERK5 +T371 UBERON:0006615 11603 11615 sinus venous +T372 UBERON:0000948 11626 11631 heart +T373 GO:0010467 11633 11643 Expression +T374 PR:000010159 11647 11651 ERK5 +T375 UBERON:0001987 11686 11694 placenta +T376 GO:0097617 11718 11731 hybridisation +T377 PR:000010159 11748 11752 ERK5 +T378 GO:0010467 11753 11763 expression +T379 UBERON:0004027 11795 11810 chorionic plate +T380 UBERON:0003946 11815 11834 labyrinthine layers +T381 GO:0010467 11847 11857 expression +T382 GO:0010467 11894 11904 expression +T383 UBERON:0000922 11921 11928 embryos +T384 PR:000010159 11953 11957 ERK5 +T385 GO:0010467 11958 11968 expression +T386 PR:000010159 12025 12029 ERK5 +T387 GO:0010467 12030 12040 expression +T388 UBERON:0000033 12057 12061 head +T389 UBERON:0002355 12066 12077 lower trunk +T390 UBERON:0000922 12085 12091 embryo +T391 UBERON:0000948 12113 12118 heart +T392 UBERON:0001987 12141 12149 placenta +T393 UBERON:0007023 12154 12159 adult +T394 NCBITaxon:10088 12160 12164 mice +T395 PR:000010159 12181 12185 ERK5 +T396 PR:000010127 12190 12194 MKK5 +T397 UBERON:0000955 12209 12214 brain +T398 UBERON:0002370 12216 12222 thymus +T399 UBERON:0002106 12227 12233 spleen +T400 UBERON:0002048 12264 12268 lung +T401 UBERON:0000945 12270 12277 stomach +T402 UBERON:0002369 12279 12292 adrenal gland +T403 UBERON:0001013 12294 12308 adipose tissue +T404 UBERON:0001264 12310 12318 pancreas +T405 UBERON:0000948 12323 12328 heart +T406 GO:0010467 12350 12360 Expression +T407 PR:000010159 12364 12368 ERK5 +T408 PR:000010127 12373 12377 MKK5 +T409 UBERON:0000922 12385 12394 embryonic +T410 GO:0009790 12385 12406 embryonic development +T411 GO:0097617 12428 12441 hybridisation +T412 UBERON:0000922 12471 12478 embryos +T413 SO:0000644 12513 12526 antisense RNA +T414 PR:000010159 12542 12546 ERK5 +T415 PR:000010127 12550 12554 MKK5 +T416 GO:0010467 12577 12587 Expression +T417 PR:000010159 12591 12595 ERK5 +T418 PR:000010127 12600 12604 MKK5 +T419 UBERON:0009748 12642 12662 cephalic neural fold +T420 UBERON:0007026 12667 12680 primitive gut +T421 GO:0010467 12690 12700 expression +T422 UBERON:0002539 12722 12736 branchial arch +T423 UBERON:0000033 12738 12746 cephalic +T424 UBERON:0002329 12758 12765 somites +T425 UBERON:0000309 12791 12800 body wall +T426 GO:0010467 12829 12839 expression +T427 PR:000010159 12843 12847 ERK5 +T428 PR:000010127 12852 12856 MKK5 +T429 GO:0010467 12877 12887 expression +T430 UBERON:0002539 12900 12914 branchial arch +T431 UBERON:0000033 12916 12920 head +T432 UBERON:0004347 12925 12934 limb buds +T433 PR:000010159 12959 12963 ERK5 +T434 GO:0010467 12964 12974 expression +T435 UBERON:0000922 12978 12984 embryo +T436 GO:0097617 13015 13028 hybridisation +T437 UBERON:0000922 13058 13065 embryos +T438 UBERON:0001987 13075 13084 placentas +T439 SO:0000644 13123 13136 antisense RNA +T440 PR:000010159 13152 13156 ERK5 +T441 UBERON:0000922 13198 13205 embryos +T442 PR:000010159 13244 13248 ERK5 +T443 GO:0010467 13249 13259 expression +T444 UBERON:0005253 13276 13295 cephalic mesenchyme +T445 UBERON:0002539 13303 13317 branchial arch +T446 UBERON:0034705 13322 13337 neuroepithelium +T447 GO:0010467 13344 13354 expression +T448 UBERON:0000948 13371 13376 heart +T449 PR:000010159 13437 13441 ERK5 +T450 GO:0010467 13442 13452 expression +T451 UBERON:0006615 13469 13481 sinus venous +T452 UBERON:0000948 13492 13497 heart +T453 UBERON:0001987 13514 13522 placenta +T454 GO:0010467 13537 13547 expression +T455 UBERON:0004027 13564 13579 chorionic plate +T456 UBERON:0003946 13584 13603 labyrinthine layers +T457 PR:000010159 13634 13638 ERK5 +T458 PR:000010127 13643 13647 MKK5 +T459 UBERON:0000922 13659 13666 embryos +T460 UBERON:0000033 13702 13706 head +T461 UBERON:0000948 13708 13713 heart +T462 UBERON:0001555 13715 13718 gut +T463 UBERON:0001987 13723 13731 placenta +T464 UBERON:0001987 13751 13759 placenta +T465 UBERON:0000922 13799 13808 embryonic +T466 PR:000010159 13867 13871 ERK5 +T467 UBERON:0000922 13875 13882 embryos +T468 UBERON:0000479 13911 13918 Tissues +T469 UBERON:0007023 13943 13948 adult +T470 NCBITaxon:10088 13959 13963 mice +T471 CHEBI:28619 14167 14177 acrylamide +T472 GO:0042571 14226 14236 antibodies +T473 PR:000010159 14253 14257 ERK5 +T474 PR:000010127 14261 14265 MKK5 +T475 UBERON:0000922 14275 14284 embryonic +T476 UBERON:0007023 14293 14298 adult +T477 PR:000000104 14322 14326 ERK1 +T478 PR:000003106 14330 14333 p38 +T479 UBERON:0007023 14372 14377 adult +T480 UBERON:0000479 14378 14384 tissue +T481 PR:000010159 14399 14403 ERK5 +T482 GO:0001525 14427 14439 angiogenesis +T483 UBERON:0001987 14444 14453 placental +T484 GO:0001890 14444 14465 placental development +T485 PR:000010159 14508 14512 ERK5 +T486 UBERON:0000922 14522 14529 embryos +T487 GO:0001568 14550 14576 formation of blood vessels +T488 UBERON:0001981 14563 14576 blood vessels +T489 UBERON:0001040 14584 14592 yolk sac +T490 UBERON:0003061 14602 14615 blood islands +T491 UBERON:0000922 14671 14678 embryos +T492 PR:000010159 14703 14707 ERK5 +T493 UBERON:0000922 14717 14724 embryos +T494 UBERON:0004537 14763 14787 network of blood vessels +T495 UBERON:0000922 14819 14826 embryos +T496 UBERON:0001981 14863 14875 blood vessel +T497 GO:0001568 14863 14885 blood vessel formation +T498 UBERON:0000922 14902 14909 embryos +T499 PR:000001904 14920 14924 CD31 +T500 UBERON:0001986 14937 14948 endothelial +T501 CL:0000115 14937 14954 endothelial cells +T502 PR:000010159 15002 15006 ERK5 +T503 UBERON:0000922 15010 15017 embryos +T504 UBERON:0004537 15041 15051;15058 15071 network of ... blood vessels +T505 UBERON:0000922 15122 15129 embryos +T506 UBERON:0001981 15136 15149 blood vessels +T507 UBERON:0001981 15193 15206 blood vessels +T508 UBERON:0002049 15232 15243;15252 15259 networks of ... vessels +T509 UBERON:0002049 15266 15285 network of vesicles +T510 UBERON:0000033 15317 15321 head +T511 UBERON:0000922 15337 15343 embryo +T512 UBERON:0001981 15384 15397 blood vessels +T513 UBERON:0000033 15418 15422 head +T514 PR:000010159 15433 15437 ERK5 +T515 UBERON:0000922 15441 15448 embryos +T516 UBERON:0000033 15505 15509 head +T517 UBERON:0001981 15533 15546 blood vessels +T518 PR:000010159 15583 15587 ERK5 +T519 UBERON:0000922 15591 15598 embryos +T520 PR:000010159 15647 15651 ERK5 +T521 UBERON:0001040 15663 15672 yolk sacs +T522 UBERON:0000922 15674 15681 Embryos +T523 GO:0007618 15707 15714 matings +T524 PR:000010159 15718 15722 ERK5 +T525 NCBITaxon:10088 15726 15730 mice +T526 UBERON:0000922 15749 15756 Embryos +T527 UBERON:0001040 15809 15817 yolk sac +T528 PR:000010159 15846 15850 ERK5 +T529 UBERON:2000084 15861 15865 yolk +T530 UBERON:0003061 15874 15887 blood islands +T531 UBERON:0001981 15916 15929 blood vessels +T532 UBERON:0001040 15949 15958 yolk sacs +T533 UBERON:0000055 15981 15988 vessels +T534 UBERON:0000055 16022 16029 vessels +T535 PR:000010159 16072 16076 ERK5 +T536 UBERON:0001040 16080 16089 yolk sacs +T537 UBERON:0001981 16133 16146 blood vessels +T538 PR:000010159 16162 16166 ERK5 +T539 UBERON:0001040 16170 16179 yolk sacs +T540 CL:0000232 16226 16241 red blood cells +T541 UBERON:0000178 16230 16235 blood +T542 PR:000010159 16255 16259 ERK5 +T543 UBERON:0000922 16263 16270 embryos +T544 UBERON:0000178 16313 16318 blood +T545 GO:0008015 16313 16330 blood circulation +T546 GO:0060047 16341 16348 beating +T547 UBERON:0000948 16349 16354 heart +T548 PR:000001904 16367 16371 CD31 +T549 UBERON:0000922 16408 16415 embryos +T550 UBERON:0000922 16437 16444 Embryos +T551 GO:0007618 16470 16476 mating +T552 PR:000001904 16498 16502 CD31 +T553 GO:0042571 16503 16511 antibody +T554 UBERON:0004537 16554 16565;16572 16585 networks of ... blood vessels +T555 UBERON:0000033 16603 16607 head +T556 PR:000010159 16638 16642 ERK5 +T557 UBERON:0000922 16646 16653 embryos +T558 UBERON:0014907 16671 16691 intersomitic vessels +T559 UBERON:0001981 16765 16778 blood vessels +T560 UBERON:0000033 16786 16790 head +T561 UBERON:0000922 16811 16818 embryos +T562 GO:0001525 16842 16854 angiogenesis +T563 UBERON:0002049 16880 16891;16900 16907 networks of ... vessels +T564 PR:000010159 16934 16938 ERK5 +T565 UBERON:0000922 16942 16949 embryos +T566 UBERON:0014907 17018 17038 intersomitic vessels +T567 PR:000010159 17061 17065 ERK5 +T568 UBERON:0000922 17069 17076 embryos +T569 PR:000010159 17201 17205 ERK5 +T570 UBERON:0000948 17246 17253 cardiac +T571 GO:0007507 17246 17265 cardiac development +T572 GO:0007507 17271 17285;17290 17295 development of ... heart +T573 UBERON:0000948 17290 17295 heart +T574 PR:000010159 17315 17319 ERK5 +T575 UBERON:0000922 17323 17330 embryos +T576 GO:0001947 17348 17358;17363 17368 looping of ... heart +T577 UBERON:0000948 17363 17368 heart +T578 UBERON:0000948 17406 17411 heart +T579 UBERON:0002349 17443 17453 myocardium +T580 UBERON:0000060 17454 17458 wall +T581 PR:000010159 17462 17466 ERK5 +T582 UBERON:0000922 17493 17500 embryos +T583 PR:000010159 17548 17552 ERK5 +T584 UBERON:0000922 17556 17563 embryos +T585 UBERON:0000948 17613 17618 heart +T586 PR:000010159 17649 17653 ERK5 +T587 UBERON:0000922 17657 17664 embryos +T588 GO:0007618 17690 17697 matings +T589 CHEBI:51686 17751 17763 haematoxylin +T590 UBERON:0000948 17828 17833 heart +T591 PR:000010159 17854 17858 ERK5 +T592 UBERON:0000922 17862 17869 embryos +T593 UBERON:0002081 17881 17887;17911 17919 atrial ... chambers +T594 UBERON:0002081 17889 17891 At +T595 UBERON:0002082 17897 17906;17911 17919 ventrical ... chambers +T596 UBERON:0002082 17908 17909 V +T597 UBERON:0002081 17942 17948 atrial +T598 UBERON:0000060 17949 17953 wall +T599 PR:000010159 17965 17969 ERK5 +T600 UBERON:0000922 17973 17980 embryos +T601 UBERON:0000948 18011 18017 hearts +T602 UBERON:0002081 18052 18058 atrium +T603 UBERON:0000060 18059 18063 wall +T604 PR:000010159 18102 18106 ERK5 +T605 UBERON:0000922 18110 18117 embryos +T606 PR:000010159 18123 18127 ERK5 +T607 UBERON:0000922 18131 18138 embryos +T608 UBERON:0002081 18169 18175 atrial +T609 UBERON:0000060 18176 18180 wall +T610 UBERON:0000948 18207 18214 cardiac +T611 http://purl.obolibrary.org/obo/MONDO_0005453 18207 18222 cardiac defects +T612 PR:000010159 18271 18275 ERK5 +T613 UBERON:0000922 18279 18286 embryos +T614 PR:000010159 18302 18306 ERK5 +T615 UBERON:0001987 18331 18340 placental +T616 GO:0001890 18331 18352 placental development +T617 PR:000010159 18396 18400 ERK5 +T618 UBERON:0001987 18404 18413 placentas +T619 CHEBI:51686 18435 18447 Haematoxylin +T620 UBERON:0001987 18499 18508 placentas +T621 PR:000010159 18572 18576 ERK5 +T622 UBERON:0000922 18580 18587 embryos +T623 UBERON:0005335 18589 18604 Chorioallantoic +T624 GO:0060710 18589 18611 Chorioallantoic fusion +T625 PR:000010159 18648 18652 ERK5 +T626 UBERON:0001987 18662 18670 placenta +T627 PR:000010159 18674 18678 ERK5 +T628 NCBITaxon:10088 18682 18686 mice +T629 UBERON:0004027 18694 18709 chorionic plate +T630 UBERON:0003946 18711 18720;18743 18749 labyrinth ... layers +T631 UBERON:0004021 18725 18749 spongiotophoblast layers +T632 UBERON:0003946 18770 18782;18795 18803 laryrinth of ... placenta +T633 PR:000010159 18787 18791 ERK5 +T634 UBERON:0001987 18836 18845 placentas +T635 UBERON:0000922 18857 18866 embryonic +T636 UBERON:0001981 18880 18893 blood vessels +T637 UBERON:0003946 18941 18956 labyrinth layer +T638 PR:000010159 18960 18964 ERK5 +T639 UBERON:0001987 18968 18977 placentas +T640 UBERON:0000922 19047 19056 embryonic +T641 UBERON:0001981 19070 19083 blood vessels +T642 UBERON:0003946 19091 19103 labyrinthine +T643 GO:0048468 19112 19126;19145 19150 Development of ... cells +T644 UBERON:0000088 19127 19138 trophoblast +T645 CL:0002488 19127 19150 trophoblast giant cells +T646 PR:000010159 19184 19188 ERK5 +T647 UBERON:0004021 19221 19245 spongiotrophoblast layer +T648 PR:000016934 19252 19256 4311 +T649 UBERON:0001987 19330 19339 placental +T650 GO:0042571 19367 19375 antibody +T651 MOP:0000780 19388 19395 cleaved +T652 PR:000018762 19388 19413 cleaved form of caspase 3 +T653 GO:0006915 19441 19450 apoptosis +T654 UBERON:0003946 19471 19480 labyrinth +T655 PR:000010159 19484 19488 ERK5 +T656 UBERON:0000922 19492 19499 embryos +T657 UBERON:0000922 19522 19529 embryos +T658 GO:0006915 19541 19550 Apoptosis +T659 UBERON:0001986 19563 19574 endothelial +T660 CL:0000115 19563 19580 endothelial cells +T661 CL:0000415 19582 19589;19602 19607 diploid ... cells +T662 UBERON:0000088 19590 19601 trophoblast +T663 CL:0000351 19590 19607 trophoblast cells +T664 UBERON:0000922 19617 19626 embryonic +T665 CL:0002321 19617 19626;19633 19638 embryonic ... cells +T666 UBERON:0000178 19627 19632 blood +T667 CL:0000081 19627 19638 blood cells +T668 MOP:0000780 19643 19650 cleaved +T669 PR:000018762 19643 19660 cleaved caspase 3 +T670 UBERON:0001987 19692 19701 placentas +T671 PR:000010159 19727 19731 ERK5 +T672 UBERON:0001987 19735 19744 placentas +T673 UBERON:0001987 19782 19790 placenta +T674 UBERON:0001987 19812 19821 Placentas +T675 PR:000010159 19872 19876 ERK5 +T676 NCBITaxon:10088 19880 19884 mice +T677 UBERON:0001987 19933 19942 placentas +T678 CHEBI:51686 19960 19972 haematoxylin +T679 PR:000010159 20121 20125 ERK5 +T680 UBERON:0000922 20129 20136 embryos +T681 UBERON:0000922 20183 20192 embryonic +T682 UBERON:0001981 20207 20220 blood vessels +T683 PR:000010159 20258 20262 ERK5 +T684 UBERON:0001987 20266 20275 placentas +T685 UBERON:0004027 20334 20349 chorionic plate +T686 UBERON:0004027 20351 20353 CP +T687 UBERON:0003946 20356 20365;20397 20403 labyrinth ... layers +T688 UBERON:0003946 20367 20368;20397 20403 L ... layers +T689 UBERON:0004021 20374 20392;20397 20403 spongiotrophoblast ... layers +T690 UBERON:0004021 20394 20395;20397 20403 S ... layers +T691 PR:000010159 20443 20447 ERK5 +T692 UBERON:0000922 20451 20458 embryos +T693 UBERON:0000922 20498 20507 embryonic +T694 UBERON:0001981 20508 20521 blood vessels +T695 PR:000010159 20556 20560 ERK5 +T696 UBERON:0001987 20564 20573 placentas +T697 UBERON:0001981 20594 20607 blood vessels +T698 UBERON:0003946 20629 20648 labyrinthine layers +T699 PR:000016934 20749 20753 4311 +T700 PR:000002312 20766 20775 caspase 3 +T701 UBERON:0001987 20795 20804 placentas +T702 CHEBI:73702 20809 20812 Wax +T703 PR:000010159 20846 20850 ERK5 +T704 UBERON:0001987 20854 20863 placentas +T705 GO:0097617 20889 20902 hybridisation +T706 SO:0000644 20911 20924 antisense RNA +T707 PR:000016934 20939 20943 4311 +T708 UBERON:0004021 20949 20973 spongiotrophoblast layer +T709 PR:000010159 21016 21020 ERK5 +T710 UBERON:0001987 21024 21033 placentas +T711 UBERON:0000922 21060 21067 embryos +T712 GO:0042571 21118 21126 antibody +T713 MOP:0000780 21144 21151 cleaved +T714 PR:000018762 21144 21161 cleaved caspase 3 +T715 MOP:0000780 21181 21188 cleaved +T716 PR:000018762 21181 21198 cleaved caspase 3 +T717 PR:000010159 21228 21232 ERK5 +T718 UBERON:0000922 21236 21243 embryos +T719 PR:000010159 21283 21287 ERK5 +T720 UBERON:0001987 21291 21300 placentas +T721 GO:0006915 21302 21311 apoptosis +T722 UBERON:0001986 21324 21335 endothelial +T723 CL:0000115 21324 21341 endothelial cells +T724 UBERON:0000088 21355 21366 trophoblast +T725 CL:0000351 21355 21372 trophoblast cells +T726 UBERON:0000922 21396 21405 embryonic +T727 CL:0002321 21396 21405;21412 21417 embryonic ... cells +T728 UBERON:0000178 21406 21411 blood +T729 CL:0000081 21406 21417 blood cells +T730 UBERON:0003946 21444 21453 labryinth +T731 GO:0006915 21458 21470;21477 21482 apoptosis of ... cells +T732 CL:0002488 21471 21482 giant cells +T733 PR:000010159 21494 21498 ERK5 +T734 GO:0060322 21522 21536;21541 21545 development of ... head +T735 UBERON:0000033 21541 21545 head +T736 UBERON:0002355 21550 21561 lower trunk +T737 PR:000010159 21585 21589 ERK5 +T738 UBERON:0000922 21593 21600 embryos +T739 UBERON:0000922 21628 21635 embryos +T740 GO:0060322 21662 21676;21681 21685 development of ... head +T741 UBERON:0000033 21681 21685 head +T742 UBERON:0002355 21690 21701 lower trunk +T743 UBERON:0000922 21717 21723 embryo +T744 UBERON:0000922 21864 21871 embryos +T745 PR:000010159 21910 21914 ERK5 +T746 UBERON:0000922 21918 21925 embryos +T747 UBERON:0002049 22060 22071 vasculature +T748 UBERON:0001987 22076 22085 placentas +T749 PR:000010159 22089 22093 ERK5 +T750 UBERON:0000922 22111 22118 embryos +T751 UBERON:0000033 22145 22149 head +T752 PR:000010159 22214 22218 ERK5 +T753 UBERON:0000922 22222 22229 embryos +T754 UBERON:0005253 22259 22285 cephalic mesenchyme tissue +T755 PR:000010159 22344 22348 ERK5 +T756 UBERON:0000922 22352 22359 embryos +T757 UBERON:0000922 22378 22385 embryos +T758 UBERON:0005253 22427 22446;22467 22473 cephalic mesenchyme ... tissue +T759 UBERON:0034705 22451 22473 neuroepithelial tissue +T760 PR:000010159 22481 22485 ERK5 +T761 UBERON:0000922 22489 22496 embryos +T762 PR:000010159 22547 22551 ERK5 +T763 UBERON:0000922 22555 22562 embryos +T764 PR:000010159 22737 22741 ERK5 +T765 UBERON:0000922 22745 22751 embryo +T766 UBERON:0000033 22798 22802 head +T767 UBERON:0000922 22816 22823 embryos +T768 UBERON:0000922 22839 22845 embryo +T769 UBERON:0005253 22885 22911 cephalic mesenchyme tissue +T770 UBERON:0000922 22948 22955 embryos +T771 UBERON:0005253 23003 23022 cephalic mesenchyme +T772 UBERON:0000922 23058 23064 embryo +T773 PR:000010159 23090 23094 ERK5 +T774 UBERON:0000922 23098 23105 embryos +T775 UBERON:0005253 23134 23153 cephalic mesenchyme +T776 UBERON:0000922 23242 23249 embryos +T777 UBERON:0005253 23254 23273 cephalic mesenchyme +T778 PR:000010159 23298 23302 ERK5 +T779 UBERON:0000922 23306 23313 embryos +T780 UBERON:0005253 23318 23337 cephalic mesenchyme +T781 UBERON:0034705 23402 23417 neuroepithelial +T782 UBERON:0005253 23454 23473 cephalic mesenchyme +T783 PR:000010159 23515 23519 ERK5 +T784 UBERON:0000922 23523 23530 embryos +T785 PR:000010159 23601 23605 ERK5 +T786 UBERON:0000922 23609 23616 embryos +T787 UBERON:0005253 23629 23648 cephalic mesenchyme +T788 UBERON:0001981 23667 23680 blood vessels +T789 UBERON:0000922 23712 23719 embryos +T790 UBERON:0001981 23743 23756 blood vessels +T791 PR:000010159 23764 23768 ERK5 +T792 UBERON:0000922 23772 23779 embryos +T793 UBERON:0003104 23846 23863 mesenchyme tissue +T794 UBERON:0003104 23918 23935 mesenchyme tissue +T795 UBERON:0000055 24000 24007 vessels +T796 UBERON:0001981 24065 24078 blood vessels +T797 UBERON:0005253 24119 24138 cephalic mesenchyme +T798 PR:000010159 24165 24169 ERK5 +T799 UBERON:0000922 24173 24180 embryos +T800 GO:0008283 24211 24222 proliferate +T801 UBERON:0000922 24344 24351 embryos +T802 GO:0006915 24373 24382 apoptosis +T803 GO:0006915 24397 24406 apoptosis +T804 UBERON:0000033 24423 24427 head +T805 UBERON:0000922 24448 24455 embryos +T806 UBERON:0000922 24465 24472 embryos +T807 GO:0006915 24489 24498 apoptosis +T808 UBERON:0008816 24520 24527;24536 24543 head of ... embryos +T809 PR:000010159 24528 24532 ERK5 +T810 UBERON:0005253 24578 24597 cephalic mesenchyme +T811 PR:000010159 24613 24617 ERK5 +T812 UBERON:0000922 24621 24628 embryos +T813 GO:0007618 24654 24661 matings +T814 CHEBI:51686 24721 24733 haematoxylin +T815 UBERON:0034705 24882 24897 neuroepithelium +T816 UBERON:0005253 24902 24921 cephalic mesenchyme +T817 PR:000010159 24940 24944 ERK5 +T818 UBERON:0000922 24948 24955 embryos +T819 UBERON:0005253 24970 24989 cephalic mesenchyme +T820 PR:000010159 25049 25053 ERK5 +T821 UBERON:0000922 25057 25064 embryos +T822 PR:000010159 25136 25140 ERK5 +T823 UBERON:0000922 25144 25151 embryos +T824 UBERON:0000922 25191 25198 embryos +T825 CHEBI:51686 25216 25228 haematoxylin +T826 UBERON:0005253 25248 25267 cephalic mesenchyme +T827 PR:000010159 25300 25304 ERK5 +T828 UBERON:0000922 25308 25315 embryos +T829 UBERON:0034705 25324 25339 neuroepithelium +T830 PR:000010159 25356 25360 ERK5 +T831 UBERON:0000922 25364 25371 embryos +T832 UBERON:0005253 25419 25438 cephalic mesenchyme +T833 UBERON:0003104 25457 25475 mesenchymal tissue +T834 PR:000010159 25483 25487 ERK5 +T835 UBERON:0000922 25491 25498 embryos +T836 UBERON:0001981 25514 25527 blood vessels +T837 PR:000010159 25569 25573 ERK5 +T838 UBERON:0000922 25577 25584 embryos +T839 UBERON:0000922 25611 25618 embryos +T840 UBERON:0005253 25658 25677 cephalic mesenchyme +T841 PR:000010159 25682 25686 ERK5 +T842 UBERON:0000922 25690 25697 embryos +T843 UBERON:0005253 25716 25735 cephalic mesenchyme +T844 UBERON:0003104 25778 25796 mesenchymal tissue +T845 PR:000010159 25819 25823 ERK5 +T846 UBERON:0000922 25827 25834 embryos +T847 GO:0006915 25872 25881 apoptosis +T848 UBERON:0008816 25889 25896;25905 25912 head of ... embryos +T849 PR:000010159 25897 25901 ERK5 +T850 GO:0006915 25927 25936 apoptosis +T851 UBERON:0000922 25946 25953 embryos +T852 GO:0006915 26045 26054 apoptosis +T853 PR:000010159 26062 26066 ERK5 +T854 UBERON:0000922 26070 26077 embryos +T855 UBERON:0000033 26151 26155 head +T856 UBERON:0000922 26192 26199 embryos +T857 UBERON:0005253 26282 26301 cephalic mesenchyme +T858 UBERON:0002355 26385 26396 lower trunk +T859 UBERON:0000948 26438 26443 heart +T860 PR:000010159 26465 26469 ERK5 +T861 UBERON:0000922 26473 26480 embryos +T862 UBERON:0000922 26493 26500 embryos +T863 UBERON:0004161 26525 26542 septum transverum +T864 PR:000010159 26571 26575 ERK5 +T865 UBERON:0000922 26579 26586 embryos +T866 UBERON:0001041 26683 26690 foregut +T867 GO:0007494 26708 26722;26727 26730;26740 26743 development of ... mid ... gut +T868 GO:0061525 26708 26722;26735 26743 development of ... hind gut +T869 UBERON:0001045 26727 26730;26740 26743 mid ... gut +T870 UBERON:0001046 26735 26743 hind gut +T871 PR:000010159 26756 26760 ERK5 +T872 UBERON:0000922 26764 26771 embryos +T873 GO:0008283 26809 26822 proliferation +T874 UBERON:0001046 26853 26857;26865 26868 hind ... gut +T875 UBERON:0001045 26861 26868 mid gut +T876 UBERON:0000328 26865 26873 gut wall +T877 UBERON:0001046 26908 26916 hind gut +T878 PR:000010159 26947 26951 ERK5 +T879 UBERON:0000922 26955 26962 embryos +T880 GO:0007618 26988 26995 matings +T881 CHEBI:51686 27049 27061 haematoxylin +T882 UBERON:0001555 27129 27133 guts +T883 PR:000010159 27137 27141 ERK5 +T884 NCBITaxon:10088 27145 27149 mice +T885 GO:0008283 27202 27215 proliferation +T886 UBERON:0001555 27223 27226 gut +T887 UBERON:0001986 27227 27238 endothelium +T888 UBERON:0000922 27283 27290 embryos +T889 PR:000010159 27339 27343 ERK5 +T890 UBERON:0000922 27347 27354 embryos +T891 PR:000010159 27410 27414 ERK5 +T892 UBERON:0000922 27426 27435 embryonic +T893 PR:000010159 27477 27481 ERK5 +T894 UBERON:0000922 27485 27492 embryos +T895 UBERON:0001987 27512 27521 placental +T896 GO:0001890 27512 27533 placental development +T897 GO:0001525 27546 27558 angiogenesis +T898 GO:0060322 27581 27595;27600 27604 development of ... head +T899 GO:0060485 27581 27595;27631 27641 development of ... mesenchyme +T900 UBERON:0000033 27600 27604 head +T901 UBERON:0005253 27622 27641 cephalic mesenchyme +T902 UBERON:0034705 27646 27661 neuroepithelium +T903 UBERON:0002355 27668 27679 lower trunk +T904 UBERON:0000922 27687 27693 embryo +T905 PR:000010159 27754 27758 ERK5 +T906 PR:000010159 27800 27804 ERK5 +T907 UBERON:0001987 27938 27947 placental +T908 GO:0001890 27938 27959 placental development +T909 GO:0001525 27964 27976 angiogenesis +T910 PR:000010159 28123 28127 ERK5 +T911 UBERON:0000922 28131 28138 embryos +T912 UBERON:0000033 28215 28219 head +T913 UBERON:0002100 28224 28229 trunk +T914 UBERON:0000922 28247 28254 embryos +T915 UBERON:0005253 28324 28343 cephalic mesenchyme +T916 UBERON:0001007 28348 28351 gut +T917 PR:000010159 28638 28642 ERK5 +T918 GO:0010467 28656 28665 expressed +T919 SO:0000831 28893 28902;28912 28916 region of ... gene +T920 PR:000010159 28907 28911 ERK5 +T921 NCBITaxon:10088 29023 29027 mice +T922 CL:0002322 29032 29040 ES cells +T923 PR:000010159 29176 29180 ERK5 +T924 UBERON:0000922 29184 29191 embryos +T925 UBERON:0000922 29254 29261 embryos +T926 UBERON:0000922 29332 29339 embryos +T927 UBERON:0000922 29356 29363 embryos +T928 GO:0060322 29412 29426;29431 29435 development of ... head +T929 UBERON:0000033 29431 29435 head +T930 UBERON:0002355 29440 29451 lower trunk +T931 UBERON:0001987 29522 29531 placental +T932 UBERON:0001987 29543 29552 Placental +T933 NCBITaxon:10088 29633 29637 mice +T934 UBERON:0001987 29672 29681 placental +T935 PR:000010159 29716 29720 ERK5 +T936 UBERON:0000922 29724 29731 embryos +T937 SO:0000704 29745 29752 genetic +T938 UBERON:0001987 29813 29822 placental +T939 GO:0001890 29813 29834 placental development +T940 UBERON:0000922 29866 29873 embryos +T941 UBERON:0000922 29909 29916 embryos +T942 PR:000003107 30054 30058 p38α +T943 UBERON:0000922 30074 30081 embryos +T944 GO:0016265 30082 30086 died +T945 UBERON:0001987 30115 30124 placental +T946 UBERON:0000922 30145 30152 embryos +T947 UBERON:0001987 30226 30234 placenta +T948 UBERON:0004027 30250 30265 chorionic plate +T949 UBERON:0003946 30267 30279 labyrinthine +T950 UBERON:0000088 30280 30291 trophoblast +T951 UBERON:0003946 30293 30302;30326 30332 labyrinth ... layers +T952 UBERON:0004021 30307 30332 spongiotrophoblast layers +T953 PR:000010159 30369 30373 ERK5 +T954 PR:000010159 30442 30446 ERK5 +T955 UBERON:0001987 30464 30472 placenta +T956 UBERON:0003946 30530 30539 labyrinth +T957 PR:000010159 30555 30559 ERK5 +T958 UBERON:0001987 30563 30572 placentas +T959 UBERON:0000922 30612 30621 embryonic +T960 UBERON:0001981 30635 30648 blood vessels +T961 UBERON:0003946 30656 30675 placental labyrinth +T962 GO:0006915 30708 30717 apoptosis +T963 PR:000010159 30736 30740 ERK5 +T964 UBERON:0001987 30744 30753 placentas +T965 PR:000010159 30797 30801 ERK5 +T966 GO:0060711 30805 30819;30824 30833 development of ... labyrinth +T967 UBERON:0003946 30824 30833 labyrinth +T968 UBERON:0005335 30838 30853 chorioallantoic +T969 UBERON:0003946 30872 30881 labyrinth +T970 UBERON:0000922 30924 30933 embryonic +T971 UBERON:0000178 30947 30952 blood +T972 PR:000010159 30978 30982 ERK5 +T973 UBERON:0001987 30986 30995 placentas +T974 UBERON:0000922 31032 31041 embryonic +T975 PR:000010159 31053 31057 ERK5 +T976 GO:0000165 31074 31095 MAP kinase signalling +T977 UBERON:0003946 31127 31136 labyrinth +T978 GO:0060711 31127 31148 labyrinth development +T979 PR:000010136 31162 31167 MEKK3 +T980 PR:000010159 31194 31198 ERK5 +T981 PR:000003106 31203 31206 p38 +T982 UBERON:0000922 31225 31234 embryonic +T983 UBERON:0003946 31277 31286 labyrinth +T984 GO:0060711 31277 31298 labyrinth development +T985 PR:000003106 31318 31321 p38 +T986 PR:000010125 31326 31330 MEK1 +T987 UBERON:0003946 31364 31373 labyrinth +T988 GO:0000165 31430 31445 MAPK signalling +T989 PR:000015413 31535 31539 Sos1 +T990 PR:000010159 31626 31630 ERK5 +T991 GO:0001525 31664 31676 angiogenesis +T992 UBERON:0000922 31684 31690 embryo +T993 PR:000010159 31704 31708 ERK5 +T994 GO:0010467 31709 31719 expression +T995 GO:0097617 31731 31744 hybridisation +T996 GO:0010467 31765 31775 expression +T997 PR:000010159 31779 31783 ERK5 +T998 UBERON:0001981 31821 31834 blood vessels +T999 GO:0010467 31852 31861 expressed +T1000 UBERON:0000922 31881 31888 embryos +T1001 GO:0010467 31934 31944 expression +T1002 CHEBI:62488 31956 31976 signalling molecules +T1003 GO:0001525 31990 32002 angiogenesis +T1004 PR:000010159 32022 32026 ERK5 +T1005 GO:0001525 32069 32081 angiogenesis +T1006 NCBITaxon:10088 32098 32102 mice +T1007 PR:000010159 32172 32176 ERK5 +T1008 UBERON:0000922 32180 32187 embryos +T1009 PR:000010159 32245 32249 ERK5 +T1010 UBERON:0000948 32264 32271 cardiac +T1011 GO:0007507 32264 32283 cardiac development +T1012 GO:0097617 32316 32329 hybridisation +T1013 UBERON:0000922 32362 32369 embryos +T1014 GO:0010467 32384 32394 expression +T1015 PR:000010159 32398 32402 ERK5 +T1016 PR:000010127 32431 32435 MKK5 +T1017 GO:0010467 32440 32449 expressed +T1018 UBERON:0000948 32457 32462 heart +T1019 GO:0010467 32497 32507 expression +T1020 UBERON:0000922 32555 32561 embryo +T1021 PR:000010159 32630 32634 ERK5 +T1022 GO:0010467 32635 32645 expression +T1023 UBERON:0000948 32665 32670 heart +T1024 UBERON:0002100 32675 32680 trunk +T1025 UBERON:0000922 32688 32694 embryo +T1026 GO:0097617 32733 32746 hybridisation +T1027 PR:000010159 32810 32814 ERK5 +T1028 GO:0010467 32815 32825 expression +T1029 UBERON:0000948 32854 32859 heart +T1030 GO:0010467 32871 32881 expression +T1031 UBERON:0000922 32901 32907 embryo +T1032 GO:0007507 33043 33057;33072 33077 development of ... heart +T1033 UBERON:0000922 33062 33071 embryonic +T1034 UBERON:0000948 33072 33077 heart +T1035 UBERON:0000922 33113 33120 embryos +T1036 UBERON:0000948 33199 33204 heart +T1037 PR:000010159 33233 33237 ERK5 +T1038 UBERON:0000948 33264 33269 heart +T1039 UBERON:0002081 33350 33356 atrial +T1040 UBERON:0000060 33357 33361 wall +T1041 PR:000010159 33386 33390 ERK5 +T1042 UBERON:0000922 33400 33407 embryos +T1043 PR:000010159 33448 33452 ERK5 +T1044 UBERON:0000948 33485 33490 heart +T1045 GO:0007507 33485 33502 heart development +T1046 PR:000010311 33555 33560 MEF2C +T1047 UBERON:0000922 33571 33578 embryos +T1048 GO:0016265 33579 33582 die +T1049 GO:0001947 33626 33633 looping +T1050 PR:000010311 33661 33666 MEF2C +T1051 PR:000010159 33729 33733 ERK5 +T1052 PR:000003106 33801 33804 p38 +T1053 PR:000010311 33846 33851 MEF2C +T1054 PR:000010159 33855 33859 ERK5 +T1055 PR:000003106 33928 33931 p38 +T1056 UBERON:0000948 33964 33971 cardiac +T1057 GO:0007507 33964 33983 cardiac development +T1058 PR:000010159 33991 33995 ERK5 +T1059 UBERON:0000948 34088 34093 heart +T1060 GO:0001947 34117 34124 looping +T1061 SO:0000704 34230 34237 genetic +T1062 NCBITaxon:10088 34249 34253 mice +T1063 PR:000010159 34276 34280 ERK5 +T1064 PR:000002032 34379 34384 Tie-2 +T1065 PR:000004023 34405 34410 Ang-1 +T1066 PR:000010159 34440 34444 ERK5 +T1067 UBERON:0000948 34497 34502 heart +T1068 PR:000010159 34653 34657 ERK5 +T1069 PR:000002082 34720 34725 erbB2 +T1070 PR:000007159 34730 34735 erbB3 +T1071 PR:000010159 34771 34775 erk5 +T1072 UBERON:0000948 34830 34835 heart +T1073 UBERON:0000948 34871 34878 cardiac +T1074 PR:000010159 34897 34901 ERK5 +T1075 SO:0000167 34956 34964 promoter +T1076 PR:000010159 35008 35012 ERK5 +T1077 UBERON:0000922 35016 35023 embryos +T1078 UBERON:0000948 35035 35042 cardiac +T1079 GO:0007507 35035 35054 cardiac development +T1080 UBERON:0000948 35088 35095 cardiac +T1081 http://purl.obolibrary.org/obo/MONDO_0005453 35088 35103 cardiac defects +T1082 PR:000010159 35116 35120 ERK5 +T1083 UBERON:0000922 35124 35131 embryos +T1084 PR:000010159 35171 35175 ERK5 +T1085 UBERON:0000948 35183 35188 heart +T1086 UBERON:0001987 35273 35282 placental +T1087 UBERON:0000948 35357 35364 cardiac +T1088 UBERON:0000922 35418 35424 embryo +T1089 UBERON:0001987 35488 35497 placental +T1090 UBERON:0000948 35508 35515 cardiac +T1091 PR:000010159 35525 35529 ERK5 +T1092 GO:0060485 35622 35636;35650 35660 development of ... mesenchyme +T1093 GO:0048565 35622 35636;35665 35668 development of ... gut +T1094 UBERON:0005253 35641 35660 cephalic mesenchyme +T1095 UBERON:0001555 35665 35668 gut +T1096 PR:000010159 35676 35680 ERK5 +T1097 UBERON:0000922 35684 35691 embryos +T1098 PR:000010159 35696 35700 ERK5 +T1099 UBERON:0000922 35704 35711 embryos +T1100 UBERON:0005253 35738 35757 cephalic mesenchyme +T1101 UBERON:0005253 35791 35810 cephalic mesenchyme +T1102 UBERON:0005253 35897 35916 cephalic mesenchyme +T1103 UBERON:0034705 35925 35940 neuroepithelium +T1104 UBERON:0000922 35957 35964 embryos +T1105 UBERON:0005253 36018 36037 cephalic mesenchyme +T1106 UBERON:0005253 36124 36143 cephalic mesenchyme +T1107 PR:000010159 36198 36202 ERK5 +T1108 GO:0097617 36242 36255 hybridisation +T1109 PR:000010159 36268 36272 ERK5 +T1110 GO:0010467 36277 36286 expressed +T1111 UBERON:0005253 36294 36313 cephalic mesenchyme +T1112 PR:000010159 36382 36386 ERK5 +T1113 UBERON:0000922 36390 36397 embryos +T1114 UBERON:0001981 36419 36431 blood vessel +T1115 GO:0001568 36419 36431;36446 36457 blood vessel ... development +T1116 UBERON:0001987 36436 36445 placental +T1117 GO:0001890 36436 36457 placental development +T1118 UBERON:0005253 36540 36559 cephalic mesenchyme +T1119 UBERON:0001555 36564 36567 gut +T1120 GO:0001525 36608 36620 angiogenesis +T1121 UBERON:0001981 36644 36657 blood vessels +T1122 UBERON:0005253 36678 36697 cephalic mesenchyme +T1123 PR:000010159 36707 36711 ERK5 +T1124 UBERON:0000922 36715 36722 embryos +T1125 UBERON:0000479 36763 36769 tissue +T1126 UBERON:0000178 36796 36801 blood +T1127 UBERON:0005253 36828 36847 cephalic mesenchyme +T1128 GO:0006915 36880 36889 apoptosis +T1129 UBERON:0000479 36902 36908 tissue +T1130 GO:0010467 37036 37046 expression +T1131 PR:000014841 37059 37073 sonic hedgehog +T1132 PR:000014898 37078 37082 Six3 +T1133 PR:000010159 37153 37157 ERK5 +T1134 GO:0006915 37175 37184 apoptosis +T1135 PR:000010159 37192 37196 ERK5 +T1136 UBERON:0000922 37200 37207 embryos +T1137 PR:000010159 37222 37226 ERK5 +T1138 GO:0065007 37246 37256 regulating +T1139 GO:0008283 37257 37261;37274 37286 cell ... poliferation +T1140 GO:0010467 37314 37324 expression +T1141 PR:000010159 37328 37332 ERK5 +T1142 PR:000010127 37360 37364 MKK5 +T1143 GO:0008283 37392 37405 proliferation +T1144 CHEBI:52290 37445 37454 mitogenic +T1145 PR:000010159 37531 37535 ERK5 +T1146 GO:0001525 37539 37551 angiogenesis +T1147 UBERON:0001987 37556 37565 placental +T1148 GO:0001890 37556 37577 placental development +T1149 PR:000010159 37606 37610 ERK5 +T1150 UBERON:0005253 37634 37653 cephalic mesenchyme +T1151 GO:0042981 37658 37671;37685 37694 regulation of ... apoptosis +T1152 PR:000010159 37787 37791 ERK5 +T1153 GO:0042571 37824 37834 Antibodies +T1154 PR:000000104 37843 37847 ERK1 +T1155 PR:000003106 37851 37854 p38 +T1156 PR:000010152 37855 37860 SAPK2 +T1157 MOP:0000780 37865 37872 cleaved +T1158 PR:000018762 37865 37882 cleaved caspase 3 +T1159 PR:000010127 37914 37918 MKK5 +T1160 GO:0042571 37919 37927 antibody +T1161 PR:000001904 37955 37959 CD31 +T1162 GO:0042571 37960 37968 antibody +T1163 PR:000010159 37990 37994 ERK5 +T1164 GO:0042571 37995 38003 antibody +T1165 PR:000010159 38054 38058 ERK5 +T1166 SO:0000040 38072 38085 genomic clone +T1167 PR:000010159 38090 38094 ERK5 +T1168 NCBITaxon:10088 38130 38135 mouse +T1169 SO:0000153 38136 38139 BAC +T1170 SO:0001026 38140 38147 genomic +T1171 NCBITaxon:10088 38164 38169 mouse +T1172 PR:000010159 38170 38174 ERK5 +T1173 SO:0000345 38175 38178 EST +T1174 SO:0000153 38195 38198 BAC +T1175 PR:000010159 38220 38224 ERK5 +T1176 SO:0001644 38313 38329 targeting vector +T1177 SO:0000147 38376 38381 exons +T1178 PR:000010159 38396 38400 ERK5 +T1179 SO:0000704 38401 38405 gene +T1180 SO:0000440 38411 38417 vector +T1181 NCBITaxon:1888 38482 38485 Sal +T1182 NCBITaxon:562 38490 38493 Eco +T1183 GO:0006266 38506 38513 ligated +T1184 SO:0000006 38519 38530 PCR product +T1185 SO:0000112 38551 38558 primers +T1186 CHEBI:7507 38613 38621 neomycin +T1187 SO:0005853 38633 38641 cassette +T1188 SO:0000112 38704 38711 primers +T1189 SO:0005853 38792 38800 cassette +T1190 SO:0001644 38814 38830 targeting vector +T1191 NCBITaxon:1823 38852 38855 Not +T1192 GO:0009294 38865 38877 transfection +T1193 NCBITaxon:10088 38883 38888 mouse +T1194 CL:0002322 38889 38897 ES cells +T1195 NCBITaxon:10088 38900 38905 Mouse +T1196 UBERON:0000922 38906 38915 embryonic +T1197 CL:0002322 38906 38926 embryonic stem cells +T1198 GO:0040007 38932 38937 grown +T1199 GO:0009294 38942 38953 transfected +T1200 UBERON:0000922 38992 39001 embryonic +T1201 CL:0000057 39002 39013 fibroblasts +T1202 NCBITaxon:10088 39027 39031 mice +T1203 CHEBI:42768 39078 39082 G418 +T1204 CHEBI:465284 39087 39098 ganciclovir +T1205 PR:000010159 39159 39163 ERK5 +T1206 SO:0001644 39164 39180 targeting vector +T1207 SO:0001644 39206 39222 targeting vector +T1208 SO:0000112 39254 39261 primers +T1209 PR:P43870 39367 39375 Hind III +T1210 NCBITaxon:2115 39380 39383 Mfe +T1211 CL:0002322 39391 39398 ES cell +T1212 UBERON:0000358 39448 39459 blastocysts +T1213 GO:0007566 39509 39518 implanted +T1214 NCBITaxon:10088 39541 39545 mice +T1215 NCBITaxon:10088 39612 39616 mice +T1216 UBERON:0010166 39678 39682 coat +T1217 PR:000010159 39762 39766 ERK5 +T1218 NCBITaxon:10088 39767 39771 mice +T1219 UBERON:0002415 39798 39802 tail +T1220 SO:0000112 39843 39850 primers +T1221 SO:0001023 40009 40016 alleles +T1222 UBERON:0000922 40032 40039 embryos +T1223 NCBITaxon:10088 40057 40061 mice +T1224 GO:0009566 40113 40126 fertilisation +T1225 GO:0007620 40156 40166 copulation +T1226 UBERON:0000922 40212 40219 Embryos +T1227 GO:0007565 40240 40248 pregnant +T1228 UBERON:0001040 40289 40298 yolk sacs +T1229 UBERON:0000922 40334 40341 embryos +T1230 GO:0097617 40371 40384 hybridisation +T1231 UBERON:0000922 40427 40434 Embryos +T1232 GO:0097617 40492 40505 hybridisation +T1233 PR:000010159 40563 40567 ERK5 +T1234 SO:0000028 40624 40626 bp +T1235 SO:0000205 40634 40640 3' utr +T1236 PR:000010127 40646 40650 MKK5 +T1237 SO:0000028 40706 40708 bp +T1238 SO:0000205 40716 40722 3' utr +T1239 SO:0000112 40756 40763 primers +T1240 SO:0000006 40879 40891 PCR products +T1241 SO:0000077 40929 40938 antisense +T1242 NCBITaxon:10760 40974 40976 T7 +T1243 SO:0001207 40974 40985 T7 promoter +T1244 UBERON:0000922 41032 41039 embryos +T1245 PR:000001904 41048 41052 CD31 +T1246 GO:0042571 41053 41061 antibody +T1247 GO:0008219 41200 41210 cell death +T1248 UBERON:0000922 41250 41257 Embryos +T1249 UBERON:0001987 41258 41266 placenta +T1250 CHEBI:16842 41281 41293 formaldehyde +T1251 CHEBI:16236 41314 41321 ethanol +T1252 CHEBI:35255 41334 41344 chloroform +T1253 CHEBI:51686 41430 41442 haematoxylin +T1254 UBERON:0002081 41459 41465 atrial +T1255 UBERON:0000060 41466 41470 wall +T1256 UBERON:0002081 41574 41588 atrial chamber +T1257 UBERON:0000060 41619 41623 wall +T1258 UBERON:0002081 41727 41741 atrial chamber +T1259 UBERON:0000922 41786 41792 embryo +T1260 PR:000010159 41816 41820 ERK5 +T1261 UBERON:0000922 41824 41831 embryos +T1262 UBERON:0000479 41864 41870 Tissue +T1263 CHEBI:9754 41896 41900 Tris +T1264 CHEBI:17883 41901 41904 HCl +T1265 CHEBI:35607 41940 41960 sodium orthovanadate +T1266 CHEBI:28741 41968 41983 sodium fluoride +T1267 CHEBI:71240 41990 42010 sodium pyrophosphate +T1268 CHEBI:17992 42019 42026 sucrose +T1269 CHEBI:9750 42037 42049 Triton X-100 +T1270 CHEBI:41218 42062 42079 2-mercaptoethanol +T1271 CHEBI:60004 42114 42122 cocktail +T1272 CHEBI:53325 42316 42330 nitrocellulose +T1273 GO:0042571 42350 42360 antibodies +T1274 PR:000000104 42369 42373 ERK1 +T1275 PR:000003106 42377 42380 p38 +T1276 PR:000010127 42385 42389 MKK5 +T1277 PR:000010159 42438 42442 ERK5 +T1278 GO:0042571 42443 42451 antibody +T1279 GO:0042571 42485 42495 antibodies +T1280 MOP:0000779 42496 42506 conjugated +T1281 NCBITaxon:3704 42510 42521 horseradish +T1282 NCBITaxon:10088 42778 42783 mouse +T1283 UBERON:0000922 42828 42835 embryos +T1284 CL:0002322 42861 42868 ES cell +T1285 UBERON:0000358 42881 42891 blastocyst +T1286 PR:000002312 42938 42947 caspase 3 +T1287 PR:000016934 43205 43209 4311 diff --git a/src/ontogpt/evaluation/craft/database/all/14675480.txt b/src/ontogpt/evaluation/craft/database/all/14675480.txt new file mode 100644 index 000000000..1c6f90758 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/14675480.txt @@ -0,0 +1,165 @@ +Knockout of ERK5 causes multiple defects in placental and embryonic development + +Abstract + +Backgroud + +ERK5 is a member of the mitogen activated protein kinase family activated by certain mitogenic or stressful stimuli in cells, but whose physiological role is largely unclear. + +Results + +To help determine the function of ERK5 we have used gene targeting to inactivate this gene in mice. Here we report that ERK5 knockout mice die at approximately E10.5. In situ hybridisation for ERK5, and its upstream activator MKK5, showed strong expression in the head and trunk of the embryo at this stage of development. Between E9.5 and E10.5, multiple developmental problems are seen in the ERK5-/- embryos, including an increase in apoptosis in the cephalic mesenchyme tissue, abnormalities in the hind gut, as well as problems in vascular remodelling, cardiac development and placental defects. + +Conclusion + +Erk5 is essential for early embryonic development, and is required for normal development of the vascular system and cell survival. + +Background + +Mitogen activated protein kinase (MAPK) cascades play important roles in many cellular processes including cell proliferation, differentiation, survival and apoptosis. They are also important for many physiological functions in several systems, including in developmental, immune and neuronal systems. At least 12 isoforms of MAPKs exist in mammalian cells, and these can be divided into 4 main groups, the 'classical' MAPKs (ERK1 and ERK2), JNKs (also referred to as SAPK1), p38s (also referred to as SAPK2, SAPK3 and SAPK4) and atypical MAPKs such as ERK3, ERK5 and ERK8. With the exception of ERK3, MAPKs are activated by dual phosphorylation on a Thr-Xaa-Tyr motif by a dual specificity MAPK kinase (MKK). MKKs are in turn activated by a MAPK kinase kinase (MKKK), which are activated in response to appropriate extracellular signals. + +ERK5 is an atypical MAPK that can be activated in vivo by a variety of stimuli, including some mitogens such as EGF, and some cellular stress such as oxidative and osmotic shock [1-3]. These stimuli activate a cascade in which the MAPK kinase kinases MEKK3 or MEKK2 activate MKK5, which in turn activates ERK5 [4,5]. Interest in the ERK5 pathway has been fuelled by reports that the activation of ERK5 by MKK5 can be blocked in vivo by the kinase inhibitors PD184352, PD 98059 and U0126. These inhibitors were developed as inhibitors of the classical MAPK cascade, and have been used extensively to study this cascade in vivo. The discovery that they can also block ERK5 activation, although at higher concentrations than are required to block the activation of ERK1 and ERK2, raised the possibility that ERK5 and ERK1/ERK2 may have some overlapping functions in vivo [6,7]. + +The physiological roles of ERK5 are still largely unclear. Overexpression of a constitutively active MKK5 in mice results in cardiac hypertrophy and death of the mice by 8 weeks of age [8]. This is suggestive of a role of ERK5 in the heart, possibly related to cardiac development. ERK5 has also been implicated in the development of smooth muscle, as ERK5 antisense oligonucleotides [9] or dominant negative ERK5 constructs [10] have been reported to block the differentiation of smooth muscle cells in cell culture models. At present little is known about the substrates for ERK5 in vivo, however it has been suggested to phosphorylate connexin 43 [11] and the transcription factor MEF2C [12-14]. Mouse knockouts of MEF2C are embryonic lethal, and MEF2C-/- embryos die due to a failure of the developing heart to undergo normal looping at E8.5-9 [15]. Knockout MEKK3 also results in embryonic lethality at E11, MEKK3-/- embryos show problems with myocardium formation, angiogenesis and placental formation [16]. While this could be consistent with a role for ERK5 in linking MEKK3 signalling to MEF2C during cardiac development, it should be noted that MEKK3 can activate other MAPK isoforms, particularly p38α (also referred to as SAPK2A) [17-20]. Knockout of p38α has been reported by several groups, and p38α-/- embryos have also been reported to show problems in cardiac development, angiogenesis and placental formation at E10-11 [21-23]. + +In order to further examine the role of ERK5 we carried out expression and gene targeting studies in mice. ERK5 knockout was found to be lethal during embryogenesis at E10.5 to E11, and here we report a detailed analysis of these embryos. While this work was in progress, both Regan et al [25] and Sohn et [26] also reported ERK5 knockouts, and the effects of these different ERK5 knockouts are considered in the discussion. + +Results + +Generation of ERK5 knockout mice + +Sequencing of the mouse ERK5 gene showed that it comprised of 7 exons spanning 5.4 kb of genomic sequence. Of these, exons 2 to 7 encoded the sequence of ERK5, while the 5' untranslated region was located in exons 1 and 2, and the 3' untranslated region in exon 7. Based on this sequence a targeting vector was designed to delete exons 4 and 5 of ERK5 in ES cells (Fig 1). Correct incorporation of this vector was confirmed by Southern screening of ES cells (Fig 1A and 1B). Germline transmission was obtained from two independent targeted ERK5 ES clones, and the ERK5+/- mice were of similar size and morphology to wild type littermates. Breeding of ERK5+/- mice gave the expected numbers of wild type and ERK5+/- mice, however no ERK5-/- mice were obtained, indicating that the ERK5 knockout is lethal during embryogenesis. To determine the point of lethality, embryos were genotyped by a PCR based method (Fig 1A and 1C) from timed matings. The expected Mendelian numbers of homozygous knockout mice were found at E9.5 and 10.5 (table 1) and at this point knockout embryos were still alive, as judged by a beating heart. In contrast, at E11.5 all homozygous ERK5 knockout embryos found had died and were undergoing reabsorption. Similar results were obtained from crosses of the ERK5 mutation onto either Balb/C or C57/Bl6 backgrounds, and from two independent ES cell clones. The deletion made in the ERK5 gene removes the sequence encoding for amino acids 133 to 712, and introduced a neomycin resistance cassette, including a polyadenylation sequence into the ERK5 gene. While exons 1 to 3 remain in the targeted gene, insertion of the neomycin cassette would be expected to interfere with normal transcription and splicing after exon 3. Should exon 3 be able to splice onto exon 6 in the targeted gene, this would result in a frame shift mutation. To confirm that the knockout blocked the production of ERK5 protein, extracts from E9.5 embryos were analysed by immunoblotting using a polyclonal antibody raised against the whole ERK5 protein. ERK5 was detected in wild type embryos and in ERK5+/- embryos, however the levels of ERK5 protein were reduced in ERK5+/- embryos compared to wild type embryos. As expected no protein was seen for ERK5 in the ERK5-/- embryos. No evidence for the production of truncated forms of ERK5 in the ERK5-/- embryos could be seen in the immunoblots (data not shown). Expression of MKK5 and other MAPK kinases (ERK1, ERK2 and p38) were unaffected by the knockout of ERK5 (Fig 1d). + +Figure 1 + +Generation of ERK5 knockout mice. A) ERK5 knockout mice were made using a targeting vector to delete exons 4 and 5 of the murine ERK5 gene through the addition of a neomycin selection cassette. A thymidine kinase cassette acts as a negative selection marker during ES cell selection. B) ES cell DNA was digested with both Hind III and Mfe I, and a Southern blot performed using a probe 3' to the targeting vector. The position of the wild type 9.5 kb fragment and targeted 3.3 kb fragment are indicated. C) DNA was isolated from E9.5 embryos and digested with Hind III and Mfe I. Southern blots were then probed with the 3' probe as described in (B)D) Soluble protein from homogenates of E9.5 embryos was run on 4–14% acrylamide gels. Immunoblotting was then carried out using antibodies which recognised ERK5, MKK5, ERK1/2 or p38. + +Table 1 + +Ratios of ERK5 adults and embryos + +* Of the E10.5 knockout embryos, 12 were class I and 16 were class II ** -/- embryos found at E11.5 were dead + +At E9.5 the appearance of homozygous ERK5 knockout embryos was similar to that of the wild type. However, between E9.5 and E10.25 some differences between the knockout embryos and wild type embryos started to become apparent (Fig 2). By E10.25 knockout embryos were clearly growth retarded compared to littermate controls, and clear morphological differences could be seen. All ERK5 knockout embryos had problems in placental and blood vessel development, and in addition to this, two distinct morphologies could be seen in knockout embryos by E10.25. The first morphology, referred to as 'class I', was characterised by severe retardation of growth, especially in the head and lower trunk region. In contrast, 'class II' embryos were less growth retarded than class I embryos, however development of the head and lower trunk was abnormal. Development of the first and second branchial arches was reduced, and the embryos developed an abnormal head shape. In addition, development of the cephalic mesenchyme appeared abnormal (Fig 2C). Changes seen in the ERK5 knockout are discussed in more detail below. + +Figure 2 + +Phenotypes of ERK5 -/- mutant embryos. Embryos were isolated from timed matings of ERK5+/- mice and genotyped by PCR analysis of the isolated yolk sac. At E9.5 (A) little difference could be observed between wild type and ERK5-/- littermates. At E9.75 (B) differences could be seen between the WT and ERK5-/- embryos in the head regions, particularly in the cephalic mesenchyme of class II embryos (red arrowhead). At E10.25 (C) ERK5-/- embryos were growth retarded compared to wild type embryos. ERK5-/- embryos showed an abnormal head shape, compared to wild type embryos (green arrow) and in class II ERK5-/- embryos also showed severe abnormalities in the cephalic mesenchyme and 1st and 2nd branchial arches (yellow arrows). Development of the hind limb buds (star) and lower trunk was also retarded in the ERK5-/- embryos. + +Expression of ERK5 and MKK5 during embryogenesis + +Analysis of the expression of ERK5, and its upstream kinase MKK5, by whole mount in situ hybridisation using antisense RNA probes showed that the expression of these kinases was dynamically regulated during embryonic development (Fig 3A,3B,3C,3D,3E). At E8.5 ERK5 expression was low and occurred mainly in the cephalic neural fold and primitive gut. At E9.5 ERK5 expression was seen in the first and second branchial arch, cephalic region, somites and lateral ridge along the body wall. By E10.5 and 11.5 ERK5 expression was also seen in the developing limb buds. As would be expected for the upstream activator of ERK5, the expression pattern of MKK5 was found to be similar to that of ERK5 from E8.5 to E12.5. Interestingly, ERK5 was found not to be highly expressed in the developing heart as judged from whole mount immunosatining. To examine this further, sections of ERK5 whole mount in situ hybridisations were taken. At E 9.75 and E10.5 strong ERK5 expression was seen in the branchial arch, cephalic mesenchyme and neuropethelial regions (Fig 4A to 4C), as well as in the limbs, hind gut, septum transversum, dorsal root ganglion, somites and tail. Only weak expression of ERK5 was seen in the heart in sections at E9.5 and E9.75, however a slightly stronger expression of ERK5 could be seen in the atrial chamber of the heart at E10.5 (Fig 4). Strong expression of ERK5 was however apparent in the sinus venous below the heart. Expression of ERK5 was also examined at E10.5 in the placenta by whole mount in situ hybridisation and sectioning. ERK5 expression was found to be highest in the chorionic plate and labyrinthine layers. As protein expression does not always exactly mirror mRNA expression, E9.5 and E10.5 embryos were also dissected and ERK5 expression examined by western blotting (Fig 5A). This showed that ERK5 expression was high in the head and lower trunk of the embryo, intermediate in the heart region and low in the placenta. In adult mice, high levels of ERK5 and MKK5 were found in brain, thymus and spleen, with lower levels present in lung, stomach, adrenal gland, adipose tissue, pancreas and heart (Fig 5B). + +Figure 3 + +Expression of ERK5 and MKK5 during embryonic development. Whole mount in situ hybridisation was carried out on wild-type embryos as described in the methods using antisense RNA probes against ERK5 or MKK5 or with no RNA probe. Expression of ERK5 and MKK5 was analysed at E8.5 was seen in the cephalic neural fold and primitive gut. At E9.5 expression was also seen in the branchial arch, cephalic region and somites and lateral ridge of the body wall. From E10.5, E11.5 to E12.5 expression of ERK5 and MKK5 increases with high expression seen in the branchial arch, head and limb buds. + +Figure 4 + +Analysis of ERK5 expression in embryo sections. Whole mount in situ hybridisation was carried out on wild-type embryos (A-C) or placentas (D) as described in the methods using antisense RNA probes against ERK5 at E9.5, E9.75 and E10.5. After staining embryos were sectioned on a vibrotone. Strong ERK5 expression was seen in the cephalic mesenchyme (star) branchial arch and neuroepithelium. Weak expression was seen in the heart at E9.5, however this increases by E10.25 (diamond). Strong ERK5 expression was seen in the sinus venous below the heart (arrow). In the placenta (D) strongest expression was seen in the chorionic plate and labyrinthine layers. + +Figure 5 + +Immunoblotting of ERK5 and MKK5. Wild type embryos (E9.5 and 10.5) were dissected and head, heart, gut and placenta were isolated. The placenta was then subdivided into upper (mainly embryonic) and lower (mainly maternal) regions. Whole wild type and ERK5-/- embryos were also isolated at E9.5. Tissues were also isolated from adult wild type mice. Samples were homogenised, insoluble material removed by centrifugation, and the concentration of soluble protein in the extract determined by a Bradford assay. Soluble protein (30 μg) were run on 4–14% acrylamide gels. Immunoblotting was then carried out using antibodies which recognise ERK5 or MKK5 for both embryonic (A) and adult (B) samples. Levels of ERK1/2, p38 and actin were also determined in the adult tissue samples (B). + +ERK5 is required for normal angiogenesis and placental development + +One of the most apparent problems in the ERK5 knockout embryos was a defect in the formation of blood vessels in the yolk sac. At E9.5 blood islands could be seen in the membranes of both WT and knockout embryos. However, by E10.25 the ERK5 knockout embryos failed to develop the highly branched network of blood vessels seen in the WT or heterozygous embryos (Fig 6). We therefore also examined blood vessel formation in the knockout embryos. At E9.75 CD31 staining of endothelial cells showed little difference between wild type and ERK5-/- embryos, and a clearly defined network of large blood vessels could be seen in both genotypes. In the wild type embryos these blood vessels continued to develop, giving rise to large blood vessels which branched down into networks of smaller vessels. This network of vesicles was especially apparent in the head regions of the embryo. In contrast much less branching of the blood vessels was apparent in the head region of ERK5-/- embryos. It should however be noted that the formation of other head structures, as well as blood vessels, was also retarded by E10.25 in the ERK5-/- embryos (Fig 7). + +Figure 6 + +Morphology of wild type and ERK5 -/- mutant yolk sacs. Embryos were isolated from timed matings of ERK5+/- mice and photographed. Embryos were then genotyped by PCR analysis of the isolated yolk sac. At E9.75 (A) wild type and ERK5-/- mutant yolk contain blood islands (arrow). By E10.25 (B), the blood vessels found in wild type yolk sacs formed distinct large vessels which branched down into smaller vessels (arrow). In contrast, the surfaces of the ERK5-/- yolk sacs became pale, and did not show the branched blood vessels. At E11.5 (C), ERK5-/- yolk sacs appeared intermittent with diffuse patches of red blood cells (arrow). The ERK5-/- embryos showed were pale and apparently devoid of blood circulation without a beating heart. + +Figure 7 + +CD31 whole-mount immunohistochemistry of embryos at E9.75 and E10.25. Embryos were isolated from timed mating and stained using an CD31 antibody as described in the Methods. At E9.75 (A) networks of large blood vessels were seen in the head regions of both wild type and ERK5-/- embryos (red arrow), and intersomitic vessels (blue arrow) were also apparent in both genotypes. By E10.25 however the blood vessels in the head region of wild type embryos had started to undergo angiogenesis to give rise to branched networks of smaller vessels. This was not seen in the ERK5-/- embryos (compare red arrows in B). Similarly more branching was seen in the intersomitic vessels in the wild type than ERK5-/- embryos at E10.25 (blue arrows). Results are representative of three independent experiments. + +As knockouts of proteins upstream of ERK5 have been reported to cause problems in cardiac development, the development of the heart was also examined. ERK5-/- embryos underwent normal looping of the heart and were able to establish the basic heart pattern. At E9.75 however, the myocardium wall of ERK5-/- was thinner than in WT embryos, and some bleeding was seen in a proportion of ERK5-/- embryos (Fig 8). + +Figure 8 + +Histological sections of the heart at E9.75. E9.75 wild type and ERK5-/- embryos were isolated from timed matings and TS paraffin sections were taken and stained with haematoxylin and eosin as described in the methods. Normal patterning of the heart was observed in the ERK5-/- embryos, with both atrial (At) and ventrical (V) chambers. The thickness of the atrial wall (arrow) in ERK5-/- embryos was thinner that in wild type hearts (A). The average thickness of the atrium wall was quantified from 4 wild type and 4 ERK5-/- embryos (B). ERK5-/- embryos had a significant decrease in atrial wall thickness (P < 0.01) + +The cardiac defects and changes in vascular remodelling seen in the ERK5-/- embryos suggested that ERK5 may also play a role in placental development, so we therefore studied the morphology of ERK5-/- placentas at E9.75 and E10.25. Haematoxylin and eosin staining of paraffin sections from E9.75 placentas (Fig 9A and 9B) showed little difference between wild type and ERK5-/- embryos. Chorioallantoic fusion was able to occur in the absence of ERK5, and the placenta of ERK5-/- mice formed chorionic plate, labyrinth and spongiotophoblast layers. At this stage, the laryrinth of the ERK5-/- placenta resembled that of the wild type placentas, with both embryonic and maternal blood vessels present. However at E10.25 (Fig 9C and 9D) the labyrinth layer in ERK5-/- placentas was thinner than in wild type and there was less intermixing between embryonic and maternal blood vessels in the labyrinthine region. Development of trophoblast giant cells did not appear to be affected by ERK5 knockout, and staining with the spongiotrophoblast layer maker 4311 [24] suggested that this layer developed normally (Fig 10A). Staining of placental sections at E10.25 with an antibody against the cleaved form of caspase 3 showed that at E10.25 more apoptosis was occuring in the labyrinth of ERK5-/- embryos compared to wild type embryos (Fig 10B). Apoptosis was seen in endothelial cells, diploid trophoblast cells and some embryonic blood cells. No cleaved caspase 3 staining was observed in E9.75 placentas from either wild type or ERK5-/- placentas. + +Figure 9 + +Histological sections of placenta at E9.75 and E10.25. Placentas were isolated from E9.75 and E10.25 wild type and ERK5-/- mice. Transverse paraffin sections were taken of the placentas and stained with haematoxylin and eosin as described. At E9.75, low (A) and high magnification (B) pictures of the TS sections are showed little difference between wild type and ERK5-/- embryos. At E9.75 both maternal (white arrowhead) and embryonic (green arrow) blood vessels, could be seen in both wild type and ERK5-/- placentas. At E10.25 low (C) and high magnification (D) showed that chorionic plate (CP), labyrinth (L) and spongiotrophoblast (S) layers are were present in both wild type and ERK5-/- embryos. At E10.25 intermixing of maternal and embryonic blood vessels (arrows) was seen, however in the ERK5-/- placentas many fewer maternal blood vessels were apparent in the labyrinthine layers. Scale bars are 0.1 mm and results are representative of three independent experiments. + +Figure 10 + +4311 in situ and caspase 3 staining of E10.25 placentas. A) Wax sections of E10.25 wild type and ERK5-/- placentas were analysed by in situ hybridisation with an antisense RNA probe against 4311. The spongiotrophoblast layer is indicated by an arrow. B)Wild type and ERK5-/- placentas were isolated from E10.25 embryos, paraffin sections taken and then stained with an antibody which recognised cleaved caspase 3. Higher numbers of cleaved caspase 3 postitive cells were seen in ERK5-/- embryos compared to wild type controls. In the ERK5-/- placentas, apoptosis was seen in endothelial cells (red arrow), trophoblast cells (green arrow) and some embryonic blood cells (yellow arrow) within the labryinth. No apoptosis of giant cells was seen. + +ERK5 is required for normal development of the head and lower trunk regions + +By E10.25 all ERK5-/- embryos, and particularly class II embryos, showed problems with the development of the head and lower trunk regions of the embryo. Superficially, between E9.5 and E10, these differences were much less apparent, however more detailed analysis of serial sections of E9.75 embryos revealed problems in these regions in ERK5-/- embryos (Figs 9,10). The timing of this is significant, because at this developmental stage relatively little difference was seen between the vasculature and placentas of ERK5-/- and wild type embryos (Figs 6,7,8,9,10). In the head region E9.75 sections, development of the lumen was retarded in ERK5-/- embryos compared to littermates. The cephalic mesenchyme tissue was less dense with larger spaces in between the cells in ERK5-/- embryos than in wild type embryos. There was also less contact between the cephalic mesenchyme and neuroepithelial tissue in the ERK5-/- embryos (Fig 11A and 11B). This phenotype was seen in all ERK5-/- embryos examined by sectioning at E9.75 when compared to wild type littermates (n = 5 for LS sections and n = 5 for TS sections), and was seen in all of the serial sections for each ERK5-/- embryo. This defect may in part explain the abnormal head shape of the embryos (compare whole embryo pictures in Fig 2B). Problems with the cephalic mesenchyme tissue were even more apparent in class II embryos at E10.25 (Fig 2C), and an apparent absence of cephalic mesenchyme could be seen clearly in the whole embryo. To confirm this, E10.25 ERK5-/- embryos were also sectioned and the cephalic mesenchyme compared to that of wild type littermates (Fig 11C and compare to Fig 2C). In wild type embryos the cephalic mesenchyme was present, however in ERK5-/- embryos the cephalic mesenchyme was almost completely absent. In addition, the thickness of the neuroepithelial layer surrounding the area were the cephalic mesenchyme should have been was much thinner in the ERK5-/- embryos when compared to wild type controls. Analysis of TS sections of E9.75 ERK5-/- embryos showed that cephalic mesenchyme did contain major blood vessels, similar to those in wild type embryos (Fig 11D and 11E). The blood vessels in the ERK5-/- embryos were however frequently ruptured giving rise to bleeding into the mesenchyme tissue. The sites of bleeding occurred where the surrounding mesenchyme tissue was absent, suggesting that the reason for the rupturing of the vessels may have been due to the lack of support provided to the blood vessels. These results suggested that while the cephalic mesenchyme was able to form in early ERK5-/- embryos (pre E9.75), it was unable to proliferate and survive through the developmental stages from E9.75 to E10.25. We therefore used whole mount TUNEL analysis of these embryos to examine levels of apoptosis. While little apoptosis was seen in the head region of wild type embryos in E9.75 embryos, high levels of apoptosis were observed in the head of ERK5-/- embryos (Fig 12). + +Figure 11 + +Analysis of cephalic mesenchyme. Wild type and ERK5-/- embryos were isolated from timed matings and LS or TS paraffin sections were taken and stained with haematoxylin and eosin as described in the methods. Analysis of LS sections at low (A) and high (B) magnification showed that there was less contact between the neuroepithelium and cephalic mesenchyme (black arrows) in ERK5-/- embryos, and that the cephalic mesenchyme was less dense with larger spaces between the cells in the ERK5-/- embryos (green arrows). Sections shown are representative of 5 wild type and 5 ERK5-/- embryos. LS sections were prepared from E10.25 embryos and stained with haematoxylin and eosin (C). The cephalic mesenchyme was almost completely absent in ERK5-/- embryos and the neuroepithelium was thinner. In ERK5-/- embryos at E9.75 the TS sections (D and E) through the cephalic mesenchyme again showed less mesenchymal tissue in the ERK5-/- embryos, however major blood vessels (arrows) were seen in both wild type and ERK5-/- embryos. In contrast to wild type embryos, where little bleeding was seen in the cephalic mesenchyme, in ERK5-/- embryos bleeding into the cephalic mesenchyme was frequently seen, especially where the mesenchymal tissue was absent (arrows in ERK5-/- embryos in D and E). + +Figure 12 + +Analysis of apoptosis in the head of ERK5-/- embryos. The level of apoptosis in E9.75 embryos were analyses by whole mount TUNEL staining. These showed that there was greatly increased apoptosis in the ERK5-/- embryos compared to wild type littermate controls. (A) Fluorescent images of the head region of whole mount TUNEL stained embryos. (B) Light microscope pictures of the same regions at the same magnification. The cephalic mesenchyme is indicated by an arrowhead. Results are representative of 3 experiments. + +In the lower trunk, the development of the region below the heart appeared abnormal in ERK5-/- embryos. In several embryos, the development of the septum transverum region was retarded in most ERK5-/- embryos (data not shown). Detailed analysis of transverse sections showed that while development of the foregut appeared normal, development of the mid and hind gut was not. In ERK5-/- embryos at E9.75 there appeared sites of overproliferation of cells in some areas of the hind to mid gut wall (Fig 13). + +Figure 13 + +Analysis of hind gut at E9.75. E9.75 wild type and ERK5-/- embryos were isolated from timed matings and TS paraffin sections were taken and stained with haematoxylin and eosin as described in the methods (A). TS sections through the guts of ERK5-/- mice showed several areas were there appeared to be hyperproliferation of the gut endothelium (arrow). This was not observed in wild type embryos. Similar results were seen in 3 wild type and 3 ERK5-/- embryos. + +Discussion + +In this report, we show that knockout of ERK5 results in embryonic lethality at around E10.25 and show that ERK5-/- embryos have problems with placental development, changes in angiogenesis and problems with the development of the head, (especially the cephalic mesenchyme and neuroepithelium), and lower trunk of the embryo. While this work was in progress, two other groups reported ERK5 knockouts. Regan et al reported that the ERK5 knockout was lethal between E9.5 to E11.5 [25], while Sohn et al reported lethality between E10.5 and E11.5 [26]. Similar effects on placental development and angiogenesis were found in both reports, and this phenotype is consistent with the effects described here. While both Sohn et al and Regan et al reported that ERK5-/- embryos were growth retarded by E10, neither study reported characterisation of the head and trunk regions of these embryos. It is therefore not possible to say if the defects we report in the cephalic mesenchyme and gut were present in these knockouts. Differences in the targeting strageties between both Sohn et al and Regan et al, and that used here may explain why some differences were seen in the phenotypes observed, as it is not possible to rule out the possibility that truncated fragments of the ERK5 protein were expressed in any one of those knockouts, which may give rise to a dominant negative effect. Interestingly however the most severe phenotype reported was that of Regan et al, and in this study the targeting used here deleted the smallest region of the ERK5 gene of all the knockouts. It should also be stressed that other differences, such as the strain and source of mice and ES cells used, may also explain differences between the phenotypes of the three knockouts. + +Interestingly we found two distinct morphologies of ERK5-/- embryos at E10.25, however the reason for this was not clear. Class I embryos were characterised by severe growth retardation compared to wild type embryos, while class II embryos were larger but had severe abnormalities in the development of the head and lower trunk. One possible explanation may relate to the degree of severity of the placental phenotype. Placental defects are a common cause of lethality at this developmental stage in knockout mice [27,28]. If the severity of these placental defects varied between individual ERK5-/- embryos due to other genetic or environmental factors, then as a result, the problems in placental development may be sufficient to kill some embryos (class I) before E10.25, but other embryos (class II) survive longer, allowing other phenotypes to become more pronounced. A similar effect has also been observed in a knockout of p38α, in which some embryos died at E11.5, presumably due to placental defects, while some embryos survived past this stage [22]. We found that the basic structures of the placenta, including the chorionic plate, labyrinthine trophoblast, labyrinth and spongiotrophoblast layers were able to form in the absence of ERK5. Histological sections did not show significant differences between ERK5-/- and wild type placenta at E9.75 (Fig 9). By E10.25 however the thickness of the labyrinth was reduced in ERK5-/- placentas, and there was less intermixing of the embryonic and maternal blood vessels in the placental labyrinth. This correlated with increased apoptosis in this region in ERK5-/- placentas (Fig 10). This is suggestive of a role for ERK5 in development of the labyrinth and chorioallantoic branching. As the labyrinth is the major site of exchange between the embryonic and maternal blood, the problem seen in the ERK5-/- placentas is likely to be sufficient to cause embryonic lethality. ERK5 is not the only MAP kinase signalling protein whose knockout affects labyrinth development. Knockout of MEKK3, an upstream activator of ERK5 and p38 [23], resulted in embryonic lethality due in part to a failure of the labyrinth development. Also knockouts of p38 and MEK1 [29] result in problems with the labyrinth, as do knockouts of several receptors known to activate MAPK signalling including LifR [30], EgfR [31], PdgfR [32,33], Met [34], and the GDP/GTP exchange factor Sos1 [35]. + +Consistent with the findings of Regan et al and Sohn et al, we also found that ERK5 knockout resulted in problems in angiogenesis in the embryo. Analysis of ERK5 expression by in situ hybridisation however showed that expression of ERK5 was not restricted to the developing blood vessels, but was instead expressed more widely in the embryos. Both Sohn et al and Regan et al report that expression of various signalling molecules important in angiogenesis were normal in the ERK5 knockout. The reason for the reduction in angiogenesis in the knockout mice is unclear, but may be related to general growth retardation seen in ERK5-/- embryos compared to wild type litermates at E10.25. + +Knockout of ERK5 also affected cardiac development. Using both whole mount in situ hybridisation and immunoblotting of dissected embryos, we show that expression of ERK5, and its upstream activator MKK5, is expressed in the heart at E9.5 to E10.25, although their expression level was low compared to other regions of the embryo (Figs 2,3,4,5). Consistent with this, Sohn et al also reported that ERK5 expression was highest in the heart and trunk of the embryo at E9 to E9.5. Using only RNA in situ hybridisation Regan at al however reported the opposite, with high levels of ERK5 expression localised to the developing heart and little expression in the rest of the embryo. The reasons for this difference between the report of Regan et al, and both our findings and those of Sohn et al is unclear. We found development of the embryonic heart was retarded compared to wild type embryos. Similar to the report of Sohn et al, we observe that basic patterning of the heart can occur in the absence of ERK5. Once formed however, the heart does not develop past it's basic patterning. In particular the thickness of the atrial wall at E9.75 was reduced in ERK5 knockout embryos (Fig 8). Interestingly, the knockout of ERK5 had much less severe effects on heart development compared to the knockout of its potential substrate MEF2C, in which embryos die at E8.5-9 due to failure to undergo normal looping. This suggests that either MEF2C has functions which are independent of its phosphorylation by ERK5 in vivo at this developmental stage, or that other kinases such as p38 can also phosphorylate the same sites on MEF2C as ERK5 in vivo. In this respect it is interesting to note that knockout of p38 resulted in similar problems in cardiac development to the ERK5 knockout. In contrast to this report, and that of Sohn et al, Regan et al reported that the heart did not undergo normal looping at E9.5. The reason for this discrepancy is not clear, but may be due to differences in targeting or the genetic strains of mice used. The knockout of ERK5 has been previously observed to have a similar phenotype to knockouts of receptor tyrosine kinase Tie-2 [46] and its ligand Ang-1 [47], which may suggest that ERK5 could function downstream of these receptors in the heart. There is however no direct evidence to demonstrate this link and further work would be needed to establish if this were true. In isolated cell lines ERK5 has been reported to be activated by the neuregulin receptors erbB2 and erbB3 [48], raising the possibility that erk5 may mediate some to the effects of neuregulins in the heart. A further possible reason for the cardiac phenotype is that ERK5 has been reported to inhibit the activity of the VEGF promoter [26], so that increased VEGF levels in the ERK5-/- embryos may affect cardiac development. A third possibility is that the cardiac defects observed in ERK5-/- embryos may not be directly due to the lack of ERK5 in the heart, and that these phenotypes may be caused wholly or in part by stress induced by the placental defects in the knockouts. It has been shown in other knockout models that cardiac phenotypes can be secondary to other problems in the embryo (for examples see [21,36]). Further work, including the use of placental rescue or cardiac specific ERK5 knockouts, will be required to fully resolve these issues. + +We also observed defects in the development of the cephalic mesenchyme and gut in the ERK5-/- embryos. In ERK5-/- embryos problems were seen in the cephalic mesenchyme from E9.75 onwards. At E9.75 the cephalic mesenchyme appeared less dense with larger spaces between the cells and less contact between the cephalic mesenchyme and the neuroepithelium. However as the embryos developed, this gradually worsened and by E10.25 the cephalic mesenchyme was essentially absent (Fig 11). Several factors suggest that the defects seen in the cephalic mesenchyme are primary phenotypes directly caused by the loss of ERK5 protein in this region. First, in stiu hybridisation showed that ERK5 was expressed in the cephalic mesenchyme from E9.75 (Fig 5). Secondly, these problems could be seen in E9.75 ERK5-/- embryos, while at this stage blood vessel and placental development appeared relatively normal in the knockouts (Fig 6,7,8,9,10), suggesting that the cephalic mesenchyme and gut defects were not secondary to a lack of angiogenesis. Consistent with this, blood vessels were present in the cephalic mesenchyme of E9.75 ERK5-/- embryos, suggesting that the problems with this tissue were not due to a lack of blood supply. The defect in the cephalic mesenchyme appeared to be due to increased apoptosis causing the tissue to be lost, rather than a problem with its initial development. Consistent with the normal initial development of this region, expression patterns of sonic hedgehog and Six3 (L. Yan, unpublished data) at E9.5 were unaffected by the knockout of ERK5. The increase in apoptosis in the ERK5-/- embryos suggests that ERK5 may be involved in regulating cell survival or poliferation. Consistent with this, overexpression of ERK5, or its upstream activator MKK5, has been shown to promote proliferation in some cell types in response to some mitogenic stimuli [1,37-39]. + +In summary these results are consistent with a role for ERK5 in angiogenesis and placental development, and show new functions for ERK5 in the survival of the cephalic mesenchyme and regulation of survival and apoptosis. Further work however will be required in order to determine the molecular details of these ERK5 functions. + +Methods + +Materials + +Antibodies against ERK1/2, p38/SAPK2 and cleaved caspase 3 were from Cell Signalling. The MKK5 antibody was from Stressgen and the CD31 antibody from Pharmhigen. The ERK5 antibody has been described previously [7]. + +Generation of ERK5 knockouts + +A genomic clone for ERK5 was obtained by screening a 129SvJ mouse BAC genomic library using a mouse ERK5 EST. Regions of the BAC corresponding to the ERK5 were subcloned by either restriction digestion or random fragmentation and sequenced. A targeting vector was designed based on this sequence to delete exons 4 to 5 of the ERK5 gene. The vector consisted of a first arm of homology (generated by cloning of a Sal I / Eco RI fragment ligated to a PCR product generated using the primers GAATTCAGATCTGTGTAAGG and AAGCTTCTGAAAATGGGAAG) then a neomycin resistance cassette, followed by a second arm of homology (generated by using the primers CATATGAGAAGAGGAAAGCCTGGGA and GCGGCCGCAGCAGGGATCAATATGT) and a thymidine kinase cassette (Fig 1). The targeting vector was linearised using Not I before transfection into mouse ES cells. + +Mouse embryonic stem cells were grown and transfected as described previously ([40]), using embryonic fibroblasts from MTK-neo mice as a feeder layer. Colonies resistant to both G418 and ganciclovir were expanded and screened for correct incorporation of the ERK5 targeting vector. A probe external to the targeting vector was generated by PCR using the primers CAAGTAGGGGACCAAGTCAAC and GGCCCAATGGAAAGGCTTCTAT. This probe was used to screen DNA double digested with Hind III and Mfe I from ES cell colonies. Positive cell lines were injected into blastocysts from a C57Bl/6 × BALB/c cross, which were then reimplanted into recipient female mice [41]. Chimeric male offspring were then bred to BALB/c or C57Bl/6 mice as indicated and transmission identified by a combination of coat colour and genotyping by Southern and PCR analysis. + +Routine gentoyping of the ERK5 mice was carried out by PCR on tail biopsies. PCR was carried out using the primers AACTAACCAACCCACCTTCCAAGAC and CACTAGTACTCCTACTGGCCCCGTA to identify wild type and AACTAACCAACCCACCTTCCAAGAC and ACCACCAAGCGAAACATCGCATCG to identify targeted alleles. + +Isolation of embryos + +Male and female mice of known genotype were placed together and time of fertilisation determined by observation of copulation plugs, and noon of that day defined as E0.5. Embryos were dissected from pregnant females at the times indicated, and the yolk sacs separated and used to genotype the embryos by PCR. + +Whole mount in situ hybridisation, immunohistochemistry and TUNEL staining + +Embryos were harvested and fixed in 4% paraformaldehyde. In situ hybridisation was carried out as described previously [42]. Probes for ERK5 (corresponding to the last 207 amino acid and first 165 bp of the 3' utr) and MKK5 (corresponding to the last 71 amino acid and first 295 bp of the 3' utr) were generated by PCR using the primers ACTAGTACTCCTACTGGC and GCTCAGGTGGCTGCTTAAG or ACTAGTAGGATTCGCCGGTCCTTC and ATCAGTGCTGCTGATAGGGCCTGAC respectively. PCR products were cloned into pBluescript to give antisense sequence when transcribed from the T7 promoter. + +Whole mount immunohistochemical analysis of embryos using a CD31 antibody as described [43]. Whole mount terminal deoxynucleotidyl transferase-mediated UTP end labelling (TUNEL) was carried out using the in situ cell death detection kit from Roche. + +Sectioning + +Embryos placenta were fixed in formaldehyde, then dehydrated in ethanol, cleared in chloroform and then embedded in paraffin as described [44]. Sections were cut and stained using haematoxylin and eosin. + +The atrial wall thickness was determined using a modified Cavalieri method [45]. Both the inner and outer areas of the atrial chamber were measured and the average wall thickness was defined as the difference between the average radius of the inner and outer areas of the atrial chamber. Between 6 and 9 sections were analysed per embryo, and 4 wild type and 4 ERK5-/- embryos were analysed. + +Immunoblotting + +Tissue was homogenised in 50 mM Tris-HCl pH 7.5, 1 mM EGTA, 1 mM EDTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 1 mM sodium pyrophosphate, 0.27 M sucrose, 1% (v/v) Triton X-100, 0.1% (v/v) 2-mercaptoethanol and complete proteinase inhibitor cocktail (Roche). Insoluble material was removed by centrifugation at 13000 g for 5 min at 4°C. Soluble lysate (30 μg) was then run on 4–12% polyacrylamide gels (Novex, Invitrogen) and transferred onto nitrocellulose membranes. Primary antibodies against ERK1/2, p38 and MKK5 were used as described by the supplier, and the ERK5 antibody was used at 0.8 μg/ml. Secondary antibodies conjugated to horseradish peroxidase were from Pierce, and detection was performed using ECL (Amsersham). + +Authors contributions + +LY was involved in all aspects of this study and was responsible for most of the experimental work. JC was responsible for genotyping and management of mouse breeding, PRA assisted with analysis of the embryos, VMT was responsible for ES cell culture and blastocyst injection and CT carried out histological and caspase 3 staining. JSCA was responsible for co-ordinating the study and drafting the paper. + +Acknowledgements + +We would like to thank Philip Cohen for many helpful discussions and critical reading of the manuscript. We would also like to thank Janet Rossant for the 4311 clone. This research was supported by grants from the UK Medical Research Council, Astra-Zeneca, Boehringer-Ingelheim, GlaxoSmithKline, NovoNordisk and Pfizer. diff --git a/src/ontogpt/evaluation/craft/database/all/14691534.ann b/src/ontogpt/evaluation/craft/database/all/14691534.ann new file mode 100644 index 000000000..d77ec369f --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/14691534.ann @@ -0,0 +1,1352 @@ +T1 PR:000028746 0 4 Pten +T2 http://purl.obolibrary.org/obo/MONDO_0004992 19 25 Cancer +T3 UBERON:0002367 45 53 Prostate +T4 PR:000028746 94 98 PTEN +T5 http://purl.obolibrary.org/obo/MONDO_0005070 99 104 tumor +T6 SO:0000704 116 120 gene +T7 http://purl.obolibrary.org/obo/MONDO_0004992 153 159 cancer +T8 UBERON:0002367 171 179 prostate +T9 http://purl.obolibrary.org/obo/MONDO_0008315 171 186 prostate cancer +T10 http://purl.obolibrary.org/obo/MONDO_0008315 188 191 CaP +T11 PR:000028746 207 211 PTEN +T12 SO:0001023 212 218 allele +T13 http://purl.obolibrary.org/obo/MONDO_0008315 259 263 CaPs +T14 PR:000028746 313 317 PTEN +T15 http://purl.obolibrary.org/obo/MONDO_0004992 337 343 cancer +T16 PR:000028746 417 421 Pten +T17 NCBITaxon:10088 422 427 mouse +T18 PR:000028746 458 462 Pten +T19 PR:000028746 473 477 Pten +T20 PR:000028746 484 488 Pten +T21 PR:000028746 494 498 Pten +T22 UBERON:0000922 541 550 embryonic +T23 PR:000028746 577 581 Pten +T24 PR:000028746 598 602 Pten +T25 UBERON:0002367 603 611 prostate +T26 PR:000028746 634 638 Pten +T27 PR:000028746 722 726 Pten +T28 PR:000028746 746 750 Pten +T29 UBERON:0000428 799 819 prostatic epithelium +T30 PR:000028746 848 852 Pten +T31 http://purl.obolibrary.org/obo/MONDO_0008315 913 916 CaP +T32 PR:000028746 979 983 Pten +T33 PR:000029189 1023 1026 Akt +T34 PR:000003090 1028 1035 p27Kip1 +T35 PR:000003041 1037 1041 mTOR +T36 PR:000007641 1047 1052 FOXO3 +T37 SO:0000704 1085 1092 genetic +T38 PR:000028746 1121 1125 PTEN +T39 http://purl.obolibrary.org/obo/MONDO_0005070 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http://purl.obolibrary.org/obo/MONDO_0004992 1780 1786 cancer +T60 PR:000028746 1812 1816 PTEN +T61 SO:0001023 1852 1859 allelic +T62 SO:0000704 1880 1884 gene +T63 PR:000028746 1912 1916 PTEN +T64 http://purl.obolibrary.org/obo/MONDO_0004992 1949 1955 cancer +T65 PR:000028746 2012 2016 PTEN +T66 SO:0001023 2017 2023 allele +T67 http://purl.obolibrary.org/obo/MONDO_0004993 2027 2037 carcinomas +T68 http://purl.obolibrary.org/obo/MONDO_0004992 2060 2067 cancers +T69 PR:000028746 2129 2133 PTEN +T70 http://purl.obolibrary.org/obo/MONDO_0005070 2179 2184 tumor +T71 PR:000028746 2220 2224 PTEN +T72 SO:0001023 2225 2231 allele +T73 GO:0010467 2252 2262 expression +T74 http://purl.obolibrary.org/obo/MONDO_0005070 2292 2297 tumor +T75 GO:0010467 2350 2360 expression +T76 http://purl.obolibrary.org/obo/MONDO_0005070 2393 2398 tumor +T77 http://purl.obolibrary.org/obo/MONDO_0004992 2422 2429 cancers +T78 http://purl.obolibrary.org/obo/MONDO_0005070 2501 2507 tumors +T79 PR:000028746 2554 2558 PTEN +T80 SO:0001023 2559 2565 allele +T81 http://purl.obolibrary.org/obo/MONDO_0008315 2592 2596 CaPs +T82 PR:000028746 2669 2673 PTEN +T83 http://purl.obolibrary.org/obo/MONDO_0004992 2712 2718 cancer +T84 PR:000028746 2839 2843 PTEN +T85 http://purl.obolibrary.org/obo/MONDO_0005070 2863 2868 tumor +T86 http://purl.obolibrary.org/obo/MONDO_0005070 2926 2931 tumor +T87 UBERON:0000483 2967 2977 epithelial +T88 http://purl.obolibrary.org/obo/MONDO_0005626 2967 2987 epithelial neoplasms +T89 SO:0000704 3092 3099 genetic +T90 http://purl.obolibrary.org/obo/MONDO_0004992 3143 3150 cancers +T91 http://purl.obolibrary.org/obo/MONDO_0005070 3212 3217 tumor +T92 NCBITaxon:10088 3278 3283 mouse +T93 SO:0000646 3285 3306 Small interfering RNA +T94 SO:0000646 3308 3313 siRNA +T95 http://purl.obolibrary.org/obo/MONDO_0005070 3394 3399 tumor +T96 PR:000003035 3419 3422 p53 +T97 GO:0030097 3454 3465 hemopoietic +T98 CL:0000037 3454 3476 hemopoietic stem cells +T99 SO:0001023 3496 3503 allelic +T100 GO:0016246 3543 3559 RNA interference +T101 GO:0016246 3561 3565 RNAi +T102 GO:0009293 3567 3579 transduction +T103 PR:000028746 3617 3621 Pten +T104 SO:0000704 3695 3699 gene +T105 UBERON:0000922 3717 3726 embryonic +T106 PR:000028746 3900 3904 Pten +T107 UBERON:0000922 3932 3941 embryonic +T108 PR:000028746 4000 4004 Pten +T109 UBERON:0000062 4054 4059 organ +T110 UBERON:0000479 4060 4066 tissue +T111 UBERON:0000062 4121 4126 organ +T112 PR:000028746 4159 4163 Pten +T113 UBERON:0007023 4170 4175 adult +T114 UBERON:0000062 4176 4181 organ +T115 NCBITaxon:9606 4236 4241 human +T116 http://purl.obolibrary.org/obo/MONDO_0004992 4242 4248 cancer +T117 PR:000028746 4285 4289 Pten +T118 NCBITaxon:10088 4365 4370 mouse +T119 UBERON:0002367 4421 4429 prostate +T120 PR:000028746 4439 4443 Pten +T121 PR:000028746 4501 4505 Pten +T122 PR:000028746 4520 4524 Pten +T123 http://purl.obolibrary.org/obo/MONDO_0005070 4558 4567 neoplasms +T124 UBERON:0002367 4611 4620 prostatic +T125 http://purl.obolibrary.org/obo/MONDO_0005193 4611 4647 prostatic intraepithelial neoplasias +T126 UBERON:0000483 4626 4636 epithelial +T127 http://purl.obolibrary.org/obo/MONDO_0005193 4649 4652 PIN +T128 PR:000028746 4750 4754 Pten +T129 http://purl.obolibrary.org/obo/MONDO_0008315 4799 4803 CaPs +T130 http://purl.obolibrary.org/obo/MONDO_0008315 4814 4818 CaPs +T131 PR:000028746 4859 4863 Pten +T132 PR:000003090 4867 4874 p27Kip1 +T133 PR:000028746 4881 4885 Pten +T134 PR:000003090 4889 4896 p27Kip1 +T135 http://purl.obolibrary.org/obo/MONDO_0005193 4977 4980 PIN +T136 http://purl.obolibrary.org/obo/MONDO_0004956 5030 5044 metastatic CaP +T137 http://purl.obolibrary.org/obo/MONDO_0005070 5142 5148 tumors +T138 GO:0009888 5160 5172 histogenesis +T139 http://purl.obolibrary.org/obo/MONDO_0005070 5186 5192 tumors +T140 http://purl.obolibrary.org/obo/MONDO_0005193 5194 5197 PIN +T141 http://purl.obolibrary.org/obo/MONDO_0008315 5203 5206 CaP +T142 PR:000028746 5210 5214 Pten +T143 NCBITaxon:10088 5218 5222 mice +T144 PR:000028746 5227 5231 Pten +T145 PR:000003090 5232 5239 p27Kip1 +T146 PR:000028746 5281 5285 Pten +T147 UBERON:0002367 5314 5322 prostate +T148 http://purl.obolibrary.org/obo/MONDO_0021259 5314 5328 prostate tumor +T149 PR:000028746 5347 5351 Pten +T150 GO:0010467 5360 5370 expression +T151 PR:000028746 5460 5464 Pten +T152 PR:000028746 5486 5490 Pten +T153 http://purl.obolibrary.org/obo/MONDO_0005070 5537 5542 tumor +T154 PR:000028746 5577 5581 Pten +T155 PR:000028746 5671 5675 Pten +T156 http://purl.obolibrary.org/obo/MONDO_0005070 5706 5716 neoplastic +T157 CHEBI:16618 5746 5786 phosphatidylinositol 3,4,5-trisphosphate +T158 CHEBI:60169 5788 5792 PIP3 +T159 PR:000028746 5827 5831 PTEN +T160 CHEBI:60169 5949 5953 PIP3 +T161 GO:0044838 5977 5986 quiescent +T162 CHEBI:18179 6059 6075 phosphoinositide +T163 PR:000028746 6116 6120 PTEN +T164 CHEBI:60169 6146 6150 PIP3 +T165 GO:0016311 6158 6175 dephosphorylation +T166 PR:000028746 6204 6208 PTEN +T167 CHEBI:60169 6239 6243 PIP3 +T168 PR:000029189 6266 6269 Akt +T169 PR:000028746 6466 6470 Pten +T170 PR:000029189 6552 6555 Akt +T171 PR:000028746 6636 6640 Pten +T172 GO:0010467 6641 6651 expression +T173 http://purl.obolibrary.org/obo/MONDO_0005070 6778 6783 tumor +T174 http://purl.obolibrary.org/obo/MONDO_0008315 6811 6814 CaP +T175 http://purl.obolibrary.org/obo/MONDO_0004992 6843 6853 malignancy +T176 UBERON:0007023 6868 6873 adult +T177 http://purl.obolibrary.org/obo/MONDO_0008315 6954 6957 CaP +T178 GO:0016265 6966 6972 deaths +T179 http://purl.obolibrary.org/obo/MONDO_0008315 7027 7030 CaP +T180 SO:0001023 7093 7099 allele +T181 PR:000028746 7103 7107 PTEN +T182 PR:000003041 7159 7163 mTOR +T183 http://purl.obolibrary.org/obo/MONDO_0005070 7312 7317 tumor +T184 http://purl.obolibrary.org/obo/MONDO_0024488 7312 7317;7328 7333 tumor grade +T185 PR:000028746 7335 7339 PTEN +T186 http://purl.obolibrary.org/obo/MONDO_0004956 7386 7401 metastatic CaPs +T187 UBERON:0002367 7478 7486 prostate +T188 SO:0000704 7529 7540 genetically +T189 PR:000028746 7569 7573 Pten +T190 http://purl.obolibrary.org/obo/MONDO_0005070 7583 7588 tumor +T191 NCBITaxon:10088 7659 7664 mouse +T192 PR:000028746 7685 7689 Pten +T193 http://purl.obolibrary.org/obo/MONDO_0008315 7729 7738 CaP tumor +T194 PR:000028746 7760 7764 Pten +T195 PR:000028746 7938 7942 Pten +T196 PR:000028746 7991 7995 Pten +T197 http://purl.obolibrary.org/obo/MONDO_0005070 8021 8026 tumor +T198 PR:000028746 8060 8064 Pten +T199 UBERON:0000922 8089 8098 embryonic +T200 NCBITaxon:10088 8197 8202 mouse +T201 PR:000028746 8231 8235 Pten +T202 PR:000028746 8292 8296 Pten +T203 SO:0001023 8375 8381 allele +T204 PR:000028746 8385 8389 Pten +T205 GO:0010467 8401 8408 express +T206 PR:000028746 8421 8425 Pten +T207 SO:0000704 8426 8430 gene +T208 GO:0016246 8499 8527 transcriptional interference +T209 PR:000028746 8601 8605 Pten +T210 SO:0000188 8606 8612 intron +T211 SO:0005853 8634 8642 cassette +T212 GO:0065007 8653 8660 control +T213 NCBITaxon:10358 8675 8678 CMV +T214 SO:0000167 8679 8687 promoter +T215 NCBITaxon:10088 8723 8728 mouse +T216 UBERON:0000922 8729 8738 embryonic +T217 CL:0002322 8729 8743;8749 8754 embryonic stem ... cells +T218 CL:0002322 8745 8747;8749 8754 ES ... cells +T219 SO:0005853 8781 8789 cassette +T220 SO:0001023 8808 8814 allele +T221 PR:000028746 8871 8875 Pten +T222 SO:0000704 8876 8880 gene +T223 GO:0010467 8909 8919 expression +T224 PR:000028746 8942 8946 Pten +T225 SO:0001023 8963 8969 allele +T226 CL:0002322 8992 8994 ES +T227 UBERON:0000358 9077 9088 blastocysts +T228 PR:000028746 9138 9142 Pten +T229 SO:0001023 9145 9151 allele +T230 CL:0002322 9182 9190 ES cells +T231 PR:000028746 9258 9262 Pten +T232 NCBITaxon:10088 9267 9271 mice +T233 PR:000028746 9277 9281 Pten +T234 NCBITaxon:10088 9285 9289 mice +T235 UBERON:0000922 9355 9364 Embryonic +T236 PR:000028746 9378 9382 Pten +T237 PR:000028746 9433 9437 Pten +T238 UBERON:0000922 9466 9475 embryonic +T239 PR:000028746 9526 9530 Pten +T240 NCBITaxon:10088 9535 9539 mice +T241 GO:0007567 9549 9553 born +T242 PR:000028746 9619 9623 Pten +T243 PR:000028746 9756 9760 Pten +T244 GO:0007567 9814 9818 born +T245 PR:000028746 9860 9864 Pten +T246 PR:000028746 9890 9894 Pten +T247 GO:0016265 9912 9916 lost +T248 GO:0007565 9924 9933 gestation +T249 PR:000028746 9991 9995 Pten +T250 PR:000028746 10017 10021 Pten +T251 UBERON:0000922 10075 10084 embryonic +T252 UBERON:0000922 10133 10142 embryonic +T253 PR:000028746 10160 10164 Pten +T254 PR:000028746 10265 10269 Pten +T255 UBERON:0002367 10343 10351 prostate +T256 PR:000028746 10394 10398 Pten +T257 PR:000029189 10437 10440 Akt +T258 CHEBI:32958 10453 10460 phospho +T259 PR:000029189 10461 10464 Akt +T260 PR:000029189 10465 10468 Akt +T261 UBERON:0000062 10493 10499 organs +T262 CL:0000001 10504 10517 primary cells +T263 PR:000028746 10523 10527 Pten +T264 PR:000028746 10557 10561 Pten +T265 PR:000028746 10566 10570 Pten +T266 NCBITaxon:10088 10630 10635 mouse +T267 UBERON:0000922 10636 10645 embryonic +T268 CL:0000057 10646 10657 fibroblasts +T269 UBERON:0000178 10677 10682 blood +T270 CL:0000081 10677 10682;10695 10700 blood ... cells +T271 CL:0000842 10683 10700 mononuclear cells +T272 GO:0005634 10687 10694 nuclear +T273 UBERON:0002367 10714 10723 prostates +T274 PR:000028746 10852 10856 Pten +T275 GO:0010467 10862 10872 expression +T276 PR:000028746 10876 10880 Pten +T277 PR:000028746 10907 10911 Pten +T278 UBERON:0002367 10961 10969 Prostate +T279 http://purl.obolibrary.org/obo/MONDO_0005043 10970 10981 Hyperplasia +T280 http://purl.obolibrary.org/obo/MONDO_0008315 10995 10998 CaP +T281 PR:000028746 11002 11006 Pten +T282 PR:000028746 11034 11038 Pten +T283 UBERON:0002367 11074 11083 prostates +T284 PR:000028746 11089 11093 Pten +T285 PR:000028746 11134 11138 Pten +T286 PR:000028746 11148 11152 Pten +T287 NCBITaxon:10088 11156 11160 mice +T288 PR:000029189 11193 11196 Akt +T289 PR:000028746 11225 11229 Pten +T290 UBERON:0002367 11293 11301 prostate +T291 PR:000028746 11319 11323 Pten +T292 PR:000028746 11338 11342 Pten +T293 PR:000028746 11438 11442 Pten +T294 NCBITaxon:10088 11488 11492 Mice +T295 CHEBI:33252 11657 11664 nuclear +T296 UBERON:0002367 11794 11802 prostate +T297 NCBITaxon:10088 11809 11813 mice +T298 http://purl.obolibrary.org/obo/MONDO_0005070 11845 11850 tumor +T299 GO:0016265 11869 11875 mortem +T300 http://purl.obolibrary.org/obo/MONDO_0005070 11956 11961 tumor +T301 PR:000028746 11977 11981 Pten +T302 PR:000028746 11998 12002 Pten +T303 NCBITaxon:10088 12006 12010 mice +T304 http://purl.obolibrary.org/obo/MONDO_0005070 12031 12037 tumors +T305 UBERON:0002367 12129 12138 prostates +T306 UBERON:0002367 12177 12186 prostates +T307 PR:000028746 12190 12194 Pten +T308 PR:000028746 12202 12206 Pten +T309 NCBITaxon:10088 12210 12214 mice +T310 PR:000028746 12296 12300 Pten +T311 http://purl.obolibrary.org/obo/MONDO_0005193 12341 12345 PINs +T312 PR:000028746 12630 12634 Pten +T313 UBERON:0002367 12651 12660 prostates +T314 NCBITaxon:10088 12670 12674 mice +T315 UBERON:0002367 12709 12717 prostate +T316 UBERON:0000998 12831 12847 seminal vesicles +T317 UBERON:0002367 12883 12891 prostate +T318 PR:000028746 12981 12985 Pten +T319 UBERON:0000483 13086 13096 epithelial +T320 http://purl.obolibrary.org/obo/MONDO_0005043 13097 13108 hyperplasia +T321 UBERON:0002367 13116 13124 prostate +T322 UBERON:0009912 13142 13147 lobes +T323 UBERON:0009912 13249 13254 lobes +T324 UBERON:0002367 13295 13311 prostatic glands +T325 NCBITaxon:10088 13334 13338 mice +T326 UBERON:0002530 13386 13392 glands +T327 UBERON:2001995 13430 13451 papillary projections +T328 GO:0005634 13512 13518 nuclei +T329 GO:0000785 13528 13537 chromatin +T330 GO:0005730 13553 13561 nucleoli +T331 UBERON:0000483 13579 13589 epithelial +T332 http://purl.obolibrary.org/obo/MONDO_0024474 13579 13599 epithelial dysplasia +T333 PR:000028746 13630 13634 Pten +T334 NCBITaxon:10088 13639 13643 mice +T335 GO:0030154 13694 13718 cellular differentiation +T336 PR:000028746 13760 13764 Pten +T337 NCBITaxon:10088 13768 13772 mice +T338 PR:000028746 13803 13807 Pten +T339 PR:000028746 13815 13819 Pten +T340 PR:000003090 13823 13830 p27Kip1 +T341 PR:000028746 13872 13876 Pten +T342 GO:0010467 13885 13895 expression +T343 http://purl.obolibrary.org/obo/MONDO_0005043 13927 13939 hyperplastic +T344 UBERON:0002367 13940 13949 prostates +T345 PR:000028746 13955 13959 Pten +T346 PR:000028746 14039 14043 Pten +T347 SO:0001023 14044 14050 allele +T348 PR:000028746 14118 14122 Pten +T349 NCBITaxon:10088 14127 14131 mice +T350 http://purl.obolibrary.org/obo/MONDO_0005070 14216 14221 tumor +T351 UBERON:0002530 14268 14273 gland +T352 GO:0040007 14278 14285 growing +T353 UBERON:0003891 14307 14313 stroma +T354 PR:000028746 14387 14391 Pten +T355 PR:000028746 14412 14416 Pten +T356 PR:000028746 14448 14452 Pten +T357 UBERON:0002367 14485 14493 prostate +T358 http://purl.obolibrary.org/obo/MONDO_0005043 14494 14505 hyperplasia +T359 http://purl.obolibrary.org/obo/MONDO_0005070 14545 14550 tumor +T360 http://purl.obolibrary.org/obo/MONDO_0005193 14579 14582 PIN +T361 http://purl.obolibrary.org/obo/MONDO_0005070 14594 14599 tumor +T362 http://purl.obolibrary.org/obo/MONDO_0008315 14611 14614 CaP +T363 SO:0000704 14634 14645 genetically +T364 NCBITaxon:10088 14657 14661 mice +T365 http://purl.obolibrary.org/obo/MONDO_0008315 14701 14704 CaP +T366 NCBITaxon:10088 14713 14718 mouse +T367 UBERON:0002367 14753 14761 Prostate +T368 PR:000028746 14771 14775 Pten +T369 PR:000028746 14821 14825 Pten +T370 UBERON:0002367 14846 14854 prostate +T371 http://purl.obolibrary.org/obo/MONDO_0008315 14876 14885 CaP tumor +T372 NCBITaxon:10088 14925 14930 mouse +T373 PR:000028746 14948 14952 Pten +T374 SO:0000147 14953 14958 exons +T375 SO:0000357 14971 14978 flanked +T376 SO:0000346 14982 14992 loxP sites +T377 SO:0005853 15041 15049 cassette +T378 PR:000028746 15102 15106 Pten +T379 NCBITaxon:10088 15136 15140 mice +T380 NCBITaxon:10088 15171 15175 mice +T381 GO:0010467 15200 15209 expressed +T382 GO:0009790 15232 15245 embryogenesis +T383 GO:0000003 15260 15273;15283 15290 production of ... progeny +T384 PR:000028746 15320 15324 Pten +T385 SO:0001023 15325 15332 alleles +T386 SO:0005853 15413 15421 cassette +T387 PR:000028746 15424 15428 Pten +T388 SO:0000359 15429 15435 floxed +T389 PR:000028746 15440 15444 Pten +T390 NCBITaxon:10088 15522 15526 mice +T391 PR:000028746 15548 15552 Pten +T392 SO:0000359 15553 15559 floxed +T393 SO:0001023 15560 15566 allele +T394 PR:000028746 15568 15572 Pten +T395 SO:0000346 15572 15576 loxP +T396 PR:000028746 15616 15620 Pten +T397 SO:0000346 15620 15624 loxP +T398 NCBITaxon:10088 15625 15629 mice +T399 GO:0007567 15635 15639 born +T400 PR:000028746 15751 15755 Pten +T401 SO:0000704 15756 15760 gene +T402 UBERON:0002367 15768 15776 prostate +T403 PR:000013530 15842 15844 PB +T404 PR:000013530 15853 15855 PB +T405 SO:0000704 15879 15883 gene +T406 GO:0065007 15897 15904 control +T407 NCBITaxon:10114 15937 15940 rat +T408 PR:000013530 15941 15949 Probasin +T409 PR:000013530 15951 15953 PB +T410 SO:0000704 15955 15959 gene +T411 SO:0000167 15960 15968 promoter +T412 PR:000013530 16013 16015 PB +T413 SO:0000704 16016 16020 gene +T414 GO:0010467 16024 16033 expressed +T415 UBERON:0000428 16041 16061 prostatic epithelium +T416 SO:0000704 16084 16088 gene +T417 CHEBI:50113 16092 16100 androgen +T418 PR:000013530 16153 16155 PB +T419 PR:000028746 16194 16198 Pten +T420 SO:0000704 16199 16203 gene +T421 UBERON:0000428 16223 16243 prostatic epithelium +T422 PR:000028746 16362 16366 Pten +T423 UBERON:0002367 16374 16382 prostate +T424 GO:0030850 16374 16396 prostate organogenesis +T425 SO:0000167 16478 16486 promoter +T426 PR:000013530 16495 16497 PB +T427 SO:0000704 16534 16538 gene +T428 SO:0000167 16564 16572 promoter +T429 PR:000013530 16579 16581 PB +T430 NCBITaxon:10114 16612 16615 rat +T431 PR:000013530 16616 16618 PB +T432 SO:0000167 16619 16627 promoter +T433 PR:000013530 16643 16645 PB +T434 PR:000013530 16704 16706 PB +T435 NCBITaxon:10088 16712 16716 mice +T436 GO:0010467 16717 16724 express +T437 PR:000013530 16774 16776 PB +T438 NCBITaxon:10088 16781 16785 mice +T439 GO:0010467 16786 16793 express +T440 PR:000028746 16908 16912 Pten +T441 UBERON:0000428 16920 16940 prostatic epithelium +T442 PR:000013530 16997 16999 PB +T443 PR:000013530 17008 17010 PB +T444 NCBITaxon:10088 17016 17020 mice +T445 PR:000028746 17039 17043 Pten +T446 SO:0000346 17043 17047 loxP +T447 PR:000013530 17077 17079 PB +T448 PR:000028746 17084 17088 Pten +T449 SO:0000346 17088 17092 loxP +T450 SO:0000346 17093 17097 loxP +T451 PR:000028746 17124 17128 Pten +T452 PR:000013530 17144 17146 PB +T453 PR:000028746 17152 17156 Pten +T454 SO:0000346 17156 17160 loxP +T455 SO:0000346 17161 17165 loxP +T456 PR:000028746 17192 17196 Pten +T457 PR:000028746 17239 17243 Pten +T458 http://purl.obolibrary.org/obo/MONDO_0005070 17253 17258 Tumor +T459 UBERON:0002367 17278 17286 Prostate +T460 PR:000028746 17327 17331 Pten +T461 UBERON:0002367 17348 17356 prostate +T462 PR:000028746 17374 17378 Pten +T463 PR:000028746 17386 17390 Pten +T464 NCBITaxon:10088 17403 17407 Mice +T465 PR:000028746 17461 17465 Pten +T466 PR:000028746 17631 17635 Pten +T467 PR:000028746 17666 17670 Pten +T468 PR:000028746 17722 17726 Pten +T469 PR:000028746 17735 17739 Pten +T470 UBERON:0002367 17799 17807 prostate +T471 UBERON:0001328 17856 17871 prostatic lobes +T472 PR:000028746 17913 17917 Pten +T473 PR:000028746 17983 17987 Pten +T474 PR:000028746 18158 18162 Pten +T475 UBERON:0000428 18183 18203 prostatic epithelium +T476 PR:000028746 18221 18225 Pten +T477 PR:000028746 18236 18240 Pten +T478 PR:000028746 18268 18272 Pten +T479 PR:000028746 18289 18293 Pten +T480 UBERON:0000428 18420 18440 prostatic epithelium +T481 PR:000028746 18518 18522 Pten +T482 UBERON:0002367 18563 18571 prostate +T483 PR:000028746 18593 18597 Pten +T484 PR:000028746 18637 18641 Pten +T485 NCBITaxon:10088 18645 18649 mice +T486 http://purl.obolibrary.org/obo/MONDO_0009237 18660 18697 focal areas of epithelial hyperplasia +T487 UBERON:0000483 18675 18685 epithelial +T488 UBERON:0002530 18713 18719 glands +T489 PR:000028746 18762 18766 Pten +T490 NCBITaxon:10088 18770 18774 mice +T491 http://purl.obolibrary.org/obo/MONDO_0005043 18798 18810 hyperplastic +T492 UBERON:0002530 18811 18817 glands +T493 UBERON:0009912 18825 18830 lobes +T494 UBERON:0002530 18925 18934 glandular +T495 UBERON:0002367 19020 19029 prostates +T496 PR:000028746 19035 19039 Pten +T497 PR:000028746 19047 19051 Pten +T498 NCBITaxon:10088 19055 19059 mice +T499 http://purl.obolibrary.org/obo/MONDO_0008315 19150 19153 CaP +T500 http://purl.obolibrary.org/obo/MONDO_0005070 19207 19213 Tumors +T501 PR:000028746 19241 19245 Pten +T502 http://purl.obolibrary.org/obo/MONDO_0000001 19304 19311 disease +T503 UBERON:0000483 19331 19340 pithelial +T504 http://purl.obolibrary.org/obo/MONDO_0005070 19372 19378 tumors +T505 UBERON:0000483 19417 19427 epithelial +T506 CL:0000066 19417 19433 epithelial cells +T507 GO:0005737 19468 19479 cytoplasmic +T508 http://purl.obolibrary.org/obo/MONDO_0005070 19536 19542 tumors +T509 http://purl.obolibrary.org/obo/MONDO_0005070 19582 19588 tumors +T510 PR:000028746 19619 19623 Pten +T511 PR:000028746 19631 19635 Pten +T512 UBERON:0000062 19707 19712 organ +T513 http://purl.obolibrary.org/obo/MONDO_0008315 19769 19773 CaPs +T514 PR:000028746 19819 19823 Pten +T515 UBERON:0001328 19849 19862 prostate lobe +T516 http://purl.obolibrary.org/obo/MONDO_0005070 19906 19916 neoplastic +T517 http://purl.obolibrary.org/obo/MONDO_0005070 19948 19953 tumor +T518 http://purl.obolibrary.org/obo/MONDO_0005070 19977 19983 tumors +T519 PR:000028746 19987 19991 Pten +T520 UBERON:0009912 20033 20037 lobe +T521 http://purl.obolibrary.org/obo/MONDO_0005070 20130 20136 tumors +T522 PR:000028746 20144 20148 Pten +T523 NCBITaxon:33208 20152 20159 animals +T524 http://purl.obolibrary.org/obo/MONDO_0005070 20205 20211 tumors +T525 PR:000028746 20219 20223 Pten +T526 NCBITaxon:10088 20227 20231 mice +T527 PR:000028746 20299 20303 Pten +T528 PR:000028746 20459 20463 Pten +T529 UBERON:0002367 20496 20504 prostate +T530 http://purl.obolibrary.org/obo/MONDO_0008315 20535 20538 CaP +T531 PR:000028746 20579 20583 Pten +T532 UBERON:0000428 20611 20631 Prostatic Epithelium +T533 PR:000028746 20689 20693 Pten +T534 UBERON:0000428 20717 20737 prostatic epithelium +T535 PR:000028746 20813 20817 Pten +T536 PR:000028746 20822 20826 Pten +T537 PR:000028746 20831 20835 Pten +T538 PR:000028746 20845 20849 Pten +T539 PR:000028746 20900 20904 Pten +T540 NCBITaxon:10088 20908 20912 mice +T541 PR:000028746 20955 20959 Pten +T542 UBERON:0002367 20991 21000 prostates +T543 PR:000028746 21049 21053 Pten +T544 GO:0050673 21072 21105 proliferation of epithelial cells +T545 UBERON:0000483 21089 21099 epithelial +T546 CL:0000066 21089 21105 epithelial cells +T547 PR:000010425 21112 21117 Ki-67 +T548 UBERON:0000428 21193 21212 prostate epithelial +T549 CL:0002231 21193 21218 prostate epithelial cells +T550 PR:000028746 21259 21263 Pten +T551 GO:0010467 21264 21274 expression +T552 GO:0008283 21363 21376 proliferation +T553 UBERON:0002367 21384 21393 prostates +T554 PR:000028746 21399 21403 Pten +T555 PR:000028746 21434 21438 Pten +T556 NCBITaxon:10088 21442 21446 mice +T557 PR:000028746 21454 21458 Pten +T558 UBERON:0000483 21510 21520 epithelial +T559 CL:0000066 21510 21526 epithelial cells +T560 UBERON:0002367 21562 21570 prostate +T561 PR:000029189 21772 21775 Akt +T562 GO:0043491 21772 21793 Akt signaling pathway +T563 PR:000028746 21895 21899 Pten +T564 PR:000028746 21951 21955 Pten +T565 PR:000028746 22095 22099 Pten +T566 PR:000029189 22166 22169 Akt +T567 PR:000003090 22171 22178 p27Kip1 +T568 PR:000003041 22180 22184 mTOR +T569 PR:000007641 22190 22195 FOXO3 +T570 PR:000029189 22330 22333 Akt +T571 PR:000028746 22389 22393 Pten +T572 CHEBI:32958 22441 22448 phospho +T573 PR:000029189 22449 22452 Akt +T574 GO:0042571 22462 22470 antibody +T575 PR:000028746 22510 22514 Pten +T576 UBERON:0002367 22536 22545 prostates +T577 GO:0042571 22579 22587 antibody +T578 PR:000028746 22632 22636 Pten +T579 UBERON:0002367 22797 22805 prostate +T580 PR:000028746 22817 22821 Pten +T581 CHEBI:32958 22887 22894 phospho +T582 PR:000029189 22895 22898 Akt +T583 GO:0005886 22938 22953 plasma membrane +T584 PR:000028746 23015 23019 Pten +T585 UBERON:0000483 23025 23035 epithelium +T586 http://purl.obolibrary.org/obo/MONDO_0005070 23045 23050 tumor +T587 http://purl.obolibrary.org/obo/MONDO_0005070 23086 23092 tumors +T588 PR:000028746 23103 23107 Pten +T589 PR:000028746 23115 23119 Pten +T590 PR:000029189 23155 23158 Akt +T591 PR:000028746 23208 23212 Pten +T592 GO:0005886 23267 23282 plasma membrane +T593 PR:000028746 23316 23320 Pten +T594 PR:000003090 23337 23344 p27Kip1 +T595 PR:000007641 23346 23351 FOXO3 +T596 PR:000003041 23357 23361 mTOR +T597 PR:000029189 23380 23390 Akt kinase +T598 PR:000003090 23420 23427 p27Kip1 +T599 PR:000007641 23432 23437 FOXO3 +T600 PR:000029189 23441 23444 Akt +T601 GO:0005634 23522 23529 nuclear +T602 GO:0051168 23522 23536 nuclear export +T603 GO:0005634 23597 23604 nuclear +T604 GO:0051170 23597 23611 nuclear import +T605 PR:000003090 23647 23654 p27Kip1 +T606 PR:000029189 23756 23759 Akt +T607 PR:000003041 23782 23786 mTOR +T608 GO:0006412 23825 23836 translation +T609 GO:0006413 23841 23863 translation initiation +T610 GO:0010467 23939 23949 expression +T611 PR:000003090 23960 23967 p27Kip1 +T612 PR:000028746 23989 23993 Pten +T613 PR:000028746 24001 24005 Pten +T614 PR:000003090 24009 24016 p27Kip1 +T615 PR:000003090 24112 24119 p27Kip1 +T616 PR:000028746 24145 24149 Pten +T617 PR:000028746 24158 24162 Pten +T618 PR:000003090 24203 24210 p27Kip1 +T619 GO:0010467 24226 24235 expressed +T620 GO:0010467 24283 24290 express +T621 PR:000007641 24346 24351 FOXO3 +T622 PR:000028746 24367 24371 Pten +T623 CHEBI:32958 24464 24471 phospho +T624 PR:000007641 24472 24477 FOXO3 +T625 GO:0042571 24478 24486 antibody +T626 PR:000028746 24565 24569 Pten +T627 PR:000028746 24577 24581 Pten +T628 CHEBI:32958 24643 24650 phospho +T629 PR:000007641 24651 24656 FOXO3 +T630 GO:0005737 24657 24668 cytoplasmic +T631 UBERON:0000428 24695 24714 prostate epithelial +T632 CL:0002231 24695 24720 prostate epithelial cells +T633 PR:000028746 24726 24730 Pten +T634 NCBITaxon:10088 24769 24773 mice +T635 PR:000028746 24796 24800 Pten +T636 PR:000003041 24846 24850 mTOR +T637 UBERON:0000428 24874 24894 prostatic epithelium +T638 PR:000028746 24898 24902 Pten +T639 PR:000028746 24967 24971 Pten +T640 PR:000028746 24996 25000 Pten +T641 PR:000028746 25017 25021 Pten +T642 UBERON:0000428 25074 25094 prostatic epithelium +T643 http://purl.obolibrary.org/obo/MONDO_0005070 25193 25198 tumor +T644 http://purl.obolibrary.org/obo/MONDO_0004992 25324 25330 cancer +T645 UBERON:0002367 25350 25358 prostate +T646 PR:000028746 25402 25406 Pten +T647 GO:0065007 25444 25455 controlling +T648 http://purl.obolibrary.org/obo/MONDO_0005070 25526 25536 neoplastic +T649 http://purl.obolibrary.org/obo/MONDO_0004992 25540 25549 malignant +T650 UBERON:0001062 25887 25897;25915 25923 anatomical ... entities +T651 UBERON:0000428 25934 25954 prostatic epithelium +T652 http://purl.obolibrary.org/obo/MONDO_0008315 25972 25975 CaP +T653 UBERON:0002367 26071 26079 prostate +T654 http://purl.obolibrary.org/obo/MONDO_0005043 26080 26091 hyperplasia +T655 http://purl.obolibrary.org/obo/MONDO_0005193 26114 26117 PIN +T656 http://purl.obolibrary.org/obo/MONDO_0005193 26131 26134 PIN +T657 http://purl.obolibrary.org/obo/MONDO_0008315 26154 26157 CaP +T658 http://purl.obolibrary.org/obo/MONDO_0008315 26169 26172 CaP +T659 UBERON:0001062 26311 26321 anatomical +T660 GO:0010467 26382 26392 expression +T661 http://purl.obolibrary.org/obo/MONDO_0005070 26405 26410 tumor +T662 SO:0000704 26422 26426 gene +T663 UBERON:0001062 26466 26476 anatomical +T664 PR:000028746 26631 26635 Pten +T665 SO:0001023 26636 26642 allele +T666 NCBITaxon:10088 26650 26655 mouse +T667 UBERON:0000428 26668 26687 prostate epithelial +T668 http://purl.obolibrary.org/obo/MONDO_0005043 26688 26699 hyperplasia +T669 PR:000003090 26729 26736 p27Kip1 +T670 NCBITaxon:10088 26744 26749 mouse +T671 NCBITaxon:9606 26774 26779 human +T672 http://purl.obolibrary.org/obo/MONDO_0010811 26780 26807 benign prostate hyperplasia +T673 UBERON:0002367 26787 26795 prostate +T674 http://purl.obolibrary.org/obo/MONDO_0010811 26809 26812 BPH +T675 UBERON:0000483 26920 26930 epithelial +T676 http://purl.obolibrary.org/obo/MONDO_0005193 26975 26978 PIN +T677 UBERON:0000483 27013 27023 epithelial +T678 UBERON:0003891 27028 27034 stroma +T679 http://purl.obolibrary.org/obo/MONDO_0005043 27058 27069 hyperplasia +T680 UBERON:0000062 27083 27088 organ +T681 http://purl.obolibrary.org/obo/MONDO_0004992 27117 27127 malignancy +T682 PR:000003090 27239 27246 p27Kip1 +T683 GO:0010467 27247 27257 expression +T684 SO:0000704 27289 27296 genetic +T685 PR:000028746 27319 27323 PTEN +T686 NCBITaxon:10088 27430 27435 mouse +T687 PR:000028746 27540 27544 Pten +T688 PR:000028746 27570 27574 Pten +T689 http://purl.obolibrary.org/obo/MONDO_0005070 27600 27605 tumor +T690 http://purl.obolibrary.org/obo/MONDO_0005193 27667 27670 PIN +T691 http://purl.obolibrary.org/obo/MONDO_0040677 27683 27701 invasive carcinoma +T692 http://purl.obolibrary.org/obo/MONDO_0008315 27790 27793 CaP +T693 PR:000028746 27855 27859 PTEN +T694 SO:0001023 27860 27866 allele +T695 PR:000028746 27899 27903 PTEN +T696 SO:0001023 27904 27910 allele +T697 http://purl.obolibrary.org/obo/MONDO_0005193 27949 27952 PIN +T698 http://purl.obolibrary.org/obo/MONDO_0040677 27964 27972;27982 27992 invasive carcinomas +T699 UBERON:0002367 27973 27981 prostate +T700 http://purl.obolibrary.org/obo/MONDO_0005159 27973 27992 prostate carcinomas +T701 CHEBI:52217 28026 28043 pharmacologically +T702 GO:0065007 28044 28054 modulating +T703 GO:0010467 28059 28069 expression +T704 PR:000028746 28087 28091 PTEN +T705 SO:0001023 28092 28098 allele +T706 PR:000028746 28149 28153 PTEN +T707 PR:000003041 28205 28209 mTOR +T708 UBERON:0002367 28252 28260 prostate +T709 http://purl.obolibrary.org/obo/MONDO_0005043 28261 28272 hyperplasia +T710 PR:000028746 28299 28303 Pten +T711 PR:000028746 28369 28373 Pten +T712 GO:0010467 28374 28384 expression +T713 http://purl.obolibrary.org/obo/MONDO_0008315 28437 28440 CaP +T714 PR:000028746 28604 28608 Pten +T715 NCBITaxon:10088 28612 28616 mice +T716 PR:000028746 28625 28629 Pten +T717 http://purl.obolibrary.org/obo/MONDO_0008315 28667 28670 CaP +T718 http://purl.obolibrary.org/obo/MONDO_0005193 28691 28694 PIN +T719 PR:000028746 28748 28752 Pten +T720 PR:000028746 28902 28906 Pten +T721 UBERON:0002367 29072 29080 prostate +T722 http://purl.obolibrary.org/obo/MONDO_0005043 29081 29092 hyperplasia +T723 SO:0000704 29141 29148 genetic +T724 PR:000028746 29174 29178 Pten +T725 PR:000028746 29279 29283 Pten +T726 PR:000028746 29423 29427 Pten +T727 UBERON:0002367 29456 29464 prostate +T728 http://purl.obolibrary.org/obo/MONDO_0005043 29465 29476 hyperplasia +T729 UBERON:0000479 29576 29583 tissues +T730 UBERON:0000062 29588 29594 organs +T731 PR:000028746 29627 29631 Pten +T732 UBERON:0000428 29653 29673 prostatic epithelium +T733 PR:000028746 29748 29752 Pten +T734 PR:000003090 29918 29925 p27Kip1 +T735 NCBITaxon:10088 29929 29933 mice +T736 http://purl.obolibrary.org/obo/MONDO_0005043 29949 29960 hyperplasia +T737 UBERON:0003891 29969 29976 stromal +T738 UBERON:0000353 29981 29992 parenchymal +T739 UBERON:0002367 30011 30019 prostate +T740 PR:000028746 30124 30128 Pten +T741 UBERON:0000428 30164 30184 prostatic epithelium +T742 GO:0008283 30199 30211 proliferates +T743 UBERON:0002367 30224 30232 prostate +T744 http://purl.obolibrary.org/obo/MONDO_0005043 30233 30244 hyperplasia +T745 NCBITaxon:7215 30344 30354 Drosophila +T746 GO:0010467 30385 30395 expression +T747 PR:000028746 30399 30403 Pten +T748 PR:000003041 30446 30449 TOR +T749 PR:000028746 30581 30585 Pten +T750 UBERON:0002367 30607 30615 prostate +T751 PR:000003090 30700 30707 p27Kip1 +T752 PR:000011248 30711 30717 Nkx3.1 +T753 GO:0010467 30725 30735 expression +T754 PR:000028746 30916 30920 Pten +T755 UBERON:0002367 30934 30942 prostate +T756 http://purl.obolibrary.org/obo/MONDO_0004992 31033 31045 malignancies +T757 PR:000028746 31112 31116 Pten +T758 UBERON:0000428 31279 31299 prostatic epithelium +T759 PR:000028746 31311 31315 Pten +T760 http://purl.obolibrary.org/obo/MONDO_0008315 31388 31391 CaP +T761 http://purl.obolibrary.org/obo/MONDO_0005193 31406 31409 PIN +T762 PR:000028746 31444 31448 PTEN +T763 PR:000003041 31511 31515 mTOR +T764 CHEBI:35222 31516 31526 inhibitors +T765 GO:0010468 31534 31547;31553 31563 modulation of ... expression +T766 PR:000028746 31548 31552 PTEN +T767 PR:000028746 31743 31747 PTEN +T768 SO:0000704 31748 31752 gene +T769 GO:0010467 31761 31771 expression +T770 NCBITaxon:9606 31775 31780 human +T771 http://purl.obolibrary.org/obo/MONDO_0008315 31781 31785 CaPs +T772 http://purl.obolibrary.org/obo/MONDO_0004992 31793 31802 cancerous +T773 PR:000028746 31957 31961 Pten +T774 NCBITaxon:10088 31982 31987 mouse +T775 UBERON:0002367 31988 31996 prostate +T776 http://purl.obolibrary.org/obo/MONDO_0008315 32056 32060 CaPs +T777 GO:0016265 32154 32160 mortem +T778 UBERON:0002048 32207 32211 lung +T779 http://purl.obolibrary.org/obo/MONDO_0005070 32212 32222 neoplastic +T780 PR:000028746 32250 32254 Pten +T781 http://purl.obolibrary.org/obo/MONDO_0005070 32353 32359 tumors +T782 PR:000028746 32427 32431 Pten +T783 UBERON:0000428 32496 32515 prostate epithelial +T784 CL:0002231 32496 32521 prostate epithelial cells +T785 http://purl.obolibrary.org/obo/MONDO_0005070 32531 32536 tumor +T786 GO:0008283 32560 32582 cellular proliferation +T787 PR:000029189 32628 32631 Akt +T788 PR:000028746 32636 32640 Pten +T789 UBERON:0000483 32646 32656 epithelial +T790 CL:0000066 32646 32662 epithelial cells +T791 http://purl.obolibrary.org/obo/MONDO_0005070 32679 32689 neoplastic +T792 http://purl.obolibrary.org/obo/MONDO_0008315 32708 32712 CaPs +T793 CHEBI:32958 32714 32721 phospho +T794 PR:000029189 32722 32725 Akt +T795 GO:0005886 32775 32790 plasma membrane +T796 PR:000029189 32859 32862 Akt +T797 PR:000029189 32930 32933 Akt +T798 PR:000028746 32958 32962 Pten +T799 UBERON:0000428 32968 32987 prostate epithelial +T800 CL:0002231 32968 32993 prostate epithelial cells +T801 PR:000003090 33076 33083 p27Kip1 +T802 GO:0010467 33084 33094 expression +T803 GO:0005737 33123 33134 cytoplasmic +T804 NCBITaxon:9606 33177 33182 human +T805 UBERON:0000310 33183 33189 breast +T806 http://purl.obolibrary.org/obo/MONDO_0007254 33183 33196 breast cancer +T807 PR:000029189 33213 33216 AKT +T808 PR:000028746 33336 33340 Pten +T809 PR:000028746 33429 33433 Pten +T810 http://purl.obolibrary.org/obo/MONDO_0005070 33454 33459 tumor +T811 UBERON:0000428 33548 33567 prostate epithelium +T812 PR:000028746 33587 33591 Pten +T813 PR:000028746 33618 33622 Pten +T814 NCBITaxon:10088 33626 33630 mice +T815 UBERON:0002367 33653 33662 prostatic +T816 PR:000028746 33697 33701 Pten +T817 UBERON:0002367 33742 33750 prostate +T818 PR:000028746 33770 33774 Pten +T819 NCBITaxon:10088 33778 33782 mice +T820 http://purl.obolibrary.org/obo/MONDO_0008315 33806 33809 CaP +T821 PR:000028746 33819 33823 Pten +T822 NCBITaxon:10088 33828 33832 mice +T823 PR:000028746 33897 33901 Pten +T824 http://purl.obolibrary.org/obo/MONDO_0005070 33902 33907 tumor +T825 PR:000028746 34057 34061 Pten +T826 PR:000028746 34103 34107 Pten +T827 PR:000028746 34118 34122 Pten +T828 NCBITaxon:10088 34126 34130 mice +T829 PR:000028746 34135 34139 Pten +T830 NCBITaxon:10088 34143 34147 mice +T831 UBERON:0002530 34191 34200 glandular +T832 PR:000028746 34271 34275 Pten +T833 PR:000028746 34376 34380 Pten +T834 NCBITaxon:10088 34384 34388 mice +T835 PR:000028746 34401 34405 Pten +T836 PR:000028746 34479 34483 Pten +T837 UBERON:0000353 34510 34520 parenchyma +T838 GO:0010467 34591 34601 expression +T839 PR:000028746 34687 34691 Pten +T840 http://purl.obolibrary.org/obo/MONDO_0005070 34692 34697 tumor +T841 PR:000028746 34741 34745 Pten +T842 UBERON:0000062 34768 34773 organ +T843 NCBITaxon:9606 34933 34938 human +T844 http://purl.obolibrary.org/obo/MONDO_0008315 34939 34942 CaP +T845 NCBITaxon:9606 34989 34994 human +T846 http://purl.obolibrary.org/obo/MONDO_0008315 34995 34998 CaP +T847 NCBITaxon:10088 35098 35103 mouse +T848 NCBITaxon:9606 35175 35180 human +T849 http://purl.obolibrary.org/obo/MONDO_0000001 35181 35188 disease +T850 http://purl.obolibrary.org/obo/MONDO_0005070 35237 35247 neoplastic +T851 SO:0000704 35271 35278 genetic +T852 http://purl.obolibrary.org/obo/MONDO_0005070 35330 35335 tumor +T853 GO:0040007 35352 35356 grow +T854 http://purl.obolibrary.org/obo/MONDO_0005070 35377 35387 neoplastic +T855 UBERON:0003891 35437 35444 stromal +T856 UBERON:0000353 35449 35460 parenchymal +T857 NCBITaxon:9606 35498 35503 human +T858 http://purl.obolibrary.org/obo/MONDO_0000001 35504 35511 disease +T859 NCBITaxon:10088 35521 35526 mouse +T860 PR:000028746 35615 35619 Pten +T861 http://purl.obolibrary.org/obo/MONDO_0008315 35623 35632 CaP tumor +T862 PR:000028746 35692 35696 Pten +T863 PR:000028746 35718 35722 Pten +T864 http://purl.obolibrary.org/obo/MONDO_0005070 35748 35753 tumor +T865 UBERON:0002367 35788 35796 prostate +T866 NCBITaxon:10088 35824 35828 Mice +T867 PR:000028746 35865 35869 Pten +T868 NCBITaxon:10088 35873 35877 mice +T869 NCBITaxon:10088 35945 35949 Mice +T870 UBERON:0002415 35975 35979 tail +T871 NCBITaxon:10088 36030 36034 mice +T872 UBERON:0002415 36092 36096 tail +T873 PR:000028746 36138 36142 Pten +T874 PR:000028746 36148 36152 Pten +T875 SO:0001023 36155 36162 alleles +T876 SO:0000112 36170 36177 primers +T877 SO:0001644 36229 36245 Targeting vector +T878 UBERON:0002367 36264 36272 prostate +T879 PR:000028746 36282 36286 Pten +T880 NCBITaxon:10088 36297 36301 mice +T881 NCBITaxon:10088 36312 36317 mouse +T882 SO:0001026 36318 36325 genomic +T883 NCBITaxon:10088 36404 36409 mouse +T884 PR:000028746 36410 36414 Pten +T885 SO:0000147 36432 36437 exons +T886 PR:P23940 36494 36499 BamHI +T887 PR:000028746 36523 36527 Pten +T888 SO:0001026 36528 36535 genomic +T889 SO:0001026 36581 36588 genomic +T890 CL:0002322 36693 36701 ES cells +T891 CHEBI:42768 36734 36738 G418 +T892 CHEBI:465284 36755 36766 gancyclovir +T893 NCBITaxon:10088 36841 36845 mice +T894 CL:0002322 36918 36925 ES cell +T895 UBERON:0000358 36975 36986 blastocysts +T896 GO:0007618 37059 37064 mated +T897 SO:0001023 37183 37189 allele +T898 UBERON:0002415 37232 37236 tail +T899 UBERON:0010166 37253 37257 coat +T900 PR:000028746 37286 37290 Pten +T901 SO:0000346 37290 37294 loxP +T902 NCBITaxon:10088 37301 37305 mice +T903 GO:0007618 37311 37316 mated +T904 NCBITaxon:10088 37342 37346 mice +T905 UBERON:0002415 37372 37376 tail +T906 NCBITaxon:10088 37483 37487 mice +T907 PR:000028746 37500 37504 Pten +T908 SO:0001023 37515 37521 allele +T909 PR:000028746 37525 37529 Pten +T910 SO:0001023 37539 37545 allele +T911 PR:000028746 37547 37551 Pten +T912 SO:0000346 37551 37555 loxP +T913 SO:0000359 37568 37574 floxed +T914 SO:0001023 37575 37581 allele +T915 PR:000028746 37583 37587 Pten +T916 SO:0000346 37587 37591 loxp +T917 GO:0007618 37653 37658 mated +T918 NCBITaxon:10088 37674 37678 mice +T919 UBERON:0002415 37683 37687 tail +T920 SO:0000112 37785 37791 primer +T921 SO:0000112 37832 37838 primer +T922 CHEBI:60004 37984 37987 Mix +T923 SO:0000112 38035 38041 primer +T924 SO:0001023 38092 38098 allele +T925 SO:0000112 38100 38106 primer +T926 PR:000028746 38153 38157 Pten +T927 SO:0000346 38157 38161 loxP +T928 SO:0000346 38162 38166 loxP +T929 NCBITaxon:10088 38167 38171 mice +T930 GO:0007618 38182 38187 mated +T931 PR:000013530 38193 38195 PB +T932 NCBITaxon:10088 38211 38215 mice +T933 PR:000013530 38247 38249 PB +T934 NCBITaxon:10088 38266 38270 mice +T935 UBERON:0002367 38304 38312 prostate +T936 PR:000028746 38322 38326 Pten +T937 NCBITaxon:33208 38370 38377 Animals +T938 UBERON:0000479 38401 38408 tissues +T939 http://purl.obolibrary.org/obo/MONDO_0005070 38475 38480 tumor +T940 UBERON:0000479 38481 38487 tissue +T941 CHEBI:16842 38523 38531 formalin +T942 CHEBI:51686 38592 38603 hematoxylin +T943 CHEBI:51686 38615 38616 H +T944 UBERON:0000922 38702 38709 embryos +T945 PR:000028746 38767 38771 Pten +T946 PR:000028746 38780 38784 Pten +T947 NCBITaxon:33208 38788 38795 animals +T948 UBERON:0000922 38797 38804 embryos +T949 SO:0000028 39321 39323 bp +T950 PR:000028746 39355 39359 Pten +T951 SO:0000147 39360 39364 exon +T952 SO:0000121 39376 39383;39462 39468 forward ... primer +T953 SO:0000147 39426 39430 exon +T954 SO:0000147 39438 39442 exon +T955 SO:0000132 39454 39468 reverse primer +T956 SO:0000006 39816 39828 PCR products +T957 CHEBI:17368 39834 39846 hypoxanthine +T958 SO:0000112 39975 39982 primers +T959 SO:0000028 40011 40013 bp +T960 UBERON:0002367 40326 40335 Prostates +T961 NCBITaxon:33208 40351 40358 animals +T962 GO:0019835 40519 40524 lysed +T963 CHEBI:9754 40534 40538 Tris +T964 CHEBI:26710 40556 40560 NaCl +T965 CHEBI:63016 40578 40583 NP-40 +T966 CHEBI:35607 40590 40611 sodium ortho-vanadate +T967 CHEBI:35607 40613 40619 Na3VO4 +T968 CHEBI:28741 40628 40631 NaF +T969 CHEBI:60004 40656 40664 cocktail +T970 CHEBI:8984 40888 40891 SDS +T971 UBERON:0000178 40914 40919 Blood +T972 UBERON:0001697 40945 40952 orbital +T973 NCBITaxon:10088 40976 40980 mice +T974 CL:0000232 40982 40991 red cells +T975 GO:0019835 40997 41002 lysed +T976 GO:0019835 41006 41011 lysis +T977 CHEBI:31206 41028 41033 NH4Cl +T978 CHEBI:81862 41041 41046 KHCO3 +T979 GO:0019835 41117 41122 lysed +T980 GO:0019835 41126 41131 lysis +T981 CHEBI:8984 41300 41303 SDS +T982 NCBITaxon:9986 41357 41363 rabbit +T983 PR:000028746 41380 41384 Pten +T984 GO:0042571 41385 41393 antibody +T985 PR:000028746 41400 41404 PTEN +T986 PR:000028746 41405 41411 MMAC-1 +T987 GO:0042571 41513 41521 antibody +T988 CHEBI:32958 41605 41612 Phospho +T989 PR:000029189 41613 41616 Akt +T990 GO:0042571 41637 41647 antibodies +T991 PR:000029189 41656 41659 Akt +T992 CHEBI:32958 41664 41671 phospho +T993 PR:000029189 41686 41689 Akt +T994 UBERON:0000479 42092 42099 Tissues +T995 PR:000003090 42204 42207 p27 +T996 NCBITaxon:10088 42208 42213 mouse +T997 GO:0009293 42239 42251 Transduction +T998 NCBITaxon:9986 42456 42462 Rabbit +T999 CHEBI:32958 42474 42481 phospho +T1000 PR:000029189 42482 42485 Akt +T1001 GO:0042571 42496 42504 antibody +T1002 NCBITaxon:9986 42583 42589 rabbit +T1003 GO:0042571 42601 42609 antibody +T1004 PR:000028746 42610 42614 PTEN +T1005 PR:000028746 42615 42620 MMAC1 +T1006 PR:000010425 42924 42929 Ki-67 +T1007 GO:0042571 42930 42938 antibody +T1008 GO:0042571 43017 43025 antibody +T1009 CHEBI:32958 43092 43099 phospho +T1010 PR:000003041 43100 43104 mTOR +T1011 GO:0042571 43105 43113 antibody +T1012 CHEBI:32958 43163 43170 phospho +T1013 PR:000007641 43171 43176 FOXO3 +T1014 CHEBI:15347 43340 43347 acetone +T1015 CHEBI:16240 43370 43374 H2O2 +T1016 CHEBI:59132 43421 43428 Antigen +T1017 CHEBI:30769 43462 43473 citric acid +T1018 MOP:0000093 43535 43547 biotinylated +T1019 GO:0042571 43558 43568 antibodies +T1020 NCBITaxon:10088 43648 43652 Mice +T1021 http://purl.obolibrary.org/obo/MONDO_0005070 43691 43696 tumor +T1022 NCBITaxon:10088 43932 43936 mice +T1023 CHEBI:6015 43960 43971 isofluorane +T1024 NCBITaxon:33208 44059 44065 animal +T1025 PR:000028746 45010 45014 Pten +T1026 PR:000013530 45084 45086 PR +T1027 NCBITaxon:10088 45102 45106 mice +T1028 http://purl.obolibrary.org/obo/MONDO_0004992 45372 45378 Cancer +T1029 UBERON:0002367 45418 45426 Prostate +T1030 http://purl.obolibrary.org/obo/MONDO_0008315 45418 45433 Prostate Cancer +T1031 UBERON:0002367 45659 45667 prostate +T1032 http://purl.obolibrary.org/obo/MONDO_0010811 45669 45672 BPH +T1033 http://purl.obolibrary.org/obo/MONDO_0010811 45675 45703 benign prostatic hyperplasia +T1034 UBERON:0002367 45682 45691 prostatic +T1035 http://purl.obolibrary.org/obo/MONDO_0008315 45705 45708 CaP +T1036 http://purl.obolibrary.org/obo/MONDO_0008315 45711 45720;45725 45733 cancer of prostate +T1037 UBERON:0002367 45725 45733 prostate +T1038 UBERON:0000922 45740 45749 embryonic +T1039 CHEBI:51686 45756 45757 H +T1040 CHEBI:51686 45762 45773 hematoxylin +T1041 CHEBI:17368 45792 45804 hypoxanthine +T1042 GO:0042571 45877 45885 antibody +T1043 NCBITaxon:10088 45893 45898 mouse +T1044 UBERON:0000922 45899 45908 embryonic +T1045 CL:0000057 45909 45919 fibroblast +T1046 CHEBI:7507 45961 45969 neomycin +T1047 PR:000013530 45971 45973 PB +T1048 PR:000013530 45976 45984 Probasin +T1049 UBERON:0000178 46004 46009 blood +T1050 CL:0000081 46004 46009;46022 46026 blood ... cell +T1051 CL:0000842 46010 46026 mononuclear cell +T1052 GO:0005634 46014 46021 nuclear +T1053 CHEBI:18179 46035 46051 phosphoinositide +T1054 http://purl.obolibrary.org/obo/MONDO_0005193 46062 46065 PIN +T1055 UBERON:0002367 46068 46077 prostatic +T1056 http://purl.obolibrary.org/obo/MONDO_0005193 46068 46103 prostatic intraepithelial neoplasia +T1057 UBERON:0000483 46083 46093 epithelial +T1058 CHEBI:60169 46105 46109 PIP3 +T1059 CHEBI:16618 46112 46152 phosphatidylinositol 3,4,5-trisphosphate +T1060 PR:000028746 46154 46158 PTEN +T1061 PR:000028746 46161 46216 phosphatase and tensin homolog deleted on chromosome 10 +T1062 SO:0000853 46184 46191 homolog +T1063 GO:0016246 46218 46222 RNAi +T1064 GO:0016246 46225 46241 RNA interference +T1065 SO:0000646 46243 46248 siRNA +T1066 SO:0000646 46251 46272 small interfering RNA +T1067 PR:000028746 46352 46356 Pten +T1068 NCBITaxon:10088 46369 46374 Mouse +T1069 SO:0001023 46465 46472 alleles +T1070 PR:000028746 46476 46480 Pten +T1071 PR:000028746 46559 46563 Pten +T1072 NCBITaxon:10088 46576 46581 mouse +T1073 PR:000028746 46616 46620 Pten +T1074 GO:0010467 46621 46631 expression +T1075 CL:0000001 46692 46704 primary cell +T1076 PR:000028746 46781 46785 Pten +T1077 GO:0010467 46786 46796 expression +T1078 PR:000029189 46855 46858 Akt +T1079 PR:000028746 46949 46953 Pten +T1080 CHEBI:32958 46964 46971 phospho +T1081 PR:000029189 46972 46975 Akt +T1082 PR:000029189 46976 46979 Akt +T1083 SO:0000147 47054 47058 exon +T1084 SO:0000121 47062 47069;47103 47110 forward ... primers +T1085 SO:0000147 47075 47079 exon +T1086 SO:0000132 47094 47101;47103 47110 reverse ... primers +T1087 PR:000028746 47115 47119 Pten +T1088 GO:0016246 47152 47180 transcriptional interference +T1089 PR:000028746 47188 47192 Pten +T1090 PR:000028746 47200 47204 Pten +T1091 PR:000028746 47220 47224 Pten +T1092 PR:000028746 47330 47334 Pten +T1093 PR:000028746 47406 47410 Pten +T1094 SO:0000704 47411 47415 Gene +T1095 NCBITaxon:10088 47419 47424 Mouse +T1096 UBERON:0002367 47425 47433 Prostate +T1097 PR:000028746 47456 47460 Pten +T1098 PR:000028746 47556 47560 Pten +T1099 SO:0005853 47617 47625 cassette +T1100 NCBITaxon:10088 47655 47660 mouse +T1101 PR:000028746 47684 47688 Pten +T1102 SO:0000359 47730 47736 floxed +T1103 SO:0000147 47737 47742 exons +T1104 PR:000013530 47770 47772 PB +T1105 PR:000013530 47780 47782 PB +T1106 NCBITaxon:10088 47799 47804 mouse +T1107 SO:0001026 47819 47826 genomic +T1108 SO:0000147 47895 47900 exons +T1109 SO:0005853 47990 47998 cassette +T1110 SO:0000346 48043 48052 loxP site +T1111 PR:000028746 48072 48076 Pten +T1112 SO:0001026 48077 48084 genomic +T1113 GO:0097617 48226 48239 hybridization +T1114 GO:0097617 48342 48355 hybridization +T1115 SO:0000112 48418 48425 primers +T1116 SO:0000359 48447 48453 floxed +T1117 SO:0001023 48454 48460 allele +T1118 PR:000028746 48462 48466 Pten +T1119 SO:0000346 48466 48470 loxP +T1120 SO:0001023 48485 48491 allele +T1121 PR:P23940 48523 48528 BamHI +T1122 PR:P23940 48530 48531 B +T1123 NCBITaxon:10088 48584 48588 mice +T1124 CL:0002322 48635 48642 ES cell +T1125 NCBITaxon:10088 48880 48885 mouse +T1126 UBERON:0002415 48886 48890 tail +T1127 NCBITaxon:10088 48923 48928 mouse +T1128 NCBITaxon:10088 48966 48971 mouse +T1129 PR:000028746 49043 49047 Pten +T1130 SO:0000346 49047 49051 loxP +T1131 SO:0000359 49062 49068 floxed +T1132 PR:000028746 49070 49074 Pten +T1133 SO:0000346 49074 49078 loxP +T1134 NCBITaxon:10088 49157 49162 mouse +T1135 UBERON:0002415 49163 49167 tail +T1136 NCBITaxon:10088 49201 49206 mouse +T1137 NCBITaxon:10088 49234 49239 mouse +T1138 UBERON:0002415 49314 49318 tail +T1139 PR:000013530 49350 49352 PB +T1140 PR:000028746 49363 49367 Pten +T1141 SO:0000346 49367 49371 loxP +T1142 PR:000028746 49386 49390 Pten +T1143 SO:0000346 49390 49394 loxP +T1144 SO:0000112 49409 49416 primers +T1145 PR:000028746 49454 49458 Pten +T1146 SO:0000346 49458 49462 loxP +T1147 PR:000028746 49492 49496 Pten +T1148 PR:000028746 49529 49533 Pten +T1149 SO:0000346 49533 49537 loxP +T1150 SO:0000346 49538 49542 loxP +T1151 UBERON:0002415 49577 49581 tail +T1152 PR:000028746 49613 49617 Pten +T1153 SO:0000346 49617 49621 loxp +T1154 PR:000013530 49636 49638 PB +T1155 PR:000028746 49648 49652 Pten +T1156 SO:0000346 49652 49656 loxP +T1157 SO:0000112 49671 49678 primers +T1158 PR:000028746 49710 49714 Pten +T1159 SO:0000346 49714 49718 loxP +T1160 PR:000028746 49740 49744 Pten +T1161 SO:0001023 49788 49794 allele +T1162 UBERON:0002415 49805 49809 tail +T1163 UBERON:0002367 49889 49897 prostate +T1164 PR:000028746 49907 49911 Pten +T1165 PR:000028746 49933 49937 Pten +T1166 SO:0000346 49937 49941 loxP +T1167 SO:0000346 49942 49946 loxP +T1168 PR:000013530 49973 49975 PB +T1169 NCBITaxon:10088 49991 49995 mice +T1170 PR:000013530 49999 50001 PB +T1171 NCBITaxon:10088 50018 50022 mice +T1172 PR:000028746 50035 50039 Pten +T1173 NCBITaxon:10088 50044 50048 Mice +T1174 http://purl.obolibrary.org/obo/MONDO_0005043 50065 50076 Hyperplasia +T1175 http://purl.obolibrary.org/obo/MONDO_0008315 50090 50093 CaP +T1176 http://purl.obolibrary.org/obo/MONDO_0005070 50109 50119 neoplastic +T1177 UBERON:0002367 50129 50137 prostate +T1178 UBERON:0000479 50143 50149 tissue +T1179 NCBITaxon:10088 50173 50177 mice +T1180 PR:000028746 50195 50199 Pten +T1181 UBERON:0001328 50266 50280 prostate lobes +T1182 PR:000028746 50371 50375 Pten +T1183 NCBITaxon:10088 50380 50384 mice +T1184 UBERON:0000998 50465 50481 seminal vesicles +T1185 UBERON:0000998 50483 50485 SV +T1186 UBERON:0018707 50523 50530 bladder +T1187 UBERON:0018707 50532 50534 Bl +T1188 UBERON:0002367 50580 50588 prostate +T1189 http://purl.obolibrary.org/obo/MONDO_0021259 50580 50595 prostate tumors +T1190 PR:000028746 50640 50644 Pten +T1191 PR:000028746 50651 50655 Pten +T1192 NCBITaxon:10088 50659 50663 mice +T1193 PR:000028746 50701 50705 Pten +T1194 NCBITaxon:10088 50710 50715 mouse +T1195 UBERON:0001329 50761 50769 AP lobes +T1196 UBERON:0000998 50788 50804 seminal vesicles +T1197 UBERON:0001329 50844 50851 AP lobe +T1198 PR:000028746 50875 50879 Pten +T1199 NCBITaxon:33208 50884 50890 animal +T1200 http://purl.obolibrary.org/obo/MONDO_0005070 50919 50929 neoplastic +T1201 http://purl.obolibrary.org/obo/MONDO_0005043 51031 51043 hyperplastic +T1202 UBERON:0002367 51044 51052 prostate +T1203 PR:000028746 51054 51058 Pten +T1204 GO:0010467 51067 51077 expression +T1205 GO:0010467 51102 51111 expressed +T1206 NCBITaxon:33208 51152 51159 animals +T1207 CHEBI:51686 51183 51184 H +T1208 http://purl.obolibrary.org/obo/MONDO_0005193 51253 51256 PIN +T1209 UBERON:0003891 51287 51294 stromal +T1210 UBERON:0000479 51295 51301 tissue +T1211 PR:000028746 51329 51333 Pten +T1212 PR:000028746 51357 51361 Pten +T1213 UBERON:0000479 51365 51371 tissue +T1214 http://purl.obolibrary.org/obo/MONDO_0005043 51383 51395 hyperplastic +T1215 PR:000028746 51409 51413 Pten +T1216 UBERON:0000479 51417 51423 tissue +T1217 PR:000028746 51495 51499 Pten +T1218 NCBITaxon:10088 51543 51548 Mouse +T1219 PR:000010425 51561 51566 Ki-67 +T1220 UBERON:0001329 51579 51586 AP lobe +T1221 GO:0008283 51619 51632 proliferation +T1222 PR:000028746 51649 51653 Pten +T1223 PR:000010425 51674 51679 Ki-67 +T1224 CHEBI:32958 51755 51762 phospho +T1225 PR:000029189 51763 51766 Akt +T1226 PR:000029189 51767 51770 Akt +T1227 UBERON:0002367 51805 51814 prostates +T1228 PR:000028746 51828 51832 Pten +T1229 NCBITaxon:33208 51836 51843 animals +T1230 CHEBI:32958 51929 51936 phospho +T1231 PR:000029189 51937 51940 Akt +T1232 GO:0042571 51941 51951 antibodies +T1233 GO:0005886 51967 51982 plasma membrane +T1234 CHEBI:32958 51999 52006 phospho +T1235 PR:000029189 52007 52010 Akt +T1236 PR:000003090 52037 52040 p27 +T1237 CHEBI:32958 52068 52075 phospho +T1238 PR:000003041 52076 52080 mTOR +T1239 CHEBI:32958 52085 52092 phospho +T1240 PR:000007641 52103 52108 FOXO3 +T1241 GO:0042571 52109 52119 antibodies +T1242 PR:000028746 52147 52151 Pten +T1243 NCBITaxon:10088 52172 52177 mouse +T1244 UBERON:0002367 52178 52187 prostates +T1245 UBERON:0002367 52237 52245 prostate +T1246 PR:000028746 52319 52323 Pten +T1247 PR:000028746 52347 52351 Pten +T1248 PR:000028746 52380 52384 Pten +T1249 NCBITaxon:10088 52408 52412 mice +T1250 UBERON:0002367 52440 52449 prostatic +T1251 PR:000028746 52487 52491 Pten +T1252 PR:000028746 52498 52502 Pten +T1253 NCBITaxon:10088 52506 52510 mice +T1254 http://purl.obolibrary.org/obo/MONDO_0008315 52539 52542 CaP +T1255 http://purl.obolibrary.org/obo/MONDO_0008315 52553 52556 CaP +T1256 http://purl.obolibrary.org/obo/MONDO_0005070 52572 52577 tumor +T1257 UBERON:0002367 52617 52633 prostatic glands +T1258 GO:0040007 52638 52645 growing +T1259 UBERON:0003891 52667 52673 stroma +T1260 PR:000028746 52712 52716 Pten +T1261 NCBITaxon:10088 52720 52724 mice +T1262 PR:000028746 52739 52743 Pten +T1263 NCBITaxon:10088 52747 52751 mice +T1264 PR:000028746 52766 52770 Pten +T1265 NCBITaxon:10088 52775 52779 mice +T1266 http://purl.obolibrary.org/obo/MONDO_0008315 52856 52859 CaP +T1267 PR:000028746 52872 52876 Pten +T1268 NCBITaxon:10088 52880 52884 Mice +T1269 http://purl.obolibrary.org/obo/MONDO_0008315 52902 52905 CaP +T1270 PR:000028746 52949 52953 Pten +T1271 PR:000028746 52962 52966 Pten +T1272 NCBITaxon:10088 52970 52974 mice +T1273 http://purl.obolibrary.org/obo/MONDO_0005070 52984 52989 tumor +T1274 CHEBI:51686 52997 52998 H +T1275 UBERON:0000428 53093 53113 prostatic epithelium +T1276 PR:000028746 53158 53162 Pten +T1277 GO:0042571 53163 53171 antibody +T1278 PR:000028746 53211 53215 Pten +T1279 PR:000028746 53224 53228 Pten +T1280 NCBITaxon:10088 53232 53236 mice +T1281 NCBITaxon:10088 53251 53255 mice +T1282 GO:0005737 53264 53275 cytoplasmic +T1283 UBERON:0000483 53301 53311 epithelial +T1284 CL:0000066 53301 53317 epithelial cells +T1285 PR:000028746 53335 53339 Pten +T1286 NCBITaxon:10088 53343 53347 mice +T1287 PR:000028746 53389 53393 Pten +T1288 NCBITaxon:10088 53397 53401 mice +T1289 UBERON:0000428 53441 53461 prostatic epithelium +T1290 UBERON:0002367 53522 53530 prostate +T1291 http://purl.obolibrary.org/obo/MONDO_0021259 53522 53536 prostate tumor +T1292 PR:000028746 53568 53572 Pten +T1293 NCBITaxon:10088 53576 53580 mice +T1294 UBERON:0002367 53632 53641 prostates +T1295 PR:000028746 53645 53649 Pten +T1296 PR:000028746 53656 53660 Pten +T1297 NCBITaxon:10088 53664 53668 mice +T1298 UBERON:0018707 53693 53700 Bladder +T1299 UBERON:0018707 53702 53704 Bl +T1300 UBERON:0000998 53710 53726 seminal vesicles +T1301 UBERON:0000998 53728 53730 SV +T1302 NCBITaxon:33208 53794 53801 animals +T1303 PR:000028746 53846 53850 Pten +T1304 PR:000028746 53894 53898 Pten +T1305 NCBITaxon:10088 53902 53906 mice +T1306 CHEBI:51686 53920 53921 H +T1307 UBERON:0002367 53949 53957 prostate +T1308 PR:000028746 53963 53967 Pten +T1309 NCBITaxon:10088 53971 53975 mice +T1310 http://purl.obolibrary.org/obo/MONDO_0005193 54014 54017 PIN +T1311 UBERON:0002367 54049 54058 prostatic +T1312 http://purl.obolibrary.org/obo/MONDO_0005082 54049 54073 prostatic adenocarcinoma +T1313 http://purl.obolibrary.org/obo/MONDO_0005070 54119 54129 neoplastic +T1314 CHEBI:51686 54185 54186 H +T1315 UBERON:0002367 54202 54211 prostates +T1316 PR:000028746 54217 54221 Pten +T1317 NCBITaxon:10088 54225 54229 mice +T1318 http://purl.obolibrary.org/obo/MONDO_0008315 54255 54258 CaP +T1319 http://purl.obolibrary.org/obo/MONDO_0008315 54288 54293 tumor +T1320 GO:0040007 54300 54307 growing +T1321 UBERON:0003891 54313 54320 stromal +T1322 PR:000028746 54339 54343 Pten +T1323 UBERON:0002367 54357 54365 Prostate +T1324 http://purl.obolibrary.org/obo/MONDO_0021259 54357 54371 Prostate Tumor +T1325 PR:000028746 54385 54389 Pten +T1326 NCBITaxon:10088 54393 54397 mice +T1327 http://purl.obolibrary.org/obo/MONDO_0005043 54406 54417 hyperplasia +T1328 http://purl.obolibrary.org/obo/MONDO_0005193 54444 54447 PIN +T1329 PR:000028746 54449 54453 Pten +T1330 NCBITaxon:10088 54458 54462 mice +T1331 http://purl.obolibrary.org/obo/MONDO_0005193 54505 54508 PIN +T1332 http://purl.obolibrary.org/obo/MONDO_0008315 54567 54570 CaP +T1333 PR:000028746 54584 54588 Pten +T1334 NCBITaxon:10088 54591 54595 mice +T1335 http://purl.obolibrary.org/obo/MONDO_0008315 54613 54616 CaP +T1336 PR:000003090 54686 54693 p27Kip1 +T1337 NCBITaxon:10088 54697 54701 mice +T1338 http://purl.obolibrary.org/obo/MONDO_0010811 54733 54736 BPH +T1339 NCBITaxon:9606 54756 54761 human +T1340 PR:000029189 54850 54853 AKT +T1341 PR:000003041 54858 54862 mTOR +T1342 PR:000028746 54888 54892 PTEN +T1343 PR:000028746 54956 54960 PTEN +T1344 GO:0010467 54961 54971 expression +T1345 SO:0001023 54996 55002 allele +T1346 http://purl.obolibrary.org/obo/MONDO_0005193 55039 55042 PIN +T1347 PR:000028746 55055 55059 PTEN +T1348 NCBITaxon:1 55063 55074 individuals +T1349 PR:000028746 55181 55185 Pten +T1350 NCBITaxon:10088 55189 55194 mouse +T1351 CHEBI:33893 55576 55584 reagents +T1352 NCBITaxon:9606 55710 55715 Human diff --git a/src/ontogpt/evaluation/craft/database/all/14691534.txt b/src/ontogpt/evaluation/craft/database/all/14691534.txt new file mode 100644 index 000000000..f7bd06902 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/14691534.txt @@ -0,0 +1,249 @@ +Pten Dose Dictates Cancer Progression in the Prostate + +Abstract + +Complete inactivation of the PTEN tumor suppressor gene is extremely common in advanced cancer, including prostate cancer (CaP). However, one PTEN allele is already lost in the vast majority of CaPs at presentation. To determine the consequence of PTEN dose variations on cancer progression, we have generated by homologous recombination a hypomorphic Pten mouse mutant series with decreasing Pten activity: Ptenhy/+ > Pten+/− > Ptenhy/− (mutants in which we have rescued the embryonic lethality due to complete Pten inactivation) > Pten prostate conditional knockout (Ptenpc) mutants. In addition, we have generated and comparatively analyzed two distinct Ptenpc mutants in which Pten is inactivated focally or throughout the entire prostatic epithelium. We find that the extent of Pten inactivation dictate in an exquisite dose-dependent fashion CaP progression, its incidence, latency, and biology. The dose of Pten affects key downstream targets such as Akt, p27Kip1, mTOR, and FOXO3. Our results provide conclusive genetic support for the notion that PTEN is haploinsufficient in tumor suppression and that its dose is a key determinant in cancer progression. + +Introduction + +The PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor gene is located on chromosome 10q23, a genomic region frequently lost in human cancers. Somatic deletions or mutations of this gene have been identified in a large fraction of tumors, frequently in prostate cancer (CaP), placing PTEN among the most commonly mutated tumor suppressor genes in human cancer (Cantley and Neel 1999; Di Cristofano and Pandolfi 2000). + +As dictated by Knudson's “two-hit” hypothesis (Knudson 1971), however, the analysis of cancer samples for mutations in PTEN has been performed searching for biallelic inactivation of the gene, which pointed at complete PTEN inactivation as a late event in cancer progression. The consequence of loss or mutation in one PTEN allele in carcinomas in situ or in primary cancers may have been underestimated. It could be postulated that if PTEN were to be haploinsufficient for some of its tumor-suppressive functions, loss of one PTEN allele or reduction in its expression may be playing a key role in tumor initiation, while further reduction of its function/expression may favor invasion and possibly tumor metastasis in advanced cancers. + +In agreement with this hypothesis, it has been reported that primary tumors often show loss or alteration of at least one PTEN allele (e.g., 70%–80% of primary CaPs; Gray et al. 1998; Whang et al. 1998), while homozygous inactivation of PTEN is generally associated with advanced cancer and metastasis (Cantley and Neel 1999; Di Cristofano and Pandolfi 2000), supporting a possible key role for progressive PTEN functional loss in tumor progression. + +The elucidation of the molecular basis for tumor initiation and progression in most epithelial neoplasms has been hindered by the lack of suitable laboratory and preclinical models that accurately reflect the genetic and histopathological progression of these cancers. Furthermore, the outcome of a progressive dose reduction in tumor suppressor function has been rarely assessed in vivo in the mouse. Small interfering RNA (siRNA) technology has more recently allowed testing the consequence of knockdown of a tumor suppressor such as p53 in specific cell types such as hemopoietic stem cells, by generating epi-allelic series of hypomorphs created by stable RNA interference (RNAi) transduction (Hemann et al. 2003). In the case of Pten, this analysis is further complicated since complete inactivation of the gene results in early embryonic lethality and aberrant developmental programs (Di Cristofano et al. 1998, 2001a; Suzuki et al. 1998; Podsypanina et al. 1999). Thus, a further unrestricted reduction of the Pten dose could still result in embryonic lethality. On the other hand, the consequence of complete Pten inactivation, even when restricted to a specific organ/tissue, could still affect the developmental program of that organ, while complete somatic loss of Pten in an adult organ would better approximate what is normally observed in human cancer. We have addressed these issues for Pten, as we describe in this article, through the generation of (i) hypomorphic mouse mutants and (ii) conditional mutants for complete prostate-specific Pten inactivation after puberty. + +We previously reported that Pten heterozygous (Pten+/−) mutants are prone to develop neoplasms of various histological origins, including prostatic intraepithelial neoplasias (PIN) after a long latency ( >14 mo) and at incomplete penetrance (40%) (Di Cristofano et al. 2001a). Pten+/− mutants, however, never develop invasive CaPs. Invasive CaPs were, by contrast, observed in compound Pten+/−/p27Kip1+/− or Pten+/−/p27Kip1−/− mutants (Di Cristofano et al. 2001a). These lesions appeared to originate in PIN lesions, which then become invasive. However, no metastatic CaP was observed in this model, also in view of early lethality due to the occurrence of concomitant tumors of various histogenesis. Analysis of tumors, PIN, and CaP in Pten+/− mice and Pten/p27Kip1 compound mutants strongly suggested that Pten may be haploinsufficient in prostate tumor suppression since Pten protein expression was never lost in these lesions. If this was indeed the case, a further reduction in the Pten dose with respect to Pten+/− mutants should impact on tumorigenesis and tumor progression. By contrast, in case Pten needed to be completely lost for phenotypic consequences to be manifest, only a complete Pten inactivation would hasten the neoplastic process. + +The discovery that phosphatidylinositol 3,4,5-trisphosphate (PIP3) is the main in vivo substrate of PTEN (Maehama and Dixon 1998) placed this phosphatase into a well-defined pathway (reviewed in Vivanco and Sawyers 2002). PIP3 levels are very low in quiescent cells, but rapidly increase upon stimulation by growth factors, through phosphoinositide 3-kinase (PI3K) activation. The role of PTEN is to keep the levels of PIP3 low by dephosphorylation at the D3 position. Loss of PTEN function results in increased PIP3 levels and subsequent Akt hyperactivation/phosphorylation (Stambolic et al. 1998; Di Cristofano et al. 1999; Backman et al. 2002; Vivanco and Sawyers 2002). It may therefore be proposed that a progressive reduction in the Pten dose could simply result in a concomitant progressive dose-dependent increase in Akt activation and its downstream molecular biological consequences. Alternatively, Pten expression levels may constitute discrete biochemical thresholds below which qualitative functional changes would occur, contributing to tumor progression and invasion. + +CaP is the most common non-skin malignancy among Western adult males. It is estimated that in 2003, approximately 220,900 new cases and 28,900 CaP-related deaths will occur in the United States. Approximately 70% of CaP patients will harbor mutation or display loss of at least one allele of PTEN ensuing in the constitutive activation of the PI3K/mTOR pathway (Gray et al. 1998; Whang et al. 1998; Di Cristofano and Pandolfi 2000). As aforementioned, those mutations are in positive correlation with tumor stage and grade, PTEN being completely lost in approximately 40% of metastatic CaPs (Gray et al. 1998; Whang et al. 1998). We therefore decided to focus on the prostate as an important model system to determine genetically whether and how the dose of Pten dictates tumor initiation and progression. + +We demonstrate in vivo, in a hypomorphic mouse mutant series, that Pten plays a crucial dose-dependent role in CaP tumor suppression and that Pten progressive inactivation leads to both quantitative and qualitative molecular and biological changes towards full-blown tumorigenesis. + +Results + +Generation of a Hypomorphic Pten Mutant Series + +We have previously reported that Pten heterozygous mutants are tumor prone and viable, while complete Pten inactivation results in embryonic lethality (Di Cristofano et al. 1998, 2001a). We therefore decided to generate and characterize a mouse mutant in which the dose of Pten would be reduced further below the levels observed in a Pten+/− mutant. To this end, we at first engineered by homologous recombination an allele of Pten that would express a wild-type Pten gene at reduced levels, taking advantage of the well-known phenomenon of transcriptional interference (McDevitt et al. 1997; Morita et al. 2003). We therefore targeted within Pten intron 3 the neomycin (Neo) cassette under the control of the strong CMV promoter (Figure 1A; see also Figure 3A) in mouse embryonic stem (ES) cells. Transcription of the Neo cassette in the recombined allele was expected to interfere with the transcription of the Pten gene, in turn resulting in lower expression levels of a wild-type Pten protein by this allele. Correctly recombined ES clones were obtained (see Figure 3B; see Materials and Methods) and injected into blastocysts for germline transmission. Mutants harboring the Ptenhy allele were obtained from recombined ES cells and were found viable, thriving, and fertile. We next intercrossed Ptenhy/+ mice with Pten+/− mice to generate a hypomorphic series (Figure 1B). + +Partial Rescue of Embryonic Lethality in Pten Hypomorphic Mutants + +The further reduction of the Pten dose could indeed result in embryonic lethality; we therefore assessed whether compound Ptenhy/− mice would be born at all. In the analyzed cohort (n = 190), viable male and female Ptenhy/− mutants were in fact obtained. However, the frequencies among various genotypes did not follow Mendelian ratios. Out of the 25% Ptenhy/− mutants expected in these crosses, only 10% were born. In particular, approximately 40% of the Ptenhy/− males and 75% of the Ptenhy/− females were lost during gestation. This strongly suggested that a further reduction of the Pten dose had occurred in Ptenhy/− mutants and that this reduction still results in embryonic lethality, albeit at incomplete penetrance (the embryonic phenotype of the Ptenhy/− will be described elsewhere; L. C. Trotman and P. P. Pandolfi, unpublished data). Enough viable Ptenhy/− males were obtained, however, and monitored for tumorigenesis in the prostate. As predicted, a significant reduction in Pten levels and the consequent increase in Akt activation (phospho-Akt/Akt ratio) were observed in organs and primary cells from Ptenhy/− mutants when compared to Pten+/−, Ptenhy/+, and wild-type littermates. In particular, we analyzed mouse embryonic fibroblasts (MEFs), peripheral blood mononuclear cells (PBMCs), and prostates of various genotypes (Figure 1C; Figure 2A and 2B; data not shown). Semiquantitative RT–PCR analysis confirmed the reduction of Pten mRNA expression in Ptenhy/− MEFs when compared to Pten+/− and wild-type cells (see Figure 1D). + +Massive Prostate Hyperplasia and Invasive CaP in Ptenhy/− Mutants + +The levels of Pten were indeed further reduced in the prostates from Ptenhy/− mutants when compared to wild-type, Ptenhy/+, and Pten+/− mice (Figure 2A and 2B). Conversely, Akt activation was increased in Ptenhy/− mutants (Figure 2B). On this premise, we therefore studied prostate tumorigenesis in Ptenhy/+ (n = 26), Pten+/− (n = 24 in this study plus n = 18, as previously reported; Di Cristofano et al. 2001a), and Ptenhy/− (n = 14) mutants on a comparative basis. Mice were followed throughout their lives according to the following experimental scheme: (i) all cohorts were subjected to serial monthly (in selected cases, biweekly) nuclear magnetic resonance imaging (MRI) (see Materials and Methods) for detection of morphological changes in the size and shape of the prostate; (ii) mice were sacrificed at or prior to tumor detection for postmortem pathological analysis or when manifest sign of distress were observed (owing to tumor codevelopment; Ptenhy/− mutants, as Pten+/− mice, in fact, developed tumors of multiple histologic origins; data not shown). Striking differences were observed in the prostates of these three cohorts over time. The prostates of Pten+/+ and Pten+/− mice were never found enlarged by MRI analysis at any age (Figure 2C; see Figure 5D). Pten+/− mutants developed moderate/low-grade PINs at incomplete penetrance (35%–40% approximately, after a long latency of >12 mo) that were only detected by serial postmortem analysis, as previously reported (Figure 2F; Di Cristofano et al. 2001a). Strikingly, however, MRI analysis immediately revealed a profound difference in the Ptenhy/− cohort. The prostates of these mice were found markedly enlarged, and prostate growths were detected starting at 3 mo (see Figure 5D). By 6–8 mo, these growths typically surpassed the size of seminal vesicles (Figure 2C and 2D). Interestingly, prostate enlargements were not accompanied by an overall increase in total body weight or size in Ptenhy/− mutants (data not shown). Postmortem pathological analysis of such mutants confirmed the marked epithelial hyperplasia of the prostate (Figure 2F). All lobes were found to be hypercellular, with prominent macroscopic enlargements of anterior and dorsolateral lobes. Microscopically, the regular and round prostatic glands observed in wild-type mice were replaced by large, irregular, and complex glands that contained multiple intraluminal papillary projections already at age 2–3 mo. Furthermore, many cells had sizeable nuclei, clumped chromatin, and prominent nucleoli, forming foci of epithelial dysplasia.The phenotype observed in the Ptenhy/− mice was characterized by a wide spectrum of levels of cellular differentiation, which was not typically observed in the Pten+/− mice. As we previously reported in Pten+/− and Pten+/−/p27Kip1−/− mutants (Di Cristofano et al. 2001a), Pten protein expression was also still retained in the hyperplastic prostates from Ptenhy/− mutants, and Southern blot analysis confirmed retention of the hypomorphic Pten allele (Figure 2E; data not shown). Importantly, approximately 25% of the Ptenhy/− mice analyzed at more than 6 mo of age showed histological signs of local invasion, with tumor cells disrupting the basement membrane of the gland and growing into the surrounding stroma (Figure 2F; see Figure 5E). Thus, strikingly, a further reduction in the Pten dose as observed in Ptenhy/− mutants when compared with Pten+/− mutants does lead to massive prostate hyperplasia at complete penetrance and accelerates tumor progression from high-grade PIN (grade 3–4 tumor as per the CaP grading system for genetically engineered mice; Park et al. 2001) to locally invasive CaP in this mouse model. + +Generation of Conditional Prostate-Specific Pten Mutants + +We next determined whether complete Pten inactivation in the prostate would further affect CaP tumor progression. To this end, we generated mouse mutants in which Pten exons 4 and 5 are flanked by loxP sites (Figure 3A; see Materials and Methods). The Neo cassette was excised from the locus in vivo by intercrossing Ptenhy/+ with EIIA-Cre transgenic mice (Lakso et al. 1996). In these mice, the Cre recombinase is expressed transiently, early in embryogenesis, allowing the production of a mosaic progeny that harbors in the germline Pten alleles in three different configurations: targeted unmodified (still retaining the Neo cassette), Pten floxed, or Pten deleted (Figure 3A). Breeding of these mosaic mutants allowed us to generate mice heterozygous for the Pten floxed allele (PtenloxP mutants; see Figure 3B). + +As expected, PtenloxP mice were born following Mendelian frequencies, viable and fertile, and were utilized for the conditional inactivation of the Pten gene in the prostate. To this end, we made use of two different Cre transgenic lines, PB-Cre and PB-Cre4, in which the Cre gene is under the control of two distinct versions of the rat Probasin (PB) gene promoter (Maddison et al. 2000; Wu et al. 2001). The PB gene is expressed in the prostatic epithelium postpuberty since the gene is androgen responsive (Matusik et al. 1986). Hence, using both PB-Cre transgenic lines, excision of the Pten gene would occur in the prostatic epithelium postpuberty. This is of relevance because it avoids any possible developmental effect due to complete inactivation of Pten during prostate organogenesis. The main difference in the two lines essentially resides in the strength of the promoter. In the PB-Cre4 line (Wu et al. 2001), the Cre gene is driven by a composite promoter, ARR2 PB, which is a derivative of the rat PB promoter from which the PB-Cre line (Maddison et al. 2000) was originally generated. PB-Cre4 mice express Cre at high levels and at high penetrance, while PB-Cre mice express Cre at lower levels and in fewer cells. This could in turn result in a more focal or more diffuse inactivation on Pten in the prostatic epithelium, as we could indeed document (Figure 4A). Also of note, PB-Cre and PB-Cre4 mice were crossed with PtenloxP mutants in order to generate PB-Cre/PtenloxP/loxP (hereafter referred to as Ptenpc1 mutants) or PB-Cre4/PtenloxP/loxP (hereafter referred to as Ptenpc2 mutants) (see Figure 3C). + +The Dose of Pten Dictates Tumor Progression in the Prostate + +We next studied the impact of complete Pten inactivation on prostate tumorigenesis in Ptenpc1 and Ptenpc2 mutants. Mice were followed over time exactly as described for the Pten hypomorphic series by serial MRI and pathological analysis over a period of approximately 18 mo (Figure 5D and 5E). Strikingly, we observed a dramatic difference in Ptenpc1/2 mutants when compared to Ptenhy/− mutants, but also, importantly, when comparing Ptenpc1 with Ptenpc2 mutants. MRI and macroscopic postmortem analysis of the prostate revealed a rapid massive enlargement of all the prostatic lobes already manifested from 2–3 mo of age in Ptenpc2 mutants at complete penetrance (see Figure 4B; Figure 5D). In Ptenpc1 mutants, the enlargement was less pronounced at early stages and undetectable by MRI, but still apparent (see Figure 4B; Figure 5D). As aforementioned, the pattern of Pten inactivation in the prostatic epithelium was different in Ptenpc1 versus Ptenpc2 mutants (see Figure 4A; Ptenpc1 focal versus Ptenpc2 widespread). This difference also correlated with a clear difference in both the morphology and proliferative rates of the prostatic epithelium in these two models (see Figure 4A; see also the following paragraph). Thus, Ptenpc2 mutants displayed a much more severe prostate enlargement than did Ptenpc1 mutants (see Figure 4B; Figure 5D). Ptenpc1 mice displayed focal areas of epithelial hyperplasia, with enlarged glands formed by relatively regular cells, while Ptenpc2 mice presented disorganized hyperplastic glands in all lobes with signs of cellular dysplasia, containing large, irregular cells, forming at times cryptic glandular formations (see Figure 4A). + +Even more strikingly, however, pathological analysis of prostates from Ptenpc1 and Ptenpc2 mice revealed in both mutants, at complete penetrance, the development of invasive and diffuse CaP after a variable latency (see Figure 4B; Figure 5E). Tumors were in both cases made of Pten null cells (data not shown). Continuity of local invasive disease with that of intraepithelial lumens was often observed. The tumors were composed of rather large, mature epithelial cells. Neuroendocrine features, such as cytoplasmic granularity and small round cells, were not seen in the tumors analyzed (see Figure 4B). Although the tumors were clearly invasive in both Ptenpc1 and Ptenpc2 mutants with disruption of the basement membrane and clear signs of organ infiltration, once again we observed differences in the CaPs from these two models. In many instances, in Ptenpc2 mutants more than one prostate lobe was completely effaced by the infiltrating neoplastic cells, suggesting a multifocal tumor origin, while invasive tumors in Ptenpc1 mutants were often affecting only one lobe at a time and the extent of the infiltration was less diffuse (see Figure 4B). In addition, tumors in the Ptenpc1 animals were well-to-moderately differentiated while tumors in the Ptenpc2 mice were in general high-grade, undifferentiated lesions. In contrast, Ptenhy/− mutants displayed a spectrum of phenotypes, from well to undifferentiated and occasionally to locally invasive phenotypes, as previously shown. + +Thus, Pten conditional inactivation in the prostate leads to invasive and diffuse CaP at complete penetrance. + +Effects of the Pten Dose on the Biology of the Prostatic Epithelium + +We studied the molecular and biological consequences of Pten dose variations in the prostatic epithelium of our various mutants. We included in the analysis age-matched (8-wk-old) Pten+/+, Pten+/−, Ptenhy/−, and Ptenpc2 males. The latter mutants were used instead of Ptenpc1 mice in view of the more widespread pattern of Pten inactivation observed in their prostates. + +First, we determined whether reduction in the Pten dose would affect proliferation of epithelial cells. Upon Ki-67 staining, we observed a progressive increase in the proliferative rates of prostate epithelial cells, which correlated with the reduction in Pten expression levels (Figure 5A). For instance, approximately 10-fold more cells were found in active proliferation in the prostates from Ptenpc2 mutants when compared with Pten+/+ mice. Thus, Pten inactivation, in a dose-dependent manner, lends to epithelial cells a dramatic growth advantage in the prostate. By contrast, MEFs of all genotypes from the hypomorphic series did not show noticeable differences in their growth rates under standard culture conditions, while showing progressive activation of the Akt signaling pathway (data not shown; see Figure 1C). This may reflect an intrinsic differential cell-type sensitivity to Pten inactivation, also in agreement with the fact that Pten hypomorphism does not result in overall increased body size and weight. + +We next assessed whether and how the progressive reduction of the Pten dose would affect the functions of key downstream targets such as Akt, p27Kip1, mTOR, and FOXO3 by immunohistochemistry (IHC) and Western blot (WB) analysis (see Materials and Methods). As aforementioned, we studied the status of Akt phosphorylation, the major known biochemical effect of Pten loss (Vivanco and Sawyers 2002), using an anti-phospho-Akt-specific antibody on cells and extracts from the various Pten mutants. Staining of prostates from various genotypes with this antibody increased proportionally to the decrease in Pten levels as confirmed by WB analysis (see Figures 2B, 5B, and 5C; data not shown). In addition, IHC analysis revealed a drastically different staining pattern in prostate cells from Ptenpc2 mutants when compared with cells from other genotypes in that phospho-Akt was found to overtly accumulate at the plasma membrane (Figure 5C). A similar staining pattern was observed both in Pten null epithelium prior to tumor occurrence and in cells from overt tumors from both Ptenpc1 and Ptenpc2 mutants (data not shown). Thus, Akt is progressively activated by a reduction in the Pten dose, which in turn results in its recruitment to the plasma membrane. + +We next studied the effects of Pten inactivation on p27Kip1, FOXO3, and mTOR, known targets of Akt kinase activity. Phosphorylation of p27Kip1 and FOXO3 by Akt results in their functional inactivation through multiple mechanisms such as nuclear export, in the case of FOXO (Brunet et al. 1999), or inhibition of nuclear import and downregulation, in the case of p27Kip1 (Mamillapalli et al. 2001; Liang et al. 2002; Shin et al. 2002; Viglietto et al. 2002). By contrast, Akt activation results in mTOR phosphorylation and increased protein translation and translation initiation (Gingras et al. 2001). We previously reported that the localization or the expression levels of p27Kip1 were not affected in Pten+/− and Pten+/−/p27Kip1−/− compound mutants (Di Cristofano et al. 2001a). By contrast, a progressive downregulation in p27Kip1 staining was observed in Ptenhy/− and Ptenpc2 mutants (Figure 5C; data not shown). p27Kip1 was apparently expressed at lower levels, and fewer cells were found to express the protein (Figure 5C). We also studied the status of FOXO3 in the various Pten mutants, analyzing both its localization and threonine phosphorylation with a specific anti-phospho-FOXO3 antibody (see Materials and Methods). While no noticeable differences were observed in Pten+/− and Ptenhy/− mutants (data not shown), increase in the levels of anti-phospho-FOXO3 cytoplasmic staining were observed in prostate epithelial cells from Ptenpc2 mutants when compared to wild-type mice (Figure 5C). Complete Pten inactivation also resulted in an increase in mTOR phosphorylation in the prostatic epithelium of Ptenpc2 mutants (Figure 5C). + +Thus, the progressive reduction of the Pten dose (and the extent of Pten inactivation in Ptenpc1/2 mutants) affects the proliferative rate of the prostatic epithelium and results in molecular changes, which in turn dictates the natural history of these lesions and tumor progression (Figure 5D and 5E). + +Discussion + +Our analysis allows a detailed deconstruction the molecular genetics underlying cancer progression in the prostate and the assessment of the key relevance of Pten and subtle variations in its dose in controlling this process. Based on our findings, we can now attribute distinct preneoplastic or malignant pathological entities to distinct molecular states. Furthermore, this new knowledge allows the reclassification, on the basis of their true molecular nature, of pathological lesions that at a superficial analysis appeared very similar (Figure 6). + +In this supposedly linear and multistep process, which separates two extremely different anatomical and pathological entities, a normal prostatic epithelium from an invasive CaP, there are, in between, a discrete number of anatomically distinct intermediary steps, such as prostate hyperplasia > displasia/low-grade PIN > high-grade PIN > locally invasive CaP > diffused CaP. These entities have been recognized before on the basis of their pathological features. The fact that we are now able to correlate these anatomical stages with specific molecular events, such as the level of expression of a single tumor suppressor gene, not only represents an integration of anatomical, descriptive findings with biological data, but also offers the opportunity of therapeutic interventions. + +We show in this study that inactivation of one Pten allele in the mouse may lead to prostate epithelial hyperplasia, as complete inactivation of p27Kip1 in the mouse leads to what resembles human benign prostate hyperplasia (BPH). However, these two lesions are very different in nature and outcome: the first, in fact, impacts only on epithelial elements and over time evolves to low-grade PIN, while the second impacts on both epithelial and stroma elements, representing hyperplasia of the whole organ, but does not evolve toward malignancy (Figure 6; Cordon-Cardo et al. 1998; Di Cristofano et al. 2001a). This obviously does not exclude that loss of p27Kip1 expression when accompanied by additional genetic events (e.g., loss of PTEN) may lead to a completely different outcome, as is also supported by our previous in vivo analysis in the mouse (Figure 6; Di Cristofano et al. 2001a). + +Moreover, our data demonstrate that a further reduction in the Pten dose, as observed in the Ptenhy/− mutants, accelerates tumor progression dramatically, eventually resulting in high-grade PIN and locally invasive carcinoma (Figure 6). This fact has very important therapeutic implications. As a large number of CaP patients (more than 80%) display at presentation loss of one PTEN allele, but do retain the other normal PTEN allele, it could be proposed that high-grade PIN or locally invasive prostate carcinomas could be prevented or treated by pharmacologically modulating the expression of the remaining PTEN allele or by antagonizing the downstream consequences of PTEN downregulation or partial inactivation (e.g., PI3K/mTOR activation; Figure 6). + +While the massive prostate hyperplasia/dysplasia observed in the Ptenhy/− mutants is not, as expected, accompanied by complete loss of Pten expression (see Figure 2E), the low penetrance of the invasive CaP in these mutants strongly suggest that additional events have to occur for this pathological transition to occur. Nevertheless, it is important to underscore that Pten+/− mice, unlike Ptenhy/− mutants, do not develop invasive CaP, but only low-grade PIN lesions (Figure 6). Thus, a further reduction of the Pten dose seems to be essential for this process. Several possibilities, not mutually exclusive, could be entertained: (i) a more severe reduction in the Pten dose could facilitate cooperative tumorigenesis by rendering the cells permissive and sensitive to the activation of additional oncogenic pathways; (ii) the massive prostate hyperplasia could facilitate the accumulation of additional genetic hits (including complete Pten loss) by simply expanding the pool of actively dividing cells; (iii) a more severe reduction in the Pten dose could protect cells from apoptotic programs that are normally triggered by the occurrence of damaging genetics events. + +The fact that Ptenhy/− mutants develop massive prostate hyperplasia not accompanied by an overall increase in body weight and size underscores the fact that different tissues and organs are differentially sensitive to Pten dose variations, the prostatic epithelium being one of the most sensitive. In agreement with this notion, MEFs from Ptenhy/− mutants do not display proliferative advantage in standard culture condition over wild-type MEFs. In this respect, it is also important to remember that, unlike p27Kip1−/− mice, which display hyperplasia of both stromal and parenchymal components in the prostate (as well as an increase in body mass; Fero et al. 1996; Kiyokawa et al. 1996; Nakayama et al. 1996), in Ptenhy/− mutants it is specifically the prostatic epithelium that actively proliferates, leading to prostate hyperplasia (Figure 6). Interestingly, this does not parallel what is observed in other model systems, such as Drosophila, where dose variations in the expression of Pten or downstream signaling components (e.g., TOR) do result in variations in body size and mass (reviewed in Oldham and Hafen 2003). + +While we and others had previously implicated Pten heterozygous loss in prostate tumorigenesis when in cooperation with additional oncogenic events (such as loss of p27Kip1 or Nkx3.1 or the expression of the large T oncogene; Di Cristofano et al. 2001a; Kwabi-Addo et al. 2001; Kim et al. 2002; Abate-Shen et al. 2003), we demonstrate in this article that complete inactivation of Pten alone in the prostate has already catastrophic consequences leading to invasive, diffuse, and highly aggressive malignancies (Figure 6). Although it cannot be formally excluded that complete Pten loss would still cooperate with additional oncogenic events toward full-blown transformation, this finding underscores once more the exquisite sensitivity of the prostatic epithelium to loss of Pten. This information should be factored in when tailoring the treatment of CaP or high-grade PIN lesions that have completely lost PTEN function. These lesions may be extremely sensitive to PI3K or mTOR inhibitors, while modulation of PTEN expression would not be a therapeutic option in these cases (Figure 6). On the basis of these findings, it would seem therefore of paramount importance to routinely assess the status of the PTEN gene and its expression in human CaPs and precancerous lesions as a key biomarker to opt for the most appropriate therapeutic or chemopreventive intervention modalities. It remains to be seen whether complete Pten inactivation in the mouse prostate also influences the metastatic potential of these invasive CaPs alone or in combination with additional oncogenic events. In this respect, while MRI and postmortem analyses have indeed revealed the presence of lung neoplastic lesions in our conditional Pten mutants, morphological and molecular analyses have so far excluded the metastatic nature of these tumors (M. Niki et al., unpublished data). + +The stepwise reduction in the Pten dose results in clear progressively quantitative changes in the prostate epithelial cells prior to tumor development. Increased cellular proliferation correlates with increased phosphorylation of Akt. In Pten null epithelial cells, both in the preneoplastic stage or in overt CaPs, phospho-Akt is found to accumulate almost exclusively at the plasma membrane (see Figure 5C). Whether this represents the extreme consequence of Akt superactivation or rather reflects a genuine qualitative change in Akt biology and function in Pten null prostate epithelial cells remains to be determined. It also remains to be resolved whether the reduction in p27Kip1 expression is also associated with its cytoplasmic relocalization, as previously reported in human breast cancer cells suffering AKT hyperactivation (Liang et al. 2002; Shin et al. 2002; Viglietto et al. 2002). Taken together, this analysis supports a Pten dose-dependent model with progressive changes at the molecular level, arguing against a Pten threshold model for tumor suppression. + +By contrast, clear qualitative morphological changes were observed in the prostate epithelium when analyzing the Pten hypomorphic series. While Pten+/− mice never display massive prostatic enlargement, further reduction to Ptenhy/− levels leads to a sharp increase in prostate mass. In addition, Pten+/− mice never develop invasive CaP, whereas Ptenhy/− mice do (Figure 6). These data clearly support a threshold model for Pten tumor suppression at the physiopathological level. + +Similarly, qualitative morphological changes are also caused by the number of cells suffering complete Pten inactivation, as observed when comparing Ptenpc1 versus Ptenpc2 mice. In Ptenpc2 mice, signs of cellular dysplasia and irregular glandular formations were frequently observed, but they were mostly absent from Ptenpc1 mutants. Along the same tenet, profound mass increase was always observed at early timepoints in Ptenpc2 mice, but not in Ptenpc1 mutants. This is likely due to the marked difference in the number of Pten null cells present in the parenchyma of these two models at puberty, reflecting the different levels of Creexpression in the two models. These observations are also consistent with a threshold model for Pten tumor suppression as a function of the number of Pten null cells in a given organ. + +From a biological standpoint, it will be challenging to determine in the future whether this “field effect” would also play a role in the natural history of human CaP. On the one hand, it could be speculated that human CaP likely arises from a single transformed cell and that therefore that the field effects observed in mouse models will not be critical determinants in the natural history of the human disease. On the other hand, the multistep nature of the neoplastic process (and hence the genetic heterogeneity of the lesion) and the fact that the tumor will eventually grow to form “fields” of neoplastic cells may suggest that these effects, as well as stromal and parenchymal interactions, are key aspects of the human disease that the mouse model can recapitulate. + +Taken together, our findings demonstrate the key importance of Pten in CaP tumor suppression and the dramatic impact that subtle changes in Pten levels and extent of Pten inactivation may have on tumor initiation and progression in the prostate. + +Materials and Methods + + + +Mice + +Generation and characterization of Pten+/− mice have been described perviously (Di Cristofano et al. 1998, 2001a). Mice were genotyped by PCR on tail DNA or by Southern blotting as shown. Hypomorphic mice were generated as described in Figure 1 and Figure 3 and tail DNA genotyped by PCR for presence of the Pten− and Ptenhy alleles (using primers 1 and 2 described below) or by Southern blotting. + +Targeting vector and generation of prostate-specific Pten-deficient mice + +A 129/Sv mouse genomic library (Stratagene, La Jolla, California, United States) was screened with a mouse Pten probe containing exons 4–6. To generate the targeting construct, a 4.1 Kb KpnI–BamHI fragment containing 5′ Pten genomic DNA and a 2.0 Kb XbaI fragment containing 3′ genomic DNA were cloned into pPNT. The targeting construct was linearized with NotI and electroporated into CJ7 ES cells. Transfectants were selected in G418 (350 μg/ml) and gancyclovir (2 μM) and expanded for Southern blot analysis using a 3′ probe. Chimeric mice were produced by microinjection of two independently generated targeted ES cell clones with normal karyotypes into E3.5 C57BL6/J blastocysts, then transferred to pseudopregnant foster mothers. Chimeric males were mated with C57BL6/J females (Jackson Laboratory, Bar Harbor, Maine, United States), and germline transmission of the mutant allele was verified by Southern blot analysis of tail DNA from agouti coat-colored F1 offspring. Next, PtenloxP-neo/+ mice were mated with EIIA-Cre transgenic mice (Lakso et al. 1996), and tail DNA from offspring was subjected to Southern blot analysis using probe 6.1. Through these crosses, mosaic mice harboring a Pten wild-type allele, a Pten targeted allele (PtenloxP-neo), and a floxed allele (Ptenloxp) in their germline were generated. These mosaic mutants were mated with wild-type mice and tail DNA from offspring subjected to Southern blot analysis using probe 6.1 and to PCR analysis using primer 1 (5′-AAAAGTTCCCCTGCTGATGATTTGT-3′) and primer 2 (5′-TGTTTTTGACCAATTAAAGTAGGCTGTG-3′). PCR conditions were 35 cycles (30 sec at 95°C, 1 min at 55°C, and 1 min at 72°C) using HotStarTaq Master Mix (Qiagen, Valencia, California, United States), primer (0.25 μM), and DNA (50 ng). To detect the deleted allele, primer 3 (5′-CCCCCAAGTCAATTGTTAGGTCTGT-3′) was used. PtenloxP/loxP mice were next mated with PB-Cre transgenic mice (Maddison et al. 2000) or male PB-Cre4 transgenic mice (Wu et al. 2001) for conditional prostate-specific Pten inactivation. + +Autopsy and histopathology + +Animals were autopsied and all tissues were examined regardless of their pathological status. Normal and tumor tissue samples were fixed in 10% buffered formalin and embedded in paraffin. Sections (5 μm) were stained with hematoxylin and eosin (H&E) according to standard protocols. + +Cells + +Primary MEFs derived from ten littermate embryos were produced as follows. MEFs were obtained by crossing Ptenhy/+ and Pten+/− animals, embryos were harvested at 13.5 dpc, and individual MEFs were produced and cultured as previously described (Di Cristofano et al. 2001b) and genotyped as described above. At passage 2, cells were harvested for WB analysis (see below) and mRNA extraction by the TRIZOL method (GIBCO–BRL, Carlsbad, California, United States) and cDNA produced using the SuperScript First-Strand Kit for RT–PCR (Invitrogen, Carlsbad, California, United States) according to the manufacturers' instructions. Semiquantitative PCR (yielding a 102 bp amplicon) was performed with a Pten exon 3-specific forward (5′-TGGATTCAAAGCATAAAAACCATTAC-3′) and an exon 4- and exon 5-specific reverse primer (5′-CAAAAGGATACTGTGCAACTCTGC-3′) using 30 cycles of 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C) with the PCR optimizer kit (Invitrogen) and buffer A according to the manufacturer's protocols. PCR amplification of the cDNAs was performed for 25, 30, and 35 cycles, and the amplified products were found to be in the linear range at 30 cycles. PCR products from hypoxanthine phosphoribosyltransferase (HPRT) control amplification with 5′-CCTGCTGGATTACATTAAAGCACTG-3′ and 5′-GTCAAGGGCATATCCAACAACAAAC-3′ primers were used as standards (352 bp amplicon). For measuring proliferative rates of all MEF genotypes, cells were seeded in 12-well plates at 3,000 cells per well in medium containing 10% FBS and fixed at 2-d intervals. Analysis and quantification were performed as previously described (Di Cristofano et al. 2001b). + +WB analysis and densitometry + +Prostates from dissected animals of all genotypes were briefly homogenized (using a Polytron homogenizer; Polytron Vertrieb, Bad Wildbad, Germany), and primary MEFs were harvested and directly lysed in 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.1% NP-40, 1 mM sodium ortho-vanadate (Na3VO4), 10 mM NaF, and protease inhibitor cocktail (Hoffmann–La Roche, Basel, Switzerland) and cleared by centrifugation; concentrations were determined by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, California, United States); and samples were taken into an SDS sample loading buffer.Blood was taken from the retro-orbital cavity of anesthetized mice, red cells were lysed in lysis buffer (0.155 M NH4Cl, 10 mM KHCO3, 1 mM EDTA), and intact cells harvested by centrifugation. PBMCs were lysed in lysis buffer as previously described (Di Cristofano et al. 2001b), and protein concentrations were determined as above. Equivalents of 50 μg of lysate per lane were used for SDS–PAGE and WB analysis. Proteins were detected using a rabbit polyclonal anti-Pten antibody (anti-PTEN/MMAC-1 Ab-2; NeoMarkers, Lab Vision Corporation, Fremont, California, United States), anti-actin monoclonal antibody (mAb) AC-74 (Sigma, St. Louis, Missouri, United States) for normalization, and the Phospho-Akt Pathway Sampler Kit antibodies against Akt and phospho-Serine 473 of Akt (Cell Signaling Technology, Beverly, Massachusetts, United States) following the manufacturers' instructions. After visualization with the ECL system (Amersham Biosciences, Little Chalfont, United Kingdom), films were scanned and band density was quantified using MacBas v2.5 (Fuji Photo Film Company, Tokyo, Japan) on a Macintosh computer (Apple Computer, Sunnyvale, California, United States). + +IHC + +Tissues were fixed in 4% paraformaldehyde and embedded in paraffin, and 8 μm-thick sections were prepared. Anti-p27 mouse mAb (catalog #610242; BD Transduction Laboratories, San Diego, California, United States) was used at 0.5 μg/ml concentration. The IHC detection was performed with the MOM kit from Vector Laboratories (Burlingame, California, United States). Rabbit polyclonal phospho-Akt (Ser 473) antibody (catalog #9270 from Cell Signaling Technology) was used in 1:50 dilution, and rabbit polyclonal antibody PTEN/MMAC1 Ab-2 (catalog #RB-072-PO from NeoMarkers) was used at 1:200 dilution. The IHC detection was performed with the ABC kit from Vector Laboratories. These staining procedures were carried out by the automated staining processor Discovery from Ventana Medical Systems (Tucson, Arizona, United States). + +Anti-Ki-67 antibody (Novocastra Laboratories Ltd., Newcastle-upon-Tyne, United Kingdom), anti-Cre antibody (Novagen, Madison, Wisconsin, United States), and polyclonal anti-phospho-mTOR antibody (Cell Signaling Technology) were purchased. Anti-phospho-FOXO3 (Thr 32) was a kind gift from Anne Brunet (Harvard University, Cambridge, Massachusetts, United States). Paraffin sections (5 μm thickness) were fixed in ice-cold acetone and incubated in 0.1% H2O2 for 15 min to inactivate internal peroxidase. Antigen retrieval was performed in 10 mM citric acid for 15 min using microwave. IHC staining was performed using biotinylated secondary antibodies followed by the Vectastain ABC Elite kit (Vector Laboratories). + +MRI analysis + +Mice were assessed individually by MRI for tumor establishment. Images were obtained on a 1.5T General Electric LX Echo Speed Signa scanner (General Electric Medical Systems, Milwaukee, Wisconsin, United States) using homemade foil solenoid coils (22 mm or 27 mm inner diameter). The mice were anesthetized with isofluorane. Images were acquired using a two-dimensional spin-echo pulse sequence. Initially, the animal was positioned in the coil in a holder, and sagittal scout (field of view, 120 mm) images obtained (TR = 300 ms, TE = 14 ms, one excitation per phase-encoding step, 256 × 128 matrix, 3.0 mm-thick slice, 1.5 mm slice separation) to localize the region of interest. Subsequently, high-quality T2-weighted fast spin-echo transverse images were obtained (TR = 3,000–5,000 ms, TEeff = 102 ms, echo train length = 12, 6–12 excitations per phase-encoding step, 1.5 mm thick slice, field of view = 40 mm, 0.5 mm slice separation, in plane resolution of 156 ×156 μm or 156 × 208 μm). + +Statistical analysis + +Survival was calculated using the Kaplan–Meier method. Log rank tests were used to compare groups by using the SPSS 10 software package (SPSS Incorporated, Chicago, Illinois, United States). + +Acknowledgements + +We thank Peter Scardino and Howard Scher for advice and discussions; Zhenbang Chen for help with genotyping and characterization of the Pten hypomorphic mutants; Lisette Anne Maddison for the genotyping of the PR-Cre transgenic mice; Katia Manova and Craig Farrell for assistance with the automated IHC procedures; Maria Socorro Jiao for assistance with pathological analysis; and Mihaela Lupu and Cornelia Matei for assistance with the MRI analysis. This study was supported, in part, by National Cancer Institute (NCI) grants (SPORE 92629 in Prostate Cancer, MMHCC CA-84292, and RO1 CA-82328) and by the I.T. Hirschl/M. Weill–Caulier Foundation to PPP and CC-C. Imaging analysis was supported by NCI grant R24CA83084 and an MSKCC NCI Core Grant to JAK. + +Abbreviations + +AP - anterior prostate + +BPH - benign prostatic hyperplasia + +CaP - cancer of the prostate + +ES - embryonic stem + +H&E - hematoxylin and eosin + +HPRT - hypoxanthine phosphoribosyltransferase + +IHC - immunohistochemistry + +mAb - monoclonal antibody + +MEF - mouse embryonic fibroblast + +MRI - magnetic resonance imaging + +Neo - neomycin + +PB - Probasin + +PBMC - peripheral blood mononuclear cell + +PI3K - phosphoinositide 3-kinase + +PIN - prostatic intraepithelial neoplasia + +PIP3 - phosphatidylinositol 3,4,5-trisphosphate + +PTEN - phosphatase and tensin homolog deleted on chromosome 10 + +RNAi - RNA interference + +siRNA - small interfering RNA + +WB - Western blot + +Figures and Tables + +Figure 1 + +Production and Analysis of a Pten Hypomorphic Mouse Series + +(A) Schematic representation of the wild-type (+), hypomorphic (hy), and null (−) alleles of Pten. (For a detailed view, see Figure 3A.) + +(B) Breeding scheme used to produce a Pten hypomorphic mouse series and predicted hierarchy of Pten expression levels. + +(C) WB analysis of MEF lysates from ten littermate primary cell cultures obtained from a single cross (indicated in [B]) confirms predicted Pten expression hierarchy in the hypomorphic series and inversely related Akt phosphorylation status (top), both verified by densitometric analysis and plotting of the Pten/actin and phospho-Akt/Akt ratios (bottom). + +(D) MEF cDNA was amplified by semiquantitative PCR with exon 3 (forward) and exon 4–5 spanning (reverse) primers for Pten. Consistent with the concept of transcriptional interference at the Pten locus, Pten mRNA levels in Ptenhy/− MEFs are clearly below the level observed in heterozygosity. Lower panels show the quantification of Pten relative to Hprt amplification. + +Figure 3 + +Conditional Knockout of the Pten Gene in Mouse Prostate + +(A) Map of wild-type Pten locus (top), targeting construct (second from top), predicted targeted locus (third from top), Pten locus after Cre-mediated excision of the Neo resistance cassette by crossing with an EIIA-Cre mouse (fourth from top), and Pten locus after Cre-mediated excision of the floxed exons 4 and 5 by crossing with a PB-Cre or PB-Cre4 transgenic mouse (bottom). The genomic sequence is depicted as a black line, with black boxes representing exons 4 and 5. Pink and yellow boxes represent the Neo resistance and the HSV thymidine kinase cassette (TK) and light blue triangles represent the loxP site, respectively. The Pten genomic fragments used as a probe for Southern blot analysis are shown (3′ probe and probe 6.1). Solid arrows represent expected fragments following hybridization with the 3′ probe upon digestion with EcoRI, and dashed arrows represent expected fragments following hybridization with the probe 6.1 upon digestion with XbaI. Locations of PCR primers to detect wild-type, floxed allele (PtenloxP), and deleted allele are shown. XbaI (X), KpnI (K), BamHI (B), and EcoRI (E) sites are shown. + +(B) Genotyping of mice. Lanes 1 and 2 show Southern blot analysis of ES cell clones with 3′ probe of control (lanes 2) and recombined clones and #176 (lane 1) after digestion with EcoRI, showing a 6 kb wild-type band (WT) and a 2.5 kb targeted band (R). Lanes 3 and 4 (control) represent Southern blot analysis of mouse tail DNA of offspring by crossing F1 mouse generated from #176 with an EIIA-Cre mouse with probe 6.1 after digestion with XbaI, showing wild-type, targeted (PtenloxP-neo), and floxed (PtenloxP) locus bands. Lanes 5, 6 (control), and 7 represent Southern blot analysis of mouse tail DNA of offspring by crossing the mouse of lane 3 with a wild-type mouse with probe 6.1 after digestion with XbaI. Lanes 8–14 show PCR analysis of tail DNA of offspring by crossing a PB-Cre4 (+), PtenloxP/+ male with a PtenloxP/+ female with primers 1 and 2. Lanes 8, 9, and 14 indicate PtenloxP/+ , lanes 10 and 13 indicate Pten+/+,and lanes 11 and 12 indicate PtenloxP/loxP. Lanes 15–17 show PCR analysis of tail DNA of offspring by crossing a Ptenloxp/+ male with a PB-Cre4(+), PtenloxP/+ female with primers 1, 2, and 3. Lane 15 indicates PtenloxP/+, lane 16 indicates Pten+/+, and lane 17 indicates that the deleted allele exists in tail DNA of this offspring. + +(C) Representation of crossing scheme used to generate prostate-specific Pten conditional mutants. PtenloxP/loxP mutants were crossed with PB-Cre transgenic mice or PB-Cre4 transgenic mice. + +Figure 2 + +Ptenhy/− Mice Display Massive Hyperplasia and Invasive CaP + +(A) IHC on preneoplastic anterior prostate (AP) tissue of 2-mo-old littermate mice shows decreasing Pten protein levels in the hypomorphic series. + +(B) WB analysis of the prostate lobes from (A) is shown on the left and their quantification is shown on the right. + +(C) MRI of Ptenhy/− mice aged 6–8 mo shows pathologic structures (encircled by dashed lines) adjacent to seminal vesicles (SV) coinciding with displacement of the bladder (Bl), features typically associated with massive prostate tumors (right). These features were never found in Pten+/+ or Pten+/− mice (left). + +(D) Macroscopic view of the Ptenhy/− mouse from (C) confirms massive enlargement of the AP lobes, but normal-sized seminal vesicles. + +(E) Quantification of WB analysis on AP lobe total lysates from the Ptenhy/− animal shown in (C) compared to preneoplastic AP of same genotype from (A) and (B), labeled 8 mo and 2 mo, respectively. Note that in the enlarged hyperplastic prostate, Pten protein expression is retained (levels are expressed relative to the corresponding wild-type animals). + +(F) Histopathology (H&E) of AP lesions at 8 mo reveals transition from low- to high-grade PIN and invasion (infiltration of stromal tissue is indicated by arrows) in Ptenhy/−, while age-matched Pten+/− tissue only shows hyperplastic features and Pten+/+ tissue is unaffected. Bars are 50 μm. + +Figure 5 + +Molecular Effects of Loss of Pten and Biological Comparison of All Generated Mouse Models + +(A) Ki-67 staining of AP lobe sections illustrates increasing proliferation with decreasing Pten levels (numbers are Ki-67-positive cells per 300 cells counted in percent; bars are 50 μm). + +(B) The phospho-Akt/Akt ratio is sharply increased in the prostates of 10-wk-old Ptenpc2 animals, as shown by densitometric quantification of WB analysis. + +(C) AP staining with anti-phospho-Akt antibodies reveals strong plasma membrane localization of phospho-Akt and apparent reduction of p27 protein detection, whereas phospho-mTOR and phospho-threonine-FOXO3 antibodies show increased staining in Ptenpc2 versus wild-type mouse prostates. Bars are 50 μm. + +(D) Kaplan–Meier curve showing prostate enlargement as visualized by MRI. Progressive rates of mass increase for Ptenpc2 (median age, 4 mo), Ptenhy/− (median age, 7 mo), and Ptenpc1 (median age, 16 mo) mice are found. In contrast, no prostatic size irregularities were detected in Pten+/+ or Pten+/− mice. + +(E) Incidence of invasive CaP. Invasive CaP was defined as tumor cells disrupting the basal membrane of prostatic glands and growing into the surrounding stroma. Full penetrance was observed in both Ptenpc1 mice as well as in Ptenpc2 mice. In contrast, Ptenhy/− mice with a follow-up of more than 6 mo displayed only 25% incidence of invasive CaP. + +Figure 4 + +Ptenpc2 Mice Develop Invasive CaP + +(A) Histopathology analysis of wild-type, Ptenpc1, and Ptenpc2 mice prior to tumor onset. H&E-stained AP (top) shows the difference in both the morphology and proliferative rates of the prostatic epithelium in these two models. IHC staining with anti-Pten antibody (bottom) was carried out on wild-type, Ptenpc1, and Ptenpc2 mice. In wild-type mice, strong cytoplasmic staining was observed in epithelial cells (arrowheads). In Ptenpc1 mice, staining was generally weak, whereas in Ptenpc2 mice, staining was completely absent in the prostatic epithelium. Original magnification, 400×. + +(B) MRI (top) shows massive prostate tumor (surrounded by dashed line) in Ptenpc2 mice (at 6 mo) and no detectable difference between the prostates of Ptenpc1 or Pten+/+ mice (at 12 mo; arrowheads). Bladder (Bl) and seminal vesicles (SV) are indicated. Macroscopic view (second from top) of the same animals confirms massive enlargement of both APs in Ptenpc2 and reveals the slightly enlarged AP of Ptenpc1 mice (encircled). H&E staining (bottom) of the prostate from Ptenpc1 mice was characterized by multiple foci of PIN lesions and by the presence of prostatic adenocarcinoma. These lesions contained well-differentiated neoplastic cells and showed focal areas of invasion (arrowheads). H&E stainings of prostates from Ptenpc2 mice showed diffuse, invasive CaP with large, undifferentiated tumor cells growing into stromal areas. + +Figure 6 + +Pten Dose Affects Prostate Tumor Progression + +Pten+/− mice develop hyperplasia, dysplasia, and low-grade PIN. Ptenhy/− mice develop at complete penetrance high-grade PIN at a young age (8–10 wk) and roughly 25% present invasive CaP around 8 mo. Ptenpc mice develop invasive CaP at complete penetrance. (See Discussion for a detailed description.) p27Kip1−/− mice, on the contrary, develop only BPH. Possibilities for human therapeutic intervention derived from our findings: in addition to inactivation of PI3K/AKT and mTOR enzymatic activities (in PTEN loss of heterozygosity condition), monitoring and elevation of PTEN expression levels of the remaining allele could not only prevent formation of PIN lesions (in PTEN+/− individuals), but could importantly also be used to counteract the progression to invasive phenotypes (as observed in Ptenhy/−mouse mutants). + +Footnotes + +Conflicts of Interest. The authors have declared that no conflicts of interest exist. + +Author Contributions. LCT, MN, ZAD, JAK, AD, AX, TVD, CC-C, and PPP conceived and designed the experiments. LCT, MN, ZAD, JAK, AD, and AX performed the experiments. LCT, MN, ZAD, JAK, AD, AX, ASBK, TVD, CC-C, and PPP analyzed the data. PR-B, NMG, TVD, and PPP contributed reagents/materials/analysis tools. LCT, MN, ZAD, and PPP wrote the paper. + +Academic Editor: Nicholas Hastie, Medical Research Council Human Genetics Unit, Western General Hospital diff --git a/src/ontogpt/evaluation/craft/database/all/14723793.ann b/src/ontogpt/evaluation/craft/database/all/14723793.ann new file mode 100644 index 000000000..0867667d6 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/14723793.ann @@ -0,0 +1,556 @@ +T1 NCBITaxon:10088 46 51 mouse +T2 SO:0000704 57 61 gene +T3 SO:0000704 142 146 gene +T4 SO:0000417 178 184 domain +T5 NCBITaxon:9606 203 209 humans +T6 SO:0000704 260 264 gene +T7 NCBITaxon:10088 316 321 mouse +T8 SO:0000853 322 331 homologue +T9 NCBITaxon:9606 339 344 human +T10 SO:0000704 350 354 gene +T11 SO:0000704 387 392 genes +T12 PR:000005643 394 399 Acdp1 +T13 PR:000005644 401 406 Acdp2 +T14 PR:000005645 408 413 Acdp3 +T15 PR:000005646 418 423 Acdp4 +T16 SO:0000028 439 441 bp +T17 SO:0000028 449 451 bp +T18 SO:0000028 459 461 bp +T19 SO:0000028 472 474 bp +T20 NCBITaxon:10088 578 583 mouse +T21 SO:0000704 589 594 genes +T22 SO:0000857 614 624 homologies +T23 NCBITaxon:9606 685 690 human +T24 SO:0000417 788 794 Domain +T25 NCBITaxon:species 840 857 taxonomic species +T26 SO:0000857 909 919 homologies +T27 NCBITaxon:2 927 935 bacteria +T28 PR:000022321 936 940 CorC +T29 SO:0000857 975 983 homology +T30 SO:0000704 1045 1050 genes +T31 GO:0010467 1062 1071 expressed +T32 UBERON:0000479 1079 1086 tissues +T33 PR:000005643 1105 1110 Acdp1 +T34 GO:0010467 1133 1142 expressed +T35 UBERON:0000955 1150 1155 brain +T36 GO:0010467 1175 1185 expression +T37 UBERON:0002113 1189 1195 kidney +T38 UBERON:0000473 1200 1206 testis +T39 PR:000005643 1226 1231 Acdp1 +T40 CL:0002608 1235 1254 hippocampus neurons +T41 GO:0005886 1298 1313 plasma membrane +T42 SO:0000704 1337 1342 genes +T43 NCBITaxon:species 1383 1390 species +T44 GO:0010467 1408 1417 expressed +T45 UBERON:0007023 1445 1450 adult +T46 UBERON:0000479 1451 1458 tissues +T47 SO:0000704 1500 1504 gene +T48 SO:0000857 1525 1533 homology +T49 NCBITaxon:2 1537 1545 bacteria +T50 PR:000022321 1546 1550 CorC +T51 GO:0005886 1594 1609 plasma membrane +T52 CHEBI:24870 1688 1691 ion +T53 GO:0006811 1688 1701 ion transport +T54 GO:0046907 1692 1704;1715 1720 transport in ... cells +T55 NCBITaxon:40674 1705 1714 mammalian +T56 SO:0000704 1784 1788 gene +T57 SO:0000417 1820 1826 domain +T58 NCBITaxon:9606 1880 1886 humans +T59 SO:0000704 1911 1915 gene +T60 NCBITaxon:species 1962 1969 species +T61 NCBITaxon:2 1983 1991 bacteria +T62 NCBITaxon:6239 2000 2010 C. elegans +T63 NCBITaxon:7227 2016 2031 D. melanogaster +T64 NCBITaxon:40674 2035 2042 mammals +T65 NCBITaxon:species 2116 2123 species +T66 SO:0000704 2176 2181 genes +T67 SO:0000704 2233 2237 gene +T68 NCBITaxon:10088 2284 2289 mouse +T69 SO:0000853 2290 2299 homologue +T70 NCBITaxon:9606 2307 2312 human +T71 SO:0000704 2318 2322 gene +T72 SO:0000704 2371 2375 gene +T73 NCBITaxon:10088 2397 2402 mouse +T74 SO:0000704 2408 2413 genes +T75 NCBITaxon:9606 2419 2424 human +T76 NCBITaxon:10088 2491 2496 mouse +T77 SO:0000345 2497 2500 EST +T78 NCBITaxon:10088 2548 2553 Mouse +T79 SO:0000345 2554 2557 EST +T80 SO:0000345 2635 2638 EST +T81 PR:000005643 2665 2670 Acdp1 +T82 PR:000005644 2683 2688 Acdp2 +T83 PR:000005645 2706 2711 Acdp3 +T84 PR:000005646 2729 2734 Acdp4 +T85 SO:0000121 2804 2818 forward primer +T86 SO:0000345 2829 2832 EST +T87 SO:0000112 2852 2858 primer +T88 SO:0000205 2899 2905 3' UTR +T89 SO:0000704 2943 2947 gene +T90 SO:0000704 3017 3022 genes +T91 NCBITaxon:10088 3078 3083 mouse +T92 NCBITaxon:9606 3088 3093 human +T93 SO:0000112 3094 3101 primers +T94 SO:0000204 3107 3113 5' UTR +T95 SO:0000153 3163 3166 BAC +T96 SO:0000704 3205 3210 genes +T97 SO:0000153 3216 3219 BAC +T98 NCBITaxon:10088 3263 3268 mouse +T99 SO:0000153 3269 3272 BAC +T100 SO:0000204 3310 3316 5' UTR +T101 PR:000005643 3385 3390 Acdp1 +T102 SO:0000704 3391 3395 gene +T103 SO:0000028 3411 3413 bp +T104 SO:0000704 3517 3522 genes +T105 PR:000005644 3524 3529 Acdp2 +T106 SO:0000028 3554 3556 bp +T107 SO:0000028 3564 3566 bp +T108 SO:0000028 3577 3579 bp +T109 UBERON:0000479 3699 3705 Tissue +T110 SO:0000704 3755 3759 gene +T111 NCBITaxon:10088 3838 3843 mouse +T112 UBERON:0000479 3844 3851 tissues +T113 SO:0000857 3905 3915 homologies +T114 SO:0000417 3962 3968 domain +T115 SO:0000147 4029 4033 exon +T116 SO:0000205 4042 4064 3' untranslated region +T117 GO:0006412 4047 4057 translated +T118 NCBITaxon:10088 4080 4085 mouse +T119 SO:0000673 4091 4099 messages +T120 CHEBI:33695 4091 4099 messages +T121 UBERON:0000479 4123 4129 tissue +T122 NCBITaxon:9606 4151 4156 human +T123 SO:0000704 4162 4167 genes +T124 PR:000005643 4169 4174 Acdp1 +T125 SO:0000673 4175 4182 message +T126 CHEBI:33695 4175 4182 message +T127 GO:0010467 4193 4202 expressed +T128 UBERON:0000955 4210 4215 brain +T129 UBERON:0002113 4223 4229 kidney +T130 UBERON:0000473 4234 4240 testis +T131 GO:0010467 4267 4277 expression +T132 PR:000005644 4279 4284 Acdp2 +T133 GO:0010467 4299 4310 expressions +T134 UBERON:0000955 4318 4323 brain +T135 UBERON:0002113 4325 4331 kidney +T136 UBERON:0002107 4336 4341 liver +T137 PR:000005644 4356 4361 Acdp2 +T138 SO:0000673 4362 4372 transcript +T139 UBERON:0004288 4396 4404 skeleton +T140 GO:0010467 4455 4465 expression +T141 UBERON:0000479 4481 4488 tissues +T142 PR:000005645 4490 4495 Acdp3 +T143 PR:000005646 4500 4505 Acdp4 +T144 GO:0010467 4533 4543 expression +T145 UBERON:0000479 4551 4558 tissues +T146 GO:0010467 4579 4590 expressions +T147 PR:000005645 4595 4600 Acdp3 +T148 UBERON:0000955 4622 4627 brain +T149 UBERON:0002113 4629 4635 kidney +T150 UBERON:0002107 4637 4642 liver +T151 UBERON:0000948 4647 4652 heart +T152 GO:0010467 4670 4681 expressions +T153 PR:000005646 4686 4691 Acdp4 +T154 UBERON:0002113 4713 4719 kidney +T155 UBERON:0002108 4721 4736 small intestine +T156 UBERON:0000473 4741 4747 testis +T157 GO:0010467 4753 4763 expression +T158 PR:000005645 4775 4780 Acdp3 +T159 UBERON:0004288 4790 4798 skeleton +T160 PR:000003676 4839 4846 β-actin +T161 GO:0010467 4861 4871 expression +T162 GO:0010467 4975 4985 expression +T163 GO:0008150 5090 5110 biological processes +T164 NCBITaxon:species 5180 5187 species +T165 SO:0000704 5235 5239 gene +T166 UBERON:0004288 5248 5250 S. +T167 UBERON:0004288 5269 5277 skeletal +T168 UBERON:0002108 5286 5294 Sm. Int. +T169 UBERON:0002108 5306 5321 small intestine +T170 PR:000005643 5457 5462 Acdp1 +T171 SO:0000704 5463 5467 gene +T172 PR:000005644 5570 5575 Acdp2 +T173 SO:0000704 5576 5580 gene +T174 PR:000005643 5614 5619 Acdp1 +T175 PR:000005645 5706 5711 Acdp3 +T176 PR:000005646 5716 5721 Acdp4 +T177 SO:0000704 5722 5727 genes +T178 SO:0000153 5759 5762 BAC +T179 SO:0000860 5841 5849 syntenic +T180 NCBITaxon:9606 5857 5862 human +T181 SO:0000857 5887 5895 homology +T182 NCBITaxon:10088 5931 5936 mouse +T183 SO:0000704 5942 5947 genes +T184 SO:0000857 5967 5977 homologies +T185 NCBITaxon:9606 6021 6026 human +T186 SO:0000704 6032 6037 genes +T187 SO:0000857 6061 6071 homologies +T188 NCBITaxon:9606 6098 6103 human +T189 PR:000005644 6104 6109 ACDP2 +T190 NCBITaxon:10088 6118 6123 mouse +T191 PR:000005644 6124 6129 Acdp2 +T192 SO:0000704 6130 6134 gene +T193 SO:0000857 6199 6207 homology +T194 SO:0000204 6227 6233 5' UTR +T195 SO:0000028 6259 6261 bp +T196 SO:0000318 6284 6295 start codon +T197 SO:0000857 6314 6324 homologies +T198 NCBITaxon:9606 6332 6337 human +T199 SO:0000853 6338 6346 homologs +T200 PR:000005644 6365 6370 Acdp2 +T201 SO:0000204 6371 6377 5' UTR +T202 NCBITaxon:9606 6416 6421 human +T203 SO:0000853 6422 6429 homolog +T204 SO:0000857 6444 6454 homologies +T205 SO:0000205 6462 6468 3' UTR +T206 SO:0000028 6483 6485 bp +T207 SO:0000319 6507 6517 stop codon +T208 SO:0000704 6557 6562 genes +T209 PR:000005646 6570 6575 Acdp4 +T210 NCBITaxon:9606 6597 6602 human +T211 SO:0000853 6603 6610 homolog +T212 SO:0000417 6635 6641 domain +T213 SO:0000857 6686 6694 homology +T214 NCBITaxon:10088 6707 6712 mouse +T215 NCBITaxon:9606 6717 6722 human +T216 SO:0000417 6755 6761 domain +T217 NCBITaxon:species 6803 6810 species +T218 NCBITaxon:2 6824 6832 bacteria +T219 NCBITaxon:6239 6841 6851 C. elegans +T220 NCBITaxon:7227 6853 6868 D. melanogaster +T221 NCBITaxon:10088 6870 6875 mouse +T222 NCBITaxon:9606 6879 6884 human +T223 SO:0000857 6965 6973 homology +T224 NCBITaxon:2 6977 6985 bacteria +T225 PR:000022321 6986 6990 CorC +T226 SO:0000857 7025 7033 homology +T227 SO:0000857 7098 7106 homology +T228 SO:0000857 7205 7213 homology +T229 SO:0000857 7244 7254 homologous +T230 NCBITaxon:2 7262 7270 bacteria +T231 PR:000022321 7271 7275 CorC +T232 SO:0000857 7556 7564 homology +T233 SO:0000857 7623 7633 homologies +T234 NCBITaxon:9606 7646 7651 human +T235 NCBITaxon:10088 7661 7666 mouse +T236 SO:0000857 7712 7720 homology +T237 SO:0000704 7760 7765 genes +T238 SO:0000417 7781 7787 domain +T239 SO:0000704 7820 7825 genes +T240 PR:000005643 7890 7895 Acdp1 +T241 PR:000005644 7908 7913 Acdp2 +T242 PR:000005645 7926 7931 Acdp3 +T243 PR:000005646 7947 7952 Acdp4 +T244 SO:0000857 8009 8019 homologies +T245 SO:0000857 8112 8122 homologies +T246 SO:0000730 8185 8189 gaps +T247 SO:0000417 8282 8288 domain +T248 NCBITaxon:species 8300 8307 species +T249 NCBITaxon:4932 8333 8357 Saccharomyces cerevisiae +T250 NCBITaxon:5478 8394 8410 Candida glabrata +T251 NCBITaxon:5141 8470 8487 Neurospora crassa +T252 SO:0000704 8499 8503 gene +T253 NCBITaxon:7227 8517 8532 D. melanogaster +T254 SO:0000704 8564 8568 gene +T255 NCBITaxon:7215 8598 8608 Drosophila +T256 SO:0001026 8609 8615 Genome +T257 NCBITaxon:7165 8661 8678 anopheles gambiae +T258 NCBITaxon:6239 8747 8768 Caenohabditis elegans +T259 PR:000022321 8770 8774 CorC +T260 NCBITaxon:2 8786 8794 bacteria +T261 NCBITaxon:70863 8840 8861 Shewanella oneidensis +T262 NCBITaxon:155919 8903 8927 Xylella fastidiosa Dixon +T263 SO:0000417 9028 9034 domain +T264 NCBITaxon:10088 9258 9263 mouse +T265 GO:0016020 9304 9312 membrane +T266 SO:0000417 9313 9320 domains +T267 SO:0000417 9339 9346 domains +T268 SO:0000417 9359 9365 domain +T269 NCBITaxon:2 9384 9392 bacteria +T270 PR:000022321 9393 9397 CorC +T271 SO:0000417 9428 9435 domains +T272 GO:0005622 9446 9459 intracellular +T273 SO:0000417 9460 9467 modules +T274 SO:0000417 9541 9548 domains +T275 GO:0046983 9549 9557 dimerise +T276 SO:0000417 9584 9590 domain +T277 SO:0000417 9639 9645 domain +T278 SO:0000417 9674 9680 domain +T279 SO:0001077 9686 9706 transmembrane region +T280 GO:0016020 9691 9699 membrane +T281 GO:0005622 9781 9794 intracellular +T282 SO:0000417 9799 9806 domains +T283 SO:0000417 9824 9830 domain +T284 SO:0000417 9872 9878 domain +T285 GO:0016020 9932 9940 membrane +T286 SO:0000417 9941 9948 domains +T287 PR:000005646 9956 9961 Acdp4 +T288 GO:0016020 9976 9984 membrane +T289 SO:0000417 9985 9992 domains +T290 SO:0001812 10093 10101 TM helix +T291 SO:0001114 10259 10264 helix +T292 PR:000005643 10349 10354 Acdp1 +T293 GO:0018032 10592 10601 amidation +T294 PR:000005644 10640 10645 Acdp2 +T295 PR:000005645 10707 10712 Acdp3 +T296 PR:000005646 10802 10807 Acdp4 +T297 GO:0018032 10883 10892 amidation +T298 GO:0042571 10913 10921 Antibody +T299 PR:000005643 10994 10999 Acdp1 +T300 SO:0000417 11093 11099 domain +T301 PR:000005643 11103 11108 Acdp1 +T302 CHEBI:59132 11194 11203 antigenic +T303 GO:0042571 11271 11281 antibodies +T304 NCBITaxon:9986 11327 11334 rabbits +T305 GO:0042571 11367 11377 antibodies +T306 NCBITaxon:10088 11417 11422 mouse +T307 UBERON:0000955 11423 11428 brain +T308 UBERON:0000479 11429 11435 tissue +T309 GO:0042571 11471 11479 antibody +T310 PR:000005643 11533 11538 Acdp1 +T311 GO:0042571 11553 11561 antibody +T312 PR:000005646 11605 11610 Acdp4 +T313 PR:000005643 11626 11631 Acdp1 +T314 GO:0042571 11728 11736 antibody +T315 GO:0042571 11870 11878 antibody +T316 PR:000005643 11891 11896 Acdp1 +T317 GO:0042571 12012 12020 antibody +T318 PR:000005643 12049 12054 Acdp1 +T319 GO:0010467 12212 12222 expression +T320 PR:000005643 12233 12238 Acdp1 +T321 GO:0010467 12288 12298 expression +T322 UBERON:0000955 12379 12384 brain +T323 UBERON:0000479 12385 12391 tissue +T324 GO:0042571 12410 12418 antibody +T325 PR:000005643 12427 12432 Acdp1 +T326 PR:000005643 12468 12474 Acdp-1 +T327 PR:000005643 12500 12505 Acdp1 +T328 GO:0042571 12517 12525 antibody +T329 PR:000005643 12575 12581 Acdp-1 +T330 GO:0010467 12642 12652 expression +T331 PR:000005643 12656 12661 Acdp1 +T332 UBERON:0000955 12669 12674 brain +T333 CL:0002608 12720 12739 hippocampus neurons +T334 NCBITaxon:10088 12754 12759 mouse +T335 UBERON:0000922 12760 12767 embryos +T336 CL:0000540 12773 12780 neurons +T337 NCBITaxon:10088 12852 12857 mouse +T338 UBERON:0001851 12858 12866 cortical +T339 CL:0002605 12858 12877 cortical astrocytes +T340 PR:000005643 12918 12923 Acdp1 +T341 GO:0042571 12944 12952 antibody +T342 PR:000005643 12985 12990 Acdp1 +T343 GO:0005886 13025 13040 plasma membrane +T344 GO:0016020 13123 13131 membrane +T345 PR:000005643 13144 13149 Acdp1 +T346 GO:0016020 13226 13234 membrane +T347 SO:0000417 13235 13242 domains +T348 UBERON:0000955 13329 13334 brain +T349 UBERON:0000479 13335 13341 tissue +T350 GO:0042571 13462 13470 antibody +T351 PR:000005643 13550 13555 Acdp1 +T352 PR:000005644 13566 13571 Acdp2 +T353 PR:000005646 13582 13587 Acdp4 +T354 PR:000005645 13600 13605 Acdp3 +T355 PR:000005643 13645 13650 Acdp1 +T356 GO:0042571 13651 13661 antibodies +T357 PR:000005643 13757 13762 Acdp1 +T358 GO:0042571 13825 13833 antibody +T359 PR:000005643 13860 13866 Acdp-1 +T360 PR:000005643 13996 14001 Acdp1 +T361 CL:0002608 14005 14024 hippocampus neurons +T362 CL:0000540 14070 14076 neuron +T363 PR:000005643 14098 14103 Acdp1 +T364 GO:0042571 14104 14112 antibody +T365 GO:0009986 14167 14174 surface +T366 CL:0000540 14182 14188 neuron +T367 SO:0000704 14279 14283 gene +T368 SO:0000704 14359 14364 genes +T369 GO:0008150 14395 14415 biological processes +T370 SO:0000704 14430 14435 genes +T371 NCBITaxon:species 14500 14507 species +T372 SO:0000704 14526 14531 genes +T373 NCBITaxon:species 14549 14556 species +T374 SO:0000704 14571 14576 genes +T375 GO:0010467 14594 14603 expressed +T376 UBERON:0007023 14631 14636 adult +T377 UBERON:0000479 14637 14644 tissues +T378 NCBITaxon:10088 14705 14710 mouse +T379 SO:0000704 14716 14720 gene +T380 SO:0000704 14808 14812 gene +T381 SO:0000857 14831 14839 homology +T382 SO:0000857 14899 14909 homologies +T383 NCBITaxon:2 14917 14925 bacteria +T384 PR:000022321 14926 14930 CorC +T385 PR:000022320 14964 14968 CorA +T386 PR:000022320 15091 15095 CorA +T387 SO:0000857 15168 15178 homologous +T388 PR:000022321 15182 15186 CorC +T389 NCBITaxon:2 15250 15258 bacteria +T390 SO:0000417 15342 15349 domains +T391 NCBITaxon:2 15368 15376 bacteria +T392 PR:000022321 15377 15381 CorC +T393 SO:0000417 15424 15431 domains +T394 SO:0000417 15439 15445 domain +T395 GO:0016020 15455 15463 membrane +T396 SO:0000417 15464 15471 domains +T397 SO:0000417 15501 15507 domain +T398 CHEBI:24870 15568 15571 ion +T399 GO:0042571 15623 15631 antibody +T400 PR:000005643 15644 15649 Acdp1 +T401 PR:000005643 15689 15694 Acdp1 +T402 GO:0005886 15729 15744 plasma membrane +T403 CL:0002608 15748 15767 hippocampus neurons +T404 NCBITaxon:9606 15801 15806 human +T405 GO:0005634 15856 15863 nucleus +T406 CL:0000540 15924 15938 neuronal cells +T407 GO:0042571 15987 15995 antibody +T408 SO:0000417 16038 16044 domain +T409 PR:000005643 16093 16098 Acdp1 +T410 GO:0042571 16110 16118 antibody +T411 PR:000005643 16135 16140 Acdp1 +T412 UBERON:0000955 16144 16149 brain +T413 UBERON:0000479 16150 16156 tissue +T414 GO:0042571 16249 16257 antibody +T415 CHEBI:24870 16317 16320 ion +T416 GO:0006811 16317 16330 ion transport +T417 GO:0046907 16321 16333;16344 16349 transport in ... cells +T418 NCBITaxon:40674 16334 16343 mammalian +T419 NCBITaxon:9606 16503 16508 human +T420 SO:0000704 16514 16519 genes +T421 SO:0000704 16641 16646 genes +T422 NCBITaxon:40674 16656 16663 mammals +T423 NCBITaxon:9606 16679 16684 human +T424 SO:0000704 16725 16729 gene +T425 NCBITaxon:1 16739 16748 organisms +T426 NCBITaxon:6239 16760 16770 C. elegans +T427 NCBITaxon:2 16783 16791 bacteria +T428 SO:0000704 16855 16860 genes +T429 NCBITaxon:species 16874 16881 species +T430 NCBITaxon:39107 16949 16955 murine +T431 SO:0000704 16961 16965 gene +T432 SO:0000704 17011 17015 gene +T433 NCBITaxon:33208 17026 17032 animal +T434 NCBITaxon:10088 17165 17170 mouse +T435 NCBITaxon:9606 17175 17180 human +T436 SO:0000704 17195 17199 gene +T437 SO:0000704 17219 17223 gene +T438 GO:0042571 17368 17378 antibodies +T439 PR:000005643 17392 17397 Acdp1 +T440 PR:000005643 17425 17430 Acdp1 +T441 GO:0042571 17442 17450 antibody +T442 PR:000005643 17485 17490 Acdp1 +T443 GO:0042571 17503 17511 antibody +T444 PR:000005643 17539 17544 Acdp1 +T445 GO:0005886 17579 17594 plasma membrane +T446 CL:0002608 17598 17617 hippocampus neurons +T447 SO:0000704 17716 17720 gene +T448 SO:0000704 17778 17782 gene +T449 NCBITaxon:9606 17813 17818 human +T450 SO:0000853 17819 17828 homologue +T451 NCBITaxon:9606 17880 17885 human +T452 NCBITaxon:10088 17948 17953 mouse +T453 SO:0000345 17954 17957 EST +T454 SO:0000345 17971 17974 EST +T455 SO:0000121 18020 18034 forward primer +T456 NCBITaxon:9606 18046 18051 human +T457 SO:0000318 18086 18097 start codon +T458 NCBITaxon:10088 18105 18110 mouse +T459 SO:0000132 18111 18125 reverse primer +T460 NCBITaxon:10088 18135 18140 mouse +T461 SO:0000345 18141 18144 EST +T462 SO:0000853 18177 18186 homologue +T463 NCBITaxon:10088 18201 18206 mouse +T464 GO:0097617 18224 18233 annealing +T465 SO:0000132 18286 18300 reverse primer +T466 NCBITaxon:10088 18310 18315 mouse +T467 SO:0000345 18316 18319 EST +T468 NCBITaxon:9606 18342 18347 human +T469 SO:0000121 18348 18362 forward primer +T470 NCBITaxon:10088 18408 18413 mouse +T471 SO:0000704 18414 18418 gene +T472 SO:0000006 18440 18452 PCR products +T473 GO:0097617 18461 18470 annealing +T474 SO:0000006 18504 18516 PCR products +T475 CHEBI:2511 18544 18551 agarose +T476 SO:0000121 18647 18661 forward primer +T477 SO:0000132 18699 18713 reverse primer +T478 SO:0000147 18806 18810 exon +T479 SO:0000204 18821 18827 5' UTR +T480 SO:0000153 18855 18858 BAC +T481 NCBITaxon:10088 18962 18967 mouse +T482 UBERON:0000479 18968 18975 tissues +T483 SO:0000704 19043 19047 gene +T484 SO:0000028 19080 19082 bp +T485 SO:0000147 19094 19098 exon +T486 SO:0000205 19107 19129 3' untranslated region +T487 GO:0006412 19112 19122 translated +T488 CHEBI:37972 19145 19148 32P +T489 SO:0000112 19171 19177 primer +T490 GO:0097617 19216 19230 Hybridizations +T491 CHEBI:8984 19324 19327 SDS +T492 CHEBI:8984 19410 19413 SDS +T493 GO:0042571 19510 19518 Antibody +T494 MOP:0000779 19566 19572 linked +T495 NCBITaxon:6462 19589 19595 limpet +T496 PR:000005643 19613 19618 Acdp1 +T497 SO:0000417 19656 19662 domain +T498 GO:0042571 19697 19707 antibodies +T499 PR:000005643 19725 19730 Acdp1 +T500 GO:0042571 19944 19954 antibodies +T501 NCBITaxon:10088 20058 20063 mouse +T502 SO:0000112 20171 20178 Primers +T503 SO:0000205 20184 20190 3' UTR +T504 NCBITaxon:10088 20281 20286 mouse +T505 SO:0001026 20287 20293 genome +T506 NCBITaxon:10088 20345 20350 Mouse +T507 SO:0000704 20468 20472 Gene +T508 SO:0000857 20502 20510 homology +T509 SO:0000110 20812 20829 sequence features +T510 CL:0000540 21003 21016 Neuronal cell +T511 CL:0002608 21050 21068 Hippocampal neuron +T512 NCBITaxon:10088 21172 21177 mouse +T513 UBERON:0000922 21178 21185 embryos +T514 UBERON:0000479 21211 21218 tissues +T515 PR:000027795 21355 21362 trypsin +T516 CL:0002608 21400 21419 hippocampal neurons +T517 NCBITaxon:10088 21489 21494 mouse +T518 UBERON:0001851 21495 21503 cortical +T519 CL:0002605 21495 21514 cortical astrocytes +T520 CL:0000540 21548 21555 neurons +T521 CHEBI:16526 21615 21618 CO2 +T522 UBERON:0001851 21620 21628 Cortical +T523 CL:0002605 21620 21639 Cortical astrocytes +T524 NCBITaxon:10088 21665 21670 mouse +T525 UBERON:0001851 21671 21679 cortices +T526 GO:0040007 21685 21690 grown +T527 CHEBI:16526 21752 21755 CO2 +T528 CHEBI:28680 21806 21826 cytosine arabinoside +T529 CL:0000540 21995 22009 neuronal cells +T530 CHEBI:75958 22131 22139 solution +T531 CHEBI:9750 22167 22179 Triton X-100 +T532 CHEBI:75958 22274 22282 solution +T533 NCBITaxon:9925 22306 22310 goat +T534 NCBITaxon:9986 22352 22358 rabbit +T535 GO:0042571 22380 22388 antibody +T536 NCBITaxon:9925 22448 22452 goat +T537 CHEBI:75958 22463 22471 solution +T538 CHEBI:52661 22506 22514 Alex 488 +T539 MOP:0000779 22515 22525 conjugated +T540 GO:0042571 22536 22544 antibody +T541 NCBITaxon:9925 22558 22562 goat +T542 CHEBI:75958 22573 22581 solution +T543 NCBITaxon:9925 22663 22667 goat +T544 CHEBI:75958 22678 22686 solution +T545 CL:0000540 22692 22706 neuronal cells +T546 CHEBI:17754 22751 22759 glycerol +T547 SO:0000704 22972 22976 Gene +T548 SO:0000704 23032 23036 gene +T549 SO:0000704 23075 23079 gene +T550 PR:000005643 23118 23123 Acdp1 +T551 PR:000005644 23136 23141 Acdp2 +T552 PR:000005645 23154 23159 Acdp3 +T553 PR:000005646 23175 23180 Acdp4 +T554 GO:0042571 23406 23414 antibody +T555 PR:000005643 23462 23467 Acdp1 +T556 CL:0000540 23484 23498 neuronal cells diff --git a/src/ontogpt/evaluation/craft/database/all/14723793.txt b/src/ontogpt/evaluation/craft/database/all/14723793.txt new file mode 100644 index 000000000..3c8ac0ebd --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/14723793.txt @@ -0,0 +1,129 @@ +Molecular cloning and characterization of the mouse Acdp gene family + +Abstract + +Background + +We have recently cloned and characterized a novel gene family named ancient conserved domain protein (ACDP) in humans. To facilitate the functional study of this novel gene family, we have cloned and characterized Acdp, the mouse homologue of the human ACDP gene family. + +Results + +The four Acdp genes (Acdp1, Acdp2, Acdp3 and Acdp4) contain 3,631 bp, 3,244 bp, 2,684 bp and 2,743 bp of cDNA sequences, and encode deduced proteins of 951, 874, 713 and 771 amino acids, respectively. The mouse Acdp genes showed very strong homologies (>90%) in both nucleotide and amino acid sequences to their human counterparts. In addition, both nucleotide and amino acid sequences within the Ancient Conserved Domain (ACD) are highly conserved in many different taxonomic species. Particularly, Acdp proteins showed very strong AA homologies to the bacteria CorC protein (35% AA identity with 55% homology), which is involved in magnesium and cobalt efflux. The Acdp genes are widely expressed in all tissues tested except for Acdp1, which is only highly expressed in the brain with low levels of expression in kidney and testis. Immunostaining of Acdp1 in hippocampus neurons revealed a predominant localization on the plasma membrane. + +Conclusion + +The Acdp genes are evolutionarily conserved in diverse species and ubiquitously expressed throughout development and adult tissues suggesting that Acdp may be an essential gene. Acdp showed strong homology to bacteria CorC protein and predominantly localized on the plasma membrane. These results suggest that Acdp is probably a family of proteins involved in ion transport in mammalian cells + +Background + +We have recently cloned and characterized a novel gene family named ancient conserved domain protein (ACDP) which encodes four protein members in humans [1]. We found that this gene family is evolutionarily conserved in diverse species ranging from bacteria, yeast, C. elegans, and D. melanogaster to mammals. The sequence conservation and the presence of multiple members within a species may imply functional importance associated with the genes. To facilitate the functional analysis of the ACDP gene family, we cloned and characterized Acdp, the mouse homologue of the human ACDP gene family. + +Results + +Molecular cloning of the Acdp gene family + +To clone the mouse Acdp genes, the human ACDP cDNA and predicted protein sequences were used to search the mouse EST database with the blastn and tblastn programs. Mouse EST markers corresponding to each Acdp member were then identified. For example, EST H3086H12-5 corresponds to Acdp1, W98010 for Acdp2, 603299135F1 for Acdp3 and BG083791 for Acdp4. A modified oligo-dT with a M13 tail was used for the RT reaction. A forward primer from each EST marker and the M13 primer (olig-dT tail) were used to amplify the 3' UTR sequence for each corresponding Acdp gene from the RT products. To obtain 5'-end coding sequences for the Acdp genes, we conducted a series nested PCR with combinations of mouse and human primers. The 5' UTR sequences were identified by directly sequencing BAC DNA containing the corresponding Acdp genes. The BAC clones were identified by screening a CITB mouse BAC DNA library (Research Genetics). The 5' UTR sequences obtained from above were further confirmed by RT-PCR. The Acdp1 gene contains 3,631 bp of nucleotide sequence and encodes a predicted protein with 951 amino acids (AA). The other three Acdp genes (Acdp2, 3 and 4) contain 3,244 bp, 2,684 bp and 2,743 bp of cDNA sequences, and encode deduced proteins of 874 amino acids, 713 amino acids and 771 amino acids, respectively. + +Tissue distribution + +Northern blot analyses of the Acdp gene family were carried out using membranes purchased from Origene. A total of 12 mouse tissues were included in the study (Fig. 1). Due to sequence homologies between each Acdp member within the conserved domain, probes for Northern bolts were PCR fragments from the last exon and the 3' untranslated region sequences. The mouse Acdp messages showed almost the same tissue distributions as the human ACDP genes. Acdp1 message is highly expressed in the brain, while kidney and testis also showed low levels of expression. Acdp2 showed higher expressions in the brain, kidney and liver. However, the Acdp2 transcript was not present in the skeleton muscle and skin, and it showed very low levels of expression in the rest of tissues. Acdp3 and Acdp4 showed different levels of expression in all tissues tested; the highest expressions for Acdp3 were observed in the brain, kidney, liver and heart, and the highest expressions for Acdp4 were observed in the kidney, small intestine and testis. The expression levels for Acdp3 and 4 in skeleton muscle were barely detectable; however, β-actin showed normal expression suggesting that the results were not a consequence of bad RNA quality (data not shown). The ubiquitous expression pattern may be taken as another indication of the functional importance of Acdp proteins in fundamental biological processes in addition to the sequence conservation in evolutionarily divergent species. + +Figure 1 + +Northern blot analyses of the Acdp gene family. S. muscle represents skeletal muscle, Sm. Int. represents small intestine. Multiple Choice Northern Blot filters were purchased from Origene. + +Chromosomal location + +Radiation hybrid mapping indicated that the Acdp1 gene maps to chromosome 19 between markers D19Mit119 (34.3 cR proximal)and D19Mit112 (13.6 cR distal). The Acdp2 gene maps slightly more distal to the Acdp1 on chromosome 19 between D19Mit9 (2.4 cR proximal) and D19Mit38 (15.1 cR distal). The Acdp3 and Acdp4 genes map to chromosome 1 within one BAC clone (RP23-294I17), proximal to marker D1Mit171 (17.4 cR). These regions are syntenic to the human counterparts. + +Sequence homology and molecular characteristics + +The mouse Acdp genes showed very strong homologies of both nucleotide and AA sequences to the human ACDP genes (Table 1). The highest homologies were observed between the human ACDP2 and the mouse Acdp2 gene (91% of nucleotide identity, 97% of AA identity and 99.4% of AA homology). In addition, the 5' UTR nucleotide sequences (20 bp of nucleotides before start codon) also showed high homologies to the human homologs, for example, the Acdp2 5' UTR sequence showed 95% identities to its human homolog. However, the homologies in the 3' UTR sequences (20 bp of nucleotides after stop codon) were much lower (40–55%) for all Acdp genes except Acdp4 (90% identity to its human homolog). The ancient conserved domain (ACD) has 55.3% of AA identity and 83.3% of homology between all mouse and human ACDP proteins (Fig. 2). The ACD domain is evolutionarily conserved in divergent species ranging from bacteria, yeast, C. elegans, D. melanogaster, mouse to human (Fig. 3). Particularly, as shown in Fig. 3, Acdp proteins showed very strong AA homology to bacteria CorC protein (35% AA identity with 55% homology), which is involved in magnesium and cobalt efflux [7]. High AA homology was also observed between the Acdp proteins and the yeast Amip3 protein (35% AA identity with 56% homology). The Amip3 is likely to be a homologous to the bacteria CorC protein. The Amip3 mutants confer resistance to copper toxicity (Personal communication with Dr. V.C. Culotte, John Hopkins Bloomberg, School of Public Health). The evolutionary relationships among those proteins are illustrated by a phylogenetic tree constructed based on the AA homology of proteins (Fig. 4). + +Table 1 + +Nucleotide and amino acid homologies (%) between human ACDP and mouse Acdp members. + +Figure 2 + +Amino acid sequence homology alignment for all of the ACDP and Acdp genes within the ACD domain. The sequence data for the Acdp genes have been deposited in GenBank under accession number AF202994 (Acdp1), AF216961 (Acdp2), AF216964 (Acdp3) and AF216963 (Acdp4). Identical amino acids or amino acids with very strong homologies among all proteins were shaded black. Identical amino acids or amino acids with very strong homologies in most of the proteins were shaded grey. Dot lines represent gaps for the alignment. + +Figure 3 + +Amino acid sequence alignment showing the conservation of ACD domain in various species. Amip3 is a protein from Saccharomyces cerevisiae (NP_014581). CanG is a protein from Candida glabrata (AAF33142). NeuC (EAA31204) is a hypothetical protein from Neurospora crassa. DroM is a gene product from D. melanogaster. The accession number for this gene is CG40084 in BDGP (Berkeley Drosophila Genome Project). AnoG represents a protein from the anopheles gambiae str. (EAA01004). CaeE (AAK77203) is a hypothetical protein from the Caenohabditis elegans. CorC represents bacteria magnesium and cobalt efflux protein from the Shewanella oneidensis. XyFD is a hypothetical protein from the Xylella fastidiosa Dixon (ZP_00038107). + +Figure 4 + +Phylogenetic tree showing relationships among proteins containing the ACD domain from figure 2 and 3. The phylogenetic tree was constructed according to the calculation of the best match for the selected sequences. Abbreviations for each protein are the same as presented in figure 3. + +We found that all mouse Acdp members contain four distinct transmembrane domains (Fig. 5), two CBS domains and a DUF21 domain that are found in bacteria CorC and yeast Amip3 proteins. CBS domains are small intracellular modules that are mostly found in 2 or four copies within a protein. Pairs of CBS domains dimerise to form a stable globular domain [8]. DUF21 (CD: pfam01959.9) is a newly defined domain with unknown function. This domain is a transmembrane region and found to be located in the N-terminus of the proteins adjacent to two intracellular CBS domains . A cNMP-binding domain (cyclic nucleotide-monophosphate-binding domain) was found in all Acdp members. + +Figure 5 + +Four transmembrane domains within Acdp4 protein. Transmembrane domains were predicted by the TMHMM program . The plot shows the posterior probabilities of inside /outside/TM helix. At the top of the plot (between 1 and 1.2) the N-best prediction is shown. The plot is obtained by calculating the total probability that a residue sits in helix, inside, or outside summed over all possible paths through the model. + +In addition, Acdp1 contains an Alanine-rich region (2–10: AAAAAAAAA), a Leucine-rich region (204–257: LLRVRPRLYGPGGDLLPPAWLRALGALLLLALSALF SGLRLSLLSLDPVELRVL), a Proline-rich region (78–130: PGPPVPAAPVPAPSLA PGENGTGDWAPRLVFIEEPPGAGGAAPSAVPTRPPGP), and two amidation sites (917–920: MGKK; 926–929: SGRK). Acdp2 has a glycine-rich region (201–222: GAGGSGSASGTVGGKGGAGVAG). Acdp3 possesses a large alanine-rich region (2–261) and a large leucine-rich region (201–299). Acdp4 contains a leucine zipper pattern (185–206: LVMVLLVLSGIFSGLNLGLMAL) and an amidation site (7–10: GGRR). + +Antibody production, Western results and subcellular localization + +Peptides from Acdp1 N- (TSFLLRVYFQPGPPATAAPVPSPT) and C- (TQQLTLSPAAVPTR) terminuses, conserved peptide from ACD domain of Acdp1 (HNIVDILFVKDLAFVDPDDCTPLLTVTRF) were commercially synthesized (Sigma Genosys). These antigenic sites were predicted by software from Sigma Genosys and polyclonal antibodies for each peptide were produced by immunizing rabbits. To test the specificity of the antibodies, we conducted Western-blot analysis of mouse brain tissue extracts. As shown in Fig. 6A, the antibody produced by C-terminal peptide specifically detected Acdp1 (lane 3). The antibody generated by N-terminal peptide recognized Acdp4 in addition to Acdp1, although the reactivity to latter was significantly higher (Fig. 6A, lane 2). As expected, the antibody produced by the conserved sequence peptide detected all Acdp proteins (Fig. 6A, lane 1). To further determine the specificity of the antibody against the Acdp1 C-terminus, we analyzed extracts of HEK293, 3T3 and PC12 cells. The results are shown in Fig. 6B. Apparently, this antibody detected a specific band of Acdp1 in all cell lysates. Of note, shown in Fig. 6B are signals of 10 μg extracts of HEK293 cell lysates, 100 μg extracts of 3T3 and PC12 cell lysates. Thus, the expression levels of Acdp1 in these cell types vary a lot, with the highest expression in HEK293 cells. Nevertheless, these immunoblot results support our analysis of brain tissue extracts that the antibody against Acdp1 C-terminus specifically recognizes Acdp-1. + +The specificity of the Acdp1 C-terminus antibody suggests the possibility of using it to localize Acdp-1 within cells. Since Northern blot revealed almost exclusive expression of Acdp1 in the brain, we examined its subcellular localization in hippocampus neurons isolated from mouse embryos. The neurons were cultured on glass coverslips coated with a confluent monolayer of mouse cortical astrocytes in dishes. Immunostaining was using the Acdp1 C-terminus specific antibody. Confocal imaging revealed that Acdp1 is predominantly localized on the plasma membrane. A series of sections of a cell at the thickness of 0.5 micrometer clearly showed membrane location of Acdp1-immunoreactivity (Fig. 7), which is consistent with the observation of transmembrane domains within the Acdp proteins. + +Figure 6 + +Fig. 6A: Immunoblot analysis of Acdp proteins in brain tissue extracts. Immunoblotting were carried out using a Western blotting detection system (ECL) (PIERCE). Lane 1, probed with antibody generated by conserved peptide. From top to bottom, each band corresponding to Acdp1 (115 kD), Acdp2 (100 kD), Acdp4 (90 kD) and Acdp3 (80 kD). Lane 2 and 3, probed with the Acdp1 antibodies generated by N-terminal and C-terminal peptides, respectively. Fig. 6B: Immunoblot analysis of Acdp1 in HEK293, 3T3 and PC12 cells. The blots were probed with the antibody against the C-terminus of Acdp-1. Lane 1, 10 μg of HEK293 cell lysates. Lane 2 and 3, 100 μg of 3T3 and PC12 cell lysates. + +Figure 7 + +Subcellular localization of Acdp1 in hippocampus neurons. A series of confocal images from a cultured neuron stained with an anti-Acdp1 antibody. The step of each imaging section is 0.5 μm, from the surface of the neuron (0 μm) to the middle plan (4.5 μm). + +Discussion + +Although the exact functions of the ACDP gene family are not yet elucidated, several lines of evidence suggest that ACDP genes may play an important role in biological processes. First, these genes are evolutionarily conserved in many phylogenetically divergent species; Second, multiple genes are present in a species; Third, these genes are ubiquitously expressed throughout development and adult tissues (unpublished data). The cloning and characterization of the mouse Acdp gene family are a very important step towards the elucidation of the functions of this multigene family. + +Sequence homology analyses revealed that Acdp proteins shared very strong AA homologies to the bacteria CorC protein and yeast Amip3 protein. CorA, B, C and D belong to a protein family involved in both influx and efflux of magnesium and cobalt. It has been shown that CorA mutants confer resistance to cobalt toxicity [9]. Amip3 appears to be a homologous to CorC protein which is involved in efflux of magnesium and cobalt in bacteria. Amip3 mutants confer resistant to copper toxicity. Acdp proteins also possess the domains that are found in bacteria CorC and yeast Amip3 proteins, such as the CBS domains, DUF21 domain and transmembrane domains. In addition, a cNMP-binding domain was found in all Acdp proteins, which is usually present in ion channels and cNMP-dependent kinases [10-13]. Using antibody produced by Acdp1 C-terminal peptide, we have shown that Acdp1 is predominantly localized on the plasma membrane in hippocampus neurons. In our previous study, we found human ACDP proteins are predominantly localized in the nucleus in HeLa cells [1]. The discrepancy for Acdp localization in neuronal cells could be caused by non-specificity for previous antibody which was produced by the recombinant ACD domain, or different cells used. In current study, the Acdp1 C-terminus antibody only recognizes Acdp1 in brain tissue extracts as well as in HEK293, 3T3 and PC12 cell lysates, suggesting the specificity of the antibody. Our observations suggested that Acdp might be involved in ion transport in mammalian cells. However, more detailed functional studies are needed to demonstrate the real functions of these proteins. + +Conclusions + +Our previous work has described human ACDP genes [1]. The new information of the current work includes: 1). It is the first time to report the existence of multiple Acdp genes in other mammals in addition to human, while Acdp appears to be a single copy gene in lower organisms such as in C. elegans, yeasts and bacteria. We have also suggested the evolutionary relationships of Acdp genes in different species (phylogenetic tree); 2). Molecular cloning and characterization of murine Acdp gene family are essential for study of this novel gene family in animal model, e.g. for generation of knockout or transgenic models; 3). We have demonstrated both DNA and amino acid conservations between mouse and human for each Acdp gene and the whole Acdp gene family, which provide important information for the possibility of functional redundancy or overlap between Acdp members; 4). We have generated antibodies specific for Acdp1 and all Acdp proteins. The Acdp1 C-terminus antibody appears to specifically recognize Acdp1. Using this antibody, we have demonstrated that Acdp1 is predominantly localized on the plasma membrane in hippocampus neurons. These results represent an important step towards the characterization of functions for the Acdp gene family. + +Methods + +cDNA cloning + +cDNA cloning of the Acdp gene family was performed based on human homologue sequences as previously reported [2]. Briefly, the human ACDP cDNAs and predicted AA sequences were used to search the mouse EST database for EST markers corresponding to each Acdp member. A forward primer within the human ACDP 5' cDNA coding region (after start codon) and a mouse reverse primer from the mouse EST marker were used to amplify the homologue sequence from mouse cDNA at very low annealing temperature (45–50°C). A nested PCR using an inside reverse primer from the mouse EST sequence and the same human forward primer was then carried out to amplify the specific mouse gene from the first round PCR products at high annealing temperature (62°C). The expected PCR products were directly excised from agarose gel and sequenced by an ABI377 automatic sequencer. The sequence was further confirmed using a forward primer from newly identified sequence and a reverse primer from known sequence. Once most of the coding sequences were identified, partial sequence of exon 1 and the 5' UTR sequences were obtained by BAC DNA sequencing. + +Northern blot analyses + +Multiple Choice Northern Blot filters containing 12 different mouse tissues were purchased from Origene. The filters were probed for each Acdp gene with a PCR fragment (around 350 bp) from last exon and the 3' untranslated region labeled with α-32P dCTP using the random primer extension system (Life Technologies). Hybridizations were carried out overnight. The filters were washed twice with washing buffer I (2×SSC, 0.1% SDS) at 42°C for 15-min, and then washed twice with washing buffer II (0.25×SSC, 0.1% SDS) at 65°C for 15-min. Washed filters were exposed to X-ray films for overnight or longer [3,4]. + +Antibody production and Western blot analyses + +Peptides linked to KLH (keyhole limpet hemacyanin) from Acdp1 N- and C-terminals and the conserved domain (ACD) were used for generation of antibodies specifically for Acdp1 and all Acdp members as reported, respectively [5]. Western blots were carried out Using ECL (PIERCE) as described previously [1]. The membranes were washed extensively after incubation with primary and secondary antibodies and were then developed with X-ray films with optimal exposure time. + +Chromosome localization + +The T31 mouse radiation hybrid panel from Research Genetics was used to map the chromosome location of each Acdp member. Primers from 3' UTR of each Acdp member were used to amplify the 100 radiation hybrid clones representing the mouse genome. The data were submitted to the Jackson Laboratory Mouse Radiation Hybrid Database for analysis. + +Sequence analyses + +Sequence assembly was performed with program Sequencher (Gene Codes Corp). Protein and DNA homology searches were carried out with tblastn, tblastx, blastp and blastn programs . Multiple sequence alignments were performed with GeneDoc and pairwise sequence alignment . Multiple programs including BCM Search Launcher , ProfileScan , sequence motif search , ExPASy and 3Dpssm were used for searching sequence features of known protein. Phylogenetic tree was constructed by Clustalw program (version 1.81) using UPGMA (Unweighted Pair Group Method using Arithmetic averages) algorithm [14]. + +Neuronal cell preparation and immunostanining + +Hippocampal neuron cultures were prepared as previously reported [6]. In brief, the hippocampuses were dissected out from mouse embryos at 16 days in utero. The tissues were then incubated for 20 min at 37°C in MEM (minimum essential medium) modified for suspension culture (Life Technologies) plus 0.25% trypsin (Life Technologies). The dissociated hippocampal neurons were plated on glass coverslips coated with a confluent monolayer of mouse cortical astrocytes obtained as described below. The neurons were maintained at 37°C in a humidified atmosphere with 5% CO2. Cortical astrocytes dissociated from newborn mouse cortices were grown in culture flasks at 37°C in a humidified atmosphere with 5% CO2 until confluent. The cells were exposed to 10-5 M cytosine arabinoside (Sigma) and cultured for additional 12–24 hrs at 37°C. After remove of the media with cellular debris, the cells were used for coating coverslips. + +For immunostaining, neuronal cells on the coverslips were first fixed in PBS containing 4% paraformaldehyde (PFA) for 12 hrs at 4°C and then incubated in a solution containing 4% PFA and 0.4% Triton X-100 at 4°C for 1 hr. After washing with PBS three times, the cells were incubated with a blocking solution containing 1:30 normal goat serum, and subsequently incubated with a rabbit polyclonal anti-ACDP antibody (1:3000) overnight at 4°C. After extensive washing with 1% goat serum PBS solution, the cells were incubated with an Alex 488 conjugated secondary antibody (1:100 in 1% goat serum PBS solution, Molecular Probes) for 3 hrs at room temperature. Following final washes with 1% goat serum PBS solution, the neuronal cells on the coverslips were cover-slipped with a glycerol-based anti-photobleach medium. The cells were viewed under a confocal microscope (Carl Zeiss). Images were captured with a CCD camera and acquired by the Scion Image software (Scion Corporation, Frederick, MD). + +Gene bank accession number + +The cDNA sequences for the Acdp gene family have already been deposited in gene bank with accession numbers AF202994 (Acdp1), AF216961 (Acdp2), AF216964 (Acdp3) and AF216963 (Acdp4). + +Authors' contributions + +This study was designed by CYW and JXS. The study was performed as follows: CYW, PY, JDS and HA contributed to the molecular cloning part of the manuscript. CYW and SP were responsible for the Acdp antibody production. JGG and JLG did immunostaining for Acdp1 localization in neuronal cells. CYW, DG and ZD carried out the Western analyses. + +Acknowledgements + +This work was partly supported by a CIGP grant to C.Y.W. diff --git a/src/ontogpt/evaluation/craft/database/all/14737183.ann b/src/ontogpt/evaluation/craft/database/all/14737183.ann new file mode 100644 index 000000000..06816a268 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/14737183.ann @@ -0,0 +1,1454 @@ +T1 NCBITaxon:10088 31 36 Mouse +T2 UBERON:0010166 37 41 Coat +T3 PR:000016145 45 50 Tbx15 +T4 NCBITaxon:33208 82 88 animal +T5 NCBITaxon:kingdom 89 96 kingdom +T6 UBERON:0010166 105 109 coat +T7 GO:0065007 200 207 control +T8 NCBITaxon:10088 278 283 mouse +T9 UBERON:0001690 301 304 ear +T10 GO:0065007 378 385 control +T11 PR:000004372 393 399 Agouti +T12 SO:0000704 400 404 gene +T13 NCBITaxon:10088 406 410 Mice +T14 PR:000004372 424 430 Agouti +T15 SO:0001023 431 437 allele +T16 UBERON:0001037 509 513 hair +T17 UBERON:0001037 533 537 hair +T18 SO:0000147 714 718 exon +T19 PR:000016145 726 731 Tbx15 +T20 SO:0000704 732 736 gene +T21 UBERON:0000922 744 753 embryonic +T22 GO:0010467 754 764 expression +T23 UBERON:0003104 779 789 mesenchyme +T24 CHEBI:26130 806 816 pigmentary +T25 UBERON:0004288 821 829 skeletal +T26 NCBITaxon:33208 864 871 animals +T27 SO:0001023 900 906 allele +T28 PR:000016145 910 915 Tbx15 +T29 PR:000016145 963 968 Tbx15 +T30 UBERON:0000922 993 1002 embryonic +T31 GO:0010467 1003 1013 expression +T32 PR:000016145 1017 1022 Tbx15 +T33 UBERON:0003104 1033 1043 mesenchyme +T34 PR:000004372 1064 1070 Agouti +T35 GO:0010467 1071 1081 expression +T36 UBERON:2000164 1085 1103 ventral mesenchyme +T37 PR:000016145 1123 1128 Tbx15 +T38 GO:0010467 1130 1140 expression +T39 PR:000004372 1144 1150 Agouti +T40 UBERON:0000922 1159 1166 embryos +T41 GO:0007567 1175 1180 natal +T42 NCBITaxon:33208 1181 1188 animals +T43 UBERON:0019248 1387 1402 early embryonic +T44 GO:0010467 1403 1413 expression +T45 PR:000016145 1417 1422 Tbx15 +T46 UBERON:0002101 1592 1596 limb +T47 UBERON:0000922 1620 1629 Embryonic +T48 GO:0010467 1630 1640 expression +T49 PR:000016145 1644 1649 Tbx15 +T50 UBERON:0003104 1666 1676 mesenchyme +T51 UBERON:0002067 1770 1776 dermis +T52 SO:0000704 1826 1830 gene +T53 UBERON:0000922 1841 1850 embryonic +T54 GO:0009790 1841 1862 embryonic development +T55 NCBITaxon:40674 1971 1978 mammals +T56 NCBITaxon:7742 2176 2186 vertebrate +T57 NCBITaxon:33208 2372 2379 animals +T58 GO:0065007 2464 2471 control +T59 NCBITaxon:7742 2531 2542 vertebrates +T60 UBERON:0004765 2582 2599 skeletal elements +T61 UBERON:0007376 2623 2632 epidermal +T62 UBERON:0004529 2633 2643 appendages +T63 CHEBI:26130 2662 2669 pigment +T64 NCBITaxon:33208 2741 2747 animal +T65 CHEBI:26130 2775 2782 pigment +T66 NCBITaxon:species 2916 2923 species +T67 NCBITaxon:33208 2942 2949 animals +T68 NCBITaxon:species 2971 2978 species +T69 CHEBI:26130 3123 3130 pigment +T70 NCBITaxon:7742 3309 3319 vertebrate +T71 CHEBI:26130 3391 3401 pigmentary +T72 UBERON:0000062 3480 3485 organ +T73 NCBITaxon:7742 3603 3613 vertebrate +T74 UBERON:0000922 3635 3641 embryo +T75 CL:0000541 3692 3704 melanoblasts +T76 UBERON:0002342 3714 3726 neural crest +T77 CHEBI:26130 3764 3771 pigment +T78 CL:0000147 3764 3777 pigment cells +T79 UBERON:0002073 3830 3844 hair follicles +T80 GO:0038001 3846 3855 paracrine +T81 GO:0065007 3864 3871 control +T82 CHEBI:26130 3884 3891 pigment +T83 UBERON:0001037 3961 3965 hair +T84 GO:0042633 3961 3971 hair cycle +T85 GO:0051904 4036 4064 movement of pigment granules +T86 CHEBI:26130 4048 4055 pigment +T87 GO:0048770 4048 4064 pigment granules +T88 CL:0000148 4072 4083 melanocytes +T89 CL:0000148 4092 4103 melanocytes +T90 CL:0000312 4107 4120 keratinocytes +T91 CHEBI:26130 4337 4344 pigment +T92 GO:0042470 4365 4375 melanosome +T93 GO:1903232 4365 4386 melanosome biogenesis +T94 GO:0065007 4480 4487 control +T95 NCBITaxon:7742 4553 4564 vertebrates +T96 UBERON:0002542 4660 4666 scales +T97 UBERON:0000022 4668 4676 feathers +T98 UBERON:0001037 4681 4685 hair +T99 UBERON:0002101 4794 4799 limbs +T100 NCBITaxon:9989 4804 4811 rodents +T101 NCBITaxon:40674 4831 4838 mammals +T102 UBERON:0001037 4872 4876 hair +T103 CHEBI:26130 4918 4925 pigment +T104 SO:0001023 4948 4955 allelic +T105 PR:000004372 4973 4979 Agouti +T106 SO:0000704 4980 4984 gene +T107 GO:0046903 5028 5036 Secreted +T108 UBERON:0000412 5040 5054 dermal papilla +T109 CL:0000346 5040 5060 dermal papilla cells +T110 UBERON:0002073 5073 5086 hair follicle +T111 PR:000004372 5109 5115 Agouti +T112 CL:0000148 5131 5142 melanocytes +T113 CHEBI:24009 5205 5214 eumelanin +T114 CHEBI:81393 5229 5240 pheomelanin +T115 PR:000004372 5242 5248 Agouti +T116 UBERON:0001037 5359 5363 hair +T117 GO:0042633 5359 5369 hair cycle +T118 GO:0065007 5451 5460 regulated +T119 GO:0010467 5461 5471 expression +T120 NCBITaxon:10088 5552 5556 mice +T121 SO:0001023 5589 5595 allele +T122 UBERON:2002284 5615 5623 markings +T123 UBERON:0002387 5635 5639 feet +T124 UBERON:0001690 5641 5645 ears +T125 UBERON:0000033 5650 5654 head +T126 UBERON:0000033 5676 5680 head +T127 UBERON:2002283 5681 5686 spots +T128 NCBITaxon:9615 5699 5702 dog +T129 NCBITaxon:10088 5726 5730 mice +T130 PR:000004372 5802 5808 Agouti +T131 SO:0001060 5814 5822 isoforms +T132 SO:0000315 5854 5885 transcriptional initiation site +T133 SO:0000445 5890 5911 5′ untranslated exons +T134 GO:0006412 5895 5905 translated +T135 UBERON:0001037 5916 5920 hair +T136 GO:0042633 5916 5926 hair cycle +T137 SO:0000673 5937 5947 transcript +T138 GO:0010467 5951 5960 expressed +T139 UBERON:0001037 6019 6023 hair +T140 SO:0000673 6059 6069 transcript +T141 GO:0010467 6073 6082 expressed +T142 UBERON:0001037 6122 6126 hair +T143 NCBITaxon:33208 6205 6212 Animals +T144 SO:0001023 6229 6235 allele +T145 GO:0010467 6236 6243 express +T146 PR:000004372 6270 6276 Agouti +T147 SO:0000673 6277 6287 transcript +T148 UBERON:0001037 6354 6359 hairs +T149 UBERON:0001037 6396 6401 hairs +T150 UBERON:0002101 6445 6449 limb +T151 UBERON:0000309 6450 6459 body wall +T152 UBERON:0004905 6460 6473 articulations +T153 UBERON:0006378 6499 6506 whisker +T154 UBERON:2001977 6507 6510 pad +T155 PR:000004372 6529 6535 Agouti +T156 SO:0001060 6536 6544 isoforms +T157 GO:0010467 6554 6563 expressed +T158 UBERON:0000922 6588 6597 embryonic +T159 CHEBI:26130 6649 6656 pigment +T160 CL:0000147 6649 6661 pigment cell +T161 GO:0030154 6657 6677 cell differentiation +T162 GO:0065007 6706 6716 regulatory +T163 SO:0005836 6706 6725 regulatory elements +T164 PR:000004372 6747 6753 Agouti +T165 SO:0001060 6754 6762 isoforms +T166 UBERON:0000922 6829 6835 embryo +T167 GO:0007567 6868 6873 birth +T168 NCBITaxon:10088 6944 6948 mice +T169 NCBITaxon:40674 7030 7037 mammals +T170 GO:0009653 7043 7056 morphogenetic +T171 CHEBI:26130 7155 7162 pigment +T172 UBERON:0002073 7171 7184 hair follicle +T173 CL:0002483 7171 7196 hair follicle melanocytes +T174 UBERON:0002067 7213 7219 dermis +T175 UBERON:0000180 7227 7232 flank +T176 UBERON:0004016 7268 7278 dermatomal +T177 UBERON:0002329 7294 7301 somites +T178 UBERON:0007529 7306 7322 loose mesenchyme +T179 UBERON:0003081 7340 7362 lateral plate mesoderm +T180 UBERON:0007023 7604 7609 adult +T181 NCBITaxon:10088 7848 7853 mouse +T182 PR:000004372 7915 7921 Agouti +T183 GO:0010467 7922 7932 expression +T184 UBERON:0002329 7977 7983 somite +T185 UBERON:0003081 7989 8002 lateral plate +T186 CL:0000148 8152 8162 melanocyte +T187 CHEBI:26130 8195 8202 pigment +T188 UBERON:0001037 8223 8227 hair +T189 UBERON:0002329 8282 8288 somite +T190 UBERON:0003081 8289 8302 lateral plate +T191 NCBITaxon:10088 8353 8358 mouse +T192 UBERON:0001690 8376 8379 ear +T193 UBERON:0000180 8446 8451 flank +T194 UBERON:0010166 8452 8456 coat +T195 PR:000004372 8509 8515 Agouti +T196 SO:0000673 8516 8526 transcript +T197 SO:0000704 8554 8558 gene +T198 SO:0001023 8585 8591 allele +T199 UBERON:0001690 8602 8605 ear +T200 PR:000016145 8638 8643 Tbx15 +T201 GO:0010467 8688 8697 expressed +T202 UBERON:0002204 8770 8792 musculoskeletal system +T203 UBERON:0000922 8794 8803 Embryonic +T204 GO:0010467 8804 8814 expression +T205 PR:000016145 8856 8861 Tbx15 +T206 UBERON:0003104 8935 8946 mesenchymal +T207 CL:0000134 8935 8952 mesenchymal cells +T208 UBERON:0000180 8971 8976 flank +T209 NCBITaxon:40674 9099 9106 mammals +T210 UBERON:0001037 9308 9312 hair +T211 UBERON:0001037 9439 9443 hair +T212 UBERON:0001037 9468 9472 hair +T213 CHEBI:26130 9624 9631 pigment +T214 CL:0000147 9624 9637 pigment cells +T215 CHEBI:26130 9662 9669 pigment +T216 GO:0010467 9697 9707 expression +T217 PR:000004372 9711 9717 Agouti +T218 UBERON:0001037 9742 9746 hair +T219 NCBITaxon:33208 9756 9763 animals +T220 NCBITaxon:10088 9975 9979 mice +T221 PR:000004372 9993 9999 Agouti +T222 UBERON:0001037 10045 10049 hair +T223 CHEBI:26130 10171 10178 pigment +T224 NCBITaxon:10088 10201 10205 mice +T225 UBERON:0001037 10251 10255 hair +T226 UBERON:0001037 10266 10270 hair +T227 UBERON:0001137 10331 10337 dorsum +T228 UBERON:0013235 10341 10348 ventrum +T229 UBERON:0000180 10362 10367 flank +T230 UBERON:0001037 10368 10373 hairs +T231 NCBITaxon:10088 10385 10389 mice +T232 CHEBI:81393 10453 10464 pheomelanin +T233 UBERON:0001037 10520 10524 hair +T234 UBERON:0001037 10564 10568 hair +T235 PR:000004372 10646 10652 Agouti +T236 UBERON:0001037 10672 10676 hair +T237 GO:0042633 10672 10682 hair-cycle +T238 UBERON:0001037 10751 10755 hair +T239 NCBITaxon:10088 10767 10771 mice +T240 UBERON:0001037 10885 10889 hair +T241 NCBITaxon:33208 10901 10908 animals +T242 PR:000004372 10937 10943 Agouti +T243 UBERON:0001037 11088 11092 hair +T244 UBERON:0001037 11175 11179 hair +T245 GO:0042633 11175 11186 hair cycles +T246 UBERON:0001037 11209 11213 hair +T247 CHEBI:26130 11257 11264 pigment +T248 NCBITaxon:10088 11290 11294 mice +T249 GO:0007567 11384 11389 natal +T250 UBERON:0002073 11420 11434 hair follicles +T251 UBERON:0002073 11483 11497 hair follicles +T252 UBERON:0001037 11710 11714 hair +T253 UBERON:0001137 11740 11745 dorsa +T254 UBERON:0013235 11750 11756 ventra +T255 UBERON:0007023 11760 11765 adult +T256 NCBITaxon:10088 11766 11770 mice +T257 NCBITaxon:10088 11794 11798 mice +T258 UBERON:0001037 11855 11860 hairs +T259 UBERON:0010513 11862 11869 zigzags +T260 UBERON:0001037 11883 11888 hairs +T261 UBERON:0010510 11890 11898 auchenes +T262 UBERON:0010511 11900 11904 awls +T263 UBERON:0010512 11910 11921 guard hairs +T264 UBERON:0001137 11926 11932 dorsum +T265 UBERON:0013235 11945 11952 ventrum +T266 UBERON:0001037 12008 12012 hair +T267 NCBITaxon:10088 12043 12047 mice +T268 NCBITaxon:10088 12128 12132 mice +T269 CHEBI:26130 12159 12166 pigment +T270 GO:0010467 12211 12221 expression +T271 PR:000004372 12225 12231 Agouti +T272 NCBITaxon:33208 12237 12244 animals +T273 SO:0001023 12267 12273 allele +T274 PR:000004372 12277 12283 Agouti +T275 UBERON:0001037 12322 12327 hairs +T276 CHEBI:25179 12346 12353 melanin +T277 UBERON:0001037 12366 12371 hairs +T278 UBERON:0010166 12423 12427 coat +T279 CHEBI:49168 12447 12451 DOPA +T280 UBERON:0002067 12559 12565 dermis +T281 NCBITaxon:10088 12595 12599 mice +T282 NCBITaxon:10088 12642 12646 mice +T283 CHEBI:49168 12691 12695 DOPA +T284 CHEBI:25179 12762 12769 melanin +T285 NCBITaxon:10088 12791 12795 mice +T286 PR:000004372 12850 12856 Agouti +T287 CHEBI:25179 12858 12865 Melanin +T288 UBERON:0001037 12888 12893 hairs +T289 CHEBI:26130 12943 12950 pigment +T290 CL:0000147 12943 12956 pigment cells +T291 UBERON:0001037 13031 13035 hair +T292 CHEBI:26130 13036 13043 pigment +T293 NCBITaxon:10088 13061 13065 mice +T294 UBERON:0001037 13094 13098 hair +T295 GO:0042633 13094 13105 hair cycles +T296 UBERON:0000113 13111 13120 adulthood +T297 UBERON:0001037 13133 13137 hair +T298 CHEBI:26130 13264 13271 pigment +T299 PR:000004372 13301 13307 Agouti +T300 UBERON:0001037 13334 13338 hair +T301 CHEBI:25179 13366 13373 melanin +T302 UBERON:0001690 13432 13435 ear +T303 UBERON:0001690 13505 13508 ear +T304 NCBITaxon:10088 13536 13541 mouse +T305 SO:0001023 13569 13575 allele +T306 NCBITaxon:33208 13860 13867 animals +T307 UBERON:0000970 13893 13897 eyes +T308 UBERON:0001819 13905 13923 palpebral fissures +T309 UBERON:0000004 13933 13938 nasal +T310 UBERON:0003129 13961 13966 skull +T311 UBERON:0001690 14068 14072 ears +T312 UBERON:0001690 14106 14109 ear +T313 SO:0001023 14131 14137 allele +T314 CHEBI:26130 14162 14169 pigment +T315 SO:0000704 14252 14259 genetic +T316 PR:000004372 14279 14280 A +T317 UBERON:0000916 14300 14305 belly +T318 UBERON:0001037 14306 14310 hair +T319 UBERON:0011270 14334 14338 back +T320 UBERON:0001037 14339 14343 hair +T321 UBERON:0000916 14349 14354 belly +T322 UBERON:0001037 14355 14359 hair +T323 UBERON:0001456 14409 14413 face +T324 CHEBI:26130 14455 14462 pigment +T325 NCBITaxon:10088 14509 14513 mice +T326 NCBITaxon:33208 14543 14550 animals +T327 UBERON:0001037 14679 14684 hairs +T328 UBERON:0002101 14758 14762 limb +T329 UBERON:0000309 14763 14772 body wall +T330 NCBITaxon:10088 14909 14913 mice +T331 UBERON:0001037 14950 14955 hairs +T332 NCBITaxon:10088 15001 15005 mice +T333 UBERON:2001463 15051 15057 stripe +T334 UBERON:0000180 15068 15073 flank +T335 NCBITaxon:10088 15097 15101 mice +T336 UBERON:0000180 15188 15193 flank +T337 CHEBI:24009 15208 15217 eumelanic +T338 NCBITaxon:33208 15342 15349 animals +T339 NCBITaxon:33208 15433 15440 animals +T340 NCBITaxon:33208 15632 15639 animals +T341 UBERON:0002101 15732 15737 limbs +T342 UBERON:1000019 15746 15753 cranium +T343 UBERON:0006378 15767 15774 whisker +T344 UBERON:2001977 15775 15778 pad +T345 CHEBI:26130 15944 15951 pigment +T346 UBERON:0001037 15995 15999 hair +T347 UBERON:0001037 16104 16108 hair +T348 UBERON:0001137 16124 16130 dorsum +T349 UBERON:0013235 16135 16142 ventrum +T350 NCBITaxon:33208 16170 16177 animals +T351 UBERON:0001037 16225 16229 hair +T352 NCBITaxon:33208 16310 16317 animals +T353 UBERON:0004288 16350 16358 skeletal +T354 UBERON:0010166 16389 16393 coat +T355 GO:0007567 16527 16532 birth +T356 GO:0042438 16566 16577 melanogenic +T357 CL:0000541 16642 16653 melanoblast +T358 UBERON:0002342 16673 16685 neural crest +T359 CL:0000148 16746 16756 melanocyte +T360 CHEBI:26130 16777 16784 pigment +T361 GO:0046148 16777 16794 pigment synthesis +T362 UBERON:0001137 16862 16868 dorsum +T363 UBERON:0000014 16906 16913;16919 16923 area of ... skin +T364 UBERON:0002101 16967 16972 limbs +T365 CHEBI:24009 17010 17019 eumelanin +T366 UBERON:0007023 17038 17043 adult +T367 NCBITaxon:33208 17044 17051 animals +T368 GO:0009953 17126 17166 establishment of dorsoventral patterning +T369 GO:0043588 17174 17190 skin development +T370 CL:0000148 17360 17370 melanocyte +T371 CHEBI:81393 17401 17412 pheomelanin +T372 UBERON:0001037 17468 17472 hair +T373 UBERON:0013235 17485 17492 ventrum +T374 UBERON:0001137 17496 17502 dorsum +T375 SO:0000704 17528 17532 gene +T376 PR:000016145 17551 17556 Tbx15 +T377 GO:0010467 17570 17579 expressed +T378 UBERON:0001690 17802 17805 ear +T379 SO:0001023 17806 17812 allele +T380 SO:0001023 17824 17830 allele +T381 NCBITaxon:10088 18058 18062 mice +T382 NCBITaxon:2 18175 18184 bacterial +T383 SO:0000153 18175 18206 bacterial artificial chromosome +T384 SO:0000153 18208 18211 BAC +T385 SO:0000149 18213 18219 contig +T386 SO:0000704 18283 18288 genes +T387 PR:000016145 18300 18305 Tbx15 +T388 PR:000016145 18333 18338 Tbx15 +T389 UBERON:0004288 18373 18381 skeletal +T390 UBERON:0000922 18472 18481 embryonic +T391 GO:0010467 18482 18492 expression +T392 UBERON:0002101 18535 18540 limbs +T393 NCBITaxon:10088 18625 18630 mouse +T394 SO:0001026 18631 18637 genome +T395 SO:0000852 18662 18673;18688 18693 portions of ... exons +T396 PR:000016145 18682 18687 Tbx15 +T397 SO:0001026 18730 18737 genomic +T398 SO:0000704 18752 18756 gene +T399 PR:000016145 18786 18790 Tbx8 +T400 PR:000016145 18836 18841 Tbx14 +T401 NCBITaxon:7742 18883 18893 vertebrate +T402 SO:0001026 18894 18901 genomes +T403 PR:000016145 18905 18910 Tbx15 +T404 SO:0001026 19035 19042 genomic +T405 NCBITaxon:10088 19069 19074 mouse +T406 SO:0001026 19075 19081 genome +T407 PR:000016145 19142 19147 Tbx15 +T408 SO:0000188 19148 19154 intron +T409 GO:0043631 19185 19200 polyadenylation +T410 SO:0000610 19185 19209 polyadenylation sequence +T411 CHEBI:17369 19237 19256 mannose-6-phosphate +T412 SO:0000336 19266 19276 pseudogene +T413 SO:0000336 19283 19285 ps +T414 SO:0000673 19378 19388 transcript +T415 PR:000016145 19508 19513 Tbx15 +T416 SO:0000417 19547 19553 domain +T417 NCBITaxon:33208 19562 19569 animals +T418 NCBITaxon:33208 19619 19626 animals +T419 SO:0001023 19675 19681 allele +T420 SO:0000336 19712 19714 ps +T421 SO:0000704 19725 19730 genes +T422 SO:0000673 19734 19745 transcripts +T423 PR:000016145 19896 19901 Tbx15 +T424 SO:0000704 19905 19909 gene +T425 UBERON:0000922 19923 19932 embryonic +T426 CL:0002322 19923 19943 embryonic stem cells +T427 SO:0001023 19958 19964 allele +T428 PR:000016145 19966 19971 Tbx15 +T429 PR:000033987 19971 19975 LacZ +T430 SO:0000243 19988 19992 IRES +T431 PR:000033987 19993 19997 LacZ +T432 SO:0005853 20007 20015 cassette +T433 SO:0000236 20034 20052 open reading frame +T434 SO:0000360 20056 20061 codon +T435 SO:0000417 20085 20091 domain +T436 NCBITaxon:33208 20105 20112 Animals +T437 SO:0001023 20143 20149 allele +T438 UBERON:0004288 20193 20201 skeletal +T439 UBERON:0001037 20218 20222 hair +T440 PR:000016145 20247 20252 Tbx15 +T441 PR:000033987 20252 20256 LacZ +T442 PR:000016145 20257 20262 Tbx15 +T443 PR:000033987 20262 20266 LacZ +T444 PR:000016145 20405 20410 Tbx15 +T445 PR:000033987 20410 20414 LacZ +T446 NCBITaxon:33208 20473 20480 animals +T447 UBERON:0000180 20572 20577 flank +T448 NCBITaxon:33208 20614 20621 animals +T449 CHEBI:26130 20678 20688 pigmentary +T450 PR:000016145 20764 20769 Tbx15 +T451 GO:0010467 20772 20782 Expression +T452 PR:000016145 20786 20791 Tbx15 +T453 PR:000004372 20796 20802 Agouti +T454 GO:0097617 20872 20885 hybridization +T455 GO:0010467 20896 20906 expression +T456 PR:000016145 20910 20915 Tbx15 +T457 UBERON:0004347 20951 20960 limb buds +T458 UBERON:0002539 20981 20997 branchial arches +T459 UBERON:0000180 20999 21005 flanks +T460 GO:0097617 21094 21104 hybridized +T461 PR:000016145 21107 21112 Tbx15 +T462 GO:0010467 21181 21191 expression +T463 UBERON:0005253 21204 21226;21231 21235 mesenchymal tissues of ... head +T464 UBERON:0005256 21204 21226;21237 21242 mesenchymal tissues of ... trunk +T465 UBERON:0009749 21204 21226;21259 21264 mesenchymal tissues of ... limbs +T466 UBERON:0003129 21315 21320 skull +T467 UBERON:0002413 21322 21340 cervical vertebrae +T468 UBERON:0002101 21346 21350 limb +T469 NCBITaxon:10088 21378 21382 mice +T470 UBERON:0001690 21412 21415 ear +T471 SO:0001023 21416 21422 allele +T472 UBERON:0000922 21496 21505 embryonic +T473 UBERON:0000180 21506 21511 flank +T474 GO:0010467 21512 21522 expression +T475 NCBITaxon:10088 21578 21582 mice +T476 UBERON:0000916 21595 21604 abdominal +T477 GO:0010467 21684 21694 expression +T478 UBERON:0003104 21714 21724 mesenchyme +T479 UBERON:0002067 21841 21847 dermis +T480 UBERON:0007529 21878 21894 loose mesenchyme +T481 UBERON:0018260 21913 21925 muscle layer +T482 PR:000016145 21927 21932 Tbx15 +T483 GO:0010467 21936 21945 expressed +T484 UBERON:0002378 21995 22012 abdominal muscles +T485 PR:000016145 22028 22033 Tbx15 +T486 GO:0010467 22037 22046 expressed +T487 UBERON:0002067 22117 22123 dermis +T488 UBERON:0005933 22146 22153 sheaths +T489 UBERON:0002073 22157 22171 hair follicles +T490 GO:0010467 22179 22189 expression +T491 UBERON:0000412 22219 22234 dermal papillae +T492 CL:0000346 22219 22240 dermal papillae cells +T493 PR:000004372 22279 22285 Agouti +T494 CHEBI:26130 22289 22296 pigment +T495 GO:0007567 22329 22334 natal +T496 UBERON:0001037 22335 22339 hair +T497 SO:0001060 22369 22376 isoform +T498 PR:000004372 22380 22386 Agouti +T499 GO:0010467 22390 22399 expressed +T500 GO:0097617 22469 22479 hybridized +T501 PR:000016145 22496 22501 Tbx15 +T502 PR:000004372 22506 22512 Agouti +T503 GO:0010467 22564 22574 expression +T504 PR:000004372 22578 22584 Agouti +T505 GO:0010467 22605 22615 expression +T506 PR:000016145 22619 22624 Tbx15 +T507 GO:0010467 22681 22691 expression +T508 PR:000016145 22729 22734 Tbx15 +T509 GO:0010467 22735 22745 expression +T510 PR:000004372 22794 22800 Agouti +T511 GO:0010467 22801 22811 expression +T512 GO:0010467 22871 22881 expression +T513 PR:000004372 22885 22891 Agouti +T514 PR:000004372 23041 23047 Agouti +T515 GO:0010467 23048 23058 expression +T516 UBERON:0000922 23103 23110 embryos +T517 GO:0010467 23138 23148 expression +T518 PR:000004372 23152 23158 Agouti +T519 NCBITaxon:33208 23196 23203 animals +T520 UBERON:0002067 23265 23271 dermis +T521 UBERON:0000412 23276 23291 dermal papillae +T522 CL:0000346 23276 23297 dermal papillae cells +T523 NCBITaxon:10088 23361 23365 mice +T524 PR:000004372 23425 23431 Agouti +T525 GO:0010467 23432 23442 expression +T526 GO:0007567 23449 23454 birth +T527 GO:0010467 23466 23476 expression +T528 PR:000016145 23480 23485 Tbx15 +T529 PR:000004372 23490 23496 Agouti +T530 PR:000016145 23576 23581 Tbx15 +T531 GO:0010467 23596 23606 expression +T532 PR:000004372 23610 23616 Agouti +T533 UBERON:0000922 23660 23669 Embryonic +T534 PR:000016145 23670 23675 Tbx15 +T535 GO:0010467 23676 23686 Expression +T536 GO:0010467 23811 23821 expression +T537 PR:000016145 23825 23830 Tbx15 +T538 UBERON:0000922 23838 23847 embryonic +T539 UBERON:0000180 23855 23860 flank +T540 UBERON:0002067 23920 23926 dermis +T541 CHEBI:26130 23953 23960 pigment +T542 PR:000004372 24007 24013 Agouti +T543 SO:0001060 24014 24021 isoform +T544 UBERON:0000922 24125 24134 embryonic +T545 UBERON:0000922 24210 24219 embryonic +T546 UBERON:0001037 24281 24285 hair +T547 GO:0010467 24307 24317 expression +T548 PR:000016145 24321 24326 Tbx15 +T549 PR:000004372 24331 24337 Agouti +T550 UBERON:0000922 24464 24471 embryos +T551 UBERON:0001037 24503 24507 hair +T552 UBERON:0000473 24546 24552 testis +T553 UBERON:0007376 24615 24624 epidermal +T554 UBERON:0000922 24728 24737 embryonic +T555 UBERON:0002067 24738 24744 dermis +T556 UBERON:0000922 24792 24801 embryonic +T557 UBERON:0000922 24817 24824 embryos +T558 UBERON:0000180 24838 24843 flank +T559 UBERON:0001037 24948 24952 hair +T560 UBERON:0000473 24959 24965 testis +T561 SO:0000704 25005 25009 gene +T562 GO:0010467 25005 25020 gene expression +T563 GO:0097617 25035 25048 hybridization +T564 UBERON:0000180 25118 25123 flank +T565 UBERON:0000180 25202 25207 flank +T566 UBERON:0000924 25232 25242 ectodermal +T567 UBERON:0003082 25306 25313 myotome +T568 UBERON:0000922 25494 25503 embryonic +T569 UBERON:0001037 25536 25540 hair +T570 UBERON:0000473 25566 25572 testis +T571 UBERON:0001037 25602 25606 hair +T572 UBERON:0001037 25641 25645 hair +T573 UBERON:0001037 25690 25694 hair +T574 UBERON:0000180 25723 25728 flank +T575 UBERON:0001037 25765 25769 hair +T576 UBERON:0001037 25806 25810 hair +T577 UBERON:0000180 25871 25876 flank +T578 UBERON:0001037 25916 25920 hair +T579 UBERON:0001037 25982 25987 hairs +T580 UBERON:0000479 26029 26035 tissue +T581 GO:0010467 26082 26091 expressed +T582 PR:000016145 26092 26097 Tbx15 +T583 PR:000004372 26106 26112 Agouti +T584 UBERON:0000180 26120 26125 flank +T585 GO:0010467 26133 26142 expressed +T586 SO:0000704 26148 26153 genes +T587 GO:0010467 26263 26273 expression +T588 PR:000016145 26295 26300 Tbx15 +T589 PR:000004372 26305 26311 Agouti +T590 GO:0043588 26401 26417 skin development +T591 GO:0010467 26431 26441 expression +T592 PR:000016145 26445 26450 Tbx15 +T593 CHEBI:26130 26537 26544 Pigment +T594 UBERON:0015178 26594 26610 Somitic Frontier +T595 UBERON:0000924 26616 26626 ectodermal +T596 UBERON:0000922 26675 26684 embryonic +T597 UBERON:0001137 26685 26691 dorsum +T598 UBERON:0000922 26696 26705 embryonic +T599 UBERON:0000180 26706 26711 flank +T600 NCBITaxon:7742 26743 26753 vertebrate +T601 UBERON:0000922 26754 26761 embryos +T602 UBERON:0002067 26876 26882 dermis +T603 UBERON:0003077 26896 26912 somitic mesoderm +T604 UBERON:0002067 26917 26923 dermis +T605 UBERON:0003081 26937 26959 lateral plate mesoderm +T606 UBERON:0015178 26993 27009 somitic frontier +T607 NCBITaxon:40674 27165 27174 mammalian +T608 UBERON:0000922 27175 27182 embryos +T609 UBERON:0002329 27184 27190 somite +T610 UBERON:0003081 27196 27209 lateral plate +T611 UBERON:0000926 27218 27226 mesoderm +T612 UBERON:0002067 27261 27267 dermis +T613 UBERON:0002101 27294 27298 limb +T614 UBERON:0000309 27299 27308 body wall +T615 UBERON:0000924 27443 27453 ectodermal +T616 UBERON:0002067 27552 27558 dermis +T617 UBERON:0003081 27572 27594 lateral plate mesoderm +T618 SO:0000902 27617 27626 transgene +T619 PR:000008703 27641 27646 Hoxb6 +T620 SO:0000167 27647 27655 promoter +T621 GO:0007565 27758 27767 gestation +T622 UBERON:0000922 27768 27775 embryos +T623 PR:000008703 27794 27799 Hoxb6 +T624 SO:0000902 27804 27813 transgene +T625 PR:000033987 27827 27831 lacZ +T626 SO:0000704 27841 27845 gene +T627 CHEBI:75055 27869 27874 X-Gal +T628 UBERON:0003081 27887 27909 lateral plate mesoderm +T629 UBERON:0003077 27918 27941 somite-derived mesoderm +T630 UBERON:0002100 27949 27954 trunk +T631 NCBITaxon:33208 28016 28023 animals +T632 CHEBI:75055 28060 28065 X-Gal +T633 UBERON:0003081 28092 28105 lateral plate +T634 UBERON:0002067 28114 28120 dermis +T635 NCBITaxon:10088 28228 28232 mice +T636 CHEBI:81393 28246 28257 pheomelanin +T637 CHEBI:24009 28387 28396 eumelanin +T638 UBERON:0002329 28401 28407 somite +T639 UBERON:0002067 28416 28422 dermis +T640 UBERON:0003081 28588 28601 lateral plate +T641 UBERON:0002329 28607 28613 somite +T642 UBERON:0002067 28622 28628 dermis +T643 UBERON:0002101 28691 28695 limb +T644 UBERON:0000309 28696 28705 body wall +T645 UBERON:0002101 28784 28788 limb +T646 UBERON:0002101 28889 28893 limb +T647 PR:000007070 28895 28905 Engrailed1 +T648 PR:000007070 28907 28910 En1 +T649 PR:000017448 28913 28918 Wnt7a +T650 PR:000009871 28924 28929 Lmx1b +T651 GO:0010467 28980 28990 expression +T652 PR:000007070 29061 29064 En1 +T653 GO:0010467 29080 29089 expressed +T654 UBERON:0000180 29108 29113 flank +T655 UBERON:0000916 29136 29145 abdominal +T656 UBERON:0001049 29177 29188 neural tube +T657 UBERON:0003077 29190 29215 somite-derived mesenchyme +T658 UBERON:0017648 29225 29242 ventral body wall +T659 GO:0097617 29276 29286 hybridized +T660 PR:000016145 29292 29297 Tbx15 +T661 UBERON:0000180 29337 29342 flank +T662 UBERON:0003081 29463 29485 lateral plate mesoderm +T663 UBERON:0015178 29549 29565 somitic frontier +T664 SO:0000704 29602 29607 genes +T665 UBERON:0002073 29656 29669 hair follicle +T666 GO:0001942 29656 29681 hair follicle development +T667 PR:000016145 29687 29692 Tbx15 +T668 SO:0000704 29705 29709 gene +T669 UBERON:0001037 29760 29764 hair +T670 UBERON:0001037 29848 29852 hair +T671 UBERON:0002100 29860 29865 trunk +T672 UBERON:0002101 29867 29872 limbs +T673 NCBITaxon:10088 29923 29927 mice +T674 UBERON:0002100 29940 29945 trunk +T675 GO:0010467 29987 29997 expression +T676 PR:000004372 30004 30010 Agouti +T677 SO:0001060 30016 30023 isoform +T678 SO:0001023 30077 30083 allele +T679 PR:000016145 30139 30144 Tbx15 +T680 PR:000016145 30235 30240 Tbx15 +T681 SO:0000704 30287 30291 gene +T682 SO:0000704 30360 30365 genes +T683 SO:0001415 30393 30413 deletion breakpoints +T684 GO:0010467 30423 30433 expression +T685 PR:000016145 30445 30450 Tbx15 +T686 SO:0001023 30535 30541 allele +T687 SO:0001023 30554 30560 allele +T688 PR:000016145 30573 30578 Tbx15 +T689 SO:0001023 30588 30594 allele +T690 GO:0010467 30662 30672 expression +T691 PR:000016145 30676 30681 Tbx15 +T692 UBERON:0002067 30764 30770 dermis +T693 CHEBI:26130 30786 30796 pigmentary +T694 UBERON:0001037 30801 30805 hair +T695 PR:000016145 30845 30850 Tbx15 +T696 GO:0010467 30851 30861 expression +T697 UBERON:0000180 30880 30885 flank +T698 UBERON:0002101 30948 30952 limb +T699 GO:0060173 30948 30964 limb development +T700 UBERON:0003081 30980 31002 lateral plate mesoderm +T701 SO:0000704 31052 31056 gene +T702 UBERON:0000922 31067 31076 embryonic +T703 GO:0009790 31067 31088 embryonic development +T704 NCBITaxon:40674 31206 31215 mammalian +T705 GO:0007601 31298 31304 visual +T706 NCBITaxon:10088 31355 31359 mice +T707 SO:0000704 31479 31483 gene +T708 GO:0010467 31479 31494 gene expression +T709 PR:000004372 31557 31563 Agouti +T710 UBERON:0000922 31577 31586 embryonic +T711 GO:0007567 31595 31600 natal +T712 UBERON:0001037 31748 31752 hair +T713 CL:0000148 31791 31802 melanocytes +T714 NCBITaxon:10088 31967 31971 mice +T715 CHEBI:26130 32038 32045 pigment +T716 CHEBI:26130 32065 32072 pigment +T717 UBERON:0000180 32127 32132 flank +T718 PR:000004372 32171 32177 Agouti +T719 UBERON:0013235 32217 32224 ventrum +T720 UBERON:0001037 32230 32234 hair +T721 CHEBI:26130 32235 32242 pigment +T722 UBERON:0013235 32308 32315 ventrum +T723 SO:0000704 32343 32350 genetic +T724 UBERON:0000180 32413 32418 flank +T725 PR:000016145 32436 32441 Tbx15 +T726 UBERON:0001037 32478 32482 hair +T727 CHEBI:26130 32491 32498 pigment +T728 GO:0010467 32512 32522 expression +T729 PR:000004372 32547 32553 Agouti +T730 SO:0001060 32554 32561 isoform +T731 UBERON:0007023 32672 32677 adult +T732 NCBITaxon:10088 32686 32690 mice +T733 NCBITaxon:10088 32760 32764 mice +T734 UBERON:0007023 32812 32818 adults +T735 UBERON:0001037 32885 32889 hair +T736 PR:000016145 32916 32921 Tbx15 +T737 SO:0000417 32963 32969 domain +T738 NCBITaxon:10088 32994 32999 mouse +T739 PR:000016001 33000 33009 Brachyury +T740 SO:0000704 33010 33014 gene +T741 UBERON:0002415 33050 33054 tail +T742 SO:0000704 33074 33079 genes +T743 UBERON:0000479 33151 33158 tissues +T744 UBERON:0000468 33163 33186 multicellular organisms +T745 NCBITaxon:1 33177 33186 organisms +T746 PR:000016145 33223 33228 Tbx15 +T747 PR:000016146 33260 33265 Tbx18 +T748 PR:000016150 33270 33275 Tbx22 +T749 NCBITaxon:7711 33315 33323 chordate +T750 SO:0000704 33358 33362 gene +T751 NCBITaxon:7740 33366 33375 amphioxus +T752 SO:0000853 33391 33398 homolog +T753 NCBITaxon:7147 33406 33409 fly +T754 SO:0001026 33410 33416 genome +T755 SO:0000704 33486 33491 genes +T756 GO:0010467 33496 33505 expressed +T757 UBERON:0002329 33562 33569 somites +T758 PR:000016146 33571 33576 Tbx18 +T759 PR:000016150 33581 33586 Tbx22 +T760 UBERON:0009749 33589 33604 limb mesenchyme +T761 PR:000016145 33606 33611 Tbx15 +T762 PR:000016146 33616 33621 Tbx18 +T763 UBERON:0003104 33641 33651 mesenchyme +T764 SO:0000704 33663 33668 genes +T765 PR:000016145 33670 33675 Tbx15 +T766 PR:000016146 33694 33699 Tbx18 +T767 PR:000016150 33703 33708 Tbx22 +T768 SO:0000704 33856 33860 gene +T769 PR:000016145 33865 33870 Tbx15 +T770 PR:000016146 33872 33877 Tbx18 +T771 PR:000016150 33883 33888 Tbx22 +T772 NCBITaxon:7735 33945 33961 cephalochordates +T773 GO:0010467 33994 34004 expression +T774 UBERON:0002101 34049 34053 limb +T775 UBERON:0002100 34062 34067 trunk +T776 NCBITaxon:7742 34081 34091 vertebrate +T777 GO:0010467 34103 34113 Expression +T778 PR:000016146 34117 34122 Tbx18 +T779 PR:000016150 34127 34132 Tbx22 +T780 UBERON:0000922 34158 34167 embryonic +T781 UBERON:0000180 34168 34173 flank +T782 UBERON:0003104 34174 34184 mesenchyme +T783 PR:000016145 34206 34211 Tbx15 +T784 UBERON:0002100 34296 34301 trunk +T785 NCBITaxon:33208 34387 34394 animals +T786 UBERON:0002101 34485 34490 limbs +T787 UBERON:0000033 34499 34503 head +T788 PR:000016150 34531 34536 Tbx22 +T789 NCBITaxon:9606 34547 34552 human +T790 http://purl.obolibrary.org/obo/MONDO_0002254 34553 34561 syndrome +T791 GO:0000805 34562 34563 X +T792 http://purl.obolibrary.org/obo/MONDO_0017938 34562 34601 X-linked cleft palate and ankyloglossia +T793 PR:000016150 34650 34655 Tbx22 +T794 GO:0010467 34656 34666 expression +T795 UBERON:0004017 34670 34680;34691 34701 periocular ... mesenchyme +T796 UBERON:0000922 34681 34690 embryonic +T797 UBERON:0000970 34797 34800 eye +T798 PR:000016145 34851 34856 Tbx15 +T799 GO:0010467 34911 34921 expression +T800 NCBITaxon:10088 34942 34947 mouse +T801 PR:000016145 34948 34953 Tbx15 +T802 NCBITaxon:9606 34987 34992 human +T803 PR:000016145 34993 34998 Tbx15 +T804 http://purl.obolibrary.org/obo/MONDO_0007051 35049 35079;35086 35094 acromegaloid facial appearance syndrome +T805 UBERON:0001456 35062 35068 facial +T806 http://purl.obolibrary.org/obo/MONDO_0007051 35081 35084;35086 35094 AFA syndrome +T807 GO:0030849 35211 35220 autosomal +T808 http://purl.obolibrary.org/obo/MONDO_0002254 35230 35238 syndrome +T809 UBERON:0001456 35256 35262 facial +T810 UBERON:0000165 35298 35302 oral +T811 UBERON:0000344 35303 35309 mucosa +T812 UBERON:0002398 35329 35334 hands +T813 http://purl.obolibrary.org/obo/MONDO_0019280 35420 35434 hypertrichosis +T814 NCBITaxon:9606 35678 35683 human +T815 PR:000016145 35684 35689 TBX15 +T816 http://purl.obolibrary.org/obo/MONDO_0009247 35699 35724 frontofacionasal syndrome +T817 GO:0030849 35738 35747 autosomal +T818 http://purl.obolibrary.org/obo/MONDO_0006025 35738 35767 autosomal recessive condition +T819 http://purl.obolibrary.org/obo/MONDO_0001008 35800 35816 blepharophimosis +T820 UBERON:0004089 35822 35829 midface +T821 UBERON:0004288 35968 35976 skeletal +T822 GO:0001501 35968 35988 skeletal development +T823 SO:0000704 36050 36054 gene +T824 PR:000003979 36055 36059 Alx3 +T825 SO:0001023 36069 36076 allelic +T826 UBERON:0001690 36089 36092 ear +T827 UBERON:0004288 36151 36159 skeletal +T828 PR:000003979 36182 36186 Alx3 +T829 PR:000003980 36190 36194 Alx4 +T830 PR:000003979 36267 36271 Alx3 +T831 PR:000016145 36311 36316 Tbx15 +T832 UBERON:0004288 36317 36325 skeletal +T833 PR:000016145 36393 36398 Tbx15 +T834 GO:0010467 36399 36409 Expression +T835 PR:000016145 36463 36468 Tbx15 +T836 CHEBI:26130 36472 36479 pigment +T837 PR:000004372 36523 36529 Agouti +T838 GO:0007567 36537 36542 natal +T839 NCBITaxon:33208 36543 36550 animals +T840 PR:000004372 36561 36567 Agouti +T841 GO:0010467 36576 36585 expressed +T842 UBERON:0000922 36593 36599 embryo +T843 UBERON:0002067 36650 36656 dermis +T844 UBERON:0000922 36697 36706 embryonic +T845 PR:000004372 36707 36713 Agouti +T846 GO:0010467 36714 36724 expression +T847 NCBITaxon:33208 36736 36743 animals +T848 PR:000016145 36783 36788 Tbx15 +T849 SO:0000704 36865 36870 genes +T850 GO:0008283 37118 37144;37147 37162 proliferative expansion of ... cell population +T851 NCBITaxon:10088 37318 37322 mice +T852 PR:000004372 37366 37372 Agouti +T853 GO:0010467 37373 37383 expression +T854 GO:0007567 37391 37396 natal +T855 NCBITaxon:33208 37405 37412 animals +T856 UBERON:0000922 37506 37515 embryonic +T857 CL:0002321 37506 37521 embryonic cells +T858 UBERON:0003104 37538 37548 mesenchyme +T859 GO:0008283 37623 37634 proliferate +T860 UBERON:0001037 37849 37853 hair +T861 NCBITaxon:10088 37871 37875 mice +T862 UBERON:0000180 37992 37997 flank +T863 UBERON:0000922 38025 38034 embryonic +T864 UBERON:0003104 38035 38045 mesenchyme +T865 GO:0010467 38047 38057 expression +T866 PR:000016145 38061 38066 Tbx15 +T867 PR:000004372 38071 38077 Agouti +T868 PR:000016145 38121 38126 Tbx15 +T869 PR:000004372 38152 38158 Agouti +T870 UBERON:0003104 38189 38199 mesenchyme +T871 PR:000016145 38225 38230 Tbx15 +T872 GO:0010467 38243 38253 expression +T873 PR:000004372 38278 38284 Agouti +T874 SO:0001060 38285 38292 isoform +T875 GO:0007567 38300 38305 natal +T876 NCBITaxon:10088 38306 38310 mice +T877 GO:0007567 38347 38352 natal +T878 GO:0010467 38353 38363 expression +T879 PR:000016145 38367 38372 Tbx15 +T880 PR:000004372 38454 38460 Agouti +T881 PR:000016145 38493 38498 Tbx15 +T882 SO:0000704 38526 38531 genes +T883 PR:000004372 38547 38553 Agouti +T884 UBERON:0001037 38561 38565 hair +T885 CL:0002483 38561 38565;38577 38587 hair ... melanocyte +T886 NCBITaxon:33208 38654 38661 animals +T887 PR:000004372 38680 38686 Agouti +T888 SO:0001023 38687 38693 allele +T889 PR:000003980 38719 38723 Alx4 +T890 PR:000004372 38737 38743 Agouti +T891 GO:0010467 38748 38757 expressed +T892 UBERON:0000922 38769 38778 embryonic +T893 UBERON:0003104 38779 38789 mesenchyme +T894 UBERON:0002073 38818 38831 hair-follicle +T895 UBERON:0002101 38843 38847 limb +T896 GO:0060173 38843 38847;38865 38876 limb ... development +T897 GO:0010467 38971 38981 expression +T898 PR:000003980 39009 39013 Alx4 +T899 PR:000003979 39026 39030 Alx3 +T900 PR:000010687 39035 39039 Msx2 +T901 UBERON:0000922 39086 39093 embryos +T902 UBERON:0002101 39158 39162 Limb +T903 PR:000016145 39183 39188 Tbx15 +T904 UBERON:0001037 39227 39231 hair +T905 UBERON:0002101 39245 39250 limbs +T906 UBERON:0001037 39297 39301 hair +T907 UBERON:0001037 39324 39328 hair +T908 CHEBI:26130 39374 39381 pigment +T909 UBERON:0002101 39404 39408 limb +T910 NCBITaxon:10088 39441 39445 mice +T911 PR:000007070 39532 39535 En1 +T912 PR:000017448 39539 39544 Wnt7a +T913 UBERON:0002101 39597 39601 limb +T914 UBERON:0001137 39607 39613 dorsum +T915 UBERON:0013235 39617 39624 ventrum +T916 UBERON:0013235 39649 39656 ventrum +T917 UBERON:0001137 39660 39666 dorsum +T918 PR:000004372 39745 39751 Agouti +T919 GO:0010467 39752 39762 expression +T920 UBERON:0001037 39782 39786 hair +T921 UBERON:0013623 39867 39875 footpads +T922 PR:000007070 39893 39896 En1 +T923 NCBITaxon:10088 39904 39908 mice +T924 CHEBI:26130 39925 39932 pigment +T925 PR:000007070 39994 39997 En1 +T926 PR:000017448 40014 40019 Wnt7a +T927 GO:0016477 40032 40041;40069 40071;40080 40085 migration ... of ... cells +T928 GO:0008283 40045 40058;40069 40071;40080 40085 proliferation ... of ... cells +T929 CHEBI:26130 40072 40079 pigment +T930 CL:0000147 40072 40085 pigment cells +T931 UBERON:0007376 40097 40106 epidermis +T932 GO:0065007 40188 40195 control +T933 UBERON:0002100 40212 40217 trunk +T934 PR:000016145 40229 40234 Tbx15 +T935 GO:0065007 40264 40271 control +T936 UBERON:0002101 40288 40292 limb +T937 PR:000009871 40307 40312 Lmx1b +T938 SO:0000417 40320 40326 domain +T939 PR:000017448 40372 40377 Wnt7a +T940 PR:000007070 40382 40385 En1 +T941 PR:000016145 40513 40518 Tbx15 +T942 PR:000009871 40523 40528 Lmx1b +T943 UBERON:0003104 40549 40560 mesenchymal +T944 CL:0000134 40549 40566 mesenchymal cells +T945 GO:0010467 40606 40616 expression +T946 UBERON:0000180 40684 40689 flank +T947 PR:000016145 40691 40696 Tbx15 +T948 UBERON:0002101 40705 40709 limb +T949 PR:000009871 40711 40716 Lmx1b +T950 PR:000009871 40777 40782 Lmx1b +T951 GO:0010467 40788 40798 expression +T952 UBERON:0002101 40813 40817 limb +T953 PR:000017448 40829 40834 Wnt7a +T954 UBERON:0000924 40868 40876 ectoderm +T955 PR:000017448 40938 40943 Wnt7a +T956 UBERON:0000924 40978 40986 ectoderm +T957 PR:000007070 40990 40993 En1 +T958 UBERON:0000924 41009 41017 ectoderm +T959 GO:0010467 41084 41094 expression +T960 UBERON:0004356 41168 41191 apical ectodermal ridge +T961 PR:000007070 41276 41279 En1 +T962 PR:000017448 41283 41288 Wnt7a +T963 UBERON:0001037 41344 41348 hair +T964 UBERON:0000924 41482 41492 ectodermal +T965 GO:0065007 41514 41521 control +T966 UBERON:0003104 41545 41555 mesenchyme +T967 UBERON:0002101 41570 41575 limbs +T968 UBERON:0002100 41593 41598 trunk +T969 UBERON:0002101 41614 41618 limb +T970 UBERON:0001911 41624 41638 mammary glands +T971 UBERON:0000483 41705 41715 epithelial +T972 UBERON:0003104 41716 41727 mesenchymal +T973 UBERON:0001911 41791 41805 mammary glands +T974 NCBITaxon:33208 41828 41835 animals +T975 GO:0065007 41892 41899 control +T976 UBERON:0002100 41931 41936 trunk +T977 UBERON:0002101 41955 41960 limbs +T978 UBERON:0011270 42066 42078 dorsal trunk +T979 UBERON:0000924 42079 42087 ectoderm +T980 GO:0010467 42120 42130 expression +T981 PR:000016145 42134 42139 Tbx15 +T982 UBERON:0011270 42143 42155 dorsal trunk +T983 UBERON:0003104 42156 42166 mesenchyme +T984 PR:000004372 42265 42271 Agouti +T985 GO:0010467 42272 42282 expression +T986 CHEBI:26130 42284 42291 pigment +T987 CL:0000147 42284 42296 pigment-cell +T988 GO:0070285 42284 42308 pigment-cell development +T989 UBERON:0001037 42314 42318 hair +T990 PR:000016145 42356 42361 Tbx15 +T991 GO:0010467 42362 42372 expression +T992 PR:000007070 42408 42411 En1 +T993 GO:0010467 42412 42422 expression +T994 UBERON:0004347 42524 42532 limb-bud +T995 UBERON:0002100 42607 42612 trunk +T996 UBERON:0000924 42613 42621 ectoderm +T997 UBERON:0002101 42645 42649 limb +T998 UBERON:0000924 42790 42798 ectoderm +T999 UBERON:0002101 42811 42815 limb +T1000 UBERON:0002101 42894 42898 limb +T1001 UBERON:0002101 42948 42952 limb +T1002 UBERON:0000926 42953 42961 mesoderm +T1003 UBERON:0002101 43079 43084 limbs +T1004 UBERON:0004356 43093 43116 apical ectodermal ridge +T1005 GO:0010467 43133 43143 expression +T1006 PR:000016145 43147 43152 Tbx15 +T1007 UBERON:0002100 43160 43165 trunk +T1008 UBERON:0002101 43181 43186 limbs +T1009 UBERON:0002101 43207 43211 limb +T1010 UBERON:0004347 43306 43315 limb buds +T1011 GO:0010467 43476 43486 expression +T1012 PR:000016145 43490 43495 Tbx15 +T1013 UBERON:2000164 43499 43517 ventral mesenchyme +T1014 PR:000016145 43637 43642 Tbx15 +T1015 GO:0010467 43643 43653 expression +T1016 UBERON:0000926 43698 43706 mesoderm +T1017 PR:000016145 43753 43758 Tbx15 +T1018 GO:0048263 43841 43873 establishment of dorsal identity +T1019 PR:000016145 43877 43882 Tbx15 +T1020 UBERON:0003104 43958 43968 mesenchyme +T1021 UBERON:0000924 43987 43995 ectoderm +T1022 PR:000016145 44024 44029 Tbx15 +T1023 NCBITaxon:40674 44039 44046 Mammals +T1024 UBERON:0015178 44060 44076 somitic frontier +T1025 UBERON:0002329 44118 44124 somite +T1026 UBERON:0003081 44140 44153 lateral plate +T1027 UBERON:0000926 44162 44170 mesoderm +T1028 GO:0007567 44343 44348 natal +T1029 NCBITaxon:33208 44349 44356 animals +T1030 UBERON:0015178 44504 44520 somitic frontier +T1031 PR:000010686 44727 44731 Msx1 +T1032 GO:0010467 44739 44749 expression +T1033 UBERON:0002329 44770 44776 somite +T1034 UBERON:0003104 44785 44796 mesenchymal +T1035 CL:0000134 44785 44802 mesenchymal cells +T1036 UBERON:0002067 44822 44828 dermis +T1037 UBERON:2001463 44841 44847 stripe +T1038 GO:0007567 44963 44968 natal +T1039 NCBITaxon:40674 44969 44978 mammalian +T1040 CHEBI:26130 45050 45057 pigment +T1041 GO:0010467 45122 45132 expression +T1042 PR:000010686 45136 45140 Msx1 +T1043 NCBITaxon:9989 45146 45153 rodents +T1044 NCBITaxon:40674 45218 45225 mammals +T1045 UBERON:0001037 45260 45264 hair +T1046 NCBITaxon:9654 45331 45339 Raccoons +T1047 NCBITaxon:55153 45341 45350 squirrels +T1048 NCBITaxon:119825 45352 45358 skunks +T1049 UBERON:2001463 45405 45412 stripes +T1050 UBERON:0015178 45503 45519 somitic frontier +T1051 PR:000010686 45536 45540 Msx1 +T1052 PR:000016145 45610 45615 Tbx15 +T1053 NCBITaxon:10088 45646 45650 mice +T1054 UBERON:0010166 45669 45673 coat +T1055 NCBITaxon:40674 45739 45746 mammals +T1056 UBERON:2002284 45755 45763 markings +T1057 NCBITaxon:9615 45783 45786 dog +T1058 NCBITaxon:42413 45851 45872 Peromyscus polionotus +T1059 NCBITaxon:subspecies 45962 45972 subspecies +T1060 NCBITaxon:10040 46081 46090 deer mice +T1061 NCBITaxon:33208 46193 46200 animals +T1062 GO:0065007 46236 46246 regulation +T1063 PR:000016145 46260 46265 Tbx15 +T1064 PR:000016145 46322 46327 Tbx15 +T1065 UBERON:0010166 46331 46335 coat +T1066 SO:0000704 46371 46378 genetic +T1067 NCBITaxon:40674 46424 46431 mammals +T1068 GO:0010467 46504 46514 expression +T1069 PR:000016145 46518 46523 Tbx15 +T1070 NCBITaxon:7742 46533 46544 vertebrates +T1071 UBERON:0004288 46606 46614 skeleton +T1072 CHEBI:26130 46630 46640 pigmentary +T1073 NCBITaxon:10088 46675 46679 Mice +T1074 NCBITaxon:10088 46685 46689 mice +T1075 PR:000008703 46876 46881 Hoxb6 +T1076 NCBITaxon:10088 46897 46901 mice +T1077 NCBITaxon:10088 47005 47009 mice +T1078 PR:000033987 47028 47032 lacZ +T1079 SO:0001023 47042 47048 allele +T1080 http://purl.obolibrary.org/obo/MONDO_0004992 47097 47103 Cancer +T1081 NCBITaxon:10088 47182 47186 mice +T1082 SO:0001023 47474 47480 allele +T1083 GO:0007618 47625 47632 matings +T1084 UBERON:0010148 47653 47657 plug +T1085 GO:0007567 47683 47690 natally +T1086 GO:0007567 47703 47708 birth +T1087 UBERON:0001037 47778 47782 hair +T1088 UBERON:0002101 47817 47821 limb +T1089 UBERON:0000014 47822 47836 region of skin +T1090 CHEBI:32139 47930 47948 sodium bicarbonate +T1091 UBERON:0001037 48029 48033 hair +T1092 UBERON:0010511 48240 48244 awls +T1093 UBERON:0010510 48249 48257 auchenes +T1094 GO:0007601 48303 48311 visually +T1095 UBERON:0010513 48334 48340;48346 48351 zigzag ... hairs +T1096 UBERON:0001037 48389 48393 hair +T1097 UBERON:0001037 48429 48434 hairs +T1098 UBERON:0001137 48460 48466 dorsum +T1099 UBERON:0013235 48473 48480 ventrum +T1100 NCBITaxon:33208 48508 48515 animals +T1101 UBERON:0010511 48619 48623 awls +T1102 UBERON:0010510 48628 48636 auchenes +T1103 CHEBI:51686 48722 48733 hematoxylin +T1104 CHEBI:49168 48749 48753 DOPA +T1105 UBERON:0002067 48768 48774 dermis +T1106 UBERON:0007376 48779 48788 epidermis +T1107 CHEBI:63004 48831 48845 sodium bromide +T1108 UBERON:0002073 48884 48898 hair follicles +T1109 UBERON:0002067 48918 48924 dermis +T1110 CHEBI:15765 48989 48995 L-DOPA +T1111 CHEBI:37586 49047 49063 sodium phosphate +T1112 CHEBI:75958 49131 49139 solution +T1113 CL:0000148 49266 49277 melanocytes +T1114 CHEBI:50913 49318 49326 fixative +T1115 NCBITaxon:10088 49493 49497 mice +T1116 NCBITaxon:33208 49578 49585 animals +T1117 NCBITaxon:33208 49738 49745 animals +T1118 SO:0000704 49755 49762 genetic +T1119 SO:0001249 49901 49913 physical map +T1120 SO:0000153 49919 49922 BAC +T1121 SO:0000040 49923 49937 genomic clones +T1122 SO:0001026 49998 50004 Genome +T1123 SO:0000153 50075 50079 BACs +T1124 SO:0000112 50204 50210 Primer +T1125 SO:0000331 50512 50515 STS +T1126 SO:0001026 50533 50540 Genomic +T1127 SO:0001026 50594 50600 Genome +T1128 SO:0001026 50652 50658 genome +T1129 SO:0000704 50723 50728 genes +T1130 CHEBI:35350 50735 50749 hydroxysteroid +T1131 PR:000008867 50776 50782 Hsd3b3 +T1132 PR:000008786 50784 50790 Hsd3b2 +T1133 PR:000008870 50792 50798 Hsd3b6 +T1134 PR:000008785 50804 50810 Hsd3b1 +T1135 CHEBI:24669 50815 50824 hydroacid +T1136 PR:000008442 50834 50838 Hao3 +T1137 PR:000017370 50872 50877 Wars2 +T1138 SO:0000704 50887 50891 gene +T1139 PR:000016145 50893 50898 Tbx15 +T1140 SO:0000704 50912 50916 gene +T1141 PR:000015456 50918 50931 4931427F14Rik +T1142 SO:0001026 50940 50946 genome +T1143 SO:0000112 50960 50967 primers +T1144 SO:0000132 51049 51063 reverse primer +T1145 SO:0000112 51117 51124 primers +T1146 SO:0000112 51245 51252 primers +T1147 SO:0000999 51274 51282 BAC ends +T1148 SO:0001026 51298 51304 genome +T1149 SO:0000153 51375 51378 BAC +T1150 SO:0000112 51467 51474 primers +T1151 SO:0000028 51575 51577 bp +T1152 SO:0000028 51620 51622 bp +T1153 SO:0000704 51663 51667 Gene +T1154 SO:0001023 51690 51696 allele +T1155 PR:000016145 51700 51705 Tbx15 +T1156 SO:0000243 51792 51796 IRES +T1157 PR:000033987 51797 51801 LacZ +T1158 SO:0005853 51806 51814 cassette +T1159 PR:P23940 51892 51897 BamHI +T1160 SO:0000315 51947 51978 transcriptional initiation site +T1161 SO:0000147 52014 52018 exon +T1162 CL:0002322 52031 52033 ES +T1163 UBERON:0000358 52063 52074 blastocysts +T1164 NCBITaxon:10088 52119 52123 mice +T1165 NCBITaxon:33208 52136 52143 animals +T1166 GO:0097617 52154 52167 hybridization +T1167 GO:0097617 52177 52190 hybridization +T1168 CHEBI:42098 52240 52251 digoxigenin +T1169 UBERON:0000922 52389 52396 Embryos +T1170 GO:0007567 52405 52410 natal +T1171 NCBITaxon:10088 52465 52469 mice +T1172 UBERON:0000922 52471 52478 Embryos +T1173 GO:0007567 52548 52553 natal +T1174 PR:000016145 52606 52611 Tbx15 +T1175 SO:0000112 52648 52655 primers +T1176 SO:0000147 52717 52722 exons +T1177 PR:000007070 52741 52744 En1 +T1178 SO:0001026 52777 52784 genomic +T1179 SO:0000112 52795 52802 primers +T1180 SO:0000147 52870 52874 exon +T1181 PR:000004372 52881 52887 Agouti +T1182 SO:0000010 52913 52927 protein-coding +T1183 UBERON:0000922 52939 52948 Embryonic +T1184 UBERON:0000922 52995 53002 embryos +T1185 CHEBI:75958 53047 53055 solution +T1186 UBERON:0000922 53061 53070 embryonic +T1187 UBERON:0000180 53101 53106 flank +T1188 UBERON:0000473 53207 53213 testes +T1189 NCBITaxon:33208 53226 53233 animals +T1190 UBERON:0000309 53321 53330 body wall +T1191 UBERON:0006983 53345 53350 point +T1192 UBERON:0002101 53377 53382 limbs +T1193 UBERON:0003916 53388 53396 fat pads +T1194 UBERON:0000473 53463 53469 testes +T1195 UBERON:0006643 53522 53536 testis capsule +T1196 UBERON:0000922 53572 53581 embryonic +T1197 UBERON:0000473 53605 53611 testes +T1198 UBERON:0003684 53640 53656 abdominal cavity +T1199 UBERON:0000309 53695 53704 body wall +T1200 NCBITaxon:10088 53734 53738 mice +T1201 UBERON:0001037 53794 53798 hair +T1202 UBERON:0000473 53822 53828 testes +T1203 UBERON:0015178 53869 53885 somitic frontier +T1204 PR:000008703 53891 53896 Hoxb6 +T1205 SO:0000902 53901 53910 transgene +T1206 GO:0010467 53967 53976 expressed +T1207 UBERON:0003081 53984 53997;54018 54026 lateral plate ... mesoderm +T1208 UBERON:0003077 54010 54026 somitic mesoderm +T1209 UBERON:0002100 54034 54039 trunk +T1210 NCBITaxon:33208 54060 54067 Animals +T1211 SO:0000902 54097 54106 transgene +T1212 SO:0000704 54129 54133 gene +T1213 UBERON:0009571 54282 54297 ventral midline +T1214 NCBITaxon:33208 54520 54527 animals +T1215 SO:0001023 54544 54550 allele +T1216 UBERON:0002329 54649 54655 somite +T1217 UBERON:0003081 54656 54669 lateral plate +T1218 PR:000004372 54892 54898 Agouti +T1219 SO:0000704 54899 54903 gene +T1220 PR:000003979 54914 54918 Alx3 +T1221 PR:000003980 54929 54933 Alx4 +T1222 PR:000007070 54946 54949 En1 +T1223 PR:000016145 54977 54982 Tbx14 +T1224 PR:000016145 54995 55000 Tbx15 +T1225 PR:000016146 55013 55018 Tbx18 +T1226 PR:000016150 55035 55040 Tbx22 +T1227 http://purl.obolibrary.org/obo/MONDO_0007051 55150 55180 acromegaloid facial appearance +T1228 UBERON:0001456 55163 55169 facial +T1229 http://purl.obolibrary.org/obo/MONDO_0009247 55195 55220 frontofacionasal syndrome +T1230 NCBITaxon:9606 55238 55243 human +T1231 http://purl.obolibrary.org/obo/MONDO_0002254 55244 55252 syndrome +T1232 GO:0000805 55253 55254 X +T1233 http://purl.obolibrary.org/obo/MONDO_0017938 55253 55292 X-linked cleft palate and ankyloglossia +T1234 PR:000008703 55504 55509 Hoxb6 +T1235 NCBITaxon:10088 55525 55529 mice +T1236 UBERON:0001690 55734 55737 ear +T1237 SO:0001023 55946 55952 allele +T1238 http://purl.obolibrary.org/obo/MONDO_0007051 55954 55957 AFA +T1239 http://purl.obolibrary.org/obo/MONDO_0007051 55960 55992 acromegaloid facial appearance +T1240 UBERON:0001456 55973 55979 facial +T1241 SO:0001023 56011 56017 allele +T1242 PR:000004372 56019 56020 A +T1243 UBERON:0000916 56030 56037 bellied +T1244 PR:000004372 56038 56044 Agouti +T1245 SO:0001023 56045 56051 allele +T1246 NCBITaxon:10088 56067 56071 mice +T1247 SO:0000153 56073 56076 BAC +T1248 NCBITaxon:2 56079 56088 bacterial +T1249 SO:0000153 56079 56110 bacterial artificial chromosome +T1250 UBERON:0001690 56125 56128 ear +T1251 SO:0001023 56129 56135 allele +T1252 UBERON:0000922 56167 56176 embryonic +T1253 PR:000007070 56187 56190 En1 +T1254 PR:000007070 56193 56203 Engrailed1 +T1255 SO:0000336 56210 56212 ps +T1256 CHEBI:17369 56215 56234 mannose-6-phosphate +T1257 SO:0000336 56244 56254 pseudogene +T1258 GO:0007567 56267 56272 natal +T1259 PR:000016145 56282 56287 Tbx15 +T1260 PR:000016145 56290 56303 T-box gene 15 +T1261 SO:0000704 56296 56300 gene +T1262 NCBITaxon:33208 56391 56398 animals +T1263 UBERON:0001037 56461 56465 hair +T1264 UBERON:0001037 56592 56596 hair +T1265 NCBITaxon:10088 56623 56627 mice +T1266 UBERON:0001037 56670 56674 hair +T1267 UBERON:0001037 56767 56771 Hair +T1268 NCBITaxon:10088 56873 56877 mice +T1269 UBERON:0010513 56898 56910 zigzag hairs +T1270 UBERON:0001137 56944 56950 dorsum +T1271 UBERON:0013235 56955 56962 ventrum +T1272 NCBITaxon:10088 56973 56977 mice +T1273 GO:0043588 57072 57088 skin development +T1274 UBERON:0001037 57159 57163 hair +T1275 CHEBI:25179 57164 57171 melanin +T1276 CHEBI:49168 57184 57188 DOPA +T1277 UBERON:0001137 57202 57208 dorsum +T1278 UBERON:0001137 57210 57211 d +T1279 UBERON:0000180 57214 57219 flank +T1280 UBERON:0000180 57221 57222 f +T1281 UBERON:0013235 57229 57236 ventrum +T1282 UBERON:0013235 57238 57239 v +T1283 NCBITaxon:10088 57260 57264 mice +T1284 UBERON:0013235 57340 57347 ventrum +T1285 UBERON:0010511 57387 57391 awls +T1286 CHEBI:49168 57436 57440 DOPA +T1287 UBERON:0002067 57449 57455 dermis +T1288 NCBITaxon:33208 57557 57564 animals +T1289 UBERON:0001037 57609 57613 hair +T1290 UBERON:0001037 57650 57655 hairs +T1291 UBERON:0001037 57679 57684 hairs +T1292 UBERON:2001463 57705 57711 stripe +T1293 UBERON:0001037 57784 57789 hairs +T1294 UBERON:0010166 57964 57969 pelts +T1295 UBERON:0002101 57990 57994 limb +T1296 UBERON:0001037 58503 58507 Hair +T1297 UBERON:0001137 58714 58720 dorsum +T1298 PR:000016145 58803 58808 Tbx15 +T1299 SO:0000704 58814 58821 Genetic +T1300 SO:0001249 58826 58838 physical map +T1301 SO:0000153 58917 58920 BAC +T1302 SO:0000149 58921 58927 contig +T1303 SO:0000331 58956 58959 STS +T1304 SO:0000357 59029 59034 flank +T1305 PR:000016145 59039 59044 Tbx15 +T1306 SO:0000336 59054 59056 ps +T1307 SO:0001026 59069 59075 genome +T1308 GO:0007126 59147 59154 meioses +T1309 PR:000016145 59207 59212 Tbx15 +T1310 SO:0000188 59213 59219 intron +T1311 SO:0000336 59243 59245 ps +T1312 SO:0001415 59264 59284 deletion breakpoints +T1313 PR:000016145 59302 59307 Tbx15 +T1314 PR:000033987 59307 59311 LacZ +T1315 SO:0001023 59312 59318 allele +T1316 SO:0000704 59334 59338 gene +T1317 SO:0001023 59381 59387 allele +T1318 SO:0000360 59461 59467 codons +T1319 SO:0001023 59546 59552 allele +T1320 UBERON:0001037 59590 59594 hair +T1321 PR:000016145 59631 59636 Tbx15 +T1322 PR:000033987 59636 59640 LacZ +T1323 GO:0010467 59727 59737 Expression +T1324 PR:000016145 59741 59746 Tbx15 +T1325 GO:0010467 59807 59817 expression +T1326 UBERON:0005253 59821 59836 head mesenchyme +T1327 UBERON:0003082 59848 59855 myotome +T1328 UBERON:0005902 59857 59866 occipital +T1329 UBERON:0004017 59872 59893 periocular mesenchyme +T1330 UBERON:0005619 59899 59912 palatal shelf +T1331 UBERON:0005434 59914 59922 cervical +T1332 UBERON:0003089 59923 59933 sclerotome +T1333 UBERON:0001823 59939 59954 nasal cartilage +T1334 UBERON:0005868 59960 59969;59985 59994 maxillary ... processes +T1335 UBERON:0005867 59974 59994 mandibular processes +T1336 UBERON:0002101 60000 60005 limbs +T1337 UBERON:0003082 60015 60022 myotome +T1338 UBERON:0003104 60035 60045 mesenchyme +T1339 UBERON:0000180 60116 60121 flank +T1340 GO:0010467 60146 60156 expression +T1341 UBERON:0003104 60168 60178 mesenchyme +T1342 UBERON:0007529 60273 60289 loose mesenchyme +T1343 UBERON:0002067 60305 60311 dermis +T1344 UBERON:0002378 60320 60329;60347 60354 abdominal ... muscles +T1345 PR:000016145 60386 60391 Tbx15 +T1346 GO:0010467 60395 60404 expressed +T1347 UBERON:0002067 60419 60425 dermis +T1348 GO:0010467 60447 60456 expressed +T1349 UBERON:0005933 60467 60474 sheaths +T1350 UBERON:0000922 60508 60517 Embryonic +T1351 GO:0010467 60518 60528 Expression +T1352 PR:000016145 60532 60537 Tbx15 +T1353 PR:000004372 60550 60556 Agouti +T1354 NCBITaxon:10088 60566 60570 Mice +T1355 PR:000016145 60582 60587 Tbx15 +T1356 PR:000004372 60599 60605 Agouti +T1357 GO:0010467 60617 60627 expression +T1358 PR:000016145 60631 60636 Tbx15 +T1359 PR:000004372 60694 60700 Agouti +T1360 GO:0010467 60742 60752 expression +T1361 SO:0000704 60762 60767 genes +T1362 PR:000016145 60783 60788 Tbx15 +T1363 GO:0010467 60789 60799 expression +T1364 PR:000004372 60861 60867 Agouti +T1365 PR:000016145 60934 60939 Tbx15 +T1366 PR:000004372 60976 60982 Agouti +T1367 PR:000004372 61034 61040 Agouti +T1368 GO:0010467 61041 61051 Expression +T1369 UBERON:0000922 61144 61151 embryos +T1370 UBERON:0002323 61167 61178 body cavity +T1371 PR:000004372 61207 61213 Agouti +T1372 GO:0010467 61214 61224 expression +T1373 PR:000004372 61314 61320 Agouti +T1374 GO:0010467 61321 61331 expression +T1375 UBERON:0000180 61423 61428 flank +T1376 PR:000004372 61456 61462 Agouti +T1377 GO:0010467 61463 61473 expression +T1378 UBERON:0002067 61510 61516 dermis +T1379 UBERON:0000922 61648 61657 Embryonic +T1380 GO:0009953 61658 61687;61693 61703 Establishment of Dorsoventral ... Patterning +T1381 UBERON:0000180 61733 61738 flank +T1382 UBERON:0000922 61774 61781 embryos +T1383 UBERON:0000473 61809 61815 testes +T1384 NCBITaxon:33208 61828 61835 animals +T1385 UBERON:0001037 61862 61866 Hair +T1386 UBERON:0000922 61930 61939 embryonic +T1387 UBERON:0001037 61969 61974 hairs +T1388 UBERON:0000922 61983 61992 embryonic +T1389 UBERON:0001037 62021 62026 hairs +T1390 UBERON:0000180 62032 62037 flank +T1391 UBERON:0000922 62038 62047 embryonic +T1392 UBERON:0001037 62101 62106 hairs +T1393 GO:0097617 62158 62171 hybridization +T1394 UBERON:0000922 62198 62207 embryonic +T1395 UBERON:0000180 62208 62213 flank +T1396 GO:0010467 62239 62249 expression +T1397 PR:000004372 62258 62264 Agouti +T1398 PR:000016145 62269 62274 Tbx15 +T1399 UBERON:0001037 62298 62303 hairs +T1400 GO:0097617 62327 62340 hybridization +T1401 UBERON:0015178 62436 62452 Somitic Frontier +T1402 UBERON:0002100 62501 62506 trunk +T1403 PR:000008703 62656 62661 Hoxb6 +T1404 NCBITaxon:10088 62668 62672 mice +T1405 CHEBI:75055 62703 62708 X-Gal +T1406 CHEBI:75055 62829 62834 X-Gal +T1407 UBERON:0002067 62850 62856 dermis +T1408 UBERON:0003081 62870 62883 lateral plate +T1409 UBERON:0002067 62891 62897 dermis +T1410 UBERON:0000926 62911 62919 mesoderm +T1411 UBERON:0015178 62933 62949 somitic frontier +T1412 UBERON:0002329 63081 63087 somite +T1413 UBERON:0002067 63096 63102 dermis +T1414 GO:0097617 63185 63198 hybridization +T1415 PR:000016145 63212 63217 Tbx15 +T1416 GO:0010467 63218 63228 expression +T1417 PR:000007070 63258 63261 En1 +T1418 GO:0010467 63262 63272 expression +T1419 UBERON:0000180 63280 63285 flank +T1420 GO:0010467 63354 63364 expression +T1421 SO:0000704 63384 63389 genes +T1422 GO:0009953 63412 63450 Acquisition of Dorsoventral Patterning +T1423 UBERON:0002100 63458 63463 Trunk +T1424 PR:000016145 63480 63485 Tbx15 +T1425 PR:000004372 63606 63612 Agouti +T1426 CL:0000148 63649 63660 melanocytes +T1427 NCBITaxon:10088 63725 63729 mice +T1428 NCBITaxon:10088 63755 63759 mice +T1429 UBERON:0001037 63769 63773 hair +T1430 CL:0002483 63769 63784 hair melanocyte +T1431 PR:000004372 63813 63819 Agouti +T1432 UBERON:0013235 63832 63839 ventrum +T1433 CL:0000148 63866 63876 melanocyte +T1434 UBERON:0001137 63918 63924 dorsum +T1435 UBERON:2001463 63936 63942 stripe +T1436 UBERON:0000180 63962 63967 flank +T1437 PR:000004372 63990 63996 Agouti +T1438 CL:0000148 64033 64044 melanocytes +T1439 PR:000016145 64066 64071 Tbx15 +T1440 UBERON:2001463 64128 64134 stripe +T1441 UBERON:2001463 64237 64243 stripe +T1442 NCBITaxon:10088 64253 64257 mice +T1443 UBERON:0002101 64288 64292 limb +T1444 UBERON:0000924 64396 64404 ectoderm +T1445 UBERON:0002100 64412 64417 trunk +T1446 UBERON:0000926 64454 64462 mesoderm +T1447 GO:0010467 64475 64485 expression +T1448 PR:000016145 64489 64494 Tbx15 +T1449 UBERON:0011270 64498 64510 dorsal trunk +T1450 UBERON:0005256 64505 64521 trunk mesenchyme +T1451 UBERON:0002067 64548 64554 dermis +T1452 PR:000016145 64584 64589 Tbx15 +T1453 UBERON:0003104 64598 64608 mesenchyme +T1454 CHEBI:33893 64943 64951 reagents diff --git a/src/ontogpt/evaluation/craft/database/all/14737183.txt b/src/ontogpt/evaluation/craft/database/all/14737183.txt new file mode 100644 index 000000000..b8cbfce45 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/14737183.txt @@ -0,0 +1,295 @@ +Dorsoventral Patterning of the Mouse Coat by Tbx15 + +Abstract + +Many members of the animal kingdom display coat or skin color differences along their dorsoventral axis. To determine the mechanisms that control regional differences in pigmentation, we have studied how a classical mouse mutation, droopy ear (deH), affects dorsoventral skin characteristics, especially those under control of the Agouti gene. Mice carrying the Agouti allele black-and-tan (at) normally have a sharp boundary between dorsal black hair and yellow ventral hair; the deH mutation raises the pigmentation boundary, producing an apparent dorsal-to-ventral transformation. We identify a 216 kb deletion in deH that removes all but the first exon of the Tbx15 gene, whose embryonic expression in developing mesenchyme correlates with pigmentary and skeletal malformations observed in deH/deH animals. Construction of a targeted allele of Tbx15 confirmed that the deH phenotype was caused by Tbx15 loss of function. Early embryonic expression of Tbx15 in dorsal mesenchyme is complementary to Agouti expression in ventral mesenchyme; in the absence of Tbx15, expression of Agouti in both embryos and postnatal animals is displaced dorsally. Transplantation experiments demonstrate that positional identity of the skin with regard to dorsoventral pigmentation differences is acquired by E12.5, which is shortly after early embryonic expression of Tbx15. Fate-mapping studies show that the dorsoventral pigmentation boundary is not in register with a previously identified dermal cell lineage boundary, but rather with the limb dorsoventral boundary. Embryonic expression of Tbx15 in dorsolateral mesenchyme provides an instructional cue required to establish the future positional identity of dorsal dermis. These findings represent a novel role for T-box gene action in embryonic development, identify a previously unappreciated aspect of dorsoventral patterning that is widely represented in furred mammals, and provide insight into the mechanisms that underlie region-specific differences in body morphology. + +Introduction + +A fundamental question in developmental biology is how adjacent regions of the vertebrate body acquire differences in their appearance or morphology. Mechanisms that establish the general body plan make use of a relatively small number of signaling pathways shared among all animals (reviewed in Pires-daSilva and Sommer 2003), but the extent to which these pathways control finer differences between body regions is not clear. Among vertebrates, differences in the shape or number of skeletal elements, altered morphology of epidermal appendages, and variation in pigment distribution combine to produce the majority of what distinguishes one animal from another. Among these, pigment patterns are an excellent system to investigate how morphological differences arise, both for different regions of the body within a species and for different animals from closely related species. In natural environments, color variation is a nearly universal mechanism for recognition, camouflage, or both; consequently, a large number of pigment patterns have been characterized from an evolutionary and ecological perspective (Boughman 2001; Jiggins et al. 2001). In the laboratory, color variation has been the subject of vertebrate genetics for more than a century (Searle 1968; Silvers 1979), and many pigmentary components have been identified whose actions are understood in a cellular or organ-based context (reviewed in Bennett and Lamoreux 2003). + +Several mechanisms may contribute to regional differences in vertebrate pigmentation. In the embryo, alterations in the determination or migration of melanoblasts from the neural crest affect the number or distribution of pigment cells in the skin (reviewed in Reedy et al. 1998). Within hair follicles, paracrine signals control the type of pigment made in specific regions of the body or at specific times during the hair cycle (reviewed in Furumura et al. 1996; Barsh et al. 2000). Finally, movement of pigment granules within melanocytes or from melanocytes to keratinocytes makes use of cellular machinery that is shared by a variety of cell types, but that can vary in different regions of the body (reviewed in Marks and Seabra 2001). However, for all of these mechanisms—white spotting, pigment-type switching, and melanosome biogenesis—more is known about the identity of the molecular components than their spatial and temporal control. + +One of the most obvious aspects of regional color variation in vertebrates is a dark dorsal surface juxtaposed to a light ventral surface, apparent in the color of skin, scales, feathers, or hair, in which the boundary between dorsal and ventral compartments is often sharp and lies in register with the limbs. In rodents and probably other mammals, this dorsoventral difference in hair color is brought about by differences in pigment type as determined by allelic variation of the Agouti gene (Bultman et al. 1992; Miller et al. 1993). Secreted by dermal papilla cells within each hair follicle (Millar et al. 1995), Agouti protein causes melanocytes in that follicle to switch from the production of brown/black eumelanin to red/yellow pheomelanin. Agouti protein has a short radius of action (Silvers and Russel 1955) and can be switched on and off during a single hair cycle (Bultman et al. 1992, 1994; Miller et al. 1993; Vrieling et al. 1994); thus, its regulated expression is thought to be responsible for the cream-colored or yellow ventral surface of mice carrying the black-and-tan (at) allele and for the yellow markings around the feet, ears, or head, i.e., tan points or head spots, of certain dog breeds. + +In laboratory mice, previous studies from our group and others identified two predominant Agouti mRNA isoforms that differ by virtue of their transcriptional initiation site and 5′ untranslated exons. A “hair cycle-specific” transcript is expressed in both dorsal and ventral skin for 2–3 days during early hair growth, while a “ventral-specific” transcript is expressed throughout the entire period of active hair growth, but only in ventral skin (Bultman et al. 1994; Vrieling et al. 1994). Animals carrying the at allele express only the ventral-specific Agouti transcript (Bultman et al. 1994; Vrieling et al. 1994) and have black dorsal hairs and cream-colored to yellow ventral hairs, with a sharp boundary at the level of the limb–body wall articulations and in the middle of the whisker pad. Ventral-specific Agouti isoforms are also expressed in developing skin from embryonic day 10.5 (E10.5) and beyond and may play a role in pigment cell differentiation (Millar et al. 1995). Thus, regulatory elements for ventral-specific Agouti isoforms are responsive to dorsoventral positional cues established in the embryo and whose effects persist after birth. + +The boundary between dorsal and ventral color compartments in at/at mice bears superficial resemblance to dorsoventral boundaries apparent for many other mammals, but morphogenetic differences between dorsal and ventral skin seem likely to include more elements than the type of pigment made by hair follicle melanocytes. In particular, dermis of the flank has at least two distinct origins: dermatomal derivatives of somites and loose mesenchyme derived from the lateral plate mesoderm (Mauger 1972; Christ et al. 1983; Olivera-Martinez et al. 2000; Nowicki et al. 2003); these lineages are established early in development and could, in principle, set up compartments whose identity contributes to dorsoventral differences in adult skin. + +To better understand the mechanisms that give rise to differences between dorsal and ventral skin and to the boundary between them, we have determined how several morphologic characteristics vary along the dorsoventral axis of the mouse and how these characteristics correspond to ventral-specific Agouti expression and the lineage boundary that distinguishes somite from lateral plate derivatives. Our results indicate that the apparent uniformity of the dorsoventral boundary represents the sum of independent mechanisms that affect melanocyte density and/or differentiation, pigment-type synthesis, and hair length; surprisingly, none of these coincide with the somite–lateral plate lineage boundary. We also make use of a classical mouse mutation, droopy ear (Curry 1959), that produces a dorsal-to-ventral transformation of flank coat color by allowing expansion of the ventral-specific Agouti transcript. By positional cloning and gene targeting, we identify an allele of droopy ear, deH, as a loss of function for Tbx15, which encodes a T-box transcription factor expressed in a dynamic and spatially restricted manner in the developing skin and musculoskeletal system. Embryonic expression and transplantation studies suggest that Tbx15 is required to establish certain characteristics of dorsal patterning in mesenchymal cells of the developing flank. These results identify a previously unappreciated aspect of dorsoventral patterning that is widely represented in furred mammals and provide insight into the mechanisms that underlie region-specific differences in body morphology. + +Results + +Morphological Components of Dorsoventral Skin Differences + +Besides the obvious change in hair color that frequently distinguishes dorsal from ventral skin, casual observation suggests there are additional differences in hair length, distribution of hair type, and skin thickness. Furthermore, dorsoventral differences in pigmentation can represent differences in the number and/or differentiated state of pigment cells, as well as the type of pigment synthesized in response to expression of Agouti. In particular, ventral hair of at/at animals can vary from cream-colored to reddish-yellow depending on age, strain background, and position along the dorsoventral axis. To evaluate the relationship among these components, we compared their features among mice of different Agouti genotypes. + +Semiquantitative measurements of hair length plotted as a function of dorsoventral position reveal that the apparent sharp boundary between dorsal and ventral pigment compartments in at/at mice coincides with a more gradual change in both hair color and hair length (Figure 1A–1D). Within the region of transition from dorsum to ventrum (Figure 1B), flank hairs from at/at mice become progressively shorter and exhibit increasing amounts of pheomelanin deposition progressing from the tip to the base of the hair. However, the region of transition for hair length is considerably broader than that for pigmentation and independent of Agouti genotype. Although hair-cycle timing varies along the rostrocaudal axis, measurements of absolute hair length for mice matched for age and rostrocaudal level are remarkably similar (Figure 1D). Furthermore, measurements of relative hair length for animals of different age, size, and Agouti genotype also are very similar when normalized to body circumference (Figure 1C). Taken together, these observations indicate that variation of hair length along the dorsoventral axis is stereotyped and maintained through multiple hair cycles, with a transition in hair length that is gradual and encompasses the pigment-type transition in at/at mice. + +Dorsal and ventral skin develop at different rates. Transverse sections of skin at postnatal day 4.5 (P4.5) exhibit dorsal hair follicles that are noticeably more developed than ventral hair follicles, along with a gradual dorsoventral decrease in dermal thickness (Figure 1F). However, differences in skin thickness disappear by 3–4 wk of age (Forsthoefel et al. 1966), and, overall, the proportion of different hair types is also similar in dorsa and ventra of adult mice. In age-matched inbred mice, we observed a small decrease in the ratio of undercoat hairs (zigzags) to overcoat hairs (auchenes, awls, and guard hairs) in dorsum compared to ventrum (Figure 1E), but there was no consistent difference in hair-type distribution for outbred mice (data not shown). + +Differences between dorsal and ventral pigmentation of at/at mice are usually attributed to pigment-type differences caused by ventral-specific expression of Agouti, but animals homozygous for a null allele of Agouti, extreme nonagouti (ae), have ventral hairs that contain less melanin than dorsal hairs, giving a slightly paler appearance to the ventral coat (Figure 1G). Using DOPA staining as an indicator of tyrosinase activity, we observed a gradual dorsoventral transition in isolated dermis preparations from P4.5 ae/ae mice (Figure 1G). By contrast, skin from at/at mice reveal an abrupt dorsoventral transition of DOPA staining, which probably reflects the additive effects of reduced melanin content (as in ae/ae mice) and downregulation of tyrosinase activity induced by Agouti. Melanin content of individual hairs is likely to be influenced both by the number of pigment cells and their follicular environment. Regardless, dorsoventral differences in hair pigment content of ae/ae mice persist throughout multiple hair cycles into adulthood, similar to hair length (but unlike skin thickness). Thus, at least three characteristics distinguish dorsal from ventral skin: differences in pigment-type synthesis (depending on Agouti genotype), differences in hair length, and differences in melanin content. + +Ventralization of Skin Morphology by the droopy ear Mutation + +Named after its effects on craniofacial morphology, droopy ear is a recessive mutation on mouse Chromosome 3; the original allele described more than 40 years ago by Curry (1959) is extinct, but a spontaneous remutation that occurred in Harwell, deH, is available through The Jackson Laboratory (Bar Harbor, Maine, United States). External craniofacial malformations are the most obvious characteristic of deH/deH animals, including widely spaced eyes, small palpebral fissures, a broad nasal area, and a shortened skull held in an elevated position, which presumably causes or contributes to the abnormal position of the ears. + +We became interested in droopy ear because the original allele was described to affect pigment pattern in a way that suggests a possible dorsal to ventral transformation: “On a genetic background (at and AW) which causes the belly hair to be lighter than the back hair, the belly hair comes up farther round the sides of the body and face” (Curry 1959). + +An abnormal dorsoventral pigment pattern is readily apparent in at/at; deH/deH mice, but comparison to nonmutant animals is more accurately described in terms of ventral, lateral, and dorsal regions (Figures 1G and 2A). The ventral region has short hairs with a gray base and cream-colored tip whose boundary coincides with the limb–body wall junction; both the appearance of this region and position of the boundary are approximately similar in at/at compared to at/at; deH/deH mice. The lateral region contains yellow hairs of progressively increasing length; in at/at mice, the lateral region appears as a thin yellow stripe along the flank, but in at/at; deH/deH mice, the lateral region is considerably expanded with a diffuse boundary along the dorsal flank, and a dorsal eumelanic region whose size is correspondingly reduced (Figure 2A and 2B). Total body size is smaller in mutant compared to nonmutant animals, but the proportion of body circumference occupied by the lateral region in mutant animals is increased about 2-fold, from 11.9% to 22.2% (Figure 2C). The proportion of the ventral cream-colored region is also expanded a small amount, 47.9% in mutant compared to 37.8% in nonmutant animals, but expansion of the lateral region, which occurs at all levels of the body, including the limbs and the cranium (but not the whisker pad), is the major feature responsible for the ventralized appearance caused by deH. + +To investigate whether deH affects other dorsoventral skin characteristics besides pigment-type switching, we examined its effects on hair length and pigmentation in an ae/ae background. Overall, deH causes a small but consistent reduction in hair length in both dorsum and ventrum; when mutant and nonmutant animals are normalized for body circumference, reduced hair length is most apparent in the lateral region (Figure 2E). Adult ae/ae; deH/deH animals exhibit body-size reduction and skeletal abnormalities, but display no coat-color phenotype (data not shown). However, ae/ae and ae/ae; deH/deH neonates are clearly distinguishable in the first few days after birth, when a dorsoventral gradient of melanogenic activity is apparent under the skin (Figure 2D). At this stage, melanoblast migration from the neural crest is mostly complete, but there is a dorsoventral gradient in melanocyte differentiation and pigment synthesis. The skin of ae/ae neonates appears uniformly dark over the entire dorsum, but in ae/ae; deH/deH neonates, the area of dark skin is more restricted, particularly above the limbs, and resembles the pattern of dorsal eumelanin in at/at; deH/deH adult animals. + +Taken together, these observations suggest that deH interferes with the establishment of dorsoventral patterning during skin development by causing dorsal expansion of a lateral region that is normally 3–5 mm in width. This same region may serve as a boundary between dorsal and ventral skin by inhibiting melanocyte differentiation, by promoting pheomelanin synthesis, and by supporting a progressive increase in hair growth from ventrum to dorsum. As described below, the gene defective in deH, Tbx15, is normally expressed in the dorsal region and therefore is likely to play a role in establishing the size and dorsal extent of the lateral region. + +Positional Cloning of deH + +As a visible marker, early linkage studies with the original droopy ear allele or the deH allele identified a map position in the middle of Chromosome 3, distal to matted and proximal to Varitint-waddler (Carter and Falconer 1951; Curry 1959; Lane and Eicher 1979; Holmes et al. 1981). We used an F2 intercross with CAST/Ei mice to localize deH to a 0.1 cM interval between D3Mit213 and 16.MMHAP32FLF1, which was refined by development of a bacterial artificial chromosome (BAC) contig and additional markers to a 1.4 Mb region that contained eight genes, including Tbx15 (Figure 3A). We considered Tbx15 as an excellent candidate for the skeletal abnormalities caused by deH, based on studies by Agulnik et al. (1998), who described its embryonic expression in the craniofacial region and developing limbs. + +Using sequence information from Agulnik et al. (1998) and the partially completed mouse genome sequence, we found that portions of several Tbx15 exons could not be amplified from deH/deH genomic DNA. The same gene was initially referred to as Tbx8 (Wattler et al. 1998) and then later renamed Tbx14, but is currently referred to in several vertebrate genomes as Tbx15 (Agulnik et al. 1998; Begemann et al. 2002). By comparing the sequence of a 1.3 kb junction fragment amplified from deH/deH genomic DNA to publicly available mouse genome sequence, we identified a 216 kb deletion that extends from Tbx15 intron 1 to 148 kb downstream of the polyadenylation sequence in a region annotated as a mannose-6-phosphate receptor pseudogene, M6pr-ps (Figure 3B and 3C). (Ludwig et al. 1992). By Northern blot analysis, we identified a fusion transcript produced from the deH chromosome (data not shown). However, the deletion removes 534 of the 602 amino acids encoded by Tbx15 (including the T-box DNA-binding domain), deH/+ animals are grossly normal, and the phenotype of deH/deH animals is identical to that described for the original allele. In addition, other than M6pr-ps, no other genes or transcripts have been annotated to the 216 kb deletion. + +While the positional cloning work was underway, one of us (A. Russ) generated an independent mutation of Tbx15 by gene targeting in embryonic stem cells. The targeted allele, Tbx15LacZ, carries an IRES-LacZ-neo cDNA cassette that disrupts the open reading frame at codon 154 early in the T-box domain (Figure 3D). Animals heterozygous for the targeted allele are completely normal with regard to size, skeletal morphology, and hair-color distribution, but Tbx15LacZ/Tbx15LacZ homozygotes were noted to exhibit reduced body size and an abnormal craniofacial appearance identical to that caused by deH. We generated Tbx15LacZ/deH compound heterozygotes; on an Aw/at background, these animals exhibited the same abnormal restriction of dorsal pigmentation at P3.5 and expanded yellow flank area as described above for deH/deH animals (see Figure 2). These observations demonstrate that the pigmentary and craniofacial characteristics of deH are caused by loss of function for Tbx15. + +Expression of Tbx15 and Agouti + +Previous studies by Agulnik et al. (1998) using whole-mount in situ hybridization described expression of Tbx15 as first detectable at E9.5 in the limb buds, progressing to the branchial arches, flanks, and craniofacial regions through E12.5. To investigate this pattern in more detail, we hybridized a Tbx15 mRNA probe to a series of transverse sections at E12.5 and observed expression in multiple mesenchymal tissues of the head, trunk, and developing limbs (Figure 4A), much of which is consistent with the skull, cervical vertebrae, and limb malformations reported for mice carrying the original droopy ear allele. + +We were particularly interested in determining the exact nature of the embryonic flank expression relative to the ventralized phenotype of adult deH/deH mice. Transverse abdominal sections from different times during development reveal a dorsolateral band of expression in the superficial mesenchyme at E11.5 that broadens both dorsally and ventrally over the next several days (Figure 4B). By E13.5, the developing dermis has become separated from the loose mesenchyme by a subcutaneous muscle layer; Tbx15 is expressed in all of these layers as well as the underlying abdominal muscles. In P3.5 skin, Tbx15 is expressed in both dorsal and ventral skin, most strongly in the condensed upper dermis and developing dermal sheaths of hair follicles; faint expression can also be detected in rare dermal papillae cells (Figure 4B). + +Although the effects of Agouti on pigment-type switching occur during postnatal hair growth, the ventral-specific isoform of Agouti is expressed in developing skin beginning at E11.5. We compared adjacent sections hybridized with probes for Tbx15 and Agouti and observed complementary patterns at E12.5, with expression of Agouti in ventral skin and expression of Tbx15 in dorsal skin (Figure 5A and 5B). The junction between expression domains is indistinct, and by E14.5, Tbx15 expression extends ventrally and overlaps extensively with Agouti expression (Figure 5C and 5D). + +We also examined the effect of deH on expression of Agouti and found no difference between mutant and nonmutant at E12.5 or E13.5 (data not shown). However, at E14.5, the normal ventral-to-dorsal gradient of Agouti expression appeared to extend more dorsally in deH/deH embryos (Figure 6A). In P4.5 skin, expression of Agouti is also extended dorsally in deH/deH animals and is most apparent in the midflank region within the upper dermis and dermal papillae cells (Figure 6B). Thus, while the pigmentation phenotype of deH/deH mice can be explained, not surprisingly, by dorsal extension of Agouti expression after birth, patterned expression of Tbx15 and Agouti are apparent some 10 days earlier, between E12.5 and E13.5, and the effects of Tbx15 deficiency on expression of Agouti can be detected by E14.5. + +Relationship of Embryonic Tbx15 Expression to Dorsal and Ventral Pigmentation Domains + +The observations described above are consistent with a model in which transient expression of Tbx15 in the embryonic dorsal flank is required to establish positional identity of the future dermis, at least with respect to pigment-type synthesis caused by the ventral-specific Agouti isoform. To further investigate this hypothesis, we carried out transplantation experiments in which pieces of embryonic skin were isolated from different dorsoventral positions. We evaluated the embryonic skin fragments for their potential to give rise to different hair colors and for their expression of Tbx15 and Agouti. + +Previous studies by Silvers and colleagues (Poole and Silvers 1976) showed that dorsal and ventral skin isolated from at/at embryos gives rise to black and yellow hair, respectively, when transplanted into testis and allowed to develop for several weeks. Furthermore, dermal–epidermal recombination experiments carried out at E14.5 demonstrated that positional identity is carried by the embryonic dermis. In a variation on this experiment, we divided embryonic skin from at/a embryos into dorsal, flank, and ventral pieces and analyzed the different pieces for their ability to give rise to black or yellow hair after testis transplantation, and, in parallel, for gene expression using in situ hybridization. For the purposes of a reproducible morphologic boundary, we divided flank from ventral skin based on a change in skin thickness and divided dorsal from flank skin at the level of an ectodermal notch that lies at the same level as the ventral extent of the myotome (Figure 7) (Huang and Christ 2000; Olivera-Martinez et al. 2000; Sudo et al. 2001; Burke and Nowicki 2003; Nowicki et al. 2003). + +We found that E12.5 is the earliest time at which embryonic ventral skin is able to produce hair when transplanted to the testis. Of the grafts that produced hair, ventral skin gave rise to yellow hair (n = 3), and dorsal skin gave rise to black hair (n = 4). Transplantation of flank skin gave rise to a patch of yellow hair juxtaposed against a patch of black hair in 85% of the successful grafts (n = 13); the remaining two flank grafts produced solely black or yellow hair. In no case did we observe intermingling of black and yellow hairs. As predicted from the experiments using tissue sections (see Figures 5 and 6), dorsal pieces expressed Tbx15 but not Agouti, while flank pieces expressed both genes (see Figure 7). Thus, dorsoventral identity for adult pigmentation is established by the time when patterned expression becomes apparent for Tbx15 and Agouti (E11.5–E12.5); furthermore, positional identity is maintained throughout later stages of skin development, even though expression of Tbx15 broadens to include ventral as well as dorsal skin. + +Relationship of the Dorsoventral Pigment Boundary to Lineage Compartments and the Lateral Somitic Frontier + +The ectodermal notch that we used to mark the boundary between embryonic dorsum and embryonic flank is a characteristic feature in vertebrate embryos. In cell lineage studies carried out in the chick system, the notch serves as a landmark for the boundary between dermis derived from somitic mesoderm and dermis derived from lateral plate mesoderm and has been termed the “lateral somitic frontier” (Olivera-Martinez et al. 2000; Sudo et al. 2001; Burke and Nowicki 2003; Nowicki et al. 2003). Although fate-mapping studies have not been carried out in mammalian embryos, somite- and lateral plate-derived mesoderm could give rise to precursors for dermis dorsal and ventral to the limb–body wall junction, respectively. However, this notion conflicts with our observation that the future pigmentation boundary lies ventral to the ectodermal notch (see Figure 7). + +To examine directly the relationship between the pigmentation boundary and dermis derived from lateral plate mesoderm, we made use of a Cre transgene driven by the Hoxb6 promoter that was developed by Kuehn and colleagues (Lowe et al. 2000). As described by Lowe et al. (2000), midgestation embryos carrying both the Hoxb6-Cre transgene and the R26R lacZ reporter gene (Soriano 1999) exhibit X-Gal staining in lateral plate mesoderm but not somite-derived mesoderm of the trunk. In whole-mount skin preparations from P1.5 or P4.5 neonatal animals, we observed a ventral band of dark X-Gal staining corresponding to lateral plate-derived dermis, which represents 63% of the total circumference (Figure 8A). However, in parallel preparations from at/at mice, the ventral pheomelanin domain represents 47% of the total skin circumference; therefore, the proportions of total skin circumference occupied by dorsal eumelanin and somite-derived dermis are 53% and 37%, respectively (Figure 8B). These results indicate that the pigmentation boundary is clearly distinct from, and more ventral to, the boundary between lateral plate- and somite-derived dermis. + +Because the pigmentation boundary lies in register with the limb–body wall junction (see Figure 2), we wondered whether mechanisms used for dorsoventral limb patterning might be related to those used to establish the pigmentation boundary. In the developing limb, Engrailed1 (En1), Wnt7a, and Lmx1b are part of a network whose restricted domains of expression help to establish dorsoventral identity (reviewed in Niswander 2003). En1 is transiently expressed in the developing flank; at E11.5, transverse abdominal sections reveal domains in the neural tube, somite-derived mesenchyme, and the ventral body wall (Figure 8C). An adjacent section hybridized with Tbx15 reveals a complementary pattern in the flank, which provides additional evidence for developmental mechanisms that establish a pigmentation boundary entirely within lateral plate mesoderm and independent of lineage restrictions imposed by the lateral somitic frontier. + +Discussion + +Several mutations and genes have been identified that affect the pattern of hair follicle development, but Tbx15 is the only gene of which we are aware that affects the pattern of hair pigmentation in different body regions. Ventral areas that normally produce yellow hair in the trunk, limbs, and craniofacial regions are expanded in deH/deH mice and, in the trunk at least, represent inappropriate dorsal expression of an Agouti mRNA isoform that is normally restricted to ventral skin. The deH allele is caused by a large deletion that removes most of the Tbx15 coding sequence, but the pleiotropic phenotype is caused by a simple loss of function for Tbx15 rather than a dominant-negative or contiguous gene effect. In particular, there is no heterozygous phenotype, no other genes lie within or close to the deletion breakpoints, and the expression pattern of Tbx15 is consistent with the spectrum of phenotypic abnormalities in both the original de allele and the deH allele. Finally, a Tbx15 targeted allele has the same phenotype as deH. + +Our results suggest that patterned expression of Tbx15 provides an instructional cue required to establish the future identity of dorsal dermis with regard to pigmentary and hair length patterning. The ventral edge of Tbx15 expression in the developing flank does not correspond to a known lineage compartment, but, like limb development, occurs within lateral plate mesoderm. These findings represent a novel role for T-box gene action in embryonic development and provide evidence for a previously unappreciated complexity to acquisition of dorsoventral positional identity in mammalian skin. + +Distinct Morphologic Regions Represent the Sum of Different Gradients + +The visual boundary between dorsal and ventral skin in at/at mice is reminiscent of other systems in which adjacent compartments enforce a binary choice between alternative patterns of gene expression and cell fate (reviewed in Dahmann and Basler 1999). However, Agouti mRNA in both embryonic and postnatal skin is distributed along a gradient whose dorsal boundary is indistinct and overlaps with two additional gradients recognized by their effects on hair length and histochemical staining for melanocytes. The three gradients are close but not congruent, and it is their proximity that gives rise to the superficial distinction between dorsal and ventral skin of at/at mice. Indeed, slight differences between the regions of transition for pigment-type switching and pigment content give rise to a subtle yellow stripe along the flank (see Figures 1, 2, and 9A). Levels of Agouti mRNA remain high throughout the entire ventrum, but hair pigment content is reduced, giving rise to a cream-colored region in the ventrum that, depending on age and genetic backgrounds, may appear more or less distinct from the yellow flank stripe. + +Loss of Tbx15 affects dorsoventral transitions of hair length, pigment content, and expression of the ventral-specific Agouti isoform; however, the former two effects are subtle and contribute little, if at all, to the abnormal pigmentation of adult deH/deH mice. Thus, despite the abnormal pattern of dark skin in neonatal deH/deH mice (e.g., Figure 2D), the most obvious feature in adults is dorsal displacement of the “boundary” between black and yellow hair (Figure 9A). + +Genetics of Tbx15 + +Named for the presence of a DNA-binding domain first identified in the mouse Brachyury gene (haploinsufficiency causes a short tail), T box–containing genes have been identified as developmental regulators in a wide spectrum of tissues and multicellular organisms (reviewed in Papaioannou 2001). The Tbx15 subfamily, which also includes Tbx18 and Tbx22, is likely to have arisen during early chordate evolution since there is a single gene in amphioxus but no obvious homolog in the fly genome (Ruvinsky et al. 2000). Consistent with this relationship, the three genes are expressed in partially overlapping patterns that include anterior somites (Tbx18 and Tbx22), limb mesenchyme (Tbx15 and Tbx18), and craniofacial mesenchyme (all three genes, Tbx15 more broadly than Tbx18 or Tbx22) (Agulnik et al. 1998; Kraus et al. 2001; Braybrook et al. 2002; Bush et al. 2002; Herr et al. 2003). These observations suggest that an ancestral gene for Tbx15, Tbx18, and Tbx22 may have been important for craniofacial development in cephalochordates, with acquisition of additional expression patterns and developmental functions in the limb and the trunk during early vertebrate evolution. Expression of Tbx18 and Tbx22 has not been reported in embryonic flank mesenchyme, which suggests that Tbx15 is the only family member involved in establishing the dorsoventral identity of the trunk. However, it would not be surprising to find some degree of functional redundancy in animals mutated for two or three of the subfamily members in other body regions, particularly the limbs and the head. For example, mutations in Tbx22 cause the human syndrome X-linked cleft palate and ankyloglossia (Braybrook et al. 2001). Despite high levels of Tbx22 expression in periocular embryonic mesenchyme (Braybrook et al. 2002; Bush et al. 2002; Herr et al. 2003), the condition does not affect the eye, perhaps because residual activity is provided by Tbx15 in the same region. + +In an initial description of the expression and map location of mouse Tbx15, Agulnik et al. (1998) suggested human Tbx15 that lies on Chromosome 1p11.1 as a candidate for acromegaloid facial appearance (AFA) syndrome, for which there is a weak positive LOD score to Chromosome 1p (Hughes et al. 1985). Originally described as a rare autosomal-dominant syndrome with progressive facial coarsening, overgrowth of the intraoral mucosa, and large, doughy hands, more recent case reports describe macrosomia, macrocephaly, or both and generalized hypertrichosis with progressive coarsening (Dallapiccola et al. 1992; Irvine et al. 1996; da Silva et al. 1998; Zelante et al. 2000). The deH phenotype exhibits little overlap with these features; instead, we suggest a more likely candidate for mutations of human TBX15 would be frontofacionasal syndrome, an unmapped autosomal recessive condition characterized by brachycephaly, blepharophimosis, and midface hypoplasia (Reardon et al. 1994). + +Two of us (S. Kuijper and F. Meijlink) became interested in the deH mutation because of its effects on skeletal development (Curry 1959) and the possibility that the aristaless-related gene Alx3 might be allelic with droopy ear (ten Berge et al. 1998). In spite of similarities between skeletal phenotypes of deH and Alx3 or Alx4 mutants, subsequent experiments (unpublished data) excluded allelism of Alx3 and deH, and a full description of the Tbx15 skeletal phenotype will be published elsewhere. + +Developmental Mechanism of Tbx15 Expression and Action in the Skin + +Our attention to the role of Tbx15 in pigment patterning was motivated by the effects of Agouti in postnatal animals. However, Agouti is also expressed in the embryo, where it provides a convenient marker of ventral dermis identity. Because an expanded domain of embryonic Agouti expression in deH/deH animals is detectable by E14.5, the effects of Tbx15 on dorsoventral patterning must occur prior to this time. Among other T-box genes whose developmental actions are at least partially understood, two general themes have emerged, one focused on the ability to specify alternative fates for an undifferentiated group of precursor cells and another focused on the ability to support proliferative expansion of a cell population whose fate is already determined (reviewed in Tada and Smith 2001). Either mechanism may apply to the apparent dorsal-to-ventral transformation in deH/deH mice. For example, while the expanded domain of Agouti expression in postnatal deH/deH animals can be traced to events that occur between E11.5 and E13.5, the underlying cause may be that embryonic cells in dorsolateral mesenchyme acquire a ventral rather than dorsal identity or that those cells fail to proliferate normally, followed by compensatory expansion of ventral cells. Cell lineage studies should provide a definitive answer, but we favor the latter hypothesis, because measurements of dorsoventral regions according to hair color in deH/deH mice revealed a small increase of the cream-colored ventral region in addition to the approximate doubling of the yellow flank region (see Figure 2). + +In embryonic mesenchyme, expression of Tbx15 and Agouti are complementary, and it is possible that Tbx15 acts directly to inhibit Agouti transcription in dorsolateral mesenchyme. However, the ability of Tbx15 to suppress expression of the ventral-specific Agouti isoform in postnatal mice is likely to be indirect, since postnatal expression of Tbx15 occurs broadly along the dorsoventral axis and overlaps extensively with that of Agouti. In either case, the targets of Tbx15 action in the skin include genes in addition to Agouti, since hair length and melanocyte distribution exhibit a demonstrable, albeit subtle, alteration in animals that carry a null Agouti allele. One potential target is Alx4, which, like Agouti, is expressed in ventral embryonic mesenchyme, and, when mutated, affects hair-follicle as well as limb and craniofacial development (Qu et al. 1997, 1998; Wu et al. 2000; Wuyts et al. 2000; Mavrogiannis et al. 2001). However, expression of ventral markers such as Alx4, as well as Alx3 and Msx2, appears to be unaffected at E11.5 in deH/deH embryos (data not shown). + +Differences and Similarities to Dorsoventral Limb Patterning + +Loss of Tbx15 also affects regional distribution of hair color in the limbs, with areas that would normally produce black hair giving rise to yellow hair instead. However, neither normal patterns of pigment-type synthesis in the limb nor their disruption in deH/deH mice correspond to obvious developmental compartments. Furthermore, losses of function for En1 or Wnt7a, which cause a partial transformation of the distal limb from dorsum to ventrum (Loomis et al. 1996) or ventrum to dorsum (Parr and McMahon 1995), respectively, have no effect on regional patterns of Agouti expression or distribution of hair-color regions (Y. Chen, unpublished data). (Ectopic pigmentation of the ventral footpads that develops in En1 mutant mice is unrelated to pigment-type synthesis and instead likely reflects a requirement for En1, independent of Wnt7a, to repress migration or proliferation (or both) of pigment cells in ventral epidermis [Cygan et al. 1997; Loomis et al. 1998].) + +These considerations notwithstanding, control of dorsoventral trunk pattern by Tbx15 shares certain features with control of dorsoventral limb patterning by Lmx1b, a LIM domain transcription factor that acts downstream of Wnt7a and En1 (Riddle et al. 1995; Vogel et al. 1995; Cygan et al. 1997; Logan et al. 1997; Loomis et al. 1998; Chen and Johnson 2002). Both Tbx15 and Lmx1b act autonomously in mesenchymal cells to promote a dorsal identity, yet have expression domains that do not correspond to cell lineage compartments in the flank (Tbx15) or the limb (Lmx1b) (Altabef et al. 1997; Michaud et al. 1997). In the case of Lmx1b, its expression in the distal limb depends on Wnt7a produced in the overlying dorsal ectoderm (Riddle et al. 1995; Cygan et al. 1997; Loomis et al. 1998). Wnt7a, in turn, is restricted to dorsal ectoderm by En1 in the ventral ectoderm (Loomis et al. 1996; Cygan et al. 1997; Logan et al. 1997), whose expression marks a lineage boundary coincident with the dorsoventral midline of the apical ectodermal ridge (Altabef et al. 1997; Michaud et al. 1997; Kimmel et al. 2000). As described above, En1 or Wnt7a mutations have not been reported to affect patterns of hair-color distribution (C. Loomis, personal communication; Parr and McMahon 1995; Loomis et al. 1996). However, the essential theme that ectodermal lineage compartments control the fate of underlying mesenchyme in developing limbs may apply to the trunk as well as the limb. The mammary glands also develop at a stereotyped dorsoventral position and depend on epithelial–mesenchymal interactions. However, the number and apparent position of the mammary glands are normal in deH/deH animals, indicating the existence of additional mechanisms that control dorsoventral patterning in the trunk as well as in the limbs. + +These ideas are summarized in the model shown in Figure 9B. We speculate that a diffusible signal from dorsal trunk ectoderm, at or prior to E11.5, promotes expression of Tbx15 in dorsal trunk mesenchyme, which then establishes dorsal positional identity of those cells as manifested by differences in Agouti expression, pigment-cell development, and hair growth. Because the ventral limit of Tbx15 expression corresponds to the dorsal limit of En1 expression and because the normal position of the pigmentation boundary lies approximately in register with the limb-bud outgrowths, we depict the position of a putative dorsoventral boundary in trunk ectoderm as coincident with the limb dorsoventral boundary. This model is consistent with studies in the chick, where distinct dorsal and ventral lineage compartments exist for ectoderm in both the limb (Altabef et al. 1997, 2000; Michaud et al. 1997; Kimmel et al. 2000) and interlimb regions (Altabef et al. 1997, 2000), but not for limb mesoderm (Altabef et al. 1997; Michaud et al. 1997). In fact, the same mechanism that determines dorsoventral position of the limbs and the apical ectodermal ridge may also act on expression of Tbx15 in the trunk, since ectopic limbs induced in the interlimb region by application of FGF beads develop along a single line that is coincident with normal limb buds (and the future pigmentation boundary) (Cohn et al. 1995; Crossley et al. 1996; Vogel et al. 1996; Altabef et al. 1997, 2000). + +Our model predicts that ectopic expression of Tbx15 in ventral mesenchyme should give rise to a dorsalized pigmentation phenotype and could be tested with gain-of-function approaches. However, Tbx15 expression is very dynamic and is restricted to dorsal mesoderm only from E11.5 to E13.5. It is possible that Tbx15 influences skin patterning in a very narrow window of development; alternatively, establishment of dorsal identity by Tbx15 may require another as-yet-unidentified factor that is only present in the mesenchyme underlying dorsal ectoderm. + +Pigmentation Patterns and Tbx15 in Other Mammals + +The lateral somitic frontier, defined as the lineage boundary between somite-derived versus lateral plate-derived mesoderm, is established during somitogenesis early in development (Mauger 1972; Christ et al. 1983; Olivera-Martinez et al. 2000; Nowicki et al. 2003), but remains distinct in postnatal animals despite the potential for extensive cell mixing (see Figure 8). However, our transplantation and fate-mapping studies demonstrate that the lateral somitic frontier lies dorsal to the pigmentation boundary and does not obviously correlate with a difference in skin morphology. An additional dorsoventral domain that is not externally apparent has emerged from studies of Msx1, whose expression marks a subgroup of somite-derived mesenchymal cells that contribute to dermis in a narrow stripe along the paraspinal region (Houzelstein et al. 2000). Thus, there exist at least three distinct boundaries in postnatal mammalian skin that are parallel to the sagittal plane, marked by differences in pigment-type synthesis, differences in cell lineage, and differences in expression of Msx1. + +In rodents, only the pigmentation boundary is evident externally, but many mammals have more complicated patterns of hair type, length, and/or color that vary along the dorsoventral axis. Raccoons, squirrels, skunks, and many different ungulates exhibit lateral stripes whose developmental origins have not been investigated, but may correspond to the lateral somitic frontier, the paraspinal Msx1 compartment, or an interaction between these domains. + +The effect of Tbx15 on pigmentation in laboratory mice is reminiscent of coat-color patterns in both selected and natural populations of other mammals. Saddle markings are common in some dog breeds, such as German shepherds, and in certain populations of Peromyscus polionotus, in which a dorsal extension of ventral depigmentation provides an adaptive advantage to subspecies that live on white sand reefs (Blair 1951; Kaufman 1974; Belk and Smith 1996). Neither German shepherds nor deer mice have craniofacial characteristics similar to the deH mutation, but the pigmentation patterns in these animals could represent alterations in the regulation or action of Tbx15 activity. From the opposite perspective, the effects of Tbx15 on coat color are only apparent in certain genetic backgrounds and may not be evident at all in mammals that lack dorsoventral pigmentation patterns. Studying the sequence and expression of Tbx15 in other vertebrates may provide additional insight into patterns that affect the skeleton as well as the pigmentary system. + +Materials and Methods + + + +Mice + +All mice were obtained originally from The Jackson Laboratory (Bar Harbor, Maine, United States), except the BA strain (Stanford Veterinary Services Center, Stanford, California, United States), Hoxb6-Cre transgenic mice (kindly provided by M. Kuehn of the National Institutes of Health, Bethesda, Maryland, United States), mice carrying the R26R lacZ reporter allele (kindly provided by P. Soriano, Fred Hutchinson Cancer Research Center, Seattle, Washington, United States), and C57BL/6J (B6) ae/ae mice (kindly provided by L. Siracusa, Jefferson Medical College, Philadelphia, Pennsylvania, United States). The deH mutation arose in the 1960s in Harwell, probably on the BN strain background (C. Beechey, personal communication). We obtained deH on a B6/EiC3H background, introduced the at allele from the BTBR strain, and have maintained the line as a mixed deH/+ × deH/+ intercross stock with periodic outcrossing to BTBR or B6. For timed matings, the morning of the plug was considered E0.5. Postnatally, the day of birth was considered to be P0.5. + +Phenotypic analysis + +For measurements of hair length and color, the entire interlimb region of skin was first dissected with a single incision at the dorsal midline and preserved with powdered sodium bicarbonate. Slices 2–2.5 mm in width were then prepared parallel to the dorsoventral axis, hair length boundaries determined from electronic images with Adobe Photoshop (San Jose, California, United States), and measurements obtained using ImageJ (National Institutes of Health). This approach samples awls and auchenes, because they are much thicker and therefore visually more predominant than zigzag underhairs. To assess dorsoventral variation in hair-type distribution, several hundred hairs were plucked from the middorsum or midventrum of 8-wk-old male BA strain animals, then sorted and categorized using a dissection microscope. No attempt was made to distinguish between awls and auchenes. + +For skin histology, 12 μm sections from paraffin-embedded tissue were stained with hematoxylin and eosin. For DOPA staining, the dermis and epidermis were split after 3 h of incubation in 2 M sodium bromide at 37°C (this preparation causes most hair follicles to remain with the dermis), individually fixed for 1 h, then rinsed and stained with 0.1% L-DOPA (Sigma, St. Louis, Missouri, United States), 0.1 M sodium phosphate buffer (pH 6.8) for 5 h at 37°C in the dark, changing the staining solution after 1 h. The samples were then fixed overnight, dehydrated, and mounted. This method is sufficient to stain interfollicular melanocytes without creating a high background. The fixative used was always 4% paraformaldehyde. + +Positional cloning + +A high-resolution map for deH was generated from an intersubspecific intercross between deH/deH and CAST/Ei mice. We initially localized deH to a 1 cM interval between D3Mit233 and D3Mit11. F2 animals carrying recombinant chromosomes between these markers whose genotype at de was indeterminate (deH/+ or +/+) were progeny-tested by crossing to deH/deH animals. Further genetic mapping established a minimal region of 0.1 cM between D3Mit213 and 16.MMHAP32FLF1; these markers were used to initiate construction of a physical map with BAC genomic clones (Research Genetics, Huntsville, Alabama, United States, and Genome Systems, St. Louis, Missouri, United States). End sequence from those BACs was used to develop SSCP markers M1 to M3, as depicted in Figure 3, and to establish a minimal physical interval of 1.4 Mb. Primer pairs used were TTCCCTCCAATAAGTTCTGGGTACC and AAGCTTGCTGCTCTGGATTCCATTTGTAG for M1, CCTTCATTTTTTTTTCAAGTAAAA and AAGCTTGGCTTAGTCCCAGTGGC for M2, CCTCCAGGAAGATCTACTAGGCAC and ATGGAAAAAAAAAAGTAAGATTGAAAG for M3, and TGGTTATCGATCTGTGGACCATTC and AAGTGAGAGAGCAGGATGGACCAC for M4 (the M4 marker represents STS 16.MMHAP32FLF1). Genomic sequence and annotations were obtained from the UCSC Genome Browser February 2003 assembly version mm3 (http://genome.ucsc.edu); the 1.4 Mb interval between M1 and M4 contains eight genes: four hydroxysteroid dehydrogenase isomerases, Hsd3b3, Hsd3b2, Hsd3b6, and Hsd3b1; an hydroacid oxidase, Hao3; a tryptophanyl-tRNA synthetase, Wars2; a T-box gene, Tbx15; and a novel gene, 4931427F14Rik. In the genome sequence, M1 primers correspond to AGGCCTCCAATAAGTTCTGGGTACC and AAGCTTGCTCTCTGGATTCCATTTGTAG, the M2 reverse primer corresponds to AAGCTTGGCTTTAGTCCCAGTGGGC, and the M3 primers correspond to CCTCCAGGAAGAATCTACTAGGCAC and AATGAAAAAAAAAAAAGTAAGATTGAAAG. Minor differences among the sequences of the primers we obtained from the BAC ends and the public genome sequence may represent strain differences or sequencing errors on the BAC DNA. + +A multiplex genotyping assay was developed to genotype for the deH deletion using primers GGAGCAGATCCAATTGCTTT, TCCATAGCCCATCTTCACAA, and CATGTCCACTTCTGCTTCCA. This PCR assay produces a 392 bp product from the deH chromosome and a 595 bp product from the nonmutant chromosome. + +Gene targeting + +A targeted allele of Tbx15 was constructed using the same approach described in Russ et al. (2000). In brief, an IRES-LacZ-neo cassette with 5′ and 3′ homology arms of 3.5 kb and 1.8 kb was inserted into a unique BamHI site that lies 479 nucleotides downstream of the transcriptional initiation site (relative to the mRNA sequence) in exon 3. Positive ES clones were injected into B6 blastocysts, and chimeric founders crossed to either B6 mice or to deH/+ animals. + +In situ hybridization + +In situ hybridization was carried out on 12-μm paraffin sections using digoxigenin-labeled RNA probes (Roche Diagnostics, Indianapolis, Indiana, United States) according to standard protocols (Wilkinson and Nieto 1993). Embryos and postnatal skin samples were obtained from intercrosses of deH/+ mice. Embryos E13.5 or younger were fixed for 24 h; those older than E13.5 and postnatal skin were fixed for 36–48 h prior to embedding. The Tbx15 probe was generated by RT–PCR using primers GGCGGCTAAAATGAGTGAAC and TGCCTGCTTTGGTGATGAT (corresponds to exons 1 and 2), and the En1 probe was generated by PCR from genomic DNA using primers ACGCACCAGGAAGCTAAAGA and AGCAACGAAAACGAAACTGG (located in the last exon). The Agouti probe corresponds to the protein-coding sequence. + +Embryonic skin transplantation + +(BTBR-at/at × B6-a/a)F1 embryos at E12.5 were dissected in sterile Tyrode's solution, and embryonic skin was divided into dorsal, flank, and ventral pieces, each 1–2 mm2 in size, as shown in Figure 7. Skin fragments were grafted to the testes of congenic animals as follows. After anesthetization with 2.5% Avertin, a 1.5-cm incision in the skin and body wall was made at a point level with the top of the limbs. The fat pads were pulled out and laid on the outside of the body, exposing the testes. Forceps were used to introduce a small hole in the testis capsule through which a piece of dissected embryonic skin was inserted, the testes were then replaced into the abdominal cavity, and the wound was closed in both the body wall and the skin. After 21 days, mice that received grafts were sacrificed and the resulting hair was dissected from the testes and examined. + +Fate-mapping the lateral somitic frontier + +The Hoxb6-Cre transgene described by Kuehn and colleagues (Lowe et al. 2000) is expressed in the lateral plate but not the somitic mesoderm of the trunk, beginning at E9.5. Animals doubly heterozygous for this transgene and the R26R reporter gene were used as a source of whole skin at P1.5 or P4.5. Skin sections parallel to the dorsoventral axis were prepared with a single incision along the ventral midline and stained for β-galactosidase activity using standard protocols at room temperature. The P1.5 sample was stained overnight and the P4.5 samples were stained for 5.5 h. Similar nonstained skin sections were prepared from animals carrying the at allele. Images of the different skin fragments were aligned and scaled, and the relative position of the somite–lateral plate and the pigmentation boundaries were measured using ImageJ. + +Supporting Information + +The GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html) accession numbers discussed in this paper are for 4931427F14Rik (AK016477), Agouti gene (L06451), Alx3 (U96109), Alx4 (AF001465), En1(L12703), M6pr-ps (X64069), Tbx14 (AF013282), Tbx15 (AF041822), Tbx18 (AF306666), and Tbx22 (NM_145224). + +The OMIM (http://www.ncbi.nlm.nih.gov/omim/) accession numbers discussed in this paper are for acromegaloid facial appearance (MIM 102150), frontofacionasal syndrome (MIM 229400) and human syndrome X-linked cleft palate and ankyloglossia (MIM 303400). + +Acknowledgements + +We thank Colin Beechey and Bruce Cattanach for providing information about the origin of deH, Cindy Loomis for communicating unpublished results, and Michael Kuehn for providing Hoxb6-Cre transgenic mice. We are especially grateful to Hirotake Ono for discussion, to Hermie Manuel and to Carla Kroon-Veenboer for technical help, and to David Kingsley for first drawing our attention to the effects of droopy ear on pigmentation. SIC is supported by a grant from The Donald E. and Delia B. Baxter Foundation. GSB is an associate investigator of the Howard Hughes Medical Institute. + +Abbreviations + +ae - extreme nonagouti allele + +AFA - acromegaloid facial appearance + +at - black-and-tan allele + +AW - white-bellied Agouti allele + +B6 - C57BL/6J mice + +BAC - bacterial artificial chromosome + +deH - droopy ear allele that arose in Harwell + +E10.5 - embryonic day 10.5 + +En1 - Engrailed1 + +M6pr-ps - mannose-6-phosphate receptor pseudogene + +P4.5 - postnatal day 4.5 + +Tbx15 - T-box gene 15 + +Figures and Tables + +Figure 1 + +Dorsoventral Skin Characteristics + +(A) Skin slices from animals of different age and genotype demonstrate similar patterns of hair-length variation along the dorsoventral axis (scale bar = 1 cm). + +(B) Enlarged area from (A), demonstrating the transition in hair length and color in at/at mice (scale bar = 0.375 cm). + +(C) Proportional hair length for (A) plotted as a function of relative position along the dorsoventral axis. + +(D) Hair length plotted as a function of absolute position along the dorsoventral axis for 8-wk-old BA strain mice. + +(E) Proportion of zigzag hairs (± SEM) differs slightly between dorsum and ventrum of inbred mice (p < 0.0001, χ2 test, n = 1,958, 1,477, 1,579, 1,502). + +(F) Differences in dorsal and ventral skin development at P4.5 (scale bar = 1 mm, upper; 200 μm, lower). + +(G) Differences in hair melanin content and DOPA staining for dorsum (d), flank (f), and ventrum (v) in ae/ae and at/at mice. The upper panel also demonstrates a cream-colored appearance of the at/at ventrum. The middle panel shows representative awls (scale bar = 100 μm). The lower panel shows DOPA-stained dermis (scale bar = 200 μm). + +Figure 2 + +The deH Pigmentation Phenotype + +(A) 10-wk-old deH/deH and nonmutant animals on a at background. A thin stripe of yellow hair normally separates the dorsal black hairs from the ventral cream hairs. In deH, the yellow stripe is extended dorsally, and the boundary between the yellow and the black hairs is fuzzier. + +(B) Skin slices taken from 1.5-mo-old deH/deH and nonmutant littermates (scale bar = 0.5 cm). + +(C) Proportion of total skin area as determined by observation of pelts taken from the interlimb region. The proportion occupied by the yellow lateral compartment (± SEM) differs between mutant and nonmutant littermate flanks (p < 0.0005, paired t-test, n = 6 pairs). There is also (data not shown) a small increase in the proportion of total skin area occupied by the ventral cream-colored compartment, 47.9 % in mutant compared to 37.8% in nonmutant (p < 0.005, paired t-test, n = 6 pairs). + +(D) On an ae/ae background, the extent of dorsal skin pigmentation is reduced in deH/deH neonates (P3.5). + +(E) Hair length in a representative pair of 1.5-mo-old deH/deH and nonmutant littermates, averaged over three skin slices at different rostrocaudal levels, and plotted as a function of the absolute distance from middorsum or the percentage of total slice length. + +Figure 3 + +Molecular Genetics of deH and Tbx15 + +(A) Genetic and physical map, as described in the text. Markers M1 to M3 are SSCP markers generated from a BAC contig of the region; marker M4 is STS 16.MMHAP32FLF1 and was also used as an SSCP marker. M2 and M3, which flank the Tbx15 and M6pr-ps on the UCSC genome browser map and lie 634 kb apart, were nonrecombinant with deH in 2340 meioses. + +(B) The deH mutation is a deletion that starts in Tbx15 intron 1 and ends in the M6pr-ps. + +(C) Sequence of deletion breakpoints. + +(D) Diagram of Tbx15LacZ allele constructed by gene targeting. As described in the text, this allele is predicted to give rise to a protein truncated after approximately 154 codons and is lacking critical residues of the T box. Heterozygotes for the targeted allele exhibit normal size, morphology, and hair-color patterns, but homozygotes and Tbx15LacZ/deH compound heterozygotes are identical to deH homozygotes. + +Figure 4 + +Developmental Expression of Tbx15 + +(A) At E12.5, transverse sections at different levels show expression in head mesenchyme (a and b); myotome, occipital, and periocular mesenchyme (b); palatal shelf, cervical sclerotome, and nasal cartilage (c); maxillary and mandibular processes (d); limbs (e); and myotome and lateral mesenchyme (e and f) (scale bars = 500 μm). + +(B) Transverse sections through the flank at different times show expression in lateral mesenchyme (E11.5), expanding dorsally at E12.5, and both ventrally and dorsally at E13.5, detectable in loose mesenchyme underlying the dermis and the abdominal and subcutaneous muscles (scale bar = 500 μm). At P3.5, Tbx15 is expressed in the entire dermis and is most strongly expressed in dermal sheaths (scale bar = 200 μm). + +Figure 5 + +Embryonic Expression of Tbx15 Compared to Agouti in at/at Mice + +(A and C) Tbx15. (B and D) Agouti. At E12.5, expression of Tbx15 in dorsal skin is approximately complementary to that of Agouti in ventral skin. At E14.5, the levels of expression for both genes are lower, but Tbx15 expression has expanded ventrally and overlaps extensively with that of Agouti. In all four panels, arrows mark the approximate ventral limit of Tbx15 and the approximate dorsal limit of Agouti (scale bars = 500 μm). + +Figure 6 + +Effect of deH on Agouti Expression + +Comparable sections from at/at; deH/deH and at/at; +/+ littermates. + +(A) At E14.5, deH/deH embryos have a smaller body cavity and loose skin within which Agouti expression appears to be shifted dorsally, as marked by arrows (scale bars = 500 μm). + +(B) At P4.5, Agouti expression in both dorsal and ventral skin is similar in deH/deH compared to nonmutant, but in the midflank region, there is increased Agouti expression in deH/deH, especially in the upper dermis (scale bars = 200 μm). Sections shown are representative of two mutant and two nonmutant samples examined at each time. + +Figure 7 + +Embryonic Establishment of Dorsoventral Skin Patterning + +Pieces of skin from dorsal, flank, and ventral regions of at/a E12.5 embryos were transplanted into the testes of congenic animals as described in the text. Hair color of the grafts was examined 3 wk later. Grafts of ventral embryonic skin (n = 3) produced yellow hairs, dorsal embryonic skin (n = 4) produced black hairs, and flank embryonic skin produced mostly (13 out of 15) black and yellow hairs in distinct regions as shown. In parallel, in situ hybridization studies revealed that the embryonic flank contains the boundary of expression between Agouti and Tbx15 (scale bars = 1 mm for hairs and 200 μm for in situ hybridization results). + +Figure 8 + +Comparison of the Dorsoventral at/at Pigmentation Boundary to the Lateral Somitic Frontier + +(A) Dorsoventral slices of skin from at the midtrunk region prepared such that the dorsal midline lies in the center of the slice. Sections were taken at P1.5 (a) or P4.5 (b–e) from at/at or R26R/+; Tg.Hoxb6-Cre/+ mice (the latter were stained with X-Gal), as described in Materials and Methods. For purposes of comparison, images were proportionally scaled. The boundary of X-Gal staining marks dermis derived from lateral plate versus dermis derived from mesoderm (the lateral somitic frontier) and lies more dorsal than the at/at pigmentation boundary. + +(B) Quantitation of mean (± SEM) dorsal pigmentation area (n = 5) and somite-derived dermis area (n = 3) shows a significant difference (p < 0.005, t-test). + +(C) RNA in situ hybridization showing that Tbx15 expression at E11.5 is complementary to En1 expression on the flank (scale bars = 200 μm). The arrow indicates the boundary between the expression domains of the two genes. + +Figure 9 + +Model for Acquisition of Dorsoventral Patterning in the Trunk and the Role of Tbx15 + +(A) A tricolor pigmentation pattern is generated by the combination of distinct mechanisms that affect distribution of Agouti mRNA and histochemical staining for melanocytes; effects of the latter mechanism by itself are evident in ae/ae mice (see Figure 1). In at/at mice, reduced hair melanocyte activity and high levels of Agouti mRNA in the ventrum lead to a cream color; as melanocyte activity gradually increases towards the dorsum, a lateral stripe is apparent on the flank. The distributions of Agouti mRNA and histochemical staining for melanocytes are both affected by Tbx15 and are externally evident by a widening of the lateral stripe and an increased proportion of total skin occupied by the cream-colored area. + +(B) The lateral yellow stripe in at/at mice lies at the same level as the limb dorsoventral boundary. As described in the text, we propose that distinct dorsoventral compartments in ectoderm of the trunk provide an instructional cue to the mesoderm, leading to expression of Tbx15 in dorsal trunk mesenchyme and acquisition of dorsal dermis character. In the absence of Tbx15, dorsal mesenchyme assumes ventral characteristics instead. + +Footnotes + +Conflicts of interest. The authors have declared that no conflicts of interest exist. + +Author contributions. SIC and GSB conceived and designed the experiments. SIC, CDVR, CC, AR, and SK performed the experiments. SIC, CDVR, CC, SK, and FM analyzed the data. YC and AR contributed reagents/materials/analysis tools. SIC and GSB wrote the paper. + +Academic Editor: Brigid L. M. Hogan, Duke University Medical Center diff --git a/src/ontogpt/evaluation/craft/database/all/15005800.ann b/src/ontogpt/evaluation/craft/database/all/15005800.ann new file mode 100644 index 000000000..eb584f996 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15005800.ann @@ -0,0 +1,1276 @@ +T1 GO:0007565 5 16 gestational +T2 UBERON:0002048 17 21 lung +T3 NCBITaxon:10088 38 43 mouse +T4 CHEBI:15440 119 127 squalene +T5 CHEBI:16113 128 139 cholesterol +T6 GO:0006695 128 152 cholesterol biosynthesis +T7 NCBITaxon:40674 170 179 mammalian +T8 UBERON:0000922 180 189 embryonic +T9 GO:0009790 180 201 embryonic development +T10 NCBITaxon:10088 212 216 mice +T11 NCBITaxon:9606 293 298 human +T12 http://purl.obolibrary.org/obo/MONDO_0000001 299 306 disease +T13 http://purl.obolibrary.org/obo/MONDO_0010035 310 336 Smith-Lemli-Opitz syndrome +T14 GO:0016265 338 341 die +T15 GO:0007567 361 366 birth +T16 GO:0016265 454 459 death +T17 UBERON:0001004 477 488 respiratory +T18 http://purl.obolibrary.org/obo/MONDO_0021113 477 496 respiratory failure +T19 UBERON:0002048 518 527 pulmonary +T20 CHEBI:16113 605 616 cholesterol +T21 UBERON:0002048 631 635 lung +T22 GO:0030324 631 647 lung development +T23 NCBITaxon:10088 657 661 mice +T24 UBERON:0000922 746 753 embryos +T25 UBERON:0002048 783 788 lungs +T26 UBERON:0002048 820 824 lung +T27 UBERON:0000922 868 875 embryos +T28 UBERON:0002048 899 903 Lung +T29 GO:0060441 899 927 Lung branching morphogenesis +T30 UBERON:0000116 1002 1010 saccular +T31 UBERON:0000922 1051 1058 embryos +T32 UBERON:0003215 1092 1100 alveolar +T33 GO:0008283 1144 1162 cell proliferation +T34 GO:0030154 1172 1190;1218 1223 differentiation of ... cells +T35 CL:0002062 1191 1223 type I alveolar epithelial cells +T36 CL:0002062 1191 1197;1225 1229 type I ... 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http://purl.obolibrary.org/obo/MONDO_0010035 3083 3109 Smith-Lemli-Opitz syndrome +T89 http://purl.obolibrary.org/obo/MONDO_0010035 3111 3115 SLOS +T90 http://purl.obolibrary.org/obo/MONDO_0000001 3162 3170 disorder +T91 CHEBI:17759 3229 3249 7-dehydrocholesterol +T92 CHEBI:16113 3293 3304 cholesterol +T93 GO:0006695 3293 3317 cholesterol biosynthesis +T94 http://purl.obolibrary.org/obo/MONDO_0002254 3354 3363 syndromes +T95 CHEBI:15440 3394 3402 squalene +T96 CHEBI:16113 3403 3414 cholesterol +T97 GO:0006695 3403 3427 cholesterol biosynthesis +T98 http://purl.obolibrary.org/obo/MONDO_0011217 3445 3460 desmosterolosis +T99 http://purl.obolibrary.org/obo/MONDO_0011816 3462 3477 lathosterolosis +T100 GO:0000805 3479 3480 X +T101 http://purl.obolibrary.org/obo/MONDO_0010556 3479 3514 X-linked chondrodysplasia punctanta +T102 http://purl.obolibrary.org/obo/MONDO_0010621 3519 3533 CHILD syndrome +T103 SO:0000704 3574 3581 Genetic +T104 NCBITaxon:10088 3651 3655 mice +T105 SO:0000704 3681 3685 gene +T106 CHEBI:16113 3714 3725 cholesterol +T107 CHEBI:16113 3765 3776 cholesterol +T108 CHEBI:17759 3787 3807 7-dehydrocholesterol +T109 CHEBI:17759 3809 3813 7DHC +T110 UBERON:0000922 3828 3837 embryonic +T111 GO:0016265 3933 3937 died +T112 GO:0007567 3956 3961 birth +T113 NCBITaxon:10088 3973 3978 mouse +T114 http://purl.obolibrary.org/obo/MONDO_0010035 3979 3983 SLOS +T115 SO:0000147 4014 4019 exons +T116 SO:0000852 4029 4041 part of exon +T117 NCBITaxon:39107 4051 4057 murine +T118 SO:0000704 4064 4068 gene +T119 UBERON:0002048 4184 4188 lung +T120 GO:0030324 4184 4200 lung development +T121 UBERON:0002299 4212 4225 lung alveolar +T122 UBERON:0001004 4374 4385 respiratory +T123 GO:0016265 4415 4420 death +T124 NCBITaxon:10088 4460 4464 mice +T125 NCBITaxon:9606 4480 4485 human +T126 http://purl.obolibrary.org/obo/MONDO_0010035 4486 4490 SLOS +T127 GO:0007567 4556 4561 natal +T128 UBERON:0001004 4577 4588 respiratory +T129 http://purl.obolibrary.org/obo/MONDO_0021113 4577 4596 respiratory failure +T130 http://purl.obolibrary.org/obo/MONDO_0010035 4625 4629 SLOS +T131 UBERON:0002048 4657 4661 lung +T132 http://purl.obolibrary.org/obo/MONDO_0010035 4706 4710 SLOS +T133 UBERON:0002048 4731 4740 pulmonary +T134 UBERON:0002048 4762 4771 pulmonary +T135 UBERON:0002012 4785 4803 pulmonary arteries +T136 UBERON:0001737 4818 4827 laryngeal +T137 UBERON:0003126 4832 4840 tracheal +T138 GO:0060438 4832 4852 tracheal development +T139 NCBITaxon:40674 4860 4869 Mammalian +T140 UBERON:0002048 4870 4874 lung +T141 GO:0007585 4942 4954 gas exchange +T142 GO:0007567 4978 4983 birth +T143 UBERON:0002048 5061 5065 Lung +T144 GO:0030324 5061 5077 Lung development +T145 GO:0061138 5113 5150 branching morphogenesis of epithelium +T146 UBERON:0000483 5140 5150 epithelium +T147 UBERON:0005169 5152 5164 interstitial +T148 GO:0001570 5187 5201 vasculogenesis +T149 GO:0030154 5203 5227 cellular differentiation +T150 NCBITaxon:10088 5288 5293 mouse +T151 UBERON:0002048 5294 5298 lung +T152 GO:0030324 5294 5310 lung development +T153 UBERON:0002048 5375 5379 lung +T154 UBERON:0000116 5420 5428 saccular +T155 UBERON:0003215 5433 5441 alveolar +T156 http://purl.obolibrary.org/obo/MONDO_0010035 5550 5554 SLOS +T157 UBERON:0000922 5602 5611 embryonic +T158 GO:0009790 5602 5623 embryonic development +T159 CHEBI:16113 5629 5640 cholesterol +T160 PR:000014841 5702 5716 sonic hedgehog +T161 GO:0007224 5708 5716;5723 5740 hedgehog ... signaling cascade +T162 PR:000014841 5718 5721 Shh +T163 UBERON:0002048 5782 5786 lung +T164 GO:0030324 5782 5798 lung development +T165 PR:000014841 5816 5819 Shh +T166 UBERON:0002048 5848 5852 lung +T167 GO:0001763 5885 5908 branching morphogenesis +T168 UBERON:0000115 5962 5977 lung epithelium +T169 GO:0010467 5988 5998 expression +T170 PR:000014841 6002 6005 Shh +T171 UBERON:0000115 6020 6035 lung epithelium +T172 UBERON:0003215 6074 6081 alveoli +T173 UBERON:0005169 6101 6120 interstitial tissue +T174 GO:0050673 6144 6160;6166 6176;6192 6197 proliferation of ... epithelial ... cells +T175 GO:0010463 6144 6160;6181 6197 proliferation of ... mesenchyme cells +T176 UBERON:0000483 6166 6176 epithelial +T177 CL:0000066 6166 6176;6192 6197 epithelial ... cells +T178 UBERON:0003104 6181 6191 mesenchyme +T179 CL:0000134 6181 6197 mesenchyme cells +T180 NCBITaxon:10088 6204 6208 Mice +T181 PR:000014841 6214 6217 Shh +T182 GO:0010467 6222 6232 expression +T183 GO:0016265 6233 6237 died +T184 GO:0007567 6259 6264 birth +T185 UBERON:0001004 6284 6295 respiratory +T186 http://purl.obolibrary.org/obo/MONDO_0021113 6284 6303 respiratory failure +T187 CHEBI:16113 6345 6356 cholesterol +T188 GO:0006695 6345 6369 cholesterol biosynthesis +T189 CHEBI:35222 6382 6392 inhibitors +T190 PR:000014841 6435 6438 Shh +T191 GO:0009790 6463 6476 embryogenesis +T192 CHEBI:15889 6497 6503 sterol +T193 GO:0009294 6553 6564 transfected +T194 PR:000014841 6565 6568 Shh +T195 CL:0000010 6572 6580;6591 6596 cultured ... cells +T196 NCBITaxon:40674 6581 6590 mammalian +T197 UBERON:0002048 6645 6649 lung +T198 GO:0030324 6645 6661 lung development +T199 UBERON:0000922 6674 6681 embryos +T200 GO:0007567 6696 6701 birth +T201 CHEBI:16113 6727 6738 cholesterol +T202 UBERON:0002048 6799 6803 lung +T203 GO:0060441 6799 6827 lung branching morphogenesis +T204 GO:0007565 6835 6846 gestational +T205 UBERON:0000116 6880 6893 lung saccular +T206 GO:0060430 6880 6905 lung saccular development +T207 UBERON:0003914 6957 6975 epithelial tubules +T208 GO:0030154 7010 7028;7057 7062 Differentiation of ... cells +T209 CL:0002063 7029 7062 type II alveolar epithelial cells +T210 CL:0002063 7029 7036;7064 7068 type II ... AECs +T211 UBERON:0003215 7037 7045 alveolar +T212 UBERON:0000483 7046 7056 epithelial +T213 CL:0002062 7109 7120 type I AECs +T214 CHEBI:16113 7155 7166 cholesterol +T215 GO:0007565 7206 7217 gestational +T216 UBERON:0002048 7218 7222 lung +T217 CL:0002062 7286 7297 type I AECs +T218 PR:000014841 7423 7426 Shh +T219 UBERON:0002048 7497 7501 lung +T220 UBERON:0000922 7525 7532 embryos +T221 GO:0010467 7546 7556 expression +T222 PR:000014841 7560 7563 Shh +T223 PR:000014841 7626 7629 Shh +T224 GO:0007565 7689 7698 gestation +T225 UBERON:0000922 7717 7724 embryos +T226 NCBITaxon:10088 7735 7739 mice +T227 GO:0007567 7745 7749 born +T228 GO:0016265 7832 7836 died +T229 GO:0007567 7873 7878 birth +T230 GO:0007567 7888 7893 birth +T231 GO:0007565 8401 8412 gestational +T232 UBERON:0000922 8451 8458 embryos +T233 UBERON:0000922 8473 8480 embryos +T234 GO:0007565 8517 8526 gestation +T235 UBERON:0000922 8653 8660 embryos +T236 UBERON:0000922 8761 8768 embryos +T237 UBERON:0000922 8820 8827 embryos +T238 GO:0007565 9152 9163 gestational +T239 UBERON:0000922 9190 9197 embryos +T240 UBERON:0000922 9244 9251 embryos +T241 UBERON:0000922 9341 9348 embryos +T242 UBERON:0000922 9490 9497 embryos +T243 UBERON:0000323 9904 9926 embryos at late stages +T244 UBERON:0002048 9937 9946 pulmonary +T245 UBERON:0000116 9971 9979 saccular +T246 UBERON:0002048 9997 10001 lung +T247 UBERON:0002048 10022 10026 lung +T248 UBERON:0002048 10168 10172 lung +T249 UBERON:0002048 10199 10204 lungs +T250 UBERON:0000922 10260 10267 embryos +T251 GO:0007567 10296 10301 birth +T252 UBERON:0002048 10356 10360 lung +T253 NCBITaxon:10088 10372 10377 mouse +T254 UBERON:0000922 10378 10385 embryos +T255 UBERON:0002048 10442 10446 lung +T256 UBERON:0000922 10466 10475 Embryonic +T257 UBERON:0002048 10482 10486 lung +T258 UBERON:0000922 10630 10637 embryos +T259 UBERON:0002048 10794 10799 lungs +T260 GO:0007567 10939 10944 birth +T261 UBERON:0009912 10960 10965 lobar +T262 UBERON:0002048 10981 10986 lungs +T263 NCBITaxon:33208 11001 11008 animals +T264 UBERON:0009856 11050 11053 sac +T265 UBERON:0002186 11202 11204 br +T266 UBERON:0002186 11206 11216 bronchiole +T267 UBERON:0001043 11218 11219 e +T268 UBERON:0001043 11221 11230 esophagus +T269 UBERON:0002012 11232 11234 pa +T270 UBERON:0002012 11236 11252 pulmonary artery +T271 UBERON:0002078 11254 11256 ra +T272 UBERON:0002078 11258 11270 right atrium +T273 UBERON:0002079 11272 11274 la +T274 UBERON:0002079 11276 11287 left atrium +T275 UBERON:0002080 11289 11291 rv +T276 UBERON:0002080 11293 11308 right ventricle +T277 UBERON:0002084 11310 11312 lv +T278 UBERON:0002084 11314 11328 left ventricle +T279 UBERON:0002048 11347 11352 lungs +T280 GO:0030324 11408 11430 organogenesis of lungs +T281 UBERON:0002048 11425 11430 lungs +T282 UBERON:0006518 11468 11484 right lung lobes +T283 UBERON:0009912 11503 11507 lobe +T284 UBERON:0000948 11521 11526 heart +T285 GO:0007567 11571 11576 birth +T286 UBERON:0001062 11651 11658 anatomy +T287 UBERON:0000922 11700 11707 embryos +T288 GO:0007567 11793 11798 birth +T289 UBERON:0002048 11831 11835 lung +T290 UBERON:0001062 11875 11883 anatomic +T291 GO:0048856 11875 11895 anatomic development +T292 UBERON:0000948 11931 11938 cardiac +T293 http://purl.obolibrary.org/obo/MONDO_0005453 11931 11946 cardiac defects +T294 GO:0007567 12100 12105 birth +T295 UBERON:0000948 12134 12141 cardiac +T296 UBERON:0002048 12310 12315 lungs +T297 UBERON:0002048 12480 12485 lungs +T298 UBERON:0000922 12500 12507 embryos +T299 UBERON:0000116 12530 12538 saccular +T300 UBERON:0003104 12565 12575 mesenchyme +T301 UBERON:0002048 12598 12603 lungs +T302 UBERON:0002048 12628 12633 Lungs +T303 NCBITaxon:33208 12647 12654 animals +T304 UBERON:0009856 12699 12702 sac +T305 UBERON:0003215 12742 12749 alveoli +T306 UBERON:0002048 12787 12792 lungs +T307 UBERON:0000922 12807 12814 embryos +T308 UBERON:0003914 12829 12847 epithelial tubules +T309 UBERON:0002048 12970 12974 lung +T310 UBERON:0002048 13014 13019 lungs +T311 UBERON:0009856 13073 13076 sac +T312 UBERON:0002048 13128 13133 lungs +T313 UBERON:0002048 13154 13159 lungs +T314 UBERON:0005169 13179 13191 Interstitial +T315 UBERON:0003104 13192 13202 mesenchyme +T316 UBERON:0002048 13228 13233 lungs +T317 UBERON:0002048 13273 13278 lungs +T318 UBERON:0003215 13305 13312 alveoli +T319 GO:0007567 13373 13378 birth +T320 UBERON:0002048 13416 13421 lungs +T321 UBERON:0000922 13433 13442 embryonic +T322 UBERON:0002048 13469 13473 lung +T323 CHEBI:51686 13496 13497 H +T324 GO:0007565 13526 13537 gestational +T325 UBERON:0000922 13747 13754 embryos +T326 UBERON:0000922 13809 13816 embryos +T327 UBERON:0003104 13987 13997 mesenchyme +T328 UBERON:0002048 14051 14056 lungs +T329 UBERON:0002048 14101 14106 lungs +T330 UBERON:0000116 14119 14127 saccular +T331 UBERON:0003104 14148 14158 mesenchyme +T332 UBERON:0002048 14220 14225 lungs +T333 UBERON:0003215 14264 14271 alveoli +T334 UBERON:0003104 14285 14295 mesenchyme +T335 UBERON:0000922 14346 14353 embryos +T336 GO:0007567 14427 14432 birth +T337 UBERON:0002048 14434 14439 lungs +T338 UBERON:0000060 14496 14502 walled +T339 UBERON:0003104 14503 14513 mesenchyme +T340 UBERON:0000483 14559 14569 epithelium +T341 UBERON:0003104 14571 14572 m +T342 UBERON:0003104 14574 14584 mesenchyme +T343 UBERON:0002012 14586 14588 PA +T344 UBERON:0002012 14590 14606 pulmonary artery +T345 UBERON:0003215 14608 14609 a +T346 UBERON:0003215 14615 14622 alveoli +T347 UBERON:0002185 14624 14625 b +T348 UBERON:0002185 14627 14634 bronchi +T349 UBERON:0002048 14679 14683 lung +T350 UBERON:0009856 14693 14696 sac +T351 UBERON:0002048 14707 14712 lungs +T352 GO:0007565 14724 14735 gestational +T353 UBERON:0009856 14784 14787 sac +T354 GO:0008283 14873 14891 cell proliferation +T355 GO:0007565 14900 14911 gestational +T356 UBERON:0002048 14921 14926 lungs +T357 CHEBI:16113 14952 14963 cholesterol +T358 GO:0006695 14952 14976 cholesterol biosynthesis +T359 GO:0008283 15002 15020 cell proliferation +T360 GO:0051301 15002 15006;15021 15029 cell ... division +T361 GO:0008219 15033 15043 cell death +T362 GO:0008283 15045 15063 Cell proliferation +T363 PR:000012421 15099 15103 PCNA +T364 CHEBI:472552 15116 15120 BrdU +T365 PR:000012421 15141 15145 PCNA +T366 CHEBI:472552 15150 15154 BrdU +T367 UBERON:0002048 15190 15195 lungs +T368 GO:0007565 15205 15216 gestational +T369 PR:000012421 15321 15325 PCNA +T370 CHEBI:472552 15330 15334 BrdU +T371 UBERON:0002048 15369 15374 lungs +T372 UBERON:0000922 15387 15394 embryos +T373 GO:0007565 15403 15414 gestational +T374 PR:000012421 15472 15476 PCNA +T375 UBERON:0000948 15513 15519 hearts +T376 UBERON:0002048 15563 15568 lungs +T377 GO:0051301 15611 15624 cell division +T378 GO:0010467 15652 15662 expression +T379 UBERON:0000115 15670 15683;15693 15697 epithelium of ... lung +T380 GO:0006915 15832 15846 cell apoptosis +T381 UBERON:0002048 15898 15903 lungs +T382 GO:0007565 15961 15972 gestational +T383 GO:0008283 15981 15999 cell proliferation +T384 GO:0051301 15981 15985;16000 16008 cell ... division +T385 UBERON:0000922 16034 16041 embryos +T386 GO:0008219 16062 16072 cell death +T387 GO:0008283 16103 16121 cell proliferation +T388 GO:0051301 16123 16136 cell division +T389 GO:0008219 16141 16151 cell death +T390 GO:0008283 16153 16171 Cell proliferation +T391 PR:000012421 16173 16177 PCNA +T392 CHEBI:472552 16201 16205 BrdU +T393 GO:0051301 16230 16243 cell division +T394 GO:0008219 16277 16287 cell death +T395 UBERON:0002048 16347 16351 lung +T396 GO:0007565 16379 16390 gestational +T397 PR:000012421 16404 16408 PCNA +T398 CHEBI:472552 16413 16417 BrdU +T399 UBERON:0000483 16440 16450 epithelial +T400 UBERON:0003104 16455 16466 mesenchymal +T401 PR:000012421 16518 16522 PCNA +T402 CHEBI:472552 16537 16541 BrdU +T403 UBERON:0002048 16586 16591 lungs +T404 UBERON:0002048 16628 16633 lungs +T405 PR:000012421 16659 16663 PCNA +T406 CHEBI:472552 16678 16682 BrdU +T407 UBERON:0002048 16698 16703 lungs +T408 PR:000012421 16752 16756 PCNA +T409 UBERON:0001133 16769 16783 cardiac muscle +T410 UBERON:0000922 16816 16823 embryos +T411 UBERON:0002048 16920 16924 lung +T412 UBERON:0002048 17045 17050 lungs +T413 UBERON:0002048 17069 17074 lungs +T414 UBERON:0002048 17146 17151 lungs +T415 UBERON:0000483 17245 17255 epithelial +T416 CHEBI:15889 17326 17332 Sterol +T417 UBERON:0002048 17355 17360 lungs +T418 NCBITaxon:10088 17380 17384 mice +T419 CHEBI:17759 17404 17409 7-DHC +T420 CHEBI:16113 17440 17451 cholesterol +T421 NCBITaxon:10088 17504 17508 mice +T422 CHEBI:16113 17542 17553 cholesterol +T423 UBERON:0002048 17572 17577 lungs +T424 NCBITaxon:10088 17590 17594 mice +T425 UBERON:0000479 17630 17636 tissue +T426 CHEBI:15889 17637 17644 sterols +T427 CHEBI:16113 17712 17723 cholesterol +T428 UBERON:0002048 17731 17736 lungs +T429 UBERON:0000922 17750 17757 embryos +T430 UBERON:0000479 17769 17775 tissue +T431 UBERON:0000479 17798 17804 tissue +T432 CHEBI:16113 17813 17824 cholesterol +T433 UBERON:0002048 17840 17845 lungs +T434 UBERON:0000922 17860 17867 embryos +T435 GO:0007565 17928 17939 gestational +T436 CHEBI:17759 17993 17998 7-DHC +T437 MOP:0000789 18051 18064 isomerization +T438 CHEBI:15889 18088 18095 sterols +T439 GO:0007567 18099 18104 birth +T440 UBERON:0002048 18121 18126 lungs +T441 NCBITaxon:10088 18145 18149 mice +T442 NCBITaxon:10088 18206 18210 mice +T443 CHEBI:15889 18224 18230 Sterol +T444 UBERON:0000922 18243 18252 embryonic +T445 UBERON:0002048 18253 18258 lungs +T446 CHEBI:15889 18260 18266 Sterol +T447 CHEBI:16113 18287 18298 cholesterol +T448 CHEBI:17759 18316 18320 7DHC +T449 UBERON:0002048 18373 18378 lungs +T450 UBERON:0000922 18419 18426 embryos +T451 UBERON:0002048 18482 18487 lungs +T452 CHEBI:15889 18520 18527 sterols +T453 UBERON:0002048 18565 18570 lungs +T454 CHEBI:16113 18599 18610 cholesterol +T455 CHEBI:15889 18698 18705 sterols +T456 CHEBI:17759 18707 18711 7DHC +T457 GO:0007565 18746 18755 gestation +T458 CHEBI:16113 18758 18769 Cholesterol +T459 CHEBI:26764 18797 18812 steroid hormone +T460 GO:0042446 18805 18822 hormone synthesis +T461 UBERON:0002369 18830 18844 adrenal glands +T462 UBERON:0002369 18846 18853 Adrenal +T463 http://purl.obolibrary.org/obo/MONDO_0010035 18914 18918 SLOS +T464 UBERON:0002048 18958 18962 lung +T465 UBERON:0012101 18978 18987 perinatal +T466 GO:0007567 18982 18987 natal +T467 CHEBI:35341 19004 19012 Steroids +T468 CHEBI:50858 19031 19046 corticosteroids +T469 UBERON:0002048 19066 19070 lung +T470 CHEBI:35195 19125 19135 surfactant +T471 UBERON:0001969 19167 19173 plasma +T472 CHEBI:16827 19174 19188 corticosterone +T473 NCBITaxon:10088 19202 19206 mice +T474 GO:0007567 19222 19227 birth +T475 NCBITaxon:10088 19242 19246 mice +T476 CHEBI:16827 19251 19265 corticosterone +T477 NCBITaxon:10088 19363 19367 mice +T478 CHEBI:16827 19379 19393 corticosterone +T479 NCBITaxon:10088 19413 19417 mice +T480 NCBITaxon:10088 19485 19489 mice +T481 GO:0016265 19495 19500 dying +T482 CHEBI:16827 19592 19606 corticosterone +T483 CHEBI:16827 19683 19697 corticosterone +T484 UBERON:0002048 19712 19716 lung +T485 UBERON:0002048 19741 19746 lungs +T486 CHEBI:35195 19751 19761 surfactant +T487 GO:0010467 19770 19780 expression +T488 CHEBI:35195 19782 19792 Surfactant +T489 PR:000014774 19782 19802 Surfactant protein C +T490 UBERON:0002048 19844 19848 lung +T491 UBERON:0000922 19887 19894 embryos +T492 GO:0007565 19923 19934 gestational +T493 PR:000015945 19981 19985 SP-A +T494 PR:000014773 19987 19991 SP-B +T495 PR:000014774 19993 19997 SP-C +T496 PR:000014775 20001 20005 SP-D +T497 NCBITaxon:10088 20075 20079 mice +T498 CHEBI:35195 20113 20123 surfactant +T499 GO:0010467 20132 20142 expression +T500 CHEBI:35195 20191 20201 Surfactant +T501 GO:0010467 20210 20220 expression +T502 UBERON:0002048 20224 20229 lungs +T503 PR:000014774 20277 20281 SP-C +T504 UBERON:0002048 20285 20290 lungs +T505 UBERON:0000922 20350 20357 embryos +T506 PR:000014774 20405 20409 SP-C +T507 UBERON:0002048 20463 20468 lungs +T508 UBERON:0003914 20514 20532 epithelial tubules +T509 UBERON:0002048 20545 20550 lungs +T510 GO:0010467 20556 20565 expressed +T511 PR:000014774 20566 20570 SP-C +T512 CL:0002063 20621 20633 type II AECs +T513 UBERON:0002048 20817 20822 lungs +T514 PR:000015945 20827 20831 SP-A +T515 PR:000014773 20833 20837 SP-B +T516 PR:000014774 20839 20843 SP-C +T517 PR:000014775 20848 20852 SP-D +T518 GO:0010467 20853 20863 expression +T519 GO:0010467 20907 20917 expression +T520 CHEBI:35195 20948 20958 surfactant +T521 UBERON:0002048 21047 21052 lungs +T522 GO:0030154 21069 21087;21115 21120 differentiation of ... cells +T523 CL:0002062 21088 21120 type I alveolar epithelial cells +T524 CL:0002062 21088 21094;21122 21126 type I ... AECs +T525 UBERON:0003215 21095 21103 alveolar +T526 UBERON:0000483 21104 21114 epithelial +T527 UBERON:0002048 21150 21154 lung +T528 UBERON:0000922 21168 21175 embryos +T529 UBERON:0000116 21189 21197 saccular +T530 CL:0002062 21223 21234 type I AECs +T531 CL:0002063 21243 21251;21273 21285 cuboidal ... type II AECs +T532 CHEBI:35195 21252 21262 surfactant +T533 UBERON:0000116 21341 21349 saccules +T534 UBERON:0002048 21382 21387 lungs +T535 UBERON:0000116 21404 21412 saccular +T536 UBERON:0012274 21444 21463 columnar epithelium +T537 CL:0002062 21486 21492 type I +T538 UBERON:0003914 21554 21572 epithelial tubules +T539 CL:0002062 21614 21625 type I AECs +T540 GO:0010467 21672 21682 expression +T541 PR:000012516 21686 21689 T1α +T542 GO:0016324 21694 21709 apical membrane +T543 UBERON:0002048 21728 21732 lung +T544 CL:0002062 21728 21744 lung type I AECs +T545 PR:000004185 21804 21808 AQP5 +T546 CL:0002062 21818 21828 type I AEC +T547 UBERON:0002048 21871 21876 lungs +T548 UBERON:0002048 21907 21912 lungs +T549 PR:000009921 21943 21950 Megalin +T550 SO:0000704 21994 21998 gene +T551 GO:0010467 22017 22026 expressed +T552 UBERON:0000922 22030 22039 embryonic +T553 CL:0002321 22030 22039;22051 22056 embryonic ... cells +T554 UBERON:0000483 22040 22050 epithelial +T555 CL:0000066 22040 22056 epithelial cells +T556 GO:0006897 22080 22089 endocytic +T557 PR:000014841 22090 22093 Shh +T558 PR:000009921 22109 22116 Megalin +T559 GO:0010467 22128 22137 expressed +T560 UBERON:0000483 22152 22162 epithelium +T561 UBERON:0002048 22187 22192 lungs +T562 CL:0002062 22205 22216 type I AECs +T563 UBERON:0000116 22238 22246 saccules +T564 PR:000009921 22278 22285 megalin +T565 UBERON:0000116 22322 22330 saccules +T566 UBERON:0000922 22343 22352 embryonic +T567 UBERON:0002048 22353 22358 lungs +T568 GO:0010467 22364 22374 expression +T569 CL:0002062 22464 22475 type I AECs +T570 UBERON:0002048 22508 22513 lungs +T571 CL:0002062 22546 22557 type I AECs +T572 UBERON:0000116 22593 22601 saccules +T573 CL:0002062 22657 22663;22686 22702 type I ... epithelial cells +T574 CL:0002062 22665 22667;22686 22702 t1 ... epithelial cells +T575 CL:0002063 22673 22680;22686 22702 type II ... epithelial cells +T576 CL:0002063 22682 22684;22686 22702 t2 ... epithelial cells +T577 UBERON:0000483 22686 22696 epithelial +T578 UBERON:0002048 22756 22761 lungs +T579 UBERON:0000116 22784 22792 saccules +T580 UBERON:0012274 22802 22821 columnar epithelial +T581 CL:0000066 22811 22827 epithelial cells +T582 UBERON:0002048 22903 22907 lung +T583 CL:0002062 22956 22967 type I cell +T584 PR:000012516 22976 22979 T1α +T585 PR:000004185 22984 22988 AQP5 +T586 UBERON:0000483 23030 23040 epithelial +T587 CL:0000066 23030 23046 epithelial cells +T588 UBERON:0002048 23060 23065 lungs +T589 UBERON:0002048 23141 23146 lungs +T590 PR:000012516 23148 23151 T1α +T591 PR:000004185 23156 23160 AQP5 +T592 PR:000009921 23227 23234 Megalin +T593 GO:0010467 23235 23245 expression +T594 PR:000009921 23247 23250 Meg +T595 UBERON:0001005 23272 23278 airway +T596 UBERON:0000483 23279 23289 epithelial +T597 CL:0000066 23279 23295 epithelial cells +T598 UBERON:0002186 23317 23327 bronchiole +T599 UBERON:0000483 23328 23338 epithelial +T600 CL:0000066 23328 23344 epithelial cells +T601 UBERON:0002048 23369 23374 lungs +T602 PR:000009921 23432 23439 megalin +T603 UBERON:0000483 23449 23459 epithelial +T604 CL:0000066 23449 23465 epithelial cells +T605 UBERON:0000116 23497 23504 sacculi +T606 UBERON:0002048 23558 23563 lungs +T607 UBERON:0000483 23591 23601 epithelial +T608 CL:0000066 23591 23607 epithelial cells +T609 PR:000009921 23630 23637 megalin +T610 GO:0042571 23638 23648 antibodies +T611 UBERON:0002048 23661 23666 lungs +T612 UBERON:0002048 23863 23868 lungs +T613 UBERON:0001981 23908 23920 blood vessel +T614 GO:0001568 23908 23932 blood vessel development +T615 UBERON:0002048 23937 23941 lung +T616 GO:0060425 23937 23964 lung structural development +T617 UBERON:0002048 23975 23979 lung +T618 GO:0030324 23975 23993 lung developmental +T619 UBERON:0013141 24008 24021 capillary bed +T620 UBERON:0003215 24075 24083 alveolar +T621 GO:0007585 24084 24096 gas-exchange +T622 UBERON:0003104 24113 24123 mesenchyme +T623 UBERON:0013141 24139 24153 capillary beds +T624 CL:0010003 24162 24166 AECs +T625 UBERON:0002048 24185 24190 lungs +T626 GO:0007565 24199 24210 gestational +T627 UBERON:0003104 24245 24255 mesenchyme +T628 UBERON:0002048 24346 24350 lung +T629 UBERON:0001981 24386 24398 blood vessel +T630 GO:0001568 24386 24410 blood vessel development +T631 PR:000027209 24435 24458 α-isoform of caveolin-1 +T632 SO:0001060 24437 24444 isoform +T633 PR:000027209 24460 24466 cav-1α +T634 CL:0000233 24472 24480 platelet +T635 PR:000001904 24472 24516 platelet endothelial cell adhesion molecular +T636 UBERON:0001986 24481 24492 endothelial +T637 CL:0000115 24481 24497 endothelial cell +T638 PR:000001904 24518 24525 PECAM-1 +T639 UBERON:0002048 24582 24587 lungs +T640 UBERON:0002048 24607 24611 lung +T641 PR:000027209 24613 24619 cav-1α +T642 GO:0010467 24623 24632 expressed +T643 UBERON:0001915 24649 24663;24675 24684;24695 24702 endothelium of ... capillary ... vessels +T644 UBERON:0004638 24649 24663;24689 24702 endothelium of ... blood vessels +T645 PR:000027209 24704 24710 Cav-1α +T646 CL:0002062 24755 24766 type I AECs +T647 CL:0002063 24812 24824 type II AECs +T648 UBERON:0000483 24850 24860 epithelium +T649 UBERON:0003104 24865 24875 mesenchyme +T650 PR:000027209 24894 24900 cav-1α +T651 PR:000027209 24915 24921 cav-1α +T652 UBERON:0001981 24953 24966 blood vessels +T653 UBERON:0001982 24971 24982 capillaries +T654 UBERON:0002048 25044 25049 lungs +T655 PR:000027209 25076 25082 Cav-1α +T656 UBERON:0000116 25166 25174 saccular +T657 UBERON:0002048 25192 25197 lungs +T658 UBERON:0002048 25237 25242 lungs +T659 UBERON:0002049 25265 25281 vascular network +T660 UBERON:0000060 25326 25331 walls +T661 UBERON:0003215 25350 25357 alveoli +T662 CL:0002062 25387 25398 type I AECs +T663 PR:000027209 25456 25462 cav-1α +T664 UBERON:0002048 25475 25480 lungs +T665 UBERON:0000922 25495 25502 embryos +T666 UBERON:0002048 25537 25546 pulmonary +T667 UBERON:0013141 25547 25560 capillary bed +T668 PR:000027209 25597 25603 cav-1α +T669 UBERON:0002048 25630 25635 lungs +T670 UBERON:0003104 25672 25682 mesenchyme +T671 UBERON:0003914 25707 25725 epithelial tubules +T672 UBERON:0000483 25737 25747 epithelial +T673 CL:0000066 25737 25753 epithelial cells +T674 PR:000001904 25818 25825 PECAM-1 +T675 UBERON:0002048 25866 25870 lung +T676 GO:0007565 25906 25917 gestational +T677 UBERON:0000922 25944 25951 embryos +T678 PR:000027209 26021 26032 caveolin-1α +T679 PR:000001904 26037 26044 PECAM-1 +T680 UBERON:0002048 26048 26052 lung +T681 PR:000027209 26063 26069 Cav-1α +T682 UBERON:0000483 26150 26160 epithelium +T683 UBERON:0003104 26165 26175 mesenchyme +T684 PR:000027209 26194 26200 cav-1α +T685 UBERON:0001981 26222 26235 blood vessels +T686 UBERON:0001982 26257 26268 capillaries +T687 PR:000027209 26323 26329 cav-1α +T688 UBERON:0002048 26400 26405 lungs +T689 PR:000027209 26424 26430 Cav-1α +T690 UBERON:0002186 26465 26475 bronchiole +T691 UBERON:0000483 26480 26490 epithelial +T692 UBERON:0001005 26546 26552 airway +T693 UBERON:0000483 26553 26563 epithelium +T694 PR:000027209 26578 26584 cav-1α +T695 UBERON:0001982 26648 26657 capillary +T696 GO:0010467 26700 26710 expression +T697 UBERON:0002048 26723 26728 lungs +T698 UBERON:0000922 26743 26750 embryos +T699 UBERON:0000922 26786 26793 embryos +T700 UBERON:0013141 26813 26826 capillary bed +T701 UBERON:0002048 26849 26854 lungs +T702 UBERON:0002048 26899 26904 lungs +T703 PR:000027209 26911 26917 cav-1α +T704 PR:000001904 26922 26929 PECAM-1 +T705 UBERON:0002049 26965 26981 vascular network +T706 UBERON:0000116 27004 27012 saccules +T707 UBERON:0000483 27064 27074 epithelial +T708 CL:0000066 27064 27080 epithelial cells +T709 UBERON:0002048 27192 27197 lungs +T710 UBERON:0016405 27207 27218;27244 27246;27260 27265 capillaries ... of ... lungs +T711 UBERON:0003104 27311 27321 mesenchyme +T712 UBERON:0003914 27345 27363 epithelial tubules +T713 UBERON:0000483 27377 27387 epithelial +T714 CL:0000066 27377 27393 epithelial cells +T715 PR:000014841 27500 27514 sonic hedgehog +T716 GO:0007224 27506 27514;27529 27538 hedgehog ... signaling +T717 GO:0010467 27515 27525 expression +T718 UBERON:0000922 27559 27566 embryos +T719 PR:000014841 27569 27572 Shh +T720 UBERON:0000922 27596 27605 embryonic +T721 GO:0009790 27596 27617 embryonic development +T722 GO:0016540 27626 27640 autoprocessing +T723 CHEBI:16113 27657 27668 cholesterol +T724 CHEBI:36357 27669 27677 molecule +T725 MOP:0000779 27678 27697 covalently attached +T726 GO:0065007 27745 27754 regulated +T727 CHEBI:16113 27803 27814 cholesterol +T728 GO:0006695 27803 27827 cholesterol biosynthesis +T729 GO:0010467 27948 27958 expression +T730 PR:000014841 27962 27965 Shh +T731 UBERON:0002048 27994 27998 lung +T732 PR:000014841 28026 28029 Shh +T733 UBERON:0002048 28060 28065 lungs +T734 PR:000014841 28109 28112 Shh +T735 GO:0042571 28123 28131 antibody +T736 PR:000014841 28163 28166 Shh +T737 UBERON:0000483 28187 28197 epithelial +T738 CL:0000066 28187 28203 epithelial cells +T739 UBERON:0000118 28207 28216 lung buds +T740 PR:000014841 28279 28282 Shh +T741 GO:0010467 28283 28293 expressing +T742 UBERON:0003079 28317 28328 floor plate +T743 UBERON:0002328 28330 28339 notochord +T744 UBERON:0001045 28344 28351 mid gut +T745 UBERON:0000922 28436 28443 embryos +T746 UBERON:0002048 28481 28485 lung +T747 GO:0007565 28511 28522 gestational +T748 GO:0007567 28539 28544 birth +T749 UBERON:0000479 28617 28624 tissues +T750 PR:000014841 28709 28712 Shh +T751 NCBITaxon:10088 28716 28721 mouse +T752 UBERON:0002048 28722 28727 lungs +T753 PR:000014841 28745 28748 Shh +T754 GO:0010467 28758 28768 expression +T755 GO:0007565 28791 28802 gestational +T756 NCBITaxon:10088 28836 28840 mice +T757 PR:000014841 28863 28866 Shh +T758 GO:0042571 28900 28908 antibody +T759 PR:000014841 28912 28915 Shh +T760 GO:0007565 28937 28948 gestational +T761 UBERON:0000922 29115 29122 embryos +T762 PR:000014841 29134 29137 Shh +T763 UBERON:0003079 29158 29169 floor plate +T764 UBERON:0003079 29171 29173 fp +T765 UBERON:0002328 29176 29185 notochord +T766 UBERON:0002328 29187 29189 nc +T767 UBERON:0001045 29202 29209 mid-gut +T768 UBERON:0001045 29211 29213 mg +T769 UBERON:0000483 29223 29233 epithelial +T770 UBERON:0000118 29234 29247 buds of lungs +T771 UBERON:0000118 29249 29251 lb +T772 PR:000014841 29264 29267 Shh +T773 UBERON:0000483 29295 29305 epithelial +T774 CL:0000066 29295 29311 epithelial cells +T775 UBERON:0000025 29351 29356 tubes +T776 UBERON:0002048 29360 29365 lungs +T777 UBERON:0000483 29405 29415 epithelial +T778 CL:0000066 29405 29421 epithelial cells +T779 UBERON:0000025 29436 29441 tubes +T780 PR:000014841 29469 29472 Shh +T781 PR:000014841 29492 29495 Shh +T782 UBERON:0000115 29516 29531 lung epithelial +T783 CL:0000082 29516 29537 lung epithelial cells +T784 UBERON:0001005 29577 29584 airways +T785 GO:0007565 29720 29731 gestational +T786 PR:000014841 29808 29811 Shh +T787 GO:0010467 29812 29822 expression +T788 UBERON:0000922 29890 29897 embryos +T789 UBERON:0000483 29922 29932 epithelial +T790 CL:0000066 29922 29938 epithelial cells +T791 UBERON:0000025 29953 29958 tubes +T792 UBERON:0002048 30005 30010 lungs +T793 PR:000014841 30109 30112 Shh +T794 UBERON:0000922 30132 30139 embryos +T795 GO:0010467 30169 30179 expression +T796 PR:000014841 30222 30225 Shh +T797 GO:0010467 30240 30250 expression +T798 GO:0065007 30259 30268 regulated +T799 PR:000014841 30272 30275 Shh +T800 UBERON:0002048 30311 30315 lung +T801 GO:0007565 30338 30349 gestational +T802 UBERON:0000922 30416 30423 embryos +T803 GO:0010467 30452 30462 expression +T804 PR:000014841 30503 30506 Shh +T805 UBERON:0000115 30547 30562 lung epithelial +T806 CL:0000082 30547 30567 lung epithelial cell +T807 GO:0010467 30568 30578 expression +T808 PR:000014841 30598 30601 Shh +T809 PR:000015288 30646 30656 smoothened +T810 PR:000015288 30658 30661 Smo +T811 SO:0000704 30707 30711 gene +T812 GO:0010467 30707 30722 gene expression +T813 PR:000014841 30738 30741 Shh +T814 GO:0010467 30771 30781 expression +T815 PR:000015288 30795 30798 Smo +T816 PR:000008026 30824 30828 Gli1 +T817 PR:000008028 30857 30861 Gli3 +T818 UBERON:0002048 30929 30934 lungs +T819 GO:0007565 30961 30972 gestational +T820 PR:000015288 31006 31009 Smo +T821 PR:000008026 31011 31015 Gli1 +T822 PR:000008028 31020 31024 Gli3 +T823 GO:0010467 31025 31035 expression +T824 UBERON:0002048 31039 31043 lung +T825 PR:000015288 31054 31064 Smoothened +T826 PR:000015288 31066 31069 Smo +T827 PR:000008026 31085 31089 Gli1 +T828 PR:000008028 31109 31113 Gli3 +T829 PR:000014841 31188 31191 Shh +T830 UBERON:0000922 31280 31287 embryos +T831 PR:000014841 31333 31336 Shh +T832 GO:0016485 31337 31355 protein processing +T833 UBERON:0000922 31368 31375 embryos +T834 PR:000014841 31383 31386 Shh +T835 GO:0010467 31387 31397 expression +T836 PR:000014841 31422 31425 Shh +T837 UBERON:0000922 31481 31488 embryos +T838 GO:0016540 31501 31515 autoprocessing +T839 PR:000014841 31519 31522 Shh +T840 CHEBI:16113 31537 31548 cholesterol +T841 PR:000014841 31581 31584 Shh +T842 PR:000014841 31586 31589 Shh +T843 GO:0009294 31622 31633 transfected +T844 PR:000014841 31651 31654 Shh +T845 PR:000014841 31712 31715 Shh +T846 PR:000014841 31722 31725 Shh +T847 GO:0010467 31729 31739 expression +T848 GO:0009294 31825 31837 Transfection +T849 PR:000014841 31866 31869 Shh +T850 MOP:0000780 31938 31945 cleaved +T851 PR:000014841 31957 31960 Shh +T852 GO:0009294 32011 32023 Transfection +T853 PR:000014841 32069 32072 Shh +T854 PR:000014841 32143 32146 Shh +T855 CHEBI:18059 32204 32209 lipid +T856 UBERON:0000479 32261 32267 tissue +T857 UBERON:0000922 32314 32321 embryos +T858 PR:000014841 32371 32374 Shh +T859 PR:000014841 32541 32544 Shh +T860 GO:0016540 32545 32559 autoprocessing +T861 CHEBI:16113 32613 32624 cholesterol +T862 UBERON:0000922 32637 32644 embryos +T863 PR:000021466 32691 32706 full-length Shh +T864 UBERON:0002048 32735 32739 lung +T865 UBERON:0000922 32766 32773 embryos +T866 PR:000014841 32805 32808 Shh +T867 PR:000014841 32839 32842 Shh +T868 GO:0016540 32843 32865 protein autoprocessing +T869 NCBITaxon:10088 32869 32874 mouse +T870 UBERON:0000922 32875 32882 embryos +T871 UBERON:0000922 32984 32991 embryos +T872 GO:0009294 33016 33027 transfected +T873 PR:000014841 33033 33036 Shh +T874 PR:000014841 33050 33053 Shh +T875 PR:000014841 33088 33091 Shh +T876 GO:0042571 33096 33106 antibodies +T877 PR:000014841 33139 33142 Shh +T878 GO:0009294 33143 33154 transfected +T879 UBERON:0000922 33182 33189 embryos +T880 PR:000014841 33220 33223 Shh +T881 PR:000014841 33275 33278 Shh +T882 PR:000014841 33330 33333 Shh +T883 PR:000014841 33353 33356 Shh +T884 GO:0009294 33357 33368 transfected +T885 PR:000014841 33415 33418 Shh +T886 GO:0009294 33421 33432 transfected +T887 PR:000014841 33466 33469 Shh +T888 CHEBI:18059 33496 33501 lipid +T889 PR:000014841 33590 33593 Shh +T890 UBERON:0000922 33631 33638 embryos +T891 PR:000014841 33728 33731 Shh +T892 UBERON:0000922 33780 33787 embryos +T893 http://purl.obolibrary.org/obo/MONDO_0010035 33837 33841 SLOS +T894 CHEBI:16113 33891 33902 cholesterol +T895 GO:0006695 33891 33915 cholesterol biosynthesis +T896 UBERON:0000922 33950 33959 embryonic +T897 GO:0009790 33950 33971 embryonic development +T898 http://purl.obolibrary.org/obo/MONDO_0000001 34027 34036 disorders +T899 UBERON:0000922 34040 34049 embryonic +T900 GO:0009790 34040 34061 embryonic development +T901 SO:0000704 34085 34090 genes +T902 CHEBI:15440 34126 34134 squalene +T903 CHEBI:16113 34144 34155 cholesterol +T904 GO:0006695 34144 34168 cholesterol biosynthesis +T905 SO:0000704 34185 34192 genetic +T906 CHEBI:16113 34235 34246 cholesterol +T907 GO:0006695 34235 34256 cholesterol synthesis +T908 CHEBI:16113 34285 34296 cholesterol +T909 UBERON:0000922 34307 34316 embryonic +T910 GO:0009790 34307 34328 embryonic development +T911 UBERON:0002048 34411 34415 lung +T912 GO:0030324 34411 34427 lung development +T913 UBERON:0000922 34440 34447 embryos +T914 UBERON:0000948 34458 34465 cardiac +T915 NCBITaxon:9606 34494 34499 human +T916 http://purl.obolibrary.org/obo/MONDO_0010035 34500 34504 SLOS +T917 UBERON:0000948 34546 34551 heart +T918 UBERON:0013768 34559 34572 great vessels +T919 UBERON:0002048 34609 34613 Lung +T920 GO:0030324 34609 34625 Lung development +T921 UBERON:0000922 34638 34645 embryos +T922 UBERON:0000118 34672 34680 lung bud +T923 GO:0060431 34672 34690 lung bud formation +T924 UBERON:0002048 34774 34778 lung +T925 UBERON:0000116 34797 34805 saccular +T926 UBERON:0003914 34869 34887 epithelial tubules +T927 UBERON:0009856 34906 34909 sac +T928 CL:0002062 34925 34936 type I AECs +T929 GO:0007565 35021 35032 gestational +T930 UBERON:0000922 35052 35059 embryos +T931 NCBITaxon:40674 35109 35118 mammalian +T932 UBERON:0004821 35119 35138 alveolar epithelium +T933 UBERON:0002048 35211 35216 lungs +T934 GO:0007567 35229 35234 natal +T935 GO:0007585 35246 35258 gas exchange +T936 CL:0002063 35270 35282 type II AECs +T937 UBERON:0002048 35294 35303 pulmonary +T938 CHEBI:35195 35304 35315 surfactants +T939 CL:0002062 35321 35332 type I AECs +T940 UBERON:0003215 35354 35361 alveoli +T941 UBERON:0003215 35381 35389 alveolar +T942 GO:0048286 35428 35450 development of alveoli +T943 UBERON:0003215 35443 35450 alveoli +T944 UBERON:0002048 35480 35484 lung +T945 GO:0060425 35480 35498 lung morphogenesis +T946 GO:0007565 35507 35518 gestational +T947 CL:0002062 35573 35584 type I AECs +T948 CL:0002063 35593 35605 type II AECs +T949 UBERON:0002048 35618 35623 lungs +T950 CL:0002062 35680 35692 type I cells +T951 GO:0010467 35708 35718 expression +T952 CL:0002062 35733 35744 type I AECs +T953 PR:000012516 35746 35749 T1α +T954 PR:000004185 35754 35758 AQP5 +T955 UBERON:0002048 35846 35851 lungs +T956 UBERON:0003104 35871 35881 mesenchyme +T957 CL:0010003 35897 35901 AECs +T958 UBERON:0002049 35911 35924 vascular beds +T959 CL:0002063 35985 35997 type II AECs +T960 SO:0000704 36020 36024 gene +T961 GO:0010467 36020 36035 gene expression +T962 CHEBI:35195 36049 36059 surfactant +T963 PR:000015945 36049 36070 surfactant proteins A +T964 PR:000014773 36049 36068;36072 36073 surfactant proteins ... B +T965 PR:000014774 36049 36068;36075 36076 surfactant proteins ... C +T966 PR:000014775 36049 36068;36080 36081 surfactant proteins ... D +T967 PR:000014774 36132 36136 SP-C +T968 UBERON:0002048 36148 36153 lungs +T969 UBERON:0000922 36168 36175 embryos +T970 GO:0007585 36192 36204 gas exchange +T971 UBERON:0003215 36239 36247 alveolar +T972 UBERON:0001004 36277 36288 respiratory +T973 http://purl.obolibrary.org/obo/MONDO_0021113 36277 36296 respiratory failure +T974 UBERON:0000922 36362 36371 embryonic +T975 CHEBI:16113 36420 36431 cholesterol +T976 CHEBI:17759 36487 36492 7-DHC +T977 UBERON:0002048 36510 36515 lungs +T978 UBERON:0000468 36547 36557 whole body +T979 UBERON:0000995 36572 36579 uterine +T980 GO:0007567 36622 36627 natal +T981 http://purl.obolibrary.org/obo/MONDO_0010035 36685 36689 SLOS +T982 GO:0007565 36784 36793 gestation +T983 NCBITaxon:33208 36806 36813 animals +T984 UBERON:0000468 36837 36847 Whole body +T985 UBERON:0002048 36866 36870 lung +T986 UBERON:0000955 36891 36896 brain +T987 UBERON:0000948 36898 36903 heart +T988 UBERON:0002113 36908 36914 kidney +T989 GO:0007565 36940 36951 gestational +T990 UBERON:0000922 36987 36994 embryos +T991 UBERON:0002048 37021 37025 lung +T992 UBERON:0000922 37059 37066 embryos +T993 UBERON:0002048 37132 37136 lung +T994 UBERON:0000062 37155 37161 organs +T995 GO:0008283 37163 37181 Cell proliferation +T996 GO:0051301 37163 37167;37186 37194 Cell ... division +T997 UBERON:0002048 37207 37212 lungs +T998 GO:0006915 37265 37279 cell apoptosis +T999 CHEBI:16113 37337 37348 cholesterol +T1000 GO:0008283 37402 37406;37418 37431 cell ... proliferation +T1001 GO:0008219 37460 37470 cell death +T1002 CHEBI:16113 37473 37484 Cholesterol +T1003 UBERON:0000922 37508 37515 embryos +T1004 PR:000014480 37529 37533 Sc5d +T1005 http://purl.obolibrary.org/obo/MONDO_0011816 37538 37553 lathosterolosis +T1006 PR:000006453 37559 37565 Dhcr24 +T1007 http://purl.obolibrary.org/obo/MONDO_0011217 37569 37584 desmosterolosis +T1008 UBERON:0000922 37586 37593 embryos +T1009 CHEBI:15889 37609 37616 sterols +T1010 CHEBI:16113 37621 37632 cholesterol +T1011 UBERON:0000922 37645 37654 embryonic +T1012 UBERON:0002048 37655 37660 lungs +T1013 UBERON:0000955 37676 37681 brain +T1014 UBERON:0002107 37686 37691 liver +T1015 CHEBI:17759 37726 37730 7DHC +T1016 CHEBI:16113 37774 37785 cholesterol +T1017 UBERON:0000479 37836 37842 tissue +T1018 CHEBI:15889 37843 37850 sterols +T1019 CHEBI:16113 37893 37904 cholesterol +T1020 UBERON:0005291 37947 37960 fetal tissues +T1021 UBERON:0005292 37977 38000 extra-embryonic tissues +T1022 UBERON:0001977 38018 38023 serum +T1023 UBERON:0001987 38025 38033 placenta +T1024 UBERON:0001040 38039 38047 yolk sac +T1025 NCBITaxon:10088 38066 38070 mice +T1026 CHEBI:16113 38104 38115 cholesterol +T1027 CHEBI:16113 38137 38148 cholesterol +T1028 PR:000006453 38207 38213 Dhcr24 +T1029 NCBITaxon:10088 38219 38223 mice +T1030 CHEBI:16113 38259 38270 cholesterol +T1031 UBERON:0001969 38274 38280 plasma +T1032 UBERON:0000479 38285 38292 tissues +T1033 CHEBI:16113 38314 38325 cholesterol +T1034 PR:000006453 38337 38343 Dhcr24 +T1035 UBERON:0005291 38349 38363 embryo tissues +T1036 UBERON:0000479 38419 38425 tissue +T1037 CHEBI:15889 38426 38433 sterols +T1038 CHEBI:16113 38525 38536 cholesterol +T1039 NCBITaxon:39107 38563 38569 murine +T1040 PR:000006453 38604 38610 Dhcr24 +T1041 NCBITaxon:10088 38614 38618 mice +T1042 NCBITaxon:10088 38659 38663 mice +T1043 CHEBI:17737 38692 38703 desmosterol +T1044 CHEBI:16113 38736 38747 cholesterol +T1045 CHEBI:16113 38767 38778 cholesterol +T1046 CHEBI:16113 38860 38871 cholesterol +T1047 GO:0006695 38860 38884 cholesterol biosynthesis +T1048 UBERON:0000922 38903 38912 embryonic +T1049 GO:0009790 38903 38924 embryonic development +T1050 PR:000014841 38985 38988 Shh +T1051 UBERON:0005597 39080 39095 lung primordium +T1052 PR:000014841 39097 39100 Shh +T1053 GO:0010467 39104 39113 expressed +T1054 GO:0001763 39143 39166 branching morphogenesis +T1055 PR:000014841 39189 39192 Shh +T1056 NCBITaxon:10088 39202 39206 mice +T1057 PR:000014841 39229 39232 Shh +T1058 GO:0046903 39234 39242 secreted +T1059 UBERON:0000483 39250 39260 epithelium +T1060 GO:0001763 39278 39301 branching morphogenesis +T1061 UBERON:0002048 39351 39355 lung +T1062 GO:0060441 39351 39379 lung branching morphogenesis +T1063 GO:0007585 39444 39456 gas-exchange +T1064 UBERON:0002048 39471 39475 lung +T1065 UBERON:0000116 39483 39491 saccular +T1066 GO:0009790 39502 39516;39526 39533 development of ... embryos +T1067 UBERON:0000922 39526 39533 embryos +T1068 PR:000014841 39565 39568 Shh +T1069 PR:000014841 39591 39594 Shh +T1070 GO:0016540 39595 39602;39618 39632 protein ... autoprocessing +T1071 GO:0010467 39603 39613 expression +T1072 UBERON:0000922 39695 39702 embryos +T1073 PR:000014841 39785 39788 Shh +T1074 CHEBI:16113 39863 39874 cholesterol +T1075 UBERON:0000922 39925 39932 embryos +T1076 PR:000014841 39963 39966 Shh +T1077 GO:0016540 39967 39981 autoprocessing +T1078 CHEBI:17759 39985 39989 7DHC +T1079 UBERON:0005291 40014 40031 embryonic tissues +T1080 CHEBI:15889 40070 40076 sterol +T1081 PR:000014841 40091 40094 Shh +T1082 PR:000014841 40157 40160 Shh +T1083 PR:000015288 40261 40271 smoothened +T1084 PR:000014841 40290 40293 Shh +T1085 PR:000014841 40406 40409 Shh +T1086 UBERON:0000922 40447 40454 embryos +T1087 GO:0010467 40615 40625 expression +T1088 UBERON:0000922 40681 40688 embryos +T1089 UBERON:0002048 40706 40710 lung +T1090 GO:0030324 40706 40722 lung development +T1091 NCBITaxon:10088 40767 40772 mouse +T1092 UBERON:0000922 40773 40782 embryonic +T1093 CL:0000057 40783 40794 fibroblasts +T1094 UBERON:0000922 40816 40823 embryos +T1095 GO:0040007 40825 40830 grown +T1096 CHEBI:18059 40834 40839 lipid +T1097 CHEBI:23456 40886 40898 cyclodextrin +T1098 PR:000014841 40920 40923 Shh +T1099 PR:000014841 40989 40992 Shh +T1100 SO:0000704 41027 41031 gene +T1101 GO:0010467 41027 41042 gene expression +T1102 GO:0010467 41103 41112 expressed +T1103 PR:000014841 41113 41116 Shh +T1104 CHEBI:15889 41136 41142 sterol +T1105 PR:000014841 41169 41172 Shh +T1106 GO:0016540 41173 41187 autoprocessing +T1107 PR:000014841 41271 41274 Shh +T1108 UBERON:0005291 41287 41304 embryonic tissues +T1109 PR:000014841 41337 41340 Shh +T1110 CHEBI:16113 41456 41467 cholesterol +T1111 GO:0006695 41456 41480 cholesterol biosynthesis +T1112 PR:000014841 41511 41514 Shh +T1113 GO:0005886 41560 41575 plasma membrane +T1114 CHEBI:16113 41602 41613 cholesterol +T1115 GO:0005886 41629 41644 plasma membrane +T1116 GO:0016020 41662 41670 membrane +T1117 CHEBI:16113 41756 41767 cholesterol +T1118 GO:0006695 41756 41777 cholesterol synthesis +T1119 PR:000014841 41890 41893 Shh +T1120 NCBITaxon:10088 42000 42004 mice +T1121 CHEBI:16113 42019 42030 Cholesterol +T1122 NCBITaxon:10088 42072 42076 mice +T1123 UBERON:0000116 42118 42131 lung saccular +T1124 CL:0002062 42176 42182;42199 42203 type I ... AECs +T1125 CL:0002063 42191 42203 type II AECs +T1126 CHEBI:16113 42280 42291 cholesterol +T1127 GO:0016020 42299 42308 membranes +T1128 CHEBI:16113 42354 42365 cholesterol +T1129 UBERON:0002048 42581 42585 lung +T1130 CHEBI:16113 42635 42646 cholesterol +T1131 UBERON:0000922 42650 42659 embryonic +T1132 GO:0009790 42650 42671 embryonic development +T1133 NCBITaxon:33208 42683 42690 Animals +T1134 NCBITaxon:10088 42696 42700 mice +T1135 NCBITaxon:33208 42719 42725 animal +T1136 NCBITaxon:33208 42831 42837 Animal +T1137 NCBITaxon:33208 42876 42883 animals +T1138 UBERON:0010148 42999 43012 vaginal plugs +T1139 UBERON:0000922 43031 43040 embryonic +T1140 GO:0007565 43060 43068 pregnant +T1141 UBERON:0000922 43094 43101 embryos +T1142 UBERON:0000995 43121 43126 uteri +T1143 UBERON:0000033 43149 43154 Crown +T1144 UBERON:0013691 43155 43159 rump +T1145 UBERON:0000922 43175 43182 embryos +T1146 UBERON:0000922 43241 43248 embryos +T1147 UBERON:0006314 43284 43289 fluid +T1148 UBERON:0002048 43296 43300 lung +T1149 UBERON:0002224 43329 43344 thoracic cavity +T1150 UBERON:0006314 43369 43374 fluid +T1151 NCBITaxon:10088 43389 43393 Mice +T1152 GO:0007567 43421 43426 birth +T1153 UBERON:0000922 43532 43539 embryos +T1154 UBERON:0002048 43543 43548 lungs +T1155 UBERON:0000479 43787 43793 Tissue +T1156 CHEBI:15889 43794 43800 sterol +T1157 UBERON:0001977 43908 43913 Serum +T1158 CHEBI:16827 43914 43928 corticosterone +T1159 UBERON:0000178 43983 43988 blood +T1160 UBERON:0000915 44142 44150 Thoracic +T1161 UBERON:0000922 44162 44169 embryos +T1162 NCBITaxon:10088 44176 44180 mice +T1163 CHEBI:50913 44214 44222 fixative +T1164 CHEBI:17790 44224 44232 methanol +T1165 CHEBI:15347 44233 44240 acetone +T1166 CHEBI:15366 44241 44252 acetic acid +T1167 CHEBI:15377 44253 44258 water +T1168 UBERON:0000479 44307 44314 Tissues +T1169 CHEBI:16236 44400 44407 ethanol +T1170 CHEBI:17578 44427 44434 toluene +T1171 CHEBI:27338 44669 44675 xylene +T1172 CHEBI:16236 44719 44726 ethanol +T1173 CHEBI:53258 44766 44780 sodium citrate +T1174 CHEBI:16240 44903 44920 hydrogen peroxide +T1175 UBERON:0001977 45015 45020 serum +T1176 GO:0042571 45063 45073 antibodies +T1177 PR:000014841 45082 45085 Shh +T1178 PR:000015288 45116 45119 Smo +T1179 PR:000008026 45133 45137 Gli1 +T1180 PR:000008028 45147 45151 Gli3 +T1181 PR:000014774 45165 45169 SP-C +T1182 PR:000027209 45183 45194 caveolin 1α +T1183 PR:000004185 45208 45219 aquaporin 5 +T1184 PR:000004185 45221 45225 AQP5 +T1185 PR:000009921 45235 45242 megalin +T1186 PR:000001904 45252 45259 PECAM-1 +T1187 PR:000012516 45314 45317 T1α +T1188 MOP:0000093 45491 45503 biotinylated +T1189 GO:0042571 45514 45524 antibodies +T1190 GO:0032991 45621 45628 complex +T1191 UBERON:0002048 45867 45871 Lung +T1192 UBERON:0000116 45894 45902 saccular +T1193 UBERON:0002048 46112 46116 lung +T1194 UBERON:0002048 46145 46149 lung +T1195 UBERON:0000116 46170 46178 saccular +T1196 UBERON:0002048 46251 46255 lung +T1197 NCBITaxon:33208 46271 46278 animals +T1198 GO:0008283 46322 46340 cell proliferation +T1199 GO:0051301 46322 46326;46341 46349 cell ... Division +T1200 GO:0008219 46322 46326;46350 46355 cell ... death +T1201 GO:0008283 46368 46386 cell proliferation +T1202 GO:0051301 46368 46372;46391 46399 cell ... division +T1203 GO:0008283 46417 46430 proliferating +T1204 PR:000012421 46417 46451 proliferating cell nuclear antigen +T1205 CHEBI:59132 46444 46451 antigen +T1206 PR:000012421 46453 46457 PCNA +T1207 CHEBI:472552 46464 46481 bromodeoxyuridine +T1208 CHEBI:472552 46483 46487 BrdU +T1209 CHEBI:15358 46522 46529 histone +T1210 PR:000027594 46522 46532 histone H3 +T1211 PR:000012421 46549 46553 PCNA +T1212 GO:0042571 46586 46596 antibodies +T1213 CHEBI:472552 46646 46650 BrdU +T1214 GO:0007565 46672 46680 pregnant +T1215 NCBITaxon:10088 46681 46685 mice +T1216 UBERON:0001179 46715 46727 peritoneally +T1217 CHEBI:472552 46758 46762 BrdU +T1218 UBERON:0000922 46809 46816 embryos +T1219 UBERON:0000995 46832 46837 uteri +T1220 CHEBI:472552 46917 46921 BrdU +T1221 GO:0042571 46922 46930 antibody +T1222 CHEBI:52302 47014 47017 Cy2 +T1223 MOP:0000779 47018 47028 conjugated +T1224 NCBITaxon:9986 47029 47035 rabbit +T1225 NCBITaxon:10088 47041 47046 mouse +T1226 GO:0042571 47047 47055 antibody +T1227 GO:0008219 47100 47110 Cell death +T1228 CHEBI:31624 47218 47229 Fluorescein +T1229 GO:0008219 47238 47248 Cell Death +T1230 UBERON:0000922 47479 47486 embryos +T1231 GO:0019835 47543 47548 lysis +T1232 CHEBI:9754 47563 47567 Tris +T1233 CHEBI:26710 47582 47586 NaCl +T1234 CHEBI:9750 47602 47614 Triton X-100 +T1235 CHEBI:18320 47623 47626 DTT +T1236 CHEBI:8102 47633 47637 PMSF +T1237 CHEBI:6426 47669 47678 leupeptin +T1238 CHEBI:7989 47683 47694 pepstatin A +T1239 UBERON:0000479 47703 47709 tissue +T1240 GO:0009294 48022 48033 transfected +T1241 GO:0009294 48050 48062 transfection +T1242 CHEBI:33893 48063 48070 reagent +T1243 GO:0010467 48099 48109 expression +T1244 PR:000021466 48141 48156 full-length Shh +T1245 PR:000014841 48187 48190 Shh +T1246 GO:0006412 48204 48217 translational +T1247 UBERON:0004535 48284 48298 Cardiovascular +T1248 GO:0009294 48388 48400 transfection +T1249 UBERON:0000922 48577 48584 embryos +T1250 CHEBI:8984 48615 48618 SDS +T1251 CHEBI:53325 48642 48656 nitrocellulose +T1252 GO:0042571 48690 48698 antibody +T1253 PR:000014841 48700 48703 Shh +T1254 NCBITaxon:9793 48741 48747 donkey +T1255 GO:0042571 48769 48777 antibody +T1256 NCBITaxon:10088 49025 49029 mice +T1257 UBERON:0002048 49030 49035 lungs +T1258 PR:000015945 49057 49061 SP-A +T1259 PR:000014773 49063 49067 SP-B +T1260 PR:000014774 49072 49076 SP-C +T1261 UBERON:0002048 49135 49144 Pulmonary +T1262 PR:000014775 49237 49241 SP-D +T1263 NCBITaxon:39107 49273 49279 murine +T1264 PR:000014775 49280 49284 SP-D +T1265 SO:0000112 49299 49306 primers +T1266 SO:0000077 49345 49354 antisense +T1267 UBERON:0000479 49519 49525 tissue +T1268 CHEBI:15889 49526 49533 sterols +T1269 UBERON:0002048 49560 49564 lung +T1270 PR:000015945 49795 49799 SP-A +T1271 PR:000014773 49801 49805 SP-B +T1272 PR:000014774 49810 49814 SP-C +T1273 PR:000014841 49843 49846 Shh +T1274 PR:000014841 49851 49854 Shh +T1275 SO:0000155 49857 49865 plasmids +T1276 UBERON:0000948 49950 49955 Heart diff --git a/src/ontogpt/evaluation/craft/database/all/15005800.txt b/src/ontogpt/evaluation/craft/database/all/15005800.txt new file mode 100644 index 000000000..e793db6ef --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15005800.txt @@ -0,0 +1,183 @@ +Late gestational lung hypoplasia in a mouse model of the Smith-Lemli-Opitz syndrome + +Abstract + +Background + +Normal post-squalene cholesterol biosynthesis is important for mammalian embryonic development. Neonatal mice lacking functional dehydrocholesterol Δ7-reductase (Dhcr7), a model for the human disease of Smith-Lemli-Opitz syndrome, die within 24 hours of birth. Although they have a number of biochemical and structural abnormalities, one cause of death is from apparent respiratory failure due to developmental pulmonary abnormalities. + +Results + +In this study, we characterized further the role of cholesterol deficiency in lung development of these mice. Significant growth retardation, beginning at E14.5~E16.5, was observed in Dhcr7-/- embryos. Normal lobation but smaller lungs with a significant decrease in lung-to-body weight ratio was noted in Dhcr7-/- embryos, compared to controls. Lung branching morphogenesis was comparable between Dhcr7-/- and controls at early stages, but delayed saccular development was visible in all Dhcr7-/- embryos from E17.5 onwards. Impaired pre-alveolar development of varying severity, inhibited cell proliferation, delayed differentiation of type I alveolar epithelial cells (AECs) and delayed vascular development were all evident in knockout lungs. Differentiation of type II AECs was apparently normal as judged by surfactant protein (SP) mRNAs and SP-C immunostaining. A significant amount of cholesterol was detectable in knockout lungs, implicating some maternal transfer of cholesterol. No significant differences of the spatial-temporal localization of sonic hedgehog (Shh) or its downstream targets by immunohistochemistry were detected between knockout and wild-type lungs and Shh autoprocessing occurred normally in tissues from Dhcr7-/- embryos. + +Conclusion + +Our data indicated that cholesterol deficiency caused by Dhcr7 null was associated with a distinct lung saccular hypoplasia, characterized by failure to terminally differentiate alveolar sacs, a delayed differentiation of type I AECs and an immature vascular network at late gestational stages. The molecular mechanism of impaired lung development associated with sterol deficiency by Dhcr7 loss is still unknown, but these results do not support the involvement of dysregulated Shh-Patched-Gli pathway in causing this defect. + +Background + +Cholesterol is a necessary membrane constituent of all mammalian, reptile and avian cells, as well as a few other organisms, but not all eukaryotic organisms. In the latter cases, such as plants and fungi, cholesterol-like sterols (phytosterols, ergosterol etc.) seem to substitute for cholesterol. The precise need for sterols in biological membranes still remains poorly defined, though its link with microdomains (variously termed rafts, caveoli etc.) may be part of the answer [1]. Although the accumulation of cholesterol in atherosclerotic plaques has led to the concept that 'too much' cholesterol may be deleterious, too little cholesterol has now been proven pathophysiological for abnormal embryonic development. Smith-Lemli-Opitz syndrome (SLOS, MIM 270400), a relative common dysmorphology disorder, is caused by mutations in DHCR7 [2-5], which encodes for 7-dehydrocholesterol Δ7-reductase and catalyzes a final step of cholesterol biosynthesis. Recognition of other dysmorphology syndromes caused by defects in the post-squalene cholesterol biosynthesis pathway, such as desmosterolosis, lathosterolosis, X-linked chondrodysplasia punctanta and CHILD syndrome [6-15], has strengthened this concept. + +Genetic disruption of the Dhcr7 results in neonatal lethality [16]. Neonatal mice homozygous for the Dhcr7 gene disruption are deficient in cholesterol and have increased accumulation of the cholesterol precursor 7-dehydrocholesterol (7DHC). Although no embryonic lethality was noted, these knockout pups exhibited a number of developmental abnormalities and died within 24 h after birth. A similar mouse SLOS model, generated by replacing exons 3, 4 and part of exon 5 of the murine Dhcr7 gene, with similar biochemical and structural defects and a 100 % neonatal lethality, has also been reported [17]. Poor lung development or diffuse lung alveolar atelectasis with cyanotic and lethargic breathing are consistent between both Dhcr7 null models [16,17], and therefore is the likely reason for the respiratory insufficiency and subsequent death of Dhcr7-/- pups. These Dhcr7 knockout mice are models for human SLOS, especially the more severely affected patients, since early postnatal lethality with respiratory failure has been reported in severe SLOS cases [4,5]. Developmental lung abnormalities are also relatively common in SLOS patients, including pulmonary hypoplasia, abnormal pulmonary lobation and pulmonary arteries, anomalies of laryngeal and tracheal development [18]. + +Mammalian lung is unique in that it is fully developed, but does not function for gas exchange until at the moment of birth; the majority of structural development and maturation takes place in utero. Lung development is a complex process that involves branching morphogenesis of epithelium, interstitial development including vasculogenesis, cellular differentiation, biochemical maturation and physical growth. Four stages of mouse lung development have been described, based upon the histological appearances of lung; pseudoglandular, canalicular, terminal saccular and alveolar stages [19]. + +Impaired hedgehog function has been proposed as a mechanism underlying malformations found in SLOS. Hedgehog family members play diverse roles in embryonic development, and cholesterol is necessary for maturation of these morphogens [20-23]. The sonic hedgehog (Shh) signaling cascade has been shown to play a central role in lung development [24-26]. Loss of Shh function resulted in severe lung defects associated with loss of branching morphogenesis, but preserved proximal-to-distal differentiation of lung epithelium [24]. Overexpression of Shh in the distal lung epithelium resulted in the absence of functional alveoli and an increase in interstitial tissue caused by an increased proliferation of both epithelial and mesenchyme cells [26]. Mice with Shh overexpression died less than 24 h after birth, probably owing to respiratory failure. It has been reported that inhibition of cholesterol biosynthesis by chemical inhibitors of Dhcr7 also led to a disturbance of the Shh signaling with abnormal embryogenesis [27,28], and severe sterol deprivation can indeed inhibit the processing of transfected Shh in cultured mammalian cells [29]. + +In the present studies, we characterized lung development in Dhcr7-/- embryos from E10.5 to birth. Our results showed that cholesterol deficiency, caused by loss of Dhcr7 activity, did not alter lung branching morphogenesis (early gestational stages, E9.5 to E14.5). However, lung saccular development was impaired, with arrested or partially developed epithelial tubules and delayed vascular development. Differentiation of type II alveolar epithelial cells (AECs) appeared normal but differentiation of type I AECs was severely impaired. Therefore, cholesterol deficiency resulted in a distinct late gestational lung hypoplasia, characterized by impaired sacculation with delayed type I AECs differentiation and immature vascular development. However, this study does not support the concept that the perturbation of Shh-Patched-Gli pathway was involved in the pathogenesis of developmental lung hypoplasia in Dhcr7-/- embryos. Patterns of expression of Shh signaling cascade members were not altered, and processing of Shh appeared normal. + +Results + +Body growth retardation at late gestation stage in Dhcr7-/- embryos + +Dhcr7-/- mice were born in the expected Mendelian ratios from heterozygous parents, but all of these pups died within 24 h and most within 14 h of birth [16]. At birth, they were clearly smaller than wild-type or heterozygous littermates. An experienced investigator can pick out knockout pups based on this along with presence of hypoxia and their lack of spontaneous movement. The breathing frequency in P0 Dhcr7-/- pups was markedly slower than control littermates (34 ± 7 breaths/min in Dhcr7-/-, n = 10 vs. 92 ± 16 breaths/min in controls, n = 32, p < 0.001) and the breathing was observed to be more irregular and shallow, with long periods of apnea. + +To determine the gestational stage at which growth of the knockout embryos was affected, embryos were harvested at various stages of gestation and their body lengths compared between Dhcr7-/- and controls. A linear increase in body length from E9.5 to E19.5 in control embryos was observed (Fig. 1). No significant differences in body length between wild-type and heterozygous embryos were noted (data not shown). In contrast, knockout embryos showed comparable body length growth until E14.5~E16.5, then began to show a slower increase in length, such that by E17.5, this was statistically significant (18.87 ± 1.29 mm in controls, n = 15 compared to 16.69 ± 1.05 mm in Dhcr7-/-, n = 7, p < 0.05, Fig. 1). + +Figure 1 + +Body length growth curves. Body length growth vs. gestational days is as shown. Control embryos (filled triangles, wild-type and heterozygous embryos were pooled together as there were no differences between these two groups) and knockout embryos (filled circles) were as shown. The growth curve showed a linear increase from E9.5 to E19.5 in controls, but deviated at ~E14.5 in Dhcr7-/- embryos. Body lengths at E17.5 (18.87 ± 1.29 mm in controls, n = 15 vs. 16.69 ± 1.05 mm in Dhcr7-/-, n = 7, p < 0.05), E18.5 (21.35 ± 1.05 mm in controls, n = 15 vs. 19.38 ± 0.9 mm in Dhcr7-/-, n = 5, p < 0.05) and E19.5 (23.4 ± 0.97 mm in controls, n = 13 vs. 20.3 ± 0.44 mm in Dhcr7-/-, n = 4, p < 0.05) were significantly decreased, compared to those in controls. Results are expressed as mean ± s.d. + +Dhcr7-/- embryos at late stages displayed pulmonary hypoplasia with delayed saccular development + +Wet lung weights, as well as lung to body weight ratios were significantly lower in Dhcr7-/- at E18.5 compared to those in wild-type but not at E13.5 (Table 1). Both body and lung weights were reduced, but lungs seemed to be more affected. A representative figure of embryos at E10.5 and of the pups at birth was shown in Fig. 2A and 2B. + +Table 1 + +Whole body and lung weights of mouse embryos + +* p < 0.05, compared to Dhcr7+/+. BW, body weight; LW, lung weight. + +Figure 2 + +Embryonic size, lung morphology and histology at various stages of development. Panel A shows the size and general morphology of wild-type (+/+) and Dhcr7-/- (-/-) embryos at E10.5, without significant difference. At P0, the knockout pups were significantly smaller and appeared hypoxic (panel B). Panel C shows dorsal views of lungs from knockout and wild-type at E13.5 and panel D those at P0. Cross-sectional examination at the cardiopulmonary level at E 20.5 (prior to birth) showed normal lobar septation, but lungs from knockout animals were smaller in size and had less distal sac space (cf. panels E and F). Original magnification: Panels A and C, 2X; panels B and D, 0.7X; E and F, 2X. Labels: r, right; l, left; b, bronchial; br, bronchiole; e, esophagus; pa, pulmonary artery; ra, right atrium; la, left atrium; rv, right ventricle; lv, left ventricle. + +We examined the lungs for any gross morphological abnormalities. The overall organogenesis of lungs was preserved in Dhcr7-/- pups; four right lung lobes and a single left lobe flanking the heart were easily seen on external examination at birth (Fig. 2D). Examination for lobation at E13.5 also showed comparable gross anatomy (Fig. 2C) between wild-type and knockout embryos. Histological analyses of the cardiopulmonary structures at E20.5, taken just before birth to avoid artifacts from lack of lung inflation, also confirmed normal gross anatomic development (Fig. 2E and 2F). Additionally, no cardiac defects were identified and this was confirmed by more detailed histological analyses (data not shown). Thus, despite the obvious hypoxia frequently observed at birth of the knockout pups, major cardiac structural abnormalities were not observed to account for this phenotype. + +No distinguishable morphological differences were discernable between Dhcr7-/- and wild-type lungs at E10.5, E13.5, E14.5 and E16.5 at low (10X) or high (40X) magnifications (Fig. 3, panel A: a-h, data for E10.5 and E13.5 not shown). However, from E17.5 onwards, lungs from knockout embryos appeared to have less saccular space and relatively more mesenchyme compared to wild-type lungs (Fig. 3, panel A: I-x). Lungs from control animals showed normal progression with formation of sac-like structures, the precursors of the alveoli (Fig. 3, panel A: i, m, q and u). In lungs from knockout embryos, expansion of epithelial tubules occurred but was significantly delayed (Fig. 3, cf. panel A: j, n, r, v with i, m, q and u). Morphometric measurements of lung sections at E17.5, E18.5, E20.5 and in lungs from neonates showed that the proportion of terminal sac space area was significantly decreased in Dhcr7-/- lungs, compared wild-type lungs (Fig. 3, panel B). Interstitial mesenchyme appeared thicker and the lungs had less septation compared to control lungs. This 'thickening' of the alveoli may explain the hypoxia frequently seen in Dhcr7-/- pups at birth. + +Figure 3 + +Histological analyses of lungs at various embryonic stages. Panel A indicates lung sections stained with H and E, and taken at various gestational stages as indicated. The first two columns are at lower magnification (10X) and the last two columns at higher magnification (40X). The first and third columns are representative sections from wild-type (+/+) embryos and the second and fourth columns from knockout (-/-) embryos. No differences were visible at E14.5 or E16.5 (panel A, a-h). Significant differences were visible from E17.5 onwards (panel A, i-x). Distal tubules showed dilation and mesenchyme thinning, with progression of septation in wild-type lungs (panel A, i, m and q), whereas the knockout lungs showed less saccular structures and more mesenchyme (panel A, j, n and r). At higher magnifications of wild-type lungs (panel A, k, o and s), developing pre-alveoli and thinning mesenchyme (arrowheads) were seen but sections from knockout embryos showed a delay in this sequence of development (panel A, l, p and t). At birth, lungs from knockout pups showed deficient septation and thick-walled mesenchyme (panel A, x, arrowheads). Labels: de, distal epithelium; m, mesenchyme; PA, pulmonary artery; a, pre-alveoli; b, bronchi. Panel B shows the morphometric analyses of lung terminal sac spaces in lungs at various gestational stages. A significant reduction in the terminal sac space was noted from E17.5 onwards. Results are expressed as mean ± s.d. + +Attenuated cell proliferation in late gestational Dhcr7-/- lungs + +Does the loss of normal cholesterol biosynthesis lead to abnormalities of cell proliferation/division or cell death? Cell proliferation was examined by immunostaining for PCNA and also by BrdU incorporation. Both PCNA and BrdU staining of wild-type and knockout lungs at early gestational stages (E13.5 and E14.5) showed comparable patterns (Fig. 4, panels A-D). However, a clear reduction of PCNA and BrdU labeling was visualized in distal lungs of knockout embryos at late gestational stages (E18.5 and E20.5, Fig. 4, panels E-H). Attenuated PCNA labeling was also noted in knockout hearts at term (Fig. 4, cf. panels I and J). When lungs were stained for pHH3, a marker of active cell division, a clear reduction of pHH3 expression in the epithelium of knockout lung was observed at E20.5 but not at E14.5 (Fig. 4, panels K, L, O and P). In contrast, using the TUNEL assay, no apparent differences in cell apoptosis rates were observed between wild-type and knockout lungs at E18.5 (Fig. 4, cf. panels M and N). Thus, at the late gestational stages, cell proliferation/division was impaired in knockout embryos with no increase in cell death rates. + +Figure 4 + +Analyses of cell proliferation, cell division and cell death. Cell proliferation (PCNA: panels A, B, E and F; BrdU: panels C, D, G and H), cell division (pHH3: panels K, L, O and P) and cell death (TUNEL: panels M and N) were analyzed by immunostaining on lung sections at early and late gestational stages. Both PCNA and BrdU labeled cells in both epithelial and mesenchymal regions were comparable at early stages (E14.5 for PCNA and E13.5 for BrdU) between wild-type (+/+) and knockout (-/-) lungs. Positively stained cells in distal lungs at late stage (E20.5 for PCNA and E18.5 for BrdU) from knockout lungs were markedly reduced, compared with wild-type. PCNA labeling of cardiac muscle was also attenuated in knockout embryos at E20.5 (cf. panels I and J). No apparent differences in TUNEL immunostaining were observed in lung sections at E18.5 between wild-type and knockout. The pHH3 immunostaining was comparable between wild-type and knockout lungs at E14.5, and the lungs from wild-type at E20.5 showed a high index of positive cells, whereas lungs from knockout showed a apparent reduced staining pattern. Note the dividing cells are mainly epithelial. Original magnification: A-D and G-N are 20X; E, F, O and P are 40X. + +Sterol content of developing lungs in Dhcr7 deficient mice + +Markedly elevated 7-DHC/8-DHC levels and reduced body cholesterol levels have been reported previously in Dhcr7-/- P0 mice [16,17]. A significant amount of cholesterol was detectable in lungs of Dhcr7-/- mice, comprising ~60% and ~30% of total tissue sterols at E13.5 and neonate, respectively. In contrast to the doubling of cholesterol in the lungs of wild type embryos (1.82 mg/g tissue at E13.5 to 3.55 mg/g tissue at P0), cholesterol content in the lungs of Dhcr7 null embryos remained at almost constant low levels between E13.5 and P0 gestational stages (Fig. 5). There was a progressive increase in 7-DHC and 8-DHC; the latter is generated as a spontaneous isomerization from the former. Total sterols at birth were reduced in lungs of the Dhcr7 null mice, but were only lower by ~30% (Fig. 5, see values for P0 mice). + +Figure 5 + +Sterol analyses of embryonic lungs. Sterol contents, including cholesterol (black bars) and 7DHC/8DHC (white bars), were quantitated by GC-MS in the lungs from wild-type (+/+) and knockout (-/-) embryos. Results are expressed as mean ± s.d and the number of lungs analyzed is as indicated. Total sterols were lower at all stages in knockout lungs, with almost no increase in cholesterol content with time (black bars), but there was a progressive increment in the precursor sterols, 7DHC/8DHC (open bars), with increasing gestation. + +Cholesterol is absolutely required for steroid hormone synthesis by the adrenal glands. Adrenal insufficiency, as has been reported in some severe cases of SLOS patients, could be responsible for the lung hypoplasia and perinatal lethality [30]. Steroids, but particularly corticosteroids, are necessary for lung maturation, especially the synthesis and secretion of surfactant components [31]. We determined plasma corticosterone levels in P0 mice within 12 h of birth. Wild type P0 mice had corticosterone levels of 158 ± 50 ng/ml (n = 11), compared with 225 ± 50 ng/ml (n = 7, p < 0.01) in Dhcr7-/- P0 mice. Thus, the corticosterone levels in Dhcr7-/- mice were not decreased, but increased by ~40%. However, since these P0 mice were dying, perhaps this increase was sub-optimal given the stress. Nevertheless, an absolute lack of corticosterone was not present in these knockout pups. + +To further rule out any effects of corticosterone deficiency on lung immaturity, we examined lungs for surfactant protein expression. Surfactant protein C, as determined by IHC, was comparable in lung sections from wild-type or Dhcr7 null embryos at either E18.5 or at E20.5 gestational ages (Fig. 6, panel A). Northern analyses for SP-A, SP-B, SP-C or SP-D showed no significantly differences between wild-type and Dhcr7 null mice at term (Fig. 6, panel B). Thus, surfactant protein expression was not altered by Dhcr7 deficiency. + +Figure 6 + +Surfactant protein expression in lungs. Panel A shows the immunostaining patterns for SP-C in lungs at E18.5 and E20.5 from wild-type (+/+) and knockout (-/-) embryos. No significant differences in the patterns of SP-C staining were evident between wild-type and knockout lungs (panel A, a-d). Note that the underdeveloped epithelial tubules in knockout lungs also expressed SP-C (panel A, b and d, arrowheads), indicating normal type II AECs differentiation. Original magnification: 10X. Panel B, left-hand panel, shows a representative result from Northern analyses of total RNA from wild-type, heterozygous and knockout P0 lungs for SP-A, SP-B, SP-C and SP-D expression. Densitometry analyses showed the relative expression (n = 3, mean ± s.d.) of these surfactant protein mRNAs were not significantly different between these three genotypes. + +Dhcr7-/- lungs showed abnormal differentiation of type I alveolar epithelial cells (AECs) + +At E20.5, the distal lung of wild type embryos consisted of saccular structures lined by flat type I AECs, and by cuboidal surfactant-producing type II AECs (Fig. 7A). In contrast, the formation of these typical saccules was impaired in Dhcr7 deficient lungs. Instead, these saccular structures were lined by a low columnar epithelium that lacked flattened type I-like cells with the presence of many undeveloped or arrested epithelial tubules (Fig. 7B). Consistent with disruption of type I AECs differentiation was the dramatic reduction of expression of T1α, an apical membrane protein marker of lung type I AECs (Fig. 7, cf. panels C and D). Decreased immunostaining for AQP5, another type I AEC marker, was also noted in Dhcr7 deficient lungs at E20.5, compared to control lungs (Fig. 7, cf. panels E and F). Megalin, a low density lipoprotein receptor (LDLR) gene family member, is expressed by embryonic epithelial cells and can function as an endocytic Shh receptor [32]. Megalin was highly expressed on the distal epithelium at E20.5 from wild-type lungs, as well as type I AECs lining the developed saccules (Fig. 7G). In contrast, though megalin staining was detected in developing saccules in knockout embryonic lungs, its expression was confined to undifferentiated cuboidal cells (Fig. 7H), further indicating a delay in type I AECs differentiation in the knockout lungs. + +Figure 7 + +Characterization of type I AECs differentiation. The normal distal saccules of wild-type (+/+) at near term (E20.5) showed typical type I (t1) and type II (t2) epithelial cells (panel A, arrowheads), but knockout (-/-) littermate lungs showed less developed saccules lined by columnar epithelial cells (panel B, black arrowheads), with partially arrested tubules in the distal lung (panel B, green arrowheads). Immunostaining for type I cell markers T1α and AQP5 demonstrated an intense staining in flat epithelial cells in wild-type lungs at E20.5 (panels C and E, arrowheads). In contrast, in knockout littermate lungs, T1α and AQP5 staining was dramatically decreased (panels D and F, arrowheads). Megalin expression (Meg) was detected in the airway epithelial cells, but not in proximal bronchiole epithelial cells (data not shown) in the lungs from both wild-type and knockout at E20.5. The elongated megalin-positive epithelial cells, lining the normally developed sacculi, were readily evident in many areas of the wild-type lungs. In contrast, the terminal epithelial cells positively stained by megalin antibodies in knockout lungs were comprised mainly of undifferentiated cuboidal cells (compare the arrowheads in G and H). Original magnification: A, B, G and H are 60X, C-F are 40X. + +Delayed vascular development in Dhcr7-/- lungs + +There is a close relationship between blood vessel development and lung structural development. At later lung developmental stages, timed capillary bed development is essential for the formation of mature alveolar gas-exchange, with a loss of mesenchyme separating the capillary beds and the AECs [19]. In Dhcr7-/- lungs at late gestational stages, the relative abundance of mesenchyme was consistent with the general arrest in sacculation. To investigate whether the delayed lung sacculation also involved abnormal blood vessel development, immunostaining for the α-isoform of caveolin-1 (cav-1α) and platelet endothelial cell adhesion molecular (PECAM-1) were performed on sections from wild-type and Dhcr7-/- lungs. In the developing lung, cav-1α is expressed strictly in the endothelium of developing capillary and blood vessels. Cav-1α is present in the terminally differentiated type I AECs, but is not detectable in its progenitors or type II AECs [33]. At E14.5, both the epithelium and mesenchyme were negative for cav-1α staining, and cav-1α was detected in the developing blood vessels and capillaries, with no differences observed between wild-type and Dhcr7-/- lungs (Fig. 8, panels A and B). Cav-1α immunostaining demonstrated that the vascular development proceeded rapidly during saccular stages in normal lungs (E17.5 and E20.5). In near-term normal lungs (E20.5), an extensive vascular network, associated with markedly thinned septation walls of well-developed alveoli and close apposition to flat type I AECs, was observed (Fig. 8, panels C and E). In contrast, the cav-1α patterns in lungs from Dhcr7-/- embryos revealed a relatively undeveloped pulmonary capillary bed (Fig. 8, panels D and F). At E20.5, cav-1α stained cells in knockout lungs were embedded in a relatively thick mesenchyme surrounding undeveloped epithelial tubules, and these epithelial cells remained cuboidal, instead of flattening out (Fig. 8, panel F). PECAM-1 staining at E20.5 confirmed the delayed lung vascular development at these late gestational stages in Dhcr7 deficient embryos (Fig. 8, cf. panels G and H). + +Figure 8 + +Immunostaining patterns for caveolin-1α and PECAM-1 in lung sections. Cav-1α staining was compared at E14.5, E17.5 and E20.5 stages of development. Both the epithelium and mesenchyme were negative for cav-1α at E14.5. Developing blood vessels (red arrowheads) and capillaries (arrows) (panels A and B) were stained positively for cav-1α, but the staining patterns between wild-type (+/+) and knockout (-/-) lungs were not altered. Cav-1α was also detected at the proximal bronchiole sub-epithelial matrices (panels A-D, black arrowheads). At E17.5, the airway epithelium, negative for cav-1α staining, was surrounded by rings of cav-1α positively stained capillary networks (panels C and D, arrows). Cav-1α expression patterns in lungs from knockout embryos at E17.5 were similar to wild-type embryos, but relative less capillary bed was noted in knockout lungs (cf. panels C and D). At E20.5 of wild-type lungs, both cav-1α and PECAM-1 staining demonstrated an extensive vascular network in the well developed saccules, with staining observed in close proximity to flat epithelial cells lining the air spaces (panels E and G, arrows). However, delayed vascular development was observed in knockout lungs, showing capillaries (panels F and H, arrows) of the knockout lungs at E20.5 were embedded in a relatively thick mesenchyme around the undeveloped epithelial tubules in which the epithelial cells were remained cuboidal (arrowheads). Original magnification: A-D are 20X; E-H are 40X. + +Is the pattern of sonic hedgehog expression or signaling altered in Dhcr7-/- embryos? + +Shh is critical for normal embryonic development and its autoprocessing, resulting in a cholesterol molecule covalently attached at its C-terminus, is a necessary step for its regulated signaling function [23,34]. Thus, disruption of cholesterol biosynthesis could conceivably disrupt its pattern of distribution and thus affect normal development. To determine whether aberrant expression of Shh contributed to the Dhcr7-/- lung phenotype, distribution of Shh in the Dhcr7-/- and wild-type lungs was assessed from E10.5 to P0 by IHC using Shh-N peptide antibody. At E10.5, the distribution of Shh was detected in the epithelial cells of lung buds (Fig. 9, left panel: C and D), as well as in the more typical Shh-expressing locations, such as the floor plate, notochord and mid gut (Fig. 9, left panel: A-D). Importantly, no differences between Dhcr7-/- and control embryos were noted. Likewise, examination of lung sections from increasing gestational ages through to birth showed no significant differences between wild-type and Dhcr7 deficient tissues. The patterns of staining observed were comparable to those previously reported for Shh in mouse lungs [25]. + +Figure 9 + +Shh and Ptch expression patterns at different gestational stages in Dhcr7-/- and wild-type mice. Left panel shows the Shh immunostaining patterns using an antibody to Shh N-peptide at various gestational stages as indicated by the legend on the y-axis. There were no significant differences noted at any of the stages examined between wild-type (+/+) and knockout (-/-) embryos. At E10.5, Shh was detected in the floor plate (fp), notochord (nc) (A and B), mid-gut (mg) and the epithelial buds of lungs (lb) (C and D). Shh staining was restricted to epithelial cells in the distal region of the primordial tubes of lungs at E13.5 and E15.5. Note that proximal epithelial cells of primordial tubes were not immunostained for Shh (E and F, arrows). Shh was detected in the lung epithelial cells on E18.5, and mainly in the conducting airways in the neonates. Original magnifications: A~H, K and L are 10X, I and J are 20X. Right panel shows the patterns of Ptch IHC at various gestational stages. Immunostaining of Ptch from E13.5 to P 0 confirmed concordance with Shh expression and was not significantly different between wild-type and knockout embryos. Note that the proximal epithelial cells of primordial tubes were also not immunostained for Ptch at E13.5 lungs (arrows). Original magnifications: A~F, I and J are 10X. G and H are 20X. + +To further investigate Shh signaling in these embryos, we examined the patterns of expression of Patched (Ptch), a cognate receptor for Shh and one whose expression is also regulated by Shh signaling. The patterns of Ptch in lung sections from various gestational stages were also indistinguishable between wild-type and knockout embryos (Fig. 9, right panels). The expression of Ptch was in concordance with that of Shh (Fig 9, left and right panels), showing lung epithelial cell expression. Downstream of the Shh-Ptch signaling cascade are proteins such as smoothened (Smo), and Gli proteins, key regulators of target-gene expression in response to Shh signaling. No differences in expression patterns for Smo (Fig. 10, upper panels), Gli1 (Fig. 10, middle panels) or Gli3 (Fig. 10, bottom panels) were noted between wild-type and knockout lungs at E13.5, E15.5 and E17.5 gestational days. + +Figure 10 + +Comparisons of Smo, Gli1 and Gli3 expression in lung sections. Smoothened (Smo, upper panel), Gli1 (middle panel) and Gli3 (bottom panel) showed a pattern of distribution similar to that seen with Shh and Ptch (compare to Fig. 7). No differences between wild-type (+/+) and knockout (-/-) embryos were evident. Original magnifications: 20X. + +Shh protein processing in Dhcr7-/- embryos + +Since Shh expression, as well as some of the Shh signaling components appeared to be normal in Dhcr7-/- embryos, endogenous autoprocessing of Shh to generate a cholesterol-modified N-terminal fragment of Shh (Shh-Np) was evaluated. COS-1 cells, transfected with full length Shh cDNA (capable of endogenous autocleavage) or a truncated Shh cDNA (Shh-N, expression of the shorter form constitutively) were used as controls (Fig. 11, tracks 1 and 2). Transfection of the full-length cDNA for Shh led to the detection of an ~20 kD protein that corresponds with the cleaved and tailed Shh (Fig. 11, track 1), as previously described [35]. Transfection of COS cells with the constitutive truncated Shh-N cDNA led to two slower migrating bands that correspond to truncated Shh, but which are thought to be generated by differences in lipid modifications, as previously demonstrated [35]. In tissue lysates from wild-type, Dhcr7+/- and Dhcr7-/- embryos at E11.5, a band running at the same position as Shh-Np (autocleaved and tailed) was detected in all of the lysates. There were no distinguishable differences between Dhcr7-/- and the wild-type samples, suggesting that Shh autoprocessing occurred normally, despite an absolute deficiency of cholesterol in knockout embryos. More importantly, no band corresponding to a full-length Shh (~45 kD) was present in the lung lysates from the knockout embryos, suggesting that processing of Shh was not impaired. + +Figure 11 + +Shh protein autoprocessing in mouse embryos. Western blotting of lysates from wild-type (track 3), heterozygous (track 4) and knockout (track 5) embryos at E11.5, or COS1 cells transfected with Shh (track 1) or Shh-N (track 2) were probed with anti-Shh-N19 antibodies. 20 μ g of lysate proteins from Shh transfected COS1 cells and 75 μ g from embryos was loaded. Both unprocessed (Shh, upper arrow ~45 kDa, faint signal) and processed (Shh-Np, lowest arrow, strong signal, track 1) forms of Shh were detected from Shh transfected COS1 cells. The two slower migrating bands in Shh-N transfected COS1 cells (track 2) represented Shh-N proteins with different lipid modifications at its N terminus. A major band running at the same position as processed Shh was detected in lysates from all the embryos (tracks 3–5). Importantly, no significant amounts of unprocessed or aberrantly migrating Shh proteins were detected in lysates from knockout embryos at this stage. + +Discussion + +The elucidation that SLOS is caused by a defect in an enzyme necessary for cholesterol biosynthesis has improved our understanding of embryonic development. This discovery has led to the identification of other disorders of embryonic development caused by mutations in genes encoding other enzymes in the post-squalene steps of cholesterol biosynthesis. Although these genetic studies now implicate the need for normal cholesterol synthesis, the mechanistic role(s) of cholesterol in normal embryonic development remains to be clarified. + +In the present study, we report the characterization of lung development in Dhcr7-/- embryos. Although cardiac abnormalities are common in human SLOS [36], no structural abnormalities of the heart or the great vessels were observed in the Dhcr7-/- pups. Lung development in Dhcr7-/- embryos at the early stages, from lung bud formation to canalicular stage, was morphologically similar to controls. Instead, a distinct lung hypoplasia at the saccular stage, characterized by arrested or partially developed distal epithelial tubules, reduced terminal sac space, delayed type I AECs differentiation and delayed vascular development, was consistently observed at late gestational stages in Dhcr7-/- embryos. At late stages of normal fetal development, the mammalian alveolar epithelium undergoes an abrupt differentiation as a part of the preparation of the lungs for the postnatal demands of gas exchange [37]. Both type II AECs, producing pulmonary surfactants, and type I AECs, lining the expanded alveoli, are important for alveolar maturation. In addition, the vascular development of alveoli is also essential for normal lung morphogenesis at late gestational stages [19]. We show here that the differentiation of type I AECs but not type II AECs in Dhcr7-/- lungs was blocked or delayed, as indicated by fewer flattened type I cells and by reduced expression of markers of type I AECs, T1α and AQP5. Delayed vascular development was also involved in the delayed sacculation of Dhcr7-/- lungs. Additionally, the mesenchyme separating the AECs from the vascular beds remained thickened. In support of normal differentiation of type II AECs, no alteration in the gene expression patterns for surfactant proteins A, B, C or D was noted, nor was the pattern of distribution of SP-C altered in lungs from knockout embryos. Thus, impaired gas exchange, as opposed to poor inflation and alveolar tension, may be the cause of respiratory failure in these pups. + +Loss of functional Dhcr7 led to a retardation of embryonic growth from E14.5~E16.5 onwards. By this stage, cholesterol levels were already lower and its immediate precursor, 7-DHC, elevated in the lungs (this study) as well as in the whole body [16,17]. Intrauterine growth retardation (IUGR) followed by postnatal failure to thrive is a universally observed phenotype in SLOS patients [2,18]. We showed here that body length was significantly reduced at later stages of gestation in Dhcr7-/- animals, compared to controls. Whole body weights and fresh lung weights (as well as brain, heart and kidney, data not shown) at late gestational stages were also lower in Dhcr7-/- embryos. Interestingly, the lower lung/body ratios at E18.5 in Dhcr7-/- embryos compared to wild-type suggested a disproportionate inhibition of lung growth than other organs. Cell proliferation and division in Dhcr7-/- lungs were markedly arrested without apparent increase in cell apoptosis. Therefore, it appears that growth retardation caused by cholesterol deficiency was primarily because of an inhibition of cell growth and proliferation, rather than an increase in cell death. + +Cholesterol is present in Dhcr7-/- embryos, as it is in Sc5d-/- (lathosterolosis) and Dhcr24-/-(desmosterolosis) embryos [15,38]. Total sterols and cholesterol in Dhcr7-/- embryonic lungs (as well as in brain and liver, data not shown) were reduced and 7DHC/8DHC elevated, but a significant amount of cholesterol was detectable, comprising ~60% and ~30% of total tissue sterols at E13.5 and neonate, respectively. Fetal cholesterol can either be synthesized endogenously in fetal tissues or accrued from extra-embryonic tissues such as maternal serum, placenta, and yolk sac [39]. Since these mice theoretically can not synthesize cholesterol, any accumulation of cholesterol must be derived from the maternal sources. Interestingly, Dhcr24 null mice are viable but contained almost no cholesterol in plasma and tissues at 3-months, whereas cholesterol content in Dhcr24 null embryo tissues accounts for ~60% at E11.5 and ~30% at E17.5, of total tissue sterols [38]. These results, as well as this study, suggest that a considerable amount of maternal cholesterol can be transferred to the murine fetus. One explanation of why the Dhcr24-/- mice are developmentally normal but Dhcr7-/- mice are affected may be because desmosterol can functionally substitute for cholesterol, whereas the other cholesterol precursors can not. + +Although a mechanistic understanding of why a deficiency in cholesterol biosynthesis leads to abnormal embryonic development is lacking, a frequently advanced explanation has been that Shh signaling, involved in early developmental patterning, may be disrupted. In the developing lung primordium, Shh is expressed initially at E9 and promotes branching morphogenesis, which is impaired in Shh knockout mice [24], suggesting that Shh, secreted by the epithelium, is critical for branching morphogenesis. In this study, the lack of evidence of abnormal lung branching morphogenesis in the early stages, but a consistent delayed maturation of the gas-exchange region of the lung in the saccular period of development of Dhcr7-/- embryos was observed, suggesting early Shh signaling was normal. Shh protein expression and autoprocessing at E11.5 was indistinguishable between wild-type and Dhcr7-/- embryos. Additionally, although these experiments were not quantitative, the abundance of Shh appeared unchanged between these genotypes. Thus, it is possible that the cholesterol levels (roughly 50% of normal levels) in Dhcr7-/- embryos were enough for completion of Shh autoprocessing or 7DHC accumulated in Dhcr7-/- embryonic tissues can also participate efficiently as a sterol adduct in the Shh processing reaction [20]. In this study, we also examined the Shh signaling pathway by immunohistochemistry of known key components of this pathway, such as patched, smoothened and Gli proteins. Shh signaling leads to the activation of transcription and increased protein levels of these components [40-42]. If Shh signaling were defective in Dhcr7-/- embryos, these could have resulted in abnormalities of the staining patterns of these components, but no such disturbance was detected. Indeed, the patterns of protein expression seemed comparable and indistinguishable from wild-type embryos at all stages of lung development examined. + +In a recent in vitro study [43], mouse embryonic fibroblasts (MEFs) from Dhcr7-/- embryos, grown in lipid-depleted culture and transiently treated with cyclodextrin, showed no affect on Shh processing. However, a deficiency in their ability to respond to Shh activation, as judged by reporter gene expression, was demonstrable. In contrast, when CHO cells, that stably expressed Shh, were subjected to sterol deprivation, an arrest of Shh autoprocessing was clearly demonstrable [29]. In the present study, we did not detect unprocessed Shh in Dhcr7-/- embryonic tissues at E11.5. Subtle differences in Shh signal transduction can not be excluded by the present study. However, we favor a model whereby the loss of normal cholesterol biosynthesis is important, not so much for Shh autocleavage, as for the loss of the correct plasma membrane milieu. Since the loss of cholesterol content in the plasma membrane (and perhaps any membrane) is likely to alter the function of receptors more sensitive to this, loss of normal cholesterol synthesis may cause a loss of signal transduction of a host of such pathways. Thus, this effect may not be limited to the Shh pathway and other targets may be more important. This hypothesis is amenable to testing in the Dhcr7 null mice. + +Conclusion + +Cholesterol deficiency caused by Dhcr7 deficiency in mice resulted in, along with IUGR, a distinct lung saccular hypoplasia with impaired differentiation of type I but not type II AECs and delayed associated vascular development. Loss of the correct amounts of cholesterol in the membranes may perturb those receptors that depend upon cholesterol for efficient signal transduction. Further investigation of which transcriptional and signal transduction pathways are more significantly affected by the loss of Dhcr7 will be critical to elucidate the mechanism of lung hypoplasia of this model, as well as the role of cholesterol in embryonic development. + +Methods + +Animals + +All mice were housed in an animal facility at 22°C with a 12 h light ~12 h dark cycle and all protocols were approved by the Institutional Animal Welfare Committee. Dhcr7 heterozygous animals used in this study have been backcrossed onto a C57BL/6J background for 11 successive generations. The presence of vaginal plugs was considered as embryonic day (E) 0.5. Timed pregnant females were sacrificed, embryos dissected from the uteri and placed in 1X PBS. Crown-rump lengths of the embryos were measured by use of an electronic digital caliper and embryos weighed, after blotting off excess fluid. Each lung was removed intact from the thoracic cavity, blotted free of excess fluid, and weighed. Mice were classified as P0 when birth was witnessed and their breathing activities visualized under a dissecting microscope. Digital images of embryos or lungs were captured using a Zeiss-1000 dissecting microscope. Genotyping was performed by PCR, as described previously [16]. And in selective cases, Southern blotting was performed for confirmation of Dhcr7 genotype. + +Biochemical measurements + +Tissue sterol compositions were analyzed by gas chromatography-mass spectrometry (GC-MS) as described previously [2,44]. Serum corticosterone levels were measured by radioimmunoassay (RIA) on the blood obtained from decapitated neonates by a commercial source (DRTC hormone Assay Core Lab, Vanderbilt University, TN). + +Histology and Immunohistochemistry + +Thoracic parts from embryos or P0 mice were fixed in either Amsterdam's fixative (methanol:acetone:acetic acid:water, 35:35:5:25 v/v) or 4% paraformaldehyde in PBS. Tissues were fixed overnight at room temperature (RT), rinsed in PBS, dehydrated in a graded ethanol series, cleared in toluene and embedded in paraffin. Five μ m sections were mounted on Superfrost-Plus slides (VWR, West Chester, PA) for histological or immunohistochemistry (IHC) analyses. + +IHC was performed using an indirect method. After deparaffination in xylene and rehydration through a graded series of ethanol, sections were heated at 95°C in 10 mM sodium citrate (pH 6.0) for an initial 15 min and for three successive 5-min periods [25]. After cooling, the sections were treated with hydrogen peroxide (3% v/v in PBS, pH7.4) for 10 min and rinsed in the PBS. Sections were blocked with 5% normal serum for 2 h, washed in PBS and incubated with antibodies against Shh-N19 (1:100), Patched (1:100), Smo-N19 (1:100), Gli1 (1:100), Gli3-N19 (1:100), SP-C C19 (1:200), caveolin 1α-N20 (1:200), aquaporin 5 (AQP5, 1:200), megalin (1:200), PECAM-1 (1:200) (Santa Cruz Biotechnology, Santa Cruz, CA) or T1α (mAB 8.1.1, 1:1000 dilution, Developmental Studies Hybridoma Bank, Iowa City, IA) overnight at 4° C in a humidified chamber. The sections were washed in PBS, incubated with biotinylated secondary antibodies (1:1000~1:5000, Santa Cruz Biotechnology) in PBS for 2 h at RT, incubated with HRP-streptavidin complex for 30 min, rinsed in PBS and visualized using a Metal Enhanced Diaminobenzidine (DAB) Substrate Kit (Pierce, Rockford, IL). Digital images were collected using a Polaroid Digital Microscope Camera mounted on an Olympus BX40 microscope. + +Lung morphometry + +Terminal saccular space areas were measured using NIH Image software (NIH, Bethesda, MD) as previously described [45]. Multiple measurements were performed on randomly selected 0.04-mm2 fields located in the distal part of the lung sections. The proportion of lung comprising terminal saccular spaces was calculated as a percentage of the total examined area of the lung section. Three animals per genotype were examined. + +Assessment of cell proliferation/Division/death + +Changes in cell proliferation and division were assessed by proliferating cell nuclear antigen (PCNA) IHC, bromodeoxyuridine (BrdU) incorporation and phosphorylated histone H3 (pHH3) IHC. The PCNA (1:2000) and pHH3-Ser28 (1:200) antibodies were obtained from Santa Cruz Biotechnology. For BrdU incorporation, timed pregnant mice at E18.5 were injected intra-peritoneally with 100 μg/gm body weight of BrdU (Sigma, St Louis, MO) 2 h prior to sacrifice, embryos dissected from uteri and processed as described above. Sections were stained with a monoclonal anti-BrdU antibody (1:100, Developmental Studies Hybridoma Bank, Iowa City, IA) and visualized with a Cy2-conjugated rabbit anti-mouse antibody using a laser-scanning confocal microscope. Cell death was investigated by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) using the Fluorescein In Situ Cell Death Detection Kit (Roche, Indianapolis, IN) according to the manufacturer's protocol. + +Western blotting + +Sample preparation and western blotting was performed according to Kawakami et al with some minor modifications [35]. Briefly, 3 embryos from each genotype were pooled, homogenized in ice cold lysis buffer (50 mM Tris-pH8.0, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5 mM DTT, 1 mM PMSF and 2 μg/ml each of aprotinin, leupeptin and pepstatin A) with a tissue homogenizer (PRO Scientific Inc., Oxford, CT) using three pulses of 10 seconds each and then subjected to Dounce homogenizer by 10 strokes. The homogenates were centrifuged at 10,000 g for 30 minutes at 4°C, supernatants collected, protein concentration determined and used for western blotting. COS1 cells were transfected using Superfect transfection reagent (Qiagen, Valencia, CA) with expression constructs encoding either the full-length Shh or the N-terminal fragment of Shh without post-translational modifications (constructs kindly provided by Dr. Pao-Tien Chuang, Cardiovascular Research Institute, University of California, San Francisco, CA), harvested 3 days after transfection and protein extracts prepared as described above. Protein concentration was determined using Bio-Rad protein assay kit (BioRad, Hercules, CA) and 20 μg (COS1 cells) and 75 μg (embryos) of lysates separated by 15 % SDS-PAGE, transferred onto nitrocellulose membrane, incubated with primary antibody (Shh-N19, 1:100) for 2 hours, followed by donkey anti-goat HRP second antibody (1:1000) and visualized by chemiluminescent detection according to manufacturer's protocols (Perkin-Elmer Life Sciences, Boston MA). + +Northern blotting + +Northern blotting was performed as previously described [46], using total RNA (20 μg) from P0 mice lungs. The cDNA probes for SP-A, SP-B and SP-C were kindly provided by Dr. Jeffery Whitsett (Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio). A cDNA probe for SP-D was amplified by RT-PCR, using murine SP-D cDNA specific primers (sense 5'-TGCCTGGTCGTGATGGACG-3', and antisense 5'-AGCACTGTCTGGAAGCCCGC-3') and confirmed by sequencing. + +Author's contributions + +HY designed the experiments. HY, ALP and JLC performed experiments. + +GST analyzed tissue sterols by GC-MS and JO performed lung morphometric analyses. HY, AW and SBP analyzed the data. HY and SBP prepared the manuscript. All authors read and approved the final manuscript. + +Acknowledgements + +We are grateful to Jeffery Whitsett for providing cDNA probes for SP-A, SP-B and SP-C and Pao-Tien Chuang for the Shh and Shh-N plasmids. This research was supported by a Beginning Grant-in-Aid 0160349U from the American Heart Association, Mid-Atlantic Consortium (HY); NIH PO1 HL52813 and NIH PO1 HD39946 (AW) and NIH HL68660 (SBP). diff --git a/src/ontogpt/evaluation/craft/database/all/15018652.ann b/src/ontogpt/evaluation/craft/database/all/15018652.ann new file mode 100644 index 000000000..17f8ef9d6 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15018652.ann @@ -0,0 +1,433 @@ +T1 PR:000006666 0 5 Dppa3 +T2 PR:000006666 8 12 Pgc7 +T3 PR:000006666 15 21 stella +T4 CL:0000586 67 76 germ cell +T5 GO:0001708 72 90 cell specification +T6 NCBITaxon:10088 94 98 mice +T7 NCBITaxon:10088 125 129 mice +T8 CL:0000586 131 141 germ cells +T9 UBERON:0000119 183 198 layers of cells +T10 UBERON:0000922 224 230 embryo +T11 PR:000006666 248 253 Dppa3 +T12 PR:000006666 269 273 Pgc7 +T13 PR:000006666 277 283 stella +T14 SO:0000704 288 292 gene +T15 GO:0010467 293 302 expressed +T16 CL:0000670 306 327 primordial germ cells +T17 GO:0007369 362 374 gastrulating +T18 UBERON:0000922 375 382 embryos +T19 CL:0000586 459 468 germ cell +T20 GO:0001708 464 482 cell specification +T21 PR:000006666 502 507 Dppa3 +T22 CL:0000586 518 527 germ cell +T23 GO:0007281 518 539 germ cell development +T24 SO:0000704 558 562 gene +T25 NCBITaxon:10088 566 571 mouse +T26 UBERON:0000922 572 581 embryonic +T27 CL:0002322 572 592 embryonic stem cells +T28 NCBITaxon:33208 614 621 animals +T29 PR:000006666 665 670 Dppa3 +T30 NCBITaxon:33208 681 688 animals +T31 UBERON:0000922 719 728 embryonic +T32 CL:0002321 719 728;744 749 embryonic ... cells +T33 UBERON:0007023 733 738 adult +T34 CL:0000586 739 749 germ cells +T35 UBERON:0000991 754 760 gonads +T36 PR:000006666 764 769 Dppa3 +T37 NCBITaxon:33208 780 787 animals +T38 UBERON:0000922 830 837 embryos +T39 PR:000006666 851 856 Dppa3 +T40 CL:0000023 867 874 oocytes +T41 UBERON:0007233 913 928 four-cell stage +T42 PR:000006666 957 962 Dppa3 +T43 GO:0040016 1002 1010 cleavage +T44 UBERON:0000107 1002 1017 cleavage stages +T45 NCBITaxon:10088 1021 1026 mouse +T46 GO:0009790 1027 1040 embryogenesis +T47 CL:0000586 1074 1083 germ cell +T48 GO:0001708 1079 1097 cell specification +T49 UBERON:0007023 1161 1166 adult +T50 NCBITaxon:40674 1167 1174 mammals +T51 GO:0009790 1223 1236 embryogenesis +T52 CL:0000586 1241 1251 germ cells +T53 CL:0000025 1272 1276 eggs +T54 CL:0000019 1281 1286 sperm +T55 CHEBI:36357 1294 1303 molecules +T56 CL:0000586 1331 1341 germ cells +T57 NCBITaxon:33208 1357 1364 animals +T58 NCBITaxon:7147 1376 1381 flies +T59 CL:0000025 1444 1447 egg +T60 GO:0009566 1455 1468 fertilization +T61 UBERON:0000922 1512 1521 embryonic +T62 CL:0002321 1512 1527 embryonic cells +T63 CL:0000586 1551 1561 germ cells +T64 NCBITaxon:40674 1572 1581 mammalian +T65 UBERON:0000922 1582 1589 embryos +T66 CL:0000586 1591 1601 germ cells +T67 UBERON:0000119 1671 1686 layers of cells +T68 UBERON:0019248 1702 1718 primitive embryo +T69 CL:0000586 1849 1858 germ cell +T70 NCBITaxon:10088 1867 1871 mice +T71 CL:0000586 1915 1924 germ cell +T72 GO:0001708 1920 1938 cell specification +T73 PR:000006666 1942 1948 stella +T74 PR:000006666 1951 1955 PGC7 +T75 PR:000006666 1958 1963 Dppa3 +T76 SO:0000704 1967 1971 gene +T77 GO:0010467 1972 1981 expressed +T78 CL:0000670 1985 2006 primordial germ cells +T79 CL:0000023 2040 2047 oocytes +T80 PR:000006666 2083 2088 Dppa3 +T81 NCBITaxon:10088 2109 2114 Mouse +T82 SO:0001026 2115 2121 Genome +T83 SO:0000704 2167 2171 gene +T84 PR:000006666 2205 2210 Dppa3 +T85 CL:0000586 2221 2230 germ cell +T86 GO:0001708 2226 2244 cell specification +T87 SO:0000704 2285 2289 gene +T88 GO:0010467 2292 2302 expression +T89 NCBITaxon:10088 2402 2406 mice +T90 PR:000006666 2415 2420 Dppa3 +T91 UBERON:0000922 2436 2443 embryos +T92 CL:0000586 2460 2470 germ cells +T93 SO:0000704 2524 2528 gene +T94 PR:000006666 2556 2561 Dppa3 +T95 NCBITaxon:10088 2572 2576 mice +T96 PR:000006666 2661 2666 Dppa3 +T97 SO:0000704 2667 2671 gene +T98 UBERON:0000922 2684 2693 embryonic +T99 CL:0002322 2684 2698;2704 2709 embryonic stem ... cells +T100 CL:0002322 2700 2702;2704 2709 ES ... cells +T101 PR:000006666 2732 2737 Dppa3 +T102 NCBITaxon:10088 2748 2752 mice +T103 SO:0000236 2791 2809 open reading frame +T104 PR:000006666 2813 2818 Dppa3 +T105 NCBITaxon:10088 2822 2827 mouse +T106 CL:0002322 2833 2841 ES cells +T107 CHEBI:24753 2853 2863 hygromycin +T108 SO:0005853 2891 2899 cassette +T109 SO:0000357 2900 2907 flanked +T110 SO:0000346 2911 2921 loxP sites +T111 SO:0005853 2949 2957 cassette +T112 GO:0010467 2997 3007 expression +T113 CL:0002322 3039 3047 ES cells +T114 PR:000006666 3076 3081 Dppa3 +T115 CL:0002322 3090 3098 ES cells +T116 NCBITaxon:10088 3130 3134 mice +T117 PR:000006666 3202 3207 Dppa3 +T118 NCBITaxon:33208 3229 3236 animals +T119 PR:000006666 3245 3250 Dppa3 +T120 PR:000006666 3257 3262 Dppa3 +T121 PR:000006666 3292 3297 Dppa3 +T122 CL:0000365 3358 3365 zygotic +T123 PR:000006666 3378 3383 Dppa3 +T124 CL:0000586 3460 3469 germ cell +T125 GO:0007281 3460 3481 germ cell development +T126 NCBITaxon:33208 3485 3492 animals +T127 PR:000006666 3501 3506 Dppa3 +T128 PR:000006666 3533 3538 Dppa3 +T129 NCBITaxon:33208 3549 3556 animals +T130 SO:0001026 3589 3596 genomic +T131 PR:000006666 3609 3614 Dppa3 +T132 SO:0000704 3620 3624 gene +T133 SO:0000147 3632 3637 exons +T134 SO:0000486 3649 3670;3675 3680;3690 3695 non-coding regions of ... first ... exons +T135 SO:0000484 3649 3670;3685 3695 non-coding regions of ... last exons +T136 CHEBI:24753 3717 3727 hygromycin +T137 CHEBI:24753 3746 3751 Hygro +T138 SO:0005853 3756 3764 cassette +T139 SO:0000236 3785 3803 open reading frame +T140 SO:0000704 3811 3815 gene +T141 SO:0005853 3856 3864 cassette +T142 SO:0000842 3892 3903;3908 3912 portions of ... gene +T143 SO:0000346 3937 3946 loxP site +T144 SO:0000112 4008 4015 primers +T145 PR:000006666 4055 4060 Dppa3 +T146 SO:0001023 4061 4068 alleles +T147 PR:000006666 4130 4135 Dppa3 +T148 NCBITaxon:33208 4145 4152 animals +T149 SO:0001023 4218 4224 allele +T150 SO:0000006 4234 4245 PCR product +T151 SO:0000028 4253 4255 bp +T152 SO:0000112 4261 4268 primers +T153 SO:0001023 4291 4297 allele +T154 PR:000006666 4299 4304 Dppa3 +T155 SO:0000006 4321 4332 PCR product +T156 SO:0000028 4340 4342 bp +T157 SO:0000112 4348 4355 primers +T158 PR:000006666 4400 4405 Dppa3 +T159 CL:0000586 4426 4435 germ cell +T160 GO:0001708 4431 4449 cell specification +T161 PR:000006666 4506 4511 Dppa3 +T162 CL:0000586 4515 4524 germ cell +T163 UBERON:0000991 4549 4555 gonads +T164 PR:000006666 4559 4564 Dppa3 +T165 PR:000006666 4571 4576 Dppa3 +T166 UBERON:0000922 4583 4590 embryos +T167 CL:0000586 4601 4611 germ cells +T168 GO:0010467 4627 4637 expression +T169 PR:000006666 4697 4702 Dppa3 +T170 UBERON:0000922 4720 4727 embryos +T171 UBERON:0000992 4753 4760 ovaries +T172 PR:000006666 4764 4769 Dppa3 +T173 PR:000006666 4776 4781 Dppa3 +T174 UBERON:0007023 4788 4793 adult +T175 GO:0010467 4802 4811 expressed +T176 PR:000013044 4812 4816 Oct4 +T177 CL:0000023 4830 4837 oocytes +T178 PR:000006666 4870 4875 Dppa3 +T179 GO:0010467 4876 4886 expression +T180 UBERON:0000991 4939 4945 gonads +T181 PR:000006666 4949 4954 Dppa3 +T182 PR:000006666 4962 4967 Dppa3 +T183 UBERON:0007023 4974 4980 adults +T184 GO:0007283 5016 5031 spermatogenesis +T185 UBERON:0001305 5045 5061 ovarian follicle +T186 GO:0001541 5045 5073 ovarian follicle development +T187 PR:000006666 5152 5157 Dppa3 +T188 PR:000006666 5164 5169 Dppa3 +T189 NCBITaxon:10088 5176 5180 mice +T190 PR:000006666 5216 5221 Dppa3 +T191 PR:000006666 5228 5233 Dppa3 +T192 PR:000006666 5326 5331 Dppa3 +T193 SO:0000704 5332 5336 gene +T194 CL:0000586 5357 5366 germ cell +T195 GO:0001708 5362 5380 cell specification +T196 CL:0000586 5400 5409 germ cell +T197 GO:0007281 5400 5421 germ cell development +T198 PR:000006666 5440 5445 Dppa3 +T199 UBERON:0000991 5450 5456 Gonads +T200 UBERON:0000922 5468 5475 embryos +T201 PR:000006666 5502 5507 Dppa3 +T202 PR:000006666 5514 5519 Dppa3 +T203 CL:0000670 5570 5591 primordial germ cells +T204 SO:0000704 5615 5619 gene +T205 GO:0010467 5615 5630 gene expression +T206 PR:000006666 5648 5653 Dppa3 +T207 PR:000006666 5660 5665 Dppa3 +T208 UBERON:0007023 5672 5677 adult +T209 UBERON:0000992 5678 5685 ovaries +T210 PR:000006666 5692 5697 Dppa3 +T211 PR:000006666 5704 5709 Dppa3 +T212 UBERON:0000473 5716 5722 testis +T213 UBERON:0000992 5731 5736 ovary +T214 SO:0000704 5822 5826 gene +T215 PR:000006666 5846 5851 Dppa3 +T216 NCBITaxon:10088 5894 5899 mouse +T217 SO:0001026 5900 5906 genome +T218 PR:000006666 5911 5916 Dppa3 +T219 SO:0000853 5917 5927 homologues +T220 SO:0000043 5951 5960;5975 5986 processed ... pseudogenes +T221 SO:0000188 5962 5968 intron +T222 PR:000006666 5990 5995 Dppa3 +T223 SO:0000853 6028 6037 homologue +T224 PR:000006666 6073 6078 Dppa3 +T225 SO:0000336 6079 6090 pseudogenes +T226 GO:0010467 6099 6108 expressed +T227 UBERON:0005291 6112 6121;6131 6138 embryonic ... tissues +T228 UBERON:0007023 6125 6130 adult +T229 PR:000006666 6158 6163 Dppa3 +T230 PR:000006666 6207 6212 Dppa3 +T231 UBERON:0019248 6240 6255 early embryonic +T232 GO:0009790 6246 6267 embryonic development +T233 PR:000006666 6296 6301 Dppa3 +T234 PR:000006666 6308 6313 Dppa3 +T235 PR:000006666 6346 6351 Dppa3 +T236 PR:000006666 6358 6363 Dppa3 +T237 PR:000006666 6464 6469 Dppa3 +T238 PR:000006666 6476 6481 Dppa3 +T239 PR:000006666 6489 6494 Dppa3 +T240 PR:000006666 6606 6611 Dppa3 +T241 SO:0000704 6648 6655 genetic +T242 GO:0007618 6690 6695 mated +T243 PR:000006666 6699 6704 Dppa3 +T244 PR:000006666 6711 6716 Dppa3 +T245 PR:000006666 6726 6731 Dppa3 +T246 PR:000006666 6843 6848 Dppa3 +T247 PR:000006666 6855 6860 Dppa3 +T248 GO:0009790 6915 6928 embryogenesis +T249 GO:0040016 6941 6949 cleavage +T250 UBERON:0000107 6941 6956 cleavage stages +T251 GO:0007566 6964 6976 implantation +T252 UBERON:0000922 7007 7014 embryos +T253 PR:000006666 7028 7033 Dppa3 +T254 CL:0000023 7044 7051 oocytes +T255 UBERON:0007232 7069 7075;7086 7091 2-cell ... stage +T256 UBERON:0007233 7079 7091 4-cell stage +T257 UBERON:0000922 7175 7182 embryos +T258 UBERON:0000922 7204 7211 embryos +T259 PR:000006666 7225 7230 Dppa3 +T260 CL:0000023 7241 7248 oocytes +T261 UBERON:0007236 7269 7281 8-cell stage +T262 UBERON:0007233 7331 7343 4-cell stage +T263 UBERON:0000922 7351 7358 embryos +T264 PR:000006666 7372 7377 Dppa3 +T265 CL:0000023 7388 7395 oocytes +T266 GO:0040016 7396 7403 cleaved +T267 CL:0000353 7420 7431 blastomeres +T268 PR:000006666 7523 7528 Dppa3 +T269 GO:0040016 7558 7566 cleavage +T270 UBERON:0000107 7558 7573 cleavage stages +T271 GO:0007566 7581 7593 implantation +T272 GO:0007566 7631 7643 implantation +T273 GO:0009790 7644 7666 development of embryos +T274 UBERON:0000922 7659 7666 embryos +T275 PR:000006666 7680 7685 Dppa3 +T276 CL:0000023 7696 7703 oocytes +T277 UBERON:0000922 7753 7760 embryos +T278 GO:0007618 7784 7791 matings +T279 UBERON:0000922 7833 7840 embryos +T280 PR:000006666 7862 7867 Dppa3 +T281 PR:000006666 7874 7879 Dppa3 +T282 UBERON:0000922 7934 7941 embryos +T283 GO:0007618 7965 7972 matings +T284 UBERON:0000358 7996 8006 blastocyst +T285 UBERON:0000922 8041 8048 embryos +T286 PR:000006666 8070 8075 Dppa3 +T287 PR:000006666 8082 8087 Dppa3 +T288 UBERON:0000358 8150 8160 blastocyst +T289 GO:0040016 8179 8185 cleave +T290 UBERON:0000922 8227 8234 embryos +T291 PR:000006666 8256 8261 Dppa3 +T292 PR:000006666 8268 8273 Dppa3 +T293 PR:000006666 8293 8298 Dppa3 +T294 PR:000013044 8307 8313 Pou5f1 +T295 PR:000013044 8331 8337 Pou5f1 +T296 UBERON:0007233 8394 8406 4-cell stage +T297 GO:0010467 8427 8434 express +T298 PR:000013044 8439 8443 Oct4 +T299 PR:000006666 8476 8481 Dppa3 +T300 CL:0000365 8489 8496 zygotic +T301 GO:0010467 8497 8507 expression +T302 PR:000013044 8511 8515 Oct4 +T303 PR:000013044 8516 8522 Pou5f1 +T304 GO:0007566 8584 8596 implantation +T305 PR:000006666 8655 8660 Dppa3 +T306 PR:000006666 8667 8672 Dppa3 +T307 PR:000006666 8692 8697 Dppa3 +T308 PR:000013044 8706 8712 Pou5f1 +T309 PR:000013044 8730 8736 Pou5f1 +T310 PR:000013044 8785 8789 Oct4 +T311 SO:0000902 8794 8803 transgene +T312 PR:000013044 8814 8818 Oct4 +T313 SO:0000167 8819 8827 promoter +T314 GO:0010467 8836 8846 expression +T315 UBERON:0000922 8882 8889 embryos +T316 UBERON:0007232 8897 8909 2-cell stage +T317 GO:0010467 8961 8971 expression +T318 PR:000013044 8979 8983 Oct4 +T319 SO:0000902 8988 8997 transgene +T320 UBERON:0000922 9008 9015 embryos +T321 GO:0010467 9033 9040 express +T322 UBERON:0000922 9103 9110 embryos +T323 GO:0009790 9199 9213;9219 9226 development of ... embryos +T324 UBERON:0000922 9219 9226 embryos +T325 PR:000006666 9240 9245 Dppa3 +T326 CL:0000023 9256 9263 oocytes +T327 CL:0000365 9303 9310 zygotic +T328 GO:0010467 9311 9321 expression +T329 PR:000013044 9325 9329 Oct4 +T330 PR:000006666 9384 9389 Dppa3 +T331 SO:0000704 9469 9473 gene +T332 PR:000006666 9517 9522 Dppa3 +T333 CL:0000586 9543 9552 germ cell +T334 GO:0001708 9548 9566 cell specification +T335 NCBITaxon:10088 9570 9574 mice +T336 NCBITaxon:40674 9596 9605 mammalian +T337 SO:0000704 9606 9610 gene +T338 SO:0000704 9614 9619 genes +T339 CL:0000586 9633 9643 germ cells +T340 PR:000006666 9670 9675 Dppa3 +T341 GO:0009790 9750 9763 embryogenesis +T342 GO:0040016 9772 9780 cleavage +T343 PR:000006666 9806 9811 Dppa3 +T344 NCBITaxon:33208 9822 9829 animals +T345 PR:000006666 9835 9840 Dppa3 +T346 NCBITaxon:10088 9899 9904 mouse +T347 SO:0001026 9905 9912 genomic +T348 PR:000006666 9943 9948 Dppa3 +T349 GO:0006413 9951 9973 translation initiation +T350 SO:0000323 9951 9978 translation initiation site +T351 SO:0000319 10012 10029 termination codon +T352 CHEBI:24753 10084 10094 hygromycin +T353 SO:0005853 10122 10130 cassette +T354 CHEBI:24753 10132 10137 Hygro +T355 SO:0000357 10142 10149 flanked +T356 SO:0000346 10157 10161 loxP +T357 CL:0002322 10204 10212 ES cells +T358 GO:0009294 10222 10233 transfected +T359 CHEBI:24753 10309 10319 hygromycin +T360 SO:0001026 10393 10400 genomic +T361 CHEBI:24753 10410 10415 Hygro +T362 SO:0005853 10419 10427 cassette +T363 GO:0009294 10454 10466 transfection +T364 CL:0002322 10470 10478 ES cells +T365 GO:0010467 10490 10500 expressing +T366 SO:0000155 10501 10508 plasmid +T367 CHEBI:465284 10528 10539 ganciclovir +T368 SO:0001026 10559 10566 genomic +T369 CL:0002322 10663 10670 ES cell +T370 UBERON:0000358 10709 10720 blastocysts +T371 NCBITaxon:33208 10743 10750 Animals +T372 SO:0000704 10803 10810 genetic +T373 SO:0000112 10824 10831 Primers +T374 SO:0001023 11025 11031 allele +T375 SO:0000006 11048 11059 PCR product +T376 SO:0000028 11067 11069 bp +T377 SO:0000112 11075 11082 primers +T378 SO:0001023 11105 11111 allele +T379 SO:0000006 11142 11153 PCR product +T380 SO:0000028 11161 11163 bp +T381 SO:0000112 11169 11176 primers +T382 NCBITaxon:10088 11245 11250 mouse +T383 UBERON:0000479 11251 11258 tissues +T384 GO:0010467 11264 11274 expression +T385 PR:000006666 11278 11283 Dppa3 +T386 PR:000013044 11285 11289 Oct4 +T387 CL:0000670 11389 11410 primordial germ cells +T388 UBERON:0000991 11412 11418 Gonads +T389 PR:000006666 11453 11458 Dppa3 +T390 PR:000006666 11466 11471 Dppa3 +T391 UBERON:0000922 11478 11485 embryos +T392 GO:0007565 11501 11510 gestation +T393 UBERON:0007023 11600 11605 adult +T394 UBERON:0000473 11606 11612 testes +T395 UBERON:0000992 11617 11624 ovaries +T396 CHEBI:75958 11657 11665 solution +T397 CHEBI:51686 11717 11728 hematoxylin +T398 PR:000013044 11755 11759 Oct4 +T399 NCBITaxon:33208 11775 11782 animals +T400 NCBITaxon:10088 11784 11788 Mice +T401 PR:000013044 11800 11804 Oct4 +T402 SO:0000902 11809 11818 transgene +T403 SO:0000987 11876 11882 linear +T404 NCBITaxon:10088 11925 11930 mouse +T405 CL:0000025 11931 11935 eggs +T406 PR:000033987 12010 12014 lacZ +T407 SO:0000704 12045 12049 gene +T408 PR:000033987 12118 12122 lacZ +T409 PR:000013044 12137 12141 Oct4 +T410 NCBITaxon:10088 12143 12147 Mice +T411 SO:0000902 12156 12165 transgene +T412 PR:000013044 12169 12175 Pou5f1 +T413 PR:000013044 12236 12240 Oct4 +T414 GO:0010467 12241 12251 expression +T415 PR:000013044 12294 12300 Pou5f1 +T416 PR:000013044 12319 12325 Pou5f1 +T417 NCBITaxon:33208 12351 12358 animals +T418 GO:0040016 12396 12404 cleavage +T419 UBERON:0000107 12396 12410 cleavage stage +T420 UBERON:0000922 12411 12418 embryos +T421 UBERON:0000922 12427 12434 embryos +T422 UBERON:0000993 12453 12461 oviducts +T423 CHEBI:46662 12554 12561 mineral +T424 CHEBI:16526 12583 12586 CO2 +T425 UBERON:0000922 12600 12607 embryos +T426 UBERON:0000995 12626 12631 uteri +T427 CL:0002322 12693 12700 ES cell +T428 UBERON:0000922 12713 12726 embryological +T429 UBERON:0000358 12780 12790 blastocyst +T430 NCBITaxon:10088 12818 12823 mouse +T431 UBERON:0000922 12828 12841 embryological +T432 http://purl.obolibrary.org/obo/MONDO_0005059 12987 12995 Leukemia +T433 http://purl.obolibrary.org/obo/MONDO_0005062 12998 13006 Lymphoma diff --git a/src/ontogpt/evaluation/craft/database/all/15018652.txt b/src/ontogpt/evaluation/craft/database/all/15018652.txt new file mode 100644 index 000000000..dc17da551 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15018652.txt @@ -0,0 +1,91 @@ +Dppa3 / Pgc7 / stella is a maternal factor and is not required for germ cell specification in mice + +Abstract + +Background + +In mice, germ cells are specified through signalling between layers of cells comprising the primitive embryo. The function of Dppa3 (also known as Pgc7 or stella), a gene expressed in primordial germ cells at the time of their emergence in gastrulating embryos, is unknown, but a recent study has claimed that it plays a central role in germ cell specification. + +Results + +To test Dppa3's role in germ cell development, we disrupted the gene in mouse embryonic stem cells and generated mutant animals. We were able to obtain viable and fertile Dppa3-deficient animals of both sexes. Examination of embryonic and adult germ cells and gonads in Dppa3-deficient animals did not reveal any defects. However, most embryos derived from Dppa3-deficient oocytes failed to develop normally beyond the four-cell stage. + +Conclusion + +We found that Dppa3 is an important maternal factor in the cleavage stages of mouse embryogenesis. However, it is not required for germ cell specification. + +Background + +Among the many specialized cell types present in adult mammals, the first to be programmed or specified during embryogenesis are germ cells, which give rise to eggs and sperm. Which molecules direct this programming of germ cells? In many other animals, including flies and worms, material known as "germ plasm" is laid down in the egg before fertilization, and its subsequent passage to a subset of embryonic cells dictates their fate as germ cells [1,2]. In mammalian embryos, germ cells are specified in a very different manner, through signalling between layers of cells comprising the primitive embryo [3,4]. + +Recently, Saitou, Barton and Surani proposed a molecular pathway by which these intercellular signals are translated into germ cell fate in mice [5,6]. Central to this proposed program of germ cell specification is stella / PGC7 / Dppa3, a gene expressed in primordial germ cells and their descendants, including oocytes [5,7,8]. Here we will use the name Dppa3, as approved by the Mouse Genome Informatics Database, when referring to this gene. Saitou and colleagues' model of Dppa3's role in germ cell specification was based on the timing and site of the gene's expression, not on functional analysis. Nonetheless, the model makes clear predictions as to the phenotype of mice lacking Dppa3 function: such embryos should not form germ cells. We tested this prediction and sought to clarify the gene's importance by generating Dppa3-deficient mice and examining their germline development. + +Results and Discussion + +We disrupted the Dppa3 gene in cultured embryonic stem (ES) cells and thereby generated Dppa3-deficient mice. Specifically, we replaced the entire open reading frame of Dppa3 in mouse V6.5 ES cells [9] with a hygromycin-thymidine kinase selection cassette flanked by loxP sites (Figure 1A). The selection cassette was subsequently removed via transient expression of Cre recombinase in targeted ES cells. The resulting heterozygous Dppa3tm1WHT/+ ES cells were used to generate chimeric mice, which transmitted the mutation to offspring. Intercrosses between Dppa3tm1WHT/+ heterozygous animals yielded Dppa3tm1WHT/Dppa3tm1WHT homozygotes as well as Dppa3tm1WHT/+ heterozygotes and +/+ offspring, demonstrating that zygotic function of Dppa3 is not essential for viability (Figure 1B). This allowed us to characterize germ cell development in animals lacking Dppa3. + +Figure 1 + +Generation of Dppa3-deficient animals. A, Schematic representation of genomic ablation of Dppa3. The gene's four exons are shown; non-coding regions of the first and last exons are shaded gray. The hygromycin-thymidine kinase (Hygro-TK) cassette replaces the entire open reading frame of the gene. Cre-mediated excision of the selection cassette leaves only the non-coding portions of the gene, together with a single loxP site (white triangle). Also shown are the locations of genotyping primers p1, p2 and p3 in wild-type and mutated Dppa3 alleles. B, PCR genotyping of the offspring of an intercross between Dppa3tm1WHT /+ animals. Inferred genotypes are shown above the gel image. The wild type allele yields a PCR product of 304 bp with primers p1 and p2. The mutant allele (Dppa3tm1WHT) yields a PCR product of 492 bp with primers p1 and p3. M, DNA molecular weight marker. + +Dppa3 is not required for germ cell specification + +Our findings do not support the proposed centrality of Dppa3 in germ cell programming. First, the gonads of Dppa3tm1WHT/Dppa3tm1WHT embryos contained germ cells, identified by expression of alkaline phosphatase, in numbers comparable to those of Dppa3tm1WHT/ + and +/+ embryos (Figure 2A). Second, the ovaries of Dppa3tm1WHT/Dppa3tm1WHT adult females expressed Oct4, a marker of oocytes [10,11], despite the absence of Dppa3 expression (Figure 2B). Third, histological examination of the gonads of Dppa3tm1WHT/ Dppa3tm1WHT adults revealed no morphological defects; spermatogenesis in males and ovarian follicle development in females appeared to be normal (Figure 2C,2D). Finally, we obtained fertile Dppa3tm1WHT/Dppa3tm1WHT mice of both sexes (though litters from Dppa3tm1WHT/Dppa3tm1WHT females were small, as described below). Each of these findings demonstrates that the Dppa3 gene is not required for germ cell specification. + +Figure 2 + +Normal germ cell development in the absence of Dppa3. A, Gonads from E12.5 embryos (above: wild type; below: Dppa3tm1WHT/Dppa3tm1WHT) stained for alkaline phosphatase to reveal primordial germ cells. B, RT-PCR analysis of gene expression in wild-type and Dppa3tm1WHT/Dppa3tm1WHT adult ovaries. C,D, Dppa3tm1WHT/Dppa3tm1WHT testis (C) and ovary (D) are histologically normal. + +Moreover, this function is not readily ascribed to a gene closely related to Dppa3. We electronically searched the sequenced mouse genome for Dppa3 homologues. We identified several processed (intron-less) pseudogenes of Dppa3, but no functional, full-length homologue. As judged by RT-PCR analysis, the Dppa3 pseudogenes are not expressed in embryonic or adult tissues (data not shown). + +Dppa3 is a potent maternal factor + +We found that Dppa3 plays an important role in early embryonic development as a maternal factor. While Dppa3tm1WHT/Dppa3tm1WHT males were fully fertile, Dppa3tm1WHT/Dppa3tm1WHT females had small litters. This was true regardless of whether such females were crossed with Dppa3tm1WHT/Dppa3tm1WHT, Dppa3tm1WHT/+ or wild type males (3.5 ± 1.5, 3.1 ± 2.1, or 3.0 ± 0.9 viable pups/litter, respectively). By contrast, Dppa3tm1WHT/+ females of the same (mixed) genetic background had large litters when mated to Dppa3tm1WHT/Dppa3tm1WHT or Dppa3tm1WHT/+ males (9.4 ± 3.5 or 10.1 ± 3.2 viable pups/litter, respectively). + +We attribute the small litters from Dppa3tm1WHT/Dppa3tm1WHT mothers to abnormalities that manifest early in embryogenesis, during the cleavage stages of pre-implantation development. While nearly all embryos derived from Dppa3-deficient oocytes developed to the 2-cell or 4-cell stage (Figure 3A,3B,3C,3D), subsequent development was severely compromised in most such embryos (Figure 3E,3F). Some embryos derived from Dppa3-deficient oocytes failed to reach the 8-cell stage and instead showed evidence of compaction at the 4-cell stage. Other embryos derived from Dppa3-deficient oocytes cleaved to form 8 to 16 blastomeres, but failed to compact (Figure 3E,3F). These observations suggest that maternally supplied Dppa3 function is important in the cleavage stages of pre-implantation development. + +Figure 3 + +Abnormal pre-implantation development of embryos derived from Dppa3–deficient oocytes. A,C, Cultured 2-cell (A) and 4-cell (C) control embryos derived from wild-type matings. B,D, Cultured 2-cell (B) and 4-cell (D) embryos produced by crossing Dppa3tm1WHT/Dppa3tm1WHT females with wild-type males. E,F, E3.5 control embryos derived from wild-type matings have progressed to the blastocyst stage (E). By contrast, most E3.5 embryos produced by crossing Dppa3tm1WHT/Dppa3tm1WHT females with wild-type males have not progressed to the blastocyst stage and instead cleave abnormally and degenerate (F). G,H, Many embryos produced by crossing Dppa3tm1WHT/Dppa3tm1WHT females with Dppa3 +/+, Tg(Pou5f1 ΔPE-GFP)10WHT/Tg(Pou5f1 ΔPE-GFP)10WHT males fail to develop normally beyond the 4-cell stage (G) but nonetheless express the Oct4-GFP marker (H). + +Might maternal Dppa3 induce zygotic expression of Oct4/Pou5f1, which encodes a transcription factor that is crucial to pre-implantation development [10,12]? To test this possibility, we crossed Dppa3tm1WHT/Dppa3tm1WHT females with Dppa3 +/+, Tg(Pou5f1 ΔPE-GFP)10WHT/Tg(Pou5f1 ΔPE-GFP)10WHT males, the latter transmitting an Oct4-GFP transgene, with the Oct4 promoter driving expression of GFP. We retrieved the resulting embryos at the 2-cell stage and cultured them in vitro for 72 hours to monitor expression of the Oct4-GFP transgene. All such embryos were observed to express the fluorescent marker, regardless of the degree to which the embryos developed or failed to develop during the culture period (Figure 3G,3H). Thus, the poor development of many embryos derived from Dppa3-deficient oocytes cannot be attributed to the absence of zygotic expression of Oct4. Further analysis of the maternal-effect phenotype of Dppa3 should illuminate the molecular and biological context and consequences of the gene's activity. + +Conclusions + +We conclude that Dppa3 is not required for germ cell specification in mice. The identity of the mammalian gene or genes that program germ cells remains an open question. Dppa3 appears to function as a maternal factor, with an important role early in embryogenesis, during cleavage. + +Methods + +Generation of Dppa3-deficient animals + +The Dppa3 targeting construct contained 1.3-kb and 3-kb segments of mouse genomic DNA, the former located 5' of Dppa3's translation initiation site and the latter located 3' of the termination codon (Figure 1). At the center of the construct was a 3-kb hygromycin-thymidine kinase selection cassette (Hygro-TK) flanked by two loxP direct repeats. V6.5 (C57BL/6 × 129/Sv)F1 ES cells [9] were transfected by electroporation, and recombined clones were selected in the presence of hygromycin (Invitrogen). Correctly targeted clones were identified by long-distance genomic PCR. The Hygro-TK cassette was removed via transient transfection of ES cells with a Cre-expressing plasmid in the presence of ganciclovir (Sigma). The final genomic structure of the resulting clones was verified by Southern analysis. Two independently targeted ES cell clones were microinjected into Balb/c blastocysts to generate chimeras. Animals used in this study were of a mixed C57BL/6 × 129/Sv genetic background. + +Primers for PCR genotyping were as follows: p1 (5' TAG CCT GGG GGT AGA CTC GGC TGT AT 3'); p2 (5' AAC GAG AAG AGA AGG GAG GGC TTC 3'); and p3 (5' TCA CAT AAA TCT GGA TCG TTG TGC ATC 3'). The wild type allele gives rise to a PCR product of 304 bp with primers p1 and p2. The mutant allele (Dppa3tm1WHT) gives rise to a PCR product of 492 bp with primers p1 and p3. + +RNA isolation and RT-PCR + +Total RNAs were isolated from mouse tissues, and expression of Dppa3, Oct4, and Gapd was assayed by RT-PCR, all as described previously[8]. + +Alkaline phosphatase staining of primordial germ cells + +Gonads were dissected from wild type and Dppa3tm1WHT /Dppa3tm1WHT embryos on day 12.5 of gestation and stained for alkaline phosphatase as described previously [13]. + +Histology + +Dissected adult testes and ovaries were fixed overnight in Bouin's solution, imbedded in paraffin, sectioned, and stained with hematoxylin and eosin. + +Generation of Oct4-GFP transgenic animals + +Mice bearing an Oct4-GFP transgene were generated by microinjection of a 14-kb Oct4Δ PE-GFP linear DNA fragment into C57Bl/6 × SJL F2 hybrid mouse eggs. This construct essentially reproduces the previously described GOF18Δ PE-lacZ construct [14] but contains a gene for enhanced green fluorescent protein (EGFP, Clontech) in place of lacZ at the ATG of Oct4. Mice bearing transgene Tg(Pou5f1 ΔPE-GFP)10WHT accurately reproduced the previously reported Oct4 expression pattern [14] and were bred to generate Tg(Pou5f1 ΔPE-GFP)10WHT /Tg(Pou5f1 ΔPE-GFP)10WHT homozygous animals. + +Isolation, culture and analysis of cleavage stage embryos + +2-cell embryos were flushed from oviducts at E1.5 and cultured for up to 72 hours in microdrops of KSOM (Specialty Media) under light mineral oil (Squibb) with 5% CO2 in air. E3.5 embryos were flushed from uteri. + +Authors' contributions + +AB conducted molecular biological, ES cell culture and embryological studies, and co-wrote the manuscript. MG carried out blastocyst injections. ML assisted in mouse and embryological studies. DP coordinated the study and co-wrote the manuscript. All authors read and approved the final manuscript. + +Acknowledgements + +A.B. was a Leukemia & Lymphoma Society Special Fellow. Supported by the Howard Hughes Medical Institute. diff --git a/src/ontogpt/evaluation/craft/database/all/15040800.ann b/src/ontogpt/evaluation/craft/database/all/15040800.ann new file mode 100644 index 000000000..c53e7bf3f --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15040800.ann @@ -0,0 +1,1733 @@ +T1 NCBITaxon:10088 2 7 mouse +T2 http://purl.obolibrary.org/obo/MONDO_0008863 17 31 sitosterolemia +T3 PR:000003573 44 49 Abcg8 +T4 PR:000003573 50 60 sterolin-2 +T5 GO:0046903 83 90 secrete +T6 UBERON:0002294 91 98 biliary +T7 CHEBI:16113 99 110 cholesterol +T8 SO:0000704 161 166 genes +T9 PR:000003572 234 239 ABCG5 +T10 PR:000003572 250 260 sterolin-1 +T11 PR:000003573 266 271 ABCG8 +T12 PR:000003573 282 292 sterolin-2 +T13 http://purl.obolibrary.org/obo/MONDO_0008863 305 319 sitosterolemia +T14 GO:0030849 328 337 autosomal +T15 http://purl.obolibrary.org/obo/MONDO_0006025 328 356 autosomal recessive disorder +T16 CHEBI:15889 360 366 sterol +T17 GO:0015918 360 378 sterol trafficking +T18 UBERON:0001969 406 412 plasma +T19 NCBITaxon:3193 413 418 plant +T20 CHEBI:15889 419 425 sterol +T21 http://purl.obolibrary.org/obo/MONDO_0008863 461 475 sitosterolemia +T22 PR:000003572 477 482 ABCG5 +T23 PR:000003572 483 493 sterolin-1 +T24 PR:000003573 498 503 ABCG8 +T25 PR:000003573 504 514 sterolin-2 +T26 NCBITaxon:9606 621 627 humans +T27 PR:000003572 660 665 ABCG5 +T28 PR:000003573 669 674 ABCG8 +T29 NCBITaxon:9606 718 724 humans +T30 GO:0065007 746 756 regulating +T31 CHEBI:15889 765 771 sterol +T32 GO:0035376 765 777 sterol entry +T33 UBERON:0002294 782 789 biliary +T34 CHEBI:15889 790 796 sterol +T35 NCBITaxon:9606 840 845 human +T36 http://purl.obolibrary.org/obo/MONDO_0000001 846 853 disease +T37 NCBITaxon:10088 895 900 mouse +T38 http://purl.obolibrary.org/obo/MONDO_0008863 910 924 sitosterolemia +T39 PR:000003573 938 943 Abcg8 +T40 PR:000003573 944 954 sterolin-2 +T41 NCBITaxon:10088 993 997 mice +T42 http://purl.obolibrary.org/obo/MONDO_0008863 1021 1035 sitosterolemia +T43 NCBITaxon:10088 1047 1051 Mice +T44 PR:000003573 1065 1070 Abcg8 +T45 UBERON:0001969 1100 1106 plasma +T46 UBERON:0000479 1111 1117 tissue +T47 NCBITaxon:3193 1118 1123 plant +T48 CHEBI:15889 1124 1130 sterol +T49 CHEBI:27693 1139 1149 sitosterol +T50 CHEBI:28623 1154 1165 campesterol +T51 http://purl.obolibrary.org/obo/MONDO_0008863 1183 1197 sitosterolemia +T52 PR:000003572 1214 1219 Abcg5 +T53 PR:000003572 1220 1230 sterolin-1 +T54 GO:0010467 1235 1244 expressed +T55 UBERON:0002107 1253 1258 liver +T56 UBERON:0000160 1263 1272 intestine +T57 PR:000003573 1276 1281 Abcg8 +T58 PR:000003573 1282 1292 sterolin-2 +T59 NCBITaxon:10088 1303 1307 mice +T60 GO:0045177 1333 1339 apical +T61 GO:0010467 1340 1350 expression +T62 PR:000003573 1364 1369 Abcg8 +T63 NCBITaxon:10088 1380 1384 mice +T64 GO:0046903 1412 1419 secrete +T65 CHEBI:16113 1420 1431 cholesterol +T66 UBERON:0001970 1437 1441 bile +T67 GO:0046903 1479 1486 secrete +T68 CHEBI:27693 1487 1497 sitosterol +T69 PR:000003573 1560 1565 Abcg8 +T70 NCBITaxon:10088 1569 1573 mice +T71 http://purl.obolibrary.org/obo/MONDO_0008863 1587 1601 sitosterolemic +T72 UBERON:0002294 1633 1640 biliary +T73 CHEBI:15889 1641 1647 sterol +T74 NCBITaxon:10088 1680 1684 mice +T75 PR:000003573 1724 1729 Abcg8 +T76 PR:000003573 1730 1740 sterolin-2 +T77 UBERON:0002294 1758 1765 biliary +T78 CHEBI:15889 1766 1772 sterol +T79 PR:000003573 1800 1805 Abcg8 +T80 PR:000003573 1806 1816 sterolin-2 +T81 UBERON:0002294 1849 1856 biliary +T82 CHEBI:16113 1857 1868 cholesterol +T83 CHEBI:27693 1884 1894 sitosterol +T84 UBERON:0002294 1902 1909 biliary +T85 CHEBI:27693 1910 1920 sitosterol +T86 http://purl.obolibrary.org/obo/MONDO_0008863 1974 1988 sitosterolemic +T87 NCBITaxon:10088 1989 1993 mice +T88 PR:000003573 2051 2056 Abcg8 +T89 PR:000003573 2057 2067 sterolin-2 +T90 UBERON:0001970 2110 2114 bile +T91 GO:0030299 2129 2142;2151 2167;2172 2181 Absorption of ... cholesterol from ... intestine +T92 CHEBI:16113 2151 2162 cholesterol +T93 UBERON:0000160 2172 2181 intestine +T94 CHEBI:16113 2206 2217 cholesterol +T95 GO:0042632 2206 2229 cholesterol homeostasis +T96 CHEBI:16113 2282 2293 cholesterol +T97 GO:0008152 2307 2316 metabolic +T98 CHEBI:16113 2386 2397 cholesterol +T99 CHEBI:16113 2406 2417 cholesterol +T100 CHEBI:15889 2418 2425 sterols +T101 NCBITaxon:3193 2434 2439 plant +T102 CHEBI:15889 2440 2447 sterols +T103 CHEBI:16113 2483 2494 cholesterol +T104 CHEBI:16113 2555 2566 cholesterol +T105 CHEBI:15889 2567 2574 sterols +T106 CHEBI:16113 2639 2650 cholesterol +T107 CHEBI:16113 2660 2671 cholesterol +T108 CHEBI:15889 2672 2679 sterols +T109 UBERON:0000160 2700 2709 intestine +T110 CHEBI:16113 2773 2784 cholesterol +T111 UBERON:0002107 2862 2867 liver +T112 GO:0046903 2868 2876 secretes +T113 CHEBI:16113 2882 2893 cholesterol +T114 UBERON:0001970 2899 2903 bile +T115 GO:0007588 2927 2934 excrete +T116 CHEBI:16113 2939 2950 cholesterol +T117 CHEBI:15889 2951 2958 sterols +T118 UBERON:0001970 2964 2968 bile +T119 NCBITaxon:9606 3083 3088 human +T120 http://purl.obolibrary.org/obo/MONDO_0000001 3089 3096 disease +T121 http://purl.obolibrary.org/obo/MONDO_0008863 3141 3155 Sitosterolemia +T122 http://purl.obolibrary.org/obo/MONDO_0008863 3183 3198 phytosterolemia +T123 GO:0030849 3210 3219 autosomal +T124 http://purl.obolibrary.org/obo/MONDO_0006025 3210 3251 autosomal, recessively inherited disorder +T125 UBERON:0000178 3278 3283 blood +T126 UBERON:0000479 3288 3294 tissue +T127 NCBITaxon:3193 3295 3300 plant +T128 CHEBI:15889 3301 3307 sterol +T129 NCBITaxon:1 3329 3340 individuals +T130 UBERON:0000043 3353 3359 tendon +T131 UBERON:4200132 3367 3375 tuberous +T132 http://purl.obolibrary.org/obo/MONDO_0005236 3376 3385 xanthomas +T133 http://purl.obolibrary.org/obo/MONDO_0005578 3420 3429 arthritis +T134 http://purl.obolibrary.org/obo/MONDO_0005311 3445 3460 atherosclerosis +T135 http://purl.obolibrary.org/obo/MONDO_0008863 3468 3482 Sitosterolemic +T136 UBERON:0001969 3521 3527 plasma +T137 NCBITaxon:3193 3528 3533 plant +T138 CHEBI:15889 3534 3540 sterol +T139 CHEBI:27693 3562 3572 sitosterol +T140 CHEBI:28623 3574 3585 campesterol +T141 CHEBI:28824 3587 3599 stigmasterol +T142 CHEBI:25212 3653 3664 metabolites +T143 CHEBI:16113 3732 3743 cholesterol +T144 NCBITaxon:1 3794 3805 individuals +T145 CHEBI:15889 3830 3837 sterols +T146 CHEBI:15889 3882 3888 sterol +T147 GO:0007588 3936 3943 excrete +T148 CHEBI:15889 3944 3951 sterols +T149 UBERON:0001970 3957 3961 bile +T150 CHEBI:27693 3981 3991 sitosterol +T151 CHEBI:16113 3996 4007 cholesterol +T152 NCBITaxon:1 4020 4031 individuals +T153 GO:0007588 4066 4075 excretion +T154 CHEBI:27693 4079 4089 sitosterol +T155 UBERON:0001970 4095 4099 bile +T156 http://purl.obolibrary.org/obo/MONDO_0008863 4108 4122 sitosterolemic +T157 NCBITaxon:1 4123 4134 individuals +T158 UBERON:0002294 4135 4142 biliary +T159 CHEBI:15889 4143 4149 sterol +T160 http://purl.obolibrary.org/obo/MONDO_0008863 4204 4218 sitosterolemia +T161 CHEBI:15889 4264 4270 sterol +T162 GO:0010467 4285 4294 expressed +T163 UBERON:0012427 4302 4325 intestinal brush border +T164 UBERON:0001283 4337 4356 hepatic canalicular +T165 SO:0000704 4375 4382 Genetic +T166 http://purl.obolibrary.org/obo/MONDO_0008863 4395 4409 sitosterolemia +T167 NCBITaxon:9606 4461 4466 human +T168 SO:0000704 4580 4585 genes +T169 CHEBI:86029 4609 4621 LXR agonists +T170 SO:0000704 4661 4666 genes +T171 PR:000003572 4668 4673 ABCG5 +T172 PR:000003573 4678 4683 ABCG8 +T173 PR:000003572 4711 4721 sterolin-1 +T174 PR:000003573 4726 4736 sterolin-2 +T175 http://purl.obolibrary.org/obo/MONDO_0008863 4763 4777 sitosterolemia +T176 PR:000003572 4830 4835 ABCG5 +T177 PR:000003573 4845 4850 ABCG8 +T178 http://purl.obolibrary.org/obo/MONDO_0000001 4908 4915 disease +T179 http://purl.obolibrary.org/obo/MONDO_0008863 4947 4961 sitosterolemia +T180 SO:0000704 5005 5010 genes +T181 PR:000003572 5097 5107 sterolin-1 +T182 PR:000003573 5112 5122 sterolin-2 +T183 GO:0016020 5286 5294 membrane +T184 SO:0000417 5295 5302 domains +T185 SO:0001077 5329 5342 transmembrane +T186 GO:0016020 5334 5342 membrane +T187 GO:0010467 5492 5502 expression +T188 PR:000003572 5506 5511 ABCG5 +T189 PR:000003573 5512 5517 ABCG8 +T190 NCBITaxon:10088 5534 5539 mouse +T191 UBERON:0002294 5570 5577 biliary +T192 CHEBI:16113 5578 5589 cholesterol +T193 CHEBI:16113 5664 5675 cholesterol +T194 PR:000003572 5709 5714 ABCG5 +T195 PR:000003573 5719 5724 ABCG8 +T196 GO:0045177 5782 5788 apical +T197 GO:0010467 5803 5813 expression +T198 GO:0045177 5839 5845 apical +T199 PR:000003572 5906 5911 Abcg5 +T200 PR:000003573 5912 5917 Abcg8 +T201 NCBITaxon:10088 5934 5938 mice +T202 CHEBI:15889 6124 6131 sterols +T203 PR:000003572 6242 6247 Abcg5 +T204 PR:000003573 6251 6256 Abcg8 +T205 NCBITaxon:10088 6266 6271 mouse +T206 PR:000003572 6294 6299 Abcg5 +T207 PR:000003573 6304 6309 Abcg8 +T208 NCBITaxon:9606 6371 6376 human +T209 http://purl.obolibrary.org/obo/MONDO_0000001 6377 6384 disease +T210 NCBITaxon:9606 6438 6443 human +T211 http://purl.obolibrary.org/obo/MONDO_0000001 6444 6451 disease +T212 SO:0000704 6503 6507 gene +T213 http://purl.obolibrary.org/obo/MONDO_0008863 6624 6638 sitosterolemia +T214 UBERON:0002294 6715 6722 biliary +T215 CHEBI:15889 6736 6743 sterols +T216 UBERON:0000160 6747 6757 intestinal +T217 GO:0050892 6747 6768 intestinal absorption +T218 CHEBI:15889 6772 6779 sterols +T219 NCBITaxon:9606 6820 6826 humans +T220 UBERON:0000062 6842 6848 organs +T221 NCBITaxon:10088 6924 6929 mouse +T222 SO:0000704 6957 6964 genetic +T223 PR:000003573 6977 6982 Abcg8 +T224 NCBITaxon:10088 6984 6988 Mice +T225 PR:000003573 7004 7009 Abcg8 +T226 NCBITaxon:9606 7058 7063 human +T227 http://purl.obolibrary.org/obo/MONDO_0000001 7064 7071 disease +T228 http://purl.obolibrary.org/obo/MONDO_0008863 7072 7086 sitosterolemia +T229 UBERON:0002294 7122 7129 biliary +T230 CHEBI:15889 7130 7136 sterol +T231 PR:000003573 7188 7193 Abcg8 +T232 NCBITaxon:33208 7204 7211 animals +T233 GO:0046903 7219 7226 secrete +T234 CHEBI:16113 7227 7238 cholesterol +T235 UBERON:0001970 7244 7248 bile +T236 CHEBI:27693 7259 7269 sitosterol +T237 SO:0001644 7309 7325 Targeting vector +T238 NCBITaxon:10088 7366 7370 mice +T239 NCBITaxon:10088 7374 7379 mouse +T240 SO:0001026 7380 7387 genomic +T241 NCBITaxon:2 7388 7397 bacterial +T242 SO:0000153 7388 7419 bacterial artificial chromosome +T243 SO:0000153 7421 7424 BAC +T244 CL:0002322 7444 7451 ES cell +T245 SO:0000112 7532 7539 primers +T246 NCBITaxon:10088 7571 7576 mouse +T247 PR:000003572 7577 7582 Abcg5 +T248 PR:000003573 7587 7592 Abcg8 +T249 SO:0000153 7638 7641 BAC +T250 SO:0001026 7682 7689 genomic +T251 PR:000003573 7707 7712 Abcg8 +T252 SO:0001026 7901 7908 genomic +T253 SO:0000147 7937 7941 exon +T254 SO:0000147 7955 7959 exon +T255 SO:0000440 8029 8035 vector +T256 SO:0001026 8053 8060 genomic +T257 SO:0000147 8090 8094 exon +T258 SO:0000147 8108 8112 exon +T259 SO:0000188 8213 8219 intron +T260 SO:0000147 8231 8235 exon +T261 SO:0000147 8250 8254 exon +T262 CHEBI:7507 8276 8284 neomycin +T263 SO:0005853 8296 8304 cassette +T264 CL:0002322 8374 8382 ES cells +T265 SO:0001644 8447 8463 targeting vector +T266 UBERON:0000922 8498 8507 embryonic +T267 CL:0000057 8508 8519 fibroblasts +T268 GO:0009294 8521 8532 Transfected +T269 CHEBI:42768 8611 8615 G418 +T270 CL:0002322 8661 8669 ES cells +T271 SO:0000121 8729 8743 forward primer +T272 SO:0005853 8788 8796 cassette +T273 SO:0000132 8802 8816 reverse primer +T274 SO:0000188 8817 8823 intron +T275 NCBITaxon:10088 8909 8914 mouse +T276 PR:000003573 8915 8920 Abcg8 +T277 SO:0000188 8921 8927 intron +T278 CL:0002322 8951 8958 ES cell +T279 CL:0002322 9009 9016 ES cell +T280 PR:P23940 9033 9038 BamHI +T281 SO:0000028 9071 9073 bp +T282 SO:0000188 9100 9106 intron +T283 SO:0000147 9113 9117 exon +T284 PR:000003573 9123 9128 Abcg8 +T285 SO:0000704 9129 9133 gene +T286 CL:0002322 9156 9164 ES cells +T287 UBERON:0000358 9196 9207 blastocysts +T288 NCBITaxon:10088 9278 9282 mice +T289 UBERON:0010166 9291 9295 coat +T290 NCBITaxon:10088 9369 9373 mice +T291 NCBITaxon:10088 9422 9426 mice +T292 PR:000003573 9430 9435 Abcg8 +T293 SO:0000704 9436 9440 gene +T294 NCBITaxon:10088 9476 9480 mice +T295 NCBITaxon:10088 9511 9515 mice +T296 NCBITaxon:10088 9589 9593 mice +T297 NCBITaxon:33208 9596 9603 Animals +T298 NCBITaxon:10088 9619 9623 mice +T299 NCBITaxon:9989 9654 9660 rodent +T300 CHEBI:33290 9661 9665 chow +T301 NCBITaxon:10088 9681 9686 mouse +T302 NCBITaxon:10114 9687 9690 rat +T303 CHEBI:16113 9729 9740 cholesterol +T304 CHEBI:28623 9751 9762 campesterol +T305 CHEBI:27693 9778 9788 sitosterol +T306 CHEBI:15377 9811 9816 water +T307 NCBITaxon:33208 9880 9887 animals +T308 NCBITaxon:33208 9933 9940 animals +T309 NCBITaxon:33208 9980 9986 Animal +T310 NCBITaxon:33208 10022 10028 Animal +T311 NCBITaxon:10088 10152 10157 Mouse +T312 UBERON:0002415 10158 10162 tail +T313 NCBITaxon:10088 10228 10233 mouse +T314 UBERON:0002415 10234 10238 tail +T315 GO:0019835 10266 10271 lysis +T316 CHEBI:9754 10286 10290 Tris +T317 CHEBI:17883 10291 10294 HCl +T318 CHEBI:26710 10323 10327 NaCl +T319 CHEBI:8984 10332 10335 SDS +T320 CHEBI:26710 10407 10411 NaCl +T321 CHEBI:75958 10436 10444 solution +T322 CHEBI:75958 10498 10506 solution +T323 CHEBI:16236 10643 10647 EtOH +T324 CHEBI:16236 10705 10709 EtOH +T325 SO:0001026 10845 10852 genomic +T326 SO:0000112 10930 10937 primers +T327 PR:000003573 10943 10948 Abcg8 +T328 SO:0000704 10949 10953 gene +T329 SO:0000112 10963 10970 primers +T330 PR:000003573 11074 11079 Abcg8 +T331 CHEBI:6636 11148 11153 MgCl2 +T332 CHEBI:62946 11161 11170 (NH4)2SO4 +T333 CHEBI:9754 11192 11196 Tris +T334 CHEBI:17883 11197 11200 HCl +T335 SO:0000112 11223 11229 primer +T336 CHEBI:15377 11231 11236 water +T337 NCBITaxon:271 11254 11257 Taq +T338 SO:0000006 11376 11388 PCR products +T339 PR:P23940 11498 11504 Bam HI +T340 CHEBI:2511 11545 11552 agarose +T341 CHEBI:25614 11576 11581 nylon +T342 GO:0097617 11619 11629 hybridized +T343 SO:0000028 11641 11643 bp +T344 SO:0000188 11670 11676 intron +T345 SO:0000147 11683 11687 exon +T346 PR:000003573 11693 11698 Abcg8 +T347 CHEBI:37972 11699 11702 32P +T348 UBERON:0002107 11794 11799 liver +T349 UBERON:0000160 11804 11814 intestinal +T350 PR:000003573 11830 11835 Abcg8 +T351 PR:000003573 11840 11845 Abcg8 +T352 PR:000003573 11853 11858 Abcg8 +T353 NCBITaxon:10088 11862 11866 mice +T354 PR:000003572 11953 11958 Abcg5 +T355 SO:0000028 11968 11970 bp +T356 PR:000003573 12037 12042 Abcg8 +T357 SO:0000028 12052 12054 bp +T358 CHEBI:25613 12056 12058 nt +T359 GO:0001171 12105 12126 Reverse Transcription +T360 GO:0000380 12166 12186 alternative splicing +T361 SO:0001023 12204 12210 allele +T362 NCBITaxon:10088 12252 12257 mouse +T363 UBERON:0002107 12258 12263 liver +T364 NCBITaxon:10088 12316 12321 mouse +T365 UBERON:0002107 12322 12328 livers +T366 GO:0001171 12342 12361 reverse transcribed +T367 SO:0000112 12546 12553 primers +T368 SO:0000028 12785 12787 bp +T369 SO:0000112 12842 12849 Primers +T370 SO:0000112 12887 12893 primer +T371 GO:0006277 12994 13010;13045 13048 amplification of ... DNA +T372 SO:0001026 13037 13044 genomic +T373 NCBITaxon:10088 13126 13131 mouse +T374 UBERON:0002107 13132 13138 livers +T375 GO:0001171 13152 13171 reverse transcribed +T376 CHEBI:51461 13509 13519 SYBR Green +T377 CHEBI:60004 13531 13534 mix +T378 GO:0001171 13660 13681 reverse transcription +T379 SO:0000112 13734 13741 primers +T380 GO:0032508 13891 13895 melt +T381 GO:0097617 13928 13937 annealing +T382 SO:0000006 14145 14157 PCR products +T383 SO:0000673 14333 14340 message +T384 CHEBI:33695 14333 14340 message +T385 SO:0000704 14344 14349 genes +T386 NCBITaxon:10088 14371 14376 mouse +T387 UBERON:0002107 14377 14382 liver +T388 SO:0000704 14695 14699 gene +T389 UBERON:0001969 15118 15124 Plasma +T390 CHEBI:18059 15125 15130 lipid +T391 UBERON:0000178 15208 15213 blood +T392 PR:000003573 15247 15252 Abcg8 +T393 PR:000003573 15257 15262 Abcg8 +T394 PR:000003573 15270 15275 Abcg8 +T395 NCBITaxon:10088 15279 15283 mice +T396 UBERON:0001697 15314 15321 orbital +T397 UBERON:0001593 15322 15335 venous plexus +T398 CHEBI:6015 15376 15386 isoflurane +T399 UBERON:0001969 15480 15486 plasma +T400 CHEBI:15889 15739 15745 Sterol +T401 UBERON:0001969 15761 15767 plasma +T402 UBERON:0000479 15772 15779 tissues +T403 NCBITaxon:10088 15823 15827 mice +T404 GO:0007631 15828 15831 fed +T405 NCBITaxon:9989 15843 15849 rodent +T406 CHEBI:33290 15850 15854 chow +T407 UBERON:0000178 15861 15866 blood +T408 UBERON:0000479 15871 15878 tissues +T409 CHEBI:6015 15900 15911 isofluorane +T410 PR:000003573 15940 15945 Abcg8 +T411 PR:000003573 15950 15955 Abcg8 +T412 PR:000003573 15963 15968 Abcg8 +T413 NCBITaxon:10088 15972 15976 mice +T414 UBERON:0000178 16001 16006 Blood +T415 UBERON:0001697 16036 16043 orbital +T416 UBERON:0001593 16044 16057 venous plexus +T417 NCBITaxon:33208 16089 16096 animals +T418 UBERON:0005434 16116 16124 cervical +T419 UBERON:0000479 16141 16147 tissue +T420 UBERON:0001969 16160 16166 Plasma +T421 CHEBI:32145 16198 16202 NaOH +T422 CHEBI:15377 16224 16229 water +T423 CHEBI:15889 16240 16247 sterols +T424 CHEBI:27750 16299 16312 ethyl acetate +T425 CHEBI:35515 16331 16344 5α-cholestane +T426 NCBITaxon:10088 16388 16393 Mouse +T427 UBERON:0000479 16394 16401 tissues +T428 UBERON:0002107 16403 16408 liver +T429 UBERON:0002106 16410 16416 spleen +T430 UBERON:0000955 16421 16426 brain +T431 CHEBI:15889 16634 16640 sterol +T432 UBERON:0000479 16661 16667 tissue +T433 CHEBI:15889 16721 16727 sterol +T434 CHEBI:29021 16799 16805 hexane +T435 UBERON:0001969 17068 17074 plasma +T436 UBERON:0000479 17079 17085 tissue +T437 CHEBI:15889 17086 17093 sterols +T438 UBERON:0000479 17137 17143 tissue +T439 GO:0042571 17171 17179 antibody +T440 PR:000003572 17223 17228 Abcg5 +T441 PR:000003572 17229 17239 sterolin-1 +T442 GO:0042571 17240 17250 antibodies +T443 PR:000003572 17325 17330 Abcg5 +T444 PR:000003572 17331 17341 sterolin-1 +T445 GO:0042571 17342 17352 antibodies +T446 UBERON:0002107 17431 17436 Liver +T447 GO:0016020 17488 17496 Membrane +T448 GO:0016020 17525 17534 membranes +T449 UBERON:0002107 17583 17588 liver +T450 UBERON:0000479 17589 17595 tissue +T451 GO:0019835 17646 17651 lysis +T452 CHEBI:9754 17665 17669 Tris +T453 CHEBI:17992 17684 17691 sucrose +T454 GO:0019835 18258 18263 lysis +T455 GO:0005886 18272 18288 Plasma membranes +T456 GO:0016020 18487 18495 membrane +T457 CHEBI:8984 18532 18535 SDS +T458 CHEBI:28619 18536 18546 acrylamide +T459 CHEBI:8984 18590 18593 SDS +T460 CHEBI:53325 18619 18633 nitrocellulose +T461 CHEBI:53424 18746 18754 Tween 20 +T462 GO:0042571 18793 18801 antibody +T463 PR:000003572 18810 18815 Abcg5 +T464 CHEBI:53424 18834 18835 T +T465 CHEBI:53424 18915 18916 T +T466 CHEBI:9754 18918 18922 Tris +T467 CHEBI:53424 18944 18952 Tween-20 +T468 CHEBI:26710 18966 18970 NaCl +T469 NCBITaxon:9925 19001 19005 goat +T470 NCBITaxon:9986 19011 19017 rabbit +T471 MOP:0000779 19018 19028 conjugated +T472 CHEBI:53424 19069 19070 T +T473 CHEBI:53424 19147 19148 T +T474 CHEBI:26710 19161 19165 NaCl +T475 CHEBI:33893 19219 19226 Reagent +T476 UBERON:0002107 19377 19382 liver +T477 UBERON:0000160 19387 19396 intestine +T478 UBERON:0000479 19397 19403 tissue +T479 CHEBI:51686 19560 19571 hematoxylin +T480 CHEBI:17790 19631 19639 methanol +T481 GO:0042571 19698 19706 antibody +T482 NCBITaxon:9925 19764 19768 goat +T483 UBERON:0001977 19769 19774 serum +T484 PR:000003572 19831 19841 sterolin-1 +T485 GO:0042571 19842 19850 antibody +T486 GO:0042571 19950 19958 antibody +T487 NCBITaxon:9925 19960 19964 goat +T488 NCBITaxon:9986 19970 19976 rabbit +T489 GO:0071735 19977 19980 IgG +T490 MOP:0000779 19981 19991 conjugated +T491 UBERON:0002107 20171 20176 Liver +T492 CHEBI:15347 20203 20210 acetone +T493 CHEBI:75958 20256 20264 solution +T494 CHEBI:53424 20278 20286 Tween-20 +T495 NCBITaxon:9925 20290 20294 goat +T496 UBERON:0001977 20295 20300 serum +T497 PR:000003572 20345 20355 sterolin-1 +T498 PR:000003549 20381 20385 Bsep +T499 CHEBI:53422 20418 20423 Tween +T500 CHEBI:53422 20491 20496 Tween +T501 GO:0042571 20515 20523 antibody +T502 NCBITaxon:9925 20525 20529 goat +T503 NCBITaxon:9986 20535 20541 rabbit +T504 CHEBI:52661 20542 20551 Alexa 488 +T505 CHEBI:51231 20687 20691 Dapi +T506 CHEBI:75958 20698 20706 solution +T507 CHEBI:51231 20819 20823 Dapi +T508 PR:000003572 20878 20883 Abcg5 +T509 PR:000003549 20888 20892 Bsep +T510 GO:0009294 21027 21039 Transfection +T511 GO:0009294 21104 21115 transfected +T512 NCBITaxon:10088 21126 21131 mouse +T513 PR:000003572 21132 21137 Abcg5 +T514 NCBITaxon:10088 21147 21152 mouse +T515 PR:000003573 21153 21158 Abcg8 +T516 GO:0010467 21159 21169 expression +T517 CHEBI:33893 21208 21215 reagent +T518 GO:0009294 21322 21334 transfection +T519 GO:0040007 21367 21372 grown +T520 CHEBI:16526 21388 21391 CO2 +T521 CHEBI:17334 21436 21446 penicillin +T522 CHEBI:17076 21458 21470 streptomycin +T523 GO:0009294 21504 21516 transfection +T524 CHEBI:33893 21562 21569 reagent +T525 CHEBI:60004 21605 21612 mixture +T526 GO:0009294 21769 21780 transfected +T527 GO:0010467 21830 21840 expression +T528 CHEBI:60004 21875 21882 mixture +T529 CHEBI:15347 21886 21893 acetone +T530 CHEBI:17790 21894 21902 methanol +T531 NCBITaxon:9925 21966 21970 goat +T532 UBERON:0001977 21971 21976 serum +T533 GO:0042571 22045 22053 antibody +T534 PR:000003572 22059 22064 Abcg5 +T535 NCBITaxon:9925 22071 22075 goat +T536 UBERON:0001977 22076 22081 serum +T537 NCBITaxon:9986 22158 22164 rabbit +T538 GO:0071736 22165 22177 IgG antibody +T539 UBERON:0002294 22285 22292 biliary +T540 CHEBI:18059 22293 22298 lipid +T541 NCBITaxon:10088 22324 22328 Mice +T542 UBERON:0001179 22356 22366 peritoneal +T543 CHEBI:119915 22388 22396 fentanyl +T544 CHEBI:49575 22421 22429 Diazepam +T545 UBERON:0000916 22435 22442 abdomen +T546 UBERON:0002394 22488 22497 bile duct +T547 UBERON:0002110 22503 22514 gallbladder +T548 UBERON:0001970 22531 22535 Bile +T549 UBERON:0001970 22610 22614 Bile +T550 UBERON:0002294 22652 22659 Biliary +T551 UBERON:0001970 22660 22664 bile +T552 CHEBI:22868 22660 22669 bile salt +T553 CHEBI:15889 22671 22677 sterol +T554 CHEBI:16247 22682 22694 phospholipid +T555 UBERON:0002294 22796 22803 biliary +T556 CHEBI:18059 22804 22809 lipid +T557 UBERON:0001970 22821 22825 bile +T558 UBERON:0001970 22876 22880 bile +T559 CHEBI:22868 22876 22885 bile salt +T560 UBERON:0004711 22996 23008 jugular vein +T561 UBERON:0001970 23081 23085 Bile +T562 UBERON:0001970 23137 23141 bile +T563 CHEBI:22868 23137 23146 bile salt +T564 CHEBI:18059 23151 23156 lipid +T565 UBERON:0002394 23224 23233 bile duct +T566 UBERON:0002110 23253 23264 gallbladder +T567 UBERON:0001970 23266 23270 bile +T568 NCBITaxon:10088 23305 23309 mice +T569 NCBITaxon:10088 23359 23363 mice +T570 CHEBI:15889 23497 23503 Sterol +T571 UBERON:0001970 23516 23520 bile +T572 CHEBI:11814 23580 23587 HMG-CoA +T573 CHEBI:16113 23592 23603 cholesterol +T574 NCBITaxon:10088 23636 23641 Mouse +T575 UBERON:0002107 23642 23647 liver +T576 UBERON:0002107 23729 23734 liver +T577 CHEBI:17992 23819 23826 sucrose +T578 CHEBI:9754 23834 23838 Tris +T579 CHEBI:64734 23847 23860 disodium EDTA +T580 CHEBI:18320 23869 23872 DTT +T581 CHEBI:32031 24029 24050 dipotassium phosphate +T582 CHEBI:32588 24059 24062 KCl +T583 CHEBI:18320 24069 24072 DTT +T584 CHEBI:64734 24079 24092 disodium EDTA +T585 CHEBI:17754 24098 24106 glycerol +T586 CHEBI:16113 24241 24252 cholesterol +T587 CHEBI:60004 24402 24409 mixture +T588 CHEBI:32031 24480 24486 K2HPO4 +T589 CHEBI:18320 24506 24509 DTT +T590 CHEBI:17154 24517 24529 nicotinamide +T591 CHEBI:36927 24541 24544 14C +T592 CHEBI:16113 24546 24557 cholesterol +T593 CHEBI:495055 24609 24623 β-cyclodextrin +T594 GO:0006741 24747 24763 NADPH-generating +T595 CHEBI:18009 24779 24784 NADP+ +T596 CHEBI:14314 24792 24811 glucose-6-phosphate +T597 CHEBI:14314 24822 24841 glucose-6-phosphate +T598 CHEBI:32035 24935 24938 KOH +T599 MOP:0000370 24948 24957 butylated +T600 CHEBI:24751 24958 24972 hydroxytoluene +T601 CHEBI:16236 24987 24996 ethanolic +T602 CHEBI:32035 24997 25016 potassium hydroxide +T603 CHEBI:29238 25019 25021 3H +T604 CHEBI:17500 25023 25044 7α-hydroxycholesterol +T605 CHEBI:15889 25151 25158 sterols +T606 CHEBI:29021 25193 25201 n-hexane +T607 CHEBI:29021 25301 25309 n-hexane +T608 CHEBI:17824 25310 25320 2-propanol +T609 CHEBI:30563 25353 25359 silica +T610 CHEBI:29021 25396 25404 n-hexane +T611 CHEBI:17824 25405 25415 2-propanol +T612 CHEBI:17500 25433 25454 7α-hydroxycholesterol +T613 CHEBI:29021 25479 25487 n-hexane +T614 CHEBI:17824 25488 25498 2-propanol +T615 CHEBI:30563 25546 25552 silica +T616 CHEBI:30563 25565 25571 Silica +T617 CHEBI:35702 25618 25631 diethyl ether +T618 CHEBI:11814 25810 25817 HMG-CoA +T619 NCBITaxon:33208 26109 26116 animals +T620 PR:000003573 26181 26186 Abcg8 +T621 PR:000003573 26187 26197 sterolin-2 +T622 NCBITaxon:10088 26208 26212 mice +T623 GO:0008380 26296 26304 splicing +T624 SO:0000147 26357 26362 exons +T625 CL:0002322 26378 26385 ES cell +T626 SO:0000155 26431 26438 plasmid +T627 CL:0002322 26471 26473 ES +T628 CL:0002322 26492 26494 ES +T629 UBERON:0000358 26529 26539 Blastocyst +T630 NCBITaxon:10088 26592 26596 mice +T631 CL:0002322 26669 26671 ES +T632 NCBITaxon:10088 26825 26829 mice +T633 NCBITaxon:33208 26903 26910 animals +T634 NCBITaxon:10088 26966 26970 mice +T635 NCBITaxon:10088 27104 27108 mice +T636 PR:000003573 27139 27144 Abcg8 +T637 GO:0010467 27146 27156 expression +T638 PR:000003572 27160 27165 Abcg5 +T639 GO:0000380 27228 27248 alternative splicing +T640 GO:0006412 27258 27271;27320 27327 production of ... protein +T641 PR:000003573 27303 27308 Abcg8 +T642 PR:000003573 27309 27319 sterolin-2 +T643 PR:000003572 27362 27367 Abcg5 +T644 PR:000003572 27368 27378 sterolin-1 +T645 GO:0001171 27409 27428 reverse transcribed +T646 UBERON:0002107 27440 27445 liver +T647 PR:000003573 27455 27460 Abcg8 +T648 NCBITaxon:10088 27471 27475 mice +T649 PR:000003573 27489 27494 Abcg8 +T650 PR:000003573 27495 27505 sterolin-2 +T651 SO:0000147 27558 27562 exon +T652 PR:000003573 27572 27577 Abcg8 +T653 NCBITaxon:10088 27588 27592 mice +T654 SO:0000112 27602 27609 primers +T655 SO:0000147 27626 27631 exons +T656 SO:0000147 27642 27647 exons +T657 SO:0000147 27660 27665 exons +T658 GO:0000380 27721 27742 alternatively spliced +T659 SO:1001187 27721 27750 alternatively spliced message +T660 CHEBI:33695 27743 27750 message +T661 PR:000003573 27827 27832 Abcg8 +T662 NCBITaxon:10088 27843 27847 mice +T663 GO:0016020 27853 27861 membrane +T664 SO:0000417 27871 27878 domains +T665 PR:000003573 27882 27887 Abcg8 +T666 PR:000003573 27888 27898 sterolin-2 +T667 SO:0000147 27914 27919 exons +T668 CHEBI:15889 27927 27933 Sterol +T669 PR:000003573 27944 27949 Abcg8 +T670 PR:000003573 27950 27960 sterolin-2 +T671 NCBITaxon:33208 27971 27978 animals +T672 NCBITaxon:3193 27989 27994 plant +T673 CHEBI:15889 27995 28002 sterols +T674 UBERON:0000479 28006 28012 tissue +T675 UBERON:0001969 28017 28023 plasma +T676 http://purl.obolibrary.org/obo/MONDO_0008863 28042 28056 sitosterolemia +T677 CHEBI:15889 28058 28064 Sterol +T678 UBERON:0001969 28127 28133 plasma +T679 UBERON:0000479 28138 28145 tissues +T680 PR:000003573 28153 28158 Abcg8 +T681 NCBITaxon:10088 28162 28166 mice +T682 UBERON:0000479 28218 28224 tissue +T683 UBERON:0001969 28282 28288 Plasma +T684 CHEBI:16113 28289 28300 cholesterol +T685 NCBITaxon:10088 28343 28347 mice +T686 NCBITaxon:10088 28424 28428 mice +T687 NCBITaxon:3193 28458 28463 Plant +T688 CHEBI:15889 28464 28471 sterols +T689 NCBITaxon:10088 28527 28531 mice +T690 PR:000003573 28568 28573 Abcg8 +T691 NCBITaxon:10088 28577 28581 mice +T692 UBERON:0001969 28583 28589 Plasma +T693 CHEBI:28623 28590 28601 campesterol +T694 CHEBI:27693 28606 28616 sitosterol +T695 PR:000003573 28658 28663 Abcg8 +T696 NCBITaxon:10088 28667 28671 mice +T697 NCBITaxon:10088 28694 28698 mice +T698 UBERON:0001969 28836 28842 plasma +T699 NCBITaxon:3193 28843 28848 plant +T700 CHEBI:15889 28849 28855 sterol +T701 NCBITaxon:10088 28898 28902 mice +T702 UBERON:0002107 28904 28909 Liver +T703 CHEBI:16113 28910 28921 cholesterol +T704 PR:000003573 28937 28942 Abcg8 +T705 NCBITaxon:10088 28946 28950 mice +T706 UBERON:0002107 28993 28998 liver +T707 UBERON:0000479 29027 29033 tissue +T708 UBERON:0000479 29069 29075 tissue +T709 CHEBI:16113 29104 29115 cholesterol +T710 CHEBI:28623 29162 29173 campesterol +T711 CHEBI:27693 29200 29210 sitosterol +T712 UBERON:0000479 29258 29264 tissue +T713 PR:000003573 29283 29288 Abcg8 +T714 NCBITaxon:10088 29301 29305 mice +T715 NCBITaxon:10088 29328 29332 mice +T716 UBERON:0000479 29377 29383 tissue +T717 UBERON:0002107 29450 29455 liver +T718 CHEBI:16113 29456 29467 cholesterol +T719 NCBITaxon:3193 29471 29476 plant +T720 CHEBI:15889 29477 29483 sterol +T721 PR:000003573 29501 29506 Abcg8 +T722 PR:000003573 29514 29519 Abcg8 +T723 NCBITaxon:10088 29523 29527 mice +T724 UBERON:0002106 29529 29535 Spleen +T725 CHEBI:15889 29536 29542 sterol +T726 UBERON:0002107 29566 29571 liver +T727 UBERON:0000955 29622 29627 brain +T728 CHEBI:15889 29628 29634 sterol +T729 UBERON:0000120 29691 29710 blood brain barrier +T730 GO:0035376 29734 29742;29749 29756 entry of ... sterols +T731 CHEBI:15889 29749 29756 sterols +T732 http://purl.obolibrary.org/obo/MONDO_0008863 29760 29774 sitosterolemia +T733 NCBITaxon:3193 29812 29817 plant +T734 CHEBI:15889 29818 29825 sterols +T735 UBERON:0000955 29833 29839 brains +T736 NCBITaxon:33208 29854 29861 animals +T737 UBERON:0000178 29874 29879 blood +T738 UBERON:0000479 29901 29907 tissue +T739 UBERON:0000479 29965 29971 tissue +T740 NCBITaxon:3193 29972 29977 plant +T741 CHEBI:15889 29978 29985 sterols +T742 UBERON:0002107 30008 30014 livers +T743 PR:000003573 30018 30023 Abcg8 +T744 NCBITaxon:10088 30040 30044 mice +T745 CHEBI:17002 30079 30101 esterified cholesterol +T746 CHEBI:27693 30165 30175 sitosterol +T747 CHEBI:28623 30179 30190 campesterol +T748 UBERON:0000479 30278 30284 tissue +T749 CHEBI:15889 30285 30291 sterol +T750 CHEBI:15889 30310 30317 sterols +T751 CHEBI:15889 30338 30345 sterols +T752 CHEBI:15889 30396 30403 sterols +T753 CHEBI:17855 30409 30422 triglycerides +T754 UBERON:0001969 30441 30447 plasma +T755 NCBITaxon:33208 30468 30475 animals +T756 GO:0007631 30476 30479 fed +T757 CHEBI:33290 30490 30494 chow +T758 CHEBI:15889 30550 30556 sterol +T759 PR:000003573 30601 30606 Abcg8 +T760 NCBITaxon:10088 30619 30623 mice +T761 CHEBI:17855 30651 30663 triglyceride +T762 NCBITaxon:10088 30742 30746 mice +T763 NCBITaxon:33208 30874 30881 animals +T764 UBERON:0001969 30889 30895 plasma +T765 CHEBI:17855 30896 30908 triglyceride +T766 PR:000003573 30923 30928 Abcg8 +T767 NCBITaxon:10088 30932 30936 mice +T768 CHEBI:39015 31182 31197 apolipoproteins +T769 PR:000004145 31182 31199 apolipoproteins B +T770 PR:000004155 31182 31197;31201 31202 apolipoproteins ... E +T771 PR:000004140 31182 31197;31204 31206 apolipoproteins ... AI +T772 PR:000004142 31182 31197;31211 31214 apolipoproteins ... AII +T773 CHEBI:17855 31241 31253 triglyceride +T774 UBERON:0002107 31293 31298 Liver +T775 SO:0000704 31299 31303 gene +T776 GO:0010467 31299 31314 gene expression +T777 PR:000003573 31345 31350 Abcg8 +T778 PR:000003573 31351 31361 sterolin-2 +T779 NCBITaxon:10088 31372 31376 mice +T780 PR:000003573 31424 31429 Abcg8 +T781 PR:000003573 31430 31440 sterolin-2 +T782 SO:0000704 31448 31453 genes +T783 GO:0065007 31459 31467 regulate +T784 CHEBI:15889 31468 31474 sterol +T785 GO:0016125 31468 31485 sterol metabolism +T786 GO:0010467 31536 31546 expression +T787 PR:000003572 31557 31562 Abcg5 +T788 PR:000003573 31564 31569 Abcg8 +T789 PR:000006148 31577 31583 Cyp7a1 +T790 PR:000003537 31585 31590 Abca1 +T791 PR:P21440 31592 31596 Mdr2 +T792 PR:000028988 31603 31608 Srebp +T793 PR:000015612 31617 31624 Srebp-2 +T794 UBERON:0002107 31637 31643 livers +T795 NCBITaxon:10088 31647 31651 mice +T796 GO:0007631 31652 31655 fed +T797 CHEBI:33290 31666 31670 chow +T798 PR:000003573 31702 31707 Abcg8 +T799 GO:0010467 31713 31723 expression +T800 PR:000003573 31756 31761 Abcg8 +T801 NCBITaxon:10088 31765 31769 mice +T802 NCBITaxon:10088 31815 31819 mice +T803 NCBITaxon:10088 31843 31847 mice +T804 GO:0010467 31897 31907 expression +T805 PR:000003572 31911 31916 Abcg5 +T806 NCBITaxon:10088 31934 31938 mice +T807 NCBITaxon:10088 31999 32003 mice +T808 NCBITaxon:10088 32056 32060 mice +T809 GO:0010467 32062 32072 Expression +T810 CHEBI:11814 32076 32083 HMG-CoA +T811 NCBITaxon:10088 32163 32167 mice +T812 NCBITaxon:9606 32222 32227 human +T813 http://purl.obolibrary.org/obo/MONDO_0000001 32247 32255 disorder +T814 UBERON:0002107 32352 32357 liver +T815 CHEBI:11814 32384 32391 HMG-CoA +T816 PR:000006148 32415 32421 Cyp7a1 +T817 CHEBI:11814 32444 32451 HMG-CoA +T818 PR:000003573 32505 32510 Abcg8 +T819 PR:000003573 32518 32523 Abcg8 +T820 NCBITaxon:10088 32527 32531 mice +T821 GO:0010467 32598 32608 expression +T822 PR:000006148 32636 32642 Cyp7a1 +T823 GO:0010467 32648 32658 expression +T824 NCBITaxon:10088 32709 32714 mouse +T825 PR:000006148 32716 32722 Cyp7a1 +T826 NCBITaxon:10088 32799 32803 mice +T827 PR:000006148 32835 32841 Cyp7a1 +T828 CHEBI:27693 32858 32868 Sitosterol +T829 CHEBI:35222 32905 32914 inhibitor +T830 PR:000006148 32918 32924 Cyp7a1 +T831 NCBITaxon:3193 32960 32965 plant +T832 CHEBI:15889 32966 32973 sterols +T833 UBERON:0002107 32981 32986 liver +T834 NCBITaxon:10088 33038 33043 mouse +T835 PR:000003572 33067 33072 Abcg5 +T836 PR:000003573 33084 33089 Abcg8 +T837 NCBITaxon:10088 33102 33106 mice +T838 PR:000003573 33125 33130 Abcg8 +T839 PR:000003573 33131 33141 sterolin-2 +T840 PR:000003572 33160 33165 Abcg5 +T841 PR:000003572 33166 33176 sterolin-1 +T842 GO:0010467 33177 33187 expression +T843 SO:0000704 33220 33227 genetic +T844 GO:0010467 33284 33294 expression +T845 GO:0042571 33313 33321 antibody +T846 NCBITaxon:10088 33325 33330 mouse +T847 PR:000003573 33331 33336 Abcg8 +T848 PR:000003573 33337 33347 sterolin-2 +T849 NCBITaxon:9986 33422 33428 rabbit +T850 GO:0042571 33440 33448 antibody +T851 NCBITaxon:10088 33452 33457 mouse +T852 PR:000003572 33458 33463 Abcg5 +T853 PR:000003572 33464 33474 sterolin-1 +T854 GO:0042571 33488 33498 antibodies +T855 UBERON:0002107 33573 33578 liver +T856 UBERON:0000160 33583 33592 intestine +T857 PR:000003573 33596 33601 Abcg8 +T858 NCBITaxon:10088 33605 33609 mice +T859 UBERON:0002107 33631 33636 liver +T860 GO:0016020 33643 33651 membrane +T861 PR:000003572 33707 33712 Abcg5 +T862 PR:000003572 33713 33723 sterolin-1 +T863 GO:0042571 33724 33734 antibodies +T864 PR:000003572 33795 33800 Abcg5 +T865 PR:000003572 33801 33811 sterolin-1 +T866 PR:000003572 33948 33953 Abcg5 +T867 PR:000003572 33954 33964 sterolin-1 +T868 GO:0042571 33973 33981 antibody +T869 GO:0042571 34027 34035 antibody +T870 NCBITaxon:33208 34107 34114 animals +T871 PR:000007078 34158 34164 Endo-H +T872 PR:000016261 34226 34237 transferrin +T873 GO:0042571 34248 34256 antibody +T874 PR:000003572 34286 34291 Abcg5 +T875 PR:000003572 34292 34302 sterolin-1 +T876 NCBITaxon:33208 34316 34323 animals +T877 GO:0042571 34409 34417 antibody +T878 PR:000007078 34443 34449 Endo-H +T879 PR:000011196 34454 34460 PNGase +T880 GO:0042571 34495 34503 antibody +T881 PR:000003572 34549 34554 Abcg5 +T882 PR:000003572 34555 34565 sterolin-1 +T883 NCBITaxon:33208 34579 34586 animals +T884 NCBITaxon:10088 34624 34629 mouse +T885 GO:0042571 34671 34681 antibodies +T886 PR:000003572 34695 34700 Abcg5 +T887 PR:000003572 34701 34711 sterolin-1 +T888 GO:0042571 34814 34824 antibodies +T889 GO:0009294 34835 34847 transfection +T890 GO:0042571 34864 34872 antibody +T891 GO:0010467 34889 34898 expressed +T892 NCBITaxon:10088 34899 34904 mouse +T893 PR:000003572 34905 34910 Abcg5 +T894 PR:000003572 34911 34921 sterolin-1 +T895 NCBITaxon:10088 34946 34951 mouse +T896 PR:000003573 34952 34957 Abcg8 +T897 PR:000003573 34958 34968 sterolin-2 +T898 GO:0009294 34988 35000 transfection +T899 PR:000003572 35026 35031 Abcg5 +T900 PR:000003573 35036 35041 Abcg8 +T901 GO:0010467 35146 35156 expression +T902 PR:000003573 35160 35165 Abcg8 +T903 GO:0010467 35175 35185 expressing +T904 PR:000003572 35186 35191 Abcg5 +T905 PR:000003572 35192 35202 sterolin-1 +T906 GO:0042571 35227 35235 antibody +T907 PR:000003573 35240 35245 Abcg8 +T908 PR:000003573 35246 35256 sterolin-2 +T909 GO:0042571 35322 35330 antibody +T910 PR:000003572 35345 35350 Abcg5 +T911 PR:000003572 35351 35361 sterolin-1 +T912 PR:000003573 35392 35397 Abcg8 +T913 PR:000003573 35398 35408 sterolin-2 +T914 UBERON:0000160 35430 35440 intestinal +T915 UBERON:0000479 35441 35447 tissue +T916 PR:000003572 35473 35478 Abcg5 +T917 PR:000003572 35479 35489 sterolin-1 +T918 PR:000003572 35528 35533 Abcg5 +T919 PR:000003572 35534 35544 sterolin-1 +T920 PR:000003572 35573 35578 Abcg5 +T921 PR:000003572 35579 35589 sterolin-1 +T922 UBERON:0000160 35597 35607 intestinal +T923 GO:0045177 35628 35634 apical +T924 CL:0000584 35669 35680 enterocytes +T925 PR:000003573 35708 35713 Abcg8 +T926 PR:000003573 35714 35724 sterolin-2 +T927 GO:0042571 35803 35811 antibody +T928 PR:000003572 35828 35833 Abcg5 +T929 PR:000003572 35834 35844 sterolin-1 +T930 GO:0042571 35877 35885 antibody +T931 PR:000003572 35980 35985 Abcg5 +T932 PR:000003572 35986 35996 sterolin-1 +T933 GO:0010467 35997 36007 expression +T934 GO:0045177 36032 36038 apical +T935 GO:0042571 36102 36110 antibody +T936 UBERON:0002107 36120 36125 liver +T937 NCBITaxon:10088 36163 36167 mice +T938 GO:0010467 36192 36202 expression +T939 PR:000003549 36206 36212 Abcb11 +T940 UBERON:0001970 36275 36279 bile +T941 CHEBI:22868 36275 36285 bile salts +T942 PR:000003572 36311 36316 Abcg5 +T943 PR:000003572 36317 36327 sterolin-1 +T944 GO:0045177 36369 36377 apically +T945 UBERON:0002107 36391 36396 liver +T946 NCBITaxon:10088 36421 36425 mice +T947 UBERON:0002107 36473 36478 liver +T948 NCBITaxon:10088 36493 36497 mice +T949 GO:0010467 36512 36522 expression +T950 PR:000003572 36532 36537 Abcg5 +T951 PR:000003572 36538 36548 sterolin-1 +T952 GO:0045177 36626 36632 apical +T953 GO:0010467 36633 36643 expression +T954 PR:000003572 36647 36652 Abcg5 +T955 PR:000003572 36653 36663 sterolin-1 +T956 NCBITaxon:10088 36680 36684 mice +T957 GO:0042571 36695 36703 antibody +T958 UBERON:0002107 36722 36727 liver +T959 NCBITaxon:10088 36763 36767 mice +T960 GO:0045177 36785 36791 apical +T961 GO:0010467 36792 36802 expression +T962 PR:000003572 36806 36811 Abcg5 +T963 PR:000003572 36812 36822 sterolin-1 +T964 UBERON:0002107 36854 36860 livers +T965 UBERON:0002294 36878 36885 Biliary +T966 UBERON:0001970 36899 36903 bile +T967 CHEBI:22868 36899 36909 bile salts +T968 CHEBI:15889 36911 36917 sterol +T969 CHEBI:16247 36922 36935 phospholipids +T970 PR:000003573 36939 36944 Abcg8 +T971 PR:000003573 36945 36955 sterolin-2 +T972 NCBITaxon:10088 36966 36970 mice +T973 http://purl.obolibrary.org/obo/MONDO_0008863 36972 36986 Sitosterolemic +T974 NCBITaxon:1 36987 36998 individuals +T975 GO:0046903 37027 37034 secrete +T976 CHEBI:16113 37035 37046 cholesterol +T977 NCBITaxon:3193 37051 37056 plant +T978 CHEBI:15889 37057 37064 sterols +T979 UBERON:0001970 37070 37074 bile +T980 NCBITaxon:10088 37109 37113 mice +T981 UBERON:0002294 37137 37144 biliary +T982 CHEBI:15889 37145 37151 sterol +T983 UBERON:0001970 37192 37196 bile +T984 CHEBI:15889 37223 37229 sterol +T985 CHEBI:16247 37234 37246 phospholipid +T986 PR:000003573 37259 37264 Abcg8 +T987 NCBITaxon:10088 37268 37272 mice +T988 NCBITaxon:10088 37311 37315 mice +T989 UBERON:0001970 37335 37339 bile +T990 CHEBI:22868 37335 37344 bile salt +T991 CHEBI:15889 37390 37396 sterol +T992 CHEBI:16247 37424 37436 phospholipid +T993 UBERON:0002294 37496 37503 biliary +T994 CHEBI:15889 37504 37510 sterol +T995 UBERON:0002294 37558 37565 biliary +T996 CHEBI:15889 37566 37572 sterol +T997 NCBITaxon:10088 37595 37599 mice +T998 CHEBI:80774 37634 37659 tauroursodeoxycholic acid +T999 CHEBI:80774 37661 37665 TUDC +T1000 NCBITaxon:10088 37668 37672 Mice +T1001 UBERON:0001970 37713 37717 bile +T1002 CHEBI:22868 37713 37722 bile salt +T1003 UBERON:0004711 37773 37785 jugular vein +T1004 CHEBI:80774 37811 37815 TUDC +T1005 UBERON:0001970 37840 37844 bile +T1006 CHEBI:22868 37840 37849 bile salt +T1007 CHEBI:80774 37937 37941 TUDC +T1008 CHEBI:16247 38035 38047 phospholipid +T1009 NCBITaxon:10088 38120 38124 mice +T1010 NCBITaxon:10088 38139 38143 mice +T1011 CHEBI:16247 38168 38180 phospholipid +T1012 UBERON:0002294 38274 38281 biliary +T1013 CHEBI:15889 38282 38288 sterol +T1014 CHEBI:15889 38312 38318 sterol +T1015 CHEBI:27693 38407 38417 sitosterol +T1016 CHEBI:16113 38421 38432 cholesterol +T1017 NCBITaxon:10088 38477 38481 mice +T1018 CHEBI:15889 38504 38510 sterol +T1019 NCBITaxon:10088 38583 38587 mice +T1020 NCBITaxon:10088 38609 38613 mice +T1021 CHEBI:15889 38615 38621 sterol +T1022 PR:000003573 38713 38718 Abcg8 +T1023 PR:000003573 38719 38729 sterolin-2 +T1024 UBERON:0002294 38758 38765 biliary +T1025 CHEBI:16113 38767 38778 cholesterol +T1026 NCBITaxon:10088 38807 38811 mice +T1027 CHEBI:15889 38813 38819 sterol +T1028 CHEBI:80774 38927 38931 TUDC +T1029 PR:000003573 38944 38949 Abcg8 +T1030 NCBITaxon:10088 38953 38957 mice +T1031 GO:0046903 38964 38972 secreted +T1032 CHEBI:15889 38978 38985 sterols +T1033 UBERON:0002294 39014 39021 biliary +T1034 CHEBI:15889 39022 39028 sterol +T1035 GO:0046903 39048 39056 secreted +T1036 UBERON:0001970 39068 39072 bile +T1037 NCBITaxon:10088 39092 39096 mice +T1038 CHEBI:80774 39127 39131 TUDC +T1039 NCBITaxon:10088 39152 39156 Mice +T1040 UBERON:0001970 39197 39201 bile +T1041 CHEBI:3098 39197 39206 bile acid +T1042 CHEBI:80774 39267 39271 TUDC +T1043 NCBITaxon:10088 39321 39325 mice +T1044 GO:0046903 39371 39378 secrete +T1045 CHEBI:16113 39379 39390 cholesterol +T1046 GO:0046903 39428 39435 secrete +T1047 CHEBI:27693 39436 39446 sitosterol +T1048 CHEBI:28623 39451 39462 campesterol +T1049 NCBITaxon:10088 39485 39489 mice +T1050 PR:000003573 39520 39525 Abcg8 +T1051 NCBITaxon:10088 39529 39533 mice +T1052 GO:0046903 39573 39580 secrete +T1053 CHEBI:15889 39585 39592 sterols +T1054 NCBITaxon:10088 39632 39636 mice +T1055 CHEBI:16113 39731 39742 cholesterol +T1056 CHEBI:16113 39784 39795 cholesterol +T1057 CHEBI:15889 39796 39803 sterols +T1058 UBERON:0002107 39856 39861 liver +T1059 GO:0046903 39878 39885 secrete +T1060 CHEBI:15889 39886 39893 sterols +T1061 SO:0000704 39941 39948 genetic +T1062 NCBITaxon:9606 39969 39974 human +T1063 http://purl.obolibrary.org/obo/MONDO_0000001 39975 39983 disorder +T1064 http://purl.obolibrary.org/obo/MONDO_0008863 39985 39999 sitosterolemia +T1065 SO:0000704 40180 40185 genes +T1066 http://purl.obolibrary.org/obo/MONDO_0008863 40270 40284 sitosterolemia +T1067 NCBITaxon:10088 40298 40303 mouse +T1068 http://purl.obolibrary.org/obo/MONDO_0008863 40313 40327 sitosterolemia +T1069 SO:0000704 40384 40389 genes +T1070 PR:000003573 40391 40396 Abcg8 +T1071 NCBITaxon:10088 40403 40408 mouse +T1072 NCBITaxon:9606 40449 40454 human +T1073 http://purl.obolibrary.org/obo/MONDO_0008863 40455 40469 sitosterolemia +T1074 UBERON:0001969 40480 40486 Plasma +T1075 UBERON:0000479 40495 40502 tissues +T1076 UBERON:0000955 40519 40524 brain +T1077 CHEBI:27693 40554 40564 sitosterol +T1078 NCBITaxon:10088 40593 40597 mice +T1079 http://purl.obolibrary.org/obo/MONDO_0008863 40622 40636 sitosterolemic +T1080 NCBITaxon:10088 40693 40697 mice +T1081 NCBITaxon:9606 40845 40851 humans +T1082 NCBITaxon:9606 40869 40875 humans +T1083 CHEBI:11814 40893 40900 HMG-CoA +T1084 PR:000006148 40915 40921 CYP7a1 +T1085 UBERON:0002107 41025 41031 livers +T1086 NCBITaxon:33208 41046 41053 animals +T1087 UBERON:0001970 41145 41149 bile +T1088 CHEBI:3098 41145 41154 bile acid +T1089 GO:0006699 41145 41164 bile acid synthesis +T1090 PR:000006148 41166 41172 CYP7a1 +T1091 CHEBI:27693 41296 41306 sitosterol +T1092 UBERON:0002294 41360 41367 Biliary +T1093 CHEBI:15889 41368 41375 sterols +T1094 PR:000003573 41379 41384 Abcg8 +T1095 NCBITaxon:10088 41395 41399 mice +T1096 NCBITaxon:10088 41450 41454 mice +T1097 UBERON:0002294 41471 41478 biliary +T1098 CHEBI:15889 41479 41485 sterol +T1099 PR:000003573 41505 41510 Abcg8 +T1100 NCBITaxon:10088 41514 41518 mice +T1101 CHEBI:15889 41554 41560 sterol +T1102 UBERON:0001970 41576 41580 bile +T1103 UBERON:0001970 41611 41615 bile +T1104 CHEBI:3098 41611 41621 bile acids +T1105 PR:000003573 41653 41658 Abcg8 +T1106 PR:000003573 41659 41669 sterolin-2 +T1107 GO:0046903 41697 41704 secrete +T1108 CHEBI:16113 41705 41716 cholesterol +T1109 UBERON:0001970 41722 41726 bile +T1110 CHEBI:27693 41750 41760 sitosterol +T1111 UBERON:0001969 41820 41826 plasma +T1112 CHEBI:27693 41827 41837 sitosterol +T1113 NCBITaxon:10088 41861 41865 mice +T1114 UBERON:0002294 41908 41915 biliary +T1115 CHEBI:27693 41916 41926 sitosterol +T1116 CHEBI:27693 42024 42034 sitosterol +T1117 UBERON:0001970 42053 42057 bile +T1118 NCBITaxon:3193 42072 42077 plant +T1119 CHEBI:15889 42078 42085 sterols +T1120 GO:0046903 42093 42101 secreted +T1121 PR:000003573 42117 42122 Abcg8 +T1122 PR:000003573 42123 42133 sterolin-2 +T1123 PR:000003573 42150 42155 Abcg8 +T1124 NCBITaxon:10088 42159 42163 mice +T1125 GO:0046903 42167 42174 secrete +T1126 UBERON:0001970 42175 42179 bile +T1127 CHEBI:22868 42175 42184 bile salt +T1128 CHEBI:16247 42189 42201 phospholipid +T1129 UBERON:0001970 42207 42211 bile +T1130 NCBITaxon:10088 42237 42241 mice +T1131 PR:000003573 42311 42316 Abcg8 +T1132 PR:000003573 42317 42327 sterolin-2 +T1133 UBERON:0002423 42345 42358 hepatobiliary +T1134 CHEBI:16113 42359 42370 cholesterol +T1135 NCBITaxon:10088 42384 42388 mice +T1136 PR:000003573 42391 42396 Abcg8 +T1137 NCBITaxon:10088 42400 42404 mice +T1138 NCBITaxon:9989 42457 42463 rodent +T1139 CHEBI:33290 42464 42468 chow +T1140 NCBITaxon:10088 42551 42556 mouse +T1141 UBERON:0001969 42608 42614 Plasma +T1142 UBERON:0002107 42619 42626 hepatic +T1143 NCBITaxon:3193 42627 42632 plant +T1144 CHEBI:15889 42633 42639 sterol +T1145 PR:000003573 42650 42655 Abcg8 +T1146 PR:000003572 42663 42668 Abcg5 +T1147 PR:000003573 42669 42674 Abcg8 +T1148 CHEBI:16113 42733 42744 cholesterol +T1149 UBERON:0001969 42768 42774 plasma +T1150 CHEBI:17855 42775 42787 triglyceride +T1151 PR:000003573 42802 42807 Abcg8 +T1152 NCBITaxon:10088 42811 42815 mice +T1153 NCBITaxon:33208 42852 42859 animals +T1154 CHEBI:17855 42879 42891 triglyceride +T1155 CHEBI:8984 43011 43014 SDS +T1156 UBERON:0002107 43124 43131 Hepatic +T1157 CHEBI:16113 43154 43165 cholesterol +T1158 PR:000003573 43190 43195 Abcg8 +T1159 NCBITaxon:10088 43199 43203 mice +T1160 NCBITaxon:10088 43226 43230 mice +T1161 CHEBI:11814 43251 43258 HMG-CoA +T1162 PR:000028988 43352 43358 Srebps +T1163 NCBITaxon:3193 43431 43436 plant +T1164 CHEBI:15889 43437 43444 sterols +T1165 PR:000028988 43471 43476 SREBP +T1166 CHEBI:27693 43489 43499 Sitosterol +T1167 PR:000006148 43535 43541 Cyp7a1 +T1168 NCBITaxon:10088 43668 43672 mice +T1169 CHEBI:27693 43693 43703 sitosterol +T1170 NCBITaxon:10088 43899 43903 mice +T1171 NCBITaxon:10088 43922 43926 mice +T1172 CHEBI:16113 43948 43959 cholesterol +T1173 CHEBI:27693 43965 43975 sitosterol +T1174 CHEBI:15889 44029 44036 sterols +T1175 NCBITaxon:9606 44083 44088 human +T1176 http://purl.obolibrary.org/obo/MONDO_0000001 44089 44097 disorder +T1177 NCBITaxon:1 44136 44147 individuals +T1178 UBERON:0001621 44168 44183 coronary artery +T1179 http://purl.obolibrary.org/obo/MONDO_0005010 44168 44191 coronary artery disease +T1180 PR:000003573 44194 44199 Abcg8 +T1181 PR:000003573 44200 44210 sterolin-2 +T1182 NCBITaxon:10088 44221 44225 mice +T1183 UBERON:0002294 44279 44286 biliary +T1184 CHEBI:15889 44287 44293 sterol +T1185 UBERON:0001970 44309 44313 bile +T1186 CHEBI:15889 44317 44323 sterol +T1187 UBERON:0001970 44364 44368 bile +T1188 CHEBI:22868 44364 44373 bile salt +T1189 GO:0007588 44374 44383 excretion +T1190 PR:000003573 44385 44390 Abcg8 +T1191 PR:000003573 44391 44401 sterolin-2 +T1192 NCBITaxon:10088 44412 44416 mice +T1193 NCBITaxon:10088 44462 44466 mice +T1194 PR:000003573 44474 44479 Abcg8 +T1195 PR:000003573 44480 44490 sterolin-2 +T1196 CHEBI:16113 44508 44519 cholesterol +T1197 NCBITaxon:3193 44552 44557 plant +T1198 CHEBI:15889 44558 44564 sterol +T1199 UBERON:0002294 44613 44620 biliary +T1200 CHEBI:15889 44621 44627 sterol +T1201 NCBITaxon:10088 44675 44679 mice +T1202 UBERON:0000479 44724 44730 tissue +T1203 UBERON:0001969 44735 44741 plasma +T1204 CHEBI:27693 44751 44761 sitosterol +T1205 NCBITaxon:10088 44794 44798 mice +T1206 CHEBI:27693 44837 44847 sitosterol +T1207 UBERON:0001970 44881 44885 bile +T1208 CHEBI:15889 44967 44974 sterols +T1209 NCBITaxon:9606 45000 45006 humans +T1210 SO:0000704 45032 45039 genetic +T1211 PR:000003572 45058 45063 ABCG5 +T1212 PR:000003573 45067 45072 ABCG8 +T1213 NCBITaxon:10088 45107 45111 mice +T1214 PR:000003573 45129 45134 Abcg8 +T1215 PR:000003573 45135 45145 sterolin-2 +T1216 CHEBI:15889 45167 45173 sterol +T1217 NCBITaxon:9989 45258 45264 rodent +T1218 CHEBI:33290 45265 45269 chow +T1219 NCBITaxon:33208 45289 45296 animals +T1220 UBERON:0001969 45331 45337 plasma +T1221 UBERON:0000479 45341 45347 tissue +T1222 CHEBI:27693 45348 45358 sitosterol +T1223 PR:000003573 45470 45475 Abcg8 +T1224 NCBITaxon:10088 45479 45483 mice +T1225 UBERON:0002294 45508 45515 biliary +T1226 CHEBI:27693 45516 45526 sitosterol +T1227 NCBITaxon:10088 45553 45557 mice +T1228 UBERON:0002294 45583 45590 biliary +T1229 CHEBI:15889 45591 45597 sterol +T1230 NCBITaxon:10088 45641 45645 mice +T1231 NCBITaxon:10088 45670 45674 mice +T1232 http://purl.obolibrary.org/obo/MONDO_0008863 45683 45697 sitosterolemic +T1233 GO:0007631 45846 45853 feeding +T1234 UBERON:0000479 45984 45990 tissue +T1235 UBERON:0001969 45995 46001 plasma +T1236 NCBITaxon:3193 46002 46007 plant +T1237 CHEBI:15889 46008 46014 sterol +T1238 NCBITaxon:33208 46038 46045 animals +T1239 NCBITaxon:33208 46096 46103 animals +T1240 NCBITaxon:3193 46114 46119 plant +T1241 CHEBI:15889 46120 46126 sterol +T1242 UBERON:0001969 46150 46156 plasma +T1243 CHEBI:15889 46157 46163 sterol +T1244 PR:000003572 46212 46217 Abcg5 +T1245 PR:000003573 46218 46223 Abcg8 +T1246 GO:0010467 46229 46239 expression +T1247 NCBITaxon:10088 46266 46271 mouse +T1248 UBERON:0002294 46296 46303 biliary +T1249 CHEBI:16113 46304 46315 cholesterol +T1250 CHEBI:4629 46365 46374 diosgenin +T1251 GO:0007631 46375 46378 fed +T1252 NCBITaxon:10088 46379 46383 mice +T1253 UBERON:0002294 46412 46419 biliary +T1254 CHEBI:16113 46420 46431 cholesterol +T1255 PR:000003572 46455 46460 Abcg5 +T1256 PR:000003573 46465 46470 Abcg8 +T1257 PR:000003572 46539 46544 Abcg5 +T1258 CHEBI:4629 46569 46578 diosgenin +T1259 PR:000003573 46589 46594 Abcg8 +T1260 UBERON:0002294 46654 46661 biliary +T1261 CHEBI:16113 46662 46673 cholesterol +T1262 UBERON:0002294 46742 46749 biliary +T1263 CHEBI:16113 46750 46761 cholesterol +T1264 PR:000003573 46804 46809 Abcg8 +T1265 NCBITaxon:10088 46820 46824 mice +T1266 PR:000003573 46846 46856 sterolin-2 +T1267 SO:0000704 46898 46905 genetic +T1268 UBERON:0001969 46916 46922 plasma +T1269 NCBITaxon:3193 46923 46928 plant +T1270 CHEBI:15889 46929 46935 sterol +T1271 GO:0065007 47009 47020 controlling +T1272 UBERON:0001969 47021 47027 plasma +T1273 NCBITaxon:3193 47028 47033 plant +T1274 CHEBI:15889 47034 47040 sterol +T1275 NCBITaxon:39107 47088 47094 murine +T1276 GO:0065007 47183 47193 regulating +T1277 UBERON:0001969 47194 47200 plasma +T1278 NCBITaxon:3193 47201 47206 plant +T1279 CHEBI:15889 47207 47213 sterol +T1280 CHEBI:15889 47294 47300 sterol +T1281 GO:0015918 47294 47312 sterol trafficking +T1282 PR:000003572 47408 47413 Abcg5 +T1283 PR:000003572 47414 47424 sterolin-1 +T1284 PR:000003573 47429 47434 Abcg8 +T1285 PR:000003573 47435 47445 sterolin-2 +T1286 PR:000003573 47502 47507 Abcg8 +T1287 PR:000003573 47508 47518 sterolin-2 +T1288 PR:000003573 47602 47607 Abcg8 +T1289 PR:000003573 47608 47618 sterolin-2 +T1290 GO:0010467 47619 47629 expression +T1291 GO:0045177 47654 47660 apical +T1292 PR:000003572 47676 47681 Abcg5 +T1293 PR:000003572 47682 47692 sterolin-1 +T1294 GO:0045177 47700 47706 apical +T1295 GO:0009986 47707 47714 surface +T1296 GO:0010467 47774 47784 expression +T1297 PR:000003572 47871 47876 Abcg5 +T1298 PR:000003572 47877 47887 sterolin-1 +T1299 GO:0042571 47888 47898 antibodies +T1300 PR:000003572 47964 47969 Abcg5 +T1301 PR:000003572 47970 47980 sterolin-1 +T1302 PR:000003573 47984 47989 Abcg8 +T1303 NCBITaxon:10088 47993 47997 mice +T1304 MOP:0002162 48020 48035 N-glycosylation +T1305 PR:000003572 48068 48073 Abcg5 +T1306 PR:000003572 48074 48084 sterolin-1 +T1307 PR:000003573 48093 48098 Abcg8 +T1308 NCBITaxon:10088 48102 48106 mice +T1309 GO:0005783 48126 48147 endoplasmic reticulum +T1310 PR:000003572 48198 48203 Abcg5 +T1311 PR:000003572 48204 48214 sterolin-1 +T1312 GO:0045177 48218 48226 apically +T1313 GO:0010467 48227 48236 expressed +T1314 PR:000003573 48244 48249 Abcg8 +T1315 NCBITaxon:10088 48260 48264 mice +T1316 PR:000003572 48288 48293 Abcg5 +T1317 PR:000003572 48294 48304 sterolin-1 +T1318 GO:0042571 48305 48313 antibody +T1319 PR:000003572 48350 48355 Abcg5 +T1320 PR:000003573 48356 48361 Abcg8 +T1321 NCBITaxon:9608 48378 48384 canine +T1322 UBERON:0002029 48385 48407 gallbladder epithelial +T1323 CL:1000415 48385 48413 gallbladder epithelial cells +T1324 GO:0005622 48445 48460 intracellularly +T1325 UBERON:0002107 48508 48513 liver +T1326 PR:000011395 48508 48530 liver X receptor alpha +T1327 CHEBI:86029 48508 48524;48538 48545 liver X receptor ... agonist +T1328 PR:000011395 48532 48536 LXRα +T1329 PR:000003572 48547 48552 Abcg5 +T1330 PR:000003572 48553 48563 sterolin-1 +T1331 PR:000003573 48568 48573 Abcg8 +T1332 PR:000003573 48574 48584 sterolin-2 +T1333 GO:0010467 48600 48609 expressed +T1334 GO:0045177 48617 48623 apical +T1335 GO:0009986 48624 48631 surface +T1336 PR:000003572 48664 48669 Abcg5 +T1337 PR:000003572 48670 48680 sterolin-1 +T1338 GO:0042571 48681 48689 antibody +T1339 GO:0045177 48860 48866 apical +T1340 NCBITaxon:10114 48878 48881 rat +T1341 PR:000015052 48882 48886 SPNT +T1342 UBERON:0004819 48900 48916 renal epithelial +T1343 CL:0002518 48900 48922 renal epithelial cells +T1344 MOP:0002162 48942 48957 N-glycosylation +T1345 PR:000003572 49033 49038 Abcg5 +T1346 PR:000003572 49039 49049 sterolin-1 +T1347 GO:0010467 49050 49060 expression +T1348 PR:000003573 49082 49087 Abcg8 +T1349 PR:000003573 49088 49098 sterolin-2 +T1350 NCBITaxon:10088 49109 49113 mice +T1351 PR:000003572 49307 49312 Abcg5 +T1352 PR:000003572 49313 49323 sterolin-1 +T1353 PR:000003573 49331 49336 Abcg8 +T1354 NCBITaxon:10088 49340 49344 mice +T1355 PR:000003572 49395 49400 Abcg5 +T1356 PR:000003572 49401 49411 sterolin-1 +T1357 NCBITaxon:10088 49422 49427 mouse +T1358 GO:0046903 49458 49465 secrete +T1359 UBERON:0002294 49466 49473 biliary +T1360 CHEBI:16113 49474 49485 cholesterol +T1361 NCBITaxon:10088 49522 49526 mice +T1362 GO:0007631 49537 49540 fed +T1363 CHEBI:86029 49543 49554 LXR agonist +T1364 PR:000003573 49567 49572 Abcg8 +T1365 GO:0010467 49578 49588 expression +T1366 GO:0046903 49603 49610 secrete +T1367 UBERON:0002294 49616 49623 biliary +T1368 CHEBI:16113 49624 49635 cholesterol +T1369 NCBITaxon:10088 49651 49655 mice +T1370 PR:000003572 49686 49691 Abcg5 +T1371 PR:000003572 49692 49702 sterolin-1 +T1372 PR:000003573 49707 49712 Abcg8 +T1373 PR:000003573 49713 49723 sterolin-2 +T1374 UBERON:0002107 49771 49778 hepatic +T1375 CHEBI:15889 49779 49786 sterols +T1376 UBERON:0001970 49792 49796 bile +T1377 PR:000003572 49846 49851 Abcg5 +T1378 NCBITaxon:10088 49862 49867 mouse +T1379 NCBITaxon:33208 50034 50040 animal +T1380 PR:000003572 50061 50066 Abcg5 +T1381 PR:000003572 50067 50077 sterolin-1 +T1382 PR:000003573 50082 50087 Abcg8 +T1383 PR:000003573 50088 50098 sterolin-2 +T1384 GO:0046903 50194 50201 secrete +T1385 UBERON:0002294 50202 50209 biliary +T1386 CHEBI:15889 50210 50217 sterols +T1387 SO:0000704 50309 50314 genes +T1388 http://purl.obolibrary.org/obo/MONDO_0008863 50378 50392 sitosterolemia +T1389 PR:000003573 50415 50420 Abcg8 +T1390 PR:000003573 50421 50431 sterolin-2 +T1391 UBERON:0002294 50453 50460 biliary +T1392 CHEBI:16113 50461 50472 cholesterol +T1393 PR:000003572 50491 50496 Abcg5 +T1394 PR:000003572 50497 50507 sterolin-1 +T1395 GO:0045177 50528 50536 apically +T1396 GO:0010467 50537 50546 expressed +T1397 PR:000003573 50570 50575 Abcg8 +T1398 PR:000003573 50576 50586 sterolin-2 +T1399 PR:000003573 50635 50640 Abcg8 +T1400 PR:000003573 50641 50651 sterolin-2 +T1401 UBERON:0002294 50671 50678 biliary +T1402 CHEBI:16113 50679 50690 cholesterol +T1403 UBERON:0002294 50711 50718 biliary +T1404 CHEBI:27693 50719 50729 sitosterol +T1405 PR:000003573 50822 50827 Abcg8 +T1406 PR:000003573 50828 50838 sterolin-2 +T1407 CHEBI:27693 50871 50881 sitosterol +T1408 UBERON:0001970 50887 50891 bile +T1409 UBERON:0001970 51032 51036 bile +T1410 SO:0000704 51087 51094 genetic +T1411 NCBITaxon:10088 51150 51154 mice +T1412 UBERON:0001970 51328 51332 bile +T1413 UBERON:0001970 51360 51364 bile +T1414 CHEBI:15889 51451 51457 sterol +T1415 UBERON:0001970 51493 51497 bile +T1416 NCBITaxon:10088 51513 51518 mouse +T1417 PR:000003549 51866 51870 Bsep +T1418 GO:0042571 51871 51879 antibody +T1419 PR:000003572 51923 51928 Abcg5 +T1420 PR:000003572 51929 51939 sterolin-1 +T1421 GO:0042571 51940 51948 antibody +T1422 NCBITaxon:33208 52012 52018 animal +T1423 UBERON:0000948 52055 52060 Heart +T1424 NCBITaxon:10088 52400 52404 mice +T1425 PR:000003573 52418 52423 Abcg8 +T1426 PR:000003573 52424 52434 sterolin-2 +T1427 PR:000003573 52472 52477 Abcg8 +T1428 PR:P23940 52527 52532 BamHI +T1429 NCBITaxon:10088 52542 52547 mouse +T1430 SO:0001026 52548 52555 genomic +T1431 CHEBI:37972 52574 52577 32P +T1432 UBERON:0002107 52764 52771 hepatic +T1433 PR:000003573 52793 52798 Abcg8 +T1434 PR:000003573 52799 52809 sterolin-2 +T1435 PR:000003573 52847 52852 Abcg8 +T1436 PR:000003573 52853 52863 sterolin-2 +T1437 PR:000003572 52899 52904 Abcg5 +T1438 PR:000003572 52905 52915 sterolin-1 +T1439 NCBITaxon:10088 52968 52972 mice +T1440 PR:000003572 53021 53026 Abcg5 +T1441 PR:000003573 53040 53045 Abcg8 +T1442 UBERON:0002107 53096 53103 hepatic +T1443 PR:000003573 53119 53124 Abcg8 +T1444 PR:000003573 53125 53135 sterolin-2 +T1445 SO:0000673 53136 53143 message +T1446 CHEBI:33695 53136 53143 message +T1447 SO:0000147 53159 53163 exon +T1448 PR:000003573 53173 53178 Abcg8 +T1449 NCBITaxon:10088 53182 53186 mice +T1450 SO:0000112 53196 53203 primers +T1451 SO:0000147 53220 53225 exons +T1452 SO:0000147 53246 53251 exons +T1453 SO:0000147 53264 53269 exons +T1454 CHEBI:35915 53314 53331 esterified sterol +T1455 UBERON:0002107 53346 53352 livers +T1456 PR:000003573 53356 53361 Abcg8 +T1457 PR:000003573 53366 53371 Abcg8 +T1458 PR:000003573 53379 53384 Abcg8 +T1459 NCBITaxon:10088 53388 53392 mice +T1460 CHEBI:15889 53394 53400 Sterol +T1461 UBERON:0002107 53413 53418 liver +T1462 NCBITaxon:10088 53450 53454 mice +T1463 GO:0007631 53455 53458 fed +T1464 CHEBI:33290 53469 53473 chow +T1465 CHEBI:15889 53532 53539 sterols +T1466 CHEBI:17002 53575 53597 Esterified cholesterol +T1467 CHEBI:27693 53678 53688 sitosterol +T1468 UBERON:0002107 53696 53702 livers +T1469 PR:000003573 53708 53713 Abcg8 +T1470 PR:000003573 53721 53726 Abcg8 +T1471 NCBITaxon:10088 53730 53734 mice +T1472 PR:000003573 53756 53761 Abcg8 +T1473 NCBITaxon:10088 53765 53769 mice +T1474 CHEBI:28623 53822 53833 campesterol +T1475 CHEBI:18059 53895 53900 Lipid +T1476 UBERON:0001969 53917 53923 plasma +T1477 PR:000003573 53927 53932 Abcg8 +T1478 PR:000003573 53937 53942 Abcg8 +T1479 PR:000003573 53950 53955 Abcg8 +T1480 NCBITaxon:10088 53959 53963 mice +T1481 NCBITaxon:10088 54005 54010 mouse +T1482 UBERON:0001969 54011 54017 plasma +T1483 CHEBI:15889 54041 54048 sterols +T1484 CHEBI:17855 54063 54075 triglyceride +T1485 CHEBI:16113 54150 54161 cholesterol +T1486 CHEBI:17855 54208 54220 triglyceride +T1487 PR:000003573 54229 54234 Abcg8 +T1488 NCBITaxon:10088 54238 54242 mice +T1489 GO:0010467 54379 54389 expression +T1490 NCBITaxon:10088 54413 54418 mouse +T1491 UBERON:0002107 54419 54425 livers +T1492 PR:000003572 54482 54487 Abcg5 +T1493 PR:000003573 54489 54494 Abcg8 +T1494 PR:000006148 54502 54508 Cyp7a1 +T1495 PR:000003537 54510 54515 Abca1 +T1496 PR:P21440 54517 54521 Mdr2 +T1497 PR:000028988 54528 54533 Srebp +T1498 PR:000015612 54541 54548 Srebp-2 +T1499 NCBITaxon:10088 54552 54557 mouse +T1500 UBERON:0002107 54558 54564 livers +T1501 PR:000003573 54570 54575 Abcg8 +T1502 PR:000003573 54592 54597 Abcg8 +T1503 PR:000003573 54620 54625 Abcg8 +T1504 NCBITaxon:10088 54653 54657 mice +T1505 PR:000003572 54695 54700 Abcg5 +T1506 PR:000003572 54701 54711 sterolin-1 +T1507 SO:0000673 54740 54747 message +T1508 CHEBI:33695 54740 54747 message +T1509 CHEBI:11814 54752 54759 HMG CoA +T1510 SO:0000673 54785 54792 message +T1511 CHEBI:33695 54785 54792 message +T1512 PR:000003573 54797 54802 Abcg8 +T1513 PR:000003573 54803 54813 sterolin-2 +T1514 NCBITaxon:10088 54843 54847 mice +T1515 CHEBI:11814 54891 54898 HMG-CoA +T1516 PR:000006148 54913 54919 CYP7a1 +T1517 UBERON:0002107 54923 54929 livers +T1518 PR:000003573 54935 54940 Abcg8 +T1519 PR:000003573 54945 54950 Abcg8 +T1520 PR:000003573 54958 54963 Abcg8 +T1521 NCBITaxon:10088 54967 54971 mice +T1522 CHEBI:11814 54987 54994 HMG-CoA +T1523 PR:000006148 55009 55015 CYP7a1 +T1524 PR:000003573 55050 55055 Abcg8 +T1525 NCBITaxon:10088 55059 55064 mouse +T1526 UBERON:0002107 55065 55070 liver +T1527 MOP:0002162 55120 55135 N-glycosylation +T1528 PR:000003572 55139 55144 Abcg5 +T1529 PR:000003572 55145 55155 sterolin-1 +T1530 PR:000003573 55168 55173 Abcg8 +T1531 NCBITaxon:10088 55177 55182 mouse +T1532 UBERON:0002107 55183 55188 liver +T1533 NCBITaxon:10088 55190 55195 Mouse +T1534 UBERON:0002107 55196 55201 liver +T1535 PR:000003572 55234 55239 Abcg5 +T1536 PR:000003572 55240 55250 sterolin-1 +T1537 PR:000007078 55272 55278 Endo-H +T1538 MOP:0001162 55359 55374 deglycosylation +T1539 UBERON:0002107 55408 55413 liver +T1540 UBERON:0001977 55440 55445 serum +T1541 PR:000016261 55542 55553 transferrin +T1542 MOP:0001162 55571 55586 deglycosylation +T1543 PR:000003572 55597 55602 Abcg5 +T1544 PR:000003572 55603 55613 sterolin-1 +T1545 NCBITaxon:10088 55626 55631 mouse +T1546 UBERON:0002107 55632 55637 liver +T1547 NCBITaxon:10088 55696 55700 mice +T1548 PR:000003573 55716 55721 Abcg8 +T1549 NCBITaxon:10088 55725 55729 mice +T1550 MOP:0001162 55788 55803 deglycosylation +T1551 NCBITaxon:10088 55805 55810 Mouse +T1552 UBERON:0002107 55811 55816 liver +T1553 PR:000003572 55851 55856 Abcg5 +T1554 PR:000003572 55857 55867 sterolin-1 +T1555 NCBITaxon:10088 55945 55949 mice +T1556 PR:000003573 55983 55988 Abcg8 +T1557 NCBITaxon:10088 55992 55996 mice +T1558 PR:000007078 56013 56019 Endo-H +T1559 NCBITaxon:10088 56092 56096 mice +T1560 PR:000003572 56098 56103 Abcg5 +T1561 PR:000003573 56104 56109 Abcg8 +T1562 UBERON:0002107 56113 56118 liver +T1563 NCBITaxon:10088 56202 56207 mouse +T1564 PR:000003572 56208 56213 Abcg5 +T1565 PR:000003572 56214 56224 sterolin-1 +T1566 GO:0010467 56225 56235 expression +T1567 UBERON:0002107 56239 56244 liver +T1568 UBERON:0000160 56249 56258 intestine +T1569 GO:0009294 56289 56300 transfected +T1570 NCBITaxon:10088 56311 56316 mouse +T1571 PR:000003572 56317 56322 Abcg5 +T1572 NCBITaxon:10088 56331 56336 mouse +T1573 PR:000003573 56337 56342 Abcg8 +T1574 GO:0010467 56366 56373 express +T1575 GO:0042571 56417 56425 antibody +T1576 SO:0000440 56438 56444 vector +T1577 GO:0009294 56445 56456 transfected +T1578 PR:000003572 56492 56497 Abcg5 +T1579 PR:000003572 56498 56508 sterolin-1 +T1580 PR:000003572 56555 56560 Abcg5 +T1581 GO:0009294 56561 56572 transfected +T1582 PR:000003572 56608 56613 Abcg5 +T1583 PR:000003572 56614 56624 sterolin-1 +T1584 GO:0042571 56625 56633 antibody +T1585 GO:0016020 56643 56651 membrane +T1586 PR:000003573 56685 56690 Abcg8 +T1587 GO:0009294 56691 56702 transfected +T1588 PR:000003572 56732 56737 Abcg5 +T1589 GO:0042571 56738 56746 antibody +T1590 PR:000003572 56793 56798 Abcg5 +T1591 PR:000003573 56803 56808 Abcg8 +T1592 GO:0009294 56812 56823 transfected +T1593 PR:000003572 56859 56864 Abcg5 +T1594 PR:000003572 56865 56875 sterolin-1 +T1595 GO:0042571 56876 56884 antibody +T1596 PR:000003572 56931 56936 Abcg5 +T1597 UBERON:0000160 56997 57006 intestine +T1598 UBERON:0001977 57036 57041 serum +T1599 PR:000003572 57064 57069 Abcg5 +T1600 PR:000003572 57070 57080 sterolin-1 +T1601 GO:0042571 57081 57089 antibody +T1602 PR:000003573 57179 57184 Abcg8 +T1603 PR:000003573 57192 57197 Abcg8 +T1604 UBERON:0000160 57201 57210 intestine +T1605 PR:000003572 57267 57272 Abcg5 +T1606 PR:000003572 57273 57283 sterolin-1 +T1607 GO:0042571 57284 57292 antibody +T1608 GO:0010467 57318 57328 expression +T1609 PR:000003573 57359 57364 Abcg8 +T1610 PR:000003573 57365 57375 sterolin-2 +T1611 NCBITaxon:10088 57392 57396 mice +T1612 UBERON:0000160 57421 57431 intestinal +T1613 GO:0042571 57506 57514 Antibody +T1614 UBERON:0002107 57527 57532 liver +T1615 UBERON:0002107 57572 57577 Liver +T1616 GO:0042571 57603 57611 antibody +T1617 PR:000003549 57615 57619 Bsep +T1618 PR:000003549 57620 57626 Abcb11 +T1619 GO:0045177 57655 57661 apical +T1620 PR:000003573 57707 57712 Abcg8 +T1621 NCBITaxon:10088 57722 57726 mice +T1622 GO:0042571 57748 57756 antibody +T1623 PR:000003572 57765 57770 Abcg5 +T1624 PR:000003572 57771 57781 sterolin-1 +T1625 PR:000003573 57826 57831 Abcg8 +T1626 UBERON:0002107 57841 57846 liver +T1627 GO:0010467 57873 57883 expression +T1628 GO:0045177 57893 57899 apical +T1629 PR:000003549 57966 57970 Bsep +T1630 PR:000003549 57971 57977 Abcb11 +T1631 UBERON:0001283 58021 58036 bile canaliculi +T1632 NCBITaxon:10088 58083 58088 mouse +T1633 PR:000003572 58089 58094 Abcg5 +T1634 PR:000003572 58095 58105 sterolin-1 +T1635 GO:0010467 58106 58116 expression +T1636 UBERON:0002107 58120 58125 liver +T1637 UBERON:0002107 58137 58142 liver +T1638 PR:000003572 58168 58173 Abcg5 +T1639 PR:000003572 58174 58184 sterolin-1 +T1640 GO:0045177 58197 58203 apical +T1641 GO:0010467 58204 58214 expression +T1642 GO:0045177 58257 58263 apical +T1643 PR:000003573 58287 58292 Abcg8 +T1644 UBERON:0002107 58296 58301 liver +T1645 PR:000003572 58336 58341 Abcg5 +T1646 PR:000003572 58342 58352 sterolin-1 +T1647 GO:0042571 58353 58361 antibody +T1648 GO:0045177 58384 58390 apical +T1649 GO:0010467 58391 58401 expression +T1650 UBERON:0001283 58469 58484 bile canaliculi +T1651 UBERON:0002394 58510 58519 bile duct +T1652 UBERON:0002294 58565 58572 Biliary +T1653 CHEBI:15889 58573 58579 sterol +T1654 CHEBI:16247 58581 58593 phospholipid +T1655 UBERON:0001970 58598 58602 bile +T1656 CHEBI:22868 58598 58607 bile salt +T1657 UBERON:0001970 58618 58622 Bile +T1658 CHEBI:22868 58618 58627 Bile salt +T1659 CHEBI:15889 58629 58635 sterol +T1660 CHEBI:16247 58640 58652 phospholipid +T1661 PR:000003573 58672 58677 Abcg8 +T1662 PR:000003573 58690 58695 Abcg8 +T1663 PR:000003573 58712 58717 Abcg8 +T1664 NCBITaxon:10088 58729 58733 mice +T1665 UBERON:0001970 58773 58777 Bile +T1666 CHEBI:22868 58773 58782 Bile salt +T1667 PR:000003573 58839 58844 Abcg8 +T1668 PR:000003573 58852 58857 Abcg8 +T1669 NCBITaxon:10088 58861 58865 mice +T1670 UBERON:0002294 58957 58964 Biliary +T1671 CHEBI:15889 58965 58971 sterol +T1672 CHEBI:16247 58976 58988 phospholipid +T1673 PR:000003573 59023 59028 Abcg8 +T1674 NCBITaxon:10088 59032 59036 mice +T1675 UBERON:0002294 59114 59121 biliary +T1676 UBERON:0001970 59122 59126 bile +T1677 CHEBI:22868 59122 59131 bile salt +T1678 CHEBI:15889 59133 59139 sterol +T1679 CHEBI:16247 59144 59156 phospholipid +T1680 PR:000003573 59185 59190 Abcg8 +T1681 PR:000003573 59203 59208 Abcg8 +T1682 PR:000003573 59225 59230 Abcg8 +T1683 NCBITaxon:10088 59242 59246 mice +T1684 UBERON:0001970 59276 59280 bile +T1685 CHEBI:22868 59276 59285 bile salt +T1686 CHEBI:80774 59317 59321 TUDC +T1687 CHEBI:80774 59378 59382 TUDC +T1688 PR:000003573 59450 59455 Abcg8 +T1689 NCBITaxon:10088 59459 59463 mice +T1690 GO:0046903 59467 59474 secrete +T1691 UBERON:0001970 59475 59479 bile +T1692 CHEBI:22868 59475 59485 bile salts +T1693 NCBITaxon:10088 59545 59549 mice +T1694 GO:0046903 59553 59560 secrete +T1695 CHEBI:15889 59561 59568 sterols +T1696 GO:0046903 59620 59627 secrete +T1697 CHEBI:16247 59628 59640 phospholipid +T1698 UBERON:0002294 59685 59692 Biliary +T1699 CHEBI:16113 59693 59704 cholesterol +T1700 CHEBI:27693 59709 59719 sitosterol +T1701 UBERON:0002294 59731 59738 Biliary +T1702 CHEBI:15889 59739 59746 sterols +T1703 PR:000003573 59779 59784 Abcg8 +T1704 PR:000003573 59797 59802 Abcg8 +T1705 PR:000003573 59818 59823 Abcg8 +T1706 NCBITaxon:10088 59835 59839 mice +T1707 UBERON:0001970 59860 59864 bile +T1708 CHEBI:22868 59860 59869 bile salt +T1709 CHEBI:80774 59905 59909 TUDC +T1710 PR:000003573 59944 59949 Abcg8 +T1711 NCBITaxon:10088 59953 59957 mice +T1712 GO:0046903 59973 59980 secrete +T1713 CHEBI:16113 59981 59992 cholesterol +T1714 UBERON:0001970 59998 60002 bile +T1715 CHEBI:80774 60015 60019 TUDC +T1716 NCBITaxon:10088 60061 60065 mice +T1717 PR:000003573 60107 60112 Abcg8 +T1718 NCBITaxon:10088 60116 60120 mice +T1719 GO:0046903 60140 60147 secrete +T1720 CHEBI:27693 60148 60158 sitosterol +T1721 CHEBI:28623 60173 60184 campesterol +T1722 NCBITaxon:10088 60209 60213 mice +T1723 GO:0046903 60245 60252 secrete +T1724 CHEBI:15889 60257 60264 sterols +T1725 CHEBI:80774 60277 60281 TUDC +T1726 SO:0000112 60381 60388 primers +T1727 NCBITaxon:10088 60428 60433 Mouse +T1728 UBERON:0001969 60434 60440 plasma +T1729 UBERON:0000479 60445 60451 tissue +T1730 CHEBI:15889 60452 60458 sterol +T1731 NCBITaxon:33208 60469 60476 Animals +T1732 GO:0007631 60554 60557 fed +T1733 CHEBI:33290 60568 60572 chow diff --git a/src/ontogpt/evaluation/craft/database/all/15040800.txt b/src/ontogpt/evaluation/craft/database/all/15040800.txt new file mode 100644 index 000000000..5f6e5487c --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15040800.txt @@ -0,0 +1,229 @@ +A mouse model of sitosterolemia: absence of Abcg8/sterolin-2 results in failure to secrete biliary cholesterol + +Abstract + +Background + +Mutations in either of two genes comprising the STSL locus, ATP-binding cassette (ABC)-transporters ABCG5 (encoding sterolin-1) and ABCG8 (encoding sterolin-2), result in sitosterolemia, a rare autosomal recessive disorder of sterol trafficking characterized by increased plasma plant sterol levels. Based upon the genetics of sitosterolemia, ABCG5/sterolin-1 and ABCG8/sterolin-2 are hypothesized to function as obligate heterodimers. No phenotypic difference has yet been described in humans with complete defects in either ABCG5 or ABCG8. These proteins, based upon the defects in humans, are responsible for regulating dietary sterol entry and biliary sterol secretion. + +Methods + +In order to mimic the human disease, we created, by a targeted disruption, a mouse model of sitosterolemia resulting in Abcg8/sterolin-2 deficiency alone. Homozygous knockout mice are viable and exhibit sitosterolemia. + +Results + +Mice deficient in Abcg8 have significantly increased plasma and tissue plant sterol levels (sitosterol and campesterol) consistent with sitosterolemia. Interestingly, Abcg5/sterolin-1 was expressed in both liver and intestine in Abcg8/sterolin-2 deficient mice and continued to show an apical expression. Remarkably, Abcg8 deficient mice had an impaired ability to secrete cholesterol into bile, but still maintained the ability to secrete sitosterol. We also report an intermediate phenotype in the heterozygous Abcg8+/- mice that are not sitosterolemic, but have a decreased level of biliary sterol secretion relative to wild-type mice. + +Conclusion + +These data indicate that Abcg8/sterolin-2 is necessary for biliary sterol secretion and that loss of Abcg8/sterolin-2 has a more profound effect upon biliary cholesterol secretion than sitosterol. Since biliary sitosterol secretion is preserved, although not elevated in the sitosterolemic mice, this observation suggests that mechanisms other than by Abcg8/sterolin-2 may be responsible for its secretion into bile. + +Background + +Absorption of dietary cholesterol from the intestine is an important part of cholesterol homeostasis and represents the initial step that allows dietary cholesterol to exert its metabolic effects. A typical western diet contains relatively equal amounts of cholesterol and non-cholesterol sterols, mainly plant sterols, of which about 55% of the dietary cholesterol is absorbed and retained compared to ~1% of the dietary non-cholesterol sterols [1-3]. Schoenheimer recognized more than 75 years ago that only cholesterol, not non-cholesterol sterols, is absorbed in the intestine, although the exact molecular mechanisms by which preferential cholesterol absorption occurs has not been fully elucidated [4]. Similarly, although the liver secretes free cholesterol into bile, it can preferentially excrete non-cholesterol sterols into bile and the mechanism(s) of this process has yet to be elucidated as well. Only recently, through the study of a rare human disease, have clues to this process been revealed. + +Sitosterolemia (MIM 210250, also known as phytosterolemia) is a rare autosomal, recessively inherited disorder characterized by elevated blood and tissue plant sterol levels [5]. Affected individuals can develop tendon and/or tuberous xanthomas, hemolytic episodes, arthralgias, arthritis, and premature atherosclerosis [6,7]. Sitosterolemic patients have diagnostically elevated plasma plant sterol levels (for example, sitosterol, campesterol, stigmasterol, and avenosterol, etc.) and their 5-saturated stanol metabolites, particularly sitostanol, with normal or only moderately increased cholesterol levels. Clinical studies have shown that affected individuals hyperabsorb all dietary sterols (thus have lost the ability to restrict the sterol species absorbed) and have lost the ability to excrete sterols into bile [8,9]. Infusion of sitosterol and cholesterol into normal individuals leads to a rapid and preferential excretion of sitosterol into bile, but in sitosterolemic individuals biliary sterol secretion is almost absent [7,9]. Previous studies on sitosterolemia have suggested a defect involving a putative sterol 'transporter' expressed in the intestinal brush border and/or the hepatic canalicular membrane [10,11]. Genetic analyses of sitosterolemia pedigrees allowed the mapping of the STSL locus to human chromosome 2p21, between D2S2294 and D2S2298 [12,13]. By using positional cloning procedures or by screening for genes induced by exposure to LXR agonists, two groups identified not one but two genes, ABCG5 and ABCG8, which encode the proteins sterolin-1 and sterolin-2, mutations of which cause sitosterolemia [14-16]. Interestingly, complete mutation in either ABCG5 alone or ABCG8 alone is not only necessary, but sufficient to cause the disease [14]. To date, no patient with sitosterolemia has been identified with mutations in both genes. Based upon these data, and the similarity of the clinical and biochemical phenotype, sterolin-1 and sterolin-2 are hypothesized to function as obligate heterodimers. This is also supported by the fact that each of these proteins is a 'half-transporter', containing six transmembrane domains, and not the classical 12 transmembrane characteristic of functional ABC transporters. The exact function of these two proteins still remains unknown but recent studies have shown that overexpression of ABCG5/ABCG8 in a transgenic mouse model results in an increased biliary cholesterol concentration and a 50% reduction in the fractional absorption of dietary cholesterol [17]. It has been suggested that ABCG5 and ABCG8 act as mutual chaperones for both maturation and correct apical targeting. Co-expression of both was required for apical trafficking in a polarized cell line, as well in vivo using Abcg5/Abcg8 double-knockout mice [18,19]. However, no functional assay has yet been developed to demonstrate whether or not the two half-transporters function as homo- or heterodimers and whether they selectively pump sterols. Likewise, the cellular localization of these two proteins has yet to be shown in a model deficient in either Abcg5 or Abcg8 alone. A mouse model disrupting both Abcg5 and Abcg8 simultaneously results in a phenotype similar to that of the human disease [20]. However, it is important to point out that the human disease is caused by complete mutation in one or the other gene, but not both simultaneously. + +We hypothesized that disruption of only one of the half-transporters would result in sitosterolemia and selective loss of either sterolin may show a differential effect on the biliary secretion of sterols or intestinal absorption of sterols. These studies are difficult to test in humans, as the target organs are relatively inaccessible. In order to test this hypothesis, we report a mouse model that has a selective genetic mutation of Abcg8. Mice homozygous for Abcg8 loss exhibit many of the characteristics of the human disease sitosterolemia. In addition, we report studies of biliary sterol secretion that show that despite forced secretion, Abcg8-deficient animals cannot secrete cholesterol into bile, although sitosterol secretion seemed unaffected. + +Methods + +Targeting vector construction and generation of knockout mice + +A mouse genomic bacterial artificial chromosome (BAC) library (CitbCJ7, ES cell line/129Sv, Research Genetics, Inc., Huntsville, AL, USA) was screened by using primers designed from the sequences of mouse Abcg5 and Abcg8 cDNA as previously reported [21]. A positive BAC clone was used as a template to amplify genomic DNA fragments of Abcg8. Long-fragment polymerase chain reaction (PCR) was performed using Expanded Long Template PCR system kit (Roche Applied Science, Indianapolis, IA, USA). An approximately 4.5 kb 'long-arm' genomic fragment containing partial exon 1 to partial exon 3 was inserted into the Pml I restriction-cloning site A of OSDUPDEL vector. The 'short-arm' genomic fragment, containing partial exon 4 to partial exon 6, was cloned into the Not I-Kpn I of cloning site B. Homologous targeting would result in complete intron 3, partial exon 3 and partial exon 4 replacement by the neomycin-resistance cassette, resulting in the disruption of the ABC Walker A motif (Figure 1a). + +ES cells (129/SvEvTac-cell line) were electroporated with the linearized targeting vector DNA and cultured on sub-confluent embryonic fibroblasts. Transfected cell colonies were selected by culturing cells in medium containing 200 μg/mL G418 (Life Technologies, Rockville, MD, USA). The ES cells were screened for homologous recombination by PCR with the forward primer, neoF (5'-GGGTCGTTTGTTCGGATCAA-3') from neo cassette, and reverse primer intron 6R (5'-ACCAGTTGGTCCTAGCTCGA-3'), which is located outside the targeting construct in mouse Abcg8 intron 6. Positively targeted ES cell clones were confirmed by Southern blotting, using ES cell DNA digested by BamHI and a probe comprising of a 562 bp PCR fragment from partial intron 2 and exon 3 of Abcg8 gene. + +Positively targeted ES cells were microinjected into C57BL6 blastocysts and transplanted into pseudopregnant recipients. Five highly chimeric mice (agouti coat color, three males and two females) were isolated and bred with C57BL/6J mice to generate germ-line transmission heterozygous mice of Abcg8 gene disruption. Heterozygous offspring mice were back-crossed to C57BL/6J mice (n > 5) to produce disrupted line and inter-crossed to generate knockout mice. + +Animals and diets + +All mice were maintained on a standard rodent chow (Harlan Teklad mouse/rat diet LM-485) which contained 61 mg/kg cholesterol, 31 mg/kg campesterol, and 105 mg/kg sitosterol, given free access to water and maintained at 25°C with a 12-h light, 12-h dark cycle. All animals were housed in the facilities for laboratory animals provided by the Division of Laboratory Animal Resources ACLAM. The Institutional Animal Care and Research Advisory Committee at Medical University of South Carolina approved all experiments. + +Genotyping by PCR + +Mouse tail DNA was isolated and purified by cutting approximately 0.5 cm of mouse tail and digesting it in 500 μl lysis buffer (50 mM Tris-HCl, pH8.0, 100 mM EDTA, 125 mM NaCl, 1% SDS, 200 μg Proteinase K) with rocking overnight at 55°C. 200 μl saturated NaCl was added into digested solution with vigorous shaking for at least 60 seconds. Mixed solution was spun down at maximum speed in top micro-centrifuge for 20 min. The supernatant was transferred to a new tube with equal volume 100% EtOH and mixed by inversion. The DNA pellet was washed in 70% EtOH and resuspended in 100–150 μl TE buffer. UV spectrophotometry and electrophoresis were used to analyze the quality and quantity of the genomic DNA. Genotyping was performed by multiplex PCR reaction using three separate primers (two Abcg8 gene specific primers, mg8-in3F 5'-CCCAATGATGAATGAGACGCT-3', mg8-R9 5'-TTTGTCACGCTGGGCCTGG-3' and neoF for identification of Abcg8 target status). The PCR reaction consisted of 1x PCR buffer (1.5 mM MgCl2, 16 mM (NH4)2SO4, 0.1 mM dNTPs, 67 mM Tris-HCl (pH8.8)) 1 μM of each primer, water and 1.0 units of Taq polymerase. The reactions were cycled as follows: 94°C for 30 sec, 60°C for 30 sec, 72°C for 1 min for 35 cycles. The PCR products were identified by electrophoresis. + +Southern blot analysis + +To confirm PCR results, DNAs were digested with Bam HI, subjected to electrophoresis in a 0.6% agarose gel and transferred to nylon membrane. Southern blot filters were hybridized with a 562 bp PCR fragment from partial intron 2 and exon 3 of Abcg8 32P-randomly-labeled probe as previously described [22]. + +Northern blot analysis + +Isolation of liver and intestinal total RNA from Abcg8+/+, Abcg8+/- and Abcg8-/- mice and Northern blot analyses were performed as previously described [23]. The probe for Abcg5 was 1964 bp (nt 136–2099, GeneBank™ accession no. AH011511) and the probe for Abcg8 was 2019 bp (nt 103–2126, GeneBank™ accession no. AH011518). + +Reverse Transcription (RT)-PCR + +To confirm that there was no alternative splicing of the disrupted allele, RT-PCR was performed on cDNAs of pooled mouse liver. Briefly, samples of total RNA (0.5 μg) from pooled mouse livers (n = 4) were reverse transcribed according to the SuperScript™ First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) using random hexamers in a final total reaction volume of 20 μl. RT-PCR was performed using primers located outside of the targeted region: F1 (CCTCAGCTGGTGAGGAGGTG) with R1 (GATGGAGAAGGTGAAGTTGCC), F2 (ATTTCCAATGACTTCCGGGAC) with R1 and F3 (CTGGAAGACGGGCTGTACACT) with R1, to produce expected product lengths of 1449, 654 and 429 bp respectively in wild-type cDNA. + +Quantitative RT-PCR + +Primers were based upon previously published primer sets [24-26] or designed using MacVector, which were designed from either mRNA or cDNA to avoid the amplification of potentially contaminating genomic DNA in the total RNA sample (Table 1). Samples of total RNA (0.5 μg) from pooled mouse livers (n = 4) were reverse transcribed according to the SuperScript™ First-Strand Synthesis System (Invitrogen, Carlsbad, CA, USA) using random hexamers with a final total reaction volume of 20 μl. Quantitative RT-PCR was performed on a PE Biosystems GeneAmp® 5700 sequence Detection System (Forest City, CA, USA). Standard reaction volume was 10 μl containing 1 × QuantiTect SYBR Green PCR master mix (Qiagen, Valencia, CA, USA), 0.002 U AmpErase UNG enzyme (PE Biosystems, Forest City, CA, USA), 0.7 μl of cDNA template from reverse transcription reaction as above and 100–500 nM of oligonucleotide primers. Initial steps of RT-PCR were two minutes at 50°C for UNG activation, followed by a 15 minute hold at 95°C. Cycles (n = 40) consisted of a 15 second melt at 95°C, followed by a 1 minute annealing/extension at 60°C. The final step was a 60°C incubation for 1 minute. At the end of the run, samples were heated to 95°C with a ramp time of 20 minutes to construct dissociation curves to ensure that single PCR products were obtained. All reactions were performed in triplicate. Threshold cycle (CT) analysis of all samples was set at 0.5 relative fluorescence units. The relative quantities of message of genes of interest from the mouse liver samples used in real-time RT-PCR were normalized to cyclophilin to compensate for variations in input RNA amounts. The data were analyzed using the comparative threshold cycle method (CT). Briefly, the CT values are averaged, from the triplicates defining a Δ-CT value calculated by taking the average CT of the gene of interest and subtracting it from the average CT of cyclophilin. The ΔΔ-CT was calculated by subtracting the average Δ-CT(calibrator) values from the Δ-CT(sample). The relative quantification was then calculated by the expression 2-AverageΔΔ-CT. The mRNA quantity for the calibrator (wild type) was expressed as 1 and all other quantities were expressed as a fold difference relative to the calibrator (wild type). + +Plasma lipid analysis by fast protein liquid chromatography + +Following 16 hrs of fasting, blood samples (n = 3) from each of the Abcg8+/+, Abcg8+/- and Abcg8-/- mice were collected from the retro-orbital venous plexus using heparinized capillary tubes under isoflurane anesthesia, and placed into precooled tubes containing 10 μl of 0.5 M EDTA. Equal volumes of plasma from each genotype were pooled and 100 μL of the pooled sample were injected and fractionated by fast protein liquid chromatography (FPLC) using a Superose 6 HR10/30 column (Pharmacia Biotech Inc., Piscataway, NJ, USA) and analyzed as described [27]. + +Sterol composition of plasma and tissues + +Following a four-hour fast of 12-week-old mice fed on regular rodent chow diet; blood and tissues were collected under isofluorane anesthesia from each of the Abcg8+/+, Abcg8+/- and Abcg8-/- mice (n = 3 for each group). Blood was collected from the retro-orbital venous plexus by heparinized glass tube. The animals were sacrificed by cervical dislocation for tissue collection. Plasma (100 μl) was saponified in 1 N NaOH for one hour, 1.5 mL water added and sterols extracted with three sequential portions of 1.5 mL ethyl acetate, containing 70 μg 5α-cholestane as an internal standard, pooled and dried. Mouse tissues (liver, spleen and brain) were weighed, homogenized in 1 mL phosphate buffer saline (PBS) with a dounce homogenizer (15 strokes at 500 rpm) and a small aliquot of the whole homogenate assayed for protein concentration. Aliquots for sterol analysis from whole tissue homogenates were extracted as described above. Dried sterol samples were derivatized as trimethylsilylethers, redissolved in 10 μL hexane and 2 μL samples were injected into a capillary column (26 m length, 0.32 mm ID, 0.45 mm OD) coated with liquid CpWAX 57 CB (Chrompak, Bridgewater, NJ, USA) in a Hewlett-Packard Gas Chromatograph equipped with a flame ionization detector [28]. Concentrations of plasma and tissue sterols were reported as mg/dL and μg/g wet weight tissue, respectively. + +Polyclonal antibody production + +Three separate polyclonal anti-Abcg5/sterolin-1 antibodies have been raised as previously described [20,29,30]. In experiments, anti-Abcg5/sterolin-1 antibodies are labelled as follows: + +1) SC – from the Patel group, 2) AMC – from the AMC Liver Center group, and 3) UTSW – from the Hobbs group. + +Membrane protein preparations + +Total membranes were prepared by taking approximately 500 mg of liver tissue cut into small pieces and homogenized in ice cold lysis buffer (5 mM Tris-pH7.5, 250 mM sucrose with protease inhibitors) by a dounce homogenizer times 10 strokes. Samples were then subjected to centrifugation – 1300 g for 10 minutes at 4°C. The supernant was saved and the pellet was resuspended and again subjected to dounce homogenization times 10 strokes then centrifuged 1000 g for 10 minutes at 4°C. This step was repeated twice each time with the supernant being collected and kept on ice. The supernants were then pooled and subjected to ultracentrifugation at 100,000 g for 60 minutes at 4°C. The supernant was removed and the pellet was resuspended in lysis buffer. Plasma membranes were prepared as previously described [31]. Protein sample concentrations were then determined by Bio-Rad protein colormetric assay per manufacturer's protocol (BioRad, Hercules, CA, USA). 50 μg of membrane proteins were then resolved on 7.5% SDS acrylamide gel. + +Immunoblotting + +Proteins resolved by SDS-PAGE were transferred to nitrocellulose membranes. Membranes were then blocked for one hour in 5% dry milk in PBS-T (Phosphate Buffered saline and 0.1% Tween 20). Blots were then probed with primary antibody against Abcg5 in 5% milk in PBS-T overnight at 4°C. The blot was then washed three times for five minutes in TBS-T (Tris Buffered Saline/0.1% Tween-20) with 150 mM NaCl, and secondarily stained with goat-anti-rabbit conjugated HRP 1:10,000 dilution in 5% milk in PBS-T for one hour. The blot was again washed three times for five minutes in TBS-T with 150 mM NaCl then covered in Western Lightning® Chemiluminescence Reagent Plus (PerkinElmer Life Sciences, Inc. Boston, MA, USA), wrapped in plastic, exposed to film and developed. + +Immunohistochemical analysis + +Snap-frozen liver and intestine tissue were cut to produce 8 μm thick frozen sections, air-dried for 30 minutes onto glass slides and stored at -80°C until required. The slides were stained with hematoxylin, rinsed three times with PBS, fixed for 10 min with cooled methanol at -20°C and then rinsed three times with PBS. Slides for antibody staining were initially blocked in PBS/0.1 M glycine/10% goat serum for 60 min at room temperature then incubated with anti-sterolin-1 antibody (1:15 dilution) overnight at 4°C. The slides were washed with 1 × PBS and incubated with secondary antibody (goat-anti-rabbit IgG conjugated with rhodamine, 1:1000 dilution) for 30 min at room temperature, washed and then observed under an Olympus BX-5 confocal microscope with Fluoview. The sections stained at the AMC Liver Center were fixed in 100% acetone for 30 min at room temperature, blocked in a solution of PBS/0.05% Tween-20/5% goat serum (GS) for one hour, then incubated with Anti-sterolin-1 (1:40 dilution) and anti-Bsep (1:200 dilution) diluted in PBS/Tween/GS, for one hour. Thereafter, slides were washed 3 × 10 min in PBS/Tween and the secondary antibody (goat-anti-rabbit Alexa 488) was applied in 1:1000 dilution in PBST/GS for one hour at room temperature. After washing for 3 × 10 min the sections were mounted in Dapi/Dapco solution. Fluorescence microscopy was done with a Leica DM RA2 microscope equipped with a Leica DC350F photo camera. For Dapi staining, 359 nm (30.38 ms exposure) was used and for Abcg5 and Bsep staining, 514 nm (for both: 1654.34 ms exposure) was used and the pictures were analyzed and overlaid using FW4000 software (Leica). + +Transfection and immunostaining of COS-1 cells + +COS-1 cells were transiently transfected with pCMV-mouse Abcg5 and pCMV-mouse Abcg8 expression constructs using the Qiagen SuperFect reagent per protocol (Valencia, CA, USA). Briefly, COS-1 cells were seeded onto a six-well plate the day prior to transfection to generate 60% confluence when grown at 37°C and 5% CO2 in DMEM supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 1 × glutamine. On the day of transfection, 2 μg of DNA was mixed with Qiagen SuperFect reagent and incubated for 10 minutes. This mixture was then added to serum-free media and placed on the cells to incubate for two hours. The cells were washed once with 1x PBS and fresh media was added. The transfected cells were then incubated for 48 hours to ensure expression and subsequently fixed with a 1:1 mixture of acetone:methanol. In preparation for immunostaining, cells were blocked with 2% goat serum in PBS for 30 minutes, then incubated with 1:50 dilution of primary antibody anti-Abcg5 in 2% goat serum-PBS for 1.5 hours, and subsequently incubated with secondary rhodamine anti-rabbit IgG antibody for 30 minutes. Cells were then observed under an Olympus BX-5 confocal microscope with Fluoview. + +Dynamic biliary lipid collection and analysis + +Mice were anaesthetized by intraperitoneal injection of 1 mL/kg fentanyl/fluanisone and 10 mg/kg Diazepam. The abdomen was opened and, after distal ligation of the bile duct, the gallbladder was cannulated. Bile sampling started directly after cannulation and was collected for 10 min. Bile flow was determined gravimetrically. Biliary bile salt, sterol and phospholipid concentrations were determined as described previously [32]. To determine maximal secretion rates of biliary lipid secretion, bile was diverted for 90 min to deplete the endogenous bile salt pool. Subsequently, tauroursodeoxycholate (Sigma Chemical Co., MO, USA) dissolved in PBS was infused into the jugular vein in stepwise increasing rates from 600–1,800 nmol/min/100 g body weight. Bile was collected in 10 min fractions and analyzed for bile salt and lipid content. In separate experiments, following distal ligation of the bile duct and cannulation of gallbladder, bile was diverted for 30 minutes while mice were infused with sterile PBS. Subsequently, the mice were infused continuously with 1,200 nmol/min/100 g body weight of tauroursodeoxycholate and two 30-minute fractions were collected. Sterol analyses of bile was performed by GC analysis as described above. + +Assay of HMG-CoA and cholesterol 7α-hydroxylase enzyme activity + +Mouse liver microsomes were prepared by differential centrifugation [33]. Briefly, 0.1–0.2 g liver was homogenized in a Potter-Elvjhem homogenizer with five volumes of buffer (0.25 M sucrose, 0.1 M Tris, 0.1 mM disodium EDTA, 0.1 mM DTT, pH 7.4). Microsomes were isolated by differential centrifugation (10,000 g to 100,000 g). The pellets were washed and resuspended in storage buffer (0.1 M dipotassium phosphate, 0.05 M KCl, 1 mM DTT, 5 mM disodium EDTA, 20% glycerol, pH 7.4) at 25–30 mg protein/ml. Protein concentrations were determined according to the method of Lowry et al. [34]. The activity of cholesterol 7α-hydroxylase (EC 1.14.12.17) was measured by an isotope incorporation method according to Shefer et al. [33] with some modifications. The reaction mixture (final volume 0.5 ml) consisted of potassium phosphate buffer (100 mM K2HPO4, 0.1 mM EDTA, 5 mM DTT, 30 mM nicotinamide, pH 7.4), [14C]-cholesterol (5 × 105 dpm) solubilized in 50 μl of 25% (wt/vol) β-cyclodextrin (final concentration 0.8%), and 50–200 μg of microsomal protein. The reaction was initiated by the addition of NADPH or an NADPH-generating system (3.4 mM NADP+, 30 mM glucose-6-phosphate, 0.3 U of glucose-6-phosphate dehydrogenase) and continued for 15 min at 37°C. The reaction was stopped with 0.5 ml of 1 N KOH, 5 μg of butylated hydroxytoluene, and 10 μl of ethanolic potassium hydroxide. [3H] 7α-hydroxycholesterol (1 × 104 dpm/5 μg) was added as an internal recovery standard. After saponification at 37°C for one hour, sterols were extracted twice with 3 ml of n-hexane and the extracts were evaporated to dryness under nitrogen. The residue was dissolved in 0.3 ml of n-hexane:2-propanol (97.3 vol/vol) and applied to a silica column (500 mg; sep-pak, by 4 ml of n-hexane:2-propanol (97.3 vol/vol)). 7α-hydroxycholesterol was eluted with 3 ml of n-hexane:2-propanol (80:20 vol:vol) and further isolated by TLC on silica gel plates (Silica gel 60, EM Science, Gibbestown, NJ, USA) with diethyl ether, and quantified by liquid scintillation counting using Ecolume (ICN Radiochemicals Irvine, CA, USA). Assays were carried out in duplicate with correction for zero-time controls. HMG-CoA reductase (EC 1.1.1.3.4) activity was determined according to the method of Xu et al. [35] with modifications as described [36]. + +Statistical analysis + +Data are shown as means ± SD. The Student's t test was used to determine the statistical significance of differences between the groups of animals. Significance was set at P values <0.05. + +Results + +Isolation of Abcg8/sterolin-2 deficient mice + +The targeting construct (Figure 1a) results in the potential disruption of normal splicing, as well as loss of some coding sequences involving exons 3 and 4. After ES cell electroporation with the linearized targeted plasmid DNA, two homologous recombinant ES clones (out of 58 ES clones screened) were identified. Blastocyst injection of these resulted in five highly chimeric mice (three males, two females). One male and one female, both from the same ES clone, showed germline transmission and the female line was used to establish a multi-generational colony bred onto a C57Bl/6J background. + +Heterozygous mice were fertile and gave rise to normal litter sizes. Breeding heterozygous animals led to wild-type, heterozygous and homozygous knockout mice in the expected Mendelian distribution (data not shown). Although not quantitative, Northern analyses showed that while the knockout mice showed no detectable mRNA for Abcg8, expression of Abcg5 appeared unaltered (Figure 1c). To exclude the possibility of alternative splicing with the production of a non-functional but truncated Abcg8/sterolin-2 protein that may serve as a chaperone for Abcg5/sterolin-1, RT-PCR was performed on cDNA reverse transcribed from total liver RNA from Abcg8-deficient mice. No mRNA for Abcg8/sterolin-2 was detected containing any sequences downstream of exon 4 in the Abcg8-deficient mice, whether primers were located in exons 4 and 13, exons 9 and 13 or exons 10 and 13 (Figure 1d,1e). This demonstrates that if an alternatively spliced message could potentially code for a truncated protein, it is not detectable in the Abcg8-deficient mice. The membrane-spanning domains of Abcg8/sterolin-2 are encoded by exons 9–13. + +Sterol levels in Abcg8/sterolin-2 deficient animals + +Elevated plant sterols in tissue and plasma are diagnostic of sitosterolemia. Sterol levels, as determined by gas chromatography (GC) analysis, in plasma and tissues of the Abcg8-/- mice are shown in Table 2. There were no differences in tissue weights between wild types, heterozygotes and knockouts. Plasma cholesterol levels of the homozygous and heterozygous mice were decreased by 52% and 26%, respectively, compared to those of wild-type mice (n = 3 all groups, Table 2). Plant sterols were almost undetectable in wild-type and heterozygous mice, but were significantly elevated in Abcg8-/- mice. Plasma campesterol and sitosterol levels were eight- and 36-fold higher in Abcg8-/- mice compared to wild-type mice (8.76 ± 2.10 and 18.52 ± 5.03 mg/dL, respectively, versus 1.14 ± 0.69 and 0.50 ± 0.55 mg/dL). There was no significant difference in the plasma plant sterol levels between heterozygous and wild-type mice. Liver cholesterol content of the Abcg8-/- mice was reduced by ~50% relative to wild-type liver (1202 ± 264 μg/g wet weight tissue, versus 2280 ± 310 μg/g wet weight tissue, respectively). The reduced cholesterol content was offset by a five-fold increase in campesterol and a 22-fold increase in sitosterol (233.9 ± 48.1 and 376.3 ± 96.1 μg/g wet weight tissue, respectively) in Abcg8-/- knockout mice compared to wild-type mice (52.2 ± 15.0 and 16.7 ± 8.6 μg/g wet weight tissue, respectively, Table 2). There were no significant differences of liver cholesterol or plant sterol contents between Abcg8+/+ and Abcg8+/- mice. Spleen sterol contents reflected the liver profiles (Table 2). No significant differences in brain sterol contents were observed, as would be expected, since the blood brain barrier is intact and prevents entry of these sterols in sitosterolemia (Table 2). There were only traces of plant sterols in the brains from knockout animals, reflecting blood contamination during tissue harvesting. + +Interestingly, the majority of the elevated tissue plant sterols are unesterified. The livers of Abcg8+/+, +/- and -/- mice have relatively similar levels of esterified cholesterol (Figure 2a). However, there is little if any esterification of sitosterol or campesterol (Figure 2b,2c) consistent with previous findings [37,38]. Thus all of the expansion of tissue sterol pools are as free sterols. + +FPLC analyses for sterols (measured enzymatically and thus reflecting total sterols) and triglycerides were performed on plasma samples from fasted animals fed a regular chow diet. No significant differences were observed for the sterol profiles (Figure 3a), but surprisingly, the Abcg8-/- knockout mice had a significantly higher triglyceride level in the LDL lipoprotein fractions compared to wild-type and heterozygous mice (Figure 3b). The size of this peak was variable between different littermate analyses, but is always increased in the knockout animals. Total plasma triglyceride levels of the Abcg8-/- mice were slightly higher (79.2 ± 14 mg/dL compared to the wild type 63.4 ± 13 mg/dL and heterozygotes 46.6 ± 12 mg/dL, n = 3 for all genotypes). Preliminary analyses of proteins in the isolated FPLC fractions did not show any qualitative changes in apolipoproteins B, E, AI, or AII. The significance of this triglyceride rich peak remains unclear at present. + +Liver gene expression and enzyme activity change in Abcg8/sterolin-2 deficient mice + +To investigate the effects of a deficiency of Abcg8/sterolin-2 on the genes that regulate sterol metabolism, quantitative RT-PCR was performed looking at the expression levels of Abcg5, Abcg8, Hmgr, Cyp7a1, Abca1, Mdr2, Lxr, Srebp-1c, and Srebp-2 mRNA in the livers of mice fed a regular chow diet (Figure 4a). As expected, Abcg8 mRNA expression levels were undetectable in the Abcg8-/- mice and were reduced by ~50% in the heterozygous mice, relative to wild-type mice. Interestingly, by quantitative RT-PCR, the mRNA expression of Abcg5 in the knock-out mice was also reduced by more than 60% compared to the wild-type mice, although no changes were noted in the heterozygous mice. Expression of HMG-CoA reductase mRNA was decreased by ~50% and ~80% in the heterozygote and knockout mice respectively, in keeping with limited observations in human patients with this disorder [7]. + +To verify whether the mRNA changes resulted in alteration of the enzyme activity changes, liver samples were analyzed for HMG-CoA reductase activity and Cyp7a1 activity (Figure 4b). HMG-CoA reductase activity was reduced by 30% and 60% in the Abcg8+/- and Abcg8-/- mice, respectively (Figure 4b), and thus reflected the changes in mRNA expression. In contrast, although the Cyp7a1 mRNA expression levels were essentially unchanged in the knockout mouse, Cyp7a1 activity was significantly decreased by 37% (P < 0.01). In the heterozygous mice, both the mRNA and activity of Cyp7a1 were decreased. Sitosterol is known to be a direct competitive inhibitor of Cyp7a1 and it is likely that the elevated plant sterols in the liver are responsible for the inhibition in the knockout mouse [39]. + +Localization of Abcg5 protein in Abcg8-/- knockout mice + +Does the loss of Abcg8/sterolin-2 result in loss of Abcg5/sterolin-1 expression, as might be predicted from the genetic studies and more recently from the in vitro and in vivo expression studies? A robust antibody to mouse Abcg8/sterolin-2 is not currently available. However, three separate groups have developed rabbit polyclonal antibody to mouse Abcg5/sterolin-1. These three antibodies were used in Western blotting and immunohistochemistry experiments on the liver and intestine of Abcg8-/- mice. Western blotting of liver total membrane preparations using the three separately-developed anti-Abcg5/sterolin-1 antibodies showed different results. As has been previously published, Abcg5/sterolin-1 exists in two separate forms, the 'immature' 75 kDa protein and a fully glycosylated 'mature' 93 kDa protein [18,30]. Using the SC anti-Abcg5/sterolin-1 peptide antibody, a 75 kDa band is detected (Figure 5a). This antibody does not detect a 'mature' 93 kDa band in either wild-type or knockout animals nor is the 75 kDa band sensitive to either Endo-H or PNGaseF (lower panel shows same aliquots probed with anti-transferrin). The AMC antibody detects the 'mature' form of Abcg5/sterolin-1 in wild-type animals but not the knockouts. Interestingly, the 'immature' 75 kDa band is detected by this antibody but the band shifts with Endo-H and PNGase F treatment (Figure 5b). The UTSW antibody detects the 'mature' and 'immature' forms of Abcg5/sterolin-1 in wild-type animals but detects no forms in the knockout mouse (Figure 5c). What does hold true for all antibodies used against Abcg5/sterolin-1 is the detection of a 75 kDa band. + +Immunohistochemistry experiments were carried out using all three antibodies. In acute transfection studies, the SC antibody recognized over-expressed mouse Abcg5/sterolin-1 in COS-1 cells, but not mouse Abcg8/sterolin-2 (Figure 6b,6c). Co-transfection of COS-1 cells with both Abcg5 and Abcg8 cDNAs did not alter the pattern of immunofluorescence, although we are not able to confirm simultaneous expression of Abcg8 in cells expressing Abcg5/sterolin-1, as we have no suitable antibody for Abcg8/sterolin-2 (Figure 6d). Nevertheless, this experiment indicates that the SC antibody can recognize Abcg5/sterolin-1 but does not cross-react with Abcg8/sterolin-2. + +Serial sections of intestinal tissue were incubated with anti-Abcg5/sterolin-1 to determine the cellular location of Abcg5/sterolin-1. The pattern of staining of Abcg5/sterolin-1 in the intestinal sections is clearly apical and localized to the villi of the enterocytes (Figure 6g,6h,6i). Loss of Abcg8/sterolin-2 does not seem to affect this pattern of staining. To further confirm that the antibody used recognizes Abcg5/sterolin-1; this pattern is blocked if the antibody is pre-incubated with the peptide to which it was raised (Figure 6f). To further confirm that Abcg5/sterolin-1 expression is preserved and may be apical, immunohistochemistry was performed in Amsterdam using the AMC antibody to stain liver sections from wild-type and knockout mice [30]. As a control, the expression of Abcb11, an ABC transporter known to be responsible for the export of bile salts, was compared to that of Abcg5/sterolin-1 (Figure 6j,6k). Again, both proteins are apically localized in liver sections from wild-type mice and this pattern is essentially unperturbed in liver from knockout mice, although the expression level of Abcg5/sterolin-1 seems to be less robust qualitatively (Figure 6l,6m). To further confirm the apical expression of Abcg5/sterolin-1 in the knockout mice, the UTSW antibody was used to stain liver sections of wild-type and knockout mice. Again, there is apical expression of Abcg5/sterolin-1 in both wild-type and knockout livers (Figure 7a,7b). + +Biliary secretion of bile salts, sterol and phospholipids in Abcg8/sterolin-2 deficient mice + +Sitosterolemic individuals have an impaired ability to secrete cholesterol and plant sterols into bile [40,41]. We analyzed the knockout mice for any alterations in biliary sterol handling. Analyses of the initial basal bile secretion showed that the sterol and phospholipid contents in Abcg8-/- mice were reduced as compared to wild-type mice, (Figure 8a,8b,8c, bile salt secretion not statistically significant, for sterol secretion P = 0.01 and for phospholipid secretion P = 0.03). + +To investigate whether the defect in biliary sterol secretion could be (partly) restored by forced biliary sterol secretion, we infused mice with stepwise increasing doses of tauroursodeoxycholic acid (TUDC). Mice were first depleted of their endogenous bile salt pools for 90 min and subsequently infused via the jugular vein with increasing doses of TUDC. As shown in Figure 8d, bile salt secretion was no different between the different genotypes during the depletion or the TUDC infusion phases of the experiment. However, a different pattern emerged for the secretion of phospholipid (Figure 8f). There was no difference between wild-type and heterozygous mice, but knockout mice showed a trend of lower phospholipid secretion during both the depletion and infusion phase. Even more dramatic was the effect on biliary sterol secretion although the sterol contents were determined enzymatically and no distinction is made as to whether this is sitosterol or cholesterol (Figure 8e, but see below). In the knockout mice, almost no stimulated sterol secretion was noted, with an intermediate phenotype in the heterozygous mice. In the heterozygous mice, sterol secretion increased to reach a maximal level of about 50% of the wild type suggesting that Abcg8/sterolin-2 is a rate-limiting step for biliary 'cholesterol' secretion. In the knockout mice, sterol secretion remained at a constant level during the depletion phase and increased minimally upon infusion of TUDC. + +Since the Abcg8-/- mice still secreted some sterols we were interested in which biliary sterol species were being secreted. Therefore bile was collected from mice during a constant infusion of TUDC and analyzed by GC. Mice were first depleted of their endogenous bile acid pools for 30 minutes then infused with a continuous dose of TUDC (1,200 nmol/min/100 g body weight). The knockout mice showed a significantly diminished ability to secrete cholesterol, yet still maintained the ability to secrete sitosterol and campesterol compared to wild-type mice (Figure 9). Surprisingly, the Abcg8+/- mice tended to show an increased ability to secrete all sterols above the levels seen in the wild-type mice, although this was not statistically significant. + +Discussion + +The mechanism by which dietary cholesterol is specifically absorbed and dietary non-cholesterol sterols primarily excluded or the mechanism(s) by which the liver can selectively secrete sterols has not been elucidated. Identification of the genetic defect(s) in a rare human disorder, sitosterolemia, where these processes are specifically disrupted, may finally have led to the identification of the 'transporters' responsible for these processes. Complete defects in one of two genes (but not both), organized in a head-to-head configuration at the STSL locus, causes sitosterolemia. We report a mouse model of sitosterolemia, with a selective, but complete, defect in one of these genes, Abcg8. This mouse reflects the known defects described in human sitosterolemia [5,7,42]. Plasma and all tissues, apart from the brain, have significantly elevated sitosterol levels. Homozygous knockout mice are viable, fertile and sitosterolemic. However, fertility seems to be reduced when homozygous mice are bred together (S Patel and J Oatis, unpublished observation). + +This model reflects many other observed changes described in limited studies in humans. For example, in humans, the activity of HMG-CoA reductase and CYP7a1 have been reported to be low [7,39]. In this study, we show that the mRNA and enzyme activities in the livers from knockout animals are also significantly reduced. Additionally, the activity of the rate-limiting enzyme for bile acid synthesis, CYP7a1, is reduced, although no changes at the mRNA level are noted. This is also in keeping with previous studies that show that sitosterol is a direct inhibitor of this enzyme in vitro [39]. + +Biliary sterols of Abcg8-deficient mice were dramatically different compared to wild-type mice. Measurement of biliary sterol secretion rates in Abcg8-/- mice demonstrated a failure to increase sterol secretion into bile despite exogenous infusion of bile acids. Furthermore, complete loss of Abcg8/sterolin-2 results in an inability to secrete cholesterol into bile, although secretion of sitosterol seemed to be preserved. Arguably, since the body pools and plasma sitosterol levels in the knockout mice are so considerably elevated, perhaps the biliary sitosterol levels could be considered to be inappropriately low. Despite this reservation, the finding that sitosterol is present in the bile suggests that plant sterols may be secreted independent of Abcg8/sterolin-2. The ability of Abcg8-/- mice to secrete bile salt and phospholipid into bile as compared to wild-type mice was not significantly impaired. Collectively these data suggest that Abcg8/sterolin-2 is necessary for hepatobiliary cholesterol secretion in mice. + +Abcg8-/- mice appeared normal and healthy maintained on a regular rodent chow diet. Their phenotypic features are very similar to those recently reported for a mouse deficient in both sterolins simultaneously [7,20]. Plasma and hepatic plant sterol levels in Abcg8-/- and Abcg5/Abcg8 knockout are increased similarly with marked decreases in cholesterol levels. Interestingly, plasma triglyceride levels of the Abcg8-/- mice were slightly higher than wild-type animals and this increased triglyceride is carried in the LDL fraction range, as measured by FPLC. The significance of this is not clear, although preliminary SDS-PAGE analyses of all the fractions failed to reveal any differences between wild-type and knockout samples. + +Hepatic levels of GC-measured cholesterol were greatly reduced in Abcg8-/- mice compared to wild-type mice, yet mRNA levels of HMG-CoA reductase and enzyme activity are also reduced without any significant change in mRNA of the Srebps. The basis for this is not clear at present, unless the increase in non-plant sterols leads to a suppression of SREBP activation. Sitosterol has been shown to directly inhibit Cyp7a1 activity in vitro and we presume this may account for the reduced enzyme activity [39]. In preliminary studies, when knockout mice are placed on a low sitosterol diet, the activity of this enzyme, as well as the mRNA levels are increased (E Klett and S Patel, unpublished observations). Another prediction, based upon the enzyme and mRNA levels in knockout mice, is that if these mice are placed on a high cholesterol/high sitosterol diet, they may show significant accumulation of both sterols in the body. This may also be relevant to the human disorder where some, although not all affected individuals, manifest premature coronary artery disease. + +Abcg8/sterolin-2 deficient mice also allow us to examine the role of this protein in biliary sterol secretion. The bile is sterol poor and upon stimulation by increasing bile salt excretion, Abcg8/sterolin-2 deficient mice cannot respond, in contrast to the wild-type mice. Thus, Abcg8/sterolin-2 is necessary for cholesterol secretion but not necessary for plant sterol secretion. It could be argued that although the biliary sterol output is comparable to that seen in wild-type mice that this is inappropriately low, since the tissue and plasma pools of sitosterol are so elevated in the knockout mice. Despite this caveat, the findings of sitosterol in significant quantities in the bile suggests that mechanisms other than via the sterolins can 'export' some of these sterols. Interestingly, although humans who are heterozygous for genetic defects in either ABCG5 or ABCG8 seem to be phenotypically normal, mice heterozygous for Abcg8/sterolin-2 deficiency show that sterol secretion is impaired, but not absent. Note that under steady-state conditions on a rodent chow diet, heterozygous animals show no significant elevations in plasma or tissue sitosterol levels, suggesting that the activity of these proteins may not be rate limiting. Furthermore, the heterozygous Abcg8+/- mice showed higher levels of biliary sitosterol relative to the wild-type mice. While the rate of total biliary sterol secretion is reduced compared to wild-type mice, since the heterozygous mice are not sitosterolemic, either the activity of these proteins is not rate limiting or other mechanisms can compensate for a 50% loss of activity. On the other hand, since feeding is intermittent, a slow, but continuous secretion during post-prandial periods could easily negate any mild temporary increase in tissue and plasma plant sterol levels in heterozygous animals. This may be amenable to testing by placing these animals on a high plant sterol diet and measuring the plasma sterol levels. + +Recently, Kosters et al. reported that Abcg5/Abcg8 mRNA expression in a variety of different mouse strains correlated with biliary cholesterol secretion rates [30]. In their studies, although diosgenin-fed mice showed a marked increase in biliary cholesterol output, mRNA levels of Abcg5 and Abcg8 were not altered. Using Western blot analyses, the protein level of Abcg5 was also not altered by diosgenin, although Abcg8 was not measured. They concluded that a parallel route for biliary cholesterol secretion might be operational, independent of the sterolins. While biliary cholesterol secretion is not completely absent in the Abcg8-deficient mice it would appear that sterolin-2 plays a major role in this process. In a genetic screen of plasma plant sterol levels, Sehayek et al. identified three loci that may be responsible for controlling plasma plant sterol levels and not one of these loci mapped to the murine STSL region [43]. Thus, there is support for at least four loci that may be involved in regulating plasma plant sterol levels. To date the STSL locus is the only one proven to be involved in dietary sterol trafficking and the identity of the others remains to be elucidated. + +We, as well as others, have proposed Abcg5/sterolin-1 and Abcg8/sterolin-2 to function as obligate heterodimers. Thus knocking out Abcg8/sterolin-2 alone is equivalent to a functional loss of both sterolins. It has been shown that Abcg8/sterolin-2 expression is required for correct apical trafficking of Abcg5/sterolin-1 to the apical surface in a polarized cell line and more recently in vivo by over-expression experiments [18,19]. In experiments shown here, using three separately developed anti-Abcg5/sterolin-1 antibodies, we have obtained inconsistent data regarding the trafficking of Abcg5/sterolin-1 in Abcg8-/- mice. Based upon classical N-glycosylation maturation it would appear that Abcg5/sterolin-1, in the Abcg8-/- mice, does not exit the endoplasmic reticulum. However, by immunohistochemistry it appears that Abcg5/sterolin-1 is apically expressed in the Abcg8-deficient mice regardless of the anti-Abcg5/sterolin-1 antibody used. Interestingly, in a report of Abcg5/Abcg8 localization in canine gallbladder epithelial cells, these two proteins were found intracellularly under baseline conditions [44]. But when given liver X receptor alpha (LXRα) agonist, Abcg5/sterolin-1 and Abcg8/sterolin-2 appeared to be expressed at the apical surface. In these studies the UTSW anti-Abcg5/sterolin-1 antibody was used. It is apparent with these conflicting data that the trafficking of these transporters is not as clear-cut. In a recent paper, Mangrivite et al. showed that the apical sorting of rat SPNT in polarized renal epithelial cells was independent of N-glycosylation [45]. Therefore, at this time, we can neither exclude the possibility that Abcg5/sterolin-1 expression is functional in our Abcg8/sterolin-2 deficient mice nor can we rule it out. We have no functional assay for these proteins at present. More extensive fractionation experiments are underway to better determine the exact compartmental location of Abcg5/sterolin-1 in the Abcg8-/- mice. In this context, Plösch et al. have described an Abcg5/sterolin-1 deficient mouse that maintains the ability to secrete biliary cholesterol to the same extent as the wild-type mice and, when fed a LXR agonist, had higher Abcg8 mRNA expression and tended to secrete more biliary cholesterol than wild-type mice [46]. It has been argued that Abcg5/sterolin-1 and Abcg8/sterolin-2 are dependent upon each other for secretion of hepatic sterols into bile [19]. Given the data presented here and from the Abcg5-deficient mouse this does not appear to be the case. Perhaps in the previously published model a non-physiologic state has been made that generates these data. Taken together, these animal models suggest that Abcg5/sterolin-1 and Abcg8/sterolin-2 have independent function in vivo or that there are proteins other than the sterolins that can secrete biliary sterols. + +Conclusions + +The major findings in this study are: (1) disruption of only one of the two genes comprising the STSL locus is necessary and sufficient to cause sitosterolemia, (2) that the loss of Abcg8/sterolin-2 leads to the loss of biliary cholesterol secretion and (3) Abcg5/sterolin-1 appears to be still apically-expressed despite the absence of Abcg8/sterolin-2. These data strongly support the direct role of Abcg8/sterolin-2 as a key player in biliary cholesterol secretion, although biliary sitosterol secretion was apparently preserved. These data suggest that there are mechanisms other than Abcg8/sterolin-2 that allow for the secretion of sitosterol into bile. + +Competing interests + +None declared. + +Authors' contributions + +ELK carried out molecular genetics studies, quantitative RT-PCR, and dynamic bile collection for Figure 9. KL carried out molecular genetic studies and made the targeting construct. The knockout mice were isolated in the NM laboratory. HY performed Northern blotting. ELK, AK, EV and MHL performed Western blotting and immunohistochemistry experiments. AK and NL performed bile collection experiments and bile characterization for Figure 8. MA performed FPLC analysis. SS, AK and RK performed GC sterol analyses. JC assisted with dynamic bile collection and mouse surgery. ROE, AG, NM, GS and SBP were responsible for supervision, data analyses of experiments and for providing funding of these experiments. ELK and SBP wrote the paper. + +Pre-publication history + +The pre-publication history for this paper can be accessed here: + + + +Acknowledgements + +We would like to thank Dr B Stieger for kindly providing anti-Bsep antibody and Dr H Hobbs for providing the UTSW anti-Abcg5/sterolin-1 antibody. We would also like to thank John Oatis, III for his excellent animal husbandry. Grants from the American Heart Association, Beginning Grant-In-Aid Mid-Atlantic Affiliate (KL), NIH Postdoctoral Training Grant T32 HL07260 (ELK), the Netherlands Organization for Scientific Research (NOW) 902-23-193 (AK) as well as the National Institutes of Health Grant HL60613 (SBP) and DK56830 (GS) supported this work. + +Figures and Tables + +Figure 1 + +Generation of mice deficient in Abcg8/sterolin-2. The targeted disruption strategy of Abcg8 is as shown (panel a). Southern blot analysis of BamHI digested mouse genomic DNA, probed with [32P]-randomly labelled probe resulting in a 6.0 kb band for wild type, a 2.7 kb band for homozygous and two bands of 5.9 and 2.6 kb for the heterozygote (panel b). Northern blot analysis of hepatic RNA showed a loss of Abcg8/sterolin-2 mRNA in the homozygote and decreased Abcg8/sterolin-2 mRNA in the heterozygote, although Abcg5/sterolin-1 mRNA appeared relatively unaffected in the knockout mice (panel c). Probes were approximately 1.9 kb for Abcg5 and 2 kb for Abcg8 (see Methods for more detail). RT-PCR analyses of hepatic cDNA showed no Abcg8/sterolin-2 message, downstream of exon 4 in the Abcg8-/- mice, whether primers were located in exons 4 and 13 (panel d), exons 9 and 13 or exons 10 and 13 (panel e). + +Figure 2 + +Free versus esterified sterol levels in the livers of Abcg8+/+, Abcg8+/- and Abcg8-/- mice. Sterol contents of liver extracts of 12-week-old female mice fed a regular chow diet, determined by GC analysis show that the majority of sterols in all genotypes are unesterified. Esterified cholesterol (panel a) remains relatively constant for each genotype. However, no esterified sitosterol in the livers from Abcg8+/+ and Abcg8+/- mice and very little from Abcg8-/- mice was detected (panel b). Small amounts of esterified campesterol were detected in each of the genotypes (panel c). + +Figure 3 + +Lipid profiles of the plasma of Abcg8+/+, Abcg8+/- and Abcg8-/- mice. Lipoproteins were separated from pooled mouse plasma samples by FPLC. Total sterols (panel a) and triglyceride (panel b) profiles of the fractions are shown. There was no difference of cholesterol profiles in the groups, but there was a small triglyceride peak in Abcg8-/- mice in fractions 21–25, corresponding to the LDL-size range, the significance of which is not known at present. + +Figure 4 + +Analyses of mRNA expression and enzyme activity in mouse livers. Panel (a) shows RT-PCR quantitation of mRNA levels for Abcg5, Abcg8, Hmgr, Cyp7a1, Abca1, Mdr2, Lxr, Srebp-1c and Srebp-2 in mouse livers from Abcg8+/+ (open bars), Abcg8+/- (hatched bars) and Abcg8-/- (filled bars). Knockout mice showed an ~60% reduction in mRNA for Abcg5/sterolin-1 and an 80% reduction in the message for HMG CoA reductase. Relatively no message for Abcg8/sterolin-2 was detected in the knockout mice. Panel (b) shows the enzyme activities for HMG-CoA reductase and CYP7a1 in livers from Abcg8+/+, Abcg8+/- and Abcg8-/- mice. Activities of HMG-CoA reductase and CYP7a1 were significantly reduced in the Abcg8-/- mouse liver (*P < 0.05, see text for discussion). + +Figure 5 + +N-glycosylation of Abcg5/sterolin-1 analyses in Abcg8-/- mouse liver. Mouse liver homogenate stained with SC anti-Abcg5/sterolin-1 after treatment with Endo-H or PNGaseF shows a 75 kDa band present in both genotypes, which is resistant to deglycosylation (panel a). Staining of wild-type liver homogenate with preimmune serum showed no detectable bands. Lower portion of panel (a) shows the same aliquots stained for anti-transferrin as a control for deglycosylation. AMC anti-Abcg5/sterolin-1 staining of mouse liver homogenate shows a 'mature' ~90 kDa band in the wild-type mice but not in the Abcg8-/- mice (panel b). A 75 kDa form is present which is sensitive to deglycosylation. Mouse liver homogenate stained with UTSW anti-Abcg5/sterolin-1 shows a 'mature' ~90 kDa band and an 'immature' 75 kDa band in the wild-type mice but no signal is detected in the Abcg8-/- mice. Treatment with Endo-H or PNGaseF results in a lower molecular weight protein in the wild-type mice. Abcg5/Abcg8-/- liver homogenate used for negative control. + +Figure 6 + +Immunohistochemical evaluation of mouse Abcg5/sterolin-1 expression in liver and intestine. COS-1 cells were transiently transfected with pCMV-mouse Abcg5 or pCMV-mouse Abcg8 constructs, allowed to express for 48 hours then fixed and incubated with antibody. pCMV empty vector transfected COS-1 cells incubated with SC anti-Abcg5/sterolin-1 showed no significant fluorescence (panel a). Abcg5 transfected COS-1 cells incubated with SC anti-Abcg5/sterolin-1 antibody showed a membrane distribution (panel b), where as Abcg8 transfected cells incubated with SC anti-Abcg5 antibody showed no significant fluorescence (panel c). Abcg5 and Abcg8 co-transfected COS-1 cells incubated with SC anti-Abcg5/sterolin-1 antibody resulted in a fluorescence pattern similar to Abcg5 alone (panel d). The yellow bar represents 20 μm. Wild-type intestine incubated with SC pre-immune serum (panel e), or SC anti-Abcg5/sterolin-1 antibody pre-incubated with the blocking peptide (panel f) showed no specific signals. Wild-type, Abcg8+/- and Abcg8-/- intestine (panels g, h and i respectively) incubated with SC anti-Abcg5/sterolin-1 antibody, showed no difference in expression patterns, despite the loss of Abcg8/sterolin-2 in the knockout mice. Single arrowhead shows intestinal villus and double arrowhead shows crypt. The yellow bar represents 50 μm. Antibody staining of liver sections was also performed at the AMC Liver Center. As a control, an antibody to Bsep/Abcb11 was used and showed a clear apical distribution in both wild-type (panel j) and Abcg8 knockout mice (panel k). Using AMC antibody against Abcg5/sterolin-1 (see text), in both wild-type (panel l) and Abcg8 knockout liver (panel m), the pattern of expression was also apical and unchanged although the signal is fainter compared to that for Bsep/Abcb11 (see text for discussion). Arrows indicate bile canaliculi. + +Figure 7 + +Immunohistochemical evaluation of mouse Abcg5/sterolin-1 expression in liver. Wild-type liver incubated with UTSW anti-Abcg5/sterolin-1 resulted in apical expression (panel a). Merged image clearly shows the apical distribution. Likewise Abcg8-/- liver incubated with the same UTSW anti-Abcg5/sterolin-1 antibody resulted in a similar apical expression pattern relative to the wild type (panel b). White arrows indicate bile canaliculi and asterisk indicates a bile duct. The yellow bar represents 50 μm. + +Figure 8 + +Biliary sterol, phospholipid and bile salt analyses. Bile salt, sterol and phospholipid contents from male Abcg8+/+ (n = 8), Abcg8+/- (n = 7), and Abcg8-/- (n = 5) mice were examined as described in Methods. Bile salt secretion following a 10 minute collection was lower in Abcg8+/- and Abcg8-/- mice compared to wild type (panel a), but these differences were not statistically significant. Biliary sterol and phospholipid were significantly reduced in the Abcg8-/- mice compared to the wild-type (panels b and c). *P < 0.05. The lower panel shows biliary bile salt, sterol and phospholipid secretion rates from female Abcg8+/+ (n = 5), Abcg8+/- (n = 4), and Abcg8-/- (n = 3) mice measured following 90-minute bile salt depletion followed by stepwise TUDC infusion as described in the text. Bars represent phase TUDC infusion rates. No differences were observed in the ability of the Abcg8-/- mice to secrete bile salts (panel d), but there is a marked inability of the knockout mice to secrete sterols (panel e) and a trend towards a reduced ability to secrete phospholipid (panel f) compared to wild type. + +Figure 9 + +Biliary cholesterol and sitosterol secretion. Biliary sterols were analyzed by GC analyses in Abcg8+/+ (n = 4), Abcg8+/- (n = 3) and Abcg8-/- (n = 4) mice following 30-minute bile salt depletion followed by a continuous TUDC infusion as described in Methods. Abcg8-/- mice were unable to secrete cholesterol into bile with forced TUDC administration relative to the wild-type mice (panel a, *P < 0.05). Interestingly, the Abcg8-/- mice were able to still secrete sitosterol (panel b) and campesterol (panel c). Heterozygous mice showed an increased ability to secrete all sterols with forced TUDC administration, although the results were not statistically significant. + +Table 1 + +Oligonucleotide primers used for quantitative RT-PCR + +Table 2 + +Mouse plasma and tissue sterol analyses + +Animals used in these studies were 12 weeks of age, a mixture of male and female and fed a regular chow diet. *P < 0.05 for -/- versus +/+ and -/- versus +/-. ND, not detectable. diff --git a/src/ontogpt/evaluation/craft/database/all/15061865.ann b/src/ontogpt/evaluation/craft/database/all/15061865.ann new file mode 100644 index 000000000..1440fe84e --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15061865.ann @@ -0,0 +1,530 @@ +T1 CHEBI:18243 0 8 Dopamine +T2 PR:000001177 0 21 Dopamine D2 receptors +T3 GO:0007612 67 75 learning +T4 NCBITaxon:10088 87 91 mice +T5 CHEBI:18243 115 123 Dopamine +T6 GO:0065007 124 134 modulation +T7 CL:0000540 138 146 neuronal +T8 UBERON:0001870 164 178 frontal cortex +T9 UBERON:0001891 180 188 midbrain +T10 UBERON:0002435 194 202 striatum +T11 GO:0051867 390 409 adaptable behaviors +T12 CHEBI:18243 434 442 dopamine +T13 GO:0007212 434 452 dopamine signaling +T14 PR:000001107 473 480;492 501 D1-like ... receptors +T15 PR:000001108 484 501 D2-like receptors +T16 PR:000001107 523 540 D1-like receptors +T17 PR:000001177 629 641 D2 receptors +T18 CHEBI:18243 697 705 dopamine +T19 PR:000001177 697 717 dopamine D2 receptor +T20 GO:0008306 752 763;777 785 associative ... learning +T21 NCBITaxon:10088 808 812 mice +T22 CHEBI:18243 845 853 dopamine +T23 PR:000001177 845 866 dopamine D2 receptors +T24 NCBITaxon:10088 880 884 mice +T25 GO:0007612 1102 1110 learning +T26 CHEBI:33290 1130 1134 food +T27 CHEBI:18243 1165 1173 dopamine +T28 PR:000001177 1165 1185 dopamine D2 receptor +T29 NCBITaxon:10088 1196 1200 mice +T30 GO:0007612 1209 1216 learned +T31 GO:0007631 1233 1240 consume +T32 CHEBI:33290 1249 1253 food +T33 GO:0007612 1329 1336 learned +T34 CHEBI:33290 1419 1423 food +T35 CHEBI:18243 1445 1453 dopamine +T36 PR:000001177 1445 1465 dopamine D2 receptor +T37 NCBITaxon:10088 1476 1480 mice +T38 NCBITaxon:10088 1531 1535 mice +T39 CHEBI:33290 1688 1692 food +T40 CHEBI:18243 1712 1720 dopamine +T41 PR:000001177 1712 1732 dopamine D2 receptor +T42 NCBITaxon:10088 1743 1747 mice +T43 NCBITaxon:10088 1895 1899 mice +T44 CHEBI:18243 1951 1959 dopamine +T45 PR:000001177 1951 1972 dopamine D2 receptors +T46 GO:0007608 1988 1997 olfaction +T47 CHEBI:18243 2110 2118 dopamine +T48 PR:000001177 2110 2130 dopamine D2 receptor +T49 NCBITaxon:10088 2141 2145 mice +T50 CHEBI:18243 2331 2339 dopamine +T51 PR:000001177 2331 2351 dopamine D2 receptor +T52 GO:0065007 2369 2379 regulating +T53 GO:0008306 2380 2391;2405 2413 associative ... learning +T54 NCBITaxon:9606 2461 2466 human +T55 http://purl.obolibrary.org/obo/MONDO_0000001 2477 2486 disorders +T56 GO:0008306 2524 2544 associative learning +T57 CHEBI:33290 2698 2702 food +T58 GO:0007612 2768 2775 learned +T59 NCBITaxon:1 2789 2797 organism +T60 GO:0007612 2857 2864 learned +T61 CHEBI:33290 2919 2923 food +T62 NCBITaxon:9989 3154 3160 rodent +T63 CHEBI:33290 3179 3183 food +T64 GO:0008306 3295 3315 associative learning +T65 CHEBI:33290 3377 3381 food +T66 CHEBI:18243 3506 3514 dopamine +T67 GO:0008306 3582 3602 associative learning +T68 GO:0007612 3642 3649 learned +T69 CHEBI:18243 3779 3787 dopamine +T70 GO:0007212 3779 3797 dopamine signaling +T71 CHEBI:18243 3820 3828 dopamine +T72 CHEBI:48561 3844 3873 dopamine receptor antagonists +T73 CHEBI:78741 3883 3889 6-OHDA +T74 CHEBI:18243 4091 4099 Dopamine +T75 GO:0007212 4091 4109 Dopamine signaling +T76 PR:000001107 4223 4231 D-1 like +T77 PR:000001175 4233 4237 D1aR +T78 PR:000001176 4239 4242;4244 4245 D1b ... 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+T129 GO:0008306 6555 6575 associative learning +T130 GO:0007612 6646 6654 learning +T131 NCBITaxon:9989 6658 6665 rodents +T132 GO:0007612 6691 6699 learning +T133 NCBITaxon:9606 6748 6753 human +T134 CHEBI:18243 6903 6911 dopamine +T135 GO:0007612 6948 6956 learning +T136 PR:000001177 6985 6988 D2R +T137 PR:000001178 6989 6992 D3R +T138 CHEBI:32168 7005 7014 sulpiride +T139 GO:0007612 7041 7048 leaning +T140 NCBITaxon:10088 7052 7056 mice +T141 GO:0050890 7114 7123 cognitive +T142 NCBITaxon:9606 7148 7153 human +T143 CHEBI:18243 7218 7226 dopamine +T144 PR:000001177 7218 7230 dopamine D2R +T145 NCBITaxon:9606 7265 7270 human +T146 PR:000001177 7347 7350 D2R +T147 GO:0007612 7409 7417 learning +T148 CHEBI:18243 7499 7507 dopamine +T149 PR:000001177 7499 7519 dopamine D2 receptor +T150 GO:0008306 7542 7553;7579 7587 associative ... learning +T151 NCBITaxon:10088 7597 7601 mice +T152 NCBITaxon:10088 7615 7619 mice +T153 CHEBI:18243 7686 7694 dopamine +T154 PR:000001177 7686 7707 dopamine D2 receptors +T155 PR:000001177 7709 7712 D2R +T156 PR:000001177 7743 7746 D2R +T157 GO:0007612 7773 7781 learning +T158 GO:0050893 7829 7847 sensory processing +T159 GO:0007612 7862 7870 learning +T160 CHEBI:18243 7900 7908 dopamine +T161 PR:000001177 7900 7912 dopamine D2R +T162 NCBITaxon:10088 7952 7957 mouse +T163 GO:0007612 7976 7981 learn +T164 CHEBI:33290 7997 8001 food +T165 GO:0051866 8043 8053 adaptively +T166 PR:000001177 8184 8187 D2R +T167 NCBITaxon:10088 8198 8202 mice +T168 GO:0007612 8203 8208 learn +T169 CHEBI:33290 8248 8252 food +T170 PR:000001177 8262 8265 D2R +T171 PR:000001177 8273 8276 D2R +T172 NCBITaxon:10088 8280 8284 mice +T173 GO:0007612 8293 8300 learned +T174 GO:0007631 8315 8322 consume +T175 CHEBI:33290 8323 8327 food +T176 GO:0040011 8418 8426 ambulate +T177 CHEBI:33290 8461 8465 food +T178 CHEBI:33290 8756 8760 food +T179 CHEBI:33290 8913 8917 food +T180 NCBITaxon:10088 8963 8967 mice +T181 PR:000001177 8979 8982 D2R +T182 NCBITaxon:10088 8986 8990 mice +T183 PR:000001177 9105 9108 D2R +T184 NCBITaxon:10088 9112 9116 mice +T185 PR:000001177 9251 9254 D2R +T186 NCBITaxon:10088 9258 9262 mice +T187 GO:0007612 9266 9271 learn +T188 CHEBI:33290 9337 9341 food +T189 GO:0007612 9382 9387 learn +T190 NCBITaxon:10088 9503 9507 Mice +T191 CHEBI:18243 9516 9524 dopamine +T192 PR:000001177 9516 9537 dopamine D2 receptors +T193 PR:000001177 9647 9650 D2R +T194 NCBITaxon:10088 9654 9658 mice +T195 GO:0007612 9711 9719 learning +T196 PR:000001177 9845 9848 D2R +T197 NCBITaxon:10088 9852 9856 mice +T198 CHEBI:33290 9999 10003 food +T199 PR:000001177 10226 10229 D2R +T200 NCBITaxon:10088 10233 10237 mice +T201 PR:000001177 10259 10262 D2R +T202 NCBITaxon:10088 10266 10270 mice +T203 PR:000001177 10292 10295 D2R +T204 NCBITaxon:10088 10299 10303 mice +T205 PR:000001177 10342 10345 D2R +T206 NCBITaxon:10088 10349 10353 mice +T207 PR:000001177 10414 10417 D2R +T208 PR:000001177 10425 10428 D2R +T209 NCBITaxon:10088 10432 10436 mice +T210 NCBITaxon:33208 10617 10624 animals +T211 PR:000001177 10725 10728 D2R +T212 NCBITaxon:10088 10732 10736 mice +T213 PR:000001177 10753 10756 D2R +T214 NCBITaxon:10088 10760 10764 mice +T215 GO:0007612 10955 10963 learning +T216 PR:000001177 11151 11154 D2R +T217 NCBITaxon:10088 11158 11162 mice +T218 NCBITaxon:10088 11236 11240 mice +T219 PR:000001177 11346 11349 D2R +T220 NCBITaxon:10088 11353 11357 mice +T221 PR:000001177 11384 11387 D2R +T222 NCBITaxon:10088 11391 11395 mice +T223 PR:000001177 11456 11459 D2R +T224 NCBITaxon:10088 11463 11467 mice +T225 CHEBI:18243 11741 11749 dopamine +T226 PR:000001177 11741 11761 dopamine D2 receptor +T227 GO:0008306 11806 11817;11831 11839 associative ... learning +T228 PR:000001177 11841 11844 D2R +T229 NCBITaxon:10088 11848 11852 mice +T230 GO:0007612 11892 11900 learning +T231 GO:0007631 11915 11922 consume +T232 CHEBI:33290 11923 11927 food +T233 GO:0007626 11959 11977 locomotor behavior +T234 CHEBI:33290 12068 12072 food +T235 PR:000001177 12130 12133 D2R +T236 NCBITaxon:10088 12137 12141 mice +T237 PR:000001177 12248 12251 D2R +T238 CL:0000540 12290 12298 neuronal +T239 PR:000001177 12392 12395 D2R +T240 NCBITaxon:10088 12399 12403 mice +T241 CHEBI:18243 12483 12491 dopamine +T242 GO:0007212 12483 12501 dopamine signaling +T243 CHEBI:18243 12515 12523 dopamine +T244 PR:000001177 12515 12528 dopamine D2Rs +T245 GO:0007612 12565 12573 learning +T246 NCBITaxon:10088 12585 12589 Mice +T247 CHEBI:18243 12613 12621 dopamine +T248 PR:000001177 12613 12625 dopamine D2R +T249 CHEBI:33290 12707 12711 food +T250 GO:0007612 12733 12740 learned +T251 CHEBI:33290 12752 12756 food +T252 NCBITaxon:10114 12868 12872 rats +T253 PR:000001177 12894 12897 D2R +T254 CHEBI:78741 12948 12954 6-OHDA +T255 GO:0008306 12976 12987;13000 13008 associative ... learning +T256 CHEBI:18243 13095 13103 dopamine +T257 PR:000001177 13128 13132 D2Rs +T258 CHEBI:33290 13183 13187 food +T259 PR:000001177 13241 13244 D2R +T260 NCBITaxon:10088 13248 13252 mice +T261 PR:000001177 13302 13306 D2Rs +T262 GO:0008306 13368 13388 associative learning +T263 GO:0008306 13478 13498 associative learning +T264 CHEBI:18243 13515 13523 dopamine +T265 PR:000001175 13515 13528 dopamine D1Rs +T266 CHEBI:18243 13563 13571 dopamine +T267 PR:000001175 13563 13576 dopamine D1Rs +T268 CHEBI:18243 13582 13590 dopamine +T269 GO:0007612 13600 13608 learning +T270 PR:000001177 13613 13617 D2Rs +T271 PR:000001177 13724 13735 dopamine D2 +T272 PR:000001178 13724 13734;13736 13737 dopamine D ... 3 +T273 CHEBI:48561 13724 13732;13738 13748 dopamine ... antagonist +T274 CHEBI:33290 13795 13799 food +T275 GO:0008306 13810 13830 associative learning +T276 GO:0007612 13903 13911 learning +T277 PR:000001175 13945 13948 D1R +T278 PR:000001177 13952 13955 D2R +T279 PR:000001177 14162 14165 D2R +T280 PR:000001177 14244 14247 D2R +T281 GO:0008306 14270 14281;14295 14303 associative ... learning +T282 PR:000001177 14387 14390 D2R +T283 GO:0040011 14406 14416 locomotion +T284 GO:0007612 14421 14429 learning +T285 PR:000001177 14499 14502 D2R +T286 GO:0007612 14560 14568 learning +T287 CHEBI:23888 14653 14658 drugs +T288 CHEBI:18243 14731 14739 dopamine +T289 PR:000001177 14731 14744 dopamine D2Rs +T290 PR:000001177 14824 14828 D2Rs +T291 GO:0008306 14832 14852 associative learning +T292 GO:0040011 14899 14908 locomotor +T293 GO:0007612 15066 15074 learning +T294 PR:000001177 15145 15149 D2Rs +T295 GO:0008306 15153 15173 associative learning +T296 PR:000001177 15325 15328 D2R +T297 NCBITaxon:9989 15344 15351 rodents +T298 PR:000001177 15481 15485 D2Rs +T299 GO:0008306 15536 15556 associative learning +T300 NCBITaxon:9989 15560 15567 rodents +T301 PR:000001177 15608 15611 D2R +T302 CHEBI:18243 15779 15787 dopamine +T303 PR:000001177 15900 15904 D2Rs +T304 PR:000001178 15906 15910 D3Rs +T305 PR:000001179 15916 15920 D4Rs +T306 NCBITaxon:10088 16027 16031 mice +T307 SO:0000704 16047 16058 genetically +T308 SO:0001023 16117 16124 alleles +T309 NCBITaxon:10088 16255 16260 mouse +T310 NCBITaxon:33208 16384 16391 animals +T311 GO:0007626 16438 16456 locomotor behavior +T312 PR:000001177 16479 16482 D2R +T313 PR:000001177 16490 16493 D2R +T314 NCBITaxon:10088 16497 16501 mice +T315 PR:000001177 16559 16562 D2R +T316 GO:0008306 16585 16605 associative learning +T317 PR:000001177 16698 16701 D2R +T318 SO:0000704 16778 16785 genetic +T319 PR:000001177 16806 16810 drd2 +T320 GO:0007608 16835 16844 olfactory +T321 PR:000001177 16863 16866 D2R +T322 NCBITaxon:10088 16870 16874 mice +T323 PR:000001177 16929 16932 D2R +T324 NCBITaxon:10088 16936 16940 mice +T325 GO:0007612 16945 16950 learn +T326 CHEBI:33290 16967 16971 food +T327 GO:0007612 17011 17018 learned +T328 CHEBI:18243 17103 17111 dopamine +T329 PR:000001177 17103 17117 dopamine D2R's +T330 GO:0030424 17156 17161 axons +T331 GO:0007608 17165 17174 olfactory +T332 UBERON:0001579 17165 17181 olfactory nerves +T333 GO:0007608 17218 17227 olfactory +T334 UBERON:0001579 17218 17233 olfactory nerve +T335 CL:1001502 17241 17256;17261 17275 mitral cells of ... olfactory bulb +T336 GO:0007608 17261 17270 olfactory +T337 UBERON:0002264 17261 17275 olfactory bulb +T338 PR:000001177 17372 17376 D2Rs +T339 GO:0007608 17384 17393 olfactory +T340 UBERON:0002264 17384 17398 olfactory bulb +T341 GO:0007608 17432 17441 olfactory +T342 GO:0046959 17484 17493 habituate +T343 GO:0007608 17504 17513 olfactory +T344 UBERON:0001579 17504 17519 olfactory nerve +T345 PR:000001177 17629 17632 D2R +T346 NCBITaxon:10088 17636 17640 mice +T347 GO:0007612 17657 17665 learning +T348 CHEBI:18243 17700 17708 dopamine +T349 PR:000001177 17700 17712 dopamine D2R +T350 PR:000001177 17827 17830 D2R +T351 NCBITaxon:10088 17834 17838 mice +T352 PR:000001177 17851 17854 D2R +T353 NCBITaxon:10088 17858 17862 mice +T354 GO:0007612 17879 17887 learning +T355 PR:000001177 18049 18052 D2R +T356 NCBITaxon:10088 18056 18060 mice +T357 CHEBI:33290 18072 18076 food +T358 PR:000001175 18166 18169 D1R +T359 PR:000001177 18201 18204 D2R +T360 NCBITaxon:10088 18208 18212 mice +T361 CHEBI:18243 18270 18278 dopamine +T362 PR:000001175 18270 18283 dopamine D1Rs +T363 NCBITaxon:10088 18291 18295 mice +T364 GO:0007612 18500 18508 learning +T365 NCBITaxon:10088 18691 18695 mice +T366 PR:000001177 18801 18804 D2R +T367 NCBITaxon:10088 18808 18812 mice +T368 CHEBI:18243 18956 18964 dopamine +T369 CHEBI:18243 18984 18992 dopamine +T370 PR:000001177 18984 18996 dopamine D2R +T371 GO:0065007 19017 19026 modulates +T372 CHEBI:18243 19182 19190 dopamine +T373 CL:0000700 19182 19196 dopamine cells +T374 GO:0008306 19260 19280 associative learning +T375 CHEBI:18243 19327 19339 dopaminergic +T376 CL:0000700 19327 19345 dopaminergic cells +T377 CHEBI:18243 19469 19481 dopaminergic +T378 CL:0000700 19469 19486 dopaminergic cell +T379 GO:0001775 19482 19497 cell activation +T380 PR:000001177 19678 19681 D2R +T381 NCBITaxon:10088 19685 19689 mice +T382 CHEBI:18243 19765 19773 dopamine +T383 PR:000001177 19765 19777 dopamine D2R +T384 CHEBI:18243 19929 19937 dopamine +T385 PR:000001177 19929 19942 dopamine D2Rs +T386 PR:000001175 19949 19952 D1R +T387 UBERON:0005382 20051 20066 dorsal striatum +T388 PR:000001177 20088 20091 D2R +T389 NCBITaxon:10088 20095 20099 mice +T390 UBERON:0002435 20159 20167 striatal +T391 GO:0045202 20168 20176 synaptic +T392 UBERON:0005382 20215 20230 dorsal striatum +T393 NCBITaxon:10088 20253 20257 mice +T394 CL:0000540 20321 20329 neuronal +T395 UBERON:0002435 20342 20350 striatal +T396 UBERON:0006798 20351 20360 efferents +T397 CHEBI:18243 20417 20425 dopamine +T398 PR:000001177 20417 20429 dopamine D2R +T399 UBERON:0002435 20449 20457 striatum +T400 UBERON:0005382 20612 20627 dorsal striatum +T401 NCBITaxon:9989 20631 20638 rodents +T402 UBERON:0001873 20669 20676 caudate +T403 http://purl.obolibrary.org/obo/MONDO_0007743 20694 20698 ADHD +T404 PR:000001177 20740 20743 D2R +T405 PR:000001177 20876 20879 D2R +T406 NCBITaxon:10088 20883 20887 mice +T407 GO:0007612 20919 20927 learning +T408 PR:000001177 21012 21016 D2Rs +T409 NCBITaxon:1 21058 21066 organism +T410 GO:0007612 21070 21078 learning +T411 GO:0007612 21115 21123 learning +T412 CHEBI:18243 21223 21231 dopamine +T413 PR:000001177 21223 21236 dopamine D2Rs +T414 NCBITaxon:10088 21345 21349 mice +T415 PR:000001177 21374 21378 D2Rs +T416 GO:0008306 21562 21582 associative learning +T417 GO:0007612 21648 21656 learning +T418 CHEBI:18243 21658 21666 dopamine +T419 CHEBI:18243 21727 21735 dopamine +T420 PR:000001177 21727 21740 dopamine D2Rs +T421 GO:0008306 21759 21779 associative learning +T422 UBERON:0002435 21781 21789 striatal +T423 GO:0045202 21790 21798 synapses +T424 UBERON:0006798 21927 21936 efferents +T425 UBERON:0001870 21953 21967 frontal cortex +T426 UBERON:0000349 21993 21999 limbic +T427 CHEBI:18243 22045 22053 dopamine +T428 PR:000001177 22045 22057 dopamine D2R +T429 UBERON:0000955 22150 22156 brains +T430 PR:000001177 22164 22167 D2R +T431 NCBITaxon:10088 22171 22175 mice +T432 CHEBI:18243 22262 22270 dopamine +T433 PR:000001175 22262 22274 dopamine D1R +T434 PR:000001177 22480 22483 D2R +T435 NCBITaxon:10088 22487 22491 mice +T436 PR:000001177 22571 22575 D2Rs +T437 GO:0008306 22579 22590;22604 22612 associative ... learning +T438 NCBITaxon:10088 22670 22674 mice +T439 GO:0044849 23163 23177 estrous cycles +T440 NCBITaxon:33208 23290 23297 animals +T441 NCBITaxon:33208 23404 23411 Animals +T442 NCBITaxon:9989 23698 23705 rodents +T443 NCBITaxon:10088 23799 23803 Mice +T444 CHEBI:33290 23816 23820 food +T445 CHEBI:18243 23870 23878 Dopamine +T446 PR:000001177 23870 23882 Dopamine D2R +T447 PR:000001177 23890 23893 D2R +T448 NCBITaxon:10088 23897 23901 mice +T449 GO:0040011 23946 23954 ambulate +T450 NCBITaxon:10088 23981 23986 mouse +T451 NCBITaxon:9989 24045 24051 rodent +T452 CHEBI:33290 24052 24056 food +T453 GO:0007631 24229 24236 consume +T454 CHEBI:33290 24241 24245 food +T455 UBERON:0002398 24291 24295 hand +T456 GO:0040011 24361 24369 ambulate +T457 CHEBI:33290 24450 24454 food +T458 NCBITaxon:10088 24524 24529 mouse +T459 CHEBI:33290 24549 24553 food +T460 CHEBI:33290 24609 24613 food +T461 GO:0007612 24763 24768 learn +T462 NCBITaxon:10088 24795 24799 mice +T463 CHEBI:33290 24935 24939 food +T464 NCBITaxon:40922 25152 25156 dill +T465 CHEBI:33290 25289 25293 food +T466 CHEBI:33290 25367 25371 food +T467 CHEBI:60004 25410 25417 mixture +T468 NCBITaxon:10088 25494 25499 mouse +T469 GO:0007631 25529 25537 consumed +T470 UBERON:0006333 25685 25690 snout +T471 UBERON:0002102 25700 25709 forelimbs +T472 NCBITaxon:10088 26061 26065 mice +T473 GO:0007612 26489 26497 learning +T474 CHEBI:18243 26552 26560 dopamine +T475 PR:000001177 26552 26565 dopamine D2Rs +T476 GO:0007612 26578 26586 learning +T477 GO:0007612 26993 27001 learning +T478 GO:0007612 27443 27451 learning +T479 GO:0007612 27560 27568 learning +T480 GO:0007612 27975 27983 learning +T481 NCBITaxon:10088 28459 28463 Mice +T482 CHEBI:18243 28472 28480 dopamine +T483 PR:000001177 28472 28493 dopamine D2 receptors +T484 NCBITaxon:10088 28548 28552 mice +T485 GO:0007612 28577 28584 learned +T486 CHEBI:33290 28597 28601 food +T487 PR:000001177 28747 28750 D2R +T488 NCBITaxon:10088 28761 28765 mice +T489 GO:0007612 29074 29082 learning +T490 CHEBI:18243 29133 29141 Dopamine +T491 PR:000001177 29133 29153 Dopamine D2 receptor +T492 GO:0008306 29227 29247 associative learning +T493 PR:000001177 29260 29263 D2R +T494 NCBITaxon:10088 29268 29272 mice +T495 PR:000001177 29314 29317 D2R +T496 NCBITaxon:10088 29321 29325 mice +T497 PR:000001177 29500 29503 D2R +T498 NCBITaxon:10088 29507 29511 mice +T499 GO:0007612 29565 29573 learning +T500 GO:0007612 29665 29673 learning +T501 PR:000001177 29736 29739 D2R +T502 PR:000001177 29747 29750 D2R +T503 NCBITaxon:10088 29754 29758 mice +T504 GO:0007612 29780 29788 learning +T505 PR:000001177 29805 29808 D2R +T506 NCBITaxon:10088 29812 29816 mice +T507 PR:000001177 29894 29897 D2R +T508 NCBITaxon:10088 29901 29905 mice +T509 PR:000001177 29952 29955 D2R +T510 NCBITaxon:10088 29959 29963 mice +T511 PR:000001177 30036 30039 D2R +T512 NCBITaxon:10088 30043 30047 mice +T513 PR:000001177 30107 30110 D2R +T514 NCBITaxon:10088 30114 30118 mice +T515 NCBITaxon:10088 30231 30235 Mice +T516 PR:000001177 30255 30259 D2Rs +T517 NCBITaxon:10088 30387 30391 mice +T518 PR:000001177 30400 30404 D2Rs +T519 NCBITaxon:10088 30415 30419 mice +T520 GO:0007612 30499 30507 learning +T521 PR:000001177 30521 30524 D2R +T522 NCBITaxon:10088 30528 30532 mice +T523 PR:000001177 30642 30645 D2R +T524 NCBITaxon:10088 30649 30653 mice +T525 PR:000001177 30680 30683 D2R +T526 NCBITaxon:10088 30687 30691 mice +T527 NCBITaxon:10088 30779 30783 mice +T528 PR:000001177 30913 30916 D2R +T529 PR:000001177 30973 30976 D2R +T530 NCBITaxon:10088 30980 30984 mice diff --git a/src/ontogpt/evaluation/craft/database/all/15061865.txt b/src/ontogpt/evaluation/craft/database/all/15061865.txt new file mode 100644 index 000000000..5a3ca7e21 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15061865.txt @@ -0,0 +1,125 @@ +Dopamine D2 receptors mediate two-odor discrimination and reversal learning in C57BL/6 mice + +Abstract + +Background + +Dopamine modulation of neuronal signaling in the frontal cortex, midbrain, and striatum is essential for processing and integrating diverse external sensory stimuli and attaching salience to environmental cues that signal causal relationships, thereby guiding goal-directed, adaptable behaviors. At the cellular level, dopamine signaling is mediated through D1-like or D2-like receptors. Although a role for D1-like receptors in a variety of goal-directed behaviors has been identified, an explicit involvement of D2 receptors has not been clearly established. To determine whether dopamine D2 receptor-mediated signaling contributes to associative and reversal learning, we compared C57Bl/6J mice that completely lack functional dopamine D2 receptors to wild-type mice with respect to their ability to attach appropriate salience to external stimuli (stimulus discrimination) and disengage from inappropriate behavioral strategies when reinforcement contingencies change (e.g. reversal learning). + +Results + +Mildly food-deprived female wild-type and dopamine D2 receptor deficient mice rapidly learned to retrieve and consume visible food reinforcers from a small plastic dish. Furthermore, both genotypes readily learned to dig through the same dish filled with sterile sand in order to locate a buried food pellet. However, the dopamine D2 receptor deficient mice required significantly more trials than wild-type mice to discriminate between two dishes, each filled with a different scented sand, and to associate one of the two odors with the presence of a reinforcer (food). In addition, the dopamine D2 receptor deficient mice repeatedly fail to alter their response patterns during reversal trials where the reinforcement rules were inverted. + +Conclusions + +Inbred C57Bl/6J mice that develop in the complete absence of functional dopamine D2 receptors are capable of olfaction but display an impaired ability to acquire odor-driven reinforcement contingencies. Furthermore, the ability of dopamine D2 receptor deficient mice to adjust their responding to a previously reinforced stimulus when unexpected outcomes are encountered is significantly impaired. These findings suggest that signaling mediated by the dopamine D2 receptor is important for regulating associative and reversal learning and may have implications for the treatment of human attention disorders. + +Background + +Reinforcement-mediated associative learning, or the ability to ascribe deterministic relationships between environmental stimuli that signal the probability of receiving a primary reinforcer (e.g. food), has been divided into 3 components or stages [1]. The first, unlearned stage is the organism's experience with a primary reinforcer that elicits some unlearned/innate physiological response (e.g. palatability of a food or satiety). The second phase involves the attachment of salience to external stimuli (discriminative stimuli) that signal whether the delivery of a primary reinforcer will or will not occur based on some emitted behavior (e.g. a rodent lever presses for food pellets only when a stimulus light is illuminated in an operant chamber). Finally, there is the maintenance of associative learning relationships over time (a stimulus light always signifies a food pellet is available subsequent to a lever press) [see refs. [1,2] for review]. Berridge and Robinson [1] have proposed that dopamine plays a pivotal role in the second phase of reinforcement-mediated associative learning by molding the "incentive salience" or learned motivational properties of conditioned stimuli. In support of this view are several studies demonstrating that the disruption of dopamine signaling via acute blockade of dopamine receptors with dopamine receptor antagonists [3-9] or 6-OHDA lesions [1,10,11] do not disrupt phase 1, the palatability or primary reinforcing characteristics of a natural reinforcer (e.g. palatability) but disrupt phase 2, the formation of incentive salience. + +Dopamine signaling is mediated at the cellular level by two major subclasses of G-protein coupled receptors that are referred to as D-1 like (D1aR, D1b/5R) or D-2 like (D2R, D3R, D4R) [12,13]. Although individual genes code for each dopamine receptor subtype, there is considerable pharmacological overlap between D1-like and D2-like receptors. Several studies have used pharmacological manipulations to investigate the contribution of dopamine receptor subtypes in various stages of associative learning [1,2,14]. Currently it is held that excitatory dopamine D1Rs mediate the acquisition and expression of several conditioned behaviors involving food reinforcers [e.g. [6,7]] as well as responding for conditioned cues predictive of cocaine delivery [9] – consistent with the interpretation that D1R mediated signaling might modulate the maintenance of stimulus-response outcomes. Despite a growing literature demonstrating disrupted associative learning following acute manipulations of dopamine D1R signaling during acquisition of associative learning tasks with food [e.g. [6,7]], the actual contribution of D2R-mediated signaling on acquisition and maintenance of associative learning in rodents is unresolved. + +While D2R antagonists have been used in the context of rodent associative learning paradigms, their lack of receptor subtype specificity [13] and motor-disrupting effects [6,8] have prevented a rigorous examination of the actual role this dopamine receptor subtype plays in associative and reversal learning in the context of an operant behavior. Past experiments seeking to explore the possible involvement of dopamine D2Rs in associative learning have utilized low to moderate doses of drugs [2,5,6] and site-specific administration [7,9] in order to avoid the confounding effect that locomotor disruption associated with acute D2R blockade can have on learning performance in rodents. Besides their lack of receptor subtype specificity, an acute exposure to commercially available D2 receptor antagonists fails to completely block signaling mediated solely by D2Rs. Moreover, acute dosing does not recapitulate the marked learning deficits produced in rodents [15,16] by chronic exposure to dopamine D2R antagonists [6,7]. + +The influence of dopamine D2R-mediated signaling on reversal learning is also poorly understood. Reversal learning can be conceptualized as the ability to recognize an unexpected consequence to a previously established associative learning rule and then alter response strategies accordingly [17,18]. Reversal learning in rodents can serve as an index of learning adaptability and alertness that correlates with human attentional-shift paradigms [17,18]. Additionally, a previous report demonstrated that excitotoxic lesions of terminal field targets of mesocortical dopamine have been shown to disrupt reversal learning [19]. Administration of the D2R/D3R antagonist, sulpiride, impairs spatial reversal leaning in mice [20] and has also been shown to impair attention and the cognitive performances of healthy human volunteers [21]. Moreover an inverse relationship between lower dopamine D2R levels and compulsive behavior in human subjects has been reported [22]. Taken together these findings suggest that D2R-mediated signaling could play a critical role in reversal learning but are insufficient to prove it. + +In an effort to establish the contribution of dopamine D2 receptor-mediated signaling to associative and non-spatial reversal learning in adult mice, we compared mice [23] that had developed in the complete absence of all functional dopamine D2 receptors (D2R-/-) to wild-type littermates (D2R+/+) in a go/no-go operant learning procedure that measures primary reinforcement, sensory processing, and reversal learning [17-19]. Here we report that dopamine D2R-mediated signaling must be intact if a mouse is to efficiently learn to associate a food reinforcer with a specific odor and then adaptively disengage inappropriate behavioral strategies following the reversal of reinforcement contingencies. + +Results + +Both wild-type and D2R deficient mice learn operant behaviors equally well + +Mildly food-deprived D2R+/+ and D2R-/- mice readily learned to locate and consume food pellets during both training sessions. Figure 1 depicts the latency for both genotypes to ambulate to a dish to retrieve an unhidden food pellet. There were no differences between genotypes to retrieve the reinforcers (F1,59 = 0.2, p > 0.65), and both groups significantly decreased the amount of time necessary to perform this task along successive trials (F4,59 = 5.74, p < 0.001). Figure 2 depicts the latency to dig for the food pellet buried in a single dish filled with unscented sand. No genotype differences were found in the ability to dig through unscented sand for a hidden food pellet (F1,128 = 1.26, p > 0.27). + +Wild-type mice outperform D2R-/- mice in an odor-driven, stimulus-discrimination, operant task + +To master the odor-driven stimulus discrimination task, D2R-/- mice required significantly more trials (Fig. 3A: t(14) = 2.20; p < 0.05) and committed more errors (Fig. 3B: t(14) = 2.92; p < 0.05) than D2R+/+ mice to learn to associate a specific odor with the presence or absence of the food reinforcer. However, both genotypes did learn the task and eventually maintained accurate discrimination for a minimum of 8 correct responses out of 10 trials. + +Mice lacking dopamine D2 receptors engage in unreinforced behavior in a perseverative manner following reversal of reinforcement contingencies + +D2R-/- mice repeatedly failed to inhibit previously established learning contingencies during reversal trials (Fig. 4A: t(14) = 3.54; p < 0.01) and committed significantly more reversal errors than D2R+/+ mice (Fig. 4B: t(14)= 3.18; p < 0.01). Categorical division of reversal errors (Ferry et al., 2000) – digging in the dish that did not contain the food pellet (S-) (error of commission; Fig. 5A) versus failing to respond within 3-min of presentation (error of omission; Fig. 5B) revealed that both genotypes chiefly committed errors of commission versus errors of omission (D2R-/- mice, U = 0.00; p < 0.01; D2R+/+ mice U = 9.00, p < 0.05), D2R-/- mice committed more commission errors than D2R+/+ mice (U = 5.00, p < 0.05), and there were no differences between D2R-/- and D2R+/+ mice in omission errors (U = 27.5, p = 0.65). Moreover, the number of commission errors committed during the first reversal session (an index of stimulus bound perseveration, where all animals are responding to the reversed contingencies for the first time; Fig. 6) indicated a deficit in the D2R-/- mice compared to the D2R+/+ mice (U = 8.5, p < 0.01). Finally, in an attempt to assess whether perseveration occurred across multiple reversal sessions (Fig. 7), we analyzed the number of commission errors emitted before relearning the reinforcement contingencies during the reversal sessions. The data depicted were collected from subjects that had not achieved the 80% performance criterion. Therefore, the number of D2R+/+ mice represented in session #1 is 8, in session #2 n = 8, session #3 n = 6 (2 mice met our criterion), in session # 4 n = 5 (three met the criterion). Following session 5, the remaining 5 D2R+/+ mice had met criteria. For the D2R-/- mice, each point on the graph represents 8 subjects. None of the D2R-/- mice achieved the criterion of 80% accuracy by session 5. For the overall 2-way ANOVA, there was a significant difference between genotypes for the number of perseverative errors (F1,51 = 16.61, p < 0.001). + +Discussion + +In this study, we sought to determine the contribution of dopamine D2 receptor-mediated signaling to the various stages of associative and reversal learning. D2R-/- mice demonstrated that they were capable of learning to locate and consume food pellets, indicating that their locomotor behavior was not detectably disrupted and their primary motivation to obtain a natural reinforcer (food pellet) was undisturbed. Rather, the impaired ability of D2R-/- mice to assign appropriate discriminative stimulus relationships in an operant discrimination task argues that D2R-mediated signaling contributes to the neuronal processes involved in attaching salience to environmental stimuli. The deficient capacity of D2R-/- mice to disengage inappropriate decision strategies strongly argues that mesolimbic dopamine signaling, mediated by dopamine D2Rs is essential for efficient reversal learning to occur. + +Mice with or without intact dopamine D2R-mediated signaling displayed similar decreases in latencies to retrieve unhidden food pellets (Fig. 1) and learned to dig for food buried in unscented sand (Fig. 2). These findings are in complete agreement with earlier studies that utilized rats and fairly selective D2R antagonists [3-8], as well as even very extensive 6-OHDA lesions [1,10,11] in associative and operant learning paradigms. Our results add to a growing literature demonstrating a negligible role of dopamine, and now, specifically, D2Rs in the unconditioned or hedonic value of natural (food) reinforcers [1,2]. + +The comparatively poor skill of D2R-/- mice during discrimination trials suggests a role for D2Rs in acquisition of appropriate S+/S- relationships in operant associative learning. Several studies have demonstrated that both the acquisition [6,7] and expression [9] of associative learning are mediated by dopamine D1Rs. Most literature reviews identify dopamine D1Rs with dopamine-mediated learning and D2Rs with motor related behaviors [e.g. [24]]. Moreover, it has been reported that acute administration of the dopamine D2/3 antagonist, raclopride, actually improves acquisition of food-motivated associative learning [6]. However, only acute administrations of antagonists were given, and learning was not measured during complete D1R or D2R blockade [6]. Significantly, the reports cited above failed to address the technical limitations of the approach: i.e. that the antagonists used lack adequate subtype specificity and only partially blocked D2R-mediated signaling thus making it impossible to rigorously assess the role of D2R-mediated signaling in associative and reversal learning. Additionally, none of these studies addressed the observation that the effects of D2R antagonists on locomotion and learning depend on whether exposure is chronic or acute [e.g. [15,16]]. + +That D2R-mediated signaling orchestrates just motor components of learning is a conclusion that is potentially biased due to the practice of avoiding doses of drugs that induce catalepsy (and therefore measuring only partial blockade of dopamine D2Rs) and single administrations [e.g. [3]]. Quite possibly, the functional role of D2Rs in associative learning might be masked because of this concern about locomotor disruption [2,6,8]. Doses of raclopride as low as 0.5 mg/kg significantly disrupt motor behavior [25], although this peripheral dose is consistently used in learning paradigms [e.g. [6]]. One might then ask: "How then could the role of D2Rs in associative learning be dissociated from motor behavior?" Seminal experiments [26-28] have clearly demonstrated that repeated administration of catalepsy-inducing doses of D2R antagonists in rodents actually leads to a striking behavioral tolerance to catalepsy. These doses have been shown to occupy well over 80% of available D2Rs [29]. Future experiments measuring acquisition of associative learning in rodents that received chronic administration of D2R antagonists and demonstrated behavioral tolerance to their motor disrupting effects would be a logical test of this hypothesis. However, the realization that multiple dopamine receptor subtypes would be concurrently targeted with the presently commercially available antagonists, such as D2Rs, D3Rs, and D4Rs would have to be rectified. We would argue that the most parsimonious approach at this time is to utilize mice that have been genetically altered such that they are lacking one or both functional alleles of the specific receptor of interest. While they do have their limitations (e.g. developmental compensation and strain effects in mouse lines not backcrossed adequately to a parental strain for a minimum of 10 generations), use of our inbred (N20 generation) animals in the present study (where no differences in locomotor behavior were detected between D2R-/- and D2R+/+ mice; Figs. 1 &2) revealed a previously unappreciated role of D2R-mediated signaling in associative learning and attention that could not be measured with the currently available, acutely administered D2R antagonists. + +A cursory analysis of our data might suggest to some that the genetic manipulation of the drd2 locus conferred a gross olfactory impairment to the D2R-/- mice. Rather, we argue that this is not likely because the D2R-/- mice did learn to retrieve the food pellets from the dishes and eventually learned to accurately discriminate odors. Recent electrophysiological data demonstrate that dopamine D2R's are located on glutamatergic terminal axons of olfactory nerves and depress excitatory input of the olfactory nerve to the mitral cells of the olfactory bulb [30,31]. Consequently, our data, and data from other studies, suggest that the complete lack of D2Rs in the olfactory bulb does not prevent transduction of olfactory stimuli; rather it affects the ability to habituate, or tune, olfactory nerve activity associated with repeatedly encountered concentrations of chemical stimuli [32]. + +The performance of D2R-/- mice during reversal learning is deficient revealing a role for dopamine D2R-mediated signaling in tasks requiring behavioral flexibility + +Perseverative behavioral patterns characterized the D2R-/- mice relative to D2R+/+ mice during reversal learning sessions (Figure 4), manifested during early reversal trials (Figure 6) and persisted across several sessions (Figure 7). These findings are significant because D2R-/- mice respond to food reinforcers and ultimately form and maintain odor-driven S+/S- relationships (a putative D1R-mediated behavior [6]) just as D2R+/+ mice (both groups achieved ≥ 80% discrimination accuracy, the dopamine D1Rs in our mice were not targeted). A future extension of this study would be to test performance over a fixed number of trials and determine patterns of error rates during acquisition of the discrimination and reversal learning tasks. This manipulation would control the number of errors based on trials performed to determine if the apparent difference in absolute number of errors demonstrated by the mutant mice (the putative performance deficit) is simply a reflection of extra trials. Nonetheless, the inability of D2R-/- mice to disengage from previously established S+/S- contingencies (responding to a discriminative stimulus; Fig. 4) strongly argues that mesolimbic dopamine, and in particular dopamine D2R-mediated signaling, modulates the process of alerting the subject that familiar contingencies are now associated unexpected consequences. + +Schultz and colleagues have demonstrated that dopamine cells display consistent tonic firing patterns during maintenance of associative learning tasks [33]. However, phasic burst activity of dopaminergic cells occurs when discrepancies between predicted and actual reinforcement contingencies transpire [14]. This robust increase of dopaminergic cell activation in response to unpredicted outcomes has been referred to as an "error" signal [14]. To date, the molecular basis of this error signal has not been identified. The inability of the D2R-/- mice to desist responding to a previously reinforced stimulus suggests that the dopamine D2R might in fact be the focal point of this error-signaling cascade. + +Electrophysiological data further support the hypothesis that signaling mediated by dopamine D2Rs tunes D1R-mediated mesocorticolimbic output. Calabresi et al. [34] demonstrated that tetanic stimulation of dorsal striatum slices prepared from D2R-/- mice is associated with enhanced EPSP and as a result increased striatal synaptic efficacy. In contrast, stimulation of dorsal striatum slices from wild-type mice resulted in IPSP activity, long-term depression, and decreased neuronal activity of striatal efferents [34]. Carlsson and colleagues [35] have speculated that dopamine D2R stimulation in the striatum serves to "brake" or diminish excitatory corticostriatal signaling and plasticity. Indeed, perseverative behavior is associated with over activity of the dorsal striatum in rodents [36] and over activity of the caudate in patients with ADHD [37] and a strong inverse correlation of D2R binding with compulsive behavior has been reported [22]. Importantly, our data indicate that the poor performances displayed by the D2R-/- mice are manifestations of reversal learning deficits and not gross motivational or sensory impairments. We therefore argue that D2Rs participate in signaling or alerting the organism of learning contingency changes during reversal learning and sculpt ongoing goal-directed behavior. + +Conclusions + +This study demonstrates that signaling by dopamine D2Rs does not mediate the hedonic value of reinforcers, supporting and refining earlier findings [1,2]. However, mice completely deficient in D2Rs demonstrated disrupted acquisition of discriminative stimulus relationships and an impaired ability to recognize changes in behavioral determinants. Therefore, we suggest that during associative learning or when unexpected reinforcement outcomes are modified (reversal learning) dopamine-driven reinforcement impulses involve signaling mediated by dopamine D2Rs. In the course of associative learning, striatal synapses [38] receive experience-driven gain [7], thus permitting newly established reinforcement-mediated signals to traverse ascending efferents en route to the frontal cortex, completing a functional limbic/motor circuit [39-41]. Possibly, the lack of dopamine D2R-mediated signaling prevents refinement of the corticostriatal reinforcement circuits in the brains of the D2R-/- mice, thus impairing their ability to form S+/S- contingencies and disengage inappropriate dopamine D1R-mediated associative responding when unexpected consequences to goal-directed behaviors happened. Consequently, we are compelled to conclude that the perseverative performance deficits demonstrated by the D2R-/- mice in the tasks used in the present study reveal a previously unreported role for D2Rs in associative and reversal learning. + +Methods + +Subjects + +The sixteen, 8–10 week old, 25–30 g mice (8 per genotype) used in this study were the congenic offspring of breeders that were descendants of the original F2 hybrid (129/Sv × C57BL/6J; Kelly et al., 1997) that was backcrossed to inbred C57Bl/6J stock (Jackson Laboratories, ME). This breeding strategy was repeated for 20 successive generations with the gender of the donor/mutant alternating with each generation. All experimental subjects were genotyped by PCR as previously described [23]. All testing occured randomly across estrous cycles. The Department of Comparative Medicine at Oregon Health and Science University approved all protocols, and all animals were maintained in accordance to National Institutes of Health's Guide for the Care and Use of Laboratory Animals. + +Experimental procedures + +Odor discrimination training and testing + +A 2-odor discrimination paradigm was chosen because it permits a measure of attentional focus in goal-directed behavior. Odor discrimination was chosen as the particular task because it is a highly robust behavior in rodents [18]. To minimize experimental error, the experimenter was blind to genotype during testing. Mice were mildly food deprived to approximately 85% ad libitum weight. Dopamine D2R-/- and D2R+/+ mice (n = 8 per group) were initially trained to ambulate the length of a polyvinyl mouse cage (30-cm) to retrieve ~30-mg of Pico® fat supplemented rodent food located in a small plastic cup (3-cm) attached to the floor of the testing apparatus with Velcro. Each subject received five trials, and the latency to locate and start to consume the food pellet was recorded manually with a standard hand-held stopwatch. Twenty-four hours later subjects were trained to ambulate towards and dig through the same 3-cm dish filled with sterile sand to locate a food pellet buried underneath the sand. Subjects received 10 trials. If a mouse failed to locate a food pellet within 10-min of the first trial, an additional food pellet was dropped on top of the dish. This manipulation usually ensured that a subject would dig through the sand to locate the original pellet and learn this task. + +The next day, mice were presented to the testing apparatus that now contained 2 dishes, each filled with different scented sand. One odor signaled that a food reinforcer was buried in that particular dish (S+) while the other odor signaled that no reinforcer was contained within that particular dish (S-). The sand was scented by adding 0.80 grams of either cinnamon or dill weed to 98.8 grams of autoclaved playground sand purchased from a local farm and garden store. Finally, 0.40 grams of finely ground food was mixed with the scented sand in order to mask any odor emitted by the food reinforcer in the S+ dish. This final mixture did not appear to be palatable to the subjects. A trial terminated when the mouse: dug in the correct dish and consumed the hidden pellet, or began to dig in the incorrect dish. Digging was considered the actual physical displacement of sand with the forepaws and/or snout. Placing forelimbs on either of the dishes and sniffing was not considered digging and was not penalized or scored. Each completed trial was separated by a 20-sec inter-trial-interval (ITI). Preliminary experiments demonstrated that an extended time-out period following an incorrect response was not required to ensure goal-directed behavior in the wild-type or mutant mice for this particular task (data not shown). Therefore, the 20-sec ITI was used to separate all trials (successful and unsuccessful). The S+ and S- scents were counterbalanced across subjects in order to control for potential scent bias (pilot data suggest there are no biases, data not shown). Following accurate discrimination, defined as 8 correct retrievals across 10 consecutive trials, reversal trials began. + +Reversal learning measurement + +We next investigated the contribution of dopamine D2Rs to reversal learning behavior by inverting the reinforcement contingencies – now the former S+ signaled no reinforcer and the previous S- now indicated reinforcement availability. This manipulation permits an evaluation of the ability to inhibit responses to previously reinforced discriminative stimuli [18]. The number of trials necessary to establish 8 out of 10 accurate responses was our definition of successful reversal learning. + +Data analysis + +The latency to retrieve a pellet from an empty dish, the latency to dig through a dish filled with unscented sand, and the number of errors of commission committed across the reversal sessions were analyzed with 2-way analyses of variance (genotype × trials). The number of trials necessary to attain criterion performance in odor discrimination (8 correct responses across 10 consecutive trials) and errors committed while learning the discrimination tasks were separated as individual testing phases (e.g. odor discrimination and reversal learning) and analyzed separately with unpaired 2-tailed t-tests. Significance was established at p < 0.05. Errors committed during reversal trials were further categorically separated as errors of commission (failing to withhold responding to S-) and errors of omission (failing to dig in either dish after 3-min had expired). Categorical errors and number of commission errors committed during the first reversal learning session were analyzed between groups with nonparametric Mann-Whitney U tests. + +Authors' contributions + +PJK designed and ran all of the experiments, analyzed the data, and co-wrote the manuscript. DKG oversaw and facilitated the experiments and co-wrote the manuscript. + +Acknowledgements + +This study was supported by grants DA07262 (PJK), DA12062 (DKG), and MH067497 (DKG). We thank Katherine M. Suchland for her excellent technical assistance. + +Figures and Tables + +Figure 1 + +Mice lacking dopamine D2 receptors acquire a goal-directed behavior similar to wild-type mice. Both genotypes readily learned to retrieve food pellets from a small dish and significantly decreased their latencies to perform this task across trials (* p < 0.001). + +Figure 2 + +Wild-type and D2R-deficient mice perform the digging task equally well. No differences in responding as a function of genotype were found, and similar latencies to retrieve the reinforcer were seen. These response patterns suggest that the training from the previous day influenced behavior, demonstrating that both genotypes are capable of learning and retaining goal-directed behaviors. + +Figure 3 + +Dopamine D2 receptor-mediated signaling contributes to the acquisition of odor discrimination/associative learning. Wild-type (D2R+/+) mice outperformed (mean + standard error) the D2R-/- mice during acquisition of (A) odor discrimination (*p < 0.05), and (B) committed significantly fewer discrimination errors (*p < 0.01) during the discrimination task. + +Figure 4 + +D2R-/- mice perseverate in unreinforced behavior during reversal learning trials. The number of necessary trials (mean + standard error) to (A) demonstrate reversal learning (*p < 0.01), and (B) reversal errors committed (*p < 0.01) by D2R-/- and D2R+/+ mice. + +Figure 5 + +Reversal learning measures reveal D2R-/- mice emit significantly more errors of commission but not errors of omission than D2R+/+ mice. Response patterns (mean + standard error) by D2R-/- mice are more suggestive of stimulus bound perseveration and not extinction. D2R-/- mice committed significantly more errors of commission (A) than D2R+/+ mice (*p < 0.01), but no differences in errors of omission (B) were observed between genotypes (p > 0.6). + +Figure 6 + +Mice lacking functional D2Rs display an inability to withhold inappropriate responses. Response patterns during first 10 reversal trials reveal deficits in mice lacking D2Rs. When the mice encountered the inverted reinforcement contingencies during the first reversal learning session, the D2R-/- mice demonstrated an almost complete inability to inhibit responding to previously reinforced stimuli compared to D2R+/+ mice (* p < 0.001). + +Figure 7 + +D2R-/- mice commit significantly more perseverative errors across reversal sessions than wild-type mice. An analysis of the time course of responding for errors of commission across the first four reversal sessions revealed that the D2R-/- committed several more errors of commission than the D2R+/+ mice (*p < 0.001). diff --git a/src/ontogpt/evaluation/craft/database/all/15070402.ann b/src/ontogpt/evaluation/craft/database/all/15070402.ann new file mode 100644 index 000000000..6ea69c6bc --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15070402.ann @@ -0,0 +1,1126 @@ +T1 PR:000010407 0 5 Mig12 +T2 http://purl.obolibrary.org/obo/MONDO_0017138 15 29 Opitz syndrome +T3 SO:0000704 30 34 gene +T4 GO:0010467 55 64 expressed +T5 UBERON:0000922 72 81 embryonic +T6 UBERON:0009571 82 97 ventral midline +T7 PR:000010406 119 123 Mid1 +T8 GO:0005874 148 160 microtubules +T9 http://purl.obolibrary.org/obo/MONDO_0017138 184 204 Opitz G/BBB syndrome +T10 SO:0000704 210 217 genetic +T11 http://purl.obolibrary.org/obo/MONDO_0003847 210 226 genetic disorder +T12 UBERON:0009571 258 265 midline 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4432 4443 microtubule +T154 GO:0005874 4513 4525 microtubules +T155 GO:0065007 4544 4553 regulated +T156 GO:0016311 4585 4602 dephosphorylation +T157 PR:000010406 4606 4610 Mid1 +T158 PR:000008941 4638 4677 α4 regulatory subunit of phosphatase 2A +T159 GO:0065007 4641 4651 regulatory +T160 GO:0000159 4663 4677 phosphatase 2A +T161 GO:0000159 4679 4683 PP2A +T162 PR:000010406 4701 4705 Mid1 +T163 GO:0005874 4711 4723 microtubules +T164 PR:000010406 4764 4768 Mid1 +T165 GO:0065007 4806 4816 regulating +T166 GO:0005874 4821 4833 microtubular +T167 GO:0000159 4834 4838 PP2A +T168 PR:000008941 4891 4893 α4 +T169 GO:0000159 4895 4899 PP2A +T170 GO:0065007 4922 4930 controls +T171 GO:0005874 4982 4993 microtubule +T172 PR:000010406 5055 5059 Mid1 +T173 GO:0010467 5138 5147 expressed +T174 UBERON:0009571 5155 5162 midline +T175 PR:000010406 5203 5207 Mid1 +T176 GO:0005874 5225 5237 microtubules +T177 PR:000010407 5267 5272 Mig12 +T178 PR:000010406 5284 5288 Mid1 +T179 PR:000010406 5335 5339 Mid1 +T180 PR:000008941 5395 5423 α4 subunit of phosphatase 2A +T181 GO:0000159 5409 5423 phosphatase 2A +T182 GO:0000159 5425 5429 PP2A +T183 PR:000010406 5453 5457 Mid1 +T184 http://purl.obolibrary.org/obo/MONDO_0017138 5481 5483 OS +T185 PR:000010406 5563 5567 Mid1 +T186 CL:0000057 5620 5630 fibroblast +T187 PR:000010406 5705 5709 MID1 +T188 SO:0001080 5764 5775 coiled-coil +T189 PR:000016651 5803 5806 RFP +T190 SO:0000417 5812 5818 domain +T191 PR:000010406 5822 5826 MID1 +T192 GO:0005874 5871 5883 microtubules +T193 SO:0000673 5990 6000 transcript +T194 SO:0000236 6030 6033 ORF +T195 SO:0000028 6041 6043 bp +T196 SO:0000028 6065 6067 bp +T197 SO:0000345 6103 6106 EST +T198 SO:0000236 6168 6171 ORF +T199 SO:0000028 6179 6181 bp +T200 SO:0000673 6235 6246 transcripts +T201 GO:0007618 6306 6312 mating +T202 PR:000010406 6459 6463 Mid1 +T203 PR:000010406 6546 6550 Mid1 +T204 SO:0001080 6566 6577 coiled-coil +T205 SO:0001080 6599 6610 Coiled-coil +T206 PR:000010406 6702 6706 MID1 +T207 SO:0001080 6732 6743 coiled-coil +T208 PR:000026474 6855 6861 TRIM19 +T209 PR:000026474 6862 6865 PML +T210 PR:000016650 6867 6873 TRIM25 +T211 PR:000016651 6874 6877 RFP +T212 PR:000016653 6879 6885 TRIM29 +T213 PR:000016653 6886 6890 ATDC +T214 SO:0000857 6914 6922 homology +T215 PR:000010406 6928 6932 Mid1 +T216 PR:000010406 6993 6997 Mid1 +T217 GO:0007618 7023 7029 mating +T218 PR:000010406 7050 7054 Mid1 +T219 PR:000010407 7055 7060 Mig12 +T220 PR:000010407 7091 7096 Mig12 +T221 SO:0000417 7137 7143 domain +T222 PR:000010407 7174 7179 Mig12 +T223 SO:0000417 7214 7220 domain +T224 PR:000023087 7222 7226 LexA +T225 PR:000010406 7266 7270 MID1 +T226 SO:0000417 7271 7278 domains +T227 PR:000023087 7292 7296 LexA +T228 SO:0000417 7309 7315 domain +T229 PR:000016651 7350 7353 RFP +T230 PR:000016651 7384 7387 RFP +T231 PR:000010407 7432 7437 Mig12 +T232 PR:000010406 7483 7487 Mid1 +T233 CHEBI:75055 7591 7596 X-gal +T234 GO:0040007 7608 7615 growing +T235 CHEBI:28260 7659 7668 galactose +T236 CHEBI:28260 7670 7673 Gal +T237 CHEBI:17234 7684 7691 glucose +T238 CHEBI:17234 7693 7696 Glu +T239 SO:0000417 7762 7769 domains +T240 SO:0001080 7771 7773 CC +T241 SO:0001080 7775 7786 coiled-coil +T242 SO:0000417 7787 7793 domain +T243 PR:000007581 7802 7813 fibronectin +T244 SO:0000417 7839 7845 domain +T245 NCBITaxon:9606 7874 7879 human +T246 NCBITaxon:9606 7881 7882 h +T247 NCBITaxon:10088 7888 7893 mouse +T248 NCBITaxon:10088 7895 7896 m +T249 PR:000010407 7898 7903 MIG12 +T250 NCBITaxon:7955 7928 7937 zebrafish +T251 PR:P47805 7938 7941 G12 +T252 NCBITaxon:9606 7950 7955 human +T253 PR:000016322 7956 7962 SPOT14 +T254 NCBITaxon:9606 8020 8025 human +T255 NCBITaxon:39107 8030 8036 murine +T256 PR:000010407 8037 8042 Mig12 +T257 NCBITaxon:9606 8105 8110 human +T258 NCBITaxon:10088 8115 8120 mouse +T259 PR:000010407 8121 8126 MIG12 +T260 PR:Q9NPA3 8176 8182 hMIG12 +T261 NCBITaxon:7955 8191 8200 zebrafish +T262 SO:0000857 8266 8274 homology +T263 NCBITaxon:9606 8284 8289 human +T264 PR:000016322 8290 8297 SPOT-14 +T265 SO:0000730 8347 8350 gap +T266 NCBITaxon:7955 8388 8397 zebrafish +T267 PR:000016322 8402 8408 SPOT14 +T268 PR:000010406 8466 8470 Mid1 +T269 PR:000010407 8471 8476 Mig12 +T270 PR:000010406 8528 8532 Mid1 +T271 GO:0042571 8545 8555 antibodies +T272 GO:0009294 8610 8621 transfected +T273 PR:000010406 8666 8670 Mid1 +T274 PR:000010406 8677 8681 MID1 +T275 PR:000010407 8700 8705 Mig12 +T276 PR:000010407 8710 8715 MIG12 +T277 GO:0009294 8750 8761 transfected +T278 GO:0042571 8780 8790 antibodies +T279 PR:000010406 8845 8849 Mid1 +T280 GO:0019814 8910 8912 Ig +T281 GO:0019814 8914 8929 immunoglobulins +T282 PR:000010406 8987 8991 Mid1 +T283 GO:0009294 9018 9029 transformed +T284 PR:000010406 9045 9049 Mid1 +T285 GO:0042571 9090 9098 antibody +T286 GO:0009294 9181 9192 transfected +T287 GO:0009294 9333 9346 transfections +T288 PR:000000020 9359 9362 Myc +T289 GO:0042571 9363 9371 antibody +T290 SO:0000343 9416 9432 sequence matches +T291 NCBITaxon:9606 9456 9461 human +T292 PR:000010407 9484 9495 STRAIT11499 +T293 NCBITaxon:10088 9532 9537 mouse +T294 SO:0000345 9616 9620 ESTs +T295 NCBITaxon:9606 9643 9648 human +T296 SO:0000704 9649 9653 gene +T297 SO:0000147 9698 9703 exons +T298 NCBITaxon:10088 9760 9765 mouse +T299 SO:0000704 9766 9770 gene +T300 GO:0000805 9808 9820 X chromosome +T301 NCBITaxon:9606 9826 9831 human +T302 NCBITaxon:10088 9869 9874 mouse +T303 SO:0000417 9989 9996 domains +T304 SO:0001080 10031 10042 coiled-coil +T305 PR:000010406 10089 10093 Mid1 +T306 SO:0000857 10125 10133 homology +T307 NCBITaxon:7955 10143 10152 zebrafish +T308 GO:0007369 10154 10166 Gastrulation +T309 PR:P47805 10154 10187 Gastrulation specific protein G12 +T310 NCBITaxon:40674 10253 10262 mammalian +T311 PR:000016322 10263 10270 SPOT-14 +T312 GO:0006631 10310 10335 metabolism of fatty acids +T313 CHEBI:35366 10324 10335 fatty acids +T314 SO:0000673 10355 10365 transcript +T315 PR:000010407 10377 10382 MIG12 +T316 PR:000010407 10387 10420 Mid1 interacting G12-like protein +T317 NCBITaxon:7955 10452 10463 Danio rerio +T318 NCBITaxon:9606 10510 10515 human +T319 NCBITaxon:10088 10520 10525 mouse +T320 PR:000010407 10526 10531 Mig12 +T321 NCBITaxon:7955 10537 10546 zebrafish +T322 PR:P47805 10547 10550 G12 +T323 NCBITaxon:9606 10560 10565 human +T324 PR:000016322 10566 10572 SPOT14 +T325 GO:0009294 10655 10666 transfected +T326 PR:000010406 10694 10698 MID1 +T327 PR:000010406 10705 10709 Mid1 +T328 PR:000010407 10739 10744 MIG12 +T329 PR:000010407 10749 10754 Mig12 +T330 PR:000010406 10813 10817 Mid1 +T331 GO:0042571 10829 10839 antibodies +T332 PR:000010406 10864 10868 Mid1 +T333 GO:0009294 10879 10890 transfected +T334 PR:000010407 10916 10921 Mig12 +T335 PR:000010407 10988 10993 Mig12 +T336 GO:0042571 11012 11020 antibody +T337 PR:000010406 11041 11045 Mid1 +T338 GO:0042571 11102 11110 antibody +T339 GO:0042571 11147 11155 antibody +T340 PR:000010406 11251 11255 Mid1 +T341 PR:000010407 11256 11261 Mig12 +T342 PR:000010407 11285 11290 Mig12 +T343 GO:0009294 11291 11302 transfected +T344 PR:000010406 11370 11374 Mid1 +T345 GO:0007618 11410 11416 mating +T346 SO:0001080 11446 11457 coiled-coil +T347 PR:000010406 11468 11472 Mid1 +T348 PR:000010407 11520 11525 Mig12 +T349 GO:0009294 11580 11591 transfected +T350 PR:000010407 11600 11605 MIG12 +T351 PR:000000020 11662 11665 Myc +T352 GO:0042571 11677 11687 antibodies +T353 SO:0001080 11732 11743 coiled-coil +T354 PR:000010407 11769 11774 Mig12 +T355 PR:000010406 11844 11848 Mid1 +T356 PR:000010407 11849 11854 Mig12 +T357 PR:000010407 11879 11884 Mig12 +T358 GO:0010467 11895 11904 expressed +T359 UBERON:0001017 11923 11926 CNS +T360 UBERON:0009571 11927 11934 midline +T361 PR:000010406 11942 11946 Mid1 +T362 http://purl.obolibrary.org/obo/MONDO_0000001 11980 11988 disorder +T363 PR:000010407 12048 12053 Mig12 +T364 PR:000010406 12058 12062 Mid1 +T365 GO:0010467 12084 12094 expression +T366 PR:000010407 12098 12103 Mig12 +T367 UBERON:0000922 12111 12120 embryonic +T368 GO:0009790 12111 12132 embryonic development +T369 PR:000010407 12138 12143 Mig12 +T370 GO:0097617 12244 12257 hybridization +T371 NCBITaxon:10088 12261 12266 mouse +T372 UBERON:0000922 12267 12274 embryos +T373 UBERON:0000922 12286 12295 embryonic +T374 GO:0010467 12317 12327 expression +T375 UBERON:0000922 12398 12407 embryonic +T376 UBERON:0001017 12484 12506 central nervous system +T377 UBERON:0001017 12508 12511 CNS +T378 UBERON:0002101 12560 12565 limbs +T379 GO:0097617 12589 12602 hybridization +T380 GO:0010467 12641 12651 expression +T381 SO:0000673 12691 12701 transcript +T382 GO:0010467 12775 12785 expression +T383 UBERON:0001017 12816 12838 central nervous system +T384 UBERON:0002028 12903 12912 hindbrain +T385 UBERON:0034705 12966 12981 neuroepithelium +T386 UBERON:0002037 12989 12999 cerebellar +T387 UBERON:0001048 13000 13009 primordia +T388 UBERON:0000988 13033 13037 pons +T389 UBERON:0001896 13069 13086 medulla oblongata +T390 UBERON:0005358 13105 13116 ventricular +T391 UBERON:0002028 13117 13126 hindbrain +T392 UBERON:0009571 13160 13175 ventral midline +T393 GO:0010467 13208 13218 expression +T394 UBERON:0002291 13248 13264;13269 13280 central canal of ... spinal cord +T395 UBERON:0003079 13303 13308;13318 13324 floor ... plates +T396 UBERON:0003054 13313 13324 roof plates +T397 UBERON:0001893 13346 13359 telencephalon +T398 PR:000010407 13361 13366 Mig12 +T399 UBERON:0003053 13392 13408 ventricular zone +T400 UBERON:0001893 13416 13429 telencephalic +T401 UBERON:0001016 13464 13478 nervous system +T402 PR:000010407 13480 13485 Mig12 +T403 SO:0000673 13486 13496 transcript +T404 UBERON:0002261 13521 13533 dorsal roots +T405 UBERON:0001675 13545 13563 trigeminal ganglia +T406 GO:0010467 13600 13610 expression +T407 PR:000010407 13614 13619 Mig12 +T408 UBERON:0000062 13659 13665 organs +T409 SO:0000673 13671 13681 transcript +T410 UBERON:0006015 13701 13717 interdigital web +T411 UBERON:0002102 13751 13760 forelimbs +T412 GO:0060173 13801 13815;13820 13825 development of ... limbs +T413 UBERON:0002101 13820 13825 limbs +T414 PR:000010407 13836 13841 Mig12 +T415 SO:0000673 13842 13852 transcript +T416 UBERON:0002222 13872 13885 perichondrium +T417 UBERON:0002544 13893 13899 digits +T418 UBERON:0000062 13924 13930 organs +T419 GO:0010467 13931 13941 expressing +T420 PR:000010407 13942 13947 Mig12 +T421 UBERON:0001120 13960 13964;13975 13988 left ... thyroid lobes +T422 UBERON:0001119 13969 13988 right thyroid lobes +T423 UBERON:0001132 13997 14015 parathyroid glands +T424 UBERON:0000989 14034 14041 phallic +T425 UBERON:0000164 14054 14070 urogenital sinus +T426 UBERON:0000159 14089 14099 anal canal +T427 UBERON:0001052 14101 14107 rectum +T428 UBERON:0000483 14117 14127 epithelium +T429 UBERON:0009958 14139 14147;14152 14159 lumen of ... bladder +T430 PR:000010407 14226 14231 Mig12 +T431 GO:0010467 14244 14251 express +T432 PR:000010406 14256 14260 Mid1 +T433 SO:0000673 14261 14271 transcript +T434 http://purl.obolibrary.org/obo/MONDO_0017138 14300 14302 OS +T435 PR:000010407 14331 14336 Mig12 +T436 GO:0010467 14337 14347 expression +T437 UBERON:0000922 14364 14373 embryonic +T438 GO:0009790 14364 14385 embryonic development +T439 GO:0097617 14411 14424 hybridization +T440 NCBITaxon:10088 14434 14439 mouse +T441 UBERON:0000922 14440 14446 embryo +T442 GO:0010467 14455 14465 expression +T443 UBERON:0001017 14473 14495 central nervous system +T444 UBERON:0002101 14518 14523 limbs +T445 NCBITaxon:10088 14588 14593 mouse +T446 UBERON:0000922 14594 14601 embryos +T447 NCBITaxon:10088 14706 14711 mouse +T448 UBERON:0000922 14712 14719 embryos +T449 PR:000010407 14728 14733 Mig12 +T450 GO:0010467 14734 14744 expression +T451 UBERON:0000988 14786 14793 pontine +T452 UBERON:0001896 14808 14825 medulla oblongata +T453 UBERON:0034705 14830 14844 neuroepithelia +T454 UBERON:0002291 14903 14928 spinal cord central canal +T455 GO:0010467 14934 14944 Expression +T456 UBERON:0000044 14965 14984 dorsal root ganglia +T457 PR:000010407 14990 14995 Mig12 +T458 SO:0000673 14996 15006 transcript +T459 UBERON:0001893 15026 15039 telencephalon +T460 UBERON:0003053 15060 15076 ventricular zone +T461 UBERON:0000062 15114 15120 organs +T462 UBERON:0002222 15129 15142 perichondrium +T463 UBERON:0002544 15150 15156 digits +T464 UBERON:0002046 15169 15176;15204 15210 thyroid ... glands +T465 UBERON:0002046 15178 15180 th +T466 UBERON:0001132 15186 15197;15204 15210 parathyroid ... glands +T467 UBERON:0001132 15199 15202 pth +T468 UBERON:0000989 15227 15234 phallic +T469 UBERON:0000164 15247 15263 urogenital sinus +T470 UBERON:0002037 15284 15286 CB +T471 UBERON:0002037 15288 15298 cerebellum +T472 UBERON:0002291 15305 15318 central canal +T473 UBERON:0034705 15319 15334 neuroepithelium +T474 UBERON:0000044 15336 15339 drg +T475 UBERON:0000044 15341 15360 dorsal root ganglia +T476 UBERON:0034705 15388 15403 neuroepithelium +T477 UBERON:0001896 15405 15406 M +T478 UBERON:0001896 15408 15425 medulla oblongata +T479 UBERON:0001896 15431 15448 medulla oblongata +T480 UBERON:0034705 15449 15464 neuroepithelium +T481 UBERON:0000988 15466 15467 P +T482 UBERON:0000988 15469 15473 pons +T483 UBERON:0002222 15475 15477 pc +T484 UBERON:0002222 15479 15492 perichondrium +T485 UBERON:0000988 15499 15506 pontine +T486 UBERON:0034705 15507 15522 neuroepithelium +T487 UBERON:0001132 15524 15527;15541 15547 pth ... glands +T488 UBERON:0001132 15529 15547 parathyroid glands +T489 UBERON:0002240 15549 15551 SC +T490 UBERON:0002240 15553 15564 spinal cord +T491 UBERON:0001893 15566 15567 T +T492 UBERON:0001893 15569 15582 telencephalon +T493 UBERON:0002046 15584 15586;15596 15601 th ... gland +T494 UBERON:0002046 15588 15601 thyroid gland +T495 UBERON:0000164 15603 15605 us +T496 UBERON:0000164 15607 15623 urogenital sinus +T497 UBERON:0003053 15625 15627 vz +T498 UBERON:0003053 15629 15645 ventricular zone +T499 PR:000010406 15648 15652 Mid1 +T500 PR:000010407 15662 15667 Mig12 +T501 GO:0005874 15675 15687 microtubules +T502 GO:0010467 15699 15709 expression +T503 PR:000010407 15739 15744 Mig12 +T504 GO:0009294 15901 15912 transfected +T505 PR:000010407 15937 15942 Mig12 +T506 PR:000010407 15962 15967 Mig12 +T507 GO:0042571 15968 15976 antibody +T508 PR:000010407 16043 16048 Mig12 +T509 GO:0005634 16072 16079 nucleus +T510 GO:0005737 16088 16097 cytoplasm +T511 PR:000010406 16237 16241 Mid1 +T512 PR:000010407 16246 16251 Mig12 +T513 GO:0010467 16317 16327 expression +T514 PR:000010407 16336 16341 Mig12 +T515 PR:000010407 16360 16365 Mig12 +T516 PR:000010407 16394 16399 Mig12 +T517 GO:0005634 16490 16497 nucleus +T518 GO:0005737 16506 16515 cytoplasm +T519 GO:0010467 16524 16534 expression +T520 PR:000010406 16543 16547 Mid1 +T521 PR:000010407 16552 16557 Mig12 +T522 GO:0005737 16606 16617 cytoplasmic +T523 PR:000010406 16695 16699 Mid1 +T524 PR:000010407 16718 16723 Mig12 +T525 GO:0010467 16741 16751 expressing +T526 GO:0009294 16788 16799 transfected +T527 PR:000010406 16811 16815 Mid1 +T528 GO:0051325 16862 16872 interphase +T529 GO:0005874 16873 16885 microtubules +T530 PR:000010407 17016 17021 Mig12 +T531 PR:000010407 17165 17170 Mig12 +T532 PR:000010406 17220 17224 Mid1 +T533 http://purl.obolibrary.org/obo/MONDO_0017138 17225 17227 OS +T534 PR:000010406 17250 17254 Mid1 +T535 PR:000010406 17270 17274 Mid1 +T536 SO:0001080 17307 17318 coiled-coil +T537 SO:0000417 17319 17325 domain +T538 GO:0005737 17345 17356 cytoplasmic +T539 PR:000026474 17404 17410 TRIM19 +T540 PR:000026474 17411 17414 PML +T541 GO:0010467 17429 17438 expressed +T542 PR:000010407 17448 17453 Mig12 +T543 PR:000010406 17502 17506 Mid1 +T544 GO:0005874 17526 17538 microtubules +T545 GO:0007049 17557 17567 cell cycle +T546 PR:000010406 17658 17662 Mid1 +T547 GO:0000235 17692 17698;17710 17722 radial ... microtubules +T548 GO:0051325 17699 17709 interphase +T549 GO:0010467 17747 17756 expressed +T550 PR:000010406 17775 17779 Mid1 +T551 PR:000010407 17784 17789 Mig12 +T552 GO:0005737 17814 17823 cytoplasm +T553 PR:000010407 17835 17840 Mig12 +T554 GO:0010467 17916 17926 expression +T555 GO:0010467 18038 18048 expression +T556 GO:0010467 18151 18160 expressed +T557 GO:0009294 18245 18256 transfected +T558 PR:000010406 18267 18271 Mid1 +T559 PR:000010406 18313 18317 Mid1 +T560 PR:000010407 18322 18327 Mig12 +T561 PR:000010407 18446 18451 Mig12 +T562 GO:0009294 18464 18475 transfected +T563 PR:000010406 18488 18492 Mid1 +T564 GO:0005874 18531 18543 microtubules +T565 PR:000010406 18545 18549 Mid1 +T566 http://purl.obolibrary.org/obo/MONDO_0017138 18561 18563 OS +T567 GO:0005737 18584 18595 cytoplasmic +T568 SO:0001080 18650 18661 coiled-coil +T569 PR:000010407 18690 18695 Mig12 +T570 GO:0010467 18800 18810 expression +T571 SO:0001080 18823 18834 coiled-coil +T572 SO:0000417 18835 18841 domain +T573 PR:000010406 18845 18849 Mid1 +T574 SO:0000417 18955 18961 domain +T575 PR:000010406 18977 18981 Mid1 +T576 GO:0010467 18987 18996 expressed +T577 PR:000010407 19002 19007 Mig12 +T578 GO:0009294 19036 19049 transfections +T579 PR:000010407 19053 19058 Mig12 +T580 PR:000026474 19064 19070 TRIM19 +T581 PR:000026474 19071 19074 PML +T582 PR:000016660 19100 19105 TRIM5 +T583 PR:000016651 19109 19115 TRIM27 +T584 PR:000010406 19161 19165 Mid1 +T585 SO:0001080 19179 19190 coiled-coil +T586 SO:0000417 19191 19197 domain +T587 PR:000010407 19231 19236 Mig12 +T588 PR:000010406 19285 19289 Mid1 +T589 GO:0005874 19295 19307 microtubular +T590 GO:0010467 19367 19377 expressing +T591 PR:000010407 19378 19383 Mig12 +T592 PR:000010406 19388 19392 Mid1 +T593 GO:0005874 19411 19423 microtubular +T594 PR:000028799 19451 19458 tubulin +T595 GO:0005874 19551 19562 microtubule +T596 GO:0010467 19588 19598 expression +T597 GO:0005634 19678 19685 nuclear +T598 PR:000010406 19714 19718 Mid1 +T599 PR:000010407 19723 19728 Mig12 +T600 GO:0005874 19746 19758 microtubules +T601 GO:0009294 19809 19820 transfected +T602 PR:000010406 19829 19833 Mid1 +T603 GO:0005874 19919 19931 microtubules +T604 GO:0042571 19961 19971 antibodies +T605 PR:000028799 19982 19989 tubulin +T606 GO:0009294 20100 20111 transfected +T607 PR:000010407 20139 20144 Mig12 +T608 PR:000010407 20164 20169 Mig12 +T609 CHEBI:45863 20243 20248 taxol +T610 MOP:0000629 20262 20273 polymerized +T611 GO:0005874 20274 20286 microtubules +T612 CHEBI:17992 20311 20318 sucrose +T613 PR:000010406 20402 20406 Mid1 +T614 PR:000010407 20408 20413 Mig12 +T615 PR:000028799 20419 20426 tubulin +T616 GO:0042571 20445 20455 antibodies +T617 GO:0009294 20467 20479 transfection +T618 PR:000010406 20498 20502 Mid1 +T619 PR:000010407 20507 20512 Mig12 +T620 MOP:0000629 20559 20570 polymerized +T621 GO:0005874 20571 20583 microtubules +T622 PR:000010407 20597 20602 Mig12 +T623 PR:000010406 20657 20661 Mid1 +T624 PR:000028799 20670 20677 tubulin +T625 PR:000010407 20689 20694 Mig12 +T626 MOP:0000629 20734 20745 polymerized +T627 PR:000028799 20746 20753 tubulin +T628 PR:000010407 20785 20790 Mig12 +T629 GO:0009294 20791 20802 transfected +T630 PR:000010407 20865 20870 Mig12 +T631 GO:0042571 20871 20879 antibody +T632 GO:0042571 21019 21027 antibody +T633 NCBITaxon:9986 21109 21116 rabbits +T634 PR:000010407 21122 21127 Mig12 +T635 PR:000010407 21198 21203 Mig12 +T636 MOP:0000629 21211 21222 polymerized +T637 GO:0005874 21223 21234 microtubule +T638 CHEBI:45863 21247 21252 taxol +T639 CHEBI:45863 21311 21316 taxol +T640 PR:000010407 21348 21353 Mig12 +T641 GO:0009294 21354 21365 transfected +T642 GO:0005874 21408 21420 microtubules +T643 GO:0005815 21442 21446 MTOC +T644 GO:0007067 21480 21487 mitotic +T645 GO:0097431 21480 21501 mitotic spindle poles +T646 GO:0005874 21555 21566 microtubule +T647 CHEBI:45863 21587 21592 taxol +T648 GO:0009294 21615 21626 transfected +T649 PR:000010406 21637 21641 Mid1 +T650 PR:000010407 21646 21651 Mig12 +T651 CHEBI:17992 21678 21685 sucrose +T652 MOP:0000629 21741 21752 polymerized +T653 PR:000028799 21753 21760 tubulin +T654 PR:000010407 21823 21828 Mig12 +T655 PR:000010406 21833 21837 Mid1 +T656 PR:000028799 21873 21880 tubulin +T657 PR:000010407 21896 21901 Mig12 +T658 GO:0005874 22049 22061 microtubular +T659 GO:0005874 22143 22155 microtubules +T660 PR:000015477 22157 22164 spastin +T661 GO:0005874 22197 22208 microtubule +T662 PR:000010407 22251 22256 Mig12 +T663 PR:000010407 22376 22381 Mig12 +T664 PR:000028799 22416 22423 tubulin +T665 GO:0005874 22473 22484 microtubule +T666 GO:0007026 22473 22498 microtubule stabilization +T667 CHEBI:61950 22473 22504 microtubule stabilization agent +T668 CHEBI:45863 22506 22511 taxol +T669 GO:0010467 22545 22554 expressed +T670 PR:000010406 22556 22560 Mid1 +T671 PR:000010407 22565 22570 Mig12 +T672 GO:0051325 22601 22611 interphase +T673 GO:0000235 22612 22631 radial microtubules +T674 GO:0009294 22678 22689 transfected +T675 PR:000010407 22690 22695 Mig12 +T676 GO:0005874 22730 22742 microtubular +T677 PR:000010407 22868 22873 Mig12 +T678 GO:0042571 22874 22882 antibody +T679 MOP:0000629 23054 23065 polymerized +T680 GO:0005874 23066 23078 microtubules +T681 PR:000010407 23128 23133 Mig12 +T682 GO:0005874 23141 23152 microtubule +T683 PR:000010406 23240 23244 Mid1 +T684 GO:0005874 23292 23304 microtubules +T685 GO:0009294 23345 23356 transfected +T686 PR:000010407 23407 23412 Mig12 +T687 GO:0005874 23422 23434 microtubules +T688 PR:000010406 23469 23473 Mid1 +T689 PR:000010407 23571 23576 Mig12 +T690 GO:0005815 23592 23596 MTOC +T691 GO:0007067 23683 23690 mitotic +T692 GO:0072686 23683 23698 mitotic spindle +T693 PR:000010406 23725 23729 Mid1 +T694 PR:000010407 23734 23739 Mig12 +T695 GO:0097427 23754 23773 microtubule bundles +T696 GO:0005874 23816 23827 microtubule +T697 PR:000010406 23865 23869 Mid1 +T698 PR:000010407 23870 23875 Mig12 +T699 GO:0005874 23907 23919 microtubular +T700 GO:0009294 23948 23959 transfected +T701 CHEBI:34892 23975 23985 nocodazole +T702 GO:0005874 23989 24000 microtubule +T703 GO:0007019 23989 24015 microtubule-depolymerizing +T704 MOP:0000630 24001 24015 depolymerizing +T705 GO:0010467 24124 24134 expression +T706 CHEBI:23888 24182 24186 drug +T707 GO:0005874 24207 24219 microtubules +T708 GO:0010467 24254 24264 expressing +T709 PR:000010406 24270 24274 Mid1 +T710 GO:0005874 24307 24319 microtubular +T711 PR:000010406 24423 24427 Mid1 +T712 PR:000010407 24428 24433 Mig12 +T713 CHEBI:34892 24485 24495 nocodazole +T714 PR:000010406 24530 24534 Mid1 +T715 PR:000010407 24539 24544 Mig12 +T716 GO:0005874 24568 24580 microtubules +T717 CHEBI:34892 24586 24596 Nocodazole +T718 PR:000010406 24628 24632 Mid1 +T719 PR:000010407 24633 24638 Mig12 +T720 PR:000028799 24660 24667 tubulin +T721 GO:0005874 24693 24705 microtubules +T722 PR:000010406 24709 24713 Mid1 +T723 GO:0009294 24721 24732 transfected +T724 GO:0005874 24781 24793 microtubules +T725 MOP:0000030 24847 24857 acetylated +T726 PR:000028799 24858 24865 tubulin +T727 GO:0042571 24866 24874 antibody +T728 PR:000028799 24907 24914 tubulin +T729 MOP:0000030 24927 24938 acetylation +T730 GO:0005874 24951 24963 microtubules +T731 GO:0042571 25030 25040 antibodies +T732 MOP:0000030 25044 25054 acetylated +T733 PR:000028799 25055 25062 tubulin +T734 PR:000010406 25076 25080 Mid1 +T735 PR:000010407 25081 25086 Mig12 +T736 CHEBI:34892 25095 25105 nocodazole +T737 GO:0005874 25148 25160 microtubules +T738 GO:0005874 25201 25213 microtubules +T739 GO:0010467 25251 25261 expressing +T740 PR:000010407 25262 25267 Mig12 +T741 CHEBI:34892 25299 25309 nocodazole +T742 GO:0005874 25339 25351 microtubular +T743 PR:000010407 25421 25426 Mig12 +T744 PR:000010406 25444 25448 Mid1 +T745 GO:0005874 25462 25474 microtubules +T746 PR:000010406 25480 25484 Mid1 +T747 PR:000010407 25485 25490 Mig12 +T748 GO:0005874 25491 25502 microtubule +T749 GO:0007026 25491 25514 microtubule-stabilizing +T750 UBERON:0009571 25594 25601 midline +T751 UBERON:0000467 25602 25609 systems +T752 http://purl.obolibrary.org/obo/MONDO_0017138 25631 25645 Opitz syndrome +T753 http://purl.obolibrary.org/obo/MONDO_0017138 25685 25699 Opitz syndrome +T754 SO:0000704 25700 25704 gene +T755 PR:000010406 25714 25718 Mid1 +T756 NCBITaxon:9606 25748 25753 human +T757 http://purl.obolibrary.org/obo/MONDO_0000001 25754 25762 disorder +T758 PR:000010406 25832 25836 Mid1 +T759 GO:0070507 25844 25878 regulation of microtubule dynamics +T760 GO:0005874 25858 25869 microtubule +T761 SO:0000704 25920 25924 gene +T762 PR:000010407 25926 25931 MIG12 +T763 PR:000010406 25948 25952 Mid1 +T764 PR:000010407 25974 25979 MIG12 +T765 SO:0000857 26001 26009 homology +T766 NCBITaxon:7955 26017 26026 zebrafish +T767 SO:0000704 26027 26031 gene +T768 GO:0007369 26046 26058 gastrulation +T769 PR:P47805 26046 26070 gastrulation protein G12 +T770 GO:0010467 26082 26091 expressed +T771 NCBITaxon:7955 26126 26134 D. rerio +T772 GO:0007369 26135 26147 gastrulation +T773 PR:000010407 26156 26161 Mig12 +T774 SO:0000854 26162 26169 paralog +T775 NCBITaxon:40674 26173 26180 mammals +T776 PR:000016322 26182 26188 SPOT14 +T777 GO:0005634 26195 26202 nuclear +T778 UBERON:0002046 26232 26239 thyroid +T779 CHEBI:60311 26232 26247 thyroid hormone +T780 GO:0065007 26252 26261 regulates +T781 CHEBI:18059 26262 26267 lipid +T782 GO:0008610 26262 26277 lipid synthesis +T783 PR:P47805 26329 26332 G12 +T784 PR:000016322 26337 26343 SPOT14 +T785 SO:0000417 26399 26406 domains +T786 PR:000010407 26473 26478 MIG12 +T787 GO:0010467 26506 26516 expression +T788 PR:000010407 26528 26533 Mig12 +T789 UBERON:0000922 26541 26550 embryonic +T790 GO:0009790 26541 26562 embryonic development +T791 PR:000010406 26590 26594 Mid1 +T792 http://purl.obolibrary.org/obo/MONDO_0017138 26686 26688 OS +T793 GO:0010467 26710 26720 expression +T794 UBERON:0009571 26728 26735 midline +T795 UBERON:0001017 26754 26776 central nervous system +T796 UBERON:0001016 26801 26813 neurological +T797 http://purl.obolibrary.org/obo/MONDO_0009022 26869 26877;26892 26894;26899 26914 agenesis of corpus callosum +T798 UBERON:0002336 26899 26914 corpus callosum +T799 UBERON:0004720 26926 26943 cerebellar vermis +T800 GO:0010467 26979 26989 expression +T801 PR:000010407 26993 26998 Mig12 +T802 UBERON:0001017 27021 27024 CNS +T803 GO:0010467 27133 27142 expressed +T804 UBERON:0001017 27150 27153 CNS +T805 UBERON:0009571 27154 27161 midline +T806 UBERON:0000970 27199 27202 eye +T807 UBERON:0000033 27263 27267 head +T808 UBERON:0009571 27268 27275 midline +T809 UBERON:0001456 27290 27294 face +T810 PR:000014841 27340 27354 Sonic hedgehog +T811 PR:000014841 27356 27359 Shh +T812 UBERON:0009571 27390 27405 ventral midline +T813 UBERON:0001049 27406 27417 neural tube +T814 GO:0065007 27433 27442 regulates +T815 GO:0009653 27447 27460 morphogenesis +T816 UBERON:0009571 27477 27484 midline +T817 UBERON:0000062 27497 27503 organs +T818 PR:000010406 27561 27565 Mid1 +T819 SO:0000704 27566 27570 gene +T820 PR:000014841 27579 27582 Shh +T821 UBERON:0009571 27604 27611 midline +T822 NCBITaxon:9031 27648 27655 chicken +T823 PR:000010407 27692 27697 Mig12 +T824 PR:000010406 27698 27702 Mid1 +T825 http://purl.obolibrary.org/obo/MONDO_0017138 27773 27775 OS +T826 GO:0010467 27777 27787 Expression +T827 UBERON:0000922 27795 27804 embryonic +T828 UBERON:0004122 27805 27815;27825 27834 urogenital ... apparatus +T829 UBERON:0001245 27820 27824 anal +T830 http://purl.obolibrary.org/obo/MONDO_0017138 27878 27880 OS +T831 http://purl.obolibrary.org/obo/MONDO_0005345 27882 27893 hypospadias +T832 http://purl.obolibrary.org/obo/MONDO_0001046 27898 27909;27921 27925 imperforate anus +T833 UBERON:0001245 27921 27925 anus +T834 UBERON:0002544 27966 27971 digit +T835 PR:000010407 27972 27977 Mig12 +T836 GO:0010467 27978 27988 expression +T837 NCBITaxon:10088 28005 28010 mouse +T838 UBERON:0000922 28011 28018 embryos +T839 http://purl.obolibrary.org/obo/MONDO_0017138 28024 28026 OS +T840 http://purl.obolibrary.org/obo/MONDO_0021002 28058 28068 syndactyly +T841 PR:000010406 28074 28078 MID1 +T842 PR:000010406 28135 28139 MID1 +T843 http://purl.obolibrary.org/obo/MONDO_0017138 28185 28187 OS +T844 SO:0000704 28230 28235 genes +T845 http://purl.obolibrary.org/obo/MONDO_0017138 28243 28245 OS +T846 PR:000010406 28309 28313 Mid1 +T847 http://purl.obolibrary.org/obo/MONDO_0017138 28361 28363 OS +T848 http://purl.obolibrary.org/obo/MONDO_0002254 28377 28386 syndromes +T849 http://purl.obolibrary.org/obo/MONDO_0017138 28417 28419 OS +T850 PR:000010407 28425 28430 Mig12 +T851 PR:000010407 28464 28469 Mig12 +T852 GO:0010467 28478 28487 expressed +T853 GO:0005874 28509 28521 microtubules +T854 GO:0005737 28597 28606 cytoplasm +T855 PR:000010407 28632 28637 Mig12 +T856 MOP:0000629 28677 28688 polymerized +T857 PR:000028799 28689 28696 tubulin +T858 GO:0010467 28746 28755 expressed +T859 PR:000010406 28761 28765 Mid1 +T860 GO:0001578 28781 28813 formation of microtubule bundles +T861 GO:0097427 28794 28813 microtubule bundles +T862 PR:000010406 28848 28852 Mid1 +T863 GO:0010467 28856 28865 expressed +T864 PR:000010406 28873 28877 Mid1 +T865 PR:000010407 28900 28905 Mig12 +T866 GO:0005874 28913 28925 microtubules +T867 PR:000010406 29021 29025 Mid1 +T868 PR:000010407 29035 29040 Mig12 +T869 GO:0032991 29107 29116 multimers +T870 GO:0005874 29147 29158 microtubule +T871 CHEBI:24433 29171 29179 moieties +T872 GO:0005874 29241 29252 microtubule +T873 GO:0097427 29293 29312 microtubule bundles +T874 GO:0005634 29346 29353 nuclear +T875 GO:0005938 29365 29373 cortical +T876 GO:0005815 29409 29413 MTOC +T877 GO:0005874 29483 29495 microtubular +T878 GO:0005874 29586 29597 microtubule +T879 GO:0051013 29586 29606 microtubule severing +T880 GO:0097427 29628 29647 microtubule bundles +T881 MOP:0000630 29665 29679 depolymerizing +T882 CHEBI:34892 29696 29706 nocodazole +T883 MOP:0000030 29728 29738 acetylated +T884 PR:000028799 29739 29746 tubulin +T885 GO:0005874 29778 29790 microtubules +T886 GO:0005874 29857 29868 microtubule +T887 GO:0005874 29901 29912 microtubule +T888 GO:0007067 29971 29978 mitotic +T889 GO:0072686 29971 29986 mitotic spindle +T890 GO:0000910 30001 30012 cytokinesis +T891 GO:2000145 30021 30045 control of cell motility +T892 PR:000013176 30055 30059 PRC1 +T893 PR:000011503 30061 30065 NuMA +T894 SO:0000857 30154 30162 homology +T895 PR:000010406 30191 30195 Mid1 +T896 PR:000007680 30197 30201 Mir1 +T897 PR:000007680 30206 30211 GLFND +T898 SO:0001080 30224 30235 coiled-coil +T899 PR:000016651 30242 30245 RFP +T900 GO:0005874 30302 30314 microtubules +T901 PR:000010406 30374 30378 Mid1 +T902 PR:000010407 30379 30384 Mig12 +T903 GO:0032991 30385 30394 complexes +T904 GO:0007067 30402 30409 mitosis +T905 PR:000010406 30411 30415 Mid1 +T906 GO:0007067 30430 30437 mitotic +T907 GO:0072686 30430 30445 mitotic spindle +T908 PR:000010407 30455 30460 Mig12 +T909 GO:0009294 30467 30478 transfected +T910 GO:0000922 30525 30538 spindle poles +T911 GO:0007067 30592 30599 mitotic +T912 GO:0010467 30610 30620 expressing +T913 GO:0010467 30766 30776 expression +T914 GO:0065007 30828 30837 regulated +T915 GO:0006913 30879 30888;30898 30927 shuttling ... between nucleus and cytoplasm +T916 PR:000010407 30892 30897 Mig12 +T917 GO:0005634 30906 30913 nucleus +T918 GO:0005737 30918 30927 cytoplasm +T919 GO:0065007 30954 30963 regulated +T920 GO:0005634 31011 31018 nucleus +T921 GO:0051325 31071 31081 interphase +T922 GO:0005874 31082 31093 microtubule +T923 PR:000010406 31103 31107 Mid1 +T924 PR:000010407 31122 31127 Mig12 +T925 GO:0005874 31131 31143 microtubules +T926 PR:000010406 31201 31205 Mid1 +T927 GO:0006412 31235 31248 translational +T928 PR:000010407 31266 31271 Mig12 +T929 GO:0065007 31278 31286 regulate +T930 GO:0005874 31359 31370 microtubule +T931 GO:0070507 31404 31429;31444 31452 regulation of microtubule ... dynamics +T932 GO:0005874 31418 31429 microtubule +T933 PR:000010406 31467 31471 Mid1 +T934 PR:000010407 31472 31477 Mig12 +T935 GO:0007049 31536 31546 cell cycle +T936 GO:0016477 31562 31576 cell migration +T937 GO:0005874 31602 31613 microtubule +T938 GO:0007026 31602 31627 microtubule stabilization +T939 http://purl.obolibrary.org/obo/MONDO_0017138 31719 31721 OS +T940 PR:000010407 31723 31728 Mig12 +T941 PR:000010406 31741 31745 Mid1 +T942 GO:0010467 31776 31785 expressed +T943 GO:0008283 31796 31809 proliferating +T944 UBERON:0000922 31810 31819 embryonic +T945 UBERON:0003053 31838 31857;31873 31878 ventricular zone of ... brain +T946 GO:0007067 31928 31935 mitosis +T947 NCBITaxon:7955 31986 31995 zebrafish +T948 GO:0007369 31996 32008 gastrulation +T949 PR:P47805 31996 32020 gastrulation protein G12 +T950 GO:0010467 32024 32033 expressed +T951 GO:0016477 32085 32099 cell migration +T952 http://purl.obolibrary.org/obo/MONDO_0017138 32207 32221 Opitz syndrome +T953 http://purl.obolibrary.org/obo/MONDO_0017138 32284 32298 Opitz syndrome +T954 SO:0000704 32299 32303 gene +T955 PR:000010407 32333 32338 Mig12 +T956 PR:000010406 32362 32366 Mid1 +T957 GO:0005874 32380 32392 microtubules +T958 PR:000010406 32437 32441 Mid1 +T959 GO:0005874 32445 32456 microtubule +T960 GO:0000226 32445 32465 microtubule dynamics +T961 PR:000010406 32467 32471 Mid1 +T962 GO:0065007 32482 32490 controls +T963 GO:0065007 32523 32533 regulation +T964 GO:0000159 32537 32541 PP2A +T965 GO:0005874 32542 32554 microtubular +T966 PR:000010407 32571 32576 Mig12 +T967 UBERON:0000922 32617 32626 embryonic +T968 GO:0009790 32617 32638 embryonic development +T969 UBERON:0009571 32642 32649 midline +T970 PR:000010406 32676 32680 Mid1 +T971 PR:000010407 32681 32686 Mig12 +T972 GO:0005874 32696 32707 microtubule +T973 GO:0070507 32696 32727 microtubule dynamics regulation +T974 http://purl.obolibrary.org/obo/MONDO_0017138 32761 32775 Opitz syndrome +T975 SO:0000155 32787 32794 Plasmid +T976 PR:000010406 32811 32815 MID1 +T977 GO:0010467 32816 32826 expression +T978 SO:0000440 32827 32834 vectors +T979 PR:000010406 32842 32846 MID1 +T980 PR:000010406 32854 32858 MID1 +T981 PR:000010406 32896 32900 MID1 +T982 SO:0000440 32985 32992 vectors +T983 SO:0000440 33038 33045 vectors +T984 PR:000010407 33082 33087 MIG12 +T985 SO:0000112 33144 33151 primers +T986 SO:0000345 33164 33168 ESTs +T987 SO:0000006 33219 33230 PCR product +T988 NCBITaxon:2759 33280 33290 eukaryotic +T989 GO:0010467 33291 33301 expression +T990 SO:0000440 33302 33309 vectors +T991 PR:000000020 33350 33353 Myc +T992 SO:0000851 33399 33408;33415 33428 region of ... coding region +T993 PR:000010407 33409 33414 MIG12 +T994 PR:000010407 33442 33447 MIG12 +T995 SO:0000440 33489 33495 vector +T996 SO:0000417 33524 33530 domain +T997 PR:000016651 33626 33629 RFP +T998 SO:0000440 33653 33659 vector +T999 PR:000023087 33678 33682 LexA +T1000 SO:0000417 33695 33701 domain +T1001 GO:0009294 33716 33727 transformed +T1002 GO:0009294 33778 33789 transformed +T1003 SO:0000417 33868 33874 domain +T1004 CHEBI:75055 33959 33964 X-gal +T1005 PR:000023087 34035 34039 LexA +T1006 SO:0000704 34056 34061 genes +T1007 PR:000033987 34063 34067 lacZ +T1008 GO:0007618 34091 34097 mating +T1009 GO:0007618 34190 34196 mating +T1010 GO:0009294 34270 34282 transfection +T1011 UBERON:0002113 34291 34297 Kidney +T1012 CL:1000497 34291 34297;34304 34309 Kidney ... cells +T1013 NCBITaxon:27592 34410 34416 bovine +T1014 UBERON:0001977 34417 34422 serum +T1015 CHEBI:16526 34440 34443 CO2 +T1016 GO:0009294 34460 34473 transfections +T1017 CHEBI:77635 34494 34511 calcium phosphate +T1018 GO:0009294 34545 34557 transfection +T1019 GO:0010467 34578 34588 expression +T1020 SO:0000440 34589 34595 vector +T1021 GO:0009294 34717 34728 transfected +T1022 GO:0042571 34768 34778 Antibodies +T1023 GO:0009294 34882 34894 transfection +T1024 CHEBI:26710 34963 34967 NaCl +T1025 CHEBI:8984 34995 34998 SDS +T1026 CHEBI:9754 35006 35010 Tris +T1027 CHEBI:17883 35011 35014 HCl +T1028 GO:0042571 35229 35237 antibody +T1029 PR:000000020 35251 35254 Myc +T1030 PR:000010406 35297 35301 Mid1 +T1031 GO:0042571 35313 35321 antibody +T1032 GO:0032991 35364 35373 complexes +T1033 PR:000029158 35389 35398 protein A +T1034 CHEBI:8984 35525 35528 SDS +T1035 CHEBI:8984 35590 35593 SDS +T1036 CHEBI:53250 35616 35620 PVDF +T1037 CHEBI:17790 35672 35680 methanol +T1038 CHEBI:9754 35708 35712 Tris +T1039 CHEBI:17883 35713 35716 HCl +T1040 CHEBI:26710 35729 35733 NaCl +T1041 CHEBI:53424 35743 35751 Tween-20 +T1042 GO:0042571 35795 35805 antibodies +T1043 PR:000000084 35831 35836 c-Myc +T1044 GO:0042571 35848 35856 antibody +T1045 GO:0042571 35892 35900 antibody +T1046 PR:000010406 35935 35939 Mid1 +T1047 GO:0042571 35951 35959 antibody +T1048 GO:0042571 35999 36007 Antibody +T1049 NCBITaxon:10088 36051 36056 mouse +T1050 NCBITaxon:9986 36065 36071 rabbit +T1051 GO:0071736 36072 36075 IgG +T1052 MOP:0000779 36076 36083 coupled +T1053 NCBITaxon:3704 36089 36100 horseradish +T1054 PR:000010407 36207 36212 Mig12 +T1055 UBERON:0001977 36217 36222 serum +T1056 PR:000010407 36261 36266 Mig12 +T1057 NCBITaxon:2 36304 36312 bacteria +T1058 GO:0042571 36343 36351 antibody +T1059 PR:000010407 36379 36384 Mig12 +T1060 MOP:0000779 36385 36404 covalently attached +T1061 PR:000010406 36668 36672 Mid1 +T1062 SO:0000417 36678 36684 domain +T1063 GO:0040007 36733 36738 grown +T1064 GO:0009294 36795 36806 transfected +T1065 CHEBI:9750 36933 36945 Triton X-100 +T1066 UBERON:0001977 36982 36987 serum +T1067 GO:0042571 37035 37045 antibodies +T1068 GO:0042571 37085 37095 antibodies +T1069 GO:0042571 37119 37129 antibodies +T1070 PR:000029158 37141 37150 protein A +T1071 PR:000010406 37176 37180 Mid1 +T1072 PR:000028799 37217 37224 tubulin +T1073 GO:0042571 37288 37296 antibody +T1074 MOP:0000030 37339 37349 acetylated +T1075 PR:000028799 37350 37357 tubulin +T1076 GO:0042571 37408 37418 antibodies +T1077 CHEBI:37926 37430 37456 fluorescein isothiocyanate +T1078 CHEBI:37926 37458 37462 FITC +T1079 MOP:0000779 37464 37474 conjugated +T1080 NCBITaxon:9986 37480 37486 rabbit +T1081 GO:0042571 37487 37497 antibodies +T1082 MOP:0000779 37556 37566 conjugated +T1083 NCBITaxon:9986 37572 37578 rabbit +T1084 CHEBI:37926 37583 37587 FITC +T1085 MOP:0000779 37588 37598 conjugated +T1086 NCBITaxon:10088 37604 37609 mouse +T1087 GO:0042571 37610 37620 antibodies +T1088 CHEBI:37987 37671 37674 Cy3 +T1089 MOP:0000779 37675 37685 conjugated +T1090 NCBITaxon:10088 37691 37696 mouse +T1091 GO:0042571 37697 37705 antibody +T1092 CHEBI:34892 37760 37770 nocodazole +T1093 CHEBI:28262 37774 37778 DMSO +T1094 GO:0005874 37859 37870 Microtubule +T1095 GO:0009294 37928 37940 transfection +T1096 GO:0009294 37985 37996 transfected +T1097 GO:0019835 38013 38018 lysed +T1098 CHEBI:39033 38045 38050 PIPES +T1099 CHEBI:6636 38075 38080 MgCl2 +T1100 CHEBI:18320 38089 38092 DTT +T1101 CHEBI:26710 38101 38105 NaCl +T1102 GO:0005829 38229 38236 Cytosol +T1103 CHEBI:45863 38437 38442 taxol +T1104 CHEBI:45863 38526 38531 taxol +T1105 CHEBI:17992 38587 38594 sucrose +T1106 MOP:0000629 38662 38673 polymerized +T1107 GO:0005874 38674 38686 microtubules +T1108 GO:0097617 38851 38864 hybridization +T1109 SO:0000028 38926 38928 bp +T1110 PR:000010407 38976 38981 MIG12 +T1111 SO:0000077 39083 39092 antisense +T1112 CHEBI:37983 39093 39096 35S +T1113 NCBITaxon:10088 39116 39121 Mouse +T1114 UBERON:0005291 39122 39135 embryo tissue +T1115 GO:0097617 39175 39188 hybridization +T1116 GO:0097617 39395 39408 hybridization +T1117 GO:0005634 39435 39441 nuclei +T1118 CHEBI:37958 39469 39472 dye +T1119 GO:0097617 39494 39507 hybridization +T1120 GO:0097617 39671 39684 hybridization +T1121 PR:000010407 39772 39777 Mig12 +T1122 GO:0042571 39787 39795 antibody +T1123 GO:0005874 39814 39825 microtubule +T1124 SO:0000440 39914 39921 vectors +T1125 GO:0007567 40455 40460 Birth +T1126 http://purl.obolibrary.org/obo/MONDO_0000839 40455 40468 Birth Defects diff --git a/src/ontogpt/evaluation/craft/database/all/15070402.txt b/src/ontogpt/evaluation/craft/database/all/15070402.txt new file mode 100644 index 000000000..99aaeee53 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15070402.txt @@ -0,0 +1,131 @@ +Mig12, a novel Opitz syndrome gene product partner, is expressed in the embryonic ventral midline and co-operates with Mid1 to bundle and stabilize microtubules + +Abstract + +Background + +Opitz G/BBB syndrome is a genetic disorder characterized by developmental midline abnormalities, such as hypertelorism, cleft palate, and hypospadias. The gene responsible for the X-linked form of this disease, MID1, encodes a TRIM/RBCC protein that is anchored to the microtubules. The association of Mid1 with the cytoskeleton is regulated by dynamic phosphorylation, through the interaction with the α4 subunit of phosphatase 2A (PP2A). Mid1 acts as an E3 ubiquitin ligase, regulating PP2A degradation on microtubules. + +Results + +In spite of these findings, the biological role exerted by the Opitz syndrome gene product is still unclear and the presence of other potential interacting moieties in the Mid1 structure prompted us to search for additional cellular partners. Through a yeast two-hybrid screening approach, we identified a novel gene, MIG12, whose protein product interacts with Mid1. We confirmed by immunoprecipitation that this interaction occurs in vivo and that it is mediated by the Mid1 coiled-coil domain. We found that Mig12 is mainly expressed in the neuroepithelial midline, urogenital apparatus, and digits during embryonic development. Transiently expressed Mig12 is found diffusely in both nucleus and cytoplasm, although it is enriched in the microtubule-organizing center region. Consistently with this, endogenous Mig12 protein is partially detected in the polymerized tubulin fraction after microtubule stabilization. When co-transfected with Mid1, Mig12 is massively recruited to thick filamentous structures composed of tubulin. These microtubule bundles are resistant to high doses of depolymerizing agents and are composed of acetylated tubulin, thus representing stabilized microtubule arrays. + +Conclusions + +Our findings suggest that Mig12 co-operates with Mid1 to stabilize microtubules. Mid1-Mig12 complexes might be implicated in cellular processes that require microtubule stabilization, such as cell division and migration. Impairment in Mig12/Mid1-mediated microtubule dynamic regulation, during the development of embryonic midline, may cause the pathological signs observed in Opitz syndrome patients. + +Background + +Opitz syndrome (OS) is a congenital disorder affecting primarily midline structures (MIM 145410 and 300000). OS patients usually present with facial anomalies, including hypertelorism and cleft lip and palate. OS also includes laryngo-tracheo-esophageal (LTE), cardiac, and genitourinary abnormalities. These symptoms show high variability even within the same family [1-5]. OS is a heterogeneous disease with an X-linked (Xp22.3) and an autosomal locus (22q11.2) [6]. The gene responsible for the X-linked form, MID1, has been identified [7]. In male OS patients, mutations have been found scattered throughout the entire length of the MID1 gene, suggesting a loss of function mechanism at the basis of this developmental phenotype. Females carrying a mutated MID1 allele usually show only hypertelorism, likely as the result of differential X-inactivation [7-11]. Interestingly, during embryonic development the murine and avian orthologs of the MID1 gene show an expression pattern that, although not highly restricted, correlates with the tissues affected in OS. Within these tissues, the mouse and chick Mid1 transcripts are preferentially enriched in areas of active proliferation [12,13]. Recently, the chick Mid1 gene has been shown to be involved in the Sonic Hedgehog pathway during the establishment of the molecular left/right asymmetry in early embryonic avian development [14]. + +MID1 encodes a protein belonging to the tripartite motif family and is composed of a RING domain, two B-Box domains, a coiled-coil region, together forming the tripartite motif, followed by a fibronectin type III (FNIII) and an RFP-like domain [7,15,16]. The tripartite motif family, also known as TRIM or RBCC, comprises multi-domain-proteins involved in the definition of cellular compartments [17]. Mid1 self-interacts and forms high molecular weight complexes that are anchored to the microtubules throughout the cell cycle [18,19]. The most frequent MID1 alterations found in OS patients affect the C-terminal portion of the protein. Mutants that reproduce these mutations show an altered microtubule association [9,18,19]. The association of the wild-type protein with microtubules is dynamic and is regulated by its phosphorylation status: dephosphorylation of Mid1, upon interaction with the α4 regulatory subunit of phosphatase 2A (PP2A) [20], displaces Mid1 from microtubules [21,22]. It has also been reported that Mid1 functions as an E3 ubiquitin ligase, regulating the microtubular PP2A catalytic subunit degradation upon interaction with α4. PP2A degradation, in turn, controls the phosphorylation status of yet to be identified microtubule-associated-proteins (MAPs) [23]. + +We have identified a novel Mid1 interacting protein through yeast two-hybrid screening. This novel protein is expressed in the midline during development and co-operates with Mid1 to stabilize the microtubules. + +Results + +Identification of Mig12 as a novel Mid1 partner + +To date, insights on the function of Mid1 in the cell have emerged from its interaction with the α4 subunit of phosphatase 2A (PP2A), however, the role of Mid1 in the pathogenesis of OS is still undetermined [21-24]. To get clues on possible biological function of Mid1, we searched for additional partners by screening a fibroblast two-hybrid library. MidM, a construct encompassing the C-terminal half of MID1, was used as a bait. This region, which comprises the coiled-coil, the FNIII repeats and the RFP-like domain of MID1, appears to be involved in the anchorage to microtubules [9,18,19]. We obtained 6 positive clones, three of which were of different lengths, belonging to a unique transcript. The largest fragment had an ORF of 514 bp, the shortest of 432 bp. We used BLAST against the nr and EST databases and we found perfectly matching clones covering an ORF of 546 bp. We derived the complete sequence from the deposited transcripts and amplified the entire cDNA. We performed an interaction-mating assay to confirm the binding. Both the full-length and the largest original clone obtained from the library specifically interact with the entire Mid1 protein (MidA) (Fig. 1A). We also found positive interaction with portions of the Mid1 protein: MidD (coiled-coil), MidH (RING-B-boxes-Coiled-coil) and with MidM, the construct used to screen the library. No interaction was observed with MID1 constructs that lack the coiled-coil region (MidF and MidC, Fig. 1A). The identified clone does not interact with other members of the TRIM family (TRIM19/PML, TRIM25/RFP, TRIM29/ATDC) that share structural homology with Mid1 [17] (data not shown). + +Figure 1 + +Identification of a novel Mid1 partner. (A) Interaction-mating assay that confirms Mid1-Mig12 interaction in yeast. B42 fl, Mig12 full-length fused to the B42 activation domain; B42 or, the largest original Mig12 clone fused to the B42 activation domain; LexA Mid, constructs encompassing different MID1 domains fused to the LexA DNA binding domain: A, full-length; C, BB; D, CC; F, RFP-like; H, R-BB-CC; M, CC-FNIII-RFP-like. Both the full-length and the original Mig12 clones specifically interact with the entire Mid1 protein and with some of its truncated mutants, MidD, MidH and MidM, as shown by yeast turning blue on X-gal plates and growing on plates lacking leucine (Leu), only when galactose (Gal), and not glucose (Glu), is used as carbon source. Abbreviations: BB, B-box1 and B-box2 domains; CC, coiled-coil domain; FNIII, fibronectin type III repeat; R, RING domain. (B) Amino acid sequence of human (h) and mouse (m) MIG12 and comparison with the zebrafish G12 and the human SPOT14 proteins. Amino acids that are identical at least in the human and murine Mig12 are in bold. Conserved amino acids are indicated in gray. The human and mouse MIG12 share 90% of similarity and 88% of identity. The hMIG12 and the zebrafish protein share 56% of similarity and 46% of identity, whereas the homology with the human SPOT-14 protein is 49% and 31%, respectively. There is a gap of 25 aa that are not present in the zebrafish and SPOT14 proteins. (C) Co-immunoprecipitation experiments showing Mid1-Mig12 interaction. Western blot (WB) analysis using anti-Mid1 and anti-HA antibodies after immunoprecipitation of HEK293 cells transiently transfected with different combination of MycGFP-tagged Mid1 (MGFP-MID1) and an HA-tagged Mig12 (HA-MIG12); + and - indicate the constructs transfected in each lane. The antibodies used for the immunoprecipitations (IP) are indicated. Mid1 indicates the band corresponding to the endogenous protein. Ig, immunoglobulins. In some experiments, we detected a trace amount of MGFP-Mid1 immunoreactivity in cells transformed with only MGFP-Mid1 and immunoprecipitated with the anti-HA antibody. This signal was always much less than that seen when both tagged constructs were transfected together. (D) The same as in (C) using the MGFP-MidM, MGFP-MidH and MGFP-MidD mutant fusions, instead of the full-length protein, in the co-transfections and an anti-Myc antibody for Western blot analysis. + +The full-length sequence matches with various anonymous human (hypothetical protein STRAIT11499, NM_021242; FLJ10386, AK001248) and mouse (AL671335, AK090003, and NM_026524 RIKEN) complete cDNA sequences and several ESTs in the databases. The human gene is located in Xp11.4 and is composed of two exons, one of which encompasses the entire coding region. The mouse gene is located in the A1.1 region of the X chromosome. The human (GenBank accession no. BK001260) and mouse (GenBank accession no. AY263385) cDNAs encode a 182- and a 181-residue-protein, respectively, displaying no known domains with the exception of a low score coiled-coil region at the C-terminus of the protein. This Mid1 interactor records the highest homology with the zebrafish 'Gastrulation specific protein G12' (NP_571410), a protein with unknown function [25], and with the mammalian SPOT-14 (NM_003251), a protein involved in the metabolism of fatty acids [26,27]. The novel transcript was dubbed MIG12 for Mid1 interacting G12-like protein, after the similarity with the Danio rerio protein. Figure 1B shows the alignment of the human and mouse Mig12, the zebrafish G12, and the human SPOT14 proteins. + +To confirm that the two proteins also interact in vivo, we transiently transfected a MycGFP-tagged version of MID1 (MGFP-Mid1) and an HA-tagged version of MIG12 (HA-Mig12) in HEK293 cells and immunoprecipitated using either anti-Mid1 or anti-HA antibodies. Immunoprecipitation of Mid1 in the co-transfected sample pulls down the HA-Mig12 protein (right panel) and, vice versa, the immunoprecipitation of Mig12 using the anti-HA antibody pulls down the MGFP-Mid1 protein (left panel) (Fig. 1C). An unrelated polyclonal antibody and a different anti-tag monoclonal antibody (anti-FLAG) did not pull down the two proteins (data not shown), confirming the specificity of Mid1-Mig12 interaction. Moreover, Mig12 transfected alone is also pulled down by immunoprecipitation of the endogenous Mid1 protein (Fig. 1C). The interaction mating experiments suggest that the coiled-coil region of Mid1 is necessary and sufficient for the binding to Mig12. MGFP tagged versions of MidM, MidH, and MidD were co-transfected with HA-MIG12 in HEK293 cells and immunoprecipitated with either anti-Myc or anti-HA antibodies. The three constructs, all encompassing the coiled-coil region, are able to bind Mig12 further confirming that, also in vivo, this region is sufficient for Mid1-Mig12 interaction (Fig. 1D). + +Mig12 is mainly expressed in the developing CNS midline + +Since Mid1 is implicated in a developmental disorder, to support a physiologically relevant interaction between Mig12 and Mid1 we analyzed the mRNA expression of Mig12 during embryonic development. The Mig12 clone originally obtained from the two-hybrid screening was used as a probe to perform mRNA in situ hybridization on mouse embryos at several embryonic stages. A ubiquitous expression pattern was found both on section and in whole mount experiments from embryonic day 9.5 (E9.5) up to E11.5. At E11.5, we detected a diffuse staining in the central nervous system (CNS) and a more restricted signal in the developing limbs by whole-mount in situ hybridization (Fig. 2A, a). An even more restricted expression pattern is observed at E14.5 when high transcript levels are detected in specific compartments (Fig. 2A, b). The strongest expression is observed in the developing central nervous system and is particularly evident in the coronal sections through the hindbrain region (Fig. 2B, a–c). The signal is observed in the neuroepithelium of the cerebellar primordia (Fig. 2B, a,b), of the pons (Fig. 2B, a, b, e), and of the medulla oblongata (Fig. 2B, c). The ventricular hindbrain signal is mainly confined to the ventral midline (Fig. 2B, a, b, c). This medial expression is maintained throughout the central canal of the spinal cord extending through the floor and roof plates (Fig. 2B, d). In the telencephalon, Mig12 signal is present in the ventricular zone of the telencephalic vesicles (Fig. 2B, f). Within the nervous system, Mig12 transcript is also detected in the dorsal roots and in the trigeminal ganglia (Fig. 2A, b; 2B, d). At this stage, expression of Mig12 is also observed in several additional organs. The transcript is observed in the interdigital web in both the developing hind- and forelimbs at E11.5 (Fig. 2A, a). At E14.5, as the development of the limbs proceeds, Mig12 transcript is detected in the perichondrium of the digits (Fig. 2B, g). The other organs expressing Mig12 include the left and right thyroid lobes and the parathyroid glands (Fig. 2B, h); the phallic part of the urogenital sinus (Fig. 2B, i); the anal canal (rectum) and the epithelium lining the lumen of the bladder (data not shown). Interestingly, many of the sites that show high Mig12 levels also express the Mid1 transcript [12,13] and are affected in OS patients [5,11]. + +Figure 2 + +Mig12 expression analysis during embryonic development. (A) Whole mount in situ hybridization on E11.5 mouse embryo showing expression in the central nervous system and in the developing limbs (blue signal, a). Coronal and sagittal sections of E14.5 entire mouse embryos (white signal) (b). (B) Details of coronal (a, b, c, d, h) and sagittal (e, f, g, i) sections of E14.5 mouse embryos. Strong Mig12 expression (red signal) is observed in isthmal (a), pontine (a, b, e) and medulla oblongata (c) neuroepithelia, and it is maintained throughout the entire region of the spinal cord central canal (d). Expression is also observed in dorsal root ganglia (d). Mig12 transcript is detected in the telencephalon at the level of the ventricular zone (f). Signal is also present in other organs: in the perichondrium of the digits (g); in the thyroid (th) and parathyroid (pth) glands (h), and in the phallic part of the urogenital sinus (i). Abbreviations: CB, cerebellum; ccn, central canal neuroepithelium; drg, dorsal root ganglia; IS, isthmus; isn, isthmal neuroepithelium; M, medulla oblongata; mn, medulla oblongata neuroepithelium; P, pons; pc, perichondrium; pnn, pontine neuroepithelium; pth, parathyroid glands; SC, spinal cord; T, telencephalon; th, thyroid gland; us, urogenital sinus; vz, ventricular zone. + +Mid1 recruits Mig12 on the microtubules + +Transient expression of either MGFP- or HA-tagged Mig12 reveals a diffuse distribution of the protein in Cos7 as well as in other cell lines (U2OS, HeLa, NIH3T3). To exclude a tag-driven mislocalization, we also transfected a non-tagged version of Mig12: the specific anti-Mig12 antibody reveals a distribution comparable to that of the tagged versions. Mig12 is present in both the nucleus and the cytoplasm and the relative abundance in the two compartments is variable (Fig. 3A). + +Figure 3 + +Immunofluorescence analyses reveal co-localization of Mid1 and Mig12 within the cell. (A) Immunofluorescence analysis after transient expression of MGFP-Mig12 (upper panel), HA-Mig12 (middle panel) and untagged Mig12 (lower panel) in Cos7 cells, revealing a diffuse distribution of the protein, in both the nucleus and the cytoplasm. (B) Co-expression of both Mid1 and Mig12 leads to co-localization of the two proteins in cytoplasmic bundles. Standard fluorescence microscopy shows formation of bundles only in Mid1 (left panels) and Mig12 (right panel) co-expressing cells. The arrow indicates a single transfected cell where Mid1 shows the classical distribution along normal interphase microtubules. (C) The co-localization is confirmed by confocal microscopy analysis in which HA-Mid protein is visible as a red signal and MGFP-Mig12 protein as a green signal; co-localization is indicated as a yellow signal in merged images. (D) Co-localization is also observed using the HA-Mig12 construct (middle panels) together with either a Mid1 OS truncated mutant (GFP-Mid1 1331insA) or a Mid1 mutant (GFP-MidD) retaining the coiled-coil domain, both localized in cytoplasmic bodies. No co-localization is observed when HA-TRIM19/PML protein is co-expressed with GFP-Mig12. The right panels represent the merged images. + +Mid1 is associated with microtubules during the entire cell cycle [18,19]. An example of its distribution is shown in figure 3B (arrow, upper panel), where Mid1 co-localizes with the normal radial interphase microtubules. Interestingly, when co-expressed in the same cell, Mid1 and Mig12 form bundles within the cytoplasm (Fig. 3B). Mig12 usually also maintains a diffused distribution whose extent depends on its expression level. As shown in the lower panels, the observed bundles show variable thickness and shape that depend on the expression levels of the two proteins. Nevertheless, these bundles are only present when the two proteins are co-expressed. In our experimental conditions we do not observe the formation of bundles in cells transfected with only Mid1 (Fig. 3B, arrow). The co-localization of Mid1 and Mig12 within the bundles has been confirmed by confocal microscopy analysis (Fig. 3C). + +We investigated the distribution of Mig12 in cells co-transfected with mutant Mid1 proteins that are not anchored to the microtubules. Mid1 C-terminal OS mutants localize to cytoplasmic bodies [9,18,19]. These mutant forms, that retain the coiled-coil region, are able to recruit Mig12 within these structures (Fig. 3D, upper panels). The same is observed using a construct that drives the expression of only the coiled-coil domain of Mid1 (Fig. 3D, middle panels). This recruitment is not observed when other TRIM proteins, that share the same domain composition of Mid1, are expressed with Mig12. This is demonstrated by co-transfections of Mig12 with TRIM19/PML (Fig. 3D, lower panels), TRIM5 or TRIM27 (data not shown). These results confirm that Mid1, through its coiled-coil domain, is able to specifically recruit Mig12 to different structures within the cell. + +Since Mid1 is a microtubular protein, we asked whether the bundles observed in cells co-expressing Mig12 and Mid1 are structures of microtubular nature. Co-localization of tubulin with the bundles, in immunofluorescence experiments, demonstrates that these structures are microtubule arrays rearranged by overexpression of the two proteins and that are often present as continuous or fragmented perinuclear rings (Fig. 4A). + +Figure 4 + +Mid1 and Mig12 co-sediment with microtubules. (A) Immunofluorescence analysis in Cos7 cells co-transfected with HA-Mid1 (left panels) and MGFPMig12 (middle panel) proteins. Coincidence of the bundles with microtubules is revealed using monoclonal antibodies against β-tubulin (right panel). These images show the different thickness and distribution of the bundles. (B) Cos7 cells were transfected with either MGFPMid and HA-Mig12 (left panel) or HA-Mig12 alone (right panel). Lysates (L) from cells were supplemented with 40 μM taxol to stabilize polymerized microtubules. After sedimentation on sucrose cushion, supernatant (S) and pellet (P) fractions were assayed for the presence of Mid1, Mig12, and tubulin using appropriate antibodies. In the co-transfection (left panel) both Mid1 and Mig12 were detected in the pellet together with the polymerized microtubules. As expected Mig12 is also present in the soluble fraction where neither Mid1 nor the tubulin are found. Mig12 is found partially associated with the polymerized tubulin fraction also in the single HA-Mig12 transfected cells (right panel). (C) Western blot analysis using the anti-Mig12 antibody reveals a 24 KDa protein in two different cell lines lysates (1, Cos7; 2, HeLa cells). To confirm specificity, incubation with the primary antibody was also performed in the presence of either the fusion protein used to immunize rabbits (GST-Mig12) or an unrelated fusion protein (GST-ur). (D) Detection of endogenous Mig12 in the polymerized microtubule fraction (+ taxol) in HeLa cells and as control in the non-treated sample (-taxol); legend as in (A). (E) Single Mig12 transfected Cos7 cells show partial localization with microtubules, particularly in the MTOC region (upper panels) and at the mitotic spindle poles (lower panels). + +To confirm these data, we performed microtubule sedimentation after taxol treatment in cells co-transfected with both Mid1 and Mig12. After fractionation on a sucrose cushion, the supernatant and the pellet containing the polymerized tubulin were assayed by immunoblot for the presence of both proteins. Mig12 and Mid1 are recovered in the pellet, where tubulin is also found. Mig12, as expected, is also present in the supernatant. This result further indicates that the bundles observed in immunofluorescence experiments are of microtubular nature (Fig. 4B, left panel). A control protein that does not associate with the microtubules, spastin Δ N [28], is not present in the microtubule fraction, confirming that the presence of Mig12 in the pellet is not due to contamination during the sedimentation process (data not shown). Moreover, the presence of Mig12 in the pellet, as well as that of tubulin, is lost when the cells are not treated with the microtubule stabilization agent, taxol (data not shown). Thus, when overexpressed, Mid1 and Mig12 have the ability to rearrange interphase radial microtubules into these structures. + +Interestingly, singly transfected Mig12 also partially sediments with the microtubular pellet, as expected to a lesser extent than the double transfectant (Fig. 4B, right panel). Since the affinity purified anti-Mig12 antibody we produced allows the specific detection of the endogenous protein in immunoblot experiments in cell line lysates, as shown in figure 4C, we carried out sedimentation of polymerized microtubules in HeLa cells to test the presence of endogenous Mig12 in the microtubule pellet. These results indicate that the protein, likely by interacting with endogenous Mid1 protein, is at least partially associated with microtubules (Fig. 4D). A closer look at some single transfected cells reveals indeed a partial co-localization of Mig12 with the microtubules, also in the absence of exogenous Mid1 (Fig. 4E). Some filaments are observed over the diffuse staining and in many cells enrichment of Mig12 protein in the MTOC region is evident (Fig. 4E, upper panels) as well as partial co-localization with the mitotic spindle (Fig. 4E, lower panels). + +Mid1 and Mig12 induce stable microtubule bundles + +To better understand the nature of these microtubule arrays, we asked what happens to the Mid1-Mig12 bundles upon disruption of the microtubular architecture. Cells were co-transfected and exposed to nocodazole, a microtubule-depolymerizing agent, for 1 hour before fixation and then analyzed by immunofluorescence. The filaments observed after overexpression of the two proteins were more resistant to the drug compared to control microtubules (Fig. 5A). In contrast, cells overexpressing only Mid1 show complete disruption of the microtubular apparatus, which is consistent with the absence of bundles (Fig. 5A, arrow). Partial disruption of the Mid1-Mig12 bundles was observed only after longer exposure to nocodazole (4 h, data not shown). + +Figure 5 + +Mid1 and Mig12 together stabilize the microtubules. (A) Nocodazole treatment does not disrupt the Mid1/Mig12 generated bundles of tubulin, whereas it disrupts the microtubules in Mid1 single transfected cells (arrow). (B) The bundles represent stable microtubules as demonstrated by perfect coincidence with the anti-acetylated tubulin antibody signal (blue). + +Modification of tubulin subunits by acetylation marks older microtubules and therefore indicates those that are more stable [29]. Specific antibodies to acetylated tubulin decorate the Mid1-Mig12 induced nocodazole-resistant bundles, thus indicating stable microtubules (Fig. 5B). The ability to stabilize the microtubules is not a characteristic of cells overexpressing Mig12 alone: in fact, treatment with nocodazole does not reveal any residual microtubular structures in these cells (data not shown). + +These data suggest that Mig12 co-operates with Mid1 to stabilize microtubules. The Mid1-Mig12 microtubule-stabilizing effect might be implicated in specific processes during the development of the midline systems that are affected in Opitz syndrome patients. + +Discussion + +The role of the Opitz syndrome gene product, Mid1, in the pathogenesis of this human disorder is still unclear [14,24]. We now present data that support a role of Mid1 in the regulation of microtubule dynamics. We report the identification of a novel gene, MIG12, that encodes a Mid1 interacting protein. MIG12 shares high sequence homology with a zebrafish gene product, the 'gastrulation protein G12', which is expressed in a narrow window of time during D. rerio gastrulation [25]. A Mig12 paralog in mammals, SPOT14, is a nuclear protein that responds to the thyroid hormone and regulates lipid synthesis [26,27]. However, the mechanism of action for both G12 and SPOT14 is still unknown. Further, the absence of recognizable domains in its peptide sequence does not allow any a priori hypothesis on MIG12 function to be drawn. + +The expression pattern of Mig12 during embryonic development is consistent with that of Mid1 [12,13]. Furthermore, this pattern overlaps with tissues whose development is defective in OS [5,9,11]. The strong expression in the midline of the developing central nervous system might be related to the neurological signs found in a high number of patients that manifest agenesis or hypoplasia of the corpus callosum and of the cerebellar vermis, and mental retardation. Moreover, expression of Mig12 in the rostral medial CNS could also be involved in the determination of proper craniofacial formation. It is well known that factors expressed in the CNS midline are implicated in resolving a single eye field into two lateral fields, an event that determines the head midline width and the face traits as reviewed in [30,31]. One of these, Sonic hedgehog (Shh), plays a crucial role in the ventral midline neural tube patterning and regulates the morphogenesis of a variety of midline and lateral organs. It is interesting to note the recent association of the Mid1 gene and the Shh pathway in the early midline and laterality specification in the chicken [14]. Interference with the correct Mig12-Mid1 pathway might be responsible for the craniofacial defects observed in OS. Expression in the embryonic urogenital and anal apparatus is also reminiscent of defects observed in OS, hypospadias and imperforate or ectopic anus. In addition, we can parallel the inter-digit Mig12 expression observed in the mouse embryos with OS manifestations, as we observed syndactyly in a MID1-mutated patient [11]. The low frequency of mutations in MID1 and the high variability of the phenotype in OS patients suggest the involvement of other genes in the OS phenotype. It is plausible that other proteins involved in the Mid1 pathway are implicated in the heterogeneity of OS (or in other syndromes showing clinical overlap with OS) and Mig12 might well be a candidate. + +When Mig12 is over-expressed, it barely decorates microtubules with a signal almost imperceptible due to its diffused distribution in the cytoplasm. Accordingly, endogenous Mig12 is partially found associated with the polymerized tubulin fraction in cell lysates. Interestingly, when co-expressed with Mid1 it induces the formation of microtubule bundles. This effect is not observed when Mid1 is expressed alone. Mid1 specifically recruits Mig12 to the microtubules and the consequent induction of bundles could be explained by the propensity of both proteins, Mid1 [18] and Mig12 (CB, GM, unpublished results), to homo-interact. The formation of multimers might tether a high number of microtubule interacting moieties that, in turn, mediate and favor the association of parallel microtubule arrays. The shape and location of these microtubule bundles is variable within the cell: perinuclear rings, sub-cortical bundles and a roundish mass in the MTOC region. In some cases, we also observed fragmentation of these thick microtubular structures (CB, GM, unpublished results) that might suggest the involvement of a putative microtubule severing activity [32]. These microtubule bundles are resistant to depolymerizing agents, such as nocodazole, and are composed of acetylated tubulin and therefore represent stable microtubules. This bundling and stabilizing effect has been observed for other microtubule binding proteins, in particular microtubule-associated-proteins (MAPs) and other proteins involved in mitotic spindle organization, cytokinesis and the control of cell motility such as, PRC1, NuMA, CLASPs, and many others [33-36]. It is worth noting that recently two proteins sharing homology with the C-terminal half of Mid1, Mir1 and GLFND that have a coiled-coil-FNIII-RFP-like structure, have been shown to bundle and stabilize microtubules [37,38]. So far, we have no indications on the behavior of Mid1-Mig12 complexes during mitosis. Mid1 decorates the mitotic spindle [18] and Mig12, when transfected alone, appears to be both associated with the spindle poles and diffused within the cell. We have never observed mitotic cells overexpressing both proteins. Whether this is due to interference with the division process is still to be clarified. + +The bundling effect observed in our over-expression system probably reflects a weaker and finely tuned-regulated process in physiological conditions. The shuttling of Mig12 between nucleus and cytoplasm might also be dynamically regulated and, in certain conditions, segregation in the nucleus might be necessary to prevent interference with the interphase microtubule network. Mid1 might recruit Mig12 to microtubules only when needed. It is possible that phosphorylation of Mid1 [21,22] and/or putative post-translational modifications of Mig12 might regulate their physiological association and the subsequent stabilization of the microtubule network. The ultimate aim of the regulation of microtubule stability and dynamics involving the Mid1-Mig12 pathway is still to be elucidated and may be connected to cell cycle progression or cell migration, events known to require microtubule stabilization [39]. Alteration of either process can be seen as possible causes of pathological signs in OS. Mig12, as well as Mid1, appears to be preferentially expressed in highly proliferating embryonic fields (e.g., the ventricular zone of the developing brain). Nevertheless, these are also cells that, after mitosis has been completed, are committed to migrate. The zebrafish gastrulation protein G12 is expressed in a restricted lineage characterized by extensive cell migration [25]; it is tempting to speculate that this process could be the one implicated in the pathogenesis of the Opitz syndrome. + +Conclusions + +We have reported the identification of a novel Opitz syndrome gene product interacting protein, Mig12, that co-operates with Mid1 to stabilize microtubules. These data are consistent with the role of Mid1 in microtubule dynamics. Mid1, in fact, controls MAP phosphorylation through the regulation of PP2A microtubular levels [23] and Mig12 may participate in this pathway. During embryonic development of midline structures, impairment in Mid1-Mig12-mediated microtubule dynamics regulation might be detrimental and lead to Opitz syndrome. + +Methods + +Plasmid constructs + +The MID1 expression vectors MycGFP-MID1 and HA-MID1 have already been reported [18]. The MID1 deletion mutants, MidC, MidD, MidF, MidH, and MidM have been excised from HA-pCDNA3 vectors [18] and cloned EcoRI/XhoI in the two-hybrid vectors pJG4-5 and pEG202 [40]. Full-length MIG12 cDNA was generated by PCR amplification, using specific primers designed on ESTs sequences, from NIH3T3 total RNA as template. The PCR product was then cloned into EcoRI and XhoI sites in the eukaryotic expression vectors pcDNA3, pcDNA3-MGFP and pcDNA3-HA. Both Myc-GFP and HA tags are positioned at N-terminus region of MIG12 coding region. Full-length MIG12 was also cloned in the pJG4-5 two-hybrid vector fused to the B42 activation domain [40]. + +Yeast two-hybrid screening + +The two-hybrid screening was performed using MIDM (CC-FNIII-RFP-like) cloned in pEG202 vector that contains the LexA DNA-binding domain. The bait was transformed into the yeast strain EGY48 that was subsequently transformed with an NIH3T3 cDNA library cloned into pJG4-5, containing the B42 activation domain. Transformants (5 × 106 independent clones) were seeded on plates containing either X-gal or lacking Leucine to select positive clones that have activated both LexA driven reporter genes (lacZ and LEU2). Interaction mating assay to confirm the positivity was performed using the same system and two different yeast mating types (EGY48 MAT α and EGY42 MAT a) as described [40]. + +Cell culture and transfection + +Monkey Kidney Cos-7 cells and HEK 293T cells were cultured in Dulbecco's modified Eagle's medium, supplemented with 10% fetal bovine serum, at 37°C in a 5% CO2 atmosphere. All transfections were carried out by calcium phosphate precipitation [41]. In a typical transfection experiment 20 μg of expression vector were used per 15-cm dish. For immunofluorescence experiments, using chamber-slides (8 wells, Nunc), 0.5 μg DNA/well were transfected. + +Immunoprecipitation, Immunoblot, and Antibodies + +In co-immunoprecipitation experiments 4.5 × 106 HEK 293T cells per 15-cm dish were seeded. 60 h after transfection cells were collected, washed and extracted with RIPA buffer (150 mM NaCl, 1% Igepal, 0.5% DOC, 0.1% SDS, 50 mM Tris-HCl pH 8) supplemented with protease inhibitors (Roche). Extracts were sonicated and centrifuged at 10000 g for 10 min at 4°C to remove cell debris. The supernatants were immunoprecipitated with either 6 μg of anti-HA antibody, 500 μl anti-Myc (9E10) hybridoma supernatant or 8 μg anti-Mid1 polyclonal antibody (H35) [18], for 3 h at 4°C and the immuno-complexes collected with protein A-Sepharose beads for 30 min. The beads were washed six times with RIPA buffer and proteins eluted from the beads by boiling in SDS loading buffer. Proteins were separated on either 10% or 12% SDS PAGE and blotted onto PVDF membranes (Amersham). The membranes were rinsed in methanol and blocked in TTBS (20 mM Tris-HCl pH 7, 50 mM NaCl and 0.1% Tween-20), 5% dry milk. Incubation with the primary antibodies was performed using anti-c-Myc monoclonal antibody (1:5 dilution), anti-HA monoclonal antibody (Roche) (1:500 dilution) and anti-Mid1 polyclonal antibody (1:250 dilution) in TTBS, 5% dry milk. Antibody binding was detected with a secondary anti-mouse or anti-rabbit IgG coupled with horseradish peroxidase, followed by visualization with the Enhanced Chemiluminescence Kit (Amersham). A specific anti-Mig12 antiserum has been raised against a full-length Mig12 protein fused to GST and produced in bacteria. Affinity purification of the antibody was performed with the GST-Mig12 covalently attached to a CNBr-activated sepharose column using standard procedures. To perform competition experiments, 20 μg of the same protein were used to compete the binding in immunoblot analysis. As non-specific competitor, the same amount of an unrelated GST fusion protein (Mid1 RING domain) was used. + +Immunofluorescence + +Cos7 cells were grown on chamber-slides (8 wells, Nunc) in DMEM, 10% FBS, and transfected as described. After 36 h, cells were fixed in 4% paraformaldehyde/PBS for 10 min at room temperature, permeabilized with 0.2% Triton X-100/PBS for 30 min, blocked with normal serum for 1 h and incubated for 3 h with the primary antibodies and 1 h with the appropriate secondary antibodies. The following primary antibodies were used: protein A-purified polyclonal anti-Mid1 (1:200 dilution), monoclonal anti-β-tubulin (1:250 dilution) (Molecular Probes), monoclonal anti-HA (CA25) antibody (1:250 dilution) (Roche), monoclonal anti-acetylated tubulin (1:200 dilution) (Sigma). The following secondary antibodies were used: fluorescein isothiocyanate (FITC)-conjugated anti-rabbit antibodies alone or both tetramethylrhodamine isothiocyanate (TRITC) conjugated anti-rabbit and FITC conjugated anti-mouse-antibodies (1:100 dilution) (Dako). For confocal microscopy, Cy3-conjugated anti-mouse antibody was used (1:200 dilution) (Amersham). When indicated, nocodazole in DMSO was added at the final concentration of 40 μM for 1 h at 37°C before fixation. + +Microtubule binding assay + +Cells were harvested either 48 hours post-transfection (Cos7 cells) or when at 80% confluence (non-transfected HeLa cells) and lysed in PEM-DNNA buffer (80 mM PIPES pH 6.8, 1 mM EGTA, 1 mM MgCl2, 0.5 mM DTT, 150 mM NaCl, 1% Igepal) supplemented with protease inhibitors, at 4°C for 1 hr. The lysate was centrifuged at 610 g for 10 min at 4°C. Cytosol was then purified by successive centrifugations at 10,000 g for 10 min, at 21,000 g for 20 min and at 100,000 g for 1 hr at 4°C. Each supernatant was then supplemented with 2 mM GTP (Roche) and 40 μM taxol (Molecular Probes) and incubated at 37°C for 30 min. Corresponding samples without taxol were also prepared. Each sample was layered over a 15% sucrose cushion and centrifuged at 30,000 g for 30 min at 30°C to sediment polymerized microtubules. The resulting supernatants were saved and the pellets were suspended in an equal volume of sample buffer for electrophoresis and immunoblot analysis. + +RNA in situ hybridization + +One of the original clones obtained from the screening (540 bp fragment whose 5' corresponds to nt 113 of the MIG12 coding region) was linearized with the appropriate restriction enzymes to transcribe either sense or antisense 35S-labeled riboprobe. Mouse embryo tissue sections were prepared and RNA in situ hybridization experiments performed as previously described [42]. Autoradiographs were exposed for 2 days. Slides were then dipped in Kodak NTB2 emulsion and exposed for 14–21 days. In the micrographs red represents the hybridization signal and blue shows the nuclei stained with Hoechst 33258 dye. Whole-mount in situ hybridization was performed using the same probe and following the protocol described in [43]. + +Authors' contributions + +CB carried out the two-hybrid screening, the RNA in situ hybridization analysis, the immunoprecipitation and immunofluorescence studies. BF produced the anti-Mig12 specific antibody and performed the microtubule sedimentation experiments. RF provided assistance in the cloning and preparation of the vectors. GM coordinated the study and wrote the paper. All authors read and approved the final manuscript. + +Acknowledgements + +We thank Salvatore Arbucci (IGB-ABT, Naples) and Francesca De Falco for assistance with the confocal microscopy and Alexandre Reymond and Alessia Errico for helpful suggestions. We are grateful to Graciana Diez-Roux, Elena Rugarli and Graziella Persico for a critical reading of the manuscript. This work was supported by the Italian Telethon Foundation and by Research Grant No. 1-FY00-700 from the March of Dimes Birth Defects Foundation. diff --git a/src/ontogpt/evaluation/craft/database/all/15207008.ann b/src/ontogpt/evaluation/craft/database/all/15207008.ann new file mode 100644 index 000000000..55683ae88 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15207008.ann @@ -0,0 +1,1246 @@ +T1 CHEBI:37527 49 55 acidic +T2 SO:0001080 56 67 coiled coil +T3 SO:0000704 68 73 genes +T4 NCBITaxon:1 123 132 organisms +T5 SO:0000704 179 183 gene +T6 SO:0000704 242 246 gene +T7 SO:0000853 292 300 homologs +T8 CHEBI:37527 346 352 acidic +T9 SO:0001080 353 364 coiled coil +T10 SO:0000704 372 376 gene +T11 NCBITaxon:1 399 408 organisms +T12 NCBITaxon:9606 423 426 man +T13 SO:0001026 621 628 genomic +T14 NCBITaxon:1 691 700 organisms +T15 SO:0001080 791 802 coiled coil +T16 NCBITaxon:9986 852 858 rabbit +T17 SO:0000855 872 882 orthologue +T18 NCBITaxon:9606 886 891 human +T19 PR:000016008 892 897 TACC3 +T20 PR:000001856 903 908 RHAMM +T21 SO:0001080 933 944 coiled coil +T22 NCBITaxon:6239 1035 1045 C. elegans +T23 NCBITaxon:7227 1050 1065 D. melanogaster +T24 NCBITaxon:9606 1215 1220 human +T25 PR:000016007 1221 1226 TACC2 +T26 GO:0005634 1234 1241 nuclear +T27 PR:000014373 1259 1263 RXRβ +T28 GO:0005813 1351 1362 centrosomal +T29 GO:0007067 1363 1370 mitotic +T30 GO:0072686 1363 1378 mitotic spindle +T31 GO:0032991 1426 1435 complexes +T32 GO:0006412 1456 1467 translation +T33 GO:0006396 1469 1483 RNA processing +T34 GO:0030674 1532 1540 bridging +T35 GO:0032991 1672 1681 complexes +T36 GO:0005634 1709 1716 nuclear +T37 PR:000017527 1736 1741 GAS41 +T38 GO:0005813 1899 1909 centrosome +T39 GO:0000785 1973 1982 chromatin +T40 GO:0006338 1973 1993 chromatin remodeling +T41 GO:0032991 2035 2044 complexes +T42 NCBITaxon:1 2096 2105 organisms +T43 SO:0000704 2149 2153 gene +T44 SO:0000704 2240 2245 genes +T45 SO:0000147 2320 2324 exon +T46 SO:0000704 2343 2347 gene +T47 SO:0000704 2358 2363 genes +T48 SO:0001026 2488 2494 genome +T49 NCBITaxon:9606 2518 2523 human +T50 NCBITaxon:10088 2525 2530 mouse +T51 NCBITaxon:10114 2532 2535 rat +T52 NCBITaxon:7215 2537 2546 fruit fly +T53 NCBITaxon:6231 2551 2560 nematodes +T54 SO:0000704 2633 2638 genes +T55 NCBITaxon:1 2676 2684 organism +T56 SO:0000704 2798 2803 genes +T57 SO:0000704 2808 2812 gene +T58 SO:0000704 2836 2840 gene +T59 NCBITaxon:1 2859 2868 organisms +T60 SO:0001026 2936 2943 genomic +T61 SO:0000704 3065 3069 gene +T62 CHEBI:37527 3126 3132 acidic +T63 SO:0001080 3133 3144 coiled coil +T64 SO:0000704 3145 3149 gene +T65 PR:000016006 3151 3156 TACC1 +T66 GO:0010467 3202 3212 expression +T67 SO:0001428 3202 3216 expression map +T68 SO:0000105 3239 3245;3252 3262 arm of ... chromosome +T69 NCBITaxon:9606 3246 3251 human +T70 SO:0000854 3348 3358;3371 3378 paralogous ... regions +T71 NCBITaxon:9606 3382 3387 human +T72 SO:0000704 3456 3460 gene +T73 SO:0000704 3540 3544 gene +T74 NCBITaxon:33317 3552 3563 protostomes +T75 NCBITaxon:6239 3564 3585 Caenorhabitis elegans +T76 NCBITaxon:7227 3591 3614 Drosophila melanogaster +T77 SO:0000704 3678 3683 genes +T78 NCBITaxon:7742 3759 3770 vertebrates +T79 SO:0000704 3824 3828 gene +T80 SO:0000704 3904 3909 genes +T81 NCBITaxon:9606 3913 3919 humans +T82 NCBITaxon:7742 3976 3986 vertebrate +T83 SO:0001026 3987 3993 genome +T84 NCBITaxon:7742 4052 4063 vertebrates +T85 SO:0000704 4111 4115 gene +T86 PR:000001451 4135 4140 FGFR4 +T87 NCBITaxon:7742 4144 4154 vertebrate +T88 SO:0001026 4155 4162 genomes +T89 SO:0000704 4196 4201 genes +T90 NCBITaxon:9606 4222 4228 humans +T91 NCBITaxon:10088 4251 4256 mouse +T92 NCBITaxon:8355 4262 4276 Xenopus laevis +T93 NCBITaxon:7227 4282 4297 D. melanogaster +T94 NCBITaxon:6239 4307 4317 C. elegans +T95 NCBITaxon:9986 4384 4405 Oryctolagus cuniculus +T96 NCBITaxon:9606 4420 4425 human +T97 PR:000001856 4426 4431 RHAMM +T98 NCBITaxon:1 4644 4653 organisms +T99 GO:0000380 4655 4676 alternatively spliced +T100 SO:0000704 4756 4761 genes +T101 SO:0001080 4795 4806 coiled coil +T102 NCBITaxon:7742 4974 4984 vertebrate +T103 SO:0001026 5067 5073 genome +T104 PR:000001856 5155 5160 RHAMM +T105 SO:0000855 5161 5172 orthologues +T106 SO:0001080 5199 5210 coiled coil +T107 NCBITaxon:species 5243 5250 species +T108 SO:0000704 5311 5316 genes +T109 NCBITaxon:1 5450 5459 organisms +T110 NCBITaxon:7742 5553 5563 vertebrate +T111 NCBITaxon:species 5564 5571 species +T112 NCBITaxon:7586 5577 5590 echinodermate +T113 NCBITaxon:7668 5591 5620 Strongylocentrotus purpuratus +T114 NCBITaxon:33317 5629 5639 protostome +T115 NCBITaxon:6960 5640 5646 insect +T116 NCBITaxon:7165 5647 5664 Anopheles gambiae +T117 SO:0000704 5734 5739 genes +T118 NCBITaxon:33208 5743 5751 metazoan +T119 SO:0001026 5811 5817 genome +T120 NCBITaxon:1 5844 5853 organisms +T121 SO:0000704 5895 5900 genes +T122 NCBITaxon:33213 6003 6013 bilaterian +T123 NCBITaxon:33208 6014 6023 metazoans +T124 NCBITaxon:6073 6033 6041 Cnidaria +T125 NCBITaxon:6040 6045 6053 Porifera +T126 NCBITaxon:1 6108 6117 organisms +T127 SO:0000417 6175 6181 domain +T128 NCBITaxon:33208 6214 6222 metazoan +T129 NCBITaxon:1 6223 6232 organisms +T130 SO:0001080 6312 6323 coiled coil +T131 SO:0001080 6457 6468 coiled coil +T132 SO:0001080 6510 6521 coiled coil +T133 PR:000001856 6553 6558 RHAMM +T134 PR:000001856 6591 6596 RHAMM +T135 NCBITaxon:7711 6747 6755 chordate +T136 SO:0000704 6786 6790 gene +T137 SO:0001026 6813 6819 genome +T138 NCBITaxon:7712 6827 6838 urochordate +T139 NCBITaxon:7719 6839 6857 Ciona intestinalis +T140 NCBITaxon:7729 6916 6935 Halocynthia rortezi +T141 SO:0000345 6936 6939 EST +T142 SO:0000704 7012 7016 gene +T143 NCBITaxon:7711 7036 7044 chordate +T144 NCBITaxon:7711 7100 7108 chordate +T145 SO:0001026 7109 7115 genome +T146 NCBITaxon:7711 7223 7231 chordate +T147 SO:0001026 7232 7238 genome +T148 SO:0000704 7313 7318 genes +T149 SO:0000704 7389 7394 genes +T150 NCBITaxon:7742 7427 7437 vertebrate +T151 SO:0001026 7438 7444 genome +T152 NCBITaxon:31031 7468 7478 pufferfish +T153 NCBITaxon:31033 7479 7496 Takifugu rubripes +T154 SO:0000704 7509 7514 genes +T155 NCBITaxon:9606 7536 7541 human +T156 PR:000016006 7542 7547 TACC1 +T157 SO:0001026 7595 7602 genomic +T158 SO:0000704 7652 7657 genes +T159 SO:0000704 7695 7699 gene +T160 PR:000016008 7718 7723 TACC3 +T161 SO:0000704 7752 7757 genes +T162 NCBITaxon:31033 7777 7788 T. rubripes +T163 SO:0000704 7804 7809 genes +T164 NCBITaxon:31033 7830 7841 T. rubripes +T165 SO:0000855 7842 7853 orthologues +T166 NCBITaxon:9606 7857 7862 human +T167 PR:000016007 7863 7868 TACC2 +T168 PR:000016008 7873 7878 TACC3 +T169 SO:0000704 7903 7908 genes +T170 PR:000016006 7960 7965 TACC1 +T171 SO:0000857 8004 8014 homologous +T172 SO:0000704 8103 8107 gene +T173 SO:0000147 8125 8130 exons +T174 NCBITaxon:31032 8162 8170 Takifugu +T175 SO:0001248 8171 8179 Scaffold +T176 SO:0000704 8253 8257 gene +T177 SO:0000147 8319 8324 exons +T178 SO:0000188 8357 8363 intron +T179 SO:0000147 8364 8368 exon +T180 SO:0000704 8407 8411 gene +T181 NCBITaxon:31031 8462 8472 pufferfish +T182 NCBITaxon:1 8552 8560 organism +T183 SO:0000704 8724 8729 genes +T184 NCBITaxon:32443 8815 8822 teleost +T185 PR:000016006 8823 8828 TACC1 +T186 SO:0001026 8869 8876 genomic +T187 NCBITaxon:32443 8910 8917 teleost +T188 NCBITaxon:31033 8955 8966 T. rubripes +T189 PR:000016008 9031 9036 TACC3 +T190 NCBITaxon:7742 9082 9092 vertebrate +T191 SO:0001026 9093 9100 genomic +T192 NCBITaxon:7742 9137 9148 vertebrates +T193 GO:0008380 9178 9184 splice +T194 PR:000016006 9197 9202 TACC1 +T195 PR:000016007 9207 9212 TACC2 +T196 NCBITaxon:10090 9216 9228 Mus musculus +T197 SO:0000855 9278 9289 orthologues +T198 PR:000016006 9293 9298 TACC1 +T199 NCBITaxon:10116 9306 9321 Rattus norvegus +T200 NCBITaxon:10088 9369 9374 mouse +T201 GO:0000805 9375 9387 chromosome X +T202 SO:0000704 9394 9398 gene +T203 NCBITaxon:10088 9425 9430 mouse +T204 PR:000016006 9431 9436 TACC1 +T205 NCBITaxon:10088 9478 9483 mouse +T206 SO:0000188 9538 9544 intron +T207 GO:0010467 9562 9572 expression +T208 NCBITaxon:10088 9599 9604 mouse +T209 SO:0000043 9681 9701 processed pseudogene +T210 PR:000016006 9714 9719 TACC1 +T211 SO:0000336 9720 9731 pseudogenes +T212 NCBITaxon:9606 9790 9795 human +T213 SO:0000028 9872 9874 bp +T214 PR:000016006 9882 9887 TACC1 +T215 SO:0000205 9888 9910 3' untranslated region +T216 GO:0006412 9893 9903 translated +T217 SO:0000336 9915 9926 pseudogenes +T218 PR:000016007 9944 9949 TACC2 +T219 PR:000016008 9953 9958 TACC3 +T220 NCBITaxon:40674 9982 9991 mammalian +T221 NCBITaxon:species 9992 9999 species +T222 NCBITaxon:7742 10022 10032 vertebrate +T223 PR:000016008 10033 10038 TACC3 +T224 SO:0000855 10039 10050 orthologues +T225 PR:000016008 10116 10121 TACC3 +T226 SO:0000855 10122 10133 orthologues +T227 SO:0000704 10225 10229 gene +T228 SO:0000704 10301 10305 gene +T229 NCBITaxon:7742 10345 10355 vertebrate +T230 NCBITaxon:31033 10392 10403 T. rubripes +T231 NCBITaxon:9989 10405 10412 rodents +T232 NCBITaxon:9606 10417 10423 humans +T233 NCBITaxon:7742 10449 10459 vertebrate +T234 PR:000016008 10460 10465 TACC3 +T235 SO:0001026 10516 10523 genomic +T236 SO:0000704 10575 10579 gene +T237 NCBITaxon:9986 10689 10710 Oryctolagus cuniculus +T238 PR:000016008 10833 10838 TACC3 +T239 SO:0000704 10888 10892 gene +T240 SO:0000673 10918 10928 transcript +T241 NCBITaxon:9606 11036 11041 human +T242 NCBITaxon:10088 11046 11051 mouse +T243 PR:000016008 11052 11057 TACC3 +T244 NCBITaxon:9986 11115 11121 rabbit +T245 PR:000016008 11122 11127 TACC3 +T246 NCBITaxon:9986 11192 11198 rabbit +T247 PR:000016008 11199 11204 TACC3 +T248 NCBITaxon:9606 11248 11253 human +T249 NCBITaxon:10088 11258 11263 mouse +T250 PR:000016008 11264 11269 TACC3 +T251 NCBITaxon:9606 11346 11351 human +T252 NCBITaxon:10088 11356 11361 mouse +T253 PR:000016008 11362 11367 TACC3 +T254 SO:0000704 11474 11479 genes +T255 SO:0000993 11495 11504 consensus +T256 SO:0000112 11521 11527 primer +T257 PR:000016008 11640 11645 TACC3 +T258 NCBITaxon:9443 11657 11665 primates +T259 NCBITaxon:9989 11670 11677 rodents +T260 SO:0000112 11690 11696 primer +T261 SO:0000112 11742 11748 primer +T262 SO:0000006 11817 11828 PCR product +T263 NCBITaxon:9986 11834 11840 rabbit +T264 UBERON:0000955 11841 11846 brain +T265 SO:0000993 11908 11917 consensus +T266 SO:0000028 11931 11933 bp +T267 SO:0000673 11959 11969 transcript +T268 GO:0000380 12126 12144 alternative splice +T269 PR:000016008 12196 12201 TACC3 +T270 SO:0000704 12202 12206 gene +T271 NCBITaxon:9986 12245 12251 rabbit +T272 SO:0000673 12263 12273 transcript +T273 NCBITaxon:9986 12286 12292 rabbit +T274 SO:0002138 12316 12336 predicted transcript +T275 PR:000016008 12349 12354 TACC3 +T276 NCBITaxon:9986 12701 12707 rabbit +T277 NCBITaxon:9986 12724 12730 rabbit +T278 PR:000016008 12731 12736 TACC3 +T279 PR:000016008 12847 12852 TACC3 +T280 SO:0000855 12853 12864 orthologues +T281 NCBITaxon:8355 12884 12898 Xenopus laevis +T282 PR:000016008 12926 12931 TACC3 +T283 NCBITaxon:10116 12945 12960 Rattus norvegus +T284 NCBITaxon:9031 12962 12975 Gallus gallus +T285 NCBITaxon:8364 12977 12996 Silurana tropicalis +T286 NCBITaxon:7955 12998 13009 Danio rerio +T287 NCBITaxon:31033 13014 13025 T. rubripes +T288 PR:000001856 13192 13197 RHAMM +T289 SO:0000704 13198 13202 gene +T290 SO:0001080 13227 13238 coiled coil +T291 SO:0000704 13239 13243 gene +T292 NCBITaxon:9606 13252 13257 Human +T293 PR:000001856 13258 13263 RHAMM +T294 NCBITaxon:9606 13428 13434 humans +T295 SO:0001080 13485 13496 coiled coil +T296 SO:0000417 13497 13503 domain +T297 SO:0000417 13516 13522 domain +T298 PR:000001856 13563 13568 RHAMM +T299 GO:0005813 13584 13594 centrosome +T300 PR:000001856 13608 13613 RHAMM +T301 NCBITaxon:7742 13786 13796 vertebrate +T302 SO:0001026 13797 13803 genome +T303 PR:000001856 13834 13839 RHAMM +T304 SO:0000855 13840 13851 orthologues +T305 SO:0000704 13864 13869 genes +T306 NCBITaxon:33208 13873 13882 metazoans +T307 SO:0001080 13925 13936 coiled coil +T308 PR:000001856 13993 13998 RHAMM +T309 SO:0000704 13999 14003 gene +T310 NCBITaxon:33511 14011 14024 deuterostomes +T311 SO:0001026 14047 14054 genomic +T312 NCBITaxon:7719 14089 14104 C. intestinalis +T313 PR:000001856 14109 14114 RHAMM +T314 SO:0000704 14115 14119 gene +T315 NCBITaxon:6960 14138 14145 insects +T316 NCBITaxon:6231 14149 14158 nematodes +T317 PR:000001856 14184 14189 RHAMM +T318 SO:0000704 14195 14200 genes +T319 NCBITaxon:33317 14220 14230 protostome +T320 NCBITaxon:33511 14231 14243 deuterostome +T321 NCBITaxon:7586 14280 14293 echinodermata +T322 NCBITaxon:7712 14294 14305 urochordate +T323 SO:0001080 14382 14393 coiled coil +T324 PR:000001856 14431 14436 RHAMM +T325 SO:0000417 14461 14467 domain +T326 SO:0001080 14523 14534 coiled coil +T327 SO:0000704 14601 14605 gene +T328 SO:0000704 14666 14671 genes +T329 SO:0000704 14707 14712 genes +T330 NCBITaxon:7742 14743 14753 vertebrate +T331 PR:000016006 14754 14759 TACC1 +T332 SO:0000704 14762 14767 genes +T333 SO:0000704 14844 14848 gene +T334 NCBITaxon:9606 14859 14865 humans +T335 NCBITaxon:10088 14867 14872 mouse +T336 NCBITaxon:6656 14881 14889 arthopod +T337 NCBITaxon:7227 14890 14905 D. melanogaster +T338 SO:0000854 14940 14950;14963 14971 paralogous ... segments +T339 NCBITaxon:1 14986 14995 organisms +T340 NCBITaxon:7742 15143 15153 vertebrate +T341 NCBITaxon:9606 15214 15219 human +T342 NCBITaxon:9606 15249 15254 human +T343 SO:0000704 15397 15401 gene +T344 SO:0000854 15495 15505;15518 15526 paralogous ... segments +T345 SO:0000704 15622 15626 gene +T346 NCBITaxon:7742 15630 15641 vertebrates +T347 PR:000001451 15689 15694 FGFR4 +T348 PR:000001856 15752 15757 RHAMM +T349 SO:0000704 15758 15762 gene +T350 NCBITaxon:9606 15800 15805 Human +T351 PR:000001856 15806 15811 RHAMM +T352 PR:000008212 15859 15863 GPX3 +T353 PR:000011245 15868 15873 NKX2E +T354 SO:0000704 15911 15916 genes +T355 NCBITaxon:9606 15920 15925 human +T356 SO:0000859 15948 15958 paralogous +T357 NCBITaxon:10088 16046 16051 mouse +T358 NCBITaxon:10114 16056 16059 rat +T359 SO:0000704 16110 16115 genes +T360 NCBITaxon:9443 16158 16165 primate +T361 NCBITaxon:9989 16166 16172 rodent +T362 SO:0000987 16196 16202 Linear +T363 SO:0000704 16219 16223 gene +T364 SO:0000704 16281 16286 genes +T365 NCBITaxon:9606 16290 16296 humans +T366 SO:0000859 16298 16308 Paralogous +T367 SO:0000704 16309 16314 genes +T368 PR:000008212 16408 16412 GPX3 +T369 PR:000011245 16417 16422 NKX2E +T370 SO:0000704 16491 16496 genes +T371 SO:0000704 16558 16563 genes +T372 SO:0005858 16599 16607;16626 16633 syntenic ... regions +T373 NCBITaxon:10088 16608 16613 mouse +T374 NCBITaxon:31033 16655 16672 Takifugu rubripes +T375 SO:0001248 16673 16682 scaffolds +T376 SO:0000857 16726 16736 homologous +T377 SO:0000704 16737 16741 gene +T378 SO:0000859 16798 16808 paralogous +T379 SO:0000704 16809 16814 genes +T380 SO:0000704 16885 16889 gene +T381 SO:0000704 16939 16943 gene +T382 GO:0065007 16944 16954 regulation +T383 SO:0000854 17006 17025 paralogous segments +T384 SO:0001026 17043 17049 genome +T385 NCBITaxon:7712 17057 17065 tunicate +T386 NCBITaxon:7719 17066 17081 C. intestinalis +T387 NCBITaxon:7742 17120 17130 vertebrate +T388 SO:0001026 17131 17137 genome +T389 NCBITaxon:31033 17157 17168 T. rubripes +T390 SO:0001026 17224 17230 genome +T391 NCBITaxon:31033 17234 17245 T. rubripes +T392 SO:0000859 17293 17303 paralogous +T393 NCBITaxon:7742 17329 17340 vertebrates +T394 SO:0000855 17369 17380 orthologues +T395 PR:000008258 17384 17390 GPRK2L +T396 PR:000013947 17395 17400 RGS12 +T397 NCBITaxon:31033 17414 17425 T. rubripes +T398 SO:0001248 17426 17434 scaffold +T399 NCBITaxon:9606 17496 17501 human +T400 NCBITaxon:31033 17512 17523 T. rubripes +T401 SO:0000855 17524 17535 orthologues +T402 PR:000001450 17539 17544 FGFR3 +T403 SO:0001026 17593 17600 genomic +T404 SO:0001248 17601 17609 scaffold +T405 NCBITaxon:9606 17661 17666 human +T406 SO:0000855 17667 17678 orthologues +T407 SO:0000704 17688 17693 genes +T408 PR:000016008 17761 17766 TACC3 +T409 PR:000007506 17771 17776 FGFRL +T410 SO:0001248 17800 17809 scaffolds +T411 SO:0000704 17869 17873 gene +T412 PR:000008260 17908 17913 GPRK6 +T413 NCBITaxon:9606 17917 17922 human +T414 NCBITaxon:31031 17965 17975 pufferfish +T415 NCBITaxon:31033 17986 17997 T. rubripes +T416 SO:0000855 17998 18009 orthologues +T417 PR:000011441 18013 18017 NSD1 +T418 PR:000001451 18019 18024 FGFR4 +T419 SO:0000704 18040 18044 gene +T420 SO:0001248 18056 18064 scaffold +T421 SO:0000704 18125 18129 gene +T422 SO:0000704 18155 18159 gene +T423 PR:000016006 18275 18280 TACC1 +T424 SO:0000704 18289 18294 genes +T425 NCBITaxon:31031 18302 18312 pufferfish +T426 SO:0001248 18348 18356 scaffold +T427 SO:0000855 18407 18418 orthologues +T428 SO:0000704 18430 18435 genes +T429 NCBITaxon:9606 18447 18452 human +T430 SO:0001248 18484 18492 scaffold +T431 PR:000016006 18525 18530 TACC1 +T432 NCBITaxon:7742 18577 18588 vertebrates +T433 SO:0000704 18622 18626 gene +T434 SO:0001248 18652 18660 scaffold +T435 SO:0001248 18689 18697 scaffold +T436 NCBITaxon:31033 18716 18727 T. rubripes +T437 SO:0000855 18728 18739 orthologues +T438 PR:000010686 18743 18747 MSX1 +T439 PR:000015790 18749 18754 STX18 +T440 SO:0000996 18772 18786 predicted gene +T441 SO:0000857 18828 18836 homology +T442 PR:000009884 18840 18844 LOXL +T443 PR:000007232 18846 18849 EVC +T444 SO:0001248 18888 18896 scaffold +T445 SO:0000704 18909 18914 genes +T446 NCBITaxon:9606 18939 18944 human +T447 PR:000016008 18996 19001 TACC3 +T448 PR:000016007 19006 19011 TACC2 +T449 SO:0001026 19071 19078 genomic +T450 SO:0000854 19148 19158;19171 19178 paralogous ... segment +T451 SO:0000859 19191 19201 paralogous +T452 SO:0000704 19257 19262 genes +T453 SO:0001026 19295 19302 genomic +T454 SO:0000149 19303 19309 contig +T455 SO:0000854 19391 19401;19414 19422 paralogous ... segments +T456 NCBITaxon:7742 19439 19450 vertebrates +T457 SO:0001026 19469 19475 genome +T458 NCBITaxon:7712 19483 19494 urochordate +T459 NCBITaxon:7719 19495 19510 C. intestinalis +T460 SO:0000855 19535 19546 orthologues +T461 PR:000030426 19560 19565 WHSC1 +T462 PR:000005841 19567 19585 carboxypeptidase Z +T463 NCBITaxon:7719 19637 19652 C. intestinalis +T464 SO:0001026 19653 19659 genome +T465 NCBITaxon:9606 19668 19673 human +T466 SO:0000855 19674 19685 orthologues +T467 SO:0000854 19710 19729 paralogous segments +T468 SO:0000704 19833 19838 genes +T469 NCBITaxon:7742 19848 19858 vertebrate +T470 SO:0000854 19859 19878 paralogous segments +T471 PR:000001856 19911 19916 RHAMM +T472 SO:0000704 19917 19922 genes +T473 NCBITaxon:7719 19926 19941 C. intestinalis +T474 SO:0000854 19992 20011 paralogous segments +T475 SO:0000704 20039 20044 genes +T476 NCBITaxon:7712 20142 20151 tunicates +T477 NCBITaxon:31033 20196 20207 T. rubripes +T478 SO:0001026 20208 20214 genome +T479 PR:000001450 20345 20350 FGFR3 +T480 SO:0000704 20385 20389 gene +T481 SO:0000704 20406 20410 gene +T482 NCBITaxon:7742 20434 20444 vertebrate +T483 NCBITaxon:61463 20487 20499 Gnanthostome +T484 SO:0000854 20574 20592 paralogous segment +T485 NCBITaxon:7737 20626 20635 amphioxus +T486 NCBITaxon:7745 20640 20647 lamprey +T487 SO:0001026 20648 20655 genomes +T488 SO:0000704 20682 20686 gene +T489 SO:0000704 20758 20763 genes +T490 NCBITaxon:31033 20832 20849 Takifugu rubripes +T491 NCBITaxon:7719 20854 20872 Ciona intestinalis +T492 SO:0001026 20896 20903 genomic +T493 SO:0001248 20904 20913 scaffolds +T494 NCBITaxon:31033 20929 20946 Takifugu rubripes +T495 SO:0000704 20969 20974 genes +T496 SO:0001248 20976 20984 Scaffold +T497 SO:0000704 21015 21019 gene +T498 SO:0000704 21029 21034 genes +T499 SO:0000853 21053 21063 homologues +T500 SO:0000855 21067 21078 orthologues +T501 SO:0000105 21098 21104;21111 21121 arm of ... chromosome +T502 NCBITaxon:9606 21105 21110 human +T503 SO:0001248 21140 21148 scaffold +T504 PR:000016006 21236 21241 TACC1 +T505 PR:000016007 21242 21247 TACC2 +T506 SO:0001248 21290 21298 scaffold +T507 SO:0000855 21319 21330 orthologues +T508 SO:0000704 21334 21339 genes +T509 SO:0000105 21368 21374;21381 21391 arm of ... chromosome +T510 NCBITaxon:9606 21375 21380 human +T511 NCBITaxon:7719 21400 21418 Ciona intestinalis +T512 SO:0000704 21439 21444 genes +T513 SO:0000854 21454 21473 paralogous segments +T514 NCBITaxon:9606 21477 21482 human +T515 SO:0001026 21515 21522 Genomic +T516 SO:0000704 21545 21550 genes +T517 NCBITaxon:9606 21607 21612 human +T518 NCBITaxon:9606 21656 21661 human +T519 PR:000016006 21662 21667 TACC1 +T520 PR:000017527 21687 21692 GAS41 +T521 SO:0000417 21731 21737 domain +T522 PR:000005512 21744 21750 ch-TOG +T523 PR:000035365 21770 21784 Aurora kinases +T524 NCBITaxon:species 21792 21799 species +T525 SO:0000147 21869 21874 exons +T526 SO:0000704 21887 21892 genes +T527 SO:0001060 21918 21925 isoform +T528 SO:0000704 21935 21939 gene +T529 SO:0001026 21963 21970 genomic +T530 SO:0001026 22005 22012 genomic +T531 SO:0000858 22048 22059 orthologous +T532 SO:0000704 22065 22070 genes +T533 NCBITaxon:9606 22074 22079 human +T534 NCBITaxon:10088 22081 22086 mouse +T535 NCBITaxon:10114 22088 22091 rat +T536 NCBITaxon:31031 22093 22103 pufferfish +T537 NCBITaxon:7719 22105 22120 C. intestinalis +T538 NCBITaxon:7227 22122 22137 D. melanogaster +T539 NCBITaxon:6239 22142 22152 C. elegans +T540 SO:0001026 22220 22227 genomic +T541 SO:0000704 22251 22255 gene +T542 NCBITaxon:10114 22276 22279 rat +T543 NCBITaxon:31031 22284 22294 pufferfish +T544 SO:0000147 22296 22301 exons +T545 GO:0006412 22357 22367 translated +T546 NCBITaxon:10088 22398 22403 mouse +T547 NCBITaxon:9606 22408 22413 human +T548 NCBITaxon:31033 22468 22479 T. rubripes +T549 SO:0001026 22481 22488 genomic +T550 CHEBI:15377 22515 22520 water +T551 NCBITaxon:31031 22521 22531 pufferfish +T552 NCBITaxon:99883 22533 22555 Tetraodon nigroviridis +T553 SO:0000147 22610 22615 exons +T554 SO:0000704 22652 22657 genes +T555 SO:0000417 22750 22756 domain +T556 NCBITaxon:6239 22828 22838 C. elegans +T557 SO:0000147 22940 22945 exons +T558 SO:0000704 22953 22957 gene +T559 NCBITaxon:1 22973 22982 organisms +T560 NCBITaxon:7227 22984 22999 D. melanogaster +T561 NCBITaxon:33511 23009 23022 deuterostomes +T562 NCBITaxon:7719 23023 23038 C. intestinalis +T563 NCBITaxon:9606 23042 23047 human +T564 SO:0000147 23091 23096 exons +T565 SO:0000704 23104 23108 gene +T566 NCBITaxon:7227 23118 23133 D. melanogaster +T567 NCBITaxon:33511 23148 23160 deuterostome +T568 SO:0000704 23161 23166 genes +T569 SO:0000417 23189 23195 domain +T570 SO:0000857 23249 23257 homology +T571 SO:0000704 23292 23296 gene +T572 SO:0000147 23316 23320 exon +T573 SO:0000855 23372 23383 orthologues +T574 PR:000016008 23433 23438 TACC3 +T575 SO:0000704 23439 23444 genes +T576 NCBITaxon:7742 23452 23463 vertebrates +T577 NCBITaxon:33511 23480 23493 deuterostomes +T578 SO:0000147 23500 23504 exon +T579 NCBITaxon:39107 23552 23558 murine +T580 PR:000016008 23559 23566 TACC3's +T581 NCBITaxon:9989 23570 23576 rodent +T582 GO:0016514 23653 23689 SWI/SNF chromatin remodeling complex +T583 GO:0006338 23661 23681 chromatin remodeling +T584 PR:000017527 23700 23705 GAS41 +T585 NCBITaxon:7742 23723 23733 vertebrate +T586 PR:000016008 23753 23758 TACC3 +T587 SO:0000855 23759 23770 orthologues +T588 NCBITaxon:10114 23868 23871 rat +T589 PR:000016008 23872 23877 TACC3 +T590 NCBITaxon:7955 23913 23924 Danio rerio +T591 SO:0001026 23990 23997 genomic +T592 PR:000016008 24015 24020 TACC3 +T593 SO:0000855 24021 24032 orthologues +T594 PR:000016008 24034 24039 TACC3 +T595 SO:0000147 24144 24149 exons +T596 NCBITaxon:7742 24166 24176 vertebrate +T597 PR:000016008 24177 24182 TACC3 +T598 SO:0000704 24183 24187 gene +T599 SO:0000417 24208 24214 domain +T600 SO:0000147 24248 24253 exons +T601 PR:000016008 24374 24379 TACC3 +T602 NCBITaxon:9606 24392 24397 human +T603 NCBITaxon:10088 24402 24407 mouse +T604 SO:0000855 24491 24502 orthologues +T605 SO:0000147 24602 24606 exon +T606 NCBITaxon:9606 24610 24615 human +T607 NCBITaxon:31031 24624 24634 pufferfish +T608 NCBITaxon:9989 24658 24665 rodents +T609 SO:0000147 24778 24782 exon +T610 NCBITaxon:10088 24790 24795 mouse +T611 NCBITaxon:10114 24800 24803 rat +T612 PR:000016008 24804 24809 TACC3 +T613 SO:0000704 24810 24815 genes +T614 NCBITaxon:10088 24869 24874 mouse +T615 PR:000016008 24875 24880 TACC3 +T616 GO:0008380 24881 24887 splice +T617 SO:0000147 24991 24995 exon +T618 NCBITaxon:9986 25319 25325 rabbit +T619 NCBITaxon:9989 25388 25394 rodent +T620 PR:000016008 25395 25400 TACC3 +T621 NCBITaxon:8292 25467 25476 amphibian +T622 NCBITaxon:8353 25477 25484 Xenopus +T623 PR:000016008 25485 25490 TACC3 +T624 GO:0000380 25505 25525 Alternative splicing +T625 NCBITaxon:7742 25529 25539 vertebrate +T626 SO:0000704 25545 25550 genes +T627 SO:0000147 25560 25564 exon +T628 SO:0000704 25619 25623 gene +T629 UBERON:0000479 25677 25683 tissue +T630 GO:0000380 25693 25713 alternative splicing +T631 SO:0000704 25719 25723 gene +T632 SO:0000704 25780 25784 gene +T633 GO:0044767 25796 25810;25814 25822 development of ... organism +T634 NCBITaxon:1 25814 25822 organism +T635 GO:0000380 25836 25856 alternative splicing +T636 PR:000016008 25860 25865 TACC3 +T637 UBERON:0000479 25913 25919 tissue +T638 GO:0008380 25929 25937 splicing +T639 PR:000016006 25957 25962 TACC1 +T640 PR:000016007 25967 25972 TACC2 +T641 SO:0000704 25973 25978 genes +T642 PR:000016007 25995 26000 TACC2 +T643 SO:0000147 26029 26033 exon +T644 GO:0008380 26085 26091 splice +T645 NCBITaxon:7742 26108 26118 vertebrate +T646 PR:000016007 26119 26124 TACC2 +T647 SO:0000704 26125 26130 genes +T648 GO:0000380 26140 26160 alternative splicing +T649 SO:0000147 26169 26173 exon +T650 PR:000016007 26229 26234 TACC2 +T651 SO:0001060 26235 26243 isoforms +T652 PR:000016007 26312 26317 TACC2 +T653 PR:000016006 26356 26361 TACC1 +T654 SO:0001060 26414 26421 isoform +T655 GO:0000380 26443 26463 Alternative splicing +T656 SO:0000167 26492 26500 promoter +T657 NCBITaxon:9606 26538 26543 human +T658 PR:000016006 26544 26549 TACC1 +T659 SO:0000704 26550 26554 gene +T660 PR:000016006 26627 26632 TACC1 +T661 SO:0001060 26633 26641 isoforms +T662 GO:0000380 26659 26679 alternative splicing +T663 SO:0000147 26683 26688 exons +T664 SO:0001060 26729 26737 isoforms +T665 SO:0000409 26820 26832 binding site +T666 PR:000009965 26837 26841 LSm7 +T667 GO:0005634 26880 26887 nuclear +T668 SO:0001528 26880 26908 nuclear localization signals +T669 SO:0000409 26913 26925 binding site +T670 PR:000017527 26930 26935 GAS41 +T671 PR:000029784 26945 26955 PCTAIRE2BP +T672 SO:0001060 26962 26970 isoforms +T673 SO:0001060 26993 27001 isoforms +T674 GO:0005737 27042 27051 cytoplasm +T675 SO:0001060 27086 27094 isoforms +T676 GO:0000785 27146 27154 chomatin +T677 GO:0006338 27146 27165 chomatin remodeling +T678 GO:0006396 27173 27187 RNA processing +T679 GO:0005634 27205 27212 nucleus +T680 PR:000016006 27249 27254 TACC1 +T681 SO:0001060 27255 27263 isoforms +T682 GO:0044237 27305 27313;27318 27328 cellular ... metabolism +T683 GO:0016070 27314 27328 RNA metabolism +T684 SO:0001060 27408 27416 isoforms +T685 UBERON:0001199 27460 27474 gastric mucosa +T686 GO:0000380 27492 27512 Alternative splicing +T687 NCBITaxon:9606 27520 27525 human +T688 PR:000016006 27526 27531 TACC1 +T689 SO:0000704 27532 27536 gene +T690 GO:0008380 27549 27555 splice +T691 NCBITaxon:9606 27590 27595 human +T692 PR:000016006 27596 27601 TACC1 +T693 GO:0008380 27650 27656 splice +T694 NCBITaxon:9606 27718 27723 human +T695 UBERON:0000955 27724 27729 brain +T696 GO:0000380 27740 27760 Alternative splicing +T697 PR:000016006 27764 27769 TACC1 +T698 NCBITaxon:9606 27777 27782 human +T699 UBERON:0000955 27783 27788 brain +T700 GO:0008380 27815 27823 splicing +T701 SO:0000198 27831 27848 untranslated exon +T702 GO:0006412 27833 27843 translated +T703 SO:0000147 27855 27859 exon +T704 SO:0000147 27965 27969 Exon +T705 GO:0008380 27978 27985 splices +T706 SO:0000147 27989 27993 exon +T707 PR:000009965 28010 28015 L-Sm7 +T708 SO:0000147 28096 28101 exons +T709 GO:0005634 28168 28175 nuclear +T710 SO:0001528 28168 28196 nuclear localization signals +T711 SO:0000417 28214 28221 domains +T712 PR:000017527 28226 28231 GAS41 +T713 PR:000029784 28236 28246 PCTAIRE2BP +T714 SO:0000417 28285 28291 domain +T715 PR:000005512 28332 28338 ch-TOG +T716 PR:000004515 28343 28358 Aurora A kinase +T717 GO:0005813 28366 28376 centrosome +T718 SO:0001026 28762 28769 genomic +T719 SO:0000417 28943 28949 domain +T720 GO:0007067 29021 29028 mitotic +T721 GO:0072686 29021 29036 mitotic spindle +T722 GO:0005813 29041 29052 centrosomal +T723 SO:0000853 29180 29189 homologue +T724 PR:P39723 29200 29206 spc-72 +T725 NCBITaxon:9606 29224 29229 human +T726 SO:0000417 29335 29341 domain +T727 GO:0005874 29352 29363 microtubule +T728 GO:0005813 29364 29375 centrosomal +T729 PR:P46675 29392 29396 stu2 +T730 PR:Q9VEZ3 29397 29401 msps +T731 PR:000005512 29402 29408 ch-TOG +T732 PR:000035365 29442 29456 Aurora kinases +T733 PR:P39723 29537 29542 spc72 +T734 PR:000016008 29555 29560 TACC3 +T735 GO:0005813 29577 29587 centrosome +T736 GO:0005813 29645 29655 centrosome +T737 GO:0007067 29660 29667 mitotic +T738 GO:0072686 29660 29675 mitotic spindle +T739 NCBITaxon:9606 29784 29789 human +T740 PR:000035365 29854 29868 Aurora kinases +T741 PR:000016006 29884 29889 TACC1 +T742 PR:000016008 29894 29899 TACC3 +T743 PR:000004515 29914 29929 Aurora A kinase +T744 PR:000016007 29939 29944 TACC2 +T745 PR:000004518 29960 29975 Aurora C kinase +T746 NCBITaxon:7711 30067 30075 chordate +T747 NCBITaxon:7742 30109 30119 vertebrate +T748 SO:0000704 30125 30130 genes +T749 NCBITaxon:7742 30157 30167 vertebrate +T750 GO:0051325 30189 30199 interphase +T751 GO:0005634 30200 30207 nucleus +T752 GO:0005813 30298 30308 centrosome +T753 GO:0005874 30313 30324 microtubule +T754 GO:0000226 30313 30333 microtubule dynamics +T755 NCBITaxon:33317 30385 30396 protostomes +T756 NCBITaxon:33511 30401 30414 deuterostomes +T757 SO:0000704 30473 30478 genes +T758 SO:0000147 30499 30504 exons +T759 NCBITaxon:6239 30565 30575 C. elegans +T760 NCBITaxon:7227 30580 30595 D. melanogaster +T761 NCBITaxon:6239 30727 30737 C. elegans +T762 PR:Q27365 30727 30744 C. elegans lin15A +T763 PR:P34427 30727 30737;30746 30751 C. elegans ... lin36 +T764 PR:000009822 30756 30761 lin37 +T765 GO:0030674 30783 30789 bridge +T766 GO:0005856 30821 30833 cytoskeleton +T767 GO:0005874 30838 30849 microtubule +T768 GO:0005840 30891 30899 ribosome +T769 PR:000043452 30905 30912 histone +T770 CHEBI:15358 30905 30912 histone +T771 GO:0000785 30925 30934 chromatin +T772 GO:0006338 30925 30945 chromatin remodeling +T773 PR:000006937 30964 30969 egr-1 +T774 PR:P90916 30974 30980 lin-53 +T775 NCBITaxon:6239 30986 30996 C. elegans +T776 SO:0000853 30997 31007 homologues +T777 NCBITaxon:9606 31015 31020 human +T778 PR:000010707 31021 31026 MTA-1 +T779 PR:000013776 31031 31037 RbAP48 +T780 PR:P34766 31081 31085 PAL1 +T781 GO:0005634 31103 31110 nuclear +T782 PR:Q965W2 31128 31134 nhr-86 +T783 NCBITaxon:7215 31205 31215 Drosophila +T784 NCBITaxon:7215 31331 31341 Drosophila +T785 GO:0016514 31342 31378 SWI/SNF chromatin remodeling complex +T786 GO:0006338 31350 31370 chromatin remodeling +T787 GO:0006281 31383 31400 DNA damage repair +T788 GO:0006281 31482 31492 DNA repair +T789 NCBITaxon:6239 31533 31543 C. elegans +T790 PR:000004642 31544 31549 BARD1 +T791 SO:0000855 31550 31560 orthologue +T792 NCBITaxon:1 31650 31659 organisms +T793 PR:P46675 31798 31802 stu2 +T794 PR:Q9VEZ3 31803 31807 msps +T795 PR:000005512 31808 31814 ch-TOG +T796 NCBITaxon:6239 31887 31897 C. elegans +T797 NCBITaxon:7227 31902 31917 D. melanogaster +T798 NCBITaxon:6239 31924 31934 C. elegans +T799 GO:0000785 32147 32156 chromatin +T800 GO:0006338 32147 32167 chromatin remodeling +T801 GO:0032991 32168 32177 complexes +T802 GO:0016514 32179 32186 SWI/SNF +T803 PR:000043452 32191 32198 histone +T804 CHEBI:15358 32191 32198 histone +T805 GO:0006281 32220 32237 DNA damage repair +T806 GO:0008380 32265 32277 RNA splicing +T807 GO:0050658 32265 32268;32279 32288 RNA ... transport +T808 GO:0006412 32293 32306 translational +T809 NCBITaxon:7742 32353 32363 vertebrate +T810 GO:0005634 32431 32438 nuclear +T811 PR:000014373 32456 32460 RXRβ +T812 SO:0000854 32601 32620 paralogous segments +T813 NCBITaxon:7742 32722 32732 Vertebrate +T814 NCBITaxon:7742 32827 32837 vertebrate +T815 SO:0000853 32838 32847 homologue +T816 PR:000035365 32911 32924 Aurora kinase +T817 SO:0000417 32987 32993 domain +T818 NCBITaxon:6239 33061 33071 C. elegans +T819 NCBITaxon:7227 33076 33091 D. melanogaster +T820 NCBITaxon:33208 33120 33127 animals +T821 NCBITaxon:7742 33236 33246 vertebrate +T822 NCBITaxon:6239 33296 33306 C. elegans +T823 PR:Q27365 33325 33331 lin15A +T824 PR:P34427 33333 33338 lin36 +T825 PR:000009822 33343 33348 lin37 +T826 SO:0000853 33381 33391 homologues +T827 NCBITaxon:7742 33395 33406 vertebrates +T828 NCBITaxon:7215 33410 33420 Drosophila +T829 SO:0000417 33461 33467 domain +T830 PR:P34427 33471 33476 lin36 +T831 PR:000016057 33664 33669 TDP43 +T832 GO:0065007 33711 33721 regulation +T833 GO:0008380 33726 33734 splicing +T834 NCBITaxon:9606 33775 33780 human +T835 SO:0000853 33781 33790 homologue +T836 NCBITaxon:9606 33849 33854 human +T837 SO:0001980 33940 33945 G-box +T838 NCBITaxon:7742 34033 34043 vertebrate +T839 NCBITaxon:7742 34088 34098 vertebrate +T840 NCBITaxon:7742 34285 34295 vertebrate +T841 GO:0005813 34398 34408 centrosome +T842 GO:0007067 34409 34416 mitotic +T843 GO:0072686 34409 34424 mitotic spindle +T844 SO:0000704 34463 34467 gene +T845 GO:0065007 34468 34478 regulation +T846 GO:0006396 34532 34546 RNA processing +T847 GO:0006412 34551 34562 translation +T848 GO:0043234 34718 34733 protein complex +T849 GO:0005813 34811 34821 centrosome +T850 GO:0072686 34842 34857 mitotic spindle +T851 GO:0090307 34842 34866 mitotic spindle assembly +T852 NCBITaxon:7742 34965 34975 vertebrate +T853 PR:000016008 34976 34981 TACC3 +T854 GO:0007067 35086 35093 mitosis +T855 PR:000004515 35128 35143 Aurora Kinase A +T856 NCBITaxon:9606 35185 35190 human +T857 SO:0000417 35280 35286 domain +T858 NCBITaxon:7742 35294 35304 vertebrate +T859 SO:0000855 35305 35316 orthologues +T860 PR:000016008 35332 35337 TACC3 +T861 NCBITaxon:species 35396 35403 species +T862 NCBITaxon:8355 35441 35450 X. laevis +T863 SO:0000410 35486 35502;35513 35520 binding site for ... protein +T864 PR:000006990 35507 35512 eIF4E +T865 GO:0006417 35561 35571;35592 35603 control of ... translation +T866 GO:0043631 35572 35587 polyadenylation +T867 NCBITaxon:8353 35611 35618 Xenopus +T868 CL:0000023 35619 35625 oocyte +T869 NCBITaxon:7742 35748 35758 vertebrate +T870 PR:000016008 35759 35764 TACC3 +T871 PR:000006990 35792 35797 eIF4E +T872 PR:000005805 35798 35802 CPEB +T873 GO:0032991 35803 35810 complex +T874 NCBITaxon:9606 35816 35821 human +T875 SO:0001060 35829 35836 isoform +T876 PR:000006990 35868 35873 eIF4E +T877 PR:000005805 35874 35878 CPEB +T878 GO:0032991 35879 35886 complex +T879 PR:000016006 35902 35907 TACC1 +T880 SO:0001060 35908 35916 isoforms +T881 GO:0008380 36008 36020 RNA splicing +T882 GO:0050658 36008 36011;36025 36034 RNA ... transport +T883 NCBITaxon:9606 36218 36223 human +T884 UBERON:0000955 36280 36285 brain +T885 PR:000043452 36350 36357 histone +T886 CHEBI:15358 36350 36357 histone +T887 PR:Q92830 36376 36383 hGCN5L2 +T888 SO:0001060 36416 36423 isoform +T889 CHEBI:26537 36427 36435 retinoid +T890 PR:000014373 36427 36448 retinoid-X receptor β +T891 SO:0000417 36491 36497 domain +T892 PR:000016007 36501 36506 TACC2 +T893 PR:000014373 36609 36613 RXRβ +T894 GO:0005634 36648 36655 nuclear +T895 PR:Q965W2 36674 36680 nhr-86 +T896 NCBITaxon:6239 36687 36697 C. elegans +T897 PR:P34427 36746 36751 lin36 +T898 NCBITaxon:33317 36788 36798 protostome +T899 NCBITaxon:1 36898 36907 organisms +T900 SO:0000417 37097 37104 domains +T901 NCBITaxon:7711 37128 37136 chordate +T902 NCBITaxon:7742 37197 37207 vertebrate +T903 PR:000004303 37303 37307 ARNT +T904 NCBITaxon:33511 37365 37377 deuterostome +T905 PR:000017527 37413 37418 GAS41 +T906 NCBITaxon:9606 37456 37461 human +T907 GO:0016514 37462 37498 SWI/SNF chromatin remodeling complex +T908 GO:0006338 37470 37490 chromatin remodeling +T909 NCBITaxon:7227 37527 37542 D. melanogaster +T910 SO:0000853 37543 37552 homologue +T911 PR:000017527 37556 37561 GAS41 +T912 SO:0000657 37720 37733 repeat region +T913 NCBITaxon:7215 37741 37751 Drosophila +T914 NCBITaxon:7742 37797 37807 vertebrate +T915 GO:0065007 37887 37897 regulatory +T916 GO:0032991 37898 37907 complexes +T917 GO:0030674 37918 37926 bridging +T918 SO:0000417 38021 38027 domain +T919 NCBITaxon:33317 38084 38095 protostomes +T920 NCBITaxon:33511 38100 38113 deuterostomes +T921 GO:0030674 38163 38171 bridging +T922 PR:Q27365 38194 38200 lin15A +T923 PR:P34427 38202 38207 lin36 +T924 PR:000009822 38211 38216 lin37 +T925 PR:Q27365 38256 38262 lin15A +T926 PR:P34427 38264 38269 lin36 +T927 PR:000009822 38274 38279 lin37 +T928 SO:0000853 38280 38290 homologues +T929 NCBITaxon:1 38301 38310 organisms +T930 GO:0032991 38394 38401 complex +T931 SO:0001026 38433 38439 genome +T932 PR:000016007 38486 38492 TACC2s +T933 CHEBI:8984 38527 38530 SDS +T934 GO:0006412 38564 38574 translated +T935 PR:000016007 38612 38617 TACC2 +T936 GO:0006412 38673 38683 translated +T937 PR:000014373 38684 38689 RXR-β +T938 PR:000016007 38802 38807 TACC2 +T939 SO:0000704 38920 38924 gene +T940 SO:0000704 38963 38967 gene +T941 SO:0000704 39059 39063 gene +T942 SO:0000853 39121 39130 homologue +T943 PR:P39723 39141 39146 spc72 +T944 GO:0005813 39171 39182 centrosomal +T945 GO:0007067 39183 39190 mitotic +T946 GO:0072686 39183 39198 mitotic spindle +T947 NCBITaxon:33317 39286 39297 protostomes +T948 NCBITaxon:33511 39302 39315 deuterostomes +T949 NCBITaxon:1 39357 39366 organisms +T950 GO:0030674 39391 39399 bridging +T951 GO:0043234 39450 39467 protein complexes +T952 GO:0006281 39480 39497 DNA damage repair +T953 GO:0006412 39507 39518 translation +T954 GO:0006396 39520 39534 RNA processing +T955 SO:0000417 39630 39637 domains +T956 NCBITaxon:7711 39661 39669 chordate +T957 NCBITaxon:7711 39690 39698 chordate +T958 GO:0032991 39803 39812 complexes +T959 GO:0005634 39840 39847 nuclear +T960 PR:000017527 39867 39872 GAS41 +T961 GO:0032991 39972 39981 complexes +T962 NCBITaxon:7742 40024 40034 vertebrate +T963 NCBITaxon:10088 40049 40054 mouse +T964 PR:000016008 40055 40060 TACC3 +T965 PR:000004303 40106 40110 ARNT +T966 CHEBI:33848 40160 40185 polyaromatic hydrocarbons +T967 NCBITaxon:10088 40191 40196 Mouse +T968 PR:000016008 40197 40202 TACC3 +T969 PR:000002091 40268 40273 STAT5 +T970 PR:000016007 40316 40321 TACC2 +T971 PR:000016008 40326 40331 TACC3 +T972 GO:0005634 40344 40351 nuclear +T973 PR:000043452 40352 40359 histone +T974 CHEBI:15358 40352 40359 histone +T975 GO:0000785 40468 40477 chromatin +T976 GO:0006338 40468 40488 chromatin remodeling +T977 NCBITaxon:9606 40524 40529 human +T978 PR:000043452 40575 40582 histone +T979 CHEBI:15358 40575 40582 histone +T980 PR:000012332 40601 40605 pCAF +T981 PR:000016006 40620 40625 TACC1 +T982 SO:0001060 40626 40634 isoforms +T983 GO:0010467 40635 40644 expressed +T984 NCBITaxon:9606 40648 40653 human +T985 UBERON:0000310 40654 40660 breast +T986 http://purl.obolibrary.org/obo/MONDO_0007254 40654 40667 breast cancer +T987 PR:000043452 40700 40707 histone +T988 CHEBI:15358 40700 40707 histone +T989 SO:0000147 40781 40785 Exon +T990 PR:000016006 40799 40804 TACC1 +T991 GO:0006396 40817 40831 RNA processing +T992 PR:000009965 40858 40863 LSm-7 +T993 PR:000015348 40868 40871 SmG +T994 GO:0000380 40884 40904 alternative splicing +T995 PR:000016006 40912 40917 TACC1 +T996 SO:0000704 40918 40922 gene +T997 PR:000016006 40949 40954 TACC1 +T998 SO:0000147 40993 40998 exons +T999 SO:0000417 41028 41035 domains +T1000 GO:0043234 41062 41079 protein complexes +T1001 GO:0008380 41150 41156 splice +T1002 PR:000016006 41233 41238 TACC1 +T1003 PR:000016007 41239 41244 TACC2 +T1004 PR:000016007 41363 41368 TACC2 +T1005 PR:000016008 41400 41405 TACC3 +T1006 PR:000017527 41423 41428 GAS41 +T1007 PR:000015262 41430 41434 INI1 +T1008 PR:000043452 41436 41443 histone +T1009 CHEBI:15358 41436 41443 histone +T1010 PR:000014373 41504 41508 RXRβ +T1011 UBERON:0000479 41540 41546 tissue +T1012 GO:0008380 41556 41564 splicing +T1013 SO:0000147 41577 41581 exon +T1014 SO:0001060 41596 41603 isoform +T1015 UBERON:0000479 41661 41667 tissue +T1016 SO:0000704 41789 41794 genes +T1017 SO:0000858 41810 41821 orthologous +T1018 PR:000001856 41842 41847 RHAMM +T1019 SO:0001026 41966 41973 genomic +T1020 NCBITaxon:31033 41998 42015 Takifugu rubripes +T1021 SO:0000704 42017 42021 gene +T1022 SO:0000853 42141 42157 homology regions +T1023 SO:0000147 42217 42222 exons +T1024 SO:0000147 42239 42244 exons +T1025 GO:0006412 42300 42310 translated +T1026 NCBITaxon:10088 42341 42346 mouse +T1027 NCBITaxon:9606 42351 42356 human +T1028 SO:0001026 42409 42416 genomic +T1029 CHEBI:15377 42443 42448 water +T1030 NCBITaxon:31031 42449 42459 pufferfish +T1031 NCBITaxon:99883 42461 42483 Tetraodon nigroviridis +T1032 SO:0000147 42538 42543 exons +T1033 NCBITaxon:7742 42593 42603 vertebrate +T1034 PR:000016008 42604 42609 TACC3 +T1035 NCBITaxon:31033 42665 42682 Takifugu rubripes +T1036 PR:000016008 42683 42688 TACC3 +T1037 NCBITaxon:7955 42729 42740 Danio rerio +T1038 PR:000016008 42741 42746 TACC3 +T1039 SO:0000854 42855 42865;42878 42886 paralogous ... segments +T1040 SO:0001248 42891 42899 scaffold +T1041 NCBITaxon:9606 42979 42984 Human +T1042 SO:0001026 42985 42991 Genome +T1043 NCBITaxon:7742 43035 43045 vertebrate +T1044 NCBITaxon:9986 43062 43068 rabbit +T1045 PR:000016008 43069 43074 TACC3 +T1046 SO:0000112 43123 43129 primer +T1047 SO:0000993 43184 43193 consensus +T1048 SO:0000112 43194 43200 primer +T1049 NCBITaxon:7742 43250 43260 vertebrate +T1050 PR:000016008 43261 43266 TACC3 +T1051 NCBITaxon:10088 43339 43344 mouse +T1052 SO:0000112 43362 43369 primers +T1053 SO:0001026 43395 43402 genomic +T1054 SO:0000317 43452 43462 cDNA clone +T1055 PR:000016006 43725 43730 TACC1 +T1056 GO:0000380 43731 43737 splice +T1057 SO:0000121 43752 43767 forward primers +T1058 SO:0000147 43789 43793 exon +T1059 SO:0000147 43830 43834 Exon +T1060 SO:0000147 43874 43878 Exon +T1061 SO:0000132 43920 43935 reverse primers +T1062 SO:0000147 43947 43951 Exon +T1063 SO:0000147 43993 43997 Exon +T1064 SO:0000147 44039 44043 Exon +T1065 SO:0000147 44085 44089 Exon +T1066 SO:0000147 44129 44133 Exon +T1067 NCBITaxon:9986 44175 44181 Rabbit +T1068 UBERON:0000955 44182 44187 brain +T1069 SO:0000610 44188 44195 poly A+ +T1070 NCBITaxon:10088 44202 44207 mouse +T1071 UBERON:0000473 44208 44214 testis +T1072 NCBITaxon:9606 44219 44224 human +T1073 UBERON:0000955 44225 44230 brain +T1074 GO:0001171 44308 44329 Reverse transcription +T1075 SO:0000610 44420 44427 poly A+ +T1076 SO:0000006 44478 44490 PCR products +T1077 GO:0009294 44553 44564 transformed +T1078 SO:0000155 44594 44601 Plasmid +T1079 SO:0000667 44602 44609 inserts +T1080 http://purl.obolibrary.org/obo/MONDO_0004992 44645 44651 Cancer +T1081 CHEBI:33694 44662 44672 Biopolymer +T1082 NCBITaxon:9606 44835 44847 Homo sapiens +T1083 PR:000016006 44848 44853 TACC1 +T1084 SO:0001060 44860 44867 isoform +T1085 NCBITaxon:10090 44882 44894 Mus musculus +T1086 PR:000016006 44895 44900 TACC1 +T1087 SO:0001060 44907 44914 isoform +T1088 NCBITaxon:10090 44929 44941 Mus musculus +T1089 PR:000016006 44942 44947 TACC1 +T1090 SO:0001060 44953 44960 isoform +T1091 NCBITaxon:10090 44975 44987 Mus musculus +T1092 NCBITaxon:9986 45007 45028 Oryctolagus cuniculus +T1093 PR:000016008 45029 45034 TACC3 +T1094 NCBITaxon:7955 45047 45058 Danio rerio +T1095 PR:000016008 45059 45064 TACC3 +T1096 NCBITaxon:10116 45162 45177 Rattus norvegus +T1097 PR:000016006 45178 45183 TACC1 +T1098 SO:0001060 45189 45196 isoform +T1099 NCBITaxon:31033 45211 45228 Takifugu rubripes +T1100 NCBITaxon:31033 45257 45274 Takifugu rubripes +T1101 NCBITaxon:10090 45294 45306 Mus musculus +T1102 NCBITaxon:10116 45326 45341 Rattus norvegus +T1103 NCBITaxon:10116 45361 45376 Rattus norvegus +T1104 NCBITaxon:31033 45396 45413 Takifugu rubripes +T1105 NCBITaxon:10116 45433 45448 Rattus norvegus +T1106 PR:000016008 45449 45454 TACC3 +T1107 NCBITaxon:9031 45467 45480 Gallus gallus +T1108 PR:000016008 45481 45486 TACC3 +T1109 NCBITaxon:8364 45499 45518 Silurana tropicalis +T1110 PR:000016008 45519 45524 TACC3 +T1111 NCBITaxon:31033 45536 45553 Takifugu rubripes +T1112 PR:000016008 45554 45559 TACC3 +T1113 NCBITaxon:31033 45572 45589 Takifugu rubripes +T1114 PR:000001856 45590 45595 RHAMM +T1115 NCBITaxon:7719 45608 45626 Ciona intestinalis +T1116 PR:000001856 45627 45632 RHAMM +T1117 NCBITaxon:31033 45645 45662 Takifugu rubripes +T1118 NCBITaxon:31033 45683 45700 Takifugu rubripes +T1119 PR:000016579 45701 45705 TPM1 +T1120 NCBITaxon:7719 45718 45736 Ciona intestinalis +T1121 PR:000009312 45737 45742 Kif3b +T1122 NCBITaxon:7719 45755 45773 Ciona intestinalis +T1123 PR:000009289 45774 45778 klp2 +T1124 SO:0001080 45885 45896 coiled coil +T1125 SO:0000417 45897 45904 domains +T1126 PR:000001856 45993 45998 RHAMM +T1127 NCBITaxon:7742 46044 46055 vertebrates +T1128 SO:0000855 46079 46090 orthologues +T1129 NCBITaxon:7712 46100 46111 urochordate +T1130 NCBITaxon:7719 46112 46130 Ciona intestinalis +T1131 NCBITaxon:7227 46132 46155 Drosophila melanogaster +T1132 NCBITaxon:6239 46157 46167 C. elegans +T1133 NCBITaxon:4932 46172 46196 Saccharomyces cerevisiae +T1134 NCBITaxon:species 46299 46306 Species +T1135 NCBITaxon:9606 46337 46339 hs +T1136 NCBITaxon:9606 46341 46353 Homo sapiens +T1137 NCBITaxon:10090 46356 46358 mm +T1138 NCBITaxon:10090 46360 46372 Mus musculus +T1139 NCBITaxon:10116 46375 46377 rn +T1140 NCBITaxon:10116 46379 46394 Rattus norvegus +T1141 NCBITaxon:9986 46397 46399 oc +T1142 NCBITaxon:9986 46401 46422 Oryctolagus cuniculus +T1143 NCBITaxon:9031 46425 46427 gg +T1144 NCBITaxon:9031 46429 46442 Gallus gallus +T1145 NCBITaxon:8355 46445 46447 xl +T1146 NCBITaxon:8355 46449 46463 Xenopus laevis +T1147 NCBITaxon:8364 46466 46468 st +T1148 NCBITaxon:8364 46470 46489 Silurana tropicalis +T1149 NCBITaxon:31033 46492 46494 tr +T1150 NCBITaxon:31033 46496 46513 Takifugu rubripes +T1151 NCBITaxon:7955 46516 46518 dr +T1152 NCBITaxon:7955 46520 46531 Danio rerio +T1153 NCBITaxon:7719 46534 46536 ci +T1154 NCBITaxon:7719 46538 46556 Ciona intestinalis +T1155 NCBITaxon:7227 46559 46561 dm +T1156 NCBITaxon:7227 46563 46578 D. melanogaster +T1157 NCBITaxon:6239 46581 46583 ce +T1158 NCBITaxon:6239 46585 46595 C. elegans +T1159 NCBITaxon:4932 46598 46600 sc +T1160 NCBITaxon:4932 46602 46626 Saccharomyces cerevisiae +T1161 PR:O75410-1 46749 46757 hsTACC1A +T1162 PR:O95359-4 46771 46779 hsTACC2l +T1163 PR:O95359-1 46791 46799 hsTACC2s +T1164 PR:Q9Y6A5 46812 46819 hsTACC3 +T1165 PR:Q9JJ11 46833 46840 mmTACC3 +T1166 PR:P39723 46909 46916 scSPC72 +T1167 PR:O75330 46930 46937 hsRHAMM +T1168 PR:Q00547 46951 46958 mmRHAMM +T1169 PR:P97779 46972 46979 rnRHAMM +T1170 PR:Q9W3V2 46993 47000 drRHAMM +T1171 PR:P09493 47078 47084 hsTPM1 +T1172 PR:P58771 47098 47104 mmTPM1 +T1173 PR:P04692 47118 47124 rnTPM1 +T1174 PR:P13104 47136 47142 drTPM1 +T1175 PR:P17536 47190 47196 scTPM1 +T1176 PR:Q9NS87 47207 47213 hsKLP2 +T1177 PR:Q7TSP2 47226 47233 rnKIF15 +T1178 PR:P46863 47265 47271 dmKLP2 +T1179 PR:Q9Y496 47305 47312 hsKIF3A +T1180 PR:P28741 47323 47330 mmKIF3A +T1181 PR:O15066 47429 47436 hsKIF3B +T1182 PR:Q61771 47450 47457 mmKIF3B +T1183 PR:P46867 47492 47499 dmKIF3B +T1184 PR:O14782 47513 47520 hsKIF3C +T1185 PR:O35066 47534 47541 mmKIF3C +T1186 PR:O55165 47555 47562 rnKIF3C +T1187 PR:000016007 48360 48365 TACC2 +T1188 PR:000016007 48381 48386 TACC2 +T1189 SO:0000440 48419 48425 vector +T1190 PR:000016007 48491 48496 TACC2 +T1191 GO:0010467 48511 48520 expressed +T1192 NCBITaxon:562 48524 48531 E. coli +T1193 CHEBI:61448 48561 48565 IPTG +T1194 CHEBI:9754 48654 48658 Tris +T1195 CHEBI:17883 48659 48662 HCl +T1196 CHEBI:26710 48678 48682 NaCl +T1197 GO:0019835 48757 48762 lysed +T1198 CHEBI:16856 48883 48894 glutathione +T1199 CHEBI:9754 48928 48932 Tris +T1200 CHEBI:17883 48933 48936 HCl +T1201 CHEBI:26710 48952 48956 NaCl +T1202 GO:0006412 49105 49116 translation +T1203 CHEBI:37983 49160 49163 35S +T1204 GO:0006412 49236 49246 translated +T1205 CHEBI:9754 49278 49282 Tris +T1206 CHEBI:17883 49283 49286 HCl +T1207 CHEBI:26710 49302 49306 NaCl +T1208 PR:000016007 49366 49371 TACC2 +T1209 CHEBI:9754 49451 49455 Tris +T1210 CHEBI:17883 49456 49459 HCl +T1211 CHEBI:26710 49475 49479 NaCl +T1212 CHEBI:9754 49597 49601 Tris +T1213 CHEBI:17883 49602 49605 HCl +T1214 CHEBI:16856 49628 49639 glutathione +T1215 CHEBI:8984 49676 49679 SDS +T1216 PR:000001856 49927 49932 RHAMM +T1217 NCBITaxon:9986 49987 49993 rabbit +T1218 PR:000016008 49994 49999 TACC3 +T1219 PR:000016006 50030 50035 TACC1 +T1220 GO:0008380 50036 50042 splice +T1221 PR:000016007 50091 50096 TACC2 +T1222 NCBITaxon:10088 50117 50122 mouse +T1223 SO:0000855 50128 50139 orthologues +T1224 NCBITaxon:31033 50191 50202 T. rubripes +T1225 SO:0000704 50203 50207 gene +T1226 SO:0000859 50385 50395 paralogous +T1227 SO:0000704 50396 50401 genes +T1228 NCBITaxon:9606 50411 50416 human +T1229 SO:0000704 50478 50483 genes +T1230 SO:0000105 50531 50537;50542 50552 arm of ... chromosome +T1231 SO:0000704 50572 50577 genes +T1232 SO:0000854 50614 50633 paralogous segments +T1233 SO:0000704 50708 50713 genes +T1234 SO:0000854 50743 50762 paralogous segments +T1235 SO:0000704 50860 50865 genes +T1236 SO:0000854 50871 50881 paralogues +T1237 SO:0000854 50970 50979 paralogue +T1238 SO:0000704 51037 51042 genes +T1239 SO:0000854 51048 51058 paralogues +T1240 SO:0001026 51124 51130 genome +T1241 SO:0000704 51247 51252 genes +T1242 SO:0000854 51258 51268 paralogues +T1243 SO:0001026 51309 51315 genome +T1244 SO:0000859 51376 51386 paralogous +T1245 http://purl.obolibrary.org/obo/MONDO_0004992 51627 51633 Cancer +T1246 http://purl.obolibrary.org/obo/MONDO_0004992 51694 51700 Cancer diff --git a/src/ontogpt/evaluation/craft/database/all/15207008.txt b/src/ontogpt/evaluation/craft/database/all/15207008.txt new file mode 100644 index 000000000..7e6cc16fc --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15207008.txt @@ -0,0 +1,163 @@ +Structure-function evolution of the Transforming acidic coiled coil genes revealed by analysis of phylogenetically diverse organisms + +Abstract + +Background + +Examination of ancient gene families can provide an insight into how the evolution of gene structure can relate to function. Functional homologs of the evolutionarily conserved transforming acidic coiled coil (TACC) gene family are present in organisms from yeast to man. However, correlations between functional interactions and the evolution of these proteins have yet to be determined. + +Results + +We have performed an extensive database analysis to determine the genomic and cDNA sequences of the TACCs from phylogenetically diverse organisms. This analysis has determined the phylogenetic relationship of the TACC proteins to other coiled coil proteins, the resolution of the placement of the rabbit TACC4 as the orthologue of human TACC3, and RHAMM as a distinct family of coiled coil proteins. We have also extended the analysis of the TACCs to the interaction databases of C. elegans and D. melanogaster to identify potentially novel TACC interactions. The validity of this modeling was confirmed independently by the demonstration of direct binding of human TACC2 to the nuclear hormone receptor RXRβ. + +Conclusion + +The data so far suggest that the ancestral TACC protein played a role in centrosomal/mitotic spindle dynamics. TACC proteins were then recruited to complexes involved in protein translation, RNA processing and transcription by interactions with specific bridging proteins. However, during evolution, the TACC proteins have now acquired the ability to directly interact with components of these complexes (such as the LSm proteins, nuclear hormone receptors, GAS41, and transcription factors). This suggests that the function of the TACC proteins may have evolved from performing assembly or coordination functions in the centrosome to include a more intimate role in the functional evolution of chromatin remodeling, transcriptional and posttranscriptional complexes in the cell. + +Background + +The evolution of complex organisms has been associated with the generation of gene families by the continual duplication of an initial relatively small set of ancestral genes. Through this process, followed by subsequent mutation, reduplication and exon shuffling between gene families, genes have evolved both discrete, and partially redundant functions with their related family members. With the completion of the genome sequencing projects of human, mouse, rat, fruit fly and nematodes, we are now in a position to ask fundamental questions in regard to how genes interact in the context of the whole organism. Thus, with the appropriate application of bioinformatics, it is now possible to trace the lineage of particular genes and gene families, with related gene families in other organisms. Furthermore, with the growing amount of large-scale proteomic and genomic data becoming publicly available, this analysis can now be extended to reveal the complex interplay between evolution of gene structure and protein function. + +The first Transforming acidic coiled coil gene, TACC1, was identified during the development of an expression map of the proximal short arm of human chromosome 8 [1]. Two additional TACC family members were subsequently identified and mapped to paralogous chromosomal regions on human chromosomes 4p16 and 10q26, physically close to members of the FGFR gene family [1-3]. This mapping data, together with identification of a single TACC gene in the protostomes Caenorhabitis elegans, and Drosophila melanogaster [4-6], led to the speculation that the ancestral FGFR and TACC genes were located physically close to each other. Thus, during the evolution of vertebrates, subsequent successive duplications of the ancestral gene cluster have given rise to three TACC family members located close to FGFR genes in humans. In accordance with the proposed quadruplication of the vertebrate genome during evolution, there is a fourth FGFR family member in vertebrates, raising the question of whether a fourth TACC gene is associated with FGFR4 in vertebrate genomes. To date, only three active TACC genes have been cloned in humans [1-3], one in each of mouse [7], Xenopus laevis [8], D. melanogaster [4], and C. elegans [5,6]. Although two additional new candidate TACC family members, Oryctolagus cuniculus TACC4 [9] and human RHAMM [10] have been proposed, their true identity and placement in the evolution of the TACC family is under debate. Thus, the identification and functional characterization of new members of the TACC family in other organisms, alternatively spliced isoforms of each TACC and comparison of the phylogenetic relationship of these genes relative to other members of the coiled coil superfamily will resolve this issue and provide clues to the evolution of TACC function. + +Results and Discussion + +In silico identification of TACC family members from vertebrate and invertebrate lineages + +Sequence similarity searches of the publicly available genome databases with the BLAST and TBLAST programs were performed to identify TACC and RHAMM orthologues, and other members of the coiled coil superfamily in a diverse set of species (Fig. 1). This identified the complete sequence of the TACC genes in representatives of five major phylogenetically distinct clades. Where possible, the construction of the TACC sequences from these organisms was also confirmed by the analysis of the cDNA databases. Several partial sequences in other vertebrate species, the echinodermate Strongylocentrotus purpuratus and the protostome insect Anopheles gambiae were also identified, suggesting an ancient conservation of the TACC genes in metazoan lineages. However, due to the relative infancy of the cDNA/genome projects for these latter organisms, complete characterization of these TACC genes could not be undertaken. No conclusion could be made about the existence of TACC-like sequence in non-bilaterian metazoans, such as Cnidaria or Porifera, due to the paucity of sequence information for these organisms, and additional definitive sequences with a defined TACC domain could not be found in other non-metazoan organisms. + +Figure 1 + +Phylogenetic analysis of the TACC family members compared to other coiled coil proteins. The phylogenetic tree was constructed as described in the Methods section. The TACC family defines a separate subfamily of coiled coil containing proteins, distinct from other coiled coil families such as the keratins, RHAMM and tropomyosins. Note that the RHAMM proteins form a separate branch more closely related to the tropomyosins and kinesin like proteins (KLP), than the TACC proteins. + +At the base of the chordate branch of life, a single TACC gene was identified in the genome of the urochordate Ciona intestinalis [11], and a partial TACC sequence from an analysis of the Halocynthia rortezi EST database [12]. This confirms the original assumption that a single TACC gene was present in the chordate ancestor. The next major event in the evolution of the chordate genome has been suggested to have occurred 687 ± 155.7 million years ago (MYA), with the first duplication of the chordate genome, and a second duplication occurring shortly thereafter. Thus, if the TACC genes were duplicated at both events, we would expect to identify four TACC genes in the most "primitive" compact vertebrate genome sequenced to date, the pufferfish Takifugu rubripes, with three genes corresponding to the human TACC1-3, and, in keeping with the proposed model for genomic duplication of the chromosomal loci for the TACC genes (discussed below), a possible fourth gene deriving from the TACC3 ancestor. Indeed, four TACC genes were identified in T. rubripes. Of these, two genes corresponded to the T. rubripes orthologues of human TACC2 and TACC3. However, the other two genes, trTACC1A and trTACC1B are clearly most related to TACC1 (Fig. 1). Although trTACC1A is highly homologous to trTACC1B, the latter encodes a significantly smaller predicted protein. The trTACC1B gene is encoded by 15 exons over approximately 7 kb of the Takifugu Scaffold 191 (see below). A search of this region using the trTACC1A sequence and gene prediction software has so far failed to identify additional exons of trTACC1B. However, given the intron/exon structure of this apparently complete gene, it appears likely that trTACC1B is active in the pufferfish, and presumably fulfils either a temporal-spatial specific function within the organism, or a distinct function from the larger trTACC1A product within the cell. Thus, based upon the surrounding chromosomal loci (see below), the trTACC1A and trTACC1B genes appear to have arisen from the duplication of the chromosomal segment containing the teleost TACC1 ancestor, during the additional partial genomic duplication that occurred in the teleost lineage. Therefore, this analysis of T. rubripes does not support the hypothesis that the region surrounding the TACC3 ancestor was included in the second round of vertebrate genomic duplication. + +Examination of higher vertebrates led to the identification of splice variants of TACC1 and TACC2 in Mus musculus, and the assembly of the previously unidentified orthologues of TACC1-3 from Rattus norvegus. In addition, the TACC1X sequence was found on mouse chromosome X. This gene is clearly related to the mouse TACC1, however, further examination revealed a mouse B1 repeat distributed over the length of the proposed intron. In addition, no expression of TACC1X was detected in mouse RNA by rt-PCR analysis (data not shown), suggesting that this sequence is a processed pseudogene. Similarly, TACC1 pseudogenes also exist spread over 22 kb of the centromeric region of human chromosome 10 and, in 8q21, a shorter region 86% identical to the final 359 bp of the TACC1 3' untranslated region. No pseudogenes corresponding to TACC2 or TACC3 were identified in any mammalian species. + +Characterization of vertebrate TACC3 orthologues + +Based upon current functional analysis, the characterization of TACC3 orthologues is likely to be pivotal to understanding the sequence and functional evolution of the TACC gene family. As indicated below, the chromosomal region containing the TACC gene precursors was duplicated twice during vertebrate evolution. Although the analysis of T. rubripes, rodents and humans so far suggests that the vertebrate TACC3 precursor was not included in the second round of genomic duplication, it could not be excluded that a TACC4 gene may have been lost during the evolution of these lineages. The cloning of a new member of the TACC family in Oryctolagus cuniculus has added to this controversy [9]. Designated TACC4, the 1.5 kb cDNA was highly related, but proposed to be distinct from TACC3. However, Northern blot data suggested that this gene produces a single 2.3 kb transcript [9], indicating that the cloned cDNA was incomplete. The degree of similarity to the published sequence of human and mouse TACC3 suggested to us that TACC4 actually represents a partial rabbit TACC3 cDNA. To test this hypothesis, we set out to clone the complete rabbit TACC3 sequence, based upon the known features of human and mouse TACC3. We have previously noted that the N-terminal and C-terminal regions of the human and mouse TACC3 proteins are highly conserved ([2], see below). Therefore, based upon the sequence identity between these genes, we designed a consensus oligonucleotide primer, T3con2, that would be suitable for the identification of the region containing the initiator methionine of the TACC3 cDNAs from primates and rodents. Using this primer, in combination with the TACC4-specific RACE primer (RACE2), initially used by Steadman et al [9], we isolated a 1.5 kb PCR product from rabbit brain cDNA by rt-PCR. In combination with 3'RACE, this generated a consensus cDNA of 2283 bp which corresponds to the transcript size of 2.3 kb detected by the "TACC4" sequence reported in Figure 4 of Steadman et al [9]. Thus, while it remains possible that the "TACC4" sequence is an alternative splice product, or is the product of reduplication of the TACC3 gene (events that would be specific to the rabbit), the only transcript detected in rabbit RNA corresponds to the predicted transcript size of the TACC3 sequence that we have identified here. Furthermore, the string of nucleotides found at the 5' end of the "TACC4" sequence is also found at the 5' ends of a number of cDNA sequences (e.g. U82468, NM_023500), that were isolated by 5'RACE, suggesting that they may correspond to an artefact of the 5'RACE methodology used in their construction. The rabbit "TACC4" and the rabbit TACC3 sequence that we have isolated are also found on the same branch of the TACC phylogenetic tree with the other TACC3 orthologues, including maskin (Xenopus laevis), and the newly identified TACC3 sequences in Rattus norvegus, Gallus gallus, Silurana tropicalis, Danio rerio and T. rubripes, reported in this manuscript (Fig. 1). Thus, it is not in a separate branch that may be expected if the sequence was a distinct TACC family member. + +Placement of the RHAMM gene in the phylogeny of the coiled coil gene family + +Human RHAMM has also been proposed to be the missing fourth member of the TACC family [10]. Evidence used in support of this claim included its chromosomal location on 5q32 in humans (discussed below), its sequence similarity in its coiled coil domain to the TACC domain and the subcellular localization of the RHAMM protein in the centrosome. However, if RHAMM were a bona fide TACC family member, then we would predict its evolution would be similar to those of other TACC family members, and fit with the proposed evolution of the vertebrate genome. Thus, we set out to identify RHAMM orthologues and related genes in metazoans, so that a more complete phylogeny of the coiled coil super family could be generated. We identified a single RHAMM gene in all deuterostomes for which cDNA and/or genomic sequence was available, including C. intestinalis. No RHAMM gene was identified in insects or nematodes. This indicates that the RHAMM/TACC genes diverged after the protostome/deuterostome split 833–933 MYA, but prior to the echinodermata/urochordate divergence (>750 MYA). Significantly, sequence and phylogenetic analysis of coiled coil proteins (Fig. 1) clearly shows that RHAMM does not contain a TACC domain and instead forms a distinct family of proteins in the coiled coil superfamily, and is not a direct descendant of the ancestral TACC gene. + +Evolution of the chromosomal segments containing the TACC genes + +The phylogenetic tree of the FGFR genes closely resembles that of the vertebrate TACC1-3 genes. Recently, detailed analyses of the chromosomal regions containing the FGFR gene family in humans, mouse and the arthopod D. melanogaster have revealed the conservation of paralogous chromosomal segments between these organisms (Fig. 2, [13], Table 1 [see Additional file 1]). This has provided further support that an ancient chromosomal segment was duplicated twice during vertebrate evolution, with the first duplication that gave rise to the human chromosome 4p16/5q32-ter and human chromosome 8p/10q23-ter ancestors occurring in the early stages after the invertebrate divergence. This suggests that the ancestral FGFR-TACC gene pair most probably arose prior to the initial duplication and subsequent divergence of these paralogous chromosomal segments, estimated to have occurred 687 ± 155.7 MYA. This has raised the suggestion that a fourth TACC gene in vertebrates would reside in the same chromosomal region as FGFR4. Indeed this hypothesis has been used in support for the RHAMM gene as a member of the TACC family [10]. Human RHAMM maps to chromosome 5q32 in a region bounded by GPX3 and NKX2E. These loci separate two clusters of genes on human chromosome 5 that are paralogous with 4p16. Interestingly, these three clusters are located on different chromosomes in mouse and rat (Fig. 2), further suggesting that this cluster of genes was transposed into this region after the primate/rodent divergence. + +Figure 2 + +Linear organization of gene clusters centering upon the chromosomal loci of the FGFR genes in humans. Paralogous genes present in at least two of the four loci are shown, with the exception of the region between GPX3 and NKX2E on chromosome 5, which appears to represent a series of intervening genes inserted after duplication of the 4p16/5q32-35 clusters, and genes mentioned in Fig. 3. Corresponding syntenic mouse chromosomal regions (mm*) are indicated. Takifugu rubripes scaffolds are shown (TR*) that contain more than one homologous gene from these clusters. Further details on the location of paralogous genes can be found in [see Additional file 1]. + +Because the conservation of gene order can also provide clues to the evolution of gene regulation, we next attempted to trace the evolution of these paralogous segments by examining the genome of the tunicate C. intestinalis [11] and the most "primitive" compact vertebrate genome sequenced to date, T. rubripes [14]. Although not fully assembled, examination of the genome of T. rubripes confirmed the presence of chromosomal segments paralogous to those found in higher vertebrates (Fig. 2). For instance, the orthologues of GPRK2L and RGS12 are found on T. rubripes scaffold 290 (emb|CAAB01000290.1), and within 300 kb of each other in human 4p16. The T. rubripes orthologues of FGFR3, LETM1 and WHSC1 are located on the same 166 kb genomic scaffold 251 (emb|CAAB01000166.1). Significantly, the three human orthologues of these genes are also located within 300 kb of each other on 4p16. Furthermore, TACC3 and FGFRL map to the overlapping scaffolds 1184/4669 (emb|CAAB01004668). Similarly, elements of these gene clusters, extending from HMP19 to GPRK6 in human chromosome 5q34-ter are also found in the pufferfish, with the T. rubripes orthologues of NSD1, FGFR4 and a RAB-like gene mapping on scaffold 407 (emb|CAAB01000407). However, there is no evidence for a gene corresponding to a TACC4 gene in any of these clusters. + +As noted above, phylogenetic analysis of the TACC sequences indicate that there are two TACC1 related genes in the pufferfish. trTACC1B is located on the 180 kb scaffold 191 (emb|CAAB01000191.1), which also contains the orthologues of several genes located in human chromosome 8p21-11. Thus, this scaffold represents the more "developed" TACC1 chromosomal segment that is evident in higher vertebrates. On the other hand, the trTACC1A gene is located in the 396 kb scaffold 12 (emb|CAAB010012.1). This scaffold also contains the T. rubripes orthologues of MSX1, STX18, D4S234E and the predicted gene LOC118711, in addition to sequences with homology to LOXL, EVC, LOC159291, and the LDB family. Thus, scaffold 12 contains genes found in the regions of human chromosome 4 and 10 that also contain the loci for TACC3 and TACC2, respectively, and may therefore more closely resemble the genomic organization resulting from the initial duplication of the ancestral paralogous chromosomal segment. + +Conserved paralogous clusters may result from the initial clustering of the genes in a relatively small ancestral genomic contig. Some evidence for the existence of "protoclusters" that could correspond to the paralogous chromosomal segments noted in higher vertebrates is present in the genome of the urochordate C. intestinalis [11]. For instance, the orthologues of FGFR, and WHSC1, carboxypeptidase Z and FLJ25359 cluster within an 85 kb region of the C. intestinalis genome and the human orthologues are still maintained in paralogous segments of 4p16, 8p and 10q (Fig. 3, [see Additional file 1]). However, it should be noted that no clusters of genes from the vertebrate paralogous segments are locate close to the TACC or RHAMM genes of C. intestinalis, indicating that the formation of the much larger paralogous segments encompassing the FGFR-TACC genes formed later in evolutionary time, or conversely have been subject to extensive rearrangement in tunicates. In combination with the examination of the T. rubripes genome, this also provides additional evidence that either the second round of duplication of the chromosomal segment that contained the FGFR3/4 ancestor did not include a TACC gene, or that such a gene was lost very early in vertebrate evolution, prior to the divergence of the Gnanthostome lineages. However, the final resolution of the initial evolution of these paralogous segment will await the sequencing of the amphioxus and lamprey genomes, which only have one FGFR gene, and therefore should only contain one copy of the other corresponding genes in this conserved segment. + +Figure 3 + +Formation of protoclusters in Takifugu rubripes and Ciona intestinalis: (A): Structure of the genomic scaffolds containing the Takifugu rubripes trTACC1A and trTACC1B genes. Scaffold 12, the site for the trTACC1A gene contains genes found with either homologues or orthologues on the distal long arm of human chromosome 10 and 4p16. This scaffold, therefore, has some of the characteristics of the predicted immediate ancestor of the TACC1/TACC2 chromosomal segment. trTACC1B is found on scaffold 191, which contains orthologues of genes found in the proximal short arm of human chromosome 8. (B): Ciona intestinalis clusters containing genes found in paralogous segments on human 8, 4p16, 10q and 5q. + +Figure 4 + +Genomic structure of the TACC genes. Conserved regions known to bind protein factors in all human TACC proteins are shown. The SDP repeat of human TACC1-3 is known to bind GAS41 ([3,15] and data not shown). The TACC domain binds ch-TOG and members of the Aurora kinases in all species examined to date. This motif is characteristically encoded by the 3' exons of the TACC genes. The size of the largest isoform for each gene is shown. + +Comparative genomic structure of the TACC family + +The genomic DNA sequences corresponding to the orthologous TACC genes of human, mouse, rat, pufferfish, C. intestinalis, D. melanogaster and C. elegans were extracted and analyzed by Genescan and BLAST to determine the genomic structure of each TACC gene. In some cases, for rat and pufferfish, exons were added or modified based on the best similarity of translated peptides to the corresponding mouse and human proteins. For regions with low sequence similarity in T. rubripes, genomic sequences from the fresh water pufferfish, Tetraodon nigroviridis were used as additional means to verify the predicted exons. + +The general structure of the TACC genes and proteins is depicted in Fig. 4. The main conserved feature of the TACC family, the TACC domain, is located at the carboxy terminus of the protein. In the case of the C. elegans TAC protein, this structure comprises the majority of the protein and is encoded by two of the three exons of the gene. In the higher organisms, D. melanogaster, and the deuterostomes C. intestinalis to human, this feature is also encoded by the final exons of the gene (five in D. melanogaster, seven in the deuterostome genes). Outside of the TACC domain, however, TACC family members show relatively little homology. It is interesting that each TACC gene contains one large exon, which shows considerable variability between TACC orthologues, and constitutes the main difference between the TACC3 genes in the vertebrates (see below). In deuterostomes, this exon contains the SDP repeat (or in the case of the murine TACC3's, a rodent-specific 24 amino acid repeat), which is responsible for the binding of the SWI/SNF chromatin remodeling complex component GAS41 [15,16]. + +Of the vertebrate TACC proteins, the TACC3 orthologues show the greatest variability in size and sequence, ranging in size from 599 amino acids for the rat TACC3 protein, to 942 amino acids in the Danio rerio protein. The reasons for these differences are apparent from the genomic structure of the TACC3 orthologues. TACC3 can be divided into three sections: a conserved N-terminal region (CNTR) of 108 amino acids, encoded by exons 2 and 3 in each vertebrate TACC3 gene, the conserved TACC domain distributed over the final seven exons, and a highly variable central region. The lack of conservation in both size and sequence of the central portion of the TACC3 proteins of human and mouse has been previously noted, and accounts for the major difference between these two orthologues [2]. The majority of this central portion, which contains the SDP repeat motifs, is encoded by one exon in human and the pufferfish (emb|CAAB01001184). In rodents, however, this region is almost entirely composed of seven 24 amino acid repeats, which are located in a single exon of the mouse and rat TACC3 genes. It has been previously reported that there are four mouse TACC3 splice variants that differ in the number of these repeats [2,7,17]. As these repeats are present in a single exon, it appears likely that these different sequences may be the result of the DNA polymerases used in the cDNA synthesis and/or PCR reaction stuttering through the repeat motif. The correct sequence, reported by Sadek et al [7], is the one used throughout the entirety of this manuscript. These repeats are not evident in the rabbit protein, or any other TACC protein, and may indicate that the rodent TACC3 has evolved distinct functions, as has already been noted for the amphibian Xenopus TACC3, maskin [8]. + +Alternative splicing in vertebrate TACC genes + +Whereas exon shuffling can drive the functional diversification of gene families over evolutionary time, the temporal and/or tissue specific alternative splicing of a gene can give rise to functional diversification of a single gene during the development of an organism. Although no alternative splicing of TACC3 has been clearly documented, both temporal and tissue specific splicing is observed in the TACC1 and TACC2 genes. In the case of TACC2, an additional large (5 kb) exon accounts for the main difference between the major splice variants of the vertebrate TACC2 genes [3]. The alternative splicing of this exon suggests a major functional difference between the two TACC2 isoforms, TACC2s and TACC2l [3], as well as a significant difference between TACC2 and its closest TACC family relative, TACC1. However, the function of this region of the TACC2l isoform is current unknown. + +Alternative splicing, together with differential promoter usage has already been noted for the human TACC1 gene [18,19]. In addition, as shown in Fig. 5, we have identified additional TACC1 isoforms that result from alternative splicing of exons 1b-4a. The functions of these different isoforms are unknown, however the region deleted from the shorter variants can include the binding site for LSm7 [20] (variants C, D, F-I), and/or the nuclear localization signals and binding site for GAS41 [15] and PCTAIRE2BP [20] (isoforms B-D, S). One of these isoforms, TACC1S is localized exclusively to the cytoplasm [19], suggesting that the shorter isoforms would not be able to interact with elements of the chomatin remodeling and/or RNA processing machinery in the nucleus. Thus, changes in the complement of TACC1 isoforms in the cell may result in alterations in cellular RNA metabolism at multiple levels, and may account for the observation that TACC1D and TACC1F isoforms are associated with tumorigenic changes in gastric mucosa [18]. + +Figure 5 + +Alternative splicing of the human TACC1 gene. (A): Seven splice variants have been identified for human TACC1 (A-F and S). We have also identified additional splice variants (G-I) from database analysis and rt-PCR analysis of human brain RNA. (B): Alternative splicing of TACC1 in the human brain. rt-PCR analysis confirms splicing of the untranslated exon 1a to exon 1, with retention of the originally defined start methionine (GB:NP_006274) (Variant A*, lanes 1 and 3). Exon 1a also splices to exon 2, removing the L-Sm7 binding motif (variant I, lanes 3,12 and 13). Variants that functionally delete exons 2 and/or 3, such as variant C (lane 15) also remove the predicted nuclear localization signals, and the binding domains for GAS41 and PCTAIRE2BP. These variants would retain the TACC domain, and therefore the potential to bind to ch-TOG and Aurora A kinase in the centrosome. Lane 1: EF/X1R, Lane 2: EF/BX647R, Lane 3: EF/6SPR, Lane 4: EF/1DR, Lane 5, EF/128R, Lane 6 X3F/X1R, Lane 7: X3F/BX647R, Lane 8: X3F/6SPR, Lane 9: X3F/1DR, Lane 10, X3F/128R, Lane 11: 1DF/X1R, Lane 12: 1DF/BX647R, Lane 13: 1DF/6SPR, Lane 14: 1DF/1DR, Lane 15: 1DF/128R, Lane 16: Biorad 1 kb+ Size ladder. + +In silico modeling of the evolution of TACC protein function + +The protein and genomic structure of the present day TACC family members suggests that the function of the ancestral TACC protein was mediated solely through the interactions of the conserved TACC domain. Using an in silico protein-protein interaction model based upon known mitotic spindle and centrosomal components, we have previously predicted a number of additional interactions that could be conserved between a functional TACC homologue in yeast, spc-72, and one or more human TACC proteins [21]. Thus, it is known that all the TACC proteins examined to date interact, via the TACC domain, with the microtubule/centrosomal proteins of the stu2/msps/ch-TOG family [5,6,22-24], and with the Aurora kinases [20,21,25]. These interactions are required for the accumulation of the D-TACC, spc72, ceTAC1 and TACC3 proteins to the centrosome [5,6,22-24]. Hence, this functional interaction with the centrosome and mitotic spindle is likely to represent the ancient, conserved function of the TACC family. However, it is apparent that the human TACC proteins also differ in their ability to interact with the Aurora kinases. For instance, TACC1 and TACC3 interact with Aurora A kinase, whereas TACC2 interacts with Aurora C kinase [21], suggesting a degree of functional specialization in the derivatives of the ancestral chordate TACC, after the radiation of the vertebrate TACC genes. + +The localization of the vertebrate TACC proteins in the interphase nucleus [15,26,27] suggests that they have additional functions outside their ancient role in the centrosome and microtubule dynamics. Thus, it seems likely that TACC family members in protostomes and deuterostomes have integrated new unique functions as the evolving TACC genes acquired additional exons. The results of the pilot large-scale proteomic analysis in C. elegans and D. melanogaster provide further suggestive evidence to this functional evolution. Yeast two hybrid analysis indicates that ceTAC directly binds to C. elegans lin15A, lin36 and lin37 [28]. These proteins bridge ceTAC to other elements of the cytoskeleton and microtubule network, as well as to components of the ribosome, the histone deacetylase chromatin remodeling machinery such as egr-1 and lin-53 (the C. elegans homologues of the human MTA-1 and RbAP48), and to transcription factors such as the PAL1 homeobox and the nuclear hormone receptor nhr-86 [28] (Fig. 6A). Similarly, large scale proteomics [29] has shown that Drosophila TACC interacts with two proteins, the RNA binding protein TBPH and CG14540 (Fig. 6B), and thus indirectly with the Drosophila SWI/SNF chromatin remodeling complex and DNA damage repair machinery. Significantly, the ceTAC protein has also recently been implicated in DNA repair through its direct interaction with the C. elegans BARD1 orthologue [30]. It should be noted that a number of interactions with the TACC proteins from these organisms have probably been missed by these large scale methods, including the well documented direct interactions with the aurora kinases and the stu2/msps/ch-TOG family. + +Figure 6 + +Functional evolution of the TACC proteins modeled in C. elegans and D. melanogaster. (A). C. elegans interaction map shows empirically defined interactions of ceTAC, and extrapolated interactions defined by [28]. (B): Using the BIND database [29], DTACC directly binds to TBPH and CG14540, and thus indirectly to chromatin remodeling complexes (SWI/SNF and histone acetyltransferases), DNA damage repair machinery (via RAD23), and RNA splicing, transport and translational machinery. (C): Predicted interaction map for vertebrate TACCs, based upon ceTAC, suggests an indirect interaction with the nuclear hormone receptor RXRβ. It is also of interest that this predicts a functional interaction with the LDB family, members of which are also found in TACC containing paralogous segments noted in Figs 2, 3 and Additional file 1. (D): Predicted TACC interaction map based upon DTACC. (E): Vertebrate TACC interactions identified to date. ? denotes uncertainty over the identity of a functional vertebrate homologue. In C, D and E, '*' denotes one or more members of the TACC or Aurora kinase family. + +Because of the evolutionary conservation of the TACC domain, we would predict that some of the functional interactions seen in C. elegans and D. melanogaster would be observed in higher animals. Phylogenetic profiling from these interaction maps suggests two similar sets of predicted interactions for vertebrate TACCs (Fig. 6C and 6D). Strikingly, however, the C. elegans specific proteins lin15A, lin36 and lin37 do not have readily discernible homologues in vertebrates or Drosophila, although the presence of a zinc finger domain in lin36 may suggest that this protein is involved directly in transcription or perform an adaptor role similar to LIM containing proteins. For the DTACC interacting proteins, TBPH corresponds to TDP43, a protein implicated in transcriptional regulation and splicing [31,32]. However, the assignment of the human homologue of CG14540 is less clear, with the closest matches in the human databases corresponding to glutamine rich transcription factors such as CREB and the G-box binding factor. + +Comparison of modeled with experimentally defined interactions of the vertebrate TACC proteins + +The interaction data for the vertebrate TACCs is relatively limited; however, interaction networks are now beginning to emerge. The results of our functional analysis, as well as other published data clearly indicate that the vertebrate TACCs interact with proteins that can be divided into two broad categories: 1) proteins with roles in centrosome/mitotic spindle dynamics, and 2) proteins involved in gene regulation, either at the level of transcription, or subsequent RNA processing and translation [3,5-7,15,19-21,24,25,33,34]. Many of these proteins do not appear to interact directly with the protostome TACCs, but would be expected to be in the same protein complex (Fig. 6C,6D). + +Significant analysis of the association of the TACCs with the centrosome and the dynamics of mitotic spindle assembly from yeast to humans has been published [5,6,21-24]. From this analysis, it seems likely that the vertebrate TACC3 protein has retained this direct ancestral function, based upon its location in these structures during mitosis [27], its strong interaction with Aurora Kinase A, and the observation that it is the only human TACC protein phosphorylated by this enzyme [21]. However, the variability of the central domain of the vertebrate orthologues, suggests that TACC3 may also have acquired additional, and in some instances, species-specific functions. For instance, in X. laevis, the maskin protein has acquired a binding site for the eIF4E protein, and thus a function in the coordinated control of polyadenylation and translation in the Xenopus oocyte [8,35]. A recent study has suggested that this function may be unique to maskin: although it is unclear whether the other vertebrate TACC3 proteins interact with the eIF4E/CPEB complex, the human TACC1A isoform is unable to interact with the eIF4E/CPEB complex. Instead, some TACC1 isoforms have evolved a related, but distinct function by directly interacting with elements of the RNA splicing and transport machinery [19]. + +To further characterize the evolving functions of the TACC proteins, we have used an unbiased yeast two hybrid screening method to identify proteins that bind to the human TACC proteins [3,34]. In a screen of a MATCHMAKER fetal brain library (BD Biosciences Clontech), in addition to isolating the histone acetyltransferase hGCN5L2 [34], we also identified the β3 isoform of retinoid-X receptor β as a protein that interacts with the TACC domain of TACC2. As shown in Fig. 7, this interaction is confirmed in vitro by GST-pull down analysis. Significantly, RXRβ is a close family relative of the nuclear hormone receptor, nhr-86, from C. elegans, which interacts with the ceTAC binding protein lin36 (Fig. 6A). This suggests that while protostome TACCs may require additional protein factors to interact with such components, the TACCs in higher organisms may have evolved the ability to directly interact with some of the proteins in the predicted interaction map (Fig. 6E). Indeed, this appears to be directly linked to the acquisition of new domains and duplication of the chordate TACC precursor. In fact, the first identified function of a vertebrate TACC protein was as a transcriptional coactivator acting through a direct interaction with the ARNT transcription factor [7]. It is also intriguing that the deuterostome specific SDP repeat interacts with GAS41, a component/accessory factor of the human SWI/SNF chromatin remodeling complex [3,15]. Although there is a D. melanogaster homologue of GAS41, dmGAS41, the large scale proteomic interaction database does not indicate a direct interaction of dmGAS41 with DTACC. This may be due to the lack of the SDP repeat region in the Drosophila TACC protein. This further suggests that the vertebrate TACCs have gained the specific ability to direct interact with transcriptional regulatory complexes, and that bridging protein(s) are no longer required. Thus, where the ceTAC protein is only composed of the TACC domain, the significantly larger TACC family members in higher protostomes and deuterostomes may have integrated one or more functions of the bridging protein (in this case lin15A, lin36 or lin37). This may also explain the absence of lin15A, lin36 and lin37 homologues in higher organisms, as they were no longer under selective evolutionary pressure to remain within the complex, and thus lost in the evolving genome. + +Figure 7 + +In vitro interaction of RXRβ3 and TACC2s. Top panel: Autoradiograph of 12% SDS polyacrylamide gel with in vitro translated RXRβ3 construct pulled down with GST-TACC2 (Lane 1) or GST (Lane 2); Lane 3: 5% input of in vitro translated RXR-β protein. Bottom two panels represent Coomassie blue stained gels of pull down experiment showing loading of GST-TACC2 and GST. + +Conclusion + +Proposed functional evolution of the TACC family + +Examination of the evolution of ancient gene families provides an insight into how gene structure relates to function. We have presented above, a detailed examination of one such gene family. The data so far suggest that the functional TACC homologue in yeast (spc72) has a specific role in centrosomal/mitotic spindle dynamics [21,22]. This ancient TACC function is conserved throughout evolution in both protostomes and deuterostomes. In addition, the TACC proteins of lower organisms appear to interact with bridging proteins that are components of several different protein complexes involved in DNA damage repair, protein translation, RNA processing and transcription. However, over the process of evolutionary time, with the acquisition of new domains and duplication of the chordate TACC precursor, the chordate TACC proteins have acquired the ability to directly interact with some of the other components of these complexes (such as the LSm proteins, nuclear hormone receptors, GAS41, accessory proteins and transcription factors), and thus evolved additional functions within these complexes. Indeed, the first assigned function of a vertebrate TACC protein, mouse TACC3, was as a transcriptional coactivator of the ARNT mediated transcriptional response to hypoxia and polyaromatic hydrocarbons [7]. Mouse TACC3 has also been reported to interact with the transcription factor STAT5 [33]. Recently, we have demonstrated that TACC2 and TACC3 can bind to nuclear histone acetyltransferases [34], further confirming a more direct role for the TACC proteins in transcriptional and chromatin remodeling events. Interestingly although all human TACC proteins can directly interact with the histone acetyltransferase pCAF in vitro, the TACC1 isoforms expressed in human breast cancer cells do not interact with this histone acetylase [34]. This may be attributable to the proposed function of the Exon 1 containing TACC1 variants in RNA processing, via the interaction with LSm-7 and SmG [19]. Thus, alternative splicing of the TACC1 gene adds further diversity to TACC1 function, as the deletion of specific exons and their associated binding domains will change the potential protein complexes with which they can associate, either directly, or by redirecting the splice variants to different subcellular compartments. With the duplication of the TACC1/TACC2 ancestor, it is apparent that an even greater functional diversity may have been introduced into the TACC family. The TACC2 protein retains the ability of TACC3 to interact with GAS41, INI1, histone acetyltransferases and transcription factors (in this case, RXRβ) (Fig. 7) [3,34]. However, the tissue specific splicing of the 5 kb exon in the TACC2l isoform [3] indicates that this protein has several temporal and tissue specific functions yet to be identified. + +Methods + +Compilation and assembly of previously uncharacterized TACC cDNAs and genes + +Corresponding orthologous sequences for TACC, RHAMM, KLP, KIF, TPM and keratins families were identified initially using the TBLASTN program [36] to search the published genomic and cDNA databases. For Takifugu rubripes, gene predictions were produced by the Ensembl automated pipeline [37] and the JGI blast server . DNA sequences covering the homology regions were extracted and analyzed by Genscan to obtain potential exons. In some cases, exons were added or modified based on the best similarity of translated peptides to the corresponding mouse and human proteins. For regions with low sequence similarity, genomic sequences from the fresh water pufferfish, Tetraodon nigroviridis were used as additional means to verify the predicted exons. Due to the variability of the central region of vertebrate TACC3 cDNAs (see text), to further confirm prediction of the Takifugu rubripes TACC3, full length cDNAs corresponding to the Danio rerio TACC3 (IMAGE clones 2639991, 2640369 and 3724452) were also obtained from A.T.C.C. and fully sequenced. Potential paralogous chromosomal segments and scaffold were identified by searching the public databases deposited at NCBI and at the Human Genome Mapping Project, Cambridge UK. + +Cloning of vertebrate TACC cDNAs + +The rabbit TACC3 was cloned by rt-PCR using the "TACC4" specific primer T4RACE2 [9] (5'-cccgaactgctccaggtaatcgatctc-3') and a consensus primer, T3con2, designed to the region encompassing the vertebrate TACC3 initiator methionine (5'-tatgagtctgcaggtcttaaacgac-3'). For cloning the mouse Tacc1X cDNA, the primers used were based upon the genomic sequence reported, and the sequence of the IMAGE cDNA clone 4933429K08: T1XF (5'-ccatgttcagtcattggcaggtc-3'), T1XF2 (5'-ctgcagaaccaacagttcaag-3'), T1XR1 (5'-agatctgtgacatcacagctc-3'), T1XR2 (5'-ctcgagtcagttagtcttatccagctt-3'), BB617F (5'-accaccaacttgagtacctg-3') and BB617R (5'-gtatcttgaactgttggttctg-3'). For analysis of TACC1 splice variants, the forward primers used were located in exon 1b: EF (5'-gagagatgcgaaatcagcg-3'), Exon 1d: X3F (5'-agtcaaagaaggcatctgcag-3'), Exon 1a: 1DF (5'-ccaagttctgcgccatggg-3'). The reverse primers used were: Exon 1: X1R (5'-ggatttggtctcggcttgcgaatc-3'), Exon 2: BX647R (5'-cttgtgattcttggcttttgg-3'), Exon 3: 6SPR (5'-gtcatcgcctcgtcctggagggc-3'), Exon 4a: 1DR (5'-aatttcacttgttcagtagtc-3'), Exon 5: 128R (5'-cctgcttctgaggatgaaaacgc-3'). Rabbit brain poly A+ mRNA, mouse testis and human brain total RNA were obtained from BD Bioscience Clontech (Palo Alto, CA, U.S.A.). Reverse transcription and PCR was performed as previously described, using either 1 μg of total RNA or 50 ng of poly A+ mRNA as template for first strand cDNA synthesis. PCR products were cloned into pCR2.1 (Invitrogen, Carlsbad CA, U.S.A.) and transformed into InvαF' competent cells. Plasmid inserts were sequenced by the Roswell Park Cancer Institute Biopolymer Core Facility. + +Deposition of nucleotide sequences + +Sequences from this article have been deposited in the GenBank database with the following accession numbers: Homo sapiens TACC1 short isoform S (AY177411), Mus musculus TACC1 short isoform S (AY177412), Mus musculus TACC1 long isoform A (AY177413), Mus musculus TACC2s (AY177410), Oryctolagus cuniculus TACC3 (AY161270), Danio rerio TACC3 (AY170618). Annotations submitted to the Third party annotation database at NCBI are as follows: Rattus norvegus TACC1 long isoform A (BK001653), Takifugu rubripes TACC1A (BK000666/BK000667), Takifugu rubripes TACC1B (BK000664), Mus musculus TACC2l (BK001495), Rattus norvegus TACC2l (BK001658), Rattus norvegus TACC2s (BK001657), Takifugu rubripes TACC2l (BK000690), Rattus norvegus TACC3 (BK001491), Gallus gallus TACC3 (BK001482), Silurana tropicalis TACC3 (BK001481),Takifugu rubripes TACC3 (BK000649), Takifugu rubripes RHAMM (BK000676), Ciona intestinalis RHAMM (BK001479), Takifugu rubripes Keratin (BK000677), Takifugu rubripes TPM1 (BAC10576), Ciona intestinalis Kif3b (BK001492), Ciona intestinalis klp2 (BK001493). + +Phylogenetic analysis + +In order to examine evolutionary relationships of proteins containing coiled coil domains, protein sequences representing the major members of this super family, including TACC, RHAMM, KLP, keratin and tropomyosin from available vertebrates and their recognizable orthologues from the urochordate Ciona intestinalis, Drosophila melanogaster, C. elegans and Saccharomyces cerevisiae were either directly retrieved from NCBI sequence databases, newly predicted or isolated (see above). Species abbreviations are as follows: hs (Homo sapiens), mm (Mus musculus), rn (Rattus norvegus), oc (Oryctolagus cuniculus), gg (Gallus gallus), xl (Xenopus laevis), st (Silurana tropicalis), tr (Takifugu rubripes), dr (Danio rerio), ci (Ciona intestinalis), dm (D. melanogaster), ce (C. elegans), sc (Saccharomyces cerevisiae). The sequences identified above and the following protein or predicted translations were used for phylogenetic analysis: hsTACC1A (NP_006274), hsTACC2l (AAO62630) hsTACC2s (AAO62629), hsTACC3 (NP_006333), mmTACC3 (Q9JJ11), xlMaskin (Q9PTG8), dmTACC (AAF52099), ceTAC1 (NP_497059), scSPC72 (NP_009352), hsRHAMM (NP_036616), mmRHAMM (NP_038580), rnRHAMM (NP_037096), drRHAMM (AAQ97980), hsKeratin (CAB76828), mmKeratin (A61368), rnKeratin (XP_235679), hsTPM1 (NP_000357), mmTPM1 (NP_077745), rnTPM1 (NP_62004, drTPM1 (NP_571180) dmTPM1 (P06754), ceTPM (NP_493540) scTPM1 (P17536), hsKLP2 (BAB03309), rnKIF15 (AAP44513), xlKLP2 (CAA08879), dmKLP2 (NP_476818), ceKLP18 (AA034669), hsKIF3A (Q9Y496), mmKIF3A (NP_032469), rnKIF3A (XP_340797), xlKIF3A (CAA08879), ceKLP11 (NP_741473), ciKIF3 (ci0100148992), hsKIF3B (NP_004789), mmKIF3B (NP_004789), rnKIF3B (XP_215883), dmKIF3B (NP_524029), hsKIF3C (NP_002245), mmKIF3C (NP_032471), rnKIF3C (NP_445938), dmKIF3C (NP_651939). + +These protein sequences were initially aligned with CLUSTAL X [38]. Minor adjustments to certain regions of the alignment for optimization purposes were made based on pairwise alignments, the output saved in PHYLIP format, after which, the distances between proteins were calculated using Poisson correction and the Unrooted trees were inferred with the NJ method and then displayed using TreeView [39]. Bootstrap values above 700 for 1000 trials are shown at the node. To validate the tree, the same sequence set was analyzed with tools in the PHYLIP package [40], using PRODIST followed by FITCH or NEIGNBOR and tree displaying using TreeView. This additional method produced trees with essentially the same topology (data not shown). + +In vitro interaction of TACC2 and RXRβ3 + +The TACC2 cDNA was cloned into GST fusion vector pGEX5X2 (Amersham Biosciences, Piscataway, NJ, USA). GST and GST-TACC2 proteins were expressed in E. coli BL21(DE3) plys "S" with 1 mM IPTG at 37°C shaker for 2 hrs. Cells (50 ml) were harvested and resuspended in 5 ml of 20 mM Tris-HCl pH.8.0, 200 mM NaCl, 1 mM EDTA pH8.0, Protease inhibitor set III (Calbiochem). The cells were lysed by sonication and lysate cleared by centrifugation at 7500 rpm at 4°C for 15 min. The cleared lysate was immobilized on glutathione sepharose beads in 3 ml of 20 mM Tris-HCl pH.8.0, 200 mM NaCl, 1 mM EDTA pH8.0). RXRβ3 cDNA was cloned into pET 28C(+) (Invitrogen, Carlsbad, CA, USA) and protein synthesized by TNT quick coupled transcription/translation system kit (Promega) and radiolabeled with 35S methionine according to manufacturer's instructions. 100 μl of in vitro translated RXRβ3 protein in 1 ml of 20 mM Tris-HCl pH.8.0, 200 mM NaCl, 1 mM EDTA pH8.0 was incubated at 4°C with immobilized GST-TACC2 or GST for 90 min. Unbound RXRβ3 was removed by washing three times with 20 mM Tris-HCl pH.8.0, 200 mM NaCl, 1 mM EDTA pH8.0. Bound proteins were eluted from the beads at room temperature for 10 min in elution buffer (100 mM Tris HCl, pH8.0, 20 mM reduced glutathione). The proteins were analyzed on 12% SDS polyacrylamide gels. Coomassie blue staining verified equal loading of GST fusion protein. Dried gels were autoradiographed. + +Authors' contributions + +I.H.S performed most of the sequence analysis and drafted the assembly of the TACC, KLP, KIF and RHAMM sequences. I.H.S. performed the cDNA isolation of the rabbit TACC3 cDNA. AK identified potential TACC1 splice variants, and the interaction between RXRβ3 and TACC2. A.M. characterized mouse TACC orthologues. P.L. performed the identification and assembly of T. rubripes gene sequences, and the phylogenetic analysis. I.H.S. conceived and designed the project and drafted the complete manuscript. + +Supplementary Material + +Additional file 1 + +Location of paralogous genes found on human 4p16, 5q31-ter, 10q23-ter, 8p and chromosome 2. Positions of genes are given in Mb from the telomere of the short arm of the chromosome. + +Blue highlighted genes (five copies) are found in all four paralogous segments, and the partially duplicated segment on chromosome 2. + +Green highlighted genes are found in either all four paralogous segments, or three of the segments and the partially duplicated region on chromosome 2. + +Pink highlighted genes have paralogues in three of the segments, suggesting either loss of the fourth copy or exclusion of one paralogue from the second round of duplication. + +Black highlighted genes (two paralogues) are found in only one of the derivatives of the second round of genome duplication, suggesting complete exclusion of both copies from the second round of duplication. + +Purple highlighted genes (two paralogues) were duplicated at the second round of genome duplication, or from interchromosomal recombination between paralogous clusters. + +Click here for file + +Acknowledgements + +We wish to thank Dr. Sei-ichi Matsui for his assistance and input in the preparation of this manuscript. This work was supported in part by developmental funds support from the Roswell Park Cancer Institute, and Core grant 2P30CA016056-27 from the National Cancer Institute. diff --git a/src/ontogpt/evaluation/craft/database/all/15238161.ann b/src/ontogpt/evaluation/craft/database/all/15238161.ann new file mode 100644 index 000000000..b72bfefff --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15238161.ann @@ -0,0 +1,1094 @@ +T1 NCBITaxon:32524 47 54 amniote +T2 UBERON:0006907 55 75 slow skeletal muscle +T3 GO:0046903 99 107 Secreted +T4 GO:0007224 108 116;122 132 Hedgehog ... signalling +T5 GO:0007224 118 120;122 132 Hh ... signalling +T6 CHEBI:62488 122 142 signalling molecules +T7 GO:0031099 191 203 regenerating +T8 UBERON:0000479 204 211 tissues +T9 NCBITaxon:7742 225 235 vertebrate +T10 UBERON:0000479 236 243 tissues +T11 UBERON:0004288 401 409 skeletal +T12 NCBITaxon:7955 449 458 zebrafish +T13 GO:0042694 479 495;503 515 specification of ... muscle cells +T14 CL:0000187 503 515 muscle cells +T15 NCBITaxon:32524 519 526 amniote +T16 UBERON:0002329 527 534 somites +T17 CL:0000056 585 594 myoblasts +T18 CL:0000187 614 622 myocytes +T19 GO:0007224 657 670 Hh signalling +T20 PR:000014841 708 722 Sonic hedgehog +T21 PR:000014841 724 727 Shh +T22 CL:0000056 761 770 myoblasts +T23 PR:000008026 780 784 Gli1 +T24 GO:0010467 785 795 expression +T25 PR:000010877 797 805 myogenin +T26 CL:0000056 937 946 myoblasts +T27 PR:000014841 970 973 Shh +T28 GO:0010467 995 1005 expression +T29 PR:000014841 1038 1041 Shh +T30 UBERON:0000023 1102 1106 wing +T31 PR:000014841 1120 1123 Shh +T32 CL:0000187 1161 1167;1183 1188 muscle ... cells +T33 CL:0000056 1237 1246 myoblasts +T34 CL:0000187 1287 1299 muscle cells +T35 GO:0010467 1300 1310 expressing +T36 PR:000014841 1352 1355 Shh +T37 PR:000014841 1357 1360 Shh +T38 GO:0010467 1366 1376 expression +T39 UBERON:0004347 1386 1394 limb bud +T40 UBERON:0006907 1468 1479 slow muscle +T41 CL:0000189 1468 1485 slow muscle fibre +T42 GO:0048747 1473 1495 muscle fibre formation +T43 GO:0010467 1552 1559 express +T44 UBERON:0004347 1618 1626 limb bud +T45 GO:0010467 1665 1675 expressing +T46 GO:0042571 1708 1716 antibody +T47 UBERON:0006907 1732 1743 slow muscle +T48 NCBITaxon:10088 1843 1847 mice +T49 PR:000014841 1863 1866 Shh +T50 UBERON:0002329 1882 1889 somitic +T51 CL:0000056 2006 2015 myoblasts +T52 GO:0010467 2104 2114 expression +T53 GO:0065007 2183 2191 regulate +T54 UBERON:0002101 2198 2202 limb +T55 UBERON:0002101 2262 2266 limb +T56 CL:0000056 2375 2384 myoblasts +T57 UBERON:0004288 2462 2470 skeleton +T58 SO:0000704 2522 2526 gene +T59 GO:0010467 2522 2537 gene expression +T60 UBERON:0005090 2607 2614 muscles +T61 GO:0010467 2649 2656 express +T62 SO:0001060 2665 2673 isoforms +T63 UBERON:0006908 2726 2737 fast muscle +T64 GO:0010467 2754 2761 express +T65 GO:0048747 2847 2859;2874 2887 formation of ... muscle fibres +T66 UBERON:0006907 2860 2864;2874 2880 slow ... muscle +T67 CL:0000189 2860 2864;2874 2887 slow ... muscle fibres +T68 UBERON:0006908 2869 2880 fast muscle +T69 CL:0000190 2869 2887 fast muscle fibres +T70 CL:0000056 2911 2919 myoblast +T71 GO:0065007 3140 3148 regulate +T72 UBERON:0002329 3198 3205 somites +T73 GO:0046903 3266 3274 secreted +T74 UBERON:0000479 3287 3294 tissues +T75 GO:0065007 3320 3330 regulating +T76 GO:0055001 3335 3344;3354 3371 formation ... of myogenic cells +T77 PR:000014841 3419 3433 Sonic hedgehog +T78 PR:000014841 3435 3438 Shh +T79 UBERON:0009571 3458 3473 ventral midline +T80 GO:0010467 3497 3507 expression +T81 UBERON:0002329 3578 3584 somite +T82 NCBITaxon:8782 3593 3598 birds +T83 NCBITaxon:10088 3603 3607 mice +T84 UBERON:0002329 3630 3637 somitic +T85 CL:0000187 3673 3686 muscle fibres +T86 UBERON:0003082 3694 3701 myotome +T87 NCBITaxon:32524 3745 3753 amniotes +T88 UBERON:0009571 3763 3778 Ventral midline +T89 GO:0055001 3823 3835;3849 3861 formation of ... muscle cells +T90 CL:0000187 3849 3861 muscle cells +T91 NCBITaxon:7955 3869 3878 zebrafish +T92 UBERON:0000922 3879 3885 embryo +T93 CL:0007016 3891 3898;3904 3909 adaxial ... cells +T94 UBERON:0006907 4003 4014 slow muscle +T95 CL:0000189 4003 4021 slow muscle fibres +T96 UBERON:0018260 4045 4053;4059 4065 layer of ... muscle +T97 UBERON:0006907 4054 4065 slow muscle +T98 UBERON:0002329 4105 4111 somite +T99 NCBITaxon:7742 4128 4139 vertebrates +T100 UBERON:0002329 4206 4212 somite +T101 SO:0000704 4242 4249 genetic +T102 UBERON:0000479 4310 4317 tissues +T103 PR:000000034 4341 4345 BMPs +T104 UBERON:0002329 4424 4431 somitic +T105 NCBITaxon:32524 4451 4459 amniotes +T106 UBERON:0000479 4512 4519 tissues +T107 UBERON:0002329 4578 4584 somite +T108 CL:0000056 4609 4617 myoblast +T109 CL:0000056 4698 4707 myoblasts +T110 GO:0006936 4713 4724 contractile +T111 PR:000014841 4829 4832 Shh +T112 UBERON:0002329 4901 4908 somitic +T113 UBERON:0004347 5018 5026 limb bud +T114 UBERON:0002101 5137 5141 limb +T115 UBERON:0004347 5204 5212 limb bud +T116 UBERON:0002329 5230 5236 somite +T117 GO:0010467 5266 5273 express +T118 SO:0000704 5274 5279 genes +T119 UBERON:0004347 5418 5426 limb bud +T120 UBERON:0004347 5472 5480 limb bud +T121 UBERON:0002329 5529 5536 somites +T122 UBERON:0004347 5602 5610 limb bud +T123 CL:0000187 5681 5693 muscle fibre +T124 UBERON:0002101 5762 5766 limb +T125 GO:0009948 5767 5797 anteroposterior axis formation +T126 UBERON:0004288 5802 5810 skeletal +T127 PR:000014841 5830 5833 Shh +T128 GO:0010467 5838 5848 expression +T129 CL:0000056 5918 5926 myoblast +T130 GO:0065007 6052 6059 control +T131 CL:0000056 6112 6121 myoblasts +T132 UBERON:0002101 6127 6132 limbs +T133 CL:0000056 6299 6307 Myoblast +T134 UBERON:0002101 6354 6358 limb +T135 CL:0000056 6384 6392 myoblast +T136 GO:0010467 6438 6448 expression +T137 UBERON:0002101 6460 6464 limb +T138 GO:0061061 6548 6560;6572 6579 formation of ... muscles +T139 UBERON:0005090 6572 6579 muscles +T140 GO:0007224 6621 6634 Hh signalling +T141 GO:0009948 6665 6695 anteroposterior axis formation +T142 UBERON:0005090 6789 6796 muscles +T143 PR:000014841 6859 6862 Shh +T144 NCBITaxon:10088 6873 6878 mouse +T145 UBERON:0002101 6879 6884 limbs +T146 UBERON:0000479 6980 6986 tissue +T147 UBERON:0002101 7149 7153 limb +T148 CL:0000189 7279 7289 slow fibre +T149 CL:0000056 7356 7365 myoblasts +T150 PR:000014841 7421 7424 Shh +T151 PR:000014841 7436 7439 Shh +T152 UBERON:0002101 7532 7537 limbs +T153 GO:0007224 7566 7579 Hh signalling +T154 PR:000014841 7680 7683 Shh +T155 UBERON:0002101 7707 7711 limb +T156 PR:000014841 7743 7746 Shh +T157 UBERON:0000023 7757 7761 wing +T158 UBERON:0004347 7789 7797 limb bud +T159 PR:000014841 7807 7810 Shh +T160 PR:000014841 7897 7900 Shh +T161 UBERON:0000023 7961 7965 Wing +T162 GO:0040007 8000 8005 grown +T163 PR:000014841 8060 8063 Shh +T164 PR:000014841 8136 8139 Shh +T165 PR:000006427 8227 8233 desmin +T166 PR:000014841 8330 8333 Shh +T167 PR:000014841 8761 8764 Shh +T168 PR:000006427 8795 8801 desmin +T169 CL:0000187 8908 8916 myocytes +T170 CL:0002372 8921 8929 myotubes +T171 GO:0010467 8979 8986 express +T172 PR:000014841 8998 9001 Shh +T173 PR:000014841 9175 9178 Shh +T174 GO:0030154 9213 9231;9256 9261 differentiation of ... cells +T175 UBERON:0004347 9238 9246 limb bud +T176 UBERON:0002101 9305 9309 limb +T177 UBERON:0002329 9314 9320 somite +T178 PR:000014841 9344 9347 Shh +T179 UBERON:0000023 9398 9402 wing +T180 UBERON:0000023 9444 9448 wing +T181 GO:0040007 9498 9503 grown +T182 PR:000014841 9560 9563 Shh +T183 PR:000006427 9670 9676 desmin +T184 PR:000014841 10007 10010 Shh +T185 PR:000006427 10078 10084 desmin +T186 GO:0010467 10085 10095 expressing +T187 PR:000014841 10128 10131 Shh +T188 GO:0008283 10261 10277;10291 10296 proliferation of ... cells +T189 GO:0005634 10377 10383 nuclei +T190 PR:000006427 10391 10397 desmin +T191 GO:0005737 10409 10418 cytoplasm +T192 GO:0005737 10448 10457 cytoplasm +T193 PR:000014841 10474 10477 Shh +T194 PR:000014841 10512 10515 Shh +T195 GO:0010467 10542 10549 express +T196 GO:0005634 10588 10594 nuclei +T197 GO:0005737 10618 10627 cytoplasm +T198 GO:0005737 10662 10671 cytoplasm +T199 GO:0010467 10739 10749 expressing +T200 GO:0010467 10831 10838 express +T201 PR:000014841 10953 10956 Shh +T202 UBERON:0004347 11028 11036 limb bud +T203 PR:000014841 11080 11083 Shh +T204 PR:000014841 11180 11183 Shh +T205 CL:0000056 11202 11210 myoblast +T206 GO:0051450 11202 11210;11237 11250 myoblast ... proliferation +T207 GO:0006915 11282 11291 apoptosis +T208 CL:0000056 11328 11337 myoblasts +T209 UBERON:0000023 11355 11359 wing +T210 UBERON:0000922 11468 11477 embryonic +T211 CL:0000056 11484 11493 myoblasts +T212 UBERON:0000023 11553 11557 wing +T213 PR:000014841 11581 11584 Shh +T214 UBERON:0007023 11612 11617 adult +T215 NCBITaxon:10088 11618 11623 mouse +T216 CL:0000056 11638 11651 myoblast cell +T217 CL:0000056 11691 11699 myoblast +T218 PR:000014841 11732 11735 Shh +T219 CHEBI:472552 11854 11858 BrdU +T220 CHEBI:51739 11912 11927 acridine orange +T221 PR:000004078 11931 11940 Annexin V +T222 PR:000014841 11996 11999 Shh +T223 CL:0000056 12026 12035 myoblasts +T224 PR:000014841 12084 12087 Shh +T225 CL:0000056 12101 12109 myoblast +T226 CL:0000056 12251 12259 myoblast +T227 PR:000014841 12326 12329 Shh +T228 CL:0000056 12392 12400 myoblast +T229 PR:000014841 12422 12425 Shh +T230 GO:0005634 12454 12460 nuclei +T231 GO:0005737 12484 12493 cytoplasm +T232 CL:0000187 12525 12533 myocytes +T233 PR:000014841 12560 12563 Shh +T234 GO:0005634 12607 12613 nuclei +T235 GO:0005737 12633 12642 cytoplasm +T236 PR:000014841 12664 12667 Shh +T237 PR:000014841 12902 12905 Shh +T238 CHEBI:472552 12920 12924 BrdU +T239 CHEBI:472552 13027 13031 BrdU +T240 GO:0097617 13055 13068 hybridisation +T241 CL:0000056 13077 13086 myoblasts +T242 PR:000008026 13182 13186 Gli1 +T243 SO:0000077 13187 13196 antisense +T244 CL:0000056 13231 13240 myoblasts +T245 CL:0000056 13334 13343 myoblasts +T246 GO:0042571 13431 13441 antibodies +T247 PR:000010875 13445 13449 MyoD +T248 PR:000010877 13451 13459 Myogenin +T249 PR:000014841 13560 13563 Shh +T250 CL:0000056 13593 13602 myoblasts +T251 PR:000014841 13623 13626 Shh +T252 SO:0000704 13681 13685 gene +T253 PR:000014841 13717 13720 Shh +T254 PR:000008026 13734 13738 gli1 +T255 SO:0000673 13739 13750 transcripts +T256 CL:0000056 13769 13778 myoblasts +T257 PR:000008027 13810 13814 gli2 +T258 GO:0010467 13815 13825 expression +T259 PR:000014841 13886 13889 Shh +T260 PR:000014841 13909 13912 Shh +T261 PR:000014841 13974 13977 Shh +T262 PR:000014841 14015 14018 Shh +T263 CL:0000056 14089 14098 myoblasts +T264 GO:0007049 14106 14116 cell cycle +T265 GO:0005634 14192 14199 nuclear +T266 PR:000010875 14200 14204 MyoD +T267 PR:000010877 14226 14234 Myogenin +T268 PR:000014841 14299 14302 Shh +T269 PR:000010875 14337 14341 MyoD +T270 GO:0005634 14371 14377 nuclei +T271 PR:000010877 14423 14431 Myogenin +T272 PR:000014841 14508 14511 Shh +T273 GO:0005634 14534 14541 nuclear +T274 PR:000010875 14542 14546 MyoD +T275 CL:0000056 14592 14601 myoblasts +T276 UBERON:0001977 14650 14655 serum +T277 PR:000014841 14658 14661 Shh +T278 CL:0000189 14683 14693 slow fibre +T279 UBERON:0000023 14710 14714 wing +T280 GO:0010467 14745 14755 expression +T281 CL:0000187 14806 14818 muscle cells +T282 GO:0010467 14827 14834 express +T283 GO:0010467 14887 14894 express +T284 GO:0005634 15070 15076 nuclei +T285 GO:0005737 15100 15109 cytoplasm +T286 GO:0005737 15143 15152 cytoplasm +T287 PR:000014841 15220 15223 Shh +T288 CL:0000187 15259 15266 myocyte +T289 CL:0002372 15267 15275 myotubes +T290 GO:0010467 15276 15286 expressing +T291 PR:000014841 15479 15482 Shh +T292 GO:0010467 15538 15548 expressing +T293 CL:0000010 15579 15587;15593 15598 cultured ... cells +T294 UBERON:0000023 15588 15592 wing +T295 GO:0010467 15599 15609 expressing +T296 CL:0000187 15647 15655 myocytes +T297 GO:0010467 15726 15733 express +T298 PR:000014841 15747 15750 Shh +T299 PR:000014841 15854 15857 Shh +T300 GO:0010467 15977 15984 express +T301 PR:000014841 16041 16044 Shh +T302 PR:000014841 16109 16112 Shh +T303 GO:0010467 16211 16220 expresses +T304 CL:0000187 16258 16266 myocytes +T305 PR:000014841 16285 16288 Shh +T306 PR:000014841 16311 16314 Shh +T307 GO:0008283 16334 16347 proliferation +T308 CL:0000187 16591 16599 myocytes +T309 PR:000014841 16693 16696 Shh +T310 GO:0042571 16768 16776 antibody +T311 PR:000014841 16798 16801 Shh +T312 PR:000014841 16865 16868 Shh +T313 PR:000014841 16980 16983 Shh +T314 GO:0010467 17023 17033 expression +T315 GO:0010467 17078 17087 expressed +T316 GO:0035425 17117 17126 autocrine +T317 PR:000014841 17168 17171 Shh +T318 CL:0000187 17246 17254 myocytes +T319 PR:000014841 17378 17381 Shh +T320 NCBITaxon:39107 17398 17404 murine +T321 CL:0000056 17405 17418 myoblast cell +T322 PR:000014841 17503 17506 Shh +T323 NCBITaxon:10088 17541 17546 Mouse +T324 CL:0000056 17551 17560 myoblasts +T325 GO:0042571 17814 17824 antibodies +T326 CL:0000187 17918 17926 myocytes +T327 GO:0005634 17933 17939 nuclei +T328 GO:0005737 17957 17966 cytoplasm +T329 CL:0000187 18008 18016 myocytes +T330 GO:0010467 18017 18027 expressing +T331 PR:000014841 18090 18093 Shh +T332 CL:0000187 18241 18249 myocytes +T333 GO:0010467 18250 18260 expressing +T334 PR:000014841 18442 18445 Shh +T335 GO:0042571 18457 18465 antibody +T336 GO:0010467 18512 18522 expression +T337 CL:0000187 18534 18542 myocytes +T338 NCBITaxon:10088 18584 18589 mouse +T339 NCBITaxon:7955 18593 18602 zebrafish +T340 PR:000014841 18603 18606 Shh +T341 PR:000014841 18747 18750 Shh +T342 GO:0007520 18780 18789;18794 18803 fusion of ... myoblasts +T343 CL:0000187 18794 18803 myoblasts +T344 PR:000014841 18818 18821 Shh +T345 CL:0002372 18872 18880 myotubes +T346 GO:0005634 18933 18941 nucleate +T347 CL:0000187 18942 18950 myocytes +T348 PR:000014841 18967 18970 Shh +T349 GO:0005634 18985 18993 nucleate +T350 CL:0000187 18994 19002 myocytes +T351 GO:0010467 19033 19040 express +T352 GO:0005634 19071 19079 nucleate +T353 CL:0002372 19080 19088 myotubes +T354 GO:0007224 19131 19144 Hh signalling +T355 PR:000014841 19169 19172 Shh +T356 NCBITaxon:39107 19205 19211 murine +T357 CL:0000056 19252 19261 myoblasts +T358 NCBITaxon:7955 19282 19291 zebrafish +T359 UBERON:0000023 19387 19391 wing +T360 GO:0010467 19423 19433 expression +T361 GO:0042571 19459 19469 antibodies +T362 UBERON:0002471 19536 19544 zeugopod +T363 UBERON:0002386 19546 19553 forearm +T364 GO:0042571 19714 19724 antibodies +T365 CL:0000187 19761 19774 muscle fibres +T366 SO:0000704 19987 19991 gene +T367 CL:0000189 20016 20027 slow fibres +T368 UBERON:0007023 20031 20036 adult +T369 NCBITaxon:9031 20037 20045 chickens +T370 GO:0042571 20083 20091 antibody +T371 GO:0010467 20101 20110 expressed +T372 GO:0010467 20175 20185 expression +T373 SO:0000704 20203 20207 gene +T374 UBERON:0000922 20257 20263 embryo +T375 GO:0042571 20317 20327 antibodies +T376 GO:0042571 20421 20429 antibody +T377 GO:0042571 20482 20492 antibodies +T378 UBERON:0005090 20655 20662 muscles +T379 UBERON:0005090 20729 20736 muscles +T380 GO:0010467 20809 20818 expressed +T381 GO:0042571 20868 20878 antibodies +T382 UBERON:0006907 20952 20963 slow muscle +T383 GO:0010467 21027 21037 expressing +T384 UBERON:0000024 21058 21066 forewing +T385 CL:0000187 21084 21096 muscle fibre +T386 UBERON:0000023 21110 21114 wing +T387 UBERON:0002471 21137 21145 zeugopod +T388 CL:0000189 21198 21202;21230 21236 slow ... fibres +T389 UBERON:0000023 21316 21321 wings +T390 GO:0030154 21339 21357;21382 21387 differentiation of ... cells +T391 UBERON:0000922 21358 21367 embryonic +T392 CL:0002321 21358 21367;21382 21387 embryonic ... cells +T393 CL:0000189 21709 21720 slow fibres +T394 CL:0000189 21785 21796 slow fibres +T395 UBERON:0002471 21914 21922 zeugopod +T396 UBERON:0005090 22058 22065 muscles +T397 CL:0000190 22099 22103;22113 22119 fast ... fibres +T398 CL:0000189 22108 22119 slow fibres +T399 UBERON:0005090 22144 22151 muscles +T400 UBERON:0001522 22191 22194 FCU +T401 CL:0000189 22205 22216 slow fibres +T402 CL:0000190 22227 22238 fast fibres +T403 UBERON:0005090 22275 22282 muscles +T404 CL:0000189 22311 22315;22334 22340 slow ... fibres +T405 CL:0000190 22329 22340 fast fibres +T406 UBERON:0007612 22370 22373 EDC +T407 UBERON:0007612 22374 22401 extensor digitorum communis +T408 UBERON:0002989 22403 22406 Anc +T409 UBERON:0002989 22407 22415 anconeus +T410 UBERON:0000311 22421 22429 extensor +T411 UBERON:0000311 22453 22461 extensor +T412 UBERON:0000311 22482 22490 extensor +T413 UBERON:0001523 22561 22564 FDP +T414 UBERON:0001523 22565 22591 flexor digitorum profundus +T415 UBERON:0001522 22626 22629 FCU +T416 UBERON:0001522 22630 22650 flexor carpi ulnaris +T417 GO:0097617 22670 22683 hybridisation +T418 PR:000013412 22688 22692 ptc1 +T419 PR:000008026 22700 22704 gli1 +T420 PR:000008027 22714 22718 gli2 +T421 PR:000014841 22806 22809 Shh +T422 UBERON:0002101 22818 22823 limbs +T423 PR:000013412 22861 22865 ptc1 +T424 PR:000008026 22870 22874 gli1 +T425 UBERON:0002101 22900 22904 limb +T426 UBERON:0000023 22933 22937 wing +T427 PR:000013412 22991 22995 ptc1 +T428 PR:000008026 23049 23053 Gli1 +T429 UBERON:0005414 23112 23115 ZPA +T430 UBERON:0002471 23137 23145 zeugopod +T431 PR:000013412 23151 23155 ptc1 +T432 PR:000013412 23220 23224 ptc1 +T433 PR:000014841 23268 23271 Shh +T434 PR:000013412 23293 23297 ptc1 +T435 PR:000014841 23362 23365 shh +T436 PR:000008026 23392 23396 gli1 +T437 GO:0010467 23397 23407 expression +T438 PR:000008027 23439 23443 gli2 +T439 PR:000008026 23454 23458 Gli1 +T440 PR:000008027 23484 23488 gli2 +T441 PR:000014841 23511 23514 Shh +T442 PR:000014841 23541 23544 shh +T443 UBERON:0001424 23627 23628 u +T444 UBERON:0001424 23630 23634 ulna +T445 UBERON:0001423 23637 23638 r +T446 UBERON:0001423 23640 23646 radius +T447 GO:0007224 23723 23736 Hh signalling +T448 UBERON:0000023 23740 23744 wing +T449 GO:0010467 23768 23778 expression +T450 PR:000008026 23782 23786 gli1 +T451 PR:000013412 23791 23795 ptc1 +T452 SO:0000704 23799 23803 gene +T453 SO:0000704 23833 23838 genes +T454 GO:0007224 23876 23889 Hh signalling +T455 PR:000013412 23896 23900 Ptc1 +T456 PR:000008026 23905 23909 gli1 +T457 GO:0010467 23921 23930 expressed +T458 UBERON:0002101 23955 23959 limb +T459 PR:000014841 23989 23992 Shh +T460 UBERON:0005414 24011 24038 zone of polarizing activity +T461 UBERON:0005414 24040 24043 ZPA +T462 PR:000013412 24058 24062 Ptc1 +T463 GO:0010467 24063 24073 expression +T464 UBERON:0000023 24086 24090 wing +T465 GO:0009888 24142 24154 histogenesis +T466 PR:000008026 24173 24177 gli1 +T467 GO:0010467 24178 24188 expression +T468 GO:0007224 24273 24286 Hh signalling +T469 PR:000008974 24349 24364 Indian hedgehog +T470 PR:000008974 24366 24369 Ihh +T471 GO:0010467 24371 24381 expression +T472 UBERON:0007688 24405 24411 anlage +T473 PR:000013412 24418 24422 Ptc1 +T474 PR:000008026 24427 24431 gli1 +T475 GO:0010467 24436 24445 expressed +T476 UBERON:0000479 24467 24474 tissues +T477 UBERON:0002222 24483 24496 perichondrium +T478 UBERON:0009749 24501 24516 limb mesenchyme +T479 CL:0000187 24555 24568 muscle fibres +T480 PR:000013412 24634 24638 ptc1 +T481 PR:000008026 24643 24647 gli1 +T482 GO:0007224 24728 24741 Hh signalling +T483 GO:0010467 24828 24838 expressing +T484 GO:0007224 24873 24886 Hh signalling +T485 PR:000014841 24914 24917 Shh +T486 GO:0010467 24923 24933 expression +T487 UBERON:0006907 24972 24983 slow muscle +T488 UBERON:0000023 25047 25051 wing +T489 UBERON:0000922 25100 25106 embryo +T490 CL:0000057 25107 25118 fibroblasts +T491 GO:0010467 25119 25129 expressing +T492 PR:000014841 25132 25135 Shh +T493 GO:0006260 25141 25152 replication +T494 NCBITaxon:11632 25163 25173 retroviral +T495 SO:0000440 25174 25180 vector +T496 UBERON:0004347 25219 25228 limb buds +T497 UBERON:0000922 25245 25252 embryos +T498 UBERON:0002544 25393 25398 digit +T499 PR:000014841 25470 25473 shh +T500 GO:0010467 25479 25489 expression +T501 PR:000013412 25491 25495 ptc1 +T502 PR:000008026 25500 25504 gli1 +T503 PR:000014841 25533 25536 Shh +T504 GO:0010467 25537 25547 expressing +T505 PR:000008027 25671 25675 gli2 +T506 SO:0000704 25699 25703 gene +T507 GO:0007224 25718 25731 Hh signalling +T508 GO:0010467 25750 25760 expression +T509 PR:000008026 25764 25768 gli1 +T510 UBERON:0002101 25787 25792 limbs +T511 PR:000014841 25804 25807 Shh +T512 GO:0010467 25813 25823 expression +T513 PR:000008027 25840 25844 gli2 +T514 PR:000013412 25862 25866 ptc1 +T515 PR:000008026 25868 25872 gli1 +T516 PR:000008027 25877 25881 gli2 +T517 GO:0010467 25882 25892 expression +T518 UBERON:0006907 25963 25974 slow muscle +T519 PR:000014841 26009 26012 Shh +T520 PR:000014841 26096 26099 Shh +T521 http://purl.obolibrary.org/obo/MONDO_0005550 26105 26114 infection +T522 UBERON:0000023 26124 26129 wings +T523 UBERON:0000922 26186 26192 embryo +T524 CL:0000057 26193 26204 fibroblasts +T525 GO:0010467 26205 26215 expressing +T526 GO:0006260 26228 26239 replication +T527 NCBITaxon:11632 26250 26260 retroviral +T528 SO:0000440 26261 26267 vector +T529 UBERON:0003104 26295 26305 mesenchyme +T530 UBERON:0000023 26321 26325 wing +T531 UBERON:0000922 26343 26350 embryos +T532 PR:000014841 26408 26411 Shh +T533 http://purl.obolibrary.org/obo/MONDO_0005550 26417 26425 infected +T534 UBERON:0000023 26432 26437 wings +T535 GO:0042571 26534 26544 antibodies +T536 UBERON:0000023 26559 26564 wings +T537 CL:0000187 26595 26608 muscle fibres +T538 PR:000014841 26618 26621 Shh +T539 UBERON:0003104 26649 26659 mesenchyme +T540 PR:000014841 26679 26682 Shh +T541 UBERON:0000023 26694 26699 wings +T542 UBERON:0002101 26935 26940 limbs +T543 CL:0000189 27205 27215 slow fibre +T544 UBERON:0002101 27310 27315 limbs +T545 UBERON:0002101 27358 27363 limbs +T546 UBERON:0002101 27371 27376 limbs +T547 UBERON:0002101 27413 27418 limbs +T548 UBERON:0002101 27445 27450 limbs +T549 UBERON:0001424 27508 27509 u +T550 UBERON:0001424 27511 27515 ulna +T551 UBERON:0001423 27518 27519 r +T552 UBERON:0001423 27521 27527 radius +T553 UBERON:0002101 27570 27575 limbs +T554 GO:0010467 27617 27627 expression +T555 PR:000014841 27631 27634 Shh +T556 UBERON:0002101 27690 27695 limbs +T557 UBERON:0001424 27764 27768 ulna +T558 UBERON:0000023 27810 27815 wings +T559 UBERON:0000023 27871 27876 wings +T560 UBERON:0002385 27907 27920 muscle tissue +T561 UBERON:0000023 28043 28048 wings +T562 PR:000014841 28163 28166 Shh +T563 PR:000014841 28214 28217 Shh +T564 UBERON:0000023 28223 28228 wings +T565 PR:000014841 28280 28283 Shh +T566 UBERON:0000023 28340 28345 wings +T567 GO:0010467 28388 28395 express +T568 UBERON:0000023 28563 28568 wings +T569 PR:000014841 28597 28600 Shh +T570 PR:000014841 28693 28696 Shh +T571 CL:0000187 28721 28734 muscle fibres +T572 UBERON:0000023 28756 28761 wings +T573 UBERON:0000023 28826 28830 wing +T574 PR:000014841 28839 28842 Shh +T575 UBERON:0006907 29007 29018 slow muscle +T576 PR:000014841 29031 29034 Shh +T577 GO:0010467 29040 29050 expression +T578 UBERON:0002101 29060 29064 limb +T579 PR:000014841 29148 29151 Shh +T580 GO:0010467 29157 29167 expression +T581 UBERON:0000922 29211 29218 embryos +T582 PR:000014841 29340 29343 Shh +T583 UBERON:0002101 29352 29357 limbs +T584 UBERON:0002101 29392 29397 limbs +T585 UBERON:0002101 29482 29487 limbs +T586 UBERON:0000023 29575 29580 wings +T587 GO:0010467 29621 29631 expression +T588 UBERON:0002470 29639 29646 autopod +T589 CL:0000187 29673 29686 muscle fibres +T590 UBERON:0002101 29721 29725 limb +T591 UBERON:0002471 29760 29768 zeugopod +T592 UBERON:0002101 29809 29814 limbs +T593 GO:0010467 29998 30008 expression +T594 PR:000014841 30160 30163 Shh +T595 UBERON:0000023 30228 30232 wing +T596 GO:0040007 30242 30247 grown +T597 GO:0097617 30332 30345 hybridisation +T598 PR:000014841 30350 30353 Shh +T599 PR:000014841 30366 30369 Shh +T600 CL:0000189 30487 30498 slow fibres +T601 UBERON:0002101 30582 30586 limb +T602 CL:0000189 30777 30788 slow fibres +T603 PR:000014841 30824 30827 Shh +T604 GO:0010467 30833 30843 expression +T605 CL:0000189 30868 30879 slow fibres +T606 GO:0010467 30979 30986 express +T607 PR:000014841 31022 31025 Shh +T608 CL:0000189 31071 31082 slow fibres +T609 CL:0000189 31133 31143 slow fibre +T610 CL:0000189 31235 31246 slow fibres +T611 UBERON:0000023 31293 31297 wing +T612 PR:000014841 31337 31340 Shh +T613 GO:0046903 31483 31492 secreting +T614 GO:0042571 31525 31533 antibody +T615 UBERON:0002101 31559 31563 limb +T616 UBERON:0002101 31678 31682 limb +T617 UBERON:0002101 32015 32020 limbs +T618 UBERON:0002101 32026 32031 limbs +T619 UBERON:0002101 32127 32132 limbs +T620 UBERON:0002471 32197 32205 zeugopod +T621 GO:0010467 32245 32255 expressing +T622 UBERON:0002101 32318 32322 limb +T623 UBERON:0002101 32362 32367 limbs +T624 UBERON:0002101 32434 32439 limbs +T625 CL:0000189 32485 32496 slow fibres +T626 UBERON:0002101 32518 32523 limbs +T627 UBERON:0002101 32602 32607 limbs +T628 UBERON:0002471 32658 32666 zeugopod +T629 UBERON:0002101 32693 32698 limbs +T630 UBERON:0002101 32704 32709 limbs +T631 UBERON:0002101 32779 32784 limbs +T632 UBERON:0002101 32805 32809 Limb +T633 UBERON:0002544 32824 32829 digit +T634 GO:0042571 32914 32922 antibody +T635 UBERON:0005414 32966 32969 ZPA +T636 UBERON:0001461 33030 33035 elbow +T637 UBERON:0002472 33039 33047 stylopod +T638 UBERON:0002472 33067 33075 stylopod +T639 UBERON:0002471 33107 33115 zeugopod +T640 UBERON:0002472 33198 33206 stylopod +T641 GO:0042571 33287 33295 antibody +T642 UBERON:0002101 33360 33365 limbs +T643 UBERON:0002471 33478 33486 zeugopod +T644 GO:0042571 33513 33521 antibody +T645 UBERON:0000023 33583 33587 wing +T646 UBERON:0000023 33710 33714 wing +T647 UBERON:0002101 33855 33859 limb +T648 UBERON:0000023 33899 33903 wing +T649 GO:0010467 33931 33941 expression +T650 UBERON:0000023 34024 34028 wing +T651 CL:0000189 34213 34224 Slow fibres +T652 UBERON:0000023 34280 34285 wings +T653 CHEBI:33893 34394 34402 reagents +T654 UBERON:0000024 34425 34433 forewing +T655 GO:0042571 34645 34653 antibody +T656 UBERON:0002101 34750 34755 limbs +T657 UBERON:0002101 34994 34999 limbs +T658 UBERON:0002101 35092 35097 limbs +T659 UBERON:0002101 35174 35179 limbs +T660 UBERON:0001461 35224 35229 elbow +T661 UBERON:0002101 35245 35249 limb +T662 UBERON:0001461 35267 35272 elbow +T663 UBERON:0002101 35351 35356 limbs +T664 NCBITaxon:39107 35402 35408 Murine +T665 UBERON:0006907 35430 35441 slow muscle +T666 CL:0000189 35430 35448 slow muscle fibres +T667 GO:0010467 35499 35509 expression +T668 UBERON:0002101 35529 35534 limbs +T669 NCBITaxon:10088 35806 35810 mice +T670 SO:0000704 35822 35827 genes +T671 NCBITaxon:10088 35836 35841 mouse +T672 CL:0000189 35848 35859 slow fibres +T673 UBERON:0002101 35889 35893 limb +T674 PR:000008974 35916 35919 Ihh +T675 UBERON:0002495 35936 35946 long bones +T676 NCBITaxon:40674 35968 35975 mammals +T677 SO:0000704 36001 36005 gene +T678 NCBITaxon:10088 36018 36022 mice +T679 CL:0000189 36036 36047 slow fibres +T680 NCBITaxon:10088 36222 36226 mice +T681 PR:000008974 36235 36238 Ihh +T682 CL:0000189 36260 36271 slow fibres +T683 UBERON:0002103 36304 36312 Hindlimb +T684 NCBITaxon:33208 36353 36360 animals +T685 UBERON:0002385 36403 36416 muscle tissue +T686 UBERON:0002102 36437 36445 forelimb +T687 UBERON:0003661 36485 36496 limb muscle +T688 UBERON:0002103 36588 36597 hindlimbs +T689 PR:000008974 36601 36604 Ihh +T690 NCBITaxon:10088 36608 36612 mice +T691 PR:000008974 36645 36648 Ihh +T692 UBERON:0003661 36675 36686 limb muscle +T693 UBERON:0002101 36739 36744 limbs +T694 GO:0007224 36754 36767 Hh signalling +T695 NCBITaxon:39107 36791 36797 Murine +T696 UBERON:0002103 36879 36888 Hindlimbs +T697 UBERON:0002102 36896 36905 forelimbs +T698 PR:000008974 36921 36924 Ihh +T699 NCBITaxon:10088 36939 36943 mice +T700 PR:000014841 37031 37034 Shh +T701 UBERON:0000922 37049 37056 embryos +T702 UBERON:0000948 37216 37221 heart +T703 UBERON:0001389 37249 37250 s +T704 UBERON:0001389 37251 37257 soleus +T705 UBERON:0001385 37259 37260 t +T706 UBERON:0001385 37261 37278 tibialis anterior +T707 UBERON:0001386 37280 37281 e +T708 UBERON:0001386 37282 37307 extensor digitorum longus +T709 UBERON:0001388 37309 37310 g +T710 UBERON:0001388 37311 37324 gastrocnemius +T711 UBERON:0000979 37326 37327 T +T712 UBERON:0000979 37328 37333 tibia +T713 UBERON:0001446 37335 37336 F +T714 UBERON:0001446 37337 37343 fibula +T715 UBERON:0001424 37345 37346 u +T716 UBERON:0001424 37347 37352 ulnar +T717 UBERON:0001423 37354 37355 r +T718 UBERON:0001423 37356 37362 radius +T719 PR:000014841 37435 37438 shh +T720 NCBITaxon:10088 37442 37446 mice +T721 UBERON:0002101 37447 37451 limb +T722 GO:0010467 37582 37592 expression +T723 UBERON:0002329 37608 37615 somitic +T724 NCBITaxon:10088 37642 37646 mice +T725 UBERON:0002329 37666 37673 somites +T726 CL:0000187 37697 37710 muscle fibres +T727 GO:0010467 37711 37721 expressing +T728 CL:0000189 37738 37749 Slow fibres +T729 UBERON:0002329 37775 37782 somites +T730 GO:0010467 37825 37835 expressing +T731 UBERON:0002329 37836 37843 somites +T732 NCBITaxon:10088 37851 37855 mice +T733 UBERON:0002329 37869 37876 somites +T734 GO:0010467 37912 37921 expressed +T735 UBERON:0000922 37922 37931 embryonic +T736 UBERON:0000922 37986 37995 embryonic +T737 CL:0000187 38039 38052 muscle fibres +T738 PR:000014841 38076 38079 shh +T739 NCBITaxon:10088 38083 38087 mice +T740 UBERON:0002329 38148 38155 somites +T741 NCBITaxon:1 38222 38233 individuals +T742 NCBITaxon:33208 38302 38309 animals +T743 NCBITaxon:33208 38337 38343 animal +T744 UBERON:0002329 38438 38445 somites +T745 PR:000014841 38460 38463 Shh +T746 UBERON:0002329 38467 38474 somites +T747 UBERON:0002101 38572 38577 limbs +T748 GO:0042571 38599 38609 antibodies +T749 NCBITaxon:32524 38670 38677 amniote +T750 CL:0000056 38928 38937 myoblasts +T751 UBERON:0004347 39036 39045 limb buds +T752 GO:0007224 39116 39129 Hh signalling +T753 UBERON:0002101 39133 39137 limb +T754 PR:000014841 39227 39230 Shh +T755 CL:0000187 39275 39288 muscle fibres +T756 PR:000014841 39337 39340 Shh +T757 GO:0030154 39359 39377;39381 39386 differentiation of ... cells +T758 PR:000014841 39427 39430 Shh +T759 CL:0000056 39440 39448 myoblast +T760 UBERON:0007023 39490 39495 adult +T761 UBERON:0002101 39587 39591 limb +T762 CL:0000010 39601 39617 cells in culture +T763 PR:000014841 39634 39637 Shh +T764 UBERON:0005090 39715 39722 muscles +T765 PR:000014841 39726 39729 Shh +T766 UBERON:0002101 39738 39742 limb +T767 PR:000014841 39811 39814 Shh +T768 UBERON:0000023 39984 39988 wing +T769 PR:000014841 40059 40062 Shh +T770 CL:0000056 40070 40078 myoblast +T771 UBERON:0002101 40153 40157 limb +T772 UBERON:0002329 40162 40169 somites +T773 PR:000014841 40173 40176 shh +T774 NCBITaxon:10088 40187 40191 mice +T775 CL:0000056 40282 40290 myoblast +T776 UBERON:0000023 40327 40331 wing +T777 CL:0000056 40363 40372 myoblasts +T778 PR:000008026 40420 40424 gli1 +T779 GO:0007224 40454 40467 Hh signalling +T780 UBERON:0002101 40488 40493 limbs +T781 PR:000013412 40542 40546 ptc1 +T782 PR:000008026 40551 40555 gli1 +T783 UBERON:0000023 40677 40681 wing +T784 UBERON:0002329 40690 40696 somite +T785 UBERON:0002101 40838 40843 limbs +T786 GO:0042571 40860 40868 antibody +T787 PR:000014841 40897 40900 Shh +T788 PR:000008974 40905 40908 Ihh +T789 PR:000008974 40968 40971 Ihh +T790 PR:000013412 41000 41004 ptc1 +T791 PR:000008026 41009 41013 gli1 +T792 GO:0010467 41014 41024 expression +T793 PR:000008974 41107 41110 Ihh +T794 GO:0010467 41111 41121 expression +T795 UBERON:0007688 41135 41141 anlage +T796 UBERON:0002102 41169 41177 forelimb +T797 PR:000008974 41192 41195 Ihh +T798 NCBITaxon:10088 41199 41203 mice +T799 PR:000008974 41298 41301 Ihh +T800 UBERON:0002103 41305 41313 hindlimb +T801 PR:000008974 41429 41432 Ihh +T802 UBERON:0002495 41452 41461 long bone +T803 PR:000014841 41565 41568 Shh +T804 NCBITaxon:39107 41623 41629 murine +T805 UBERON:0003661 41630 41641 limb muscle +T806 PR:000008026 41673 41677 gli1 +T807 GO:0010467 41678 41688 expression +T808 PR:000014841 41703 41706 Shh +T809 UBERON:0002101 41802 41806 limb +T810 PR:000014841 41911 41914 Shh +T811 NCBITaxon:7955 41919 41928 zebrafish +T812 UBERON:0002329 41929 41936 somites +T813 GO:0007224 41957 41970 Hh signalling +T814 SO:0000704 42024 42029 genes +T815 PR:000008974 42105 42108 Ihh +T816 PR:000014841 42113 42116 Shh +T817 PR:000014841 42290 42293 Shh +T818 CL:0000857 42330 42343 slow myoblast +T819 UBERON:0000023 42383 42387 wing +T820 CL:0000187 42388 42396 myocytes +T821 GO:0010467 42397 42404 express +T822 PR:000014841 42437 42440 Shh +T823 PR:000014841 42471 42474 Shh +T824 GO:0010467 42480 42490 expression +T825 UBERON:0000023 42559 42563 wing +T826 PR:000014841 42617 42620 Shh +T827 NCBITaxon:10088 42624 42628 mice +T828 UBERON:0002329 42668 42675 somites +T829 PR:000014841 42798 42801 Shh +T830 GO:0010467 42848 42855 express +T831 SO:0001026 43018 43025 genomic +T832 NCBITaxon:7955 43038 43047 zebrafish +T833 NCBITaxon:8353 43052 43059 Xenopus +T834 UBERON:0000922 43060 43067 embryos +T835 GO:0007224 43076 43089 Hh signalling +T836 CL:0000189 43124 43135 slow fibres +T837 NCBITaxon:32524 43242 43250 amniotes +T838 NCBITaxon:7742 43278 43289 vertebrates +T839 CL:0000189 43291 43301 slow fibre +T840 NCBITaxon:32524 43337 43345 amniotes +T841 GO:0007224 43361 43374 Hh signalling +T842 CL:0000189 43419 43423;43433 43439 slow ... fibres +T843 CL:0000190 43428 43439 fast fibres +T844 UBERON:0002101 43626 43630 limb +T845 PR:000014841 43742 43745 Shh +T846 GO:0005622 43767 43780 intracellular +T847 GO:0035556 43767 43791 intracellular signalling +T848 PR:000014841 43900 43903 Shh +T849 PR:000014841 43993 43996 Shh +T850 PR:000014841 44046 44049 Shh +T851 UBERON:0000479 44054 44061 tissues +T852 GO:0046903 44067 44074 secrete +T853 PR:000014841 44292 44295 Shh +T854 GO:0006915 44375 44384 apoptosis +T855 GO:0008219 44425 44435 cell death +T856 PR:000014841 44464 44467 Shh +T857 UBERON:0000023 44528 44532 wing +T858 GO:0010467 44641 44651 expression +T859 CL:0000010 44753 44761;44784 44789 cultured ... cells +T860 NCBITaxon:7955 44762 44771 zebrafish +T861 CL:0000353 44772 44789 blastomeric cells +T862 PR:000014841 44832 44835 Shh +T863 GO:0007224 44933 44946 Hh signalling +T864 NCBITaxon:7955 44950 44959 zebrafish +T865 GO:0008219 45002 45012 cell death +T866 GO:0006915 45196 45205 apoptosis +T867 CL:0000056 45430 45438 myoblast +T868 CL:0000056 45499 45508 myoblasts +T869 GO:0010467 45831 45841 expression +T870 GO:0005634 45902 45908 nuclei +T871 CL:0002372 45923 45930 myotube +T872 PR:000014841 46059 46062 Shh +T873 PR:000014841 46123 46126 Shh +T874 PR:000014841 46194 46197 Shh +T875 CL:0000056 46220 46229 myoblasts +T876 PR:000014841 46263 46266 Shh +T877 CL:0000056 46371 46380 myoblasts +T878 GO:0065007 46381 46389 regulate +T879 PR:000014841 46408 46411 Shh +T880 CL:0000056 46548 46556 Myoblast +T881 CL:0000056 46601 46609 myoblast +T882 UBERON:0004347 46730 46738 limb bud +T883 CL:0000056 46739 46748 myoblasts +T884 PR:000014841 46770 46773 Shh +T885 PR:000014841 46784 46787 Shh +T886 GO:0030154 46818 46833;46851 46853;46863 46868 differentiation ... of ... cells +T887 PR:000014841 46931 46934 Shh +T888 CL:0000056 46984 46993 myoblasts +T889 PR:000014841 47028 47031 Shh +T890 UBERON:0002101 47048 47052 limb +T891 GO:0010467 47122 47129 express +T892 CL:0000056 47195 47204 myoblasts +T893 PR:000014841 47212 47215 Shh +T894 CL:0000056 47403 47411 myoblast +T895 UBERON:0003661 47441 47453 limb muscles +T896 UBERON:0005090 47480 47487 muscles +T897 NCBITaxon:7215 47586 47596 Drosophila +T898 CL:0000189 47622 47633 slow fibres +T899 CL:0000056 47682 47691 myoblasts +T900 CL:0000056 47746 47754 myoblast +T901 PR:000014841 47822 47825 Shh +T902 GO:0010467 47831 47841 expression +T903 PR:000014841 47892 47895 Shh +T904 CL:0000056 47915 47923 myoblast +T905 GO:0051716 48063 48074;48084 48089 response of ... cells +T906 UBERON:0002471 48105 48113 zeugopod +T907 GO:0042571 48207 48215 antibody +T908 PR:000014841 48222 48225 Shh +T909 NCBITaxon:10088 48229 48233 mice +T910 GO:0007224 48252 48265 Hh signalling +T911 UBERON:0002329 48305 48312 somitic +T912 CL:0000056 48351 48359 myoblast +T913 UBERON:0002101 48420 48425 limbs +T914 PR:000014841 48473 48476 Shh +T915 PR:000014841 48563 48566 Shh +T916 GO:0010467 48572 48582 expression +T917 CL:0000189 48592 48603 slow fibres +T918 GO:0010467 48652 48662 expression +T919 CL:0000056 48729 48738 myoblasts +T920 UBERON:0002471 48777 48785 zeugopod +T921 UBERON:0002101 48897 48901 limb +T922 CL:0000056 49030 49038 myoblast +T923 CL:0000056 49222 49231 myoblasts +T924 PR:000014841 49349 49352 Shh +T925 GO:0051450 49364 49390 proliferation of myoblasts +T926 CL:0000056 49381 49390 myoblasts +T927 UBERON:0004347 49408 49417 limb buds +T928 UBERON:0002101 49485 49490 limbs +T929 PR:000014841 49499 49502 Shh +T930 UBERON:0002101 49511 49516 limbs +T931 UBERON:0002101 49595 49599 limb +T932 UBERON:0000479 49600 49606 tissue +T933 PR:000014841 49608 49611 Shh +T934 GO:0010467 49617 49627 expression +T935 UBERON:0002101 49635 49639 limb +T936 PR:000013412 49674 49678 ptc1 +T937 PR:000008026 49683 49687 gli1 +T938 UBERON:0000479 49739 49745 tissue +T939 PR:000014841 49774 49777 Shh +T940 UBERON:0002329 49880 49886 somite +T941 UBERON:0000023 49934 49938 wing +T942 GO:0042571 49952 49960 antibody +T943 UBERON:0002101 49969 49973 limb +T944 UBERON:0000479 50069 50075 tissue +T945 PR:000014841 50134 50137 Shh +T946 CL:0000056 50141 50150 myoblasts +T947 CHEBI:52290 50264 50273 mitogenic +T948 PR:000014841 50284 50287 Shh +T949 NCBITaxon:7955 50311 50320 zebrafish +T950 PR:000014841 50322 50325 Shh +T951 CHEBI:52290 50335 50342 mitogen +T952 UBERON:0006907 50347 50358 slow muscle +T953 PR:000014841 50412 50417 sonic +T954 PR:000014841 50440 50443 Shh +T955 UBERON:0000922 50464 50471 embryos +T956 GO:0010467 50477 50487 expressing +T957 PR:000014841 50488 50491 Shh +T958 UBERON:0000023 50525 50529 wing +T959 PR:000014841 50559 50562 Shh +T960 CL:0000056 50583 50592 myoblasts +T961 CHEBI:52290 50596 50605 mitogenic +T962 PR:000014841 50617 50620 Shh +T963 CL:0000056 50624 50633 myoblasts +T964 CL:0000056 50725 50733 myoblast +T965 CHEBI:52290 50734 50742 mitogens +T966 PR:000014841 50785 50788 Shh +T967 PR:000000034 50802 50806 BMPs +T968 GO:0008283 50810 50817 amplify +T969 PR:000000034 50856 50859 BMP +T970 PR:000008974 50873 50876 Ihh +T971 GO:0008283 50894 50907 proliferation +T972 PR:000014841 50984 50987 Shh +T973 GO:0010467 50993 51003 expression +T974 UBERON:0002101 51007 51012 limbs +T975 GO:0007224 51074 51087 Hh signalling +T976 CL:0000056 51091 51099 myoblast +T977 GO:0051450 51091 51113 myoblast proliferation +T978 CL:0000056 51234 51243 myoblasts +T979 CL:0000056 51291 51299 myoblast +T980 GO:0051450 51291 51313 myoblast proliferation +T981 UBERON:0002101 51378 51382 limb +T982 CL:0000056 51478 51487 myoblasts +T983 GO:0007224 51825 51838 Hh signalling +T984 CL:0000056 51899 51908 myoblasts +T985 GO:0007224 51942 51955 Hh signalling +T986 PR:000008026 51990 51994 gli1 +T987 GO:0010467 51995 52005 expression +T988 CL:0000056 52069 52078 myoblasts +T989 CL:0000056 52259 52267 myoblast +T990 UBERON:0004347 52321 52329 limb bud +T991 GO:0007224 52331 52344 Hh signalling +T992 GO:0046903 52488 52497 secreting +T993 GO:0042571 52520 52528 antibody +T994 UBERON:0006907 52576 52580;52596 52602 slow ... muscle +T995 UBERON:0006908 52590 52594;52596 52602 fast ... muscle +T996 GO:0010467 52640 52650 expression +T997 UBERON:0006907 52673 52684 slow muscle +T998 UBERON:0004347 52694 52702 limb bud +T999 UBERON:0006907 52787 52798 slow muscle +T1000 UBERON:0002101 52811 52815 limb +T1001 CL:0000189 52912 52922 slow fibre +T1002 UBERON:0002101 53018 53022 limb +T1003 UBERON:0002102 53063 53072 forelimbs +T1004 UBERON:0000922 53080 53086 embryo +T1005 CL:0000057 53087 53098 fibroblasts +T1006 GO:0009294 53110 53121 transfected +T1007 PR:000014841 53127 53130 Shh +T1008 GO:0006260 53139 53150 replication +T1009 NCBITaxon:11632 53161 53171 retrovirus +T1010 PR:Q91035 53194 53198 cShh +T1011 UBERON:0000922 53392 53398 embryo +T1012 UBERON:0000023 53405 53409 wing +T1013 UBERON:0004288 53490 53498 skeletal +T1014 UBERON:0000024 53563 53571 forewing +T1015 UBERON:0000922 53610 53617 Embryos +T1016 UBERON:0005090 53780 53787 muscles +T1017 UBERON:0000922 53790 53797 Embryos +T1018 CHEBI:17790 53821 53829 methanol +T1019 CHEBI:17992 53881 53888 sucrose +T1020 CHEBI:60004 53911 53918 mixture +T1021 CHEBI:17992 53926 53933 sucrose +T1022 UBERON:0000023 54005 54010 wings +T1023 GO:0042571 54086 54094 antibody +T1024 PR:000014841 54305 54308 Shh +T1025 GO:0042571 54378 54388 antibodies +T1026 GO:0042571 54471 54481 antibodies +T1027 CHEBI:15956 54501 54507 biotin +T1028 MOP:0000093 54501 54518 biotin-conjugated +T1029 NCBITaxon:9796 54519 54524 horse +T1030 NCBITaxon:10088 54538 54543 mouse +T1031 GO:0071735 54544 54547 IgG +T1032 CHEBI:15956 54554 54560 biotin +T1033 MOP:0000093 54554 54571 biotin-conjugated +T1034 NCBITaxon:9925 54572 54576 goat +T1035 NCBITaxon:10088 54590 54595 mouse +T1036 GO:0071753 54596 54599 IgM +T1037 GO:0097617 54664 54677 hybridisation +T1038 UBERON:0000024 54718 54726 forewing +T1039 UBERON:0005090 54727 54734 muscles +T1040 UBERON:0002101 54778 54782 limb +T1041 UBERON:0002102 54896 54905 forelimbs +T1042 UBERON:0000922 54924 54931 embryos +T1043 UBERON:0002101 54997 55002 Limbs +T1044 PR:000027795 55048 55055 trypsin +T1045 NCBITaxon:9796 55529 55534 horse +T1046 UBERON:0001977 55535 55540 serum +T1047 UBERON:0000922 55559 55565 embryo +T1048 PR:000014841 55651 55654 Shh +T1049 http://purl.obolibrary.org/obo/MONDO_0005550 55655 55663 infected +T1050 CL:0000057 55674 55685 fibroblasts +T1051 CL:0000057 55713 55724 fibroblasts +T1052 NCBITaxon:27592 55917 55923 bovine +T1053 UBERON:0001977 55924 55929 serum +T1054 UBERON:0001977 55956 55961 serum +T1055 CHEBI:17790 56026 56034 methanol +T1056 GO:0042571 56133 56143 antibodies +T1057 PR:000006427 56208 56214 desmin +T1058 CL:0000056 56254 56263 myoblasts +T1059 CL:0002372 56268 56276 myotubes +T1060 PR:000006427 56351 56357 desmin +T1061 GO:0005634 56439 56446 nuclear +T1062 GO:0005634 56553 56559 nuclei +T1063 PR:000014841 56654 56657 Shh +T1064 NCBITaxon:10088 56680 56685 mouse +T1065 CL:0000056 56686 56695 myoblasts +T1066 UBERON:0001977 57057 57062 serum +T1067 CHEBI:33282 57068 57079 antibiotics +T1068 PR:000014841 57335 57338 Shh +T1069 CHEBI:17790 57601 57609 methanol +T1070 CHEBI:33893 57711 57719 reagents +T1071 GO:0005634 57869 57875 nuclei +T1072 GO:0005737 57895 57904 cytoplasm +T1073 CL:0000187 57959 57967 myocytes +T1074 CHEBI:472552 58011 58028 Bromodeoxyuridine +T1075 GO:0097617 58135 58148 hybridisation +T1076 CHEBI:17790 58232 58240 methanol +T1077 CHEBI:42098 58254 58265 digoxigenin +T1078 PR:000014841 58404 58407 Shh +T1079 GO:0010467 58413 58423 expression +T1080 UBERON:0004347 58427 58436 limb buds +T1081 NCBITaxon:10088 58441 58446 mouse +T1082 PR:000014841 58472 58475 Shh +T1083 GO:0010467 58481 58491 expression +T1084 GO:0042571 58783 58793 antibodies +T1085 GO:0042571 58811 58821 antibodies +T1086 NCBITaxon:10088 58847 58852 mouse +T1087 PR:000014841 58853 58856 Shh +T1088 NCBITaxon:7955 58901 58910 zebrafish +T1089 PR:000014841 58911 58914 Shh +T1090 PR:000008974 59001 59004 Ihh +T1091 NCBITaxon:10088 59008 59012 mice +T1092 PR:000014841 59030 59033 Shh +T1093 NCBITaxon:10088 59037 59041 mice +T1094 GO:0007568 59179 59185 Ageing diff --git a/src/ontogpt/evaluation/craft/database/all/15238161.txt b/src/ontogpt/evaluation/craft/database/all/15238161.txt new file mode 100644 index 000000000..11a03c934 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15238161.txt @@ -0,0 +1,169 @@ +Hedgehog can drive terminal differentiation of amniote slow skeletal muscle + +Abstract + +Background + +Secreted Hedgehog (Hh) signalling molecules have profound influences on many developing and regenerating tissues. Yet in most vertebrate tissues it is unclear which Hh-responses are the direct result of Hh action on a particular cell type because Hhs frequently elicit secondary signals. In developing skeletal muscle, Hhs promote slow myogenesis in zebrafish and are involved in specification of medial muscle cells in amniote somites. However, the extent to which non-myogenic cells, myoblasts or differentiating myocytes are direct or indirect targets of Hh signalling is not known. + +Results + +We show that Sonic hedgehog (Shh) can act directly on cultured C2 myoblasts, driving Gli1 expression, myogenin up-regulation and terminal differentiation, even in the presence of growth factors that normally prevent differentiation. Distinct myoblasts respond differently to Shh: in some slow myosin expression is increased, whereas in others Shh simply enhances terminal differentiation. Exposure of chick wing bud cells to Shh in culture increases numbers of both muscle and non-muscle cells, yet simultaneously enhances differentiation of myoblasts. The small proportion of differentiated muscle cells expressing definitive slow myosin can be doubled by Shh. Shh over-expression in chick limb bud reduces muscle mass at early developmental stages while inducing ectopic slow muscle fibre formation. Abundant later-differentiating fibres, however, do not express extra slow myosin. Conversely, Hh loss of function in the limb bud, caused by implanting hybridoma cells expressing a functionally blocking anti-Hh antibody, reduces early slow muscle formation and differentiation, but does not prevent later slow myogenesis. Analysis of Hh knockout mice indicates that Shh promotes early somitic slow myogenesis. + +Conclusions + +Taken together, the data show that Hh can have direct pro-differentiative effects on myoblasts and that early-developing muscle requires Hh for normal differentiation and slow myosin expression. We propose a simple model of how direct and indirect effects of Hh regulate early limb myogenesis. + +Background + +Each muscle in a developing chick limb acquires a unique character from its inception [1]. Fibres form by the terminal differentiation of dividing myoblasts that elongate in particular orientations to form specific attachments to the skeleton. Simultaneously, the fibres of each muscle take on gene expression patterns characteristic of their future function. For example, those muscles destined to maintain body posture express certain isoforms of slow myosin from their inception, whereas future fast muscle regions fail to express this slow myosin [2]. It has been suggested that distinct cell lineages underlie the formation of slow and fast muscle fibres, and much evidence for myoblast heterogeneity has been obtained from studies both in vitro and in vivo [[3-7], reviewed in [8]]. Nevertheless, it is clear that for fibres to undergo differentiation at the appropriate time and place extrinsic cues must regulate muscle patterning. + +Work on muscle patterning in somites over the past decade has shown that various protein factors secreted by adjacent tissues act as extrinsic signals regulating the formation and fate of myogenic cells [[9], reviewed in [10-12]]. One such factor is Sonic hedgehog (Shh), derived from the ventral midline, which is required for expression of markers of the earliest population of myogenic cells in the medial somite of both birds and mice [13-15]. These medial somitic cells contribute to the early-born muscle fibres of the myotome, but their subsequent fate is not known in amniotes [16,17]. Ventral midline Hedgehog (Hh) signals are also required for formation of the earliest muscle cells in the zebrafish embryo, the adaxial slow cells [[18,19], reviewed in [20]]. The fate of these cells is known, they generate a population of slow muscle fibres that migrate to form a layer of slow muscle that covers the lateral surface of the somite [21,22]. In all vertebrates examined, a second myogenic cell population arises in the lateral somite by a distinct Hh-independent genetic pathway in response to signals from more lateral and dorsal tissues. Signals such as FGFs, BMPs and WNTs and their antagonists are prime candidates for patterning of lateral somitic cells, at least in amniotes [reviewed in [8,9,23,24]]. Wnt proteins from dorsal tissues are also implicated in medial myogenesis [25-30]. + +In the somite, induction of precursor myoblast populations is occurring close in space and time to terminal differentiation of myoblasts into contractile fibres. This makes analysis of the precise effects of extrinsic signals hard to determine. For example, Shh can promote both primary myogenesis and subsequent cell survival in somitic explants and in vivo, but the precise target cell populations are unclear [13,15,31-33]. In contrast, in the limb bud myogenic induction and terminal differentiation are temporally and spatially separated. Myogenic cells of the limb derive from a population of precursors that migrates into the limb bud from the lateral somite [34-36]. These cells already express genes required for myogenesis prior to their migration [37,38]. Evidence suggests that several distinct populations of myogenic cells enter the limb bud [5,39,40]. Thus, muscle formation within the limb bud omits some of the early steps that occur in the somites. Consequently, we chose the somewhat simpler and more accessible limb bud to analyse the effects of Hh on the differentiation and patterning of muscle fibre types. + +Previous work has shown that early manipulations that alter limb anteroposterior axis formation and skeletal pattern, including Shh mis-expression, change muscle and fibre type pattern in parallel [41,42]. Moreover, myoblast clones appear uncommitted to a particular character either early or late in myogenesis [43,44] indicating that local signals control fibre pattern. Nevertheless, implantation of cloned myoblasts into limbs can alter fibre pattern, although in such experiments it is difficult to rule out implantation of cells that are already undergoing terminal differentiation [45,46]. Myoblast implantation at later stages shows that local limb signals can re-programme myoblast differentiation [47,48]. It is clear that Hh expression within the limb at these later stages does not have a spatial pattern that is well correlated with formation of individual muscles or fibre types. Nevertheless, augmenting Hh signalling in a way that does not affect anteroposterior axis formation severely disrupts muscle patterning and differentiation leading to enlarged but disorganised muscles [49-51]. Conversely, a dramatic loss of muscle is observed in Shh-deficient mouse limbs [52]. Interpretation of these Hh results has differed, possibly because muscle is not the only tissue affected. Moreover, these studies do not distinguish direct effects of Hh on myogenic cells from indirect effects acting via non-myogenic cell populations in the limb. In the current paper, we use both in vitro and in vivo approaches to analyse the effect of Hh on muscle differentiation and slow fibre formation. We establish definitively using in vitro cultures that myoblasts can be directly induced to terminal differentiation by Shh. Moreover, Shh enhances slow myosin accumulation. With these in vitro results in mind, in vivo analysis of limbs with increased or decreased Hh signalling indicates that Hh is a muscle differentiation factor that promotes early slow myogenesis. + +Results + +Shh promotes myogenesis in limb monolayer culture + +We examined Shh action on wing myogenic cells by exposing limb bud cells to Shh in monolayer cultures, in which we hoped the effects of secondary signals elicited by Shh action on non-myogenic cell populations would be minimized. Wing buds were dissociated at HH22 and grown in growth medium either in the presence or absence of Shh-containing conditioned medium. After two days (the equivalent of HH28), Shh-treated cultures are noticeably more dense (Fig. 1A). Immunohistochemical detection of desmin, a marker of myogenic cells, shows that myogenic and non-myogenic cells increase in parallel on Shh exposure, so that the proportion of myogenic cells remains unaltered (Fig. 1B). However, control cultures seeded at higher density show a reduced overall proportion of myogenic cells compared to low density control cultures, suggesting that interactions between cells in high density cultures repress myogenic cell accumulation. Nevertheless, even at high density, the proportion of myogenic cells is maintained on exposure to Shh, demonstrating an increase in desmin+ cell number (compare Fig. 1A,1B). We next examined pan-MyHC immunoreactivity, a marker of differentiated myocytes and myotubes. In all control cultures, ~50% of myogenic cells express MyHC. With Shh, this proportion increases to ~70% in low and ~85% in high density cultures, equivalent to a threefold increase in absolute numbers of differentiated cells (Fig. 1C). Thus, Shh enhances both number and terminal differentiation of chick limb bud myogenic cells, confirming results on explant cultures of limb and somite [32,49,53]. + +Figure 1 + +Shh induces terminal differentiation and slow MyHC in wing primary cultures. Dissociated cells HH22 wing bud cells were plated at low or high density and grown for two days in the absence (white bars) or presence of Shh-conditioned medium (ShhQT6 CM, black bars) or control CM (QT6 CM, grey bars) and the number of total (A), desmin-reactive (B), pan-MyHC-reactive (C) and slow MyHC-reactive (D) cells determined. Data from two low and one high density experiment are presented (mean ± SEM, derived by addition of fractional errors, numbers above columns are dishes for the numerator). A. Irrespective of plating density, the number of cells in dishes exposed to Shh was ~70% greater than that of control dishes. B. The proportion of desmin-expressing myogenic cells was unchanged by Shh, at either low or high cell density. Note that the proportion of myogenic cells declined at high density, consistent with faster proliferation of non-myogenic cells. C. The proportion of differentiated myogenic cells (defined as the fraction of nuclei within desmin-containing cytoplasm that were in MyHC-containing cytoplasm) increased with Shh, irrespective of density. D. With Shh more differentiated cells express slow MyHC (defined as the fraction of nuclei within MyHC-containing cytoplasm that were in slow MyHC-containing cytoplasm). Note that even the high density culture has more total slow MyHC expressing cells because the same proportion of an increased number of differentiated cells express slow MyHC. ** P ≤ 0.002 compared to control(s), t-test on cell numbers after correcting for change in numerator. + +Shh can directly induce muscle terminal differentiation + +Experiments using limb bud cells do not exclude an indirect effect of Shh, as non-myogenic cell types are present in the culture. Nor do such experiments resolve whether Shh directly promotes myoblast terminal differentiation, proliferation or both, or acts by preventing apoptosis. We next tried to isolate clones of myoblasts from early chick wing buds, but despite repeated attempts this proved impossible, probably due to the known difficulty of cloning embryonic chick myoblasts combined with the low abundance of myogenic cells in early wing buds. So the effect of Shh exposure was tested on the adult mouse-derived C2C12 myoblast cell line (Fig. 2). All three C2C12-derived myoblast lines tested [54,55] respond to Shh treatment by increased terminal differentiation (Fig. 2A,2B). No change in cell density is apparent (Fig. 2A), nor is BrdU incorporation altered (Fig. 2C). No change in TUNEL, acridine orange or Annexin V staining is ever observed (data not shown). Therefore, Shh can act directly on these myoblasts to promote terminal differentiation. + +Figure 2 + +Shh increases C2 myoblast differentiation. C2 cells were exposed to ShhQT6 conditioned or control QT6 conditioned medium during both growth and differentiation. A. C2 myoblast lines C2/4 and C2C12 show enhanced differentiation in response to Shh. B. Quantitative comparison of enhanced differentiation of C2 myoblast lines in response to Shh calculated as the number of nuclei within MyHC-containing cytoplasm (i.e. number of differentiated myocytes). Note that the effect of Shh appears more dramatic if the proportion of nuclei in MyHC-containing cytoplasm is measured, because Shh-treated wells have the same or fewer total cells. For example, in one experiment C2/4 cells showed around 44% differentiation compared to ~6% in controls although across all experiments cell numbers were not significantly affected by Shh treatment. C. BrdU staining of C2/4 cells treated with ShhQT6 or control QT6 conditioned growth medium prior to a 2 hour BrdU pulse. D. In situ mRNA hybridisation on C2/4 myoblasts treated for one day with ShhQT6 conditioned growth medium revealed increased reactivity with a Gli1 antisense, but not sense probe, compared to myoblasts exposed to conditioned medium from control QT6 cells. E. Immunocytochemical analysis of C2/4 myoblasts similarly exposed to ShhQT6 conditioned or control QT6-conditioned growth medium using antibodies to MyoD, Myogenin and pan-MyHC. + +Members of the Gli family of transcription factors are both targets and mediators of Shh signalling [56]. Thus, if C2 myoblasts respond directly to Shh they would be expected to show elevated levels of gli gene transcription. The addition of Shh up-regulates gli1 transcripts in over 50% of C2 myoblasts (Fig. 2D), but does not induce gli2 expression (data not shown). Thus, C2 cells appear to be responsive to Shh through the normal Shh response pathway. + +To examine the mechanisms involved in the Shh response we determined the effect of Shh on C2 cells in growth factor-rich medium, which normally maintains C2 myoblasts in the cell cycle and inhibits terminal differentiation. Control cultures have low levels of nuclear MyoD protein, very little Myogenin and only rare MyHC-containing cells. However, after two days of Shh treatment, C2 cells show enhanced MyoD protein accumulation in many nuclei and a markedly higher frequency of groups of Myogenin immunoreactive cells, some of which also contain detectable MyHC (Fig. 2E). Shh, therefore, can drive nuclear MyoD accumulation and terminal differentiation of myoblasts even in the presence of growth factors found in serum. + +Shh can directly promote slow fibre formation + +When wing bud cultures are analysed for expression of slow MyHC, the vast majority of differentiated muscle cells fail to express detectable slow MyHC, just as nascent fibres do not express slow MyHC at the initiation of myogenesis in vivo (see below). In control low density cultures, a small proportion of differentiated cells (1–2%, defined as the proportion of nuclei within MyHC-containing cytoplasm that are in slow MyHC-containing cytoplasm) contain slow MyHC, detected by A4.840 immunoreactivity (Fig. 1D). Shh exposure doubles the proportion of myocyte/myotubes expressing slow MyHC to ~4%, which corresponds to a six-fold increase in absolute numbers of slow MyHC-reactive cells (Fig. 1D). However, in high density culture, slow MyHC is suppressed in controls and Shh fails to induce an increase in the proportion of cells expressing slow MyHC. + +The small rise in cultured wing cells expressing slow MyHC from ~1–2% to ~4% of total myocytes suggested that a sub-population of myogenic cells might be induced to express slow MyHC by Shh. However, as with overall terminal differentiation, selective cell survival and/or indirect effects of Shh could not be ruled out. To avoid these criticisms, we again turned to C2 cells. Two lines of C2 cells, C2C12 and C2/4, express very little slow MyHC after three days differentiation. Shh exposure did not induce slow MyHC in these cells, despite their Shh responsiveness (Fig. 2 and data not shown). However, another line of C2C12 cells, designated C2X, expresses a low level of slow MyHC in 5–15% of myocytes in the absence of Shh (Fig. 3). Exposure to Shh during two days of proliferation in growth medium and a subsequent three day period in differentiation medium enhances differentiation to a similar extent to that in other C2C12 lines (Figs 2B and 3A,3B,3D). In addition, the frequency and intensity of slow MyHC-reactivity in myocytes increases dramatically to above 30% of differentiated cells (Fig. 3A,3C,3D). Both effects of Shh conditioned medium on C2X cells are blocked by addition of the anti-Hh antibody 5E1, confirming that Shh is the inducing component of the medium (Fig. 3B,3C). Purified Shh also up-regulates slow MyHC (Fig. 3C). Despite the ability of 5E1 to block the activity of exogenously applied Shh, it does not reduce baseline slow MyHC expression, suggesting that the low level of slow MyHC expressed in these cells is not due to autocrine exposure to a Hh signal (Fig. 3C). After Shh exposure, a very similar proportion of mono-, di-, tri- and tetranucleate myocytes contain slow MyHC (Fig. 3D), indicating that increased fusion is not responsible for the extra slow MyHC. We conclude that Shh can act on this murine myoblast cell line to induce both terminal differentiation and slow MyHC accumulation. + +Figure 3 + +Shh promotes slow differentiation. A. Mouse C2X myoblasts were treated with control QT6 or ShhQT6 conditioned medium for two days growth and three days differentiation. Dual immunofluorescent analysis with pan MyHC (A4.1025, upper panels) and slow MyHC (A4.840, lower panels). Insets: anti-slow MyHC monoclonal antibodies A4.840 and BA-D5 confirm the induction of slow MyHC [61]. B, C. The number of differentiated myocytes, i.e. nuclei in MyHC-positive cytoplasm (B) and the proportion of differentiated myocytes expressing slow MyHC (C) was determined under various treatment regimes. Shh conditioned medium significantly increased (p < 0.001, t-test, n = 18 replicate wells in 3 experiments) both differentiation and the proportion of myocytes expressing slow MyHC compared to either untreated cells or control conditioned medium. This effect was blocked by addition of the 5E1 (1:300 diluted from 1.9 mg/ml) functionally-blocking anti-Shh monoclonal antibody, although basal differentiation and slow MyHC expression in control myocytes was unaffected. Purified preparations of mouse or zebrafish Shh N-terminal fragment also significantly induce differentiation and slow MyHC (p < 0.01, t test, n = 11 replicate wells in 2 experiments). D. Shh enhances differentiation and fusion of C2X myoblasts (left panel). Shh enhances slow MyHC accumulation in all classes of myotubes (right panel). Note that although the number of mononucleate myocytes is unaltered by Shh exposure, mononucleate myocytes are as efficiently induced to express slow MyHC as more mature multinucleate myotubes. + +Initial slow myogenesis correlates with Hh signalling in vivo + +The ability of Shh to enhance slow myogenesis in a murine cell line and a subset of chick primary myoblasts, just as it does in zebrafish, prompted us to re-examine the effects of Hh in vivo. First, we characterised developing chick wing buds with respect to slow MyHC expression using several monoclonal antibodies (Fig. 4). As we have described previously [47], myogenesis in the zeugopod (forearm region) commences at HH25 in both ventral and dorsal muscle mass (DMM) (Fig. 4B). At HH27/28, slow MyHC is detectable in both muscle masses. Two anti-slow MyHC antibodies, Na8 and A4.840, detect a subset of muscle fibres within the DMM running from the posterior of proximal DMM to the middle of the distal DMM (Fig. 4A,4B,4C,4D,4E,4F,4G,4H,4I,4J,4K). This pattern is similar to that previously reported for the product of the SMHC2 gene, a definitive marker of slow fibres in adult chickens [42,57]. By contrast, anti-slow MyHC antibody BA-D5 is expressed in most, if not all, early fibres, consistent with the reported expression pattern of SMHC1 gene product in all early primary fibres of the chick embryo (Fig. 4E,4I) [58]. At HH31, all three anti-slow MyHC antibodies react in both DMM and VMM, with a pattern approximately reciprocal with a neonatal/fast MyHC antibody N3.36, consistent with previous results using other antibodies [4]. Slow MyHC is most abundant in the posterior proximal (Fig. 4) and medial distal (data not shown) regions of the former DMM, which have split into individual muscles (Fig. 4L,4M,4N). Thus, BA-D5 immunoreactivity is lost within some muscles of the dorsal compartment, but is retained in the region that initially expressed the Na8 and A4.840-reactive MyHCs. Consequently, antibodies Na8 and/or A4.840 were used in all subsequent studies to mark definitive slow muscle. + +Figure 4 + +Early formation of a restricted group of slow MyHC-expressing fibres in the chick forewing. A. Schematic of muscle fibre types in the wing DMM of the HH28 chick zeugopod showing the location of sections in C-H. Definitive slow (red) and non-slow (green) fibres are indicated. B. Immunohistochemistry of transverse cryosection of HH25 chick wings showing earliest differentiation of embryonic MyHC-reactive cells. No fibre type differences were detected at this stage (data not shown). C-K. Serial cryosections at proximal (C-G) and distal (H-K) positions (as indicated in panel A) of HH28 transverse cryosections stained for pan-MyHC (C,D,E), and slow MyHC-reactive BA-D5 (E,I), A4.840 (F,J) and Na8 (G,K). Proximally the definitive slow fibres are located in posterior DMM (cf D with F, C with G). Distally, slow fibres are localised to the central region of the DMM (cf H with I-K). L-N. Serial transverse cryosections of HH32 proximal zeugopod with pan-MyHC (L) revealing muscle splitting, N3.36-reactive neonatal/fast MyHC (M) and definitive A4.840-reactive slow MyHC (N). Some muscles, such as the EMU, contained both fast and slow fibres (M,N arrowheads). Other muscles, such as the superficial region of the FCU, had many slow fibres but fewer fast fibres (M,N; right-pointing arrows). Other muscles, such as the PP/PS, had few slow but numerous fast fibres (M,N; left-pointing arrows). EDC extensor digitorum communis; Anc anconeus; EMU extensor metacarpi ulnaris; EIL extensor indicis longus; EMR extensor metacarpi radialis; PS pronator superficialis; PP pronator profundus; FDP flexor digitorum profundus; UMV ulni metacarpalis ventralis; FCU flexor carpi ulnaris. O-V. In situ mRNA hybridisation for ptc1 (O-Q), gli1 (R-T) or gli2 (U,V) and subsequent MyHC staining with MF20 (P,Q,S-V) in control (O,P,R,S,U) and RCAS/Shh (Q,T,V) limbs. Wholemount in situ at HH22-26 shows ptc1 and gli1 mRNA in distal/posterior limb (O,R; dorsal views of right wing bud, anterior is up). By HH28, an additional zone of ptc1 mRNA is present proximally in the posterior DMM (O). Gli1 mRNA is also accumulating in distinct zones away from the ZPA (R). Sections of the zeugopod show ptc1 mRNA in both muscle and non-muscle regions, with restriction of ptc1 signal to regions without MyHC stain. RCAS/Shh implant up-regulates ptc1 throughout the dorsal region around the implant (Q, inset shows shh mRNA in/near DMM). Normal gli1 expression (S) is partially reciprocal to gli2 mRNA (U). Gli1 mRNA is up-regulated and gli2 reduced around a RCAS/Shh implant (T,V; inset shows shh mRNA in/near DMM). Dorsal is up and posterior to the left in panels B-N,P,Q,S-V. (u) ulna, (r) radius. Scale bar = 600 μm (A-K), 250 μm (L-N). + +To examine the role of endogenous Hh signalling in wing myogenesis we analysed expression of gli1 and ptc1, a gene encoding a Hh receptor. Both genes are themselves downstream targets of Hh signalling [56]. Ptc1 and gli1 are highly expressed in posterior and distal limb regions from HH21-28, due to Shh deriving from the zone of polarizing activity (ZPA; Fig. 4O,4R). Ptc1 expression declines in wing cells as they leave the progress zone and commence histogenesis (Fig. 4O). Distal gli1 expression is more extensive but declines similarly (Fig. 4R). However, by HH27 new regions of Hh signalling arise around the posterior region of the DMM, perhaps because Indian hedgehog (Ihh) expression commences in cartilage anlage [59]. Ptc1 and gli1 are expressed in both non-myogenic tissues such as perichondrium and limb mesenchyme, and in the myogenic zone surrounding muscle fibres (Fig. 4P,4S). It is striking that the muscle region with highest ptc1 and gli1 mRNA roughly corresponds to the early slow zone, although it is also clear that Hh signalling in this region is not restricted to myogenic cells and that there are many fibres not expressing slow MyHC in the region of strong Hh signalling (Fig. 4F,4G,4O,4P,4R,4S). + +Shh over-expression delays myogenesis and induces ectopic slow muscle 48 hours after grafting + +To investigate the influence of Hh on wing muscle formation, we implanted pellets of chick embryo fibroblasts expressing a Shh/RCAS replication competent retroviral vector into the dorsal surface of HH22 chick limb buds and allowed the embryos to develop for two days until HH27-28 (Figs 4P,4Q,4R,4S,4T,4U,4V,5). Previous work had shown that this implantation regime does not disrupt digit pattern as do earlier and more anterior/distal implants [42,49]. After shh over-expression, ptc1 and gli1 mRNAs are up-regulated near Shh-expressing cells in a broader and more anterior region in and around the DMM than in contralateral controls (Fig. 4Q,4T). Analysis of gli2, another Hh-responsive gene implicated in Hh signalling, shows reciprocal expression to gli1 in un-manipulated limbs (Fig. 4U). Shh over-expression does not induce gli2 (Fig. 4V). Thus, ptc1, gli1 and gli2 expression suggest that the posterior DMM receives Hh signals around the time of slow muscle initiation at HH27, and that RCAS/Shh implantation augments this signal and expands it into the anterior DMM. + +Figure 5 + +Shh/RCAS-infection of chick wings blocks early muscle differentiation. A. Pellet of chick embryo fibroblasts expressing theShh/RCAS replication-competent retroviral vector is grafted into the dorsal mesenchyme of chick right wing buds at HH22 and embryos were maintained to HH27-28. B-G. Contralateral (B-D) and Shh/RCAS infected (E-G) wings were serially sectioned and stained for pan-MyHC (B,E), anti-slow MyHC (C,F), and anti-Hh (D,G) antibodies. Control left wings have a normal distribution of muscle fibres and lack Shh staining in muscle-forming mesenchyme (B-D) whereas some Shh/RCAS right wings show a loss of pan-MyHC-reactive cells (E). Ectopic slow MyHC is present in the anterior dorsal muscle mass (F, arrow). H-J. Fibre numbers counted in adjacent sections at comparable levels of control contralateral and operated HH27-28 limbs. The values presented must be regarded as an imprecise reflection of absolute fibre numbers because resolution of small fibres from adjacent larger fibres was difficult and varied depending on the orientation of fibres within the section. Total fibre numbers (H), slow fibre numbers (I) and the proportion of fibres that contained slow MyHC (J) are presented from four limbs showing normal DMM extent (Increased slow limbs), five limbs showing reduced DMM (Reduced muscle limbs) and the pooled data (All limbs). Dorsal is up and posterior to the left in panels B-G. (u) ulna, (r) radius. Scale bar = 500 μm. + +Similarly implanted limbs were serially-sectioned and analysed for expression of Shh protein, slow MyHC and pan MyHC. Control contralateral limbs show a broad DMM, with slow MyHC in the posterior portion above the ulna condensation (Fig. 5B,5C). Among treated wings, three classes of outcome are observed. In ~50% (8/15) wings, there was a complete loss of muscle tissue in the posterior region of the dorsal zone and reduced muscle in the anterior region (Fig. 5E,5H). In another ~30% (4/15) wings, the DMM had altered shape and a variable reduction in the total number of differentiated fibres in the region of Shh accumulation (Fig. 5E,5H). The three remaining Shh/RCAS wings revealed no phenotype, correlated with low ectopic Shh protein and young age (data not shown). In all affected wings, the anterior DMM that would not normally express any slow MyHC contained significant levels of slow MyHC, such that the proportion of all fibres that contained slow MyHC was doubled (Fig. 5F,5I,5J). In most operated wings showing a muscle phenotype, Shh was detectable in or close to the DMM (Fig. 5G, arrowhead). The location of highest ectopic Shh correlated with loss of muscle fibres in severely-affected wings (Fig. 5E,5F,5G; arrowheads). Thus, exposure of developing chick wing buds to Shh leads to a reduction in total muscle differentiation 48 hours after grafting, as already reported [50,51]. Despite this reduced myogenesis, we found an increase of slow muscle. + +Prolonged Shh over-expression enhances limb and muscle size without increasing slow + +To examine the longer term consequence of Shh over-expression on muscle formation we permitted implanted embryos to develop for three or four days to HH30 or 32 when significantly more fibres have formed in controls. At these stages, Shh-treated limbs are obviously bigger than control limbs (Fig. 6 compare A to D and G to K for HH30 and 32, respectively). At HH30, operated limbs (Fig. 6D,6E) show increased DMM area and altered muscle splitting, compared to control wings (Fig. 6A,6B). By HH32, analysis of MyHC expression in the autopod shows a large increase in muscle fibres compared to contralateral control limb (data not shown). In contrast, in zeugopod, expansion of the DMM is variable: some limbs have enhanced muscle mass whereas others simply appear disorganized, with muscle splitting less clear cut than in the VMM (data not shown). Thus, as reported previously [60], Hh over-expression ultimately perturbs muscle splitting and enhances terminal differentiation. + +Figure 6 + +Later-forming fibres do not accumulate slow MyHC in response to Shh. Dorsal muscle mass from contralateral (A-C) and operated (D-F) wing implants grown to HH30 and stained for Pan MyHC (MF20, A,D) and slow MyHC (Na8, B,E) after in situ hybridisation for Shh mRNA (C,F). Shh mRNA is widespread in the dorsal muscle mass, which is expanded and poorly split (A,D). In contralateral muscle most slow fibres are in the medial region of the dorsal/posterior muscle block (B). In the operated limb, slow MyHC is more abundant in the dorsal region, with numerous cells in ectopic lateral locations (arrows, E). Note that the ventral region of the dorsal muscle mass has few if any ectopic slow fibres. + +Concerning slow differentiation, Shh over-expression increases the number of slow fibres at HH30 (Fig. 6B,6E). This change may relate to the increase in total fibres as many fibres do not express slow MyHC despite proximity to the Shh source (Fig. 6A,6D). By HH32, no increase in slow fibres, as a proportion of total fibres, is detected and slow fibre pattern appears normal (data not shown). Thus, the induction of an increased proportion of slow fibres was not a continuing process within the chick wing bud, despite the continued presence of Shh. + +Blockade of Hh reduces slow muscle differentiation + +To address the role of endogenous Hh in muscle patterning, we implanted hybridoma cells secreting a functionally-blocking anti-Hh antibody into the proximal dorsal limb at HH 21-24 and examined subsequent muscle differentiation (Fig. 7). Anti-Hh hybridoma cells cause a reduction in limb cross sectional area by 15% (P < 0.001, n = 14, 0.64 to 0.55 mm2), whereas control hybridoma cells have no effect. Overall DMM area is also reduced at HH27/28 but without obvious change in location or shape. However, the initial appearance of slow MyHC is more severely reduced or blocked entirely compared to contralateral control limbs (7/9 limbs at HH27/29, Figs 7A,7B,7C,7D,8). The effect of the implant is generally more severe in younger limbs, but in all affected cases extended over at least 480 μm of the zeugopod (Fig. 8). We next quantified slow MyHC-expressing fibres in at least four sections spaced by 120 μm within each limb in comparison to contralateral control limbs at the same proximodistal level. At HH27/28 or 28 anti-Hh treated limbs showed a 79% (± 12% SEM, n = 4) reduction in slow fibres. More mature treated limbs at HH28/29 showed a lesser reduction of 27% (± 19% SEM, n = 5). In even older limbs (HH30), there is a reduction in DMM extent in the zeugopod region of anti-Hh treated limbs (4/4 limbs, Fig. 7E,7F,7G,7H). However, slow MyHC is not noticably reduced (4/4 limbs, Fig. 7E,7F,7G,7H). Limb outgrowth and digit formation are unaffected at any stage (data not shown), suggesting that the anti-Hh antibody does not reach sufficient titre to prevent ZPA activity. Although implants were generally found within the elbow or stylopod region, changes in stylopod muscle are less marked than in zeugopod (data not shown), consistent with a diminished role for Hh in the later stages of stylopod muscle formation. Control hybridoma cells (encoding a high-titre IgG1 anti-MyHC antibody N2.261) have no significant effect on any parameter examined (6 limbs, Fig. 7I,7J). Thus, Hh is required for normal initiation of slow myogenesis and early muscle differentiation in zeugopod. + +Figure 7 + +Anti-Hedgehog antibody delays slow MyHC and reduces muscle differentiation in chick wing bud. Implants of anti-Hh (5E1; A,B,E,F) or control anti-MyHC (N2.261; I,J) hybridoma cells were placed into HH21-24 chick wing buds and analysed two days later at HH27-30 for pan MyHC (A4.1025; A,C,E,G,I) or slow MyHC (Na8; B,D,F,H,J), in parallel with contralateral limb controls (C,D,G,H). A-D. A less mature wing shows failure of slow MyHC expression (arrows) with little effect on overall muscle differentiation. E-H. A more mature wing showing reduced muscle differentiation, but slow MyHC is present in the residual muscle mass. I,J. Control implants have no effect on timing, extent or type of muscle differentiation. Slow fibres are less abundant in some regions, as in unmanipulated wings (red arrows, G-J). In this example, the Cellagen block containing hybridoma cells detected by the secondary reagents is located within the forewing region (arrowheads) but does not disrupt muscle pattern. Dorsal up, posterior to left. Contralateral images have been reversed to aid comparison. + +Figure 8 + +Preferential inhibition of slow myogenesis by anti-Hh antibody. Schematic drawings of 120 μm spaced sections from each of the seven affected anti-Hh implanted limbs and aligned contralateral controls. Black outline shows the DMM, red stipple indicates slow MyHC. Drawings were made from identical low magnification images of adjacent sections reacted immunohistochemically for A4.1025 or Na8. Two young limbs (HH27/28) show marked reduction in slow MyHC, but little effect on DMM area. Slightly older limbs show a reduction in slow MyHC accompanying a diminished muscle mass. In two limbs, the Cellagen implant is present within the elbow region. Distal limb at top of stack, elbow at base. Within each section dorsal is up and anterior to left. Contralateral limbs are flipped horizontally to aid comparison. + +Murine Hh knockouts contain slow muscle fibres + +The early loss, but later recovery, of slow MyHC expression in anti-Hh treated limbs raised the possibility that the implant might lose effectiveness with time in vivo. However, later implants had lesser effects on muscle growth (4/4, data not shown), arguing that Hh has less role in later myogenesis. To examine the issue more definitively, we turned to mice lacking Hh genes. In the mouse, many slow fibres arise in deep regions of the limb near to the source of Ihh from developing long bones [61,62]. As in other mammals, only a single slow MyHC gene is known in mice, but primary slow fibres do fall into two distinct populations with different innervation and fate: deep fibres remain slow, superficial ones turn fast [61,63]. We, therefore, examined myogenesis in mice lacking Ihh at a stage when deep slow fibres display their unique character. Hindlimb elongation is severely reduced in these animals, and this is accompanied by a decrease in muscle tissue (Fig. 9A). However, forelimb growth is relatively normal, and so is limb muscle content and pattern (Fig. 9B). No obvious lack of slow MyHC is observed in either fore- or hindlimbs of Ihh-/- mice (Fig. 9A,9B). Thus, ablation of Ihh alone permits fairly good limb muscle patterning, similar to the situation in older chick limbs in which Hh signalling is reduced. + +Figure 9 + +Murine Hh knockouts have inefficient differentiation and delayed slow myogenesis. A, B. Hindlimbs (A) or forelimbs (B) from E18.5 Ihh-/- or sibling mice were cryo-sectioned and stained for slow (A4.840) and fast (N3.36) MyHC. C. Whole E9.5 Shh-/- or sibling embryos were cryo-sectioned and stained serially for pan (A4.1025) or slow (A4.840) MyHC. Comparable anteroposterior levels are shown, based on the orientation of the heart elsewhere in the sections. s soleus, t tibialis anterior, e extensor digitorum longus, g gastrocnemius, T tibia, F fibula, u ulnar, r radius, A anterior, P posterior, D dorsal, V ventral, L lateral, M medial. + +In shh-/- mice limb outgrowth and muscle growth is severely curtailed preventing meaningful analysis of muscle pattern [52]. So we examined slow MyHC expression in the reduced somitic muscle. In wild-type E9.5 mice, around 12 rostral somites contain differentiated muscle fibres expressing MyHC (Fig. 9C). Slow fibres were observed in rostral somites, but not in the two-three caudalmost MyHC-expressing somites (n = 6 mice). In rostral somites, many fibres lacked slow MyHC, but expressed embryonic MyHC (Fig. 9C and data not shown). This confirms that embryonic MyHC is acquired before slow MyHC in early muscle fibres [64]. In sections from shh-/- mice, fewer differentiated fibres were present, but around eight somites contained differentiated muscle which was often mis-oriented (6/6 individuals; Fig. 9C). Among residual fibres slow MyHC was undetectable in most animals (5/6; Fig. 9C). The single animal containing slow MyHC was developmentally more advanced, based on the presence of MyHC in more somites. Thus lack of Shh in somites leads to reduced early differentiation and delayed slow MyHC accummulation, as observed in chick limbs treated with anti-Hh antibodies. + +Discussion + +Hitherto, all studies of the actions of Hh on amniote muscle have failed to rule out indirect effects deriving, for example, from Hh eliciting secondary signals from adjacent non-muscle cells. Here we show that Hh can directly promote terminal differentiation and slow MyHC accumulation by at least some myoblasts in cell culture. We find that Hh is required for the earliest definitive slow myogenesis in chick limb buds and use this new understanding to develop a simple model of a role of Hh signalling in limb myogenesis. + +Hh directly induces muscle differentiation + +The results presented show that Shh can promote the terminal differentiation of muscle fibres both in vivo and in vitro. Our observation that Shh promotes terminal differentiation of C2 cells (Fig. 2) definitively demonstrates that Shh can be a myoblast differentiation factor, at least on this adult muscle-derived cell line. This effect occurs even in the presence of growth factors. Early limb myogenic cells in culture also respond to Shh exposure by increased differentiation (Fig. 1). We also observed increase of muscles in Shh-treated limb (Fig. 6). This is consistent with the numerous reports showing that Shh increases muscle differentiation in explant cultures or in vivo [14,25,33,52,53,65]. Conversely, and crucially, we find that local reduction of Hh function in the chick wing reduces muscle differentiation (Figs 7,8). This suggests that lack of Shh-driven myoblast differentiation may contribute to the severe reduction of muscle in early limb and somites in shh deficient mice [14,52]. It is, therefore, highly likely that one action of Hh is the direct promotion of myoblast differentiation in developing chick wing bud. + +A direct action of Hh on myoblasts is also supported by the rapid accumulation of gli1 mRNA, a downstream target of Hh signalling, in C2 cells and in limbs. In unmanipulated chicks, significant levels of ptc1 and gli1 mRNA accumulate in muscle masses, being highest in the posterior DMM (Fig. 4). This suggests that myogenic cells in both wing DMM and somite are exposed to Hh signals around the time of their first differentiation [13,49,59,66,67]. + +Which Hh could promote muscle differentiation in limbs? As the anti-Hh antibody blocks the function of both Shh and Ihh [68], our in vivo manipulations do not address this issue. Ihh is an obvious candidate, as ptc1 and gli1 expression are up-regulated in the posterior DMM at the time and location of commencement of Ihh expression in cartilage anlage around HH27 [59]. However, forelimb myogenesis in Ihh-/- mice appears relatively normal, although we can not rule out undetected transient defects. Even in Ihh-/- hindlimb, where muscle differentiation is greatly reduced, it is impossible to ascribe this reduction to a direct action of Ihh because failure of long bone elongation could prevent muscle growth through lack of stretch-induced hypertrophy signals. Similarly, Shh is not absolutely required for the initiation of some murine limb muscle differentiation [52]. However, gli1 expression suggests that Shh signalling extends quite far into what we have called the pre-myogenic zone [47] in the distal limb until at least HH27/28 (Fig. 4). So differentiating myogenic cells may also be exposed to low levels of Shh. In zebrafish somites, distinct levels of Hh signalling elicited by the combined action of at least three Hh genes lead to different myogenic outcomes [18,69-72]. So the additive effects of Ihh and Shh, perhaps having different effects at particular overall concentrations, likely contribute to the sculpting of muscle differentiation. + +Hh and slow myogenesis + +We found that Shh has a consistent positive effect on slow myoblast differentiation. Differentiating chick wing myocytes express slow MyHC more frequently after Shh exposure in vitro (Fig. 1) or Shh over-expression in vivo (Fig. 5). Conversely, an early effect of blocking Hh in the wing is failure of slow MyHC accumulation (Figs 7,8). And Shh-/- mice have delayed slow MyHC accumulation in somites, perhaps due to loss of an early fibre population (Fig. 9). Lastly, one line of C2 cells accumulates more slow MyHC after Shh exposure (Fig. 3). Other C2 lines that do not express significant levels of slow MyHC in control conditions fail to up-regulate slow MyHC in response to Hh, perhaps indicating that Hh is unable to open the slow MyHC genomic locus. Both zebrafish and Xenopus embryos require Hh signalling to make some early populations of slow fibres, but not others [73,74]. This argues strongly that Hh-driven slow myogenesis is an ancestral character of amniotes. Nevertheless, as in lower vertebrates, slow fibre formation does eventually occur in amniotes with defective Hh signalling. Indeed, considering the complex pattern of slow and fast fibres in older muscle, it is clear that many factors in addition to Hh must be involved in establishing the pattern. Our evidence suggests that Hh acts primarily during the earliest stages of limb myogenesis. + +What is the relationship between the differentiation-promoting and slow MyHC-promoting actions of Shh on C2 cells? Whereas intracellular signalling and terminal differentiation was triggered rapidly in all C2 cells, slow MyHC up-regulation required longer Shh exposure. Therefore, one can argue that differential cell survival could account for the Shh-dependent increase in slow MyHC, particularly as Shh, or tissues that secrete it, have been shown to promote survival of some myogenic cells [31,32,52,75]. However, we think a purely survival effect is unlikely, for several reasons. First, slow MyHC accumulation in single cells is greater with Shh, suggesting induction rather than simply enhanced survival. Second, assays for apoptosis in our C2 cultures revealed very little cell death, and this was unaffected by Shh exposure (unpublished result). Third, blockade of Hh in the wing bud reduces slow MyHC without a proportional reduction in differentiated muscle (Figs 7,8). Fourth, Hh over-expression in vivo induces ectopic slow while simultaneously reducing total differentiation (Fig. 5). Fifth, in cultured zebrafish blastomeric cells, some of which spontaneously form muscle, Shh induces conversion to a slow fate without affecting cell survival [76]. Conversely, reduction of Hh signalling in zebrafish prevents slow myogenesis without inducing cell death [69]. As altered cell survival does not explain the differentiation promoting activity of Hh, it seems unnecessary to invoke it in regard to slow myogenesis. In C2 cells, blockade of apoptosis appears unlikely to explain the slow promoting activity of Hh, leaving promotion of slow differentiation as the prime explanation. In vivo, the potential combination of direct and indirect effects of Hh, possibly on several myoblast subsets, make attribution of direct effects to Hh action on myoblasts impossible (see below). Nevertheless, our in vitro findings highlight direct induction of slow differentiation by Hh as a mechanism requiring serious consideration. + +Could simply enhancing terminal differentiation account for the increase in slow? In vivo manipulations fail to reveal a correlation between increased slow expression and enhanced differentiation (Fig. 5). Nor do the number of nuclei in a cultured myotube (a rough assay of maturity) predict whether slow MyHC is induced (Fig. 3). Indeed, C2/4 cells differentiate well in response to Shh but fail to show the up-regulation of slow MyHC elicited by Shh in C2X cells, which differentiate less extensively with or without Shh. In addition, not all myoblasts in any line respond similarly to Shh exposure. It seems probable, therefore, that both intrinsic and micro-environmental differences between myoblasts regulate their response to Shh and could influence whether the response is simply terminal differentiation, or includes other events, such as slow MyHC accumulation. + +Myoblast hetereogeneity of response to Hh + +Intrinsic myoblast heterogeneity, possibly based on cell lineage, may also influence Hh response. As with C2 cells, not all cultured chick limb bud myoblasts respond similarly to Shh exposure. Shh efficiently enhanced terminal differentiation from 50% to ~80% of myogenic cells, showing that at least 30% of myogenic cells are likely to be Shh-responsive. However, only a few percent of chick myoblasts acquired slow MyHC in response to Shh (Fig. 1). Early limb myogenic cells have distinct clonally-heritable tendencies to either express slow MyHC or not do so [3,45,48,77]. We suggest that, while most myoblasts may be Shh sensitive, sub-populations may respond differently based on their intrinsic capacity. This view parallels that of Stockdale and colleagues based on experiments showing differences in the myoblast populations forming distinct limb muscles [6,7]. Fibres in distinct muscles differ in slow MyHC from their inception [1]. So it is possible, by analogy with the situation in Drosophila [8] that the increase in slow fibres reflects a change in muscle identity of founder myoblasts, rather than a direct induction of slow MyHC. Altered myoblast identity could contribute to the failure of muscle splitting after Shh over-expression in vivo. Thus, by showing differential effects of Shh on distinct clonal myoblast lines that parallel those in primary cultures and in vivo (see below), our findings indicate that cell intrinsic differences determine the response of myogenic cells to Hh. + +In the zeugopod, the earliest muscle differentiation is reduced, but not ablated, by introduction of anti-Hh antibody or in Shh-/- mice (Figs 7,8; [52]). Hh signalling is required for some but not all early somitic myogenesis [15]. This shows that some myoblast populations do not require Hh for differentiation. In later limbs, blocking Hh reduces fibre formation and extra Shh augments differentiation (Figs 5,6,7,8). Similarly, Hh blockade reduces slow MyHC and Shh over-expression augments slow fibres early, but has little or no effect on slow MyHC expression in later muscle. Taken together, these observations indicate that myoblasts generating the earliest fibres in the zeugopod (before about HH28) may respond differently to Hh from those contributing to DMM growth after this stage. Many limb signals other than Hhs undoubtedly influence muscle pattern and likely affect the response to Hh [78,79]. Resolution of whether myoblast lineage or environmental effects underlie this difference will be important. + +Indirect proliferative effects of Hh on myogenic cells + +Direct pro-differentiative effects of Hh on some myoblasts do not rule out other direct or indirect effects of Hh. Our results confirm and extend previous reports showing that Shh can induce proliferation of myoblasts [49,50], both in limb buds and in primary cultures. However, other cell types are affected in limbs because Shh-treated limbs are bigger (Fig. 6). There is good evidence for effects of Hh on non-myogenic limb tissue. Shh over-expression causes limb hypertrophy with up-regulation of ptc1 and gli1 outside muscle masses and increase in non-myogenic tissue area (Figs 5,6). Similarly, Shh causes growth of non-myogenic as well as myogenic cells in our chick primary cultures (Fig. 1) and in somite explants [32]. Conversely, inhibition of Hh in wing with anti-Hh antibody reduces limb growth in addition to reducing muscle mass size: this effect is first noticeable in non-muscle tissue (Figs 7,8). Nobody has reported a proliferative effect of Shh on myoblasts independently of a proliferative effect on other cells. On the contrary, in the C2 cell line we clearly found no mitogenic effect of Shh (Fig. 2). Moreover, in zebrafish, Shh is not a mitogen for slow muscle precursors: muscle differentiation is delayed in the sonic-you mutants that lack Shh [69] and induced in embryos over-expressing Shh [18,19]. Therefore, either chick wing cells respond differently to Shh compared with other myoblasts or mitogenic effects of Shh on myoblasts are indirect. It is highly likely that Hh action on non-myogenic cells leads to release of myoblast mitogens. One hypothesis has already proposed that Shh acts through BMPs to amplify the number of myogenic cells [50] and BMP induction by Ihh causes cartilage proliferation indirectly [68]. So the temporary inhibition of terminal differentiation by Shh over-expression in limbs (Fig. 5; [50]) may be a consequence of an indirect effect of Hh signalling on myoblast proliferation. + +Combining our results with published data, we propose a model in which Hh promotes muscle differentiation directly in myoblasts within the muscle masses. But Hh also promotes myoblast proliferation indirectly by eliciting muscle growth factors from non-myogenic limb cells. This hypothesis explains how Hh may contribute to growth of muscle masses by increasing myoblasts at the edge, where proliferative signals from non-myogenic cells would predominate over the direct differentiative signal. Deeper within the muscle mass, pro-differentiative signals including Hh would be in the ascendant, adding new primary fibres at the periphery of the existing differentiated zone. At early stages distinct levels of Hh signalling may trigger slow myogenesis, possibly in sub-populations of myoblasts. Once muscle splitting commences Hh signalling declines, as indicated by reduced gli1 expression, and other influences probably determine the decision of later myoblasts to divide or differentiate. Muscle-specific ablation of Hh responsiveness will be required to test this hypothesis definitively. + +Conclusions + +We show that Hh can directly promote myoblast differentiation, at least in vitro. In vivo in chick limb bud, Hh signalling is occurring at the right time and place to affect early slow myogenesis. We introduce a new methodology, Cellagen implants of hybridoma cells secreting functionally-blocking antibody, and show that Hh is required for proper early slow (but not fast) muscle differentiation. Conversely, Hh over-expression induces ectopic early slow muscle in chick limb bud. Neither gain nor loss of later Hh function affects differentiation of later-formed slow muscle. Thus early limb Hh levels promote slow myogenesis, but are unlikely to be solely responsible for the details of slow fibre pattern. The data suggest a simple model of how direct and indirect effects of Hh sculpt early limb myogenesis. + +Methods + +Manipulated chick forelimbs + +Chick embryo fibroblasts (CEF) were transfected with Shh/RCAS, a replication-competent retrovirus containing the entire cShh coding sequence. Anti-Hh 5E1 hybridoma cells (~2 × 104) were embedded in a 10 μl Cellagen block. Pellets (~100 μm diameter) or block fragments (2 μl) were implanted into Rhode Island Red chick embryo right wing buds at Hamburger and Hamilton stages (HH) 21-24, avoiding complications due to skeletal pattern alteration by grafting on the dorsal side in the future forewing region, as previously described [49]. Embryos were maintained in a humidifier at 37°C, for 2–4 days and analysed at HH27-29 (E6) pre-splitting, and HH30-32 (E7/8) mid/post-splitting of DMM into its component muscles. + +Embryos were fixed at -20°C in methanol, rehydrated in graded PBS, soaked for 2 hrs in 20% sucrose, transferred to a 2:1 mixture of 20% sucrose and Tissue-Tek cryoprotectant (Bright), experimental and contralateral wings aligned and frozen in a single block and cryosectioned. Primary monoclonal antibody supernatants of A4.1025 [80], BA-D5, A4.840 and N3.36 [81] were diluted 1:10 [61,80]. EB165 and Na8 ascites, gifts of E. Bandman (University of California, Davis) were used at 1:5000 [57,82]. To detect ectopic Shh protein, 5E1 supernatant [83] was dilutied 1:10. MF20 and most other antibodies used in this study are available from Developmental Studies Hybridoma Bank. First antibodies were detected with biotin-conjugated horse-derived anti-mouse IgG, or a biotin-conjugated goat-derived anti-mouse IgM (Vector) and ABC Vectastain kit as described [18]. In situ mRNA hybridisation was after [49]. Identification of chick forewing muscles was according to [84] and staging based on limb and muscle mass morphology according to [41]. + +Primary cultures + +Following the methods of Stockdale [3,85], both forelimbs were removed from embryos around HH22 in Dulbecco's modified Eagle's medium (DMEM, Sigma). Limbs were washed with sterile PBS, incubated with trypsin:EDTA (Gibco) for 10 mins, dissociated by trituration and the cells washed, filtered through two 80 μm pore filters (Gibco) and pre-plated on a 90 mm collagen-coated dish for 10 min at 37°C. After this incubation period, 30–40% of cells stuck to the dish but fewer than 1:1000 were myogenic. Non-adherent cells were collected and plated in triplicate at either 2 × 105 (low density) or 4 × 105 (high density) cells per Nunc 35 mm plate in either unconditioned DMEM with 10% horse serum (HS) and 2% chick embryo extract (CEE) or medium that had been conditioned for 24 hours on 90% confluent RCAS/Shh infected QT6 quail fibroblasts (ShhQT6) or the parent QT6 fibroblasts [49,86]. Fresh medium (conditioned or not) was added after 24 hours, and the cells fixed 50–55 hrs after plating. Prior to conditioning QT6 and ShhQT6 cells were maintained in DMEM 10% foetal bovine serum (FBS -Gibco) and 2% chick serum (Gibco). Cultures were washed in PBS, fixed for 5 mins in -20°C methanol, rehydrated in PBS and stained as for the cryosections. Replicate dishes were singly stained with antibodies A4.840 to detect slow MyHC, A4.1025 to detect pan-MyHC and anti-desmin (Sigma, 1:500 dilution) to detect both myoblasts and myotubes. Dual immunofluorescence showed that all MyHC-reactive cells are strongly desmin-reactive and that immunohistochemistry is more sensitive (data not shown). Total nuclear (cell) numbers were counted on a Zeiss Axioplan 2 microscope in ten separate 10x fields on each dish. All nuclei within immunohistochemically-stained cells were counted on each dish. + +Growth and addition of Shh conditioned medium to mouse myoblasts + +C2 cells were obtained from three sources i) C2C12 from ATCC, ii) C2/4 from Y. Nabeshima and iii) C2X which arose in late passage cultures of C2C12 from the lab of H. Blau. All were maintained on plastic by standard procedures prior to plating on collagen-coated glass chamber slides (16-well, LAB-TEK, Nalge Nunc International, USA) in DMEM 10% FBS, 2% chick serum with antibiotics and differentiated by switching to DMEM 2% HS. Conditioned medium was created by incubating QT6 or ShhQT6 cells for 36 hours with either growth or differentiation medium. Each QT6 cell culture was used to condition only a single batch of medium. Purified Shh was synthesised in vitro and the biologically active proportion of the protein in each preparation is unknown, low and varies between batches (P. Ingham and T. Jessell personal communication) so no meaningful concentration can be given. C2 cells were fixed with methanol, stained by dual immunofluorescence with A4.840, A4.1025 and/or BA-D5 using class-specific secondary reagents (Jackson) and viewed under epifluorescence on a Zeiss Axiophot. Unless otherwise stated quantitation of differentiation was by scoring the number of nuclei in MyHC-containing cytoplasm (i.e. the number of C2 cells that differentiated into myocytes, whether or not these subsequently fused). Bromodeoxyuridine was added for the last two hours of a 24 hour culture in QT6 or QT6Shh conditioned growth medium. In situ hybridisation was performed on chamber slide cultures fixed with 4% paraformaldehyde followed by methanol and employed digoxigenin-labelled riboprobes essentially as described [87]. + +Authors' contributions + +XL performed the C2 experiments. CSB analysed chick cultures, Shh over-expression in limb buds and mouse mutants. HB repeated the Shh over-expression, performed wholemount in situs and did the Hh knockdown. MAB and DD implanted RCASShh cells and did section in situs. SMH conceived and coordinated the study and wrote the manuscript with help from DD. All authors approved the final manuscript. + +Acknowledgment + +We thank Everett Bandman for antibodies, Tom Jessell for antibodies, 5E1 hybridoma cells and mouse Shh protein, Peter Currie and Philip Ingham for zebrafish Shh protein, Alex Joyner, Andrew Lumsden and Susanne Dietrich for cDNAs, Andy McMahon for Ihh-/- mice, Chin Chiang for Shh-/- mice and Abi Jensen, Graham Dunn, Pete Currie and Phil Ingham for advice. SMH was supported by MRC and EU QLK6-2000-530, XL by an EC Biomed 2 Ageing grant, and DD by the CNRS. CSB held a MRC PhD studentship and part of this work was reported in his PhD thesis (London University, 1999). diff --git a/src/ontogpt/evaluation/craft/database/all/15314655.ann b/src/ontogpt/evaluation/craft/database/all/15314655.ann new file mode 100644 index 000000000..d68bc8a3b --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15314655.ann @@ -0,0 +1,1276 @@ +T1 NCBITaxon:7955 4 13 Zebrafish +T2 PR:Q6E2N3 14 23 moonshine +T3 SO:0000704 24 28 Gene +T4 PR:000016655 37 75 Transcriptional Intermediary Factor 1γ +T5 GO:0030097 103 116 Hematopoiesis +T6 GO:0030097 128 141 Hematopoiesis +T7 GO:0065007 214 222 regulate +T8 NCBITaxon:7955 301 310 zebrafish +T9 PR:Q6E2N3 311 320 moonshine +T10 PR:Q6E2N3 322 325 mon +T11 SO:0000704 327 331 gene +T12 UBERON:0000922 358 367 embryonic +T13 GO:0035162 358 367;378 391 embryonic ... hematopoiesis +T14 UBERON:0007023 372 377 adult +T15 CL:0000232 413 427 red blood cell +T16 http://purl.obolibrary.org/obo/MONDO_0020113 413 435 red blood cell aplasia +T17 UBERON:0000178 417 422 blood +T18 PR:Q6E2N3 452 455 mon +T19 NCBITaxon:7955 468 477 zebrafish +T20 SO:0000855 478 486 ortholog +T21 NCBITaxon:40674 490 499 mammalian +T22 PR:000016655 500 538 transcriptional intermediary factor 1γ +T23 PR:000016655 540 545 TIF1γ +T24 PR:000016655 551 557 TRIM33 +T25 GO:0030097 642 655 hematopoietic +T26 CL:0008001 642 672 hematopoietic progenitor cells +T27 PR:Q6E2N3 676 679 mon +T28 GO:0010467 696 703 express +T29 GO:0030097 721 734 hematopoietic +T30 PR:000007857 768 773 gata1 +T31 GO:0006915 787 796 apoptosis +T32 PR:Q6E2N3 814 817 mon +T33 SO:0001023 825 832 alleles +T34 SO:0001587 845 866 premature stop codons +T35 GO:0010467 881 891 expression +T36 PR:000016655 905 910 tif1γ +T37 UBERON:0000922 924 933 embryonic +T38 GO:0035162 924 947 embryonic hematopoiesis +T39 PR:Q6E2N3 962 965 mon +T40 CL:0000365 1005 1012 zygotic +T41 PR:000016655 1013 1018 tif1γ +T42 GO:0010467 1024 1034 expression +T43 UBERON:2000083 1046 1062 ventral mesoderm +T44 GO:0030097 1070 1083 hematopoietic +T45 CL:0000037 1070 1093 hematopoietic stem cell +T46 GO:0048468 1089 1093;1109 1118 cell ... formation +T47 PR:000007857 1128 1133 gata1 +T48 GO:0010467 1134 1144 expression +T49 PR:000016655 1182 1187 tif1γ +T50 CL:0000764 1256 1265 erythroid +T51 NCBITaxon:39107 1289 1295 murine +T52 CL:0000764 1296 1310 erythroid cell +T53 PR:000016655 1334 1339 Tif1γ +T54 GO:0005634 1374 1381 nuclear +T55 GO:0010467 1392 1402 expression +T56 CL:0000764 1420 1434 erythroid cell +T57 GO:0048469 1430 1445 cell maturation +T58 GO:0030154 1534 1552;1567 1572 differentiation of ... cells +T59 GO:0030097 1553 1566 hematopoietic +T60 CL:0000988 1553 1572 hematopoietic cells +T61 NCBITaxon:7742 1576 1587 vertebrates +T62 GO:0030097 1604 1617 Hematopoiesis +T63 GO:0008283 1656 1674 cell proliferation +T64 GO:0030154 1679 1697;1738 1743 differentiation of ... cells +T65 CL:0000080 1761 1772;1793 1798 circulating ... cells +T66 CL:0000232 1773 1776;1787 1798 red ... blood cells +T67 CL:0000738 1781 1798 white blood cells +T68 UBERON:0000178 1787 1792 blood +T69 GO:0030097 1822 1835 Hematopoiesis +T70 NCBITaxon:7742 1839 1850 vertebrates +T71 NCBITaxon:7955 1857 1866 zebrafish +T72 NCBITaxon:9606 1870 1876 humans +T73 CL:0000034 1944 1948;1963 1968 stem ... cells +T74 UBERON:0000922 1995 2004 embryonic +T75 UBERON:0000178 2051 2056 blood +T76 CL:0000081 2051 2062 blood cells +T77 GO:0030097 2172 2185 hematopoiesis +T78 UBERON:2000083 2202 2218 ventral mesoderm +T79 UBERON:0000922 2276 2285 embryonic +T80 UBERON:0003061 2286 2299 blood islands +T81 GO:0030097 2323 2336 hematopoiesis +T82 UBERON:0000922 2357 2366 embryonic +T83 CL:0000232 2367 2379 erythrocytes +T84 CL:0000235 2384 2395 macrophages +T85 GO:0030097 2432 2445 hematopoiesis +T86 CL:0000034 2472 2482 stem cells +T87 UBERON:0000922 2518 2527 embryonic +T88 UBERON:0000947 2528 2533 aorta +T89 UBERON:0000991 2534 2539 gonad +T90 UBERON:0000080 2540 2551 mesonephros +T91 GO:0060216 2566 2590 definitive hematopoietic +T92 CL:0000037 2577 2601 hematopoietic stem cells +T93 UBERON:0007023 2670 2675 adult +T94 UBERON:0000178 2676 2681 blood +T95 CL:0000081 2676 2687 blood cells +T96 CL:0000232 2710 2722 erythrocytes +T97 CL:0000763 2724 2737 myeloid cells +T98 CL:0000542 2743 2754 lymphocytes +T99 SO:0000704 2778 2785 genetic +T100 SO:0000704 2811 2816 genes +T101 GO:0065007 2822 2829 control +T102 GO:0030097 2830 2843 hematopoiesis +T103 NCBITaxon:7955 2854 2863 zebrafish +T104 SO:0000704 2938 2945 genetic +T105 UBERON:0000178 2979 2984 blood +T106 NCBITaxon:7955 2989 2998 zebrafish +T107 GO:0010467 3022 3029 express +T108 CL:0000764 3034 3043 erythroid +T109 PR:000007857 3065 3070 gata1 +T110 UBERON:0000922 3083 3092 embryonic +T111 GO:0035162 3083 3106 embryonic hematopoietic +T112 NCBITaxon:7955 3162 3171 zebrafish +T113 SO:0000704 3172 3177 genes +T114 PR:Q6E2N3 3178 3187 moonshine +T115 PR:Q6E2N3 3189 3192 mon +T116 SO:0001023 3238 3244 allele +T117 PR:Q6E2N3 3326 3329 mon +T118 SO:0000704 3330 3334 gene +T119 GO:0060215 3362 3381;3403 3416 primitive embryonic ... hematopoiesis +T120 UBERON:0000922 3372 3381 embryonic +T121 GO:0060216 3386 3396;3403 3416 definitive ... hematopoiesis +T122 UBERON:0007023 3397 3402 adult +T123 CL:0000764 3448 3463 erythroid cells +T124 CL:0000038 3465 3491 Erythroid progenitor cells +T125 PR:Q6E2N3 3495 3498 mon +T126 GO:0010467 3542 3549 express +T127 GO:0030097 3567 3580 hematopoietic +T128 GO:0006915 3615 3624 apoptosis +T129 PR:Q6E2N3 3661 3664 mon +T130 SO:0000704 3665 3669 gene +T131 NCBITaxon:7955 3677 3686 zebrafish +T132 SO:0000855 3687 3695 ortholog +T133 NCBITaxon:40674 3699 3708 mammalian +T134 PR:000016655 3709 3747 transcriptional intermediary factor 1γ +T135 PR:000016655 3749 3754 TIF1γ +T136 NCBITaxon:7742 3965 3975 vertebrate +T137 PR:000016649 3989 3990 α +T138 PR:000016652 3992 3993 β +T139 PR:000016655 3999 4000 γ +T140 GO:0005634 4012 4019 nuclear +T141 SO:0000417 4074 4080 domain +T142 SO:0001080 4149 4160 coiled-coil +T143 SO:0000417 4161 4167 domain +T144 NCBITaxon:3193 4215 4220 plant +T145 GO:0000785 4283 4292 chromatin +T146 GO:0006338 4283 4303 chromatin remodeling +T147 PR:000016649 4313 4318 TIF1α +T148 GO:0005634 4378 4385 nuclear +T149 CHEBI:26536 4463 4476 retinoic acid +T150 PR:000016652 4507 4512 TIF1β +T151 SO:0000417 4606 4612 domain +T152 PR:000016655 4704 4709 TIF1γ +T153 GO:0005634 4750 4757 nuclear +T154 SO:0000417 4776 4783 domains +T155 PR:000016655 4914 4919 TIF1γ +T156 PR:000016649 4972 4977 TIF1α +T157 PR:000016652 4991 4996 TIF1β +T158 NCBITaxon:39107 5021 5027 murine +T159 PR:000016649 5028 5033 Tif1α +T160 PR:000016655 5038 5043 Tif1γ +T161 SO:0000704 5044 5049 genes +T162 SO:0000704 5081 5085 gene +T163 NCBITaxon:10088 5129 5134 mouse +T164 PR:000016652 5161 5166 Tif1β +T165 GO:0007566 5187 5199 implantation +T166 GO:0009790 5200 5213 embryogenesis +T167 UBERON:0000926 5218 5226 mesoderm +T168 PR:000016655 5476 5481 tif1γ +T169 UBERON:0000922 5521 5530 embryonic +T170 GO:0035162 5521 5530;5541 5554 embryonic ... hematopoiesis +T171 UBERON:0007023 5535 5540 adult +T172 NCBITaxon:7742 5558 5569 vertebrates +T173 PR:000016655 5617 5622 tif1γ +T174 UBERON:0000922 5663 5672 embryonic +T175 GO:0035162 5663 5686 embryonic hematopoiesis +T176 PR:000016655 5692 5697 tif1γ +T177 SO:0000704 5698 5702 gene +T178 GO:0010467 5706 5715 expressed +T179 UBERON:2000083 5732 5748 ventral mesoderm +T180 GO:0030097 5753 5766 hematopoietic +T181 CL:0000764 5802 5811 erythroid +T182 PR:000016655 5831 5836 Tif1γ +T183 GO:0016604 5875 5889 nuclear bodies +T184 NCBITaxon:10088 5906 5911 mouse +T185 UBERON:0000922 5912 5918 embryo +T186 CL:0000057 5919 5930 fibroblasts +T187 http://purl.obolibrary.org/obo/MONDO_0017858 5935 5950 erythroleukemia +T188 PR:000016655 6008 6013 Tif1γ +T189 CL:0000764 6037 6051 erythroid cell +T190 GO:0048468 6047 6063 cell development +T191 NCBITaxon:7955 6092 6101 Zebrafish +T192 PR:Q6E2N3 6102 6105 mon +T193 SO:0000704 6106 6110 Gene +T194 GO:0060319 6133 6142;6158 6172 Primitive ... Erythropoiesis +T195 GO:0060318 6147 6172 Definitive Erythropoiesis +T196 PR:Q6E2N3 6205 6208 mon +T197 SO:0000704 6209 6213 gene +T198 GO:0030097 6260 6273 hematopoietic +T199 SO:0000704 6274 6278 gene +T200 GO:0010467 6274 6289 gene expression +T201 GO:0006915 6294 6303 apoptosis +T202 NCBITaxon:7955 6307 6316 zebrafish +T203 PR:Q6E2N3 6328 6331 mon +T204 UBERON:0000922 6339 6346 embryos +T205 GO:0009790 6355 6368 embryogenesis +T206 NCBITaxon:7955 6381 6390 zebrafish +T207 PR:Q6E2N3 6391 6394 mon +T208 CL:0000232 6411 6426 red blood cells +T209 UBERON:0000178 6415 6420 blood +T210 CL:0000232 6428 6432 RBCs +T211 PR:Q6E2N3 6506 6509 mon +T212 GO:0010467 6527 6537 expression +T213 PR:000007857 6541 6546 gata1 +T214 GO:0030097 6550 6563 hematopoietic +T215 CL:0000988 6550 6569 hematopoietic cells +T216 UBERON:0002329 6586 6592 somite +T217 UBERON:0000922 6621 6628 embryos +T218 CL:0000764 6701 6716 erythroid cells +T219 GO:0012501 6725 6746 programmed cell death +T220 UBERON:0002329 6759 6765 somite +T221 GO:0009566 6784 6797 fertilization +T222 UBERON:0002329 6838 6845 somites +T223 PR:000007857 6847 6852 gata1 +T224 GO:0010467 6853 6863 expression +T225 GO:0030097 6904 6917 hematopoietic +T226 PR:000007857 6943 6948 gata1 +T227 PR:000016043 6950 6953 scl +T228 PR:000007858 6955 6960 gata2 +T229 PR:000001811 6966 6972 ikaros +T230 UBERON:0000922 6997 7006 embryonic +T231 UBERON:0003061 7007 7019 blood island +T232 GO:0030097 7062 7075 hematopoietic +T233 CL:0000988 7062 7081 hematopoietic cells +T234 GO:0009790 7128 7142;7154 7161 development of ... embryos +T235 PR:Q6E2N3 7143 7146 mon +T236 UBERON:0000922 7154 7161 embryos +T237 GO:0008219 7192 7202 cell death +T238 GO:0010467 7213 7223 expression +T239 PR:000010799 7227 7232 c-myb +T240 PR:000003457 7237 7241 rag1 +T241 PR:Q6E2N3 7263 7266 mon +T242 CL:0000763 7287 7294 myeloid +T243 CL:0000232 7365 7369 RBCs +T244 PR:Q6E2N3 7373 7376 mon +T245 UBERON:0008897 7415 7418 fin +T246 UBERON:0002415 7428 7432 tail +T247 UBERON:0003104 7433 7443 mesenchyme +T248 PR:Q6E2N3 7484 7487 mon +T249 GO:0097152 7519 7549 apoptosis of mesenchymal cells +T250 UBERON:0003104 7532 7543 mesenchymal +T251 CL:0000134 7532 7549 mesenchymal cells +T252 UBERON:0002100 7557 7562 trunk +T253 UBERON:0002533 7567 7575 tail bud +T254 PR:Q6E2N3 7616 7619 mon +T255 SO:0000704 7620 7624 gene +T256 GO:0048468 7653 7664;7678 7680;7717 7722 development ... of ... cells +T257 GO:0014031 7653 7664;7678 7680;7737 7754 development ... of ... mesenchymal cells +T258 CL:0000038 7696 7722 erythroid progenitor cells +T259 UBERON:0003104 7737 7748 mesenchymal +T260 CL:0000134 7737 7754 mesenchymal cells +T261 GO:0060216 7774 7798 definitive hematopoiesis +T262 UBERON:0007023 7828 7833 adult +T263 NCBITaxon:7955 7834 7843 zebrafish +T264 PR:Q6E2N3 7844 7847 mon +T265 PR:Q6E2N3 7870 7873 mon +T266 PR:Q6E2N3 7960 7963 mon +T267 UBERON:0000178 8007 8012 blood +T268 UBERON:0000922 8017 8024 embryos +T269 SO:0001023 8040 8047 alleles +T270 UBERON:0000113 8059 8068 adulthood +T271 UBERON:0007023 8070 8075 Adult +T272 PR:Q6E2N3 8076 8079 mon +T273 UBERON:0000948 8093 8100 cardiac +T274 http://purl.obolibrary.org/obo/MONDO_0002280 8143 8149 anemia +T275 NCBITaxon:7955 8206 8215 zebrafish +T276 UBERON:0007023 8221 8226 adult +T277 GO:0030097 8235 8248 hematopoiesis +T278 UBERON:0002113 8256 8262 kidney +T279 CL:0000764 8305 8314 erythroid +T280 CL:0000763 8330 8337 myeloid +T281 PR:Q6E2N3 8413 8416 mon +T282 CL:0000547 8482 8497 proerythroblast +T283 GO:0030154 8528 8546;8555 8560 differentiation of ... cells +T284 CL:0000763 8547 8560 myeloid cells +T285 PR:Q6E2N3 8618 8621 mon +T286 SO:0000704 8622 8626 gene +T287 GO:0060319 8652 8661;8677 8691 primitive ... erythropoiesis +T288 GO:0060318 8666 8691 definitive erythropoiesis +T289 PR:Q6E2N3 8724 8727 mon +T290 NCBITaxon:7955 8735 8744 Zebrafish +T291 SO:0000855 8745 8753 Ortholog +T292 NCBITaxon:40674 8757 8766 Mammalian +T293 PR:000016655 8767 8772 TIF1γ +T294 PR:Q6E2N3 8792 8795 mon +T295 SO:0000704 8796 8800 gene +T296 PR:Q6E2N3 8941 8944 mon +T297 SO:0001023 8950 8956 allele +T298 PR:Q6E2N3 8962 8965 mon +T299 SO:0000704 8973 8977 gene +T300 SO:0001830 9175 9213 amplified fragment length polymorphism +T301 SO:0001830 9215 9219 AFLP +T302 SO:0000704 9345 9349 gene +T303 SO:0000154 9435 9469 P1 bacterial artificial chromosome +T304 NCBITaxon:2 9438 9447 bacterial +T305 SO:0000154 9477 9480 PAC +T306 SO:0000704 9521 9528 genetic +T307 SO:0000154 9614 9617 PAC +T308 SO:0000357 9624 9629 flank +T309 SO:0000704 9643 9650 genetic +T310 GO:0007126 9716 9723 meioses +T311 SO:0000154 9874 9877 PAC +T312 NCBITaxon:7955 9902 9911 zebrafish +T313 SO:0001026 9912 9919 genomic +T314 SO:0000149 9929 9935 contig +T315 SO:0000996 9996 10005;10028 10032 predicted ... gene +T316 NCBITaxon:7955 10006 10015 zebrafish +T317 SO:0000154 10039 10042 PAC +T318 GO:0097617 10047 10057 hybridized +T319 UBERON:0002113 10063 10069 kidney +T320 SO:0000704 10152 10156 gene +T321 PR:Q6E2N3 10163 10166 mon +T322 SO:0000704 10167 10171 gene +T323 CHEBI:23357 10227 10236 cofactors +T324 PR:Q6E2N3 10280 10283 mon +T325 NCBITaxon:9606 10303 10308 human +T326 PR:000016655 10309 10313 TIFγ +T327 SO:0000333 10406 10421 exon boundaries +T328 NCBITaxon:7955 10448 10457 zebrafish +T329 NCBITaxon:9606 10462 10467 human +T330 SO:0000704 10468 10473 genes +T331 PR:Q6E2N3 10498 10501 mon +T332 NCBITaxon:7955 10511 10520 zebrafish +T333 SO:0000860 10558 10566 syntenic +T334 NCBITaxon:9606 10584 10589 human +T335 PR:000016655 10622 10627 TIF1γ +T336 SO:0000704 10628 10632 gene +T337 SO:0000858 10678 10689 orthologous +T338 SO:0000704 10690 10694 gene +T339 PR:000011416 10712 10716 NRAS +T340 NCBITaxon:9606 10745 10750 human +T341 NCBITaxon:7955 10755 10764 zebrafish +T342 NCBITaxon:7955 10859 10868 zebrafish +T343 PR:Q6E2N3 10869 10872 mon +T344 SO:0000704 10873 10877 gene +T345 SO:0000855 10892 10900 ortholog +T346 NCBITaxon:9606 10908 10913 human +T347 PR:000016655 10914 10919 TIF1γ +T348 SO:0000704 10920 10924 gene +T349 CHEBI:23995 10946 10963 ethyl-nitrosourea +T350 CHEBI:23995 10965 10968 ENU +T351 SO:0001023 11003 11010 alleles +T352 PR:Q6E2N3 11014 11017 mon +T353 SO:0001587 11064 11084 premature stop codon +T354 PR:Q6E2N3 11090 11093 mon +T355 PR:Q6E2N3 11104 11107 mon +T356 SO:0001023 11113 11120 alleles +T357 CL:0000080 11153 11164;11171 11176 circulating ... cells +T358 UBERON:0000178 11165 11170 blood +T359 CL:0000081 11165 11176 blood cells +T360 PR:Q6E2N3 11195 11198 mon +T361 SO:0001023 11203 11209 allele +T362 CL:0000080 11221 11232;11239 11244 circulating ... cells +T363 UBERON:0000178 11233 11238 blood +T364 CL:0000081 11233 11244 blood cells +T365 CL:0000232 11297 11301 RBCs +T366 UBERON:0000922 11350 11357 embryos +T367 PR:Q6E2N3 11386 11389 mon +T368 SO:0001023 11394 11400 allele +T369 SO:0001587 11423 11443 premature stop codon +T370 PR:Q6E2N3 11594 11597 mon +T371 SO:0000704 11598 11602 gene +T372 GO:0006412 11688 11699 translation +T373 CL:0000558 11715 11727 reticulocyte +T374 GO:0006412 11764 11775 translation +T375 UBERON:0002548 11844 11850 larval +T376 PR:Q6E2N3 11868 11871 mon +T377 SO:0001023 11876 11882 allele +T378 PR:Q6E2N3 11916 11919 mon +T379 GO:0010467 11932 11942 expression +T380 PR:Q6E2N3 11985 11988 mon +T381 SO:0001023 11989 11996 alleles +T382 PR:000016655 12022 12027 Tif1γ +T383 PR:Q6E2N3 12057 12060 mon +T384 PR:000016655 12103 12108 tif1γ +T385 GO:0010467 12112 12121 expressed +T386 GO:0030097 12125 12138 hematopoietic +T387 UBERON:0000926 12139 12147 mesoderm +T388 NCBITaxon:7955 12166 12175 zebrafish +T389 UBERON:0000922 12176 12183 embryos +T390 GO:0097617 12207 12220 hybridization +T391 PR:000016655 12234 12239 tif1γ +T392 GO:0010467 12248 12257 expressed +T393 UBERON:0000922 12297 12303 embryo +T394 UBERON:0000108 12311 12326 blastula stages +T395 GO:0007369 12335 12347 gastrulation +T396 GO:0090504 12352 12359 epiboly +T397 CL:0000365 12368 12375 zygotic +T398 GO:0010467 12376 12386 expression +T399 PR:Q6E2N3 12390 12393 mon +T400 UBERON:0002541 12431 12440 germ ring +T401 UBERON:0002533 12445 12453 tail bud +T402 UBERON:0002329 12464 12470 somite +T403 PR:000016655 12494 12499 tif1γ +T404 GO:0010467 12500 12510 expression +T405 UBERON:2000083 12555 12562;12571 12579 ventral ... mesoderm +T406 UBERON:0003081 12563 12579 lateral mesoderm +T407 UBERON:0000178 12603 12608 blood +T408 GO:0010467 12618 12627 expresses +T409 CL:0000034 12628 12637 stem cell +T410 PR:000016043 12628 12680 stem cell leukemiahematopoietic transcription factor +T411 GO:0030097 12646 12659 hematopoietic +T412 PR:000016043 12682 12685 scl +T413 GO:0010467 12740 12747 express +T414 PR:000016655 12748 12753 tif1γ +T415 PR:000016043 12758 12761 scl +T416 UBERON:0000922 12795 12804 embryonic +T417 UBERON:0003061 12805 12817 blood island +T418 PR:000016655 12845 12850 tif1γ +T419 SO:0000704 12851 12855 gene +T420 GO:0010467 12871 12880 expressed +T421 UBERON:0001017 12888 12910 central nervous system +T422 UBERON:0005256 12926 12939;12944 12949 mesenchyme of ... trunk +T423 UBERON:0002415 12954 12958 tail +T424 PR:Q6E2N3 12971 12974 mon +T425 PR:000016655 13021 13026 tif1γ +T426 UBERON:0000479 13039 13046 tissues +T427 GO:0000184 13063 13094 nonsense-mediated message decay +T428 SO:0000673 13081 13088 message +T429 CHEBI:33695 13081 13088 message +T430 NCBITaxon:7955 13102 13111 zebrafish +T431 PR:000016655 13112 13117 tif1γ +T432 GO:0010467 13134 13143 expressed +T433 UBERON:2000083 13147 13163 ventral mesoderm +T434 UBERON:0000922 13216 13225 embryonic +T435 GO:0030097 13238 13251 hematopoietic +T436 GO:0006915 13279 13288 apoptosis +T437 PR:Q6E2N3 13292 13295 mon +T438 GO:0010467 13326 13336 expression +T439 NCBITaxon:7955 13340 13349 zebrafish +T440 PR:Q6E2N3 13350 13353 mon +T441 NCBITaxon:10088 13357 13362 mouse +T442 PR:000016655 13363 13368 Tif1γ +T443 NCBITaxon:10088 13389 13394 Mouse +T444 PR:000016655 13395 13400 Tif1γ +T445 GO:0010467 13411 13420 expressed +T446 CL:0000764 13424 13433 erythroid +T447 UBERON:0011919 13434 13450;13455 13463 blood islands of ... yolk sac +T448 GO:0010467 13488 13497 expressed +T449 UBERON:0002107 13511 13516 liver +T450 UBERON:0000479 13547 13554 tissues +T451 UBERON:0001017 13570 13592 central nervous system +T452 NCBITaxon:7955 13645 13654 zebrafish +T453 PR:Q6E2N3 13655 13658 mon +T454 NCBITaxon:10088 13663 13668 mouse +T455 PR:000016655 13669 13674 Tif1γ +T456 SO:0000855 13679 13688 orthologs +T457 GO:0030097 13710 13723 hematopoiesis +T458 NCBITaxon:40674 13737 13746 mammalian +T459 PR:000016655 13747 13752 TIF1γ +T460 PR:000016649 13798 13803 Tif1α +T461 NCBITaxon:7955 13874 13883 zebrafish +T462 PR:000016655 13900 13905 tif1γ +T463 NCBITaxon:7955 13913 13922 zebrafish +T464 GO:0010467 13923 13932 expressed +T465 SO:0000345 13923 13945 expressed sequence tag +T466 SO:0000345 13947 13950 EST +T467 SO:0000112 13975 13982 primers +T468 UBERON:0000922 14026 14035 embryonic +T469 NCBITaxon:7955 14089 14098 zebrafish +T470 SO:0000855 14099 14107 ortholog +T471 NCBITaxon:9606 14111 14116 human +T472 PR:000016649 14117 14122 TIF1α +T473 NCBITaxon:7955 14193 14202 zebrafish +T474 PR:000016649 14203 14208 tif1α +T475 SO:0000345 14209 14213 ESTs +T476 SO:0000860 14253 14261 syntenic +T477 NCBITaxon:9606 14279 14284 human +T478 PR:000016649 14316 14321 TIF1α +T479 SO:0000704 14322 14326 gene +T480 SO:0000858 14375 14386 orthologous +T481 SO:0000704 14387 14391 gene +T482 PR:000014615 14409 14415 SEMA3A +T483 NCBITaxon:9606 14444 14449 human +T484 NCBITaxon:7955 14454 14463 zebrafish +T485 UBERON:0000922 14509 14518 embryonic +T486 GO:0010467 14519 14529 expression +T487 PR:000016649 14541 14546 tif1α +T488 PR:000016655 14555 14560 tif1γ +T489 GO:0097617 14572 14585 hybridization +T490 NCBITaxon:40674 14592 14601 mammalian +T491 PR:000016649 14602 14607 TIF1α +T492 SO:0000996 14665 14674;14691 14695 predicted ... gene +T493 NCBITaxon:7955 14675 14684 zebrafish +T494 PR:000016655 14685 14690 tif1γ +T495 GO:0010467 14707 14716 expressed +T496 UBERON:0002329 14742 14749 somites +T497 NCBITaxon:7955 14751 14760 zebrafish +T498 PR:000016649 14761 14766 tif1α +T499 GO:0010467 14804 14814 expression +T500 GO:0030097 14849 14862 hematopoietic +T501 UBERON:0000926 14863 14871 mesoderm +T502 PR:000016655 14882 14887 tif1γ +T503 PR:000016649 14906 14911 tif1α +T504 GO:0010467 14922 14931 expressed +T505 UBERON:0000922 14955 14961 embryo +T506 UBERON:0003061 14988 15001 blood islands +T507 PR:000016649 15014 15019 tif1α +T508 GO:0010467 15025 15034 expressed +T509 PR:000016655 15058 15063 tif1γ +T510 GO:0010467 15137 15147 Expression +T511 PR:000016655 15151 15156 tif1γ +T512 GO:0030097 15165 15178 Hematopoiesis +T513 PR:Q6E2N3 15182 15185 mon +T514 NCBITaxon:7955 15237 15246 zebrafish +T515 PR:000016655 15247 15252 tif1γ +T516 SO:0000704 15253 15257 gene +T517 PR:Q6E2N3 15281 15284 mon +T518 UBERON:0000922 15315 15321 embryo +T519 PR:Q6E2N3 15401 15404 mon +T520 GO:0048821 15444 15456;15467 15479 formation of ... erythrocytes +T521 UBERON:0000922 15457 15466 embryonic +T522 CL:0000232 15467 15479 erythrocytes +T523 UBERON:0000922 15500 15507 embryos +T524 UBERON:0000922 15543 15552 embryonic +T525 GO:0048568 15543 15552;15567 15580 embryonic ... organogenesis +T526 PR:Q6E2N3 15621 15624 mon +T527 CL:0000232 15770 15774 RBCs +T528 UBERON:0008897 15864 15867 fin +T529 UBERON:0003104 15909 15920 mesenchymal +T530 CL:0000134 15909 15926 mesenchymal cells +T531 http://purl.obolibrary.org/obo/MONDO_0002280 15966 15972 anemia +T532 GO:0010467 15997 16007 expression +T533 PR:Q6E2N3 16011 16014 mon +T534 UBERON:0000178 16042 16047 blood +T535 CL:0000081 16042 16052 blood cell +T536 UBERON:0000922 16074 16081 embryos +T537 PR:Q6E2N3 16138 16141 mon +T538 UBERON:0000178 16210 16215 blood +T539 UBERON:0000922 16235 16242 embryos +T540 PR:Q6E2N3 16282 16285 mon +T541 GO:0030097 16332 16345 hematopoiesis +T542 GO:0030218 16379 16393 Erythropoiesis +T543 PR:Q6E2N3 16397 16400 mon +T544 PR:000016655 16429 16434 tif1γ +T545 GO:0010467 16435 16445 expression +T546 CL:0000764 16449 16464 erythroid cells +T547 SO:0000704 16525 16529 gene +T548 GO:0010467 16525 16540 gene expression +T549 GO:0030097 16544 16557 hematopoietic +T550 CL:0000988 16544 16563 hematopoietic cells +T551 UBERON:0007023 16625 16630 adult +T552 NCBITaxon:7955 16631 16640 zebrafish +T553 UBERON:0007132 16641 16654 kidney marrow +T554 PR:000007857 16672 16677 gata1 +T555 SO:0000902 16710 16719 transgene +T556 PR:Q6E2N3 16732 16735 mon +T557 UBERON:0000922 16743 16750 embryos +T558 PR:000007857 16777 16782 gata1 +T559 SO:0000902 16787 16796 transgene +T560 PR:000007857 16814 16819 gata1 +T561 SO:0000167 16820 16828 promoter +T562 GO:0010467 16842 16852 expression +T563 CL:0000764 16896 16911 erythroid cells +T564 UBERON:0000922 16954 16961 embryos +T565 UBERON:0000922 16970 16979 embryonic +T566 CL:0002321 16970 16979;16986 16991 embryonic ... cells +T567 UBERON:0000178 16980 16985 blood +T568 CL:0000081 16980 16991 blood cells +T569 CL:0000080 16986 17006 cells in circulation +T570 GO:0018995 17042 17046 host +T571 UBERON:0000922 17047 17054 embryos +T572 UBERON:0000922 17099 17106 embryos +T573 CL:0000080 17139 17159 cells in circulation +T574 CL:0000764 17409 17424 erythroid cells +T575 CL:0000080 17445 17465 cells in circulation +T576 UBERON:0000922 17507 17514 embryos +T577 CL:0000080 17542 17562 cells in circulation +T578 UBERON:0000178 17589 17594 blood +T579 CL:0000081 17589 17600 blood cells +T580 UBERON:0000922 17611 17618 embryos +T581 UBERON:0000178 17653 17658 blood +T582 CL:0000081 17653 17664 blood cells +T583 GO:0036268 17706 17710 swim +T584 UBERON:0006860 17706 17719 swim bladders +T585 GO:0016265 17797 17802 dying +T586 PR:Q6E2N3 17857 17860 mon +T587 UBERON:0000113 17896 17905 adulthood +T588 PR:000007857 17975 17980 gata1 +T589 PR:000016655 18074 18079 tif1γ +T590 CL:0000764 18112 18127 erythroid cells +T591 GO:0030097 18148 18161 hematopoietic +T592 UBERON:0000479 18162 18169 tissues +T593 UBERON:0005256 18179 18195 trunk mesenchyme +T594 UBERON:0001016 18203 18217 nervous system +T595 UBERON:0000922 18240 18246 embryo +T596 PR:000016655 18258 18263 Tif1γ +T597 GO:0005634 18276 18283 Nuclear +T598 GO:0065007 18308 18317 Regulated +T599 PR:000016655 18371 18376 Tif1γ +T600 NCBITaxon:9986 18420 18426 rabbit +T601 UBERON:0001977 18442 18447 serum +T602 NCBITaxon:9606 18508 18513 human +T603 PR:000016655 18514 18519 TIF1γ +T604 NCBITaxon:10088 18524 18529 mouse +T605 PR:000016655 18530 18535 Tif1γ +T606 NCBITaxon:10088 18559 18564 mouse +T607 UBERON:0000922 18565 18571 embryo +T608 CL:0000057 18572 18582 fibroblast +T609 GO:0005634 18583 18589 nuclei +T610 PR:000016655 18604 18609 Tif1γ +T611 UBERON:0001977 18614 18619 serum +T612 PR:000016655 18638 18643 Tif1γ +T613 GO:0005634 18666 18673 nuclear +T614 PR:000016655 18712 18717 Tif1γ +T615 GO:0005634 18766 18773 nuclear +T616 PR:000016649 18812 18817 Tif1α +T617 PR:000016652 18847 18852 TIF1β +T618 PR:000016652 18909 18914 TIF1β +T619 GO:0000792 18931 18946 heterochromatin +T620 CHEBI:26536 18969 18982 retinoic acid +T621 UBERON:0001977 18996 19001 serum +T622 GO:0010467 19057 19067 expression +T623 GO:0065007 19096 19105 regulated +T624 GO:0005634 19176 19183 nuclear +T625 PR:000016655 19202 19207 Tif1γ +T626 GO:0000792 19246 19261 heterochromatin +T627 PR:000005086 19263 19267 HP1α +T628 CHEBI:51231 19280 19284 DAPI +T629 PR:000016655 19327 19332 Tif1γ +T630 CL:0000836 19358 19371 promyelocytic +T631 PR:000026474 19358 19380 promyelocytic leukemia +T632 GO:0016605 19358 19393;19400 19414 promyelocytic leukemia gene product ... nuclear bodies +T633 SO:0000704 19381 19385 gene +T634 PR:000026474 19395 19398 PML +T635 GO:0016605 19395 19398;19400 19414 PML ... nuclear bodies +T636 GO:0006281 19416 19426 DNA repair +T637 GO:1990391 19416 19436 DNA repair complexes +T638 PR:000010562 19450 19455 Mre11 +T639 GO:0032991 19476 19485 complexes +T640 PR:000008313 19497 19503 TFII-B +T641 GO:0010467 19545 19555 expression +T642 PR:000016655 19559 19564 Tif1γ +T643 GO:0030154 19584 19602;19607 19612 differentiation of ... cells +T644 NCBITaxon:39107 19616 19622 murine +T645 http://purl.obolibrary.org/obo/MONDO_0017858 19623 19638 erythroleukemia +T646 CL:0000232 19688 19700 erythrocytes +T647 PR:000007857 19708 19713 Gata1 +T648 CHEBI:50114 19714 19722 estrogen +T649 CHEBI:50114 19776 19784 estrogen +T650 PR:000016655 19855 19860 Tif1γ +T651 GO:0010467 19869 19879 expression +T652 CL:0000764 19904 19913 erythroid +T653 NCBITaxon:7955 19987 19996 zebrafish +T654 PR:Q6E2N3 19997 20000 mon +T655 GO:0010467 20006 20016 expression +T656 GO:0048469 20043 20056;20081 20086 maturation of ... cells +T657 CL:0000764 20071 20086 erythroid cells +T658 NCBITaxon:39107 20124 20130 murine +T659 http://purl.obolibrary.org/obo/MONDO_0017858 20131 20146 erythroleukemia +T660 PR:000016655 20173 20178 Tif1γ +T661 GO:0010467 20187 20196 expressed +T662 GO:0005634 20200 20207 nuclear +T663 PR:000016655 20242 20247 Tif1γ +T664 GO:0005634 20279 20286 nuclear +T665 PR:000016655 20416 20421 Tif1γ +T666 GO:0005634 20440 20447 nuclear +T667 CL:0000764 20461 20470 erythroid +T668 NCBITaxon:7955 20505 20514 zebrafish +T669 GO:0065007 20581 20592 controlling +T670 SO:0000704 20593 20597 gene +T671 GO:0010467 20593 20608 gene expression +T672 GO:0030097 20616 20629 hematopoiesis +T673 SO:0000704 20709 20716 genetic +T674 SO:0001023 20796 20803 alleles +T675 NCBITaxon:7955 20811 20820 zebrafish +T676 SO:0000704 20821 20825 gene +T677 PR:Q6E2N3 20840 20849 moonshine +T678 PR:Q6E2N3 20916 20919 mon +T679 SO:0000704 20920 20924 gene +T680 PR:000016655 20998 21003 Tif1γ +T681 GO:0030097 21012 21025 hematopoietic +T682 PR:Q6E2N3 21044 21047 mon +T683 SO:0000704 21048 21052 Gene +T684 NCBITaxon:7955 21065 21074 Zebrafish +T685 SO:0000855 21075 21083 Ortholog +T686 NCBITaxon:40674 21087 21096 Mammalian +T687 PR:000016655 21097 21102 TIF1γ +T688 NCBITaxon:7955 21185 21194 zebrafish +T689 PR:Q6E2N3 21195 21198 mon +T690 SO:0000704 21199 21203 gene +T691 SO:0000855 21229 21237 ortholog +T692 NCBITaxon:40674 21241 21250 mammalian +T693 PR:000016655 21251 21256 Tif1γ +T694 PR:000016655 21258 21263 Tif1γ +T695 SO:0000704 21291 21298 genetic +T696 SO:0000154 21350 21353 PAC +T697 NCBITaxon:7955 21437 21446 zebrafish +T698 PR:000016655 21447 21452 tif1γ +T699 SO:0000188 21506 21512 intron +T700 SO:0000147 21513 21517 exon +T701 SO:0000996 21544 21553;21582 21587 predicted ... genes +T702 SO:0000858 21554 21565 orthologous +T703 NCBITaxon:9606 21566 21571 human +T704 NCBITaxon:10088 21576 21581 mouse +T705 NCBITaxon:7955 21589 21598 Zebrafish +T706 PR:000016655 21599 21604 tif1γ +T707 NCBITaxon:7955 21631 21640 zebrafish +T708 SO:0000860 21654 21662 syntenic +T709 NCBITaxon:9606 21680 21685 human +T710 PR:000016655 21710 21715 TIF1γ +T711 PR:000016655 21750 21755 tif1γ +T712 SO:0001023 21777 21784 alleles +T713 PR:Q6E2N3 21788 21791 mon +T714 SO:0001587 21812 21833 premature stop codons +T715 GO:0006402 21838 21848 mRNA decay +T716 PR:000016655 21863 21868 tif1γ +T717 PR:000016655 21869 21874 Tif1γ +T718 GO:0010467 21885 21894 expressed +T719 GO:0030097 21898 21911 hematopoietic +T720 CL:0000988 21898 21917 hematopoietic cells +T721 GO:0009790 21929 21942 embryogenesis +T722 NCBITaxon:7955 21951 21960 zebrafish +T723 NCBITaxon:10088 21965 21970 mouse +T724 GO:0010467 22012 22022 expression +T725 PR:000016655 22036 22041 tif1γ +T726 GO:0030097 22067 22080 hematopoiesis +T727 PR:Q6E2N3 22084 22087 mon +T728 GO:0035162 22117 22133;22144 22151 hematopoiesis in ... embryos +T729 UBERON:0000922 22144 22151 embryos +T730 NCBITaxon:7955 22202 22211 zebrafish +T731 SO:0000855 22212 22220 ortholog +T732 PR:000016649 22224 22229 tif1α +T733 GO:0010467 22255 22264 expressed +T734 NCBITaxon:7955 22268 22277 zebrafish +T735 UBERON:0000922 22278 22285 embryos +T736 NCBITaxon:40674 22291 22300 mammalian +T737 PR:000016649 22301 22306 TIF1α +T738 PR:000016655 22444 22449 Tif1γ +T739 GO:0030097 22472 22485 hematopoietic +T740 CL:0000988 22472 22491 hematopoietic cells +T741 NCBITaxon:7955 22513 22522 zebrafish +T742 NCBITaxon:40674 22527 22536 mammalian +T743 PR:000016655 22594 22599 Tif1γ +T744 SO:0000855 22600 22609 orthologs +T745 PR:000016649 22665 22670 Tif1α +T746 PR:000016652 22732 22737 Tif1β +T747 SO:0000855 22738 22746 ortholog +T748 NCBITaxon:7955 22765 22774 zebrafish +T749 NCBITaxon:31032 22778 22782 fugu +T750 SO:0001026 22783 22789 genome +T751 SO:0000345 22793 22796 EST +T752 PR:000016652 22828 22833 Tif1β +T753 SO:0000417 22849 22855 domain +T754 NCBITaxon:32523 22914 22923 tetrapods +T755 SO:0001026 22963 22969 genome +T756 NCBITaxon:7955 23048 23057 zebrafish +T757 PR:Q6E2N3 23058 23061 mon +T758 PR:000016655 23104 23109 Tif1γ +T759 NCBITaxon:9606 23201 23206 human +T760 NCBITaxon:10088 23211 23216 mouse +T761 GO:0030097 23217 23230 hematopoiesis +T762 PR:000016655 23246 23251 tif1γ +T763 GO:0006915 23258 23267 Apoptosis +T764 CL:0000764 23271 23280 Erythroid +T765 GO:0030097 23313 23326 hematopoietic +T766 SO:0000704 23327 23331 gene +T767 GO:0010467 23327 23342 gene expression +T768 GO:0006915 23344 23353 apoptosis +T769 PR:Q6E2N3 23379 23382 mon +T770 CL:0000764 23415 23424 erythroid +T771 GO:0012501 23531 23552 programmed cell death +T772 GO:0010467 23573 23583 expression +T773 PR:000007857 23587 23592 gata1 +T774 CL:0000764 23639 23654 erythroid cells +T775 PR:Q6E2N3 23658 23661 mon +T776 PR:000007857 23733 23738 gata1 +T777 GO:0010467 23739 23749 expression +T778 CL:0000445 23766 23781 apoptotic cells +T779 UBERON:0002329 23805 23811 somite +T780 GO:0030097 23852 23865 hematopoietic +T781 SO:0000704 23866 23870 gene +T782 GO:0010467 23866 23881 gene expression +T783 GO:0010467 23903 23913 expression +T784 SO:0000704 23924 23929 genes +T785 PR:000016043 23941 23944 scl +T786 PR:000007858 23949 23954 gata2 +T787 GO:0030097 23974 23987 hematopoietic +T788 CL:0000037 23974 23998 hematopoietic stem cells +T789 GO:0060215 24003 24026 primitive hematopoietic +T790 UBERON:0000922 24069 24078 embryonic +T791 UBERON:0003061 24079 24092 blood islands +T792 GO:0030097 24146 24159 hematopoietic +T793 CL:0000988 24146 24165 hematopoietic cells +T794 CL:0000764 24209 24218 erythroid +T795 CL:0000764 24260 24275 erythroid cells +T796 GO:0006915 24288 24297 apoptosis +T797 PR:000007857 24310 24315 gata1 +T798 CL:0000764 24326 24341 erythroid cells +T799 GO:0030218 24395 24409 erythropoiesis +T800 http://purl.obolibrary.org/obo/MONDO_0002280 24421 24427 anemia +T801 PR:Q6E2N3 24483 24486 mon +T802 UBERON:0007023 24487 24493 adults +T803 PR:000016655 24514 24519 tif1γ +T804 GO:0060216 24540 24564 definitive hematopoiesis +T805 CL:0000365 24586 24593 zygotic +T806 PR:Q6E2N3 24608 24611 mon +T807 PR:000016655 24672 24677 Tif1γ +T808 GO:0010467 24690 24699 expressed +T809 NCBITaxon:7955 24700 24709 zebrafish +T810 PR:000016655 24710 24715 Tif1γ +T811 GO:0030097 24734 24747 hematopoiesis +T812 GO:0048513 24768 24781 organogenesis +T813 CL:0000025 24847 24851 eggs +T814 PR:Q6E2N3 24922 24925 mon +T815 NCBITaxon:7955 24940 24949 zebrafish +T816 CL:0000365 25031 25038 zygotic +T817 PR:Q6E2N3 25039 25042 mon +T818 PR:000016655 25110 25115 tif1γ +T819 GO:0030218 25133 25147 erythropoiesis +T820 GO:0030097 25197 25210 hematopoietic +T821 GO:0030097 25226 25239 hematopoietic +T822 PR:Q6E2N3 25253 25256 mon +T823 CL:0000764 25287 25302 erythroid cells +T824 NCBITaxon:10088 25316 25321 mouse +T825 PR:000007857 25322 25327 Gata1 +T826 UBERON:0000922 25337 25344 embryos +T827 NCBITaxon:7955 25349 25358 zebrafish +T828 PR:000007857 25371 25376 gata1 +T829 UBERON:0000922 25385 25392 embryos +T830 SO:0000704 25476 25483 genetic +T831 PR:Q6E2N3 25505 25508 mon +T832 PR:000007857 25513 25518 gata1 +T833 GO:0030218 25554 25568 erythropoiesis +T834 PR:000007857 25588 25593 gata1 +T835 PR:Q6E2N3 25604 25607 mon +T836 UBERON:0000922 25626 25633 embryos +T837 PR:000016655 25651 25656 tif1γ +T838 PR:000007857 25667 25672 gata1 +T839 UBERON:0000922 25684 25691 embryos +T840 GO:0030097 25709 25722 hematopoiesis +T841 PR:000016655 25791 25796 Tif1γ +T842 PR:000007857 25801 25806 Gata1 +T843 SO:0000704 25954 25959 genes +T844 PR:000007857 26033 26038 gata1 +T845 PR:000016655 26043 26048 tif1γ +T846 SO:0000704 26121 26128 genetic +T847 PR:000007857 26148 26153 gata1 +T848 PR:000016655 26158 26163 tif1γ +T849 GO:0065007 26185 26193 regulate +T850 SO:0000704 26194 26198 gene +T851 UBERON:0000178 26220 26225 blood +T852 CL:0000081 26220 26231 blood cells +T853 PR:000016655 26246 26251 Tif1γ +T854 GO:0060319 26255 26264;26280 26294 Primitive ... Erythropoiesis +T855 GO:0060318 26269 26294 Definitive Erythropoiesis +T856 PR:000016655 26334 26339 tif1γ +T857 CHEBI:23357 26368 26376 cofactor +T858 CL:0000764 26385 26394 erythroid +T859 GO:0010468 26412 26422;26437 26452 control of ... gene expression +T860 GO:0030097 26423 26436 hematopoietic +T861 SO:0000704 26437 26441 gene +T862 PR:000016655 26481 26486 Tif1γ +T863 GO:0030097 26549 26562 hematopoietic +T864 CL:0008001 26549 26579 hematopoietic progenitor cells +T865 PR:000016649 26596 26601 TIF1α +T866 PR:000016652 26626 26631 TIF1β +T867 SO:0000704 26761 26765 gene +T868 PR:000016649 26809 26814 TIF1α +T869 PR:000016652 26819 26824 TIF1β +T870 GO:0000785 26898 26907 chromatin +T871 GO:0065007 26940 26950 regulatory +T872 GO:0032991 26951 26960 complexes +T873 PR:000016655 26962 26967 Tif1γ +T874 GO:0005634 26987 26994 nuclear +T875 GO:0016604 27115 27129 nuclear bodies +T876 PR:000026474 27141 27144 PML +T877 GO:0016605 27141 27151 PML bodies +T878 PR:000016655 27169 27174 Tif1γ +T879 GO:0016604 27184 27198 nuclear bodies +T880 GO:0065007 27206 27215 regulated +T881 GO:0006412 27223 27236 translational +T882 PR:000026474 27297 27300 PML +T883 PR:000026474 27309 27312 PML +T884 GO:0016605 27309 27328 PML nuclear domains +T885 PR:000016655 27422 27427 Tif1γ +T886 GO:0032991 27447 27456 complexes +T887 CL:0000764 27579 27588 erythroid +T888 PR:000016655 27656 27661 Tif1γ +T889 GO:0005634 27686 27693 nuclear +T890 PR:000016655 27736 27741 Tif1γ +T891 GO:0065007 27745 27753 regulate +T892 UBERON:0000178 27754 27759 blood +T893 CL:0000081 27754 27764 blood cell +T894 GO:0048468 27760 27776 cell development +T895 NCBITaxon:7955 27804 27813 Zebrafish +T896 NCBITaxon:10088 27818 27823 mouse +T897 NCBITaxon:7955 27845 27854 Zebrafish +T898 SO:0001023 27919 27926 alleles +T899 PR:Q6E2N3 27927 27930 mon +T900 PR:Q6E2N3 27941 27944 mon +T901 CHEBI:23995 27993 27996 ENU +T902 PR:Q6E2N3 28059 28062 mon +T903 SO:0001023 28067 28073 allele +T904 GO:0007618 28198 28204 mating +T905 UBERON:0000922 28293 28300 embryos +T906 GO:0097617 28349 28362 hybridization +T907 UBERON:0000922 28380 28387 embryos +T908 GO:0097617 28461 28474 hybridization +T909 NCBITaxon:10088 28478 28483 mouse +T910 UBERON:0000922 28484 28491 embryos +T911 SO:0001026 28543 28550 Genomic +T912 SO:0001830 28578 28582 AFLP +T913 SO:0000112 28725 28732 primers +T914 SO:0000704 28737 28744 genetic +T915 PR:Q6E2N3 28780 28783 mon +T916 GO:0010467 28816 28826 expression +T917 SO:0001183 28839 28850 morpholinos +T918 PR:Q6E2N3 28888 28891 mon +T919 SO:0000440 28950 28956 vector +T920 UBERON:0007132 29106 29119 kidney marrow +T921 UBERON:0007023 29145 29150 adult +T922 PR:000007857 29151 29156 gata1 +T923 NCBITaxon:27592 29238 29244 bovine +T924 UBERON:0001977 29245 29250 serum +T925 UBERON:0002063 29274 29287 sinus venosus +T926 PR:Q6E2N3 29299 29302 mon +T927 UBERON:0000922 29324 29331 embryos +T928 UBERON:0007132 29353 29366 kidney marrow +T929 UBERON:0000922 29391 29397 embryo +T930 UBERON:0000922 29423 29430 embryos +T931 UBERON:0002548 29622 29628 larvae +T932 GO:0042571 29692 29702 Antibodies +T933 UBERON:0001977 29741 29745 sera +T934 NCBITaxon:9606 29758 29763 human +T935 PR:000016655 29775 29780 TIF1γ +T936 NCBITaxon:9986 29823 29830 rabbits +T937 NCBITaxon:10088 29922 29927 Mouse +T938 UBERON:0000922 29928 29937 embryonic +T939 CL:0000057 29938 29949 fibroblasts +T940 GO:0040007 29950 29955 grown +T941 PR:000005086 29994 29998 HP1α +T942 PR:000016655 30051 30056 Tif1γ +T943 UBERON:0001977 30061 30065 sera +T944 UBERON:0001977 30202 30207 serum +T945 GO:0042571 30261 30271 antibodies +T946 GO:0042571 30356 30366 antibodies +T947 CHEBI:51231 30493 30497 DAPI +T948 GO:0030154 30688 30708 cell differentiation +T949 NCBITaxon:7955 30836 30845 zebrafish +T950 PR:000007857 30870 30875 gata1 +T951 SO:0000902 30880 30889 transgene +T952 UBERON:0000922 30903 30910 embryos +T953 GO:0030218 30925 30939 erythropoiesis +T954 PR:Q6E2N3 30963 30966 mon +T955 UBERON:0000922 31007 31014 embryos +T956 CL:0000232 31068 31072 RBCs +T957 GO:0008283 31125 31136 proliferate +T958 CL:0000232 31178 31190 erythrocytes +T959 NCBITaxon:7955 31248 31257 zebrafish +T960 UBERON:0002548 31258 31264 larvae +T961 PR:Q6E2N3 31300 31303 mon +T962 CL:0000080 31347 31367 Cells in Circulation +T963 CL:0000232 31508 31520 Erythrocytes +T964 PR:Q6E2N3 31562 31565 mon +T965 PR:Q6E2N3 31678 31681 mon +T966 CL:0000080 31725 31745 Cells in Circulation +T967 CL:0000232 31896 31908 Erythrocytes +T968 PR:Q6E2N3 31961 31964 mon +T969 SO:0000704 32137 32142 genes +T970 SO:0000704 32147 32151 gene +T971 NCBITaxon:7147 32189 32192 fly +T972 NCBITaxon:9606 32211 32216 human +T973 PR:000016649 32217 32222 TIF1α +T974 NCBITaxon:9606 32233 32238 human +T975 PR:000016652 32239 32244 TIF1β +T976 NCBITaxon:9606 32255 32260 human +T977 PR:000016655 32261 32265 TIFγ +T978 NCBITaxon:9606 32276 32281 human +T979 PR:000016655 32282 32287 TIF1γ +T980 PR:Q6E2N3 32298 32301 mon +T981 NCBITaxon:10088 32313 32318 mouse +T982 PR:000016649 32319 32324 Tif1α +T983 NCBITaxon:10088 32335 32340 mouse +T984 PR:000016652 32341 32346 Tif1β +T985 NCBITaxon:10088 32363 32368 mouse +T986 PR:000016655 32369 32374 Tif1γ +T987 NCBITaxon:7955 32410 32419 zebrafish +T988 PR:Q6E2N3 32420 32423 mon +T989 PR:000016655 32424 32429 tif1γ +T990 PR:000016649 32434 32439 tif1α +T991 SO:0001023 32764 32770 allele +T992 PR:Q6E2N3 32774 32777 mon +T993 http://purl.obolibrary.org/obo/MONDO_0004992 32855 32861 Cancer +T994 SO:0001830 33159 33163 AFLP +T995 SO:0001830 33166 33204 amplified fragment length polymorphism +T996 CHEBI:23995 33206 33209 ENU +T997 CHEBI:23995 33212 33229 ethyl-nitrosourea +T998 SO:0000345 33231 33234 EST +T999 GO:0010467 33237 33246 expressed +T1000 SO:0000345 33237 33259 expressed sequence tag +T1001 GO:0009566 33310 33323 fertilization +T1002 PR:Q6E2N3 33356 33359 mon +T1003 PR:Q6E2N3 33362 33371 moonshine +T1004 SO:0000154 33373 33376 PAC +T1005 PR:000026474 33378 33381 PML +T1006 CL:0000836 33384 33397 promyelocytic +T1007 PR:000026474 33384 33406 promyelocytic leukemia +T1008 SO:0000704 33407 33411 gene +T1009 CL:0000232 33421 33425 RBCs +T1010 CL:0000232 33428 33443 red blood cells +T1011 UBERON:0000178 33432 33437 blood +T1012 PR:000016043 33445 33448 scl +T1013 CL:0000034 33451 33460 stem cell +T1014 PR:000016043 33451 33469 stem cell leukemia +T1015 NCBITaxon:7955 33576 33585 Zebrafish +T1016 PR:Q6E2N3 33586 33589 mon +T1017 GO:0060215 33621 33644 Primitive Hematopoiesis +T1018 UBERON:2000083 33687 33694;33705 33715 ventral ... mesodermal +T1019 CL:0000222 33705 33721 mesodermal cells +T1020 GO:0006915 33730 33739 apoptosis +T1021 PR:Q6E2N3 33754 33757 mon +T1022 UBERON:0000922 33770 33777 embryos +T1023 GO:0097617 33799 33812 hybridization +T1024 PR:000007857 33816 33821 gata1 +T1025 UBERON:0002329 33849 33855 somite +T1026 UBERON:0000105 33856 33861 stage +T1027 UBERON:0000922 33875 33882 embryos +T1028 GO:0006915 33938 33947 apoptosis +T1029 UBERON:0002100 33966 33971 trunk +T1030 UBERON:0002415 33976 33980 tail +T1031 GO:0030097 34005 34018 hematopoietic +T1032 CL:0000988 34005 34024 hematopoietic cells +T1033 UBERON:0000922 34032 34041 embryonic +T1034 UBERON:0003061 34042 34054 blood island +T1035 GO:0097617 34107 34120 hybridization +T1036 PR:000016043 34141 34144 scl +T1037 PR:000007858 34146 34151 gata2 +T1038 PR:000007857 34153 34158 gata1 +T1039 PR:000001811 34160 34166 ikaros +T1040 PR:000010799 34172 34175 myb +T1041 PR:Q6E2N3 34179 34182 mon +T1042 GO:0010467 34197 34207 Expression +T1043 PR:000010799 34211 34214 myb +T1044 UBERON:0003061 34241 34254 blood islands +T1045 CL:0000764 34276 34291 erythroid cells +T1046 UBERON:0000922 34297 34306 embryonic +T1047 CL:0000235 34307 34318 macrophages +T1048 GO:0010467 34351 34361 expression +T1049 PR:000003457 34365 34369 rag1 +T1050 UBERON:0002370 34373 34379 thymic +T1051 CL:0000084 34380 34387 T-cells +T1052 PR:Q6E2N3 34406 34409 mon +T1053 GO:0009566 34429 34442 fertilization +T1054 UBERON:0000922 34494 34501 embryos +T1055 NCBITaxon:7955 34514 34523 Zebrafish +T1056 PR:Q6E2N3 34524 34527 mon +T1057 GO:0060216 34564 34588 Definitive Hematopoiesis +T1058 UBERON:0007023 34590 34595 Adult +T1059 PR:Q6E2N3 34623 34626 mon +T1060 PR:Q6E2N3 34653 34656 mon +T1061 UBERON:0007023 34673 34678 adult +T1062 UBERON:0007023 34802 34807 adult +T1063 CL:0000764 34903 34918 erythroid cells +T1064 CL:0000547 34970 34986 proerythroblasts +T1065 PR:Q6E2N3 34994 34997 mon +T1066 PR:Q6E2N3 35063 35066 mon +T1067 SO:0000704 35067 35071 Gene +T1068 NCBITaxon:7955 35075 35084 Zebrafish +T1069 PR:000016655 35085 35090 tif1γ +T1070 SO:0001249 35096 35108 Physical map +T1071 PR:Q6E2N3 35116 35119 mon +T1072 NCBITaxon:7955 35129 35138 zebrafish +T1073 PR:Q6E2N3 35265 35268 mon +T1074 SO:0001830 35298 35302 AFLP +T1075 SO:0000154 35357 35360 PAC +T1076 SO:0000154 35385 35389 PACS +T1077 PR:Q6E2N3 35426 35429 mon +T1078 SO:0000154 35476 35479 PAC +T1079 PR:Q6E2N3 35525 35528 mon +T1080 SO:0000704 35529 35533 gene +T1081 SO:0000154 35540 35543 PAC +T1082 NCBITaxon:7955 35605 35614 zebrafish +T1083 PR:000016655 35615 35620 tif1γ +T1084 NCBITaxon:7955 35760 35769 zebrafish +T1085 NCBITaxon:7955 35771 35782 Danio rerio +T1086 NCBITaxon:7955 35784 35786 Dr +T1087 PR:000016655 35789 35794 Tif1γ +T1088 PR:000016649 35799 35804 Tif1α +T1089 NCBITaxon:9606 35861 35866 human +T1090 NCBITaxon:9606 35868 35870 Hs +T1091 PR:000016649 35872 35877 TIF1α +T1092 PR:000016652 35879 35884 TIF1β +T1093 PR:000016655 35890 35895 TIF1γ +T1094 NCBITaxon:10088 35897 35902 mouse +T1095 NCBITaxon:10090 35904 35906 Mm +T1096 PR:000016649 35908 35913 Tif1α +T1097 PR:000016652 35915 35920 Tif1β +T1098 PR:000016655 35926 35931 Tif1γ +T1099 NCBITaxon:7147 35938 35941 fly +T1100 NCBITaxon:7227 35943 35945 Dm +T1101 PR:000016655 36002 36007 Tif1γ +T1102 NCBITaxon:3193 36107 36112 plant +T1103 SO:0000417 36254 36261 domains +T1104 NCBITaxon:7955 36265 36274 zebrafish +T1105 NCBITaxon:9606 36279 36284 human +T1106 PR:000016655 36285 36290 TIF1γ +T1107 CHEBI:23995 36390 36393 ENU +T1108 PR:Q6E2N3 36476 36479 mon +T1109 PR:000016655 36480 36485 tif1γ +T1110 SO:0000704 36486 36490 Gene +T1111 GO:0010467 36501 36510 Expressed +T1112 GO:0030097 36514 36527 Hematopoietic +T1113 UBERON:0000926 36528 36536 Mesoderm +T1114 GO:0097617 36550 36563 hybridization +T1115 NCBITaxon:7955 36567 36576 zebrafish +T1116 UBERON:0000922 36577 36584 embryos +T1117 UBERON:0000922 36603 36612 embryonic +T1118 GO:0010467 36613 36623 expression +T1119 PR:000016655 36627 36632 tif1γ +T1120 PR:000016655 36634 36639 tif1γ +T1121 GO:0010467 36653 36662 expressed +T1122 GO:0010467 36693 36703 expression +T1123 PR:000016655 36707 36712 tif1γ +T1124 UBERON:2000083 36716 36723;36732 36740 ventral ... mesoderm +T1125 UBERON:0003081 36724 36740 lateral mesoderm +T1126 UBERON:0002329 36774 36780 somite +T1127 UBERON:0002329 36837 36844 somites +T1128 PR:000016655 36846 36851 tif1γ +T1129 GO:0010467 36864 36873 expressed +T1130 UBERON:0000926 36896 36904 mesoderm +T1131 GO:0010467 36915 36922 express +T1132 PR:000016043 36923 36926 scl +T1133 PR:000016655 36938 36943 tif1γ +T1134 GO:0010467 36947 36956 expressed +T1135 UBERON:0000955 36972 36977 brain +T1136 UBERON:0002240 36979 36990 spinal cord +T1137 UBERON:0002100 36992 36997 trunk +T1138 UBERON:0002415 37003 37007 tail +T1139 UBERON:0003104 37008 37018 mesenchyme +T1140 GO:0030097 37052 37065 hematopoietic +T1141 CL:0000988 37052 37071 hematopoietic cells +T1142 UBERON:0003061 37079 37091 blood island +T1143 NCBITaxon:7955 37093 37102 Zebrafish +T1144 PR:000016649 37103 37108 tif1α +T1145 GO:0010467 37125 37134 expressed +T1146 UBERON:0000479 37171 37178 tissues +T1147 PR:000016655 37197 37202 tif1γ +T1148 PR:000016649 37204 37209 Tif1α +T1149 GO:0010467 37220 37229 expressed +T1150 UBERON:0002329 37239 37245 somite +T1151 GO:0030097 37256 37269 hematopoietic +T1152 UBERON:0000926 37270 37278 mesoderm +T1153 GO:0010467 37293 37302 expressed +T1154 GO:0010467 37324 37334 expression +T1155 UBERON:0003061 37342 37355 blood islands +T1156 GO:0010467 37357 37367 Expression +T1157 PR:000016043 37371 37374 scl +T1158 UBERON:0002329 37383 37390 somites +T1159 UBERON:0000922 37417 37426 embryonic +T1160 UBERON:0003061 37427 37439 blood island +T1161 PR:000016655 37457 37462 tif1γ +T1162 GO:0010467 37463 37473 expression +T1163 GO:0097617 37488 37501 hybridization +T1164 NCBITaxon:10088 37505 37510 mouse +T1165 UBERON:0000922 37511 37518 embryos +T1166 GO:0010467 37533 37543 expression +T1167 PR:000016655 37547 37552 Tif1γ +T1168 UBERON:0000922 37556 37565 embryonic +T1169 UBERON:0011919 37588 37610 yolk sac blood islands +T1170 UBERON:0000922 37623 37632 embryonic +T1171 GO:0010467 37663 37673 expression +T1172 UBERON:0000922 37681 37686 fetal +T1173 UBERON:0002107 37687 37692 liver +T1174 GO:0010467 37711 37721 expression +T1175 UBERON:0000922 37729 37738 embryonic +T1176 UBERON:0000955 37739 37744 brain +T1177 UBERON:0002240 37746 37758 spinal chord +T1178 UBERON:0001555 37760 37763 gut +T1179 GO:0010467 37792 37802 expression +T1180 PR:000016655 37816 37821 tif1γ +T1181 UBERON:0000922 37861 37870 Embryonic +T1182 GO:0035162 37861 37884 Embryonic Hematopoiesis +T1183 PR:Q6E2N3 37888 37891 mon +T1184 PR:Q6E2N3 37905 37908 mon +T1185 PR:000016655 37974 37979 Tif1γ +T1186 CL:0000232 38029 38033 RBCs +T1187 NCBITaxon:7955 38078 38087 zebrafish +T1188 CHEBI:82321 38103 38116 o-dianisidine +T1189 PR:Q6E2N3 38152 38155 mon +T1190 UBERON:0000178 38196 38201 blood +T1191 PR:000016655 38240 38245 tif1γ +T1192 GO:0030218 38259 38273 erythropoiesis +T1193 UBERON:0000922 38284 38291 embryos +T1194 CHEBI:82321 38293 38306 o-dianisidine +T1195 UBERON:0002548 38315 38321 larvae +T1196 UBERON:0000178 38362 38367 blood +T1197 UBERON:0000055 38371 38378 vessels +T1198 NCBITaxon:7955 38414 38423 zebrafish +T1199 PR:000007857 38448 38453 gata1 +T1200 SO:0000902 38458 38467 transgene +T1201 UBERON:0000922 38481 38488 embryos +T1202 GO:0030218 38503 38517 erythropoiesis +T1203 PR:Q6E2N3 38541 38544 mon +T1204 UBERON:0000922 38603 38610 embryos +T1205 CL:0000232 38664 38668 RBCs +T1206 GO:0008283 38727 38738 proliferate +T1207 CL:0000232 38779 38791 erythrocytes +T1208 UBERON:0000948 38832 38838 hearts +T1209 NCBITaxon:7955 38867 38876 zebrafish +T1210 NCBITaxon:40674 38907 38916 Mammalian +T1211 PR:000016655 38917 38922 Tif1γ +T1212 GO:0016604 38944 38958 Nuclear Bodies +T1213 GO:0000792 38973 38988 Heterochromatin +T1214 NCBITaxon:10088 39037 39042 mouse +T1215 UBERON:0000922 39043 39052 embryonic +T1216 CL:0002321 39043 39052;39064 39068 embryonic ... cell +T1217 CL:0000057 39053 39068 fibroblast cell +T1218 GO:0005634 39064 39076 cell nucleus +T1219 PR:000016655 39098 39103 Tif1γ +T1220 GO:0042571 39104 39112 antibody +T1221 GO:0042571 39143 39151 antibody +T1222 PR:000005086 39169 39173 HP1α +T1223 CHEBI:51231 39200 39204 DAPI +T1224 GO:0005634 39240 39247 nucleus +T1225 PR:000016655 39258 39263 Tif1γ +T1226 PR:000005086 39293 39297 HP1α +T1227 CHEBI:51231 39301 39305 DAPI +T1228 GO:0000792 39318 39333 heterochromatin +T1229 PR:000005086 39340 39344 HP1α +T1230 CHEBI:51231 39349 39353 DAPI +T1231 NCBITaxon:10088 39382 39387 mouse +T1232 http://purl.obolibrary.org/obo/MONDO_0017858 39388 39403 erythroleukemia +T1233 GO:0010467 39410 39417 express +T1234 PR:000016655 39433 39438 Tif1γ +T1235 GO:0010467 39485 39495 Expression +T1236 PR:000016655 39499 39504 Tif1γ +T1237 PR:000007857 39522 39527 Gata1 +T1238 CL:0000764 39538 39547 erythroid +T1239 CHEBI:16469 39570 39581 β-estradiol +T1240 PR:000007857 39604 39609 Gata1 +T1241 GO:0010467 39643 39653 expression +T1242 PR:000016655 39657 39662 tif1γ +T1243 PR:Q6E2N3 39676 39679 mon +T1244 GO:0030097 39689 39702 Hematopoietic +T1245 PR:000016655 39725 39730 tif1γ +T1246 UBERON:0000922 39785 39792 embryos +T1247 PR:Q6E2N3 39829 39832 mon +T1248 UBERON:0000922 39833 39840 embryos +T1249 CL:0000080 39842 39859 circulating cells +T1250 UBERON:0000922 39905 39912 embryos +T1251 CHEBI:82321 39941 39954 o-dianisidine +T1252 CL:0000232 39986 39990 RBCs +T1253 UBERON:0000922 39999 40006 embryos +T1254 CL:0000080 40035 40052 circulating cells +T1255 UBERON:0000922 40106 40113 embryos +T1256 UBERON:0000922 40197 40204 embryos +T1257 GO:0030097 40255 40268 Hematopoiesis +T1258 PR:Q6E2N3 40289 40292 mon +T1259 UBERON:0000922 40302 40309 Embryos +T1260 CL:0000764 40328 40343 Erythroid Cells +T1261 UBERON:0007132 40367 40380 kidney marrow +T1262 UBERON:0007023 40392 40397 adult +T1263 PR:000007857 40398 40403 gata1 +T1264 NCBITaxon:7955 40445 40454 zebrafish +T1265 UBERON:0000922 40455 40461 embryo +T1266 UBERON:0000922 40497 40504 embryos +T1267 CL:0000764 40547 40562 erythroid cells +T1268 UBERON:0000922 40616 40623 embryos +T1269 CL:0000080 40659 40679 cells in circulation +T1270 UBERON:0000922 40757 40764 embryos +T1271 CHEBI:33893 41207 41215 reagents +T1272 NCBITaxon:7955 41561 41570 zebrafish +T1273 PR:Q6E2N3 41571 41580 moonshine +T1274 SO:0000704 41581 41585 gene +T1275 PR:000016655 41594 41632 transcriptional intermediary factor 1γ +T1276 GO:0030097 41660 41673 hematopoiesis diff --git a/src/ontogpt/evaluation/craft/database/all/15314655.txt b/src/ontogpt/evaluation/craft/database/all/15314655.txt new file mode 100644 index 000000000..c7d1edb3c --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15314655.txt @@ -0,0 +1,237 @@ +The Zebrafish moonshine Gene Encodes Transcriptional Intermediary Factor 1γ, an Essential Regulator of Hematopoiesis + +Abstract + +Hematopoiesis is precisely orchestrated by lineage-specific DNA-binding proteins that regulate transcription in concert with coactivators and corepressors. Mutations in the zebrafish moonshine (mon) gene specifically disrupt both embryonic and adult hematopoiesis, resulting in severe red blood cell aplasia. We report that mon encodes the zebrafish ortholog of mammalian transcriptional intermediary factor 1γ (TIF1γ) (or TRIM33), a member of the TIF1 family of coactivators and corepressors. During development, hematopoietic progenitor cells in mon mutants fail to express normal levels of hematopoietic transcription factors, including gata1, and undergo apoptosis. Three different mon mutant alleles each encode premature stop codons, and enforced expression of wild-type tif1γ mRNA rescues embryonic hematopoiesis in homozygous mon mutants. Surprisingly, a high level of zygotic tif1γ mRNA expression delineates ventral mesoderm during hematopoietic stem cell and progenitor formation prior to gata1 expression. Transplantation studies reveal that tif1γ functions in a cell-autonomous manner during the differentiation of erythroid precursors. Studies in murine erythroid cell lines demonstrate that Tif1γ protein is localized within novel nuclear foci, and expression decreases during erythroid cell maturation. Our results establish a major role for this transcriptional intermediary factor in the differentiation of hematopoietic cells in vertebrates. + +Introduction + +Hematopoiesis involves the coordinated processes of cell proliferation and differentiation of a relatively small number of progenitor cells into billions of circulating red and white blood cells (Thisse and Zon 2002). Hematopoiesis in vertebrates, from zebrafish to humans, is an evolutionarily conserved program that produces two waves of stem or progenitor cells that differ both in their embryonic origins and in the lineages of differentiated blood cells produced (Palis and Yoder 2001; Orkin and Zon 2002; Galloway and Zon 2003). The first, or primitive, wave of hematopoiesis originates from ventral mesoderm and gives rise to progenitor cells that differentiate in embryonic blood islands. The primitive wave of hematopoiesis produces a burst of embryonic erythrocytes and macrophages. The second, or definitive, wave of hematopoiesis arises from self-renewing stem cells that develop primarily in the intraembryonic aorta–gonad–mesonephros region. These definitive hematopoietic stem cells seed the later developing marrow spaces, to produce all lineages of adult blood cells, including definitive erythrocytes, myeloid cells, and lymphocytes. + +We have undertaken a genetic approach to characterize genes that control hematopoiesis using the zebrafish as a model system (Thisse and Zon 2002). As part of a large-scale forward genetic screen, we previously identified bloodless zebrafish mutants that failed to express the erythroid transcription factor gata1 normally in embryonic hematopoietic precursors (Ransom et al. 1996). We named one of these zebrafish genes moonshine (mon), and another group named a noncomplementing allele vampire (Weinstein et al. 1996). + +Here, we have determined that mutations in the mon gene cause a disruption in both primitive embryonic and definitive adult hematopoiesis, resulting in a severe loss of erythroid cells. Erythroid progenitor cells in mon mutants are initially present, but fail to express normal levels of hematopoietic transcription factors and undergo apoptosis. + +Positional cloning identifies the mon gene as the zebrafish ortholog of mammalian transcriptional intermediary factor 1γ (TIF1γ), a member of the TIF1 family of transcriptional coactivators and corepressors (Le Douarin et al. 1995; Friedman et al. 1996; Kim et al. 1996; Venturini et al. 1999; Peng et al. 2002). The three members of the vertebrate TIF1 family (α, β, and γ) are large nuclear proteins that each contain an N-terminal RBCC or TRIM domain (Reymond et al. 2001) composed of a RING finger, two B-boxes, and a coiled-coil domain. TIF1 family members also contain a C-terminal plant homeodomain finger and bromodomain that are characteristic of chromatin remodeling factors. TIF1α has been shown to associate with a variety of ligand-bound nuclear hormone receptors (Le Douarin et al. 1995) and function as a coactivator for retinoic acid receptors (Zhong et al.1999). TIF1β has been shown to act as a corepressor for the large family of Krüppel-associated box (KRAB) domain zinc-finger transcription factors (Friedman et al. 1996; Abrink et al. 2001). In contrast, TIF1γ does not associate directly with either nuclear receptors or KRAB domains that bind to the other TIF1 family members (Venturini et al. 1999; Abrink et al. 2001). Biochemical studies also demonstrate that TIF1γ forms both homo-oligomers and hetero-oligomers with TIF1α but not with TIF1β (Peng et al. 2002). The murine Tif1α and Tif1γ genes have not yet been subjected to gene targeting experiments, whereas analysis of mouse mutants demonstrates that Tif1β is required for postimplantation embryogenesis and mesoderm induction in particular (Cammas et al. 2000). Taken together, these studies suggest that a major function of TIF1 family members is to link DNA-binding proteins with other coactivators or corepressors during development. + +Our studies establish that tif1γ functions as an essential regulator of embryonic and adult hematopoiesis in vertebrates. Cell transplantation studies demonstrate that tif1γ acts in a cell-autonomous manner during embryonic hematopoiesis. The tif1γ gene is expressed specifically in ventral mesoderm and hematopoietic progenitors, then downregulated as erythroid maturation occurs. Tif1γ protein localizes to a novel class of nuclear bodies in both primary mouse embryo fibroblasts and erythroleukemia cell lines. Taken together, our studies demonstrate that Tif1γ is required for normal erythroid cell development and survival. + +Results + +The Zebrafish mon Gene Is Essential for Both Primitive and Definitive Erythropoiesis + +In order to determine when the mon gene is required in development, we first examined hematopoietic gene expression and apoptosis in zebrafish homozygous mon mutant embryos. During embryogenesis, homozygous zebrafish mon mutants have no red blood cells (RBCs) visible in circulation (Ransom et al. 1996; Weinstein et al. 1996). The mon mutants initiate expression of gata1 in hematopoietic cells around the five-somite stage, similar to wild-type embryos (data not shown); however, based on TUNEL staining, the differentiating erythroid cells undergo programmed cell death from the 12-somite stage to 22 h postfertilization (hpf) (Figure 1A and 1B, arrows). At 12 somites, gata1 expression is only slightly reduced. By 18–22 hpf, hematopoietic-specific markers such as gata1, scl, gata2, and ikaros are not detected in the embryonic blood island (Figure 1A and 1B; unpublished data). The hematopoietic cells are thus correctly specified early during the development of mon mutant embryos, but these precursors undergo cell death. Based on expression of c-myb and rag1 (Figure 1B, arrows), mon mutants have normal myeloid and lymphoid development, respectively. In addition to the deficit of RBCs in mon mutants, there is a prominent loss of fin-fold and tail mesenchyme (Ransom et al. 1996). TUNEL staining of mon mutants demonstrates extensive apoptosis of mesenchymal cells in the trunk and tail bud regions (Figure 1A and 1B, arrows). The mon gene is thus required for normal development and survival of both committed erythroid progenitor cells and posterior mesenchymal cells. + +We next examined definitive hematopoiesis in rare surviving homozygous adult zebrafish mon mutants. Mutations in mon are generally lethal by 10 to 14 d of development (Ransom et al. 1996), although rare mon homozygous mutants (approximately 1 in 500 bloodless embryos) of all tested alleles survive to adulthood. Adult mon mutants show cardiac hypertrophy, presumably due to the severe anemia leading to a high output state (Figure 2). In wild-type zebrafish, the adult site of hematopoiesis is the kidney (Al Adhami and Kunz 1977), which contains erythroid, lymphoid, and myeloid populations at various stages of differentiation (Bennett et al. 2001). In mon homozygous mutants, there is a severe block in maturation at the proerythroblast stage (Figure 2), whereas the differentiation of myeloid cells is normal (unpublished data). This demonstrates that the mon gene product acts during both primitive and definitive erythropoiesis. + +Positional Cloning Identifies mon as the Zebrafish Ortholog of Mammalian TIF1γ + +We identified the mon gene by positional cloning using a panel of 2,200 diploid mutants collected from Tübingen background (TU)/WIK strain hybrid parents carrying the montg234 allele. The mon mutant gene was positioned on Chromosome 8 between microsatellite markers z987 and z11001 (Figure 3A) (Knapik et al. 1998). For positional cloning purposes, over 12,000 polymorphic markers were screened using amplified fragment length polymorphism (AFLP) (Ransom and Zon 1999), and 36 markers within the interval were isolated. One of these, MA3, was found to be 0.3 cM from the gene (Figure 3A) and was utilized as the starting point of a chromosomal walk. A critical P1 bacterial artificial chromosome clone (PAC), 107N19, was obtained that spanned the genetic interval. Two simple sequence conformation polymorphism (SSCP) markers found on this PAC clone flank the critical genetic interval. The marker 80M12-T7 maps two recombinants out of 4,400 meioses telomeric of the mutation, and the marker 157J23-T7 maps one recombinant centromic of the mutation (Figure 3A). The end sequences and SSCP markers of PAC 107N19 are found in the zebrafish genomic sequence contig ctg23107 (http://www.ensembl.org/Danio_rerio/) containing a predicted zebrafish TIF1 family gene. This PAC was hybridized to a kidney cDNA library, resulting in the isolation of four clones that represented the same gene. + +The mon gene encodes a member of the TIF1 family of transcriptional cofactors (Figure 3B and 3C). The coding sequence of mon is most similar to human TIFγ (Le Douarin et al. 1995; Friedman et al. 1996; Venturini et al. 1999), and the locations of exon boundaries are conserved between the zebrafish and human genes (unpublished data). The mon locus on zebrafish Chromosome 8 is also predicted to be syntenic to the region of human Chromosome 1p that contains the TIF1γ gene based on the conserved locations of 12 other orthologous gene pairs, including NRAS, mapped to these regions in human and zebrafish (Barbazuk et al. 2000). Therefore, based on sequence similarity and chromosomal location, the zebrafish mon gene is the likely ortholog of the human TIF1γ gene. + +We have identified ethyl-nitrosourea (ENU)-induced point mutations in three alleles of mon (Figure 3C and 3D), each of which generates a premature stop codon. The montb222b and montg234 alleles have a severe phenotype with no circulating blood cells. In contrast, the monm262 allele has 10–100 circulating blood cells by 48 hpf, in comparison to the approximately 3,000 RBCs in the circulation of wild-type or heterozygous embryos at the same time point. The monm262 allele was found to encode a premature stop codon at position E40, which would encode a putative protein of only 40 amino acids. Although this mutation would be expected to lead to a complete loss of mon gene product, another methionine is found downstream at amino acid position 104. In vitro translation experiments in reticulocyte lysates demonstrate reinitiation of translation from this methionine (unpublished data). Therefore, the hypomorphic larval phenotype of the monm262 allele is likely due to partial loss of mon function or expression. The presence of mutations in each of the mon alleles indicates that defective Tif1γ function is the cause of the mon phenotype. + +In order to determine whether tif1γ is expressed in hematopoietic mesoderm, we next examined zebrafish embryos by whole-mount in situ hybridization (Figure 4A). tif1γ mRNA is expressed maternally and is found throughout the embryo during blastula stages. During gastrulation and epiboly stages, zygotic expression of mon is highest in the mesendoderm of the germ ring. At tail bud and early somite stages a high level of tif1γ expression delineates a horseshoe-shaped population of ventral/lateral mesoderm that will give rise to blood and also expresses stem cell leukemiahematopoietic transcription factor (scl) (Liao et al. 1997). This group of cells continues to express tif1γ and scl while it converges and forms the embryonic blood island (Detrich et al. 1995). The tif1γ gene is also highly expressed in the central nervous system as well as the mesenchyme of the trunk and tail. Homozygous montg234 mutants have a greatly reduced amount of tif1γ mRNA in all tissues consistent with nonsense-mediated message decay. Thus, zebrafish tif1γ is specifically expressed in ventral mesoderm and putative hemangioblasts prior to and during the embryonic stages when hematopoietic progenitors are undergoing apoptosis in mon mutants. We also compared the expression of zebrafish mon to mouse Tif1γ (Figure 4A and 4B). Mouse Tif1γ is highly expressed in erythroid blood islands of the yolk sac, and it is subsequently expressed in the fetal liver at a high level, and in other tissues, including the central nervous system. Taken together these results strongly suggest that zebrafish mon and mouse Tif1γ are orthologs that function during hematopoiesis. + +Given that mammalian TIF1γ has been shown to form hetero-oligomers with Tif1α (Peng et al. 2002), we searched for additional TIF1 family members in zebrafish to compare with tif1γ. Using zebrafish expressed sequence tag (EST) sequences, we designed primers to RT-PCR amplify a TIF1-related cDNA from embryonic 10-hpf and 24-hpf RNA. This cDNA encodes a predicted zebrafish ortholog of human TIF1α based on predicted amino acid sequences (see Figure 3B). In addition, zebrafish tif1α ESTs map to LG4 in a region predicted to be syntenic to the region of human Chromosome 7 that contains the TIF1α gene based on the conserved locations of eight other orthologous gene pairs, including SEMA3A, mapped to these regions in human and zebrafish (Barbazuk et al. 2000). We next compared the embryonic expression pattern of tif1α mRNA to tif1γ by in situ hybridization. Like mammalian TIF1α (Le Douarin et al. 1995; Niederreither et al. 1999), the predicted zebrafish tif1γ gene is broadly expressed (see Figure 4A). At five somites, zebrafish tif1α does not display the relatively high expression in the horseshoe-shaped region of hematopoietic mesoderm seen with tif1γ. At later stages, tif1α is evenly expressed throughout most of the embryo, including the developing blood islands. Therefore, tif1α is coexpressed in the same cells with tif1γ and may therefore be available to form hetero-oligomers in vivo. + +Forced Expression of tif1γ Rescues Hematopoiesis in mon Mutants + +To further confirm that a mutation in the zebrafish tif1γ gene is responsible for the mon mutant phenotype we performed embryo rescue experiments (Figure 5A; Table 1). Microinjection of synthetic wild-type mon mRNA at the one-cell stage rescues the formation of embryonic erythrocytes in genotyped mutant embryos without causing obvious defects in embryonic patterning or organogenesis. At 4 d of development, 70% (n = 10) of montg234 mutants show significant (greater than 200 cells in comparison to a wild-type estimate of 3,000 cells) rescue of circulating hemoglobinized RBCs in comparison to control sibling mutants (n = 75). Based on the correction of the jagged fin-fold phenotype (Ransom et al. 1996), the mesenchymal cells are rescued to a similar extent as the anemia (unpublished data). Overexpression of mon did not result in expanded blood cell numbers in wild-type embryos and was not toxic at doses that rescue the phenotype of mon mutants (unpublished data). Since there were no expanded or ectopic blood populations in the embryos, these rescue experiments suggest that mon functions as a permissive factor required for hematopoiesis. + +Marrow Transplantation Rescues Erythropoiesis in mon Mutants + +The high levels of tif1γ expression in erythroid cells suggest that it functions as a cell-autonomous regulator of gene expression in hematopoietic cells. In order to test this hypothesis, we transplanted wild-type adult zebrafish kidney marrow cells carrying a gata1:green fluorescent protein (GFP) transgene into 48-hpf mon mutant embryos (Figure 5B; Table 2). The gata1:GFP transgene makes use of the gata1 promoter to drive GFP expression and can thus be used to mark donor-derived erythroid cells (Long et al. 1997). Untransplanted mutant embryos have no embryonic blood cells in circulation. Following transplantation, mutant host embryos were observed daily for 2 wk. Of 191 mutant embryos injected, 129 (68%) showed GFP+ cells in circulation 2 d later. Many recipients showed robust increases in donor-derived cells over the observation period. Of 81 recipients initially scored as having less than ten GFP+ cells at day 2 posttransplant, 13 (16%) of these demonstrated a marked increase in erythroid cells with 100–1,000 GFP+ cells in circulation 6 d later. By day 10, these transplanted embryos showed approximately 3,000 cells in circulation, similar to the number of blood cells in normal embryos. Despite robust reconstitution of blood cells, mutant recipients did not inflate their swim bladders and thus failed to survive longer than nontransplanted sibling controls, all dying by 3 wk of age. In contrast, 13/35 (37%) heterozygous montg234 transplants survived to early adulthood. Similar transplants of wild-type cells can fully rescue vlad tepes (gata1) mutants (Traver et al. 2003). Therefore, the results of cell transplantations suggests that tif1γ plays a cell-autonomous role in erythroid cells, and its role in nonhematopoietic tissues, such as trunk mesenchyme or the nervous system, is also required for embryo survival. + +Tif1γ in Punctate Nuclear Foci Is Developmentally Regulated + +In order to examine the subcellular distribution of Tif1γ protein, we generated an affinity-purified rabbit polyclonal antiserum directed against the C-terminal 15 amino acids conserved in human TIF1γ and mouse Tif1γ. Immunofluorescence of mouse embryo fibroblast nuclei with the anti-Tif1γ antiserum demonstrates that Tif1γ is localized in small nuclear foci (Figure 6A). The localization of Tif1γ protein appears different from the more diffuse nuclear patterns typically seen in studies of Tif1α (Remboutsika et al. 2002) or TIF1β (Cammas et al. 2002). A recent report demonstrates that TIF1β associates with heterochromatin-containing foci after retinoic acid treatment or serum starvation (Cammas et al. 2002). Thus, localization or expression of the TIF1 proteins may be regulated during distinct developmental processes or by environmental cues. The nuclear foci that contain Tif1γ do not colocalize with two markers of heterochromatin, HP1α protein and DAPI staining of DNA (Figure 6A). Furthermore, Tif1γ does not colocalize with promyelocytic leukemia gene product (PML) nuclear bodies, DNA repair complexes that contain Mre11, or transcriptional complexes containing TFII-B (unpublished data). We next examined the expression of Tif1γ protein during the differentiation of G1E cells, a murine erythroleukemia cell line that can terminally differentiate into erythrocytes when a Gata1:estrogen receptor fusion protein is stabilized in response to estrogen exposure (Weiss et al. 1997). Western blot analysis demonstrated that Tif1γ protein expression decreases with terminal erythroid differentiation (Figure 6B). Consistent with this finding, after 24 hpf, zebrafish mon mRNA expression falls during the terminal maturation of the primitive erythroid cells (unpublished data). In two different murine erythroleukemia cell lines (MEL and G1E), Tif1γ is also expressed in nuclear foci, and even though the overall Tif1γ protein level is reduced, this nuclear foci localization does not change with differentiation (unpublished data). This provides further support for the hypothesis that Tif1γ acts within novel nuclear foci, during erythroid differentiation. + +Discussion + +The zebrafish is an excellent model system to elucidate the molecular machinery controlling gene expression during hematopoiesis (Thisse and Zon 2002; Galloway and Zon 2003). As part of a large-scale forward genetic screen, we originally identified a complementation group of independent mutant alleles in the zebrafish gene that we named moonshine (Ransom et al. 1996). Positional cloning was used to identify the mon gene, establishing a critical role for a transcriptional intermediary factor, Tif1γ, during hematopoietic development. + +The mon Gene Encodes the Zebrafish Ortholog of Mammalian TIF1γ + +Our results strongly support the conclusion that we have positionally cloned the zebrafish mon gene correctly, and it is the ortholog of mammalian Tif1γ. Tif1γ is present in the critical genetic interval encompassing a single approximately 50-kb PAC clone defined by linkage analysis (see Figure 3). Sequence analysis indicates that zebrafish tif1γ is most similar in predicted amino acid sequence and intron/exon structure compared to the predicted orthologous human and mouse genes. Zebrafish tif1γ is located in a region of zebrafish Chromosome 8 syntenic to the region of human Chromosome 1 containing TIF1γ. We identified point mutations in tif1γ from three different alleles of mon that each result in premature stop codons and mRNA decay. In addition, tif1γ/Tif1γ is highly expressed in hematopoietic cells throughout embryogenesis in both zebrafish and mouse (see Figure 4). And as predicted, forced expression of wild-type tif1γ mRNA efficiently rescues hematopoiesis in mon mutants and does not perturb hematopoiesis in wild-type embryos (see Figure 5). We have also cloned the predicted zebrafish ortholog of tif1α, which is more uniformly expressed in zebrafish embryos like mammalian TIF1α (Le Douarin et al. 1995; Niederreither et al. 1999) (see Figures 3A and 4A) and may therefore be available to form hetero-oligomers with Tif1γ protein in developing hematopoietic cells. Comparing available zebrafish and mammalian TIF1-predicted amino acid sequences, it appears that the Tif1γ orthologs are the most highly conserved family members while the Tif1α sequences are relatively more divergent. We have not found a Tif1β ortholog, thus far, in the zebrafish or fugu genome or EST sequences. It is possible that Tif1β, like the KRAB domain transcription factors it binds to, may be present only in tetrapods (Urrutia 2003). However, more complete genome sequences will be needed to confirm this hypothesis. Based on our analysis of zebrafish mon mutants, it is reasonable to predict that Tif1γ, the most evolutionarily conserved TIF1 family member, plays a similarly essential role in human and mouse hematopoiesis. + +Mutations in tif1γ Cause Apoptosis of Erythroid Progenitors + +Our examination of hematopoietic gene expression, apoptosis, and marrow histology in mon mutants demonstrates that early erythroid progenitors are formed in homozygous mutants, but they fail to properly differentiate and instead undergo programmed cell death (see Figure 1). The expression of gata1 appears to initiate normally in the committed erythroid cells of mon mutants. However, the cells are abnormal prior to the complete loss of gata1 expression. TUNEL-positive apoptotic cells are abundant by the 12-somite stage of development, and by 22 hpf all hematopoietic gene expression is extinguished. The expression of marker genes, including scl and gata2, characteristic of hematopoietic stem cells and primitive hematopoietic progenitors, are also not detected in the embryonic blood islands of mutants at 22 hpf. This indicates that the mutant hematopoietic cells are not blocked prior to commitment to the erythroid lineage, but instead develop as abnormal erythroid cells and undergo apoptosis, similar to gata1-deficient erythroid cells (Fujiwara et al. 1996; Lyons et al. 2002). Defective erythropoiesis and severe anemia were also observed in rare surviving homozygous mutant mon adults, demonstrating that tif1γ is also required in definitive hematopoiesis (see Figure 2). + +The zygotic phenotypes of mon mutants may not reveal the function of maternally inherited Tif1γ. Maternally expressed zebrafish Tif1γ may play roles in hematopoiesis or other aspects of organogenesis that are not detectable due to the presence of wild-type mRNA in eggs laid by heterozygous mothers. Analysis of the offspring of homozygous mon mutant female zebrafish will aid in defining the function of this maternal mRNA. The present analysis of zygotic mon mutants provides data that are consistent with the conclusion that tif1γ is essential for erythropoiesis but do not rule out essential functions in other hematopoietic lineages. + +The hematopoietic phenotype of mon mutants resembles the loss of erythroid cells seen in both mouse Gata1 knockout embryos and zebrafish vlad tepes (gata1) mutant embryos (Fujiwara et al. 1996; Lyons et al. 2002). In an effort to determine if there is a genetic relationship between mon and gata1, we tested their ability to rescue erythropoiesis. Both injection of gata1 mRNA into mon homozygous mutant embryos and injection of tif1γ mRNA into gata1 knock-down embryos failed to rescue hematopoiesis (unpublished data). We also tested for a direct interaction between Tif1γ and Gata1 proteins by coimmunoprecipitation and yeast two-hybrid assays and found no association (unpublished data). Although the mutations in each of these genes arrest cells at a similar stage of development, our results suggest that gata1 and tif1γ act independently. This does not rule out the possibility that parallel genetic pathways involving gata1 and tif1γ, operating together, regulate gene transcription within blood cells. + +The Role of Tif1γ in Primitive and Definitive Erythropoiesis + +Taken together, our data suggest that tif1γ is required as a permissive cofactor for the erythroid lineage-specific control of hematopoietic gene expression. We reasonably predict that Tif1γ protein functions as a transcriptional intermediary factor in hematopoietic progenitor cells given that both TIF1α (Zhong et al. 1999) and TIF1β (Friedman et al. 1996; Abrink et al. 2001) have been shown to act as intermediary factors that positively or negatively regulate gene transcription. These studies indicate that TIF1α and TIF1β act as scaffolds that link different classes of DNA-binding proteins and chromatin-associated proteins into larger regulatory complexes. Tif1γ is detected within nuclear foci (see Figure 6), which, based on our analysis, do not appear to correspond to several types of previously described nuclear bodies, including PML bodies. Localization of Tif1γ to these nuclear bodies may be regulated by posttranslational modification such as SUMO modification that is required for PML to form PML nuclear domains (Zhong et al. 2000a, 2000b; Best et al. 2002). These foci may serve as assembly points where Tif1γ forms multisubunit complexes with DNA-binding transcription factors and their other essential coactivators or corepressors, during the early stages of erythroid differentiation. It will be important to determine the identity of Tif1γ-interacting proteins in nuclear foci and establish how they function with Tif1γ to regulate blood cell development. + +Materials and Methods + + + +Zebrafish and mouse strains and studies + +Zebrafish were maintained and staged as described (Westerfield 1998). The alleles montb222b and montg234 were generated in a large-scale screen for ENU-induced mutations (Ransom et al. 1996) on the TU, whereas the monm262 allele was derived on the AB strain and was originally called vampire (Weinstein et al. 1996). Mapping strains were constructed by mating to WIK or SJD polymorphic strains. Linkage analysis was performed on haploid or diploid embryos obtained from TU/SJD or TU/WIK hybrids. In situ hybridization and stainings of embryos were done as described (Thompson et al. 1998; Liao et al. 2002). In situ hybridization of mouse embryos was performed as described (Kingsley et al. 2001). Genomic DNA isolation, genotyping, AFLP analysis, and chromosomal walking were each performed as previously described (Brownlie et al. 1998; Ransom and Zon 1999). A complete list of primers for genetic mapping, RT-PCR, and sequencing of mon are available on request. + +mRNA expression constructs, morpholinos, and microinjection + +The full-length mon cDNA was subcloned into EcoRI and XhoI sites in the pCS2+ vector. Synthetic mRNA was transcribed in vitro, and microinjection was performed essentially as described (Liao et al. 2002). + +Cell transplantation + +Whole kidney marrow cells were isolated from adult gata1:EGFP transgenic donors, resuspended in 0.9X phosphate-buffered saline + 5% fetal bovine serum, and injected into the sinus venosus of 2-d-old montg234 −/− and control embryos. Between 102 and 103 kidney marrow cells were injected per embryo. Individual transplanted embryos were anesthetized and visualized daily under an inverted fluorescent microscope (DM-IRE2; Leica, Wetzlar, Germany) for GFP+ cells over a span of 12 d. On day 13 posttransplant, all surviving larvae (12/129; 9%) were placed in tanks and monitored for survival. + +Antibodies, immunostaining, and immunoblots + +Antisera against the human C-terminal TIF1γ sequence RRKRLKSDERPVHIK was generated in rabbits (Genemed Synthesis, South San Francisco, California, United States) and affinity purified. Mouse embryonic fibroblasts grown on coverslips were immunostained with HP1α (Chemicon, Temecula, California, United States) and Tif1γ antisera simultaneously. In brief, cells were fixed in 4% paraformaldehyde for 5 min, washed with phosphate-buffered saline, and blocked with 5% serum (PBST) for 30 min. After incubation with the primary antibodies (PBST, 60 min) cells were washed three times with PBST and incubated with secondary antibodies (Jackson Laboratory, Bar Harbor, Maine, United States) followed by three washes in PBST. Cells were embedded with Vectashield/DAPI and analyzed using an Axioplan 2 microscope (Zeiss, Jena, Germany). Digital images were processed using the Volocity 1.0 software (Improvision, Lexington, Massachusetts, United States). G1E cell differentiation experiments were performed essentially as described (Weiss et al. 1997). + +Supporting Information + +Transplantation of wild-type zebrafish marrow cells carrying a gata1:GFP transgene into 2-d-old embryos reconstitutes erythropoiesis, but not viability, in montg234 homozygous mutants. Movies of live embryos at day 3 posttransplant highlight less than 100 GFP+ RBCs in circulation. Transplanted cells were observed to proliferate, resulting in thousands of donor-derived erythrocytes 7 d later. Movies present GFP-fluorescent images of live zebrafish larvae. + +Video S1 + +Untransplanted Control montg234 Homozygous Mutants Had No Fluorescent Cells in Circulation at 3 Days of Development + +(13.7 MB MOV). + +Click here for additional data file. + +Video S2 + +One Day after Transplantation, Less Than 100 GFP+ Erythrocytes Were Visible in the Circulation of Three montg234 Homozygous Mutants + +(11.3 MB MOV). + +Click here for additional data file. + +Video S3 + +Untransplanted Control montg234 Homozygous Mutants Had No Fluorescent Cells in Circulation at 9 Days of Development + +(7.9 MB MOV). + +Click here for additional data file. + +Video S4 + +Seven Days after Transplantation, Thousands of Donor-Derived Erythrocytes Were Visible in the Circulation of a Representative montg234 Homozygous Mutant + +(11.2 MB MOV) + +Click here for additional data file. + +Accession Numbers + +The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the genes and gene products discussed in this paper are fly bonus (AAF19646), human TIF1α (015164), human TIF1β (Q13263), human TIFγ (Q9UPN9), human TIF1γ (Q9UPN9), mon (AY59853), mouse Tif1α (Q64127), mouse Tif1β (AAH58391), and mouse Tif1γ (NP444400). + +The cDNA sequences of zebrafish mon/tif1γ and tif1α have been deposited in GenBank under the accession numbers AY598453 and AY598454, respectively. + +Acknowledgements + +We thank A. Davidson, J. Amatruda, and J. Christian for critical review of this manuscript; J. Postlethwait and W. Talbot for helpful discussions and experimental advice; B. Weinstein for the gift of the m262 allele of mon; and D. Giarla for administrative assistance. DGR was funded by the American Cancer Society and an award to Oregon Health and Science University by the Howard Hughes Medical Institute (HHMI) Biomedical Research Support Program for Medical Schools. LIZ and SHO are investigators of the HHMI. This work was supported by grants from the National Institutes of Health. + +Abbreviations + +AFLP - amplified fragment length polymorphism + +ENU - ethyl-nitrosourea + +EST - expressed sequence tag + +GFP - green fluorescent protein + +hpf - hours postfertilization + +KRAB - Krüppel-associated box + +mon - moonshine; PAC + +PML - promyelocytic leukemia gene product + +RBCs - red blood cells + +scl - stem cell leukemia; SSCP + +TIF - transcriptional intermediary factor + +TU - Tübingen background + +Figures and Tables + +Figure 1 + +Zebrafish mon Mutants Have Severe Defects in Primitive Hematopoiesis + +(A) Whole-mount TUNEL assays reveal that ventral-posterior mesodermal cells undergo apoptosis in homozygous montg234 mutant embryos. Whole-mount in situ hybridization of gata1 detected at the 12- and 18-somite stage in genotyped embryos. Posterior views, anterior to the left. + +(B) Extensive apoptosis is visible in the trunk and tail (arrowhead) and also in hematopoietic cells of the embryonic blood island at 22 h of development (arrow). Whole-mount in situ hybridization at 22 hpf including scl, gata2, gata1, ikaros, and myb in montg234 mutants. Expression of myb is greatly reduced in the blood islands because of a loss of erythroid cells, but embryonic macrophages are still present (arrows). The expression of rag1 in thymic T-cells appears normal in mon mutants at 5 d postfertilization (arrow heads). Lateral views of 22 hpf and 5-d-old embryos. + +Figure 2 + +Zebrafish mon Mutants Also Have Severe Defects in Definitive Hematopoiesis + +Adult phenotype of wild-type and mon mutants. A rare surviving montb222 homozygous adult shows significant cardiomegaly in comparison to a wild-type age-matched control. Wright–Giemsa stained marrow of wild-type adult in comparison to a homozygous mutant. Note the dramatic reduction of terminally differentiated erythroid cells and the presence of abnormally large megaloblastic proerythroblasts in the montb222 mutant marrow. + +Figure 3 + +Positional Cloning Identifies the mon Gene as Zebrafish tif1γ + +(A) Physical map of the mon locus on zebrafish Chromosome 8. Microsatellite markers z987 and z11001 were used to initially identify recombinants in a panel of 2,200 diploid montg234 homozygous mutants. The AFLP marker MA3 was used to initiate a chromosomal walk in PAC libraries. The critical PACS that were isolated to encompass the mon locus are indicated by numbers above bar. The PAC 107N19 defines the critical interval for the mon gene. This PAC was used as a probe to screen cDNA libraries and to identify zebrafish tif1γ cDNAs. Numbers below the bar indicate the number of recombinants identified by SSCP analysis. + +(B) Clustal-W–generated phylogentic tree of zebrafish (Danio rerio [Dr]) Tif1γ and Tif1α peptide sequences in comparison to TIF1 family members: human (Hs) TIF1α, TIF1β, and TIF1γ; mouse (Mm) Tif1α, Tif1β, and Tif1γ;; and fly (Dm) bonus. + +(C) Diagrams illustrating the structure of the Tif1γ-predicted peptide and the three identified point mutants. RING finger (RING), B-boxes (B1 and B2), plant homeodomain finger (PHD) and bromodomain (BROMO). Numbers below the first diagram indicate the percent identity shared between each of these domains in zebrafish and human TIF1γ. The predicted truncated proteins are indicated. + +(D) DNA sequence chromatograms showing the three ENU-induced point mutants in comparison to wild-type control sequences + +Figure 4 + +The mon/tif1γ Gene Is Highly Expressed in Hematopoietic Mesoderm + +(A) In situ hybridization of zebrafish embryos demonstrating the embryonic expression of tif1γ. tif1γ is initially expressed as a maternal mRNA. Increased expression of tif1γ in ventral-lateral mesoderm begins between the one- to three-somite stages and increases through early development. By five somites, tif1γ is strongly expressed in lateral stripes of mesoderm that also express scl. At 22 hpf tif1γ is expressed broadly in the brain, spinal cord, trunk, and tail mesenchyme, but is at much higher levels in hematopoietic cells of the blood island. Zebrafish tif1α is also broadly expressed but relatively more uniform in most tissues, in comparison to tif1γ. Tif1α is weakly expressed at early somite stages in hematopoietic mesoderm and uniformly expressed at 22 hpf, including expression in the blood islands. Expression of scl at five somites and 22 hpf highlights the embryonic blood island in comparison to tif1γ expression. + +(B) In situ hybridization of mouse embryos detects broad expression of Tif1γ at embryonic day 8.5 including the yolk sac blood islands (arrow). AT embryonic day 12.5, there is high level expression in the fetal liver (arrow) and broad expression in the embryonic brain, spinal chord, gut, and muscle. + +Figure 5 + +Overexpression of Wild-Type tif1γ mRNA or Marrow Transplantation Rescues Embryonic Hematopoiesis in mon Mutants + +(A) montg234 mutants are rescued by injection of mRNA-encoding wild-type Tif1γ protein. At 4 d of development, large numbers of RBCs are visible in the circulation of wild-type zebrafish, shown here by o-dianisidine staining of hemoglobin. Uninjected monttg234 homozygous mutants are completely bloodless. Injection of 100 pg of wild-type tif1γ mRNA rescues erythropoiesis in mutant embryos. o-dianisidine-stained larvae are shown in ventral views to highlight blood in vessels. + +(B) Transplantation of wild-type zebrafish marrow cells carrying a gata1:GFP transgene into 2-d-old embryos reconstitutes erythropoiesis, but not viability, in montg234 homozygous mutants. Still frames from movies of live embryos at day 3 posttransplant highlight less than 100 GFP+ RBCs in circulation (top). Transplanted cells were observed to proliferate resulting in thousands of donor-derived erythrocytes 7 d later (bottom). Arrows indicate the hearts of control and transplanted zebrafish. See Videos S1–S4. + +Figure 6 + +Mammalian Tif1γ Protein Localizes to Nuclear Bodies Distinct from Heterochromatin + +(A) Deconvolved immunofluorescence images of a mouse embryonic fibroblast cell nucleus stained with an anti-Tif1γ antibody and stained with a monoclonal antibody directed against HP1α. This is also compared to DAPI staining. The merged images of the nucleus show that Tif1γ does not colocalize with the HP1α or DAPI staining of heterochromatin while HP1α and DAPI staining overlap. + +(B) G1ER mouse erythroleukemia cells express high levels of Tif1γ protein as detected by Western blot analysis. Expression of Tif1γ decreases during Gata1-dependent erythroid maturation induced by β-estradiol treatment to induce a Gata1–ER fusion protein. + +Table 1 + +Overexpression of tif1γ mRNA Rescues mon Mutants: Hematopoietic Phenotypes + +Synthetic tif1γ mRNA (100 pg) was injected at the one-cell stage into embryos of the indicated genotypes. For the mon embryos, circulating cells where counted each day through 4 d, when the embryos were fixed and stained with o-dianisidine to detect hemoglobin in mature RBCs. Normal embryos contain approximately 3,000 circulating cells at these time points. Results are given as number of embryos with the indicated phenotype. Numbers in parentheses represent percentage of total embryos analyzed + +Table 2 + +Marrow Transplantation Rescues Hematopoiesis But Not Survival in mon Mutants: Embryos with Transplanted Erythroid Cells + +Between 100 and 1,000 kidney marrow cells from adult gata1:EGFP transgenic donors were injected per zebrafish embryo at 48 hpf. Individual transplanted embryos were anesthetized and visualized for GFP+ erythroid cells. By 10 d posttransplantation the indicated number of embryos had an estimated 100 to 3,000 GFP+ cells in circulation. At 3 mo the indicated number of fish were alive. The relative percentage of embryos is shown in parentheses + +Footnotes + +Conflicts of interest. The authors have declared that no conflicts of interest exist. + +Author contributions. DGR, NB, KN, DT, CB, NST, YZ, JP, SHO, and LIZ conceived and designed the experiments. DGR, NB, KN, DT, CB, NST, NPL, WJS, CAL, CH, BAB, and PDK performed the experiments. DGR, NB, KN, DT, CB, NST, NPL, YZ, JP, SHO, and LIZ analyzed the data. DGR, NB, KN, DT, NST, YZ, BAB, SL, and JP contributed reagents/materials/analysis tools. DGR, NB, KN, DT, and LIZ wrote the paper. + +Academic Editor: William Talbot, Stanford University + +¤Current address: Department of Cell and Developmental Biology, Oregon Health and Science University, Portland, Oregon, United States of America + +Citation: Ransom DG, Bahary N, Niss K, Traver D, Burns C, et al. (2004) The zebrafish moonshine gene encodes transcriptional intermediary factor 1γ, an essential regulator of hematopoiesis. 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+T414 NCBITaxon:10088 22953 22957 mice +T415 SO:0001026 23154 23160 genome +T416 PR:000004126 23212 23216 Apcs +T417 SO:0000704 23217 23221 gene +T418 GO:0000785 23288 23297 chromatin +T419 GO:0042571 23298 23300 Ab +T420 PR:000004126 23399 23403 Apcs +T421 NCBITaxon:10088 23407 23411 mice +T422 SO:0000704 23429 23440 genetically +T423 GO:0042571 23538 23541 Abs +T424 PR:000004126 23620 23624 Apcs +T425 SO:0000704 23680 23685 genes +T426 SO:0001026 23753 23760 genomic +T427 NCBITaxon:10088 23853 23857 mice +T428 SO:0001023 24022 24029 alleles +T429 SO:0000704 24040 24045 genic +T430 http://purl.obolibrary.org/obo/MONDO_0007915 24060 24063 SLE +T431 PR:000004126 24175 24179 Apcs +T432 SO:0000704 24180 24184 gene +T433 http://purl.obolibrary.org/obo/MONDO_0000001 24193 24200 disease +T434 SO:0001026 24325 24331 genome +T435 SO:0000704 24403 24407 gene +T436 GO:0042571 24493 24496 Abs +T437 NCBITaxon:10088 24553 24557 mice +T438 SO:0000704 24585 24590 genes +T439 http://purl.obolibrary.org/obo/MONDO_0004670 24646 24651 lupus +T440 NCBITaxon:10088 24676 24680 mice +T441 PR:000001479 24689 24696 FcγRIIB +T442 PR:000001337 24747 24750 CR1 +T443 PR:000025662 24797 24822 decay-accelerating factor +T444 PR:000025662 24824 24828 CD55 +T445 UBERON:0002405 24872 24878 immune +T446 NCBITaxon:10088 24906 24910 mice +T447 UBERON:0000922 24959 24968 embryonic +T448 CL:0002322 24959 24979 embryonic stem cells +T449 SO:0000704 25027 25034 genetic +T450 NCBITaxon:39107 25118 25124 murine +T451 http://purl.obolibrary.org/obo/MONDO_0007915 25135 25138 SLE +T452 SO:0000704 25173 25177 gene +T453 SO:0000704 25234 25238 gene +T454 NCBITaxon:33208 25248 25255 animals +T455 SO:0000704 25278 25283 genes +T456 SO:0001023 25325 25331 allele +T457 GO:0005634 25401 25408 nuclear +T458 PR:000004126 25468 25472 Apcs +T459 NCBITaxon:10088 25476 25480 mice +T460 NCBITaxon:10088 25529 25533 mice +T461 http://purl.obolibrary.org/obo/MONDO_0002462 25559 25577 glomerulonephritis +T462 PR:000004126 25612 25616 Apcs +T463 NCBITaxon:10088 25620 25624 mice +T464 NCBITaxon:10088 25652 25656 mice +T465 PR:000004126 25699 25703 Apcs +T466 GO:0019882 25729 25742;25747 25755 processing of ... antigens +T467 PR:000004126 25813 25817 Apcs +T468 http://purl.obolibrary.org/obo/MONDO_0005556 25867 25882 lupus nephritis +T469 GO:0010467 25908 25918 expression +T470 NCBITaxon:9606 25926 25931 human +T471 PR:000005897 25932 25950 C-reactive protein +T472 GO:0006953 25955 25966 acute-phase +T473 PR:000004126 25994 25998 Apcs +T474 http://purl.obolibrary.org/obo/MONDO_0005556 26050 26065 lupus nephritis +T475 UBERON:0002405 26118 26124 immune +T476 GO:0032991 26125 26134 complexes +T477 UBERON:0001225 26142 26154 renal cortex +T478 PR:000005897 26253 26271 C-reactive protein +T479 http://purl.obolibrary.org/obo/MONDO_0007915 26306 26309 SLE +T480 NCBITaxon:9606 26313 26319 humans +T481 NCBITaxon:10088 26368 26372 mice +T482 PR:000004126 26381 26385 Apcs +T483 NCBITaxon:10088 26389 26393 mice +T484 SO:0000704 26610 26615 genes +T485 UBERON:0000062 26651 26656 organ +T486 http://purl.obolibrary.org/obo/MONDO_0005556 26690 26705 lupus nephritis +T487 NCBITaxon:10088 26722 26726 mice +T488 SO:0000704 26783 26788 genes +T489 http://purl.obolibrary.org/obo/MONDO_0004670 26816 26821 lupus +T490 PR:000004126 26887 26891 Apcs +T491 UBERON:0002113 26915 26920 renal +T492 SO:0001026 27028 27035 genomes +T493 http://purl.obolibrary.org/obo/MONDO_0007915 27058 27061 SLE +T494 SO:0000704 27098 27102 gene +T495 UBERON:0002405 27172 27178 immune +T496 SO:0000704 27201 27208 genetic +T497 NCBITaxon:10088 27248 27252 Mice +T498 NCBITaxon:10088 27263 27267 mice +T499 NCBITaxon:33208 27390 27396 animal +T500 NCBITaxon:10088 27474 27478 mice +T501 NCBITaxon:10088 27559 27563 mice +T502 NCBITaxon:10088 27603 27607 mice +T503 PR:000004126 27613 27617 Apcs +T504 NCBITaxon:10088 27620 27624 mice +T505 PR:000004126 27710 27714 Apcs +T506 NCBITaxon:10088 27718 27722 mice +T507 PR:000004126 27755 27759 Apcs +T508 NCBITaxon:10088 27763 27767 mice +T509 SO:0000704 27779 27786 genetic +T510 PR:000004126 27803 27807 Apcs +T511 NCBITaxon:33208 27811 27818 animals +T512 PR:000004126 27924 27928 Apcs +T513 NCBITaxon:10088 27939 27943 mice +T514 NCBITaxon:10088 28026 28030 mice +T515 NCBITaxon:10088 28266 28270 mice +T516 NCBITaxon:10088 28399 28403 mice +T517 PR:000004126 28654 28658 Apcs +T518 NCBITaxon:10088 28662 28666 mice +T519 SO:0001026 28723 28729 genome +T520 PR:000004126 28741 28745 Apcs +T521 PR:000004126 29027 29031 Apcs +T522 NCBITaxon:10088 29034 29038 mice +T523 NCBITaxon:33208 29064 29071 animals +T524 NCBITaxon:10088 29109 29113 mice +T525 NCBITaxon:33208 29149 29156 Animals +T526 NCBITaxon:33208 29215 29221 animal +T527 UBERON:0001977 29307 29311 Sera +T528 GO:0042571 29384 29387 Abs +T529 GO:0071735 29399 29402 IgG +T530 GO:0042571 29484 29487 Abs +T531 NCBITaxon:46909 29536 29554 Crithidia luciliae +T532 UBERON:0001977 29603 29608 Serum +T533 GO:0042571 29724 29727 Abs +T534 GO:0000785 29746 29755 chromatin +T535 NCBITaxon:10088 30005 30010 mouse +T536 UBERON:0001977 30114 30119 serum +T537 PR:000004126 30156 30160 Apcs +T538 NCBITaxon:9940 30197 30202 sheep +T539 NCBITaxon:10088 30208 30213 mouse +T540 PR:000004126 30214 30218 Apcs +T541 NCBITaxon:9986 30223 30229 rabbit +T542 NCBITaxon:10088 30235 30240 mouse +T543 PR:000004126 30241 30245 Apcs +T544 GO:0042571 30246 30249 Abs +T545 GO:0006953 30439 30450 acute-phase +T546 UBERON:0001977 30451 30456 serum +T547 PR:000004126 30487 30491 Apcs +T548 PR:000004126 30506 30510 Apcs +T549 NCBITaxon:10088 30514 30519 mouse +T550 UBERON:0001977 30520 30525 serum +T551 NCBITaxon:10088 30595 30599 mice +T552 GO:0016265 30621 30625 died +T553 UBERON:0002113 30695 30701 kidney +T554 CHEBI:75958 30733 30741 solution +T555 CHEBI:29149 30796 30809 periodic acid +T556 CHEBI:33893 30817 30824 reagent +T557 UBERON:0005749 30968 30983 glomerular tuft +T558 UBERON:0000074 30998 31007 glomeruli +T559 UBERON:0005749 31069 31084 glomerular tuft +T560 UBERON:0000074 31099 31108 glomeruli +T561 UBERON:0000074 31119 31129 glomerular +T562 UBERON:0000074 31170 31179 glomeruli +T563 UBERON:0000074 31216 31225 glomeruli +T564 SO:0001026 31807 31814 genomic +T565 GO:0030849 31907 31916 autosomes +T566 CHEBI:33893 31953 31961 reagents +T567 CHEBI:6636 31980 31985 MgCl2 +T568 SO:0000112 31997 32004 primers +T569 NCBITaxon:10088 32090 32094 mice +T570 SO:0000112 32101 32108 primers +T571 CHEBI:4883 32140 32156 ethidium bromide +T572 CHEBI:2511 32165 32172 agarose +T573 CHEBI:8984 32184 32187 SDS +T574 SO:0000771 32242 32245 QTL +T575 SO:0000771 32265 32268 QTL +T576 NCBITaxon:10088 32397 32401 mice +T577 GO:0042571 32461 32464 Abs +T578 SO:0000771 32530 32533 QTL +T579 SO:0000704 33018 33023 genes +T580 SO:0000704 33028 33032 gene +T581 PR:000004126 33070 33074 Apcs +T582 PR:000001337 33097 33101 CD35 +T583 PR:000001338 33102 33106 CD21 +T584 PR:000025662 33129 33133 CD55 +T585 PR:000001962 33160 33172 FAS receptor +T586 SO:0000704 33173 33177 gene +T587 PR:000001479 33204 33210 FcγRII +T588 NCBITaxon:33208 33408 33414 animal +T589 GO:0042571 33638 33640 Ab +T590 GO:0042571 33643 33651 antibody +T591 GO:0005634 33664 33671 nuclear +T592 GO:0042571 33672 33680 antibody +T593 PR:000004126 33710 33714 Apcs +T594 PR:000004126 33720 33724 Apcs +T595 NCBITaxon:10088 33735 33739 mice +T596 PR:000004126 33741 33745 Apcs +T597 UBERON:0001977 33748 33753 serum +T598 PR:000004126 33748 33773 serum amyloid P component +T599 CHEBI:60425 33754 33761 amyloid +T600 SO:0000704 33774 33778 gene +T601 SO:0000985 33788 33803 double-stranded +T602 SO:0000771 33834 33837 QTL +T603 SO:0000771 33840 33866 quantitative trait linkage +T604 http://purl.obolibrary.org/obo/MONDO_0007915 33868 33871 SLE +T605 http://purl.obolibrary.org/obo/MONDO_0007915 33874 33902 systemic lupus erythematosus +T606 SO:0000984 33917 33932 single-stranded +T607 GO:0000785 34010 34019 Chromatin +T608 GO:0042571 34020 34023 Abs +T609 PR:000004126 34045 34049 Apcs +T610 NCBITaxon:10088 34053 34057 Mice +T611 NCBITaxon:33208 34117 34124 animals +T612 GO:0042571 34475 34478 Abs +T613 NCBITaxon:10088 34520 34524 mice +T614 PR:000004126 34582 34586 Apcs +T615 NCBITaxon:33208 34589 34596 animals +T616 SO:0000771 34977 34980 QTL +T617 GO:0042571 35028 35031 Abs +T618 SO:0000771 35502 35505 QTL +T619 GO:0000785 35540 35549 Chromatin +T620 GO:0042571 35550 35553 Abs +T621 GO:0042571 35941 35943 Ab +T622 PR:000004126 35994 35998 Apcs +T623 NCBITaxon:10088 36002 36006 mice +T624 NCBITaxon:10088 36074 36079 mouse +T625 NCBITaxon:33208 36118 36125 animals +T626 UBERON:0001977 36156 36161 Serum +T627 PR:000004126 36238 36242 Apcs +T628 NCBITaxon:10088 36247 36251 mice +T629 NCBITaxon:33208 36305 36312 animals +T630 GO:0000785 36446 36455 chromatin +T631 GO:0042571 36456 36458 Ab +T632 NCBITaxon:10088 36607 36611 mice +T633 SO:0000771 36714 36717 QTL +T634 GO:0000785 36749 36758 Chromatin +T635 GO:0042571 36775 36778 Abs +T636 GO:0042571 37226 37228 Ab +T637 NCBITaxon:10088 37265 37269 mice +T638 PR:000004126 37279 37283 Apcs +T639 NCBITaxon:10088 37288 37292 mice +T640 NCBITaxon:10088 37333 37337 mice +T641 NCBITaxon:10088 37381 37386 mouse +T642 NCBITaxon:33208 37424 37431 animals +T643 UBERON:0001977 37461 37466 Serum +T644 GO:0000785 37536 37545 chromatin +T645 GO:0042571 37550 37552 Ab +T646 NCBITaxon:10088 37583 37587 mice +T647 GO:0042571 37603 37605 Ab +T648 GO:0042571 37729 37731 Ab +T649 UBERON:0001977 37740 37745 Serum +T650 UBERON:0002113 37869 37874 Renal +T651 NCBITaxon:10088 37908 37912 mice +T652 PR:000004126 37922 37926 Apcs +T653 NCBITaxon:10088 37930 37934 mice +T654 NCBITaxon:10088 37975 37979 mice +T655 UBERON:0002113 38065 38071 kidney +T656 http://purl.obolibrary.org/obo/MONDO_0002462 38105 38123 glomerulonephritis +T657 http://purl.obolibrary.org/obo/MONDO_0002462 38125 38143 Glomerulonephritis +T658 GO:0000785 38256 38265 Chromatin +T659 GO:0042571 38281 38284 Abs +T660 NCBITaxon:10088 38306 38310 Mice +T661 PR:000004126 38366 38370 Apcs +T662 NCBITaxon:33208 38374 38381 animals +T663 GO:0042571 38657 38660 Abs +T664 PR:000004126 38664 38668 Apcs +T665 NCBITaxon:10088 38711 38715 Mice +T666 PR:000004126 38749 38753 Apcs +T667 UBERON:0001977 38891 38896 serum +T668 GO:0016265 38900 38905 death +T669 NCBITaxon:10088 38913 38917 mice +T670 UBERON:0002113 39009 39015 Kidney +T671 PR:000004126 39028 39032 Apcs +T672 NCBITaxon:10088 39075 39079 Mice +T673 UBERON:0002113 39085 39090 renal +T674 SO:0001026 39319 39325 Genome +T675 PR:000004126 39351 39355 Apcs +T676 NCBITaxon:10088 39398 39402 Mice +T677 NCBITaxon:10088 39857 39862 Mouse +T678 SO:0001026 39863 39869 Genome +T679 NCBITaxon:10088 39880 39885 Mouse +T680 SO:0001026 39886 39892 Genome +T681 NCBITaxon:10088 40540 40544 mice +T682 SO:0000704 40588 40592 gene +T683 NCBITaxon:10088 40602 40606 mice diff --git a/src/ontogpt/evaluation/craft/database/all/15314659.txt b/src/ontogpt/evaluation/craft/database/all/15314659.txt new file mode 100644 index 000000000..3a8f3baf5 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15314659.txt @@ -0,0 +1,223 @@ +Spontaneous Autoimmunity in 129 and C57BL/6 Mice—Implications for Autoimmunity Described in Gene-Targeted Mice + +Abstract + +Systemic lupus erythematosus (SLE) is a multisystem autoimmune disorder in which complex genetic factors play an important role. Several strains of gene-targeted mice have been reported to develop SLE, implicating the null genes in the causation of disease. However, hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, develop spontaneous autoimmunity. Furthermore, the genetic background markedly influences the autoimmune phenotype of SLE in gene-targeted mice. This suggests an important role in the expression of autoimmunity of as-yet-uncharacterised background genes originating from these parental mouse strains. Using genome-wide linkage analysis, we identified several susceptibility loci, derived from 129 and C57BL/6 mice, mapped in the lupus-prone hybrid (129 × C57BL/6) model. By creating a C57BL/6 congenic strain carrying a 129-derived Chromosome 1 segment, we found that this 129 interval was sufficient to mediate the loss of tolerance to nuclear antigens, which had previously been attributed to a disrupted gene. These results demonstrate important epistatic modifiers of autoimmunity in 129 and C57BL/6 mouse strains, widely used in gene targeting. These background gene influences may account for some, or even all, of the autoimmune traits described in some gene-targeted models of SLE. + +Introduction + +Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterised by the production of autoantibodies (auto-Abs) against a wide spectrum of self-antigens, mainly from subcellular compartments, especially the cell nucleus. Genetic predisposition is an important contributor to susceptibility to SLE in both humans and animals (Vyse and Todd 1996; Harley et al. 1998; Theofilopoulos and Kono 1999; Wakeland et al. 2001). Genes in multiple pathways participate in mediating disease pathogenesis, and epistatic interactions amongst these genes influence the expression of disease. In this context, both genetic linkage studies in spontaneous lupus-prone models and synthetic murine models of autoimmunity generated by targeted disruption of specific genes modulating the immune system have widely been used to investigate the complexity of SLE. + +The best-studied strains of mice that spontaneously develop a lupus-like pathology are the New Zealand Black/New Zealand White hybrid strain (NZB/WF1); the MRL/Mp lpr/lpr strain, which carries the lpr mutation of the FAS receptor gene; and the BXSB strain, which carries the Y chromosome autoimmune accelerator (Yaa) gene (Theofilopoulos and Dixon 1985). Extensive genetic mapping studies in all three strains have identified multiple intervals associated with disease susceptibility. Interestingly, the majority of the intervals detected are strain-specific, confirming the genetic complexity of the disease and indicating the presence of extensive heterogeneity in the genes contributing to the pathogenesis of the disease. However, some susceptibility loci have been mapped to similar chromosome locations in different strains, suggesting that at least some susceptibility may be shared amongst lupus-prone strains. Amongst these shared susceptibility loci, the most striking are loci on distal Chromosome 1, for which important contributing genes have been found in New Zealand and BXSB models (Theofilopoulos and Kono 1999; Wakeland et al. 2001). + +Although considerable efforts have been made to identify the genes responsible for the development of the disease, with the exception of the lpr mutation, none of the genetic contributions to disease in the three well-documented murine SLE strains have yet been fully resolved at the molecular or protein level. Thus, targeted genetic disruption of candidate genes encoding proteins of the immune system has been extensively used to examine their role in immune regulation. However, the most surprising result of this powerful approach has been the high frequency with which such mutations have been associated with an autoimmune phenotype. In this regard, it is of note that hybrid strains between 129 and C57BL/6 mice, widely used in the generation of gene-targeted mice, are spontaneously predisposed to development of humoral autoimmunity with low levels of glomerulonephritis (Obata et al. 1979; Botto et al. 1998; Bickerstaff et al. 1999; Santiago-Raber et al. 2001). Furthermore, the genetic background markedly influences the autoimmune phenotype in gene-targeted mice (Bolland and Ravetch 2000; Santiago-Raber et al. 2001; Mitchell et al. 2002). These observations led to the hypothesis that the autoimmune phenotype described in some gene-targeted mice might be due primarily to combinations of as-yet-uncharacterised background genes, originating from 129 and C57BL/6 mice strains, interacting or not with the mutated allele. To test this, we conducted a genome-wide scan analysis of two large cohorts of (129 × C57BL/6)F2 mice, one of which carried a mutation in the serum amyloid P component gene (Apcs). The Apcs-deficient mice (Apcs−/−) were chosen as an example of a gene-targeted strain previously reported to develop a lupus-like disease on the hybrid genetic background (129 × C57BL/6); autoimmunity in Apcs−/− mice persists even after backcrossing the mutated gene onto C57BL/6 (Bickerstaff et al. 1999). We chose this targeted gene in particular to study since the Apcs gene is located on Chromosome 1, approximately 94 cM from the centromere, within a region where several lupus-susceptibility loci, designated Sle1 (Morel et al. 2001), Nba2 (Drake et al. 1995; Vyse et al. 1997), and Bxs3 (Hogarth et al. 1998; Haywood et al. 2000), have been mapped in NZW, NZB, and BXSB lupus-prone strains, respectively. This region contains several genes, including those encoding FcγRII, the complement receptor CR1/2 (CD35/CD21), and the decay-accelerating factor CD55 (Prodeus et al. 1998; Bolland and Ravetch 2000; Miwa et al. 2002; Wu et al. 2002), which have each been implicated in the causation of SLE when inactivated by gene-targeting in 129 embryonic stem cells. + +Here we show that there are multiple genetic loci, derived from both 129 and C57BL/6 mice, contributing to autoimmunity. Furthermore, a 129-derived interval on distal Chromosome 1, when transferred onto the C57BL/6 genome, a combination commonly created by backcrossing onto C57BL/6 a gene that has been inactivated in 129 embryonic stem cells, was sufficient to cause humoral autoimmunity in its own right, irrespective of the presence of the mutated Apcs gene. These results demonstrate important epistatic interactions between genes from 129 and C57BL/6 genomes on the development of autoimmunity and illustrate the important effects of background genes in the analysis and interpretation of autoimmune phenotypes associated with targeted genetic disruptions. + +Results + +Disease Traits in (129 × C57BL/6)F2 and (129 × C57BL/6)F2.Apcs−/− Mice + +To investigate the genetic basis of the lupus-like disease observed in the (129 × C57BL/6) hybrid mice, we generated two large cohorts of (129 × C57BL/6)F2 animals, one carrying a mutation in the Apcs gene, and monitored them for 1 y under identical environmental conditions. Since female mice in the original reports showed a higher penetrance of disease, the present study was conducted only on female mice. The results of the phenotypic analysis at 1 y of age are summarised in Tables 1 and 2. As previously reported (Botto et al. 1998), the wild-type (129 × C57BL/6)F2 mice developed lupus traits with elevated levels of auto-Abs, starting from 6 mo of age (data not shown), and histological evidence of proliferative glomerulonephritis. In agreement with our previous observations (Bickerstaff et al. 1999), the titres of anti-nuclear Abs (ANAs) and anti-chromatin Ab were considerably greater in the (129 × C57BL/6)F2.Apcs−/− mice compared with the strain-matched controls. However, in contrast to our original findings, the levels of the other two disease serological markers analysed (anti-single-stranded DNA [ssDNA] and anti-double-stranded DNA [dsDNA] Abs) and the severity of the renal pathology were not different between the two experimental groups. In view of the possibility of an association between the fixed 129-derived segment flanking the mutated Apcs gene and the autoimmune traits observed, the genome-wide linkage analysis of the two experimental cohorts was carried out separately. + +Mapping of Loci Predisposing to Lupus in the Hybrid Strain (129 × C57BL/6) + +Mice were genotyped with 143 microsatellite markers distributed throughout the autosomes such that 98% of the genes were within 20 cM of an informative marker. A summary of the genome-wide linkage analysis for each of the disease traits measured is shown in Table 3. The areas of linkage were defined according to the parental origin, 129 or C57BL/6. Only linkages identified in both experimental groups are reported in Table 3, with the exception of the Chromosome 1 distal segment, where the linkage analysis could not be applied to the (129 × C57BL/6)F2.Apcs−/− mice as this region was of fixed 129 origin. Chromosomes where linkages were present only in one of the two cohorts are shown in Figures 1–3. + +The quantitative trait linkage (QTL) analysis identified several intervals on Chromosome 1 with linkage to disease serological markers, and these regions were all derived from the 129 mouse strain (see Table 3; Figures 4 and 5). Interestingly, whilst ANA and anti-chromatin Ab levels showed suggestive or significant linkages only to the telomeric region of Chromosome 1, with an estimated peak occurring at a position approximately 90 cM near the Apcs gene, anti-dsDNA or anti-ssDNA Ab production was also linked to other segments on Chromosome 1, indicating a more complex genetic contribution from the 129 mouse strain. + +Guided by these observations, we investigated whether the increased levels of ANA and anti-chromatin Ab observed in the Apcs−/− mice were caused by a gene(s) within the fixed 129 region surrounding the mutated Apcs gene, rather than caused by the mutated Apcs gene itself. We compared the levels of these auto-Abs between all (129 × C57BL/6)F2.Apcs−/− mice and a group of 33 wild-type mice that were selected for being homozygous 129 in the region of Chromosome 1 between microsatellites D1Mit105 and D1Mit 223 (80–106 cM) (Figure 6A–6D). In contrast to the results reported in Table 1, this comparison showed no significant differences between the two experimental groups. This result demonstrates that, most likely, the 129-derived region and not the lack of Apcs was mediating the production of ANA and anti-chromatin Ab. Consistent with this explanation, we found that the 129 mice have significantly higher levels of Apcs in circulation compared with the C57BL/6 mice (median, 83 mg/l; range, 25–208; n = 16 versus median, 5 mg/l; range, 4–9; n = 10, respectively; p < 0.0001). The C57BL/6 strain has previously been reported to be one of the murine strains defined as low Apcs producers (Pepys et al. 1979; Baltz et al. 1980). In addition, sequence analysis of the entire Apcs coding region in both strains failed to identify any coding sequence polymorphisms in the Apcs gene (data not shown), indicating that a structural variant of the protein is unlikely to be the explanation for our findings. This is consistent with a previous report by Drake et al. (1996) that showed no Apcs coding sequence differences amongst several autoimmune and nonautoimmune murine strains. + +In addition to the 129-derived segments, in both cohorts the C57BL/6 strain contributed to the autoimmune traits with one major susceptibility locus on Chromosome 3. A genomic region between D3Mit40 and D3Mit13, with an estimated peak at position approximately 51 cM, showed a significant linkage to ANA production and weaker linkages to anti-ssDNA and anti-chromatin production (see Table 3; Figure 7). The high frequency of autoimmune phenotype in the (129 × C57BL/6) hybrid genetic background and its absence in either of the inbred parental strains imply that there are essential interactions between 129- and C57BL/6-derived alleles for the expression of autoimmunity. We investigated further the effects of genes from the C57BL/6 background by repeating the linkage analysis in (129 × C57BL/6)F2 mice, whilst controlling for the very strong 129 effect on distal Chromosome 1, as previously described (Zeng 1994). The results of this analysis showed that the statistical support for the linkage of the C57BL/6 locus on Chromosome 3 for ANA increased from logarithm of odds (LOD) 5.4 to LOD 6.4. + +In contrast to these strong associations with disease serological markers, the QTL analysis identified only two potential linkages to glomerulonephritis: one in the wild-type mice on Chromosome 7 across a 10 cM region between D7Mit246 (15 cM) and D7Mit145 (26.5 cM) of 129 origin (LOD 2.86, p = 0.0013), and one on Chromosome 17 between D17Mit100 (11.7 cM) and D17Mit216 (29.4 cM) from the C57BL/6 strain (LOD 1.3, p = 0.049 and LOD 1.67, p = 0.021 in the wild-type and Apcs−/− mice, respectively). Histological evidence of glomerulonephritis was only found in approximately 20% of the mice in each cohort, which reduces the power of the QTL analysis for this disease trait. + +Production of a C57BL/6.129 Chromosome 1 Congenic Line and Its Phenotypic Analysis + +We generated a C57BL/6 congenic line carrying the telomeric region of Chromosome 1 from the 129 mouse strain, in order to dissect the complex polygenic disease phenotype of the (C57BL/6 × 129/Sv)F2 hybrid strain into its individual genetic components. The 129 interval was backcrossed seven times onto C57BL/6, and at each generation the presence or absence of the Chromosome 1 interval was determined with several microsatellite markers. Each backcrossed generation was screened with more than three markers per chromosome to facilitate the removal of unselected 129 genomic regions. At the end of the backcrossing, the 129-derived Chromosome 1 interval in the congenic mice extended from microsatellite marker D1Mit105 to D1Mit223 (80–106 cM), which encompasses the most important 129 interval identified by the linkage studies in the (C57BL/6 × 129/Sv)F2 mice. + +Female Chromosome 1 congenic mice (C57BL/6.129[D1Mit105–223]), together with sex-matched Apcs−/− mice backcrossed onto C57BL/6 for ten generations (C57BL/6.Apcs−/−) and C57BL/6 controls, were monitored for the presence of lupus. In the C57BL/6.Apcs−/− mice, the 129 genome around the Apcs locus was mapped as a stretch of approximately 17 cM, positioned from 87.9 cM (D1Mit15) to 105 cM (D1Mit17). Thus, the congenic line carried a similar 129 region (80–106 cM) to the one present in the C57BL/6.Apcs−/− mice (87.9–105 cM). At 1 y of age, all animals were sacrificed, the auto-Abs assessed, and the renal histology examined. The results of this analysis are shown in Figure 8. As previously reported (Bickerstaff et al. 1999), the levels of auto-Abs were markedly increased in the C57BL/6.Apcs−/− compared to the wild-type C57BL/6 controls. However, the C57BL/6.129(D1Mit105–223) animals also expressed high levels of auto-Abs, and these titres were not different from those detected in the matched congenic mice containing a null mutation of the Apcs gene. These results clearly demonstrated that epistatic interactions between 129 loci on Chromosome 1 and C57BL/6 genes were sufficient to mediate the loss of tolerance to nuclear autoantigens. However, in contrast to the serological data, the histological assessment of the kidneys showed evidence of markedly increased glomerulonephritis in the C57BL/6.Apcs−/− compared to both control groups (Figure 9), suggesting that the lack of Apcs, when combined with other C57BL/6 susceptibility alleles, can induce the development of severe renal damage. + +Discussion + +There is accumulating evidence that background genes may influence the expression of autoimmunity in gene-targeted mice. Here we report what is to our knowledge the first systematic study that has examined this in the 129 and C57BL/6 mouse strains, widely used for gene targeting. Our results demonstrate interacting loci between 129 and C57BL/6 mice that can cause the expression of a powerful autoimmune phenotype in these animals, in the absence of any gene-targeted mutations. We also developed a congenic mouse strain bearing a portion of 129 Chromosome 1 on a C57BL/6 background and showed that this wild-type congenic line expressed striking anti-nuclear autoimmunity. By comparing this Chromosome 1 congenic strain with matched congenic mice lacking the Apcs gene, we demonstrated that serum amyloid P component deficiency influences the severity of glomerulonephritis, but is not the prime mover in the induction of anti-nuclear autoimmunity, contrary to our own original interpretation of our data (Bickerstaff et al. 1999). The same consideration applies to other genes located in the same Chromosome 1 region that have been implicated in the development of SLE when inactivated by gene-targeting in 129 embryonic stem cells and then backcrossed onto a pure genetic background (Bolland and Ravetch 2000; Miwa et al. 2002; Wu et al. 2002). For each, there has to be a question as to whether the anti-nuclear autoimmunity is due to the gene-targeted mutant gene or to the normal 129 genes expressed in the same region as the targeted gene. + +The influence of background genes on the development of spontaneous autoimmune disease is well known, especially with respect to the lpr and Yaa disease-susceptibility genes. In MRL/Mp mice, the presence of the lpr gene accelerates the development of high level and broad-spectrum auto-Ab production and lethal glomerulonephritis, in addition to marked lymphoproliferative disease. In contrast, homozygosity of the lpr gene in other strains such as C57BL/6, AKR, LG/J, and C3H leads only to auto-Ab production (Izui et al. 1984). Similarly, the Y-chromosome-linked Yaa gene in BXSB and MRL/Mp males enhances the rapid development of auto-Abs and glomerulonephritis (Izui et al. 1988; Merino et al. 1989). However, in the C57BL/6 background, the Yaa gene does not lead to an autoimmune phenotype (Izui et al. 1988). Not surprisingly, important effects of the genetic background on the expression of autoimmunity have also been reported in gene-targeted mice (Bolland and Ravetch 2000; Santiago-Raber et al. 2001; Mitchell et al. 2002). Thus, SLE exists as a complex-trait disorder in which specific combinations of susceptibility alleles are required for the expression of the full phenotype. + +Through the use of microsatellite marker maps, the identification of murine SLE susceptibility intervals in experimental crosses has been made possible. These mapping studies have shown that the disease expression in relation to the inheritance of the different alleles followed a threshold liability pattern in which a positive phenotype depended upon the presence of multiple discrete susceptibility loci with no single locus being a prerequisite factor. We have employed the same approach to analyse the genetic basis of disease inheritance in the (129 × C57BL/6) hybrid strain, the most common genetic background in gene-targeted mice. Although spontaneous autoimmunity has not been documented in either of the pure 129 or C57BL/6 strains, a spontaneous lupus-like phenotype has been described in (129 × C57BL/6) hybrid strains (Obata et al. 1979; Botto et al. 1998; Bickerstaff et al. 1999; Santiago-Raber et al. 2001), suggesting that the predisposition in these hybrid mice may arise as a result of the interaction between specific combinations of alleles inherited from both the 129 and C57BL/6 parental strains. + +This was confirmed by the mapping study reported here. We showed that there are multiple genetic loci contributing to the disease and these are derived from both 129 and C57BL/6 mice. We demonstrated that a 129-derived segment of Chromosome 1 was strongly linked to the expression of auto-Abs. This region is probably capable of causing the initiation of a humoral autoimmune response to nuclear antigens; however, this response does not occur in the absence of C57BL/6 genes. In support of this, we identified a C57BL/6 segment on Chromosome 3, which may interact with the 129 genes on Chromosome 1 to mediate the loss of tolerance. Interestingly, although the C57BL/6 SLE-susceptibility region on Chromosome 3 is novel, disease-modifying alleles derived from C57BL/10 and C57BL/6 strains have been mapped to a portion of Chromosome 3 close to the region identified in this study (Morel et al. 1999; Haywood et al. 2000). Furthermore, the region on Chromosome 7 associated with the development of lupus nephritis has been linked to the same trait in other murine models of SLE (Santiago et al. 1998; Morel et al. 1999; Xie et al. 2002), suggesting the possibility of shared susceptibility loci. + +Taken together our results suggest a complex genetic contribution from the (129 × C57BL/6) hybrid background genome, with both enhancing as well as inhibitory loci from the 129 mouse, in addition to genes promoting autoimmunity from the C57BL/6 mice. The impact that these interacting loci may have on the lupus-like disease present in several gene-targeted animals was further assessed by comparing Apcs−/− mice with Chromosome 1 genetically matched controls. + +In the context of SLE susceptibility, one of the most consistently mapped non-MHC regions of the mouse genome is the telomeric Chromosome 1 segment, where several disease loci, designated Sle1 (Morel et al. 2001), Nba2 (Drake et al. 1995; Rozzo et al. 1996; Vyse et al. 1997), and Bxs3 (Hogarth et al. 1998), have been mapped in lupus-prone strains. Moreover, this region of mouse Chromosome 1 is orthologous to a region on human Chromosome 1q22–1q25, which has also been linked with human SLE (Moser et al. 1998). + +The Apcs gene is one of the candidate genes known to lie within this region. The human serum amyloid P component binds avidly to DNA, chromatin, and apoptotic cells in physiological conditions in vitro (Pepys 1974; Pepys and Butler 1987; Butler et al. 1990) and also to exposed chromatin and apoptotic cells in vivo (Hintner et al. 1988; Breathnach et al. 1989; Familian et al. 2001). We have previously reported that (129 × C57BL/6).Apcs−/− mice spontaneously produce a wide range of ANAs and develop significant immune complex glomerulonephritis (Bickerstaff et al. 1999). On the basis of these observations, it was postulated that Apcs, by altering the clearance of chromatin, contributes to the pathogenesis of SLE. However, in this study we found that only ANA and anti-chromatin Ab levels were significantly increased in the (129 × C57BL/6)F2.Apcs−/− mice. A possible explanation for this discrepancy may lie in the fact that the Apcs−/− mice analysed in the original study were generated from a limited number of founders and that this may have caused a nonrandom inheritance of the loci from the parental strains. Furthermore, the whole-genome analysis identified the 129 region surrounding the Apcs gene as the main locus contributing to the development of ANA and anti-chromatin Ab. In agreement with this, when we carried out a selective comparison between the (129 × C57BL/6)F2.Apcs−/− mice and Chromosome 1 genetically matched controls, we failed to detect any significant difference in the levels of these two auto-Abs. These findings, taken together, indicated that the phenotype associated with Apcs deficiency was caused by the presence of unaltered 129 genes from the telomeric region of Chromosome 1 operating in the C57BL/6 genomic background. Strong supportive evidence for this was provided by the analysis of the C57BL/6 mice congenic for this 129 region. + +The generation and analysis of congenic strains have successfully been used to dissect the contribution of individual susceptibility alleles to a multigenic trait such as SLE. We adopted the same strategy to investigate the relative contribution of the 129 Chromosome 1 segment and the Apcs gene to each disease trait. Using this approach, we demonstrated that the 129 interval on distal Chromosome 1, when transferred onto the C57BL/6 genome, a combination commonly created by backcrossing onto C57BL/6 a mutated gene located in that chromosomal region, was sufficient to mediate the production of auto-Abs. In this context, it is of note that several strains of mice with targeted mutations of genes encoded in this region have been reported to express a lupus-like illness, including mice lacking FcγRIIB (Bolland and Ravetch 2000), complement receptors (CR1/2) (Prodeus et al. 1998; Wu et al. 2002), and decay-accelerating factor (CD55) (Miwa et al. 2002). In each case, the autoimmune phenotype was described in mice in which the null mutation was generated in 129 embryonic stem cells and then backcrossed to the C57BL/6 or another genetic background. Thus, in view of our findings, one may postulate that in each of these murine models of SLE, the effects of the targeted null gene were irrelevant. Similar conclusions may apply to other gene-targeted animals carrying mutations of genes mapped in the 129-derived susceptibility allele on Chromosome 7 (O'Keefe et al. 1996, 1999). + +The expression of anti-nuclear autoimmunity was identical comparing the congenic with the Apcs−/− mice. The only difference in phenotype between these mice was in the expression of glomerulonephritis, which was more pronounced in the Apcs−/− mice compared with the congenic mice. Although these findings demonstrate that Apcs is not implicated in the processing of autoantigens, as it had previously been suggested, they indicate that Apcs might still play an important protective role in lupus nephritis. In support of this, the expression of the human C-reactive protein, an acute-phase protein closely related to Apcs, has been shown to delay the onset and severity of lupus nephritis in the NZB/W strain by preventing the deposition of immune complexes in the renal cortex (Szalai et al. 2003). Consistent with this, a polymorphism associated with reduced basal level of C-reactive protein has been reported to be linked to SLE in humans (Russell et al. 2004). However, as the congenic mice and the Apcs−/− mice carried similar but not identical 129 regions on Chromosome 1, an alternative explanation for our findings may still lay in the numerous and complex synergistic and counteractive interactions between 129 and C57BL/6 genes involved in self-tolerance and end organ damage. Thus, whilst the lack of lupus nephritis in the congenic mice is consistent with the need for multiple susceptibility genes for the full expression of lupus, further studies will be required to fully elucidate the role of Apcs in the pathogenesis of renal damage. + +In summary, our findings demonstrate the impact of epistatic interactions between 129 and C57BL/6 genomes on the development of SLE and illustrate how these background gene effects may lead to incorrect interpretations when analysing the autoimmune phenotype of specific genetic disruptions. + +Materials and Methods + + + +Mice. + +All the mice were females. Wild-type C57BL/6 and 129/Sv (129S6, according to the revised nomenclature) were bred and maintained in the animal care facility at Imperial College, London, United Kingdom. (129 × C57BL/6)F1 mice were generated by intercrossing the two wild-type strains and (129 × C57BL/6)F2 mice by interbreeding the (129 × C57BL/6)F1 mice. The Apcs−/−mice were generated as previously reported (Botto et al. 1997), and the (129 × C57BL/6)F2.Apcs−/− mice were generated by intercrossing Apcs−/− mice on the 129 genetic background with Apcs−/− animals backcrossed onto C57BL/6 for ten generations. A total of 141 (129 × C57BL/6)F2 and 158 (129 × C57BL/6)F2.Apcs−/− female mice were produced and monitored for 1 y. Wild-type congenic C57BL/6.129(D1Mit105–223) mice were generated by backcrossing the 129 interval between microsatellites D1Mit105 and D1Mit223 (80 cM to 106 cM) onto the C57BL/6 strain. Inherited 129 regions were mapped with microsatellite markers polymorphic between 129 and C57BL/6 mice (see below). After seven generations of backcrossing, siblings were intercrossed to generate C57BL/6.129(D1Mit105–223) congenic mice homozygous for the 129 Chromosome 1 interval. Inside 129 markers at positions 81.6 cM (D1Mit159) and 105 cM (D1Mit17), respectively, and an outside C57BL/6 marker at position 74.3cM (D1Mit159) were used to further define the interval. In the C57BL/6.Apcs−/− mice (backcrossed onto C57BL/6 for ten generations), the 129 genome around the Apcs locus was mapped as a segment from 87.9 cM (D1Mit15) to 105 cM (D1Mit17). In this analysis, the inside 129 markers were at positions 93 cM (D1Mit36) and 99.7 cM (D1Mit115) and the outside C57BL/6 markers at positions 81.6 cM (D1Mit159) and 106 cM (D1Mit223). Along with 28 C57BL/6.Apcs−/−mice and 30 C57BL/6 wild-type animals, 26 C57BL/6.129(D1Mit105–223) female mice −/−were followed up to 1 y of age. Animals were maintained in specific pathogen-free conditions. All animal procedures were in accordance with institutional guidelines. + +Serological analyses. + +Sera, collected at 6 and 12 mo of age, were assayed for the presence of auto-Abs. Levels of IgG ANA were sought by indirect immunofluorescence using Hep-2 cells, and anti-dsDNA Abs were detected by indirect immunofluorescence on Crithidia luciliae as previously described (Mitchell et al. 2002). Serum samples were screened at a 1:80 (ANA) or 1:20 (anti-dsDNA) dilution and the positive samples titrated to endpoint. Abs to ssDNA and anti-chromatin were measured by ELISA, as previously described (Mitchell et al. 2002). Samples were screened at a 1:100 dilution, and the results were expressed in arbitrary ELISA units (AEUs) relative to a standard positive sample (derived from an MRL/Mp.lpr/lpr mouse), which was assigned a value of 100. For interplate comparison, serial dilutions of a positive control serum sample were included on each plate. Apcs levels were assessed by ELISA using sheep anti-mouse Apcs and rabbit anti-mouse Apcs Abs (Calbiochem, Nottigham, United Kingdom). Samples were screened at a 1:3,000 dilution, and the results were expressed in milligrams per liters, referring to a standard curve derived from an acute-phase serum with a known concentration of Apcs (Calbiochem). Apcs−/− mouse serum was included as a negative control. + +Histological analysis. + +All the mice, except the few that died before the end of the experiment, were sacrificed at 1 y of age, and kidney portions were fixed in Bouin's solution and paraffin embedded, and sections were stained with periodic acid–Schiff reagent. Glomerular histology was graded in a blinded fashion as follows: grade 0, normal; grade 1, hypercellularity involving greater than 50% of the glomerular tuft in 25%–50% of glomeruli; grade 2, hypercellularity involving greater than 50% of the glomerular tuft in 50%–75% of glomeruli; grade 3, glomerular hypercellularity in greater than 75% of glomeruli or crescents in greater than 25% of glomeruli. + +Statistical analysis + +Non-parametric data are expressed as median with range of values in parentheses. All statistics were calculated using GraphPad PrismTM version 3.0 for Windows (GraphPad Software, San Diego, California, United States). Non-parametric tests were applied throughout, with differences being considered significant for p values < 0.05. The Mann–Whitney test was used for comparison of two groups, whilst for analysis of three groups the Kruskal–Wallis test with Dunn's multiple comparison test was used. + +Genotypic analysis + +Genotyping was carried out by PCR of genomic DNA using 143 polymorphic markers (list available on request) distributed throughout all 19 autosomes. PCRs were performed using standard reagents containing 1.5 mM MgCl2 and 0.4 μM primers. Microsatellite markers were screened for size polymorphisms between 129 and C57BL/6 mice. Only primers with differences detectable on ethidium bromide-stained agarose gels or on SDS-polyacrylamide gels were used. + +Linkage analysis + +The QTL program MAPMANAGER.QTL (ftp://mcbio.med.buffalo.edu/pub/MapMgr/) was used, and the two experimental groups were examined independently. Only data from mice at 12 mo of age were analysed. Log transformations of auto-Abs levels resulted in more normalised distribution and were used in QTL mapping. LOD thresholds for suggestive and significant linkages were determined by using a cohort- and trait-specific permutation test (1,000 permutations). The average threshold for suggestive, significant, and highly significant linkages were LOD ≥ 2.1 (p ≤ 7.8 × 10−3), LOD ≥ 3.6 (p ≤ 2.4 × 10−4), and LOD ≥ 5 (p ≤ 1 × 10−5), respectively (Manly and Olson 1999). + +Supporting Information + +Accession Numbers + +The LocusLink (http://www.ncbi.nlm.nih.gov/LocusLink/) ID numbers for the genes and gene products discussed in this paper are Apcs (LocusLink ID 20219), CD35/CD21 (LocusLink ID 12902), CD55 (LocusLink ID 13136), the FAS receptor gene (LocusLink ID 14102), and FcγRII (LocusLink ID 14130). + +Acknowledgements + +We are grateful to M. Lewis for the processing of the samples for histology and to F. Reid and D. Mitchell for their help. We thank all of the staff in the animal facility for their technical assistance. This work was supported by the Wellcome Trust (grant number 061438). JCH was a recipient of a fellowship from the National Institute of Health, Spain (BEFI 99/9212). + +Abbreviations + +Ab - antibody + +ANA - anti-nuclear antibody + +AEU - arbitrary ELISA unit + +Apcs−/− - Apcs-deficient mice + +Apcs - serum amyloid P component gene + +dsDNA - double-stranded DNA + +LOD - logarithm of odds + +QTL - quantitative trait linkage + +SLE - systemic lupus erythematosus + +ssDNA - anti-single-stranded DNA + +Figures and Tables + +Figure 1 + +Linkage of Chromosome 2 with ANA and Anti-Chromatin Abs in (129 × C57BL/6)F2.Apcs−/− Mice + +These associations were not detected in (129 × C57BL/6)F2 animals. Centimorgan positions were deduced by interval mapping, anchoring marker locations to data from http://www.informatics.jax.org. Dotted lines and the dashed line indicate the threshold over which linkages were considered suggestive or significant, respectively, as defined in Materials and Methods. + +Figure 3 + +Linkage of Chromosome 4 with Anti-dsDNA Abs + +The estimated peak in (129 × C57BL/6)F2 mice was at position 51.3 cM, whilst in the (129 × C57BL/6)F2.Apcs−/−animals it was was at position 71 cM, indicating that most likely these were two independent loci. Centimorgan positions were deduced by interval mapping, anchoring marker locations to data from http://www.informatics.jax.org. Dotted lines the indicate threshold over which linkage was considered suggestive, as defined in Materials and Methods. + +Figure 4 + +Interval Mapping Scans Showing QTL on Chromosome 1 with Anti-dsDNA and Anti-ssDNA Abs + +Centimorgan positions were deduced by interval mapping, anchoring marker locations to data from http://www.informatics.jax.org. Dotted lines indicate the threshold over which linkage was considered suggestive, dashed lines indicate the threshold over which linkage was considered significant, and dotted/dashed lines indicate highly significant linkage, as defined in Materials and Methods. See Table 3 for additional details. + +Figure 5 + +Interval Mapping Scans Showing QTL on Chromosome 1 with ANA and Anti-Chromatin Abs + +Centimorgan positions were deduced by interval mapping, anchoring marker locations to data from http://www.informatics.jax.org. Dotted lines indicate the threshold over which linkage was considered suggestive, and dashed lines indicate the threshold over which linkage was considered significant, as defined in Materials and Methods. See Table 3 for additional details. + +Figure 6 + +Auto-Ab Profiles + +(A) ANA titres in the (129 × C57BL/6)F2.Apcs−/− mice and (129 × C57BL/6)F2 at 1 y of age. A small circle represents one mouse; a large circle, a variable number of animals, as indicated in parentheses. Serum samples were titrated to endpoint. + +(B) ANA titres in the (129 × C57BL/6)F2.Apcs −/− mice and a selected number of wild-type (129 × C57BL/6)F2 animals carrying the Chromosome 1 region between D1Mit105 and D1Mit223 (80–106 cM) of 129 origin. The symbols are as in (A). + +(C and D) Anti-chromatin Ab levels expressed in AEUs related to a standard positive sample, which was assigned a value of 100 AEU. The comparison is between the same groups of mice as in (A) and (B), respectively. The symbols are as in (A). + +Figure 7 + +Interval Mapping Scans Showing QTL on Chromosome 3 with ANA, Anti-Chromatin, and Anti-ssDNA Abs + +See Table 3 for additional details. Centimorgan positions were deduced by interval mapping, anchoring marker locations to data from http://www.informatics.jax.org. Dotted lines indicate the threshold over which linkage was considered suggestive, the dashed line indicate the threshold over which linkage was considered significant, and dotted/dashed lines indicate highly significant linkage, as defined in Materials and Methods. + +Figure 8 + +Auto-Ab Profiles + +(A) ANA titres in C57BL/6 mice, C57BL/6.Apcs −/− mice, and C57BL/6.129(D1Mit105–223) congenic mice at 1 y of age. Small symbols represent one mouse; large symbols, a variable number of animals as indicated in parentheses. Serum sample were titrated to endpoint. + +(B and C) Anti-ssDNA (B) and anti-chromatin (C) Ab levels in the same cohorts of mice as in (A). The Ab levels are expressed in AEUs related to a standard positive sample, which was assigned a value of 100 AEU. + +(D) Anti-dsDNA Ab levels. Serum samples were screened at 1:20. Samples that were positive were titrated to endpoint. The symbols are as in (A). + +Figure 9 + +Renal Histological Assessment + +C57BL/6 mice, C57BL/6.Apcs−/− mice, and C57BL/6.129(D1Mit105–223) congenic mice were sacrificed at 1 y of age to obtain age-matched autopsy specimens. Bouin's fixed kidney sections were scored blinded for glomerulonephritis. Glomerulonephritis was graded on a 0–3 scale (see Materials and Methods for details). + +Figure 2 + +Linkage of Chromosome 8 with Anti-Chromatin and Anti-dsDNA Abs in (129 × C57BL/6)F2 Mice + +These linkages were not detected in (129 × C57BL/6)F2.Apcs−/− animals. Centimorgan positions were deduced by interval mapping, anchoring marker locations to data from http://www.informatics.jax.org. Dotted lines indicate the threshold over which linkage was considered suggestive, as defined in Materials and Methods. + +Table 1 + +Spontaneous Auto-Abs in Apcs−/− and Wild-Type (129 × C57BL/6)F2 Female Mice + +Significant differences between Apcs−/− and wild-type controls by Mann–Whitney U test. The numbers tested for different phenotypes are not equal due to the limited amount of serum or death of the mice before the end of the experiment. NS, not significant + +Table 2 + +Histological Assessment of Kidney Sections in Apcs−/− and Wild-Type (129 × C57BL/6)F2 Female Mice + +The renal sections were scored on a 0–3 scale on the intensity and extent of the histopathological changes as described in Materials and Methods. The difference between the two experimental groups was not significant + +Table 3 + +Summary of Genome-Wide Linkage Analysis in Apcs−/− and Wild-Type (129 × C57BL/6)F2 Female Mice + +a Suggestive linkage + +b Significant linkage + +c Highly significant linkage as defined in the material and method section + +Chr, Chromosome; Est. Peak, Estimated Peak; LOD, logarithm of odds; N/A, not applicable + +Chromosomes where linkages were not present in both experimental groups are not illustrated. See Materials and Methods for details. The oligonucleotide sequences and approximate positions of the microsatellite markers used were taken from the Mouse Genome Database, Mouse Genome Informatics, Jackson Laboratory, Bar Harbor, Maine, United States (http://www.informatics.jax.org) + +Footnotes + +Conflicts of interest. The authors have declared that no conflicts of interest exist. + +Author contributions. MJW, TJV, and MB conceived and designed the experiments. AEB, KLR, JW, JC-H, and RJR performed the experiments. AEB, KLR, RJR, and HTC analyzed the data. MB wrote the paper. + +Academic Editor: David Nemazee, Scripps Research Institute + +¤ Current address: The Wellcome Trust, London, United Kingdom + +Citation: Bygrave AE, Rose KL, Cortes-Hernandez J, Warren J, Rigby RJ, et al. (2004) Spontaneous autoimmunity in 129 and C57BL/6 mice—Implications for autoimmunity described in gene-targeted mice. PLoS Biol 2(8): e243. diff --git a/src/ontogpt/evaluation/craft/database/all/15320950.ann b/src/ontogpt/evaluation/craft/database/all/15320950.ann new file mode 100644 index 000000000..6738119a4 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15320950.ann @@ -0,0 +1,940 @@ +T1 PR:000001979 8 14 KCNJ10 +T2 GO:0010467 23 33 expression +T3 http://purl.obolibrary.org/obo/MONDO_0010134 90 106 Pendred syndrome +T4 NCBITaxon:10088 107 112 mouse +T5 http://purl.obolibrary.org/obo/MONDO_0010134 142 158 Pendred syndrome +T6 GO:0030849 169 178 autosomal +T7 http://purl.obolibrary.org/obo/MONDO_0006025 169 197 autosomal-recessive disorder +T8 http://purl.obolibrary.org/obo/MONDO_0021140 215 225 congenital +T9 http://purl.obolibrary.org/obo/MONDO_0005397 239 245 goiter +T10 PR:000015039 273 280 SLC26A4 +T11 PR:000015039 298 305 pendrin +T12 PR:000015039 348 355 pendrin +T13 NCBITaxon:10088 375 379 mice +T14 PR:000015039 391 398 Slc26a4 +T15 PR:000015039 422 429 Slc26a4 +T16 SO:0000704 430 434 gene +T17 PR:000015039 436 443 Slc26a4 +T18 GO:0010467 459 469 Expression +T19 PR:000015039 473 480 pendrin +T20 GO:0010467 548 558 Expression +T21 CHEBI:29103 655 657 K+ +T22 UBERON:0002282 750 755 stria +T23 CL:0002492 750 770 stria marginal cells +T24 UBERON:0000479 809 815 Tissue +T25 PR:000015039 935 942 Pendrin +T26 UBERON:0001844 960 967 cochlea +T27 GO:0016324 971 990;1009 1014 apical membranes of ... cells +T28 UBERON:0028194 991 1008 spiral prominence +T29 UBERON:0002282 1043 1059 stria vascularis +T30 UBERON:0008832 1064 1076 outer sulcus +T31 UBERON:0001852 1093 1102 Endolymph +T32 PR:000015039 1113 1120 Slc26a4 +T33 NCBITaxon:10088 1124 1128 mice +T34 UBERON:0000479 1147 1153 tissue +T35 CL:0002670 1191 1197;1205 1215 type I ... fibrocytes +T36 CL:0002666 1191 1195;1202 1215 type ... II fibrocytes +T37 PR:000015039 1230 1237 Slc26a4 +T38 NCBITaxon:10088 1241 1245 mice +T39 CL:0000646 1311 1321 basal cell +T40 CHEBI:29103 1337 1339 K+ +T41 PR:000001979 1348 1354 KCNJ10 +T42 CL:0002486 1368 1386 intermediate cells +T43 UBERON:0002282 1388 1404 Stria vascularis +T44 CHEBI:26130 1414 1423 pigmented +T45 CHEBI:26519 1449 1461 free radical +T46 CL:0000646 1474 1484 basal cell +T47 CL:0002486 1510 1528 intermediate cells +T48 PR:000001979 1533 1539 KCNJ10 +T49 PR:000001979 1562 1568 KCNJ10 +T50 UBERON:0001852 1589 1602 Endolymphatic +T51 CHEBI:29103 1603 1605 K+ +T52 GO:0016020 1637 1645 membrane +T53 CHEBI:29103 1669 1671 K+ +T54 CHEBI:29103 1710 1712 K+ +T55 PR:000000728 1721 1726 KCNQ1 +T56 PR:000009255 1731 1736 KCNE1 +T57 CHEBI:29101 1738 1741 Na+ +T58 CHEBI:29103 1747 1749 K+ +T59 PR:000014925 1764 1771 SLC12A2 +T60 GO:0005921 1780 1792 gap junction +T61 PR:000007998 1793 1797 GJB2 +T62 PR:000015039 1849 1856 pendrin +T63 PR:000001979 1888 1894 KCNJ10 +T64 GO:0010467 1903 1913 expression +T65 http://purl.obolibrary.org/obo/MONDO_0010134 2001 2017 Pendred syndrome +T66 http://purl.obolibrary.org/obo/MONDO_0010134 2032 2048 Pendred syndrome +T67 GO:0030849 2072 2081 autosomal +T68 http://purl.obolibrary.org/obo/MONDO_0006025 2072 2100 autosomal-recessive disorder +T69 http://purl.obolibrary.org/obo/MONDO_0005397 2131 2137 goiter +T70 http://purl.obolibrary.org/obo/MONDO_0002254 2147 2155 syndrome +T71 SO:0000704 2190 2194 gene +T72 PR:000015039 2195 2202 SLC26A4 +T73 PR:000015039 2232 2239 pendrin +T74 http://purl.obolibrary.org/obo/MONDO_0021140 2257 2267 congenital +T75 UBERON:0000033 2346 2350 head +T76 http://purl.obolibrary.org/obo/MONDO_0005397 2395 2401 Goiter +T77 http://purl.obolibrary.org/obo/MONDO_0005397 2470 2476 goiter +T78 CHEBI:16382 2508 2514 iodide +T79 UBERON:0012246 2531 2547 follicular lumen +T80 CHEBI:16382 2573 2579 iodide +T81 GO:1904200 2573 2596;2608 2616 iodide transport across ... membrane +T82 GO:0016324 2601 2619;2645 2650 apical membrane of ... cells +T83 UBERON:0002046 2620 2633 thyroid gland +T84 CL:0002257 2620 2650 thyroid gland epithelial cells +T85 UBERON:0000483 2634 2644 epithelial +T86 CHEBI:49706 2667 2678 perchlorate +T87 UBERON:0002279 2710 2729 vestibular aqueduct +T88 http://purl.obolibrary.org/obo/MONDO_0010134 2779 2795 Pendred syndrome +T89 PR:000015039 2802 2809 Pendrin +T90 CHEBI:22563 2816 2821 anion +T91 CHEBI:17996 2851 2854 Cl- +T92 CHEBI:16382 2856 2858 I- +T93 CHEBI:17544 2860 2866 HCO3 - +T94 CHEBI:15740 2871 2878 formate +T95 GO:0010467 2886 2896 Expression +T96 UBERON:0001846 2919 2928 inner ear +T97 UBERON:0002046 2933 2946 thyroid gland +T98 http://purl.obolibrary.org/obo/MONDO_0005397 2998 3004 goiter +T99 PR:000015039 3027 3034 pendrin +T100 GO:0010467 3035 3045 expression +T101 UBERON:0002113 3068 3074 kidney +T102 UBERON:0001911 3080 3093 mammary gland +T103 UBERON:0000995 3100 3106 uterus +T104 UBERON:0000473 3113 3119 testes +T105 UBERON:0001987 3129 3137 placenta +T106 GO:0010467 3147 3157 expression +T107 UBERON:0007023 3180 3185 adult +T108 UBERON:0000955 3186 3191 brain +T109 GO:0010467 3249 3259 Expression +T110 PR:000015039 3263 3270 pendrin +T111 UBERON:0001846 3283 3292 inner ear +T112 UBERON:0001844 3340 3347 cochlea +T113 UBERON:0001862 3353 3373 vestibular labyrinth +T114 UBERON:0002223 3382 3399 endolymphatic sac +T115 PR:000015039 3429 3436 pendrin +T116 GO:0010467 3445 3455 expression +T117 http://purl.obolibrary.org/obo/MONDO_0010134 3526 3542 Pendred syndrome +T118 PR:000015039 3617 3624 pendrin +T119 PR:000015039 3701 3708 pendrin +T120 GO:0010467 3743 3753 expression +T121 GO:0007605 3818 3825 hearing +T122 http://purl.obolibrary.org/obo/MONDO_0010134 3906 3922 Pendred syndrome +T123 PR:000015039 3980 3987 pendrin +T124 GO:0042571 4008 4016 antibody +T125 PR:000015039 4044 4051 Slc26a4 +T126 NCBITaxon:10088 4055 4059 mice +T127 NCBITaxon:10088 4101 4106 mouse +T128 PR:000015039 4107 4114 Slc26a4 +T129 SO:0000704 4115 4119 gene +T130 PR:000015039 4188 4195 pendrin +T131 PR:000015039 4225 4232 Slc26a4 +T132 NCBITaxon:10088 4236 4240 mice +T133 UBERON:0001852 4287 4300 endolymphatic +T134 UBERON:0001845 4305 4318 perilymphatic +T135 CHEBI:29103 4319 4321 K+ +T136 UBERON:0007023 4360 4365 adult +T137 NCBITaxon:10088 4366 4370 mice +T138 PR:000015039 4408 4415 Slc26a4 +T139 PR:000015039 4429 4436 Slc26a4 +T140 SO:0000704 4454 4458 gene +T141 PR:000015039 4463 4470 pendrin +T142 NCBITaxon:10088 4481 4486 mouse +T143 PR:000015039 4557 4564 Slc26a4 +T144 PR:000015039 4584 4591 Slc26a4 +T145 NCBITaxon:10088 4595 4599 mice +T146 CL:0000034 4657 4666 stem cell +T147 PR:000015039 4695 4702 Slc26a4 +T148 PR:000015039 4710 4717 Slc26a4 +T149 UBERON:0010166 4757 4761 coat +T150 PR:000015039 4840 4847 Slc26a4 +T151 PR:000015039 4855 4862 Slc26a4 +T152 GO:0010467 4895 4905 Expression +T153 GO:0006813 4987 5002 transport of K+ +T154 CHEBI:29103 5000 5002 K+ +T155 CHEBI:29103 5075 5077 K+ +T156 NCBITaxon:33208 5241 5247 Animal +T157 NCBITaxon:33208 5330 5337 Animals +T158 CHEBI:7984 5368 5388 sodium pentobarbital +T159 UBERON:0000948 5415 5424 cardially +T160 CHEBI:17996 5441 5444 Cl- +T161 CHEBI:84997 5469 5481 Na-gluconate +T162 CHEBI:75958 5482 5490 solution +T163 UBERON:0001678 5523 5537 Temporal bones +T164 UBERON:0001844 5555 5563 cochleae +T165 UBERON:0001845 5573 5586 perilymphatic +T166 CHEBI:17992 5639 5646 sucrose +T167 CHEBI:46793 5677 5696 polyethylene glycol +T168 UBERON:0006723 5702 5710 modiolar +T169 CHEBI:9750 5797 5805 Triton-X +T170 CHEBI:9750 5811 5813 TX +T171 NCBITaxon:27592 5823 5829 bovine +T172 UBERON:0001977 5830 5835 serum +T173 PR:000003918 5830 5843 serum albumin +T174 GO:0042571 5897 5907 antibodies +T175 CHEBI:9750 5915 5917 TX +T176 NCBITaxon:9986 5933 5939 rabbit +T177 PR:000015039 5945 5952 pendrin +T178 NCBITaxon:10114 5972 5975 rat +T179 PR:000016364 5981 5985 ZO-1 +T180 NCBITaxon:9925 6019 6023 goat +T181 PR:000000728 6029 6034 KCNQ1 +T182 NCBITaxon:9986 6091 6097 rabbit +T183 PR:000009255 6103 6108 KCNE1 +T184 NCBITaxon:9986 6146 6152 rabbit +T185 PR:000001979 6158 6164 KCNJ10 +T186 NCBITaxon:9986 6183 6189 rabbit +T187 PR:000014925 6195 6202 SLC12A2 +T188 NCBITaxon:9986 6226 6232 rabbit +T189 PR:000007998 6238 6249 connexin 26 +T190 CHEBI:9750 6312 6314 TX +T191 GO:0042571 6372 6382 antibodies +T192 CHEBI:9750 6411 6413 TX +T193 NCBITaxon:9793 6429 6435 donkey +T194 NCBITaxon:9986 6441 6447 rabbit +T195 CHEBI:52661 6448 6457 Alexa 488 +T196 NCBITaxon:9031 6459 6466 chicken +T197 NCBITaxon:10114 6472 6475 rat +T198 CHEBI:51248 6476 6485 Alexa 594 +T199 NCBITaxon:9031 6491 6498 chicken +T200 NCBITaxon:9925 6504 6508 goat +T201 CHEBI:51248 6509 6518 Alexa 594 +T202 GO:0005884 6552 6567 Actin filaments +T203 CHEBI:52661 6601 6610 Alexa 488 +T204 MOP:0000779 6611 6621 conjugated +T205 CHEBI:8040 6622 6632 phalloidin +T206 CHEBI:9750 6699 6701 TX +T207 NCBITaxon:33208 7028 7035 Animals +T208 CHEBI:7984 7066 7086 sodium pentobarbital +T209 UBERON:0002113 7105 7112 Kidneys +T210 UBERON:0000955 7117 7122 brain +T211 CHEBI:17997 7161 7163 N2 +T212 UBERON:0001678 7165 7179 Temporal bones +T213 UBERON:0001846 7217 7226 inner ear +T214 UBERON:0000479 7227 7234 tissues +T215 UBERON:0002282 7273 7289 stria vascularis +T216 UBERON:0006725 7298 7313 spiral ligament +T217 UBERON:0000395 7323 7337 spiral ganglia +T218 UBERON:0002227 7377 7391 organ of Corti +T219 UBERON:0000479 7473 7480 tissues +T220 PR:000015039 7542 7549 Slc26a4 +T221 UBERON:0002280 7602 7610 otoconia +T222 UBERON:0002214 7618 7634 utricular macula +T223 SO:0000407 7792 7800 18S rRNA +T224 UBERON:0002113 7815 7822 kidneys +T225 UBERON:0000955 7827 7832 brain +T226 UBERON:0002282 7960 7976 stria vascularis +T227 UBERON:0000395 7981 7995 spiral ganglia +T228 NCBITaxon:33208 8104 8111 animals +T229 CHEBI:51461 8199 8211 SYBR green I +T230 SO:0000673 8232 8243 Transcripts +T231 SO:0000407 8247 8255 18S rRNA +T232 CHEBI:29103 8273 8275 K+ +T233 PR:000001979 8285 8291 KCNJ10 +T234 PR:000000728 8293 8298 KCNQ1 +T235 PR:000000731 8303 8308 KCNQ4 +T236 SO:0000704 8330 8334 gene +T237 SO:0000112 8344 8351 primers +T238 GO:0032508 8565 8572 melting +T239 SO:0000112 8676 8682 primer +T240 GO:0032508 8699 8706 melting +T241 GO:0032508 8768 8772 melt +T242 GO:0032508 8892 8896 melt +T243 CHEBI:2511 8909 8916 agarose +T244 SO:0000112 8994 9001 Primers +T245 CHEBI:36357 9012 9021 molecules +T246 CHEBI:36357 9104 9113 molecules +T247 CHEBI:36357 9200 9209 molecules +T248 CHEBI:36357 9477 9486 molecules +T249 SO:0000407 9558 9566 18S rRNA +T250 SO:0000407 9616 9624 18S rRNA +T251 UBERON:0000479 9646 9652 tissue +T252 SO:0001026 9662 9669 Genomic +T253 UBERON:0001846 9687 9696 inner ear +T254 NCBITaxon:33208 9788 9795 Animals +T255 CHEBI:7984 9826 9846 sodium pentobarbital +T256 UBERON:0002282 9865 9881 Stria vascularis +T257 UBERON:0006725 9890 9905 spiral ligament +T258 UBERON:0002282 9965 9970 stria +T259 UBERON:0000483 9980 9990 epithelium +T260 UBERON:0000483 10114 10124 epithelium +T261 NCBITaxon:33208 10538 10545 Animals +T262 CHEBI:26714 10595 10606 sodium salt +T263 UBERON:0001852 10658 10671 endolymphatic +T264 CHEBI:29103 10673 10675 K+ +T265 UBERON:0002226 10814 10830 basilar membrane +T266 UBERON:0002282 10887 10903 stria vascularis +T267 CHEBI:29103 10939 10941 K+ +T268 CHEBI:75958 10982 10991 solutions +T269 CHEBI:36916 11004 11010 cation +T270 CHEBI:29103 11012 11014 K+ +T271 CHEBI:29101 11019 11022 Na+ +T272 CHEBI:29103 11078 11080 K+ +T273 CHEBI:29101 11096 11099 Na+ +T274 CHEBI:29103 11288 11290 K+ +T275 CHEBI:29101 11299 11302 Na+ +T276 CHEBI:29101 11369 11372 Na+ +T277 CHEBI:29101 11377 11380 Na+ +T278 UBERON:0001844 11475 11482 cochlea +T279 UBERON:0001969 11484 11490 Plasma +T280 CHEBI:29103 11491 11493 K+ +T281 UBERON:0000178 11531 11536 blood +T282 GO:0097617 11754 11767 hybridization +T283 UBERON:0001844 11775 11782 cochlea +T284 PR:000015039 11802 11809 pendrin +T285 GO:0010467 11818 11827 expressed +T286 UBERON:0028194 11873 11890 spiral prominence +T287 UBERON:0000060 11906 11910 wall +T288 UBERON:0008832 11918 11933 external sulcus +T289 PR:000015039 11968 11975 pendrin +T290 GO:0010467 11984 11994 expression +T291 UBERON:0001678 12068 12082 temporal bones +T292 PR:000015039 12094 12101 Slc26a4 +T293 NCBITaxon:10088 12106 12110 mice +T294 GO:0042571 12132 12140 antibody +T295 GO:0042571 12183 12191 antibody +T296 CHEBI:59132 12218 12227 antigenic +T297 GO:0010467 12261 12271 expression +T298 PR:000015039 12275 12282 pendrin +T299 UBERON:0008832 12308 12320 outer sulcus +T300 UBERON:0000483 12321 12331 epithelial +T301 CL:0000066 12321 12337 epithelial cells +T302 GO:0097617 12365 12378 hybridization +T303 GO:0016324 12412 12431;12469 12474 apical membranes of ... cells +T304 UBERON:0028194 12432 12449 spiral prominence +T305 UBERON:0000483 12458 12468 epithelial +T306 CL:0000066 12458 12474 epithelial cells +T307 GO:0016324 12482 12501;12517 12522 apical membranes of ... cells +T308 UBERON:0002282 12540 12556 stria vascularis +T309 PR:000015039 12590 12597 pendrin +T310 GO:0046903 12648 12655 secrete +T311 CHEBI:17544 12656 12662 HCO3 - +T312 UBERON:0001852 12668 12677 endolymph +T313 PR:000015039 12679 12686 Pendrin +T314 CHEBI:17544 12696 12702 HCO3 - +T315 GO:0015701 12696 12712 HCO3 - transport +T316 UBERON:0002113 12746 12752 kidney +T317 CHEBI:17544 12758 12764 HCO3 - +T318 UBERON:0001844 12772 12779 cochlea +T319 CHEBI:16526 12802 12805 CO2 +T320 UBERON:0002282 12839 12845 strial +T321 CL:0002486 12839 12864 strial intermediate cells +T322 CHEBI:16526 12874 12877 CO2 +T323 GO:0008152 12901 12914 metabolically +T324 UBERON:0002282 12929 12934 stria +T325 CL:0002492 12929 12949 stria marginal cells +T326 PR:000015039 12974 12981 pendrin +T327 CHEBI:17544 13005 13011 HCO3 - +T328 CHEBI:17544 13054 13060 HCO3 - +T329 UBERON:0002282 13064 13080 stria vascularis +T330 PR:000015039 13150 13157 pendrin +T331 CHEBI:17544 13161 13167 HCO3 - +T332 UBERON:0001852 13183 13192 endolymph +T333 PR:000015039 13229 13236 pendrin +T334 PR:000000728 13238 13243 KCNQ1 +T335 PR:000016364 13245 13249 ZO-1 +T336 GO:0031941 13254 13261 F actin +T337 UBERON:0001844 13265 13272 cochlea +T338 UBERON:0001862 13277 13297 vestibular labyrinth +T339 PR:000015039 13301 13308 Slc26a4 +T340 PR:000015039 13316 13323 Slc26a4 +T341 NCBITaxon:10088 13327 13331 mice +T342 UBERON:0001844 13348 13355 cochlea +T343 UBERON:0006106 13386 13394;13403 13407 cochlear ... wall +T344 UBERON:0001853 13435 13442 utricle +T345 UBERON:0004043 13472 13479 ampulla +T346 UBERON:0002281 13494 13496 RM +T347 UBERON:0002281 13498 13517 Reissner's membrane +T348 CL:0002492 13545 13548 SMC +T349 UBERON:0002282 13550 13556 strial +T350 CL:0002492 13550 13571 strial marginal cells +T351 UBERON:0002282 13573 13575 SV +T352 UBERON:0002282 13577 13593 stria vascularis +T353 UBERON:0006725 13595 13597 SL +T354 UBERON:0006725 13599 13614 spiral ligament +T355 CL:0000646 13636 13638 BC +T356 CL:0000646 13640 13651 basal cells +T357 UBERON:0028194 13653 13655 SP +T358 UBERON:0028194 13657 13674 spiral prominence +T359 UBERON:0000483 13675 13685 epithelial +T360 CL:0000066 13675 13691 epithelial cells +T361 UBERON:0008832 13713 13725 outer sulcus +T362 UBERON:0000483 13726 13736 epithelial +T363 CL:0000066 13726 13742 epithelial cells +T364 CL:0000609 13744 13747 VHC +T365 CL:0000609 13749 13770 vestibular hair cells +T366 CL:0000846 13808 13811 VDC +T367 CL:0000846 13813 13834 vestibular dark cells +T368 CL:0000646 13844 13855 basal cells +T369 UBERON:0002282 13881 13897 stria vascularis +T370 UBERON:0000483 13932 13942 epithelial +T371 CL:0000066 13932 13948 epithelial cells +T372 PR:000015039 13965 13972 pendrin +T373 GO:0010467 13973 13983 expression +T374 UBERON:0028194 13987 14004 spiral prominence +T375 UBERON:0002282 14015 14031 stria vascularis +T376 PR:000000728 14059 14064 KCNQ1 +T377 CHEBI:29103 14068 14070 K+ +T378 GO:0010467 14087 14096 expressed +T379 UBERON:0002282 14100 14106 strial +T380 CL:0002492 14100 14121 strial marginal cells +T381 PR:000016364 14127 14131 ZO-1 +T382 CL:0000646 14170 14181 basal cells +T383 UBERON:0002282 14223 14239 stria vascularis +T384 PR:000015039 14285 14292 pendrin +T385 GO:0010467 14296 14305 expressed +T386 UBERON:0000483 14349 14359 epithelial +T387 CL:0000066 14349 14365 epithelial cells +T388 UBERON:0002282 14369 14385 stria vascularis +T389 UBERON:0002282 14398 14404 strial +T390 CL:0002492 14398 14419 strial marginal cells +T391 UBERON:0028194 14449 14466 spiral prominence +T392 UBERON:0002281 14471 14490 Reissner's membrane +T393 GO:0097617 14511 14524 hybridization +T394 UBERON:0001862 14532 14552 vestibular labyrinth +T395 PR:000015039 14568 14575 pendrin +T396 GO:0010467 14584 14593 expressed +T397 GO:0010467 14691 14701 expression +T398 PR:000015039 14709 14716 pendrin +T399 GO:0016324 14732 14750;14775 14780 apical membrane of ... cells +T400 UBERON:0001853 14788 14795 utricle +T401 UBERON:0004043 14800 14808 ampullae +T402 PR:000000728 14845 14850 KCNQ1 +T403 PR:000015039 14869 14876 pendrin +T404 GO:0010467 14877 14887 expression +T405 CL:0000846 14995 15016 vestibular dark cells +T406 GO:0010467 15055 15065 expression +T407 PR:000000728 15069 15074 KCNQ1 +T408 PR:000009255 15079 15084 KCNE1 +T409 GO:0016324 15094 15110 apical membranes +T410 PR:000015039 15126 15133 pendrin +T411 GO:0010467 15134 15144 expression +T412 GO:0048839 15152 15166;15177 15186 development of ... inner ear +T413 NCBITaxon:10088 15171 15176 mouse +T414 UBERON:0001846 15177 15186 inner ear +T415 UBERON:0000922 15213 15222 embryonic +T416 UBERON:0001846 15287 15297 inner ears +T417 PR:000015039 15301 15308 Slc26a4 +T418 PR:000015039 15316 15323 Slc26a4 +T419 NCBITaxon:10088 15327 15331 mice +T420 PR:000015039 15371 15378 Slc26a4 +T421 NCBITaxon:10088 15382 15386 mice +T422 UBERON:0011078 15416 15435 endolymphatic space +T423 UBERON:0000113 15455 15464 adulthood +T424 CL:0000855 15486 15504 sensory hair cells +T425 UBERON:0001844 15512 15519 cochlea +T426 GO:0007567 15544 15549 natal +T427 UBERON:0001844 15652 15660 cochlear +T428 CL:0000855 15698 15716 sensory hair cells +T429 UBERON:0001860 15752 15770 endolymphatic duct +T430 UBERON:0001852 15781 15794 endolymphatic +T431 CHEBI:29103 15795 15797 K+ +T432 CL:0000855 15835 15844 hair cell +T433 PR:000015039 15907 15914 Slc26a4 +T434 GO:0007605 16044 16051 hearing +T435 UBERON:0001852 16145 16158 endolymphatic +T436 CHEBI:29103 16159 16161 K+ +T437 PR:000015039 16206 16213 Slc26a4 +T438 NCBITaxon:10088 16217 16221 mice +T439 UBERON:0001852 16301 16314 endolymphatic +T440 CHEBI:29103 16315 16317 K+ +T441 UBERON:0007023 16346 16351 adult +T442 PR:000015039 16352 16359 Slc26a4 +T443 PR:000015039 16409 16416 Slc26a4 +T444 PR:000015039 16424 16431 Slc26a4 +T445 NCBITaxon:10088 16435 16439 mice +T446 UBERON:0001845 16454 16467 perilymphatic +T447 UBERON:0001969 16481 16487 plasma +T448 CHEBI:29103 16488 16490 K+ +T449 PR:000015039 16507 16514 Slc26a4 +T450 PR:000015039 16540 16547 Slc26a4 +T451 PR:000015039 16645 16652 Slc26a4 +T452 NCBITaxon:10088 16656 16660 mice +T453 http://purl.obolibrary.org/obo/MONDO_0010134 16705 16721 Pendred syndrome +T454 CL:0000855 16782 16792 hair cells +T455 CHEBI:29103 16881 16883 K+ +T456 UBERON:0002282 16919 16935 stria vascularis +T457 PR:000015039 16939 16946 Slc26a4 +T458 PR:000015039 16954 16961 Slc26a4 +T459 NCBITaxon:10088 16965 16969 mice +T460 CHEBI:29103 17001 17003 K+ +T461 UBERON:0001852 17022 17031 endolymph +T462 UBERON:0001845 17036 17045 perilymph +T463 UBERON:0002819 17053 17057;17075 17077;17082 17089 apex ... of ... cochlea +T464 UBERON:0002819 17059 17060;17075 17077;17082 17089 A ... of ... cochlea +T465 UBERON:0014626 17066 17070;17075 17077;17082 17089 base ... of ... cochlea +T466 UBERON:0014626 17072 17073;17075 17077;17082 17089 B ... of ... cochlea +T467 UBERON:0002282 17171 17187 stria vascularis +T468 PR:000015039 17191 17198 Slc26a4 +T469 PR:000015039 17206 17213 Slc26a4 +T470 NCBITaxon:10088 17217 17221 mice +T471 UBERON:0002282 17234 17250 stria vascularis +T472 UBERON:0005411 17263 17278;17283 17290 bony capsule of ... cochlea +T473 UBERON:0002501 17292 17294 OW +T474 UBERON:0002501 17296 17307 oval window +T475 UBERON:0002502 17309 17311 RW +T476 UBERON:0002502 17313 17325 round window +T477 UBERON:0002282 17335 17351 stria vascularis +T478 UBERON:0002282 17374 17390 stria vascularis +T479 NCBITaxon:10088 17417 17421 mice +T480 UBERON:0002282 17566 17582 stria vascularis +T481 UBERON:0006106 17598 17605;17610 17617 wall of ... cochlea +T482 CL:0000646 17665 17675 basal cell +T483 UBERON:0002282 17687 17703 stria vascularis +T484 CHEBI:29103 17711 17713 K+ +T485 PR:000001979 17722 17728 KCNJ10 +T486 CL:0002486 17740 17758 intermediate cells +T487 CL:0000646 17788 17799 basal cells +T488 GO:0005921 17821 17834 gap junctions +T489 CL:0002492 17841 17855 Marginal cells +T490 UBERON:0002282 17859 17875 stria vascularis +T491 UBERON:0001852 17907 17916 endolymph +T492 CHEBI:29103 17928 17930 K+ +T493 UBERON:0002282 17945 17951 strial +T494 UBERON:0001852 17963 17972 endolymph +T495 CHEBI:29103 17986 17988 K+ +T496 PR:000001979 18023 18029 KCNJ10 +T497 CHEBI:29103 18030 18032 K+ +T498 PR:000015039 18115 18122 Slc26a4 +T499 NCBITaxon:10088 18126 18130 mice +T500 CL:0002486 18160 18178 intermediate cells +T501 UBERON:0002282 18194 18210 stria vascularis +T502 CL:0002486 18228 18246 intermediate cells +T503 CL:0002486 18313 18331 Intermediate cells +T504 UBERON:0002282 18335 18351 stria vascularis +T505 UBERON:0002282 18419 18435 stria vascularis +T506 PR:000015039 18439 18446 Slc26a4 +T507 NCBITaxon:10088 18450 18454 mice +T508 CL:0002486 18486 18504 intermediate cells +T509 UBERON:0002282 18549 18565 stria vascularis +T510 PR:000015039 18587 18594 Slc26a4 +T511 PR:000015039 18606 18613 Slc26a4 +T512 NCBITaxon:10088 18617 18621 mice +T513 PR:000015039 18710 18717 Slc26a4 +T514 NCBITaxon:10088 18721 18725 mice +T515 UBERON:0002282 18754 18770 stria vascularis +T516 UBERON:0000395 18775 18789 spiral ganglia +T517 PR:000015039 18811 18818 Slc26a4 +T518 PR:000015039 18857 18864 Slc26a4 +T519 NCBITaxon:10088 18868 18872 mice +T520 UBERON:0000479 18903 18910 tissues +T521 SO:0000407 18932 18940 18S rRNA +T522 GO:0010467 18961 18971 expression +T523 PR:000001979 18975 18981 KCNJ10 +T524 UBERON:0002282 19002 19018 stria vascularis +T525 NCBITaxon:10088 19049 19053 mice +T526 SO:0000407 19130 19138 18S rRNA +T527 CHEBI:36357 19139 19148 molecules +T528 UBERON:0000395 19224 19238 spiral ganglia +T529 PR:000015039 19267 19274 Slc26a4 +T530 NCBITaxon:10088 19278 19282 mice +T531 PR:000015039 19374 19381 Slc26a4 +T532 NCBITaxon:10088 19385 19389 mice +T533 GO:0010467 19471 19481 Expression +T534 PR:000001979 19485 19491 KCNJ10 +T535 UBERON:0002282 19511 19527 stria vascularis +T536 UBERON:0000395 19532 19546 spiral ganglia +T537 PR:000015039 19556 19563 Slc26a4 +T538 NCBITaxon:10088 19567 19571 mice +T539 PR:000015039 19605 19612 Slc26a4 +T540 NCBITaxon:10088 19616 19620 mice +T541 PR:000000728 19650 19655 KCNQ1 +T542 PR:000000731 19660 19665 KCNQ4 +T543 UBERON:0002282 19732 19748 stria vascularis +T544 UBERON:0000395 19753 19767 spiral ganglia +T545 PR:000000728 19815 19820 KCNQ1 +T546 GO:0010467 19824 19833 expressed +T547 UBERON:0002282 19850 19866 stria vascularis +T548 UBERON:0000395 19879 19893 spiral ganglia +T549 PR:000000731 19914 19919 KCNQ4 +T550 GO:0010467 19923 19932 expressed +T551 UBERON:0000395 19949 19963 spiral ganglia +T552 UBERON:0002282 19976 19992 stria vascularis +T553 PR:000000728 20020 20025 KCNQ1 +T554 CHEBI:36357 20031 20040 molecules +T555 UBERON:0002282 20044 20060 stria vascularis +T556 UBERON:0000395 20089 20103 spiral ganglia +T557 PR:000000731 20123 20128 KCNQ4 +T558 CHEBI:36357 20134 20143 molecules +T559 UBERON:0000395 20147 20161 spiral ganglia +T560 UBERON:0002282 20189 20205 stria vascularis +T561 PR:000001979 20255 20261 KCNJ10 +T562 PR:000000728 20266 20271 KCNQ1 +T563 UBERON:0002282 20329 20345 stria vascularis +T564 UBERON:0000395 20350 20364 spiral ganglia +T565 PR:000001979 20431 20437 KCNJ10 +T566 PR:000000728 20442 20447 KCNQ1 +T567 GO:0010467 20453 20463 expression +T568 UBERON:0002282 20467 20483 stria vascularis +T569 UBERON:0000395 20488 20502 spiral ganglia +T570 PR:000015039 20506 20513 Slc26a4 +T571 PR:000015039 20521 20528 Slc26a4 +T572 NCBITaxon:10088 20532 20536 mice +T573 UBERON:0002282 20585 20601 stria vascularis +T574 NCBITaxon:10088 20626 20631 mouse +T575 SO:0000407 20721 20729 18S rRNA +T576 SO:0000407 20768 20771 18S +T577 SO:0000407 20803 20806;20815 20819 18S ... rRNA +T578 SO:0000653 20811 20819 28S rRNA +T579 PR:000015039 20877 20884 Slc26a4 +T580 NCBITaxon:10088 20888 20892 mice +T581 UBERON:0002280 20962 20970 otoconia +T582 UBERON:0002214 20978 20994 utricular macula +T583 UBERON:0004721 21004 21005 A +T584 UBERON:0004721 21007 21024 crista ampullaris +T585 UBERON:0002214 21026 21027 U +T586 UBERON:0002214 21029 21045 utricular macula +T587 PR:000001979 21134 21140 KCNJ10 +T588 PR:000000728 21142 21147 KCNQ1 +T589 PR:000000731 21152 21157 KCNQ4 +T590 SO:0000407 21179 21187 18S rRNA +T591 UBERON:0002282 21226 21228 SV +T592 UBERON:0002282 21230 21246 stria vascularis +T593 UBERON:0000395 21248 21250 SG +T594 UBERON:0000395 21252 21266 spiral ganglia +T595 PR:000001979 21289 21295 KCNJ10 +T596 PR:000000728 21300 21305 KCNQ1 +T597 PR:000015039 21320 21327 Slc26a4 +T598 PR:000015039 21349 21356 Slc26a4 +T599 NCBITaxon:10088 21360 21364 mice +T600 PR:000001979 21383 21389 KCNJ10 +T601 UBERON:0002282 21398 21414 stria vascularis +T602 PR:000015039 21418 21425 Slc26a4 +T603 NCBITaxon:10088 21429 21433 mice +T604 CL:0002486 21460 21478 intermediate cells +T605 GO:0010467 21492 21502 Expression +T606 PR:000001979 21510 21516 KCNJ10 +T607 PR:000015039 21547 21554 Slc26a4 +T608 NCBITaxon:10088 21558 21562 mice +T609 GO:0010467 21611 21621 expression +T610 PR:000001979 21629 21635 KCNJ10 +T611 UBERON:0002282 21658 21674 stria vascularis +T612 UBERON:0000395 21689 21703 spiral ganglia +T613 PR:000001979 21733 21739 KCNJ10 +T614 CHEBI:29103 21740 21742 K+ +T615 UBERON:0002282 21754 21770 stria vascularis +T616 PR:000001979 21876 21882 KCNJ10 +T617 UBERON:0001844 21890 21897 cochlea +T618 PR:000015039 21901 21908 Slc26a4 +T619 PR:000015039 21916 21923 Slc26a4 +T620 NCBITaxon:10088 21927 21931 mice +T621 UBERON:0001844 21948 21955 cochlea +T622 UBERON:0002295 22011 22022 scala media +T623 UBERON:0002281 22041 22060 Reissner's membrane +T624 UBERON:0000060 22085 22089 wall +T625 UBERON:0000395 22094 22108 spiral ganglia +T626 GO:0010467 22150 22160 Expression +T627 PR:000001979 22164 22170 KCNJ10 +T628 PR:000015039 22174 22181 Slc26a4 +T629 NCBITaxon:10088 22185 22189 mice +T630 UBERON:0002282 22204 22220 stria vascularis +T631 UBERON:0000395 22238 22253 spiral ganglion +T632 UBERON:0002281 22261 22263 RM +T633 UBERON:0002281 22265 22284 Reissner's membrane +T634 UBERON:0002282 22286 22288 SV +T635 UBERON:0002282 22290 22306 stria vascularis +T636 UBERON:0028194 22308 22310 SP +T637 UBERON:0028194 22312 22329 spiral prominence +T638 UBERON:0006725 22331 22333 SL +T639 UBERON:0006725 22335 22350 spiral ligament +T640 UBERON:0000395 22372 22374 SG +T641 UBERON:0000395 22376 22391 spiral ganglion +T642 UBERON:0002295 22461 22472 scala media +T643 UBERON:0002281 22497 22516 Reissner's membrane +T644 UBERON:0002227 22553 22567 organ of Corti +T645 UBERON:0000479 22596 22602 Tissue +T646 UBERON:0002282 22613 22629 stria vascularis +T647 SO:0000407 22690 22698 18S rRNA +T648 CHEBI:36357 22699 22708 molecules +T649 UBERON:0000479 22774 22780 tissue +T650 CL:0002486 22823 22841 intermediate cells +T651 UBERON:0000479 22889 22895 tissue +T652 CL:0002670 22952 22958;22966 22976 type I ... fibrocytes +T653 CL:0002666 22952 22956;22963 22976 type ... II fibrocytes +T654 UBERON:0028194 22978 22995 Spiral prominence +T655 PR:000015039 22999 23006 Slc26a4 +T656 NCBITaxon:10088 23010 23014 mice +T657 UBERON:0006725 23035 23050 spiral ligament +T658 UBERON:0000479 23116 23122 tissue +T659 SO:0000407 23222 23230 18S rRNA +T660 CHEBI:36357 23231 23240 molecules +T661 UBERON:0000395 23248 23262 spiral ganglia +T662 UBERON:0002282 23341 23357 stria vascularis +T663 CL:0002492 23448 23462 marginal cells +T664 GO:0031941 23514 23521 F-actin +T665 GO:0010467 23522 23532 expression +T666 CL:0002492 23534 23548 Marginal cells +T667 UBERON:0001844 23622 23630 cochlear +T668 UBERON:0000479 23631 23637 tissue +T669 PR:000015039 23648 23655 Slc26a4 +T670 PR:000015039 23663 23670 Slc26a4 +T671 NCBITaxon:10088 23674 23678 mice +T672 UBERON:0002282 23723 23739 stria vascularis +T673 UBERON:0002282 23741 23743 SV +T674 GO:0009986 23821 23831;23841 23846 surface of ... cells +T675 CL:0002492 23832 23846 marginal cells +T676 UBERON:0028194 23861 23878 spiral prominence +T677 UBERON:0028194 23880 23882 SP +T678 UBERON:0008832 23974 23986 outer sulcus +T679 UBERON:0008832 23988 23990 OS +T680 UBERON:0002282 24016 24032 stria vascularis +T681 UBERON:0006725 24047 24062 spiral ligament +T682 UBERON:0006725 24064 24066 SL +T683 UBERON:0028194 24153 24170 spiral prominence +T684 UBERON:0006725 24197 24212 spiral ligament +T685 UBERON:4200230 24294 24304;24309 24313 surface of ... bone +T686 UBERON:0008831 24352 24364 inner sulcus +T687 UBERON:0008831 24366 24368 IS +T688 UBERON:0002384 24459 24476 connective tissue +T689 NCBITaxon:33208 24513 24520 animals +T690 CL:0002492 24572 24580;24591 24595 marginal ... cell +T691 CL:0000646 24585 24595 basal cell +T692 PR:000015039 24611 24618 Slc26a4 +T693 NCBITaxon:10088 24622 24626 mice +T694 GO:0031941 24663 24670 F actin +T695 UBERON:0002282 24688 24704 stria vascularis +T696 CL:0000646 24733 24743 basal cell +T697 CL:0002492 24767 24780 marginal cell +T698 GO:0031941 24932 24939 F actin +T699 CHEBI:26130 24977 24984 pigment +T700 GO:0048770 24977 24993 pigment granules +T701 CL:0002492 25052 25065 marginal cell +T702 CL:0002492 25075 25078 SMC +T703 CL:0000646 25100 25110 basal cell +T704 CL:0000646 25120 25122 BC +T705 CL:0002492 25147 25160 marginal cell +T706 CL:0000646 25175 25185 basal cell +T707 CL:0002492 25399 25412 marginal cell +T708 CL:0000646 25430 25440 basal cell +T709 CL:0002492 25566 25574;25585 25589 marginal ... cell +T710 CL:0000646 25579 25589 basal cell +T711 PR:000015039 25602 25609 Slc26a4 +T712 PR:000015039 25617 25624 Slc26a4 +T713 NCBITaxon:10088 25628 25632 mice +T714 GO:0031941 25669 25676 F actin +T715 UBERON:0002282 25696 25712 stria vascularis +T716 CL:0002492 25800 25813 marginal cell +T717 CL:0000646 25822 25832 basal cell +T718 PR:000015039 25849 25856 Slc26a4 +T719 NCBITaxon:10088 25860 25864 mice +T720 PR:000015039 25890 25897 Slc26a4 +T721 NCBITaxon:10088 25901 25905 mice +T722 GO:0031941 25925 25932 F actin +T723 UBERON:0002282 25963 25979 stria vascularis +T724 PR:000015039 25985 25992 Slc26a4 +T725 NCBITaxon:10088 25996 26000 mice +T726 CL:0000646 26029 26039 basal cell +T727 CL:0002492 26063 26076 marginal cell +T728 CL:0002492 26215 26228 marginal cell +T729 CL:0002492 26238 26241 SMC +T730 CL:0000646 26261 26271 basal cell +T731 CL:0000646 26281 26283 BC +T732 CL:0000646 26306 26316 basal cell +T733 CL:0002492 26329 26342 marginal cell +T734 CL:0002492 26466 26479 marginal cell +T735 CL:0000646 26495 26505 basal cell +T736 PR:000015039 26619 26626 Pendrin +T737 GO:0010467 26627 26637 expressing +T738 UBERON:0000483 26646 26656 epithelial +T739 CL:0000066 26646 26662 epithelial cells +T740 UBERON:0028194 26670 26687 spiral prominence +T741 CL:0000646 26724 26735 basal cells +T742 UBERON:0000483 26827 26837 epithelial +T743 CL:0000066 26827 26843 epithelial cells +T744 PR:000015039 26894 26901 Slc26a4 +T745 NCBITaxon:10088 26905 26909 mice +T746 GO:0010467 27062 27072 expression +T747 PR:000016364 27076 27080 ZO-1 +T748 GO:0031941 27088 27095 F-actin +T749 GO:0032991 27133 27142 complexes +T750 PR:000016364 27144 27148 ZO-1 +T751 GO:0031941 27153 27160 F-actin +T752 GO:0010467 27161 27171 expression +T753 UBERON:0000119 27194 27202;27209 27214 layer of ... cells +T754 CL:0000646 27203 27214 basal cells +T755 GO:0005911 27228 27239;27246 27256;27276 27281 junction of ... cells with ... cells +T756 CL:0000646 27240 27251 basal cells +T757 UBERON:0000483 27265 27275 epithelial +T758 CL:0000066 27265 27281 epithelial cells +T759 PR:000015039 27285 27292 Slc26a4 +T760 NCBITaxon:10088 27296 27300 mice +T761 NCBITaxon:10088 27324 27328 mice +T762 CL:0000646 27477 27487 basal cell +T763 UBERON:0001852 27519 27532 endolymphatic +T764 UBERON:0001845 27537 27550 perilymphatic +T765 CHEBI:29103 27551 27553 K+ +T766 PR:000015039 27584 27591 Slc26a4 +T767 NCBITaxon:10088 27595 27599 mice +T768 UBERON:0002282 27614 27630 stria vascularis +T769 GO:0046903 27643 27650 secrete +T770 CHEBI:29103 27651 27653 K+ +T771 CHEBI:29103 27714 27716 K+ +T772 UBERON:0001852 27749 27762 endolymphatic +T773 CHEBI:29103 27763 27765 K+ +T774 PR:000015039 27820 27827 Slc26a4 +T775 NCBITaxon:10088 27831 27835 mice +T776 CL:0000855 27865 27883 sensory hair cells +T777 CHEBI:29103 27919 27921 K+ +T778 GO:0046903 27922 27926 exit +T779 UBERON:0001852 27932 27941 endolymph +T780 UBERON:0002282 27990 28006 stria vascularis +T781 PR:000015039 28010 28017 Slc26a4 +T782 GO:0046903 28021 28029 secretes +T783 CHEBI:29103 28030 28032 K+ +T784 CHEBI:3213 28067 28077 bumetanide +T785 UBERON:0002282 28104 28120 stria vascularis +T786 GO:0016324 28132 28150;28167 28172 apical membrane of ... cells +T787 UBERON:0002282 28151 28157 strial +T788 CL:0002492 28151 28172 strial marginal cells +T789 GO:0010467 28192 28202 expression +T790 PR:000000728 28219 28224 KCNQ1 +T791 PR:000009255 28226 28231 KCNE1 +T792 PR:000014925 28236 28243 SLC12A2 +T793 CHEBI:29103 28269 28271 K+ +T794 PR:000007998 28301 28305 GJB2 +T795 PR:000007998 28307 28311 Cx26 +T796 CHEBI:29103 28348 28350 K+ +T797 CHEBI:29103 28365 28367 K+ +T798 CHEBI:3213 28413 28423 bumetanide +T799 CHEBI:77608 28427 28440 loop-diuretic +T800 CHEBI:29101 28459 28462 Na+ +T801 CHEBI:29103 28468 28470 K+ +T802 PR:000014925 28485 28492 SLC12A2 +T803 CHEBI:3213 28499 28509 Bumetanide +T804 PR:000015039 28548 28555 Slc26a4 +T805 PR:000015039 28563 28570 Slc26a4 +T806 NCBITaxon:10088 28574 28578 mice +T807 PR:000015039 28646 28653 Slc26a4 +T808 NCBITaxon:10088 28657 28661 mice +T809 PR:000000728 28714 28719 KCNQ1 +T810 PR:000009255 28724 28729 KCNE1 +T811 GO:0046903 28747 28756 secretory +T812 CHEBI:29103 28757 28759 K+ +T813 GO:0016324 28794 28812;28829 28834 apical membrane of ... cells +T814 UBERON:0002282 28813 28819 strial +T815 CL:0002492 28813 28834 strial marginal cells +T816 CHEBI:29101 28840 28843 Na+ +T817 CHEBI:29103 28849 28851 K+ +T818 PR:000014925 28866 28873 SLC12A2 +T819 GO:0016323 28899 28922;28939 28944 basolateral membrane of ... cells +T820 UBERON:0002282 28923 28929 strial +T821 CL:0002492 28923 28944 strial marginal cells +T822 UBERON:0002282 28959 28975 stria vascularis +T823 GO:0005921 28985 28997 gap junction +T824 PR:000007998 29006 29010 GJB2 +T825 UBERON:0006725 29024 29039 spiral ligament +T826 PR:000015039 29043 29050 Slc26a4 +T827 PR:000015039 29058 29065 Slc26a4 +T828 NCBITaxon:10088 29069 29073 mice +T829 PR:000000728 29103 29108 KCNQ1 +T830 PR:000009255 29113 29118 KCNE1 +T831 CL:0000846 29149 29170 vestibular dark cells +T832 GO:0010467 29182 29192 Expression +T833 PR:000000728 29196 29201 KCNQ1 +T834 UBERON:0002282 29213 29229 stria vascularis +T835 PR:000015039 29233 29240 Slc26a4 +T836 NCBITaxon:10088 29244 29248 mice +T837 PR:000000728 29283 29288 KCNQ1 +T838 GO:0010467 29294 29304 expression +T839 CHEBI:29103 29435 29437 K+ +T840 UBERON:0002282 29451 29457 strial +T841 CL:0002492 29451 29472 strial marginal cells +T842 GO:0005921 29492 29504 gap junction +T843 CHEBI:29103 29514 29516 K+ +T844 PR:000000728 29561 29566 KCNQ1 +T845 PR:000009255 29568 29573 KCNE1 +T846 PR:000014925 29575 29582 SLC12A2 +T847 PR:000007998 29587 29591 GJB2 +T848 UBERON:0006106 29599 29607;29616 29620 cochlear ... wall +T849 PR:000015039 29624 29631 Slc26a4 +T850 PR:000015039 29639 29646 Slc26a4 +T851 NCBITaxon:10088 29650 29654 mice +T852 CL:0002492 29674 29677 SMC +T853 UBERON:0002282 29679 29685 strial +T854 CL:0002492 29679 29700 strial marginal cells +T855 UBERON:0002282 29702 29704 SV +T856 UBERON:0002282 29706 29722 stria vascularis +T857 CL:0000646 29724 29726 BC +T858 CL:0000646 29728 29739 basal cells +T859 UBERON:0006725 29741 29743 SL +T860 UBERON:0006725 29745 29760 spiral ligament +T861 PR:000015039 29802 29809 pendrin +T862 GO:0010467 29810 29820 expression +T863 UBERON:0001844 29828 29835 cochlea +T864 UBERON:0001862 29840 29860 vestibular labyrinth +T865 UBERON:0001852 29881 29894 endolymphatic +T866 CHEBI:29103 29895 29897 K+ +T867 PR:000015039 29981 29988 Slc26a4 +T868 NCBITaxon:10088 29992 29996 mice +T869 GO:0010467 30049 30056 express +T870 PR:000001979 30057 30063 KCNJ10 +T871 CL:0002486 30073 30091 Intermediate cells +T872 UBERON:0002282 30100 30116 stria vascularis +T873 CHEBI:16526 30131 30134 CO2 +T874 CHEBI:17544 30138 30144 HCO3 - +T875 GO:0098754 30152 30163 detoxifying +T876 CHEBI:26519 30164 30177 free radicals +T877 CHEBI:16526 30188 30191 CO2 +T878 CHEBI:26519 30196 30209 free radicals +T879 GO:0005739 30248 30260 mitochondria +T880 GO:0008152 30268 30281 metabolically +T881 UBERON:0002282 30296 30302 strial +T882 CL:0002492 30296 30317 strial marginal cells +T883 CL:0002486 30319 30337 Intermediate cells +T884 CHEBI:16526 30375 30378 CO2 +T885 CHEBI:17544 30382 30388 HCO3 - +T886 GO:0098754 30413 30421 detoxify +T887 CHEBI:26519 30422 30435 free radicals +T888 CHEBI:26519 30464 30476 free radical +T889 CL:0002486 30485 30503 intermediate cells +T890 CHEBI:16856 30513 30524 glutathione +T891 CHEBI:25179 30529 30536 melanin +T892 CHEBI:26130 30537 30544 pigment +T893 PR:000015039 30585 30592 pendrin +T894 GO:0046903 30604 30611 secrete +T895 CHEBI:17544 30612 30618 HCO3 - +T896 UBERON:0001852 30624 30633 endolymph +T897 CHEBI:17544 30665 30671 HCO3 - +T898 UBERON:0002282 30706 30712 strial +T899 GO:0005576 30726 30739 extracellular +T900 CHEBI:26519 30767 30779 free radical +T901 GO:0006750 30850 30863;30879 30890 production of ... glutathione +T902 CHEBI:16856 30879 30890 glutathione +T903 CHEBI:26519 30936 30948 free radical +T904 NCBITaxon:10088 31001 31005 mice +T905 PR:000015039 31014 31021 pendrin +T906 CHEBI:26519 31116 31128 free radical +T907 GO:0005829 31186 31195 cytosolic +T908 CHEBI:26519 31219 31231 free radical +T909 PR:000001979 31263 31269 KCNJ10 +T910 GO:0010467 31278 31288 expression +T911 UBERON:0002282 31292 31298 strial +T912 CL:0002486 31292 31317 strial intermediate cells +T913 GO:0010467 31332 31342 expression +T914 CHEBI:29103 31352 31354 K+ +T915 GO:0065007 31385 31395 controlled +T916 GO:0005829 31403 31412 cytosolic +T917 CHEBI:26519 31420 31433 free radicals +T918 GO:0008152 31452 31461 metabolic +T919 PR:000001979 31505 31511 KCNJ10 +T920 CHEBI:29103 31512 31514 K+ +T921 UBERON:0002282 31526 31532 strial +T922 CL:0002486 31526 31551 strial intermediate cells +T923 PR:000015039 31666 31673 Slc26a4 +T924 NCBITaxon:10088 31677 31681 mice +T925 http://purl.obolibrary.org/obo/MONDO_0010134 31710 31726 Pendred syndrome +T926 PR:000001979 31761 31767 KCNJ10 +T927 PR:000015039 31786 31793 pendrin +T928 GO:0010467 31794 31804 expression +T929 UBERON:0002282 31808 31824 stria vascularis +T930 CHEBI:16856 31900 31911 glutathione +T931 CHEBI:16856 31927 31930 GSH +T932 CHEBI:16856 31932 31943 glutathione +T933 UBERON:0002282 32243 32259 stria vascularis +T934 UBERON:0001852 32435 32448 endolymphatic +T935 CHEBI:29103 32449 32451 K+ +T936 UBERON:0002282 32507 32513 strial +T937 CL:0002492 32507 32528 strial marginal cells +T938 NCBITaxon:10088 32565 32569 mice +T939 PR:000015039 32578 32585 pendrin +T940 GO:0042571 32595 32603 antibody diff --git a/src/ontogpt/evaluation/craft/database/all/15320950.txt b/src/ontogpt/evaluation/craft/database/all/15320950.txt new file mode 100644 index 000000000..f6b32b893 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15320950.txt @@ -0,0 +1,137 @@ +Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model + +Abstract + +Background + +Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4+/+) or lack a complete Slc26a4 gene (Slc26a4-/-). + +Methods + +Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic K+ concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry. + +Results + +Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in Slc26a4-/- mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4-/- mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K+ channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K+ concentrations were normal and membrane proteins necessary for K+ secretion were present, including the K+ channel KCNQ1 and KCNE1, Na+/2Cl-/K+ cotransporter SLC12A2 and the gap junction GJB2. + +Conclusions + +These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome. + +Background + +Pendred syndrome is a relatively common autosomal-recessive disorder characterized by deafness and goiter [1]. The syndrome is caused by mutations of the PDS gene SLC26A4, which codes for the protein pendrin [2]. Deafness is congenital and generally profound although sometimes late in onset and provoked by light head injury. Vestibular dysfunction is uncommon. Goiter is variable and generally develops around puberty [3]. The cause of goiter appears to be an impairment of iodide fixation in the follicular lumen due to a reduced rate of iodide transport across the apical membrane of thyroid gland epithelial cells [4]. A positive perchlorate discharge test and an enlarged vestibular aqueduct appear to be the most reliable clinical signs of Pendred syndrome [5]. + +Pendrin is an anion exchanger that can transport Cl-, I-, HCO3 - and formate [6,7]. Expression has been found in the inner ear and thyroid gland consistent with the clinical signs of deafness and goiter [2,3,8]. In addition, pendrin expression has been found in the kidney [9], mammary gland [10], uterus [11], testes [12] and placenta [13]. No expression was found in fetal or adult brain, consistent with a peripheral cause of deafness [2,11]. + +Expression of pendrin mRNA in the inner ear has been found in several places including the cochlea, the vestibular labyrinth and the endolymphatic sac [8]. The precise location of pendrin protein expression, however, has not yet been determined. The variability of deafness in Pendred syndrome and the observation that deafness is sometimes late in onset suggest that pendrin dysfunction may not be the direct cause of deafness. It is conceivable that pendrin dysfunction favors changes in the expression levels of proteins that are critical for the maintenance of the hearing function. Detailed studies aimed at identifying the direct cause of deafness in Pendred syndrome have recently become possible due to the generation of a pendrin-specific polyclonal antibody [9] and the development of Slc26a4-/- mice, which bear a targeted disruption of the mouse Slc26a4 gene [14]. The aim in the present study was to determine the location of pendrin and the cause of deafness in Slc26a4-/- mice. + +Methods + +The endocochlear potential and the endolymphatic and perilymphatic K+ concentrations were measured in young adult mice (1–4 month of age) that either have (Slc26a4+/+) or lack (Slc26a4-/-) a functional gene for pendrin [14]. The mouse strain 129Sv/Ev (Taconic, Germantown, NY) was chosen as the source of Slc26a4+/+ controls, since Slc26a4-/- mice were propagated in the this strain and generated using a stem cell line derived from 129Sv/Ev. Slc26a4+/+ and Slc26a4-/- were agouti. They did not differ in coat color. Differences in pigmentation were verified using coisogenic age-matched Slc26a4+/+ and Slc26a4-/- that were bred in parallel. + +Expression of key proteins involved in the generation of the endocochlear potential and the transport of K+ was studied using confocal immunocytochemistry and quantitative RT-PCR. K+ secretion and the generation of the endocochlear potential were measured using electrophysiological techniques. All experiments were approved by the Institutional Animal Care and Use Committee of Kansas State University. + +Confocal immunocytochemistry + +Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.) and transcardially perfused with a Cl--free phosphate-buffered Na-gluconate solution containing 4% paraformaldehyde. Temporal bones were removed and cochleae fixed by perilymphatic perfusion, decalcified in EDTA, processed through a sucrose gradient and infiltrated with polyethylene glycol. Mid-modiolar cryosections (12 μm, CM3050S, Leica, Nussloch, Germany) were blocked in PBS with 0.2% Triton-X (PBS-TX) and 10% bovine serum albumin. Slides were incubated overnight at 4°C with primary antibodies in PBS-TX with 1–3% BSA [rabbit anti-pendrin, 1:500 (h766–780); rat anti-ZO-1, 1:100 (Chemicon, Temecula, CA); goat anti-KCNQ1, 1:200 (C20, Santa Cruz Biotechnology, Santa Cruz, CA), rabbit anti-KCNE1, 1:200 (Alomone, Jerusalem, Israel); rabbit anti-KCNJ10, 1:300 (Alomone); rabbit anti-SLC12A2, 1:100 (Chemicon); and rabbit anti-connexin 26, 1:100 (Zymed, San Francisco, CA)]. Slides were washed in PBS-TX and incubated for 1 h at 25°C with appropriate secondary antibodies at a 1:1000 dilution in PBS-TX with 1–3% BSA [donkey anti-rabbit Alexa 488, chicken anti-rat Alexa 594, and chicken anti-goat Alexa 594 (Molecular Probes, Eugene, OR)]. Actin filaments were visualized by staining with Alexa 488 conjugated phalloidin (Molecular Probes). After incubation, slides were washed with PBS-TX, mounted with FluorSave (Calbiochem, La Jolla, CA), and viewed by confocal microscopy (LSM 5 Pascal or LSM 510 Meta, Carl Zeiss, Jena or Göttingen, Germany). Laser scanning brightfield images were collected to document structural preservation, for morphometric analysis and for analysis of pigmentation. + +Quantitative RT-PCR + +Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.). Kidneys and brain were removed and pulverized in liquid N2. Temporal bones were removed and two preparations of inner ear tissues were obtained by microdissection: (1) stria vascularis without spiral ligament, and (2) spiral ganglia including the surrounding bone and the organ of Corti. The dissection medium was changed twice during the microdissection and isolated tissues were washed three times to minimize cross-contamination. The Slc26a4-/-genotype was verified by the observation of large otoconia in the utricular macula [14]. Total RNA was isolated and residual DNA contamination was removed by enzymatic digestion (RNeasy micro, Qiagen, Valencia, CA). Quality and quantity of 18S rRNA obtained from kidneys and brain were determined (Nano Assay, 2100 Bioanalyzer, Agilent, Palo Alto, CA). Further, the quality of RNA preparations obtained from stria vascularis and spiral ganglia was assessed (Pico Assay, 2100 Bioanalyzer). Real time RT-PCR was performed on RNA obtained from individual animals (OneStep RT-PCR, Qiagen; Smart Cycler, Cepheid, Sunnyvale, CA) in the presence of 0.2× SYBR green I (Molecular Probes). Transcripts of 18S rRNA and mRNA for the K+ channels KCNJ10, KCNQ1 and KCNQ4 were amplified using gene-specific primers (Table 1). RT was performed for 30 min at 50°C and 15 min at 95°C. PCR consisted of 50 cycles of 1 min at 60°C, 1 min at 72°C, 7s heating to hot-measurement temperature, 13s hot-measurement at 3–5°C below product melting temperature (Table 1) and 1 min at 94°C. Hot-measurements were performed to eliminate the detection of primer-dimers that had melting temperatures between 72 and 75°C [15]. PCR was followed by a melt (60 to 95°C). The generation of a single product of appropriate size was routinely checked by the presence of a single melt peak and by agarose gel-electrophoresis. Product identity was confirmed by sequencing. + +Table 1 + +Primers + +Template molecules (T) were quantified according to T = P / (F ^Ct) where P is the number of product molecules, F is the fidelity of the reaction and Ct is the cycle at which the number of product molecules reaches a chosen threshold [15]. Fidelity (F) was obtained from the slope (S) of the log-linear phase of the growth curve via a best-fit fifth-order polynomial: F = 7.39 + 3.80 × S + 1.05 × S2 + 0.15 × S3 + 11.38 × 10-3 * S4 + 3.39 × 10-4 × S5. The number of product molecules at threshold (PCt) was determined by amplification of known amounts of 18S rRNA according to PCt = T × F ^Ct. Quantifications of 18S rRNA were used to compare tissue amounts. Genomic contamination of inner ear samples was assessed to be <0.02% by omission of the RT step. + +In vitro electrophysiology + +Animals were deeply anesthetized with sodium pentobarbital (100 mg/kg i.p.). Stria vascularis without spiral ligament was obtained by microdissection. Currents generated by the stria marginal epithelium were recorded [16]. A Pt-Ir wire microelectrode with a Pt-black tip was positioned 20–30 μm from the apical surface of the epithelium and vibrated at 200–400 Hz by piezo-electric bimorph elements (Applicable Electronics, Forest Dale, MA; ASET version 1.05, Science Wares, East Falmouth, MA). A Pt-black electrode (26-gauge) served as reference in the bath chamber. The signals from the phase-sensitive detectors were digitized (16 bit) at a rate of 0.5 Hz. The output was expressed as current density at the electrode. + +In situ electrophysiology + +Animals were anesthetized with inactin (thiobutabarbital sodium salt, 140 mg/kg ip). The endocochlear potential and the endolymphatic [K+] were measured with double-barreled microelectrodes [17]. Measurements were made in the basal turn by a round-window approach through the basilar membrane and in the apical turn after thinning the bone over the stria vascularis and picking a small hole (~30 μm). K+-selective electrodes were calibrated in solutions of constant cation (K+ and Na+) concentration of 150 mM. The minor selectivity of the K+ electrodes for Na+ produced a nonlinearity in the calibration curve, which was closely fitted by the Nicolski equation using nonlinear curve-fitting software (OriginLab, Northampton, MA): V = Vi + S × log ([K+] + A × [Na+]), where Vi is an offset term, S is slope, A is selectivity, and [Na+] is Na+ concentration. Calibrations were made immediately after withdrawal of the electrodes from the cochlea. Plasma K+ concentrations were obtained using a blood analyzer (Stat Profile M, Nova Biomedical, Waltham, MA). + +Data are presented as mean ± sem; n denotes the number of experiments. Differences were considered significant when p < 0.05. + +Results and Discussion + +In situ hybridization in the cochlea has suggested that pendrin mRNA is expressed in cells that reside immediately beneath the spiral prominence on the lateral wall of the external sulcus [8]. To determine the location of pendrin protein expression, we performed confocal immunocytochemistry on cryosections prepared from temporal bones of normal (Slc26a4+/+) mice using an established antibody [3]. Staining was absent when the primary antibody was pre-absorbed with the antigenic peptide (data not shown). Strong expression of pendrin was observed not only in outer sulcus epithelial cells, as predicted from in situ hybridization data, but also in root cells, in apical membranes of spiral prominence surface epithelial cells and in apical membranes of spindle-shaped cells that are part of stria vascularis (Fig. 1a,1b,1c). The presence of pendrin in spindle-shaped cells suggests that these cells secrete HCO3 - into endolymph. Pendrin-mediated HCO3 - transport has previously been shown in the kidney [9]. HCO3 - in the cochlea may be generated from CO2 by carbonic anhydrase located in strial intermediate cells [18,19]. CO2 may be supplied by the metabolically highly active stria marginal cells. It is conceivable that pendrin dysfunction interrupts HCO3 - secretion and leads to an accumulation of HCO3 - in stria vascularis. Preliminary data (Wangemann et al., unpublished) support a role for pendrin in HCO3 - secretion into endolymph. + +Figure 1 + +Protein localization of pendrin, KCNQ1, ZO-1 and F actin in cochlea and vestibular labyrinth of Slc26a4+/+ and Slc26a4-/- mice. a: Overview of cochlea; bar = 100 μm. b-f: Detail of cochlear lateral wall; bar = 10 μm. g: Detail of utricle; bar = 10 μm. h-i: Detail of ampulla; bar = 10 μm. RM, Reissner's membrane; SC, spindle-shaped cells, SMC, strial marginal cells; SV, stria vascularis; SL, spiral ligament; LIM, spiral limbus. BC, basal cells; SP, spiral prominence epithelial cells; RC, root cells; OS, outer sulcus epithelial cells; VHC, vestibular hair cells; VTC, vestibular transitional cells; VDC, vestibular dark cells; arrows, basal cells at the top and bottom of stria vascularis form tight junctions with surface epithelial cells. + +The extent of pendrin expression in spiral prominence cells and stria vascularis was determined by labeling KCNQ1, a K+ channel that is expressed in strial marginal cells, and ZO-1, a tight junction protein that labels basal cells and thereby delineates the boundaries of stria vascularis. Dual labeling experiments demonstrated that pendrin is expressed in spindle-shaped cells, which are surface epithelial cells in stria vascularis adjacent to strial marginal cells near the borders of both the spiral prominence and Reissner's membrane (Fig. 1c). + +In situ hybridization in the vestibular labyrinth suggested that pendrin mRNA is expressed in non-sensory cells [8]. Using confocal immunocytochemistry on cryosections, we observed strong expression of the pendrin protein in the apical membrane of vestibular transitional cells in the utricle and ampullae (Fig. 1g,1h,1i). Dual labeling with KCNQ1 demonstrated that pendrin expression was clearly limited to vestibular transitional cells and did not extend to other non-sensory cells such as vestibular dark cells, which were clearly identified by the expression of KCNQ1 and KCNE1 in their apical membranes. + +The onset of pendrin expression during development of the mouse inner ear has been determined to be embryonic day (ED) 13 [14]. Morphologically detectable differences in the inner ears of Slc26a4+/+ and Slc26a4-/- mice become evident as early as ED 15, when Slc26a4-/- mice start to develop an enlarged endolymphatic space that persists into adulthood [14]. Interestingly, sensory hair cells in the cochlea appear normal until postnatal day (PD) 7 but show clear evidence of degeneration by PD 15 [14]. These observations suggest that the cochlear environment supports the survival of sensory hair cells in spite of the enlargement of the endolymphatic duct. A normal endolymphatic K+ concentration, which is critical for hair cell survival [20], is established at PD 3 [21] and may persist in Slc26a4-/- at least until PD 7. The time period between PD 7 and 15 is the time when the endocochlear potential develops at the onset of hearing [22]. We hypothesized that a lack of a normal endocochlear potential or an alteration of the endolymphatic K+ concentration could account for deafness in Slc26a4-/- mice. Measurements revealed that the endocochlear potential was absent but that the endolymphatic K+ concentration was normal in adult Slc26a4-/- (Fig. 2a). No significant differences between Slc26a4+/+ and Slc26a4-/- mice were found in perilymphatic (Fig. 2a) or plasma K+ concentrations (Slc26a4+/+, 4.9 ± 0.3 mM, n = 6; Slc26a4-/-, 5.1 ± 0.3 mM, n = 6). These observations suggest that a primary event leading to deafness in Slc26a4-/- mice, and potentially in patients suffering from Pendred syndrome, is the loss of the endocochlear potential. Degeneration of hair cells is probably a response to the loss of the endocochlear potential. + +Figure 2 + +Potential, K+ concentrations and pigmentation of stria vascularis in Slc26a4+/+ and Slc26a4-/- mice. a: Endocochlear potential and K+ concentrations in endolymph and perilymph at the apex (A) and base (B) of the cochlea. Numbers adjacent to symbols denote number of measurements. b-d: Pigmentation of stria vascularis in Slc26a4+/+ and Slc26a4-/- mice. b: View of stria vascularis through the bony capsule of the cochlea. OW, oval window, RW, round window; arrows, stria vascularis. c-d: Whole-mounts of stria vascularis isolated from age-matched mice. c: Laser-scanning images, bar = 10 μm, d: Quantification of pigmentation based on optical density. + +The endocochlear potential is generated by stria vascularis in the lateral wall of the cochlea [17,23]. The potential is generated across the basal cell barrier of stria vascularis by the K+ channel KCNJ10 located in intermediate cells [24], which are connected to basal cells by a high density of gap junctions [25]. Marginal cells of stria vascularis, which form the barrier toward endolymph, transport K+ from the intrastrial space into endolymph and keep the K+ concentration low adjacent to the KCNJ10 K+ channel [26]. To determine the cause of the loss of the endocochlear potential in Slc26a4-/- mice, we first determined whether intermediate cells are present in stria vascularis, since a loss of intermediate cells is known to lead to a loss of the endocochlear potential [27,28]. Intermediate cells of stria vascularis were visualized by their pigmentation. Pigmentation was present in stria vascularis of Slc26a4-/- mice (Fig. 2b), which suggests that intermediate cells are present. Interestingly, pigmentation of stria vascularis was much stronger in Slc26a4-/- than in Slc26a4+/+ mice. + +To determine in greater detail the cause of the loss of the endocochlear potential in Slc26a4-/- mice, we isolated total RNA from stria vascularis and spiral ganglia of young (1–4 month) Slc26a4+/+ and both young and old (~12 month) Slc26a4-/- mice, assessed amounts of isolated tissues by quantification of 18S rRNA, and quantified the expression of KCNJ10 mRNA. Quantities of stria vascularis isolated from these different mice were similar, since no significant differences were found in the numbers of 18S rRNA molecules (log(rRNA) = 9.46 ± 0.08, n = 17, pooled data). In contrast, quantities of spiral ganglia isolated from young and old Slc26a4-/- mice (log(rRNA) = 9.04 ± 0.18, n = 4 and 9.29 ± 0.15, n = 6) were significantly smaller than in Slc26a4+/+ mice (log(rRNA) = 9.48 ± 0.19, n = 7), consistent with morphometric data (see below). Expression of KCNJ10 mRNA was normal in stria vascularis and spiral ganglia of young Slc26a4-/- mice but significantly reduced in old Slc26a4-/- mice (Fig. 3). Quantifications of KCNQ1 and KCNQ4 mRNA were used to assess possible cross-contamination between the stria vascularis and spiral ganglia preparations based on the assumptions that (1) KCNQ1 is expressed in cells of the stria vascularis but not the spiral ganglia preparation and (2) KCNQ4 is expressed in cells of the spiral ganglia but not the stria vascularis preparation. The number of KCNQ1 mRNA molecules in stria vascularis was 24-fold greater than in spiral ganglia, and the number of KCNQ4 mRNA molecules in spiral ganglia was 5-fold greater than in stria vascularis. These observations validate our measurements of KCNJ10 and KCNQ1 by demonstrating that the microdissected preparations of stria vascularis and spiral ganglia were 98% and 78% pure, respectively. + +Figure 3 + +Quantification of KCNJ10 and KCNQ1 mRNA expression in stria vascularis and spiral ganglia of Slc26a4+/+ and Slc26a4-/- mice. a: Electropherogram of total RNA isolated from stria vascularis microdissected from one mouse. The amount of total RNA was obtained from the total integral (shaded) and the amount of 18S rRNA was obtained from the integral of the 18S peak. Sharp peaks representing 18S and 28S rRNA demonstrate the high quality of RNA. Insert: Genotype of Slc26a4-/- mice was verified by the observation of one or few very large rhomboedric otoconia in the utricular macula (arrow). A, crista ampullaris; U, utricular macula. Scale bar: 100 μm. b: Example of real-time RT-PCR data used for quantification of 18S, KCNJ10, KCNQ1 and KCNQ4. Known quantities of 18S rRNA were used to calibrate the threshold. SV, stria vascularis; SG, spiral ganglia. c: Quantification of KCNJ10 and KCNQ1 mRNA in young Slc26a4+/+ and young and old Slc26a4-/- mice. + +The presence of KCNJ10 mRNA in stria vascularis of Slc26a4-/- mice supports the finding that intermediate cells are present. Expression of the KCNJ10 protein was assessed in young Slc26a4-/- mice by confocal immunocytochemistry. Interestingly, expression of the KCNJ10 protein was absent in stria vascularis but normal in spiral ganglia (Fig. 4). The absence of the KCNJ10 K+ channel in stria vascularis is sufficient to explain the loss of the endocochlear potential [17]. + +Figure 4 + +Protein localization of KCNJ10 in the cochlea of Slc26a4+/+ and Slc26a4-/- mice. a: Overview of cochlea; bar = 100 μm. Compare to Fig. 1a to note the enlarged scala media and the distended Reissner's membrane. b-c: Detail of lateral wall and spiral ganglia (insert); main bar: 10 μm, insert: 5 μm. Expression of KCNJ10 in Slc26a4-/- mice was absent in stria vascularis but unchanged in spiral ganglion cells. RM, Reissner's membrane, SV, stria vascularis; SP, spiral prominence; SL, spiral ligament; LIM, spiral limbus; SG, spiral ganglion. + +Histological evaluation of cryosections revealed an enlargement of scala media with a large bulging of Reissner's membrane and an apparent degeneration of the organ of Corti, as described earlier [14]. Tissue height of stria vascularis was normal (Fig. 5), consistent with the similar numbers of 18S rRNA molecules in isolated preparations (see above). The absence of a change in tissue height is consistent with the presence of intermediate cells [28]. Further, we observed an apparent loss of tissue masses in areas that are normally occupied primarily by type I and II fibrocytes. Spiral prominence in Slc26a4-/- mice was less prominent, spiral ligament thinner and spiral limbus flatter (Fig. 5). The observation that tissue masses were lost in the spiral limbus region is consistent with the finding of a reduced number of 18S rRNA molecules in the spiral ganglia preparation (see above). In addition, we observed an apparent degeneration of stria vascularis, including an increase in pigmentation and an irregular pattern of the tight junctions of marginal cells (Fig. 2c, 6,7). Tight junctions were visualized by F-actin expression. Marginal cells appeared to form a continuous layer. + +Figure 5 + +Morphometric analysis of cochlear tissue masses in Slc26a4+/+ and Slc26a4-/- mice. a: locations of measurements. Thickness of stria vascularis (SV) was obtained as average of three distance measurements perpendicular to the surface of marginal cells. Thickness of spiral prominence (SP) was measured perpendicular to a tangential line (dashed) that connects the surface of the outer sulcus (OS) with the basal layer of stria vascularis. Thickness of spiral ligament (SL) was measured perpendicular to the tangential line as distance between the surface of spiral prominence and the interface between spiral ligament and bone (B). Thickness of spiral limbus (LIM) was obtained perpendicular to the surface of the bone as a tangential line that touches the inner sulcus (IS) and reaches from the surface of the spiral limbus to the interface between spiral limbus connective tissue and bone. b: Summary. Data from 7–8 animals contributed to each column. + +Figure 6 + +Analysis of marginal and basal cell barriers by in Slc26a4-/- mice. Tight junctions were visualized by F actin. Whole-mounts of stria vascularis were viewed either from the basal cell side (a-f) or from the marginal cell side (g-l). Bright field images verify that the same area was viewed from either side (b and h). Colored bright field images were mixed with images of F actin staining to indicate the position of pigment granules (d, j, f and l). Focus was varied to either visualize the marginal cell barrier (SMC, c-d and i-j) or the basal cell barrier (BC, e-f and k-l). Both the marginal cell (e-f) and the basal cell barrier (i-f) appeared to be intact. It was critical for this finding that pigmentation did not block the path of the laser. Blockage of the laser by pigmentation produces the untrue impression of a discontinuous marginal cell barrier (c-d) or basal cell barrier (k-l). Comparison of images is aided by marking a significant area with a star. Bars = 10 μm. + +Figure 7 + +Analysis of marginal and basal cell barriers in Slc26a4+/+ and Slc26a4-/- mice. Tight junctions were visualized by F actin in whole-mounts of stria vascularis. Bright field images were taken to evaluate pigmentation (a, d and g). Note the intact marginal cell (b) and basal cell (c) barriers in Slc26a4+/+ mice. Minimal pigmentation of Slc26a4+/+ mice did not compromise F actin localization. Whole-mounts of stria vascularis from Slc26a4-/- mice were viewed either from the basal cell side (e-f) or from the marginal cell side (h-i). Bright field images verify that the same area was viewed from either side (d and g). Focus was varied to either visualize the marginal cell barrier (SMC, b,e and h) or the basal cell barrier (BC, c,f and i). Both the basal cell (f) and the marginal cell (h) barriers appeared to be intact. Blockage of the laser by pigmentation produces the untrue impression of 'holes' in the marginal cell barrier (e) or basal cell barrier (i). Comparison of images is aided by marking three significant areas with colored stars. Bars = 10 μm. + +Pendrin-expressing surface epithelial cells in the spiral prominence region are located in an area where basal cells, which are interconnected by tight junctions, form additional tight junctions with surface epithelial cells [29]. A discontinuity of this complex junction in Slc26a4-/- mice would explain the absence of the endocochlear potential. To evaluate the presence of this connection, we determined by confocal immunocytochemistry the expression of ZO-1 and of F-actin, which associate with tight junction complexes. ZO-1 and F-actin expression revealed a continuous layer of basal cells, including a junction of basal cells with surface epithelial cells in Slc26a4-/- mice, as observed in normal mice (Fig. 1c,1d,1e,1f,6,7). These observations make it unlikely that the primary cause of the loss of the endocochlear potential is a compromise in the basal cell barrier. + +The observation that endolymphatic and perilymphatic K+ concentrations were normal in Slc26a4-/- mice suggests that stria vascularis was able to secrete K+ in spite of the apparent signs of degeneration. The rate of K+ secretion necessary to maintain endolymphatic K+ concentration, however, may be less than necessary in Slc26a4+/+ mice given the reduced numbers of sensory hair cells, which provide a major pathway for K+ exit from endolymph [30,31]. In order to substantiate the view that stria vascularis in Slc26a4-/- secretes K+, we measured the magnitude of the bumetanide-sensitive current exiting stria vascularis across the apical membrane of strial marginal cells and determined the expression of the proteins KCNQ1, KCNE1 and SLC12A2, which are essential for K+ secretion [20,32,33], and of GJB2 (Cx26), which is thought to contribute to K+ cycling [23]. K+ secretion is known to be sensitive to 10-5 M bumetanide, a loop-diuretic that inhibits the Na+/2Cl-/K+ cotransporter SLC12A2 [26]. Bumetanide-sensitive currents were found in both Slc26a4+/+ and Slc26a4-/- mice although the magnitude of the current was significantly smaller in Slc26a4-/- mice (22 ± 6 μA/cm2, n = 4 versus 14 ± 2 μA/cm2, n = 4). KCNQ1 and KCNE1, subunits of the secretory K+ channel, were co-localized in the apical membrane of strial marginal cells; the Na+/2Cl-/K+ cotransporter SLC12A2, which is located in the basolateral membrane of strial marginal cells, was found in stria vascularis; and the gap junction protein GJB2 was found in spiral ligament of Slc26a4+/+ and Slc26a4-/- mice (Fig. 8). Co-localization of KCNQ1 and KCNE1 proteins was also observed in vestibular dark cells (Fig. 1i). Expression of KCNQ1 protein in stria vascularis of Slc26a4-/- mice is consistent with the finding of KCNQ1 mRNA expression (Fig. 3). These observations make it unlikely that the primary cause of the loss of the endocochlear potential is a compromise of K+ secretion by strial marginal cells or a compromise of gap junction mediated K+ cycling. + +Figure 8 + +Protein localization of KCNQ1, KCNE1, SLC12A2 and GJB2 in the cochlear lateral wall of Slc26a4+/+ and Slc26a4-/- mice. a-f: bars: 10 μm. SMC, strial marginal cells; SV, stria vascularis; BC, basal cells; SL, spiral ligament. + +Conclusions + +We described locations of pendrin expression in the cochlea and vestibular labyrinth and detected normal endolymphatic K+ concentrations in spite of an enlargement of this fluid compartment. We found that Slc26a4-/- mice lack the endocochlear potential because they do not express KCNJ10 protein. Intermediate cells protect stria vascularis by converting CO2 to HCO3 - and by detoxifying free radicals (Fig. 9). CO2 and free radicals are generated by the large numbers of mitochondria in the metabolically highly active strial marginal cells. Intermediate cells employ carbonic anhydrase to convert CO2 to HCO3 - [18,19] and catalase to detoxify free radicals. To protect themselves from free radical damage, intermediate cells generate glutathione and melanin pigment [34-37]. It is conceivable that loss of pendrin, which may secrete HCO3 - into endolymph, results in an accumulation of HCO3 - and an alkalinization of the intrastrial spaces. This extracellular alkalinization may enhance free radical stress, since it may inhibit the uptake of cysteine and thereby limit production of the protective glutathione [38]. Support for the hypothesis of enhanced free radical stress comes from the observed hyperpigmentation in mice lacking pendrin. Strial hyperpigmentation has also been observed in other conditions that are associated with free radical stress, such as acoustic trauma [37]. Alterations in the cytosolic pH in conjunction with free radical stress may lead to the loss of KCNJ10 protein expression in strial intermediate cells. Function and expression of other K+ channels has been shown to be controlled by the cytosolic pH and free radicals, which encode the metabolic state of the cell [39]. Suppression of the KCNJ10 K+ channel in strial intermediate cells, which is essential for the generation of the endocochlear potential, is probably the direct cause of deafness in Slc26a4-/- mice and patients suffering from Pendred syndrome. + +Figure 9 + +Model for the loss of KCNJ10 in the absence of pendrin expression in stria vascularis. Cys, cysteine, Glu, glutamate, Gly, glycine, CA, carbonic anhydrase, GST, glutathione-S-transferase, GSH, glutathione. + +Competing interests + +None declared. + +Authors' contributions + +PW designed and coordinated the immunocytochemical, morphometrical and molecular biological experiments. EMI and BA carried out confocal immunocytochemistry on cryosections. SJ and SVJ carried out confocal microscopy on whole-mounts of stria vascularis. SVJ and RJM carried out quantitative RT-PCR. DCM designed and coordinated electrophysiological experiments. TW carried out measurements of the endocochlear potential and the endolymphatic K+ concentration. JHL carried out current measurements on strial marginal cells. SMW, LAE, IER and EDG provided the mice and the pendrin-specific antibody. PW and DCM conceived the study. PW wrote the manuscript. All authors read and approved the final manuscript. + +Pre-publication history + +The pre-publication history for this paper can be accessed here: + + + +Acknowledgements + +This work was supported by NIH-R01-DC01098 (PW), NIH-R01-DC00212 (DCM), NIH-R01-DK52935 (SMW) and Core facilities funded by NIH-P20-RR017686 (Confocal Microfluorometry and Microscopy Core, Molecular Biology Core) are gratefully acknowledged. diff --git a/src/ontogpt/evaluation/craft/database/all/15328533.ann b/src/ontogpt/evaluation/craft/database/all/15328533.ann new file mode 100644 index 000000000..6539180ef --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15328533.ann @@ -0,0 +1,539 @@ +T1 GO:0065007 0 10 Regulation +T2 CL:0000187 14 26 Muscle Fiber +T3 PR:000013057 57 62 PPARδ +T4 GO:0043500 117 153 adaptive muscle fiber transformation +T5 CL:0000187 126 138 muscle fiber +T6 GO:0005739 173 186 mitochondrial +T7 SO:0000704 232 236 gene +T8 GO:0010467 232 247 gene expression +T9 NCBITaxon:10088 369 374 mouse +T10 GO:0010467 490 500 expression +T11 GO:0005777 525 535 peroxisome +T12 PR:000013057 525 569 peroxisome proliferator-activated receptor δ +T13 PR:000013057 571 576 PPARδ +T14 UBERON:0004288 581 589 skeletal +T15 CL:0002211 650 670 type I muscle fibers +T16 NCBITaxon:10088 695 699 mice +T17 PR:000013057 705 710 PPARδ +T18 CHEBI:73730 705 718 PPARδ agonist +T19 CL:0002211 737 749 type I fiber +T20 SO:0000704 750 754 gene +T21 GO:0010467 750 765 gene expression +T22 SO:0000704 801 812 genetically +T23 http://purl.obolibrary.org/obo/MONDO_0011122 851 858 obesity +T24 GO:0008152 873 882 metabolic +T25 UBERON:0004288 1097 1105 Skeletal +T26 CL:0008002 1097 1119 Skeletal muscle fibers +T27 CL:0002211 1148 1154;1201 1207 type I ... fibers +T28 MOP:0000568 1156 1165 oxidative +T29 CL:0002210 1156 1165;1201 1207 oxidative ... fibers +T30 CL:0000189 1166 1170;1201 1207 slow ... fibers +T31 CL:0002212 1175 1182;1201 1207 type II ... fibers +T32 GO:0006096 1184 1194 glycolytic +T33 CL:0000190 1184 1194;1201 1207 glycolytic ... fibers +T34 CL:0000190 1195 1199;1201 1207 fast ... fibers +T35 GO:0006936 1255 1266 contraction +T36 GO:0008152 1268 1278 metabolism +T37 CL:0002211 1311 1324 Type I fibers +T38 GO:0005739 1329 1341 mitochondria +T39 MOP:0000568 1362 1371 oxidative +T40 GO:0045333 1362 1382 oxidative metabolism +T41 CL:0002212 1494 1508 Type II fibers +T42 CL:0002214 1534 1537 IIa +T43 CL:0002215 1548 1551 IIb +T44 CL:0002215 1553 1568 Type IIb fibers +T45 GO:0005739 1595 1608 mitochondrial +T46 MOP:0000568 1621 1630 oxidative +T47 GO:0006096 1648 1669 glycolytic metabolism +T48 MOP:0000568 1738 1747 oxidative +T49 GO:0006936 1752 1763 contraction +T50 CL:0002214 1777 1785 type IIa +T51 CL:0002211 1806 1812 type I +T52 CL:0002215 1806 1810;1817 1820 type ... IIb +T53 UBERON:0007023 1896 1901 Adult +T54 UBERON:0004288 1902 1910 skeletal +T55 GO:0065007 2028 2038 modulation +T56 CL:0000100 2042 2052 motoneuron +T57 CL:0000187 2176 2188 muscle fiber +T58 CL:0002215 2194 2202 type IIb +T59 CL:0002214 2206 2214 type IIa +T60 CL:0002211 2219 2225 type I +T61 GO:0019722 2256 2281 calcium signaling pathway +T62 PR:000004978 2309 2319 calmodulin +T63 CHEBI:23357 2362 2370 cofactor +T64 GO:0005777 2371 2381 Peroxisome +T65 PR:000013059 2371 2434 Peroxisome proliferator-activated receptor-gamma coactivator 1α +T66 PR:000013059 2436 2442 PGC-1α +T67 GO:0006936 2626 2637 contractile +T68 GO:0008152 2642 2651 metabolic +T69 SO:0000704 2652 2657 genes +T70 CL:0000187 2684 2696 Muscle fiber +T71 http://purl.obolibrary.org/obo/MONDO_0011122 2741 2748 obesity +T72 http://purl.obolibrary.org/obo/MONDO_0005015 2753 2761 diabetes +T73 NCBITaxon:9989 2777 2784 rodents +T74 CL:0002211 2843 2856 type I fibers +T75 http://purl.obolibrary.org/obo/MONDO_0011122 2880 2885 obese +T76 UBERON:0004288 2896 2904 skeletal +T77 MOP:0000568 2946 2955 oxidative +T78 GO:0006096 2976 2986 glycolytic +T79 CL:0002211 3027 3040 type I fibers +T80 http://purl.obolibrary.org/obo/MONDO_0005015 3129 3137 diabetic +T81 MOP:0000568 3235 3244 oxidative +T82 CL:0002210 3235 3251 oxidative fibers +T83 PR:000045358 3273 3280 insulin +T84 CL:0000136 3300 3309 adipocyte +T85 GO:0005777 3393 3403 peroxisome +T86 PR:000013057 3393 3435;3443 3444 peroxisome proliferator-activated receptor ... δ +T87 PR:000013057 3437 3441;3443 3444 PPAR ... δ +T88 UBERON:0001013 3500 3514 adipose tissue +T89 CHEBI:15904 3561 3582 long-chain fatty-acid +T90 GO:0006635 3572 3594 fatty-acid β-oxidation +T91 MOP:0000568 3585 3594 oxidation +T92 PR:000013057 3624 3629 PPARδ +T93 SO:0001060 3654 3661 isoform +T94 UBERON:0004288 3673 3681 skeletal +T95 PR:000013057 3763 3768 PPARδ +T96 GO:0048747 3821 3833;3852 3865 formation of ... muscle fibers +T97 CL:0002211 3845 3865 type I muscle fibers +T98 http://purl.obolibrary.org/obo/MONDO_0011122 3970 3977 obesity +T99 PR:000013057 4014 4019 PPARδ +T100 CL:0000187 4029 4041 Muscle Fiber +T101 PR:000013057 4068 4073 PPARδ +T102 CL:0000187 4077 4089 muscle fiber +T103 GO:0010467 4120 4130 expression +T104 PR:000013056 4174 4179 PPARα +T105 SO:0001060 4186 4194 isoforms +T106 PR:000013057 4247 4252 PPARδ +T107 CL:0000187 4266 4279 muscle fibers +T108 CL:0002211 4320 4326 type I +T109 UBERON:0001389 4335 4341 soleus +T110 CL:0002212 4355 4362 type II +T111 UBERON:0001386 4376 4401 extensor digitorum longus +T112 CL:0002211 4406 4412 type I +T113 CL:0002212 4417 4424 type II +T114 UBERON:0001388 4439 4452 gastrocnemius +T115 GO:0010467 4472 4482 expression +T116 PR:000013059 4517 4523 PGC-1α +T117 PR:000013057 4688 4693 PPARδ +T118 GO:0065007 4697 4704 control +T119 CL:0000187 4708 4720 muscle fiber +T120 GO:0005739 4736 4749 mitochondrial +T121 NCBITaxon:10088 4775 4779 mice +T122 GO:0010467 4780 4790 expressing +T123 SO:0000902 4793 4802 transgene +T124 PR:P06492 4830 4834 VP16 +T125 SO:0000417 4846 4852 domain +T126 PR:000013057 4896 4901 PPARδ +T127 GO:0065007 4909 4916 control +T128 NCBITaxon:9606 4931 4936 human +T129 PR:000003674 4937 4953 α-skeletal actin +T130 UBERON:0004288 4939 4947 skeletal +T131 SO:0000167 4954 4962 promoter +T132 SO:0000167 5020 5028 promoter +T133 PR:P06492 5083 5087 VP16 +T134 PR:000013057 5088 5093 PPARδ +T135 SO:0000902 5094 5103 transgene +T136 GO:0010467 5120 5129 expressed +T137 UBERON:0004288 5133 5141 skeletal +T138 UBERON:0000948 5175 5180 heart +T139 CL:0000187 5219 5232 muscle fibers +T140 PR:P06492 5248 5252 VP16 +T141 PR:000013057 5253 5258 PPARδ +T142 GO:0010467 5259 5269 expression +T143 UBERON:0001388 5339 5359 gastrocnemius muscle +T144 PR:P06492 5361 5365 VP16 +T145 PR:000013057 5366 5371 PPARδ +T146 PR:000013057 5441 5446 PPARδ +T147 PR:000013057 5519 5524 PPARδ +T148 NCBITaxon:10088 5551 5555 mice +T149 PR:000013057 5644 5649 PPARδ +T150 CL:0002211 5686 5698 type I fiber +T151 GO:0010467 5732 5741 expresses +T152 PR:000013057 5759 5764 PPARδ +T153 CL:0002211 5786 5792 Type I +T154 CL:0002212 5834 5841 type II +T155 GO:0010467 5944 5953 expressed +T156 MOP:0000568 5957 5966 oxidative +T157 CL:0002210 5957 5980 oxidative muscle fibers +T158 UBERON:0005090 5996 6003 muscles +T159 NCBITaxon:10088 6022 6026 mice +T160 CL:0002211 6099 6105;6109 6115 type I ... fibers +T161 CL:0002212 6099 6103;6106 6115 type ... II fibers +T162 UBERON:0002103 6123 6131 hindlimb +T163 CL:0000187 6199 6211 muscle fiber +T164 UBERON:0001388 6243 6263 gastrocnemius muscle +T165 CL:0002211 6314 6327 type I fibers +T166 MOP:0000568 6355 6364 oxidative +T167 CL:0002210 6355 6371 oxidative fibers +T168 GO:0005739 6400 6413 mitochondrial +T169 GO:0005739 6509 6522 mitochondrial +T170 CHEBI:10545 6538 6546 electron +T171 MOP:0000615 6538 6555 electron transfer +T172 PR:000002199 6557 6569 cytochrome c +T173 CHEBI:18070 6557 6569 cytochrome c +T174 CHEBI:18070 6574 6586 cytochrome c +T175 PR:000025365 6574 6594;6601 6603 cytochrome c oxidase ... II +T176 PR:000005776 6574 6594;6608 6610 cytochrome c oxidase ... IV +T177 PR:000025365 6596 6599;6601 6603 COX ... II +T178 PR:000005776 6596 6599;6608 6610 COX ... IV +T179 CHEBI:35366 6616 6626 fatty-acid +T180 GO:0006635 6616 6638 fatty-acid β-oxidation +T181 MOP:0000568 6629 6638 oxidation +T182 PR:000013057 6742 6747 PPARδ +T183 PR:000013059 6773 6779 PGC-1α +T184 CL:0000187 6807 6819 muscle fiber +T185 GO:0005739 6831 6844 mitochondrial +T186 GO:0005739 7005 7018 mitochondrial +T187 SO:0001026 7019 7025 genome +T188 PR:000025365 7034 7039 COXII +T189 NCBITaxon:10088 7062 7066 mice +T190 GO:0005739 7080 7093 Mitochondrial +T191 SO:0001032 7080 7097 Mitochondrial DNA +T192 UBERON:0001388 7124 7144 gastrocnemius muscle +T193 NCBITaxon:10088 7163 7167 mice +T194 GO:0005739 7226 7239 mitochondrial +T195 CL:0000187 7296 7308 muscle fiber +T196 CL:0002211 7420 7432 type I fiber +T197 PR:000002199 7465 7477 cytochrome c +T198 CHEBI:18070 7465 7477 cytochrome c +T199 PR:000010690 7465 7475;7482 7483 cytochrome ... b +T200 CHEBI:38551 7465 7475;7482 7483 cytochrome ... b +T201 GO:0006936 7549 7560 contraction +T202 PR:000016504 7569 7586 troponin I (slow) +T203 CL:0002211 7590 7602 type I fiber +T204 GO:0006936 7687 7698 contraction +T205 PR:000016505 7707 7724 troponin I (fast) +T206 CL:0002212 7728 7741 type II fiber +T207 PR:000013057 7849 7854 PPARδ +T208 SO:0000704 7883 7888 genes +T209 NCBITaxon:10088 7946 7950 mice +T210 NCBITaxon:10088 8003 8007 mice +T211 PR:000013057 8017 8022 PPARδ +T212 CHEBI:73726 8040 8048 GW501516 +T213 SO:0000704 8064 8069 genes +T214 CL:0000189 8074 8084 slow fiber +T215 GO:0006936 8085 8096 contractile +T216 GO:0005739 8107 8120 mitochondrial +T217 MOP:0000568 8139 8148 oxidation +T218 CL:0000187 8249 8261 muscle fiber +T219 CL:0002211 8280 8286 type I +T220 PR:000013057 8335 8340 PPARδ +T221 CL:0000187 8351 8363 Muscle Fiber +T222 PR:000013057 8374 8379 PPARδ +T223 http://purl.obolibrary.org/obo/MONDO_0011122 8397 8404 Obesity +T224 http://purl.obolibrary.org/obo/MONDO_0011122 8451 8456 obese +T225 NCBITaxon:1 8457 8468 individuals +T226 MOP:0000568 8480 8489 oxidative +T227 CL:0002210 8480 8496 oxidative fibers +T228 MOP:0000568 8528 8537 oxidative +T229 CL:0002210 8528 8544 oxidative fibers +T230 http://purl.obolibrary.org/obo/MONDO_0011122 8570 8577 obesity +T231 GO:0007631 8619 8622 fed +T232 NCBITaxon:10088 8638 8642 mice +T233 NCBITaxon:10088 8792 8796 mice +T234 NCBITaxon:33208 8901 8908 animals +T235 NCBITaxon:10088 8937 8941 mice +T236 http://purl.obolibrary.org/obo/MONDO_0011122 9124 9129 obese +T237 NCBITaxon:10088 9154 9158 mice +T238 UBERON:0010410 9262 9278 inguinal fat pad +T239 NCBITaxon:10088 9331 9335 mice +T240 MOP:0000568 9377 9386 oxidative +T241 CHEBI:24384 9456 9464 glycogen +T242 CHEBI:17855 9478 9490 triglyceride +T243 NCBITaxon:10088 9531 9535 mice +T244 CHEBI:17234 9589 9596 glucose +T245 NCBITaxon:10088 9652 9656 mice +T246 PR:000013057 9722 9727 PPARδ +T247 CHEBI:73730 9722 9735 PPARδ agonist +T248 CHEBI:73726 9736 9744 GW501516 +T249 CHEBI:73726 9755 9763 GW501516 +T250 SO:0000704 9798 9803 genes +T251 CL:0002211 9808 9828 type I muscle fibers +T252 CHEBI:17234 9958 9965 glucose +T253 CL:0000187 10033 10045 muscle fiber +T254 PR:000013057 10081 10086 PPARδ +T255 CHEBI:73730 10081 10094 PPARδ agonist +T256 SO:0000902 10112 10121 transgene +T257 http://purl.obolibrary.org/obo/MONDO_0011122 10152 10159 obesity +T258 PR:000013057 10176 10181 PPARδ +T259 MOP:0000568 10220 10229 oxidative +T260 CL:0002211 10306 10319 type I fibers +T261 CL:0002211 10428 10441 type I fibers +T262 PR:000013057 10468 10473 PPARδ +T263 GO:0010467 10474 10484 expression +T264 SO:0000704 10600 10611 genetically +T265 NCBITaxon:10088 10811 10815 Mice +T266 NCBITaxon:10088 10939 10943 mice +T267 NCBITaxon:10088 11064 11068 mice +T268 NCBITaxon:10088 11398 11402 mice +T269 CL:0000187 11483 11495 muscle fiber +T270 SO:0000704 11531 11542 genetically +T271 CL:0000187 11552 11564 muscle fiber +T272 PR:000013057 11671 11676 PPARδ +T273 PR:000013057 11740 11745 PPARδ +T274 NCBITaxon:10088 11751 11755 mice +T275 PR:000013057 11933 11938 PPARδ +T276 PR:000013057 12015 12020 PPARδ +T277 GO:0065007 12058 12066 regulate +T278 CL:0000187 12067 12079 muscle fiber +T279 NCBITaxon:10088 12134 12138 mice +T280 GO:0010467 12203 12213 expression +T281 PR:000013057 12238 12243 PPARδ +T282 MOP:0000568 12291 12300 oxidation +T283 GO:0005739 12310 12323 mitochondrial +T284 GO:0006412 12340 12353;12391 12399 production of ... proteins +T285 CL:0002211 12366 12378 type I fiber +T286 GO:0006936 12379 12390 contractile +T287 CL:0000187 12424 12436 muscle fiber +T288 MOP:0000568 12491 12500 oxidation +T289 PR:000013057 12512 12517 PPARδ +T290 CL:0000187 12684 12696 muscle fiber +T291 MOP:0000568 12752 12761 oxidative +T292 CL:0002211 12811 12830 type I muscle fiber +T293 http://purl.obolibrary.org/obo/MONDO_0011122 12903 12910 obesity +T294 CL:0000187 12964 12976 muscle fiber +T295 CL:0000100 13057 13069 motor neuron +T296 GO:0008152 13095 13104 metabolic +T297 PR:000013057 13201 13206 PPARδ +T298 NCBITaxon:10088 13483 13487 mice +T299 GO:0010467 13488 13498 expressing +T300 PR:000004978 13519 13529 calmodulin +T301 PR:000013059 13551 13557 PGC-1α +T302 PR:000013057 13627 13632 PPARδ +T303 PR:000013057 13805 13810 PPARδ +T304 CL:0000187 13856 13868 muscle fiber +T305 GO:0010467 13940 13950 expression +T306 PR:000013057 13964 13969 PPARδ +T307 http://purl.obolibrary.org/obo/MONDO_0011122 14040 14047 obesity +T308 MOP:0000568 14077 14086 oxidation +T309 PR:000013057 14227 14232 PPARδ +T310 PR:000013057 14279 14284 PPARδ +T311 PR:000013057 14404 14409 PPARδ +T312 PR:000013057 14541 14546 PPARδ +T313 UBERON:0000479 14550 14557 tissues +T314 CHEBI:35366 14598 14608 fatty-acid +T315 CHEBI:35366 14638 14649 Fatty acids +T316 CHEBI:25212 14660 14671 metabolites +T317 PR:000013057 14685 14690 PPARδ +T318 GO:0010467 14735 14745 expression +T319 PR:000013059 14749 14755 PGC-1α +T320 PR:000013057 14796 14801 PPARδ +T321 PR:000013059 14869 14875 PGC-1α +T322 PR:000013057 14903 14908 PPARδ +T323 UBERON:0002385 14912 14925 muscle tissue +T324 PR:000013057 15023 15028 PPARδ +T325 PR:000013057 15157 15162 PPARδ +T326 GO:0065007 15171 15181 regulatory +T327 CL:0000187 15254 15266 muscle fiber +T328 UBERON:0004288 15314 15322 Skeletal +T329 GO:0065007 15349 15357 regulate +T330 CHEBI:35366 15369 15379 fatty-acid +T331 GO:0006631 15369 15379;15392 15402 fatty-acid ... metabolism +T332 CHEBI:17234 15384 15391 glucose +T333 GO:0006006 15384 15402 glucose metabolism +T334 NCBITaxon:10088 15417 15421 mice +T335 MOP:0000568 15437 15446 oxidative +T336 CL:0002210 15437 15453 oxidative fibers +T337 http://purl.obolibrary.org/obo/MONDO_0011122 15488 15495 obesity +T338 CHEBI:17234 15500 15507 glucose +T339 http://purl.obolibrary.org/obo/MONDO_0001076 15500 15519 glucose intolerance +T340 PR:000013057 15604 15609 PPARδ +T341 PR:000013057 15645 15650 PPARδ +T342 CHEBI:17234 15682 15689 glucose +T343 GO:0006006 15682 15700 glucose metabolism +T344 PR:000045358 15761 15768 insulin +T345 UBERON:0004288 15817 15825 skeletal +T346 GO:0005739 15864 15877 mitochondrial +T347 PR:000013057 15940 15945 PPARδ +T348 GO:0005739 15959 15972 mitochondrial +T349 MOP:0000568 15988 15997 oxidative +T350 PR:000013057 16021 16026 PPARδ +T351 GO:0065007 16050 16057 control +T352 PR:000045358 16061 16068 insulin +T353 GO:0007568 16094 16099 aging +T354 PR:000013057 16136 16141 PPARδ +T355 GO:0065007 16193 16201 regulate +T356 CL:0000187 16202 16214 muscle fiber +T357 http://purl.obolibrary.org/obo/MONDO_0011122 16230 16237 obesity +T358 PR:000045358 16250 16257 insulin +T359 SO:0000704 16430 16441 genetically +T360 NCBITaxon:33208 16481 16488 Animals +T361 SO:0000417 16511 16517 domain +T362 PR:P06492 16581 16585 VP16 +T363 SO:0001817 16596 16604 in frame +T364 NCBITaxon:10088 16628 16633 mouse +T365 PR:000013057 16634 16639 PPARδ +T366 PR:P06492 16645 16649 VP16 +T367 PR:000013057 16650 16655 PPARδ +T368 NCBITaxon:9606 16697 16702 human +T369 PR:000003674 16703 16719 α-skeletal actin +T370 UBERON:0004288 16705 16713 skeletal +T371 SO:0000167 16720 16728 promoter +T372 NCBITaxon:10633 16778 16782 SV40 +T373 SO:0000188 16783 16789 intron +T374 SO:0000610 16790 16806 poly(A) sequence +T375 SO:0000902 16812 16821 transgene +T376 CL:0000365 16871 16878 zygotes +T377 NCBITaxon:10088 16891 16895 mice +T378 CHEBI:33290 17024 17028 chow +T379 NCBITaxon:10088 17050 17054 mice +T380 PR:000013057 17115 17120 PPARδ +T381 NCBITaxon:10088 17126 17130 mice +T382 NCBITaxon:10088 17178 17182 Mice +T383 GO:0007631 17188 17191 fed +T384 CHEBI:33290 17210 17214 chow +T385 CHEBI:73726 17476 17484 GW501516 +T386 CHEBI:36357 17485 17493 compound +T387 NCBITaxon:10088 17498 17502 mice +T388 SO:0000704 17559 17563 Gene +T389 GO:0010467 17559 17574 Gene expression +T390 NCBITaxon:10088 17611 17616 Mouse +T391 SO:0000345 17617 17620 EST +T392 GO:0042571 17742 17752 Antibodies +T393 GO:0005634 17890 17897 nuclear +T394 NCBITaxon:10088 17999 18003 mice +T395 NCBITaxon:10088 18291 18295 mice +T396 CL:0000187 18348 18360 Muscle fiber +T397 GO:0005739 18372 18385 mitochondrial +T398 SO:0001032 18372 18389 mitochondrial DNA +T399 CL:0000187 18401 18413 Muscle fiber +T400 CHEBI:37958 18467 18470 dye +T401 GO:0005739 18534 18546 mitochondria +T402 GO:0005739 18584 18597 Mitochondrial +T403 SO:0001032 18584 18601 Mitochondrial DNA +T404 CHEBI:2511 18634 18641 agarose +T405 NCBITaxon:10088 18680 18684 mice +T406 NCBITaxon:10088 18992 18996 mice +T407 NCBITaxon:10088 19307 19311 mice +T408 PR:000003674 19564 19580 α-skeletal actin +T409 UBERON:0004288 19566 19574 skeletal +T410 SO:0000167 19581 19589 promoter +T411 NCBITaxon:4097 19949 19956 Tobacco +T412 http://purl.obolibrary.org/obo/MONDO_0000001 19965 19972 Disease +T413 CHEBI:18070 20493 20505 cytochrome c +T414 PR:000005836 20515 20520 mCPT1 +T415 PR:000005836 20523 20562 muscle carnitine palmitoyltransferase-1 +T416 CHEBI:17126 20530 20539 carnitine +T417 PR:000013059 20564 20570 PGC-1α +T418 GO:0005777 20573 20583 Peroxisome +T419 PR:000013059 20573 20636 Peroxisome proliferator-activated receptor-gamma coactivator 1α +T420 GO:0005777 20645 20655 peroxisome +T421 GO:0010467 20745 20755 Expression +T422 PR:000013057 20770 20775 PPARδ +T423 PR:P06492 20780 20784 VP16 +T424 PR:000013057 20785 20790 PPARδ +T425 SO:0000902 20791 20800 Transgene +T426 UBERON:0005090 20849 20856 muscles +T427 NCBITaxon:10088 20886 20890 mice +T428 GO:0097617 20895 20905 hybridized +T429 UBERON:0001386 20929 20932 EDL +T430 UBERON:0001386 20934 20959 extensor digitorum longus +T431 UBERON:0001388 20961 20967 Gastro +T432 UBERON:0001388 20969 20982 gastrocnemius +T433 GO:0005634 20996 21003 nuclear +T434 UBERON:0005090 21040 21047 muscles +T435 PR:000013057 21099 21104 PPARδ +T436 GO:0042571 21105 21113 antibody +T437 GO:0010467 21178 21188 Expression +T438 PR:P06492 21196 21200 VP16 +T439 PR:000013057 21201 21206 PPARδ +T440 SO:0000902 21207 21216 transgene +T441 UBERON:0000479 21228 21235 tissues +T442 UBERON:0000479 21266 21272 tissue +T443 GO:0097617 21277 21287 hybridized +T444 PR:P06492 21295 21299 VP16 +T445 UBERON:0001388 21312 21332 Gastrocnemius muscle +T446 GO:0005634 21353 21360 Nuclear +T447 UBERON:0001388 21397 21417 gastrocnemius muscle +T448 NCBITaxon:10088 21436 21440 mice +T449 GO:0042571 21508 21518 antibodies +T450 PR:000013057 21581 21586 PPARδ +T451 GO:0042571 21587 21595 antibody +T452 MOP:0000568 21643 21652 Oxidative +T453 CL:0002211 21643 21666 Oxidative Type I Fibers +T454 NCBITaxon:10088 21685 21689 Mice +T455 UBERON:0005090 21701 21708 Muscles +T456 NCBITaxon:10088 21723 21727 mice +T457 NCBITaxon:10088 21768 21772 mice +T458 CL:0002212 21814 21821 type II +T459 UBERON:0011905 21822 21838 plantaris muscle +T460 CL:0002211 21840 21853 Type I fibers +T461 PR:000013057 21902 21907 PPARδ +T462 SO:0000704 21916 21921 Genes +T463 CL:0002211 21934 21947 Type I Fibers +T464 GO:0005739 21961 21974 Mitochondrial +T465 UBERON:0001388 22028 22048 gastrocnemius muscle +T466 SO:0000704 22172 22176 gene +T467 SO:0001026 22198 22205 genomic +T468 UBERON:0001388 22237 22257 gastrocnemius muscle +T469 PR:000025365 22321 22326 COXII +T470 GO:0005739 22328 22341 mitochondrial +T471 SO:0001026 22342 22348 genome +T472 PR:000013829 22362 22367 MCIP1 +T473 GO:0005634 22369 22376 nuclear +T474 SO:0001026 22377 22383 genome +T475 UBERON:0001388 22427 22447 gastrocnemius muscle +T476 NCBITaxon:10088 22484 22488 mice +T477 GO:0005739 22520 22533 mitochondrial +T478 SO:0001032 22520 22537 mitochondrial DNA +T479 CHEBI:2511 22571 22578 agarose +T480 GO:0005739 22610 22623 mitochondrial +T481 SO:0001032 22610 22627 mitochondrial DNA +T482 NCBITaxon:10088 22656 22660 mice +T483 CL:0000187 22705 22717 muscle fiber +T484 GO:0005739 22730 22743 mitochondrial +T485 UBERON:0001388 22797 22817 gastrocnemius muscle +T486 NCBITaxon:10088 22849 22853 mice +T487 PR:000013057 22883 22888 PPARδ +T488 CHEBI:73730 22883 22896 PPARδ agonist +T489 CHEBI:73726 22897 22905 GW501516 +T490 UBERON:0001388 22957 22977 gastrocnemius muscle +T491 http://purl.obolibrary.org/obo/MONDO_0011122 23054 23061 Obesity +T492 NCBITaxon:10088 23080 23084 Mice +T493 GO:0007631 23182 23185 fed +T494 NCBITaxon:10088 23292 23296 mice +T495 GO:0007631 23394 23401 feeding +T496 UBERON:0010410 23421 23437 inguinal fat pad +T497 CHEBI:24384 23539 23547 glycogen +T498 CHEBI:17855 23564 23576 triglyceride +T499 NCBITaxon:10088 23592 23596 mice +T500 GO:0007631 23619 23626 feeding +T501 CHEBI:17234 23641 23648 Glucose +T502 NCBITaxon:10088 23665 23669 Mice +T503 GO:0007631 23692 23699 feeding +T504 CHEBI:17234 23743 23750 glucose +T505 UBERON:0000178 23797 23802 blood +T506 CHEBI:17234 23803 23810 glucose +T507 PR:000013057 23874 23879 PPARδ +T508 CHEBI:73730 23874 23888 PPARδ Agonists +T509 http://purl.obolibrary.org/obo/MONDO_0011122 23900 23907 Obesity +T510 NCBITaxon:10088 23970 23974 mice +T511 GO:0007631 23980 23983 fed +T512 CHEBI:73726 24031 24039 GW501516 +T513 UBERON:0001388 24091 24111 gastrocnemius muscle +T514 NCBITaxon:10088 24176 24180 mice +T515 NCBITaxon:10088 24234 24238 mice +T516 CHEBI:73726 24343 24351 GW501516 +T517 UBERON:0000479 24380 24386 tissue +T518 NCBITaxon:10088 24399 24403 mice +T519 UBERON:0008337 24434 24442 inguinal +T520 GO:0000003 24455 24467 reproductive +T521 UBERON:0003693 24483 24498 retroperitoneal +T522 CHEBI:17234 24509 24516 Glucose +T523 NCBITaxon:10088 24533 24537 Mice +T524 CHEBI:17234 24604 24611 glucose +T525 UBERON:0000178 24653 24658 Blood +T526 CHEBI:17234 24659 24666 glucose +T527 PR:000013057 24727 24732 PPARδ +T528 GO:0065007 24733 24742 Regulates +T529 NCBITaxon:10088 24815 24819 mice +T530 PR:000013057 25023 25028 PPARδ +T531 NCBITaxon:10088 25034 25038 mice +T532 PR:000013057 25059 25064 PPARδ +T533 NCBITaxon:10088 25070 25074 mice +T534 PR:000013057 25217 25222 PPARδ +T535 UBERON:0004288 25226 25234 skeletal +T536 CHEBI:33893 25537 25545 reagents +T537 GO:0065007 25730 25740 Regulation +T538 CL:0000187 25744 25756 muscle fiber +T539 PR:000013057 25787 25792 PPARδ diff --git a/src/ontogpt/evaluation/craft/database/all/15328533.txt b/src/ontogpt/evaluation/craft/database/all/15328533.txt new file mode 100644 index 000000000..4caf75b0d --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15328533.txt @@ -0,0 +1,179 @@ +Regulation of Muscle Fiber Type and Running Endurance by PPARδ + +Abstract + +Endurance exercise training can promote an adaptive muscle fiber transformation and an increase of mitochondrial biogenesis by triggering scripted changes in gene expression. However, no transcription factor has yet been identified that can direct this process. We describe the engineering of a mouse capable of continuous running of up to twice the distance of a wild-type littermate. This was achieved by targeted expression of an activated form of peroxisome proliferator-activated receptor δ (PPARδ) in skeletal muscle, which induces a switch to form increased numbers of type I muscle fibers. Treatment of wild-type mice with PPARδ agonist elicits a similar type I fiber gene expression profile in muscle. Moreover, these genetically generated fibers confer resistance to obesity with improved metabolic profiles, even in the absence of exercise. These results demonstrate that complex physiologic properties such as fatigue, endurance, and running capacity can be molecularly analyzed and manipulated. + +Introduction + +Skeletal muscle fibers are generally classified as type I (oxidative/slow) or type II (glycolytic/fast) fibers. They display marked differences in respect to contraction, metabolism, and susceptibility to fatigue. Type I fibers are mitochondria-rich and mainly use oxidative metabolism for energy production, which provides a stable and long-lasting supply of ATP, and thus are fatigue-resistant. Type II fibers comprise three subtypes, IIa, IIx, and IIb. Type IIb fibers have the lowest levels of mitochondrial content and oxidative enzymes, rely on glycolytic metabolism as a major energy source, and are susceptible to fatigue, while the oxidative and contraction functions of type IIa and IIx lie between type I and IIb (Booth and Thomason 1991; Berchtold et al. 2000; Olson and Williams 2000). Adult skeletal muscle shows plasticity and can undergo conversion between different fiber types in response to exercise training or modulation of motoneuron activity (Booth and Thomason 1991, Jarvis et al. 1996; Pette 1998; Olson and Williams 2000; Hood 2001). This conversion of muscle fiber from type IIb to type IIa and type I is likely to be mediated by a calcium signaling pathway that involves calcineurin, calmodulin-dependent kinase, and the transcriptional cofactor Peroxisome proliferator-activated receptor-gamma coactivator 1α (PGC-1α) (Naya et al. 2000; Olson and Williams 2000; Lin et al. 2002; Wu et al. 2002). However, the targeted transcriptional factors directly responsible for reprogramming the fiber-specific contractile and metabolic genes remain to be identified. + +Muscle fiber specification appears to be associated with obesity and diabetes. For instance, rodents that gain the most weight on high-fat diets possess fewer type I fibers (Abou et al. 1992). In obese patients, skeletal muscle has been observed to have reduced oxidative capacity, increased glycolytic capacity, and a decreased percentage of type I fibers (Hickey et al. 1995; Tanner et al. 2002). Similar observations have been made in type 2 diabetic patients (Lillioja et al. 1987; Hickey et al. 1995). Recently, it has been shown that increasing oxidative fibers can lead to improved insulin action and reduced adipocyte size (Luquet et al. 2003; Ryder et al. 2003). + +We have previously established that peroxisome proliferator-activated receptor (PPAR) δ is a major transcriptional regulator of fat burning in adipose tissue through activation of enzymes associated with long-chain fatty-acid β-oxidation (Wang et al. 2003). Although PPARδ is the predominant PPAR isoform present in skeletal muscle, its in vivo function has not been determined. Our current study uncovers PPARδ as the first transcription factor able to drive the formation of functional type I muscle fibers, whose activation entrains complex pathways both enhancing physical performance and creating a state of obesity resistance. + +Results + +Activation of PPARδ Leads to Muscle Fiber Transformation + +A role of PPARδ in muscle fiber was suggested by its enhanced expression—at levels 10-fold and 50-fold greater than PPARα and γ isoforms, respectively (unpublished data). An examination of PPARδ in different muscle fibers reveals a significantly higher level in type I muscle (soleus) relative to type II–rich muscle (extensor digitorum longus) or type I and type II mixed muscle (gastrocnemius) (Figure 1A); this expression pattern closely resembles that of PGC-1α (Lin et al. 2002). A similar pattern but with more pronounced differences was found at the protein level (Figure 1B). + +To directly assess the role of activation of PPARδ in control of muscle fiber plasticity and mitochondrial biogenesis, we generated mice expressing a transgene in which the 78-amino-acid VP16 activation domain was fused to the N-terminus of full-length PPARδ, under control of the 2.2-kb human α-skeletal actin promoter. In agreement with the previous characterization of this promoter (Brennan and Hardeman 1993; Clapham et al. 2000), the VP16-PPARδ transgene was selectively expressed in skeletal muscle, with 10-fold less in the heart (Figure 1C). Among different types of muscle fibers, the levels of VP16-PPARδ expression appeared to be similar (unpublished data). As shown in Figure 1D for gastrocnemius muscle, VP16-PPARδ fusion protein was produced at a level similar to that of endogenous PPARδ in wild-type littermates. Interestingly, the level of endogenous muscle PPARδ protein in the transgenic mice was much higher than in the control littermates. The substantial increase of endogenous PPARδ may have been caused by a switch to type I fiber (see below), which intrinsically expresses higher levels of PPARδ (Figure 1A and 1B). + +Type I muscle can be readily distinguished from type II or mixed muscle by its red color, because of its high concentration of myoglobin, a protein typically expressed in oxidative muscle fibers. We found that muscles in the transgenic mice appeared redder (Figure 2A), which is particularly evident in the mixed type I/II fibers of the hindlimb (Figure 2B). Indeed, metachromatic staining revealed a substantial muscle fiber transformation (Figure 2C). In gastrocnemius muscle, we estimated that there was a 2-fold increase of type I fibers. A diagnostic component of oxidative fibers is their high myoglobin and mitochondrial content, which is supported by the mRNA analysis shown in Figure 3A. In addition to myoglobin, mitochondrial components for electron transfer (cytochrome c and cytochrome c oxidase [COX] II and IV) and fatty-acid β-oxidation enzymes were elevated (Figure 3A; unpublished data). These effects appear to be direct consequences of PPARδ activation, as levels of PGC-1α, a coactivator involved in muscle fiber switch and mitochondrial biogenesis (Wu et al. 1999; Lehman et al. 2000; Lin et al. 2002), remained unchanged. Southern blot analysis detected a substantially higher copy number of the mitochondrial genome–encoded COXII DNA in the transgenic mice (Figure 3B). Mitochondrial DNA was increased 2.3-fold in gastrocnemius muscle of the transgenic mice (Figure 3C). These results reveal a marked stimulation of mitochondrial biogenesis and further support the idea that there is a muscle fiber switch. This conclusion was also confirmed by Western blot analysis. As shown in Figure 3D, the characteristic type I fiber proteins, such as myoglobin and cytochrome c and b, were significantly increased. More importantly, the specialized contraction protein troponin I (slow) of type I fiber was robustly induced; this was accompanied by a marked reduction of the specialized contraction protein troponin I (fast) of type II fiber, indicating a high degree of fiber transformation. We next examined whether acute activation of endogenous PPARδ would induce similar target genes. In agreement with the chronic effects in the transgenic mice, we found that, after treatment of wild-type C57B6J mice with the PPARδ-specific agonist GW501516 for only 10 d, genes for slow fiber contractile proteins, mitochondrial biogenesis, and β-oxidation were all upregulated (Figure 3E). This indicates that rapid, systematic, and coordinated changes of muscle fiber properties toward type I can be achieved by activation of the endogenous PPARδ pathway. + +Muscle Fiber Switch by PPARδ Protects Against Obesity + +A number of previous studies have shown that obese individuals have fewer oxidative fibers, implying that the presence of oxidative fibers alone may play a part in obesity resistance. To test this possibility, we fed the transgenic mice and their wild-type littermates with a high-fat diet for 97 d. Although the initial body weights of the two groups were very similar, the transgenic mice had gained less than 50% at day 47, and only one-third at day 97, of the weight gained by the wild-type animals (Figure 4A). The transgenic mice displayed significantly higher oxygen consumption on the high-fat diet than the control littermates (unpublished data). By the end of this experiment, the control littermates became obese, whereas the transgenic mice still maintained a normal body weight and fat mass composition (Figure 4A). A histological analysis of inguinal fat pad revealed a much smaller cell size in the transgenic mice (Figure 4B), due to the increased muscle oxidative capacity. While there was no significant difference in intramuscular glycogen content, the triglyceride content was much less in the transgenic mice (Figure 4C and 4D), which may explain their improved glucose tolerance (Figure 4E). We also placed wild-type C57BJ6 mice on the high-fat diet and treated them with either vehicle or the PPARδ agonist GW501516 for 2 mo. GW501516 produced a sustained induction of genes for type I muscle fibers; this, at least in part, resulted in an only 30% gain in body weight, a dramatically reduced fat mass accumulation, and improved glucose tolerance, compared to the vehicle-treated group (Figure 5). Thus, muscle fiber conversion by stimulation with the PPARδ agonist or the activated transgene has a protective role against obesity. + +Activation of PPARδ Enhances Physical Performance + +Muscle oxidative capacity is a crucial factor for determining endurance and fatigue. Indeed, type I fibers adaptively generated through exercise training are considered to be fatigue resistant. However, whether the type I fibers generated molecularly via PPARδ expression can contribute to enhanced performance in the absence of previous training is unclear. In fact, the consequence of genetically induced fiber switch on running capacity has to our knowledge never been evaluated. We thus compared exercise performance between untrained, body-weight-matched transgenic and wild-type littermates. Mice were run on oxygen-infused, enclosed treadmills until exhaustion. Strikingly, the running time and distance the transgenic mice were able to sustain were increased by 67% and 92%, respectively (Figure 6A; also see Videos S1 and S2). The transgenic mice ran about 1 h longer than the controls, which translates to nearly a kilometer further. No significant differences in muscle mass (unpublished data) and daily activity (total counts of activity per hour: 1618 ± 209 for transgenic versus 1987 ± 301 for wild-type, p > 0.35, n = 4) were observed between the transgenic and control mice. Thus, the remarkable increase in endurance is the physiologic manifestation of muscle fiber transformation. This suggests that genetically directed muscle fiber switch is physiologically and functionally relevant. In addition, we looked at what effect the absence of PPARδ function has on exercise endurance. In the treadmill test, the PPARδ-null mice could sustain only 38% of the running time and 34% of the distance of their age- and weight-matched wild-type counterparts (Figure 6B). These results further support a role for PPARδ in enhancement of physical performance. + +Discussion + +Our data reveal that a PPARδ-mediated transcriptional pathway can regulate muscle fiber specification, enabling the generation of a strain of mice with a “long-distance running” phenotype. We show that targeted expression of an activated form of PPARδ produces profound and coordinated increases in oxidation enzymes, mitochondrial biogenesis, and production of specialized type I fiber contractile proteins—the three hallmarks for muscle fiber type switching (Figure 6C). While induction of muscle oxidation enzymes by PPARδ has been seen both in vivo and in vitro (Muoio et al. 2002; Dressel et al. 2003; Luquet et al. 2003; Tanaka et al. 2003; Wang et al. 2003), its effects shown here on muscle fiber switching are unexpected. These progressive changes in oxidative capacity in conjunction with eventual changes in type I muscle fiber lead to a dramatically improved exercise profile and protection against obesity. This does not solely depend on achieving a directed muscle fiber type switch but also requires all the associated changes in neural innervation, motor neuron function, and peripheral metabolic adaptation to enable a new integrated physiological response. Accordingly, activation of muscle PPARδ essentially recapitulates the effects of exercise training even in the absence of training itself. To our knowledge, this has not been directly described for any other transcriptional factor. + +The muscle phenotypes described here are remarkably similar to those of transgenic mice expressing either calcineurin, calmodulin-dependent kinase, or PGC-1α (Naya et al. 2000; Lin et al. 2002; Wu et al. 2002), indicating that PPARδ could be one of the hypothetical downstream transcription factors of these pathways. It is important to note that, from our ligand and gain-of-function transgenic studies, PPARδ needs to be activated in order to direct the muscle fiber switch. Indeed, in a recent report by Luquet et al. (2003), simple overexpression of wild-type PPARδ in muscle was found not to be sufficient to promote a fiber switch or obesity resistance, although certain oxidation enzymes were increased. This supports the model in Figure 6C that the activating signal or ligand, but not the receptor, is limiting. Thus, PPARδ activation, rather than merely an increase of PPARδ levels, is an essential element for fiber switching and its associated functional manifestations. How might endogenous PPARδ become activated naturally by exercise training? First, it is possible that exercise generates or increases endogenous ligands for PPARδ as tissues are undergoing substantial increases in fatty-acid internalization and burning. Fatty acids and their metabolites can activate PPARδ. A second model is that exercise may induce expression of PGC-1α (Goto et al. 2000) and thereby activate PPARδ. This is consistent with previous work in which we have shown that PGC-1α physically associates with PPARδ in muscle tissue and can powerfully activate it even in the absence of ligands (Wang et al. 2003). Alternatively, PPARδ may be activated by a distal upstream signaling component such as a kinase cascade. Further dissecting the interactions between PPARδ and its regulatory components will be necessary to fully understand the molecular basis of muscle fiber determination pertinent to exercise training. + +Skeletal muscle is a major site to regulate whole-body fatty-acid and glucose metabolism. We show that mice with increased oxidative fibers are resistant to high-fat-induced obesity and glucose intolerance. Moreover, ligand studies provide compelling evidence that activation of endogenous PPARδ can produce similar effects. Might PPARδ have any beneficial effects on glucose metabolism in the lean condition? This has not been explored; however, insulin resistance in the elderly is confined mostly to skeletal muscle and may be due to reduction of mitochondrial number and/or function (Petersen et al. 2003). The ability of PPARδ to stimulate mitochondrial biogenesis and oxidative function suggests that PPARδ could be important for control of insulin resistance during normal aging. Together, these data indicate that PPARδ and its ligands comprise a key molecular switch to regulate muscle fiber specification, obesity resistance, insulin sensitivity, and, most surprisingly, physical endurance. This work demonstrates that complex physiologic properties such as fatigue, endurance, and running capacity can be genetically manipulated. + +Materials and Methods + + + +Animals. + +The transactivation domain (78 amino acid residues, corresponding to residues 413–490) of VP16 was fused in frame with the N-terminus of mouse PPARδ. The VP16-PPARδ fusion cDNA was placed downstream of the human α-skeletal actin promoter (Brennan and Hardeman 1993), and upstream of the SV40 intron/poly(A) sequence. The transgene was purified and injected into C57BL/6J × CBA F1 zygotes. Transgenic mice were backcrossed with C57BL/6J for two generations. Wild-type littermates were used as controls throughout the study. On normal chow diet, the transgenic mice and control littermates used here had similar body weights. PPARδ-null mice were previously generated (Barak et al. 2002). Mice were fed either a standard chow with 4% (w/w) fat content (Harlan Teklad, Harlan, Indianapolis, Indiana, United States) or a high-fat diet containing 35% (w/w) fat content (product F3282, Bioserv, Frenchtown, New Jersey, United States) as indicated. For ligand experiments, we synthesized the GW501516 compound and mice were orally gavaged daily (10 mg/kg or vehicle alone). + +Gene expression analysis and physiological studies + +Mouse EST clones were obtained from ATCC (Manassas, Virginia, United States), verified by sequencing, and used as Northern probes. Antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, California, United States). Total muscle protein extracts (Lin et al. 2002) and nuclear proteins (Wang et al. 2003) were prepared as described. + +Prior to the exercise performance test, the mice were accustomed to the treadmill (Columbus Instruments, Columbus, Ohio, United States) with a 5-min run at 7 m/min once per day for 2 d. The exercise test regimen was 10 m/min for the first 60 min, followed by 1 m/min increment increases at 15-min intervals. Exhaustion was defined when mice were unable to avoid repetitive electrical shocks. + +Muscle fiber typing and mitochondrial DNA isolation + +Muscle fiber typing was essentially performed using metachromatic dye–ATPase methods as described (Ogilvie and Feeback 1990). Muscle mitochondria were isolated (Scholte et al. 1997). Mitochondrial DNA was prepared and analyzed on 1% agarose gel. + +Statistical analysis + +Number of mice for each group used in experiments is indicated in figure legends. Values are presented as mean ± SEM. A two-tailed Student's t test was used to calculate p-values. + +Supporting Information + +Video S1 + +Beginning of Running Test + +This video shows the exercise performance of a representative of the transgenic mice (right chamber) and a representative of wild-type control littermates (left chamber) on the treadmill 15 min into the exercise challenge. + +(52.4 MB MOV). + +Click here for additional data file. + +Video S2 + +Running Test 90 Min Later + +This video shows the exercise performance of a representative of the transgenic mice (right chamber) and a representative of wild-type control littermates (left chamber) on the treadmill 90 min into the exercise challenge. + +(41.7 MB MOV). + +Click here for additional data file. + +Acknowledgements + +We thank Dr. M. Downes for providing the α-skeletal actin promoter; Dr. G. D. Barish for comments on the manuscript; M.A. Lawrence for histology; Dr. S. Pfaff for the use of microscopes and photographic equipment; M. Lieberman and K.L. Schnoeker for photography; J.M. Shelton for advice on fiber staining; and E. Stevens and E. Ong for administrative assistance. YXW was supported by a postdoctoral fellowship from California Tobacco-Related Disease Research Program. JH was supported by the BK21 program, Ministry of Education, Korea. HK was supported by grant M1–0311-00–0145 from the Molecular and Cellular BioDiscovery Research Program, Ministry of Science and Technology, Korea. RME is an Investigator of the Howard Hughes Medical Institute at the Salk Institute for Biological Studies and March of Dimes Chair in Molecular and Developmental Biology. This work was supported by the Howard Hughes Medical Institute and the Hillblom Foundation. + +Abbreviations + +COX - cytochrome c oxidase + +mCPT1 - muscle carnitine palmitoyltransferase-1 + +PGC-1α - Peroxisome proliferator-activated receptor-gamma coactivator 1α + +PPAR - peroxisome proliferator-activated receptor + +UCP - uncoupling protein + +Figures and Tables + +Figure 1 + +Expression of Endogenous PPARδ and VP16-PPARδ Transgene in Muscle + +(A) Pooled RNA isolated from various muscles of five wild-type male C57B6 mice was hybridized with indicated probes. EDL, extensor digitorum longus; Gastro, gastrocnemius. + +(B) Pooled nuclear proteins (15 μg/lane) isolated from muscles of five wild-type male C57B6 were probed with anti-PPARδ antibody. RNA polymerase II (Pol II) is shown as a loading control. + +(C) Expression of the VP16-PPARδ transgene in various tissues. 10 μg of total RNA from each tissue was hybridized with a VP16 cDNA probe. Gastrocnemius muscle was used here. + +(D) Nuclear proteins (15 μg/lane) isolated from gastrocnemius muscle of the transgenic mice (TG) and the wild-type littermates (WT) were probed with indicated antibodies. The upper, nonspecific band that cross-reacted with the anti-PPARδ antibody serves a loading control. + +Figure 2 + +Increased Oxidative Type I Fibers in the Transgenic Mice + +(A and B) Muscles in transgenic mice (TG) are redder than those in wild-type mice (WT). + +(C) Metachromatic staining of the type II plantaris muscle. Type I fibers are stained dark blue. + +Figure 3 + +Activation of PPARδ Induces Genes Typical for Type I Fibers and Promotes Mitochondrial Biogenesis + +(A) Total RNA (10 μg/lane) prepared from gastrocnemius muscle of transgenic (TG) and wild-type (WT) littermates was probed with indicated probes. The fold increase of induction of each gene is shown. + +(B) Total genomic DNA (10 μg/lane) prepared from gastrocnemius muscle was digested with Nco1 and subjected to Southern analysis with COXII (mitochondrial genome–encoded) and MCIP1 (nuclear genome–encoded) DNA probes. + +(C) Equal amounts of gastrocnemius muscle were collected from both transgenic mice and control littermates. Total mitochondrial DNA was isolated and separated on 1% agarose gel. The relative abundance of mitochondrial DNA in transgenic and wild-type mice is presented. + +(D) Western blot analysis of muscle fiber markers and mitochondrial components. Each lane was loaded with 80 μg of total gastrocnemius muscle extracts. + +(E) Wild-type C57B6 mice were treated with vehicle or PPARδ agonist GW501516 for 10 d. Total RNA (10 μg/lane) prepared from the gastrocnemius muscle was probed with indicated probes. + +Figure 4 + +Resistance to High-Fat-Induced Obesity in the Transgenic Mice + +(A) Seven-week-old transgenic (TG) and wild-type (WT) littermates (n = 5–6 for each group) were fed with a high-fat diet for 97 d. Left panel shows net body weight gain, which was calculated for individual mice and then averaged. Right panel shows the body weights before (Day 0) and after (Day 97) high-fat feeding. + +(B) Histology of inguinal fat pad in the transgenic and wild-type littermates under a high-fat diet for 2 mo. + +(C and D) Intramuscular glycogen content (C) and triglyceride content (D) of mice in (A) after high-fat feeding (n = 6). + +(E) Glucose tolerance test. Mice in (A) after high-fat feeding were fasted for 6 h and then injected with glucose at a concentration of 1g/kg body weight. Then blood glucose levels were measured periodically over 2 h (n = 6). + +Figure 5 + +PPARδ Agonists Counteract Obesity Induced by High-Fat Diet + +(A) Eleven-week-old wild-type C57B6 mice were fed a high-fat diet in combination with vehicle or GW501516 for 57 d. Total RNA (10 μg/lane) prepared from the gastrocnemius muscle was probed with indicated probes. + +(B) Net body weight gain for mice in (A) after treatment was calculated for individual mice and averaged. Initial body weights were 28.54 ± 1.04 g for vehicle group (n = 5) and 28.86 ± 0.80 g for GW501516 group (n = 5). + +(C) Various tissue weights for mice in (A) after treatment. ifat, inguinal fat; rdfat, reproductive fat; retrofat, retroperitoneal fat. + +(D) Glucose tolerance test. Mice in (A) after treatment were fasted for 6 h and then injected with glucose at a concentration of 1g/kg body weight. Blood glucose levels were then measured periodically over 2 h. + +Figure 6 + +PPARδ Regulates Exercise Endurance + +(A) Enhanced exercise performance in the transgenic mice. Fourteen-week-old male transgenic and wild-type littermates with similar body weights (n = 4 for each group) were subjected to a forced treadmill exercise test. + +(B) Compromised exercise performance in PPARδ-null mice. Two-month-old male PPARδ-null mice and wild-type controls with similar body weights (n = 6 for each group) were subjected to a forced treadmill exercise test. + +(C) Functions of PPARδ in skeletal muscle. + +Footnotes + +Conflicts of interest. The authors have declared that no conflicts of interest exist. + +Author contributions. YXW and RME conceived and designed the experiments. YXW, CLZ, RTY, MCN, and CRBO performed the experiments. YXW, CLZ, and RME analyzed the data. HKC, JH, and HK contributed reagents/materials/analysis tools. YXW and RME wrote the paper. + +Academic Editor: Steve O'Rahilly, University of Cambridge + +Citation: Wang YX, Zhang CL, Yu RT, Cho HK, Nelson MC, et al. (2004) Regulation of muscle fiber type and running endurance by PPARδ. PLoS Biol 2(10): e294. diff --git a/src/ontogpt/evaluation/craft/database/all/15328538.ann b/src/ontogpt/evaluation/craft/database/all/15328538.ann new file mode 100644 index 000000000..e87edc15d --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15328538.ann @@ -0,0 +1,682 @@ +T1 UBERON:0004288 8 16 Skeletal +T2 PR:000008555 24 30 HIF-1α +T3 UBERON:0004288 138 146 skeletal +T4 GO:0006754 224 241 production of ATP +T5 CHEBI:25212 326 337 metabolites +T6 GO:0006096 339 349 Glycolysis +T7 NCBITaxon:33208 395 402 animals +T8 GO:0008152 413 422 metabolic +T9 GO:0065007 434 443 regulated +T10 PR:000008555 500 527 hypoxia-inducible factor 1α +T11 PR:000008555 529 535 HIF-1α +T12 PR:000008555 563 569 HIF-1α +T13 GO:0065007 573 583 regulating +T14 UBERON:0004288 584 592 skeletal +T15 UBERON:0000479 613 619 tissue +T16 SO:0000704 645 649 gene +T17 UBERON:0004288 673 681 skeletal +T18 GO:0010467 730 740 expression +T19 SO:0000704 744 749 genes +T20 UBERON:0004288 781 789 skeletal +T21 PR:000008555 797 803 HIF-1α +T22 NCBITaxon:10088 813 817 mice +T23 PR:000008555 819 825 HIF-1α +T24 GO:0006096 857 867 glycolytic +T25 GO:0005739 959 971 mitochondria +T26 UBERON:0005090 979 986 muscles +T27 PR:000008555 990 996 HIF-1α +T28 CHEBI:30769 1022 1033 citric acid +T29 GO:0006099 1022 1039 citric acid cycle +T30 CHEBI:35366 1054 1064 fatty acid +T31 GO:0019395 1054 1074 fatty acid oxidation +T32 MOP:0000568 1065 1074 oxidation +T33 GO:0006096 1126 1144 glycolytic pathway +T34 CHEBI:24996 1262 1269 lactate +T35 UBERON:0001977 1277 1282 serum +T36 PR:000008555 1297 1303 HIF-1α +T37 GO:0008152 1314 1323 metabolic +T38 GO:0006096 1340 1350 glycolysis +T39 MOP:0000568 1362 1371 oxidation +T40 PR:000008555 1428 1434 HIF-1α +T41 PR:000008555 1514 1520 HIF-1α +T42 NCBITaxon:33208 1603 1610 animals +T43 NCBITaxon:9606 1666 1672 humans +T44 http://purl.obolibrary.org/obo/MONDO_0000001 1676 1684 diseases +T45 UBERON:0004288 1711 1719 skeletal +T46 GO:0005980 1727 1741 glycogenolysis +T47 GO:0006096 1746 1756 glycolysis +T48 GO:0019222 1837 1854 metabolic control +T49 UBERON:0002385 1953 1966 muscle tissue +T50 UBERON:0004288 2115 2123 skeletal +T51 UBERON:0000479 2194 2200 tissue +T52 GO:0015671 2231 2246 oxygen delivery +T53 GO:0008152 2309 2319 metabolism +T54 GO:0008152 2376 2385 metabolic +T55 UBERON:0004288 2443 2451 skeletal +T56 PR:000008555 2502 2529 hypoxia-inducible factor 1α +T57 PR:000008555 2531 2537 HIF-1α +T58 PR:000008555 2643 2649 HIF-1α +T59 UBERON:0000479 2783 2789 tissue +T60 GO:0065007 2875 2885 regulation +T61 GO:0008152 2889 2898 metabolic +T62 GO:0015671 2953 2964 oxygenation +T63 UBERON:0004288 2970 2978 skeletal +T64 CHEBI:28358 3110 3121 lactic acid +T65 GO:0006096 3143 3161 glycolytic pathway +T66 GO:0008152 3165 3175 metabolism +T67 GO:0006096 3211 3229 glycolytic pathway +T68 GO:0065007 3413 3421 controls +T69 UBERON:0004288 3422 3430 skeletal +T70 NCBITaxon:10088 3460 3465 mouse +T71 UBERON:0004288 3466 3474 skeletal +T72 UBERON:0000479 3486 3492 tissue +T73 PR:000008555 3514 3520 HIF-1α +T74 SO:0001023 3561 3567 allele +T75 SO:0000704 3575 3579 gene +T76 NCBITaxon:10088 3627 3632 mouse +T77 UBERON:0004288 3685 3693 skeletal +T78 PR:000005515 3710 3732 muscle creatine kinase +T79 CHEBI:16919 3717 3725 creatine +T80 PR:000005515 3734 3737 MCK +T81 SO:0000167 3739 3747 promoter +T82 GO:0010467 3762 3772 expression +T83 SO:0000704 3796 3800 gene +T84 GO:0065007 3862 3872 regulation +T85 NCBITaxon:9606 3996 4001 human +T86 GO:0008152 4002 4011 metabolic +T87 http://purl.obolibrary.org/obo/MONDO_0020123 4002 4022 metabolic myopathies +T88 NCBITaxon:10088 4057 4061 mice +T89 UBERON:0004288 4071 4079 skeletal +T90 PR:000008555 4087 4093 HIF-1α +T91 SO:0000704 4094 4098 gene +T92 PR:000008555 4112 4118 HIF-1α +T93 SO:0000902 4295 4304 transgene +T94 PR:000008555 4459 4465 HIF-1α +T95 UBERON:0001388 4483 4496 gastrocnemius +T96 NCBITaxon:10088 4509 4513 mice +T97 SO:0000359 4533 4545 loxP-flanked +T98 SO:0001023 4546 4552 allele +T99 SO:0000902 4569 4578 transgene +T100 GO:0010467 4582 4591 expressed +T101 UBERON:0000948 4612 4619 cardiac +T102 UBERON:0000479 4620 4626 tissue +T103 UBERON:0000948 4632 4639 cardiac +T104 UBERON:0000948 4734 4741 cardiac +T105 CL:0000746 4734 4749 cardiac myocyte +T106 PR:000008555 4772 4778 HIF-1α +T107 GO:0001944 4861 4876 vascularization +T108 GO:0005739 4955 4967 mitochondria +T109 PR:000008555 5034 5040 HIF-1α +T110 CL:0002214 5108 5123 type IIA fibers +T111 UBERON:0001389 5131 5145 soleus muscles +T112 UBERON:0000178 5180 5185 blood +T113 PR:000008555 5217 5223 HIF-1α +T114 NCBITaxon:10088 5247 5251 mice +T115 PR:000008555 5315 5321 HIF-1α +T116 SO:0000704 5332 5336 gene +T117 GO:0010467 5332 5347 gene expression +T118 SO:0000704 5402 5407 genes +T119 CHEBI:17234 5420 5427 glucose +T120 GO:0015758 5420 5437 glucose transport +T121 GO:0006006 5420 5427;5442 5452 glucose ... metabolism +T122 UBERON:0001986 5463 5474 endothelial +T123 CHEBI:17234 5540 5547 glucose +T124 PR:000015066 5540 5561 glucose transporter 4 +T125 PR:000015066 5563 5568 GLUT4 +T126 CHEBI:17234 5591 5598 glucose +T127 CHEBI:17234 5686 5693 glucose +T128 PR:000012583 5713 5756 muscle-specific form of phosphofructokinase +T129 PR:000012583 5758 5763 PFK-M +T130 CHEBI:61304 5766 5782 phosphoglycerate +T131 CHEBI:24996 5801 5808 lactate +T132 PR:000009739 5801 5824 lactate dehydrogenase-A +T133 PR:000009739 5826 5831 LDH-A +T134 PR:000008555 5937 5943 HIF-1α +T135 PR:000008555 5972 5978 HIF-1α +T136 UBERON:0004288 6028 6036 skeletal +T137 GO:0006096 6128 6138 glycolytic +T138 PR:000008555 6171 6177 HIF-1α +T139 PR:000008555 6415 6421 HIF-1α +T140 NCBITaxon:10088 6441 6445 mice +T141 PR:000008555 6463 6469 HIF-1α +T142 GO:0006096 6551 6561 glycolytic +T143 NCBITaxon:10088 6613 6617 mice +T144 PR:000008555 6622 6628 HIF-1α +T145 CHEBI:21008 6674 6688 phosphoglucose +T146 CHEBI:17138 6761 6787 glyceraldehyde 3-phosphate +T147 UBERON:0005090 6855 6862 muscles +T148 CHEBI:15361 6899 6907 pyruvate +T149 NCBITaxon:10088 6996 7000 mice +T150 PR:000008555 7125 7131 HIF-1α +T151 UBERON:0005090 7135 7142 muscles +T152 CHEBI:24996 7262 7269 lactate +T153 NCBITaxon:10088 7280 7284 mice +T154 PR:000008555 7297 7303 HIF-1α +T155 PR:000008555 7378 7384 HIF-1α +T156 GO:0006096 7530 7540 glycolytic +T157 CHEBI:25212 7541 7552 metabolites +T158 PR:000008555 7583 7589 HIF-1α +T159 CHEBI:17234 7645 7652 glucose +T160 GO:0046323 7645 7659 glucose uptake +T161 NCBITaxon:33208 7667 7674 animals +T162 CHEBI:17234 7743 7750 glucose +T163 GO:0006096 7827 7845 glycolytic pathway +T164 GO:0005980 7922 7936 glycogenolysis +T165 CHEBI:17287 7976 7991 phosphocreatine +T166 CHEBI:17287 7993 7996 PCr +T167 CHEBI:17287 8044 8047 PCr +T168 PR:000008555 8061 8067 HIF-1α +T169 CHEBI:24996 8211 8218 lactate +T170 PR:000008555 8240 8246 HIF-1α +T171 NCBITaxon:10088 8258 8262 mice +T172 CHEBI:24996 8292 8299 lactate +T173 GO:0008152 8671 8680 metabolic +T174 PR:000008555 8708 8714 HIF-1α +T175 GO:0006096 8741 8751 glycolytic +T176 MOP:0000568 8869 8878 oxidative +T177 PR:000008555 8895 8901 HIF-1α +T178 MOP:0000568 8930 8939 oxidative +T179 http://purl.obolibrary.org/obo/MONDO_0005336 8977 8987 myopathies +T180 GO:0006096 9005 9015 glycolysis +T181 GO:0005980 9019 9033 glycogenolysis +T182 http://purl.obolibrary.org/obo/MONDO_0009295 9045 9072 phosphofructokinase disease +T183 http://purl.obolibrary.org/obo/MONDO_0009295 9074 9078 PFKD +T184 http://purl.obolibrary.org/obo/MONDO_0009293 9084 9101 McArdle's disease +T185 CHEBI:30769 9207 9218 citric acid +T186 GO:0006099 9207 9224 citric acid cycle +T187 PR:000008555 9236 9242 HIF-1α +T188 PR:000008555 9304 9310 HIF-1α +T189 GO:0005739 9324 9337 mitochondrial +T190 MOP:0000568 9432 9441 oxidative +T191 GO:0005739 9458 9470 mitochondria +T192 GO:0005739 9518 9531 mitochondrial +T193 CHEBI:20060 9539 9559 beta-hydroxyacyl CoA +T194 PR:000008555 9594 9600 HIF-1α +T195 CHEBI:15846 9699 9703 NAD+ +T196 GO:0005739 9754 9767 mitochondrial +T197 MOP:0000568 9768 9777 oxidation +T198 GO:0019395 9768 9792 oxidation of fatty acids +T199 CHEBI:35366 9781 9792 fatty acids +T200 MOP:0000568 9838 9847 oxidative +T201 CHEBI:24996 9899 9906 lactate +T202 http://purl.obolibrary.org/obo/MONDO_0009295 9939 9943 PFKD +T203 CHEBI:24996 10246 10253 lactate +T204 PR:000008555 10302 10308 HIF-1α +T205 CHEBI:24996 10376 10383 lactate +T206 MOP:0000568 10447 10456 oxidative +T207 GO:0045333 10447 10467 oxidative metabolism +T208 UBERON:0004288 10471 10479 skeletal +T209 GO:0006096 10530 10540 glycolytic +T210 MOP:0000568 10783 10792 oxidative +T211 GO:0006754 10793 10807 ATP production +T212 CHEBI:24384 10860 10868 glycogen +T213 PR:000008555 10964 10970 HIF-1α +T214 NCBITaxon:10088 10984 10988 mice +T215 PR:000008555 10993 10999 HIF-1α +T216 PR:000008555 11092 11098 HIF-1α +T217 NCBITaxon:33208 11158 11165 animals +T218 GO:0036268 11179 11187 swimming +T219 PR:000008555 11233 11239 HIF-1α +T220 GO:0036268 11290 11298 swimming +T221 PR:000008555 11440 11446 HIF-1α +T222 PR:000008555 11604 11610 HIF-1α +T223 UBERON:0001015 11780 11793 muscle groups +T224 CL:0000187 11780 11786;11798 11804 muscle ... fibers +T225 NCBITaxon:10088 11954 11958 mice +T226 CL:0000190 12128 12157 fast-twitch glycolytic fibers +T227 GO:0006096 12140 12150 glycolytic +T228 GO:0006936 12162 12173 contraction +T229 MOP:0000568 12237 12246 oxidative +T230 GO:0006936 12273 12284 contraction +T231 NCBITaxon:33208 12292 12299 animals +T232 MOP:0000568 12338 12347 oxidation +T233 GO:0036268 12424 12432 swimming +T234 NCBITaxon:10088 12480 12484 mice +T235 PR:000008555 12534 12540 HIF-1α +T236 NCBITaxon:10088 12589 12593 mice +T237 PR:000008555 12662 12668 HIF-1α +T238 GO:0006096 12811 12821 glycolytic +T239 CL:0000190 12811 12828 glycolytic fibers +T240 UBERON:0001004 12884 12895 respiratory +T241 GO:0007585 12884 12904 respiratory exchange +T242 http://purl.obolibrary.org/obo/MONDO_0009295 12996 13000 PFKD +T243 http://purl.obolibrary.org/obo/MONDO_0009293 13005 13022 McArdle's disease +T244 http://purl.obolibrary.org/obo/MONDO_0005336 13047 13056 myopathic +T245 PR:000008555 13184 13190 HIF-1α +T246 SO:0001060 13226 13233 isoform +T247 CHEBI:16919 13237 13245 creatine +T248 UBERON:0001977 13262 13267 serum +T249 UBERON:0004288 13302 13310 skeletal +T250 NCBITaxon:10088 13363 13367 mice +T251 PR:000008555 13467 13473 HIF-1α +T252 PR:000008555 13512 13518 HIF-1α +T253 PR:000008555 13716 13722 HIF-1α +T254 NCBITaxon:10088 13791 13795 mice +T255 UBERON:0001388 13836 13849 gastrocnemius +T256 UBERON:0000479 13850 13856 tissue +T257 PR:000008555 13942 13948 HIF-1α +T258 UBERON:0000479 13952 13958 tissue +T259 UBERON:0000479 13967 13973 tissue +T260 UBERON:0000479 14003 14009 tissue +T261 GO:0008283 14014 14027 proliferating +T262 PR:000012421 14014 14052 proliferating cellular nuclear antigen +T263 CHEBI:59132 14045 14052 antigen +T264 PR:000012421 14054 14058 PCNA +T265 GO:0005634 14083 14089 nuclei +T266 GO:0051301 14129 14142 cell division +T267 PR:000008555 14146 14152 HIF-1α +T268 PR:000008555 14194 14200 HIF-1α +T269 UBERON:0000479 14233 14239 tissue +T270 http://purl.obolibrary.org/obo/MONDO_0009295 14289 14293 PFKD +T271 http://purl.obolibrary.org/obo/MONDO_0009293 14298 14315 McArdle's disease +T272 CHEBI:24384 14372 14380 glycogen +T273 UBERON:0001977 14395 14400 serum +T274 CHEBI:24996 14401 14408 lactate +T275 CHEBI:16919 14545 14553 creatine +T276 UBERON:0001977 14568 14573 serum +T277 http://purl.obolibrary.org/obo/MONDO_0009295 14628 14632 PFKD +T278 CHEBI:17287 14767 14770 PCr +T279 GO:0006936 14790 14801 contraction +T280 GO:0006096 14899 14909 glycolytic +T281 PR:000008555 14924 14930 HIF-1α +T282 CHEBI:17234 14970 14977 glucose +T283 GO:0046323 14970 14984 glucose uptake +T284 PR:000008555 14996 15002 HIF-1α +T285 CHEBI:17234 15020 15027 glucose +T286 NCBITaxon:10088 15122 15126 mice +T287 CHEBI:17234 15152 15159 glucose +T288 CHEBI:29149 15188 15201 Periodic acid +T289 UBERON:0000479 15227 15233 tissue +T290 NCBITaxon:10088 15239 15243 mice +T291 CHEBI:24384 15302 15310 glycogen +T292 UBERON:0005090 15329 15336 muscles +T293 PR:000008555 15342 15348 HIF-1α +T294 PR:000008555 15420 15426 HIF-1α +T295 GO:0006110 15604 15628 regulation of glycolysis +T296 NCBITaxon:10088 15658 15662 mice +T297 http://purl.obolibrary.org/obo/MONDO_0005336 15847 15856 myopathic +T298 http://purl.obolibrary.org/obo/MONDO_0002254 15857 15866 syndromes +T299 NCBITaxon:9606 15870 15876 humans +T300 NCBITaxon:10088 15904 15909 Mouse +T301 NCBITaxon:10088 15932 15936 Mice +T302 PR:000008555 15957 15963 HIF-1α +T303 SO:0000359 15964 15976 loxP-flanked +T304 SO:0001023 15977 15983 allele +T305 NCBITaxon:10088 15984 15989 mouse +T306 PR:000005515 16094 16097 MCK +T307 SO:0000902 16102 16111 transgene +T308 SO:0000359 16195 16207 loxP-flanked +T309 PR:000008555 16208 16214 HIF-1α +T310 SO:0001023 16215 16221 allele +T311 PR:000005515 16234 16237 MCK +T312 SO:0000902 16242 16251 transgene +T313 NCBITaxon:33208 16364 16371 animals +T314 PR:000008555 16407 16413 HIF-1α +T315 NCBITaxon:10088 16424 16428 Mice +T316 UBERON:0002415 16492 16496 tail +T317 UBERON:0001388 16539 16552 gastrocnemius +T318 UBERON:0000948 16554 16559 heart +T319 UBERON:0002107 16561 16566 liver +T320 UBERON:0000995 16572 16578 uterus +T321 SO:0000359 16598 16610 loxP-flanked +T322 PR:000008555 16611 16617 HIF-1α +T323 PR:000005515 16631 16634 MCK +T324 NCBITaxon:10088 16648 16652 mice +T325 PR:000008555 16654 16660 HIF-1α +T326 CHEBI:60004 16739 16742 Mix +T327 GO:0097617 17029 17035 anneal +T328 PR:000008555 17096 17102 HIF-1α +T329 PR:000009232 17117 17122 c-Jun +T330 PR:000008555 17135 17141 HIF-1α +T331 SO:0000112 17156 17163 primers +T332 SO:0000121 17191 17205 forward primer +T333 SO:0001030 17217 17218 F +T334 SO:0000132 17250 17264 reverse primer +T335 SO:0001031 17276 17277 R +T336 CHEBI:10545 17384 17392 electron +T337 UBERON:0001388 17417 17430 gastrocnemius +T338 CHEBI:51686 17485 17496 hematoxylin +T339 CHEBI:15377 17540 17545 water +T340 CHEBI:15377 17563 17568 water +T341 CHEBI:16236 17584 17591 ethanol +T342 CHEBI:51686 17619 17630 Hematoxylin +T343 http://purl.obolibrary.org/obo/MONDO_0004992 17716 17722 Cancer +T344 CHEBI:10545 17908 17916 Electron +T345 UBERON:0001388 17965 17985 gastrocnemius muscle +T346 CHEBI:64276 18018 18032 glutaraldehyde +T347 CHEBI:62956 18042 18059 sodium cacodylate +T348 CHEBI:37958 18350 18353 dye +T349 NCBITaxon:10088 18528 18532 mice +T350 PR:000008555 18537 18543 HIF-1α +T351 NCBITaxon:33208 18914 18920 animal +T352 CHEBI:15379 19004 19006 O2 +T353 CHEBI:16526 19011 19014 CO2 +T354 CHEBI:15379 19095 19097 O2 +T355 CHEBI:16526 19111 19114 CO2 +T356 GO:0008152 19216 19225 metabolic +T357 GO:0036268 19410 19418 swimming +T358 PR:000008555 19467 19473 HIF-1α +T359 CHEBI:15377 19523 19528 water +T360 NCBITaxon:33208 19611 19617 animal +T361 GO:0008152 19717 19726 metabolic +T362 UBERON:0003701 19742 19757 Achilles tendon +T363 NCBITaxon:10088 19803 19807 mice +T364 GO:0006936 19876 19887 contractile +T365 UBERON:0005090 19895 19902 Muscles +T366 UBERON:0001322 19958 19971 sciatic nerve +T367 GO:0006936 20026 20038 contractions +T368 GO:0006936 20119 20130 contraction +T369 UBERON:0005090 20258 20265 muscles +T370 CHEBI:24996 20323 20330 lactate +T371 CHEBI:17287 20332 20347 phosphocreatine +T372 CHEBI:24384 20353 20361 glycogen +T373 UBERON:0001388 20498 20518 gastrocnemius muscle +T374 NCBITaxon:10088 20529 20534 mouse +T375 SO:0000704 20596 20600 gene +T376 GO:0010467 20596 20611 gene expression +T377 SO:0000704 20624 20628 gene +T378 GO:0010467 20624 20639 gene expression +T379 UBERON:0001388 20676 20689 gastrocnemius +T380 UBERON:0000479 20690 20696 tissue +T381 PR:000008555 20720 20726 HIF-1α +T382 GO:0001171 20792 20813 Reverse transcription +T383 GO:0001171 21013 21034 Reverse transcription +T384 SO:0000112 21049 21056 primers +T385 PR:000012607 21089 21094 PGK-1 +T386 SO:0000132 21096 21110 reverse primer +T387 SO:0001031 21116 21117 R +T388 SO:0000121 21148 21162 forward primer +T389 SO:0001030 21168 21169 F +T390 CHEBI:51657 21252 21257 TAMRA +T391 CHEBI:32958 21260 21269 phosphate +T392 PR:000017284 21279 21285 VEGF-A +T393 SO:0000132 21287 21301 reverse primer +T394 SO:0001031 21308 21309 R +T395 SO:0000121 21337 21351 forward primer +T396 SO:0001030 21358 21359 F +T397 PR:000015066 21454 21459 GLUT4 +T398 SO:0000132 21461 21475 reverse primer +T399 PR:000015066 21477 21483 GLUT-4 +T400 SO:0001031 21484 21485 R +T401 SO:0000121 21514 21528 forward primer +T402 PR:000015066 21530 21536 GLUT-4 +T403 SO:0001030 21537 21538 F +T404 PR:000015066 21575 21581 GLUT-4 +T405 PR:000012583 21632 21637 PFK-M +T406 SO:0000132 21639 21653 reverse primer +T407 PR:000012583 21655 21660 PFK-M +T408 SO:0001031 21661 21662 R +T409 SO:0000121 21693 21707 forward primer +T410 PR:000012583 21709 21714 PFK-M +T411 SO:0001030 21715 21716 F +T412 PR:000012583 21752 21757 PFK-M +T413 PR:000009739 21811 21816 LDH-A +T414 SO:0000132 21818 21832 reverse primer +T415 PR:000009739 21834 21839 LDH-A +T416 SO:0001031 21840 21841 R +T417 SO:0000121 21871 21885 forward primer +T418 PR:000009739 21887 21892 LDH-A +T419 SO:0001030 21893 21894 F +T420 PR:000009739 21931 21936 LDH-A +T421 SO:0000704 22001 22005 gene +T422 GO:0010467 22001 22016 gene expression +T423 NCBITaxon:10088 22051 22055 mice +T424 NCBITaxon:10088 22146 22150 mice +T425 NCBITaxon:10088 22311 22315 mice +T426 SO:0000704 22399 22403 gene +T427 GO:0010467 22399 22414 gene expression +T428 UBERON:0000479 22425 22431 Tissue +T429 NCBITaxon:10088 22477 22481 mice +T430 CHEBI:78682 22641 22666 fructose 1,6-bisphosphate +T431 CHEBI:16919 22901 22909 Creatine +T432 UBERON:0001977 22918 22923 serum +T433 CHEBI:24996 22924 22931 lactate +T434 CHEBI:16919 22969 22977 Creatine +T435 UBERON:0001977 23011 23016 serum +T436 NCBITaxon:10088 23025 23029 mice +T437 PR:000008555 23034 23040 HIF-1α +T438 CHEBI:16919 23144 23152 Creatine +T439 SO:0001060 23160 23168 isoforms +T440 UBERON:0001977 23243 23248 Serum +T441 CHEBI:24996 23249 23256 lactate +T442 UBERON:0000178 23332 23337 blood +T443 UBERON:0000948 23350 23357 cardiac +T444 NCBITaxon:10088 23379 23383 mice +T445 PR:000008555 23392 23398 HIF-1α +T446 NCBITaxon:10088 23528 23532 mice +T447 NCBITaxon:33208 23568 23574 Animal +T448 CHEBI:17234 23612 23619 Glucose +T449 NCBITaxon:33208 23638 23645 Animals +T450 CHEBI:17234 23705 23712 glucose +T451 NCBITaxon:33208 23766 23773 animals +T452 CHEBI:17234 23802 23809 glucose +T453 NCBITaxon:33208 23853 23860 animals +T454 UBERON:0000178 23909 23914 Blood +T455 UBERON:0002415 23934 23938 tail +T456 UBERON:0001969 24029 24035 plasma +T457 UBERON:0001969 24037 24043 Plasma +T458 UBERON:0000178 24044 24049 blood +T459 CHEBI:17234 24050 24057 glucose +T460 CHEBI:17234 24092 24099 Glucose +T461 CL:0000187 24162 24175 muscle fibers +T462 UBERON:0000366 24236 24242;24250 24256 flexor ... muscle +T463 CHEBI:29108 24336 24340 Ca2+ +T464 NCBITaxon:33208 24426 24433 animals +T465 NCBITaxon:10088 24580 24584 Mice +T466 NCBITaxon:10088 24709 24713 mice +T467 UBERON:0000479 24759 24765 Tissue +T468 CHEBI:51686 24798 24809 hematoxylin +T469 PR:000012421 24843 24847 PCNA +T470 CHEBI:20060 25270 25290 beta-hydroxyacyl CoA +T471 PR:000015066 25329 25334 GLUT4 +T472 CHEBI:17234 25337 25344 glucose +T473 PR:000015066 25337 25358 glucose transporter 4 +T474 PR:000008555 25360 25366 HIF-1α +T475 PR:000008555 25369 25396 hypoxia-inducible factor 1α +T476 PR:000008555 25398 25404 HIF-1α +T477 UBERON:0004288 25410 25418 skeletal +T478 PR:000008555 25426 25432 HIF-1α +T479 NCBITaxon:10088 25442 25447 mouse +T480 PR:000009739 25477 25482 LDH-A +T481 CHEBI:24996 25485 25492 lactate +T482 PR:000009739 25485 25508 lactate dehydrogenase-A +T483 PR:000005515 25510 25513 MCK +T484 PR:000005515 25516 25538 muscle creatine kinase +T485 CHEBI:16919 25523 25531 creatine +T486 CHEBI:29149 25546 25559 periodic acid +T487 PR:000012421 25568 25572 PCNA +T488 GO:0008283 25575 25588 proliferating +T489 PR:000012421 25575 25613 proliferating cellular nuclear antigen +T490 CHEBI:59132 25606 25613 antigen +T491 CHEBI:17287 25615 25618 PCr +T492 CHEBI:17287 25621 25636 phosphocreatine +T493 http://purl.obolibrary.org/obo/MONDO_0009295 25638 25642 PFKD +T494 http://purl.obolibrary.org/obo/MONDO_0009295 25645 25672 phosphofructokinase disease +T495 PR:000012583 25674 25679 PFK-M +T496 PR:000012583 25682 25725 muscle-specific form of phosphofructokinase +T497 CHEBI:61304 25733 25749 phosphoglycerate +T498 CHEBI:15361 25763 25771 pyruvate +T499 UBERON:0001004 25786 25797 respiratory +T500 GO:0007585 25786 25806 respiratory exchange +T501 UBERON:0001986 25830 25841 endothelial +T502 UBERON:0000948 25924 25931 Cardiac +T503 PR:000008555 25932 25938 HIF-1α +T504 PR:000008555 25947 25953 HIF-1α +T505 PR:000005515 25954 25957 MCK +T506 GO:0005739 25962 25975 Mitochondrial +T507 NCBITaxon:10088 25989 25993 Mice +T508 UBERON:0000948 26002 26009 cardiac +T509 PR:000008555 26010 26016 HIF-1α +T510 NCBITaxon:10088 26076 26080 mice +T511 PR:000005515 26137 26140 MCK +T512 NCBITaxon:10088 26145 26149 mice +T513 PR:000008555 26172 26178 HIF-1α +T514 UBERON:0004288 26182 26190 skeletal +T515 UBERON:0000948 26203 26210 cardiac +T516 UBERON:0000479 26211 26217 tissue +T517 NCBITaxon:10088 26224 26228 Mice +T518 UBERON:0004288 26237 26245 skeletal +T519 PR:000008555 26253 26259 HIF-1α +T520 GO:0005739 26305 26318 mitochondrial +T521 GO:0005739 26356 26368 mitochondria +T522 CHEBI:10545 26373 26381 electron +T523 PR:000008555 26455 26461 HIF-1α +T524 NCBITaxon:10088 26473 26477 Mice +T525 PR:000008555 26533 26539 HIF-1α +T526 NCBITaxon:10088 26567 26571 mice +T527 PR:000008555 26597 26603 HIF-1α +T528 UBERON:0004288 26607 26615 skeletal +T529 GO:0015671 26639 26654 oxygen carrying +T530 UBERON:0000178 26671 26676 blood +T531 NCBITaxon:10088 26728 26732 mice +T532 PR:000008555 26737 26743 HIF-1α +T533 UBERON:0000178 26764 26769 blood +T534 CHEBI:25212 26814 26824 Metabolite +T535 GO:0006096 26871 26881 Glycolytic +T536 UBERON:0001388 26915 26936 gastrocnemius muscles +T537 UBERON:0001388 27034 27047 gastrocnemius +T538 NCBITaxon:33208 27062 27069 animals +T539 PR:000008555 27071 27077 HIF-1α +T540 GO:0006096 27301 27319 glycolytic pathway +T541 PR:000008555 27378 27384 HIF-1α +T542 CHEBI:17234 27410 27417 glucose +T543 PR:000008555 27464 27470 HIF-1α +T544 UBERON:0005090 27481 27488 muscles +T545 CHEBI:17234 27525 27532 glucose +T546 GO:0046323 27525 27539 glucose uptake +T547 CHEBI:17234 27584 27591 glucose +T548 PR:000008555 27655 27661 HIF-1α +T549 CHEBI:24384 27683 27691 glycogen +T550 NCBITaxon:10088 27703 27707 mice +T551 CHEBI:24384 27709 27717 Glycogen +T552 CHEBI:24384 27804 27812 glycogen +T553 PR:000008555 27866 27872 HIF-1α +T554 GO:0008152 27899 27910 metabolized +T555 CHEBI:24384 27916 27924 glycogen +T556 PR:000008555 28007 28013 HIF-1α +T557 CHEBI:17287 28031 28034 PCr +T558 NCBITaxon:10088 28073 28077 mice +T559 CHEBI:17287 28097 28100 PCr +T560 PR:000008555 28124 28130 HIF-1α +T561 GO:0008152 28135 28146 metabolized +T562 CHEBI:17287 28328 28331 PCr +T563 PR:000008555 28369 28375 HIF-1α +T564 NCBITaxon:10088 28395 28399 mice +T565 CHEBI:17287 28570 28573 PCr +T566 PR:000008555 28601 28607 HIF-1α +T567 CHEBI:17287 28695 28698 PCr +T568 GO:0006754 28703 28717 ATP generation +T569 PR:000008555 28842 28848 HIF-1α +T570 PR:000008555 28966 28972 HIF-1α +T571 NCBITaxon:33208 28984 28991 animals +T572 CHEBI:24996 29027 29034 lactate +T573 CHEBI:24996 29178 29185 lactate +T574 PR:000008555 29223 29229 HIF-1α +T575 CHEBI:29108 29267 29271 Ca2+ +T576 CL:0000187 29292 29305 Muscle Fibers +T577 CL:0000187 29393 29406 muscle fibers +T578 UBERON:0000366 29447 29453;29461 29467 flexor ... muscle +T579 PR:000008555 29558 29564 HIF-1α +T580 CHEBI:29108 29588 29592 Ca2+ +T581 PR:000008555 29617 29623 HIF-1α +T582 CHEBI:29108 29692 29696 Ca2+ +T583 CHEBI:29108 29761 29765 Ca2+ +T584 CHEBI:47867 29766 29775 indicator +T585 CHEBI:29108 29871 29875 Ca2+ +T586 MOP:0000568 29893 29902 Oxidative +T587 GO:0045333 29893 29913 Oxidative Metabolism +T588 UBERON:0001977 29918 29923 Serum +T589 CHEBI:24996 29924 29931 Lactate +T590 GO:0019249 29924 29942 Lactate Production +T591 PR:000008555 29946 29952 HIF-1α +T592 NCBITaxon:10088 29964 29968 Mice +T593 PR:000008555 29974 29980 HIF-1α +T594 GO:0006099 30051 30062 Krebs cycle +T595 GO:0065007 30075 30084 regulated +T596 MOP:0000568 30171 30180 oxidative +T597 GO:0005980 30224 30238 glycogenolytic +T598 GO:0006096 30242 30252 glycolytic +T599 http://purl.obolibrary.org/obo/MONDO_0005336 30253 30263 myopathies +T600 PR:000008555 30293 30299 HIF-1α +T601 MOP:0000568 30394 30401 oxidize +T602 CHEBI:35366 30402 30413 fatty acids +T603 UBERON:0001977 30450 30455 serum +T604 CHEBI:24996 30456 30463 lactate +T605 PR:000008555 30484 30490 HIF-1α +T606 NCBITaxon:10088 30598 30602 Mice +T607 PR:000008555 30608 30614 HIF-1α +T608 GO:0036268 30645 30653 swimming +T609 GO:0036268 30689 30693 swim +T610 PR:000008555 30766 30772 HIF-1α +T611 NCBITaxon:10088 30808 30812 mice +T612 PR:000008555 31021 31027 HIF-1α +T613 NCBITaxon:10088 31060 31064 mice +T614 NCBITaxon:10088 31147 31151 mice +T615 NCBITaxon:10088 31237 31241 mice +T616 GO:0006096 31313 31323 glycolytic +T617 CL:0000190 31313 31330 glycolytic fibers +T618 NCBITaxon:10088 31361 31365 mice +T619 PR:000008555 31445 31451 HIF-1α +T620 NCBITaxon:10088 31504 31508 mice +T621 PR:000008555 31591 31597 HIF-1α +T622 NCBITaxon:10088 31638 31642 mice +T623 PR:000008555 31647 31653 HIF-1α +T624 NCBITaxon:33208 31699 31706 animals +T625 PR:000008555 31806 31812 HIF-1α +T626 PR:000008555 31971 31977 HIF-1α +T627 NCBITaxon:33208 32073 32080 animals +T628 PR:000008555 32250 32256 HIF-1α +T629 CHEBI:51686 32289 32300 hematoxylin +T630 UBERON:0001388 32323 32344 gastrocnemius muscles +T631 NCBITaxon:10088 32370 32374 mice +T632 PR:000008555 32447 32453 HIF-1α +T633 UBERON:0005090 32457 32464 muscles +T634 UBERON:0005090 32480 32487 muscles +T635 PR:000012421 32505 32509 PCNA +T636 UBERON:0001388 32522 32543 gastrocnemius muscles +T637 NCBITaxon:10088 32559 32563 mice +T638 GO:0031099 32606 32618 regeneration +T639 PR:000008555 32622 32628 HIF-1α +T640 PR:000012421 32649 32653 PCNA +T641 GO:0005634 32663 32669 nuclei +T642 UBERON:0001388 32695 32716 gastrocnemius muscles +T643 NCBITaxon:10088 32723 32727 mice +T644 PR:000008555 32740 32746 HIF-1α +T645 PR:000008555 32797 32803 HIF-1α +T646 PR:000012421 32834 32838 PCNA +T647 GO:0005634 32848 32854 nuclei +T648 PR:000008555 33037 33043 HIF-1α +T649 CHEBI:17234 33104 33111 Glucose +T650 CHEBI:24384 33126 33134 Glycogen +T651 UBERON:0000178 33196 33201 blood +T652 CHEBI:17234 33202 33209 glucose +T653 PR:000008555 33220 33226 HIF-1α +T654 NCBITaxon:10088 33237 33241 mice +T655 CHEBI:17234 33266 33273 glucose +T656 UBERON:0000178 33347 33352 blood +T657 CHEBI:17234 33353 33360 glucose +T658 CHEBI:17234 33376 33383 glucose +T659 UBERON:0001388 33454 33474 gastrocnemius muscle +T660 NCBITaxon:10088 33483 33487 mice +T661 PR:000008555 33492 33498 HIF-1α +T662 PR:000008555 33504 33510 HIF-1α +T663 CHEBI:24384 33567 33575 glycogen +T664 PR:000008555 33599 33605 HIF-1α +T665 UBERON:0000479 33617 33624 Tissues +T666 PR:000008555 33669 33675 HIF-1α +T667 UBERON:0001388 33715 33735 Gastrocnemius Muscle +T668 UBERON:0001389 33787 33800 Soleus Muscle +T669 SO:0000704 33868 33872 Gene +T670 GO:0010467 33868 33883 Gene Expression +T671 GO:0010467 33892 33902 Expression +T672 SO:0000704 33952 33956 gene +T673 SO:0000704 34031 34035 gene +T674 GO:0010467 34031 34046 gene expression +T675 GO:0006096 34147 34157 Glycolytic +T676 UBERON:0001388 34186 34207 Gastrocnemius Muscles +T677 CHEBI:17138 34253 34279 glyceraldehyde 3-phosphate +T678 CHEBI:21008 34300 34314 phosphoglucose +T679 PR:000008555 34344 34350 HIF-1α +T680 CHEBI:33893 34763 34771 reagents +T681 UBERON:0004288 34967 34975 skeletal +T682 PR:000008555 34983 34989 HIF-1α diff --git a/src/ontogpt/evaluation/craft/database/all/15328538.txt b/src/ontogpt/evaluation/craft/database/all/15328538.txt new file mode 100644 index 000000000..30466f625 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15328538.txt @@ -0,0 +1,266 @@ +Loss of Skeletal Muscle HIF-1α Results in Altered Exercise Endurance + +Abstract + +The physiological flux of oxygen is extreme in exercising skeletal muscle. Hypoxia is thus a critical parameter in muscle function, influencing production of ATP, utilization of energy-producing substrates, and manufacture of exhaustion-inducing metabolites. Glycolysis is the central source of anaerobic energy in animals, and this metabolic pathway is regulated under low-oxygen conditions by the transcription factor hypoxia-inducible factor 1α (HIF-1α). To determine the role of HIF-1α in regulating skeletal muscle function, we tissue-specifically deleted the gene encoding the factor in skeletal muscle. Significant exercise-induced changes in expression of genes are decreased or absent in the skeletal-muscle HIF-1α knockout mice (HIF-1α KOs); changes in activities of glycolytic enzymes are seen as well. There is an increase in activity of rate-limiting enzymes of the mitochondria in the muscles of HIF-1α KOs, indicating that the citric acid cycle and increased fatty acid oxidation may be compensating for decreased flow through the glycolytic pathway. This is corroborated by a finding of no significant decreases in muscle ATP, but significantly decreased amounts of lactate in the serum of exercising HIF-1α KOs. This metabolic shift away from glycolysis and toward oxidation has the consequence of increasing exercise times in the HIF-1α KOs. However, repeated exercise trials give rise to extensive muscle damage in HIF-1α KOs, ultimately resulting in greatly reduced exercise times relative to wild-type animals. The muscle damage seen is similar to that detected in humans in diseases caused by deficiencies in skeletal muscle glycogenolysis and glycolysis. Thus, these results demonstrate an important role for the HIF-1 pathway in the metabolic control of muscle function. + +Introduction + +During exercise in normoxia, the partial pressure of oxygen in muscle tissue has been shown to dip to as low as 3.1 mm Hg, whereas in the capillary, it remains at 38 mm Hg (Hoppeler et al. 2003). In order to maintain effort, skeletal muscle exertion must be able to rely on pathways designed to help the tissue cope with oxygen stress after oxygen delivery capacity is exceeded. A switch between aerobic and nonaerobic metabolism during strenuous exertion requires mechanisms to adjust metabolic function, and this need is acute in extended exertion in skeletal muscle. It is clear that the transcription factor hypoxia-inducible factor 1α (HIF-1α) is an essential factor in maintenance of ATP levels in cells (Seagroves et al. 2001). In fact, although HIF-1α is typically thought of as acting only during hypoxia, its loss has an effect on both normoxic and hypoxic ATP levels in a number of tissue types (Seagroves et al. 2001; Cramer et al. 2003), and this implicates the factor in regulation of metabolic function even during conditions of normal physiologic oxygenation. + +In skeletal muscle, signaling of fatigue has been studied extensively, and signaling of exhaustion involves, to some degree, elevated systemic lactic acid, a by-product of the glycolytic pathway of metabolism (Myers and Ashley 1997). Thus, the glycolytic pathway is intrinsically involved in muscle function and fatigue, and this in turn is linked to the response to hypoxia. To understand how the primary hypoxia-responsive transcription factor controls skeletal muscle function, we targeted mouse skeletal muscle for tissue-specific deletion of HIF-1α via the use of a conditionally targeted allele of the gene (Ryan et al. 2000; Schipani et al. 2001). This mouse strain was crossed into a strain transgenic for the skeletal-muscle-specific muscle creatine kinase (MCK) promoter, which drives expression of the cre recombinase gene (Bruning et al. 1998; Sauer 1998). We found that loss of the regulation of hypoxic response in muscle has a profound effect on the function of the muscle during exertion, with effects that mimic human metabolic myopathies. + +Results/Discussion + +In 4-mo–old mice with the skeletal-muscle HIF-1α gene knocked out (HIF-1α KOs), the frequency of excision was evaluated through real-time PCR techniques. We saw deletion frequencies consistent with those described previously for this cre recombinase transgene (Bruning et al. 1998) with some variation in penetration; mean frequency of deletion was 54.9%, with the highest frequency of muscle-specific deletion of HIF-1α being 72% in the gastrocnemius of 4-mo–old mice homozygous for the loxP-flanked allele (Table 1). This transgene is expressed at a lower level in cardiac tissue, and cardiac deletion was detected (Table 1); however, none of the phenotypes described below were seen in cardiac myocyte-specific deletions of HIF-1α (Figure 1A). Gross muscle sections were evaluated histologically to evaluate both vascularization and fiber type (Tables 2 and 3), and ultrastructurally to determine number of mitochondria (Figure 1B). No changes were detected in any of these features in HIF-1α KOs, except for a slight but statistically significant decrease in type IIA fibers in the soleus muscles (Table 3). Similar hematocrit and blood hemoglobin levels were seen in HIF-1α KOs and wild-type (WT) mice (Figure 2). + +As can be seen in Table 4, significant changes in HIF-1α–dependent gene expression occur in muscle during exercise, including changes in genes involved in glucose transport and metabolism. Vascular endothelial growth factor (VEGF), which increases vascular permeability, and glucose transporter 4 (GLUT4), the muscle-specific glucose transporter, show increased levels in exercise and likely increase the availability of glucose to the muscle. The muscle-specific form of phosphofructokinase (PFK-M), phosphoglycerate kinase (PGK), and lactate dehydrogenase-A (LDH-A) are also up-regulated at the mRNA level by exercise, and this up-regulation is inhibited by the loss of HIF-1α, further demonstrating that HIF-1α is important for transcriptional response during skeletal muscle activity. + +In Table 5, we show the changes in enzymatic activity in a number of key glycolytic enzymes affected by deletion of HIF-1α. As can be seen from the data, several of the enzymes assayed showed a decrease in activity in response to exercise. In particular, the activity of one of the key rate-limiting enzymes, PFK, was significantly lower following exercise in HIF-1α KOs compared to WT mice, indicating that HIF-1α KOs may have difficulty maintaining optimal PFK activity. The responses of other glycolytic enzymes to exercise were fairly similar between WT mice and HIF-1α KOs. These include no significant changes in phosphoglucose isomerase activity and significant, yet similar, decreases in aldolase, glyceraldehyde 3-phosphate dehydrogenase, and PGK activities. An exception to this is that WT muscles were able to significantly increase pyruvate kinase (PK) activity (see Table 4; p < 0.05). LDH activity was also increased in the WT mice, although the level did not reach statistical significance. Activities of both PK and LDH were not significantly changed in HIF-1α KO muscles following exercise. Increased activities of PK, and subsequently LDH, could be expected to lead to increased levels of lactate in the WT mice relative to HIF-1α KOs.In Figure 3A, it can be seen that the decrease in PFK activity in the HIF-1α KOs is correlated with a trend approaching significance (p = 0.10) toward an increased amount of hexose monophosphates (HMPs), which are pre-PFK glycolytic metabolites, following stimulation of the HIF-1α KO muscle. This increase was not due to differences in glucose uptake, since animals of both genotypes were able to significantly increase intramuscular glucose to a similar degree (Figure 3B). Consistent with decreased flow through the glycolytic pathway, however, the increased amount of HMPs was correlated with increased muscle glycogenolysis (Figure 3C) and increased depletion of phosphocreatine (PCr) (Figure 3D), with a resultant decrease in the PCr/ATP ratio in HIF-1α KO muscle (Figure 3E), although there was only a nonsignificant drop in overall muscle ATP concentrations (Figure 3F). Intramuscular levels of lactate did increase in both HIF-1α KOs and WT mice during stimulation, although lactate accumulation did not differ significantly between them (Figure 3G). In order to evaluate whether these changes had any effect on overall muscle force, we measured force and calcium release in isolated single fibers; as can be seen in Figure 4A and 4B, there were no significant changes in these parameters, indicating that the muscle can compensate at this level for the metabolic changes induced by loss of HIF-1α. + +Given altered levels of glycolytic throughput without significant changes in intramuscular ATP levels, it is likely that there is increased activity of oxidative pathways in the HIF-1α KO muscle. Increased muscle oxidative activity is typical in patients with myopathies involving muscle glycolysis or glycogenolysis, including phosphofructokinase disease (PFKD) and McArdle's disease (Vissing et al. 1996). We analyzed the activity of citrate synthase (CS), a key allosteric enzyme of the citric acid cycle, in WT and HIF-1α KO muscle (Figure 5A), and found that it was up-regulated in HIF-1α KOs. CS is a mitochondrial enzyme that responds to decreases in ATP concentration allosterically, allowing for increased oxidative activity in the mitochondria. In addition, significant up-regulation of the mitochondrial enzyme beta-hydroxyacyl CoA dehydrogenase (B-HAD) was seen in HIF-1α KO muscle (Figure 5B). B-HAD is also affected by energy levels in the cell, and decreases in NADH/NAD+ concentration ratios cause the enzyme to increase mitochondrial oxidation of fatty acids (Nelson and Cox 2000). Increased activity of oxidative pathways in the muscle should result in more rapid lactate clearance, as in fact occurs in PFKD patients during exercise; this phenomenon gives rise to a “second wind” in these patients, and under some circumstances allows for an increase in exercise endurance (Vissing et al. 1996; Haller and Vissing 2002), although this was disputed in one recent study (Haller and Vissing 2004). This decreased lactate accumulation postexercise clearly occurs in the HIF-1α KOs, as can be seen in Figure 5C. This systemically lower level of lactate postexercise indicates that there may be a shift toward a more oxidative metabolism in skeletal muscle. + +As mentioned above, patients with muscle glycolytic deficiencies demonstrate both increased exercise-induced muscle damage and a “second wind”; the latter phenomenon allows them to exercise for extended periods of time at submaximal levels. This is thought to be due to an increase in rates of oxidative ATP production, and a decreased utilization of and need for muscle glycogen (Vissing et al. 1996; Haller and Vissing 2002). To assess whether this is also the case in the HIF-1α KOs, both WT mice and HIF-1α KOs were subjected to endurance tests to assess muscle function. To first determine whether HIF-1α KOs were capable of extended activity during exercise, the animals were given a swimming endurance test. As can be seen in Figure 6A, HIF-1α KOs were capable of significantly longer-duration swimming activity when compared to matched WT controls (p < 0.05). + +Further testing was done to determine the parameters of this increased endurance. HIF-1α KOs were run on an enclosed treadmill, with a 5° incline and an initial velocity of 10 m/min, with an increase in velocity every 5 min. In their first runs, HIF-1α KOs again had significantly greater endurance, as shown by their consistently longer run times compared to WT controls (p < 0.01, Figure 6B). + +As it has been shown that muscle groups and fibers respond differently to eccentric exercise (i.e., downhill running) than to concentric exercise (i.e., uphill running) (Nardone and Schieppati 1988), mice from both genotypes were run on a 10° decline with the same velocity and time parameters as in the uphill runs. Eccentric exercises have been shown to recruit primarily fast-twitch glycolytic fibers for contraction, as opposed to the traditional recruitment of slower, smaller, oxidative motor units in concentric contraction, where animals with an increased capacity for muscle oxidation would be at an advantage (Nardone and Schieppati 1988). Now, the trend from swimming and uphill running tests was reversed, with WT mice able to run for a significantly longer time than HIF-1α KOs (p < 0.05, Figure 6C). Within genotypes, WT mice ran for significantly longer times downhill than uphill (p < 0.01); HIF-1α KOs did the reverse, and ran for significantly shorter times downhill than uphill (p < 0.05). Substrate utilization confirms the shift toward glycolytic fibers in downhill running; both genotypes had higher average respiratory exchange ratio (RER) values when running downhill compared with running uphill (Figure 6D and 6E). + +PFKD and McArdle's disease demonstrate significant myopathic effects in muscle, including soreness and cramping induced by bouts of exercise. After 1 d of recovery from endurance testing, HIF-1α KOs had increased levels of the MM isoform of creatine kinase in their serum (unpublished data), indicative of skeletal muscle damage. To further investigate this finding, mice were run on a treadmill daily for 4 d. By the second day, the trend for increased endurance in the HIF-1α KOs was absent, and by the final day, HIF-1α KOs were running for significantly shorter times than they had on the first day (p < 0.01, Figure 7A). In addition, a repeated measures ANOVA performed on run times showed that the response of the HIF-1α KOs to the protocol was significantly different than that of the WT mice (p < 0.05). Histological examination of gastrocnemius tissue following 1 d of recovery revealed significantly greater amounts of muscle damage in HIF-1α KO tissue than WT tissue (Figure 7B). Staining of the tissue for proliferating cellular nuclear antigen (PCNA) and counts of positive nuclei (Olive et al. 1995) also revealed more cell division in HIF-1α KOs than in WTs, another indication that HIF-1α KOs had been subject to greater tissue damage (Figure 7C and 7D). + +As noted above, both PFKD and McArdle's disease are marked by increased resting intramuscular levels of glycogen, a failure of serum lactate to rise during exertion, an exercise-induced “second wind,” and signs of muscle damage following exertion, including elevated levels of creatine kinase in the serum (Tarui et al. 1965; Layzer et al. 1967). In addition, PFKD is characterized by elevated levels of HMPs (Tarui et al. 1965; Layzer et al. 1967; Argov et al. 1987; Grehl et al. 1998) and greater PCr utilization during contraction (Argov et al. 1987; Grehl et al. 1998). We see many of these hallmarks of muscle deficiencies in glycolytic processing in HIF-1α KOs. The effects are not likely due to glucose uptake, as WT and HIF-1α KO intramuscular glucose levels were not different at rest or following stimulation (see Figure 3B), and both types of mice responded similarly to a glucose tolerance test (Figure 8A). Periodic acid–Schiff (PAS) staining of tissue from mice of both genotypes gave further demonstration of increased glycogen levels in resting muscles from HIF-1α KOs (Figure 8B). + +Given the differences in performance observed in the HIF-1α KOs in eccentric and concentric exercise, it is clear that the HIF-1 pathway and hypoxic response have a central role in determining the capacity for work and endurance through regulation of glycolysis. It is also clear that these mice will provide an important model system to investigate the physiology of muscle response during work and oxygen depletion, and may be useful as a model for a group of very debilitating myopathic syndromes in humans. + +Materials and Methods + + + +Mouse strains and crosses. + +Mice were generated from HIF-1α loxP-flanked allele mouse stocks backcrossed into a C57Bl6/J background. These were crossed into a C57Bl6/J strain containing the MCK/cre transgene. Controls were in all cases littermates that were genotyped as containing only the loxP-flanked HIF-1α allele or only the MCK/cre transgene. No phenotypic differences were seen in the two controls, so they were considered interchangeably as WT control animals. + +Genotyping and real-time PCR for HIF-1α deletion + +Mice from the above crosses were genotyped using DNA extracted from tail sections. DNA was then extracted from the gastrocnemius, heart, liver, and uterus of eight 4-mo–old, loxP-flanked HIF-1α–positive and MCK/cre–positive mice. HIF-1α levels were measured by real-time PCR analysis using the Universal PCR Master Mix Kit (Applied Biosystems, Foster City, California, United States) and the ABI Prism 7700 Sequence Detector (Applied Biosystems). Conditions for the PCR were one 10-min incubation at 95 °C (polymerase activation), followed by 40 cycles of 15 s at 95 °C (denaturation) and 1 min at 60 °C (anneal/extend). The degree of excision was calculated by comparing HIF-1α DNA levels to c-Jun DNA levels. HIF-1α real-time PCR primers and probe were as follows: forward primer, HIFLOX501/F 5′-CTATGGAGGCCAGAAGAGGGTAT-3′; reverse primer, HIFLOX574/R 5′-CCCACATCAGGTGGCTCATAA-3′; probe, HIFLOX/P 5′-(6FAM)AGATCCCTTGAAGCTAG(MGBNFQ)-3′. + +Muscle histology and electron microscopy. + +Paraffined gastrocnemius sections were deparaffinized and stained with Gill II hematoxylin. Sections were then washed successively in water, a bluing agent, water again, and 95% ethanol, and restained with eosin. Hematoxylin and eosin staining was performed by the University of California at San Diego (UCSD) Cancer Center Histology Resource (La Jolla, California, United States). Imaging was performed on sections mounted on slides using Cytoseal 60 (VWR, West Chester, Pennsylvania, United States). Electron microscopy was performed by standard methods on gastrocnemius muscle. Briefly, fixation was by 25.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). Postfix was in 1% osmium tetroxide. The section was stained in 2% uranyl acetate in sodium maleate buffer (pH 5.2), then placed in Epon resin (VWR, West Chester, Pennsylvania, United States), and cured overnight at 60 °C. Fiber typing was performed using the metachromatic dye ATPase method (Ogilvie and Feeback 1990). PAS staining was performed as has been described (Bancroft and Stevens 1996). + +Assessment of exhaustion. + +Untrained, age-matched WT mice and HIF-1α KOs (WT, n = 10; KO, n = 14) were run either on an Omnipacer treadmill (Columbus Instruments, Columbus, Ohio, United States) or on an enclosed-chamber modular treadmill (Columbus Instruments) with a 5° incline at an initial velocity of 10 m/min. Velocity was increased by 2 m/min every 5 min during the assessment. Exhaustion was determined to be the point at which the animal would not resume running when provoked through a low-voltage power grid. Gas flow (O2 and CO2) into and out of the enclosed chamber treadmill was monitored using the Paramax O2 sensor and a CO2 sensor (Columbus Instruments) and analyzed using Oxymax software (Columbus Instruments) to determine metabolic parameters. The downhill running assessment (WT, n = 8; KO, n = 6) was carried out in the enclosed-chamber modular treadmill at a 10° decline using the same protocol as above. + +In the swimming exhaustion assessment, a second group of WT and HIF-1α KOs (n = 8 for each class) was placed in a 30 °C water bath with mild turbulence. Exhaustion was determined to be the point at which the animal experienced three successive periods below the surface of more than 3 s. + +Isolated stimulation and metabolic analysis. + +The Achilles tendon was surgically freed from live, anesthetized mice (WT, n = 8; KO, n = 6) and attached to a force transducer to record contractile force. Muscles were electrically stimulated through excitation of the sciatic nerve. Stimulation was in the form of 8–10-V direct titanic contractions using 200-ms trains at 70 Hz with 0.2 ms duration. Initial frequency of tetanic contraction was one every 8 s and was increased every 2 minutes to one every 4 s and one every 3 s, up to the end point of 6 min. Isolated muscles were then immediately harvested and snap-frozen for ATP, lactate, phosphocreatine, and glycogen analyses. Samples were freeze-dried and analyzed by enzymatic assay as has been previously described (Bergmeyer 1974). The unstimulated gastrocnemius muscle from each mouse was used as a resting control. + +Real-time PCR measurement of gene expression. + +For basal gene expression levels, total RNA was isolated from gastrocnemius tissue from seven WT and five HIF-1α KOs using RNA-Bee (Tel-Test, Friendswood, Texas, United States). Reverse transcription was performed using the Superscript First Stand Synthesis System for RT-PCR (Invitrogen, Carlsbad, California, United States). Amplification was performed using the ABIPrism 7700 as described above. Reverse transcription real-time PCR primers and probes were as follows. For PGK-1: reverse primer, PGK/R 5′-CAGGACCATTCCAAACAATCTG-3′; forward primer, PGK/F 5′-CTGTGGTACTGAGAGCAGCAAGA-3′; probe, PGK/P 5′-(6∼FAM)TAGCTCGACCCA-CAGCCTCGGCATAT(TAMRA)-(phosphate)-3′. For VEGF-A: reverse primer, VEGF/R 5′-ATCCGCATGATCTGCATGG-3′; forward primer, VEGF/F 5′-AGTCCCATGAAGTGATCAAGTTCA-3; probe, VEGF/P (6∼FAM)TGCCCACGTCAGAGAGCAACATCAC(BHQ∼6∼FAM). For GLUT4: reverse primer, GLUT-4/R 5′-CCCATGCCGACAATGAAGTT-3′; forward primer, GLUT-4/F 5′-TGTGGCCTTCTTTGAGATTGG-3′; probe, GLUT-4/P 5′(6-FAM)TGGCCCCATTCCCTGGTTCATT(BHQ1-Q)-3′. For PFK-M: reverse primer, PFK-M/R 5′-AAGTCGTGCAGATGGTGTTCAG-3′; forward primer, PFK-M/F 5′-GCCACGGTTTCCAATAACGT-3′; probe, PFK-M/P 5′-(6-FAM)CCTGGGTCAGACTTCAGCATCGGG(BHQ1-Q)-3′. For LDH-A: reverse primer, LDH-A/R 5′-ATGCACCCGCCTAAGGTTCTT-3′; forward primer, LDH-A/F 5′-TGCCTACGAGGTGATCAAGCT-3′; probe, LDH-A/P 5′-(6- FAM)TGGCAGACTTGGCTGAGAGCAT(BHQ1-Q)-3′. + +For changes in gene expression due to exercise, age-matched male mice (WT, n = 5; KO, n = 6) were run on a treadmill at 25 m/min for 30 min. Following the run, mice were euthanized and RNA was isolated and analyzed as described above. + +Analysis of enzyme activity levels + +For changes in enzyme activity levels with exercise, mice (WT, n = 5; KO, n = 12) were run on a treadmill using the same protocol as for the gene expression analysis. Tissue was harvested after the run and from resting mice (WT, n = 6; KO, n = 10), and enzymes were extracted and analyzed spectrophotometrically as has been described (Reichmann et al. 1983), with the exception that fructose 1,6-bisphosphate was replaced with fructose 2,6-bisphosphate for stabilization of PFK. Units of activity were normalized to milligrams of total protein using a BCA protein quantification kit (Pierce Biotechnology, Rockford, Illinois, United States). + +Creatine kinase, serum lactate, hematocrit, and hemoglobin levels. + +Creatine kinase levels were analyzed from serum from WT mice and HIF-1α KOs 24 h after running-induced exhaustion using a kit from Sigma (St. Louis, Missouri, United States). Creatine kinase isoforms were analyzed enzymatically and then fractionated by gel electrophoresis. Serum lactate levels were analyzed by the UCSD Comparative Neuromuscular Laboratory from blood obtained by cardiac puncture from six WT mice and six HIF-1α KOs following 25 min running time on the treadmill ramp at 25 m/min. Hematocrit and hemoglobin levels were measured from resting mice (WT, n = 6; KO, n = 4) by the UCSD Animal Care Program Diagnostic Laboratory. + +Glucose tolerance curve. + +Animals were assigned into either a sham (WT, n = 5; KO, n = 4) or glucose tolerance group (WT, n = 8; KO, n = 8). Experimental animals were injected with 0.3 g/ml glucose in PBS to achieve a dosage of 2 g/kg. Sham animals were injected with an equivalent amount of PBS. Blood was drawn from the tail at time intervals of 0, 15, 30, 60, and 120 min. Samples were then centrifuged to isolate plasma. Plasma blood glucose was quantified using the Infinity Glucose Kit (Sigma). + +Calcium uptake measurements. + +Intact individual muscle fibers (WT, n = 6; KO, n = 4) were mechanically dissected from the flexor brevis muscle and loaded with fura-2. Fibers were then stimulated while force generation and Ca2+ release were monitored. + +Four-day endurance test + +Endurance was tested by running 24 animals (WT, n = 10; KO, n = 14) on the Omnipacer Treadmill or the enclosed-chamber modular treadmill using the same exhaustion protocol described above. Mice ran according to this protocol every day for 4 d with a minimum of 22 h of rest between trials. Following the fourth trial, mice were given 24 h of rest and then euthanized. Tissue was harvested and stained using hematoxylin-eosin (as described above) and α-PCNA (Pharmingen, San Diego, California, United States) combined with a DAB Kit (Vector Labs, Burlingame, California, United States). + +Statistical analysis + +Statistical analyses (unpaired Student's t-test, Mann-Whitney test, ANOVA) were carried out using StatView software (SAS Institute, Cary, North Carolina, United States) or Prism software (GraphPad Software, San Diego, California, United States). + +Abbreviations + +B-HAD - beta-hydroxyacyl CoA dehydrogenase + +CS - citrate synthase + +GLUT4 - glucose transporter 4 + +HIF-1α - hypoxia-inducible factor 1α + +HIF-1α KO - skeletal-muscle HIF-1α knockout mouse + +HMP - hexose monophosphate + +LDH-A - lactate dehydrogenase-A + +MCK - muscle creatine kinase + +PAS - periodic acid–Schiff + +PCNA - proliferating cellular nuclear antigen + +PCr - phosphocreatine + +PFKD - phosphofructokinase disease + +PFK-M - muscle-specific form of phosphofructokinase + +PGK - phosphoglycerate kinase + +PK - pyruvate kinase + +RER - respiratory exchange ratio + +VEGF - vascular endothelial growth factor + +WT - wild-type + +Figures and Tables + +Figure 1 + +Exercise Capacity of Cardiac HIF-1α KOs and HIF-1α/MCK/cre Mitochondrial Density + +(A) Mice lacking cardiac HIF-1α perform no differently in endurance running trials than WT mice, showing that the increase in exercise capacity seen in MCK/Cre mice is due to deletion of HIF-1α in skeletal muscle, not cardiac tissue. + +(B) Mice lacking skeletal muscle HIF-1α have a slight but nonsignificant increase in mitochondrial density as measured by the number of mitochondria per electron microscope field of view. + +Figure 2 + +Hematocrit and Hemoglobin Levels in HIF-1α KOs and WT Mice + +(A) Hematocrit levels are virtually identical in both HIF-1α KOs (n = 3) and WT (n = 4) mice, indicating that loss of HIF-1α in skeletal muscle does not affect oxygen carrying capacity of the blood. + +(B) In addition to similar hematocrit levels, WT mice and HIF-1α KOs have very close blood hemoglobin levels. + +Figure 3 + +Intramuscular Metabolite Levels at Rest and Following Stimulation + +(A) Glycolytic intermediates were measured from gastrocnemius muscles following the isolated stimulation protocol. Resting values represent levels in the unstimulated gastrocnemius from the same animals. HIF-1α KOs had a trend toward greater accumulated levels of HMPs during the stimulation protocol, although the difference did not reach statistical significance (p = 0.10). This difference could be indicative of a blockage in the glycolytic pathway at PFK. + +(B) No significant differences were seen between HIF-1α KOs and WT intramuscular glucose levels at rest or following stimulation. Both HIF-1α KO and WT muscles were able to significantly increase glucose uptake, leading to greater levels of intramuscular glucose in response to stimulation (WT, p < 0.001; KO, p < 0.05). + +(C) HIF-1α KOs have more stored glycogen than do WT mice. Glycogen levels were measured following the same stimulation protocol as in (B). The change in glycogen from rest to poststimulation was also greater in the HIF-1α KOs, indicating that they metabolized more glycogen in response to stimulation (p < 0.01; *p < 0.05, WT at rest vs. KO at rest). + +(D) HIF-1α KOs utilize more PCr in response to stimulation than do WT mice. Similar levels of PCr were seen at rest, but HIF-1α KOs metabolized significantly more during stimulation (p < 0.05) and had much lower levels following the protocol (**p < 0.01, WT poststimulation vs. KO poststimulation). + +(E) A trend toward lower PCr/ATP concentration ratios was seen in HIF-1α KOs relative to WT mice following stimulation, although the difference did not quite reach statistical significance (p < 0.10). A trend toward a greater drop from rest to poststimulation in the PCr/ATP ratio was also seen in HIF-1α KOs following stimulation (p < 0.10), indicating that they had to rely more heavily on PCr for ATP generation. + +(F) Slight but nonsignificant differences were seen in whole-muscle ATP levels at rest or following stimulation. Although HIF-1α KOs exhibited altered substrate utilization, they were able to meet their ATP demands during the protocol. + +(G) Both HIF-1α KOs and WT animals produced significant intramuscular lactate during the stimulation protocol; however, there was no significant difference in the amount produced by either genotype. Resting intramuscular lactate levels were also similar for WTs and HIF-1α KOs. + +Figure 4 + +Force Generation and Ca2+ Release in Isolated Muscle Fibers during Stimulation + +(A) No differences were seen in total force generation in isolated muscle fibers. Mechanically dissected fibers from the flexor brevis muscle were subjected to a fatiguing protocol. Neither initial nor final forces differed between HIF-1α KO and WT fibers. + +(B) Ca2+ release and reuptake in HIF-1α KO and WT fibers was not different during the stimulation protocol. Ca2+ levels were measured in individual fibers through use of fura-2 Ca2+ indicator. The altered substrate utilization did not affect the ability of the fibers to maintain proper Ca2+ flux. + +Figure 5 + +Oxidative Metabolism and Serum Lactate Production in HIF-1α KOs and WT Mice + +(A) HIF-1α KOs have higher resting levels of CS activity. CS is an enzyme in the Krebs cycle that can be regulated allosterically by ATP levels. Increased CS activity is indicative of increased muscle oxidative capacity, which is common in patients with glycogenolytic or glycolytic myopathies (#p < 0.10, KO vs. WT). + +(B) HIF-1α KOs have higher resting levels of B-HAD activity, which is indicative of a greater ability to oxidize fatty acids (**p < 0.01, WT vs. KO). + +(C) Lower serum lactate levels were seen in HIF-1α KOs following a timed 25-minute run (*p < 0.05, WT vs. KO). + +Figure 6 + +Endurance Capabilities of Untrained Mice + +(A) HIF-1α KOs have greater endurance in swimming tests as shown by their ability to swim on average more than 45 min longer than WT (*p < 0.05, WT vs. KO). + +(B) HIF-1α KOs have greater endurance than WT mice in uphill running tests. Although only a 10-min difference is seen between run times, it is to be noted that because of the protocol, this 10 min included two velocity increases (**p < 0.01, WT vs. KO). + +(C) HIF-1α KOs have less endurance than WT mice in downhill running tests. The same protocol was used as in Figure 4A, except the mice were run on a 10° decline (*p < 0.05, WT vs. KO). + +(D) RER uphill vs. downhill in WT mice. As would be expected from eccentric exercises relying more heavily on glycolytic fibers, the RER values are higher in mice running downhill than in those running uphill. + +(E) RER uphill vs. downhill in HIF-1α KOs. Once again, higher RER values are observed for mice running downhill than those running uphill. + +Figure 7 + +Increased Muscle Damage in HIF-1α KOs Following Repeated Exercise + +(A) WT mice and HIF-1α KOs underwent a 4-d endurance test, in which animals were run to exhaustion on each of four successive days with a minimum of 22 h rest between trials. HIF-1α KOs demonstrated initially greater endurance under the protocol; however, by the second day, their endurance advantage was eliminated, and by the fourth day, HIF-1α KOs were running for a significantly shorter time (**p < 0.01) than on the first day, while WT animals were running for approximately similar times as on the first day. Repeated measures ANOVA revealed that the decrease in performance on each successive day was unique to HIF-1α KOs (p < 0.05). + +(B) Example of hematoxylin and eosin staining of gastrocnemius muscles after 1 d of recovery by mice after the 4-d endurance test. Evidence of greater damage can be seen in HIF-1α KO muscles compared to WT muscles. + +(C) Example of PCNA staining of gastrocnemius muscles from exercised mice, demonstrating increased levels of muscle regeneration in HIF-1α KOs. + +(D) Number of PCNA-positive nuclei per square millimeter in gastrocnemius muscles of WT mice (n = 5) and HIF-1α KOs (n = 7) that ran repeatedly for 4 d. Although HIF-1α KOs have almost twice as many PCNA-positive nuclei per square millimeter, the difference is not significant, because of wild variations in that population. F-test analysis of the data reveals that the variance is much greater in the HIF-1α KO population than the WT population (p < 0.05). + +Figure 8 + +Glucose Tolerance and Glycogen Storage + +(A) No significant differences were seen in resting blood glucose levels in HIF-1α KOs or WT mice. Following injection of glucose at a dosage of 2 g/kg, no differences were seen in the maximum levels of blood glucose or the rate of glucose disappearance in either genotype. + +(B) Representative PAS staining of gastrocnemius muscle from WT mice and HIF-1α KOs. HIF-1α KOs demonstrate darker staining, indicating more stored glycogen. + +Table 1 + +Excision of HIF-1α in Various Tissues + +Deletion levels are the average percent of HIF-1α deleted ± SE + +Table 2 + +Fiber Typing of Gastrocnemius Muscle + +Values are percent ± SE + +Table 3 + +Fiber Typing of Soleus Muscle + +Values are percent ± SE + +* p < 0.05, WT vs. KO + +Table 4 + +Relative Gene Expression Levels + +Expression levels are means relative to resting WT for each gene ± SE + +Percent increase indicates percent increase of postexercise average gene expression over resting average + +*p < 0.05, rest vs. postexercise; **p < 0.01, rest vs. postexercise + +Table 5 + +Glycolytic Enzyme Activity Levels from Gastrocnemius Muscles + +Activities are in U/mg protein ± SE + +GAPDH, glyceraldehyde 3-phosphate dehydrogenase; PGI, phosphoglucose isomerase + +*p < 0.05, WT vs. HIF-1α KO for given exercised or resting state; **p < 0.05, rest vs. postexercise within given genotype + +Footnotes + +Conflicts of interest. The authors have declared that no conflicts of interest exist. + +Author contributions. RSJ conceived and designed the experiments. SDM, RAH, MJK, IMO, WM, RPH, and FJG performed the experiments. SDM, RAH, MJK, IMO, MCH, PDW, FJG, and RSJ analyzed the data. CRK and RSJ contributed reagents/materials/analysis tools. SDM and RSJ wrote the paper. + +Academic Editor: Michael Bate, University of Cambridge + +Citation: Mason SD, Howlett RA, Kim MJ, Olfert IM, Hogan MC, et al. (2004) Loss of skeletal muscle HIF-1α results in altered exercise endurance. PLoS Biol 2(10): e288. + diff --git a/src/ontogpt/evaluation/craft/database/all/15345036.ann b/src/ontogpt/evaluation/craft/database/all/15345036.ann new file mode 100644 index 000000000..92d07211c --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15345036.ann @@ -0,0 +1,1920 @@ +T1 CHEBI:18303 4 22 phosphatidylserine +T2 PR:000009218 4 31 phosphatidylserine receptor +T3 GO:0009790 63 76 embryogenesis +T4 CL:0000445 88 102 apoptotic cell +T5 GO:0043277 88 110 apoptotic cell removal +T6 GO:0043277 134 165 Phagocytosis of apoptotic cells +T7 CL:0000445 150 165 apoptotic cells +T8 NCBITaxon:33208 184 190 animal +T9 UBERON:0002405 204 210 immune +T10 GO:0019725 224 244 cellular homeostasis +T11 CHEBI:18303 250 268 phosphatidylserine +T12 PR:000009218 250 277 phosphatidylserine receptor +T13 PR:000009218 279 284 Ptdsr +T14 CL:0000234 289 299 phagocytes +T15 GO:0043652 343 372 engulfment of apoptotic cells +T16 CL:0000445 357 372 apoptotic cells +T17 CHEBI:18303 453 471 phosphatidylserine +T18 PR:000009218 453 480 phosphatidylserine receptor +T19 PR:000009218 509 514 Ptdsr +T20 SO:0000704 515 519 gene +T21 NCBITaxon:10088 527 532 mouse +T22 PR:000009218 556 561 Ptdsr +T23 NCBITaxon:10088 574 578 mice +T24 UBERON:0012101 586 595 perinatal +T25 GO:0007567 590 595 natal +T26 UBERON:0002113 673 679 kidney +T27 UBERON:0000160 681 690 intestine +T28 UBERON:0002107 692 697 liver +T29 UBERON:0002048 702 707 lungs +T30 GO:0009790 715 728 embryogenesis +T31 UBERON:0000970 740 743 eye +T32 GO:0001654 740 755 eye development +T33 UBERON:0000966 807 814 retinal +T34 UBERON:0000970 878 882 eyes +T35 PR:000009218 884 889 Ptdsr +T36 NCBITaxon:10088 894 898 mice +T37 UBERON:0001782 966 994 retinal-pigmented epithelium +T38 CHEBI:26130 974 983 pigmented +T39 UBERON:0001707 998 1012 nasal cavities +T40 CL:0000445 1047 1061 apoptotic cell +T41 GO:0043277 1047 1071 apoptotic cell clearance +T42 GO:0043652 1111 1140 engulfment of apoptotic cells +T43 CL:0000445 1125 1140 apoptotic cells +T44 PR:000009218 1155 1160 Ptdsr +T45 NCBITaxon:10088 1170 1174 mice +T46 PR:000009218 1180 1185 Ptdsr +T47 CL:0000235 1196 1207 macrophages +T48 GO:0019221 1252 1270 cytokine signaling +T49 CL:0000445 1294 1309 apoptotic cells +T50 CHEBI:16412 1318 1336 lipopolysaccharide +T51 PR:000009218 1351 1356 Ptdsr +T52 GO:0048568 1378 1389;1410 1412;1422 1449 development ... of ... organs during embryogenesis +T53 UBERON:0000062 1422 1428 organs +T54 CL:0000445 1462 1476 apoptotic cell +T55 GO:0043277 1462 1484 apoptotic cell removal +T56 PR:000009218 1486 1491 Ptdsr +T57 SO:0000704 1591 1595 gene +T58 PR:000009218 1607 1612 Ptdsr +T59 CL:0000445 1633 1647 apoptotic cell +T60 GO:0043277 1633 1657 apoptotic cell clearance +T61 CL:0000235 1661 1672 macrophages +T62 GO:0065007 1707 1717 regulation +T63 CL:0000235 1721 1731 macrophage +T64 CHEBI:18303 1811 1829 phosphatidylserine +T65 PR:000009218 1811 1838 phosphatidylserine receptor +T66 CL:0000445 1862 1876 apoptotic cell +T67 GO:0043277 1862 1886 apoptotic cell clearance +T68 GO:0012501 1901 1922 Programmed cell death +T69 GO:0006915 1927 1936 apoptosis +T70 GO:0007275 1965 1979;1991 2014 development of ... multicellular organisms +T71 UBERON:0000468 1991 2014 multicellular organisms +T72 NCBITaxon:1 2005 2014 organisms +T73 GO:0065007 2052 2063 controlling +T74 NCBITaxon:40674 2233 2242 mammalian +T75 UBERON:0000922 2243 2252 embryonic +T76 GO:0009790 2243 2264 embryonic development +T77 GO:0008219 2287 2297 cell death +T78 UBERON:0012101 2320 2336 perinatal period +T79 GO:0007567 2324 2329 natal +T80 CL:0000445 2342 2357 Apoptotic cells +T81 CL:0000234 2446 2456 phagocytes +T82 GO:0065007 2494 2503 regulated +T83 CHEBI:60816 2559 2570 immunogenic +T84 GO:0005622 2571 2584 intracellular +T85 GO:0016265 2628 2633 dying +T86 UBERON:0000104 2655 2663 lifespan +T87 NCBITaxon:1 2670 2678 organism +T88 GO:0043277 2686 2717 Phagocytosis of apoptotic cells +T89 CL:0000445 2702 2717 apoptotic cells +T90 GO:0043652 2837 2866 engulfment of apoptotic cells +T91 CL:0000445 2851 2866 apoptotic cells +T92 CHEBI:67079 2900 2917;2940 2949 anti-inflammatory ... mediators +T93 CHEBI:35705 2922 2949 immunosuppressive mediators +T94 CL:0000445 3093 3108 apoptotic cells +T95 GO:0006909 3135 3145 phagocytic +T96 CL:0000445 3189 3204 apoptotic cells +T97 SO:0000704 3215 3222 genetic +T98 NCBITaxon:7215 3234 3244 Drosophila +T99 NCBITaxon:6239 3249 3271 Caenorhabditis elegans +T100 GO:0006909 3314 3324 phagocytic +T101 GO:0043652 3373 3402 engulfment of apoptotic cells +T102 CL:0000445 3387 3402 apoptotic cells +T103 NCBITaxon:7742 3455 3466 vertebrates +T104 GO:0043277 3528 3559 phagocytosis of apoptotic cells +T105 CL:0000445 3544 3559 apoptotic cells +T106 PR:000001905 3651 3655 CD36 +T107 PR:000001885 3657 3661 SR-A +T108 PR:000001889 3666 3670 CD14 +T109 GO:0034683 3715 3719 αvβ3 +T110 PR:000002058 3777 3781 CD91 +T111 PR:000004984 3786 3798 calreticulin +T112 CHEBI:36357 3837 3846 molecules +T113 GO:0006909 3859 3871 phagocytosis +T114 CL:0000445 3922 3936 apoptotic cell +T115 CL:0000445 4038 4052 apoptotic cell +T116 GO:0043277 4038 4062 apoptotic cell clearance +T117 UBERON:0002405 4112 4118 immune +T118 NCBITaxon:10088 4148 4152 mice +T119 CHEBI:36357 4162 4171 molecules +T120 CL:0000445 4176 4190 apoptotic cell +T121 GO:0043652 4176 4190;4207 4213 apoptotic cell ... uptake +T122 NCBITaxon:10088 4263 4267 mice +T123 NCBITaxon:10088 4313 4317 mice +T124 SO:0000704 4366 4370 gene +T125 PR:000010329 4371 4374 Mer +T126 NCBITaxon:10088 4403 4407 mice +T127 PR:000016293 4416 4434 transglutaminase 2 +T128 PR:000010367 4438 4480 milk fat globule epidermal growth factor 8 +T129 UBERON:0007376 4455 4464 epidermal +T130 PR:000010367 4482 4488 MFG-E8 +T131 CHEBI:16247 4520 4532 phospholipid +T132 CHEBI:18303 4533 4551 phosphatidylserine +T133 CHEBI:18303 4553 4555 PS +T134 GO:0005886 4585 4600 plasma membrane +T135 CL:0000445 4604 4619 apoptotic cells +T136 GO:0006915 4683 4692 apoptosis +T137 CL:0000445 4764 4778 apoptotic cell +T138 GO:0043277 4764 4778;4795 4802 apoptotic cell ... removal +T139 GO:0009986 4821 4833 cell-surface +T140 GO:0030674 4838 4846 bridging +T141 CHEBI:36357 4847 4856 molecules +T142 CHEBI:18303 4883 4885 PS +T143 CL:0000445 4889 4904 apoptotic cells +T144 UBERON:0001977 4924 4929 serum +T145 PR:000004157 4939 4956 β2-glycoprotein 1 +T146 CHEBI:17089 4942 4954 glycoprotein +T147 PR:000013269 4961 4970 protein S +T148 SO:0000704 5007 5011 gene +T149 PR:000007853 5020 5025 GAS-6 +T150 GO:0006956 5032 5053 complement activation +T151 PR:000010367 5098 5104 MFG-E8 +T152 PR:000004071 5115 5124 annexin I +T153 CL:0000234 5162 5172 phagocytes +T154 CHEBI:18303 5194 5196 PS +T155 GO:0030674 5197 5205 bridging +T156 CHEBI:36357 5206 5215 molecules +T157 PR:000007853 5269 5274 GAS-6 +T158 PR:000010329 5320 5323 Mer +T159 PR:000010367 5333 5339 MFG-E8 +T160 GO:0034683 5377 5381 αvβ3 +T161 CHEBI:36357 5397 5406 molecules +T162 CHEBI:18303 5417 5419 PS +T163 PR:000011634 5453 5508 lectin-like oxidized low-density lipoprotein receptor-1 +T164 MOP:0000568 5465 5473 oxidized +T165 PR:000011634 5510 5515 LOX-1 +T166 PR:000001905 5545 5549 CD36 +T167 PR:000002064 5554 5558 CD68 +T168 CHEBI:36357 5628 5636 molecule +T169 CHEBI:18303 5655 5657 PS +T170 CHEBI:18303 5693 5711 phosphatidylserine +T171 PR:000009218 5693 5720 phosphatidylserine receptor +T172 PR:000009218 5722 5727 Ptdsr +T173 PR:000009218 5772 5777 Ptdsr +T174 GO:0043652 5794 5819 uptake of apoptotic cells +T175 CL:0000445 5804 5819 apoptotic cells +T176 PR:000009218 5834 5839 Ptdsr +T177 GO:0006909 5849 5861 phagocytosis +T178 CHEBI:18303 5899 5901 PS +T179 CHEBI:18303 5917 5919 PS +T180 CHEBI:36357 5928 5936 molecule +T181 PR:000004078 5937 5946 annexin V +T182 PR:000009218 5958 5963 Ptdsr +T183 GO:0042571 5964 5972 antibody +T184 PR:000009218 6004 6009 Ptdsr +T185 CHEBI:18303 6013 6015 PS +T186 CL:0000445 6019 6034 apoptotic cells +T187 CHEBI:67079 6088 6115 anti-inflammatory mediators +T188 PR:000000182 6127 6156 transforming growth factor-β1 +T189 PR:000000182 6158 6164 TGF-β1 +T190 CL:0000233 6167 6175 platelet +T191 GO:0030168 6167 6186 platelet-activating +T192 CHEBI:44811 6167 6193 platelet-activating factor +T193 CHEBI:44811 6195 6198 PAF +T194 CHEBI:15551 6205 6221 prostaglandin E2 +T195 PR:000009218 6272 6277 Ptdsr +T196 CL:0000235 6347 6358 macrophages +T197 CL:0000445 6364 6379 apoptotic cells +T198 UBERON:0002405 6428 6434 immune +T199 GO:0006955 6428 6444 immune responses +T200 CL:0000445 6448 6463 apoptotic cells +T201 PR:000009218 6509 6514 Ptdsr +T202 GO:0005634 6537 6549 cell nucleus +T203 GO:0005634 6555 6562 nuclear +T204 GO:0005634 6608 6615 nuclear +T205 SO:0001528 6608 6636 nuclear localization signals +T206 PR:000009218 6682 6687 Ptdsr +T207 GO:0005634 6695 6707 cell nucleus +T208 NCBITaxon:6083 6766 6771 Hydra +T209 GO:0005634 6794 6801 nuclear +T210 PR:000009218 6823 6828 Ptdsr +T211 GO:0005634 6867 6874 nuclear +T212 PR:000009218 6891 6896 Ptdsr +T213 NCBITaxon:6083 6900 6905 Hydra +T214 UBERON:0000483 6906 6916 epithelial +T215 CL:0000066 6906 6922 epithelial cells +T216 GO:0043277 6943 6974 phagocytosis of apoptotic cells +T217 CL:0000445 6959 6974 apoptotic cells +T218 PR:000009218 7044 7049 Ptdsr +T219 GO:0016020 7073 7081 membrane +T220 CL:0000445 7095 7109 apoptotic cell +T221 PR:000009218 7187 7192 Ptdsr +T222 SO:0000704 7215 7219 gene +T223 GO:0010467 7215 7230 gene-expression +T224 SO:0000704 7235 7239 gene +T225 NCBITaxon:10088 7261 7265 mice +T226 UBERON:0012101 7269 7280 perinatally +T227 GO:0007567 7273 7280 natally +T228 PR:000009218 7314 7319 Ptdsr +T229 NCBITaxon:10088 7329 7333 mice +T230 PR:000009218 7339 7344 Ptdsr +T231 UBERON:0000922 7355 7362 embryos +T232 UBERON:0000479 7393 7399 tissue +T233 UBERON:0000062 7404 7409 organ +T234 PR:000009218 7569 7574 Ptdsr +T235 NCBITaxon:10088 7584 7588 mice +T236 GO:0043277 7719 7747 clearance of apoptotic cells +T237 CL:0000445 7732 7747 apoptotic cells +T238 PR:000009218 7812 7817 Ptdsr +T239 NCBITaxon:10088 7828 7832 mice +T240 GO:0006915 7857 7866 apoptosis +T241 PR:000009218 7892 7897 Ptdsr +T242 NCBITaxon:10088 7908 7912 mice +T243 CHEBI:18303 7958 7976 phosphatidylserine +T244 PR:000009218 7958 7985 phosphatidylserine receptor +T245 PR:000009218 7986 7991 Ptdsr +T246 SO:0001023 8013 8019 allele +T247 NCBITaxon:10088 8027 8032 mouse +T248 SO:0000704 8036 8040 gene +T249 PR:000009218 8106 8111 Ptdsr +T250 NCBITaxon:10088 8121 8125 mice +T251 UBERON:0000922 8150 8159 embryonic +T252 CL:0002322 8150 8164;8170 8175 embryonic stem ... cells +T253 CL:0002322 8166 8168;8170 8175 ES ... cells +T254 SO:0000704 8180 8184 gene +T255 PR:000009218 8219 8224 Ptdsr +T256 SO:0001023 8230 8236 allele +T257 SO:0000704 8266 8273 genetic +T258 NCBITaxon:10088 8317 8322 mouse +T259 PR:000009218 8338 8343 Ptdsr +T260 PR:000009218 8376 8381 Ptdsr +T261 PR:000009218 8401 8406 Ptdsr +T262 NCBITaxon:10088 8410 8414 mice +T263 PR:000009218 8476 8481 Ptdsr +T264 NCBITaxon:10088 8486 8490 mice +T265 PR:000009218 8532 8537 Ptdsr +T266 NCBITaxon:10088 8548 8552 mice +T267 PR:000009218 8569 8574 Ptdsr +T268 GO:0010467 8575 8585 expression +T269 PR:000009218 8589 8594 Ptdsr +T270 UBERON:0000922 8599 8606 embryos +T271 NCBITaxon:10088 8740 8744 mice +T272 UBERON:0000922 8893 8902 embryonic +T273 GO:0009790 8893 8914 embryonic development +T274 PR:000009218 8931 8936 Ptdsr +T275 UBERON:0000922 9016 9023 embryos +T276 UBERON:0000922 9083 9090 embryos +T277 PR:000009218 9112 9117 Ptdsr +T278 NCBITaxon:10088 9120 9124 mice +T279 UBERON:0000922 9148 9155 embryos +T280 GO:0007565 9173 9184 gestational +T281 PR:000009218 9220 9225 Ptdsr +T282 UBERON:0000922 9247 9254 embryos +T283 UBERON:0000922 9344 9353 embryonic +T284 PR:000009218 9395 9400 Ptdsr +T285 UBERON:0000922 9406 9413 embryos +T286 PR:000009218 9485 9490 Ptdsr +T287 UBERON:0000922 9494 9501 embryos +T288 UBERON:0000922 9563 9570 embryos +T289 UBERON:0000033 9722 9726 head +T290 http://purl.obolibrary.org/obo/MONDO_0016064 9780 9792 cleft palate +T291 UBERON:0000033 9806 9810 head +T292 UBERON:0000970 9863 9866 eye +T293 GO:0001654 9863 9878 eye development +T294 UBERON:0000922 9924 9931 embryos +T295 NCBITaxon:33208 9946 9953 animals +T296 UBERON:0000970 10014 10018 eyes +T297 PR:000009218 10056 10061 Ptdsr +T298 PR:000009218 10069 10074 Ptdsr +T299 UBERON:0000922 10116 10123 embryos +T300 PR:000009218 10228 10233 Ptdsr +T301 UBERON:0000922 10238 10245 embryos +T302 PR:000009218 10283 10288 Ptdsr +T303 NCBITaxon:10088 10298 10302 mice +T304 GO:0007567 10312 10316 born +T305 GO:0007618 10353 10360 matings +T306 PR:000009218 10383 10388 Ptdsr +T307 GO:0016265 10423 10427 died +T308 GO:0007567 10435 10443 delivery +T309 GO:0007567 10468 10473 birth +T310 PR:000009218 10475 10480 Ptdsr +T311 http://purl.obolibrary.org/obo/MONDO_0016064 10601 10613 cleft palate +T312 UBERON:0000033 10624 10628 head +T313 UBERON:0000970 10647 10651 eyes +T314 PR:000009218 10706 10711 Ptdsr +T315 SO:0000704 10712 10716 gene +T316 UBERON:0012101 10729 10738 perinatal +T317 GO:0007567 10733 10738 natal +T318 GO:0010467 10803 10813 Expression +T319 PR:000009218 10817 10822 Ptdsr +T320 GO:0009790 10830 10843 embryogenesis +T321 UBERON:0007023 10851 10856 adult +T322 UBERON:0000479 10857 10864 tissues +T323 UBERON:0012101 10879 10888 perinatal +T324 GO:0007567 10883 10888 natal +T325 PR:000009218 10914 10919 Ptdsr +T326 PR:000009218 11012 11017 Ptdsr +T327 GO:0010467 11021 11030 expressed +T328 PR:000009218 11084 11089 Ptdsr +T329 SO:0000673 11090 11101 transcripts +T330 CL:0002322 11105 11113 ES cells +T331 UBERON:0000922 11118 11125 embryos +T332 GO:0010467 11206 11216 expression +T333 PR:000009218 11229 11234 Ptdsr +T334 GO:0010467 11253 11263 expression +T335 PR:000009218 11332 11337 Ptdsr +T336 SO:0000704 11344 11348 gene +T337 NCBITaxon:10088 11363 11368 mouse +T338 PR:000009218 11391 11396 Ptdsr +T339 SO:0000704 11397 11401 gene +T340 CL:0002322 11407 11414 ES cell +T341 SO:0000842 11478 11487;11492 11496 region of ... gene +T342 PR:000009218 11529 11534 Ptdsr +T343 GO:0010467 11535 11545 expression +T344 CHEBI:75055 11549 11554 X-Gal +T345 UBERON:0000922 11580 11587 embryos +T346 PR:000009218 11675 11680 Ptdsr +T347 GO:0010467 11681 11691 expression +T348 UBERON:0000062 11704 11710 organs +T349 PR:000009218 11763 11768 Ptdsr +T350 UBERON:0000922 11773 11780 embryos +T351 PR:000009218 11799 11804 Ptdsr +T352 GO:0010467 11805 11815 expression +T353 UBERON:0001049 11834 11845 neural tube +T354 UBERON:0002329 11847 11854 somites +T355 UBERON:0000948 11856 11861 heart +T356 UBERON:0001555 11863 11866 gut +T357 UBERON:0002539 11871 11887 branchial arches +T358 PR:000009218 11911 11916 Ptdsr +T359 GO:0010467 11917 11927 expression +T360 UBERON:0001016 11960 11974 nervous system +T361 UBERON:0001890 12010 12019 forebrain +T362 UBERON:0002028 12021 12030 hindbrain +T363 UBERON:0001049 12035 12046 neural tube +T364 GO:0009790 12065 12078 embryogenesis +T365 PR:000009218 12095 12100 Ptdsr +T366 GO:0010467 12101 12111 expression +T367 UBERON:0004347 12153 12162 limb buds +T368 UBERON:0000970 12167 12171 eyes +T369 PR:000009218 12185 12190 Ptdsr +T370 GO:0010467 12191 12201 expression +T371 UBERON:0000970 12276 12280 eyes +T372 UBERON:0004347 12314 12323 limb buds +T373 UBERON:0001049 12325 12336 neural tube +T374 UBERON:0000955 12341 12346 brain +T375 CHEBI:75055 12383 12388 X-Gal +T376 UBERON:0000922 12397 12404 embryos +T377 GO:0010467 12435 12445 expression +T378 UBERON:0001049 12461 12472 neural tube +T379 UBERON:0004061 12510 12522 mantle layer +T380 GO:0010467 12530 12540 expression +T381 UBERON:0001049 12567 12578 neural tube +T382 UBERON:0003054 12597 12607 roof plate +T383 GO:0010467 12621 12631 Expression +T384 UBERON:0000044 12635 12654 dorsal root ganglia +T385 UBERON:0001049 12670 12681 neural tube +T386 UBERON:0002329 12693 12700 somites +T387 PR:000009218 12715 12720 Ptdsr +T388 GO:0010467 12725 12734 expressed +T389 UBERON:0002329 12750 12756 somite +T390 UBERON:0003082 12768 12775 myotome +T391 UBERON:0004016 12777 12786 dermatome +T392 UBERON:0003089 12791 12801 sclerotome +T393 GO:0010467 12815 12825 Expression +T394 UBERON:0002329 12845 12852 somites +T395 GO:0010467 12875 12885 expression +T396 UBERON:0001066 12951 12971 intervertebral discs +T397 UBERON:0000970 13023 13026 eye +T398 PR:000009218 13052 13057 Ptdsr +T399 GO:0010467 13058 13068 expression +T400 UBERON:0005425 13076 13090;13095 13105 inner layer of ... neural cup +T401 UBERON:0003902 13141 13154 neural retina +T402 PR:000009218 13169 13174 Ptdsr +T403 GO:0010467 13175 13185 expression +T404 CL:0002228 13206 13230 primary lens fiber cells +T405 PR:000009218 13301 13306 Ptdsr +T406 GO:0010467 13310 13319 expressed +T407 UBERON:0002113 13358 13364 kidney +T408 UBERON:0002048 13369 13374 lungs +T409 GO:0010467 13383 13393 expression +T410 PR:000009218 13428 13433 Ptdsr +T411 GO:0010467 13434 13444 expression +T412 GO:0048513 13485 13499;13506 13512 development of ... organs +T413 UBERON:0000062 13506 13512 organs +T414 GO:0097617 13527 13540 Hybridization +T415 UBERON:0000479 13555 13561 tissue +T416 SO:0000673 13594 13604 transcript +T417 UBERON:0000479 13637 13643 tissue +T418 UBERON:0007023 13656 13661 adult +T419 NCBITaxon:10088 13662 13666 mice +T420 GO:0010467 13699 13709 expression +T421 UBERON:0000473 13726 13732 testis +T422 UBERON:0002370 13734 13740 thymus +T423 UBERON:0002113 13742 13748 kidney +T424 UBERON:0002107 13750 13755 liver +T425 GO:0010467 13787 13797 expression +T426 UBERON:0002048 13801 13805 lung +T427 UBERON:0002108 13807 13822 small intestine +T428 UBERON:0002106 13824 13830 spleen +T429 UBERON:0000945 13832 13839 stomach +T430 UBERON:0004288 13844 13852 skeletal +T431 PR:000009218 13867 13872 Ptdsr +T432 GO:0010467 13889 13898 expressed +T433 GO:0009790 13910 13923 embryogenesis +T434 UBERON:0007023 13931 13936 adult +T435 UBERON:0000479 13937 13944 tissues +T436 PR:000009218 13977 13982 Ptdsr +T437 UBERON:0000479 14006 14012 tissue +T438 UBERON:0000062 14017 14022 organ +T439 PR:000009218 14069 14074 Ptdsr +T440 UBERON:0000062 14078 14083 organ +T441 GO:0048513 14078 14095 organ development +T442 PR:000009218 14129 14134 Ptdsr +T443 UBERON:0000922 14151 14158 embryos +T444 UBERON:0000467 14222 14235 organ systems +T445 UBERON:0000062 14279 14284 organ +T446 UBERON:0000479 14289 14295 tissue +T447 UBERON:0002048 14337 14342 lungs +T448 UBERON:0002113 14344 14351 kidneys +T449 UBERON:0000160 14356 14365 intestine +T450 UBERON:0002048 14367 14372 Lungs +T451 UBERON:0003215 14435 14442 alveoli +T452 UBERON:0002041 14456 14472 Terminal bronchi +T453 UBERON:0002187 14456 14464;14477 14488 Terminal ... bronchioles +T454 UBERON:0000483 14548 14558 epithelial +T455 CL:0000067 14548 14575 epithelial cells with cilia +T456 GO:0005929 14570 14575 cilia +T457 GO:0009986 14591 14603 cell surface +T458 UBERON:0003215 14641 14648 alveoli +T459 UBERON:0002186 14652 14663 bronchioles +T460 PR:000009218 14680 14685 Ptdsr +T461 UBERON:0002048 14691 14696 lungs +T462 UBERON:0002048 14730 14734 lung +T463 UBERON:0003104 14799 14809 mesenchyme +T464 UBERON:0000479 14872 14878 tissue +T465 PR:000009218 14898 14903 Ptdsr +T466 UBERON:0000922 14908 14915 embryos +T467 UBERON:0002113 14933 14940 kidneys +T468 UBERON:0002113 14954 14961 Kidneys +T469 PR:000009218 14967 14972 Ptdsr +T470 UBERON:0000922 14977 14984 embryos +T471 UBERON:0000074 15049 15058 glomeruli +T472 UBERON:0001230 15064 15080 Bowman's capsule +T473 UBERON:0001232 15085 15103 collecting tubules +T474 UBERON:0000483 15124 15134 epithelial +T475 CL:0000066 15124 15140 epithelial cells +T476 PR:000009218 15167 15172 Ptdsr +T477 UBERON:0002113 15183 15190 kidneys +T478 UBERON:0000074 15210 15219 glomeruli +T479 UBERON:0001232 15234 15252 collecting tubules +T480 UBERON:0003104 15323 15333 mesenchyme +T481 PR:000009218 15349 15354 Ptdsr +T482 UBERON:0002113 15359 15366 kidneys +T483 UBERON:0000479 15391 15397 tissue +T484 UBERON:0000160 15436 15445 intestine +T485 PR:000009218 15476 15481 Ptdsr +T486 UBERON:0000922 15486 15493 embryos +T487 UBERON:0000009 15563 15572 submucosa +T488 UBERON:0000922 15599 15606 embryos +T489 UBERON:0000160 15620 15630 intestinal +T490 GO:0030154 15631 15655 cellular differentiation +T491 UBERON:0001809 15691 15710 intramural ganglion +T492 UBERON:0018260 15751 15766 muscular layers +T493 CL:0000540 15773 15787 neuronal cells +T494 UBERON:0000160 15809 15818 intestine +T495 PR:000009218 15822 15827 Ptdsr +T496 UBERON:0000922 15832 15839 embryos +T497 PR:000009218 15868 15873 Ptdsr +T498 NCBITaxon:10088 15878 15882 mice +T499 UBERON:0000955 15916 15921 brain +T500 UBERON:0000033 15972 15976 head +T501 UBERON:0000479 16016 16022 tissue +T502 UBERON:0003129 16035 16040 skull +T503 UBERON:0000955 16130 16135 brain +T504 UBERON:0000479 16136 16142 tissue +T505 UBERON:0000955 16162 16167 brain +T506 UBERON:0000479 16168 16174 tissue +T507 UBERON:0003129 16194 16199 skull +T508 UBERON:0003129 16227 16232 skull +T509 UBERON:0000955 16287 16292 brain +T510 UBERON:0000479 16293 16299 tissue +T511 NCBITaxon:10088 16310 16314 mice +T512 UBERON:0001851 16352 16360 cortical +T513 UBERON:0000955 16406 16411 brain +T514 PR:000009218 16442 16447 Ptdsr +T515 NCBITaxon:10088 16458 16463 mouse +T516 UBERON:0002048 16595 16600 lungs +T517 GO:0007567 16604 16609 birth +T518 PR:000009218 16611 16616 Ptdsr +T519 UBERON:0002048 16620 16625 lungs +T520 PR:000009218 16786 16791 Ptdsr +T521 NCBITaxon:10088 16802 16806 mice +T522 UBERON:0000922 16833 16842 embryonic +T523 UBERON:0002048 16843 16847 lung +T524 UBERON:0002048 16883 16887 lung +T525 GO:0007567 16902 16907 birth +T526 PR:000009218 16952 16957 Ptdsr +T527 NCBITaxon:10088 16962 16966 mice +T528 GO:0016265 16967 16970 die +T529 UBERON:0001004 16976 16987 respiratory +T530 http://purl.obolibrary.org/obo/MONDO_0021113 16976 16995 respiratory failure +T531 GO:0030218 17089 17119 erythropoietic differentiation +T532 CL:0000549 17126 17144 early erythroblast +T533 UBERON:0002107 17158 17163 liver +T534 http://purl.obolibrary.org/obo/MONDO_0002280 17222 17228 anemic +T535 PR:000009218 17264 17269 Ptdsr +T536 NCBITaxon:10088 17274 17278 mice +T537 PR:000009218 17289 17294 Ptdsr +T538 UBERON:0000970 17334 17340 ocular +T539 GO:0001654 17334 17352 ocular development +T540 UBERON:0000970 17390 17393 eye +T541 PR:000009218 17464 17469 Ptdsr +T542 UBERON:0000970 17516 17520 eyes +T543 UBERON:0000922 17674 17681 embryos +T544 UBERON:0000922 17735 17742 embryos +T545 UBERON:0000970 17784 17787 eye +T546 UBERON:0001781 17836 17845;17850 17856 layers of ... retina +T547 UBERON:0001781 17974 17988 retinal layers +T548 PR:000009218 17992 17997 Ptdsr +T549 UBERON:0000922 18002 18009 embryos +T550 UBERON:0000922 18046 18053 embryos +T551 UBERON:0000966 18143 18150 retinal +T552 PR:000009218 18165 18170 Ptdsr +T553 PR:000009218 18179 18184 Ptdsr +T554 UBERON:0000922 18189 18196 embryos +T555 UBERON:0001781 18220 18234 retinal layers +T556 PR:000009218 18251 18256 Ptdsr +T557 NCBITaxon:10088 18266 18270 mice +T558 NCBITaxon:33208 18334 18341 animals +T559 GO:0007567 18347 18352 natal +T560 UBERON:0000966 18394 18401 retinal +T561 PR:000009218 18426 18431 Ptdsr +T562 PR:000009218 18439 18444 Ptdsr +T563 NCBITaxon:10088 18449 18453 mice +T564 UBERON:0001781 18504 18518 retinal layers +T565 NCBITaxon:10088 18555 18559 mice +T566 PR:000009218 18561 18566 Ptdsr +T567 NCBITaxon:33208 18577 18584 animals +T568 GO:0030154 18634 18658 cellular differentiation +T569 UBERON:0001781 18676 18690 retinal layers +T570 UBERON:0000966 18713 18720 retinal +T571 UBERON:0001791 18750 18770 inner granular layer +T572 PR:000009218 18798 18803 Ptdsr +T573 NCBITaxon:33208 18814 18821 animals +T574 UBERON:0001781 18941 18954 retinal layer +T575 NCBITaxon:33208 18966 18973 animals +T576 PR:000009218 19027 19032 Ptdsr +T577 UBERON:0000970 19049 19055 ocular +T578 UBERON:0000966 19105 19112 retinal +T579 PR:000009218 19143 19148 Ptdsr +T580 UBERON:0000922 19153 19160 embryos +T581 UBERON:0000970 19211 19215 eyes +T582 UBERON:0000922 19258 19265 embryos +T583 UBERON:0000922 19273 19280 embryos +T584 UBERON:0003072 19317 19328 optical cup +T585 CHEBI:26130 19401 19410 pigmented +T586 CL:0000529 19401 19427 pigmented epithelial cells +T587 UBERON:0000483 19411 19421 epithelial +T588 UBERON:0001707 19435 19447 nasal cavity +T589 PR:000009218 19455 19460 Ptdsr +T590 NCBITaxon:10088 19470 19474 mice +T591 CHEBI:26130 19565 19574 pigmented +T592 CL:0000147 19565 19580 pigmented cells +T593 UBERON:0000483 19597 19607 epithelium +T594 UBERON:0001764 19615 19630 maxillary sinus +T595 UBERON:0001782 19658 19686 retinal-pigmented epithelium +T596 CHEBI:26130 19666 19675 pigmented +T597 UBERON:0000970 19783 19786 eye +T598 GO:0008283 19828 19841 proliferation +T599 UBERON:0003104 19853 19871 mesenchymal tissue +T600 CHEBI:26130 19910 19919 pigmented +T601 UBERON:0000483 19920 19930 epithelium +T602 UBERON:0000922 20058 20065 embryos +T603 UBERON:0000970 20107 20113 ocular +T604 PR:000009218 20131 20136 Ptdsr +T605 NCBITaxon:10088 20147 20151 mice +T606 CL:0009004 20196 20208 retinal cell +T607 UBERON:0001781 20196 20203;20209 20215 retinal ... layers +T608 UBERON:0000119 20204 20215 cell layers +T609 UBERON:0001791 20234 20254 inner granular layer +T610 PR:000009218 20324 20329 Ptdsr +T611 NCBITaxon:10088 20334 20338 mice +T612 UBERON:0000970 20385 20388 eye +T613 UBERON:0001707 20403 20417 nasal cavities +T614 GO:0043277 20420 20432;20447 20465 Phagocytosis ... of apoptotic cells +T615 GO:0043277 20437 20465 clearance of apoptotic cells +T616 CL:0000445 20450 20465 apoptotic cells +T617 PR:000009218 20479 20484 Ptdsr +T618 NCBITaxon:10088 20495 20499 mice +T619 PR:000009218 20524 20529 Ptdsr +T620 GO:0043277 20563 20591 clearance of apoptotic cells +T621 CL:0000445 20576 20591 apoptotic cells +T622 GO:0008219 20629 20639 cell death +T623 UBERON:0006012 20655 20673 interdigital areas +T624 UBERON:0002101 20692 20697 limbs +T625 GO:0006915 20699 20711;20725 20730 Apoptosis of ... cells +T626 UBERON:0002544 20717 20724 digital +T627 UBERON:0010328 20745 20768 mesenchyme of limb buds +T628 UBERON:0002544 20988 20995 digital +T629 GO:0008219 20996 21006 cell death +T630 UBERON:0005417 21010 21014;21024 21033 fore ... limb buds +T631 UBERON:0005418 21019 21033 hind limb buds +T632 PR:000009218 21039 21044 Ptdsr +T633 PR:000009218 21061 21066 Ptdsr +T634 NCBITaxon:10088 21079 21083 mice +T635 GO:0008219 21235 21245 cell death +T636 GO:0060033 21261 21271 regression +T637 UBERON:0006015 21279 21295 interdigital web +T638 PR:000009218 21372 21377 Ptdsr +T639 GO:0043277 21417 21439;21453 21458 clearance of apoptotic ... cells +T640 CL:0000445 21430 21439;21453 21458 apoptotic ... cells +T641 UBERON:0002544 21445 21452 digital +T642 UBERON:0002101 21466 21470 limb +T643 GO:0060173 21466 21482 limb development +T644 GO:0043277 21516 21542 removal of apoptotic cells +T645 CL:0000445 21527 21542 apoptotic cells +T646 PR:000009218 21558 21563 Ptdsr +T647 NCBITaxon:10088 21568 21572 mice +T648 PR:000026878 21611 21630 activated caspase 3 +T649 PR:000026878 21632 21638 aCasp3 +T650 UBERON:0000062 21664 21670 organs +T651 UBERON:0000479 21675 21682 tissues +T652 GO:0006915 21689 21698 apoptosis +T653 UBERON:0000479 21723 21729 tissue +T654 GO:0048771 21723 21740 tissue remodeling +T655 PR:000026878 21836 21842 aCasp3 +T656 PR:000009218 21913 21918 Ptdsr +T657 UBERON:0000922 21923 21930 embryos +T658 GO:0006915 22052 22061 apoptosis +T659 UBERON:0000062 22073 22079 organs +T660 UBERON:0000479 22084 22091 tissues +T661 UBERON:0000479 22093 22099 Tissue +T662 GO:0048771 22093 22113 Tissue restructuring +T663 GO:0012501 22117 22138 programmed cell death +T664 UBERON:0001049 22192 22203 neural tube +T665 UBERON:0001807 22240 22261 paravertebral ganglia +T666 CL:0000445 22286 22301 apoptotic cells +T667 UBERON:0000479 22326 22333 tissues +T668 PR:000009218 22334 22339 Ptdsr +T669 GO:0010467 22350 22359 expressed +T670 CL:0000445 22444 22459 apoptotic cells +T671 PR:000009218 22463 22468 Ptdsr +T672 PR:000009218 22476 22481 Ptdsr +T673 UBERON:0000922 22486 22493 embryos +T674 UBERON:0002113 22532 22538 kidney +T675 CL:0000445 22540 22555 apoptotic cells +T676 PR:000009218 22572 22577 Ptdsr +T677 PR:000009218 22585 22590 Ptdsr +T678 UBERON:0000922 22595 22602 embryos +T679 CL:0000445 22681 22696 apoptotic cells +T680 CL:0000445 22782 22796 apoptotic cell +T681 GO:0043277 22782 22806 apoptotic cell clearance +T682 UBERON:0000922 22844 22853 embryonic +T683 GO:0009790 22844 22865 embryonic development +T684 NCBITaxon:10088 22889 22893 mice +T685 CL:0000445 22926 22941 apoptotic cells +T686 PR:000026878 23015 23021 aCasp3 +T687 UBERON:0002370 23057 23063 thymus +T688 UBERON:0000948 23065 23070 heart +T689 UBERON:0001103 23072 23081 diaphragm +T690 UBERON:0005294 23083 23096 genital ridge +T691 UBERON:0000970 23098 23102 eyes +T692 UBERON:0000966 23107 23113 retina +T693 CL:0000445 23166 23180 apoptotic cell +T694 GO:0043277 23166 23188 apoptotic cell removal +T695 PR:000009218 23192 23197 Ptdsr +T696 NCBITaxon:10088 23202 23206 mice +T697 GO:0043277 23267 23290 clearance of dead cells +T698 UBERON:0002048 23298 23302 lung +T699 GO:0030324 23298 23314 lung development +T700 PR:000009218 23318 23323 Ptdsr +T701 NCBITaxon:10088 23334 23338 mice +T702 GO:0006915 23364 23373 apoptosis +T703 GO:0043277 23388 23402 cell clearance +T704 PR:000009218 23410 23415 Ptdsr +T705 NCBITaxon:10088 23425 23429 mice +T706 UBERON:0002048 23437 23441 lung +T707 PR:000026878 23455 23461 aCasp3 +T708 UBERON:0002048 23470 23474 lung +T709 UBERON:0000479 23475 23481 tissue +T710 PR:000009218 23487 23492 Ptdsr +T711 PR:000009218 23500 23505 Ptdsr +T712 NCBITaxon:10088 23510 23514 mice +T713 GO:0006915 23549 23558 apoptosis +T714 UBERON:0002048 23594 23598 lung +T715 GO:0060425 23594 23612 lung morphogenesis +T716 CL:0000445 23700 23715 apoptotic cells +T717 PR:000009218 23719 23724 Ptdsr +T718 PR:000009218 23733 23738 Ptdsr +T719 NCBITaxon:10088 23743 23747 mice +T720 UBERON:0000479 23803 23809 tissue +T721 UBERON:0002048 23822 23827 lungs +T722 PR:000009218 23833 23838 Ptdsr +T723 NCBITaxon:10088 23849 23853 mice +T724 CL:0000775 23933 23944 neutrophils +T725 UBERON:0002048 23963 23972 pulmonary +T726 PR:000009218 24021 24026 Ptdsr +T727 NCBITaxon:10088 24037 24041 mice +T728 CL:0000235 24063 24074 macrophages +T729 GO:0006915 24106 24115 apoptosis +T730 GO:0009790 24136 24149 embryogenesis +T731 CL:0000235 24206 24216 macrophage +T732 GO:0009986 24217 24224 surface +T733 PR:000001813 24232 24237 F4/80 +T734 PR:000026878 24246 24252 aCasp3 +T735 CL:0000235 24300 24311 macrophages +T736 CL:0000445 24317 24332 apoptotic cells +T737 UBERON:0005291 24351 24368 embryonic tissues +T738 CL:0000445 24370 24385 apoptotic cells +T739 CL:0000235 24390 24401 macrophages +T740 GO:0043277 24607 24633 removal of apoptotic cells +T741 CL:0000445 24618 24633 apoptotic cells +T742 CL:0000235 24660 24671 macrophages +T743 CL:0000445 24739 24753 apoptotic cell +T744 GO:0043277 24739 24763 apoptotic cell clearance +T745 PR:000009218 24767 24772 Ptdsr +T746 UBERON:0000922 24783 24790 embryos +T747 GO:0043277 24836 24867 phagocytosis of apoptotic cells +T748 CL:0000445 24852 24867 apoptotic cells +T749 CL:0000235 24948 24959 macrophages +T750 PR:000009218 24965 24970 Ptdsr +T751 NCBITaxon:10088 24980 24984 mice +T752 CL:0000445 25018 25032 apoptotic cell +T753 GO:0043652 25018 25039 apoptotic cell uptake +T754 GO:0006909 25063 25075 phagocytosis +T755 UBERON:0002107 25094 25099 liver +T756 CL:0000235 25108 25119 macrophages +T757 GO:0006909 25149 25161 phagocytosis +T758 GO:0006909 25169 25181 Phagocytosis +T759 CL:0000893 25195 25205 thymocytes +T760 UBERON:0001977 25298 25303 serum +T761 GO:0006909 25317 25329 phagocytosis +T762 CL:0000445 25414 25428 apoptotic cell +T763 GO:0043652 25414 25435 apoptotic cell uptake +T764 PR:000009218 25444 25449 Ptdsr +T765 PR:000009218 25458 25463 Ptdsr +T766 CL:0000235 25467 25478 macrophages +T767 CL:0000445 25514 25528 apoptotic cell +T768 GO:0043652 25514 25539 apoptotic cell engulfment +T769 CL:0000445 25657 25671 apoptotic cell +T770 GO:0043652 25657 25678 apoptotic cell uptake +T771 PR:000009218 25682 25687 Ptdsr +T772 CL:0000235 25692 25703 macrophages +T773 GO:0006909 25718 25730 phagocytosis +T774 CL:0000235 25782 25793 macrophages +T775 CL:0000445 25812 25827 apoptotic cells +T776 CL:0000235 25856 25867 macrophages +T777 GO:0006909 25904 25916 Phagocytosed +T778 CHEBI:51657 25918 25947 5-carboxytetramethylrhodamine +T779 CHEBI:51657 25950 25955 TAMRA +T780 CL:0000445 25966 25981 apoptotic cells +T781 PR:000001813 26032 26037 F4/80 +T782 CL:0000235 26046 26057 macrophages +T783 CL:0000235 26136 26146 macrophage +T784 PR:000009218 26158 26163 Ptdsr +T785 PR:000009218 26171 26176 Ptdsr +T786 CL:0000235 26236 26247 macrophages +T787 CL:0000445 26266 26281 apoptotic cells +T788 GO:0006909 26327 26339 phagocytosed +T789 CL:0000445 26340 26355 apoptotic cells +T790 CL:0000235 26360 26370 macrophage +T791 GO:0006909 26372 26384 phagocytotic +T792 PR:000009218 26421 26426 Ptdsr +T793 CL:0000235 26430 26441 macrophages +T794 CL:0000445 26490 26499;26507 26512 apoptotic ... cells +T795 CL:0000235 26532 26543 macrophages +T796 PR:000009218 26565 26570 Ptdsr +T797 CL:0000235 26581 26592 macrophages +T798 CL:0000445 26624 26639 apoptotic cells +T799 GO:0006909 26680 26692 phagocytosis +T800 GO:0012501 26721 26742 programmed cell death +T801 PR:000009218 26745 26750 Ptdsr +T802 CL:0000235 26838 26848 macrophage +T803 GO:0042116 26838 26860 macrophage stimulation +T804 GO:0043277 26906 26937 phagocytosis of apoptotic cells +T805 CL:0000445 26922 26937 apoptotic cells +T806 PR:000009218 26965 26970 Ptdsr +T807 GO:0065007 27004 27014 regulating +T808 CL:0000445 27086 27101 apoptotic cells +T809 CL:0000235 27105 27116 macrophages +T810 PR:000009218 27151 27156 Ptdsr +T811 CL:0000235 27161 27172 macrophages +T812 GO:0043652 27228 27256 ingestion of apoptotic cells +T813 CL:0000445 27241 27256 apoptotic cells +T814 PR:000000182 27280 27286 TGF-β1 +T815 PR:000001471 27291 27305 interleukin-10 +T816 PR:000001471 27307 27312 IL-10 +T817 CHEBI:16412 27346 27364 lipopolysaccharide +T818 CHEBI:16412 27366 27369 LPS +T819 CL:0000445 27403 27418 apoptotic cells +T820 PR:000000182 27438 27444 TGF-β1 +T821 PR:000001471 27449 27454 IL-10 +T822 PR:000009218 27506 27511 Ptdsr +T823 CL:0000235 27516 27527 macrophages +T824 GO:0046903 27541 27548 secrete +T825 GO:0043652 27588 27616 ingestion of apoptotic cells +T826 CL:0000445 27601 27616 apoptotic cells +T827 PR:000009218 27715 27720 Ptdsr +T828 CL:0000235 27776 27787 macrophages +T829 UBERON:0002405 27799 27805 immune +T830 GO:0043652 27850 27879 engulfment of apoptotic cells +T831 CL:0000445 27864 27879 apoptotic cells +T832 PR:000009218 27943 27948 Ptdsr +T833 CL:0000235 27953 27964 macrophages +T834 PR:000009218 27991 27996 Ptdsr +T835 PR:000009218 28004 28009 Ptdsr +T836 NCBITaxon:10088 28014 28018 mice +T837 CHEBI:16412 28024 28027 LPS +T838 http://purl.obolibrary.org/obo/MONDO_0005070 28051 28056 tumor +T839 PR:000000134 28051 28074 tumor necrosis factor-α +T840 PR:000000134 28076 28081 TNF-α +T841 PR:000009218 28139 28144 Ptdsr +T842 CL:0000235 28149 28160 macrophages +T843 PR:000000134 28189 28194 TNF-α +T844 CL:0000235 28214 28225 macrophages +T845 PR:000000134 28245 28250 TNF-α +T846 CHEBI:16412 28292 28295 LPS +T847 CHEBI:16412 28406 28409 LPS +T848 PR:000000134 28454 28459 TNF-α +T849 GO:1990774 28454 28467 TNF-α release +T850 PR:000009218 28471 28476 Ptdsr +T851 CL:0000235 28481 28492 macrophages +T852 GO:0043652 28512 28541 engulfment of apoptotic cells +T853 CL:0000445 28526 28541 apoptotic cells +T854 CHEBI:16412 28568 28571 LPS +T855 CL:0000445 28573 28588 apoptotic cells +T856 PR:000000134 28616 28621 TNF-α +T857 PR:000009218 28661 28666 Ptdsr +T858 CL:0000235 28677 28688 macrophages +T859 PR:000000134 28702 28707 TNF-α +T860 CHEBI:16412 28731 28734 LPS +T861 GO:0042116 28764 28790 stimulation of macrophages +T862 CL:0000235 28779 28790 macrophages +T863 CHEBI:16412 28796 28799 LPS +T864 CL:0000445 28804 28819 apoptotic cells +T865 CHEBI:16412 28888 28891 LPS +T866 PR:000000134 28900 28905 TNF-α +T867 GO:1990774 28900 28913 TNF-α release +T868 PR:000009218 28917 28922 Ptdsr +T869 CL:0000235 28927 28938 macrophages +T870 CL:0000445 28982 28997 apoptotic cells +T871 CL:0000235 29048 29059 macrophages +T872 PR:000001393 29138 29151 interleukin-6 +T873 CL:0000576 29156 29164 monocyte +T874 PR:000002122 29156 29190 monocyte chemoattractant protein-1 +T875 PR:000009218 29252 29257 Ptdsr +T876 CL:0000235 29277 29288 macrophages +T877 GO:0043652 29360 29389 engulfment of apoptotic cells +T878 CL:0000445 29374 29389 apoptotic cells +T879 PR:000009218 29391 29396 Ptdsr +T880 GO:0050663 29442 29452;29480 29489 release of ... cytokines +T881 CHEBI:16412 29513 29516 LPS +T882 CHEBI:16412 29549 29552 LPS +T883 CL:0000445 29557 29572 apoptotic cells +T884 PR:000009218 29590 29595 Ptdsr +T885 CL:0000235 29606 29617 macrophages +T886 GO:0046903 29656 29663 secrete +T887 PR:000009218 29715 29720 Ptdsr +T888 UBERON:0000467 29769 29782 organ systems +T889 CHEBI:18303 29859 29877 phosphatidylserine +T890 PR:000009218 29859 29886 phosphatidylserine receptor +T891 PR:000009218 29888 29893 Ptdsr +T892 SO:0000704 29895 29899 gene +T893 NCBITaxon:10088 29912 29916 mice +T894 PR:000009218 29943 29948 Ptdsr +T895 UBERON:0000062 30005 30011 organs +T896 UBERON:0000479 30016 30023 tissues +T897 GO:0009790 30031 30044 embryogenesis +T898 PR:000009218 30158 30163 Ptdsr +T899 NCBITaxon:10088 30174 30178 mice +T900 NCBITaxon:10088 30210 30215 mouse +T901 SO:0000147 30237 30242 exons +T902 SO:0000147 30268 30273 exons +T903 CHEBI:7507 30327 30335 neomycin +T904 SO:0005853 30346 30354 cassette +T905 PR:000009218 30360 30365 Ptdsr +T906 NCBITaxon:10088 30375 30380 mouse +T907 SO:0000704 30401 30408 genetic +T908 PR:000009218 30498 30503 Ptdsr +T909 SO:0001023 30509 30515 allele +T910 PR:000009218 30650 30655 Ptdsr +T911 NCBITaxon:10088 30665 30669 mice +T912 PR:000009218 30723 30728 Ptdsr +T913 UBERON:0012101 30749 30758 perinatal +T914 GO:0007567 30753 30758 natal +T915 PR:000009218 30885 30890 Ptdsr +T916 NCBITaxon:10088 30901 30906 mouse +T917 SO:0000704 30957 30964 genetic +T918 PR:000009218 31098 31103 Ptdsr +T919 NCBITaxon:10088 31113 31118 mouse +T920 UBERON:0000062 31244 31250 organs +T921 PR:000009218 31260 31265 Ptdsr +T922 GO:0010467 31269 31278 expressed +T923 GO:0009790 31293 31306 embryogenesis +T924 UBERON:0000113 31319 31328 adulthood +T925 GO:0060441 31349 31375;31380 31395 branching morphogenesis of ... lung epithelium +T926 UBERON:0000115 31380 31395 lung epithelium +T927 PR:000009218 31412 31417 Ptdsr +T928 UBERON:0002048 31422 31427 lungs +T929 UBERON:0000483 31440 31450 epithelial +T930 UBERON:0002113 31502 31509 kidneys +T931 UBERON:0001230 31547 31563 Bowman's capsule +T932 UBERON:0001232 31624 31642 collecting tubules +T933 UBERON:0000160 31681 31690 intestine +T934 UBERON:0000160 31785 31795 intestinal +T935 UBERON:0000479 31796 31803 tissues +T936 PR:000009218 31807 31812 Ptdsr +T937 NCBITaxon:10088 31822 31826 mice +T938 UBERON:0001809 31869 31884 enteric ganglia +T939 UBERON:0001135 31907 31927 smooth muscle tissue +T940 UBERON:0002113 31955 31961 kidney +T941 UBERON:0000160 31966 31975 intestine +T942 PR:000009218 32018 32023 Ptdsr +T943 PR:000009218 32119 32124 Ptdsr +T944 UBERON:0000922 32128 32135 embryos +T945 GO:0007567 32151 32156 birth +T946 UBERON:0000062 32225 32231 organs +T947 GO:0007565 32271 32280 gestation +T948 PR:000009218 32318 32323 Ptdsr +T949 UBERON:0002048 32328 32333 lungs +T950 UBERON:0002048 32359 32364 lungs +T951 NCBITaxon:10088 32381 32385 mice +T952 UBERON:0003215 32445 32452 alveoli +T953 UBERON:0002186 32457 32468 bronchioles +T954 GO:0012501 32505 32526 programmed cell death +T955 UBERON:0002048 32534 32538 lung +T956 GO:0030324 32534 32550 lung development +T957 PR:000009218 32568 32573 Ptdsr +T958 NCBITaxon:10088 32583 32587 mice +T959 GO:0009790 32599 32612 embryogenesis +T960 PR:000026878 32665 32671 aCasp3 +T961 GO:0006915 32686 32695 apoptosis +T962 UBERON:0002048 32719 32723 lung +T963 GO:0060425 32719 32737 lung morphogenesis +T964 CL:0000445 32805 32820 apoptotic cells +T965 PR:000009218 32824 32829 Ptdsr +T966 NCBITaxon:33208 32853 32860 animals +T967 CL:0000445 32901 32916 apoptotic cells +T968 UBERON:0002048 32924 32928 lung +T969 UBERON:0000479 32929 32936 tissues +T970 GO:0043277 33037 33059;33087 33092 clearance of apoptotic ... cells +T971 CL:0000445 33050 33059;33087 33092 apoptotic ... cells +T972 UBERON:0003104 33060 33071 mesenchymal +T973 CL:0000134 33060 33071;33087 33092 mesenchymal ... cells +T974 UBERON:0000483 33076 33086 epithelial +T975 CL:0000066 33076 33092 epithelial cells +T976 UBERON:0002048 33113 33117 lung +T977 GO:0060425 33113 33131 lung morphogenesis +T978 PR:000009218 33135 33140 Ptdsr +T979 NCBITaxon:10088 33151 33155 mice +T980 UBERON:0002048 33220 33224 lung +T981 GO:0030324 33220 33236 lung development +T982 GO:0009790 33244 33257 embryogenesis +T983 GO:0060428 33272 33284;33289 33304 formation of ... epithelial lung +T984 UBERON:0000115 33289 33304 epithelial lung +T985 GO:0001763 33309 33332 branching morphogenesis +T986 UBERON:0000062 33423 33428 organ +T987 UBERON:0007688 33429 33435 anlage +T988 UBERON:0000062 33657 33662 organ +T989 GO:0009790 33763 33776 embryogenesis +T990 UBERON:0001911 33786 33799 mammary gland +T991 GO:0030879 33786 33809 mammary gland formation +T992 UBERON:0002048 33828 33833 lungs +T993 GO:0006915 33926 33935 apoptosis +T994 UBERON:0002048 33966 33971 lungs +T995 PR:000009218 33975 33980 Ptdsr +T996 PR:000009218 34120 34125 Ptdsr +T997 GO:0016265 34134 34137 die +T998 UBERON:0001004 34141 34152 respiratory +T999 UBERON:0002048 34153 34157 lung +T1000 CHEBI:35195 34221 34231 surfactant +T1001 GO:0010467 34232 34242 expression +T1002 PR:000009218 34256 34261 Ptdsr +T1003 NCBITaxon:33208 34272 34279 animals +T1004 GO:0002070 34311 34324;34363 34379 maturation of ... epithelial cells +T1005 CHEBI:35195 34325 34335 surfactant +T1006 CL:0002063 34346 34379 type II alveolar epithelial cells +T1007 UBERON:0004821 34354 34373 alveolar epithelial +T1008 UBERON:0002048 34384 34388 lung +T1009 GO:0016265 34451 34456 death +T1010 PR:000009218 34460 34465 Ptdsr +T1011 NCBITaxon:10088 34473 34477 mice +T1012 PR:000009218 34544 34549 Ptdsr +T1013 PR:000009218 34592 34597 Ptdsr +T1014 UBERON:0004535 34648 34662 cardiovascular +T1015 PR:000009218 34733 34738 Ptdsr +T1016 NCBITaxon:10088 34748 34752 mice +T1017 GO:0016265 34753 34756 die +T1018 UBERON:0000948 34783 34788 heart +T1019 GO:0007507 34783 34800 heart development +T1020 UBERON:0000970 35035 35038 eye +T1021 GO:0001654 35035 35050 eye development +T1022 PR:000009218 35073 35078 Ptdsr +T1023 SO:0000704 35079 35083 gene +T1024 PR:000009218 35085 35090 Ptdsr +T1025 UBERON:0000922 35101 35108 embryos +T1026 GO:0003406 35231 35243;35252 35280 formation of ... retinal-pigmented epithelium +T1027 UBERON:0001782 35252 35280 retinal-pigmented epithelium +T1028 CHEBI:26130 35260 35269 pigmented +T1029 GO:0008283 35298 35311 proliferation +T1030 UBERON:0003104 35326 35336 mesenchyme +T1031 UBERON:0001707 35344 35356 nasal cavity +T1032 NCBITaxon:10088 35475 35480 mouse +T1033 UBERON:0000970 35528 35531 eye +T1034 UBERON:0000966 35566 35573 retinal +T1035 GO:0060041 35566 35585 retinal development +T1036 GO:0007565 35619 35628 gestation +T1037 GO:0009653 35656 35669 morphogenesis +T1038 UBERON:0001791 35677 35705 inner granular retinal layer +T1039 GO:0009790 35725 35738 embryogenesis +T1040 GO:0010467 35808 35818 expression +T1041 PR:000009218 35834 35839 Ptdsr +T1042 SO:0000704 35840 35844 gene +T1043 PR:000009218 35857 35862 Ptdsr +T1044 GO:0010467 35866 35875 expressed +T1045 UBERON:0001016 35908 35922 nervous system +T1046 UBERON:0001890 35983 35992 forebrain +T1047 GO:0010467 36000 36010 expression +T1048 UBERON:0000966 36053 36059 retina +T1049 PR:000009218 36076 36081 Ptdsr +T1050 UBERON:0000970 36130 36136 ocular +T1051 GO:0048592 36130 36150 ocular morphogenesis +T1052 UBERON:0000970 36191 36194 eye +T1053 GO:0003408 36206 36229 formation of optic cups +T1054 UBERON:0003072 36219 36229 optic cups +T1055 UBERON:0000970 36243 36246 eye +T1056 GO:0001654 36243 36256 eye-formation +T1057 GO:0030900 36295 36309;36314 36323 development of ... forebrain +T1058 GO:0043584 36295 36309;36340 36344 development of ... nose +T1059 UBERON:0001890 36314 36323 forebrain +T1060 UBERON:0000004 36340 36344 nose +T1061 UBERON:0001890 36420 36429 forebrain +T1062 UBERON:0000004 36434 36439 nasal +T1063 PR:000009218 36454 36459 Ptdsr +T1064 UBERON:0000922 36469 36476 embryos +T1065 UBERON:0000922 36554 36560 embryo +T1066 PR:000009218 36595 36600 Ptdsr +T1067 GO:0065007 36620 36630 regulation +T1068 UBERON:0001890 36667 36676 forebrain +T1069 PR:000009218 36707 36712 Ptdsr +T1070 UBERON:0000970 36753 36756 eye +T1071 GO:0001654 36753 36766 eye formation +T1072 PR:000009218 36807 36812 Ptdsr +T1073 NCBITaxon:10088 36822 36826 mice +T1074 UBERON:0000966 36858 36865 retinal +T1075 UBERON:0004529 36866 36877 protrusions +T1076 UBERON:0000966 36941 36948 retinal +T1077 CL:0009004 36941 36953 retinal cell +T1078 UBERON:0000119 36949 36960 cell layers +T1079 UBERON:0000970 37028 37031 eye +T1080 GO:0043277 37085 37111 removal of apoptotic cells +T1081 CL:0000445 37096 37111 apoptotic cells +T1082 UBERON:0000970 37119 37122 eye +T1083 GO:0001654 37119 37134 eye development +T1084 CL:0000445 37205 37219 apoptotic cell +T1085 GO:0043277 37205 37229 apoptotic cell clearance +T1086 GO:0006915 37274 37283 apoptosis +T1087 NCBITaxon:10088 37301 37306 mouse +T1088 UBERON:0000966 37307 37314 retinal +T1089 GO:0060041 37307 37326 retinal development +T1090 CL:0000445 37357 37371 apoptotic cell +T1091 GO:0006915 37357 37377 apoptotic cell death +T1092 GO:0008219 37442 37452 cell death +T1093 UBERON:0003072 37484 37493 optic cup +T1094 GO:0006915 37535 37544 apoptosis +T1095 GO:0007567 37584 37589 birth +T1096 GO:0007567 37604 37609 natal +T1097 GO:0007567 37632 37637 natal +T1098 UBERON:0005425 37709 37714;37725 37734;37739 37748 inner ... layers of ... optic cup +T1099 UBERON:0005424 37719 37734;37739 37748 outer layers of ... optic cup +T1100 UBERON:0000970 37758 37761 eye +T1101 GO:0001654 37758 37773 eye development +T1102 UBERON:0000966 37797 37804 retinal +T1103 CL:0009004 37797 37809 retinal cell +T1104 GO:0006915 37805 37819 cell apoptosis +T1105 GO:0007567 37840 37847 natally +T1106 CL:0000540 37908 37916 neuronal +T1107 GO:0008219 37943 37953 cell death +T1108 UBERON:0000966 37968 37975 retinal +T1109 GO:0060041 37968 37987 retinal development +T1110 UBERON:0001781 37998 38012 retinal layers +T1111 UBERON:0001791 38031 38051 inner granular layer +T1112 PR:000009218 38121 38126 Ptdsr +T1113 GO:0007567 38190 38195 natal +T1114 GO:0043277 38196 38220;38235 38240 elimination of apoptotic ... cells +T1115 CL:0000445 38211 38220;38235 38240 apoptotic ... cells +T1116 CL:0000210 38221 38240 photoreceptor cells +T1117 PR:000009218 38244 38249 Ptdsr +T1118 CL:0000235 38259 38269 macrophage +T1119 PR:000009218 38380 38385 Ptdsr +T1120 GO:0042571 38386 38394 antibody +T1121 UBERON:0000955 38496 38501 brain +T1122 PR:000009218 38523 38528 Ptdsr +T1123 NCBITaxon:10088 38533 38537 mice +T1124 UBERON:0000955 38568 38573 brain +T1125 PR:000009218 38622 38627 Ptdsr +T1126 NCBITaxon:10088 38635 38639 mice +T1127 UBERON:0000955 38717 38722 brain +T1128 PR:000002194 38760 38765 Apaf1 +T1129 NCBITaxon:10088 38775 38779 mice +T1130 CL:0000445 38890 38905 apoptotic cells +T1131 GO:0030263 38909 38917 pyknotic +T1132 UBERON:0034705 38939 38954 neuroepithelium +T1133 PR:000009218 38958 38963 Ptdsr +T1134 PR:000009218 38972 38977 Ptdsr +T1135 NCBITaxon:10088 38981 38985 mice +T1136 GO:0008219 39001 39011 cell death +T1137 GO:0043277 39026 39048;39067 39072 clearance of apoptotic ... cells +T1138 CL:0000445 39039 39048;39067 39072 apoptotic ... cells +T1139 UBERON:0000955 39108 39113 brain +T1140 PR:000009218 39169 39174 Ptdsr +T1141 UBERON:0000479 39198 39204 tissue +T1142 GO:0007565 39248 39257 gestation +T1143 UBERON:0000062 39332 39338 organs +T1144 PR:000009218 39342 39347 Ptdsr +T1145 NCBITaxon:10088 39357 39361 mice +T1146 UBERON:0000479 39387 39393 tissue +T1147 CL:0000445 39444 39458 apoptotic cell +T1148 GO:0043277 39444 39468 apoptotic cell clearance +T1149 PR:000009218 39503 39508 Ptdsr +T1150 PR:000009218 39561 39566 Ptdsr +T1151 UBERON:0000479 39619 39625 tissue +T1152 PR:000009218 39714 39719 Ptdsr +T1153 NCBITaxon:10088 39730 39734 mice +T1154 PR:000009218 39796 39801 Ptdsr +T1155 GO:0010467 39802 39812 expression +T1156 UBERON:0000479 39833 39839 tissue +T1157 PR:000009218 39858 39863 Ptdsr +T1158 GO:0043277 39889 39917 clearance of apoptotic cells +T1159 CL:0000445 39902 39917 apoptotic cells +T1160 PR:000009218 39948 39953 Ptdsr +T1161 GO:0043652 39988 40013 uptake of apoptotic cells +T1162 CL:0000445 39998 40013 apoptotic cells +T1163 CL:0000445 40032 40046 apoptotic cell +T1164 GO:0043277 40032 40056 apoptotic cell clearance +T1165 PR:000009218 40068 40073 Ptdsr +T1166 UBERON:0000922 40078 40085 embryos +T1167 GO:0043277 40111 40137 removal of apoptotic cells +T1168 CL:0000445 40122 40137 apoptotic cells +T1169 PR:000009218 40172 40177 Ptdsr +T1170 UBERON:0000479 40226 40233 tissues +T1171 UBERON:0000062 40238 40244 organs +T1172 PR:000009218 40248 40253 Ptdsr +T1173 PR:000009218 40261 40266 Ptdsr +T1174 NCBITaxon:33208 40271 40278 animals +T1175 UBERON:0000922 40300 40309 embryonic +T1176 GO:0009790 40300 40321 embryonic development +T1177 GO:0043652 40366 40391 uptake of apoptotic cells +T1178 CL:0000445 40376 40391 apoptotic cells +T1179 GO:0006909 40437 40449 phagocytosis +T1180 GO:0043652 40499 40524 uptake of apoptotic cells +T1181 CL:0000445 40509 40524 apoptotic cells +T1182 PR:000009218 40528 40533 Ptdsr +T1183 CL:0000235 40538 40549 macrophages +T1184 CL:0000235 40570 40581 macrophages +T1185 PR:000009218 40698 40703 Ptdsr +T1186 CL:0000445 40707 40721 apoptotic cell +T1187 GO:0043277 40707 40731 apoptotic cell clearance +T1188 CHEBI:18303 40839 40857 phosphatidylserine +T1189 PR:000009218 40839 40866 phosphatidylserine receptor +T1190 SO:0001023 40872 40878 allele +T1191 NCBITaxon:6239 40882 40892 C. elegans +T1192 NCBITaxon:10088 40926 40931 mouse +T1193 CL:0000445 40964 40979 apoptotic cells +T1194 PR:000009218 40983 40988 Ptdsr +T1195 NCBITaxon:33208 41010 41017 animals +T1196 UBERON:0000479 41050 41057 tissues +T1197 UBERON:0002048 41132 41136 lung +T1198 UBERON:0001891 41138 41146 midbrain +T1199 UBERON:0000966 41151 41157 retina +T1200 GO:0007565 41174 41183 gestation +T1201 CL:0000445 41199 41214 apoptotic cells +T1202 GO:0006915 41327 41336 apoptosis +T1203 UBERON:0001781 41349 41362 retina layers +T1204 GO:0007567 41392 41399 natally +T1205 GO:0006915 41410 41419 apoptosis +T1206 GO:0042592 41481 41492 homeostasis +T1207 UBERON:0000115 41496 41511 lung epithelium +T1208 GO:0007567 41518 41523 birth +T1209 http://purl.obolibrary.org/obo/MONDO_0000001 41530 41553 pathological conditions +T1210 UBERON:0002048 41564 41573 pulmonary +T1211 UBERON:0002048 41602 41606 lung +T1212 GO:0030324 41602 41618 lung development +T1213 GO:0007567 41634 41639 natal +T1214 GO:0006915 41649 41658 apoptosis +T1215 CL:0000445 41713 41728 apoptotic cells +T1216 UBERON:0002048 41742 41746 lung +T1217 UBERON:0000479 41747 41753 tissue +T1218 GO:0009790 41765 41778 embryogenesis +T1219 PR:000009218 41782 41787 Ptdsr +T1220 PR:000009218 41795 41800 Ptdsr +T1221 NCBITaxon:10088 41805 41809 mice +T1222 UBERON:0002107 41867 41872 liver +T1223 UBERON:0002370 41877 41883 thymus +T1224 PR:000009218 41926 41931 Ptdsr +T1225 PR:000009218 41939 41944 Ptdsr +T1226 UBERON:0000922 41949 41956 embryos +T1227 PR:000009218 42032 42037 Ptdsr +T1228 UBERON:0000922 42048 42055 embryos +T1229 PR:000001813 42108 42113 F4/80 +T1230 CL:0000235 42123 42134 macrophages +T1231 PR:000009218 42155 42160 Ptdsr +T1232 UBERON:0000922 42165 42172 embryos +T1233 GO:0006909 42227 42239 phagocytosed +T1234 PR:000001813 42304 42309 F4/80 +T1235 CL:0000235 42478 42489 macrophages +T1236 UBERON:0000479 42499 42506 tissues +T1237 GO:0009790 42578 42591 embryogenesis +T1238 CL:0000235 42593 42603 macrophage +T1239 GO:0043277 42613 42641 clearance of apoptotic cells +T1240 CL:0000445 42626 42641 apoptotic cells +T1241 GO:0043277 42700 42726 removal of apoptotic cells +T1242 CL:0000445 42711 42726 apoptotic cells +T1243 UBERON:0000479 42736 42743 tissues +T1244 GO:0012501 42750 42771 programmed cell death +T1245 GO:0009790 42807 42820 embryogenesis +T1246 UBERON:0005294 42848 42861 genital ridge +T1247 UBERON:0000991 42869 42874 gonad +T1248 GO:0035262 42869 42888 gonad morphogenesis +T1249 UBERON:0001049 42916 42927 neural tube +T1250 CL:0000445 42967 42982 apoptotic cells +T1251 CL:0000235 42987 42998 macrophages +T1252 GO:0043277 43035 43063 clearance of apoptotic cells +T1253 CL:0000445 43048 43063 apoptotic cells +T1254 CL:0000235 43133 43144 macrophages +T1255 GO:0006915 43187 43196 apoptosis +T1256 PR:000009218 43273 43278 Ptdsr +T1257 CL:0000235 43354 43364 macrophage +T1258 PR:000001944 43369 43374 Sfpi1 +T1259 UBERON:0000922 43384 43391 embryos +T1260 GO:0030097 43419 43432 hematopoietic +T1261 PR:000001944 43471 43475 PU.1 +T1262 GO:0043277 43487 43518 phagocytosis of apoptotic cells +T1263 CL:0000445 43503 43518 apoptotic cells +T1264 GO:0009790 43526 43539 embryogenesis +T1265 UBERON:0003104 43568 43579 mesenchymal +T1266 CHEBI:18303 43614 43632 phosphatidylserine +T1267 GO:0006909 43714 43725 phagocytose +T1268 CL:0000445 43726 43741 apoptotic cells +T1269 CL:0000445 43768 43782 apoptotic cell +T1270 GO:0043277 43768 43792 apoptotic cell clearance +T1271 PR:000009218 43865 43870 Ptdsr +T1272 GO:0043652 43956 43981 uptake of apoptotic cells +T1273 CL:0000445 43966 43981 apoptotic cells +T1274 PR:000009218 43985 43990 Ptdsr +T1275 CL:0000235 43995 44006 macrophages +T1276 GO:0006909 44030 44042 phagocytosis +T1277 UBERON:0002107 44070 44075 liver +T1278 CL:0000235 44084 44095 macrophages +T1279 CL:0000861 44158 44166;44178 44189 elicited ... macrophages +T1280 UBERON:0001179 44167 44177 peritoneal +T1281 CL:0000581 44167 44189 peritoneal macrophages +T1282 PR:000009218 44217 44222 Ptdsr +T1283 GO:0030097 44227 44240 hematopoietic +T1284 CL:0000037 44227 44251 hematopoietic stem cells +T1285 CL:0000235 44359 44369 macrophage +T1286 PR:000009218 44430 44435 Ptdsr +T1287 GO:0030097 44492 44505 hematopoiesis +T1288 GO:0042116 44552 44562;44579 44593 activation ... of macrophages +T1289 CL:0000235 44582 44593 macrophages +T1290 PR:000009218 44665 44670 Ptdsr +T1291 PR:000009218 44738 44743 Ptdsr +T1292 NCBITaxon:10088 44753 44757 mice +T1293 SO:0000704 44849 44856 genetic +T1294 CL:0000445 44891 44905 apoptotic cell +T1295 GO:0043652 44891 44916 apoptotic cell engulfment +T1296 CL:0000861 44946 44966 elicited macrophages +T1297 NCBITaxon:10088 44998 45002 mice +T1298 CL:0000445 45035 45049 apoptotic cell +T1299 GO:0043652 45035 45056 apoptotic cell uptake +T1300 GO:0006911 45197 45220 phagocytotic engulfment +T1301 PR:000009218 45233 45238 Ptdsr +T1302 CL:0000235 45249 45260 macrophages +T1303 PR:000009218 45310 45315 Ptdsr +T1304 PR:000009218 45323 45328 Ptdsr +T1305 CL:0000235 45333 45344 macrophages +T1306 CHEBI:16412 45425 45428 LPS +T1307 CL:0000445 45433 45448 apoptotic cells +T1308 GO:0001775 45478 45497 cellular activation +T1309 CHEBI:35224 45502 45510 effector +T1310 PR:000009218 45538 45543 Ptdsr +T1311 CL:0000235 45552 45563 macrophages +T1312 CL:0000235 45621 45631 macrophage +T1313 GO:0042116 45621 45642 macrophage activation +T1314 PR:000009218 45664 45669 Ptdsr +T1315 GO:0005634 45746 45753 nuclear +T1316 PR:000009218 45774 45779 Ptdsr +T1317 SO:0000704 45854 45861 genetic +T1318 CHEBI:18303 45890 45908 phosphatidylserine +T1319 PR:000009218 45890 45917 phosphatidylserine receptor +T1320 NCBITaxon:6239 45930 45940 C. elegans +T1321 PR:Q9GYI4 45996 46001 psr-1 +T1322 NCBITaxon:6239 46007 46017 C. elegans +T1323 SO:0000853 46018 46025 homolog +T1324 PR:000009218 46029 46034 Ptdsr +T1325 GO:0043652 46053 46075 cell-corpse engulfment +T1326 PR:Q9GYI4 46085 46090 psr-1 +T1327 GO:0016246 46091 46095 RNAi +T1328 PR:Q9GYI4 46249 46254 psr-1 +T1329 PR:Q9NHC3 46311 46316 Ced-2 +T1330 PR:000039485 46318 46324 Crk II +T1331 PR:000006616 46334 46342 Dock 180 +T1332 PR:Q03206 46345 46351 Ced-10 +T1333 PR:000013660 46353 46358 Rac 1 +T1334 PR:Q8STE5 46364 46370 Ced-12 +T1335 GO:0043652 46396 46418 cell-corpse engulfment +T1336 PR:Q9GYI4 46484 46489 psr-1 +T1337 GO:0010467 46540 46550 expressing +T1338 NCBITaxon:6239 46639 46649 C. elegans +T1339 PR:000009218 46740 46745 Ptdsr +T1340 GO:0043277 46754 46785 phagocytosis of apoptotic cells +T1341 CL:0000445 46770 46785 apoptotic cells +T1342 PR:000009218 46811 46816 Ptdsr +T1343 GO:0042571 46817 46825 antibody +T1344 GO:0042571 46849 46857 antibody +T1345 PR:000009218 46870 46875 Ptdsr +T1346 PR:000009218 46991 46996 Ptdsr +T1347 GO:0016020 47007 47015 membrane +T1348 NCBITaxon:9606 47148 47153 human +T1349 PR:000009218 47163 47168 Ptdsr +T1350 CHEBI:36357 47169 47178 molecules +T1351 CHEBI:18303 47193 47211 phosphatidylserine +T1352 PR:000009218 47298 47303 Ptdsr +T1353 PR:000009218 47338 47343 Ptdsr +T1354 CL:0000235 47354 47365 macrophages +T1355 CL:0000057 47370 47381 fibroblasts +T1356 PR:000009218 47548 47553 Ptdsr +T1357 PR:000009218 47615 47620 Ptdsr +T1358 PR:000009218 47667 47672 Ptdsr +T1359 SO:0000317 47673 47684 cDNA clones +T1360 GO:0042571 47716 47724 antibody +T1361 GO:0016020 47771 47779 membrane +T1362 GO:0042571 47875 47883 antibody +T1363 PR:000009218 48031 48036 Ptdsr +T1364 UBERON:0000479 48101 48108 tissues +T1365 GO:0009790 48116 48129 embryogenesis +T1366 PR:000009218 48143 48148 Ptdsr +T1367 GO:0009887 48214 48230;48239 48245 morphogenesis of ... organs +T1368 UBERON:0000062 48239 48245 organs +T1369 GO:0043277 48311 48339 clearance of apoptotic cells +T1370 CL:0000445 48324 48339 apoptotic cells +T1371 PR:000009218 48379 48384 Ptdsr +T1372 NCBITaxon:10088 48395 48399 mice +T1373 PR:000009218 48470 48475 Ptdsr +T1374 CHEBI:18303 48515 48533 phosphatidylserine +T1375 CHEBI:18303 48579 48597 phosphatidylserine +T1376 CL:0000445 48613 48627 apoptotic cell +T1377 GO:0043652 48613 48638 apoptotic cell engulfment +T1378 CHEBI:18303 48699 48717 phosphatidylserine +T1379 GO:0030674 48718 48726 bridging +T1380 PR:000009218 48774 48779 Ptdsr +T1381 CL:0000235 48784 48795 macrophages +T1382 GO:0006909 48834 48845 phagocytose +T1383 CL:0000445 48846 48861 apoptotic cells +T1384 PR:000009218 49015 49020 Ptdsr +T1385 SO:0001644 49095 49111 targeting vector +T1386 PR:000009218 49130 49135 Ptdsr +T1387 SO:0000704 49149 49153 gene +T1388 NCBITaxon:10088 49159 49163 mice +T1389 SO:0001644 49165 49181 Targeting vector +T1390 PR:000009218 49185 49190 Ptdsr +T1391 NCBITaxon:2 49202 49211 bacterial +T1392 SO:0000153 49202 49233 bacterial artificial chromosome +T1393 SO:0000153 49235 49238 BAC +T1394 SO:0001026 49345 49352 genomic +T1395 SO:0000153 49353 49356 BAC +T1396 PR:P23940 49421 49426 BamHI +T1397 PR:000009218 49458 49463 Ptdsr +T1398 SO:0001416 49474 49476;49484 49500 5' ... flanking regions +T1399 SO:0001417 49481 49500 3' flanking regions +T1400 SO:0000153 49525 49528 BAC +T1401 SO:0000147 49580 49585 exons +T1402 PR:000009218 49602 49607 Ptdsr +T1403 SO:0000704 49608 49612 gene +T1404 SO:0000359 49638 49650 loxP-flanked +T1405 CHEBI:7507 49651 49659 neomycin +T1406 SO:0005853 49671 49684 gene cassette +T1407 CL:0002322 49721 49729 ES cells +T1408 CL:0002322 49775 49783 ES cells +T1409 GO:0009294 49789 49800 transfected +T1410 SO:0001644 49822 49838 targeting vector +T1411 CHEBI:42768 49857 49861 G418 +T1412 CL:0002322 49863 49870 ES-cell +T1413 CHEBI:42768 49891 49895 G418 +T1414 PR:000009218 49996 50001 Ptdsr +T1415 NCBITaxon:10088 50018 50022 mice +T1416 PR:000009218 50098 50103 Ptdsr +T1417 CL:0002322 50108 50116 ES cells +T1418 UBERON:0000358 50129 50140 blastocysts +T1419 GO:0007618 50240 50245 mated +T1420 CL:0002322 50291 50298 ES-cell +T1421 SO:0001023 50391 50397 allele +T1422 SO:0001026 50439 50446 genomic +T1423 UBERON:0010166 50462 50466 coat +T1424 PR:000009218 50488 50493 Ptdsr +T1425 SO:0000704 50494 50498 gene +T1426 CL:0002322 50545 50552 ES-cell +T1427 SO:0000704 50575 50579 gene +T1428 SO:0000440 50585 50591 vector +T1429 PR:000009218 50599 50604 Ptdsr +T1430 PR:000009218 50725 50730 Ptdsr +T1431 CL:0002322 50746 50753 ES-cell +T1432 SO:0000704 50787 50791 gene +T1433 SO:0000188 50800 50806 intron +T1434 SO:0000147 50818 50823 exons +T1435 PR:000009218 50840 50845 Ptdsr +T1436 SO:0000704 50846 50850 gene +T1437 NCBITaxon:10088 50861 50865 mice +T1438 UBERON:0000358 50909 50920 blastocysts +T1439 GO:0007618 50988 50993 mated +T1440 SO:0000704 51058 51062 gene +T1441 SO:0001023 51068 51074 allele +T1442 GO:0010467 51091 51101 expression +T1443 UBERON:0000922 51191 51198 embryos +T1444 NCBITaxon:33208 51202 51209 animals +T1445 SO:0001026 51274 51281 Genomic +T1446 UBERON:0005631 51312 51336 extraembryonic membranes +T1447 UBERON:0002415 51340 51344 tail +T1448 UBERON:0002415 51371 51375 tail +T1449 SO:0001026 51428 51435 genomic +T1450 PR:000009218 51539 51544 Ptdsr +T1451 SO:0001023 51545 51551 allele +T1452 SO:0000121 51571 51585 forward primer +T1453 SO:0000132 51622 51636 reverse primer +T1454 SO:0005850 51675 51681;51684 51688 primer ... site +T1455 SO:0005850 51727 51733;51736 51740 primer ... site +T1456 SO:0000112 51767 51773 primer +T1457 SO:0000028 51821 51823 bp +T1458 PR:000009218 51843 51848 Ptdsr +T1459 NCBITaxon:10088 51852 51856 mice +T1460 PR:000009218 51870 51875 Ptdsr +T1461 PR:000009218 51909 51914 Ptdsr +T1462 SO:0001023 51915 51921 allele +T1463 SO:0001026 51923 51930 genomic +T1464 SO:0000112 51960 51966 primer +T1465 SO:0000132 51973 51987 reverse primer +T1466 SO:0005853 52063 52071 cassette +T1467 SO:0000028 52093 52095 bp +T1468 NCBITaxon:10088 52121 52125 mice +T1469 SO:0001023 52168 52174 allele +T1470 NCBITaxon:10088 52218 52222 mice +T1471 SO:0001026 52252 52259 genomic +T1472 PR:P23940 52300 52305 BamHI +T1473 CHEBI:2511 52404 52411 agarose +T1474 CHEBI:25614 52434 52439 nylon +T1475 GO:0097617 52517 52527 hybridized +T1476 SO:0000357 52543 52551 flanking +T1477 PR:P23940 52564 52569 BamHI +T1478 GO:0097617 52581 52591 hybridized +T1479 PR:000009218 52599 52604 Ptdsr +T1480 SO:0000357 52617 52625 flanking +T1481 NCBITaxon:10088 52774 52778 mice +T1482 GO:0097617 52800 52810 hybridized +T1483 SO:0000357 52822 52830 flanking +T1484 NCBITaxon:10088 52960 52964 mice +T1485 UBERON:0000922 53031 53038 embryos +T1486 CHEBI:33893 53052 53059 reagent +T1487 UBERON:0000922 53163 53170 embryos +T1488 CHEBI:25614 53209 53214 nylon +T1489 SO:0000871 53250 53260 polyA+ RNA +T1490 GO:0097617 53323 53333 hybridized +T1491 PR:000009218 53355 53360 Ptdsr +T1492 SO:0000121 53406 53420 forward primer +T1493 SO:0000132 53452 53466 reverse primer +T1494 GO:0097617 53539 53549 hybridized +T1495 PR:000003676 53557 53564 β-actin +T1496 UBERON:0000922 53697 53704 embryos +T1497 GO:0010467 53713 53720 express +T1498 PR:000009218 53721 53726 Ptdsr +T1499 UBERON:0000922 53756 53763 embryos +T1500 GO:0010467 53764 53773 expressed +T1501 PR:000009218 53798 53803 Ptdsr +T1502 UBERON:0000922 53834 53841 Embryos +T1503 GO:0019835 53892 53897 lysis +T1504 CHEBI:78708 53928 53940 Nonidet P-40 +T1505 CHEBI:9177 53947 53966 sodium deoxycholate +T1506 CHEBI:8984 53973 53976 SDS +T1507 CHEBI:60004 54000 54008 cocktail +T1508 CHEBI:8984 54090 54093 SDS +T1509 CHEBI:53250 54152 54156 PVDF +T1510 GO:0042571 54268 54276 antibody +T1511 PR:000009218 54280 54285 Ptdsr +T1512 PR:000009218 54287 54290 PSR +T1513 PR:000003676 54359 54366 β-actin +T1514 GO:0042571 54439 54449 antibodies +T1515 MOP:0000779 54450 54460 conjugated +T1516 NCBITaxon:3704 54464 54475 horseradish +T1517 NCBITaxon:33208 54646 54652 Animal +T1518 NCBITaxon:10088 54702 54706 mice +T1519 NCBITaxon:10088 54813 54817 mice +T1520 GO:0007631 54941 54944 fed +T1521 PR:000009218 55050 55055 Ptdsr +T1522 NCBITaxon:10088 55093 55097 mice +T1523 PR:000009218 55136 55141 Ptdsr +T1524 NCBITaxon:10088 55148 55153 mouse +T1525 UBERON:0000922 55232 55239 embryos +T1526 NCBITaxon:10088 55270 55274 mice +T1527 PR:000009218 55312 55317 Ptdsr +T1528 UBERON:0010148 55370 55383 vaginal plugs +T1529 UBERON:0010148 55408 55412 plug +T1530 UBERON:0000922 55444 55451 Embryos +T1531 CHEBI:50913 55555 55563 fixative +T1532 UBERON:0005631 55565 55590 Extra-embryonic membranes +T1533 PR:000009218 55626 55631 Ptdsr +T1534 UBERON:0000922 55636 55643 embryos +T1535 UBERON:0000922 55756 55763 Embryos +T1536 CHEBI:16842 55852 55860 formalin +T1537 CHEBI:30879 55900 55907 alcohol +T1538 CHEBI:51686 56013 56024 hematoxylin +T1539 CHEBI:51686 56036 56037 H +T1540 PR:000009218 56119 56124 Ptdsr +T1541 CL:0000445 56192 56207 apoptotic cells +T1542 CL:0000235 56212 56223 macrophages +T1543 PR:000026878 56230 56236 aCasp3 +T1544 GO:0042571 56241 56249 antibody +T1545 PR:000026878 56263 56282 activated caspase 3 +T1546 PR:000001813 56324 56329 F4/80 +T1547 GO:0042571 56377 56387 antibodies +T1548 MOP:0000093 56485 56497 biotinylated +T1549 GO:0042571 56508 56518 antibodies +T1550 CHEBI:75050 56585 56595 chromogens +T1551 CHEBI:51686 56631 56642 hematoxylin +T1552 UBERON:0004347 56733 56742 limb buds +T1553 UBERON:0000922 56779 56786 embryos +T1554 UBERON:0002107 56896 56901 liver +T1555 CL:0000235 56910 56921 macrophages +T1556 UBERON:0002107 56937 56943 livers +T1557 UBERON:0000922 56962 56969 embryos +T1558 UBERON:0002107 57101 57106 liver +T1559 CL:0000235 57419 57429 macrophage +T1560 PR:000005930 57419 57455 macrophage colony-stimulating factor +T1561 CL:0000235 57457 57458 M +T1562 PR:000005930 57457 57462 M-CSF +T1563 UBERON:0000479 57523 57529 tissue +T1564 CHEBI:16526 57561 57564 CO2 +T1565 PR:000005930 57658 57663 M-CSF +T1566 CL:0000235 57766 57776 Macrophage +T1567 GO:0006909 57777 57789 phagocytosis +T1568 CL:0000235 57839 57850 macrophages +T1569 CL:0000445 57973 57982;57990 57995 apoptotic ... cells +T1570 CL:0000893 58005 58015 thymocytes +T1571 UBERON:0002370 58040 58046 thymus +T1572 NCBITaxon:10088 58076 58080 mice +T1573 CHEBI:51657 58095 58100 TAMRA +T1574 GO:0006915 58117 58126 apoptosis +T1575 CHEBI:15738 58174 58187 staurosporine +T1576 GO:0006915 58273 58282 apoptosis +T1577 UBERON:0002370 58309 58315 thymic +T1578 CHEBI:18303 58446 58448 PS +T1579 GO:0009986 58458 58465 surface +T1580 CHEBI:37926 58528 58532 FITC +T1581 PR:000004078 58533 58542 annexin V +T1582 CHEBI:51240 58547 58563 propidium iodide +T1583 CL:0000893 58588 58598 thymocytes +T1584 GO:0006909 58683 58695 Phagocytosis +T1585 CHEBI:16526 58739 58742 CO2 +T1586 GO:0043652 58782 58807 uptake of apoptotic cells +T1587 CL:0000445 58792 58807 apoptotic cells +T1588 GO:0006909 58891 58903 phagocytosed +T1589 GO:0006909 58922 58934 phagocytosis +T1590 CL:0000893 58948 58958 thymocytes +T1591 CL:0000235 58960 58971 macrophages +T1592 PR:000001813 59118 59123 F4/80 +T1593 GO:0042571 59124 59132 antibody +T1594 GO:0042571 59167 59175 antibody +T1595 MOP:0000779 59176 59183 coupled +T1596 CHEBI:52661 59187 59196 Alexa 488 +T1597 CL:0000893 59282 59292 thymocytes +T1598 GO:0006909 59355 59367 phagocytosis +T1599 CL:0000235 59408 59419 macrophages +T1600 CL:0000235 59450 59461 macrophages +T1601 CL:0000893 59490 59500 thymocytes +T1602 GO:0006909 59506 59518 phagocytotic +T1603 GO:0006909 59576 59588 phagocytotic +T1604 CL:0000235 59653 59664 macrophages +T1605 CL:0000235 59679 59690 macrophages +T1606 CL:0000235 59741 59752 macrophages +T1607 CL:0000235 59915 59925 macrophage +T1608 CL:0000893 59989 59999 thymocytes +T1609 CHEBI:16412 60068 60071 LPS +T1610 CL:0000445 60084 60099 apoptotic cells +T1611 PR:000001471 60146 60151 IL-10 +T1612 PR:000000182 60153 60159 TGF-β1 +T1613 PR:000000134 60163 60168 TNF-α +T1614 PR:000000134 60207 60212 TNF-α +T1615 CHEBI:16412 60301 60304 LPS +T1616 PR:000000134 60358 60363 TNF-α +T1617 NCBITaxon:10088 60365 60370 Mouse +T1618 PR:000000134 60371 60376 TNF-α +T1619 PR:000000182 60430 60436 TGF-β1 +T1620 PR:000000182 60450 60456 TGF-β1 +T1621 PR:000001471 60536 60541 IL-10 +T1622 NCBITaxon:10088 60609 60614 Mouse +T1623 CL:0000445 60991 61006 apoptotic cells +T1624 CL:0000235 61011 61022 macrophages +T1625 UBERON:0000922 61053 61060 embryos +T1626 PR:000009218 61120 61125 Ptdsr +T1627 CL:0000235 61137 61148 macrophages +T1628 PR:000009218 61176 61181 Ptdsr +T1629 NCBITaxon:10088 61191 61195 mice +T1630 CL:0000445 61284 61299 apoptotic cells +T1631 CL:0000235 61304 61315 macrophages +T1632 UBERON:0000922 61346 61353 embryos +T1633 PR:000009218 61413 61418 Ptdsr +T1634 CL:0000235 61430 61441 macrophages +T1635 PR:000009218 61469 61474 Ptdsr +T1636 NCBITaxon:10088 61484 61488 mice +T1637 NCBITaxon:10088 61756 61760 mice +T1638 PR:000009218 61795 61800 Ptdsr +T1639 CL:0002322 61838 61845 ES cell +T1640 UBERON:0000358 61846 61856 blastocyst +T1641 CHEBI:10545 61893 61901 electron +T1642 SO:0001026 62056 62063 genomic +T1643 UBERON:0000948 62101 62106 Heart +T1644 UBERON:0002048 62108 62112 Lung +T1645 UBERON:0000178 62118 62123 Blood +T1646 CL:0002322 62153 62160 ES cell +T1647 SO:0000704 62161 62165 gene +T1648 NCBITaxon:9606 62260 62265 human +T1649 NCBITaxon:10088 62347 62352 mouse +T1650 CHEBI:18303 62442 62460 phosphatidylserine +T1651 PR:000009218 62442 62469 phosphatidylserine receptor +T1652 SO:0000704 62470 62474 gene +T1653 PR:000009218 62480 62485 Ptdsr +T1654 SO:0000704 62486 62490 gene +T1655 CL:0002322 62539 62547 ES cells +T1656 SO:0000147 62575 62580 exons +T1657 NCBITaxon:39107 62597 62603 murine +T1658 PR:000009218 62604 62609 Ptdsr +T1659 SO:0000704 62610 62614 gene +T1660 SO:0000359 62640 62652 loxP-flanked +T1661 CHEBI:7507 62653 62661 neomycin +T1662 SO:0000704 62681 62685 gene +T1663 SO:0000717 62714 62727 reading frame +T1664 SO:0000200 62752 62766 Coding exons I +T1665 SO:0000147 62809 62814 exons +T1666 SO:0000061 62834 62851 Restriction sites +T1667 PR:P23940 62870 62875 BamHI +T1668 SO:0001026 63058 63065 genomic +T1669 PR:000009218 63105 63110 Ptdsr +T1670 NCBITaxon:33208 63120 63127 animals +T1671 PR:P23940 63143 63148 BamHI +T1672 GO:0097617 63153 63163 hybridized +T1673 PR:000009218 63238 63243 Ptdsr +T1674 SO:0001023 63244 63250 allele +T1675 PR:P23940 63278 63283 BamHI +T1676 PR:000009218 63323 63328 Ptdsr +T1677 SO:0001023 63329 63335 allele +T1678 PR:P23940 63360 63365 BamHI +T1679 PR:000009218 63404 63409 Ptdsr +T1680 SO:0001023 63410 63416 allele +T1681 UBERON:0000922 63440 63447 embryos +T1682 NCBITaxon:33208 63452 63459 animals +T1683 PR:000009218 63494 63499 Ptdsr +T1684 SO:0001023 63534 63540 allele +T1685 SO:0000112 63550 63556 primer +T1686 PR:000009218 63655 63660 Ptdsr +T1687 PR:000009218 63668 63673 Ptdsr +T1688 UBERON:0000922 63678 63685 embryos +T1689 PR:000009218 63761 63766 Ptdsr +T1690 PR:000009218 63774 63779 Ptdsr +T1691 UBERON:0000922 63784 63791 embryos +T1692 PR:000009218 63800 63805 Ptdsr +T1693 GO:0042571 63815 63823 antibody +T1694 GO:0007567 63876 63881 birth +T1695 NCBITaxon:10088 63986 63990 mice +T1696 PR:000009218 64009 64014 Ptdsr +T1697 UBERON:0000922 64020 64027 embryos +T1698 UBERON:0001890 64052 64066 prosencephalic +T1699 UBERON:0001890 64081 64090 forebrain +T1700 UBERON:0000970 64157 64161 eyes +T1701 UBERON:0000033 64227 64231 head +T1702 http://purl.obolibrary.org/obo/MONDO_0002280 64301 64307 anemia +T1703 GO:0010467 64347 64357 Expression +T1704 PR:000009218 64370 64375 Ptdsr +T1705 UBERON:0000922 64383 64392 embryonic +T1706 GO:0009790 64383 64404 embryonic development +T1707 PR:000009218 64462 64467 Ptdsr +T1708 SO:0000704 64468 64472 gene +T1709 NCBITaxon:10088 64478 64483 mouse +T1710 GO:0010467 64498 64508 expression +T1711 UBERON:0000922 64531 64540 embryonic +T1712 GO:0065007 64586 64596 regulatory +T1713 SO:0005836 64586 64605 regulatory elements +T1714 SO:0000704 64613 64617 gene +T1715 CHEBI:7507 64655 64663 neomycin +T1716 GO:0010467 64698 64708 expression +T1717 SO:0005853 64709 64717 cassette +T1718 PR:000009218 64834 64839 Ptdsr +T1719 SO:0000704 64840 64844 gene +T1720 GO:0007565 64865 64874 gestation +T1721 GO:0010467 64876 64886 Expression +T1722 PR:000009218 64890 64895 Ptdsr +T1723 UBERON:0003714 64910 64924 neural tissues +T1724 UBERON:0002329 64929 64936 somites +T1725 UBERON:0002539 64945 64961 branchial arches +T1726 UBERON:0002101 64978 64983 limbs +T1727 UBERON:0000948 64989 64994 heart +T1728 UBERON:0007026 65000 65013 primitive gut +T1729 UBERON:0000970 65033 65036 eye +T1730 UBERON:0000922 65088 65095 embryos +T1731 GO:0010467 65105 65115 expression +T1732 PR:000009218 65119 65124 Ptdsr +T1733 UBERON:0001049 65136 65147 neural tube +T1734 UBERON:0034705 65162 65179 neural epithelium +T1735 UBERON:0002329 65185 65192 somites +T1736 UBERON:0000970 65202 65206 eyes +T1737 GO:0010467 65208 65218 Expression +T1738 UBERON:0000970 65226 65229 eye +T1739 UBERON:0003902 65258 65272 neural retinal +T1740 CL:0002319 65258 65264;65282 65287 neural ... cells +T1741 CL:0009004 65265 65272;65282 65287 retinal ... cells +T1742 CL:0002222 65277 65287 lens cells +T1743 GO:0010467 65293 65303 Expression +T1744 UBERON:0007023 65316 65321 adult +T1745 UBERON:0000479 65322 65329 tissues +T1746 GO:0010467 65348 65358 Expression +T1747 PR:000009218 65362 65367 Ptdsr +T1748 PR:000003676 65460 65467 β-actin +T1749 GO:0097617 65468 65481 hybridization +T1750 PR:000009218 65601 65606 Ptdsr +T1751 UBERON:0000062 65610 65616 organs +T1752 GO:0009790 65624 65637 embryogenesis +T1753 UBERON:0000922 65655 65662 embryos +T1754 PR:000009218 65673 65678 Ptdsr +T1755 UBERON:0000922 65720 65729 embryonic +T1756 CHEBI:51686 65829 65830 H +T1757 UBERON:0002048 65857 65862 lungs +T1758 PR:000009218 65870 65875 Ptdsr +T1759 UBERON:0000922 65880 65887 embryos +T1760 UBERON:0003215 65935 65942 alveoli +T1761 UBERON:0000483 65958 65968 epithelium +T1762 UBERON:0002186 65975 65986 bronchioles +T1763 UBERON:0002048 66034 66039 lungs +T1764 UBERON:0000074 66055 66064 glomeruli +T1765 UBERON:0002113 66081 66087 kidney +T1766 PR:000009218 66095 66100 Ptdsr +T1767 UBERON:0000922 66105 66112 embryos +T1768 UBERON:0001232 66160 66178 collecting tubules +T1769 UBERON:0005306 66226 66235 blastemas +T1770 UBERON:0002115 66272 66279 jejunum +T1771 UBERON:0001809 66287 66305 intramural ganglia +T1772 PR:000009218 66309 66314 Ptdsr +T1773 UBERON:0000922 66318 66325 embryos +T1774 UBERON:0000009 66369 66378 submucosa +T1775 UBERON:0000955 66408 66413 Brain +T1776 PR:000009218 66446 66451 Ptdsr +T1777 UBERON:0000922 66455 66462 embryos +T1778 UBERON:0001898 66498 66510 hypothalamus +T1779 UBERON:0003129 66531 66536 skull +T1780 UBERON:0001716 66538 66554 secondary palate +T1781 UBERON:0005356 66577 66591 Rathke's pouch +T1782 UBERON:0001851 66626 66632 cortex +T1783 UBERON:0000922 66671 66678 embryos +T1784 PR:000009218 66712 66717 Ptdsr +T1785 UBERON:0002048 66722 66727 lungs +T1786 GO:0048286 66758 66778 formation of alveoli +T1787 GO:0060435 66758 66770;66795 66806 formation of ... bronchioles +T1788 UBERON:0003215 66771 66778 alveoli +T1789 UBERON:0002186 66795 66806 bronchioles +T1790 UBERON:0002107 66839 66844 liver +T1791 CL:0000556 66872 66886 megakaryocytes +T1792 GO:0030218 66966 66980 erythropoietic +T1793 CL:0000232 67003 67015 erythrocytes +T1794 GO:0005773 67032 67040 vacuoles +T1795 CHEBI:24384 67052 67060 glycogen +T1796 GO:0008152 67094 67105 metabolized +T1797 UBERON:0012101 67109 67120 perinatally +T1798 GO:0007567 67113 67120 natally +T1799 GO:0016265 67121 67126 dying +T1800 PR:000009218 67127 67132 Ptdsr +T1801 NCBITaxon:33208 67137 67144 animals +T1802 PR:000009218 67269 67274 Ptdsr +T1803 UBERON:0000966 67279 67286 retinas +T1804 PR:000009218 67342 67347 Ptdsr +T1805 UBERON:0000966 67352 67358 retina +T1806 UBERON:0000966 67486 67492 retina +T1807 PR:000009218 67509 67514 Ptdsr +T1808 UBERON:0000922 67518 67525 embryos +T1809 UBERON:0001789 67535 67555 outer granular layer +T1810 UBERON:0001789 67557 67560 OGL +T1811 UBERON:0001790 67563 67584 outer plexiform layer +T1812 UBERON:0001790 67586 67589 OPL +T1813 UBERON:0001791 67592 67612 inner granular layer +T1814 UBERON:0001791 67614 67617 IGL +T1815 UBERON:0001795 67623 67644 inner plexiform layer +T1816 UBERON:0001795 67646 67649 IPL +T1817 UBERON:0001791 67666 67669 IGL +T1818 PR:000009218 67673 67678 Ptdsr +T1819 UBERON:0000966 67683 67690 retinas +T1820 PR:000009218 67826 67831 Ptdsr +T1821 UBERON:0000966 67836 67843 retinas +T1822 UBERON:0000966 67887 67893 retina +T1823 PR:000009218 67897 67902 Ptdsr +T1824 NCBITaxon:33208 67907 67914 animals +T1825 UBERON:0000970 68007 68010 eye +T1826 GO:0001654 68007 68022 eye development +T1827 UBERON:0000970 68044 68047 eye +T1828 PR:000009218 68052 68057 Ptdsr +T1829 UBERON:0000922 68062 68069 embryos +T1830 PR:000009218 68091 68096 Ptdsr +T1831 UBERON:0000922 68101 68108 embryos +T1832 UBERON:0000970 68149 68153 eyes +T1833 CHEBI:51686 68186 68187 H +T1834 UBERON:0000922 68237 68244 embryos +T1835 UBERON:0003072 68307 68316 optic cup +T1836 UBERON:0001782 68431 68459 retinal-pigmented epithelium +T1837 CHEBI:26130 68439 68448 pigmented +T1838 UBERON:0001764 68467 68482 maxillary sinus +T1839 GO:0043473 68500 68521 deposition of pigment +T1840 CHEBI:26130 68514 68521 pigment +T1841 GO:0008283 68594 68607 proliferation +T1842 UBERON:0000479 68622 68629 tissues +T1843 UBERON:0001764 68666 68681 maxillary sinus +T1844 GO:0012501 68738 68759 programmed cell death +T1845 CL:0000235 68779 68790 macrophages +T1846 GO:0043277 68798 68824 removal of apoptotic cells +T1847 CL:0000445 68809 68824 apoptotic cells +T1848 PR:000009218 68842 68847 Ptdsr +T1849 UBERON:0000922 68851 68858 embryos +T1850 UBERON:0004347 68901 68910 limb buds +T1851 PR:000009218 68930 68935 Ptdsr +T1852 UBERON:0000922 68940 68947 embryos +T1853 CL:0000445 69010 69025 apoptotic cells +T1854 GO:0060033 69047 69057 regression +T1855 UBERON:0006015 69065 69081 interdigital web +T1856 PR:000026878 69120 69139 activated caspase 3 +T1857 PR:000026878 69141 69147 aCasp3 +T1858 PR:000009218 69183 69188 Ptdsr +T1859 UBERON:0000922 69193 69200 embryos +T1860 CL:0000445 69215 69230 apoptotic cells +T1861 UBERON:0001049 69238 69249 neural tube +T1862 UBERON:0000080 69261 69272 mesonephros +T1863 UBERON:0001807 69298 69319 paravertebral ganglia +T1864 UBERON:0000479 69327 69333 Tissue +T1865 CL:0000445 69367 69382 apoptotic cells +T1866 PR:000009218 69500 69505 Ptdsr +T1867 UBERON:0000922 69509 69516 embryos +T1868 CL:0000235 69566 69576 macrophage +T1869 PR:000001813 69601 69606 F4/80 +T1870 UBERON:0001807 69651 69672 paravertebral ganglia +T1871 UBERON:0000922 69716 69723 embryos +T1872 CL:0000235 69738 69749 macrophages +T1873 CL:0000445 69784 69799 apoptotic cells +T1874 UBERON:0000922 69807 69816 embryonic +T1875 GO:0009790 69807 69828 embryonic development +T1876 GO:0043277 69963 69994 Phagocytosis of apoptotic cells +T1877 CL:0000445 69979 69994 apoptotic cells +T1878 UBERON:0002107 70004 70009 liver +T1879 CL:0000235 70018 70029 macrophages +T1880 PR:000009218 70076 70081 Ptdsr +T1881 UBERON:0000922 70086 70093 embryos +T1882 CHEBI:51657 70124 70129 TAMRA +T1883 CL:0000893 70154 70164 thymocytes +T1884 CHEBI:15738 70179 70192 staurosporine +T1885 NCBITaxon:10088 70208 70212 mice +T1886 PR:000001813 70235 70240 F4/80 +T1887 CL:0000235 70250 70261 Macrophages +T1888 GO:0006909 70285 70297 phagocytosed +T1889 CL:0000445 70298 70313 apoptotic cells +T1890 GO:0006909 70350 70362 phagocytosis +T1891 CL:0000445 70366 70381 apoptotic cells +T1892 PR:000009218 70398 70403 Ptdsr +T1893 CL:0000235 70407 70418 macrophages +T1894 CL:0000235 70464 70475 macrophages +T1895 CL:0000445 70494 70509 apoptotic cells +T1896 GO:0006915 70526 70535 apoptosis +T1897 CHEBI:15738 70556 70569 staurosporine +T1898 CL:0000445 70634 70649 apoptotic cells +T1899 GO:0006909 70650 70662 phagocytosed +T1900 CL:0000235 70673 70684 macrophages +T1901 GO:0006909 70736 70748 phagocytosed +T1902 CL:0000235 70753 70763 macrophage +T1903 GO:0043277 70812 70838 removal of apoptotic cells +T1904 CL:0000445 70823 70838 apoptotic cells +T1905 PR:000009218 70861 70866 Ptdsr +T1906 CHEBI:16412 70941 70959 lipopolysaccharide +T1907 CHEBI:16412 70961 70964 LPS +T1908 CL:0000445 70970 70985 apoptotic cells +T1909 PR:000009218 71012 71017 Ptdsr +T1910 UBERON:0000922 71022 71029 embryos +T1911 CHEBI:16412 71070 71073 LPS +T1912 CL:0000445 71086 71101 apoptotic cells +T1913 CHEBI:16412 71138 71141 LPS +T1914 CL:0000445 71146 71161 apoptotic cells +T1915 CHEBI:16412 71174 71177 LPS +T1916 PR:000000134 71289 71294 TNF-α +T1917 PR:000000182 71299 71305 TGF-β1 +T1918 PR:000001471 71335 71340 IL-10 +T1919 PR:000009218 71665 71670 Ptdsr +T1920 NCBITaxon:10088 71675 71679 mice diff --git a/src/ontogpt/evaluation/craft/database/all/15345036.txt b/src/ontogpt/evaluation/craft/database/all/15345036.txt new file mode 100644 index 000000000..a3cfb203b --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15345036.txt @@ -0,0 +1,221 @@ +The phosphatidylserine receptor has essential functions during embryogenesis but not in apoptotic cell removal + +Abstract + +Background + +Phagocytosis of apoptotic cells is fundamental to animal development, immune function and cellular homeostasis. The phosphatidylserine receptor (Ptdsr) on phagocytes has been implicated in the recognition and engulfment of apoptotic cells and in anti-inflammatory signaling. To determine the biological function of the phosphatidylserine receptor in vivo, we inactivated the Ptdsr gene in the mouse. + +Results + +Ablation of Ptdsr function in mice causes perinatal lethality, growth retardation and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis. Moreover, eye development can be severely disturbed, ranging from defects in retinal differentiation to complete unilateral or bilateral absence of eyes. Ptdsr -/- mice with anophthalmia develop novel lesions, with induction of ectopic retinal-pigmented epithelium in nasal cavities. A comprehensive investigation of apoptotic cell clearance in vivo and in vitro demonstrated that engulfment of apoptotic cells was normal in Ptdsr knockout mice, but Ptdsr-deficient macrophages were impaired in pro- and anti-inflammatory cytokine signaling after stimulation with apoptotic cells or with lipopolysaccharide. + +Conclusion + +Ptdsr is essential for the development and differentiation of multiple organs during embryogenesis but not for apoptotic cell removal. Ptdsr may thus have a novel, unexpected developmental function as an important differentiation-promoting gene. Moreover, Ptdsr is not required for apoptotic cell clearance by macrophages but seems to be necessary for the regulation of macrophage cytokine responses. These results clearly contradict the current view that the phosphatidylserine receptor primarily functions in apoptotic cell clearance. + +Background + +Programmed cell death, or apoptosis, is required for the normal development of almost all multicellular organisms and is a physiological mechanism for controlling cell number; as a result, structures that are no longer needed are deleted during development and abnormal cells are eliminated [1,2]. Most of the cells produced during mammalian embryonic development undergo physiological cell death before the end of the perinatal period [3]. Apoptotic cells are removed rapidly and efficiently as intact cells or apoptotic bodies by professional phagocytes or by neighboring cells. This highly regulated process prevents the release of potentially noxious or immunogenic intracellular materials and constitutes the fate of most dying cells throughout the lifespan of an organism [4,5]. Phagocytosis of apoptotic cells is very distinct from other engulfment processes that result, for example, in the clearance of microorganisms, because engulfment of apoptotic cells triggers the secretion of potent anti-inflammatory and immunosuppressive mediators, whereas pathogen recognition causes the release of pro-inflammatory signals [6]. + +Almost all cell types can recognize, respond to, and ingest apoptotic cells by using specific sets of phagocytic receptors that bind to specific ligands on apoptotic cells. Detailed genetic studies in Drosophila and Caenorhabditis elegans have recently yielded evidence that basic phagocytic mechanisms and pathways for the recognition and engulfment of apoptotic cells are highly conserved throughout phylogeny [7,8]. In vertebrates, a number of receptors have been identified that can mediate phagocytosis of apoptotic cells. These include, for example, scavenger receptors and pattern recognition receptors such as CD36, SR-A and CD14, integrins such as the vitronectin receptor αvβ3, and members of the collectin family and their receptors CD91 and calreticulin [9-13]. The individual roles of these molecules in binding, phagocytosis or transduction of anti-inflammatory signals upon apoptotic cell recognition have not been well defined, however [5,6,14]. The importance of efficient mechanisms for apoptotic cell clearance in vivo is supported by the observation that autoimmune responses can be provoked in mice when key molecules for apoptotic cell recognition and uptake are missing. This has been reported for knockout mice lacking the complement protein C1q [15], for mice with a mutation in the tyrosine kinase receptor gene Mer [16] and, more recently, in mice lacking transglutaminase 2 or milk fat globule epidermal growth factor 8 (MFG-E8) [17,18]. + +The exposure of the phospholipid phosphatidylserine (PS) in the outer leaflet of the plasma membrane of apoptotic cells has been described as one of the hallmarks of the induction of apoptosis and is considered to be one of the most important signals required for apoptotic cell recognition and removal [19]. A number of cell-surface and bridging molecules can interact with exposed PS on apoptotic cells. These include the serum proteins β2-glycoprotein 1 and protein S [20,21], the growth-arrest-specific gene product GAS-6 [22], complement activation products [23], the milk fat globule protein MFG-E8 [24], and annexin I [25]. In most cases the receptors on phagocytes that recognize these PS-bridging molecules have not been defined, but it has been reported that GAS-6 is a ligand for the tyrosine kinase receptor Mer and that MFG-E8 can bind to the vitronectin receptor αvβ3 [16,24]. Other molecules that bind PS with varying specificity are the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) and the scavenger receptors CD36 and CD68 (for review see [5] and references therein). + +The best-characterized molecule so far that binds PS in a stereo-specific manner is the phosphatidylserine receptor (Ptdsr) [26]. In vitro, it has been shown that the Ptdsr can mediate the uptake of apoptotic cells and that such Ptdsr-mediated phagocytosis can be inhibited through addition of PS liposomes, the PS-binding molecule annexin V or an anti-Ptdsr antibody [26]. Moreover, the binding of Ptdsr to PS on apoptotic cells has been reported to be important for the release of anti-inflammatory mediators, including transforming growth factor-β1 (TGF-β1), platelet-activating factor (PAF), and prostaglandin E2 [26,27]. These data supported the hypothesis that Ptdsr fulfils a role as a crucial signaling switch after the engagement of macrophages with apoptotic cells and is thereby fundamental for preventing local immune responses to apoptotic cells before their clearance [28]. + +Very recently, Ptdsr has been found in the cell nucleus. Its nuclear localization is mediated by five independent nuclear localization signals, each of which alone is capable of targeting Ptdsr to the cell nucleus [29]. Moreover, an additional study performed recently in Hydra showed an exclusively nuclear localization for the Ptdsr protein [30]. Most interestingly, the nuclear localization of Ptdsr in Hydra epithelial cells did not change upon phagocytosis of apoptotic cells. These reports challenge the original hypothesis, according to which Ptdsr is an exclusively transmembrane receptor for apoptotic cell recognition and anti-inflammatory signaling. + +To examine further the role of Ptdsr in vivo, we performed gene-expression and gene-targeting studies in mice. A perinatally lethal phenotype was observed in Ptdsr-knockout mice, and Ptdsr-deficient embryos displayed multiple defects in tissue and organ differentiation. While this work was in progress, both Li et al. [31] and Kunisaki et al. [32] also reported the generation and phenotypic characterization of Ptdsr-knockout mice. Of note, although some of their results were confirmed in our study, we found a fundamentally different phenotype with regard to clearance of apoptotic cells. Moreover, our study revealed marked and unexpected findings in Ptdsr-deficient mice that are not related to apoptosis. + +Results + +Generation of Ptdsr-deficient mice + +To investigate in vivo the functions of the phosphatidylserine receptor Ptdsr, we generated a null allele in the mouse by gene targeting (Figure 1a,1b,1c). In contrast to previously described Ptdsr-knockout mice [31,32], we used Bruce4 embryonic stem (ES) cells for gene targeting [33], thus generating a Ptdsr-null allele in a pure, isogenic C57BL/6J genetic background. The newly established knockout mouse line was named Ptdsrtm1Gbf (hereafter referred to as Ptdsr -/-). Heterozygous Ptdsr+/- mice were viable and fertile and showed no obvious abnormalities. Ptdsr +/- mice were intercrossed to generate homozygous Ptdsr-deficient mice. The absence of Ptdsr expression in Ptdsr -/- embryos was confirmed by RT-PCR (data not shown), and by northern and western blotting analyses (Figure 1d,e). Interbreeding of heterozygous mice showed that the mutation was lethal, since homozygous mutants were not detected in over 100 analyzed litters at weaning. To determine the stages of embryonic development affected by the Ptdsrtm1Gbf mutation, timed breedings were followed by PCR genotyping (Figure 1c) of embryos. We recovered fewer than the expected number of homozygous embryos from intercrosses of Ptdsr+/-mice. From a total of 1,031 embryos analyzed between gestational day (E) 9.5 and E18.5, 198 (19.2%) Ptdsr-deficient homozygous embryos were harvested, indicating that the introduced mutation is associated with a low rate of embryonic lethality in utero. + +From E9.5 to E12.5, Ptdsr -/- embryos were viable and of normal size. At E13.5 and thereafter, however, most Ptdsr -/-embryos showed morphological abnormalities (Table 1). All homozygous embryos harvested were growth-retarded from E13.5 onwards, had a pale appearance, and displayed multiple developmental dysmorphologies. These included various head and craniofacial malformations, such as exencephaly, cleft palate and abnormal head shape (Figure 1f,g). Gross inspection revealed that eye development was severely affected in 14.1% of homozygous embryos. The affected animals displayed a complete unilateral or bilateral absence of the eyes (Table 1) that was never detected in Ptdsr +/+ or Ptdsr +/- littermates. Furthermore, homozygous embryos harvested between E12.5 and E15.5 had subcutaneous edema (Figure 1f,g). Because we were able to recover Ptdsr -/- embryos until E18.5, we investigated whether Ptdsr-knockout mice could be born alive. Careful observation of timed matings allowed us to recover Ptdsr -/- neonates, but homozygous pups died during delivery or within minutes after birth. Ptdsr-deficient neonates were also growth-retarded, had a pale appearance and displayed various malformations. These included cleft palate, abnormal head shape, absence of eyes and edematous skin (Figure 1h). Thus, deletion of the Ptdsr gene resulted in perinatal lethality with variable severity and penetrance of phenotypes. + +Expression of Ptdsr during embryogenesis and in adult tissues + +The observed perinatal lethality indicates that Ptdsr plays an important role during development. Analysis by RT-PCR (data not shown) showed that Ptdsr is expressed early in development, because we were able to detect Ptdsr transcripts in ES cells and embryos at all developmental stages. To analyze in more detail the temporal and spatial expression patterns of Ptdsr, and to correlate expression patterns with observed pathological malformations, we made use of a Ptdsr-β-geo gene-trap reporter mouse line generated from a Ptdsr gene-trap ES cell clone. This line has an insertion of β-galactosidase in the 3' region of the gene (Figure 2a). + +We first examined Ptdsr expression by X-Gal staining in heterozygous embryos staged from E9.5 to E12.5. These developmental stages were chosen so as to investigate Ptdsr expression in affected organs prior to the onset of pathological malformations in Ptdsr -/- embryos. At E9.5 we found Ptdsr expression in the developing neural tube, somites, heart, gut and branchial arches (Figure 2b). At E10.5, Ptdsr expression remained high in the developing nervous system, with most intense staining in the forebrain, hindbrain and neural tube. At this stage of embryogenesis, high levels of Ptdsr expression could also be detected in the developing limb buds and eyes (Figure 2b). Ptdsr expression was altered at E12.5, with most intensive β-galactosidase staining in the eyes, developing condensations of the limb buds, neural tube and brain (Figure 2b). Transverse sections of X-Gal-stained embryos at E12.5 showed an asymmetric expression pattern in the neural tube with intense staining of the central mantle layer but no expression in the dorsal part of the neural tube (for example, the roof plate; Figure 2c). Expression in dorsal root ganglia lateral to the neural tube and in the somites was observed; Ptdsr was expressed throughout the somite structure (myotome, dermatome and sclerotome; Figure 2d). Expression boundaries between somites were evident, with no expression in the segmental interzones, which correspond to the prospective intervertebral discs (Figure 2d). Transverse sections of the developing eye at E12.5 revealed strong Ptdsr expression in the inner layer of the neural cup, which will later develop into the neural retina. Furthermore, Ptdsr expression was detected in the primary lens fiber cells of the developing lens (Figure 2e). We carefully investigated whether Ptdsr is expressed from E10.5 to E12.5 in the developing kidney and lungs, but no expression could be detected indicating that Ptdsr expression is required only at later stages in the development of these organs (see below). + +Hybridization of a multiple-tissue northern blot revealed a single transcript of about 1.8 kb in almost every tissue analyzed in adult mice (Figure 2f). The most prominent expression was observed in testis, thymus, kidney, liver and skin, with moderate to low expression in lung, small intestine, spleen, stomach and skeletal muscle. Thus, Ptdsr is ubiquitously expressed throughout embryogenesis and in adult tissues, although at different levels. + +Ptdsr is required for normal tissue and organ differentiation + +We next examined the role of Ptdsr in organ development. Serial histological sections of Ptdsr -/- and control embryos were taken to perform a detailed morphological analysis of all organ systems during development. A significant delay in organ and tissue differentiation was observed at E16.5 in lungs, kidneys and intestine. Lungs of control littermates were properly developed with expanding alveoli (Figure 3a). Terminal bronchi and bronchioles were already well developed, and terminally differentiated epithelial cells with cilia on the luminal cell surface were present. In contrast, almost no alveoli or bronchioles were present in Ptdsr -/- lungs, indicating a delay or arrest in lung sacculation and expansion. Instead, we observed an abundance of mesenchyme that appeared highly immature (Figure 3g). A similar delay in tissue differentiation of Ptdsr -/- embryos was found in the kidneys (Figure 3h). Kidneys from Ptdsr+/+ embryos were well developed at E16.5, showing terminally differentiated glomeruli with Bowman's capsule and collecting tubules lined with cuboidal epithelial cells (Figure 3b). In contrast, Ptdsr-deficient kidneys had only primitive glomeruli at E16.5, and collecting tubules were less well-developed. Instead, a large amount of undifferentiated mesenchyme was present in Ptdsr -/- kidneys (Figure 3h). A delay in tissue differentiation was also found in the intestine at this stage of development. Ptdsr -/- embryos displayed improperly developed villi and an underdeveloped or absent submucosa (Figure 3i). In wild-type embryos (Figure 3c), intestinal cellular differentiation was already highly organized, with intramural ganglion cells between the external and internal muscular layers. Such neuronal cells were absent from the intestine of Ptdsr -/- embryos (Figure 3i), however. + +Some Ptdsr -/- mice (4.5 %) also displayed extensive brain malformations that resulted in externally visible head abnormalities, with occasional ectopic tissue outside the skull or exencephaly (Figure 1f,h). Histological analysis revealed an extensive hyperplasia of brain tissue with herniation of brain tissue either through the skull-cap or through the ventral skull (Figure 3d,j). In the most severe cases, expansion of brain tissue in mutant mice resulted in further perturbations of cortical structures (Figure 3d,j). Of note, a similar brain phenotype was observed in the Ptdsr-deficient mouse line generated by Li and colleagues [31]. + +In contrast to the study of Li et al. [31], however, we found almost normally developed lungs at birth. Ptdsr -/-lungs showed, in comparison to wild-type, only a slight delay in maturation and were fully ventilated in neonates in most cases (Figure 3e,k). This demonstrates that Ptdsr-deficient mice can overcome the delay in embryonic lung differentiation and display normal lung morphology at birth. Thus, it would appear highly unlikely that Ptdsr -/- mice die from respiratory failure. Consistent with the observations of Kunisaki and colleagues [32], we found severely blocked erythropoietic differentiation at an early erythroblast stage in the liver (Figure 3f,3l), suggesting an explanation for the grossly anemic appearance that we observed in our Ptdsr -/- mice. + +Loss of Ptdsr activity is associated with defects in ocular development and can lead to formation of ectopic eye structures + +By gross morphology we could differentiate two classes of Ptdsr mutants: those that appeared normal with both eyes present (Figure 4) and those that were severely affected and displayed uni- or bilateral anophthalmia (Figure 5). + +Analysis of normal or mildly affected embryos revealed no differences between mutant and wild-type embryos in the differentiation of the developing eye until E16.5. In both genotypes, inner and outer layers of the retina displayed a comparable differentiation status, as shown, for example, at E12.5 (Figure 4a,e). At day E16.5, however, retinal layers in Ptdsr -/- embryos were much thinner than in wild-type embryos, contained fewer cells and were greatly reduced in size (Figure 4b,f). Comparison of the retinal structures of Ptdsr +/+ and Ptdsr -/- embryos revealed that all four retinal layers were present in Ptdsr-knockout mice at E16.5 (Figure 4b,f). At E18.5 (Figure 4c,g) and in neonatal animals (postnatal day P0; Figure 4d,h), the differences in retinal differentiation between Ptdsr+/+ and Ptdsr -/- mice were still evident, but the size reduction of the retinal layers was less pronounced in the knockout mice. Ptdsr-deficient animals seem to have compensated for the marked delay in cellular differentiation and expansion of retinal layers. Close examination of retinal structures revealed that the inner granular layer was still less expanded in Ptdsr-deficient animals, however, and that it contained fewer cells and was still severely underdeveloped in comparison with the corresponding retinal layer in control animals (Figure 4c,4g and 4d,4h). Thus, even mildly affected Ptdsr -/- mutants had ocular malformations with defects in differentiation of retinal structures. + +We next examined Ptdsr -/- embryos that displayed unilateral or bilateral absence of eyes (Figure 5a) by serial sectioning of whole embryos. These embryos showed complex malformations of the optical cup, including absence of the lens (Figure 5b). Most surprisingly, we found pigmented epithelial cells in the nasal cavity of all Ptdsr-knockout mice with anophthalmia that were analyzed histopathologically. We could identify black-colored pigmented cells embedded in the epithelium of the maxillary sinus that resembled presumptive retinal-pigmented epithelium (Figure 5b,c). Examination of consecutive serial sections revealed the formation of a primitive eye structure, with induction and subsequent proliferation of ectopic mesenchymal tissue immediately adjacent to the displaced pigmented epithelium (Figure 5d). This structure was clearly induced ectopically, and we failed to identify similar changes in any of the wild-type embryos. In summary, we observed a wide range of ocular malformations in Ptdsr-deficient mice that ranged from differentiation defects in retinal cell layers (for example, the inner granular layer) in mildly affected homozygotes to anophthalmia in severely affected Ptdsr -/- mice that was associated with induction of ectopic eye structures in nasal cavities. + +Phagocytosis and clearance of apoptotic cells is normal in Ptdsr-deficient mice + +We next tested whether Ptdsr is functionally required for the clearance of apoptotic cells. We started with an investigation of cell death in vivo in the interdigital areas of the developing limbs. Apoptosis of interdigital cells in the distal mesenchyme of limb buds occurs most prominently from developmental stages E12.0 to E13.5 and can be easily examined in situ by whole-mount terminal deoxynucleotide transferase-mediated UTP end-labeling (TUNEL). We compared the pattern of interdigital cell death in fore and hind limb buds from Ptdsr -/- (n = 3) and Ptdsr +/+ (n = 3) mice at E12.5 and E13.5. No differences in accumulation of TUNEL-positive cell corpses were observed between the two genotypes (Figure 6a). The kinetics of cell death occurrence and regression of the interdigital web was similar in wild-type and mutant littermates, providing no evidence that Ptdsr-deficiency is associated with impaired clearance of apoptotic interdigital cells during limb development. + +To investigate further whether removal of apoptotic cells is impaired in Ptdsr -/- mice, we stained immunohistochemically for activated caspase 3 (aCasp3) and analyzed additional organs and tissues where apoptosis plays a crucial role in tissue remodeling during development. Starting at E12.5, we analyzed and compared the number and distribution of aCasp3-positive cells in over 140 serial sections of three wild-type and six Ptdsr -/- embryos in consecutive and corresponding sections. The sagittal sections were separated by 5 μm, allowing a detailed analysis of apoptosis in several organs and tissues. Tissue restructuring by programmed cell death occurred most notably within the ventral part of the neural tube (Figure 6b,f) and in the developing paravertebral ganglia (Figure 6d,h) with many apoptotic cells being present. In these tissues Ptdsr is highly expressed at E12.5 (Figure 2c) but we observed no difference in the number or distribution of apoptotic cells in Ptdsr+/+ and Ptdsr -/- embryos. The same was true for the developing kidney: apoptotic cells were present in Ptdsr+/+ and Ptdsr -/- embryos, in limited numbers, but we failed to detect any differences in the number of apoptotic cells between the genotypes (Figure 6c,6g). Furthermore, when we continued our analysis of apoptotic cell clearance in vivo at E16.5, E17.5 and E18.5 of embryonic development as well as in neonatal mice, the number and distribution of apoptotic cells was similar in both genotypes. As already observed at E12.5, analysis of aCasp3-stained sections of the developing thymus, heart, diaphragm, genital ridge, eyes and retina convincingly showed that there was no impairment in apoptotic cell removal in Ptdsr -/- mice. Moreover, because Li and colleagues [31] reported impaired clearance of dead cells during lung development in Ptdsr-deficient mice, we examined the rate of apoptosis induction and cell clearance in our Ptdsr-knockout mice in the lung. Analysis of aCasp3-stained lung tissue from Ptdsr+/+ and Ptdsr -/- mice at E17.5 and P0 demonstrated that apoptosis was an extremely rare event during lung morphogenesis at this stage. In addition, there were no differences in the number or distribution of apoptotic cells in Ptdsr -/- and Ptdsr +/+ mice. Furthermore, we were unable to detect any evidence of tissue necrosis in lungs from Ptdsr-deficient mice. In contrast to the report of Li et al. [31], we never observed recruitment of neutrophils or other signs of pulmonary inflammation at any stage of development in our Ptdsr-deficient mice. + +To analyze whether macrophages are recruited into areas where apoptosis is prominent during embryogenesis, we stained consecutive serial sections either with the macrophage surface marker F4/80 or with aCasp3. Surprisingly, there was no co-localization of macrophages with apoptotic cells. In virtually all embryonic tissues, apoptotic cells and macrophages were localized in different compartments (Figure 6e,6i; and see also Additional data file 1, Figure S1). This suggests that at this stage of development it is mainly neighboring cells that are involved in removal of apoptotic cells, rather than professional macrophages. In summary, our analysis in vivo did not reveal any impairment in apoptotic cell clearance in Ptdsr-deficient embryos during development and further suggests that phagocytosis of apoptotic cells is mainly mediated by non-professional 'bystander' cells. + +To determine whether macrophages from Ptdsr-knockout mice were impaired in the efficacy of apoptotic cell uptake in vitro, we performed phagocytosis assays with fetal-liver-derived macrophages (FLDMs) and quantified their phagocytosis rates. Phagocytosis of apoptotic thymocytes was investigated at 60, 90 and 120 minutes after addition of target cells in the absence of serum. Analysis of phagocytosis rates by flow cytometric analysis (FACS) revealed no differences in the efficacy of apoptotic cell uptake between Ptdsr -/- and Ptdsr+/+ macrophages and demonstrated no differences in apoptotic cell engulfment between selected time points (data not shown). To re-examine and further independently validate the result of normal apoptotic cell uptake by Ptdsr -/- macrophages, we performed phagocytosis assays for 60 min and determined the percentage of macrophages that had engulfed apoptotic cells, in a total of at least 300 macrophages counted by fluorescence microscopy. Phagocytosed, 5-carboxytetramethylrhodamine- (TAMRA-) labeled apoptotic cells were identified as being engulfed by inclusion in F4/80-labeled macrophages. Analysis was done independently by three investigators who were not aware of macrophage genotypes (Ptdsr -/- or Ptdsr+/+). Again, no differences were found in the percentage of macrophages that had engulfed apoptotic cells (Figure 7a,c,e) or in the relative number of phagocytosed apoptotic cells per macrophage (phagocytotic index; Figure 7f). Moreover, single Ptdsr -/-macrophages could be identified that had engulfed even more apoptotic target cells than had wild-type macrophages (Figure 7b,d). Thus, Ptdsr-deficient macrophages had a normal ability to ingest apoptotic cells and were not impaired in recognition or phagocytosis of cells that had undergone programmed cell death. + +Ptdsr-deficiency results in reduced production of pro- and anti-inflammatory cytokines after macrophage stimulation + +In addition to its suggested importance for phagocytosis of apoptotic cells, it has been proposed that Ptdsr fulfils a second crucial role in regulating and maintaining a non-inflammatory environment upon the recognition of apoptotic cells by macrophages [26]. We therefore tested whether Ptdsr -/- macrophages were able to release anti-inflammatory cytokines after ingestion of apoptotic cells. We examined levels of TGF-β1 and interleukin-10 (IL-10) after stimulation of FLDMs with lipopolysaccharide (LPS), with and without co-culture of apoptotic cells. Quantification of TGF-β1 and IL-10 levels after 22 hours of culture demonstrated that Ptdsr -/- macrophages were able to secrete these anti-inflammatory cytokines upon ingestion of apoptotic cells, although at a slightly lower level than wild-type (Figure 8a,b). This indicates that ablation of Ptdsr function does not compromise in general the ability of macrophages to release immune-suppressive cytokines after recognition and engulfment of apoptotic cells. + +To analyze whether pro-inflammatory signaling is affected in Ptdsr -/- macrophages, we stimulated FLDMs from Ptdsr+/+ and Ptdsr -/- mice with LPS and measured levels of tumor necrosis factor-α (TNF-α) at different time points after stimulation (Figure 8c). Ptdsr -/- macrophages produced significantly less TNF-α than did wild-type macrophages. The difference in TNF-α secretion was first visible after 3 h of LPS stimulation and became more prominent during the course of the experiment (for example, after 9 h and 12 h of LPS stimulation; Figure 8c). To analyze whether TNF-α release by Ptdsr -/- macrophages can be affected by engulfment of apoptotic cells, we stimulated FLDMs with LPS, apoptotic cells or both. Quantification of TNF-α levels by ELISA after 22 h showed that Ptdsr-deficient macrophages release less TNF-α after stimulation with LPS alone, and also after double stimulation of macrophages with LPS and apoptotic cells (Figure 8d). Moreover, the double stimulation demonstrated that the LPS-induced TNF-α release by Ptdsr -/- macrophages could be inhibited by co-administration of apoptotic cells to an extent comparable to that seen in wild-type macrophages. Similar results were obtained when other pro-inflammatory cytokines, such as interleukin-6 and monocyte chemoattractant protein-1, were analyzed (data not shown). These results indicate that Ptdsr is not required in macrophages for the inhibition of pro-inflammatory signaling after recognition and engulfment of apoptotic cells. Ptdsr-deficiency does, however, affect the overall release of pro- and anti-inflammatory cytokines after stimulation with LPS and after double treatment with LPS and apoptotic cells, indicating that Ptdsr-deficient macrophages have a reduced capacity to produce or secrete pro- and anti-inflammatory cytokines. + +Discussion + +Ptdsr is required for the differentiation of multiple organ systems during development + +In this study, we have generated a null mutation in the phosphatidylserine receptor (Ptdsr) gene in C57BL/6J mice. We show that ablation of Ptdsr results in profound differentiation defects in multiple organs and tissues during embryogenesis, although with variable penetrance. While this work was in progress, two other groups reported the generation of Ptdsr-deficient mice [31,32]. In all three knockout mouse lines, the first two exons ([31] and this study) or exons one to three [32] were deleted by replacement with a neomycin-selection cassette. The Ptdsr-knockout mouse lines differ in the genetic background in which the mutation was generated and maintained, however. In our case, the Ptdsr-null allele was generated in an isogenic C57BL/6J background, whereas Li et al. [31] and Kunisaki et al. [32] investigated the phenotype of their Ptdsr-knockout mice in a mixed 129 × C57BL/6 background. The ablation of Ptdsr function results in perinatal lethality in all cases, but there are interesting differences in severity or expressivities of phenotypes among the different Ptdsr-deficient mouse lines. This might be due either to differences in genetic background or because the phenotypes that have been investigated in this study have not been analyzed in such detail before. + +In the Ptdsr-knockout mouse line reported here, growth retardation started from E12.5 onwards and was associated with delayed differentiation in several organs in which Ptdsr is expressed either during embryogenesis or later in adulthood. At E16.5 almost no branching morphogenesis of the lung epithelium was observed in Ptdsr -/- lungs. Similarly, epithelial structures were only partially developed in mutant kidneys, without terminal differentiation of Bowman's capsule and with a severe reduction in the number of differentiated collecting tubules. Likewise, the differentiation of the intestine was also severely delayed at this developmental stage. When compared with wild-type controls, intestinal tissues of Ptdsr knockout mice appeared unstructured, with an absence of enteric ganglia and of differentiated smooth muscle tissue. Interestingly, defects in kidney and intestine differentiation were not described in the Ptdsr-knockouts generated by Li et al. [31] and Kunisaki et al. [32]. Surprisingly, when we examined Ptdsr-/- embryos shortly before birth (E18.5) or neonatally, we found only mild differentiation delays in organs that appeared severely affected at mid-gestation. This 'recovery' was most visible in Ptdsr -/- lungs: at P0 we found expanded lungs in the knockout mice that showed normal branching patterns, with differentiated alveoli and bronchioles. + +We investigated the occurrence of programmed cell death during lung development in wild-type and Ptdsr-knockout mice throughout embryogenesis (E16.5 to P0). Comparative immunohistochemistry for aCasp3 revealed that apoptosis is a rare event during lung morphogenesis. Furthermore, we failed to detect any differences in the number of apoptotic cells in Ptdsr-knockout and wild-type animals in the rare cases where we could detect apoptotic cells within lung tissues. These findings are contrary to the results reported by Li et al. [31], who suggested that impaired clearance of apoptotic mesenchymal and epithelial cells causes a failure in lung morphogenesis in Ptdsr-deficient mice. In contrast, our findings are in line with the current view on lung development during embryogenesis. Accordingly, formation of the epithelial lung via branching morphogenesis can be subdivided into a series of sequential steps that involve: first, formation of the organ anlage in the form of a placode; second, primary bud formation by placode invagination; third, branch initiation and branch outgrowth; fourth, further reiteration of the branching process; and fifth, terminal differentiation of organ-specific proximal and distal structures [34,35]. In contrast to other invagination processes during embryogenesis, such as mammary gland formation, the lumen of the lungs is expanded by successive branching events, branch outgrowth and elongation, rather than by apoptosis [34,36]. Finally, because the lungs of Ptdsr -/-neonates were almost fully expanded and appeared normal in structure in comparison to wild-type littermates, it is highly unlikely that Ptdsr mutants die of respiratory lung failure. In addition, Li and colleagues [31] demonstrated that surfactant expression is normal in Ptdsr-deficient animals, supporting the idea of normal maturation of surfactant-producing type II alveolar epithelial cells and lung function. Other defects must therefore be responsible for the death of Ptdsr-mutant mice. The frequently observed subcutaneous edema of various extents in Ptdsr-deficient homozygotes gave us a hint that Ptdsr-deficiency and lethality might be associated with cardiovascular problems. Indeed, very recently we have obtained strong evidence that Ptdsr-knockout mice die as a result of defects in heart development that are associated with specific cardiopulmonary malformations; (J.E. Schneider, J.B., S.D. Bamfort, A.D.G., C. Broadbent, K. Clarke, S. Neubauer, A.L. and S. Battacharya, unpublished observations). + +In addition, we demonstrate that eye development requires a functional Ptdsr gene. Ptdsr-deficient embryos can be roughly divided into two categories. The first, severely affected group develops anophthalmia that correlates with formation of ectopic retinal-pigmented epithelium and induction of proliferation of underlying mesenchyme in the nasal cavity. This phenotype represents a completely novel lesion that to our knowledge has not been described before in any other mouse mutant. The second group shows normal external eye structures, although in this case retinal development is temporally delayed during mid-gestation, with persistent, abnormal morphogenesis of the inner granular retinal layer at later stages of embryogenesis. A possible explanation for these two phenotypes can be found in the expression pattern of the Ptdsr gene. Initially, Ptdsr is expressed throughout the whole developing nervous system, with exceptionally high levels in the anterior part of the forebrain. Later expression becomes more restricted to the developing retina and lens. Thus, Ptdsr might play an important role in early events of ocular morphogenesis, such as establishment and bisection of eye fields and formation of optic cups. These early eye-formation steps are closely interconnected with development of the forebrain [37,38] and the nose [39-41]. + +Interestingly, we occasionally observed serious malformations of forebrain and nasal structures in Ptdsr-knockout embryos that were associated with bilateral anophthalmia (see for example the mutant embryo in Figure 1g). This suggests that Ptdsr is involved in the regulation of differentiation processes within forebrain regions, and that ablation of Ptdsr function might secondarily affect early eye formation. Li et al. [31] found smaller lenses in Ptdsr-knockout mice and described the formation of retinal protrusions, although anophthalmia and specific differentiation defects of retinal cell layers were not reported in their study. Li et al. proposed [31] that the eye phenotype they observed could be explained by failed removal of apoptotic cells during eye development, but we think that the observed defects are unrelated to a failure of apoptotic cell clearance. A recent comprehensive kinetic analysis of apoptosis induction during mouse retinal development described four major peaks of apoptotic cell death [42]. This study demonstrated that there is an initial phase of cell death during the invagination of the optic cup (E10.5), followed by subsequent waves of apoptosis induction immediately before and after birth (E18.5 to postnatal day P2), and from postnatal days P9 to P10 and P14 to P16 [42]. Thus, besides the formation of the inner and outer layers of the optic cup in early eye development, other major phases of retinal cell apoptosis take place only postnatally and correspond to important periods in the establishment of neuronal connections. Furthermore, cell death during normal retinal development occurs in retinal layers distinct from the inner granular layer where we observed the most pronounced differentiation defects in the Ptdsr -/- mutants described here. Other studies that connect the postnatal elimination of apoptotic photoreceptor cells to Ptdsr-mediated macrophage engulfment [43] should be interpreted with extreme caution as these studies were based on the monoclonal anti-Ptdsr antibody mAb 217G8E9 [26,43] (see below). + +Consistent with the results of Li et al. [31], we found particular brain malformations in our Ptdsr -/- mice. Exencephaly and hyperplastic brain phenotypes were observed at a low penetrance in Ptdsr-mutant mice (less then 4.5% of homozygotes), but these do not resemble to any extent the brain-overgrowth phenotypes of caspase- or Apaf1-knockout mice ([44], and references therein) in that we failed to identify any differences in the number or distribution of apoptotic cells or pyknotic cell clusters in the neuroepithelium of Ptdsr -/- and Ptdsr+/+ mice. Thus, reduced cell death or diminished clearance of apoptotic neural progenitor cells is unlikely to be the cause of the brain hyperplasia. + +In summary, our studies demonstrate that Ptdsr is required for normal tissue differentiation, especially during the mid-gestation period when we observed the most severe differentiation delays in several organs of Ptdsr-knockout mice. The multiple defects in tissue differentiation cannot be explained by failure of apoptotic cell clearance, as this process is normal in our Ptdsr-knockout line. This result therefore indicates that Ptdsr has a novel, hitherto unexpected, role in promoting tissue maturation and terminal differentiation. Additional studies with conditionally targeted Ptdsr-deficient mice are required to investigate the role of spatial and temporal Ptdsr expression and function during tissue differentiation. + +Ptdsr is not essential for the clearance of apoptotic cells + +Our studies demonstrate that Ptdsr is not a primary receptor for the uptake of apoptotic cells. Investigation of apoptotic cell clearance in vivo in Ptdsr -/- embryos conclusively showed that removal of apoptotic cells is not compromised by ablation of Ptdsr function. Comparative analysis of ten different tissues and organs in Ptdsr+/+ and Ptdsr -/- animals at several stages of embryonic development and in neonates failed to identify impaired uptake of apoptotic cells at any time during development. Furthermore, phagocytosis assays in vitro demonstrated a completely normal uptake of apoptotic cells by Ptdsr -/- macrophages, with some knockout macrophages showing loads even higher than wild-type of engulfed dead cells. These results are contrary to the expected role of Ptdsr in apoptotic cell clearance and to the reported findings of Li et al. [31] and Kunisaki et al. [32], as well as to a study done with a phosphatidylserine receptor null allele in C. elegans [45]. In previous studies in the mouse, the distribution and amount of apoptotic cells in Ptdsr-knockout and control animals were investigated in only a few tissues and at one [31] or two [32] developmental stages. Li et al. [31] examined lung, midbrain and retina at day E17.5 of gestation and identified apoptotic cells by TUNEL staining. Their findings must be interpreted with caution because remodeling of cellular structures by apoptosis in specific retina layers is known to occur mainly postnatally [42], and apoptosis plays an important physiological role in the maintenance and homeostasis of lung epithelium after birth or in pathological conditions involving pulmonary inflammation and not during lung development [46]. This postnatal role for apoptosis is in accordance with our data, as we rarely observed apoptotic cells in retina or lung tissue throughout embryogenesis in Ptdsr+/+ and Ptdsr -/- mice. Kunisaki et al. [32] analyzed TUNEL-stained sections of liver and thymus at days E13.5 and E16.5 of development in Ptdsr+/- and Ptdsr -/- embryos and found reduced rather than increased numbers of TUNEL-positive cells in Ptdsr-deficient embryos. Using co-localization of TUNEL-positive cells with F4/80-positive macrophages they suggested that Ptdsr -/- embryos exhibited a three-fold increase in the frequency of unphagocytosed TUNEL-positive cells together with a severely reduced number of F4/80-positive cells. These results must be interpreted very carefully, however, as it is technically difficult to unambiguously identify engulfed target cells in individual macrophages in solid tissues by fluorescence microscopy. + +In addition, our data suggest that during embryogenesis, macrophage-mediated clearance of apoptotic cells is not the only - or even the primary - mechanism for the removal of apoptotic cells. In many tissues where programmed cell death occurs as a prominent event during embryogenesis, such as remodeling of the genital ridge during gonad morphogenesis and differentiation of the neural tube, we found almost no co-localization of apoptotic cells and macrophages. This indicates that in these cases clearance of apoptotic cells is directly mediated by neighboring 'bystander' cells rather than by macrophages that have been recruited into areas where apoptosis occurs. Obviously these in vivo clearance mechanisms are not compromised by Ptdsr-deficiency in our knockout mutant. This finding is in line with studies in macrophageless Sfpi1-knockout embryos that are deficient for the hematopoietic-lineage-specific transcription factor PU.1. Here, the phagocytosis of apoptotic cells during embryogenesis is taken over by 'stand-in' mesenchymal neighbors [47]. As recognition of phosphatidylserine is thought to be a universal engulfment mechanism for all cells that are able to phagocytose apoptotic cells, it is very striking that apoptotic cell clearance mediated by non-professional bystander cells is also not compromised by Ptdsr-deficiency. + +In contrast to Li et al. [31], we did not observe any impairment in the uptake of apoptotic cells by Ptdsr -/- macrophages in vitro. We performed phagocytosis assays in vitro with fetal-liver-derived macrophages, while in their assays, Li and colleagues used thioglycollate-elicited peritoneal macrophages after adoptive transfer of Ptdsr -/- hematopoietic stem cells. The different results obtained in the two studies are puzzling; they might be due to the use of different macrophage or cell populations. We and Kunisaki et al. [32] found that Ptdsr-deficiency is to some extent associated with defects in hematopoiesis. Thus, it seems possible that recruitment and activation/differentiation of macrophages after adoptive transfer and thioglycollate elicitation are affected by Ptdsr-deficiency. We do not think that the different results observed in Ptdsr-knockout mice in a mixed C57BL/6 × 129 background and in a pure C57BL/6J background can be attributed to genetic background effects: comparison of apoptotic cell engulfment efficacies of thioglycollate-elicited macrophages from 129P2/OlaHsd and C57BL/6J mice did not show any differences in apoptotic cell uptake (J.B. and A.L., unpublished observations). Moreover, in contrast to our studies, neither Li et al. [31] nor Kunisaki et al. [32] determined phagocytotic engulfment indexes for Ptdsr-deficient macrophages. + +Interestingly, we observed differences between Ptdsr+/+ and Ptdsr -/- macrophages in the secretion of pro- and anti-inflammatory cytokines after stimulation with LPS and apoptotic cells. This provides evidence that cellular activation and effector mechanisms are impaired in Ptdsr-deleted macrophages. It remains to be determined which classical pathways of macrophage activation and function involve Ptdsr. This is especially important in light of recent findings that demonstrated nuclear localization of the Ptdsr protein [29]. + +Most strikingly, the recently published data regarding the genetic ablation or perturbation of phosphatidylserine receptor function in C. elegans are also contradictory. Wang et al. [45] reported that psr-1, the C. elegans homolog of Ptdsr, is important for cell-corpse engulfment, whereas psr-1 RNAi studies performed by Arur et al. [25] yielded, in this respect, no phenotype. Moreover, Wang and colleagues hypothesized on the basis of their data that psr-1 might act to transduce an engulfment signal upstream of Ced-2 (Crk II), Ced-5 (Dock 180), Ced-10 (Rac 1) and Ced-12 (Elmo) in one of the two cell-corpse engulfment pathways in the worm [45]. But the loss-of-function phenotype of psr-1 mutants and the complementation phenotypes in overexpressing transgenic worms shown by Wang et al. [45] are rather weak as compared to the classical C. elegans engulfment mutants [8]. + +Many previous functional studies that reported a requirement for Ptdsr for the phagocytosis of apoptotic cells used the monoclonal anti-Ptdsr antibody mAb 217G8E9 [26]. This antibody was used in Ptdsr binding and blocking experiments, as well as in subcellular localization studies, which led to the conclusion that Ptdsr is a transmembrane receptor critical for signal transduction at the engulfment interface. More recently it was used in binding assays to show that the human and worm Ptdsr molecules can recognize phosphatidylserine [45]. In the course of the study presented here, we stained immunohistochemically for Ptdsr with mAb 217G8E9 on wild-type and Ptdsr-deficient macrophages and fibroblasts (see Additional data file 1, Figure S2 and data not shown). To our surprise, we observed similar staining patterns with cells of both genotypes. Furthermore, using a Ptdsr-peptide array we found that mAb 217G8E9 can bind weakly to a Ptdsr peptide, explaining the original isolation of Ptdsr cDNA clones by phage display [26]; but the antibody mainly recognizes additional, as-yet unknown, membrane-associated protein(s) (see Additional data file 1, Figure S2). Experiments that have used this antibody should therefore be interpreted with great caution as they might come to be viewed in a different light. + +Conclusion + +Our results demonstrate that Ptdsr is essential for the differentiation and maturation of multiple tissues during embryogenesis. Ablation of Ptdsr function results in neonatal lethality and severe defects in the morphogenesis of several organs. The developmental malformations cannot be explained by impaired clearance of apoptotic cells, a process that proved to be normal in Ptdsr-deficient mice. This opens up the possibility either that there is an as-yet unknown Ptdsr receptor, which might act as a primary phosphatidylserine recognition receptor, or that recognition of phosphatidylserine and subsequent apoptotic cell engulfment and anti-inflammatory signaling are mainly mediated through phosphatidylserine bridging proteins and their cognate receptors. Although Ptdsr -/- macrophages were not impaired in their ability to phagocytose apoptotic cells, they showed reduced cytokine responses after stimulation. Further work will be required to determine the molecular mechanisms of these newly recognized Ptdsr functions during development. + +Materials and methods + +Construction of the targeting vector and generation of Ptdsr-knockout and gene-trap mice + +Targeting vector + +A Ptdsr-containing bacterial artificial chromosome (BAC) clone (GenBank accession number AC091694; RP-23-316F3) was isolated by sequence homology from a C57BL/6J genomic BAC library (RP-23; BACPAC Resources, Oakland, USA). A 14.5 kb KpnI/BamHI fragment containing the entire Ptdsr locus and 5' and 3' flanking regions was subcloned from this BAC clone and a 1.9 kb RsrII/AatII fragment containing exons I and II of the Ptdsr gene was replaced by a 1.2 kb loxP-flanked neomycin-resistance gene cassette (neo). + +Homologous recombination in ES cells and generation of germ-line chimeras + +Bruce4 ES cells were transfected with KpnI-linearized targeting vector and selected with G418. ES-cell clones resistant to G418 were isolated and analyzed by Southern blot analysis for homologous recombination events within the Ptdsr locus. Chimeric mice were produced by microinjection of two independent homologous recombinant (Ptdsr+/-) ES cells into BALB/c blastocysts and transfer to pseudopregnant foster mothers followed by development to term. Chimeric males were mated with C57BL/6J females. From the two selected ES-cell clones, one successfully contributed to the germ-line. Germ-line transmission of the mutant allele was verified by PCR and Southern blot of genomic DNA from black coat-color F1 offspring. + +Ptdsr gene-trap and generation of germ-line chimeras + +An ES-cell line carrying a β-geo gene-trap vector in the Ptdsr locus was identified by searching the BayGenomics database (BayGenomics, San Francisco, USA; [48]) with the full-length Ptdsr cDNA. A single ES-cell line was identified carrying the gene-trap in intron V, between exons V and VI of the Ptdsr gene. Chimeric mice were generated by microinjection into CB20 blastocysts and transfer to pseudopregnant foster mothers. Chimeric males were mated with 129P2/OlaHsd females. Germ-line transmission of the mutant gene-trap allele was verified by expression analysis using β-galactosidase staining and RT-PCR. + +Genotype analysis + +The genotypes of embryos or animals were determined by PCR analysis and confirmed by Southern blot. Genomic DNA for PCR was prepared from extraembryonic membranes or tail clips using a non-organic tail-DNA extraction protocol [49]. High molecular weight genomic DNA for Southern blotting was prepared according to standard protocols. For PCR analysis the wild-type Ptdsr allele was detected using forward primer 1 (5'-GACACTGTCCATGGCAAACAC-3') and reverse primer 2 (5'-TAAAGTCGCCTTCCAGAAGATT-3'). The primer 1 site is located 5' to the deletion and the primer 2 site within the deletion. This primer pair amplified a fragment of approximately 300 bp from wild-type and Ptdsr+/- mice but not from Ptdsr -/-mutants. To detect the mutant Ptdsr allele, genomic DNA was also amplified using primer 1 and reverse primer 3 (5'-CCACACGCGTCACCTTAATA-3'), which corresponds to a sequence in the neo cassette. In this case, a 500 bp fragment was detected in mice heterozygous or homozygous for the mutant allele, while no signal was detected in wild-type mice. For Southern blot analysis, genomic DNA (30 μg) was digested overnight with BamHI (30 U; Roche Diagnostics GmbH, Mannheim, Germany) and ScaI (30 U; Roche), fractionated on a 0.8 % agarose gel, transferred to a nylon membrane (Hybond N; Amersham Biosciences Europe GmbH, Freiburg, Germany) and hybridized with 5' and 3' flanking probes. The BamHI digest was hybridized with a Ptdsr-specific 5' flanking probe, and Southern blot gave a single 17.2 kb band for wild-type (+/+), an 11.6 kb band for homozygous (-/-) and both bands for heterozygous (+/-) mice. The ScaI digest was hybridized using a 3' flanking probe, and Southern blot gave a single 12.4 kb band for wild-type, a 17.2 kb band for homozygous and both bands for heterozygous mice. + +Northern blot analysis + +Total RNA was isolated from homogenized embryos using TRIZOL reagent (Invitrogen GmbH, Karlsruhe, Germany). For northern blots, either total RNA (30 μg) was extracted from embryos, electrophoresed and transferred to a nylon membrane (Hybond N; Amersham) or a polyA+ RNA northern blot (OriGene Technologies Inc., Rockville, USA) was hybridized using as the probe a Ptdsr fragment amplified from wild-type cDNA using forward primer 5'-GTTCCAGCTCGTCAGACTCG-3' and reverse primer 5'-TGCCCCTAAGACATGACCAC-3'. In all experiments the same membrane was re-hybridized with a β-actin probe (OriGene) to confirm that equivalent RNA levels were present in each lane. Northern blotting indicated that homozygous mutant embryos did not express Ptdsr mRNA and heterozygous mutant embryos expressed only reduced amounts of Ptdsr mRNA. + +Western blot analysis + +Embryos (E13.5) for protein isolation were homogenized in lysis buffer containing 1 × PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS and protease inhibitor cocktail (CompleteMini; Roche). Equal amounts (25 μg) of protein lysate were separated by SDS-polyacrylamide gel electrophoresis and transferred onto a PVDF membrane (Millipore, Billerica, USA) according to standard protocols. Western blots were done using a specific antibody to Ptdsr (PSR N-20, sc-11632; Santa Cruz Biotechnology Inc., Santa Cruz, USA) and β-actin (ab-6276; Abcam, Cambridge, UK) as described by the supplier. Secondary antibodies conjugated to horseradish peroxidase were from Santa Cruz and Abcam, used as described by the supplier, and detection was performed with an enhanced chemiluminescence system (ECLPlus; Amersham). + +Animal experiments + +Wild-type C57BL/6J and 129P2/OlaHsd mice were obtained from Jackson Laboratories (Bar Harbor, USA) and Harlan UK (Bicester, UK), respectively. All mice were housed in individually ventilated cages in a specific pathogen-free environment with a 12 h light-dark cycle and were fed a regular unrestricted diet. The GBF's routine surveillance program screened for selected pathogens. The Ptdsrtm1Gbf mutant was crossed to C57BL/6J mice to establish the co-isogenic C57BL/6J-Ptdsrtm1Gbf mouse line. All studies were approved by the appropriate authorities. + +Isolation of embryos + +Heterozygous male and female mice were intercrossed in order to obtain Ptdsr-deficient progeny. Females were daily monitored for vaginal plugs, and noon of the day of plug detection was defined as E0.5. Embryos at indicated time points were dissected in sterile PBS, washed in ice-cold PBS and transferred to cold fixative. Extra-embryonic membranes were kept and used for genotyping. Ptdsr -/- embryos and their wild-type littermates were used for experiments. + +Histology, TUNEL staining and immunohistochemistry + +Embryos for histology and immunohistochemistry were harvested and fixed in 10% neutral-buffered formalin, dehydrated through a graded series of alcohol, embedded in paraffin, sagittally sectioned at 5 μm intervals, and every fifth section was processed for hematoxylin and eosin (H&E) staining according to standard protocols. Remaining sections of wild-type and Ptdsr -/- specimens were used for immunohistochemistry. For detection of apoptotic cells and macrophages, anti-aCasp3 (an antibody specific for activated caspase 3; R&D Systems, Minneapolis, USA) and anti-F4/80 (Serotec GmBH, Düsseldorf, Germany; #MCA 1957) antibodies were used as described by the supplier. Detection was performed using indirect streptavidin with biotinylated secondary antibodies and cobalt-enhanced diaminobenzidine (brown) or fast-red (red) as chromogens. Sections were counterstained with hematoxylin. For whole-mount terminal deoxynucleotidyl transferase-mediated UTP end labeling (TUNEL), limb buds were dissected from E12.5 and E13.5 embryos, fixed in 4% paraformaldehyde and processed for analysis as previously described [50]. + +Preparation of fetal liver-derived macrophages (FLDMs) + +Fetal livers were excised from embryos at E12.5 and E13.5, respectively, washed in PBS and dissociated enzymatically for 60 min at 37°C. The digestion buffer (150 μl per liver) comprised 0.6 U/ml dispase I (Roche), 0.1% collagenase D (Roche), 10 U DNase (Roche), and 20% FCS in PBS. X-Vivo 15 medium (Cambrex, East Rutherford, USA) was added to the resulting cell suspension, and after centrifugation (200 × g; 3 min) cells were resuspended in X-Vivo 15 medium supplemented with 50 ng/ml macrophage colony-stimulating factor (M-CSF; Sigma-Aldrich, St. Louis, USA) and cultured on non-treated tissue-culture dishes at 37°C with 5% CO2. Every second or third day the medium was changed by centrifugation. Following withdrawal of M-CSF on day 6 after excision, adherent cells were cultured for an additional 24-48 h in X-Vivo 15 medium. + +Macrophage phagocytosis assays + +For preparation of monolayer cultures of macrophages, FLDMs were plated on glass coverslips in 24 well plates (2 × 105 cells per well) in X-Vivo 15 medium. For preparation of apoptotic target cells, primary thymocytes were harvested from the thymus of 4- to 8-week-old C57BL/6J mice, stained with TAMRA for 15 min, and apoptosis was induced either by treating cells with 5 μM staurosporine in medium for 4 h at 37°C or by culturing cells in medium overnight. The efficacy of apoptosis induction was compared in thymic target cells and controls by FACS analysis. On average, 60% of the cells of the resulting population were apoptotic, with exposed PS on their surface, and less than 5% of the cells were necrotic, as confirmed by FITC-annexin V and propidium iodide staining. The apoptotic thymocytes obtained were washed with PBS and added to the prepared FLDM cultures (ratio 10:1). Phagocytosis was then allowed to proceed at 37°C and 5% CO2. After the indicated time periods, the uptake of apoptotic cells by FLDMs was stopped by intensive washing of co-cultures with cold PBS to remove unphagocytosed cells. To measure phagocytosis of apoptotic thymocytes, macrophages were further processed for immunofluorescence analysis. Cells were fixed in 4% paraformaldehyde, blocked in 0.5% BSA/PBS and stained with an anti-F4/80 antibody (Serotec) followed by a secondary antibody coupled to Alexa 488 (Molecular Probes Inc., Eugene, USA). Coverslips were mounted on slides and engulfed thymocytes were enumerated by fluorescence microscopy. The percentage of phagocytosis was calculated by counting at least 300 macrophages and determining the number of macrophages that had engulfed apoptotic thymocytes. The phagocytotic index was calculated according to the following formula: phagocytotic index = (total number of engulfed cells/total number of counted macrophages) × (number of macrophages containing engulfed cells/total number of counted macrophages) × 100. The experiments were performed at least three times, each time in triplicate, and the counting was done by three different investigators. + +Measurement of macrophage cytokine production + +Monolayer cultures of FLDMs and apoptotic thymocytes were prepared as described above. FLDMs were incubated with medium, LPS (10 ng/ml), apoptotic cells (ratio 1:10) or both for the determination of IL-10, TGF-β1 or TNF-α levels after co-culture for 22 h. For TNF-α quantification at various time points, FLDMs were cultured with a high concentration of LPS (100 ng/ml). Culture supernatants were harvested and TNF-α (Mouse TNF-α OptEIA set; BD Biosciences, Heidelberg, Germany) and TGF-β1 (Quantikine, TGF-β1 immunoassay; R&D Systems) were measured by ELISA as described by the supplier. IL-10 in culture supernatants was determined by a cytometric bead assay (Mouse inflammation CBA; BD Biosciences) as indicated in the manual. Data are presented as mean ± SEM from at least three independent experiments, each carried out in triplicate. Analysis of the results used the Wilcoxon-signed rank test; p values below 0.05 were considered significant. + +Additional data files + +Additional data file 1 contains: Figure S1 showing the localization of apoptotic cells and macrophages in the subcutis of developing embryos; and Figure S2 showing immunohistochemical staining of the Ptdsr protein in macrophages derived from wild-type and Ptdsr-knockout mice. + +Supplementary Material + +Additional data file 1 + +Figure S1 showing the localization of apoptotic cells and macrophages in the subcutis of developing embryos; and Figure S2 showing immunohistochemical staining of the Ptdsr protein in macrophages derived from wild-type and Ptdsr-knockout mice + +Click here for additional data file + +Acknowledgements + +We thank Rudi Balling (GBF Research Center) and Shoumo Bhattacharya (University of Oxford) for many helpful and stimulating discussions. We thank Evi Wollscheid-Lengeling (GBF) for help with harvest of neonatal mice, Ronald Frank (GBF) for providing Ptdsr peptide arrays, Maria Ebel (GBF) for ES cell blastocyst injections, Manfred Rohde (GBF) for electron microscopy, Kurt Dittmar (GBF) for help with confocal microscopy and Bastian Pasche (GBF) for critical reading of the manuscript. We thank BayGenomics, a genomic consortium funded by the US National Heart, Lung, and Blood Institute, for providing the ES cell gene-trap line RRJ099. This work was supported in part by the EU project EUMORPHIA, "Understanding human molecular physiology and pathology through integrated functional genomics in the mouse model" (QLG2-CT-2002-00930). + +Figures and Tables + +Figure 1 + +Targeted inactivation of the phosphatidylserine receptor gene. (a) Ptdsr gene-targeting strategy. Homologous recombination in ES cells results in the deletion of exons I and II of the murine Ptdsr gene through replacement of a loxP-flanked neomycin phosphotransferase gene (neo), thereby ablating the reading frame of the encoded protein. Coding exons I-VI are shown as filled boxes, and deleted exons are colored green. Restriction sites are: A, AatII; B, BamHI; EI, EcoRI; EV, EcoRV; K, KpnI; R, RsrII; S, SacII; Sc, ScaI, X, XhoI. The probe sites are red boxes labeled: C, 5' outside probe; D, 3' outside probe. (b) Southern blot analysis of genomic DNA extracted from wild-type (+/+) and Ptdsr+/- (+/-) animals, digested with BamHI and hybridized with the 5' outside probe to confirm germ-line transmission of the mutant Ptdsr allele. 'Wild-type' indicates the BamHI fragment of 17.2 kb from the wild-type Ptdsr allele; 'mutant' indicates the BamHI fragment of 11.6 kb from the targeted Ptdsr allele. (c) PCR genotyping of embryos and animals from intercrosses of heterozygous Ptdsr+/- using a wild-type and a mutant allele-specific primer combination, respectively. (d) Northern blot analysis of total RNA isolated from E13.5 wild-type, Ptdsr+/- and Ptdsr -/- embryos. (e) Western blot analysis of protein from homogenates of E13.5 wild-type, Ptdsr+/- and Ptdsr -/- embryos using a Ptdsr-specific antibody. Developmental abnormalities at (f,g) E15.5 and (h) birth; in this and all subsequent figures wild-type littermates are located on the left and homozygous mutant mice on the right. The Ptdsr -/- embryos show exencephaly (f) or prosencephalic hernia in the forebrain region (arrowhead, neonate 2; h), uni-or bilateral absence of the eyes (f,g and neonate 2 in h, and arrow, neonate 3 in h), an abnormal head shape with proboscis (g), edema (arrowheads in f and g), and general anemia (asterisk, neonate 3 in h). + +Figure 2 + +Expression analysis of Ptdsr during embryonic development. (a) Schematic representation of the construction of the Ptdsr gene-trap mouse line used for expression analysis at different embryonic stages. Gray and bright blue boxes represent regulatory elements of the gene-trap, and β-geo, the β-galactosidase/neomycin phosphotransferase fusion protein-expression cassette [48,51]. Restriction enzyme nomenclature is as in Figure 1 (b) Whole-mount β-galactosidase staining of heterozygous Ptdsr gene-trap embryos at mid-gestation. Expression of Ptdsr is highest in neural tissues and somites, in the branchial arches, the developing limbs, the heart, the primitive gut and the developing eye. (c-e) Sectioning of E12.5 β-galactosidase-stained embryos confirms expression of Ptdsr in (c) the neural tube; (inset in c) neural epithelium; (d) somites; and (e) eyes. Expression in the eye is restricted to developing neural retinal and lens cells. (f) Expression analysis of adult tissues by northern blot. Expression of Ptdsr in the muscle (asterisk) was detected only on long-term exposures of the filter (> 48 h). A β-actin hybridization was used to confirm equal loading of RNA samples. Scale bar, 100 μm. + +Figure 3 + +Histological analysis of wild-type and Ptdsr -/-organs during embryogenesis. (a-f) Wild-type embryos and (g-l) Ptdsr -/- littermates were isolated at various embryonic stages, serially sectioned sagittally and analyzed for developmental abnormalities in detail after H&E staining. At E16.5, the lungs of (g) Ptdsr -/- embryos had sacculation just starting, and well-formed alveoli (asterisks) or epithelium-lined bronchioles (arrows) were scarce compared to (a) wild-type lungs. At E16.5, the glomeruli (arrows) in the kidney of (h) Ptdsr -/- embryos were underdeveloped compared to (b) wild-type, collecting tubules (arrowheads) were missing and undifferentiated blastemas (asterisks) were more abundant. The jejunum had no intramural ganglia in Ptdsr -/-embryos (i; and arrows in c); and a well-developed submucosa (asterisk in c) was missing. Brain sections at E18.5 show that (j) Ptdsr -/-embryos may have herniation (arrow) of the hypothalamus through the ventral skull (secondary palate), most likely through Rathke's pouch, and a severe malformation of the cortex (asterisks) compared to (d) wild-type embryos. At E18.5, (e) wild-type and (k) Ptdsr -/- lungs showed normal sacculation and formation of alveoli (asterisks) and bronchioles (arrow). (f) Wild-type neonatal liver had significant numbers of megakaryocytes (arrows), compared to (l) homozygous mutant littermates, and higher numbers of erythropoietic islands and of mature erythrocytes. Hepatocellular vacuoles are due to glycogen stores (asterisks) that were not metabolized in perinatally dying Ptdsr -/- animals, in contrast to wild-type newborns. Scale bar, 100 μm, except for (d) and (j), 1 mm. + +Figure 4 + +Morphology of wild-type and Ptdsr -/- retinas. Serial sagittal sections of (a-d) wild-type and (e-h) Ptdsr -/- retina were analyzed for developmental abnormalities at (a,e) E12.5, (b,f) E16.5, (c,g) E18.5, and (d,h) P0. Normal patterning of the retina was observed in Ptdsr -/-embryos, with an outer granular layer (OGL), outer plexiform layer (OPL), inner granular layer (IGL) and inner plexiform layer (IPL). Note that the IGL in Ptdsr -/- retinas is less thick than that in wild-type littermates in comparing (c,g) and (d,h). Morphometric analysis (numbered lines) of wild-type and Ptdsr -/- retinas confirmed the initial finding of a thinner retina in Ptdsr -/- animals than in wild-type (all values in μm). Scale bar, 50 μm. + +Figure 5 + +Histological analysis of eye development in severely affected eyeless Ptdsr -/- embryos. (a) In anophthalmic Ptdsr -/- embryos, unilateral or bilateral absence of the eyes could be detected. (b-d) Serial H&E-stained sagittal sections of homozygous mutant embryos at (b) E17.5 and (c,d) E18.5 show complex malformation of the optic cup and lack of any lens structure. Careful examination of adjacent sections (b-d) reveals an ectopic misplacement of retinal-pigmented epithelium in the maxillary sinus. Not only is the deposition of pigment clearly visible (higher magnification insets) but also the induction of proliferation of underlying tissues and the change in morphology of the maxillary sinus (d). Scale bar, 100 μm in (b-d). + +Figure 6 + +Analysis of programmed cell death and involvement of macrophages in the removal of apoptotic cells in wild-type and Ptdsr -/-embryos. (a) Whole-mount TUNEL staining (blue) of limb buds from wild-type and Ptdsr -/- embryos at E13.5 show no differences in the amount or localization of apoptotic cells during the beginning regression of the interdigital web. Serial sagittal sections stained for activated caspase 3 (aCasp3; red) in (b-d) wild-type and (f-h) Ptdsr -/- embryos at E12.5 show apoptotic cells in the neural tube (b,f), the mesonephros (c,g) and the developing paravertebral ganglia (d,h). Tissue distribution and total number of apoptotic cells was indistinguishable between genotypes and was confirmed by the comparison of consecutive sections of wild-type and Ptdsr -/-embryos from different developmental stages. Analysis of macrophage numbers and location by F4/80 staining (brown) of consecutive sections in paravertebral ganglia of (e) wild-type and (i) homozygous mutant embryos revealed that macrophages (arrows) are not located close to apoptotic cells during embryonic development. (For comparison, see also Additional data file 1, Figure S1, with the online version of this article). Scale bar, 100 μm. + +Figure 7 + +Phagocytosis of apoptotic cells by fetal liver-derived macrophages (FLDMs). FLDMs from (a,b) wild-type and (c,d) Ptdsr -/- embryos were cultured for 60 min with TAMRA-stained (red) apoptotic thymocytes (treated with staurosporine) from C57BL/6J mice and then stained with F4/80 (green). Macrophages of both genotypes have phagocytosed apoptotic cells (arrowheads). (e) Quantification of phagocytosis of apoptotic cells by wild-type or Ptdsr -/-macrophages revealed no differences in the percentage of macrophages that had engulfed apoptotic cells, whether or not apoptosis had been induced by staurosporine. Microscopic analysis (b,d) and quantification of the number of apoptotic cells phagocytosed by single macrophages and (f) calculation of the average number of cells phagocytosed per macrophage failed to reveal differences in the efficacy of removal of apoptotic cells between wild-type and Ptdsr -/- FLDMs. + +Figure 8 + +Cytokine production by FLDMs upon stimulation with lipopolysaccharide (LPS) and apoptotic cells. FLDMs from wild-type and Ptdsr -/- embryos were incubated (a,b,d) with medium (0), LPS (10 ng/ml), apoptotic cells (ratio 1:10) or in combination with LPS and apoptotic cells or (c) with LPS (100 ng/ml) alone. Culture supernatants were harvested after 22 h (a,b,d) or at the indicated time points (c). TNF-α and TGF-β1 were quantified by ELISA and IL-10 by cytometric bead array (CBA) assay. Data are presented as mean ± SEM from at least three independent experiments, each carried out in triplicate. *, significant difference between genotypes, p < 0.05; **, significant difference between genotypes, p < 0.01; Wilcoxon-signed rank test. + +Table 1 + +Penetrance of phenotypes in Ptdsr -/- mice from E9.5 to E18.5, as detected by gross morphology + +Subsets of the major categories of malformation are indicated by indentation. diff --git a/src/ontogpt/evaluation/craft/database/all/15492776.ann b/src/ontogpt/evaluation/craft/database/all/15492776.ann new file mode 100644 index 000000000..07dabea01 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15492776.ann @@ -0,0 +1,1668 @@ +T1 PR:000000034 0 3 BMP +T2 GO:0030509 0 22 BMP Receptor Signaling +T3 GO:0007567 43 48 natal +T4 UBERON:0010996 64 83 Articular Cartilage +T5 UBERON:0010996 95 114 Articular cartilage +T6 NCBITaxon:9606 212 218 people +T7 http://purl.obolibrary.org/obo/MONDO_0005578 232 241 arthritis +T8 UBERON:0001474 349 354 bones +T9 UBERON:0004905 358 363 joint +T10 SO:0000704 431 435 gene +T11 GO:0010467 431 446 gene expression +T12 UBERON:0000479 468 474 tissue +T13 GO:0065007 512 522 regulatory +T14 NCBITaxon:10088 544 549 mouse +T15 PR:000000281 550 554 Gdf5 +T16 SO:0000704 555 559 gene +T17 GO:0060349 563 581 bone morphogenetic +T18 PR:000000034 563 589 bone morphogenetic protein +T19 PR:000000034 591 594 BMP +T20 NCBITaxon:10088 626 631 mouse +T21 SO:0000704 688 693 genes +T22 UBERON:0004905 721 727 joints +T23 GO:0010467 729 739 Expression +T24 PR:000000281 764 768 Gdf5 +T25 NCBITaxon:2 769 778 bacterial +T26 SO:0000153 769 800 bacterial artificial chromosome +T27 SO:0000363 856 862;870 875 floxed ... genes +T28 UBERON:0004905 890 896 joints +T29 UBERON:0004905 914 919 joint +T30 UBERON:0007023 932 937 adult +T31 UBERON:0010996 938 957 articular cartilage +T32 UBERON:0001484 967 980 joint capsule +T33 PR:000000034 1027 1030 BMP +T34 GO:0030509 1027 1049 BMP receptor signaling +T35 UBERON:0004905 1053 1058 joint +T36 NCBITaxon:10088 1072 1076 Mice +T37 PR:000000035 1100 1106 Bmpr1a +T38 GO:0016265 1120 1123 die +T39 GO:0009790 1133 1146 embryogenesis +T40 SO:0000359 1191 1197 floxed +T41 PR:000000035 1198 1204 Bmpr1a +T42 SO:0001023 1205 1211 allele +T43 PR:000000281 1221 1225 Gdf5 +T44 UBERON:0000922 1251 1260 embryonic +T45 GO:0007567 1285 1290 birth +T46 GO:0007567 1299 1304 natal +T47 NCBITaxon:10088 1320 1324 mice +T48 PR:000000035 1337 1343 Bmpr1a +T49 SO:0000704 1344 1348 gene +T50 UBERON:0004905 1352 1361 articular +T51 UBERON:0004905 1376 1382 joints +T52 PR:000000035 1427 1433 Bmpr1a +T53 UBERON:0010996 1462 1481 articular cartilage +T54 UBERON:0004905 1493 1499 joints +T55 NCBITaxon:10088 1543 1547 mice +T56 GO:0007567 1554 1559 birth +T57 NCBITaxon:9606 1584 1589 human +T58 http://purl.obolibrary.org/obo/MONDO_0005178 1590 1604 osteoarthritis +T59 PR:000000281 1606 1610 Gdf5 +T60 NCBITaxon:10088 1615 1619 mice +T61 SO:0000704 1682 1687 genes +T62 UBERON:0004905 1691 1700 articular +T63 PR:000000034 1710 1713 BMP +T64 GO:0030509 1710 1732 BMP receptor signaling +T65 GO:0009888 1764 1775;1789 1791;1801 1808 development ... of ... tissues +T66 GO:0009888 1780 1791;1801 1808 creation of ... tissues +T67 UBERON:0000479 1801 1808 tissues +T68 UBERON:0010996 1846 1865 articular cartilage +T69 GO:0007567 1872 1877 birth +T70 SO:0000704 1879 1886 Genetic +T71 PR:000000034 1916 1919 BMP +T72 GO:0030509 1916 1938 BMP receptor signaling +T73 NCBITaxon:9606 1974 1979 human +T74 http://purl.obolibrary.org/obo/MONDO_0005178 1980 1994 osteoarthritis +T75 PR:000000034 2033 2036 BMP +T76 GO:0030509 2033 2055 BMP receptor signaling +T77 UBERON:0004905 2144 2149 joint +T78 UBERON:0010996 2189 2208 articular cartilage +T79 UBERON:0001474 2218 2223 bones +T80 UBERON:0002217 2227 2242 synovial joints +T81 UBERON:0010996 2378 2397 articular cartilage +T82 http://purl.obolibrary.org/obo/MONDO_0005578 2415 2424 arthritic +T83 http://purl.obolibrary.org/obo/MONDO_0000001 2425 2433 diseases +T84 UBERON:0007023 2496 2502 adults +T85 NCBITaxon:9606 2534 2540 people +T86 http://purl.obolibrary.org/obo/MONDO_0005578 2560 2569 arthritis +T87 NCBITaxon:9606 2615 2621 people +T88 UBERON:0010996 2757 2776 articular cartilage +T89 UBERON:0004905 2818 2823 joint +T90 http://purl.obolibrary.org/obo/MONDO_0006816 2818 2833 joint disorders +T91 UBERON:0004905 2876 2881 Joint +T92 GO:0009790 2906 2919 embryogenesis +T93 UBERON:0004288 2996 3004 skeletal +T94 GO:0012501 3031 3052 Programmed cell death +T95 UBERON:0004905 3206 3211 joint +T96 UBERON:0004288 3230 3238 skeleton +T97 UBERON:0010996 3453 3472 articular cartilage +T98 UBERON:0004905 3493 3498 joint +T99 UBERON:0004905 3638 3643 joint +T100 PR:000017452 3655 3660 Wnt14 +T101 GO:0010467 3664 3673 expressed +T102 UBERON:0004905 3704 3710 joints +T103 GO:0010467 3752 3762 expression +T104 UBERON:0004905 3772 3777 joint +T105 GO:0010467 3794 3803 expressed +T106 UBERON:0002101 3828 3832 limb +T107 GO:0060349 3883 3901 bone morphogenetic +T108 PR:000000034 3883 3909 bone morphogenetic protein +T109 PR:000000034 3911 3914 BMP +T110 GO:0046903 3926 3934 secreted +T111 CHEBI:62488 3935 3954 signaling molecules +T112 GO:0010467 3964 3973 expressed +T113 UBERON:0004905 4000 4006 joints +T114 SO:0000704 4049 4054 genes +T115 PR:000000281 4055 4059 Gdf5 +T116 PR:000007924 4061 4065 Gdf6 +T117 PR:000000282 4067 4071 Gdf7 +T118 PR:000000164 4073 4077 Bmp2 +T119 PR:000000165 4083 4087 Bmp4 +T120 PR:000000281 4192 4196 Gdf5 +T121 GO:0010467 4197 4207 expression +T122 UBERON:0004905 4252 4258 joints +T123 UBERON:0004905 4316 4321 joint +T124 PR:000000281 4353 4357 Gdf5 +T125 PR:000007924 4381 4385 Gdf6 +T126 SO:0000704 4386 4390 gene +T127 UBERON:0004905 4415 4421 joints +T128 CHEBI:36357 4482 4491 molecules +T129 UBERON:0004905 4514 4519 joint +T130 PR:000000164 4601 4605 Bmp2 +T131 PR:000000165 4609 4613 Bmp4 +T132 UBERON:0019248 4620 4635 early embryonic +T133 UBERON:0004905 4689 4694 joint +T134 UBERON:0007023 4862 4867 adult +T135 UBERON:0004905 4868 4873 joint +T136 PR:000000034 4899 4902 BMP +T137 GO:0030509 4899 4912 BMP signaling +T138 UBERON:0007023 4939 4944 adult +T139 UBERON:0010996 4945 4964 articular cartilage +T140 PR:000000034 5180 5184 BMPs +T141 GO:0043235 5201 5213 complexes of +T142 GO:0016020 5246 5254 membrane +T143 GO:0043235 5279 5288 receptors +T144 PR:000000034 5295 5298 BMP +T145 GO:0032991 5314 5323 complexes +T146 PR:000000034 5480 5483 BMP +T147 UBERON:0004288 5504 5512 skeletal +T148 PR:000000035 5525 5531 BMPR1A +T149 PR:000000036 5536 5542 BMPR1B +T150 PR:000000164 5568 5572 BMP2 +T151 PR:000000165 5574 5578 BMP4 +T152 PR:000000281 5584 5588 GDF5 +T153 PR:000000281 5599 5603 GDF5 +T154 PR:000000036 5630 5636 BMPR1B +T155 GO:0010467 5769 5778 expressed +T156 UBERON:0002101 5829 5834 limbs +T157 PR:000000035 5836 5842 Bmpr1a +T158 GO:0010467 5843 5853 expression +T159 UBERON:0004905 5876 5881 joint +T160 UBERON:0002222 5894 5907 perichondrium +T161 UBERON:0004905 5913 5922 articular +T162 CL:0000743 5934 5959 hypertrophic chondrocytes +T163 UBERON:0009585 5965 5993 interdigital limb mesenchyme +T164 PR:000000036 6010 6016 Bmpr1b +T165 GO:0010467 6017 6027 expression +T166 CL:0000335 6049 6059;6077 6094 condensing ... mesenchymal cells +T167 UBERON:0003104 6077 6088 mesenchymal +T168 UBERON:0004905 6113 6118 joint +T169 UBERON:0002222 6131 6144 perichondrium +T170 UBERON:0004905 6154 6163 articular +T171 PR:000000036 6274 6280 Bmpr1b +T172 SO:0000704 6281 6285 gene +T173 NCBITaxon:10088 6301 6305 mice +T174 UBERON:0004905 6331 6336 joint +T175 NCBITaxon:10088 6383 6387 mice +T176 PR:000000281 6396 6400 Gdf5 +T177 PR:000000035 6480 6486 Bmpr1a +T178 UBERON:0000922 6499 6508 embryonic +T179 GO:0007369 6536 6548 gastrulation +T180 NCBITaxon:10088 6574 6578 mice +T181 PR:000000165 6597 6601 Bmp4 +T182 SO:0000359 6666 6672 floxed +T183 SO:0001023 6673 6680 alleles +T184 PR:000000035 6694 6700 Bmpr1a +T185 UBERON:0004905 6781 6786 joint +T186 SO:0000704 6841 6848 genetic +T187 SO:0000704 6887 6892 genes +T188 UBERON:0004905 6909 6914 joint +T189 UBERON:0000479 6915 6922 tissues +T190 UBERON:0004905 6975 6980 joint +T191 UBERON:0000479 7038 7044 tissue +T192 GO:0010467 7054 7064 expression +T193 PR:000000281 7080 7084 Gdf5 +T194 SO:0000704 7085 7089 gene +T195 SO:0000346 7108 7112 loxP +T196 PR:000000281 7133 7137 Gdf5 +T197 GO:0010467 7185 7192 express +T198 SO:0000704 7193 7198 genes +T199 UBERON:0004905 7202 7208 joints +T200 NCBITaxon:10088 7230 7234 mice +T201 SO:0000704 7281 7286 genes +T202 UBERON:0002217 7326 7340 synovial joint +T203 UBERON:0000211 7356 7365 ligaments +T204 UBERON:0001484 7373 7386 joint capsule +T205 UBERON:0010996 7419 7438 articular cartilage +T206 PR:000000281 7440 7444 Gdf5 +T207 UBERON:0019248 7476 7491 early embryonic +T208 PR:000000035 7523 7529 Bmpr1a +T209 UBERON:0004905 7582 7587 joint +T210 GO:0012501 7638 7659 programmed cell death +T211 UBERON:0006015 7663 7685 webbing between digits +T212 PR:000000035 7702 7708 Bmpr1a +T213 GO:0007567 7734 7739 natal +T214 UBERON:0010996 7755 7774 articular cartilage +T215 UBERON:0004288 7798 7806 skeleton +T216 PR:000000281 7811 7815 Gdf5 +T217 PR:000000035 7820 7826 Bmpr1a +T218 SO:0000359 7826 7831 floxP +T219 NCBITaxon:10088 7832 7836 mice +T220 UBERON:0010996 7838 7857 articular cartilage +T221 GO:0010467 7907 7917 expression +T222 GO:0007567 7957 7962 birth +T223 http://purl.obolibrary.org/obo/MONDO_0005178 8027 8041 osteoarthritis +T224 PR:000000034 8095 8098 BMP +T225 GO:0030509 8095 8108 BMP signaling +T226 GO:0007567 8151 8156 natal +T227 UBERON:0010996 8157 8176 articular cartilage +T228 GO:0030510 8187 8200;8205 8226 modulation of ... BMP signaling pathway +T229 PR:000000034 8205 8208 BMP +T230 UBERON:0004905 8257 8262 joint +T231 http://purl.obolibrary.org/obo/MONDO_0006816 8257 8270 joint disease +T232 SO:0000704 8282 8289 Genetic +T233 SO:0000704 8325 8330 Genes +T234 UBERON:0004905 8334 8339 Joint +T235 SO:0000704 8414 8419 genes +T236 UBERON:0000982 8437 8451 skeletal joint +T237 NCBITaxon:10088 8490 8494 mice +T238 GO:0010467 8498 8505 express +T239 UBERON:0004905 8536 8542 joints +T240 PR:000000281 8555 8559 Gdf5 +T241 SO:0000704 8565 8569 gene +T242 GO:0010467 8579 8588 expressed +T243 UBERON:0004765 8618 8635 skeletal elements +T244 UBERON:0000922 8643 8652 embryonic +T245 GO:0009790 8643 8652;8659 8668 embryonic ... formation +T246 UBERON:0004905 8653 8658 joint +T247 NCBITaxon:2 8672 8681 bacterial +T248 SO:0000153 8672 8703 bacterial artificial chromosome +T249 SO:0000153 8705 8708 BAC +T250 PR:000000281 8725 8729 Gdf5 +T251 NCBITaxon:2 8780 8788 bacteria +T252 SO:0005853 8801 8809 cassette +T253 SO:0000243 8823 8851 internal ribosome entry site +T254 GO:0005840 8832 8840 ribosome +T255 SO:0000243 8853 8857 IRES +T256 NCBITaxon:9606 8859 8864 human +T257 UBERON:0001987 8865 8874 placental +T258 PR:000003969 8865 8895 placental alkaline phosphatase +T259 PR:P05187 8897 8902 hPLAP +T260 GO:0006413 8913 8930 translation start +T261 SO:0000318 8913 8935 translation start site +T262 PR:000000281 8939 8943 Gdf5 +T263 SO:0000153 8971 8974 BAC +T264 NCBITaxon:10088 9017 9021 mice +T265 PR:000000281 9037 9041 Gdf5 +T266 NCBITaxon:10088 9057 9061 mice +T267 SO:0000902 9078 9087 transgene +T268 GO:0010467 9088 9098 expression +T269 NCBITaxon:10088 9162 9166 mice +T270 GO:0010467 9185 9195 expression +T271 PR:000033987 9199 9203 lacZ +T272 SO:0000616 9234 9264 transcriptional stop sequences +T273 GO:0010467 9326 9336 expression +T274 SO:0000902 9344 9353 transgene +T275 PR:P05187 9366 9371 HPLAP +T276 PR:000033987 9422 9426 LACZ +T277 PR:000033987 9484 9488 LACZ +T278 GO:0010467 9489 9499 expression +T279 UBERON:0004905 9513 9519 joints +T280 PR:P05187 9547 9552 HPLAP +T281 GO:0010467 9553 9563 expression +T282 UBERON:0004905 9586 9591 joint +T283 PR:P05187 9616 9621 HPLAP +T284 GO:0010467 9622 9632 expression +T285 PR:000000281 9640 9644 Gdf5 +T286 PR:000033987 9737 9741 LACZ +T287 GO:0010467 9742 9752 expression +T288 UBERON:0004905 9786 9792 joints +T289 UBERON:0002544 9800 9806 digits +T290 PR:000000281 9886 9890 Gdf5 +T291 SO:0000902 9895 9904 transgene +T292 GO:0010467 9905 9914 expresses +T293 UBERON:0004905 9974 9979 joint +T294 NCBITaxon:10088 9997 10001 mice +T295 PR:000033987 10020 10024 lacZ +T296 NCBITaxon:10088 10059 10063 mice +T297 PR:000033987 10103 10107 lacZ +T298 UBERON:0004905 10146 10152 Joints +T299 UBERON:0002101 10167 10172 limbs +T300 UBERON:0016884 10230 10244 shoulder joint +T301 UBERON:0001490 10264 10275 elbow joint +T302 UBERON:0004905 10318 10323 joint +T303 UBERON:0004905 10515 10520 joint +T304 UBERON:0002101 10540 10545 limbs +T305 PR:000033987 10547 10551 LACZ +T306 UBERON:0000922 10572 10581 embryonic +T307 UBERON:0004905 10620 10626 joints +T308 UBERON:0001467 10642 10650 shoulder +T309 UBERON:0001465 10655 10659 knee +T310 PR:000033987 10690 10694 LACZ +T311 GO:0010467 10695 10705 expression +T312 UBERON:0003657 10751 10760;10765 10770 joints of ... limbs +T313 GO:0010467 10848 10858 expression +T314 UBERON:0000922 10864 10870 embryo +T315 UBERON:0000922 10874 10880 embryo +T316 UBERON:0000922 10905 10912 embryos +T317 UBERON:0009552 10947 10957 fingertips +T318 UBERON:0000922 10981 10987 embryo +T319 UBERON:0000922 11038 11044 embryo +T320 UBERON:0004905 11093 11099 joints +T321 PR:000033987 11110 11114 LACZ +T322 GO:0010467 11192 11202 expression +T323 PR:000033987 11206 11210 LACZ +T324 UBERON:0004905 11229 11234 joint +T325 UBERON:0001485 11316 11326 knee joint +T326 UBERON:0006658 11353 11370 phalangeal joints +T327 UBERON:0004288 11422 11430 skeleton +T328 GO:0010467 11445 11455 expression +T329 PR:000033987 11459 11463 LACZ +T330 UBERON:0004905 11495 11501 joints +T331 GO:0007567 11579 11584 natal +T332 UBERON:0002217 11585 11600 synovial joints +T333 UBERON:0010996 11615 11634 articular cartilage +T334 UBERON:0001484 11636 11649 joint capsule +T335 GO:0010467 11768 11778 expression +T336 PR:000000281 11782 11786 Gdf5 +T337 UBERON:0000922 11815 11824 embryonic +T338 GO:0009790 11815 11836 embryonic development +T339 UBERON:0007023 11864 11869 Adult +T340 GO:0010467 11870 11880 expression +T341 PR:000000281 11897 11901 Gdf5 +T342 SO:0000704 11902 11906 gene +T343 PR:000000281 11942 11946 Gdf5 +T344 GO:0010467 11947 11957 expression +T345 UBERON:0007023 11990 11995 adult +T346 UBERON:0010996 11996 12015 articular cartilage +T347 UBERON:0003657 12142 12153 limb joints +T348 PR:000033987 12177 12181 lacZ +T349 GO:0010467 12182 12192 expression +T350 PR:000033987 12213 12217 LACZ +T351 UBERON:0004347 12282 12290 limb bud +T352 PR:000033987 12314 12318 LACZ +T353 UBERON:0001757 12360 12370 ear pinnae +T354 UBERON:0002228 12372 12376 ribs +T355 UBERON:0000975 12378 12385 sternum +T356 UBERON:0000479 12387 12394 tissues +T357 UBERON:0001456 12402 12406 face +T358 UBERON:0002616 12417 12427;12432 12437 regions of ... brain +T359 UBERON:0001948 12417 12427;12442 12453 regions of ... spinal cord +T360 GO:0007567 12489 12494 birth +T361 PR:000033987 12496 12500 LACZ +T362 GO:0010467 12509 12518 expressed +T363 UBERON:0000043 12522 12529 tendons +T364 UBERON:0001130 12548 12564 vertebral column +T365 UBERON:0000043 12577 12584 tendons +T366 UBERON:0004452 12592 12597 wrist +T367 UBERON:0004454 12602 12607 ankle +T368 UBERON:0000043 12618 12624 tendon +T369 PR:000033987 12684 12688 LACZ +T370 GO:0010467 12697 12706 expressed +T371 UBERON:0002073 12714 12728 hair follicles +T372 UBERON:0001867 12730 12743 ear cartilage +T373 UBERON:0002516 12763 12778;12783 12793 growth plate of ... long bones +T374 UBERON:0002616 12799 12810;12815 12820 portions of ... brain +T375 UBERON:0001948 12799 12810;12825 12836 portions of ... spinal cord +T376 NCBITaxon:10088 12902 12906 mice +T377 PR:000033987 12957 12961 LACZ +T378 GO:0010467 12962 12972 expression +T379 UBERON:0000479 13017 13024 tissues +T380 NCBITaxon:33208 13032 13038 animal +T381 GO:0010467 13059 13069 expression +T382 SO:0000902 13077 13086 transgene +T383 PR:P05187 13109 13114 HPLAP +T384 UBERON:0004905 13158 13164 joints +T385 NCBITaxon:33208 13168 13175 animals +T386 PR:000033987 13238 13242 LACZ +T387 GO:0010467 13243 13253 expression +T388 NCBITaxon:33208 13310 13317 animals +T389 GO:0010467 13328 13338 expression +T390 PR:000033987 13436 13440 LACZ +T391 GO:0010467 13441 13451 expression +T392 NCBITaxon:33208 13475 13482 animals +T393 NCBITaxon:33208 13579 13586 animals +T394 SO:0000704 13663 13668 genes +T395 PR:000000281 13748 13752 Gdf5 +T396 PR:000000035 13757 13763 Bmpr1a +T397 SO:0000359 13763 13768 floxP +T398 NCBITaxon:33208 13769 13776 Animals +T399 UBERON:0000113 13788 13797 Adulthood +T400 UBERON:0001690 13803 13806 Ear +T401 UBERON:0004905 13821 13826 Joint +T402 PR:000000281 13853 13857 Gdf5 +T403 PR:000000034 13889 13892 BMP +T404 GO:0030509 13889 13902 BMP signaling +T405 UBERON:0004905 13917 13922 joint +T406 PR:000000281 13936 13940 Gdf5 +T407 NCBITaxon:10088 13956 13960 mice +T408 NCBITaxon:33208 13974 13981 animals +T409 SO:0000359 14005 14011 floxed +T410 SO:0001023 14012 14018 allele +T411 PR:000000035 14026 14032 Bmpr1a +T412 SO:0001023 14107 14113 allele +T413 SO:0000147 14266 14270 exon +T414 PR:000000035 14278 14284 Bmpr1a +T415 SO:0000704 14285 14289 gene +T416 NCBITaxon:10088 14305 14309 mice +T417 PR:000000281 14332 14336 Gdf5 +T418 SO:0000902 14341 14350 transgene +T419 PR:000000035 14419 14425 Bmpr1a +T420 SO:0000359 14425 14430 floxP +T421 SO:0001023 14431 14437 allele +T422 SO:0001023 14452 14458 allele +T423 PR:000000035 14466 14472 Bmpr1a +T424 PR:000000281 14544 14548 Gdf5 +T425 PR:000000035 14553 14559 Bmpr1a +T426 SO:0000359 14559 14564 floxP +T427 NCBITaxon:10088 14586 14590 mice +T428 UBERON:0000113 14619 14628 adulthood +T429 PR:000000281 14647 14651 Gdf5 +T430 UBERON:0019248 14678 14693 early embryonic +T431 NCBITaxon:33208 14727 14734 animals +T432 PR:000000035 14763 14769 Bmpr1a +T433 PR:000000281 14811 14815 Gdf5 +T434 PR:000000035 14820 14826 Bmpr1a +T435 SO:0000359 14826 14831 floxP +T436 NCBITaxon:10088 14832 14836 mice +T437 NCBITaxon:10088 14896 14900 mice +T438 UBERON:0001690 14913 14917 ears +T439 UBERON:0000033 14955 14960 heads +T440 PR:000000034 15057 15060 BMP +T441 GO:0030509 15057 15070 BMP signaling +T442 UBERON:0001691 15113 15125 external ear +T443 NCBITaxon:10088 15129 15133 mice +T444 PR:000000035 15201 15207 Bmpr1a +T445 UBERON:0001690 15236 15239 ear +T446 GO:0010467 15251 15258 express +T447 PR:000000281 15263 15267 Gdf5 +T448 SO:0000902 15272 15281 transgene +T449 NCBITaxon:10088 15295 15299 mice +T450 UBERON:0000479 15317 15323 tissue +T451 http://purl.obolibrary.org/obo/MONDO_0021002 15324 15334 syndactyly +T452 UBERON:0008439 15351 15366;15388 15397;15404 15408 webbing between ... digits of ... feet +T453 UBERON:0003631 15371 15376;15388 15397;15404 15408 first ... digits of ... feet +T454 UBERON:0003632 15381 15397;15404 15408 second digits of ... feet +T455 UBERON:0002102 15468 15477 forelimbs +T456 UBERON:0002398 15502 15510 forefeet +T457 UBERON:0002387 15538 15546 hindfeet +T458 NCBITaxon:33208 15565 15572 animals +T459 UBERON:0004288 15588 15596 skeletal +T460 UBERON:0004288 15620 15628 skeletal +T461 UBERON:0004454 15664 15670 ankles +T462 UBERON:0004905 15672 15678 joints +T463 UBERON:0001474 15725 15730 bones +T464 UBERON:0001453 15781 15801 second distal tarsal +T465 UBERON:4200011 15819 15838 central tarsal bone +T466 NCBITaxon:33208 15869 15875 animal +T467 UBERON:0004905 15987 15993 joints +T468 GO:0031012 16069 16075 matrix +T469 UBERON:0004905 16174 16183 articular +T470 UBERON:0004129 16205 16231 cartilaginous growth plate +T471 PR:000000035 16286 16292 Bmpr1a +T472 UBERON:0004905 16344 16350 joints +T473 UBERON:0004454 16358 16370 ankle region +T474 GO:0051216 16386 16396;16412 16414;16425 16434 generation ... of ... cartilage +T475 UBERON:0010996 16415 16434 articular cartilage +T476 UBERON:0003657 16449 16458;16463 16467 joints of ... limb +T477 UBERON:0009585 16513 16536 Interdigital mesenchyme +T478 GO:0006915 16563 16572 apoptosis +T479 UBERON:0000922 16580 16589 embryonic +T480 GO:0009790 16580 16601 embryonic development +T481 PR:000000034 16639 16642 BMP +T482 PR:000000021 16663 16669 Noggin +T483 GO:0010467 16689 16699 expression +T484 PR:000000034 16721 16724 BMP +T485 UBERON:0002101 16830 16835 Limbs +T486 PR:000000281 16839 16843 Gdf5 +T487 PR:000000035 16848 16854 Bmpr1a +T488 SO:0000359 16854 16859 floxP +T489 UBERON:0000922 16867 16874 embryos +T490 UBERON:0006015 16903 16923 interdigital webbing +T491 UBERON:0001463 16936 16941;16969 16978;16985 16994 first ... digits of ... forelimbs +T492 UBERON:0003622 16946 16952;16969 16978;16985 16994 second ... digits of ... forelimbs +T493 UBERON:0007023 17092 17097 adult +T494 UBERON:0002101 17098 17102 limb +T495 UBERON:0000479 17128 17134 tissue +T496 UBERON:0006052 17166 17177 fifth digit +T497 PR:000033987 17210 17214 LACZ +T498 GO:0010467 17215 17225 expression +T499 PR:000000281 17229 17233 Gdf5 +T500 UBERON:0000922 17252 17259 embryos +T501 UBERON:0003695 17328 17356 metacarpal-phalangeal joints +T502 UBERON:0006016 17369 17396;17401 17417;17434 17440 interdigital region between ... first and second ... digits +T503 GO:0010467 17485 17495 expression +T504 PR:000033987 17499 17503 LACZ +T505 UBERON:0006052 17559 17570 fifth digit +T506 UBERON:0009596 17695 17726;17731 17754 interdigital mesenchyme between ... first and second digits +T507 UBERON:0006052 17782 17793 fifth digit +T508 UBERON:0003104 17803 17813 mesenchyme +T509 GO:0016265 17843 17848 dying +T510 UBERON:0000479 17883 17889 tissue +T511 UBERON:0002101 17916 17921 limbs +T512 CHEBI:15358 17949 17956 histone +T513 PR:000027594 17949 17959 histone H3 +T514 GO:0008283 17968 17981 proliferating +T515 UBERON:0006015 18061 18082;18104 18110 webbed region between ... digits +T516 UBERON:0006048 18087 18092;18104 18110 first ... digits +T517 UBERON:0006049 18097 18110 second digits +T518 GO:0010467 18129 18138 expressed +T519 PR:000033987 18139 18143 LACZ +T520 PR:000000281 18147 18151 Gdf5 +T521 PR:000000035 18156 18162 Bmpr1a +T522 SO:0000359 18162 18167 floxP +T523 UBERON:0000922 18175 18182 embryos +T524 PR:000000035 18237 18243 BMPR1A +T525 GO:0012501 18270 18291 programmed cell death +T526 UBERON:0009585 18295 18318 interdigital mesenchyme +T527 GO:0008283 18362 18373 proliferate +T528 PR:000000035 18392 18398 BMPR1A +T529 UBERON:0004905 18428 18433 Joint +T530 UBERON:0004454 18447 18460 Ankle Regions +T531 PR:000000035 18466 18472 Bmpr1a +T532 SO:0000704 18473 18477 gene +T533 GO:0010467 18481 18490 expressed +T534 UBERON:0004905 18529 18535 joints +T535 GO:0097617 18573 18586 hybridization +T536 SO:0000704 18603 18607 gene +T537 GO:0010467 18616 18625 expressed +T538 UBERON:0001488 18647 18659 ankle joints +T539 UBERON:0010996 18676 18695 articular cartilage +T540 UBERON:0002544 18707 18712 digit +T541 UBERON:0004905 18713 18719 joints +T542 PR:000033987 18741 18745 LACZ +T543 UBERON:0001488 18816 18828 ankle joints +T544 UBERON:0001488 18915 18926 ankle joint +T545 GO:0007567 18968 18973 natal +T546 NCBITaxon:33208 18981 18988 animals +T547 UBERON:0004905 19011 19016 joint +T548 GO:0010467 19024 19034 expression +T549 PR:000000281 19083 19087 Gdf5 +T550 SO:0000704 19088 19092 gene +T551 GO:0010467 19105 19114 expressed +T552 UBERON:0004905 19149 19154 joint +T553 SO:0000704 19186 19190 gene +T554 GO:0031012 19235 19241 matrix +T555 PR:000003266 19243 19249 Col2a1 +T556 PR:000003266 19319 19325 Col2a1 +T557 UBERON:0004905 19367 19372 joint +T558 UBERON:0001447 19411 19417 tarsal +T559 PR:000000281 19421 19425 Gdf5 +T560 PR:000000035 19430 19436 Bmpr1a +T561 SO:0000359 19436 19441 floxP +T562 PR:000000281 19480 19484 Gdf5 +T563 GO:0010467 19485 19495 expression +T564 UBERON:0004905 19552 19557 joint +T565 UBERON:0001447 19647 19658 ankle bones +T566 GO:0007567 19666 19671 natal +T567 UBERON:0004288 19679 19688 skeletons +T568 UBERON:0004288 19734 19742 skeletal +T569 UBERON:0000922 19761 19770 embryonic +T570 GO:0009790 19761 19782 embryonic development +T571 UBERON:0004454 19827 19832 ankle +T572 UBERON:0010996 19855 19874 Articular Cartilage +T573 UBERON:0004905 19884 19890 Joints +T574 UBERON:0004905 19900 19906 joints +T575 PR:000000035 19910 19916 Bmpr1a +T576 NCBITaxon:10088 19938 19942 mice +T577 UBERON:0000922 19944 19953 embryonic +T578 UBERON:0004288 19970 19978 skeletal +T579 PR:000000281 20018 20022 Gdf5 +T580 UBERON:0002544 20080 20085 digit +T581 GO:0008219 20135 20145 cell death +T582 GO:0008283 20149 20167 cell proliferation +T583 UBERON:0003695 20189 20210;20236 20242 metacarpal-phalangeal ... joints +T584 UBERON:0003696 20214 20242 metatarsal-phalangeal joints +T585 PR:000033987 20307 20311 LACZ +T586 GO:0010467 20312 20322 expression +T587 UBERON:0006658 20344 20366 interphalangeal joints +T588 UBERON:0004905 20375 20384 articular +T589 GO:0010467 20437 20447 expression +T590 PR:000003266 20451 20457 Col2a1 +T591 PR:000000281 20459 20463 Gdf5 +T592 PR:000000036 20468 20474 Bmpr1b +T593 UBERON:0004905 20491 20500 articular +T594 UBERON:0003221 20516 20525 phalanges +T595 GO:0007567 20566 20571 birth +T596 UBERON:0002544 20573 20578 digit +T597 UBERON:0004905 20579 20585 joints +T598 NCBITaxon:33208 20641 20648 animals +T599 CL:0000138 20650 20662 chondrocytes +T600 UBERON:0004905 20680 20689 articular +T601 GO:0031012 20739 20745 matrix +T602 CHEBI:37396 20807 20820 proteoglycans +T603 UBERON:0004905 20883 20892 articular +T604 GO:0010467 20906 20915 expressed +T605 PR:000003266 20931 20937 Col2a1 +T606 PR:000003606 20942 20950 Aggrecan +T607 PR:000003606 20952 20955 Agg +T608 SO:0000704 20962 20967 genes +T609 GO:0031012 21020 21026 matrix +T610 GO:0006915 21084 21102 cellular apoptosis +T611 GO:0008283 21084 21092;21106 21119 cellular ... proliferation +T612 UBERON:0004905 21176 21185 articular +T613 GO:0010467 21245 21255 expression +T614 PR:000010208 21259 21269 Matrilin-4 +T615 PR:000010208 21271 21275 Mat4 +T616 SO:0000704 21280 21284 gene +T617 GO:0010467 21285 21294 expressed +T618 UBERON:0004905 21319 21328 articular +T619 UBERON:0004905 21368 21374 joints +T620 NCBITaxon:33208 21423 21430 animals +T621 PR:000010208 21449 21453 Mat4 +T622 UBERON:0010996 21484 21503 articular cartilage +T623 UBERON:0004905 21522 21528 joints +T624 GO:0010467 21569 21579 expression +T625 PR:000033987 21583 21587 LACZ +T626 UBERON:0004905 21599 21608 articular +T627 UBERON:0004905 21687 21696 articular +T628 SO:0000704 21802 21806 gene +T629 GO:0010467 21802 21817 gene expression +T630 PR:000000035 21840 21846 Bmpr1a +T631 GO:0051216 21879 21888;21906 21908;21919 21928 formation ... of ... cartilage +T632 UBERON:0010996 21909 21928 articular cartilage +T633 GO:0007567 21945 21950 birth +T634 UBERON:0004905 22000 22009 articular +T635 NCBITaxon:33208 22028 22035 animals +T636 GO:0010467 22041 22051 expression +T637 PR:000003266 22055 22061 Col2a1 +T638 UBERON:0004905 22089 22098 articular +T639 UBERON:0001435 22115 22122 carpals +T640 UBERON:0002374 22124 22135 metacarpals +T641 UBERON:0003221 22141 22150 phalanges +T642 UBERON:0002398 22158 22166 forefeet +T643 UBERON:0004905 22228 22237 articular +T644 UBERON:0001447 22247 22254 tarsals +T645 UBERON:0001448 22259 22270 metatarsals +T646 UBERON:0002387 22278 22286 hindfeet +T647 PR:000003266 22323 22329 Col2a1 +T648 GO:0010467 22330 22340 expression +T649 UBERON:0004905 22374 22383 articular +T650 GO:0010467 22497 22507 expression +T651 PR:000003606 22511 22514 Agg +T652 SO:0000704 22523 22528 genes +T653 GO:0010467 22538 22547 expressed +T654 UBERON:0010996 22563 22582 articular cartilage +T655 CL:1001607 22563 22588 articular cartilage cells +T656 PR:000003328 22590 22600 Collagen 3 +T657 PR:000003328 22602 22608 Col3a1 +T658 PR:000005693 22614 22625 Collagen 10 +T659 PR:000005693 22627 22634 Col10a1 +T660 PR:000000034 22701 22704 BMP +T661 GO:0030509 22701 22714 BMP signaling +T662 CL:0000138 22727 22739 chondrocytes +T663 PR:000003263 22779 22789 Collagen 1 +T664 PR:000003264 22791 22797 Col1a1 +T665 GO:0010467 22799 22809 expression +T666 GO:0008283 22820 22833 proliferation +T667 CL:0000057 22871 22881 fibroblast +T668 GO:0010467 22964 22974 expression +T669 PR:000003264 22978 22984 Col1a1 +T670 UBERON:0010996 22995 23014 articular cartilage +T671 GO:0008283 23023 23036 proliferation +T672 UBERON:0004905 23053 23062 articular +T673 NCBITaxon:33208 23097 23104 animals +T674 PR:000033987 23142 23146 LACZ +T675 GO:0010467 23154 23164 expression +T676 UBERON:0010996 23186 23205 articular cartilage +T677 CL:1001607 23186 23211 articular cartilage cells +T678 UBERON:0004905 23250 23259 articular +T679 CL:0000138 23260 23272 chondrocytes +T680 UBERON:0002516 23274 23286 growth plate +T681 CL:0000138 23287 23299 chondrocytes +T682 CL:0000062 23305 23316 osteoblasts +T683 PR:000000034 23383 23386 BMP +T684 GO:0030509 23383 23396 BMP signaling +T685 UBERON:0010996 23477 23496 articular cartilage +T686 PR:000030444 23511 23522 Osteocalcin +T687 PR:000003264 23527 23533 Col1a1 +T688 GO:0010467 23534 23544 expression +T689 CL:0000062 23564 23575 osteoblasts +T690 PR:000000035 23630 23636 BMPR1A +T691 GO:0007567 23665 23670 natal +T692 UBERON:0004905 23671 23676 joint +T693 UBERON:0010996 23677 23696 articular cartilage +T694 GO:0010467 23709 23719 expression +T695 SO:0000704 23728 23733 genes +T696 GO:0031012 23778 23784 matrix +T697 PR:000015435 23820 23824 Sox9 +T698 GO:0010467 23879 23889 expression +T699 GO:0031012 23903 23923 extracellular matrix +T700 GO:0031012 23925 23928 ECM +T701 SO:0000704 23930 23935 genes +T702 PR:000003606 23946 23949 Agg +T703 GO:0031012 24014 24020 matrix +T704 SO:0000704 24021 24025 gene +T705 PR:000003266 24026 24032 Col2a1 +T706 GO:0010467 24136 24146 expression +T707 GO:0031012 24165 24171 matrix +T708 SO:0000704 24179 24184 genes +T709 PR:000000035 24188 24194 Bmpr1a +T710 NCBITaxon:10088 24202 24206 mice +T711 PR:000015435 24212 24216 SOX9 +T712 UBERON:0004905 24257 24266 articular +T713 NCBITaxon:10088 24351 24355 mice +T714 UBERON:0007616 24396 24404 Synovial +T715 PR:000000035 24495 24501 Bmpr1a +T716 UBERON:0001484 24560 24573 joint capsule +T717 UBERON:0004905 24582 24588 joints +T718 UBERON:0004454 24610 24622 ankle region +T719 UBERON:0004905 24654 24660 joints +T720 GO:0040007 24693 24697 grew +T721 UBERON:0004905 24707 24712 joint +T722 UBERON:0010996 24774 24793 articular cartilage +T723 GO:0010467 24880 24890 expression +T724 PR:000005693 24894 24901 Col10a1 +T725 CL:0000138 24929 24941 chondrocytes +T726 UBERON:0004905 24957 24966 articular +T727 PR:000005693 25065 25072 Col10a1 +T728 GO:0010467 25073 25083 expression +T729 GO:0010467 25184 25194 expression +T730 PR:000005693 25198 25205 Col10a1 +T731 PR:000033987 25236 25240 LACZ +T732 GO:0001503 25302 25314 ossification +T733 UBERON:0006773 25302 25320 ossification front +T734 PR:000005693 25328 25335 Col10a1 +T735 GO:0010467 25348 25357 expressed +T736 CL:0000138 25370 25385 cartilage cells +T737 UBERON:0010996 25421 25440 articular cartilage +T738 UBERON:0001132 25534 25545 parathyroid +T739 PR:000013433 25534 25569 parathyroid hormone-related protein +T740 UBERON:0004905 25603 25612 articular +T741 UBERON:0004905 25738 25747 articular +T742 CL:0000138 25848 25860 chondrocytes +T743 UBERON:0004905 25882 25891 articular +T744 UBERON:0007616 25907 25915 synovial +T745 CL:0000226 25968 25985 mononuclear cells +T746 GO:0005634 25972 25979 nuclear +T747 CL:0000214 25997 26009 synoviocytes +T748 CL:0000235 26013 26024 macrophages +T749 GO:0009986 26081 26088 surface +T750 GO:0007567 26110 26115 natal +T751 CL:0000775 26136 26147 neutrophils +T752 UBERON:0007616 26225 26233 synovial +T753 UBERON:0007616 26307 26315 synovial +T754 GO:0065007 26331 26340 regulated +T755 PR:000000034 26344 26347 BMP +T756 GO:0030509 26344 26357 BMP signaling +T757 UBERON:0007616 26374 26382 synovium +T758 UBERON:0004288 26424 26432 skeletal +T759 UBERON:0001447 26489 26496 tarsals +T760 UBERON:0010996 26515 26534 articular cartilage +T761 UBERON:0010996 26570 26589 Articular Cartilage +T762 UBERON:0002544 26593 26598 Digit +T763 UBERON:0001485 26603 26614 Knee Joints +T764 UBERON:0004454 26631 26643 ankle region +T765 NCBITaxon:10088 26732 26736 mice +T766 http://purl.obolibrary.org/obo/MONDO_0005178 26766 26780 osteoarthritis +T767 UBERON:0004905 26810 26819 articular +T768 NCBITaxon:33208 26878 26885 animals +T769 PR:000003606 26911 26914 Agg +T770 PR:000005693 26919 26924 Col10 +T771 GO:0010467 26925 26935 expression +T772 UBERON:0004905 26963 26972 articular +T773 UBERON:0002398 26988 26996 forefeet +T774 UBERON:0002387 27001 27009 hindfeet +T775 UBERON:0010996 27134 27153 articular cartilage +T776 GO:0010467 27299 27309 expression +T777 PR:000030444 27313 27324 Osteocalcin +T778 PR:000003264 27326 27332 Col1a1 +T779 GO:0031012 27337 27343 matrix +T780 PR:000010473 27337 27362 matrix metalloprotease-13 +T781 UBERON:0003840 27413 27421;27426 27434 joint of ... hindlimb +T782 UBERON:0001465 27440 27444 knee +T783 UBERON:0001487 27502 27513 foot joints +T784 GO:0031012 27540 27546 matrix +T785 UBERON:0004905 27616 27621 joint +T786 GO:0007567 27678 27683 natal +T787 PR:000003266 27715 27721 Col2a1 +T788 PR:000003606 27726 27729 Agg +T789 GO:0010467 27730 27740 expression +T790 GO:0010467 27795 27805 expression +T791 PR:000015435 27809 27813 Sox9 +T792 UBERON:0001465 27862 27866 knee +T793 UBERON:0004765 27867 27884 skeletal elements +T794 UBERON:0001995 27928 27946 fibrocartilaginous +T795 UBERON:0000387 27947 27955 meniscus +T796 UBERON:0000981 27981 27986 femur +T797 UBERON:0000979 27991 27996 tibia +T798 UBERON:0000387 28072 28080 meniscus +T799 UBERON:0000387 28253 28261 meniscus +T800 NCBITaxon:33208 28315 28322 animals +T801 UBERON:0004383 28395 28411 tibial epiphysis +T802 UBERON:0001465 28447 28452 knees +T803 NCBITaxon:33208 28463 28470 animals +T804 UBERON:0002516 28515 28527 growth plate +T805 UBERON:0004905 28532 28541 articular +T806 UBERON:0010996 28584 28603 Articular cartilage +T807 NCBITaxon:33208 28637 28644 animals +T808 UBERON:0004905 28853 28862 articular +T809 PR:000033987 28914 28918 LACZ +T810 PR:000000035 28972 28978 Bmpr1a +T811 UBERON:0010996 29012 29031 articular cartilage +T812 UBERON:0001465 29101 29106 knees +T813 UBERON:0004905 29122 29131 articular +T814 UBERON:0001474 29147 29152 bones +T815 UBERON:0000981 29160 29165 femur +T816 UBERON:0000979 29170 29175 tibia +T817 UBERON:0004383 29238 29250;29255 29260 epiphysis of ... tibia +T818 UBERON:0004129 29304 29326 growth plate cartilage +T819 UBERON:0001474 29373 29377 bone +T820 UBERON:0004384 29493 29505;29510 29515 epiphysis of ... femur +T821 http://purl.obolibrary.org/obo/MONDO_0005178 29613 29627 osteoarthritis +T822 UBERON:0010996 29745 29764 articular cartilage +T823 PR:000000035 29805 29811 Bmpr1a +T824 PR:000033987 29830 29834 LACZ +T825 UBERON:0004905 29937 29942 joint +T826 http://purl.obolibrary.org/obo/MONDO_0005578 29943 29952 arthritis +T827 NCBITaxon:33208 30051 30058 animals +T828 PR:000000281 30060 30064 Gdf5 +T829 PR:000000035 30069 30075 Bmpr1a +T830 SO:0000359 30075 30080 floxP +T831 NCBITaxon:33208 30088 30095 animals +T832 NCBITaxon:10088 30284 30288 mice +T833 UBERON:0004905 30368 30374 joints +T834 UBERON:0002544 30382 30388 digits +T835 UBERON:0001448 30426 30428 MT +T836 UBERON:0004302 30429 30431 P1 +T837 UBERON:0004905 30432 30437 joint +T838 UBERON:0004302 30502 30504 P1 +T839 UBERON:4100005 30505 30507 P2 +T840 UBERON:0004905 30508 30513 joint +T841 SO:0000704 30615 30619 gene +T842 GO:0010467 30615 30630 gene expression +T843 NCBITaxon:10088 30665 30669 mice +T844 PR:000000035 30687 30693 BMPR1A +T845 GO:0007567 30721 30726 natal +T846 UBERON:0010996 30742 30761 articular cartilage +T847 PR:000000034 30806 30809 BMP +T848 GO:0030509 30806 30819 BMP signaling +T849 GO:0009790 30911 30924 embryogenesis +T850 PR:000000034 30955 30958 BMP +T851 GO:0016265 30976 30981 death +T852 PR:000000281 31017 31021 Gdf5 +T853 UBERON:0000922 31066 31075 embryonic +T854 PR:000000035 31089 31095 Bmpr1a +T855 UBERON:0002101 31181 31185 limb +T856 GO:0060173 31181 31185;31199 31210 limb ... development +T857 UBERON:0004288 31190 31198 skeletal +T858 GO:0001501 31190 31210 skeletal development +T859 UBERON:0002101 31229 31233 limb +T860 PR:000000035 31269 31275 Bmpr1a +T861 PR:000000281 31281 31285 Gdf5 +T862 UBERON:0006015 31305 31327 webbing between digits +T863 UBERON:0004905 31337 31342 joint +T864 UBERON:0004454 31382 31387 ankle +T865 UBERON:0010996 31413 31432 articular cartilage +T866 GO:0007567 31439 31444 birth +T867 http://purl.obolibrary.org/obo/MONDO_0005578 31466 31475 arthritis +T868 PR:000000034 31526 31529 BMP +T869 GO:0030509 31526 31539 BMP signaling +T870 UBERON:0006012 31547 31559 interdigital +T871 GO:0006915 31560 31569 apoptosis +T872 GO:0060173 31577 31591;31596 31600 development of ... limb +T873 UBERON:0002101 31596 31600 limb +T874 PR:000000034 31660 31663 BMP +T875 GO:0030509 31660 31681 BMP signaling pathway +T876 PR:000000034 31842 31845 BMP +T877 GO:0030509 31842 31855 BMP signaling +T878 UBERON:0006012 31872 31884 interdigital +T879 GO:0006915 31885 31894 apoptosis +T880 PR:000000035 31913 31919 Bmpr1a +T881 PR:000000035 31995 32001 Bmpr1a +T882 UBERON:0004905 32052 32057 joint +T883 UBERON:0004905 32111 32116 joint +T884 UBERON:0001447 32131 32139;32144 32149 bones in ... ankle +T885 PR:000000034 32193 32196 BMP +T886 PR:000000281 32205 32209 Gdf5 +T887 PR:000007924 32214 32218 Gdf6 +T888 PR:000000036 32224 32230 Bmpr1b +T889 NCBITaxon:9606 32252 32257 human +T890 PR:000000021 32258 32264 Noggin +T891 UBERON:0004905 32393 32398 joint +T892 UBERON:0002101 32438 32443 limbs +T893 UBERON:0004905 32449 32454 joint +T894 PR:000000034 32506 32509 BMP +T895 GO:0030509 32506 32517 BMP pathway +T896 PR:000000034 32547 32550 BMP +T897 GO:0030509 32547 32560 BMP signaling +T898 UBERON:0004905 32593 32598 joint +T899 UBERON:0001062 32617 32627 anatomical +T900 UBERON:0004905 32645 32651 joints +T901 PR:000000035 32677 32683 Bmpr1a +T902 PR:000000281 32702 32706 Gdf5 +T903 GO:0010467 32707 32717 expression +T904 UBERON:0004905 32739 32744 joint +T905 UBERON:0004454 32765 32777 ankle region +T906 PR:000000034 32825 32828 BMP +T907 GO:0030509 32825 32838 BMP signaling +T908 UBERON:0004905 32852 32858 joints +T909 GO:0010467 32876 32886 expression +T910 PR:000000034 32896 32899 BMP +T911 UBERON:0004454 32922 32928 ankles +T912 PR:000000281 32974 32978 Gdf5 +T913 SO:0000704 32994 32998 gene +T914 UBERON:0004454 33015 33021 ankles +T915 UBERON:0004905 33032 33037 joint +T916 GO:0010467 33065 33075 expression +T917 PR:P05187 33083 33088 HPLAP +T918 PR:000000281 33116 33120 Gdf5 +T919 GO:0065007 33121 33128 control +T920 PR:000033987 33152 33156 LACZ +T921 GO:0010467 33165 33174 expressed +T922 PR:000000281 33185 33189 Gdf5 +T923 SO:0000704 33259 33263 gene +T924 GO:0010467 33259 33274 gene expression +T925 UBERON:0002544 33311 33316 digit +T926 PR:000000035 33364 33370 Bmpr1a +T927 PR:000000281 33424 33428 Gdf5 +T928 PR:000000035 33513 33519 Bmpr1a +T929 UBERON:0004905 33523 33528 joint +T930 GO:0006402 33573 33581;33589 33593 decay of ... mRNA +T931 GO:0030163 33573 33581;33598 33605 decay of ... protein +T932 PR:000000035 33582 33588 Bmpr1a +T933 PR:000000281 33611 33615 Gdf5 +T934 PR:000000035 33672 33678 Bmpr1a +T935 SO:0000704 33759 33766 genetic +T936 PR:000000035 33776 33782 Bmpr1a +T937 UBERON:0004905 33811 33816 joint +T938 UBERON:0004905 33863 33872 articular +T939 SO:0000704 33885 33889 gene +T940 GO:0010467 33885 33900 gene expression +T941 GO:0007567 33919 33924 birth +T942 PR:000000035 33926 33932 Bmpr1a +T943 NCBITaxon:33208 33943 33950 animals +T944 UBERON:0010996 34009 34028 articular cartilage +T945 GO:0007568 34061 34064 age +T946 PR:000000034 34093 34096 BMP +T947 GO:0030509 34093 34115 BMP receptor signaling +T948 UBERON:0010996 34167 34186 articular cartilage +T949 GO:0007567 34198 34203 natal +T950 UBERON:0010996 34213 34232 Articular cartilage +T951 UBERON:0002217 34255 34270 synovial joints +T952 UBERON:0004288 34308 34316 skeleton +T953 UBERON:0000113 34352 34361 adulthood +T954 UBERON:0010996 34389 34408 articular cartilage +T955 UBERON:0004905 34412 34417 joint +T956 UBERON:0001437 34523 34541 ends of long bones +T957 UBERON:0010996 34563 34582 articular cartilage +T958 PR:000000035 34591 34597 Bmpr1a +T959 GO:0008283 34667 34680 proliferation +T960 GO:0010467 34696 34703 express +T961 PR:000003264 34704 34710 Col1a1 +T962 GO:0010467 34729 34736 express +T963 PR:000015435 34737 34741 SOX9 +T964 GO:0065007 34772 34782 regulating +T965 GO:0010467 34783 34793 expression +T966 GO:0031012 34832 34838 matrix +T967 GO:0031012 34914 34920 matrix +T968 NCBITaxon:33208 34953 34960 animals +T969 GO:0030166 34985 34997;35015 35028 synthesis of ... proteoglycans +T970 PR:000003266 34998 35004 Col2a1 +T971 PR:000003606 35006 35009 Agg +T972 CHEBI:37396 35015 35028 proteoglycans +T973 PR:000000035 35041 35047 BMPR1A +T974 UBERON:0010996 35068 35087 articular cartilage +T975 GO:0010467 35115 35125 expression +T976 GO:0044420 35133 35147 ECM components +T977 PR:000015435 35177 35181 SOX9 +T978 GO:0010467 35219 35228 expressed +T979 PR:000003266 35265 35271 Col2a1 +T980 PR:000015435 35390 35394 SOX9 +T981 GO:0006468 35456 35479 protein phosphorylation +T982 GO:0010467 35489 35499 expression +T983 PR:000026780 35525 35531 L-SOX5 +T984 PR:000015432 35536 35540 SOX6 +T985 SO:0000704 35631 35636 genes +T986 NCBITaxon:9031 35652 35659 chicken +T987 UBERON:0002544 35660 35665 digit +T988 PR:000015435 35689 35693 Sox9 +T989 PR:000000036 35722 35728 Bmpr1b +T990 PR:000026780 35734 35740 L-Sox5 +T991 PR:000015432 35751 35755 Sox6 +T992 GO:0031012 35774 35780 matrix +T993 PR:000003266 35803 35809 Col2a1 +T994 PR:000003606 35814 35817 Agg +T995 SO:0000704 35899 35903 gene +T996 GO:0010467 35899 35914 gene expression +T997 PR:000000035 35927 35933 Bmpr1a +T998 NCBITaxon:10088 35944 35948 mice +T999 PR:000000035 35963 35969 BMPR1A +T1000 PR:000015435 36010 36014 SOX9 +T1001 GO:0006412 36023 36036 translational +T1002 PR:000026780 36072 36078 L-Sox5 +T1003 PR:000015432 36082 36086 Sox6 +T1004 GO:0010467 36112 36122 expression +T1005 GO:0044420 36126 36140 ECM components +T1006 PR:000000164 36190 36194 BMP2 +T1007 GO:0010467 36236 36246 expression +T1008 PR:000015432 36250 36254 Sox6 +T1009 UBERON:0000479 36258 36264 tissue +T1010 CL:0000010 36265 36278 culture cells +T1011 GO:0010467 36370 36380 expression +T1012 PR:000026780 36384 36390 L-Sox5 +T1013 PR:000015432 36394 36398 Sox6 +T1014 GO:0007567 36406 36411 natal +T1015 UBERON:0010996 36412 36431 articular cartilage +T1016 PR:000015435 36471 36475 SOX9 +T1017 CHEBI:33893 36503 36511 reagents +T1018 GO:0007567 36617 36622 natal +T1019 PR:000026780 36690 36696 L-Sox5 +T1020 PR:000015432 36700 36705 Sox-6 +T1021 GO:0007567 36739 36744 birth +T1022 GO:0065007 36879 36888 regulated +T1023 PR:000000034 36892 36895 BMP +T1024 GO:0030509 36892 36905 BMP signaling +T1025 PR:000015435 36921 36925 SOX9 +T1026 SO:0000704 36943 36948 genes +T1027 UBERON:0010996 36952 36971 articular cartilage +T1028 PR:000000365 36986 36991 Smad3 +T1029 GO:0010467 36995 37005 expression +T1030 PR:000000046 37027 37055 transforming growth factor β +T1031 PR:000000046 37057 37062 TGF-β +T1032 UBERON:0010996 37102 37121 articular cartilage +T1033 PR:000000046 37207 37211 TGFβ +T1034 GO:0007179 37207 37211;37228 37237 TGFβ ... signaling +T1035 PR:000000034 37224 37227 BMP +T1036 GO:0030509 37224 37237 BMP signaling +T1037 UBERON:0010996 37268 37287 articular cartilage +T1038 PR:000000035 37357 37363 Bmpr1a +T1039 UBERON:0010996 37371 37390 articular cartilage +T1040 GO:0044420 37408 37422 ECM components +T1041 PR:000000046 37484 37488 TGFβ +T1042 GO:0007179 37484 37488;37497 37506 TGFβ ... signaling +T1043 PR:000000034 37493 37496 BMP +T1044 GO:0030509 37493 37506 BMP signaling +T1045 UBERON:0010996 37560 37579 articular cartilage +T1046 PR:000000034 37591 37595 BMPs +T1047 UBERON:0010996 37703 37722 articular cartilage +T1048 GO:0051216 37744 37763 cartilage formation +T1049 GO:0031099 37815 37825 regenerate +T1050 UBERON:0007023 37847 37852 adult +T1051 NCBITaxon:33208 37853 37860 animals +T1052 UBERON:0010996 37990 38009 articular cartilage +T1053 PR:000000035 38035 38041 BMPR1A +T1054 CHEBI:36357 38082 38090 molecule +T1055 UBERON:0010996 38239 38258 articular cartilage +T1056 GO:0007567 38266 38271 natal +T1057 PR:000000035 38289 38295 Bmpr1a +T1058 UBERON:0010996 38308 38327 articular cartilage +T1059 UBERON:0004905 38366 38375 articular +T1060 UBERON:0004905 38396 38401 joint +T1061 http://purl.obolibrary.org/obo/MONDO_0005578 38438 38447 arthritis +T1062 PR:000000035 38460 38466 Bmpr1a +T1063 NCBITaxon:10088 38477 38481 mice +T1064 PR:000000034 38521 38524 BMP +T1065 GO:0030509 38521 38534 BMP signaling +T1066 NCBITaxon:9606 38554 38559 human +T1067 UBERON:0004905 38560 38565 joint +T1068 http://purl.obolibrary.org/obo/MONDO_0006816 38560 38573 joint disease +T1069 http://purl.obolibrary.org/obo/MONDO_0005178 38575 38589 Osteoarthritis +T1070 SO:0000704 38621 38628 genetic +T1071 SO:0000704 38672 38679 genetic +T1072 NCBITaxon:9606 38788 38794 Humans +T1073 PR:000000035 38851 38857 BMPR1A +T1074 http://purl.obolibrary.org/obo/MONDO_0017380 38886 38904 juvenile polyposis +T1075 http://purl.obolibrary.org/obo/MONDO_0005178 38959 38973 osteoarthritis +T1076 NCBITaxon:9606 38984 38990 people +T1077 NCBITaxon:10088 39035 39039 mice +T1078 SO:0001023 39088 39094 allele +T1079 PR:000000035 39098 39104 Bmpr1a +T1080 http://purl.obolibrary.org/obo/MONDO_0005178 39137 39151 osteoarthritis +T1081 http://purl.obolibrary.org/obo/MONDO_0005578 39229 39238 arthritis +T1082 NCBITaxon:9606 39253 39259 humans +T1083 SO:0000704 39415 39420 genes +T1084 PR:000000034 39455 39458 BMP +T1085 GO:0030509 39455 39476 BMP signaling pathway +T1086 PR:000000166 39492 39496 BMP5 +T1087 SO:0000704 39497 39501 gene +T1088 NCBITaxon:9606 39505 39510 human +T1089 PR:000000363 39555 39560 MADH1 +T1090 SO:0000704 39561 39565 gene +T1091 NCBITaxon:9606 39569 39574 human +T1092 PR:000000037 39648 39653 BMPR2 +T1093 NCBITaxon:9606 39666 39671 human +T1094 NCBITaxon:9606 39732 39737 human +T1095 http://purl.obolibrary.org/obo/MONDO_0005178 39738 39752 osteoarthritis +T1096 SO:0000704 39797 39802 genes +T1097 http://purl.obolibrary.org/obo/MONDO_0000001 39854 39861 disease +T1098 SO:0001645 39880 39895 genetic markers +T1099 PR:000000034 39901 39904 BMP +T1100 GO:0030509 39901 39914 BMP signaling +T1101 http://purl.obolibrary.org/obo/MONDO_0005178 39955 39969 osteoarthritis +T1102 http://purl.obolibrary.org/obo/MONDO_0000001 40058 40065 disease +T1103 UBERON:0002217 40079 40094 synovial joints +T1104 SO:0000704 40124 40135 genetically +T1105 UBERON:0002217 40145 40160 synovial joints +T1106 NCBITaxon:7742 40217 40227 vertebrate +T1107 PR:000000281 40245 40249 Gdf5 +T1108 SO:0000704 40299 40303 gene +T1109 GO:0010467 40299 40314 gene expression +T1110 UBERON:0004905 40344 40353 articular +T1111 SO:0000704 40411 40416 genes +T1112 UBERON:0000479 40426 40433 tissues +T1113 SO:0000363 40473 40479;40487 40491 floxed ... gene +T1114 GO:0010467 40540 40550 expression +T1115 SO:0000704 40556 40560 gene +T1116 UBERON:0004905 40585 40591 joints +T1117 SO:0000704 40630 40634 gene +T1118 UBERON:0004905 40647 40656 articular +T1119 http://purl.obolibrary.org/obo/MONDO_0000001 40788 40795 disease +T1120 UBERON:0004905 40810 40816 joints +T1121 UBERON:0004905 40880 40885 joint +T1122 http://purl.obolibrary.org/obo/MONDO_0006816 40880 40894 joint diseases +T1123 PR:000000281 40936 40940 Gdf5 +T1124 NCBITaxon:10088 40956 40960 mice +T1125 NCBITaxon:10088 40964 40969 mouse +T1126 SO:0000153 40980 40983 BAC +T1127 SO:0000153 41039 41042 BAC +T1128 PR:000000281 41052 41056 Gdf5 +T1129 SO:0000153 41069 41072 BAC +T1130 NCBITaxon:562 41129 41136 E. coli +T1131 GO:0005634 41165 41172 nuclear +T1132 SO:0000155 41205 41212 plasmid +T1133 SO:0000243 41253 41257 IRES +T1134 PR:P05187 41258 41263 hPLAP +T1135 SO:0000155 41270 41277 plasmid +T1136 SO:0000318 41327 41341 ATG start site +T1137 PR:000000281 41345 41349 Gdf5 +T1138 SO:0000028 41371 41373 bp +T1139 SO:0000147 41387 41391 exon +T1140 PR:000000281 41395 41399 Gdf5 +T1141 PR:000000281 41430 41434 GDF5 +T1142 SO:0000006 41513 41524 PCR product +T1143 SO:0000061 41554 41571 restriction sites +T1144 SO:0000028 41590 41592 bp +T1145 SO:0001026 41599 41606 genomic +T1146 PR:000000281 41607 41611 Gdf5 +T1147 SO:0000318 41635 41661 ATG translation start site +T1148 GO:0006413 41639 41656 translation start +T1149 SO:0000121 41663 41677 forward primer +T1150 SO:0001031 41718 41725 reverse +T1151 SO:0000243 41829 41833 IRES +T1152 PR:P05187 41834 41839 hPLAP +T1153 SO:0000006 41868 41879 PCR product +T1154 PR:P05187 41931 41936 hPLAP +T1155 GO:0006415 41937 41953 translation stop +T1156 SO:0000319 41937 41958 translation stop site +T1157 SO:0000121 41960 41974 forward primer +T1158 SO:0001031 42021 42028 reverse +T1159 SO:0000006 42124 42135 PCR product +T1160 PR:000000281 42165 42169 Gdf5 +T1161 SO:0001026 42170 42177 genomic +T1162 SO:0000852 42198 42205;42216 42220 part of ... exon +T1163 SO:0001014 42198 42205;42236 42242 part of ... intron +T1164 SO:0000121 42248 42262 forward primer +T1165 SO:0000147 42296 42300 exon +T1166 SO:0000132 42337 42351 reverse primer +T1167 NCBITaxon:10760 42364 42366 T7 +T1168 SO:0000167 42367 42375 promoter +T1169 SO:0000440 42383 42389 vector +T1170 SO:0000357 42425 42431 flanks +T1171 SO:0000188 42436 42444 intronic +T1172 SO:0000121 42456 42470 forward primer +T1173 SO:0001031 42510 42517 reverse +T1174 SO:0000440 42719 42725 vector +T1175 SO:0000153 42880 42883 BAC +T1176 SO:0000153 42949 42952 BAC +T1177 NCBITaxon:33208 42988 42995 animals +T1178 SO:0000346 42999 43008 loxP site +T1179 SO:0000153 43024 43034 BAC vector +T1180 SO:0001026 43123 43129 genome +T1181 SO:0000153 43143 43146 BAC +T1182 CHEBI:63039 43167 43171 CsCl +T1183 SO:0000667 43215 43221 insert +T1184 SO:0000440 43231 43237 vector +T1185 CHEBI:17992 43268 43275 sucrose +T1186 SO:0000440 43365 43371 vector +T1187 SO:0000667 43428 43434 insert +T1188 SO:0000440 43460 43466 vector +T1189 CHEBI:9754 43517 43521 Tris +T1190 SO:0000667 43661 43667 insert +T1191 GO:0045120 43718 43728 pronucleus +T1192 GO:0009566 43732 43742 fertilized +T1193 CL:0000025 43743 43747 eggs +T1194 NCBITaxon:10088 43759 43763 mice +T1195 NCBITaxon:10088 43820 43824 mice +T1196 SO:0000112 43867 43874 primers +T1197 SO:0000028 43966 43968 bp +T1198 SO:0000153 44011 44021 BAC vector +T1199 SO:0000440 44028 44034 vector +T1200 SO:0000112 44044 44051 primers +T1201 SO:0000028 44138 44140 bp +T1202 PR:000000281 44165 44169 Gdf5 +T1203 NCBITaxon:10088 44174 44178 mice +T1204 GO:0007618 44234 44241 Matings +T1205 PR:000033987 44266 44270 LACZ +T1206 NCBITaxon:10088 44280 44284 mice +T1207 PR:000033987 44350 44354 LACZ +T1208 PR:P05187 44359 44364 HPLAP +T1209 UBERON:0000922 44374 44381 embryos +T1210 UBERON:0000922 44397 44404 embryos +T1211 PR:000033987 44482 44486 LACZ +T1212 PR:000000035 44652 44658 Bmpr1a +T1213 NCBITaxon:10088 44666 44670 mice +T1214 PR:000000035 44672 44678 Bmpr1a +T1215 SO:0000359 44688 44694 floxed +T1216 SO:0001023 44695 44702 alleles +T1217 NCBITaxon:10088 44829 44833 Mice +T1218 SO:0000359 44856 44862 floxed +T1219 SO:0001023 44863 44870 alleles +T1220 GO:0007618 44886 44891 mated +T1221 PR:000000281 44895 44899 Gdf5 +T1222 NCBITaxon:10088 44904 44908 mice +T1223 NCBITaxon:10088 44945 44949 mice +T1224 NCBITaxon:33208 45027 45034 animals +T1225 GO:0007618 45074 45081 matings +T1226 UBERON:0004288 45095 45103 skeletal +T1227 NCBITaxon:10088 45148 45152 mice +T1228 UBERON:0001690 45184 45188 ears +T1229 NCBITaxon:33208 45214 45221 animals +T1230 UBERON:0010887 45328 45334 tragus +T1231 UBERON:0016467 45339 45349 antitragus +T1232 UBERON:0001757 45391 45397 pinnae +T1233 NCBITaxon:10088 45428 45432 mice +T1234 NCBITaxon:33208 45457 45464 animals +T1235 NCBITaxon:10088 45621 45626 mouse +T1236 UBERON:0001448 45687 45689 MT +T1237 UBERON:0004302 45690 45692 P1 +T1238 UBERON:0004302 45697 45699 P1 +T1239 UBERON:4100005 45700 45702 P2 +T1240 UBERON:0004905 45703 45709 joints +T1241 UBERON:0003632 45717 45738 second hindlimb digit +T1242 NCBITaxon:33208 45765 45772 animals +T1243 UBERON:0004905 45804 45809 joint +T1244 NCBITaxon:33208 45996 46003 animals +T1245 NCBITaxon:10088 46026 46030 mice +T1246 PR:000000281 46109 46113 Gdf5 +T1247 PR:000000035 46122 46128 Bmpr1a +T1248 PR:000000035 46169 46175 Bmpr1a +T1249 SO:0000359 46175 46180 floxP +T1250 GO:0008219 46325 46335 Cell death +T1251 GO:0008283 46325 46329;46340 46353 Cell ... proliferation +T1252 UBERON:0002101 46362 46367 Limbs +T1253 NCBITaxon:33208 46392 46399 animals +T1254 UBERON:0000479 46514 46520 tissue +T1255 GO:0008219 46561 46571 Cell Death +T1256 CHEBI:31624 46587 46598 Fluorescein +T1257 CHEBI:53424 46697 46705 Tween-20 +T1258 NCBITaxon:9925 46711 46715 goat +T1259 UBERON:0001977 46716 46721 serum +T1260 NCBITaxon:9986 46778 46784 rabbit +T1261 CHEBI:32958 46790 46797 phospho +T1262 CHEBI:15358 46798 46805 histone +T1263 PR:000027594 46798 46808 histone-H3 +T1264 GO:0042571 46809 46817 antibody +T1265 GO:0007067 46825 46832 Mitosis +T1266 GO:0007067 46923 46930 mitosis +T1267 CHEBI:37987 46932 46935 Cy3 +T1268 NCBITaxon:9986 46949 46955 rabbit +T1269 GO:0042571 46966 46974 antibody +T1270 GO:0042571 46998 47006 antibody +T1271 GO:0005634 47008 47019 Cell nuclei +T1272 CHEBI:51231 47038 47042 DAPI +T1273 UBERON:0001062 47202 47212 anatomical +T1274 GO:0005634 47266 47273 nuclear +T1275 CHEBI:37987 47302 47305 Cy3 +T1276 GO:0005634 47314 47320 nuclei +T1277 NCBITaxon:33208 47412 47419 animals +T1278 UBERON:0003695 47456 47477;47504 47510 metacarpal-phalangeal ... joints +T1279 UBERON:0003696 47482 47510 metatarsal-phalangeal joints +T1280 UBERON:0004905 47586 47591 joint +T1281 UBERON:0006052 47621 47632 fifth digit +T1282 UBERON:0009551 47672 47678;47683 47688 tip of ... digit +T1283 UBERON:0000479 47740 47746 tissue +T1284 UBERON:0000479 47837 47843 Tissue +T1285 NCBITaxon:33208 47849 47856 animals +T1286 CHEBI:17992 48050 48057 sucrose +T1287 CHEBI:17992 48089 48096 sucrose +T1288 UBERON:0000479 48168 48174 Tissue +T1289 NCBITaxon:33208 48180 48187 animals +T1290 CHEBI:17992 48333 48340 sucrose +T1291 CHEBI:75958 48346 48355 solutions +T1292 UBERON:0000479 48419 48426 tissues +T1293 NCBITaxon:10088 48442 48446 mice +T1294 UBERON:0000479 48494 48500 Tissue +T1295 CHEBI:82468 48586 48596 Fast Green +T1296 CHEBI:51686 48610 48621 hematoxylin +T1297 PR:000033987 48691 48695 LACZ +T1298 CHEBI:75055 48710 48715 X-Gal +T1299 CHEBI:15377 48825 48830 water +T1300 GO:0005634 48853 48860 Nuclear +T1301 CHEBI:15377 48898 48903 water +T1302 GO:0097617 49011 49024 hybridization +T1303 MOP:0000030 49130 49141 acetylation +T1304 GO:0097617 49350 49363 hybridization +T1305 UBERON:0005291 49390 49406 embryonic tissue +T1306 CHEBI:60004 49451 49454 mix +T1307 SO:0000704 49500 49505 genes +T1308 PR:000000035 49538 49544 Bmpr1a +T1309 PR:000003266 49568 49574 Col2a1 +T1310 PR:000005693 49601 49608 Col10a1 +T1311 PR:000000281 49629 49633 Gdf5 +T1312 PR:000030444 49661 49672 Osteocalcin +T1313 PR:000015431 49700 49704 Sox5 +T1314 PR:000015432 49709 49713 Sox6 +T1315 PR:000003606 49780 49783 Agg +T1316 PR:000000164 49822 49826 Bmp2 +T1317 PR:000000165 49831 49835 Bmp4 +T1318 PR:000003264 49871 49877 Col1a1 +T1319 PR:000000036 49916 49922 Bmpr1b +T1320 PR:000003328 49924 49930 Col3a1 +T1321 PR:000010208 49936 49940 Mat4 +T1322 SO:0000345 49963 49967 ESTs +T1323 NCBITaxon:27592 50187 50193 bovine +T1324 CHEBI:16240 50368 50385 hydrogen peroxide +T1325 CHEBI:17790 50394 50402 methanol +T1326 CHEBI:53424 50448 50455 Tween20 +T1327 NCBITaxon:9925 50461 50465 goat +T1328 NCBITaxon:27592 50475 50481 bovine +T1329 UBERON:0001977 50482 50487 serum +T1330 GO:0042571 50530 50540 antibodies +T1331 CHEBI:53424 50556 50564 Tween 20 +T1332 NCBITaxon:9925 50570 50574 goat +T1333 NCBITaxon:27592 50584 50590 bovine +T1334 UBERON:0001977 50591 50596 serum +T1335 CHEBI:15956 50616 50622 Biotin +T1336 GO:0042571 50641 50651 antibodies +T1337 GO:0042571 50790 50800 antibodies +T1338 NCBITaxon:9925 50826 50830 goat +T1339 NCBITaxon:10088 50836 50841 mouse +T1340 PR:000010473 50842 50847 MMP13 +T1341 NCBITaxon:9986 50918 50924 rabbit +T1342 NCBITaxon:9606 50930 50935 human +T1343 PR:000015435 50936 50940 SOX9 +T1344 NCBITaxon:9986 50979 50985 rabbit +T1345 PR:000015435 51006 51010 SOX9 +T1346 PR:000015435 51012 51016 SOX9 +T1347 SO:0000704 51166 51171 genes +T1348 PR:000000281 51200 51204 Gdf5 +T1349 PR:000000035 51220 51226 Bmpr1a +T1350 SO:0000155 51303 51310 plasmid +T1351 SO:0000243 51344 51348 IRES +T1352 PR:P05187 51349 51354 hPLAP +T1353 SO:0000155 51366 51373 plasmid +T1354 GO:0042571 51423 51433 antibodies +T1355 NCBITaxon:1 51449 51460 individuals +T1356 PR:000000164 51520 51524 Bmp2 +T1357 PR:000000165 51529 51533 Bmp4 +T1358 PR:000003264 51572 51578 Col1a1 +T1359 PR:000015431 51604 51608 Sox5 +T1360 PR:000015432 51613 51617 Sox6 +T1361 NCBITaxon:10088 51710 51714 mice +T1362 UBERON:0007616 51757 51765 synovial +T1363 http://purl.obolibrary.org/obo/MONDO_0005578 52009 52018 Arthritis +T1364 SO:0000153 52190 52193 BAC +T1365 NCBITaxon:2 52196 52205 bacterial +T1366 SO:0000153 52196 52227 bacterial artificial chromosome +T1367 PR:000000035 52229 52235 Bmpr1a +T1368 GO:0060349 52238 52256 bone morphogenetic +T1369 PR:000000035 52238 52276 bone morphogenetic protein receptor 1a +T1370 UBERON:0000922 52278 52279 E +T1371 UBERON:0000922 52290 52298 embyonic +T1372 GO:0031012 52313 52316 ECM +T1373 GO:0031012 52319 52339 extracellular matrix +T1374 PR:000000281 52372 52376 Gdf5 +T1375 PR:000000281 52413 52417 Gdf5 +T1376 PR:000000281 52420 52451 growth differentiation factor 5 +T1377 PR:P05187 52453 52458 hPLAP +T1378 NCBITaxon:9606 52461 52466 human +T1379 UBERON:0001987 52467 52476 placental +T1380 PR:000003969 52467 52497 placental alkaline phosphatase +T1381 SO:0000243 52499 52503 IRES +T1382 SO:0000243 52506 52534 internal ribosome entry site +T1383 GO:0005840 52515 52523 ribosome +T1384 PR:000033987 52567 52571 lacZ +T1385 PR:000000046 52600 52605 TGF-β +T1386 PR:000000046 52608 52636 transforming growth factor β +T1387 SO:0000704 52770 52777 Genetic +T1388 SO:0000704 52794 52798 Gene +T1389 UBERON:0004905 52827 52833 Joints +T1390 SO:0000153 52848 52851 BAC +T1391 PR:000000281 52861 52865 Gdf5 +T1392 SO:0000243 52902 52906 IRES +T1393 PR:P05187 52907 52912 hPLAP +T1394 GO:0006413 52922 52939 translation start +T1395 SO:0000318 52922 52944 translation start site +T1396 PR:000000281 52948 52952 Gdf5 +T1397 NCBITaxon:10088 52981 52985 mice +T1398 PR:000000281 53064 53068 Gdf5 +T1399 PR:000033987 53126 53130 lacZ +T1400 GO:0010467 53131 53141 expression +T1401 SO:0001023 53169 53175 allele +T1402 PR:000033987 53181 53185 LACZ +T1403 UBERON:0001690 53230 53233 ear +T1404 UBERON:0001690 53235 53237 ea +T1405 UBERON:0016884 53247 53256;53261 53269 joints of ... shoulder +T1406 UBERON:0001490 53247 53256;53275 53280 joints of ... elbow +T1407 UBERON:0001491 53247 53256;53287 53292 joints of ... wrist +T1408 UBERON:0001485 53247 53256;53298 53302 joints of ... knee +T1409 UBERON:0001488 53247 53253;53308 53313 joints ... ankle +T1410 UBERON:0016884 53271 53272 s +T1411 UBERON:0001490 53282 53284 eb +T1412 UBERON:0001491 53294 53295 w +T1413 UBERON:0001485 53304 53305 k +T1414 UBERON:0001488 53315 53316 a +T1415 UBERON:0002412 53319 53327 vertebra +T1416 UBERON:0003221 53338 53347 phalanges +T1417 NCBITaxon:10088 53379 53384 mouse +T1418 UBERON:0000922 53385 53391 embryo +T1419 UBERON:0002103 53403 53411 hindlimb +T1420 PR:P05187 53440 53445 HPLAP +T1421 GO:0010467 53446 53456 expression +T1422 SO:0000902 53466 53475 transgene +T1423 PR:000033987 53503 53507 LACZ +T1424 GO:0010467 53508 53518 expression +T1425 SO:0001023 53544 53550 allele +T1426 UBERON:0009767 53618 53648 proximal interphalangeal joint +T1427 PR:P05187 53673 53678 HPLAP +T1428 UBERON:0006658 53734 53755 interphalangeal joint +T1429 PR:P05187 53783 53788 HPLAP +T1430 PR:000033987 53793 53797 LACZ +T1431 GO:0010467 53798 53808 expression +T1432 UBERON:0004905 53849 53854 joint +T1433 UBERON:0002544 53862 53867 digit +T1434 UBERON:0002102 53904 53912 forelimb +T1435 PR:000033987 53949 53953 LACZ +T1436 GO:0010467 53963 53972 expressed +T1437 UBERON:0003221 53980 53990 phalangeal +T1438 UBERON:0004905 53991 53997 joints +T1439 UBERON:0000043 54062 54069 tendons +T1440 UBERON:0003221 54118 54128 phalangeal +T1441 UBERON:0004905 54129 54134 joint +T1442 UBERON:0002103 54143 54151 hindlimb +T1443 PR:000033987 54203 54207 LACZ +T1444 GO:0010467 54208 54218 expression +T1445 UBERON:0000479 54240 54247 tissues +T1446 UBERON:0004905 54262 54268 joints +T1447 UBERON:0010996 54270 54289 articular cartilage +T1448 UBERON:0000211 54323 54332 ligaments +T1449 PR:000000035 54434 54440 Bmpr1a +T1450 GO:0060033 54465 54475 Regression +T1451 GO:0006915 54480 54489 Apoptosis +T1452 UBERON:0002101 54517 54521 Limb +T1453 UBERON:0002102 54547 54555 forelimb +T1454 UBERON:0002102 54588 54596 forelimb +T1455 UBERON:0006015 54609 54631 webbing between digits +T1456 UBERON:0006048 54625 54633 digits 1 +T1457 UBERON:0006049 54625 54631;54638 54639 digits ... 2 +T1458 UBERON:0000479 54663 54669 tissue +T1459 UBERON:0006052 54690 54697 digit 5 +T1460 PR:000000281 54713 54717 Gdf5 +T1461 PR:000033987 54730 54734 lacZ +T1462 GO:0010467 54735 54745 expression +T1463 UBERON:0002102 54768 54776 forelimb +T1464 PR:000033987 54785 54789 LACZ +T1465 UBERON:0003695 54809 54837 metacarpal-phalangeal joints +T1466 UBERON:0006048 54847 54855 digits 1 +T1467 UBERON:0006049 54847 54853;54860 54861 digits ... 2 +T1468 UBERON:0006052 54904 54911 digit 5 +T1469 UBERON:0002103 54950 54959 hindlimbs +T1470 GO:0006915 54968 54977 apoptosis +T1471 GO:0008283 55019 55032 proliferation +T1472 CHEBI:15358 55060 55067 histone +T1473 PR:000027594 55060 55070 histone H3 +T1474 UBERON:0006048 55147 55155 digits 1 +T1475 UBERON:0006049 55147 55153;55160 55161 digits ... 2 +T1476 GO:0007067 55236 55243 mitotic +T1477 GO:0007067 55334 55341 mitotic +T1478 UBERON:0006052 55379 55390 fifth digit +T1479 GO:0006915 55397 55406 apoptosis +T1480 GO:0008283 55428 55441 proliferation +T1481 UBERON:0006012 55539 55551 interdigital +T1482 UBERON:0000479 55552 55558 tissue +T1483 GO:0060033 55563 55572 regressed +T1484 UBERON:0000479 55614 55620 tissue +T1485 PR:000000281 55707 55711 Gdf5 +T1486 PR:000000035 55756 55762 Bmpr1a +T1487 GO:0010467 55786 55796 expression +T1488 PR:000033987 55800 55804 LACZ +T1489 PR:000000281 55840 55844 Gdf5 +T1490 PR:000000035 55870 55876 Bmpr1a +T1491 PR:000000035 55923 55929 Bmpr1a +T1492 SO:0000359 55929 55934 floxP +T1493 PR:000000281 55963 55967 Gdf5 +T1494 PR:000033987 55998 56002 lacZ +T1495 GO:0010467 56003 56013 expression +T1496 NCBITaxon:10088 56062 56066 mice +T1497 CHEBI:16866 56114 56126 alizarin red +T1498 UBERON:0004454 56145 56150 Ankle +T1499 UBERON:0004905 56200 56205 joint +T1500 UBERON:0004454 56224 56229 Ankle +T1501 UBERON:0004905 56311 56316 joint +T1502 UBERON:0001447 56363 56370 tarsals +T1503 UBERON:0003696 56407 56434 metatarsal/phalangeal joint +T1504 UBERON:0004905 56464 56473 articular +T1505 UBERON:0002516 56523 56535 growth plate +T1506 UBERON:0002102 56562 56570 forelimb +T1507 UBERON:0002102 56584 56592 forelimb +T1508 UBERON:0006015 56598 56613;56635 56640 webbing between ... digit +T1509 UBERON:0006048 56618 56623;56635 56640 first ... digit +T1510 UBERON:0006049 56628 56640 second digit +T1511 PR:000000035 56671 56677 Bmpr1a +T1512 GO:0010467 56681 56690 Expressed +T1513 UBERON:0004905 56694 56700 Joints +T1514 UBERON:0004905 56731 56736 Joint +T1515 UBERON:0004454 56754 56766 Ankle Region +T1516 UBERON:0001447 56783 56794 ankle bones +T1517 NCBITaxon:10088 56812 56817 mouse +T1518 UBERON:0001474 56819 56824 bones +T1519 UBERON:0001448 56881 56892 metatarsals +T1520 UBERON:0001447 56923 56935 tarsal bones +T1521 UBERON:0001447 56948 56959 tarsal bone +T1522 UBERON:0002395 56961 56963 ta +T1523 UBERON:0002395 56965 56970 talus +T1524 UBERON:0001450 56972 56974 ca +T1525 UBERON:0001450 56976 56985 calcaneus +T1526 GO:0097617 57006 57019 hybridization +T1527 PR:000000035 57042 57048 Bmpr1a +T1528 GO:0010467 57052 57061 expressed +T1529 UBERON:0001488 57065 57076 ankle joint +T1530 UBERON:0004905 57123 57132 articular +T1531 UBERON:0003221 57148 57158 phalangeal +T1532 UBERON:0004905 57159 57165 joints +T1533 PR:000000281 57225 57229 Gdf5 +T1534 PR:000033987 57242 57246 LACZ +T1535 GO:0010467 57247 57257 expression +T1536 UBERON:0004905 57283 57289 joints +T1537 UBERON:0002544 57297 57303 digits +T1538 SO:0000704 57332 57336 gene +T1539 GO:0010467 57332 57347 gene expression +T1540 PR:000033987 57357 57361 LACZ +T1541 UBERON:0000922 57428 57435 embryos +T1542 NCBITaxon:10088 57448 57452 mice +T1543 UBERON:0001488 57462 57474 ankle joints +T1544 PR:000003266 57534 57538 Col2 +T1545 GO:0010467 57544 57551 express +T1546 PR:000000281 57552 57556 Gdf5 +T1547 GO:0010467 57577 57584 express +T1548 PR:000033987 57585 57589 LACZ +T1549 UBERON:0000922 57654 57661 embryos +T1550 UBERON:0004905 57681 57686 joint +T1551 PR:000003266 57718 57722 Col2 +T1552 GO:0010467 57723 57733 expression +T1553 UBERON:0001447 57776 57782 tarsal +T1554 UBERON:0003651 57790 57803 metatarsal II +T1555 PR:000000281 57830 57834 Gdf5 +T1556 GO:0010467 57835 57845 expression +T1557 UBERON:0004905 57885 57890 joint +T1558 UBERON:0001447 57938 57945 tarsals +T1559 GO:0010467 57963 57970 express +T1560 PR:000003266 57971 57975 Col2 +T1561 UBERON:0004905 57994 57999 joint +T1562 GO:0010467 58041 58051 expression +T1563 PR:000000281 58055 58059 Gdf5 +T1564 UBERON:0004288 58075 58083 skeletal +T1565 PR:000000035 58170 58176 Bmpr1a +T1566 GO:0010467 58201 58211 Expression +T1567 GO:0044420 58215 58229 ECM Components +T1568 UBERON:0011002 58233 58252 Articular Cartilage +T1569 GO:0097617 58262 58275 hybridization +T1570 PR:000033987 58279 58283 LACZ +T1571 UBERON:0003695 58322 58350 metacarpal-phalangeal joints +T1572 UBERON:0001447 58373 58379 tarsal +T1573 UBERON:0003651 58382 58395 metatarsal II +T1574 UBERON:0004905 58396 58401 joint +T1575 NCBITaxon:10088 58422 58426 mice +T1576 GO:0007567 58431 58436 birth +T1577 UBERON:0011002 58438 58457 articular cartilage +T1578 PR:000003266 58548 58552 Col2 +T1579 GO:0010467 58553 58563 expression +T1580 PR:000010208 58572 58576 Mat4 +T1581 GO:0010467 58577 58587 expression +T1582 UBERON:0011002 58602 58621 articular cartilage +T1583 PR:000033987 58676 58680 LACZ +T1584 GO:0010467 58681 58691 expression +T1585 UBERON:0011002 58744 58763 articular cartilage +T1586 UBERON:0003695 58818 58846 metacarpal-phalangeal joints +T1587 NCBITaxon:10088 58854 58858 mice +T1588 GO:0007567 58876 58881 birth +T1589 UBERON:0011002 58883 58902 articular cartilage +T1590 GO:0010467 58977 58986 expresses +T1591 PR:000003266 58987 58991 Col2 +T1592 PR:000003606 58997 59000 Agg +T1593 PR:000015435 59010 59014 SOX9 +T1594 UBERON:0004905 59040 59049 articular +T1595 GO:0010467 59149 59159 expression +T1596 PR:000003266 59163 59167 Col2 +T1597 PR:000003606 59176 59179 Agg +T1598 PR:000015435 59213 59217 SOX9 +T1599 UBERON:0004905 59273 59282 articular +T1600 PR:000033987 59294 59298 LACZ +T1601 GO:0010467 59299 59309 expression +T1602 UBERON:0004905 59362 59371 articular +T1603 UBERON:0004905 59469 59478 Articular +T1604 UBERON:0004454 59550 59556 Ankles +T1605 PR:000000035 59560 59566 Bmpr1a +T1606 NCBITaxon:10088 59574 59578 Mice +T1607 UBERON:0001447 59612 59618 tarsal +T1608 UBERON:0003651 59621 59634 metatarsal II +T1609 UBERON:0004905 59635 59640 joint +T1610 NCBITaxon:10088 59652 59656 mice +T1611 PR:000033987 59668 59672 LACZ +T1612 UBERON:0004905 59747 59756 articular +T1613 UBERON:0007616 59774 59782 synovial +T1614 CL:0000214 59774 59788 synovial cells +T1615 GO:0097617 59849 59862 hybridization +T1616 PR:000005693 59869 59874 Col10 +T1617 GO:0010467 59875 59885 expression +T1618 UBERON:0004905 59955 59964 articular +T1619 UBERON:0004905 60047 60052 joint +T1620 PR:000033987 60071 60075 LACZ +T1621 GO:0010467 60076 60086 expressing +T1622 UBERON:0004905 60100 60109 articular +T1623 PR:000005693 60146 60151 Col10 +T1624 PR:000005693 60192 60197 Col10 +T1625 GO:0010467 60198 60208 expression +T1626 UBERON:0001484 60344 60357 joint capsule +T1627 UBERON:0004905 60378 60383 joint +T1628 PR:000000035 60521 60527 Bmpr1a +T1629 UBERON:0011002 60547 60566 Articular Cartilage +T1630 UBERON:0002544 60600 60606 Digits +T1631 UBERON:0001465 60611 60616 Knees +T1632 GO:0007568 60620 60625 Aging +T1633 NCBITaxon:10088 60626 60630 Mice +T1634 UBERON:0003696 60664 60692 metatarsal-phalangeal joints +T1635 NCBITaxon:10088 60710 60714 mice +T1636 UBERON:0011002 60716 60735 Articular cartilage +T1637 UBERON:0004905 60828 60837 articular +T1638 PR:000033987 60948 60952 LACZ +T1639 GO:0010467 60953 60963 expression +T1640 UBERON:0004905 61016 61025 articular +T1641 UBERON:0001485 61076 61087 knee joints +T1642 NCBITaxon:33208 61115 61122 animals +T1643 UBERON:0000981 61130 61132 fe +T1644 UBERON:0000981 61134 61139 femur +T1645 UBERON:0000979 61141 61143 ti +T1646 UBERON:0000979 61145 61150 tibia +T1647 UBERON:0002516 61152 61154 gp +T1648 UBERON:0002516 61156 61168 growth plate +T1649 GO:0007567 61188 61193 birth +T1650 UBERON:0004383 61213 61229 tibial epiphysis +T1651 UBERON:0004905 61279 61288 articular +T1652 PR:000033987 61438 61442 LACZ +T1653 UBERON:0004905 61503 61512 articular +T1654 PR:000033987 61552 61556 LACZ +T1655 PR:000000035 61636 61642 Bmpr1a +T1656 UBERON:0004383 61722 61738 tibial epiphysis +T1657 UBERON:0004905 61782 61791 articular +T1658 PR:000033987 61877 61881 LACZ +T1659 UBERON:0004905 61936 61945 articular +T1660 UBERON:0004755 61990 62005 skeletal tissue +T1661 UBERON:0000387 62050 62058 meniscal +T1662 UBERON:0001437 62140 62149 epiphyses +T1663 CHEBI:33893 62517 62525 reagents +T1664 CHEBI:52217 62688 62703 Pharmaceuticals +T1665 PR:000000034 62816 62819 BMP +T1666 GO:0030509 62816 62838 BMP receptor signaling +T1667 GO:0007567 62859 62864 natal +T1668 UBERON:0011002 62880 62899 articular cartilage diff --git a/src/ontogpt/evaluation/craft/database/all/15492776.txt b/src/ontogpt/evaluation/craft/database/all/15492776.txt new file mode 100644 index 000000000..6167b1a00 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15492776.txt @@ -0,0 +1,243 @@ +BMP Receptor Signaling Is Required for Postnatal Maintenance of Articular Cartilage + +Abstract + +Articular cartilage plays an essential role in health and mobility, but is frequently damaged or lost in millions of people that develop arthritis. The molecular mechanisms that create and maintain this thin layer of cartilage that covers the surface of bones in joint regions are poorly understood, in part because tools to manipulate gene expression specifically in this tissue have not been available. Here we use regulatory information from the mouse Gdf5 gene (a bone morphogenetic protein [BMP] family member) to develop new mouse lines that can be used to either activate or inactivate genes specifically in developing joints. Expression of Cre recombinase from Gdf5 bacterial artificial chromosome clones leads to specific activation or inactivation of floxed target genes in developing joints, including early joint interzones, adult articular cartilage, and the joint capsule. We have used this system to test the role of BMP receptor signaling in joint development. Mice with null mutations in Bmpr1a are known to die early in embryogenesis with multiple defects. However, combining a floxed Bmpr1a allele with the Gdf5-Cre driver bypasses this embryonic lethality, and leads to birth and postnatal development of mice missing the Bmpr1a gene in articular regions. Most joints in the body form normally in the absence of Bmpr1a receptor function. However, articular cartilage within the joints gradually wears away in receptor-deficient mice after birth in a process resembling human osteoarthritis. Gdf5-Cre mice provide a general system that can be used to test the role of genes in articular regions. BMP receptor signaling is required not only for early development and creation of multiple tissues, but also for ongoing maintenance of articular cartilage after birth. Genetic variation in the strength of BMP receptor signaling may be an important risk factor in human osteoarthritis, and treatments that mimic or augment BMP receptor signaling should be investigated as a possible therapeutic strategy for maintaining the health of joint linings. + +Introduction + +Thin layers of articular cartilage line the bones of synovial joints and provide a smooth, wear-resistant structure that reduces friction and absorbs impact forces (Brandt et al. 1998). Loss or damage to articular cartilage is a hallmark of arthritic diseases and is one of the most common reasons that both young and old adults seek medical care. Millions of people are afflicted with arthritis, and it ultimately affects more than half of people over the age of 65 (Badley 1995; Yelin and Callahan 1995). A better understanding of the molecular mechanisms that create and maintain articular cartilage is crucial for discovering the causes of joint disorders and providing useful medical treatments. + +Joint formation begins during embryogenesis, when stripes of high cell density called interzones form across developing skeletal precursors (Haines 1947). Programmed cell death occurs within the interzone, and a three-layered interzone forms that has two layers of higher cell density flanking a region of lower cell density. Non-joint precursors of the skeleton typically develop into cartilage, which hypertrophies and is replaced by bone. However, cells within the high-density layers of the interzone are excluded from this process and develop into the permanent layers of articular cartilage found in the mature joint (Mitrovic 1978). + +Studies over the last 10 y have begun to elucidate some of the signaling pathways that contribute to the early stages of joint formation. Wnt14 is expressed in stripes at the sites where joints will form, and it is capable of inducing expression of other joint markers when misexpressed at new locations in the limb (Hartmann and Tabin 2001). Several members of the bone morphogenetic protein (BMP) family of secreted signaling molecules are also expressed in stripes at sites where joints will form, including those encoded by the genes Gdf5, Gdf6, Gdf7, Bmp2, and Bmp4 (Storm and Kingsley 1996; Wolfman et al. 1997; Francis-West et al. 1999; Settle et al. 2003). Of these, Gdf5 expression is most strikingly limited to regions where joints will develop and is one of the earliest known markers of joint formation. Mutations in either Gdf5 or the closely related Gdf6 gene also block formation of joints at specific locations, providing strong evidence that these molecules are essential for the joint formation process (Storm et al. 1994; Settle et al. 2003). However, mutations in Bmp2 or Bmp4 cause early embryonic lethality, making it difficult to test their role in joint formation (Winnier et al. 1995; Zhang and Bradley 1996). + +Much less is known about how signaling pathways function during the subsequent maturation and maintenance of adult joint structures. Importantly, BMP signaling components are present in adult articular cartilage, suggesting that they may function during the late development or maintenance of this critical structure (Erlacher et al. 1998; Chubinskaya et al. 2000; Muehleman et al. 2002; Bau et al. 2002; Bobacz et al. 2003). + +BMPs bind tetrameric complexes of two type I and two type II transmembrane serine-threonine kinase receptors. Upon BMP binding, these complexes transduce a signal by phosphorylating members of the Smad family of transcription factors (Massague 1996). Recent experiments have implicated two different BMP type I receptors in skeletal patterning, BMPR1A and BMPR1B. Both receptors can bind BMP2, BMP4, and GDF5, although GDF5 shows higher affinity for BMPR1B (Koenig et al. 1994; ten Dijke et al. 1994; Yamaji et al. 1994; Nishitoh et al. 1996; Chalaux et al. 1998). Both receptors are also expressed in dynamic patterns during normal development. In limbs, Bmpr1a expression becomes restricted to joint interzones, perichondrium, periarticular cartilage, hypertrophic chondrocytes, and interdigital limb mesenchyme. In comparison, Bmpr1b expression is seen primarily in condensing precartilaginous mesenchymal cells, regions flanking joint interzones, perichondrium, and periarticular cartilage (Dewulf et al. 1995; Mishina et al. 1995; Zou et al. 1997; Baur et al. 2000). Null mutations in the Bmpr1b gene produce viable mice with defects in bone and joint formation that closely resemble those seen in mice missing Gdf5 (Storm and Kingsley 1996; Baur et al. 2000; Yi et al. 2000). Null mutations in Bmpr1a cause early embryonic lethality, with defects in gastrulation similar to those seen in mice with mutations in Bmp4 (Mishina et al. 1995; Winnier et al. 1995). Recent studies with floxed alleles suggest that Bmpr1a is also required for many later developmental events, but its roles in bone and joint formation have not yet been tested (Mishina 2003). + +A genetic system for activating or inactivating genes specifically in joint tissues would be particularly useful for further studies of joint formation and maintenance. Here we take advantage of the tissue-specific expression pattern of the Gdf5 gene to engineer a Cre/loxP system (Nagy 2000), Gdf5-Cre, that can be used to remove or ectopically express genes in joints. Tests with reporter mice show that this system is capable of modifying genes in all of the structures of the mature synovial joint, including the ligaments of the joint capsule, the synovial membrane, and the articular cartilage. Gdf5-Cre recombination bypasses the early embryonic lethality of null mutations in Bmpr1a, and shows that this receptor is required for early joint formation at some locations and for initiation of programmed cell death in webbing between digits. Interestingly, Bmpr1a is also required for postnatal maintenance of articular cartilage throughout most of the skeleton. In Gdf5-Cre/Bmpr1afloxP mice, articular cartilage initially forms normally, but subsequently loses expression of several key cartilage markers after birth. It ultimately fibrillates and degenerates, resulting in severe osteoarthritis and loss of mobility. These experiments suggest that BMP signaling is required for normal maintenance of postnatal articular cartilage, and that modulation of the BMP signaling pathway may play an important role in joint disease. + +Results + +Genetic System for Testing the Function of Genes in Joint Development + +To generate a general system capable of specifically testing genes for functions in skeletal joint development, we engineered transgenic mice to express Cre recombinase in developing joints (Figure 1). Gdf5 is a gene strongly expressed in stripes across developing skeletal elements during embryonic joint formation. A bacterial artificial chromosome (BAC) containing the Gdf5 locus was modified by homologous recombination in bacteria to insert a cassette encoding Cre-internal ribosome entry site (IRES)-human placental alkaline phosphatase (hPLAP) into the translation start site of Gdf5 (Figure 1A). This modified BAC was then used to make lines of transgenic mice. The resulting Gdf5-Cre transgenic mice were tested for transgene expression and Cre recombinase activity by crossing them to R26R reporter mice that activate the expression of lacZ after Cre-mediated removal of transcriptional stop sequences (Soriano 1999). The resulting progeny were analyzed both for expression of the transgene by assaying HPLAP activity and for recombination of DNA by assaying LACZ activity. The progeny from all three lines showed strong LACZ expression primarily in joints, and in two of three lines HPLAP expression could also be seen in joint regions. Interestingly, HPLAP expression in the Gdf5-Cre transgenic GAC(A) line used for all subsequent breeding experiments was seen to precede LACZ expression during successive development of joints in the digits (Figure 1C) (unpublished data). These experiments clearly demonstrate that the Gdf5-Cre transgene expresses Cre recombinase and causes DNA recombination in developing joint regions. + +GAC(A) mice were crossed with lacZ ROSA26 Cre reporter strain (R26R) mice to analyze the pattern of Cre-mediated lacZ recombination throughout development. Joints in developing limbs begin forming in a proximal-distal pattern such that the shoulder joint forms prior to the elbow joint. In addition, three major stages of early joint development have been defined by histology as (1) interzone formation, (2) three-layer interzone formation, and (3) cavitation (Mitrovic 1978). Consistent with the proximal-distal pattern of joint development in the limbs, LACZ activity is seen at embryonic day 12.5 (E12.5) in the more proximal joints, including the shoulder and knee (unpublished data). By E14.5, LACZ expression is typically seen in all but the most distal joints of the limbs (Figure 1B and 1C), but with some variability in both strength and extent of expression from embryo to embryo. The strongest-staining embryos often have additional staining in fingertips (not seen in the E14.5 embryo in Figure 1C, but clearly detectable in the E13.5 embryo shown in Figure 2). Sections through developing joints show that LACZ is present in many cells at the interzone stage (unpublished data). However, expression of LACZ in nearly 100% of joint cells is not achieved until the three-layer interzone stage (for example, in the knee joint at E14.5 or in any of the phalangeal joints at E16.5 (unpublished data). Within the developing skeleton, Cre-mediated expression of LACZ remains strikingly specific to joints throughout development. Furthermore, it is seen in all the structures of postnatal synovial joints including the articular cartilage, joint capsule, and synovial membrane (Figure 1D and 1E) (unpublished data). These patterns are consistent with the well-established expression of Gdf5 in interzone regions during embryonic development (Storm and Kingsley 1996). Adult expression patterns of the Gdf5 gene are not as well characterized, but Gdf5 expression has previously been detected in adult articular cartilage using both RT-PCR and immunocytochemistry (Chang et al. 1994; Erlacher et al. 1998; Bobacz et al. 2002). + +Other sites besides limb joints also have Cre-mediated lacZ expression. Starting at E13.5, LACZ activity is detected in an anterior and posterior domain of the limb bud (Figure 2C). At E14.5, LACZ activity is detectable in the developing ear pinnae, ribs, sternum, tissues in the face, and some regions of the brain and spinal cord (Figure 1B) (unpublished data). At birth, LACZ is also expressed in tendons running along the vertebral column, regions of tendons in the wrist and ankle, and some tendon insertions (Figure 1D) (unpublished data). By 5 wk of age, LACZ is also expressed in the hair follicles, ear cartilage, some cells in the growth plate of the long bones, and portions of the brain and spinal cord (unpublished data). Surprisingly, 23 of 63, or 37% of transgenic mice analyzed also show some degree of wider “ectopic” LACZ expression, which can extend throughout many different tissues in the animal. However, sustained expression of the transgene itself, as assayed by HPLAP activity, is still restricted primarily to joints in animals that show evidence of more generalized recombination based on LACZ expression (unpublished data). This suggests that in a fraction of animals, sporadic expression of Cre at some time early in development is sufficient to lead to both ectopic recombination and LACZ expression. While the fraction of animals with broader recombination patterns must be tracked and accounted for during experiments, these animals offer the potential benefit of revealing additional new functions of target genes that could be subsequently studied with additional site-specific Cre drivers. + +Gdf5-Cre/Bmpr1afloxP Animals Survive to Adulthood with Ear, Webbing, and Joint Defects + +We next used the Gdf5-Cre system to test the role of BMP signaling during normal joint development. Gdf5-Cre transgenic mice were bred to animals carrying a conditional floxed allele of the Bmpr1a locus (Mishina et al. 2002), usually in the presence of the R26R reporter allele to facilitate simultaneous visualization of Cre-mediated recombination patterns (see typical cross in Figure 3). PCR amplification confirmed that a key exon of the Bmpr1a gene was deleted in mice that also carried the Gdf5-Cre transgene (unpublished data). Previous studies have shown that the recombined Bmpr1afloxP allele mimics a null allele of the Bmpr1a locus when transmitted through the germline (Mishina et al. 2002). The Gdf5-Cre/Bmpr1afloxP conditional knockout mice were viable and survived to adulthood, showing that the Gdf5-Cre driver can bypass the early embryonic lethality previously reported in animals with a null mutation in the Bmpr1a locus (Mishina et al. 1995). + +The viable Gdf5-Cre/Bmpr1afloxP mice showed several phenotypes. First, the conditional knockout mice had shorter ears that often lay flatter against their heads than controls (controls 13.1 ± 0.1 mm long, n = 38; mutants 11.8 ± 0.2 mm, n = 11; p < 0.0001). BMP signaling is known to be required for growth of the external ear of mice (Kingsley et al. 1992), and this phenotype likely reflects loss of Bmpr1a function in the fraction of ear cells that express the Gdf5-Cre transgene. Most mutant mice also showed soft tissue syndactyly or retention of webbing between the first and second digits of their feet, a phenotype that was more frequent and more severe in the forelimbs (201 of 220, or 91%, of forefeet and 109 of 220, or 50%, of hindfeet). Finally, mutant animals showed obvious skeletal changes in whole-mount skeletal preparations. At some sites in the ankles, joints seemed to be missing entirely, with fusion of bones that would normally be separate. For example, the second distal tarsal was fused to the central tarsal bone in every conditional knockout animal examined (18 of 18), a phenotype not observed in controls (zero of 18) (Figure 3B and 3C). At other locations, joints had clearly formed but showed dramatic loss of staining with the cartilage matrix marker Alcian blue (Figure 3B–3E) (unpublished data). Normal Alcian blue staining was seen in non-articular regions, such as the cartilaginous growth plate (Figure 3D and 3E, asterisk). These data suggest that Bmpr1a function is required for the formation of specific joints in the ankle region and for either generation or maintenance of articular cartilage in most other joints of the limb. + +Developmental Origin of Webbing Phenotype + +Interdigital mesenchyme is normally eliminated by apoptosis during embryonic development, a process that can be stimulated by BMP beads, inhibited by Noggin, or blocked by overexpression of dominant-negative BMP receptors (Garcia-Martinez et al. 1993; Yokouchi et al. 1996; Zou and Niswander 1996; Guha et al. 2002). Limbs of Gdf5-Cre/Bmpr1afloxP mutant embryos showed obvious retention of interdigital webbing between the first and second, but not other, digits of E14.5 forelimbs (Figure 2A and 2B), a pattern that corresponds to the presence or absence of webbing seen in the adult limb. They also showed excess tissue on the posterior margin of the fifth digit (Figure 2B, arrow). Analysis of LACZ expression in Gdf5-Cre/R26R reporter embryos showed that Cre-mediated recombination has occurred by E13.5 in the metacarpal-phalangeal joints, and in the interdigital region between the first and second, but not other, digits. In addition, a domain of recombination and expression of LACZ is also reproducibly seen in the posterior half of the fifth digit (Figure 2C). Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining of interdigital mesenchyme between the first and second digits (Figure 2D and 2E) and the fifth digit flanking mesenchyme showed a decreased number of dying cells in the regions where excess tissue is retained in the mutant limbs. Numbers of phosphorylated histone H3-labeled proliferating cells were also elevated in these regions (Figure 2F). Most cells found in the webbed region between the first and second digits at E15.5 strongly expressed LACZ in Gdf5-Cre/Bmpr1afloxP mutant embryos (Figure 2H). These data suggest that regional loss of BMPR1A receptor signaling blocks programmed cell death in interdigital mesenchyme, and that the recombined cells survive and proliferate in the absence of BMPR1A signaling. + +Failure of Early Joint Formation in Ankle Regions + +The Bmpr1a gene is expressed in the interzone region of developing joints at E13.5 (Baur et al. 2000). In situ hybridization showed that the gene is also expressed in the interzones of ankle joints and prospective articular cartilage regions of digit joints at E15.5 (Figure 4). LACZ staining indicated that Cre-mediated recombination begins to occur in ankle joints around E14.5, and is extensive by E15.5 (Figure 4G and 4J) (unpublished data). In the ankle joint regions that were obviously fused in postnatal mutant animals, alterations in early joint marker expression could also be seen by E15.5. At this stage, the Gdf5 gene is normally expressed in stripes that mark the sites of joint formation (Figure 4F), and the gene for the major collagen protein of cartilage matrix (Col2a1) is down-regulated in the interzone region (Figure 4E). In contrast, Col2a1 staining extended completely through the joint region between the second and central tarsal of Gdf5-Cre/Bmpr1afloxP mutants (Figure 4H, black arrow), and Gdf5 expression was seen only as a small notch extending into where the joint should be forming (Figure 4I, bracket). These data suggest that the fusions seen between ankle bones in postnatal mutant skeletons are the result of incomplete segmentation of skeletal precursors during embryonic development, a defect confined to some locations in the ankle. + +Failure to Maintain Articular Cartilage in Other Joints + +In most joints of Bmpr1a conditional knockout mice, embryonic segmentation of skeletal precursors occurred normally. Although Gdf5-Cre-mediated recombination was seen as early as E13.5 in digit interzone regions (see Figure 2C), no changes in cell death or cell proliferation could be seen in the metacarpal-phalangeal or metatarsal-phalangeal joints at E13.5 or E14.5 (unpublished data). Similarly, although clear LACZ expression was seen by E15.5 in interphalangeal joints and periarticular regions (Figure 4D), no difference in morphology or expression of Col2a1, Gdf5, or Bmpr1b was seen in the articular regions of the phalanges at these stages (unpublished data). + +At birth, digit joints were generally indistinguishable from those in control animals; chondrocytes were abundant in articular regions and were surrounded by typical cartilage matrix with normal staining by Safranin O, a histological stain for proteoglycans (Figure 5). At this stage, both wild-type and mutant cells in articular regions also expressed high levels of Col2a1 and Aggrecan (Agg), the genes encoding the major structural proteins of cartilage matrix (Figure 5B and 5G) (unpublished data). No alterations in cellular apoptosis or proliferation were observed (unpublished data). + +To determine whether articular cells were properly specified in mutants, we also analyzed expression of Matrilin-4 (Mat4), a gene expressed specifically in the periarticular and perichondral regions of developing joints (Klatt et al. 2001). In both control and mutant animals, transcription of Mat4 was clearly detectable in the articular cartilage layers of newborn joints (Figure 5D and 5I). In all experiments, expression of LACZ throughout articular regions indicated that Cre-mediated recombination had occurred throughout the articular regions (Figure 5C, 5H, 5E, and 5J). The normal histological appearance, staining properties, and marker gene expression patterns suggest that Bmpr1a is not required for the initial formation or specification of articular cartilage. + +By 1 wk after birth, obvious differences began to be detected in the articular regions of mutant animals. The expression of Col2a1 was reduced throughout the articular surfaces of the carpals, metacarpals, and phalanges of the forefeet (unpublished data). Less severe reductions were also seen in articular cells of tarsals and metatarsals in the hindfeet (unpublished data). By 2 wk of age, Col2a1 expression was reduced in most cells of the articular region (Figure 5L and 5Q), accompanied by markedly reduced Safranin O staining (Figure 5K and 5P), and decreased expression of Agg and two genes normally expressed in more mature articular cartilage cells, Collagen 3 (Col3a1) and Collagen 10 (Col10a1) (Figure 5M and 5R) (unpublished data) (Eyre 2002). Inhibition of BMP signaling in cultured chondrocytes has previously been reported to induce Collagen 1 (Col1a1) expression, increase proliferation, and result in cells with flattened, fibroblast-like morphology (Enomoto-Iwamoto et al. 1998). However, we saw no increase in the expression of Col1a1 in mutant articular cartilage, and no proliferation was detected in articular cells of either mutant or control animals (unpublished data). While recombined LACZ marker expression was detected in most articular cartilage cells, it was also observed in scattered subarticular chondrocytes, growth plate chondrocytes, and osteoblasts (Figure 5O and 5T) (unpublished data). Although this implies that BMP signaling was defective in multiple cell types, the observed defects were confined to the articular cartilage. For example, Osteocalcin and Col1a1 expression appeared normal in osteoblasts (unpublished data). Together, these data suggest that BMPR1A activity is required in postnatal joint articular cartilage to maintain expression of many genes encoding structural components of cartilage matrix. + +Previous studies have shown that Sox9 is required for normal cartilage differentiation, for expression of cartilage extracellular matrix (ECM) genes including Agg, and is a direct transcriptional regulator of the key cartilage matrix gene Col2a1 (Bell et al. 1997; Lefebvre et al. 1997; Bi et al. 1999; Sekiya et al. 2000). Notably, despite reduced expression of many cartilage matrix marker genes in Bmpr1a mutant mice, the SOX9 protein was present at normal levels in articular regions at all stages examined, including newborn, 2-wk-old, 7-wk-old, and 9-mo-old mice (Figure 5N and 5S) (unpublished data). + +Synovial Hypertrophy, Cartilage Erosion, and Accelerated Cartilage Maturation + +Conditional loss of Bmpr1a led to marked hypertrophy of the synovial membrane in the joint capsule of some joints, particularly in the ankle region. In the most severely affected joints, the expanded synovial membrane grew into the joint space and was associated with obvious loss or erosion of the articular cartilage (Figure 6A and 6B, asterisks, arrows). Accelerated cartilage maturation and increased expression of Col10a1 was frequently seen in the chondrocytes underlying the articular erosions (Figure 6C and 6D, brackets) (unpublished data). Interestingly, the regions of increased Col10a1 expression did not correspond to the regions that had undergone Cre-mediated recombination. Instead, increased expression of Col10a1 was seen in a zone of largely LACZ-negative cells stretching from the cartilage adjacent to the ossification front (where Col10a1 is normally expressed in maturing cartilage cells), toward the regions where surface articular cartilage was severely eroded or missing (Figure 6A and 6B, arrowheads). Previous studies suggest that parathyroid hormone-related protein, a diffusible signal made in the articular surface, may normally inhibit maturation of underlying cartilage (Vortkamp et al. 1996; Weir et al. 1996). Local loss of the articular surface could remove this inhibition and lead to a cell-nonautonomous acceleration of maturation in chondrocytes underlying points of articular erosion. + +This synovial hypertrophy is associated with increased numbers of mononuclear cells resembling synoviocytes or macrophages, cell types that are difficult to distinguish even with surface markers at early postnatal stages. However, no neutrophils were observed, suggesting that there is little inflammation. At later stages synovial hypertrophy is reduced. Further work will be needed to determine whether synovial development is regulated by BMP signaling, or whether the synovium becomes enlarged as a response to nearby skeletal malformations (such as fusion of the second and central tarsals or defects in the articular cartilage). + +Noninflammatory Degeneration of Articular Cartilage in Digit and Knee Joints + +Outside of the ankle region, little or no evidence was seen for expansion of the synovial membrane. Instead, mutant mice showed histological signs of osteoarthritis, such as fibrillation of the articular surface (Figure 7). As previously seen in 1- and 2-wk-old animals, Safranin O staining and Agg and Col10 expression were all reduced in mutant articular regions of the forefeet and hindfeet by 7 wk of age, and the beginning signs of cartilage loss were observed (unpublished data). By 9 mo of age, many regions of articular cartilage were completely missing or extremely fibrillated, leaving regions of exposed bone on the surface (Figure 7A–7D). No alterations were seen in the expression of Osteocalcin, Col1a1, or matrix metalloprotease-13 at either 7 wk or 9 mo. + +The major weight-bearing joint of the hindlimb, the knee, showed changes that closely paralleled that seen in the foot joints. All markers of cartilage matrix looked similar to controls at E16.5, suggesting that early stages of joint formation were not disrupted (unpublished data). By postnatal day 7, Safranin O staining and Col2a1 and Agg expression were clearly reduced in the mutant, despite continued expression of Sox9 (unpublished data). The overall shape of mutant knee skeletal elements appeared similar to controls, although the fibrocartilaginous meniscus that resides between the femur and tibia appeared much less dense in mutants at E16.5. Some cartilage formed in the meniscus region, but the size of these elements was greatly reduced and contained abundant cells with fibrous, noncartilaginous appearance (unpublished data). This reduction of the meniscus can also be seen in sections from 7-wk- and 9-mo-old animals (Figure 7E, 7H, 7K, and 7N, arrows). + +At 7 wk of age the normally domed tibial epiphysis was flattened and depressed in the knees of mutant animals, markedly reducing the distance between the growth plate and articular surface (Figure 7E and 7H, vertical bar). Articular cartilage was also thinner than in control animals, showed nearly complete absence of Safranin O staining, and was either acellular or beginning to fibrillate in many regions (Figure 7F and 7I). The few large Safranin O-stained cells still apparent in mutant articular regions appeared to correspond in position to rare LACZ-negative cells in adjacent sections, suggesting that Bmpr1a is required cell-autonomously in articular cartilage (Figure 7I and 7J, white arrowheads). By 9 mo, large areas of mutant knees were devoid of articular cells, and the bones of the femur and tibia appeared to rub directly against each other. Furthermore, the epiphysis of the tibia was extremely depressed, to the point that growth plate cartilage was almost exposed through the surface of the bone (Figure 7K, 7L, 7N, and 7O). In addition, mutants at 7 wk and 9 mo showed subchondral sclerosis, especially in the epiphysis of the femur (Figure 7E, 7H, 7K, and 7N, asterisks). While subchondral sclerosis is commonly seen in cases of osteoarthritis, it is unclear in this case whether the sclerosis is mainly a response of bone formation to compensate for decreased articular cartilage, or whether it is the effect of loss of Bmpr1a signaling in some LACZ-positive cells that are also observed in these regions (unpublished data). + +The histological signs of joint arthritis were accompanied by functional impairments in both grasping ability and range of motion in mutant animals. Gdf5-Cre/Bmpr1afloxP mutant animals showed a highly significantly reduced ability to grasp and remain suspended on a slender rod (mean suspension time: controls 38 ± 6 s, n = 39; mutants 6 ± 3 s, n = 11; p < 0.0001). Mutant mice also showed a clear decrease in the maximum range of mobility of two different joints in the digits, as assayed by passive manipulation (MT/P1 joint: controls 100 ± 0°, n = 26; mutants 82 ± 3°, n = 8; p < 0.0003; P1/P2 joint: controls 152 ± 1°, n = 23; mutants 140 ± 5°, n = 6; p < 0.05). The structural, histological, marker gene expression, and functional changes in mutant mice demonstrate that BMPR1A is required for normal postnatal maintenance of articular cartilage. + +Discussion + +Previous studies suggest that BMP signaling is involved in a large number of developmental events. Many of these events occur early in embryogenesis, and complete inactivation of BMP receptors causes death by E9.5 (Mishina et al. 1995). The Gdf5-Cre recombination system bypasses the early embryonic lethality of Bmpr1a mutations, and provides important new information about the role of this receptor in limb and skeletal development. + +The three major limb phenotypes revealed by eliminating Bmpr1a with Gdf5-driven Cre include webbing between digits, lack of joint formation at specific locations in the ankle, and failure to maintain articular cartilage after birth, resulting in severe arthritis. Previous studies have shown that manipulation of BMP signaling alters interdigital apoptosis during development of the limb, but no experiment has identified a specific member of the BMP signaling pathway that is required for this process (Yokouchi et al. 1996; Zou and Niswander 1996; Zou et al. 1997; Guha et al. 2002). Our new loss-of-function data confirm that BMP signaling is required for interdigital apoptosis and suggests that Bmpr1a is a critical component for mediating this signal. + +At some sites, loss of Bmpr1a function leads to a defect in the early stages of joint formation, resulting in a complete failure to form a joint and fusion of bones in the ankle. Mutations in two different ligands in the BMP family, Gdf5 and Gdf6, the Bmpr1b receptor, and in the human Noggin locus (Storm and Kingsley 1996; Gong et al. 1999; Baur et al. 2000; Yi et al. 2000; Settle et al. 2003) also produce defects in joint formation at specific locations in the limbs. The joint defects associated with multiple components of the BMP pathway provide strong evidence that BMP signaling is required for early stages of joint formation at some anatomical locations. + +Most joints still form normally when Bmpr1a is knocked out in Gdf5 expression domains. The lack of joint fusions outside the ankle region could be due to differences in requirement for BMP signaling in different joints, to compensating expression of other BMP receptors outside the ankles, or to differences in the detailed timing of Gdf5-Cre stimulated gene inactivation in ankles and other joint regions. Comparison of the expression of the HPLAP marker (driven directly by Gdf5 control elements) and the R26R LACZ marker (expressed following Gdf5-Cre recombination) suggests that recombination-stimulated changes in gene expression may be delayed for a 0.5–1 d in the digit region (see Figure 1C). In addition, levels of Bmpr1a mRNA and protein may persist for some time following Gdf5-Cre stimulated recombination, making it possible to bypass an early requirement for Bmpr1a in joint formation at some locations. + +Following the decay of Bmpr1a mRNA and protein, the Gdf5-Cre strategy should result in permanent inactivation of Bmpr1a function in recombined cells. This system thus provides one of the first strong genetic tests of Bmpr1a function at later stages of joint development. Despite the normal appearance of articular regions and gene expression immediately after birth, Bmpr1a-deficient animals are unable to maintain the normal differentiated state of articular cartilage as they continue to develop and age. These results suggest that BMP receptor signaling is essential for continued health and integrity of articular cartilage in the postnatal period. + +Articular cartilage is a key component of synovial joints and is one of the few regions in the skeleton where cartilage is maintained into adulthood. Despite the importance of articular cartilage in joint health and mobility, little is known about the factors that create and maintain it in thin layers at the ends of long bones. In our experiments, articular cartilage lacking Bmpr1a retains some normal characteristics, in that it maintains a very low proliferation rate, does not express Col1a1, and continues to express SOX9, a major transcription factor regulating expression of structural components of cartilage matrix. However, several of the most prominent structural components of cartilage matrix fail to be maintained in mutant animals, resulting in decreased synthesis of Col2a1, Agg, and proteoglycans. Therefore, BMPR1A appears to maintain articular cartilage primarily through inducing expression of key ECM components. + +It is interesting that the SOX9 transcription factor continues to be expressed in mutant cartilage despite loss of Col2a1, a direct target of this transcription factor (Bell et al. 1997; Lefebvre et al. 1997). Previous studies suggest that SOX9 activity can be modified by protein kinase A (PKA)-dependent protein phosphorylation, or by coexpression of two related proteins, L-SOX5 and SOX6 (Lefebvre et al. 1998; Huang et al. 2000). In addition, close examination of the order of genes induced during chicken digit formation reveals that Sox9 turns on first, followed by Bmpr1b with L-Sox5, and then Sox6 and the cartilage matrix structural components Col2a1 and Agg (Chimal-Monroy et al. 2003). These results, together with the altered pattern of gene expression seen in our Bmpr1a-deficient mice, suggest that BMPR1A signaling may normally act to stimulate SOX9 by post-translational protein modification, or to induce L-Sox5 or Sox6 in cartilage to maintain expression of ECM components. These models are consistent with the ability of BMP2 to both increase PKA activity and induce expression of Sox6 in tissue culture cells (Lee and Chuong 1997; Fernandez-Lloris et al. 2003). Although we have tried to monitor the expression of L-Sox5 or Sox6 in postnatal articular cartilage, and test the phosphorylation state of SOX9 using previously described reagents (Lefebvre et al. 1998; Huang et al. 2000), we have been unable to obtain specific signal at the late postnatal stages required (unpublished data). Furthermore, null mutations in L-Sox5 or Sox-6 cause lethality at or soon after birth, and no effect on cartilage maintenance has been reported (Smits et al. 2001). However, it seems likely that these or other processes regulated by BMP signaling cooperate with SOX9 to induce target genes in articular cartilage. + +Mutation of Smad3 or expression of dominant negative transforming growth factor β (TGF-β) type II receptor also disrupts normal articular cartilage maintenance (Serra et al. 1997; Yang et al. 2001). Both manipulations should disrupt TGFβ rather than BMP signaling, and both manipulations cause articular cartilage to hypertrophy and be replaced by bone. In contrast, our analysis of Bmpr1a mutant articular cartilage showed a loss of ECM components, but no signs of hypertrophy or bone replacement. Therefore, TGFβ and BMP signaling are playing distinct but necessary roles to maintain articular cartilage. + +Although BMPs were originally isolated on the basis of their ability to induce ectopic bone formation, their presence in articular cartilage and strong effect on cartilage formation has stimulated interest in using them to repair or regenerate cartilage defects in adult animals (Chang et al. 1994; Erlacher et al. 1998; Edwards and Francis-West 2001; Chubinskaya and Kuettner 2003). The failure to maintain articular cartilage in the absence of normal BMPR1A function suggests that ligands or small molecule agonists that interact specifically with this receptor subtype may be particularly good candidates for designing new approaches to maintain or heal articular cartilage at postnatal stages. + +Lack of Bmpr1a function in articular cartilage results in severe fibrillation of the articular surface and loss of joint mobility. The development of severe arthritis symptoms in Bmpr1a-deficient mice raises the possibility that defects in BMP signaling also contribute to human joint disease. Osteoarthritis is known to have a significant genetic component, but it likely involves multiple genetic factors that have been difficult to identify (Spector et al. 1996; Felson et al. 1998; Hirsch et al. 1998). Humans that are heterozygous for loss-of-function mutations in BMPR1A are known to be at risk for juvenile polyposis (Howe et al. 2001; Zhou et al. 2001), but the risk of osteoarthritis for these people has not been reported. However, the control mice used in this study were heterozygous for a null allele of Bmpr1a, and they showed little sign of osteoarthritis even late in life. Several chromosome regions have been previously linked to arthritis phenotypes in humans using either association studies in populations or linkage studies in families. It is interesting to note that several of these chromosome regions contain genes encoding different members of the BMP signaling pathway, including the BMP5 gene on human chromosome 6p12 (Loughlin et al. 2002), the MADH1 gene on human chromosome 4q26–4q31 (Leppavuori et al. 1999; Kent et al. 2002), and the BMPR2 receptor on human chromosome 2q33 (Wright et al. 1996). The complex nature of human osteoarthritis suggests that interactions between multiple genes may be involved in modifying susceptibility to the disease. The inclusion of genetic markers near BMP signaling components may help identify additional osteoarthritis susceptibility loci and facilitate the search for causative mutations. + +Development and disease processes in synovial joints have been difficult to study genetically, because synovial joints are generated and function at relatively late stages of vertebrate development. The Gdf5-Cre system provides a new method for restricting gene expression or inactivation primarily to articular regions, thus avoiding the pleiotropic functions of many genes in other tissues. Depending on the configuration of the floxed target gene, this system can be used to either activate the expression of a gene primarily in developing joints (ssee Figure 1B–1D), or to inactivate gene function in articular regions (see Figure 3). Additional studies with this system should greatly enhance our knowledge of the development, function, and disease mechanisms of joints, and may bring us closer to better prevention and treatment of joint diseases. + +Materials and Methods + + + +Generation of Gdf5-Cre transgenic mice + +A mouse 129x1/SvJ BAC library (Invitrogen) was screened to identify a 140-kb BAC from the Gdf5 locus. This BAC was modified using a homologous recombination system in E. coli (Yang et al. 1997) to place nuclear-localized Cre recombinase (from plasmid pML78, gift of Gail Martin) followed by IRES-hPLAP (from plasmid 1726, gift of Oliver Bogler) directly behind the ATG start site of Gdf5. In the process, 583 bp of the first exon of Gdf5 was removed and no functional GDF5 protein is predicted to be produced. The 5′ homology arm was subcloned from a PCR product tailed with XhoI and Bsp120I restriction sites that contains 781 bp of 5′ genomic Gdf5 sequence ending at the ATG translation start site (forward primer 5′-CTGTCTCGAGATGAGGTGGAGGTGAAGACCCC-3′; reverse 5′-GTTTGGGCCCATCCTCTGGCCAGCCGCTG-3′). Cre was subcloned from a 1.1-kb Bsp120I/EcoRI fragment of pML78. IRES hPLAP was subcloned from a 2.1-kb PCR product tailed with EcoRI and SpeI sites that contains the hPLAP translation stop site (forward primer 5′-ATCTCTCGAGGAATTCTCCACCATATTGCCGTCTTTTG-3′; reverse 5′-AGAACTCGAGACTAGTCGGGACACTCAGGGAGTAGTGG-3′). The 3′ homology arm was subcloned from a 0.8-kb PCR product amplified from a 0.9-kb XhoI Gdf5 genomic subclone containing part of the first exon and downstream intron. The forward primer contains the 3′ end of the first exon and is tailed with a SpeI site; the reverse primer is from the T7 promoter of the vector containing the 0.9-kb subclone and flanks the intronic XhoI site (forward primer 5′-CTAAACTAGTCACCAGCTTTATTGACAAAGG-3′; reverse 5′-GATTTCTAGAGTAATACGACTCACTATAGGGC-3′). The targeting construct was built and verified in pBSSK (Stratagene, La Jolla, California, United States), then digested with XhoI and subcloned into pSV1, the vector used for homologous recombination (Yang et al. 1997). Southern blotting, PCR, and DNA sequence analysis confirmed the appropriate targeting construct and BAC modifications were made (unpublished data). + +Before the modified BAC was injected to produce transgenic animals, a loxP site present in the BAC vector, pBeloBAC11, was removed to prevent the addition of undesired Cre target sites into the genome. To do this, BAC DNA was prepared by CsCl separation, digested with NotI to free the insert from the vector, and size-fractionated over a sucrose gradient. Aliquots of fractions were run on a pulse-field gel and Southern blotted using vector-specific DNA as a probe. Fractions containing unsheared insert and almost no detectable vector DNA were dialyzed in microinjection buffer (10 mM Tris [pH 7.4] with 0.15 mM EDTA [pH 8.0]) using Centriprep-30 concentrators (Millipore, Billerica, Massachusetts, United States). This purified insert DNA was adjusted to 1 ng/μl and injected into the pronucleus of fertilized eggs from FVB/N mice by the Stanford Transgenic Facility. Transgenic founder mice were identified by PCR using Cre-specific primers 5′-GCCTGCATTACCGGTCGATGCAACGA-3′ and 5′-GTGGCAGATGGCGCGGCAACACCATT-3′, which amplify a 725-bp product, and were assessed for absence of BAC vector using vector-specific primers 5′-CGGAGTCTGATGCGGTTGCGATG-3′ and 5′-AGTGCTGTTCCCTGGTGCTTCCTC-3′, which amplify a 465-bp product. Three lines of Gdf5-Cre mice were established and maintained on the FVB background. Matings with R26R Cre-inducible LACZ reporter mice (Soriano 1999) were used to test for Cre activity. + +Staining for LACZ and HPLAP on whole embryos or sections of embryos was accomplished following established protocols (Lobe et al. 1999). The red LACZ substrate (see Figure 1E) is 6-chloro-3-indoxyl-beta-D-galactopyranoside (Biosynth International, Naperville, Illinois, United States). + +General characterization of Bmpr1a mutant mice + +Bmpr1a null and floxed alleles (Ahn et al. 2001; Mishina et al. 2002) were obtained on a mixed 129 and C57BL/6 background and maintained by random breeding. Mice carrying the null and floxed alleles were typically mated to Gdf5-Cre mice as shown in Figure 3. The resulting mice are on a mixed 129; C57Bl/6; FVB/N background, with both controls and mutant animals generated as littermates from the same matings. Whole-mount skeletal preparations were made from 34- to 36-d-old mice (Lufkin et al. 1992). Pairs of ears from euthanized 6-mo-old animals were removed, pinned, photographed, projected, and measured from the base of the curve formed between the tragus and antitragus to the farthest point at the edge of the pinnae. Grasping ability in 6-mo-old mice was measured by placing animals on a slender rod and timing how long they could remain suspended on the rod, to a maximum time allowed of 2 min. Data from five consecutive trials for each mouse were averaged. Range of motion assays were conducted on the MT/P1 and P1/P2 joints of the second hindlimb digit from euthanized 18-wk-old animals. Forceps were used to bend the joint to its natural stopping position, and the resulting angle was measured to the nearest 10° under 12.5× magnification using a 360° reticule. Analysis described in this section occurred on animals lacking R26R. Control mice included all nonmutant genotypes generated by Parent 1 being heterozygous for Gdf5-Cre and Bmpr1anull and Parent 2 being heterozygous for Bmpr1afloxP (see Figure 3). All statistical analysis used the Student's t-test or Welch's t-test, and values listed are mean ± standard error of the mean. + +Cell death and proliferation assays + +Limbs from mutant and control animals at E13.5 and E14.5 were dissected and frozen in OCT (Sakura Finetek,Torrence, CA, United States). Cryosections of tissue were assayed by TUNEL using the In Situ Cell Death Detection Kit, Fluorescein (Roche, Basel, Switzerland). Following TUNEL, slides were washed in PBS, blocked with PBS + 0.05% Tween-20 + 5% goat serum, washed again, and incubated with a 1:200 dilution of a rabbit anti-phospho-histone-H3 antibody called Mitosis Marker (Upstate Biotechnology, Lake Placid, New York, United States) to identify cells in mitosis. Cy3-labeled anti-rabbit secondary antibody was used to detect the antibody. Cell nuclei were labeled with DAPI, and slides were mounted in Vectamount (Vector Laboratories, Burlingame, California, United States) and visualized at 100× magnification. The area of selected anatomical sites were measured, and the number of TUNEL-labeled nuclear fragments and the number of Cy3-labeled nuclei were counted from three 10-μm sections spanning 50 μm, from three control and three mutant animals. The number of labeled cells in the metacarpal-phalangeal and metatarsal-phalangeal joints was counted in a 290 μm × 365 μm rectangle placed around the center of the joint. The posterior region of the fifth digit was defined by drawing a line from the tip of the digit down 2.15 mm and across to the lateral edge of the tissue. For this analysis, the R26R Cre reporter was not present. + +Histology and histochemistry + +Tissue from animals ranging from stages E14.5 to P14 was prepared for analysis by fixing in 4% paraformaldehyde (PFA) in PBS for 45 min to 4 h depending on the stage; washing three times in PBS, once in PBS + 15% sucrose for 1 h, and once in PBS + 30% sucrose for 2 h to overnight depending on the stage; and then freezing in OCT. Tissue from animals aged 7 wk to 9 mo was processed similarly to earlier stages except that it was decalcified in 0.5 M EDTA (pH 7.4) for 4 d prior to incubating in sucrose. All solutions were prechilled and used at 4 °C with agitation, and skin from tissues of P0 or older mice was lacerated or removed prior to processing. + +Tissue was then cryosectioned at 12 μm and processed. Staining of sections with Safranin O, Fast Green, and Harris' hematoxylin was carried out using standard histological procedures. Detection of LACZ activity with X-Gal was performed as described (Lobe et al. 1999) and was followed by refixing in 4% PFA, rinsing with deionized water, counterstaining with Nuclear Fast Red (Vector Labs), rinsing with water again, and then mounting in Aquamount (Lerner Labs, Pittsburgh, Pennsylvania, United States). + +RNA in situ hybridization was performed as described (Storm and Kingsley 1996), with the following modifications: (1) Prior to the acetylation step, sections were incubated with 10–20 μg/ml proteinase K for 30 s to 7 min at room temperature (depending on the developmental stage), followed by refixing in 4% PFA and washing three times in PBS; (2) prehybridization step was skipped, and (3) embryonic tissue sections used a different color development mix (Thut et al. 2001). Probes for the following genes have been published previously: Bmpr1a (Mishina et al. 1995), Col2a1 (Metsaranta et al. 1991), Col10a1 (Apte et al. 1992), Gdf5 (Storm and Kingsley 1996), Osteocalcin (Celeste et al. 1986), and Sox5 and Sox6 (Lefebvre et al. 1998). The following probe templates were gifts: Agg, Dr. Vicki Rosen, Genetics Institute; Bmp2 and Bmp4, Arend Sidow, Stanford University; Col1a1, Bjorn Olsen, Harvard Medical School; Bmpr1b, Col3a1, and Mat4 probes were made from ESTs with IMAGE clone numbers 5056341, 478480, and 406027, respectively (Invitrogen, Carlsbad, California, United States). + +Sections for immunohistochemistry were fixed in 4% PFA, then digested with 942–2,000 U/ml type IV-S bovine hyaluronindase (Sigma, St. Louis, Missouri, United States) in PBS (pH 5) at 37 °C for 30 min to 2 h depending on the stage. Slides were then washed in PBS, treated with 0.3% hydrogen peroxide in 100% methanol for 30 min, washed, blocked with PBS + 0.05% Tween20 + 5% goat or fetal bovine serum, washed again, and incubated with primary antibodies in PBS + 0.05% Tween 20 + 1% goat or fetal bovine serum overnight at 4 °C. Biotin-labeled secondary antibodies (Vector Labs) were tagged with HRP using the Vectastain Elite ABC kit (Vector Labs) followed by detection with DAB (Vector Labs). Primary antibodies and dilutions used were: goat anti-mouse MMP13, 1:100 (Chemicon International, Temecula, California, United States); rabbit anti-human SOX9, 1:500 (Morais da Silva et al. 1996); rabbit anti-phosphorylated-SOX9 (SOX9.P), 1:10–1:250 (Huang et al. 2000). + +Supporting Information + +Accession Numbers + +GenBank (http://www.ncbi.nih.gov/Genbank/) accession numbers for the genes discussed in this paper are Gdf5 (AC084323) and Bmpr1a (NM_009758). + +Acknowledgements + +We thank Gail Martin for the Cre construct (plasmid pML78) and Oliver Bogler for the IRES-hPLAP construct (plasmid 1726); Bjorn Olsen and Benoit de Crombrugghe for antibodies; the following individuals for in situ probe templates: Sophie Candille, Arend Sidow (Bmp2 and Bmp4), Vicki Rosen (Agg), and Bjorn Olsen (Col1a1); Véronique Lefebvre for Sox5 and Sox6 probe templates and useful discussions; Michelle Johnson for help with phenotypic assays on mice; Dr. Corrine Davis for help in evaluating synovial sections; Rebecca Rountree for Adobe Photoshop and Illustrator tips; and members of the Kingsley lab for helpful comments on the manuscript. This work was supported by an NIH predoctoral training grant (RR), a postdoctoral fellowship from the Arthritis Foundation (MS), and grants from the National Institutes of Health (DK). Dr. Kingsley is an associate investigator of the Howard Hughes Medical Institute. + +Abbreviations + +BAC - bacterial artificial chromosome + +Bmpr1a - bone morphogenetic protein receptor 1a + +E[number] - embyonic day [number] + +ECM - extracellular matrix + +GAC - transgenic line carrying Gdf5-alkaline phosphatase-Cre construct + +Gdf5 - growth differentiation factor 5 + +hPLAP - human placental alkaline phosphatase + +IRES - internal ribosome entry site + +PFA - paraformaldehyde + +R26R - lacZ ROSA26 Cre reporter strain + +TGF-β - transforming growth factor β + +TUNEL - terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling + +Figures and Tables + +Figure 1 + +A Genetic System to Drive Gene Recombination in Developing Joints + +(A) A 140-kb BAC from the Gdf5 locus was modified by inserting Cre-IRES-hPLAP into the translation start site of Gdf5 and used to make transgenic mice. Not to scale. See Materials and Methods for details. + +(B–E) Visualization of Gdf5-Cre driven recombination patterns based on activation of lacZ expression from the R26R Cre reporter allele. (B) LACZ activity is visible as blue staining in the ear (ea) and the joints of the shoulder (s), elbow (eb), wrist (w), knee (k), ankle (a), vertebra (vj), and phalanges (black arrowheads) of an E14.5 mouse embryo. (C) E14.5 hindlimb double-stained to show both HPLAP expression from the transgene (grey/purple staining) and LACZ expression from the rearranged R26R allele (blue staining). Note that both markers are visible in the oldest, proximal interphalangeal joint (black arrowhead), only HPLAP activity is visible in the more recently formed medial interphalangeal joint (black arrow), and neither HPLAP nor LACZ expression is visible in the youngest, most distal joint of the digit (white arrowhead). (D) Newborn (P0) forelimb with skin partially removed showing LACZ activity expressed in all phalangeal joints (red Salmon gal staining, black arrowheads) and regions of some tendons (asterisk). (E) Section through the most distal phalangeal joint of a P0 hindlimb stained with Alcian blue to mark cartilage showing LACZ expression (stained red) in all tissues of developing joints: articular cartilage (black arrowhead), precursors of ligaments and synovial membranes (black arrow), and cells where cavitation is occurring (asterisk). + +Figure 2 + +Bmpr1a Is Required for Webbing Regression and Apoptosis in Specific Regions of the Limb + +(A and B) Control E14.5 forelimb (A) compared to a, E14.5 mutant forelimb (B) showing webbing between digits 1 and 2 (arrowheads) and extra tissue at the posterior of digit 5 (arrows). + +(C) Gdf5-Cre induced lacZ expression from R26R in an E13.5 forelimb showing LACZ staining (blue) in metacarpal-phalangeal joints, between digits 1 and 2 (arrowhead), and in a region posterior to digit 5 (arrow). + +(D and E) Sections of E14.5 hindlimbs showing apoptosis visualized by TUNEL staining (green) and proliferation visualized by staining for histone H3 phosphorylation (red). Controls show strong, uniform TUNEL staining between digits 1 and 2 (D, arrowhead) while mutants show patchy TUNEL staining interspersed with mitotic cells in similar regions (E). Scale bar = 200 μm. + +(F) Quantitation of TUNEL staining and mitotic cells in the posterior region of the fifth digit shows apoptosis is reduced 30% while proliferation is increased 20% (asterisks indicate statistically significant difference). + +(G and H) By E15.5, interdigital tissue has regressed in controls (G, arrowhead). In contrast, tissue remains in mutants at this location, primarily derived from cells that have undergone Gdf5-Cre-mediated recombination that inactivates Bmpr1a function and activates expression of LACZ (H). Scale bar = 75 μm. + +Figure 3 + +Gdf5-Cre-Mediated Deletion of Bmpr1a + +(A) Breeding strategy simultaneously deletes Bmpr1afloxP and allows visualization of Gdf5-Cre-mediated recombination by lacZ expression from R26R. + +(B–E) 5-week-old mutant and control mice stained with Alcian blue to mark cartilage and alizarin red to mark bone. (B) Ankle of control with strong blue staining lining each joint (arrowheads). (C) Ankle of mutant showing an absence of blue staining in most regions (arrowheads) and a joint fusion between the central (c) and second (2) tarsals (arrow). (D) Control and (E) mutant metatarsal/phalangeal joint which lacks blue staining in articular regions (arrowheads) but retains staining in the growth plate (asterisks). + +(F) Control forelimb. + +(G) Mutant forelimb with webbing between the first and second digit (black arrowhead). + +Figure 4 + +Bmpr1a Is Expressed in Joints and Is Required for Continued Joint Formation in the Ankle Region + +(A) Diagram of ankle bones from a wild-type mouse; bones fusing in mutant are colored red. Roman numerals II–IV, metatarsals; 2, 3, and 4/5, distal row of tarsal bones; c, central tarsal bone; ta, talus; ca, calcaneus. + +(B and C) In situ hybridization at E15.5 showing that Bmpr1a is expressed in ankle joint interzones (B, arrowheads) and in the forming articular regions of the phalangeal joints (C, arrowheads). + +(D) Near adjacent section to (C) showing Gdf5-Cre induced LACZ expression from R26R in the forming joints of the digits (arrowheads). + +(E–J) Marker gene expression and R26R LACZ staining patterns on near adjacent sections of control and mutant embryos. In control mice at E15.5 ankle joints are clearly delineated as regions that have down-regulated Col2 (E), express Gdf5 throughout (F), and express LACZ in most cells (G; white arrowheads and black arrows). In mutant embryos at the same stage, joint formation is incomplete. Faint Col2 expression can be seen connecting a medial region of tarsal 2 with metatarsal II (H, white arrowhead), and Gdf5 expression does not extend all the way across the joint at this location (I, white arrowhead). Between tarsals c and 2, mutants express Col2 across the normal joint-forming region (H, black arrow) and lack expression of Gdf5 at sites where skeletal fusions are observed (I, black arrow and bracket). (J) Scale bar = 100 μm. + +Figure 5 + +Bmpr1a Is Required to Maintain Expression of ECM Components in Articular Cartilage + +In situ hybridization or LACZ staining on near adjacent sections of metacarpal-phalangeal joints (A–C and F–H) and the tarsal 2-metatarsal II joint (D–E and I–J) of P0 mice. At birth, articular cartilage of controls (A–E) and mutants (F–J) appears similar by Safranin O staining (A and F), and Col2 expression (B, G). Mat4 expression confirms that articular cartilage is initially specified in mutants (D andI, brackets). LACZ expression confirms Cre-mediated recombination has occurred in articular cartilage (C, H, E, and J). (K–T) Near adjacent sections of the metacarpal-phalangeal joints of P14 mice. Two weeks after birth, articular cartilage of controls stains with pericellular Safranin O (orange staining, K), and expresses Col2 (L), Agg (M), and SOX9 (N). In contrast, mutant articular cells are smaller and more densely packed, lack pericellular Safranin O staining (P), have reduced expression of Col2 (Q) and Agg (R), but retain normal levels of SOX9 protein (S, brackets; dashed line marks faint edges of articular surfaces). LACZ expression confirms Cre-mediated recombination has occurred in articular cells (O ansd T, brackets). (A and K) Scale bar = 75 μm. + +Figure 6 + +Synovial Membrane Expansion, Articular Surface Erosion, and Accelerated Maturation of Underlying Cartilage in Ankles of Bmpr1a Mutant Mice + +Near adjacent sections from the tarsal 2-metatarsal II joint of 7-d-old mice. (A and B) LACZ staining (blue) shows Cre-mediated recombination is largely restricted to articular (arrowheads) and synovial cells (asterisks) in both controls and mutants. (C and D) In situ hybridization shows Col10 expression expands in mutants toward regions of synovial membrane expansion and articular surface erosion (brackets and arrows). This may be a cell nonautonomous effect of joint damage, since the LACZ expressing cells at the articular surface do not show upregulation of Col10 (arrowheads) and the region of expanded Col10 expression is largely made up of cells that have not undergone Cre-mediated recombination. Note the formation of a cartilaginous bridge along the joint capsule of the mutant where joint formation is disrupted at earlier stages (B, white arrowhead, and Figure 3, white arrowheads). (A) Scale bar = 75 μm. + +Figure 7 + +Loss of Bmpr1a Signaling Leads to Articular Cartilage Fibrillation and Degeneration in Digits and Knees of Aging Mice + +(A–D) Near adjacent sections of metatarsal-phalangeal joints from 9 month old mice. Articular cartilage of controls is complete and stains strongly with Safranin O (A, orange stain). In contrast, articular cells of mutants are severely fibrillated or absent with much reduced staining of Safranin O (C, arrowheads). LACZ expression confirms Cre-mediated recombination has occurred in articular cells (B and D). + +(E–P) Sagittal sections through knee joints of 7-wk- (E–J) or 9-mo-old animals (K–P); fe, femur; ti, tibia; gp, growth plate. Seven weeks after birth, the height of the tibial epiphysis is reduced in mutants (E and H, bars), and their articular layer stains poorly with Safranin O, is fibrillated, and is strikingly thinner (F and I, black arrowhead, and brackets). Near adjacent sections with LACZ staining confirm Cre-mediated recombination has occurred in articular cells (G and J). Note that in mutants, LACZ is absent in cells adjacent to those that do stain with Safranin O, suggesting Bmpr1a may act cell autonomously (I and J, white arrowheads). At 9 mo old, the mutant tibial epiphysis is extremely thin (K and N, bars), and the articular layer is completely absent, leaving bone to rub directly on bone (L and O, bracket). LACZ staining shows Cre-mediated recombination occurred in articular cells of controls (M) and in some remaining skeletal tissue of mutants (P). Also note aberrantly formed meniscal cartilage in mutants (E, H, K, and N, arrows), and increased sclerosis in mutant epiphyses (E, H, K, and N, asterisks). + +(A and K) Scale bar = 50 μm; (I) scale bar = 300 μm. + +Footnotes + +Conflicts of interest. The authors have declared that no conflicts of interest exist. + +Author contributions. RBR, MS, and DMK conceived and designed the experiments. RBR and MEM performed the experiments. RBR, HC, and DMK analyzed the data. MS, HC, VH, and YM contributed reagents/materials/analysis tools. RBR and DMK wrote the paper. + +Academic Editor: Lee Niswander, University of Colorado Health Sciences Center + +¤ Current address: ARTEMIS Pharmaceuticals, an Exelixis Company, Köln, Germany + +Citation: Rountree RB, Schoor M, Chen H, Marks ME, Harley V, et al. (2004) BMP receptor signaling is required for postnatal maintenance of articular cartilage. PLoS Biol 2(11): e355. diff --git a/src/ontogpt/evaluation/craft/database/all/15550985.ann b/src/ontogpt/evaluation/craft/database/all/15550985.ann new file mode 100644 index 000000000..467bb3d96 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15550985.ann @@ -0,0 +1,1097 @@ +T1 PR:000011459 27 31 NT-3 +T2 GO:0019230 35 49 Proprioceptive +T3 GO:0030424 50 54 Axon +T4 GO:0007411 50 63 Axon Guidance +T5 PR:000011459 75 89 Neurotrophin-3 +T6 PR:000011459 91 95 NT-3 +T7 GO:0019230 113 127 proprioceptive +T8 CL:1001451 113 134 proprioceptive neuron +T9 SO:0000704 174 178 gene +T10 PR:000002193 179 182 Bax +T11 PR:000011459 186 190 NT-3 +T12 NCBITaxon:10088 200 204 mice +T13 CL:0000540 219 226 neurons +T14 GO:0030424 263 267 axon +T15 PR:000011459 293 297 NT-3 +T16 PR:000011471 309 313 TrkC +T17 GO:0030424 346 351 axons +T18 UBERON:0000044 357 376 dorsal root ganglia +T19 GO:0060384 463 472 innervate +T20 UBERON:0003718 493 508 muscle spindles +T21 PR:000011471 524 528 TrkC +T22 GO:0030424 538 543 axons +T23 UBERON:0005090 570 577 muscles +T24 GO:0019230 590 604 proprioceptive +T25 GO:0030424 605 610 axons +T26 UBERON:0001948 629 639;644 655 regions of ... spinal cord +T27 UBERON:0009571 675 682 midline +T28 PR:000011459 734 738 NT-3 +T29 UBERON:0000044 742 762 dorsal root ganglion +T30 GO:0030424 763 768 axons +T31 PR:000011459 808 812 NT-3 +T32 GO:0030424 838 842 axon +T33 GO:0007411 838 851 axon guidance +T34 CHEBI:36357 852 860 molecule +T35 PR:000011459 944 958 Neurotrophin-3 +T36 PR:000011459 960 964 NT-3 +T37 GO:0019230 1010 1024 proprioceptive +T38 CL:0000100 1035 1048 motor neurons +T39 NCBITaxon:10088 1090 1094 Mice +T40 PR:000011459 1108 1112 NT-3 +T41 PR:000011471 1144 1148 TrkC +T42 PR:000011471 1156 1160 TrkC +T43 CL:0000540 1170 1176 neuron +T44 PR:000014365 1207 1212 Runx3 +T45 http://purl.obolibrary.org/obo/MONDO_0000437 1228 1234 ataxia +T46 UBERON:0003718 1266 1281 muscle spindles +T47 GO:0019230 1295 1309 proprioceptive +T48 CL:1001451 1295 1317 proprioceptive neurons +T49 UBERON:0000044 1321 1340 dorsal root ganglia +T50 UBERON:0000044 1342 1346 DRGs +T51 GO:0030424 1357 1362 axons +T52 PR:000011459 1509 1513 NT-3 +T53 GO:0010467 1517 1526 expressed +T54 UBERON:0002240 1542 1553 spinal cord +T55 UBERON:0004347 1573 1582 limb buds +T56 UBERON:0003718 1616 1631 muscle spindles +T57 GO:0030424 1732 1737 axons +T58 CL:0002372 1757 1765 myotubes +T59 UBERON:0003718 1779 1793 muscle spindle +T60 UBERON:0001021 1836 1841 nerve +T61 NCBITaxon:9031 1870 1877 chicken +T62 UBERON:0000922 1878 1884 embryo +T63 UBERON:0002101 1886 1890 limb +T64 PR:000011459 1909 1913 NT-3 +T65 GO:0042571 1914 1922 antibody +T66 UBERON:0004347 1939 1948 limb buds +T67 PR:000011471 1972 1976 TrkC +T68 CL:0000540 1986 1993 neurons +T69 CL:0000100 2023 2036 motor neurons +T70 PR:000011459 2068 2072 NT-3 +T71 GO:0045202 2246 2254 synaptic +T72 GO:0060004 2255 2261 reflex +T73 PR:000011459 2297 2301 NT-3 +T74 GO:0030424 2340 2345 axons +T75 PR:000011471 2417 2421 TrkC +T76 UBERON:0000044 2431 2434 DRG +T77 CL:0000540 2435 2442 Neurons +T78 PR:000002193 2458 2461 Bax +T79 PR:000011459 2462 2466 NT-3 +T80 NCBITaxon:10088 2483 2487 Mice +T81 NCBITaxon:10088 2489 2493 Mice +T82 PR:000002193 2523 2526 Bax +T83 PR:000021998 2572 2585 neurotrophins +T84 PR:000002193 2606 2609 Bax +T85 CL:0000101 2620 2635 sensory neurons +T86 PR:000021998 2654 2667 neurotrophins +T87 GO:0030424 2754 2760 axonal +T88 PR:000011459 2778 2782 NT-3 +T89 PR:000002193 2800 2803 Bax +T90 NCBITaxon:10088 2818 2822 mice +T91 NCBITaxon:10088 2833 2837 mice +T92 PR:000011459 2861 2865 NT-3 +T93 PR:000002193 2870 2873 Bax +T94 SO:0000704 2874 2879 genes +T95 GO:0019230 2894 2908 proprioceptive +T96 GO:0030424 2909 2927 axonal projections +T97 PR:000011459 2933 2937 NT-3 +T98 GO:0016265 2953 2957 died +T99 GO:0007567 2976 2981 birth +T100 PR:000011469 3021 3025 TrkA +T101 PR:000011471 3026 3030 TrkC +T102 PR:000011471 3136 3140 TrkC +T103 PR:000011459 3206 3210 NT-3 +T104 UBERON:0000922 3218 3227 embryonic +T105 UBERON:0000044 3255 3258 DRG +T106 GO:0010467 3265 3275 expressing +T107 PR:000011469 3283 3287 TrkA +T108 PR:000011471 3291 3295 TrkC +T109 PR:000002193 3354 3357 Bax +T110 NCBITaxon:33208 3361 3368 animals +T111 GO:0007567 3391 3396 natal +T112 GO:0010467 3420 3429 expressed +T113 PR:000011471 3430 3434 TrkC +T114 PR:000011459 3443 3447 NT-3 +T115 NCBITaxon:33208 3451 3458 animals +T116 PR:000002193 3466 3469 Bax +T117 SO:0000704 3473 3480 genetic +T118 GO:0010467 3511 3520 expressed +T119 PR:000011469 3521 3525 TrkA +T120 PR:000011471 3530 3534 TrkC +T121 PR:000011469 3635 3639 TrkA +T122 PR:000011471 3644 3648 TrkC +T123 UBERON:0000044 3690 3694 DRGs +T124 NCBITaxon:33208 3698 3705 animals +T125 PR:000002193 3751 3754 Bax +T126 PR:000011459 3755 3759 NT-3 +T127 UBERON:0000044 3772 3776 DRGs +T128 PR:000011469 3781 3785 TrkA +T129 PR:000011471 3786 3790 TrkC +T130 PR:000002193 3818 3821 Bax +T131 UBERON:0000044 3827 3831 DRGs +T132 PR:000011459 3858 3862 NT-3 +T133 UBERON:0000044 3868 3872 DRGs +T134 PR:000011471 3886 3890 TrkC +T135 CL:0000540 3900 3907 neurons +T136 PR:000002193 3919 3922 Bax +T137 GO:0010467 4004 4014 expression +T138 GO:0019230 4022 4036 proprioceptive +T139 PR:000013502 4054 4065 Parvalbumin +T140 PR:000013502 4067 4069 PV +T141 PR:000011459 4085 4089 NT-3 +T142 CL:0000100 4129 4142 Motor Neurons +T143 PR:000011469 4144 4148 TrkA +T144 PR:000011471 4149 4153 TrkC +T145 UBERON:0002240 4177 4188 spinal cord +T146 PR:000011469 4227 4231 TrkA +T147 UBERON:0002240 4302 4313 spinal cord +T148 PR:000011471 4323 4327 TrkC +T149 UBERON:0002240 4356 4360 cord +T150 UBERON:0002257 4399 4412 ventral horns +T151 PR:000002193 4447 4450 Bax +T152 UBERON:0000922 4454 4461 embryos +T153 PR:000011471 4487 4491 TrkC +T154 GO:0010467 4492 4502 expression +T155 PR:000011459 4506 4510 NT-3 +T156 UBERON:0002240 4514 4525 spinal cord +T157 GO:0019230 4558 4572 proprioceptive +T158 UBERON:0002240 4594 4605 spinal cord +T159 PR:000011471 4607 4611 TrkC +T160 UBERON:0002240 4647 4658 spinal cord +T161 PR:000002193 4724 4727 Bax +T162 PR:000011471 4777 4781 TrkC +T163 GO:0030424 4889 4903;4908 4913 projections of ... axons +T164 UBERON:0000044 4904 4907 DRG +T165 CHEBI:35204 4934 4940 tracer +T166 CHEBI:52029 4941 4944 DiI +T167 PR:000002193 4962 4965 Bax +T168 GO:0019230 4975 4989 proprioceptive +T169 UBERON:0002240 5019 5030 spinal cord +T170 UBERON:0002257 5072 5084 ventral horn +T171 CL:0000100 5121 5134 motor neurons +T172 UBERON:0010405 5142 5162 lateral motor column +T173 CHEBI:52029 5212 5215 DiI +T174 UBERON:0002240 5248 5259 spinal cord +T175 PR:000011459 5263 5267 NT-3 +T176 GO:0019230 5344 5358 proprioceptive +T177 GO:0019230 5433 5447 proprioceptive +T178 UBERON:0000934 5580 5592 ventral cord +T179 UBERON:0010405 5606 5626 lateral motor column +T180 UBERON:0009571 5661 5676 ventral midline +T181 UBERON:0009571 5695 5702 midline +T182 UBERON:0000934 5739 5751 ventral cord +T183 PR:000011459 5815 5819 NT-3 +T184 CL:0000100 5837 5849 motor neuron +T185 CHEBI:52029 5906 5909 DiI +T186 UBERON:0002240 5955 5967 spinal cords +T187 UBERON:0002256 6019 6031 Dorsal horns +T188 CHEBI:52029 6066 6069 DiI +T189 UBERON:0001021 6102 6107 nerve +T190 PR:000011194 6102 6121 nerve growth factor +T191 GO:0019233 6132 6143 nociceptive +T192 GO:0030424 6144 6149 axons +T193 PR:000002193 6161 6164 Bax +T194 UBERON:0000922 6168 6175 embryos +T195 GO:0019230 6177 6191 proprioceptive +T196 UBERON:0002257 6216 6228 ventral horn +T197 CL:0000100 6229 6242 motor neurons +T198 UBERON:0002257 6275 6288 ventral horns +T199 PR:000011459 6296 6300 NT-3 +T200 UBERON:0000922 6304 6311 embryos +T201 PR:000002193 6359 6362 Bax +T202 PR:000011459 6363 6367 NT-3 +T203 CHEBI:52029 6380 6383 DiI +T204 UBERON:0002240 6419 6430 spinal cord +T205 UBERON:0009571 6457 6464 midline +T206 UBERON:0002257 6480 6492 ventral horn +T207 GO:0019230 6591 6605 proprioceptive +T208 UBERON:0002257 6625 6640;6659 6670 ventral horn of ... spinal cord +T209 PR:000002193 6645 6648 Bax +T210 PR:000011459 6649 6653 NT-3 +T211 GO:0030424 6736 6741 axons +T212 CL:0000100 6754 6766 motor neuron +T213 GO:0030425 6767 6776 dendrites +T214 UBERON:0002257 6784 6796 ventral horn +T215 GO:0060004 6822 6828 reflex +T216 UBERON:0024914 6833 6840 circuit +T217 GO:0030424 6909 6914 axons +T218 CL:0000100 6919 6932 motor neurons +T219 PR:000011459 6951 6955 NT-3 +T220 PR:000011459 6983 6987 NT-3 +T221 GO:0030424 6999 7003 axon +T222 GO:0007411 6999 7013 axon targeting +T223 GO:0030424 7047 7051 axon +T224 PR:000011459 7066 7070 NT-3 +T225 NCBITaxon:33208 7172 7179 animals +T226 UBERON:0003718 7197 7204 spindle +T227 UBERON:0001388 7224 7244 gastrocnemius muscle +T228 UBERON:0003718 7246 7261 Muscle spindles +T229 PR:000002193 7302 7305 Bax +T230 NCBITaxon:33208 7309 7316 animals +T231 GO:0019230 7372 7386 Proprioceptive +T232 GO:0005883 7407 7420 neurofilament +T233 PR:000011120 7407 7422 neurofilament-M +T234 GO:0005883 7424 7426 NF +T235 PR:000011120 7424 7428 NF-M +T236 GO:0042571 7430 7438 antibody +T237 GO:0042571 7525 7533 antibody +T238 UBERON:0003718 7651 7666 muscle spindles +T239 PR:000011459 7670 7674 NT-3 +T240 NCBITaxon:33208 7678 7685 animals +T241 PR:000002193 7728 7731 Bax +T242 NCBITaxon:33208 7735 7742 animals +T243 UBERON:0003718 7752 7760 spindles +T244 UBERON:0003718 7774 7782 spindles +T245 NCBITaxon:33208 7811 7818 animals +T246 GO:0010467 7824 7834 expressing +T247 PR:000011459 7835 7839 NT-3 +T248 PR:000011459 7885 7889 NT-3 +T249 UBERON:0003718 7923 7938 muscle spindles +T250 UBERON:0002101 7960 7965 limbs +T251 UBERON:0003718 8000 8015 Muscle spindles +T252 PR:000011471 8039 8043 TrkC +T253 GO:0042571 8044 8052 antibody +T254 PR:000002193 8063 8066 Bax +T255 NCBITaxon:33208 8070 8077 animals +T256 PR:000011459 8090 8094 NT-3 +T257 PR:000011459 8130 8134 NT-3 +T258 NCBITaxon:33208 8149 8156 animals +T259 GO:0005883 8158 8160 NF +T260 PR:000011120 8158 8162 NF-M +T261 UBERON:0005090 8199 8206 muscles +T262 UBERON:0005090 8217 8224 muscles +T263 NCBITaxon:33208 8234 8241 animals +T264 UBERON:0006134 8272 8284 nerve fibers +T265 PR:000011471 8306 8310 TrkC +T266 UBERON:0005090 8336 8343 muscles +T267 GO:0005883 8365 8367 NF +T268 PR:000011120 8365 8369 NF-M +T269 GO:0030424 8405 8410 axons +T270 UBERON:0003718 8423 8438 muscle spindles +T271 GO:0019230 8501 8515 proprioceptive +T272 GO:0030424 8516 8521 axons +T273 PR:000011459 8584 8588 NT-3 +T274 UBERON:0003718 8654 8668 muscle spindle +T275 GO:0005883 8705 8707 NF +T276 PR:000011120 8705 8709 NF-M +T277 CHEBI:52029 8736 8739 DiI +T278 UBERON:0003718 8788 8802 muscle spindle +T279 UBERON:0003718 8900 8908 spindles +T280 PR:000002193 8946 8949 Bax +T281 PR:000011459 8966 8970 NT-3 +T282 UBERON:0000922 8974 8981 embryos +T283 PR:000002193 8995 8998 Bax +T284 PR:000011459 8999 9003 NT-3 +T285 UBERON:0005090 9016 9023 muscles +T286 UBERON:0003718 9039 9047 spindles +T287 UBERON:0003718 9076 9083 spindle +T288 UBERON:0000978 9115 9118 leg +T289 UBERON:0000922 9125 9131 embryo +T290 CHEBI:52029 9177 9180 DiI +T291 UBERON:0003718 9244 9259 muscle spindles +T292 PR:000002193 9302 9305 Bax +T293 UBERON:0005090 9311 9318 muscles +T294 PR:000011459 9343 9347 NT-3 +T295 NCBITaxon:33208 9361 9368 animals +T296 CHEBI:52029 9379 9382 DiI +T297 UBERON:0005090 9431 9438 muscles +T298 UBERON:0003718 9497 9512 muscle spindles +T299 PR:000011469 9531 9535 TrkA +T300 PR:000011471 9536 9540 TrkC +T301 GO:0010467 9541 9551 expression +T302 UBERON:0001323 9565 9577 tibial nerve +T303 UBERON:0001388 9615 9635 gastrocnemius muscle +T304 UBERON:0004264 9651 9658;9663 9672 skin of ... lower leg +T305 PR:000011459 9677 9681 NT-3 +T306 PR:000011469 9687 9691 TrkA +T307 GO:0030424 9701 9706 axons +T308 UBERON:0001323 9728 9740 tibial nerve +T309 PR:000011471 9759 9763 TrkC +T310 GO:0030424 9773 9778 axons +T311 PR:000011469 9793 9797 TrkA +T312 PR:000011471 9803 9807 TrkC +T313 GO:0030424 9816 9821 axons +T314 PR:000002193 9846 9849 Bax +T315 NCBITaxon:33208 9868 9875 animals +T316 GO:0019230 9925 9939 proprioceptive +T317 GO:0030424 9940 9945 axons +T318 UBERON:0001021 9983 10000 peripheral nerves +T319 GO:0060384 10015 10024 innervate +T320 UBERON:0005090 10038 10045 muscles +T321 PR:000011459 10064 10068 NT-3 +T322 PR:000011459 10071 10075 NT-3 +T323 UBERON:0000044 10101 10104 DRG +T324 GO:0030424 10105 10110 Axons +T325 PR:000011459 10149 10153 NT-3 +T326 GO:0030424 10192 10197 axons +T327 GO:0019230 10241 10255 Proprioceptive +T328 GO:0030424 10256 10261 axons +T329 NCBITaxon:10088 10265 10269 mice +T330 UBERON:0002315 10280 10291 gray matter +T331 UBERON:0002240 10299 10310 spinal cord +T332 UBERON:0009571 10349 10356 midline +T333 CL:0000100 10378 10391 motor neurons +T334 UBERON:0000044 10464 10467 DRG +T335 PR:000011459 10482 10486 NT-3 +T336 NCBITaxon:27592 10555 10561 bovine +T337 UBERON:0001977 10562 10567 serum +T338 PR:000003918 10562 10575 serum albumin +T339 UBERON:0000044 10639 10642 DRG +T340 GO:0030424 10643 10648 axons +T341 PR:000011459 10687 10691 NT-3 +T342 GO:0050918 10763 10778 chemoattraction +T343 PR:000011459 10938 10942 NT-3 +T344 PR:000002193 10951 10954 Bax +T345 UBERON:0000045 10960 10967 ganglia +T346 GO:0050918 10987 11002 chemoattraction +T347 PR:000011459 11024 11028 NT-3 +T348 PR:000011471 11052 11056 TrkC +T349 PR:000001405 11065 11071 p75NTR +T350 UBERON:0000044 11117 11120 DRG +T351 PR:000001405 11143 11149 p75NTR +T352 NCBITaxon:10088 11153 11157 mice +T353 GO:0030424 11168 11173 Axons +T354 UBERON:0000045 11183 11190 ganglia +T355 GO:0050918 11210 11225 chemoattraction +T356 PR:000011459 11238 11242 NT-3 +T357 PR:000011471 11290 11294 TrkC +T358 MOP:0000779 11305 11315 conjugated +T359 GO:0071735 11319 11322 IgG +T360 PR:000011471 11341 11345 TrkC +T361 PR:000011459 11397 11401 NT-3 +T362 PR:000011471 11457 11461 TrkC +T363 GO:0050918 11469 11484 chemoattraction +T364 PR:000011459 11562 11566 NT-3 +T365 UBERON:0000044 11665 11669 DRGs +T366 UBERON:0000045 11724 11731 ganglia +T367 GO:0030424 11873 11878 axons +T368 UBERON:0000044 11944 11947 DRG +T369 UBERON:0002240 11948 11959 spinal cord +T370 PR:000011459 11983 11987 NT-3 +T371 UBERON:0009571 12015 12022 midline +T372 UBERON:0002240 12030 12041 spinal cord +T373 PR:000011459 12062 12066 NT-3 +T374 CHEBI:52029 12142 12145 DiI +T375 UBERON:0000044 12167 12171 DRGs +T376 UBERON:0002240 12210 12221 spinal cord +T377 GO:0040007 12245 12252 growing +T378 PR:000011459 12265 12269 NT-3 +T379 UBERON:0002240 12432 12443 spinal cord +T380 UBERON:0002315 12496 12507 gray matter +T381 GO:0030424 12531 12536 axons +T382 GO:0030424 12600 12605 Axons +T383 UBERON:0002315 12630 12641 gray matter +T384 UBERON:0002240 12657 12668 spinal cord +T385 GO:0040007 12669 12673 grow +T386 UBERON:0009571 12686 12693 midline +T387 PR:000011459 12726 12730 NT-3 +T388 GO:0030424 12747 12751 axon +T389 PR:000011459 12771 12775 NT-3 +T390 UBERON:0000044 12799 12803 DRGs +T391 UBERON:0002240 12818 12829 spinal cord +T392 UBERON:0002240 12861 12872 spinal cord +T393 PR:000011459 12886 12890 NT-3 +T394 UBERON:0000044 12947 12950 DRG +T395 GO:0030424 12951 12956 axons +T396 PR:000011459 13010 13014 NT-3 +T397 GO:0030424 13064 13069 axons +T398 UBERON:0000922 13118 13127 embryonic +T399 NCBITaxon:10088 13128 13133 mouse +T400 UBERON:0005868 13134 13151 maxillary process +T401 UBERON:0001675 13155 13174 trigeminal ganglion +T402 CL:0000540 13175 13182 neurons +T403 PR:000011459 13230 13234 NT-3 +T404 UBERON:0000955 13239 13244 brain +T405 PR:000004716 13239 13272 brain-derived neurotrophic factor +T406 PR:000004716 13274 13278 BDNF +T407 GO:0030424 13376 13381 axons +T408 UBERON:0002101 13385 13389 limb +T409 GO:0040007 13420 13424 grow +T410 PR:000021998 13433 13445 neurotrophin +T411 PR:000021998 13537 13549 neurotrophin +T412 GO:0042571 13568 13578 antibodies +T413 GO:0030424 13617 13621 axon +T414 UBERON:0002101 13641 13645 limb +T415 NCBITaxon:10088 13661 13665 mice +T416 GO:0010467 13678 13685 express +T417 PR:000011459 13686 13690 NT-3 +T418 PR:000011141 13701 13707 nestin +T419 SO:0000167 13708 13716 promoter +T420 UBERON:0001017 13724 13746 central nervous system +T421 GO:0019230 13766 13780 proprioceptive +T422 PR:000011459 13864 13868 NT-3 +T423 GO:0010467 13869 13879 expression +T424 UBERON:0002240 13887 13898 spinal cord +T425 PR:000011459 13973 13977 NT-3 +T426 UBERON:0002257 14036 14049 ventral horns +T427 GO:0019230 14053 14067 proprioceptive +T428 GO:0019230 14115 14129 proprioceptive +T429 NCBITaxon:9031 14155 14162 chicken +T430 UBERON:0000922 14163 14170 embryos +T431 PR:000011459 14210 14214 NT-3 +T432 GO:0042571 14215 14223 antibody +T433 UBERON:0002240 14233 14244 spinal cord +T434 GO:0042571 14359 14367 antibody +T435 PR:000011459 14384 14388 NT-3 +T436 GO:0030424 14571 14576 axons +T437 PR:000011459 14623 14627 NT-3 +T438 GO:0010467 14637 14647 expression +T439 PR:000011459 14651 14655 NT-3 +T440 PR:000021998 14714 14726 neurotrophin +T441 PR:000004716 14735 14739 BDNF +T442 GO:0030424 14800 14805 axons +T443 GO:0060384 14806 14817 innervating +T444 UBERON:0001690 14818 14821 ear +T445 SO:0000704 14853 14857 gene +T446 PR:000004716 14888 14892 BDNF +T447 GO:0010467 14893 14903 expression +T448 PR:000011459 14922 14926 NT-3 +T449 SO:0000167 14927 14935 promoter +T450 GO:0030424 14948 14953 axons +T451 PR:000004716 14990 14994 BDNF +T452 UBERON:0001844 15002 15009 cochlea +T453 GO:0010467 15024 15033 expressed +T454 PR:000011459 15034 15038 NT-3 +T455 GO:0060384 15052 15062 innervated +T456 GO:0030424 15072 15077 axons +T457 PR:000011459 15087 15091 NT-3 +T458 NCBITaxon:10508 15125 15135 adenovirus +T459 GO:0010467 15145 15155 expression +T460 GO:0030424 15171 15175 axon +T461 GO:0031099 15190 15202 regeneration +T462 GO:0030424 15296 15302 axonal +T463 UBERON:0002707 15317 15330 corticospinal +T464 GO:0030424 15331 15336 axons +T465 UBERON:0007023 15348 15353 adult +T466 UBERON:0002240 15354 15365 spinal cord +T467 GO:0030424 15406 15411 axons +T468 UBERON:0009571 15443 15450 midline +T469 PR:000011459 15463 15467 NT-3 +T470 UBERON:0002240 15501 15512 spinal cord +T471 PR:000011459 15610 15614 NT-3 +T472 CL:0000100 15684 15696 motor neuron +T473 PR:000011459 15724 15728 NT-3 +T474 NCBITaxon:33208 15732 15739 animals +T475 GO:0010467 15748 15758 expressing +T476 PR:000011459 15759 15763 NT-3 +T477 UBERON:0005090 15785 15792 muscles +T478 PR:000011459 15842 15846 NT-3 +T479 GO:0019230 15880 15894 proprioceptive +T480 CL:1001451 15880 15902 proprioceptive neurons +T481 PR:000011459 15906 15910 NT-3 +T482 NCBITaxon:33208 15914 15921 animals +T483 GO:0030424 15933 15939 axonal +T484 GO:0007411 15933 15951 axonal pathfinding +T485 UBERON:0002240 15959 15970 spinal cord +T486 PR:000011459 16003 16007 NT-3 +T487 UBERON:0002240 16035 16047 spinal cords +T488 NCBITaxon:33208 16057 16064 animals +T489 CL:0000100 16103 16116 motor neurons +T490 PR:000011459 16144 16148 NT-3 +T491 UBERON:0002240 16166 16177 spinal cord +T492 PR:000011459 16233 16237 NT-3 +T493 UBERON:0002257 16256 16269 ventral horns +T494 NCBITaxon:10088 16279 16283 mice +T495 PR:000002193 16368 16371 Bax +T496 PR:000011459 16372 16376 NT-3 +T497 NCBITaxon:10088 16387 16391 mice +T498 GO:0019230 16468 16482 proprioceptive +T499 GO:0019230 16556 16570 proprioceptive +T500 UBERON:0002240 16611 16622 spinal cord +T501 PR:000013502 16684 16686 PV +T502 CHEBI:52029 16706 16709 DiI +T503 UBERON:0001021 16726 16743 peripheral nerves +T504 PR:000013502 16759 16761 PV +T505 PR:000002193 16798 16801 Bax +T506 PR:000011459 16802 16806 NT-3 +T507 PR:000021998 16851 16864 neurotrophins +T508 GO:0019230 16910 16924 proprioceptive +T509 GO:0030424 16925 16930 axons +T510 PR:000013502 16939 16941 PV +T511 SO:0000704 16956 16960 gene +T512 UBERON:0001021 16981 16986 nerve +T513 PR:000011194 16981 17000 nerve growth factor +T514 GO:0030424 17012 17017 axons +T515 PR:000013502 17083 17085 PV +T516 PR:000002193 17104 17107 Bax +T517 PR:000011459 17108 17112 NT-3 +T518 GO:0019230 17152 17166 proprioceptive +T519 GO:0030424 17167 17172 axons +T520 UBERON:0001388 17238 17258 gastrocnemius muscle +T521 GO:0010467 17289 17299 expressing +T522 PR:000011471 17300 17304 TrkC +T523 UBERON:0007023 17316 17321 adult +T524 NCBITaxon:10114 17322 17325 rat +T525 UBERON:0000044 17326 17329 DRG +T526 PR:000011471 17460 17464 TrkC +T527 GO:0010467 17565 17574 expressed +T528 GO:0030424 17588 17593 axons +T529 GO:0019230 17644 17658 proprioceptive +T530 CL:1001451 17644 17666 proprioceptive neurons +T531 PR:000011459 17685 17689 NT-3 +T532 PR:000011471 17693 17697 TrkC +T533 NCBITaxon:10088 17703 17707 mice +T534 PR:000011471 17723 17727 TrkC +T535 UBERON:0001323 17744 17756 tibial nerve +T536 GO:0019230 17775 17789 proprioceptive +T537 GO:0030424 17790 17795 axons +T538 PR:000011459 17884 17888 NT-3 +T539 UBERON:0003718 17929 17936 spindle +T540 GO:0019230 17960 17974 proprioceptive +T541 GO:0030424 17975 17980 axons +T542 CHEBI:52029 18012 18015 DiI +T543 UBERON:0000044 18032 18035 DRG +T544 GO:0030424 18036 18041 axons +T545 UBERON:0002240 18049 18060 spinal cord +T546 GO:0019230 18116 18130 proprioceptive +T547 GO:0030424 18131 18136 axons +T548 UBERON:0000957 18162 18169 laminae +T549 UBERON:0000934 18190 18202 ventral cord +T550 PR:000002193 18226 18229 Bax +T551 PR:000011459 18230 18234 NT-3 +T552 CHEBI:52029 18273 18276 DiI +T553 GO:0030424 18325 18330 axons +T554 UBERON:0002240 18352 18363 spinal cord +T555 GO:0030424 18450 18455 axons +T556 GO:0043195 18469 18485 terminal boutons +T557 GO:0030424 18588 18593 axons +T558 UBERON:0009571 18603 18618 ventral midline +T559 GO:0043195 18671 18687 terminal boutons +T560 PR:000007223 18817 18821 Er81 +T561 PR:000007227 18826 18830 Pea3 +T562 GO:0010467 18836 18845 expressed +T563 UBERON:0000044 18849 18852 DRG +T564 CL:0000540 18853 18860 neurons +T565 CL:0000100 18872 18885 motor neurons +T566 CL:0000187 18903 18916 muscle fibers +T567 UBERON:0002240 18941 18952 spinal cord +T568 PR:000007223 18956 18960 Er81 +T569 NCBITaxon:10088 18964 18968 mice +T570 GO:0030424 18978 18992;19008 19013 projections of ... axons +T571 GO:0019230 18993 19007 proprioceptive +T572 GO:0030424 19047 19052 axons +T573 UBERON:0000934 19068 19080 ventral cord +T574 PR:000002193 19149 19152 Bax +T575 PR:000011459 19153 19157 NT-3 +T576 NCBITaxon:10088 19168 19172 mice +T577 PR:000007223 19201 19205 Er81 +T578 NCBITaxon:10088 19209 19213 mice +T579 PR:000011459 19242 19246 NT-3 +T580 PR:000007223 19255 19259 Er81 +T581 GO:0010467 19260 19270 expression +T582 UBERON:0000044 19274 19277 DRG +T583 PR:000007223 19329 19333 Er81 +T584 GO:0010467 19339 19349 expression +T585 PR:000011459 19392 19396 NT-3 +T586 PR:000002193 19404 19407 Bax +T587 PR:000011459 19408 19412 NT-3 +T588 NCBITaxon:10088 19423 19427 mice +T589 GO:0010467 19486 19496 expression +T590 NCBITaxon:10088 19514 19518 mice +T591 UBERON:0000044 19582 19585 DRG +T592 GO:0010467 19592 19599 express +T593 PR:000007223 19625 19629 Er81 +T594 GO:0030424 19645 19650 axons +T595 GO:0040007 19651 19655 grow +T596 UBERON:0002240 19694 19705 spinal cord +T597 PR:000002193 19709 19712 Bax +T598 PR:000011459 19713 19717 NT-3 +T599 NCBITaxon:10088 19728 19732 mice +T600 PR:000007223 19757 19761 Er81 +T601 NCBITaxon:10088 19765 19769 mice +T602 GO:0019230 19798 19812 proprioceptive +T603 GO:0030424 19813 19818 axons +T604 UBERON:0002240 19848 19859 spinal cord +T605 GO:0030424 19896 19901 axons +T606 UBERON:0002240 19938 19949 spinal cord +T607 CL:0000100 19977 19990 motor neurons +T608 NCBITaxon:10088 20074 20078 mice +T609 CL:0000100 20120 20133 motor neurons +T610 UBERON:0000934 20141 20153 ventral cord +T611 NCBITaxon:10088 20164 20168 mice +T612 PR:000013502 20169 20171 PV +T613 GO:0030424 20193 20198 axons +T614 UBERON:0002257 20206 20219 ventral horns +T615 CL:0000100 20251 20264 motor neurons +T616 NCBITaxon:10088 20285 20289 mice +T617 PR:000011459 20291 20295 NT-3 +T618 GO:0046903 20296 20304 secreted +T619 GO:0019230 20339 20353 proprioceptive +T620 GO:0030424 20354 20359 axons +T621 UBERON:0010405 20373 20394 lateral motor columns +T622 PR:000011459 20439 20443 NT-3 +T623 GO:0010467 20460 20469 expressed +T624 UBERON:0002240 20485 20496 spinal cord +T625 CL:0000100 20517 20530 motor neurons +T626 NCBITaxon:10088 20593 20597 mice +T627 PR:000011459 20599 20603 NT-3 +T628 GO:0010467 20604 20614 expression +T629 UBERON:0002240 20630 20641 spinal cord +T630 UBERON:0000922 20703 20712 embryonic +T631 NCBITaxon:10088 20713 20717 mice +T632 PR:000011459 20727 20731 NT-3 +T633 UBERON:0002257 20744 20760;20765 20776 ventral horns of ... spinal cord +T634 GO:0010467 20834 20843 expressed +T635 CL:0000100 20854 20867 motor neurons +T636 UBERON:0007023 20876 20881 adult +T637 UBERON:0002240 20882 20893 spinal cord +T638 CL:0000100 20901 20914 motor neurons +T639 GO:0010467 20915 20922 express +T640 PR:000011459 20938 20942 NT-3 +T641 CL:0000125 20967 20971 glia +T642 GO:0010467 20978 20985 express +T643 NCBITaxon:10088 21104 21108 mice +T644 PR:000011459 21114 21118 NT-3 +T645 GO:0010467 21124 21134 expression +T646 UBERON:0001948 21146 21156;21161 21172 regions of ... spinal cord +T647 PR:000011459 21218 21222 NT-3 +T648 GO:0030424 21242 21246 axon +T649 GO:0007411 21242 21255 axon guidance +T650 GO:0019230 21259 21273 proprioceptive +T651 GO:0030424 21274 21279 axons +T652 UBERON:0002240 21287 21298 spinal cord +T653 GO:0050918 21345 21360 chemoattraction +T654 UBERON:0000044 21364 21367 DRG +T655 CL:0000540 21368 21375 neurons +T656 PR:000011459 21400 21404 NT-3 +T657 PR:000002193 21435 21438 Bax +T658 PR:000001405 21451 21454 p75 +T659 UBERON:0000044 21460 21463 DRG +T660 PR:000021998 21506 21519 neurotrophins +T661 GO:0030424 21538 21542 axon +T662 GO:0007411 21538 21551 axon guidance +T663 CHEBI:36357 21552 21561 molecules +T664 GO:0030424 21594 21598 axon +T665 PR:000011459 21611 21615 NT-3 +T666 PR:000011459 21698 21702 NT-3 +T667 GO:0030424 21728 21733 axons +T668 GO:0030424 21841 21846 axons +T669 PR:000011459 21924 21928 NT-3 +T670 GO:0030424 21959 21964 axons +T671 PR:000021998 21990 22002 neurotrophin +T672 GO:0030424 22026 22032 axonal +T673 GO:0045202 22133 22141 synaptic +T674 GO:0060004 22150 22156 reflex +T675 PR:000017442 22171 22176 Wnt-3 +T676 GO:0030424 22203 22207 axon +T677 UBERON:0002240 22235 22246 spinal cord +T678 CHEBI:36357 22278 22286 molecule +T679 PR:000015235 22288 22293 Slit2 +T680 GO:0010467 22295 22304 expressed +T681 UBERON:0009571 22312 22319 midline +T682 CL:0000100 22327 22340 motor neurons +T683 GO:0030424 22383 22389 axonal +T684 UBERON:0000044 22462 22465 DRG +T685 CL:0000540 22466 22473 neurons +T686 GO:0010467 22474 22481 express +T687 GO:0019230 22523 22537 proprioceptive +T688 GO:0030424 22538 22543 axons +T689 PR:000015235 22616 22621 Slit2 +T690 PR:000011459 22650 22654 NT-3 +T691 UBERON:0000044 22666 22669 DRG +T692 GO:0030424 22670 22675 axons +T693 UBERON:0001675 22767 22777 trigeminal +T694 GO:0030424 22778 22783 axons +T695 UBERON:0002298 22791 22800 brainstem +T696 PR:000015235 22838 22843 Slit2 +T697 GO:0019230 22892 22905 propioceptive +T698 GO:0030424 22906 22911 axons +T699 UBERON:0000934 22919 22931 ventral cord +T700 UBERON:0009571 22943 22950 midline +T701 PR:000013502 22998 23000 PV +T702 GO:0010467 23001 23011 expression +T703 PR:000002193 23015 23018 Bax +T704 PR:000011459 23019 23023 NT-3 +T705 NCBITaxon:10088 23027 23031 mice +T706 PR:000013502 23046 23048 PV +T707 GO:0010467 23049 23059 expression +T708 GO:0030424 23092 23096 axon +T709 GO:0007411 23092 23106 axon targeting +T710 UBERON:0003718 23111 23125 muscle spindle +T711 GO:0030424 23224 23228 axon +T712 GO:0007411 23224 23240 axon pathfinding +T713 GO:0045202 23255 23263 synaptic +T714 GO:0060004 23264 23270 reflex +T715 UBERON:0003718 23281 23295 muscle spindle +T716 PR:000013502 23331 23333 PV +T717 NCBITaxon:10088 23337 23341 mice +T718 GO:0030424 23491 23497 axonal +T719 GO:0007411 23491 23507 axonal targeting +T720 PR:000002193 23519 23522 Bax +T721 PR:000011459 23523 23527 NT-3 +T722 NCBITaxon:10088 23538 23542 mice +T723 PR:000013502 23575 23577 PV +T724 GO:0010467 23578 23588 expression +T725 GO:0019230 23592 23606 proprioceptive +T726 PR:000011459 23648 23652 NT-3 +T727 GO:0030424 23675 23679 axon +T728 GO:0007411 23675 23688 axon guidance +T729 GO:0019230 23697 23711 proprioceptive +T730 GO:0030424 23712 23717 axons +T731 GO:0030424 23792 23796 axon +T732 GO:0007411 23792 23805 axon guidance +T733 GO:0030424 23834 23839 axons +T734 PR:000011459 23882 23886 NT-3 +T735 CHEBI:36357 23907 23915 molecule +T736 GO:0097374 23937 23946;23970 23986 targeting ... of sensory axons +T737 GO:0030424 23981 23986 axons +T738 UBERON:0002240 24002 24013 spinal cord +T739 PR:000011459 24015 24019 NT-3 +T740 GO:0030424 24062 24066 axon +T741 GO:0007411 24062 24075 axon guidance +T742 CHEBI:36357 24076 24085 molecules +T743 GO:0065007 24092 24102 regulating +T744 GO:0010467 24103 24113 expression +T745 GO:0019230 24138 24152 proprioceptive +T746 CL:1001451 24138 24159 proprioceptive neuron +T747 GO:0030424 24200 24204 axon +T748 GO:0007411 24200 24213 axon guidance +T749 PR:000002193 24284 24287 Bax +T750 PR:000011459 24381 24385 NT-3 +T751 NCBITaxon:33208 24460 24467 animals +T752 NCBITaxon:33208 24505 24512 animals +T753 SO:0000704 24535 24540 genes +T754 SO:0000112 24577 24584 Primers +T755 PR:000002193 24598 24601 Bax +T756 SO:0000112 24720 24727 Primers +T757 PR:000011459 24741 24745 NT-3 +T758 PR:000002193 24907 24910 Bax +T759 PR:000011459 24919 24923 NT-3 +T760 PR:000002193 24976 24979 Bax +T761 PR:000011459 24980 24984 NT-3 +T762 NCBITaxon:10088 24995 24999 mice +T763 PR:000001405 25047 25050 p75 +T764 UBERON:0002415 25127 25131 tail +T765 NCBITaxon:33208 25148 25155 animals +T766 SO:0000112 25166 25173 primers +T767 SO:0000112 25260 25267 primers +T768 PR:000001405 25353 25356 p75 +T769 UBERON:0000922 25369 25378 embryonic +T770 UBERON:0010148 25399 25403 plug +T771 NCBITaxon:33208 25559 25565 Animal +T772 NCBITaxon:33208 25675 25682 animals +T773 PR:000011469 25685 25689 TrkA +T774 PR:000011471 25690 25694 TrkC +T775 UBERON:0002240 25725 25736 spinal cord +T776 CHEBI:60004 25792 25800 cocktail +T777 NCBITaxon:9986 25804 25810 rabbit +T778 PR:000011469 25816 25820 TrkA +T779 NCBITaxon:9925 25825 25829 goat +T780 PR:000011471 25835 25839 TrkC +T781 GO:0042571 25840 25850 antibodies +T782 CHEBI:60004 25909 25917 cocktail +T783 CHEBI:37987 25921 25924 CY3 +T784 MOP:0000779 25925 25935 conjugated +T785 NCBITaxon:9793 25936 25942 donkey +T786 NCBITaxon:9986 25948 25954 rabbit +T787 CHEBI:37926 25959 25963 FITC +T788 MOP:0000779 25964 25974 conjugated +T789 NCBITaxon:9793 25975 25981 donkey +T790 NCBITaxon:9925 25987 25991 goat +T791 GO:0042571 25992 26002 antibodies +T792 CHEBI:9750 26075 26086 TritonX-100 +T793 NCBITaxon:9793 26102 26108 donkey +T794 UBERON:0001977 26109 26114 serum +T795 PR:000013502 26120 26122 PV +T796 NCBITaxon:10088 26183 26188 mouse +T797 PR:000013502 26194 26196 PV +T798 GO:0042571 26197 26205 antibody +T799 CHEBI:31624 26275 26286 fluorescein +T800 NCBITaxon:10088 26287 26292 mouse +T801 NCBITaxon:10088 26296 26301 mouse +T802 PR:000011469 26397 26401 TrkA +T803 PR:000011471 26407 26411 TrkC +T804 UBERON:0003718 26519 26533 Muscle spindle +T805 UBERON:0001388 26546 26566 Gastrocnemius muscle +T806 UBERON:0000978 26655 26658 leg +T807 UBERON:0001388 26675 26695 gastrocnemius muscle +T808 UBERON:0001323 26701 26713 tibial nerve +T809 GO:0042571 26790 26798 antibody +T810 UBERON:0003718 26839 26846 spindle +T811 SO:0001060 26886 26893 isoform +T812 NCBITaxon:10088 26918 26923 mouse +T813 NCBITaxon:10088 26927 26932 mouse +T814 NCBITaxon:9986 26969 26975 rabbit +T815 GO:0005883 26981 26983 NF +T816 PR:000011120 26981 26985 NF-M +T817 GO:0042571 26986 26994 antibody +T818 CHEBI:37987 27010 27013 CY3 +T819 MOP:0000779 27014 27024 conjugated +T820 NCBITaxon:9925 27025 27029 goat +T821 NCBITaxon:9986 27035 27041 rabbit +T822 GO:0042571 27042 27050 antibody +T823 CHEBI:52029 27123 27126 DiI +T824 UBERON:0000044 27151 27155 DRGs +T825 UBERON:0000922 27163 27170 embryos +T826 UBERON:0001388 27213 27233 gastrocnemius muscle +T827 CHEBI:52029 27302 27305 DiI +T828 UBERON:0002240 27317 27329 Spinal cords +T829 UBERON:0000044 27358 27362 DRGs +T830 CL:0000100 27418 27431 motor neurons +T831 CHEBI:52029 27449 27515 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate +T832 CHEBI:52029 27517 27520 DiI +T833 UBERON:0000044 27588 27592 DRGs +T834 UBERON:0002240 27602 27614 spinal cords +T835 CHEBI:9754 27928 27932 Tris +T836 UBERON:0000044 27979 27982 DRG +T837 PR:000002193 28025 28028 Bax +T838 PR:000001405 28039 28042 p75 +T839 NCBITaxon:10088 28048 28053 mouse +T840 UBERON:0000922 28054 28061 embryos +T841 UBERON:0000044 28097 28101 DRGs +T842 CHEBI:15366 28250 28261 acetic acid +T843 UBERON:0000045 28367 28375 ganglion +T844 PR:000011459 28607 28611 NT-3 +T845 PR:000021998 28776 28788 neurotrophin +T846 UBERON:0000045 28885 28893 ganglion +T847 UBERON:0001977 28993 28998 Serum +T848 UBERON:0000044 29068 29071 DRG +T849 UBERON:0001977 29095 29100 serum +T850 PR:000011471 29148 29152 TrkC +T851 CHEBI:52217 29167 29182 Pharmaceuticals +T852 UBERON:0002240 29288 29300 Spinal cords +T853 UBERON:0000044 29315 29319 DRGs +T854 NCBITaxon:10088 29358 29363 mouse +T855 UBERON:0000922 29364 29371 embryos +T856 UBERON:0000479 29420 29426 Tissue +T857 UBERON:0000479 29459 29465 Tissue +T858 PR:000011459 29535 29539 NT-3 +T859 UBERON:0009571 29646 29653 midline +T860 UBERON:0002240 29661 29672 spinal cord +T861 UBERON:0001977 29724 29729 serum +T862 CHEBI:16526 29818 29821 CO2 +T863 CHEBI:52029 29903 29906 DiI +T864 UBERON:0000044 29928 29932 DRGs +T865 CHEBI:37958 29992 29995 dye +T866 CL:0000100 30126 30138 Motor Neuron +T867 UBERON:0000922 30171 30178 Embryos +T868 PR:000002193 30201 30204 Bax +T869 PR:000011459 30205 30209 NT-3 +T870 CL:0000100 30246 30259 motor neurons +T871 UBERON:0002260 30300 30312 ventral root +T872 GO:0030424 30340 30345 axons +T873 UBERON:0000044 30366 30370 DRGs +T874 UBERON:0000922 30380 30386 embryo +T875 GO:0019230 30395 30409 proprioceptive +T876 GO:0030424 30410 30415 axons +T877 CL:0000100 30427 30439 motor neuron +T878 GO:0030425 30440 30449 dendrites +T879 UBERON:0002257 30457 30469 ventral horn +T880 GO:0045202 30479 30487 synapses +T881 PR:000002193 30494 30497 Bax +T882 PR:000011459 30498 30502 NT-3 +T883 UBERON:0002240 30515 30526 spinal cord +T884 UBERON:0002240 30570 30581 spinal cord +T885 UBERON:0009571 30607 30614 midline +T886 UBERON:0002257 30630 30642 ventral horn +T887 CL:0000100 30665 30677 motor neuron +T888 GO:0030425 30678 30687 dendrites +T889 GO:0019230 30752 30766 proprioceptive +T890 CL:0000100 30785 30798 motor neurons +T891 GO:0030424 30887 30892 axons +T892 CL:0000100 30912 30925 motor neurons +T893 GO:0005883 31053 31055 NF +T894 PR:000011120 31053 31057 NF-M +T895 UBERON:0001388 31086 31106 Gastrocnemius Muscle +T896 CL:0000187 31126 31132;31143 31149 muscle ... fibers +T897 UBERON:0006134 31137 31149 nerve fibers +T898 UBERON:0003718 31167 31182 muscle spindles +T899 UBERON:0001021 31252 31257 nerve +T900 PR:000011459 31477 31481 NT-3 +T901 NCBITaxon:33208 31485 31492 animals +T902 PR:000011469 31511 31515 TrkA +T903 PR:000011471 31520 31524 TrkC +T904 GO:0042571 31525 31535 antibodies +T905 GO:0042571 31562 31570 antibody +T906 NCBITaxon:33208 31635 31642 animals +T907 UBERON:0001091 31872 31878 Dental +T908 PR:000004716 31940 31944 BDNF +T909 UBERON:0000955 31947 31952 brain +T910 PR:000004716 31947 31980 brain-derived neurotrophic factor +T911 NCBITaxon:27592 31988 31994 bovine +T912 UBERON:0001977 31995 32000 serum +T913 PR:000003918 31995 32008 serum albumin +T914 UBERON:0000044 32010 32013 DRG +T915 UBERON:0000044 32016 32036 dorsal root ganglion +T916 UBERON:0000922 32042 32051 embryonic +T917 GO:0005883 32072 32074 NF +T918 PR:000011120 32072 32076 NF-M +T919 GO:0005883 32079 32092 neurofilament +T920 PR:000011120 32079 32094 neurofilament-M +T921 PR:000011459 32096 32100 NT-3 +T922 PR:000011459 32103 32117 neurotrophin-3 +T923 GO:0007567 32127 32132 natal +T924 PR:000013502 32169 32171 PV +T925 PR:000013502 32174 32185 parvalbumin +T926 PR:000011471 32187 32191 TrkC +T927 PR:000011471 32208 32212 TrkC +T928 MOP:0000779 32223 32233 conjugated +T929 GO:0071735 32237 32240 IgG +T930 PR:000011469 32305 32309 TrkA +T931 PR:000011471 32310 32314 TrkC +T932 PR:000013502 32319 32321 PV +T933 UBERON:0000044 32346 32349 DRG +T934 UBERON:0002240 32354 32365 Spinal Cord +T935 PR:000011469 32382 32386 TrkA +T936 PR:000011471 32405 32409 TrkC +T937 PR:000013502 32415 32417 PV +T938 GO:0010467 32452 32462 expression +T939 PR:000011469 32469 32473 TrkA +T940 PR:000011471 32474 32478 TrkC +T941 PR:000011471 32506 32510 TrkC +T942 CL:0000540 32520 32527 neurons +T943 PR:000011459 32551 32555 NT-3 +T944 PR:000011469 32592 32596 TrkA +T945 PR:000011471 32597 32601 TrkC +T946 UBERON:0000044 32626 32629 DRG +T947 PR:000013502 32636 32638 PV +T948 UBERON:0000044 32660 32663 DRG +T949 PR:000011471 32673 32677 TrkC +T950 GO:0010467 32701 32708 express +T951 PR:000013502 32709 32711 PV +T952 PR:000011469 32727 32731 TrkA +T953 PR:000011471 32756 32760 TrkC +T954 UBERON:0000044 32796 32800 DRGs +T955 PR:000002193 32909 32912 Bax +T956 PR:000011459 32922 32926 NT-3 +T957 PR:000011471 32955 32959 TrkC +T958 PR:000011469 32989 32993 TrkA +T959 PR:000011471 32994 32998 TrkC +T960 UBERON:0002240 33021 33032 spinal cord +T961 GO:0030424 33127 33145 Axonal Projections +T962 UBERON:0002240 33153 33164 Spinal Cord +T963 CHEBI:52029 33171 33174 DiI +T964 UBERON:0000044 33187 33190 DRG +T965 UBERON:0000044 33210 33213 DRG +T966 CL:1001451 33210 33236 DRG proprioceptive neurons +T967 GO:0019230 33214 33228 proprioceptive +T968 GO:0060384 33254 33263 innervate +T969 CL:0000100 33264 33277 motor neurons +T970 GO:0030424 33307 33312 axons +T971 UBERON:0009571 33338 33353 ventral midline +T972 UBERON:0009571 33370 33377 midline +T973 GO:0045202 33424 33432 synaptic +T974 GO:0060004 33433 33439 reflex +T975 PR:000011459 33496 33500 NT-3 +T976 CL:0000100 33527 33540 motor neurons +T977 UBERON:0003718 33565 33580 muscle spindles +T978 UBERON:0009571 33653 33660 midline +T979 GO:0043195 33686 33701 synaptic bouton +T980 GO:0030424 33772 33777 Axons +T981 CHEBI:52029 33791 33794 DiI +T982 UBERON:0000044 33807 33810 DRG +T983 CHEBI:52029 33823 33826 DiI +T984 UBERON:0002240 33848 33859 spinal cord +T985 GO:0019230 33868 33882 proprioceptive +T986 GO:0030424 33883 33888 axons +T987 CL:0000100 33911 33924 motor neurons +T988 UBERON:0002257 33940 33955;33960 33971 ventral horn of ... spinal cord +T989 PR:000002193 33995 33998 Bax +T990 UBERON:0002240 34004 34015 spinal cord +T991 PR:000011459 34022 34026 NT-3 +T992 UBERON:0002240 34032 34043 spinal cord +T993 GO:0019233 34082 34093 nociceptive +T994 GO:0030424 34094 34099 axons +T995 UBERON:0002256 34107 34118 dorsal horn +T996 UBERON:0002240 34184 34195 spinal cord +T997 PR:000002193 34202 34205 Bax +T998 PR:000011459 34206 34210 NT-3 +T999 UBERON:0002240 34223 34234 spinal cord +T1000 UBERON:0002240 34276 34287 spinal cord +T1001 GO:0040007 34300 34304 grow +T1002 CL:0000100 34317 34330 motor neurons +T1003 UBERON:0009571 34361 34368 midline +T1004 UBERON:0003718 34409 34424 Muscle Spindles +T1005 UBERON:0001388 34428 34448 Gastrocnemius Muscle +T1006 PR:000011469 34453 34457 TrkA +T1007 PR:000011471 34458 34462 TrkC +T1008 UBERON:0001323 34479 34491 Tibial Nerve +T1009 GO:0005883 34503 34505 NF +T1010 PR:000011120 34503 34507 NF-M +T1011 UBERON:0001388 34565 34585 gastrocnemius muscle +T1012 UBERON:0003718 34606 34621 muscle spindles +T1013 GO:0005883 34651 34653 NF +T1014 PR:000011120 34651 34655 NF-M +T1015 UBERON:0001388 34717 34737 gastrocnemius muscle +T1016 GO:0005883 34750 34752 NF +T1017 PR:000011120 34750 34754 NF-M +T1018 UBERON:0001388 34816 34836 gastrocnemius muscle +T1019 UBERON:0005090 34857 34864 muscles +T1020 UBERON:0003718 34886 34901 muscle spindles +T1021 UBERON:0003718 34918 34925 spindle +T1022 UBERON:0003718 34998 35013 Muscle spindles +T1023 CHEBI:52029 35026 35029 DiI +T1024 UBERON:0000044 35051 35054 DRG +T1025 UBERON:0001388 35056 35076 Gastrocnemius muscle +T1026 CL:0000187 35134 35147 muscle fibers +T1027 UBERON:0003718 35154 35169 Muscle spindles +T1028 PR:000011471 35182 35186 TrkC +T1029 UBERON:0001388 35216 35236 gastrocnemius muscle +T1030 PR:000011469 35249 35253 TrkA +T1031 PR:000011471 35264 35268 TrkC +T1032 UBERON:0001323 35299 35311 tibial nerve +T1033 PR:000011471 35333 35337 TrkC +T1034 PR:000011459 35369 35373 NT-3 +T1035 PR:000011469 35488 35492 TrkA +T1036 PR:000011471 35497 35501 TrkC +T1037 UBERON:0003718 35591 35606 muscle spindles +T1038 GO:0061642 35673 35691;35700 35705 Chemoattraction of ... Axons +T1039 UBERON:0000044 35696 35699 DRG +T1040 GO:0030424 35700 35705 Axons +T1041 PR:000011459 35715 35719 NT-3 +T1042 UBERON:0000044 35767 35770 DRG +T1043 PR:000011459 35776 35780 NT-3 +T1044 UBERON:0000044 35802 35805 DRG +T1045 PR:000002193 35833 35836 Bax +T1046 UBERON:0000044 35842 35845 DRG +T1047 PR:000011459 35851 35855 NT-3 +T1048 PR:000001405 35874 35877 p75 +T1049 UBERON:0000044 35883 35886 DRG +T1050 PR:000011459 35892 35896 NT-3 +T1051 UBERON:0000044 35918 35921 DRG +T1052 PR:000011459 35927 35931 NT-3 +T1053 PR:000011471 35948 35952 TrkC +T1054 UBERON:0000044 35979 35982 DRG +T1055 PR:000011459 35988 35992 NT-3 +T1056 UBERON:0000045 36051 36058 ganglia +T1057 GO:0050918 36131 36146 Chemoattraction +T1058 PR:000011459 36155 36159 NT-3 +T1059 UBERON:0002240 36180 36191 Spinal Cord +T1060 UBERON:0000044 36192 36195 DRG +T1061 PR:000011459 36221 36225 NT-3 +T1062 UBERON:0009571 36245 36252 midline +T1063 UBERON:0002240 36263 36274 spinal cord +T1064 GO:0030424 36283 36288 axons +T1065 UBERON:0000044 36309 36313 DRGs +T1066 GO:0040007 36348 36355 growing +T1067 UBERON:0002240 36417 36428 spinal cord +T1068 UBERON:0002240 36463 36474 spinal cord +T1069 UBERON:0002240 36504 36515 spinal cord +T1070 GO:0030424 36529 36534 axons +T1071 UBERON:0002240 36559 36570 spinal cord +T1072 GO:0030424 36651 36656 axons +T1073 PR:000011459 36707 36711 NT-3 +T1074 GO:0030424 36732 36737 axons +T1075 PR:000011459 36791 36795 NT-3 +T1076 GO:0030424 36820 36825 axons +T1077 UBERON:0002240 36862 36873 spinal cord +T1078 UBERON:0002240 37015 37026 spinal cord +T1079 UBERON:0000044 37027 37030 DRG +T1080 UBERON:0002240 37104 37115 spinal cord +T1081 UBERON:0002020 37147 37158 gray matter +T1082 PR:000011459 37197 37201 NT-3 +T1083 UBERON:0009571 37226 37233 midline +T1084 GO:0030424 37241 37246 axons +T1085 GO:0040007 37247 37251 grow +T1086 PR:000011459 37264 37268 NT-3 +T1087 PR:000011459 37275 37279 NT-3 +T1088 GO:0030424 37295 37299 axon +T1089 UBERON:0000044 37316 37320 DRGs +T1090 UBERON:0002240 37335 37346 spinal cord +T1091 GO:0040007 37372 37379 growing +T1092 UBERON:0001019 37428 37441 nerve bundles +T1093 UBERON:0001016 37928 37942 Nervous System +T1094 PR:000011459 38090 38094 NT-3 +T1095 GO:0019230 38098 38112 proprioceptive +T1096 GO:0030424 38113 38117 axon +T1097 GO:0007411 38113 38126 axon guidance diff --git a/src/ontogpt/evaluation/craft/database/all/15550985.txt b/src/ontogpt/evaluation/craft/database/all/15550985.txt new file mode 100644 index 000000000..81b36d93a --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15550985.txt @@ -0,0 +1,255 @@ +A Chemoattractant Role for NT-3 in Proprioceptive Axon Guidance + +Abstract + +Neurotrophin-3 (NT-3) is required for proprioceptive neuron survival. Deletion of the proapoptotic gene Bax in NT-3 knockout mice rescues these neurons and allows for examination of their axon growth in the absence of NT-3 signaling. TrkC-positive peripheral and central axons from dorsal root ganglia follow proper trajectories and arrive in close proximity to their targets but fail to innervate them. Peripherally, muscle spindles are absent and TrkC-positive axons do not enter their target muscles. Centrally, proprioceptive axons branch in ectopic regions of the spinal cord, even crossing the midline. In vitro assays reveal chemoattractant effects of NT-3 on dorsal root ganglion axons. Our results show that survival factor NT-3 acts as a short-distance axon guidance molecule for muscle sensory afferents as they approach their proper targets. + +Introduction + +Neurotrophin-3 (NT-3) is a key requirement for the development of proprioceptive inputs to motor neurons (Chen and Frank 1999; Chen et al. 2003). Mice deficient in NT-3, its tyrosine kinase receptor, TrkC, or in TrkC-positive neuron-specific transcription factor Runx3 display severe ataxia associated with the absence of muscle spindles, and loss of proprioceptive neurons in dorsal root ganglia (DRGs) or their axons (Ernfors et al. 1994; Klein et al. 1994; Tessarollo et al. 1994; Fariñas et al. 1996; Liebl et al. 1997; Inoue et al. 2002; Levanon et al. 2002). NT-3 is expressed in the ventral spinal cord, in the developing limb buds, and in intrafusal bag fibers of muscle spindles later in development (Copray and Brouwer 1994; Fariñas et al. 1996; Tojo et al. 1996). When sensory axons contact developing myotubes, they induce muscle spindle differentiation, forming ring-like spiral nerve endings around them. In the chicken embryo, limb ablations or anti-NT-3 antibody injections into limb buds lead to elimination of TrkC-positive neurons and decreased innervation of motor neurons (Oakley et al. 1995, 1997). Is NT-3 only a chemotrophic survival factor for muscle sensory afferents, or does it have additional roles in the development of the proprioceptors and the establishment of the monosynaptic reflex arc? Here we provide evidence that NT-3 acts as a chemoattractant for sensory axons during the final phase of their target-directed pathfinding. + +Results + +TrkC-Positive DRG Neurons Are Rescued in Bax/NT-3 Double Knockout Mice + +Mice lacking proapoptotic protein Bax allow for distinguishing survival effects of neurotrophins from other effects. Bax-deficient sensory neurons no longer require neurotrophins for survival (White et al. 1998; Patel et al. 2000), thus they can be used to examine axonal effects. We bred NT-3 heterozygote and Bax knockout (KO) mice to obtain mice with double KO of both NT-3 and Bax genes, and examined proprioceptive axonal projections. All NT-3 and double KOs died within 48 h after birth (Tessarollo et al. 1994). We performed TrkA/TrkC double immunohistochemistry (Huang et al. 1999), enabling detection of both proteins in the same sample. TrkC-positive cells (Figure 1A) and fibers (Figure 1E) were absent in NT-3 KOs at embryonic day (E) 15. Two subsets of DRG cells expressing either TrkA or TrkC were detected in double KOs, similar to wild-type (WT) or Bax KO animals. Surprisingly, at postnatal day (P) 0, a few cells expressed TrkC even in NT-3 KO animals in the Bax+/+ genetic background, and some cells co-expressed TrkA and TrkC, regardless of the genotype (Figure 1B). In order to quantify our results, we analyzed the ratio of TrkA and TrkC immunopositive cells from four different DRGs of animals of different genotypes. At all ages studied, Bax/NT-3 double null DRGs had TrkA/TrkC ratios similar to those of Bax null DRGs, and higher than those of NT-3 null DRGs (Figure 1D). TrkC-positive neurons rescued by Bax deletion, however, failed to differentiate properly, as evidenced by the lack of expression of the proprioceptive molecular marker Parvalbumin (PV) (Figure 1C). + +NT-3 Is Necessary for Proper Innervation of Motor Neurons + +TrkA/TrkC-positive fibers in the spinal cord could be detected at E15 (Figure 1E). TrkA-positive fibers were restricted to and terminated in the dorsolateral spinal cord, whereas TrkC-positive fibers entered the cord dorsomedially, and descended into the ventral horns in WT (Ozaki and Snider 1997) and Bax KO embryos. There was no detectable TrkC expression in NT-3 KO spinal cord, indicating complete absence of proprioceptive fibers. In double KO spinal cord, TrkC-positive fibers entered the dorsal spinal cord and descended medially in a manner similar to that seen in WT or Bax KO cases. However, it was not possible to follow TrkC immunolabeled fibers all the way to their terminal zones in any of the cases. Next we examined the central projections of DRG axons with the lipophilic tracer DiI at P0. In WT and Bax KO pups, proprioceptive afferents entered the dorsal spinal cord and followed a medial course towards the ventral horn. They then turned laterally towards motor neurons in the lateral motor column, where they branched and terminated (Figure 2A). DiI labeling was confined to dorsal spinal cord in NT-3 KOs (Figure 2A), as reported earlier, consistent with a complete absence of proprioceptive innervation (Ernfors et al. 1994; Tessarollo et al. 1994). In double KOs, proprioceptive afferents initially followed a trajectory similar to that of WT counterparts, but most of them failed to project all the way to the ventral cord and into the lateral motor column. Instead, they arborized near the ventral midline; some crossed the midline and extended into the contralateral ventral cord (Figure 2A and 2C). In order to distinguish between a role for NT-3 in initiation of motor neuron innervation and a role for maintenance, we repeated the DiI labeling at E17. Innervation patterns of E17 spinal cords (Figure 3) were similar to those at P0 (Figure 2). Dorsal horns of all genotypes were filled with DiI-labeled fibers corresponding to nerve growth factor–dependent nociceptive axons. In WT and Bax KO embryos, proprioceptive fibers extended towards ventral horn motor neurons (Figure 3A and 3B), whereas the ventral horns of the NT-3 KO embryos were devoid of innervation (Figure 3C). In the Bax/NT-3 double KOs, DiI-labeled fibers entered the ventral spinal cord, but extended towards the midline instead of the ventral horn (Figure 3D), in a pattern similar to that observed at P0. Our data point to a complete absence of proprioceptive innervation of the ventral horn of the Bax/NT-3 null spinal cord throughout the developmental stages investigated. As the sensory axons never reach motor neuron dendrites in the ventral horn (Figure S1), the stretch reflex arc circuit is not established. The failure to initiate contact between sensory axons and motor neurons in the absence of NT-3 suggests a requirement for NT-3 for proper axon targeting in addition to a role in sensory axon maintenance. + +NT-3 Is Required for Proper Peripheral Innervation + +In order to study peripheral innervation in double KO animals, we investigated spindle development in the gastrocnemius muscle. Muscle spindles could be identified easily in P0 WT and Bax KO animals by their characteristic morphology (Figure 4A and 4B). Proprioceptive fibers labeled with neurofilament-M (NF-M) antibody formed ring-like spiral endings wrapped around intrafusal bag fibers labeled with S46 antibody, specific for slow developmental myosin heavy chain protein (Miller et al. 1985). As reported earlier, there were no muscle spindles in NT-3 KO animals (Ernfors et al. 1994). On the other hand, Bax KO animals had more spindles than WT, and spindles were in clusters similar to animals over-expressing NT-3 in the muscle (Wright et al. 1997). Although NT-3-dependent cells were rescued, no muscle spindles were detected in the limbs of double KOs (Figure 4A and 4B). Muscle spindles could be observed with TrkC antibody in WT and Bax KO animals, but not in NT-3 or double KOs (Figure 4E). In both NT-3 and double KO animals, NF-M-labeled fibers could be detected in muscles, thus the muscles of these animals were not completely devoid of nerve fibers. Since there were no TrkC-positive fibers in these muscles, we think that these NF-M-labeled fibers correspond to motor axons. Absence of muscle spindles might be due to a failure in initiation of differentiation by proprioceptive axons, or a failure of maturation and maintenance in the absence of NT-3. To distinguish between these two possibilities, we investigated muscle spindle development at E15 and E17 with S46/NF-M immunostaining as well as DiI labeling. No structures with the characteristic muscle spindle shape could be detected in any of the genotypes at E15 (Figure S2). However, numerous developing spindles could be identified at E17 in WT and Bax KOs, but not in NT-3 KO embryos (Figure 4C). Bax/NT-3 double null muscles were devoid of spindles (Figure 4C), except for one spindle-like structure observed in one leg of an embryo (Figure 4C, inset, denoted by the asterisk). DiI labeling through the DRGs at E17 yielded similar results, with muscle spindles identified in parallel sections in WT and Bax null muscles (Figure 4D), but not in NT-3 or double KO animals. Although DiI-labeled fibers could be detected in double null muscles, they never formed ring-like structures characteristic of muscle spindles. We also examined TrkA/TrkC expression at P0 in the tibial nerve, which carries sensory fibers to the gastrocnemius muscle as well as the skin of the lower leg. In NT-3 KOs, TrkA-positive axons could be seen in the tibial nerve but there were no TrkC-positive axons; in contrast, TrkA- and TrkC-labeled axons are both present in WT, Bax KO, and double KO animals (Figure 4F). These results suggest that although proprioceptive axons follow proper trajectories in distal peripheral nerves, they fail to innervate their target muscles in the absence of NT-3. + +NT-3 Is a Chemoattractant for DRG Axons In Vitro + +To test the hypothesis that NT-3 acts as a chemoattractant for sensory axons, we performed a series of in vitro assays. Proprioceptive axons in mice enter the gray matter in the spinal cord and advance ventrally parallel to the midline by E13 and reach the motor neurons by E15 (Ozaki and Snider 1994). We co-cultured collagen-embedded E13 WT DRG explants with NT-3-soaked sepharose beads (n = 26). Control cultures were set up using bovine serum albumin (BSA)– or phosphate buffer saline (PBS)–soaked beads (n = 12). DRG axons began extending towards the localized NT-3 source by the end of the first day and consistently displayed a strong chemoattraction by 3 d in vitro, whereas they did not show such preference for BSA-loaded control beads (Figure 5A and 5B). This attraction was not due to survival support of NT-3 because Bax null ganglia displayed the same chemoattraction (Figure 5C; n = 16). NT-3 may act through either TrkC, or the p75NTR. We repeated the co-culture experiments with DRG explants derived from p75NTR KO mice (n = 18). Axons of these ganglia also showed strong chemoattraction towards the NT-3 beads (Figure 5D). Finally, we used diffusible TrkC receptors conjugated to IgG constant regions (TrkC-Fc) added to the medium (n = 6) to deplete soluble NT-3 from the collagen gels (Figure 5E). In the presence of TrkC-Fc the chemoattraction was completely blocked, demonstrating that the effect we see is specific for NT-3. In order to investigate the active range of our beads, we have repeated the cultures with WT E13 DRGs by placing the beads at increasing distances from the ganglia (n = 4 each) (Figure 5F). There was still preferred growth towards the bead at 1,200μm, the longest distance studied, although the number of axons and the extent of growth were not as robust. Next, we set up E13 DRG spinal cord explant cultures using NT-3-loaded beads placed at the midline at mid-spinal cord level as an ectopic NT-3 source (n = 15). Control cultures were set using PBS-loaded beads (n = 6). DiI labeling through the DRGs revealed numerous fibers entering the spinal cord at ectopic regions and growing towards the NT-3 beads (Figure 6A), surrounding the beads and forming bundles around them (Figure 6C–6E). In control cultures, all labeled fibers were directed towards the dorsal spinal cord and terminated there, where they normally enter the gray matter at E13 (Figure 6B). No axons were observed around the PBS-loaded control beads (Figure 6F). Axons that normally enter the gray matter through dorsal spinal cord grow towards the midline when presented with a localized NT-3 source, and new axon growth towards the NT-3 bead is initiated from DRGs, entering the spinal cord at ectopic loci at lateral mid-spinal cord (Figure 6G). NT-3 is therefore capable of acting as a chemoattractant for DRG axons. + +Discussion + +Relatively few studies have implicated NT-3 as a chemoattractant agent for sensory and motor axons. Previously noted chemoattractant action of the embryonic mouse maxillary process on trigeminal ganglion neurons (Lumsden and Davies 1986) is now attributed to NT-3 and brain-derived neurotrophic factor (BDNF) (O'Connor et al. 1999). Recently, Tucker et al. (2001) showed that developing sensory and motor axons in limb slice cultures preferentially grow towards neurotrophin-soaked beads instead of following their normal trajectories. Conversely, beads soaked with neurotrophin function-blocking antibodies led to reduction of sensory and motor axon growth towards the limb. In transgenic mice, which over-express NT-3 under the nestin promoter in the central nervous system, the course of the proprioceptive afferents are altered and directed towards the regions with high levels of ectopic NT-3 expression in the spinal cord (Ringstedt et al. 1997). Ringstedt et al. considered the possibility that NT-3 may play a chemoattractant role during the innervation of ventral horns by proprioceptive afferents. However, earlier findings of normal proprioceptive afferent trajectories in chicken embryos despite injection of function-blocking NT-3 antibody into the spinal cord (Oakley et al. 1995) led them to discount this possibility. In that study, though, assays on the effectiveness of antibody perturbation on NT-3-dependent cell survival showed that effectiveness was significant (approximately 90%) but not complete (Oakley et al. 1995). Ringstedt et al. (1997) argue that while central sensory axons may still navigate properly in the absence of NT-3, ectopic expression of NT-3 disrupts their targeting. Recently, another member of the neurotrophin family, BDNF, has been suggested to act as a chemoattractant for sensory axons innervating ear (Tessarollo et al. 2004). In a gene replacement strategy in which BDNF expression was driven by the NT-3 promoter, vestibular axons rerouted towards ectopic sources of BDNF in the cochlea that normally expressed NT-3 and were not innervated by these axons. Ectopic NT-3 supplied using osmotic pumps and adenovirus-mediated expression induce sensory axon growth during regeneration (Zhang et al. 1998, Bradbury et al. 1999, Oudega et al. 1999, Ramer et al. 2002), and induce axonal plasticity of corticospinal axons in injured adult spinal cord (Zhou et al. 2003), where the sprouting axons from the intact site cross the midline towards the NT-3 source on the lesion side of the spinal cord. Our present results are in agreement with these observations, and provide further evidence that NT-3 acts as a chemoattractant for sensory afferents. + +Previously, normal motor neuron innervation was rescued in NT-3 KO animals by over-expressing NT-3 selectively in their muscles (Wright et al. 1997). It appears that peripheral NT-3 alone is sufficient for rescuing proprioceptive neurons in NT-3 KO animals and proper axonal pathfinding in the spinal cord. However, it is unclear whether NT-3 is absent from the ventral spinal cords of these animals. The possibility has been raised that motor neurons may retrogradely transport NT-3 in muscle to the spinal cord (Chen and Frank 1999). Assays to determine whether any NT-3 is present in the ventral horns of these mice would be informative. + +Recently, Patel et al. (2003) reported their observations on Bax/NT-3 double KO mice they bred. Their results are significantly different from ours. They see no proprioceptive afferents in the periphery of the double knockouts and note that central proprioceptive afferents terminate in the intermediate spinal cord without extending ventrally. Their observations are based on PV immunostaining and DiI labeling of the peripheral nerves. We noted that PV immunolabeling is diminished in our Bax/NT-3 double KOs. In various manipulations of the neurotrophins it has been noted that molecular markers for proprioceptive axons such as PV or calcitonin gene-related peptide for nerve growth factor–responsive axons are compromised (Ringstedt et al. 1997; Patel et al. 2000), thus PV immunostaining in Bax/NT-3 double KOs cannot reveal the extent of proprioceptive axons in the periphery. + +Seventy-three percent of retrogradely labeled gastrocnemius muscle afferents were reported to be expressing TrkC RNA in the adult rat DRG, although some of these (about 10%) may represent cutaneous innervation (McMahon et al. 1994). Presently, it is not clear whether TrkC protein is also made by cutaneous afferents and if so at what stage in development this receptor is expressed by cutaneous axons. Based on the available evidence showing that all proprioceptive neurons are eliminated in NT-3 or TrkC null mice, we think that TrkC staining in the tibial nerve mostly represents proprioceptive axons in the vicinity of their peripheral target. Altogether, our findings suggest a role for NT-3 in initiation of muscle innervation and spindle differentiation by the proprioceptive axons. + +Patel et al. (2003) examined DiI-labeled central DRG axons in the spinal cord of three E17 and two P0 cases, and report that central proprioceptive axons stop in the intermediate laminae, never entering the ventral cord. We have examined nine Bax/NT-3 double KO cases, and often incomplete DiI labeling gives the impression that there are no axons reaching the ventral spinal cord. We have also seen cases similar to theirs (n = 4), but at higher magnification these axons did not have terminal boutons and were not completely labeled. However, with complete fills (n = 5), it was possible to trace these axons into the ventral midline and across to the contralateral side, and visualize terminal boutons at their tips (see Figure 3C). Previously, Arber et al. (2000) reported that members of the Ets family of transcription factors, Er81 and Pea3, are expressed by DRG neurons as well as motor neurons and their target muscle fibers. They found that in the spinal cord of Er81 KO mice, ventral projections of proprioceptive axons were mostly absent, and very few axons made it to the ventral cord. Patel et al. (2003) note that the phenotype they observed in their Bax/NT-3 double KO mice is quite similar to that of Er81 KO mice. They provide evidence that NT-3 induces Er81 expression in DRG explants in vitro. Patel et al. (2003) report that Er81 mRNA expression is diminished (but not abolished) in both NT-3 KO and Bax/NT-3 double KO mice, while their immunohistochemistry shows much less protein expression in the double KO mice. It is highly possible that a small but considerable number of DRG cells express the transcription factor Er81 and that their axons grow beyond the intermediate levels of the spinal cord in Bax/NT-3 double KO mice. While the phenotype of Er81 KO mice is quite dramatic, and most proprioceptive axons stop within the intermediate spinal cord, it is important to note that a few axons still find their way to the ventral spinal cord and target properly to the motor neurons (Arber et al. 2000). + +Patel et al. (2003) also present observations from islet2DTA mice, which lack a significant portion of the motor neurons in the ventral cord. In these mice PV immunostaining shows axons in the ventral horns. Patel et al. argue that since motor neurons are absent in these mice, NT-3 secreted by them could not be a signal for proprioceptive axons to enter the lateral motor columns. However, there is no evidence showing that NT-3 mRNA or protein expressed in the ventral spinal cord is exclusively from motor neurons, and there are no available data indicating that in islet2DTA mice, NT-3 expression in the ventral spinal cord is abolished (Yang et al. 2001; Pun et al. 2002). Studies in embryonic mice reported NT-3 mRNA in the ventral horns of the spinal cord, but it is not definitive that both mRNA and protein are expressed solely by motor neurons. In the adult spinal cord, while motor neurons express high levels of NT-3, other cells, including glia, also express it (Zhou and Rush 1994; Dreyfus et al. 1999; Buck et al. 2000). Our present results, along with those from transgenic mice with NT-3 over-expression in ectopic regions of the spinal cord (Ringstedt et al. 1997), argue for a role of NT-3 in chemoattractant axon guidance of proprioceptive axons in the spinal cord. + +Finally, in culture assays, we see a strong chemoattraction of DRG neurons to localized sources of NT-3. This response is seen in WT, Bax null and in p75 null DRG explants, and in the absence of any other neurotrophins or target-derived axon guidance molecules. Furthermore, in vitro, sensory axon response to NT-3 does not appear to be dose-dependent (Ringstedt et al. 1997; Tucker et al. 2001). NT-3 is capable of attracting axons along distances of up to 1 mm in collagen gel matrix, covering the physiological range it needs to attract axons during development. Previous studies with exogenous or local applications of NT-3 to developing primary sensory axons have indicated that this neurotrophin can attract and induce axonal branching (Ulupınar et al. 2000; Özdinler and Erzurumlu 2001, Özdinler et al. 2004). + +Along the monosynaptic stretch reflex pathway, only Wnt-3 has been implicated as an axon arborization factor in the spinal cord (Krylova et al. 2002). Another molecule, Slit2, expressed in the midline and by motor neurons (Wang et al. 1999) is capable of inducing axonal branching (Nguyen Ba-Charvet et al. 2001; Özdinler and Erzurumlu 2002). DRG neurons express Robo receptors, which bind to Slits, and proprioceptive axons are therefore capable of responding to Slit signals (Wang et al. 1999). Slit2 does not cause repulsion of NT-3-responsive DRG axons in vitro (Nguyen Ba-Charvet et al. 2001), but causes ectopic branching and arborization of trigeminal axons in the brainstem (Özdinler and Erzurumlu 2002). Thus, Slit2 might also be involved in terminal branching of propioceptive axons in the ventral cord and in the midline branching observed in our double KOs. + +Lack of PV expression in Bax/NT-3 KO mice suggests that PV expression might be responsible for proper axon targeting and muscle spindle differentiation. Presently we cannot completely rule out this possibility. However, no defects in axon pathfinding along the monosynaptic reflex arc or in muscle spindle differentiation have been noted in PV KO mice, which develop normally and show no apparent changes in their behavior or physical activity (Schwaller et al. 1999). These observations suggest that axonal targeting defects in Bax/NT-3 double KO mice cannot be simply due to lack of PV expression in proprioceptive cells. + +Our present results suggest that NT-3 acts as a short-range axon guidance cue for proprioceptive axons centrally and peripherally, as they navigate to their targets using other axon guidance cues. In its absence, these axons terminate in inappropriate loci. However, NT-3 may not be the only molecule that plays a role in targeting and terminal branching of sensory axons in the ventral spinal cord. NT-3 most likely acts cooperatively with other axon guidance molecules or by regulating expression of yet to be identified proprioceptive neuron-specific receptors/ligands for numerous axon guidance cues. + +Materials and Methods + + + +Generation of double KOs. + +We crossed Bax KO females on a C57BL/6 background (Jackson Laboratory, Bar Harbor, Maine, United States) to NT-3 heterozygote males on a 129 Sv background to generate double heterozygote animals. Progeny was genotyped with PCR, and animals heterozygous for both genes were bred to obtain the double KOs. Primers used for the Bax locus were R661, GTT GAC CAG AGT GGC GTA GG; R662, CCG CTT CCA TTG CTC AGC GG; and R663, GAG CTG ATC AGA ACC ATC ATG. Primers used for the NT-3 locus were NT3A, CGT GGT GAG GTT CTA TTG GCT AC; NT3B, CAG AGC ACC CTG CCC AAA GCA GAG; NT3R, CCT TGA CAA TAC TGA ATG CC; and NEOF, GGG AAC TTC CTG ACT AGG. WT, Bax KO, and NT-3 KO littermates were used as controls. A total of 11 Bax/NT-3 double KO mice were analyzed, of these nine were P0 pups. The p75 colony on a 129 S1 background was received from Jackson Laboratory. We used tail DNA to genotype animals using the primers IMR0013, CTT GGG TGG AGA GGC TAT TC; IMR0014, AGG TGA GAT GAC AGG AGA TC (generic neo primers); IMR0710, TGT TAC GTT CTC TGA CGT GGT GAG; and IMR0711, TCA GCC CAG GGT GTG CAC TC (p75 locus). For embryonic experiments, day of plug positivity was considered E0. All of the protocols used in this study were approved by the Louisiana State University Health Sciences Center Institutional Animal Care and Use Committee and conformed to the National Institutes of Health guidelines for use of experimental animals. + +TrkA/TrkC immunohistochemistry. + +Frozen spinal cord sections (10 μm thick) were blocked and incubated in a cocktail of rabbit anti-TrkA and goat anti-TrkC antibodies (gift of Dr. Reichardt; Huang et al. 1999), followed by a cocktail of CY3 conjugated donkey anti-rabbit and FITC conjugated donkey anti-goat antibodies (Chemicon, Temecula, California, United States) in the presence of 0.3% TritonX-100 and 10% normal donkey serum. For PV immunohistochemistry, sections were reacted with monoclonal mouse anti-PV antibody (Sigma, St. Louis, Missouri, United States), and developed by Vector fluorescein mouse on mouse kit (Vector Laboratories, Burlingame, California, United States). For quantification purposes, TrkA- and TrkC-labeled cells from four different DRGs of each genotype and age studied were counted, and ratios plotted. + +Muscle spindle detection. + +Gastrocnemius muscle was dissected out in P0 pups and sectioned longitudinally, or, in some cases, the whole leg at the level of gastrocnemius muscle with tibial nerve was sectioned in cross section. Sections were incubated with monoclonal S46 antibody (gift of Dr. Stockdale) reactive to the spindle-specific slow-tonic myosin heavy chain isoform and developed by Vector mouse on mouse kit as described above, followed by rabbit anti-NF-M antibody (Chemicon) and CY3 conjugated goat anti-rabbit antibody (Chemicon) in a sequential double-labeling protocol. In another method, DiI crystals were placed in DRGs of E17 embryos from different genotypes (n = 2), and the gastrocnemius muscle was isolated and sectioned into 40-μm-thick slices on a vibratome. + +DiI labeling. + +Spinal cords were dissected out with the DRGs attached, motor root was cut to prevent backfilling of motor neurons, and crystals of 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI; Molecular Probes, Eugene, Oregon, United States) were inserted in DRGs. Labeled spinal cords were incubated at 37 °C for 8 d and sectioned on a vibrotome at 100 μm. Sections were observed under the fluorescent microscope and photographed, and images were transferred to Adobe Photoshop, inverted, and adjusted for brightness and contrast. Photoconverted in 0.15% DAB (3,3′-diaminobenzidine; Sigma) in 0.1M Tris buffer (pH 8.2). + +In vitro co-culture assay. + +DRG explants were derived from Swiss Webster, Bax null, and p75 null mouse embryos (E13). For collagen matrix assays, DRGs were dissected out under a stereomicroscope using tungsten needles. Collagen matrix was prepared with 430 μl of collagen (3 mg/ml dissolved in 0.1M acetic acid, Sigma), 50 μl of 10X DMEM medium, and 2.5 μl of 0.8 M NaCa2, and the pH was adjusted to 7.5. Individual ganglion explants were placed in 24-well plates and covered with freshly prepared collagen. Sepharose beads with an average diameter of 150 μm were used. Beads were washed twice with PBS, air dried, and loaded with 10, 20, 50, or 100 ng/μl NT-3 (Collaborative Research, Chemicon) at 4 °C overnight with constant shaking. For negative control, beads were loaded with BSA (10–100 ng/μl) or PBS. Either a single neurotrophin-loaded bead or a single BSA (or PBS)–loaded bead was implanted about 200–1,200 μm away from the ganglion explant. Collagen-embedded cultures were then placed at 33 °C for 15 min for the matrix to harden. Serum-free culture medium was then added to each well. In cultures with WT DRG and control beads, 10% serum was added to ensure viability of the explants. TrkC-Fc (Regeneron Pharmaceuticals, Tarrytown, New York, United States) was added (20 μg/ml) into the culture medium. + +Explant co-cultures + +Spinal cords with attached DRGs were dissected out from Swiss Webster mouse embryos at E13, and sectioned into 300-μm-thick slices. Tissue slices were placed on Millicell Tissue Culture Inserts (Millipore, Billrica, Massachusetts, United States). NT-3-loaded (n = 15) or PBS-loaded (n = 6) sepharose beads were prepared as described above, and placed in the midline at mid-spinal cord level. Inserts were placed in six-well plates with serum-free medium at the bottom of the wells, and kept at 33 °C for 3 d in the presence of 5% CO2. Cultures were then fixed with 4% paraformaldehyde in PBS, and small crystals of DiI were placed into the DRGs. Samples were incubated in a 37-°C incubator, allowing the dye to diffuse, and photographed under a Nikon (Tokyo, Japan) inverted epifluoresence microscope. + +Supporting Information + +Figure S1 + +Motor Neuron Innervation Initiated in WT E17 Embryos Cannot Be Detected in Bax/NT-3 Double KOs + +In some of the samples, motor neurons were labeled by backfilling through the ventral root in addition to the sensory axons labeled through the DRGs. + +(A) WT embryo showing proprioceptive axons contacting motor neuron dendrites in the ventral horn, forming synapses. + +(B) Bax/NT-3 double null spinal cord. Although labeled fibers enter the ventral spinal cord, they extend towards the midline instead of the ventral horn and never contact the motor neuron dendrites. + +(C) High-power image of the inset in (A). Arrows point to the proprioceptive fibers contacting motor neurons (asterisk). + +(D) High-power image of the inset in (B). Notice that there are no sensory axons contacting labeled motor neurons (asterisk). + +Scale bar: 100 μm (A and B), 50 μm (C and D). + +(24 MB TIF). + +Click here for additional data file. + +Figure S2 + +S46/NF-M Immunohistochemistry at E15 Gastrocnemius Muscle + +Although numerous muscle and nerve fibers were labeled, no muscle spindles could be identified because the characteristic morphology of sensory nerve ending wrapped around muscle bag fiber had not begun to develop in any of the genotypes yet. Scale bar: 25 μm. + +(13 MB TIF). + +Click here for additional data file. + +Acknowledgements + +We thank L. Tessarollo for providing NT-3 KO animals, L. Reichardt for TrkA and TrkC antibodies, F. Stockdale for the S46 antibody, K. Muneoka for sepharose beads, and B. King for maintenance of animals used in this study. We particularly thank E. Frank for many helpful comments and discussions about this study and for sharing unpublished data from his laboratory. Supported by National Institutes of Health/National Institute of Dental and Craniofacial Research grant P01 DE07734. + +Abbreviations + +BDNF - brain-derived neurotrophic factor + +BSA - bovine serum albumin + +DRG - dorsal root ganglion + +E - embryonic day + +KO - knockout + +NF-M - neurofilament-M + +NT-3 - neurotrophin-3 + +P - postnatal day + +PBS - phosphate buffer saline + +PV - parvalbumin + +TrkC-Fc - diffusible TrkC receptors conjugated to IgG constant regions + +WT - wild-type + +Figures and Tables + +Figure 1 + +TrkA/TrkC and PV Immunohistochemistry in DRG and Spinal Cord + +Red represents TrkA, green represents TrkC (and PV in [C]), and yellow represents co-expression. + +(A) TrkA/TrkC immunostaining in E15 DRG. TrkC-positive neurons normally eliminated in NT-3 KOs are rescued in double KOs. + +(B) TrkA/TrkC immunostaining at P0 in DRG. + +(C) PV immunostaining in P0 DRG. Rescued TrkC-positive cells fail to express PV. + +(D) Ratio of TrkA-immunopositive cells to TrkC-immunopositive cells in E15 and P0 DRGs. Data are presented as percentage of cells with standard deviation. Double KOs always had similar ratios to Bax KOs, and NT-3 KOs had the least amount of TrkC-positive cells, if any. + +(E) TrkA/TrkC immunostaining in E15 spinal cord. Arrow points to group Ia fibers. Dorsal is up. + +Scale bar: 50 μm (A–C), 1 mm (E). + +Figure 2 + +Axonal Projections in the Spinal Cord after DiI Labeling of DRG at P0 + +(A) Rescued DRG proprioceptive neurons fail to properly innervate motor neurons in double KOs. Instead, some axons are directed towards the ventral midline; they cross the midline and branch. + +(B) Schematic drawing of the monosynaptic reflex arc as it normally develops. Small black dots represent NT-3 released centrally by the motor neurons and peripherally by the muscle spindles. + +(C) High-power magnification of the inset in (A). Arrow points to the midline, and arrowheads point to synaptic bouton-like structures. + +Scale bar: 1 mm (A), 400 μm (C). + +Figure 3 + +Sensory Axons Labeled with DiI through the DRG at E17 + +(A) DiI-labeled fibers in WT spinal cord. Notice proprioceptive axons extending towards the motor neurons located in the ventral horn of the spinal cord in cross section. + +(B) Bax null spinal cord. + +(C) NT-3 null spinal cord. Stained fibers are restricted to the nociceptive axons in the dorsal horn, as evidenced by the complete absence of labeling in the ventral spinal cord. + +(D) Bax/NT-3 double null spinal cord. Although fibers extend into the ventral spinal cord, they never grow towards the motor neurons, but are directed towards the midline instead. + +Scale bar: 100 μm. + +Figure 4 + +Muscle Spindles in Gastrocnemius Muscle and TrkA/TrkC Staining in the Tibial Nerve at P0 + +(A) NF-M (red) and S46 (green) immunostaining in cross section of gastrocnemius muscle at P0. There are no muscle spindles detected in double KOs. + +(B) NF-M (red) and S46 (green) immunostaining in parallel sections of gastrocnemius muscle at P0. + +(C) NF-M (red) and S46 (green) immunostaining in parallel sections of gastrocnemius muscle at E17. Double null muscles are mostly devoid of muscle spindles, except for one spindle-like structure detected (shown in inset, denoted by the asterisk). + +(D) Muscle spindles detected by DiI labeling through the DRG. Gastrocnemius muscle is sectioned at 40 μm thickness in parallel plane to the muscle fibers. + +(E) Muscle spindles detected by TrkC staining in cross section of gastrocnemius muscle at P0. + +(F) TrkA (red) and TrkC (green) immunostaining in the tibial nerve cross section at P0. TrkC-positive fibers are missing in NT-3 KOs. Red-green overlap (yellow) is due to the thickness of the section and overlapping of red- and green-labeled (TrkA and TrkC) fibers present at different focal depths, rather than co-localization. + +Arrows indicate muscle spindles. + +Scale bar: 50 μm (A, B, D), 25 μm (C, E), 75 μm (F). + +Figure 5 + +Chemoattraction of E13 DRG Axons to Local NT-3 Observed by In Vitro Co-Culture Assays + +(A) WT DRG with NT-3-loaded bead. + +(B) WT DRG with BSA-loaded bead. + +(C) Bax null DRG with NT-3-loaded bead. + +(D) p75 null DRG with NT-3-loaded bead. + +(E) WT DRG with NT-3-loaded bead and TrkC-Fc in the medium. + +(F) WT DRG with NT-3 loaded beads placed at increasing distances away from the ganglia (range, 500–1,200 μm). + +Scale bar: 150 μm (A–E), 350 μm (F). + +Figure 6 + +Chemoattraction Towards NT-3 Beads Placed in E13 Spinal Cord DRG Explant Co-Cultures + +(A) NT-3 bead placed in the midline of E13 WT spinal cord. Notice axons labeled through the DRGs (circled with black dashed lines) growing towards the bead (circled with white dashed lines) enter the spinal cord at ectopic loci instead of dorsal spinal cord. + +(B) PBS-loaded bead in E13 spinal cord. All labeled axons extend along the dorsal spinal cord, where they terminate. + +(C) High-power image of the bead in (A). Notice labeled axons surrounding the bead. + +(D) High-power image of an NT-3-loaded bead. Notice axons bundled around the bead. + +(E) High-power image of an NT-3-loaded bead. Notice the axons approaching the bead via the dorsal spinal cord. + +(F) High-power image of a PBS-loaded bead. No labeled fibers were observed around control beads. + +(G) Summary of our observations from E13 spinal cord DRG organotypic cultures. In control cultures fibers extend along the dorsal spinal cord, where they normally enter the gray matter at E13. In the presence of an ectopic NT-3 source localized at the midline, these axons grow towards the NT-3 bead. NT-3 also initiates axon growth from the DRGs, entering the spinal cord at ectopic lateral loci, growing towards the bead, surrounding the bead, forming nerve bundles, and branching around it. + +Scale bar, 175 μm (A and B), 100 μm (C–F). + +Footnotes + +Conflicts of interest. The authors have declared that no conflicts of interest exist. + +Author contributions. BG and RE conceived and designed the experiments. BG, AEM, and PHO performed the experiments. BG, PHO, and RE analyzed the data. BG and RE wrote the paper. + +Academic Editor: Joshua R. Sanes, Harvard University + +¤Current address: Massachusetts General Hospital, Harvard Medical School Center for Nervous System Repair, Boston, Massachusetts, United States of America + +Citation: Genç B, Özdinler PH, Mendoza AE, Erzurumlu RS (2004) A chemoattractant role for NT-3 in proprioceptive axon guidance. 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PR:000014774 9154 9158 SP-C +T290 PR:000003244 9159 9164 C-Raf +T291 PR:000004619 9169 9174 BAG-1 +T292 PR:000004619 9234 9239 BAG-1 +T293 GO:0065007 9247 9257 regulation +T294 UBERON:0000922 9311 9320 embryonic +T295 PR:000004619 9330 9335 BAG-1 +T296 UBERON:0000922 9341 9348 embryos +T297 PR:000026878 9383 9402 activated caspase-3 +T298 CHEBI:78897 9407 9418 trypan blue +T299 GO:0006915 9495 9504 apoptosis +T300 UBERON:0002107 9512 9518 livers +T301 PR:000004619 9522 9527 BAG-1 +T302 UBERON:0000922 9531 9538 embryos +T303 GO:0008283 9568 9581 Proliferation +T304 PR:000004619 9621 9626 BAG-1 +T305 NCBITaxon:10088 9640 9644 mice +T306 PR:000004619 9711 9716 BAG-1 +T307 GO:0010467 9717 9727 expression +T308 NCBITaxon:33208 9744 9751 animals +T309 GO:0008283 9772 9790 cell proliferation +T310 http://purl.obolibrary.org/obo/MONDO_0004972 9798 9806 adenomas +T311 GO:0008283 9821 9834 proliferating +T312 PR:000012421 9821 9851 proliferating cellular antigen +T313 CHEBI:59132 9844 9851 antigen +T314 PR:000012421 9853 9857 PCNA +T315 GO:0008283 9929 9942 proliferating +T316 http://purl.obolibrary.org/obo/MONDO_0004972 9943 9950 adenoma +T317 PR:000014774 9965 9969 SP-C +T318 PR:000003244 9970 9975 C-Raf +T319 NCBITaxon:33208 9980 9987 animals +T320 PR:000004619 10018 10023 BAG-1 +T321 http://purl.obolibrary.org/obo/MONDO_0004972 10065 10072 adenoma +T322 PR:000010425 10092 10097 Ki-67 +T323 GO:0008283 10107 10120 proliferation +T324 PR:000004770 10132 10137 Bmi-1 +T325 GO:0000785 10141 10150 chromatin +T326 GO:0010467 10170 10179 expressed +T327 CL:0000034 10183 10193 stem cells +T328 PR:000004619 10220 10225 BAG-1 +T329 UBERON:0002048 10279 10283 lung +T330 http://purl.obolibrary.org/obo/MONDO_0004972 10360 10368 adenomas +T331 PR:000014774 10372 10376 SP-C +T332 PR:000003244 10377 10382 C-Raf +T333 NCBITaxon:33208 10387 10394 animals +T334 PR:000004619 10425 10430 BAG-1 +T335 GO:0000165 10453 10471;10476 10493 signalling through ... mitogenic cascade +T336 CHEBI:52290 10476 10485 mitogenic +T337 PR:000004619 10518 10523 BAG-1 +T338 http://purl.obolibrary.org/obo/MONDO_0004972 10546 10553 adenoma +T339 http://purl.obolibrary.org/obo/MONDO_0005070 10575 10582 Tumours +T340 PR:000004619 10789 10794 BAG-1 +T341 GO:0010467 10843 10853 expression +T342 http://purl.obolibrary.org/obo/MONDO_0005070 10881 10887 tumour +T343 http://purl.obolibrary.org/obo/MONDO_0004992 10926 10932 cancer +T344 GO:0065007 10992 11001 regulated +T345 PR:000004619 11133 11138 BAG-1 +T346 GO:0010467 11139 11149 expression +T347 http://purl.obolibrary.org/obo/MONDO_0005070 11171 11177 tumour +T348 GO:0006915 11187 11196 apoptosis +T349 http://purl.obolibrary.org/obo/MONDO_0004972 11247 11254 adenoma +T350 PR:000004619 11283 11288 BAG-1 +T351 PR:000003244 11304 11309 C-Raf +T352 PR:000008812 11313 11318 Hsc70 +T353 PR:000003425 11319 11324 Hsp70 +T354 PR:000004619 11429 11434 BAG-1 +T355 SO:0001060 11435 11443 isoforms +T356 PR:000004619 11473 11478 BAG-1 +T357 NCBITaxon:10088 11489 11494 mouse +T358 SO:0001060 11503 11511 isoforms +T359 PR:000004619 11515 11520 BAG-1 +T360 PR:000025855 11522 11525 p50 +T361 PR:000025856 11530 11533 p32 +T362 UBERON:0000922 11578 11585 embryos +T363 PR:000004619 11609 11614 BAG-1 +T364 UBERON:0000948 11647 11652 heart +T365 PR:000004619 11677 11682 BAG-1 +T366 GO:0006915 11717 11726 apoptosis +T367 PR:000004619 11819 11824 BAG-1 +T368 PR:000003244 11854 11859 C-Raf +T369 GO:0005741 11867 11895 outer mitochondrial membrane +T370 PR:000003244 11926 11931 C-Raf +T371 GO:0006915 11938 11947 apoptosis +T372 PR:000002184 11972 11975 BAD +T373 GO:0010467 12014 12023 expressed +T374 PR:000003244 12024 12029 C-Raf +T375 GO:0032991 12076 12083 complex +T376 PR:000003425 12089 12094 Hsp70 +T377 GO:0032991 12135 12142 complex +T378 PR:000025350 12148 12153 Hsp90 +T379 PR:000004619 12189 12194 BAG-1 +T380 GO:0065007 12210 12218 regulate +T381 GO:0005739 12261 12273 mitochondria +T382 PR:000004619 12475 12480 BAG-1 +T383 CHEBI:35222 12481 12490 inhibitor +T384 http://purl.obolibrary.org/obo/MONDO_0004992 12534 12540 cancer +T385 SO:0000704 12590 12597 genetic +T386 PR:000004619 12628 12633 BAG-1 +T387 GO:0010467 12634 12644 expression +T388 GO:0016246 12663 12700 RNA interference-based gene silencing +T389 SO:0000704 12686 12690 gene +T390 PR:000004619 12719 12724 BAG-1 +T391 GO:0010467 12729 12739 expression +T392 NCBITaxon:9606 12761 12766 human +T393 http://purl.obolibrary.org/obo/MONDO_0005070 12767 12774 tumours +T394 CHEBI:23888 12781 12786 Drugs +T395 SO:0000409 12808 12820 binding site +T396 PR:000008812 12824 12829 Hsc70 +T397 PR:000003425 12830 12835 Hsp70 +T398 PR:000004619 12916 12921 BAG-1 +T399 SO:0000417 12938 12944 domain +T400 PR:000004619 12987 12992 BAG-1 +T401 CHEBI:35222 12993 13003 inhibitors +T402 SO:0000417 13081 13087 domain +T403 PR:000004619 14070 14075 BAG-1 +T404 PR:000003244 14103 14108 C-Raf +T405 http://purl.obolibrary.org/obo/MONDO_0004972 14116 14123 adenoma +T406 http://purl.obolibrary.org/obo/MONDO_0004972 14136 14143 Adenoma +T407 PR:000014774 14158 14162 SP-C +T408 PR:000003244 14163 14168 C-Raf +T409 NCBITaxon:10088 14173 14177 mice +T410 PR:000004619 14179 14184 BAG-1 +T411 PR:000004619 14199 14204 Bag-1 +T412 PR:000004619 14237 14242 BAG-1 +T413 http://purl.obolibrary.org/obo/MONDO_0004972 14276 14283 Adenoma +T414 NCBITaxon:10088 14334 14338 mice +T415 http://purl.obolibrary.org/obo/MONDO_0004972 14418 14425 Adenoma +T416 UBERON:0002048 14438 14443 lungs +T417 NCBITaxon:10088 14460 14464 mice +T418 NCBITaxon:10088 14516 14520 mice +T419 CHEBI:51686 14587 14598 hematoxylin +T420 UBERON:0002048 14625 14630 lungs +T421 PR:000014774 14636 14640 SP-C +T422 PR:000003244 14641 14646 C-Raf +T423 NCBITaxon:10088 14662 14666 mice +T424 PR:000004619 14680 14685 BAG-1 +T425 PR:000004619 14705 14710 BAG-1 +T426 PR:000004619 14766 14771 BAG-1 +T427 GO:0010467 14772 14782 expression +T428 UBERON:0002048 14790 14794 lung +T429 PR:000014774 14798 14802 SP-C +T430 PR:000003244 14803 14808 C-Raf +T431 NCBITaxon:10088 14824 14828 mice +T432 GO:0010467 14872 14882 expression +T433 PR:000004619 14886 14891 BAG-1 +T434 UBERON:0002048 14899 14904 lungs +T435 PR:000014774 14920 14924 SP-C +T436 PR:000003244 14925 14930 C-Raf +T437 NCBITaxon:10088 14946 14950 mice +T438 PR:000004619 14994 14999 BAG-1 +T439 PR:000004619 15054 15059 BAG-1 +T440 GO:0010467 15060 15070 expression +T441 PR:000004619 15076 15081 BAG-1 +T442 UBERON:0000922 15093 15102 embryonic +T443 UBERON:0002107 15112 15117 liver +T444 UBERON:0002107 15143 15148 liver +T445 GO:0042571 15260 15268 antibody +T446 PR:000004619 15345 15350 BAG-1 +T447 PR:000014774 15369 15373 SP-C +T448 PR:000003244 15374 15379 C-Raf +T449 NCBITaxon:10088 15395 15400 mouse +T450 UBERON:0002048 15401 15405 lung +T451 http://purl.obolibrary.org/obo/MONDO_0008903 15401 15412 lung cancer +T452 UBERON:0000479 15413 15419 tissue +T453 GO:0006915 15442 15451 apoptosis +T454 PR:000012421 15469 15473 PCNA +T455 http://purl.obolibrary.org/obo/MONDO_0005070 15487 15492 tumor +T456 PR:000014774 15502 15506 SP-C +T457 PR:000004619 15527 15532 BAG-1 +T458 NCBITaxon:10088 15546 15550 mice +T459 GO:0006915 15608 15617 apoptosis +T460 GO:0042571 15627 15635 antibody +T461 PR:000026878 15649 15668 activated caspase-3 +T462 PR:000012421 15674 15678 PCNA +T463 http://purl.obolibrary.org/obo/MONDO_0004972 15720 15727 adenoma +T464 PR:000014774 15751 15755 SP-C +T465 PR:000003244 15756 15761 C-Raf +T466 NCBITaxon:10088 15777 15781 mice +T467 PR:000004619 15799 15804 BAG-1 +T468 NCBITaxon:10088 15865 15869 mice +T469 PR:000004619 15971 15976 BAG-1 +T470 PR:000003244 15981 15986 C-Raf +T471 PR:000004619 16102 16107 BAG-1 +T472 PR:000003244 16137 16142 C-Raf +T473 GO:0005741 16150 16178 outer mitochondrial membrane +T474 PR:000003244 16209 16214 C-Raf +T475 GO:0006915 16221 16230 apoptosis diff --git a/src/ontogpt/evaluation/craft/database/all/15560850.txt b/src/ontogpt/evaluation/craft/database/all/15560850.txt new file mode 100644 index 000000000..30f9c99ab --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15560850.txt @@ -0,0 +1,101 @@ +BAG-1 haplo-insufficiency impairs lung tumorigenesis + +Abstract + +Background + +BAG-1 is a multifunctional co-chaperone of heat shock proteins (Hsc70/Hsp70) that is expressed in most cells. It interacts with Bcl-2 and Raf indicating that it might connect protein folding with other signaling pathways. Evidence that BAG-1 expression is frequently altered in human cancers, in particular in breast cancer, relative to normal cells has been put forward but the notion that overexpression of BAG-1 contributes to poor prognosis in tumorigenesis remains controversial. + +Methods + +We have evaluated the effect of BAG-1 heterozygosity in mice in a model of non-small-cell lung tumorigenesis with histological and molecular methods. We have generated mice heterozygous for BAG-1, carrying a BAG-1 null allele, that in addition express oncogenic, constitutively active C-Raf kinase (SP-C C-Raf BxB) in type II pneumocytes. SP-C C-Raf BxB mice develop multifocal adenomas early in adulthood. + +Results + +We show that BAG-1 heterozygosity in mice impairs C-Raf oncogene-induced lung adenoma growth. Lung tumor initiation was reduced by half in BAG-1 heterozygous SP-C C-Raf BxB mice compared to their littermates. Tumor area was reduced by 75% in 4 month lungs of BAG-1 haploinsufficient mice compared to mice with two BAG-1 copies. Whereas BAG-1 heterozygosity did not affect the rate of cell proliferation or signaling through the mitogenic cascade in adenoma cells, it increased the rate of apoptosis. + +Conclusion + +Reduced BAG-1 expression specifically targets tumor cells to apoptosis and impairs tumorigenesis. Our data implicate BAG-1 as a key player in oncogenic transformation by Raf and identify it as a potential molecular target for cancer treatment. + +Background + +BAG-1 is a multifunctional protein that is expressed in most cells. Originally identified as a Bcl-2 binding protein [1], other interaction partners of BAG-1 were described, including the serine threonine kinase C-Raf [2]. The C-terminal "BAG domain" of BAG-1 mediates the interaction with the Hsc70 and Hsp70 heat shock proteins [3], molecular chaperones that bind proteins in non-native states assisting them to reach a functional active conformation [4]. BAG-1 acts as a nucleotide exchange factor in this activation cycle [3]. The above findings indicated that BAG-1 might connect protein folding with other signaling pathways. Signaling networks promoting cell growth and proliferation are frequently deregulated in cancer [5]. The classical mitogenic cascade transmits stimuli from growth factor receptors via Ras, Raf, MEK and ERK to the cell nucleus [6]. C-Raf, like A- and B-Raf kinases also act at the outer membrane of mitochondria to augment cell survival [7,8]. Previously we had observed the stimulation of C-Raf kinase activity by BAG-1 in vitro [2]. Ras and B-Raf mutations have been found in various human cancers [9,10]. Evidence that BAG-1 expression is frequently altered in human cancers, in particular in breast cancer, relative to normal cells has been put forward but the notion that overexpression of BAG-1 contributes to poor prognosis in tumorigenesis remains controversial [11]. + +Methods + +Animals + +Mice used in these studies were generated and maintained according to protocols approved by the animal care and use committee at University of Würzburg. To inactivate the BAG-1 gene, we constructed a vector where exons 1 and 2 are replaced with a neomycin resistance gene. A phage clone with a 15-kb genomic insert from mouse strain 129/Sv spanning all seven exons of BAG-1 was identified and characterised using standard methods. The targeting construct contained 1,1-kb from the BAG-1 locus upstream of the neomycin resistance gene of plasmid pPNT [12] and 6-kb downstream. The upstream arm of 1,1 kb is located 5' to the start codon in the first exon of BAG-1 and the 3' arm of 6 kb is located downstream of exon 2. The mutation was introduced into embryonic stem cells by homologous recombination. Positive clones were identified by Southern blot analysis. Germline transmitting chimeras were obtained and bred to C57BL/6 mice. Further details will be described elsewhere. Heterozygous BAG-1 mice were genotyped by a PCR assay. The targeted BAG-1 allele was detected with primers P1 (5'-GAG TCT CCC GAT CCC TTT TCC), located upstream of exon 1, and P2 (5'-GAT TCG CAG CGC ATC GCC TT), located in the neomycin resistance gene, yielding a product of 600 base pairs. BAG-1 heterozygous mice were backcrossed at least three times onto C57BL/6 background before crossing with SP-C C-Raf BxB mice. Lung tumour mice expressing oncogenic C-Raf BxB were backcrossed at least six times onto C57BL/6 background. + +Western blot + +For the analysis of BAG-1 expression, lung lysates of the indicated genotypes were separated on 12,5% polyacrylamide-SDS (sodium dodecyl sulphate) gel, transferred to nitrocellulose Protran BA83 membrane (Schleicher&Schüll) and probed with rabbit anti-BAG-1 (FL-274) antibody (1:250, Santa Cruz Biotechnology). Amounts of protein were determined by Bradford protein assay to ensure equal protein loading for the analysis. Blots were developed using the appropriate horseradish peroxidase coupled secondary antibody and the ECL system (Amersham Pharmacia Biotech). Subsequently, the membrane was stripped and reprobed with rabbit antibody to glyceraldehyde 3 phosphate dehydrogense (1:2000, ab9485, Abcam Ltd.). + +Histopathology and immunohistochemistry + +Animals were sacrificed and lungs were fixed under 25 cm water pressure with 4% paraformaldehyde and embedded in paraffin. 5 μm sections were stained with hematoxylin and eosin and analysed. Pictures were taken using a Leica DMLA microscope and a Hitachi HV-C20A colour camera. Immunohistochemical staining to detect activated caspase-3, phospho-ERK (extracellular signal-regulated kinase), PCNA (proliferating cell nuclear antigen) have been described elsewhere [13]. Apoptotic, PCNA and p-ERK indices were determined by evaluating randomly chosen adenomas or fields of normal lung in 3–4 sections and determining the percentage of positive cells per 2000 cells at ×400. + +Results and discussion + +BAG-1 heterozygosity impairs C-Raf driven tumorigenesis + +In order to assess the functional role of BAG-1 on tumorigenesis, we have generated a null allele of BAG-1. To inactivate the BAG-1 gene, exons 1 and 2 were replaced with a neomycin resistance gene. This strategy was chosen to disrupt the expression of all known isoforms of BAG-1 which are generated by alternate translation initiation of a single mRNA; the start codons are present in exons 1 and 2. Western blot analysis of liver protein extracts of BAG-1 deficient embryos showed the complete loss of all BAG-1 protein isoforms. Embryos homozygous for this allele died at midgestation at around E13,5, but the heterozygous animals (BAG-1+/-) are normal. A comprehensive description of the BAG-1-/- phenotype is subject of another manuscript. + +Previously, we had generated a lung cancer mouse model by targeting constitutively active C-Raf kinase (SP-C C-Raf BxB) to the lung [14]. These mice develop multifocal adenomas early in adulthood. Based on the observation, that BAG-1 can activate C-Raf [2], we asked whether heterozygosity for BAG-1 would affect C-Raf BxB driven adenoma growth. We observed that lung tumour initiation was reduced by half in 1, 2 and 4 months old BAG-1+/- mice transgenic for SP-C C-Raf BxB compared to their BAG-1+/+ littermates. Tumour area was reduced by 75% in 4 month lungs of BAG-1 haploinsufficient mice compared to mice with two BAG-1 copies, see Figure 1. The histological picture emphasises the difference in adenoma formation between a representative SP-C C-Raf BxB/BAG-1+/+ and SP-C C-Raf BxB/BAG-1+/- lung. The difference in the staining intensity of the two lung sections derives mainly from the observation that the adenoma cells have a tendency to bind more intensively hematoxylin and eosin compared to normal lung cells. Thus, reduction of the BAG-1 gene dosage impairs the oncogenic activity of C-Raf in vivo. + +Reduced BAG-1 expression in BAG-1 heterozygous lungs + +Quantitative immunoblots demonstrated that the specific BAG-1 protein concentration in the lungs of BAG-1+/- mice was half the amount of BAG-1+/+ littermates, see Figure 2a. Moreover, immunohistochemical staining showed that BAG-1 was expressed in adenoma cells, see Figure 2b. There was no obvious difference in the BAG-1 immunohistochemistry of SP-C C-RafBxB/BAG-1+/+ and SP-C C-RafBxB/BAG-1+/- lungs. + +Tumour cells of BAG-1 heterozygous mice show increased apoptosis + +Concerning the molecular mechanism how a reduction of the BAG-1 protein expression in the heterozygous mice would impair tumorigenesis, we determined the fraction of apoptotic cells. Staining for activated caspase-3 revealed indistinguishable apoptosis in healthy regions of the lung of 1 month old SP-C C-Raf BxB mice with either one or two BAG-1 alleles, in line with the unaltered, normal lung structure of BAG-1+/- mice. In the adenomas, however, we observed a significant increase of apoptotic cells in BAG-1+/- SP-C C-Raf BxB mice compared with their SP-C C-Raf BxB/BAG-1+/+ littermates, see Figure 3a. This mechanism of action of BAG-1 on the regulation of cell survival is compatible with the phenotype of embryonic day 12,5 BAG-1 null embryos. Immunohistochemical staining for activated caspase-3 and trypan blue staining of dissociated cells showed hypocellularity and elevated levels of apoptosis in the livers of BAG-1-/- embryos (unpublished observations). + +Proliferation and p-ERK signalling are unaffected in BAG-1 heterozygous mice + +To exclude the alternative mechanism that the decreased level of BAG-1 expression in heterozygous animals would cause reduced cell proliferation in the adenomas, we performed proliferating cellular antigen (PCNA) staining. No significant differences were observed in the fraction of proliferating adenoma cells between SP-C C-Raf BxB animals heterozygous or wild type for BAG-1, see Figure 3b. Also, the percentages of adenoma cells positive for Ki-67, another proliferation marker and Bmi-1, a chromatin-associated protein expressed in stem cells, were not affected by the BAG-1 heterozygosity (not shown). Furthermore, staining of lung sections for phosphorylated ERK revealed no quantitative differences in the adenomas of SP-C C-Raf BxB animals heterozygous or wild type for BAG-1, see Figure 3c. Thus, signalling through the mitogenic cascade was not affected by the BAG-1 heterozygosity in the adenoma cells. + +Conclusions + +Tumours often are highly dependent on signalling pathways promoting cell growth or survival and may become hypersensitive to downregulation of key components within these signalling cascades. This study identifies BAG-1 as a protein specifically required at wild type expression levels for the survival of tumour cells and reveals it as potential anticancer target. Since many key components of survival pathways are regulated by interaction with (co-)chaperones [15], our finding is not without precedent but novel insofar as we have uncovered that reduced BAG-1 expression specifically targets tumour cells to apoptosis and impairs tumorigenesis. Whether this effect on adenoma cell survival requires that BAG-1 interacts with C-Raf or Hsc70/Hsp70 or with both partners requires additional studies. Questions concerning specific roles of the different BAG-1 isoforms were not addressed with this BAG-1 deficient mouse as both isoforms of BAG-1, p50 and p32 are absent in protein extracts of knock-out embryos. Another setting where BAG-1 has a physiological role is the heart, where up-regulation of BAG-1 after ischemia rescues cells from apoptosis [16]. + +A possible model combining the findings of this report and other data indicates that BAG-1 functions as an activator of C-Raf at the outer mitochondrial membrane where enzymatically activated C-Raf finds apoptosis-related targets such as BAD [17], see Figure 4. We can purify overexpressed C-Raf either in an enzymatically inactive form in a complex with Hsp70 or in an enzymatically active form in a complex with Hsp90/50 (unpublished observations), and BAG-1 is proposed to regulate this activation with ATP generated in the mitochondria. Experiments dealing with this questions are currently ongoing. + +Therefore, the therapeutic efficacy of a standard chemotherapeutic agent [13] should be increased dramatically by co-application with a BAG-1 inhibitor, since it would target the adaptability of cancer cells to environmental stress and overcome their genetic plasticity. One way to reduce BAG-1 expression is through use of RNA interference-based gene silencing, in particular as BAG-1 overexpression has been observed in human tumours [11]. Drugs that bind to the ATP binding site of Hsc70/Hsp70 might also be expected to be effective as they would inhibit the interaction of BAG-1 with the ATPase domain of heat shock proteins. Such new specific BAG-1 inhibitors may be identified, aided by the known three-dimensional structure of the BAG domain [18,19]. + +Competing interests + +The author(s) declare that they have no competing interests. + +Authors' contributions + +RG caried out the molecular and histological studies and participated in the design and co-ordination of the study. BWK carried out the histological and immunohisto-chemical studies. GC participated in the histological and immunohistochemical experiments. URR participated in the design and co-ordination of the study. All authors read and approved the final manuscript. + +Pre-publication history + +The pre-publication history for this paper can be accessed here: + + + +Acknowledgements + +We thank L. Fedorov, N. Gribanow, D. Heim, S. Hilz and T. Potapenko and Y. Yang for support. This work was supported by Deutsche Krebshilfe – Mildred Scheel Foundation (grants 10-1793-Ra7; 10-1935-Ra8) and by the DFG (grants TR17-TPB7; -TPZ2). G. Camarero was a postdoctoral fellow supported by Spanish Government (Ministerio de Educación y Cultura). + +Figures and Tables + +Figure 1 + +BAG-1 haplo-insufficiency delays C-Raf driven adenoma growth. (a) Adenoma initiation in SP-C C-Raf BxB mice (BAG-1+/+) and their Bag-1 haplo-insufficient littermates (BAG-1+/-) at 1, 2 and 4 months of age. Adenoma foci values represent mean ± s.e. from at least 4 mice of each genotype analyzed in a blinded fashion by two independent readers. (b) Adenoma area in the lungs of 4 months old mice. Each value represents mean ± s.e. from at least 4 mice of each genotype analyzed in a blinded fashion. (c-d) Examples of hematoxylin-eosin stained sections of lungs from SP-C C-Raf BxB transgenic mice wildtype for BAG-1 in comparison to a BAG-1 heterozygous littermate. Scale bar, 200 μm. + +Figure 2 + +BAG-1 expression in the lung of SP-C C-Raf BxB transgenic mice (a) Lanes 1–8 show immunoblotting data for expression of BAG-1 in the lungs of 8 month old SP-C C-Raf BxB transgenic mice heterozygous (+/-) or homozygous (+/+) for BAG-1 as indicated below the lanes. Lane 9 shows absence of BAG-1 expression in a BAG-1 null (-/-) embryonic day 12,5 liver extract; lane 10 control liver. The markers along the left indicate relative molecular mass. The same blots were subsequently reacted with an antibody against GAPDH to demonstrate protein equal loading and are shown below. (b) BAG-1 immunostaining in SP-C C-Raf BxB transgenic mouse lung cancer tissue. + +Figure 3 + +Increased apoptosis but no change in PCNA and p-ERK in tumor cells of SP-C C-RafBxB transgenic BAG-1 heterozygous mice (a-c) Quantification of immunohistochemical staining for apoptosis using an antibody that detects activated caspase-3 (a), PCNA (b) and phosphorylated ERK (p-ERK, c) of adenoma cells from 1-month-old SP-C C-Raf BxB transgenic mice of the indicated BAG-1 genotype. Each value represents mean ± s.e. from at least 4 mice of each genotype analyzed in three different experiments. + +Figure 4 + +Model for cooperative action of BAG-1 and C-Raf in tumorigenesis A possible model combining the findings of this report and other data is shown. It indicates that BAG-1 functions as an activator of C-Raf at the outer mitochondrial membrane where enzymatically activated C-Raf finds apoptosis-related targets (for details see text). diff --git a/src/ontogpt/evaluation/craft/database/all/15588329.ann b/src/ontogpt/evaluation/craft/database/all/15588329.ann new file mode 100644 index 000000000..4e6cb1846 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15588329.ann @@ -0,0 +1,889 @@ +T1 GO:0016020 0 8 Membrane +T2 GO:0055085 0 20 Membrane trafficking +T3 GO:0005739 25 38 mitochondrial +T4 PR:000022190 61 70 subunit c +T5 UBERON:0002037 87 97 cerebellar +T6 http://purl.obolibrary.org/obo/MONDO_0019262 112 151 juvenile neuronal ceroid lipofuscinosis +T7 CL:0000540 121 129 neuronal +T8 http://purl.obolibrary.org/obo/MONDO_0019262 175 179 JNCL +T9 http://purl.obolibrary.org/obo/MONDO_0024237 197 206;224 249 inherited neurodegenerative disease +T10 PR:000005591 282 286 CLN3 +T11 PR:000005591 319 327 battenin +T12 GO:0005739 360 373 mitochondrial +T13 PR:000022190 360 397 mitochondrial ATP synthase, subunit c +T14 UBERON:0001017 421 424 CNS +T15 CL:0000117 421 432 CNS neurons +T16 PR:000005591 458 462 Cln3 +T17 NCBITaxon:10088 478 482 mice +T18 http://purl.obolibrary.org/obo/MONDO_0019262 511 515 JNCL +T19 GO:0010467 526 533 express +T20 PR:000005591 541 549 battenin +T21 http://purl.obolibrary.org/obo/MONDO_0019262 562 566 JNCL +T22 http://purl.obolibrary.org/obo/MONDO_0019262 637 641 JNCL +T23 CL:0000540 654 668 neuronal cells +T24 NCBITaxon:10088 690 695 mouse +T25 UBERON:0002037 696 705 cerebella +T26 UBERON:0002037 746 748 Cb +T27 PR:000005591 748 752 Cln3 +T28 CL:0000540 793 801 neuronal +T29 PR:000010124 857 862 MAP-2 +T30 PR:000006252 867 871 NeuN +T31 CL:0000540 882 888 neuron +T32 UBERON:0002037 912 914 Cb +T33 PR:000005591 914 918 Cln3 +T34 GO:0010467 941 948 express +T35 PR:000005591 970 978 battenin +T36 PR:000022190 1020 1036 ATPase subunit c +T37 PR:000006025 1102 1113 cathepsin D +T38 GO:0005764 1208 1217 lysosomal +T39 GO:0006897 1257 1268 endocytosis +T40 GO:0005739 1294 1306 mitochondria +T41 MOP:0000568 1391 1400 oxidative +T42 PR:000005591 1461 1469 battenin +T43 GO:0005622 1486 1499 intracellular +T44 GO:0046907 1486 1499;1509 1520 intracellular ... trafficking +T45 GO:0016020 1500 1508 membrane +T46 GO:0055085 1500 1520 membrane trafficking +T47 GO:0005739 1525 1538 mitochondrial +T48 http://purl.obolibrary.org/obo/MONDO_0019262 1615 1619 JNCL +T49 http://purl.obolibrary.org/obo/MONDO_0000001 1620 1627 disease +T50 CL:0000540 1664 1672 neuronal +T51 http://purl.obolibrary.org/obo/MONDO_0019262 1696 1735 Juvenile neuronal ceroid lipofuscinosis +T52 CL:0000540 1705 1713 neuronal +T53 http://purl.obolibrary.org/obo/MONDO_0019262 1737 1741 JNCL +T54 http://purl.obolibrary.org/obo/MONDO_0019262 1747 1761 Batten disease +T55 http://purl.obolibrary.org/obo/MONDO_0024237 1780 1789;1806 1832 inherited neurodegenerative disorder +T56 http://purl.obolibrary.org/obo/MONDO_0001941 1862 1871 blindness +T57 GO:0050890 1893 1902 cognitive +T58 GO:0016265 1922 1927 death +T59 SO:0000704 1945 1952 genetic +T60 http://purl.obolibrary.org/obo/MONDO_0000001 1966 1973 disease +T61 http://purl.obolibrary.org/obo/MONDO_0019262 1998 2002 JNCL +T62 SO:0001026 2016 2023 genomic +T63 PR:000005591 2044 2048 CLN3 +T64 SO:0000704 2049 2053 gene +T65 SO:0000147 2072 2077 exons +T66 SO:0000188 2102 2110 intronic +T67 http://purl.obolibrary.org/obo/MONDO_0019262 2195 2199 JNCL +T68 GO:0044754 2253 2266 autolysosomes +T69 PR:000022190 2288 2300;2305 2339 subunit c of ... mitochondrial ATP synthase complex +T70 GO:0005753 2305 2339 mitochondrial ATP synthase complex +T71 UBERON:0001017 2396 2399 CNS +T72 CL:0000117 2396 2407 CNS neurons +T73 CL:0000540 2437 2445 neuronal +T74 UBERON:0001016 2467 2481 nervous system +T75 PR:000022190 2503 2512 subunit c +T76 http://purl.obolibrary.org/obo/MONDO_0019262 2529 2533 JNCL +T77 http://purl.obolibrary.org/obo/MONDO_0000001 2534 2541 disease +T78 CL:0000540 2585 2593 neuronal +T79 http://purl.obolibrary.org/obo/MONDO_0000001 2613 2620 disease +T80 PR:000005591 2652 2656 CLN3 +T81 PR:000005591 2674 2682 battenin +T82 PR:000005591 2696 2700 CLN3 +T83 PR:000005591 2704 2710 cln3 p +T84 GO:0010467 2748 2757 expressed +T85 GO:0016020 2770 2778 membrane +T86 GO:0005764 2813 2821 lysosome +T87 GO:0031982 2832 2841 vesicular +T88 PR:000005591 2862 2870 Battenin +T89 PR:Q9US09 2926 2930 btn1 +T90 PR:000005591 2942 2946 CLN3 +T91 SO:0000855 2947 2955 ortholog +T92 PR:000005591 2973 2981 battenin +T93 GO:0005764 2985 2994 lysosomal +T94 GO:0006885 2995 3009 pH homeostasis +T95 GO:0006865 3014 3034 amino acid transport +T96 http://purl.obolibrary.org/obo/MONDO_0019262 3056 3060 JNCL +T97 PR:000005591 3078 3086 battenin +T98 SO:0000704 3123 3134 genetically +T99 http://purl.obolibrary.org/obo/MONDO_0019262 3143 3147 JNCL +T100 NCBITaxon:10088 3148 3153 mouse +T101 PR:000005591 3161 3165 Cln3 +T102 NCBITaxon:10088 3181 3185 mice +T103 http://purl.obolibrary.org/obo/MONDO_0019262 3210 3214 JNCL +T104 GO:0010467 3228 3235 express +T105 PR:000005591 3259 3267 battenin +T106 SO:0001060 3268 3275 isoform +T107 GO:0042571 3300 3310 antibodies +T108 PR:000005591 3355 3359 Cln3 +T109 NCBITaxon:10088 3375 3379 mice +T110 http://purl.obolibrary.org/obo/MONDO_0019262 3402 3406 JNCL +T111 http://purl.obolibrary.org/obo/MONDO_0000001 3412 3419 disease +T112 UBERON:0012101 3426 3435 perinatal +T113 GO:0007567 3430 3435 natal +T114 PR:000022190 3445 3454 subunit c +T115 CL:0000540 3504 3512 neuronal +T116 http://purl.obolibrary.org/obo/MONDO_0019262 3593 3597 JNCL +T117 GO:0005739 3635 3648 mitochondrial +T118 PR:000022190 3635 3658 mitochondrial subunit c +T119 UBERON:0001017 3701 3704 CNS +T120 CL:0000117 3701 3712 CNS neurons +T121 CL:0000540 3747 3760 neuronal cell +T122 http://purl.obolibrary.org/obo/MONDO_0019262 3808 3812 JNCL +T123 GO:0008150 3825 3845 biological processes +T124 UBERON:0002037 3878 3888 cerebellar +T125 CL:1001611 3878 3897 cerebellar neuronal +T126 PR:000005591 3924 3928 Cln3 +T127 NCBITaxon:10088 3944 3948 mice +T128 UBERON:0002037 3961 3963 Cb +T129 PR:000005591 3963 3967 Cln3 +T130 http://purl.obolibrary.org/obo/MONDO_0000001 4018 4025 disease +T131 GO:0043227 4043 4062 membrane organelles +T132 GO:0016020 4072 4080 membrane +T133 GO:0055085 4072 4092 membrane trafficking +T134 GO:0005739 4105 4118 mitochondrial +T135 UBERON:0002037 4152 4154 Cb +T136 SO:0000704 4198 4209 genetically +T137 UBERON:0002037 4218 4228 cerebellar +T138 http://purl.obolibrary.org/obo/MONDO_0019262 4229 4233 JNCL +T139 SO:0000704 4268 4275 genetic +T140 CL:0000540 4277 4283 neuron +T141 http://purl.obolibrary.org/obo/MONDO_0019262 4292 4296 JNCL +T142 CL:0000120 4334 4349 granule neurons +T143 GO:0007567 4368 4373 natal +T144 UBERON:0002037 4385 4394 cerebella +T145 PR:000005591 4426 4430 Cln3 +T146 NCBITaxon:10088 4446 4450 mice +T147 CL:0000001 4479 4491 Primary cell +T148 CL:0000120 4514 4529 granule neurons +T149 GO:0009293 4535 4545 transduced +T150 NCBITaxon:11632 4551 4561 retroviral +T151 SO:0000440 4562 4568 vector +T152 SO:0005853 4589 4597 cassette +T153 NCBITaxon:10633 4630 4634 SV40 +T154 CHEBI:59132 4643 4650 antigen +T155 CHEBI:42768 4662 4666 G418 +T156 PR:000011141 4777 4783 nestin +T157 PR:000007939 4808 4812 GFAP +T158 NCBITaxon:10633 4993 4997 SV40 +T159 CHEBI:59132 5006 5013 antigen +T160 GO:0010467 5014 5024 expression +T161 GO:0051301 5046 5059 cell division +T162 CL:0000540 5167 5175 neuronal +T163 CHEBI:60004 5192 5200 cocktail +T164 CL:0000540 5225 5231 neuron +T165 PR:000011141 5275 5281 nestin +T166 GO:0010467 5282 5292 expression +T167 PR:000010124 5324 5328 MAP2 +T168 PR:000006252 5333 5337 NeuN +T169 GO:0010467 5338 5348 expression +T170 GO:0010467 5371 5381 expression +T171 PR:000004967 5406 5415 calbindin +T172 CL:0000540 5438 5446 Neuronal +T173 GO:0010467 5454 5464 expression +T174 UBERON:0002037 5468 5470 Cb +T175 PR:000005591 5470 5474 Cln3 +T176 UBERON:0002037 5504 5506 Cb +T177 PR:000005591 5506 5510 Cln3 +T178 GO:0042571 5554 5564 antibodies +T179 UBERON:0002037 5575 5577 Cb +T180 PR:000005591 5577 5581 Cln3 +T181 PR:000011141 5604 5610 nestin +T182 GO:0010467 5611 5621 expression +T183 PR:000007939 5634 5638 GFAP +T184 GO:0010467 5639 5649 expression +T185 CL:0000540 5673 5681 neuronal +T186 CHEBI:60004 5742 5750 cocktail +T187 UBERON:0002037 5766 5768 Cb +T188 PR:000005591 5768 5772 Cln3 +T189 CL:0000540 5791 5797 neuron +T190 GO:0044297 5828 5839 cell bodies +T191 GO:0042995 5857 5866 processes +T192 PR:000010124 5872 5876 MAP2 +T193 PR:000006252 5885 5889 NeuN +T194 GO:0010467 5894 5904 expression +T195 UBERON:0002037 5920 5922 Cb +T196 PR:000005591 5922 5926 Cln3 +T197 CL:0000121 5957 5972 Purkinje neuron +T198 PR:000004967 5980 5989 calbindin +T199 UBERON:0002037 5995 5997 Cb +T200 PR:000005591 5997 6001 Cln3 +T201 UBERON:0002037 6014 6016 Cb +T202 PR:000005591 6016 6020 Cln3 +T203 UBERON:0002037 6167 6169 Cb +T204 PR:000005591 6169 6173 Cln3 +T205 GO:0010467 6186 6193 express +T206 PR:000005591 6201 6209 battenin +T207 http://purl.obolibrary.org/obo/MONDO_0019262 6222 6226 JNCL +T208 UBERON:0002037 6254 6256 Cb +T209 PR:000005591 6257 6261 Cln3 +T210 http://purl.obolibrary.org/obo/MONDO_0019262 6298 6302 JNCL +T211 PR:000005591 6336 6340 Cln3 +T212 NCBITaxon:10088 6356 6360 mice +T213 GO:0010467 6361 6368 express +T214 PR:000005591 6378 6382 Cln3 +T215 GO:0008380 6388 6394 splice +T216 PR:000005591 6415 6423 battenin +T217 GO:0042571 6460 6468 antibody +T218 UBERON:0002037 6545 6547 Cb +T219 PR:000005591 6547 6551 Cln3 +T220 PR:000005591 6581 6589 battenin +T221 PR:000005591 6651 6655 Cln3 +T222 SO:0001060 6661 6669 isoforms +T223 PR:000005591 6786 6790 Cln3 +T224 UBERON:0000955 6806 6811 brain +T225 PR:000005591 6886 6894 battenin +T226 UBERON:0002037 6917 6919 Cb +T227 PR:000005591 6919 6923 Cln3 +T228 GO:0005737 6965 6976 cytoplasmic +T229 GO:0031982 6978 6987 vesicular +T230 GO:0005764 7083 7092 lysosomal +T231 PR:000002060 7101 7106 Lamp1 +T232 GO:0005769 7146 7160 early endosome +T233 PR:000006901 7146 7170 early endosome antigen 1 +T234 CHEBI:59132 7161 7168 antigen +T235 PR:000006901 7172 7176 EEA1 +T236 GO:0005770 7186 7200 late endosomal +T237 PR:000013639 7209 7213 Rab7 +T238 GO:0055037 7263 7282 recycling endosomes +T239 PR:000016261 7301 7312 transferrin +T240 PR:000002060 7366 7371 Lamp1 +T241 PR:000006901 7376 7380 EEA1 +T242 UBERON:0002037 7440 7442 Cb +T243 PR:000005591 7442 7446 Cln3 +T244 GO:0005634 7474 7481 nuclear +T245 PR:000013639 7522 7526 Rab7 +T246 UBERON:0002037 7578 7580 Cb +T247 PR:000005591 7580 7584 Cln3 +T248 UBERON:0002037 7620 7622 Cb +T249 PR:000005591 7622 7626 Cln3 +T250 CHEBI:60004 7651 7658 mixture +T251 PR:000005591 7662 7666 Cln3 +T252 SO:0001023 7705 7711 allele +T253 SO:0001023 7727 7733 allele +T254 PR:000005591 7835 7839 Cln3 +T255 UBERON:0002037 7873 7875 Cb +T256 PR:000005591 7875 7879 Cln3 +T257 PR:000005591 7892 7896 Cln3 +T258 SO:0001030 7903 7910 forward +T259 SO:0000147 7912 7916 Exon +T260 SO:0001031 7920 7927 reverse +T261 SO:0000006 7931 7943 PCR products +T262 UBERON:0000955 8019 8024 brain +T263 UBERON:0000955 8044 8049 Brain +T264 SO:0000006 8067 8088 PCR reaction products +T265 CHEBI:4883 8120 8136 ethidium-bromide +T266 CHEBI:2511 8145 8152 agarose +T267 SO:0000006 8172 8183 PCR product +T268 GO:0008380 8302 8308 splice +T269 PR:000005591 8330 8338 Battenin +T270 GO:0005764 8343 8352 lysosomal +T271 GO:0005768 8357 8366 endosomal +T272 UBERON:0002037 8414 8416 Cb +T273 PR:000005591 8416 8420 Cln3 +T274 UBERON:0002037 8427 8437 cerebellar +T275 UBERON:0002037 8489 8491 Cb +T276 PR:000005591 8491 8495 Cln3 +T277 UBERON:0002037 8523 8525 Cb +T278 PR:000005591 8525 8529 Cln3 +T279 UBERON:0002037 8544 8554 cerebellar +T280 GO:0005764 8602 8611 lysosomes +T281 PR:000002060 8613 8619 Lamp 1 +T282 GO:0005769 8622 8637 early endosomes +T283 PR:000006901 8639 8643 EEA1 +T284 GO:0005770 8650 8664 late endosomes +T285 PR:000013639 8666 8670 Rab7 +T286 PR:000006901 8720 8724 EEA1 +T287 PR:000013639 8752 8756 Rab7 +T288 PR:000013639 8889 8893 Rab7 +T289 PR:000002060 8940 8946 Lamp 1 +T290 UBERON:0002037 9007 9009 Cb +T291 PR:000005591 9009 9013 Cln3 +T292 PR:000006901 9081 9085 EEA1 +T293 PR:000013639 9090 9094 Rab7 +T294 PR:000002060 9112 9118 Lamp 1 +T295 PR:000002060 9192 9198 Lamp 1 +T296 PR:000006901 9203 9207 EEA1 +T297 PR:000013639 9241 9245 Rab7 +T298 UBERON:0002037 9297 9299 Cb +T299 PR:000005591 9299 9303 Cln3 +T300 UBERON:0002037 9342 9344 Cb +T301 PR:000005591 9344 9348 Cln3 +T302 UBERON:0002037 9513 9515 Cb +T303 PR:000005591 9515 9519 Cln3 +T304 PR:000022190 9562 9571 subunit c +T305 GO:0016234 9572 9581 inclusion +T306 GO:0070841 9572 9591 inclusion formation +T307 UBERON:0002037 9692 9694 Cb +T308 PR:000005591 9694 9698 Cln3 +T309 PR:000022190 9714 9723 subunit c +T310 PR:000022190 9888 9897 subunit c +T311 UBERON:0002037 10056 10058 Cb +T312 PR:000005591 10058 10062 Cln3 +T313 CHEBI:10545 10134 10142 electron +T314 GO:0016234 10149 10159 inclusions +T315 GO:0005776 10199 10213 autophagosomes +T316 GO:0031045 10229 10250 dense core structures +T317 GO:0005739 10265 10277 mitochondria +T318 GO:0031982 10296 10304 vesicles +T319 GO:0016234 10316 10332 Inclusion bodies +T320 GO:0005776 10337 10351 autophagosomes +T321 PR:000022190 10446 10455 Subunit c +T322 UBERON:0002037 10483 10485 Cb +T323 PR:000005591 10485 10489 Cln3 +T324 UBERON:0002037 10496 10506 cerebellar +T325 PR:000022190 10526 10535 Subunit c +T326 UBERON:0002037 10622 10624 Cb +T327 PR:000005591 10624 10628 Cln3 +T328 UBERON:0002037 10671 10673 Cb +T329 PR:000005591 10673 10677 Cln3 +T330 PR:000022190 10700 10709 subunit c +T331 UBERON:0002037 10756 10758 Cb +T332 PR:000005591 10758 10762 Cln3 +T333 PR:000022190 10801 10810 subunit c +T334 PR:000022190 10926 10935 subunit c +T335 PR:000022190 11012 11021 subunit c +T336 PR:000022190 11235 11244 subunit c +T337 PR:000022190 11256 11261 sub c +T338 UBERON:0002037 11305 11307 Cb +T339 PR:000005591 11307 11311 Cln3 +T340 PR:000022190 11363 11372 subunit c +T341 CHEBI:18070 11461 11473 cytochrome c +T342 PR:000005776 11461 11492 cytochrome c oxidase subunit IV +T343 PR:000005776 11496 11500 cox4 +T344 GO:0016234 11522 11532 inclusions +T345 UBERON:0002037 11569 11571 Cb +T346 PR:000005591 11571 11575 Cln3 +T347 GO:0005776 11606 11619 autophagosome +T348 GO:0016020 11640 11648 membrane +T349 GO:0005739 11686 11698 mitochondria +T350 CHEBI:10545 11705 11713 electron +T351 GO:0031045 11705 11725 electron dense cores +T352 GO:0031982 11766 11775 vesicular +T353 CHEBI:10545 11796 11804 electron +T354 GO:0016234 11811 11820 inclusion +T355 GO:0005739 11874 11875 M +T356 GO:0005739 11877 11889 mitochondria +T357 UBERON:0002037 11927 11929 Cb +T358 PR:000005591 11929 11933 Cln3 +T359 PR:000005591 11950 11954 Cln3 +T360 NCBITaxon:10088 11970 11974 mice +T361 PR:000006025 11983 11994 cathepsin D +T362 PR:000022190 12042 12051 subunit c +T363 PR:000006025 12094 12105 cathepsin D +T364 PR:000022190 12143 12165 ATP synthase subunit c +T365 GO:0005764 12185 12193 lysosome +T366 PR:000006025 12216 12227 cathepsin D +T367 UBERON:0002037 12267 12269 Cb +T368 PR:000005591 12269 12273 Cln3 +T369 PR:000005591 12290 12294 Cln3 +T370 NCBITaxon:10088 12301 12305 mice +T371 PR:000006025 12317 12328 cathepsin D +T372 GO:0042571 12329 12337 antibody +T373 PR:000006025 12380 12391 cathepsin D +T374 SO:0001060 12392 12400 isoforms +T375 UBERON:0002037 12445 12447 Cb +T376 PR:000005591 12447 12451 Cln3 +T377 GO:0005634 12479 12486 nuclear +T378 GO:0031982 12500 12509 vesicular +T379 PR:000006025 12510 12521 cathepsin D +T380 GO:0005764 12631 12639 lysosome +T381 UBERON:0002037 12674 12676 Cb +T382 PR:000005591 12676 12680 Cln3 +T383 PR:000006025 12694 12705 cathepsin D +T384 GO:0031982 12728 12737 vesicular +T385 GO:0005634 12751 12758 nuclear +T386 UBERON:0002037 12820 12822 Cb +T387 PR:000005591 12822 12826 Cln3 +T388 PR:000005591 12842 12846 Cln3 +T389 UBERON:0000479 12853 12859 tissue +T390 PR:000006025 12908 12919 cathepsin D +T391 SO:0001060 12920 12928 isoforms +T392 PR:000006025 12940 12951 Cathepsin D +T393 SO:0001060 12952 12960 isoforms +T394 SO:0000984 13062 13074 single chain +T395 SO:0000985 13129 13141 double-chain +T396 UBERON:0002037 13188 13190 Cb +T397 PR:000005591 13190 13194 Cln3 +T398 PR:000005591 13210 13214 Cln3 +T399 UBERON:0000479 13221 13227 tissue +T400 PR:000006025 13418 13429 cathepsin D +T401 PR:000005591 13490 13494 Cln3 +T402 NCBITaxon:10088 13501 13505 mice +T403 UBERON:0002037 13510 13512 Cb +T404 PR:000005591 13512 13516 Cln3 +T405 http://purl.obolibrary.org/obo/MONDO_0000001 13596 13603 disease +T406 PR:000006025 13643 13654 Cathepsin D +T407 UBERON:0002037 13711 13713 Cb +T408 PR:000005591 13713 13717 Cln3 +T409 UBERON:0002037 13776 13778 Cb +T410 PR:000005591 13778 13782 Cln3 +T411 PR:000006025 13815 13826 cathepsin D +T412 GO:0042571 13827 13835 antibody +T413 PR:000006025 13884 13895 cathepsin D +T414 UBERON:0002037 13914 13916 Cb +T415 PR:000005591 13916 13920 Cln3 +T416 GO:0005634 13959 13966 nuclear +T417 GO:0005737 13971 13982 cytoplasmic +T418 PR:000006025 14000 14011 Cathepsin D +T419 UBERON:0002037 14033 14035 Cb +T420 PR:000005591 14035 14039 Cln3 +T421 GO:0005634 14085 14092 nuclear +T422 GO:0005737 14104 14115 cytoplasmic +T423 UBERON:0002037 14155 14157 Cb +T424 PR:000005591 14157 14161 Cln3 +T425 PR:000006025 14197 14208 Cathepsin D +T426 PR:000005591 14265 14269 Cln3 +T427 UBERON:0000479 14285 14291 tissue +T428 UBERON:0002037 14295 14297 Cb +T429 PR:000005591 14297 14301 Cln3 +T430 PR:000006025 14349 14360 cathepsin D +T431 UBERON:0000479 14434 14440 tissue +T432 PR:000005591 14573 14577 Cln3 +T433 UBERON:0002037 14588 14590 Cb +T434 PR:000005591 14590 14594 Cln3 +T435 SO:0000985 14682 14694 double-chain +T436 SO:0000984 14739 14751 single-chain +T437 PR:000006025 14794 14805 cathepsin D +T438 PR:000022190 14923 14932 subunit c +T439 CHEBI:76219 14948 14959 fluorogenic +T440 PR:000006025 14976 14987 cathepsin D +T441 UBERON:0002037 15068 15070 Cb +T442 PR:000005591 15070 15074 Cln3 +T443 PR:000006025 15277 15288 cathepsin D +T444 UBERON:0002037 15342 15344 Cb +T445 PR:000005591 15344 15348 Cln3 +T446 UBERON:0002037 15451 15453 Cb +T447 PR:000005591 15453 15457 Cln3 +T448 GO:0043227 15484 15503 membrane organelles +T449 PR:000006025 15546 15557 cathepsin D +T450 GO:0016020 15568 15576 membrane +T451 GO:0055085 15568 15588 membrane trafficking +T452 UBERON:0002037 15615 15617 Cb +T453 PR:000005591 15617 15621 Cln3 +T454 GO:0043227 15705 15724 membrane organelles +T455 GO:0005783 15777 15779 ER +T456 GO:0005801 15781 15790 cis-Golgi +T457 GO:0005802 15796 15807 trans-Golgi +T458 CHEBI:16249 15900 15917 protein disulfide +T459 PR:000008131 15935 15940 GM130 +T460 GO:0005764 15985 15994 lysosomal +T461 PR:000002061 16020 16026 Lamp 2 +T462 UBERON:0002037 16074 16076 Cb +T463 PR:000005591 16076 16080 Cln3 +T464 GO:0005764 16161 16170 lysosomes +T465 GO:0005634 16212 16219 nuclear +T466 UBERON:0002037 16246 16248 Cb +T467 PR:000005591 16248 16252 Cln3 +T468 GO:0005764 16259 16268 lysosomes +T469 GO:0031982 16299 16307 vesicles +T470 GO:0005737 16350 16359 cytoplasm +T471 PR:000002060 16382 16388 Lamp 1 +T472 PR:000002060 16459 16465 Lamp 1 +T473 PR:000002061 16470 16476 Lamp 2 +T474 UBERON:0002037 16539 16541 Cb +T475 PR:000005591 16541 16545 Cln3 +T476 GO:0005764 16638 16647 lysosomes +T477 CHEBI:37958 16752 16755 dye +T478 CHEBI:37527 16790 16796 acidic +T479 GO:0005764 16850 16859 lysosomal +T480 GO:0005764 16907 16916 lysosomal +T481 http://purl.obolibrary.org/obo/MONDO_0019262 16947 16951 JNCL +T482 PR:000002061 16988 16994 Lamp 2 +T483 UBERON:0002037 17032 17034 Cb +T484 PR:000005591 17034 17038 Cln3 +T485 GO:0005764 17045 17054 lysosomes +T486 GO:0005764 17055 17064 Lysosomal +T487 UBERON:0002037 17102 17104 Cb +T488 PR:000005591 17104 17108 Cln3 +T489 PR:000002061 17152 17158 Lamp 2 +T490 GO:0042571 17159 17167 antibody +T491 CHEBI:37958 17190 17193 dye +T492 GO:0005634 17226 17233 nuclear +T493 GO:0005764 17244 17253 lysosomes +T494 GO:0005764 17268 17277 lysosomes +T495 GO:0071944 17285 17297;17308 17313 periphery of ... cells +T496 UBERON:0002037 17315 17317 Cb +T497 PR:000005591 17317 17321 Cln3 +T498 UBERON:0002037 17398 17400 Cb +T499 PR:000005591 17400 17404 Cln3 +T500 GO:0031982 17441 17449 vesicles +T501 GO:0005634 17472 17479 nuclear +T502 PR:000002061 17492 17498 Lamp 2 +T503 GO:0005634 17581 17588 nuclear +T504 UBERON:0002037 17614 17616 Cb +T505 PR:000005591 17616 17620 Cln3 +T506 CHEBI:37958 17717 17720 dye +T507 UBERON:0002037 17747 17749 Cb +T508 PR:000005591 17749 17753 Cln3 +T509 GO:0005769 17873 17887 early endosome +T510 PR:000006901 17896 17900 EEA1 +T511 GO:0006907 17946 17969 fluid-phase endocytosis +T512 UBERON:0002037 18001 18003 Cb +T513 PR:000005591 18003 18007 Cln3 +T514 CHEBI:52071 18036 18043 dextran +T515 CHEBI:37926 18044 18048 FITC +T516 CHEBI:52071 18119 18126 dextran +T517 CHEBI:37926 18127 18131 FITC +T518 GO:0006897 18200 18209 endocytic +T519 GO:0030139 18200 18218 endocytic vesicles +T520 GO:0005634 18250 18257 nuclear +T521 UBERON:0002037 18286 18288 Cb +T522 PR:000005591 18288 18292 Cln3 +T523 CHEBI:52071 18342 18349 dextran +T524 CHEBI:37926 18350 18354 FITC +T525 GO:0031982 18384 18392 vesicles +T526 GO:0005737 18418 18427 cytoplasm +T527 GO:0006897 18452 18463 Endocytosis +T528 UBERON:0002037 18506 18508 Cb +T529 PR:000005591 18508 18512 Cln3 +T530 CHEBI:52071 18525 18532 Dextran +T531 CHEBI:37926 18533 18537 FITC +T532 UBERON:0002037 18587 18589 Cb +T533 PR:000005591 18589 18593 Cln3 +T534 UBERON:0002037 18640 18642 Cb +T535 PR:000005591 18642 18646 Cln3 +T536 UBERON:0002037 18681 18683 Cb +T537 PR:000005591 18683 18687 Cln3 +T538 CHEBI:52071 18718 18725 dextran +T539 CHEBI:37926 18726 18730 FITC +T540 GO:0005634 18759 18766 nuclear +T541 GO:0031982 18777 18786 vesicular +T542 GO:0031982 18818 18826 vesicles +T543 CHEBI:52071 18871 18878 dextran +T544 CHEBI:37926 18879 18883 FITC +T545 UBERON:0002037 18912 18914 Cb +T546 PR:000005591 18914 18918 Cln3 +T547 GO:0031982 19002 19010 vesicles +T548 GO:0005634 19025 19032 nuclear +T549 PR:000022190 19147 19156 subunit c +T550 GO:0005739 19162 19175 mitochondrial +T551 GO:0000422 19218 19255 autophagic engulfment of mitochondria +T552 GO:0005739 19243 19255 mitochondria +T553 UBERON:0002037 19285 19287 Cb +T554 PR:000005591 19287 19291 Cln3 +T555 GO:0005739 19303 19316 mitochondrial +T556 GO:0005739 19342 19355 Mitochondrial +T557 UBERON:0002037 19383 19385 Cb +T558 PR:000005591 19385 19389 Cln3 +T559 UBERON:0002037 19483 19485 Cb +T560 PR:000005591 19485 19489 Cln3 +T561 GO:0005739 19496 19508 mitochondria +T562 PR:000008813 19536 19541 grp75 +T563 GO:0005739 19617 19629 mitochondria +T564 GO:0005739 19717 19729 mitochondria +T565 GO:0005739 19786 19799 Mitochondrial +T566 UBERON:0002037 19836 19838 Cb +T567 PR:000005591 19838 19842 Cln3 +T568 UBERON:0002037 19939 19941 Cb +T569 PR:000005591 19941 19945 Cln3 +T570 CHEBI:16240 20070 20087 hydrogen peroxide +T571 GO:0008152 20164 20174 metabolism +T572 MOP:0000568 20179 20188 oxidative +T573 GO:0005739 20250 20263 mitochondrial +T574 UBERON:0002037 20287 20289 Cb +T575 PR:000005591 20289 20293 Cln3 +T576 GO:0005739 20318 20331 Mitochondrial +T577 UBERON:0002037 20398 20400 Cb +T578 PR:000005591 20400 20404 Cln3 +T579 UBERON:0002037 20477 20479 Cb +T580 PR:000005591 20479 20483 Cln3 +T581 GO:0005739 20490 20503 mitochondrial +T582 GO:0005743 20550 20578 inner mitochondrial membrane +T583 PR:000008813 20587 20592 grp75 +T584 GO:0005739 20628 20640 mitochondria +T585 UBERON:0002037 20669 20671 Cb +T586 PR:000005591 20671 20675 Cln3 +T587 GO:0005739 20713 20725 mitochondria +T588 UBERON:0002037 20727 20729 Cb +T589 PR:000005591 20729 20733 Cln3 +T590 GO:0005739 20762 20775 Mitochondrial +T591 UBERON:0002037 20854 20856 Cb +T592 PR:000005591 20856 20860 Cln3 +T593 GO:0005739 20867 20879 mitochondria +T594 UBERON:0002037 21001 21003 Cb +T595 PR:000005591 21003 21007 Cln3 +T596 UBERON:0002037 21090 21092 Cb +T597 PR:000005591 21092 21096 Cln3 +T598 UBERON:0002037 21148 21150 Cb +T599 PR:000005591 21150 21154 Cln3 +T600 UBERON:0002037 21320 21322 Cb +T601 PR:000005591 21322 21326 Cln3 +T602 CHEBI:16240 21528 21545 hydrogen peroxide +T603 UBERON:0002037 21577 21579 Cb +T604 PR:000005591 21579 21583 Cln3 +T605 MOP:0000568 21627 21636 oxidative +T606 CHEBI:16240 21647 21664 hydrogen peroxide +T607 UBERON:0002037 21723 21725 Cb +T608 PR:000005591 21725 21729 Cln3 +T609 CHEBI:16240 21787 21791 H2O2 +T610 UBERON:0002037 21812 21814 Cb +T611 PR:000005591 21814 21818 Cln3 +T612 CHEBI:16240 21877 21881 H2O2 +T613 UBERON:0002037 21976 21978 Cb +T614 PR:000005591 21978 21982 Cln3 +T615 UBERON:0002037 21989 21999 cerebellar +T616 SO:0000704 22036 22047 genetically +T617 CL:0000540 22057 22063 neuron +T618 http://purl.obolibrary.org/obo/MONDO_0019262 22089 22093 JNCL +T619 UBERON:0002037 22106 22108 Cb +T620 PR:000005591 22108 22112 Cln3 +T621 GO:0010467 22125 22132 express +T622 PR:000005591 22140 22148 battenin +T623 http://purl.obolibrary.org/obo/MONDO_0019262 22153 22157 JNCL +T624 GO:0005739 22167 22180 mitochondrial +T625 PR:000022190 22167 22197 mitochondrial ATPase subunit c +T626 GO:0007569 22217 22231 aging of cells +T627 GO:0010008 22303 22312;22323 22331 endosomal ... membrane +T628 GO:0016197 22303 22312;22332 22343 endosomal ... trafficking +T629 GO:0005765 22313 22331 lysosomal membrane +T630 GO:0007041 22313 22322;22332 22343 lysosomal ... trafficking +T631 GO:0055085 22323 22343 membrane trafficking +T632 GO:0005739 22356 22369 mitochondrial +T633 PR:000022190 22396 22405 subunit c +T634 http://purl.obolibrary.org/obo/MONDO_0019262 22432 22436 JNCL +T635 http://purl.obolibrary.org/obo/MONDO_0019262 22508 22512 JNCL +T636 http://purl.obolibrary.org/obo/MONDO_0000001 22513 22520 disease +T637 CL:0000540 22557 22565 neuronal +T638 PR:000006025 22586 22597 cathepsin D +T639 UBERON:0002037 22640 22642 Cb +T640 PR:000005591 22642 22646 Cln3 +T641 PR:000005591 22663 22667 Cln3 +T642 NCBITaxon:10088 22674 22678 mice +T643 GO:0031982 22703 22712 vesicular +T644 GO:0016192 22703 22724 vesicular trafficking +T645 GO:0005764 22732 22741 lysosomal +T646 PR:000006025 22771 22782 cathepsin D +T647 PR:000005591 22811 22815 CLN3 +T648 GO:0010467 22820 22830 expression +T649 GO:0005764 22856 22865 lysosomal +T650 PR:000006025 22873 22884 cathepsin D +T651 GO:0005764 22906 22915 lysosomal +T652 GO:0006885 22916 22930 pH homeostasis +T653 http://purl.obolibrary.org/obo/MONDO_0019262 22947 22951 JNCL +T654 PR:000006023 22983 22994 cathepsin B +T655 PR:000016585 23003 23007 CLN2 +T656 PR:000016585 23024 23028 TPPI +T657 http://purl.obolibrary.org/obo/MONDO_0019262 23050 23054 JNCL +T658 PR:000006025 23090 23101 cathepsin D +T659 UBERON:0002037 23154 23156 Cb +T660 PR:000005591 23156 23160 Cln3 +T661 PR:000006025 23174 23185 cathepsin D +T662 PR:000006025 23252 23263 cathepsin D +T663 PR:000022190 23300 23309 subunit c +T664 http://purl.obolibrary.org/obo/MONDO_0019262 23326 23330 JNCL +T665 GO:0007569 23333 23341;23366 23371 Aging of ... cells +T666 UBERON:0002037 23353 23355 Cb +T667 PR:000005591 23355 23359 Cln3 +T668 PR:000022190 23435 23444 subunit c +T669 GO:0043227 23463 23481 membrane organelle +T670 PR:000022190 23502 23511 subunit c +T671 UBERON:0002037 23539 23541 Cb +T672 PR:000005591 23541 23545 Cln3 +T673 GO:0007568 23591 23596 aging +T674 GO:0005764 23612 23621 Lysosomal +T675 GO:0005768 23626 23635 endosomal +T676 GO:0005739 23675 23687 mitochondria +T677 UBERON:0002037 23770 23772 Cb +T678 PR:000005591 23772 23776 Cln3 +T679 GO:0016020 23823 23831 membrane +T680 GO:0055085 23823 23843 membrane trafficking +T681 PR:000022190 23881 23890 subunit c +T682 GO:0005764 23921 23929 lysosome +T683 http://purl.obolibrary.org/obo/MONDO_0000001 23966 23973 disease +T684 PR:000022190 23992 24001 subunit c +T685 GO:0005739 24016 24029 Mitochondrial +T686 http://purl.obolibrary.org/obo/MONDO_0019262 24078 24082 JNCL +T687 NCBITaxon:33208 24102 24108 animal +T688 GO:0006914 24165 24174 autophagy +T689 GO:0005764 24178 24187 lysosomal +T690 PR:000005591 24245 24253 battenin +T691 GO:0005739 24276 24289 mitochondrial +T692 GO:0005739 24331 24344 mitochondrial +T693 GO:0015031 24371 24380;24396 24398;24413 24421 transport ... of ... proteins +T694 GO:0034982 24385 24421 processing of mitochondrial proteins +T695 GO:0005739 24399 24412 mitochondrial +T696 UBERON:0002037 24437 24439 Cb +T697 PR:000005591 24439 24443 Cln3 +T698 CL:0000540 24444 24452 neuronal +T699 PR:000005591 24469 24477 battenin +T700 GO:0005769 24506 24511;24521 24530 early ... endosomes +T701 GO:0005770 24516 24530 late endosomes +T702 PR:000005591 24532 24540 Battenin +T703 UBERON:0002037 24570 24572 Cb +T704 PR:000005591 24572 24576 Cln3 +T705 CL:0000540 24583 24591 neuronal +T706 GO:0005768 24683 24692 endosomal +T707 PR:000005591 24719 24727 battenin +T708 PR:000005591 24823 24827 CLN3 +T709 PR:000005591 24828 24836 battenin +T710 GO:0005770 24902 24916 late endosomes +T711 GO:0005764 24921 24930 lysosomes +T712 CL:0000540 24938 24946 neuronal +T713 GO:0005764 24965 24974 lysosomes +T714 GO:0005768 24997 25006 endosomes +T715 CL:0000540 25017 25024 neurons +T716 PR:000005591 25059 25067 battenin +T717 GO:0031982 25091 25100 vesicular +T718 GO:0016020 25131 25139 membrane +T719 GO:0055085 25131 25151 membrane trafficking +T720 GO:0031982 25185 25194 vesicular +T721 GO:0016192 25185 25204 vesicular transport +T722 GO:0006906 25185 25194;25212 25218 vesicular ... fusion +T723 GO:0006897 25220 25229 Endocytic +T724 GO:0005764 25234 25243 lysosomal +T725 GO:0005739 25273 25286 mitochondrial +T726 GO:0000422 25273 25296 mitochondrial autophagy +T727 GO:0016020 25359 25367 membrane +T728 GO:0055085 25359 25379 membrane trafficking +T729 GO:0005739 25384 25397 mitochondrial +T730 UBERON:0002037 25431 25433 Cb +T731 PR:000005591 25433 25437 Cln3 +T732 CL:0000540 25484 25492 neuronal +T733 GO:0031988 25539 25555 membrane vesicle +T734 GO:0016192 25548 25565 vesicle transport +T735 GO:0008152 25585 25595 metabolism +T736 CL:0000540 25618 25625 neurons +T737 PR:000005591 25641 25649 battenin +T738 UBERON:0002037 25719 25721 Cb +T739 PR:000005591 25721 25725 Cln3 +T740 UBERON:0002037 25732 25742 cerebellar +T741 PR:000005591 25800 25808 battenin +T742 http://purl.obolibrary.org/obo/MONDO_0019262 25822 25826 JNCL +T743 GO:0042571 25851 25859 Antibody +T744 CHEBI:33893 25878 25886 reagents +T745 PR:000011141 25888 25894 Nestin +T746 NCBITaxon:10114 25902 25905 Rat +T747 PR:000002060 25921 25927 Lamp 1 +T748 PR:000002061 25955 25961 Lamp 2 +T749 GO:0042571 25997 26007 antibodies +T750 PR:000022190 26194 26203 subunit c +T751 GO:0042571 26204 26212 antibody +T752 GO:0042571 26309 26319 antibodies +T753 PR:000007939 26337 26341 GFAP +T754 PR:000004967 26370 26379 calbindin +T755 PR:000006252 26397 26401 NeuN +T756 NCBITaxon:10633 26424 26428 SV40 +T757 CHEBI:59132 26431 26438 antigen +T758 PR:000006025 26486 26497 cathepsin D +T759 CHEBI:18070 26542 26554 cytochrome c +T760 PR:000005776 26542 26573 cytochrome c oxidase subunit IV +T761 PR:000005776 26575 26579 cox4 +T762 PR:000008131 26665 26670 GM130 +T763 GO:0009293 26684 26696 Transduction +T764 PR:000028799 26706 26713 tubulin +T765 PR:000008813 26733 26738 grp75 +T766 GO:0005769 26759 26773 early endosome +T767 PR:000006901 26759 26783 early endosome antigen-1 +T768 CHEBI:59132 26774 26781 antigen +T769 PR:000006901 26785 26789 EEA1 +T770 PR:000013639 26828 26833 rab 7 +T771 GO:0042571 26897 26907 antibodies +T772 MOP:0000779 26971 26981 conjugated +T773 GO:0042571 26992 27002 antibodies +T774 CHEBI:15956 27089 27095 biotin +T775 CHEBI:52071 27137 27144 dextran +T776 CHEBI:37926 27145 27149 FITC +T777 CHEBI:52117 27163 27185 Lysotracker Red DND-99 +T778 UBERON:0002037 27262 27264 Cb +T779 PR:000005591 27264 27268 Cln3 +T780 UBERON:0002037 27269 27279 cerebellar +T781 CL:1001611 27269 27288 cerebellar neuronal +T782 PR:000005591 27306 27310 Cln3 +T783 NCBITaxon:10088 27326 27330 mice +T784 UBERON:0002037 27498 27508 cerebellar +T785 CL:0001031 27498 27523 cerebellar granule neuron +T786 GO:0007567 27552 27557 natal +T787 UBERON:0002037 27569 27578 cerebella +T788 CHEBI:17234 27666 27673 glucose +T789 UBERON:0002415 27675 27679 Tail +T790 SO:0001026 27713 27720 genomic +T791 PR:000027795 27759 27766 Trypsin +T792 UBERON:0002037 27859 27868 cerebella +T793 UBERON:0002037 27986 27995 cerebella +T794 CHEBI:16526 28032 28035 CO2 +T795 CL:0000120 28040 28054 granule neuron +T796 NCBITaxon:27592 28142 28148 bovine +T797 UBERON:0001977 28149 28154 serum +T798 CHEBI:32588 28196 28199 KCl +T799 http://purl.obolibrary.org/obo/MONDO_0005550 28202 28211 Infection +T800 NCBITaxon:11632 28259 28269 retrovirus +T801 GO:0009293 28270 28281 transducing +T802 CHEBI:59132 28326 28333 antigen +T803 CHEBI:7507 28350 28358 neomycin +T804 SO:0005853 28370 28378 cassette +T805 http://purl.obolibrary.org/obo/MONDO_0005550 28425 28434 infection +T806 CHEBI:42768 28581 28585 G418 +T807 http://purl.obolibrary.org/obo/MONDO_0005550 28652 28661 infection +T808 UBERON:0002037 28840 28842 Cb +T809 PR:000005591 28842 28846 Cln3 +T810 UBERON:0002037 28859 28861 Cb +T811 CHEBI:16526 28968 28971 CO2 +T812 CL:0000540 29075 29083 Neuronal +T813 CHEBI:60004 29152 29160 cocktail +T814 PR:000007479 29171 29176 α-FGF +T815 CHEBI:48518 29185 29189 IBMX +T816 CHEBI:37537 29198 29201 TPA +T817 CHEBI:42471 29209 29218 forskolin +T818 CHEBI:18243 29225 29233 dopamine +T819 SO:0001026 29267 29274 Genomic +T820 UBERON:0002415 29298 29302 tail +T821 PR:000005591 29365 29369 Cln3 +T822 SO:0001023 29385 29391 allele +T823 SO:0000112 29426 29433 primers +T824 SO:0000028 29523 29525 bp +T825 SO:0000112 29544 29551 primers +T826 SO:0000028 29645 29647 bp +T827 PR:000005591 29797 29801 Cln3 +T828 SO:0000112 29809 29816 primers +T829 PR:000022190 29870 29879 Subunit c +T830 CHEBI:16842 30285 30297 formaldehyde +T831 PR:000022190 30392 30401 subunit c +T832 CHEBI:64276 30522 30536 glutaraldehyde +T833 CHEBI:16223 30566 30576 cacodylate +T834 GO:0042571 30870 30878 antibody +T835 GO:0040007 31042 31047 grown +T836 CHEBI:16842 31125 31137 formaldehyde +T837 CHEBI:17790 31187 31195 methanol +T838 CHEBI:15347 31196 31203 acetone +T839 GO:0042571 31258 31266 antibody +T840 GO:0042571 31458 31466 antibody +T841 CHEBI:9750 31478 31490 Triton X-100 +T842 NCBITaxon:27592 31536 31542 bovine +T843 UBERON:0001977 31543 31548 serum +T844 PR:000003918 31543 31556 serum albumin +T845 GO:0042571 31594 31602 antibody +T846 GO:0042571 31651 31659 antibody +T847 CHEBI:9754 32220 32224 Tris +T848 CHEBI:9750 32237 32249 Triton X-100 +T849 GO:0016020 32251 32259 membrane +T850 CHEBI:7989 32342 32353 pepstatin A +T851 CHEBI:6426 32382 32391 leupeptin +T852 CHEBI:8984 32636 32639 SDS +T853 CHEBI:9754 32702 32706 tris +T854 CHEBI:46760 32707 32714 tricine +T855 CHEBI:8984 32715 32718 SDS +T856 PR:000022190 32743 32752 subunit c +T857 PR:000006025 32782 32793 Cathepsin D +T858 UBERON:0000479 32817 32823 tissue +T859 CHEBI:9754 32992 32996 Tris +T860 CHEBI:9750 33011 33023 Triton X-100 +T861 PR:000006025 33285 33296 cathepsin D +T862 CHEBI:76219 33316 33327 Fluorogenic +T863 PR:000006025 33338 33349 Cathepsin D +T864 GO:0005764 33725 33734 Lysosomal +T865 GO:0006897 33748 33764 endocytic uptake +T866 GO:0040007 33854 33859 grown +T867 CHEBI:52071 33982 33989 dextran +T868 CHEBI:37926 33990 33994 FITC +T869 CHEBI:37958 34168 34171 dye +T870 CHEBI:16842 34202 34214 formaldehyde +T871 GO:0005739 34409 34421 mitochondria +T872 GO:0005739 34566 34578 mitochondria +T873 GO:0005739 34636 34648 mitochondria +T874 GO:0005739 34912 34925 mitochondrial +T875 CHEBI:33893 35374 35381 Reagent +T876 GO:0019835 35409 35419 cell lysis +T877 CHEBI:16240 35718 35735 Hydrogen peroxide +T878 CHEBI:16240 35924 35941 hydrogen peroxide +T879 CHEBI:16240 36010 36027 hydrogen peroxide +T880 GO:0008283 36126 36144 Cell Proliferation +T881 GO:0005739 36356 36369 mitochondrial +T882 NCBITaxon:10239 36522 36527 virus +T883 GO:0042571 36900 36908 antibody +T884 PR:000022190 36912 36945 subunit c of mitochondrial ATPase +T885 GO:0005739 36925 36938 mitochondrial +T886 CHEBI:10545 37005 37013 Electron +T887 GO:0006897 37124 37135 endocytosis +T888 PR:000006025 37213 37224 cathepsin D +T889 http://purl.obolibrary.org/obo/MONDO_0019262 37349 37363 Batten Disease diff --git a/src/ontogpt/evaluation/craft/database/all/15588329.txt b/src/ontogpt/evaluation/craft/database/all/15588329.txt new file mode 100644 index 000000000..93d5b0d39 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15588329.txt @@ -0,0 +1,153 @@ +Membrane trafficking and mitochondrial abnormalities precede subunit c deposition in a cerebellar cell model of juvenile neuronal ceroid lipofuscinosis + +Abstract + +Background + +JNCL is a recessively inherited, childhood-onset neurodegenerative disease most-commonly caused by a ~1 kb CLN3 mutation. The resulting loss of battenin activity leads to deposition of mitochondrial ATP synthase, subunit c and a specific loss of CNS neurons. We previously generated Cln3Δex7/8 knock-in mice, which replicate the common JNCL mutation, express mutant battenin and display JNCL-like pathology. + +Results + +To elucidate the consequences of the common JNCL mutation in neuronal cells, we used P4 knock-in mouse cerebella to establish conditionally immortalized CbCln3 wild-type, heterozygous, and homozygous neuronal precursor cell lines, which can be differentiated into MAP-2 and NeuN-positive, neuron-like cells. Homozygous CbCln3Δex7/8 precursor cells express low levels of mutant battenin and, when aged at confluency, accumulate ATPase subunit c. Recessive phenotypes are also observed at sub-confluent growth; cathepsin D transport and processing are altered, although enzyme activity is not significantly affected, lysosomal size and distribution are altered, and endocytosis is reduced. In addition, mitochondria are abnormally elongated, cellular ATP levels are decreased, and survival following oxidative stress is reduced. + +Conclusions + +These findings reveal that battenin is required for intracellular membrane trafficking and mitochondrial function. Moreover, these deficiencies are likely to be early events in the JNCL disease process and may particularly impact neuronal survival. + +Background + +Juvenile neuronal ceroid lipofuscinosis (JNCL), or Batten disease, is a recessively inherited childhood-onset neurodegenerative disorder characterized by progressive blindness, seizures, motor and cognitive decline, and early death [1]. The primary genetic defect (>80% disease chromosomes) leading to JNCL is a 1.02 kb genomic DNA deletion in the CLN3 gene, which eliminates exons 7 and 8 and surrounding intronic DNA, predicting a non-functional protein product [2]. + +The pathological hallmark of JNCL is autofluorescent ceroid lipofuscin deposits within autolysosomes that are enriched in subunit c of the mitochondrial ATP synthase complex [3-5]. Remarkably, these deposits are not only found in CNS neurons but are also abundant in non-neuronal cells outside of the nervous system. The relationship of subunit c deposits to the JNCL disease process, and the underlying reason for the neuronal specificity of the disease remain poorly understood. + +The CLN3-encoded protein (battenin, also called CLN3 or cln3 p) is a highly conserved, ubiquitously expressed, multi-pass membrane protein [6] that localizes to the lysosome and other vesicular compartments [7-9]. Battenin function remains to be elucidated, although studies of btn1, the yeast CLN3 ortholog, have implicated battenin in lysosomal pH homeostasis and amino acid transport [10,11]. + +To explore JNCL pathogenesis and battenin function, we previously generated a genetically precise JNCL mouse model. Cln3Δex7/8 knock-in mice harbor the ~1 kb common JNCL mutation and express a non-truncated mutant battenin isoform that is detectable with antibodies recognizing C-terminal epitopes. Homozygous Cln3Δex7/8 knock-in mice exhibit a progressive JNCL-like disease, with perinatal onset of subunit c deposition in many cell types and later onset of neuronal dysfunction and behavioral deficits [12]. These findings suggest that the major JNCL defect leads to abnormal turnover of mitochondrial subunit c, in a manner that selectively compromises CNS neurons. + +Currently, there is no suitable neuronal cell system to investigate the impact of the common JNCL mutation on biological processes. Therefore, we have established cerebellar neuronal precursor cell lines from Cln3Δex7/8 knock-in mice. Homozygous CbCln3Δex7/8 cells exhibit pathological hallmarks of the disease, and a survey of membrane organelles revealed membrane trafficking defects and mitochondrial dysfunction in homozygous mutant CbCln3Δex7/8 cells. + +Results + +Generation of a genetically precise cerebellar JNCL cell model + +To generate a precise genetic, neuron-derived JNCL cell culture system, we immortalized granule neurons cultured from postnatal day 4 (P4) cerebella of homozygous and heterozygous Cln3Δex7/8 knock-in mice, and wild-type littermates. Primary cell cultures enriched for granule neurons were transduced with retroviral vector bearing a selection cassette and temperature-sensitive tsA58 SV40 large T antigen. Growth in G418 containing medium at the permissive temperature (33°C) allowed for selection and isolation of multiple clonal nestin-positive (Fig. 1a), and GFAP-negative (Fig. 1b), cell lines for each genotype. No genotype specific differences were observed in cellular morphology or doubling time (~46 hours) (data not shown). As expected, SV40 large T antigen expression was rapidly lost and cell division ceased when cells were shifted to the non-permissive temperature (39°C) (data not shown). Upon addition of neuronal differentiation cocktail, precursor cells became neuron-like in morphology and exhibited decreased nestin expression (data not shown) and increased MAP2 and NeuN expression (Fig. 1c,1d), but not expression of the Purkinje marker, calbindin (Fig. 1e). + +Figure 1 + +Neuronal marker expression in CbCln3+/+ cells Characterization of CbCln3+/+ cells by immunofluorescence with marker antibodies is shown. CbCln3+/+ precursors exhibit nestin expression (a) but not GFAP expression (b), consistent with a neuronal precursor identity. Upon stimulation with a differentiation cocktail (see Methods), CbCln3+/+ cells achieved neuron-like morphology, with rounded cell bodies and extension of processes, and MAP2 (c) and NeuN (d) expression was increased. CbCln3+/+ cells are negative for the Purkinje neuron marker calbindin (e). CbCln3+/Δex7/8 and CbCln3Δex7/8/Δex7/8 cell lines exhibited identical marker immunofluorescence results. a, b) 20 × magnification; c, d, e) 40 × magnification. + +Homozygous CbCln3Δex7/8 cells express mutant battenin and display JNCL-like pathology + +Homozygous Cb Cln3Δex7/8 cells were first examined for JNCL-like characteristics. Homozygous Cln3Δex7/8 knock-in mice express multiple Cln3 mRNA splice variants and mutant battenin protein that is detectable by batp1 antibody recognizing C-terminal epitopes [12]. To assess this molecular phenotype in CbCln3Δex7/8 cells, RT-PCR and anti-battenin (batp1) immunostaining were performed. As shown in Figure 2, Cln3 mRNA isoforms in wild-type and homozygous cells were similar to those observed in total RNA isolated from wild-type or homozygous Cln3Δex7/8 knock-in brain, respectively (Fig. 2). In addition, batp1 immunostaining detected mutant battenin product in homozygous CbCln3Δex7/8 cells, in a similar albeit reduced cytoplasmic, vesicular staining pattern as that seen in wild-type cells. Batp1 signal exhibited some overlap with the lysosomal marker, Lamp1, but had more significant overlap with early endosome antigen 1 (EEA1) and the late endosomal marker, Rab7 (Fig. 3). Only limited overlap was observed with recycling endosomes, as determined by transferrin receptor co-staining (data not shown). Intriguingly, Lamp1 and EEA1 immunocytochemical distribution were altered in homozygous CbCln3Δex7/8 cells, with less perinuclear clustering than in wild-type cells, and Rab7 staining was frequently less intense in homozygous CbCln3Δex7/8 cells (Fig. 3). Heterozygous CbCln3Δex7/8 cells contained a mixture of Cln3 mRNA products from both the wild-type allele and the mutant allele, and batp1 signal was similar to that seen in wild-type cells (data not shown). + +Figure 2 + +RT-PCR of Cln3 mRNA in wild-type and homozygous CbCln3Δex7/8 cells Cln3 Exon1-forward, Exon 15-reverse RT-PCR products are shown, from total wild-type (+/+) or homozygous mutant (Δex7/8/Δex7/8) brain and cell line RNA. Brain and cell line RT-PCR reaction products had identical band patterns on ethidium-bromide stained agarose gels. Wild-type RT-PCR product was a single ~1.6 kb band and mutant products were ~1.6, ~1.5, ~1.4, ~1.35, and ~1.3 kb, representing multiple mutant splice variants. + +Figure 3 + +Battenin and lysosomal and endosomal marker co-staining in wild-type and homozygous CbCln3Δex7/8 cerebellar precursor cells Batp1 immunostaining of wild-type (CbCln3+/+) and homozygous mutant (CbCln3Δex7/8/Δex7/8) cerebellar precursor cells is shown, with co-staining for lysosomes (Lamp 1), early endosomes (EEA1), and late endosomes (Rab7). Significant overlap of Batp1 signal (red) with EEA1 (green, middle panels) and Rab7 (green, bottom panels) can be seen as yellow when the two channels are merged (Merge). The degree of Batp1 overlap is greatest with Rab7. Only limited overlap between Batp1 (red) and Lamp 1 (green, top panels) can be seen. Batp1 signal in homozygous CbCln3Δex7/8 cells is significantly reduced, but significant overlap with EEA1 and Rab7, and very little Lamp 1 overlap, can be seen as yellow in the respective merged panels. Notably, Lamp 1 and EEA1 localization appear altered, and Rab7 staining was frequently less intense in homozygous CbCln3Δex7/8 cells. Wild-type and homozygous CbCln3Δex7/8 confocal images were captured with identical exposure settings. 60 × magnification. + +During sub-confluent growth conditions, neither wild-type nor homozygous CbCln3Δex7/8 cells displayed autofluorescence or subunit c inclusion formation (data not shown). However, when cells were aged at confluency (3+ days post-confluency), homozygous CbCln3Δex7/8 cellular subunit c levels were elevated beyond normal wild-type levels by immunostaining (Fig. 4a) and immunoblot analysis (Fig. 4b). Autofluorescent signal sometimes overlapped with subunit c signal, but also was elevated more diffusely (Fig. 4a). Moreover, although multilamellar "fingerprint" profiles were not detected, confluency-aged homozygous CbCln3Δex7/8 cells displayed numerous ultrastructural abnormalities including electron dense inclusions characteristic of lipofuscin and large autophagosomes that contained dense core structures, degenerating mitochondria, and many smaller vesicles (Fig. 4c). Inclusion bodies and autophagosomes were infrequently observed in confluency-aged wild-type cultures (data not shown). + +Figure 4 + +Subunit c accumulation in homozygous CbCln3Δex7/8 cerebellar precursor cells a. Subunit c immunostaining and autofluorescence of 7-day confluency-aged wild-type and homozygous CbCln3Δex7/8 cells is shown. Wild-type cultures (CbCln3+/+) exhibited limited subunit c immunostaining and autofluorescence. However, CbCln3Δex7/8/Δex7/8 cells contained numerous subunit c puncta. Autofluorescence (7 days AF) was also significantly elevated (right panels), although limited overlap with subunit c puncta was observed (arrows). 40 × magnification. b. Immunoblot analysis of subunit c protein at sub-confluency or 7-day confluency incubation is shown. Total protein extracts from sub-confluency wild-type (+/+) and homozygous mutant (Δex7/8/Δex7/8) cultures contained approximately equal levels of subunit c protein (α-sub c). 7-day confluency extract from homozygous CbCln3Δex7/8 cells (Δex7/8/Δex7/8) had elevated levels of subunit c protein (~1.5X), relative to wild-type extract (+/+). Protein levels were normalized to cytochrome c oxidase subunit IV (α-cox4). c. TEM analysis of inclusions in 7-day confluency-aged homozygous CbCln3Δex7/8 cells is shown. A large autophagosome contained by double membrane (arrows) is filled with degenerating mitochondria (Md), electron dense cores (left and right of *) and other smaller vesicular structures. A large electron-dense inclusion, with a lipofuscin (Ln) appearance, is also present. M, mitochondria. 10,000 × magnification. + +Homozygous CbCln3Δex7/8 cells and Cln3Δex7/8 knock-in mice process cathepsin D abnormally + +We next investigated the basis for subunit c accumulation, testing the hypothesis that cathepsin D is abnormal since it is required for ATP synthase subunit c degradation in the lysosome [13]. We first tested cathepsin D transport and processing in homozygous CbCln3Δex7/8 cells and Cln3Δex7/8 mice using anti-cathepsin D antibody that recognizes unprocessed and processed cathepsin D isoforms. Immunostaining of wild-type and homozygous CbCln3Δex7/8 cells revealed a perinuclear and punctate vesicular cathepsin D distribution, consistent with its transport and processing through the secretory pathway and delivery to the lysosome (Fig. 5a). However, in homozygous CbCln3Δex7/8 cells, cathepsin D distribution was less vesicular and more perinuclear-clustered than in wild-type cells. Immunoblots of homozygous CbCln3Δex7/8 cell and Cln3Δex7/8 tissue extracts also showed altered relative levels of cathepsin D isoforms (Fig. 5b). Cathepsin D isoforms, identified by relative molecular weights, represent the ~45 kDa precursor, the ~43 kDa intermediate single chain form of the enzyme, and the 31 kDa heavy chain of the double-chain form of the mature enzyme [14]. In homozygous CbCln3Δex7/8 cell and Cln3Δex7/8 tissue extracts, the precursor and heavy chains were reduced, and the single chain was slightly elevated compared to wild-type extracts. The cellular growth media did not contain altered levels of cathepsin D, indicating enzyme secretion was not affected. Heterozygous Cln3Δex7/8 mice and CbCln3Δex7/8 cells were indistinguishable from wild-type, as expected for a recessive disease phenotype (data not shown). + +Figure 5 + +Cathepsin D localization and processing in wild-type and homozygous CbCln3Δex7/8 cells a. Immunostaining of wild-type and homozygous CbCln3Δex7/8 precursor cells with anti-cathepsin D antibody, recognizing unprocessed and processed forms of cathepsin D protein is shown. CbCln3+/+ cells (left panel) exhibited a perinuclear and cytoplasmic punctate signal. Cathepsin D signal in homozygous CbCln3Δex7/8 cells (right panel) was more often perinuclear, with less cytoplasmic punctate signal, compared to wild-type CbCln3+/+ cells. 40 × magnification. b. α-Cathepsin D-probed immunoblots of total wild-type versus homozygous Cln3Δex7/8 knock-in tissue or CbCln3Δex7/8 cellular extracts are shown. The ~45 kDa cathepsin D band, representing precursor, was the predominant band in wild-type (wt) tissue and cellular extracts, with lower levels of mature enzyme (single chain, ~43 kDa, and heavy chain, ~31 kDa). Conversely, homozygous Cln3Δex7/8 and CbCln3Δex7/8 mutant (m) extracts exhibited reduced levels of precursor and heavy chain of the double-chain form of the enzyme, with elevated levels of single-chain mature enzyme. + +The impact of the altered cathepsin D processing on enzymatic activity was next tested to determine if altered enzymatic activity accounts for inefficient subunit c turnover. In a fluorogenic in vitro assay, cathepsin D activity in total cellular extracts was not significantly altered in homozygous CbCln3Δex7/8 cells (376 ± 89 RFU/μg total protein), versus wild-type cells (324 ± 58 RFU/μg total protein), although a consistent trend towards increased enzymatic activity in mutant cells was observed. Thus, cathepsin D transport and processing are disrupted in homozygous CbCln3Δex7/8 cells in a manner such that enzymatic activity appears to be relatively unaffected. + +Homozygous CbCln3Δex7/8 cells show abnormal membrane organelles + +The abnormal transport and processing of cathepsin D suggested membrane trafficking disruptions in homozygous CbCln3Δex7/8 cells; therefore, we surveyed the subcellular distribution and morphology of membrane organelles. Components of the secretory pathway, including the ER, cis-Golgi, and trans-Golgi, did not appear altered from wild-type appearance when labeled with the respective markers, protein disulfide isomerase (PDI), GM130, and VVL (data not shown). By contrast, the lysosomal markers, Lysotracker and Lamp 2 had significantly altered signal in homozygous CbCln3Δex7/8 cells, versus wild-type cells. Wild-type cells exhibited brightly stained lysosomes that were large and clustered in the perinuclear region whereas homozygous CbCln3Δex7/8 lysosomes were lightly stained, smaller vesicles that were more diffusely scattered in the cytoplasm of the cell (Fig. 6). Lamp 1 distribution was also altered, as previously noted (Fig. 3). However, Lamp 1 and Lamp 2 total protein levels were similar in wild-type and homozygous CbCln3Δex7/8 cells by immunoblot analysis, indicating the altered signal likely reflects dispersed lysosomes or altered localization and/or epitope availability (data not shown). It is noteworthy that Lysotracker dye, which selectively accumulates in acidic compartments, exhibited the most marked reduction in lysosomal labeling. This observation may reflect altered lysosomal pH, an established finding in JNCL [10,15]. + +Figure 6 + +Lysotracker and Lamp 2 labeling of wild-type and homozygous CbCln3Δex7/8 lysosomes Lysosomal labeling of wild-type and homozygous CbCln3Δex7/8 precursor cells with lysotracker and Lamp 2 antibody is shown. Lysotracker dye (top panels) labeled large, perinuclear-clustered lysosomes and scattered lysosomes in the periphery of wild-type cells (CbCln3+/+). Lysotracker stain was dramatically reduced in homozygous mutant cells (CbCln3Δex7/8/Δex7/8), with smaller labeled vesicles and less apparent perinuclear clustering. Lamp 2 (bottom panels) immunostaining also showed reduced signal intensity with less perinuclear clustering in homozygous CbCln3Δex7/8 cells, although the effect was somewhat less dramatic than that observed with Lysotracker dye. Wild-type and homozygous CbCln3Δex7/8 confocal images were captured with identical exposure settings. 60 × magnification. + +Consistent with the altered early endosome marker (EEA1) signal observed by immunostaining (Fig. 3), fluid-phase endocytosis was also altered in homozygous CbCln3Δex7/8 cells, as measured by dextran-FITC uptake (Fig. 7). Following a 15-minute incubation in media containing dextran-FITC, wild-type and heterozygote cells displayed brightly stained, large endocytic vesicles that were clustered in the perinuclear region. However, homozygous CbCln3Δex7/8 cells were less brightly stained with most dextran-FITC signal localizing to smaller vesicles scattered throughout the cytoplasm of the cell. + +Figure 7 + +Endocytosis in wild-type, heterozygous and homozygous CbCln3Δex7/8 cells Dextran-FITC uptake in wild-type, heterozygous and homozygous CbCln3Δex7/8 precursor cells is shown. In wild-type (CbCln3+/+, left panel) and heterozygous (CbCln3+/Δex7/8, middle panel) cells, dextran-FITC label was observed in a perinuclear-clustered vesicular pattern with scattered labeled vesicles also present in the periphery. In contrast, dextran-FITC label of homozygous mutant (CbCln3Δex7/8/Δex7/8, right panel) cells was reduced overall and exhibited smaller stained vesicles with less perinuclear clustering. Confocal images were captured with identical exposure settings. 40 × magnification. + +Finally, because subunit c is a mitochondrial protein and its turnover proceeds through autophagic engulfment of mitochondria [13], we analyzed homozygous CbCln3Δex7/8 cell mitochondrial morphology and function. Mitochondrial distribution in homozygous CbCln3Δex7/8 cells was indistinguishable from wild-type and heterozygous cells; however, homozygous CbCln3Δex7/8 mitochondria appeared more elongated by grp75 marker immunostaining and TEM analysis (Fig. 8a). 72% of homozygous mutant mitochondria were greater than 0.6 μm in length (range = 0.26 μm to 2.75 μm), while fewer wild-type mitochondria (51%) reached this length (range = 0.15 μm to 2.29 μm). Mitochondrial width was not altered in homozygous CbCln3Δex7/8 cells (data not shown). Moreover, compared to wild-type or heterozygous cells, homozygous CbCln3Δex7/8 cells had significantly reduced cellular ATP levels (1.3 fold less, Fig. 8b) and exhibited reduced survival following hydrogen peroxide treatment (~50% of wild-type survival, Fig. 8c), suggesting impaired energy metabolism and oxidative stress response. Taken together, these data support impaired mitochondrial function in homozygous CbCln3Δex7/8 cells. + +Figure 8 + +Mitochondrial morphology and function in wild-type, heterozygous and homozygous CbCln3Δex7/8 cells a. Confocal and TEM micrographs of wild-type and homozygous CbCln3Δex7/8 mitochondrial morphology are shown. Immunostaining with the inner mitochondrial membrane marker, grp75 (top panels) highlighted elongated mitochondria in homozygous mutant cells (CbCln3Δex7/8/Δex7/8), relative to wild-type mitochondria (CbCln3+/+) (insets, zoom = 2.75x). Mitochondrial distribution was not altered from the wild-type pattern. Elongated homozygous CbCln3Δex7/8 mitochondria were also observed by TEM analysis. 60 × magnification. b. Cellular ATP levels in wild-type, heterozygous and homozygous CbCln3Δex7/8 precursor cells are shown. Wild-type (open bar) and heterozygous (gray bar) CbCln3Δex7/8 cells contained ~39 μM ATP, while homozygous CbCln3Δex7/8 cells (black bar) contained ~1.3 fold reduced levels of ATP (~30 μM), which was statistically significant in a t-test (p < 0.0001). Wild-type and heterozygous CbCln3Δex7/8 cellular ATP levels were not statistically different from each other (p > 0.4). A representative of triplicate experiments is shown (n = 6 in each experiment). c. Cell survival following 24-hour hydrogen peroxide treatment is shown. Homozygous CbCln3Δex7/8 cells were ~2-fold more sensitive to oxidative stress by hydrogen peroxide treatment. Wild-type (circle) and heterozygous (triangle) CbCln3Δex7/8 cells exhibited ~50% survival rates with 75–100 μM H2O2, whereas homozygous CbCln3Δex7/8 cells (squares) had a ~50% survival rate with 50 μM H2O2. A representative of triplicate experiments is shown (n = 4 in each experiment). + +Discussion + +CbCln3Δex7/8 cerebellar precursor cells represent the first genetically accurate neuron-derived culture model of JNCL. Homozygous CbCln3Δex7/8 cells express mutant battenin and JNCL-hallmark mitochondrial ATPase subunit c accumulation, upon aging of cells at confluency. Moreover, this is the first study to indicate recessive endosomal/lysosomal membrane trafficking defects and mitochondrial dysfunction that precedes subunit c deposition in an accurate JNCL model. Importantly, these defects are likely to be early events in the JNCL disease process and may particularly impact neuronal function. + +Abnormal cathepsin D localization and processing in homozygous CbCln3Δex7/8 cells and Cln3Δex7/8 mice likely reflects altered vesicular trafficking and/or lysosomal pH, which is known to impact cathepsin D processing [14,16]. Indeed, CLN3 overexpression in HEK-293 cells altered lysosomal pH and cathepsin D processing [17], and lysosomal pH homeostasis is disrupted in JNCL [10,15]. It is noteworthy that cathepsin B and the CLN2-encoded enzyme, TPPI, are also altered in JNCL [18-20]. Nevertheless, despite the cathepsin D protein alterations that are observed in homozygous CbCln3Δex7/8 cells, cathepsin D enzymatic activity does not appear to be reduced. Thus, decreased cathepsin D activity is unlikely to account for subunit c accumulation in JNCL. + +Aging of homozygous CbCln3Δex7/8 cells at confluency is necessary to induce significantly accumulated subunit c protein. However, membrane organelle disruptions precede subunit c accumulation in homozygous CbCln3Δex7/8 cells, since they are observed without aging at confluency. Lysosomal and endosomal size and distribution are altered, and mitochondria are abnormally elongated and functionally compromised in sub-confluent homozygous CbCln3Δex7/8 cultures. These observations argue that membrane trafficking defects do not result from excessive subunit c accumulation compromising the lysosome, but rather are early events in the disease process preceding subunit c accumulation. Mitochondrial abnormalities, which have also been reported in JNCL patients and other animal models [21-23], may result from ineffective turnover by autophagy, a lysosomal-targeted pathway [24]. Alternatively, or simultaneously, battenin deficiency may impact mitochondrial function upstream of turnover, affecting mitochondrial biogenesis and/or altered transport and processing of mitochondrial proteins. + +In wild-type CbCln3 neuronal precursor cells battenin primarily co-localizes with early and late endosomes. Battenin immunostaining in homozygous CbCln3Δex7/8 neuronal precursors is significantly reduced in abundance, but mutant signal also co-localizes with endosomal markers suggesting mutant battenin protein with C-terminal epitopes is trafficked similar to wild-type protein. In other studies, CLN3/battenin protein localization has been reported to partially overlap with late endosomes and lysosomes in non-neuronal cells [7], and to lysosomes, synaptosomes [8] and endosomes [9,25] in neurons. These data jointly indicate that battenin resides in a subset of vesicular compartments linking multiple membrane trafficking pathways, perhaps functioning in vesicular transport and/or fusion. Endocytic and lysosomal-targeted pathways, including mitochondrial autophagy, are specifically implicated in this study. + +Conclusions + +The membrane trafficking and mitochondrial deficits uncovered in homozygous CbCln3Δex7/8 cells are likely to particularly impact neuronal function. Neurotransmission heavily relies on membrane vesicle transport, and a high-energy metabolism may further sensitize neurons to the loss of battenin activity. Thus, our panel of wild-type, heterozygous, and homozygous CbCln3Δex7/8 cerebellar cells provide an ideal model system to further elucidate battenin function and JNCL pathogenesis. + +Methods + +Antibody and cell staining reagents + +Nestin (clone Rat 401, 2 μg/ml), Lamp 1 (clone 1D4B, 6 μg/ml), and Lamp 2 (clone Abl-93; 6 μg/ml) monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, maintained by The University of Iowa, Department of Biological Sciences. Batp1 (1 μg/ml) was previously described [12]. Anti-subunit c antibody (0.7 μg/ml) was kindly provided by Dr. Kominami (Juntendo University, Tokyo, Japan). Additional antibodies were as follows: GFAP, 1:2000 (DAKO Corporation); calbindin, 1:5000 (Sigma); NeuN, 10 μg/ml (Chemicon); SV40 T antigen (Pab 101), 2 μg/ml (Santa Cruz Biotechnology); cathepsin D (C-20), 2 μg/ml (Santa Cruz Biotechnology); cytochrome c oxidase subunit IV (cox4), 1:1000 (BD Biosciences Clontech); PDI (H-160), 2 μg/ml (Santa Cruz Biotechnology); GM130, 1 μg/ml (BD Transduction Labs); α-tubulin, 1:15,000 (Sigma); grp75, 1:200 (Stressgen); early endosome antigen-1 (EEA1), 2 μg/ml (Santa Cruz Biotechnology); rab 7, 4 μg/ml (Santa Cruz Biotechnology). All fluorescent secondary antibodies were obtained from Jackson ImmunoResearch Laboratories and HRP-conjugated secondary antibodies were obtained from Amersham Biosciences. Additional cell markers were as follows: VVL-biotin, 1:2000 (Vector Laboratories), 10,000 MW dextran-FITC, 1 mg/ml and Lysotracker Red DND-99, 500 nM (Molecular Probes). + +Generation, maintenance and differentiation of CbCln3 cerebellar neuronal precursor lines + +Cln3Δex7/8 knock-in mice have been previously described [12]. Littermate pups from heterozygote crosses were used for primary culture establishment, by previously described methods that yield cerebellar granule neuron-enriched cultures [26]. Postnatal day 4 (P4) cerebella were dissected in Hank's Balanced Salt Solution (HBSS, Sigma), supplemented with 35 mM glucose. Tail biopsies were also collected for genomic DNA isolation and genotypic analysis. Trypsin/EDTA (10 mg/ml, Sigma) and DNase I (100 μg/ml, Sigma), suspended in HBSS, helped dissociate cerebella for primary culture plating onto 0.01% poly-ornithine (Sigma) coated 100 mm dishes. Primary cultures from individual cerebella were cultured overnight at 37°C, 5% CO2, in granule neuron growth media (Dulbecco's Modified Eagle Medium [DMEM, Gibco BRL #11995-065], 10% fetal bovine serum [Sigma #F-2442], supplemented with 24 mM KCl). Infection was performed the following day with defective retrovirus transducing the temperature-sensitive tsA58/U19 large T antigen and a selection neomycin-resistance cassette [27], as previously described [28]. Following infection, cultures were shifted to the tsA58/U19 permissive growth temperature of 33°C and selection was in the same growth media as above, with 200 μg/ml G418. Conditionally immortalized colonies were isolated 3–9 weeks post-infection and expanded for frozen stocks and further sub-clone isolation. Multiple clonal lines were obtained for each genotype and all phenotypes were confirmed in at least 2 independent CbCln3 cell lines. CbCln3 cell lines were maintained on 0.01% poly-ornithine coated dishes at 30–90% confluency, in 33°C and 5% CO2 atmosphere. Passage number was recorded (up to ~20 passages), but had no apparent impact on phenotype. Neuronal differentiation was as previously described [29] with the following cocktail: 10 ng/ml α-FGF, 250 μM IBMX, 200 nM TPA, 50 μM forskolin, 5 μM dopamine (Sigma). + +Genotyping and RT-PCR + +Genomic DNA was extracted from tail biopsies and cell pellets as described (Cotman et al., 2002). Cln3Δex7/8 knock-in allele PCR genotyping was with wild-type primers, WtF (5'-CAGCATCTCCTCAGGGCTA-3') and WtR (5'-CCAACATAGAAAGTAGGGTGTGC-3') to yield a ~250 bp band and knock-in primers, 552F (5'-GAGCTTTGTTCTGGTTGCCTTC-3') and Ex9RA (5'-GCAGTCTCTGCCTCGTTTTCT-3') to yield a ~500 bp band. PCR cycling conditions were 95°C for 30 seconds, 58°C for 30 seconds, and 72°C for 35 seconds, repeated for 34 cycles. Total RNA isolation and Cln3 RT-PCR primers and procedures have been previously described [12]. + +Subunit c accumulation assay + +Cells were seeded into 4-well chamber-slides (Falcon) at a density of 5 × 104 cells per well for microscopy studies, or into 100 mm dishes (Falcon) at a density of 5 × 105 cells per dish for protein extraction. Cells were typically >95% confluent one day post-plating, and the following day was considered 1-day post-confluency. At the indicated times, cells were either fixed with 4% formaldehyde in phosphate buffered saline (PBS), pH 7.4, for 20 minutes and processed for autofluorescence/subunit c immunostaining, or cell pellets were collected for total protein extraction. + +Alternatively, cells were fixed with 2.5% glutaraldehyde/2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.4 for 1 hour and subsequently post-fixed and processed for TEM analysis as described [12]. In confocal microscopy studies, autofluorescent signal was observed over multiple wavelengths. For co-staining, settings were reduced such that autofluorescent signal did not contribute to antibody label signal. + +Immunostaining and Immunoblot analysis + +For immunostaining, cells were seeded at a density of 3–5 × 104 cells per well in 4-well chamber-slides and grown overnight at 33°C, unless indicated otherwise. Fixation was with ice-cold 4% formaldehyde in PBS, pH 7.4, for 20 minutes, or with ice-cold methanol/acetone (1:1) for 10 minutes at -20°C followed by air-drying (antibody-dependent). Cells were washed with PBS at least 2 times, 5 minutes per wash, between each of the following steps of the staining procedure: 0.1 M glycine in PBS for 5 minutes, 0.05% or 0.1% (antibody-dependent) Triton X-100 (Fisher Scientific) in PBS for 5 minutes, 2% bovine serum albumin (BSA) in PBS for 30 minutes, primary antibody diluted in 2% BSA/PBS for 90 minutes, secondary antibody diluted in 2% BSA/PBS for 60 minutes. All incubations were carried out at room temperature. Following staining procedures, slides were coverslipped with Vectashield mounting medium (Vector Laboratories) and analyzed on a BioRad Radiance 2100 confocal microscope (Biorad), with identical exposure settings for wild-type and mutant like images. All comparisons of wild-type and mutant staining were performed in Adobe Photoshop with identical brightness and contrast adjustments. + +Total proteins were isolated from cell pellets by extraction with ice-cold 20 mM Tris, pH 7.4, 1% Triton X-100 (membrane-research grade, Roche), plus protease inhibitors (Complete mini tablet, 0.7 μg/ml pepstatin A, 2 μg/ml aprotinin, 5 μg/ml leupeptin [Roche]). Following homogenization through a 25-gauge needle (~10 passes), extracts were centrifuged at 1000 × g for 10 minutes, at 4°C, to remove debris. Typically, 20–40 μg of protein (determined by Bio-rad Dc Protein Assay) was separated by SDS-PAGE, for subsequent immunoblotting, as described [12]. 16.5% tris-tricine SDS-PAGE gels were used for subunit c immunoblotting experiments. + +Cathepsin D activity assay + +100 mm tissue culture dishes, which were approximately 80–90% confluent, were washed briefly with ice-cold PBS, and total protein extracts were isolated by scraping cells into 10 mM Tris, pH 7.4, 0.1% Triton X-100 followed by incubation on ice, for 20 minutes. The insoluble material was centrifuged at 14,000 g, the supernatant was isolated, and protein concentration was determined using the Bio-rad Dc Protein Assay. 50–70 μg of total protein extract were used to measure cathepsin D activity using the Fluorogenic Innozyme™ Cathepsin D Immunocapture Activity Assay Kit (EMD Biosciences) according to the manufacturer's recommendations. Relative fluorescence was measured using an Analyst AD plate reader (Molecular Devices) with the following filters and settings: excitation filter, 360-35; emission filter, 400-20, Flash lamp with 100 readings/well, 100 ms between readings, and 100,000 μs integration time. + +Lysosomal staining and endocytic uptake + +Cells were seeded at a density of 3–5 × 104 cells per well in 4-well chamber-slides and grown overnight at 33°C. Growth media was exchanged for fresh, pre-warmed growth media containing 500 nM Lysotracker or 1 mg/ml dextran-FITC, and cells were incubated at 33°C for 45 minutes or 15 minutes, respectively. Following labeling, cells were immediately placed on ice and washed for 10 minutes in ice-cold dye-free media, and fixed with 4% formaldehyde in PBS, for 20 minutes on ice. Chambers were removed and slides were coverslipped with Vectashield mounting media for confocal microscopy analysis, as described above. + +Morphometric analysis of mitochondria + +TEM photomicrographs (10,000 × – 40,000 × magnification) were taken from random grid fields. For length measurements, the longest side of each mitochondria was measured in centimeters, and along the length of the mitochondria, width measurements were taken every 2.5–4 mm (dependent on the magnification of the micrograph image). Following measurement, all numbers were normalized to reflect one magnification and data was analyzed using Microsoft Excel software. To ascertain unmagnified mitochondrial size, final measurement data, in centimeters, was converted to nanometers according to scale bar representation. + +ATP measurement + +ATP was measured by using the CellTiter-GLO® Luminescent Cell Viability kit (Promega), according to the manufacturer's recommendations. Briefly, cells were plated in a black opaque-walled 96 well plate (Packard Bioscience) at a density of 20000/well and incubated at 33°C overnight. The following day, CellTiter-GLO® Reagent was added to each well and cell lysis was induced by mixing 2 minutes. An ATP standard curve was prepared in the same plate. Before recording luminescence with a microplate luminometer (MicroLumat Plus LB 96V, Berthold Techonologies), the plate was dark adapted for 10 minutes at room temperature to stabilize the luminescence signal. + +Hydrogen peroxide treatment assay + +Cells were plated at a density of 10,000 cells/well in 96-well plates and incubated at 33°C overnight. The following day, fresh media containing varying concentrations of hydrogen peroxide was dispersed to each well. Cells were incubated in the presence of hydrogen peroxide for 24 hours, at 33°C, and viability was measured using the CellTiter-96® AQueous Non-Radioactive Cell Proliferation Assay (Promega), according to the manufacturer's specifications. + +Authors' contributions + +EF participated in establishment and characterization of cell lines and performed ATP determinations. PW participated in mitochondrial analysis and immunocytochemistry. JE, TL-N, AMT, and HG participated in genotypic and additional phenotypic analysis of cell lines. DR and EC generated virus-conditioned medium for conditional immortalization of cells. MEM co-conceived of the study and assisted on drafting of the manuscript. SLC co-conceived of the study, participated in establishment and phenotypic analysis of cell lines, and drafted the manuscript. All authors read and approved the final manuscript. + +Acknowledgements + +The authors thank Dr. E. Kominami for antibody to subunit c of mitochondrial ATPase, L. Trakimas and M. Ericsson of the Harvard Medical School Electron Microscope Facility, Dr. David Sulzer for assistance with TEM analysis, Dr. Sylvie Breton for assistance with endocytosis experiments, and Dr. Andre Bernards and James Follen for assistance with the cathepsin D activity assay. This work was supported by NIH/NINDS grant # NS 33648. Dr. S.L. Cotman received fellowship funding from the Batten Disease Support and Research Association (BDSRA). diff --git a/src/ontogpt/evaluation/craft/database/all/15615595.ann b/src/ontogpt/evaluation/craft/database/all/15615595.ann new file mode 100644 index 000000000..7a799e7cf --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15615595.ann @@ -0,0 +1,954 @@ +T1 UBERON:0000948 18 25 cardiac +T2 NCBITaxon:10088 43 47 mice +T3 PR:000009218 56 61 Ptdsr +T4 http://purl.obolibrary.org/obo/MONDO_0005453 152 176 Congenital heart defects +T5 UBERON:0000948 163 168 heart +T6 http://purl.obolibrary.org/obo/MONDO_0005550 197 207 infectious +T7 GO:0016265 217 222 death +T8 SO:0000704 236 243 Genetic +T9 NCBITaxon:10088 259 264 mouse +T10 SO:0000704 298 303 genes +T11 UBERON:0000948 343 348 heart +T12 GO:0007507 343 360 heart development +T13 http://purl.obolibrary.org/obo/MONDO_0005453 365 389 congenital heart disease +T14 UBERON:0000948 376 381 heart +T15 NCBITaxon:39107 413 419 murine +T16 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UBERON:0002063 13385 13388 svs +T408 UBERON:0006765 13391 13414 left superior vena cava +T409 UBERON:0006765 13416 13421 l-svc +T410 UBERON:0002016 13424 13438 pulmonary vein +T411 UBERON:0002016 13440 13443 pvn +T412 UBERON:0001514 13446 13462 descending aorta +T413 UBERON:0001514 13464 13468 d-ao +T414 UBERON:0002135 13471 13477;13492 13498 mitral ... valves +T415 UBERON:0002134 13482 13498 tricuspid valves +T416 UBERON:0002135 13500 13502 mv +T417 UBERON:0002134 13504 13506 tv +T418 UBERON:0004155 13513 13536 secondary atrial septum +T419 UBERON:0004155 13538 13541 sas +T420 UBERON:0005956 13544 13548;13559 13585 left ... ventricular outflow tracts +T421 UBERON:0005953 13553 13585 right ventricular outflow tracts +T422 UBERON:0005956 13587 13591 lvot +T423 UBERON:0005953 13593 13597 rvot +T424 UBERON:0002146 13600 13615 pulmonary valve +T425 UBERON:0002146 13617 13619 pv +T426 UBERON:0005440 13626 13639 arterial duct +T427 UBERON:0005440 13641 13643 ad +T428 UBERON:0002012 13652 13668 pulmonary artery +T429 UBERON:0000922 13700 13706 embryo +T430 UBERON:0000922 13730 13736 embryo +T431 UBERON:0002369 13826 13833 adrenal +T432 UBERON:0000922 13855 13861 embryo +T433 UBERON:0000922 13900 13907 embryos +T434 UBERON:0000922 13933 13939 embryo +T435 UBERON:0000922 13992 13999 embryos +T436 UBERON:0000922 14039 14045 embryo +T437 UBERON:0001233 14081 14100 right adrenal gland +T438 UBERON:0001233 14102 14105 rad +T439 UBERON:0004539 14123 14135 right kidney +T440 UBERON:0004539 14137 14139 rk +T441 UBERON:0000922 14156 14162 embryo +T442 UBERON:0002167 14168 14178 right lung +T443 UBERON:0002167 14180 14182 rl +T444 UBERON:0001233 14218 14237 right adrenal gland +T445 PR:000005506 14243 14249 Cited2 +T446 UBERON:0000922 14253 14259 embryo +T447 UBERON:0000922 14291 14297 embryo +T448 UBERON:0000922 14321 14327 embryo +T449 UBERON:0000948 14397 14404 Cardiac +T450 NCBITaxon:10088 14422 14426 mice +T451 PR:000009218 14435 14440 Ptdsr +T452 UBERON:0000922 14478 14484 embryo +T453 UBERON:0000922 14527 14534 embryos +T454 NCBITaxon:10088 14604 14608 mice +T455 CHEBI:18303 14621 14639 phosphatidylserine +T456 PR:000009218 14621 14648 phosphatidylserine receptor +T457 PR:000009218 14650 14655 Ptdsr +T458 SO:0000704 14689 14693 gene +T459 UBERON:0000922 14707 14716 embryonic +T460 CL:0002322 14707 14727 embryonic stem cells +T461 PR:000009218 14734 14739 Ptdsr +T462 GO:0005634 14745 14752 nuclear +T463 GO:0048568 14809 14820;14841 14843;14853 14880 development ... of ... organs during embryogenesis +T464 UBERON:0000062 14853 14859 organs +T465 PR:000009218 14902 14907 Ptdsr +T466 NCBITaxon:10088 14929 14933 mice +T467 UBERON:0012101 14941 14950 perinatal +T468 GO:0007567 14945 14950 natal +T469 UBERON:0002113 15039 15045 kidney +T470 UBERON:0000160 15047 15056 intestine +T471 UBERON:0002107 15058 15063 liver +T472 UBERON:0002048 15068 15073 lungs +T473 GO:0009790 15081 15094 embryogenesis +T474 PR:000009218 15114 15119 Ptdsr +T475 UBERON:0000922 15123 15130 embryos +T476 UBERON:0000970 15147 15153 ocular +T477 GO:0030097 15178 15192 haematopoietic +T478 PR:000009218 15261 15266 Ptdsr +T479 UBERON:0012101 15326 15335 perinatal +T480 GO:0007567 15330 15335 natal +T481 PR:000009218 15349 15354 Ptdsr +T482 NCBITaxon:10088 15358 15362 mice +T483 PR:000009218 15419 15424 Ptdsr +T484 NCBITaxon:10088 15435 15440 mouse +T485 PR:000009218 15509 15514 Ptdsr +T486 UBERON:0000922 15518 15525 embryos +T487 NCBITaxon:10088 15606 15611 mouse +T488 UBERON:0004535 15650 15664 cardiovascular +T489 PR:000009218 15732 15737 Ptdsr +T490 NCBITaxon:10088 15748 15752 mice +T491 UBERON:0000922 15768 15775 embryos +T492 PR:000009218 15784 15789 Ptdsr +T493 UBERON:0000922 15855 15861 embryo +T494 PR:000009218 15888 15893 Ptdsr +T495 UBERON:0000922 15897 15904 embryos +T496 UBERON:0000948 15909 15916 cardiac +T497 UBERON:0002094 15947 15965 ventricular septal +T498 http://purl.obolibrary.org/obo/MONDO_0002070 15947 15973 ventricular septal defects +T499 http://purl.obolibrary.org/obo/MONDO_0018089 15975 16004 double outlet right ventricle +T500 UBERON:0004145 15982 15988 outlet +T501 UBERON:0002080 15989 16004 right ventricle +T502 UBERON:0002012 16010 16026 pulmonary artery +T503 http://purl.obolibrary.org/obo/MONDO_0020419 16010 16037 pulmonary artery hypoplasia +T504 PR:000009218 16075 16080 Ptdsr +T505 UBERON:0000922 16084 16091 embryos +T506 UBERON:0000948 16096 16103 cardiac +T507 UBERON:0000922 16159 16165 embryo +T508 UBERON:0000948 16224 16231 cardiac +T509 http://purl.obolibrary.org/obo/MONDO_0005453 16224 16239 cardiac defects +T510 PR:000009218 16247 16252 Ptdsr +T511 NCBITaxon:10088 16256 16260 mice +T512 UBERON:0000922 16319 16326 embryos +T513 UBERON:0000948 16376 16381 heart +T514 http://purl.obolibrary.org/obo/MONDO_0005453 16376 16389 heart defects +T515 UBERON:0000922 16434 16440 embryo +T516 UBERON:0000922 16501 16507 embryo +T517 PR:000009218 16521 16526 Ptdsr +T518 NCBITaxon:10088 16536 16541 mouse +T519 PR:000009218 16548 16553 Ptdsr +T520 PR:000009218 16610 16615 Ptdsr +T521 SO:0000359 16639 16651 loxP-flanked +T522 CHEBI:7507 16652 16660 neomycin +T523 SO:0005853 16671 16679 cassette +T524 PR:000009218 16717 16722 Ptdsr +T525 NCBITaxon:10358 16754 16757 CMV +T526 NCBITaxon:10088 16770 16775 mouse +T527 PR:000009218 16797 16802 Ptdsr +T528 NCBITaxon:10088 16821 16826 mouse +T529 PR:000009218 16846 16851 Ptdsr +T530 UBERON:0000922 16855 16862 embryos +T531 PR:000009218 16894 16899 Ptdsr +T532 PR:000009218 16909 16914 Ptdsr +T533 UBERON:0000922 16918 16925 embryos +T534 UBERON:0002094 16936 16954 ventricular septal +T535 http://purl.obolibrary.org/obo/MONDO_0002070 16936 16962 ventricular septal defects +T536 PR:000009218 16988 16993 Ptdsr +T537 UBERON:0000922 16997 17004 embryos +T538 UBERON:0000948 17054 17061 cardiac +T539 UBERON:0000922 17124 17131 embryos +T540 PR:000009218 17153 17158 Ptdsr +T541 NCBITaxon:10088 17162 17166 mice +T542 PR:000009218 17175 17180 Ptdsr +T543 UBERON:0000922 17264 17270 embryo +T544 UBERON:0000922 17310 17317 embryos +T545 UBERON:0000922 17342 17348 embryo +T546 UBERON:0000948 17493 17500 cardiac +T547 PR:000009218 17518 17523 Ptdsr +T548 UBERON:0000922 17527 17534 embryos +T549 UBERON:0000922 17547 17553 embryo +T550 UBERON:0000915 17575 17583 thoracic +T551 UBERON:0000948 17605 17610 heart +T552 UBERON:0000922 17648 17655 embryos +T553 UBERON:0002084 17678 17682;17693 17703 left ... ventricles +T554 UBERON:0002080 17687 17703 right ventricles +T555 UBERON:0002084 17705 17707 lv +T556 UBERON:0002080 17709 17711 rv +T557 UBERON:0002094 17734 17757 interventricular septum +T558 UBERON:0002094 17759 17762 ivs +T559 UBERON:0002079 17769 17773;17784 17789 left ... atria +T560 UBERON:0002078 17778 17789 right atria +T561 UBERON:0002079 17791 17793 la +T562 UBERON:0002078 17795 17797 ra +T563 UBERON:0004154 17836 17857 primary atrial septum +T564 UBERON:0004154 17859 17862 pas +T565 PR:000009218 17913 17918 Ptdsr +T566 UBERON:0000922 17922 17929 embryos +T567 UBERON:0002094 17939 17957 ventricular septal +T568 http://purl.obolibrary.org/obo/MONDO_0002070 17939 17965 ventricular septal defects +T569 http://purl.obolibrary.org/obo/MONDO_0002070 17967 17970 VSD +T570 UBERON:0000922 18085 18092 embryos +T571 UBERON:0000948 18129 18136 Cardiac +T572 UBERON:0002370 18155 18161 thymus +T573 PR:000009218 18176 18181 Ptdsr +T574 UBERON:0000922 18185 18192 embryos +T575 UBERON:0001496 18249 18264 ascending aorta +T576 UBERON:0000948 18331 18336 heart +T577 UBERON:0000922 18352 18358 embryo +T578 UBERON:0002084 18376 18380;18391 18401 left ... ventricles +T579 UBERON:0002080 18385 18401 right ventricles +T580 UBERON:0002084 18403 18405 lv +T581 UBERON:0002080 18407 18409 rv +T582 UBERON:0002094 18432 18455 interventricular septum +T583 UBERON:0002094 18457 18460 ivs +T584 UBERON:0002079 18467 18471;18482 18487 left ... atria +T585 UBERON:0002078 18476 18487 right atria +T586 UBERON:0002079 18489 18491 la +T587 UBERON:0002078 18493 18495 ra +T588 UBERON:0003126 18506 18513 trachea +T589 UBERON:0003126 18515 18517 tr +T590 UBERON:0001496 18543 18558 ascending aorta +T591 UBERON:0001496 18560 18564 a-ao +T592 UBERON:0005956 18582 18612 left ventricular outflow tract +T593 UBERON:0005956 18614 18618 lvot +T594 UBERON:0002137 18629 18641 aortic valve +T595 UBERON:0002137 18643 18647 ao-v +T596 UBERON:0001508 18674 18685 aortic arch +T597 UBERON:0001508 18687 18691 ao-a +T598 UBERON:0001514 18710 18726 descending aorta +T599 UBERON:0001514 18728 18732 d-ao +T600 UBERON:0002012 18739 18755 pulmonary artery +T601 UBERON:0002012 18757 18759 pa +T602 UBERON:0005953 18777 18808 right ventricular outflow tract +T603 UBERON:0005953 18810 18814 rvot +T604 UBERON:0005440 18838 18851 arterial duct +T605 UBERON:0005440 18853 18855 ad +T606 UBERON:0001514 18874 18890 descending aorta +T607 PR:000009218 18924 18929 Ptdsr +T608 UBERON:0000922 18933 18939 embryo +T609 UBERON:0000948 18959 18964 heart +T610 UBERON:0002094 18972 18990 ventricular septal +T611 http://purl.obolibrary.org/obo/MONDO_0002070 18972 18997 ventricular septal defect +T612 http://purl.obolibrary.org/obo/MONDO_0002070 18999 19002 VSD +T613 UBERON:0000947 19009 19014 aorta +T614 UBERON:0002080 19031 19046 right ventricle +T615 UBERON:0002012 19052 19068 pulmonary artery +T616 UBERON:0001514 19104 19120 descending aorta +T617 UBERON:0005440 19122 19135 arterial duct +T618 PR:000009218 19200 19205 Ptdsr +T619 UBERON:0000922 19209 19215 embryo +T620 UBERON:0002094 19227 19245 ventricular septal +T621 http://purl.obolibrary.org/obo/MONDO_0002070 19227 19252 ventricular septal defect +T622 http://purl.obolibrary.org/obo/MONDO_0002070 19254 19257 VSD +T623 UBERON:0000947 19264 19269 aorta +T624 http://purl.obolibrary.org/obo/MONDO_0002070 19284 19287 VSD +T625 http://purl.obolibrary.org/obo/MONDO_0018089 19303 19332 double-outlet right ventricle +T626 UBERON:0004145 19310 19316 outlet +T627 UBERON:0002080 19317 19332 right ventricle +T628 PR:000009218 19361 19366 Ptdsr +T629 PR:000009218 19374 19379 Ptdsr +T630 UBERON:0000922 19383 19390 embryos +T631 UBERON:0005483 19408 19416;19421 19427 lobes of ... thymus +T632 UBERON:0005483 19429 19431 th +T633 UBERON:0005440 19438 19451 arterial duct +T634 UBERON:0002012 19459 19475 pulmonary artery +T635 PR:000009218 19483 19488 Ptdsr +T636 UBERON:0000922 19492 19498 embryo +T637 UBERON:0000922 19536 19542 embryo +T638 UBERON:0000922 19577 19583 embryo +T639 UBERON:0000922 19598 19604 embryo +T640 UBERON:0000922 19632 19638 embryo +T641 UBERON:0000922 19659 19666 embryos +T642 UBERON:0000922 19746 19752 embryo +T643 UBERON:0002370 19757 19763 thymus +T644 PR:000009218 19862 19867 Ptdsr +T645 UBERON:0000922 19875 19882 embryos +T646 UBERON:0002370 20030 20036 thymus +T647 UBERON:0000922 20051 20057 embryo +T648 PR:000009218 20085 20090 Ptdsr +T649 UBERON:0000922 20094 20101 embryos +T650 UBERON:0000922 20150 20157 embryos +T651 UBERON:0000922 20364 20371 embryos +T652 UBERON:0000948 20408 20415 Cardiac +T653 PR:000009218 20433 20438 Ptdsr +T654 UBERON:0000922 20442 20449 embryos +T655 UBERON:0000922 20476 20483 Embryos +T656 CHEBI:51686 20556 20567 hematoxylin +T657 UBERON:0000922 20636 20642 embryo +T658 UBERON:0000948 20658 20665 cardiac +T659 UBERON:0001062 20679 20686 anatomy +T660 UBERON:0002084 20692 20696;20707 20717 left ... ventricles +T661 UBERON:0002080 20701 20717 right ventricles +T662 UBERON:0002084 20719 20721 lv +T663 UBERON:0002080 20723 20725 rv +T664 UBERON:0002094 20748 20771 interventricular septum +T665 UBERON:0002094 20773 20776 ivs +T666 UBERON:0001496 20783 20798 ascending aorta +T667 UBERON:0001496 20800 20804 a-ao +T668 UBERON:0002084 20822 20836 left ventricle +T669 UBERON:0001508 20858 20869 aortic arch +T670 UBERON:0001508 20871 20875 ao-a +T671 UBERON:0001514 20894 20910 descending aorta +T672 UBERON:0001514 20912 20916 d-ao +T673 UBERON:0002012 20923 20939 pulmonary artery +T674 UBERON:0002012 20941 20943 pa +T675 UBERON:0002080 20961 20976 right ventricle +T676 UBERON:0002146 20985 21000 pulmonary valve +T677 UBERON:0002146 21002 21004 pv +T678 UBERON:0005440 21027 21040 arterial duct +T679 UBERON:0005440 21042 21044 ad +T680 UBERON:0001514 21063 21079 descending aorta +T681 UBERON:0002079 21085 21089;21100 21105 left ... atria +T682 UBERON:0002078 21094 21105 right atria +T683 UBERON:0002079 21107 21109 la +T684 UBERON:0002078 21111 21113 ra +T685 UBERON:0003126 21116 21123 trachea +T686 UBERON:0003126 21125 21127 tr +T687 UBERON:0002177 21130 21149 right main bronchus +T688 UBERON:0002177 21151 21154 rmb +T689 UBERON:0001043 21160 21169 esophagus +T690 UBERON:0001043 21171 21173 es +T691 UBERON:0000922 21210 21216 embryo +T692 UBERON:0002094 21235 21253 ventricular septal +T693 http://purl.obolibrary.org/obo/MONDO_0002070 21235 21260 ventricular septal defect +T694 http://purl.obolibrary.org/obo/MONDO_0002070 21262 21265 VSD +T695 UBERON:0000922 21292 21298 embryo +T696 UBERON:0000947 21320 21325 aorta +T697 UBERON:0002012 21330 21346 pulmonary artery +T698 UBERON:0002080 21362 21377 right ventricle +T699 http://purl.obolibrary.org/obo/MONDO_0018089 21379 21408 double outlet right ventricle +T700 UBERON:0004145 21386 21392 outlet +T701 UBERON:0002080 21393 21408 right ventricle +T702 UBERON:0005440 21424 21437 arterial duct +T703 UBERON:0002012 21445 21461 pulmonary artery +T704 UBERON:0000947 21493 21498 aorta +T705 UBERON:0002012 21512 21528 pulmonary artery +T706 http://purl.obolibrary.org/obo/MONDO_0020419 21512 21539 pulmonary artery hypoplasia +T707 UBERON:0002137 21545 21557 aortic valve +T708 UBERON:0002137 21559 21562 aov +T709 UBERON:0000922 21626 21632 embryo +T710 http://purl.obolibrary.org/obo/MONDO_0002070 21646 21649 VSD +T711 UBERON:0000947 21651 21656 aorta +T712 UBERON:0002080 21674 21689 right ventricle +T713 http://purl.obolibrary.org/obo/MONDO_0018089 21691 21720 double outlet right ventricle +T714 UBERON:0004145 21698 21704 outlet +T715 UBERON:0002080 21705 21720 right ventricle +T716 UBERON:0005440 21747 21760 arterial duct +T717 UBERON:0000922 21846 21853 embryos +T718 UBERON:0002370 21916 21922 thymic +T719 PR:000009218 21937 21942 Ptdsr +T720 UBERON:0000922 21946 21953 embryos +T721 UBERON:0002370 21984 21990 thymus +T722 UBERON:0000922 21995 22001 embryo +T723 PR:000009218 22029 22034 Ptdsr +T724 UBERON:0000922 22038 22045 embryos +T725 UBERON:0000922 22086 22093 embryos +T726 UBERON:0000922 22107 22113 Embryo +T727 UBERON:0000922 22182 22188 embryo +T728 PR:000009218 22237 22242 Ptdsr +T729 UBERON:0000922 22246 22253 embryos +T730 UBERON:0002370 22285 22291 thymus +T731 PR:000009218 22321 22326 Ptdsr +T732 UBERON:0000922 22330 22337 embryos +T733 UBERON:0000922 22365 22371 embryo +T734 PR:000009218 22484 22489 Ptdsr +T735 UBERON:0000922 22493 22500 embryos +T736 GO:0010467 22506 22516 expression +T737 PR:000009218 22524 22529 Ptdsr +T738 SO:0000704 22530 22534 gene +T739 UBERON:0000948 22542 22547 heart +T740 GO:0007507 22542 22559 heart development +T741 PR:000009218 22580 22585 Ptdsr +T742 SO:0000704 22586 22590 gene +T743 NCBITaxon:10088 22605 22610 mouse +T744 CHEBI:75055 22628 22633 X-Gal +T745 UBERON:0000922 22659 22666 embryos +T746 PR:000009218 22721 22726 Ptdsr +T747 GO:0010467 22727 22737 expression +T748 UBERON:0000948 22745 22750 heart +T749 UBERON:0004125 22804 22816 compact zone +T750 UBERON:0000440 22825 22834 trabeculi +T751 PR:000009218 22899 22904 Ptdsr +T752 UBERON:0000948 22908 22914 hearts +T753 UBERON:0004125 22996 23008 compact zone +T754 UBERON:0000440 23027 23036 trabeculi +T755 PR:000009218 23073 23078 Ptdsr +T756 UBERON:0001133 23107 23119 heart muscle +T757 PR:000009218 23211 23216 Ptdsr +T758 UBERON:0004535 23254 23268 cardiovascular +T759 GO:0072358 23254 23280 cardiovascular development +T760 UBERON:0001133 23295 23309 cardiac muscle +T761 PR:000009218 23350 23355 Ptdsr +T762 GO:0010467 23356 23366 expression +T763 UBERON:0000922 23374 23383 embryonic +T764 UBERON:0000948 23384 23389 heart +T765 PR:000009218 23422 23427 Ptdsr +T766 UBERON:0000922 23433 23440 embryos +T767 CHEBI:75055 23452 23457 X-Gal +T768 PR:000009218 23508 23513 Ptdsr +T769 GO:0010467 23514 23524 expression +T770 UBERON:0000948 23552 23557 heart +T771 CHEBI:75055 23586 23591 X-Gal +T772 UBERON:0000922 23600 23607 embryos +T773 GO:0010467 23640 23650 expression +T774 PR:000009218 23654 23659 Ptdsr +T775 UBERON:0002349 23667 23677 myocardial +T776 UBERON:0000060 23678 23682 wall +T777 GO:0010467 23715 23725 expression +T778 UBERON:0002349 23786 23796 Myocardial +T779 UBERON:0000060 23797 23801 wall +T780 PR:000009218 23819 23824 Ptdsr +T781 UBERON:0000922 23828 23835 embryos +T782 UBERON:0000922 23903 23910 embryos +T783 UBERON:0002349 23950 23960 myocardial +T784 UBERON:0000060 23961 23965 wall +T785 UBERON:0004125 23967 23979 compact zone +T786 UBERON:0002349 23998 24008 myocardial +T787 UBERON:0000948 24041 24046 heart +T788 NCBITaxon:10088 24111 24116 mouse +T789 NCBITaxon:9606 24127 24132 human +T790 UBERON:0000948 24242 24249 cardiac +T791 UBERON:0002075 24254 24262 visceral +T792 GO:0007565 24285 24294 gestation +T793 NCBITaxon:10088 24295 24300 mouse +T794 UBERON:0000922 24301 24308 embryos +T795 UBERON:0000922 24321 24327 embryo +T796 GO:0007565 24386 24395 gestation +T797 http://purl.obolibrary.org/obo/MONDO_0000839 24423 24441 congenital defects +T798 GO:0007565 24478 24487 gestation +T799 NCBITaxon:9606 24525 24530 human +T800 http://purl.obolibrary.org/obo/MONDO_0000839 24531 24555 congenital malformations +T801 NCBITaxon:10088 24575 24580 mouse +T802 http://purl.obolibrary.org/obo/MONDO_0000001 24611 24619 diseases +T803 NCBITaxon:10088 24746 24751 mouse +T804 UBERON:0000922 24752 24759 embryos +T805 UBERON:0000922 24842 24848 embryo +T806 NCBITaxon:39107 25094 25100 murine +T807 UBERON:0000922 25101 25110 embryonal +T808 UBERON:0012101 25114 25123 perinatal +T809 GO:0007567 25118 25123 natal +T810 NCBITaxon:10088 25140 25145 mouse +T811 SO:0000704 25146 25150 gene +T812 GO:0007565 25174 25185 gestational +T813 PR:000009218 25372 25377 Ptdsr +T814 UBERON:0000922 25381 25388 embryos +T815 UBERON:0000948 25422 25427 heart +T816 http://purl.obolibrary.org/obo/MONDO_0005453 25422 25435 heart defects +T817 UBERON:0000922 25556 25563 embryos +T818 SO:0000704 25616 25623 genetic +T819 UBERON:0000922 25698 25705 embryos +T820 NCBITaxon:33208 25814 25820 animal +T821 UBERON:0000922 25886 25893 embryos +T822 NCBITaxon:33208 25921 25927 animal +T823 GO:0007565 26033 26042 gestation +T824 SO:0000704 26073 26078 genes +T825 SO:0001023 26168 26175 alleles +T826 SO:0000704 26179 26184 genes +T827 SO:0001026 26247 26253 genome +T828 CHEBI:23995 26259 26262 ENU +T829 NCBITaxon:10088 26290 26295 mouse +T830 UBERON:0000922 26781 26788 embryos +T831 UBERON:0000922 26827 26833 embryo +T832 UBERON:0000922 26907 26914 embryos +T833 UBERON:0000922 26983 26989 embryo +T834 UBERON:0000922 27356 27362 embryo +T835 CHEBI:50905 27429 27440 teratogenic +T836 CHEBI:23888 27441 27446 drugs +T837 PR:000009218 27468 27473 Ptdsr +T838 UBERON:0000948 27477 27482 heart +T839 GO:0007507 27477 27494 heart development +T840 CHEBI:18303 27577 27595 phosphatidylserine +T841 PR:000009218 27577 27604 phosphatidylserine receptor +T842 GO:0065007 27608 27619 controlling +T843 UBERON:0002094 27620 27638 ventricular septal +T844 GO:0003281 27620 27638;27684 27695 ventricular septal ... development +T845 UBERON:0004145 27640 27653 outflow tract +T846 UBERON:0002012 27655 27671 pulmonary artery +T847 GO:0060840 27665 27671;27684 27695 artery ... development +T848 UBERON:0002370 27677 27683 thymus +T849 GO:0048538 27677 27695 thymus development +T850 PR:000009218 27732 27737 Ptdsr +T851 UBERON:0000948 27771 27778 cardiac +T852 GO:0007507 27771 27778;27790 27801 cardiac ... development +T853 UBERON:0002370 27783 27789 thymus +T854 GO:0048538 27783 27801 thymus development +T855 PR:000009218 27887 27892 Ptdsr +T856 PR:000009218 27908 27913 Ptdsr +T857 GO:0043277 27946 27974 clearance of apoptotic cells +T858 CL:0000445 27959 27974 apoptotic cells +T859 GO:0006915 28012 28021 apoptosis +T860 CL:0000445 28036 28050 apoptotic cell +T861 GO:0043277 28036 28060 apoptotic cell clearance +T862 PR:000009218 28064 28069 Ptdsr +T863 PR:000009218 28077 28082 Ptdsr +T864 UBERON:0000922 28086 28093 embryos +T865 UBERON:0000948 28101 28106 heart +T866 GO:0007507 28101 28118 heart development +T867 CL:0000445 28179 28194 apoptotic cells +T868 PR:000009218 28294 28299 Ptdsr +T869 CL:0000445 28320 28334 apoptotic cell +T870 GO:0043277 28320 28344 apoptotic cell clearance +T871 PR:000009218 28411 28416 Ptdsr +T872 UBERON:0002342 28431 28443 neural crest +T873 GO:0048538 28475 28489;28540 28546 development of ... thymus +T874 UBERON:0004145 28494 28515 cardiac outflow tract +T875 UBERON:0001508 28517 28530 aortic arches +T876 UBERON:0002370 28540 28546 thymus +T877 PR:000009218 28556 28561 Ptdsr +T878 UBERON:0000922 28572 28579 embryos +T879 UBERON:0000160 28585 28595 intestinal +T880 UBERON:0000045 28596 28603 ganglia +T881 UBERON:0002342 28641 28653 neural crest +T882 PR:000009218 28682 28687 Ptdsr +T883 NCBITaxon:10088 28691 28695 mice +T884 UBERON:0002342 28719 28731 neural crest +T885 PR:000009218 28774 28779 Ptdsr +T886 UBERON:0000948 28850 28855 heart +T887 http://purl.obolibrary.org/obo/MONDO_0005453 28850 28863 heart defects +T888 NCBITaxon:9606 28867 28873 humans +T889 UBERON:0000922 28931 28937 embryo +T890 NCBITaxon:39107 28980 28986 murine +T891 NCBITaxon:9606 28998 29003 human +T892 http://purl.obolibrary.org/obo/MONDO_0019512 29004 29036 congenital cardiac malformations +T893 UBERON:0000948 29015 29022 cardiac +T894 PR:000009218 29082 29087 Ptdsr +T895 UBERON:0000948 29112 29119 cardiac +T896 GO:0007507 29112 29131 cardiac development +T897 PR:000009218 29200 29205 Ptdsr +T898 UBERON:0000948 29225 29230 heart +T899 GO:0007507 29225 29242 heart development +T900 UBERON:0000922 29265 29271 embryo +T901 NCBITaxon:10088 29336 29341 mouse +T902 NCBITaxon:10088 29499 29504 mouse +T903 SO:0000704 29555 29562 genetic +T904 UBERON:0000948 29599 29606 cardiac +T905 GO:0007507 29599 29618 cardiac development +T906 http://purl.obolibrary.org/obo/MONDO_0005453 29623 29648 congenital heart diseases +T907 UBERON:0000948 29634 29639 heart +T908 NCBITaxon:10088 29660 29664 Mice +T909 PR:000005506 29666 29672 Cited2 +T910 PR:000003035 29682 29687 Trp53 +T911 PR:000009218 29701 29706 Ptdsr +T912 NCBITaxon:10088 29710 29714 mice +T913 UBERON:0000922 29756 29763 embryos +T914 UBERON:0010148 29813 29825 vaginal plug +T915 UBERON:0000922 29828 29834 Embryo +T916 UBERON:0000922 29848 29855 Embryos +T917 CHEBI:2511 29934 29941 agarose +T918 CHEBI:33252 30062 30069 nuclear +T919 UBERON:0002102 30116 30124 forelimb +T920 UBERON:0000922 30147 30153 embryo +T921 UBERON:0000922 30218 30225 embryos +T922 UBERON:0002101 30236 30241 limbs +T923 UBERON:0002415 30249 30254 tails +T924 UBERON:0000922 30293 30299 embryo +T925 UBERON:0000922 30326 30333 embryos +T926 UBERON:0000922 30406 30412 embryo +T927 UBERON:0000922 30478 30484 embryo +T928 UBERON:0000922 31388 31395 embryos +T929 UBERON:0000922 31574 31581 embryos +T930 UBERON:0000922 31606 31613 embryos +T931 UBERON:0000922 31798 31805 embryos +T932 UBERON:0000922 31824 31831 embryos +T933 UBERON:0000479 32113 32119 tissue +T934 UBERON:0000479 32186 32192 Tissue +T935 UBERON:0000922 32366 32373 Embryos +T936 CHEBI:16236 32393 32400 ethanol +T937 CHEBI:73702 32423 32426 wax +T938 CHEBI:51686 32459 32470 hematoxylin +T939 CHEBI:75055 32483 32488 X-Gal +T940 UBERON:0000922 32501 32508 embryos +T941 UBERON:0000922 32510 32517 Embryos +T942 UBERON:0005631 32541 32565 extraembryonic membranes +T943 GO:0010467 32612 32622 Expression +T944 PR:000009218 32626 32631 Ptdsr +T945 UBERON:0000922 32661 32668 embryos +T946 CHEBI:75055 32682 32687 X-Gal +T947 UBERON:0000922 32725 32732 embryos +T948 UBERON:0000922 32868 32874 embryo +T949 PR:000009218 32910 32915 Ptdsr +T950 UBERON:0000922 32945 32952 embryos +T951 UBERON:0000922 33036 33045 embryonic +T952 PR:000009218 33104 33109 Ptdsr +T953 UBERON:0000922 33140 33147 embryos +T954 UBERON:0000948 33573 33578 Heart diff --git a/src/ontogpt/evaluation/craft/database/all/15615595.txt b/src/ontogpt/evaluation/craft/database/all/15615595.txt new file mode 100644 index 000000000..42f51f49b --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15615595.txt @@ -0,0 +1,131 @@ +Identification of cardiac malformations in mice lacking Ptdsr using a novel high-throughput magnetic resonance imaging technique + +Abstract + +Background + +Congenital heart defects are the leading non-infectious cause of death in children. Genetic studies in the mouse have been crucial to uncover new genes and signaling pathways associated with heart development and congenital heart disease. The identification of murine models of congenital cardiac malformations in high-throughput mutagenesis screens and in gene-targeted models is hindered by the opacity of the mouse embryo. + +Results + +We developed and optimized a novel method for high-throughput multi-embryo magnetic resonance imaging (MRI). Using this approach we identified cardiac malformations in phosphatidylserine receptor (Ptdsr) deficient embryos. These included ventricular septal defects, double-outlet right ventricle, and hypoplasia of the pulmonary artery and thymus. These results indicate that Ptdsr plays a key role in cardiac development. + +Conclusions + +Our novel multi-embryo MRI technique enables high-throughput identification of murine models for human congenital cardiopulmonary malformations at high spatial resolution. The technique can be easily adapted for mouse mutagenesis screens and, thus provides an important new tool for identifying new mouse models for human congenital heart diseases. + +Background + +Congenital malformations are a major cause of death in childhood, and are typically characterized by lesions that do not compromise fetal survival. For instance, congenital heart disease (CHD) typically consists of lesions such as ventricular and atrial septal defects, which are compatible with fetal hemodynamics [1]. Although human genetic studies have identified some genes that cause congenital cardiac malformations, the molecular and developmental mechanisms underlying most of these defects remain largely unknown. Despite the high incidence of CHD (~1% of live births), only a handful of genes have been identified that when mutated, result in congenital heart disease [2-4]. + +The mouse is a particularly good model for studying mechanisms of cardiac diseases as its anatomy and development resembles that of the human more closely than any other genetically tractable organism. Importantly, the mouse is amenable to genotype-driven approaches such as transgenic knockouts of defined candidate genes [5], genome wide mutagenesis approaches using gene-trap [6] or transposon insertion screens [7], and high-throughput phenotype-driven screens that rely, for instance, on N-ethyl-N-nitrosourea (ENU) mutagenesis [8,9]. However, high-throughput cardiovascular genomic approaches in the mouse have been hampered by the paucity of phenotyping tools that allow efficient identification of complex cardiac malformations. As mouse embryos are opaque, late developmental defects are particularly difficult to identify. For instance, cardiac septal defects or outflow tract abnormalities can only be confidently identified after 14.5 days post coitum (dpc) when cardiac and outflow tract septation are completed in normal embryos. The identification of malformations in late gestation embryos typically relies on serial histological sectioning, which is extremely labor intensive. Furthermore, this often results in the irretrievable loss of 3D information, which is essential for the interpretation of complex cardiac malformations. In addition, standard pathological analysis is not amenable to high-throughput phenotype screening protocols that are required for any mutagenesis screen aiming at the functional dissection of the developmental biology of cardiac diseases. Therefore, new technological approaches must be harnessed that allow an efficient phenotyping of heart defects and also of subtle cardiac abnormalities that are at danger of being overseen in traditional histopathology screens. This is even more important in the light of upcoming new endeavors in functional mouse genome annotation [10]. Currently, new large-scale mouse mutagenesis screens are being set up in the US and in Europe that aim to produce heritable mutations in every gene in the mouse genome [11-13]. To make full use of these new mouse mutant resources more precise and efficient phenotyping methods are urgently needed [12-14]. The success of genome wide saturation mutagenesis screens depends therefore on improved phenotyping, and new high-resolution imaging approaches for mouse mutants are one of the most important which need to be established. + +We previously reported the development of fast gradient-echo MRI of single mouse embryos [15-17]. This resulted in the acquisition of a 3D dataset in under 9 hours, with an experimental image resolution of 25 × 25 × 26 μm/voxel. We showed that MRI is capable of accurately identifying normal embryonal structures, and cardiac and adrenal malformations in knockout mouse embryos, and we have validated this technique by performing in depth histological examinations of imaged embryos [15-17]. These experiments showed that single-embryo MRI could correctly identify all cardiac lesions (atrial septal defects, ventricular septal defects, outflow tract defects such as double-outlet right ventricle, and aortic arch defects) except those under 20 μm – which is below the resolution of the MRI technique. As this method images single embryos in overnight runs, it still lacks the throughput required for phenotype-driven mutagenesis screens. For instance, in a typical recessive ENU mutagenesis screen, to screen 50 ENU mutant lines using a 3-generation breeding scheme would require the analysis of ~1200 embryos [18]. We now report the development of a method of imaging up to 32 embryos simultaneously in a single unattended overnight run, at high spatial resolution. Allowing ~30 minutes per embryo, the analysis of 1200 embryos would take 75 working days for a single trained individual. We show that this high-throughput multi-embryo MRI technique can be used to rapidly identify unsuspected embryonal cardiac and visceral malformations. Using this technique we could identify a novel, and hitherto unsuspected role for the phosphatidylserine receptor (Ptdsr) in controlling ventricular septal, outflow tract and pulmonary artery development. In addition, we found thymus hypoplasia in Ptdsr-deficient embryos. These findings suggest that a novel Ptdsr-mediated pathway is required for cardiac and thymus development. + +Results + +Multi-embryo imaging + +We modified our previously described fast gradient echo magnetic resonance imaging technique [15-17] to image embryos embedded in four to eight layers (16 – 32 embryos total) in 28 mm nuclear magnetic resonance tubes using a single quadrature driven birdcage coil (Figure 1a). Preparation and embedding of embryos typically took less than an hour. In initial experiments we imaged up to 16 embryos simultaneously in overnight runs of <9 hours, with an experimental resolution of 51 × 51 × 39 μm. Subsequently, we used a custom made optimized probe with an increased sensitivity range to enhance imaging throughput. This allowed us to image 32 embryos simultaneously (Figure 1a,b), but with a larger matrix size and an increased field-of-view in the long axis of the tube. For these experiments we imaged embryos for ~12 hours, and achieved an improved experimental resolution of 43 × 43 × 36 μm. The optimized coil used for 32 embryos MRI (Figure 1) has a sensitivity range in z-direction of close to 50 mm. The artefacts seen at both ends in the longitudinal image (Figure 1b) are caused by B1-inhomogeneities at the end-rings of the coil. However, accurate image analysis for the entire data set remains possible if the height of the embryo stack does not exceed approximately 47 mm as demonstrated in the corresponding axial views of the top and the bottom layer (Figure 1c,d). The resolution achieved with the multi-embryo MRI technique allowed us to visualize the heart, cardiac septa, central nervous system, and visceral organs in fine detail (Figure 1d–f), in embryos taken from each layer. The data are permanently archived on DVDs, for subsequent analysis. Construction of 3D reconstructions of the heart typically takes ~4 hours, but was not necessary for the identification of cardiovascular defects. + +Figure 1 + +High-throughput high-resolution magnetic resonance microscopy. (a) Stack of 32 embryos embedded in a NMR tube. (b) Section through the long axis of the NMR tube showing embryos in eight layers. (c) Sagittal section through layer 8 showing the four embryos in this layer. (d–f) Transverse, sagittal, and coronal sections through individual embryos in layers 5, 1 and 4 respectively. The voxel size is 25.4 × 25.4 × 24.4 μm. Structures indicated are the spinal cord (sc), the right and left lungs, atria and ventricles (rl, ll, ra, la, rv, lv), primary atrial and interventricular septa (pas, ivs), mitral valve (mv), midbrain roof (mbr), midbrain (mb), mesencephalic vesicle (mes), thalamus (tha), hypothalamus (hy), pons (po), cerebellum (c), medulla oblongata (mo), pituitary (pit), tongue (t), thymus (th), left superior vena cava and main bronchus (lsvc, lmb), aorta (ao), liver (li), stomach (s), left adrenal and kidney (lad, lk), pancreas (pa), intestines (i), umbilical hernia (uh), aqueduct of Sylvius (aq), fourth ventricle (fv), inner ear (ie), larynx (lar), right ventricular outflow tract (rvot), spleen (sp), and testes (te). Scale bars = 500 μm; axes: d – dorsal; v – ventral; r – right; l – left; a – anterior, p – posterior. + +Sensitivity and specificity of multi-embryo imaging + +To assess the sensitivity and specificity of multi-embryo imaging in comparison to single-embryo imaging, we used a model of Cited2 deficiency [19]. Embryos lacking Cited2 (Cited2-/-) have diverse cardiac malformations, including atrial and ventricular septal defects, outflow tract and aortic arch malformations, and adrenal agenesis [15,17,19]. As the Trp53-repressor gene Cdkn2aP19ARF is a target of Cited2 [20], we also examined embryos lacking both Cited2 and Trp53 to determine if this would rescue the heart and adrenal defects in Cited2-/- mice. We imaged 50 embryos using the multi-embryo technique in the 16-embryo mode. Embryonal genotypes included 12 wild-type, 13 Cited2+/-, 14 Cited2-/-, three Cited2-/-: Trp53-/-, four Cited2-/-: Trp53+/-, two Trp53+/-, and two Trp53-/-. We analyzed the data from each embryo for cardiac malformations without knowledge of the genotype. This typically took a maximum of 30 minutes per embryo. We scored each embryo for atrial and ventricular septal defects (ASD, VSD), outflow tract (e.g. double-outlet right ventricle, common arterial trunk), and aortic arch malformations (e.g. right-sided or bilateral aortic arch, retroesophageal subclavian artery), and for adrenal agenesis. Each embryo was then re-imaged singly at high resolution, and the data re-analyzed as before. In this group we identified 20 embryos with ASD, 19 with VSD, 18 with outflow tract defects, 11 with aortic arch defects, and 21 with bilateral adrenal agenesis, using high-resolution single embryo imaging. In comparison to single embryo imaging, the overall sensitivity and specificity of multi-embryo imaging for cardiac malformations was 88% and 92% respectively. For ASD (18 identified by single embryo imaging) the sensitivity and specificity was 85% and 95%; for VSD 94% and 94%; for outflow tract malformations 94% and 100%; and for aortic arch malformations 91% and 100% respectively (Figure 2). For bilateral adrenal agenesis, the sensitivity was 100% and specificity was 95% (Figure 3). Embryos lacking both Cited2 and Trp53 had cardiovascular defects and adrenal agenesis, indicating that Trp53 does not play a major role in the genesis of these defects in mice lacking Cited2. These results indicate that multi-embryo MRI is a potentially powerful high-throughput tool for efficiently characterizing cardiovascular malformations and identifying other defects in organogenesis. + +Figure 2 + +Identification of septal, outflow tract, and aortic arch malformations using multi-embryo MRI (a – e') Images of transverse sections from 5 Cited2-/- embryos obtained using the multi-embryo technique (a–e) compared with images from the same embryos obtained subsequently using the single embryo technique (a'–e'). (a, a') Section showing left and right atria and ventricles (la, ram, live, rave). The atria are separated by the primary atria septum (pas), which is deficient at its ventral margin creating an osmium premium type of atria septal defect (ASD-P). (b, b') Section showing a ventricular septal defect (VSD) in the interventricular septum (ivs). (c, c') Section showing double outlet right ventricle, wherein the ascending aorta (a-ao) and the pulmonary artery (pa) both arise from the right ventricle (rv). The aortic valve (ao-v) is indicated. (d, d') Section showing a right-sided aortic arch (ao-a) passing to the right of the trachea (tr) and the esophagus (es). (e, e') Section showing bilateral aortic arches (ao-a) forming a vascular ring around the trachea (tr) and the esophagus (es). Also indicated are the thymus (th) and the right superior vena cava (r-svc). (f – j) Serial transverse sections through a wild-type heart obtained using single embryo MRI, demonstrating corresponding normal structures, including the systemic venous sinus (svs), left superior vena cava (l-svc), pulmonary vein (pvn), descending aorta (d-ao), mitral and tricuspid valves (mv, tv), the secondary atrial septum (sas), left and right ventricular outflow tracts (lvot, rvot), pulmonary valve (pv), and arterial duct (ad) of the pulmonary artery. Scale bars = 635 μm for multi-embryo, and 317 μm for single embryo images; axes: d – dorsal; v – ventral; r – right; l – left. + +Figure 3 + +Identification of adrenal agenesis using multi-embryo MRI Images of coronal sections from 2 embryos obtained using the multi-embryo technique (a, b) compared with images from the same embryos obtained subsequently using the single embryo technique (a', b'). (a, a') Normal right adrenal gland (rad) anterior to the right kidney (rk) in a wild-type embryo. The right lung (rl) is indicated. (b, b') Agenesis of right adrenal gland in a Cited2-/- embryo. Scale bars = 635 μm for multi-embryo, and 317 μm for single embryo images; axes: d – dorsal; v – ventral; a – anterior, p – posterior. + +Cardiac malformations in mice lacking Ptdsr + +We next evaluated the role of multi-embryo MRI in analyzing unexplained lethality in embryos generated in collaborating laboratories. Recently, we have generated mice lacking the phosphatidylserine receptor (Ptdsr-/-) on a C57BL/6J background, by gene targeting in embryonic stem cells [21]. Ptdsr is a nuclear protein of unknown function, which is essential for the development and differentiation of multiple organs during embryogenesis [21-24]. Ablation of Ptdsr function in knockout mice causes perinatal lethality, growth retardation [21,22,24] and a delay in terminal differentiation of the kidney, intestine, liver and lungs during embryogenesis [21]. In addition, Ptdsr-/- embryos develop complex ocular lesions [21] as well as haematopoietic defects [24]. However, as many malformations have been described in Ptdsr mutants, none of those detected could explain the observed perinatal lethality of Ptdsr-/- mice. In the process of phenotypical characterization of our Ptdsr-deficient mouse line, we frequently observed subcutaneous edema of varying sizes in Ptdsr-/- embryos by gross inspection ([21] and Figure 4). As the development of edema in various mouse mutants is frequently associated with cardiovascular defects [25] we started to investigate if this also holds true for Ptdsr-deficient mice. We examined 8 embryos lacking Ptdsr, and 8 littermate wild-type or heterozygous controls using multi-embryo MRI. We found that 5 of 8 Ptdsr-/- embryos had cardiac malformations, which included ventricular septal defects, double outlet right ventricle, and pulmonary artery hypoplasia (Figure 5). None of the wild-type or Ptdsr+/- embryos had cardiac malformations. These findings were confirmed on single embryo imaging (Figure 6). Furthermore, to verify the identified cardiac defects in the Ptdsr-/- mice we performed serial transverse sectioning of all analyzed embryos. In all cases, we could recognize again the same heart defects that were identified before using the multi-embryo MRI technique (Figure 7). In addition, we analyzed by multi-embryo MRI a second Ptdsr-knockout mouse line (Ptdsrtm1.1 Gbf), which is identical to the initially analyzed Ptdsr mutant except that the loxP-flanked neomycin selection cassette was removed by breeding the original Ptdsrtm1 Gbf knockout line [21] to a CMV-Cre deleter mouse line [26]. From this Ptdsrtm1.1 Gbf knockout mouse line we analyzed 8 Ptdsr-/- embryos, and as littermate controls, 3 Ptdsr+/+ and 1 Ptdsr+/- embryos. We found ventricular septal defects in five out of the eight Ptdsr-/- embryos (data not shown). Again we found no evidence for cardiac malformations in wild-type or heterozygous littermate control embryos. + +Figure 4 + +Edema in Ptdsr-/- mice (a) The Ptdsr-/- mutant (15.5 dpc) is growth retarded and the severe edema along the back of the embryo is visible. (b, c) Sagital sections of embryos at 16.5 dpc. The mutant embryo (c) exhibits massive subcutaneous edema compared to a wild-type (b) littermate. Scale bar = 100 μm in (b) and (c). + +Figure 5 + +Identification of cardiac malformations in Ptdsr-/- embryos using multi-embryo MRI (a–e) Transverse thoracic sections showing the heart of heterozygous or wild-type control embryos from each litter. The left and right ventricles (lv, rv) are separated by the interventricular septum (ivs). The left and right atria (la, ra) are also indicated, separated by the primary atrial septum (pas). (f–i) Corresponding sections through littermate Ptdsr-/- embryos, showing ventricular septal defects (VSD). Scale bar = 635 μm; axes: d – dorsal; v – ventral; r – right; l – left; a – anterior, p – posterior. Individual embryos are indicated by number. + +Figure 6 + +Cardiac malformations and thymus hypoplasia in Ptdsr-/- embryos. (a–c) Transverse and oblique (through the plane of the ascending aorta) sections, and 3D reconstruction (left-ventral oblique view) of a heart of a wild-type embryo at 15.5 dpc. The left and right ventricles (lv, rv) are separated by the interventricular septum (ivs). The left and right atria (la, ra), and the trachea (tr) are also indicated. The ascending aorta (a-ao) arises from the left ventricular outflow tract (lvot), via the aortic valve (ao-v), and continues on as the aortic arch (ao-a), which joins the descending aorta (d-ao). The pulmonary artery (pa) arises from the right ventricular outflow tract (rvot), and continues as the arterial duct (ad), which joins the descending aorta. (d–f) Corresponding images of a Ptdsr-/- embryo, showing a smaller heart with a ventricular septal defect (VSD). The aorta arises from the right ventricle. The pulmonary artery is small and its connection to the descending aorta (arterial duct) could not be identified. (g–i) Corresponding images of another Ptdsr-/- embryo, showing a ventricular septal defect (VSD). The aorta overrides the VSD resulting in a double-outlet right ventricle. (j, k) Coronal sections of Ptdsr+/+ and Ptdsr-/- embryos, showing the two lobes of the thymus (th). The arterial duct of the pulmonary artery in the Ptdsr-/- embryo is narrowed. (l) Correlation between embryo weight and volume. Scattergram of embryo weight versus embryo volume measured from multi-embryo MRI datasets for 16 embryos using Amira. The co-efficient of regression (r) is indicated. (m, n,) Absolute embryo and thymus volumes (μl) were measured from the MRI datasets from 5 wild-type (wt), 3 heterozygote (h), and 8 Ptdsr-/- (m) embryos at 15.5 dpc. There was no significant difference in the wild-type and heterozygote data, which were therefore pooled together (wt/h). (o) Relative thymus volumes (% of embryo volume) were calculated as Ptdsr-/- embryos were slightly smaller than littermate wild-type embryos. The data are represented as mean ± S.D. The probability of a type I error (P) is indicated. Scale bars = 317 μm; axes: r – right; l – left; d – dorsal; v – ventral; a – anterior, p – posterior. Individual embryos are indicated by number. + +Figure 7 + +Cardiac malformations in Ptdsr-/- embryos: analysis using histology Embryos analyzed by MRI (Figure 3) were sectioned transversely and stained with hematoxylin and eosin. (a–c) Serial caudal to cranial sections of the wild-type embryo showing normal cardiac and vascular anatomy. The left and right ventricles (lv, rv) are separated by the interventricular septum (ivs). The ascending aorta (a-ao) arises from the left ventricle, continues on as the aortic arch (ao-a), which joins the descending aorta (d-ao). The pulmonary artery (pa) arises from the right ventricle via the pulmonary valve (pv) and continues as the arterial duct (ad), which joins the descending aorta. The left and right atria (la, ra), trachea (tr), right main bronchus (rmb) and esophagus (es) are indicated. (d) Section through embryo 33 indicating the ventricular septal defect (VSD). (e, f) Sections through embryo 55 showing that both aorta and pulmonary artery arise from the right ventricle (double outlet right ventricle), and that the arterial duct of the pulmonary artery is narrow in comparison to the aorta – indicating pulmonary artery hypoplasia. The aortic valve (aov) is indicated. (g–i) Serial caudal to cranial sections through embryo 35 showing a VSD, aorta arising from the right ventricle (double outlet right ventricle), and a severely narrowed arterial duct. Scale bars = 500 μm; axes: r – right; l – left; d – dorsal; v – ventral. Individual embryos are indicated by number. + +We also observed a modest degree of thymic hypoplasia in Ptdsr-/- embryos (Figure 6k). To confirm this, thymus and embryo volumes were measured in 8 Ptdsr-/- embryos and 8 wild-type or heterozygous control embryos at 15.5 dpc. Embryo volume measured from the MRI datasets correlated very strongly with embryo weight (Figure 6l), and was modestly reduced in Ptdsr-/- embryos (Figure 6m). The volume of the thymus was significantly reduced in Ptdsr-/- embryos, even after correction for embryo volume, to 58% of the control value (Figure 6n,o). To correlate the identified cardiopulmonary malformations in Ptdsr-/- embryos with expression of the Ptdsr gene during heart development, we made use of our Ptdsr gene-trap reporter mouse line [21]. Using X-Gal staining in heterozygous embryos staged between 9.5 dpc and 12.5 dpc we found specific Ptdsr expression in the heart starting at 10.5 dpc and getting more defined to the compact zone and the trabeculi from 11.5 dpc onwards (Figure 8). Furthermore, when we analyzed Ptdsr-/- hearts by histopathology at 16.5 dpc we observed a severe differentiation defect in the compact zone as well as in the trabeculi (Figure 9), thus demonstrating that Ptdsr is in addition required for heart muscle differentiation at later stages of development. Taken together these results indicate that Ptdsr plays a hitherto unsuspected role in cardiovascular development as well as in cardiac muscle differentiation. + +Figure 8 + +Analysis of Ptdsr expression in the embryonic heart (a, b) Staining of heterozygous Ptdsr-βgeo-embryos [21] using X-Gal at 10.5 dpc (a) and 11.5 dpc (b). (a) At 10.5 dpc Ptdsr expression can be seen throughout the heart. (b) Transverse sections of X-Gal stained embryos at 11.5 dpc showed an increased expression of Ptdsr in the myocardial wall and a beginning decrease of the expression in the trabeculation. Scale bar = 100 μm in (b). + +Figure 9 + +Myocardial wall malformations in Ptdsr-/- embryos (a, b) Sagital sections of wild-type (a) and homozygous mutant (b) embryos at 16.5 dpc revealed a thinning of the myocardial wall (compact zone) and an increased myocardial trabeculation (b) in the mutant heart. Scale bar = 100 μm. + +Discussion + +Utility of MRI in identifying mouse models of human malformations + +Our results show that it is possible to efficiently identify and quantitate relatively subtle cardiac and visceral malformations in late gestation mouse embryos using multi-embryo MRI. We have deliberately optimized our technique at late gestation in order to identify those congenital defects that allow survival through most of gestation. Importantly, these defects resemble human congenital malformations, and would provide mouse models for the study of these diseases. Our method represents a simpler alternative to the multi-coil approach published recently [27,28] in which up to eight fixed mouse embryos were imaged simultaneously, with a resolution of 200 μm. In comparison, the multi-embryo method described here has a higher experimental resolution of 43 μm. Notably, it requires substantial developmental and financial effort to equip an experimental MR-system with multiple coil and receive capability. + +Role of MRI in investigating murine embryonal or perinatal lethality + +Many mouse gene knockouts display late gestational lethality, but incomplete analysis and loss of 3D information consequent to histological sectioning, results in major developmental malformations being frequently missed. As shown here, Ptdsr-/- embryos on a C57BL/6J background develop heart defects. This was not noted in recent reports [22,24], and emphasizes the need not only for completely examining several mutant embryos, but also repeating these examinations in different genetic backgrounds. A major advantage of MRI is the ability to easily ship fixed embryos from referring laboratories to the laboratory that performs the MRI analysis. This minimizes the expense of animal relocation, re-derivation, and breeding required to generate the embryos, and significantly reduces animal experimentation. + +Role of MRI in high-throughput phenotype driven screens + +MRI screens performed at late gestation would be expected to identify genes that affect later aspects of development, and identify hypomorphic and haploinsufficient alleles of genes that affect earlier steps of development. Published data from genome-wide ENU mutagenesis screens in the mouse indicate that ~30% progeny carry a heritable recessive phenotype, making 3-generation recessive screens the method of choice for identifying developmental malformations [18]. At least 24 3rd generation progeny per 1st generation mutant are typically screened, resulting in a >78% probability of identifying at least one fully penetrant recessive homozygous mutant. A typical recessive screen (e.g. 50 – 100 first generation mutants per year) would require the analysis of ~1200 – 2400 embryos per year. Our results show that multi-embryo MRI is eminently suitable for such throughput. Although, screening of 32 embryos overnight requires typically about 16 hours of data analysis, multi-embryo MRI mutagenesis screens can be easily performed at a reasonable scale if multiple operators analyze the data in parallel. As the data are permanently stored on DVDs, and can be analyzed easily by commercially available software, they can be without difficulty disseminated to specialists for further and more detailed analysis. Another powerful application of multi-embryo MRI will likely be the investigation and screening of potentially teratogenic drugs. + +Functional role of Ptdsr in heart development + +Our results presented here indicate a new, and hitherto unsuspected role for the phosphatidylserine receptor in controlling ventricular septal, outflow tract, pulmonary artery, and thymus development. This finding suggests that a novel Ptdsr-mediated pathway is required for cardiac and thymus development. Recently, we have demonstrated that in contrast to previously reported hypothetical Ptdsr functions, the Ptdsr protein is not required for the clearance of apoptotic cells [21]. Moreover, detailed analysis of apoptosis induction and apoptotic cell clearance in Ptdsr+/+ and Ptdsr-/- embryos during heart development did not reveal any difference in the number and location of apoptotic cells between the genotypes (J.B., A.D.G. and A.L. unpublished observations). This further excludes that Ptdsr has any function in apoptotic cell clearance and points to other developmental mechanisms that are affected by Ptdsr ablation. The neural crest plays an important role in the development of the cardiac outflow tract, aortic arches, and the thymus [29]. As Ptdsr-deficient embryos lack intestinal ganglia [21] which are also derived from the neural crest, these results suggest that Ptdsr-/- mice may have an underlying neural crest defect. Importantly, dysfunction of these Ptdsr-mediated pathways during development could also potentially result in heart defects in humans. + +Conclusions + +Our results validate the utility of multi-embryo MRI for high-throughput identification of murine models for human congenital cardiac malformations, and using this technique we have shown that Ptdsr is essential for normal cardiac development. Further experiments are needed to define exactly in which pathways Ptdsr is involved during heart development. We expect that multi-embryo MRI will be an important technology for future phenotype-driven mouse mutagenesis screens. The technology can be easily implemented at standard MRI imaging centers, thus allowing by collaboration with individual researchers or mouse mutagenesis centers, a high-throughput functional genetic dissection of mechanisms underlying cardiac development and congenital heart diseases. + +Methods + +Mice + +Cited2-/- [19], Trp53-/- [30], and Ptdsr-/- mice [21] have been described previously. All embryos were harvested at 15 days after detection of the vaginal plug. + +Embryo preparation + +Embryos were fixed in 4% paraformaldehyde at 4°C for ~1 week, and then embedded in 1% agarose (Seakem) containing 2 mM gadolinium-diethylenetriamine pentaacetic anhydride (Gd-DTPA, Magnevist, Schering UK) in 28 mm nuclear magnetic resonance tubes (Figure 1). The left forelimb was removed from each embryo to facilitate the identification of the left side. In addition, embryos had other limbs and/or tails removed before embedding so that each embryo in a given layer (of four embryos) could be unequivocally identified. + +Magnetic resonance imaging + +Single embryo imaging was performed as described previously [15-17]. For multi-embryo imaging, we used the same 11.7 Tesla (500 MHz) vertical magnet (Magnex Scientific, Oxon, UK). This was interfaced to a Bruker Avance console (Bruker Medical, Ettlingen, Germany) equipped with a shielded gradient system with a maximal gradient strength of 548 mTesla/m (Magnex Scientific, Oxon, UK), and quadrature-driven birdcage type coils with an inner diameter of 28 mm (Rapid Biomedical, Würzburg, Germany). Compressed air at room temperature was used to reduce the heating induced by the gradients. A 3D spoiled gradient echo sequence (echo time 10 ms), a π/2 excitation pulse with rectangular pulse shape, (π/2 = 100 μs), was used with a short repetition time (30 ms) to obtain strong T1 contrast. A matrix size of 512 × 512 × 768 (bandwidth: 130 Hz/pixel) at a field of view of 26 × 26 × 30 mm achieved an experimental resolution of 51 × 51 × 39 μm when imaging up to 16 specimens. In case of 32 embryos, a matrix size of 608 × 608 × 1408 at field of view of 26 × 26 × 50 mm, yielded an experimental resolution of 43 × 43 × 36 μm. The total experimental time was ~8.75 hours for 16 embryos, and ~12.3 hours for 32 embryos (typically overnight runs) whereby each phase encoding step was averaged four times. + +Data reconstruction and analysis + +The raw MR data were reconstructed into a stack of 1024 (for 16 embryos), or 2048 (for 32 embryos) 2D TIFF files (16 bit pixel resolution, 2 or 4 GB total size) using purpose-written software as described previously [16]. The TIFF files were analyzed using Amira 3.1(TGS Europe, Mérignac Cedex, France). 3D reconstructions were performed using the Image Segmentation Editor, and tissue volumes for morphometric analysis were measured using the Measure Tissue Statistics tool available in Amira 3.1. The probability (p) of a Type I error was calculated using a 2-sample equal variance 2-tailed t-test in Microsoft Excel. + +Histology + +Embryos were dehydrated in ethanol, embedded in paraffin wax, and sections were stained with hematoxylin and eosin. + +X-Gal staining of embryos + +Embryos were dissected free of extraembryonic membranes and then fixed in 4% paraformaldehyde at 4°C. Expression of Ptdsr was detected by staining the embryos overnight in X-Gal according to standard protocols. The embryos were postfixed in 4% paraformaldehyde and processed for documentation or histology. + +Authors' contribution + +J.E.S. developed the multi-embryo MRI technique, J.B. generated both Ptdsr knockout lines and harvested embryos for MRI and histopathological analysis, S.D.B developed the sample preparation for embryonic MRI, A.D.G. carried out the histopathological analysis of Ptdsr-/- mutants, C.B. prepared the embryos for MRI, K.C. and S.N. assisted the experimental development, S.B. analyzed the MRI and histopathological data, A.L. and S.B. were responsible for the co-ordination of the study and the drafting of the paper. + +Acknowledgments + +We thank Tim Bardsley and Neil Hoggarth for help with information technology, and Titus Lanz (Rapid Biomedical) for developing the MR probe. These studies were funded by the Wellcome Trust, British Heart Foundation, and the EU project EUMORPHIA (QLG2-CT-2002-00930). S.B. is a Wellcome Trust Senior Fellow in Clinical Science. diff --git a/src/ontogpt/evaluation/craft/database/all/15619330.ann b/src/ontogpt/evaluation/craft/database/all/15619330.ann new file mode 100644 index 000000000..ae1de7908 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15619330.ann @@ -0,0 +1,595 @@ +T1 PR:000043452 50 57 histone +T2 CHEBI:15358 50 57 histone +T3 NCBITaxon:10088 88 92 mice +T4 SO:0000704 186 197 genetically +T5 NCBITaxon:1 317 326 organisms +T6 NCBITaxon:10088 414 419 mouse +T7 UBERON:0000922 493 502 embryonic +T8 CL:0002322 493 512 embryonic stem cell +T9 NCBITaxon:9606 614 619 human +T10 PR:000043452 620 627 histone +T11 CHEBI:15358 620 627 histone +T12 GO:0000786 704 715 nucleosomes +T13 NCBITaxon:10088 719 723 mice +T14 UBERON:0000922 755 764 embryonic +T15 CL:0002322 755 775 embryonic stem cells +T16 NCBITaxon:10088 806 810 mice +T17 GO:0000785 862 871 chromatin +T18 GO:0010467 891 901 expression +T19 SO:0000902 918 927 transgene +T20 GO:0010467 928 938 expression +T21 NCBITaxon:33208 1029 1036 animals +T22 GO:0007067 1124 1131 mitosis +T23 GO:0007126 1135 1142 meiosis +T24 CHEBI:30212 1282 1288 photon +T25 GO:0022403 1345 1354;1359 1369 stages of ... cell cycle +T26 GO:0051325 1396 1406 interphase +T27 GO:0007067 1425 1432 mitosis +T28 GO:0008219 1436 1446 cell death +T29 PR:000043452 1473 1480 histone +T30 CHEBI:15358 1473 1480 histone +T31 GO:0035327 1532 1548 active chromatin +T32 GO:0007601 2150 2156 visual +T33 SO:0000704 2371 2382 genetically +T34 CHEBI:30212 2545 2552 photons +T35 UBERON:0001062 2816 2826 anatomical +T36 GO:0010467 3194 3204 expressing +T37 GO:0010467 3340 3347 express +T38 GO:0043226 3778 3787 organelle +T39 GO:0005634 3793 3800 nucleus +T40 GO:0005634 3949 3956 nucleus +T41 NCBITaxon:10088 4099 4103 mice +T42 SO:0000704 4206 4217 genetically +T43 GO:0022403 4290 4299;4304 4314 phases of ... cell cycle +T44 NCBITaxon:2759 4403 4413 eukaryotic +T45 PR:000043452 4427 4435 histones +T46 CHEBI:15358 4427 4435 histones +T47 GO:0005634 4475 4482 nuclear +T48 PR:000043452 4493 4500 Histone +T49 CHEBI:15358 4493 4500 Histone +T50 GO:0000785 4583 4592 chromatin +T51 GO:0005634 4702 4709 nuclear +T52 SO:0001528 4702 4732 nuclear localization sequences +T53 SO:0001528 4734 4737 nls +T54 PR:000043452 4740 4747 histone +T55 CHEBI:15358 4740 4747 histone +T56 GO:0051301 4882 4895 cell division +T57 GO:0005635 4905 4921 nuclear envelope +T58 SO:0001528 4951 4954 nls +T59 PR:000033987 4984 4988 lacZ +T60 GO:0007067 5101 5108 mitosis +T61 PR:000043452 5142 5150 histones +T62 CHEBI:15358 5142 5150 histones +T63 GO:0005634 5193 5199 nuclei +T64 NCBITaxon:33208 5219 5226 animals +T65 NCBITaxon:6231 5238 5252 nematode worms +T66 NCBITaxon:7215 5254 5265 fruit flies +T67 NCBITaxon:7955 5270 5279 zebrafish +T68 NCBITaxon:9606 5337 5342 human +T69 CHEBI:15358 5343 5350 histone +T70 PR:000036509 5343 5354 histone H2B +T71 GO:0035327 5393 5409 active chromatin +T72 GO:0007059 5433 5447;5462 5473 segregation of ... chromosomes +T73 http://purl.obolibrary.org/obo/MONDO_0004992 5477 5483 cancer +T74 GO:0010467 5521 5531 expression +T75 NCBITaxon:10088 5584 5588 mice +T76 GO:0007049 5641 5651 cell cycle +T77 PR:000043452 5781 5788 histone +T78 CHEBI:15358 5781 5788 histone +T79 PR:000043452 5914 5921 histone +T80 CHEBI:15358 5914 5921 histone +T81 UBERON:0000922 5960 5969 embryonic +T82 CL:0002322 5960 5974;5980 5985 embryonic stem ... cells +T83 CL:0002322 5976 5978;5980 5985 ES ... cells +T84 NCBITaxon:10088 5990 5994 mice +T85 NCBITaxon:9606 6058 6063 human +T86 PR:000036509 6064 6067 H2B +T87 CL:0002322 6238 6246 ES cells +T88 NCBITaxon:10088 6250 6254 mice +T89 PR:000036509 6421 6424 H2B +T90 SO:0000440 6487 6494 vectors +T91 SO:0000167 6513 6521 promoter +T92 SO:0000704 6569 6573 gene +T93 GO:0010467 6569 6584 gene expression +T94 CL:0002322 6588 6596 ES cells +T95 UBERON:0000922 6598 6605 embryos +T96 UBERON:0007023 6610 6615 adult +T97 NCBITaxon:10088 6616 6620 mice +T98 CL:0002322 6685 6693 ES cells +T99 GO:0010467 6709 6719 expressing +T100 PR:000036509 6723 6726 H2B +T101 CL:0002322 6762 6769 ES cell +T102 GO:0010467 6796 6806 expressing +T103 PR:000036509 6807 6810 H2B +T104 PR:000036509 6897 6900 H2B +T105 PR:000036509 6912 6915 H2B +T106 GO:0010467 6929 6939 expression +T107 PR:000036509 7121 7124 H2B +T108 PR:000036509 7135 7138 H2B +T109 NCBITaxon:10088 7285 7289 mice +T110 PR:000036509 7392 7395 H2B +T111 GO:0010467 7401 7411 expressing +T112 CL:0002322 7412 7420 ES cells +T113 PR:000043452 7471 7478 histone +T114 CHEBI:15358 7471 7478 histone +T115 GO:0007067 7575 7582 mitotic +T116 GO:0051325 7632 7642 interphase +T117 GO:0000785 7643 7652 chromatin +T118 GO:0005634 7657 7664 nuclear +T119 GO:0007067 7723 7730 mitosis +T120 GO:0098763 7757 7773 stage of mitosis +T121 GO:0051301 7791 7804 cell division +T122 GO:0010467 7876 7885 expressed +T123 GO:0007049 7915 7925 cell cycle +T124 GO:0005634 7988 7995 nuclear +T125 GO:0098763 8030 8047 phases of mitosis +T126 CL:0002322 8056 8064 ES cells +T127 GO:0007067 8145 8152 mitotic +T128 CL:0002322 8177 8185 ES cells +T129 CL:0002322 8222 8230 ES cells +T130 PR:000036509 8273 8276 H2B +T131 CL:0002322 8293 8301 ES cells +T132 GO:0007067 8313 8320 mitosis +T133 GO:0051324 8371 8379 prophase +T134 GO:0000910 8383 8394 cytokinesis +T135 UBERON:0014374 8455 8470 embryoid bodies +T136 GO:0005634 8500 8506 nuclei +T137 GO:0010467 8604 8614 expressing +T138 PR:000036509 8619 8622 H2B +T139 CL:0002322 8722 8730 ES cells +T140 GO:0010467 8731 8741 expressing +T141 PR:000036509 8746 8749 H2B +T142 PR:000009799 8829 8832 LIF +T143 PR:000009799 8866 8869 LIF +T144 GO:0000785 8891 8900 chromatin +T145 CL:0002322 8922 8930 ES cells +T146 GO:0010467 8946 8956 expressing +T147 PR:000036509 8959 8962 H2B +T148 PR:000036509 9042 9045 H2B +T149 CL:0002322 9051 9058 ES cell +T150 GO:0005634 9103 9109 nuclei +T151 GO:0051323 9113 9122 metaphase +T152 GO:0051323 9150 9159 metaphase +T153 GO:0007067 9195 9202 mitotic +T154 GO:0072686 9195 9210 mitotic spindle +T155 UBERON:0014374 9512 9525 embryoid body +T156 CL:0002322 9539 9547 ES cells +T157 GO:0010467 9563 9573 expressing +T158 PR:000036509 9576 9579 H2B +T159 GO:0005634 9622 9628 nuclei +T160 GO:0051322 9637 9645 anaphase +T161 GO:0051326 9648 9657 telophase +T162 GO:0005634 9690 9697 nuclear +T163 GO:0006915 9737 9746 apoptosed +T164 CL:0002322 9821 9829 ES cells +T165 GO:0010467 9845 9855 expressing +T166 PR:000036509 9860 9863 H2B +T167 GO:0051325 9933 9943 interphase +T168 GO:0005634 9944 9950 nuclei +T169 GO:0007067 9954 9961 mitotic +T170 GO:0005634 9962 9969 nucleus +T171 GO:0030263 9993 10001 pycnotic +T172 GO:0005634 10002 10009 nucleus +T173 GO:0007067 10078 10085 mitosis +T174 GO:0005634 10242 10249 nuclear +T175 GO:0051322 10331 10339 anaphase +T176 GO:0000910 10343 10354 cytokinesis +T177 GO:0051324 10398 10406 prophase +T178 GO:0051326 10410 10419 telophase +T179 GO:0051324 10487 10495 prophase +T180 GO:0000910 10499 10510 cytokinesis +T181 GO:0010467 10605 10615 expression +T182 PR:000036509 10622 10625 H2B +T183 NCBITaxon:10088 10644 10648 mice +T184 NCBITaxon:10088 10718 10722 mice +T185 GO:0010467 10738 10748 expressing +T186 PR:000036509 10749 10752 H2B +T187 SO:0000902 10786 10795 transgene +T188 NCBITaxon:33208 10845 10852 animals +T189 GO:0010467 10875 10885 expression +T190 SO:0000902 10933 10942 transgene +T191 NCBITaxon:10088 11019 11023 mice +T192 UBERON:0000104 11086 11094 lifespan +T193 GO:0007067 11202 11209 mitosis +T194 GO:0007126 11213 11220 meiosis +T195 NCBITaxon:10088 11262 11267 mouse +T196 UBERON:0000922 11268 11275 embryos +T197 UBERON:0007023 11280 11285 adult +T198 UBERON:0000062 11286 11292 organs +T199 GO:0010467 11317 11327 expression +T200 PR:000036509 11335 11338 H2B +T201 CL:0002242 11367 11382 nucleated cells +T202 GO:0010467 11465 11474 expressed +T203 NCBITaxon:10088 11529 11534 mouse +T204 UBERON:0000922 11535 11542 embryos +T205 GO:0000785 11579 11588 chromatin +T206 UBERON:0001062 11737 11747 anatomical +T207 GO:0051325 11798 11808 interphase +T208 GO:0000785 11809 11818 chromatin +T209 GO:0007067 11834 11841 mitotic +T210 GO:0005634 11870 11876 nuclei +T211 NCBITaxon:33208 12125 12132 animals +T212 NCBITaxon:10088 12174 12179 mouse +T213 UBERON:0000922 12180 12187 embryos +T214 UBERON:0007233 12195 12207 4-cell stage +T215 UBERON:0000358 12213 12223 blastocyst +T216 UBERON:0000109 12243 12257 gastrula stage +T217 UBERON:0000922 12565 12571 embryo +T218 GO:0007566 12586 12598 implantation +T219 GO:0007566 12613 12625 implantation +T220 NCBITaxon:10088 12626 12631 mouse +T221 UBERON:0000922 12632 12639 embryos +T222 GO:0010467 12672 12681 expressed +T223 PR:000036509 12682 12685 H2B +T224 GO:0007566 12814 12826 implantation +T225 UBERON:0000922 12827 12833 embryo +T226 CL:0000353 12846 12857 blastomeres +T227 GO:0051323 12896 12905 metaphase +T228 UBERON:0000922 13032 13038 embryo +T229 UBERON:0000922 13134 13140 embryo +T230 UBERON:0000358 13257 13267 blastocyst +T231 UBERON:0000922 13274 13280 embryo +T232 UBERON:0000087 13282 13297 Inner cell mass +T233 UBERON:0000087 13299 13302 ICM +T234 UBERON:0000087 13405 13408 ICM +T235 UBERON:0000922 13564 13570 embryo +T236 GO:0005634 13614 13620 nuclei +T237 GO:0051324 13624 13632 prophase +T238 CL:1000274 13672 13680;13691 13704 cells of ... trophectoderm +T239 UBERON:0006265 13685 13704 mural trophectoderm +T240 CL:1000274 13706 13714;13725 13738 Cells of ... trophectoderm +T241 UBERON:0006280 13719 13738 polar trophectoderm +T242 UBERON:0000087 13761 13776 inner cell mass +T243 UBERON:0000358 13848 13858 blastocyst +T244 UBERON:0000922 13952 13958 embryo +T245 UBERON:0004341 14067 14073 streak +T246 UBERON:0000922 14081 14087 embryo +T247 UBERON:0000922 14206 14212 embryo +T248 UBERON:0000922 14270 14279 embryonic +T249 UBERON:0000922 14281 14283 Em +T250 UBERON:0000922 14294 14303 embryonic +T251 UBERON:0000922 14324 14330 embryo +T252 CL:0000352 14395 14403;14408 14416 Cells of ... epiblast +T253 CL:0000223 14395 14403;14447 14455 Cells of ... endoderm +T254 UBERON:0002532 14408 14416 epiblast +T255 UBERON:0004877 14438 14455 visceral endoderm +T256 GO:0005634 14532 14539 nuclear +T257 GO:0007067 14561 14568 mitosis +T258 UBERON:0000922 14609 14615 embryo +T259 UBERON:0000922 14901 14908 embryos +T260 UBERON:0007023 14913 14918 adult +T261 UBERON:0000062 14919 14925 organs +T262 GO:0005634 15338 15345 nuclear +T263 PR:000036509 15373 15376 H2B +T264 UBERON:0000922 15492 15501 embryonic +T265 UBERON:0000922 15514 15520 embryo +T266 CL:0000223 15522 15530;15546 15554 cells of ... endoderm +T267 CL:0000222 15522 15530;15556 15564 cells of ... mesoderm +T268 CL:0000221 15522 15530;15579 15587 cells of ... ectoderm +T269 UBERON:0005439 15535 15554 definitive endoderm +T270 UBERON:0000926 15556 15564 mesoderm +T271 UBERON:0000924 15569 15587 embryonic ectoderm +T272 GO:0005634 15632 15639 nuclear +T273 UBERON:0008816 15827 15834;15844 15850 head of ... embryo +T274 GO:0005634 15905 15911 nuclei +T275 GO:0005634 16039 16045 nuclei +T276 UBERON:0002328 16069 16078 notochord +T277 UBERON:0003104 16095 16105 mesenchyme +T278 UBERON:0000925 16110 16118 endoderm +T279 UBERON:0009145 16126 16143 pharyngeal region +T280 GO:0051301 16231 16244 cell division +T281 GO:0008219 16249 16259 cell death +T282 PR:000036509 16339 16342 H2B +T283 GO:0007566 16355 16367 implantation +T284 NCBITaxon:10088 16368 16373 mouse +T285 UBERON:0000922 16374 16381 embryos +T286 UBERON:0000922 16407 16416 embryonic +T287 UBERON:0000922 16435 16441 embryo +T288 UBERON:0000922 16850 16856 embryo +T289 UBERON:0002532 16871 16879 epiblast +T290 UBERON:0000926 16881 16889 mesoderm +T291 UBERON:0004877 16891 16908 visceral endoderm +T292 UBERON:0003062 16913 16917 node +T293 GO:0005634 16973 16980 nuclear +T294 UBERON:0000922 17300 17306 embryo +T295 UBERON:0000033 17356 17360 head +T296 UBERON:0002028 17405 17414 hindbrain +T297 UBERON:0007122 17419 17438 1st branchial pouch +T298 UBERON:0002328 17759 17768 notochord +T299 UBERON:0003068 17781 17795 axial mesoderm +T300 CL:0000222 17787 17795;17811 17816 mesoderm ... cells +T301 UBERON:0003104 17800 17810 mesenchyme +T302 CL:0000134 17800 17816 mesenchyme cells +T303 UBERON:0004117 17889 17904 branchial pouch +T304 UBERON:0000925 17924 17932 endoderm +T305 CL:0000223 17924 17932;17948 17953 endoderm ... cells +T306 UBERON:0003104 17937 17947 mesenchyme +T307 CL:0000134 17937 17953 mesenchyme cells +T308 GO:0007067 18072 18079 mitotic +T309 GO:0005634 18080 18086 nuclei +T310 GO:0030263 18107 18115 pycnotic +T311 GO:0005634 18116 18122 nuclei +T312 UBERON:0000924 18124 18127 ect +T313 UBERON:0000924 18129 18137 ectoderm +T314 UBERON:0000925 18139 18141 en +T315 UBERON:0000925 18143 18151 endoderm +T316 UBERON:0009746 18153 18155 hf +T317 UBERON:0009746 18157 18165 headfold +T318 UBERON:0000926 18167 18170 mes +T319 UBERON:0000926 18172 18180 mesoderm +T320 UBERON:0002328 18182 18186 noto +T321 UBERON:0002328 18188 18197 notochord +T322 UBERON:0000062 18238 18244 organs +T323 UBERON:0007023 18250 18255 adult +T324 NCBITaxon:33208 18256 18263 animals +T325 NCBITaxon:33208 18322 18329 animals +T326 GO:0010467 18330 18340 expressing +T327 GO:0065007 18379 18389 regulation +T328 SO:0000167 18401 18409 promoter +T329 UBERON:0000062 18465 18471 organs +T330 UBERON:0007023 18486 18491 adult +T331 NCBITaxon:33208 18492 18499 animals +T332 GO:0005634 18568 18575 nuclear +T333 GO:0007067 18668 18675 mitosis +T334 GO:0005634 18680 18687 nuclear +T335 UBERON:0000922 18716 18723 embryos +T336 UBERON:0000062 18810 18816 organs +T337 PR:000036509 18825 18828 H2B +T338 UBERON:0007023 18834 18839 adult +T339 NCBITaxon:10088 18840 18844 mice +T340 UBERON:0000062 18882 18888 organs +T341 UBERON:0007023 18907 18912 adult +T342 PR:000036509 18934 18937 H2B +T343 NCBITaxon:33208 18948 18954 animal +T344 GO:0005634 18981 18988 nuclear +T345 GO:0010467 18999 19009 expression +T346 PR:000043452 19017 19024 histone +T347 CHEBI:15358 19017 19024 histone +T348 UBERON:0000062 19072 19077 organ +T349 GO:0005737 19191 19202 cytoplasmic +T350 UBERON:0000955 19289 19294 brain +T351 UBERON:0000948 19347 19352 heart +T352 UBERON:0000101 19399 19408 lung lobe +T353 UBERON:0002113 19458 19464 kidney +T354 UBERON:0000955 19589 19594 brain +T355 UBERON:0000948 19599 19604 heart +T356 UBERON:0002113 19657 19663 kidney +T357 UBERON:0002185 19732 19736 Bron +T358 UBERON:0002185 19738 19746 bronchus +T359 UBERON:0000074 19748 19752 glom +T360 UBERON:0000074 19754 19763 glomeruus +T361 UBERON:0000958 19765 19768 med +T362 UBERON:0000958 19770 19777 medulla +T363 UBERON:0003037 19779 19783 sept +T364 UBERON:0003037 19785 19791 septum +T365 UBERON:0000084 19793 19795 ub +T366 UBERON:0000084 19797 19809 ureteric bud +T367 UBERON:0002082 19811 19814 ven +T368 UBERON:0002082 19816 19825 ventricle +T369 GO:0048870 20018 20031 cell movement +T370 GO:0051301 20018 20022;20033 20041 cell ... division +T371 GO:0008219 20018 20022;20047 20052 cell ... death +T372 CL:0002322 20125 20133 ES cells +T373 UBERON:0000922 20138 20145 embryos +T374 PR:000036509 20515 20518 H2B +T375 CL:0002322 20524 20532 ES cells +T376 PR:000036509 20641 20644 H2B +T377 GO:0007566 20653 20665 implantation +T378 UBERON:0000922 20672 20679 embryos +T379 UBERON:0000922 20760 20767 embryos +T380 UBERON:0000922 20790 20797 embryos +T381 UBERON:0000358 20839 20850 blastocysts +T382 CHEBI:30212 20874 20880 photon +T383 UBERON:0000109 20949 20963 gastrula-stage +T384 NCBITaxon:10088 20964 20969 mouse +T385 UBERON:0000922 20970 20976 embryo +T386 GO:0009653 21000 21013 morphogenetic +T387 GO:0007369 21040 21052 gastrulation +T388 GO:0008283 21408 21426 cell proliferation +T389 GO:0005634 21495 21502 nuclear +T390 UBERON:0000479 21607 21613 tissue +T391 NCBITaxon:10088 21771 21776 mouse +T392 PR:000036509 21782 21785 H2B +T393 CL:0002322 21802 21810 ES cells +T394 GO:0007566 21815 21827 implantation +T395 GO:0007566 21836 21848 implantation +T396 UBERON:0000922 21849 21856 embryos +T397 CL:0002322 21936 21944 ES cells +T398 GO:0010467 21960 21970 expressing +T399 PR:000036509 21978 21981 H2B +T400 SO:0000902 21987 21996 transgene +T401 GO:0007067 22290 22297 mitosis +T402 GO:0005634 22324 22331 nuclear +T403 GO:0006915 22376 22385 apoptosis +T404 PR:000036509 22469 22472 H2B +T405 GO:0007566 22492 22504 implantation +T406 UBERON:0000922 22505 22512 embryos +T407 GO:0001824 22583 22595;22600 22610 formation of ... blastocyst +T408 UBERON:0000358 22600 22610 blastocyst +T409 UBERON:0000922 22622 22629 embryos +T410 CHEBI:30212 22668 22674 photon +T411 PR:000036509 22690 22693 H2B +T412 GO:0007369 22710 22722 gastrulation +T413 GO:0007566 22733 22745 implantation +T414 UBERON:0000922 22746 22753 embryos +T415 GO:0051301 22802 22815 cell division +T416 UBERON:0004877 22840 22857 visceral endoderm +T417 UBERON:0002532 22880 22888 epiblast +T418 UBERON:0000926 22927 22935 mesoderm +T419 UBERON:0004341 22955 22971 primitive streak +T420 GO:0000785 23131 23140 chromatin +T421 PR:000043452 23151 23158 histone +T422 CHEBI:15358 23151 23158 histone +T423 GO:0048864 23207 23220;23242 23246;23252 23257 generation of ... stem ... cells +T424 UBERON:0000922 23232 23241 embryonic +T425 CL:0002322 23232 23246;23252 23257 embryonic stem ... cells +T426 CL:0002322 23248 23250;23252 23257 ES ... cells +T427 NCBITaxon:10088 23262 23266 mice +T428 GO:0010467 23285 23295 expression +T429 NCBITaxon:10088 23330 23334 mice +T430 SO:0000704 23415 23426 genetically +T431 NCBITaxon:40674 23437 23446 mammalian +T432 NCBITaxon:1 23453 23461 organism +T433 http://purl.obolibrary.org/obo/MONDO_0000001 23527 23534 disease +T434 UBERON:0000922 23570 23577 embryos +T435 UBERON:0007023 23582 23587 adult +T436 UBERON:0000479 23588 23595 tissues +T437 UBERON:0001062 23711 23721 anatomical +T438 GO:0010467 23906 23916 expressing +T439 UBERON:0001062 24008 24018 anatomical +T440 NCBITaxon:10088 24129 24134 mouse +T441 PR:000036509 24573 24576 H2B +T442 CHEBI:15358 25037 25044 histone +T443 PR:000036509 25037 25048 histone H2B +T444 PR:000036509 25281 25284 H2B +T445 UBERON:0000922 25301 25308 embryos +T446 PR:000036509 25342 25345 H2B +T447 UBERON:0000922 25362 25368 embryo +T448 UBERON:0000922 25504 25510 embryo +T449 UBERON:0009746 25597 25606 headfolds +T450 UBERON:0004341 25627 25643 primitive streak +T451 UBERON:0004340 25657 25666 allantois +T452 UBERON:0000922 25861 25867 embryo +T453 NCBITaxon:10088 26062 26067 mouse +T454 GO:0010467 26091 26101 expressing +T455 CHEBI:15358 26119 26126 histone +T456 PR:000036509 26119 26130 histone H2B +T457 UBERON:0001062 26175 26182 anatomy +T458 NCBITaxon:10088 26272 26276 mice +T459 NCBITaxon:1 26322 26331 organisms +T460 SO:0001026 26618 26624 genome +T461 GO:0030261 26673 26696 chromosome condensation +T462 CHEBI:33893 26708 26716 reagents +T463 GO:0030261 26788 26812 chromosomal condensation +T464 GO:0098763 26840 26857 phases of mitosis +T465 GO:0000785 26900 26909 chromatin +T466 GO:0031498 26900 26923 chromatin fragmentation +T467 http://purl.obolibrary.org/obo/MONDO_0000001 26991 26998 disease +T468 PR:000036509 27040 27043 H2B +T469 NCBITaxon:10088 27060 27064 mice +T470 PR:000036509 27089 27092 H2B +T471 SO:0000704 27156 27167 genetically +T472 NCBITaxon:10088 27189 27193 mice +T473 GO:0008150 27519 27539 biological processes +T474 NCBITaxon:33208 27624 27631 animals +T475 GO:0010467 27632 27642 expressing +T476 SO:0000704 27643 27654 genetically +T477 GO:0008150 27763 27783 biological processes +T478 SO:0001026 27971 27978 genomic +T479 NCBITaxon:9606 28287 28292 human +T480 CHEBI:15358 28293 28300 histone +T481 PR:000036509 28293 28304 histone H2B +T482 SO:0000704 28305 28309 gene +T483 SO:0001026 28338 28345 genomic +T484 PR:000036509 28475 28478 H2B +T485 SO:0000155 28509 28517 plasmids +T486 SO:0000155 28599 28607 plasmids +T487 PR:000036509 28822 28825 H2B +T488 PR:000036509 28836 28839 H2B +T489 PR:000036509 28855 28858 H2B +T490 SO:0000440 28877 28884 vectors +T491 GO:0009294 28910 28922 transfection +T492 CL:0002322 28945 28953 ES cells +T493 GO:0009294 28969 28981 Transfection +T494 CHEBI:33893 28982 28989 Reagent +T495 PR:000036509 29074 29077 H2B +T496 PR:000036509 29089 29092 H2B +T497 PR:000036509 29195 29198 H2B +T498 GO:0005634 29223 29230 nuclear +T499 GO:0008283 29341 29354 proliferation +T500 GO:0007067 29363 29370 mitotic +T501 CL:0002322 29418 29425 ES cell +T502 GO:0010467 29447 29457 expressing +T503 PR:000036509 29458 29461 H2B +T504 SO:0000988 29547 29555 circular +T505 SO:0000155 29568 29575 plasmid +T506 CHEBI:17939 29602 29611 puromycin +T507 CHEBI:17939 29624 29633 Puromycin +T508 CHEBI:23888 29702 29706 drug +T509 SO:0000902 30110 30119 transgene +T510 GO:0010467 30120 30130 expression +T511 CL:0000034 30151 30160 stem cell +T512 CL:0000034 30258 30267 stem cell +T513 CL:0002322 30279 30287 ES cells +T514 GO:0040007 30293 30298 grown +T515 CHEBI:5291 30302 30309 gelatin +T516 PR:000009799 30329 30332 LIF +T517 CL:0002322 30355 30363 ES cells +T518 GO:0040007 30369 30374 grown +T519 PR:000009799 30425 30428 LIF +T520 UBERON:0014374 30453 30466 embryoid body +T521 UBERON:0014374 30489 30504 embryoid bodies +T522 UBERON:0000479 30525 30531 tissue +T523 PR:000036509 30632 30635 H2B +T524 GO:0010467 30669 30678 expressed +T525 NCBITaxon:10088 30719 30723 mice +T526 PR:000036509 30732 30735 H2B +T527 GO:0010467 30741 30751 expressing +T528 CL:0002322 30752 30760 ES cells +T529 UBERON:0000358 30810 30821 blastocysts +T530 NCBITaxon:10088 30907 30911 mice +T531 SO:0000902 31169 31178 transgene +T532 SO:0000366 31231 31250 site of integration +T533 SO:0000704 31279 31283 gene +T534 NCBITaxon:33208 31298 31305 animals +T535 CHEBI:26130 31489 31498 pigmented +T536 NCBITaxon:33208 31500 31506 animal +T537 UBERON:0002415 31507 31512 tails +T538 GO:0097617 31554 31565 hybridizing +T539 UBERON:0000922 31596 31603 Embryos +T540 UBERON:0000062 31608 31614 organs +T541 CHEBI:46756 31633 31638 HEPES +T542 UBERON:0001977 31685 31690 serum +T543 UBERON:0000479 31727 31733 tissue +T544 NCBITaxon:10088 31828 31833 mouse +T545 UBERON:0000922 31834 31841 embryos +T546 NCBITaxon:10114 31857 31860 rat +T547 UBERON:0001977 31861 31866 serum +T548 CHEBI:17544 31891 31902 bicarbonate +T549 CHEBI:16526 32024 32027 CO2 +T550 GO:0005737 32091 32102 cytoplasmic +T551 UBERON:0000922 32244 32251 Embryos +T552 UBERON:0000479 32276 32282 tissue +T553 UBERON:0000922 32479 32486 embryos +T554 UBERON:0000479 32518 32525 tissues +T555 UBERON:0007023 32545 32551 adults +T556 UBERON:0000922 32707 32713 embryo +T557 UBERON:0000922 32862 32869 embryos +T558 CHEBI:30212 33283 33289 photon +T559 CHEBI:30212 33662 33668 photon +T560 CHEBI:33341 33686 33694 Titanium +T561 CHEBI:30212 33794 33800 photon +T562 CHEBI:30212 33884 33890 photon +T563 PR:000036509 34402 34405 H2B +T564 NCBITaxon:10088 34421 34425 mice +T565 PR:000036509 34633 34636 H2B +T566 UBERON:0000358 34647 34657 blastocyst +T567 UBERON:0000358 34835 34845 blastocyst +T568 UBERON:0003062 35079 35083 node +T569 PR:000036509 35106 35109 H2B +T570 UBERON:0000922 35125 35131 embryo +T571 UBERON:0002328 35293 35302 notochord +T572 PR:000036509 35318 35321 H2B +T573 UBERON:0000922 35338 35344 embryo +T574 PR:000036509 35518 35521 H2B +T575 UBERON:0000922 35538 35544 embryo +T576 PR:000036509 35704 35707 H2B +T577 CL:0002322 35718 35726 ES cells +T578 PR:000036509 35887 35890 H2B +T579 GO:0007566 35905 35916 mplantation +T580 UBERON:0000922 35917 35924 embryos +T581 PR:000036509 36086 36089 H2B +T582 GO:0007566 36104 36116 implantation +T583 UBERON:0000922 36117 36123 embryo +T584 PR:000036509 36294 36297 H2B +T585 UBERON:0000922 36313 36319 embryo +T586 UBERON:0009746 36501 36509 headfold +T587 PR:000036509 36540 36543 H2B +T588 UBERON:0000922 36559 36565 embryo +T589 UBERON:0004340 36767 36776 allantois +T590 UBERON:0002415 36791 36795 tail +T591 PR:000036509 36826 36829 H2B +T592 UBERON:0000922 36845 36851 embryo +T593 SO:0000155 37041 37048 plasmid +T594 http://purl.obolibrary.org/obo/MONDO_0004992 37100 37106 Cancer +T595 UBERON:0000948 37335 37340 Heart diff --git a/src/ontogpt/evaluation/craft/database/all/15619330.txt b/src/ontogpt/evaluation/craft/database/all/15619330.txt new file mode 100644 index 000000000..6b6fe38d2 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15619330.txt @@ -0,0 +1,183 @@ +Dynamic in vivo imaging and cell tracking using a histone fluorescent protein fusion in mice + +Abstract + +Background + +Advances in optical imaging modalities and the continued evolution of genetically-encoded fluorescent proteins are coming together to facilitate the study of cell behavior at high resolution in living organisms. As a result, imaging using autofluorescent protein reporters is gaining popularity in mouse transgenic and targeted mutagenesis applications. + +Results + +We have used embryonic stem cell-mediated transgenesis to label cells at sub-cellular resolution in vivo, and to evaluate fusion of a human histone protein to green fluorescent protein for ubiquitous fluorescent labeling of nucleosomes in mice. To this end we have generated embryonic stem cells and a corresponding strain of mice that is viable and fertile and exhibits widespread chromatin-localized reporter expression. High levels of transgene expression are maintained in a constitutive manner. Viability and fertility of homozygous transgenic animals demonstrates that this reporter is developmentally neutral and does not interfere with mitosis or meiosis. + +Conclusions + +Using various optical imaging modalities including wide-field, spinning disc confocal, and laser scanning confocal and multiphoton excitation microscopy, we can identify cells in various stages of the cell cycle. We can identify cells in interphase, cells undergoing mitosis or cell death. We demonstrate that this histone fusion reporter allows the direct visualization of active chromatin in situ. Since this reporter segments three-dimensional space, it permits the visualization of individual cells within a population, and so facilitates tracking cell position over time. It is therefore attractive for use in multidimensional studies of in vivo cell behavior and cell fate. + +Background + +Macro- and microscopic imaging are pivotal readouts in the field of biology both for determining the normal (baseline) course of events and for observing the effects of experimental perturbations and natural aberrations [1]. Recent advances in microscopic imaging make it possible to routinely gain visual access to samples hundreds of microns thick [2]. The emergence of green fluorescent protein (GFP) as a reporter has opened up many new experimental approaches that were not previously possible [2-4]. GFP and other genetically-encoded autofluorescent protein reporters have a number of properties that make them ideal for multidimentional imaging of living specimens: no substrate (except photons) is required to generate signal, they have a high signal-to-noise ratio, are non-toxic, stable at 37°C and resistant to photobleaching. Moreover they are available in an increasingly large compendium of spectrally-distinct variants. + +To construct high-resolution anatomical models of normal, mutant and pathological situations, we must establish technologies to identify and follow individual cells in three-dimensional (3D) space and in 3D over time, in four-dimensions (4D). Unfortunately, native fluorescent proteins permit tracking the position of any given cell over time only if the population of tagged cells is distributed among non-expressing cells by virtue of lineage or in a mosaic experimental situation [5-9]. In situations where groups, or all cells in a 3D field of view express a fluorescent protein label, information on the behavior of individual cells cannot be discerned. Therefore an approach is required where 3D space is segmented at cellular resolution. This is most easily achieved if each cell can be marked with an easily identifiable tag that is visible at subcellular resolution [10,11]. Since it exhibits low autofluorescence, and is a single, universal and volumetrically constrained cellular organelle, the nucleus is ideal for such labeling [12-14]. + +Our goal was to take advantage of this feature and to develop a non-invasive fluorescent protein marker of the nucleus for in toto imaging (all cells within the multidimensional space being imaged – discussed in Ref. [11]) of individual cells in situ in living mice [10-12]. For the unequivocal identification of individual cells, we sought a developmentally neutral, genetically-encoded autofluorescent protein-based marker that labels DNA during all phases of the cell cycle while preserving cell morphology and behavior. + +As the principal structural proteins of eukaryotic chromosomes, histones are attractive targets for fluorescent nuclear labeling. Histone tagged fluorescent protein fusions have previously been shown to incorporate into chromatin without any adverse effects on the viability of cells in culture [15]. When compared to reporters containing nuclear localization sequences (nls), histone fusions exhibit an improved signal-to-noise ratio and have the distinct advantage of signal remaining bound to the target even during cell division when the nuclear envelope has broken down. In contrast nls-tagged markers (both GFP and lacZ) become dispersed throughout a cell during division, making it difficult to distinguish individual cells during mitosis. + +To date GFP fusions to several histones have been generated and used for labeling nuclei in live transgenic animals, including nematode worms, fruit flies and zebrafish [13,14,16,17]. One of these is a fusion between EGFP and human histone H2B which was developed in order to label active chromatin and used to follow the segregation of double minute chromosomes in cancer cells [15]. We have investigated the expression and germline transmission of this type of fusion in mice and established its usefulness not only for imaging cell cycle dynamics [18], but also for tracking cells in living specimens. Moreover unlike native GFP variants this subcellularly localized histone fusion was found to withstand fixation while retaining both fluorescence and subcellular localization. + +Results + +To evaluate histone-tagged fluorescent protein fusions in embryonic stem (ES) cells and mice, we generated constructs comprising an N-terminally positioned human H2B sequence followed at the C-terminus by sequences for various fluorescent proteins both GFP and DsRed-based. We previously reported that DsRed1 was not amenable to use in ES cells or mice [22], however several improved DsRed variants have recently become available [10]. We therefore chose to evaluate DsRed2 and DsRedExpress as part of this study. + +The H2B-fluorescent protein fusions we generated were introduced into vectors utilizing the CAG promoter [19] designed to drive high-level constitutive gene expression in ES cells, embryos and adult mice [20]. Standard protocols were used to establish stable lines of ES cells constitutively expressing an H2B fusion [20-22]. Several transgenic ES cell lines were generated each expressing H2B-EGFP at strong homogenous levels [23]. However, even though we did recover lines with H2B-DsRed2 and H2B-DsRedExpress expression [24], subsequent maintenance of these lines in culture revealed a continued reduction and heterogeneity in fluorescence. We were unable to establish lines with sustained homogenous H2B-DsRed2 or H2B-DsRedExpress fluorescence. Moreover our recent data suggest that mRFP1 [10,25], a rapidly-maturing monomeric form of DsRed, is amenable to use in mice, both in its native form and as a part of functional fusion proteins (AKH unpublished observations). + +H2B-EGFP expressing ES cells are shown in Fig. 1. It noteworthy that with this histone fusion we observed a high signal-to-noise ratio and so could achieve high-resolution imaging of mitotic chromosomes (pink arrowheads), various states of interphase chromatin and nuclear debris (yellow arrowheads). Moreover for cells undergoing mitosis we could also discern the stage of mitosis and the plane of cell division (Fig. 1b inset). Previous work indicated that a similar fusion protein expressed in HeLa cells did not affect cell cycle progression [15], and accordingly not only could we visualize nuclear dynamics and identify the various phases of mitosis in live ES cells [26] (Fig. 2), but in doing so, we did not observe any change in growth rate or mitotic index in the transgenic ES cells compared to non-transgenic parental ES cells (data not shown). By imaging several CAG::H2B-EGFP transgenic ES cells undergoing mitosis (n = 30) we calculated the progression from early prophase to cytokinesis to take less than one hour (Fig. 2). Furthermore imaging of embryoid bodies demonstrated that individual nuclei could be discerned from a three-dimensional population of densely packed cells all of which were expressing the H2B-EGFP marker (Fig. 1c). No loss of fluorescence was observed with prolonged in vitro passage of the ES cells expressing the H2B-EGFP fusion in the absence of positive selection in the presence or absence of LIF (t > 3 months in the presence of LIF). + +Figure 1 + +Imaging chromatin in living transgenic ES cells constitutively expressing a H2B-EGFP fusion protein. (a) Bright-field and (b) dark-field micrographs of a CAG::H2B-EGFP ES cell colony. The inset shows a detail with three nuclei in metaphase (pink arrowheads) with the metaphase plates orientated differently. The mitotic spindle of the cell at the top is closely aligned to the z-y plane whereas those for the lower two cells are more closely aligned with the x-z planes. (c) Rendered stack (3-D reconstruction) of sequential optical slices acquired using spinning disc confocal methodology, projected as a fixed angle view of an embryoid body comprised of ES cells constitutively expressing a H2B-EGFP fusion. Pink arrowheads indicate two nuclei in late-anaphase – telophase. Yellow arrowhead points to the nuclear remnant of a cell that has necrosed or apoptosed. (d – f) High-power sequential optical sections each (1 μm apart) through ES cells constitutively expressing the H2B-EGFP fusion, taken using laser scanning confocal methodology showing interphase nuclei, a mitotic nucleus (pink arrowhead) and a pycnotic nucleus (yellow arrowhead). + +Figure 2 + +Live imaging the progression through mitosis. Laser scanning confocal x-y images taken at a single z-plane at five minute intervals for one hour. Note that not all green fluorescence (corresponding to nuclear material) will be represented in the plane being imaged. A cell progressing from anaphase to cytokinesis (pink arrowheads). A cell progressing from prophase to telophase (blue arrowheads). The average time taken to transition from early prophase to cytokinesis was calculated to be approximately 1 hour (n = 30). + +We next tested the effects of widespread expression of an H2B fusion protein in mice. We generated germ line chimeras and established transgenic lines of mice constitutively expressing H2B-EGFP. We were able to breed this transgene to homozygosity, resulting in viable and fertile animals exhibiting widespread expression with no overt morphological abnormalities. The transgene has been maintained for over three years in a breeding colony of homozygous mice with no apparent effect on viability, breeding performance or lifespan. We therefore infer that this fusion protein is developmentally neutral and does not interfere with either mitosis or meiosis. Wide-field microscopic analysis of both mouse embryos and adult organs demonstrates widespread expression of the H2B-EGFP fusion in all types of nucleated cells. + +We used laser scanning confocal microscopy [10,11] to image this constitutively expressed transgenic reporter at subcellular resolution in live mouse embryos. Such non-invasive visualization of chromatin in living preparations allowed us to acquire high-magnification sequential optical sections (z-stacks) that can be used to generate high-resolution anatomical volumetric (3-dimensional) images with details of interphase chromatin in addition to mitotic chromosomes and fragmenting nuclei. To do this, stacks of sequential optical sections are reconstructed into 3-dimensional projections. This methodology can be used to generate 3-dimensional (3D) image sets not only of cells propagated in culture but also of cells in situ in living animals and is illustrated here by imaging whole mouse embryos at the 4-cell stage, the blastocyst stage, and the pre-gastrula stage (Fig. 3 and Additional Files 1 and 2). These data sets can be computationally manipulated in various ways, for example for the visualization of individual xy slices from a z-stack, rendered images from the full, or part of a z-stack, and color-coded depth projections of a z-stack (Fig. 3). + +Figure 3 + +Live embryo imaging of preimplantation and early postimplantation mouse embryos hemizygous for a constitutively expressed H2B-EGFP fluorescent fusion. (a) Single confocal optical section fluorescence overlay on a bright-field image of a 5-cell stage pre-implantation embryo. Two of the blastomeres are dividing synchronously and are in metaphase (pink arrowheads in b). (b) Dark-field projection of the entire rendered z-stack of x-y sections (n = 19), through the entire embryo shown in panel a. (c) Color-coded depth projection of the entire z-stack of x-y images for the embryo shown in the previous panels. (d) Single confocal optical section fluorescence overlay on a bright-field image of a blastocyst stage embryo. Inner cell mass (ICM) is to the top left corner and second polar body is on the bottom left, juxtaposed to the edge of the ICM. (e) Dark-field projection of half the rendered z-stack of x-y sections (n = 40, sections 1–19 were used for generating the projection), spanning half the embryo shown in panel d. Condensed chromosomes of nuclei in prophase (pink arrowheads) can be seen in three cells of the mural trophectoderm. Cells of the polar trophectoderm (green arrowhead) and inner cell mass (blue arrowhead) can also be distinguished by position within the half-blastocyst reconstruction. (f) Color-coded depth projection of the entire z-stack of x-y images for the embryo shown in the previous two panels. (g-h) Saggital views and rendered z-stacks of x-y images of an E5.75 (pre-streak stage) embryo. (g) Single optical confocal section fluorescence overlay on a bright-field image positioned half the way through the embryo. The brackets on the left illustrate the position of the embryonic (Em) and extraembryonic (Ex) regions of the embryo. (h) The same optical section with only the fluorescence image. Cells of the epiblast (blue arrowhead) and visceral endoderm (green arrowhead) can clearly be distinguished on the basis of position and nuclear morphology. Cells in mitosis can readily be distinguished within the embryo (pink arrowhead). (i) Color-coded depth projection of the stack of serial sections (n = 60), part of the series of which is shown in the previous two panels. Color-coded z-scale (upper right) applies to all projections and denotes distances along the z-axis (0–120 μm). + +Data on older embryos and adult organs illustrates that larger specimens can be imaged, however not in their entirety given current limitations in optical imaging capabilities. Instead of imaging the whole specimen, larger samples are positioned so that data can be acquired from regions of interest, which can then be acquired in a tiled manner and computationally re-aligned in image acquisition and processing software. + +Our data demonstrates that nuclear morphology afforded by the H2B-EGFP fusion can be used to identify different cell types. In both the raw data, and a rotated rendered stack of an embryonic day (E) 7.5 embryo, cells of the definitive endoderm, mesoderm and embryonic ectoderm can be distinguished solely on the basis of nuclear morphology and orientation in addition to their expected position (Fig. 4b–h and Additional File 3). Low magnification rendered z-stacks taken from a transversely cut section through the head of an E10.5 embryo (Fig. 4i) reveal the stereotypical 3D organization of nuclei within the region imaged (Fig. 4j), and electronically magnified views of this image illustrate a characteristic apposition of nuclei both in and around the notochord, and within the mesenchyme and endoderm of the pharyngeal region (Fig. 4k and 4l and Additional Files 4 and 5), in addition to providing information on cell division and cell death (pink and yellow arrowheads, respectively in Fig. 4l). + +Figure 4 + +Live imaging H2B-EGFP in postimplantation mouse embryos. (a) Lateral view of the embryonic region of an E7.5 embryo (anterior to the left) with box depicting the region imaged in b and double-headed arrow depicting the x-y layering of the z-stack. (b-d) single optical x-y sections of fluorescence overlayed on bright-field images acquired at the same focal plane. Each panel is 60 μm apart from the preceding panel. These panels comprise x-y images in the z-stack depicted in panel a. The different layers of this stage of embryo including the epiblast, mesoderm, visceral endoderm and node can be distinguished on the basis of both position and nuclear morphology. (e-h) projection of a rendered z-stack of (x-y) sections (n = 90) of the dark-field component of the sections taken in the series schematized in a and of the raw data shown in b. (e) 0° rotation, (f) 60° rotation, (g) 120° rotation, and (h) 180° rotation views. (i) low-magnification frontal view of an E11 embryo that has had a transverse cut made to remove the head. The box depicts the region (at the ventral hindbrain and 1st branchial pouch) subject to laser scanning confocal imaging, with the double-headed arrow depicting the x-y layering of the acquired z-stack. (j) rendered (z-) stack of sections (n = 200, i.e. 400 μm depth) taken through the boxed region. (k) rendered stack of top 50 x-y sections (100 μm depth) taken from the region imaged around the notochord (comprising axial mesoderm and mesenchyme cells). (l) rendered stack of top 50 sections (100 μm depth) taken around the branchial pouch region (comprising endoderm and mesenchyme cells). The sections used to generate the rendered stacks in panels k and l were electronically magnified. Pink arrowheads, mitotic nuclei; yellow arrowheads, pycnotic nuclei; ect, ectoderm, en, endoderm, hf, headfold, mes, mesoderm, noto, notochord. + +Wide-field microscopic examination of organs from adult animals revealed widespread fluorescence as has been reported for animals expressing native fluorescent proteins under the regulation of the CAG promoter [20,22,27]. Laser scanning confocal imaging of various organs obtained from adult animals was used to generate high-resolution images revealing stereotypical nuclear positions, reflecting different cell types and revealing other subcellular details, such as mitosis and nuclear fragments, also observed in embryos (Fig. 5 and Additional Files 3, 4, 5). + +Figure 5 + +High resolution live imaging of the organs of CAG::H2B-EGFP adult mice. Confocal images of freshly isolated organs from a 6 week old adult male hemizygous CAG::H2B-EGFP Tg/+ animal illustrate the widespread nuclear localized expression of the histone fusion. A transverse cut was made through each organ and the cut surface was placed closest to the objective lens and imaged. Cell tracker orange was used as a vital cytoplasmic counter stain. The panels show rendered confocal z-stacks imaged through 80 μm of the brain using a 20x plan-apo objective (a-c), 568 μm of the heart using a 5x fluar objective (d-f), 142 μm of a lung lobe using a 5x fluar objective (g-i) and 346 μm of a kidney using a 5x fluar objective low power view (j-l), and high power view (m-o). Insets in panels a and d show the region of the brain and heart imaged, respectively. High resolution images of the kidney (m-o) illustrate electronic magnification of the data shown in j-l. Bron, bronchus; glom, glomeruus; med, medulla; sept, septum; ub, ureteric bud; ven, ventricle. Areas of increased fluorescence in the red channel are an artefact due to saturated pixels in regions of the sample closest to the objective. + +Finally we investigated whether we could follow cell movement, division, and death in time-lapse experiments using various imaging modalities. We cultured ES cells and embryos on the stages various each of which had been modified to permit culture under physiological conditions. The different types of data routinely generated using different optical imaging modalities that are widely used and commercially available are illustrated in Figure 6. Spinning disc confocal microscopy [1] was used for short-term high-resolution 4D imaging of CAG::H2B-EGFP ES cells (Fig. 6 and Additional File 6), wide-field microscopy was used for long-term low-resolution imaging of CAG::H2B-EGFP preimplantation stage embryos (Fig. 6 and Additional File 7). Note that development proceeds normally in most embryos, and that some of the embryos imaged are undergoing cavitation to form blastocysts [28] (arrowheads). Two-photon excitation microscopy [10,29,30] was used to image cells in a whole gastrula-stage mouse embryo without perturbing the morphogenetic movements associated with gastrulation (Fig. 6 and Additional File 8). Cells can clearly be followed through the successive time points in each of these experimental situations ranging from a few minutes (short-term) to 24 hours (long-term) time-lapse duration. These studies reflect the range of resolutions at which information can be acquired using a marker of this type. We observed normal cell proliferation throughout the course of these imaging experiments and no excessive nuclear fragmentation. Also, because the on-stage cultures were comparable to parallel cultures maintained in a tissue culture incubator, we conclude that the outcome of the cultures was not affected by the various imaging modalities. + +Figure 6 + +Dynamic time-lapse imaging of mouse CAG::H2B-EGFP transgenic ES cells, preimplantation and postimplantation embryos using different imaging modalities. (a) Rendered confocal stacks of transgenic ES cells constitutively expressing a CAG::H2B-EGFP transgene representing a 25 minute time-lapse recording of images acquired using a spinning disc confocal scan head. x-y sections with a z-interval of 0.2 μm were taken at a rate of 10/second over a total z-stack of 40 μm. Cells can be traced through the 4D rendered stack. Cells entering or completing mitosis (pink arrowheads) and the nuclear remnant of a cell that has either undergone apoptosis or necrosis (yellow arrowhead) are clearly visible. (b) Wide-field imaging of CAG::H2B-EGFP transgenic preimplantation embryos. This 24 hour image sequence illustrates cavitation leading up to the formation of the blastocyst in several embryos (violet arrowheads). (c) Rendered two-photon stacks of CAG::H2B-EGFP transgenic gastrulation stage postimplantation embryos. This 40 minute time-lapse sequence illustrates cell division and tracking within the visceral endoderm (green arrowhead) and epiblast (blue arrowheads) and the movement of mesoderm emanating from the primitive streak, which is positioned to the right, out of the field of view. Scale bar in a = 10 μm, b = 100 μm and c = 50 μm. + +Discussion + +Here we report the evaluation of a chromatin localized histone fusion fluorescent reporter in vivo through the generation of transgenic embryonic stem (ES) cells and mice having widespread expression of this reporter. + +The transgenic mice that we have generated provide a new tool for high-resolution live imaging of a genetically tractable mammalian model organism [10,11]. They represent a resource for analyzing development and disease at the subcellular level in cells, embryos and adult tissues. The marker used facilitates the acquisition of in vivo data and allows it to be integrated onto a high-resolution anatomical framework. This type of multidimensional data is complex and thus difficult to digitize and compile into a standardized and integratable format. In toto imaging of fluorescent protein expressing specimens on a large-scale could be used for generating high-resolution digitally recorded anatomical databases where the baseline (wild-type) cell behaviors and cell fates can be contrasted to those observed in mouse mutants. However, developing in toto imaging technologies for acquiring large amounts of data will necessitate improving the speed and throughput of microscopic image acquisition and analysis. This would also be coincident with the ongoing development of improved computational approaches to mine and integrate this type of data [discussed in ref. [11]]. + +Much of the information generated using a fluorescent fusion reporter such as the H2B-EGFP fusion is analogous to conventional histology [21,31] except that this mode of data acquisition optically sections a sample (circumventing the need to physical section), excels in permitting computational 3D reconstructions of spatial information, and can additionally be coupled to time-lapse imaging for the capture, processing and quantitation of 4D information (Fig. 6 and Additional Files). Also, unlike conventional GFP-based reporters [20,22], the histone H2B-EGFP fusion is resilient to fixation, so samples can be processed and stored for extended periods of time without compromising signal integrity or specificity (Fig. 7). + +Figure 7 + +High-resolution 3-dimensional imaging of fixed CAG::H2B-EGFP transgenic embryos. Confocal images of an E8.5 CAG::H2B-EGFP transgenic embryo fixed in 4% paraformaldehyde for 72 hours, then washed, stored and imaged in PBS. Low-magnification views and reconstructions of whole embryo (a-c). Boxes in a designate region imaged in d and g. High-magnification views of the headfolds (d-f) and posterior primitive streak and proximal allantois (g-i). Single xy images (a, d and g) from the z-stacks used to computationally render the data sets. These images are overlayed onto the bright field channel so as to display the outline of the embryo. Rotations through the rendered z-stacks displayed at 45° intervals (b, e and h). Color-coded depth projections of each of the z-stacks (c, f and i). + +The future development and availability of mouse strains constitutively expressing spectral variant histone H2B fusions should prove useful for visualizing anatomy and tracking different populations of cells in multiple dimensions at high-resolution in mice as has previously been demonstrated in other organisms which are classically perceived as being more amenable to in vitro culture and optical imaging [13,16]. They can also be used as tagged populations of cells in chimeras [8], in addition to transplantation and cell isolation experiments [32]. Also, since fluorescence is proportional to genome content and the fluorescence intensity reflects chromosome condensation state, the reagents we have generated should permit the study of alterations in ploidy and chromosomal condensation including determination of phases of mitosis [26]. Additionally, real-time analysis of chromatin fragmentation as well as the effects of mutations on chromosome stability during disease processes can be investigated using CAG::H2B-EGFP transgenic mice. + +Conclusions + +The CAG::H2B-EGFP strain that we have generated takes in vivo imaging using genetically-encoded reporters in mice to sub-cellular resolution. The development of additional strains permitting spectrally-distinct high-resolution live cell in vivo imaging, coupled to advances in optical imaging modalities and the development of improved computational methods to mine imaging data should pave the way for a multidimensional understanding of biological processes. + +It is anticipated that in the future, in vivo imaging approaches using transgenic animals expressing genetically-encoded fluorescent proteins will not only provide high-resolution information on cell behavior in specific biological processes [12,33], but more importantly it may lead to an exponential increase in the available multidimensional in vivo biological information which could mirror the recent explosion of available genomic data. Therefore recent advances in live imaging will need to be paired with developments in computational biology, as appropriate informatics methods will need to be developed and implemented in order to mine, present and integrate this type of in vivo biological data. + +Methods + +The coding sequence for the human histone H2B gene (X57127) was amplified from genomic DNA by PCR using Pfx Polymerase (Invitrogen). The resulting product was cloned into pCR4 TOPO (Invitrogen) to generate pH2B. The H2B fragment was then cloned into plasmids pEGFP-N1, pDsRed2-N1 pDsRedExpress-N1 (BD Biosciences, Inc) in order to generate plasmids pH2B-EGFP, pH2B-DsRed2 and pH2B-DsRedExpress (oligonucleotide sequences are available upon request). The resulting fusions were then re-amplified by PCR and cloned into the XhoI site of pCAGGS [19] to generate pCX-H2B-EGFP, pCX-H2B-DsRed2 and pCX-H2B-DsRedExpress. All vectors were tested by transient transfection of Cos-7 cells and R1 ES cells using Fugene 6 Transfection Reagent as per manufacturer's recommendations (Roche) and electroporation respectively. The H2B-DsRed2 and H2B-DsRedExpress fusions failed to produce sustained homogenous levels of fluorescent signal, however the H2B-EGFP fusion gave strong nuclear-localized fluorescence throughout the extended culture period without perturbing cell morphology, the rate of proliferation, or the mitotic index (Fig. 2 and data not shown). + +Transgenic ES cell lines constitutively expressing H2B-EGFP were generated by co-electroporation of the linearized reporter construct and a circular PGK-Puro-pA plasmid [34] conferring transient puromycin resistance. Puromycin selection was carried out as described previously [20,22]. Briefly, drug selection was initiated 24–36 hours after electroporation, maintained for 5 days, after which time it was replaced with non-selection media for a further 24–48 hours. Fluorescent colonies were identified and picked under an epifluorescence microscope (Nikon SMZ1500). Clones were passaged in 96-well plates, and scored for maintenance and extent of fluorescence. Those exhibiting homogeneous and robust transgene expression in vitro under both stem cell conditions and conditions employed to promote their differentiation were maintained further. For stem cell conditions ES cells were grown on gelatin in the presence of LIF. For differentiation, ES cells were grown on bacteriological Petri dishes in the absence of LIF for 2–5 days to promote embryoid body formation. Thereafter embryoid bodies were re-plated onto tissue culture dishes in the presence of factors promoting directed differentiation. + +To assess whether an H2B fusion can continue to be widely expressed and transmitted through the germline of mice we used H2B-EGFP expressing ES cells for chimera generation by injection into C57BL/6 blastocysts using standard procedures [21]. Chimeras were bred to outbred ICR and inbred 129/Tac mice (Taconic, Germantown, NY) for germline transmission and subsequent maintenance of the lines. Two independent clones were taken germline giving indistinguishable results. We therefore focused on one of the transgenic lines. After germline transmission, this transgene was maintained at homozygosity, suggesting that the site of integration is not perturbing essential gene function. All animals retained widespread homogenous fluorescence for at least five subsequent generations. Homozygotes were distinguished from heterozygotes either by increased fluorescence in newborn (unpigmented) animal tails, by breeding, or by intensity of an EGFP hybridizing fragment on a Southern blot. + +Embryos and organs were dissected in HEPES buffered DMEM media containing 10% fetal calf serum, then cultured either in a standard tissue culture incubator or on a microscope stage under standard conditions promoting the culture of mouse embryos [35,36] in 50% rat serum: 50% DMEM buffered with bicarbonate and maintained under physiological conditions in a closed temperature-controlled, humidified and oxygenated (95% air, 5% CO2) chamber (Bioptechs Inc. or Solent Sci Ltd. or home-made). For cytoplasmic staining, samples were incubated in Cell Tracker Orange (Molecular Probes; 1:500 dilution in dissecting or culture media) for 10–20 minutes. Embryos were kept in a standard tissue culture incubator at 37°C during staining. Samples were then washed twice with warm dissecting or culture media prior to imaging. + +All images shown (except Fig. 7) are of living hemizygous (Tg/+) embryos or freshly dissected (unfixed) tissues obtained from Tg/+ adults and maintained under physiological conditions. Increased fluorescence and a higher signal-to-noise ratio was observed in homozygous (Tg/Tg) specimens. The embryo presented in Figure 7 was fixed in 4% paraformaldehyde at 4°C for 72 hours, then washed, stored and imaged in PBS. Similar results were obtained in embryos fixed for up to two weeks. + +Wide-field images were acquired on a Nikon SMZ1500 stereo-dissecting microscope or Nikon Eclipse 5000 inverted microscope equipped with epifluorescent illumination. Spinning disc confocal data was acquired on an UltraView RS3 (Perkin-Elmer Systems) fitted on a Zeiss Axiovert 200M microscope with illumination from a 488 nm Argon laser (Melles Griot). Laser scanning confocal and multiphoton excitation data were taken on a Zeiss LSM510 NLO on a Zeiss Axioscop 2 FS MOT microscope. Objective lenses used on the Axiovert 200M and Axioscop 2 were plan-apochromat 63x/NA1.4, C-apochromat 40x/NA1.2, a plan-apochromat 20x/NA0.75 and a fluar 5x/NA0.25. For laser scanning microscopy GFP was excited using either a 488 nm Argon laser (Lassos, Inc) at 488 nm (for single-photon excitation) or a Titanium:Sapphire laser (Coherent Mira 900F with Verdi 5W pump laser) tuned between 860 and 890 nm (for two-photon excitation). Cell Tracker Orange was excited using a 543 nm HeNe laser (for single-photon use). + +Images were acquired as z-stacks comprising sequential x-y sections taken at 0.1–2 μm z-intervals. Raw data was processed using a variety of packages including Zeiss AIM software (Carl Zeiss Microsystems at ), Image J (NIH at ) and Volocity (Improvision at ). Each image series was re-animated using software to make the time-lapse movies that are available as additional files. Both rendered volume and time-lapse movies were assembled in QuickTime Player (Apple Computer, Inc at ). + +Appendix + +The CAG::H2B-EGFP strain of mice generated in this study will be made available through the Jackson Laboratories Induced Mutant Resource (JAX IMR at ). + +Supplementary Material + +Additional File 1 + +Rotating 3D projection of a whole live CAG::H2B-EGFP Tg/+ blastocyst. Supplementary to stills presented in Fig. 3. This file was assembled at 6 frames/second. + +Click here for file + +Additional File 2 + +Rotating 3D projection of a (electronic) half blastocyst. Generated from the same raw data set used to compile Supplementary File 1. Supplementary to stills presented in Fig. 3. This file was assembled at 6 frames/second. + +Click here for file + +Additional File 3 + +Rotating 3D projection the node region of a live CAG::H2B-EGFP Tg/+ E7.5 embryo. Supplementary to stills presented in Fig. 4. This file was assembled at 6 frames/second. + +Click here for file + +Additional File 4 + +Rotating 3D projection of the notochord of a live CAG::H2B-EGFP Tg/+ E10.5 embryo. Supplementary to Fig. 4. This file was assembled at 6 frames/second. + +Click here for file + +Additional File 5 + +Rotating 3D projection of the branchial region of a live CAG::H2B-EGFP Tg/+ E10.5 embryo. Supplementary to stills presented in Fig. 4. This file was assembled at 6 frames/second. + +Click here for file + +Additional File 6 + +Time-lapse sequence of CAG::H2B-EGFP Tg/+ ES cells. Supplementary to stills presented in Fig. 6. This file was assembled at 12 frames/second. + +Click here for file + +Additional File 7 + +Time-lapse sequence of CAG::H2B-EGFP Tg/+ preimplantation embryos. Supplementary to stills presented in Fig. 6. This file was assembled at 6 frames/second. + +Click here for file + +Additional File 8 + +Time-lapse sequence of a CAG::H2B-EGFP Tg/+ postimplantation embryo. Supplementary to stills presented in Fig. 6. This file was assembled at 6 frames/second. + +Click here for file + +Additional File 9 + +Rotating 3D projection of a fixed CAG::H2B-EGFP Tg/+ E8.5 embryo. Supplementary to stills presented in Fig. 7. This file was assembled at 6 frames/second. + +Click here for file + +Additional File 10 + +High-magnification rotating 3D projection of the headfold region of the same fixed CAG::H2B-EGFP Tg/+ E8.5 embryo as shown in Movie 7. Supplementary to stills presented in Fig. 7. This file was assembled at 6 frames/second. + +Click here for file + +Additional File 11 + +High-magnification rotating 3D projection of the allantois and posterior tail region of the same fixed CAG::H2B-EGFP Tg/+ E8.5 embryo as shown in Movie 7. Supplementary to stills presented in Fig. 7. This file was assembled at 6 frames/second. + +Click here for file + +Acknowledgements + +We thank J.-I. Miyazaki for the pCAGGS plasmid, and T. Swayne at the Herbert Irving Comprehensive Cancer Center Optical Microscopy Facility for instruction and assistance with laser scanning microscopy data acquisition and processing. This work was supported by NIH grants GM60561 and HD33082 (VEP). AKH was a fellow of the American Heart Association during part of this work. diff --git a/src/ontogpt/evaluation/craft/database/all/15630473.ann b/src/ontogpt/evaluation/craft/database/all/15630473.ann new file mode 100644 index 000000000..f1c9485b8 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15630473.ann @@ -0,0 +1,1669 @@ +T1 PR:000000183 30 36 TGF-β2 +T2 PR:000015308 41 46 Snail +T3 UBERON:0002073 50 63 Hair Follicle +T4 GO:0031069 50 77 Hair Follicle Morphogenesis +T5 GO:0048513 110 123 organogenesis +T6 UBERON:0010136 160 176 epithelial sheet +T7 UBERON:0001037 255 259 hair +T8 GO:0009653 264 277 morphogenesis +T9 NCBITaxon:10088 312 317 mouse +T10 CL:0000312 338 351 keratinocytes +T11 GO:0005576 393 406 extracellular +T12 GO:0008283 451 464 proliferation +T13 PR:000000183 519 548 transforming growth factor β2 +T14 PR:000015308 619 624 Snail +T15 CHEBI:52290 646 653 mitogen +T16 UBERON:0007376 713 722 epidermis +T17 PR:000015308 724 729 Snail +T18 GO:0010467 733 743 expression +T19 GO:0008283 758 771 proliferation +T20 PR:000001447 820 830 E-cadherin +T21 GO:0005911 948 966 cell-cell contacts +T22 GO:0000187 1012 1027 MAPK activation +T23 GO:0008283 1167 1180 proliferation +T24 UBERON:0001037 1358 1362 hair +T25 NCBITaxon:40674 1420 1429 Mammalian +T26 GO:0009653 1455 1468 morphogenesis +T27 GO:0048513 1601 1613;1619 1625 formation of ... organs +T28 UBERON:0000062 1619 1625 organs +T29 UBERON:0002048 1637 1642 lungs +T30 UBERON:0002240 1644 1655 spinal cord +T31 UBERON:0001911 1657 1671 mammary glands +T32 UBERON:0002073 1677 1691 hair follicles +T33 UBERON:0000483 1723 1733 epithelial +T34 CL:0000066 1723 1739 epithelial cells +T35 UBERON:0010136 1812 1828 epithelial sheet +T36 UBERON:0003104 1863 1873 mesenchyme +T37 GO:0009653 1883 1896 morphogenesis +T38 UBERON:0000483 1951 1961 epithelium +T39 UBERON:0003104 1966 1976 mesenchyme +T40 UBERON:0000062 2008 2013 organ +T41 GO:0065007 2036 2042 govern +T42 PR:000000034 2120 2145 bone morphogenic proteins +T43 PR:000000034 2147 2151 BMPs +T44 PR:000000046 2154 2183 transforming growth factor βs +T45 PR:000000046 2185 2191 TGF-βs +T46 CL:0000057 2198 2208 fibroblast +T47 GO:0009986 2254 2266 cell surface +T48 GO:0016020 2272 2280 membrane +T49 GO:0005576 2298 2306 external +T50 CHEBI:62488 2307 2326 signaling molecules +T51 GO:0035556 2344 2376 cascades of intracellular events +T52 GO:0005622 2356 2369 intracellular +T53 SO:0000704 2406 2410 gene +T54 GO:0010467 2406 2421 gene expression +T55 GO:0009653 2568 2581 morphogenetic +T56 CHEBI:35224 2783 2792 effectors +T57 UBERON:0010136 2970 2986 epithelial sheet +T58 GO:0008283 3042 3055;3091 3093;3103 3108 proliferation ... of ... cells +T59 GO:0005576 3178 3191 extracellular +T60 UBERON:0000483 3192 3202 epithelial +T61 UBERON:0003104 3203 3214 mesenchymal +T62 GO:0001709 3272 3288;3298 3308 determination of ... cell fates +T63 UBERON:0000062 3427 3433 organs +T64 UBERON:0002073 3435 3449 hair follicles +T65 UBERON:0005153 3491 3505 epithelial bud +T66 NCBITaxon:40674 3517 3526 Mammalian +T67 UBERON:0019204 3527 3542 skin epithelium +T68 UBERON:0000924 3583 3593 ectodermal +T69 CL:0000221 3583 3599 ectodermal cells +T70 UBERON:0003104 3633 3644 mesenchymal +T71 CL:0000134 3633 3650 mesenchymal cells +T72 UBERON:0000483 3724 3734 epithelial +T73 UBERON:0003104 3735 3746 mesenchymal +T74 UBERON:0000483 3823 3833 epithelial +T75 CL:0000066 3823 3839 epithelial cells +T76 UBERON:0003104 3886 3897 mesenchymal +T77 CL:0000134 3886 3903 mesenchymal cells +T78 UBERON:0000412 3937 3951 dermal papilla +T79 UBERON:0002073 3994 4007 hair follicle +T80 UBERON:0000412 4062 4076 dermal papilla +T81 UBERON:0002067 4138 4144 dermis +T82 UBERON:0000483 4292 4302 epithelial +T83 UBERON:0003104 4307 4318 mesenchymal +T84 PR:000000034 4341 4344 BMP +T85 PR:000000021 4363 4369 noggin +T86 PR:000009751 4401 4427 lymphoid enhancer factor-1 +T87 SO:0000165 4410 4418 enhancer +T88 PR:000002198 4428 4437 β-catenin +T89 PR:000009751 4439 4444 LEF-1 +T90 PR:000002198 4445 4454 β-catenin +T91 SO:0000704 4465 4469 gene +T92 UBERON:0011817 4495 4511 follicle placode +T93 SO:0000704 4638 4642 gene +T94 PR:000001447 4652 4662 E-cadherin +T95 UBERON:0000483 4681 4691 epithelial +T96 PR:000001327 4692 4700 cadherin +T97 GO:0016020 4721 4729 membrane +T98 GO:0005913 4738 4770 intercellular adherens junctions +T99 GO:0005912 4772 4775 AJs +T100 PR:000001447 4815 4825 E-cadherin +T101 NCBITaxon:10088 4856 4861 mouse +T102 UBERON:0002073 4868 4881 hair follicle +T103 GO:0031069 4868 4895 hair follicle morphogenesis +T104 PR:000001447 4924 4934 E-cadherin +T105 GO:0065007 4974 4983 governing +T106 GO:0009653 5028 5041 morphogenesis +T107 PR:000009751 5052 5057 LEF-1 +T108 PR:000001447 5059 5069 E-cadherin +T109 PR:000002198 5084 5093 β-catenin +T110 PR:000001447 5135 5145 E-cadherin +T111 PR:000002198 5146 5155 β-catenin +T112 GO:0032991 5156 5165 complexes +T113 GO:0030041 5226 5246 actin polymerization +T114 MOP:0000629 5232 5246 polymerization +T115 GO:0005912 5287 5290 AJs +T116 GO:0005856 5309 5321 cytoskeleton +T117 UBERON:0010136 5332 5348 epithelial sheet +T118 PR:P20154 5396 5401 Lin-1 +T119 PR:000009116 5403 5408 Isl-1 +T120 PR:P09088 5410 5415 Mec-3 +T121 PR:000009231 5430 5435 Ajuba +T122 PR:000018242 5453 5458 zyxin +T123 CHEBI:52290 5559 5566 mitogen +T124 GO:0005912 5636 5639 AJs +T125 GO:0005856 5676 5688 cytoskeletal +T126 GO:0005634 5714 5721 nuclear +T127 GO:0005737 5726 5737 cytoplasmic +T128 PR:000001447 5799 5809 E-cadherin +T129 GO:0065007 5954 5963 governing +T130 PR:000001447 5964 5974 E-cadherin +T131 SO:0000167 5975 5983 promoter +T132 PR:000009751 6042 6047 LEF-1 +T133 PR:000002198 6048 6057 β-catenin +T134 SO:0000409 6058 6070 binding site +T135 PR:000015308 6099 6104 Snail +T136 PR:000015309 6105 6109 Slug +T137 PR:000015308 6199 6204 Snail +T138 PR:000001447 6228 6238 E-cadherin +T139 UBERON:0000483 6261 6271 epithelial +T140 GO:0001837 6261 6298 epithelial to mesenchymal transitions +T141 UBERON:0003104 6275 6286 mesenchymal +T142 GO:0001837 6300 6304 EMTs +T143 UBERON:0000483 6361 6371 epithelial +T144 CL:0000066 6361 6377 epithelial cells +T145 CL:0000219 6383 6389;6402 6407 motile ... cells +T146 UBERON:0003104 6390 6401 mesenchymal +T147 CL:0000134 6390 6407 mesenchymal cells +T148 GO:0001837 6447 6450 EMT +T149 PR:000001447 6459 6469 E-cadherin +T150 PR:000001327 6652 6660 cadherin +T151 GO:0010467 6661 6671 expression +T152 PR:000015308 6702 6707 Snail +T153 PR:000009751 6746 6751 LEF-1 +T154 PR:000002198 6752 6761 β-catenin +T155 PR:000001447 6787 6797 E-cadherin +T156 SO:0000167 6798 6806 promoter +T157 PR:000015308 6863 6868 Snail +T158 GO:0010467 6872 6881 expressed +T159 UBERON:0001037 6911 6915 hair +T160 PR:000001447 6936 6946 E-cadherin +T161 GO:0008283 6981 6994 proliferation +T162 PR:000015308 7019 7024 Snail +T163 UBERON:0001037 7244 7248 hair +T164 PR:000001447 7293 7303 E-cadherin +T165 GO:0008283 7315 7328 proliferation +T166 PR:000015308 7401 7406 Snail +T167 UBERON:0001037 7414 7418 hair +T168 GO:0016055 7474 7487 Wnt signaling +T169 PR:000000034 7492 7495 BMP +T170 PR:000009751 7508 7513 LEF-1 +T171 PR:000002198 7514 7523 β-catenin +T172 PR:000015308 7561 7566 Snail +T173 SO:0000704 7567 7571 gene +T174 GO:0010467 7567 7582 gene expression +T175 UBERON:0000922 7586 7595 embryonic +T176 CL:0000312 7601 7614 keratinocytes +T177 SO:0000704 7671 7675 gene +T178 PR:000000183 7708 7714 TGF-β2 +T179 PR:000000364 7719 7789 small phenotype– and mothers against decapentaplegic–related protein 2 +T180 PR:000000364 7791 7796 SMAD2 +T181 PR:000015308 7833 7838 Snail +T182 SO:0000704 7839 7843 gene +T183 GO:0010467 7839 7854 gene expression +T184 UBERON:0019204 7858 7873 skin epithelium +T185 PR:000000183 7893 7899 TGF-β2 +T186 PR:000015308 7914 7919 Snail +T187 SO:0000704 7920 7924 gene +T188 GO:0010467 7920 7935 gene expression +T189 UBERON:0005086 7937 7950 hair placodes +T190 PR:000015308 8041 8046 Snail +T191 GO:0001708 8078 8101 cell fate specification +T192 GO:0008283 8155 8168 proliferation +T193 PR:000015308 8194 8199 Snail +T194 GO:0010467 8221 8230 Expressed +T195 UBERON:0001037 8250 8254 Hair +T196 GO:0009653 8277 8290 Morphogenesis +T197 PR:000015308 8301 8306 Snail +T198 GO:0001837 8358 8362 EMTs +T199 GO:0005911 8479 8502 intercellular junctions +T200 PR:000015308 8555 8560 Snail +T201 UBERON:0000483 8623 8633 epithelial +T202 UBERON:0002073 8665 8678 hair follicle +T203 PR:000015308 8745 8750 Snail +T204 PR:000009751 8772 8777 LEF-1 +T205 PR:000002198 8778 8787 β-catenin +T206 PR:000001447 8807 8817 E-cadherin +T207 GO:0010467 8818 8828 expression +T208 CL:0000312 8832 8845 keratinocytes +T209 PR:000009751 8896 8901 LEF-1 +T210 PR:000002198 8902 8911 β-catenin +T211 UBERON:0002073 8915 8928 hair follicle +T212 GO:0031069 8915 8942 hair follicle morphogenesis +T213 PR:000015308 8983 8988 Snail +T214 PR:000015309 8989 8993 Slug +T215 PR:000015308 9083 9088 Snail +T216 GO:0010467 9094 9104 expression +T217 GO:0009790 9129 9142 embryogenesis +T218 UBERON:0002073 9157 9171 hair follicles +T219 PR:000015308 9234 9239 Snail +T220 GO:0010467 9248 9257 expressed +T221 GO:0097617 9303 9316 hybridization +T222 PR:000015308 9350 9355 Snail +T223 SO:0000205 9356 9378 3′ untranslated region +T224 SO:0000205 9356 9358;9380 9383 3′ ... UTR +T225 GO:0006412 9361 9371 translated +T226 UBERON:0000922 9386 9395 Embryonic +T227 GO:0009653 9462 9475 morphogenesis +T228 PR:000015308 9522 9527 Snail +T229 GO:0010467 9528 9538 expression +T230 GO:0097617 9607 9620 hybridization +T231 UBERON:0000483 9645 9655 epithelium +T232 UBERON:0001037 9667 9671 hair +T233 GO:0097617 9805 9818 hybridization +T234 PR:000015308 9856 9861 Snail +T235 GO:0010467 9867 9877 expression +T236 GO:0097617 9927 9937 hybridized +T237 GO:0009653 10001 10014 morphogenesis +T238 GO:0097617 10039 10050 Hybridizing +T239 UBERON:0001037 10051 10055 hair +T240 GO:0097617 10107 10118 hybridizing +T241 PR:000015308 10198 10203 Snail +T242 GO:0010467 10209 10219 expression +T243 GO:0042571 10271 10279 antibody +T244 SO:0000417 10368 10375 domains +T245 GO:0042571 10417 10425 antibody +T246 CL:0000312 10475 10488 keratinocytes +T247 CL:0000312 10540 10553 keratinocytes +T248 GO:0010467 10566 10576 expressing +T249 PR:000015308 10605 10610 Snail +T250 GO:0042571 10636 10644 antibody +T251 PR:000015308 10708 10713 Snail +T252 UBERON:0000922 10753 10762 embryonic +T253 NCBITaxon:10088 10763 10768 mouse +T254 UBERON:0002073 10837 10850 hair follicle +T255 GO:0031069 10837 10864 hair follicle morphogenesis +T256 UBERON:0010164 10908 10912 hair +T257 NCBITaxon:10088 10920 10925 mouse +T258 PR:000015308 11032 11037 Snail +T259 UBERON:0007376 11062 11071 epidermis +T260 GO:0045165 11152 11165;11178 11183;11210 11212;11220 11229 commitment of ... cells ... to ... cell fate +T261 CL:0000221 11178 11186;11201 11209 cells of ... ectoderm +T262 UBERON:0000924 11191 11209 embryonic ectoderm +T263 UBERON:0001037 11215 11219 hair +T264 CL:0002559 11215 11224 hair cell +T265 GO:0097617 11271 11284 hybridization +T266 PR:000015308 11299 11304 Snail +T267 GO:0042571 11305 11313 antibody +T268 UBERON:0002073 11327 11331;11345 11354 hair ... follicles +T269 PR:000015308 11430 11435 Snail +T270 GO:0042571 11436 11444 antibody +T271 PR:000015308 11517 11522 Snail +T272 GO:0010467 11550 11559 expressed +T273 CL:0000312 11563 11576 keratinocytes +T274 PR:000015308 11666 11671 Snail +T275 GO:0097617 11680 11693 hybridization +T276 PR:000015308 11719 11724 Snail +T277 GO:0010467 11725 11735 expression +T278 UBERON:0001037 11743 11747 hair +T279 PR:000015308 11807 11812 Snail +T280 GO:0005634 11842 11851;11865 11870 nuclei of ... cells +T281 UBERON:0001037 11856 11860 hair +T282 PR:000015308 11917 11922 Snail +T283 PR:000015308 11999 12004 Snail +T284 GO:0009653 12077 12090 morphogenesis +T285 PR:000015308 12092 12097 Snail +T286 UBERON:0010512 12127 12138 guard hairs +T287 UBERON:0001037 12168 12173 hairs +T288 NCBITaxon:10088 12235 12240 mouse +T289 UBERON:0010166 12241 12245 coat +T290 GO:0042571 12304 12314 antibodies +T291 GO:0008283 12327 12340 proliferating +T292 GO:0005634 12341 12348 nuclear +T293 CHEBI:59132 12349 12356 antigen +T294 PR:000010425 12357 12361 Ki67 +T295 PR:000015308 12377 12382 Snail +T296 GO:0010467 12383 12393 expression +T297 GO:0008283 12465 12478 proliferation +T298 GO:0042571 12534 12544 antibodies +T299 GO:0008283 12625 12638 proliferating +T300 PR:000010425 12640 12644 Ki67 +T301 UBERON:0001037 12708 12712 hair +T302 PR:000015308 12744 12749 Snail +T303 PR:000010425 12764 12768 Ki67 +T304 UBERON:0001037 12797 12801 hair +T305 PR:000009751 12850 12855 LEF-1 +T306 PR:000002198 12856 12865 β-catenin +T307 UBERON:0005086 12901 12913 hair placode +T308 UBERON:0001037 12950 12954 hair +T309 GO:0010629 12970 12988;13000 13010 down-regulation in ... expression +T310 PR:000001447 12989 12999 E-cadherin +T311 GO:0010467 13049 13059 Expression +T312 PR:000015308 13063 13068 Snail +T313 UBERON:0007376 13080 13089 Epidermal +T314 GO:0008283 13095 13108 proliferation +T315 NCBITaxon:10088 13143 13148 Mouse +T316 PR:000015308 13177 13182 Snail +T317 GO:0010467 13183 13193 expression +T318 UBERON:0001037 13210 13214 hair +T319 GO:0008283 13242 13255 proliferation +T320 GO:0010467 13311 13321 expressing +T321 PR:000015308 13322 13327 Snail +T322 NCBITaxon:10088 13341 13346 mouse +T323 UBERON:0001003 13347 13361 skin epidermis +T324 PR:000015308 13397 13402 Snail +T325 PR:000015308 13458 13463 Snail +T326 PR:000009454 13503 13506 K14 +T327 PR:000015308 13507 13512 Snail +T328 NCBITaxon:33208 13520 13527 animals +T329 GO:0010467 13545 13554 expressed +T330 SO:0000902 13559 13568 transgene +T331 NCBITaxon:10088 13609 13613 Mice +T332 SO:0000902 13634 13643 transgene +T333 GO:0016265 13669 13673 died +T334 GO:0007567 13694 13699 birth +T335 NCBITaxon:10088 13737 13741 mice +T336 SO:0000902 13751 13760 transgene +T337 PR:000015308 13814 13819 Snail +T338 SO:0000704 13820 13824 gene +T339 GO:0010467 13820 13835 gene expression +T340 GO:0007567 13962 13967 birth +T341 PR:000015308 13973 13978 Snail +T342 NCBITaxon:33208 13982 13989 animals +T343 GO:0040007 13990 13994 grew +T344 UBERON:0002415 14049 14054 tails +T345 NCBITaxon:10088 14122 14126 mice +T346 UBERON:0007376 14161 14170 epidermal +T347 PR:000015308 14250 14255 Snail +T348 GO:0010467 14302 14312 expressing +T349 GO:0005634 14313 14320 nuclear +T350 PR:000015308 14331 14336 Snail +T351 GO:0008283 14402 14415 proliferation +T352 GO:0042571 14432 14442 antibodies +T353 GO:0008283 14455 14468 proliferating +T354 GO:0005634 14469 14476 nuclear +T355 CHEBI:59132 14477 14484 antigen +T356 PR:000010425 14485 14489 Ki67 +T357 GO:0010467 14611 14621 expressing +T358 UBERON:0010402 14658 14675 suprabasal layers +T359 GO:0010467 14724 14734 Expression +T360 PR:000015308 14742 14747 Snail +T361 SO:0000902 14748 14757 transgene +T362 UBERON:0007376 14823 14832 epidermis +T363 GO:0008283 14900 14913 proliferating +T364 PR:000015308 14926 14931 Snail +T365 GO:0010467 14932 14942 expression +T366 UBERON:0002026 14971 14977;14983 14990 layers ... spinous +T367 PR:000009481 15078 15087 keratin 5 +T368 PR:000009481 15089 15091 K5 +T369 UBERON:0002025 15131 15142 basal layer +T370 PR:000015308 15183 15188 Snail +T371 NCBITaxon:10088 15194 15198 mice +T372 GO:0007369 15215 15227 gastrulation +T373 PR:000015308 15276 15281 Snail +T374 GO:0043588 15294 15310 skin development +T375 GO:0010467 15351 15361 expression +T376 PR:000015308 15365 15370 Snail +T377 PR:000009481 15400 15402 K5 +T378 PR:000010425 15404 15408 Ki67 +T379 UBERON:0010402 15420 15432 suprabasally +T380 GO:0008283 15489 15502 proliferation +T381 UBERON:0000483 15572 15582 epithelial +T382 UBERON:0007376 15617 15626 epidermal +T383 PR:000015308 15674 15679 Snail +T384 GO:0010467 15684 15693 expressed +T385 GO:0042571 15945 15955 antibodies +T386 GO:0030056 15982 15996 hemidesmosomes +T387 CL:0002187 16031 16052 basal epidermal cells +T388 UBERON:0007376 16037 16046 epidermal +T389 UBERON:0001037 16186 16190 hair +T390 UBERON:0000922 16198 16207 embryonic +T391 NCBITaxon:10088 16220 16224 mice +T392 PR:000015308 16230 16235 Snail +T393 GO:0010467 16248 16257 expressed +T394 UBERON:0007376 16310 16319 epidermis +T395 UBERON:0002067 16329 16335 dermis +T396 UBERON:0007023 16359 16364 adult +T397 NCBITaxon:33208 16368 16375 animals +T398 UBERON:0007376 16457 16466 epidermis +T399 UBERON:0007376 16520 16529 Epidermal +T400 GO:0008283 16535 16548 proliferation +T401 GO:0005912 16572 16574 AJ +T402 PR:000015308 16587 16592 Snail +T403 NCBITaxon:10088 16596 16600 Mice +T404 PR:000001447 16617 16627 E-cadherin +T405 SO:0000167 16628 16636 promoter +T406 PR:000015308 16660 16665 Snail +T407 PR:000001447 16715 16725 E-cadherin +T408 PR:000015308 16748 16753 Snail +T409 GO:0010467 16754 16764 expressing +T410 UBERON:0001037 16765 16769 hair +T411 PR:000001447 16802 16812 E-cadherin +T412 GO:0005912 16823 16825 AJ +T413 UBERON:0007376 16872 16881 epidermis +T414 PR:000015308 16891 16896 Snail +T415 PR:000001447 16971 16981 E-cadherin +T416 GO:0042571 17052 17060 antibody +T417 GO:0005912 17084 17086 AJ +T418 PR:000002198 17097 17106 β-catenin +T419 PR:000009231 17111 17116 Ajuba +T420 PR:000002198 17216 17225 β-catenin +T421 PR:000009231 17230 17235 Ajuba +T422 GO:0005737 17259 17270 cytoplasmic +T423 GO:0005911 17286 17303 cell-cell borders +T424 UBERON:0007376 17358 17367 epidermis +T425 UBERON:0001690 17452 17455 ear +T426 PR:000015308 17490 17495 Snail +T427 GO:0010467 17509 17518 expressed +T428 UBERON:0001137 17558 17562 back +T429 UBERON:0001690 17572 17575 ear +T430 PR:000001447 17600 17610 E-cadherin +T431 PR:000002198 17662 17671 β-catenin +T432 PR:000009231 17676 17681 Ajuba +T433 PR:000015308 17951 17956 Snail +T434 GO:0005912 17977 17979 AJ +T435 GO:0034333 17977 17989 AJ formation +T436 PR:000001447 18016 18026 E-cadherin +T437 SO:0000704 18027 18031 gene +T438 GO:0010467 18027 18042 gene expression +T439 GO:0010467 18154 18164 expression +T440 PR:000001447 18168 18178 E-cadherin +T441 PR:000015308 18182 18187 Snail +T442 GO:0010467 18188 18198 expressing +T443 CL:0000312 18199 18212 keratinocytes +T444 GO:0009294 18249 18260 transfected +T445 CL:0000312 18261 18274 keratinocytes +T446 GO:0010467 18275 18285 expressing +T447 PR:000015308 18296 18301 Snail +T448 PR:000001447 18322 18332 E-cadherin +T449 GO:0005911 18350 18367 cell-cell borders +T450 GO:0010467 18371 18381 expression +T451 PR:000001447 18405 18415 E-cadherin +T452 GO:0005911 18433 18449 cell-cell border +T453 PR:000001447 18466 18476 E-cadherin +T454 GO:0005911 18507 18523 cell-cell border +T455 GO:0010467 18592 18602 expression +T456 PR:000015308 18674 18679 Snail +T457 PR:000001447 18783 18793 E-cadherin +T458 GO:0006412 18814 18825 translation +T459 GO:0005912 18877 18879 AJ +T460 GO:0016020 18920 18929 membranes +T461 GO:0005911 18934 18957 intercellular junctions +T462 UBERON:0019204 19063 19078 skin epithelium +T463 UBERON:0001037 19101 19105 hair +T464 PR:000015308 19266 19271 Snail +T465 UBERON:0007376 19275 19284 epidermis +T466 UBERON:0001037 19289 19293 hair +T467 GO:0005912 19393 19395 AJ +T468 PR:000015308 19481 19486 Snail +T469 UBERON:0007376 19490 19499 epidermis +T470 PR:000001447 19579 19589 E-cadherin +T471 PR:000015308 19609 19614 Snail +T472 GO:0008283 19636 19649 proliferation +T473 GO:0009294 19693 19704 transfected +T474 CL:0000312 19705 19718 keratinocytes +T475 GO:0010467 19719 19729 expressing +T476 PR:000015308 19730 19735 Snail +T477 GO:0010467 19826 19836 expression +T478 PR:000001447 19840 19850 E-cadherin +T479 PR:000015308 19856 19861 Snail +T480 GO:0005912 19952 19954 AJ +T481 GO:0051235 19991 20002 sequestered +T482 GO:0005886 20010 20025 plasma membrane +T483 GO:0008283 20047 20060 proliferation +T484 GO:0005912 20084 20087 AJs +T485 PR:000001447 20162 20172 E-cadherin +T486 GO:0051170 20180 20193;20207 20209;20214 20221 translocation ... to ... nucleus +T487 PR:000002198 20197 20206 β-catenin +T488 GO:0005634 20214 20221 nucleus +T489 SO:0000704 20247 20252 genes +T490 GO:0065007 20262 20271 regulated +T491 PR:000009751 20279 20284 LEF-1 +T492 CL:0000084 20285 20291 T cell +T493 GO:0005634 20364 20371 nuclear +T494 PR:000015308 20403 20408 Snail +T495 UBERON:0007376 20412 20421 epidermis +T496 SO:0000704 20493 20497 gene +T497 PR:000002198 20524 20533 β-catenin +T498 GO:0005634 20583 20590 nuclear +T499 PR:000002198 20591 20600 β-catenin +T500 UBERON:0007376 20611 20620 epidermis +T501 GO:0007618 20626 20632 mating +T502 PR:000015308 20637 20642 Snail +T503 NCBITaxon:10088 20646 20650 mice +T504 NCBITaxon:10088 20679 20684 mouse +T505 PR:000009751 20715 20720 LEF-1 +T506 PR:000002198 20725 20734 β-catenin +T507 GO:0005737 20799 20810 cytoplasmic +T508 PR:000009231 20811 20816 Ajuba +T509 PR:000015308 20886 20891 Snail +T510 UBERON:0007376 20895 20904 epidermis +T511 PR:000009231 20968 20973 Ajuba +T512 PR:000008224 20998 21036 growth factor receptor-bound protein-2 +T513 PR:000008224 21038 21043 Grb-2 +T514 PR:P26675 21045 21061 son of sevenless +T515 PR:P26675 21063 21066 Sos +T516 GO:0000187 21132 21150 activation of MAPK +T517 PR:000009231 21238 21243 Ajuba +T518 PR:000015308 21286 21291 Snail +T519 GO:0010467 21292 21302 expressing +T520 UBERON:0007376 21303 21312 epidermis +T521 PR:000009231 21329 21334 Ajuba +T522 PR:000008224 21364 21369 Grb-2 +T523 PR:000015308 21422 21427 Snail +T524 NCBITaxon:10088 21431 21435 mice +T525 NCBITaxon:33208 21482 21489 animals +T526 UBERON:0007376 21560 21569 epidermis +T527 PR:000008224 21581 21586 Grb-2 +T528 PR:000009231 21587 21592 Ajuba +T529 PR:000015308 21813 21818 Snail +T530 PR:000001447 21847 21857 E-cadherin +T531 PR:000009231 21904 21909 Ajuba +T532 PR:000008224 21927 21932 Grb-2 +T533 PR:P26675 21933 21936 Sos +T534 PR:000008224 21966 21971 Grb-2 +T535 PR:P26675 21972 21975 Sos +T536 PR:000009231 21994 21999 Ajuba +T537 CL:0000312 22062 22074 keratinocyte +T538 GO:0010467 22085 22095 expression +T539 PR:000009231 22099 22104 Ajuba +T540 CL:0000312 22202 22215 keratinocytes +T541 UBERON:0001977 22230 22235 serum +T542 CL:0000312 22244 22257 keratinocytes +T543 GO:0009294 22275 22286 transfected +T544 PR:000009231 22295 22300 Ajuba +T545 GO:0010467 22301 22311 expression +T546 SO:0000440 22312 22318 vector +T547 GO:0009294 22392 22403 transfected +T548 PR:000009454 22413 22416 K14 +T549 SO:0000902 22425 22434 transgene +T550 CHEBI:35222 22523 22532 inhibitor +T551 PR:000008224 22566 22571 Grb-2 +T552 PR:P26675 22572 22575 Sos +T553 PR:000009231 22631 22636 Ajuba +T554 SO:0000417 22647 22653 domain +T555 PR:000008224 22690 22695 Grb-2 +T556 PR:000027234 22698 22701 Src +T557 SO:0000857 22702 22710 homology +T558 SO:0000417 22713 22719 domain +T559 SO:0000417 22735 22741 domain +T560 GO:0010467 22750 22759 expressed +T561 UBERON:0001977 22763 22768 serum +T562 CL:0000312 22777 22790 keratinocytes +T563 CHEBI:35222 22881 22890 inhibitor +T564 PR:000008224 22911 22916 Grb-2 +T565 PR:P26675 22917 22920 Sos +T566 GO:0005829 22993 23002 cytosolic +T567 PR:000009231 23003 23008 Ajuba +T568 PR:000009231 23017 23022 Ajuba +T569 SO:0000417 23033 23039 domain +T570 PR:000008224 23059 23064 Grb-2 +T571 PR:P26675 23065 23068 Sos +T572 PR:000015308 23233 23238 Snail +T573 GO:0010467 23239 23249 expression +T574 GO:0008283 23254 23267 proliferation +T575 UBERON:0019204 23286 23301 skin epithelium +T576 GO:0005912 23339 23342 AJs +T577 GO:0010468 23507 23520;23527 23542 Regulation of ... Gene Expression +T578 PR:000015308 23521 23526 Snail +T579 SO:0000704 23527 23531 Gene +T580 PR:000000183 23572 23578 TGF-β2 +T581 UBERON:0001037 23607 23611 Hair +T582 PR:000015308 23639 23644 Snail +T583 GO:0010467 23650 23660 expression +T584 UBERON:0001037 23668 23672 hair +T585 GO:0065007 23723 23733 regulating +T586 PR:000015308 23738 23743 Snail +T587 SO:0000704 23744 23748 gene +T588 GO:0005576 23763 23776 extracellular +T589 GO:0010467 23826 23836 expression +T590 PR:000015308 23850 23855 Snail +T591 PR:000000034 23906 23910 BMPs +T592 PR:000000046 23922 23928 TGF-βs +T593 UBERON:0001037 23942 23946 hair +T594 PR:000015308 23980 23985 Snail +T595 GO:0010467 23993 24002 expressed +T596 CL:0000312 24020 24033 keratinocytes +T597 GO:0046903 24039 24046 secrete +T598 PR:000000034 24054 24058 BMPs +T599 GO:0016055 24113 24116;24127 24136 Wnt ... signaling +T600 PR:000000046 24121 24126 TGF-β +T601 GO:0007179 24121 24136 TGF-β signaling +T602 PR:000015308 24167 24172 Snail +T603 PR:000017443 24258 24264 Wnt-3a +T604 CL:0000312 24284 24297 keratinocytes +T605 PR:000000034 24344 24347 BMP +T606 CHEBI:35222 24348 24357 inhibitor +T607 PR:000000021 24358 24364 noggin +T608 PR:000009751 24380 24385 LEF-1 +T609 GO:0010467 24386 24396 expression +T610 PR:000001447 24448 24458 E-cadherin +T611 SO:0000167 24459 24467 promoter +T612 PR:000009751 24482 24487 LEF-1 +T613 PR:000002198 24488 24497 β-catenin +T614 SO:0000704 24517 24521 gene +T615 PR:000015308 24550 24555 Snail +T616 GO:0010467 24556 24566 expression +T617 PR:000000021 24659 24665 noggin +T618 UBERON:0002073 24669 24682 hair follicle +T619 GO:0065007 24775 24784 governing +T620 PR:000015308 24785 24790 Snail +T621 GO:0010467 24791 24801 expression +T622 CL:0000312 24805 24818 keratinocytes +T623 PR:000000182 24821 24827 TGF-β1 +T624 PR:000015308 24853 24858 Snail +T625 CL:0000182 24877 24888 hepatocytes +T626 UBERON:0000948 24893 24898 heart +T627 CL:0000312 24912 24925 keratinocytes +T628 PR:000000182 24936 24942 TGF-β1 +T629 CL:0000312 24952 24964 keratinocyte +T630 UBERON:0002073 25048 25061 hair follicle +T631 PR:000000046 25127 25132 TGF-β +T632 SO:0000704 25133 25138 genes +T633 PR:000000183 25149 25155 TGF-β2 +T634 UBERON:0001037 25203 25207 hair +T635 PR:000000183 25267 25273 TGF-β2 +T636 GO:0065007 25295 25305 regulating +T637 PR:000015308 25306 25311 Snail +T638 GO:0010467 25312 25322 expression +T639 CL:0000312 25326 25339 keratinocytes +T640 UBERON:0002025 25358 25372;25377 25386 basal layer of ... epidermis +T641 CL:0000312 25481 25494 keratinocytes +T642 UBERON:0000483 25586 25596 epithelial +T643 CL:0000066 25586 25602 epithelial cells +T644 CL:0000312 25657 25670 keratinocytes +T645 PR:000000183 25700 25706 TGF-β2 +T646 PR:000015308 25749 25754 Snail +T647 PR:000015308 25794 25799 Snail +T648 PR:000015308 25898 25903 Snail +T649 PR:000000183 25989 25995 TGF-β2 +T650 PR:000000046 26095 26100 TGF-β +T651 CHEBI:35224 26241 26249 effector +T652 PR:000000364 26250 26255 SMAD2 +T653 PR:000015308 26257 26262 Snail +T654 GO:0010467 26263 26273 expression +T655 PR:000015308 26326 26331 Snail +T656 GO:0010467 26332 26342 expression +T657 PR:000000364 26374 26379 SMAD2 +T658 PR:000015308 26482 26487 Snail +T659 GO:0010467 26488 26498 expression +T660 PR:000015308 26549 26554 Snail +T661 SO:0000167 26555 26563 promoter +T662 CL:0000312 26576 26589 keratinocytes +T663 PR:000000183 26612 26618 TGF-β2 +T664 SO:0000902 26684 26693 transgene +T665 GO:0065007 26741 26748 control +T666 SO:0000167 26776 26784 promoter +T667 SO:0000315 26814 26843 transcription initiation site +T668 SO:0000842 26839 26846;26863 26867 site of ... gene +T669 NCBITaxon:10088 26851 26856 mouse +T670 PR:000015308 26857 26862 Snail +T671 GO:0009294 26892 26904 transfection +T672 CL:0000312 26906 26919 keratinocytes +T673 PR:000000183 26938 26944 TGF-β2 +T674 SO:0000902 26974 26983 transgene +T675 PR:000015308 27044 27049 Snail +T676 PR:000000183 27145 27151 TGF-β2 +T677 PR:000015308 27163 27168 Snail +T678 SO:0000167 27169 27177 promoter +T679 PR:000015308 27301 27306 Snail +T680 SO:0000167 27307 27315 promoter +T681 CL:0000312 27383 27396 keratinocytes +T682 PR:000015308 27414 27419 Snail +T683 SO:0000167 27420 27428 promoter +T684 PR:000015308 27476 27481 Snail +T685 PR:000000183 27562 27568 TGF-β2 +T686 PR:000000183 27631 27637 TGF-β2 +T687 SO:0000409 27680 27695 binding element +T688 PR:000015308 27729 27734 Snail +T689 SO:0000315 27735 27759 transcription start site +T690 CL:0000312 27821 27834 keratinocytes +T691 PR:000000183 27836 27842 TGF-β2 +T692 PR:000015308 27911 27916 Snail +T693 SO:0000704 27917 27921 gene +T694 PR:000015308 27947 27952 Snail +T695 SO:0000167 27993 28001 promoter +T696 PR:000015308 28028 28033 Snail +T697 SO:0000704 28034 28038 gene +T698 PR:000000183 28064 28070 TGF-β2 +T699 CL:0000312 28088 28101 keratinocytes +T700 PR:000015308 28139 28144 Snail +T701 UBERON:0002073 28183 28196 hair follicle +T702 GO:0031069 28183 28210 hair follicle morphogenesis +T703 UBERON:0000922 28214 28223 embryonic +T704 NCBITaxon:10088 28224 28229 mouse +T705 PR:000000183 28252 28258 TGF-β2 +T706 PR:000015308 28281 28286 Snail +T707 UBERON:0001037 28300 28304 hair +T708 PR:000000183 28354 28360 TGF-β2 +T709 GO:0010467 28365 28374 expressed +T710 UBERON:0001037 28392 28396 hair +T711 PR:000000183 28454 28460 TGF-β2 +T712 GO:0042571 28461 28469 antibody +T713 UBERON:0001037 28489 28493 hair +T714 NCBITaxon:10088 28606 28610 mice +T715 PR:000000183 28628 28634 TGF-β2 +T716 CHEBI:35224 28693 28701 effector +T717 PR:000000183 28705 28711 TGF-β2 +T718 GO:0010467 28740 28749 expressed +T719 PR:000000183 28765 28771 TGF-β2 +T720 UBERON:0001037 28778 28782 hair +T721 PR:000000183 28855 28861 TGF-β2 +T722 SO:0001060 28862 28869 isoform +T723 GO:0010467 28878 28888 expression +T724 PR:000000182 28897 28903 TGF-β1 +T725 PR:000000183 28908 28914 TGF-β2 +T726 UBERON:0001037 28929 28933 hair +T727 PR:000015308 29004 29009 Snail +T728 PR:000000183 29014 29020 TGF-β2 +T729 PR:000015308 29048 29053 Snail +T730 GO:0010467 29054 29064 expression +T731 PR:000000183 29068 29074 TGF-β2 +T732 UBERON:0001037 29080 29084 hair +T733 PR:000015308 29126 29131 Snail +T734 PR:000000183 29170 29176 TGF-β2 +T735 UBERON:0000922 29182 29189 embryos +T736 PR:000015308 29315 29320 Snail +T737 PR:000000183 29350 29356 TGF-β2 +T738 UBERON:0001037 29362 29366 hair +T739 UBERON:0001037 29437 29441 hair +T740 PR:000009454 29504 29507 K14 +T741 PR:000000364 29508 29513 Smad2 +T742 NCBITaxon:10088 29517 29521 mice +T743 PR:000000046 29546 29551 TGF-β +T744 GO:0007179 29546 29561 TGF-β signaling +T745 PR:000015308 29576 29581 Snail +T746 GO:0007567 29667 29672 natal +T747 PR:000000183 29754 29760 TGF-β2 +T748 PR:000015308 29800 29805 Snail +T749 SO:0000704 29806 29810 gene +T750 GO:0010467 29806 29821 gene expression +T751 UBERON:0001037 29864 29868 hair +T752 PR:000015308 29918 29923 Snail +T753 UBERON:0001037 29994 29998 hair +T754 PR:000000183 30020 30026 TGF-β2 +T755 PR:000000183 30129 30135 TGF-β2 +T756 UBERON:0000483 30175 30185 epithelial +T757 GO:0009653 30243 30256 morphogenesis +T758 PR:000000183 30340 30346 TGF-β2 +T759 GO:0042571 30395 30405 antibodies +T760 PR:000009140 30414 30425 β4 integrin +T761 CL:0000312 30452 30464 keratinocyte +T762 PR:000000183 30545 30551 TGF-β2 +T763 GO:0008283 30611 30624 proliferation +T764 UBERON:0001037 30655 30659 hair +T765 PR:000010425 30684 30688 Ki67 +T766 PR:000000183 30747 30753 TGF-β2 +T767 PR:000015308 30765 30770 Snail +T768 GO:0010467 30771 30781 expression +T769 GO:0010467 30857 30867 expression +T770 SO:0000704 30871 30876 genes +T771 PR:000015308 30909 30914 Snail +T772 PR:000015308 30941 30946 Snail +T773 PR:000001447 30974 30984 E-cadherin +T774 SO:0000704 30985 30989 gene +T775 PR:000001447 31029 31039 E-cadherin +T776 PR:000000183 31043 31049 TGF-β2 +T777 UBERON:0001037 31084 31088 hair +T778 PR:000000183 31097 31103 TGF-β2 +T779 GO:0005576 31257 31270 extracellular +T780 PR:000000021 31287 31293 noggin +T781 GO:0065003 31315 31328;31361 31368 generation of ... complex +T782 PR:000009751 31331 31336 LEF-1 +T783 PR:000002198 31337 31346 β-catenin +T784 GO:0032991 31361 31368 complex +T785 PR:000001447 31380 31390 E-cadherin +T786 UBERON:0001037 31421 31425 hair +T787 PR:000000183 31488 31494 TGF-β2 +T788 GO:0005634 31515 31522 nuclear +T789 PR:000009751 31523 31528 LEF-1 +T790 PR:000002198 31533 31542 β-catenin +T791 GO:0016055 31572 31575;31583 31600 Wnt ... signaling pathway +T792 PR:000000021 31576 31582 noggin +T793 UBERON:0002073 31644 31657 hair follicle +T794 GO:0031069 31644 31671 hair follicle morphogenesis +T795 PR:000000183 31673 31679 TGF-β2 +T796 PR:000000021 31710 31716 noggin +T797 UBERON:0001037 31743 31747 hair +T798 PR:000015308 31786 31791 Snail +T799 SO:0000704 31792 31796 gene +T800 GO:0010467 31792 31807 gene expression +T801 PR:000000183 31809 31815 TGF-β2 +T802 PR:000001447 31887 31897 E-cadherin +T803 GO:0009653 32000 32013 morphogenesis +T804 UBERON:0000483 32056 32066 epithelium +T805 UBERON:0003104 32071 32081 mesenchyme +T806 GO:0065007 32082 32088 govern +T807 GO:0005634 32193 32200 nuclear +T808 GO:0005829 32205 32214 cytosolic +T809 UBERON:0000062 32281 32286 organ +T810 GO:0009887 32281 32300 organ morphogenesis +T811 GO:0008283 32566 32574;32587 32600 cellular ... proliferation +T812 GO:0030154 32566 32574;32606 32621 cellular ... differentiation +T813 UBERON:0001037 32708 32712 hair +T814 UBERON:0002073 32771 32784 Hair Follicle +T815 GO:0031069 32771 32798 Hair Follicle Morphogenesis +T816 UBERON:0001037 32818 32822 hair +T817 GO:0009653 32827 32840 morphogenesis +T818 UBERON:0000483 32882 32892 epithelium +T819 PR:000000034 32897 32900 BMP +T820 UBERON:0003104 32940 32950 mesenchyme +T821 GO:0005667 32981 33009 transcription factor complex +T822 PR:000002198 33020 33029 β-catenin +T823 PR:000009751 33034 33039 LEF-1 +T824 UBERON:0002073 33090 33103 hair follicle +T825 PR:000014841 33126 33140 Sonic hedgehog +T826 PR:000014841 33142 33145 Shh +T827 PR:000000183 33151 33157 TGF-β2 +T828 GO:0009653 33206 33219 morphogenesis +T829 PR:000002198 33240 33249 β-catenin +T830 UBERON:0001037 33315 33319 hair +T831 PR:000009751 33354 33359 LEF-1 +T832 PR:000014841 33361 33364 Shh +T833 PR:000000183 33369 33375 TGF-β2 +T834 UBERON:0010512 33440 33450 guard hair +T835 GO:0009653 33452 33465 morphogenesis +T836 UBERON:0001037 33520 33525 hairs +T837 GO:0065007 33548 33566 regulatory control +T838 UBERON:0010512 33568 33579 Guard hairs +T839 PR:000009751 33603 33608 LEF-1 +T840 PR:000000183 33613 33619 TGF-β2 +T841 GO:0010467 33662 33669 express +T842 PR:000015308 33670 33675 Snail +T843 PR:000001447 33736 33746 E-cadherin +T844 GO:0065007 33750 33759 regulated +T845 UBERON:0010512 33763 33774 guard hairs +T846 PR:000015308 33834 33839 Snail +T847 PR:000015309 33863 33867 Slug +T848 PR:000027825 33871 33876 twist +T849 PR:000002198 33928 33937 β-catenin +T850 PR:000009751 33968 33973 LEF-1 +T851 PR:000015643 33978 33981 Sry +T852 PR:000000046 34179 34185 TGF-βs +T853 CL:0000312 34221 34234 keratinocytes +T854 GO:0007049 34244 34254 cell cycle +T855 PR:000000183 34273 34279 TGF-β2 +T856 GO:0040007 34331 34338 growing +T857 GO:0044851 34355 34364;34375 34385 phases of ... hair cycle +T858 UBERON:0007023 34369 34374 adult +T859 UBERON:0001037 34375 34379 hair +T860 GO:0006915 34502 34511 apoptosis +T861 PR:000000182 34567 34573 TGF-β1 +T862 GO:0040007 34612 34619 growing +T863 GO:0007567 34633 34638 natal +T864 UBERON:0002073 34639 34653 hair follicles +T865 PR:000000183 34655 34661 TGF-β2 +T866 UBERON:0000922 34684 34693 embryonic +T867 PR:000000183 34777 34783 TGF-β2 +T868 UBERON:0000922 34786 34795 embryonic +T869 GO:0010467 34796 34806 expression +T870 PR:000000183 34835 34841 TGF-β2 +T871 PR:000000046 34997 35003 TGF-βs +T872 PR:000000183 35023 35029 TGF-β2 +T873 PR:000010425 35047 35051 Ki67 +T874 GO:0010467 35052 35062 expression +T875 GO:0000187 35067 35082 MAPK activation +T876 UBERON:0002073 35124 35137 hair follicle +T877 CL:0000312 35138 35151 keratinocytes +T878 PR:000000183 35196 35202 TGF-β2 +T879 PR:000000046 35266 35279 TGF-β factors +T880 SO:0000704 35528 35533 genes +T881 PR:000000183 35575 35581 TGF-β2 +T882 GO:0008283 35585 35598 proliferation +T883 UBERON:0001037 35610 35614 hair +T884 http://purl.obolibrary.org/obo/MONDO_0005096 35681 35704 squamous cell carcinoma +T885 http://purl.obolibrary.org/obo/MONDO_0024879 35708 35728 metastatic carcinoma +T886 CL:0000312 35774 35787 keratinocytes +T887 PR:000015308 35833 35838 Snail +T888 SO:0000704 35839 35843 gene +T889 PR:000000046 35869 35874 TGF-β +T890 GO:0007179 35869 35884 TGF-β signaling +T891 PR:000015308 35933 35938 Snail +T892 SO:0000704 35939 35943 gene +T893 GO:0010467 35939 35954 gene expression +T894 UBERON:0001037 35985 35989 hair +T895 UBERON:0000483 36030 36040 epithelial +T896 GO:0008283 36046 36059 proliferation +T897 PR:000015308 36064 36069 Snail +T898 SO:0000902 36070 36079 transgene +T899 GO:0010467 36080 36090 expression +T900 PR:000000183 36148 36154 TGF-β2 +T901 PR:000015308 36159 36164 Snail +T902 PR:000000183 36214 36220 TGF-β2 +T903 PR:000015308 36222 36227 Snail +T904 GO:0010467 36228 36238 expression +T905 UBERON:0001037 36266 36270 hair +T906 PR:000009454 36296 36299 K14 +T907 PR:000000364 36300 36305 Smad2 +T908 PR:000015308 36312 36317 Snail +T909 PR:000000183 36404 36410 TGF-β2 +T910 PR:000015308 36460 36465 Snail +T911 PR:000015308 36509 36514 Snail +T912 GO:0010467 36529 36538 expressed +T913 UBERON:0002073 36546 36559 hair follicle +T914 GO:0031069 36546 36573 hair follicle morphogenesis +T915 GO:0010467 36630 36640 expression +T916 PR:000015308 36647 36652 Snail +T917 GO:0007567 36660 36665 natal +T918 UBERON:0000483 36722 36732 epithelial +T919 GO:0001837 36722 36759 epithelial to mesenchymal transitions +T920 UBERON:0003104 36736 36747 mesenchymal +T921 PR:000015308 36807 36812 Snail +T922 GO:0010467 36813 36823 expression +T923 UBERON:0002073 36858 36871 hair follicle +T924 GO:0031069 36858 36885 hair follicle morphogenesis +T925 UBERON:0010166 36926 36935 hair coat +T926 PR:000009454 36939 36942 K14 +T927 PR:000015308 36943 36948 Snail +T928 NCBITaxon:10088 36952 36956 mice +T929 UBERON:0001037 37076 37081 hairs +T930 UBERON:0007376 37115 37124 epidermis +T931 NCBITaxon:10088 37211 37215 mice +T932 PR:000009454 37243 37246 K14 +T933 SO:0000167 37247 37255 promoter +T934 UBERON:0002025 37282 37296;37301 37310 basal layer of ... epidermis +T935 UBERON:0005942 37319 37336;37343 37345;37350 37363 outer root sheath ... of ... hair follicle +T936 UBERON:0005942 37338 37341;37343 37345;37350 37363 ORS ... of ... hair follicle +T937 UBERON:0000922 37410 37419 embryonic +T938 UBERON:0001037 37420 37424 hair +T939 GO:0007567 37452 37457 natal +T940 UBERON:0001037 37458 37462 hair +T941 PR:000009454 37485 37488 K14 +T942 SO:0000167 37489 37497 promoter +T943 UBERON:0005942 37525 37528 ORS +T944 UBERON:0007376 37534 37543 epidermis +T945 UBERON:0005942 37619 37622 ORS +T946 PR:000015308 37634 37639 Snail +T947 PR:000015308 37702 37707 Snail +T948 GO:0010467 37746 37756 expression +T949 GO:0065007 37794 37804 regulatory +T950 GO:0065007 37821 37827 govern +T951 GO:0005912 37828 37830 AJ +T952 GO:0034333 37828 37840 AJ formation +T953 GO:0008283 37857 37870 proliferation +T954 UBERON:0005942 37878 37890 follicle ORS +T955 NCBITaxon:10088 37964 37968 mice +T956 PR:000015308 38010 38015 Snail +T957 UBERON:0000483 38079 38089 Epithelial +T958 GO:0008283 38106 38119 Proliferation +T959 UBERON:0001037 38127 38131 Hair +T960 UBERON:0002073 38181 38194 hair follicle +T961 GO:0031069 38181 38208 hair follicle morphogenesis +T962 PR:000001447 38210 38220 E-cadherin +T963 SO:0000704 38221 38225 gene +T964 GO:0010467 38221 38236 gene expression +T965 PR:000009751 38403 38408 LEF-1 +T966 PR:000002198 38409 38418 β-catenin +T967 GO:0005667 38419 38447 transcription factor complex +T968 PR:000009751 38502 38507 LEF-1 +T969 SO:0000409 38512 38524 binding site +T970 PR:000001447 38532 38542 E-cadherin +T971 SO:0000167 38543 38551 promoter +T972 PR:000009751 38628 38633 LEF-1 +T973 PR:000002198 38634 38643 β-catenin +T974 PR:000001447 38676 38686 E-cadherin +T975 SO:0000704 38687 38691 gene +T976 GO:0010467 38687 38702 gene expression +T977 CL:0000312 38711 38724 keratinocytes +T978 PR:000015308 38781 38786 Snail +T979 CL:0000312 38826 38839 keratinocytes +T980 PR:000015308 38888 38893 Snail +T981 GO:0010467 38897 38906 expressed +T982 PR:000000021 38986 38992 noggin +T983 UBERON:0000922 38998 39007 embryonic +T984 PR:000009751 39014 39019 LEF-1 +T985 GO:0010467 39020 39030 expression +T986 PR:000009751 39064 39069 LEF-1 +T987 PR:000002198 39070 39079 β-catenin +T988 SO:0000704 39089 39093 gene +T989 PR:000001447 39164 39174 E-cadherin +T990 GO:0016055 39221 39224;39232 39241 Wnt ... signaling +T991 PR:000000021 39225 39231 noggin +T992 GO:0065007 39245 39255 regulating +T993 GO:0009653 39279 39292 morphogenesis +T994 SO:0000704 39310 39314 gene +T995 PR:000015308 39372 39377 Snail +T996 GO:0010629 39416 39434;39446 39461 down-regulation in ... gene expression +T997 PR:000001447 39435 39445 E-cadherin +T998 SO:0000704 39446 39450 gene +T999 PR:000009454 39532 39535 K14 +T1000 PR:000015308 39536 39541 Snail +T1001 UBERON:0007376 39545 39554 epidermis +T1002 GO:0010629 39574 39592;39604 39614 down-regulation in ... expression +T1003 PR:000001447 39593 39603 E-cadherin +T1004 PR:000001447 39714 39724 E-cadherin +T1005 UBERON:0002073 39772 39785 hair follicle +T1006 GO:0031069 39772 39799 hair follicle morphogenesis +T1007 UBERON:0007376 39834 39843 epidermal +T1008 GO:0008283 39849 39862 proliferation +T1009 PR:000009454 39875 39878 K14 +T1010 PR:000015308 39879 39884 Snail +T1011 GO:0008283 39935 39948 proliferation +T1012 PR:000015308 39953 39958 Snail +T1013 PR:000000183 39962 39968 TGF-β2 +T1014 UBERON:0001037 39974 39978 hair +T1015 PR:000001447 40032 40042 E-cadherin +T1016 GO:0009653 40059 40072 morphogenesis +T1017 GO:0065007 40214 40224 regulation +T1018 PR:000001447 40228 40238 E-cadherin +T1019 PR:000015308 40240 40245 Snail +T1020 GO:0005912 40312 40314 AJ +T1021 PR:000009231 40363 40368 Ajuba +T1022 PR:000008224 40431 40436 Grb-2 +T1023 CL:0000312 40500 40513 keratinocytes +T1024 GO:0010467 40586 40593 express +T1025 PR:000015308 40594 40599 Snail +T1026 PR:000009231 40601 40606 Ajuba +T1027 PR:000008224 40636 40641 Grb-2 +T1028 CL:0000312 40679 40692 keratinocytes +T1029 PR:000015308 40732 40737 Snail +T1030 GO:0009294 40738 40749 transfected +T1031 PR:000009231 40753 40758 Ajuba +T1032 GO:0009294 40759 40770 transfected +T1033 CL:0000312 40771 40784 keratinocytes +T1034 PR:000008224 40857 40862 Grb-2 +T1035 PR:000009231 40878 40883 Ajuba +T1036 GO:0005912 40922 40924 AJ +T1037 GO:0008283 40983 40996 proliferation +T1038 UBERON:0000483 41009 41019 epithelial +T1039 CL:0000066 41009 41019;41063 41067 epithelial ... cell +T1040 NCBITaxon:9608 41039 41045 canine +T1041 UBERON:0002113 41046 41052 kidney +T1042 PR:000015308 41075 41080 Snail +T1043 GO:0007049 41105 41115 cell cycle +T1044 PR:000015308 41243 41248 Snail +T1045 UBERON:0000483 41265 41275 epithelial +T1046 PR:000015308 41316 41321 Snail +T1047 PR:000015308 41448 41453 Snail +T1048 GO:0010467 41454 41464 expression +T1049 NCBITaxon:10088 41539 41544 mouse +T1050 GO:0005912 41843 41845 AJ +T1051 GO:0008283 41879 41892 proliferation +T1052 UBERON:0000483 41969 41979 epithelial +T1053 GO:0008283 41980 41993 proliferation +T1054 GO:0005912 42018 42020 AJ +T1055 GO:0008283 42343 42356 proliferation +T1056 UBERON:0000483 42360 42370 epithelial +T1057 GO:0002009 42360 42384 epithelial morphogenesis +T1058 CHEBI:33893 42616 42624 Reagents +T1059 GO:0042571 42634 42644 antibodies +T1060 PR:000001447 42664 42674 E-cadherin +T1061 PR:000002198 42726 42735 β-catenin +T1062 PR:000028799 42744 42751 tubulin +T1063 PR:000009231 42797 42802 Ajuba +T1064 PR:000009140 42877 42888 β4 integrin +T1065 PR:000009140 42889 42894 CD104 +T1066 GO:0005610 42950 42959 laminin 5 +T1067 PR:000009481 43036 43038 K5 +T1068 PR:000009450 43040 43042 K1 +T1069 PR:000009882 43044 43052 loricrin +T1070 PR:000009167 43066 43076 involucrin +T1071 PR:000007551 43078 43087 fillagrin +T1072 PR:000008224 43206 43211 Grb-2 +T1073 PR:000005245 43273 43283 P-cadherin +T1074 PR:000017298 43379 43387 vimentin +T1075 PR:000010425 43437 43441 Ki67 +T1076 PR:000000183 43643 43649 TGF-β2 +T1077 CHEBI:37926 43717 43721 FITC +T1078 CHEBI:51247 43724 43733 Texas Red +T1079 MOP:0000779 43743 43753 conjugated +T1080 GO:0042571 43764 43774 antibodies +T1081 MOP:0000093 43851 43863 Biotinylated +T1082 GO:0042571 43874 43884 antibodies +T1083 PR:000015308 44015 44020 Snail +T1084 GO:0042571 44021 44029 antibody +T1085 NCBITaxon:10140 44047 44058 Guinea pigs +T1086 NCBITaxon:39107 44111 44117 murine +T1087 PR:000015308 44118 44123 Snail +T1088 NCBITaxon:9606 44198 44203 human +T1089 PR:000000183 44204 44210 TGF-β2 +T1090 PR:000000183 44292 44298 TGF-β2 +T1091 NCBITaxon:10088 44371 44375 Mice +T1092 PR:000009454 44381 44384 K14 +T1093 PR:000015308 44385 44390 Snail +T1094 NCBITaxon:10088 44394 44399 mouse +T1095 PR:000015308 44441 44446 Snail +T1096 SO:0000155 44450 44457 plasmid +T1097 PR:P23940 44525 44530 BamHI +T1098 PR:000009454 44563 44566 K14 +T1099 SO:0000440 44567 44573 vector +T1100 GO:0005634 44627 44634 nucleus +T1101 UBERON:0000922 44638 44645 embryos +T1102 NCBITaxon:10088 44655 44659 mice +T1103 PR:000009454 44665 44668 K14 +T1104 PR:000000364 44669 44675 Smad 2 +T1105 NCBITaxon:10088 44679 44684 mouse +T1106 PR:000000183 44723 44729 TGF-β2 +T1107 NCBITaxon:10088 44744 44749 mouse +T1108 PR:000014841 44777 44780 shh +T1109 NCBITaxon:10088 44784 44789 mouse +T1110 NCBITaxon:10088 44806 44811 mouse +T1111 CL:0000312 44917 44930 keratinocytes +T1112 GO:0019835 44956 44962 lysing +T1113 GO:0019835 44972 44977 lysis +T1114 CHEBI:63016 44989 44994 NP-40 +T1115 CHEBI:9177 44999 45018 sodium deoxycholate +T1116 CHEBI:9754 45026 45030 Tris +T1117 CHEBI:17996 45031 45033 Cl +T1118 CHEBI:26710 45051 45055 NaCl +T1119 CHEBI:35607 45072 45087 sodium vanadate +T1120 CHEBI:8102 45094 45123 phenylmethylsulfonyl fluoride +T1121 UBERON:0000479 45202 45208 tissue +T1122 UBERON:0000479 45217 45223 tissue +T1123 CHEBI:9750 45358 45370 Triton X-100 +T1124 CHEBI:26710 45402 45406 NaCl +T1125 CHEBI:9177 45411 45430 sodium deoxycholate +T1126 CHEBI:8984 45441 45444 SDS +T1127 GO:0042571 45818 45826 antibody +T1128 GO:0019835 45996 46001 lysis +T1129 GO:0042571 46038 46046 antibody +T1130 CHEBI:59132 46047 46054 antigen +T1131 CHEBI:8984 46139 46142 SDS +T1132 CHEBI:53325 46167 46181 nitrocellulose +T1133 CL:0000312 46379 46392 keratinocytes +T1134 GO:0009294 46467 46480 transfections +T1135 CHEBI:33893 46511 46518 reagent +T1136 SO:0000167 46662 46670 promoter +T1137 NCBITaxon:6134 46869 46876 Runella +T1138 GO:0009294 46894 46905 transfected +T1139 GO:0009294 46932 46944 transfection +T1140 CL:0000312 47174 47187 keratinocytes +T1141 UBERON:0001977 47193 47198 serum +T1142 UBERON:0001977 47276 47281 serum +T1143 PR:000000021 47316 47322 noggin +T1144 NCBITaxon:39107 47396 47402 murine +T1145 PR:000015308 47403 47408 Snail +T1146 SO:0000167 47409 47417 promoter +T1147 SO:0000121 47447 47461 forward primer +T1148 SO:0000132 47543 47557 reverse primer +T1149 NCBITaxon:10088 47630 47635 mouse +T1150 SO:0001026 47636 47643 genomic +T1151 SO:0000006 47667 47678 PCR product +T1152 GO:0006266 47770 47777 ligated +T1153 SO:0000440 47797 47803 vector +T1154 SO:0000167 47859 47867 promoter +T1155 SO:0000440 47964 47970 vector +T1156 SO:0000409 48068 48083 binding element +T1157 SO:0000121 48183 48197 forward primer +T1158 SO:0000132 48258 48272 reverse primer +T1159 PR:000015308 48346 48351 Snail +T1160 GO:0097617 48360 48373 hybridization +T1161 SO:0000205 48401 48407 3′ UTR +T1162 SO:0000121 48425 48439 forward primer +T1163 SO:0000132 48480 48494 reverse primer +T1164 SO:0001026 48531 48538 genomic +T1165 SO:0000006 48562 48573 PCR product +T1166 GO:0006266 48595 48602 ligated +T1167 SO:0000440 48622 48628 vector +T1168 SO:0000417 48642 48648 domain +T1169 PR:000009231 48652 48657 Ajuba +T1170 SO:0000440 48758 48764 vector +T1171 GO:0097617 48800 48813 hybridization +T1172 SO:0000440 48833 48839 vector +T1173 SO:0000837 48853 48862;48870 48873 region of ... UTR +T1174 SO:0000205 48867 48873 3′ UTR +T1175 PR:000015308 48877 48882 Snail +T1176 CHEBI:42098 48918 48929 digoxigenin +T1177 SO:0000077 48948 48957 antisense +T1178 PR:P23940 49026 49031 BamH1 +T1179 GO:0097617 49052 49066 hybridizations +T1180 NCBITaxon:10088 49115 49120 mouse +T1181 UBERON:0000922 49121 49128 embryos +T1182 GO:0097617 49201 49211 hybridized +T1183 GO:0097617 49243 49253 hybridized +T1184 CHEBI:42098 49345 49348 dig +T1185 GO:0042571 49356 49364 antibody +T1186 CHEBI:7586 49446 49449 NBT +T1187 CHEBI:75508 49454 49458 BCIP +T1188 UBERON:0000479 49541 49547 Tissue +T1189 GO:0042571 49740 49750 antibodies +T1190 UBERON:0000479 49752 49758 Tissue +T1191 GO:0042571 49955 49963 antibody +T1192 PR:000015308 49986 49991 Snail +T1193 CHEBI:59132 50026 50033 antigen +T1194 CHEBI:53258 50054 50068 sodium citrate +T1195 CHEBI:59132 50082 50089 Antigen +T1196 CL:0000312 50292 50305 keratinocytes +T1197 UBERON:0000479 50314 50320 tissue +T1198 SO:0000112 50422 50429 primers +T1199 SO:0000112 50475 50482 primers +T1200 PR:000015308 50501 50506 Snail +T1201 SO:0001030 50507 50514 forward +T1202 PR:000015308 50544 50549 Snail +T1203 SO:0001031 50550 50557 reverse +T1204 SO:0001030 50591 50598 forward +T1205 SO:0001031 50641 50648 reverse +T1206 GO:0042571 50773 50783 antibodies +T1207 PR:000015308 50812 50817 Snail +T1208 NCBITaxon:33208 50947 50953 animal +T1209 PR:000000183 51014 51020 TGF-β2 +T1210 PR:000009454 51028 51031 K14 +T1211 PR:000000364 51032 51037 Smad2 +T1212 NCBITaxon:10088 51038 51042 mice +T1213 CHEBI:33893 51132 51140 reagents +T1214 GO:0005912 51510 51512 AJ +T1215 GO:0005912 51515 51532 adherens junction +T1216 PR:000000034 51534 51537 BMP +T1217 PR:000000034 51540 51564 bone morphogenic protein +T1218 GO:0001837 51566 51569 EMT +T1219 UBERON:0000483 51572 51582 epithelial +T1220 GO:0001837 51572 51608 epithelial to mesenchymal transition +T1221 UBERON:0003104 51586 51597 mesenchymal +T1222 UBERON:0000922 51622 51631 embryonic +T1223 CL:0000057 51652 51662 fibroblast +T1224 PR:000008224 51678 51683 Grb-2 +T1225 PR:000008224 51686 51724 growth factor receptor-bound protein-2 +T1226 PR:000009450 51745 51747 K1 +T1227 PR:000009450 51750 51759 keratin 1 +T1228 PR:000009481 51761 51763 K5 +T1229 PR:000009481 51766 51775 keratin 5 +T1230 PR:000009751 51792 51797 LEF-1 +T1231 PR:000009751 51800 51826 lymphoid enhancer factor-1 +T1232 SO:0000165 51809 51817 enhancer +T1233 CHEBI:52290 51848 51855 mitogen +T1234 UBERON:0005942 51882 51885 ORS +T1235 UBERON:0005942 51888 51905 outer root sheath +T1236 GO:0007567 51952 51957 natal +T1237 PR:000014841 52001 52004 Shh +T1238 PR:000014841 52007 52021 sonic hedgehog +T1239 PR:P26675 52102 52105 Sos +T1240 PR:P26675 52108 52124 son of sevenless +T1241 PR:000015643 52132 52135 Sry +T1242 CL:0000084 52156 52162 T cell +T1243 PR:000000046 52188 52193 TGF-β +T1244 PR:000000046 52196 52224 transforming growth factor β +T1245 SO:0000167 52244 52252 promoter +T1246 SO:0000203 52254 52257 UTR +T1247 SO:0000203 52260 52279 untranslated region +T1248 GO:0006412 52262 52272 translated +T1249 PR:000015308 52327 52332 Snail +T1250 GO:0010467 52336 52345 Expressed +T1251 UBERON:0001037 52365 52369 Hair +T1252 GO:0009653 52381 52394 Morphogenesis +T1253 UBERON:0000922 52396 52403 Embryos +T1254 CHEBI:36357 52440 52448 compound +T1255 GO:0097617 52545 52559 hybridizations +T1256 PR:000015308 52565 52570 Snail +T1257 SO:0000077 52580 52589 antisense +T1258 UBERON:0007376 52673 52682 epidermis +T1259 UBERON:0007376 52684 52687 epi +T1260 UBERON:0002067 52694 52700 dermis +T1261 UBERON:0002067 52702 52705 der +T1262 PR:000015308 52724 52729 Snail +T1263 GO:0010467 52734 52744 expression +T1264 UBERON:0001037 52764 52768 hair +T1265 GO:0009653 52791 52804 morphogenesis +T1266 UBERON:0001037 52831 52835 hair +T1267 GO:0010467 52861 52871 Expression +T1268 PR:000015308 52875 52880 Snail +T1269 UBERON:0001037 52904 52908 hair +T1270 CL:0000312 52958 52971 keratinocytes +T1271 GO:0009294 52972 52983 transfected +T1272 GO:0010467 52995 53005 expression +T1273 SO:0000440 53006 53012 vector +T1274 PR:000009454 53014 53017 K14 +T1275 PR:000009454 53035 53038 K14 +T1276 SO:0000167 53039 53047 promoter +T1277 SO:0000440 53060 53066 vector +T1278 PR:000015308 53085 53090 Snail +T1279 PR:000009454 53092 53095 K14 +T1280 PR:000015308 53096 53101 Snail +T1281 NCBITaxon:33208 53140 53147 animals +T1282 CHEBI:8984 53221 53224 SDS +T1283 PR:000015308 53315 53320 Snail +T1284 UBERON:0001977 53336 53341 serum +T1285 PR:000028799 53371 53378 tubulin +T1286 GO:0010467 53432 53442 expression +T1287 PR:000015308 53446 53451 Snail +T1288 GO:0005634 53467 53482 nuclei of cells +T1289 UBERON:0001037 53494 53498 hair +T1290 UBERON:0007376 53546 53555 epidermis +T1291 UBERON:0007376 53557 53560 epi +T1292 PR:000015308 53571 53576 Snail +T1293 GO:0010467 53577 53587 expression +T1294 UBERON:0002073 53648 53661 hair follicle +T1295 PR:000015308 53721 53726 Snail +T1296 GO:0010467 53734 53743 expressed +T1297 PR:000015308 53778 53783 Snail +T1298 GO:0010467 53787 53796 expressed +T1299 UBERON:0001037 53804 53808 hair +T1300 PR:000010425 53925 53929 Ki67 +T1301 GO:0008283 53953 53966 proliferating +T1302 UBERON:0002025 54004 54018;54023 54032 basal layer of ... epidermis +T1303 UBERON:0002073 54048 54062 hair follicles +T1304 PR:000009140 54069 54075 β4 int +T1305 GO:0030056 54113 54127 hemidesmosomal +T1306 PR:000009140 54128 54139 integrin β4 +T1307 GO:0045178 54159 54172 base of cells +T1308 GO:0008283 54337 54350 proliferating +T1309 UBERON:0007376 54368 54377 epidermis +T1310 UBERON:0001037 54382 54386 hair +T1311 UBERON:0001037 54447 54451 hair +T1312 GO:0005610 54491 54500 laminin 5 +T1313 GO:0005610 54502 54506 lam5 +T1314 PR:000001447 54558 54568 E-cadherin +T1315 PR:000001447 54570 54575 E-cad +T1316 GO:0005912 54593 54596 AJs +T1317 GO:0040007 54625 54632 growing +T1318 GO:0005911 54638 54655 cell-cell borders +T1319 PR:000001447 54686 54696 E-cadherin +T1320 GO:0010467 54734 54744 expression +T1321 PR:000015308 54748 54753 Snail +T1322 NCBITaxon:10088 54757 54762 Mouse +T1323 UBERON:0001003 54763 54777 Skin Epidermis +T1324 GO:0008283 54794 54807 proliferation +T1325 NCBITaxon:10088 54846 54850 mice +T1326 PR:000009454 54862 54865 K14 +T1327 PR:000015308 54866 54871 Snail +T1328 SO:0000902 54872 54881 transgene +T1329 GO:0010467 54952 54962 expression +T1330 SO:0000902 54970 54979 transgene +T1331 UBERON:0001003 54983 54997 skin epidermis +T1332 PR:000009454 55076 55079 K14 +T1333 PR:000015308 55080 55085 Snail +T1334 NCBITaxon:10088 55089 55093 mice +T1335 UBERON:0002415 55119 55123 tail +T1336 NCBITaxon:10088 55183 55187 mice +T1337 PR:000009454 55247 55250 K14 +T1338 SO:0000167 55251 55259 promoter +T1339 UBERON:0001723 55276 55282 tongue +T1340 UBERON:0002424 55287 55302 oral epithelium +T1341 CHEBI:33290 55349 55353 food +T1342 GO:0007631 55349 55360 food intake +T1343 NCBITaxon:10088 55399 55403 mice +T1344 CHEBI:51686 55430 55441 Hematoxylin +T1345 UBERON:0007376 55556 55565 epidermis +T1346 UBERON:0007376 55567 55570 epi +T1347 UBERON:0002073 55585 55598 hair follicle +T1348 UBERON:0002073 55600 55602 hf +T1349 UBERON:0007376 55631 55640 epidermis +T1350 GO:0042571 55707 55717 antibodies +T1351 PR:000015308 55793 55798 Snail +T1352 PR:000015308 55818 55823 Snail +T1353 GO:0010467 55824 55834 expression +T1354 UBERON:0007376 55877 55886 epidermis +T1355 UBERON:0007376 55929 55938 epidermis +T1356 GO:0042571 56100 56110 antibodies +T1357 PR:000009481 56143 56152 keratin 5 +T1358 PR:000009481 56163 56165 K5 +T1359 UBERON:0002025 56194 56208;56213 56222 basal layer of ... epidermis +T1360 GO:0010467 56258 56267 expressed +T1361 GO:0007567 56275 56280 natal +T1362 UBERON:0007376 56281 56290 epidermis +T1363 GO:0008283 56340 56353 proliferation +T1364 GO:0042571 56372 56382 antibodies +T1365 GO:0008283 56531 56544 proliferating +T1366 PR:000010425 56546 56550 Ki67 +T1367 PR:000010425 56594 56598 Ki67 +T1368 UBERON:0010402 56641 56651 suprabasal +T1369 PR:000010425 56659 56663 Ki67 +T1370 PR:000015308 56699 56704 Snail +T1371 PR:000015308 56802 56807 Snail +T1372 GO:0010467 56808 56818 Expressing +T1373 UBERON:0007376 56822 56831 Epidermis +T1374 NCBITaxon:10088 56893 56897 mice +T1375 PR:000015308 56951 56956 Snail +T1376 GO:0010467 56973 56982 expressed +T1377 GO:0042571 57034 57044 antibodies +T1378 GO:0042571 57089 57099 Antibodies +T1379 UBERON:0007376 57130 57139 epidermal +T1380 PR:000009481 57169 57171 K5 +T1381 UBERON:0002025 57175 57182 basally +T1382 GO:0010467 57183 57192 expressed +T1383 PR:000009450 57203 57205 K1 +T1384 UBERON:0010402 57209 57219 suprabasal +T1385 GO:0010467 57229 57238 expressed +T1386 UBERON:0002026 57242 57255 spinous layer +T1387 CL:0000649 57242 57261 spinous layer cells +T1388 PR:000009167 57264 57274 involucrin +T1389 PR:000009167 57276 57279 Inv +T1390 UBERON:0010402 57283 57295 suprabasally +T1391 GO:0010467 57296 57305 expressed +T1392 GO:0070268 57306 57315 cornified +T1393 GO:0001533 57306 57324 cornified envelope +T1394 UBERON:0002026 57348 57355;57369 57374 spinous ... layer +T1395 CL:0000649 57348 57355;57369 57380 spinous ... layer cells +T1396 UBERON:0002069 57360 57374 granular layer +T1397 CL:0000712 57360 57380 granular layer cells +T1398 PR:000009882 57383 57391 loricrin +T1399 PR:000009882 57393 57396 Lor +T1400 GO:0070268 57400 57409 cornified +T1401 GO:0001533 57400 57418 cornified envelope +T1402 GO:0010467 57427 57436 expressed +T1403 UBERON:0002069 57444 57458 granular layer +T1404 PR:000007551 57465 57474 filaggrin +T1405 PR:000007551 57476 57479 Fil +T1406 GO:0045095 57504 57521 keratin filaments +T1407 UBERON:0002069 57529 57543 granular layer +T1408 UBERON:0002027 57548 57563 stratum corneum +T1409 PR:000009481 57598 57600 K5 +T1410 UBERON:0010402 57601 57613 suprabasally +T1411 PR:000009450 57637 57639 K1 +T1412 UBERON:0010402 57649 57659 suprabasal +T1413 GO:0042571 57826 57836 antibodies +T1414 UBERON:0007376 57893 57902 epidermis +T1415 GO:0005634 58094 58101 nuclear +T1416 UBERON:0007376 58161 58170 epidermis +T1417 GO:0042571 58243 58251 antibody +T1418 GO:0030056 58307 58321 hemidesmosomal +T1419 PR:000015308 58457 58462 Snail +T1420 PR:000015308 58577 58582 Snail +T1421 GO:0005912 58606 58609 AJs +T1422 GO:0008283 58630 58643 proliferation +T1423 NCBITaxon:10088 58704 58708 mice +T1424 PR:000015308 58762 58767 Snail +T1425 GO:0010467 58784 58793 expressed +T1426 GO:0042571 58818 58828 Antibodies +T1427 GO:0005912 58846 58848 AJ +T1428 PR:000001447 58870 58880 E-cadherin +T1429 PR:000001447 58882 58887 E-cad +T1430 GO:0016020 58899 58907 membrane +T1431 PR:000002198 58922 58931 β-catenin +T1432 PR:000002198 58933 58938 β-cat +T1433 PR:000001447 58953 58963 E-cadherin +T1434 GO:0005912 58967 58970 AJs +T1435 CHEBI:23357 59021 59029 cofactor +T1436 PR:000009751 59051 59056 LEF-1 +T1437 GO:0005634 59077 59084 nucleus +T1438 PR:000002198 59124 59133 β-catenin +T1439 PR:000009231 59138 59143 Ajuba +T1440 PR:000018242 59165 59170 zyxin +T1441 PR:000009231 59176 59181 Ajuba +T1442 GO:0015629 59234 59252 actin cytoskeleton +T1443 PR:000008224 59270 59275 Grb-2 +T1444 PR:P26675 59327 59330 Sos +T1445 GO:0007265 59362 59365;59371 59388 Ras ... signaling cascade +T1446 GO:0000165 59366 59388 MAPK signaling cascade +T1447 PR:000015308 59393 59398 Snail +T1448 GO:0010467 59399 59409 expressing +T1449 PR:000001447 59461 59466 E-cad +T1450 PR:000009231 59520 59525 Ajuba +T1451 PR:000002198 59552 59561 β-catenin +T1452 PR:000009231 59566 59571 Ajuba +T1453 GO:0016020 59633 59641 membrane +T1454 GO:0005737 59719 59730 cytoplasmic +T1455 PR:000002198 59731 59740 β-catenin +T1456 PR:000009231 59744 59749 Ajuba +T1457 UBERON:0001137 59817 59821 back +T1458 UBERON:0001690 59826 59829 ear +T1459 GO:0042571 59837 59847 Antibodies +T1460 PR:000005245 59874 59879 P-cad +T1461 PR:000005245 59895 59905 P-cadherin +T1462 GO:0010467 59913 59923 expression +T1463 UBERON:0002073 59931 59944 hair follicle +T1464 PR:000028799 59972 59979 tubulin +T1465 PR:000028799 59995 60002 tubulin +T1466 PR:000001447 60075 60085 E-cadherin +T1467 GO:0010467 60197 60206 expressed +T1468 PR:000015308 60207 60212 Snail +T1469 PR:000015308 60258 60263 Snail +T1470 GO:0010467 60305 60315 expression +T1471 PR:000001447 60319 60329 E-cadherin +T1472 CL:0000312 60331 60344 Keratinocytes +T1473 GO:0009294 60350 60361 transfected +T1474 PR:000015308 60384 60389 Snail +T1475 PR:000015308 60391 60396 Snail +T1476 PR:000015308 60425 60430 Snail +T1477 PR:000001447 60439 60443 Ecad +T1478 GO:0009294 60481 60493 transfection +T1479 GO:0005912 60587 60589 AJ +T1480 GO:0034333 60587 60599 AJ formation +T1481 GO:0042571 60625 60635 antibodies +T1482 PR:000015308 60725 60730 Snail +T1483 GO:0009294 60731 60742 transfected +T1484 CL:0000312 60743 60755 keratinocyte +T1485 GO:0009294 60778 60789 transfected +T1486 PR:000001447 60819 60829 E-cadherin +T1487 CL:0000312 60833 60846 keratinocytes +T1488 GO:0010467 60847 60857 expressing +T1489 PR:000015308 60858 60863 Snail +T1490 CL:0000312 60895 60908 Keratinocytes +T1491 GO:0009294 60914 60925 transfected +T1492 SO:0000440 60939 60945 vector +T1493 PR:000009454 60947 60950 K14 +T1494 PR:000015308 60956 60961 Snail +T1495 PR:000015308 60970 60975 Snail +T1496 PR:000001447 60982 60987 E-cad +T1497 UBERON:0001977 61015 61020 serum +T1498 GO:0042571 61095 61105 antibodies +T1499 PR:000015308 61146 61151 Snail +T1500 PR:000001447 61156 61166 E-cadherin +T1501 PR:000028799 61181 61188 tubulin +T1502 PR:000009231 61216 61221 Ajuba +T1503 PR:000008224 61237 61242 Grb-2 +T1504 PR:000009454 61348 61351 K14 +T1505 PR:000015308 61352 61357 Snail +T1506 NCBITaxon:10088 61365 61369 mice +T1507 PR:000009454 61412 61415 K14 +T1508 NCBITaxon:33208 61457 61464 animals +T1509 PR:000008224 61548 61553 Grb-2 +T1510 GO:0042571 61554 61562 antibody +T1511 GO:0042571 61586 61594 antibody +T1512 CHEBI:8984 61667 61670 SDS +T1513 PR:000009231 61712 61717 Ajuba +T1514 PR:000008224 61727 61732 Grb-2 +T1515 GO:0042571 61733 61743 antibodies +T1516 PR:000009231 61766 61771 Ajuba +T1517 GO:0005912 61830 61832 AJ +T1518 SO:0000902 61878 61887 Transgene +T1519 GO:0010467 61888 61898 expression +T1520 PR:000009231 61909 61914 Ajuba +T1521 PR:000008224 61922 61927 Grb-2 +T1522 SO:0000417 61940 61946 domain +T1523 PR:000009231 61960 61965 Ajuba +T1524 CL:0000312 61969 61982 keratinocytes +T1525 NCBITaxon:10088 62050 62055 mouse +T1526 CL:0000312 62056 62069 keratinocytes +T1527 GO:0009294 62075 62086 transfected +T1528 PR:000009454 62109 62112 K14 +T1529 GO:0010467 62113 62123 expression +T1530 SO:0000440 62124 62130 vector +T1531 PR:000009454 62132 62135 K14 +T1532 GO:0010467 62145 62155 expression +T1533 SO:0000440 62156 62162 vector +T1534 PR:000015308 62171 62176 Snail +T1535 PR:000009231 62190 62195 Ajuba +T1536 SO:0000417 62212 62218 domain +T1537 PR:000009231 62222 62227 Ajuba +T1538 CHEBI:35222 62268 62277 inhibitor +T1539 CHEBI:35222 62279 62282 inh +T1540 PR:000008224 62322 62327 Grb-2 +T1541 PR:P26675 62332 62335 Sos +T1542 GO:0009294 62346 62358 transfection +T1543 CHEBI:8984 62408 62411 SDS +T1544 GO:0042571 62448 62458 antibodies +T1545 PR:000009231 62486 62491 Ajuba +T1546 SO:0000417 62531 62537 domain +T1547 PR:000015308 62544 62549 Snail +T1548 PR:000000183 62562 62568 TGF-β2 +T1549 PR:000000021 62582 62588 noggin +T1550 PR:000015308 62610 62615 Snail +T1551 GO:0010467 62616 62626 Expression +T1552 GO:0016055 62652 62655;62667 62676 Wnt ... signaling +T1553 PR:000000021 62660 62666 noggin +T1554 PR:000015308 62687 62692 Snail +T1555 CL:0000312 62705 62718 keratinocytes +T1556 NCBITaxon:10088 62728 62733 mouse +T1557 CL:0000312 62734 62747 keratinocytes +T1558 PR:000000021 62778 62784 noggin +T1559 PR:000009751 62892 62897 LEF-1 +T1560 PR:000002198 62898 62907 β-catenin +T1561 PR:000001447 62946 62956 E-cadherin +T1562 SO:0000167 62957 62965 promoter +T1563 PR:000015308 63059 63064 Snail +T1564 PR:000009751 63066 63071 LEF-1 +T1565 PR:000002198 63073 63082 β-catenin +T1566 PR:000028799 63088 63095 tubulin +T1567 CL:0000312 63111 63124 keratinocytes +T1568 GO:0009294 63125 63136 transfected +T1569 PR:000009454 63142 63145 K14 +T1570 PR:000015308 63146 63151 Snail +T1571 PR:000015308 63188 63193 Snail +T1572 GO:0010467 63194 63204 expression +T1573 PR:000000183 63211 63217 TGF-β2 +T1574 PR:000015308 63229 63234 Snail +T1575 CL:0000312 63252 63265 keratinocytes +T1576 PR:000000183 63320 63326 TGF-β2 +T1577 PR:000000183 63351 63357 TGF-β2 +T1578 PR:000015308 63438 63443 Snail +T1579 PR:000028799 63498 63505 tubulin +T1580 PR:000015308 63530 63535 Snail +T1581 GO:0010467 63536 63546 expression +T1582 PR:000000183 63568 63574 TGF-β2 +T1583 PR:000015308 63624 63629 Snail +T1584 GO:0010467 63635 63645 expression +T1585 PR:000000183 63672 63678 TGF-β2 +T1586 CL:0000312 63761 63774 keratinocytes +T1587 PR:000000183 63788 63794 TGF-β2 +T1588 SO:0000112 63901 63907 primer +T1589 PR:000015308 63926 63931 Snail +T1590 PR:000015308 63959 63964 Snail +T1591 GO:0010467 63970 63980 expression +T1592 PR:000015308 64013 64018 Snail +T1593 PR:000000183 64033 64039 TGF-β2 +T1594 PR:000015308 64084 64089 Snail +T1595 SO:0000167 64090 64098 promoter +T1596 CL:0000312 64125 64138 Keratinocytes +T1597 GO:0009294 64144 64155 transfected +T1598 PR:000015308 64200 64205 Snail +T1599 SO:0000167 64206 64214 promoter +T1600 SO:0000167 64237 64241 prom +T1601 SO:0000409 64293 64305 binding site +T1602 SO:0000167 64310 64314 prom +T1603 GO:0009294 64328 64340 transfection +T1604 PR:000000046 64373 64378 TGF-β +T1605 PR:000000183 64399 64405 TGF-β2 +T1606 PR:000000183 64678 64684 TGF-β2 +T1607 PR:000015308 64708 64713 Snail +T1608 GO:0010467 64714 64724 Expression +T1609 GO:0065007 64729 64737 Regulate +T1610 GO:0008283 64738 64751 Proliferation +T1611 PR:000001447 64756 64766 E-Cadherin +T1612 UBERON:0001037 64774 64778 Hair +T1613 PR:000000183 64801 64807 TGF-β2 +T1614 UBERON:0000922 64823 64830 embryos +T1615 GO:0010467 64849 64859 expression +T1616 PR:000000183 64863 64869 TGF-β2 +T1617 UBERON:0007376 64907 64916 epidermis +T1618 UBERON:0002067 64921 64927 dermis +T1619 UBERON:0001037 64968 64972 hair +T1620 PR:000015308 64990 64995 Snail +T1621 PR:000015308 65005 65010 Snail +T1622 UBERON:0001037 65041 65045 hair +T1623 PR:000015308 65082 65087 Snail +T1624 GO:0010467 65088 65098 expression +T1625 PR:000009454 65124 65127 K14 +T1626 PR:000000364 65128 65133 Smad2 +T1627 PR:000000364 65146 65151 SMAD2 +T1628 NCBITaxon:10088 65179 65183 mice +T1629 GO:0042571 65185 65193 Antibody +T1630 PR:000028799 65197 65204 tubulin +T1631 PR:000009454 65259 65262 K14 +T1632 PR:000000364 65263 65268 Smad2 +T1633 NCBITaxon:10088 65272 65277 mouse +T1634 PR:000000046 65320 65325 TGF-β +T1635 GO:0007179 65320 65335 TGF-β signaling +T1636 GO:0008283 65349 65362 Proliferation +T1637 PR:000010425 65371 65375 Ki67 +T1638 PR:000000183 65412 65418 TGF-β2 +T1639 PR:000000183 65468 65474 TGF-β2 +T1640 UBERON:0001037 65480 65484 hair +T1641 PR:000001447 65508 65518 E-cadherin +T1642 GO:0016055 65524 65527;65539 65557 Wnt ... signaling pathways +T1643 PR:000000021 65532 65538 noggin +T1644 PR:000000183 65582 65588 TGF-β2 +T1645 UBERON:0001037 65594 65598 hair +T1646 GO:0005634 65602 65609 nuclear +T1647 PR:000009751 65610 65615 LEF-1 +T1648 GO:0005634 65624 65631 nuclear +T1649 PR:000002198 65632 65641 β-catenin +T1650 GO:0010467 65656 65665 expressed +T1651 CHEBI:33893 65853 65861 reagents +T1652 PR:000008288 66018 66024 GSK-3β +T1653 GO:0065007 66025 66033 controls +T1654 PR:000015308 66034 66039 Snail +T1655 PR:000008288 66122 66128 GSK-3β +T1656 GO:0016055 66130 66133;66145 66154 Wnt ... signaling +T1657 PR:000000183 66138 66144 TGF-β2 +T1658 PR:000015308 66173 66178 Snail +T1659 PR:000008288 66249 66255 GSK-3β +T1660 PR:000015308 66267 66272 Snail +T1661 PR:000001447 66290 66300 E-cadherin +T1662 PR:000015308 66317 66322 Snail +T1663 PR:000008288 66327 66333 GSK-3β +T1664 GO:0065007 66366 66377 controlling +T1665 UBERON:0001037 66378 66382 hair +T1666 PR:000000183 66509 66515 TGF-β2 +T1667 PR:000015308 66520 66525 Snail +T1668 UBERON:0002073 66529 66542 hair follicle +T1669 GO:0031069 66529 66556 hair follicle morphogenesis diff --git a/src/ontogpt/evaluation/craft/database/all/15630473.txt b/src/ontogpt/evaluation/craft/database/all/15630473.txt new file mode 100644 index 000000000..fe394f91a --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15630473.txt @@ -0,0 +1,309 @@ +A Signaling Pathway Involving TGF-β2 and Snail in Hair Follicle Morphogenesis + +Abstract + +In a common theme of organogenesis, certain cells within a multipotent epithelial sheet exchange signals with their neighbors and develop into a bud structure. Using hair bud morphogenesis as a paradigm, we employed mutant mouse models and cultured keratinocytes to dissect the contributions of multiple extracellular cues in orchestrating adhesion dynamics and proliferation to shape the cluster of cells involved. We found that transforming growth factor β2 signaling is necessary to transiently induce the transcription factor Snail and activate the Ras-mitogen-activated protein kinase (MAPK) pathway in the bud. In the epidermis, Snail misexpression leads to hyperproliferation and a reduction in intercellular adhesion. When E-cadherin is transcriptionally down-regulated, associated adhesion proteins with dual functions in signaling are released from cell-cell contacts, a process which we demonstrate leads to Ras-MAPK activation. These studies provide insights into how multipotent cells within a sheet are stimulated to undergo transcriptional changes that result in proliferation, junctional remodeling, and bud formation. This novel signaling pathway further weaves together the web of different morphogens and downstream transcriptional events that guide hair bud formation within the developing skin. + +Introduction + +Mammalian development involves the morphogenesis of complex three-dimensional structures from seemingly uniform sheets or masses of cells. A simple bud-like structure initiates the formation of many organs, including lungs, spinal cord, mammary glands, and hair follicles [1]. The multipotent, adhering epithelial cells are typically attached to an underlying basal lamina that polarizes the epithelial sheet and separates it from surrounding mesenchyme. Budding morphogenesis is guided by a reciprocal exchange of signals between epithelium and mesenchyme to specify the identity of the organ that will form and to govern its growth. + +At the helm of these molecular communication pathways are Wnts, bone morphogenic proteins (BMPs), transforming growth factor βs (TGF-βs), and fibroblast growth factors (FGFs). Through activation of cell surface transmembrane receptors, these external signaling molecules trigger distinct cascades of intracellular events that culminate in changes in gene expression, growth, and differentiation [2]. How this constellation of signals collaborates in tailoring each budding process so that it executes a distinct morphogenetic program has yet to be comprehensively defined. However, the process appears to be patterned at the initial stages of bud formation, since the relative importance of these pathways and their downstream effectors differ as buds begin to develop and cell fates are specified. + +The development of a bud requires a number of coordinated changes in the behavior of the targeted cells within an epithelial sheet. The process must be accompanied by alterations in the proliferation, polarity, shape, and adhesiveness of selected cells, as well as by modifications in their underlying basal lamina. Thus, extracellular epithelial-mesenchymal crosstalk must be intricately orchestrated to couple the determination of distinct cell fates with the contemporaneous remodeling of the physical and structural properties of the cell. + +Among the few dispensable organs, hair follicles offer an excellent model system to study epithelial bud formation. Mammalian skin epithelium begins as a single sheet of multipotent ectodermal cells. During development, specialized mesenchymal cells populate the skin in a spatially defined pattern to initiate the complex epithelial-mesenchymal crosstalk that will specify the bud [3]. Once committed, a small cluster of epithelial cells, the placode, instructs a group of underlying mesenchymal cells to condense and form the nascent dermal papilla, which will be a permanent fixture of the hair follicle. Subsequent exchanges between the placode and nascent dermal papilla result in further growth of the follicle into the underlying dermis, or down-growth, and eventual differentiation into the six concentric layers of the mature follicle. + +Previously, we delineated how two respective epithelial and mesenchymal signals, Wnts and the BMP-inhibitory factor noggin, function in concert to induce lymphoid enhancer factor-1/β-catenin (LEF-1/β-catenin)-mediated gene transcription within the follicle placode [4]. The downstream changes elicited through convergence of these two early signaling pathways include down-regulation of the gene encoding E-cadherin, the prototypical epithelial cadherin that forms the transmembrane core of intercellular adherens junctions (AJs) [5]. We subsequently showed that when E-cadherin is transgenically elevated in mouse skin, hair follicle morphogenesis is blocked, suggesting that E-cadherin down-regulation is a critical event in governing the adhesion dynamics necessary for budding morphogenesis [4]. Like LEF-1, E-cadherin also binds to β-catenin. At sites of cell-cell contact, however, E-cadherin-β-catenin complexes recruit α-catenin, which in turn coordinates the associated actin polymerization dynamics necessary to stabilize nascent AJs and integrate the cytoskeleton across an epithelial sheet [6,7,8]. α-Catenin also binds to the class III Lin-1, Isl-1, Mec-3 (LIM) protein Ajuba (a member of the zyxin family of proteins), which appears to function dually in both adhesion and in activation of the Ras-mitogen-activated protein kinase (MAPK) pathway [9,10]. Through these links, AJs appear able to couple adhesion with cytoskeletal dynamics as well as with nuclear and cytoplasmic signaling. This provides a framework for conceptualizing why E-cadherin levels appear to impact upon a plethora of developmental processes (reviewed in [11]). + +As we probed more deeply into the underlying mechanisms governing E-cadherin promoter activity, we were intrigued by the close proximity of the LEF-1/β-catenin binding site to a site known to bind the Snail/Slug family of zinc finger transcriptional repressor proteins [12,13,14,15]. Both activity of Snail and down-regulation of E-cadherin play pivotal roles in epithelial to mesenchymal transitions (EMTs), typified by the transformation of polarized, adhering epithelial cells into motile mesenchymal cells [16,17]. Bud formation differs from an EMT in that E-cadherin activity needs to be down-regulated but not prevented, so that adhesive junctions are remodeled rather than quantitatively impaired. Supportive of an underlying ability to fine-tune cadherin expression at the transcriptional level, Snail seems to have an additive effect with LEF-1/β-catenin in negatively modulating E-cadherin promoter activity [4]. + +In the present study, we discovered that Snail is expressed briefly at an early stage of hair bud formation, when E-cadherin down-regulation and activation of proliferation take place. Thereafter, Snail disappears and remains absent during subsequent follicle down-growth and maturation. This exquisite pattern appears to be functionally relevant since altering it in vivo correspondingly affects features associated with hair bud formation, including down-regulation of E-cadherin, increased proliferation, and repressed terminal differentiation. Although the temporal spike of Snail in the hair bud is reflected at the mRNA level and seems to follow Wnt signaling and BMP inhibition, LEF-1/β-catenin activation does not appear to induce Snail gene expression in embryonic skin keratinocytes. In contrast, we provide in vitro, transgenic (Tg), and gene targeting evidence to show that TGF-β2 and small phenotype– and mothers against decapentaplegic–related protein 2 (SMAD2) signaling are upstream inducers of Snail gene expression in skin epithelium. In the absence of TGF-β2 signaling and Snail gene expression, hair placodes can form, but further follicle down-growth is blocked. Our studies point to the view that Snail likely functions downstream of cell fate specification, at a stage where the bud begins to exhibit enhanced proliferation and migration. + +Results + +Snail mRNA and Protein Are Expressed Transiently at the Hair Bud Stage of Follicle Morphogenesis + +Although Snail family members are most frequently associated with EMTs, they also participate in many malignant processes involving a down-regulation but not a quantitative abrogation of intercellular junctions [18]. The range of developmental processes in which Snail family members have been implicated thus includes the type of epithelial remodeling that is observed in hair follicle bud formation. Given our prior observation that exogenously added Snail can participate with LEF-1/β-catenin in down-regulating E-cadherin expression in keratinocytes [4], coupled with the established requirement for LEF-1/β-catenin in hair follicle morphogenesis [4,19], we turned to addressing whether Snail/Slug family members might also participate in the process. + +PCR analyses identified transient Snail mRNA expression during a period of skin embryogenesis when waves of hair follicles are forming (unpublished data).To pinpoint specifically where Snail mRNA is expressed in the developing skin, we conducted in situ hybridization using a cRNA probe unique to the Snail 3′ untranslated region (UTR). Embryonic day 17.5 (E17.5) was chosen, since the multiple waves of follicle morphogenesis occurring at this time enabled us to evaluate Snail expression at different stages of the process. As shown in Figure 1A, specific hybridization was detected within the epithelium of nascent hair buds. By contrast, as follicles progressed further through their development (e.g., germ and peg stages), they exhibited no signs of hybridization (Figure 1A). The transient nature of Snail mRNA expression during follicle development was most apparent in hybridized skin sections containing follicles from two different waves of morphogenesis (as shown in Figure 1). Hybridizing hair buds from a later wave appeared juxtaposed with nonhybridizing follicles from an earlier wave. + +To determine whether this transient nature of Snail mRNA expression is reflected at the protein level, we generated an antibody against the N-terminal sequence that resides upstream of the more conserved zinc finger domains. As judged by Western blot analysis, the antibody did not detect endogenous proteins from cultured keratinocytes, but it did yield a band of the expected size from keratinocytes transiently expressing a hemagglutinin (HA)-tagged Snail protein (Figure 1B). The antibody also recognized a band corresponding to the size of endogenous Snail (approximately 28 kDa) in lysates from embryonic mouse skin, the temporal appearance of which corresponded to the waves of hair follicle morphogenesis from E15.5 to newborn when over 90% of the hair on the mouse is formed (Figure 1B). Consistent with the Western blot data, immunohistochemical analysis did not detect Snail in single-layered E13.5 epidermis (Figure 1C) nor in the placode, which is the earliest morphological sign of the commitment of multipotent cells of the embryonic ectoderm to a hair cell fate (Figure 1D). Consistent with the in situ hybridization results, anti-Snail antibody labeled only hair buds and not follicles at more mature stages of development (Figure 1E). Taken together, the anti-Snail antibody appeared to be specific for its target protein. It did not detect other Snail family members known to be expressed in keratinocytes and/or skin (unpublished data). Furthermore, the immunohistochemical data paralleled our Snail in situ hybridization data revealing transient Snail expression at the hair bud stage (Figure 1A). + +As judged by immunohistochemistry, Snail protein was localized to the nuclei of the hair bud cells (Figure 1E). This feature was consistent with Snail's known function as a transcriptional repressor [12,13]. Additionally, anti-Snail labeling was detected in only three of the four major waves of follicle morphogenesis. Snail was not found in the buds of guard hairs that are the earliest of all hairs to form (at E13.5), and which constitute less than 5% of the mouse coat (unpublished data). + +As judged by immunofluorescence with antibodies against the proliferating nuclear antigen Ki67, the timing of Snail expression coincided with the stage at which the developing follicle enhanced its proliferation and down-growth (Figure 1F). Immunohistochemistry with antibodies against the active (phosphorylated) form of MAPK (pMAPK) marked a subset of the proliferating (Ki67-positive) cells, and pMAPK-positive cells were enriched in the hair bud (Figure 1G). The timing of Snail induction and Ki67 and pMAPK enrichment in the hair bud appeared to follow closely the induction of LEF-1/β-catenin activity, known to initiate in the hair placode stage [20]. However, like placodes, hair buds exhibited down-regulation in E-cadherin expression (Figure 1H; see also [4]). + +Sustained Expression of Snail Results in Epidermal Hyperproliferation and Differentiation Defects in Tg Mouse Skin + +The striking spike of Snail expression coincident with hair bud formation and enhanced proliferation prompted us to examine the consequences of ectopically expressing Snail elsewhere in mouse skin epidermis. To distinguish Tg from endogenous Snail, we used the HA-epitope, shown previously not to alter Snail's transcriptional activity [12]. Of 20 K14-Snail[HA] Tg animals generated, three expressed the transgene and all exhibited analogous phenotypes. Mice that integrated the transgene at the single-cell stage died at or shortly after birth. The three surviving full-Tg founder mice harbored transgene integrations that gave stable transmission of mosaic Snail gene expression through the germline. Progressively poor health necessitated our sacrificing most offspring from these lines within a year of birth. + +As Snail Tg animals grew, they became distinguished by their small size, short tails, and flaky skin (Figure 2A). Histological analyses of 3-d old (P3) mice revealed mosaic patches marked by epidermal thickening (Figure 2B). The mosaic morphology was reflected at the level of Tg Snail protein, with only the hyperthickened regions expressing nuclear HA-tagged Snail (Figure 2C). These hyperthickened areas were marked by excessive proliferation, as revealed by antibodies against the proliferating nuclear antigen Ki67 (Figure 2D and 2E). Activated, pMAPK-positive cells were also prevalent in these areas (Figure 2F and 2G), as were cells expressing keratin 6, a keratin induced in the suprabasal layers of hyperproliferative skin (Figure 2H and 2I). + +Expression of the Snail transgene did not block terminal differentiation in the hyperproliferative epidermis, but it distorted it markedly (Figure 3A–3H). Typical of most hyperproliferating conditions, Snail expression led to a large expansion in layers with spinous and granular morphology. Additionally, however, was a marked and variable expansion of keratin 5 (K5), normally restricted to the innermost basal layer (see Figure 3). Although the failure of Snail-null mice to develop past gastrulation [21] precluded our ability to study the loss of Snail function in skin development, a good correlation emerged between the expression of Snail protein and the extension of K5, Ki67, and pMAPK suprabasally (compare data in Figures 2 and 3). + +The changes in hyperproliferation and differentiation were not initially accompanied by gross signs of epithelial invaginations. With age, however, epidermal folds and undulations developed in areas where Snail was expressed, and proliferative markers persisted in these regions (Figure 3I and 3J; anti-cyclin D staining). The undulations were accompanied by partial dissolution of the underlying basement membrane (Figure 3K and 3L). Aberrant staining was also observed with antibodies against components of the hemidesmosomes, which provide strong adhesion of basal epidermal cells to the underlying basal lamina (Figure 3M and 3N). Interestingly, similar types of alterations occur in the basement membrane in the hair bud of embryonic and newborn mice when Snail is normally expressed. The fact that the basement membrane separating the epidermis from the dermis is altered only in the adult Tg animals suggests the involvement of intermediary factors not as readily available in the epidermis as they are in the follicle. + +Possible Links between Epidermal Hyperproliferation and Down-regulation of AJ Proteins in Snail Tg Mice + +Given that the E-cadherin promoter is a direct target for Snail-mediated repression in vitro [4,12,13], and that E-cadherin was down-regulated in Snail-expressing hair buds, we examined the status of E-cadherin and other AJ proteins within regions of hyperproliferative epidermis where Tg Snail was present (Figure 4A). In these regions, immunofluorescence staining of E-cadherin and α-catenin were markedly diminished. In contrast, the intensity of antibody staining for two other AJ proteins, β-catenin and Ajuba, was still strong. Interestingly, however, despite appreciable immunofluorescence, localization of β-catenin and Ajuba appeared to be largely cytoplasmic rather than at cell-cell borders (Figure 4A insets). + +Architectural differences in the epidermis made Western blot analyses somewhat difficult to gauge. However, in regions such as ear skin, where the highest levels of Snail protein were expressed, the effects were accentuated. In both back skin and ear skin, overall levels of E-cadherin and α-catenin were reduced, under conditions where β-catenin and Ajuba levels remained unchanged relative to controls (Figure 4B). Taken together, these data were consistent with our results obtained from immunofluorescence microscopy. + +A priori, the decrease in α-catenin levels could be due to either direct transcriptional repression by Snail or perturbations in AJ formation caused by the decrease in E-cadherin gene expression. To distinguish between these possibilities, we tested whether α-catenin levels could be restored by exogenous expression of E-cadherin in Snail-expressing keratinocytes. As shown in Figure 4C, transiently transfected keratinocytes expressing HA-tagged Snail displayed a loss of E-cadherin and α-catenin at cell-cell borders. Coexpression of exogenous HA-tagged E-cadherin not only enabled cell-cell border localization of E-cadherin protein, but also rescued the cell-cell border staining of α-catenin (Figure 4C). The ability to restore α-catenin expression and localization under these conditions argues against the notion that Snail transcriptionally represses α-catenin. Rather, the findings are consistent with a previous report that E-cadherin is required for the translation of α-catenin mRNA [22]. + +Despite the reductions in AJ markers, Tg skin still displayed sealed membranes and intercellular junctions that were largely intact, as judged by ultrastructural analyses (unpublished data). In this respect, the skin epithelium resembled that of the hair bud, where the down-regulation in junction proteins is permissive for cell-cell remodeling without abrogating intercellular adhesion. + +The similarities between Snail Tg epidermis and hair buds extended to the hyperproliferative state, leading us to wonder whether the down-regulation of AJ proteins might contribute to this condition. Given the increase in pMAPK staining in Snail Tg epidermis (see Figure 2G), we used pMAPK levels as our assay to test whether the loss of E-cadherin contributed to the Snail-mediated increase in proliferation. Consistent with our in vivo observations, transfected keratinocytes expressing Snail exhibited a substantial increase in pMAPK levels relative to control cells (Figure 4D). Coexpression of E-cadherin with Snail appeared to abrogate this effect. Together, these findings raised the possibility that an AJ-associated protein that is normally sequestered at the plasma membrane may participate in a proliferation signaling pathway when AJs are deconstructed. + +Numerous studies have correlated a down-regulation of E-cadherin with a translocation of β-catenin to the nucleus and a transactivation of genes that are regulated by the LEF-1/T cell factor (TCF) family of DNA binding proteins [23,24,25]. The presence of nuclear cyclin D in hyperproliferative Snail Tg epidermis was particularly intriguing since prior studies have reported cyclin D gene as a direct target of TCF/β-catenin transcription [26]. This said, we did not detect nuclear β-catenin in our Tg epidermis, and mating the Snail Tg mice against the TOPGal reporter mouse [20] gave no signs of ectopic LEF-1/Tcf/β-catenin activity (unpublished data). + +We next turned to the presence of cytoplasmic Ajuba for a possible mechanistic link to the proliferative increase in our Snail Tg epidermis. In addition to its documented ability to bind α-catenin [10], Ajuba can also associate with growth factor receptor-bound protein-2 (Grb-2)/son of sevenless (Sos), the nucleotide exchange factor for Ras, which is upstream from activation of MAPK [9]. Given the increase in pMAPK staining in Tg skin, we examined the possibility that Ajuba might have changed its binding partner in Snail-expressing epidermis. Interestingly, Ajuba was readily detected in anti-Grb-2 immunoprecipitates of protein lysates from skins of Snail Tg mice but not from the corresponding wild-type (WT) animals (Figure 4E). When these experiments were repeated with α-catenin-null epidermis, a similar Grb-2-Ajuba association was detected, and again, this interaction was not detected in the protein extracts from control littermate skin (Figure 4E). Together, these data demonstrate that the reduction in α-catenin levels, either by Snail-mediated down-regulation of E-cadherin or by α-catenin conditional targeting, allows Ajuba to interact with Grb-2/Sos. + +If the competition between Grb-2/Sos and α-catenin for Ajuba is functionally relevant to the hyperproliferative state of a keratinocyte, then overexpression of Ajuba would be expected to bypass the competition and promote activation of the Ras-MAPK pathway in WT keratinocytes. Indeed, when serum-starved keratinocytes were transiently transfected with an Ajuba expression vector, the levels of pMAPK were not only elevated but also comparable to those transfected with the K14-HASnail transgene (Figure 4F). This activation was abolished when cells were treated with a small peptide inhibitor that specifically interrupts the Grb-2/Sos interaction (Figure 4F; see lanes marked “inh”) [27]. + +Ajuba's pre-LIM domain is the segment that associates with Grb-2's Src-homology 3 domain [9]. When this domain was overexpressed in serum-starved keratinocytes, a comparable elevation in pMAPK was observed (Figure 4F). As expected, the small peptide inhibitor that interrupts the Grb-2/Sos association blocked the effects. These data suggested that by elevating cytosolic Ajuba levels, Ajuba's pre-LIM domain may associate with Grb-2/Sos in a manner that stimulates its nucleotide exchange activity and leads to activation of the Ras-MAPK pathway. Although this pathway provides one mechanism by which Snail expression and proliferation may be coupled in skin epithelium, proliferative circuitries involving AJs are known to be complex and often interwoven. Future studies will be needed to systematically dissect these putative intricacies at a molecular level. + +Probing the Regulation of Snail Gene Expression Reveals an Essential Link to TGF-β2 Signaling in the Developing Hair Bud + +The temporal spike of Snail mRNA expression in the hair bud prompted us to consider what factor(s) may be regulating the Snail gene. A variety of extracellular signals have an impact on the cell type-specific expression of different Snail family members, and many of them, including Wnts, BMPs, FGFs, and TGF-βs, also affect hair bud development [2,16,28]. Since Snail is not expressed in cultured skin keratinocytes that secrete active BMPs and FGFs (see Figure 1B), we focused our attention on Wnt and TGF-β signaling as more likely candidates for Snail induction in this cell type. + +Previously, we showed that effective transmission of a Wnt-3a signal in cultured keratinocytes can be achieved through their exposure to the BMP inhibitor noggin, which induces LEF-1 expression [4]. In vitro, these conditions down-regulated the E-cadherin promoter and induced a LEF-1/β-catenin-sensitive reporter gene, TOPFLASH [4]. In contrast, Snail expression was not induced by these conditions (Figure 5A). Thus, despite essential roles for Wnts and noggin in hair follicle specification [4,29,30], our studies did not support an essential role for these signals in governing Snail expression in keratinocytes. + +TGF-β1 has been shown to induce Snail family members in hepatocytes and heart [15, 31]. In keratinocytes, however, TGF-β1 inhibits keratinocyte growth and seems to be involved in triggering the destructive phase of the cycling hair follicle [32]. Of the loss-of-function mutations generated in each of the TGF-β genes, only the TGF-β2 null state blocked follicle development at the hair bud stage [32]. Thus, we turned towards addressing whether TGF-β2 might be involved in regulating Snail expression in keratinocytes isolated from the basal layer of the epidermis. Though there is no cell culture system available to specifically study placodal cells, these keratinocytes are their progenitors and are the closest approximation available to study the behavior of epithelial cells of the placode. + +Interestingly, treatment of cultured keratinocytes with as little as 5 ng/ml of TGF-β2 caused a rapid and transient induction of Snail (Figure 5B). Following this treatment, Snail protein was detected within 30 min, peaked at 2 h, and then declined thereafter. The induction of Snail appeared to be specific for the active form of the growth factor, as pretreatment of TGF-β2 for 10 min at 100 °C obliterated the response [Figure 5B, lanes marked (–)]. By contrast, although TGF-β receptor activation remained elevated during the duration of the experiment (as measured by the sustained phosphorylation of the downstream effector SMAD2) Snail expression could not be maintained (Figure 5B). Thus, although Snail expression correlated with phosphorylated SMAD2 (pSMAD2) induction, its decline seemed to rely on secondary downstream events. + +The rapid kinetics of Snail expression were reflected at the mRNA level, suggesting that Snail promoter activity in keratinocytes might be sensitive to TGF-β2 signaling (Figure 5C). To test this possibility, we engineered a transgene driving the β-galactosidase reporter under the control of approximately 2.2 kb of promoter sequence located 5′ from the transcription initiation site of the mouse Snail gene. At 2 d after transient transfection, keratinocytes were treated with TGF-β2 (t = 0) and then assayed for transgene activity over the same time course in which we had observed Snail protein induction. The results of this experiment are presented in Figure 5D. + +Within 0.5 h of TGF-β2 treatment, Snail promoter activity had increased 3-fold, and by 2 h, it peaked to approximately 10-fold over control levels (Figure 5D). Thereafter, Snail promoter activity rapidly returned to the basal levels seen in unstimulated keratinocytes. The kinetics of Snail promoter activity closely paralleled those observed for Snail protein induction. Moreover, the stimulatory effects appeared to be specific to TGF-β2, since they were abrogated either by heat inactivation of the TGF-β2 protein or by mutation of a putative SMAD binding element located about 1.8 kb 5′ from the Snail transcription start site (Figure 5D). Taken together, these results suggested that in keratinocytes, TGF-β2 signaling results in a pSMAD2-dependent transient activation of the Snail gene, and that maintenance of Snail protein relies, in part, upon sustained promoter activity. + +The brevity of Snail gene and protein induction in TGF-β2 treated cultured keratinocytes resembled the temporal appearance of Snail mRNA and protein at the initiation of hair follicle morphogenesis in embryonic mouse skin. To test whether TGF-β2 might be required for Snail induction in hair bud formation in vivo, we first analyzed whether TGF-β2 was expressed in or around the hair bud. Consistent with previous observations [33], an anti-TGF-β2 antibody labeled developing hair buds (Figure 6A). This labeling appeared to be specific as judged by the lack of staining in follicle buds from mice homozygous for a TGF-β2 null mutation (Figure 6A; [34]). Moreover, the downstream effector of TGF-β2 signaling, pSMAD2, was also expressed in WT, but not TGF-β2-null, hair buds (Figure 6B). Together, these data underscore the importance of the TGF-β2 isoform despite expression of both TGF-β1 and TGF-β2 in developing hair buds at this stage. + +To further explore the possible relation between Snail and TGF-β2, we examined the status of Snail expression in TGF-β2-null hair buds. As judged by immunohistochemistry, Snail protein was absent from E17.5 skin of TGF-β2-null embryos but not from that of control littermates (Figure 6C). This effect appeared to be exerted at the transcriptional level, since Snail mRNAs were also not found in TGF-β2 null hair buds under conditions in which the signal was readily detected in the hair buds of littermate skin (Figure 6D). + +Conversely, in 2-wk-old K14-Smad2 Tg mice, which display elevated TGF-β signaling in skin [35], Snail protein was readily detected by Western blot analyses, where it was not found in postnatal skin (Figure 6E). Taken together, these results provide compelling evidence that TGF-β2 is functionally important for inducing Snail gene expression in a pSMAD-dependent manner in developing hair buds. Whether pMARK activity also contributes to Snail induction was not addressed in the present study [15]. + +Although some hair buds still formed in TGF-β2 null skin, their number was reduced by approximately 50% [32]. Thus, although the pathway mediated by TGF-β2 signaling impacts the earliest step of epithelial invagination, it does not appear to be essential for bud morphogenesis. Consistent with this notion, basement membrane remodeling still took place in the TGF-β2-null buds, as judged by immunofluorescence with antibodies against β4 integrin, an integral component of keratinocyte-mediated adhesion to its underlying basement membrane (Figure 6F). In contrast, TGF-β2 signaling appeared to be an important factor for the early proliferation that occurs in the developing hair buds, as judged by anti-Ki67 and anti-pMAPK immunofluorescence (Figure 6F and 6G). + +If TGF-β2 stimulates Snail expression in developing buds, loss of this morphogen would be expected to affect the expression of genes that are typically repressed by Snail. Since a major target for Snail-mediated repression is the E-cadherin gene [12,13], we investigated the status of E-cadherin in TGF-β2-null buds. As shown in Figure 6H, hair buds in TGF-β2 null skin displayed elevated immunofluorescence staining relative to their WT counterparts. + +Previously we demonstrated that the concerted action of the extracellular signals Wnt and noggin are required for the generation of a LEF-1/β-catenin transcription complex to repress E-cadherin transcription at the onset of hair fate specification. As shown in Figure 6I and 6J, both WT and TGF-β2 null buds exhibited nuclear LEF-1 and β-catenin localization, signs that the Wnt-noggin signaling pathway was intact. These data suggest that during hair follicle morphogenesis, TGF-β2 functions subsequently to Wnt/noggin-mediated determination of hair fate. Moreover, through activation of Snail gene expression, TGF-β2 appears to work in tandem with these other morphogens to down-regulate E-cadherin levels, which contributes to the activation of proliferative circuitries. + +Discussion + +During budding morphogenesis, intersecting signaling networks from the epithelium and mesenchyme govern transcriptional, adhesive, polarity, and motility programs in these select groups of cells. The dynamic nuclear and cytosolic changes that take place during this time form the cornerstone for organ morphogenesis. Two major challenges in understanding the mechanisms underlying a particular budding process are to order the temporal sequence of external cues involved and then to dissect how the cells of the developing bud translate these signals into the downstream events of cellular remodeling, proliferation, and differentiation. Our studies here provide some insights into how these events are orchestrated during hair bud formation in developing skin. + +Signaling during Early Hair Follicle Morphogenesis + +Recent studies on hair bud morphogenesis suggest that Wnt signals likely from the epithelium and BMP inhibitory signals from the underlying mesenchyme converge to produce an active transcription factor complex involving β-catenin and LEF-1, which in turn plays a key role in specifying the hair follicle fate [4,29,30,36,37]. Sonic hedgehog (Shh) and TGF-β2 signaling also play essential roles in follicle morphogenesis, but in contrast to β-catenin null skin, in which follicle invaginations are absent [30], some hair buds still form in the absence of LEF-1, Shh, or TGF-β2 [32,38]. These likely reflect the first wave of follicle (i.e., guard hair) morphogenesis, which accounts for a small number (fewer than 5%) of hairs and is under distinct regulatory control. Guard hairs form in the absence of LEF-1 and TGF-β2, and we have found that they also fail to express Snail at the budding stage of development (unpublished data). How E-cadherin is regulated in guard hairs remains to be determined. Several candidates include other Snail family members such as Slug or twist, or alternatively, transcription factors involving β-catenin and a different member of the LEF-1/TCF/Sry-type HMG box (commonly known as SOX) family [39,40]. Further investigation will be required to determine whether the signaling pathway we have elucidated here is a theme with multiple variations. + +TGF-βs are known to promote withdrawal of keratinocytes from the cell cycle [41]. Hence, when TGF-β2 protein was detected at the transition between the growing and destructive phases of the adult hair cycle, research initially and naturally focused on a role for this family member in cessation of growth and/or triggering apoptosis ([42] and references therein). However, in contrast to TGF-β1-null skin, which exhibits an extended growing phase of postnatal hair follicles, TGF-β2-null skin displays an embryonic block in follicle bud progression [32]. Although this phenotype is consistent with TGF-β2's embryonic expression patterns [33], about 50% of TGF-β2 null buds appear unable to progress to the down-growth phase, a feature that cannot be explained readily on the basis of previously established effects of TGF-βs. + +Our finding that TGF-β2 is upstream from Ki67 expression and MAPK activation lends further support to the notion that hair follicle keratinocytes at this early stage of development react to TGF-β2 signaling in a fashion opposite to that typically expected for TGF-β factors. This said, based upon pSMAD2 immunohistochemistry, the immediate steps of downstream signaling appeared to be intact. Thus, we surmise that the proliferative outcome is likely to be rooted in differences in the repertoire of activated SMAD target genes. In this regard, the positive effects of TGF-β2 on proliferation within the hair bud may be more analogous to what has been seen in progression of squamous cell carcinoma to metastatic carcinoma [43] rather than that typically observed for keratinocytes [44,45,46]. + +The prior identification of the Snail gene as a potential target of TGF-β signaling [15] was intriguing, given the temporal wave of Snail gene expression that occurs in the developing hair bud. The additional correlation between epithelial hyperproliferation and Snail transgene expression further fostered our interest in a possible link between TGF-β2 and Snail. Our functional studies demonstrate that without TGF-β2, Snail expression is abolished in the mutant hair buds, and conversely, in K14-Smad2 skin, Snail is ectopically activated. Moreover, our in vitro studies indicate that even sustained TGF-β2 exposure may cause only a transient induction of Snail, offering a possible explanation as to why Snail is so briefly expressed during hair follicle morphogenesis. An additional point worth mentioning is that prolonged expression of Tg Snail in postnatal skin resulted in morphological and biochemical signs of epithelial to mesenchymal transitions (unpublished data), underscoring why transient Snail expression may be so important during normal hair follicle morphogenesis [18]. + +At first glance, the sparsity in hair coat of K14-Snail Tg mice seemed indicative of a defect in follicle formation (see Figure 2A). Closer inspection, however, revealed that not all hairs penetrated the hyperthickened Tg epidermis. Several factors may contribute to the seemingly normal follicle development in these mice. One obvious factor is the K14 promoter, which is elevated in the basal layer of the epidermis and the outer root sheath (ORS) of the hair follicle, but is markedly down-regulated in developing embryonic hair buds as well as in the postnatal hair progenitor cells. The K14 promoter is also less active in the ORS than epidermis and hence this might also account for the lack of apparent response of the ORS to ectopic Snail. Additional contributing factors could be the multiplicity of Snail family members and their differential expression, the saturation, and/or diversity of regulatory mechanisms that govern AJ formation, migration, and proliferation in the follicle ORS. Distinguishing between these possibilities must await the generation of mice harboring skin-specific loss-of-function Snail mutations. + +Links between Signaling, Transcriptional Cascades, Epithelial Remodeling, and Proliferation in the Hair Bud + +Previously, we discovered that early during hair follicle morphogenesis, E-cadherin gene expression is down-regulated concomitantly with the invagination of developing bud cells into the skin [4]. Because the timing of this event correlated with the activation of a LEF-1/β-catenin transcription factor complex [20], we were intrigued by the presence of a putative LEF-1/TCF binding site in the E-cadherin promoter. This prompted an investigation that subsequently led to our discovery that LEF-1/β-catenin can contribute to repression of E-cadherin gene expression in skin keratinocytes [4]. In the course of these studies, we also noted that Snail can also contribute to this process in keratinocytes in vitro, and our present studies revealed that Snail is expressed at the right place and time to be physiologically relevant in the process. + +In noggin-null embryonic skin, LEF-1 expression and subsequent activation of the LEF-1/β-catenin reporter gene is abrogated in the developing placodes. The corresponding failure of E-cadherin down-regulation underscores the importance of Wnt/noggin signaling in regulating this event in follicle morphogenesis [4]. Conditional gene targeting studies will be necessary to establish whether Snail family members also contribute to the down-regulation in E-cadherin gene expression that occurs during follicle formation. However, it is intriguing that K14-Snail Tg epidermis displayed a marked down-regulation in E-cadherin expression, thereby demonstrating its potential to do so in skin. Our prior findings showed that by elevating E-cadherin levels or by conditionally ablating α-catenin, hair follicle morphogenesis can be impaired [4,7]. The marked epidermal hyperproliferation seen in the K14-Snail Tg skin, coupled with the converse suppression of proliferation and Snail in TGF-β2-null hair buds led us to wonder whether the down-regulation of E-cadherin during follicle morphogenesis might have a direct impact on elevating the proliferative state of these cells. + +Our Tg studies suggested that, at least in part through its regulation of E-cadherin, Snail is able to influence the subcellular localization of a variety of AJ-associated proteins. One of these appears to be Ajuba, which was previously shown to have the dual capacity to bind Grb-2 as well as α-catenin [9,10]. Our studies revealed that in skin keratinocytes that either harbor a conditional null mutation in α-catenin or that overexpress Snail, Ajuba develops an interaction with Grb-2 that is otherwise not observed in WT keratinocytes. The corresponding abilities of either Snail-transfected or Ajuba-transfected keratinocytes to exhibit elevated activation of the Ras-MAPK pathway suggest that the Grb-2 association of Ajuba under conditions of reduced levels of AJ proteins may be directly relevant to the parallel in hyperproliferation. + +In stable epithelial (i.e., Madin-Darby canine kidney, or MDCK) cell lines, Snail has been shown to block cell cycle progression and promote motility and shape changes for invasion [47]. While our in vivo studies are consistent with a role for Snail in motility and epithelial remodeling, they differ with respect to Snail's apparent proliferative effects. A priori, this could be simply due to variations in the response of different cell types to Snail expression. Alternatively, these differences may be relevant to the benefit of using mouse models to reveal functions not always recapitulated in stable cell line models. Future studies should highlight the underlying reasons for these opposing results. + +Irrespective of these differences, our in vivo studies do not stand alone, as there are many situations in which a down-regulation in AJ proteins correlate with enhanced proliferation. In fact, a myriad of diverse mechanisms have been implicated in activating epithelial proliferation upon down-regulation of AJ proteins [7,23,24,48]. Sifting through these converging pathways is likely to be a difficult and painstaking process. This said, by identifying the status of different players involved in specific cell types and at specific stages in development, our mechanistic understanding of how intercellular remodeling is linked to proliferation in epithelial morphogenesis should begin to surface in the future. Elucidating the molecular mechanisms through which these networks converge is also a prerequisite for understanding how these processes go awry during tumorigenesis. + +Materials and Methods + + + +Reagents + +Primary antibodies used were against: E-cadherin (M. Takeichi, Kyoto University, Japan); α-catenin, β-catenin, pMAPK, tubulin (Sigma, St. Louis, Missouri, United States), Ajuba (G. Longmore, Washington University, St. Louis, Missouri, United States); β4 integrin/CD104 (BD Pharmingen, San Diego, California, United States), laminin 5 (R. Burgeson, Harvard University, Cambridge, Massachusetts, United States), K5, K1, loricrin (Fuchs Lab), involucrin, fillagrin (Covance, Berkeley, California, United States), MAPK, pSMAD2 (Cell Signaling, Beverly, Massachusetts, United States); Grb-2 (Santa Cruz Biotech, Santa Cruz, California, United States); P-cadherin (Zymed Laboratories, South San Francisco, California, United States); HA (Roche Biochemicals), vimentin (Chemicon, Temecula, California, United States), Ki67 (Novo Castra, Newcastle Upon Tyne, United Kingdom), keratin 6 (P. Coulombe, John Hopkins University, Baltimore, Maryland, United States), cyclin D (Oncogene, San Diego, California, United States), and TGF-β2 (L. Gold, New York University, New York, New York, United States). FITC-, Texas Red-, or HRP-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, Pennsylvania, United States). Biotinylated secondary antibodies were from Vector Labs (Burlingame, California, United States). Dilutions were according to the manufacturer's recommendation. The Snail antibody was generated in Guinea pigs by inoculating them with the N-terminal sequence of murine Snail fused to GST (Covance, Princeton, New Jersey, United States). Recombinant human TGF-β2 was purchased from R&D (Minneapolis, Minnesota, United States). Heat inactivated TGF-β2 was generated by heating the recombinant protein at 100 °C for 10 min. + +Mice + +The K14-Snail Tg mouse was generated by digesting the pcDNA3-mm Snail-HA plasmid (G. de Herreros, Universitat Pompeu, Fabra, Barcelona, Spain) with BamHI and NotI and subcloned into the K14 vector [49]. The linearized construct was injected into the nucleus of embryos from CD1 mice. The K14-Smad 2 Tg mouse was reported in Ito et al., 2001. The TGF-β2 knockout (KO) mouse was described in [34]. The shh KO mouse [38] and TOPGal mouse [20] have previously been reported. + +Western blot and immunoprecipitation + +Protein extracts from primary keratinocytes were generated either by lysing cells in lysis buffer (1% NP-40, 1% sodium deoxycholate, 20 mM Tris-Cl [pH 7.4], 140 mM NaCl containing 1 mM sodium vanadate, 2 mM phenylmethylsulfonyl fluoride, and protease inhibitors) or directly in Laemmli bβuffer and boiled. For skin tissue: Frozen tissue was pulverized in a liquid nitrogen-cooled Gevebesmascher and the powder scraped into a chilled microcentrifuge tube. RIPA buffer (1% Triton X-100 in PBS with 10 mM EDTA, 150 mN NaCl, 1% sodium deoxycholate, and 0.1% SDS) and protease inhibitors or Laemmli buffer was added. The cell suspension was sonicated three times for 15 s and centrifuged at 14,000 rpm at 4 °C. The supernatant was separated from the pellet and used in the experiments. Extracts subjected to immunoprecipitation were precleared with Protein G Sepharose (Amersham, Piscataway, New York, United States) and incubated with antibody with rocking overnight at 4 °C. Protein G Sepharose was added and samples were incubated for 1 h at 4 °C with rocking. Samples were washed three times for 5 min each in lysis buffer, and the Protein G Sepharose-antibody-antigen pellet was resuspended in Laemmli buffer and boiled for 10 min. Samples were run on SDS-PAGE and transferred to nitrocellulose membrane (Schleicher and Schuell Bioscience, Keene, New Hampshire, United States). Western blot signals were developed using the enhanced chemiluminescence kit from Amersham + +Cell culture + +Primary keratinocytes were culture in low-calcium medium as previously described [4]. Transient transfections were carried out with FuGENE6 reagent (Roche, Indianapolis, Indiana, United States) according to the manufacturer's protocol. Measurement of β-galactosidase or luciferase levels in promoter activity studies were carried out with the Galacto-Lite assay kit (TROPIX, Bedford, Massachusetts, United States) and the Dual luciferase (Promega, Madison, Wisconsin, United States), respectively. Runella luciferase was cotransfected into cells to correct for transfection efficiency. Experiments were done in triplicate and repeated at least three times. Measurements were done on a luminometer (MGM Instruments, Hamden, Connecticut, United States). For experiments measuring phosphorylation of MAPK, keratinocytes were serum starved for 3 h prior to harvesting of cells by incubation in medium lacking serum. Treatment of cells with Wnt- and noggin-conditioned medium was previously described [4]. + +Constructs + +The 2.2-kb murine Snail promoter was generated by PCR using a forward primer with an XbaI linker sequence, 5′-TCTAGAATTGTTTGCTGCTGTATGGTCTTC-3′, along with a reverse primer with a BglII linker sequence, 5′-AGATCTGTTGGCCAGAGCGACCTAG-GTAG-3′, and mouse genomic DNA as a template. The PCR product was purified with the Gel Extraction Kit (Qiagen, Valencia, California, United States) and ligated into pCRII-TOPO TA vector (Invitrogen, Carlsbad, California, United States). The promoter was verified by sequencing and digested with XbaI and BglII and subcloned into the pβ-gal BASIC vector (BD Biosciences Clontech, Palo Alto, California, United States). The point mutations in the SMAD binding element was generated with the Quik-Change Kit (Stratagene, La Jolla, California, United States) using the forward primer 5′-GGGCGGGCTTAGGTGTTTTCATTTACTCTTGAGGAAAAGCTTGGC-3′ and the reverse primer 5′-GCTTTT-CCTCAAGAGTAAATGAAAACACCTAAGCCCGCCCTGCCC-3′. The probes for the Snail in situ hybridization were generated against the 3′ UTR by PCR using the forward primer 5′-ACCTTCTCCCGCATGTCCTTGCTCC-3′ and the reverse primer 5′-CTGCTGAGGCATGGTTACAGCTGG-3′, and genomic DNA as a template. The PCR product was gel purified and ligated into pCRII-TOPO TA vector. The pre-LIM domain of Ajuba was generated essentially as described [9], but was fused to GFP by subcloning from the pEGFP-N1 20 vector (BD Biosciences Clontech) + +In situ hybridization + +The pCRII-TOPO TA vector containing a region of the 3′ UTR of Snail was used as a template to generate digoxigenin-labeled sense and antisense riboprobes (Roche). The respective probes were obtained by XhoI and BamH1 digestions. In situ hybridizations were performed on 10-μm thick sections of E17.5 mouse embryos. The sections were fixed with 4% PFA for 10 min at room temperature, prehybridized at room temperature for 4.5 h, hybridized with the probe (2 μg/ml) at 55 °C for 12–14 h, blocked with 10% NGS, and treated with anti-dig Fab-AP antibody (Roche #1093274) at a 1:2,500 dilution for 3 h. The sections were incubated with NBT and BCIP until adequate signal was detected. + +Immunofluorescence and immunohistochemistry + +Tissue samples for immunofluorescence were frozen in OCT and sectioned 10 μm thick on a cryostat. Sections were fixed in 4% paraformaldehyde for 10 min at room temperature, blocked, and stained with antibodies. Tissue samples for immunohistochemistry were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraffin. Samples were sectioned on a microtome (10 μm thick) and rehydrated prior to staining with antibody. Samples stained with Snail, pMAPK, pSmad2, and cyclin D were antigen unmasked with 10 mM sodium citrate (pH 6) in an Antigen Retriever 2100 (Pickcell Laboratories, Leiden, Netherlands). The DAB substrate kit (Vector Labs) was used according to manufacturer's instructions to develop the signal. + +RT-PCR + +RNA was extracted from keratinocytes or skin tissue with Trizol (Invitrogen) according to the manufacturer's protocol. cDNA was generated using oligo-dT primers and the Superscript II kit (Invitrogen). The primers used for PCR were Snail forward: 5′-CAGCTGGCCAGGCTCTCGGT-3′; Snail reverse: 5′-GCGAGGGCCTCCGGAGCA-3′; GAPDH forward 5′-CGTAGACAAAATGGTGAAGGTCGG-3′; and GAPDH reverse: 5′-AAGCAGTTGGTGGTGCAGGATG-3′. + +Acknowledgements + +We thank M Takeichi, P Coulombe, G Longmore, and L Gold for sharing their antibodies, and AG de Herreros for the Snail construct. L Degenstein and L Polak from the Fuchs laboratory provided outstanding technical assistance with transgenic work and animal husbandry. T Doetschman and Y Chai are acknowledged for the TGF-β2 KO and K14-Smad2 mice, respectively. We thank additional members of the Fuchs lab for generously sharing their reagents and ideas. CJ was partially supported by a fellowship from the Helen Hay Whitney Foundation. EF is an investigator at the Howard Hughes Medical Institute. This work was supported by the Howard Hughes Medical Institute and a grant from the National Institutes of Health. + +Competing interests. The authors have declared that no competing interests exist. + +Abbreviations + +AJ - adherens junction + +BMP - bone morphogenic protein + +EMT - epithelial to mesenchymal transition + +E[number] - embryonic day [number] + +FGF - fibroblast growth factor + +Grb-2 - growth factor receptor-bound protein-2 + +HA - hemaglutinin + +K1 - keratin 1 + +K5 - keratin 5 + +KO - knockout + +LEF-1 - lymphoid enhancer factor-1 + +LIM - Lin-1 + +MAPK - mitogen-activated protein kinase + +ORS - outer root sheath + +pMAPK - phosphorylated MAPK + +P[number] - postnatal day [number] + +pSMAD - phosphorylated SMAD + +Shh - sonic hedgehog + +SMADs - small phenotype– and mothers against decapentaplegic–related proteins + +Sos - son of sevenless + +SOX - Sry-type HMG box + +TCF - T cell factor + +Tg - transgenic + +TGF-β - transforming growth factor β + +TOP - TCF-optimal-promoter + +UTR - untranslated region + +WT - wild-type + +Figures and Tables + +Figure 1 + +Snail Is Expressed Exclusively in the Hair Bud during Morphogenesis + +Embryos were either frozen in OCT embedding compound (A, F, and H) or embedded in paraffin (C, D, E, and G), and then sectioned (8 μm). + +(A) In situ hybridizations with Snail sense or antisense cRNA probes. Black dotted lines demarcate the basement membrane that separates the epidermis (epi) from dermis (der). Arrows point to Snail RNA expression, restricted to the hair bud stage of follicle morphogenesis. It was not seen in later hair germ or peg stages. + +(B) Expression of Snail protein coincides with hair development. Protein extracts were prepared from keratinocytes transfected with empty expression vector (K14), containing the K14 promoter or with the vector driving HA-tagged Snail (K14-Snail); or from whole skin from E13.5 to P5 animals, including newborn (nb). Equal amounts of proteins were then resolved by SDS-PAGE through 12% gels and subjected to Western blotting using either an affinity-purified Snail polyclonal antiserum, which we generated, or anti-tubulin (loading control). + +(C–E) Immunohistochemistry shows expression of Snail protein in the nuclei of cells within the hair and skin. (C) E13.5 skin with a single layered epidermis (epi) shows no Snail expression. (D) The first morphological sign that cells have adopted a hair follicle fate is a cluster of cells called a placode in E16.5 skin. Snail is not expressed at this stage of development. (E) Snail is expressed in the hair bud of E17.5 skin but not in later stages of development such as the germ or peg. + +(F) Immunofluorescence with anti-Ki67 (green) identifies the proliferating cells of the skin, restricted to the basal layer of the epidermis and developing hair follicles. Anti-β4 int labeling reveals the presence of the hemidesmosomal integrin β4, restricted to the base of cells adhering to the underlying basement membrane. The white dotted line marks the outermost surface of the skin. + +(G) Immunohistochemistry with pMAPK marks a subset of proliferating cells within the epidermis and hair bud. Anti-pMAPK labeling was consistently robust within the hair bud. + +(H) Immunofluorescence with anti-laminin 5 (lam5), which demarcates the basement membrane, and anti-E-cadherin (E-cad), a component of AJs. At the leading edge of the growing bud, cell-cell borders show markedly diminished anti-E-cadherin labeling (arrowheads). + +Figure 2 + +Misexpression of Snail in Mouse Skin Epidermis Results in Hyperproliferation + +Three different surviving Tg founder mice harbored a K14-Snail transgene that was integrated into a locus that resulted in inheritable, mosaic expression of the transgene in skin epidermis. All displayed similar abnormalities, as did their offspring. + +(A) P16 WT and K14-Snail Tg mice. Insets denote magnified tail segments, which displayed a mosaic, flaky appearance in Tg mice. Size differences appeared with age, and are likely due to K14-promoter activity in the tongue and oral epithelium, resulting in progressive defects and reduced food intake. Hence, skin sections from young (P3) mice were analyzed (B–I). + +(B) Hematoxylin- and eosin-stained Tg skin section. Double arrows demarcate the border of mosaic histology, with seemingly normal epidermis (epi) and a mature hair follicle (hf) at left and hyperthickened epidermis at right. + +(C) Immunofluorescence of Tg skin section labeled with antibodies as color-coded on frame. Double arrows demarcate the border of mosaic anti-Snail (green), revealing Snail expression coincident with regions of hyperthickened epidermis (at left) and absent in regions of normal epidermis (at right). + +(D–I) Sections of P3 WT or Tg skin (affected region) subjected to either immunofluorescence (D, E, H, and I) or immunohistochemistry (F and G) with antibodies as indicated on the panel. Anti-keratin 5 indicates K5, normally restricted to the basal layer of the epidermis; anti-keratin 6 detects keratin 6, expressed in postnatal epidermis under conditions such as wounding, in which hyperproliferation occurs. All other antibodies are as in the legend to Figure 2. Comparison of D and E provide representative examples that illustrate that pMAPK is found in only a subset of all proliferating (Ki67-positive) cells. Note also the presence of Ki67- (E) and pMAPK-positive (G) cells in some suprabasal areas; Ki67-positive cells colabeled with anti-Snail (E). + +Figure 3 + +Alterations in the Differentiation Program and Basement Membrane Organization in Snail-Expressing Tg Epidermis + +(A–H) Immunofluorescence of skin sections from P3 WT and Tg mice. Shown are affected areas of Tg skin; in areas where Snail protein was not expressed, stainings were normal. Sections were labeled with antibodies as indicated and color-coded on each frame. Antibodies are against markers of normal epidermal differentiation, and include K5 (a basally expressed keratin), K1 (a suprabasal keratin, expressed in spinous layer cells), involucrin (Inv; a suprabasally expressed cornified envelope protein found in upper spinous and granular layer cells), loricrin (Lor; a cornified envelope protein expressed in the granular layer), and filaggrin (Fil; a protein that bundles keratin filaments in the granular layer and stratum corneum). Note abnormal extension of anti-K5 suprabasally, often present in anti-K1 positive suprabasal Tg cells. + +(I–N) Immunohistochemistry (I and J) or immunofluorescence (K–N) of sections of P30 Wt (I, K, and M) and Tg (J, L, and N) (affected areas) skins using the antibodies indicated. Note that with age, affected areas of the Tg epidermis became increasingly undulating, often exhibiting papilloma-like invaginations (J). Insets in I and J are magnified views of the boxed areas, illustrating the absence (Wt) or presence (Tg) of nuclear anti-cyclin D staining. With age, affected areas of the Tg epidermis also displayed perturbations within the basement membrane, as judged by antibody labeling against either basement membrane (K and L) or hemidesmosomal (M and N) components. Double arrows in L demarcate mosaic zones, revealing that perturbations were restricted to hyperthickened, i.e., Snail-positive zones (to left of double arrows). Other abbreviations are as noted in the legend to Figure 2. + +Figure 4 + +Snail-Mediated Remodeling of AJs Contributes to Hyperproliferation + +(A) Immunofluorescence of skin sections from P30 Wt and Tg mice. Shown are affected areas of Tg skin; in areas where Snail protein was not expressed, stainings were normal. Antibodies used are against AJ proteins and include E-cadherin (E-cad), the transmembrane core protein; β-catenin (β-cat), which binds E-cadherin at AJs and which can also participate as a transcription cofactor when associated with LEF-1/TCF proteins in the nucleus; α-catenin (α-cat) which binds to both β-catenin and Ajuba, a close relative of zyxin; and Ajuba, which can associate with proteins that bind to the actin cytoskeleton, as well as with Grb-2, a mediator of the GTP nucleotide-exchange protein Sos, involved in activation of the Ras-MAPK signaling cascade. In Snail-expressing Tg regions, there was a reduced staining with anti-E-cad and anti-α-cat and a more diffuse staining with anti-Ajuba. Insets in the panels for β-catenin and Ajuba staining are magnified views of the boxed areas. Arrows mark membrane localization of the protein and asterisks mark cells with elevated levels of cytoplasmic β-catenin or Ajuba. + +(B) Western blot analyses of protein extracts from P30 Wt and Tg back and ear skins. Antibodies are as in (A) except anti-P-cad, which detects P-cadherin, whose expression in the hair follicle was not affected, and anti-tubulin, which detects tubulin, a control for equal protein loadings. Note that the reductions seen in E-cadherin and α-catenin are likely to be underestimates of the actual differences in affected regions, since the Tg skin expressed Snail mosaically. + +(C) In the presence of elevated Snail, α-catenin levels can be restored by overexpression of E-cadherin. Keratinocytes were transfected with either HA-tagged Snail (Snail[HA]; images on the left) or Snail(HA) and Ecad(HA) (images on the right). 2 d after transfection, cells were switched from low-calcium growth medium to high-calcium medium for 6 h to induce AJ formation. Cells were stained with antibodies as indicated on the panels. Arrowheads point to sites of intercellular contact between a Snail-transfected keratinocyte and its neighboring untransfected cell. + +(D) Reintroduction of E-cadherin in keratinocytes expressing Snail returns pMAPK to basal levels. Keratinocytes were transfected with control vector (K14), or Snail(HA), or Snail(HA) + E-cad(HA). After 2 d, cells were serum starved for 4 h and whole cell lysates were made and Western blotted with antibodies to pMAPK, HA to recognize the HA-tagged Snail and E-cadherin protein, 20or tubulin as a loading control. + +(E) Ajuba interacts with Grb-2 under conditions where α-catenin levels are reduced. Protein extracts were made from skins of P30 Wt and K14-Snail Tg P30 mice (blots on the left) and of newborn Wt and K14-Cre/α-catenin (fl/fl) conditionally null animals (blots on the right) [7]. Equal amounts of protein extracts were treated with anti-Grb-2 antibody (+) or control isotype antibody (–), and following centrifugation, immunoprecipitates were subjected to SDS-PAGE and Western blot analysis with anti-Ajuba and anti-Grb-2 antibodies. Note the presence of Ajuba only under conditions where levels of α-catenin and other AJ proteins were aberrantly low or absent. + +(F) Transgene expression of excess Ajuba or the Grb-2-interacting domain (pre-LIM) of Ajuba in keratinocytes results in the activation of the Ras-MAPK pathway. Primary newborn mouse keratinocytes were transfected with either the empty K14 expression vector (K14), or the expression vector driving Snail, full length Ajuba, or the pre-LIM domain of Ajuba in the absence or presence of a peptide inhibitor (inh) that disrupts the interaction between Grb-2 and Sos. 48 h posttransfection, protein extracts were prepared and subjected to SDS-PAGE and Western blot analyses with antibodies against pMAPK, total MAPK, Ajuba (also recognizing the smaller, pre-LIM domain), and Snail. + +Figure 5 + +TGF-β2, but Not Wnt/noggin, Transiently Induces Snail Expression In Vitro + +(A) Failure of Wnt and noggin signaling to induce Snail in cultured keratinocytes. Primary mouse keratinocytes were treated with Wnt- and/or noggin-conditioned medium (+) or the corresponding control medium (–). These conditions are known to activate the LEF-1/β-catenin reporter TOPGal and down-regulate the E-cadherin promoter (see [4] for details). Using Western blot analyses, cellular proteins were then analyzed for Snail, LEF-1, β-catenin, and tubulin. Proteins from keratinocytes transfected with K14-Snail were used as a positive control for Snail expression. + +(B) TGF-β2 can induce Snail protein. Primary keratinocytes were treated for the indicated times with recombinant TGF-β2 (+) or heat inactivated TGF-β2 (–).Total cellular proteins were then isolated and analyzed by Western blot for Snail, pSMAD2 (reflective of activated TGF- signaling), and tubulin. Note the activation of Snail expression, peaking at 2 h post-TGF-β2 treatment and then disappearing thereafter. + +(C) Snail mRNA expression is transiently induced by TGF-β2. The experiment in (B) was repeated, and this time, total RNAs were isolated from keratinocytes treated with TGF-β2 for the indicated times. RT-PCR was then used with (+) or without (–) reverse transcriptase (RT) and with primer sets specific for Snail and GAPDH mRNAs. Note that Snail mRNA expression also peaked at 2 h, paralleling Snail protein. + +(D) TGF-β2 treatment results in enhanced activity of a Snail promoter-β-galactosidase reporter. Keratinocytes were transfected with a β-galactosidase reporter driven by a Snail promoter that is either WT (wt prom) or harbors a mutation in a putative pSMAD2/pSMAD4 binding site (mt prom). At 2 d posttransfection, cells were treated with either TGF-β or heat-inactivated TGF-β2 (inact) for the times indicated. β-galactosidase assays were then conducted, and results are reported as fold increase over a basal level of activity of 1. The experiment was repeated three times in triplicate, and error bars reflect variations in the results. + +Figure 6 + +TGF-β2 Is Necessary to Induce Snail Expression and Regulate Proliferation and E-Cadherin in the Hair Bud + +(A–D) Skins from TGF-β2 WT or KO E17.5 embryos were analyzed for expression of TGF-β2 protein (A), which is present in the epidermis and dermis as previously described [33] and in the hair bud, pSMAD2 (B), Snail (C), and Snail mRNA (D). Arrows point to the hair buds. + +(E) Western blot analyses of Snail expression in the skins of 2-wk-old K14-Smad2 transgenic (SMAD2 TG) and WT littermate (WT) mice. Antibody to tubulin was used as a control for equal protein loadings. The K14-Smad2 Tg mouse was previously shown to possess activated TGF-β signaling [35]. + +(F–G) Proliferation markers Ki67 (F) and pMAPK (G) are diminished in TGF-β2-null hair relative to its WT counterpart. + +(H–J) TGF-β2-null hair fails to down-regulate E-cadherin (H). Wnt and noggin signaling pathways are still intact in the TGF-β2 null hair as nuclear LEF-1 (I) and nuclear β-catenin (J) are still expressed. + +Footnotes + +Author contributions. CJ and EF conceived and designed the experiments. CJ, PL, and PK performed the experiments. CJ and EF analyzed the data. MA, RH, YC, and EF contributed reagents/materials/analysis tools. CJ and EF wrote the paper. + +Note Added in Proof + +Our results are particularly interesting in light of the recent implication that GSK-3β controls Snail's stability and subcellular localization [50]. Since Wnts are known to deactivate GSK-3β, Wnt and TGF-β2 signaling may contribute to Snail's transient induction and accumulation. Moreover, since inhibition of GSK-3β results in Snail upregulation and E-cadherin downregulation, Snail and GSK-3β may function at a crossroads in controlling hair bud development. + +Citation: Jamora C, Lee P, Kocieniewski P, Azhar M, Hosokawa R, et al. (2004) A signaling pathway involving TGF-β2 and Snail in hair follicle morphogenesis. 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expressed +T64 CL:0000210 2052 2066 photoreceptors +T65 UBERON:0007023 2115 2120 adult +T66 PR:000005904 2133 2136 Crx +T67 SO:0000704 2137 2141 gene +T68 GO:0010467 2137 2152 gene expression +T69 PR:000012086 2182 2186 Otx2 +T70 GO:0010467 2240 2249 expressed +T71 CL:0000210 2259 2278 photoreceptor cells +T72 CL:0000604 2287 2305 rod photoreceptors +T73 PR:000005904 2307 2310 Crx +T74 PR:000011432 2343 2346 Nrl +T75 GO:0010467 2404 2414 expression +T76 UBERON:0000966 2422 2428 retina +T77 CL:0000604 2432 2450 rod photoreceptors +T78 CL:0000210 2461 2474 photoreceptor +T79 SO:0000704 2484 2489 genes +T80 PR:000005904 2504 2507 Crx +T81 SO:0000409 2508 2524 binding elements +T82 GO:0065007 2534 2544 regulatory +T83 SO:0005836 2534 2552 regulatory regions +T84 PR:000001245 2569 2578 rhodopsin +T85 PR:000005904 2616 2619 Crx +T86 NCBITaxon:9606 2654 2659 human +T87 http://purl.obolibrary.org/obo/MONDO_0000001 2660 2668 diseases +T88 http://purl.obolibrary.org/obo/MONDO_0001941 2682 2691 blindness +T89 CL:0000573 2703 2707 cone +T90 http://purl.obolibrary.org/obo/MONDO_0007362 2703 2723 cone-rod dystrophy 2 +T91 CL:0000604 2708 2711 rod +T92 http://purl.obolibrary.org/obo/MONDO_0019200 2735 2755 retinitis pigmentosa +T93 http://purl.obolibrary.org/obo/MONDO_0018998 2766 2769 LCA +T94 PR:000005904 2800 2803 Crx +T95 GO:0030154 2852 2867;2885 2887;2902 2907 differentiation ... of ... cells +T96 CL:0000210 2888 2907 photoreceptor cells +T97 http://purl.obolibrary.org/obo/MONDO_0018998 2916 2919 LCA +T98 SO:0000704 2939 2946 genetic +T99 http://purl.obolibrary.org/obo/MONDO_0003847 2939 2954 genetic disease +T100 CL:0000210 2958 2972 photoreceptors +T101 GO:0007601 3068 3074 vision +T102 GO:0007567 3080 3085 birth +T103 UBERON:0000966 3100 3107 retinal +T104 SO:0000704 3117 3122 genes +T105 PR:000005904 3132 3135 Crx +T106 http://purl.obolibrary.org/obo/MONDO_0018998 3163 3166 LCA +T107 PR:000008353 3187 3193 GUCY2D +T108 PR:000014179 3200 3205 RPE65 +T109 PR:000003876 3212 3218 AIPL-1 +T110 PR:000005848 3225 3230 CRB-1 +T111 PR:000014182 3241 3249 RPGRIP-1 +T112 SO:0000018 3302 3325 genetically linked loci +T113 SO:0000704 3332 3337 genes +T114 PR:000005904 3369 3372 Crx +T115 http://purl.obolibrary.org/obo/MONDO_0018998 3386 3389 LCA +T116 PR:000005904 3498 3501 Crx +T117 SO:0001023 3572 3578 allele +T118 http://purl.obolibrary.org/obo/MONDO_0018998 3688 3691 LCA +T119 http://purl.obolibrary.org/obo/MONDO_0000001 3736 3743 disease +T120 NCBITaxon:9606 3818 3823 human +T121 http://purl.obolibrary.org/obo/MONDO_0018998 3824 3827 LCA +T122 UBERON:0010230 3892 3898 globes +T123 UBERON:0007023 3902 3908 adults +T124 http://purl.obolibrary.org/obo/MONDO_0018998 3914 3917 LCA +T125 UBERON:0000479 3929 3935 tissue +T126 UBERON:0000970 4017 4020 eye +T127 NCBITaxon:33208 4053 4059 Animal +T128 http://purl.obolibrary.org/obo/MONDO_0018998 4071 4074 LCA +T129 http://purl.obolibrary.org/obo/MONDO_0000001 4181 4188 disease +T130 http://purl.obolibrary.org/obo/MONDO_0018998 4204 4207 LCA +T131 SO:0000704 4228 4239 genetically +T132 http://purl.obolibrary.org/obo/MONDO_0000001 4254 4262 disorder +T133 NCBITaxon:10088 4275 4280 mouse +T134 CL:0000210 4357 4370 photoreceptor +T135 GO:0042461 4357 4382 photoreceptor development +T136 http://purl.obolibrary.org/obo/MONDO_0000001 4443 4450 disease +T137 CL:0000210 4452 4471 photoreceptor cells +T138 CL:0000540 4515 4523 neuronal +T139 CL:0000210 4541 4560 Photoreceptor cells +T140 GO:0045177 4592 4596 apex +T141 CL:0000540 4604 4611 neurons +T142 GO:0016020 4619 4629 membranous +T143 GO:0001750 4630 4643 outer segment +T144 GO:0007602 4685 4710 phototransduction cascade +T145 PR:000001245 4742 4751 rhodopsin +T146 GO:0001750 4833 4847 outer segments +T147 CL:0000210 4873 4887 photoreceptors +T148 GO:0045202 4903 4911 synaptic +T149 GO:0042995 4947 4959;4983 4988 processes of ... cells +T150 CL:0000745 4960 4970;4983 4988 horizontal ... cells +T151 CL:0000103 4975 4988 bipolar cells +T152 CL:0000604 4998 5001 Rod +T153 GO:0044317 4998 5011 Rod spherules +T154 GO:0045202 5038 5045 synapse +T155 CL:0000604 5051 5054 rod +T156 GO:0030425 5063 5072 dendrites +T157 GO:0043679 5077 5091 axonal endings +T158 CL:0000745 5095 5111 horizontal cells +T159 GO:0045202 5118 5125 synapse +T160 GO:0098793 5178 5189 presynaptic +T161 GO:0005737 5190 5199 cytoplasm +T162 CL:0000573 5201 5205 Cone +T163 GO:0044316 5201 5214 Cone pedicles +T164 GO:0045202 5233 5241 synapses +T165 GO:0030425 5251 5260 dendrites +T166 CL:0000749 5264 5266;5272 5285 on ... bipolar cells +T167 CL:0000573 5267 5271 cone +T168 CL:0000745 5290 5306 horizontal cells +T169 GO:0030425 5336 5345 dendrites +T170 CL:0000750 5349 5352;5358 5371 off ... bipolar cells +T171 CL:0000573 5353 5357 cone +T172 GO:0065007 5385 5395 regulating +T173 GO:0007416 5400 5412;5431 5439 formation of ... synapses +T174 CL:0000210 5417 5430 photoreceptor +T175 GO:0045202 5431 5439 synapses +T176 CL:0000210 5477 5490 photoreceptor +T177 GO:0045202 5491 5499 synaptic +T178 PR:000017084 5509 5513 HRG4 +T179 PR:000005904 5536 5539 Crx +T180 SO:0001679 5563 5598 transcriptional regulatory sequence +T181 GO:0065007 5579 5589 regulatory +T182 http://purl.obolibrary.org/obo/MONDO_0018998 5621 5624 LCA +T183 NCBITaxon:33208 5625 5631 animal +T184 UBERON:0000966 5674 5681 retinal +T185 CL:0000540 5742 5750 neuronal +T186 GO:0045202 5776 5783 synapse +T187 GO:0007416 5776 5793 synapse formation +T188 http://purl.obolibrary.org/obo/MONDO_0018998 5904 5907 LCA +T189 UBERON:0000966 5977 5983 retina +T190 PR:000005904 5987 5990 Crx +T191 NCBITaxon:10088 5994 5998 mice +T192 UBERON:0000966 6035 6042 retinas +T193 PR:000005904 6078 6081 Crx +T194 CL:0000210 6085 6099 photoreceptors +T195 GO:0001750 6132 6145 outer segment +T196 GO:0045202 6219 6227 synaptic +T197 PR:000005904 6275 6278 Crx +T198 CL:0000210 6282 6295 photoreceptor +T199 GO:0007416 6296 6310 synaptogenesis +T200 GO:0045202 6381 6388 synapse +T201 GO:0007416 6381 6398 synapse formation +T202 http://purl.obolibrary.org/obo/MONDO_0018998 6402 6405 LCA +T203 GO:0001750 6445 6458 outer segment +T204 UBERON:0003926 6445 6464 outer segment layer +T205 PR:000005904 6468 6471 Crx +T206 NCBITaxon:10088 6475 6479 mice +T207 NCBITaxon:10088 6532 6536 mice +T208 PR:000005904 6565 6568 Crx +T209 PR:000005904 6618 6621 Crx +T210 NCBITaxon:10088 6625 6629 mice +T211 PR:000005904 6724 6727 Crx +T212 CL:0000210 6731 6744 photoreceptor +T213 GO:0008594 6731 6758 photoreceptor morphogenesis +T214 UBERON:0000966 6770 6777 retinae +T215 PR:000005904 6783 6786 Crx +T216 NCBITaxon:10088 6790 6794 mice +T217 CHEBI:10545 6828 6836 electron +T218 GO:0007567 6856 6861 natal +T219 PR:000005904 6881 6884 Crx +T220 CL:0000210 6888 6902 photoreceptors +T221 GO:0001750 6920 6934 outer segments +T222 PR:000005904 6952 6955 Crx +T223 UBERON:0000966 6959 6966 retinas +T224 GO:0001750 6995 7008 outer segment +T225 UBERON:0003926 6995 7014 outer segment layer +T226 PR:000005904 7028 7031 Crx +T227 CL:0000210 7035 7049 photoreceptors +T228 GO:0001917 7054 7068 inner segments +T229 CL:0000210 7101 7114 photoreceptor +T230 PR:000005904 7135 7138 Crx +T231 GO:0001917 7155 7169 inner segments +T232 GO:0001917 7232 7246 inner segments +T233 GO:0005739 7319 7331 mitochondria +T234 GO:0001917 7382 7395 inner segment +T235 GO:0019835 7407 7412 lysis +T236 GO:0005773 7450 7458 vacuoles +T237 GO:0005739 7471 7483 mitochondria +T238 CHEBI:10545 7526 7534 electron +T239 UBERON:0000966 7559 7565 retina +T240 PR:000005904 7580 7583 Crx +T241 PR:000005904 7595 7598 Crx +T242 UBERON:0000966 7602 7609 retinas +T243 CHEBI:26130 7615 7624 pigmented +T244 UBERON:0000483 7625 7635 epithelium +T245 GO:0001750 7637 7639 os +T246 GO:0001750 7641 7655 outer segments +T247 GO:0001917 7657 7659 is +T248 GO:0001917 7661 7675 inner segments +T249 UBERON:0001789 7677 7680 onl +T250 UBERON:0001789 7682 7701 outer nuclear layer +T251 GO:0005634 7688 7695 nuclear +T252 CL:0000210 7707 7720 photoreceptor +T253 GO:0005634 7721 7727 nuclei +T254 CHEBI:10545 7783 7791 electron +T255 GO:0001750 7810 7823 outer segment +T256 UBERON:0003926 7810 7829 outer segment layer +T257 PR:000005904 7833 7836 Crx +T258 UBERON:0000966 7840 7846 retina +T259 GO:0001917 7855 7872;7880 7894 Inner segments of ... photoreceptors +T260 PR:000005904 7873 7876 Crx +T261 CL:0000210 7880 7894 photoreceptors +T262 GO:0005739 7912 7924 mitochondria +T263 GO:0005739 7926 7927 m +T264 PR:000005904 7954 7957 Crx +T265 CHEBI:26130 7978 7987 pigmented +T266 UBERON:0000483 7988 7998 epithelium +T267 GO:0001917 8000 8002 is +T268 GO:0001917 8004 8018 inner segments +T269 UBERON:0001789 8020 8023 onl +T270 UBERON:0001789 8025 8044 outer nuclear layer +T271 GO:0005634 8031 8038 nuclear +T272 CL:0000210 8065 8078 Photoreceptor +T273 GO:0001917 8065 8093 Photoreceptor inner segments +T274 GO:0001750 8065 8078;8098 8112 Photoreceptor ... outer segments +T275 GO:0032391 8129 8157 non-motile connecting cilium +T276 GO:0005874 8210 8221 microtubule +T277 PR:000005904 8272 8275 Crx +T278 UBERON:0000966 8279 8286 retinas +T279 GO:0032391 8315 8331 connecting cilia +T280 GO:0032391 8377 8393 connecting cilia +T281 GO:0005874 8441 8452 microtubule +T282 GO:0032391 8494 8511 connecting cilium +T283 GO:0001917 8712 8725 inner segment +T284 GO:0042384 8737 8749 ciliogenesis +T285 PR:000005904 8776 8779 Crx +T286 CL:0000210 8783 8797 photoreceptors +T287 PR:000005904 8811 8814 Crx +T288 UBERON:0000966 8818 8825 retinas +T289 UBERON:0001782 8830 8858 retinal pigmented epithelium +T290 CHEBI:26130 8838 8847 pigmented +T291 CHEBI:10545 8967 8975 electron +T292 PR:000005904 8990 8993 Crx +T293 UBERON:0000966 8997 9003 retina +T294 CHEBI:10545 9035 9043 electron +T295 PR:000005904 9058 9061 Crx +T296 GO:0001750 9079 9092 outer segment +T297 UBERON:0003926 9079 9098 outer segment layer +T298 GO:0042384 9116 9128 ciliogenesis +T299 CL:0000210 9136 9149 photoreceptor +T300 UBERON:0001787 9136 9158;9166 9172 photoreceptor layer of ... retina +T301 PR:000005904 9159 9162 Crx +T302 GO:0032391 9174 9200 Nonmotile connecting cilia +T303 GO:0032391 9271 9288 Connecting cilium +T304 GO:0005874 9324 9334 microtuble +T305 GO:0005929 9351 9357 cilium +T306 GO:0005929 9364 9370 cilium +T307 GO:0005634 9407 9419 cell nucleus +T308 GO:0005634 9421 9422 n +T309 GO:0030263 9434 9442 pyknotic +T310 GO:0031012 9470 9476 matrix +T311 GO:0031012 9478 9480 mx +T312 GO:0031988 9550 9569 membranous vesicles +T313 CL:0000210 9595 9608 photoreceptor +T314 GO:0001917 9648 9662 inner segments +T315 GO:0032391 9688 9715 Nonmotile connecting cilium +T316 PR:000005904 9741 9744 Crx +T317 CL:0000210 9748 9761 photoreceptor +T318 GO:0005874 9812 9823 microtubule +T319 CHEBI:10545 9866 9874 electron +T320 GO:0031988 9895 9914 membranous vesicles +T321 GO:0001917 9951 9965 inner segments +T322 PR:000005904 9969 9972 Crx +T323 CL:0000210 9976 9990 photoreceptors +T324 GO:0001917 10012 10026 inner segments +T325 UBERON:0001773 10043 10050 scleral +T326 CHEBI:26130 10065 10074 pigmented +T327 UBERON:0000483 10075 10085 epithelium +T328 GO:0001750 10153 10167 outer segments +T329 PR:000005904 10169 10172 Crx +T330 UBERON:0000966 10176 10183 retinas +T331 GO:0001750 10233 10246 outer segment +T332 UBERON:0003926 10233 10252 outer segment layer +T333 GO:0031012 10287 10293 matrix +T334 GO:0005634 10372 10378 nuclei +T335 GO:0005634 10449 10455 nuclei +T336 GO:0030263 10465 10473 pyknotic +T337 GO:0000792 10525 10540 heterochromatin +T338 CL:0000210 10560 10574 photoreceptors +T339 CL:0000210 10643 10657 photoreceptors +T340 GO:0001750 10702 10715 outer segment +T341 UBERON:0003926 10702 10721 outer segment layer +T342 GO:0031982 10742 10750 vesicles +T343 GO:0001917 10834 10848 inner segments +T344 CHEBI:10545 10862 10870 electron +T345 GO:0001917 10935 10949 inner segments +T346 GO:0008594 11008 11024;11032 11046 morphogenesis of ... photoreceptors +T347 PR:000005904 11025 11028 Crx +T348 CL:0000210 11032 11046 photoreceptors +T349 GO:0001750 11063 11076 outer segment +T350 UBERON:0003926 11063 11082 outer segment layer +T351 CHEBI:10545 11106 11114 electron +T352 PR:000005904 11160 11163 Crx +T353 UBERON:0000966 11167 11174 retinas +T354 CL:0000210 11176 11189 photoreceptor +T355 GO:0001917 11176 11204 photoreceptor inner segments +T356 GO:0032391 11206 11222 connecting cilia +T357 GO:0001750 11250 11263 outer segment +T358 PR:000005904 11300 11303 Crx +T359 UBERON:0000966 11307 11313 retina +T360 GO:0032391 11335 11352 connecting cilium +T361 GO:0001917 11377 11391 inner segments +T362 CHEBI:10545 11489 11497 electron +T363 PR:000005904 11583 11586 Crx +T364 PR:000005904 11594 11597 Crx +T365 CL:0000210 11601 11615 photoreceptors +T366 GO:0001750 11636 11650 outer segments +T367 PR:000005904 11665 11668 Crx +T368 CL:0000210 11672 11686 photoreceptors +T369 GO:0045177 11748 11752 apex +T370 PR:000005904 11773 11776 Crx +T371 UBERON:0000966 11780 11787 retinae +T372 CL:0000210 11810 11824 photoreceptors +T373 GO:0032391 11852 11868 connecting cilia +T374 GO:0001750 11877 11891 outer segments +T375 PR:000005904 11923 11926 Crx +T376 CL:0000210 11930 11944 photoreceptors +T377 GO:0032391 11999 12016 connecting cilium +T378 GO:0001750 12067 12080 outer segment +T379 GO:0001750 12167 12181 outer segments +T380 PR:000005904 12208 12211 Crx +T381 PR:000005904 12277 12280 Crx +T382 CL:0000210 12284 12298 photoreceptors +T383 GO:0032391 12315 12331 connecting cilia +T384 PR:000005904 12382 12385 Crx +T385 CL:0000210 12389 12403 photoreceptors +T386 GO:0001750 12597 12610 outer segment +T387 PR:000005904 12628 12631 Crx +T388 NCBITaxon:10088 12639 12644 mouse +T389 CL:0000210 12683 12696 photoreceptor +T390 GO:0042995 12697 12706 appendage +T391 GO:0001750 12734 12747 outer segment +T392 GO:0001750 12770 12783 Outer segment +T393 GO:0009653 12784 12797 morphogenesis +T394 PR:000005904 12801 12804 Crx +T395 CL:0000210 12808 12822 photoreceptors +T396 CHEBI:10545 12833 12841 electron +T397 CL:0000210 12867 12881 photoreceptors +T398 UBERON:0001773 12898 12905 scleral +T399 CHEBI:26130 12920 12929 pigmented +T400 UBERON:0000483 12930 12940 epithelium +T401 PR:000005904 12973 12976 Crx +T402 PR:000005904 12998 13001 Crx +T403 PR:000005904 13031 13034 Crx +T404 UBERON:0000966 13038 13044 retina +T405 GO:0032391 13055 13071 connecting cilia +T406 GO:0032391 13076 13078 cc +T407 GO:0001750 13116 13130 outer segments +T408 PR:000005904 13145 13148 Crx +T409 GO:0001750 13152 13165 outer segment +T410 GO:0001750 13196 13210 outer segments +T411 GO:0001750 13244 13246 os +T412 GO:0001750 13272 13274 os +T413 PR:000005904 13280 13283 Crx +T414 CL:0000210 13287 13301 photoreceptors +T415 GO:0032391 13307 13323 connecting cilia +T416 GO:0032391 13363 13379 connecting cilia +T417 GO:0001750 13428 13441 outer segment +T418 GO:0032391 13469 13486 connecting cilium +T419 GO:0032391 13497 13499 cc +T420 PR:000005904 13533 13536 Crx +T421 GO:0032391 13590 13606 connecting cilia +T422 PR:000005904 13620 13623 Crx +T423 GO:0032391 13631 13633 cc +T424 CHEBI:10545 13679 13687 electron +T425 PR:000005904 13703 13706 Crx +T426 CL:0000210 13710 13724 photoreceptors +T427 CL:0000210 13732 13745 photoreceptor +T428 UBERON:0001787 13732 13751 photoreceptor layer +T429 CL:0000210 13763 13776 Photoreceptor +T430 UBERON:0001787 13763 13785;13793 13799 Photoreceptor layer of ... retina +T431 PR:000005904 13786 13789 Crx +T432 GO:0001750 13822 13835 outer segment +T433 CL:0000210 13869 13882 photoreceptor +T434 GO:0032391 13869 13899 photoreceptor connecting cilia +T435 GO:0032391 13901 13903 cc +T436 PR:000005904 13910 13913 Crx +T437 CL:0000210 13917 13931 photoreceptors +T438 GO:0032391 13942 13958 connecting cilia +T439 GO:0032391 13960 13962 cc +T440 GO:0001750 14002 14015 outer segment +T441 PR:000005904 14106 14109 Crx +T442 CL:0000210 14113 14127 photoreceptors +T443 PR:000001245 14144 14153 rhodopsin +T444 CL:0000210 14175 14189 photoreceptors +T445 PR:000001245 14191 14200 Rhodopsin +T446 CL:0000210 14224 14237 photoreceptor +T447 SO:0000704 14247 14252 genes +T448 GO:0010467 14259 14269 expression +T449 PR:000005904 14308 14311 Crx +T450 UBERON:0000966 14315 14322 retinae +T451 SO:0000704 14379 14384 genes +T452 GO:0001750 14451 14465 outer segments +T453 CL:0000210 14475 14489 Photoreceptors +T454 NCBITaxon:10088 14512 14516 mice +T455 UBERON:0001773 14542 14549 scleral +T456 PR:000005904 14582 14585 Crx +T457 CL:0000210 14589 14603 photoreceptors +T458 CHEBI:10545 14665 14673 electron +T459 PR:000001245 14710 14719 rhodopsin +T460 CL:0000210 14727 14741 photoreceptors +T461 UBERON:0001773 14766 14773 scleral +T462 CHEBI:26130 14788 14797 pigmented +T463 UBERON:0000483 14798 14808 epithelium +T464 GO:0032391 14818 14820 cc +T465 GO:0032391 14822 14839 connecting cilium +T466 GO:0001917 14841 14843 is +T467 GO:0001917 14845 14858 inner segment +T468 PR:000005904 14880 14883 Crx +T469 CL:0000210 14918 14931 photoreceptor +T470 GO:0010467 14992 15002 expression +T471 SO:0001023 15026 15032 allele +T472 PR:000005904 15036 15039 Crx +T473 CL:0000604 15054 15058 rods +T474 CL:0000604 15085 15088 rod +T475 GO:0044317 15085 15098 rod spherules +T476 UBERON:0001790 15106 15127 outer plexiform layer +T477 UBERON:0001790 15129 15132 OPL +T478 GO:0001750 15138 15152 outer segments +T479 CL:0000210 15208 15221 photoreceptor +T480 GO:0045202 15222 15230 synapses +T481 PR:000005904 15247 15250 Crx +T482 UBERON:0000966 15254 15261 retinas +T483 PR:000005904 15266 15269 Crx +T484 UBERON:0000966 15273 15280 retinas +T485 CL:0000604 15302 15305 rod +T486 GO:0044317 15302 15315 rod spherules +T487 GO:0044317 15347 15355 sperules +T488 GO:0045202 15452 15459 synapse +T489 GO:0042995 15524 15538;15550 15555 processes from ... cells +T490 CL:0000745 15539 15555 horizontal cells +T491 GO:0045202 15592 15600 synaptic +T492 GO:0030425 15647 15656 dendrites +T493 CL:0000751 15660 15677 rod bipolar cells +T494 CL:0000573 15739 15743 Cone +T495 GO:0044316 15770 15778 pedicles +T496 GO:0045202 15810 15818 synapses +T497 CL:0000745 15847 15857;15870 15874 horizontal ... cell +T498 CL:0000103 15862 15874 bipolar cell +T499 GO:0042995 15870 15884 cell processes +T500 GO:0044316 15891 15898 pedicle +T501 GO:0044317 15969 15978 spherules +T502 PR:000005904 15982 15985 Crx +T503 UBERON:0000966 15989 15996 retinas +T504 UBERON:0001790 16028 16034;16042 16049 OPL of ... retinas +T505 PR:000005904 16035 16038 Crx +T506 CL:0000210 16051 16064 photoreceptor +T507 GO:0042995 16128 16137 Processes +T508 GO:0008021 16149 16166 synaptic vesicles +T509 GO:0044456 16251 16269 synapse components +T510 GO:0044317 16292 16301 spherules +T511 GO:0044316 16306 16314 pedicles +T512 GO:0005886 16441 16456 plasma membrane +T513 GO:0005634 16481 16488 nuclear +T514 CHEBI:10545 16536 16544 electron +T515 UBERON:0001790 16564 16585 outer plexiform layer +T516 PR:000005904 16589 16592 Crx +T517 UBERON:0000966 16596 16603 retinas +T518 PR:000005904 16612 16615 Crx +T519 UBERON:0000966 16619 16625 retina +T520 CL:0000604 16647 16650 rod +T521 GO:0044317 16647 16660 rod spherules +T522 GO:0044317 16713 16722 spherules +T523 GO:0045202 16776 16783 synapse +T524 CL:0000573 16809 16813 Cone +T525 GO:0044316 16841 16849 pedicles +T526 GO:0045202 16881 16889 synapses +T527 PR:000005904 16973 16976 Crx +T528 UBERON:0001790 17028 17049;17056 17058;17066 17073 outer plexiform layer ... of ... retinas +T529 UBERON:0001790 17051 17054;17056 17058;17066 17073 OPL ... of ... retinas +T530 PR:000005904 17059 17062 Crx +T531 CL:0000210 17075 17088 photoreceptor +T532 GO:0044316 17157 17165 pedicles +T533 GO:0044317 17170 17179 spherules +T534 GO:0044456 17308 17326 synapse components +T535 UBERON:0001790 17532 17535 opl +T536 UBERON:0001790 17537 17558 outer plexiform layer +T537 CHEBI:10545 17602 17610 electron +T538 UBERON:0001790 17630 17651 outer plexiform layer +T539 PR:000005904 17655 17658 Crx +T540 UBERON:0000966 17662 17669 retinas +T541 PR:000005904 17682 17685 Crx +T542 CL:0000604 17689 17692 rod +T543 GO:0044317 17689 17702 rod spherules +T544 GO:0045202 17735 17742 synapse +T545 GO:0044317 17772 17780 spherule +T546 PR:000005904 17823 17826 Crx +T547 CL:0000604 17830 17833 rod +T548 CL:0000604 17881 17882 r +T549 GO:0042995 17889 17898 processes +T550 CL:0000745 17905 17921 horizontal cells +T551 CL:0000745 17923 17924 h +T552 CL:0000604 17941 17944 rod +T553 GO:0042995 17970 17977 process +T554 GO:0098794 17983 17995 postsynaptic +T555 GO:0030425 18004 18012 dendrite +T556 GO:0008021 18074 18091 synaptic vesicles +T557 GO:0030135 18097 18111 coated vesicle +T558 GO:0005886 18133 18146 cell membrane +T559 UBERON:0001790 18180 18186;18194 18201 OPL of ... retinas +T560 PR:000005904 18187 18190 Crx +T561 CL:0000210 18203 18216 photoreceptor +T562 GO:0008021 18287 18304 synaptic vesicles +T563 GO:0044456 18398 18416 synapse components +T564 GO:0044317 18439 18448 spherules +T565 GO:0044316 18453 18461 pedicles +T566 GO:0097060 18621 18638 synaptic membrane +T567 GO:0005634 18710 18717 nuclear +T568 CL:0000745 18730 18731 H +T569 CL:0000745 18733 18748 horizontal cell +T570 CL:0000103 18750 18751 B +T571 CL:0000103 18753 18765 bipolar cell +T572 GO:0005634 18767 18768 N +T573 GO:0005634 18770 18777 nucleus +T574 PR:000005904 18927 18930 Crx +T575 CL:0000210 18934 18948 photoreceptors +T576 PR:000005904 18969 18972 Crx +T577 http://purl.obolibrary.org/obo/MONDO_0018998 19009 19037 Leber's congenital amaurosis +T578 http://purl.obolibrary.org/obo/MONDO_0000001 19117 19124 disease +T579 PR:000005904 19178 19181 Crx +T580 UBERON:0000966 19185 19191 retina +T581 GO:0001750 19218 19231 outer segment +T582 GO:0009653 19232 19245 morphogenesis +T583 GO:0001750 19308 19321 outer segment +T584 PR:000005904 19337 19340 Crx +T585 CL:0000210 19344 19358 photoreceptors +T586 GO:0045202 19394 19401 synapse +T587 GO:0007416 19394 19411 synapse formation +T588 GO:0007416 19451 19465 synaptogenesis +T589 NCBITaxon:33208 19479 19485 animal +T590 http://purl.obolibrary.org/obo/MONDO_0018998 19495 19498 LCA +T591 PR:000005904 19501 19504 Crx +T592 CL:0000210 19508 19522 photoreceptors +T593 GO:0001750 19539 19552 outer segment +T594 GO:0009653 19553 19566 morphogenesis +T595 PR:000005904 19581 19584 Crx +T596 SO:0000704 19618 19622 gene +T597 GO:0001750 19649 19662 outer segment +T598 PR:000013290 19725 19728 RDS +T599 SO:0000704 19729 19733 gene +T600 PR:000001245 19745 19754 rhodopsin +T601 GO:0001750 19781 19794 outer segment +T602 PR:000005904 19840 19843 Crx +T603 CL:0000210 19847 19860 photoreceptor +T604 GO:0008594 19847 19874 photoreceptor morphogenesis +T605 GO:0031982 19906 19915 Vesicular +T606 PR:000005904 19930 19933 Crx +T607 CL:0000210 19937 19951 photoreceptors +T608 PR:000013290 20017 20020 rds +T609 NCBITaxon:10088 20035 20040 mouse +T610 GO:0031982 20079 20087 vesicles +T611 GO:0030397 20104 20116;20131 20140 breakdown of ... membranes +T612 GO:0042622 20117 20140 outer segment membranes +T613 GO:0001750 20181 20195 outer segments +T614 GO:0005902 20274 20284 microvilli +T615 CL:0000636 20288 20300 Müller cells +T616 PR:000001245 20425 20434 rhodopsin +T617 GO:0031982 20452 20460 vesicles +T618 PR:000001245 20503 20512 rhodopsin +T619 GO:0031982 20555 20563 vesicles +T620 GO:0001917 20587 20601 inner segments +T621 CL:0000210 20629 20643 photoreceptors +T622 GO:0001750 20696 20709 outer segment +T623 GO:0032391 20740 20757 connecting cilium +T624 PR:000013290 20804 20807 RDS +T625 PR:000014157 20812 20817 ROM-1 +T626 PR:000001119 20860 20865 opsin +T627 GO:0001750 20967 20981 outer segments +T628 PR:000014157 20997 21002 ROM-1 +T629 NCBITaxon:10088 21006 21010 mice +T630 GO:0001750 21032 21046 outer segments +T631 PR:000005904 21070 21073 Crx +T632 GO:0065007 21129 21137 controls +T633 SO:0000704 21138 21143 genes +T634 GO:0001750 21213 21226 outer segment +T635 PR:000001245 21248 21257 rhodopsin +T636 SO:0000704 21342 21346 gene +T637 GO:0010467 21342 21357 gene expression +T638 SO:0000704 21405 21410 genes +T639 GO:0010467 21437 21447 expression +T640 PR:000005904 21457 21460 Crx +T641 NCBITaxon:10088 21464 21468 mice +T642 PR:000001245 21484 21493 rhodopsin +T643 GO:0010467 21494 21504 expression +T644 PR:000005904 21532 21535 Crx +T645 NCBITaxon:33208 21539 21546 animals +T646 NCBITaxon:10088 21613 21617 mice +T647 GO:0010467 21642 21652 expression +T648 PR:000001245 21666 21675 rhodopsin +T649 CL:0000604 21684 21687 rod +T650 PR:000001245 21749 21758 rhodopsin +T651 GO:0010467 21759 21769 expression +T652 PR:000001245 21798 21807 rhodopsin +T653 GO:0010467 21808 21818 expression +T654 NCBITaxon:7215 21869 21879 Drosophila +T655 NCBITaxon:7215 21885 21895 Drosophila +T656 PR:000001245 21897 21906 rhodopsin +T657 PR:P06002 21908 21913 ninaE +T658 GO:0010467 21918 21927 expressed +T659 CL:0000687 21931 21948 photoreceptors R1 +T660 CL:0000705 21931 21945;21949 21951 photoreceptors ... R6 +T661 PR:P06002 21956 21961 ninaE +T662 GO:0016028 21980 21990 rhabdomere +T663 NCBITaxon:7742 22017 22027 vertebrate +T664 GO:0001750 22028 22042 outer segments +T665 CL:0000687 22064 22066;22070 22084 R1 ... photoreceptors +T666 CL:0000705 22067 22084 R6 photoreceptors +T667 PR:000001245 22123 22132 rhodopsin +T668 NCBITaxon:10088 22136 22140 mice +T669 PR:000001245 22233 22242 rhodopsin +T670 GO:0010467 22243 22253 expression +T671 GO:0016028 22261 22271 rhabdomere +T672 GO:0042052 22261 22283 rhabdomere development +T673 PR:P06002 22288 22293 ninaE +T674 NCBITaxon:7147 22299 22304 flies +T675 PR:P06002 22308 22313 ninaE +T676 SO:0000902 22314 22323 transgene +T677 GO:0065007 22334 22341 control +T678 SO:0000167 22358 22366 promoter +T679 PR:000001245 22468 22477 rhodopsin +T680 CL:0000210 22535 22548 photoreceptor +T681 CL:0000210 22583 22596 photoreceptor +T682 GO:0010467 22618 22628 expression +T683 PR:000001245 22708 22717 rhodopsin +T684 GO:0010467 22718 22728 expression +T685 GO:0016028 22801 22811 rhabdomere +T686 GO:0061541 22801 22825 rhabdomere morphogenesis +T687 NCBITaxon:10114 22878 22881 rat +T688 PR:000001245 22890 22899 rhodopsin +T689 CL:0000604 22969 22973 rods +T690 GO:0065007 23006 23016 regulation +T691 PR:000001245 23020 23029 rhodopsin +T692 GO:0065007 23057 23064 control +T693 PR:000001245 23105 23114 rhodopsin +T694 PR:000005904 23138 23141 Crx +T695 GO:0065007 23149 23159 regulating +T696 GO:0001750 23160 23173 outer segment +T697 GO:0009653 23174 23187 morphogenesis +T698 PR:000005904 23264 23267 Crx +T699 NCBITaxon:7215 23280 23290 Drosophila +T700 PR:P22810 23294 23297 Otd +T701 SO:0000704 23344 23349 genes +T702 PR:000005904 23359 23362 Crx +T703 SO:0001023 23406 23412 allele +T704 NCBITaxon:7215 23416 23426 Drosophila +T705 PR:P22810 23427 23430 otd +T706 PR:P22810 23432 23435 otd +T707 CL:0000210 23440 23453 photoreceptor +T708 GO:0008594 23440 23467 photoreceptor morphogenesis +T709 SO:0000704 23528 23533 genes +T710 PR:000005904 23558 23561 Crx +T711 GO:0010467 23578 23587 expressed +T712 PR:000005904 23612 23615 Crx +T713 UBERON:0000966 23619 23625 retina +T714 PR:000001245 23635 23644 rhodopsin +T715 CL:0000210 23715 23729 photoreceptors +T716 GO:0010467 23739 23749 expression +T717 CHEBI:18059 23799 23804 lipid +T718 GO:0006629 23799 23815 lipid metabolism +T719 GO:0006457 23817 23832 protein folding +T720 GO:0001750 23957 23970 outer segment +T721 GO:0097617 23980 23993 hybridization +T722 SO:0000704 24031 24036 genes +T723 GO:0001917 24100 24113 inner segment +T724 GO:0001750 24162 24175 outer segment +T725 SO:0000704 24218 24223 genes +T726 GO:0001750 24251 24264 outer segment +T727 CL:0000210 24303 24317 photoreceptors +T728 GO:0042384 24357 24369 ciliogenesis +T729 http://purl.obolibrary.org/obo/MONDO_0018998 24379 24382 LCA +T730 SO:0000704 24383 24387 gene +T731 PR:000005848 24389 24393 CRB1 +T732 SO:0000704 24409 24413 gene +T733 PR:000005850 24414 24418 CRB3 +T734 GO:0042384 24444 24456 ciliogenesis +T735 NCBITaxon:7215 24486 24496 Drosophila +T736 SO:0000853 24497 24506 homologue +T737 PR:000005848 24510 24514 CRB1 +T738 PR:P10040 24516 24522 Crumbs +T739 CL:0000210 24547 24560 photoreceptor +T740 GO:0008594 24547 24574 photoreceptor morphogenesis +T741 NCBITaxon:10088 24618 24623 mouse +T742 PR:000005848 24634 24638 CRB1 +T743 NCBITaxon:10088 24648 24653 mouse +T744 GO:0001750 24674 24688 outer segments +T745 CL:0000210 24740 24753 photoreceptor +T746 GO:0007416 24771 24785 synaptogenesis +T747 PR:000005848 24819 24823 CRB1 +T748 PR:000005904 24828 24831 Crx +T749 http://purl.obolibrary.org/obo/MONDO_0018998 24857 24860 LCA +T750 UBERON:0000966 24932 24939 retinal +T751 GO:0060041 24932 24951 retinal development +T752 GO:0007416 24954 24968 Synaptogenesis +T753 PR:000005904 24985 24988 Crx +T754 CL:0000210 24992 25006 photoreceptors +T755 PR:000005904 25012 25015 Crx +T756 NCBITaxon:10088 25019 25024 mouse +T757 CL:0000210 25069 25082 photoreceptor +T758 GO:0045202 25083 25091 synapses +T759 NCBITaxon:10088 25128 25133 mouse +T760 CL:0000210 25166 25179 photoreceptor +T761 CL:0000210 25219 25233 photoreceptors +T762 PR:000001245 25268 25277 rhodopsin +T763 PR:000001245 25279 25282 Rho +T764 CHEBI:23447 25288 25305 cyclic nucleotide +T765 PR:000000696 25288 25328 cyclic nucleotide-gated channel, alpha-3 +T766 PR:000000696 25330 25335 CNGA3 +T767 NCBITaxon:10088 25353 25357 mice +T768 PR:000001245 25359 25362 Rho +T769 PR:000000696 25367 25372 CNGA3 +T770 GO:0045202 25378 25386 synapses +T771 CL:0000210 25459 25472 photoreceptor +T772 GO:0007416 25473 25487 synaptogenesis +T773 GO:0001750 25516 25529 outer segment +T774 NCBITaxon:10088 25651 25655 mice +T775 GO:0007602 25681 25698 phototransduction +T776 NCBITaxon:10088 25719 25723 mice +T777 CL:0000210 25757 25770 photoreceptor +T778 GO:0045202 25771 25778 synapse +T779 GO:0044456 25805 25821 synapse elements +T780 GO:0001750 25858 25871 outer segment +T781 CL:0000210 25936 25949 photoreceptor +T782 GO:0007602 25995 26012 phototransduction +T783 GO:0045202 26017 26024 synapse +T784 GO:0007416 26017 26034 synapse formation +T785 PR:000005904 26124 26127 Crx +T786 CL:0000210 26131 26145 photoreceptors +T787 GO:0045202 26165 26173 synaptic +T788 GO:0001750 26206 26219 outer segment +T789 PR:000005904 26265 26268 Crx +T790 CL:0000210 26285 26298 photoreceptor +T791 GO:0045202 26299 26306 synapse +T792 GO:0007416 26299 26316 synapse formation +T793 GO:0065007 26329 26339 regulating +T794 SO:0000704 26375 26380 genes +T795 GO:0010467 26442 26452 expression +T796 GO:0098793 26463 26475 pre-synaptic +T797 PR:000009311 26505 26510 KIF3a +T798 PR:000015849 26512 26515 SV2 +T799 PR:000015890 26521 26534 synaptophysin +T800 PR:000005904 26595 26598 Crx +T801 UBERON:0000479 26614 26620 tissue +T802 SO:0000704 26729 26734 genes +T803 SO:0000704 26783 26788 genes +T804 SO:0000704 26875 26880 genes +T805 GO:0010467 26881 26890 expressed +T806 CL:0000210 26894 26908 photoreceptors +T807 GO:0010467 26945 26955 expression +T808 PR:000005904 26969 26972 Crx +T809 NCBITaxon:10088 26976 26981 mouse +T810 CL:0000210 27037 27050 photoreceptor +T811 GO:0008594 27037 27064 photoreceptor morphogenesis +T812 SO:0000704 27082 27087 genes +T813 GO:0010467 27102 27111 expressed +T814 CL:0000210 27115 27128 photoreceptor +T815 PR:000008536 27217 27256 HGF-regulated tyrosine kinase substrate +T816 GO:0065007 27221 27230 regulated +T817 PR:000005877 27262 27267 CRIPT +T818 PR:000015894 27281 27296 synaptotagmin 1 +T819 SO:0000704 27354 27358 gene +T820 PR:000005904 27385 27388 Crx +T821 UBERON:0000966 27392 27398 retina +T822 PR:000017084 27402 27406 HRG4 +T823 SO:0000853 27410 27419 homologue +T824 NCBITaxon:6239 27427 27437 C. elegans +T825 PR:Q10658 27438 27444 Unc119 +T826 SO:0000704 27445 27449 gene +T827 GO:0097470 27524 27538 ribbon synapse +T828 SO:0000704 27660 27665 genes +T829 GO:0044430 27684 27705 cytoskeletal elements +T830 GO:0005874 27732 27743 microtubule +T831 PR:000010142 27732 27764 microtubule associated protein 4 +T832 PR:000005385 27785 27794 cofilin 1 +T833 PR:000005904 27803 27806 Crx +T834 UBERON:0000966 27810 27816 retina +T835 SO:0000704 27879 27884 genes +T836 GO:0065007 27913 27923 regulating +T837 GO:0045202 27924 27932 synaptic +T838 SO:0000704 27962 27967 genes +T839 GO:0097470 28085 28099 ribbon synapse +T840 CL:0000210 28111 28124 photoreceptor +T841 UBERON:0000966 28179 28186 retinal +T842 GO:0060041 28179 28198 retinal development +T843 PR:000009655 28206 28225 laminin beta2 chain +T844 NCBITaxon:10088 28236 28241 mouse +T845 NCBITaxon:10088 28288 28292 mice +T846 PR:000009655 28301 28320 laminin beta2 chain +T847 NCBITaxon:10088 28331 28335 mice +T848 GO:0001750 28355 28368 outer segment +T849 PR:000005904 28424 28427 Crx +T850 NCBITaxon:10088 28431 28435 mice +T851 GO:0001750 28441 28455 outer segments +T852 CL:0000210 28492 28505 photoreceptor +T853 PR:000009655 28534 28547 laminin beta2 +T854 PR:000005904 28610 28613 Crx +T855 NCBITaxon:10088 28617 28621 mice +T856 UBERON:0001790 28627 28651;28672 28679 outer plexiform layer of ... retinas +T857 PR:000009655 28656 28661 beta2 +T858 GO:0045202 28721 28729 synapses +T859 GO:0045202 28795 28803 synaptic +T860 CHEBI:36357 28869 28878 molecules +T861 CL:0000210 28891 28904 photoreceptor +T862 GO:0008594 28891 28918 photoreceptor morphogenesis +T863 PR:000009655 28952 28965 laminin beta2 +T864 PR:000005904 29031 29034 Crx +T865 PR:000005904 29042 29045 Crx +T866 PR:000009655 29109 29122 laminin beta2 +T867 PR:000005904 29138 29141 Crx +T868 NCBITaxon:10088 29145 29149 mice +T869 http://purl.obolibrary.org/obo/MONDO_0018998 29166 29169 LCA +T870 PR:000005904 29171 29174 Crx +T871 CL:0000210 29204 29217 photoreceptor +T872 http://purl.obolibrary.org/obo/MONDO_0000001 29218 29226 diseases +T873 NCBITaxon:9606 29242 29247 human +T874 http://purl.obolibrary.org/obo/MONDO_0001941 29248 29257 blindness +T875 CL:0000573 29259 29263 cone +T876 http://purl.obolibrary.org/obo/MONDO_0007362 29259 29278 cone-rod dystrophy2 +T877 CL:0000604 29264 29267 rod +T878 http://purl.obolibrary.org/obo/MONDO_0018998 29280 29308 Leber's congenital amaurosis +T879 http://purl.obolibrary.org/obo/MONDO_0019200 29314 29334 retinitis pigmentosa +T880 CL:0000573 29363 29367 cone +T881 http://purl.obolibrary.org/obo/MONDO_0015993 29363 29383 cone-rod dystrophies +T882 CL:0000604 29368 29371 rod +T883 http://purl.obolibrary.org/obo/MONDO_0015993 29385 29389 CRDs +T884 CL:0000573 29420 29424 cone +T885 GO:0007601 29434 29440 vision +T886 UBERON:0000104 29464 29468 life +T887 CL:0000604 29518 29521 rod +T888 GO:0007601 29531 29537 vision +T889 http://purl.obolibrary.org/obo/MONDO_0019200 29556 29558 RP +T890 CL:0000604 29590 29593 rod +T891 CL:0000573 29624 29628 cone +T892 GO:0007601 29638 29644 vision +T893 SO:0000704 29673 29678 genes +T894 NCBITaxon:9606 29695 29700 human +T895 SO:0000704 29701 29708 genetic +T896 http://purl.obolibrary.org/obo/MONDO_0001941 29709 29718 blindness +T897 GO:0010467 29736 29745 expressed +T898 CL:0000210 29785 29799 photoreceptors +T899 GO:0001750 29821 29834 outer segment +T900 GO:0007602 29881 29898 phototransduction +T901 GO:0001750 29902 29915 outer segment +T902 CL:0000604 29963 29966 rod +T903 SO:0000704 29976 29981 genes +T904 PR:000001245 29991 30000 rhodopsin +T905 CL:0000573 30021 30025 cone +T906 http://purl.obolibrary.org/obo/MONDO_0019200 30042 30044 RP +T907 PR:000005904 30074 30077 Crx +T908 CL:0000210 30177 30190 photoreceptor +T909 http://purl.obolibrary.org/obo/MONDO_0000001 30191 30198 disease +T910 http://purl.obolibrary.org/obo/MONDO_0018998 30201 30204 LCA +T911 http://purl.obolibrary.org/obo/MONDO_0000001 30210 30217 disease +T912 CL:0000210 30249 30262 photoreceptor +T913 PR:000005904 30352 30355 Crx +T914 NCBITaxon:10088 30359 30364 mouse +T915 http://purl.obolibrary.org/obo/MONDO_0000001 30426 30434 disorder +T916 http://purl.obolibrary.org/obo/MONDO_0000001 30468 30476 disorder +T917 PR:000005904 30483 30486 Crx +T918 http://purl.obolibrary.org/obo/MONDO_0018998 30561 30564 LCA +T919 NCBITaxon:9606 30568 30573 human +T920 UBERON:0000479 30574 30580 tissue +T921 UBERON:0007023 30604 30609 adult +T922 http://purl.obolibrary.org/obo/MONDO_0018998 30624 30627 LCA +T923 NCBITaxon:33208 30688 30694 animal +T924 http://purl.obolibrary.org/obo/MONDO_0018998 30705 30708 LCA +T925 GO:0009888 30788 30800;30809 30815 formation of ... tissue +T926 UBERON:0000966 30801 30808 retinal +T927 UBERON:0000479 30809 30815 tissue +T928 NCBITaxon:9606 30840 30845 human +T929 UBERON:0000479 30846 30852 tissue +T930 NCBITaxon:9606 30868 30873 human +T931 UBERON:0000966 30882 30888 retina +T932 PR:000014179 30903 30908 RPE65 +T933 UBERON:0000966 30949 30956 retinae +T934 CL:0000210 31041 31054 photoreceptor +T935 UBERON:0001787 31041 31060 photoreceptor layer +T936 GO:0045202 31120 31128 synaptic +T937 UBERON:0000966 31139 31146 retinal +T938 CL:0000210 31191 31204 photoreceptor +T939 GO:0045202 31205 31213 synapses +T940 NCBITaxon:9606 31312 31317 human +T941 UBERON:0000479 31318 31324 tissue +T942 http://purl.obolibrary.org/obo/MONDO_0018998 31375 31378 LCA +T943 NCBITaxon:33208 31392 31398 animal +T944 GO:0001750 31477 31490 outer segment +T945 GO:0009653 31491 31504 morphogenesis +T946 GO:0007416 31506 31520 synaptogenesis +T947 http://purl.obolibrary.org/obo/MONDO_0018998 31577 31580 LCA +T948 NCBITaxon:10088 31592 31596 Mice +T949 PR:000005904 31598 31601 Crx +T950 NCBITaxon:10088 31605 31609 mice +T951 PR:000001245 31653 31661 Rhodosin +T952 NCBITaxon:10088 31667 31671 mice +T953 PR:000013290 31735 31738 Rds +T954 NCBITaxon:10088 31739 31743 mice +T955 CHEBI:10545 31797 31805 electron +T956 PR:000005904 31829 31832 Crx +T957 CHEBI:16842 31874 31886 formaldehyde +T958 CHEBI:64276 31896 31910 glutaraldehyde +T959 GO:0007567 31926 31931 natal +T960 UBERON:0000970 31944 31948 eyes +T961 UBERON:0000964 31979 31985 cornea +T962 UBERON:0000970 32013 32016 eye +T963 CHEBI:50913 32037 32045 fixative +T964 CHEBI:16842 32050 32062 formaldehyde +T965 CHEBI:64276 32072 32086 glutaraldehyde +T966 UBERON:0001773 32117 32123 sclera +T967 UBERON:0000966 32159 32166 retinas +T968 CHEBI:64276 32237 32251 glutaraldehyde +T969 CL:0000210 32355 32368 photoreceptor +T970 GO:0001750 32355 32383 photoreceptor outer segments +T971 UBERON:0000479 32404 32410 tissue +T972 UBERON:0000479 32465 32471 tissue +T973 CHEBI:30059 32521 32543 potassium ferrocyanide +T974 CHEBI:60004 32544 32551 mixture +T975 CHEBI:75211 32697 32708 tannic acid +T976 CHEBI:16223 32718 32728 cacodylate +T977 CHEBI:10545 32932 32940 electron +T978 CHEBI:10545 32963 32971 electron +T979 UBERON:0001782 33031 33057 retinal pigment epithelium +T980 UBERON:0003902 33115 33128 neural retina +T981 UBERON:0000966 33130 33137 Retinae +T982 PR:000005904 33143 33146 Crx +T983 PR:000001245 33151 33160 rhodopsin +T984 PR:000013290 33165 33168 RDS +T985 UBERON:0000970 33182 33186 eyes +T986 CHEBI:75958 33228 33236 solution +T987 CHEBI:64276 33256 33270 glutaraldehyde +T988 CHEBI:16842 33280 33292 formaldehyde +T989 UBERON:0000479 33311 33317 Tissue +T990 CHEBI:16223 33340 33350 cacodylate +T991 CHEBI:16236 33396 33403 ethanol +T992 UBERON:0000479 33405 33412 Tissues +T993 CHEBI:16526 33455 33469 carbon dioxide +T994 CHEBI:10545 33625 33633 electron +T995 CHEBI:10545 33704 33712 electron +T996 CHEBI:10545 33747 33755 electron +T997 PR:000005904 33822 33825 Crx +T998 NCBITaxon:10088 33829 33834 mouse +T999 GO:0001750 34197 34211 outer segments diff --git a/src/ontogpt/evaluation/craft/database/all/15676071.txt b/src/ontogpt/evaluation/craft/database/all/15676071.txt new file mode 100644 index 000000000..398c5b31f --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15676071.txt @@ -0,0 +1,129 @@ +Synaptogenesis and outer segment formation are perturbed in the neural retina of Crx mutant mice + +Abstract + +Background + +In Leber's congenital amaurosis (LCA), affected individuals are blind, or nearly so, from birth. This early onset suggests abnormal development of the neural retina. Mutations in genes that affect the development and/or function of photoreceptor cells have been found to be responsible in some families. These examples include mutations in the photoreceptor transcription factor, Crx. + +Results + +A Crx mutant strain of mice was created to serve as a model for LCA and to provide more insight into Crx's function. In this study, an ultrastructural analysis of the developing retina in Crx mutant mice was performed. Outer segment morphogenesis was found to be blocked at the elongation stage, leading to a failure in production of the phototransduction apparatus. Further, Crx-/- photoreceptors demonstrated severely abnormal synaptic endings in the outer plexiform layer. + +Conclusions + +This is the first report of a synaptogenesis defect in an animal model for LCA. These data confirm the essential role this gene plays in multiple aspects of photoreceptor development and extend our understanding of the basic pathology of LCA. + +Background + +Photoreceptor cells play a primary role in vision by capturing light energy and converting it into neural stimuli. These sensory neurons are a shared element in all organisms capable of sensing light. In humans, genetic diseases of the eye are common and the primary site of disease is most frequently photoreceptors (for review see [1-3]). + +Photoreceptors have been well studied, particularly with respect to the biochemistry and physiology of phototransduction. Insight into the development of vertebrate photoreceptors, however, has lagged behind our understanding of function. Only recently have the first molecular mechanisms regulating photoreceptor development been identified (for review see, [2,4]). Crx (cone-rod homeobox) is an otx-family homeobox gene expressed predominantly in photoreceptors, from early in their development through to the adult ages [5-7]. Crx gene expression is critically dependent upon Otx2, another member of the same homeobox family which is expressed in early photoreceptor cells [8]. In rod photoreceptors, Crx appears to work in concert with Nrl, a leucine zipper protein that is also restricted in its expression in the retina to rod photoreceptors [9]. Many photoreceptor-specific genes have putative Crx-binding elements in their regulatory regions [10], including rhodopsin [11] and arrestin [12]. Mutations in Crx have been associated with several human diseases that lead to blindness, including cone-rod dystrophy 2 [6,13,14], retinitis pigmentosa [14], and LCA [14-16]. Based on these data, Crx was hypothesized to play a critical role in the differentiation and maintainance of photoreceptor cells [5,7]. + +LCA is the most severe genetic disease of photoreceptors (see [17], for recent review). Affected infants exhibit a complete or near complete absence of vision from birth. Mutations in retinal specific genes, such as Crx, have been associated with LCA [14,15], as well as GUCY2D [18], RPE65 [19], AIPL-1 [20], CRB-1 [21], and RPGRIP-1 [22]. There also may be as many as three additional genetically linked loci where genes have not been identified [23]. Crx mutations in LCA are varied, and include a putative dominant mutation that is proposed to encode a dominant-negative form of Crx [14,15]. Recessive mutations also have been reported and at least one allele encodes a protein with decreased DNA-binding activity [16]. Histopathological and ultrastructural studies of LCA should enable a better understanding of the disease process, and the design of suitable therapies. Few such studies exist for human LCA (reviewed in [17]) and the majority of such studies examine the globes of adults with LCA, after the tissue has undergone secondary changes. Only a single study exists where the developing eye of an infant was examined [24]. Animal models for LCA have recently been reported and have already served to broaden our understanding of the pathology of this disease [25-28]. Since LCA is a clinically and genetically heterogeneous disorder, additional mouse models are in order to allow a full understanding of the many ways in which photoreceptor development can go awry. + +In addition to their importance as a locus of disease, photoreceptor cells serve as an excellent model for studies in neuronal differentiation. Photoreceptor cells are highly polarized. At their apex, these neurons have a membranous outer segment, which contains proteins involved in the phototransduction cascade. Loss of function mutations in rhodopsin [29], or the structural protein, peripherin [30], result in an inability to form outer segments. At the other extremity, photoreceptors terminate with synaptic endings that make contact with the processes of horizontal and bipolar cells [31,32]. Rod spherules establish an invaginating synapse with rod bipolar dendrites and axonal endings of horizontal cells. This synapse is characterized by the presence of a ribbon in the presynaptic cytoplasm. Cone pedicles make invaginating synapses with the dendrites of on-cone bipolar cells and horizontal cells and basal junctions with the dendrites of off-cone bipolar cells. The factors regulating the formation of the photoreceptor synapses are completely unknown. At least one photoreceptor synaptic protein, HRG4, contains a potential Crx target sequence in its transcriptional regulatory sequence [33]. + +Few studies of LCA animal models have extended their examination of retinal pathology to the ultrastructural level. Certain features of neuronal differentiation, such as synapse formation, can be detected definitively at this level of analysis. With the hope of understanding the neuropathology of LCA in greater detail, we have analyzed the differentiation of the outer retina in Crx-/- mice at the ultrastructural level. These retinas exhibit several prominent defects. Crx-/- photoreceptors demonstrate a complete block in outer segment formation at the elongation stage. Further, these cells exhibit abnormal synaptic morphology, thereby broadening the function of Crx to photoreceptor synaptogenesis. This represents the first report strongly implicating the process of synapse formation in LCA. + +Results + +Multiple pathologies in the outer segment layer in Crx-/- mice + +A standard knock-out protocol was used to generate mice in which the homeodomain of Crx-/- was targeted and deleted. Generation of these Crx-/- mice has been reported elsewhere [34]. In this study, in order to characterize further the role of Crx in photoreceptor morphogenesis, the outer retinae from Crx-/- mice were examined using transmission electron microscopy. At postnatal day 21 (P21), when Crx+/+ photoreceptors exhibited robust outer segments (Figure 1A, os), Crx-/- retinas were without a recognizable outer segment layer (Figure 1B). Crx-/- photoreceptors had inner segments, demonstrating at least limited photoreceptor polarization in the Crx mutant, but the inner segments were extremely short (Figure 2). Furthermore, the majority of inner segments showed some morphological differentiation, having approximately as many mitochondria as the control (Figure 1 and 2). Occasionally, an inner segment undergoing lysis was noted, appearing swollen or with vacuoles and swollen mitochondria (data not shown). + +Figure 1 + +Transmission electron microscopy of the outer retina at P21 in (A) Crx+/+ and (B) Crx-/- retinas. pe, pigmented epithelium. os, outer segments. is, inner segments. onl, outer nuclear layer with photoreceptor nuclei. Scale bar = 5 μm for A and B. + +Figure 2 + +Transmission electron micrograph of the outer segment layer of Crx-/- retina at P21. Inner segments of Crx-/- photoreceptors exhibit numerous mitochondria (m indicated by arrow) as in Crx+/+ (Figure 1A). pe, pigmented epithelium. is, inner segments. onl, outer nuclear layer. Scale bar = 2 μm. + +Photoreceptor inner segments and outer segments are joined by a non-motile connecting cilium that exhibits a characteristic 9 + 0 arrangement of microtubule doublets when viewed in cross-section. At P21, in Crx-/- retinas, numerous cross sections of connecting cilia were noted (Figure 3A and 3B). Sporadically, connecting cilia contained other than the typical complement of microtubule doublets. For example, in Figure 3A, the connecting cilium labelled by arrowhead 1, shows 7 + 0 doublets. The majority exhibited the characteristic 9 + 0 doublets (arrowhead 2 and 3 in Figure 3A and Figure 3B). These observations indicate that in addition to inner segment formation, ciliogenesis is also largely intact in Crx-/- photoreceptors. Further, in Crx-/- retinas the retinal pigmented epithelium (PE) appeared normal, at least up to P21 (data not shown), the oldest age examined. + +Figure 3 + +Transmission electron micrograph of Crx-/- retina at P21 (A and B), and scanning electron micrograph of Crx-/- at P10 (C) of outer segment layer. (A) Evidence of ciliogenesis in the photoreceptor layer of Crx-/- retina. Nonmotile connecting cilia were observed in cross section (arrowheads 1,2, and 3, for examples). Connecting cilium 1 (arrowhead 1) demonstrated seven microtuble doublets, while cilium 2 and cilium 3 exhibited nine. In A, a displaced cell nucleus (n) appearing pyknotic and abnormal deposition of matrix (mx) material of unknown identity were seen, along with large amounts of membranous vesicles (arrow) which filled the photoreceptor space and appeared to be released from inner segments. Scale bar = 3.7 μm. (B) Nonmotile connecting cilium in cross section, from a Crx-/- photoreceptor, demonstrating characteristic 9+0 radial array of microtubule doublets. Scale bar = 88 nm. (C) Scanning electron micrograph (SEM) of membranous vesicles (arrow shows one example) shed from inner segments of Crx-/- photoreceptors at P10. Figure shows inner segments viewed from the scleral side with the pigmented epithelium removed. Scale bar = 1 μm. + +In addition to the complete absence of outer segments, Crx-/- retinas exhibited three other notable pathologies in the outer segment layer. First, an abnormal deposition of matrix of unknown identity was noted (Figure 3A, mx). Second, sporadically displaced nuclei were found residing in the space abutting the PE. Occasionally, these nuclei appeared pyknotic (Figure 3A, n); but, more frequently exhibited the heterochromatin pattern typical of photoreceptors (data not shown), strongly suggesting that they belonged to ectopic photoreceptors. The third pathological entity noted in the outer segment layer were numerous small vesicles (Figure 3A arrow) 100 to 200 nm in diameter. They appeared to be emerging from the inner segments, as scanning electron microscopic images showed spherical structures budding from the inner segments (Figure 3C, arrow). + +In order to characterize further the morphogenesis of Crx-/- photoreceptors, the developing outer segment layer was viewed by scanning electron microscopy at P7, P14 and P21 (Figure 4). In Crx+/+ retinas, photoreceptor inner segments, connecting cilia, and the first rudimentary outer segment structures were noted at P7. In the Crx-/- retina, only an occassional connecting cilium was noted emerging from inner segments at this stage (Figure 4A and 4B). This observation was confirmed by comparison with transmission electron micrographs (Figure 5). These differences are the earliest noted differences between Crx+/+ and Crx-/- photoreceptors. At P14, elongating outer segments were noted on Crx+/+ photoreceptors, occasionally demonstrating a paddle-like structure at their apex (Figure 4C, os). In Crx-/- retinae, the vast majority of photoreceptors at this stage demonstrated connecting cilia without outer segments (Figure 4D, cc). Sporadically, Crx-/- photoreceptors would exhibit an irregular structure extending from a connecting cilium (Figure 4D, cc*) perhaps representing a malformed outer segment. Such structures were also observed at P21 (Figure 4F, cc*). These putative, abnormal outer segments were only rarely noted in Crx+/+ at P14, and never at P21 (Figure 4C and 4E, cc*). Further, in Crx-/- photoreceptors, unusually long connecting cilia were noted (Figure 4F, cc). Serial examination of Crx-/- photoreceptors at P7, P10, P14, and P21 by TEM, demonstrated a distinctive lack of any structure interpretable as orderly stacks of discs or forming discs. These data demonstrate a complete absence of normal outer segment formation in the Crx mutant mouse, and the arrest of development of the photoreceptor appendage at the elongation stage of outer segment formation. + +Figure 4 + +Outer segment morphogenesis in Crx-/- photoreceptors. Scanning electron microscopy of developing photoreceptors viewed from the scleral side with the pigmented epithelium removed at P7, P14, and P21 for Crx+/+ (A, C, and E) and Crx-/- (B, D, F) littermates. In Crx+/+ retina, numerous connecting cilia (A, cc) were evident at P7 with rudimentary outer segments. After P7, in Crx+/+ outer segment elongation occurs. Initially, outer segments have a paddle-like structure (C, os) which is later shed (E, os). In Crx-/- photoreceptors, few connecting cilia were observed at P7 (B, cc). After P7, connecting cilia were more numerous and occasionally a malformed outer segment was noted extending from a connecting cilium (D and F, cc*). These were rarely observed in Crx+/+ and only at P14 (C, cc*). At P21, abnormally long connecting cilia are noted in Crx-/- (F, cc). Scale bars = 10 μm + +Figure 5 + +Transmission electron micrographs of Crx-/- photoreceptors in the photoreceptor layer at P7. (A) Photoreceptor layer of Crx+/+ retina demonstrating nascent outer segment structures (arrow) emerging from photoreceptor connecting cilia (cc). (B) Crx-/- photoreceptors exhibited connecting cilia (cc) at this early stage, however, nascent outer segment structures were not observed. Scale Bar = 1 μm. + +Finally, the morphology of the malformed Crx-/- photoreceptors was compared to rhodopsin-/- and peripherin-/- photoreceptors. Rhodopsin and peripherin are two photoreceptor-specific genes whose expression is significantly downregulated in the Crx-/- retinae [10,34,35]. Loss of function mutations in each of these genes separately have been reported to result in a failure to elaborate outer segments [29,30]. Photoreceptors from these two mutant mice examined by SEM from the scleral side appeared highly similar to Crx-/- photoreceptors (compare Figure 4F to Figure 6A and 6B). + +Figure 6 + +Scanning electron microscopy of peripherin-/- (A) and rhodopsin-/- (B) photoreceptors at P21, viewed from the scleral side with the pigmented epithelium removed. cc, connecting cilium. is, inner segment. Scale bar = 10 μm. + +Crx is necessary for the formation of photoreceptor terminals + +In a previous study, we demonstrated that forced expression of a dominant-negative allele of Crx in developing rods blocked formation of both rod spherules in the outer plexiform layer (OPL) and outer segments [7]. To expand on these studies, the ultrastructure of photoreceptor synapses was examined in Crx-/- retinas. In Crx+/+ retinas at P21, newly mature rod spherules were abundant (Figure 7A). The sperules were blunt or club-shaped structures with a single ribbon associated with a single invaginating synapse (Figure 7A, arrow indicates one example; Figure 8A and 8B). Two processes from horizontal cells were situated on either side of the synaptic ridge (Figure 8B, labelled H) and one or more dendrites of rod bipolar cells occupied a central position (Figure 8B, bipolar labelled B). Cone terminals are large, flat pedicles that exhibit many invaginating synapses containing separate sets of horizontal and bipolar cell processes. Each pedicle contains numerous ribbons. These terminals were much less common than spherules in Crx+/+ retinas at P21 (Figure 7, box). In the OPL of Crx-/- retinas, photoreceptor terminals were highly disorganized at P21 (Figure 7B, arrows). Processes containing synaptic vesicles and ribbon-like structures were apparent, suggesting at least limited generation of synapse components. However, well formed spherules and pedicles were not observed. In addition, many terminals appeared to contain multiple ribbons (Figure 8C and 8D, r) not tethered to the plasma membrane and occasionally in perinuclear positions (Figure 8D). + +Figure 7 + +Transmission electron micrographs of the outer plexiform layer in Crx-/- retinas. (A) In Crx+/+ retina at P21, newly formed rod spherules were abundant (arrow demonstrates one example). The spherules were club-shaped and contained a single invaginating synapse with one ribbon complex. Cone terminals form large, flat pedicles that contain many invaginating synapses with separate ribbon structures. These terminals were more scarce, but apparent in Crx+/+ retinas at P21 (one example in box). (B) In the outer plexiform layer (OPL) of Crx-/- retinas, photoreceptor terminals appeared highly disorganized at P21 (arrows). Well-formed pedicles and spherules were not evident. Putative terminals containing ribbon-like structures were apparent, suggesting at least limited generation of synapse components. Many terminals appeared to contain multiple ribbon-like structures, instead of a singule ribbon. For example, terminal 1 and 2 contained two ribbons each, whereas terminal 3 appeared to contain only one. opl, outer plexiform layer. Scale bar = 2 μm. + +Figure 8 + +Transmission electron micrographs of the outer plexiform layer in Crx-/- retinas at P21. (A) Crx+/+ rod spherules contained a single invaginating synapse with one ribbon complex. The spherule was a blunt or club-shaped structure. (B) Crx+/+ rod terminals contained a single ribbon structure (r). Two processes, from horizontal cells (h), contacted the rod laterally. An additional process, the postsynaptic bipolar dendrite (b), was situated more centrally. Terminals were filled with synaptic vesicles. One coated vesicle originatinf from the cell membrane was observed (arrow). (C) In the OPL of Crx-/- retinas, photoreceptor terminals appeared highly disorganized. Putative terminals containing synaptic vesicles and ribbon-like structures were apparent (arrows), suggesting at least limited generation of synapse components. However, well formed spherules and pedicles were not observed. Further, many terminals appeared to contain multiple ribbon-like structures (r). The majority of these ribbons were not associated with the synaptic membrane, but instead were found free floating and, in some instances, were perinuclear (D, arrow). H, horizontal cell; B, bipolar cell; N, nucleus; r, ribbon. (A) Scale bar = 500 nm, (B) Scale bar = 250 nm, (C and D) Scale bar = 500 nm. + +Discussion + +In this study, an ultrastructural analysis of Crx-/- photoreceptors was carried out. As Crx mutations have been associated with Leber's congenital amaurosis, the findings in this study broaden our understanding of the pathology of this disease. Two prominent pathologies were characterized in the Crx-/- retina: (1) An absolute block in outer segment morphogenesis was noted, with the block occuring at the elongation stage of outer segment formation; (2) Crx-/- photoreceptors exhibited a severe perturbation in synapse formation. This represents the first report of a synaptogenesis defect in an animal model of LCA. + +Crx-/- photoreceptors cannot complete outer segment morphogenesis + +Mutations in Crx represent one of a collection of gene mutations that lead to an outer segment formation defect. Homozygous null mutations in the peripherin/RDS gene [36] or in rhodopsin [29] lead to a failure of outer segment formation. The deficits in peripherin-/- and Crx-/- photoreceptor morphogenesis were found to be very similar. Vesicular structures in Crx-/- photoreceptors were observed that were similar to those previously noted in the rds/peripherin-/- mouse. It was initially proposed that these vesicles were due to the breakdown of outer segment membranes that were not properly recruited to the outer segments in the absence of peripherin, or were from the result of the breakdown of the microvilli of Müller cells [30]. Strong support in favor of the former explanation was provided by Nir and colleagues who demonstrated the presence of rhodopsin protein in these vesicles using immunoelectron microscopy against a rhodopsin epitope [37]. Further, as shown here, the vesicles appear to bud from the inner segments themselves. + +In developing photoreceptors, an extraordinary growth process occurs whereby the outer segment is generated from the nascent connecting cilium (see [38] and references therein). Peripherin/RDS and ROM-1 proteins (localized in disc rims) and the opsin proteins (localized throughout the discs) have important roles in the structural integrity of mature outer segments (see [39,29]). ROM-1-/- mice produce disorganized outer segments with large disks [40]. Crx, by virtue of being a transcription factor, presumably controls genes that are responsible for the building and perhaps maintenance of the outer segment structure, including rhodopsin and peripherin. Using northern blots [34], microarrays [10], and serial analysis of gene expression (SAGE) [35], we have defined a large number of genes that are altered in their expression level in Crx-/- mice. We found that rhodopsin expression was severely diminished in Crx-/- animals, and peripherin mRNA was reduced by approximately 30%. Transgenic mice with variable levels of expression of wild type rhodopsin exhibit rod degeneration [41], indicating the importance of the level of rhodopsin expression. In addition, the timing of rhodopsin expression may be very important, as indicated by studies in Drosophila. + +In Drosophila, rhodopsin (ninaE) is expressed in photoreceptors R1–R6. In ninaE null mutants, the rhabdomere, a structure analogous to vertebrate outer segments, fails to develop in R1–R6 photoreceptors [42], reminiscent of the situation in rhodopsin-/- mice [29]. An intriguing experiment by Kumar et al. [43] demonstrated a temporal requirement for rhodopsin expression during rhabdomere development. In ninaE null flies, a ninaE transgene under the control of a heat shock promoter was subjected to various temperature shifts during development. Heat shock during the normal time of rhodopsin onset resulted in substantial and long-lasting rescue of photoreceptor structure and transient rescue of photoreceptor physiology. However, expression shortly before or after this critical period failed to rescue, suggesting that rhodopsin expression during a discrete window of time in development is essential for proper rhabdomere morphogenesis. This result is consistent with observations in the rat wherein rhodopsin onset occurs with strict timing in the developmental history of most rods in vivo [44]. Thus, through its regulation of rhodopsin levels, or perhaps through control of the kinetics of the up-regulation of rhodopsin beginning at about P6, Crx may be regulating outer segment morphogenesis. The similarty of the two cases may extend further. At present, the closest Crx relative in Drosophila is Otd, the founding member of the class of homeobox genes to which Crx belongs. Interestingly, in one hypomorphic allele of Drosophila otd, otduvi, photoreceptor morphogenesis is also disrupted [45]. + +We found that there are many other genes that are dependent upon Crx. Those that are expressed at a lower level in the Crx-/- retina, such as rhodopsin and peripherin, comprise many that are either enriched or specific to photoreceptors in their expression [35]. They include enzymes that are important in lipid metabolism, protein folding and transport, as well as in other processes that one might envision would be important in building a structure such as the outer segment. In situ hybridization using probes from this collection of genes has revealed that some of them have their RNA localized to the inner segment, a finding typical for proteins targeted to the outer segment. Future analyses of the function of these genes might reveal their role in outer segment biogenesis. + +Finally, polarization of photoreceptors was found to be largely intact, as was ciliogenesis. Another LCA gene, CRB1, and a related gene CRB3, have been implicated in ciliogenesis in in vitro models [46]. The Drosophila homologue of CRB1, Crumbs, has been implicated in photoreceptor morphogenesis [47]. However, the spontaneously occurring mouse mutant in CRB1, the Rd8 mouse, develops shortened outer segments that subsequently degenerate [48], suggesting that photoreceptor polarization and synaptogenesis are intact in this mutant. While CRB1 and Crx have been both linked to LCA, further work is necessary to determine if their function is linked in retinal development. + +Synaptogenesis is perturbed in Crx-/- photoreceptors + +The Crx-/- mouse demonstrates the most severe abnormality of photoreceptor synapses reported to date. The peripherin-/- mouse develops a normal complement of photoreceptor terminals which then degenerate as the photoreceptors are lost [30]. Also, similarly in rhodopsin (Rho) and cyclic nucleotide-gated channel, alpha-3 (CNGA3) double knockout mice (Rho-/-, CNGA3-/-), synapses are reported to form normally [49]. These observations demonstrate that photoreceptor synaptogenesis can occur in the absence of outer segment formation. In keeping with this observation is the fact that some electroretinogram activity is present in peripherin-/- mice, suggesting that minimal phototransduction is present in these mice, enough to drive activity at the photoreceptor synapse. In vitro studies wherein synapse elements are formed in the absence of proper outer segment development and, therefore, possible absence of light-dependent photoreceptor activity, have indicated the independence of phototransduction and synapse formation, at least for the initial stages [50,51]. These data then suggest that the fact that the Crx-/- photoreceptors do not have proper synaptic endings is not due to a lack of outer segment formation. A more likely explanation is that Crx plays a role in photoreceptor synapse formation, perhaps by regulating directly, or indirectly, important genes in this process. Using immunohistochemistry, we examined the expression of common pre-synaptic terminal proteins, including KIF3a, SV2, and synaptophysin, and were unable to observe qualitative differences between Crx-/- and control tissue at P14 (data not shown). Examination of their RNA levels by SAGE showed no significant difference for all 3 genes, though very few tags were recovered from these genes and thus the analysis of RNA levels may not be significant [35]. However, since other genes expressed in photoreceptors were significantly altered in their expression level in the Crx-/- mouse, there are many candidates that could be important for photoreceptor morphogenesis. Tags from three genes from proteins expressed in photoreceptor terminals were found to be decreased in a statistically significant fashion, namely the HGF-regulated tyrosine kinase substrate, the CRIPT protein, and synaptotagmin 1 (Blackshaw and Cepko, unpublished data). An example of a gene that was increased in the Crx-/- retina is HRG4 (a homologue of the C. elegans Unc119 gene) (Blackshaw and Cepko, unpublished data) which encodes a component of the ribbon synapse [33]. The fact that it is upregulated might indicate a response to the lack of proper terminal structures. Several other genes encoding putative cytoskeletal elements also were increased (e.g. microtubule associated protein 4) or decreased (e.g. cofilin 1) in the Crx-/- retina, with P values of <.005. It is not known whether any of these genes are involved in building or regulating synaptic structures, but they are now genes that might lead to a better understanding of the construction and function of the relatively unique structure of the ribbon synapse. + +Abnormal photoreceptor terminal formation was noted in a study that examined retinal development in the laminin beta2 chain-deficient mouse [52]. Several pathologies were noted in these mice. First, laminin beta2 chain-deficient mice displayed abnormal outer segment elongation, but a more mild phenotype than that of the Crx-/- mice; the outer segments were reduced by 50% in length. Also photoreceptor terminals were perturbed in laminin beta2 mutants, but again the phenotype was more subtle then that of Crx-/- mice. The outer plexiform layer of the beta2-deficient retinas demonstrated only 7% normal invaginating synapses, while the remainder had various pathologies, including floating synaptic ribbons, as seen here. The mechanistic relationship of these two molecules, if any, in photoreceptor morphogenesis is unknown to date. The mRNA for laminin beta2 was not detected in the SAGE study of the relative RNA levels in Crx-/- and Crx+/+ and thus we cannot comment on whether the levels of RNA for laminin beta2 were altered. + +Crx-/- mice are a model for LCA + +Crx has been implicated in three photoreceptor diseases that result in human blindness, cone-rod dystrophy2, Leber's congenital amaurosis, and retinitis pigmentosa (for review, see [53]). The cone-rod dystrophies (CRDs) are characterized by loss of cone-mediated vision in the first decade of life or later, with concomitant or subsequent loss of rod-mediated vision [54]. Conversely, RP is notable for initial loss of rod function, followed by loss of cone-mediated vision [55]. The majority of known genes responsible for human genetic blindness, encode proteins expressed almost exclusively, or exclusively, in photoreceptors, particularly in the outer segment [35]. Many of these proteins are required for phototransduction or outer segment structure. The mechanisms whereby mutations in rod-specific genes, such as rhodopsin, lead eventually to cone degeneration in RP remain obscure. Mutations in Crx were the first, and still one of a very few examples of a transcription factor mutation leading to photoreceptor disease. + +LCA is a disease in which there is little or no photoreceptor function in infancy; thereby, likely developmental in etiology ([17,56] for review). The Crx-/- mouse may be an excellent model for studying the pathology of this disorder, particularly the subtype of the disorder where Crx mutations are involved. The vast majority of histopathological studies of LCA in human tissue have been derived from adult patients with LCA where secondary changes are likely to be present. Indeed in animal models of LCA, secondary reactive and/or degenerative changes occur early after the abnormal formation of retinal tissue [57]. The only study in human tissue derived from a human 33-week retina with proposed RPE65 mutations was reported to have abnormal retinae at this early stage [24]. These authors report cell loss, including thinning of the photoreceptor layer. In addition, they claim in the text to have seen aberrant synaptic and inner retinal organization, although their examination of photoreceptor synapses unfortunately are not presented in the data section of the paper. Given the scarcity of available human tissue, the characterization of the primary pathology of LCA will require animal models. In the current study, we present data that argue that, in addition to outer segment morphogenesis, synaptogenesis may also be critically impaired in at least a subset of LCA. + +Methods + +Mice + +Crx-/- mice were generated as reported elsewhere [34]. Rhodosin-null mice [29] were obtained from Paul Sieving (University of Michigan). Rds mice were acquired from Jackson Laboratory. + +Transmission electron microscopy + +Littermate Crx-/- and wildtype pups were perfused in 1% formaldehyde and 0.5% glutaraldehyde at various postnatal stages. The eyes were then enucleated, and the cornea and lens were removed. The eye cup was immersed in fixative (1% formaldehyde and 2.5% glutaraldehyde) for 3 to 4 hours at 4°C. The sclera was then partially removed and the retinas were sliced into small pieces and fixed (1% paraformaldehyde and 2.5% glutaraldehyde) overnight at 4°C. These procedures were found optimal for maintaining the structural integrity of the photoreceptor outer segments. + +After fixing, the tissue was washed 2X in PBS for thirty minutes per wash. The tissue was then postfixed in a 1% osmium tetroxide/1.5% potassium ferrocyanide mixture for 2 hours at 4°C. Staining was carried out for 30 minutes in 1% uranyl acetate in maleate buffer (pH = 6.0) at room temperature followed by 1% tannic acid in 0.1 M cacodylate buffer (pH = 7.4) for thirty minutes. The specimens were then dehydrated and embedded in Epon/Araldite. Thin sections were stained with uranyl acetate and lead citrate, and examined in a Jeol JEM-1200EX electron microscope. + +Scanning electron microscopy + +Specimens used for SEM required removal of the retinal pigment epithelium (PE), enabling visualization of the outer surface of the neural retina. Retinae from Crx-/-, rhodopsin-/-, RDS, or wildtype eyes were dissected free from PE in a dispase solution and fixed in 1.25% glutaraldehyde and 1.0% formaldehyde overnight at 4°C. Tissue was then washed 5X in cacodylate buffer and dehydrated in ascending grades of ethanol. Tissues were subsequently critical point dried in carbon dioxide. All specimens were mounted and coated with sublimated gold-palladium by the sputtering technique. Micrographs were obtained with a Jeol JSM-35CF scanning electron microscope. + +Authors' contributions + +EM and ER conducted transmission electron microscopy. EM performed scanning electron microscopy. TF and EM generated, characterized and maintained the Crx-/- mouse line. CC participated in the design and coordination of the study and all data analysis. EM and CC drafted the manuscript. All authors read and approved the final manuscript. + +Acknowledgements + +We would like to thank Heather Regan and Dr. Susumu Ito for helping with ultrastructural analyses and Dr. David Papermaster for helpful discussions on the formation of outer segments. diff --git a/src/ontogpt/evaluation/craft/database/all/15760270.ann b/src/ontogpt/evaluation/craft/database/all/15760270.ann new file mode 100644 index 000000000..6eb9266c9 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15760270.ann @@ -0,0 +1,1909 @@ +T1 PR:000013059 0 6 PGC-1α +T2 UBERON:0000467 31 37 System +T3 GO:0008152 45 54 Metabolic +T4 GO:0065007 105 112 Control +T5 UBERON:0002107 117 124 Hepatic +T6 http://purl.obolibrary.org/obo/MONDO_0004790 117 134 Hepatic Steatosis +T7 SO:0000704 150 154 gene +T8 GO:0005777 196 206 peroxisome +T9 PR:000013059 196 255 peroxisome proliferator-activated receptor-γ coactivator-1α +T10 PR:000013059 257 263 PGC-1α +T11 NCBITaxon:10088 281 285 mice +T12 PR:000013059 287 293 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GO:0005739 1827 1840 mitochondrial +T66 GO:0065007 1900 1910 regulatory +T67 GO:0065007 1925 1932 control +T68 GO:0010467 1948 1958 expression +T69 GO:0005634 1962 1969 nuclear +T70 SO:0000087 1962 1969;1988 1993 nuclear ... genes +T71 GO:0005739 1974 1987 mitochondrial +T72 SO:0000088 1974 1993 mitochondrial genes +T73 GO:0005739 2006 2019 mitochondrial +T74 GO:0008152 2031 2041 metabolism +T75 GO:0005777 2127 2137 peroxisome +T76 PR:000013059 2127 2171;2180 2194 peroxisome proliferator-activated receptor-γ ... coactivator-1α +T77 PR:000013059 2173 2178;2180 2194 PPARγ ... coactivator-1α +T78 PR:000013059 2196 2202 PGC-1α +T79 GO:0065007 2239 2249 regulatory +T80 GO:0031323 2292 2311;2319 2329 control of cellular ... metabolism +T81 GO:0005739 2341 2354 mitochondrial +T82 PR:000013059 2386 2392 PGC-1α +T83 UBERON:0001348 2441 2454 brown adipose +T84 GO:0060612 2495 2505 adipogenic +T85 GO:0005634 2506 2513 nuclear +T86 PR:000013058 2523 2528 PPARγ +T87 PR:000013159 2601 2626 PGC-1 related coactivator +T88 PR:000013159 2628 2631 PRC +T89 PR:000013060 2641 2647 PGC-1β +T90 PR:000013059 2655 2661 PGC-1α +T91 GO:0005634 2712 2719 nuclear +T92 GO:0005634 2727 2734 nuclear +T93 GO:0044237 2778 2786;2794 2804 cellular ... metabolism +T94 PR:000013059 2810 2816 PGC-1α +T95 UBERON:0000479 2875 2881 tissue +T96 GO:0010467 2891 2901 expression +T97 GO:0005739 2993 3006 mitochondrial +T98 GO:0006754 3007 3010;3019 3029 ATP ... production +T99 GO:0031649 3014 3029 heat production +T100 PR:000013059 3039 3045 PGC-1α +T101 UBERON:0001348 3061 3081 brown adipose tissue +T102 UBERON:0001348 3083 3086 BAT +T103 UBERON:0000948 3089 3094 heart +T104 UBERON:0006907 3096 3123 slow-twitch skeletal muscle +T105 UBERON:0002113 3129 3135 kidney +T106 UBERON:0000479 3140 3147 tissues +T107 GO:0005739 3167 3180 mitochondrial +T108 GO:0010467 3194 3204 expression +T109 SO:0000704 3212 3216 gene +T110 PR:000013059 3226 3232 PGC-1α +T111 PR:000013059 3369 3375 PGC-1α +T112 GO:0019222 3407 3417;3425 3435 control of ... metabolism +T113 GO:0010467 3493 3503 expression +T114 PR:000013059 3527 3533 PGC-1α +T115 GO:0005739 3559 3572 mitochondrial +T116 GO:0065007 3588 3596 regulate +T117 GO:0005739 3597 3610 mitochondrial +T118 GO:0045333 3611 3622 respiratory +T119 PR:000013059 3640 3646 PGC-1α +T120 GO:0005739 3678 3691 mitochondrial +T121 PR:000017035 3678 3712 mitochondrial uncoupling protein-1 +T122 PR:000017035 3714 3719 UCP-1 +T123 UBERON:0001348 3724 3727 BAT +T124 GO:0005634 3758 3765 nuclear +T125 PR:000013058 3784 3789 PPARγ +T126 UBERON:0002046 3794 3801 thyroid +T127 GO:0010467 3831 3841 expression +T128 GO:0060612 3853 3863 adipogenic +T129 NCBITaxon:40674 3877 3886 mammalian +T130 PR:000013059 3916 3922 PGC-1α +T131 GO:0005739 3933 3946 mitochondrial +T132 GO:0005634 4016 4023 nuclear +T133 PR:000011422 4016 4045 nuclear respiratory factors 1 +T134 GO:0045333 4024 4035 respiratory +T135 PR:000011422 4053 4058 NRF-1 +T136 GO:0005739 4071 4084 mitochondrial +T137 PR:000016262 4071 4107 mitochondrial transcription factor A +T138 PR:000016262 4109 4113 Tfam +T139 GO:0005739 4150 4163 mitochondrial +T140 GO:0006264 4150 4167;4186 4197 mitochondrial DNA ... replication +T141 UBERON:0000948 4229 4236 cardiac +T142 CL:0000746 4229 4245 cardiac myocytes +T143 UBERON:0000948 4268 4274 hearts +T144 NCBITaxon:10088 4289 4293 mice +T145 GO:0010467 4321 4331 expression +T146 PR:000013059 4335 4341 PGC-1α +T147 GO:0005739 4351 4364 mitochondrial +T148 GO:0010467 4400 4410 expression +T149 PR:000013059 4414 4420 PGC-1α +T150 UBERON:0004288 4424 4432 skeletal +T151 NCBITaxon:10088 4454 4458 mice +T152 GO:0005739 4468 4481 mitochondrial +T153 GO:0048747 4504 4516;4570 4583 formation of ... muscle fibers +T154 GO:0005739 4517 4530 mitochondrial +T155 CL:0002211 4536 4542;4570 4583 type I ... muscle fibers +T156 MOP:0000568 4544 4553 oxidative +T157 CL:0002210 4544 4553;4570 4583 oxidative ... muscle fibers +T158 CL:0000189 4556 4567;4570 4583 slow-twitch ... muscle fibers +T159 PR:000013059 4632 4638 PGC-1α +T160 GO:0005739 4662 4675 mitochondrial +T161 PR:000013059 4721 4727 PGC-1α +T162 GO:0042592 4735 4754 homeostatic control +T163 GO:0019222 4747 4757;4774 4784 control of ... metabolism +T164 PR:000013059 4786 4792 PGC-1α +T165 GO:0065007 4811 4819 regulate +T166 UBERON:0002107 4832 4839 hepatic +T167 GO:0006094 4840 4853 gluconeogenic +T168 SO:0000704 4854 4859 genes +T169 GO:0010467 4914 4924 expression +T170 PR:000013059 4928 4934 PGC-1α +T171 GO:0005739 4950 4963 mitochondrial +T172 UBERON:0004288 4983 4991 skeletal +T173 NCBITaxon:9606 5002 5008 humans +T174 PR:000045358 5014 5021 insulin +T175 http://purl.obolibrary.org/obo/MONDO_0005015 5037 5045 diabetes +T176 SO:0000694 5071 5102 single nucleotide polymorphisms +T177 NCBITaxon:9606 5114 5119 human +T178 PR:000013059 5120 5126 PGC-1α +T179 SO:0000704 5127 5131 gene +T180 http://purl.obolibrary.org/obo/MONDO_0011122 5170 5177 obesity +T181 http://purl.obolibrary.org/obo/MONDO_0005044 5179 5191 hypertension +T182 http://purl.obolibrary.org/obo/MONDO_0005015 5197 5205 diabetes +T183 PR:000013059 5308 5314 PGC-1α +T184 GO:0065007 5329 5339 regulating +T185 GO:0007567 5344 5349 natal +T186 GO:0008152 5357 5367 metabolism +T187 PR:000013059 5395 5401 PGC-1α +T188 GO:0008152 5413 5422 metabolic +T189 GO:0042592 5423 5434 homeostasis +T190 GO:0005739 5436 5449 mitochondrial +T191 NCBITaxon:10088 5594 5598 mice +T192 PR:000013059 5629 5635 PGC-1α +T193 SO:0000704 5636 5640 gene +T194 PR:000013059 5657 5663 PGC-1α +T195 NCBITaxon:10088 5667 5671 mice +T196 PR:000013059 5689 5695 PGC-1α +T197 GO:0007567 5730 5735 natal +T198 GO:0005739 5756 5769 mitochondrial +T199 PR:000013059 5834 5840 PGC-1α +T200 GO:0008152 5878 5887 metabolic +T201 UBERON:0000062 5909 5915 organs +T202 GO:0051866 5936 5968 adaptation to physiologic stress +T203 GO:0007567 5980 5985 natal +T204 PR:000013059 6020 6026 PGC-1α +T205 SO:0000704 6027 6031 Gene +T206 NCBITaxon:10088 6035 6039 Mice +T207 SO:0000704 6058 6062 gene +T208 SO:0001644 6063 6079 targeting vector +T209 SO:0000147 6104 6109 exons +T210 NCBITaxon:39107 6125 6131 murine +T211 PR:000013059 6132 6138 PGC-1α +T212 SO:0000704 6139 6143 gene +T213 SO:0000147 6382 6386 exon +T214 SO:0000147 6409 6414 exons +T215 SO:0001023 6555 6561 allele +T216 PR:000013059 6644 6650 PGC-1α +T217 SO:0000704 6651 6655 gene +T218 SO:0000673 6691 6701 transcript +T219 UBERON:0000948 6753 6758 heart +T220 UBERON:0000479 6769 6776 tissues +T221 PR:000013059 6780 6786 PGC-1α +T222 NCBITaxon:10088 6790 6794 mice +T223 SO:0000704 6902 6906 gene +T224 SO:0000112 6941 6948 primers +T225 SO:0000833 6976 6985;7002 7012 region of ... transcript +T226 PR:000013059 6990 6996 PGC-1α +T227 SO:0000704 6997 7001 gene +T228 SO:0000333 7028 7032;7037 7043 exon ... border +T229 PR:000013059 7071 7077 PGC-1α +T230 NCBITaxon:10088 7081 7085 mice +T231 SO:0000333 7094 7098;7103 7109 exon ... border +T232 PR:000013059 7147 7153 PGC-1α +T233 NCBITaxon:10088 7157 7161 mice +T234 SO:0000147 7168 7172 exon +T235 UBERON:0000948 7202 7207 heart +T236 UBERON:0001348 7212 7215 BAT +T237 PR:000013059 7242 7248 PGC-1α +T238 NCBITaxon:10088 7252 7256 mice +T239 SO:0000147 7286 7290 exon +T240 PR:000013059 7323 7329 PGC-1α +T241 NCBITaxon:10088 7333 7337 mice +T242 SO:0000333 7354 7358;7365 7371 exon ... border +T243 PR:000013059 7448 7454 PGC-1α +T244 NCBITaxon:10088 7458 7462 mice +T245 PR:000013059 7498 7504 PGC-1α +T246 NCBITaxon:10088 7508 7512 mice +T247 SO:0000673 7541 7551 transcript +T248 PR:000013059 7565 7571 PGC-1α +T249 GO:0005634 7626 7633 nuclear +T250 UBERON:0001348 7684 7687 BAT +T251 PR:000013059 7691 7697 PGC-1α +T252 NCBITaxon:10088 7701 7705 mice +T253 GO:0010467 7803 7813 expression +T254 PR:000013059 7817 7823 PGC-1α +T255 UBERON:0001348 7827 7830 BAT +T256 PR:000013059 7847 7853 PGC-1α +T257 GO:0010467 7939 7949 expression +T258 SO:0000704 7957 7962 genes +T259 PR:000013060 8010 8016 PGC-1β +T260 PR:000013159 8021 8024 PRC +T261 UBERON:0000948 8059 8064 heart +T262 PR:000013059 8068 8074 PGC-1α +T263 NCBITaxon:10088 8078 8082 mice +T264 SO:0000704 8158 8162 gene +T265 PR:000013059 8193 8199 PGC-1α +T266 SO:0001023 8205 8211 allele +T267 PR:000013059 8245 8251 PGC-1α +T268 NCBITaxon:10088 8255 8259 Mice +T269 http://purl.obolibrary.org/obo/MONDO_0011122 8284 8291 Obesity +T270 PR:000013059 8307 8313 PGC-1α +T271 NCBITaxon:10088 8318 8322 mice +T272 PR:000013059 8345 8351 PGC-1α +T273 GO:0016265 8494 8500 deaths +T274 PR:000013059 8541 8547 PGC-1α +T275 PR:000013059 8555 8561 PGC-1α +T276 GO:0007567 8631 8636 birth +T277 PR:000013059 8705 8711 PGC-1α +T278 NCBITaxon:10088 8715 8719 mice +T279 PR:000013059 8744 8750 PGC-1α +T280 PR:000013059 8807 8813 PGC-1α +T281 PR:000013059 8821 8827 PGC-1α +T282 PR:000013059 8970 8976 PGC-1α +T283 NCBITaxon:10088 8980 8984 mice +T284 PR:000013059 9009 9015 PGC-1α +T285 PR:000013059 9096 9102 PGC-1α +T286 NCBITaxon:10088 9106 9110 mice +T287 PR:000013059 9168 9174 PGC-1α +T288 NCBITaxon:10088 9178 9182 mice +T289 CHEBI:33290 9222 9226 food +T290 GO:0007631 9222 9233 food intake +T291 PR:000013059 9435 9441 PGC-1α +T292 NCBITaxon:10088 9445 9449 mice +T293 PR:000013059 9481 9487 PGC-1α +T294 PR:000013059 9747 9753 PGC-1α +T295 NCBITaxon:10088 9757 9761 mice +T296 NCBITaxon:10088 9803 9807 mice +T297 UBERON:0000062 9921 9926 organ +T298 GO:0005739 9974 9987 mitochondrial +T299 GO:0008152 9995 10005 metabolism +T300 GO:0007567 10014 10019 natal +T301 UBERON:0000062 10038 10044 organs +T302 UBERON:0000948 10061 10066 heart +T303 UBERON:0004288 10098 10106 skeletal +T304 UBERON:0001388 10126 10139 gastrocnemius +T305 UBERON:0001389 10144 10150 soleus +T306 MOP:0000568 10169 10178 oxidative +T307 UBERON:0001385 10179 10196 tibialis anterior +T308 PR:000013059 10242 10248 PGC-1α +T309 NCBITaxon:10088 10252 10256 mice +T310 PR:000013059 10291 10297 PGC-1α +T311 UBERON:0000955 10393 10398 brain +T312 UBERON:0002107 10400 10405 liver +T313 UBERON:0002113 10407 10413 kidney +T314 UBERON:0001348 10419 10422 BAT +T315 UBERON:0000479 10528 10535 tissues +T316 GO:0005739 10546 10559 mitochondrial +T317 UBERON:0000948 10589 10594 heart +T318 UBERON:0006907 10599 10626 slow-twitch skeletal muscle +T319 PR:000013059 10661 10667 PGC-1α +T320 NCBITaxon:10088 10671 10675 mice +T321 GO:0005739 10694 10707 Mitochondrial +T322 PR:000013059 10721 10727 PGC-1α +T323 NCBITaxon:10088 10731 10735 Mice +T324 GO:0007567 10837 10842 natal +T325 UBERON:0000948 10843 10848 heart +T326 UBERON:0004288 10853 10861 skeletal +T327 PR:000013059 10876 10882 PGC-1α +T328 NCBITaxon:10088 10886 10890 mice +T329 GO:0031012 10958 10978 extracellular matrix +T330 UBERON:0000479 10986 10993 tissues +T331 PR:000013059 11008 11014 PGC-1α +T332 NCBITaxon:10088 11018 11022 mice +T333 PR:000013059 11071 11077 PGC-1α +T334 GO:0005739 11081 11094 mitochondrial +T335 GO:0005739 11132 11145 mitochondrial +T336 UBERON:0000479 11177 11184 tissues +T337 CHEBI:10545 11186 11194 Electron +T338 GO:0005739 11243 11255 mitochondria +T339 UBERON:0001389 11259 11272 soleus muscle +T340 PR:000013059 11276 11282 PGC-1α +T341 NCBITaxon:10088 11286 11290 mice +T342 PR:000013059 11325 11331 PGC-1α +T343 CHEBI:10545 11389 11397 electron +T344 UBERON:0001389 11456 11462 soleus +T345 GO:0005739 11463 11475 mitochondria +T346 PR:000013059 11503 11509 PGC-1α +T347 NCBITaxon:10088 11513 11517 mice +T348 PR:000013059 11530 11536 PGC-1α +T349 GO:0030016 11579 11591 myofibrillar +T350 GO:0005739 11643 11656 mitochondrial +T351 GO:0010467 11697 11707 expression +T352 GO:0005634 11711 11718 nuclear +T353 SO:0000087 11711 11724 nuclear genes +T354 GO:0005739 11755 11768 mitochondrial +T355 CHEBI:10545 11769 11777 electron +T356 MOP:0000615 11769 11787 electron transport +T357 PR:000002199 11789 11801 cytochrome c +T358 CHEBI:18070 11789 11801 cytochrome c +T359 CHEBI:4056 11806 11816 cytochrome +T360 MOP:0000568 11833 11842 oxidative +T361 GO:0006119 11833 11858 oxidative phosphorylation +T362 PR:000004454 11860 11888 beta subunit of ATP synthase +T363 UBERON:0001389 11893 11906 soleus muscle +T364 PR:000013059 11910 11916 PGC-1α +T365 NCBITaxon:10088 11920 11924 mice +T366 PR:000013059 11939 11945 PGC-1α +T367 GO:0010467 11976 11986 expression +T368 PR:000016262 11990 11994 Tfam +T369 PR:000013059 12004 12010 PGC-1α +T370 GO:0005739 12030 12043 mitochondrial +T371 GO:0006264 12030 12059 mitochondrial DNA replication +T372 PR:000013059 12093 12099 PGC-1α +T373 UBERON:0001389 12103 12109 soleus +T374 GO:0005739 12159 12172 mitochondrial +T375 UBERON:0001389 12229 12235 soleus +T376 GO:0005739 12267 12280 mitochondrial +T377 UBERON:0000948 12328 12333 heart +T378 UBERON:0001348 12337 12340 BAT +T379 PR:000013059 12344 12350 PGC-1α +T380 NCBITaxon:10088 12354 12358 mice +T381 GO:0005739 12401 12414 mitochondrial +T382 UBERON:0004288 12443 12451 skeletal +T383 PR:000013059 12462 12468 PGC-1α +T384 NCBITaxon:10088 12472 12476 mice +T385 GO:0005739 12478 12491 mitochondrial +T386 GO:0045333 12492 12503 respiration +T387 UBERON:0000479 12530 12536 tissue +T388 UBERON:0001389 12558 12571 soleus muscle +T389 UBERON:0001389 12576 12582 soleus +T390 PR:000013059 12586 12592 PGC-1α +T391 NCBITaxon:10088 12596 12600 mice +T392 GO:0045333 12651 12662 respiration +T393 GO:0045333 12756 12767 respiration +T394 CHEBI:25675 12794 12804 oligomycin +T395 GO:0045333 12879 12890 respiration +T396 GO:0006754 12894 12908 ATP production +T397 PR:000013059 12942 12948 PGC-1α +T398 NCBITaxon:10088 12952 12956 mice +T399 GO:0005739 13032 13045 mitochondrial +T400 UBERON:0004288 13071 13079 Skeletal +T401 PR:000013059 13099 13105 PGC-1α +T402 NCBITaxon:10088 13109 13113 Mice +T403 GO:0005739 13134 13147 mitochondrial +T404 GO:0045333 13159 13170 respiratory +T405 UBERON:0004288 13183 13191 skeletal +T406 UBERON:0004288 13230 13238 skeletal +T407 GO:0040011 13289 13298 locomotor +T408 PR:000013059 13375 13381 PGC-1α +T409 NCBITaxon:10088 13390 13394 mice +T410 GO:0040011 13442 13453 ambulations +T411 PR:000013059 13499 13505 PGC-1α +T412 PR:000013059 13603 13609 PGC-1α +T413 NCBITaxon:10088 13613 13617 mice +T414 PR:000013059 13706 13712 PGC-1α +T415 NCBITaxon:10088 13716 13720 mice +T416 PR:000013059 14115 14121 PGC-1α +T417 NCBITaxon:10088 14125 14129 mice +T418 GO:0001966 14205 14216 thigmotaxis +T419 UBERON:0025534 14350 14362 sensorimotor +T420 PR:000013059 14380 14386 PGC-1α +T421 NCBITaxon:10088 14390 14394 mice +T422 PR:000013059 14430 14436 PGC-1α +T423 NCBITaxon:10088 14440 14444 mice +T424 PR:000013059 14449 14455 PGC-1α +T425 UBERON:0025534 14594 14606 sensorimotor +T426 PR:000013059 14636 14642 PGC-1α +T427 NCBITaxon:10088 14646 14650 mice +T428 PR:000013059 14665 14671 PGC-1α +T429 NCBITaxon:10088 14675 14679 mice +T430 PR:000013059 14743 14749 PGC-1α +T431 UBERON:0004288 15074 15082 skeletal +T432 PR:000013059 15139 15145 PGC-1α +T433 NCBITaxon:10088 15149 15153 mice +T434 PR:000013059 15172 15178 PGC-1α +T435 NCBITaxon:10088 15182 15186 mice +T436 PR:000013059 15271 15277 PGC-1α +T437 NCBITaxon:10088 15281 15285 mice +T438 PR:000013059 15369 15375 PGC-1α +T439 NCBITaxon:10088 15379 15383 mice +T440 PR:000013059 15407 15413 PGC-1α +T441 NCBITaxon:10088 15417 15421 mice +T442 PR:000013059 15490 15496 PGC-1α +T443 NCBITaxon:10088 15500 15504 mice +T444 PR:000013059 15814 15820 PGC-1α +T445 NCBITaxon:10088 15824 15828 mice +T446 CHEBI:15379 15845 15847 O2 +T447 PR:000013059 15876 15882 PGC-1α +T448 CHEBI:15379 15911 15913 O2 +T449 UBERON:0001389 16042 16055 soleus muscle +T450 GO:0005739 16150 16163 mitochondrial +T451 GO:0006754 16164 16178 ATP production +T452 PR:000013059 16301 16307 PGC-1α +T453 NCBITaxon:10088 16311 16315 mice +T454 PR:000013059 16320 16326 PGC-1α +T455 GO:0006936 16463 16475 contractions +T456 PR:000013059 16508 16514 PGC-1α +T457 NCBITaxon:10088 16518 16522 mice +T458 PR:000013059 16549 16555 PGC-1α +T459 UBERON:0004288 16654 16662 skeletal +T460 GO:0005739 16670 16683 mitochondrial +T461 PR:000013059 16722 16728 PGC-1α +T462 GO:0043501 16757 16786 adaptation of skeletal muscle +T463 UBERON:0004288 16771 16779 skeletal +T464 UBERON:0000948 16840 16846 Hearts +T465 PR:000013059 16850 16856 PGC-1α +T466 NCBITaxon:10088 16860 16864 Mice +T467 PR:000013059 16866 16872 PGC-1α +T468 GO:0010467 16873 16883 expression +T469 UBERON:0000948 16899 16904 heart +T470 UBERON:0000479 16908 16914 tissue +T471 GO:0005739 16938 16951 mitochondrial +T472 GO:0008152 16959 16969 metabolism +T473 GO:0007567 17015 17020 natal +T474 UBERON:0000104 17021 17025 life +T475 NCBITaxon:40674 17033 17051 mammalian organism +T476 PR:000013059 17092 17098 PGC-1α +T477 NCBITaxon:10088 17102 17106 mice +T478 UBERON:0004151 17168 17175 chamber +T479 UBERON:0002082 17185 17196 ventricular +T480 UBERON:0000948 17250 17257 Cardiac +T481 GO:0008152 17273 17282 metabolic +T482 PR:000013059 17396 17402 PGC-1α +T483 NCBITaxon:10088 17406 17410 mice +T484 PR:000013059 17677 17683 PGC-1α +T485 NCBITaxon:33208 17695 17702 animals +T486 PR:000013059 17780 17786 PGC-1α +T487 NCBITaxon:10088 17790 17794 mice +T488 PR:000013059 17896 17902 PGC-1α +T489 PR:000013059 17949 17955 PGC-1α +T490 NCBITaxon:10088 17959 17963 mice +T491 UBERON:0002084 18027 18043 left ventricular +T492 UBERON:0000948 18070 18075 heart +T493 UBERON:0000948 18154 18159 heart +T494 PR:000013059 18172 18178 PGC-1α +T495 NCBITaxon:10088 18182 18186 mice +T496 UBERON:0002084 18313 18329 left ventricular +T497 PR:000013059 18373 18379 PGC-1α +T498 NCBITaxon:10088 18383 18387 mice +T499 PR:000013059 18401 18407 PGC-1α +T500 NCBITaxon:10088 18411 18415 mice +T501 UBERON:0000948 18542 18549 cardiac +T502 UBERON:0004288 18653 18661 skeletal +T503 UBERON:0000948 18701 18708 cardiac +T504 UBERON:0000948 18723 18729 hearts +T505 PR:000013059 18733 18739 PGC-1α +T506 PR:000013059 18747 18753 PGC-1α +T507 NCBITaxon:10088 18757 18761 mice +T508 UBERON:0000948 18810 18816 Hearts +T509 PR:000013059 18831 18837 PGC-1α +T510 NCBITaxon:10088 18841 18845 mice +T511 UBERON:0000948 18862 18869 cardiac +T512 UBERON:0000948 18876 18883 cardiac +T513 PR:000013059 18941 18947 PGC-1α +T514 NCBITaxon:10088 18951 18955 mice +T515 UBERON:0000948 19017 19024 cardiac +T516 UBERON:0000948 19051 19058 cardiac +T517 UBERON:0000948 19106 19111 heart +T518 UBERON:0000948 19149 19156 cardiac +T519 PR:000013059 19171 19177 PGC-1α +T520 NCBITaxon:10088 19181 19185 mice +T521 UBERON:0000948 19334 19339 heart +T522 UBERON:0002082 19349 19360 ventricular +T523 CHEBI:4670 19442 19452 dobutamine +T524 UBERON:0002082 19509 19520 ventricular +T525 CHEBI:4670 19544 19554 dobutamine +T526 PR:000013059 19570 19576 PGC-1α +T527 PR:000013059 19584 19590 PGC-1α +T528 NCBITaxon:10088 19594 19598 mice +T529 PR:000013059 19634 19640 PGC-1α +T530 NCBITaxon:10088 19644 19648 mice +T531 UBERON:0000948 19683 19688 heart +T532 PR:000013059 19825 19831 PGC-1α +T533 UBERON:0000948 19835 19841 hearts +T534 GO:0002027 19877 19889 chronotropic +T535 UBERON:0000948 19981 19986 heart +T536 GO:0006936 20037 20048 contractile +T537 PR:000013059 20063 20069 PGC-1α +T538 NCBITaxon:10088 20073 20077 Mice +T539 GO:0031649 20098 20109 Thermogenic +T540 PR:000013059 20120 20126 PGC-1α +T541 GO:0005739 20176 20189 mitochondrial +T542 GO:0045333 20190 20201 respiratory +T543 GO:0031649 20237 20252 heat production +T544 UBERON:0001348 20256 20259 BAT +T545 PR:000013059 20286 20292 PGC-1α +T546 GO:0031649 20325 20336 thermogenic +T547 PR:000013059 20347 20353 PGC-1α +T548 PR:000013059 20361 20367 PGC-1α +T549 NCBITaxon:10088 20371 20375 mice +T550 PR:000013059 20475 20481 PGC-1α +T551 NCBITaxon:10088 20485 20489 mice +T552 PR:000013059 20685 20691 PGC-1α +T553 NCBITaxon:10088 20695 20699 mice +T554 PR:000013059 20748 20754 PGC-1α +T555 GO:0031649 20782 20793 thermogenic +T556 NCBITaxon:10088 20832 20836 mice +T557 NCBITaxon:10088 20874 20878 mice +T558 CHEBI:18059 20938 20943 lipid +T559 UBERON:0001348 20954 20957 BAT +T560 GO:0031649 21011 21022 thermogenic +T561 PR:000013059 21046 21052 PGC-1α +T562 NCBITaxon:10088 21056 21060 mice +T563 CHEBI:18059 21077 21082 lipid +T564 CHEBI:10545 21122 21130 Electron +T565 GO:0005739 21171 21184 mitochondrial +T566 UBERON:0001348 21215 21218 BAT +T567 PR:000013059 21233 21239 PGC-1α +T568 PR:000013059 21247 21253 PGC-1α +T569 NCBITaxon:10088 21257 21261 mice +T570 UBERON:0001348 21336 21339 BAT +T571 CHEBI:17855 21340 21352 triglyceride +T572 PR:000017035 21412 21417 UCP-1 +T573 GO:0005739 21458 21471 mitochondrial +T574 GO:0045333 21472 21483 respiratory +T575 UBERON:0001348 21515 21518 BAT +T576 PR:000017035 21520 21525 UCP-1 +T577 SO:0000704 21526 21530 gene +T578 PR:000013059 21573 21579 PGC-1α +T579 UBERON:0001348 21622 21625 BAT +T580 PR:000017035 21626 21631 UCP-1 +T581 PR:000013059 21660 21666 PGC-1α +T582 PR:000013059 21674 21680 PGC-1α +T583 NCBITaxon:10088 21684 21688 mice +T584 PR:000013059 21729 21735 PGC-1α +T585 GO:0010467 21778 21788 expression +T586 PR:000017035 21792 21797 UCP-1 +T587 GO:0005739 21871 21884 mitochondrial +T588 GO:0045333 21885 21896 respiration +T589 GO:0031649 21932 21943 thermogenic +T590 PR:000013059 21960 21966 PGC-1α +T591 NCBITaxon:10088 21970 21974 mice +T592 GO:0031649 21977 21990 Thermogenesis +T593 NCBITaxon:9989 21994 22001 rodents +T594 GO:0005739 22013 22026 mitochondrial +T595 GO:0065007 22051 22058 control +T596 PR:000013059 22202 22208 PGC-1α +T597 NCBITaxon:10088 22212 22216 mice +T598 PR:000013059 22419 22425 PGC-1α +T599 PR:000013059 22437 22443 PGC-1α +T600 NCBITaxon:10088 22447 22451 mice +T601 GO:0008152 22497 22506 metabolic +T602 UBERON:0001348 22519 22522 BAT +T603 PR:000013059 22608 22614 PGC-1α +T604 NCBITaxon:10088 22620 22624 mice +T605 GO:0005739 22665 22678 mitochondrial +T606 GO:0045333 22679 22690 respiratory +T607 UBERON:0002107 22720 22727 Hepatic +T608 http://purl.obolibrary.org/obo/MONDO_0004790 22720 22737 Hepatic Steatosis +T609 PR:000013059 22741 22747 PGC-1α +T610 NCBITaxon:10088 22751 22755 Mice +T611 PR:000013059 22790 22796 PGC-1α +T612 UBERON:0002107 22808 22815 hepatic +T613 GO:0008152 22816 22825 metabolic +T614 CHEBI:35366 22846 22856 fatty acid +T615 GO:0019395 22846 22866 fatty acid oxidation +T616 MOP:0000568 22857 22866 oxidation +T617 GO:0006094 22871 22886 gluconeogenesis +T618 UBERON:0002107 22919 22926 hepatic +T619 CHEBI:35366 23028 23038 fatty acid +T620 GO:0019395 23028 23048 fatty acid oxidation +T621 MOP:0000568 23039 23048 oxidation +T622 GO:0006094 23053 23066 gluconeogenic +T623 UBERON:0002107 23076 23081 liver +T624 GO:0007631 23095 23098 fed +T625 UBERON:0002107 23115 23121 livers +T626 PR:000013059 23129 23135 PGC-1α +T627 NCBITaxon:10088 23139 23143 mice +T628 PR:000013059 23269 23275 PGC-1α +T629 NCBITaxon:10088 23279 23283 mice +T630 UBERON:0002107 23301 23308 hepatic +T631 http://purl.obolibrary.org/obo/MONDO_0004790 23301 23318 hepatic steatosis +T632 CHEBI:10545 23374 23382 electron +T633 UBERON:0002107 23415 23420 liver +T634 CHEBI:17855 23421 23433 triglyceride +T635 CHEBI:17855 23435 23438 TAG +T636 UBERON:0001969 23488 23494 plasma +T637 CHEBI:17855 23495 23508 triglycerides +T638 CHEBI:35366 23517 23528 fatty acids +T639 GO:0007631 23554 23557 fed +T640 UBERON:0002107 23665 23672 hepatic +T641 http://purl.obolibrary.org/obo/MONDO_0004790 23665 23682 hepatic steatotic +T642 CL:0000182 23693 23704 hepatocytes +T643 PR:000013059 23724 23730 PGC-1α +T644 NCBITaxon:10088 23734 23738 mice +T645 CHEBI:30823 23756 23762 Oleate +T646 PR:000013059 23801 23807 PGC-1α +T647 CL:0000182 23811 23822 hepatocytes +T648 CHEBI:18059 23843 23848 lipid +T649 CHEBI:29238 23914 23916 3H +T650 CHEBI:7896 23917 23926 palmitate +T651 MOP:0000568 23927 23936 oxidation +T652 PR:000013059 23971 23977 PGC-1α +T653 CL:0000182 23981 23992 hepatocytes +T654 PR:000013059 24005 24011 PGC-1α +T655 CL:0000182 24015 24026 hepatocytes +T656 CHEBI:30823 24076 24082 oleate +T657 PR:000013059 24170 24176 PGC-1α +T658 CL:0000182 24180 24191 hepatocytes +T659 CHEBI:18059 24242 24247 lipid +T660 GO:0006869 24284 24301 delivery of lipid +T661 CHEBI:18059 24296 24301 lipid +T662 UBERON:0002107 24359 24366 hepatic +T663 GO:0005739 24367 24380 mitochondrial +T664 CHEBI:35366 24381 24391 fatty acid +T665 GO:0019395 24381 24401 fatty acid oxidation +T666 MOP:0000568 24392 24401 oxidation +T667 SO:0000704 24409 24413 gene +T668 GO:0010467 24409 24424 gene expression +T669 PR:000013059 24458 24464 PGC-1α +T670 PR:000013059 24544 24550 PGC-1α +T671 NCBITaxon:10088 24554 24558 mice +T672 GO:0010467 24578 24588 expression +T673 SO:0000704 24604 24609 genes +T674 SO:0000704 24646 24651 genes +T675 SO:0000704 24656 24660 gene +T676 GO:0010467 24656 24671 gene expression +T677 UBERON:0002107 24728 24735 hepatic +T678 GO:0010467 24736 24746 expression +T679 SO:0000704 24762 24767 genes +T680 CHEBI:35366 24789 24799 fatty acid +T681 MOP:0000568 24804 24813 oxidation +T682 PR:000005836 24815 24819 MCPT +T683 GO:0007631 24891 24894 fed +T684 MOP:0000568 25008 25017 oxidation +T685 CL:0000182 25025 25036 hepatocytes +T686 PR:000013059 25044 25050 PGC-1α +T687 NCBITaxon:10088 25054 25058 mice +T688 GO:0005739 25082 25095 mitochondrial +T689 GO:0045333 25096 25107 respiratory +T690 PR:000013059 25147 25153 PGC-1α +T691 CL:0000182 25157 25168 hepatocytes +T692 GO:0045333 25242 25253 respiration +T693 UBERON:0002107 25356 25363 hepatic +T694 http://purl.obolibrary.org/obo/MONDO_0004790 25356 25373 hepatic steatosis +T695 MOP:0000568 25400 25409 oxidation +T696 GO:0005739 25417 25430 mitochondrial +T697 GO:0045333 25431 25442 respiratory +T698 UBERON:0002107 25470 25475 liver +T699 SO:0000704 25476 25480 gene +T700 GO:0010467 25476 25491 gene expression +T701 CHEBI:35366 25546 25556 fatty acid +T702 GO:0019395 25546 25566 fatty acid oxidation +T703 MOP:0000568 25557 25566 oxidation +T704 PR:000013059 25582 25588 PGC-1α +T705 NCBITaxon:10088 25592 25596 mice +T706 CHEBI:15889 25653 25659 sterol +T707 SO:0001861 25653 25678 sterol regulatory element +T708 PR:000028988 25653 25694 sterol regulatory element binding protein +T709 GO:0065007 25660 25670 regulatory +T710 PR:000028988 25699 25704 SREBP +T711 PR:000028988 25783 25788 SREBP +T712 SO:0000704 25807 25811 gene +T713 CHEBI:15541 25812 25824 stearoyl-CoA +T714 PR:000015921 25837 25841 SCD1 +T715 PR:000013059 25861 25867 PGC-1α +T716 NCBITaxon:10088 25871 25875 mice +T717 GO:0010467 25900 25910 expression +T718 SO:0000704 25918 25922 gene +T719 CHEBI:18035 25932 25943 diglyceride +T720 CHEBI:17855 26001 26004 TAG +T721 GO:0019432 26001 26014 TAG synthesis +T722 PR:000013059 26087 26093 PGC-1α +T723 NCBITaxon:10088 26097 26101 mice +T724 MOP:0000568 26168 26177 oxidation +T725 CHEBI:17855 26197 26200 TAG +T726 GO:0019432 26197 26210 TAG synthesis +T727 PR:000013059 26240 26246 PGC-1α +T728 NCBITaxon:10088 26250 26254 mice +T729 CHEBI:29238 26304 26306 3H +T730 CHEBI:17754 26307 26315 glycerol +T731 CHEBI:17855 26335 26338 TAG +T732 CL:0000182 26367 26378 hepatocytes +T733 CHEBI:29238 26380 26382 3H +T734 CHEBI:17855 26383 26386 TAG +T735 CL:0000182 26429 26440 hepatocytes +T736 PR:000013059 26455 26461 PGC-1α +T737 NCBITaxon:10088 26465 26469 mice +T738 PR:000013059 26482 26488 PGC-1α +T739 CHEBI:17855 26530 26533 TAG +T740 GO:0019432 26530 26543 TAG synthesis +T741 PR:000013059 26567 26573 PGC-1α +T742 CL:0000182 26579 26590 hepatocytes +T743 UBERON:0002107 26669 26676 hepatic +T744 http://purl.obolibrary.org/obo/MONDO_0004790 26669 26686 hepatic steatosis +T745 http://purl.obolibrary.org/obo/MONDO_0011122 26704 26709 Obese +T746 PR:000013059 26728 26734 PGC-1α +T747 NCBITaxon:10088 26738 26742 Mice +T748 PR:000045358 26758 26765 Insulin +T749 PR:000013059 26822 26828 PGC-1α +T750 SO:0000694 26829 26860 single nucleotide polymorphisms +T751 SO:0001024 26865 26875 haplotypes +T752 PR:000045358 26909 26916 insulin +T753 http://purl.obolibrary.org/obo/MONDO_0005015 26932 26940 diabetes +T754 PR:000045358 26990 26997 insulin +T755 http://purl.obolibrary.org/obo/MONDO_0005015 27012 27020 diabetic +T756 CHEBI:17234 27061 27068 glucose +T757 PR:000045358 27082 27089 insulin +T758 PR:000013059 27122 27128 PGC-1α +T759 NCBITaxon:10088 27132 27136 mice +T760 CHEBI:17234 27138 27145 Glucose +T761 NCBITaxon:10088 27192 27196 mice +T762 UBERON:0000178 27235 27240 blood +T763 CHEBI:17234 27241 27248 glucose +T764 PR:000013059 27267 27273 PGC-1α +T765 PR:000013059 27281 27287 PGC-1α +T766 PR:000013059 27342 27348 PGC-1α +T767 NCBITaxon:10088 27352 27356 mice +T768 CHEBI:17234 27397 27404 glucose +T769 PR:000045358 27419 27426 insulin +T770 CHEBI:17234 27480 27487 Glucose +T771 PR:000013059 27527 27533 PGC-1α +T772 NCBITaxon:10088 27537 27541 mice +T773 PR:000013059 27638 27644 PGC-1α +T774 NCBITaxon:10088 27648 27652 mice +T775 PR:000013059 27670 27676 PGC-1α +T776 NCBITaxon:10088 27680 27684 mice +T777 PR:000013059 27703 27709 PGC-1α +T778 NCBITaxon:10088 27713 27717 mice +T779 CHEBI:17234 27746 27753 glucose +T780 NCBITaxon:10088 27779 27783 mice +T781 NCBITaxon:9989 27796 27802 rodent +T782 CHEBI:33290 27803 27807 chow +T783 CHEBI:17234 27831 27838 glucose +T784 GO:0042593 27831 27850 glucose homeostasis +T785 PR:000013059 27888 27894 PGC-1α +T786 PR:000013059 27902 27908 PGC-1α +T787 NCBITaxon:10088 27912 27916 mice +T788 CHEBI:33290 27941 27945 chow +T789 PR:000013059 28063 28069 PGC-1α +T790 PR:000013059 28077 28083 PGC-1α +T791 PR:000013059 28125 28131 PGC-1α +T792 NCBITaxon:10088 28135 28139 mice +T793 CHEBI:17234 28183 28190 glucose +T794 PR:000045358 28204 28211 insulin +T795 PR:000013059 28238 28244 PGC-1α +T796 NCBITaxon:10088 28248 28252 mice +T797 PR:000013059 28373 28379 PGC-1α +T798 NCBITaxon:10088 28383 28387 mice +T799 PR:000045358 28403 28410 insulin +T800 PR:000013059 28437 28443 PGC-1α +T801 NCBITaxon:10088 28447 28451 mice +T802 CHEBI:17234 28461 28468 glucose +T803 PR:000045358 28482 28489 insulin +T804 NCBITaxon:10088 28508 28512 mice +T805 UBERON:0001017 28566 28588 Central Nervous System +T806 PR:000013059 28592 28598 PGC-1α +T807 NCBITaxon:10088 28602 28606 Mice +T808 UBERON:0000479 28625 28632 tissues +T809 PR:000013059 28640 28646 PGC-1α +T810 NCBITaxon:10088 28650 28654 mice +T811 UBERON:0000955 28688 28693 brain +T812 PR:000013059 28742 28748 PGC-1α +T813 UBERON:0000955 28752 28757 brain +T814 UBERON:0000479 28758 28764 tissue +T815 UBERON:0000956 28803 28820 cerebral cortical +T816 CL:0010012 28803 28829 cerebral cortical neuronal +T817 CL:0000540 28879 28885 neuron +T818 UBERON:0001872 28913 28926 parietal lobe +T819 PR:000013059 28928 28934 PGC-1α +T820 CL:0000540 28950 28957 neurons +T821 PR:000013059 28969 28975 PGC-1α +T822 CL:0000540 28991 28998 neurons +T823 UBERON:0002606 29069 29077 neuropil +T824 CL:0000598 29093 29110 pyramidal neurons +T825 UBERON:0000956 29137 29152 cerebral cortex +T826 PR:000013059 29171 29177 PGC-1α +T827 NCBITaxon:10088 29181 29185 mice +T828 PR:000013059 29198 29204 PGC-1α +T829 NCBITaxon:10088 29208 29212 mice +T830 CL:0000127 29252 29262 astrocytic +T831 CL:0000125 29271 29276 glial +T832 PR:000007939 29271 29302 glial fibrillary acidic protein +T833 CHEBI:37527 29288 29294 acidic +T834 CL:0000127 29345 29355 astrocytic +T835 GO:0097449 29345 29365 astrocytic processes +T836 PR:000013059 29369 29375 PGC-1α +T837 NCBITaxon:10088 29379 29384 mouse +T838 UBERON:0000956 29385 29400 cerebral cortex +T839 CL:0000540 29449 29457 neuronal +T840 UBERON:0016530 29511 29526 parietal cortex +T841 UBERON:0002606 29552 29560 neuropil +T842 CL:0000540 29565 29572 neurons +T843 PR:000013059 29580 29586 PGC-1α +T844 UBERON:0002420 29590 29603 basal ganglia +T845 UBERON:0001873 29605 29612 caudate +T846 UBERON:0001874 29617 29624 putamen +T847 CL:0000125 29710 29715 glial +T848 PR:000007939 29710 29741 glial fibrillary acidic protein +T849 CHEBI:37527 29727 29733 acidic +T850 CL:0000127 29757 29767 astrocytic +T851 GO:0097449 29757 29777 astrocytic processes +T852 UBERON:0002298 29847 29856 brainstem +T853 CL:0000121 29887 29895;29908 29920 Purkinje ... cell neurons +T854 CL:0000120 29900 29920 granule cell neurons +T855 PR:000013059 29944 29950 PGC-1α +T856 UBERON:0002129 29954 29971 cerebellar cortex +T857 CL:0000129 29981 29991 microglial +T858 GO:0061518 29981 30005 microglial proliferation +T859 UBERON:0014930 30010 30022 perivascular +T860 CL:0000542 30023 30034 lymphocytic +T861 PR:000013059 30078 30084 PGC-1α +T862 UBERON:0001017 30088 30091 CNS +T863 PR:000013059 30129 30135 PGC-1α +T864 UBERON:0001872 30139 30147 parietal +T865 UBERON:0000956 30148 30163 cerebral cortex +T866 CL:0000540 30206 30213 neurons +T867 UBERON:0002606 30218 30226 neuropil +T868 GO:0005773 30241 30249 Vacuoles +T869 GO:0016020 30275 30285 membranous +T870 UBERON:0001851 30323 30331 cortical +T871 CL:0010012 30323 30339 cortical neurons +T872 GO:0005773 30373 30381 vacuoles +T873 UBERON:0002606 30456 30464 neuropil +T874 GO:0006909 30478 30488 phagocytic +T875 CL:0000234 30478 30494 phagocytic cells +T876 GO:0098793 30496 30507 presynaptic +T877 UBERON:0001021 30508 30513 nerve +T878 GO:0044297 30540 30544 soma +T879 GO:0043204 30562 30572 perikaryal +T880 PR:000013059 30679 30685 PGC-1α +T881 GO:0008152 30766 30785 metabolic processes +T882 CHEBI:35366 30796 30806 fatty acid +T883 GO:0019395 30796 30816 fatty acid oxidation +T884 MOP:0000568 30807 30816 oxidation +T885 CHEBI:10545 30818 30826 electron +T886 MOP:0000615 30818 30836 electron transport +T887 MOP:0000568 30842 30851 oxidative +T888 GO:0006119 30842 30867 oxidative phosphorylation +T889 GO:0010467 30880 30890 expression +T890 PR:000013059 30894 30900 PGC-1α +T891 GO:0005739 30910 30923 mitochondrial +T892 PR:000011422 30990 30995 NRF-1 +T893 SO:0000910 31012 31018 orphan +T894 GO:0005634 31019 31026 nuclear +T895 CHEBI:50114 31036 31044 estrogen +T896 PR:000007208 31036 31063 estrogen-related receptor α +T897 PR:000013059 31135 31141 PGC-1α +T898 GO:0008152 31175 31194 metabolic processes +T899 GO:0005739 31205 31218 mitochondrial +T900 SO:0000704 31259 31263 gene +T901 NCBITaxon:10088 31276 31280 mice +T902 PR:000013059 31300 31306 PGC-1α +T903 UBERON:0000922 31335 31346 embryologic +T904 GO:0009790 31335 31358 embryologic development +T905 GO:0005739 31388 31401 mitochondrial +T906 PR:000013059 31477 31483 PGC-1α +T907 GO:0065007 31519 31527 regulate +T908 GO:0007567 31532 31537 natal +T909 GO:0005739 31538 31551 mitochondrial +T910 GO:0044237 31565 31573;31581 31591 cellular ... metabolism +T911 NCBITaxon:1 31618 31626 organism +T912 GO:0008152 31642 31651 metabolic +T913 GO:0007567 31670 31675 natal +T914 GO:0005739 31696 31709 mitochondrial +T915 UBERON:0006907 31742 31769 slow-twitch skeletal muscle +T916 PR:000013059 31773 31779 PGC-1α +T917 NCBITaxon:10088 31783 31787 mice +T918 GO:0005739 31797 31810 mitochondrial +T919 GO:0045333 31811 31822 respiratory +T920 UBERON:0004288 31873 31881 skeletal +T921 UBERON:0002107 31893 31898 liver +T922 PR:000013059 31902 31908 PGC-1α +T923 NCBITaxon:10088 31912 31916 mice +T924 UBERON:0000948 31939 31944 heart +T925 UBERON:0001389 31949 31962 soleus muscle +T926 UBERON:0000479 31964 31971 tissues +T927 GO:0005739 31994 32007 mitochondrial +T928 GO:0065007 32047 32054 control +T929 PR:000013059 32091 32097 PGC-1α +T930 NCBITaxon:10088 32101 32105 mice +T931 PR:000013059 32116 32122 PGC-1α +T932 NCBITaxon:10088 32126 32130 mice +T933 MOP:0000568 32222 32231 oxidative +T934 PR:000013059 32300 32306 PGC-1α +T935 GO:0005739 32347 32360 mitochondrial +T936 GO:0007567 32420 32425 natal +T937 NCBITaxon:10088 32488 32492 mice +T938 PR:000013059 32501 32507 PGC-1α +T939 GO:0007567 32570 32575 natal +T940 UBERON:0004288 32601 32609 skeletal +T941 PR:000013059 32643 32649 PGC-1α +T942 NCBITaxon:10088 32653 32657 mice +T943 PR:000013059 32794 32800 PGC-1α +T944 NCBITaxon:10088 32804 32808 mice +T945 UBERON:0000948 32891 32898 cardiac +T946 PR:000013059 32914 32920 PGC-1α +T947 NCBITaxon:10088 32924 32928 mice +T948 UBERON:0000948 33015 33020 heart +T949 UBERON:0000948 33080 33087 cardiac +T950 UBERON:0000948 33088 33093 heart +T951 GO:0008152 33275 33285 metabolism +T952 UBERON:0002351 33289 33299 sinus node +T953 PR:000013059 33310 33316 PGC-1α +T954 PR:000013059 33405 33411 PGC-1α +T955 NCBITaxon:10088 33415 33419 mice +T956 GO:0031649 33554 33567 thermogenesis +T957 PR:000017035 33601 33606 UCP-1 +T958 UBERON:0001348 33615 33618 BAT +T959 CHEBI:37886 33638 33656 adrenergic agonist +T960 GO:0031649 33708 33719 thermogenic +T961 UBERON:0000479 33720 33726 tissue +T962 PR:000013059 33744 33750 PGC-1α +T963 NCBITaxon:10088 33754 33758 mice +T964 GO:0031649 33780 33791 thermogenic +T965 GO:0005739 33837 33850 mitochondrial +T966 GO:0045333 33851 33862 respiration +T967 UBERON:0001348 33866 33869 BAT +T968 GO:0007567 33955 33960 natal +T969 UBERON:0000104 33961 33965 life +T970 NCBITaxon:33208 33967 33974 Animals +T971 PR:000013059 34149 34155 PGC-1α +T972 GO:0019222 34206 34216;34224 34234 control of ... metabolism +T973 UBERON:0002107 34272 34279 hepatic +T974 http://purl.obolibrary.org/obo/MONDO_0004790 34272 34289 hepatic steatosis +T975 PR:000013059 34329 34335 PGC-1α +T976 NCBITaxon:10088 34339 34343 mice +T977 GO:0007567 34362 34367 natal +T978 GO:0008152 34382 34391 metabolic +T979 PR:000013059 34452 34458 PGC-1α +T980 NCBITaxon:10088 34462 34466 mice +T981 CL:0000182 34484 34494 hepatocyte +T982 CHEBI:17855 34495 34507 triglyceride +T983 CHEBI:7896 34553 34562 palmitate +T984 MOP:0000568 34563 34572 oxidation +T985 CL:0000182 34595 34606 hepatocytes +T986 PR:000013059 34625 34631 PGC-1α +T987 NCBITaxon:10088 34635 34639 mice +T988 CHEBI:18059 34667 34672 lipid +T989 CHEBI:35366 34718 34728 fatty acid +T990 GO:0019395 34718 34738 fatty acid oxidation +T991 MOP:0000568 34729 34738 oxidation +T992 PR:000013059 34748 34754 PGC-1α +T993 CL:0000182 34760 34771 hepatocytes +T994 GO:0010467 34795 34805 expression +T995 PR:000013059 34809 34815 PGC-1α +T996 SO:0000704 34828 34833 genes +T997 GO:0005739 34846 34859 mitochondrial +T998 CHEBI:35366 34860 34870 fatty acid +T999 GO:0019395 34860 34880 fatty acid oxidation +T1000 MOP:0000568 34871 34880 oxidation +T1001 GO:0005739 34891 34904 mitochondrial +T1002 GO:0045333 34905 34916 respiratory +T1003 CHEBI:17855 34967 34979 triglyceride +T1004 GO:0019432 34967 34989 triglyceride synthesis +T1005 GO:0010467 35024 35034 expression +T1006 SO:0000704 35038 35043 genes +T1007 PR:000028988 35053 35058 SREBP +T1008 PR:000015921 35066 35071 SCD-1 +T1009 UBERON:0002107 35093 35100 hepatic +T1010 GO:0008610 35101 35110 lipogenic +T1011 PR:000013059 35172 35178 PGC-1α +T1012 NCBITaxon:10088 35182 35186 mice +T1013 CHEBI:17855 35293 35305 triglyceride +T1014 GO:0019432 35293 35315 triglyceride synthesis +T1015 PR:000013059 35451 35457 PGC-1α +T1016 SO:0000704 35479 35484 genes +T1017 CL:0000182 35525 35535 hepatocyte +T1018 CHEBI:35366 35536 35546 fatty acid +T1019 PR:000013059 35632 35638 PGC-1α +T1020 GO:0005634 35655 35662 nuclear +T1021 PR:000011396 35672 35675 FXR +T1022 PR:000028988 35701 35706 SREBP +T1023 GO:0010467 35710 35720 expression +T1024 CHEBI:17855 35725 35737 triglyceride +T1025 GO:0019432 35725 35747 triglyceride synthesis +T1026 CL:0000182 35779 35789 hepatocyte +T1027 GO:0005739 35790 35803 mitochondrial +T1028 GO:0045333 35804 35815 respiratory +T1029 GO:0008610 35853 35862 lipogenic +T1030 CL:0000182 35883 35893 hepatocyte +T1031 CHEBI:17855 35894 35906 triglyceride +T1032 UBERON:0002107 35948 35955 hepatic +T1033 GO:0015908 35956 35979 delivery of fatty acids +T1034 CHEBI:35366 35968 35979 fatty acids +T1035 PR:000013059 36051 36057 PGC-1α +T1036 NCBITaxon:10088 36061 36065 mice +T1037 GO:0007568 36228 36233 aging +T1038 PR:000013059 36304 36310 PGC-1α +T1039 NCBITaxon:10088 36314 36318 mice +T1040 GO:0065007 36390 36397 control +T1041 CHEBI:33290 36441 36445 food +T1042 GO:0007631 36441 36452 food intake +T1043 PR:000013059 36482 36488 PGC-1α +T1044 NCBITaxon:10088 36492 36496 mice +T1045 GO:0005739 36577 36590 mitochondrial +T1046 PR:000013059 36655 36661 PGC-1α +T1047 NCBITaxon:10088 36665 36669 mice +T1048 PR:000013059 36709 36715 PGC-1α +T1049 SO:0000704 36716 36720 gene +T1050 http://purl.obolibrary.org/obo/MONDO_0011122 36739 36746 obesity +T1051 NCBITaxon:9606 36750 36756 humans +T1052 PR:000013059 36833 36839 PGC-1α +T1053 NCBITaxon:10088 36843 36847 mice +T1054 GO:0065007 36972 36979 control +T1055 CHEBI:17234 37032 37039 glucose +T1056 http://purl.obolibrary.org/obo/MONDO_0001076 37032 37051 glucose intolerance +T1057 PR:000045358 37055 37062 insulin +T1058 PR:000013059 37081 37087 PGC-1α +T1059 NCBITaxon:33208 37091 37098 animals +T1060 CHEBI:33290 37111 37115 chow +T1061 PR:000013059 37134 37140 PGC-1α +T1062 NCBITaxon:10088 37144 37148 mice +T1063 CHEBI:17234 37159 37166 glucose +T1064 PR:000045358 37180 37187 insulin +T1065 PR:000013059 37203 37209 PGC-1α +T1066 GO:0007631 37227 37236 consuming +T1067 GO:0010467 37351 37361 expression +T1068 PR:000013059 37365 37371 PGC-1α +T1069 NCBITaxon:9606 37375 37380 human +T1070 http://purl.obolibrary.org/obo/MONDO_0005015 37381 37389 diabetic +T1071 UBERON:0004288 37390 37398 skeletal +T1072 GO:0019222 37458 37478 metabolic regulatory +T1073 PR:000013059 37517 37523 PGC-1α +T1074 NCBITaxon:10088 37534 37538 mice +T1075 PR:000013059 37588 37594 PGC-1α +T1076 PR:000045358 37661 37668 insulin +T1077 NCBITaxon:10088 37740 37744 mice +T1078 PR:000013059 37757 37763 PGC-1α +T1079 CHEBI:17234 37811 37818 glucose +T1080 http://purl.obolibrary.org/obo/MONDO_0001076 37811 37830 glucose intolerance +T1081 PR:000013059 37870 37876 PGC-1α +T1082 NCBITaxon:10088 37880 37884 mice +T1083 UBERON:0001017 37931 37953 central nervous system +T1084 UBERON:0000955 37996 38002 brains +T1085 PR:000013059 38006 38012 PGC-1α +T1086 NCBITaxon:10088 38016 38020 mice +T1087 CL:0000598 38070 38087 pyramidal neurons +T1088 CL:0010012 38080 38090;38095 38110 neurons of ... cerebral cortex +T1089 UBERON:0000956 38095 38110 cerebral cortex +T1090 CL:0000127 38160 38170 astrocytes +T1091 UBERON:0002420 38178 38191 basal ganglia +T1092 PR:000013059 38291 38297 PGC-1α +T1093 CHEBI:18059 38325 38330 lipid +T1094 GO:0006629 38325 38341 lipid metabolism +T1095 GO:0016020 38353 38361 membrane +T1096 GO:0044091 38353 38371 membrane synthesis +T1097 CL:0000129 38493 38503 microglial +T1098 UBERON:0001017 38521 38543 central nervous system +T1099 UBERON:0001016 38560 38570 neurologic +T1100 PR:000013059 38603 38609 PGC-1α +T1101 NCBITaxon:10088 38613 38617 mice +T1102 UBERON:0000104 38643 38647 life +T1103 UBERON:0025534 38696 38708 sensorimotor +T1104 PR:000013059 38721 38727 PGC-1α +T1105 NCBITaxon:10088 38731 38735 mice +T1106 PR:000013059 38852 38858 PGC-1α +T1107 NCBITaxon:10088 38862 38866 mice +T1108 UBERON:0000010 38889 38899;38911 38925 peripheral ... nervous system +T1109 UBERON:0001017 38903 38925 central nervous system +T1110 GO:0040011 39028 39037 locomotor +T1111 UBERON:0000955 39093 39098 brain +T1112 PR:000013059 39111 39117 PGC-1α +T1113 NCBITaxon:10088 39121 39125 mice +T1114 UBERON:0001016 39175 39185 neurologic +T1115 GO:0008152 39227 39236 metabolic +T1116 PR:000013059 39258 39264 PGC-1α +T1117 NCBITaxon:10088 39270 39274 mice +T1118 NCBITaxon:10088 39355 39360 mouse +T1119 PR:000013059 39379 39385 PGC-1α +T1120 SO:0000704 39386 39390 gene +T1121 PR:000013059 39443 39449 PGC-1α +T1122 PR:000013059 39564 39570 PGC-1α +T1123 CL:0000182 39621 39631 hepatocyte +T1124 GO:0045333 39632 39643 respiration +T1125 UBERON:0001016 39655 39665 neurologic +T1126 PR:000013059 39771 39777 PGC-1α +T1127 NCBITaxon:10088 39781 39785 mice +T1128 GO:0007567 39824 39829 natal +T1129 GO:0006094 39890 39905 gluconeogenesis +T1130 UBERON:0000178 39923 39928 blood +T1131 CHEBI:17234 39929 39936 glucose +T1132 GO:0010467 40014 40024 expression +T1133 PR:000005308 40036 40068 CCAAT-enhancer-binding protein β +T1134 PR:000005309 40036 40066;40073 40074 CCAAT-enhancer-binding protein ... δ +T1135 SO:0000165 40042 40050 enhancer +T1136 GO:0006094 40083 40096 gluconeogenic +T1137 SO:0000704 40097 40102 genes +T1138 CHEBI:18021 40112 40131 phosphoenolpyruvate +T1139 CHEBI:17234 40150 40157 glucose +T1140 PR:000013059 40208 40214 PGC-1α +T1141 NCBITaxon:10088 40218 40222 mice +T1142 PR:000013059 40323 40329 PGC-1α +T1143 NCBITaxon:10088 40333 40337 mice +T1144 http://purl.obolibrary.org/obo/MONDO_0011122 40441 40448 obesity +T1145 PR:000045358 40453 40460 insulin +T1146 PR:000013059 40502 40508 PGC-1α +T1147 NCBITaxon:10088 40519 40523 mice +T1148 http://purl.obolibrary.org/obo/MONDO_0011122 40568 40575 obesity +T1149 PR:000045358 40619 40626 insulin +T1150 PR:000013059 40661 40667 PGC-1α +T1151 NCBITaxon:10088 40671 40675 mice +T1152 PR:000045358 40781 40788 insulin +T1153 PR:000013059 40812 40818 PGC-1α +T1154 NCBITaxon:10088 40822 40826 mice +T1155 NCBITaxon:10088 40887 40891 mice +T1156 GO:0005634 40904 40911 nuclear +T1157 CHEBI:50114 40921 40929 estrogen +T1158 PR:000007208 40921 40948 estrogen-related receptor α +T1159 PR:000013059 40968 40974 PGC-1α +T1160 http://purl.obolibrary.org/obo/MONDO_0011122 41011 41018 obesity +T1161 PR:000013059 41043 41049 PGC-1α +T1162 NCBITaxon:10088 41055 41059 mice +T1163 PR:000013059 41078 41084 PGC-1α +T1164 NCBITaxon:10088 41088 41092 mice +T1165 UBERON:0002107 41143 41150 hepatic +T1166 http://purl.obolibrary.org/obo/MONDO_0004790 41143 41160 hepatic steatotic +T1167 NCBITaxon:10088 41195 41200 mouse +T1168 UBERON:0001016 41237 41247 neurologic +T1169 PR:000013059 41311 41317 PGC-1α +T1170 NCBITaxon:10088 41321 41325 mice +T1171 GO:0040011 41354 41363 locomotor +T1172 NCBITaxon:33208 41598 41605 animals +T1173 UBERON:0004288 41670 41678 skeletal +T1174 UBERON:0000948 41690 41697 cardiac +T1175 PR:000013059 41899 41905 PGC-1α +T1176 NCBITaxon:10088 41916 41921 mouse +T1177 SO:0000704 41972 41979 genetic +T1178 GO:0007567 42124 42129 natal +T1179 PR:000013059 42153 42159 PGC-1α +T1180 NCBITaxon:10088 42163 42167 mice +T1181 UBERON:0002107 42282 42287 liver +T1182 UBERON:0000479 42298 42305 tissues +T1183 SO:0000704 42369 42373 gene +T1184 PR:000013059 42434 42440 PGC-1α +T1185 NCBITaxon:10088 42444 42448 mice +T1186 SO:0000147 42489 42494 exons +T1187 CL:0000023 42502 42509 oocytes +T1188 PR:000013059 42515 42521 PGC-1α +T1189 NCBITaxon:10088 42525 42529 mice +T1190 SO:0001644 42656 42672 targeting vector +T1191 SO:0000147 42692 42696 exon +T1192 SO:0000147 42707 42712 exons +T1193 SO:0000147 42726 42730 exon +T1194 SO:0000673 42795 42805 transcript +T1195 SO:0000673 42872 42882 transcript +T1196 SO:0000333 42897 42901;42906 42912 exon ... border +T1197 PR:000013059 43027 43033 PGC-1α +T1198 PR:000013059 43218 43224 PGC-1α +T1199 GO:0005634 43292 43299 nuclear +T1200 SO:0000417 43321 43328 domains +T1201 SO:0000417 43363 43369 domain +T1202 PR:000013059 43391 43397 PGC-1α +T1203 NCBITaxon:10088 43426 43430 mice +T1204 PR:000013059 43589 43595 PGC-1α +T1205 NCBITaxon:10088 43599 43603 mice +T1206 PR:000013059 43642 43648 PGC-1α +T1207 NCBITaxon:10088 43686 43691 mouse +T1208 PR:000013059 43800 43806 PGC-lα +T1209 GO:0051866 43827 43845 adaptive responses +T1210 GO:0007567 43868 43873 natal +T1211 PR:000013059 43981 43987 PGC-1α +T1212 GO:0019222 44091 44101;44109 44119 control of ... metabolism +T1213 GO:0050794 44091 44101;44138 44156 control of ... cellular processes +T1214 PR:000013059 44200 44206 PGC-1α +T1215 GO:0008152 44283 44292 metabolic +T1216 GO:0008152 44315 44324 metabolic +T1217 GO:0007567 44344 44349 natal +T1218 PR:000013059 44375 44381 PGC-1α +T1219 NCBITaxon:10088 44387 44391 mice +T1220 PR:000013059 44523 44529 PGC-1α +T1221 PR:000013059 44591 44597 PGC-1α +T1222 NCBITaxon:10088 44601 44605 mice +T1223 NCBITaxon:39107 44631 44637 murine +T1224 GO:0008152 44686 44696 metabolism +T1225 http://purl.obolibrary.org/obo/MONDO_0011122 44700 44707 obesity +T1226 http://purl.obolibrary.org/obo/MONDO_0005015 44709 44717 diabetes +T1227 UBERON:0002107 44719 44726 hepatic +T1228 http://purl.obolibrary.org/obo/MONDO_0004790 44719 44736 hepatic steatosis +T1229 http://purl.obolibrary.org/obo/MONDO_0005267 44742 44753;44758 44763 diseases of heart +T1230 http://purl.obolibrary.org/obo/MONDO_0020120 44742 44753;44765 44780 diseases of skeletal muscle +T1231 http://purl.obolibrary.org/obo/MONDO_0002602 44742 44753;44786 44808 diseases of central nervous system +T1232 UBERON:0000948 44758 44763 heart +T1233 UBERON:0004288 44765 44773 skeletal +T1234 UBERON:0001017 44786 44808 central nervous system +T1235 PR:000013059 44850 44856 PGC-1α +T1236 SO:0000704 44857 44861 gene +T1237 NCBITaxon:10088 44865 44869 mice +T1238 SO:0000153 44873 44876 BAC +T1239 SO:0000040 44877 44890 genomic clone +T1240 NCBITaxon:39107 44906 44912 murine +T1241 PR:000013059 44913 44919 PGC-1α +T1242 SO:0000704 44920 44924 gene +T1243 SO:0001026 44949 44956 genomic +T1244 SO:0000147 45063 45067 exon +T1245 SO:0000040 45093 45106 genomic clone +T1246 SO:0000121 45110 45119 5′ primer +T1247 SO:0000028 45185 45187 bp +T1248 SO:0000147 45200 45204 exon +T1249 SO:0000061 45243 45259 restriction site +T1250 SO:0000132 45295 45304 3′ primer +T1251 PR:P23940 45317 45322 BamH1 +T1252 SO:0000147 45444 45448 exon +T1253 SO:0000121 45536 45538;45546 45553 5′ ... primers +T1254 SO:0000132 45543 45553 3′ primers +T1255 SO:0001644 45681 45697 targeting vector +T1256 CL:0002322 45766 45774 ES cells +T1257 CHEBI:42768 45797 45801 G418 +T1258 http://purl.obolibrary.org/obo/MONDO_0004992 45862 45868 Cancer +T1259 CL:0002322 45876 45883 ES Cell +T1260 UBERON:0000358 46215 46225 blastocyst +T1261 GO:0007618 46241 46246 mated +T1262 NCBITaxon:10088 46259 46263 mice +T1263 UBERON:0002415 46328 46332 tail +T1264 NCBITaxon:33208 46454 46460 animal +T1265 NCBITaxon:33208 46474 46480 animal +T1266 NCBITaxon:33208 46628 46635 animals +T1267 NCBITaxon:33208 46674 46680 Animal +T1268 NCBITaxon:33208 46723 46730 Animals +T1269 PR:000013059 46801 46807 PGC-1α +T1270 PR:000013059 46815 46821 PGC-1α +T1271 NCBITaxon:10088 46825 46829 mice +T1272 UBERON:0000479 46851 46858 tissues +T1273 UBERON:0000479 46912 46918 Tissue +T1274 PR:000013059 47114 47120 PGC-1α +T1275 PR:000013059 47128 47134 PGC-1α +T1276 NCBITaxon:10088 47138 47142 mice +T1277 PR:000013059 47191 47197 PGC-1α +T1278 PR:000013059 47205 47211 PGC-1α +T1279 NCBITaxon:10088 47215 47219 mice +T1280 CHEBI:33290 47274 47278 food +T1281 UBERON:0001052 47321 47327 rectal +T1282 NCBITaxon:10088 47373 47377 Mice +T1283 NCBITaxon:10088 47457 47461 mice +T1284 UBERON:0000479 47482 47489 tissues +T1285 NCBITaxon:33208 47553 47560 animals +T1286 CHEBI:15377 47590 47595 water +T1287 CHEBI:33290 47608 47612 Food +T1288 UBERON:0000479 47655 47662 tissues +T1289 NCBITaxon:10088 47723 47727 mice +T1290 NCBITaxon:33208 47954 47960 Animal +T1291 NCBITaxon:10088 48066 48070 mice +T1292 PR:000013060 48451 48457 PGC-1β +T1293 PR:000013159 48462 48465 PRC +T1294 PR:000017035 48555 48560 UCP-1 +T1295 UBERON:0001389 48673 48686 soleus muscle +T1296 UBERON:0001348 48688 48691 BAT +T1297 UBERON:0000948 48697 48702 heart +T1298 PR:000013059 48717 48723 PGC-1α +T1299 PR:000013059 48730 48734 PGCα +T1300 NCBITaxon:10088 48738 48742 mice +T1301 GO:0001171 48747 48766 reverse transcribed +T1302 GO:0001171 48779 48800 reverse transcription +T1303 CHEBI:33893 48801 48809 reagents +T1304 CHEBI:33893 48947 48955 reagents +T1305 NCBITaxon:10088 49017 49022 mouse +T1306 SO:0000112 49032 49038 primer +T1307 SO:0000704 49074 49078 gene +T1308 GO:0010467 49074 49089 gene expression +T1309 SO:0000112 49120 49127 primers +T1310 PR:000017035 49132 49137 UCP-1 +T1311 SO:0000112 49181 49187 primer +T1312 UBERON:0001389 49277 49283 soleus +T1313 UBERON:0001348 49285 49288 BAT +T1314 UBERON:0000948 49294 49299 heart +T1315 SO:0000704 49300 49304 gene +T1316 GO:0010467 49300 49315 gene expression +T1317 NCBITaxon:9989 49328 49334 Rodent +T1318 SO:0000112 49335 49342 primers +T1319 UBERON:0002107 49408 49413 liver +T1320 SO:0000704 49414 49418 gene +T1321 GO:0010467 49414 49429 gene expression +T1322 SO:0001026 49472 49479 genomic +T1323 SO:0000704 49571 49575 Gene +T1324 GO:0097617 49649 49662 hybridization +T1325 GO:0005739 49939 49952 Mitochondrial +T1326 GO:0045333 49953 49964 respiration +T1327 GO:0005739 49974 49987 Mitochondrial +T1328 GO:0045333 49988 49999 respiration +T1329 CHEBI:26605 50016 50023 saponin +T1330 UBERON:0001389 50032 50038 soleus +T1331 CHEBI:28201 50097 50105 rotenone +T1332 NCBITaxon:10088 50162 50166 mice +T1333 CHEBI:28142 50190 50205 chloral hydrate +T1334 UBERON:0001389 50234 50240 Soleus +T1335 CHEBI:6636 50340 50345 MgCl2 +T1336 CHEBI:14434 50353 50362 imidazole +T1337 CHEBI:39010 50374 50377 MES +T1338 CHEBI:15891 50385 50392 taurine +T1339 CHEBI:17287 50412 50415 PCr +T1340 CHEBI:63036 50422 50428 KH2PO4 +T1341 CHEBI:18320 50437 50440 DTT +T1342 CHEBI:26605 50478 50485 saponin +T1343 CHEBI:6636 50635 50640 MgCl2 +T1344 CHEBI:14434 50648 50657 imidazole +T1345 CHEBI:39010 50668 50671 MES +T1346 CHEBI:15891 50679 50686 taurine +T1347 CHEBI:63036 50693 50699 KH2PO4 +T1348 CHEBI:18320 50708 50711 DTT +T1349 GO:0045333 50736 50747 Respiration +T1350 GO:0045333 50900 50911 respiration +T1351 GO:0045333 50946 50957 respiration +T1352 GO:0005741 51026 51054 outer mitochondrial membrane +T1353 PR:000002199 51093 51105 cytochrome c +T1354 CHEBI:18070 51093 51105 cytochrome c +T1355 GO:0045333 51140 51151 respiration +T1356 CHEBI:25675 51200 51210 oligomycin +T1357 GO:0045333 51254 51265 respiration +T1358 CHEBI:15379 51307 51309 O2 +T1359 GO:0045333 51318 51329 Respiration +T1360 CHEBI:15379 51359 51361 O2 +T1361 PR:000045358 51381 51388 Insulin +T1362 CHEBI:17234 51393 51400 glucose +T1363 CHEBI:17234 51418 51425 Glucose +T1364 PR:000045358 51430 51437 Insulin +T1365 NCBITaxon:10088 51506 51510 mice +T1366 NCBITaxon:10088 51569 51573 mice +T1367 CHEBI:75958 51599 51607 solution +T1368 CHEBI:17634 51611 51620 D-glucose +T1369 NCBITaxon:10088 51640 51644 mice +T1370 NCBITaxon:9606 51673 51678 human +T1371 CHEBI:5931 51673 51678;51687 51694 human ... insulin +T1372 PR:000045358 51687 51694 insulin +T1373 UBERON:0002415 51788 51792 Tail +T1374 CHEBI:17234 51799 51806 glucose +T1375 CHEBI:17234 51874 51881 GLUCOSE +T1376 NCBITaxon:10088 51997 52001 mice +T1377 CHEBI:10545 52415 52423 electron +T1378 UBERON:0001389 52436 52449 Soleus muscle +T1379 UBERON:0002107 52454 52459 liver +T1380 CHEBI:64276 52501 52515 glutaraldehyde +T1381 CHEBI:62956 52548 52565 sodium cacodylate +T1382 UBERON:0000479 52578 52585 tissues +T1383 CHEBI:16236 52646 52653 ethanol +T1384 CHEBI:10545 52709 52717 electron +T1385 UBERON:0001133 52730 52737;52751 52757 Cardiac ... muscle +T1386 UBERON:0004288 52742 52750 skeletal +T1387 GO:0005739 52758 52771 mitochondrial +T1388 GO:0030016 52776 52788 myofibrillar +T1389 CHEBI:10545 52827 52835 electron +T1390 NCBITaxon:33208 52887 52893 animal +T1391 GO:0005739 53030 53042 mitochondria +T1392 GO:0030016 53046 53056 myofibrils +T1393 CHEBI:10545 53077 53085 electron +T1394 UBERON:0000955 53114 53119 brain +T1395 UBERON:0000479 53121 53127 tissue +T1396 UBERON:0001851 53193 53199 cortex +T1397 CHEBI:10545 53315 53323 electron +T1398 CHEBI:51686 53340 53341 H +T1399 UBERON:0000955 53366 53371 brain +T1400 UBERON:0000956 53383 53398 cerebral cortex +T1401 UBERON:0002298 53400 53409 brainstem +T1402 UBERON:0002037 53415 53425 cerebellum +T1403 CHEBI:30879 53471 53478 alcohol +T1404 NCBITaxon:10088 53553 53558 mouse +T1405 CL:0000182 53559 53569 hepatocyte +T1406 NCBITaxon:10088 53599 53604 mouse +T1407 CL:0000182 53605 53616 hepatocytes +T1408 PR:000013059 53641 53647 PGC-1α +T1409 PR:000013059 53655 53661 PGC-1α +T1410 NCBITaxon:10088 53665 53669 mice +T1411 CHEBI:35366 53701 53711 Fatty acid +T1412 GO:0019395 53701 53721 Fatty acid oxidation +T1413 MOP:0000568 53712 53721 oxidation +T1414 CHEBI:17855 53726 53738 triglyceride +T1415 GO:0019432 53726 53748 triglyceride synthesis +T1416 CHEBI:17855 53811 53823 Triglyceride +T1417 GO:0019432 53811 53833 Triglyceride synthesis +T1418 CHEBI:7896 53888 53897 Palmitate +T1419 MOP:0000568 53898 53907 oxidation +T1420 CHEBI:29238 53942 53944 3H +T1421 CHEBI:15756 53946 53959 palmitic acid +T1422 GO:0045333 54032 54043 respiration +T1423 CHEBI:26605 54174 54181 saponin +T1424 GO:0045333 54183 54194 Respiration +T1425 CHEBI:28201 54277 54285 rotenone +T1426 GO:0045333 54287 54298 Respiration +T1427 CHEBI:15379 54328 54330 O2 +T1428 GO:0040011 54373 54382 locomotor +T1429 UBERON:0025534 54393 54405 sensorimotor +T1430 NCBITaxon:10088 54493 54497 mice +T1431 GO:0040011 55205 55216 ambulations +T1432 GO:0040011 55407 55418 ambulations +T1433 NCBITaxon:10088 55477 55481 mice +T1434 NCBITaxon:33208 55614 55620 animal +T1435 UBERON:0025534 55662 55674 sensorimotor +T1436 NCBITaxon:10088 55824 55829 mouse +T1437 NCBITaxon:33208 55963 55969 animal +T1438 UBERON:0000033 56018 56022 head +T1439 NCBITaxon:10088 56176 56180 mice +T1440 NCBITaxon:33208 56284 56290 animal +T1441 NCBITaxon:10088 56329 56334 mouse +T1442 NCBITaxon:10088 56510 56515 mouse +T1443 NCBITaxon:10088 56685 56690 mouse +T1444 NCBITaxon:10088 56851 56856 mouse +T1445 NCBITaxon:10088 57020 57025 mouse +T1446 NCBITaxon:10088 57177 57182 mouse +T1447 PR:000013059 57306 57312 PGC-1α +T1448 PR:000013059 57328 57334 PGC-1α +T1449 NCBITaxon:10088 57346 57350 mice +T1450 NCBITaxon:10088 57679 57683 mice +T1451 NCBITaxon:33208 57878 57885 Animals +T1452 GO:0008152 57907 57916 metabolic +T1453 NCBITaxon:10088 57973 57977 Mice +T1454 NCBITaxon:33208 58148 58155 animals +T1455 NCBITaxon:10088 58286 58291 mouse +T1456 NCBITaxon:33208 58395 58401 animal +T1457 CHEBI:15379 58404 58406 O2 +T1458 NCBITaxon:33208 58476 58483 Animals +T1459 CHEBI:6121 58507 58515 ketamine +T1460 UBERON:0001389 58533 58546 soleus muscle +T1461 UBERON:0000978 58568 58571 leg +T1462 CHEBI:75958 58623 58631 solution +T1463 CHEBI:15379 58649 58651 O2 +T1464 CHEBI:16526 58659 58662 CO2 +T1465 CHEBI:75958 58685 58693 solution +T1466 CHEBI:15377 58718 58723 water +T1467 GO:0006936 58902 58910 contract +T1468 UBERON:0007023 59634 59639 Adult +T1469 NCBITaxon:10088 59647 59651 mice +T1470 NCBITaxon:10088 59857 59862 mouse +T1471 UBERON:0000948 60001 60008 cardiac +T1472 UBERON:0007023 60129 60134 adult +T1473 NCBITaxon:10088 60135 60139 mice +T1474 UBERON:0001179 60174 60186 peritoneally +T1475 CHEBI:9561 60197 60214 thiopental sodium +T1476 NCBITaxon:10088 60231 60235 mice +T1477 UBERON:0005396 60303 60317 carotid artery +T1478 UBERON:0003126 60352 60359 trachea +T1479 UBERON:0002084 60512 60526 left ventricle +T1480 UBERON:0002137 60549 60561 aortic valve +T1481 CHEBI:4670 60678 60688 dobutamine +T1482 CHEBI:37886 60705 60723 adrenergic agonist +T1483 NCBITaxon:10088 60950 60955 mouse +T1484 UBERON:0000948 60956 60961 heart +T1485 NCBITaxon:10088 60990 60995 mouse +T1486 UBERON:0000948 60996 61001 heart +T1487 UBERON:0007023 61064 61069 Adult +T1488 NCBITaxon:10088 61070 61074 mice +T1489 NCBITaxon:33208 61148 61155 Animals +T1490 CHEBI:7984 61202 61222 sodium pentobarbital +T1491 UBERON:0000948 61227 61233 Hearts +T1492 CHEBI:17544 61289 61300 bicarbonate +T1493 CHEBI:75958 61307 61315 solution +T1494 CHEBI:26710 61324 61328 NaCl +T1495 CHEBI:32139 61336 61342 NaHCO3 +T1496 CHEBI:32588 61351 61354 KCl +T1497 CHEBI:63036 61363 61369 KH2PO4 +T1498 CHEBI:3312 61378 61383 CaCl2 +T1499 CHEBI:17234 61392 61399 glucose +T1500 PR:000045358 61417 61424 insulin +T1501 UBERON:0000948 61436 61442 Hearts +T1502 UBERON:0000947 61473 61478 aorta +T1503 UBERON:0002079 61542 61553 left atrial +T1504 CHEBI:75958 61619 61627 solution +T1505 CHEBI:7896 61646 61655 palmitate +T1506 CHEBI:35366 61668 61678 fatty acid +T1507 CHEBI:75958 61798 61806 solution +T1508 UBERON:0000948 61840 61847 cardiac +T1509 UBERON:0000947 61856 61862 aortic +T1510 UBERON:0000948 61898 61903 heart +T1511 UBERON:0000948 62182 62189 Cardiac +T1512 UBERON:0000948 62255 62262 cardiac +T1513 UBERON:0025534 62361 62373 sensorimotor +T1514 UBERON:0025534 62545 62557 sensorimotor +T1515 PR:000013059 63170 63176 PGC-1α +T1516 NCBITaxon:10088 63180 63184 Mice +T1517 PR:000013059 63221 63227 PGC-1α +T1518 PR:000013059 63243 63249 PGC-1α +T1519 NCBITaxon:10088 63268 63272 mice +T1520 PR:000013059 63579 63585 PGC-1α +T1521 NCBITaxon:10088 63589 63593 Mice +T1522 PR:000013059 63944 63950 PGC-1α +T1523 NCBITaxon:10088 63954 63958 mice +T1524 PR:000013059 64083 64089 PGC-1α +T1525 NCBITaxon:10088 64093 64097 Mice +T1526 NCBITaxon:10088 64139 64143 mice +T1527 GO:0007631 64149 64152 fed +T1528 PR:000013059 64283 64289 PGC-1α +T1529 PR:000013059 64305 64311 PGC-1α +T1530 NCBITaxon:10088 64324 64328 mice +T1531 PR:000013059 64421 64427 PGC-1α +T1532 NCBITaxon:10088 64431 64435 Mice +T1533 http://purl.obolibrary.org/obo/MONDO_0011122 64475 64482 Obesity +T1534 PR:000013059 64500 64506 PGC-1α +T1535 PR:000013059 64522 64528 PGC-1α +T1536 NCBITaxon:10088 64540 64544 mice +T1537 GO:0007631 64550 64553 fed +T1538 PR:000013059 64809 64815 PGC-1α +T1539 SO:0000112 64914 64921 Primers +T1540 NCBITaxon:10088 64936 64941 mouse +T1541 SO:0000112 64962 64969 primers +T1542 SO:0000440 65137 65143 vector +T1543 SO:0001644 65190 65206 targeting vector +T1544 NCBITaxon:10088 65305 65310 mouse +T1545 CL:0002322 65344 65351 ES cell +T1546 NCBITaxon:10088 65356 65361 mouse +T1547 CHEBI:10545 65516 65524 electron +T1548 UBERON:0000948 65569 65576 cardiac +T1549 CHEBI:17855 65627 65630 TAG +T1550 GO:0007586 65877 65886 Digestive +T1551 http://purl.obolibrary.org/obo/MONDO_0004335 65877 65895 Digestive Diseases +T1552 http://purl.obolibrary.org/obo/MONDO_0005147 66063 66080 Juvenile Diabetes +T1553 UBERON:0000948 66126 66131 Heart +T1554 UBERON:0001348 66287 66290 BAT +T1555 UBERON:0001348 66293 66313 brown adipose tissue +T1556 UBERON:0001179 66402 66416 peritoneal(ly) +T1557 GO:0005634 66424 66431 nuclear +T1558 GO:0045333 66432 66443 respiratory +T1559 PR:000013058 66458 66463 PPARγ +T1560 GO:0005777 66484 66494 peroxisome +T1561 PR:000013159 66528 66531 PRC +T1562 PR:000013159 66534 66559 PGC-1 related coactivator +T1563 CHEBI:15541 66567 66578 steroyl CoA +T1564 PR:000028988 66591 66596 SREBP +T1565 CHEBI:15889 66599 66605 sterol +T1566 SO:0001861 66599 66624 sterol regulatory element +T1567 PR:000028988 66599 66640 sterol regulatory element binding protein +T1568 GO:0065007 66606 66616 regulatory +T1569 CHEBI:17855 66676 66679 TAG +T1570 CHEBI:17855 66682 66694 triglyceride +T1571 PR:000016262 66696 66700 Tfam +T1572 GO:0005739 66703 66716 mitochondrial +T1573 PR:000016262 66703 66739 mitochondrial transcription factor A +T1574 PR:000013059 66829 66835 PGC-1α +T1575 SO:0000704 66836 66840 Gene +T1576 SO:0000704 66863 66867 gene +T1577 SO:0000842 66890 66899;66918 66922 region of ... gene +T1578 NCBITaxon:39107 66904 66910 murine +T1579 PR:000013059 66911 66917 PGC-1α +T1580 SO:0000147 66934 66939 exons +T1581 SO:0000061 66988 67018 restriction endonuclease sites +T1582 SO:0005853 67087 67095 cassette +T1583 PR:000013059 67115 67121 PGC-1α +T1584 SO:0000704 67122 67126 gene +T1585 SO:0001644 67258 67274 targeting vector +T1586 PR:000013059 67283 67289 PGC-1α +T1587 SO:0000704 67290 67294 gene +T1588 PR:000013059 67370 67376 PGC-1α +T1589 SO:0000704 67377 67381 gene +T1590 SO:0000147 67412 67416 exon +T1591 SO:0000412 67560 67581 restriction fragments +T1592 UBERON:0000922 67671 67680 embryonic +T1593 CL:0002322 67671 67691 embryonic stem cells +T1594 CL:0002322 67693 67696 ESC +T1595 UBERON:0002415 67723 67727 tail +T1596 GO:0097617 67778 67788 hybridized +T1597 PR:000013059 67837 67843 PGC-1α +T1598 PR:000013059 67854 67860 PGC-1α +T1599 PR:000013059 67875 67881 PGC-1α +T1600 UBERON:0000948 67993 67999 hearts +T1601 PR:000013059 68022 68028 PGC-1α +T1602 PR:000013059 68072 68078 PGC-1α +T1603 PR:000013060 68109 68115 PGC-1β +T1604 PR:000013159 68120 68123 PRC +T1605 CHEBI:4883 68156 68172 Ethidium bromide +T1606 SO:0000407 68185 68202 18S ribosomal RNA +T1607 GO:0005840 68189 68198 ribosomal +T1608 CHEBI:51461 68263 68273 Sybr green +T1609 PR:000013059 68294 68300 PGC-1α +T1610 SO:0000673 68301 68312 transcripts +T1611 SO:0000112 68319 68325 primer +T1612 SO:0000333 68350 68362 exon borders +T1613 SO:0000147 68379 68383 exon +T1614 SO:0000112 68388 68394 primer +T1615 SO:0000673 68423 68433 transcript +T1616 SO:0000147 68453 68457 exon +T1617 SO:0000112 68462 68468 primer +T1618 SO:0000673 68493 68503 transcript +T1619 UBERON:0000479 68568 68575 tissues +T1620 PR:000013059 68616 68622 PGC-1α +T1621 PR:000013059 68630 68636 PGC-1α +T1622 SO:0000147 68668 68672 exon +T1623 SO:0000673 68739 68750 transcripts +T1624 UBERON:0001348 68893 68896 BAT +T1625 PR:000013059 69011 69017 PGC-1α +T1626 GO:0042571 69018 69026 antibody +T1627 PR:000013059 69051 69057 PGC-1α +T1628 GO:0010467 69063 69072 expressed +T1629 CL:0002495 69076 69101 neonatal cardiac myocytes +T1630 UBERON:0000948 69085 69092 cardiac +T1631 NCBITaxon:10508 69111 69121 adenoviral +T1632 SO:0000440 69122 69128 vector +T1633 PR:000013059 69133 69139 PGC-1α +T1634 UBERON:0000479 69261 69267 Tissue +T1635 http://purl.obolibrary.org/obo/MONDO_0011122 69334 69341 Obesity +T1636 PR:000013059 69345 69351 PGC-1α +T1637 NCBITaxon:10088 69355 69359 Mice +T1638 PR:000013059 69473 69479 PGC-1α +T1639 PR:000013059 69487 69493 PGC-1α +T1640 NCBITaxon:10088 69497 69501 mice +T1641 PR:000013059 69540 69546 PGC-1α +T1642 NCBITaxon:10088 69550 69554 mice +T1643 PR:000013059 69581 69587 PGC-1α +T1644 PR:000013059 69711 69717 PGC-1α +T1645 NCBITaxon:10088 69721 69725 mice +T1646 PR:000013059 69825 69831 PGC-1α +T1647 PR:000013059 69839 69845 PGC-1α +T1648 NCBITaxon:10088 69849 69853 mice +T1649 PR:000013059 69991 69997 PGC-1α +T1650 NCBITaxon:10088 70001 70005 mice +T1651 PR:000013059 70099 70105 PGC-1α +T1652 PR:000013059 70113 70119 PGC-1α +T1653 NCBITaxon:10088 70123 70127 mice +T1654 PR:000013059 70239 70245 PGC-1α +T1655 NCBITaxon:10088 70249 70253 mice +T1656 GO:0005739 70275 70288 Mitochondrial +T1657 UBERON:0006907 70302 70329 Slow-Twitch Skeletal Muscle +T1658 PR:000013059 70333 70339 PGC-1α +T1659 NCBITaxon:10088 70343 70347 Mice +T1660 CHEBI:10545 70368 70376 electron +T1661 UBERON:0001389 70391 70404 soleus muscle +T1662 PR:000013059 70426 70432 PGC-1α +T1663 PR:000013059 70440 70446 PGC-1α +T1664 NCBITaxon:10088 70450 70454 mice +T1665 GO:0005739 70539 70552 mitochondrial +T1666 GO:0005739 70554 70558 Mito +T1667 GO:0030016 70564 70576 myofibrillar +T1668 GO:0030016 70578 70581 Myo +T1669 CHEBI:10545 70614 70622 electron +T1670 NCBITaxon:33208 70662 70669 animals +T1671 PR:000013059 70750 70756 PGC-1α +T1672 SO:0000704 70773 70777 Gene +T1673 GO:0010467 70773 70788 Gene expression +T1674 GO:0005634 70836 70843 nuclear +T1675 SO:0000087 70836 70843;70862 70867 nuclear ... genes +T1676 GO:0005739 70848 70861 mitochondrial +T1677 SO:0000088 70848 70867 mitochondrial genes +T1678 GO:0005739 70902 70915 mitochondrial +T1679 GO:0008152 70916 70926 metabolism +T1680 GO:0005739 70931 70944 mitochondrial +T1681 PR:000002199 70957 70969 cytochrome C +T1682 CHEBI:18070 70957 70969 cytochrome C +T1683 PR:000002199 70971 70977 Cyto c +T1684 CHEBI:18070 70971 70977 Cyto c +T1685 PR:000004454 70980 70994 ATP synthase β +T1686 PR:000016262 70996 71000 Tfam +T1687 CHEBI:4056 71006 71016 cytochrome +T1688 PR:000013059 71219 71225 PGC-1α +T1689 GO:0005739 71242 71255 Mitochondrial +T1690 GO:0045333 71256 71267 respiration +T1691 CHEBI:26605 71329 71336 saponin +T1692 UBERON:0001389 71379 71385 soleus +T1693 PR:000013059 71389 71395 PGC-1α +T1694 PR:000013059 71403 71409 PGC-1α +T1695 NCBITaxon:10088 71413 71417 mice +T1696 NCBITaxon:33208 71495 71502 animals +T1697 CHEBI:28201 71568 71576 rotenone +T1698 GO:0045333 71667 71678 respiration +T1699 CHEBI:25675 71692 71702 oligomycin +T1700 PR:000013059 71716 71722 PGC-1α +T1701 NCBITaxon:10088 71726 71730 Mice +T1702 UBERON:0004288 71751 71759 Skeletal +T1703 PR:000013059 71900 71906 PGC-1α +T1704 PR:000013059 71922 71928 PGC-1α +T1705 NCBITaxon:10088 71941 71945 mice +T1706 GO:0040011 72016 72027 ambulations +T1707 GO:0040011 72099 72108 locomotor +T1708 PR:000013059 72301 72307 PGC-1α +T1709 PR:000013059 72351 72357 PGC-1α +T1710 PR:000013059 72365 72371 PGC-1α +T1711 NCBITaxon:10088 72375 72379 mice +T1712 PR:000013059 72542 72548 PGC-1α +T1713 NCBITaxon:10088 72614 72618 mice +T1714 PR:000013059 72812 72818 PGC-1α +T1715 UBERON:0001389 72892 72905 soleus muscle +T1716 PR:000013059 72933 72939 PGC-1α +T1717 PR:000013059 72955 72961 PGC-1α +T1718 NCBITaxon:10088 72973 72977 mice +T1719 PR:000013059 73122 73128 PGC-1α +T1720 UBERON:0000948 73160 73167 Cardiac +T1721 PR:000013059 73202 73208 PGC-1α +T1722 NCBITaxon:10088 73212 73216 Mice +T1723 PR:000013059 73258 73264 PGC-1α +T1724 PR:000013059 73280 73286 PGC-1α +T1725 NCBITaxon:10088 73305 73309 mice +T1726 PR:000013059 73428 73434 PGC-1α +T1727 NCBITaxon:10088 73438 73442 mice +T1728 UBERON:0000948 73614 73619 heart +T1729 UBERON:0002082 73675 73686 ventricular +T1730 CHEBI:37886 73852 73870 adrenergic agonist +T1731 CHEBI:4670 73871 73881 dobutamine +T1732 PR:000013059 73899 73905 PGC-1α +T1733 PR:000013059 73921 73927 PGC-1α +T1734 NCBITaxon:10088 73939 73943 mice +T1735 UBERON:0005396 74037 74051 carotid artery +T1736 UBERON:0002084 74061 74075 left ventricle +T1737 UBERON:0000948 74115 74120 Heart +T1738 UBERON:0002082 74160 74171 ventricular +T1739 CHEBI:4670 74251 74261 dobutamine +T1740 PR:000013059 74286 74292 PGC-1α +T1741 NCBITaxon:10088 74296 74300 Mice +T1742 GO:0031649 74321 74332 Thermogenic +T1743 PR:000013059 74347 74353 PGC-1α +T1744 PR:000013059 74370 74376 PGC-1α +T1745 NCBITaxon:10088 74389 74393 mice +T1746 UBERON:0001052 74443 74449 rectal +T1747 UBERON:0001348 74694 74697 BAT +T1748 PR:000017035 74708 74713 UCP-1 +T1749 SO:0000673 74714 74724 transcript +T1750 PR:000017035 74784 74788 UCP1 +T1751 CHEBI:4883 74791 74807 Ethidium bromide +T1752 CHEBI:4883 74809 74815 Eth Br +T1753 GO:0005840 74829 74838 ribosomal +T1754 PR:000017035 74900 74905 UCP-1 +T1755 SO:0000673 74906 74916 transcript +T1756 PR:000014237 75012 75016 36B4 +T1757 SO:0000673 75017 75027 transcript +T1758 CHEBI:37886 75067 75085 adrenergic agonist +T1759 UBERON:0001348 75162 75165 BAT +T1760 GO:0045333 75176 75187 respiration +T1761 CHEBI:37886 75196 75214 adrenergic agonist +T1762 PR:000013059 75256 75262 PGC-1α +T1763 PR:000013059 75278 75284 PGC-1α +T1764 NCBITaxon:10088 75303 75307 mice +T1765 UBERON:0002107 75428 75435 Hepatic +T1766 http://purl.obolibrary.org/obo/MONDO_0004790 75428 75445 Hepatic Steatosis +T1767 PR:000013059 75458 75464 PGC-1α +T1768 NCBITaxon:10088 75468 75472 Mice +T1769 GO:0001889 75505 75519;75527 75532 development of ... liver +T1770 UBERON:0002107 75527 75532 liver +T1771 PR:000013059 75536 75542 PGC-1α +T1772 NCBITaxon:10088 75546 75550 mice +T1773 UBERON:0002107 75627 75632 liver +T1774 PR:000013059 75644 75650 PGC-1α +T1775 NCBITaxon:10088 75654 75658 mice +T1776 GO:0007631 75665 75668 fed +T1777 CHEBI:18059 75732 75737 lipid +T1778 CHEBI:10545 75759 75767 electron +T1779 UBERON:0002107 75787 75792 liver +T1780 PR:000013059 75798 75804 PGC-1α +T1781 PR:000013059 75812 75818 PGC-1α +T1782 NCBITaxon:10088 75822 75826 mice +T1783 CHEBI:18059 75889 75894 lipid +T1784 UBERON:0002107 75919 75924 liver +T1785 CHEBI:17855 75925 75928 TAG +T1786 PR:000013059 75939 75945 PGC-1α +T1787 PR:000013059 75961 75967 PGC-1α +T1788 NCBITaxon:10088 75979 75983 mice +T1789 GO:0007631 75990 75993 fed +T1790 CL:0000182 76045 76056 Hepatocytes +T1791 PR:000013059 76071 76077 PGC-1α +T1792 NCBITaxon:10088 76081 76085 Mice +T1793 MOP:0000568 76102 76111 Oxidative +T1794 CL:0000182 76157 76168 hepatocytes +T1795 CHEBI:30823 76205 76211 oleate +T1796 CHEBI:30823 76230 76236 oleate +T1797 CHEBI:29238 76244 76246 3H +T1798 CHEBI:7896 76247 76256 palmitate +T1799 MOP:0000568 76257 76266 oxidation +T1800 CHEBI:29238 76274 76276 3H +T1801 CHEBI:7896 76277 76286 palmitate +T1802 MOP:0000568 76287 76296 oxidation +T1803 CL:0000182 76317 76328 hepatocytes +T1804 PR:000013059 76343 76349 PGC-1α +T1805 PR:000013059 76357 76363 PGC-1α +T1806 NCBITaxon:10088 76367 76371 mice +T1807 CHEBI:30823 76432 76438 oleate +T1808 CHEBI:30823 76444 76450 oleate +T1809 CL:0000182 76533 76544 hepatocytes +T1810 NCBITaxon:10088 76552 76556 mice +T1811 MOP:0000568 76599 76608 oxidation +T1812 PR:000013059 76656 76662 PGC-1α +T1813 PR:000013059 76721 76727 PGC-1α +T1814 NCBITaxon:10088 76731 76735 mice +T1815 PR:000013059 76760 76766 PGC-1α +T1816 GO:0045333 76809 76820 respiration +T1817 CL:0000182 76842 76853 hepatocytes +T1818 PR:000013059 76868 76874 PGC-1α +T1819 PR:000013059 76890 76896 PGC-1α +T1820 NCBITaxon:10088 76908 76912 mice +T1821 CHEBI:28201 76929 76937 rotenone +T1822 PR:000013059 76991 76997 PGC-1α +T1823 CHEBI:17855 77007 77010 TAG +T1824 GO:0019432 77007 77020 TAG synthesis +T1825 CL:0000182 77039 77050 hepatocytes +T1826 CHEBI:17855 77076 77079 TAG +T1827 GO:0019432 77076 77089 TAG synthesis +T1828 CHEBI:17754 77097 77105 glycerol +T1829 CL:0000182 77152 77163 hepatocytes +T1830 PR:000013059 77178 77184 PGC-1α +T1831 PR:000013059 77200 77206 PGC-1α +T1832 NCBITaxon:10088 77218 77222 mice +T1833 PR:000013059 77265 77271 PGC-1α +T1834 PR:000013059 77304 77310 PGC-1α +T1835 NCBITaxon:10088 77314 77318 Mice +T1836 CHEBI:17234 77328 77335 Glucose +T1837 PR:000045358 77349 77356 Insulin +T1838 PR:000013059 77379 77385 PGC-1α +T1839 CHEBI:17234 77429 77436 glucose +T1840 PR:000013059 77485 77491 PGC-1α +T1841 PR:000013059 77507 77513 PGC-1α +T1842 NCBITaxon:10088 77525 77529 mice +T1843 CHEBI:33290 77553 77557 chow +T1844 PR:000013059 77580 77586 PGC-1α +T1845 PR:000013059 77602 77608 PGC-1α +T1846 NCBITaxon:10088 77621 77625 mice +T1847 CHEBI:33290 77691 77695 chow +T1848 CHEBI:17234 77722 77729 glucose +T1849 PR:000013059 77746 77752 PGC-1α +T1850 NCBITaxon:10088 77756 77760 mice +T1851 PR:000013059 77931 77937 PGC-1α +T1852 NCBITaxon:10088 77941 77945 mice +T1853 UBERON:0001017 78004 78026 Central Nervous System +T1854 PR:000013059 78030 78036 PGC-1α +T1855 NCBITaxon:10088 78040 78044 Mice +T1856 UBERON:0000956 78097 78112 cerebral cortex +T1857 PR:000013059 78125 78131 PGC-1α +T1858 NCBITaxon:10088 78135 78139 mice +T1859 UBERON:0002606 78179 78187 neuropil +T1860 CL:0000540 78211 78219 neuronal +T1861 GO:0043204 78220 78229 perikarya +T1862 PR:000013059 78251 78257 PGC-1α +T1863 NCBITaxon:10088 78261 78265 mice +T1864 CHEBI:51686 78267 78278 hematoxylin +T1865 GO:0005773 78385 78393 vacuoles +T1866 GO:0016020 78405 78415 membranous +T1867 UBERON:0000956 78453 78468 cerebral cortex +T1868 PR:000013059 78489 78495 PGC-1α +T1869 NCBITaxon:10088 78499 78504 mouse +T1870 PR:000013059 78522 78528 PGC-1α +T1871 UBERON:0000948 78565 78572 Cardiac +T1872 UBERON:0000948 78615 78621 Hearts +T1873 PR:000013059 78625 78631 PGC-1α +T1874 PR:000013059 78639 78645 PGC-1α +T1875 NCBITaxon:10088 78649 78653 Mice +T1876 GO:0008152 78705 78714 Metabolic +T1877 SO:0000704 78715 78719 Gene +T1878 GO:0010467 78715 78730 Gene Expression +T1879 UBERON:0002107 78734 78739 Liver +T1880 GO:0007631 78743 78746 Fed +T1881 PR:000013059 78758 78764 PGC-1α +T1882 PR:000013059 78772 78778 PGC-1α +T1883 NCBITaxon:10088 78782 78786 Mice +T1884 GO:0007631 78964 78967 fed +T1885 PR:000013059 78968 78974 PGC-1α +T1886 NCBITaxon:10088 78978 78982 mice +T1887 GO:0007631 79002 79005 fed +T1888 NCBITaxon:10088 79006 79010 mice +T1889 PR:000013059 79051 79057 PGC-1α +T1890 NCBITaxon:10088 79061 79065 mice +T1891 PR:000005835 79097 79104 L-CPT I +T1892 UBERON:0002107 79106 79111 liver +T1893 PR:000005835 79106 79147 liver-type carnitine palmitoyltransferase +T1894 CHEBI:17126 79117 79126 carnitine +T1895 CHEBI:61907 79155 79176 medium-chain acyl-CoA +T1896 PR:000028988 79192 79197 SREBP +T1897 CHEBI:15889 79199 79205 sterol +T1898 SO:0001861 79199 79224 sterol regulatory element +T1899 PR:000028988 79199 79240 sterol regulatory element binding protein +T1900 GO:0065007 79206 79216 regulatory +T1901 CHEBI:15541 79247 79258 steroyl-CoA +T1902 CHEBI:35366 79276 79286 fatty acid +T1903 CHEBI:18035 79347 79361 diacylglycerol +T1904 PR:000013059 79799 79805 PGC-1α +T1905 UBERON:0000467 79830 79836 system +T1906 GO:0008152 79844 79853 metabolic +T1907 GO:0065007 79904 79911 control +T1908 UBERON:0002107 79916 79923 hepatic +T1909 http://purl.obolibrary.org/obo/MONDO_0004790 79916 79933 hepatic steatosis diff --git a/src/ontogpt/evaluation/craft/database/all/15760270.txt b/src/ontogpt/evaluation/craft/database/all/15760270.txt new file mode 100644 index 000000000..76f02b3a0 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15760270.txt @@ -0,0 +1,391 @@ +PGC-1α Deficiency Causes Multi-System Energy Metabolic Derangements: Muscle Dysfunction, Abnormal Weight Control and Hepatic Steatosis + +Abstract + +The gene encoding the transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) was targeted in mice. PGC-1α null (PGC-1α−/−) mice were viable. However, extensive phenotyping revealed multi-system abnormalities indicative of an abnormal energy metabolic phenotype. The postnatal growth of heart and slow-twitch skeletal muscle, organs with high mitochondrial energy demands, is blunted in PGC-1α−/− mice. With age, the PGC-1α−/− mice develop abnormally increased body fat, a phenotype that is more severe in females. Mitochondrial number and respiratory capacity is diminished in slow-twitch skeletal muscle of PGC-1α−/− mice, leading to reduced muscle performance and exercise capacity. PGC-1α−/− mice exhibit a modest diminution in cardiac function related largely to abnormal control of heart rate. The PGC-1α−/− mice were unable to maintain core body temperature following exposure to cold, consistent with an altered thermogenic response. Following short-term starvation, PGC-1α−/− mice develop hepatic steatosis due to a combination of reduced mitochondrial respiratory capacity and an increased expression of lipogenic genes. Surprisingly, PGC-1α−/− mice were less susceptible to diet-induced insulin resistance than wild-type controls. Lastly, vacuolar lesions were detected in the central nervous system of PGC-1α−/− mice. These results demonstrate that PGC-1α is necessary for appropriate adaptation to the metabolic and physiologic stressors of postnatal life. + +Introduction + +Mitochondrial functional capacity is dynamically regulated to meet the diverse energy demands imposed on the mammalian organism following birth. Postnatal mitochondrial biogenesis involves multiple signaling and transcriptional regulatory pathways that control the coordinate expression of nuclear and mitochondrial genes involved in mitochondrial structure, metabolism, and proliferation [1]. Recent evidence points toward a transcriptional coactivator, peroxisome proliferator-activated receptor-γ (PPARγ) coactivator-1α (PGC-1α), as an integrator of the molecular regulatory circuitry involved in the transcriptional control of cellular energy metabolism, including mitochondrial function and biogenesis [1,2]. PGC-1α was discovered in a yeast two-hybrid screen for brown adipose-specific factors that interact with the adipogenic nuclear receptor PPARγ [2]. Subsequently, two additional PGC-1 family members were identified, PGC-1 related coactivator (PRC) [3] and PGC-1β [4,5]. PGC-1α serves as a direct transcriptional coactivator of nuclear and nonnuclear receptor transcription factors involved in cellular energy metabolism [6]. PGC-1α is distinct among most coactivators in that it exhibits a tissue-enriched expression pattern and is highly inducible by physiologic conditions known to increase the demand for mitochondrial ATP or heat production [2,6,7]. PGC-1α is enriched in brown adipose tissue (BAT), heart, slow-twitch skeletal muscle, and kidney—all tissues with high-capacity mitochondrial systems. The expression of the gene encoding PGC-1α is rapidly induced by cold exposure, short-term exercise, and fasting [2,8,9,10,11,12,13,14,15]. These latter observations suggest that PGC-1α is involved in the physiologic control of energy metabolism. + +Several lines of evidence, based on the results of overexpression studies, indicate that PGC-1α is sufficient to promote mitochondrial biogenesis and regulate mitochondrial respiratory capacity. First, PGC-1α activates the transcription of mitochondrial uncoupling protein-1 (UCP-1) in BAT through interactions with the nuclear hormone receptors PPARγ and thyroid receptor [2]. Second, forced expression studies in adipogenic and myogenic mammalian cell lines demonstrated that PGC-1α activates mitochondrial biogenesis through a group of transcription factor targets including nuclear respiratory factors 1 and 2 (NRF-1 and -2) and mitochondrial transcription factor A (Tfam), key transcriptional regulators of mitochondrial DNA transcription and replication [8]. Third, studies in primary cardiac myocytes in culture and in the hearts of transgenic mice have demonstrated that overexpression of PGC-1α promotes mitochondrial biogenesis [10,16]. Lastly, forced expression of PGC-1α in skeletal muscle of transgenic mice triggers mitochondrial proliferation and the formation of mitochondrial-rich type I, oxidative (“slow-twitch”) muscle fibers [17]. Collectively, these results indicate that PGC-1α is sufficient to drive mitochondrial biogenesis. + +Recent evidence also implicates PGC-1α in the homeostatic control of systemic energy metabolism. PGC-1α has been shown to regulate several key hepatic gluconeogenic genes [18,19,20,21]. Recent studies have also shown altered expression of PGC-1α and downstream mitochondrial target pathways in skeletal muscle of humans with insulin resistance and diabetes [22,23,24]. In addition, single nucleotide polymorphisms within the human PGC-1α gene have been shown to be associated with obesity, hypertension, and diabetes [25,26,27,28,29,30]. + +The gain-of-function studies described to date provide compelling evidence that PGC-1α is capable of regulating postnatal energy metabolism. However, the necessity of PGC-1α for energy metabolic homeostasis, mitochondrial biogenesis, development, and growth can only be addressed using loss-of-function strategies. To this end, we have established and characterized mice with targeted deletion of the PGC-1α gene. Our studies of PGC-1α−/− mice demonstrate that PGC-1α is not absolutely required for prenatal viability including mitochondrial biogenesis. However, our findings indicate that the coactivator PGC-1α serves a critical role in the normal metabolic function of multiple organs and for appropriate adaptation to physiologic stress during postnatal life. + +Results + +Disruption of the PGC-1α Gene in Mice + +A neomycin-based gene targeting vector was generated to delete exons 4 and 5 of the murine PGC-1α gene. The targeting event resulted in a 3′ homologous recombination with insertion of the remainder of the construct (Figure 1A). The insertion/recombination event was confirmed by Southern blotting and DNA sequencing. The insertion caused an exon 3 duplication between exons 5 and 6 that creates a coding region frameshift resulting in a premature termination at amino acid 255. Germline transmission of the mutant allele was confirmed using Southern blotting (Figure 1B) and PCR (unpublished data). The PGC-1α gene disruption resulted in an unstable transcript that could not be detected by RNA blot analysis in heart and other tissues in PGC-1α−/− mice (Figure 1C and unpublished data). Quantitative RT-PCR was utilized to further evaluate the efficacy of the gene targeting. For these studies, PCR primers were designed to amplify a region of the PGC-1α gene transcript containing the exon 5–6 border (predicted to be absent in PGC-1α−/− mice) or the exon 5–3 border (predicted to be present only in the PGC-1α−/− mice). The exon 5–6 amplicon was detected in heart and BAT of wild-type (WT) but not PGC-1α−/− mice (Figure 1D). Conversely, the exon 5–3 product was present only in PGC-1α−/− mice (Figure 1D). An exon 10–11 border amplicon (predicted to be present in both genotypes) was detected in WT and PGC-1α−/− mice, but was greatly diminished in the PGC-1α−/− mice, indicating that the mutant transcript is unstable. PGC-1α protein was not detected in whole cell (Figure 1E) or nuclear protein extracts (unpublished data) isolated from BAT of PGC-1α−/− mice under basal conditions or in response to cold exposure, a condition known to markedly induce the expression of PGC-1α in BAT. Smaller mutant PGC-1α proteins were also not detected by Western blot analysis (unpublished data). Lastly, expression of the genes encoding the other known PGC-1 family members, PGC-1β and PRC, was not significantly altered in heart of PGC-1α−/− mice (Figure 1C). Taken together, these results support the conclusion that the gene targeting event resulted in a PGC-1α null allele. + +General Characteristics of the PGC-1α−/− Mice: Age- and Sex-Dependent Obesity + +Heterozygous (PGC-1α+/−) mice were bred to generate PGC-1α−/− offspring. The observed genotype ratios of the offspring were consistent with the expected Mendelian ratios (unpublished data). Unexpected deaths of the offspring were not observed, and PGC-1α+/− and PGC-1α−/− offspring appeared normal. Total body weights obtained 1 wk after birth revealed a 15%–20% reduction in total body mass for male and female PGC-1α−/− mice relative to sex-matched PGC-1α+/+ littermates (Figure 2). The weight decrement between PGC-1α−/− and PGC-1α+/+ littermates disappeared by 3 wk of age (Figure 2A). At 18 wk of age, body weight was modestly but significantly greater in male and female PGC-1α−/− mice compared to sex-matched PGC-1α+/+ controls (Figure 2A). This weight difference was also significant for female PGC-1α−/− mice at 24 wk of age (Figure 2A). The abnormal weight gain in PGC-1α−/− mice was not associated with differences in food intake (unpublished data) or alterations in general activity as monitored for 48 h (Figure S1). Percent body fat, as determined by dual-energy X-ray absorption (DEXA), was greater in 18- and 24-wk-old female PGC-1α−/− mice compared to age-matched female PGC-1α+/+ counterparts, indicating that the body weight difference was due, at least in part, to increased body fat (Figure 2A). Lean mass was not significantly different between the genotypes (unpublished data). Although DEXA did not detect excess body fat in male PGC-1α−/− mice at 18 or 24 wk of age, older male mutant mice (over 7 mo of age) accumulated more body fat than male WT controls (Figure 2A and unpublished data). + +Individual organ weights were assessed, given the importance of mitochondrial energy metabolism for postnatal growth in certain organs. The weights of heart and slow-twitch fiber-enriched skeletal muscles, including gastrocnemius and soleus, but not the less oxidative tibialis anterior, were significantly lower in male and female PGC-1α−/− mice compared with age and sex-matched PGC-1α+/+ controls at 3 and 8 wk of age (Figure 2B and unpublished data). In contrast, the weights of brain, liver, kidney, and BAT were not significantly different between the genotypes at the 3-wk time point (Figure 2B). Thus, certain tissues with high mitochondrial energy requirements, such as heart and slow-twitch skeletal muscle, exhibit modest growth defects in PGC-1α−/− mice. + +Abnormal Muscle Mitochondrial Phenotype in PGC-1α−/− Mice + +General histologic analyses were performed to begin to evaluate the mild growth defect found in postnatal heart and skeletal muscle of the PGC-1α−/− mice. There were no obvious abnormalities in cellularity, cell size, or extracellular matrix in the tissues of 1–2-mo-old PGC-1α−/− mice (unpublished data). Given the important role of PGC-1α in mitochondrial function and biogenesis, we examined mitochondrial ultrastructure in the relevant tissues. Electron microscopic analysis revealed fewer and smaller mitochondria in soleus muscle of PGC-1α−/− mice compared to sex- and age- matched PGC-1α+/+ controls (Figure 3A). Quantitative morphometry of the electron micrographs confirmed that the cellular volume density of soleus mitochondria was significantly lower in PGC-1α−/− mice compared to PGC-1α+/+ controls independent of changes in the myofibrillar component (Figure 3B). Consistent with a defect in mitochondrial biogenesis, we found a reduction in the expression of nuclear genes encoding proteins involved in mitochondrial electron transport (cytochrome c and cytochrome oxidase IV) and oxidative phosphorylation (beta subunit of ATP synthase) in soleus muscle of PGC-1α−/− mice compared with PGC-1α+/+ controls. In addition, the expression of Tfam, a known PGC-1α target involved in mitochondrial DNA replication/transcription, was diminished in PGC-1α−/− soleus, providing one potential mechanism for defective mitochondrial biogenesis (Figure 3C). In contrast to the results with soleus, no significant differences in mitochondrial ultrastructure or volume density were noted in heart or BAT of PGC-1α−/− mice (unpublished data). + +To determine whether mitochondrial function was altered in the skeletal muscle of PGC-1α−/− mice, mitochondrial respiration rates were measured using tissue strips prepared from soleus muscle. In soleus of PGC-1α−/− mice, a significant defect in state 3 (ADP-stimulated) respiration, but not state 2 (basal), was detected using succinate as the substrate (Figure 3D). State 4 respiration rates (in the presence of oligomycin) were also similar between the genotypes, indicating that the coupling of respiration to ATP production was not significantly altered in PGC-1α−/− mice. These results are consistent with the modest but significant reduction in mitochondrial volume density. + +Altered Skeletal Muscle Function in PGC-1α−/− Mice + +The abnormality in mitochondrial number and respiratory function in skeletal muscle led us to further evaluate the skeletal muscle phenotype. As an initial step, we measured locomotor activity levels over a 1-h period using a high-resolution photobeam system. PGC-1α−/− male mice exhibited a significantly lower mean number of ambulations and rearings during the hour compared to the PGC-1α+/+ age-matched controls (Figure 4). However, an analysis of exploratory behavior showed that the PGC-1α−/− mice were reluctant to go into the center of the “field” compared to controls. Specifically, PGC-1α−/− mice made significantly fewer entries into, spent significantly less time in, and traveled a significantly shorter distance in the central area of the “field,” although differences in distance traveled in the peripheral zone of the “field” was not significantly different between groups (Figure S2). These data suggest that the general activity level may have been affected by the reluctance of the PGC-1α−/− mice to go into the central area of the field and thus remain in the periphery (thigmotaxis), possibly reflecting altered emotionality such as increased fear. + +A battery of tests was performed to further evaluate the general sensorimotor phenotype of the PGC-1α−/− mice. No differences were found between PGC-1α−/− mice and PGC-1α+/+ controls on the ledge, platform, walking initiation, and 60° and 90° inclined screen tests (unpublished data), suggesting that several sensorimotor functions were intact in the PGC-1α−/− mice. However, the PGC-1α−/− mice were unable to remain on an inverted screen for as long as the PGC-1α+/+ controls (Figure 4A). Since the groups did not differ on the times it took to turn around and climb to the top of 60° and 90° inclined screens, the differences on the inverted screen test suggest that impaired strength rather than deficits in coordination were responsible for these differences. + +To further evaluate the skeletal muscle phenotype, exercise capacity was assessed in the PGC-1α−/− mice. To this end, the PGC-1α−/− mice were exercised on a motorized treadmill apparatus using a run-to-exhaustion format. PGC-1α−/− mice (6–8 mo of age) exhibited a markedly reduced capacity to sustain running exercise (PGC-1α−/− mice, 64 ± 6 s; age-matched PGC-1α+/+ mice, 586 ± 104 s; Figure 4B). The same result was obtained with younger PGC-1α−/− mice, i.e., at 3.5 mo of age (unpublished data). To quantify aerobic exercise capacity, VO2max (maximum oxygen consumption, measured in milliliters of oxygen per kilogram of body weight per minute) was measured with the treadmill-running protocol using indirect calorimetry. VO2max was significantly lower for the PGC-1α−/− mice (120.9 ± 2.0 ml O2 · kg−1 · min−1) compared to PGC-1α+/+ controls (141.6 ± 2.1 ml O2 · kg−1 · min−1) (Figure 4B). To directly evaluate muscle fatigability, the force response to repetitive stimulation of isolated soleus muscle was determined. The capacity to generate force following a series of tetani is dependent upon mitochondrial ATP production. During the initial phase of the stimulation period, there was no difference in force generation in muscles isolated from PGC-1α−/− mice and PGC-1α+/+ controls. However, fatigue resistance index, defined as the percent of initial force generated following a 2-min series of fatiguing contractions, was significantly lower in the PGC-1α−/− mice (14.6 ± 1.5%) compared to PGC-1α+/+ controls (24.8 ± 2.9%) (Figure 4C). These results, together with the observed abnormalities in skeletal muscle mitochondrial structure and function, indicate that PGC-1α is necessary for functional adaptation of skeletal muscle to physiologic demands. + +Functional Abnormalities in Hearts of PGC-1α−/− Mice + +PGC-1α expression is enriched in heart, a tissue that relies heavily on mitochondrial energy metabolism to maintain pump function throughout the postnatal life of the mammalian organism. Echocardiographic screening studies of PGC-1α−/− mice at ages 4–6 mo did not reveal any significant differences in chamber sizes or ventricular function compared to WT controls (unpublished data). Cardiac functional and metabolic reserve was evaluated using exercise echocardiographic stress testing (EST). Given that the exercise capacity of PGC-1α−/− mice is diminished, a series of preliminary treadmill exercise studies were performed to define a reasonable exercise duration for run-to-exhaustion to be used as a target duration for the EST. Based on the results of these studies, an EST regimen was performed in which PGC-1α+/+ control animals were exercised for a duration of 60 s to match the predicted average for the PGC-1α−/− mice (ages 6–8 mo). Echocardiographic images were obtained immediately following 60 s of exercise for the PGC-1α+/+ controls or at the point of exhaustion for PGC-1α−/− mice (mean 60 ± 6.1 s, range 45–90 s). Echocardiographic-determined left ventricular fractional shortening and heart rate were monitored for the 10-min period immediately post exercise. The mean heart rate of the PGC-1α−/− mice exhibited an inappropriate decline during the post exercise period (Figure 5A). In addition, echocardiographically determined left ventricular fractional shortening was decreased in the PGC-1α−/− mice, but not the PGC-1α+/+ mice during the first 4 min of the post exercise period (Figure 5A). + +The results of the EST did not distinguish between a primary cardiac abnormality versus effects secondary to the exhaustion caused by reduced exercise tolerance related to skeletal muscle dysfunction. To directly assess cardiac function, the hearts of PGC-1α−/− and PGC-1α+/+ mice were isolated and perfused in the working mode. Hearts isolated from PGC-1α−/− mice generated lower cardiac work (cardiac output multiplied by peak systolic pressure) compared to PGC-1α+/+ mice at identical loading conditions (Table 1). This reduction in cardiac work was due to a reduced cardiac output (Table 1). The relative contribution of heart rate and stroke volume to diminished cardiac output in the PGC-1α−/− mice could not be delineated, because both were decreased but neither to a significant degree (Table 1). To further distinguish between abnormalities in heart rate and ventricular function, in vivo hemodynamic response to the β1,α1-adrenergic-selective agonist dobutamine was evaluated using a miniaturized Millar catheter. The ventricular functional response to dobutamine was similar in PGC-1α+/+ and PGC-1α−/− mice (Figure 5B, right graph). However, PGC-1α−/− mice exhibited a significantly blunted heart rate response to β-adrenergic stimulation (Figure 5B, left graph). Taken together with the EST, these results strongly suggest that the PGC-1α−/− hearts are unable to mount an appropriate chronotropic response to exercise and other physiologic stimuli that activate β-adrenergic input to the heart. However, our results did not reveal evidence for contractile dysfunction. + +PGC-1α−/− Mice Exhibit an Abnormal Thermogenic Response + +PGC-1α has been implicated as an inducible regulator of mitochondrial respiratory uncoupling, an important source of heat production in BAT [2]. To determine whether PGC-1α is necessary for an appropriate thermogenic response, PGC-1α+/+ and PGC-1α−/− mice were subjected to cold exposure (4 °C) for a 5-h period while core body temperature was monitored. PGC-1α−/− mice exhibited a markedly abnormal drop in core temperature compared to the WT controls (Figure 6A). Specifically, the mean decline in core temperature was greater than 12 °C at the 5-h time point in PGC-1α−/− mice, compared to an approximately 3 °C decrement in PGC-1α+/+ controls. Although this thermogenic phenotype was consistently present in mice aged 28–37 d, it was absent in older mice (unpublished data). + +The histologic appearance and neutral lipid stores of BAT were assessed as an initial step to characterize the thermogenic phenotype exhibited by PGC-1α−/− mice. Histologic and lipid quantification studies were performed. Electron microscopic analyses indicated that the mitochondrial ultrastructure was similar in BAT isolated from PGC-1α−/− and PGC-1α+/+ mice before and after cold exposure (unpublished data). In addition, levels of BAT triglyceride were similar between the two genotypes (unpublished data). UCP-1 is a cold-inducible protein involved in mitochondrial respiratory uncoupling to generate heat in BAT. UCP-1 gene transcription is known to be activated by PGC-1α [2]. Surprisingly, basal and cold-induced BAT UCP-1 mRNA levels were similar in PGC-1α+/+ and PGC-1α−/− mice (Figure 6B). These results suggest that PGC-1α is not necessary for the induction of the expression of UCP-1 with cold exposure, and that other factors, such as reduced capacity for mitochondrial respiration, likely contribute to the abnormal thermogenic response in the PGC-1α−/− mice. + +Thermogenesis in rodents related to mitochondrial uncoupling is under the control of β3-adrenergic receptor coupled signaling. Accordingly, the in vivo oxygen consumption response to β3-adrenergic stimulation was examined in PGC-1α−/− mice. For these experiments, VO2 (oxygen consumption) was measured following administration of the β3-agonist BRL 37344 using indirect calorimetry. VO2 was significantly increased in response to BRL 3744 in PGC-1α+/+ but not PGC-1α−/− mice (Figure 6C). These results indicate that the metabolic response of BAT to an acute stimulus such as cold and/or β3-adrenergic stimulation is altered in the PGC-1α null mice, likely related to reduced capacity for mitochondrial respiratory uncoupling. + +Fasting-Induced Hepatic Steatosis in PGC-1α−/− Mice + +Previous studies have implicated PGC-1α in several hepatic metabolic functions including fatty acid oxidation and gluconeogenesis [18,19,20,21]. Accordingly, the hepatic phenotype was evaluated under basal conditions and following a 24-h fast, a stimulus known to induce fatty acid oxidation and gluconeogenic rates in liver. Under basal fed conditions, the livers of the PGC-1α−/− mice appeared grossly normal and did not exhibit histologic abnormalities (unpublished data). However, following a 24 h-fast, the PGC-1α−/− mice exhibited marked hepatic steatosis as determined by gross inspection, oil red O staining, electron microscopy, and measurements of liver triglyceride (TAG) levels (Figure 7). There were no differences in plasma triglycerides or free fatty acids between the genotypes in fed or fasted states (unpublished data). To further investigate the mechanisms involved in the fasting-induced hepatic steatotic response, hepatocytes were isolated from PGC-1α−/− mice and WT controls. Oleate loading experiments revealed that the PGC-1α−/− hepatocytes accumulated neutral lipid to a significantly greater extent than the WT cells (Figure 8A). 3H-palmitate oxidation rates were significantly lower in PGC-1α−/− hepatocytes compared to PGC-1α+/+ hepatocytes under basal conditions and following exposure to oleate (Figure 8B). Taken together, these latter results indicate a cell-autonomous defect in PGC-1α−/− hepatocytes that results in an inability to maintain cellular lipid balance in the context of increased delivery of lipid such as occurs with fasting. + +PPAR, a known regulator of hepatic mitochondrial fatty acid oxidation enzyme gene expression, is a target for coactivation by PGC-1α [31]. Therefore, we sought to determine whether the steatotic phenotype of the PGC-1α−/− mice related to reduced expression of PPAR target genes. To this end, a survey of candidate genes and gene expression profiling experiments were performed. Surprisingly, the hepatic expression of PPAR target genes involved in cellular fatty acid and oxidation (MCPT and MCAD) were not significantly different between the genotypes under fed or fasted conditions (Table 2). Next, we performed experiments to determine whether the reduced capacity for fat oxidation in the hepatocytes of the PGC-1α−/− mice was related to altered mitochondrial respiratory function. Compared to the WT controls, PGC-1α−/− hepatocytes exhibited a modest but significant reduction in both state 2 and state 3 respiration rates (Figure 8C). These results identify one potential mechanism responsible for the fasting-induced hepatic steatosis: reduced capacity for fat oxidation due to mitochondrial respiratory dysfunction. + +Although the liver gene expression profiling studies did not reveal abnormalities in the fatty acid oxidation pathway in the PGC-1α−/− mice, several interesting differences in the activity of the sterol regulatory element binding protein-1c (SREBP-1c) pathway were noted. Specifically, the fasting-mediated down-regulation of SREBP-1c and its target gene stearoyl-CoA desaturase (SCD1), was abolished in PGC-1α−/− mice (Table 2). Furthermore, expression of the gene encoding diglyceride acyltransferase (DGAT), which catalyzes the last step in TAG synthesis, was activated at baseline and induced by fasting to a greater level in PGC-1α−/− mice (Table 2). These results suggest that, in addition to a defect in oxidation, components of the TAG synthesis pathway are activated in the PGC-1α−/− mice. To evaluate this possibility directly, rates of 3H-glycerol incorporation into TAG were determined in isolated hepatocytes. 3H-TAG incorporation was increased nearly 50% in hepatocytes isolated from PGC-1α−/− mice compared to PGC-1α+/+ controls (Figure 8D), confirming that TAG synthesis rates are increased in PGC-1α null hepatocytes, identifying a second potential mechanism contributing to the fasting-induced hepatic steatosis. + +Despite a Mild Obese Phenotype, Female PGC-1α−/− Mice Do Not Exhibit Insulin Resistance + +Recent studies have suggested that specific PGC-1α single nucleotide polymorphisms and haplotypes may influence the development of insulin resistance and diabetes [27,30] and that PGC-1 activity is diminished in insulin-resistant and diabetic muscle [22,23]. Accordingly, peripheral glucose disposal and insulin responsiveness were examined in PGC-1α−/− mice. Glucose tolerance testing of 2-mo-old male and female mice revealed no significant difference in blood glucose excursion between PGC-1α+/+ and PGC-1α−/− groups (unpublished data). Given that older female PGC-1α−/− mice develop an increase in body fat stores, glucose tolerance and insulin responsiveness were further evaluated in this group. Glucose tolerance testing in 4.5-mo-old female PGC-1α−/− mice revealed that, despite increased body weight [mean ± standard error of the mean (SEM) weight of PGC-1α+/+ mice = 22.4 ± 0.79 g; PGC-1α−/− mice = 25.2 ± 1.04 g), PGC-1α−/− mice exhibited similar levels of glucose tolerance compared to WT mice on standard rodent chow (Figure 9). To examine glucose homeostasis in response to high-fat diet, female PGC-1α+/+ and PGC-1α−/− mice were placed on high-fat chow (43% calories from fat) for 6 wk starting at 8 wk of age. The weight gained on the high-fat diet was similar for the PGC-1α+/+ and PGC-1α−/− groups (Figure S3). Surprisingly, the PGC-1α−/− mice on a high-fat diet were significantly more glucose-tolerant and insulin-sensitive compared to the PGC-1α+/+ mice (Figure 9B). Taken together, these results indicate that, despite excess body fat under standard conditions, the female PGC-1α−/− mice do not exhibit insulin resistance. Moreover, the PGC-1α−/− mice are more glucose-tolerant and insulin-sensitive than WT mice on a high-fat diet. + +Structural Abnormalities of the Central Nervous System in PGC-1α−/− Mice + +In surveying the tissues of the PGC-1α−/− mice, structural abnormalities of the brain were observed. Light microscopic examination of PGC-1α−/− brain tissue samples demonstrated a well-preserved cerebral cortical neuronal complement, a result confirmed by measurement of neuron density in sections of the parietal lobe (PGC-1α+/+, 1,261 ± 91 neurons/mm2 versus PGC-1α−/−, 1,299 ± 82 neurons/mm2; not significant). Patchy areas of microvacuolation involving the neuropil and individual pyramidal neurons of the deep layers of the cerebral cortex were noted in the PGC-1α−/− mice but not the PGC-1α+/+ mice (Figure 10A). Immunolocalization of an astrocytic marker, glial fibrillary acidic protein, failed to show an increase in numbers of astrocytic processes in PGC-1α−/− mouse cerebral cortex (unpublished data). The hippocampus also showed neuronal microvacuolation, albeit to a lesser degree than the parietal cortex. Microvacuolation of the neuropil and neurons of the PGC-1α−/− basal ganglia (caudate and putamen) was also noted in association with a patchy increase in the number and intensity of glial fibrillary acidic protein-immunoreactive astrocytic processes (unpublished data). Areas of microvacuolation also involved multiple brainstem regions. Only rare vacuolated Purkinje and granule cell neurons were identified in the PGC-1α−/− cerebellar cortex. Neither microglial proliferation nor perivascular lymphocytic inflammatory infiltrates were noted in the PGC-1α−/− CNS. + +Ultrastructural examination of the PGC-1α−/− parietal cerebral cortex confirmed the presence of microvacuolated neurons and neuropil (Figure 10B). Vacuoles containing aggregates of membranous material were present in a subset of cortical neurons. Subcellular localization of the vacuoles was difficult to establish; some may represent vacuolated elements of the neuropil, material in phagocytic cells, presynaptic nerve terminals compressing the soma, or genuine intraperikaryal deposits. + +Discussion + +Previous studies using gain-of-function strategies have shown that the coactivator PGC-1α is capable of coactivating an array of transcription factors involved in energy metabolic processes including fatty acid oxidation, electron transport, and oxidative phosphorylation [6]. Forced expression of PGC-1α triggers mitochondrial biogenesis by activating a complex circuitry of factors including NRF-1, NRF-2, and the orphan nuclear receptor estrogen-related receptor α [23,32]. However, gain-of-function strategies cannot determine whether PGC-1α is essential for critical energy metabolic processes including mitochondrial biogenesis and function. Using targeted gene deletion in mice, we show here that PGC-1α is not essential for normal embryologic development or the fundamental events of mitochondrial biogenesis. However, several lines of evidence support the conclusion that PGC-1α is necessary for the programs that regulate postnatal mitochondrial function and cellular energy metabolism, processes that equip the organism for the energy metabolic rigors of the postnatal environment. First, mitochondrial volume density is diminished in slow-twitch skeletal muscle of PGC-1α−/− mice. Second, mitochondrial respiratory capacity is modestly but significantly altered in skeletal muscle and liver of PGC-1α−/− mice. Third, the growth of heart and soleus muscle, tissues with high reliance on mitochondrial energy production, is blunted. Fourth, control of body fat mass is abnormal in the PGC-1α−/− mice. Finally, PGC-1α−/− mice do not respond normally to a variety of physiologic and dietary stresses known to increase oxidative energy demands. Taken together, these results strongly suggest that PGC-1α is necessary for the terminal stages of mitochondrial maturation necessary to meet the energy demands of the postnatal environment. + +Extensive phenotypic analyses demonstrated that mice lacking PGC-1α are unable to cope with physiologic stressors relevant to postnatal survival. For example, a skeletal muscle phenotype was unveiled in PGC-1α−/− mice under conditions in which energy supply becomes limiting. This was most clearly demonstrated by the profound abnormalities exhibited by PGC-1α−/− mice with exercise-to-exhaustion and repetitive muscle stimulation studies. Similarly, cardiac performance of PGC-1α−/− mice was compromised following severe exertion. This effect was largely due to an abnormal heart rate response. The basis for the observed abnormalities of cardiac heart rate, including a blunted response to β-adrenergic stimulation, is unknown, but could be related to the effects of late-stage growth arrest and corresponding derangements in energy metabolism on sinus node function. PGC-1α was first identified as a coactivator in BAT [2]. Indeed, we found that exposure of the PGC-1α−/− mice to cold, another relevant physiologic stress, resulted in an untoward drop in core body temperature consistent with an abnormality in thermogenesis despite normal cold induction of UCP-1 mRNA in BAT. Studies with a β3-adrenergic agonist confirmed that the peak oxygen consumption rate in thermogenic tissue is diminished in PGC-1α−/− mice. We propose that the thermogenic phenotype is related to reduced capacity for mitochondrial respiration in BAT. Interestingly, this phenotype was only evident during a rather narrow window of postnatal life. Animals at an older age did not exhibit cold intolerance, possibly due to the insulating properties of increased body mass. Collectively, these results demonstrate the importance of PGC-1α as a key transducer of physiologic stimuli to the control of energy metabolism. + +The observation of fasting-induced hepatic steatosis is another example of the inability of PGC-1α−/− mice to respond to postnatal environmental metabolic demands. Following short-term starvation, we found that the PGC-1α−/− mice developed marked hepatocyte triglyceride accumulation. Further analysis revealed that palmitate oxidation rates were reduced in hepatocytes isolated from the PGC-1α−/− mice, which would predispose to lipid accumulation. Surprisingly, the reduction in fatty acid oxidation rates in PGC-1α null hepatocytes was not due to altered expression of PGC-1α/PPAR target genes involved in mitochondrial fatty acid oxidation. However, mitochondrial respiratory rates were diminished. In addition, we found that triglyceride synthesis was abnormally activated, and the expression of genes encoding SREBP-1c and SCD-1, key proteins in the hepatic lipogenic pathway, failed to be appropriately down-regulated in fasted PGC-1α−/− mice. The mechanism involved in this latter finding is unknown. Indeed, the relative contribution of increased triglyceride synthesis rates to the steatotic phenotype cannot be fully discerned from our data, given that this response could reflect the direct effects of PGC-1α deficiency on target genes or a secondary compensatory response to hepatocyte fatty acid accumulation. Consistent with the former possibility, recent evidence indicates that PGC-1α coactivates the nuclear receptor FXR, a negative regulator of SREBP-1c expression and triglyceride synthesis [33]. We conclude that reduced hepatocyte mitochondrial respiratory capacity, and possibly activation of lipogenic programs, result in hepatocyte triglyceride accumulation in the context of increased hepatic delivery of fatty acids such as occurs with fasting. + +We found that after 18 wk of age, female PGC-1α−/− mice exhibit a mild but significantly abnormal weight increase associated with increased fat stores. Lean mass was unchanged at the time points examined. With further aging, a modest but significant increase in body fat was also noted in male PGC-1α−/− mice (unpublished data). The basis for the observed abnormalities in weight control is unknown. We did not find differences in food intake or activity levels in female PGC-1α−/− mice. It is possible that a reduction in systemic energy utilization, related to the mitochondrial dysfunction, leads to increased fat mass and weight gain in the PGC-1α−/− mice. Interestingly, an association between PGC-1α gene polymorphisms and obesity in humans has been recently reported [26]. Clearly, future studies of male and female PGC-1α−/− mice in pure-strain backgrounds over a range of ages will be necessary to fully investigate the observed abnormalities in weight control and fat distribution. + +We did not find evidence for glucose intolerance or insulin resistance in the PGC-1α−/− animals on standard chow. Moreover, female PGC-1α−/− mice were more glucose-tolerant and insulin-sensitive than PGC-1α+/+ controls when consuming a high-fat diet. These findings are surprising, given the results of several recent studies demonstrating reduced expression of PGC-1α in human diabetic skeletal muscle [24,34]. It is certainly possible that compensatory metabolic regulatory mechanisms have been activated in the PGC-1α-deficient mice, accounting for this observation. Alternatively, PGC-1α could serve as a coactivator of factors that mediate diet-induced insulin resistance. Consistent with this notion, we and others have shown that mice lacking the PGC-1α target PPAR exhibit resistance to diet-induced glucose intolerance [21,35,36]. + +Histologic surveys of the PGC-1α−/− mice revealed ultrastructural abnormalities in the central nervous system. Inspection of sections prepared from the brains of PGC-1α−/− mice revealed patchy areas of microvacuolation in the pyramidal neurons of the cerebral cortex, accompanied by a mild increase in the number of astrocytes in the basal ganglia. The basis for this interesting but relatively nonspecific finding is unknown. It is possible that PGC-1α plays an important role in lipid metabolism related to membrane synthesis. Alternatively, the normal process of cellular debris turnover could be altered due to a defect in the energetics of the microglial component of the central nervous system. Although overt neurologic dysfunction was not apparent in PGC-1α−/− mice during the first 6 mo of life (no group differences were found on five of six sensorimotor tests), the PGC-1α−/− mice showed clear deficits on the inverted screen test. These deficits are likely due to impaired muscle strength in the PGC-1α−/− mice, but contributions by peripheral or central nervous system determinants (or both) could be contributory. Moreover, evidence of altered emotionality from the 1-h locomotor activity test also suggests the possibility of altered brain function in PGC-1α−/− mice. It will be of interest to determine whether the neurologic abnormalities contribute to the systemic metabolic abnormalities of the PGC-1α null mice. + +During the preparation of this manuscript, Lin et al. reported an independent mouse line in which the PGC-1α gene was targeted [37]. Phenotypic comparison of the our PGC-1α-deficient line with that of Lin et al. reveals a number of similarities and several interesting differences. Both PGC-1α-deficient lines exhibit cold intolerance, reduced hepatocyte respiration rates, and neurologic lesions. However, a number of interesting differences are notable. First, in contrast to Lin et al., the PGC-1α−/− mice described here do not exhibit any postnatal mortality. Second, we did not find evidence for a defect in gluconeogenesis based on fasting blood glucose levels (unpublished data). In addition, whereas Lin et al. found an abnormal expression profile forCCAAT-enhancer-binding protein β and δ and the gluconeogenic genes encoding phosphoenolpyruvate carboxykinase and glucose-6-phosphatase at baseline and with fasting in the PGC-1α−/− mice, we did not (unpublished data). Third, we found evidence for an age-related increase in body fat in PGC-1α−/− mice (females earlier than males), whereas Lin et al. identified a male-specific resistance to diet-induced obesity and insulin resistance. We have also found that male PGC-1α-deficient mice are somewhat protected against diet-induced obesity (Figure S4). However, we observed that the insulin-sensitive phenotype of the female PGC-1α−/− mice occurred in the context of normal weight gain with high-fat diet. These latter results indicate that the insulin-sensitive phenotype of PGC-1α−/− mice cannot be fully explained by a lean phenotype. Of interest, mice lacking the nuclear receptor estrogen-related receptor α, a known target of PGC-1α, exhibit resistance to diet-induced obesity similar to that of male PGC-1α null mice [38]. Fourth, the PGC-1α−/− mice described here exhibit a dramatic fasting-induced hepatic steatotic phenotype, whereas the Lin et al. mouse does not. Fifth, Lin et al. found a neurologic phenotype in males characterized by hyperactivity, whereas the PGC-1α−/− mice described here show reduced locomotor activity. However, it should be noted that we did not study activity levels over an extended period of time in males as did Lin et al., so it is possible that our findings reflect an emotional disturbance that manifests only when the animals are placed in a new environment. Finally, we report significant skeletal muscle and cardiac functional abnormalities (although the report by Lin et al. did not address these phenotypes, so this may not represent a true difference). + +The reasons for the interesting differences between the two PGC-1α-deficient mouse lines are not clear. It is possible that distinct genetic backgrounds related to hybrid strains confer different degrees of secondary compensatory responses. In addition, the incompletely penetrant postnatal mortality noted in the PGC-1α−/− mice reported by Lin et al. could have resulted in a selection bias toward greater levels of compensatory responses in liver and other tissues in the surviving group. It is also possible that the method of gene targeting led to different phenotypes. Lin et al. generated PGC-1α−/− mice by Cre recombinase-mediated excision of exons 3–5 in oocytes. The PGC-1α−/− mice described here were generated by a targeting event that involved a 3′ homologous recombination leading to an insertion of the targeting vector including an extra exon 3 between exons 5 and 6. The exon 3 insertion, which was confirmed by RT-PCR, results in a mutant transcript that encodes a truncated protein. We were unable to detect normal transcript containing an exon 5–6 border, indicating that the targeting was accurate and complete. In addition, we could not detect full-length or smaller PGC-1α proteins by Western blotting. However, we cannot exclude the possibility that the sensitivity of the immunoblotting was not high enough to pick up a small amount of mutant (truncated) PGC-1α protein that could have some activity, given that it would contain nuclear receptor-interacting domains and the amino-terminal activation domain. If small amounts of PGC-1α activity are present in the mice reported here, it could explain some of the observed differences between the models. However, the bulk of data presented here support the conclusion that the PGC-1α−/− mice described are completely deficient in PGC-1α. Future direct comparison of the two mouse lines in pure background strains will be of interest. + +In summary, this body of work provides evidence that PGC-lα is critical for the adaptive responses necessary to meet postnatal energy demands. Our results also suggest a broader role for inducible transcriptional coactivators such as PGC-1α in transducing cellular signals triggered by physiologic and developmental cues to the transcriptional control of energy metabolism and other dynamic cellular processes. In this regard, the inducible coactivator PGC-1α serves as a transcriptional “booster” to augment the capacity of downstream metabolic pathways critical for metabolic maturation and postnatal growth. Indeed, although PGC-1α null mice survive in the protected environment of the laboratory, our results indicate that in the rigors of a typical external environment, PGC-1α would be necessary for survival. Lastly, we propose that the PGC-1α−/− mice should serve as a useful murine model to investigate the role of altered energy metabolism in obesity, diabetes, hepatic steatosis, and diseases of the heart, skeletal muscle, and central nervous system. + +Materials and Methods + + + +Targeting the PGC-1α gene in mice + +A BAC genomic clone containing the murine PGC-1α gene, isolated from an Sv129 genomic library, was obtained from Incyte Genomics (Palo Alto, California, United States). A 3-kb region spanning exon 3 was amplified from the genomic clone. A 5′ primer was designed to amplify a fragment with the 5′ end beginning 732 bp upstream of exon 3 just upstream of an endogenous Kpn1 restriction site (5′-AGTTTCCTTAGCAACTTCATA-3′). The 3′ primer contained a BamH1 site engineered by mutating the bases shown in lowercase (5′-AAGGATTTTAgGATccCAGTAC-3′). A second fragment downstream of exon 5 was amplified. In this latter amplicon, Not1 and Xho1 sites were engineered into the 5′ and 3′ primers, respectively (5′-TGGAGTgcGGCCGCTGGGA-3′ and 5′-AAAGAGTCTCgAgAATAGTTTCT-3′). The fragments were cloned into p1339-PGK-Neomycin targeting vector. The construct was linearized with Xho1 and electroporated into RW4 ES cells (Sv129 derived) using G418 selection. The electroporation was performed by the Siteman Cancer Center ES Cell Core at Washington University (St. Louis, Missouri, United States). The clones were screened by Southern blot using an Xba1 digest (see Figure 1A and 1B). One clone out of approximately 400 screened was positive for the homologous recombination on the 3′ end and an insertion on the 5′ end. This clone was injected into a C57BL6/J blastocyst. Chimeras were mated to C57BL6/J mice and germline transmission was confirmed by Southern blotting of tail DNA (see Figure 1B). All experiments were performed using sex- and age-matched or littermate controls as noted. + +General animal studies + +All animal experiments and euthanasia protocols were conducted in strict accordance with the National Institutes of Health guidelines for humane treatment of animals and were reviewed and approved by the Animal Care Committee of Washington University. + +Animals were weighed at different time points. Male and female 3- to 8-wk-old PGC-1α+/+ and PGC-1α−/− mice were euthanized, and tissues were dissected and weighed on an analytical balance. Tissue weights were corrected for total body weight before comparison. DEXA studies were performed as previously described [39] using a Lunar PIXIMUS DEXA system at 10, 18, and 24 wk in male and female PGC-1α+/+ and PGC-1α−/− mice. For cold exposure experiments, male and female PGC-1α+/+ and PGC-1α−/− mice were singly housed and placed at 4 °C for 5 h without food. Core body temperatures were monitored by rectal probe at baseline and every hour thereafter. Mice were monitored at least every 30 min to check for lethargy. At the end of 5 h, mice were sacrificed and tissues harvested for RNA and protein extraction. For fasting studies, animals were singly housed and given water ad libitum. Food was removed from cages in the morning and tissues harvested at 24 h for RNA and histology. Photography of the mice was performed by MedPic at Washington University School of Medicine. 48-h activity monitoring was performed by JAX Services (The Jackson Laboratory, West Sacramento, California, United States) using a Comprehensive Laboratory Animal Monitoring System (CLAMS, Columbus Instruments, Columbus, Ohio, United States). Briefly, 3-mo-old female mice were acclimated for 17 h before data collection. Data were collected every 30 min. Total beam breaks in the XY direction were tabulated for the 12-h light and dark cycles and compared across genotypes. + +RNA, DNA, and protein analyses + +Total RNA was isolated by the RNAzol method (Tel-Test, Friendswood, Texas) and Northern blotting was performed as previously described [40]. The PGC-1β and PRC cDNAs were generous gifts from Bruce Spiegelman and Richard Scarpulla, respectively. The UCP-1 cDNA was a gift from Daniel Ricquier. RT-PCR was performed as described [41]. In brief, total RNA isolated from soleus muscle, BAT, and heart of 1–2-mo-old PGC-1α+/+ or PGCα−/− mice was reverse transcribed with Taqman reverse transcription reagents (Applied Biosystems, Foster City, California, United States). Reactions were performed in triplicate in 96-well format using Taqman core reagents and a Prism 7500 Sequence Detector (Applied Biosystems). The mouse-specific primer-probe sets used to detect specific gene expression can be found in Table S1. The primers for UCP-1 have been previously described [42]. Actin primer-probe set (Applied Biosystems) was included in a separate well and used to normalize the soleus, BAT, and heart gene expression data. GAPDH Rodent primers (Applied Biosystems) were used in the same well to normalize the liver gene expression data. + +For Southern blot studies, 5 μg of genomic DNA was digested with Pst1 or Xba1, electrophoresed on a 0.8% TAE gel and transferred to a Gene Screen (Perkin Elmer, Wellesley, California, United States) membrane for hybridization. Western blotting was performed as described [43] using the Enhanced Chemiluminescence detection system (Amersham Pharmacia Biotech, Piscataway, New Jersey, United States). Ponceau S (Sigma, St. Louis, Missouri, United States) staining of the membrane was used as a control. + +Mitochondrial respiration studies + +Mitochondrial respiration was assessed in saponin-skinned soleus fibers with succinate as substrate and in the presence of rotenone as previously described [44]. In brief, 3-mo-old female mice were anesthetized with chloral hydrate (400 mg/kg of body weight). Soleus fibers were separated and then transferred to a buffer (2.77 mM K2Ca-EGTA, 7.23 mM K2EGTA, 6.56 mM MgCl2, 20 mM imidazole, 53.3 mM K-MES, 20 mM taurine, 5.3 mM ATP, 15 mM PCr, 3 mM KH2PO4, 0.5 mM DTT [pH 7.1]) supplemented with 50 μg/ml saponin and permeabilized for 30 min at 4 °C with gentle stirring. Fibers were then washed twice for 10 min each (2.77 mM K2Ca-EGTA, 7.23 mM K2EGTA, 1.38 mM MgCl2, 20 mM imidazole, 100 mM K-MES, 20 mM taurine, 3 mM KH2PO4, 0.5 mM DTT, 2 mg/ml BSA [pH 7.1]). Respiration was measured at 25 °C using an optical probe (Oxygen FOXY Probe, Ocean Optics, Dunedin, Florida, United States). Following measurement of basal state 2 respiration, maximal (ADP-stimulated) state 3 respiration was determined by exposing fibers to 1 mM ADP. The integrity of the outer mitochondrial membrane was assessed by adding 8 μM exogenous cytochrome c to ADP-stimulated fibers. State 4 respiration (uncoupled) was evaluated following addition of oligomycin (1 μg/ml). The solubility of oxygen in the respiration buffer at 25 °C was taken as 246.87 nmol O2 · ml−1. Respiration rates were expressed as nmol O2 · min−1 · mgdw−1. + +Insulin and glucose tolerance tests + +Glucose and Insulin tolerance tests were performed as described [35]. Prior to studies, mice were fasted overnight (GTT) or 6 h (ITT). In GTT studies, mice were injected with a 10% solution of D-glucose (1 g/kg). For ITT, mice received an IP injection of human regular insulin (Eli Lilly, Indianapolis, Indiana, United States) at a dose of 0.75 units/kg of body weight. Tail blood glucose was determined at 0, 30, 60, and 120 min after challenge using a B-GLUCOSE Analyzer (Hemacue AB, Angelholm, Sweden). + +Indirect calorimetry + +Oxygen consumption rates (VO2) of 5-wk-old female mice were measured using a Columbus Instruments Oxymax System. Resting baseline oxygen consumption rates were assessed for at least 1.0 h. For β3-adrenergic stimulation studies, BRL 37344 (Sigma) was dissolved in sterile saline and injected IP (2 μg/g of body weight) [45]. Postagonist assessment of oxygen consumption was recorded for an additional 1.0 h, with data collected at the 40-min time point. + +Histology and electron microscopy + +Soleus muscle and liver were dissected and fixed overnight in 2% glutaraldehyde, 1% paraformaldehyde, and 0.08% sodium cacodylate buffer. The tissues were postfixed in 1% osmium tetroxide, dehydrated in graded ethanol, embedded in Poly Bed plastic resin, and sectioned for electron microscopy. Cardiac and skeletal muscle mitochondrial and myofibrillar volume densities were determined from electron micrographs as described previously [10]. For each animal, three different fields at the magnification of 7500× were quantified in blinded fashion. Data were expressed as mean volume density of mitochondria or myofibrils in each field. + +For electron microscopic analysis of the brain, tissue was prepared as previously described [46]. Ultrathin sections of cortex were cut onto formvar-coated slot grids stained with uranyl acetate and lead citrate and examined with a JEOL 1200 electron microscope. For H&E staining, sections of brain, including cerebral cortex, brainstem, and cerebellum, were dehydrated in graded concentrations of alcohol and embedded in paraffin from which 5-μm sections were prepared. + +Primary mouse hepatocyte studies + +Primary cultures of mouse hepatocytes were prepared from male PGC-1α+/+ and PGC-1α−/− mice essentially as described [47]. Fatty acid oxidation and triglyceride synthesis experiments were commenced 2–3 h after the cells were plated. Triglyceride synthesis studies, were performed as previously described [47]. Palmitate oxidation rates were quantified using [9,10-3H]-palmitic acid as described [48] and corrected for total cellular protein content. For respiration studies, cells were spun down prior to plating and resuspended in a permeabilization buffer (described above) containing 50 μg/ml saponin. Respiration studies were performed in the presence of 5 mM succinate in the presence of 10 μM rotenone. Respiration rates were expressed as nmol O2 · min−1 · mg of protein−1. + +Evaluation of locomotor activity, sensorimotor capabilities, and muscle function + +To evaluate general activity levels and muscle use, mice were evaluated over a 1-h period in transparent (47.6 cm × 25.4 cm × 20.6 cm) polystyrene enclosures as previously described [49] using a high-resolution photobeam system (Motor Monitor, Hamilton-Kinder, Poway, California, United States). Each enclosure was surrounded by a frame containing a 4 × 8 matrix of photocell pairs, the output of which was fed to an on-line computer. The system software (Hamilton-Kinder) was used to define a 33 cm × 11 cm central zone and a peripheral or surrounding zone that was 5.5 cm wide with the sides of the cage being the outermost boundary. This peripheral area extended along the entire perimeter of the cage. Variables that were analyzed included the total number of ambulations, as well as the number of entries, the time spent, and the distance traveled in the center area as well as the distance traveled in the periphery surrounding the center. The total number of ambulations and rearings were recorded. For the inverted screen test, mice were placed on a wire mesh grid (16 squares per 10 cm) and the screen was inverted to 180°. A maximum score of 60 s was given if an animal did not fall. + +The tests included in the sensorimotor battery [50] and accompanying protocols were designed as follows. (1) Inclined screen and inverted screen tests: For the inclined screen tests, each mouse was placed on top of an elevated (47 cm above the floor) wire mesh grid (16 squares per 10 cm) that was inclined to 60° or 90°. Each animal was placed in the middle of the screen with its head oriented down and was timed for how long it remained on the screen and how long it took to climb to the top of the screen. For the inverted screen test, mice were placed as above and then the screen was inverted to 180°. A maximum score of 60 s was given if an animal did not fall; (2) Platform test: Each mouse was timed for how long it remained on an elevated (47 cm above the floor) circular platform (1.0 cm thick and 3.0 cm in diameter). A maximum score of 60 s was assigned if the mouse remained on the platform for the maximum amount of time or if it could climb down on a very thin pole that supported the platform, without falling; (3) Ledge test: Each mouse was timed for how long it could maintain its balance on a 0.75-cm wide Plexiglas ledge without falling (60 s maximum). A score of 60 s was also assigned if the mouse traversed the entire length (51 cm) of the Plexiglas ledge and returned to the starting place in less than 60 s without falling; (4) Walking initiation test: Each mouse was placed in the middle of a square outlined by white cloth tape (21 cm × 21 cm) on a smooth black surface of a large tabletop. The time it took each mouse to leave the square (place all four paws outside of the tape) was recorded. The maximum time allowed was 60 s. + +6–8-mo-old PGC-1α+/+ (n = 4) and PGC-1α−/− (n = 8) mice were run to exhaustion employing a motorized, speed controlled, modular treadmill system (Columbus Instruments). The treadmill was equipped with an electric shock stimulus and an adjustable inclination angle. Running velocity was set at 35 m/min, with a level inclination angle. + +VO2max studies + +VO2max was determined while the mice were running on a treadmill using an open flow system (Columbus Instruments Oxymax System). All measurements of oxygen consumption took place at an elevation of 150 m (ambient PBAR = 745 torr). Animals were placed into the metabolic chamber for 3–5 min to allow the system to equilibrate. Mice were then induced to run up an 18° incline at a speed of 40 m/min using a shock grid in the rear of the chamber. The speed was increased by 5 m/min every 2 min until the animals were unable to continue. Maximal effort was determined when oxygen uptake did not increase with power output and subsequently the mouse failed to maintain effort. VO2max was calculated using the averaged values over 1 min during which the animal's O2 consumption reached a plateau. + +Isolated muscle stimulation studies + +Animals were anesthetized with ketamine/xylazine and the soleus muscle was removed from one leg. Upon removal, the muscle was suspended in a Krebs solution aerated with 95% O2 and 5% CO2. The muscle and Krebs solution were suspended within a water bath maintained at 37 °C, and the muscle was anchored to a Grass (West Warwick, Rhode Island, United States) isometric force transducer (model FTO3C). Muscles were stimulated to contract with a Grass stimulator (model S88) generating a field stimulus through electrodes located at both ends of the muscle. Force-voltage (maximal force at about 100 V) and length-tension relationships were determined using single twitch stimuli. The stimulator then delivered repeating trains of stimuli at one per second at 40 Hz for 2 min. Each train lasted 330 ms, and were digitally recorded using MacLab (AD Instruments, Colorado Springs, Colorado, United States). Fatigue resistance was calculated as the ratio of the force generated by the last tetanus divided by the highest force generated multiplied by 100 to give the percent of force generation that remained after the fatigue protocol. + +Exercise echocardiography + +Adult female mice (6–8 mo old) were exercised on the motorized treadmill using the run-to-exhaustion settings described above for 60 s or until exhaustion, whichever came first. Immediately following its treadmill run, the mouse was subjected to serial echocardiography using an Acuson Sequoia Echocardiography System performed as previously described [51]. + +In vivo cardiac hemodynamic studies + +Hemodynamic studies were performed as previously described with some modifications [52]. In brief, adult mice (10–12 wk) were anesthetized intraperitoneally (IP) with thiopental sodium (60 mg/kg). The mice were intubated and ventilated with a Harvard ventilator. The right carotid artery was isolated in the region of the trachea and cannulated with a 1.4-French high-fidelity micromanometer catheter (Millar Instruments, Houston, Texas, United States), which was inserted into the left ventricle retrograde across the aortic valve. Hemodynamic measurements were recorded at baseline and 3 min following continuous infusion of incremental doses of dobutamine (β1, β2, and α1-adrenergic agonist) up to 32 ng · gBW−1· min−1 [53]. Continuous pressure-volume data were acquired and digitized with the BioBench computer software data acquisition system (National Instruments, Austin, Texas, United States). + +Isolated working mouse heart perfusion + +Isolated working mouse heart perfusion was based on a previously described procedure [54]. Adult mice (4–7 mo old) were heparinized (100 units IP) 10 min prior to anesthesia. Animals were then deeply anesthetized with 5–10 mg of sodium pentobarbital IP. Hearts were excised and placed in an ice-cold Krebs-Henseleit bicarbonate (KHB) solution [118 mM NaCl, 25 mM NaHCO3, 4.7 mM KCl, 1.2 mM KH2PO4, 2.5 mM CaCl2, 5.0 mM glucose, and 100 units/L insulin (pH 7.4)]. Hearts were cannulated first via the aorta and perfused retrogradely by the Langendorff method. Following left atrial cannulation, perfusion was switched to the working mode with KHB solution containing 1.2 mM palmitate bound to 3% fatty acid-free BSA with a preload pressure of 11.5 mm Hg and an afterload pressure of 50 mm Hg for 60 min with oxygenated buffer solution. Functional measurements, namely cardiac output, aortic flows, peak systolic pressure, and heart rate were acquired every 10 min using inline flow probes (Transonic Systems, Ithaca, New York, United States), a pressure transducer (TSD 104A, BIOPAC Systems, Santa Barbara, California, United States) and data acquired with the MP100 system from AcqKnowledge (BIOPAC Systems). Cardiac work was calculated as the product of peak systolic pressure and cardiac output. + +Statistics + +Data were analyzed using T-tests or ANOVAs (measures of general activity and sensorimotor battery). The level of significance was set at p < 0.05 in all cases. Data are reported as mean values ± SEM, unless otherwise noted. The ANOVA model used to analyze each sensorimotor test included one between-subjects variable (genotype), and one within-subjects variable (trials). When ANOVAs with repeated measures were conducted, the Huynh-Feldt (H-F) adjustment of alpha levels was used for all within-subjects effects containing more than two levels, in order to protect against violations of the sphericity/compound symmetry assumptions underlying this ANOVA model. In addition, Bonferroni correction was used when appropriate to help maintain prescribed alpha levels (e.g., p < 0.05) when multiple comparisons were conducted. + +Supporting Information + +Figure S1 + +Activity Levels in Female PGC-1α−/− Mice Is Unchanged + +Using a CLAMS system, PGC-1α+/+ (n = 4) and PGC-1α−/− (n = 3) female mice were monitored for 48 h after a 17-h period of acclimation. XY beam breaks were tabulated over the 12-h light and dark cycles as denoted on the bottom. The bars represent mean (± SEM) beam breaks per each 12-h cycle. + +(342 KB EPS). + +Click here for additional data file. + +Figure S2 + +Altered Emotionality in PGC-1α−/− Mice + +An analysis of exploratory behavior included the number of entries into the center of the cage (upper left), the time spent in the center of the cage in seconds (sec) (upper right), the distance traveled in the center of the cage in meters (m) (lower left) as well as the distance traveled in the periphery (lower right). * p < 0.05 compared to the PGC-1α+/+ mice. + +(620 KB EPS). + +Click here for additional data file. + +Figure S3 + +No Difference in Weight Gain on a High-Fat Diet in Female PGC-1α−/− Mice Compared to WT Controls + +8-wk-old female mice were fed a diet high in fat (43% calories from fat) for 6 wk. The change in body weight (grams) after 6 wk on a high-fat diet is shown for PGC-1α+/+ (n = 8) and PGC-1α−/− (n = 11) mice. NS, not significant. + +(264 KB EPS). + +Click here for additional data file. + +Figure S4 + +Male PGC-1α−/− Mice Are Somewhat Resistant to Diet-Induced Obesity + +Male and female PGC-1α+/+ (n ≥ 6) and PGC-1α−/− (n ≥ 6) mice were fed a high-fat diet (43% calories from fat) beginning at 4 wk of age. Body weight was monitored weekly as shown on the graph on the left. The mean (± SEM) change in body weight is shown in the bar graph on the right. *, significant difference compared to the PGC-1α+/+ controls, p < 0.05. + +(794 KB EPS). + +Click here for additional data file. + +Table S1 + +Probes and Primers + +Sequences of mouse-specific probes and primers used for real-time RT-PCR. + +(25 KB DOC). + +Click here for additional data file. + +Accession Numbers + +The GenBank (http://www.ncbi.nlm.nih.gov/) accession numbers of the vector discussed in this paper is p1339-PGK-Neomycin targeting vector (AF335420). + +Acknowledgements + +We would like to thank Deanna Young and Karen Hemker for help with mouse husbandry, Carolyn Mansfield for ES cell and mouse genotyping, Anthony Nardi for assistance with the studies to assess general activity and muscle strength, Bill Kraft for expert technical assistance with electron microscopy, Carla Weinheimer for performing cardiac catheterizations, Trey Coleman for performing the TAG assays, Krista Olson for expert technical assistance with the Langendorff method, and Mary Wingate for assistance with manuscript preparation. This work was supported by National Institutes of Health grants RO1 DK45416, RO1 HL58427, PO1 HL57278, Digestive Diseases Research Core Center (P30 DK52574), Clinical Nutrition Research Unit Core Center (P30 DK56341), and K08 AG24844. SB is supported by a postdoctoral fellowship from the Juvenile Diabetes Foundation. EDA is supported by the American Heart Association (Established Investigator) and RO1 HL70070. + +Competing interests. The authors have declared that no competing interests exist. + +Abbreviations + +BAT - brown adipose tissue + +DEXA - dual-energy X-ray absorption + +EST - echocardiographic stress testing + +IP - intraperitoneal(ly) + +NRF - nuclear respiratory factor + +PGC - PPARγ coactivator + +PPAR - peroxisome proliferator-activated receptor + +PRC - PGC-1 related coactivator + +SCD - steroyl CoA desaturase + +SREBP - sterol regulatory element binding protein + +SEM - standard error of the mean + +TAG - triglyceride + +Tfam - mitochondrial transcription factor A + +UCP - uncoupling protein + +WT - wild-type + +Figures and Tables + +Figure 1 + +Deletion of the PGC-1α Gene + +(A) Schematic of the gene targeting strategy. A region of the murine PGC-1α gene containing exons 3–6 is shown schematically at the top. Relevant restriction endonuclease sites are also shown. The targeting construct containing a neomycin (Neo) cassette is shown below the PGC-1α gene with dashed lines indicating the regions targeted for homologous recombination. Homologous recombination between the 3′ end of the targeting vector and the PGC-1α gene is indicated by the solid lines. The targeting construct inserted into the PGC-1α gene resulting in a duplication of exon 3 and incorporation of the targeting construct DNA into the final recombinant as shown. Probes used for the Southern blot studies and relevant restriction fragments predicted by digestion of the recombinant are also shown. + +(B) Southern blot analysis of embryonic stem cells (ESC) (digested with Xba1) and tail DNA (digested with Pst1) is shown. The blots were hybridized with the probes shown in Figure 1A. Results for PGC-1α+/+ (+/+), PGC-1α+/− (+/−), and PGC-1α−/− (−/−) genotypes are shown as denoted at the bottom. + +(C) Northern blot analysis using RNA isolated from the hearts of the three relevant PGC-1α genotypes (as in Figure 1B) is shown using PGC-1α cDNA as a probe. In addition, PGC-1β and PRC cDNA probes were used as shown. Ethidium bromide staining of 18S ribosomal RNA is shown at the bottom. + +(D) Quantitative real time RT-PCR (Sybr green) was used to detect PGC-1α transcripts using primer sets crossing different exon borders as denoted. The exon 5–3 primer set detects only the mutant transcript (−/−), whereas the exon 5–6 primer set detects only the WT transcript. The values represent arbitrary units for RNA isolated from the tissues shown for the three amplicons comparing PGC-1α+/+ and PGC-1α−/−. N.D. = not detectable. The exon 10–11 amplicon was evaluated to assess levels of mutant versus WT transcripts. The values represent arbitrary units normalized to actin control. + +(E) Western blot analysis using whole-cell protein extracts prepared from BAT under basal conditions and following exposure to 4 °C for 8 h. The signal shown was obtained with polyclonal anti-PGC-1α antibody [10]. An epitope-tagged PGC-1α, overexpressed in neonatal cardiac myocytes using an adenoviral vector (Ad-PGC-1α), is shown as a positive control. The Ponceau S stain of the protein gel is shown at the bottom. + +Figure 2 + +Evidence for Tissue-Specific Growth Abnormalities and Mild Sex-Limited, Age-Dependent Obesity in PGC-1α−/− Mice + +(A) The bars represent total body weight for the ages indicated for male (left graph) and female (center graph) PGC-1α+/+ and PGC-1α−/− mice. The body weight (BW) of the 1-wk-old PGC-1α−/− mice was normalized to that of PGC-1α+/+ littermates, which was assigned a value of 100 (left axis). For the 3-, 18-, and 24-wk time points, absolute weights of PGC-1α−/− mice were compared to age-matched controls (right axis). Percent fat as determined by DEXA scanning for PGC-1α+/+ and PGC-1α−/− mice (right graph). The results represent n = 4 (males) and n ≥ 11 (females) for each genotype at 24 wk. * p < 0.05 compared to corresponding PGC-1α+/+ mice. + +(B) The bars represent organ weights corrected to body weight for 3-wk-old male and female PGC-1α+/+ and PGC-1α−/− mice. The error bars represent ± SEM. Results represent n ≥ 14 for each group. * p < 0.05 compared to corresponding PGC-1α+/+ mice. + +Figure 3 + +Abnormal Mitochondrial Phenotype in Slow-Twitch Skeletal Muscle of PGC-1α−/− Mice + +(A) Representative electron micrograph of soleus muscle from 1-mo-old female PGC-1α+/+ and PGC-1α−/− mice. + +(B) Quantitative morphometric measurements of the cellular volume density for the mitochondrial (Mito) and myofibrillar (Myo) fractions based on analysis of electron micrographs (three sections from three animals per group). The bars represent mean ± SEM. * p < 0.05 compared to corresponding PGC-1α+/+ values. + +(C) Gene expression data. The results of real-time PCR analysis of nuclear and mitochondrial genes involved in various components of mitochondrial metabolism and mitochondrial biogenesis: cytochrome C (Cyto c), ATP synthase β, Tfam, and cytochrome oxidase IV (COX IV). Eight littermate pairs were used for analysis at 1–2 mo of age and normalized to the WT value, which was assigned a value of 100, in each case. * p < 0.05 compared to corresponding PGC-1α+/+ values. + +(D) Mitochondrial respiration rates as determined by oxygen consumption (VO2) performed on saponin-permeabilized muscle strips prepared from soleus of PGC-1α+/+ and PGC-1α−/− mice (as described in Materials and Methods). The results are based on six female animals in each group, using succinate as a substrate in the presence of rotenone. Mean values (± SEM) are shown for state 2 (basal), state 3 (ADP-stimulated), and state 4 respiration (presence of oligomycin). + +Figure 4 + +PGC-1α−/− Mice Exhibit an Abnormal Skeletal Muscle Functional Phenotype + +(A) Measures of general activity and muscle strength. General activity levels were measured in 3.5-mo-old male PGC-1α+/+ (n = 8) and PGC-1α−/− (n = 11) mice using a photobeam system as described in Materials and Methods. Total ambulations (left graph), and rearings (center graph) provide a general measure of locomotor activity. Time spent on an inverted screen (right graph) represents a general measure of extremity muscle strength. The results from two trials are shown. * p < 0.05 compared to corresponding PGC-1α+/+. + +(B) Exercise studies. Male 6–8-mo-old PGC-1α−/− and PGC-1α+/+ mice were subjected to a run-to-exhaustion protocol on a motorized treadmill (left graph) as described in Materials and Methods. * p < 0.001 compared to corresponding PGC-1α+/+ values. VO2max measurements were determined for 2-mo-old male mice for each genotype using a motorized treadmill at an elevation of 150 m and indirect calorimetry set-up (right graph) as described in Materials and Methods. * p < 0.05 compared to corresponding PGC-1α+/+ values. + +(C) Time course of fatigue following repeated stimulation of soleus muscle is shown for 4-mo-old male PGC-1α−/− (n = 5) and PGC-1α+/+ (n = 5) mice (left graph). The mean percent force remaining at 2 min (Fatigue Resistance Index) is shown (right graph). * p < 0.05 compared to corresponding PGC-1α+/+ values. + +Figure 5 + +Abnormal Cardiac Response to Physiologic Stress in PGC-1α−/− Mice + +(A) Exercise echocardiographic studies. PGC-1α+/+ (n = 4) and PGC-1α−/− (n = 8) female mice aged 6–8 mo were subjected to an exercise protocol on a motorized treadmill. This protocol was designed such that the PGC-1α−/− mice ran to exhaustion based on the results of the exercise studies shown in Figure 4. Accordingly, an exercise regimen of 60 s was used for both groups. The graphs depict the heart rate (left graph) and echocardiographically-determined ventricular fractional shortening (FS) as a percent (right graph). Responses were monitored for 10 min immediately post exercise. + +(B) In vivo hemodynamic response to the β1,α1-adrenergic agonist dobutamine. Male and female PGC-1α+/+ (n = 6) and PGC-1α−/− (n = 6) mice at 10–12 wk of age were anesthetized and a 1.4-French Millar catheter was placed through the carotid artery into the left ventricle as described in Materials and Methods. Heart rate (left graph) and a measurement of ventricular systolic performance, dP/dt (right graph), were measured following infusion of dobutamine. * p < 0.05. + +Figure 6 + +PGC-1α−/− Mice Exhibit an Abnormal Thermogenic Response + +(A) PGC-1α+/+ (n = 15) and PGC-1α−/− (n = 21) mice aged 28–37 d were subjected to cold (4 °C). Core rectal temperature was monitored over a 5-h period. The change in core temperature ± SEM is shown in the graph (left) as a function of time. * p < 0.05. + +(B) Representative Northern blot analysis (blot and gel at top) performed with RNA isolated from BAT to detect UCP-1 transcript at baseline (RT) and after 5 h of exposure to cold (4 °C) (UCP1). Ethidium bromide (Eth Br) staining of ribosomal RNA is shown as a control. Quantitative real-time RT-PCR for UCP-1 transcript is shown on the graph at the bottom. The values represent mean arbitrary units normalized to a 36B4 transcript (control). + +(C) Altered response to β3-adrenergic agonist. To evaluate the oxygen consumption (VO2) in response to the stimulation of BAT uncoupled respiration, the β3-adrenergic agonist BRL 37344 was administered to littermate PGC-1α+/+ (n = 5) and PGC-1α−/− (n = 5) female mice followed by measurement of VO2 by indirect calorimetry. Mean ± SEM VO2 is shown. * p < 0.05. + +Figure 7 + +Fasting-Induced Hepatic Steatosis Develops in PGC-1α−/− Mice + +(A) The photograph depicts the development of a pale liver in PGC-1α−/− mice subjected to a 24-h fast. + +(B) Oil red O staining of histologic sections of liver taken from PGC-1α−/− mice under fed and 24 h fasted conditions. The red staining indicates neutral lipid. + +(C) Representative electron micrographs of the liver from PGC-1α+/+ and PGC-1α−/− mice following a 24-h fast. The droplets are indicative of neutral lipid accumulation. + +(D) Mean liver TAG levels in PGC-1α+/+ (n = 5) and PGC-1α−/− (n = 5) mice under fed and 24-h fasted conditions. * p < 0.05. + +Figure 8 + +Hepatocytes Isolated from PGC-1α−/− Mice Exhibit Reduced Oxidative Capacity + +(A) Oil red O staining of isolated hepatocytes exposed to BSA alone (BSA) or 50 μM oleate complexed to BSA (oleate). + +(B) 3H-palmitate oxidation rates. 3H-palmitate oxidation rates determined in hepatocytes isolated from PGC-1α+/+ and PGC-1α−/− mice under cell culture conditions containing BSA or BSA + 50 μM oleate (2:1 oleate/BSA ratio). Values were derived from ten sets of triplicates for each group using hepatocytes from 5 mice of each genotype. The bars represent mean oxidation rates (n = 100) normalized to the condition of PGC-1α+/+ in BSA alone. * p < 0.05 compared to the corresponding PGC-1α+/+ mice. † p < 0.05 compared to PGC-1α+/+ with BSA treatment. + +(C) State 2 and 3 respiration rates determined for hepatocytes isolated from PGC-1α+/+ (n = 3) and PGC-1α−/− (n = 3) mice using succinate/rotenone as a substrate. * p < 0.05 compared to corresponding PGC-1α+/+. + +(D) TAG synthesis rates in isolated hepatocytes. The bars represent mean TAG synthesis rates (glycerol incorporation, see Materials and Methods) for hepatocytes isolated from PGC-1α+/+ (n = 6) and PGC-1α−/− (n = 6) mice. * p < 0.05 compared to the corresponding PGC-1α+/+ condition. + +Figure 9 + +Female PGC-1α−/− Mice Are More Glucose Tolerant and Insulin Sensitive Compared to PGC-1α+/+ on High-Fat Diet + +(A) At 4.5 mo of age, glucose tolerance testing (GTT) was performed on female PGC-1α+/+ (n = 6) and PGC-1α−/− (n = 6) mice maintained on standard chow. + +(B) At 8 wk of age, PGC-1α+/+ (n = 8) and PGC-1α−/− (n = 11) mice were provided a diet containing 43% of its calories from fat (HF chow). The graphs depict blood glucose levels ± SEM in PGC-1α−/− mice during GTT (left graph) and ITT (right graph) studies. Studies were performed 5 wk (GTT) and 6 wk (ITT) after the initiation of the high-fat diet. * p < 0.05 compared to PGC-1α+/+ mice at the same time point. + +Figure 10 + +Neuropathology of the Central Nervous System of PGC-1α−/− Mice + +(A) Light microscopic appearance of representative cerebral cortex of 2-mo-old PGC-1α−/− mice demonstrates marked vacuolation of the neuropil (arrows) and scattered neuronal perikarya, which are absent in PGC-1α+/+ mice (hematoxylin and eosin). The scale bar shown is applicable to all sections. + +(B) Ultrastructural appearance of typical vacuoles containing membranous debris, denoted by the arrow, in the cerebral cortex of a representative PGC-1α−/− mouse in comparison to PGC-1α+/+ (magnification 4000×). + +Table 1 + +Cardiac Hemodynamics Measured in Isolated Working Hearts of PGC-1α+/+ and PGC-1α−/− Mice + +Values represent mean ± SEM + +a p < 0.05 + +Table 2 + +Metabolic Gene Expression in Liver of Fed and Fasted PGC-1α+/+ and PGC-1α−/− Mice + +Values represent mean (± SEM) (n ≥ 6 for each group) mRNA levels as determined by real-time RT-PCR corrected for GAPDH signal intensity and normalized (to 1.0) to the value of fed PGC-1α+/+ mice + +a p < 0.05 versus fed mice of the same genotype + +b p < 0.05 versus PGC-1α+/+ mice of the same dietary treatment + +L-CPT I, liver-type carnitine palmitoyltransferase; MCAD, medium-chain acyl-CoA dehydrogenase; SREBP, sterol regulatory element binding protein; SCD, steroyl-CoA desaturase; FAS, fatty acid synthase; GPAT, glycerol-3-phosphate acyltransferase; DGAT, diacylglycerol acyltransferase + +Footnotes + +Author contributions. TCL, JJL, BNF, PJS, and DPK conceived and designed the experiments. TCL, JJL, BNF, PJS, ARW, SB, MC, DFW, NS, CBM, ZC, DMM, RES, and EDA performed the experiments. TCL, JJL, BNF, PJS, ARW, SB, MC, DFW, NS, CBM, ZC, JOH, DMM, RES, JES, EDA, CFS, and DPK analyzed the data. TCL, BNF, and DPK wrote the paper. + +Citation: Leone TC, Lehman JJ, Finck BN, Schaeffer PJ, Wende AR, et al. (2005) PGC-1α-deficiency causes multi-system energy metabolic derangements: Muscle dysfunction, abnormal weight control and hepatic steatosis. PLoS Biol 3(4): e101. diff --git a/src/ontogpt/evaluation/craft/database/all/15784609.ann b/src/ontogpt/evaluation/craft/database/all/15784609.ann new file mode 100644 index 000000000..04fdb2d82 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15784609.ann @@ -0,0 +1,720 @@ +T1 SO:0000902 26 35 transgene +T2 GO:0010467 36 46 expression +T3 NCBITaxon:10088 50 54 mice +T4 CHEBI:27902 119 131 tetracycline +T5 NCBITaxon:10088 190 195 mouse +T6 UBERON:0000479 223 229 tissue +T7 CHEBI:27902 306 318 tetracycline +T8 CHEBI:27902 341 353 tetracycline +T9 SO:0000902 388 398 transgenes +T10 GO:0010467 424 434 expression +T11 SO:0000902 493 502 transgene +T12 GO:0010467 535 545 expression +T13 GO:0010467 718 728 expression +T14 UBERON:0001986 745 756 endothelial +T15 PR:000017284 772 778 VEGF-A +T16 UBERON:0000922 787 796 embryonic +T17 GO:0009790 787 808 embryonic development +T18 GO:0007567 817 824 natally +T19 UBERON:0007023 828 833 adult +T20 NCBITaxon:10088 834 838 mice +T21 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12883 animals +T258 CL:0000540 12889 12897 neuronal +T259 UBERON:0001047 12909 12919 glomerulus +T260 GO:0010467 12929 12939 expression +T261 UBERON:0000922 13010 13016 embryo +T262 PR:000011141 13021 13027 Nestin +T263 UBERON:0001047 13063 13073 glomerulus +T264 PR:000011366 13078 13085 Podocin +T265 SO:0000359 13138 13150 loxP-flanked +T266 GO:0010467 13192 13202 expression +T267 UBERON:0019248 13256 13261;13273 13279 early ... embryo +T268 PR:000017284 13323 13329 VEGF-A +T269 CHEBI:50845 13439 13450 doxycycline +T270 SO:0000243 13498 13502 IRES +T271 PR:000017284 13519 13525 VEGF-A +T272 UBERON:0000922 13534 13541 embryos +T273 CHEBI:50845 13628 13639 doxycycline +T274 GO:0007565 13665 13673 pregnant +T275 GO:0007618 13705 13711 mating +T276 UBERON:0000922 13751 13758 embryos +T277 UBERON:0000922 13850 13857 embryos +T278 CL:0000232 13894 13909 red blood cells +T279 UBERON:0000178 13898 13903 blood +T280 CL:0000232 13911 13915 RBCs +T281 UBERON:0001040 13944 13952 yolk sac +T282 UBERON:0000922 13969 13976 embryos +T283 PR:000017284 14037 14043 VEGF-A +T284 UBERON:0003061 14086 14098 blood island +T285 UBERON:0001040 14118 14126 yolk sac +T286 PR:000017284 14147 14153 VEGF-A +T287 GO:0010467 14158 14168 expression +T288 UBERON:0003061 14228 14241 blood islands +T289 UBERON:0001040 14249 14257 yolk sac +T290 CL:0000765 14290 14302 erythroblast +T291 UBERON:0003061 14339 14352 blood islands +T292 UBERON:0000922 14387 14394 embryos +T293 UBERON:0000925 14418 14428 endodermal +T294 UBERON:0000926 14433 14443 mesodermal +T295 UBERON:0003061 14480 14493 blood islands +T296 CHEBI:50845 14523 14534 doxycycline +T297 UBERON:0000922 14543 14550 embryos +T298 UBERON:0003061 14561 14574 blood islands +T299 UBERON:0000922 14624 14631 embryos +T300 CHEBI:50845 14686 14697 doxycycline +T301 SO:0000902 14836 14846 transgenes +T302 SO:0000243 14885 14889 IRES +T303 PR:000017284 14906 14912 VEGF-A +T304 UBERON:0000922 14935 14942 embryos +T305 GO:0097617 14966 14979 hybridization +T306 GO:0010467 15062 15072 expression +T307 GO:0048477 15230 15239 oogenesis +T308 PR:000017284 15306 15312 VEGF-A +T309 http://purl.obolibrary.org/obo/MONDO_0005070 15342 15347 tumor +T310 GO:0010467 15354 15364 expressing +T311 PR:000017284 15380 15386 VEGF-A +T312 NCBITaxon:10088 15410 15414 mice +T313 UBERON:0002405 15460 15466 immune +T314 CL:0000738 15460 15471 immune cell +T315 UBERON:0002370 15510 15516 thymus +T316 UBERON:0002107 15529 15534 liver +T317 NCBITaxon:9606 15573 15578 human +T318 http://purl.obolibrary.org/obo/MONDO_0002254 15579 15587 syndrome +T319 http://purl.obolibrary.org/obo/MONDO_0004717 15597 15613 peliosis hepatis +T320 NCBITaxon:38323 15658 15677 Bartonella henselae +T321 http://purl.obolibrary.org/obo/MONDO_0005550 15678 15687 infection +T322 CHEBI:50113 15709 15717 androgen +T323 http://purl.obolibrary.org/obo/MONDO_0004992 15751 15758 cancers +T324 CHEBI:50845 15790 15801 doxycycline +T325 PR:000017284 15818 15824 VEGF-A +T326 GO:0010467 15829 15839 expression +T327 SO:0000243 15952 15956 IRES +T328 PR:000017284 15973 15979 VEGF-A +T329 NCBITaxon:10088 15988 15992 mice +T330 UBERON:0000113 16015 16024 adulthood +T331 CHEBI:50845 16042 16053 doxycycline +T332 NCBITaxon:10088 16117 16121 mice +T333 GO:0007567 16127 16131 born +T334 CHEBI:50845 16281 16292 doxycycline +T335 GO:0042756 16300 16308 drinking +T336 CHEBI:15377 16309 16314 water +T337 SO:0000243 16360 16364 IRES +T338 PR:000017284 16381 16387 VEGF-A +T339 NCBITaxon:10088 16396 16400 mice +T340 NCBITaxon:10088 16476 16480 mice +T341 UBERON:0001456 16523 16527 face +T342 UBERON:0001690 16529 16533 ears +T343 UBERON:0002387 16538 16542 feet +T344 CHEBI:50845 16574 16585 doxycycline +T345 NCBITaxon:10088 16639 16643 mice +T346 GO:0016265 16648 16652 died +T347 UBERON:0000062 16746 16752 organs +T348 NCBITaxon:10088 16807 16811 mice +T349 CHEBI:50845 16841 16852 doxycycline +T350 CHEBI:23888 16887 16891 drug +T351 CHEBI:50845 17104 17115 doxycycline +T352 UBERON:0000922 17173 17180 embryos +T353 SO:0000243 17191 17195 IRES +T354 PR:000017284 17212 17218 VEGF-A +T355 UBERON:0007023 17241 17246 adult +T356 NCBITaxon:10088 17247 17252 mouse +T357 GO:0016265 17253 17257 died +T358 CHEBI:50845 17265 17276 doxycycline +T359 NCBITaxon:10088 17298 17303 mouse +T360 GO:0010467 17353 17363 expression +T361 CL:0000365 17378 17385 zygotic +T362 UBERON:0000178 17489 17494 blood +T363 UBERON:0002107 17502 17507 liver +T364 UBERON:0002370 17551 17557 thymus +T365 UBERON:0000029 17581 17592 lymph nodes +T366 UBERON:0000467 17664 17677 organ systems +T367 UBERON:0000178 17689 17694 blood +T368 UBERON:0000160 17702 17711 intestine +T369 UBERON:0002107 17760 17765 liver +T370 SO:0000243 17807 17811 IRES +T371 PR:000017284 17828 17834 VEGF-A +T372 UBERON:0007023 17843 17848 adult +T373 NCBITaxon:10088 17849 17853 mice +T374 UBERON:0002107 17880 17887 hepatic +T375 CHEBI:50845 17916 17927 Doxycycline +T376 SO:0000243 17971 17975 IRES +T377 PR:000017284 17992 17998 VEGF-A +T378 UBERON:0007023 18007 18012 adult +T379 UBERON:0002107 18013 18019 livers +T380 UBERON:0000178 18089 18094 blood +T381 UBERON:0002107 18125 18130 liver +T382 UBERON:0002107 18159 18164 liver +T383 http://purl.obolibrary.org/obo/MONDO_0004717 18159 18173 liver peliosis +T384 UBERON:0001281 18213 18230 hepatic sinusoids +T385 UBERON:0002107 18268 18273 liver +T386 UBERON:0003909 18321 18331 sinusoidal +T387 CL:0002262 18321 18349 sinusoidal endothelial cells +T388 UBERON:0001986 18332 18343 endothelial +T389 UBERON:0014399 18384 18400 sinusoidal space +T390 UBERON:0002107 18427 18432 liver +T391 UBERON:0002107 18472 18477 liver +T392 http://purl.obolibrary.org/obo/MONDO_0004717 18472 18486 liver peliosis +T393 NCBITaxon:10088 18524 18528 mice +T394 http://purl.obolibrary.org/obo/MONDO_0005070 18558 18563 tumor +T395 GO:0010467 18570 18580 expressing +T396 PR:000017284 18605 18611 VEGF-A +T397 UBERON:0002107 18709 18714 liver +T398 UBERON:0003909 18757 18767 sinusoidal +T399 UBERON:0001986 18768 18779 endothelium +T400 UBERON:0002107 18787 18792 liver +T401 PR:000017284 18837 18843 VEGF-A +T402 PR:000017284 18883 18889 VEGF-A +T403 UBERON:0003909 18920 18930 sinusoidal +T404 UBERON:0002049 18931 18942 vasculature +T405 PR:000007563 18948 18952 Flt1 +T406 CL:0000182 18980 18990 hepatocyte +T407 PR:000008534 18980 19004 hepatocyte growth factor +T408 PR:000008534 19006 19009 HGF +T409 CL:0000182 19058 19069 hepatocytes +T410 PR:000017284 19112 19118 VEGF-A +T411 UBERON:0002107 19141 19146 liver +T412 PR:000003918 19178 19185 albumin +T413 PR:000017284 19253 19259 VEGF-A +T414 UBERON:0002107 19302 19307 liver +T415 PR:000017284 19443 19449 VEGF-A +T416 UBERON:0002107 19475 19480 liver +T417 UBERON:0002370 19487 19493 thymus +T418 SO:0000243 19537 19541 IRES +T419 PR:000017284 19558 19564 VEGF-A +T420 UBERON:0007023 19573 19578 adult +T421 NCBITaxon:10088 19579 19583 mice +T422 UBERON:0002370 19613 19619 thymic +T423 UBERON:0001851 19653 19661 cortical +T424 CL:0000767 19676 19686 basophilic +T425 CL:0000893 19687 19697 thymocytes +T426 UBERON:0000483 19711 19721 epitheloid +T427 UBERON:0000958 19727 19741 medullary zone +T428 UBERON:0002370 19799 19804 thymi +T429 UBERON:0002370 19941 19946 thymi +T430 PR:000017284 19950 19956 VEGF-A +T431 UBERON:0007023 19976 19982 adults +T432 UBERON:0002370 20103 20108 thymi +T433 UBERON:0000958 20160 20169 medullary +T434 UBERON:0001851 20174 20182 cortical +T435 CL:0000767 20198 20208 basophilic +T436 UBERON:0001851 20209 20217 cortical +T437 CL:0000893 20248 20258 thymocytes +T438 UBERON:0002370 20344 20349 thymi +T439 NCBITaxon:33208 20425 20431 animal +T440 UBERON:0002370 20499 20504 thymi +T441 NCBITaxon:33208 20551 20558 animals +T442 CHEBI:50845 20604 20615 doxycycline +T443 NCBITaxon:33208 20700 20707 animals +T444 UBERON:0002370 20836 20842 thymic +T445 PR:000017284 20953 20959 VEGF-A +T446 CL:0000084 20967 20973 T-cell +T447 GO:0048468 20969 20985 cell development +T448 GO:0010467 21016 21026 expression +T449 PR:000017284 21030 21036 VEGF-A +T450 CL:0000765 21109 21122 erythroblasts +T451 UBERON:0001040 21269 21277 yolk sac +T452 PR:000017284 21307 21313 VEGF-A +T453 GO:0035425 21318 21327 autocrine +T454 PR:000017284 21405 21411 VEGF-A +T455 GO:0030097 21447 21460 hematopoietic +T456 GO:0030097 21540 21553 hematopoietic +T457 UBERON:0001016 21606 21620 nervous system +T458 PR:000017284 21650 21656 VEGF-A +T459 GO:0010467 21657 21667 expression +T460 PR:000011141 21763 21769 Nestin +T461 SO:0000902 21774 21783 transgene +T462 SO:0001023 21811 21817 allele +T463 PR:000017284 21821 21827 VEGF-A +T464 UBERON:0001851 21857 21865 cortical +T465 UBERON:0000055 21866 21872 vessel +T466 PR:000011141 21905 21911 Nestin +T467 SO:0000243 21933 21937 IRES +T468 PR:000017284 21954 21960 VEGF-A +T469 UBERON:0000922 21987 21994 embryos +T470 UBERON:0002240 22069 22080 spinal cord +T471 UBERON:0000955 22085 22090 brain +T472 CHEBI:50845 22109 22120 doxycycline +T473 GO:0007565 22170 22179 pregnancy +T474 CHEBI:50845 22207 22218 doxycycline +T475 UBERON:0000922 22255 22262 embryos +T476 UBERON:0000113 22294 22303 adulthood +T477 NCBITaxon:10088 22331 22335 mice +T478 CHEBI:50845 22345 22356 doxycycline +T479 UBERON:0000055 22472 22478 vessel +T480 UBERON:0007023 22533 22538 adult +T481 NCBITaxon:10088 22539 22544 mouse +T482 UBERON:0000955 22545 22551 brains +T483 UBERON:0000055 22595 22601 vessel +T484 UBERON:0001851 22649 22655 cortex +T485 UBERON:0007023 22730 22735 adult +T486 UBERON:0001017 22736 22739 CNS +T487 UBERON:0002049 22740 22751 vasculature +T488 PR:000017284 22755 22761 VEGF-A +T489 CHEBI:50845 22823 22834 doxycycline +T490 PR:000017284 22856 22862 VEGF-A +T491 UBERON:0007023 22905 22910 adult +T492 UBERON:0001016 22911 22925 nervous system +T493 PR:000011366 22981 22988 Podocin +T494 SO:0000243 23010 23014 IRES +T495 PR:000017284 23031 23037 VEGF-A +T496 NCBITaxon:10088 23064 23068 mice +T497 CHEBI:50845 23082 23093 doxycycline +T498 http://purl.obolibrary.org/obo/MONDO_0003634 23143 23154 proteinuria +T499 PR:000003918 23156 23163 albumin +T500 http://purl.obolibrary.org/obo/MONDO_0003634 23273 23284 proteinuria +T501 http://purl.obolibrary.org/obo/MONDO_0003634 23302 23313 proteinuria +T502 GO:0003094 23368 23389 glomerular filtration +T503 UBERON:0005777 23368 23397 glomerular filtration barrier +T504 UBERON:0000074 23429 23439 glomerulus +T505 UBERON:0000178 23470 23475 blood +T506 PR:000003918 23496 23503 albumin +T507 UBERON:0000178 23505 23510 blood +T508 GO:0007596 23505 23519 blood clotting +T509 CHEBI:35222 23527 23537 inhibitors +T510 CHEBI:18059 23542 23548 lipids +T511 http://purl.obolibrary.org/obo/MONDO_0000831 23583 23593 thrombotic +T512 GO:0007596 23583 23593 thrombotic +T513 NCBITaxon:10088 23620 23624 mice +T514 UBERON:0002113 23715 23721 kidney +T515 PR:000017284 23742 23748 VEGF-A +T516 GO:0010467 23749 23759 expression +T517 CL:0000653 23763 23772 podocytes +T518 UBERON:0000479 23878 23884 tissue +T519 NCBITaxon:10088 23909 23914 mouse +T520 CHEBI:27902 23950 23962 tetracycline +T521 SO:0000902 24009 24018 transgene +T522 GO:0010467 24019 24029 expression +T523 SO:0000704 24127 24131 gene +T524 SO:0000902 24298 24308 transgenes +T525 GO:0010468 24377 24398 control of expression +T526 NCBITaxon:10088 24534 24539 mouse +T527 UBERON:0000479 24557 24563 tissue +T528 GO:0010467 24616 24625 expressed +T529 SO:0000167 24771 24779 promoter +T530 GO:0010467 24874 24884 expression +T531 SO:0000167 24901 24909 promoter +T532 GO:0010467 25019 25029 expression +T533 SO:0000704 25139 25144 genes +T534 UBERON:0001062 25156 25166 anatomical +T535 SO:0000902 25304 25314 transgenes +T536 SO:0001026 25326 25333 genomic +T537 GO:0010467 25385 25395 expression +T538 SO:0000243 25575 25579 IRES +T539 SO:0000440 25619 25626 vectors +T540 http://purl.obolibrary.org/obo/MONDO_0004992 25679 25685 Cancer +T541 SO:0000243 26125 26129 IRES +T542 SO:0000902 26135 26144 transgene +T543 SO:0000147 26196 26201 exons +T544 SO:0000704 26216 26220 gene +T545 SO:0000346 26274 26284 loxP sites +T546 SO:0000366 26290 26305 insertion point +T547 SO:0000061 26331 26348 restriction sites +T548 SO:0001026 26486 26493 genomic +T549 CL:0002322 26503 26511 ES cells +T550 GO:0097617 26534 26544 hybridized +T551 GO:0009294 26668 26679 transfected +T552 SO:0000155 26687 26694 plasmid +T553 GO:0010467 26717 26727 expression +T554 SO:0005853 26728 26737 cassettes +T555 CHEBI:17939 26741 26750 puromycin +T556 CHEBI:27902 26788 26799 Tetracyclin +T557 PR:000033987 26813 26817 lacZ +T558 GO:0009294 26860 26871 transfected +T559 SO:0000155 26879 26886 plasmid +T560 PR:000033987 26906 26910 lacZ +T561 SO:0000902 26911 26920 transgene +T562 CHEBI:50845 26929 26940 doxycycline +T563 CHEBI:50845 26968 26979 doxycycline +T564 SO:0000346 27051 27055 loxP +T565 CHEBI:27902 27067 27079 tetracycline +T566 NCBITaxon:10088 27113 27117 Mice +T567 SO:0000243 27189 27193 IRES +T568 SO:0000879 27199 27210 bicistronic +T569 SO:0000902 27211 27220 transgene +T570 NCBITaxon:10088 27266 27270 mice +T571 UBERON:0000479 27284 27290 tissue +T572 SO:0000902 27304 27313 transgene +T573 CHEBI:50845 27320 27331 doxycycline +T574 SO:0000704 27363 27367 GENE +T575 SO:0000243 27476 27480 IRES +T576 SO:0000704 27497 27501 GENE +T577 GO:0010467 27533 27540 express +T578 SO:0000704 27611 27615 GENE +T579 GO:0010467 27638 27648 expressing +T580 GO:0010467 27670 27680 expression +T581 CHEBI:50845 27739 27750 doxycycline +T582 GO:0010467 27778 27788 expression +T583 SO:0000704 27802 27806 GENE +T584 CHEBI:50845 27827 27838 doxycycline +T585 GO:0010467 27896 27906 expression +T586 SO:0000704 27921 27925 gene +T587 PR:000017284 27949 27955 VEGF-A +T588 UBERON:0001016 27995 28009 nervous system +T589 CL:0000653 28013 28021 podocyte +T590 CHEBI:50845 28054 28065 doxycycline +T591 UBERON:0000479 28078 28084 Tissue +T592 GO:0010467 28094 28104 expression +T593 SO:0000243 28117 28121 IRES +T594 SO:0000879 28127 28138 bicistronic +T595 SO:0000902 28139 28148 transgene +T596 PR:000017284 28205 28211 VEGF-A +T597 GO:0065007 28227 28236 regulated +T598 CHEBI:50845 28240 28251 doxycycline +T599 SO:0000243 28315 28319 IRES +T600 PR:000017284 28336 28342 VEGF-A +T601 UBERON:0000922 28356 28362 embryo +T602 UBERON:0001040 28367 28375 yolk sac +T603 CHEBI:50845 28394 28405 doxycycline +T604 CHEBI:51686 28492 28493 H +T605 UBERON:0001040 28524 28532 yolk sac +T606 CHEBI:50845 28552 28563 doxycycline +T607 UBERON:0000922 28583 28590 embryos +T608 UBERON:0001040 28635 28643 yolk sac +T609 UBERON:0000055 28644 28650 vessel +T610 CL:0000232 28688 28692 RBCs +T611 UBERON:0003061 28704 28716 blood island +T612 UBERON:0003061 28718 28720 BI +T613 UBERON:0008776 28744 28762 primitive endoderm +T614 UBERON:0008776 28764 28765 e +T615 UBERON:0000926 28771 28779 mesoderm +T616 UBERON:0000926 28781 28782 m +T617 UBERON:0001040 28798 28806 yolk sac +T618 UBERON:0000922 28836 28842 embryo +T619 UBERON:0001040 28847 28855 yolk sac +T620 CHEBI:50845 28881 28892 doxycycline +T621 CHEBI:51686 28979 28980 H +T622 UBERON:0001040 29011 29019 yolk sac +T623 CHEBI:50845 29044 29055 doxycycline +T624 UBERON:0000922 29078 29085 embryos +T625 UBERON:0000055 29125 29131 vessel +T626 UBERON:0001040 29151 29159 yolk sac +T627 UBERON:0001040 29161 29163 ys +T628 UBERON:0000922 29169 29175 embryo +T629 UBERON:0000922 29177 29178 e +T630 CL:0000232 29206 29209 RBC +T631 UBERON:0003061 29227 29239 blood island +T632 UBERON:0003061 29241 29243 BI +T633 CL:0000765 29297 29310 erythroblasts +T634 CL:0000765 29312 29314 EB +T635 GO:0010467 29374 29384 expression +T636 UBERON:0001040 29392 29400 yolk sac +T637 UBERON:0000922 29405 29411 embryo +T638 UBERON:0000922 29437 29444 embryos +T639 PR:000011141 29474 29480 Nestin +T640 SO:0000243 29502 29506 IRES +T641 PR:000017284 29523 29529 VEGF-A +T642 UBERON:0000922 29538 29545 embryos +T643 CHEBI:50845 29581 29592 doxycycline +T644 UBERON:0001016 29602 29616 nervous system +T645 GO:0010467 29626 29636 expression +T646 UBERON:0000922 29685 29692 embryos +T647 CHEBI:50845 29756 29767 doxycycline +T648 GO:0007565 29779 29788 pregnancy +T649 PR:000017284 29794 29800 VEGF-A +T650 UBERON:0000922 29813 29819 embryo +T651 UBERON:0001016 29860 29874 nervous system +T652 CL:0000653 29923 29931 podocyte +T653 PR:000011366 29947 29954 Podocin +T654 UBERON:0002107 29980 29985 Liver +T655 http://purl.obolibrary.org/obo/MONDO_0004717 29980 29994 Liver peliosis +T656 UBERON:0002370 30014 30020 thymus +T657 CHEBI:50845 30058 30069 doxycycline +T658 PR:000017284 30086 30092 VEGF-A +T659 GO:0010467 30097 30107 expression +T660 UBERON:0007023 30111 30116 adult +T661 SO:0000243 30145 30149 IRES +T662 PR:000017284 30166 30172 VEGF-A +T663 NCBITaxon:10088 30181 30185 mice +T664 CHEBI:51686 30187 30188 H +T665 UBERON:0002107 30216 30222 livers +T666 UBERON:0007023 30244 30249 adult +T667 NCBITaxon:10088 30250 30254 mice +T668 PR:000017284 30265 30271 VEGF-A +T669 CHEBI:50845 30290 30293 DOX +T670 PR:000017284 30306 30312 VEGF-A +T671 CHEBI:50845 30327 30330 DOX +T672 CHEBI:50845 30335 30346 doxycycline +T673 CHEBI:50845 30366 30376 doxycyclin +T674 UBERON:0002107 30388 30394 livers +T675 UBERON:0002107 30409 30416 hepatic +T676 UBERON:0002049 30448 30459 vasculature +T677 UBERON:0002107 30471 30478 hepatic +T678 UBERON:0001979 30479 30486 venules +T679 UBERON:0001979 30488 30489 V +T680 UBERON:0001281 30495 30512 hepatic sinusoids +T681 UBERON:0001281 30514 30516 HS +T682 CHEBI:50845 30527 30538 doxycycline +T683 UBERON:0002107 30554 30560 livers +T684 UBERON:0002107 30594 30601 hepatic +T685 UBERON:0001281 30641 30658 hepatic sinusoids +T686 UBERON:0001281 30660 30662 HS +T687 UBERON:0000178 30684 30689 blood +T688 UBERON:0003909 30762 30772 sinusoidal +T689 UBERON:0001986 30773 30784 endothelial +T690 CHEBI:51686 30818 30819 H +T691 PR:000017284 30847 30853 VEGF-A +T692 CHEBI:50845 30872 30875 DOX +T693 PR:000017284 30888 30894 VEGF-A +T694 CHEBI:50845 30909 30912 DOX +T695 UBERON:0002370 30914 30920 thymus +T696 UBERON:0007023 30942 30947 adult +T697 NCBITaxon:10088 30948 30952 mice +T698 CHEBI:50845 30962 30973 doxycycline +T699 UBERON:0002370 30988 30994 thymus +T700 UBERON:0002370 31008 31014 thymic +T701 UBERON:0001851 31048 31054 cortex +T702 UBERON:0001851 31062 31064 cx +T703 CL:0000767 31086 31096 basophilic +T704 CL:0000542 31097 31108 lymphocytes +T705 UBERON:0002124 31122 31137 medullary layer +T706 UBERON:0002124 31139 31140 m +T707 CL:0000542 31162 31173 lymphocytes +T708 UBERON:0000483 31191 31201 epithelial +T709 CHEBI:50845 31213 31224 Doxycycline +T710 UBERON:0002370 31240 31246 thymus +T711 UBERON:0005483 31289 31301 thymic lobes +T712 UBERON:0005483 31360 31371 thymic lobe +T713 NCBITaxon:10088 31444 31448 mice +T714 UBERON:0002370 31474 31480 thymus +T715 UBERON:0001851 31502 31510 cortical +T716 CL:0000767 31561 31571 basophilic +T717 CL:0000893 31572 31582 thymocytes +T718 CHEBI:50845 31796 31807 doxycycline +T719 UBERON:0007023 31856 31861 adult +T720 NCBITaxon:10088 31862 31866 mice diff --git a/src/ontogpt/evaluation/craft/database/all/15784609.txt b/src/ontogpt/evaluation/craft/database/all/15784609.txt new file mode 100644 index 000000000..91b4a258c --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15784609.txt @@ -0,0 +1,107 @@ +Conditional and inducible transgene expression in mice through the combinatorial use of Cre-mediated recombination and tetracycline induction + +Abstract + +Here we describe a triple transgenic mouse system, which combines the tissue specificity of any Cre-transgenic line with the inducibility of the reverse tetracycline transactivator (rtTA)/tetracycline-responsive element (tet-O)-driven transgenes. To ensure reliable rtTA expression in a broad range of cell types, we have targeted the rtTA transgene into the ROSA26 locus. The rtTA expression, however, is conditional to a Cre recombinase-mediated excision of a STOP region from the ROSA26 locus. We demonstrate the utility of this technology through the inducible expression of the vascular endothelial growth factor (VEGF-A) during embryonic development and postnatally in adult mice. Our results of adult induction recapitulate several different hepatic and immune cell pathological phenotypes associated with increased systemic VEGF-A protein levels. This system will be useful for studying genes in which temporal control of expression is necessary for the discovery of the full spectrum of functions. The presented approach abrogates the need to generate tissue-specific rtTA transgenes for tissues where well-characterized Cre lines already exist. + +INTRODUCTION + +Cellular diversity in a developing organism is achieved by spatial and temporal regulation of gene expression determined by genetic programs and inductive signals. As a consequence, genes have spatially and temporally acting multiple functions, which are difficult to dissect without proper genetic tools providing similar complexity of control of expression—disruption or overexpression of a gene of interest (1,2). Uncontrolled transgenic expression of a given gene in all tissues or even in restricted cell types may be associated with embryonic lethality that precludes further studies at later stages of development or in adults. + +Cell type-/tissues-specific mutation of a gene can be achieved by a two-step process; introduction of loxP sites around a functionally essential genomic part followed by a cell type-specific Cre recombinase-mediated excision of the loxP flanked sequence. The same strategy can be used for cell type-specific overexpression of a transgene, when a strong overall expressing promoter is separated from the coding region of a gene of interest by loxP flanked ‘STOP’ sequences (1,3–5). In both scenarios, a Cre recombinase transgene provides spatial control. However, once Cre expression has been switched on and recombination has occurred, the resultant change in gene expression is, in most cases, irreversible. Tetracycline-inducible transgenic systems [tetracycline transactivator (tTA) or ‘Tet-Off’ and reverse tetracycline transactivator (rtTA) or ‘Tet-On’] allow for reversible temporal regulation of transgene expression (6,7). Of the two systems, rtTA is better suited for rapid induction of gene expression. + +In the following report, we describe a novel system that allows for the spatial and temporal regulation of transgene expression in vivo by combining any of the existing Cre recombinase transgenic lines with the reverse tTA (rtTA)–tet-O system. We targeted rtTA into the widely expressed ROSA26 locus, in a way where rtTA expression is conditional to a Cre-mediated excision event (8). Furthermore, we demonstrate the tightness and inducibility of this approach by systemic and cell type-specific (neuronal and podocyte) inducible expression of a dosage-sensitive gene, VEGF-A (9). The presented mouse line can be used to achieve spatially and temporally controlled transgene expression in a wide variety of settings simply by crossing to any existing mice carrying cell type-specific Cre recombinase and tet-O-regulatable responder genes. + +MATERIALS AND METHODS + +DNA constructs + +An EcoRI fragment carrying the rtTA coding sequence and an N-terminal nuclear localization signal was excised from the plasmid pSPC-rtTA (10), blunted by Klenow enzyme and inserted into the pCALL2-IRES-EGFP plasmid (a gift from Dr Corrine Lobe) at a blunted XhoI site. The resultant vector (pCALL2-rtTA-IRES-EGFP) was first digested with BglII, filled in by Klenow polymerase and then cut with NotI, and the fragment containing rtTA-IRES-EGFP was isolated. The pBigT vector (11) was digested first with SalI, filled in and subsequently digested with NotI. The above fragment was inserted into this vector resulting in pBigT-rtTA-IRES-EGFP. The whole insert of the latter vector was released by AscI–PacI double digest and inserted into pROSA26-PA vector (11) digested by the same enzymes. The resultant target vector was named pROSA26-rtTA (Figure 1A). Before introduction into mouse embryonic stem (ES) cells, the vector was linearized with XhoI. + +The murine podocin promoter was amplified from genomic murine DNA as described previously (12) and cloned upstream of the NLS-Cre transgene (a gift from B. Sauer). + +ES cell manipulation + +R1 mouse ES cells (13) were maintained and manipulated as described elsewhere (14). In brief, 20 μg of linearized target vector was electroporated into 107 cells using 500 μF and 250 V settings on Gene Pulser (Bio-Rad). After 24 h, the cells were selected for neomycin resistance in G418 (170 μg/ml) for 8 days. The resistant colonies were picked, expanded and split in 96-well plates, and the master plates were frozen and stored at −80°C until genetic characterization identified the correctly targeted clones. Some of these targeted clones were thawed from the master plates, expanded and subjected to further procedures described below. + +To activate rtTA expression from the ROSA26 locus, the transfection of the selected targeted clones with the pCAGGS-Cre-PGK-puro vector (a gift from C. Lobe) was performed using Lipofectamine 2000 (Invitrogen). An aliquot of 2 μg circular plasmid was transfected into cells growing in a 35 mm diameter dish. During the transfection, OPTI-MEM medium was used. Puromycin selection (1.25 μg/ml) was applied, and resistant colonies expanded. Green fluorescence was detected as described previously (15). + +The pBI-3 plasmid (a gift from H. Bujard) contains a bi-directional tetracycline-responsive element followed by the lacZ coding sequence. This vector was introduced into ES-cells by lipofection according to the protocol above. After 5 h of transfection, doxycycline (Sigma) was added to the medium in differing concentrations. The day after lipofection, the cells were passaged 1:5 and cultured for a further 1–2 days in the presence of doxycyline (100 ng/ml), and then stained by using X-gal. + +Generation of transgenic animals + +Chimeric mice were generated by aggregation of targeted ES-cells with eight-cell stage embryos as described previously (16). Germline transmitting chimeric males were crossed with outbred ICR females and the offspring were genotyped by Southern blotting and PCR for the transmission of the transgene. + +The Cre-recombinase transgenic founder lines were generated as described previously (17). Regarding the Podocin-Cre transgene four individual founder lines were crossed with the Z/EG reporter strain (15) to determine the degree and timing of Cre-mediated DNA excision in podocytes. + +Genotyping by Southern blotting + +To detect targeting of the ROSA26 locus, genomic DNA was isolated from the ES cells grown on 96-well plates (18), digested with EcoRV and the fragments separated on 0.7% agarose gel. Southern blotting was performed as described previously (19). Briefly, the DNA was transferred onto a nitrocellulose membrane (Hybond N; Amersham) and hybridized with a probe complementary to a sequence upstream of the 5′ homology arm of the vector (external probe) or with a probe complementary to the neo gene (internal probe) (Figure 1A and B). The radioactive signal was detected by phosphorimaging. To detect the mutation in mice, 10 μg genomic DNA was extracted from mouse tail biopsies and analyzed by Southern hybridization as above. + +Genotyping by PCR + +PCR was performed in 25 μl reaction mixture containing standard PCR buffer, 1.0 mM MgCl2, 200 μM dNTPs, 200 nM primers, 5 U Taq polymerase and 100 ng genomic DNA isolated from mouse ear biopsies. The primers for detection of the targeted ROSA26 allele containing the STOP cassette were as follows: ROSA5, GAGTTCTCTGCTGCCTCCTG; and RTTA3, AAGACCGCGAAGAGTTTGTC. The reaction resulted in a 215 bp band. The wild-type ROSA26 allele was detected by an amplicon of 322 bp using primers ROSA5 and ROSA3: CGAGGCGGATACAAGCAATA. The PCR program was as follows: initial denaturation for 1 min at 94°C followed by 35 cycles of 1 min at 94°C, 45 s at 62°C (knock-in allele) or 64°C (wild-type allele) and 1 min at 72°C and a final extension of 5 min at 72°C. PCR products were detected on 2% agarose gel. + +Histology + +The X-gal staining of ES cells was performed as described previously (15). Embryonic and adult tissue samples were dissected and fixed overnight in 4% PFA. The following day, the samples were washed extensively with 1× PBS, dehydrated through graded alcohol washes and embedded in paraffin wax as described previously (20). Sections (7 μm) were deparaffinized and stained with Harris' Hematoxylin and Eosin (Sigma Immunochemicals). + +RESULTS AND DISCUSSION + +Generation of the ROSA26-rtTA knock-in ES cell line + +We inserted an artificial exon between the first and the second exons of the mouse ROSA26 locus by gene-targeting. Of the 140 G418 resistant colonies picked and genotyped, 12 (8%) were correctly targeted (Figure 1A and B). In the artificial exon, a loxP site-flanked selectable marker (neo) precedes two coding sequences separated by an internal ribosomal entry site (IRES). The upstream sequence is coding for rtTA with a nuclear localization signal, and the downstream sequence is coding for enhanced green fluorescent protein (EGFP). Translation of the latter is ensured by the IRES sequence. Before Cre-excision, the three consecutive polyadenylation signals of the selectable marker terminate mRNA synthesis and, therefore, the downstream coding sequences are not expressed (8). After Cre-mediated deletion of the loxP flanked sequence, however, rtTA and EGFP are expressed by the ROSA26 promoter. Presence of doxycycline results in the formation of an active transcriptional activator and the activation of the responder transgene (see Figure 2). + +Assessment of tetracycline-inducibility in vitro + +To test the inducibility of the system in vitro, we removed the loxP flanked sequence by transfecting 10 targeted ES cell lines with a plasmid expressing Cre recombinase as well as puromycin acyltransferase (pCAGGS-Cre-PGK-Puro). We isolated stable integrants using puromycin selection and verified that they displayed green fluorescence while the parental cell lines were not fluorescent (Figure 1C and data not shown). There was no difference among the different targeted cell lines. Next, we expanded the fluorescent Cre-excised subclones of two original lines (1C12 and 1H6) and transfected them with a vector containing a lacZ transgene driven by a rtTA-responsive promoter (tet-O-LacZ). (We did not use any selection for transfectants in this experiment.) We did not observe any X-gal activity in the absence of the inducer (Figure 1D). When the medium was supplemented with 100 ng/ml doxycycline, LacZ positive cells were detected (Figure 1E). + +Inducible overexpression of VEGF-A in vivo + +Two ES-cell clones were used to generate chimeric mice by ES cells ⇔ embryo aggregation. In both experiments, germline transmission was obtained. Heterozygous animals of both lines were healthy, fertile and had a normal lifespan. Heterozygotes from one of the lines (1C12) were intercrossed, resulting in litters of pups with a Mendelian distribution of the F2 genotypes. Homozygous transgenic animals were healthy and fertile and were used to maintain this line in the homozygous state. These homozygous, Cre-conditional ROSA26-rtTA mice were used in consecutive experiments. + +In order to test the in vivo ‘silent but inducible’ nature of the conditional ROSA26-rtTA-IRES-EGFP transgene and rtTA protein activity, male mice that were homozygous for the Cre-conditional ROSA26-rtTA allele were bred with female mice that were double heterozygous for a tet-O-VEGF-A-164 responder transgene (21) and either the ubiquitous Cre-deletor line (pCAGGS-CreTg/+) (5) or a nervous system specific (Nestin-CreTg/+) (22) or a podocyte specific (Podocin-CreTg/+) Cre line. Figure 2 describes how the three transgenes work to unite the Cre/loxP and tetracycline inducible systems. + +Since the pCAGGS-Cre transgene expresses Cre in all the cells of the early embryo, it activates an overall rtTA and EGFP expression as shown for a E9.5 day embryo in Figure 3B and E. On the other hand, breeding with the Nestin-Cre or Podocin-Cre line resulted in embryos/animals with neuronal lineage or glomerulus-specific expression of rtTA and EGFP, respectively, as shown in Figure 3G for a E10.5 day embryo for Nestin-Cre and in Figure 3J for a newborn glomerulus for Podocin-Cre. In the absence of Cre-mediated excision of the loxP-flanked PGK-neo-pA sequence blocks rtTA and EGFP expression. + +We made use of the tight dosage sensitivity of the early developing embryo to increased (9,23) or decreased levels of VEGF-A (20,23–26) to demonstrate the correct activation of our inducible system. In the absence of the rtTA-inducer doxycycline, triple transgenic pCAGGS-CreTg/+, ROSA26-rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+ embryos (Figure 3A and B) develop normally and show no phenotype. However, in the presence of doxycycline that was administered to pregnant females starting one day after mating (E1.5), all of these triple transgenic embryos showed lethality (Figure 3D and E and see Table 1) at E9.5. The phenotypes of these mutant embryos were quite severe with no primitive red blood cells (RBCs) observed in the developing yolk sac (Figure 3D) and embryos were blocked in development. Unlike the original homozygous VEGF-A mutants that showed a severe reduction of blood island development in the yolk sac (24,25), ubiquitous VEGF-A-164 expression in this triple transgenic inducible system led to enlarged blood islands in the yolk sac that were filled with nucleated erythroblast progenitors (Figure 3F). The normal blood islands that developed in the non-induced embryos showed clearly defined endodermal and mesodermal layers ‘sandwiching’ the developing blood islands (Figure 3C); however, in the doxycycline-induced embryos, aberrant blood islands formed (Figure 3F). All the 10 triple transgenic embryos showed the same mutant phenotype described above upon doxycycline administration to their mothers (Table 1). Out of the 22 remaining littermates that were either single or double transgenic for the three transgenes, 20 appeared totally normal. Two rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+, Cre-negative embryos (as judged by Southern hybridization and PCR, data not shown), however, were also EGFP expressers indicating that rtTA expression activation (Cre-excision) occurred by maternally produced Cre protein. This known phenomenon is a consequence of high-Cre production and accumulation during oogenesis (27). + +The ubiquitous systemic administration or induction of the VEGF-A-164 protein, or injection of tumor cells expressing high levels of VEGF-A-164 protein into adult mice, results in severe edema (28–30), defects in immune cell function including destruction of the thymus (31–33) and liver pathologies that closely resemble the human syndrome known as peliosis hepatis that has been described in association with Bartonella henselae infection, long-term high-dose androgen therapy, or rarely with advanced cancers (34). In order to determine if doxycycline-induced overall VEGF-A-164 expression could mimic some or all of these previously described phenotypes, triple transgenic pCAGGS-CreTg/+, ROSA26-rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+ mice were allowed to reach adulthood and a regimen of doxycycline administration was followed (Table 1). These triple transgenic mice were born in a normal Mendelian frequency and showed no sign of any phenotypic abnormalities (Figure 4A–C and G–I, and data not shown). Upon administration of doxycycline in the drinking water to 3–4 month old pCAGGS-CreTg/+, ROSA26-rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+ mice, several phenotypes became apparent already after 2 days of treatment. The mice showed signs of edema and erythema of the face, ears and feet (data not shown). By 5 days of doxycycline administration, three of the eight triple transgenic mice had died and three were so moribund and sickly in appearance that they had to be euthanized and their organs were harvested for further histological analysis. Two mice seemed more resistant to the doxycycline administration and were given the drug for an additional 4 days at which time they too became moribund and were as well sacrificed. Of the 10 single and double transgenic littermates, 9 were completely normal and showed no adverse side affects to the doxycycline administration. Similar to what was observed in the E9.5 embryos, one rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+, Cre-negative adult mouse died during doxycycline administration. This mouse, however, was EGFP positive, indicating the rtTA expression activation by zygotic (maternal) Cre protein (27). Upon autopsy, the most obvious gross phenotypes observed were an extended blood filled liver and a dramatic decrease in the size of the thymus together with enlarged lymph nodes and signs of hyper-vascularity/permeability and edema in several other organ systems as well as blood in the intestine (data not shown). + +Histological analysis of the liver of uninduced pCAGGS-CreTg/+, ROSA26-rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+ adult mice (control) revealed normal hepatic architecture (Figure 4A–C). Doxycycline-treated mutant pCAGGS-CreTg/+, ROSA26-rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+ adult livers showed a dramatic ‘peliosis-like’ phenotype (Figure 4D) and signs of blood pooling in large areas of the liver (arrows in Figure 4E). This liver peliosis phenotype is characterized by enlarged hepatic sinusoids and a total disruption of the normal liver architecture (Figure 4D–F) as well as detached sinusoidal endothelial cells that are ‘sloughing-off’ into the sinusoidal space (arrow in Figure 4F). The liver pathology described here resembles the liver peliosis-like pathology recently described in mice that had been engrafted with tumor cells expressing high levels of systemic VEGF-A-164 protein (34). In addition, LeCouter et al. (35) have observed similar (although less severe) liver enlargement phenotypes and effects on the sinusoidal endothelium of the liver with systemic administration of recombinant VEGF-A protein. It was also demonstrated that VEGF-A at lower doses can act on the sinusoidal vasculature (via Flt1) to influence secretion of hepatocyte growth factor (HGF) and may have a beneficial protective effect on hepatocytes under times of cytotoxic stress (35). Our VEGF-A inducible system with liver-specific Cre lines such as the albumin-Cre line (36) will be useful in determining the optimal dosages of VEGF-A required for its beneficial action on the liver under times of cytotoxic stress and may at higher induction levels be a useful model system to unravel the molecular mechanisms behind VEGF-A's harmful effects on the liver. + +The thymus of non-induced pCAGGS-CreTg/+, ROSA26-rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+ adult mice (control) displayed a normal thymic architecture with a well-defined cortical layer rich in basophilic thymocytes and a normal epitheloid-rich medullary zone (Figure 4G–I). Total average cell counts for two control thymi were 1.33 × 108 cells/ml (1.05 × 108 and 1.6 × 108 cells/ml in the two different controls). Representative histological analysis of the thymi of VEGF-A-164-induced mutant adults (Table 1) revealed a dramatic decrease in cellularity that correlated with its decreased size (Figure 4J–L). The mutant thymi lacked major histological distinctions between the medullary and cortical layers and the basophilic cortical layer that is usually rich in thymocytes was almost completely absent (Figure 4L). Total average cell counts for three mutant thymi were 1.37 × 107 cells/ml (3.9 × 107, 8.1 × 105 and 1.14 × 106 cells/ml per animal). These numbers may under-represent the acellularity of the mutant thymi as two of the higher values were from the two animals that survived the initial five-day period of doxycycline treatment. The lower cell count of 8.1 × 105 cells/ml from one of the original sick animals that had to be euthanized in the first five-day period may be more representative of the acellularity shown in Figure 4J–L. The thymic atrophy presented in this paper phenocopies previous reports concerning the detrimental effects that systemic VEGF-A has on T-cell development (33). In addition, ubiquitous expression of VEGF-A-164 during early development seems to give rise to increased numbers of erythroblasts and may inhibit the differentiation of these progenitors. From this study, it is not clear whether this phenotype is caused by alterations in the yolk sac environment or by artificial VEGF-A-164 autocrine loop created. We are currently investigating the molecular mechanisms behind VEGF-A's suppressive effects on different hematopoietic populations through the use of this transgenic system together with additional hematopoietic lineage-specific Cre lines (37,38). + +The developing nervous system is also sensitive to altered VEGF-A expression, We have recently demonstrated that downregulation of the level of this growth factor, using a Nestin-Cre transgene and a conditional targeted allele of VEGF-A, have severe consequences to cortical vessel density and structure (39). The Nestin-CreTg/+, ROSA26-rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+ triple transgenic embryos presented here developed vascular abnormalities by E12.5, associated with spinal cord and brain hemorrhages, when doxycycline was administered to their mother from 1.5 dpc of pregnancy (Figure 3H and I). Without doxycycline administration to the mother, these embryos developed normally and reached adulthood. Interestingly, when these mice received doxycycline for 5 days at 4 months of age, they did not show any obvious gross behavioral or phenotypic abnormalities. Initial vessel and histological analysis from two of the six treated adult mouse brains did not reveal any striking differences in vessel architecture or structural organization of the cortex (data not shown). This result may indicate an increased resistance of the adult CNS vasculature to VEGF-A. We are currently further examining the potential effects of doxycycline-induced increases of VEGF-A-164 and its effects on the developing and adult nervous system using this triple transgenic system. + +In contrast, the Podocin-CreTg/+, ROSA26-rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+ triple transgenic mice treated with doxycycline for 7 days starting at 4 months of age developed proteinuria (albumin >5 g/l, data not shown). The presence of albuminuria in concentrations >3 g/l is designated ‘nephrotic range proteinuria’. This degree of proteinuria represents loss of the permeselective function of the glomerular filtration barrier. Increased permeability in the glomerulus leads to the loss of critical blood proteins, including albumin, blood clotting factor inhibitors and lipids, and is associated with edema and thrombotic events in patients. These mice are currently under detailed phenotypic analysis to identify the nature of changes in the kidney caused by induction VEGF-A expression in podocytes. + +Table 1 displays the results of all the experiments described above. In summary, by combining existing tissue-specific Cre-transgenic mouse strains to define spatial, and the tetracycline-inducible system to define temporal aspect of transgene expression, we have developed a versatile system which allows quick and efficient investigation of multiple gene functions. This aim has also been addressed by others using hormone-activated Cre proteins (40,41). The major advantage of these earlier efforts lie in that only two transgenes have to be combined. However, our system allows for complete on/off control of expression. In addition, the existing inducible systems cannot be combined with and take advantage of the large number of existing Cre-transgenic mouse strains defining tissue/cell type-specificities. Our results show that when expressed from the ROSA26 locus, the baseline activity of rtTA in the absence of inducer is not sufficient to cause significant activation of a responsive promoter. This is in agreement with the findings of Wutz and Jaenisch (42), who used constitutive rtTA expression from the ROSA26 promoter. We expect though that the ROSA26 locus placed rtTA could be a limitation by providing only a given level of expression for rtTA, which might not be high enough to reach sufficient level of induction of different responder tet-O-genes in certain anatomical areas or cell types. Therefore, an obvious future improvement of the presented system would be the establishment of Cre conditional rtTA transgenes in several genomic positions permissive to different level of overall expression. Our ROSA26 placed Cre-conditional system could be the first member of this envisioned useful series. + +Acknowledgements + +We are grateful to Dr C. G. Lobe for providing the pCALL2-IRES-EGFP and to Dr H. Bujard for the pBI-3 vectors. These studies were supported in part by a National Cancer Institute of Canada grant (NCIC grant 21335). J.H. was a recipient of an NCIC postdoctoral fellowship. A.N. is a senior Canadian Institutes of Health Research (CIHR) scientist. S.E.Q. is supported by CIHR grant no. 62931. Funding to pay the Open Access publication charges for this article was provided by NCIC 021335. + +Conflict of interest statement. None declared. + +Figures and Tables + +Figure 1 + +Targeted insertion of a conditional rtTA-IRES-EGFP transgene into the ROSA26 locus. (A) Targeting strategy. The exons of the ROSA26 gene are depicted as numbered boxes, and the triangles as loxP sites. The insertion point (XbaI site), informative restriction sites and diagnostic fragments are also shown. Abbreviations are explained in the text. Drawing is not to scale. (B) Southern-blot analysis of genomic DNA from ES cells digested by EcoRV and hybridized with a 5′ external probe (left) and neo probe (right) (C) After Cre-excision, cells show green fluroescence. Line 1C12 was transfected with a plasmid carrying Cre and puro expression cassettes. A puromycin-resistant colony is shown. (D and E) Tetracyclin-induction of lacZ in Cre-excised 1C12 cells. The cells were transfected with a plasmid containing a tet-O-lacZ transgene. (D) No doxycycline induction. (E) Cells after doxycycline administration (100 ng/ml for 2 days). + +Figure 2 + +Operation of the Cre/loxP-dependent, tetracycline inducible transgenic system. (A) Mice that are homozygous for the targeted insertion of the conditional rtTA-IRES-EGFP bicistronic transgene at ROSA26 can be bred with double transgenic mice that carry a tissue-specific Cre transgene and a doxycycline-inducible rtTA-dependent tet-O-GENE responder line. A total of 25% of the pups will be triple transgenic (SpecPromoterCreTg/+, ROSA26-STOP-rtTA-IRES-EGFPTg/+, tet-O-GENETg/+). (B) In cells that do not express Cre, neither rtTA nor EGFP protein is generated; therefore, the tet-O-GENE is silent. (C) In Cre-expressing cells, rtTA and EGFP expression is turned on. However, in the absence of an inducer (e.g. doxycycline), rtTA cannot activate the expression of the tet-O-GENE target. Addition of doxycycline results in the formation of an active transactivator and expression of the target gene. In the present study, VEGF-A-164 was induced either ubiquitously or nervous system or podocyte specifically in the presence of doxycycline. + +Figure 3 + +Tissue-specific expression of the rtTA-IRES-EGFP bicistronic transgene is Cre-dependent and the rtTA trans-activation of tet-O-VEGF-A-164 is tightly regulated by doxycycline. (A–C) Images of triple transgenic pCAGGS-CreTg/+, ROSA26-rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+ E9.5 embryo and yolk sac in the absence of doxycycline: (A), Whole mount bright field microscopy; (B), GFP fluorescence microscopy; and (C), H&E histological section of the yolk sac. In the absence of doxycycline, triple transgenic embryos develop normally. Arrow in (A) shows normal yolk sac vessel with primitive hemoglobin-containing RBCs and normal blood island (BI) formation between the primitive endoderm (e) and mesoderm (m) layers of the yolk sac (C). (D–F) Triple transgenic embryo and yolk sac, the mother treated with doxycycline. (D), Whole mount bright field microscopy; (E), GFP fluorescence microscopy; and (F), H&E histological section of the yolk sac. (D) In the presence of doxycycline the triple transgenic embryos show a lethal phenotype with no proper vessel development in the yolk sac (ys) and embryo (e), no hemoglobin-containing RBC (D) and abnormal blood island (BI) structures that are filled with excessive nucleated erythroblasts (EB). (B and E) The overall EGFP signal marks the overall rtTA expression in the yolk sac and embryo of the triple transgenic embryos. (G) E10.5 triple transgenic Nestin-CreTg/+, ROSA26-rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+ embryos develop normally in the absence of doxycycline and show nervous system specific expression of EGFP/rtTA. (H and I) E12.5 triple transgenic embryos when the mother was either non-treated (H) or treated (I) with doxycycline during the pregnancy. The VEGF-A-164 induced embryo (I) shows hemorrhages in the developing nervous system (arrows). (J) Immunostaining for EGFP shows the podocyte specificity of Podocin-Cre excision. + +Figure 4 + +Liver peliosis-like phenotype and thymus degeneration are associated with the doxycycline-induced overall VEGF-A-164 expression in adult pCAGGS-CreTg/+, ROSA26-rtTA-IRES-EGFPTg/+, tet-O-VEGF-A-164Tg/+ mice. H&E histological analysis of livers of triple transgenic adult mice of (A–C): VEGF-A-164 non-induced (−DOX) and (D–F): VEGF-A-164 induced (+DOX) by doxycycline treatment. Without doxycyclin treatment, livers have a normal hepatic architecture with well-defined vasculature and normal hepatic venules (V) and hepatic sinusoids (HS) (C). The doxycycline-treated mutant livers show major disruptions of normal hepatic architecture with a severe dilation of hepatic sinusoids (HS) (F) and evidence of blood engorgement and pooling [arrows in (E)]. Arrow in (F) shows evidence of sinusoidal endothelial sloughing and detachment. (G–I): H&E histological analysis of VEGF-A-164 non-induced (−DOX) and (J–L): VEGF-A-164 induced (+DOX) thymus of triple transgenic adult mice. Without doxycycline treatment the thymus shows normal thymic architecture with a well-defined cortex layer (cx) that is packed with basophilic lymphocytes and a normal medullary layer (m) that contains fewer lymphocytes but an extensive epithelial framework. Doxycycline-treated mutant thymus shows massive degeneration with all three thymic lobes fitting into a single optical field (J) compared with one thymic lobe fitting into the optic field at the same magnification (G) from control mice. In addition, the mutant thymus lacks a well-defined cortical layer owing to a dramatically decreased number of basophilic thymocytes seen at higher magnifications (L) compared with (I). (A, D, G and J), 50× magnification; (B and E), 125× magnification; (H and K), 250× magnification; and (C, F, I and L), 500× magnification. + +Table 1 + +Summary of doxycycline induction experiments during development and in adult mice diff --git a/src/ontogpt/evaluation/craft/database/all/15819996.ann b/src/ontogpt/evaluation/craft/database/all/15819996.ann new file mode 100644 index 000000000..248fa4c00 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15819996.ann @@ -0,0 +1,1105 @@ +T1 GO:0005634 0 7 Nuclear +T2 PR:000004080 24 34 Annexin A7 +T3 NCBITaxon:39107 42 48 murine +T4 UBERON:0000955 49 54 brain +T5 GO:0007420 49 66 brain development +T6 PR:000004080 90 100 Annexin A7 +T7 CHEBI:16247 199 212 phospholipids +T8 CHEBI:29108 232 241 Ca2+-ions +T9 CHEBI:29108 278 282 Ca2+ +T10 GO:0042592 283 294 homeostasis +T11 NCBITaxon:10088 316 320 mice +T12 CHEBI:29108 336 340 Ca2+ +T13 CL:0000127 361 371 astrocytes +T14 PR:000004080 411 421 Annexin A7 +T15 UBERON:0000955 429 434 brain +T16 GO:0007420 429 446 brain development +T17 PR:000004080 515 525 Annexin A7 +T18 NCBITaxon:10088 541 546 mouse +T19 GO:0009790 547 560 embryogenesis +T20 UBERON:0001017 579 601 central nervous system +T21 UBERON:0007023 613 618 adult +T22 NCBITaxon:10088 619 624 mouse +T23 UBERON:0000955 625 630 brain +T24 PR:000004080 642 652 Annexin A7 +T25 GO:0010467 656 665 expressed +T26 UBERON:0000955 693 698 brain +T27 GO:0005737 751 760 cytoplasm +T28 GO:0005634 764 771 nucleus +T29 UBERON:0007023 793 798 adult +T30 UBERON:0001017 799 802 CNS +T31 PR:000004080 836 846 Annexin A7 +T32 PR:000004080 906 916 Annexin A7 +T33 GO:0005829 937 944 cytosol +T34 UBERON:0000922 974 983 embryonic +T35 GO:0005829 1043 1050 cytosol +T36 UBERON:0003053 1089 1117 ventricular germinative zone +T37 UBERON:0002285 1134 1151 lateral ventricle +T38 UBERON:0000922 1166 1175 embryonic +T39 PR:000004080 1185 1195 Annexin A7 +T40 UBERON:0001950 1250 1260 neopallium +T41 GO:0005634 1281 1288 nucleus +T42 CL:0000540 1290 1304 Neuronal cells +T43 UBERON:0007023 1325 1330 adult +T44 UBERON:0000955 1331 1336 brain +T45 PR:000004080 1345 1355 Annexin A7 +T46 GO:0005634 1363 1370 nucleus +T47 CL:0000125 1380 1385 glial +T48 PR:000007939 1380 1411 glial fibrillary acidic protein +T49 CHEBI:37527 1397 1403 acidic +T50 PR:000007939 1413 1417 GFAP +T51 CL:0000127 1428 1438 astrocytes +T52 GO:0005737 1455 1466 cytoplasmic +T53 GO:0005634 1471 1478 nuclear +T54 GO:0005634 1505 1512 nuclear +T55 PR:000004080 1513 1523 Annexin A7 +T56 GO:0005654 1559 1570 nucleoplasm +T57 GO:0005634 1585 1591 nuclei +T58 CL:0000540 1606 1614;1630 1634 neuronal ... cell +T59 CL:0000127 1619 1634 astroglial cell +T60 GO:0051170 1678 1691;1706 1715 translocation ... to nuclei +T61 PR:000004080 1695 1705 Annexin A7 +T62 GO:0005634 1709 1724 nuclei of cells +T63 NCBITaxon:39107 1734 1740 murine +T64 UBERON:0000955 1741 1746 brain +T65 GO:0007420 1741 1758 brain development +T66 PR:000004080 1779 1789 Annexin A7 +T67 GO:0005634 1793 1802;1812 1817 nuclei of ... cells +T68 CL:0000540 1803 1817 neuronal cells +T69 UBERON:0007023 1825 1830 adult +T70 NCBITaxon:33208 1831 1837 animal +T71 PR:000004080 1851 1861 Annexin A7 +T72 GO:0005634 1865 1874;1911 1916 nuclei of ... cells +T73 CL:0000540 1902 1916 neuronal cells +T74 CHEBI:62643 2018 2050 negatively charged phospholipids +T75 CHEBI:29108 2056 2060 Ca2+ +T76 SO:0000417 2164 2170 domain +T77 SO:0000417 2195 2201 domain +T78 SO:0000417 2267 2273 domain +T79 CHEBI:29108 2378 2382 Ca2+ +T80 SO:0000409 2383 2395 binding site +T81 CHEBI:16247 2420 2432 phospholipid +T82 PR:000004080 2653 2663 Annexin A7 +T83 GO:0042583 2755 2774 chromaffin granules +T84 GO:0061025 2779 2798 fusion of membranes +T85 GO:0016020 2789 2798 membranes +T86 CHEBI:16247 2803 2816 phospholipids +T87 CHEBI:29108 2836 2845 Ca2+-ions +T88 GO:0010467 2851 2861 Expression +T89 PR:000004080 2903 2913 Annexin A7 +T90 UBERON:0000479 2935 2942 tissues +T91 GO:0005829 2976 2983 cytosol +T92 GO:0016020 3010 3020 membranous +T93 GO:0005634 3066 3076;3096 3101 nucleus of ... cells +T94 UBERON:0002369 3077 3084 adrenal +T95 CL:1000426 3077 3101 adrenal chromaffin cells +T96 GO:0000380 3126 3147 alternatively spliced +T97 SO:0005853 3148 3156 cassette +T98 SO:0000147 3157 3161 exon +T99 PR:000004080 3180 3190 Annexin A7 +T100 SO:0001060 3191 3199 isoforms +T101 SO:0001060 3258 3266 isoforms +T102 SO:0000417 3294 3300 domain +T103 UBERON:0000479 3315 3321 tissue +T104 GO:0010467 3331 3341 expression +T105 SO:0001060 3362 3369 isoform +T106 UBERON:0000479 3388 3395 tissues +T107 UBERON:0001134 3407 3422 skeletal muscle +T108 SO:0001060 3441 3448 isoform +T109 UBERON:0001133 3473 3485 Heart muscle +T110 UBERON:0000955 3487 3492 brain +T111 UBERON:0000479 3493 3499 tissue +T112 CL:0000232 3504 3519 red blood cells +T113 UBERON:0000178 3508 3513 blood +T114 SO:0001060 3533 3541 isoforms +T115 PR:000004080 3613 3623 Annexin A7 +T116 CL:0000056 3639 3647 myoblast +T117 GO:0005829 3734 3741 cytosol +T118 GO:0016020 3746 3754 membrane +T119 GO:0016020 3828 3836 membrane +T120 PR:000004080 3926 3936 Annexin A7 +T121 CL:0000232 3972 3987 red blood cells +T122 UBERON:0000178 3976 3981 blood +T123 CL:0000233 3989 3997 platelet +T124 GO:0070527 3989 4009 platelet aggregation +T125 CL:0000127 4053 4063 astrocytic +T126 CHEBI:29108 4064 4068 Ca2+ +T127 PR:000004080 4076 4086 Annexin A7 +T128 UBERON:0000948 4134 4141 cardiac +T129 CHEBI:29108 4164 4168 Ca2+ +T130 GO:0042592 4169 4180 homeostasis +T131 PR:000004080 4263 4273 Annexin A7 +T132 UBERON:0000955 4281 4286 brain +T133 GO:0007420 4281 4298 brain development +T134 PR:000004080 4371 4381 Annexin A7 +T135 UBERON:0000955 4400 4405 brain +T136 NCBITaxon:10088 4409 4413 mice +T137 UBERON:0000922 4414 4421 embryos +T138 UBERON:0007023 4453 4458 adult +T139 NCBITaxon:10088 4459 4464 mouse +T140 UBERON:0000955 4465 4470 brain +T141 PR:000004080 4482 4492 Annexin A7 +T142 GO:0010467 4496 4505 expressed +T143 NCBITaxon:10088 4519 4524 mouse +T144 UBERON:0000922 4525 4531 embryo +T145 GO:0010467 4555 4565 expression +T146 PR:000004080 4569 4579 Annexin A7 +T147 CL:0002322 4583 4591 ES cells +T148 NCBITaxon:10088 4627 4631 mice +T149 NCBITaxon:10088 4657 4662 mouse +T150 UBERON:0000922 4663 4672 embryonic +T151 GO:0009790 4663 4684 embryonic development +T152 CL:0002322 4846 4854 ES-cells +T153 UBERON:0000922 4862 4871 embryonic +T154 GO:0000380 4956 4976 alternative splicing +T155 SO:0000205 4984 5002 untranslated 3'end +T156 GO:0006412 4986 4996 translated +T157 PR:000004080 5036 5046 Annexin A7 +T158 UBERON:0000922 5071 5080 embryonic +T159 PR:000003676 5106 5113 β-actin +T160 PR:000003676 5250 5257 β-actin +T161 CL:0002322 5286 5294 ES cells +T162 GO:0010467 5295 5302 express +T163 PR:000004080 5320 5330 Annexin A7 +T164 SO:0001060 5331 5338 isoform +T165 UBERON:0000948 5378 5383 heart +T166 UBERON:0000479 5384 5390 tissue +T167 GO:0010467 5429 5439 Expression +T168 PR:000004080 5443 5453 Annexin A7 +T169 CL:0002322 5494 5502 ES-cells +T170 UBERON:0000922 5507 5514 embryos +T171 PR:000004080 5559 5569 annexin A7 +T172 SO:0000673 5576 5587 Transcripts +T173 PR:000003676 5674 5681 β-actin +T174 NCBITaxon:39107 5743 5749 Murine +T175 CL:0002322 5750 5758 ES cells +T176 CL:0002322 5760 5762 ES +T177 GO:0010467 5764 5771 express +T178 PR:000004080 5787 5797 Annexin A7 +T179 SO:0001060 5798 5805 isoform +T180 SO:0001060 5812 5820 isoforms +T181 GO:0010467 5825 5834 expressed +T182 http://purl.obolibrary.org/obo/MONDO_0005072 5838 5851 neuroblastoma +T183 UBERON:0000948 5874 5879 heart +T184 UBERON:0000948 5881 5884 HRT +T185 UBERON:0000955 5890 5895 brain +T186 UBERON:0000479 5896 5902 tissue +T187 UBERON:0001017 5904 5907 CNS +T188 UBERON:0007023 5914 5919 adult +T189 NCBITaxon:10088 5920 5924 mice +T190 CL:0002322 5953 5961 ES cells +T191 UBERON:0000948 5963 5968 heart +T192 UBERON:0000955 5973 5978 brain +T193 CHEBI:8984 5996 5999 SDS +T194 CHEBI:28619 6040 6050 acrylamide +T195 CHEBI:53325 6071 6085 nitrocellulose +T196 MOP:0000779 6183 6190 coupled +T197 GO:0042571 6201 6209 antibody +T198 PR:000004080 6344 6354 Annexin A7 +T199 SO:0001060 6426 6434 isoforms +T200 PR:000004080 6438 6448 Annexin A7 +T201 UBERON:0004013 6495 6503 cylinder +T202 PR:000004080 6530 6540 Annexin A7 +T203 GO:0010467 6541 6551 expression +T204 UBERON:0000924 6559 6567 ectoderm +T205 PR:000004080 6587 6597 Annexin A7 +T206 UBERON:0005291 6628 6638;6643 6649 tissues of ... embryo +T207 PR:000004080 6706 6716 Annexin A7 +T208 UBERON:0000923 6769 6780 germ layers +T209 UBERON:0034705 6859 6874 neuroepithelium +T210 UBERON:0005062 6882 6893 neural fold +T211 UBERON:0001049 6882 6888;6898 6902 neural ... tube +T212 PR:000004080 6980 6990 Annexin A7 +T213 GO:0005829 7040 7047 cytosol +T214 PR:000004080 7161 7171 Annexin A7 +T215 NCBITaxon:10088 7198 7203 mouse +T216 UBERON:0000922 7204 7211 embryos +T217 UBERON:0000922 7233 7239 embryo +T218 CL:0000025 7248 7251 egg +T219 UBERON:0004013 7248 7260 egg cylinder +T220 UBERON:0000087 7276 7291 inner cell mass +T221 UBERON:0000924 7313 7321 ectoderm +T222 UBERON:0000119 7335 7343;7353 7358 layer of ... cells +T223 UBERON:0000925 7344 7352 endoderm +T224 CL:0000223 7344 7358 endoderm cells +T225 CHEBI:37987 7452 7455 Cy3 +T226 MOP:0000779 7456 7466 conjugated +T227 NCBITaxon:10088 7472 7477 mouse +T228 GO:0071735 7478 7481 IgG +T229 PR:000004080 7483 7493 Annexin A7 +T230 GO:0010467 7497 7506 expressed +T231 CL:0000025 7533 7536 egg +T232 UBERON:0004013 7533 7545 egg cylinder +T233 UBERON:0000925 7576 7584 endoderm +T234 UBERON:0000924 7614 7622 ectoderm +T235 GO:0005634 7628 7634 nuclei +T236 UBERON:0002405 7649 7655 immune +T237 CHEBI:37987 7708 7711 Cy3 +T238 GO:0042571 7712 7720 antibody +T239 PR:000004080 7733 7743 Annexin A7 +T240 GO:0010467 7744 7754 expression +T241 UBERON:0001049 7771 7782 neural tube +T242 UBERON:0005062 7798 7809 neural fold +T243 UBERON:0000922 7817 7823 embryo +T244 PR:000004080 7866 7876 Annexin A7 +T245 CHEBI:52661 7947 7962 Alexa Fluor 488 +T246 MOP:0000779 7963 7973 conjugated +T247 NCBITaxon:10088 7979 7984 mouse +T248 GO:0071735 7985 7988 IgG +T249 PR:000004080 8005 8015 Annexin A7 +T250 UBERON:0034705 8052 8067 neuroepithelium +T251 UBERON:0001049 8075 8086 neural tube +T252 UBERON:0003842 8091 8111 lumen of neural tube +T253 UBERON:0034705 8149 8164 neuroepithelium +T254 PR:000004080 8175 8185 Annexin A7 +T255 GO:0010467 8189 8198 expressed +T256 GO:0005829 8206 8213 cytosol +T257 PR:000004080 8235 8245 Annexin A7 +T258 GO:0005829 8270 8277 cytosol +T259 UBERON:0000922 8299 8305 embryo +T260 UBERON:0001049 8318 8329 neural tube +T261 CHEBI:52661 8419 8434 Alexa Fluor 488 +T262 MOP:0000779 8435 8445 conjugated +T263 NCBITaxon:10088 8451 8456 mouse +T264 GO:0071735 8457 8460 IgG +T265 CHEBI:52661 8503 8518 Alexa Fluor 488 +T266 GO:0042571 8519 8527 antibody +T267 PR:000004080 8547 8557 Annexin A7 +T268 UBERON:0000922 8602 8611 embryonic +T269 UBERON:0000955 8612 8617 brain +T270 PR:000004080 8688 8698 Annexin A7 +T271 NCBITaxon:10088 8717 8722 mouse +T272 UBERON:0000955 8723 8728 brain +T273 UBERON:0000922 8780 8789 embryonic +T274 UBERON:0000955 8790 8795 brain +T275 UBERON:0003053 8849 8877 ventricular germinative zone +T276 UBERON:0002285 8894 8911 lateral ventricle +T277 PR:000004080 8994 9004 Annexin A7 +T278 GO:0005829 9032 9039 cytosol +T279 PR:000004080 9154 9164 Annexin A7 +T280 GO:0005829 9172 9179 cytosol +T281 GO:0005634 9239 9246 nuclear +T282 PR:000004080 9259 9269 Annexin A7 +T283 CL:0000540 9348 9355 neurons +T284 GO:0040007 9387 9394 growing +T285 UBERON:0001950 9395 9412 neopallium cortex +T286 CL:0000125 9430 9441 glial cells +T287 UBERON:0002316 9454 9466 white matter +T288 UBERON:0007023 9474 9479 adult +T289 UBERON:0001851 9480 9486 cortex +T290 UBERON:0001950 9520 9537 neopallial cortex +T291 GO:0005634 9553 9560 nuclear +T292 UBERON:0003053 9585 9613 ventricular germinative zone +T293 GO:0005829 9640 9647 cytosol +T294 GO:0005634 9692 9699 nucleus +T295 PR:000004080 9740 9750 Annexin A7 +T296 UBERON:0000922 9754 9761 embryos +T297 UBERON:0000922 9801 9810 embryonic +T298 UBERON:0000955 9811 9816 brain +T299 PR:000004080 9857 9867 Annexin A7 +T300 CHEBI:52661 9888 9903 Alexa Fluor 488 +T301 MOP:0000779 9904 9914 conjugated +T302 GO:0042571 9925 9933 antibody +T303 PR:000007939 9969 9973 GFAP +T304 UBERON:0001950 10004 10022 cerebral neocortex +T305 UBERON:0002285 10043 10060 lateral ventricle +T306 GO:0005829 10243 10250 cytosol +T307 PR:000004080 10321 10331 Annexin A7 +T308 GO:0005829 10345 10352 cytosol +T309 GO:0005634 10375 10382 nuclear +T310 UBERON:0003053 10471 10499 ventricular germinative zone +T311 UBERON:0001950 10513 10530 neopallial cortex +T312 PR:000007939 10642 10646 GFAP +T313 UBERON:0002285 10687 10704 lateral ventricle +T314 UBERON:0001851 10736 10744 cortical +T315 UBERON:0003053 10782 10793;10822 10827 ventricular ... zones +T316 CL:0000540 10898 10905 neurons +T317 UBERON:0007023 10909 10914 adult +T318 NCBITaxon:10088 10915 10920 mouse +T319 UBERON:0000955 10921 10926 brain +T320 GO:0005634 10943 10950 nuclear +T321 PR:000004080 10963 10973 Annexin A7 +T322 PR:000004080 10980 10990 Annexin A7 +T323 SO:0001060 10991 10999 isoforms +T324 UBERON:0007023 11013 11018 adult +T325 UBERON:0000955 11019 11024 brain +T326 UBERON:0000479 11025 11031 tissue +T327 PR:000004080 11108 11118 Annexin A7 +T328 GO:0005634 11132 11139 nuclear +T329 CL:0000540 11149 11156 neurons +T330 PR:000007939 11180 11184 GFAP +T331 GO:0005737 11235 11246 cytoplasmic +T332 GO:0005634 11251 11258 nuclear +T333 CL:0000127 11268 11278 astrocytes +T334 PR:000007939 11280 11284 GFAP +T335 NCBITaxon:39107 11322 11328 murine +T336 UBERON:0000955 11329 11334 brain +T337 UBERON:0001950 11343 11352 neocortex +T338 UBERON:0001950 11354 11363 isocortex +T339 PR:000004080 11365 11375 Annexin A7 +T340 GO:0005634 11401 11407 nuclei +T341 CL:0000540 11411 11418 neurons +T342 UBERON:0002301 11430 11446 cortical laminae +T343 UBERON:0016538 11478 11495 cortex temporalis +T344 UBERON:0007023 11518 11523 adult +T345 UBERON:0000955 11524 11529 brain +T346 CL:0000127 11537 11547 astrocytes +T347 PR:000007939 11583 11587 GFAP +T348 GO:0005634 11642 11649 nuclear +T349 PR:000004080 11650 11660 Annexin A7 +T350 UBERON:0001950 11666 11677 neocortical +T351 CL:0000540 11678 11684 neuron +T352 CL:0000127 11737 11752 astrocytic cell +T353 UBERON:0005390 11760 11787 neocortical molecular layer +T354 GO:0005737 11851 11860 cytoplasm +T355 UBERON:0001950 11914 11923 isocortex +T356 GO:0005634 11966 11973 nucleus +T357 GO:0005829 11978 11985 cytosol +T358 CL:0000598 12009 12025 pyramidal neuron +T359 PR:000007939 12061 12065 GFAP +T360 PR:000004080 12093 12103 Annexin A7 +T361 GO:0005634 12139 12146 nucleus +T362 PR:000004080 12169 12179 Annexin A7 +T363 CL:0000540 12194 12201 neurons +T364 CL:0000127 12206 12216 astrocytes +T365 UBERON:0016538 12224 12241 cortex temporalis +T366 UBERON:0002421 12246 12267 hippocampal formation +T367 NCBITaxon:10088 12284 12288 mice +T368 UBERON:0016538 12319 12336 cortex temporalis +T369 PR:000004080 12349 12359 Annexin A7 +T370 GO:0010467 12360 12370 expression +T371 UBERON:0002361 12387 12391 pial +T372 CL:0000540 12403 12410 neurons +T373 UBERON:0002301 12422 12441 isocortical laminae +T374 UBERON:0002316 12477 12489 white matter +T375 PR:000007939 12530 12534 GFAP +T376 UBERON:0002313 12556 12575;12605 12607;12612 12623 Stratum pyramidalis ... of ... hippocampus +T377 UBERON:0001885 12591 12607;12612 12623 dentate gyrus of ... hippocampus +T378 PR:000004080 12709 12719 Annexin A7 +T379 GO:0042571 12799 12807 antibody +T380 PR:000004080 12830 12840 Annexin A7 +T381 CL:0000598 12844 12861 pyramidal neurons +T382 UBERON:0005392 12863 12889 lamina pyramidalis externa +T383 UBERON:0016538 12898 12918 isocortex temporalis +T384 CL:0000540 12926 12933 neurons +T385 PR:000007939 13002 13006 GFAP +T386 PR:000004080 13017 13026 AnnexinA7 +T387 GO:0005634 13084 13091 nucleus +T388 PR:000004080 13154 13164 Annexin A7 +T389 GO:0005634 13168 13174 nuclei +T390 CL:0000540 13178 13185 neurons +T391 GO:0005737 13252 13261 cytoplasm +T392 GO:0005634 13266 13272 nuclei +T393 CL:0000127 13276 13286 astrocytes +T394 PR:000007939 13315 13319 GFAP +T395 CL:0000598 13364 13381 pyramidal neurons +T396 PR:000004080 13430 13440 Annexin A7 +T397 GO:0005634 13456 13463 nucleus +T398 CL:0000540 13486 13493 neurons +T399 PR:000004080 13558 13563 AnxA7 +T400 NCBITaxon:10088 13567 13572 mouse +T401 GO:0042571 13611 13619 antibody +T402 GO:0005634 13635 13642 nuclear +T403 UBERON:0000479 13677 13683 tissue +T404 PR:000004080 13741 13746 AnxA7 +T405 UBERON:0000955 13750 13755 brain +T406 GO:0042571 13777 13785 antibody +T407 PR:000007939 13880 13884 GFAP +T408 GO:0042571 13885 13893 antibody +T409 GO:0042571 13953 13961 antibody +T410 UBERON:0002421 13971 13992 hippocampal formation +T411 PR:000004080 14015 14025 Annexin A7 +T412 UBERON:0001885 14079 14092 dentate gyrus +T413 CL:0000127 14109 14119 astrocytic +T414 CL:0000127 14144 14154 astrocytes +T415 GO:0005634 14213 14220 nuclear +T416 PR:000004080 14221 14231 Annexin A7 +T417 GO:0005634 14274 14281 nucleus +T418 GO:0005737 14290 14299 cytoplasm +T419 CL:0000598 14339 14356 pyramidal neurons +T420 PR:000004080 14416 14426 Annexin A7 +T421 GO:0005634 14457 14463 nuclei +T422 GO:0042571 14486 14496 antibodies +T423 GO:0005634 14559 14566 nuclear +T424 GO:0042571 14638 14646 antibody +T425 UBERON:0000955 14663 14668 brain +T426 PR:000004080 14691 14696 AnxA7 +T427 NCBITaxon:10088 14700 14704 mice +T428 PR:000004080 14717 14727 Annexin A7 +T429 PR:000004080 14773 14778 AnxA7 +T430 UBERON:0000955 14782 14787 brain +T431 GO:0042571 14879 14887 antibody +T432 PR:000004080 14904 14914 Annexin A7 +T433 UBERON:0000955 14926 14931 brain +T434 UBERON:0000479 14942 14949 tissues +T435 PR:000004080 14957 14962 AnxA7 +T436 NCBITaxon:10088 14966 14971 mouse +T437 NCBITaxon:10088 15040 15045 mouse +T438 GO:0042571 15171 15179 antibody +T439 PR:000004080 15203 15213 Annexin A7 +T440 UBERON:0002037 15236 15246 cerebellum +T441 GO:0005634 15258 15264 Nuclei +T442 CL:0000540 15268 15275 neurons +T443 GO:0043025 15392 15414 cell bodies of neurons +T444 CL:0000540 15407 15414 neurons +T445 GO:0005634 15464 15470 nuclei +T446 CL:0000540 15474 15481 neurons +T447 CL:0000127 15483 15493 Astrocytes +T448 PR:000004080 15516 15526 Annexin A7 +T449 GO:0005634 15534 15540 nuclei +T450 GO:0005737 15545 15554 cytoplasm +T451 UBERON:0002361 15591 15595 pial +T452 UBERON:0002316 15634 15646 white matter +T453 PR:000004080 15686 15696 Annexin A7 +T454 CL:0000127 15706 15716 astrocytes +T455 PR:000004080 15838 15848 Annexin A7 +T456 GO:0005634 15859 15865 nuclei +T457 GO:0005737 15883 15892 cytoplasm +T458 GO:0005886 15904 15919 plasma membrane +T459 CL:0000121 15923 15937 Purkinje cells +T460 GO:0030425 15969 15978 dendrites +T461 UBERON:0002129 16007 16024 cerebellar cortex +T462 PR:000004080 16049 16059 Annexin A7 +T463 UBERON:0000955 16100 16105 brain +T464 PR:000004080 16122 16127 AnxA7 +T465 NCBITaxon:10088 16131 16136 mouse +T466 PR:000004080 16162 16172 Annexin A7 +T467 GO:0043005 16176 16184 neurites +T468 GO:0030424 16186 16191 axons +T469 CL:0000121 16236 16249 Purkinje-cell +T470 UBERON:0000119 16245 16255 cell layer +T471 GO:0030424 16283 16288 axons +T472 CL:0000121 16296 16310 Purkinje-cells +T473 UBERON:0006798 16329 16337 efferent +T474 GO:0043005 16338 16346 neurites +T475 UBERON:0014540 16371 16382 lamina alba +T476 UBERON:0002316 16384 16396 white matter +T477 GO:0044301 16425 16440 climbing fibers +T478 UBERON:0002317 16473 16496 cerebellar white matter +T479 PR:000004080 16509 16519 Annexin A7 +T480 CL:0000127 16531 16541 astrocytes +T481 GO:0043005 16546 16554 neurites +T482 PR:000004080 16567 16577 Annexin A7 +T483 UBERON:0002037 16600 16610 cerebellum +T484 UBERON:0007023 16614 16619 adult +T485 NCBITaxon:10088 16620 16624 mice +T486 UBERON:0002037 16655 16665 cerebellum +T487 PR:000004080 16678 16688 Annexin A7 +T488 GO:0010467 16689 16699 expression +T489 CL:0000712 16710 16718;16723 16741 cells of ... stratum granulosum +T490 PR:000007939 16804 16808 GFAP +T491 UBERON:0004081 16838 16846;16851 16861 folia of ... cerebellum +T492 PR:000004080 16887 16896 AnnexinA7 +T493 GO:0042571 16897 16905 antibody +T494 UBERON:0000119 17066 17074;17084 17089 layer of ... cells +T495 CL:0000121 17075 17089 Purkinje-cells +T496 CL:0000121 17182 17196 Purkinje-cells +T497 PR:000004080 17220 17230 Annexin A7 +T498 GO:0005634 17289 17295 nuclei +T499 CL:0000540 17299 17306 neurons +T500 GO:0005634 17354 17361 nucleus +T501 GO:0043025 17367 17376 cell body +T502 PR:000004080 17380 17389 AnnexinA7 +T503 GO:0030425 17414 17423 dendrites +T504 CL:0000121 17431 17444 Purkinje-cell +T505 PR:000004080 17486 17491 AnxA7 +T506 NCBITaxon:10088 17495 17500 mouse +T507 PR:000004080 17506 17515 AnnexinA7 +T508 GO:0030424 17528 17533 axons +T509 CL:0000121 17590 17603 Purkinje-cell +T510 UBERON:0000119 17599 17609 cell layer +T511 GO:0010467 17708 17718 Expression +T512 PR:000004080 17722 17732 Annexin A7 +T513 UBERON:0007023 17740 17745 adult +T514 NCBITaxon:9606 17746 17751 human +T515 UBERON:0001950 17752 17761 isocortex +T516 NCBITaxon:9606 17770 17775 human +T517 UBERON:0016530 17776 17794 parietal neocortex +T518 NCBITaxon:1 17803 17814 individuals +T519 UBERON:0002361 17861 17865 pial +T520 CL:0000127 17866 17876 astrocytes +T521 PR:000004080 17901 17911 Annexin A7 +T522 GO:0005737 17919 17928 cytoplasm +T523 GO:0005634 17932 17939 nuclear +T524 PR:000004080 17952 17962 Annexin A7 +T525 CL:0000127 17985 17995 astrocytes +T526 CL:0000598 18007 18024 Pyramidal neurons +T527 PR:000004080 18067 18077 Annexin A7 +T528 GO:0005886 18085 18100 plasma membrane +T529 GO:0043204 18110 18120 perikaryon +T530 GO:0097440 18139 18154 apical dendrite +T531 CL:0000540 18172 18179 neurons +T532 PR:000004080 18200 18210 Annexin A7 +T533 GO:0005634 18220 18226 nuclei +T534 GO:0097440 18228 18244 Apical dendrites +T535 PR:000004080 18311 18321 Annexin A7 +T536 PR:000004080 18357 18367 Annexin A7 +T537 NCBITaxon:9606 18375 18380 human +T538 UBERON:0000955 18389 18394 brain +T539 PR:000027795 18435 18442 trypsin +T540 PR:000004080 18455 18465 Annexin A7 +T541 NCBITaxon:9606 18484 18489 human +T542 UBERON:0001950 18490 18499 isocortex +T543 CL:0000598 18505 18522 Pyramidal neurons +T544 GO:0097440 18540 18556 apical dendrites +T545 GO:0042571 18602 18610 antibody +T546 PR:000004080 18619 18629 Annexin A7 +T547 NCBITaxon:9606 18637 18642 human +T548 UBERON:0016530 18643 18658 parietal cortex +T549 GO:0030425 18666 18675 dendritic +T550 UBERON:0002361 18757 18761 pial +T551 CL:0000127 18762 18772 astrocytes +T552 GO:0005737 18834 18845 cytoplasmic +T553 CL:0000127 18856 18866 astrocytes +T554 GO:0005634 18876 18883 nuclear +T555 PR:000004080 18934 18944 Annexin A7 +T556 GO:0005886 18961 18974 cell membrane +T557 GO:0043204 18982 18992 perikaryon +T558 GO:0097440 19014 19029 apical dendrite +T559 GO:0042571 19086 19094 antibody +T560 CL:0000540 19182 19189 neurons +T561 PR:000004080 19275 19285 Annexin A7 +T562 GO:0005634 19289 19295 nuclei +T563 CL:0000540 19301 19309;19325 19330 neuronal ... cells +T564 CL:0000127 19314 19330 astroglial cells +T565 GO:0005634 19336 19343 nuclear +T566 PR:000004080 19360 19370 Annexin A7 +T567 NCBITaxon:10088 19374 19378 mice +T568 UBERON:0007023 19432 19437 adult +T569 CL:0000540 19438 19445 neurons +T570 UBERON:0000955 19497 19502 brain +T571 PR:000027795 19534 19541 trypsin +T572 GO:0042571 19549 19557 antibody +T573 GO:0042571 19607 19617 antibodies +T574 CHEBI:16842 19651 19663 formaldehyde +T575 CHEBI:17790 19674 19682 Methanol +T576 CL:0000010 19695 19709 cultured cells +T577 CHEBI:59132 19739 19746 Antigen +T578 CHEBI:16842 19760 19768 formalin +T579 UBERON:0000479 19797 19803 tissue +T580 GO:0006508 19840 19851 proteolytic +T581 GO:0042571 20006 20014 antibody +T582 CHEBI:59132 20056 20064 antigens +T583 CHEBI:59132 20155 20162 antigen +T584 PR:000004080 20202 20212 Annexin A7 +T585 GO:0005634 20216 20222 nuclei +T586 GO:0005634 20268 20274 nuclei +T587 GO:0005654 20376 20387 nucleoplasm +T588 GO:0005654 20403 20414 nucleoplasm +T589 GO:0005634 20433 20439 nuclei +T590 GO:0005654 20456 20467 nucleoplasm +T591 CHEBI:8984 20516 20519 SDS +T592 PR:000004080 20592 20602 Annexin A7 +T593 GO:0005654 20610 20621 nucleoplasm +T594 CL:0000540 20625 20633;20672 20676 neuronal ... cell +T595 CL:0000127 20656 20666;20672 20676 astroglial ... cell +T596 SO:0001060 20713 20720 isoform +T597 GO:0005654 20758 20769 nucleoplasm +T598 GO:0042571 20798 20808 antibodies +T599 PR:000037206 20817 20822 LAP2α +T600 PR:000007050 20824 20830 Emerin +T601 PR:000028799 20835 20842 tubulin +T602 GO:0005654 20872 20883 nucleoplasm +T603 PR:000037206 20892 20897 LAP2α +T604 GO:0016020 20913 20921 membrane +T605 SO:0001060 20928 20935 isoform +T606 PR:000029585 20939 20943 LAP2 +T607 GO:0031965 21001 21018 nuclear membranes +T608 PR:000007050 21027 21033 Emerin +T609 GO:0005737 21041 21050 cytoplasm +T610 PR:000028799 21059 21066 tubulin +T611 CL:0000540 21088 21096;21112 21116 neuronal ... cell +T612 CL:0000127 21101 21116 astroglial cell +T613 PR:000004080 21123 21133 Annexin A7 +T614 GO:0005634 21159 21166 nucleus +T615 UBERON:0000955 21298 21303 brain +T616 PR:000004080 21342 21352 Annexin A7 +T617 PR:000027795 21387 21394 trypsin +T618 GO:0005634 21407 21414 Nuclear +T619 PR:000004080 21431 21441 Annexin A7 +T620 PR:000004080 21461 21471 Annexin A7 +T621 GO:0005634 21477 21483 nuclei +T622 CL:0000540 21487 21495;21532 21536 neuronal ... cell +T623 CL:0000127 21516 21526;21532 21536 astroglial ... cell +T624 GO:0031965 21625 21642 nuclear membranes +T625 GO:0031965 21644 21645 m +T626 GO:0005654 21651 21662 nucleoplasm +T627 GO:0005654 21664 21665 p +T628 CHEBI:8984 21685 21688 SDS +T629 PR:000004080 21716 21726 Annexin A7 +T630 PR:000004080 21728 21733 AnxA7 +T631 SO:0001060 21735 21743 isoforms +T632 GO:0005634 21769 21779;21794 21799 nucleus of ... cells +T633 SO:0001060 21819 21826 isoform +T634 PR:000037206 21871 21876 LAP2α +T635 GO:0016020 21890 21898 membrane +T636 SO:0001060 21905 21912 isoform +T637 PR:000007050 21915 21921 Emerin +T638 PR:000007050 21923 21925 EM +T639 PR:000028799 21940 21947 tubulin +T640 PR:000028799 21949 21951 TB +T641 GO:0005654 21984 21995 nucleoplasm +T642 PR:000037206 21997 22002 LAP2α +T643 GO:0031965 22005 22022 nuclear membranes +T644 PR:000007050 22024 22030 Emerin +T645 GO:0005737 22041 22050 cytoplasm +T646 PR:000028799 22052 22059 tubulin +T647 GO:0005654 22086 22097 nucleoplasm +T648 CHEBI:9750 22243 22255 Triton X-100 +T649 PR:000027795 22273 22280 trypsin +T650 PR:000004080 22312 22322 Annexin A7 +T651 CHEBI:51231 22345 22349 DAPI +T652 PR:000004080 22434 22444 Annexin A7 +T653 NCBITaxon:10088 22452 22457 mouse +T654 UBERON:0001017 22519 22541 central nervous system +T655 GO:0009790 22549 22562 embryogenesis +T656 GO:0010467 22621 22631 expression +T657 PR:000004080 22635 22645 Annexin A7 +T658 UBERON:0005291 22653 22670 embryonic tissues +T659 UBERON:0002346 22745 22760 neural ectoderm +T660 UBERON:0001016 22812 22826 nervous system +T661 PR:000004080 22833 22843 Annexin A7 +T662 GO:0005829 22875 22882 cytosol +T663 UBERON:0000925 22884 22894 Endodermal +T664 CL:0000223 22884 22894;22917 22922 Endodermal ... cells +T665 UBERON:0000926 22906 22916 mesodermal +T666 CL:0000222 22906 22922 mesodermal cells +T667 UBERON:0000955 23001 23006 brain +T668 PR:000004080 23069 23079 Annexin A7 +T669 UBERON:0002301 23094 23101;23115 23117;23122 23139 stratum ... of ... neopallial cortex +T670 UBERON:0002285 23161 23178 lateral ventricle +T671 PR:000004080 23215 23225 Annexin A7 +T672 GO:0005829 23240 23247 cytosol +T673 PR:000004080 23325 23335 Annexin A7 +T674 GO:0005634 23343 23359 nucleus of cells +T675 UBERON:0001950 23405 23415 neopallium +T676 GO:0010467 23431 23441 expression +T677 PR:000004080 23454 23464 Annexin A7 +T678 UBERON:0003053 23489 23517 ventricular germinative zone +T679 UBERON:0001950 23552 23561 neocortex +T680 PR:000016471 23630 23640 Tenascin-C +T681 CL:0000127 23650 23660 astroglial +T682 CL:0000681 23702 23720 radial glial cells +T683 CL:0000540 23796 23803 neurons +T684 CL:0000540 23836 23843 Neurons +T685 GO:0007567 23866 23873 natally +T686 UBERON:0003053 23881 23912 proliferative ventricular layer +T687 UBERON:0001950 23920 23937 neopallial cortex +T688 UBERON:0001851 24011 24019 cortical +T689 UBERON:0000119 24020 24031 cell layers +T690 GO:0048469 24070 24085 cell maturation +T691 PR:000004080 24153 24163 Annexin A7 +T692 UBERON:0007023 24218 24223 adult +T693 UBERON:0000955 24224 24229 brain +T694 GO:0005634 24254 24261 nuclear +T695 PR:000004080 24279 24289 Annexin A7 +T696 CL:0000540 24293 24300 neurons +T697 CL:0000127 24309 24319 astrocytes +T698 GO:0005829 24337 24346 cytosolic +T699 GO:0005634 24360 24367 nuclear +T700 CL:0000598 24387 24404 pyramidal neurons +T701 UBERON:0001950 24412 24421 isocortex +T702 CL:0000121 24426 24443;24448 24458 Purkinje-cells of ... cerebellum +T703 UBERON:0002037 24448 24458 cerebellum +T704 GO:0005829 24471 24480 cytosolic +T705 GO:0043005 24529 24537 neurites +T706 GO:0030425 24542 24551 dendrites +T707 GO:0010467 24585 24595 expression +T708 PR:000004080 24599 24609 Annexin A7 +T709 NCBITaxon:9606 24613 24618 human +T710 UBERON:0016538 24619 24637 temporal neocortex +T711 http://purl.obolibrary.org/obo/MONDO_0005027 24704 24712 epilepsy +T712 PR:000004080 24731 24741 Annexin A7 +T713 SO:0001060 24742 24750 isoforms +T714 GO:0005737 24769 24778 cytoplasm +T715 GO:0005634 24783 24789 nuclei +T716 CL:0000127 24793 24803 astrocytes +T717 CL:0000540 24813 24820 neurons +T718 UBERON:0001954 24890 24902 Ammon's horn +T719 UBERON:0016538 24931 24949 temporal neocortex +T720 UBERON:0000479 24950 24956 tissue +T721 NCBITaxon:39107 25068 25074 murine +T722 UBERON:0000955 25075 25080 brain +T723 GO:0010467 25182 25192 expression +T724 NCBITaxon:10088 25205 25210 mouse +T725 NCBITaxon:9606 25215 25220 human +T726 NCBITaxon:9606 25260 25265 human +T727 UBERON:0000955 25266 25271 brain +T728 NCBITaxon:9606 25313 25318 human +T729 UBERON:0016530 25319 25334 parietal cortex +T730 UBERON:0000955 25346 25351 brain +T731 CL:0000127 25357 25367 astroglial +T732 GO:0010467 25368 25378 expression +T733 PR:000004080 25382 25392 Annexin A7 +T734 CL:0000127 25430 25440 astrocytes +T735 GO:0005634 25455 25462 nuclear +T736 CL:0000598 25473 25490 Pyramidal neurons +T737 PR:000004080 25532 25542 Annexin A7 +T738 GO:0030425 25570 25579 dendrites +T739 CL:0000540 25620 25627 neurons +T740 UBERON:0000479 25688 25694 tissue +T741 UBERON:0001871 25759 25772 temporal lobe +T742 http://purl.obolibrary.org/obo/MONDO_0005115 25759 25782 temporal lobe epilepsia +T743 NCBITaxon:9606 25813 25818 human +T744 UBERON:0000955 25819 25824 brain +T745 UBERON:0000479 25825 25831 tissue +T746 NCBITaxon:10088 25901 25905 mice +T747 CL:0000010 25933 25947 cultured cells +T748 GO:0006915 25954 25958;25969 25978 cell ... apoptosis +T749 CHEBI:29108 26033 26037 Ca2+ +T750 CHEBI:24869 26038 26047 ionophore +T751 PR:000004080 26048 26058 Annexin A7 +T752 GO:0005737 26081 26090 cytoplasm +T753 GO:0005886 26094 26112 cellular membranes +T754 PR:000004080 26158 26168 Annexin A7 +T755 CL:0000540 26186 26193 neurons +T756 NCBITaxon:9606 26201 26206 human +T757 UBERON:0000955 26215 26220 brain +T758 GO:0005886 26260 26277 cellular membrane +T759 GO:0016020 26314 26322 membrane +T760 GO:0006915 26393 26402 apoptosis +T761 PR:000004078 26432 26441 AnnexinA5 +T762 GO:0005634 26459 26466 nuclear +T763 PR:000004080 26467 26477 Annexin A7 +T764 NCBITaxon:39107 26481 26487 murine +T765 UBERON:0000955 26488 26493 brain +T766 PR:000004080 26544 26549 AnxA7 +T767 NCBITaxon:10088 26553 26558 mouse +T768 GO:0005634 26615 26622 nucleus +T769 PR:000004080 26629 26639 Annexin A7 +T770 SO:0001060 26640 26648 isoforms +T771 UBERON:0000955 26662 26667 brain +T772 UBERON:0000479 26668 26674 tissue +T773 GO:0010467 26689 26698 expressed +T774 CL:0000540 26702 26709 neurons +T775 CL:0000127 26714 26724 astrocytes +T776 GO:0010467 26831 26841 expression +T777 UBERON:0000955 26845 26850 brain +T778 PR:000004080 26857 26867 Annexin A7 +T779 SO:0001060 26868 26876 isoforms +T780 UBERON:0001133 26905 26917 heart muscle +T781 CL:0000232 26922 26937 red blood cells +T782 UBERON:0000178 26926 26931 blood +T783 PR:000004080 26979 26989 annexin A7 +T784 SO:0000704 26990 26994 gene +T785 NCBITaxon:10088 27061 27065 mice +T786 PR:000004080 27123 27133 Annexin A7 +T787 CL:0000127 27197 27207 astrocytes +T788 PR:000004080 27216 27221 AnxA7 +T789 NCBITaxon:10088 27225 27230 mouse +T790 CHEBI:29108 27235 27239 Ca2+ +T791 GO:0019722 27235 27259 Ca2+-dependent signaling +T792 CL:0000127 27359 27369 astrocytic +T793 CHEBI:29108 27370 27374 Ca2+ +T794 PR:000004080 27440 27450 Annexin A7 +T795 CHEBI:29108 27464 27468 Ca2+ +T796 CHEBI:29108 27495 27499 Ca2+ +T797 GO:0042592 27500 27511 homeostasis +T798 CL:0000540 27516 27523 neurons +T799 CHEBI:29108 27524 27533 Ca2+ ions +T800 PR:000004080 27660 27670 Annexin A7 +T801 GO:0065007 27678 27688 regulation +T802 CHEBI:29108 27698 27702 Ca2+ +T803 UBERON:0000948 27808 27813 heart +T804 PR:000004080 27899 27909 Annexin A7 +T805 UBERON:0000948 27942 27949 cardiac +T806 CHEBI:29108 27972 27976 Ca2+ +T807 GO:0042592 27977 27988 homeostasis +T808 GO:0009790 28030 28043 embryogenesis +T809 PR:000004073 28066 28077 Annexin A11 +T810 UBERON:0002315 28094 28108;28127 28138 gray matter of ... spinal cord +T811 NCBITaxon:10114 28113 28116 rat +T812 UBERON:0000922 28117 28126 embryonic +T813 GO:0005634 28159 28166 nuclear +T814 PR:000004073 28183 28194 Annexin A11 +T815 GO:0005634 28237 28243 nuclei +T816 UBERON:0007023 28251 28256 adult +T817 UBERON:0002240 28257 28268 spinal cord +T818 GO:0051170 28307 28317;28349 28351;28356 28363 relocation ... to ... nucleus +T819 PR:000004080 28321 28331 Annexin A7 +T820 GO:0005829 28341 28348 cytosol +T821 GO:0005634 28356 28363 nucleus +T822 UBERON:0005291 28380 28389;28399 28405 embryonic ... tissue +T823 CL:0000540 28390 28398 neuronal +T824 PR:000004080 28411 28421 Annexin A7 +T825 CHEBI:29108 28505 28509 Ca2+ +T826 PR:000004080 28537 28547 Annexin A7 +T827 GO:0016020 28551 28560 membranes +T828 PR:000004080 28627 28637 Annexin A7 +T829 PR:000015619 28646 28652 sorcin +T830 GO:0005886 28764 28779 plasma membrane +T831 GO:0005737 28788 28797 cytoplasm +T832 GO:0005856 28825 28837 cytoskeleton +T833 PR:000004071 28839 28850 Annexins A1 +T834 PR:000004075 28839 28847;28852 28854 Annexins ... A2 +T835 PR:000004077 28839 28847;28856 28858 Annexins ... A4 +T836 PR:000004078 28839 28847;28860 28862 Annexins ... A5 +T837 PR:000004073 28839 28847;28867 28870 Annexins ... A11 +T838 GO:0005634 28933 28940 nucleus +T839 NCBITaxon:9606 28966 28971 human +T840 UBERON:0001471 28972 28980 foreskin +T841 CL:1001608 28972 28992 foreskin fibroblasts +T842 PR:000004071 29011 29021 Annexin A1 +T843 PR:000004077 29011 29018;29023 29025 Annexin ... A4 +T844 PR:000004078 29011 29018;29030 29032 Annexin ... A5 +T845 GO:0005634 29056 29063 nucleus +T846 GO:0005829 29100 29107 cytosol +T847 GO:0005622 29122 29135 intracellular +T848 CHEBI:29108 29136 29140 Ca2+ +T849 GO:0031965 29184 29200 nuclear membrane +T850 CHEBI:29108 29250 29254 Ca2+ +T851 GO:0005634 29273 29279 nuclei +T852 CL:0000057 29287 29298 fibroblasts +T853 GO:0009294 29354 29365 transfected +T854 GO:0005622 29390 29403 intracellular +T855 CHEBI:29108 29404 29408 Ca2+ +T856 PR:000004080 29459 29469 Annexin A7 +T857 GO:0005654 29499 29510 nucleoplasm +T858 GO:0031965 29518 29534 nuclear membrane +T859 GO:0005634 29582 29589 nuclear +T860 SO:0001528 29582 29609 nuclear localization signal +T861 GO:0005634 29633 29640 nuclear +T862 GO:0051170 29633 29647 nuclear import +T863 PR:000004073 29678 29689 Annexin A11 +T864 GO:0005634 29708 29715 nuclear +T865 SO:0000409 29789 29801 binding side +T866 PR:000014413 29823 29832 calcyclin +T867 PR:000004073 29886 29897 Annexin A11 +T868 PR:000014413 29902 29908 S100A6 +T869 GO:0005635 29925 29941 nuclear envelope +T870 GO:0005634 29949 29956 nuclear +T871 GO:0005634 30038 30045 nuclear +T872 GO:0065007 30087 30096 regulated +T873 PR:000004075 30156 30166 Annexin A2 +T874 GO:0065007 30170 30180 controlled +T875 GO:0051235 30184 30197 sequestration +T876 PR:000004075 30205 30210 AnxA2 +T877 PR:000014403 30211 30214 p11 +T878 GO:0032991 30215 30222 complex +T879 GO:0065007 30223 30232 modulated +T880 GO:0005634 30261 30268 nuclear +T881 GO:0051168 30261 30275 nuclear export +T882 SO:0001531 30261 30282 nuclear export signal +T883 PR:000004075 30296 30301 AnxA2 +T884 PR:000004075 30336 30346 Annexin A2 +T885 GO:0005634 30354 30361 nucleus +T886 PR:000014403 30402 30405 p11 +T887 SO:0000112 30459 30465 primer +T888 GO:0043234 30478 30493 protein complex +T889 GO:0005634 30582 30589 nuclear +T890 GO:0001775 30610 30626 cell stimulation +T891 CHEBI:29108 30635 30639 Ca2+ +T892 GO:0065007 30665 30675 regulating +T893 GO:0006260 30676 30691 DNA replication +T894 PR:000004080 30697 30707 Annexin A7 +T895 GO:0005634 30862 30869 nuclear +T896 SO:0001528 30862 30889 nuclear localization signal +T897 PR:000004080 30893 30903 Annexin A7 +T898 PR:000004080 30930 30940 Annexin A7 +T899 GO:0005634 30948 30955 nuclear +T900 GO:0051170 31012 31025;31040 31049 translocation ... to nuclei +T901 PR:000004080 31029 31039 Annexin A7 +T902 GO:0005634 31043 31052;31069 31074 nuclei of ... cells +T903 NCBITaxon:39107 31093 31099 murine +T904 UBERON:0000955 31100 31105 brain +T905 UBERON:0007023 31114 31119 adult +T906 UBERON:0000955 31120 31125 brain +T907 PR:000004080 31126 31136 Annexin A7 +T908 GO:0005634 31163 31169 nuclei +T909 CL:0000540 31173 31180 neurons +T910 CL:0000127 31182 31192 Astrocytes +T911 UBERON:0002037 31194 31204 cerebellar +T912 CL:0000121 31194 31219 cerebellar Purkinje-cells +T913 UBERON:0001950 31224 31235 neocortical +T914 CL:0000598 31236 31253 pyramidal neurons +T915 PR:000004080 31270 31280 Annexin A7 +T916 GO:0005829 31288 31295 cytosol +T917 GO:0005634 31307 31313 nuclei +T918 PR:000004080 31353 31363 Annexin A7 +T919 PR:000004080 31428 31438 Annexin A7 +T920 GO:0005634 31471 31477 nuclei +T921 PR:000004080 31574 31584 Annexin A7 +T922 GO:0005634 31590 31596 nuclei +T923 CL:0000540 31600 31608;31619 31623 neuronal ... cell +T924 CL:0000125 31613 31623 glial cell +T925 NCBITaxon:33208 31641 31648 Animals +T926 UBERON:0000479 31653 31659 tissue +T927 NCBITaxon:10088 31686 31690 mice +T928 UBERON:0010148 31723 31735 vaginal plug +T929 NCBITaxon:33208 31777 31783 animal +T930 NCBITaxon:33208 31875 31882 Animals +T931 GO:0007565 31912 31920 Pregnant +T932 UBERON:0005434 31948 31956 cervical +T933 UBERON:0000922 32021 32028 embryos +T934 UBERON:0000995 32063 32069 uterus +T935 UBERON:0000922 32081 32088 embryos +T936 NCBITaxon:33208 32192 32199 animals +T937 UBERON:0000955 32240 32245 brain +T938 NCBITaxon:10088 32268 32272 mice +T939 UBERON:0000922 32308 32315 embryos +T940 UBERON:0000479 32320 32327 tissues +T941 CHEBI:75958 32379 32387 solution +T942 UBERON:0000479 32416 32422 tissue +T943 CHEBI:16236 32465 32472 ethanol +T944 NCBITaxon:9606 32627 32632 Human +T945 UBERON:0000955 32633 32638 brain +T946 UBERON:0000479 32639 32645 tissue +T947 NCBITaxon:9606 32652 32657 human +T948 UBERON:0000955 32666 32672 brains +T949 GO:0016265 32762 32768 mortem +T950 CHEBI:15377 32805 32812 aqueous +T951 CHEBI:75958 32813 32821 solution +T952 CHEBI:16842 32825 32837 formaldehyde +T953 UBERON:0016530 32869 32887 parietal neocortex +T954 GO:0042571 33217 33227 Antibodies +T955 GO:0042571 33229 33239 Antibodies +T956 SO:0000417 33302 33308 domain +T957 NCBITaxon:10088 33312 33317 mouse +T958 PR:000004080 33318 33328 Annexin A7 +T959 GO:0071735 33343 33346 IgG +T960 PR:000004080 33384 33394 Annexin A7 +T961 SO:0001060 33395 33403 isoforms +T962 GO:0042571 33447 33455 antibody +T963 NCBITaxon:10088 33464 33469 mouse +T964 PR:000004080 33470 33480 Annexin A7 +T965 UBERON:0001977 33512 33517 serum +T966 CL:0000127 33537 33546 astrocyte +T967 GO:0005882 33556 33577 intermediate filament +T968 CL:0000125 33578 33583 glial +T969 PR:000007939 33578 33609 glial fibrillary acidic protein +T970 CHEBI:37527 33595 33601 acidic +T971 PR:000007939 33611 33615 GFAP +T972 PR:000028799 33638 33645 tubulin +T973 PR:000007050 33668 33674 Emerin +T974 PR:000037206 33717 33722 LAP2α +T975 GO:0042571 33862 33870 antibody +T976 CHEBI:52661 33880 33893 AlexaFluor488 +T977 MOP:0000779 33894 33904 conjugated +T978 NCBITaxon:9925 33905 33909 goat +T979 NCBITaxon:10088 33915 33920 mouse +T980 GO:0071735 33921 33924 IgG +T981 CHEBI:37987 33973 33976 Cy3 +T982 NCBITaxon:9925 33985 33989 goat +T983 NCBITaxon:10088 33995 34000 mouse +T984 GO:0071735 34001 34004 IgG +T985 GO:0042571 34022 34032 antibodies +T986 PR:000004080 34050 34060 Annexin A7 +T987 GO:0042571 34061 34069 antibody +T988 PR:000007939 34079 34083 GFAP +T989 GO:0042571 34084 34092 antibody +T990 CHEBI:51766 34112 34127 Alexa Fluor 568 +T991 MOP:0000779 34128 34138 conjugated +T992 NCBITaxon:9925 34139 34143 goat +T993 NCBITaxon:9986 34149 34155 rabbit +T994 GO:0071735 34156 34159 IgG +T995 PR:000004080 34206 34216 Annexin A7 +T996 NCBITaxon:10088 34240 34245 mouse +T997 GO:0071735 34246 34249 IgG +T998 GO:0042571 34260 34270 antibodies +T999 NCBITaxon:10088 34274 34279 mouse +T1000 UBERON:0000479 34280 34286 tissue +T1001 GO:0042571 34324 34334 antibodies +T1002 MOP:0000779 34356 34363 coupled +T1003 CHEBI:52661 34367 34381 Alexa Fluor488 +T1004 CHEBI:27338 34459 34465 xylene +T1005 CHEBI:16236 34552 34559 ethanol +T1006 GO:0042571 34564 34572 antibody +T1007 CHEBI:26710 34624 34628 NaCl +T1008 CHEBI:32588 34637 34640 KCl +T1009 CHEBI:34683 34649 34656 Na2HPO4 +T1010 CHEBI:63036 34665 34671 KH2PO4 +T1011 UBERON:0000479 34686 34692 tissue +T1012 PR:000027795 34740 34747 trypsin +T1013 CHEBI:16842 34787 34799 formaldehyde +T1014 SO:0000409 34883 34896 binding sites +T1015 CHEBI:5291 34969 34977 gelatine +T1016 NCBITaxon:9925 35000 35004 goat +T1017 UBERON:0001977 35005 35010 serum +T1018 GO:0042571 35054 35064 antibodies +T1019 GO:0042571 35232 35240 antibody +T1020 CHEBI:15377 35334 35339 water +T1021 GO:0042571 35432 35440 antibody +T1022 PR:000004080 35470 35480 annexin A7 +T1023 NCBITaxon:10088 35490 35495 mouse +T1024 NCBITaxon:9606 35644 35649 human +T1025 UBERON:0000955 35650 35655 brain +T1026 NCBITaxon:9606 35679 35684 human +T1027 UBERON:0000955 35685 35690 brain +T1028 PR:000004080 35748 35758 Annexin A7 +T1029 GO:0042571 35794 35802 antibody +T1030 PR:000027795 35872 35879 Trypsin +T1031 CHEBI:52302 35917 35931 Carbocyanine 2 +T1032 CHEBI:37987 35917 35929;35936 35937 Carbocyanine ... 3 +T1033 NCBITaxon:10088 35951 35956 mouse +T1034 GO:0071735 35957 35960 IgG +T1035 GO:0042571 35971 35981 antibodies +T1036 GO:0042571 36042 36050 antibody +T1037 NCBITaxon:39107 36244 36250 murine +T1038 http://purl.obolibrary.org/obo/MONDO_0005072 36251 36264 neuroblastoma +T1039 NCBITaxon:10114 36290 36293 rat +T1040 NCBITaxon:10114 36338 36341 rat +T1041 http://purl.obolibrary.org/obo/MONDO_0018177 36342 36354 glioblastoma +T1042 CHEBI:9750 36453 36465 Triton X-100 +T1043 UBERON:0000479 36550 36556 tissue +T1044 PR:000027795 36586 36593 trypsin +T1045 GO:0005634 36620 36627 nuclear +T1046 PR:000004080 36628 36638 Annexin A7 +T1047 CL:0000010 36640 36654 Cultured cells +T1048 CHEBI:17992 36746 36753 sucrose +T1049 CHEBI:9754 36761 36765 Tris +T1050 CHEBI:3312 36779 36784 CaCl2 +T1051 CHEBI:62964 36791 36801 Mg-acetate +T1052 CHEBI:63016 36821 36825 NP40 +T1053 CHEBI:18320 36832 36835 DTT +T1054 CHEBI:8102 36844 36848 PMSF +T1055 CHEBI:63016 37150 37154 NP40 +T1056 GO:0005634 37217 37223 nuclei +T1057 GO:0005654 37255 37266 nucleoplasm +T1058 GO:0005634 37286 37292 nuclei +T1059 CHEBI:46756 37329 37334 Hepes +T1060 CHEBI:6636 37351 37356 MgCl2 +T1061 CHEBI:32588 37364 37367 KCl +T1062 CHEBI:17754 37389 37398 glycerine +T1063 CHEBI:18320 37407 37410 DTT +T1064 CHEBI:8102 37419 37423 PMSF +T1065 GO:0005634 37425 37431 nuclei +T1066 CHEBI:46756 37552 37557 Hepes +T1067 CHEBI:6636 37574 37579 MgCl2 +T1068 CHEBI:32588 37588 37591 KCl +T1069 CHEBI:17754 37613 37622 glycerine +T1070 CHEBI:18320 37631 37634 DTT +T1071 CHEBI:8102 37643 37647 PMSF +T1072 CHEBI:63016 37652 37656 NP40 +T1073 CHEBI:60004 37677 37685 cocktail +T1074 CHEBI:75958 37703 37711 solution +T1075 CHEBI:75958 37780 37788 solution +T1076 GO:0005654 37871 37882 nucleoplasm +T1077 GO:0005654 37957 37968 Nucleoplasm +T1078 GO:0005634 37973 37980 nuclear +T1079 CHEBI:8984 38006 38009 SDS +T1080 GO:0019835 38089 38094 lysed +T1081 CHEBI:8984 38098 38101 SDS +T1082 CHEBI:8984 38150 38153 SDS +T1083 MOP:0000779 38244 38251 coupled +T1084 GO:0042571 38262 38272 antibodies +T1085 UBERON:0000479 38391 38397 Tissue +T1086 PR:000004080 38429 38439 Annexin A7 +T1087 NCBITaxon:10088 38448 38453 mouse +T1088 CL:0002322 38454 38462 ES cells +T1089 UBERON:0000922 38467 38474 embryos +T1090 UBERON:0000922 38539 38545 Embryo +T1091 UBERON:0000479 38555 38561 Tissue +T1092 NCBITaxon:10088 38731 38736 mouse +T1093 CL:0002322 38759 38767 ES cells +T1094 GO:0097617 38830 38840 hybridized +T1095 CHEBI:37972 38865 38868 32P +T1096 PR:000004080 38896 38906 annexin A7 +T1097 PR:000003676 38929 38936 β-actin +T1098 CHEBI:75958 38978 38986 solution +T1099 PR:000004080 39194 39204 Annexin A7 +T1100 GO:0042571 39205 39215 antibodies +T1101 UBERON:0000955 39220 39225 brain +T1102 PR:000004080 39242 39247 AnxA7 +T1103 NCBITaxon:10088 39258 39263 mouse +T1104 NCBITaxon:9606 39335 39340 human +T1105 UBERON:0000955 39349 39354 brain diff --git a/src/ontogpt/evaluation/craft/database/all/15819996.txt b/src/ontogpt/evaluation/craft/database/all/15819996.txt new file mode 100644 index 000000000..7b64157be --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15819996.txt @@ -0,0 +1,145 @@ +Nuclear localization of Annexin A7 during murine brain development + +Abstract + +Background + +Annexin A7 is a member of the annexin protein family, which is characterized by its ability to interact with phospholipids in the presence of Ca2+-ions and which is thought to function in Ca2+-homeostasis. Results from mutant mice showed altered Ca2+-wave propagation in astrocytes. As the appearance and distribution of Annexin A7 during brain development has not been investigated so far, we focused on the distribution of Annexin A7 protein during mouse embryogenesis in the developing central nervous system and in the adult mouse brain. + +Results + +Annexin A7 is expressed in cells of the developing brain where a change in its subcellular localization from cytoplasm to nucleus was observed. In the adult CNS, the subcellular distribution of Annexin A7 depends on the cell type. By immunohistochemistry analysis Annexin A7 was detected in the cytosol of undifferentiated cells at embryonic days E5–E8. At E11–E15 the protein is still present in the cytosol of cells predominantly located in the ventricular germinative zone surrounding the lateral ventricle. Later on, at embryonic day E16, Annexin A7 in cells of the intermediate and marginal zone of the neopallium translocates to the nucleus. Neuronal cells of all areas in the adult brain present Annexin A7 in the nucleus, whereas glial fibrillary acidic protein (GFAP)-positive astrocytes exhibit both, a cytoplasmic and nuclear staining. The presence of nuclear Annexin A7 was confirmed by extraction of the nucleoplasm from isolated nuclei obtained from neuronal and astroglial cell lines. + +Conclusion + +We have demonstrated a translocation of Annexin A7 to nuclei of cells in early murine brain development and the presence of Annexin A7 in nuclei of neuronal cells in the adult animal. The role of Annexin A7 in nuclei of differentiating and mature neuronal cells remains elusive. + +Background + +Annexins form a family of structurally related proteins, which bind to negatively charged phospholipids in a Ca2+-dependent manner [1]. They are characterized by a bipartite structure with a conserved C-terminal core domain and a unique N-terminal domain that varies in length and amino acid composition. The C-terminal domain is formed by either a four- or eightfold repeat of approximately 70 amino acids, each repeat carrying a Ca2+-binding site, and is responsible for phospholipid binding. The N-terminal regions are thought to confer functional diversity to the annexin proteins [2]. The biochemical features in vitro were analyzed extensively, but the in vivo functions of annexins remain unclear. + +Annexin A7, the first annexin to be described, was isolated as the agent that mediated aggregation of chromaffin granules and fusion of membranes and phospholipids in the presence of Ca2+-ions [3]. Expression studies demonstrated the distribution of Annexin A7 in a wide variety of tissues and cells mainly enriched in the cytosol in close association with membranous structures, but it was also described in the nucleus of adrenal chromaffin cells [4]. The presence of an alternatively spliced cassette exon gives rise to two Annexin A7 isoforms corresponding in molecular mass to 47 kDa and 51 kDa. The isoforms differ in their N-terminal domain and exhibit a tissue-specific expression pattern. The 47 kDa isoform is present in all tissues except for skeletal muscle, where the 51 kDa isoform is exclusively present. Heart muscle, brain tissue and red blood cells contain both isoforms [5-8]. Previous studies indicated that the subcellular localization of Annexin A7 changes during myoblast differentiation. In undifferentiated cells the protein is equally distributed between cytosol and membrane fractions while in differentiated cells it is exclusively present in the membrane fraction [7]. Reports by Clemen et al. [9] and Herr et al. [8,10] demonstrated roles for Annexin A7 in shape and osmotic resistance of red blood cells, platelet aggregation velocity, and in the velocity of spreading astrocytic Ca2+-waves. Annexin A7 is also involved in the maintenance of regular cardiac electrophysiology and Ca2+-homeostasis [Schrickel et al., submitted]. Detailed reports on appearance and distribution of Annexin A7 during brain development are not available. In the present study we focus on the distribution of Annexin A7 in the developing brain of mice embryos between E5 and E16, and in the adult mouse brain. + +Results + +Annexin A7 is expressed in the early mouse embryo + +First we examined the expression of Annexin A7 in ES cells (Bruce4, established from C57BL/6J mice) and the early stages of mouse embryonic development at the mRNA level by northern blot analysis and at the protein level by Western blotting and immunohistochemistry, respectively. Northern blot analysis shows in ES-cells and at embryonic days E7, E11, E15, and E17 two mRNA species of 1.8 kb and 2.4 kb, which result from alternative splicing in the untranslated 3'end (Fig. 1A) [11]. We found similar Annexin A7 mRNA levels in the four embryonic stages. Reprobing with a β-actin probe verified equal loading; the appearance of a faster migrating mRNA species in addition to the 2.0 kb species is characteristic for β-actin [12]. On the protein level, ES cells express only the smaller Annexin A7 isoform of 47 kDa (Fig. 1B, neuro-2a cells and heart tissue are included for control). + +Figure 1 + +Expression of Annexin A7. (A) Northern blot analysis of RNA from ES-cells and embryos E7, E11, E15, E17 probed with a full length annexin A7 cDNA. Transcripts of 1.8 and 2.4 kb are detected in all stages examined (upper panel). Reprobing with a β-actin probe verified equal loading (lower panel, 2.0 kb band). (B) Murine ES cells (ES) express only the small Annexin A7 isoform. Both isoforms are expressed in neuroblastoma neuro-2a cells (N2A), heart (HRT) and brain tissue (CNS) from adult mice. Proteins from neuro-2a and ES cells, heart and brain were resolved by SDS polyacrylamide gel electrophoresis (12% acrylamide) and transferred to nitrocellulose membranes. The Western blot was probed with mAb 203–217 followed by incubation with a peroxidase coupled secondary antibody. Detection was with enhanced chemiluminescence. + +Immunohistochemistry confirmed the presence of the protein during early development. Annexin A7 immunoreactivity using mAb 203–217, which specifically recognizes both isoforms of Annexin A7, can be observed in the two cell types of the cylinder stage at E5 with a weaker Annexin A7 expression in the ectoderm (Fig. 2A–C). At E8 Annexin A7 can be detected in almost all tissues of the embryo (data not shown). A correlation between the presence of Annexin A7 and the origin of a cell type from one of the three germ layers is not apparent. We specifically noted the localization of the protein in the neuroepithelium of the neural fold and tube (Fig. 2D–F, proximal, E8; Fig. 2G–I, distal, E13). At the subcellular level, Annexin A7 was present in punctate structures mainly in the cytosol of all examined cells (Fig. 2E,F). This distribution was also observed during subsequent development. + +Figure 2 + +Annexin A7 immunoreactivity in early mouse embryos. (A) Phase contrast, embryo E5: The egg cylinder consists of an inner cell mass (a) representing the ectoderm and an outer layer of endoderm cells (b). (B) Immunostaining of the paraffin section was performed using purified mAb 203–217 and Cy3-conjugated anti-mouse IgG. Annexin A7 is expressed in both cell types of the egg cylinder with a strong staining of the endoderm and a weaker staining of the ectoderm. The nuclei are devoid of immune reactions. (C) Negative control using the secondary Cy3-antibody only. (D-F) Annexin A7 expression in the proximal neural tube (D) and nearby neural fold (E,F), embryo E8, transverse section. Immunolabeling of Annexin A7 was performed with purified mAb 203–217 and visualization was with an Alexa Fluor 488-conjugated anti-mouse IgG. (D) An intense Annexin A7 immunostaining is detectable in the neuroepithelium of the neural tube (a, lumen of neural tube). (E,F) Higher magnifications of the neuroepithelium show that Annexin A7 is expressed in the cytosol. Arrowheads point to Annexin A7 immunoreactivity in the cytosol. (G) Phase contrast, embryo E13, caudal neural tube. (H) Immunostaining of the paraffin section was performed using purified mAb 203–217 and Alexa Fluor 488-conjugated anti-mouse IgG. (I) Negative control using the secondary Alexa Fluor 488-antibody only. Bar, 20 μm. + +Annexin A7 changes its subcellular localization in the embryonic brain at E16 + +Next we analysed the cellular and subcellular distribution of Annexin A7 in the developing mouse brain (Fig. 3). At E13–E16 in transverse sections of the embryonic brain, most of the immunoreactive cells are present in the ventricular germinative zone surrounding the lateral ventricle (Fig. 3A, overview E16; Fig. 3H, overview E13). A closer inspection revealed that Annexin A7 is mainly localized in the cytosol at E13 (Fig. 3B,E). During further development the immature cells have rounded up two days later, but they retain Annexin A7 in the cytosol at E15 (Fig. 3C,F). At E16 we observed the first prominent nuclear staining of Annexin A7 (Fig. 3D,G) in cells of the intermediate zone (Fig. 3I, oval), which contains neurons radially migrating towards the growing neopallium cortex [13,14] and also glial cells forming the white matter of the adult cortex. Cells located marginally in the neopallial cortex also exhibit a nuclear stain. Labelling in the ventricular germinative zone is less prominent and the cytosol is no longer more strongly stained than the nucleus. + +Figure 3 + +Subcellular localization of Annexin A7 in embryos E13, E15 and E16. Paraffin sections of embryonic brain were stained with purified mAb 203–217. Annexin A7 was visualized with Alexa Fluor 488-conjugated secondary antibody. (A) Overview, at E16 the immature GFAP-negative cells of the forming cerebral neocortex (b) surrounding the lateral ventricle (a) are strongly stained; square, a higher magnification of this area is given in (I). (B) Higher magnification of the earlier stage E13 (H) shows, that the cells are stained in the cytosol (arrowhead). (C) Two days later at E15 the cells have rounded up, and Annexin A7 stays in the cytosol. (D) At E16 the first nuclear staining becomes apparent (arrowhead) in cells of the intermediate zone located between ventricular germinative zone and marginal neopallial cortex as seen in (I), oval. (E-G) Confocal microscopy confirms the results in B-D. (H) Overview, at E13 the immature GFAP-negative cells are strongly stained; a, lateral ventricle. (I) Higher magnification of a cortical section of (A, square) demonstrating ventricular, intermediate, and marginal zones; oval, a higher magnification of this area is given in (D,G). + +Mature neurons in adult mouse brain show an intense nuclear staining of Annexin A7 + +Both Annexin A7 isoforms are found in adult brain tissue (Fig. 1B). In general, we observed two characteristic staining patterns for Annexin A7, a prominent nuclear stain in neurons (determined by lack of GFAP-immunoreactivity, localization, morphology) and a cytoplasmic and nuclear stain in astrocytes (GFAP-positive) in all areas of the mature murine brain. In the neocortex (isocortex) Annexin A7 was strongly enriched in nuclei of neurons of all six cortical laminae (Fig. 4A, section derived from cortex temporalis). For control, in the adult brain type-1 astrocytes can be characterized by a positive GFAP-stain (Fig. 4B). Fig. 4F demonstrates the presence of nuclear Annexin A7 in a neocortical neuron of the external granular layer (layer II) and in an astrocytic cell of the neocortical molecular layer (layer I) which additionally exhibits an intense signal in the cytoplasm and in cellular branches. A further cell type of the isocortex showed a strong immunoreactivity in both, nucleus and cytosol, and was identified as pyramidal neuron based on morphology and absence of GFAP immunoreactivity. However, Annexin A7 apparently is more enriched in the nucleus (Fig. 4E). + +Figure 4 + +Annexin A7 is present in neurons and astrocytes of the cortex temporalis and hippocampal formation of 10-weeks-old mice. (A) Low magnification of the cortex temporalis presents an Annexin A7 expression in cells of the pial border, in neurons of all six isocortical laminae, and a weak signal in the adjacent white matter. (B) Corresponding section stained with GFAP. (C) Staining in the Stratum pyramidalis (a) and in the dentate gyrus of the hippocampus; square, a higher magnification of acorresponding area is given in (G,H). An intense Annexin A7 immunostaining is detectable. (D) Corresponding section stained with secondary antibody only. (E) Presence of Annexin A7 in pyramidal neurons (lamina pyramidalis externa) of the isocortex temporalis. These neurons were identified based on their morphology, distribution and lack of GFAP staining. AnnexinA7 exhibits a punctate staining, which is pronounced in the nucleus (arrowhead). (F) Higher magnification of image (A) also shows Annexin A7 in nuclei of neurons (lamina granularis externa (corpuscularis), arrowhead) and in the cytoplasm and nuclei of astrocytes (lamina molecularis, arrow; GFAP-confirmed). (G) Higher magnification of the pyramidal neurons in the hippocampus confirms the presence of the Annexin A7 protein in the nucleus (arrowhead) of mature neurons. (H) To further confirm this, a similar section derived from an AnxA7-/- mouse was stained with the annexin specific antibody and lacked the nuclear signal. The residual stain of the tissue is unspecific, as it is also observed in controls of the AnxA7-/- brain omitting the primary antibody (data not shown). All paraffin sections were stained with mAb 203–217 (A, C, E, F, G) or anti-GFAP-antibody (B). The hippocampal control section (D) lacks the primary antibody. + +In the hippocampal formation we detected prominent Annexin A7 immunostaining in the stratum pyramidalis and in the dentate gyrus and also a weak astrocytic staining (Fig. 4C). The astrocytes are mainly localized in the area between cells exhibiting nuclear Annexin A7 staining and show the protein also in the nucleus and the cytoplasm (data not shown). When we analyzed the pyramidal neurons in the hippocampus at a higher magnification we found that Annexin A7 again mainly localized to the nuclei (Fig. 4G). Polyclonal antibodies gave a similar staining pattern (data not shown). The intense nuclear staining was absent in controls either stained only with the secondary antibody (Fig. 4D) or in brain sections derived from AnxA7-/- mice stained for Annexin A7 (Fig. 4H). The residual staining seen in the AnxA7-/- brain is unspecific as it is also present in corresponding negative controls lacking the primary antibody. The absence of Annexin A7 protein in brain and other tissues of the AnxA7-/- mouse has been verified in [10]. In another actual study of the knock out mouse we detected a thickened basal membrane and a widened intercellular space. This may cause unspecific binding of the secondary antibody. + +An equal staining of Annexin A7 was also found in the cerebellum (Fig. 5A). Nuclei of neurons in the stratum granulosum show the most prominent staining (Fig. 5C,D). In the stratum moleculare, which is poor in cell bodies of neurons, only a few dots appear which also correspond to nuclei of neurons. Astrocytes are also positive for Annexin A7 in the nuclei and cytoplasm (data not shown). The signal of the pial border and the prominent stain of the white matter tracts (laminae medullares) are due to Annexin A7 positive astrocytes (Fig. 5 A–C). Higher magnification of the boundary layer between stratum moleculare and granulosum (Fig. 5D) revealed an Annexin A7 signal in nuclei, but also in the cytoplasm and at the plasma membrane of Purkinje cells (Fig. 5E). The typical pair of dendrites points to the margin of the cerebellar cortex. The specificity of the Annexin A7 signal was further confirmed in similar brain sections of the AnxA7-/- mouse (Fig. 5F). Fig. 5G shows Annexin A7 in neurites (axons) connecting the laminae medullares with the Purkinje-cell layer. Most likely these are the axons of the Purkinje-cells. Apart from these efferent neurites the laminae medullares (lamina alba, white matter) contain afferent mossy and climbing fibers. Thus, the intense stain of the cerebellar white matter arises from Annexin A7 located in astrocytes and neurites. + +Figure 5 + +Annexin A7 immunostaining in the cerebellum of adult mice. (A) Low magnification of the cerebellum presents an Annexin A7 expression mainly in cells of the stratum granulosum and laminae medullares. (B) Corresponding section stained for GFAP. (C) Higher magnification of folia of the cerebellum, where a polyclonal anti-AnnexinA7 antibody was used; square, a higher magnification of acorresponding area stained with mAb 203–217 is given in (D). Between stratum granulosum and stratum moleculare the layer of Purkinje-cells (stratum neuronorum piriformium (ganglionare)) can be observed. (D) Staining of the band of Purkinje-cells (arrows). The positive Annexin A7-stain in the stratum granulosum is due to staining of the nuclei of neurons. (E) In addition to an intense staining of the nucleus, the cell body is AnnexinA7-positive including both dendrites of the Purkinje-cell shown. (F) Corresponding section from an AnxA7-/- mouse. (G) AnnexinA7 staining of axons (arrowheads) running from the laminae medullares to the Purkinje-cell layer located in the round end of a convolution. Sections A, D, E, F, G were stained with mAb 203–217. + +Expression of Annexin A7 in the adult human isocortex + +In the human parietal neocortex of aged individuals without any neuropathological alterations, subpial astrocytes exhibited a staining of Annexin A7 in the cytoplasm. A nuclear presence of Annexin A7 was limited to single astrocytes (Fig. 6B). Pyramidal neurons, predominantly those of layer V exhibited Annexin A7 at the plasma membrane of their perikaryon as well as of the apical dendrite (Fig. 6A,C). The neurons lacked a signal for Annexin A7 in their nuclei. Apical dendrites within the molecular layer also indicated a positive staining for Annexin A7 (Fig. 6B). The staining pattern of Annexin A7 in the human autopsy brain did not change after pre-treatment with trypsin. + +Figure 6 + +Annexin A7 immunostaining in human isocortex. (A) Pyramidal neurons (bold arrow) and apical dendrites (small arrows) were clearly labeled with the antibody against Annexin A7 in the human parietal cortex. (B) A dendritic staining was also seen in the molecular layer (small arrows). In addition the subpial astrocytes were weakly labeled (double headed arrows). The staining was cytoplasmic, only few astrocytes showed a nuclear staining as well (inset). (C) Enlargement of (A): Annexin A7 was seen at the cell membrane of the perikaryon (bold arrow) and the apical dendrite (small arrows). (D) A blank control lacking the primary antibody did not show a specific labeling, but autofluorescent lipofuscin was detectable in the neurons (arrowheads). Bar, (A) 70 μm, (B) 50 μm, (B-inset, C) 17 μm, (D) 20 μm. + +Presence of Annexin A7 in nuclei from neuronal and astroglial cells + +The nuclear localisation of Annexin A7 in mice observed in immature cells at E16 and differentiated adult neurons could be only detected as a strong signal when the brain sections were pre-treated with trypsin before antibody staining. This procedure is thought to allow the antibodies to access epitopes masked by the formaldehyde fixation. Methanol fixation of cultured cells led to inconsistent results. Antigen retrieval in formalin-fixed and paraffin-embedded tissue sections employs various heating or proteolytic pre-treatment methods [[15-17]; Ein Handbuch für die Histologie, dianova GmbH, Hamburg, Germany]. These methods can result in moderate or strong specific antibody staining, but the detectability of other antigens might be decreased. Moreover, the optimal pre-treatment has to be individualised for each antigen. + +To verify the presence of endogenous Annexin A7 in nuclei, we used a biochemical approach and purified nuclei from neuro-2a, PC-12, and C6 cells and treated them with a hypotonic extraction buffer to obtain the nucleoplasm (Fig. 7A). The nucleoplasm and the remaining nuclei, from which the nucleoplasm has been extracted partially, were subjected to SDS-PAGE and Western blotting. Western blots probed with mAb 203–217 showed Annexin A7 in the nucleoplasm of neuronal (neuro-2a, PC-12) and astroglial (C6) cell lines (Fig. 7A), however, the large isoform could only be clearly extracted with nucleoplasm from C6 cells. For control, antibodies against LAP2α, Emerin and tubulin were used to verify that the nucleoplasm (marker:LAP2α, which is anon-membrane-bound isoform of LAP2) was successfully extracted and also not contaminated by nuclear membranes (marker:Emerin) or by cytoplasm (marker:tubulin). Likewise, in these neuronal and astroglial cell lines Annexin A7 had been observed in the nucleus by immunofluorescence (Fig. 7B and data not shown). We noticed no difference between PC-12 and neuro-2a cells, however, as for the brain sections, we found an increase of the Annexin A7 staining after pre-treatment with trypsin. + +Figure 7 + +Nuclear localization of Annexin A7. (A) Extraction of Annexin A7 from nuclei of neuronal (PC-12, N2A) and an astroglial (C6) cell line using a hypotonic buffer. Samples of total cells (c), and corresponding amounts of nuclear membranes (m) and nucleoplasm (p) were subjected to SDS-PAGE and Western blotting. Annexin A7 (AnxA7) isoforms are extractable from the nucleus of the indicated cells (47 kDa and 51 kDa isoform, arrowhead). For control, immunoblotting of LAP2α (75 kDa; non-membrane-bound isoform), Emerin (EM, 34 kDa), and tubulin (TB, 55 kDa) which are specific for nucleoplasm (LAP2α), nuclear membranes (Emerin), and the cytoplasm (tubulin) are shown. Note that the nucleoplasm is only partially extractable. (B) Immunofluorescence images of the PC-12 cells used. Cells were fixed with paraformaldehyde, permeabilized with Triton X-100, pretreated with trypsin as indicated, and stained with Annexin A7 specific mAb 203–217, DAPI in blue, bar 10 μm. + +Discussion + +In the present study we explored the appearance of Annexin A7 during mouse development at the mRNA and protein level and focused on the central nervous system during embryogenesis. Northern blot and immunohistochemistry analysis show the expression of Annexin A7 in all embryonic tissues from day E5 on, the earliest day studied. Differentiating cells, like the neural ectoderm, which is the origin of the cells belonging to the nervous system, show Annexin A7 immunoreactivity mainly in the cytosol. Endodermal as well as mesodermal cells exhibit a similar subcellular localization of the protein. + +In the developing brain we noted a striking change in the subcellular distribution of Annexin A7. Cells in the stratum germinativum of the neopallial cortex, which surrounds the lateral ventricle, show at E13 and E15 a staining for Annexin A7 mainly in the cytosol. But this staining largely disappears and at the following day E16 we detect Annexin A7 in the nucleus of cells in the intermediate and marginal zone of the neopallium. The different expression patterns of Annexin A7-positive cells from the ventricular germinative zone to the marginal zone of the later neocortex observed in this study are similar to the developmental patterns of Tenascin-C-positive astroglial precursors following the guidance of the radial glial cells [18]. Moreover, the patterns resemble that of migrating and differentiated neurons described by Berry et al. [13]. Neurons that are generated prenatally in the proliferative ventricular layer of the neopallial cortex subsequently migrate through the intermediate zone to form the different cortical cell layers in a declining inside-out gradient of cell maturation. These observations suggested that the subcellular localization of Annexin A7 depends on developmental stage and cell type. + +In the adult brain we generally observed a nuclear localization for Annexin A7 in neurons whereas astrocytes exhibited both a cytosolic as well as a nuclear staining. However, pyramidal neurons of the isocortex and Purkinje-cells of the cerebellum exhibited a cytosolic stain of intermediate intensity including their neurites and dendrites. We have previously reported the expression of Annexin A7 in human temporal neocortex and hippocampus obtained from neurosurgery for therapy-refractory epilepsy and found the two Annexin A7 isoforms restricted to the cytoplasm and nuclei of astrocytes, whereas neurons lacked any signal [19]. The hippocampal area showed typical signs of Ammon's horn sclerosis, but the adjacent temporal neocortex tissue did not show any histopathological alterations. This data is in discrepancy with our actual observation in the murine brain. To determine if this is due to different methods in immunofluorescence staining or indeed different expression patterns in mouse and human, we repeated the immunofluorescence of human brain. This time we investigated sections from human parietal cortex of autopsy brain. The astroglial expression of Annexin A7 could be confirmed, although not all astrocytes exhibited the nuclear staining. Pyramidal neurons however indicated a distinct staining of Annexin A7 most prominent along their dendrites. The different results obtained for the neurons may be explained by the fact that we previously used frozen tissue sections or that they were derived from patients suffering from temporal lobe epilepsia. On the other hand the actual human brain tissue used was from aged patients and did not correspond to the age of the mice included in this study. In cultured cells after cell damage or apoptosis (unpublished observations) or in cells treated with a Ca2+-ionophore Annexin A7 translocated from the cytoplasm to cellular membranes [19]. We therefore favor the hypothesis that Annexin A7 in the sensitive neurons of the human autopsy brain may have similarly translocated to the cellular membrane. This property of translocation and membrane binding is common to all annexins and commercially available kits for apoptosis detection employ recombinant AnnexinA5. The presence of nuclear Annexin A7 in murine brain was confirmed by controls using sections from the AnxA7-/- mouse and by a biochemical extraction of the protein from the nucleus. Both Annexin A7 isoforms described in brain tissue seemingly are expressed by neurons and astrocytes, which was shown using total cell extracts of cultured neuo-2a, PC-12, and C6 cells. In addition to their expression in brain, both Annexin A7 isoforms have only been described in heart muscle and red blood cells [5-8]. + +Although the inactivation of the annexin A7 gene did not interfere with the viability and development of knock out mice [10], their generation allowed us to address the role of Annexin A7 in specific cell types [8,9]. Indeed, when we analyzed primary astrocytes from an AnxA7-/- mouse for Ca2+-dependent signaling processes, we found that they exhibited a significantly increased velocity of mechanically induced astrocytic Ca2+-waves as compared to wild type [9]. This led us to propose, that Annexin A7 can act as a Ca2+-buffer and is involved in Ca2+-homeostasis. In neurons Ca2+ ions play major roles in various physiological and pathophysiological processes [20-25]. One can speculate about an involvement of Annexin A7 in the regulation of these Ca2+-dependent processes, propositions that need further investigation. However, such roles are confirmed for heart function by studies of Schrickel et al. [submitted], who described an involvement of Annexin A7 in the maintenance of a regular cardiac electrophysiology and Ca2+-homeostasis. + +An altered subcellular location during embryogenesis was also reported for Annexin A11. The developing gray matter of the rat embryonic spinal cord exhibited primarily nuclear localization of Annexin A11, while immunoreactivity was lost from the nuclei in the adult spinal cord [26]. In contrast, our studies show a relocation of Annexin A7 from the cytosol to the nucleus in cells of the embryonic neuronal tissue. The Annexin A7 distribution is determined by a variety of factors. An important one appears to be Ca2+, which promotes binding of Annexin A7 to membranes and also allows aggregation of annexins [27]. Binding partners of Annexin A7 such as sorcin might represent additional factors [28]. Although the members of the annexin family are generally found at the plasma membrane, in the cytoplasm or in association with the cytoskeleton, Annexins A1, A2, A4, A5 and A11 have been described to be localized at least partially in the nucleus [26,29,30]. Studies with human foreskin fibroblasts demonstrated that Annexin A1, A4 and A5 are all present in the nucleus at higher concentration than in the cytosol [31]. Raising intracellular Ca2+ led to relocation of these annexins to the nuclear membrane. An important role for annexins in mediating the Ca2+-signal within the nuclei of the fibroblasts was proposed. These results mirror studies with stably transfected C6 cells, in which high intracellular Ca2+-concentrations induced a marked redistribution of Annexin A7 from its localization in the nucleoplasm to the nuclear membrane [19]. + +None of the annexins contains a typical nuclear localization signal and their mechanism of nuclear import remains to be elucidated. For Annexin A11 it was shown that nuclear localization is mediated by its N-terminal region, which also contains a binding side for the S100 protein calcyclin [29]. More recently Tomas and Moss [32] showed, that Annexin A11 and S100A6 assemble at the nuclear envelope during nuclear breakdown. Their role in this process is not known. In general it seems that the nuclear localization of the annexins is actively regulated. For example the nucleocytoplasmic compartmentalization of Annexin A2 is controlled by sequestration of the AnxA2/p11 complex modulated by phosphorylation and by a nuclear export signal found in the AnxA2 3–12 region [33]. One function of Annexin A2 in the nucleus, that appears not to involve binding of p11, has been suggested by its purification as part of a primer recognition protein complex that enhances DNA polymeraseα-activity in vitro [34]. The annexins may participate in a nuclear response to initial cell stimulation or to a Ca2+-transient, presumably by regulating DNA replication. For Annexin A7 such a pathway is very speculative at the moment. Future studies are however directed by these findings and will concentrate on the identification of the nuclear localization signal of Annexin A7 as well as on the role of Annexin A7 in the nuclear compartment. + +Conclusion + +In this article we report the translocation of Annexin A7 to nuclei of differentiating cells in the developing murine brain. In the adult brain Annexin A7 generally was detected in nuclei of neurons. Astrocytes, cerebellar Purkinje-cells and neocortical pyramidal neurons exhibited both, Annexin A7 in the cytosol and in the nuclei. Thus, the subcellular localisation of Annexin A7 depends on the developmental stage and the cell type. A role of Annexin A7 as well as of other annexins in nuclei remains speculative. The results obtained by immunofluorescence were confirmed by extraction of Annexin A7 from nuclei of neuronal and glial cell lines. + +Methods + +Animals and tissue preparation + +129SV strain mice were used. The day on which the vaginal plug was confirmed was defined as E0. All the animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23). Pregnant females were sacrificed by cervical dislocation at various intervals between E5 and E17. Before E9, embryos were embedded within the maternal uterus. After E9, embryos were removed from their membranes and embedded separately. Experiments were performed with four to six animals at each developmental stage. The mature brain used was derived from mice of three to four month of age. The embryos and tissues were fixed in freshly prepared 4% paraformaldehyde solution for 2–12 h depending on the tissue thickness [35]. They were dehydrated with ethanol and embedded in paraffin according to standard procedures. Microtome sections were cut at 7–10 μm, placed on slides and stored until further processing. + +Human brain tissue + +Five human autopsy brains of male and female patients without any neuropathological changes (age 61–77 years; post mortem interval: 12–72 h) were fixed in an aqueous solution of formaldehyde for three weeks. Blocks of the parietal neocortex were embedded in paraffin and microtomed at 12 μm thickness. Sections were stained with aldehydefuchsin Darrow-red to confirm the absence of neuropathological alterations. All procedures were conducted in accordance with the Declaration of Helsinki and approved by the ethics committee of the University of Bonn Medical Center. + +Antibodies + +Antibodies used in this study were mAb 203–217 directed against the core domain of mouse Annexin A7 [6] (purified IgG, 1:50) specifically recognizing both Annexin A7 isoforms [[6,10], and data not shown], a polyclonal antibody against mouse Annexin A7 [10] (1:200), a polyclonal antiserum raised against the astrocyte-specific intermediate filament glial fibrillary acidic protein (GFAP; DAKO, 1:400), anti-β-tubulin (Sigma, 1:1000), anti-Emerin (novocastra, 1:2000), and polyclonal anti-LAP2α (kindly provided by Ronald Foisner, 1:5000). + +Immunofluorescence microscopy + +Immunohistochemical staining using mAb 203–217 as the primary antibody was with AlexaFluor488-conjugated goat anti-mouse IgG (Molecular Probes, Leiden, The Nether-lands) or Cy3-labeled goat anti-mouse IgG as the secondary antibodies. Polyclonal anti-Annexin A7 antibody and anti-GFAP-antibody were combined with Alexa Fluor 568-conjugated goat anti-rabbit IgG (Molecular Probes). The specific labelling of Annexin A7 detected with the anti-mouse-IgG secondary antibodies in mouse tissue sections was confirmed using primary antibodies, which were directly coupled to Alexa Fluor488. + +The paraffin-embedded sections were first treated three times for 2 min in xylene to remove paraffin and then rehydrated step by step with descending concentrations of ethanol for antibody staining [35]. After three washings in PBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.5 mM KH2PO4, pH 7.4), the tissue sections were incubated in PBS containing 0.1% trypsin for 10 min at room temperature to free formaldehyde-crosslinked epitopes [15-17]. After additional three washes with PBS, non-specific binding sites were blocked by incubating the slides with PBG (0.5 % BSA, 0.045 % fish gelatine in PBS) in 10% normal goat serum (NGS) for 2 h. Incubation with the primary antibodies was performed at 4°C overnight diluted in PBG supplemented with 1 % NGS. After five washes in PBG for 5 min each, the slides were incubated for 1 h with the secondary antibody at room temperature, washed three times in PBG and three times in PBS, 5 min each, rinsed in water and embedded in gelvatol [36]. For control, sections were incubated only with the secondary antibody or sections obtained from an annexin A7 knockout mouse were used [10]. Samples were examined with a confocal laser scan microscope (Leica TCS SP; Leica, Bensheim, FRG). + +Immunofluorescence microscopy of human brain + +The paraffin-embedded human brain sections were deparaffinzed likewise. Immunostaining for Annexin A7 was performed using the monoclonal antibody mAb 203–217. Microwave pre-treatment was used for all sections [15]. Trypsin pre-treatment was used as indicated. Carbocyanine 2 and 3 labeled anti-mouse IgG secondary antibodies (Dianova, Hamburg, Germany) were used to detect the primary antibody and showed similar staining patterns. Blank controls were performed, too. + +Cell culture and growth conditions + +The cell lines used were cultured according to the instructions provided by ATCC: murine neuroblastoma neuro-2a (ATCC CCL-131), rat pheochromocytoma PC-12 (ATCC CRL-1721), and rat glioblastoma C6 (ATCC CCL-107). One day before fixation with 4%paraformaldehyde and permeabilization with 0.5% Triton X-100, cells were seeded on coverslips. Immunostaining was performed as described for the tissue sections with or without the trypsin treatment. + +Extraction of nuclear Annexin A7 + +Cultured cells were trypsinized, counted and washed once in PBS. They were resuspended in buffer1 (0.32 M sucrose, 10 mM Tris, pH8.0, 3 mM CaCl2, 2 mM Mg-acetate, 0.1 mM EDTA, 0.5% NP40, 1 mM DTT, 0.5 mM PMSF; 1 × 107cells/400μl buffer) and sonified on ice (sonifier UP200S, dr.hielscher, 40 s, amplitude 50%, cycle 0.5). This step was repeated until no more intact cells were observed by light microscopy. The resulting homogenate was centrifuged at 500 g for 5 minutes, washed twice in 1 ml buffer 1 without NP40 and centrifuged again. The resulting pellet contains purified nuclei. For partial extraction of the nucleoplasm according to [37], nuclei were resuspended in buffer 2 (20 mM Hepes, pH 7.9, 1.5 mM MgCl2, 20 mM KCl, 0.2 mM EDTA, 25%v/v glycerine, 0.5 mM DTT, 0.5 mM PMSF; nuclei of 107cells/30 μl buffer) and incubated on ice for 30 minutes. An equal volume of buffer 3 was added in aliquots (20 mM Hepes, pH 7.9, 1.5 mM MgCl2, 800 mM KCl, 0.2 mM EDTA, 25%v/v glycerine, 0.5 mM DTT, 0.5 mM PMSF, 1% NP40, protease inhibitor cocktail (Sigma)) and the solution was incubated while shaking for further 30 minutes on ice. Then the solution was centrifuged for 15 minutes at 14.000 g, the resulting supernatant was used as nucleoplasm, and the pellet was washed once and adjusted to an equal volume with PBS. Nucleoplasm and nuclear pellet were subjected to SDS-PAGE and Western blotting. + +Western blotting + +Cells or cellular fractions were lysed in SDS sample buffer and homogenates were subjected to SDS-PAGE. Western blotting was carried out using the method of Towbin et al. [38]. Peroxidase-coupled secondary antibodies (Sigma) and the supersignal enhanced chemiluminescence system (Pierce, Rockford, IL, USA) were used. + +Embryo Multiple Tissue Northern Blot + +The presence of Annexin A7 mRNA in mouse ES cells and embryos E7, E11, E15 and E17 was analyzed by northern blot analysis. An Embryo Multiple Tissue Northern Blot membrane was obtained from Clontech Laboratories (Heidelberg, FRG). It contained approximately 2 μg of poly(A)+ RNA per lane from four different stages of mouse development. RNA from ES cells was isolated with the RNeasy kit (Qiagen). The membranes were hybridized with a random-primed [α-32P]-dATP-labelled full length annexin A7 cDNA probe and with a β-actin specific probe for control in ExpressHyb solution according to the manufacturer's directions. + +Authors' contributions + +MR, SIRG, and CSC planned and carried out the experiments, evaluated the results, and drafted the manuscript. CH provided polyclonal anti-Annexin A7 antibodies and brain sections of the AnxA7 knock out mouse, and contributed to the analysis of data. DT provided the sections the human autopsy brain, analyzed them by immunofluorescence, and contributed to the analysis of data. AAN conceived of the studies and participated in its design. All authors read and approved the manuscript. + +Acknowledgements + +We thank Maria Stumpf, Rolf Müller and Berthold Gassen for technical assistance, Drs. Andreas Hasse and Rolf Schröder for helpful discussion and Bettina Lauss for help with the manuscript. This work is supported by the Center of Molecular Medicine Cologne. diff --git a/src/ontogpt/evaluation/craft/database/all/15836427.ann b/src/ontogpt/evaluation/craft/database/all/15836427.ann new file mode 100644 index 000000000..3681cc412 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15836427.ann @@ -0,0 +1,1670 @@ +T1 UBERON:0000044 42 45 DRG +T2 CL:0000540 46 53 Neurons +T3 PR:000007227 140 144 Pea3 +T4 UBERON:0000044 188 208 dorsal root ganglion +T5 CL:1001451 188 208;215 222;240 247 dorsal root ganglion ... sensory ... neurons +T6 UBERON:0000044 210 213 DRG +T7 CL:1001451 210 213;215 222;240 247 DRG ... sensory ... neurons +T8 UBERON:0001130 227 233 spinal +T9 CL:0011001 227 247 spinal motor neurons +T10 GO:0010467 281 291 expression +T11 GO:0065007 292 300 controls +T12 CL:0000540 317 325 neuronal +T13 SO:0000704 422 426 gene +T14 GO:0010467 422 437 gene expression +T15 CL:0000101 477 491 sensory neuron +T16 SO:0000704 520 527 genetic +T17 NCBITaxon:10088 544 549 mouse +T18 GO:0010467 570 580 expression +T19 UBERON:0000044 584 587 DRG +T20 CL:1001451 584 603 DRG sensory neurons +T21 GO:0030424 613 631 axonal projections +T22 UBERON:0000044 769 772 DRG +T23 CL:1001451 769 788 DRG sensory neurons +T24 CL:0000540 933 941 neuronal +T25 GO:0042551 933 952 neuronal maturation +T26 CL:0000540 969 977 Neuronal +T27 CL:0000540 1047 1054 neurons +T28 GO:0010467 1055 1062 express +T29 GO:0007409 1146 1174 long-distance axon outgrowth +T30 GO:0030424 1160 1164 axon +T31 GO:0007416 1219 1233 synaptogenesis +T32 CL:0000540 1261 1269 neuronal +T33 GO:0010467 1314 1324 expression +T34 GO:0010467 1402 1412 expression +T35 GO:0007049 1430 1440 cell cycle +T36 GO:0010467 1475 1485 expression +T37 GO:0007049 1574 1584 cell cycle +T38 CL:0000540 1634 1642 neuronal +T39 UBERON:0024914 1634 1642;1663 1670 neuronal ... circuit +T40 CL:1001451 1728 1766 sensory neurons of dorsal root ganglia +T41 CL:1001451 1728 1746;1768 1771 sensory neurons of ... DRG +T42 UBERON:0000044 1747 1766 dorsal root ganglia +T43 UBERON:0000044 1768 1771 DRG +T44 CL:0000540 1848 1856 neuronal +T45 GO:0065007 1947 1954 control +T46 CL:0000540 1958 1966 neuronal +T47 GO:0065007 2066 2076 regulating +T48 CL:0000540 2094 2102 neuronal +T49 SO:0000704 2143 2150 genetic +T50 NCBITaxon:10088 2276 2280 mice +T51 PR:000021998 2299 2311 neurotrophin +T52 GO:0038179 2299 2321 neurotrophin signaling +T53 SO:0000704 2362 2366 gene +T54 PR:000002193 2367 2370 Bax +T55 PR:000021998 2425 2437 neurotrophin +T56 GO:0038179 2425 2447 neurotrophin signaling +T57 GO:0065007 2448 2456 controls +T58 GO:0019233 2498 2509 nociceptive +T59 CL:0000198 2498 2509;2514 2521 nociceptive ... neurons +T60 UBERON:0000044 2510 2513 DRG +T61 GO:0065007 2530 2537 control +T62 CL:0000540 2614 2621 neurons +T63 CL:0000540 2672 2679 neurons +T64 GO:0007049 2689 2699 cell cycle +T65 NCBITaxon:7215 2736 2746 Drosophila +T66 NCBITaxon:7742 2751 2762 vertebrates +T67 NCBITaxon:7215 2874 2884 Drosophila +T68 PR:000000034 2897 2900 BMP +T69 GO:0065007 2932 2939 control +T70 CL:0000110 2991 3010 peptidergic neurons +T71 GO:0010467 3011 3021 expressing +T72 PR:P29673 3022 3030 Apterous +T73 PR:Q9VDZ3 3035 3042 Squeeze +T74 NCBITaxon:7742 3055 3066 vertebrates +T75 GO:0010467 3139 3149 expression +T76 PR:000007223 3183 3187 Er81 +T77 PR:000007227 3192 3196 Pea3 +T78 UBERON:0000044 3200 3203 DRG +T79 CL:1001451 3200 3219 DRG sensory neurons +T80 CL:0000100 3224 3236 motor neuron +T81 CL:0000540 3268 3275 neurons +T82 GO:0007067 3293 3300 mitotic +T83 PR:000007223 3345 3349 Er81 +T84 GO:0010467 3350 3360 expression +T85 GO:0019230 3364 3378 proprioceptive +T86 PR:000011459 3427 3441 neurotrophin 3 +T87 PR:000011459 3443 3447 NT-3 +T88 GO:0065007 3477 3484 control +T89 UBERON:0001130 3501 3507 spinal +T90 GO:0045202 3512 3520 synaptic +T91 UBERON:0024914 3521 3528 circuit +T92 PR:000007223 3544 3548 Er81 +T93 GO:0019230 3559 3581 proprioceptive sensory +T94 CL:1001451 3559 3588 proprioceptive sensory neuron +T95 PR:000007227 3609 3613 Pea3 +T96 CL:0000100 3624 3636 motor neuron +T97 PR:000007223 3714 3718 Er81 +T98 SO:0000704 3748 3752 gene +T99 PR:000021998 3785 3797 neurotrophin +T100 GO:0038179 3785 3807 neurotrophin signaling +T101 GO:0019230 3818 3832 proprioceptive +T102 UBERON:0002257 3862 3881 ventral spinal cord +T103 GO:0045202 3904 3924 synaptic connections +T104 CL:0000100 3930 3943 motor neurons +T105 GO:0010467 4036 4046 expression +T106 CL:0000540 4141 4149 neuronal +T107 GO:0042551 4141 4160 neuronal maturation +T108 GO:0010467 4179 4189 expression +T109 GO:0007067 4214 4221 mitotic +T110 CL:0000540 4222 4229 neurons +T111 CL:0000101 4258 4272 sensory neuron +T112 NCBITaxon:10088 4325 4330 mouse +T113 CL:0000101 4482 4496 sensory neuron +T114 GO:0019230 4655 4677 proprioceptive sensory +T115 CL:1001451 4655 4685 proprioceptive sensory neurons +T116 GO:0042995 4781 4792 projections +T117 UBERON:0002240 4800 4811 spinal cord +T118 GO:0007067 4860 4867 mitotic +T119 UBERON:0000044 4868 4871 DRG +T120 CL:0000540 4872 4879 neurons +T121 UBERON:0000044 4898 4901 DRG +T122 CL:0000540 4902 4908 neuron +T123 PR:000021998 4942 4954 neurotrophin +T124 GO:0043005 4967 4974 neurite +T125 SO:0000704 5015 5019 gene +T126 GO:0010467 5015 5030 gene expression +T127 GO:0010467 5149 5159 expression +T128 CL:0000540 5260 5268 neuronal +T129 GO:0042551 5260 5279 neuronal maturation +T130 UBERON:0024914 5260 5268;5284 5291 neuronal ... circuit +T131 GO:0014041 5417 5427;5440 5459 control of ... neuronal maturation +T132 CL:0000540 5440 5448 neuronal +T133 GO:0019230 5491 5505 proprioceptive +T134 CL:1001451 5491 5517 proprioceptive DRG neurons +T135 UBERON:0000044 5506 5509 DRG +T136 CHEBI:35224 5541 5550 effectors +T137 GO:0065007 5551 5560 regulated +T138 CL:0000540 5675 5682 neurons +T139 PR:000007223 5684 5688 Er81 +T140 GO:0065007 5689 5697 controls +T141 GO:0019230 5698 5712 proprioceptive +T142 GO:0010467 5821 5830 expressed +T143 PR:000007223 5862 5866 Er81 +T144 GO:0010467 5867 5877 expression +T145 PR:000007223 5905 5909 Er81 +T146 CL:0000101 5935 5959 afferent sensory neurons +T147 GO:0007067 6059 6066 mitotic +T148 GO:0010467 6067 6077 expression +T149 CL:0000101 6118 6132 sensory neuron +T150 PR:000007243 6151 6154 EWS +T151 PR:000007227 6155 6159 Pea3 +T152 PR:000007223 6172 6176 Er81 +T153 GO:0065007 6189 6200 Controlling +T154 GO:0042995 6213 6224 Projections +T155 PR:000007223 6314 6318 Er81 +T156 GO:0019230 6326 6340 proprioceptive +T157 GO:0042995 6361 6372 projections +T158 UBERON:0002257 6382 6401 ventral spinal cord +T159 PR:000007223 6403 6407 Er81 +T160 PR:000007227 6409 6413 Pea3 +T161 PR:000007228 6419 6422 Erm +T162 PR:000007227 6438 6442 Pea3 +T163 PR:000007223 6623 6627 Er81 +T164 PR:000007227 6717 6721 Pea3 +T165 PR:000007228 6726 6729 Erm +T166 GO:0019230 6746 6760 proprioceptive +T167 GO:0042995 6770 6781 projections +T168 UBERON:0002257 6808 6823;6828 6839 ventral horn of ... spinal cord +T169 NCBITaxon:10088 6896 6900 mice +T170 PR:000007243 6924 6927 EWS +T171 PR:000007227 6928 6932 Pea3 +T172 SO:0001021 6936 6947 break-point +T173 SO:0000417 6990 6996 domain +T174 PR:000007243 7004 7017 Ewing sarcoma +T175 http://purl.obolibrary.org/obo/MONDO_0012817 7004 7017 Ewing sarcoma +T176 PR:000007243 7019 7022 EWS +T177 http://purl.obolibrary.org/obo/MONDO_0012817 7019 7022 EWS +T178 SO:0000704 7024 7028 gene +T179 PR:000007227 7037 7041 Pea3 +T180 SO:0000417 7054 7060 domain +T181 PR:000007223 7079 7083 Er81 +T182 GO:0009294 7158 7170 transfection +T183 PR:000007243 7178 7181 EWS +T184 PR:000007227 7182 7186 Pea3 +T185 PR:000007223 7233 7237 Er81 +T186 PR:000007227 7241 7245 Pea3 +T187 PR:000007243 7351 7354 EWS +T188 PR:000007227 7355 7359 Pea3 +T189 SO:0000409 7393 7406 binding sites +T190 SO:0000155 7423 7430 plasmid +T191 SO:0000409 7450 7462 binding-site +T192 GO:0010467 7493 7503 Expression +T193 PR:000007223 7507 7511 Er81 +T194 UBERON:0000044 7515 7518 DRG +T195 CL:0000540 7519 7526 neurons +T196 UBERON:0000922 7530 7537 embryos +T197 PR:000007243 7564 7567 EWS +T198 PR:000007227 7568 7572 Pea3 +T199 PR:000007223 7580 7584 Er81 +T200 PR:000007223 7592 7596 Er81 +T201 PR:000007243 7596 7599 EWS +T202 PR:000007227 7600 7604 Pea3 +T203 GO:0010467 7643 7653 expression +T204 PR:000013502 7691 7702 Parvalbumin +T205 PR:000013502 7704 7706 PV +T206 GO:0019230 7711 7725 proprioceptive +T207 PR:000007223 7787 7791 Er81 +T208 PR:000007223 7844 7848 Er81 +T209 PR:000007243 7848 7851 EWS +T210 PR:000007227 7852 7856 Pea3 +T211 UBERON:0000922 7859 7866 embryos +T212 UBERON:0000044 7901 7904 DRG +T213 CL:0000540 7905 7911 neuron +T214 PR:000007243 7947 7950 EWS +T215 PR:000007227 7951 7955 Pea3 +T216 GO:0019230 7959 7973 proprioceptive +T217 PR:000007223 8028 8032 Er81 +T218 PR:000007243 8036 8039 EWS +T219 PR:000007227 8040 8044 Pea3 +T220 CL:0000540 8065 8073 neuronal +T221 GO:0010467 8093 8103 expression +T222 GO:0019230 8107 8121 proprioceptive +T223 SO:0000704 8140 8145 genes +T224 PR:000007223 8147 8151 Er81 +T225 PR:000007243 8151 8154 EWS +T226 PR:000007227 8155 8159 Pea3 +T227 NCBITaxon:10088 8162 8166 mice +T228 GO:0019230 8214 8228 proprioceptive +T229 CL:1001451 8214 8242 proprioceptive afferent cell +T230 GO:0044297 8238 8249 cell bodies +T231 UBERON:0000044 8261 8264 DRG +T232 GO:0010467 8282 8292 expression +T233 SO:0000704 8304 8309 genes +T234 GO:0010467 8319 8328 expressed +T235 GO:0019230 8332 8346 proprioceptive +T236 GO:0010467 8374 8384 expression +T237 SO:0000704 8388 8393 genes +T238 GO:0010467 8407 8416 expressed +T239 GO:0019230 8420 8434 proprioceptive +T240 GO:0010467 8516 8526 expression +T241 PR:000007243 8530 8533 EWS +T242 PR:000007227 8534 8538 Pea3 +T243 PR:000007223 8592 8596 Er81 +T244 SO:0000704 8641 8645 gene +T245 GO:0010467 8641 8656 gene expression +T246 GO:0019230 8664 8678 proprioceptive +T247 GO:0019230 8731 8745 proprioceptive +T248 GO:0042995 8755 8766 projections +T249 UBERON:0002257 8776 8795 ventral spinal cord +T250 PR:000007223 8799 8803 Er81 +T251 NCBITaxon:10088 8811 8815 mice +T252 GO:0010467 8828 8838 expression +T253 PR:000007243 8842 8845 EWS +T254 PR:000007227 8846 8850 Pea3 +T255 UBERON:0001130 8867 8873 spinal +T256 GO:0042995 8883 8894 projections +T257 GO:0030424 8898 8904 axonal +T258 PR:000013502 8917 8919 PV +T259 GO:0030424 8960 8964 axon +T260 PR:000013502 9002 9004 PV +T261 GO:0010467 9005 9015 expression +T262 UBERON:0000044 9019 9022 DRG +T263 CL:0000540 9023 9030 neurons +T264 CHEBI:52071 9114 9121 dextran +T265 UBERON:0002261 9129 9141 dorsal roots +T266 GO:0042995 9210 9221 projections +T267 UBERON:0002257 9231 9246;9251 9262 ventral horn of ... spinal cord +T268 PR:000007223 9266 9270 Er81 +T269 PR:000007243 9270 9273 EWS +T270 PR:000007227 9274 9278 Pea3 +T271 NCBITaxon:10088 9281 9285 mice +T272 UBERON:0002257 9317 9329 ventral horn +T273 PR:000007223 9366 9370 Er81 +T274 PR:000007243 9370 9373 EWS +T275 PR:000007227 9374 9378 Pea3 +T276 NCBITaxon:10088 9381 9385 mice +T277 PR:000014963 9393 9399 vGlut1 +T278 PR:000007223 9431 9435 Er81 +T279 NCBITaxon:10088 9443 9447 mice +T280 GO:0045202 9482 9490 synapses +T281 CL:0000100 9516 9529 motor neurons +T282 PR:000007223 9548 9552 Er81 +T283 PR:000007243 9552 9555 EWS +T284 PR:000007227 9556 9560 Pea3 +T285 NCBITaxon:10088 9563 9567 mice +T286 GO:0005622 9582 9595 intracellular +T287 UBERON:0001377 9623 9633 quadriceps +T288 CL:0000100 9634 9647 motor neurons +T289 UBERON:0001021 9669 9675 nerves +T290 GO:0060384 9676 9687 innervating +T291 UBERON:0001377 9692 9709 quadriceps muscle +T292 UBERON:0001015 9703 9715 muscle group +T293 UBERON:0001377 9778 9788 quadriceps +T294 CL:0000100 9789 9802 motor neurons +T295 PR:000007223 9831 9835 Er81 +T296 PR:000007243 9835 9838 EWS +T297 PR:000007227 9839 9843 Pea3 +T298 NCBITaxon:10088 9846 9850 mice +T299 PR:000007223 9897 9901 Er81 +T300 PR:000007243 9901 9904 EWS +T301 PR:000007227 9905 9909 Pea3 +T302 PR:000007223 9990 9994 Er81 +T303 PR:000007243 9996 9999 EWS +T304 PR:000007227 10000 10004 Pea3 +T305 GO:0008150 10028 10046 biological process +T306 UBERON:0000957 10058 10065 laminar +T307 UBERON:0002257 10089 10108 ventral spinal cord +T308 GO:0007416 10117 10138 formation of synapses +T309 GO:0045202 10130 10138 synapses +T310 CL:0000100 10144 10157 motor neurons +T311 GO:0010467 10246 10256 expression +T312 UBERON:0000044 10272 10275 DRG +T313 CL:0000540 10276 10283 neurons +T314 GO:0010467 10297 10307 Expression +T315 PR:000007243 10311 10314 EWS +T316 PR:000007227 10315 10319 Pea3 +T317 UBERON:0000044 10323 10326 DRG +T318 CL:0000540 10327 10334 Neurons +T319 GO:0030424 10344 10361 Axonal Projection +T320 GO:0019230 10431 10445 proprioceptive +T321 GO:0010467 10480 10489 expressed +T322 PR:000007243 10490 10493 EWS +T323 PR:000007227 10494 10498 Pea3 +T324 UBERON:0000044 10502 10505 DRG +T325 CL:0000540 10506 10513 neurons +T326 GO:0007067 10542 10549 mitotic +T327 NCBITaxon:10088 10568 10573 mouse +T328 SO:0000704 10574 10581 genetic +T329 SO:0005853 10658 10666 cassette +T330 SO:0000357 10667 10674 flanked +T331 SO:0000346 10678 10688 loxP sites +T332 SO:0005853 10700 10709 cassettes +T333 PR:000010173 10735 10738 Tau +T334 NCBITaxon:10088 10772 10776 mice +T335 GO:0010467 10791 10801 expressing +T336 PR:000007243 10809 10812 EWS +T337 PR:000007227 10813 10817 Pea3 +T338 GO:0016020 10823 10831 membrane +T339 GO:0030424 10883 10901 axonal projections +T340 UBERON:0000044 10905 10908 DRG +T341 CL:0000540 10909 10916 neurons +T342 UBERON:0000922 10936 10943 Embryos +T343 PR:000009116 10964 10968 Isl1 +T344 PR:000010173 10978 10981 Tau +T345 PR:000007243 10981 10984 EWS +T346 PR:000007227 10985 10989 Pea3 +T347 PR:000009116 10995 10999 Isl1 +T348 PR:000010173 11009 11012 Tau +T349 SO:0001023 11019 11026 alleles +T350 PR:000010173 11069 11072 Tau +T351 SO:0001023 11073 11079 allele +T352 UBERON:0000044 11102 11105 DRG +T353 CL:0000540 11106 11113 neurons +T354 GO:0019230 11125 11139 proprioceptive +T355 PR:000007243 11224 11227 EWS +T356 PR:000007227 11228 11232 Pea3 +T357 GO:0010467 11233 11243 expression +T358 GO:0007067 11258 11265 mitotic +T359 UBERON:0000044 11266 11269 DRG +T360 CL:0000540 11270 11277 neurons +T361 GO:0042995 11311 11322 projections +T362 UBERON:0002240 11332 11343 spinal cord +T363 PR:000010173 11354 11357 Tau +T364 SO:0001023 11364 11370 allele +T365 PR:000001843 11376 11380 Thy1 +T366 SO:0000167 11381 11389 promoter +T367 PR:000015890 11397 11410 synaptophysin +T368 GO:0010467 11453 11463 expression +T369 UBERON:0000044 11486 11489 DRG +T370 CL:1001451 11486 11505 DRG sensory neurons +T371 UBERON:0000922 11509 11518 embryonic +T372 PR:000001843 11533 11537 Thy1 +T373 GO:0019230 11587 11601 proprioceptive +T374 GO:0042995 11611 11622 projections +T375 PR:000010173 11665 11668 Tau +T376 PR:000007243 11668 11671 EWS +T377 PR:000007227 11672 11676 Pea3 +T378 PR:000009116 11679 11683 Isl1 +T379 UBERON:0000922 11689 11696 embryos +T380 UBERON:0002240 11718 11729 spinal cord +T381 UBERON:0002261 11791 11802 dorsal root +T382 GO:0042995 11947 11958 projections +T383 UBERON:0002261 11971 11982 dorsal root +T384 PR:000010173 11997 12000 Tau +T385 PR:000007243 12000 12003 EWS +T386 PR:000007227 12004 12008 Pea3 +T387 PR:000009116 12011 12015 Isl1 +T388 UBERON:0000922 12021 12028 embryos +T389 CHEBI:52071 12064 12071 dextran +T390 UBERON:0000044 12091 12094 DRG +T391 UBERON:0000922 12159 12166 embryos +T392 UBERON:0001130 12195 12201 spinal +T393 PR:000010173 12360 12363 Tau +T394 PR:000007243 12363 12366 EWS +T395 PR:000007227 12367 12371 Pea3 +T396 PR:000009116 12374 12378 Isl1 +T397 UBERON:0000922 12384 12391 embryos +T398 GO:0040007 12486 12490 grow +T399 GO:0042995 12551 12562 projections +T400 PR:000010173 12579 12582 Tau +T401 PR:000007243 12582 12585 EWS +T402 PR:000007227 12586 12590 Pea3 +T403 PR:000009116 12593 12597 Isl1 +T404 UBERON:0000922 12603 12610 embryos +T405 GO:0042995 12846 12857 projections +T406 PR:000007243 12874 12877 EWS +T407 PR:000007227 12878 12882 Pea3 +T408 GO:0010467 12883 12893 expression +T409 UBERON:0000044 12897 12900 DRG +T410 CL:0000540 12901 12908 neurons +T411 GO:0030424 12924 12929 axons +T412 PR:000010173 12933 12936 Tau +T413 PR:000007243 12936 12939 EWS +T414 PR:000007227 12940 12944 Pea3 +T415 PR:000009116 12947 12951 Isl1 +T416 UBERON:0000922 12957 12964 embryos +T417 UBERON:0002464 13004 13016 nerve trunks +T418 GO:0030424 13052 13056 axon +T419 UBERON:0003718 13183 13198 muscle spindles +T420 PR:000010173 13202 13205 Tau +T421 PR:000007243 13205 13208 EWS +T422 PR:000007227 13209 13213 Pea3 +T423 PR:000009116 13216 13220 Isl1 +T424 UBERON:0000922 13226 13233 embryos +T425 GO:0010467 13316 13326 expression +T426 SO:0000704 13330 13335 genes +T427 CL:0000187 13360 13373 muscle fibers +T428 PR:000006939 13382 13386 Egr3 +T429 GO:0010467 13453 13463 expression +T430 PR:000007243 13467 13470 EWS +T431 PR:000007227 13471 13475 Pea3 +T432 GO:0019230 13506 13520 proprioceptive +T433 GO:0042995 13530 13541 projections +T434 UBERON:0002257 13551 13570 ventral spinal cord +T435 GO:0010467 13583 13593 expression +T436 UBERON:0000044 13630 13633 DRG +T437 CL:0000540 13634 13641 neurons +T438 GO:0042995 13675 13686 projections +T439 UBERON:0002240 13696 13707 spinal cord +T440 PR:000007243 13754 13757 EWS +T441 PR:000007227 13758 13762 Pea3 +T442 GO:0010467 13763 13773 Expression +T443 PR:000021998 13783 13795 Neurotrophin +T444 GO:0043005 13821 13828 Neurite +T445 PR:000007243 13945 13948 EWS +T446 PR:000007227 13949 13953 Pea3 +T447 CL:0000540 14142 14150 neuronal +T448 GO:0043005 14173 14180 neurite +T449 UBERON:0000044 14194 14197 DRG +T450 CL:0000540 14198 14205 neurons +T451 UBERON:0000044 14330 14333 DRG +T452 PR:000010173 14362 14365 Tau +T453 PR:000007243 14365 14368 EWS +T454 PR:000007227 14369 14373 Pea3 +T455 PR:000009116 14376 14380 Isl1 +T456 UBERON:0000922 14386 14393 embryos +T457 PR:000011194 14413 14416 NGF +T458 PR:000011459 14420 14424 NT-3 +T459 PR:000021998 14446 14459 neurotrophins +T460 CL:0000540 14473 14481 neuronal +T461 GO:0043005 14495 14502 neurite +T462 UBERON:0000044 14605 14608 DRG +T463 CL:0000540 14609 14616 neurons +T464 UBERON:0000044 14672 14675 DRG +T465 CL:0000540 14709 14717 neuronal +T466 GO:0043005 14731 14738 neurite +T467 PR:000011194 14762 14765 NGF +T468 GO:0043005 14851 14858 neurite +T469 GO:0040007 14897 14902 grown +T470 PR:000011459 14922 14926 NT-3 +T471 GO:0019230 14955 14969 proprioceptive +T472 GO:0043005 15011 15018 neurite +T473 UBERON:0000044 15084 15087 DRG +T474 CL:0000540 15088 15095 neurons +T475 PR:000010173 15110 15113 Tau +T476 PR:000007243 15113 15116 EWS +T477 PR:000007227 15117 15121 Pea3 +T478 PR:000009116 15124 15128 Isl1 +T479 UBERON:0000922 15134 15141 embryos +T480 GO:0043005 15258 15266 neurites +T481 GO:0043005 15303 15310 neurite +T482 CL:0000540 15325 15333 neuronal +T483 PR:000011194 15393 15396 NGF +T484 PR:000011459 15400 15404 NT-3 +T485 PR:000021998 15446 15458 neurotrophin +T486 UBERON:0000044 15473 15476 DRG +T487 CL:0000540 15477 15484 neurons +T488 GO:0010467 15485 15495 expressing +T489 PR:000007243 15496 15499 EWS +T490 PR:000007227 15500 15504 Pea3 +T491 PR:000010173 15514 15517 Tau +T492 NCBITaxon:10088 15598 15602 mice +T493 GO:0010467 15631 15640 expressed +T494 PR:000013502 15650 15652 PV +T495 GO:0010467 15676 15686 expression +T496 PR:000010173 15697 15700 Tau +T497 PR:000013502 15707 15709 PV +T498 PR:000013502 15733 15735 PV +T499 GO:0019230 15737 15751 proprioceptive +T500 CL:1001451 15737 15763 proprioceptive DRG neurons +T501 UBERON:0000044 15752 15755 DRG +T502 GO:0010467 15790 15800 expression +T503 PR:000013502 15804 15806 PV +T504 UBERON:0000044 15886 15889 DRG +T505 PR:000010173 15904 15907 Tau +T506 PR:000007243 15907 15910 EWS +T507 PR:000007227 15911 15915 Pea3 +T508 PR:000013502 15918 15920 PV +T509 PR:000010173 15930 15933 Tau +T510 PR:000013502 15940 15942 PV +T511 NCBITaxon:10088 15948 15952 mice +T512 PR:000011459 16001 16005 NT-3 +T513 UBERON:0000044 16032 16035 DRG +T514 CL:0000540 16036 16043 neurons +T515 GO:0043005 16086 16094 neurites +T516 PR:000011459 16119 16123 NT-3 +T517 GO:0016265 16138 16142 died +T518 PR:000011459 16161 16165 NT-3 +T519 UBERON:0000044 16268 16271 DRG +T520 CL:0000540 16272 16279 neurons +T521 GO:0043005 16318 16325 neurite +T522 PR:000021998 16359 16371 neurotrophin +T523 GO:0038179 16359 16381 neurotrophin signaling +T524 UBERON:0000044 16413 16416 DRG +T525 CL:0000540 16440 16448 neuronal +T526 UBERON:0000044 16461 16464 DRG +T527 CL:0000540 16465 16472 neurons +T528 PR:000010173 16478 16481 Tau +T529 PR:000007243 16481 16484 EWS +T530 PR:000007227 16485 16489 Pea3 +T531 PR:000009116 16492 16496 Isl1 +T532 UBERON:0000922 16502 16509 embryos +T533 CL:0000540 16587 16595 neuronal +T534 UBERON:0000044 16619 16622 DRG +T535 NCBITaxon:10088 16637 16641 mice +T536 SO:0000704 16669 16673 gene +T537 PR:000002193 16674 16677 Bax +T538 PR:000002193 16718 16721 Bax +T539 UBERON:0000044 16725 16728 DRG +T540 CL:0000540 16729 16736 neurons +T541 GO:0043005 16794 16801 neurite +T542 PR:000002193 16815 16818 Bax +T543 UBERON:0000044 16822 16825 DRG +T544 CL:0000540 16826 16833 neurons +T545 UBERON:0000044 16893 16896 DRG +T546 PR:000010173 16902 16905 Tau +T547 PR:000007243 16905 16908 EWS +T548 PR:000007227 16909 16913 Pea3 +T549 PR:000009116 16916 16920 Isl1 +T550 UBERON:0000922 16926 16933 embryos +T551 PR:000002193 17001 17004 Bax +T552 UBERON:0000044 17008 17011 DRG +T553 CL:0000540 17012 17019 neurons +T554 PR:000021998 17146 17158 neurotrophin +T555 CL:0000540 17171 17179 neuronal +T556 GO:0010467 17190 17200 expression +T557 PR:000007243 17204 17207 EWS +T558 PR:000007227 17208 17212 Pea3 +T559 GO:0007067 17227 17234 mitotic +T560 CL:0000540 17235 17242 neurons +T561 GO:0043005 17257 17264 neurite +T562 PR:000021998 17280 17292 neurotrophin +T563 PR:000021998 17354 17366 neurotrophin +T564 GO:0038179 17354 17384 neurotrophin signaling cascade +T565 UBERON:0000044 17385 17388 DRG +T566 CL:0000540 17389 17396 neurons +T567 PR:000010173 17402 17405 Tau +T568 PR:000007243 17405 17408 EWS +T569 PR:000007227 17409 17413 Pea3 +T570 PR:000009116 17416 17420 Isl1 +T571 UBERON:0000922 17426 17433 embryos +T572 PR:000021998 17473 17486 neurotrophins +T573 GO:0010467 17503 17513 expression +T574 PR:000021998 17517 17529 neurotrophin +T575 PR:000010173 17543 17546 Tau +T576 PR:000007243 17546 17549 EWS +T577 PR:000007227 17550 17554 Pea3 +T578 PR:000009116 17557 17561 Isl1 +T579 UBERON:0000922 17567 17574 embryos +T580 GO:0010467 17595 17605 expression +T581 PR:000021998 17613 17625 neurotrophin +T582 PR:000011469 17636 17640 TrkA +T583 PR:000011470 17642 17646 TrkB +T584 PR:000011471 17652 17656 TrkC +T585 UBERON:0000044 17707 17710 DRG +T586 UBERON:0000922 17724 17731 embryos +T587 PR:000010173 17755 17758 Tau +T588 PR:000007243 17758 17761 EWS +T589 PR:000007227 17762 17766 Pea3 +T590 PR:000009116 17769 17773 Isl1 +T591 UBERON:0000922 17779 17786 embryos +T592 GO:0010467 17814 17824 expression +T593 PR:000011469 17828 17832 TrkA +T594 PR:000011470 17834 17838 TrkB +T595 PR:000011471 17844 17848 TrkC +T596 UBERON:0000044 17852 17855 DRG +T597 CL:0000540 17856 17863 neurons +T598 PR:000021997 17905 17917 Trk receptor +T599 GO:0010467 17918 17928 expression +T600 UBERON:0000044 17932 17935 DRG +T601 PR:000010173 17939 17942 Tau +T602 PR:000007243 17942 17945 EWS +T603 PR:000007227 17946 17950 Pea3 +T604 PR:000009116 17953 17957 Isl1 +T605 UBERON:0000922 17963 17970 embryos +T606 CL:0000540 18041 18048 neurons +T607 PR:000021997 18139 18151 Trk receptor +T608 GO:0010467 18152 18162 expression +T609 PR:000010173 18166 18169 Tau +T610 PR:000007243 18169 18172 EWS +T611 PR:000007227 18173 18177 Pea3 +T612 PR:000009116 18180 18184 Isl1 +T613 UBERON:0000922 18190 18197 embryos +T614 GO:0008219 18238 18248 cell death +T615 UBERON:0000044 18272 18275 DRG +T616 GO:0006915 18314 18323 apoptosis +T617 UBERON:0000922 18366 18373 embryos +T618 UBERON:0000044 18403 18406 DRG +T619 PR:000010173 18410 18413 Tau +T620 PR:000007243 18413 18416 EWS +T621 PR:000007227 18417 18421 Pea3 +T622 PR:000009116 18424 18428 Isl1 +T623 UBERON:0000922 18434 18441 embryos +T624 CL:0000540 18533 18540 neurons +T625 UBERON:0002836 18544 18554 lumbar DRG +T626 PR:000010173 18558 18561 Tau +T627 PR:000007243 18561 18564 EWS +T628 PR:000007227 18565 18569 Pea3 +T629 PR:000009116 18572 18576 Isl1 +T630 UBERON:0000922 18582 18589 embryos +T631 CHEBI:472552 18699 18703 BrdU +T632 UBERON:0000044 18759 18762 DRG +T633 CL:0000540 18763 18770 neurons +T634 PR:000010173 18774 18777 Tau +T635 PR:000007243 18777 18780 EWS +T636 PR:000007227 18781 18785 Pea3 +T637 PR:000009116 18788 18792 Isl1 +T638 UBERON:0000922 18798 18805 embryos +T639 GO:0007049 18818 18828 cell cycle +T640 CHEBI:472552 18833 18837 BrdU +T641 PR:000033987 18839 18843 LacZ +T642 UBERON:0000922 18858 18865 embryos +T643 UBERON:0000044 18993 18996 DRG +T644 CL:0000540 18997 19004 neurons +T645 PR:000010173 19010 19013 Tau +T646 PR:000007243 19013 19016 EWS +T647 PR:000007227 19017 19021 Pea3 +T648 PR:000009116 19024 19028 Isl1 +T649 UBERON:0000922 19034 19041 embryos +T650 GO:0007067 19054 19061 mitotic +T651 GO:0008219 19114 19124 cell death +T652 PR:000021997 19156 19169 Trk receptors +T653 GO:0010467 19237 19247 expression +T654 GO:0010941 19288 19301;19323 19333 regulation of ... cell death +T655 CL:0000540 19302 19310 neuronal +T656 UBERON:0000044 19355 19358 DRG +T657 PR:000010173 19362 19365 Tau +T658 PR:000007243 19365 19368 EWS +T659 PR:000007227 19369 19373 Pea3 +T660 PR:000009116 19376 19380 Isl1 +T661 UBERON:0000922 19386 19393 embryos +T662 PR:000029189 19456 19459 Akt +T663 PR:000029189 19462 19465 Akt +T664 UBERON:0000044 19484 19487 DRG +T665 CL:0000540 19561 19569 neuronal +T666 PR:000002307 19625 19629 Bcl2 +T667 PR:000002193 19644 19647 Bax +T668 GO:0010467 19715 19725 expression +T669 PR:000002307 19754 19758 Bcl2 +T670 PR:000002385 19774 19780 Bcl-xl +T671 PR:000002307 19785 19789 Bcl2 +T672 PR:000002307 19853 19857 Bcl2 +T673 PR:000002385 19865 19871 Bcl-xl +T674 CL:0000540 20001 20009 neuronal +T675 UBERON:0000044 20022 20025 DRG +T676 CL:0000540 20026 20033 neurons +T677 PR:000010173 20037 20040 Tau +T678 PR:000007243 20040 20043 EWS +T679 PR:000007227 20044 20048 Pea3 +T680 PR:000009116 20051 20055 Isl1 +T681 UBERON:0000922 20061 20068 embryos +T682 PR:000021997 20087 20099 Trk receptor +T683 GO:0010467 20100 20110 expression +T684 UBERON:0000044 20170 20173 DRG +T685 CL:0000540 20174 20181 Neurons +T686 CL:0000540 20198 20206 Neuronal +T687 CL:0000540 20253 20261 neuronal +T688 GO:0043005 20275 20282 neurite +T689 GO:0010467 20327 20337 expression +T690 PR:000007243 20341 20344 EWS +T691 PR:000007227 20345 20349 Pea3 +T692 SO:0000704 20406 20410 gene +T693 GO:0010467 20406 20421 gene expression +T694 NCBITaxon:10088 20430 20434 mice +T695 PR:000007243 20451 20454 EWS +T696 PR:000007227 20455 20459 Pea3 +T697 GO:0010467 20460 20470 expression +T698 NCBITaxon:10088 20475 20479 mice +T699 GO:0010467 20493 20503 expression +T700 PR:000007243 20507 20510 EWS +T701 PR:000007227 20511 20515 Pea3 +T702 UBERON:0000044 20532 20535 DRG +T703 CL:1001451 20532 20551 DRG sensory neurons +T704 PR:000007223 20585 20589 Er81 +T705 GO:0010467 20590 20600 expression +T706 SO:0000704 20695 20702 genetic +T707 GO:0010467 20723 20733 expression +T708 PR:000007243 20737 20740 EWS +T709 PR:000007227 20741 20745 Pea3 +T710 GO:0019230 20749 20763 proprioceptive +T711 PR:000007223 20822 20826 Er81 +T712 PR:000007243 20826 20829 EWS +T713 PR:000007227 20830 20834 Pea3 +T714 PR:000010173 20841 20844 Tau +T715 PR:000007243 20844 20847 EWS +T716 PR:000007227 20848 20852 Pea3 +T717 PR:000013502 20855 20857 PV +T718 UBERON:0000922 20863 20870 embryos +T719 GO:0010467 20891 20901 expression +T720 PR:000011471 20905 20909 TrkC +T721 SO:0000704 20913 20917 gene +T722 UBERON:0000044 20935 20938 DRG +T723 CL:0000540 20939 20946 neurons +T724 PR:000010173 20950 20953 Tau +T725 PR:000007243 20953 20956 EWS +T726 PR:000007227 20957 20961 Pea3 +T727 PR:000009116 20964 20968 Isl1 +T728 UBERON:0000922 20974 20981 embryos +T729 GO:0010467 21015 21025 expression +T730 PR:000011471 21029 21033 TrkC +T731 UBERON:0000044 21074 21077 DRG +T732 CL:0000540 21078 21085 neurons +T733 PR:000007223 21089 21093 Er81 +T734 PR:000007243 21093 21096 EWS +T735 PR:000007227 21097 21101 Pea3 +T736 PR:000010173 21108 21111 Tau +T737 PR:000007243 21111 21114 EWS +T738 PR:000007227 21115 21119 Pea3 +T739 PR:000013502 21122 21124 PV +T740 UBERON:0000922 21130 21137 embryos +T741 PR:000013502 21173 21175 PV +T742 GO:0010467 21184 21193 expressed +T743 UBERON:0000044 21197 21200 DRG +T744 CL:0000540 21201 21208 neurons +T745 PR:000010173 21212 21215 Tau +T746 PR:000007243 21215 21218 EWS +T747 PR:000007227 21219 21223 Pea3 +T748 PR:000009116 21226 21230 Isl1 +T749 UBERON:0000922 21236 21243 embryos +T750 GO:0010467 21264 21273 expressed +T751 GO:0019230 21277 21291 proprioceptive +T752 PR:000007223 21324 21328 Er81 +T753 PR:000007223 21328 21331 EWS +T754 PR:000007227 21332 21336 Pea3 +T755 UBERON:0000922 21339 21346 embryos +T756 SO:0000704 21398 21403 genes +T757 UBERON:0000044 21441 21444 DRG +T758 CL:0000540 21445 21452 neurons +T759 PR:000010173 21456 21459 Tau +T760 PR:000007243 21459 21462 EWS +T761 PR:000007227 21463 21467 Pea3 +T762 PR:000009116 21470 21474 Isl1 +T763 UBERON:0000922 21480 21487 embryos +T764 PR:000004968 21500 21510 Calretinin +T765 PR:000004967 21515 21524 Calbindin +T766 GO:0010467 21565 21574 expressed +T767 UBERON:0000044 21596 21599 DRG +T768 CL:0000540 21600 21607 neurons +T769 PR:000007223 21622 21626 Er81 +T770 PR:000007243 21626 21629 EWS +T771 PR:000007227 21630 21634 Pea3 +T772 PR:000010173 21642 21645 Tau +T773 PR:000007243 21645 21648 EWS +T774 PR:000007227 21649 21653 Pea3 +T775 PR:000013502 21656 21658 PV +T776 UBERON:0000922 21664 21671 embryos +T777 UBERON:0000044 21758 21761 DRG +T778 CL:0000540 21762 21769 neurons +T779 PR:000010173 21773 21776 Tau +T780 PR:000007243 21776 21779 EWS +T781 PR:000007227 21780 21784 Pea3 +T782 PR:000009116 21787 21791 Isl1 +T783 UBERON:0000922 21797 21804 embryos +T784 UBERON:0000044 21854 21857 DRG +T785 CL:0000540 21858 21865 neurons +T786 PR:000010173 21869 21872 Tau +T787 PR:000007243 21872 21875 EWS +T788 PR:000007227 21876 21880 Pea3 +T789 PR:000009116 21883 21887 Isl1 +T790 UBERON:0000922 21893 21900 embryos +T791 UBERON:0000044 22026 22029 DRG +T792 CL:0000540 22030 22037 neurons +T793 PR:000007243 22066 22069 EWS +T794 PR:000007227 22070 22074 Pea3 +T795 GO:0010467 22075 22084 expressed +T796 UBERON:0000044 22178 22181 DRG +T797 CL:0000540 22182 22189 neurons +T798 GO:0010467 22204 22214 expression +T799 PR:000007243 22218 22221 EWS +T800 PR:000007227 22222 22226 Pea3 +T801 PR:000010501 22233 22236 Hb9 +T802 NCBITaxon:10088 22240 22244 mice +T803 GO:0010467 22297 22307 expression +T804 PR:000010501 22311 22314 Hb9 +T805 UBERON:0003075 22318 22330 neural plate +T806 UBERON:0000044 22348 22351 DRG +T807 CL:0000540 22352 22359 neurons +T808 UBERON:0001460 22363 22371 brachial +T809 CL:0000540 22401 22408 neurons +T810 PR:000010173 22455 22458 Tau +T811 PR:000007243 22458 22461 EWS +T812 PR:000007227 22462 22466 Pea3 +T813 PR:000010501 22469 22472 Hb9 +T814 PR:000010173 22482 22485 Tau +T815 PR:000010501 22492 22495 Hb9 +T816 UBERON:0000922 22501 22508 embryos +T817 GO:0010629 22535 22552;22566 22576 downregulation of ... expression +T818 PR:000021997 22553 22565 Trk receptor +T819 PR:000004968 22596 22606 Calretinin +T820 CL:0000540 22636 22643 neurons +T821 PR:000010173 22704 22707 Tau +T822 PR:000010501 22714 22717 Hb9 +T823 UBERON:0000922 22723 22730 embryos +T824 GO:0010467 22891 22901 expression +T825 PR:000007243 22905 22908 EWS +T826 PR:000007227 22909 22913 Pea3 +T827 CL:0000540 22926 22933 neurons +T828 SO:0000704 23018 23022 gene +T829 GO:0010467 23018 23033 gene expression +T830 CL:0000540 23035 23043 neuronal +T831 GO:0043005 23058 23065 neurite +T832 GO:0010467 23210 23220 expression +T833 GO:0007067 23234 23241 mitotic +T834 CL:0000540 23242 23250 neuronal +T835 GO:0010467 23346 23356 expression +T836 CL:0000540 23396 23404 neuronal +T837 UBERON:0024914 23396 23404;23425 23432 neuronal ... circuit +T838 UBERON:0000044 23467 23470 DRG +T839 CL:0000540 23471 23478 neurons +T840 SO:0000704 23601 23605 gene +T841 GO:0010467 23601 23616 gene expression +T842 GO:0030424 23621 23627 axonal +T843 GO:0065007 23737 23747 controlled +T844 GO:0023056 23749 23764;23790 23799 upregulation of ... signaling +T845 GO:0065007 23846 23855 regulated +T846 CL:0000540 23935 23942 neurons +T847 CL:0000540 24010 24018 neuronal +T848 GO:0042551 24010 24029 neuronal maturation +T849 CL:0000540 24126 24134 neuronal +T850 PR:000021997 24218 24230 Trk receptor +T851 GO:0010467 24231 24241 expression +T852 PR:000007223 24301 24305 Er81 +T853 GO:0010467 24306 24316 expression +T854 CL:0000540 24345 24353 neuronal +T855 PR:000021998 24381 24394 neurotrophins +T856 GO:0010629 24421 24438;24444 24454 downregulation of ... expression +T857 PR:000011471 24439 24443 TrkC +T858 GO:0019230 24458 24472 proprioceptive +T859 GO:0007067 24626 24633 mitotic +T860 CL:0000540 24634 24642 neuronal +T861 PR:000021997 24667 24679 Trk receptor +T862 GO:0010467 24680 24690 expression +T863 GO:0030424 24787 24805 axonal projections +T864 PR:000011469 24822 24835 TrkA receptor +T865 PR:000002193 24849 24852 Bax +T866 NCBITaxon:10088 24860 24864 mice +T867 GO:0042995 24902 24913 projections +T868 GO:0042995 24971 24982 projections +T869 PR:000011459 25037 25041 NT-3 +T870 GO:0019230 25098 25112 proprioceptive +T871 GO:0042995 25122 25133 projections +T872 GO:0042995 25297 25308 projections +T873 UBERON:0000044 25317 25320 DRG +T874 CL:0000540 25321 25328 neurons +T875 PR:000007223 25344 25348 Er81 +T876 GO:0010467 25349 25359 expression +T877 GO:0019230 25363 25377 proprioceptive +T878 GO:0065007 25391 25401 controlled +T879 PR:000011459 25416 25420 NT-3 +T880 GO:0030424 25424 25429 axons +T881 UBERON:0005090 25459 25466 muscles +T882 GO:0019230 25513 25527 proprioceptive +T883 CL:1001451 25513 25535 proprioceptive neurons +T884 GO:0007067 25548 25555 mitotic +T885 GO:0010628 25607 25622;25648 25658 upregulation of ... expression +T886 CL:0000540 25680 25688 neuronal +T887 UBERON:0000044 25755 25758 DRG +T888 CL:1001451 25755 25774 DRG sensory neurons +T889 CL:0000100 25797 25809 motor neuron +T890 GO:0065007 25874 25885 controlling +T891 CL:0000540 25886 25894 neuronal +T892 GO:0042551 25886 25905 neuronal maturation +T893 CL:0000540 25923 25930 neurons +T894 NCBITaxon:7215 25934 25944 Drosophila +T895 GO:0010467 25955 25965 expression +T896 PR:000000034 25984 25987 BMP +T897 CL:0000540 26102 26109 neurons +T898 GO:0010467 26135 26145 expression +T899 PR:P29673 26179 26187 Apterous +T900 PR:Q9VDZ3 26192 26199 Squeeze +T901 NCBITaxon:7215 26223 26233 Drosophila +T902 NCBITaxon:7742 26238 26249 vertebrates +T903 GO:0010467 26315 26325 expression +T904 CL:0000540 26375 26383 neuronal +T905 GO:0042551 26375 26394 neuronal maturation +T906 UBERON:0000044 26447 26450 DRG +T907 CL:0000540 26451 26458 neurons +T908 GO:0065007 26514 26524 controlled +T909 CL:0000540 26619 26627 neuronal +T910 CL:0000540 26807 26815 neuronal +T911 GO:0010467 26911 26921 expression +T912 SO:0000704 27048 27053 genes +T913 CHEBI:23357 27076 27085 cofactors +T914 GO:0065007 27327 27334 control +T915 SO:0000704 27354 27359 genes +T916 CHEBI:23357 27479 27488 cofactors +T917 GO:0006412 27505 27518 translational +T918 PR:000007243 27559 27562 EWS +T919 PR:000007227 27568 27572 Pea3 +T920 CHEBI:23357 27629 27638 cofactors +T921 PR:000007243 27699 27702 EWS +T922 PR:000007227 27703 27707 Pea3 +T923 GO:0065007 27926 27936 regulation +T924 GO:0007067 27949 27956 mitotic +T925 UBERON:0000044 27957 27960 DRG +T926 CL:0000540 27961 27968 neurons +T927 GO:0065007 28082 28092 regulation +T928 CL:0000540 28096 28104 neuronal +T929 GO:0030424 28148 28166 axonal projections +T930 GO:0008283 28279 28292 proliferating +T931 GO:0065007 28407 28414 control +T932 SO:0000704 28442 28447 genes +T933 CL:0000540 28474 28482 neuronal +T934 NCBITaxon:7215 28508 28518 Drosophila +T935 CL:0000031 28519 28529 neuroblast +T936 GO:0014019 28519 28540 neuroblast generation +T937 PR:P05084 28567 28576 Hunchback +T938 GO:0065007 28577 28585 controls +T939 GO:0014018 28586 28599;28620 28622;28634 28645 specification ... of ... neuroblasts +T940 CL:0000031 28634 28645 neuroblasts +T941 CL:0000031 28672 28683 neuroblasts +T942 PR:P05084 28770 28779 Hunchback +T943 GO:0010467 28780 28790 expression +T944 GO:0030097 29004 29017 hematopoietic +T945 GO:0065007 29255 29262 control +T946 SO:0000704 29299 29304 genes +T947 CL:0000540 29335 29343 neuronal +T948 GO:0007067 29391 29398 mitotic +T949 CL:0000540 29399 29406 neurons +T950 GO:0065007 29486 29493 control +T951 CL:0000540 29510 29518 neuronal +T952 UBERON:0024914 29510 29527 neuronal circuits +T953 NCBITaxon:10088 29580 29584 mice +T954 NCBITaxon:10088 29589 29594 mouse +T955 PR:000007223 29605 29609 Er81 +T956 PR:000007243 29609 29612 EWS +T957 PR:000007227 29613 29617 Pea3 +T958 NCBITaxon:10088 29618 29622 mice +T959 PR:000007223 29707 29711 Er81 +T960 NCBITaxon:10088 29715 29719 mice +T961 SO:0001644 29738 29754 targeting vector +T962 PR:000007243 29778 29781 EWS +T963 PR:000007227 29782 29786 Pea3 +T964 SO:0001817 29800 29808 in frame +T965 SO:0000318 29829 29838 start ATG +T966 SO:0000147 29844 29848 exon +T967 PR:000007223 29858 29862 Er81 +T968 SO:0001026 29863 29870 genomic +T969 CL:0002322 29922 29930 ES cells +T970 PR:000007243 29932 29935 EWS +T971 PR:000007227 29936 29940 Pea3 +T972 PR:000007243 29996 29999 EWS +T973 SO:0000417 30012 30018 domain +T974 PR:000007227 30022 30026 Pea3 +T975 SO:0000112 30037 30043 primer +T976 PR:000007223 30081 30085 Er81 +T977 PR:000007243 30085 30088 EWS +T978 PR:000007227 30089 30093 Pea3 +T979 SO:0001023 30094 30100 allele +T980 PR:000010173 30189 30192 Tau +T981 PR:000010173 30201 30204 Tau +T982 PR:000007243 30204 30207 EWS +T983 PR:000007227 30208 30212 Pea3 +T984 NCBITaxon:10088 30213 30217 mice +T985 SO:0000346 30219 30222 lox +T986 SO:0000346 30228 30231 lox +T987 SO:0000243 30237 30241 IRES +T988 SO:0001528 30242 30245 NLS +T989 PR:000033987 30246 30250 LacZ +T990 SO:0000346 30258 30261 lox +T991 SO:0000346 30267 30270 lox +T992 PR:000007243 30271 30274 EWS +T993 PR:000007227 30275 30279 Pea3 +T994 SO:0000243 30280 30284 IRES +T995 SO:0001528 30285 30288 NLS +T996 PR:000033987 30289 30293 LacZ +T997 SO:0005853 30307 30316 cassettes +T998 SO:0000147 30338 30342 exon +T999 PR:000010173 30352 30355 Tau +T1000 SO:0001026 30356 30363 genomic +T1001 SO:0000318 30386 30395 start ATG +T1002 SO:0001644 30415 30432 targeting vectors +T1003 CL:0002322 30504 30511 ES cell +T1004 CL:0002322 30666 30674 ES cells +T1005 PR:000010173 30706 30709 Tau +T1006 PR:000013502 30744 30746 PV +T1007 NCBITaxon:10088 30750 30754 mice +T1008 NCBITaxon:10088 30756 30761 mouse +T1009 SO:0000040 30762 30776 genomic clones +T1010 SO:0001026 30814 30821 genomic +T1011 SO:0001026 30896 30903 genomic +T1012 NCBITaxon:10088 30921 30926 mouse +T1013 PR:000013502 30927 30929 PV +T1014 SO:0000243 30949 30953 IRES +T1015 SO:0005853 30971 30979 cassette +T1016 SO:0000205 31009 31015 3′ UTR +T1017 SO:0000147 31019 31023 exon +T1018 CL:0002322 31031 31038 ES cell +T1019 SO:0001026 31158 31165 genomic +T1020 PR:P43870 31171 31178 HindIII +T1021 CL:0002322 31230 31238 ES cells +T1022 UBERON:0000085 31303 31323 morula stage embryos +T1023 NCBITaxon:10088 31353 31357 mice +T1024 SO:0001023 31386 31393 alleles +T1025 NCBITaxon:33208 31439 31446 animals +T1026 SO:0000704 31461 31468 genetic +T1027 PR:000001843 31502 31506 Thy1 +T1028 NCBITaxon:10088 31523 31527 mice +T1029 NCBITaxon:10088 31617 31621 mice +T1030 UBERON:0019248 31627 31642 early embryonic +T1031 GO:0010467 31643 31653 expression +T1032 PR:000009116 31668 31672 Isl1 +T1033 PR:000010501 31680 31683 Hb9 +T1034 NCBITaxon:10088 31687 31692 mouse +T1035 PR:000002193 31733 31736 Bax +T1036 NCBITaxon:33208 31740 31747 animals +T1037 GO:0007565 31824 31835 pregnancies +T1038 UBERON:0000922 31860 31867 embryos +T1039 SO:0000155 32008 32016 plasmids +T1040 NCBITaxon:11886 32075 32078 RSV +T1041 NCBITaxon:11886 32134 32137 RSV +T1042 PR:000007223 32138 32142 Er81 +T1043 NCBITaxon:11886 32148 32151 RSV +T1044 PR:000007243 32152 32155 EWS +T1045 PR:000007227 32156 32160 Pea3 +T1046 NCBITaxon:11886 32195 32198 RSV +T1047 PR:000007223 32199 32203 Er81 +T1048 NCBITaxon:11886 32212 32215 RSV +T1049 PR:000007243 32216 32219 EWS +T1050 PR:000007227 32220 32224 Pea3 +T1051 PR:000007223 32269 32273 Er81 +T1052 PR:000007243 32277 32280 EWS +T1053 PR:000007227 32281 32285 Pea3 +T1054 NCBITaxon:11886 32321 32324 RSV +T1055 SO:0005853 32367 32375 cassette +T1056 PR:000007227 32419 32423 Pea3 +T1057 SO:0000409 32424 32437 binding sites +T1058 PR:000007227 32578 32582 Pea3 +T1059 SO:0000409 32583 32596 binding sites +T1060 SO:0000155 32626 32633 plasmid +T1061 GO:0009294 32651 32663 transfection +T1062 GO:0009294 32745 32756 transfected +T1063 CHEBI:35224 32805 32813 effector +T1064 SO:0000155 32814 32822 plasmids +T1065 NCBITaxon:11886 32827 32830 RSV +T1066 NCBITaxon:11886 32842 32845 RSV +T1067 PR:000007223 32846 32850 Er81 +T1068 NCBITaxon:11886 32859 32862 RSV +T1069 PR:000007243 32863 32866 EWS +T1070 PR:000007227 32867 32871 Pea3 +T1071 SO:0000155 32893 32901 plasmids +T1072 PR:000033987 33025 33029 LacZ +T1073 PR:000033987 33101 33105 LacZ +T1074 GO:0097617 33161 33174 hybridization +T1075 GO:0097617 33213 33226 hybridization +T1076 GO:0097617 33260 33270 hybridized +T1077 CHEBI:42098 33277 33288 digoxigenin +T1078 NCBITaxon:10088 33326 33331 mouse +T1079 PR:000011469 33332 33336 TrkA +T1080 PR:000011470 33340 33344 TrkB +T1081 NCBITaxon:10114 33349 33352 rat +T1082 PR:000011471 33353 33357 TrkC +T1083 GO:0042571 33384 33394 Antibodies +T1084 NCBITaxon:9986 33431 33437 rabbit +T1085 PR:000007223 33443 33447 Er81 +T1086 NCBITaxon:9986 33454 33460 rabbit +T1087 PR:000007227 33466 33470 Pea3 +T1088 NCBITaxon:9986 33477 33483 rabbit +T1089 PR:000013502 33489 33491 PV +T1090 NCBITaxon:9986 33498 33504 rabbit +T1091 NCBITaxon:9986 33566 33572 rabbit +T1092 PR:000004967 33578 33587 Calbindin +T1093 NCBITaxon:9986 33589 33595 rabbit +T1094 PR:000004968 33601 33611 Calretinin +T1095 NCBITaxon:9986 33646 33652 rabbit +T1096 NCBITaxon:9986 33712 33718 rabbit +T1097 PR:000014963 33724 33730 vGlut1 +T1098 NCBITaxon:9986 33772 33778 rabbit +T1099 PR:000013041 33784 33789 Brn3a +T1100 NCBITaxon:9986 33813 33819 rabbit +T1101 PR:000011469 33825 33829 TrkA +T1102 PR:000001405 33835 33838 p75 +T1103 NCBITaxon:9986 33868 33874 rabbit +T1104 PR:000014365 33880 33885 Runx3 +T1105 CHEBI:33893 33917 33924 reagent +T1106 NCBITaxon:9986 33927 33933 rabbit +T1107 NCBITaxon:10088 33969 33974 mouse +T1108 GO:0005883 33980 33993 neurofilament +T1109 NCBITaxon:9940 34065 34070 sheep +T1110 NCBITaxon:9925 34118 34122 goat +T1111 PR:000033987 34128 34132 LacZ +T1112 NCBITaxon:9925 34139 34143 goat +T1113 PR:000011471 34149 34153 TrkC +T1114 NCBITaxon:10140 34187 34197 guinea pig +T1115 PR:000009116 34203 34207 Isl1 +T1116 MOP:0000093 34261 34273 biotinylated +T1117 CL:0000445 34314 34329 apoptotic cells +T1118 UBERON:0000044 34339 34342 DRG +T1119 UBERON:0000044 34472 34475 DRG +T1120 CHEBI:472552 34527 34531 BrdU +T1121 PR:000033987 34560 34564 LacZ +T1122 GO:0043005 34680 34694;34703 34710 projections of ... neurons +T1123 CL:0000101 34695 34710 sensory neurons +T1124 MOP:0000779 34722 34732 conjugated +T1125 CHEBI:52071 34733 34740 dextran +T1126 UBERON:0002836 34785 34791;34797 34800 lumbar ... DRG +T1127 UBERON:0002858 34793 34795;34797 34800 L3 ... DRG +T1128 UBERON:0002836 34830 34849 lumbar dorsal roots +T1129 UBERON:0004619 34851 34853 L3 +T1130 GO:0007567 34862 34867 natal +T1131 NCBITaxon:33208 34920 34927 animals +T1132 CHEBI:51217 35062 35073 fluorophore +T1133 MOP:0000779 35074 35084 conjugated +T1134 GO:0042571 35095 35105 antibodies +T1135 GO:0097617 35227 35240 hybridization +T1136 UBERON:0000044 35493 35496 DRG +T1137 UBERON:0002836 35509 35519 lumbar DRG +T1138 UBERON:0000922 35555 35562 embryos +T1139 PR:000021998 35817 35830 neurotrophins +T1140 PR:000011194 35860 35863 NGF +T1141 PR:000011459 35886 35890 NT-3 +T1142 UBERON:0000044 35910 35913 DRG +T1143 UBERON:0002836 36074 36084 Lumbar DRG +T1144 UBERON:0000922 36096 36103 embryos +T1145 GO:0019835 36186 36191 lysed +T1146 GO:0019835 36204 36209 lysis +T1147 CHEBI:8984 36324 36327 SDS +T1148 GO:0042571 36373 36383 antibodies +T1149 PR:000029189 36392 36395 Akt +T1150 PR:000029189 36399 36402 Akt +T1151 PR:000002193 36436 36439 Bax +T1152 PR:000002385 36441 36447 Bcl-xl +T1153 PR:000002307 36520 36524 Bcl2 +T1154 GO:0005622 36886 36899 intracellular +T1155 UBERON:0001377 36927 36937 quadriceps +T1156 CL:0000100 36938 36951 motor neurons +T1157 CHEBI:32588 37000 37003 KCl +T1158 UBERON:0001021 37059 37064 nerve +T1159 GO:0045202 37125 37133 synaptic +T1160 UBERON:0001377 37151 37161 quadriceps +T1161 UBERON:0001021 37162 37167 nerve +T1162 GO:0045202 37308 37316 synaptic +T1163 SO:0000704 37520 37524 Gene +T1164 GO:0010467 37520 37535 Gene Expression +T1165 UBERON:0000044 37548 37551 DRG +T1166 CL:0000540 37552 37559 Neurons +T1167 PR:000007223 37563 37567 Er81 +T1168 PR:000007243 37567 37570 EWS +T1169 PR:000007227 37571 37575 Pea3 +T1170 NCBITaxon:10088 37576 37580 Mice +T1171 PR:000011471 37594 37598 TrkC +T1172 GO:0010467 37599 37609 expression +T1173 GO:0097617 37621 37634 hybridization +T1174 PR:000014365 37650 37655 Runx3 +T1175 PR:000013041 37672 37677 Brn3A +T1176 PR:000009116 37694 37698 Isl1 +T1177 PR:000001405 37715 37718 p75 +T1178 PR:000011469 37735 37739 TrkA +T1179 PR:000004968 37777 37787 Calretinin +T1180 PR:000004968 37789 37791 CR +T1181 PR:000033987 37812 37816 LacZ +T1182 UBERON:0002836 37871 37881 lumbar DRG +T1183 PR:000007223 37885 37889 Er81 +T1184 PR:000007223 37913 37917 Er81 +T1185 PR:000007243 37917 37920 EWS +T1186 PR:000007227 37921 37925 Pea3 +T1187 UBERON:0000922 37942 37949 embryos +T1188 GO:0019230 38097 38111 Proprioceptive +T1189 CL:0000100 38155 38168 Motor Neurons +T1190 PR:000007223 38172 38176 Er81 +T1191 PR:000007243 38176 38179 EWS +T1192 PR:000007227 38180 38184 Pea3 +T1193 GO:0005622 38231 38244 intracellular +T1194 GO:0045202 38282 38290 synaptic +T1195 UBERON:0001377 38300 38310 quadriceps +T1196 CL:0000100 38311 38324 motor neurons +T1197 UBERON:0001377 38369 38379 quadriceps +T1198 UBERON:0001021 38380 38385 nerve +T1199 PR:000007223 38407 38411 Er81 +T1200 PR:000007243 38411 38414 EWS +T1201 PR:000007227 38415 38419 Pea3 +T1202 NCBITaxon:33208 38433 38440 animals +T1203 GO:0045202 38459 38467 synaptic +T1204 NCBITaxon:10088 38648 38652 Mice +T1205 GO:0010467 38653 38663 Expressing +T1206 PR:000007243 38664 38667 EWS +T1207 PR:000007227 38668 38672 Pea3 +T1208 GO:0007067 38695 38702 Mitotic +T1209 UBERON:0000044 38703 38706 DRG +T1210 CL:0000540 38707 38714 Neurons +T1211 PR:000010173 38756 38759 Tau +T1212 SO:0001026 38760 38767 genomic +T1213 SO:0000147 38859 38864 Exons +T1214 PR:000010173 38908 38911 Tau +T1215 SO:0000318 38912 38923 start codon +T1216 SO:0000147 38927 38931 exon +T1217 PR:000010173 39059 39062 Tau +T1218 SO:0005853 39123 39132 cassettes +T1219 SO:0000147 39146 39150 exon +T1220 PR:000010173 39196 39199 Tau +T1221 SO:0000318 39200 39211 start codon +T1222 SO:0005853 39238 39247 cassettes +T1223 GO:0010467 39270 39280 expression +T1224 PR:000007243 39284 39287 EWS +T1225 PR:000007227 39288 39292 Pea3 +T1226 SO:0001528 39297 39300 NLS +T1227 PR:000033987 39301 39305 LacZ +T1228 SO:0001528 39333 39336 NLS +T1229 PR:000033987 39337 39341 LacZ +T1230 SO:0000141 39430 39459 transcriptional stop sequence +T1231 SO:0000357 39460 39467 flanked +T1232 SO:0000346 39471 39481 loxP sites +T1233 GO:0010467 39491 39501 expression +T1234 SO:0000902 39520 39530 transgenes +T1235 SO:0000318 39542 39554 start codons +T1236 PR:000010173 39601 39604 Tau +T1237 PR:000007243 39604 39607 EWS +T1238 PR:000007227 39608 39612 Pea3 +T1239 PR:000010173 39619 39622 Tau +T1240 SO:0001026 39629 39636 genomic +T1241 SO:0001023 39662 39668 allele +T1242 SO:0000141 39715 39744 transcriptional stop sequence +T1243 SO:0005853 39752 39761 cassettes +T1244 PR:000010173 39782 39785 Tau +T1245 GO:0010467 39804 39814 Expression +T1246 PR:000007243 39818 39821 EWS +T1247 PR:000007227 39822 39826 Pea3 +T1248 SO:0001528 39831 39834 NLS +T1249 PR:000033987 39835 39839 LacZ +T1250 SO:0001528 39858 39861 NLS +T1251 PR:000033987 39862 39866 LacZ +T1252 CL:0000540 39893 39900 neurons +T1253 GO:0010467 39914 39924 expressing +T1254 PR:000010173 39945 39948 Tau +T1255 GO:0010467 39985 39995 Expression +T1256 PR:000009116 39999 40003 Isl1 +T1257 PR:000007243 40019 40022 EWS +T1258 PR:000007227 40023 40027 Pea3 +T1259 PR:000033987 40051 40055 LacZ +T1260 UBERON:0000044 40099 40102 DRG +T1261 CL:0000540 40103 40110 neurons +T1262 PR:000010173 40131 40134 Tau +T1263 PR:000007243 40134 40137 EWS +T1264 PR:000007227 40138 40142 Pea3 +T1265 PR:000009116 40145 40149 Isl1 +T1266 PR:000010173 40166 40169 Tau +T1267 PR:000009116 40176 40180 Isl1 +T1268 UBERON:0000922 40192 40199 embryos +T1269 NCBITaxon:10088 40340 40344 Mice +T1270 PR:000013502 40383 40385 PV +T1271 SO:0001026 40386 40393 genomic +T1272 SO:0000147 40401 40406 Exons +T1273 SO:0000147 40464 40468 exon +T1274 SO:0000318 40484 40495 start codon +T1275 SO:0000147 40506 40510 exon +T1276 SO:0000319 40526 40536 stop codon +T1277 PR:000013502 40653 40655 PV +T1278 SO:0000243 40690 40694 IRES +T1279 SO:0005853 40699 40707 cassette +T1280 GO:0006412 40726 40739 translational +T1281 SO:0000319 40726 40750 translational stop codon +T1282 PR:000013502 40754 40756 PV +T1283 CL:0002322 40791 40799 ES cells +T1284 PR:000013502 40832 40834 PV +T1285 SO:0001026 40877 40884 genomic +T1286 GO:0010467 40934 40944 Expression +T1287 PR:000033987 40964 40968 LacZ +T1288 PR:000013502 40981 40983 PV +T1289 PR:000010173 40999 41002 Tau +T1290 PR:000013502 41009 41011 PV +T1291 NCBITaxon:10088 41017 41021 mice +T1292 PR:000013502 41050 41052 PV +T1293 CL:0000540 41054 41061 neurons +T1294 GO:0010467 41064 41071 express +T1295 SO:0000704 41190 41194 Gene +T1296 GO:0010467 41190 41205 Gene Expression +T1297 PR:000007243 41244 41247 EWS +T1298 PR:000007227 41248 41252 Pea3 +T1299 UBERON:0000044 41256 41259 DRG +T1300 CL:0000540 41260 41267 Neurons +T1301 PR:000013502 41301 41303 PV +T1302 PR:000004968 41315 41325 Calretinin +T1303 PR:000004967 41341 41350 Calbindin +T1304 GO:0010467 41361 41371 expression +T1305 UBERON:0002836 41381 41391 lumbar DRG +T1306 PR:000010173 41415 41418 Tau +T1307 PR:000007243 41418 41421 EWS +T1308 PR:000007227 41422 41426 Pea3 +T1309 PR:000009116 41429 41433 Isl1 +T1310 UBERON:0000922 41445 41452 embryos +T1311 PR:000007223 41592 41596 Er81 +T1312 PR:000007243 41596 41599 EWS +T1313 PR:000007227 41600 41604 Pea3 +T1314 NCBITaxon:10088 41607 41611 mice +T1315 PR:000014365 42038 42043 Runx3 +T1316 GO:0042571 42044 42054 antibodies +T1317 PR:000007243 42123 42126 EWS +T1318 PR:000007227 42127 42131 Pea3 +T1319 SO:0000704 42169 42173 gene +T1320 PR:000010173 42232 42235 Tau +T1321 UBERON:0000044 42625 42628 DRG +T1322 UBERON:0000044 42631 42651 dorsal root ganglion +T1323 UBERON:0000044 42631 42642;42652 42659 dorsal root ... ganglia +T1324 UBERON:0000922 42665 42674 embryonic +T1325 PR:000007243 42680 42683 EWS +T1326 PR:000007243 42686 42699 Ewing sarcoma +T1327 GO:0016020 42708 42716 membrane +T1328 PR:000011459 42753 42757 NT-3 +T1329 PR:000011459 42760 42774 neurotrophin 3 +T1330 GO:0007567 42784 42789 natal +T1331 PR:000013502 42795 42797 PV +T1332 PR:000013502 42800 42811 Parvalbumin +T1333 PR:000015890 42821 42834 synaptophysin +T1334 PR:000007223 42907 42911 Er81 +T1335 PR:000007243 42915 42918 EWS +T1336 PR:000007227 42919 42923 Pea3 +T1337 PR:000007223 42943 42947 Er81 +T1338 PR:000007243 42947 42950 EWS +T1339 PR:000007227 42951 42955 Pea3 +T1340 NCBITaxon:10088 42963 42967 mice +T1341 PR:000007223 43002 43006 Er81 +T1342 SO:0001026 43007 43014 genomic +T1343 SO:0000147 43092 43097 Exons +T1344 PR:000007223 43141 43145 Er81 +T1345 SO:0000318 43146 43157 start codon +T1346 SO:0000147 43161 43165 exon +T1347 PR:000007223 43287 43291 Er81 +T1348 PR:000007243 43295 43298 EWS +T1349 PR:000007227 43299 43303 Pea3 +T1350 PR:000007243 43331 43334 EWS +T1351 PR:000007227 43335 43339 Pea3 +T1352 SO:0001817 43340 43348 in frame +T1353 SO:0000318 43369 43380 start codon +T1354 PR:000007223 43388 43392 Er81 +T1355 SO:0000147 43402 43406 exon +T1356 PR:000007223 43470 43474 Er81 +T1357 PR:000007243 43474 43477 EWS +T1358 PR:000007227 43478 43482 Pea3 +T1359 SO:0001026 43541 43548 genomic +T1360 SO:0001023 43574 43580 allele +T1361 SO:0000112 43586 43592 primer +T1362 PR:000007243 43600 43603 EWS +T1363 SO:0001023 43660 43666 allele +T1364 SO:0000112 43674 43680 primer +T1365 SO:0001023 43744 43750 allele +T1366 PR:000007223 43776 43780 Er81 +T1367 GO:0010467 43781 43791 expression +T1368 UBERON:0002836 43795 43805 lumbar DRG +T1369 CL:0000540 43806 43813 neurons +T1370 PR:000007223 43838 43842 Er81 +T1371 PR:000007223 43855 43859 Er81 +T1372 PR:000007243 43859 43862 EWS +T1373 PR:000007227 43863 43867 Pea3 +T1374 UBERON:0000922 43874 43881 embryos +T1375 PR:000009116 43931 43935 Isl1 +T1376 GO:0010467 43936 43946 expression +T1377 UBERON:0000044 43965 43968 DRG +T1378 PR:000013502 43977 43979 PV +T1379 GO:0010467 43980 43990 expression +T1380 UBERON:0002836 43994 44004 lumbar DRG +T1381 PR:000007223 44029 44033 Er81 +T1382 PR:000007223 44046 44050 Er81 +T1383 PR:000007243 44050 44053 EWS +T1384 PR:000007227 44054 44058 Pea3 +T1385 UBERON:0000922 44065 44072 embryos +T1386 GO:0010467 44182 44192 expression +T1387 SO:0000994 44243 44252;44269 44274 consensus ... sites +T1388 SO:0001429 44257 44274 DNA-binding sites +T1389 SO:0000167 44313 44321 promoter +T1390 GO:0009294 44337 44349 transfection +T1391 PR:000007223 44353 44357 Er81 +T1392 PR:000007243 44382 44385 EWS +T1393 PR:000007227 44386 44390 Pea3 +T1394 GO:0019230 44513 44527 Proprioceptive +T1395 GO:0042995 44537 44548 Projections +T1396 UBERON:0002257 44558 44577 Ventral Spinal Cord +T1397 PR:000007223 44581 44585 Er81 +T1398 PR:000007243 44585 44588 EWS +T1399 PR:000007227 44589 44593 Pea3 +T1400 GO:0042995 44643 44654 projections +T1401 UBERON:0005462 44658 44664 lumbar +T1402 UBERON:0004619 44671 44673 L3 +T1403 PR:000013502 44677 44679 PV +T1404 UBERON:0000044 44681 44684 DRG +T1405 CL:0000540 44685 44692 neurons +T1406 UBERON:0000044 44706 44709 DRG +T1407 CHEBI:52071 44771 44778 dextran +T1408 UBERON:0002261 44793 44805 dorsal roots +T1409 PR:000007223 44859 44863 Er81 +T1410 PR:000007223 44882 44886 Er81 +T1411 PR:000007243 44886 44889 EWS +T1412 PR:000007227 44890 44894 Pea3 +T1413 NCBITaxon:10088 44907 44911 mice +T1414 UBERON:0002240 44961 44972 spinal cord +T1415 PR:000014963 44993 44999 vGlut1 +T1416 UBERON:0002257 45027 45039 ventral horn +T1417 PR:000007223 45063 45067 Er81 +T1418 PR:000007223 45080 45084 Er81 +T1419 PR:000007243 45084 45087 EWS +T1420 PR:000007227 45088 45092 Pea3 +T1421 NCBITaxon:10088 45099 45103 mice +T1422 GO:0019230 45239 45253 proprioceptive +T1423 GO:0042995 45263 45274 projections +T1424 UBERON:0002257 45291 45310 ventral spinal cord +T1425 PR:000007223 45338 45342 Er81 +T1426 PR:000007223 45355 45359 Er81 +T1427 PR:000007243 45359 45362 EWS +T1428 PR:000007227 45363 45367 Pea3 +T1429 NCBITaxon:10088 45374 45378 mice +T1430 UBERON:0000044 45380 45383 DRG +T1431 CL:0000100 45415 45428 motor neurons +T1432 GO:0042995 45565 45576 Projections +T1433 GO:0010467 45593 45603 Expression +T1434 PR:000007243 45607 45610 EWS +T1435 PR:000007227 45611 45615 Pea3 +T1436 UBERON:0000044 45619 45622 DRG +T1437 CL:0000540 45623 45630 Neurons +T1438 GO:0042995 45680 45691 projections +T1439 UBERON:0002240 45709 45720 spinal cord +T1440 PR:000010173 45744 45747 Tau +T1441 PR:000007243 45747 45750 EWS +T1442 PR:000007227 45751 45755 Pea3 +T1443 PR:000009116 45758 45762 Isl1 +T1444 UBERON:0000922 45774 45781 embryos +T1445 GO:0010467 45875 45885 expression +T1446 PR:000010173 45895 45898 Tau +T1447 PR:000001843 45930 45934 Thy1 +T1448 SO:0000902 45940 45949 transgene +T1449 GO:0042995 46015 46026 projections +T1450 UBERON:0002240 46036 46047 spinal cord +T1451 UBERON:0002240 46143 46154 spinal cord +T1452 PR:000010173 46158 46161 Tau +T1453 PR:000007243 46161 46164 EWS +T1454 PR:000007227 46165 46169 Pea3 +T1455 PR:000009116 46172 46176 Isl1 +T1456 UBERON:0000922 46182 46189 embryos +T1457 GO:0042995 46235 46246 projections +T1458 PR:000010173 46348 46351 Tau +T1459 PR:000007243 46351 46354 EWS +T1460 PR:000007227 46355 46359 Pea3 +T1461 PR:000009116 46362 46366 Isl1 +T1462 UBERON:0000922 46378 46385 embryos +T1463 GO:0010467 46459 46469 expression +T1464 PR:000010173 46479 46482 Tau +T1465 PR:000006939 46505 46509 Egr3 +T1466 GO:0010467 46510 46520 expression +T1467 CL:0000187 46535 46548 muscle fibers +T1468 GO:0097617 46563 46576 hybridization +T1469 GO:0042995 46675 46686 projections +T1470 UBERON:0002240 46699 46710 spinal cord +T1471 PR:000010173 46738 46741 Tau +T1472 PR:000007243 46741 46744 EWS +T1473 PR:000007227 46745 46749 Pea3 +T1474 PR:000009116 46752 46756 Isl1 +T1475 UBERON:0000922 46772 46779 embryos +T1476 CHEBI:52071 46821 46828 dextran +T1477 UBERON:0000044 46846 46849 DRG +T1478 UBERON:0005462 46851 46857 lumbar +T1479 UBERON:0004619 46864 46866 L3 +T1480 GO:0030424 47039 47057 axonal projections +T1481 PR:000021998 47204 47216 Neurotrophin +T1482 GO:0043005 47229 47236 Neurite +T1483 UBERON:0000044 47259 47262 DRG +T1484 CL:0000540 47263 47270 Neurons +T1485 GO:0010467 47271 47281 Expressing +T1486 PR:000007243 47282 47285 EWS +T1487 PR:000007227 47286 47290 Pea3 +T1488 UBERON:0002836 47311 47321 lumbar DRG +T1489 PR:000010173 47352 47355 Tau +T1490 PR:000007243 47355 47358 EWS +T1491 PR:000007227 47359 47363 Pea3 +T1492 PR:000009116 47366 47370 Isl1 +T1493 PR:000002193 47394 47397 Bax +T1494 UBERON:0000922 47415 47422 embryos +T1495 PR:000011194 47506 47509 NGF +T1496 PR:000011459 47527 47531 NT-3 +T1497 GO:0010467 47563 47573 expression +T1498 GO:0005883 47577 47590 neurofilament +T1499 GO:0030424 47604 47621 axonal extensions +T1500 UBERON:0000044 47654 47657 DRG +T1501 CL:0000540 47658 47665 Neurons +T1502 GO:0010467 47666 47676 Expressing +T1503 PR:000007243 47677 47680 EWS +T1504 PR:000007227 47681 47685 Pea3 +T1505 PR:000021998 47711 47724 Neurotrophins +T1506 UBERON:0002836 47745 47755 lumbar DRG +T1507 PR:000010173 47761 47764 Tau +T1508 PR:000013502 47771 47773 PV +T1509 PR:000010173 47793 47796 Tau +T1510 PR:000007243 47796 47799 EWS +T1511 PR:000007227 47800 47804 Pea3 +T1512 PR:000013502 47807 47809 PV +T1513 UBERON:0000922 47825 47832 embryos +T1514 PR:000011459 47912 47916 NT-3 +T1515 GO:0010467 47944 47954 expression +T1516 GO:0005883 47958 47971 neurofilament +T1517 PR:000033987 47982 47986 LacZ +T1518 GO:0030424 48008 48025 axonal extensions +T1519 PR:000013502 48042 48044 PV +T1520 GO:0010467 48045 48055 expressing +T1521 GO:0019230 48056 48070 proprioceptive +T1522 PR:000021997 48121 48133 Trk Receptor +T1523 GO:0010467 48134 48144 Expression +T1524 UBERON:0000044 48171 48174 DRG +T1525 CL:0000540 48175 48182 Neurons +T1526 GO:0097617 48236 48249 hybridization +T1527 PR:000011469 48262 48266 TrkA +T1528 PR:000011470 48278 48282 TrkB +T1529 PR:000011471 48298 48302 TrkC +T1530 GO:0010467 48313 48323 expression +T1531 UBERON:0002836 48333 48343 lumbar DRG +T1532 PR:000010173 48367 48370 Tau +T1533 PR:000007243 48370 48373 EWS +T1534 PR:000007227 48374 48378 Pea3 +T1535 PR:000009116 48381 48385 Isl1 +T1536 UBERON:0000922 48397 48404 embryos +T1537 UBERON:0002836 48433 48443 lumbar DRG +T1538 PR:000010173 48462 48465 Tau +T1539 PR:000009116 48472 48475 Isl +T1540 PR:000010173 48496 48499 Tau +T1541 PR:000007243 48499 48502 EWS +T1542 PR:000007227 48503 48507 Pea3 +T1543 PR:000009116 48510 48514 Isl1 +T1544 UBERON:0000922 48534 48541 embryos +T1545 CL:0000540 48550 48563 neuronal cell +T1546 GO:0070997 48550 48569 neuronal cell death +T1547 GO:0008283 48610 48614;48628 48641 cell ... proliferation +T1548 PR:000033987 48654 48658 LacZ +T1549 CHEBI:472552 48740 48744 BrdU +T1550 PR:000033987 48753 48757 LacZ +T1551 CL:0000540 48944 48952 neuronal +T1552 UBERON:0000044 48996 48999 DRG +T1553 UBERON:0005462 49003 49009 lumbar +T1554 UBERON:0004617 49017 49019 L1 +T1555 UBERON:0004621 49023 49025 L5 +T1556 UBERON:0002836 49121 49131 lumbar DRG +T1557 PR:000010173 49160 49163 Tau +T1558 PR:000007243 49163 49166 EWS +T1559 PR:000007227 49167 49171 Pea3 +T1560 PR:000009116 49174 49178 Isl1 +T1561 UBERON:0000922 49190 49197 embryos +T1562 GO:0042571 49218 49228 antibodies +T1563 PR:000029189 49230 49233 Akt +T1564 PR:000029189 49237 49240 Akt +T1565 PR:000002193 49274 49277 Bax +T1566 PR:000002307 49279 49283 Bcl2 +T1567 PR:000002385 49289 49295 Bcl-xl +T1568 SO:0000704 49530 49534 Gene +T1569 GO:0010467 49530 49545 Gene Expression +T1570 PR:000011471 49631 49635 TrkC +T1571 GO:0010467 49636 49646 expression +T1572 GO:0097617 49658 49671 hybridization +T1573 PR:000004968 49682 49692 Calretinin +T1574 PR:000033987 49703 49707 LacZ +T1575 GO:0010467 49716 49726 expression +T1576 UBERON:0002836 49767 49777 lumbar DRG +T1577 PR:000010173 49802 49805 Tau +T1578 PR:000007243 49805 49808 EWS +T1579 PR:000007227 49809 49813 Pea3 +T1580 PR:000009116 49816 49820 Isl1 +T1581 PR:000007223 49837 49841 Er81 +T1582 PR:000007243 49841 49844 EWS +T1583 PR:000007227 49845 49849 Pea3 +T1584 PR:000010173 49867 49870 Tau +T1585 PR:000007243 49870 49873 EWS +T1586 PR:000007227 49874 49878 Pea3 +T1587 PR:000013502 49881 49883 PV +T1588 UBERON:0000922 49899 49906 embryos +T1589 GO:0065007 49944 49954 regulation +T1590 PR:000011471 49958 49962 TrkC +T1591 PR:000004968 49996 50006 Calretinin +T1592 GO:0010467 50036 50046 expression +T1593 PR:000007243 50086 50089 EWS +T1594 PR:000007227 50090 50094 Pea3 +T1595 GO:0010467 50095 50105 expression +T1596 UBERON:0000044 50109 50112 DRG +T1597 CL:0000540 50113 50120 neurons +T1598 GO:0007049 50160 50170 cell cycle +T1599 PR:000007243 50215 50218 EWS +T1600 PR:000007227 50219 50223 Pea3 +T1601 PR:000007223 50244 50248 Er81 +T1602 GO:0010467 50299 50309 expression +T1603 PR:000013502 50319 50321 PV +T1604 GO:0010467 50344 50354 expression +T1605 PR:000010173 50364 50367 Tau +T1606 GO:0010467 50426 50436 expression +T1607 PR:000011471 50440 50444 TrkC +T1608 PR:000004968 50449 50459 Calretinin +T1609 SO:0000704 50561 50565 Gene +T1610 GO:0010467 50561 50576 Gene Expression +T1611 GO:0010467 50610 50620 Expression +T1612 PR:000011469 50624 50628 TrkA +T1613 PR:000011471 50649 50653 TrkC +T1614 PR:000033987 50676 50680 LacZ +T1615 UBERON:0002836 50697 50707 lumbar DRG +T1616 PR:000010173 50711 50714 Tau +T1617 PR:000010501 50721 50724 Hb9 +T1618 PR:000010173 50744 50747 Tau +T1619 PR:000007243 50747 50750 EWS +T1620 PR:000007227 50751 50755 Pea3 +T1621 PR:000010501 50758 50761 Hb9 +T1622 UBERON:0000922 50777 50784 embryos +T1623 GO:0010467 50793 50803 Expression +T1624 PR:000004968 50807 50817 Calretinin +T1625 PR:000033987 50827 50831 LacZ +T1626 PR:000009116 50843 50847 Isl1 +T1627 UBERON:0001460 50880 50888 brachial +T1628 UBERON:0002836 50899 50905;50912 50915 lumbar ... DRG +T1629 PR:000010173 50919 50922 Tau +T1630 PR:000010501 50929 50932 Hb9 +T1631 PR:000010173 50959 50962 Tau +T1632 PR:000007243 50962 50965 EWS +T1633 PR:000007227 50966 50970 Pea3 +T1634 PR:000010501 50973 50976 Hb9 +T1635 UBERON:0000922 50999 51006 embryos +T1636 CL:0000540 51071 51079 Neuronal +T1637 GO:0048665 51071 51093 Neuronal Specification +T1638 GO:0010628 51264 51279;51301 51311 upregulation of ... expression +T1639 GO:0048665 51319 51335;51340 51347 specification of ... neurons +T1640 UBERON:0000044 51336 51339 DRG +T1641 CL:0000540 51340 51347 neurons +T1642 CL:0000540 51368 51376 neuronal +T1643 UBERON:0024914 51368 51376;51397 51404 neuronal ... circuit +T1644 GO:0010467 51422 51432 Expression +T1645 PR:000007243 51436 51439 EWS +T1646 PR:000007227 51440 51444 Pea3 +T1647 PR:000007223 51465 51469 Er81 +T1648 UBERON:0001062 51487 51497 anatomical +T1649 PR:000007223 51518 51522 Er81 +T1650 NCBITaxon:10088 51526 51530 mice +T1651 GO:0010467 51549 51559 expression +T1652 PR:000011471 51563 51567 TrkC +T1653 PR:000004968 51579 51589 Calretinin +T1654 PR:000004968 51591 51593 CR +T1655 GO:0019230 51616 51630 proprioceptive +T1656 GO:0031175 51725 51741;51746 51766 establishment of ... neuronal projections +T1657 UBERON:0000044 51742 51745 DRG +T1658 CL:0000540 51746 51754 neuronal +T1659 GO:0043005 51746 51766 neuronal projections +T1660 SO:0000704 51796 51800 gene +T1661 GO:0010467 51796 51811 gene expression +T1662 PR:000004968 51840 51842 CR +T1663 PR:000011471 51871 51875 TrkC +T1664 GO:0048665 51940 51956;51980 51987 specification of ... neurons +T1665 GO:0019230 51957 51979 proprioceptive sensory +T1666 CL:1001451 51957 51987 proprioceptive sensory neurons +T1667 CL:0000540 52006 52014 neuronal +T1668 CL:0000540 52202 52210 neuronal +T1669 UBERON:0000044 52528 52531 DRG +T1670 CL:0000540 52532 52539 neurons diff --git a/src/ontogpt/evaluation/craft/database/all/15836427.txt b/src/ontogpt/evaluation/craft/database/all/15836427.txt new file mode 100644 index 000000000..1374389d1 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15836427.txt @@ -0,0 +1,303 @@ +A Developmental Switch in the Response of DRG Neurons to ETS Transcription Factor Signaling + +Abstract + +Two ETS transcription factors of the Pea3 subfamily are induced in subpopulations of dorsal root ganglion (DRG) sensory and spinal motor neurons by target-derived factors. Their expression controls late aspects of neuronal differentiation such as target invasion and branching. Here, we show that the late onset of ETS gene expression is an essential requirement for normal sensory neuron differentiation. We provide genetic evidence in the mouse that precocious ETS expression in DRG sensory neurons perturbs axonal projections, the acquisition of terminal differentiation markers, and their dependence on neurotrophic support. Together, our findings indicate that DRG sensory neurons exhibit a temporal developmental switch that can be revealed by distinct responses to ETS transcription factor signaling at sequential steps of neuronal maturation. + +Introduction + +Neuronal differentiation is a protracted process during which newly generated neurons express distinct cellular and molecular programs at precise times during their maturation: long-distance axon outgrowth, subsequent terminal branching, and finally synaptogenesis. Many important aspects of neuronal character appear to be acquired through the expression of transcription factors at progenitor cell stages, whereas others depend on expression immediately upon cell cycle exit [1]. But whether the orderly expression and activity of transcriptional programs at much later developmental stages, well after cell cycle exit, is an essential step in the progression of neuronal differentiation and circuit assembly has yet to be resolved. + +The differentiation of sensory neurons of dorsal root ganglia (DRG) has been studied extensively with respect to inductive events that specify neuronal fate [2,3], as well as the involvement of late target-derived neurotrophic factors in the control of neuronal survival [4]. Recent evidence has begun to emerge that target-derived factors are also involved in regulating later aspects of neuronal differentiation [5,6,7]. In particular, genetic experiments have addressed the survival-independent role of neurotrophic factors during development by exploiting strains of mice defective both in neurotrophin signaling and in the function of the proapoptotic gene Bax [8,9]. These studies, for example, have revealed that neurotrophin signaling controls the acquisition of peptidergic traits in nociceptive DRG neurons and the control of target innervation [8,9]. + +The onset of some transcriptional programs in neurons, however, has also been shown to occur long after neurons exit the cell cycle. An emerging principle from work in Drosophila and vertebrates is that target-derived factors play a crucial role in the induction of these transcriptional programs [10]. In Drosophila, retrograde BMP signals from the target region control the terminal differentiation of a subpopulation of peptidergic neurons expressing Apterous and Squeeze [11,12]. In vertebrates, peripheral neurotrophic signals have been shown to direct the onset of expression of the ETS transcription factors Er81 and Pea3 in DRG sensory neurons and motor neuron pools several days after these neurons have become post-mitotic [9,13,14,15,16]. Moreover, the induction of Er81 expression in proprioceptive afferents is known to be mediated by peripheral neurotrophin 3 (NT-3) [9]. These two ETS proteins control late aspects of spinal monosynaptic circuit assembly, with Er81 directing proprioceptive sensory neuron differentiation and Pea3 directing motor neuron pool differentiation, respectively [14,15]. In particular, in the absence of Er81, achieved by mutation in the gene or by deprivation of peripheral neurotrophin signaling, group Ia proprioceptive afferents fail to invade the ventral spinal cord and to make effective synaptic connections with motor neurons [9,14]. + +The involvement of target-derived signals in induction of ETS transcription factor expression raises the question of the necessity for the observed delay in the onset of ETS signaling for neuronal maturation. Would precocious expression of ETS proteins in post-mitotic neurons also direct the appropriate sensory neuron developmental programs? In this study, we have used mouse genetics to test this general idea, by investigating whether the precise timing of onset of ETS transcription factor signaling is essential for normal sensory neuron development. We have assessed the biological effects of inducing ETS signaling either at the correct developmental time, or precociously. We find that within proprioceptive sensory neurons, the late onset of ETS signaling is essential for the establishment of normal sensory afferent projections in the spinal cord. Precocious initiation of ETS signaling in post-mitotic DRG neurons leads to abnormal DRG neuron differentiation characterized by neurotrophin-independent neurite outgrowth and inappropriate profiles of gene expression. Our findings reveal that target-triggered inductive signals provide an effective means of ensuring the late onset of expression of transcription factors, and thus an orderly temporal transcriptional sequence that is crucial for neuronal maturation and circuit assembly. + +Results + +To test the hypothesis that a temporal delay in the onset of transcriptional programs is crucial for the control of appropriate neuronal maturation, we studied the development of proprioceptive DRG neurons, since transcriptional effectors regulated by target-derived signals, as well as some of their downstream biological actions, have been identified for these neurons. Er81 controls proprioceptive afferent connectivity [14], and we therefore sought to identify an ETS transcriptional regulator that, when expressed over the normal time course of Er81 expression, is able to substitute for Er81 function within group Ia afferent sensory neurons. With this reference point, we then designed experiments to examine the effects of precocious post-mitotic expression of the same ETS transcription factor on sensory neuron differentiation. + +EWS-Pea3 Can Replace Er81 Function in Controlling Ia Afferent Projections + +We first defined an ETS transcription regulator that is able to replace the function of Er81 within proprioceptive afferents to direct projections into the ventral spinal cord. Er81, Pea3, and Erm constitute the Pea3 subfamily of ETS transcription factors, show a high degree of amino acid identity, and bind to very similar DNA target sequences [17,18,19]. Nevertheless, when introduced into the Er81 locus in analogy to a previously used targeting strategy (data not shown; [14]), neither Pea3 nor Erm could rescue Ia proprioceptive afferent projections to extensively invade the ventral horn of the spinal cord (data not shown). These findings prompted us to analyze mice in which we integrated EWS-Pea3, a break-point fusion product between the amino-terminal domain of the Ewing sarcoma (EWS) gene and the Pea3 DNA binding domain [20,21], into the Er81 locus (Figure 1). We found that in a luciferase-enzyme-based cell culture transfection assay, EWS-Pea3 showed stronger transactivation activity than Er81 or Pea3 (Figure 1J; data not shown), in agreement with previous studies [22,23,24]. Moreover, transactivation by EWS-Pea3 was abolished by mutation of ETS-binding sites in the reporter plasmid, demonstrating ETS-binding-site dependence (data not shown). + +Expression of Er81 in DRG neurons of embryos containing integration of EWS-Pea3 in the Er81 locus (Er81EWS-Pea3/−) was abolished (Figure 1E), and the expression level of the calcium-binding protein Parvalbumin (PV) in proprioceptive afferents, which is decreased approximately 5- to 10-fold in Er81 mutants [14], was comparable to wild-type levels in Er81EWS-Pea3/− embryos (Figure 1F–1H). To further define DRG neuron differentiation in the presence of EWS-Pea3 in proprioceptive afferents in vivo, we assessed whether replacement of Er81 by EWS-Pea3 had an influence on neuronal survival or on the expression of proprioceptive-afferent-specific genes. Er81EWS-Pea3/− mice did not differ from wild-type in the number of proprioceptive afferent cell bodies within the DRG of L1 to L5, the expression of several genes normally expressed by proprioceptive afferents, and the lack of expression of genes not normally expressed in proprioceptive afferents (Figure S1; data not shown). Together, these findings suggest that the expression of EWS-Pea3 from the normal time of onset mimics the function of Er81 as assessed by induction and maintenance of gene expression within proprioceptive afferents. + +To determine the extent of rescue of Ia proprioceptive afferent projections into the ventral spinal cord of Er81 mutant mice achieved by expression of EWS-Pea3, we traced intraspinal afferent projections by axonal labeling of PV (Figure 2A–2C). In addition, to analyze axon ingrowth independent of the level of PV expression in DRG neurons, we used anterograde labeling of afferent fibers by applying fluorescently labeled dextran to cut dorsal roots (Figure 2D–2F). Using both assays, we found extensive rescue of the projections into the ventral horn of the spinal cord in Er81EWS-Pea3/− mice (Figure 2C and 2F). Within the ventral horn, Ia afferents in both wild-type and Er81EWS-Pea3/− mice formed vGlut1+ terminals that were absent in Er81 mutant mice (Figure 2G–2I). To assess whether synapses between Ia afferents and motor neurons are functional in Er81EWS-Pea3/− mice, we performed intracellular recordings from identified quadriceps motor neurons after stimulation of nerves innervating the quadriceps muscle group. We found no significant difference in the input amplitude to quadriceps motor neurons when comparing wild-type to Er81EWS-Pea3/− mice (Figure S2; wild-type, 10.6 ± 0.9 mV, n = 11; Er81EWS-Pea3/−, 10.9 ± 1 mV, n = 8). Together, these findings suggest that in the absence of Er81, EWS-Pea3 can direct the complex biological process of correct laminar termination within the ventral spinal cord and the formation of synapses with motor neurons (Figure 2J–2L), thus identifying an ETS transcription factor suitable for heterochronic expression experiments in DRG neurons. + +Precocious Expression of EWS-Pea3 in DRG Neurons Leads to Axonal Projection Defects + +To address the consequences of precocious ETS signaling for proprioceptive afferent differentiation, we next expressed EWS-Pea3 in DRG neurons as soon as they became post-mitotic. We used a binary mouse genetic system based on Cre-recombinase-mediated excision of a transcriptional stop cassette flanked by loxP sites. Targeting cassettes were integrated into the Tau locus to generate two strains of mice conditionally expressing either EWS-Pea3 or a membrane-targeted green fluorescent protein (mGFP) to trace axonal projections of DRG neurons (Figure S3; [25]). Embryos positive for either Isl1Cre/+ and TauEWS-Pea3/+ or Isl1Cre/+ and TaumGFP/+ alleles showed efficient activation of the silent Tau allele in 95% or more of all DRG neurons, including proprioceptive afferents, at all segmental levels (Figure S3). + +We first assessed the influence of EWS-Pea3 expression in early post-mitotic DRG neurons on the establishment of afferent projections into the spinal cord using the TaumGFP/+ allele or a Thy1-promoter-driven synaptophysin green fluorescent protein (spGFP) with an expression profile restricted to DRG sensory neurons at embryonic day (E) 13.5 (Thy1spGFP; [25]) (Figure 3). In contrast to wild-type proprioceptive afferent projections (Figure 3A–3C), GFP+ sensory afferents in TauEWS-Pea3/+ Isl1Cre/+ embryos failed to invade the spinal cord and instead were found in an extreme lateral position at the dorsal root entry zone, a phenotype observed at least up to E18.5 (Figure 3A–3C and 3G–3I; data not shown). We next visualized the path of sensory afferent projections towards the dorsal root entry zone in TauEWS-Pea3/+ Isl1Cre/+ embryos by injecting fluorescently labeled dextran into an individual DRG (L3; n = 3; Figure 3M–3Q). Sensory afferents in E13.5 wild-type embryos bifurcated at their lateral spinal entry point, and projected rostrally and caudally over six or more segmental levels while gradually approaching the midline (Figure 3M). Sensory afferents in TauEWS-Pea3/+ Isl1Cre/+ embryos also bifurcated at the entry point, although approximately 5% of afferent fibers continued to grow towards the midline (Figure 3O and 3Q). While rostro-caudal projections were present in TauEWS-Pea3/+ Isl1Cre/+ embryos, afferent fibers failed to approach the midline at distal segments and continued to occupy an extreme lateral position (Figure 3O), consistent with the analysis of transverse sections. + +We next examined the establishment of peripheral projections upon precocious EWS-Pea3 expression in DRG neurons. While sensory axons in TauEWS-Pea3/+ Isl1Cre/+ embryos reached the skin and established major nerve trunks by E16.5, only rudimentary sensory axon branching was established within the skin (Figure 3D and 3J). In addition, there was a significant reduction in the number of muscle spindles in TauEWS-Pea3/+ Isl1Cre/+ embryos (approximately 25% of wild-type complement; n = 3) as assessed by innervation and expression of genes specific for intrafusal muscle fibers such as Egr3 (Figure 3E, 3F, 3K, and 3L; [26]). In summary, whereas isochronic expression of EWS-Pea3 promoted the establishment of proprioceptive afferent projections into the ventral spinal cord, precocious expression of the same ETS signaling factor in DRG neurons interfered with establishment of projections into the spinal cord as well as to peripheral targets. + +Precocious EWS-Pea3 Expression Promotes Neurotrophin-Independent Survival and Neurite Outgrowth + +To begin to address the cellular and molecular mechanisms involved in the distinct biological actions of EWS-Pea3 at different developmental stages, we first turned to in vitro culture experiments. These experiments permit assessment of whether precocious ETS transcription factor signaling influences neuronal survival and in vitro neurite outgrowth of DRG neurons, two parameters prominently influenced by target-derived neurotrophic factors and their receptors. + +We cultured E13.5 whole DRG explants from wild-type and TauEWS-Pea3/+ Isl1Cre/+ embryos in the presence of NGF or NT-3 or in the absence of neurotrophins and analyzed neuronal survival and neurite outgrowth on matrigel substrate after 48 h in vitro. Without neurotrophic support, very few wild-type DRG neurons survived (Figure 4A). In contrast, culturing wild-type DRG with neurotrophic factors led to neuronal survival and neurite outgrowth. Addition of NGF, which supports survival of cutaneous afferents, resulted in straight and unbranched neurite outgrowth (Figure 4B), while cultures grown in the presence of NT-3, which supports survival of proprioceptive afferents, resulted in a highly branched neurite outgrowth pattern after 48 h in vitro (Figure 4C). Surprisingly, DRG neurons isolated from TauEWS-Pea3/+ Isl1Cre/+ embryos and cultured without neurotrophic support survived after 48 h in vitro and had established long and highly branched neurites (Figure 4D). Neither the pattern of neurite outgrowth nor neuronal survival changed significantly after application of either NGF or NT-3 (Figure 4E and 4F). + +To directly compare neurotrophin dependence of DRG neurons expressing EWS-Pea3 from the Tau locus at a precocious versus isochronic time of onset, we generated a strain of mice in which Cre recombinase is expressed from the PV locus (Figure S4). The expression of GFP in TaumGFP/+ PVCre/+ was restricted to PV+ proprioceptive DRG neurons and mirrored the onset of expression of PV at approximately E14 (Figure S4; data not shown). We next cultured E14.5 whole DRG explants from TauEWS-Pea3/+ PVCre/+ and TaumGFP/+ PVCre/+ mice for 48 h in vitro in the presence or absence of NT-3 (Figure 5). We found that DRG neurons from both genotypes survived and extended neurites only in the presence of NT-3, whereas they died in the absence of NT-3 (Figure 5). Together, these findings suggest that only precocious but not isochronic ETS signaling in DRG neurons is capable of uncoupling survival and neurite outgrowth from a requirement for neurotrophin signaling normally observed in wild-type DRG. + +To determine whether neuronal survival of DRG neurons from TauEWS-Pea3/+ Isl1Cre/+ embryos in the absence of neurotrophic support is sufficient to explain the observed neuronal outgrowth, we analyzed DRG isolated from mice mutant in the proapoptotic gene Bax [27]. Consistent with previous results, Bax−/− DRG neurons survived without neurotrophic support [28]. In contrast, neurite outgrowth of Bax−/− DRG neurons was significantly less (see Figure 4G) than that of either DRG from TauEWS-Pea3/+ Isl1Cre/+ embryos cultured in the absence of neurotrophic support (see Figure 4D) or Bax−/− DRG neurons cultured in the presence of neurotrophic support (see Figure 4H and 4I). These findings suggest that in addition to mediating neurotrophin-independent neuronal survival, expression of EWS-Pea3 in early post-mitotic neurons also promotes neurite outgrowth in a neurotrophin-independent manner. + +To begin to assess at which step of the neurotrophin signaling cascade DRG neurons from TauEWS-Pea3/+ Isl1Cre/+ embryos become unresponsive to the addition of neurotrophins, we assayed the expression of neurotrophin receptors in TauEWS-Pea3/+ Isl1Cre/+ embryos (Figure 6). Whereas expression of the neurotrophin receptors TrkA, TrkB, and TrkC marks afferents of distinct sensory modalities in DRG of wild-type embryos (Figure 6A–6C) [4,29], TauEWS-Pea3/+ Isl1Cre/+ embryos showed complete absence of expression of TrkA, TrkB, and TrkC in DRG neurons at E16.5 (Figure 6G–6I). This absence of Trk receptor expression in DRG of TauEWS-Pea3/+ Isl1Cre/+ embryos provides a likely explanation for the lack of responsiveness of these neurons to the addition of neurotrophic factors. + +We next assayed whether the complete absence of Trk receptor expression in TauEWS-Pea3/+ Isl1Cre/+ embryos had an influence on naturally occurring cell death in vivo using TUNEL on DRG sections. Surprisingly, we found that apoptosis was decreased by approximately 50% (n = 3 embryos, average of >50 sections) in DRG of TauEWS-Pea3/+ Isl1Cre/+ embryos in comparison to wild-type (Figure 6D, 6J, and 6M). Quantitative analysis of the number of neurons in lumbar DRG of TauEWS-Pea3/+ Isl1Cre/+ embryos revealed a significant increase to approximately 170% of wild-type levels (Figure 6E, 6K, and 6N). Moreover, BrdU pulse-chase experiments ruled out the possibility that DRG neurons in TauEWS-Pea3/+ Isl1Cre/+ embryos reenter the cell cycle (no BrdU+/LacZ+ cells, n = 3 embryos, analysis of >50 sections each; Figure 6F and 6L). Together with the in vitro culture experiments, these findings suggest that DRG neurons from TauEWS-Pea3/+ Isl1Cre/+ embryos remain post-mitotic but fail to become sensitive to naturally occurring cell death, and survive in the absence of Trk receptors and neurotrophic support. + +We next analyzed whether changes in the expression of proteins known to be involved in the regulation of neuronal survival or cell death could be detected in DRG of TauEWS-Pea3/+ Isl1Cre/+ embryos. We found no significant quantitative changes in the level of Akt/p-Akt or CREB/p-CREB in DRG (Figure 6O and 6P) both of which have been shown to be key regulators of neuronal survival [29]. Moreover, the level of the proapoptotic Bcl2 family member Bax was not significantly reduced (Figure 6O and 6P). In contrast, the expression level of the anti-apoptotic Bcl2 family members Bcl-xl and Bcl2 was significantly increased when compared to wild-type levels (Bcl2, 157%; Bcl-xl, 259%; average of n = 3 independent experiments; Figure 6O and 6P), providing a potential molecular explanation for the enhanced neuronal survival of DRG neurons of TauEWS-Pea3/+ Isl1Cre/+ embryos in the absence of Trk receptor expression [30]. + +Only Precocious but Not Isochronic ETS Signaling in DRG Neurons Interferes with Neuronal Fate Acquisition + +The observed differences in neuronal survival and neurite outgrowth between precocious and isochronic expression of EWS-Pea3 prompted us to perform a direct comparative analysis of gene expression between mice with precocious EWS-Pea3 expression and mice in which the expression of EWS-Pea3 is initiated in DRG sensory neurons from the time of normal onset of Er81 expression. Moreover, to rule out the possibility that a differential effect may be due to the different genetic strategies by which expression of EWS-Pea3 in proprioceptive afferents is achieved, we performed this analysis both in Er81EWS-Pea3/− and TauEWS-Pea3/+ PVCre/+ embryos. + +We first analyzed expression of TrkC, a gene downregulated in DRG neurons of TauEWS-Pea3/+ Isl1Cre/+ embryos (Figure 7A and 7B). The level of expression of TrkC was indistinguishable from wild-type in DRG neurons of Er81EWS-Pea3/− and TauEWS-Pea3/+ PVCre/+ embryos (Figure 7A, 7C, and 7D). Moreover, PV was not expressed in DRG neurons of TauEWS-Pea3/+ Isl1Cre/+ embryos (Figure S5) but was expressed by proprioceptive afferents in both wild-type and Er81EWS-Pea3/− embryos (see Figures 1 and S5) [14]. We also found several genes that were ectopically upregulated in DRG neurons of TauEWS-Pea3/+ Isl1Cre/+ embryos (Figure 7). Calretinin and Calbindin, two different calcium-binding proteins expressed by subpopulations of DRG neurons in wild-type, Er81EWS-Pea3/−, and TauEWS-Pea3/+ PVCre/+ embryos (Figure 7E, 7G, and 7H; data not shown) [31,32], were induced in more than 95% of all DRG neurons of TauEWS-Pea3/+ Isl1Cre/+ embryos (Figures 7F and S5). These findings suggest that DRG neurons in TauEWS-Pea3/+ Isl1Cre/+ embryos fail to differentiate to a normal fate and instead acquire an aberrant identity distinct from any subpopulation of wild-type DRG neurons. Finally, to assess whether EWS-Pea3 expressed precociously acts exclusively cell-autonomously or whether it may also influence neighboring DRG neurons, we activated expression of EWS-Pea3 using Hb9Cre mice [33]. Due to a transient and rostro-caudally graded expression of Hb9 at neural plate stages, very few DRG neurons at brachial levels and increasingly more neurons progressing caudally undergo recombination in TauEWS-Pea3/+ Hb9Cre/+ and TaumGFP/+ Hb9Cre/+ embryos (Figure 8). Nevertheless, downregulation of Trk receptor expression or upregulation of Calretinin is restricted exclusively to neurons that have undergone recombination and cannot be observed in TaumGFP/+ Hb9Cre/+ embryos (Figure 8). Together, these results and the findings obtained from in vitro culture experiments (see Figures 4 and 5) demonstrate that precocious or isochronic expression of EWS-Pea3 in the same neurons leads to significantly different cell-autonomous cellular responses with respect to gene expression, neuronal survival, and neurite outgrowth (Figure 9). + +Discussion + +Target-derived signals exhibit a conserved role in the induction of defined programs of transcription factor expression late in post-mitotic neuronal differentiation [10]. This study provides evidence that the late onset of transcription factor expression is essential for many later aspects of neuronal differentiation and circuit formation. Our data indicate that DRG neurons undergo a temporal change in their competence to respond to ETS transcription factor signaling, as assessed by changes in gene expression and axonal target invasion (Figure 9). Our findings argue for the necessity of target-induced, and therefore temporally controlled, upregulation of ETS transcription factor signaling. More generally, they suggest that temporally regulated activation of transcriptional programs coupled to a particular fate induced in neurons at early developmental stages represents an important mechanism of neuronal maturation. + +One striking observation of this study is that precocious induction of ETS signaling promotes neuronal survival without a requirement for neurotrophic support and in complete absence of Trk receptor expression. In contrast, ETS signaling at the normal time of onset of Er81 expression does not result in enhanced neuronal survival in the absence of neurotrophins and also does not lead to downregulation of TrkC expression in proprioceptive afferents. These findings demonstrate very distinct activities of one transcriptional regulator at different developmental steps within a committed post-mitotic neuronal lineage. The absence of Trk receptor expression upon precocious induction of ETS signaling can only partially explain the observed phenotype in axonal projections. Elimination of TrkA receptor signaling in Bax mutant mice perturbs establishment of peripheral projections of cutaneous afferents, whereas establishment of central projections does not appear to be affected [8]. In the absence of NT-3 signaling, development of central as well as peripheral proprioceptive afferent projections is perturbed [9]. In contrast, upon precocious induction of ETS signaling, we found more pronounced defects in the establishment of central rather than peripheral projections for all DRG neurons. + +Induction of Er81 expression in proprioceptive afferents is controlled by peripheral NT-3 as axons reach the vicinity of target muscles, and thus occurs only approximately 3 d after proprioceptive neurons become post-mitotic [9,14]. This temporally delayed and target-induced upregulation of ETS transcription factor expression several days after a neuronal lineage of a specific identity first emerges is not restricted to DRG sensory neurons, but is also found in motor neuron pools [13]. Target-derived factors have also been implicated in controlling neuronal maturation of predetermined neurons in Drosophila, in which expression of members of the BMP family in the target region is essential for the induction of mature peptidergic properties in a subpopulation of neurons marked by the coordinate expression of the two transcription factors Apterous and Squeeze [11,12]. Thus, both in Drosophila and vertebrates, target-derived factors appear to act permissively to induce the expression of transcriptional programs involved in terminal neuronal maturation. + +Our findings are compatible with a model in which DRG neurons acquire their mature fate by sequential and temporally controlled addition of lineage-specific features (Figure 9). Target-derived factors act on predetermined neuronal lineages to switch their developmental programs to become compatible with processes such as target invasion and branching. Such a transition state in the acquisition of a defined neuronal fate would be accompanied by the induction of appropriate transcriptional programs through the expression of specific transcription factors. Mechanisms such as chromosomal remodeling that restrict or expand access to certain target genes [34] or activation by cofactors responsible for changing the action of particular transcription factors [35] could represent possible mechanisms by which the downstream transcriptional profile of a transcription factor could be temporally shifted towards the selection and control of distinct target genes. Interestingly, several ETS transcription factors are activated through release of autoinhibition via interaction with cofactors and/or via post-translational modifications [35,36,37]. The fusion of EWS with Pea3 could circumvent a need for activation through specific cofactors while still maintaining ETS site dependence, thus rendering EWS-Pea3 less sensitive to the cellular context than endogenous ETS transcription factors. Using this fusion protein, our experiments demonstrate a profound change in the action of ETS signaling at the level of transcriptional regulation within post-mitotic DRG neurons over time. Moreover, the observed transcriptional shift in ETS signaling is paired with the onset of appropriate regulation of neuronal subtype specification and establishment of axonal projections into the target area. + +Recent experiments addressing the temporal constraints of transcription factor action in proliferating neural progenitor cells adds to the idea that defined temporal windows, during which transcription factors act to control distinct downstream target genes, are of key importance to neuronal fate acquisition. During Drosophila neuroblast generation, the transcription factor Hunchback controls specification and differentiation of early-born neuroblasts [38]. Over time, however, neuroblasts progressively lose their competence to generate cells of an early fate in response to Hunchback expression [39]. These findings thus also argue for a change in cellular competence to respond to a specific transcription factor over time albeit in an early precursor context. More generally, during the differentiation of hematopoietic lineages, several transcription factors have also been shown to exhibit distinct functions at progressive steps of lineage specification [40]. Analysis of the mechanisms by which transcription factor programs can be shifted over time to control different complements of downstream genes and thus different aspects of neuronal and cellular fates in progenitor cells or post-mitotic neurons may provide further insight into the way in which transcription factors act to control the assembly of neuronal circuits. + +Materials and Methods + + + +Generation of transgenic mice and mouse genetics + +Er81EWS-Pea3 mice were generated following a strategy similar to that described for the generation of Er81NLZ mice [14]. In brief, a targeting vector with a cDNA coding for EWS-Pea3 was inserted in frame with the endogenous start ATG into exon 2 of the Er81 genomic locus and used for homologous recombination in W95 ES cells. EWS-Pea3 represents a fusion gene between the amino terminal of EWS and the ETS domain of Pea3 [20]. The primer pair used to specifically detect the Er81EWS-Pea3 allele was 5′-CAGCCACTGCACCTACAAGAC-3′ and 5′-CTTCCTGCTTGATGTCTCCTTC-3′. For the generation of TaumGFP and TauEWS-Pea3 mice, lox-STOP-lox-mGFP-IRES-NLS-LacZ-pA and lox-STOP-lox-EWS-Pea3-IRES-NLS-LacZ-pA targeting cassettes were integrated into exon 2 of the Tau genomic locus (the endogenous start ATG was removed in the targeting vectors; details available upon request). mGFP was provided by P. Caroni [25]. ES cell recombinants were screened by Southern blot analysis using the probe in the 5′ region as described previously [41]. Frequency of recombination in 129/Ola ES cells was approximately 1/3 for both Tau constructs. For the generation of PVCre mice, mouse genomic clones were obtained by screening a 129SV/J genomic library (Incyte, Wilmington, Delaware, United States). For details on the genomic structure of the mouse PV locus see [42]. An IRES-Cre-pA targeting cassette [33] was integrated into the 3′ UTR of exon 5, and ES cell recombinants were screened with a 5′ probe (oligos, 5′-GAGATGACCCAGCCAGGATGCCTC-3′ and 5′-CTGACCACTCTCGCTCCGGTGTCC-3′; genomic DNA, HindIII digest). The frequency of recombination in 129/Ola ES cells was approximately 1/20. Recombinant clones were aggregated with morula stage embryos to generate chimeric founder mice that transmitted the mutant alleles. In all experiments performed in this study, animals were of mixed genetic background (129/Ola and C57Bl6). Thy1spGFP transgenic mice were generated in analogy to De Paola et al. [25], and for these experiments a strain of mice with early embryonic expression was selected. Isl1Cre and Hb9Cre mouse strains have been described [33,43] and Bax+/− animals were from Jackson Laboratory (Bar Harbor, Maine, United States) [27]. Timed pregnancies were set up to generate embryos of different developmental stages with all genotypes described throughout the study. + +Transcriptional transactivation assays + +The following plasmids were used for transcriptional transactivation assays: pRc/RSV (Invitrogen, Carlsbad, California, United States), pRc/RSV-Er81, pRc/RSV-EWS-Pea3, pTP-5xETS, and pTP-5xETSmut. pRc/RSV-Er81 and pRc/RSV-EWS-Pea3 were obtained by insertion of the cDNAs for Er81 or EWS-Pea3 (gift from J. A. Hassell) into pRc/RSV. pTP-5xETS was constructed by inserting a cassette of five repetitive copies of high-affinity Pea3 binding sites (5′-GCCGGAAGC-3′) [18,19] into a modified version of pTK-Luc. pTP-5xETSmut was generated as pTP-5xETS but using a mutated complement of the Pea3 binding sites (5′-GCCTATGGC-3′). A control plasmid to normalize for transfection efficiency (placZ) and pTK-Luc were a gift from D. Kressler. COS-7 cells were co-transfected with 1–1.2 μg of total DNA including one of the effector plasmids pRc/RSV-empty, pRc/RSV-Er81, or pRc/RSV-EWS-Pea3; one of the reporter plasmids pTP-5xETS or pTP-5xETSmut; and placZ. Cells were harvested after 25 h and processed for assays to determine luciferase and LacZ activity as described previously [44]. Luciferase values normalized to LacZ activity are referred to as luciferase units. + +In situ hybridization and immunohistochemistry + +For in situ hybridization analysis, cryostat sections were hybridized using digoxigenin-labeled probes [45] directed against mouse TrkA or TrkB, or rat TrkC (gift from L. F. Parada). Antibodies used in this study were as follows: rabbit anti-Er81 [14], rabbit anti-Pea3 [14], rabbit anti-PV [14], rabbit anti-eGFP (Molecular Probes, Eugene, Oregon, United States), rabbit anti-Calbindin, rabbit anti-Calretinin (Swant, Bellinzona, Switzerland), rabbit anti-CGRP (Chemicon, Temecula, California, United States), rabbit anti-vGlut1 (Synaptic Systems, Goettingen, Germany), rabbit anti-Brn3a (gift from E. Turner), rabbit anti-TrkA and -p75 (gift from L. F. Reichardt), rabbit anti-Runx3 (Kramer and Arber, unpublished reagent), rabbit anti-Rhodamine (Molecular Probes), mouse anti-neurofilament (American Type Culture Collection, Manassas, Virginia, United States), sheep anti-eGFP (Biogenesis, Poole, United Kingdom), goat anti-LacZ [14], goat anti-TrkC (gift from L. F. Reichardt), and guinea pig anti-Isl1 [14]. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) to detect apoptotic cells in E13.5 DRG on cryostat sections was performed as described by the manufacturer (Roche, Basel, Switzerland). Quantitative analysis of TUNEL+ DRG cells was performed essentially as described [27]. BrdU pulse-chase experiments and LacZ wholemount stainings were performed as previously described [46]. For anterograde tracing experiments to visualize projections of sensory neurons, rhodamine-conjugated dextran (Molecular Probes) was injected into single lumbar (L3) DRG at E13.5 or applied to whole lumbar dorsal roots (L3) at postnatal day (P) 5 using glass capillaries. After injection, animals were incubated for 2–3 h (E13.5) or overnight (P5). Cryostat sections were processed for immunohistochemistry as described [14] using fluorophore-conjugated secondary antibodies (1:1,000, Molecular Probes). Images were collected on an Olympus (Tokyo, Japan) confocal microscope. Images from in situ hybridization experiments were collected with an RT-SPOT camera (Diagnostic Instruments, Sterling Heights, Michigan, United States), and Corel (Eden Prairie, Minnesota, United States) Photo Paint 10.0 was used for digital processing of images. + +In vitro cultures of DRG + +Individual lumbar DRG were dissected from E13.5 or E14.5 embryos and placed on Matrigel (BD Biosciences, San Jose, California, United States) coated coverslips in DMEM/F12 (Gibco, San Diego, California, United States), 2 mM L-Gln (Gibco), N2 (Gibco), and 1 mg/ml BSA (Sigma, St. Louis, Missouri, United States) without neurotrophins, or supplemented with either NGF (100 ng/ml, Gibco) or NT-3 (20 ng/ml, Sigma). DRG explants (n ≥ 20 for each condition) were cultured for 48 h, processed for immunocytochemistry, and analyzed using confocal microscopy. + +Western blot analysis + +Lumbar DRG from E16.5 embryos were isolated, mechanically disrupted, homogenized using glass beads (Sigma), and lysed in standard lysis buffer supplemented with protease and phosphatase inhibitors as described [47]. Protein extracts were resolved by SDS-PAGE, and immunoblotting was performed using antibodies against Akt, p-Akt (Ser473), CREB, p-CREB (Ser133), Bax, Bcl-xl (Cell Signaling Technology, Beverly, Massachusetts, United States), and Bcl2 (BD Pharmingen, San Diego, California, United States). For quantification, films (X-OMAT AR, Eastman Kodak, Rochester, New York, United States) were scanned and densitometry was performed using IMAGEQUANT 5.2 (Molecular Dynamics, Amersham, Uppsala, Sweden). + +Electrophysiology + +Electrophysiological analysis was performed as previously described [48]. Briefly, intracellular recordings from identified quadriceps motor neurons were made using sharp electrodes (75–120 MΩ, 3M KCl). Average responses (10–20 trials) from suprathreshold nerve stimulation (1.5 times the strength that evokes maximal monosynaptic response) of the quadriceps nerve were acquired with LTP software [49]. Only cells with stable resting potentials more negative than −50 mV were considered for analysis. Monosynaptic amplitudes were determined offline using custom routines in the Matlab environment (The Mathworks, Natick, Massachusetts, United States) as previously described [48]. + +Supporting Information + +Figure S1 + +Gene Expression Analysis in DRG Neurons of Er81EWS-Pea3 Mice + +Analysis of TrkC expression by in situ hybridization (A and E), and Runx3 (red; B and F), Brn3A (red; C and G), Isl1 (red; D and H), p75 (red; I and M), TrkA (red; J and N), CGRP (red; K and O), Calretinin (CR; red; L and P), and LacZ (green; B–D and F–P) by immunohistochemistry on E16.5 lumbar DRG of Er81NLZ/+ (A–D and I–L) and Er81EWS-Pea3/− (E–H and M–P) embryos. + +Scale bar: (A, B, E, F, L, and P), 70 μm; (C, D, G, H, I–K, and M–O), 75 μm. + +(4.9 MB CDR). + +Click here for additional data file. + +Figure S2 + +Ia Proprioceptive Afferents Make Functional Connections with Motor Neurons in Er81EWS-Pea3 Mutants + +(A and B) Representative traces from intracellular recordings measuring Ia afferent monosynaptic input to quadriceps motor neurons evoked by suprathreshold stimulation of the quadriceps nerve in wild-type (A) and Er81EWS-Pea3/− mutant (B) animals. + +(C) Average monosynaptic amplitudes (± standard error of the mean) from all recorded cells (wild-type, n = 11; mutant, n = 8). + +(20 KB CDR). + +Click here for additional data file. + +Figure S3 + +Generation of Mice Expressing EWS-Pea3 or mGFP in Early Post-Mitotic DRG Neurons + +(A) Top panel shows organization of the Tau genomic locus in the region targeted by homologous recombination in analogy to Tucker et al. [41]. Exons 1–3 are shown as light blue boxes, and the Tau start codon in exon 2 is indicated as ATG. The probe used to detect homologous recombination is shown as a grey box. Middle and bottom panels show Tau locus after homologous recombination to integrate targeting cassettes (green) into exon 2 with coincident elimination the endogenous Tau start codon. The integrated targeting cassettes allow for conditional expression of EWS-Pea3 and NLS-LacZ (NLZ) (middle) or mGFP and NLS-LacZ (bottom) upon Cre-recombinase-mediated activation. In the absence of Cre recombinase, a transcriptional stop sequence flanked by loxP sites inhibits expression of the respective transgenes from their start codons (ATG in grey). + +(B) Southern blot analysis of TauEWS-Pea3/+ and TaumGFP/+ genomic DNA to detect the mutant allele. + +(C) In the presence of Cre recombinase, the transcriptional stop sequence in the cassettes integrated into the Tau locus is removed. Expression of EWS-Pea3 and NLS-LacZ (top) or mGFP and NLS-LacZ (bottom) can now occur in neurons coincidently expressing Cre recombinase and Tau (indicated as ATG in green). + +(D–L) Expression of Isl1 (D, G, and J), EWS-Pea3 (E and H), GFP (K), or LacZ (F, I, and L), in E12 (D–I) or E13.5 (J–L) DRG neurons of wild-type (D–F), TauEWS-Pea3/+ Isl1Cre/+ (G–I), and TaumGFP/+ Isl1Cre/+ (J–L) embryos. + +Scale bar: (D–F), 40 μm; (G–I), 35 μm; (J–K), 50 μm. + +(1.5 MB CDR). + +Click here for additional data file. + +Figure S4 + +Generation of PVCre Mice + +(A) Above is the organization of the PV genomic locus. Exons are schematically illustrated as light blue boxes, where exon 2 contains the start codon (ATG) and exon 5 contains the stop codon (STOP). Probe to screen for homologous recombination is shown as grey box. Below is a schematic diagram to show the PV locus after the integration of an IRES-Cre cassette (green) 3′ to the translational stop codon of PV using homologous recombination in ES cells. + +(B) Southern blot analysis of PVCre wild-type (+/+) and heterozygous (+/−) genomic DNA using the probe indicated in (A). + +(C and D) Expression of GFP (green) and LacZ (red; C) or PV (red; D) in P0 TaumGFP/+ PVCre/+ mice. Note that more than 90% of PV+ neurons coexpress GFP (D; data not shown). + +Scale bar: (C and D), 50 μm. + +(2 MB CDR). + +Click here for additional data file. + +Figure S5 + +Gene Expression Analysis upon Precocious Induction of EWS-Pea3 in DRG Neurons + +Immunohistochemical analysis of PV (A and D), Calretinin (B and E), and Calbindin (C and F) expression on E16.5 lumbar DRG of wild-type (A–C) and TauEWS-Pea3/+ Isl1Cre/+ (D–F) embryos. + +Scale bar: 80 μm. + +(950 KB CDR). + +Click here for additional data file. + +Acknowledgements + +The generation and initial characterization of Er81EWS-Pea3/− mice was performed in the laboratory of Thomas Jessell at Columbia University, with expert assistance from Barbara Hahn and Monica Mendelsohn. We thank Thomas Jessell for encouragement and many stimulating discussions and thank him and Pico Caroni for helpful comments on the manuscript. We thank Jean-Francois Spetz, Patrick Kopp, Bernard Kuchemann, and Monika Mielich for excellent technical assistance, Ina Kramer for providing Runx3 antibodies, Pico Caroni for providing mGFP cDNA, John A. Hassell for providing EWS-Pea3 cDNA and fruitful discussions on ETS gene function, and Y. A. Barde and K. Tucker for providing the Tau targeting construct. SH, EV, MS, TP, CL, DRL, and SA were supported by a grant from the Swiss National Science Foundation, by the Kanton of Basel-Stadt, and by the Novartis Research Foundation. In addition, DRL was supported by a research fellowship from the Roche Research Foundation. + +Conflict of interest. The authors have declared that no conflicts of interests exist. + +Abbreviations + +DRG - dorsal root ganglion/ganglia + +E - embryonic day + +EWS - Ewing sarcoma + +mGFP - membrane-targeted green fluorescent protein + +NT-3 - neurotrophin 3 + +P - postnatal day + +PV - Parvalbumin + +spGFP - synaptophysin green fluorescent protein + +Figures and Tables + +Figure 1 + +Replacement of Er81 by EWS-Pea3 + +(A) Generation of Er81EWS-Pea3 mutant mice. Above is the organization of the Er81 genomic locus in the region targeted by homologous recombination in analogy to [14]. Exons 1–4 are shown as light blue boxes, and the Er81 start codon in exon 2 is indicated as ATG. The probe used to detect homologous recombination is shown as a grey box. Below is replacement of Er81 by EWS-Pea3 through the integration of EWS-Pea3 in frame with the endogenous start codon of the Er81 locus in exon 2 (in analogy to [14]). + +(B) PCR and Southern blot analysis of Er81EWS-Pea3 wild-type (+/+), heterozygous (+/−), and homozygous (−/−) genomic DNA to detect the mutant allele. PCR primer pairs (EWS-Pea3ki) were used to detect specifically the recombined allele, and a primer pair in exon2 was used to detect the presence of the wild-type allele [14]. + +(C–E) Analysis of Er81 expression in lumbar DRG neurons of E16.5 wild-type (C), Er81−/− (D), and Er81EWS-Pea3/− (E) embryos. Inset in lower right corner of each panel shows Isl1 expression in the respective DRG. + +(F–H) PV expression in lumbar DRG of E16.5 wild-type (F), Er81−/− (G), and Er81EWS-Pea3/− (H) embryos. Confocal scans were performed with equal gain intensity. + +(J) Transcriptional transactivation of luciferase expression from a minimal reporter construct containing five consensus ETS DNA-binding sites (GCCGGAAGC; [18,19]) and a minimal TK promoter upon transient transfection of Er81 (n ≥ 7; 3.03 ± 0.66) or EWS-Pea3 (n ≥ 7; 20.3 ± 2.7). Relative luciferase activity normalized to control (Con). + +Scale bar: 80 μm. + +Figure 2 + +Rescue of Ia Proprioceptive Afferent Projections into the Ventral Spinal Cord in Er81EWS-Pea3 Mutants + +(A–F) Morphological analysis of central projections at lumbar level L3 of PV+ DRG neurons (A–C) or all DRG sensory afferents after application of fluorescently labeled dextran to individual dorsal roots (D–F) in P0.5 (A–C) or P5 (D–F) wild-type (A and D), Er81−/− (B and E), and Er81EWS-Pea3/− (C and F) mice. Red dotted line indicates intermediate level of spinal cord. + +(G–I) Analysis of vGlut1 immunocytochemistry in the ventral horn of P0.5 wild-type (G), Er81−/− (H), and Er81EWS-Pea3/− (I) mice. Yellow dotted box in (A) indicates size of images shown in (G–I). + +(J–L) Schematic summary diagrams of the morphological rescue of Ia proprioceptive afferent projections (blue) into the ventral spinal cord observed in wild-type (J), Er81−/− (K), and Er81EWS-Pea3/− (L) mice. DRG indicated by dotted grey line; motor neurons are shown in black. + +Scale bar: (A–C), 150 μm; (D–F), 160 μm; (G–I), 70 μm. + +Figure 3 + +Defects in the Establishment of Sensory Afferent Projections upon Precocious Expression of EWS-Pea3 in DRG Neurons + +(A–C and G–I) Visualization of sensory afferent projections (green) into the spinal cord of wild-type (A–C) and TauEWS-Pea3/+ Isl1Cre/+ (G–I) embryos at E13.5 (A, C, G, and I) and E16.5 (B and H) by Cre-recombinase-mediated activation of mGFP expression from the Tau locus (A, B, G, and H) or by a Thy1spGFP transgene (C and I; [25]). Grey arrows indicate normal pattern of afferent projections into the spinal cord, whereas red arrows show aberrant accumulation of sensory afferents at the lateral edge of the spinal cord in TauEWS-Pea3/+ Isl1Cre/+ embryos. + +(D–F and J–L) Analysis of sensory afferent projections (green) into the skin (D and J) or muscle (E and K; red, α-Bungarotoxin, BTX) of wild-type (D–F) and TauEWS-Pea3/+ Isl1Cre/+ (J–L) embryos at E16.5 by Cre-recombinase-mediated activation of mGFP (D, E, J, and K) expression from the Tau locus. (F and L) show Egr3 expression in intrafusal muscle fibers using in situ hybridization (consecutive sections to [E and K] are shown). + +(M–Q) Analysis of bifurcation of sensory afferent projections towards the spinal cord in E13.5 wild-type (M) and TauEWS-Pea3/+ Isl1Cre/+ (O and Q) embryos after injection of fluorescently labeled dextran (green) into one DRG (lumbar level L3). Confocal scanning plane for (M and O) is schematically illustrated in (N). Inset in (O) is also shown at a deeper confocal scanning plane (P and Q) to visualize aberrant axonal projections. + +Scale bar: (A and G), 60 μm; (B and H), 80 μm; (C and I), 100 μm; (D and J), 160 μm; (E, F, K, and L), 70 μm; (M, O, and Q), 240 μm. + +Figure 4 + +Neurotrophin-Independent Neurite Outgrowth In Vitro of DRG Neurons Expressing EWS-Pea3 Precociously + +E13.5 lumbar DRG from wild-type (A, B, and C), TauEWS-Pea3/+ Isl1Cre/+ (D, E, and F), or Bax−/− (G, H, and I) embryos cultured for 48 h without neurotrophic support (A, D, and G) or in the presence of NGF (B, E, and H) or NT-3 (C, F, and I) were stained for expression of neurofilament to visualize axonal extensions. + +Scale bar: 130 μm. + +Figure 5 + +DRG Neurons Expressing EWS-Pea3 Isochronically Depend on Neurotrophins for Survival + +E14.5 lumbar DRG from TaumGFP/+ PVCre/+ (A and B) and TauEWS-Pea3/+ PVCre/+ (C and D) embryos cultured for 48 h without neurotrophic support (A and C) or in the presence of NT-3 (B and D) were stained for expression of neurofilament (red) and LacZ (green) to visualize axonal extensions and survival of PV-expressing proprioceptive afferents. + +Scale bar: 150 μm. + +Figure 6 + +Loss of Trk Receptor Expression and Increased Survival in DRG Neurons upon Precocious ETS Signaling + +(A–C and G–I) In situ hybridization analysis of TrkA (A and G), TrkB (B and H), and TrkC (C and I) expression in E16.5 lumbar DRG of wild-type (A–C) and TauEWS-Pea3/+ Isl1Cre/+ (G–I) embryos. + +(D–F and J–L) Analysis of lumbar DRG of wild-type (D), TaumGFP/+ IslCre/+ (E and F), and TauEWS-Pea3/+ Isl1Cre/+ (J, K, and L) embryos for (1) neuronal cell death at E13.5 by TUNEL (green; D and J), (2) cell survival and proliferation at E16.5 by LacZ (blue) wholemount staining (E and K; lumbar levels L1 and L2 are shown), and (3) BrdU (green)/LacZ (red) double labeling (F and L). + +(M and N) Quantitative analysis (n ≥ 3 independent experiments) of the mean number of apoptotic events relative to wild-type levels is shown in (M) and neuronal survival in (N) as percent of wild-type of DRG at lumbar levels L1 to L5 as quantified on serial sections. + +(O) Western blot analysis of protein extracts isolated from lumbar DRG of E16.5 wild-type (wt) and TauEWS-Pea3/+ Isl1Cre/+ (mut) embryos using the following antibodies: Akt, p-Akt (Ser473), CREB, p-CREB (Ser133), Bax, Bcl2, and Bcl-xl. + +(P) Quantitative analysis of protein levels relative to wild-type in percent is shown on the right (n = 3 independent experiments). + +Scale bar: (A–C and G–I), 35 μm; (D and J), 40 μm; (E and K), 200 μm; (F and L), 50 μm. + +Figure 7 + +Gene Expression Analysis upon Induction of Precocious or Isochronic ETS Signaling + +(A–H) Analysis of TrkC expression by in situ hybridization (A–D), or Calretinin (red) and LacZ (green) expression by immunohistochemistry (E–H), on E16.5 lumbar DRG of wild-type (A and E), TauEWS-Pea3/+ Isl1Cre/+ (B and F), Er81EWS-Pea3/− (C and G), and TauEWS-Pea3/+ PVCre/+ (D and H) embryos. + +(I) Summary diagram illustrating deregulation of TrkC (red arrows, downregulation) and Calretinin (green arrows, upregulation) expression upon precocious (B and F) induction of EWS-Pea3 expression in DRG neurons (B and F; E10–E11, i.e., shortly after cell cycle exit, E9.5–E10). In contrast, activation of EWS-Pea3 from the endogenous Er81 locus (C and G; E12.5–E13) or via Cre recombinase expression from the PV locus activating late expression from the Tau locus (D and H; E14.5) does not interfere with the normal expression of TrkC and Calretinin (shown in grey). + +Scale bar: (A–D), 65 μm; (E–H), 80 μm. + +Figure 8 + +Precocious ETS Signaling Induces Gene Expression Changes Cell-Autonomously + +(A–D) Expression of TrkA (A and C; green) or TrkC (B and D; green), and LacZ (red), in E16.5 lumbar DRG of TaumGFP/+ Hb9Cre/+ (A and B) and TauEWS-Pea3/+ Hb9Cre/+ (C and D) embryos. + +(E–L) Expression of Calretinin (green), LacZ (red), and Isl1 (F, J, H, and L; blue) in E16.5 brachial (E–H) and lumbar (I–L) DRG of TaumGFP/+ Hb9Cre/+ (E, F, I, and J) and TauEWS-Pea3/+ Hb9Cre/+ (G, H, K, and L) embryos. + +Scale bar: (A–D), 80 μm; (E–L), 70 μm. + +Figure 9 + +Progressive Neuronal Specification Is Paralleled by a Developmental Shift in Response to ETS Transcription Factor Signaling + +Schematic summary diagram illustrating the importance of temporally appropriate upregulation of transcription factor expression during specification of DRG neurons for late aspects of neuronal differentiation and circuit assembly. + +(A–D) Expression of EWS-Pea3 from the endogenous Er81 locus can rescue anatomical defects observed in Er81−/− mice, and no change in expression of TrkC (green) or Calretinin (CR; grey) is observed in proprioceptive afferents (A, B, and D). In contrast, precocious ETS signaling leads to severe defects in the establishment of DRG neuronal projections accompanied by inappropriate gene expression changes (C; upregulation of CR (red) and downregulation of TrkC [grey]). + +(E) Precocious ETS signaling (red) during progressive specification of proprioceptive sensory neurons leads to aberrant neuronal differentiation (red dashed line). In contrast, the isochronic, target-induced (green; peripheral signal) onset of ETS transcription factor signaling (black) induces appropriate terminal neuronal differentiation (blue). + +Footnotes + +Author contributions. SH and SA conceived and designed the experiments. SH, EV, MS, TP, CL, DRL, and SA performed the experiments. SA wrote the paper. + +Citation: Hippenmeyer S, Vrieseling E, Sigrist M, Portmann T, Laengle C, et al. (2005) A developmental switch in the response of DRG neurons to ETS transcription factor signaling. PLoS Biol 3(5): e159. diff --git a/src/ontogpt/evaluation/craft/database/all/15850489.ann b/src/ontogpt/evaluation/craft/database/all/15850489.ann new file mode 100644 index 000000000..3225aafa9 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15850489.ann @@ -0,0 +1,834 @@ +T1 CHEBI:16865 0 4 GABA +T2 PR:000007778 0 25 GABAA receptor γ2 subunit +T3 NCBITaxon:10088 36 40 mice +T4 http://purl.obolibrary.org/obo/MONDO_0005618 55 62 anxiety +T5 CHEBI:22720 112 127 benzodiazepines +T6 CHEBI:16865 151 174 Gamma-aminobutyric acid +T7 CHEBI:16865 193 197 GABA +T8 NCBITaxon:40674 245 254 mammalian +T9 UBERON:0000955 255 260 brain +T10 GO:0065007 269 278 modulated +T11 CHEBI:35717 294 302;312 317 sedative ... drugs +T12 CHEBI:22720 328 343 benzodiazepines +T13 CHEBI:38867 348 359 anesthetics +T14 CHEBI:16865 390 394 GABA +T15 CHEBI:23888 445 449 drug +T16 PR:000007778 492 502 γ2 subunit +T17 GO:0010467 513 522 expressed +T18 UBERON:0000955 538 543 brain +T19 PR:000007778 552 554 γ2 +T20 NCBITaxon:10088 564 568 mice +T21 CHEBI:49575 612 620 diazepam +T22 GO:0016265 625 628 die +T23 UBERON:0012101 629 640 perinatally +T24 GO:0007567 633 640 natally +T25 PR:000007778 655 657 γ2 +T26 NCBITaxon:10088 674 678 mice +T27 http://purl.obolibrary.org/obo/MONDO_0005618 709 716 anxiety +T28 PR:000007778 772 782 γ2 subunit +T29 NCBITaxon:33208 805 811 animal +T30 CHEBI:23888 812 816 drug +T31 SO:0000704 833 837 gene +T32 NCBITaxon:10088 866 871 mouse +T33 PR:000007778 893 895 γ2 +T34 GO:0010467 896 906 expression +T35 PR:000007778 914 916 γ2 +T36 NCBITaxon:10088 927 931 mice +T37 NCBITaxon:10088 953 957 mice +T38 CHEBI:7507 986 994 neomycin +T39 SO:0005853 1006 1014 cassette +T40 SO:0000188 1020 1026 intron +T41 PR:000007778 1036 1038 γ2 +T42 SO:0000704 1039 1043 gene +T43 NCBITaxon:10088 1055 1059 mice +T44 PR:000007778 1099 1109 γ2 subunit +T45 PR:000007778 1145 1147 γ2 +T46 SO:0000704 1148 1152 gene +T47 GO:0010467 1148 1163 gene expression +T48 NCBITaxon:10088 1193 1197 mice +T49 PR:000007778 1276 1278 γ2 +T50 CHEBI:52217 1322 1337 Pharmacological +T51 UBERON:0000955 1378 1383 brain +T52 CHEBI:22720 1426 1440 benzodiazepine +T53 NCBITaxon:10088 1487 1491 mice +T54 NCBITaxon:10088 1538 1542 mice +T55 http://purl.obolibrary.org/obo/MONDO_0005618 1562 1569 anxiety +T56 NCBITaxon:10088 1670 1674 mice +T57 CHEBI:22720 1726 1740 benzodiazepine +T58 CHEBI:49575 1754 1762 diazepam +T59 CHEBI:6931 1764 1773 midazolam +T60 CHEBI:10125 1778 1786 zolpidem +T61 CHEBI:16236 1798 1805 ethanol +T62 CHEBI:7983 1810 1823 pentobarbital +T63 PR:000007778 1858 1860 γ2 +T64 NCBITaxon:10088 1871 1876 mouse +T65 PR:000007778 1904 1906 γ2 +T66 NCBITaxon:10088 1923 1927 mice +T67 GO:0010467 1965 1975 expressing +T68 NCBITaxon:10088 1976 1981 mouse +T69 CHEBI:7507 2037 2045 neomycin +T70 SO:0000704 2057 2061 gene +T71 SO:0000188 2067 2073 intron +T72 PR:000007778 2083 2085 γ2 +T73 SO:0000704 2086 2090 gene +T74 PR:000007778 2122 2124 γ2 +T75 GO:0010467 2149 2159 expression +T76 PR:000007778 2163 2165 γ2 +T77 http://purl.obolibrary.org/obo/MONDO_0005618 2176 2183 anxiety +T78 CHEBI:22720 2259 2273 benzodiazepine +T79 GO:0045202 2315 2323 synaptic +T80 http://purl.obolibrary.org/obo/MONDO_0005618 2372 2379 anxiety +T81 CHEBI:23888 2415 2419 drug +T82 CHEBI:16865 2478 2482 GABA +T83 CHEBI:16865 2508 2512 GABA +T84 NCBITaxon:40674 2559 2568 mammalian +T85 UBERON:0001017 2569 2591 central nervous system +T86 GO:0065007 2603 2612 modulated +T87 CHEBI:35717 2628 2636;2662 2667 sedative ... drugs +T88 CHEBI:16865 2673 2677 GABA +T89 CHEBI:24870 2777 2780 ion +T90 CHEBI:16865 2844 2848 GABA +T91 PR:000007766 2911 2913 α1 +T92 PR:000007772 2917 2919 β1 +T93 PR:000007777 2923 2925 γ1 +T94 PR:000007775 2929 2930 δ +T95 PR:000007776 2932 2933 ε +T96 PR:000007780 2935 2936 π +T97 PR:000007781 2942 2943 θ +T98 CHEBI:16865 2989 2993 GABA +T99 CHEBI:23888 3054 3058 drug +T100 SO:0000704 3132 3143 genetically +T101 NCBITaxon:10088 3155 3159 mice +T102 PR:000007778 3172 3182 γ2 subunit +T103 GO:0010467 3193 3202 expressed +T104 UBERON:0007023 3229 3234 adult +T105 UBERON:0000955 3235 3240 brain +T106 UBERON:0002240 3245 3256 spinal cord +T107 CHEBI:16865 3294 3298 GABA +T108 GO:0045202 3330 3338 synaptic +T109 GO:0043113 3339 3352;3359 3361 clustering of ... 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NCBITaxon:10088 9927 9932 mouse +T279 CL:0001031 10008 10031 cerebellar granule cell +T280 UBERON:0002956 10008 10037 cerebellar granule cell layer +T281 PR:000007778 10056 10058 γ2 +T282 CHEBI:16865 10085 10089 GABA +T283 CHEBI:37983 10103 10106 35S +T284 UBERON:0000955 10147 10152 brain +T285 CL:0001031 10238 10261 cerebellar granule cell +T286 UBERON:0002956 10238 10267 cerebellar granule cell layer +T287 CHEBI:16865 10280 10284 GABA +T288 NCBITaxon:33208 10368 10375 animals +T289 NCBITaxon:10088 10461 10465 mice +T290 http://purl.obolibrary.org/obo/MONDO_0005618 10589 10596 anxiety +T291 NCBITaxon:10088 10712 10716 mice +T292 http://purl.obolibrary.org/obo/MONDO_0005618 10866 10873 anxiety +T293 NCBITaxon:10088 10912 10916 mice +T294 GO:0040011 11015 11025 locomotion +T295 http://purl.obolibrary.org/obo/MONDO_0005618 11066 11073 anxiety +T296 NCBITaxon:10088 11125 11129 mice +T297 NCBITaxon:10088 11279 11283 mice +T298 GO:0040011 11307 11316 locomotor +T299 NCBITaxon:10088 11348 11352 mice +T300 NCBITaxon:33208 11453 11460 animals +T301 http://purl.obolibrary.org/obo/MONDO_0005618 11498 11505 anxiety +T302 PR:000007778 11539 11549 γ2 subunit +T303 CHEBI:22720 11586 11600 benzodiazepine +T304 CHEBI:35717 11623 11631;11641 11646 sedative ... drugs +T305 GO:0060013 11679 11694 righting reflex +T306 CHEBI:35717 11730 11738;11748 11753 sedative ... drugs +T307 CHEBI:49575 11821 11829 diazepam +T308 CHEBI:6931 11831 11840 midazolam +T309 CHEBI:10125 11842 11850 zolpidem +T310 CHEBI:16236 11852 11859 ethanol +T311 CHEBI:7983 11864 11877 pentobarbital +T312 PR:000007778 11903 11905 γ2 +T313 NCBITaxon:10088 11922 11926 mice +T314 SO:0000704 11932 11936 gene +T315 PR:000007778 11984 11986 γ2 +T316 NCBITaxon:10088 11997 12001 mice +T317 SO:0000346 12036 12046 loxP sites +T318 SO:0000357 12052 12057 flank +T319 SO:0000147 12058 12062 exon +T320 PR:000007778 12072 12074 γ2 +T321 SO:0000704 12075 12079 gene +T322 SO:0000346 12117 12127 loxP sites +T323 SO:0000359 12166 12172 floxed +T324 PR:000007778 12173 12175 γ2 +T325 NCBITaxon:10088 12176 12180 mice +T326 GO:0007618 12186 12191 mated +T327 GO:0010467 12217 12227 expressing +T328 NCBITaxon:10088 12239 12244 mouse +T329 SO:0000902 12296 12305 transgene +T330 GO:0010467 12335 12344 expressed +T331 NCBITaxon:9606 12345 12350 human +T332 PR:000003676 12351 12361 beta actin +T333 SO:0000167 12362 12370 promoter +T334 SO:0000193 12377 12410 Restriction fragment polymorphism +T335 SO:0001026 12467 12474 genomic +T336 SO:0000346 12487 12497 loxP sites +T337 GO:0097617 12534 12544 hybridized +T338 SO:0001023 12601 12607 allele +T339 SO:0000147 12686 12690 exon +T340 SO:0001023 12712 12718 allele +T341 NCBITaxon:10088 12720 12724 mice +T342 SO:0000902 12748 12757 transgene +T343 PR:000007778 12794 12796 γ2 +T344 SO:0001023 12797 12803 allele +T345 SO:0000359 12807 12808 f +T346 GO:0007618 12815 12820 mated +T347 NCBITaxon:10088 12824 12828 mice +T348 SO:0000359 12850 12856 floxed +T349 PR:000007778 12873 12875 γ2 +T350 SO:0001023 12876 12882 allele +T351 SO:0000359 12886 12887 F +T352 GO:0007618 12931 12937 mating +T353 SO:0000359 12989 12990 f +T354 SO:0000359 12991 12992 f +T355 SO:0000359 13085 13086 f +T356 SO:0000359 13087 13088 f +T357 NCBITaxon:10088 13089 13093 mice +T358 GO:0016265 13094 13098 died +T359 GO:0007567 13120 13125 birth +T360 SO:0000359 13131 13132 f +T361 SO:0000359 13133 13134 f +T362 NCBITaxon:10088 13135 13140 mouse +T363 GO:0007567 13160 13165 natal +T364 PR:000007778 13201 13203 γ2 +T365 SO:0000359 13204 13205 f +T366 SO:0000359 13206 13207 f +T367 NCBITaxon:10088 13208 13212 mice +T368 PR:000007778 13258 13260 γ2 +T369 SO:0000147 13322 13326 exon +T370 SO:0000346 13392 13402 loxP sites +T371 SO:0000147 13419 13423 exon +T372 PR:000007778 13442 13444 γ2 +T373 SO:0000704 13445 13449 gene +T374 SO:0000704 13464 13471 Genetic +T375 CHEBI:16865 13490 13494 GABA +T376 CHEBI:16865 13542 13546 GABA +T377 CHEBI:23888 13598 13603 drugs +T378 CHEBI:16865 13647 13651 GABA +T379 NCBITaxon:10088 13662 13667 mouse +T380 GO:0010467 13695 13705 expression +T381 PR:000007778 13713 13723 γ2 subunit +T382 PR:000007778 13772 13774 γ2 +T383 GO:0016265 13882 13885 die +T384 NCBITaxon:10088 13913 13917 mice +T385 GO:0010467 13972 13982 expression +T386 PR:000007778 13986 13988 γ2 +T387 NCBITaxon:10088 14028 14032 mice +T388 PR:000007778 14101 14103 γ2 +T389 NCBITaxon:10088 14146 14150 mice +T390 NCBITaxon:10088 14177 14181 mice +T391 GO:0010467 14215 14225 expressing +T392 NCBITaxon:10088 14241 14246 mouse +T393 NCBITaxon:10088 14268 14272 mice +T394 NCBITaxon:10088 14351 14355 mice +T395 SO:0000704 14361 14368 genetic +T396 PR:000007778 14388 14401;14406 14413 γ2 subunit of ... GABAA-R +T397 CHEBI:16865 14406 14410 GABA +T398 PR:000007778 14465 14467 γ2 +T399 PR:000007778 14495 14497 γ2 +T400 NCBITaxon:10088 14507 14511 mice +T401 NCBITaxon:10088 14526 14530 mice +T402 PR:000007778 14556 14558 γ2 +T403 CHEBI:10125 14575 14583 zolpidem +T404 CHEBI:109895 14604 14615 β-carboline +T405 GO:0008380 14665 14671 splice +T406 PR:000007778 14683 14685 γ2 +T407 NCBITaxon:10088 14707 14711 mice +T408 GO:0010467 14717 14724 express +T409 GO:0008380 14750 14756 splice +T410 PR:000007778 14773 14783 γ2 subunit +T411 SO:0000704 14906 14913 genetic +T412 PR:000007778 14932 14934 γ2 +T413 PR:000007778 14963 14965 γ2 +T414 PR:000007778 14989 14991 γ2 +T415 SO:0000188 15046 15054 intronic +T416 SO:0001023 15121 15128 alleles +T417 SO:0001023 15162 15169 alleles +T418 SO:0000704 15217 15221 gene +T419 SO:0000704 15299 15303 gene +T420 SO:0000188 15326 15334 Intronic +T421 SO:0001023 15372 15379 alleles +T422 SO:0000976 15395 15402 cryptic +T423 GO:0008380 15403 15411 splicing +T424 SO:0000704 15431 15435 gene +T425 GO:0008380 15480 15488 splicing +T426 SO:0000704 15512 15516 gene +T427 GO:0010467 15512 15527 gene expression +T428 GO:0010467 15639 15649 expression +T429 SO:0000704 15668 15672 gene +T430 SO:0001023 15733 15740 alleles +T431 PR:000007778 15781 15783 γ2 +T432 NCBITaxon:10088 15804 15808 mice +T433 PR:000007778 15855 15857 γ2 +T434 NCBITaxon:10088 15970 15974 mice +T435 PR:000007778 16014 16016 γ2 +T436 NCBITaxon:10088 16049 16053 mice +T437 PR:000007778 16058 16060 γ2 +T438 GO:0010467 16210 16220 expression +T439 NCBITaxon:10088 16249 16253 mice +T440 GO:0010467 16424 16434 expression +T441 PR:000007778 16610 16612 γ2 +T442 GO:0010467 16739 16749 expression +T443 SO:0001023 16895 16901 allele +T444 SO:0000188 16924 16932 intronic +T445 SO:0000704 16987 16991 gene +T446 GO:0010467 16987 17002 gene expression +T447 PR:000007778 17091 17101 γ2 subunit +T448 CHEBI:16865 17113 17117 GABA +T449 http://purl.obolibrary.org/obo/MONDO_0005618 17186 17193 anxiety +T450 NCBITaxon:10088 17232 17236 mice +T451 PR:000007778 17389 17391 γ2 +T452 NCBITaxon:10088 17421 17425 mice +T453 CHEBI:16865 17488 17492 GABA +T454 http://purl.obolibrary.org/obo/MONDO_0005618 17538 17555 anxiety disorders +T455 NCBITaxon:9606 17559 17565 humans +T456 NCBITaxon:10088 17580 17585 mouse +T457 UBERON:0000955 17733 17739 brains +T458 PR:000007778 17745 17747 γ2 +T459 NCBITaxon:10088 17758 17762 mice +T460 CHEBI:22720 17815 17829 benzodiazepine +T461 CHEBI:22720 17878 17892 benzodiazepine +T462 SO:0000409 17893 17906 binding sites +T463 PR:000007778 17956 17970;17975 17982 γ2 subunits of ... GABAA-R +T464 CHEBI:16865 17975 17979 GABA +T465 PR:000007778 18062 18064 γ2 +T466 NCBITaxon:10088 18074 18078 mice +T467 PR:000007778 18109 18119 γ2 subunit +T468 CHEBI:22720 18149 18163 benzodiazepine +T469 PR:000007778 18202 18204 γ2 +T470 NCBITaxon:10088 18214 18218 mice +T471 CHEBI:29238 18246 18248 3H +T472 UBERON:0000955 18275 18280 brain +T473 PR:000007778 18323 18325 γ2 +T474 UBERON:0000955 18409 18415 brains +T475 CHEBI:22720 18469 18483 benzodiazepine +T476 PR:000007778 18501 18503 γ2 +T477 PR:000007778 18573 18575 γ2 +T478 CHEBI:29238 18601 18603 3H +T479 CHEBI:7035 18604 18612 muscimol +T480 PR:000007778 18676 18686 γ2 subunit +T481 PR:000007775 18726 18735 δ subunit +T482 CHEBI:29238 18773 18775 3H +T483 CHEBI:7035 18776 18784 muscimol +T484 UBERON:0000955 18803 18808 brain +T485 CHEBI:24870 18849 18852 ion +T486 CHEBI:37983 18869 18872 35S +T487 CHEBI:16865 18920 18924 GABA +T488 PR:000007778 18950 18961 γ2 subunits +T489 PR:000007778 18980 18982 γ2 +T490 UBERON:0000955 19080 19085 brain +T491 CHEBI:16865 19136 19140 GABA +T492 UBERON:0000955 19210 19215 brain +T493 GO:0032800 19232 19245;19252 19254 production of ... Rs +T494 CHEBI:16865 19246 19250 GABA +T495 CHEBI:16865 19418 19422 GABA +T496 CHEBI:16865 19478 19482 GABA +T497 GO:0045202 19561 19569 synaptic +T498 PR:000007778 19615 19625 γ2 subunit +T499 PR:000007778 19660 19662 γ2 +T500 NCBITaxon:10088 19672 19676 mice +T501 UBERON:0000955 19686 19691 brain +T502 GO:0045202 19724 19732 synaptic +T503 GO:0045202 19742 19750 synaptic +T504 GO:0045202 19778 19786 synaptic +T505 CHEBI:16865 19787 19791 GABA +T506 CHEBI:22720 19909 19923 benzodiazepine +T507 CHEBI:49575 19937 19945 diazepam +T508 CHEBI:10125 19947 19955 zolpidem +T509 CHEBI:6931 19960 19969 midazolam +T510 NCBITaxon:10088 20002 20006 mice +T511 CHEBI:22720 20090 20104 benzodiazepine +T512 PR:000007778 20202 20204 γ2 +T513 CHEBI:16865 20216 20220 GABA +T514 CHEBI:22720 20264 20279 benzodiazepines +T515 NCBITaxon:10088 20281 20285 Mice +T516 PR:000007778 20307 20309 γ2 +T517 CHEBI:22720 20325 20339 benzodiazepine +T518 NCBITaxon:10088 20370 20374 mice +T519 CHEBI:49575 20423 20431 diazepam +T520 NCBITaxon:10088 20447 20451 mice +T521 PR:000007778 20477 20479 γ2 +T522 CHEBI:10125 20508 20516 zolpidem +T523 CHEBI:22720 20548 20562 benzodiazepine +T524 CHEBI:22720 20616 20630 benzodiazepine +T525 NCBITaxon:10088 20680 20684 mice +T526 CHEBI:22720 20730 20744 benzodiazepine +T527 CHEBI:30225 20780 20788 positron +T528 NCBITaxon:9606 20820 20826 humans +T529 CHEBI:22720 20868 20882 benzodiazepine +T530 CHEBI:6539 20912 20921 lorazepam +T531 CHEBI:10125 20926 20934 zolpidem +T532 NCBITaxon:33208 21098 21104 animal +T533 CHEBI:52217 21105 21120 pharmacological +T534 PR:000007778 21170 21172 γ2 +T535 PR:000007778 21224 21226 γ2 +T536 CHEBI:16865 21238 21242 GABA +T537 NCBITaxon:10088 21284 21288 mice +T538 CHEBI:22720 21318 21332 benzodiazepine +T539 SO:0000409 21333 21345 binding site +T540 PR:000007766 21362 21364 α1 +T541 PR:000007767 21369 21371 α2 +T542 CHEBI:16865 21383 21387 GABA +T543 CHEBI:22720 21412 21426 benzodiazepine +T544 PR:000007778 21492 21494 γ2 +T545 NCBITaxon:10088 21505 21509 mice +T546 PR:000007766 21549 21551 α1 +T547 PR:000007767 21580 21582 α2 +T548 PR:000007768 21586 21588 α3 +T549 SO:0001060 21609 21617 isoforms +T550 PR:000007778 21650 21652 γ2 +T551 NCBITaxon:10088 21663 21667 mice +T552 CHEBI:16865 21690 21694 GABA +T553 GO:0042571 21850 21860 antibodies +T554 PR:000007767 21869 21871 α2 +T555 PR:000007768 21876 21878 α3 +T556 CHEBI:10125 21908 21916 zolpidem +T557 CHEBI:16865 22043 22047 GABA +T558 GO:0045202 22073 22080 synapse +T559 PR:000007778 22119 22121 γ2 +T560 PR:000008174 22126 22134 gephyrin +T561 NCBITaxon:10088 22182 22186 mice +T562 CHEBI:23888 22197 22201 drug +T563 GO:0045202 22243 22251 synaptic +T564 GO:0043113 22252 22265;22272 22274 clustering of ... Rs +T565 CHEBI:16865 22266 22270 GABA +T566 CHEBI:22720 22324 22339 benzodiazepines +T567 CHEBI:22720 22365 22380 benzodiazepines +T568 GO:0045202 22401 22409 synaptic +T569 CHEBI:16865 22410 22414 GABA +T570 UBERON:0000955 22463 22468 brain +T571 PR:000007778 22531 22533 γ2 +T572 UBERON:0000955 22548 22553 brain +T573 NCBITaxon:10088 22567 22571 mice +T574 CHEBI:23888 22622 22627 drugs +T575 PR:000007778 22681 22683 γ2 +T576 NCBITaxon:10088 22703 22707 mice +T577 PR:000007778 22825 22827 γ2 +T578 NCBITaxon:33208 22852 22858 animal +T579 PR:000007778 22928 22930 γ2 +T580 CHEBI:22720 22971 22985 benzodiazepine +T581 PR:000007766 23076 23078 α1 +T582 NCBITaxon:10088 23084 23088 mice +T583 CHEBI:22720 23101 23115 benzodiazepine +T584 NCBITaxon:33208 23145 23151 animal +T585 CHEBI:6931 23167 23176 midazolam +T586 CHEBI:49575 23206 23214 diazepam +T587 NCBITaxon:10088 23240 23244 mice +T588 GO:0008380 23276 23282 splice +T589 PR:000007778 23294 23296 γ2 +T590 CHEBI:22720 23311 23325 benzodiazepine +T591 CHEBI:6931 23403 23412 midazolam +T592 CHEBI:10125 23417 23425 zolpidem +T593 NCBITaxon:10088 23464 23468 mice +T594 PR:000007774 23481 23491 β3 subunit +T595 CHEBI:22720 23503 23517 benzodiazepine +T596 NCBITaxon:33208 23546 23552 animal +T597 CHEBI:6931 23553 23562 midazolam +T598 SO:0000704 23691 23702 genetically +T599 NCBITaxon:10088 23714 23719 mouse +T600 PR:000007778 23754 23756 γ2 +T601 NCBITaxon:10088 23801 23805 mice +T602 PR:000007778 23875 23877 γ2 +T603 NCBITaxon:10088 23888 23892 mice +T604 http://purl.obolibrary.org/obo/MONDO_0005618 23922 23929 anxiety +T605 NCBITaxon:10088 23979 23983 mice +T606 CHEBI:22720 24034 24048 benzodiazepine +T607 SO:0000704 24082 24093 genetically +T608 NCBITaxon:10088 24105 24110 mouse +T609 PR:000007778 24150 24152 γ2 +T610 NCBITaxon:10088 24169 24174 mouse +T611 GO:0010467 24208 24218 expressing +T612 NCBITaxon:10088 24245 24250 mouse +T613 SO:0000704 24281 24292 genetically +T614 NCBITaxon:10088 24301 24305 mice +T615 NCBITaxon:10088 24338 24343 mouse +T616 SO:0001026 24344 24351 genomic +T617 SO:0000147 24376 24381 Exons +T618 CHEBI:16865 24394 24398 GABA +T619 PR:000007778 24394 24404 GABAA-R γ2 +T620 SO:0000704 24405 24409 gene +T621 SO:0001026 24447 24453 Genome +T622 SO:0000346 24511 24520 loxP site +T623 SO:0000028 24573 24575 bp +T624 SO:0000147 24582 24586 Exon +T625 SO:0000704 24637 24641 gene +T626 PR:000012607 24647 24652 PGK-1 +T627 SO:0000167 24653 24661 promoter +T628 GO:0043631 24666 24681 polyadenylation +T629 SO:0000551 24666 24689 polyadenylation signals +T630 SO:0000346 24729 24738 loxP site +T631 SO:0000028 24775 24777 bp +T632 SO:0000147 24784 24788 Exon +T633 SO:0000704 24800 24804 gene +T634 PR:000007778 24849 24851 γ2 +T635 GO:0010467 24897 24907 expression +T636 SO:0005853 24908 24916 cassette +T637 PR:000007778 24969 24971 γ2 +T638 SO:0001026 24972 24979 genomic +T639 UBERON:0000922 25061 25070 embryonic +T640 CL:0002322 25061 25081 embryonic stem cells +T641 CHEBI:42768 25135 25139 G418 +T642 CHEBI:465284 25193 25204 gancyclovir +T643 SO:0000704 25253 25257 gene +T644 SO:0001026 25312 25319 genomic +T645 GO:0097617 25340 25350 hybridized +T646 PR:000007778 25450 25452 γ2 +T647 CHEBI:7507 25584 25592 neomycin +T648 SO:0005853 25593 25601 cassette +T649 SO:0001644 25635 25651 targeting vector +T650 PR:000007778 25778 25780 γ2 +T651 UBERON:0000922 25812 25821 embryonic +T652 CL:0002322 25812 25831 embryonic stem cell +T653 UBERON:0000358 25888 25899 blastocysts +T654 NCBITaxon:10088 25920 25924 mice +T655 GO:0007618 25945 25950 mated +T656 SO:0001023 26019 26025 allele +T657 SO:0000359 26027 26028 F +T658 NCBITaxon:10088 26058 26062 mice +T659 SO:0000359 26127 26128 F +T660 SO:0000359 26129 26130 F +T661 NCBITaxon:10088 26137 26141 mice +T662 CL:0002322 26199 26206 ES cell +T663 NCBITaxon:10088 26218 26222 mice +T664 NCBITaxon:10088 26349 26353 mice +T665 UBERON:0005434 26373 26381 cervical +T666 UBERON:0000955 26423 26428 brain +T667 CHEBI:33893 26442 26449 reagent +T668 CHEBI:16842 26513 26525 formaldehyde +T669 CHEBI:2511 26529 26536 agarose +T670 CHEBI:37972 26620 26623 32P +T671 PR:000007778 26632 26634 γ2 +T672 NCBITaxon:9606 26680 26685 human +T673 PR:000003676 26686 26693 β-actin +T674 PR:000007778 26854 26856 γ2 +T675 GO:0097617 26857 26870 hybridization +T676 GO:0097617 26884 26897 hybridization +T677 CHEBI:50913 27131 27139 fixative +T678 CHEBI:49826 27160 27169 periodate +T679 UBERON:0000955 27229 27235 brains +T680 NCBITaxon:9986 27381 27387 rabbit +T681 PR:000007778 27393 27395 γ2 +T682 CHEBI:9750 27434 27446 Triton X-100 +T683 CHEBI:22885 27523 27529 biotin +T684 CHEBI:35696 27697 27712 cobalt chloride +T685 CHEBI:86151 27717 27740 nickel ammonium sulfate +T686 CHEBI:75050 27744 27754 chromogens +T687 CHEBI:16240 27759 27776 hydrogen peroxide +T688 CHEBI:63248 27780 27787 oxidant +T689 GO:0042571 27844 27852 antibody +T690 GO:0042571 27882 27890 antibody +T691 NCBITaxon:10088 27958 27962 mice +T692 UBERON:0000955 28005 28011 brains +T693 NCBITaxon:10088 28141 28146 mouse +T694 UBERON:0000955 28147 28153 brains +T695 CHEBI:5291 28190 28197 gelatin +T696 NCBITaxon:10088 28334 28339 mouse +T697 CHEBI:29238 28466 28468 3H +T698 CHEBI:29238 28482 28484 3H +T699 CHEBI:7035 28485 28493 muscimol +T700 CHEBI:37983 28500 28503 35S +T701 CHEBI:15377 28590 28595 water +T702 CHEBI:9754 28621 28625 Tris +T703 CHEBI:17883 28626 28629 HCl +T704 CHEBI:26710 28664 28668 NaCl +T705 CHEBI:29238 28677 28679 3H +T706 CHEBI:37983 28696 28699 35S +T707 CHEBI:9754 28744 28748 Tris +T708 CHEBI:29238 28774 28776 3H +T709 CHEBI:7035 28777 28785 muscimol +T710 CHEBI:37983 28964 28967 35S +T711 CHEBI:29238 29024 29026 3H +T712 CHEBI:7035 29027 29035 muscimol +T713 CHEBI:29238 29080 29082 3H +T714 CHEBI:49575 29132 29140 diazepam +T715 CHEBI:37983 29290 29293 35S +T716 CHEBI:29238 29304 29306 3H +T717 CHEBI:29238 29325 29327 3H +T718 CHEBI:7035 29328 29336 muscimol +T719 CHEBI:15377 29399 29404 water +T720 CHEBI:29238 29475 29477 3H +T721 CHEBI:36927 29484 29487 14C +T722 CHEBI:25218 29489 29501 methacrylate +T723 CHEBI:5103 29601 29611 flumazenil +T724 CHEBI:8206 29660 29672 picrotoxinin +T725 CHEBI:16865 29692 29696 GABA +T726 CHEBI:29238 29709 29711 3H +T727 CHEBI:37983 29725 29728 35S +T728 CHEBI:29238 29739 29741 3H +T729 CHEBI:7035 29742 29750 muscimol +T730 UBERON:0000955 29807 29812 brain +T731 CHEBI:29238 29936 29938 3H +T732 CHEBI:37983 29954 29957 35S +T733 CHEBI:29238 30058 30060 3H +T734 CHEBI:7035 30061 30069 muscimol +T735 CHEBI:29238 30083 30085 3H +T736 CHEBI:16865 30200 30204 GABA +T737 SO:0000409 30319 30332 binding sites +T738 CHEBI:23888 30357 30361 Drug +T739 GO:0060013 30433 30448 righting reflex +T740 CHEBI:35717 30472 30480;30490 30495 sedative ... drugs +T741 CHEBI:49575 30497 30505 Diazepam +T742 CHEBI:6931 30525 30534 midazolam +T743 CHEBI:7983 30591 30604 pentobarbital +T744 CHEBI:10125 30638 30646 zolpidem +T745 CHEBI:16236 30682 30689 ethanol +T746 UBERON:0001179 30750 30762 peritoneally +T747 CHEBI:23888 30800 30805 Drugs +T748 GO:0060013 30909 30924 righting reflex +T749 NCBITaxon:10088 30926 30930 mice +T750 NCBITaxon:10088 31008 31013 mouse +T751 GO:0060013 31026 31031 right +T752 GO:0060013 31276 31291 righting reflex +T753 http://purl.obolibrary.org/obo/MONDO_0005618 31361 31368 anxiety +T754 NCBITaxon:10088 31428 31432 mice +T755 NCBITaxon:10088 31513 31518 mouse +T756 NCBITaxon:10088 31767 31772 mouse +T757 NCBITaxon:10088 32063 32067 mice +T758 NCBITaxon:10088 32132 32136 mice +T759 GO:0040011 32219 32227 traveled +T760 SO:0000704 33021 33025 Gene +T761 PR:000007778 33043 33056;33061 33068 γ2 subunit of ... GABAA-R +T762 CHEBI:16865 33061 33065 GABA +T763 PR:000007778 33088 33090 γ2 +T764 NCBITaxon:10088 33101 33105 mice +T765 PR:000007778 33146 33148 γ2 +T766 NCBITaxon:10088 33159 33163 mice +T767 SO:0000704 33195 33199 gene +T768 SO:0001644 33205 33221 targeting vector +T769 PR:000007778 33252 33254 γ2 +T770 SO:0000359 33256 33257 F +T771 SO:0001023 33288 33295 alleles +T772 SO:0000061 33342 33359 restriction sites +T773 SO:0000147 33361 33366 exons +T774 SO:0000346 33392 33402 loxP sites +T775 SO:0000155 33421 33428 plasmid +T776 SO:0005853 33502 33511 cassettes +T777 UBERON:0002415 33568 33572 tail +T778 GO:0097617 33587 33597 hybridizes +T779 SO:0001023 33655 33661 allele +T780 SO:0001023 33708 33714 allele +T781 SO:0000359 33830 33831 F +T782 SO:0000359 33832 33833 F +T783 UBERON:0007023 33835 33840 adult +T784 UBERON:0000955 33847 33852 brain +T785 PR:000007778 33881 33883 γ2 +T786 NCBITaxon:9606 33894 33899 human +T787 PR:000003676 33900 33907 β-actin +T788 PR:000007778 34016 34018 γ2 +T789 PR:000003676 34023 34030 β-actin +T790 GO:0097617 34031 34044 hybridization +T791 NCBITaxon:10088 34102 34106 mice +T792 PR:000007778 34501 34514;34519 34526 γ2 subunit of ... GABAA-R +T793 CHEBI:16865 34519 34523 GABA +T794 NCBITaxon:10088 34598 34602 mice +T795 PR:000007778 34640 34642 γ2 +T796 GO:0007608 34663 34672 olfactory +T797 UBERON:0002264 34663 34677 olfactory bulb +T798 UBERON:0002264 34679 34681 OB +T799 UBERON:0000956 34684 34699 cerebral cortex +T800 UBERON:0000956 34701 34703 CC +T801 UBERON:0002038 34724 34740 substantia nigra +T802 UBERON:0002038 34742 34744 SN +T803 UBERON:0002037 34751 34761 cerebellum +T804 UBERON:0002037 34763 34765 CB +T805 PR:000007778 34803 34805 γ2 +T806 UBERON:0000955 34834 34839 brain +T807 NCBITaxon:10088 34926 34930 mice +T808 NCBITaxon:10088 34968 34972 mice +T809 NCBITaxon:10088 35096 35100 mice +T810 NCBITaxon:10088 35116 35120 mice +T811 CHEBI:16236 35310 35317 ethanol +T812 CHEBI:7983 35319 35332 pentobarbital +T813 CHEBI:10125 35334 35342 zolpidem +T814 CHEBI:6931 35344 35353 midazolam +T815 CHEBI:49575 35358 35366 diazepam +T816 CHEBI:16865 35447 35451 GABA +T817 SO:0000409 35462 35475 binding sites +T818 NCBITaxon:10088 35523 35527 mice +T819 NCBITaxon:10088 35577 35581 mice +T820 CHEBI:29238 35632 35634 3H +T821 CHEBI:37983 35660 35663 35S +T822 UBERON:0001851 35785 35788 Ctx +T823 UBERON:0001851 35790 35796 cortex +T824 UBERON:0001897 35798 35800 Th +T825 UBERON:0001897 35802 35810 thalamus +T826 UBERON:0005383 35829 35832 CPu +T827 UBERON:0005383 35834 35849 caudate putamen +T828 UBERON:0001946 35851 35853 IC +T829 UBERON:0001946 35855 35874 inferior colliculus +T830 UBERON:0002956 35876 35878 Gr +T831 CL:0001031 35880 35903 cerebellar granule cell +T832 UBERON:0002956 35880 35909 cerebellar granule cell layer +T833 UBERON:0002974 35911 35914 Mol +T834 UBERON:0002974 35916 35942 cerebellar molecular layer diff --git a/src/ontogpt/evaluation/craft/database/all/15850489.txt b/src/ontogpt/evaluation/craft/database/all/15850489.txt new file mode 100644 index 000000000..91d7eb8e2 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15850489.txt @@ -0,0 +1,147 @@ +GABAA receptor γ2 subunit knockdown mice have enhanced anxiety-like behavior but unaltered hypnotic response to benzodiazepines + +Abstract + +Background + +Gamma-aminobutyric acid type A receptors (GABAA-Rs) are the major inhibitory receptors in the mammalian brain and are modulated by a number of sedative/hypnotic drugs including benzodiazepines and anesthetics. The significance of specific GABAA-Rs subunits with respect to behavior and in vivo drug responses is incompletely understood. The γ2 subunit is highly expressed throughout the brain. Global γ2 knockout mice are insensitive to the hypnotic effects of diazepam and die perinatally. Heterozygous γ2 global knockout mice are viable and have increased anxiety-like behaviors. To further investigate the role of the γ2 subunit in behavior and whole animal drug action, we used gene targeting to create a novel mouse line with attenuated γ2 expression, i.e., γ2 knockdown mice. + +Results + +Knockdown mice were created by inserting a neomycin resistance cassette into intron 8 of the γ2 gene. Knockdown mice, on average, showed a 65% reduction of γ2 subunit mRNA compared to controls; however γ2 gene expression was highly variable in these mice, ranging from 10–95% of normal. Immunohistochemical studies demonstrated that γ2 protein levels were also variably reduced. Pharmacological studies using autoradiography on frozen brain sections demonstrated that binding of the benzodiazepine site ligand Ro15-4513 was decreased in mutant mice compared to controls. Behaviorally, knockdown mice displayed enhanced anxiety-like behaviors on the elevated plus maze and forced novelty exploration tests. Surprisingly, mutant mice had an unaltered response to hypnotic doses of the benzodiazepine site ligands diazepam, midazolam and zolpidem as well as ethanol and pentobarbital. Lastly, we demonstrated that the γ2 knockdown mouse line can be used to create γ2 global knockout mice by crossing to a general deleter cre-expressing mouse line. + +Conclusion + +We conclude that: 1) insertion of a neomycin resistance gene into intron 8 of the γ2 gene variably reduced the amount of γ2, and that 2) attenuated expression of γ2 increased anxiety-like behaviors but did not lead to differences in the hypnotic response to benzodiazepine site ligands. This suggests that reduced synaptic inhibition can lead to a phenotype of increased anxiety-like behavior. In contrast, normal drug effects can be maintained despite a dramatic reduction in GABAA-R targets. + +Background + +GABAA-Rs are the major inhibitory receptors in the mammalian central nervous system and can be modulated by a number of sedative, hypnotic and anesthetic drugs [1]. GABAA-Rs are putatively pentameric complexes and are members of a 'superfamily' of related ligand-gated ion channels. There are a variety of subunit families that make up GABAA-Rs; a total of seventeen distinct subunits have been cloned, α1–6, β1–4, γ1–3, δ, ε, π, and θ. Much is known of the subunits that comprise GABAA-Rs, but the contribution of individual subunits to in vivo drug responses is only beginning to be understood, largely through the use of genetically engineered mice [2-4]. + +The γ2 subunit is highly expressed throughout developing and adult brain and spinal cord, is a component of almost 60% of all GABAA-Rs [5,6], and is required for synaptic clustering of GABAA-Rs [7]. In vitro electrophysiologic studies have demonstrated an obligatory role of the γ2 subunit for benzodiazepine responsiveness [8]. Gunther et al. created and studied mice that globally lacked all γ2 subunits of the GABAA-R [9]. Mice homozygous for the γ2 knockout died in the perinatal period with rare survivors reaching postnatal day 18. Knockouts appeared normal at birth, but developed sensorimotor abnormalities characterized by hyperactivity, impaired grasping and righting reflex, and abnormal gait. Pharmacologically, knockout of γ2 resulted in a severe deficit in benzodiazepine binding sites (~94% reduction compared to control) while GABA binding sites were only slightly reduced. Behaviorally, in the few mice that survived long enough to be tested, diazepam failed to induce sedation and loss of the righting reflex at doses that were fully effective in wild type mice [9]. γ2 heterozygous knockout mice were normally viable, had a ~50% reduction in γ2 protein levels, and displayed increased anxiety-like behaviors [10]. Drug responses of heterozygous γ2 global knockouts have not been reported, except for the high efficacy of low diazepam doses to induce anxiolysis [10]. + +We set out to make mice with markedly attenuated expression of the γ2 gene (i.e., γ2 knockdown mice) that would be viable and therefore useful for studies of basic behaviors and drug induced responses. We used gene targeting and embryonic stem cell technologies to create this mouse line by inserting a neomycin resistance gene (NEO) into intron 8 of the γ2 genomic locus. Insertion of NEO into intronic DNA has previously been used to create hypomorphic alleles of several genes [11-23]. In those studies, expression of the hypomorphic allele ranged from 5–25% of control; substantially lower than the 50% reduction typically observed in heterozygous knockouts. The γ2 knockdown mouse line described in the present report had a highly variable reduction in γ2 mRNA and protein levels. γ2 knockdown mice were normally viable, had increased anxiety-like behaviors, but did not differ in the hypnotic response to benzodiazepines. In addition, since exon 8 of the γ2 gene was flanked by loxP sites in this mouse line, these mice could also be converted into a global knockout mouse by crossing to a general deleter Cre-expressing transgenic mouse line. + +Results + +Production of gene targeted mice + +Four out of 330 embryonic stem cell clones displayed the predicted restriction fragment length polymorphisms (data not shown) indicative of the targeting event presented in Fig 1A. Because the targeting event results in a γ2 locus in which exon 8 is flanked by loxP sites, we refer to this locus as being floxed (F) in contrast to the endogenous wild type allele (+). Two correctly targeted cell lines, 26L6 and 26C8 yielded germ-line competent chimeric males. Chimeras were mated to C57BL/6J females. Offspring that were +/F were interbred to produce +/+, +/F, and F/F mice. Mice were genotyped at weaning using Southern blot analysis as indicated in Fig 1B. In this analysis, Probe A hybridized to a 9.2 kB BglII fragment from the wild type γ2 allele and a 3.4 kB fragment from the targeted allele. Of 724 mice genotyped, 196 (27%) were +/+, 372 (51%) were +/F, and 156 (22%) were F/F. These values are in accord with the 1:2:1 genotype frequency as expected by Mendelian genetics. Thus, homozygous floxed mice were normally viable. They were also healthy and overtly indistinguishable from wild type mice. + +Northern blot analysis + +Semi-quantitative northern blots were used to compare the amount of γ2 mRNA in wild type and F/F animals. Hybridization of adult brain RNA with a γ2 cDNA probe yielded an intense 2.8 kB band in wild type mice. In contrast, RNA from F/F mice hybridized less intensely in most mice, however the attenuation within this group was highly variable. A sample northern blot is shown in Figure 2A. We semi-quantitatively determined γ2 mRNA amounts by comparing γ2 band densities between wild type (n = 23) and F/F (n = 36) mice following normalization with β-actin. To normalize results over four different blots, the ratio of wild type band densities on each blot was averaged and normalized to 100. All samples on each blot were then compared to the average of wild type band densities. Values are shown in Figure 2B. Mean wild type ratio was 100 whereas mean ratio in F/F samples was 35 with high variability including several mice with values that overlapped with wild type values. Thus, γ2 mRNA levels in knockdown mice were dramatically attenuated on average, but the levels varied significantly between mice. + +Immunohistochemistry + +Immunohistochemical staining of sagittal sections probed with a γ2-specific antibody was used to assess γ2 protein amount and distribution in the brain. A sampling of wild type (Fig 3A) and knockdown (Fig 3B) brain sections are displayed. In three of the four knockdown mouse sections shown, there was a marked overall reduction of γ2 immunoreactivity in all areas of the brain except the outer layer of the olfactory bulb. However, staining intensity was variable between knockdown mice, with one having near wild type intensity (Fig. 3B, d). A total of 10 F/F mice were studied. Seven out of 10 had a marked decrease in γ2 staining compared to wild type controls, and three out of 10 had near wild-type or intermediate levels of staining. Adjacent sections from these same mice showed no change in α1 or β2/3 immunoreactivity (CPM and ALD, unpublished observations). + +Pharmacological characterization + +Autoradiography on brain cryostat sections with various ligands binding to selective sites of the GABAA-R revealed clear differences in the homozygous γ2 knockdowns as compared to heterozygous and wild type mice (Table 1). Total flumazenil-sensitive [3H]Ro15-4513 binding to benzodiazepine sites was reduced in the knockdown in most brain regions. The reduced binding in the 5 mice analyzed ranged on the average from 25 to 57%. In several regions, the heterozygous mice also showed significant reduction of the binding, but clearly less than the homozygous knockdown samples. A small proportion of the reduction in the total binding was apparently due to reduction in diazepam-insensitive α4 and α6 subunit-containing receptors, this component being mostly affected in the cerebellar granule cell layer. + +GABA-sensitive [3H]muscimol binding to GABA agonist sites was not altered in any brain region (Table 1). + +Picrotoxinin-sensitive [35S]TBPS binding to the GABAA-R channels was little affected between the mouse lines: basal binding was increased in the hippocampus and decreased in the cerebellar granule cell layer of the homozygous γ2 knockdowns (Table 1). The GABA-insensitive [35S]TBPS binding was increased all over the brain, but this component still made up less than 10% of the basal binding, except for the cerebellar granule cell layer. There, the GABA-insensitive component was 20% as compared to 10% in the heterozygous and wild type animals. + +Behavioral characterization + +We examined the behavioral phenotype of the knockdown mice using the elevated plus maze and forced exploration in a novel open field. The elevated plus maze is widely used to assess anxiety-like behavior in response to the aversive stimulus of an elevated exposed space. On this test apparatus, knockdown mice had a 74% reduction in open arm entries (Fig. 4A) and an 83% reduction in time on open arms (Fig. 4B) compared to controls suggesting an increase in anxiety-like behavior. In contrast, knockdown mice had the same number of total arm entries as wild types (Fig. 4C) indicating that an alteration in locomotion does not account for the differences in anxiety-like behavioral measures on this assay. + +Knockdown mice were also tested for behavioral response to an aversive, brightly lit open field test apparatus using the forced novelty exploration test. Knockdown mice had a 28% reduction in locomotor activity compared to wild type mice when placed into this test arena (Fig. 4D). This reduction in exploratory behavior in the knockdown animals is also indicative of an increase in anxiety-like behavior. + +Knockdown of the γ2 subunit did not alter hypnotic responses to benzodiazepine site ligands or other sedative/hypnotic drugs tested. We measured the loss of righting reflex to assess the acute sensitivity to sedative/hypnotic drugs; no significant differences were found in response to injection of diazepam, midazolam, zolpidem, ethanol, or pentobarbital (Fig. 5). + +Production of γ2 global knockout mice + +The gene targeting strategy that we used to produce the γ2 knockdown mice also resulted in the insertion of loxP sites that flank exon 8 of the γ2 gene (see Fig. 1A). To determine if these loxP sites were functional in vivo, heterozygous floxed γ2 mice were mated to a general deleter cre-expressing transgenic mouse line (JAX Laboratory, stock #003376) where the cre transgene was driven by a ubiquitously expressed human beta actin promoter [24]. Restriction fragment polymorphism analysis by Southern blotting confirmed the deletion of genomic DNA between loxP sites. As predicted from Fig. 1A, Probe A hybridized to a 6.6 kb BglII fragment from the recombined knockout allele (data not shown). + +To determine if cre-mediated recombination and deletion of exon 8 resulted in a null allele, mice hemizygous for the cre transgene and heterozygous for the recombined γ2 allele (+/f) were mated to mice heterozygous for the floxed, non-recombined γ2 allele (+/F). 10.2% (14 of 137) of offspring from this mating strategy were homozygous for the recombined locus (f/f). This ratio is in agreement with the 12.5% predicted from Mendalian genetics. 13 of the 14 f/f mice died within three days of birth. One f/f mouse survived until postnatal day 21. This neonatal lethality of γ2 f/f mice is consistent with those found in the global γ2 knockouts of Günther et al. [9] which also had a deletion of exon 8. Therefore, we conclude that cre-mediated recombination of the loxP sites and deletion of exon 8 inactivates the γ2 gene. + +Discussion + +Genetic dissection of the GABAA-R system is yielding remarkable insights into GABAA-R biology and the mechanisms of action of various drugs. Here we report the establishment of a new GABAA-R mutant mouse model, one with attenuated expression of the γ2 subunit that averages ~35% of normal levels. This novel γ2 knockdown model has several unique features that make it useful. First, compared to global knockouts which die as neonates [9], knockdown mice have normal viability. Secondly, the graded levels of expression of γ2 (range 10–95% of normal) in the mutant mice could be useful in studies correlating phenotype with the amount of γ2 protein present. Finally, global knockout mice can be created from these mice by crossing to a Cre recombinase expressing global deleter mouse line. Therefore, the mice reported here represent a useful addition to the rapidly expanding arsenal of mice with genetic alterations in the γ2 subunit of the GABAA-R. In addition to homozygous and heterozygous global γ2 knockouts [9], conditional γ2 knockout mice [25], knockin mice with a point mutation in γ2 that eliminates zolpidem and inverse agonist β-carboline sensitivity [26,27], global knockout of the long splice variant of γ2 [28], and transgenic mice that express either the long or short splice variants of the γ2 subunit [29] have been described. + +An important unexpected finding from this study was the extremely variable degree to which the genetic alteration of the γ2 locus changed the amount of γ2 product produced. This γ2 knockdown model was produced by insertion of NEO into intronic DNA. This strategy has been used previously to create hypomorphic alleles [11-23]. Creation of hypomorphic alleles has been useful in determining function of the gene that has been targeted, especially in cases where complete disruption of the gene is lethal [12,14-16]. Intronic insertion of NEO creates hypomorphic alleles either through cryptic splicing signals in the NEO gene that lead to a frameshift or alterations in splicing, or by down regulating gene expression through an unknown mechanism [14-16]. In the majority of these studies, the authors' reported a 5–25% level of expression from the targeted gene. However, in none of these previously described hypomorphic alleles was extensive variability reported. The γ2 targeted homozygous mice in this study clearly display a wide range of γ2 mRNA and protein levels. Semi-quantitative northern blot analysis revealed that 4 out of 5 homozygous knockdown mice showed, on average, a 70% reduction of γ2 mRNA levels, whereas 1 out of 5 mice had γ2 mRNA at a level that was similar to wild type. Immunohistochemical data were consistent with northern blot data. The reason for this highly variable expression within homozygous knockdown mice has yet to be determined. The mixed background of Strain 129S1/X1 and C57BL6/J may not be the cause given that other studies have not observed substantial variability in expression using similar background strains [11,18,20]. Gender as a cause of variability also is not likely given that a set of three male knockdown siblings exhibited the full range of γ2 levels; one being near wild type levels, another being at an intermediate level, and the third exhibiting severely attenuated expression. Further study to determine the cause of this variability needs to be undertaken. Nevertheless, investigators who desire to create a hypomorphic allele by inserting NEO into intronic DNA should take into consideration the variability of gene expression observed in this study. The extent of variability may be locus dependent. + +Knockdown of γ2 subunit containing GABAA-Rs resulted in behavioral changes that are indicative of increased anxiety-like behavior. These mutant knockdown mice showed reduced exploratory behavior for a novel open field apparatus and the open arms of the elevated plus maze. Similar changes have been observed in γ2 global heterozygous knockout mice [10]. Together, these results strengthen the possibility that GABAA-R dysfunction may be an underlying cause of anxiety disorders in humans. These mutant mouse models may be useful for dissecting the etiology of this pervasive condition and for developing effective therapeutic interventions. + +As expected, brains from γ2 knockdown mice demonstrated significantly decreased binding of the benzodiazepine site ligand, Ro15-4513. This was expected since benzodiazepine binding sites are located at the interface of select alpha and γ2 subunits of the GABAA-R [30]. The reduction observed in Ro15-4513 binding agree with those from global γ2 knockout mice [9] in that deficiency of the γ2 subunit abolishes the binding of the benzodiazepine-site ligands. The global heterozygous γ2 knockout mice have a reduction in total [3H]Ro15-4513 binding in most brain regions [31], and the present data on the γ2 knockdown show a very similar pattern of reduced binding. However, the 5 knockdown brains that we examined had actually a greater reduction in benzodiazepine binding than the γ2 heterozygous global knockouts published earlier [31]. Similar to the γ2 heterozygous knockouts, [3H]muscimol binding was not affected, indicating that the reduction of the γ2 subunit levels is not compensated by increased δ subunit that is largely responsible for the [3H]muscimol binding signal in brain sections [32]. The basal binding of the ion channel ligand [35S]TBPS is usually reduced and its sensitivity to GABA increased by addition of γ2 subunits [31]. Both in the γ2 heterozygous global knockout and in our homozygous knockdown, TBPS binding was increased in some brain regions, and, more consistently, there emerged a "GABA-insensitive" receptor population with widespread distribution in the brain. This indicates production of GABAA-Rs with an αβ configuration. These receptors bind strongly the agonist and have reduced channel conductance [33], perhaps reflecting only partial agonist efficacy of GABA. This would explain the reduced allosteric efficacy of GABA in abolishing TBPS binding. In addition, these receptors have most likely non-synaptic, non-clustered localization as they lack the γ2 subunit [10,33]. Therefore, the partially γ2 depleted mice may have brain region-selective alterations in synaptic and extrasynaptic receptors, and the reduced synaptic GABAA-R function might correlate with the anxious phenotype. However, surprisingly, the high-dose hypnotic effects of the benzodiazepine site ligands diazepam, zolpidem and midazolam were unchanged in the knockdown mice. + +Several hypotheses can be generated to explain the discrepancy between decreased benzodiazepine ligand binding and unchanged hypnotic effects. One possibility is that only a threshold level of γ2 containing GABAA-Rs are required for hypnotic responses to benzodiazepines. Mice that completely lack γ2 had only 6% of benzodiazepine binding compared to wild type mice and were insensitive to the hypnotic effects of diazepam [9]. Likewise, mice with a point mutation in γ2 that eliminated response to zolpidem eliminated the effects of this benzodiazepine site ligand [26]. It seems likely that the 20–40% of benzodiazepine sensitive receptors that remain in the knockdown mice are enough to mediate the hypnotic effect of benzodiazepine site ligands. This is supported by positron emission tomography studies in humans, in which only a small proportion of the benzodiazepine sites need to be occupied by lorazepam and zolpidem to induce clinical effects such as sedation [34,35]. There may thus be spare receptors that can be measured by biochemical techniques but are not needed for whole animal pharmacological effects. Another hypothesis is that knockdown of γ2 does not result in a proportional reduction in all γ2 containing GABAA-Rs. Recent studies conducted in knockin mice with a point mutation at the benzodiazepine binding site have attributed α1 and α2 containing GABAA-Rs to the mediation of benzodiazepine-induced sedation and anxiolysis, respectively [4,36-38]. Perhaps γ2 knockdown mice have disproportionate decreases in non-α1 containing receptors, e.g., α2 or α3 containing receptor isoforms. It may be possible that in our γ2 knockdown mice, the number of α2βxγ2 GABAA-Rs is greatly reduced while the number of α1βxγ2 receptors is not, which remains to be studied. This could be tested by immunohistochemical methods using antibodies against α2 and α3 or by testing for changes in zolpidem insensitive binding. Changes in subunit trafficking may also play a role. Essrich et al. [7] established strong evidence that GABAA-Rs are clustered at the synapse through indirect interactions between γ2 and gephyrin. Enough clustering may remain in the knockdown mice such that drug response does not change. Alternatively, synaptic clustering of GABAA-Rs may not be important for behavioral responses to benzodiazepines, and it is possible that benzodiazepines potentiate the extrasynaptic GABAA-R responses determined by electrophysiology in brain slices [39]. It is also possible that the high variability in γ2 levels in the brain of knockdown mice masked any change in behavioral response to these drugs. In hindsight, it would have been useful to quantify γ2 levels in the same mice that were used for the behavioral sensitivity studies. It would be interesting to see if sensitivity correlated with γ2 levels on an individual animal basis. This type of approach could be exploited in future studies of γ2 receptor function. Lastly, decreases in benzodiazepine binding may not directly translate into changes in in vivo insensitivity. For example, in α1 null mice, changes in benzodiazepine binding did not change whole animal sensitivity to midazolam but increased sensitivity to diazepam [40,41]. In contrast, in mice that selectively lack the long splice variant of γ2, affinity for benzodiazepine site ligands is increased and behavioral response to the sedative effects of midazolam and zolpidem is also increased [42]. Similarly, in mice lacking the β3 subunit, decreased benzodiazepine binding and decreased whole animal midazolam sensitivity were observed [43]. Further study is required to examine these possibilities. + +Conclusion + +We have produced a novel genetically engineered mouse line that exhibits a reduction in γ2 mRNA levels that is highly variable between mice (range 10–95% of control) but averages ~35% of control levels. These γ2 knockdown mice are viable and have enhanced anxiety-like behavioral abnormalities. Surprising, these mice are normally sensitive to the hypnotic effects of benzodiazepine site ligands. Lastly, this novel genetically engineered mouse line can also be easily converted to a γ2 global knockout mouse line by simply crossing to a cre-expressing global deleter transgenic mouse line. + +Methods + +Generation of genetically altered mice + +A 13.6 kB BglII Strain 129/SvJ mouse genomic DNA fragment containing Exons 8–10 of the GABAA-R γ2 gene was subcloned from a P1 phage clone (Genome Systems, St. Louis, MO). An oligonucleotide containing a loxP site and an EcoRV site was inserted into a NcoI site 647 bp 5' to Exon 8. A blunted fragment containing a NEO resistance gene with PGK-1 promoter and polyadenylation signals (obtained from pPNT [44]) fused with a loxP site was inserted into a HincII site 855 bp 3' of Exon 8. The NEO gene was inserted in the opposite orientation to γ2 transcription. A PGK driven thymidine kinase expression cassette (obtained from pPNT [44]) was then cloned 3' to the γ2 genomic DNA. The targeting construct was linearized with SalI and electroporated into R1 embryonic stem cells [45] following previously described procedures [46]. G418 (270 μg/ml; Life Technologies, Gaithersburg, MD) and gancyclovir (2 μM; Sigma) resistant cells were screened for gene targeting by Southern blot analysis of BglII digested genomic DNA. Fragments were hybridized with a 5' probe that was external to the targeting construct (see Fig. 1). Proper targeting of the γ2 locus was confirmed by Southern blot analysis of EcoRV and SstI digested DNA. Additional Southern blot analysis with probes to the neomycin cassette or internal to the 3' arm of the targeting vector (with 1–3 restriction enzymes) were also conducted (results not shown). All results were consistent with a correctly targeted γ2 locus. + +Two correctly targeted embryonic stem cell clones (26C8 and 26L6) were microinjected into C57BL/6J blastocysts to produce chimeric mice. Male chimeras were mated to C57BL/6J females. Agouti offspring heterozygous for the targeted allele (F/+) were interbred to produce mice that were wild type (+/+), heterozygous, or homozygous mutants (F/F). The mice used in studies reported here were derived from the 26L6 ES cell clone. All mice were housed under conditions of lights on at 07:00 and lights off at 19:00. + +Northern blot analysis + +Ten to fourteen week old mice were sacrificed by cervical dislocation. RNA was isolated from whole brain using Trizol reagent (Invitrogen). 20 μg of total RNA was electrophoresed in a 1.9% formaldehyde/1% agarose gel and blotted onto Hybond-N (Amersham Pharmacia). Blots were first probed with a 32P-labeled γ2 cDNA probe then re-hybrizided with a control human β-actin probe (Clontech). Autoradiographs were digitally photographed and band densities were measured using Kodak 1D Image Analysis Software. Ratio between density of γ2 hybridization versus actin hybridization was recorded. + +Immunocytochemistry + +The procedure has been described elsewhere [47]. Briefly, wild type and homozygous mutants were deeply anesthetized with Avertin and transcardially perfused with Dextran-Phosphate Buffer (PB) then fixative consisting of 0.01M periodate/0.075M lysine/4% paraformaldehyde in 0.1M PB (pH 7.4). The brains were frozen and sliced in parasaggital sections (25 μm) with a freezing microtome. Slices were incubated overnight at 4°C with affinity-purified rabbit anti-γ2 [48,49] in 0.1M PB (pH 7.4) with 0.3% Triton X-100, at a concentration of (1:100). The sections were processed using an avidin-biotin-peroxidase system (Vectastain Elite; Novocastra, Burlingame, CA). Peroxidase reaction was carried out with 3-3' diaminobenzidine tetrahydrochloride in the presence of cobalt chloride and nickel ammonium sulfate as chromogens and hydrogen peroxide as oxidant. Controls were performed either by omitting the primary antibody or by incubating the primary antibody with the corresponding peptide [50]. + +Pharmacology + +Eight-week-old mice were sacrificed by decapitation and whole brains were rapidly dissected out and frozen on dry ice. For autoradiography, 14-μm horizontal serial cryostat sections were cut from 5 mouse brains of each genotype, thaw-mounted onto gelatin-coated object glasses, and stored frozen under desiccant at -20°C. All experiments were carried out in parallel fashion with respect to mouse lines, eliminating any day-to-day variation between the assays. The autoradiographic procedures for regional localization of [3H]Ro 15-4513, [3H]muscimol, and [35S]TBPS binding were as described in [51]. Briefly, sections were preincubated in an ice-water bath for 15 min in 50 mM Tris-HCl (pH 7.4) supplemented with 120 mM NaCl in the [3H]Ro 15-4513 and [35S]TBPS autoradiographic assays, and in 0.31 M Tris-citrate (pH 7.1) in the [3H]muscimol assay. All radioligands were purchased from Perkin Elmer Life Sciences, Inc. (Boston, MA, USA). + +The final incubation in respective preincubation buffer was performed with 6 nM [35S]TBPS at room temperature for 90 min, assays with 10 nM [3H]muscimol at 0–4°C for 30 min, and assays with 10 nM [3H]Ro 15-4513 in the presence and absence of 100 μM diazepam (Orion Pharma, Espoo, Finland) at 0–4°C for 60 min. After incubation, sections were washed 3 × 15 s or 2 × 30 s in an ice-cold incubation buffer in [35S]TBPS and [3H]Ro 15-4513 or in [3H]muscimol assay, respectively. Sections were then dipped into distilled water, air-dried under a fan at room temperature, and exposed with plastic [3H]- or [14C]-methacrylate standards to Kodak Biomax MR films for 1 to 8 weeks. Nonspecific binding was determined with 10 μM flumazenil (Hoffmann-La Roche, Basel, Switzerland), 100 μM picrotoxinin (Sigma) and 100 μM GABA (Sigma) in [3H]Ro 15-4513, [35S]TBPS and [3H]muscimol assays, respectively. Binding densities in the relevant brain areas were quantitated with MCID M5-imaging software (Imaging Research Inc., Ontario, Canada) and converted to nCi/mg (for 3H) or nCi/g (for 35S) radioactivity values on the basis of the simultaneously exposed standards. + +The concentrations of [3H]muscimol (10 nM) and [3H]Ro 15-4513 (10 nM) were greater than or equal to the dissociation constants for a range of recombinant and native GABAA receptors [8,52-54]. Therefore, the autoradiographic images should represent the density rather than affinity of binding sites. + +Hypnotic sensitivity + +Drug induced hypnosis was assessed by measuring the duration of the loss of righting reflex in response to various sedative/hypnotic drugs. Diazepam (25 mg/kg; Sigma), midazolam (45 mg/kg and 75 mg/kg; ESI Lederle, Philadelphia, PA), pentobarbital (45 mg/kg; Abbott, Chicago, IL), zolpidem (60 mg/kg; Searle, Malvern, PA) or ethanol (3.5 mg/kg; Pharmco, Brookfield, CT) were administered intraperitoneally to wild type and homozygous mutants. Drugs were diluted in saline such that the injection volume was 20 μL per gram body weight. After losing the righting reflex, mice were placed in a plastic V-shaped trough and the time was recorded. When the mouse was able to right itself three times in 30 s, the measure of hypnotic effect was over. Body temperature was maintained with aid of a heat lamp. Assays were performed in a blinded manner with respect to genotype. Effect of genotype on the duration of the loss of righting reflex was compared using Student's t-test. + +Elevated plus-maze test + +Basal anxiety-like behavior was tested using the elevated plus-maze. All mice were between 7 and 9 weeks of age and were tested between 09:00 and 11:00. Each mouse was placed on the central platform of the maze, facing an open arm and allowed to freely explore the maze for 5 min under ambient room light. Open-arm and closed-arm entries and the cumulative time spent on the open and closed arms was recorded. A mouse was considered to be on the central platform or on an arm when all four paws were within its perimeter. The percent open-arm entries, total number of entries, and percent time in open-arms was determined. Data were analyzed using one-way ANOVA. + +Forced exploration + +Basal motor activity of mice was determined using the forced exploration test. 8–10 week old mice were placed into a walled arena (43.2 cm × 43.2 cm × 30.5 cm) for 5 min. Distance traveled (cm) was measured automatically using an Activity Monitor (Med Associates, St. Albans, VT). All tests were performed between 12:00 and 15:00. Effect of genotype on basal motor activity was compared using Student's t-test. + +Authors' contributions + +CPM and ALD carried out the immunohistochemical studies. ERK carried out the ligand autoradiography studies. All other experiments were performed by DC and GEH. All authors contributed to composing and editing the manuscript and have read and approved the final version. + +Acknowledgements + +Carolyn Ferguson and Ed Mallick are gratefully acknowledged for superb technical support. This work was supported by NIH grants AA10422, GM52035, GM47818, NS039287, the Sigrid Juselius Foundation, and the Academy of Finland. + +Figures and Tables + +Figure 1 + +Gene targeting of the γ2 subunit of the GABAA-R and development of γ2 knockdown mice. (A) Targeting strategy used to produce γ2 knockdown mice. Relevant region of endogenous gene (+), targeting vector, correctly targeted knockdown γ2 (F), and cre-recombined knockout alleles (f) are shown. Relative locations of relevant restriction sites, exons (numbered orange boxes), loxP sites (blue triangles), plasmid backbone (wavy line), and positive (NEO) and negative (PGK-TK) selection cassettes are shown. (B) Southern Blot analysis of BglII digested tail DNAs. Probe A hybridizes to a 9.2 kb BglII fragment from the wild type endogenous allele and a 3.4 kb BglII fragment from the targeted allele. + +Figure 2 + +Northern blot analysis. (A) Sample northern blot analysis of wild type (+/+) and homozygous knockdown (F/F) adult whole brain total RNA hybrizided with a γ2 cDNA or a human β-actin probe as a loading control. (B) Densitometric analysis of northern blots. The ratio of band density between γ2 and β-actin hybridization for wild type (n = 23) and homozygous knockdown (n = 36) mice was plotted. On each individual blot, the ratio of wild type band densities was averaged and normalized to 100. All samples on each blot were then compared to the average of wild type band densities on that blot. A total of 4 different blots were included in this analysis. The horizontal bar in each column represents the mean for that genotype. + +Figure 3 + +Immunohistochemical distribution of γ2 subunit of the GABAA-R in sections from individual (A) wild type and (B) homozygous knockdown mice. Note the high level of abundance of γ2 immunoreactivity in olfactory bulb (OB), cerebral cortex (CC), hippocampus (HP), substantia nigra (SN), and cerebellum (CB) of wild type samples. The amount of γ2 is variably reduced in many brain regions of the knockdown samples. + +Figure 4 + +Behavioral characterization of knockdown mice. (A-C) Elevated plus maze. Knockdown mice (A) entered open arms less often and (B) for less time, however, (C) total entries into arms did not differ from wild type mice. (D) Knockdown mice were also less active in the forced exploration test. The bars are means ± SEM. *p < .01, **p < .001. + +Figure 5 + +Hypnotic sensitivity. No significant differences in the hypnotic effects of ethanol, pentobarbital, zolpidem, midazolam, or diazepam were observed. The bars are means ± SEM. + +Table 1 + +Autoradiographic analysis of GABAA receptor binding sites in horizontal sections of wild type and mutant mice. + +The data are means ± standard deviations for 5 mice in each genotype, and are expressed as nCi/mg for 3H-ligands and as nCi/g for 35S-ligand. Significance of the difference from wild type (Tukey-Kramer test after ANOVA): aP < 0.05, bP < 0.01, cP < 0.001. Ctx, cortex; Th, thalamus; Hi, hippocampus; CPu, caudate putamen; IC, inferior colliculus; Gr, cerebellar granule cell layer; Mol, cerebellar molecular layer. diff --git a/src/ontogpt/evaluation/craft/database/all/15876356.ann b/src/ontogpt/evaluation/craft/database/all/15876356.ann new file mode 100644 index 000000000..24afa7f58 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15876356.ann @@ -0,0 +1,748 @@ +T1 http://purl.obolibrary.org/obo/MONDO_0000437 0 6 Ataxia +T2 UBERON:0001021 11 27 peripheral nerve +T3 PR:000003718 47 53 ADAM22 +T4 NCBITaxon:10088 64 68 mice +T5 PR:000003718 92 98 ADAM22 +T6 SO:0000704 123 127 gene +T7 GO:0010467 160 169 expressed +T8 UBERON:0001016 182 197 nervous systems +T9 PR:000003718 215 221 ADAM22 +T10 GO:0031012 341 347 matrix +T11 PR:000003718 406 412 ADAM22 +T12 SO:0000704 426 430 gene +T13 PR:000003718 453 459 ADAM22 +T14 NCBITaxon:10088 469 473 mice +T15 PR:000003718 485 491 ADAM22 +T16 NCBITaxon:10088 502 506 mice +T17 GO:0007567 590 595 birth +T18 http://purl.obolibrary.org/obo/MONDO_0000437 620 626 ataxia +T19 GO:0016265 661 665 died +T20 UBERON:0000956 768 783 cerebral cortex +T21 UBERON:0002037 787 797 cerebellum +T22 UBERON:0001021 864 881 peripheral nerves +T23 PR:000003718 951 957 ADAM22 +T24 UBERON:0001016 1012 1026 nervous system +T25 PR:000003718 1048 1054 ADAM22 +T26 NCBITaxon:9606 1109 1114 human +T27 http://purl.obolibrary.org/obo/MONDO_0000001 1115 1123 diseases +T28 http://purl.obolibrary.org/obo/MONDO_0005027 1132 1141 epileptic +T29 http://purl.obolibrary.org/obo/MONDO_0005244 1155 1176 peripheral neuropathy +T30 GO:0016020 1247 1255 membrane +T31 SO:0000417 1271 1277 domain +T32 SO:0000417 1323 1329 domain +T33 SO:0000417 1353 1359 domain +T34 NCBITaxon:40674 1415 1422 mammals +T35 GO:0009566 1471 1484 fertilization +T36 GO:0022008 1501 1513 neurogenesis +T37 GO:0006508 1517 1528 proteolysis +T38 GO:0005576 1617 1630 extracellular +T39 SO:0000417 1631 1638 domains +T40 GO:0016020 1642 1650 membrane +T41 PR:000001279 1705 1711 ADAM17 +T42 PR:000001279 1713 1717 TACE +T43 MOP:0000780 1737 1743 cleave +T44 http://purl.obolibrary.org/obo/MONDO_0005070 1774 1780 tumour +T45 PR:000000134 1774 1802 tumour necrosis factor alpha +T46 CHEBI:28304 1809 1816 heparin +T47 PR:000008459 1809 1867 heparin-binding epidermal growth factor-like growth factor +T48 UBERON:0007376 1825 1834 epidermal +T49 PR:000016285 1878 1910 transforming growth factor alpha +T50 PR:000001279 1927 1933 ADAM17 +T51 NCBITaxon:10088 1939 1943 mice +T52 PR:000001279 1963 1969 ADAM17 +T53 GO:0009790 1985 1998 embryogenesis +T54 GO:0031012 2111 2117 matrix +T55 PR:000003715 2300 2305 ADAM2 +T56 PR:000003732 2315 2320 ADAM3 +T57 CL:0000019 2363 2374 spermatozoa +T58 CL:0000025 2380 2383 egg +T59 PR:000003715 2416 2421 ADAM2 +T60 PR:000003732 2426 2431 ADAM3 +T61 SO:0000417 2525 2531 domain +T62 PR:000003710 2670 2676 ADAM11 +T63 PR:000003718 2678 2684 ADAM22 +T64 PR:000003719 2689 2695 ADAM23 +T65 SO:0000704 2696 2701 genes +T66 GO:0010467 2723 2733 expression +T67 NCBITaxon:9606 2741 2746 human +T68 NCBITaxon:39107 2751 2757 murine +T69 UBERON:0001016 2758 2773 nervous systems +T70 PR:000003719 2911 2917 ADAM23 +T71 GO:0009986 2946 2958 cell surface +T72 GO:0034683 2980 3003 alpha-v beta-3 integrin +T73 PR:000002042 2988 2994 beta-3 +T74 PR:000003719 3043 3049 ADAM23 +T75 SO:0000704 3050 3054 gene +T76 NCBITaxon:10088 3062 3067 mouse +T77 GO:0016265 3089 3094 death +T78 http://purl.obolibrary.org/obo/MONDO_0000437 3111 3117 ataxia +T79 GO:0016265 3157 3162 death +T80 NCBITaxon:10088 3171 3176 mouse +T81 GO:0031012 3238 3244 matrix +T82 UBERON:0001016 3265 3279 nervous system +T83 PR:000003719 3298 3304 ADAM23 +T84 PR:000003718 3325 3331 ADAM22 +T85 PR:000003719 3336 3342 ADAM23 +T86 SO:0000857 3356 3366 homologous +T87 GO:0005576 3386 3399 extracellular +T88 SO:0000417 3400 3407 domains +T89 SO:0000417 3548 3554 domain +T90 PR:000003718 3598 3604 ADAM22 +T91 UBERON:0001016 3662 3676 nervous system +T92 PR:000003719 3686 3692 ADAM23 +T93 PR:000003718 3738 3744 ADAM22 +T94 PR:000003718 3772 3778 Adam22 +T95 SO:0000704 3779 3783 gene +T96 NCBITaxon:10088 3793 3797 mice +T97 PR:000003718 3823 3829 ADAM22 +T98 NCBITaxon:10088 3840 3844 mice +T99 NCBITaxon:10088 3846 3850 Mice +T100 PR:000003718 3889 3895 Adam22 +T101 SO:0000704 3896 3900 gene +T102 SO:0000319 4042 4059 termination codon +T103 SO:0000147 4078 4082 exon +T104 SO:0001062 4092 4103 pro-protein +T105 SO:0000417 4104 4110 domain +T106 PR:000003718 4143 4149 ADAM22 +T107 SO:0001023 4198 4204 allele +T108 SO:0001062 4216 4227 pro-protein +T109 SO:0000417 4228 4234 domain +T110 PR:000003137 4299 4304 Furin +T111 PR:000003718 4406 4412 ADAM22 +T112 PR:000003718 4456 4462 ADAM22 +T113 GO:0042571 4551 4559 antibody +T114 GO:0005737 4582 4593 cytoplasmic +T115 SO:0000417 4594 4603;4615 4622 domain of ... protein +T116 PR:000003718 4608 4614 ADAM22 +T117 GO:0007567 4685 4690 birth +T118 GO:0007567 4791 4796 natal +T119 UBERON:0002101 4945 4950 limbs +T120 http://purl.obolibrary.org/obo/MONDO_0000437 4996 5002 ataxia +T121 GO:0016265 5025 5028 die +T122 PR:000003718 5521 5527 ADAM22 +T123 GO:0009790 5549 5562 embryogenesis +T124 GO:0016265 5718 5722 died +T125 GO:0016265 5765 5769 died +T126 GO:0007567 5872 5877 birth +T127 PR:000003718 6020 6026 ADAM22 +T128 NCBITaxon:10088 6037 6041 mice +T129 GO:0010467 6076 6086 expression +T130 PR:000003718 6090 6096 ADAM22 +T131 NCBITaxon:9606 6109 6114 human +T132 NCBITaxon:10088 6119 6124 mouse +T133 UBERON:0000955 6125 6130 brain +T134 PR:000003718 6199 6205 ADAM22 +T135 SO:0000673 6206 6216 transcript +T136 UBERON:0007023 6224 6229 adult +T137 NCBITaxon:10088 6230 6235 mouse +T138 UBERON:0001017 6236 6239 CNS +T139 GO:0097617 6259 6272 hybridisation +T140 CHEBI:37983 6302 6305 35S +T141 PR:000003718 6345 6351 ADAM22 +T142 GO:0010467 6361 6370 expressed +T143 UBERON:0007023 6386 6391 adult +T144 NCBITaxon:10088 6392 6397 mouse +T145 UBERON:0001017 6398 6401 CNS +T146 UBERON:0002037 6437 6447 cerebellar +T147 CL:0001031 6437 6461 cerebellar granule cells +T148 UBERON:0002421 6466 6487 hippocampal formation +T149 UBERON:0002240 6496 6507 spinal cord +T150 GO:0097617 6509 6522 hybridisation +T151 UBERON:0002020 6552 6563 grey matter +T152 PR:000003718 6585 6591 ADAM22 +T153 SO:0000673 6592 6603 transcripts +T154 UBERON:0001017 6611 6614 CNS +T155 PR:000003710 6646 6652 ADAM11 +T156 CL:0000540 6660 6668 neuronal +T157 GO:0010467 6669 6679 expression +T158 PR:000003718 6735 6741 ADAM22 +T159 GO:0010467 6747 6757 expression +T160 CL:0000540 6761 6769 neuronal +T161 NCBITaxon:10088 6777 6782 mouse +T162 UBERON:0001017 6783 6786 CNS +T163 UBERON:0002037 6882 6892 cerebellum +T164 GO:0010467 6948 6958 expression +T165 PR:000003718 6962 6968 ADAM22 +T166 UBERON:0002037 6982 6992 cerebellar +T167 CL:0001031 6982 7006 cerebellar granule cells +T168 CL:0000120 7008 7020 granule cell +T169 CL:0000121 7051 7064 Purkinje cell +T170 PR:000004967 7077 7086 calbindin +T171 NCBITaxon:10088 7111 7116 mouse +T172 UBERON:0002421 7145 7166 Hippocampal formation +T173 CL:0000540 7233 7246 neuronal cell +T174 GO:0001764 7233 7256 neuronal cell migration +T175 NCBITaxon:10088 7288 7293 mouse +T176 PR:000010228 7324 7327 MBP +T177 UBERON:0000345 7329 7335 myelin +T178 PR:000010228 7329 7349 myelin basic protein +T179 CHEBI:22695 7336 7341 basic +T180 UBERON:0002037 7381 7391 cerebellum +T181 UBERON:0002240 7406 7417 spinal cord +T182 NCBITaxon:10088 7570 7575 mouse +T183 UBERON:0000955 7576 7581 brain +T184 UBERON:0002240 7631 7642 spinal cord +T185 UBERON:0001021 7647 7664 peripheral nerves +T186 UBERON:0000345 7735 7741 myelin +T187 UBERON:0000345 7775 7781 myelin +T188 UBERON:0000345 7790 7796 myelin +T189 UBERON:0001322 7817 7831 sciatic nerves +T190 UBERON:0001645 7846 7863 trigeminal nerves +T191 UBERON:0002240 7937 7948 spinal cord +T192 CL:0002573 7982 7994 Schwann cell +T193 NCBITaxon:10088 8050 8054 mice +T194 CL:0000218 8080 8105 myelinating Schwann cells +T195 GO:0030424 8110 8115 axons +T196 CHEBI:10545 8117 8125 electron +T197 UBERON:0001322 8159 8172 sciatic nerve +T198 CL:0002573 8197 8210 Schwann cells +T199 UBERON:0000345 8229 8235 myelin +T200 NCBITaxon:10088 8298 8302 mice +T201 GO:0030424 8360 8365 axons +T202 UBERON:0001322 8434 8447 sciatic nerve +T203 GO:0005634 8480 8486 nuclei +T204 PR:000010228 8521 8524 MBP +T205 CL:0002573 8581 8593 Schwann cell +T206 GO:0014010 8727 8743;8748 8761 proliferation of ... Schwann cells +T207 CL:0002573 8748 8761 Schwann cells +T208 PR:000003718 8826 8832 ADAM22 +T209 NCBITaxon:10088 8843 8847 mice +T210 PR:000003718 8856 8862 ADAM22 +T211 SO:0000673 8863 8873 transcript +T212 UBERON:0000010 8901 8926 peripheral nervous system +T213 NCBITaxon:9606 8943 8948 human +T214 PR:000003718 8949 8955 ADAM22 +T215 GO:0008380 8975 8983 splicing +T216 SO:0000147 9026 9031 exons +T217 SO:0000147 9095 9099 exon +T218 SO:0000147 9101 9105 exon +T219 NCBITaxon:10088 9117 9122 mouse +T220 NCBITaxon:10088 9188 9193 mouse +T221 PR:000003718 9194 9200 ADAM22 +T222 SO:0000673 9201 9212 transcripts +T223 NCBITaxon:10088 9238 9243 mouse +T224 SO:0001026 9244 9250 genome +T225 SO:0000857 9260 9268 homology +T226 NCBITaxon:10088 9308 9313 mouse +T227 SO:0001026 9314 9320 genome +T228 SO:0000149 9321 9327 contig +T229 NCBITaxon:10088 9360 9365 mouse +T230 PR:000003718 9366 9372 Adam22 +T231 SO:0000704 9373 9377 gene +T232 SO:0000149 9387 9393 contig +T233 SO:0000147 9422 9427 exons +T234 NCBITaxon:9606 9477 9482 human +T235 PR:000003718 9483 9489 ADAM22 +T236 SO:0001060 9490 9497 isoform +T237 SO:0000673 9547 9558 transcripts +T238 SO:0000112 9572 9579 primers +T239 SO:0000147 9596 9600 exon +T240 NCBITaxon:10088 9631 9636 mouse +T241 UBERON:0002037 9637 9647 cerebellum +T242 SO:0001060 9768 9775 isoform +T243 SO:0001060 9804 9811 isoform +T244 SO:0000147 9854 9858 exon +T245 SO:0000147 9867 9872 exons +T246 UBERON:0007023 9950 9955 adult +T247 NCBITaxon:10088 9956 9961 mouse +T248 UBERON:0000479 9962 9969 tissues +T249 UBERON:0002240 9983 9994 spinal cord +T250 UBERON:0000044 9996 10016 dorsal root ganglion +T251 UBERON:0000044 10018 10021 DRG +T252 UBERON:0001322 10024 10037 sciatic nerve +T253 CL:0000001 10042 10058;10067 10072 cultured primary ... cells +T254 CL:0002573 10059 10072 Schwann cells +T255 UBERON:0000479 10137 10143 tissue +T256 UBERON:0001322 10274 10287 sciatic nerve +T257 CL:0000010 10310 10318;10327 10332 cultured ... cells +T258 CL:0002573 10319 10332 Schwann cells +T259 UBERON:0000479 10404 10411 tissues +T260 UBERON:0000044 10451 10454 DRG +T261 SO:0000147 10539 10543 exon +T262 SO:0000673 10574 10585 transcripts +T263 SO:0001026 10717 10724 genomic +T264 SO:0000147 10768 10773 exons +T265 SO:0000147 10775 10780 exons +T266 SO:0000147 10839 10844 exons +T267 SO:0000357 10850 10857 flanked +T268 SO:0000188 10874 10881 introns +T269 SO:0000147 10908 10912 Exon +T270 PR:000003718 10988 10994 ADAM22 +T271 SO:0000673 10995 11006 transcripts +T272 SO:0000147 11051 11056 exons +T273 SO:0000147 11095 11100 exons +T274 SO:0000147 11185 11190 exons +T275 SO:0000717 11222 11235 reading frame +T276 PR:000003718 11354 11360 ADAM22 +T277 SO:0000673 11361 11372 transcripts +T278 UBERON:0001017 11410 11413 CNS +T279 SO:0000147 11431 11435 exon +T280 UBERON:0000044 11441 11444 DRG +T281 SO:0000147 11462 11466 exon +T282 CL:0002573 11476 11488 Schwann-cell +T283 SO:0000147 11504 11508 exon +T284 PR:000003718 11559 11565 ADAM22 +T285 GO:0010467 11569 11578 expressed +T286 UBERON:0000010 11664 11667 PNS +T287 PR:000003718 11732 11738 ADAM22 +T288 NCBITaxon:10088 11753 11757 mice +T289 PR:000003718 11774 11780 Adam22 +T290 SO:0000704 11781 11785 gene +T291 PR:000003718 11787 11793 ADAM22 +T292 NCBITaxon:10088 11804 11808 mice +T293 http://purl.obolibrary.org/obo/MONDO_0000437 11826 11832 ataxia +T294 GO:0016265 11847 11852 death +T295 GO:0016265 11984 11989 death +T296 NCBITaxon:10088 12188 12192 mice +T297 GO:0016265 12232 12236 died +T298 UBERON:0001851 12271 12279 cortical +T299 http://purl.obolibrary.org/obo/MONDO_0017094 12271 12289 cortical dysplasia +T300 UBERON:0000955 12314 12319 brain +T301 GO:0007420 12314 12331 brain development +T302 http://purl.obolibrary.org/obo/MONDO_0002254 12347 12355 syndrome +T303 NCBITaxon:10088 12375 12379 mice +T304 CL:0000540 12392 12398 neuron +T305 PR:000005259 12408 12429 activator of cyclin 5 +T306 PR:000005259 12431 12434 p35 +T307 CL:0000540 12466 12474 neuronal +T308 GO:0001764 12466 12484 neuronal migration +T309 PR:000003718 12506 12512 ADAM22 +T310 GO:0010467 12524 12533 expressed +T311 GO:0009986 12541 12553 cell surface +T312 GO:0031012 12604 12610 matrix +T313 PR:000003718 12659 12665 ADAM22 +T314 CL:0000540 12690 12698 neuronal +T315 GO:0001764 12690 12708 neuronal migration +T316 NCBITaxon:10088 12835 12840 mouse +T317 UBERON:0000955 12841 12846 brain +T318 PR:000003718 12879 12885 ADAM22 +T319 CL:0000540 12889 12897 neuronal +T320 GO:0001764 12889 12907 neuronal migration +T321 GO:0016265 12960 12965 death +T322 UBERON:0000104 12986 12990 life +T323 UBERON:0002410 13027 13051 autonomic nervous system +T324 UBERON:0001021 13065 13071 nerves +T325 GO:0002087 13078 13085 control +T326 UBERON:0001004 13137 13155 respiration system +T327 GO:0007567 13218 13223 birth +T328 UBERON:0000345 13242 13248 myelin +T329 NCBITaxon:10114 13264 13267 rat +T330 PR:000012879 13307 13326 proteolipid protein +T331 PR:000012879 13328 13331 PLP +T332 SO:0000704 13333 13337 gene +T333 http://purl.obolibrary.org/obo/MONDO_0000437 13348 13354 ataxia +T334 GO:0016265 13383 13387 dies +T335 GO:0007567 13409 13414 natal +T336 NCBITaxon:10114 13429 13432 rat +T337 GO:0016265 13440 13445 death +T338 UBERON:0002298 13482 13492 brain stem +T339 GO:0002087 13516 13548 autonomic control of respiration +T340 GO:0016265 13617 13622 death +T341 PR:000003718 13626 13632 ADAM22 +T342 NCBITaxon:10088 13643 13647 mice +T343 PR:000003718 13692 13698 ADAM22 +T344 NCBITaxon:10088 13709 13713 mice +T345 UBERON:0001322 13804 13811;13827 13833 sciatic ... nerves +T346 UBERON:0001645 13816 13833 trigeminal nerves +T347 UBERON:0000345 13844 13850 myelin +T348 UBERON:0002240 13868 13879 spinal cord +T349 UBERON:0000955 13888 13893 brain +T350 UBERON:0001021 13984 14001 peripheral nerves +T351 NCBITaxon:10088 14018 14022 mice +T352 CL:0002573 14035 14047 Schwann cell +T353 UBERON:0001322 14075 14088 sciatic nerve +T354 CL:0000218 14105 14134 pro-myelinating Schwann cells +T355 PR:000010228 14151 14154 MBP +T356 CL:0000218 14225 14253 pro-myelinating Schwann cell +T357 UBERON:0000345 14282 14288 myelin +T358 PR:000003718 14313 14319 ADAM22 +T359 CL:0002573 14347 14359 Schwann cell +T360 GO:0014010 14347 14359;14388 14401 Schwann cell ... proliferation +T361 GO:0030154 14355 14375 cell differentiation +T362 PR:000003718 14457 14463 ADAM22 +T363 NCBITaxon:10088 14474 14478 mice +T364 SO:0000417 14588 14595 domains +T365 PR:000009131 14632 14639 alpha-6 +T366 GO:0034675 14632 14655 alpha-6 beta-1 integrin +T367 PR:000002040 14640 14646 beta-1 +T368 CL:0002573 14663 14675 Schwann cell +T369 UBERON:0000345 14703 14709 myelin +T370 GO:0005607 14739 14748 laminin-2 +T371 UBERON:0000345 14779 14785 myelin +T372 GO:0005607 14805 14814 laminin-2 +T373 NCBITaxon:10088 14851 14855 mice +T374 NCBITaxon:9606 14860 14866 humans +T375 PR:000009131 14887 14894 alpha-6 +T376 GO:0034675 14887 14910 alpha-6 beta-1 integrin +T377 PR:000002040 14895 14901 beta-1 +T378 PR:000006267 14915 14927 dystroglycan +T379 UBERON:0000345 14969 14975 myelin +T380 PR:000002040 15031 15046 beta-1 integrin +T381 PR:000009658 15051 15066 laminin gamma-1 +T382 CL:0002573 15070 15082 Schwann cell +T383 GO:0005607 15103 15112 laminin-2 +T384 GO:0030424 15177 15182 axons +T385 UBERON:0000345 15205 15211 myelin +T386 PR:000003718 15270 15276 ADAM22 +T387 NCBITaxon:10088 15287 15291 mice +T388 GO:0030424 15346 15351 axons +T389 UBERON:0001021 15369 15375 nerves +T390 UBERON:0002211 15380 15385 roots +T391 PR:000003718 15402 15408 ADAM22 +T392 GO:0065007 15419 15428 modulates +T393 UBERON:0000345 15490 15496 myelin +T394 PR:000003718 15546 15552 ADAM22 +T395 NCBITaxon:10088 15563 15567 mice +T396 PR:000003718 15584 15590 ADAM22 +T397 UBERON:0001017 15627 15630 CNS +T398 UBERON:0000010 15639 15642 PNS +T399 PR:000003718 15683 15689 ADAM22 +T400 NCBITaxon:9606 15705 15710 human +T401 http://purl.obolibrary.org/obo/MONDO_0000001 15711 15719 diseases +T402 http://purl.obolibrary.org/obo/MONDO_0005027 15728 15736 epilepsy +T403 http://purl.obolibrary.org/obo/MONDO_0005244 15741 15762 peripheral neuropathy +T404 NCBITaxon:9606 15768 15773 human +T405 PR:000003718 15774 15780 ADAM22 +T406 SO:0000704 15781 15785 gene +T407 SO:0000147 15861 15866 exons +T408 UBERON:0001016 15954 15966 neurological +T409 http://purl.obolibrary.org/obo/MONDO_0005071 15954 15976 neurological disorders +T410 PR:000003718 16055 16061 ADAM22 +T411 NCBITaxon:9606 16134 16139 human +T412 UBERON:0001016 16140 16152 neurological +T413 http://purl.obolibrary.org/obo/MONDO_0005071 16140 16161 neurological diseases +T414 PR:000003718 16192 16198 Adam22 +T415 SO:0000704 16199 16203 gene +T416 SO:0001644 16204 16220 targeting vector +T417 SO:0001026 16232 16239 genomic +T418 SO:0000147 16263 16268 exons +T419 PR:000003718 16293 16299 Adam22 +T420 SO:0000704 16300 16304 gene +T421 SO:0001026 16333 16340 genomic +T422 CHEBI:60004 16384 16387 mix +T423 SO:0000112 16412 16419 primers +T424 SO:0000147 16438 16442 exon +T425 NCBITaxon:10088 16474 16479 mouse +T426 PR:000003718 16480 16486 Adam22 +T427 SO:0000704 16487 16491 gene +T428 SO:0005853 16516 16524 cassette +T429 SO:0000147 16530 16534 exon +T430 PR:000003718 16544 16550 Adam22 +T431 SO:0000704 16551 16555 gene +T432 SO:0001026 16610 16617 genomic +T433 SO:0005853 16683 16691 cassette +T434 SO:0005853 16715 16723 cassette +T435 PR:000003718 16802 16808 Adam22 +T436 NCBITaxon:10088 16819 16823 mice +T437 SO:0001644 16840 16856 targeting vector +T438 UBERON:0000922 16885 16894 embryonic +T439 CL:0002322 16885 16899;16905 16910 embryonic stem ... cells +T440 CL:0002322 16901 16903;16905 16910 ES ... cells +T441 SO:0001026 16977 16984 genomic +T442 SO:0000121 17029 17043 forward primer +T443 SO:0005853 17101 17109 cassette +T444 SO:0000132 17119 17133 reverse primer +T445 SO:0001644 17190 17206 targeting vector +T446 CL:0002322 17414 17416 ES +T447 GO:0009566 17443 17453 fertilized +T448 NCBITaxon:10088 17458 17463 mouse +T449 CL:0000025 17464 17468 eggs +T450 UBERON:0007236 17476 17492 eight-cell stage +T451 GO:0007618 17527 17532 mated +T452 NCBITaxon:10088 17589 17593 mice +T453 NCBITaxon:10088 17632 17636 mice +T454 http://purl.obolibrary.org/obo/MONDO_0000437 17642 17648 ataxic +T455 NCBITaxon:10088 17674 17678 mice +T456 NCBITaxon:10088 17744 17749 Mouse +T457 SO:0001026 17750 17757 genomic +T458 UBERON:0002107 17817 17822 liver +T459 PR:P23940 17841 17846 BamHI +T460 SO:0001026 17856 17863 genomic +T461 CHEBI:37972 17889 17892 32P +T462 PR:P23940 17915 17920 BamHI +T463 SO:0001026 17921 17928 genomic +T464 SO:0000147 17964 17969 exons +T465 GO:0042571 17980 17988 Antibody +T466 PR:000010228 18016 18019 MBP +T467 GO:0005737 18057 18068 cytoplasmic +T468 SO:0000417 18069 18075 domain +T469 NCBITaxon:9606 18084 18089 human +T470 PR:000003718 18090 18096 ADAM22 +T471 SO:0001060 18097 18104 isoform +T472 PR:000003718 18191 18197 ADAM22 +T473 CHEBI:44557 18254 18257 NTA +T474 CHEBI:60809 18371 18379 adjuvant +T475 CHEBI:60004 18404 18411 mixture +T476 NCBITaxon:9986 18441 18448 rabbits +T477 UBERON:0001977 18458 18463 serum +T478 PR:000003718 18494 18500 ADAM22 +T479 PR:000010228 18531 18534 MBP +T480 PR:000003718 18541 18547 ADAM22 +T481 MOP:0000779 18559 18566 coupled +T482 GO:0042571 18616 18626 antibodies +T483 GO:0042571 18661 18671 antibodies +T484 GO:0042571 18770 18778 antibody +T485 NCBITaxon:9606 18785 18790 human +T486 NCBITaxon:10088 18795 18800 mouse +T487 PR:000003718 18801 18807 ADAM22 +T488 GO:0042571 18884 18892 antibody +T489 NCBITaxon:10088 18946 18951 mouse +T490 UBERON:0000479 18952 18959 tissues +T491 PR:000003718 19000 19006 ADAM22 +T492 PR:000003718 19022 19028 Adam22 +T493 NCBITaxon:10088 19033 19037 mice +T494 UBERON:0002037 19091 19101 cerebellum +T495 NCBITaxon:10088 19128 19133 mouse +T496 GO:0019835 19198 19208 cell lysis +T497 CHEBI:9754 19223 19227 Tris +T498 CHEBI:17883 19228 19231 HCl +T499 CHEBI:26710 19248 19252 NaCl +T500 CHEBI:63016 19257 19262 NP-40 +T501 CHEBI:8984 19400 19403 SDS +T502 CHEBI:53325 19431 19445 nitrocellulose +T503 GO:0042571 19500 19508 antibody +T504 GO:0005737 19521 19532 cytoplasmic +T505 SO:0000417 19533 19539 domain +T506 PR:000003718 19543 19549 ADAM22 +T507 GO:0042571 19570 19580 antibodies +T508 NCBITaxon:3704 19602 19613 horseradish +T509 GO:0042571 19641 19649 antibody +T510 CL:0000001 19731 19738;19747 19751 Primary ... cell +T511 CL:0002573 19739 19751 Schwann cell +T512 CL:0000001 19761 19768;19777 19782 Primary ... cells +T513 CL:0002573 19769 19782 Schwann cells +T514 NCBITaxon:10088 19822 19826 mice +T515 UBERON:0001322 19898 19912 sciatic nerves +T516 CHEBI:17234 20011 20018 glucose +T517 CHEBI:2682 20097 20106 fungizone +T518 CHEBI:42471 20132 20141 forskolin +T519 UBERON:0001322 20248 20262 sciatic nerves +T520 CL:0000010 20607 20615;20624 20629 cultured ... cells +T521 CL:0002573 20616 20629 Schwann cells +T522 UBERON:0007023 20721 20726 Adult +T523 NCBITaxon:10088 20740 20744 mice +T524 UBERON:0002037 20799 20809 cerebellum +T525 UBERON:0002240 20811 20822 spinal cord +T526 UBERON:0001322 20824 20837 sciatic nerve +T527 UBERON:0000044 20839 20842 DRG +T528 CL:0000010 20847 20855;20864 20869 cultured ... cells +T529 CL:0002573 20856 20869 Schwann cells +T530 SO:0000112 20987 20993 primer +T531 PR:000003718 21148 21154 ADAM22 +T532 SO:0000112 21164 21171 primers +T533 GO:0008380 21183 21191 splicing +T534 GO:0005737 21208 21219 cytoplasmic +T535 SO:0000417 21220 21226 domain +T536 SO:0000121 21230 21244 forward primer +T537 GO:0016020 21282 21290 membrane +T538 SO:0000417 21291 21297 domain +T539 SO:0000132 21304 21318 reverse primer +T540 SO:0000147 21346 21350 exon +T541 SO:0000112 21356 21363 primers +T542 SO:0001030 21406 21413 forward +T543 SO:0001031 21456 21463 reverse +T544 NCBITaxon:10088 21514 21518 Mice +T545 CHEBI:35702 21542 21553 ethyl ether +T546 CHEBI:64276 21599 21613 glutaraldehyde +T547 CHEBI:75958 21614 21622 solution +T548 CHEBI:28304 21635 21642 heparin +T549 UBERON:0001322 21674 21688 Sciatic nerves +T550 UBERON:0001645 21690 21707 trigeminal nerves +T551 UBERON:0000955 21709 21714 brain +T552 UBERON:0002240 21719 21730 spinal cord +T553 CHEBI:16842 21778 21786 formalin +T554 UBERON:0002240 21792 21803 spinal cord +T555 UBERON:0001021 21814 21819 nerve +T556 CHEBI:16236 21902 21909 ethanol +T557 CHEBI:10545 22058 22066 electron +T558 CHEBI:16236 22176 22183 ethanol +T559 CHEBI:10545 22226 22234 electron +T560 NCBITaxon:33208 22300 22306 Animal +T561 CHEBI:9750 22395 22407 Triton X-100 +T562 CHEBI:75958 22473 22481 solution +T563 GO:0042571 22510 22520 antibodies +T564 NCBITaxon:10088 22583 22588 mouse +T565 PR:000004967 22594 22608 calbindin 28 K +T566 NCBITaxon:10088 22636 22641 mouse +T567 PR:000010228 22647 22650 MBP +T568 NCBITaxon:10088 22708 22713 mouse +T569 PR:000006252 22719 22724 Neu N +T570 NCBITaxon:9986 22744 22750 rabbit +T571 GO:0005634 22787 22793 Nuclei +T572 CHEBI:51231 22827 22831 DAPI +T573 PR:000010228 22841 22844 MBP +T574 GO:0042571 22988 22998 antibodies +T575 CHEBI:37987 23000 23003 Cy3 +T576 NCBITaxon:9793 23012 23018 donkey +T577 NCBITaxon:9986 23024 23030 rabbit +T578 GO:0071735 23031 23034 IgG +T579 CHEBI:37926 23069 23073 FITC +T580 NCBITaxon:9793 23083 23089 donkey +T581 NCBITaxon:10088 23095 23100 mouse +T582 GO:0071735 23101 23104 IgG +T583 GO:0097617 23265 23278 hybridisation +T584 GO:0097617 23288 23301 hybridisation +T585 CHEBI:37983 23344 23347 35S +T586 SO:0000028 23382 23384 bp +T587 NCBITaxon:10088 23388 23393 mouse +T588 PR:000003718 23394 23400 ADAM22 +T589 SO:0000318 23432 23446 initiating ATG +T590 SO:0000440 23517 23523 vector +T591 SO:0000155 23537 23545 plasmids +T592 SO:0000077 23570 23579 antisense +T593 NCBITaxon:10760 23618 23620 T7 +T594 PR:P00573 23618 23635 T7 RNA polymerase +T595 CHEBI:37983 23642 23645 35S +T596 UBERON:0000955 23658 23663 brain +T597 UBERON:0002240 23668 23679 spinal cord +T598 NCBITaxon:10088 23712 23716 mice +T599 GO:0097617 23759 23772 hybridisation +T600 SO:0000155 24111 24118 plasmid +T601 GO:0042571 24133 24141 antibody +T602 GO:0097617 24299 24312 hybridisation +T603 NCBITaxon:10088 24378 24382 mice +T604 PR:000003718 24761 24767 Adam22 +T605 SO:0001026 24777 24784 genomic +T606 PR:000003718 24812 24818 Adam22 +T607 SO:0001023 24819 24825 allele +T608 SO:0001023 24884 24890 allele +T609 PR:000003718 24911 24917 ADAM22 +T610 GO:0010467 24918 24928 expression +T611 SO:0000319 24965 24982 termination codon +T612 SO:0005853 24996 25004 cassette +T613 SO:0000147 25010 25014 exon +T614 SO:0005853 25028 25036 cassette +T615 SO:0001644 25055 25071 targeting vector +T616 PR:P23940 25208 25213 BamHI +T617 NCBITaxon:10088 25283 25288 mouse +T618 SO:0001026 25289 25296 genomic +T619 SO:0001023 25347 25353 allele +T620 SO:0001023 25368 25374 allele +T621 PR:000003718 25493 25499 ADAM22 +T622 GO:0010467 25500 25510 expression +T623 NCBITaxon:10088 25518 25523 mouse +T624 UBERON:0002037 25524 25534 cerebellum +T625 PR:000003718 25547 25553 ADAM22 +T626 PR:000003718 25569 25575 Adam22 +T627 UBERON:0002037 25589 25599 cerebellum +T628 PR:000003718 25621 25627 ADAM22 +T629 GO:0005737 25632 25643 cytoplasmic +T630 SO:0000417 25644 25650 domain +T631 GO:0042571 25663 25671 antibody +T632 PR:000003718 25715 25721 Adam22 +T633 NCBITaxon:10088 25728 25732 mice +T634 GO:0007567 25740 25745 natal +T635 PR:000003718 25764 25770 Adam22 +T636 NCBITaxon:10088 25777 25781 mice +T637 PR:000003718 25842 25848 Adam22 +T638 NCBITaxon:10088 25855 25859 mice +T639 UBERON:0002103 25910 25919 hindlimbs +T640 CL:0000540 25932 25940 Neuronal +T641 PR:000003718 25941 25947 ADAM22 +T642 GO:0010467 25953 25963 expression +T643 UBERON:0001017 25971 25974 CNS +T644 PR:000003718 25993 25999 ADAM22 +T645 GO:0097617 26027 26040 hybridisation +T646 CHEBI:37983 26056 26059 35S +T647 NCBITaxon:10088 26140 26145 mouse +T648 UBERON:0000955 26146 26151 brain +T649 UBERON:0002240 26156 26167 spinal cord +T650 SO:0000077 26194 26203 antisense +T651 UBERON:0002037 26286 26296 cerebellum +T652 UBERON:0002240 26362 26373 spinal cord +T653 PR:000003718 26393 26399 ADAM22 +T654 UBERON:0002020 26425 26436 grey matter +T655 GO:0007399 26460 26476 neurodevelopment +T656 UBERON:0001017 26484 26487 CNS +T657 PR:000003718 26491 26497 ADAM22 +T658 NCBITaxon:10088 26508 26512 mice +T659 UBERON:0002037 26539 26549 cerebellum +T660 NCBITaxon:10088 26565 26569 mice +T661 GO:0007567 26626 26631 natal +T662 PR:000004967 26656 26665 calbindin +T663 PR:000010228 26686 26689 MBP +T664 CHEBI:51231 26737 26741 DAPI +T665 CL:0002608 26826 26845 Hippocampal neurons +T666 PR:000006252 26867 26872 Neu N +T667 GO:0042571 26873 26881 antibody +T668 UBERON:0001130 26893 26899 Spinal +T669 UBERON:0000345 26900 26906 myelin +T670 PR:000010228 26923 26926 MBP +T671 UBERON:0001021 27124 27141 peripheral nerves +T672 PR:000003718 27145 27151 ADAM22 +T673 NCBITaxon:10088 27162 27166 mice +T674 UBERON:0001322 27213 27227 sciatic nerves +T675 UBERON:0001645 27240 27257 trigeminal nerves +T676 UBERON:0002240 27273 27284 spinal cord +T677 UBERON:0002179 27286 27303 lateral funiculus +T678 GO:0007567 27346 27351 natal +T679 PR:000003718 27407 27413 ADAM22 +T680 NCBITaxon:10088 27424 27429 mouse +T681 UBERON:0000345 27441 27447 myelin +T682 UBERON:0000345 27459 27465 myelin +T683 UBERON:0001021 27473 27489 peripheral nerve +T684 UBERON:0006134 27484 27496 nerve fibres +T685 UBERON:0001130 27513 27519 spinal +T686 UBERON:0006135 27520 27537 myelinated fibres +T687 CHEBI:10545 27565 27573 Electron +T688 UBERON:0001322 27598 27612 sciatic nerves +T689 CHEBI:10545 27614 27622 Electron +T690 UBERON:0001322 27642 27656 sciatic nerves +T691 PR:000003718 27662 27668 Adam22 +T692 PR:000003718 27681 27687 Adam22 +T693 NCBITaxon:10088 27696 27700 mice +T694 GO:0007567 27708 27713 natal +T695 UBERON:0000345 27763 27769 myelin +T696 UBERON:0000345 27791 27797 myelin +T697 PR:000003718 27816 27822 ADAM22 +T698 NCBITaxon:10088 27833 27838 mouse +T699 GO:0030424 27848 27853 axons +T700 GO:0030154 27913 27931;27951 27956 differentiation of ... cells +T701 CL:0002573 27943 27956 Schwann cells +T702 UBERON:0001322 27985 27999 sciatic nerves +T703 GO:0007567 28034 28039 natal +T704 PR:000010228 28064 28067 MBP +T705 CHEBI:51231 28138 28142 DAPI +T706 UBERON:0001322 28171 28184 sciatic nerve +T707 CHEBI:51231 28197 28201 DAPI +T708 PR:000003718 28311 28317 Adam22 +T709 SO:0000704 28318 28322 gene +T710 UBERON:0000479 28337 28343 tissue +T711 SO:0000673 28353 28364 transcripts +T712 CHEBI:2511 28432 28439 agarose +T713 SO:0000028 28474 28476 bp +T714 UBERON:0002037 28492 28502 cerebellum +T715 UBERON:0002240 28507 28518 spinal cord +T716 UBERON:0000044 28523 28543 dorsal root ganglion +T717 UBERON:0001322 28548 28561 sciatic nerve +T718 CL:0000010 28566 28574;28583 28588 cultured ... cells +T719 CL:0002573 28575 28588 Schwann cells +T720 CHEBI:15377 28603 28608 water +T721 SO:0000147 28613 28617 Exon +T722 NCBITaxon:10088 28638 28643 mouse +T723 PR:000003718 28644 28650 Adam22 +T724 SO:0000704 28651 28655 gene +T725 SO:0000147 28687 28692 exons +T726 SO:0000673 28702 28712 transcript +T727 SO:0000858 28714 28725 orthologous +T728 NCBITaxon:9606 28733 28738 human +T729 PR:000003718 28739 28745 ADAM22 +T730 SO:0001060 28746 28753 isoform +T731 SO:0000673 28756 28766 transcript +T732 UBERON:0000479 29057 29063 tissue +T733 UBERON:0002037 29079 29081 Cb +T734 UBERON:0002037 29083 29093 cerebellum +T735 UBERON:0002240 29095 29097 Sp +T736 UBERON:0002240 29099 29110 spinal cord +T737 UBERON:0000044 29112 29115 DRG +T738 UBERON:0000044 29117 29137 dorsal root ganglion +T739 UBERON:0001322 29139 29141 SN +T740 UBERON:0001322 29143 29156 sciatic nerve +T741 CL:0000010 29163 29171;29180 29185 cultured ... cells +T742 CL:0002573 29172 29185 Schwann cells +T743 PR:000003718 29226 29232 Adam22 +T744 NCBITaxon:10088 29296 29300 mice +T745 NCBITaxon:10088 29327 29331 mice +T746 GO:0007567 29369 29374 natal +T747 GO:0007567 29437 29442 natal +T748 GO:0007567 29543 29548 natal diff --git a/src/ontogpt/evaluation/craft/database/all/15876356.txt b/src/ontogpt/evaluation/craft/database/all/15876356.txt new file mode 100644 index 000000000..f8b21120b --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15876356.txt @@ -0,0 +1,149 @@ +Ataxia and peripheral nerve hypomyelination in ADAM22-deficient mice + +Abstract + +Background + +ADAM22 is a member of the ADAM gene family, but the fact that it is expressed only in the nervous systems makes it unique. ADAM22's sequence similarity to other ADAMs suggests it to be an integrin binder and thus to have a role in cell-cell or cell-matrix interactions. To elucidate the physiological functions of ADAM22, we employed gene targeting to generate ADAM22 knockout mice. + +Results + +ADAM22-deficient mice were produced in a good accordance with the Mendelian ratio and appeared normal at birth. After one week, severe ataxia was observed, and all homozygotes died before weaning, probably due to convulsions. No major histological abnormalities were detected in the cerebral cortex or cerebellum of the homozygous mutants; however, marked hypomyelination of the peripheral nerves was observed. + +Conclusion + +The results of our study demonstrate that ADAM22 is closely involved in the correct functioning of the nervous system. Further analysis of ADAM22 will provide clues to understanding the mechanisms of human diseases such as epileptic seizures and peripheral neuropathy. + +Background + +ADAM (A Disintegrin And Metalloprotease) is a family of membrane-spanning multi-domain proteins containing a metalloproteinase-like domain and a disintegrin-like domain. Currently, more than 30 ADAMs have been identified in mammals. Their biological activities implicate ADAMs in fertilization, myogenesis and neurogenesis by proteolysis and adhesion. Some types of ADAM are catalytically active metalloproteases and shed the extracellular domains of membrane-bound growth factors or receptors [1,2]. For example, ADAM17 (TACE) has been shown to cleave several substrates, including tumour necrosis factor alpha[3,4], heparin-binding epidermal growth factor-like growth factor [6,7] and transforming growth factor alpha [8]. Studies of ADAM17-null mice have revealed that ADAM17 is critical in embryogenesis and plays an essential role in the supply of growth factors [6,8]. ADAMs are also involved in cell-cell or cell-matrix adhesion through their interaction with integrins or syndecans. More than 10 ADAMs have been shown to support integrin-mediated cell adhesion in vitro [9]. It has been reported that ADAM2-null and ADAM3-null male mutants are infertile and their spermatozoa lack egg-binding abilities [10-12]. Both ADAM2 and ADAM3 are not metalloproteases because they lack catalytic site sequences in their metalloprotease domain. These studies clearly showed that non-proteinase members of ADAMs also have significant roles in vivo. + +We have reported the findings of ADAM11, ADAM22 and ADAM23 genes and their restricted expression in the human and murine nervous systems [13-15]. Sequence analysis suggests that they are not metalloproteases, since they all lack a catalytic motif. It has been reported that ADAM23 protein is localised to the cell surface [16], interacts with alpha-v beta-3 integrin heterodimer [17] and the disruption of ADAM23 gene in the mouse results in premature death associated with ataxia and tremor [18]. Although the cause of death in this mouse is unknown, for these phenotypes, impaired cell-cell or cell-matrix interactions in the nervous system caused by loss of ADAM23 may be responsible. ADAM22 and ADAM23 share highly homologous sequences in their extracellular domains. Especially, it is evident in their putative integrin binding loop sequences, CR(E/D)AVN(E/D)CD, which is located centre of the disintegrin domain. These findings led us to hypothesize that ADAM22 is an integrin binder and plays an important role in the nervous system, as does ADAM23. To determine the physiological functions of ADAM22, we generated and analysed Adam22 gene-targeted mice. + +Results + +Generation of ADAM22-deficient mice + +Mice carrying a targeted mutation in their Adam22 gene were generated by homologous recombination (Fig. 1A). Correct targeting events were confirmed by Southern blot analysis (Fig. 1B). Since the termination codon was introduced in exon 8 in the pro-protein domain, only the truncated form of the ADAM22 protein would be synthesized from this targeted allele. Because a pro-protein domain is always removed in the mature functional ADAM-proteins by the Furin-like proteases and is thought to be non-functional itself, we considered that this truncated form of ADAM22 protein has no function. Absence of mature ADAM22 protein in homozygous mutants was confirmed by Western blot analysis using the specific antibody, which recognizes the cytoplasmic domain of the ADAM22 protein (Fig. 1C). Homozygous mutants showed no noticeable defects at birth and were indistinguishable from wild-type or heterozygous littermates during the first week. At postnatal day 10 (P10), most of the homozygous mutants were distinguishable by abnormalities such as reduced body weight and uncoordinated movements of their limbs. After P10, all homozygotes displayed severe ataxia (Fig. 2) and began to die. To measure the survival rate and body weight of each genotype precisely, we backcrossed heterozygous male mutants to C57BL/6 females more than 6 times. The resulting (N 6) heterozygous males and females were intercrossed and the produced offspring were analysed. The numbers of survivors of each genotype every 5 days are shown in Table 1. At P10, the ratio of each genotype was in close accordance with the Mendelian ratio (20.5% +/+, 55.1% +/-, 24.4% -/- ; n = 78). This result shows that ADAM22 is not essential for embryogenesis. At P10, the average body weight of homozygous mutants was approximately half that of wild-type and heterozygous littermates (Table 2). Homozygous mutants died one by one after P10, and all homozygotes died before P20. Of more than 100 homozygotes we have produced, none have survived more than 25 days after birth. Meanwhile, heterozygous mutants looked normal, were fertile, and survived for more than one year without obvious defects. + +Histopathology of ADAM22-deficient mice + +We have reported the predominant expression of ADAM22 mRNA in the human and mouse brain by Northern blot analysis [13,14]. To determine the distribution of ADAM22 transcript in the adult mouse CNS precisely, in situ hybridisation analysis was performed using 35S-labeled RNA probe. As shown in Fig. 3, ADAM22 mRNA was expressed throughout the adult mouse CNS. Strong signal was detected in the cerebellar granule cells and hippocampal formation. In the spinal cord, hybridisation signal was restricted to the grey matter. The distribution of ADAM22 transcripts in the CNS was quite similar with that of ADAM11, whose neuronal expression has been reported [19]. These results suggest that the ADAM22 mRNA expression is neuronal in the mouse CNS. + +Based on the mRNA distribution pattern, we performed immunohistopathological analysis of the cerebellum and hippocampus extensively. Despite the high level of expression of ADAM22 mRNAs in the cerebellar granule cells, granule cell layer was normally formed and Purkinje cell morphology (calbindin-staining) of the mutant mouse looked intact (Figs. 4A–D). Hippocampal formation was also normally formed (Figs. 4I–J). These results suggest that neuronal cell migration was not impaired in the mutant mouse. Myelin-formation detected by MBP (myelin basic protein) staining of the mutant in the cerebellum (Fig. 4H) and spinal cord (Fig. 4L) was also indistinguishable with the wild-type littermate (Figs. 4G,K). In summary, we could not find any signs of abnormalities in the mutant mouse brain by the light microscopic examination. + +Next, the spinal cord and peripheral nerves of each genotype were analysed by toluidine blue stain, which reveals myelin formation. Surprisingly, lack of myelin or thin myelin was observed in the sciatic nerves (Fig. 5B) and trigeminal nerves (Fig. 5D) in homozygous mutants. Because no lesions were observed in the spinal cord (Fig. 5F), it was suggested that Schwann cell specific myelination defect occurred in the homozygous mice. To analyse the state of myelinating Schwann cells and axons, electron microscopic (EM) analysis of the sciatic nerve was performed (Fig. 6). Schwann cells formed thin or no myelin in the homozygous mutant (Fig. 6B). In contrast, heterozygous mice showed complete myelination (Fig. 6A). Morphology of the axons looked normal in each genotype. Immunohistochemical analysis of the sciatic nerve showed that increased number of nuclei (Fig. 7B) and reduced staining of MBP (Fig. 7D) were remarkable in the homozygous mutant. The Schwann cell marker, S100 staining signal was intensely observed in the homozygote as well as wild-type (Figs. 7E, F). These results suggest that proliferation of the Schwann cells was not impaired but differentiation is severely delayed in the ADAM22-deficient mice. + +Novel ADAM22 transcript variants isolated from the peripheral nervous system + +In the case of human ADAM22, isolation of five splicing variants and existence of two terminating exons have been reported [13,20,21]. However, the latter terminating exon (exon 31) of the mouse species has not been identified yet. To isolate the long form of mouse ADAM22 transcripts, at first, we sought the mouse genome by BLAST homology search. Search results showed that the mouse genome contig NT_039297 contained most of the mouse Adam22 gene. In this contig sequence, we found putative exons 30 and 31, those sequences were quite similar to human ADAM22 isoform 1 (GenBank # NM_021723). To confirm existence of transcripts, we designed primers on the putative exon 31 and performed RT-PCR using mouse cerebellum mRNA as a template. TA-cloning of the amplified fragments and sequencing analysis showed that 5 of 8 clones were type 1 isoform (G01), 2 clones were type 3 isoform (G03) and 1 clone (G08) contained a novel exon between exons 29 and 30. Next, we performed RT-PCR analysis of mRNAs purified from several adult mouse tissues, such as the spinal cord, dorsal root ganglion (DRG), sciatic nerve and cultured primary Schwann cells. Interestingly, multiple DNA fragments were amplified from each tissue (Fig. 8), and length of major fragments in each lane was different. For example, length of major amplified PCR fragments from the sciatic nerve (Fig. 8A, lane 5) and cultured Schwann cells (lane 6) were almost identical, and were shorter than those from other tissues. In contrast, major fragments from the DRG (lane 4) were longer than others. We performed TA-cloning and sequencing to analyse exon organization of the amplified transcripts (Fig. 8C). Forty seven clones were isolated in total and were classified in 16 independent clones (G01 – G23). Comparison with the genomic sequence revealed the existence of 8 novel exons (exons 27S, 27L, 29.1, 29.3, 29.5, 29.7, 30 and 31). These novel exons were flanked by well-defined introns that obey the GT-AG rule. Exon organization of each clone was shown in Fig. 8C. Characteristic feature of ADAM22 transcripts is tissue specific insertion or skipping of exons. Because the number of nucleotides of exons 26, 27, 27L, 29.3, 29.5 and 29.7 is a multiple of 3, insertion or skipping of these exons will not change the downstream reading frame. Known peptide motives were not found in the newly isolated sequences. This RT-PCR analysis is summarized as follows: ADAM22 transcripts were roughly subdivided in 3 groups, CNS-form (containing exon 27), DRG-form (containing exon 27L) and Schwann-cell-form (skipping exon 26 and 27). From these results, we concluded that ADAM22 is expressed in a cell-type specific manner, and plays an essential role in myelinogenesis in the PNS. + +Discussion + +In the present study, we examined the function of ADAM22 by generating mice that lacked the Adam22 gene. ADAM22-deficient mice exhibited severe ataxia and premature death. In contrast, heterozygous mutants were healthy, fertile and survived more than 1 year without obvious abnormalities. The cause of death in the homozygous mutants is not clear; however, it is likely to be due to multiple seizures. This is because convulsive seizures were occasionally observed in homozygotes, after which the suffered mice appeared exhausted and, in most cases, died on the next day. It is known that cortical dysplasia resulting from aberrant brain development causes seizure syndrome [22]. For example, mice lacking the neuron-specific activator of cyclin 5, p35, exhibit seizures and a severe neuronal migration defect [23]. Because ADAM22 protein is expressed on the cell surface and is likely to be involved in cell-cell or cell-matrix interactions, we hypothesized that depletion of ADAM22 would generate aberrant neuronal migration. Histochemical analysis was performed to verify this hypothesis. However, no marked abnormalities were observed in the mutant mouse brain, ruling out a critical role for ADAM22 in neuronal migration. Another possible explanation is that the premature death in first 2 weeks of life is caused by the dysfunction of the autonomic nervous system. Especially, nerves which control the breathing would be very important, because the respiration system undergoes significant maturation in the first 2–3 weeks after birth. For example, the myelin-deficient (MD) rat, which carries a point mutation in the proteolipid protein (PLP) gene, exhibits ataxia, tremor, dysmyelination and dies at approximately postnatal day 21. In MD rat, early death is caused by the dysfunction of the brain stem which is essential for autonomic control of respiration during hypoxia [24]. Further study is needed to verify the cause of death in ADAM22-deficient mice. + +Another interesting phenotype observed in ADAM22-deficient mice was hypomyelinogenesis. Histopathological analysis revealed marked hypomyelination in the sciatic and trigeminal nerves. However, myelin formation in the spinal cord and the brain was normal. It was therefore evident that hypomyelinogenesis occurred specifically in the peripheral nerves in the knockout mice, suggesting Schwann cell dysfunction. In the mutant sciatic nerve, the density of pro-myelinating Schwann cells (S100-positive, MBP-negative) was markedly higher than that seen in heterozygotes. Higher pro-myelinating Schwann cell density and marked delay in myelin formation indicate that ADAM22 plays an important role in Schwann cell differentiation, but not in proliferation. + +Investigations on why hypomyelinogenesis occurred in ADAM22-deficient mice centre on integrin. This is because several ADAMs have been shown to interact with integrins via disintegrin domains [9], and it has been suggested that alpha-6 beta-1 integrin on the Schwann cell plays an important role in myelin formation [25]. In addition, laminin-2 is a key player in peripheral myelin formation, because laminin-2 deficiency causes dysmyelination in mice and humans, and its receptors, alpha-6 beta-1 integrin and dystroglycan, are also shown to be deeply involved in myelin formation [26,27]. The conditional knockout studies of beta-1 integrin and laminin gamma-1 in Schwann cell suggested that both laminin-2 and integrins are essential for segregation of large bundles of axons in the early stage of myelin formation [25,28]. In contrast, the phenotype seen in the ADAM22-deficient mice was quite different: for example, unsorted bundles of axons were not seen in nerves and roots. We assume that ADAM22 partially modulates integrin-mediated signals or is involved at a later stage of myelin formation. + +Conclusion + +In summary, we generated ADAM22-deficient mice and proved that ADAM22 plays an essential role in both the CNS and the PNS. The results of this study suggest that ADAM22 is involved in human diseases such as epilepsy and peripheral neuropathy. The human ADAM22 gene is relatively large, at 300 kb in length, and is comprised of more than 30 exons. It is assigned on 7q21, where several chromosomal aberrations that are accompanied by neurological disorders have been identified [29,30]. Further detailed molecular function analysis of ADAM22 may lead to progress in uncovering the mechanisms that underlie certain human neurological diseases. + +Methods + +Construction of an Adam22 gene-targeting vector + +The 18-kb genomic DNA fragments covering exons 5, 6, 7, 8 and 9 of the Adam22 gene were amplified from C57BL/6 genomic DNA by PCR using Expand Hi-Fidelity enzyme mix (Roche Diagnostics) and primers designed for each exon. To generate a mutation in the mouse Adam22 gene, we inserted the PGKneo cassette into exon 8 of the Adam22 gene. The final targeting construct consisted of a 10.5-kb genomic DNA fragment that was interrupted by the insertion of the PGKneo cassette and contained a MC1/TK cassette as a negative selection marker against random integration [5]. + +Generation of Adam22 deficient mice + +The linearised targeting vector was electroporated into TT2 embryonic stem (ES) cells [31]. Homologous recombinants were selected by PCR. The extracted genomic DNA from each clone was amplified using the forward primer AGN2: 5'-GCCTGCTTGCCGAATATCATGGTGGAAAAT-3' in the PGKneo cassette, and the reverse primer MFP065R: 5'-ACTATTTCTGTGATGAGGGCACAGCATC-3' outside the targeting vector. Homologous recombined DNA was efficiently amplified by 35 cycles of the following amplification steps (94°C-30 s and 68°C-5 min). The targeting efficiency of this construct was about 4%. Correctly targeted ES clones were injected into fertilized ICR mouse eggs at the eight-cell stage. The resulting male chimeras were mated with C57BL/6N females, and heterozygous male and female mice were interbred to generate homozygous mice. The ataxic phenotypes of homozygous mice were observed in two independent lines. + +Southern blot analysis + +Mouse genomic DNA used for Southern blot analysis was extracted from the liver of each genotype. BamHI-digested genomic DNA was probed with the [32P]-labelled 0.4-kb SpeI-BamHI genomic fragment, which is located between exons 8 and 9. + +Antibody production + +His-tagged and MBP-fused recombinant protein containing cytoplasmic domain (cp) of human ADAM22 isoform 1 (position: 756–906, 151 amino acids in length) were produced in E. coli. His-tagged ADAM22-cp protein was purified in denatured condition using Ni-NTA resin (Qiagen GmbH) and dialysed in PBS. Precipitated protein was recovered and was mixed with Freund's complete adjuvant (Invitrogen), then, the mixture was used for immunization of rabbits. The antiserum raised against the His-tagged ADAM22-cp protein was incubated with MBP-fused ADAM22-cp protein coupled to Affi-Gel 10 beads (Bio-Rad). Beads with bound antibodies were washed in PBS, and the bound antibodies were eluted with 100 mM glycine, pH 3.0, and neutralized immediately. Using the affinity purified antibody, both human and mouse ADAM22 proteins were detected by Western blot analysis. However, unfortunately the antibody was not suitable for immunohistochemical analysis of mouse tissues. + +Western blot analysis + +The absence of ADAM22 protein in the Adam22 -/- mice was confirmed by Western blot analysis. Briefly, the cerebellum was isolated from a P14.5 mouse of each genotype and homogenized with a Polytron homogenizer in cell lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% NP-40, Complete protease inhibitors [Roche Diagnostics]). After removal of cell debris by centrifugation, the supernatant was separated on 10% SDS-PAGE, and transferred to a nitrocellulose membrane. The blot was then incubated with polyclonal antibody against the cytoplasmic domain of ADAM22 (at 1:1,000). Bound antibodies were visualized with horseradish peroxidase-labelled second antibody and a ECL-plus chemiluminescence detection system (Amersham Biosciences Corp.). + +Primary Schwann cell culture + +Primary Schwann cells were prepared from 4-month-old C57BL/6 mice according to Manent's protocol [32] with minor modifications. Briefly, sciatic nerves were removed and incubated for 7 days in the pre-treatment medium, which consisted of D-MEM (high glucose) supplemented with 10% FCS, 50 mg/ml gentamicin (Invitrogen Corp.), 2.5 μg/ml fungizone (Invitrogen Corp.), 2 μM forskolin (EMD Biosciences Inc.) and 10 ng/ml recombinant heregulin-beta1 (R&D Systems). For dissociation, cultured sciatic nerves were cut into pieces and incubated at 37°C for 3 hours in Opti-MEM medium (Invitrogen Corp.) containing 130 U/ml collagenase type I (Invitrogen Corp.) and 0.4 U/ml dispase II (Roche Diagnostics). Dissociated cells were resuspended in the pre-treatment medium and plated on Poly-D-Lysine/Laminin coated plate (BD Biosciences). The purity of the cultured Schwann cells, as determined by indirect immunofluorescence analysis, approached 90 %. + +RT-PCR analysis + +Adult C57BL/6 male mice were used in this study. Total RNAs purified from the cerebellum, spinal cord, sciatic nerve, DRG and cultured Schwann cells using TRIzol (Invitrogen Corp.) and RNeasy kit (Qiagen GmbH) were analysed by RT-PCR using SuperScript II and random primer (Invitrogen Corp.), and PCR amplification (40 cycles; 94°C-30 s, 60°C-30 s and 68°C-5 min) with Expand Hi-Fidelity DNA polymerase (Roche Diagnostics) and ADAM22-specific primers. To detect splicing variants in the cytoplasmic domain, a forward primer was placed just upstream of the transmembrane domain and a reverse primer was set on the terminating exon. The primers used in this study were as follows. ISH04-forward: 5'-AACAGGCACTGGACAGGGGCTGAC-3' and ISH04-reverse: 5'-AATGGATGTCTCCCATAGCCTGGC-3'. + +Histopathology + +Mice were anesthetized with ethyl ether. Whole-body perfusion by 2% paraformaldehyde-glutaraldehyde solution followed by heparin-included saline was performed. Sciatic nerves, trigeminal nerves, brain and spinal cord were removed and fixed in 10% neutral-buffered formalin. The spinal cord and other nerve blocks were washed and postfixed with 2% osmium tetroxide, and were dehydrated in ethanol and equilibrated in Epon. Epon embedded semithin sections were stained with toluidine blue and were subjected to light microscopic examination. For electron microscopic analysis, thin sections were cut using an ultramicrotome, stained with 1.5% uranylacetate in 50% ethanol and 0.8% lead citrate, and analysed using electron microscope. All procedures were conducted according to the Eisai Animal Care Committee's guidelines. + +Immunohistochemistry + +Frozen sections were rinsed in 0.1% Triton X-100/PBS at room temperature for 1 hour, and were blocked in BLOCKACE solution (Dai-nippon). The following antibodies were incubated overnight in 0.1% BLOCKACE at 4°C: + +monoclonal mouse anti-calbindin 28 K (Sigma, 1:200); monoclonal mouse anti-MBP (SMI-99, Sternberger Monoclonals Inc., 1:50); monoclonal mouse anti-Neu N (CHEMICON, 1:100); rabbit anti-S100 polyclonal (DAKO, 1:200). Nuclei were counterstained with 1 μg/ml DAPI (Sigma). MBP staining was performed after microwave epitope retrieval in 10 mmol/L citrate buffer (pH 6). Sections were incubated for 1 hour with secondary antibodies: Cy3-labeled donkey anti-rabbit IgG (Jackson Immuno Research, 1:200), FITC-labelled donkey anti-mouse IgG (Jackson Immuno Research, 1:200). Sections were photographed with an Olympus microscope (Olympus IX71) equipped with a high-resolution CCD camera. + +RNA in situ hybridisation + +In situ hybridisation on frozen sections were carried out using 35S-labelled RNA probes. Briefly, 610 bp of mouse ADAM22 cDNA (position: 1351–1960 from initiating ATG) was cloned into the pBluescript II SK(+) or the pBluescript II KS(+) vector. Using these plasmids as templates, sense and antisense labelled RNA probes were generated by T7 RNA polymerase and [α35S]UTP. Frozen brain and spinal cord from 2 month-old C57BL/6 female mice were used in this analysis. Pretreatment, hybridisation, RNase treatment and washing was carried out following the protocol described in the literature [33]. Dehydrated slides were attached to imaging plates for 48 hours and the autoradiograms were analysed using a Bio-Image Analyser (BAS3000, Fuji Photo Film). + +Authors' contributions + +KS is leading this project, and performed cDNA cloning, plasmid construction, antibody production, immunohistochemical study, and wrote the manuscript. KH and JK conducted histopathological experiments. TH analysed mRNA distribution by in situ hybridisation. ET, NM, MI, TO and KY contributed to the generation of knockout mice. TN supervised the project. All authors read and approved the manuscript. + +Acknowledgements + +We are grateful to Isao Tanaka, Masayuki Okada, Yoshiharu Mizui, Kappei Tsukahara, Hiroyuki Kato, Hiroo Ogura and Junro Kuromitsu (Eisai Co., Ltd.), and Shin-ichi Murase (Virginia University) for the fruitful discussions and advice. + +Figures and Tables + +Figure 1 + +Targeted mutation of Adam22. (A) The genomic structure of the wild-type Adam22 allele (top), the targeting construct (middle) and the disrupted allele (bottom) are shown. ADAM22 expression was disrupted by the insertion of a termination codon and a PGKneo cassette into exon 8. An MC1/TK cassette at the end of the targeting vector allows for negative selection. The 3' probe represents the position of the external probe used for Southern blot analysis, and expected BamHI [B] fragments are indicated by arrows. (B) Southern blot analysis of mouse genomic DNA. The expected DNA fragments for the wild-type allele and disrupted allele are 7.5-kb and 2.5-kb, respectively. +/+, wild-type; +/-, heterozygote; -/-, homozygote. (C) Western blot analysis of ADAM22 expression in the mouse cerebellum. Absence of ADAM22 protein in the Adam22 (-/-) mutant cerebellum was shown using anti-ADAM22-cp (cytoplasmic domain) polyclonal antibody. + +Figure 2 + +Uncoordinated movements in the Adam22 (-/-) mice at postnatal day 18 (P18). (A) Adam22 (-/-) mice were smaller than their wild-type (+/+) littermates. (B, C) Adam22 (-/-) mice at P18 were unable to support themselves on their hindlimbs. + +Figure 3 + +Neuronal ADAM22 mRNA expression in the CNS. To determine the ADAM22 mRNA distribution, in situ hybridisation analysis using 35S-labeled probe was performed. Coronal (A, B) and sagittal (C, D) sections of the mouse brain and spinal cord (E) were shown. Using the antisense probe (A, C), strong signals were obtained, especially in the hippocampus and the cerebellum, while no signals was detected by the sense probe (B, D). In the spinal cord, autoradiograms of ADAM22 mRNA was detected in the grey matter (E). + +Figure 4 + +Normal neurodevelopment in the CNS of ADAM22-deficient mice. Sagittal sections of the cerebellum from wild-type mice (A, C, E, G) and homozygous mutants (B, D, F, H) at postnatal day 13 were stained for calbindin (C and D; green) or MBP (G and H; green), and were counterstained with DAPI (A, B, E, F; blue). Significant abnormalities were not observed in the homozygotes. Hippocampal neurons were stained by anti-Neu N antibody (I and J). Spinal myelin was analysed by MBP staining (K and L). These analyses showed no obvious differences between homozygotes (J, L) and wild-type littermates (I, K). Bar: (A, B, E, F) 100 μm, (K, L) 250 μm. + +Figure 5 + +Hypomyelination of peripheral nerves in ADAM22-deficient mice. Epon embedded semithin cross-sections of the sciatic nerves (A, B), the trigeminal nerves (C, D) and the spinal cord [lateral funiculus] (E, F) of the indicated genotypes at postnatal day 10 were stained with toluidine blue. Note that the ADAM22-deficient mouse shows thin myelin or lack of myelin in the peripheral nerve fibres (B, D), but the spinal myelinated fibres are intact (F). + +Figure 6 + +Electron microscopic analysis of sciatic nerves. Electron micrographs of the sciatic nerves from Adam22 +/- (A) and Adam22 -/- (B) mice at postnatal day 10 are shown. In the heterozygote (A), thick myelin was formed, while no myelin was formed in the ADAM22-deficient mouse (B). The axons looked normal in each genotype. + +Figure 7 + +Marked delay in differentiation of the mutant Schwann cells. Transverse sections of the sciatic nerves of the indicated genotypes at postnatal day 13 were stained for MBP (C and D; green) or S100 (E and F; red), and were counterstained with DAPI (A, B; blue). In the mutant sciatic nerve, density of DAPI-signals were greatly increased (B) compared with those of the wild-type (A). Bar: (A, B) = 50 μm. + +Figure 8 + +Adam22 gene structure and tissue specific transcripts. (A) RT-PCR analysis. Amplified DNA fragments were analysed by 1 % agarose gel electrophoresis. Lanes 1. 100 bp DNA ladder; 2. cerebellum; 3. spinal cord; 4. dorsal root ganglion; 5. sciatic nerve; 6. cultured Schwann cells; 7. distilled water (B) Exon organization of the mouse Adam22 gene is illustrated. Boxes indicate exons. The G01 transcript (orthologous to the human ADAM22 isoform 1 transcript) is composed of grey boxes. Boxes in black indicate non-coding region. (C) Summary of the isolated clones. The nucleotide sequence data have been deposited with the DDBJ/EMBL/GenBank Data Libraries under the accession numbers described in the table. (D) Number of clones isolated from each tissue is summarized. Cb; cerebellum, Sp; spinal cord, DRG; dorsal root ganglion, SN; sciatic nerve, cSC; cultured Schwann cells. + +Table 1 + +Genotyping of the progeny of Adam22 heterozygous intercrosses. + +From heterozygous intercrosses, 78 mice were produced. Numbers of mice in each genotype were counted on postnatal day 10, 15 and 20. No homozygotes were survived more than postnatal day 20. +/+, wild-type; +/-, heterozygote; -/-, homozygote. + +Table 2 + +Body weight of progeny at postnatal day 10.5 + ++/+, wild-type; +/-, heterozygote; -/-, homozygote. diff --git a/src/ontogpt/evaluation/craft/database/all/15882093.ann b/src/ontogpt/evaluation/craft/database/all/15882093.ann new file mode 100644 index 000000000..ae0e65f04 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15882093.ann @@ -0,0 +1,1779 @@ +T1 CL:0000540 0 8 Neuronal +T2 GO:0001764 0 18 Neuronal Migration +T3 UBERON:0001893 55 68 Telencephalon +T4 PR:000015417 79 83 SOX1 +T5 CL:0000540 206 214 neuronal +T6 GO:0010467 272 281 expressed +T7 UBERON:0000204 310 331 ventral telencephalon +T8 GO:0065007 351 358 control +T9 CL:0000540 359 367 neuronal +T10 GO:0048665 359 381 neuronal specification +T11 GO:0007067 497 504 mitotic +T12 CL:0000540 505 512 neurons +T13 PR:000015417 569 573 SOX1 +T14 GO:0010467 611 620 expressed +T15 PR:000015426 699 703 SOX2 +T16 PR:000015428 708 712 SOX3 +T17 GO:0022008 752 764 neurogenesis +T18 PR:000015417 766 770 SOX1 +T19 GO:0010467 788 797 expressed +T20 UBERON:0001893 833 846 telencephalic +T21 CL:0000540 847 854 neurons +T22 UBERON:0005403 875 891 ventral striatum +T23 UBERON:0005403 893 895 VS +T24 CL:0000540 904 911 neurons +T25 PR:000015417 927 931 Sox1 +T26 NCBITaxon:10088 944 948 mice +T27 PR:000015417 1010 1014 SOX1 +T28 PR:000015417 1110 1114 Sox1 +T29 SO:0001023 1120 1126 allele +T30 GO:0010467 1127 1137 expressing +T31 CL:0000540 1203 1211 neuronal +T32 PR:000015417 1261 1265 SOX1 +T33 CL:0000540 1287 1294 neurons +T34 GO:0001764 1359 1371;1395 1402 migration of ... neurons +T35 PR:000015417 1376 1380 Sox1 +T36 GO:0010467 1381 1391 expressing +T37 UBERON:0005403 1392 1394 VS +T38 CL:0000540 1395 1402 neurons +T39 GO:0010467 1418 1428 expressing +T40 PR:000012318 1429 1433 Pax6 +T41 PR:000015417 1471 1475 SOX1 +T42 PR:000015417 1493 1497 Sox1 +T43 GO:0010467 1498 1508 expressing +T44 CL:0000540 1509 1516 neurons +T45 UBERON:0005403 1560 1562 VS +T46 PR:000015417 1580 1584 SOX1 +T47 GO:0007067 1604 1611 mitotic +T48 UBERON:0005403 1639 1641 VS +T49 CL:0000540 1642 1650 neuronal +T50 NCBITaxon:10088 1674 1678 mice +T51 PR:000015417 1688 1692 Sox1 +T52 GO:0010467 1693 1703 expression +T53 UBERON:0000204 1724 1745 ventral telencephalic +T54 UBERON:0005403 1781 1783 VS +T55 CL:0000540 1784 1791 neurons +T56 NCBITaxon:10088 1799 1803 mice +T57 UBERON:0005403 1829 1831 VS +T58 PR:000015417 1849 1853 SOX1 +T59 GO:0010467 1854 1864 expression +T60 UBERON:0005403 1901 1903 VS +T61 CL:0000540 1937 1944 neurons +T62 PR:000015417 1954 1958 Sox1 +T63 GO:0010467 1959 1969 expression +T64 UBERON:0005403 2013 2015 VS +T65 PR:000015417 2032 2036 Sox1 +T66 GO:0010467 2037 2047 expression +T67 UBERON:0005403 2084 2086 VS +T68 CL:0000540 2087 2095 neuronal +T69 GO:0001764 2087 2095;2109 2118 neuronal ... migration +T70 GO:0007067 2152 2159 mitotic +T71 PR:000015417 2207 2211 SOX1 +T72 GO:0010467 2268 2278 expression +T73 CL:0000540 2291 2299 neuronal +T74 UBERON:0001893 2436 2449 telencephalon +T75 UBERON:0000203 2477 2484 pallial +T76 UBERON:0000204 2499 2509 subpallial +T77 UBERON:0000956 2547 2562 cerebral cortex +T78 UBERON:0002420 2582 2595 basal ganglia +T79 UBERON:0000922 2615 2624 embryonic +T80 UBERON:0000204 2625 2635 subpallium +T81 UBERON:0004529 2654 2665 protrusions +T82 UBERON:0004023 2670 2690 ganglionic eminences +T83 CL:0000540 2718 2725 neurons +T84 UBERON:0004023 2743 2763 ganglionic eminences +T85 GO:0007608 2796 2805 olfactory +T86 UBERON:0002264 2796 2810 olfactory bulb +T87 UBERON:0001950 2829 2838 neocortex +T88 CL:0000540 2900 2907 neurons +T89 UBERON:0004025 2915 2942 lateral ganglionic eminence +T90 UBERON:0004025 2944 2947 LGE +T91 UBERON:0005382 2958 2964;2977 2985 dorsal ... striatum +T92 UBERON:0005403 2969 2985 ventral striatum +T93 UBERON:0005403 2987 2989 VS +T94 UBERON:0005403 2996 2998 VS +T95 UBERON:0001873 3012 3019 caudate +T96 UBERON:0001874 3021 3028 putamen +T97 UBERON:0001882 3030 3047 nucleus accumbens +T98 GO:0007608 3053 3062 olfactory +T99 UBERON:0001883 3053 3071 olfactory tubercle +T100 UBERON:0001883 3073 3075 OT +T101 GO:0065007 3084 3091 control +T102 GO:0050890 3118 3127 cognitive +T103 GO:0065007 3212 3219 control +T104 CL:0000540 3255 3262 neurons +T105 SO:0000704 3305 3309 Gene +T106 GO:0010467 3305 3320 Gene-expression +T107 PR:000012318 3405 3409 PAX6 +T108 PR:000008309 3420 3424 GSH2 +T109 UBERON:0005403 3497 3499 VS +T110 CL:0000540 3500 3507 neurons +T111 UBERON:0004025 3558 3561 LGE +T112 CHEBI:22695 3577 3582 basic +T113 SO:0001114 3583 3588 helix +T114 SO:0001114 3594 3599 helix +T115 PR:000004362 3614 3619 MASH1 +T116 CL:0000540 3660 3667 neurons +T117 UBERON:0004025 3675 3678 LGE +T118 NCBITaxon:10088 3718 3723 mouse +T119 CL:0000540 3766 3773 neurons +T120 UBERON:0001893 3781 3794 telencephalon +T121 UBERON:0005403 3814 3816 VS +T122 CL:0000540 3817 3824 neurons +T123 PR:000008309 3845 3849 GSH2 +T124 PR:000004362 3854 3859 MASH1 +T125 GO:0065007 3860 3867 control +T126 UBERON:0005403 3868 3870 VS +T127 GO:0010467 3931 3940 expressed +T128 GO:0007067 3948 3955 mitotic +T129 CL:0000540 4035 4043 neuronal +T130 UBERON:0000204 4060 4081 ventral telencephalon +T131 CL:0000540 4132 4138 neuron +T132 GO:0010467 4148 4158 expression +T133 GO:0065007 4249 4257 regulate +T134 SO:0000417 4346 4353 domains +T135 SO:0000704 4380 4385 genes +T136 NCBITaxon:40674 4389 4396 mammals +T137 GO:0010467 4420 4429 expressed +T138 UBERON:0001016 4448 4462 nervous system +T139 PR:000015417 4536 4540 SOX1 +T140 PR:000015426 4542 4546 SOX2 +T141 PR:000015428 4552 4556 SOX3 +T142 SO:0000857 4660 4668 homology +T143 GO:0010467 4709 4718 expressed +T144 UBERON:0034705 4726 4741 neuroepithelium +T145 UBERON:0001017 4753 4775 central nervous system +T146 GO:0007417 4753 4775;4782 4793 central nervous system ... development +T147 UBERON:0001017 4777 4780 CNS +T148 GO:0007417 4777 4780;4782 4793 CNS ... development +T149 CL:0000047 4904 4921 neural stem cells +T150 CL:0000034 5001 5011 stem cells +T151 CL:0000047 5078 5094 neural stem cell +T152 GO:0022008 5129 5141 neurogenesis +T153 PR:000015417 5190 5194 SOX1 +T154 GO:0022008 5204 5216 neurogenesis +T155 GO:0007049 5221 5231 cell cycle +T156 NCBITaxon:10088 5252 5256 mice +T157 PR:000015417 5275 5279 Sox1 +T158 PR:000015428 5288 5292 Sox3 +T159 NCBITaxon:10088 5302 5306 mice +T160 PR:000015426 5316 5320 Sox2 +T161 SO:0001023 5321 5327 allele +T162 CL:0000540 5454 5462 neuronal +T163 GO:0010467 5503 5512 expressed +T164 CL:0000540 5536 5543 neurons +T165 NCBITaxon:10088 5578 5582 mice +T166 GO:0007067 5655 5662 mitotic +T167 PR:000015417 5710 5714 SOX1 +T168 UBERON:0005403 5845 5847 VS +T169 CL:0000540 5848 5855 neurons +T170 http://purl.obolibrary.org/obo/MONDO_0005027 5894 5902 epilepsy +T171 PR:000015417 5939 5943 SOX1 +T172 GO:0008283 5977 5990 proliferation +T173 CL:0000540 6010 6018 neuronal +T174 PR:000015426 6082 6086 SOX2 +T175 PR:000015428 6091 6095 SOX3 +T176 GO:0010467 6110 6119 expressed +T177 PR:000015417 6125 6129 SOX1 +T178 NCBITaxon:10088 6155 6159 mice +T179 CL:0000540 6177 6183 neuron +T180 GO:0010467 6193 6203 expression +T181 PR:000015417 6207 6211 Sox1 +T182 UBERON:0000204 6219 6240 ventral telencephalon +T183 UBERON:0005403 6263 6265 VS +T184 CL:0000540 6266 6273 neurons +T185 CL:0000540 6314 6321 neurons +T186 PR:000015417 6360 6364 Sox1 +T187 GO:0010467 6365 6375 expression +T188 CL:0000540 6379 6386 neurons +T189 UBERON:0000204 6394 6415 ventral telencephalon +T190 UBERON:0005403 6452 6454 VS +T191 PR:000015417 6483 6487 SOX1 +T192 GO:0007067 6504 6511 mitotic +T193 UBERON:0005403 6563 6565 VS +T194 CL:0000540 6575 6583 neuronal +T195 GO:0001764 6575 6593 neuronal migration +T196 GO:0007067 6678 6685 mitotic +T197 PR:000015417 6725 6729 SOX1 +T198 PR:000015417 6741 6745 SOX1 +T199 GO:0009888 6767 6779 Histogenesis +T200 UBERON:0005403 6787 6789 VS +T201 PR:000015417 6821 6825 Sox1 +T202 GO:0010467 6826 6836 expression +T203 UBERON:0007023 6859 6864 adult +T204 UBERON:0000955 6865 6870 brain +T205 NCBITaxon:10088 6874 6878 mice +T206 SO:0001023 6984 6990 allele +T207 PR:000015417 7006 7010 Sox1 +T208 SO:0001817 7088 7096 in-frame +T209 PR:000015417 7106 7110 SOX1 +T210 SO:0000236 7111 7129 open reading frame +T211 NCBITaxon:10088 7143 7147 Mice +T212 PR:000015417 7163 7167 Sox1 +T213 PR:000015417 7185 7189 SOX1 +T214 NCBITaxon:10088 7249 7253 mice +T215 PR:000015417 7285 7289 SOX1 +T216 PR:000015417 7305 7309 Sox1 +T217 http://purl.obolibrary.org/obo/MONDO_0005027 7347 7356 epileptic +T218 CHEBI:75055 7406 7411 X-gal +T219 PR:000015417 7425 7429 Sox1 +T220 UBERON:0000922 7449 7456 embryos +T221 SO:0001023 7488 7494 allele +T222 GO:0097617 7530 7543 hybridization +T223 PR:000015417 7548 7552 SOX1 +T224 GO:0042571 7553 7561 antibody +T225 PR:000015417 7613 7617 SOX1 +T226 UBERON:0005403 7642 7644 VS +T227 GO:0010467 7662 7672 expression +T228 PR:000015417 7684 7688 Sox1 +T229 UBERON:0000955 7750 7756 brains +T230 UBERON:0000922 7762 7771 embryonic +T231 GO:0007567 7792 7797 natal +T232 CHEBI:75055 7815 7820 X-gal +T233 UBERON:0001017 7854 7857 CNS +T234 CHEBI:75055 7859 7864 X-gal +T235 PR:000015417 7881 7885 Sox1 +T236 CHEBI:75055 8047 8052 X-gal +T237 NCBITaxon:10088 8076 8080 mice +T238 NCBITaxon:33208 8108 8115 animals +T239 NCBITaxon:10088 8141 8145 mice +T240 PR:000015417 8172 8176 Sox1 +T241 SO:0001023 8182 8189 alleles +T242 PR:000015417 8197 8201 Sox1 +T243 SO:0001023 8248 8254 allele +T244 PR:000015417 8256 8260 Sox1 +T245 GO:0010467 8285 8292 express +T246 CHEBI:75055 8335 8340 X-gal +T247 UBERON:0001890 8374 8383 forebrain +T248 PR:000015417 8385 8389 Sox1 +T249 GO:0010467 8393 8402 expressed +T250 UBERON:0003053 8418 8434 ventricular zone +T251 UBERON:0003053 8436 8438 VZ +T252 UBERON:0004922 8444 8463 subventricular zone +T253 UBERON:0004922 8465 8468 SVZ +T254 CL:0000540 8477 8484 neurons +T255 UBERON:0000935 8496 8515 anterior commissure +T256 UBERON:0001882 8546 8563 nucleus accumbens +T257 UBERON:0002435 8619 8627 striatal +T258 UBERON:0007651 8628 8635 bridges +T259 UBERON:0001883 8702 8704 OT +T260 UBERON:0002361 8723 8727 pial +T261 CHEBI:75055 8737 8742 X-gal +T262 CL:0000540 8752 8759 neurons +T263 UBERON:0001883 8781 8783 OT +T264 GO:0007567 8847 8852 birth +T265 PR:000015417 8876 8880 Sox1 +T266 CHEBI:75055 8906 8911 X-gal +T267 UBERON:0003053 8936 8938 VZ +T268 UBERON:0004922 8939 8942 SVZ +T269 PR:000015417 8981 8985 Sox1 +T270 CHEBI:75055 9042 9047 X-gal +T271 UBERON:0000935 9074 9093 anterior commissure +T272 UBERON:0002435 9123 9131 striatal +T273 UBERON:0007651 9132 9139 bridges +T274 UBERON:0001883 9148 9150 OT +T275 PR:000015417 9176 9180 Sox1 +T276 UBERON:0000955 9186 9191 brain +T277 CL:0000540 9273 9280 neurons +T278 GO:0016265 9281 9284 die +T279 GO:0006915 9321 9330 apoptosis +T280 GO:0008219 9378 9388 cell death +T281 CHEBI:75055 9434 9439 X-gal +T282 UBERON:0000204 9477 9498 ventral telencephalon +T283 GO:0007567 9517 9522 natal +T284 UBERON:0000955 9523 9528 brain +T285 PR:000015417 9579 9583 Sox1 +T286 UBERON:0000955 9647 9652 brain +T287 CL:0000540 9686 9693 neurons +T288 UBERON:0012170 9708 9715;9720 9737 core of ... nucleus accumbens +T289 GO:0010467 9738 9745 express +T290 PR:000015417 9746 9750 Sox1 +T291 PR:000015417 9842 9846 SOX1 +T292 PR:000015417 9881 9885 SOX1 +T293 GO:0009888 9902 9914 histogenesis +T294 UBERON:0001883 9922 9924 OT +T295 GO:0008283 9971 9984 Proliferation +T296 GO:0022008 9989 10001 Neurogenesis +T297 UBERON:0001883 10014 10016 OT +T298 CL:0000540 10017 10025 Neuronal +T299 PR:000015417 10060 10064 SOX1 +T300 PR:000015417 10112 10116 SOX1 +T301 PR:000015426 10130 10134 SOX2 +T302 PR:000015428 10139 10143 SOX3 +T303 GO:0022008 10157 10169 neurogenesis +T304 PR:000015426 10186 10190 SOX2 +T305 PR:000015428 10195 10199 SOX3 +T306 GO:0022008 10210 10222 neurogenesis +T307 PR:000015417 10260 10264 SOX1 +T308 UBERON:0002435 10323 10331 striatum +T309 PR:000016819 10349 10361 βIII-tubulin +T310 PR:000016819 10363 10367 TuJ1 +T311 GO:0042571 10369 10377 antibody +T312 CL:0000540 10410 10417 neurons +T313 UBERON:0004025 10474 10477 LGE +T314 PR:000016819 10479 10484 TuJ 1 +T315 PR:000015417 10572 10576 Sox1 +T316 PR:000015417 10629 10633 SOX1 +T317 CL:0000540 10680 10686 neuron +T318 CL:0000540 10773 10780 neurons +T319 UBERON:0007023 10819 10824 adult +T320 GO:0010467 10841 10851 expression +T321 UBERON:0002435 10855 10863 striatal +T322 PR:000012544 10880 10896 preproenkephalin +T323 PR:000007786 10901 10906 Gad65 +T324 UBERON:0000922 10945 10954 embryonic +T325 PR:000013040 10994 10998 Brn4 +T326 UBERON:0001882 11137 11154 nucleus accumbens +T327 UBERON:0001883 11155 11157 OT +T328 UBERON:0002435 11183 11191 striatum +T329 PR:000015417 11197 11201 Sox1 +T330 GO:0010467 11202 11212 expression +T331 UBERON:0034705 11262 11277 neuroepithelium +T332 GO:0008283 11301 11314 proliferation +T333 UBERON:0000204 11374 11395 ventral telencephalon +T334 CHEBI:472552 11405 11428 5-bromo-2′-deoxyuridine +T335 CHEBI:472552 11430 11434 BrdU +T336 GO:0008283 11449 11462 proliferating +T337 PR:000015417 11491 11495 Sox1 +T338 UBERON:0000922 11501 11508 embryos +T339 UBERON:0000955 11541 11547 brains +T340 UBERON:0003053 11604 11606 VZ +T341 UBERON:0004922 11607 11610 SVZ +T342 GO:0008283 11649 11662 proliferation +T343 PR:000015417 11700 11704 SOX1 +T344 GO:0008283 11736 11749 proliferation +T345 GO:0007049 11785 11795 cell cycle +T346 UBERON:0004025 11803 11806 LGE +T347 PR:000015417 11847 11851 SOX1 +T348 GO:0008283 11877 11890 proliferation +T349 CL:0000540 11917 11925 neuronal +T350 GO:0001764 12011 12023;12027 12034 migration of ... neurons +T351 UBERON:0005403 12024 12026 VS +T352 CL:0000540 12027 12034 neurons +T353 UBERON:0001883 12058 12060 OT +T354 CL:0000540 12061 12068 Neurons +T355 PR:000015417 12103 12107 SOX1 +T356 CL:0000540 12145 12152 neurons +T357 PR:000015417 12168 12172 Sox1 +T358 UBERON:0001883 12178 12180 OT +T359 GO:0010467 12230 12237 express +T360 PR:000015417 12238 12242 Sox1 +T361 UBERON:0000922 12293 12300 embryos +T362 CHEBI:472552 12304 12308 BrdU +T363 GO:0008283 12346 12359 proliferating +T364 PR:000015417 12388 12392 Sox1 +T365 UBERON:0002435 12402 12410 striatal +T366 SO:0000704 12418 12422 gene +T367 GO:0010467 12418 12433 gene expression +T368 UBERON:0000922 12461 12470 embryonic +T369 GO:0007567 12488 12493 birth +T370 UBERON:0000922 12543 12552 embryonic +T371 UBERON:0001883 12609 12611 OT +T372 CL:0000540 12612 12619 neurons +T373 NCBITaxon:10088 12659 12664 mouse +T374 UBERON:0005403 12689 12705 ventral striatal +T375 CL:0002613 12697 12713 striatal neurons +T376 GO:0007567 12757 12762 birth +T377 NCBITaxon:10114 12844 12847 rat +T378 UBERON:0000922 12883 12890 embryos +T379 CHEBI:472552 12892 12896 BrdU +T380 UBERON:0000922 12946 12953 embryos +T381 GO:0007567 13009 13014 birth +T382 CL:0000540 13062 13069 neurons +T383 PR:000015417 13124 13128 Sox1 +T384 UBERON:0000922 13134 13141 embryos +T385 CHEBI:472552 13195 13199 BrdU +T386 GO:0007608 13235 13244 olfactory +T387 UBERON:0002894 13235 13251 olfactory cortex +T388 UBERON:0005403 13305 13307 VS +T389 PR:000015417 13422 13426 Sox1 +T390 GO:0007567 13436 13441 natal +T391 UBERON:0000955 13442 13447 brain +T392 UBERON:0002435 13452 13460 striatal +T393 CHEBI:472552 13497 13501 BrdU +T394 CHEBI:75055 13572 13577 X-gal +T395 UBERON:0002435 13611 13619 striatum +T396 PR:000015417 13682 13686 SOX1 +T397 CL:0000540 13709 13716 neurons +T398 UBERON:0002435 13794 13802 striatum +T399 UBERON:0004025 13838 13841 LGE +T400 PR:000015417 13881 13885 SOX1 +T401 UBERON:0005403 13920 13922 VS +T402 CL:0000540 13923 13930 neurons +T403 UBERON:0004025 13975 13978 LGE +T404 GO:0010467 14077 14087 expression +T405 UBERON:0004025 14139 14142 LGE +T406 UBERON:0001883 14187 14189 OT +T407 CL:0000540 14190 14198 neuronal +T408 GO:0048665 14190 14212 neuronal specification +T409 PR:000012318 14277 14281 PAX6 +T410 PR:000008309 14286 14290 GSH2 +T411 GO:0010467 14295 14304 expressed +T412 UBERON:0000203 14327 14334 pallial +T413 UBERON:0000204 14339 14349 subpallial +T414 UBERON:0018264 14375 14385 dorsal LGE +T415 UBERON:0005403 14466 14468 VS +T416 PR:000008309 14503 14507 GSH2 +T417 NCBITaxon:10088 14513 14517 mice +T418 UBERON:0001883 14541 14543 OT +T419 CL:0000540 14544 14551 neurons +T420 UBERON:0018264 14579 14589 dorsal LGE +T421 PR:000012318 14602 14606 PAX6 +T422 UBERON:0004025 14634 14637 LGE +T423 PR:000012318 14661 14665 PAX6 +T424 PR:000008309 14670 14674 GSH2 +T425 UBERON:0001883 14730 14732 OT +T426 GO:0042571 14764 14772 antibody +T427 UBERON:0000922 14797 14804 embryos +T428 PR:000015417 14839 14843 SOX1 +T429 GO:0010467 14865 14875 expression +T430 PR:000008309 14879 14883 GSH2 +T431 PR:000012318 14884 14888 PAX6 +T432 UBERON:0018264 14913 14923 dorsal LGE +T433 PR:000012318 14981 14985 PAX6 +T434 GO:0010467 14986 14996 expressing +T435 GO:0007067 15001 15008 mitotic +T436 UBERON:0005403 15080 15082 VS +T437 PR:000015417 15142 15146 SOX1 +T438 PR:000012318 15162 15166 PAX6 +T439 GO:0007067 15180 15187 mitotic +T440 PR:000012318 15259 15263 PAX6 +T441 PR:000015417 15269 15273 SOX1 +T442 GO:0010467 15274 15284 expressing +T443 CL:0000540 15285 15292 neurons +T444 UBERON:0001883 15314 15316 OT +T445 GO:0042571 15333 15341 antibody +T446 UBERON:0000955 15396 15401 brain +T447 PR:000012318 15419 15423 PAX6 +T448 PR:000015417 15428 15432 SOX1 +T449 GO:0042571 15433 15443 antibodies +T450 PR:000015417 15466 15470 Sox1 +T451 GO:0010467 15471 15481 expressing +T452 PR:000015417 15495 15499 Sox1 +T453 UBERON:0000955 15512 15517 brain +T454 GO:0042571 15538 15546 antibody +T455 PR:000012318 15590 15594 PAX6 +T456 PR:000015417 15599 15603 SOX1 +T457 GO:0010467 15621 15630 expressed +T458 GO:0007067 15680 15687 mitotic +T459 UBERON:0004025 15701 15704 LGE +T460 GO:0010467 15710 15720 expression +T461 PR:000012318 15767 15771 Pax6 +T462 GO:0010467 15772 15782 expressing +T463 CL:0000540 15783 15790 neurons +T464 GO:0010467 15825 15835 expressing +T465 PR:000015417 15836 15840 Sox1 +T466 UBERON:0001883 15868 15870 OT +T467 GO:0007608 15875 15884 olfactory +T468 UBERON:0002894 15875 15891 olfactory cortex +T469 PR:000015417 15923 15927 Sox1 +T470 NCBITaxon:10088 15933 15937 mice +T471 GO:0007067 15947 15954 mitotic +T472 PR:000012318 15955 15959 Pax6 +T473 GO:0010467 15960 15970 expressing +T474 UBERON:0005403 16009 16011 VS +T475 PR:000012318 16132 16136 Pax6 +T476 GO:0010467 16137 16147 expressing +T477 CL:0000540 16148 16155 neurons +T478 PR:000015417 16174 16178 Sox1 +T479 CL:0000540 16184 16191 neurons +T480 GO:0010467 16216 16226 expressing +T481 PR:000012318 16232 16236 PAX6 +T482 PR:000015417 16262 16266 Sox1 +T483 SO:0001023 16271 16277 allele +T484 UBERON:0004025 16313 16316 LGE +T485 CL:0000681 16363 16375 radial glial +T486 UBERON:0003053 16428 16430 VZ +T487 UBERON:0002361 16438 16442 pial +T488 GO:0001764 16499 16519 migration of neurons +T489 CL:0000540 16512 16519 neurons +T490 CHEBI:75055 16543 16548 X-gal +T491 GO:0042571 16584 16592 antibody +T492 NCBITaxon:10088 16596 16600 mice +T493 PR:000015417 16614 16618 Sox1 +T494 SO:0001023 16623 16629 allele +T495 GO:0005737 16659 16670 cytoplasmic +T496 PR:000015417 16690 16694 SOX1 +T497 GO:0010467 16695 16705 expressing +T498 CL:0000681 16730 16742 radial glial +T499 CL:0000681 16806 16817 radial glia +T500 PR:000015417 16825 16829 Sox1 +T501 UBERON:0004025 16835 16838 LGE +T502 GO:0042571 16853 16861 antibody +T503 PR:000012318 16988 16992 Pax6 +T504 GO:0010467 16993 17003 expressing +T505 CL:0000540 17004 17011 neurons +T506 UBERON:0004025 17019 17022 LGE +T507 PR:000015417 17030 17034 Sox1 +T508 NCBITaxon:10088 17040 17044 mice +T509 CL:0000681 17100 17112 radial glial +T510 CL:0000681 17143 17154 radial glia +T511 UBERON:0000204 17180 17201 ventral telencephalon +T512 PR:000004362 17233 17238 MASH1 +T513 PR:000006527 17271 17275 DLX1 +T514 UBERON:0004025 17281 17284 LGE +T515 PR:000004362 17313 17318 MASH1 +T516 NCBITaxon:10088 17322 17326 mice +T517 UBERON:0002435 17384 17392 striatal +T518 CL:0002613 17384 17400 striatal neurons +T519 UBERON:0001883 17424 17426 OT +T520 UBERON:0001882 17431 17448 nucleus accumbens +T521 PR:000008309 17468 17472 Gsh2 +T522 NCBITaxon:10088 17478 17482 mice +T523 UBERON:0001883 17515 17517 OT +T524 PR:000006527 17535 17539 Dlx1 +T525 GO:0010467 17540 17550 expression +T526 UBERON:0004025 17554 17557 LGE +T527 GO:0010467 17600 17610 expression +T528 SO:0000704 17624 17629 genes +T529 PR:000015417 17633 17637 Sox1 +T530 UBERON:0000922 17643 17650 embryos +T531 UBERON:0004025 17745 17748 LGE +T532 PR:000015417 17778 17782 SOX1 +T533 PR:000015417 17823 17827 SOX1 +T534 UBERON:0004025 17891 17894 LGE +T535 PR:000015417 17908 17912 Sox1 +T536 GO:0010467 17913 17923 Expression +T537 PR:000015426 17944 17948 Sox2 +T538 SO:0000167 17949 17957 Promoter +T539 SO:0000704 18023 18028 genes +T540 GO:0010467 18033 18042 expressed +T541 GO:0007067 18129 18136 mitotic +T542 UBERON:0005403 18165 18167 VS +T543 GO:0042571 18174 18182 antibody +T544 SO:0000704 18208 18213 genes +T545 PR:000015426 18236 18240 SOX2 +T546 PR:000015428 18246 18250 SOX3 +T547 CL:0000540 18260 18267 neurons +T548 GO:0010467 18319 18329 expressing +T549 PR:000015417 18330 18334 SOX1 +T550 SO:0000704 18396 18401 genes +T551 PR:000015417 18429 18433 Sox1 +T552 UBERON:0004025 18482 18485 LGE +T553 GO:0007067 18490 18497 mitotic +T554 PR:000015417 18547 18551 SOX1 +T555 UBERON:0005403 18587 18589 VS +T556 GO:0007067 18619 18626 mitotic +T557 NCBITaxon:10088 18751 18755 mice +T558 GO:0010467 18761 18768 express +T559 PR:000015417 18769 18773 Sox1 +T560 UBERON:0004025 18806 18809 LGE +T561 CL:0000540 18810 18817 neurons +T562 PR:000015426 18854 18858 Sox2 +T563 GO:0010467 18865 18874 expressed +T564 PR:000015417 18880 18884 Sox1 +T565 UBERON:0004025 18927 18930 LGE +T566 CL:0000540 18931 18938 neurons +T567 NCBITaxon:10088 18954 18958 mice +T568 GO:0010467 18964 18971 express +T569 PR:000015417 18972 18976 Sox1 +T570 PR:000015426 18997 19001 Sox2 +T571 SO:0000167 19002 19010 promoter +T572 PR:000015417 19040 19044 Sox1 +T573 PR:000015426 19049 19053 Sox2 +T574 GO:0010467 19054 19064 expression +T575 UBERON:0003053 19072 19074 VZ +T576 UBERON:0004922 19075 19078 SVZ +T577 UBERON:0004025 19086 19089 LGE +T578 UBERON:0001893 19117 19130 telencephalic +T579 GO:0042571 19145 19155 antibodies +T580 SO:0000704 19176 19181 genes +T581 GO:0005634 19212 19219 nuclear +T582 PR:000015417 19281 19285 SOX1 +T583 PR:000015426 19291 19295 SOX2 +T584 PR:000015426 19364 19368 SOX2 +T585 SO:0000236 19369 19387 open reading frame +T586 PR:000015417 19401 19405 SOX1 +T587 PR:000015426 19436 19440 Sox2 +T588 SO:0001023 19441 19447 allele +T589 SO:0001023 19469 19475 allele +T590 PR:000015426 19477 19481 Sox2 +T591 GO:0010467 19502 19509 express +T592 PR:000015417 19519 19523 Sox1 +T593 SO:0000243 19545 19574 internal ribosomal entry site +T594 GO:0005840 19554 19563 ribosomal +T595 SO:0000243 19576 19580 IRES +T596 PR:000015417 19617 19621 SOX1 +T597 PR:000015426 19629 19633 Sox2 +T598 SO:0001023 19635 19641 allele +T599 SO:0000357 19646 19653 flanked +T600 SO:0000346 19657 19667 LoxP sites +T601 NCBITaxon:10088 19757 19762 mouse +T602 SO:0001023 19785 19791 allele +T603 PR:000015426 19800 19804 Sox2 +T604 PR:000015426 19840 19844 Sox2 +T605 GO:0010467 19857 19866 expresses +T606 SO:0000704 19890 19894 gene +T607 PR:000015426 19904 19908 Sox2 +T608 SO:0000167 19909 19917 promoter +T609 PR:000015417 19938 19942 SOX1 +T610 PR:000015426 19953 19957 Sox2 +T611 PR:000015426 19979 19983 Sox2 +T612 NCBITaxon:10088 19987 19991 mice +T613 PR:000015417 20057 20061 SOX1 +T614 GO:0010467 20067 20077 expression +T615 GO:0010467 20112 20122 expression +T616 PR:000015426 20148 20152 Sox2 +T617 GO:0010467 20165 20174 expressed +T618 CHEBI:75055 20231 20236 X-gal +T619 UBERON:0000922 20249 20256 embryos +T620 PR:000015426 20283 20287 Sox2 +T621 SO:0001023 20288 20295 alleles +T622 PR:000015426 20297 20301 Sox2 +T623 PR:000015426 20310 20314 Sox2 +T624 GO:0010467 20317 20324 express +T625 UBERON:0001017 20337 20340 CNS +T626 PR:000015417 20361 20365 SOX1 +T627 PR:000015426 20396 20400 Sox2 +T628 SO:0001023 20402 20408 allele +T629 PR:000015417 20418 20422 SOX1 +T630 GO:0042571 20423 20431 antibody +T631 PR:000015417 20456 20460 SOX1 +T632 PR:000015426 20508 20512 SOX2 +T633 GO:0010467 20535 20545 expression +T634 UBERON:0003309 20566 20580;20585 20597 floor plate of ... diencephalon +T635 GO:0010467 20787 20797 expression +T636 PR:000015417 20817 20821 Sox1 +T637 SO:0001023 20822 20829 alleles +T638 UBERON:0003053 20831 20833 VZ +T639 UBERON:0004922 20834 20837 SVZ +T640 GO:0010467 20869 20879 expression +T641 SO:0001023 20909 20915 allele +T642 PR:000015426 20932 20936 Sox2 +T643 SO:0001023 20938 20944 allele +T644 PR:000015417 20954 20958 SOX1 +T645 CL:0000540 20978 20985 neurons +T646 PR:000015426 21008 21012 SOX2 +T647 PR:000015417 21051 21055 SOX1 +T648 CL:0000540 21074 21081 neurons +T649 GO:0010467 21087 21094 express +T650 SO:0000704 21100 21105 genes +T651 NCBITaxon:10088 21144 21148 mice +T652 GO:0010467 21172 21182 expression +T653 PR:000015417 21274 21278 SOX1 +T654 GO:0010467 21338 21348 expressing +T655 PR:000015426 21349 21353 Sox2 +T656 SO:0000704 21415 21420 genes +T657 PR:000015417 21473 21477 Sox1 +T658 GO:0010467 21483 21493 Expression +T659 UBERON:0005403 21526 21528 VS +T660 UBERON:0001883 21529 21531 OT +T661 CL:0000540 21532 21540 Neuronal +T662 GO:0048665 21532 21559 Neuronal Fate Specification +T663 GO:0010467 21612 21622 expression +T664 PR:000015417 21626 21630 Sox1 +T665 PR:000015426 21652 21656 Sox2 +T666 NCBITaxon:10088 21660 21664 mice +T667 NCBITaxon:10088 21706 21710 mice +T668 GO:0001764 21730 21742;21766 21773 migration of ... neurons +T669 PR:000015417 21747 21751 Sox1 +T670 PR:000015426 21752 21756 Sox2 +T671 CL:0000540 21766 21773 neurons +T672 UBERON:0005403 21781 21783 VS +T673 CHEBI:75055 21788 21793 X-gal +T674 NCBITaxon:10088 21812 21816 mice +T675 PR:000015426 21826 21830 Sox2 +T676 PR:000015426 21844 21848 Sox2 +T677 PR:000015417 21853 21857 Sox1 +T678 PR:000015426 21881 21885 Sox2 +T679 PR:000015417 21890 21894 Sox1 +T680 PR:000015417 21911 21915 Sox1 +T681 SO:0001023 21937 21944 alleles +T682 PR:000015426 21979 21983 Sox2 +T683 PR:000015417 21991 21995 Sox1 +T684 GO:0010467 22011 22018 express +T685 PR:000015417 22019 22023 Sox1 +T686 NCBITaxon:10088 22063 22067 mice +T687 PR:000015426 22092 22096 Sox2 +T688 SO:0001023 22097 22103 allele +T689 PR:000015417 22126 22130 Sox1 +T690 PR:000015426 22140 22144 Sox2 +T691 CL:0000540 22150 22157 neurons +T692 UBERON:0000204 22165 22186 ventral telencephalon +T693 PR:000015417 22190 22194 Sox1 +T694 PR:000015426 22224 22228 Sox2 +T695 NCBITaxon:10088 22244 22248 mice +T696 UBERON:0000955 22284 22289 brain +T697 PR:000015426 22295 22299 Sox2 +T698 CL:0000540 22309 22316 neurons +T699 PR:000015417 22355 22359 Sox1 +T700 UBERON:0000479 22410 22416 tissue +T701 CHEBI:75055 22445 22450 X-gal +T702 PR:000015417 22470 22474 Sox1 +T703 PR:000015426 22554 22558 Sox2 +T704 GO:0042571 22585 22593 antibody +T705 PR:000015426 22625 22629 Sox2 +T706 CL:0000540 22635 22642 neurons +T707 UBERON:0001883 22661 22663 OT +T708 UBERON:0005403 22706 22708 VS +T709 CL:0000540 22709 22717 neuronal +T710 GO:0010467 22753 22763 expression +T711 PR:000015417 22767 22771 Sox1 +T712 UBERON:0004025 22775 22778 LGE +T713 CL:0000540 22779 22786 neurons +T714 GO:0001764 22841 22850;22866 22868;22873 22880 migration ... of ... neurons +T715 UBERON:0004025 22869 22872 LGE +T716 CL:0000540 22873 22880 neurons +T717 GO:0010467 22881 22891 expressing +T718 PR:000015426 22905 22909 Sox2 +T719 SO:0000167 22910 22918 promoter +T720 PR:000015417 22984 22988 Sox1 +T721 SO:0001023 22989 22996 alleles +T722 PR:000015417 23024 23028 Sox1 +T723 UBERON:0005403 23112 23114 VS +T724 CL:0000540 23115 23122 neurons +T725 UBERON:0002435 23136 23144 striatal +T726 CHEBI:18243 23162 23170 dopamine +T727 PR:000013121 23162 23204 dopamine and cAMP-regulated phosphoprotein +T728 CHEBI:17489 23175 23179 cAMP +T729 GO:0065007 23180 23189 regulated +T730 PR:000013121 23206 23214 DARPP-32 +T731 GO:0007567 23223 23228 natal +T732 PR:000015426 23271 23275 Sox2 +T733 NCBITaxon:10088 23279 23283 mice +T734 GO:0010467 23346 23356 expression +T735 PR:000015417 23360 23364 SOX1 +T736 UBERON:0001883 23397 23399 OT +T737 CL:0000540 23400 23408 neuronal +T738 GO:0048665 23400 23422 neuronal specification +T739 PR:000015417 23425 23429 Sox1 +T740 GO:0010467 23430 23440 Expression +T741 UBERON:0001883 23469 23471 OT +T742 CL:0000540 23472 23478 Neuron +T743 PR:000015417 23520 23524 SOX1 +T744 PR:000015417 23573 23577 Sox1 +T745 PR:000015426 23583 23587 Sox2 +T746 NCBITaxon:10088 23591 23595 mice +T747 PR:000015417 23601 23605 Sox1 +T748 PR:000015426 23613 23617 Sox2 +T749 NCBITaxon:10088 23621 23625 mice +T750 PR:000015426 23666 23670 Sox2 +T751 PR:000015417 23696 23700 Sox1 +T752 SO:0001023 23701 23708 alleles +T753 PR:000015417 23710 23714 Sox1 +T754 UBERON:0001883 23737 23739 OT +T755 PR:000015417 23750 23754 Sox1 +T756 UBERON:0000922 23758 23765 embryos +T757 PR:000015417 23791 23795 Sox1 +T758 SO:0001023 23807 23813 allele +T759 PR:000015417 23834 23838 SOX1 +T760 GO:0010467 23857 23866 expressed +T761 PR:000015426 23880 23884 Sox2 +T762 SO:0001023 23886 23892 allele +T763 GO:0007067 23940 23947 mitotic +T764 UBERON:0004025 23948 23951 LGE +T765 GO:0010467 23973 23983 expression +T766 PR:000015417 24004 24008 Sox1 +T767 SO:0001023 24016 24022 allele +T768 UBERON:0001883 24044 24046 OT +T769 CL:0000540 24059 24066 neurons +T770 PR:000015417 24084 24088 Sox1 +T771 UBERON:0001883 24108 24110 OT +T772 CL:0000540 24111 24118 neurons +T773 CHEBI:75055 24124 24129 X-gal +T774 UBERON:0001883 24192 24194 OT +T775 UBERON:0000922 24203 24210 embryos +T776 PR:000015426 24227 24231 Sox2 +T777 SO:0001023 24233 24239 allele +T778 GO:0010467 24245 24254 expresses +T779 UBERON:0004025 24273 24276 LGE +T780 GO:0007067 24281 24288 mitotic +T781 PR:000015426 24296 24300 Sox2 +T782 GO:0010467 24306 24316 expression +T783 PR:000015417 24343 24347 Sox1 +T784 CHEBI:75055 24392 24397 X-gal +T785 CHEBI:75055 24434 24439 X-gal +T786 UBERON:0005403 24464 24466 VS +T787 UBERON:0000955 24508 24513 brain +T788 CHEBI:75055 24624 24629 X-gal +T789 PR:000015417 24693 24697 SOX1 +T790 GO:0042571 24698 24706 antibody +T791 CHEBI:75055 24755 24760 X-gal +T792 UBERON:0001883 24795 24797 OT +T793 CL:0000540 24798 24805 neurons +T794 PR:000015417 24822 24826 Sox1 +T795 NCBITaxon:10088 24833 24837 mice +T796 PR:000015417 24896 24900 Sox1 +T797 UBERON:0000922 24906 24913 embryos +T798 PR:000015417 24915 24919 Sox1 +T799 PR:000015426 24933 24937 Sox2 +T800 PR:000015417 25005 25009 Sox1 +T801 UBERON:0000922 25015 25022 embryos +T802 UBERON:0001883 25038 25040 OT +T803 PR:000015426 25069 25073 Sox2 +T804 SO:0001023 25075 25081 allele +T805 PR:000015417 25086 25090 SOX1 +T806 PR:000015417 25141 25145 SOX1 +T807 NCBITaxon:10088 25184 25188 mice +T808 PR:000015417 25245 25249 SOX1 +T809 GO:0042571 25250 25258 antibody +T810 PR:000015417 25267 25271 Sox1 +T811 UBERON:0000922 25278 25285 embryos +T812 PR:000015417 25296 25300 SOX1 +T813 UBERON:0003053 25316 25318 VZ +T814 UBERON:0001883 25363 25365 OT +T815 CL:0000540 25366 25373 neurons +T816 PR:000015417 25408 25412 Sox1 +T817 UBERON:0000922 25427 25434 embryos +T818 PR:000015417 25436 25440 SOX1 +T819 GO:0010467 25441 25451 expression +T820 UBERON:0000922 25504 25511 embryos +T821 PR:000015417 25513 25517 SOX1 +T822 UBERON:0003053 25545 25547 VZ +T823 CL:0000540 25592 25599 neurons +T824 UBERON:0004025 25607 25610 LGE +T825 NCBITaxon:10088 25629 25633 mice +T826 PR:000015417 25644 25648 Sox1 +T827 GO:0007567 25660 25664 born +T828 UBERON:0000970 25676 25680 eyes +T829 PR:000015417 25802 25806 Sox1 +T830 NCBITaxon:10088 25812 25816 mice +T831 UBERON:0000955 25842 25847 brain +T832 NCBITaxon:10088 25860 25864 mice +T833 PR:000013121 25888 25896 DARPP-32 +T834 GO:0042571 25897 25905 antibody +T835 PR:000015417 25918 25922 SOX1 +T836 UBERON:0002435 25935 25943 striatal +T837 UBERON:0001883 25983 25985 OT +T838 CL:0000540 25986 25993 neurons +T839 UBERON:0002435 26021 26029 striatal +T840 UBERON:0005403 26052 26054 VS +T841 PR:000015417 26118 26122 SOX1 +T842 GO:0010467 26123 26133 expression +T843 UBERON:0005403 26176 26178 VS +T844 UBERON:0001883 26179 26181 OT +T845 CL:0000540 26182 26188 neuron +T846 GO:0048665 26182 26207 neuron fate specification +T847 PR:000015417 26244 26248 SOX1 +T848 GO:0007067 26256 26263 mitotic +T849 PR:000015417 26303 26307 Sox1 +T850 PR:000015426 26308 26312 Sox2 +T851 GO:0010467 26313 26323 Expression +T852 CL:0000540 26327 26334 Neurons +T853 UBERON:0005403 26376 26378 VS +T854 NCBITaxon:10088 26402 26406 mice +T855 PR:000015417 26421 26425 Sox1 +T856 PR:000015417 26439 26443 Sox1 +T857 PR:000015417 26450 26454 Sox1 +T858 SO:0001023 26465 26472 alleles +T859 GO:0001764 26506 26518;26541 26548 migration of ... neurons +T860 PR:000015426 26523 26527 Sox2 +T861 UBERON:0004025 26537 26540 LGE +T862 CL:0000540 26541 26548 neurons +T863 NCBITaxon:10088 26596 26600 mice +T864 PR:000015426 26614 26618 Sox2 +T865 SO:0001023 26622 26628 allele +T866 PR:000015417 26671 26675 Sox1 +T867 PR:000015426 26676 26680 Sox2 +T868 UBERON:0004025 26697 26700 LGE +T869 CL:0000540 26701 26708 neurons +T870 UBERON:0005403 26725 26727 VS +T871 PR:000015417 26738 26742 Sox1 +T872 SO:0001023 26754 26761 alleles +T873 NCBITaxon:10088 26781 26785 mice +T874 CHEBI:75055 26796 26801 X-gal +T875 CL:0000540 26827 26834 neurons +T876 NCBITaxon:10088 26879 26883 mice +T877 PR:000015417 26900 26904 Sox1 +T878 SO:0001023 26907 26914 alleles +T879 GO:0010467 26929 26939 expression +T880 PR:000015426 26966 26970 Sox2 +T881 SO:0001023 26972 26978 allele +T882 UBERON:0004025 27001 27004 LGE +T883 NCBITaxon:10088 27014 27018 mice +T884 CL:0000540 27040 27047 neurons +T885 UBERON:0001883 27081 27083 OT +T886 UBERON:0001883 27184 27186 OT +T887 PR:000015417 27187 27191 SOX1 +T888 CL:0000540 27192 27199 neurons +T889 GO:0010467 27240 27250 expression +T890 PR:000015417 27254 27258 Sox1 +T891 CL:0000540 27262 27269 neurons +T892 UBERON:0004025 27277 27280 LGE +T893 UBERON:0001883 27328 27330 OT +T894 PR:000015417 27360 27364 Sox1 +T895 GO:0048665 27383 27407 specification of neurons +T896 CL:0000540 27400 27407 neurons +T897 UBERON:0000204 27415 27436 ventral telencephalon +T898 GO:0010467 27504 27513 expressed +T899 GO:0008283 27524 27537 proliferating +T900 CL:0000540 27643 27651 neuronal +T901 UBERON:0000204 27676 27697 ventral telencephalon +T902 GO:0010467 27711 27721 expression +T903 GO:0001764 27811 27823;27845 27852 migration of ... neurons +T904 CL:0000540 27845 27852 neurons +T905 UBERON:0005403 27873 27875 VS +T906 PR:000015417 27884 27888 SOX1 +T907 GO:0010467 27889 27899 expression +T908 GO:0007067 27939 27946 mitotic +T909 GO:0001764 27987 27996;28014 28016;28023 28030 migration ... of ... neurons +T910 CL:0000540 28023 28030 neurons +T911 GO:0010467 28045 28055 expressing +T912 PR:000012318 28056 28060 Pax6 +T913 PR:000015417 28092 28096 SOX1 +T914 UBERON:0005403 28106 28108 VS +T915 CL:0000540 28109 28116 neurons +T916 PR:000015417 28135 28139 SOX1 +T917 CL:0000540 28153 28160 neurons +T918 GO:0065007 28164 28171 control +T919 CL:0000540 28315 28322 neurons +T920 GO:0001764 28338 28358 Migration of Neurons +T921 CL:0000540 28351 28358 Neurons +T922 UBERON:0005403 28366 28368 VS +T923 GO:0001764 28410 28430 migration of neurons +T924 CL:0000540 28423 28430 neurons +T925 UBERON:0000204 28438 28459 ventral telencephalon +T926 GO:0010467 28497 28507 expression +T927 CL:0000540 28543 28550 neurons +T928 UBERON:0005403 28559 28561 VS +T929 UBERON:0005382 28566 28581 dorsal striatum +T930 PR:000015417 28602 28606 SOX1 +T931 UBERON:0005403 28648 28650 VS +T932 CL:0000540 28651 28658 neurons +T933 UBERON:0001883 28699 28701 OT +T934 UBERON:0001881 28707 28725 islands of Calleja +T935 UBERON:0001882 28735 28752 nucleus accumbens +T936 CL:0000540 28808 28815 neurons +T937 GO:0010467 28816 28826 expressing +T938 PR:000015417 28827 28831 Sox1 +T939 GO:0007567 28883 28888 natal +T940 UBERON:0001893 28950 28963 telencephalon +T941 CL:0000540 28981 28988 neurons +T942 PR:000015417 29073 29077 SOX1 +T943 CL:0000540 29095 29102 neurons +T944 UBERON:0005403 29110 29112 VS +T945 PR:000015417 29134 29138 Sox1 +T946 GO:0010467 29139 29149 expressing +T947 CL:0000540 29150 29157 neurons +T948 UBERON:0001883 29165 29167 OT +T949 UBERON:0001881 29176 29194 islands of Calleja +T950 PR:000015417 29203 29207 SOX1 +T951 UBERON:0001882 29277 29294 nucleus accumbens +T952 GO:0010467 29319 29328 expresses +T953 CL:0000540 29339 29346 neurons +T954 UBERON:0002435 29363 29371 striatal +T955 GO:0007608 29387 29396 olfactory +T956 UBERON:0002894 29387 29403 olfactory cortex +T957 GO:0010467 29416 29423 express +T958 PR:000015417 29424 29428 Sox1 +T959 CL:0000540 29478 29485 neurons +T960 UBERON:0005403 29497 29499 VS +T961 CL:0000540 29580 29587 neurons +T962 UBERON:0001883 29611 29613 OT +T963 GO:0007608 29637 29646 olfactory +T964 UBERON:0002894 29637 29653 olfactory cortex +T965 GO:0010467 29659 29668 expresses +T966 PR:000012318 29669 29673 Pax6 +T967 PR:000015417 29683 29687 Sox1 +T968 PR:000015417 29707 29711 SOX1 +T969 CL:0000540 29719 29726 neurons +T970 UBERON:0001883 29798 29800 OT +T971 CL:0000540 29801 29808 neurons +T972 PR:000015417 29853 29857 Sox1 +T973 CL:0000540 29863 29870 neurons +T974 GO:0010467 29891 29898 express +T975 PR:000015417 29925 29929 Sox1 +T976 GO:0010467 29930 29940 expressing +T977 UBERON:0001883 29941 29943 OT +T978 CL:0000540 29944 29951 neurons +T979 CL:0000540 30013 30020 neurons +T980 UBERON:0005403 30138 30140 VS +T981 PR:000015417 30159 30163 SOX1 +T982 CL:0000540 30190 30198 neuronal +T983 PR:000015417 30310 30314 SOX1 +T984 NCBITaxon:33208 30325 30332 animals +T985 PR:000015417 30340 30344 SOX1 +T986 SO:0000857 30402 30410 homology +T987 SO:0000417 30479 30486 domains +T988 GO:0010467 30504 30513 expressed +T989 UBERON:0004025 30566 30569 LGE +T990 PR:000015417 30594 30598 SOX1 +T991 GO:0048665 30606 30622;30629 30636 specification of ... neurons +T992 UBERON:0001883 30623 30625 OT +T993 UBERON:0005403 30626 30628 VS +T994 CL:0000540 30629 30636 neurons +T995 SO:0000704 30669 30674 genes +T996 GO:0010467 30688 30698 expression +T997 UBERON:0034705 30706 30721 neuroepithelium +T998 UBERON:0003053 30796 30798 VZ +T999 UBERON:0003053 30821 30823 VZ +T1000 UBERON:0004025 30831 30834 LGE +T1001 GO:0065007 30839 30849 controlled +T1002 UBERON:0004025 30869 30872 LGE +T1003 PR:Q24533 30912 30920 Dichaete +T1004 NCBITaxon:7215 30930 30940 Drosophila +T1005 SO:0000855 30941 30950 orthologs +T1006 NCBITaxon:7742 30958 30968 vertebrate +T1007 SO:0000704 30981 30986 genes +T1008 NCBITaxon:7215 31068 31078 Drosophila +T1009 SO:0000704 31090 31095 genes +T1010 SO:0000704 31115 31126 genetically +T1011 SO:0000704 31169 31174 genes +T1012 CL:0000031 31193 31203 neuroblast +T1013 UBERON:0000934 31224 31243 ventral nerve chord +T1014 NCBITaxon:7742 31268 31278 vertebrate +T1015 SO:0000855 31279 31288 orthologs +T1016 PR:000008308 31308 31312 Gsh1 +T1017 PR:000011242 31327 31333 Nkx2.2 +T1018 NCBITaxon:10088 31361 31366 mouse +T1019 PR:000008309 31368 31372 Gsh2 +T1020 GO:0010467 31376 31385 expressed +T1021 UBERON:0003053 31393 31395 VZ +T1022 UBERON:0004922 31396 31399 SVZ +T1023 PR:000015417 31410 31414 Sox1 +T1024 UBERON:0005403 31451 31453 VS +T1025 CL:0000540 31454 31461 neurons +T1026 SO:0000704 31473 31477 gene +T1027 NCBITaxon:7215 31602 31612 Drosophila +T1028 UBERON:0004025 31650 31653 LGE +T1029 PR:000008308 31665 31669 GSH1 +T1030 UBERON:0000204 31722 31743 ventral telencephalic +T1031 CL:0000540 31744 31752 neuronal +T1032 CL:0000540 31768 31775 neurons +T1033 UBERON:0005403 31783 31785 VS +T1034 UBERON:0002435 31829 31837 striatal +T1035 GO:0007567 31916 31921 natal +T1036 UBERON:0004025 31933 31936 LGE +T1037 UBERON:0001883 31966 31968 OT +T1038 UBERON:0005403 31969 31971 VS +T1039 UBERON:0004025 31979 31982 LGE +T1040 PR:000008309 32071 32075 Gsh2 +T1041 NCBITaxon:10088 32081 32085 mice +T1042 UBERON:0001883 32116 32118 OT +T1043 CL:0000540 32119 32126 neurons +T1044 UBERON:0004025 32168 32171 LGE +T1045 GO:0010467 32201 32211 expression +T1046 PR:000006527 32215 32219 Dlx1 +T1047 UBERON:0004025 32225 32228 LGE +T1048 PR:000015417 32251 32255 Sox1 +T1049 NCBITaxon:10088 32261 32265 mice +T1050 CL:0000540 32271 32279 neuronal +T1051 PR:000008309 32316 32320 Gsh2 +T1052 UBERON:0001883 32371 32373 OT +T1053 UBERON:0005403 32374 32376 VS +T1054 CL:0000540 32377 32384 neurons +T1055 CHEBI:472552 32395 32399 BrdU +T1056 UBERON:0004025 32413 32416 LGE +T1057 PR:000008309 32463 32467 Gsh2 +T1058 PR:000006527 32469 32473 Dlx1 +T1059 PR:000004362 32477 32482 Mash1 +T1060 PR:000012318 32488 32492 Pax6 +T1061 PR:000015417 32497 32501 Sox1 +T1062 UBERON:0000955 32507 32513 brains +T1063 CHEBI:75055 32590 32595 X-gal +T1064 PR:000015417 32605 32609 Sox1 +T1065 CHEBI:472552 32634 32638 BrdU +T1066 CL:0000540 32647 32654 neurons +T1067 UBERON:0003037 32697 32703 septum +T1068 UBERON:0002435 32712 32720 striatum +T1069 UBERON:0005403 32750 32752 VS +T1070 UBERON:0001883 32753 32755 OT +T1071 CL:0000540 32756 32763 neurons +T1072 UBERON:0005403 32782 32784 VS +T1073 GO:0010467 32850 32860 expression +T1074 PR:000016819 32864 32868 TuJ1 +T1075 CL:0000540 32891 32898 neurons +T1076 PR:000015417 32938 32942 SOX1 +T1077 PR:000015417 33008 33012 SOX1 +T1078 CL:0000540 33052 33059 neurons +T1079 UBERON:0005403 33094 33096 VS +T1080 PR:000015417 33167 33171 Sox1 +T1081 CL:0000540 33177 33184 neurons +T1082 PR:000015417 33250 33254 SOX1 +T1083 CL:0000540 33264 33271 neurons +T1084 UBERON:0005403 33355 33357 VS +T1085 CL:0000540 33358 33364 Neuron +T1086 PR:000015417 33395 33399 Sox1 +T1087 GO:0010467 33400 33410 expression +T1088 CL:0000540 33414 33421 neurons +T1089 GO:0007067 33479 33486 mitotic +T1090 GO:0010467 33505 33515 expression +T1091 PR:000015417 33519 33523 Sox1 +T1092 UBERON:0004025 33549 33552 LGE +T1093 CL:0000540 33553 33560 neurons +T1094 PR:000015426 33562 33566 Sox2 +T1095 NCBITaxon:10088 33570 33574 mice +T1096 GO:0010467 33575 33582 express +T1097 PR:000015417 33583 33587 Sox1 +T1098 PR:000015426 33604 33608 Sox2 +T1099 SO:0001023 33609 33616 alleles +T1100 PR:000015426 33627 33631 Sox2 +T1101 SO:0001023 33633 33639 allele +T1102 NCBITaxon:33208 33654 33661 animals +T1103 PR:000015417 33681 33685 Sox1 +T1104 SO:0001023 33696 33703 alleles +T1105 PR:000015417 33712 33716 SOX1 +T1106 GO:0010467 33717 33727 expression +T1107 PR:000015426 33743 33747 SOX2 +T1108 UBERON:0005403 33765 33767 VS +T1109 UBERON:0001883 33768 33770 OT +T1110 CL:0000540 33810 33817 neurons +T1111 NCBITaxon:10088 33842 33846 mice +T1112 UBERON:0005403 33884 33886 VS +T1113 UBERON:0001883 33887 33889 OT +T1114 CL:0000540 33890 33897 neurons +T1115 PR:000015417 33964 33968 Sox1 +T1116 NCBITaxon:10088 33974 33978 mice +T1117 UBERON:0001883 33986 33988 OT +T1118 NCBITaxon:10088 33999 34003 mice +T1119 PR:000015417 34029 34033 Sox1 +T1120 GO:0010467 34144 34154 expression +T1121 PR:000015417 34158 34162 Sox1 +T1122 PR:000015426 34172 34176 Sox2 +T1123 SO:0000167 34177 34185 promoter +T1124 UBERON:0001883 34261 34263 OT +T1125 UBERON:0005403 34264 34266 VS +T1126 CL:0000540 34267 34273 neuron +T1127 NCBITaxon:10088 34294 34298 mice +T1128 GO:0010467 34325 34335 expression +T1129 PR:000015417 34339 34343 SOX1 +T1130 UBERON:0000955 34397 34402 brain +T1131 UBERON:0001883 34412 34414 OT +T1132 PR:000015417 34445 34449 SOX1 +T1133 GO:0042571 34450 34458 antibody +T1134 NCBITaxon:33208 34485 34491 animal +T1135 PR:000015417 34527 34531 SOX1 +T1136 GO:0010467 34605 34615 expression +T1137 PR:000015417 34619 34623 SOX1 +T1138 UBERON:0003053 34639 34641 VZ +T1139 UBERON:0004922 34642 34645 SVZ +T1140 UBERON:0000922 34649 34656 embryos +T1141 PR:000015417 34674 34678 Sox1 +T1142 GO:0010467 34694 34703 expressed +T1143 PR:000015426 34713 34717 Sox2 +T1144 PR:000015417 34766 34770 Sox1 +T1145 SO:0001023 34771 34777 allele +T1146 PR:000015417 34781 34785 Sox1 +T1147 UBERON:0005403 34851 34853 VS +T1148 UBERON:0001883 34854 34856 OT +T1149 PR:000015417 34875 34879 Sox1 +T1150 GO:0010467 34880 34890 expression +T1151 GO:0007067 34898 34905 mitotic +T1152 UBERON:0004025 34973 34976 LGE +T1153 GO:0007067 34981 34988 mitotic +T1154 NCBITaxon:10088 35003 35007 mice +T1155 PR:000015417 35022 35026 SOX1 +T1156 GO:0010467 35027 35037 expression +T1157 PR:000015426 35047 35051 Sox2 +T1158 SO:0001023 35053 35059 allele +T1159 UBERON:0005403 35075 35077 VS +T1160 CL:0000540 35108 35115 neurons +T1161 UBERON:0005403 35174 35176 VS +T1162 GO:0048665 35202 35236 specification of neuronal identity +T1163 CL:0000540 35219 35227 neuronal +T1164 CL:0000540 35278 35286 neuronal +T1165 PR:000015417 35351 35355 SOX1 +T1166 UBERON:0000955 35392 35397 brain +T1167 GO:0065007 35448 35458 controlled +T1168 GO:0010467 35466 35476 expression +T1169 GO:0007067 35509 35516 mitotic +T1170 PR:000015417 35585 35589 SOX1 +T1171 GO:0010467 35590 35600 expression +T1172 NCBITaxon:10088 35620 35625 mouse +T1173 GO:0010467 35678 35688 expression +T1174 SO:0000704 35705 35710 genes +T1175 PR:000015417 35735 35739 SOX1 +T1176 GO:0007067 35771 35778 mitotic +T1177 GO:0030154 35779 35799 cell differentiation +T1178 GO:0007067 35893 35900 mitotic +T1179 GO:0010467 35922 35932 expression +T1180 SO:0000704 35974 35978 Gene +T1181 SO:0000704 36000 36004 gene +T1182 PR:000015417 36027 36031 Sox1 +T1183 SO:0000147 36039 36043 exon +T1184 PR:000015417 36173 36177 SOX1 +T1185 GO:0043631 36319 36334 polyadenylation +T1186 SO:0000551 36319 36341 polyadenylation signal +T1187 UBERON:0000479 36344 36350 Tissue +T1188 CHEBI:465284 36426 36437 gancyclovir +T1189 SO:0001644 36466 36482 targeting vector +T1190 SO:0000167 36531 36540 promoters +T1191 PR:000015417 36583 36587 Sox1 +T1192 GO:0010467 36604 36613 expressed +T1193 UBERON:0000922 36617 36626 embryonic +T1194 CL:0002322 36617 36631;36637 36642 embryonic stem ... cells +T1195 CL:0002322 36633 36635;36637 36642 ES ... cells +T1196 CHEBI:42768 36673 36677 G418 +T1197 CL:0002322 36810 36817 ES cell +T1198 UBERON:0001062 36902 36912 anatomical +T1199 NCBITaxon:10088 36946 36950 mice +T1200 SO:0000704 36960 36967 genetic +T1201 PR:000015417 36980 36984 Sox1 +T1202 NCBITaxon:10088 36991 36995 mice +T1203 GO:0007618 37001 37006 mated +T1204 NCBITaxon:10088 37012 37016 mice +T1205 PR:000015417 37074 37078 Sox1 +T1206 PR:000015417 37089 37093 Sox1 +T1207 GO:0010467 37109 37116 express +T1208 PR:000015426 37143 37147 Sox2 +T1209 SO:0000440 37160 37166 vector +T1210 PR:000015417 37257 37261 Sox1 +T1211 PR:000015417 37286 37290 SOX1 +T1212 SO:0000236 37291 37309 open reading frame +T1213 SO:0000357 37314 37321 flanked +T1214 SO:0000346 37325 37329 LoxP +T1215 SO:0005853 37330 37339 cassettes +T1216 SO:0000243 37366 37370 IRES +T1217 SO:0000610 37376 37381 polyA +T1218 SO:0000155 37392 37399 plasmid +T1219 SO:0000440 37466 37472 vector +T1220 CL:0002322 37522 37530 ES cells +T1221 CL:0002322 37634 37642 ES cells +T1222 NCBITaxon:33208 37742 37749 animals +T1223 PR:000015426 37763 37767 Sox2 +T1224 SO:0001023 37776 37782 allele +T1225 PR:000015426 37811 37815 Sox2 +T1226 GO:0010467 37818 37827 expressed +T1227 PR:000015417 37828 37832 Sox1 +T1228 PR:000015426 37848 37852 Sox2 +T1229 GO:0010467 37865 37874 expressed +T1230 PR:000015417 37893 37897 SOX1 +T1231 PR:000015426 37921 37925 Sox2 +T1232 SO:0001023 37934 37940 allele +T1233 GO:0045120 37958 37968 pronuclear +T1234 SO:0000155 37996 38003 plasmid +T1235 GO:0010467 38004 38014 expressing +T1236 SO:0001023 38126 38132 allele +T1237 PR:000015426 38136 38140 Sox2 +T1238 PR:000015426 38169 38173 Sox2 +T1239 CHEBI:75055 38238 38243 X-gal +T1240 GO:0097617 38265 38278 hybridization +T1241 UBERON:0007023 38330 38335 adult +T1242 NCBITaxon:10088 38336 38341 mouse +T1243 UBERON:0000955 38342 38348 brains +T1244 PR:000015417 38407 38411 Sox1 +T1245 UBERON:0000922 38440 38447 embryos +T1246 UBERON:0000922 38511 38520 embryonic +T1247 UBERON:0000955 38521 38526 brain +T1248 UBERON:0000922 38564 38573 embryonic +T1249 UBERON:0000955 38574 38579 brain +T1250 PR:000006527 38617 38621 Dlx1 +T1251 PR:000013040 38635 38639 Brn4 +T1252 SO:0000440 38725 38731 vector +T1253 UBERON:0000955 38990 38996 brains +T1254 CHEBI:472552 39042 39046 BrdU +T1255 CHEBI:75958 39068 39076 solution +T1256 CHEBI:472552 39080 39084 BrdU +T1257 CHEBI:75958 39166 39174 solution +T1258 UBERON:0001179 39244 39261 peritoneal cavity +T1259 GO:0007565 39265 39273 pregnant +T1260 NCBITaxon:10088 39274 39278 mice +T1261 UBERON:0000955 39349 39355 Brains +T1262 UBERON:0000955 39417 39423 brains +T1263 CHEBI:73702 39492 39495 wax +T1264 GO:0042571 39652 39662 antibodies +T1265 PR:000012318 39702 39706 PAX6 +T1266 PR:000008309 39741 39745 GSH2 +T1267 PR:000016819 39818 39822 TuJ1 +T1268 PR:000015417 39893 39897 SOX1 +T1269 PR:000015426 39906 39910 SOX2 +T1270 PR:000015428 39923 39927 SOX3 +T1271 PR:000004362 39962 39967 MASH1 +T1272 PR:000013121 40022 40030 DARPP-32 +T1273 UBERON:0005291 40112 40128 embryonic tissue +T1274 UBERON:0000955 40244 40250 brains +T1275 CHEBI:17992 40307 40314 sucrose +T1276 NCBITaxon:9925 40464 40468 goat +T1277 UBERON:0001977 40469 40474 serum +T1278 CHEBI:9750 40484 40496 Triton X-100 +T1279 GO:0042571 40550 40560 antibodies +T1280 CHEBI:75958 40585 40593 solution +T1281 GO:0042571 40684 40694 antibodies +T1282 CHEBI:37926 40696 40700 FITC +T1283 CHEBI:75958 40738 40746 solution +T1284 PR:000015417 40840 40844 SOX1 +T1285 CHEBI:51766 40884 40892 Alexa568 +T1286 NCBITaxon:9925 40893 40897 goat +T1287 NCBITaxon:9986 40903 40909 rabbit +T1288 GO:0042571 40910 40918 antibody +T1289 NCBITaxon:9986 41016 41022 rabbit +T1290 GO:0042571 41033 41041 antibody +T1291 PR:000015417 41136 41140 SOX1 +T1292 GO:0042571 41141 41149 antibody +T1293 PR:000015426 41201 41205 SOX2 +T1294 GO:0042571 41206 41214 antibody +T1295 CHEBI:75958 41227 41235 solution +T1296 CHEBI:9750 41244 41256 Triton X-100 +T1297 CHEBI:52661 41306 41314 Alexa488 +T1298 NCBITaxon:9925 41315 41319 goat +T1299 NCBITaxon:9986 41325 41331 rabbit +T1300 GO:0042571 41332 41340 antibody +T1301 PR:000015417 41396 41400 SOX1 +T1302 PR:000015426 41405 41409 SOX2 +T1303 GO:0042571 41410 41420 antibodies +T1304 UBERON:0000479 41480 41487 tissues +T1305 PR:000015417 41494 41498 SOX1 +T1306 PR:000015426 41515 41519 SOX2 +T1307 GO:0042571 41593 41601 antibody +T1308 GO:0042571 41689 41699 antibodies +T1309 PR:000008309 41961 41965 GSH2 +T1310 PR:000012318 41966 41970 PAX6 +T1311 PR:000015417 42035 42039 SOX1 +T1312 GO:0010467 42092 42102 expression +T1313 PR:000008309 42106 42110 GSH2 +T1314 PR:000012318 42123 42127 PAX6 +T1315 UBERON:0000955 42180 42186 brains +T1316 CHEBI:51231 42241 42245 DAPI +T1317 GO:0005634 42253 42260 nuclear +T1318 UBERON:0001851 42268 42270 Cx +T1319 UBERON:0001851 42272 42278 cortex +T1320 UBERON:0004025 42280 42283 lge +T1321 UBERON:0004025 42285 42312 lateral ganglionic eminence +T1322 UBERON:0004024 42314 42317 mge +T1323 UBERON:0004024 42319 42345 medial ganglionic eminence +T1324 UBERON:0005403 42472 42474 VS +T1325 PR:000012318 42475 42479 Pax6 +T1326 GO:0010467 42480 42490 Expressing +T1327 CL:0000540 42491 42498 Neurons +T1328 PR:000015417 42517 42521 SOX1 +T1329 UBERON:0000204 42523 42544 Ventral telencephalic +T1330 UBERON:0000955 42563 42568 brain +T1331 GO:0042571 42591 42601 antibodies +T1332 PR:000012318 42603 42607 PAX6 +T1333 UBERON:0007023 42637 42642 adult +T1334 PR:000015417 42654 42658 SOX1 +T1335 PR:000012318 42671 42675 PAX6 +T1336 PR:000015417 42698 42702 SOX1 +T1337 PR:000012318 42748 42752 Pax6 +T1338 GO:0010467 42753 42763 expressing +T1339 UBERON:0001883 42792 42794 OT +T1340 UBERON:0000204 42809 42830 ventral telencephalon +T1341 PR:000015417 42845 42849 Sox1 +T1342 NCBITaxon:10088 42854 42858 mice +T1343 PR:000015417 42881 42885 Sox1 +T1344 PR:000012318 42929 42933 Pax6 +T1345 GO:0010467 42934 42944 expressing +T1346 PR:000012318 42952 42956 PAX6 +T1347 PR:000015417 42961 42965 SOX1 +T1348 GO:0010467 42973 42982 expressed +T1349 GO:0007067 43012 43019 mitotic +T1350 PR:000015417 43156 43160 Sox1 +T1351 GO:0010467 43161 43171 Expressing +T1352 UBERON:0003053 43172 43174 VZ +T1353 CL:0000681 43191 43203 Radial Glial +T1354 GO:0042571 43268 43276 antibody +T1355 UBERON:0000955 43295 43300 brain +T1356 NCBITaxon:10088 43314 43318 mice +T1357 PR:000015417 43332 43336 Sox1 +T1358 SO:0001023 43341 43347 allele +T1359 UBERON:0001851 43356 43364 cortical +T1360 UBERON:0003053 43365 43381 ventricular zone +T1361 GO:0005737 43409 43420 cytoplasmic +T1362 PR:000015417 43440 43444 SOX1 +T1363 GO:0010467 43445 43455 expressing +T1364 CL:0000681 43480 43492 radial glial +T1365 CL:0000681 43591 43602 Radial Glia +T1366 UBERON:0004025 43610 43613 LGE +T1367 PR:000015417 43646 43650 SOX1 +T1368 UBERON:0000955 43660 43665 brain +T1369 PR:000015417 43698 43702 Sox1 +T1370 NCBITaxon:10088 43714 43718 mice +T1371 GO:0042571 43750 43758 antibody +T1372 CL:0000681 43762 43773 radial glia +T1373 PR:000015417 43894 43898 SOX1 +T1374 PR:000015426 43903 43907 SOX2 +T1375 GO:0005634 43920 43926 Nuclei +T1376 UBERON:0004025 43934 43937 LGE +T1377 UBERON:0003053 43938 43940 VZ +T1378 UBERON:0000955 44000 44005 brain +T1379 UBERON:0004025 44035 44038 LGE +T1380 PR:000015417 44052 44056 SOX1 +T1381 PR:000015426 44071 44075 SOX2 +T1382 GO:0042571 44086 44096 antibodies +T1383 GO:0005634 44195 44202 nuclear +T1384 UBERON:0004025 44429 44432 LGE +T1385 PR:000015417 44471 44475 SOX1 +T1386 CHEBI:472552 44477 44481 BrdU +T1387 UBERON:0004025 44639 44642 LGE +T1388 UBERON:0003053 44644 44646 VZ +T1389 UBERON:0004922 44647 44650 SVG +T1390 UBERON:0000203 44663 44670 pallial +T1391 UBERON:0003053 44671 44673 VZ +T1392 UBERON:0000955 44816 44821 brain +T1393 UBERON:0000479 44906 44912 tissue +T1394 GO:0008283 44962 44975 proliferation +T1395 UBERON:0004025 44987 44990 LGE +T1396 UBERON:0003053 44991 44993 VZ +T1397 UBERON:0004922 44994 44997 SVZ +T1398 CHEBI:472552 45014 45018 BrdU +T1399 UBERON:0004025 45083 45086 LGE +T1400 UBERON:0003053 45087 45089 VZ +T1401 UBERON:0004922 45090 45093 SVZ +T1402 CHEBI:51686 45124 45135 hematoxylin +T1403 CHEBI:472552 45168 45172 BrdU +T1404 UBERON:0000203 45195 45202 pallial +T1405 UBERON:0003053 45203 45205 VZ +T1406 UBERON:0004922 45206 45209 SVZ +T1407 NCBITaxon:10088 45262 45266 mice +T1408 PR:000013040 45502 45506 Brn4 +T1409 PR:000006527 45520 45524 Dlx1 +T1410 PR:000015417 45556 45560 Sox1 +T1411 PR:000015426 45578 45582 Sox2 +T1412 GO:0042571 45628 45638 antibodies +T1413 NCBITaxon:10088 46033 46037 mice +T1414 NCBITaxon:10088 46085 46089 mice +T1415 CHEBI:22695 46374 46379 basic +T1416 SO:0001114 46380 46385 helix +T1417 SO:0001114 46391 46396 helix +T1418 CHEBI:472552 46398 46402 BrdU +T1419 CHEBI:472552 46405 46428 5-bromo-2′-deoxyuridine +T1420 UBERON:0001017 46430 46433 CNS +T1421 UBERON:0001017 46436 46458 central nervous system +T1422 PR:000013121 46460 46468 DARPP-32 +T1423 CHEBI:18243 46471 46479 dopamine +T1424 PR:000013121 46471 46513 dopamine and cAMP-regulated phosphoprotein +T1425 CHEBI:17489 46484 46488 cAMP +T1426 GO:0065007 46489 46498 regulated +T1427 UBERON:0000922 46527 46536 embryonic +T1428 UBERON:0000922 46556 46565 embryonic +T1429 SO:0000243 46572 46576 IRES +T1430 SO:0000243 46579 46608 internal ribosomal entry site +T1431 GO:0005840 46588 46597 ribosomal +T1432 UBERON:0004025 46610 46613 LGE +T1433 UBERON:0004025 46616 46643 lateral ganglionic eminence +T1434 UBERON:0001883 46645 46647 OT +T1435 GO:0007608 46650 46659 olfactory +T1436 UBERON:0001883 46650 46668 olfactory tubercle +T1437 GO:0007567 46686 46691 natal +T1438 UBERON:0004922 46706 46709 SVZ +T1439 UBERON:0004922 46712 46731 subventricular zone +T1440 PR:000016819 46733 46737 TuJ1 +T1441 PR:000016819 46745 46757 βIII-tubulin +T1442 GO:0042571 46758 46766 antibody +T1443 UBERON:0003053 46768 46770 VZ +T1444 UBERON:0003053 46773 46789 ventricular zone +T1445 UBERON:0005403 46791 46793 VS +T1446 UBERON:0005403 46796 46812 ventral striatum +T1447 NCBITaxon:10088 46876 46881 Mouse +T1448 PR:000015417 46882 46886 Sox1 +T1449 SO:0001023 46891 46897 Allele +T1450 PR:000015417 46925 46929 SOX1 +T1451 UBERON:0005403 46952 46954 VS +T1452 CL:0000540 46955 46962 Neurons +T1453 PR:000015417 46998 47002 Sox1 +T1454 PR:P23940 47091 47092 B +T1455 PR:P23940 47094 47099 BamHI +T1456 PR:000015417 47136 47140 SOX1 +T1457 SO:0000147 47141 47145 exon +T1458 SO:0001026 47227 47234 genomic +T1459 GO:0097617 47240 47250 hybridized +T1460 CHEBI:75055 47314 47319 X-gal +T1461 PR:000015417 47324 47328 SOX1 +T1462 GO:0042571 47329 47337 antibody +T1463 PR:000015417 47350 47354 Sox1 +T1464 PR:000015417 47376 47380 Sox1 +T1465 GO:0010467 47385 47395 expression +T1466 CHEBI:75055 47410 47415 X-gal +T1467 PR:000015417 47464 47468 Sox1 +T1468 SO:0000704 47469 47473 gene +T1469 PR:000015417 47516 47520 SOX1 +T1470 GO:0042571 47521 47529 antibody +T1471 UBERON:0000922 47568 47575 embryos +T1472 UBERON:0000204 47623 47644 ventral telencephalon +T1473 CHEBI:75055 47707 47712 X-gal +T1474 PR:000015417 47736 47740 Sox1 +T1475 SO:0000167 47741 47749 promoter +T1476 PR:000015417 47771 47775 Sox1 +T1477 UBERON:0001890 47782 47791 forebrain +T1478 GO:0007567 47813 47818 birth +T1479 GO:0016477 47839 47851;47868 47873 migration of ... cells +T1480 PR:000015417 47852 47856 Sox1 +T1481 GO:0010467 47857 47867 expressing +T1482 UBERON:0003053 47883 47885 VZ +T1483 UBERON:0001883 47905 47907 OT +T1484 UBERON:0002435 47919 47927 striatal +T1485 UBERON:0007651 47928 47935 bridges +T1486 PR:000015417 47960 47964 Sox1 +T1487 UBERON:0001890 47972 47981 forebrain +T1488 CHEBI:75055 48002 48007 X-gal +T1489 UBERON:0001883 48024 48026 OT +T1490 UBERON:0002435 48035 48043 striatal +T1491 UBERON:0007651 48044 48051 bridges +T1492 UBERON:0000935 48077 48096 anterior commissure +T1493 PR:000015417 48191 48195 Sox1 +T1494 CL:0000540 48201 48208 Neurons +T1495 CHEBI:75055 48210 48215 X-gal +T1496 NCBITaxon:10088 48228 48233 mouse +T1497 UBERON:0001890 48234 48244 forebrains +T1498 UBERON:0001890 48275 48284 forebrain +T1499 PR:000015417 48371 48375 Sox1 +T1500 NCBITaxon:10088 48382 48386 mice +T1501 PR:000015417 48405 48409 Sox1 +T1502 NCBITaxon:10088 48417 48421 mice +T1503 UBERON:0001883 48474 48476 OT +T1504 CHEBI:75055 48512 48517 X-gal +T1505 UBERON:0000955 48569 48574 brain +T1506 UBERON:0002435 48594 48602 striatum +T1507 UBERON:0003037 48607 48613 septum +T1508 UBERON:0001881 48686 48704 islands of Calleja +T1509 UBERON:0001882 48706 48708 an +T1510 UBERON:0001882 48710 48727 accumbens nucleus +T1511 UBERON:0000119 48741 48752 cell layers +T1512 UBERON:0001883 48760 48762 OT +T1513 UBERON:0001881 48777 48795 islands of Calleja +T1514 GO:0007608 48810 48819 olfactory +T1515 UBERON:0002265 48810 48825 olfactory tract +T1516 UBERON:0002667 48827 48830 lsn +T1517 UBERON:0002667 48832 48854 lateral septal nucleus +T1518 UBERON:0002264 48856 48858 ob +T1519 GO:0007608 48860 48869 olfactory +T1520 UBERON:0002264 48860 48874 olfactory bulb +T1521 UBERON:0002590 48876 48878 PC +T1522 GO:0007608 48880 48889 olfactory +T1523 UBERON:0002590 48880 48907 olfactory (piriform) cortex +T1524 UBERON:0002435 48909 48910 S +T1525 UBERON:0002435 48912 48920 striatum +T1526 UBERON:0002435 48926 48934 striatal +T1527 UBERON:0007651 48935 48941 bridge +T1528 GO:0008283 48990 49003 Proliferation +T1529 GO:0022008 49008 49020 Neurogenesis +T1530 UBERON:0001883 49033 49035 OT +T1531 CL:0000540 49036 49044 Neuronal +T1532 PR:000015417 49079 49083 SOX1 +T1533 UBERON:0000955 49093 49098 brain +T1534 UBERON:0000204 49117 49138 ventral telencephalon +T1535 PR:000015417 49162 49166 Sox1 +T1536 UBERON:0000922 49178 49185 embryos +T1537 PR:000016819 49187 49191 TuJ1 +T1538 CL:0000540 49253 49261 neuronal +T1539 UBERON:0000922 49278 49285 embryos +T1540 GO:0097617 49295 49308 hybridization +T1541 PR:000013040 49320 49324 Brn4 +T1542 UBERON:0001883 49420 49422 OT +T1543 UBERON:0000955 49452 49457 brain +T1544 UBERON:0001883 49477 49479 OT +T1545 UBERON:0001893 49481 49494 Telencephalic +T1546 PR:000015417 49535 49539 Sox1 +T1547 UBERON:0000922 49566 49575 embryonic +T1548 UBERON:0000955 49576 49582 brains +T1549 CHEBI:472552 49608 49612 BrdU +T1550 UBERON:0003053 49715 49717 VZ +T1551 UBERON:0004922 49718 49721 SVZ +T1552 CHEBI:472552 49764 49768 BrdU +T1553 UBERON:0018264 49841 49851 dorsal LGE +T1554 GO:0008283 49916 49929 proliferation +T1555 GO:0008283 49980 49993 proliferation +T1556 UBERON:0000955 50021 50027 brains +T1557 CHEBI:472552 50070 50074 BrdU +T1558 CL:0000540 50283 50290 Neurons +T1559 UBERON:0005403 50309 50311 VS +T1560 PR:000015417 50330 50334 SOX1 +T1561 CHEBI:472552 50354 50358 BrdU +T1562 GO:0008283 50371 50384 proliferating +T1563 UBERON:0001890 50409 50418 forebrain +T1564 UBERON:0001883 50505 50507 OT +T1565 CHEBI:472552 50543 50547 BrdU +T1566 CHEBI:472552 50663 50667 BrdU +T1567 UBERON:0001883 50826 50828 OT +T1568 GO:0007608 50864 50873 olfactory +T1569 UBERON:0002894 50864 50880 olfactory cortex +T1570 CL:0000540 50896 50903 neurons +T1571 GO:0007608 50932 50941 olfactory +T1572 UBERON:0002590 50932 50959 olfactory (piriform) cortex +T1573 UBERON:0001883 50997 50999 OT +T1574 UBERON:0002435 51008 51016 striatal +T1575 UBERON:0007651 51017 51024 bridges +T1576 CL:0000540 51047 51054 neurons +T1577 UBERON:0005403 51081 51083 VS +T1578 UBERON:0001881 51175 51193 islands of Calleja +T1579 UBERON:0004025 51302 51305 LGE +T1580 PR:000015417 51335 51339 SOX1 +T1581 UBERON:0000955 51386 51391 brain +T1582 UBERON:0000203 51404 51410;51423 51436 dorsal ... telencephalic +T1583 UBERON:0000204 51415 51436 ventral telencephalic +T1584 PR:000015417 51468 51472 Sox1 +T1585 UBERON:0000922 51484 51491 embryos +T1586 PR:000012318 51493 51497 PAX6 +T1587 PR:000008309 51502 51506 GSH2 +T1588 UBERON:0018264 51534 51544 dorsal LGE +T1589 GO:0010467 51579 51589 expression +T1590 PR:000015417 51617 51621 SOX1 +T1591 PR:000012318 51657 51661 PAX6 +T1592 PR:000015417 51733 51737 SOX1 +T1593 PR:000012318 51738 51742 PAX6 +T1594 UBERON:0000955 51756 51761 brain +T1595 PR:000012318 51791 51795 PAX6 +T1596 PR:000015417 51807 51811 Sox1 +T1597 UBERON:0000955 51818 51823 brain +T1598 UBERON:0005403 51832 51834 VS +T1599 PR:000012318 51873 51877 PAX6 +T1600 CL:0000540 51887 51894 neurons +T1601 UBERON:0005403 51914 51916 VS +T1602 PR:000015417 51924 51928 Sox1 +T1603 UBERON:0000955 51935 51940 brain +T1604 PR:000004362 51953 51958 MASH1 +T1605 UBERON:0004025 51986 51989 LGE +T1606 PR:000015417 52011 52015 Sox1 +T1607 UBERON:0000955 52021 52026 brain +T1608 PR:000006527 52096 52100 Dlx1 +T1609 GO:0010467 52101 52111 expressing +T1610 GO:0097617 52142 52155 hybridization +T1611 UBERON:0000955 52197 52203 brains +T1612 PR:000015426 52279 52283 SOX2 +T1613 PR:000015428 52288 52292 SOX3 +T1614 UBERON:0004025 52312 52315 LGE +T1615 CL:0000540 52316 52323 Neurons +T1616 PR:000015417 52328 52332 SOX1 +T1617 PR:000015426 52333 52337 SOX2 +T1618 GO:0010467 52341 52351 Expression +T1619 UBERON:0004025 52355 52358 LGE +T1620 UBERON:0004025 52413 52416 LGE +T1621 UBERON:0000922 52468 52475 embryos +T1622 GO:0042571 52513 52521 antibody +T1623 PR:000015417 52552 52556 SOX1 +T1624 PR:000015426 52581 52585 SOX2 +T1625 PR:000015428 52599 52603 SOX3 +T1626 PR:000015417 52626 52630 SOX1 +T1627 PR:000015426 52641 52645 SOX2 +T1628 UBERON:0001883 52688 52690 OT +T1629 UBERON:0004025 52704 52707 LGE +T1630 CL:0000540 52731 52738 neurons +T1631 GO:0010467 52739 52749 expressing +T1632 PR:000015417 52750 52754 SOX1 +T1633 PR:000015426 52770 52774 SOX2 +T1634 PR:000015428 52789 52793 SOX3 +T1635 GO:0010467 52821 52831 expression +T1636 PR:000015417 52839 52843 SOX1 +T1637 PR:000015426 52848 52852 SOX2 +T1638 PR:000015426 52977 52981 Sox2 +T1639 SO:0001023 52983 52989 Allele +T1640 PR:000015417 52999 53003 SOX1 +T1641 PR:000015426 53007 53011 Sox2 +T1642 GO:0010467 53021 53031 Expression +T1643 PR:000015426 53084 53088 SOX2 +T1644 PR:000015417 53116 53120 SOX1 +T1645 SO:0000243 53125 53129 IRES +T1646 PR:000015417 53215 53219 Sox1 +T1647 SO:0000346 53247 53257 LoxP sites +T1648 SO:0000243 53281 53285 IRES +T1649 SO:0001026 53368 53375 genomic +T1650 GO:0097617 53381 53391 hybridized +T1651 PR:000015417 53552 53556 SOX1 +T1652 UBERON:0000922 53601 53608 embryos +T1653 PR:000015426 53620 53624 Sox2 +T1654 PR:000015426 53642 53646 Sox2 +T1655 GO:0010467 53670 53680 expression +T1656 PR:000015417 53684 53688 SOX1 +T1657 UBERON:0001894 53696 53708 diencephalon +T1658 UBERON:0005870 53730 53739 nasal pit +T1659 UBERON:0005870 53741 53743 np +T1660 UBERON:0005403 53784 53786 VS +T1661 CL:0000540 53787 53794 Neurons +T1662 NCBITaxon:10088 53812 53816 Mice +T1663 GO:0010467 53822 53832 Expressing +T1664 PR:000015417 53833 53837 SOX1 +T1665 SO:0001023 53853 53859 Allele +T1666 CHEBI:75055 53868 53873 X-gal +T1667 UBERON:0000204 53912 53933 ventral telencephalon +T1668 NCBITaxon:10088 53940 53944 mice +T1669 PR:000015426 54002 54006 Sox2 +T1670 GO:0010467 54007 54017 expressing +T1671 UBERON:0001883 54018 54020 OT +T1672 CL:0000540 54021 54028 neurons +T1673 PR:000015417 54034 54038 SOX1 +T1674 PR:000015417 54054 54058 SOX1 +T1675 PR:000015426 54071 54075 Sox2 +T1676 NCBITaxon:10088 54079 54083 mice +T1677 PR:000015417 54103 54107 Sox1 +T1678 SO:0001023 54108 54115 alleles +T1679 CL:0000540 54177 54184 neurons +T1680 GO:0010467 54185 54195 expressing +T1681 PR:000015426 54196 54200 Sox2 +T1682 PR:000015417 54216 54220 Sox1 +T1683 GO:0010467 54250 54260 expression +T1684 PR:000013121 54332 54340 DARPP-32 +T1685 UBERON:0000204 54385 54406 ventral telencephalon +T1686 PR:000015426 54414 54418 Sox2 +T1687 PR:000015417 54426 54430 Sox1 +T1688 NCBITaxon:10088 54457 54461 mice +T1689 GO:0001764 54507 54519;54523 54530 migration of ... neurons +T1690 UBERON:0001883 54520 54522 OT +T1691 CL:0000540 54523 54530 neurons +T1692 UBERON:0000935 54532 54534 AC +T1693 UBERON:0000935 54536 54555 anterior commissure +T1694 PR:000015417 54588 54592 Sox1 +T1695 GO:0010467 54593 54603 Expression +T1696 UBERON:0001883 54657 54659 OT +T1697 UBERON:0005403 54660 54662 VS +T1698 CL:0000540 54663 54670 Neurons +T1699 CHEBI:75055 54678 54683 X-gal +T1700 UBERON:0000204 54722 54743 ventral telencephalon +T1701 PR:000015417 54752 54756 Sox1 +T1702 GO:0010467 54762 54772 expressing +T1703 UBERON:0001883 54773 54775 OT +T1704 CL:0000540 54788 54795 neurons +T1705 UBERON:0000922 54809 54816 embryos +T1706 PR:000015417 54836 54840 Sox1 +T1707 SO:0001023 54841 54847 allele +T1708 PR:000015417 54849 54853 Sox1 +T1709 PR:000015417 54879 54883 Sox1 +T1710 PR:000015417 54910 54914 Sox1 +T1711 PR:000015426 54923 54927 Sox2 +T1712 CHEBI:75055 54960 54965 X-gal +T1713 CL:0000540 54974 54981 neurons +T1714 UBERON:0005403 55001 55003 VS +T1715 PR:000015426 55048 55052 Sox2 +T1716 SO:0001023 55054 55060 allele +T1717 UBERON:0001883 55071 55073 OT +T1718 CL:0000540 55074 55080 neuron +T1719 UBERON:0000922 55105 55112 embryos +T1720 GO:0010467 55114 55124 Expression +T1721 PR:000015426 55142 55146 Sox2 +T1722 SO:0001023 55148 55154 allele +T1723 CHEBI:75055 55210 55215 X-gal +T1724 PR:000015417 55253 55257 SOX1 +T1725 UBERON:0000922 55280 55287 embryos +T1726 UBERON:0000955 55325 55331 brains +T1727 PR:000015417 55383 55387 SOX1 +T1728 GO:0010467 55388 55398 expression +T1729 GO:0010467 55460 55469 expressed +T1730 PR:000015426 55479 55483 Sox2 +T1731 SO:0001023 55485 55491 allele +T1732 PR:000015417 55527 55531 Sox1 +T1733 SO:0001023 55542 55549 alleles +T1734 PR:000015417 55557 55561 Sox1 +T1735 GO:0010467 55615 55625 expression +T1736 UBERON:0001883 55667 55669 OT +T1737 CL:0000540 55670 55677 neurons +T1738 PR:000013121 55690 55697 DARPP32 +T1739 UBERON:0000955 55724 55729 brain +T1740 PR:000015417 55744 55748 Sox1 +T1741 PR:000015426 55752 55756 Sox2 +T1742 PR:000015417 55768 55772 Sox1 +T1743 PR:000015426 55778 55782 Sox2 +T1744 NCBITaxon:10088 55794 55798 mice +T1745 UBERON:0005403 55818 55820 VS +T1746 CL:0000540 55821 55828 neurons +T1747 UBERON:0001883 55855 55857 OT +T1748 UBERON:0000935 55879 55898 anterior commissure +T1749 UBERON:0002894 55900 55902 OC +T1750 GO:0007608 55904 55913 olfactory +T1751 UBERON:0002894 55904 55920 olfactory cortex +T1752 PR:000015417 55976 55980 Sox1 +T1753 GO:0010467 55981 55991 Expression +T1754 GO:0007067 55999 56006 mitotic +T1755 UBERON:0004025 56007 56010 LGE +T1756 CL:0000540 56035 56043 Neuronal +T1757 GO:0001764 56035 56053 Neuronal Migration +T1758 UBERON:0005403 56061 56063 VS +T1759 CHEBI:75055 56065 56070 X-gal +T1760 UBERON:0000204 56109 56130 ventral telencephalon +T1761 NCBITaxon:10088 56137 56141 mice +T1762 GO:0001764 56157 56177 migration of neurons +T1763 CL:0000540 56170 56177 neurons +T1764 GO:0010467 56178 56188 expressing +T1765 PR:000015417 56189 56193 SOX1 +T1766 PR:000015426 56203 56207 Sox2 +T1767 SO:0001023 56209 56215 allele +T1768 PR:000015417 56272 56276 Sox1 +T1769 SO:0000704 56287 56292 genes +T1770 UBERON:0002435 56320 56328 striatal +T1771 UBERON:0007651 56329 56336 bridges +T1772 NCBITaxon:10088 56353 56357 mice +T1773 CHEBI:33893 56623 56631 reagents +T1774 CL:0000034 56699 56708 Stem Cell +T1775 http://purl.obolibrary.org/obo/MONDO_0000001 56760 56768 Diseases +T1776 CL:0000540 57045 57053 Neuronal +T1777 GO:0001764 57045 57063 Neuronal migration +T1778 UBERON:0001893 57100 57113 telencephalon +T1779 PR:000015417 57124 57128 SOX1 diff --git a/src/ontogpt/evaluation/craft/database/all/15882093.txt b/src/ontogpt/evaluation/craft/database/all/15882093.txt new file mode 100644 index 000000000..2013eb5f1 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15882093.txt @@ -0,0 +1,325 @@ +Neuronal Migration and Ventral Subtype Identity in the Telencephalon Depend on SOX1 + +Abstract + +Little is known about the molecular mechanisms and intrinsic factors that are responsible for the emergence of neuronal subtype identity. Several transcription factors that are expressed mainly in precursors of the ventral telencephalon have been shown to control neuronal specification, but it has been unclear whether subtype identity is also specified in these precursors, or if this happens in postmitotic neurons, and whether it involves the same or different factors. SOX1, an HMG box transcription factor, is expressed widely in neural precursors along with the two other SOXB1 subfamily members, SOX2 and SOX3, and all three have been implicated in neurogenesis. SOX1 is also uniquely expressed at a high level in the majority of telencephalic neurons that constitute the ventral striatum (VS). These neurons are missing in Sox1-null mutant mice. In the present study, we have addressed the requirement for SOX1 at a cellular level, revealing both the nature and timing of the defect. By generating a novel Sox1-null allele expressing β-galactosidase, we found that the VS precursors and their early neuronal differentiation are unaffected in the absence of SOX1, but the prospective neurons fail to migrate to their appropriate position. Furthermore, the migration of non-Sox1-expressing VS neurons (such as those expressing Pax6) was also affected in the absence of SOX1, suggesting that Sox1-expressing neurons play a role in structuring the area of the VS. To test whether SOX1 is required in postmitotic cells for the emergence of VS neuronal identity, we generated mice in which Sox1 expression was directed to all ventral telencephalic precursors, but to only a very few VS neurons. These mice again lacked most of the VS, indicating that SOX1 expression in precursors is not sufficient for VS development. Conversely, the few neurons in which Sox1 expression was maintained were able to migrate to the VS. In conclusion, Sox1 expression in precursors is not sufficient for VS neuronal identity and migration, but this is accomplished in postmitotic cells, which require the continued presence of SOX1. Our data also suggest that other SOXB1 members showing expression in specific neuronal populations are likely to play continuous roles from the establishment of precursors to their final differentiation. + +Introduction + +The telencephalon is subdivided into dorsal (pallial) and ventral (subpallial) territories, which give rise to the cerebral cortex and the underlying basal ganglia, respectively. The embryonic subpallium consists of large protrusions—the ganglionic eminences. Several distinct types of neurons originate in the ganglionic eminences, and some migrate as far as the olfactory bulb, hippocampus, and neocortex [1–3], while others contribute more locally. The majority of neurons of the lateral ganglionic eminence (LGE) form the dorsal and ventral striatum (VS). The VS includes the caudate, putamen, nucleus accumbens, and olfactory tubercle (OT), which control various aspects of motor, cognitive, and emotional functions [4,5]. Little is known about the molecular mechanisms that control the emergence of various groups of neurons with distinct identities in this region. + +Gene-expression studies and loss-of-function mutations in homeodomain transcription factors such as PAX6 [6,7] and GSH2/1 [8–13] confirm fate-mapping findings [14–16] that the majority of the VS neurons are specified within the progenitor domain of the LGE. The proneural basic helix-loop-helix (bHLH) factor MASH1 also marks the precursors of early-born neurons in the LGE progenitor domain, and its loss in the mouse leads to a deficit of both precursors and neurons of the telencephalon, including loss of VS neurons [17,18]. Therefore, GSH2 and MASH1 control VS precursor patterning and specification, but as they are not expressed in postmitotic cells it remained unknown to what extent they are involved in the emergence of neuronal subtypes in the ventral telencephalon, and whether different transcription factors with neuron-specific expression are required. + +The SOX proteins constitute a family of transcription factors [19,20] that regulate transcription through their ability to bind to specific DNA sequences via their HMG box domains [21–24]. There are 20 Sox genes in mammals, and at least half are expressed in the developing nervous system [20,24]; however, their role in neural development is poorly understood. SOX1, SOX2, and SOX3 constitute the SOXB1 subfamily and share more than 95% identity within their HMG boxes and significant homology outside [25,26]. All three proteins are expressed in the neuroepithelium throughout central nervous system (CNS) development [25,27], and as they tend to be down-regulated upon neural differentiation they have been used as markers for neural stem cells and precursors [28,29]. Several studies suggest that SOXB1 factors function in stem cells and precursors to maintain broad developmental potential [30] and neural stem cell identity [30–32] by counteracting neurogenesis. Contradictory evidence, however, suggests that SOX1 promotes neurogenesis and cell cycle exit [33]. However, mice that are null for Sox1 [34] or Sox3 [35], or mice with one Sox2 allele deleted and the other hypomorphic [36], exhibit phenotypes associated with the loss of or functional deficit of only specific neuronal populations. As these SOXB1 factors are expressed in both precursors and neurons that are affected in these mutant mice, it was not known whether their function is required in precursors, postmitotic cells, or both. + +We have previously shown that SOX1 is essential for the terminal differentiation of lens fibers and the activation of γ-crystallins [37], and for the development of VS neurons, the lack of which is associated with epilepsy [34]. Here, we show that absence of SOX1 has no effect on the generation, proliferation, and patterning of neuronal precursors. This is probably due to functional compensation by SOX2 and SOX3, which are co-expressed with SOX1 in precursors. Moreover, mice lacking only the neuron-specific expression of Sox1 in the ventral telencephalon still fail to develop VS neurons, revealing its requirement within these neurons. Consistent with this, maintenance of Sox1 expression in neurons of the ventral telencephalon is sufficient to direct them to the VS, confirming the adequacy of SOX1 function in postmitotic cells for their migration and identity. Therefore, VS-specific neuronal migration and subtype identity most likely is initiated in precursors but is completed in postmitotic cells by transcription factors such as SOX1. + +Results + +SOX1 Is Essential for the Histogenesis of the VS + +To generate a detailed map of Sox1 expression in the developing and adult brain of mice, and to perform comparative studies between homozygotes and heterozygotes, we generated a novel targeted allele referred to as Sox1βgeo. This contains an insertion of β-galactosidase-neo (βgeo) fusion protein in-frame with the SOX1 open reading frame (Figure 1A). Mice homozygous for Sox1βgeo are null for SOX1 and exhibit the same phenotype as the previously described mice, which carry a deletion of the SOX1 coding region (Sox1M1) [34,37], namely, lens defects and epileptic seizures. Staining for β-galactosidase activity (X-gal staining) in Sox1βgeo/+ heterozygous embryos matches that for the wild-type allele as revealed by whole-mount in situ hybridization and SOX1 antibody staining (Figure 1B–1E). + +To elucidate the role of SOX1 in the formation of the VS, we compared the expression pattern of Sox1 in heterozygous (Figure 1F–1I) and homozygous (Figure 1J–1M) brains from embryonic day 14 (E14) to postnatal day 0 (P0) using X-gal staining. Throughout much of the CNS, X-gal staining in the Sox1βgeo/βgeo homozygotes is double the intensity observed in heterozygotes (data not shown). To perform comparative histological studies, we equalized the levels of X-gal staining in homozygous mice with those of heterozygous animals by generating homozygous mice that harbor two different Sox1-null alleles: βgeo (Sox1βgeo) and the previously described M1-targeted allele (Sox1M1) [37], which does not express β-galactosidase. + +Our analysis shows (via X-gal staining) that in the developing forebrain, Sox1 is expressed throughout the ventricular zone (VZ) and subventricular zone (SVZ) and in neurons around the anterior commissure region, where the prospective nucleus accumbens forms (red arrowheads in Figure 1K and 1M), and in the striatal bridges that link this intermediate cluster of cells with the prospective OT region toward the pial surface. X-gal-positive neurons start populating the OT area as early as E14 and continue to accumulate at least until birth (Figure 1F–1I). In the Sox1βgeo/M1-null mutants, the X-gal staining pattern of the VZ/SVZ is indistinguishable from that of the Sox1βgeo/+ heterozygotes, and there is no obvious deficit of X-gal-positive cells around the anterior commissure. On the other hand, both the striatal bridges and the OT layers are absent in the Sox1-null brain at all developmental stages (compare Figure 1F–1I to 1J–1M). It is unlikely that neurons die en masse in this region, because an apoptosis assay did not reveal any evidence of increased cell death in the mutant (data not shown). In addition, X-gal staining is increased throughout the ventral telencephalon in the mutant postnatal brain (red arrowheads in Figure 2), suggesting that the Sox1-null cells are not correctly specified and contribute to other brain regions. Interestingly, although neurons that form the core of the nucleus accumbens express Sox1 highly, they form normally (Figure 2F and red arrowheads in Figure 1) and do not depend on SOX1 for their development. Therefore, SOX1 is required for histogenesis of the OT throughout its development. + +Normal Precursor Proliferation and Neurogenesis but Loss of OT Neuronal Differentiation in the Absence of SOX1 + +Studies with conflicting results suggest that SOX1 either, like SOX2 and SOX3, counteracts neurogenesis [32] or, unlike SOX2 and SOX3, promotes neurogenesis [33]. To examine whether the loss of SOX1 affects general neural differentiation in the area of the striatum, we used an anti-βIII-tubulin (TuJ1) antibody, which is a marker for immature neurons [38], at E13, a critical time of differentiation in the LGE. TuJ 1 immunocytochemistry did not reveal any obvious general differentiation problems in the Sox1 mutants (Figure 3A and 3B), suggesting that loss of SOX1 alone is not sufficient to compromise general neuron differentiation and maturity. The differentiation and distribution of specific mature neurons was examined in our previous study at adult stages with the expression of striatal markers such as preproenkephalin and Gad65/67 [34], and in our current study, at embryonic stages with additional markers such as Brn4 [39] (Figure 3C and 3D) and Robo [40] (Figure 3E and 3F). This analysis revealed a differentiation defect restricted in the region of the nucleus accumbens/OT, and not the rest of the striatum. + +As Sox1 expression is associated with dividing cells throughout the neuroepithelium, we examined whether a proliferation defect could partially account for the cell deficit in the ventral telencephalon. We used 5-bromo-2′-deoxyuridine (BrdU) to label all proliferating precursors in wild-type and Sox1-null embryos at E13–E15, and harvested their brains 1 h later. Most of the dividing cells were found in the VZ/SVZ, and there was no increase or ectopic proliferation (Figure 3G–3L; Table S1). Therefore, SOX1 is unlikely to be required for proliferation or the exit of precursors from the cell cycle in the LGE. Collectively, the above data show that SOX1 is not essential for the proliferation of precursors and general neuronal differentiation. However, it is required specifically for the differentiation and/or migration of VS neurons. + +Early- and Late-Born OT Neurons Fail to Migrate in the Absence of SOX1 + +To investigate the possibility that neurons migrate in the Sox1-null OT regions, but are not visible because they do not express Sox1βgeo and other differentiation markers, we exposed embryos to BrdU. This way, we permanently marked all proliferating precursors independently of Sox1 or other striatal-marker gene expression and followed them at later embryonic stages and after birth (Figure 4). Labeling the precursors at different embryonic days also provided information on the birthdates of the OT neurons, which were previously unknown for the mouse. Specifically, birth of ventral striatal neurons commences early at E13 and continues until birth (Figure 4A, 4C, 4E, and 4G; data not shown) and is consistent with data from the rat [41,42]. Furthermore, in wild-type embryos, BrdU exposure between E13 and E16 with examination of embryos either 72 h later (Figure 4A; data not shown) or after birth (Figure 4C, 4E, and 4G) showed that early-born neurons migrate more laterally than those born later. + +In the Sox1-null embryos, the presence and distribution of cells labeled with BrdU between E13 and E16 shows that the olfactory cortex is largely normal (bracket in Figure 4D), but in the VS area the number of labeled cells was found to be greatly reduced (Figure 4B, 4D, 4F, and 4H). Furthermore, in the Sox1-null postnatal brain the striatal mantle is more densely populated by BrdU-labeled cells (Figure 4D–4H), consistent with the general increase of X-gal staining observed throughout the striatum (see Figure 2). The above data suggest that in the absence of SOX1, early- and late-born neurons fail to migrate to the appropriate position to form the ventral areas of the striatum. + +The Generation and Patterning of LGE Precursors Is Normal in the Absence of SOX1 + +It is known that the majority of VS neurons derive from precursors that are born in the LGE [10,15,43]. To investigate whether the defect is in the patterning of precursors, we examined the expression pattern of various transcription factors that mark LGE progenitors and are known to have a role in OT neuronal specification (Figures 5, S1, and S2). + +The homeodomain transcription factors PAX6 and GSH2 are expressed, respectively, in the pallial and subpallial precursor domains of the dorsal LGE. The boundary between them has been shown to be essential for the patterning of VS precursors [10,11]. Specifically, GSH2-null mice do not form early-born OT neurons, and the precursors of the dorsal LGE are lost as PAX6 expands ventrally into the LGE. However, loss of both PAX6 and GSH2 restores dorsoventral patterning and partially rescues OT formation [9–11]. Using double antibody immunohistochemistry in embryos at E12–E16, we found that loss of SOX1 has no effect on the expression of GSH2/PAX6 and the boundary in the dorsal LGE (Figures 5A, 5B, and S1). In addition, at this boundary, PAX6-expressing postmitotic cells form a stream (arrow in Figure 5A) that extends laterally to the VS (Figures 5A, 5C, S2E, and S2F) [6,7,44]. In the absence of SOX1, the stream of PAX6-positive postmitotic cells is normal (arrow in Figures 5B and S2F), but to characterize the PAX6- and SOX1-expressing neurons in the region of the OT, we used double antibody immunohistochemistry. Specifically, for the wild-type brain sections we used PAX6 and SOX1 antibodies, but to visualize the Sox1-expressing cells in the Sox1βgeo/M1-null brain sections we used an antibody for β-galactosidase. + +Our data showed that PAX6 and SOX1 proteins were co-expressed in progenitors (Figures S2E and S2F), but in postmitotic cells of the LGE this expression became mutually exclusive (Figure 5C and 5D). Pax6-expressing neurons were clustered laterally to those expressing Sox1, at the border between the OT and olfactory cortex (Figures 5C, S2A, and S2C). In Sox1-null mice, the postmitotic Pax6-expressing cells were distributed throughout the VS area (Figures 5D, S2B, and S2D), suggesting structural disorganization. It is unlikely that these ectopically localized Pax6-expressing neurons are mis-specified Sox1-null neurons, because they should be expressing both PAX6 and βgeo from the mutant Sox1βgeo allele, but they do not (Figure 5D). + +The LGE structure consists of neural progenitors with radial glial characteristics, having fibers that extend from the VZ to the pial surface. These cells also provide the substrate for the migration of neurons [45,46]. Staining with X-gal (see Figure 1) and β-galactosidase antibody in mice carrying the Sox1βgeo allele allowed visualization of the cytoplasmic compartment of the SOX1-expressing progenitors, which have radial glial morphology (Figures S2F and S3). We examined the morphology of radial glia in the Sox1-null LGE using the RC2 antibody [47], but we did not find any difference from wild-type (Figure S4). Therefore, we conclude that the abnormal distribution of Pax6-expressing neurons in the LGE of the Sox1-null mice is unlikely to be caused by abnormal morphology of the radial glial fibers or substantial loss of radial glia-like precursors. + +In the ventral telencephalon, the bHLH transcription factor MASH1 [18] and the homeodomain factor DLX1 mark LGE precursors [8]. Ablation of MASH1 in mice causes loss of specific subpopulations of precursors and striatal neurons that contribute to the OT and nucleus accumbens [18]. In addition, Gsh2-null mice, which also fail to develop the OT, exhibit reduced Dlx1 expression in LGE precursors [9]. We therefore examined the expression of these two genes in Sox1-null embryos, but found no difference (Figure 5E–5H), indicating that there is no deficit of early or late LGE precursors in the absence of SOX1. Collectively, the above data show that SOX1 is not required for patterning, generation, and maintenance of LGE precursors. + +Sox1 Expression from the Endogenous Sox2 Promoter Can Be Tolerated In Vivo + +It has been shown that all three SoxB1 genes are expressed [25,27] and share similar functions in neural precursors [31,32]. However, in the postmitotic cells of the mantle and the VS area, antibody staining for each of the genes at E15 indicated that SOX2- and SOX3-positive neurons represent a very small population compared to that expressing SOX1 (Figure 6A–6C). Therefore, it is likely that the other SoxB1 genes compensate for the loss of Sox1 in precursors, whereas they cannot do so in the LGE postmitotic cells. Nevertheless, it remained unknown whether SOX1 functions solely in precursors for VS fate specification or in postmitotic cells for maintaining this fate and the emergence of specific subtype identity and migration. To address this, we generated mice that express Sox1 mainly in precursors and not in LGE neurons. We took advantage of the fact that Sox2 is co-expressed with Sox1 in precursors but it is down-regulated in LGE neurons, and generated mice that express Sox1 from the endogenous Sox2 promoter. We confirmed the overlap of Sox1 and Sox2 expression in the VZ/SVZ of the LGE by staining serial coronal telencephalic sections with antibodies for each of the two genes and counter-staining with the nuclear stain TOTO at E14 (Figure S5), and by performing double anti-SOX1 and -SOX2 immunohistochemistry at E13 (Figure 6D–6L). + +The replacement of the SOX2 open reading frame with that of SOX1 was achieved by targeting the Sox2 allele (Figure 7A). The new allele, Sox2R, was engineered to express not only Sox1 but also βgeo via an internal ribosomal entry site (IRES). Furthermore, the coding region of SOX1 in the Sox2R allele was flanked by LoxP sites, which can be deleted using Cre-mediated recombination [48]. In this way, we generated a mouse line carrying another allele, termed Sox2βgeo2 (but referred to hereafter as Sox2βgeo), which expresses only the βgeo reporter gene from the Sox2 promoter (Figure 7A) and not SOX1. Like the Sox2βgeo/+ heterozygotes, Sox2R/+ mice were viable, fertile, and phenotypically normal, indicating that SOX1 over-expression in precursors, as well as ectopic expression in other locations where Sox2 is uniquely expressed, does not cause any obvious developmental abnormality. + +X-gal staining of embryos showed that both targeted Sox2 alleles (Sox2βgeo and Sox2R) express βgeo in the CNS, but to verify that SOX1 protein was produced from the Sox2R allele, we used SOX1 antibody staining. We found that SOX1 protein was ectopically present at sites where SOX2 normally shows unique expression—for example, in the floor plate of the diencephalon (arrowheads in Figure 7B and 7C) and in the sensory placodes (arrows in Figure 7D and 7E). The intensity of the immunostaining at ectopic sites was comparable to the staining in areas with expression from two wild-type Sox1 alleles (VZ/SVZ), indicating that the level of expression was similar to the wild-type allele. Therefore, the Sox2R allele produces SOX1 ectopically in all neurons uniquely positive for SOX2 and increases the endogenous level of SOX1 in precursors and neurons that express both genes, without causing an obvious defect in mice. The fact that ectopic expression does not cause any obvious phenotype suggests either that the partner factors required for SOX1 target specificity [19] are absent in those cells uniquely expressing Sox2 or that the two proteins are interchangeable, sharing target genes. Further experiments are required to clarify this. + +Sox1 Over-Expression in Precursors Does Not Increase VS/OT Neuronal Fate Specification + +To investigate more subtle defects due to the over-expression of Sox1 in precursors of the Sox2R/+ mice, we examined several litters (n > 10) of mice and visualized the migration of the Sox1/Sox2-positive neurons in the VS via X-gal staining. Newborn mice carrying Sox2R/+ with two (Sox2R/+, Sox1+/+; Figure 8A) or one (Sox2R/+, Sox1M1/+; Figure 8B) Sox1 endogenous wild-type alleles were compared with those carrying Sox2βgeo/+, Sox1+/+ that do not express Sox1 ectopically (Figure 8C). All the above mice have only one wild-type Sox2 allele. We also compared the Sox1-βgeo and Sox2-βgeo neurons of the ventral telencephalon in Sox1βgeo/+ (Figure 8C and 8D) and Sox2R/+ (Figure 8A) mice, respectively. In this area of the brain, the Sox2-positive neurons are far fewer than those positive for Sox1. Therefore, for comparison purposes, we used thin tissue sections (80 μm) with short X-gal staining (3 h) for Sox1-βgeo, and thicker sections (100–150 μm) with a long staining period (48 h) for Sox2-βgeo. Consistent with the antibody staining data (see Figure 6B), Sox2-βgeo neurons contribute to the OT, indicating that they are a subset of the VS neuronal population. Therefore, the ectopic expression of Sox1 in LGE neurons is expected to be very limited. More importantly, the migration and the number of LGE neurons expressing βgeo via the Sox2 promoter were found to be the same regardless of the number of endogenous Sox1 alleles or the ectopic presence of Sox1 (compare Figure 8A, 8B, and 8C). To further investigate the differentiation of the VS neurons, we used the striatal-specific markers dopamine and cAMP-regulated phosphoprotein (DARPP-32) at postnatal stages and found them to be unaffected in Sox2R/+ mice (Figure 8E and 8F). The data therefore indicate that the over-expression of SOX1 in precursors does not increase OT neuronal specification. + +Sox1 Expression in Precursors Cannot Rescue OT Neuron Development + +To address directly whether SOX1 function is essential in precursors, we crossed Sox1M1/+, Sox2R/+ mice with Sox1βgeo/+, Sox2+/+ mice and examined whether offspring carrying Sox2R/+ without any wild-type Sox1 alleles (Sox1M1/βgeo) could develop OT. In these Sox1R/+ embryos that carry no endogenous Sox1 functional allele (termed here HoHe), SOX1 is expected to be expressed only via the Sox2R allele in precursors and become down-regulated in postmitotic LGE cells. However, βgeo expression from the endogenous Sox1 mutant allele marks precursors and OT prospective neurons. We followed the Sox1M1/βgeo prospective OT neurons with X-gal to determine whether they were capable of contributing to the OT in HoHe embryos (Figure 9). The Sox2R allele also expresses βgeo; however, in LGE postmitotic cells, Sox2-βgeo expression is much less than that of Sox1-βgeo and is not very visible by short (3 h) X-gal staining (only a slight increase of X-gal staining is seen in the VS; Figure 9C compared to Figure 9B). + +Each brain was split into left and right hemispheres, and coronal sections of the left were used for short staining with X-gal (Figure 9A–9C) whereas sections of the right were stained with SOX1 antibody (Figure 9D and 9E). The hemispheres stained for X-gal showed characteristic staining of OT neurons in heterozygous Sox1βgeo/+ mice (red arrowheads in Figure 9A), but the hemispheres of the Sox1-null embryos (Sox1βgeo/M1) with Sox2R/+ (Figure 9B) or without (Figure 9C) did not. This indicates that Sox1-null embryos do not develop OT despite the presence of the Sox2R allele and SOX1 protein in progenitors. To verify the presence of SOX1 protein in the precursors of the HoHe mice, we examined the other hemisphere that was stained with SOX1 antibody. In the Sox1βgeo/+ embryos, we found SOX1 present in the VZ (yellow arrows in Figure 9D and 9E) and the OT neurons (red arrowheads in Figure 9D). In Sox1βgeo/M1 (null) embryos, SOX1 expression was completely absent (data not shown); in the HoHe embryos, SOX1 protein was present in the VZ (yellow arrow in Figure 9E) and in very few neurons of the LGE (Figure 9E). HoHe mice, like the Sox1-nulls, are born with small eyes, and around weaning age develop seizures associated with lethality, which, if anything, is increased compared to that of Sox1-null mice (data not shown). In the brain of P10 HoHe mice, we used staining with DARPP-32 antibody (which is a SOX1-independent striatal marker) to investigate the recovery of OT neurons, and found staining in the striatal mantle but not in the VS (Figure 9G compared to Figure 9F). We therefore concluded that SOX1 expression in precursors is not sufficient to rescue VS/OT neuron fate specification, and that the continued presence of SOX1 in postmitotic cells is required for their identity. + +Sox1/Sox2 Expression in Neurons Is Sufficient for Their Migration to the VS + +We have shown that in mice carrying two (Sox1+/+), or one (Sox1M1/+), Sox1 wild-type alleles (see Figure 8A, 8B, and 8C), the migration of the Sox2-positive LGE neurons is not overtly different from that observed in mice carrying the Sox2R/+ allele. However, it remained unknown whether the Sox1/Sox2 double-positive LGE neurons migrated to the VS when both Sox1 endogenous alleles were missing (HoHe mice). We used X-gal staining to follow these neurons in several litters (n > 10), including HoHe mice, which have two Sox1M1 alleles and thus βgeo expression exclusively driven by the Sox2R allele. We found that in the LGE of these mice, the double-positive neurons are generated and migrate to the OT area (compare Figure 10A and 10B), but this area is compacted in the absence of the majority of the OT/SOX1 neurons. The above data show that the continued expression of Sox1 in neurons of the LGE is sufficient to direct their migration to the OT in the absence of endogenous Sox1. + +Discussion + +The specification of neurons in the ventral telencephalon has been shown to depend on several transcription factors that are expressed mainly in proliferating precursors. However, it was unknown to what degree specification in precursors included the emergence of neuronal subtype identity in the ventral telencephalon, and whether expression of additional transcription factors was required. We showed that the differentiation and migration of early- and late-born neurons that constitute the VS require SOX1 expression not only in precursors but also in postmitotic cells. Furthermore, in this region, the migration and organization of other neurons such as those expressing Pax6 also depend on the presence of SOX1-positive VS neurons. The finding that SOX1 functions in neurons to control migration and identity is novel and suggests that the other SOXB1 factors, in addition to their roles in precursors, have similar functions in neurons. + +Identity and Migration of Neurons in the VS + +The development of subtype identity and migration of neurons in the ventral telencephalon has not been well characterized. The expression of differentiation markers reveals neurons in both VS and dorsal striatum, but we showed that SOX1 specifically marks a large population of VS neurons that form the principal layer II of the OT, the islands of Calleja, and the nucleus accumbens (see Figures 1 and 2). In addition, we showed that the neurons expressing Sox1 are born continuously from E13 until the first postnatal week and that these migrate to a ventrolateral region of the telencephalon, with later-born neurons positioned progressively to more medial positions (see Figure 4). In the absence of SOX1, the majority of neurons of the VS fail to develop. All Sox1-expressing neurons of the OT and the islands of Calleja require SOX1 for their development, but it is essential only for the shell of the nucleus accumbens, although the core also expresses it. While neurons of the adjacent striatal mantle and the olfactory cortex that do not express Sox1 develop normally in its absence, other groups of neurons within the VS appear to be disorganized. Specifically, we identified a distinct population of neurons located lateral to the OT at the border with the olfactory cortex that expresses Pax6, but not Sox1. In the absence of SOX1, these neurons migrate into more medial positions, occupying the space of the missing OT neurons (see Figure 5). These are not mis-specified Sox1-null neurons because they do not express βgeo. This indicates that Sox1-expressing OT neurons play a non-cell-autonomous role in the organization of other neurons in this region, including the production of essential signals for migration. Most likely, the disorganization of the VS in the absence of SOX1 results in abnormal local neuronal connectivity, which in turn leads to the abnormal (epileptiform) electrophysiological behavior observed in the SOX1-deficient animals [34]. + +SOX1 Function in Precursors + +SOXB1 factors share considerable homology in both their DNA binding and C-terminal transcriptional activation domains, and they are co-expressed in precursors. It is therefore possible that in the LGE precursors, the role of SOX1 in the specification of OT/VS neurons is redundant. However, as SoxB1 genes have a broad expression in the neuroepithelium, we have to assume that their specific function at different areas of the VZ, and particularly the VZ of the LGE, is controlled by the presence of LGE-specific partner factors. SoxNeuro and Dichaete, the two Drosophila orthologs of the vertebrate SoxB1 group genes, also show overlapping functions during neural development [49]. Furthermore, in Drosophila, these two genes have been shown to genetically interact with the dorsoventral patterning genes ind (intermediate neuroblast defective) and vnd (ventral nerve chord defective) [50,51]. The vertebrate orthologs of ind and vnd are Gsh1/2 [52,53] and Nkx2.2 [54], respectively. In the mouse, Gsh2 is expressed in the VZ/SVZ, and like Sox1, its loss results in a reduction of VS neurons. As target gene specificity of SOX proteins depends on partnering with other transcription factors [20], our work, along with the data from Drosophila, supports the hypothesis that in the LGE precursors GSH1/2 may act as partners for SOXB1 factors to initiate ventral telencephalic neuronal identity. + +The neurons of the VS area occupy approximately a quarter of the striatal mass [55], and migrate there over a period of at least 10 d (E13 to first postnatal week). The LGE precursors that generate the OT/VS in the LGE are expected to have an equivalent representation during this period of development. In Gsh2-null mice, which are missing early-born OT neurons, there is a deficit of precursors in the LGE, readily seen by the reduced expression of Dlx1/2 in LGE precursors [9–11]. In Sox1-null mice, the neuronal deficit is more severe than that in Gsh2 mutants, as it includes both early- and late-born OT/VS neurons. However, BrdU labeling and LGE precursor-specific marker analysis, including Gsh2, Dlx1/2, Mash1, and Pax6, in Sox1-null brains at different stages did not show any deficit in precursors. The increase of X-gal-stained (Sox1-βgeo; see Figure 2) and BrdU-labeled neurons (see Figure 4D and 4F) in the area of the septum and the striatum supports the notion that the VS/OT neurons are born but lack VS subtype identity to migrate toward ventral positions. The normal expression of TuJ1, a marker of immature neurons, excluded the possibility that loss of SOX1 delays or enhances differentiation. Therefore, in the absence of SOX1, the precursors are there and generate neurons, but these fail to migrate to the VS because they assume different identity and position. The finding that Sox1-null neurons contribute widely to different areas argues that the presence of SOX1 provides neurons with ventral identity and the ability to migrate to ventral regions. + +Emergence of VS Neuron Identity + +To test the role of Sox1 expression in neurons and to determine whether ventral identity emerges in postmitotic cells, we limited expression of Sox1 largely to precursors of LGE neurons. Sox2R/+ mice express Sox1 from one of the Sox2 alleles. When the Sox2R allele is present in animals with no endogenous Sox1 wild-type alleles (HoHe), SOX1 expression mimics that of SOX2—being present in VS/OT precursors but largely absent from the neurons they give rise to. HoHe mice also fail to develop the majority of VS/OT neurons (see Figure 9) and exhibit an equally severe phenotype to that of Sox1-null mice in the OT. As these mice reproduce faithfully the Sox1-null phenotype without any evidence of a partial rescue, it is unlikely that this is the result of incomplete expression of Sox1 from the Sox2 promoter in the precursors. However, to exclude the possibility that the failure of OT/VS neuron development in HoHe mice was due to a low level of expression of SOX1 protein in precursors, we used one hemisphere of the brain to assay OT development and the other for SOX1 antibody staining, linking in each animal the phenotype with the presence of SOX1 protein in precursors. We found no difference in the extent and level of expression of SOX1 protein in the VZ/SVZ of embryos with one copy of Sox1, whether it is expressed from the Sox2 locus in HoHe (see Figure 9E) or the endogenous Sox1 allele in Sox1βgeo/+ heterozygotes (see Figure 9D). Therefore, the emergence of VS/OT identity requires Sox1 expression in postmitotic cells. Consistent with the above findings, the small population of LGE postmitotic cells in HoHe mice that maintain SOX1 expression from the Sox2R allele migrate to the VS. However, the number of these neurons is small and cannot rescue the deficit in the area of the VS. In conclusion, although specification of neuronal identity is initiated in precursors, emergence of neuronal subtype and ventral migration require the continued presence of SOX1. Our findings suggest that in other brain areas, subtype identity and migration may also be controlled by the expression of transcription factors in postmitotic cells. + +The current study, along with our previous one showing that SOX1 expression in the lens of the mouse is responsible for terminal differentiation and the expression of γ-crystallin genes [37], has revealed that SOX1 has important functions in postmitotic cell differentiation at two distinct sites. It is possible that the other SOXB1 factors have similar roles in postmitotic cells in which their expression is maintained. + +Materials and Methods + + + +Gene targeting. + +The βgeo gene was inserted into the Sox1 single exon as previously described [37]. The resulting targeted locus produces a fusion protein consisting of the first 50 amino acids from SOX1 (which excludes the HMG box) followed by ten amino acids that are encoded by a synthetic linker sequence, and the βgeo sequence (including a polyadenylation signal). Tissue culture was carried out as described before [37], omitting the addition of gancyclovir for negative selection. The targeting vector did not contain any other selectable markers or promoters, and the targeting frequency was 1/52. As Sox1 is not normally expressed in embryonic stem (ES) cells, we used the minimum level of G418 for selection. Positive recombinants were identified by Southern blotting, using a 3′ 1-kb external probe on an EcoRI digest. Three ES cell clones were obtained, and one was successfully passed through to the germ line. All anatomical investigations were performed on mice of mixed genetic background. Sox1βgeo/+ mice were mated with mice that were heterozygous for the previously described [37] Sox1 deletion (Sox1M1) and did not express β-galactosidase. + +For the Sox2 replacement vector, the 5′ and 3′ homologies used were the same as described before [30]. A SmaI-XhoI 2.2-kb Sox1 fragment containing the SOX1 open reading frame was flanked by LoxP cassettes followed by the NotI-SalI IRES–βgeo-polyA fragment (plasmid gift from Dr. A. Smith, University of Edinburgh). The replacement vector was linearized with SalI and electroporated into ES cells. Positive recombinants were identified by Southern blotting as described before [30]. Several targeted ES cells were isolated at a frequency of 1/20 and gave germ-line transmission of the mutation. Heterozygous animals carrying the Sox2Sox1βgeo allele (referred to in the text as Sox2R) expressed Sox1 and βgeo where Sox2 is normally expressed. + +Deletion of the SOX1 coding region from the Sox2Sox1βgeo allele was achieved via pronuclear injection of a supercoiled plasmid expressing Cre-recombinase (gift from Dr. K. Rajewsky, Harvard Medical School). Although in the text we refer to this new allele as Sox2βgeo, it is officially named Sox2βgeo2 to distinguish it from the one previously described [30]. + +X-gal staining and in situ hybridization. + +For β-galactosidase staining, fetal, newborn, or adult mouse brains were processed as previously described [30]. Detection of Sox1 mRNA was performed in whole embryos, as described previously [27,34]. The probes that were used on embryonic brain slices were generated by RT-PCR from embryonic brain cDNA. The position of the probes was Dlx1 (nt 41–573), Brn4 (nt 199–541), and Robo (nt 8–779). All fragments were cloned into a suitable cloning vector (pGET-easy, Promega, Madison, Wisconsin, United States), and were re-amplified using a sense oligonucleotide and an oligonucleotide upstream of either the T7 or SP6 sites. The resulting products were gel-purified, and 40 ng was used for probe synthesis. The brains were processed as described before [27,34]. + +BrdU labeling + +A 25 mg/ml solution of BrdU (Sigma, St. Louis, Missouri, United States) was made in PBS warmed to 37 °C. The solution was sterilized through a 0.2-μm syringe filter and injected into the peritoneal cavity of pregnant mice (0.1 ml per 25 g of body weight, to give a final dosage of 0.1 mg/g). Brains were harvested 1 or 72 h after the injection, or on P16. The brains were fixed in 4% PFA in PBS at 4 °C overnight, embedded in paraffin wax, and cut into 5-μm thick sections. The sections were processed for immunohistochemistry as previously described [18]. + +Immunohistochemistry. + +The source of antibodies and the dilutions used are as follows: PAX6, 1:10 (gift from Dr. J. Briscoe); GSH2, 1:5,000 (gift from Dr. K. Campbell); β-galactosidase 1:2,000 (Cappel); TuJ1, 1:1,000 (Novus Biologicals, Littleton, Colorado, United States); and SOX1, 1:500; SOX2, 1:500; and SOX3, 1:500 (gift from Dr. T. Edlund). MASH1, 1:2 (gift from Dr. F. Guillemot) [18], RC2 [56], and DARPP-32 [57] were used as previously described. For single or double immunofluorescence, embryonic tissue was fixed either for 1 h in 4% PFA in PBS at room temperature or for 15 min in MEMFA as described before [58]. The brains were then washed in PBS, cryoprotected overnight in 30% sucrose in PBS at 4 °C, embedded in OCT (Raymond Lamb), and cut in 10-μm and 15-μm sections using a cryostat. Sections were rehydrated in PBS, blocked in 4% goat serum and 0.1% Triton X-100 in PBS, and incubated overnight at 4 °C with primary antibodies diluted in the blocking solution. After incubation, the slides were washed in PBS and incubated with fluorescent secondary antibodies (FITC- or TRITC-labeled, 1:200 in blocking solution) for 1 h at room temperature. + +Slides for double immunolabeling were first immunostained for SOX1 as described above and visualized with Alexa568 goat anti-rabbit antibody (1:500, Molecular Probes, Eugene, Oregon, United States), and then incubated with unlabeled anti-rabbit secondary antibody (1:100, Dako, Glostrup, Denmark) for 1 h at room temperature to block existing unlabeled anti-SOX1 antibody. Subsequently, the slides were incubated with anti-SOX2 antibody in blocking solution without Triton X-100 for 48 h at room temperature and visualized with Alexa488 goat anti-rabbit antibody (1:500, Molecular Probes). The cross-reactivity of the SOX1 and SOX2 antibodies when using immunohistochemistry was excluded by looking at tissues where SOX1 (lens; [37]) or SOX2 (see Figure 7B–7E) are uniquely present, and in Western blots where each antibody recognizes a different size band (data not shown). After incubation with the secondary antibodies, the slides were washed in PBS and the sections were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, California, United States) and observed under either a fluorescent or a confocal microscope. + +Supporting Information + +Figure S1 + +The GSH2/PAX6 Boundary Is Unaffected throughout Development in the Absence of SOX1 + +Similar to earlier stages (E12; see Figure 5), the expression of GSH2 (green) and PAX6 (red) protein in wild-type (A, C, and E) and mutant brains (B, D, and F) is the same at E14 and E15, as shown by DAPI (blue) nuclear stain. Cx, cortex; lge, lateral ganglionic eminence; mge, medial ganglionic eminence. Scale bar = 200 μm for (C) and (D). + +(10 MB TIF). + +Click here for additional data file. + +Figure S2 + +Abnormal Distribution of VS Pax6-Expressing Neurons in the Absence of SOX1 + +Ventral telencephalic region of coronal brain sections stained with antibodies: PAX6 (brown) at E16 (A and B) and adult (C and D); SOX1 (green) and PAX6 (red) at E14 (E); and SOX1 and β-galactosidase (green) at E15 (F). Note Pax6-expressing cells are excluded from the OT region in the ventral telencephalon in wild-type (Sox1+/+) mice but not in the mutant(Sox1−/−). Arrows indicate ectopically localized Pax6-expressing cells. PAX6 and SOX1 are co-expressed in precursors but not in postmitotic cells in both mutant and wild-type. Scale bar = 500 μm for (C) and (D). + +(10 MB TIF). + +Click here for additional data file. + +Figure S3 + +Sox1-Expressing VZ Precursors Have Radial Glial Morphology + +Detail from immunofluorescence with β-galactosidase antibody of an E15 coronal brain section from mice carrying the Sox1βgeo allele. In the cortical ventricular zone, this staining reveals the cytoplasmic compartment of the SOX1-expressing progenitors, which have radial glial morphology. + +(3.34 MB TIF). + +Click here for additional data file. + +Figure S4 + +The Distribution of Radial Glia in the LGE Is Unaffected in the Absence of SOX1 + +Coronal brain sections of wild-type (+/+) and Sox1-null (−/−) mice at E16. Immunostained with RC2 antibody (a radial glia marker) showing no differences. + +(2.5 MB TIF). + +Click here for additional data file. + +Figure S5 + +Widespread Presence of SOX1 and SOX2 Proteins in Nuclei of the LGE VZ + +Immunofluorescence (green) of E14-stage wild-type coronal brain sections at the level of the LGE stained with SOX1 (C and G) and SOX2 (D and H) antibodies and visualized under a confocal microscope. (A, B, and E–H) are stained with TOTO red-fluorescent nuclear stain. (C, E, and G) and (D, F, and H) show high magnification of the area indicated in the rectangle in (A) and (B), respectively. + +(10 MB TIF). + +Click here for additional data file. + +Table S1 + +No Difference in the Number of LGE Dividing Precursors in the Absence of SOX1 + +BrdU-positive cells were counted using the Openlab image analysis program (Improvision, Coventry, United Kingdom). Measurements were performed in the area of the LGE (VZ/SVG) and of the pallial VZ. All data are represented as mean ± standard error of the mean. Cell counts were done in at least three different slides (sections) from each brain and in at least three separate optical fields in each slide (n = 4). To correct for tissue thickness and to obtain a better estimate of the proliferation within the LGE VZ/SVZ, the numbers of BrdU-positive cells were expressed as a ratio of the total number of LGE VZ/SVZ cells that were counted after hematoxylin staining, and as a ratio to the BrdU-positive cells of the pallial VZ/SVZ. Comparisons were made between wild-type and mutant mice using the unpaired Student's t-test (p < 0.05). + +(21 KB DOC). + +Click here for additional data file. + +Accession Numbers + +The GenBank (http://www.ncbi.nlm.nih.gov/Genebank/) accession numbers for the entities discussed in this paper are Brn4 (NM 008901), Dlx1 (NM 010053), Robo (MMU 17793), Sox1 (MN 009233), and Sox2 (MN 011443). + +Acknowledgements + +We thank for antibodies Drs. K. Campbell (Children's Hospital Research Foundation, Cincinnati, Ohio), T. Edlund (University of Umea, Sweden), and F. Guillemot (National Institute for Medical Research Medical Research [NIMR MRC], London). For comments on the manuscript we thank J. Briscoe (NIMR MRC) and J. Corbin (Georgetown University, Washington, DC). We are grateful to Z. Webster for the generation of transgenic mice and M. Delahaye for technical support with the mice. This work was supported by the MRC, the Wellcome Trust (grant 062197 to AC), and a Marie Curie Fellowship of the European Community Program (contract QLGA-CT-2001–50880 to AE). + +Competing interests. The authors have declared that no competing interests exist. + +Abbreviations + +bHLH - basic helix-loop-helix + +BrdU - 5-bromo-2′-deoxyuridine + +CNS - central nervous system + +DARPP-32 - dopamine and cAMP-regulated phosphoprotein + +E[number] - embryonic day [number] + +ES - embryonic stem + +IRES - internal ribosomal entry site + +LGE - lateral ganglionic eminence + +OT - olfactory tubercle + +P[number] - postnatal day [number] + +SVZ - subventricular zone + +TuJ1 - anti-βIII-tubulin antibody + +VZ - ventricular zone + +VS - ventral striatum + +βgeo - β-galactosidase-neo + +Figures and Tables + +Figure 1 + +The Mouse Sox1βgeo Allele Reveals the Requirement of SOX1 in the Development of VS Neurons + +(A) Strategy for targeting of the Sox1 locus by insertion of βgeo. Restriction enzymes: RV, EcoRV; K, KpnI; E, EcoRI; S, SpeI; B, BamHI. Yellow boxes indicate βgeo, green, SOX1 exon, and blue lines indicate fragments appearing in Southern blots of EcoR1-digested genomic DNA, hybridized with the external probe, which is shown with red lines. + +(B–E) X-gal and SOX1 antibody staining of Sox1βgeo/+. Comparison of Sox1βgeo expression visualized by X-gal staining (B and D) and the endogenous wild-type Sox1 gene visualized by whole-mount in situ (C) and SOX1 antibody staining (E). (B and C) show E9-stage embryos and (D and E) show coronal sections of newborn ventral telencephalon. + +(F–M) 100-μm coronal sections (Vibratome) were stained with X-gal to identify cells with Sox1 promoter activity. (F–I) show Sox1βgeo/+ forebrain sections from E13 to birth (P0) showing normal migration of Sox1-expressing cells from the VZ to the site of the OT, including striatal bridges. (J–M) show sections of Sox1βgeo/M1 forebrain, showing absence of X-gal staining in the OT and the striatal bridges. Red arrowheads show the anterior commissure. + +Scale bar = 500 μm for (B) and (C) and 300 μm for (D–M). + +Figure 2 + +Ectopic Distribution of Sox1-Null Neurons + +X-gal staining of mouse forebrains at P16. (A and B) show intact forebrain viewed from the ventral surface, and (C–F) show 150-μm coronal Vibratome sections for Sox1βgeo/+ mice (A, C, and E) and Sox1βgeo/M1 mice (B, D, and E). Red arrows indicate the width of the OT. Red arrowheads indicate increased X-gal staining at more medial and posterior areas of the brain in (B), and in the striatum and septum in (D) and (F). White arrowheads indicate islands other than the medial islands of Calleja. an, accumbens nucleus; I, II, III, cell layers of the OT; ICjM, medial islands of Calleja; lot, lateral olfactory tract; lsn, lateral septal nucleus; ob, olfactory bulb; PC, olfactory (piriform) cortex; S, striatum; sb, striatal bridge Scale bar = 500 μm. + +Figure 3 + +Normal Precursor Proliferation and Neurogenesis but Loss of OT Neuronal Differentiation in the Absence of SOX1 + +Coronal brain sections from the ventral telencephalon of wild-type (+/+) and Sox1-null (−/−) embryos. TuJ1 immunolabeling (A and B) at E13 shows no difference in early neuronal differentiation embryos; in situ hybridization at E16 for Brn4 (C and D) and Robo (E and F) shows absence of differentiation in the mutant at the prospective OT area. Red arrow in wild-type brain sections indicates OT. Telencephalic sections of wild-type (G, I, and K) and Sox1-null mutant (H, J, and L) embryonic brains were harvested 1 h after BrdU injection at E13 (G and H), E14 (I and J), and E15 (K and L) to detect actively dividing cells of the VZ/SVZ. Positive cells were visualized with anti-BrdU immunofluorescence (G–J) or with DAB staining (K and L). (K and L) show dorsal LGE area at high magnification. No differences were detected in the proliferation precursors at all stages examined, and no ectopic proliferation was observed in the mutant brains. Measurements and statistical analysis of BrdU-positive cells were performed on the DAB-stained sections, showing no significant differences (see Table S1). Scale bar = 300 μm (A and B), 300 μm (C–F), 500 μm (G–J), 500 μm (K and L). + +Figure 4 + +Failure of Neurons to Migrate to the VS in the Absence of SOX1 + +This figure shows BrdU labeling of proliferating cells in the developing forebrain. Immunohistochemistry was performed on 5-μm coronal sections, cut at the level of the OT. + +(A and B) Sections at E17, after BrdU injection at E14. White arrowheads in (A and B) indicate streams of migrating cells. + +(C–H) Sections at P16, after BrdU injection at E13 (C and D), E14 (E and F), or E16 (G and H). The DAB reaction product (C–H) was viewed under dark-field illumination. “II” is layer II of the OT, and the red bracket indicates the olfactory cortex. Note E13-born neurons contribute laterally to the olfactory (piriform) cortex, and medially to the layer II of the OT and the striatal bridges (red arrow). E14-born neurons contribute to more medial VS structures than E15- and E16-born cells, which contribute almost exclusively to the medial islands of Calleja (red arrowheads). + +Scale bar = 300 μm (A and B), 1 mm (C–H). + +Figure 5 + +Normal Generation and Patterning of LGE Precursors in the Absence of SOX1 + +(A and B) Immunocytochemistry and on coronal brain sections of dorsal and ventral telencephalic markers in wild-type (+/+) and Sox1-null (−/−) embryos. PAX6 and GSH2 immunocytochemistry in the dorsal LGE at E12 shows no difference at the expression boundary in the absence of SOX1; the arrows point at the stream of PAX6-positive cells emanating from the boundary. + +Double immunostaining for SOX1/PAX6 in wild-type brain (C), and for β-galactosidase/PAX6 (D) in the Sox1βgeo/− brain, at the VS area at E15. Note the presence of the PAX6-positive neurons in the area of the VS in the Sox1βgeo/− brain. + +(E and F) MASH1 immunocytochemistry in the LGE of wild-type (E) and Sox1-null brain (F), at E13. No changes are detected. + +(G and H) The distribution of Dlx1-expressing cells, as detected by in situ hybridization, is similar in both wild-type and mutant brains. + +Scale bar = 300 μm (A and B), 200 μm (C–F), 150 μm (G and H). + +Figure 6 + +SOX2 and SOX3 Down-Regulation in LGE Neurons and SOX1/SOX2 Co-Expression in LGE Precursors + +Immunofluorescence of coronal sections at LGE levels in (A–C) E15- and (D–L) E13-stage wild-type embryos visualized on a confocal microscope: antibody staining for (A, D, G, and J) SOX1 (red), (B, E, H, and K) SOX2 (green), (C) SOX3 (green), (D–L) double SOX1 (red) and SOX2 (green), and (F, I, and L) merged. In the OT area and the LGE mantle, there are more neurons expressing SOX1 (A and J) than SOX2 (B and K) and SOX3 (C). Note the extensive co-expression of the SOX1 and SOX2 in precursors (D–I). (G–I) are higher magnifications of the areas within the rectangles. Scale bar = 300 μm. + +Figure 7 + +The Sox2R Allele Delivers SOX1 in Sox2-Specific Expression Sites + +(A) Strategy for targeted replacement of the SOX2 coding region with that of SOX1 and IRES-βgeo. Restriction enzymes: S, SalI; E, EcoRI; Sm, SmaI; X, Xho. Green boxes indicate Sox1; black arrowheads indicate LoxP sites; yellow boxes indicate IRES βgeo; blue lines indicate fragments appearing in Southern blots of EcoR1-digested genomic DNA, hybridized with the external probe, which is shown with red lines. Black arrows show the locus after recombination, homologous and Cre-mediated where is indicated. + +(B–E) SOX1 immunostaining of frontal sections from E10 embryos. (B and D) Sox2+/+ and (C and E) Sox2R/+ showing the ectopic expression of SOX1 in the diencephalon (arrowheads) and the nasal pit (np) at E13. + +Figure 8 + +The Distribution of VS Neurons Is Unaffected in Mice Over-Expressing SOX1 from the Sox2R Allele + +(A–D), X-gal staining of coronal sections from the ventral telencephalon of P0 mice. Note that there is no difference in the distribution of Sox2-expressing OT neurons with SOX1 (A) or without SOX1 (C), and in Sox2R/+ mice with two wild-type Sox1 alleles (A) or one (B). Comparison of the number and distribution of neurons expressing Sox2βgeo in (C) and Sox1βgeo in (D) shows overlapping expression. (A–C) show 150-μm sections, and (D) shows a 80-μm section. + +(E and F) DARPP-32 immunostaining of coronal sections from the ventral telencephalon of P10 Sox2R/+ and Sox1βgeo/+ single heterozygous mice, showing no difference in the generation and migration of OT neurons. AC, anterior commissure. Scale bar = 100 μm. + +Figure 9 + +Sox1 Expression in Precursors Is Not Sufficient for the Emergence of OT/VS Neurons + +(A–C) X-gal staining of coronal sections from the ventral telencephalon showing Sox1-βgeo-expressing OT-prospective neurons at E16-stage embryos with one wild-type Sox1 allele, Sox1βgeo/+, in (A), and none, Sox1βgeo/M1, in (B), and HoHe (Sox1βgeo/M1, Sox2R/+) in (C). Note the absence of X-gal-stained neurons in the area of the VS (red arrowheads), indicating failure of the Sox2R allele to rescue OT neuron development in the HoHe embryos. Expression of βgeo from the Sox2R allele in the HoHe (C) may account for the slight increase of X-gal staining compared to (B). + +(D and E) SOX1 immunostaining at E16 embryos performed on the other halves of the brains of (A and C), respectively. Note that the level of SOX1 expression in the precursors (yellow arrows) is the same, whether it is expressed from the Sox2R allele in the HoHe (E) or from one of the Sox1 wild-type alleles in the Sox1 single heterozygotes (D). Note in the HoHe (E), this expression is not sufficient for the development of OT neurons. + +(F and G) DARPP32 immunostaining of coronal brain sections from Sox1+/+ Sox2R/+ (F) and Sox1M1/M1 Sox2R/+ (G) P10 mice indicating loss of VS neurons. + +Red arrowheads indicate OT; red arrows indicate anterior commissure. OC, olfactory cortex. Scale bar = 500 μm (A–E), 1 mm (F and G). + +Figure 10 + +Sox1 Expression in Postmitotic LGE Cells Is Sufficient for Neuronal Migration in the VS + +X-gal staining of coronal sections from the ventral telencephalon of P0 mice indicating the migration of neurons expressing SOX1 from the Sox2R allele in the presence (A) and absence (B; HoHe) of endogenous Sox1 wild-type genes. Black arrow points at the striatal bridges forming in HoHe mice. Scale bar = 100 μm. + +Footnotes + +Author contributions. AE, IK, SM, HW, and VE conceived and designed the experiments. AE, IK, SM, HW, PA, MD, and VE performed the experiments. AE, IK, SM, HW, and VE analyzed the data. AE, IK, SM, HW, DK, AC, RL, and VE contributed reagents/materials/analysis tools. VE wrote the paper. + +¤a Current address: Stem Cell Biology Laboratory, Wolfson Centre for Age-Related Diseases, King's College, London, United Kingdom + +¤b Current address: The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus + +¤c Current address: Nature Reviews Neuroscience, London, United Kingdom + +Citation: Ekonomou A, Kazanis I, Malas S, Wood H, Alifragis P, et al. (2005) Neuronal migration and ventral subtype identity in the telencephalon depend on SOX1. PLoS Biol 3(6): e186. diff --git a/src/ontogpt/evaluation/craft/database/all/15917436.ann b/src/ontogpt/evaluation/craft/database/all/15917436.ann new file mode 100644 index 000000000..43209e7a7 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15917436.ann @@ -0,0 +1,875 @@ +T1 NCBITaxon:10088 36 41 mouse +T2 PR:000010246 42 46 MCM4 +T3 GO:0097373 42 50 MCM4/6/7 +T4 GO:0006270 103 113;129 147 initiation ... of DNA replication +T5 GO:0006271 118 147 elongation of DNA replication +T6 NCBITaxon:40674 215 224 mammalian +T7 PR:000010246 225 229 Mcm4 +T8 GO:0097373 225 241 Mcm4/6/7 complex +T9 SO:0000984 285 300 single-stranded +T10 PR:000010246 423 427 Mcm4 +T11 GO:0097373 423 431 Mcm4/6/7 +T12 SO:0000984 489 504 single-stranded +T13 PR:000010246 767 771 Mcm4 +T14 GO:0097373 767 775 Mcm4/6/7 +T15 PR:000010246 830 834 Mcm4 +T16 GO:0097373 830 838 Mcm4/6/7 +T17 GO:0005657 865 869 fork +T18 SO:0000984 906 921 single-stranded +T19 GO:0000732 1092 1111 Strand displacement +T20 PR:000010246 1115 1119 Mcm4 +T21 GO:0097373 1115 1123 Mcm4/6/7 +T22 SO:0000985 1163 1169 duplex +T23 NCBITaxon:40674 1304 1313 mammalian +T24 PR:000010246 1314 1318 Mcm4 +T25 GO:0097373 1314 1322 Mcm4/6/7 +T26 SO:0000028 1369 1380 base-paired +T27 NCBITaxon:2759 1492 1502 eukaryotic +T28 GO:0006260 1503 1514 replicative +T29 MOP:0000619 1565 1575 hydrolysis +T30 GO:0032508 1625 1641 duplex unwinding +T31 MOP:0000619 1736 1746 hydrolysis +T32 GO:0032508 1763 1786 unwinding of duplex DNA +T33 SO:0000985 1776 1782 duplex +T34 GO:0042555 1828 1854 minichromosome maintenance +T35 GO:0042555 1856 1859 Mcm +T36 NCBITaxon:10633 1985 2000 simian virus 40 +T37 CHEBI:59132 2009 2016 antigen +T38 NCBITaxon:562 2022 2038 Escherichia coli +T39 PR:000022460 2039 2043 DnaB +T40 NCBITaxon:2157 2052 2060 Archaeal +T41 NCBITaxon:2759 2119 2129 eukaryotic +T42 GO:0042555 2130 2133 Mcm +T43 PR:000010246 2173 2177 Mcm4 +T44 GO:0097373 2173 2181 Mcm4/6/7 +T45 GO:0042555 2213 2216 Mcm +T46 PR:000010242 2213 2218 Mcm 2 +T47 PR:000010249 2213 2216;2219 2220 Mcm ... 7 +T48 GO:0032991 2345 2354 complexes +T49 GO:0042555 2361 2364 MCM +T50 PR:000010246 2397 2401 Mcm4 +T51 GO:0097373 2397 2413 Mcm4/6/7 complex +T52 PR:000010246 2496 2500 Mcm4 +T53 PR:000010248 2502 2506 Mcm6 +T54 PR:000010249 2511 2515 Mcm7 +T55 PR:000010242 2586 2590 Mcm2 +T56 PR:000010243 2594 2598 Mcm3 +T57 PR:000010246 2638 2642 Mcm4 +T58 GO:0097373 2638 2654 Mcm4/6/7 complex +T59 GO:0000785 2759 2768 Chromatin +T60 SO:0000704 2800 2807 genetic +T61 NCBITaxon:4932 2828 2852 Saccharomyces cerevisiae +T62 GO:0042555 2877 2880 Mcm +T63 GO:0022616 2932 2952 DNA chain elongation +T64 GO:0006260 2964 2975 replicative +T65 NCBITaxon:4896 3047 3072 Schizosaccharomyces pombe +T66 NCBITaxon:10088 3077 3082 mouse +T67 PR:000010246 3083 3087 Mcm4 +T68 GO:0097373 3083 3101 Mcm4/6/7 complexes +T69 GO:0005657 3133 3139 forked +T70 SO:0000985 3173 3179 duplex +T71 SO:0000028 3195 3197 bp +T72 PR:000010246 3207 3211 Mcm4 +T73 GO:0097373 3207 3215 Mcm4/6/7 +T74 GO:0005657 3225 3229 fork +T75 CHEBI:59132 3341 3348 antigen +T76 MOP:0000619 3409 3419 hydrolysis +T77 NCBITaxon:40674 3434 3443 mammalian +T78 PR:000010246 3444 3448 Mcm4 +T79 GO:0097373 3444 3452 Mcm4/6/7 +T80 SO:0000984 3483 3498 single-stranded +T81 GO:0042555 3576 3579 Mcm +T82 GO:0006260 3620 3631 replication +T83 SO:0000296 3620 3639 replication origins +T84 NCBITaxon:2759 3650 3660 eukaryotes +T85 GO:0042555 3730 3733 Mcm +T86 GO:0006260 3794 3805 replication +T87 GO:0005657 3794 3811 replication forks +T88 NCBITaxon:10088 3895 3900 mouse +T89 PR:000010246 3901 3905 Mcm4 +T90 GO:0097373 3901 3909 Mcm4/6/7 +T91 GO:0005657 3933 3939 forked +T92 GO:0042555 4022 4025 Mcm +T93 GO:0042555 4163 4166 Mcm +T94 NCBITaxon:40674 4232 4241 mammalian +T95 PR:000010246 4242 4246 Mcm4 +T96 GO:0097373 4242 4250 Mcm4/6/7 +T97 SO:0000984 4270 4285 single-stranded +T98 SO:0000984 4382 4397 single-stranded +T99 PR:000010246 4421 4425 Mcm4 +T100 GO:0097373 4421 4429 Mcm4/6/7 +T101 SO:0000985 4491 4497 duplex +T102 GO:0006260 4578 4589 replication +T103 SO:0001026 4604 4610 genome +T104 CHEBI:33893 4636 4644 Reagents +T105 SO:0000984 4754 4769 single-stranded +T106 SO:0000988 4770 4778 circular +T107 NCBITaxon:10665 4784 4786 T4 +T108 CHEBI:15986 4787 4801 polynucleotide +T109 PR:000012924 4787 4808 polynucleotide kinase +T110 GO:0042571 4905 4907 Ab +T111 GO:0042571 4909 4917 antibody +T112 CHEBI:2511 4919 4926 agarose +T113 NCBITaxon:10633 4961 4965 SV40 +T114 CHEBI:59132 4968 4975 antigen +T115 NCBITaxon:10442 4994 5005 baculovirus +T116 http://purl.obolibrary.org/obo/MONDO_0005550 5006 5014 infected +T117 NCBITaxon:6960 5015 5021 insect +T118 PR:000023591 5046 5050 PriA +T119 GO:0010467 5308 5318 Expression +T120 NCBITaxon:10088 5339 5344 mouse +T121 PR:000010246 5345 5349 Mcm4 +T122 GO:0097373 5345 5361 Mcm4/6/7 complex +T123 NCBITaxon:10442 5379 5392 baculoviruses +T124 GO:0010467 5393 5403 expressing +T125 PR:000010246 5409 5413 Mcm4 +T126 PR:000010248 5414 5418 Mcm6 +T127 PR:000010249 5422 5426 Mcm7 +T128 NCBITaxon:6960 5491 5497 insect +T129 GO:0010467 5629 5639 expression +T130 PR:000010246 5647 5651 Mcm4 +T131 GO:0097373 5647 5655 Mcm4/6/7 +T132 http://purl.obolibrary.org/obo/MONDO_0100128 5687 5697 coinfected +T133 NCBITaxon:10442 5715 5728 baculoviruses +T134 GO:0010467 5729 5739 expressing +T135 PR:000010246 5749 5753 Mcm4 +T136 PR:000010248 5754 5758 Mcm6 +T137 GO:0010467 5778 5788 expressing +T138 PR:000010249 5793 5797 Mcm7 +T139 http://purl.obolibrary.org/obo/MONDO_0005550 5836 5845 infection +T140 PR:000010246 5863 5867 Mcm4 +T141 GO:0097373 5863 5881 Mcm4/6/7 complexes +T142 SO:0000985 6075 6081 duplex +T143 GO:0097617 6113 6122 annealing +T144 SO:0000984 6161 6176 single-stranded +T145 SO:0000988 6177 6185 circular +T146 GO:0005657 6467 6471 fork +T147 GO:0097617 6500 6509 annealing +T148 GO:0005657 6581 6585 fork +T149 GO:0006272 6590 6593;6600 6609 3′- ... extension +T150 GO:0006273 6597 6609 5′-extension +T151 GO:0097617 6685 6693 annealed +T152 PR:000009861 6702 6710 lamin B2 +T153 PR:000009861 6747 6755 lamin B2 +T154 SO:0000296 6756 6762 origin +T155 PR:000000084 6838 6843 c-myc +T156 PR:000000084 6851 6856 c-myc +T157 GO:0032508 7023 7029 melted +T158 GO:0097617 7095 7103 annealed +T159 CHEBI:60004 7162 7169 mixture +T160 CHEBI:9754 7195 7199 Tris +T161 CHEBI:17883 7200 7203 HCl +T162 CHEBI:6636 7220 7225 MgCl2 +T163 CHEBI:26710 7236 7240 NaCl +T164 SO:0000985 7451 7457 duplex +T165 SO:0000984 7474 7489 single-stranded +T166 SO:0000988 7490 7498 circular +T167 SO:0000985 7526 7532 duplex +T168 GO:0097617 7627 7638 hybridizing +T169 SO:0000440 7673 7679 vector +T170 GO:0097617 7681 7689 annealed +T171 SO:0000984 7697 7712 single-stranded +T172 SO:0000988 7713 7721 circular +T173 CHEBI:37972 7790 7793 32P +T174 SO:0000985 7909 7915 duplex +T175 PR:000010246 8020 8024 Mcm4 +T176 GO:0097373 8020 8036 Mcm4/6/7 complex +T177 CHEBI:60004 8065 8072 mixture +T178 CHEBI:8984 8153 8156 SDS +T179 SO:0000984 8315 8330 single-stranded +T180 SO:0000988 8331 8339 circular +T181 GO:0097617 8476 8485 annealing +T182 PR:P23940 8573 8578 BamHI +T183 SO:0000667 8620 8626 insert +T184 PR:000010246 8700 8704 Mcm4 +T185 GO:0097373 8700 8708 Mcm4/6/7 +T186 CHEBI:60004 8764 8772 mixtures +T187 CHEBI:46756 8798 8803 HEPES +T188 CHEBI:32145 8804 8808 NaOH +T189 CHEBI:32954 8825 8839 sodium acetate +T190 CHEBI:62964 8847 8858 Mg(CH3COO)2 +T191 CHEBI:18320 8865 8868 DTT +T192 CHEBI:27575 8893 8900 ATP-γ-S +T193 CHEBI:17754 8979 8987 glycerol +T194 CHEBI:60004 9002 9010 mixtures +T195 CHEBI:62964 9073 9090 magnesium acetate +T196 CHEBI:17754 9098 9106 glycerol +T197 CHEBI:60004 9213 9221 mixtures +T198 CHEBI:8984 9452 9455 SDS +T199 CHEBI:60004 9663 9671 mixtures +T200 CHEBI:46756 9696 9701 HEPES +T201 CHEBI:32145 9702 9706 NaOH +T202 CHEBI:32954 9723 9737 sodium acetate +T203 CHEBI:62964 9745 9756 Mg(CH3COO)2 +T204 CHEBI:18320 9763 9766 DTT +T205 CHEBI:27575 9791 9798 ATP-γ-S +T206 CHEBI:3312 9807 9812 CaCl2 +T207 CHEBI:37972 9825 9828 32P +T208 PR:000010246 9883 9887 Mcm4 +T209 GO:0097373 9883 9891 Mcm4/6/7 +T210 CHEBI:60004 10017 10025 mixtures +T211 CHEBI:75958 10130 10138 solution +T212 CHEBI:26710 10157 10172 sodium chloride +T213 CHEBI:8984 10177 10180 SDS +T214 CHEBI:3312 10317 10322 CaCl2 +T215 MOP:0000780 10421 10429 Cleavage +T216 CHEBI:32145 10487 10491 NaOH +T217 CHEBI:60004 10497 10504 mixture +T218 CHEBI:32954 10591 10605 sodium acetate +T219 CHEBI:15882 10641 10647 phenol +T220 CHEBI:35255 10648 10658 chloroform +T221 CHEBI:16236 10698 10705 ethanol +T222 CHEBI:16236 10746 10753 ethanol +T223 CHEBI:16199 10870 10874 urea +T224 PR:000010246 10915 10919 Mcm4 +T225 GO:0097373 10915 10931 Mcm4/6/7 complex +T226 SO:0000984 10961 10976 single-stranded +T227 PR:000010246 10990 10994 Mcm4 +T228 GO:0097373 10990 10998 Mcm4/6/7 +T229 SO:0000985 11048 11054 duplex +T230 SO:0000984 11081 11096 single-stranded +T231 PR:000010246 11181 11185 Mcm4 +T232 GO:0097373 11181 11189 Mcm4/6/7 +T233 PR:000010246 11286 11290 Mcm4 +T234 GO:0097373 11286 11294 Mcm4/6/7 +T235 CHEBI:27575 11314 11321 ATP-γ-S +T236 MOP:0000780 11444 11453 cleavages +T237 SO:0000028 11478 11480 bp +T238 SO:0000985 11481 11487 duplex +T239 MOP:0000780 11543 11552 cleavages +T240 SO:0000984 11582 11597 single-stranded +T241 SO:0000985 11647 11662 double-stranded +T242 SO:0000984 11746 11761 single-stranded +T243 PR:000010246 11834 11838 Mcm4 +T244 GO:0097373 11834 11842 Mcm4/6/7 +T245 PR:000010246 11877 11881 Mcm4 +T246 GO:0097373 11877 11885 Mcm4/6/7 +T247 SO:0000984 11987 12002 single-stranded +T248 SO:0000984 12067 12082 single-stranded +T249 GO:0042555 12118 12129 Mcm complex +T250 SO:0000984 12195 12210 single-stranded +T251 PR:000010246 12242 12246 Mcm4 +T252 GO:0097373 12242 12250 Mcm4/6/7 +T253 SO:0000028 12407 12416 base-pair +T254 SO:0000985 12417 12423 duplex +T255 PR:000010246 12584 12588 Mcm4 +T256 GO:0097373 12584 12592 Mcm4/6/7 +T257 GO:0006260 12607 12618 replication +T258 NCBITaxon:10633 12737 12741 SV40 +T259 CHEBI:59132 12744 12751 antigen +T260 GO:0005657 12822 12826 fork +T261 SO:0000984 12863 12878 single-stranded +T262 SO:0000028 12906 12908 bp +T263 SO:0000985 12909 12915 duplex +T264 SO:0000985 12936 12942 duplex +T265 SO:0000984 12946 12959 single-strand +T266 GO:0042555 12987 12990 Mcm +T267 PR:000010246 13050 13054 Mcm4 +T268 GO:0097373 13050 13058 Mcm4/6/7 +T269 SO:0000984 13092 13107 single-stranded +T270 SO:0000985 13159 13165 duplex +T271 GO:0006260 13203 13214 replication +T272 GO:0005657 13203 13219 replication fork +T273 GO:0065003 13222 13234;13261 13270 Formation of ... complexes +T274 PR:000010246 13252 13256 Mcm4 +T275 GO:0097373 13252 13260 Mcm4/6/7 +T276 PR:000010246 13353 13357 Mcm4 +T277 GO:0097373 13353 13361 Mcm4/6/7 +T278 SO:0000984 13487 13502 single-stranded +T279 SO:0000984 13535 13550 single-stranded +T280 GO:0042555 13622 13633 Mcm complex +T281 PR:000010246 13674 13678 Mcm4 +T282 GO:0097373 13674 13682 Mcm4/6/7 +T283 SO:0000984 13780 13795 single-stranded +T284 SO:0000984 14069 14077 unpaired +T285 PR:000010246 14147 14151 Mcm4 +T286 GO:0097373 14147 14155 Mcm4/6/7 +T287 PR:000010246 14191 14195 Mcm4 +T288 GO:0097373 14191 14199 Mcm4/6/7 +T289 CHEBI:27575 14287 14294 ATP-γ-S +T290 GO:0032991 14304 14311 complex +T291 GO:0065003 14304 14321 complex formation +T292 CHEBI:60004 14348 14356 mixtures +T293 GO:0032991 14572 14581 complexes +T294 PR:000010246 14587 14591 Mcm4 +T295 GO:0097373 14587 14595 Mcm4/6/7 +T296 PR:000010246 14698 14702 Mcm4 +T297 GO:0097373 14698 14706 Mcm4/6/7 +T298 GO:0032991 14741 14750 complexes +T299 SO:0000984 15032 15047 single-stranded +T300 GO:0042555 15137 15140 Mcm +T301 PR:000010246 15198 15202 Mcm4 +T302 GO:0097373 15198 15214 Mcm4/6/7 complex +T303 SO:0000984 15354 15369 single-stranded +T304 SO:0000984 15501 15516 single-stranded +T305 PR:000010246 15699 15703 Mcm4 +T306 GO:0097373 15699 15715 Mcm4/6/7 complex +T307 SO:0000985 15788 15794 duplex +T308 SO:0000984 15854 15869 single-stranded +T309 GO:0032508 15906 15918;15926 15929 unwinding of ... DNA +T310 PR:000010246 15933 15937 Mcm4 +T311 GO:0097373 15933 15941 Mcm4/6/7 +T312 PR:000010246 15968 15972 Mcm4 +T313 GO:0097373 15968 15976 Mcm4/6/7 +T314 SO:0000985 16067 16073 duplex +T315 PR:000010246 16243 16247 Mcm4 +T316 GO:0097373 16243 16251 Mcm4/6/7 +T317 SO:0000984 16302 16317 single-stranded +T318 PR:000010246 16549 16553 Mcm4 +T319 GO:0097373 16549 16557 Mcm4/6/7 +T320 PR:000010246 16654 16658 Mcm4 +T321 GO:0097373 16654 16662 Mcm4/6/7 +T322 SO:0000985 16960 16966 duplex +T323 GO:0005657 17017 17021 fork +T324 CHEBI:36357 17298 17307 molecules +T325 PR:000010246 17315 17319 Mcm4 +T326 GO:0097373 17315 17323 Mcm4/6/7 +T327 PR:000010246 17396 17400 Mcm4 +T328 GO:0097373 17396 17404 Mcm4/6/7 +T329 GO:0005657 17408 17414 forked +T330 GO:0006272 17419 17422;17429 17438 3′- ... extension +T331 GO:0006273 17426 17438 5′-extension +T332 GO:0005657 17528 17534 forked +T333 GO:0005657 17549 17553 fork +T334 GO:0005657 17604 17608 fork +T335 GO:0005657 17641 17645 fork +T336 GO:0005657 17671 17675 fork +T337 GO:0006273 17696 17708 5′-extension +T338 GO:0006272 17712 17724 3′-extension +T339 PR:000010246 17754 17758 Mcm4 +T340 GO:0097373 17754 17762 Mcm4/6/7 +T341 GO:0005657 17774 17778 fork +T342 GO:0005657 17788 17792 fork +T343 GO:0005657 17841 17845 fork +T344 PR:000023591 17891 17895 PriA +T345 GO:0005657 17936 17940 fork +T346 GO:0005657 17972 17976 fork +T347 PR:000010246 18014 18018 Mcm4 +T348 GO:0097373 18014 18022 Mcm4/6/7 +T349 GO:0006273 18037 18049 5′-extension +T350 GO:0006272 18054 18066 3′-extension +T351 GO:0005657 18120 18124 fork +T352 GO:0005657 18135 18139 fork +T353 PR:000010246 18157 18161 Mcm4 +T354 GO:0097373 18157 18165 Mcm4/6/7 +T355 GO:0005657 18177 18181 fork +T356 GO:0032991 18223 18230 complex +T357 PR:000010246 18292 18296 Mcm4 +T358 GO:0097373 18292 18308 Mcm4/6/7 complex +T359 SO:0000984 18367 18382 single-stranded +T360 SO:0000984 18490 18505 single-stranded +T361 GO:0005657 18518 18522 fork +T362 GO:0005657 18558 18562 fork +T363 GO:0005657 18572 18576 fork +T364 PR:000010246 18604 18608 Mcm4 +T365 GO:0097373 18604 18612 Mcm4/6/7 +T366 GO:0005657 18659 18663 fork +T367 SO:0000985 18688 18694 duplex +T368 SO:0000985 18721 18736 double-stranded +T369 PR:000010246 18788 18792 Mcm4 +T370 GO:0097373 18788 18796 Mcm4/6/7 +T371 PR:000023591 18820 18824 PriA +T372 GO:0005657 18888 18892 fork +T373 GO:0005657 18918 18922 fork +T374 GO:0005657 18963 18967 fork +T375 GO:0005657 19016 19020 fork +T376 PR:000010246 19086 19090 Mcm4 +T377 GO:0097373 19086 19102 Mcm4/6/7 complex +T378 SO:0000984 19112 19127 single-stranded +T379 SO:0000984 19193 19208 single-stranded +T380 GO:0006272 19219 19231 3′-extension +T381 PR:000010246 19267 19271 Mcm4 +T382 GO:0097373 19267 19275 Mcm4/6/7 +T383 GO:0006272 19307 19319 3′-extension +T384 NCBITaxon:2157 19342 19350 archaeal +T385 GO:0042555 19351 19354 Mcm +T386 NCBITaxon:4894 19378 19385;19398 19403 fission ... yeast +T387 GO:0007114 19390 19397 budding +T388 NCBITaxon:4892 19390 19403 budding yeast +T389 GO:0042555 19404 19417 Mcm complexes +T390 GO:0006272 19450 19462 3′-extension +T391 NCBITaxon:10088 19564 19569 mouse +T392 PR:000010246 19570 19574 Mcm4 +T393 GO:0097373 19570 19578 Mcm4/6/7 +T394 PR:000010246 19630 19634 Mcm4 +T395 GO:0097373 19630 19638 Mcm4/6/7 +T396 GO:0006272 19664 19676 3′-extension +T397 GO:0006272 19710 19722 3′-extension +T398 PR:000010246 19811 19815 Mcm4 +T399 GO:0097373 19811 19819 Mcm4/6/7 +T400 GO:0006272 19916 19928 3′-extension +T401 PR:000010246 19958 19963 Mcm 4 +T402 PR:000010248 19958 19961;19964 19965 Mcm ... 6 +T403 PR:000010249 19958 19961;19966 19967 Mcm ... 7 +T404 GO:0097373 19958 19967 Mcm 4/6/7 +T405 PR:000010246 20095 20099 Mcm4 +T406 GO:0097373 20095 20103 Mcm4/6/7 +T407 GO:0006272 20149 20161 3′-extension +T408 GO:0006273 20197 20209 5′-extension +T409 PR:000010246 20220 20224 Mcm4 +T410 GO:0097373 20220 20228 Mcm4/6/7 +T411 GO:0042555 20266 20269 Mcm +T412 GO:0006272 20321 20333 3′-extension +T413 GO:0005657 20423 20427 fork +T414 NCBITaxon:40674 20534 20543 mammalian +T415 GO:0042555 20544 20547 Mcm +T416 SO:0000984 20613 20628 single-stranded +T417 GO:0042555 20689 20692 Mcm +T418 GO:0042555 20826 20829 Mcm +T419 SO:0000985 20865 20871 duplex +T420 SO:0000984 20876 20891 single-stranded +T421 SO:0000028 20912 20914 bp +T422 SO:0000985 20915 20921 duplex +T423 NCBITaxon:10088 21055 21060 mouse +T424 PR:000010246 21061 21065 Mcm4 +T425 GO:0097373 21061 21069 Mcm4/6/7 +T426 PR:000010246 21125 21129 Mcm4 +T427 GO:0097373 21125 21133 Mcm4/6/7 +T428 GO:0097617 21155 21163 annealed +T429 GO:0042555 21653 21656 Mcm +T430 GO:0097617 21716 21722 anneal +T431 GO:0042555 21761 21764 Mcm +T432 SO:0000985 21982 21988 duplex +T433 PR:000010246 22089 22093 Mcm4 +T434 GO:0097373 22089 22105 Mcm4/6/7 complex +T435 GO:0042555 22228 22231 Mcm +T436 GO:0042555 22611 22614 Mcm +T437 PR:000000084 22685 22690 c-myc +T438 SO:0000296 22691 22697 origin +T439 GO:0042555 22707 22710 Mcm +T440 NCBITaxon:9606 22822 22827 human +T441 PR:000009861 22828 22836 lamin B2 +T442 SO:0000296 22837 22843 origin +T443 GO:0042555 22910 22913 Mcm +T444 GO:0006260 23055 23066 replication +T445 SO:0000296 23055 23074 replication origins +T446 GO:0042555 23135 23138 Mcm +T447 PR:000000084 23215 23220 c-myc +T448 GO:0032508 23265 23278 DNA unwinding +T449 NCBITaxon:9606 23302 23307 human +T450 PR:000000084 23308 23313 c-myc +T451 SO:0000296 23314 23320 origin +T452 PR:000000084 23344 23349 c-myc +T453 SO:0000984 23383 23391 unpaired +T454 PR:000000084 23409 23414 c-myc +T455 PR:000000084 23464 23469 c-myc +T456 PR:000000084 23575 23580 c-myc +T457 SO:0000296 23581 23587 origin +T458 PR:000010246 23663 23667 Mcm4 +T459 GO:0097373 23663 23671 Mcm4/6/7 +T460 PR:000009861 23686 23694 lamin B2 +T461 PR:000000084 23716 23721 c-myc +T462 PR:000000084 23742 23747 c-myc +T463 PR:000000084 23836 23841 c-myc +T464 SO:0000296 23842 23848 origin +T465 GO:0042555 23895 23898 Mcm +T466 PR:000010246 23989 23993 Mcm4 +T467 GO:0097373 23989 23997 Mcm4/6/7 +T468 GO:0042555 24150 24153 Mcm +T469 PR:000010246 24197 24201 MCM4 +T470 GO:0097373 24197 24205 MCM4/6/7 +T471 PR:000000084 24269 24274 c-myc +T472 PR:000000084 24292 24297 c-myc +T473 PR:000009861 24342 24350 lamin B2 +T474 GO:0042555 24553 24556 Mcm +T475 GO:0032508 24597 24610 DNA unwinding +T476 PR:000010246 24614 24618 Mcm4 +T477 GO:0097373 24614 24622 Mcm4/6/7 +T478 SO:0000985 24668 24674 duplex +T479 SO:0000984 24723 24738 single-stranded +T480 GO:0042555 24797 24800 Mcm +T481 GO:0032508 24890 24913 unwinding of duplex DNA +T482 SO:0000985 24903 24909 duplex +T483 SO:0000985 24985 24991 duplex +T484 GO:0005657 25092 25096 fork +T485 GO:0005657 25111 25115 fork +T486 SO:0000985 25153 25159 duplex +T487 PR:000010246 25263 25267 Mcm4 +T488 GO:0097373 25263 25271 Mcm4/6/7 +T489 GO:0005657 25289 25293 fork +T490 PR:000010246 25378 25382 Mcm4 +T491 GO:0097373 25378 25386 Mcm4/6/7 +T492 SO:0000984 25396 25411 single-stranded +T493 GO:0005657 25454 25458 fork +T494 PR:000010246 25491 25495 Mcm4 +T495 GO:0097373 25491 25499 Mcm4/6/7 +T496 NCBITaxon:10633 25530 25534 SV40 +T497 CHEBI:59132 25537 25544 antigen +T498 GO:0005657 25695 25699 fork +T499 GO:0005657 25718 25722 fork +T500 SO:0000985 25823 25829 duplex +T501 GO:0005657 25841 25845 fork +T502 GO:0005657 25863 25867 fork +T503 GO:0005657 25885 25889 fork +T504 GO:0005657 25910 25914 fork +T505 SO:0000985 25962 25968 duplex +T506 SO:0000985 26106 26112 duplex +T507 SO:0000984 26122 26137 single-stranded +T508 SO:0000988 26138 26146 circular +T509 SO:0000985 26208 26214 duplex +T510 SO:0000985 26241 26247 duplex +T511 SO:0000440 26292 26298 vector +T512 GO:0097617 26381 26392 hybridizing +T513 PR:000010246 26428 26432 Mcm4 +T514 GO:0097373 26428 26436 Mcm4/6/7 +T515 SO:0000985 26459 26465 duplex +T516 PR:000010246 26810 26814 Mcm4 +T517 GO:0097373 26810 26826 Mcm4/6/7 complex +T518 GO:0005657 26869 26873 fork +T519 SO:0000985 26901 26907 duplex +T520 GO:0042555 26951 26954 Mcm +T521 SO:0000028 27029 27034 pairs +T522 SO:0000985 27067 27073 duplex +T523 PR:000010246 27120 27124 Mcm4 +T524 GO:0097373 27120 27128 Mcm4/6/7 +T525 PR:000010246 27145 27149 Mcm4 +T526 GO:0097373 27145 27153 Mcm4/6/7 +T527 SO:0000028 27218 27229 base-paired +T528 GO:0005657 27309 27313 fork +T529 SO:0000028 27418 27427 base pair +T530 SO:0000028 27458 27467 base pair +T531 SO:0000985 27471 27477 duplex +T532 SO:0000028 27542 27551 base pair +T533 GO:0005657 27586 27590 fork +T534 SO:0000985 27657 27663 duplex +T535 PR:000010246 27689 27693 Mcm4 +T536 GO:0097373 27689 27697 Mcm4/6/7 +T537 GO:0005657 27771 27775 fork +T538 GO:0005657 27837 27841 fork +T539 GO:0005657 27912 27916 fork +T540 SO:0000985 27973 27979 duplex +T541 SO:0000028 28008 28017 base pair +T542 PR:000010246 28051 28055 Mcm4 +T543 GO:0097373 28051 28059 Mcm4/6/7 +T544 GO:0006260 28147 28158 replication +T545 GO:0005657 28147 28164 replication forks +T546 GO:0006260 28225 28236 replication +T547 SO:0000296 28225 28244 replication origins +T548 GO:0006260 28284 28295 replicative +T549 GO:0006270 28413 28442 initiation of DNA replication +T550 GO:0042555 28468 28471 Mcm +T551 NCBITaxon:2759 28500 28510 eukaryotic +T552 GO:0006260 28511 28522 replicative +T553 NCBITaxon:10088 28620 28625 mouse +T554 PR:000010246 28626 28630 Mcm4 +T555 GO:0097373 28626 28634 Mcm4/6/7 +T556 GO:0005657 28670 28674 fork +T557 PR:000010246 28734 28738 Mcm4 +T558 GO:0097373 28734 28742 Mcm4/6/7 +T559 GO:0006270 28775 28804 Initiation of DNA replication +T560 GO:0032508 28834 28855 melting of duplex DNA +T561 SO:0000985 28845 28851 duplex +T562 GO:0006260 28861 28872 replication +T563 SO:0000296 28861 28880 replication origins +T564 GO:0006260 28908 28919 replication +T565 GO:0005657 28908 28925 replication forks +T566 GO:0032508 28938 28944 melted +T567 NCBITaxon:2 28986 28994 bacteria +T568 PR:000010246 29029 29033 Mcm4 +T569 GO:0097373 29029 29037 Mcm4/6/7 +T570 GO:0005657 29120 29125 forks +T571 PR:000010246 29147 29151 Mcm4 +T572 GO:0097373 29147 29155 Mcm4/6/7 +T573 SO:0000985 29212 29218 duplex +T574 GO:0042555 29257 29260 Mcm +T575 SO:0000984 29287 29302 single-stranded +T576 PR:000010246 29343 29347 Mcm4 +T577 GO:0097373 29343 29359 Mcm4/6/7 complex +T578 SO:0000984 29425 29440 single-stranded +T579 PR:000010246 29539 29543 Mcm4 +T580 GO:0097373 29539 29547 Mcm4/6/7 +T581 SO:0000984 29602 29610 unpaired +T582 GO:0065003 29674 29685;29705 29712 assembly of ... complex +T583 NCBITaxon:10633 29746 29750 SV40 +T584 CHEBI:59132 29753 29760 antigen +T585 SO:0000984 29810 29825 single-stranded +T586 PR:000010246 29985 29989 Mcm4 +T587 GO:0097373 29985 29993 Mcm4/6/7 +T588 SO:0000984 30018 30033 single-stranded +T589 GO:0005657 30139 30143 fork +T590 PR:000010246 30149 30153 Mcm4 +T591 GO:0097373 30149 30165 Mcm4/6/7 complex +T592 SO:0000984 30192 30207 single-stranded +T593 SO:0000984 30523 30538 single-stranded +T594 CHEBI:59132 30644 30651 antigen +T595 PR:000010246 30718 30722 Mcm4 +T596 GO:0097373 30718 30726 Mcm4/6/7 +T597 GO:0005657 30743 30747 fork +T598 NCBITaxon:10088 30808 30813 mouse +T599 NCBITaxon:2157 30818 30824 archea +T600 GO:0042555 30825 30828 MCM +T601 CHEBI:10545 30845 30853 electron +T602 NCBITaxon:10633 30927 30931 SV40 +T603 CHEBI:59132 30940 30947 antigen +T604 NCBITaxon:10088 31009 31014 mouse +T605 GO:0042555 31015 31018 Mcm +T606 PR:000010246 31076 31080 Mcm4 +T607 GO:0097373 31076 31084 Mcm4/6/7 +T608 PR:000010246 31139 31143 Mcm4 +T609 GO:0097373 31139 31147 Mcm4/6/7 +T610 SO:0000984 31185 31200 single-stranded +T611 SO:0000984 31254 31269 single-stranded +T612 GO:0032508 31284 31296;31301 31311 Unwinding of ... duplex DNA +T613 SO:0000985 31301 31307 duplex +T614 SO:0000984 31340 31355 single-stranded +T615 GO:0005657 31440 31444 fork +T616 GO:0006272 31453 31465 3′-extension +T617 GO:0005657 31479 31483 fork +T618 GO:0005657 31491 31495 fork +T619 GO:0006273 31507 31519 5′-extension +T620 SO:0000984 31572 31587 single-stranded +T621 PR:000010246 31599 31603 Mcm4 +T622 GO:0097373 31599 31607 Mcm4/6/7 +T623 GO:0097617 31621 31630 annealing +T624 SO:0000988 31652 31660 circular +T625 SO:0000984 31661 31676 single-stranded +T626 NCBITaxon:10088 31701 31706 mouse +T627 PR:000010246 31707 31711 Mcm4 +T628 GO:0097373 31707 31715 Mcm4/6/7 +T629 GO:0006272 31726 31738 3′-extension +T630 NCBITaxon:2157 31756 31764 archaeal +T631 GO:0042555 31765 31768 Mcm +T632 PR:000010246 31789 31793 Mcm4 +T633 GO:0097373 31789 31797 Mcm4/6/7 +T634 NCBITaxon:4896 31803 31810 S.pombe +T635 NCBITaxon:4932 31815 31827 S.cerevisiae +T636 NCBITaxon:2157 31844 31852 archaeal +T637 GO:0042555 31853 31856 Mcm +T638 GO:0005657 31870 31874 fork +T639 NCBITaxon:2759 31883 31893 eukaryotic +T640 PR:000010246 31894 31898 Mcm4 +T641 GO:0097373 31894 31902 Mcm4/6/7 +T642 SO:0000985 31937 31952 double-stranded +T643 GO:0042555 32001 32004 Mcm +T644 SO:0000985 32034 32040 duplex +T645 NCBITaxon:40674 32082 32091 mammalian +T646 PR:000010246 32092 32096 Mcm4 +T647 GO:0097373 32092 32100 Mcm4/6/7 +T648 GO:0006260 32215 32226 replication +T649 SO:0000296 32215 32234 replication origins +T650 GO:0042555 32260 32263 Mcm +T651 NCBITaxon:40674 32316 32325 mammalian +T652 GO:0006260 32326 32341 DNA replication +T653 NCBITaxon:9606 32400 32405 human +T654 GO:0006260 32406 32417 replication +T655 SO:0000296 32406 32425 replication origins +T656 PR:000010246 32438 32442 Mcm4 +T657 GO:0097373 32438 32455 Mcm4/6/7 helicase +T658 PR:000009861 32462 32470 lamin-B2 +T659 PR:000000084 32475 32480 c-myc +T660 SO:0000296 32481 32488 origins +T661 GO:0042555 32523 32526 Mcm +T662 GO:0042555 32591 32594 Mcm +T663 GO:0006270 32602 32628 DNA replication initiation +T664 PR:000000084 32641 32646 c-myc +T665 SO:0000984 32741 32756 single-stranded +T666 GO:0042555 32789 32792 Mcm +T667 PR:000010246 33132 33136 Mcm4 +T668 GO:0097373 33132 33140 Mcm4/6/7 +T669 SO:0000984 33212 33227 single-stranded +T670 PR:000010246 33408 33412 Mcm4 +T671 GO:0097373 33408 33416 Mcm4/6/7 +T672 SO:0000984 33474 33489 single-stranded +T673 GO:0042555 33671 33674 Mcm +T674 GO:0065007 33851 33861 regulation +T675 GO:0006270 33884 33913 initiation of DNA replication +T676 GO:0006260 34018 34029 replication +T677 SO:0000296 34018 34036 replication origin +T678 SO:0001026 34044 34050 genome +T679 PR:000010246 34086 34090 Mcm4 +T680 GO:0097373 34086 34094 Mcm4/6/7 +T681 GO:0005657 34139 34143 fork +T682 SO:0000984 34173 34188 single-stranded +T683 GO:0042555 34224 34227 Mcm +T684 GO:0032508 34306 34329 unwinding of duplex DNA +T685 SO:0000985 34319 34325 duplex +T686 SO:0000028 34373 34377 pair +T687 SO:0000985 34385 34391 duplex +T688 GO:0042555 34428 34431 Mcm +T689 SO:0000985 34451 34457 Duplex +T690 SO:0000028 34482 34487 pairs +T691 GO:0005657 34515 34519 fork +T692 NCBITaxon:10633 34595 34599 SV40 +T693 CHEBI:59132 34602 34609 antigen +T694 GO:0042555 34623 34626 Mcm +T695 SO:0000985 34677 34683 duplex +T696 SO:0000988 34702 34710 circular +T697 SO:0000984 34711 34726 single-stranded +T698 SO:0000985 34741 34747 duplex +T699 PR:000010246 34787 34791 Mcm4 +T700 GO:0097373 34787 34795 Mcm4/6/7 +T701 SO:0000985 34938 34944 duplex +T702 GO:0042555 34955 34958 Mcm +T703 SO:0000988 34978 34986 circular +T704 SO:0000984 34987 35002 single-stranded +T705 GO:0005657 35078 35082 fork +T706 GO:0042555 35155 35158 Mcm +T707 SO:0000985 35242 35248 duplex +T708 GO:0042555 35295 35298 Mcm +T709 SO:0000985 35369 35375 duplex +T710 GO:0005657 35391 35395 fork +T711 SO:0000028 35423 35433 base pairs +T712 SO:0000985 35564 35570 duplex +T713 PR:000010246 35609 35613 Mcm4 +T714 GO:0097373 35609 35617 Mcm4/6/7 +T715 SO:0000028 35673 35678 pairs +T716 SO:0000985 35748 35754 duplex +T717 SO:0000028 35848 35857 base pair +T718 GO:0042555 35887 35890 Mcm +T719 SO:0000028 35944 35954 base pairs +T720 PR:000010246 35992 35996 Mcm4 +T721 GO:0097373 35992 36000 Mcm4/6/7 +T722 NCBITaxon:40674 36085 36094 mammalian +T723 PR:000010246 36095 36099 Mcm4 +T724 GO:0097373 36095 36103 Mcm4/6/7 +T725 NCBITaxon:2759 36159 36169 eukaryotic +T726 GO:0006260 36170 36181 replicative +T727 GO:0006270 36201 36230 initiation of DNA replication +T728 GO:0042555 36232 36235 Mcm +T729 GO:0032508 36299 36305 melted +T730 SO:0000296 36306 36312 origin +T731 GO:0032508 36349 36372 unwinding of duplex DNA +T732 SO:0000985 36362 36368 duplex +T733 SO:0000296 36428 36454 origins of DNA replication +T734 GO:0006260 36439 36454 DNA replication +T735 CHEBI:59132 36481 36488 antigen +T736 GO:0032508 36662 36668 melted +T737 GO:0042555 36681 36684 Mcm +T738 GO:0033202 36784 36802 helicase complexes +T739 SO:0000296 36855 36862 origins +T740 GO:0032508 36891 36912 melting of duplex DNA +T741 SO:0000985 36902 36908 duplex +T742 GO:0005656 36953 36958 preRC +T743 GO:0000785 36995 37004 chromatin +T744 PR:000010250 37095 37099 Mcm8 +T745 GO:0005657 37153 37157 fork +T746 PR:000005215 37185 37190 Cdc45 +T747 PR:000005218 37244 37248 Cdc7 +T748 PR:000010246 37331 37335 Mcm4 +T749 GO:0097373 37331 37339 Mcm4/6/7 +T750 SO:0000985 37351 37357 duplex +T751 PR:000010246 37413 37417 Mcm4 +T752 GO:0097373 37413 37421 Mcm4/6/7 +T753 GO:0006260 37460 37471 replicative +T754 GO:0006260 37488 37499 replication +T755 GO:0005657 37488 37505 replication forks +T756 PR:000010246 37530 37534 Mcm4 +T757 GO:0097373 37530 37546 Mcm4/6/7 complex +T758 SO:0000985 37583 37589 duplex +T759 GO:0006260 37590 37605 DNA replication +T760 GO:0005657 37590 37611 DNA replication forks +T761 PR:000010246 37613 37617 Mcm4 +T762 GO:0097373 37613 37621 Mcm4/6/7 +T763 GO:0042555 37704 37707 Mcm +T764 PR:000005215 37718 37723 Cdc45 +T765 GO:0000811 37733 37737 GINS +T766 PR:000010250 37777 37781 Mcm8 +T767 GO:0006260 37856 37867 replicating +T768 SO:0001026 37879 37885 genome +T769 PR:000010246 38485 38489 Mcm4 +T770 GO:0097373 38485 38493 Mcm4/6/7 +T771 GO:0005657 38526 38530 fork +T772 CHEBI:37972 38630 38633 32P +T773 PR:000010246 38741 38745 Mcm4 +T774 GO:0097373 38741 38749 Mcm4/6/7 +T775 CHEBI:60004 38768 38776 mixtures +T776 GO:0005657 38862 38866 fork +T777 PR:000010246 38993 38997 Mcm4 +T778 GO:0097373 38993 39001 Mcm4/6/7 +T779 SO:0000984 39134 39149 Single-stranded +T780 SO:0000985 39154 39160 duplex +T781 PR:000010246 39630 39634 Mcm4 +T782 GO:0097373 39630 39638 Mcm4/6/7 +T783 GO:0097617 39747 39755 annealed +T784 SO:0000028 39800 39802 bp +T785 NCBITaxon:10088 39854 39859 mouse +T786 PR:000010246 39860 39864 Mcm4 +T787 GO:0097373 39860 39868 Mcm4/6/7 +T788 SO:0000984 39901 39916 single-stranded +T789 NCBITaxon:10088 40007 40012 mouse +T790 PR:000010246 40013 40017 Mcm4 +T791 GO:0097373 40013 40021 Mcm4/6/7 +T792 PR:000010246 40157 40161 Mcm4 +T793 GO:0097373 40157 40165 Mcm4/6/7 +T794 CHEBI:37972 40309 40312 32P +T795 SO:0000984 40399 40405 single +T796 PR:000010246 40450 40454 Mcm4 +T797 GO:0097373 40450 40458 Mcm4/6/7 +T798 PR:000010246 40468 40472 Mcm4 +T799 GO:0097373 40468 40476 Mcm4/6/7 +T800 SO:0000984 40561 40569 unpaired +T801 PR:000010246 40893 40897 Mcm4 +T802 GO:0097373 40893 40909 Mcm4/6/7 complex +T803 PR:000010246 40965 40969 Mcm4 +T804 GO:0032991 40965 40973 Mcm4/6/7 +T805 PR:000010246 41149 41153 Mcm4 +T806 GO:0097373 41149 41157 Mcm4/6/7 +T807 GO:0005657 41169 41173 fork +T808 PR:000010246 41227 41231 Mcm4 +T809 GO:0097373 41227 41235 Mcm4/6/7 +T810 GO:0005657 41248 41252 fork +T811 PR:000010246 41399 41403 Mcm4 +T812 GO:0097373 41399 41407 Mcm4/6/7 +T813 PR:000023591 41485 41489 PriA +T814 GO:0032991 41724 41733 complexes +T815 CHEBI:37972 41921 41924 32P +T816 PR:000010246 41992 41996 Mcm4 +T817 GO:0097373 41992 42000 Mcm4/6/7 +T818 GO:0006273 42004 42007;42015 42024 5′- ... extension +T819 GO:0006272 42012 42024 3′-extension +T820 PR:000010246 42084 42088 Mcm4 +T821 GO:0097373 42084 42092 Mcm4/6/7 +T822 GO:0006272 42118 42130 3′-extension +T823 GO:0006273 42243 42255 5′-extension +T824 GO:0006272 42257 42269 3′-extension +T825 GO:0005657 42276 42282 forked +T826 PR:000010246 42352 42356 Mcm4 +T827 GO:0097373 42352 42368 Mcm4/6/7 complex +T828 CHEBI:37972 42480 42483 32P +T829 SO:0000985 42606 42612 duplex +T830 PR:000010246 42627 42631 Mcm4 +T831 GO:0097373 42627 42635 Mcm4/6/7 +T832 SO:0000985 42705 42711 duplex +T833 SO:0000984 42738 42753 single-stranded +T834 SO:0000988 42754 42762 circular +T835 CHEBI:37972 42867 42870 32P +T836 GO:0097617 42894 42902 annealed +T837 SO:0000985 42995 43001 duplex +T838 PR:000010246 43157 43161 Mcm4 +T839 GO:0097373 43157 43173 Mcm4/6/7 complex +T840 PR:000010246 43235 43239 Mcm4 +T841 GO:0097373 43235 43243 Mcm4/6/7 +T842 NCBITaxon:9606 43287 43292 human +T843 PR:000000084 43293 43298 c-myc +T844 SO:0000296 43299 43305 origin +T845 PR:000010246 43334 43338 Mcm4 +T846 GO:0097373 43334 43342 Mcm4/6/7 +T847 PR:000000084 43409 43414 c-myc +T848 PR:000009861 43420 43428 Lamin B2 +T849 PR:000000084 43433 43438 c-myc +T850 SO:0000984 43462 43470 unpaired +T851 PR:000010246 43629 43633 Mcm4 +T852 GO:0097373 43629 43637 Mcm4/6/7 +T853 PR:000010246 43762 43766 Mcm4 +T854 GO:0097373 43762 43778 Mcm4/6/7 complex +T855 SO:0000985 43867 43873 duplex +T856 PR:000010246 43905 43909 Mcm4 +T857 GO:0097373 43905 43913 Mcm4/6/7 +T858 PR:000010246 43975 43979 Mcm4 +T859 GO:0097373 43975 43983 Mcm4/6/7 +T860 GO:0005657 44020 44024 fork +T861 SO:0000985 44077 44083 duplex +T862 SO:0000984 44119 44134 single-stranded +T863 SO:0000988 44135 44143 circular +T864 PR:000010246 44276 44280 Mcm4 +T865 GO:0097373 44276 44284 Mcm4/6/7 +T866 NCBITaxon:10633 44334 44338 SV40 +T867 CHEBI:59132 44341 44348 antigen +T868 SO:0000985 44498 44504 duplex +T869 PR:000010246 44615 44619 Mcm4 +T870 GO:0097373 44615 44623 Mcm4/6/7 +T871 CHEBI:37972 44923 44926 32P +T872 SO:0000985 44954 44960 duplex +T873 CHEBI:37972 44972 44975 32P +T874 GO:0097617 44999 45007 annealed +T875 SO:0000028 45051 45053 bp diff --git a/src/ontogpt/evaluation/craft/database/all/15917436.txt b/src/ontogpt/evaluation/craft/database/all/15917436.txt new file mode 100644 index 000000000..bf879a988 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15917436.txt @@ -0,0 +1,161 @@ +DNA binding and helicase actions of mouse MCM4/6/7 helicase + +Abstract + +Helicases play central roles in initiation and elongation of DNA replication. We previously reported that helicase and ATPase activities of the mammalian Mcm4/6/7 complex are activated specifically by thymine-rich single-stranded DNA. Here, we examined its substrate preference and helicase actions using various synthetic DNAs. On a bubble substrate, Mcm4/6/7 makes symmetric dual contacts with the 5′-proximal 25 nt single-stranded segments adjacent to the branch points, presumably generating double hexamers. Loss of thymine residues from one single-strand results in significant decrease of unwinding efficacy, suggesting that concurrent bidirectional unwinding by a single double hexameric Mcm4/6/7 may play a role in efficient unwinding of the bubble. Mcm4/6/7 binds and unwinds various fork and extension structures carrying a single-stranded 3′-tail DNA. The extent of helicase activation depends on the sequence context of the 3′-tail, and the maximum level is achieved by DNA with 50% or more thymine content. Strand displacement by Mcm4/6/7 is inhibited, as the GC content of the duplex region increases. Replacement of cytosine–guanine pairs with cytosine–inosine pairs in the duplex restored unwinding, suggesting that mammalian Mcm4/6/7 helicase has difficulties in unwinding stably base-paired duplex. Taken together, these findings reveal important features on activation and substrate preference of the eukaryotic replicative helicase. + +INTRODUCTION + +DNA helicases couple the hydrolysis of nucleotide or deoxynucleotide triphosphate to duplex unwinding (1). Coordination of a set of subactivities including nucleotide binding, DNA binding and ATP hydrolysis is required for unwinding of duplex DNA (1,2). The hexameric helicases, to which minichromosome maintenance (Mcm) helicase belongs, are known to assemble a ring-like structure (3–5). Whereas most of DNA helicases of this group, including simian virus 40 large T-antigen (6), Escherichia coli DnaB (7) and Archaeal Mcm (4,8,9), are composed of a single subunit, the active eukaryotic Mcm helicases are composed of two trimeric Mcm4/6/7 proteins (10–14). + +All the six Mcm 2–7 proteins contain highly conserved DNA-dependent ATPase motifs in their central regions (15,16). Among the several stable subcomplexes which MCM proteins can generate, only the Mcm4/6/7 complex has been shown to possess an intrinsic DNA helicase activity (10–14,17–18). While Mcm4, Mcm6 and Mcm7 proteins make distinct contribution to its helicase activity (11–13), Mcm2 or Mcm3/5 inhibit the helicase activity of the Mcm4/6/7 complex by converting its double trimer structure into a heterotetramer or heteropentamer, respectively (3,11). Chromatin immunoprecipitation assays and genetic characterization in Saccharomyces cerevisiae strongly suggested that Mcm is involved not only in initiation but also in the DNA chain elongation stage as a replicative helicase (19,20). Consistent with this notion, the processivity of the Schizosaccharomyces pombe and mouse Mcm4/6/7 complexes is significantly stimulated on forked DNA structures and it can unwind duplex DNA of 400–500 bp (13,14). Mcm4/6/7 binds to fork and bubble structures in an ATP-dependent manner, and generates a double-hexameric complex, as was shown for T-antigen (13,14,21). Recently, we reported that the helicase and ATP hydrolysis activities of mammalian Mcm4/6/7 are specifically activated by single-stranded DNA containing stretches of thymine residues and proposed a novel model that Mcm may play a crucial role in selection of replication origins in higher eukaryotes (13). + +In this report, in order to clarify the mode of action of the Mcm helicase and obtain insight into how it may function at the replication forks in vivo, we have conducted detailed analyses of helicase action and DNA binding of mouse Mcm4/6/7 helicase using various forked and bubble substrate DNAs. We specifically addressed sequence requirement for the Mcm helicase activation, mode of interaction with DNA substrates and whether the thymine sequences are required for continuous activation of Mcm helicase during the unwinding process. The results indicate that mammalian Mcm4/6/7 primarily binds to single-stranded DNA region, and that the extent of helicase activation is related to the thymine content of the single-stranded segment. Unexpectedly, Mcm4/6/7 helicase is not capable of efficiently unwinding the GC-rich duplex segment, suggesting that some other mechanism may be required for completion of replication of the entire genome. + +MATERIALS AND METHODS + +Reagents + +Labeled and unlabeled dNTPs/rNTPs and Sequenase were purchased from Amersham Pharmacia. M13mp18 and M13mp19 single-stranded circular DNA, T4 polynucleotide kinase, DNase I and the Klenow fragment were from TAKARA, Nuclease P1 was from Roche, and anti-FLAG M2 Ab (antibody)-agarose and FLAG peptide were from Sigma. SV40 T-antigen was purified from baculovirus-infected insect cells as reported (22). PriA helicase protein was provided by Dr Taku Tanaka (23). Oligonucleotides were purchased from Hokkaido system science Co., Ltd (Hokkaido, Japan), and were purified on polyacrylamide gel, followed by purification by Sep-Pak Plus C18 cartridge column (Waters). + +Expression and purification of mouse Mcm4/6/7 complex + +The recombinant baculoviruses expressing His6-Mcm4/Mcm6 or Mcm7-FLAG proteins were previously described (11). Sf9 and High five insect cells were cultured at 27°C in Sf-900 II SFM (Life Technologies, Inc.) and EX-CELL 405 medium (JRH Biosciences), respectively. For expression of the Mcm4/6/7 proteins, High five cells were coinfected with recombinant baculoviruses expressing the His6-Mcm4/Mcm6 proteins and those expressing the Mcm7-FLAG, and were collected at 48 h post-infection. The recombinant Mcm4/6/7 complexes were purified as previously described (12). + +DNA substrates + +The sequences for all the oligonucleotides used for constructions of DNA substrates are listed in Tables 1 and 2. The partial heteroduplex substrates were constructed by annealing labeled oligonucleotides with M13mp18 single-stranded circular DNA or its derivative. The labeled substrates were purified by Sepharose CL4B column chromatography (Amersham Pharmacia Biotech). + +The DNA bubble substrates were assembled from two partially complementary oligonucleotides with top and bottom strand sequences (Table 1). T-tailed Y-fork substrates were prepared by annealing two oligonucleotides (dT30 and dT60 series). To construct the arrested fork and 3′- or 5′-extension substrates, various combinations of oligonucleotides shown in Table 1 were annealed. Bub-82/lamin B2 containing the T-rich strand of the lamin B2 origin region was constructed as previously described (13). The bubble substrate, c-myc/DUE or c-myc/DUE-C, contains sequences (GenBank accession number K01908) from nucleotides 722 to 853 or those from 828 to 747 (complementary strand), respectively, in the central melted region. For each substrate, labeled oligonucleotide (3 pmol) was annealed with the unlabeled oligonucleotide (6 pmol) in a reaction mixture (50 μl) containing 20 mM Tris–HCl (pH 7.5), 10 mM MgCl2 and 25 mM NaCl, which were heated to 95°C, kept at 67°C for 1 h, and then allowed to slowly cool down to 37°C. The assembled substrates were purified from polyacrylamide gel by elution into TE buffer (24). + +The partial heteroduplex substrates on a single-stranded circular DNA with varied lengths of duplex regions were prepared by extending DNA chains from the 3′ end of the dT40-37mer (37mer region hybridizing at positions 6289–6326 of M13mp18 vector) annealed to the single-stranded circular DNA. DNA chains were elongated with Sequenase in the presence of [α-32P]dGTP, followed by extension after addition of ddGTP and all four dNTPs, resulting in substrates containing labeled duplex regions of varied lengths (13). The substrate (5 fmol) was first incubated with indicated amount of the Mcm4/6/7 complex at 37°C for 1 h in reaction mixture as described (11), and then reactions were stopped by addition of EDTA (20 mM), SDS (0.1%) and 2 μg proteinase K, followed by incubation for 20 min. Aliquots were electrophoresed on a 6.5% polyacrylamide gel in 1× TBE at 300 V for 2.5 h. The single-stranded circular DNAs used were M13mp18 and M13mp19+G-rich. The latter was constructed by inserting a G-rich repeat sequence (GGGGCGTGGGC)6, prepared by annealing of two oligonucleotides (5′-GATCC-[GGGGCGTGGGC]6-3′/5′-GATCC-[GCCCACGCCCC]6-3′) at the BamHI site of M13mp19 and the sequences of the insert were confirmed. + +DNA helicase and gel shift assays + +In gel shift assays, Mcm4/6/7 proteins were incubated at 30°C for 30 min in reaction mixtures (15 μl) containing 25 mM HEPES–NaOH (pH 7.5), 50 mM sodium acetate, 10 mM Mg(CH3COO)2, 1 mM DTT, 0.25 μg/ml BSA, 0.5 mM ATP-γ-S and labeled substrates of the amount indicated. After addition of 2 μl of 50% glycerol, the reaction mixtures were directly applied on a polyacrylamide gel containing 6 mM magnesium acetate and 5% glycerol in 0.5× TBE, and electrophoresis was conducted at room temperature. For DNA helicase assays, the reaction mixtures of the gel shift assays, as described above, were incubated at 30°C for 15 min, and then the ATP was added at 10 mM, followed by incubation at 37°C for additional 30 min. The reactions were terminated by addition of EDTA (20 mM), SDS (0.1%) and 2 μg proteinase K, and were incubated for additional 15 min. The samples were separated by electrophoresis on nondenaturing polyacrylamide gel in 1× TBE. + +Nuclease protection assays + +The reaction mixtures (25 μl) contained 25 mM HEPES–NaOH (pH 7.5), 50 mM sodium acetate, 10 mM Mg(CH3COO)2, 1 mM DTT, 0.25 mg/ml BSA, 0.5 mM ATP-γ-S, 0.5 mM CaCl2, 10 fmol of 32P-labeled bubble substrate, and the indicated amount of Mcm4/6/7 proteins. After incubation at 30°C for 30 min, the indicated amount of DNase I (TAKARA Biomedical, Japan) was added, and the mixtures were incubated at room temperature for 1 min. DNase I was inactivated by the addition of 250 μl of stop solution (20 mM EDTA, 0.2M sodium chloride, 1% SDS and 12.5 μg/ml yeast RNA). For nuclease P1 footprinting assay, binding reactions were carried out under the same conditions except that CaCl2 was omitted. P1 nuclease (0.3 U, Roche) was added and incubation was continued at 37°C for 1 min. Cleavage was stopped by addition of 50 μl of 25 mM EDTA and 0.4 M NaOH; the mixture was incubated at room temperature for 10 min followed by the addition of 12 μl of 3 M sodium acetate (pH 4.8). Proteins were removed by phenol–chloroform extraction, and DNAs were collected by ethanol precipitation followed by wash with 70% ethanol. The digested DNA was subjected to electrophoresis through a 10 or 12% polyacrylamide sequencing gel containing 7 M urea, followed by autoradiography. + +RESULTS + +Mcm4/6/7 complex primarily interacts with the single-stranded DNA segment + +Mcm4/6/7 binds to ‘bubble’ DNA substrates and unwinds the duplex segments when the central single-stranded segment contains thymine stretches (13). In order to examine the mode of binding of Mcm4/6/7 on bubble DNAs, we conducted nuclease protection assays using labeled bubble substrate DNAs and Mcm4/6/7 in the presence of ATP-γ-S which permits binding to substrate DNA but does not mobilize the helicase. At an optimum concentration of DNase I, strong cleavages were detected on the 21 bp duplex regions at both ends of the substrates, whereas weaker cleavages were detected on the central single-stranded region, consistent with preference of DNase I to double-stranded DNA. With the Bub66/T-rich substrate, strong protection was detected on the entire single-stranded regions of both top and bottom strands with increasing concentration of Mcm4/6/7, consistent with high affinity of Mcm4/6/7 to T-rich bubble sequences (Figure 1A). Similar, but less significant protection was detected on the single-stranded DNA segment with P1 nucleases (Figure 1A), which is specific to single-stranded DNA. The results indicate that the Mcm complex is loaded onto bubble DNA through binding to both strands of the single-stranded region. Careful examination of Mcm4/6/7 footprints on a bubble indicated strong protection on the 5′-half of the top strand and moderate protection on the remaining 3′-half, as well as on several base-pair duplex segment adjacent to the branch point (Figure 1A and data not shown). A similar pattern of protection was observed also on the bottom strand. This suggests that Mcm4/6/7 may bind to a replication bubble substrate with a 2-fold symmetry as a double hexamer (Figure 1A, see the lower scheme), in a manner similar to SV40 T-antigen (21). + +Nuclease protection assays were conducted also on a T-tailed Y-fork structure (Figure 1B). Not only the single-stranded tail region but also the 7 bp duplex DNA adjacent to the duplex-to-single-strand junction were protected by Mcm binding from DNaseI digestion. These results indicate that Mcm4/6/7 protein primarily interacts with single-stranded DNA region and that the interaction also spans the duplex segments near the branchpoint of the replication fork. + +Formation of double hexameric Mcm4/6/7 complexes facilitates helicase actions on bubble substrates + +The above results suggest that Mcm4/6/7 generates a double hexameric complex on a bubble, each hexamer of which makes symmetric contact with the 5′-proximal central single-stranded segment. This predicts that the single-stranded segment needs to be of a sufficient length in order to accommodate the Mcm complex. In fact, we previously showed that the Mcm4/6/7 mainly forms a single hexameric complex on bubble-20 (a bubble substrate with 20 nt long central single-stranded segment) and a double hexameric complex on bubble-60 DNA (13). In order to determine the minimum requirement for double-hexamer formation, a set of synthetic bubble-like substrates (Bub-T10, Bub-T20, Bub-T30, Bub-T40 and Bub-T50) that differed in the length of the central unpaired segment were constructed, and the binding and helicase activities of Mcm4/6/7 on these substrates were examined. Mcm4/6/7 was incubated with a radiolabeled synthetic bubble substrate in the presence of 0.5 mM ATP-γ-S to allow complex formation. A half of these reaction mixtures were analyzed for DNA binding in gel shift assays (Figure 2A), and the remainder was further incubated in the presence of 10 mM ATP to measure DNA helicase activity (Figure 2B). Bub-10 generated only a low level of complexes with Mcm4/6/7, and Bub-20 generated primarily a single mobility-shifted form, which may contain a single hexamer of Mcm4/6/7. On Bub-30, significant amount of complexes containing a double hexamer were generated in addition to those containing a single hexamer (Figure 2A) (13,21). The amounts of double hexamer complexes increased on Bub-T40 and Bub-T50 with concomitant decrease of single hexamer complexes (Figure 2A). The results indicate that a single-stranded segment of at least 40 nt long is required for efficient loading of the double-hexameric Mcm helicase on a bubble substrate. + +In DNA helicase assays, Mcm4/6/7 complex did not show helicase activity on either Bub-10 or Bub-20 substrate. The efficiency of displacement slightly improved as the length of the single-stranded segment increased up to 30 nt. On Bub-T40 and Bub-T50, on the other hand, significant displacement was observed, indicating that a single-stranded segment of at least 40 nt long is required for efficient helicase actions on bubble substrates (Figure 2B). Taken together, the above results are consistent with the notion that the Mcm4/6/7 complex, loaded onto DNA as a double hexameric complex, efficiently unwinds the duplex region of the substrate DNA. + +Both strands of thymine-rich single-stranded segments are required for efficient unwinding of bubble DNA by Mcm4/6/7 + +Symmetric binding of the Mcm4/6/7 double hexamer to bubble suggests that each hexamer catalyzes unwinding of one of the two duplex segments. We asked whether activation of both hexamers is required for unwinding of the bubble substrate. The preferred bubble substrate which is efficiently unwound by Mcm4/6/7 contains thymine stretches on both strands of the single-stranded segment. We constructed a hetero-bubble (Bub66/T–G-rich) in which one strand is T-rich (same as Bub66/T-rich) and the other strand is G-rich (same as Bub66/G-rich) (13), and helicase assays were conducted with increasing amount of Mcm4/6/7. Only weak unwinding was detected on this substrate compared to Bub66/T-rich (Figure 3A). Since Mcm4/6/7 can bind to Bub66/T–G-rich and forms a double hexameric complex (data not shown), the defective unwinding is not due to inefficient loading of the helicase onto the substrate. If one hexamer interacting with the thymine-rich strand dissociates from the other hexamer and displaces only one of the duplex segments, then it would result in generation of Y-fork structures which could not be further displaced due to the presence of G-rich 3′-tail and would appear on the gel. However, we did not detect such intermediates during the course of the experiment (Figure 3B), suggesting that bubble can be displaced efficiently only when two molecules of the Mcm4/6/7 hexamers are simultaneously activated. + +Binding and helicase actions of Mcm4/6/7 on forked and 3′- or 5′-extension substrates + +In order to examine more precisely the interaction and helicase actions with forked substrates, Y-fork substrates containing a nascent leading strand (A-fork[3′]), nascent lagging strand (A-fork[5′]), or both of them (A-fork[3′,5′]), as well as 5′-extension or 3′-extension structures were constructed. Mcm4/6/7 bound to A-fork[3′] or A-fork[5′] with affinity similar to that of a simple Y-fork (Figure 4A and data not shown). In contrast, PriA helicase, used as a control, bound to A-fork[3′] more efficiently than to A-fork[5′], as reported previously (23,25). Mcm4/6/7 bound also to 5′-extension and 3′-extension, albeit with slightly reduced affinity compared to A-fork[3′] and A-fork[5′] (Figure 4A). Mcm4/6/7 bound to A-fork[3′,5′] with much lower affinity, and the complex gave rise to a single band. These findings indicate that the Mcm4/6/7 complex is loaded onto DNA preferentially and efficiently through single-stranded region, regardless of the presence of 3′- or 5′-terminus. + +In contrast, only the substrate containing a 3′ single-stranded DNA tail (A-fork[5′]) was efficiently unwound and A-fork[3′] or A-fork[3′,5′], was not unwound by Mcm4/6/7 (Figure 4B). Absence of helicase actions on A-fork[3′,5′] composed only of duplex regions is expected since double-stranded DNA is not able to activate the ATPase activity of Mcm4/6/7 (10). In contrast, the PriA, a structure-specific helicase used as a control, binds at the fork junction and unwinds the fork and the nascent lagging strand DNA on A-fork[5′], or unwinds the nascent lagging strand on A-fork[3′,5′], as reported before (25). These results indicate that the Mcm4/6/7 complex binds to single-stranded DNA and that its helicase is activated only in the presence of a single-stranded 3′-tail. + +3′-extension with poly(dT) tail is displaced by Mcm4/6/7 + +It has been reported that the 3′-extension can be unwound by the archaeal Mcm homohexamer but not by fission and budding yeast Mcm complexes (14,26,27). We have constructed 3′-extension substrates containing poly(dT), poly(dA), poly(dC) and poly(dG) 3′-tails in order to examine whether mouse Mcm4/6/7 can displace these substrates. We first found that Mcm4/6/7 binds to dT-, dC- and dG-3′-extension with similar affinity, and to dA-3′-extension with much lower affinity (Figure 5A). This is consistent with the previous results that Mcm4/6/7 binds to 50mer oligo-dT, -dC and -dG, but not to a 50mer oligo-dA (13). We found that dT-tailed 3′-extension was displaced efficiently by Mcm 4/6/7 while almost no or very little activity was detected on other substrates (Figure 5B and C). This is in sharp contrast to yeast Mcm4/6/7 helicases which are not capable of unwinding 3′-extension substrates. Furthermore, dT-tailed 5′-extension, to which Mcm4/6/7 could bind, was not displaced by the Mcm helicase. The addition of the 5′-tail to dT-tailed 3′-extension further stimulated the displacement (Figure 5D), confirming the previous report that the fork structures facilitate the helicase action. Thus, these results further strengthen our conclusion that the mammalian Mcm helicase is generally activated by the thymine-stretch on the 3′-single-stranded tail. + +The effects of the thymine content of the 3′-tail on Mcm helicase activity + +We next examined more systematically the effect of the compositions of the thymine residues of the 3′-tail on the Mcm helicase activity. A partial heteroduplex M13 single-stranded DNA containing a 37 bp duplex region in the presence or absence of the 50 nt long 3′-tail of various nucleotide compositions were constructed and the unwinding by mouse Mcm4/6/7 was examined (Figure 6A). Due to its low processivity, Mcm4/6/7 barely displaced the annealed 37mer oligonucleotide without a 3′-tail (37mer; Figure 6A). Only weak helicase activity was observed on the substrate carrying the 3′-tail of 25 repeats of TA dinucleotide (37-TA25; Figure 6A), whereas strong helicase activity was observed on 37-TTA17. Increase of thymine content on the 3′-tail did not further increase the extent of unwinding (37-TTTA13, 37-TTTTA10 and 37-T50; Figure 6A), suggesting that 17 repeats of TT dinucleotides (67% thymine) may be sufficient for activation of Mcm helicase. It should be noted that the TA25 tail would self-anneal and this would prevent the loading of Mcm, since the 3′-tail containing TTAA13 or TTTAAA8 did not enhance the unwinding either (data not shown). Therefore, we next examined the effects of repeating sequences of thymine and cytosine (Figure 6B). Partial heteroduplex substrates containing the 3′-tail of TC25, TTCC13, TTTCCC8, TTTTCCCC6, TTCC17 were displaced by the Mcm4/6/7 complex to the extent similar to that achieved by TTA17, suggesting that 50% thymine content is sufficient for full activation of Mcm helicase. TTCC13 and TTCC17 were displaced to a similar extent, consistent with the earlier result that a 40 nt tail is sufficient for activation (13). However, the efficiency of the displacement significantly decreased as the thymine content of the 3′-tail dropped to 33%, as in TAA17 or TCC17 (Figure 6B and C). Thus, the minimal thymine content required for activation of the Mcm helicase may be somewhere between 33 and 50%. + +DUE sequences from the c-myc origin activate Mcm helicase + +Our previous studies have shown that the sequences containing periodic (dT)n tracts derived from the human lamin B2 origin (48% thymine content) can serve as an efficient activator for the Mcm helicase on a bubble, and replacement of thymines with guanines abolished the helicase activation (13,28). In order to examine other natural replication origins with different thymine contents for the ability to activate Mcm helicase on a bubble substrate, we constructed new bubble substrates, Bub82/c-myc, containing sequences derived from the DUE (DNA unwinding element) region of the human c-myc origin which is essential for c-myc replicator activity (29,30). The unpaired segment in Bub82/c-myc/DUE contains one strand of DUE and that in Bub82/c-myc/DUE-C contains another strand. This DUE is believed to coincide with the initially unwound region of the c-myc origin (29), and its deletion substantially reduced the replicator activity (30). Mcm4/6/7 unwound Bub82/lamin B2 (thymine 48%), Bub82/c-myc/DUE (37%) and Bub82/c-myc/DUE-C (39%) with similar efficiency (Figure 7A), indicating that the sequences from the c-myc origin also can serve as efficient activators of the Mcm helicase. This result also indicates that sequences with 37% thymine content can activate Mcm4/6/7 to displace 24 nt duplex on both sides in a bubble substrate, suggesting that the not only thymine content but also the sequence context may affect the Mcm helicase activation. + +In gel shift assays, MCM4/6/7 bound to all the three bubbles, although the affinity to Bub82/c-myc/DUE and to Bub82/c-myc/DUE-C is slightly lower than that to Bub-82/lamin B2 (Figure 7B). Therefore, helicase and DNA binding activities do not necessarily correlate with each other. This was also indicated by our previous results that guanine and cytosine stretches can bind to Mcm but cannot activate the helicase (13). + +DNA unwinding by Mcm4/6/7 is inhibited by the GC-rich sequences on the duplex segment + +Our results indicate that thymine-rich single-stranded DNA is required for initial loading and activation of the Mcm helicase. However, it is not known whether thymine sequences are required for processive unwinding of duplex DNA. Therefore, we have examined whether the nucleotide composition of the duplex region affects its unwinding activity. To address this issue, we constructed a series of T-tailed Y-fork structures (T-fork) containing various sequences in the duplex segment. They carried varied contents of cytosine residues on the 3′-tail strand. In gel shift assays, Mcm4/6/7 bound to these Y-fork substrates with identical affinity (Figure 8A), consistent with the notion that the Mcm4/6/7 binds to single-stranded tails. However, in DNA helicase assays, T-fork/(C:G)49 was hardly displaced by Mcm4/6/7, but was readily displaced by SV40 T-antigen DNA helicase (Figure 8B). When thymine or adenine is inserted as every third nucleotide (repeats of CCT or CCA), the extent of unwinding increased (T-fork/(CCT:GGA)16 and T-fork/(CCA:GGT)16). The efficiency of unwinding is roughly correlated with the content of GC pairs in the duplex segment [T-fork/(CCAA:GGTT)12, T-fork/(CCTT:GGAA)12, T-fork/(CAAA:GTTT)12 and T-fork/(CTTT:GAAA)12; Figure 8B]. It appears that the duplex segment containing <50% GC pairs is efficiently unwound (Figure 8C). + +We next examined the effect of GC-rich segments on a partial heteroduplex DNA on a single-stranded circular DNA. We have constructed two sets of 5′-tailed partial heteroduplex DNA substrates containing duplex regions of variable lengths; one on M13mp18 vector and the other on M13mp19 containing a 66 nt long G-rich segment downstream of the hybridizing oligonucleotide. We found that the Mcm4/6/7 helicase can displace duplex DNA up to 350 nt long on its own on the former substrate (Figure 8D, left), consistent with previous results (13). In contrast, displacement was inhibited over the GC-rich segments on the latter substrate (Figure 8D, right panel), although displacement up to 250 nt in length, albeit at a reduced level, was observed at a high concentration of Mcm4/6/7 complex. This result is consistent with that on Y-fork and indicates that GC-rich duplex segment is inhibitory for unwinding by the Mcm helicase. These results may indicate that the presence of thymine–adenine pairs with a certain frequency in the duplex region may facilitate continuous unwinding by Mcm4/6/7. Alternatively, Mcm4/6/7 is not efficient enough to displace thermodynamically stable GC-base-paired segment. + +In order to distinguish these two possibilities, we designed a new T-fork substrate containing inosine (I) residue instead of guanosine residue. The thermal stability of the I:C base pair is lower than that of the G:C base pair in duplex DNA due to loss of one hydrogen bond and is even lower than A:T base pair (31). We constructed the T-tailed fork substrates containing the (GCC:CGG)10, (GCC:CIG)10 or (GAA:CTT)10 duplex DNA segment (Figure 8E). Mcm4/6/7 hardly displaced the 31 nt long duplex of GCC:CGG repeats, whereas the T-fork/(GAA:CTT)10 was displaced efficiently, as described above. T-fork/(GCC:CIG)10 was displaced with efficiency much greater than that of T-fork/(GCC:CGG)10, indicating that the thermostability of the duplex segment, not the lack of AT base pair, is responsible for inability of Mcm4/6/7 to displace the duplex. + +DISCUSSION + +DNA helicase is a central factor for operation of replication forks. It also plays a crucial role in the initiation step at the replication origins. Therefore, the elucidation of how the replicative helicases interact with DNA substrate and how they are activated is crucial for understanding the molecular basis of initiation of DNA replication. It is now believed that Mcm is a major component of the eukaryotic replicative helicase. In this report, we have conducted detailed analyses on binding and helicase actions of mouse Mcm4/6/7 using various substrates including fork and bubble structures. + +Modes of binding and activation of Mcm4/6/7 helicase on a bubble structure + +Initiation of DNA replication is associated with localized melting of duplex DNA near replication origins. Helicases are loaded onto replication forks through the melted region, induced by initiator binding, in bacteria (32). We previously reported that Mcm4/6/7 can be loaded onto a bubble-like structure and can serve as a DNA helicase at the forks (13). The ability of Mcm4/6/7 to unwind the bubble substrate but not the conventional duplex DNA (Figures 2 and 13) indicates that Mcm can be loaded through the single-stranded segment of the bubble. Furthermore, the Mcm4/6/7 complex displays marked preference for thymine-rich sequences within the single-stranded segment for helicase activation (13). The results in this report indicate that helicase action of Mcm4/6/7 on synthetic bubbles may depend on the presence of an unpaired region of sufficient length (at least 40 nt), which may permit assembly of a double-hexameric complex on the substrate DNA, similar to SV40 T-antigen protein (Figure 2A) (21). When one strand of the single-stranded segment in T-rich bubble was replaced by guanine-rich sequences, the efficiency of unwinding was significantly reduced. + +Our footprinting analyses showed that Mcm4/6/7 strongly protects 25 nt single-stranded DNA segment adjacent to each branch point and proximal to 5′ ends of both strands of the bubble. At each fork, one Mcm4/6/7 complex is likely to encircle the single-stranded DNA strand and two hexamers may bind symmetrically to the bubble substrate, forming a double hexameric structure on the bubble (see schematic drawing of Figure 1A). Efficient unwinding into both directions may require simultaneous activation of both hexamers which may sit at the center while extruding the unwound single-stranded DNA through the rings. This may closely resemble the proposed modes of binding and helicase actions of T-antigen (21). Although we cannot totally exclude the possibility that one Mcm4/6/7 hexamer at each fork unwinds the duplex independently, ring-shaped structures of mouse and archea MCM, as revealed by electron microscopy, bear much similarity to the recently solved structure of the SV40 large T-antigen (4,6), and are in favor of the double-hexameric structure of mouse Mcm on a bubble DNA. + +Substrate and sequence requirement for Mcm4/6/7 helicase activation + +DNA binding assays indicate that Mcm4/6/7 binds to those substrates containing single-stranded DNA regions regardless of the presence or absence of single-stranded 3′ or 5′ end. Unwinding of the duplex DNA depends on translocation of single-stranded DNA from 3′ to 5′ direction. This is most clearly shown by its helicase action on A-fork[5′] and 3′-extension but not on A-fork[3′], A-fork[3′,5′] nor 5′-extension. However, it does not necessarily require 3′ end of single-stranded DNA, since Mcm4/6/7 can displace annealing oligonucleotide on a circular single-stranded DNA. The ability of the mouse Mcm4/6/7 to unwind 3′-extension is shared by the archaeal Mcm helicase but not by Mcm4/6/7 from S.pombe and S.cerevisiae (14,26,27). The archaeal Mcm can unwind A-fork[3′] but eukaryotic Mcm4/6/7 cannot, since the former binds to double-stranded DNA but the latter does not (5,27). While yeast Mcm helicases can translocate on duplex DNA, such activity was not observed with mammalian Mcm4/6/7 (data not shown). + +Occurrence of AT-rich sequences, with asymmetric distribution of adenine and thymine, near the replication origins, lead us to propose that Mcm may play a role in selection of initiation sites of mammalian DNA replication, and prompted us to examine the ability of sequences from human replication origins to activate Mcm4/6/7 helicase. Both lamin-B2 and c-myc origins served as efficient activator for Mcm helicase in vitro. Consistent with it, site-specific loading of Mcm in the DNA replication initiation zone of the c-myc was recently reported (33). + +We have examined in detail the effect of sequence context of the single-stranded DNA on the helicase activity of Mcm. The results indicate that thymine content of 50% is sufficient for the maximum helicase activity. The efficiency of displacement decreased as the thymine content of the 3′-tail dropped to 33% (Figure 6). The stretches of thymine residues may not be necessarily required, since repeats of TC dinucleotides served as a potent activator for Mcm4/6/7. We also noticed that the presence of a secondary structure within the single-stranded DNA is inhibitory for helicase action. Nuclease footprinting assays indicated that binding was interfered by the secondary structure (data not shown). Thus, we have concluded that Mcm4/6/7 helicase is most efficiently activated by non-structured single-stranded DNA with thymine content of 50% or more, although significant stimulation is observed also by DNA with less thymine content (Figure 7), suggesting that the sequence specificity for Mcm helicase activation is rather relaxed and that the extent of the activation may depend on the sequence context. This would be reasonable given the flexibility and differential regulation of site selection for initiation of DNA replication during development or in various cell types, as well as the variability in initiation potential of each replication origin on the genome even within the single cell type. + +Mcm4/6/7 helicase during processive unwinding at the fork + +The specific requirement of single-stranded thymine residues for activation of Mcm helicase prompted us to examine whether they are required also for processive unwinding of duplex DNA. Our results indicated that increase of GC pair in the duplex segment significantly inhibited the Mcm helicase activity. Duplex DNA composed only of GC pairs (10 repeats of CGG) on a Y-fork was not displaced at all, while the same template was readily displaced by SV40 T-antigen (Figure 8B). Mcm helicase was inhibited by the presence of GC-rich duplex segment also on a circular single-stranded partial heteroduplex substrate. However, on this substrate, Mcm4/6/7 was able to displace DNA past the GC-rich region, albeit to a limited extent, when it was added at a high concentration. On the partial heteroduplex template, Mcm is loaded onto the circular single-stranded DNA of 6.4 kb, while it is loaded onto the 50 nt long 3′-tail DNA on the Y-fork. Thus, the difference of helicase actions may reflect the efficiency of Mcm loading. Alternatively, the presence of ‘random’ sequence at the initially unwound duplex segment in the former template may engage the Mcm helicase in a more active conformation which can displace the GC-rich duplex segment. + +On Y-fork substrates, increase of AT base pairs in the duplex (10 repeats of CTT) restored the unwinding. These results indicate either that thymine residues are required on the duplex DNA for continuous unwinding, or that Mcm4/6/7 is simply not efficient enough to unwind the stable GC pairs. Replacement of the central guanosine with inosine in the CGG repeat duplex DNA resulted in displacement (Figure 8E) (31), suggesting that the continuous presence of AT base pair may not be essential for the Mcm helicase function and that the thermostability of GC base pairs is inhibitory for helicase action of Mcm4/6/7. + +The results described in this manuscript reveal potentially important features of mammalian Mcm4/6/7 helicase, which is likely to be a key component of the eukaryotic replicative helicase. Prior to initiation of DNA replication, Mcm helicase may adopt a double hexameric complex at the partially melted origin region, and may catalyze concurrent unwinding of duplex DNA into both directions, while stably associated with the origins of DNA replication. This is similar to the T-antigen model originally proposed by Smelkova and Borowiec (21). We propose that, only when both hexamers are activated by the interacting thymine-rich sequences present within the melted region, the Mcm helicase is mobilized and initiation takes place. A crucial question is how these double hexameric helicase complexes are generated and turn into active helicases at the origins. This process would involve melting of duplex DNA, which may be facilitated by binding of preRC components in the context of proper chromatin structures, or by other unwinding factors including a topoisomerase or a newly identified Mcm8 helicase (34–36). It may also require association of fork-assisting proteins such as Cdc45 (37,38) as well as phosphorylation events by Cdk and Cdc7 kinases (39,40). One unexpected finding of this study is low helicase activity of Mcm4/6/7 on GC-rich duplex DNA. This, in conjunction with the low processivity of Mcm4/6/7 helicase, strongly indicates that the replicative helicase at the replication forks would require more than Mcm4/6/7 complex. During the processive unwinding of duplex DNA replication forks, Mcm4/6/7 may be further stimulated by interaction with other proteins, including remaining Mcm subunits, Cdc45 (37,38), GINS (41,42), DNA polymerase subunits (43), Mcm8 (35,36), etc. to become a truly processive and potent helicase capable of replicating the entire genome. + +Acknowledgements + +We thank Dr Taku Tanaka for preparations of some of the substrate DNAs used in this study and for useful suggestions, and also the members of our laboratory for helpful discussion. This work was supported in part by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT) awarded to Z.Y. and H.M. Funding to pay the Open Access publication charges for this article was provided by MEXT. + +Conflict of interest statement. None declared. + +Figures and Tables + +Figure 1 + +Nuclease protection analyses of binding of Mcm4/6/7 protein to synthetic bubble and fork substrates. (A) The Bub66/T-rich substrate (4 fmol), the top strand or bottom strand of which were 32P-labeled at the 5′ end, were incubated with 0 (lane 1), 25 ng (lane 2), 50 ng (lane 3) or 75 ng (lane 4) of Mcm4/6/7 protein. Reaction mixtures were then treated with 0.11 U of DNase I or 0.3 U of nuclease P1. (B) The T-tailed Y-fork/random substrate (4 fmol) was incubated with 0 (lane 1), 15 ng (lane 2), 30 ng (lane 3), 60 ng (lane 4) or 120 ng (lane 5) of Mcm4/6/7 protein, and then treated with 0.037 U of DNase I or 0.3 U of nuclease P1. The reaction products were separated on denaturing PAGE. Single-stranded and duplex regions of the substrates used in the assays are indicated along the gel. In (A), top and bottom strands are indicated by boldface and normal lines, respectively. Regions of strong and moderate protection are indicated by bold and normal gray lines, respectively, along the substrate structure. The drawing at the bottom of (A) shows summary of the protection pattern (protected regions indicated by gray bold lines) and predicted binding modes of the double hexameric Mcm4/6/7 (shown by pale gray ovals) on the bubble substrate. The star marks represent the radioactive 5′ ends of the annealed oligonucleotides. M, radiolabeled 10 and 50 bp ladder. + +Figure 2 + +Binding and helicase actions of mouse Mcm4/6/7 on bubble substrates containing single-stranded segments of varied lengths. Gel shift (A) and DNA helicase (B) assays were performed with mouse Mcm4/6/7 on synthetic bubble DNAs (4 fmol), Bub-T10, Bub-T20, Bub-T30, Bub-T40 and Bub-T50 (Table 1). Lanes 1–4 contain 0, 25, 50 and 100 ng of Mcm4/6/7 protein, respectively. B, boiled substrate. The drawing in (A) shows a schematic representation of the substrates used. The asterisk indicates 32P-labeled 5′-terminus. + +Figure 3 + +Effect of the loss of thymine-rich sequences from one single-strand of the T-rich bubble on unwinding by Mcm4/6/7. (A) The Mcm4/6/7 helicase activity was measured by using bubble DNA substrate (4 fmol) containing an unpaired segment of two thymine-rich sequences (Bub66/T-rich) or that containing thymine- and guanine-rich sequence on each strand (Bub66/T–G-rich). The drawings under the panels show schematic representations of the substrates used in this assay. B, boiled substrate; lane 1, no protein; lanes 2–4 contain 25, 50 and 100 ng of the Mcm4/6/7 complex, respectively. (B) Helicase assays were conducted with Mcm4/6/7 (50 ng) on the bubble substrates indicated for various time. Quantification of displaced substrates is presented in the graphs. + +Figure 4 + +DNA binding and helicase actions of Mcm4/6/7 on various fork substrates. (A) Gel-shift assays were conducted with Mcm4/6/7 on specific fork and extension substrates (3 fmol) as indicated. (B) DNA helicase assays were conducted on various substrates (3 fmol) used in (A). The amounts of Mcm4/6/7 added were 0 (lane 1), 25 (lane 2), 50 (lane 3) and 100 ng (lane 4). Lane 5, PriA helicase at 10 nM. B, boiled substrate. The drawings show schematic representation of the substrates used in the assays. Arrows a–d indicate displaced products, and their structures are indicated below the panel. The positions of the complexes containing a single hexamer or double hexamer are indicated. In the schematic drawings of the substrates, the labeled oligonucleotides are shown as bold lines, and the asterisks indicate 32P-labeled 5′-termini. + +Figure 5 + +DNA binding and helicase actions of Mcm4/6/7 on 5′- and 3′-extension substrates. DNA-binding (A) and helicase (B) activities of Mcm4/6/7 were examined on various 3′-extension substrates (4 fmol) as shown. (C) Quantification of the displaced substrates in (B). (D) DNA helicase assays on 5′-extension, 3′-extension and Y-forked substrates. Lanes 1–4 are reactions with 0, 25, 50 and 100 ng of the Mcm4/6/7 complex, respectively. Schematic drawings of the substrates used in the assays are also shown. The star marks indicate 32P-labeled 5′-termini. + +Figure 6 + +The effects of the nucleotide compositions of the 3′-tail on displacement of partial heteroduplex substrates by Mcm4/6/7. (A) DNA helicase assays were performed with 3′-tailed partial heteroduplex helicase substrates (on a single-stranded circular DNA; 4 fmol) carrying various nucleotide sequences in the 3′-tail as shown. The asterisks represent the 32P-labeled 5′ ends of the annealed oligonucleotides. (B) DNA helicase assays were performed with similar sets of partial heteroduplex helicase substrates carrying the 3′-tails as shown. (C) Quantification of displaced oligonucleotides in (B). Lanes 1–4 contain 0, 25, 50 and 100 ng of the Mcm4/6/7 complex, respectively. B, boiled substrate. + +Figure 7 + +Activation of Mcm4/6/7 DNA helicase by sequences derived from the human c-myc origin. (A) DNA helicase assays of Mcm4/6/7 were conducted using Bub-82 bubble substrates (6 fmol) containing c-myc/DUE, Lamin B2 and c-myc/DUE-C sequences in the unpaired segment, the thymine contents of which are 48, 37 and 39%, respectively. Quantification of displaced substrates is presented in the graph. (B) DNA binding of Mcm4/6/7 was examined in gel shift assays using the same set of substrates. Lanes 1–4 are reactions with 0, 25, 50 and 100 ng of the Mcm4/6/7 complex, respectively. B, boiled substrate. + +Figure 8 + +Effect of nucleotide compositions of the duplex segments on helicase action of Mcm4/6/7. DNA binding (A) and helicase (B, D and E) activities of the Mcm4/6/7 helicase were examined on various Y-fork DNAs (3 fmol) carrying different nucleotides in the duplex region as shown (A, B and E) or on single-stranded circular partial heteroduplex DNA substrates (5 fmol) (D). (A, B and E) Lane 1, no protein added; lanes 2–4 contain 25, 50 and 100 ng of the Mcm4/6/7 protein, respectively; lane 5 [in (B)], 50 ng of SV40 T-antigen. (C) Quantification of the displaced substrates in (B). (D) Helicase assays on dT40-Nmer/M13mp18 and dT40-Nmer/M13mp19 + G-rich carrying the labeled duplex regions of varied lengths. Lane 1, no protein added; lanes 2–7 contain 25, 50, 75, 125, 200 and 300 ng of the Mcm4/6/7 protein, respectively. Lanes 8 and 9, boiled M13mp18 and M13mp19 + G-rich substrates, respectively. The drawings show schematic representations of the substrates used in the assays. The red segment in bold face in (D) indicates the 66 nt long G-rich segment. The asterisks represent radiaoactive [α-32P]dGTP incorporated into the duplex segment or 32P-labeled 5′ ends of the annealed oligonucleotides. M [in (D)], denatured 50 bp ladder DNA marker; and I [in (E)], inosine. B, boiled substrate. + +Table 1 + +Constructions of helicase substrates used in this study + +The numbers with ‘#’ refer to oligonucleotides whose sequences are given in Table 2. + +Table 2 + +List of oligonucleotides used in this study diff --git a/src/ontogpt/evaluation/craft/database/all/15921521.ann b/src/ontogpt/evaluation/craft/database/all/15921521.ann new file mode 100644 index 000000000..9472e15c5 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15921521.ann @@ -0,0 +1,732 @@ +T1 SO:0000704 10 17 genetic +T2 http://purl.obolibrary.org/obo/MONDO_0009061 35 50 cystic fibrosis +T3 UBERON:0000160 51 61 intestinal +T4 NCBITaxon:10088 88 93 mouse +T5 http://purl.obolibrary.org/obo/MONDO_0009061 151 166 cystic fibrosis +T6 http://purl.obolibrary.org/obo/MONDO_0009061 197 212 cystic fibrosis +T7 PR:000001044 197 248 cystic fibrosis transmembrane conductance regulator +T8 GO:0055085 213 238 transmembrane conductance +T9 GO:0016020 218 226 membrane +T10 PR:000001044 250 254 CFTR +T11 SO:0000704 256 260 gene +T12 SO:0000704 342 347 genes +T13 UBERON:0000160 353 363 intestines +T14 PR:000001044 367 371 Cftr +T15 NCBITaxon:10088 382 386 mice +T16 http://purl.obolibrary.org/obo/MONDO_0009061 388 390 CF +T17 NCBITaxon:10088 391 395 mice +T18 PR:000001044 397 401 Cftr +T19 UBERON:0000912 447 452 mucus +T20 UBERON:0007109 493 501 meconium +T21 http://purl.obolibrary.org/obo/MONDO_0054868 493 507 meconium ileus +T22 UBERON:0000160 519 529 intestinal +T23 http://purl.obolibrary.org/obo/MONDO_0004565 519 541 intestinal obstruction +T24 http://purl.obolibrary.org/obo/MONDO_0002254 542 550 syndrome +T25 SO:0000704 596 603 genetic +T26 NCBITaxon:10088 622 626 mice +T27 http://purl.obolibrary.org/obo/MONDO_0009061 671 673 CF +T28 NCBITaxon:10088 674 678 mice +T29 NCBITaxon:10088 703 708 mouse +T30 CHEBI:33290 709 713 chow +T31 http://purl.obolibrary.org/obo/MONDO_0043579 720 735;740 755 inflammation of small intestine +T32 UBERON:0002108 740 755 small intestine +T33 http://purl.obolibrary.org/obo/MONDO_0009061 855 857 CF +T34 UBERON:0000160 858 868 intestinal +T35 http://purl.obolibrary.org/obo/MONDO_0009061 906 908 CF +T36 NCBITaxon:10088 909 913 mice +T37 SO:0000704 925 932 genetic +T38 http://purl.obolibrary.org/obo/MONDO_0009061 988 990 CF +T39 NCBITaxon:10088 991 995 mice +T40 UBERON:0000160 1061 1071 intestinal +T41 http://purl.obolibrary.org/obo/MONDO_0009061 1072 1074 CF +T42 http://purl.obolibrary.org/obo/MONDO_0009061 1086 1088 CF +T43 NCBITaxon:10088 1089 1093 mice +T44 GO:0007631 1162 1165 fed +T45 NCBITaxon:10088 1170 1175 mouse +T46 CHEBI:33290 1176 1180 chow +T47 UBERON:0000160 1195 1205 intestinal +T48 http://purl.obolibrary.org/obo/MONDO_0002269 1195 1218 intestinal inflammation +T49 SO:0000704 1265 1270 genes +T50 UBERON:0000912 1324 1329 mucus +T51 UBERON:0001983 1350 1367 intestinal crypts +T52 SO:0001026 1609 1615 genome +T53 SO:0001023 1625 1631 allele +T54 http://purl.obolibrary.org/obo/MONDO_0009061 1969 1971 CF +T55 NCBITaxon:10088 1972 1977 mouse +T56 UBERON:0002108 1978 1993 small intestine +T57 NCBITaxon:33208 1998 2004 animal +T58 SO:0000704 2059 2064 genes +T59 SO:0000704 2129 2136 genetic +T60 http://purl.obolibrary.org/obo/MONDO_0009061 2154 2156 CF +T61 UBERON:0000160 2157 2167 intestinal +T62 http://purl.obolibrary.org/obo/MONDO_0009061 2192 2207 Cystic fibrosis +T63 http://purl.obolibrary.org/obo/MONDO_0009061 2209 2211 CF +T64 http://purl.obolibrary.org/obo/MONDO_0009061 2243 2258 cystic fibrosis +T65 PR:000001044 2243 2294 cystic fibrosis transmembrane conductance regulator +T66 GO:0055085 2259 2284 transmembrane conductance +T67 GO:0016020 2264 2272 membrane +T68 PR:000001044 2296 2300 CFTR +T69 SO:0000704 2302 2306 gene +T70 PR:000001044 2373 2377 CFTR +T71 UBERON:0000483 2416 2426 epithelial +T72 GO:0006821 2427 2443 transport of Cl- +T73 GO:0015701 2427 2439;2448 2453 transport of ... 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7265 gene +T203 GO:0010467 7261 7276 gene expression +T204 GO:0010467 7278 7288 Expression +T205 SO:0000704 7306 7311 genes +T206 http://purl.obolibrary.org/obo/MONDO_0009061 7342 7344 CF +T207 NCBITaxon:10088 7345 7349 mice +T208 SO:0000704 7367 7374 genetic +T209 CL:0000097 7388 7397 Mast cell +T210 PR:000010903 7388 7408 Mast cell protease 2 +T211 PR:000010903 7410 7415 Mcpt2 +T212 CL:0000097 7447 7457 mast cells +T213 CL:0000097 7467 7477 mast cells +T214 http://purl.obolibrary.org/obo/MONDO_0009061 7506 7508 CF +T215 NCBITaxon:10088 7509 7514 mouse +T216 UBERON:0000160 7515 7524 intestine +T217 PR:000009913 7526 7554 Leucine-rich α2 glycoprotein +T218 CHEBI:17089 7542 7554 glycoprotein +T219 PR:000009913 7556 7560 Lrg1 +T220 CL:0000775 7599 7610 neutrophils +T221 http://purl.obolibrary.org/obo/MONDO_0009061 7646 7648 CF +T222 NCBITaxon:10088 7649 7654 mouse +T223 UBERON:0000160 7655 7664 intestine +T224 SO:0000704 7675 7679 gene +T225 PR:000009913 7697 7744 leucine-rich high endothelial cell glycoprotein +T226 UBERON:0001986 7715 7726 endothelial +T227 CL:0000115 7715 7731 endothelial cell +T228 CHEBI:17089 7732 7744 glycoprotein +T229 PR:000009913 7746 7750 Lrhg +T230 UBERON:0001986 7794 7805 endothelial +T231 UBERON:0001979 7806 7813 venules +T232 UBERON:0000479 7843 7850 tissues +T233 GO:0030097 7880 7893 Hematopoietic +T234 CL:0000988 7880 7898 Hematopoietic cell +T235 SO:0000673 7899 7909 transcript +T236 UBERON:0000178 7941 7946 blood +T237 CL:0000081 7941 7951 blood cell +T238 GO:0008283 7947 7965 cell proliferation +T239 GO:0010467 7974 7984 expression +T240 http://purl.obolibrary.org/obo/MONDO_0009061 8016 8018 CF +T241 NCBITaxon:10088 8019 8024 mouse +T242 UBERON:0002108 8025 8040 small intestine +T243 UBERON:0001977 8042 8047 Serum +T244 PR:000015919 8042 8058 Serum amyloid A3 +T245 CHEBI:60425 8048 8055 amyloid +T246 PR:000015919 8060 8064 SAA3 +T247 GO:0006953 8078 8089 acute phase +T248 SO:0000704 8090 8094 gene +T249 GO:0010467 8103 8113 expression +T250 UBERON:0013636 8117 8134 villus epithelial +T251 CL:0000066 8124 8140 epithelial cells +T252 http://purl.obolibrary.org/obo/MONDO_0009061 8164 8166 CF +T253 UBERON:0000160 8167 8176 intestine +T254 PR:000015394 8178 8212 Suppressor of cytokine signaling 3 +T255 GO:0019221 8192 8210 cytokine signaling +T256 PR:000015394 8214 8219 SOCS3 +T257 SO:0000704 8251 8255 gene +T258 PR:000025748 8280 8283 JAK +T259 GO:0007259 8280 8296 JAK-STAT pathway +T260 PR:000001933 8284 8288 STAT +T261 GO:0010467 8305 8315 expression +T262 http://purl.obolibrary.org/obo/MONDO_0009061 8339 8341 CF +T263 UBERON:0000160 8342 8351 intestine +T264 PR:000006534 8353 8359 Muclin +T265 PR:000006534 8375 8380 dmbt1 +T266 GO:0010467 8388 8398 expression +T267 http://purl.obolibrary.org/obo/MONDO_0009061 8424 8426 CF +T268 UBERON:0000160 8427 8436 intestine +T269 GO:0009986 8446 8458 cell surface +T270 CHEBI:17089 8459 8471 glycoprotein +T271 UBERON:0000483 8492 8502 epithelial +T272 CHEBI:36357 8514 8522 molecule +T273 PR:000010903 8567 8572 Mcpt2 +T274 http://purl.obolibrary.org/obo/MONDO_0009061 8590 8592 CF +T275 NCBITaxon:10088 8593 8597 mice +T276 PR:000010903 8722 8727 Mcpt2 +T277 GO:0010467 8728 8738 expression +T278 http://purl.obolibrary.org/obo/MONDO_0009061 8747 8749 CF +T279 PR:000009913 8799 8803 Lrg1 +T280 PR:000009913 8804 8808 Lrhg +T281 GO:0010467 8809 8819 expression +T282 http://purl.obolibrary.org/obo/MONDO_0009061 8855 8857 CF +T283 NCBITaxon:10088 8858 8862 mice +T284 http://purl.obolibrary.org/obo/MONDO_0009061 8955 8957 CF +T285 PR:000015919 9007 9011 SAA3 +T286 http://purl.obolibrary.org/obo/MONDO_0009061 9049 9051 CF +T287 NCBITaxon:10088 9052 9056 mice +T288 PR:000015394 9172 9177 SOCS3 +T289 http://purl.obolibrary.org/obo/MONDO_0009061 9212 9214 CF +T290 NCBITaxon:10088 9215 9219 mice +T291 PR:000006534 9384 9390 Muclin +T292 GO:0010467 9398 9407 expressed +T293 http://purl.obolibrary.org/obo/MONDO_0009061 9429 9431 CF +T294 UBERON:0000160 9432 9441 intestine +T295 GO:0010467 9496 9506 expression +T296 http://purl.obolibrary.org/obo/MONDO_0009061 9516 9518 CF +T297 NCBITaxon:10088 9519 9523 mice +T298 GO:0010467 9605 9614 expressed +T299 http://purl.obolibrary.org/obo/MONDO_0009061 9636 9638 CF +T300 NCBITaxon:10088 9639 9643 mice +T301 http://purl.obolibrary.org/obo/MONDO_0009061 9699 9701 CF +T302 GO:0010467 9702 9712 expression +T303 SO:0000704 9798 9805 genetic +T304 SO:0000704 9833 9837 gene +T305 GO:0010467 9833 9848 gene expression +T306 http://purl.obolibrary.org/obo/MONDO_0009061 9852 9854 CF +T307 NCBITaxon:10088 9855 9860 mouse +T308 UBERON:0002108 9861 9876 small intestine +T309 GO:0010467 9882 9892 expression +T310 SO:0000704 9955 9959 gene +T311 SO:0000112 9969 9976 primers +T312 http://purl.obolibrary.org/obo/MONDO_0009061 10063 10065 CF +T313 NCBITaxon:10088 10066 10070 mice +T314 http://purl.obolibrary.org/obo/MONDO_0009061 10098 10100 CF +T315 http://purl.obolibrary.org/obo/MONDO_0009061 10151 10153 CF +T316 http://purl.obolibrary.org/obo/MONDO_0009061 10181 10183 CF +T317 SO:0000704 10309 10314 genes +T318 NCBITaxon:10088 10339 10343 mice +T319 http://purl.obolibrary.org/obo/MONDO_0009061 10378 10380 CF +T320 NCBITaxon:10088 10381 10385 mice +T321 SO:0000704 10497 10501 gene +T322 SO:0000704 10558 10562 gene +T323 GO:0010467 10558 10573 gene expression +T324 NCBITaxon:33208 10697 10704 animals +T325 GO:0071514 10737 10747 imprinting +T326 http://purl.obolibrary.org/obo/MONDO_0009061 10750 10752 CF +T327 NCBITaxon:10088 10753 10757 mice +T328 UBERON:0000160 10792 10802 intestinal +T329 UBERON:0000912 10803 10808 mucus +T330 http://purl.obolibrary.org/obo/MONDO_0009061 10872 10874 CF +T331 NCBITaxon:10088 10875 10880 mouse +T332 UBERON:0002108 10881 10896 small intestine +T333 UBERON:0000912 10920 10925 mucus +T334 UBERON:0001983 10929 10946 intestinal crypts +T335 GO:0016265 11011 11016 death +T336 NCBITaxon:10088 11026 11030 mice +T337 CHEBI:33290 11051 11055 chow +T338 CHEBI:29149 11073 11086 periodic acid +T339 UBERON:0002108 11150 11165 small intestine +T340 UBERON:0000479 11166 11173 tissues +T341 UBERON:0002108 11203 11218 small intestine +T342 SO:0000704 11248 11255 genetic +T343 UBERON:0001983 11286 11303 intestinal crypts +T344 UBERON:0013636 11420 11437 villus epithelium +T345 CL:0000160 11466 11478 goblet cells +T346 UBERON:0000479 11518 11525 tissues +T347 UBERON:0000160 11544 11553 intestine +T348 http://purl.obolibrary.org/obo/MONDO_0009061 11557 11559 CF +T349 NCBITaxon:10088 11560 11564 mice +T350 SO:0000704 11575 11582 genetic +T351 UBERON:0001983 11620 11626 crypts +T352 UBERON:0000912 11652 11657 mucus +T353 http://purl.obolibrary.org/obo/MONDO_0009061 11669 11671 CF +T354 NCBITaxon:10088 11672 11676 mice +T355 UBERON:0000912 11710 11715 mucus +T356 http://purl.obolibrary.org/obo/MONDO_0009061 11734 11736 CF +T357 NCBITaxon:10088 11737 11741 mice +T358 UBERON:0000912 11790 11795 mucus +T359 http://purl.obolibrary.org/obo/MONDO_0009061 11799 11801 CF +T360 NCBITaxon:10088 11802 11806 mice +T361 NCBITaxon:10088 11849 11854 mouse +T362 NCBITaxon:10088 11858 11863 mouse +T363 NCBITaxon:10088 11873 11877 mice +T364 UBERON:0001983 11905 11911 crypts +T365 UBERON:0000912 11928 11933 mucus +T366 UBERON:0001983 11942 11947 crypt +T367 NCBITaxon:10088 11986 11990 mice +T368 UBERON:0000912 12006 12011 mucus +T369 UBERON:0001983 12019 12025 crypts +T370 UBERON:0000912 12063 12068 mucus +T371 http://purl.obolibrary.org/obo/MONDO_0009061 12108 12110 CF +T372 NCBITaxon:10088 12111 12115 mice +T373 UBERON:0000912 12191 12196 mucus +T374 UBERON:0000912 12211 12216 mucus +T375 UBERON:0000912 12250 12255 mucus +T376 http://purl.obolibrary.org/obo/MONDO_0009061 12290 12292 CF +T377 NCBITaxon:10088 12293 12297 mice +T378 UBERON:0001983 12331 12336 crypt +T379 UBERON:0000912 12355 12360 mucus +T380 http://purl.obolibrary.org/obo/MONDO_0009061 12386 12388 CF +T381 NCBITaxon:10088 12389 12393 mice +T382 UBERON:0002108 12458 12473 small intestine +T383 http://purl.obolibrary.org/obo/MONDO_0009061 12491 12493 CF +T384 NCBITaxon:10088 12494 12498 mice +T385 SO:0000704 12516 12523 genetic +T386 UBERON:0000479 12537 12543 Tissue +T387 UBERON:0002108 12655 12670 small intestine +T388 http://purl.obolibrary.org/obo/MONDO_0009061 12711 12713 CF +T389 http://purl.obolibrary.org/obo/MONDO_0009061 12781 12783 CF +T390 UBERON:0000479 12833 12839 tissue +T391 UBERON:0001983 12867 12873 crypts +T392 http://purl.obolibrary.org/obo/MONDO_0009061 12928 12930 CF +T393 UBERON:0000479 12931 12937 tissue +T394 UBERON:0001983 12964 12969 crypt +T395 UBERON:0000912 13026 13031 mucus +T396 http://purl.obolibrary.org/obo/MONDO_0009061 13050 13052 CF +T397 NCBITaxon:10088 13053 13057 mice +T398 UBERON:0001983 13087 13093 crypts +T399 UBERON:0001983 13151 13157 crypts +T400 http://purl.obolibrary.org/obo/MONDO_0009061 13194 13196 CF +T401 NCBITaxon:10088 13197 13201 mice +T402 UBERON:0001983 13246 13252 crypts +T403 UBERON:0001983 13283 13288 crypt +T404 UBERON:0000912 13302 13307 mucus +T405 http://purl.obolibrary.org/obo/MONDO_0009061 13339 13341 CF +T406 NCBITaxon:10088 13342 13346 mice +T407 SO:0000704 13489 13496 genetic +T408 UBERON:0000912 13587 13592 mucus +T409 http://purl.obolibrary.org/obo/MONDO_0009061 13600 13602 CF +T410 UBERON:0002108 13603 13618 small intestine +T411 SO:0000704 13691 13695 gene +T412 GO:0010467 13691 13706 gene expression +T413 GO:0010467 13753 13763 expression +T414 UBERON:0000160 13771 13781 intestinal +T415 CL:0000160 13782 13793 goblet cell +T416 SO:0000704 13800 13804 gene +T417 PR:000010764 13806 13810 Muc2 +T418 UBERON:0000160 13825 13834 intestine +T419 NCBITaxon:10088 13839 13843 mice +T420 PR:000010764 13874 13878 Muc2 +T421 http://purl.obolibrary.org/obo/MONDO_0009061 13962 13964 CF +T422 NCBITaxon:10088 13965 13969 mice +T423 PR:000010764 14021 14025 Muc2 +T424 UBERON:0000912 14141 14146 mucus +T425 http://purl.obolibrary.org/obo/MONDO_0009061 14163 14165 CF +T426 PR:000010764 14197 14201 Muc2 +T427 http://purl.obolibrary.org/obo/MONDO_0009061 14214 14216 CF +T428 UBERON:0002108 14217 14232 small intestine +T429 http://purl.obolibrary.org/obo/MONDO_0009061 14296 14298 CF +T430 UBERON:0000912 14354 14359 mucus +T431 SO:0000704 14407 14411 gene +T432 GO:0010467 14407 14422 gene expression +T433 http://purl.obolibrary.org/obo/MONDO_0009061 14425 14427 CF +T434 NCBITaxon:10088 14428 14432 mice +T435 PR:000001044 14532 14536 Cftr +T436 NCBITaxon:10088 14542 14546 mice +T437 NCBITaxon:10088 14725 14729 mice +T438 NCBITaxon:10088 14907 14911 mice +T439 http://purl.obolibrary.org/obo/MONDO_0009061 15113 15115 CF +T440 NCBITaxon:10088 15116 15120 mice +T441 PR:000001044 15148 15152 Cftr +T442 PR:000001044 15197 15201 Cftr +T443 NCBITaxon:10088 15332 15336 mice +T444 NCBITaxon:10088 15393 15397 mice +T445 PR:000001044 15470 15474 Cftr +T446 PR:000001044 15517 15521 Cftr +T447 NCBITaxon:10088 15652 15656 mice +T448 NCBITaxon:10088 15713 15717 mice +T449 GO:0016265 15784 15789 death +T450 http://purl.obolibrary.org/obo/MONDO_0009061 15793 15795 CF +T451 NCBITaxon:10088 15796 15800 mice +T452 UBERON:0000160 15804 15814 intestinal +T453 http://purl.obolibrary.org/obo/MONDO_0004565 15804 15826 intestinal obstruction +T454 UBERON:0000160 15832 15842 intestinal +T455 http://purl.obolibrary.org/obo/MONDO_0004565 15832 15854 intestinal obstruction +T456 NCBITaxon:10088 15876 15880 mice +T457 GO:0007631 15885 15888 fed +T458 NCBITaxon:10088 15904 15909 mouse +T459 CHEBI:33290 15910 15914 chow +T460 http://purl.obolibrary.org/obo/MONDO_0009061 15931 15933 CF +T461 NCBITaxon:10088 15934 15938 mice +T462 UBERON:0000912 16021 16026 mucus +T463 UBERON:0002108 16047 16062 small intestine +T464 CHEBI:33290 16098 16102 chow +T465 NCBITaxon:10088 16115 16119 Mice +T466 CHEBI:33290 16204 16208 chow +T467 NCBITaxon:10088 16262 16266 mice +T468 CHEBI:33290 16288 16292 chow +T469 http://purl.obolibrary.org/obo/MONDO_0009061 16323 16325 CF +T470 NCBITaxon:10088 16326 16330 mice +T471 GO:0016265 16352 16356 died +T472 CHEBI:33290 16383 16387 chow +T473 http://purl.obolibrary.org/obo/MONDO_0009061 16467 16469 CF +T474 NCBITaxon:10088 16470 16474 mice +T475 GO:0016265 16499 16503 died +T476 SO:0000704 16535 16542 genetic +T477 http://purl.obolibrary.org/obo/MONDO_0009061 16557 16559 CF +T478 NCBITaxon:10088 16560 16565 mouse +T479 CHEBI:33290 16584 16588 chow +T480 NCBITaxon:10088 16590 16594 Mice +T481 GO:0007631 16687 16690 fed +T482 NCBITaxon:10088 16706 16711 mouse +T483 CHEBI:33290 16712 16716 chow +T484 GO:0016265 16747 16753 Deaths +T485 NCBITaxon:10088 16789 16793 mice +T486 GO:0016265 16835 16840 death +T487 http://purl.obolibrary.org/obo/MONDO_0009061 16892 16894 CF +T488 NCBITaxon:10088 16895 16899 mice +T489 GO:0016265 16950 16955 death +T490 GO:0016265 17015 17020 death +T491 http://purl.obolibrary.org/obo/MONDO_0009061 17026 17028 CF +T492 NCBITaxon:10088 17029 17033 mice +T493 http://purl.obolibrary.org/obo/MONDO_0009061 17068 17070 CF +T494 NCBITaxon:10088 17071 17075 mice +T495 GO:0016265 17102 17106 died +T496 SO:0001023 17247 17254 alleles +T497 SO:0000704 17268 17275 genetic +T498 UBERON:0002415 17332 17336 tail +T499 NCBITaxon:10088 17408 17413 mouse +T500 NCBITaxon:10088 17423 17427 Mice +T501 NCBITaxon:10088 17483 17487 mice +T502 http://purl.obolibrary.org/obo/MONDO_0009061 17588 17590 CF +T503 NCBITaxon:10088 17591 17595 mice +T504 SO:0001023 17635 17642 alleles +T505 NCBITaxon:10088 17662 17666 mice +T506 SO:0001023 17671 17678 alleles +T507 NCBITaxon:10088 17725 17729 mice +T508 PR:000001044 17818 17822 Cftr +T509 SO:0000704 17823 17827 gene +T510 http://purl.obolibrary.org/obo/MONDO_0009061 17867 17869 CF +T511 NCBITaxon:10088 17870 17874 mice +T512 http://purl.obolibrary.org/obo/MONDO_0009061 17980 17982 CF +T513 NCBITaxon:10088 17983 17987 mice +T514 http://purl.obolibrary.org/obo/MONDO_0009061 18215 18217 CF +T515 NCBITaxon:10088 18218 18222 mice +T516 NCBITaxon:10088 18253 18257 mice +T517 SO:0001023 18278 18285 alleles +T518 NCBITaxon:10088 18308 18312 mice +T519 SO:0001023 18325 18332 alleles +T520 NCBITaxon:10088 18357 18361 mice +T521 SO:0001023 18382 18389 alleles +T522 SO:0001026 18402 18408 Genome +T523 http://purl.obolibrary.org/obo/MONDO_0009061 18430 18432 CF +T524 NCBITaxon:10088 18433 18437 mice +T525 SO:0001023 18511 18518 alleles +T526 NCBITaxon:10088 18522 18526 mice +T527 SO:0000704 18540 18547 genetic +T528 SO:0001023 18693 18700 alleles +T529 NCBITaxon:10088 18816 18820 mice +T530 SO:0000704 18939 18944 genes +T531 http://purl.obolibrary.org/obo/MONDO_0009061 19056 19058 CF +T532 UBERON:0000160 19059 19069 intestinal +T533 CHEBI:17996 19099 19107 chloride +T534 PR:000001044 19155 19159 CFTR +T535 SO:0000704 19278 19283 genes +T536 UBERON:0006314 19356 19361 fluid +T537 GO:0042044 19356 19371 fluid transport +T538 PR:000002004 19373 19378 Kcnj9 +T539 PR:000001979 19397 19403 Kcnj10 +T540 PR:000002005 19422 19427 Kcnj5 +T541 PR:000000782 19448 19453 Kcnc2 +T542 SO:0000704 19475 19479 gene +T543 NCBITaxon:10088 19499 19504 Mouse +T544 SO:0001026 19505 19511 Genome +T545 http://purl.obolibrary.org/obo/MONDO_0009061 19565 19567 CF +T546 http://purl.obolibrary.org/obo/MONDO_0009061 19612 19614 CF +T547 SO:0000704 19684 19689 genes +T548 UBERON:0002405 19723 19736 immune system +T549 PR:000001949 19815 19821 Tnfsf4 +T550 CL:0000084 19874 19880 T cell +T551 GO:0042110 19874 19891 T cell activation +T552 SO:0000704 19916 19921 genes +T553 PR:000001415 19923 19927 Sele +T554 PR:000001318 19929 19933 Sell +T555 PR:000001432 19935 19939 Selp +T556 UBERON:0002405 19979 19985 immune +T557 CL:0000738 19979 19990 immune cell +T558 UBERON:0000479 20018 20025 tissues +T559 UBERON:0002405 20051 20057 immune +T560 CL:0000738 20051 20062 immune cell +T561 GO:0009986 20058 20070 cell surface +T562 PR:000001833 20100 20106 slamf1 +T563 SO:0000704 20165 20169 gene +T564 PR:000002140 20170 20174 Xcl1 +T565 GO:0010467 20199 20208 expressed +T566 CL:0000097 20212 20222 mast cells +T567 CL:0000542 20236 20247 lymphocytes +T568 GO:0019814 20262 20276 immunoglobulin +T569 SO:0000704 20289 20294 genes +T570 PR:000007442 20296 20301 Fcrl3 +T571 PR:000001481 20303 20309 Fcgr2b +T572 PR:000001483 20315 20320 Fcgr3 +T573 PR:000007432 20340 20346 Fcer1g +T574 PR:000007431 20366 20372 Fcer1a +T575 PR:000001156 20416 20420 Tlr5 +T576 PR:000010488 20437 20441 Mmp3 +T577 PR:000001004 20471 20474 CD4 +T578 CL:0000542 20476 20487 lymphocytes +T579 PR:000010489 20494 20498 Mmp7 +T580 CL:0000510 20529 20540 Paneth cell +T581 CHEBI:82761 20562 20571 defensins +T582 PR:000001467 20579 20584 Icam1 +T583 CL:0000542 20620 20630 lymphocyte +T584 UBERON:0000479 20658 20665 tissues +T585 PR:000009345 20672 20676 Kitl +T586 CL:0000034 20716 20725 stem cell +T587 PR:000009345 20716 20732 stem cell factor +T588 CL:0000097 20753 20762 mast cell +T589 GO:0030154 20758 20778 cell differentiation +T590 GO:0042571 20826 20834 antibody +T591 PR:000010030 20856 20860 Lyzs +T592 CL:0000510 20888 20899 Paneth cell +T593 GO:0005618 20921 20931 cell walls +T594 NCBITaxon:2 20935 20943 bacteria +T595 PR:000000017 20950 20954 Ifng +T596 http://purl.obolibrary.org/obo/MONDO_0009061 21016 21018 CF +T597 PR:000001383 21053 21057 Il22 +T598 PR:000001335 21077 21086;21109 21133 member of ... IL-10 interleukin family +T599 PR:000002088 21148 21153 Stat2 +T600 SO:0000704 21160 21165 genes +T601 GO:0005622 21216 21229 intracellular +T602 GO:0035556 21216 21248 intracellular signaling pathways +T603 SO:0000771 21305 21308 QTL +T604 http://purl.obolibrary.org/obo/MONDO_0009061 21345 21347 CF +T605 NCBITaxon:10088 21348 21353 mouse +T606 GO:0007567 21593 21598 natal +T607 SO:0000704 21667 21672 genes +T608 http://purl.obolibrary.org/obo/MONDO_0009061 21732 21734 CF +T609 NCBITaxon:10088 21735 21740 mouse +T610 UBERON:0000160 21741 21751 intestinal +T611 GO:0045087 21785 21812 innate type immune response +T612 UBERON:0002405 21797 21803 immune +T613 CL:0000097 21832 21842 mast cells +T614 CL:0000775 21847 21858 neutrophils +T615 SO:0000704 21864 21869 genes +T616 PR:000009345 21923 21927 Kitl +T617 SO:0000704 21928 21932 gene +T618 GO:0030154 21948 21966;21972 21977 differentiation of ... cells +T619 CL:0000097 21967 21977 mast cells +T620 http://purl.obolibrary.org/obo/MONDO_0009061 21983 21985 CF +T621 NCBITaxon:10088 21986 21990 mice +T622 CL:0000097 22038 22048 mast cells +T623 GO:0010467 22095 22105 expression +T624 PR:000010903 22109 22114 Mcpt2 +T625 CL:0000775 22131 22142 neutrophils +T626 PR:000001467 22161 22166 Icam1 +T627 GO:0072672 22219 22247 extravasation of neutrophils +T628 CL:0000775 22236 22247 neutrophils +T629 UBERON:0000479 22287 22293 tissue +T630 UBERON:0002405 22304 22310 immune +T631 GO:0006955 22304 22320 immune responses +T632 UBERON:0000912 22350 22355 mucus +T633 http://purl.obolibrary.org/obo/MONDO_0009061 22376 22378 CF +T634 UBERON:0000160 22379 22388 intestine +T635 UBERON:0000912 22408 22413 mucus +T636 http://purl.obolibrary.org/obo/MONDO_0009061 22444 22446 CF +T637 UBERON:0000479 22447 22454 tissues +T638 UBERON:0006314 22489 22494 fluid +T639 CHEBI:37527 22516 22522 acidic +T640 UBERON:0000062 22561 22567 organs +T641 UBERON:0000912 22623 22628 mucus +T642 http://purl.obolibrary.org/obo/MONDO_0009061 22632 22634 CF +T643 CHEBI:35224 22663 22671 effector +T644 CHEBI:36357 22672 22681 molecules +T645 CL:0000097 22694 22704 mast cells +T646 CL:0000775 22709 22720 neutrophils +T647 CHEBI:18295 22722 22731 histamine +T648 CHEBI:26333 22744 22758 prostaglandins +T649 UBERON:0000912 22798 22803 mucus +T650 http://purl.obolibrary.org/obo/MONDO_0009061 22860 22862 CF +T651 NCBITaxon:10088 22909 22913 mice +T652 SO:0001023 22950 22957 alleles +T653 http://purl.obolibrary.org/obo/MONDO_0043579 23099 23114;23122 23137 inflammation of small intestine +T654 http://purl.obolibrary.org/obo/MONDO_0009061 23119 23121 CF +T655 UBERON:0002108 23122 23137 small intestine +T656 UBERON:0002405 23167 23173 immune +T657 CL:0000738 23167 23179 immune cells +T658 UBERON:0000912 23183 23188 mucus +T659 SO:0000704 23204 23209 genes +T660 CL:0000097 23258 23267 mast cell +T661 CL:0000775 23272 23282 neutrophil +T662 http://purl.obolibrary.org/obo/MONDO_0009061 23349 23351 CF +T663 GO:0010467 23475 23485 expression +T664 SO:0000704 23498 23503 genes +T665 http://purl.obolibrary.org/obo/MONDO_0009061 23518 23520 CF +T666 UBERON:0000160 23521 23531 intestinal +T667 NCBITaxon:33208 23565 23572 Animals +T668 PR:000001044 23588 23592 Cftr +T669 NCBITaxon:10088 23598 23602 mice +T670 SO:0000704 23620 23627 genetic +T671 NCBITaxon:10088 23833 23837 mice +T672 NCBITaxon:10088 23928 23932 Mice +T673 SO:0000704 23977 23981 gene +T674 NCBITaxon:10088 24089 24093 mice +T675 PR:000001044 24137 24141 Cftr +T676 NCBITaxon:10088 24147 24151 mice +T677 SO:0001026 24185 24191 genome +T678 NCBITaxon:10088 24227 24231 mice +T679 NCBITaxon:10088 24280 24284 mice +T680 NCBITaxon:10088 24305 24309 mice +T681 SO:0001023 24337 24344 alleles +T682 PR:000001044 24356 24360 Cftr +T683 PR:000001044 24383 24387 Cftr +T684 PR:000001044 24397 24401 Cftr +T685 PR:000001044 24419 24423 Cftr +T686 NCBITaxon:10088 24432 24436 Mice +T687 NCBITaxon:10088 24532 24536 mice +T688 UBERON:0000160 24633 24643 intestinal +T689 http://purl.obolibrary.org/obo/MONDO_0004565 24633 24655 intestinal obstruction +T690 http://purl.obolibrary.org/obo/MONDO_0009061 24671 24673 CF +T691 NCBITaxon:10088 24674 24678 mice +T692 NCBITaxon:10088 24773 24777 mice +T693 NCBITaxon:10088 24806 24811 mouse +T694 CHEBI:33290 24812 24816 chow +T695 NCBITaxon:10088 24865 24869 Mice +T696 CHEBI:33290 24925 24929 chow +T697 NCBITaxon:10088 24981 24985 mice +T698 NCBITaxon:10088 25018 25022 Mice +T699 SO:0000704 25172 25179 Genetic +T700 SO:0001026 25210 25216 Genome +T701 NCBITaxon:10088 25364 25368 mice +T702 NCBITaxon:10088 25380 25385 mouse +T703 UBERON:0002415 25386 25390 tail +T704 SO:0000207 25431 25466 simple sequence length polymorphism +T705 SO:0000207 25468 25472 SSLP +T706 SO:0000112 25501 25508 primers +T707 SO:0001023 25540 25547 alleles +T708 GO:0030849 25708 25717 autosomes +T709 SO:0000704 25735 25739 gene +T710 GO:0010467 25735 25750 gene expression +T711 UBERON:0002108 25792 25807 small intestine +T712 GO:0010467 25889 25899 expression +T713 SO:0000704 25912 25917 genes +T714 SO:0000112 25949 25956 primers +T715 GO:0010467 26004 26014 expression +T716 SO:0000704 26036 26040 gene +T717 http://purl.obolibrary.org/obo/MONDO_0009061 26063 26065 CF +T718 NCBITaxon:10088 26066 26071 mouse +T719 UBERON:0002108 26072 26087 small intestine +T720 GO:0010467 26094 26104 Expression +T721 UBERON:0000160 26118 26128 intestinal +T722 PR:000010764 26136 26140 Muc2 +T723 SO:0000121 26170 26177;26260 26267 forward ... primers +T724 SO:0000132 26217 26224;26260 26267 reverse ... primers +T725 UBERON:0002108 26285 26300 small intestine +T726 UBERON:0000479 26402 26409 tissues +T727 CHEBI:29149 26548 26561 periodic acid +T728 SO:0000704 26610 26614 Gene +T729 GO:0010467 26610 26625 Gene expression +T730 PR:000001044 26888 26892 Cftr +T731 NCBITaxon:10088 26898 26902 mice +T732 NCBITaxon:10088 27368 27373 mouse diff --git a/src/ontogpt/evaluation/craft/database/all/15921521.txt b/src/ontogpt/evaluation/craft/database/all/15921521.txt new file mode 100644 index 000000000..8e8c3d2c1 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15921521.txt @@ -0,0 +1,137 @@ +Potential genetic modifiers of the cystic fibrosis intestinal inflammatory phenotype on mouse chromosomes 1, 9, and 10 + +Abstract + +Background + +Although cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the severity of disease is highly variable indicating the influence of modifier genes. The intestines of Cftr deficient mice (CF mice: Cftrtm1Unc) are prone to obstruction by excessive mucus accumulation and are used as a model of meconium ileus and distal intestinal obstruction syndrome. This phenotype is strongly dependent on the genetic background of the mice. On the C57Bl/6 background, the majority of CF mice cannot survive on solid mouse chow, have inflammation of the small intestine, and are about 30% smaller than wild type littermates. In this work potential modifier loci of the CF intestinal phenotype were identified. + +Results + +CF mice on a mixed genetic background (95% C57Bl/6 and 5% 129Sv) were compared to CF mice congenic on the C57Bl/6 background for several parameters of the intestinal CF phenotype. CF mice on the mixed background exhibit significantly greater survival when fed dry mouse chow, have reduced intestinal inflammation as measured by quantitative RT-PCR for marker genes, have near normal body weight gain, and have reduced mucus accumulation in the intestinal crypts. There was an indication of a gender effect for body weight gain: males did not show a significant improvement at 4 weeks of age, but were of normal weight at 8 weeks, while females showed improvement at both 4 and 8 weeks. By a preliminary genome-wide PCR allele scanning, three regions were found to be potentially associated with the milder phenotype. One on chr.1, defined by marker D1Mit36, one on chr. 9 defined by marker D9Mit90, and one on chr. 10, defined by marker D10Mit14. + +Conclusion + +Potential modifier regions were found that have a positive impact on the inflammatory phenotype of the CF mouse small intestine and animal survival. Identification of polymorphisms in specific genes in these regions should provide important new information about genetic modifiers of the CF intestinal phenotype. + +Background + +Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene [1]. Different mutations have a range of effects on the levels of CFTR protein and its proper functioning in epithelial transport of Cl- and HCO3- [2,3]. The severity of the pancreatic phenotype in human CF is well correlated with the extent of impaired CFTR function caused by specific mutations. Loss of CFTR function results in destruction of the exocrine tissue and eventual pancreatic insufficiency. On the other hand, the effects of CF on organs including the airways and intestines is less well correlated with specific CFTR mutations and their effects on CFTR protein function [4-8]. This indicates that other genes are likely to be important as modifiers of the CF phenotype. + +With the exception of pancreatic insufficiency resulting in impaired digestion, other aspects of CF are less readily related to loss of CFTR function. Nutritional problems can persist even with adequate oral enzyme supplementation [9] and neutralization of gastric acid to improve lipase function [10], and may involve both impaired digestion and absorption of nutrients [11]. Inadequate absorption or assimilation of nutrients appears to be of greater importance because even with adequate oral enzyme supplementation nutrition is rarely fully corrected [11]. There is also excessive mucus accumulation in the CF intestine, and inappropriate inflammation is common [12]. Mucus is involved in obstruction of the gut which occurs frequently in CF infants (called meconium ileus, MI) and adults (called distal intestinal obstruction syndrome, DIOS) [11,13]. And, similar to CF airways, there is also an inflammation of the CF intestines [14,15]. These changes are less directly related to specific mutations in the CFTR gene and are likely related to other differences in individual genetic makeup. + +Previous work using human patients and genetically altered mice has identified some modifier genes and have advanced our understanding of CF pathophysiology [4]. In one study using CF mice on different genetic backgrounds, a region on mouse chromosome 7 was shown to ameliorate intestinal blockage and the effect was in part due to a calcium-regulated Cl-channel which compensated for loss of CFTR function [16,17]. Marker haplotypes of the syntenic region of human chromosome 19q13 were also shown to be associated with the risk of MI in CF patients [18]. In other work, a region on mouse chr. 6 was strongly associated with lung inflammation, consisting of mononuclear cell interstitial infiltration and fibrosis in CF mouse airways; and other loci on chr. 1, 2, 10, and 17 were also linked to the airway phenotype [19]. + +In this work, CF mice on a mixed strain background were found to have a less severe CF phenotype compared to CF mice congenic on the C57Bl/6 background. There were no differences in the pancreatic phenotype comparing CF mice on the different backgrounds [20]. However, mice on the mixed background seemed more robust than CF mice on the B6 background which prompted us to characterize them in greater detail. Genome wide allele scanning was used to begin identification of regions associated with the less severe intestinal phenotype. Future identification of specific genes should further our understanding of the complex intestinal CF phenotype. + +Results and discussion + +CF mice on the mixed background have improved body weight gain + +Body mass was recorded for male and female mice at 4 and 8 weeks of age. On the B6 background, female and male mice are about 30% smaller than wild type mice at both 4 and 8 weeks of age (Fig. 1A, B). By comparison, female CF mice on the mixed background were not significantly smaller than wild type mice at 4 weeks of age (Fig. 1A). The improved body weight of female CF mice on the mixed background was maintained at 8 weeks of age. Male CF mice on the mixed background at 4 weeks of age were significantly smaller than wild type and not significantly larger than CF mice congenic on the B6 background (Fig. 1B). By 8 weeks of age, CF males on the mixed background were 12% smaller but this was not significantly different compared to wild type mice (Fig. 1B). + +Figure 1 + +Effect of genetic background on body weights of CF mice. (A) Female and (B) male mice were weighed at 4 and 8 weeks of age. Data are means ± SEM. (*) P < 0.001 vs other three groups by ANOVA with a post-hoc Tukey's test. There were no significant differences for any of the other comparisons. Data were obtained from: 65 wild type and 26 CF B6 females, 36 wild type and 8 CF mixed background females, 68 wild type and 30 CF B6 males, and 37 wild type and 6 CF mixed background males. + +CF mice on the mixed background have reduced expression of inflammatory markers + +Previous work showed that CF mice on the B6 background have an innate-type inflammation of the small intestine [21]. To determine whether the mixed genetic background affected expression of inflammatory marker genes, quantitative, real-time RT-PCR was used to measure gene expression. Expression of the following genes was compared in wild type and CF mice on the different genetic backgrounds. Mast cell protease 2 (Mcpt2) is a marker of differentiated mast cells [22] and mast cells are more abundant in the B6 CF mouse intestine. Leucine-rich α2 glycoprotein (Lrg1, [23]) is a marker of differentiating neutrophils, which are more numerous in the B6 CF mouse intestine. The same gene is also known as leucine-rich high endothelial cell glycoprotein (Lrhg) and has been shown to be a marker of high endothelial venules (HEV) [24] which increase in tissues during inflammation [25,26]. Hematopoietic cell transcript 1 (HemT1, [27]) is a marker of blood cell proliferation and its expression is strongly elevated in the B6 CF mouse small intestine. Serum amyloid A3 (SAA3, [28]) is an acute phase gene and its expression in villus epithelial cells is increased in the B6 CF intestine. Suppressor of cytokine signaling 3 (SOCS3, [29]) is an anti-inflammatory gene that interacts with the JAK-STAT pathway and its expression in increased in the B6 CF intestine. Muclin (also known as dmbt1, [30]) expression is upregulated in the B6 CF intestine; it is a cell surface glycoprotein postulated to be an epithelial protective molecule [21,31]. + +Consistent with previous results, Mcpt2 was increased in CF mice on the B6 background by over 9-fold compared to wild type (Fig. 2A). By contrast, there was not a significant difference in Mcpt2 expression between CF and wild type on the mixed background (Fig. 2A). Lrg1/Lrhg expression was increased more than 20-fold in CF mice on the B6 background compared to wild type, but there was no significant difference between CF and wild type on the mixed background (Fig. 2B). SAA3 mRNA was about 3.5-fold increased in CF mice on the B6 background, but was not significantly different compared to wild type on the mixed background (Fig. 2C). SOCS3 was more than 2-fold increased in CF mice on the B6 background compared to wild type, and on the mixed background was only 1.5-fold greater than wild type, and the difference was not significant (Fig. 2D). Muclin is overexpressed almost 3-fold in the CF intestine on the B6 background, but on the mixed background the expression level in CF mice was not significantly different than wild type (Fig. 2E). Finally, HemT1 was overexpressed almost 20-fold in B6 CF mice compared to wild type, and on the mixed background the CF expression level was not statistically different from wild type (Fig. 2F). + +Figure 2 + +Effect of genetic background on inflammatory gene expression in CF mouse small intestine. RNA expression levels were determined by quantitative real-time RT-PCR using gene-specific primers. Data are expressed relative to GAPDH mRNA, which does not vary between wild type and CF mice. Data are means ± SEM. (*) CF vs wild type on the B6 background, P < 0.005; (+) CF on the mixed background vs CF on the B6 background, P < 0.05 by ANOVA with a post-hoc Tukey's test. There were no significant differences for any of the 6 genes comparing: wild type B6 mice vs wild type mixed background; or CF mice on the mixed background vs wild type on either background. There were 8–11 samples analyzed per group for each gene. + +Because of the gender differences in body weight, the gene expression data were analyzed by gender but there was no significant difference between females and males. With the limited number of animals, there was also no evidence for imprinting. + +CF mice on the mixed background have less intestinal mucus accumulation + +The most striking histological difference in the CF mouse small intestine is the accumulation of mucus in intestinal crypts which is associated with the lethal obstruction that results in death of these mice on a standard solid chow diet [32]. Using periodic acid Schiff's staining for neutral mucins, histological analysis of small intestine tissues was performed. The wild type small intestine on both the B6 and the mixed genetic backgrounds were similar. The intestinal crypts were very small with only traces of PAS-reactivity in the lumen (Fig. 3A and 3C, respectively). The surfaces of the villus epithelium were moderately stained and goblet cells were strongly stained in the wild type tissues. In contrast, the intestine of CF mice on the B6 genetic background exhibited greatly dilated crypts filled with PAS-reactive mucus (Fig. 3B). CF mice on the mixed background had less mucus accumulation than CF mice on the B6 background (Fig. 3D-F). The amount of mucus in CF mice on the mixed background was variable from mouse to mouse. In some mice (Fig. 3D), only occasional crypts had accumulated mucus and the crypt lumina were not very dilated. In some mice there was more mucus in the crypts (Fig. 3E), while others had moderate mucus accumulation (Fig. 3F). A total of six CF mice on the mixed background were examined histologically, and three had little mucus, one had some mucus, and two had moderate amounts of mucus. Despite the variability, all the CF mice on the mixed background had less crypt dilation and less mucus accumulation compared to CF mice on the B6 background. + +Figure 3 + +Histological appearance of the small intestine of wild type and CF mice on the different genetic backgrounds. Tissue was paraffin embedded and stained with PAS for neutral mucins. The sections are from the middle portion of the small intestine (A) Wild type on the B6 background. (B) CF on the B6 background. (C) Wild type on the mixed background. (D-F) CF on the mixed background. (A, C) In the wild type tissue from both backgrounds, the crypts are small and have narrow lumina (arrows). (B) In the CF tissue on the B6 background, the crypt lumina are greatly dilated and filled with PAS-reactive mucus (arrows). In some CF mice on the mixed background, the crypts are not apparently different than wild type (D), and the crypts are normal appearing (arrows). Some CF mice on the mixed background had mildly affected crypts (E), while others had greater crypt dilation and mucus accumulation (F). Overall, the CF mice on the mixed background were less severely affected compared to those on the B6 background. + +Thus, it appears that whatever the nature of the genetic differences between the two background strains, they affect secretion and accumulation of mucus in the CF small intestine. To determine if the difference could be accounted for by altered mucin gene expression, quantitative RT-PCR was used to measure mRNA expression of the intestinal goblet cell mucin gene, Muc2. In wild type intestine, B6 mice had 0.079 +/- 0.024 copies of Muc2/GAPDH (n = 8), and on the mixed background the level was 0.077 +/- 0.012 (n = 11). CF mice on the B6 background had 0.059 +/- 0.024 copies of Muc2 mRNA per GAPDH (n = 11), and on the mixed background the level was 0.056 +/- 0.010 (n = 11). Despite the increased mucus accumulation in CF, there is a slight decrease in Muc2 mRNA in the CF small intestine (P < 0.0001), as previously reported [33]. However, the milder CF phenotype on the mixed background, which includes less mucus accumulation, does not involve decreased mucin gene expression. + +CF mice on the mixed background have improved survival + +As reported by others [34], the expected number of Cftr null mice on the B6 background that survived to weaning was significantly less than that expected from Mendelian genetics (Tables 1 and 2). The degree of significance was greater for male mice (Tables 1 and 2). In contrast, on the mixed background, the distribution of genotypes of female offspring was not significantly different from the expected (Tables 1). For male mice on the mixed background, the P-value was less significant but still different compared to the B6 males (Table 2). These data indicate that the mixed background is associated with increased survival of CF mice. + +Table 1 + +Distribution of Cftr genotypes in female offspring from breeding Cftr heterozygotes on the B6 and mixed backgrounds. + +Statistical analysis was by Chi-square. (*) P-value if the sample size for the B6 mice is adjusted to be equal to that of the mixed background mice, assuming the same distribution of genotypes. + +Table 2 + +Distribution of Cftr genotypes in male offspring from breeding Cftr heterozygotes on the B6 and mixed backgrounds. + +Statistical analysis was by Chi-square. (*) P-value if the sample size for the B6 mice is adjusted to be equal to that of the mixed background mice, assuming the same distribution of genotypes. + +The major cause of death in CF mice is intestinal obstruction, and intestinal obstruction is worsened when the mice are fed standard solid mouse chow [35]. Since the CF mice on the mixed background had better weight gain than on the B6 background and less mucus accumulation in the small intestine, their ability to survive on solid chow was tested. Mice were maintained on the liquid diet until 8 weeks of age, and then switched to solid chow for up to eight weeks. As shown in Fig. 4, wild type mice had 100% survival on chow, as expected. The majority of CF mice on the B6 background died within 2–3 weeks on solid chow, with about 60% mortality over the 8 week period. In contrast, only 22% of the CF mice on the mixed background died (Fig. 4). + +Figure 4 + +Effect of genetic background on CF mouse survival on solid chow. Mice were maintained on the liquid diet (Peptamen) until 8 weeks of age, at which time they were fed standard solid mouse chow for up to 8 additional weeks. Deaths were recorded as they occurred and mice in obvious distress were sacrificed and 'death' recorded as the subsequent day. By log-rank test, CF mice on the mixed background had significantly earlier death compared to wild type (P = 0.035), and significantly later death than CF mice on the B6 background (P = 0.025). CF mice on the B6 background also died significantly earlier than wild type (P < 0.0005). + +Identification of potential modifier loci + +To determine the contributions of B6 and 129 alleles to the mixed genetic background, and to identify potential modifier regions, tail DNA was analyzed by PCR for markers of polymorphisms between these two mouse strains. Mice congenic on the B6 background, which were derived from mice originally on a B6/129 mixed background, were also analyzed to confirm they are congenic B6. Twelve CF mice on the B6 background averaged 99.5% B6 alleles. One of the twelve mice had alleles from both strains at chr.9, 40 cM. All twelve mice had both B6 and 129 markers on chromosome 6, 1 cM which is probably due to the targeted Cftr gene which is at chr.6, 3.1 cM [36]. + +Three CF mice on the mixed background were initially analyzed and were found to be 95% B6, 5% 129. A second group of 8 CF mice was analyzed and the markers used were refined to focus on the chromosomal regions showing variations from the B6 strain. The differences found from the two analyses are combined in Table 3. The differences in the mixed strain CF mice were at chr.1, 92 cM (9 of 11 mice had both B6 and 129 alleles); chr.9, 9 cM (all 11 mice had two 129 alleles); and chr.10, 65 cM (10 mice had both B6 and 129 alleles). + +Table 3 + +Genome scanning analysis of CF mice on the mixed background. + +The table shows the distribution of B6 and 129 alleles in mice from the two genetic backgrounds. An initial analysis was performed on 3 samples and a second analysis on 8 more samples. Only the markers that were not uniformly B6 alleles are shown. Some of the markers were refined based on the initial analysis, so not all markers were used for all 11 mice. (*) Eleven samples were analyzed but one sample was ambiguous. + +Because the spacing of markers used was about 12 cM, genes within 75% of this interval on either side of the markers were looked at for potential relevance to the milder CF intestinal phenotype. None of the known chloride channels that might substitute for the missing CFTR are in the regions of the three chromosomes associated with the milder phenotype. There are several potassium channel genes in the identified regions that potentially could affect electrolyte and fluid transport: Kcnj9 (chr.1, 94.2 cM), Kcnj10 (chr.1, 93.5 cM), Kcnj5 (chr.9, 11 cM), and Kcnc2 (chr.10, 62 cM). All gene names are from the Mouse Genome Informatics website . + +Inflammation is a hallmark of CF, and whether there is an inherent defect in CF that predisposes to excessive inflammation is controversial. Several genes involved in inflammation and the immune system are located in the regions of the markers identified: TNF superfamily members Tnfsf4, 6, and 8 (chr.1, 84.9–85 cM) which are involved in T cell activation [37,38]; three selectin genes (Sele, Sell, Selp, chr.1, 86.6 cM) which are involved in immune cell infiltration into inflamed tissues [39]; several members of immune cell surface proteins of the Slam family (slamf1, 2, 5, 6, and 9; chr.1, 89.5–93.3 cM) [40]; the chemokine gene Xcl1 (chr.1, 87 cM) which is expressed by mast cells and recruits lymphocytes [41]; several immunoglobulin Fc receptor genes (Fcrl3, Fcgr2b, and Fcgr3 at chr.1, 92.3 cM; Fcer1g at chr.1, 93.3 cM; Fcer1a at chr.1, 94.2 cM); the flagellin receptor Tlr5 (chr.1, 98 cM); Mmp3 (chr.9, 1 cM) which recruits CD4+ lymphocytes [42]; Mmp7 (chr.9, 1 cM) which activates Paneth cell-derived cryptdins (α-defensins) [43]; Icam1 (chr.9, 7 cM) which is involved in lymphocyte infiltration into inflamed tissues [44]; Kitl (chr.10, 57 cM) which is also known as stem cell factor, and is crucial for mast cell differentiation [45]; Im5 (chr.10, 65 cM) which is involved in antibody-responsiveness [46]; Lyzs (chr.10, 66 cM) which is a Paneth cell product that digests cell walls of bacteria [47]; Ifng (chr.10, 67 cM) which is an important inflammatory signal in CF as well as other conditions [48]; Il22 (chr.10, 67 cM), a member of the anti-inflammatory IL-10 interleukin family [49]; and the Stat2 and 6 genes (chr.10, 70 cM) which are important components of intracellular signaling pathways [50]. + +Also near the identified markers are a number of QTL associated with body weight: Cfbw1, CF mouse body weight at chr.1, 85 cM; Obq9, obesity 9 at chr.1, 88 cM; Bw8q1, body weight 8 at chr.1, 100 cM; Lbm6, lean body mass 6 at chr.9, 7.7 cM; Bwtq4, body weight 4 at chr.9, 8 cM; Bgeq8, body growth early 8 at chr.10, 57 cM; and Pbwg5, postnatal body weight growth 5 at chr.10, 68 cM. + +Clearly, there are numerous genes in the three regions identified in this study. Because the CF mouse intestinal phenotype is characterized by an innate type immune response, with increases in mast cells and neutrophils, the genes that affect these cells are of special interest. The Kitl gene is crucial for differentiation of mast cells, and CF mice on the mixed background have much fewer mature mast cells than on the B6 background as revealed by less expression of Mcpt2. Similarly, for neutrophils the selectins and Icam1 are of interest, as these proteins are required for extravasation of neutrophils from the circulation into the inflamed tissue. + +Altered immune responses may also relate to excessive mucus accumulation in the CF intestine. It is unclear why mucus accumulates to high levels in CF tissues. In part it may be due to reduced fluid secretion and a more acidic environment in the lumina of affected organs. However, there is also evidence for hypersecretion of mucus in CF [12], and it is likely that effector molecules released by mast cells and neutrophils (histamine, proteases, prostaglandins) have an important role in stimulating mucus secretion. + +Conclusion + +This work demonstrated that the CF inflammatory phenotype is much less severe in mice with a small contribution of 129/Sv alleles. A preliminary analysis identified regions on chr.1, 9, and 10 are that are potentially associated with the milder phenotype. Because of the inflammation of the CF small intestine, and the possible effects of immune cells on mucus secretion, the genes in the identified regions which are involved in mast cell and neutrophil differentiation and behavior are of special interest as potential CF modifiers. Future work should focus on narrowing down these regions and determining if there are polymorphisms that affect expression of specific genes that make the CF intestinal phenotype less severe. + +Methods + +Animals + +Wild type and Cftr null mice on two different genetic backgrounds were used in this study. One group was congenic on the C57Bl/6 (B6) background, as previously described [21]. The other group was on a mixed background of about 95% B6 and 5% 129/Sv (129). The mice on the mixed background originated as part of a recently published study [20] as follows. Mice carrying a targeted mutation of the gastrin gene on a mixed B6/129 background [51] were bred for eight generations onto the B6 background. The gastrin(+/-) mice were then crossed for six generations with Cftr(+/-) mice congenic on the B6 background. A genome scan at this point showed that the mice were about 95% B6 and 5% 129 (see below). These mice were bred to obtain mice wild type for both gastrin alleles and either Cftr homozygous wild type [Cftr(+/+)] or Cftr homozygous null [Cftr(-/-)]. + +Mice were genotyped at 2 weeks of age by PCR as previously described [21]. Unless otherwise stated, mice were maintained on a complete elemental liquid diet (Peptamen; Nestle Deerfield, IL) to prevent intestinal obstruction that occurs in CF mice [52]. Wild type littermates were maintained on the same diet. In some experiments, 8 week old mice were transferred onto solid mouse chow instead of Peptamen, and survival was recorded. Mice with apparent distress were sacrificed and survival on chow was recorded as the following day. Male and female mice were used at 6–16 weeks of age. Mice were kept in a specific pathogen-free facility in barrier-top cages. All procedures were approved by the University of Kansas Medical Center IACUC. + +Genetic background determination + +The Genome Scanning Service of The Jackson Laboratory (Bar Harbor, ME) was used to determine the contributions of C57Bl/6 and 129/Sv strains in the interbred mice. Pieces of mouse tail were sent to The Jackson Laboratory for simple sequence length polymorphism (SSLP) PCR analysis with the DMit primers specific for B6 and 129 strain alleles . The SSLP panel consists of 108 mapped markers designed to distinguish between B6 and 129 strains. The markers are spaced 12–13 cM apart and span the nineteen autosomes. + +Measurement of gene expression + +Total RNA was extracted from the entire small intestine as previously described [21]. Quantitative, real-time RT-PCR was used to measure expression of specific genes using the previously described primers [21]. Values were normalized to GAPDH mRNA and expression of this housekeeping gene is not altered in the CF mouse small intestine [21]. Expression of the major intestinal mucin, Muc2, was also measured using the forward (5'-GAC TTC GAT GGA CAC TGC TC-3') and reverse (5'-CAC GGT GTT TAT CTA CCA AC-3') primers. + +Histology + +The small intestine was flushed with phosphate buffered saline and immersion fixed overnight in 4% paraformaldehyde. The tissues were then prepared for paraffin embedding and sectioning by a commercial service (HSRL, Woodstock, VA). Sections (5 μm) were stained with periodic acid Schiff's (PAS) for neutral mucins. + +Statistics + +Gene expression and body weight data were compared by ANOVA with a post-hoc Tukey's analysis (Systat software, Chicago, IL). Survival data were analyzed by a log-rank test for P values (PEPI software, ). The distributions of genotypes of pups surviving to weaning from breeding Cftr(+/-) mice were compared to that expected by Mendelian genetics using Chi-square analysis. For all statistical tests, P < 0.05 was considered significant. + +Authors' contributions + +RCD oversaw the project, performed the histological analysis, performed statistical analyses, and contributed to writing the manuscript. ON performed the quantitative RT-PCR analysis, and contributed to writing the manuscript. + +Acknowledgements + +We thank Larysa Stroganova for maintenance of the mouse colonies and PCR genotyping. Supported by National Institutes of Health grant DK 56791. diff --git a/src/ontogpt/evaluation/craft/database/all/15938754.ann b/src/ontogpt/evaluation/craft/database/all/15938754.ann new file mode 100644 index 000000000..93024aa8f --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15938754.ann @@ -0,0 +1,591 @@ +T1 SO:0001024 0 10 Haplotypes +T2 PR:000036337 18 23 Tas2r +T3 CHEBI:15854 63 70 quinine +T4 GO:0050909 71 76 taste +T5 NCBITaxon:10088 99 103 mice +T6 GO:0050913 144 158 bitter-tasting +T7 CHEBI:36357 159 168 compounds +T8 GO:0050909 176 185 gustatory +T9 UBERON:0001033 176 192 gustatory system +T10 NCBITaxon:33208 213 220 animals +T11 CHEBI:33290 258 262 food +T12 GO:0050909 315 320 taste +T13 PR:000036337 336 340 T2Rs +T14 GO:0010467 352 361 expressed +T15 GO:0050909 376 381 taste +T16 CL:0000209 376 396 taste receptor cells +T17 UBERON:0001723 404 410 tongue +T18 CHEBI:15854 561 568 quinine +T19 CHEBI:17883 569 582 hydrochloride +T20 CHEBI:16150 605 613 benzoate +T21 GO:0050909 662 667 taste +T22 NCBITaxon:10088 710 715 mouse +T23 SO:0000771 741 765 quantitative trait locus +T24 SO:0000771 767 770 QTL +T25 GO:0050909 776 781 taste +T26 SO:0000771 808 811 QTL +T27 SO:0000704 879 884 genes +T28 PR:000036337 894 898 T2Rs +T29 PR:000036337 900 906 Tas2rs +T30 PR:000036337 909 915 Tas2rs +T31 SO:0000694 965 996 single nucleotide polymorphisms +T32 SO:0000694 998 1002 SNPs +T33 CHEBI:15854 1058 1065 quinine +T34 CHEBI:15854 1089 1096 quinine +T35 PR:000036337 1226 1231 Tas2r +T36 SO:0001023 1232 1239 alleles +T37 PR:000036337 1288 1293 Tas2r +T38 SO:0001023 1294 1301 alleles +T39 SO:0001023 1362 1369 alleles +T40 PR:000036337 1388 1393 Tas2r +T41 SO:0001024 1420 1429 haplotype +T42 CHEBI:15854 1451 1458 quinine +T43 GO:0050909 1520 1525 taste +T44 SO:0000771 1557 1560 QTL +T45 CHEBI:15854 1565 1572 quinine +T46 GO:0050909 1573 1578 taste +T47 PR:000036337 1596 1599 T2R +T48 CHEBI:15854 1685 1692 quinine +T49 GO:0050909 1693 1698 taste +T50 SO:0000195 1766 1778 coding exons +T51 PR:000036337 1786 1791 Tas2r +T52 GO:0050913 1867 1879 bitter taste +T53 PR:000036337 1905 1908 T2R +T54 NCBITaxon:33208 1965 1972 Animals +T55 GO:0050909 1981 1990 gustatory +T56 UBERON:0001033 1981 1997 gustatory system +T57 CHEBI:33290 2027 2031 food +T58 GO:0050916 2054 2067 sweet-tasting +T59 CHEBI:33290 2068 2073 foods +T60 GO:0050913 2131 2145 bitter-tasting +T61 CHEBI:33290 2146 2151 foods +T62 GO:0010467 2263 2272 expressed +T63 GO:0050909 2294 2299 taste +T64 CL:0000209 2294 2314 taste receptor cells +T65 CL:0000209 2316 2320 TRCs +T66 GO:0050909 2329 2338 gustatory +T67 UBERON:0002926 2329 2349 gustatory epithelium +T68 GO:0001580 2378 2407;2415 2421;2450 2455 detection and transduction of ... bitter ... taste +T69 GO:0046535 2378 2407;2426 2431;2450 2455 detection and transduction of ... umami ... taste +T70 GO:0001582 2378 2413;2450 2455 detection and transduction of sweet ... taste +T71 PR:000036336 2457 2461 T1Rs +T72 GO:0050916 2466 2472;2482 2489 sweet- ... tasting +T73 GO:0050917 2476 2489 umami-tasting +T74 PR:000036337 2509 2513 T2Rs +T75 GO:0050913 2518 2532 bitter-tasting +T76 CHEBI:36357 2533 2542 compounds +T77 SO:0000704 2556 2561 genes +T78 PR:000036337 2574 2578 T2Rs +T79 PR:000036337 2584 2590 Tas2rs +T80 NCBITaxon:40674 2636 2645 mammalian +T81 SO:0001026 2646 2653 genomes +T82 GO:0050913 2715 2727 bitter taste +T83 NCBITaxon:10088 2754 2758 mice +T84 PR:000036337 2776 2782 Tas2rs +T85 NCBITaxon:9606 2848 2853 human +T86 PR:000036337 2854 2860 Tas2rs +T87 SO:0000336 2874 2885 pseudogenes +T88 NCBITaxon:10088 2902 2907 mouse +T89 PR:000036337 2908 2914 Tas2rs +T90 SO:0000336 2928 2939 pseudogenes +T91 NCBITaxon:10088 2952 2956 mice +T92 GO:0010467 3064 3074 expression +T93 SO:0000704 3126 3130 gene +T94 GO:0050913 3146 3158 bitter taste +T95 SO:0000771 3199 3222 quantitative trait loci +T96 SO:0000771 3224 3227 QTL +T97 GO:0007631 3276 3282 intake +T98 NCBITaxon:10088 3308 3313 mouse +T99 CHEBI:15854 3334 3341 quinine +T100 CHEBI:15854 3343 3346 Qui +T101 CHEBI:27641 3360 3373 cyclohexamide +T102 CHEBI:27641 3375 3378 Cyx +T103 SO:0000771 3453 3456 QTL +T104 NCBITaxon:10088 3464 3469 mouse +T105 PR:000036337 3569 3575 Tas2rs +T106 SO:0001026 3591 3597 genome +T107 SO:0000771 3890 3893 QTL +T108 GO:0050913 3934 3946 bitter taste +T109 SO:0000771 3947 3951 QTLs +T110 GO:0007631 3994 4005 consumption +T111 GO:0007631 4058 4067 ingestive +T112 GO:0050913 4171 4183 bitter taste +T113 SO:0000771 4267 4271 QTLs +T114 GO:0050913 4275 4287 bitter taste +T115 CHEBI:59941 4397 4408 amphiphilic +T116 CHEBI:36357 4416 4425 compounds +T117 CHEBI:15854 4435 4442 quinine +T118 CHEBI:16150 4458 4466 benzoate +T119 GO:0050909 4482 4487 taste +T120 CL:0000209 4482 4502 taste receptor cells +T121 CHEBI:15854 4539 4546 Quinine +T122 CHEBI:15854 4590 4597 quinine +T123 CHEBI:29103 4623 4625 K+ +T124 CHEBI:27732 4645 4653 caffeine +T125 GO:0050913 4663 4677 bitter-tasting +T126 GO:0005622 4707 4720 intracellular +T127 PR:000036337 4784 4787 T2R +T128 PR:000036337 4802 4805 T2R +T129 GO:0050909 4898 4903 taste +T130 GO:0050909 4954 4959 taste +T131 NCBITaxon:10088 5045 5049 mice +T132 CHEBI:15854 5073 5080 quinine +T133 CHEBI:17883 5081 5094 hydrochloride +T134 CHEBI:16150 5117 5125 benzoate +T135 SO:0000771 5229 5232 QTL +T136 GO:0050909 5242 5247 taste +T137 PR:000036337 5319 5324 Tas2r +T138 SO:0000704 5325 5330 genes +T139 PR:000036337 5387 5392 Tas2r +T140 SO:0001023 5393 5399 allele +T141 SO:0000704 5488 5493 genes +T142 PR:000036337 5553 5559 Tas2rs +T143 SO:0001024 5578 5587 haplotype +T144 GO:0050909 5614 5619 taste +T145 GO:0050909 5643 5648 Taste +T146 SO:0000771 5682 5685 QTL +T147 GO:0050913 5690 5702 bitter taste +T148 GO:0007631 5717 5728 consumption +T149 GO:0007631 5787 5796 ingestive +T150 NCBITaxon:10088 5942 5946 mice +T151 GO:0050909 5970 5975 taste +T152 GO:0050909 5995 6000 taste +T153 NCBITaxon:10088 6058 6062 mice +T154 CHEBI:36357 6191 6199 compound +T155 GO:0050909 6253 6258 taste +T156 NCBITaxon:10088 6400 6404 mice +T157 CHEBI:36357 6503 6512 compounds +T158 NCBITaxon:10088 6635 6639 mice +T159 NCBITaxon:10088 6684 6688 mice +T160 GO:0050909 6776 6781 taste +T161 NCBITaxon:10088 6904 6908 mice +T162 NCBITaxon:10088 6936 6940 mice +T163 CHEBI:15377 7046 7051 water +T164 CHEBI:16150 7107 7115 benzoate +T165 CHEBI:8502 7122 7126 PROP +T166 CHEBI:8502 7128 7148 6-n-propylthiouracil +T167 CHEBI:8502 7150 7152 PR +T168 CHEBI:15854 7158 7165 quinine +T169 CHEBI:17883 7166 7179 hydrochloride +T170 NCBITaxon:10088 7372 7376 mice +T171 GO:0050909 7506 7511 taste +T172 GO:0050909 7944 7949 taste +T173 SO:0000704 8333 8338 genic +T174 GO:0065007 8339 8346 control +T175 NCBITaxon:10088 8364 8368 mice +T176 NCBITaxon:10088 8411 8415 mice +T177 GO:0050909 8535 8540 taste +T178 SO:0000771 8702 8705 QTL +T179 SO:0000771 8803 8807 QTLs +T180 GO:0050909 8831 8836 taste +T181 SO:0001026 8949 8955 genome +T182 SO:0001026 9022 9028 genome +T183 SO:0000771 9044 9047 QTL +T184 SO:0001026 9147 9153 genome +T185 SO:0000771 9169 9172 QTL +T186 SO:0000771 9230 9233 QTL +T187 SO:0001026 9281 9287 genome +T188 SO:0000771 9338 9341 QTL +T189 GO:0050909 9351 9356 taste +T190 NCBITaxon:10088 9360 9365 mouse +T191 SO:0000771 9443 9446 QTL +T192 SO:0000771 9488 9491 QTL +T193 GO:0050909 9527 9532 taste +T194 SO:0000771 9592 9595 QTL +T195 SO:0001026 9648 9654 genome +T196 SO:0000771 9692 9695 QTL +T197 SO:0000771 9829 9832 QTL +T198 NCBITaxon:10088 10231 10235 mice +T199 GO:0050909 10274 10279 taste +T200 SO:0000771 10413 10416 QTL +T201 SO:0000771 10434 10437 QTL +T202 NCBITaxon:10088 10496 10501 mouse +T203 SO:0001026 10815 10821 genome +T204 SO:0000771 10890 10893 QTL +T205 SO:0000704 10965 10970 genes +T206 CL:0000623 11043 11062 natural killer cell +T207 PR:000036337 11090 11114 T2R-type taste receptors +T208 GO:0050909 11099 11104 taste +T209 PR:000036337 11120 11125 Tas2r +T210 SO:0000704 11126 11131 genes +T211 PR:000036337 11150 11154 T2Rs +T212 GO:0010467 11317 11327 expression +T213 GO:0050909 11331 11336 taste +T214 CL:0000209 11331 11351 taste receptor cells +T215 GO:0050913 11402 11416 bitter-tasting +T216 CHEBI:36357 11417 11426 compounds +T217 PR:000036337 11471 11477 Tas2rs +T218 GO:0050909 11528 11533 taste +T219 SO:0000771 11546 11549 QTL +T220 SO:0000771 11571 11574 QTL +T221 SO:0001026 11827 11833 genome +T222 SO:0000771 11866 11869 QTL +T223 GO:0050913 11961 11973 bitter taste +T224 PR:000036337 11994 12000 Tas2rs +T225 NCBITaxon:10088 12123 12128 mouse +T226 SO:0001026 12129 12135 genome +T227 PR:000036337 12233 12238 Tas2r +T228 PR:000036337 12267 12272 Tas2r +T229 SO:0000704 12273 12278 genes +T230 PR:000036337 12319 12325 Tas2rs +T231 SO:0000704 12418 12423 genes +T232 UBERON:0001836 12446 12454 salivary +T233 PR:000013555 12465 12469 Prp2 +T234 PR:000013555 12474 12478 Prh1 +T235 SO:0001026 12577 12583 genome +T236 PR:000036337 12586 12589 T2R +T237 SO:0001023 12590 12597 alleles +T238 PR:000036337 12616 12622 Tas2rs +T239 GO:0050909 12654 12659 taste +T240 SO:0000771 12672 12675 QTL +T241 PR:000036337 12713 12718 Tas2r +T242 SO:0000704 12719 12724 genes +T243 PR:000036337 12782 12787 Tas2r +T244 SO:0001023 12788 12794 allele +T245 SO:0000336 12800 12810 pseudogene +T246 NCBITaxon:10088 12882 12886 mice +T247 SO:0000195 12925 12936 coding exon +T248 PR:000036337 12945 12950 Tas2r +T249 SO:0001023 12951 12957 allele +T250 GO:0065007 13091 13101 regulatory +T251 SO:0005836 13091 13109 regulatory regions +T252 PR:000036337 13118 13123 Tas2r +T253 SO:0001023 13124 13130 allele +T254 GO:0010467 13139 13149 expression +T255 PR:000036337 13348 13353 Tas2r +T256 GO:0050909 13384 13389 taste +T257 PR:000036337 13435 13441 Tas2rs +T258 PR:000036337 13469 13474 Tas2r +T259 SO:0000336 13475 13486 pseudogenes +T260 NCBITaxon:10088 13550 13554 mice +T261 SO:0000357 13623 13631 flanking +T262 PR:000036337 13667 13672 Tas2r +T263 PR:000036337 13736 13741 Tas2r +T264 SO:0001026 13766 13773 genomic +T265 SO:0000006 13779 13791 PCR products +T266 SO:0000440 13820 13827 vectors +T267 SO:0000855 13885 13896 orthologues +T268 PR:000036337 13939 13944 Tas2r +T269 SO:0001023 13945 13952 alleles +T270 PR:000016893 13963 13971 Tas2r106 +T271 PR:000016904 13976 13984 Tas2r124 +T272 PR:000016902 14067 14075 Tas2r120 +T273 SO:0001026 14108 14115 genomic +T274 PR:000036337 14219 14224 Tas2r +T275 NCBITaxon:10088 14242 14246 mice +T276 SO:0001023 14255 14262 alleles +T277 PR:000016890 14264 14272 Tas2r103 +T278 PR:000016900 14277 14285 Tas2r117 +T279 SO:0000704 14407 14412 genes +T280 SO:0000336 14420 14431 pseudogenes +T281 PR:000036337 14465 14471 Tas2rs +T282 PR:000036337 14528 14534 Tas2rs +T283 SO:0001023 14559 14566 alleles +T284 NCBITaxon:10088 14580 14584 mice +T285 SO:0000694 14594 14625 single nucleotide polymorphisms +T286 SO:0000195 14645 14657 coding exons +T287 PR:000036337 14724 14730 Tas2rs +T288 GO:0005576 14834 14847 extracellular +T289 PR:000036337 14861 14865 T2Rs +T290 SO:0001023 14895 14902 Allelic +T291 PR:000036337 14937 14943 Tas2rs +T292 SO:0001023 14955 14962 alleles +T293 PR:000036337 14971 14977 Tas2rs +T294 SO:0000006 15053 15065 PCR products +T295 PR:000016892 15067 15075 Tas2r105 +T296 PR:000016899 15077 15085 Tas2r116 +T297 SO:0000006 15135 15146 PCR product +T298 PR:000016902 15148 15156 Tas2r120 +T299 PR:000036337 15197 15202 Tas2r +T300 SO:0000858 15420 15431 orthologous +T301 GO:0050909 15552 15557 taste +T302 SO:0000771 15558 15561 QTL +T303 PR:000036337 15583 15588 Tas2r +T304 SO:0001023 15589 15596 alleles +T305 GO:0050909 15622 15627 taste +T306 SO:0001026 15675 15682 genomic +T307 GO:0050909 15756 15761 taste +T308 PR:000036337 15779 15784 Tas2r +T309 SO:0001023 15785 15792 alleles +T310 PR:000036337 15954 15959 Tas2r +T311 SO:0000006 15960 15971 PCR product +T312 SO:0001023 16002 16008 allele +T313 SO:0000704 16035 16040 genes +T314 PR:000016891 16042 16050 Tas2r104 +T315 PR:000016898 16052 16060 Tas2r114 +T316 PR:000016896 16065 16073 Tas2r110 +T317 PR:000016902 16145 16153 Tas2r120 +T318 NCBITaxon:10088 16185 16189 mice +T319 SO:0000006 16208 16219 PCR product +T320 SO:0000704 16263 16267 gene +T321 PR:000036337 16465 16470 Tas2r +T322 PR:000036337 16591 16596 Tas2r +T323 SO:0000704 16597 16601 gene +T324 GO:0050909 16634 16639 taste +T325 PR:000036337 16693 16698 Tas2r +T326 SO:0001024 16719 16728 haplotype +T327 PR:000036337 16781 16786 Tas2r +T328 SO:0001024 16807 16816 haplotype +T329 NCBITaxon:10088 16830 16834 mice +T330 SO:0000195 16840 16851 coding exon +T331 PR:000036337 16866 16872 Tas2rs +T332 SO:0001026 16922 16929 genomic +T333 PR:000036337 16966 16971 Tas2r +T334 SO:0001023 17049 17055 allele +T335 SO:0001023 17067 17073 allele +T336 PR:000036337 17177 17182 Tas2r +T337 GO:0050909 17213 17218 taste +T338 PR:000036337 17315 17320 Tas2r +T339 SO:0001024 17321 17330 haplotype +T340 SO:0001024 17335 17344 haplotype +T341 SO:0001024 17356 17365 haplotype +T342 NCBITaxon:10088 17377 17381 mice +T343 NCBITaxon:10088 17461 17465 mice +T344 GO:0050909 17507 17512 taste +T345 PR:000036337 17555 17560 Tas2r +T346 SO:0001024 17561 17570 haplotype +T347 PR:000036337 17657 17662 Tas2r +T348 SO:0001024 17663 17672 haplotype +T349 GO:0050909 17795 17804 gustatory +T350 UBERON:0001033 17795 17811 gustatory system +T351 NCBITaxon:40674 17815 17822 mammals +T352 GO:0050913 17876 17890 bitter-tasting +T353 GO:0050909 18058 18063 taste +T354 CL:0000209 18058 18078 taste receptor cells +T355 GO:0001775 18087 18106 cellular activation +T356 UBERON:0001017 18128 18150 central nervous system +T357 SO:0000771 18207 18210 QTL +T358 GO:0050909 18277 18282 taste +T359 NCBITaxon:10088 18313 18317 mice +T360 SO:0000771 18324 18327 QTL +T361 SO:0000771 18393 18396 QTL +T362 CHEBI:15854 18401 18408 quinine +T363 GO:0007631 18409 18415 intake +T364 CHEBI:15854 18417 18420 Qui +T365 GO:0050909 18446 18451 taste +T366 GO:0065007 18475 18485 regulating +T367 CHEBI:15854 18486 18493 quinine +T368 GO:0007631 18545 18556 consumption +T369 GO:0050913 18560 18574 bitter-tasting +T370 GO:0050909 18626 18631 taste +T371 CHEBI:15854 18727 18734 quinine +T372 GO:0050909 18735 18740 taste +T373 SO:0000771 18741 18744 QTL +T374 PR:000036337 18804 18809 Tas2r +T375 SO:0000704 18810 18815 genes +T376 SO:0000704 18835 18840 genes +T377 SO:0000704 18886 18891 genes +T378 UBERON:0001836 18917 18925 salivary +T379 PR:000013555 18936 18940 Prp2 +T380 PR:000013555 18945 18949 Prh1 +T381 GO:0050913 18999 19011 bitter taste +T382 PR:000036337 19018 19024 Tas2rs +T383 SO:0000704 19088 19095 gene(s) +T384 GO:0010467 19114 19124 expression +T385 GO:0050909 19128 19133 taste +T386 CL:0000209 19128 19148 taste receptor cells +T387 SO:0000704 19157 19164 genetic +T388 GO:0050913 19234 19246 bitter taste +T389 CHEBI:15854 19291 19298 quinine +T390 PR:000036337 19313 19317 T2Rs +T391 GO:0016020 19385 19393 membrane +T392 CHEBI:15854 19447 19454 quinine +T393 CHEBI:15854 19520 19527 quinine +T394 CHEBI:36916 19564 19572 cationic +T395 GO:0006812 19564 19585 cationic conductances +T396 CHEBI:29103 19600 19602 K+ +T397 GO:0050909 19615 19620 taste +T398 CL:0000209 19615 19635 taste receptor cells +T399 CHEBI:15854 19675 19682 quinine +T400 GO:0050909 19683 19688 taste +T401 PR:000036337 19700 19703 T2R +T402 CHEBI:15854 19792 19799 quinine +T403 SO:0000704 20044 20049 genic +T404 CHEBI:15854 20060 20067 quinine +T405 GO:0050909 20068 20073 taste +T406 SO:0000771 20102 20105 QTL +T407 PR:000036337 20143 20148 Tas2r +T408 SO:0000704 20149 20154 genes +T409 SO:0000704 20185 20190 genes +T410 CHEBI:24870 20200 20203 ion +T411 SO:0000771 20345 20348 QTL +T412 CHEBI:15854 20362 20369 quinine +T413 GO:0050909 20370 20375 taste +T414 GO:0050913 20474 20486 bitter taste +T415 PR:000036337 20588 20593 Tas2r +T416 SO:0000771 20740 20743 QTL +T417 PR:000036337 20840 20846 Tas2rs +T418 CHEBI:15854 20859 20866 quinine +T419 GO:0050909 20867 20872 taste +T420 PR:000036337 20997 21003 Tas2rs +T421 PR:000036337 21067 21072 Tas2r +T422 SO:0000704 21110 21117 genetic +T423 SO:0000704 21166 21171 genes +T424 GO:0050909 21184 21189 taste +T425 PR:000016062 21212 21218 Tas1r3 +T426 SO:0000704 21219 21223 gene +T427 GO:0050916 21262 21267;21278 21283 sweet ... taste +T428 GO:0050917 21272 21283 umami taste +T429 PR:000036337 21350 21355 Tas2r +T430 CHEBI:46261 21400 21419 phenylthiocarbamide +T431 CHEBI:46261 21421 21424 PTC +T432 GO:0050909 21426 21431 taste +T433 NCBITaxon:9606 21447 21453 humans +T434 SO:0000704 21497 21502 genes +T435 CHEBI:32111 21513 21522 saccharin +T436 CHEBI:46261 21526 21529 PTC +T437 GO:0050909 21530 21535 taste +T438 SO:0000854 21568 21578 paralogues +T439 GO:0050913 21584 21596 bitter taste +T440 SO:0000704 21609 21616 genetic +T441 SO:0000704 21663 21668 genes +T442 CHEBI:62488 21689 21708 signaling molecules +T443 PR:000036337 21726 21729 T2R +T444 SO:0000771 21782 21785 QTL +T445 CHEBI:8502 21790 21794 PROP +T446 SO:0000771 21874 21877 QTL +T447 CHEBI:15854 21882 21889 quinine +T448 GO:0050909 21890 21895 taste +T449 PR:000036337 21944 21950 Tas2rs +T450 PR:000036337 22032 22038 Tas2rs +T451 NCBITaxon:10088 22085 22089 mice +T452 GO:0050909 22132 22137 taste +T453 CHEBI:36357 22158 22167 compounds +T454 CHEBI:27641 22182 22195 cyclohexamide +T455 PR:000036337 22250 22256 Tas2rs +T456 SO:0000417 22354 22361 domains +T457 PR:000036337 22486 22492 Tas2rs +T458 GO:0005576 22514 22527 extracellular +T459 PR:000036337 22640 22644 T2Rs +T460 PR:000036337 22726 22729 T2R +T461 SO:0001816 22926 22939 nonsynonymous +T462 SO:0000855 22962 22973 orthologues +T463 PR:000036337 23045 23051 Tas2rs +T464 NCBITaxon:9606 23055 23061 humans +T465 NCBITaxon:9604 23063 23073 great apes +T466 NCBITaxon:9527 23078 23095 old world monkeys +T467 PR:000036337 23109 23115 Tas2rs +T468 NCBITaxon:10088 23207 23212 mouse +T469 NCBITaxon:species 23242 23249 species +T470 NCBITaxon:10088 23340 23344 mice +T471 PR:000036337 23421 23426 Tas2r +T472 SO:0001024 23427 23437 haplotypes +T473 NCBITaxon:10088 23454 23459 mouse +T474 PR:000036337 23513 23518 Tas2r +T475 NCBITaxon:10088 23545 23550 mouse +T476 NCBITaxon:species 23551 23558 species +T477 NCBITaxon:subspecies 23562 23572 subspecies +T478 GO:0050913 23698 23712 bitter-tasting +T479 CHEBI:15854 23723 23730 quinine +T480 GO:0050909 23748 23753 taste +T481 SO:0000704 23791 23796 genic +T482 NCBITaxon:10088 23806 23810 mice +T483 CHEBI:15854 23845 23852 quinine +T484 GO:0050909 23853 23858 taste +T485 PR:000036337 23907 23910 T2R +T486 PR:000036337 23927 23932 Tas2r +T487 SO:0000704 23933 23938 genes +T488 NCBITaxon:10088 24015 24019 mice +T489 SO:0001024 24053 24062 haplotype +T490 CHEBI:15854 24084 24091 quinine +T491 GO:0050909 24092 24097 taste +T492 PR:000036337 24139 24142 T2R +T493 NCBITaxon:10088 24178 24183 mouse +T494 PR:000036337 24206 24210 T2Rs +T495 PR:000036337 24328 24331 T2R +T496 GO:0010467 24332 24342 expressing +T497 GO:0050909 24361 24366 taste +T498 CL:0000209 24361 24372 taste cells +T499 NCBITaxon:10088 24440 24444 Mice +T500 CHEBI:75958 24449 24458 solutions +T501 UBERON:0007023 24475 24480 adult +T502 NCBITaxon:10088 24497 24501 mice +T503 NCBITaxon:10088 24652 24656 mice +T504 NCBITaxon:10088 24791 24795 mice +T505 GO:0007618 24879 24885 mating +T506 NCBITaxon:10088 24922 24926 mice +T507 CHEBI:33290 25015 25019 food +T508 NCBITaxon:9989 25033 25039 rodent +T509 GO:0050909 25047 25052 Taste +T510 CHEBI:33893 25100 25107 reagent +T511 CHEBI:17992 25125 25132 Sucrose +T512 CHEBI:16150 25145 25153 benzoate +T513 CHEBI:8502 25155 25175 6-n-propylthiouracil +T514 CHEBI:15854 25180 25187 quinine +T515 CHEBI:17883 25188 25201 hydrochloride +T516 CHEBI:75958 25263 25271 solution +T517 CHEBI:15377 25310 25315 water +T518 GO:0050909 25325 25330 taste +T519 NCBITaxon:33208 25379 25385 animal +T520 NCBITaxon:33208 25437 25443 Animal +T521 CHEBI:15377 25717 25722 water +T522 NCBITaxon:10088 25742 25746 mice +T523 CHEBI:15377 25835 25840 water +T524 NCBITaxon:10088 25875 25879 mice +T525 CHEBI:15377 25983 25988 water +T526 NCBITaxon:10088 26079 26084 mouse +T527 NCBITaxon:10088 26154 26158 mice +T528 NCBITaxon:10088 26309 26313 Mice +T529 GO:0050909 26368 26373 taste +T530 CHEBI:8502 26427 26431 PROP +T531 CHEBI:17992 26471 26478 sucrose +T532 CHEBI:15377 26681 26686 water +T533 NCBITaxon:10088 26715 26719 mice +T534 NCBITaxon:10088 26804 26809 mouse +T535 CHEBI:15377 26895 26900 water +T536 CHEBI:15377 26982 26987 water +T537 CHEBI:15377 27008 27013 water +T538 NCBITaxon:10088 27115 27119 mice +T539 NCBITaxon:10088 27257 27261 mice +T540 CHEBI:17992 27265 27272 sucrose +T541 CHEBI:36357 27356 27364 compound +T542 CHEBI:15377 27397 27402 water +T543 NCBITaxon:33208 27420 27427 animals +T544 CHEBI:15377 27535 27540 water +T545 CHEBI:15377 27622 27627 water +T546 NCBITaxon:10088 27668 27672 mice +T547 CHEBI:15377 27706 27711 water +T548 GO:0050909 27728 27733 taste +T549 GO:0007608 27749 27758 olfactory +T550 NCBITaxon:10088 27792 27796 mice +T551 SO:0000771 27876 27879 QTL +T552 NCBITaxon:10088 27927 27931 mice +T553 SO:0000771 28244 28268 quantitative trait locus +T554 SO:0000771 28270 28273 QTL +T555 SO:0001026 28508 28514 genome +T556 SO:0000771 28664 28667 QTL +T557 PR:000036337 28795 28798 T2R +T558 SO:0001023 28799 28806 alleles +T559 SO:0001026 28905 28911 genome +T560 SO:0000357 28950 28958 flanking +T561 SO:0000147 28983 28987 exon +T562 PR:000036337 28988 28993 Tas2r +T563 SO:0001026 29026 29033 genomic +T564 SO:0000006 29206 29218 PCR products +T565 CHEBI:33694 29336 29346 Biopolymer +T566 SO:0000061 29510 29527 restriction sites +T567 SO:0001023 29574 29581 alleles +T568 PR:000036337 29666 29671 Tas2r +T569 SO:0001026 29693 29700 genomic +T570 PR:000016902 29735 29743 Tas2r120 +T571 SO:0000006 29762 29773 PCR product +T572 SO:0001023 29810 29816 allele +T573 CHEBI:15854 29886 29893 quinine +T574 GO:0050909 29894 29899 taste +T575 SO:0000771 29900 29903 QTL +T576 PR:000036337 29918 29923 Tas2r +T577 SO:0000704 29924 29929 genes +T578 SO:0000771 30039 30042 QTL +T579 CHEBI:15854 30100 30107 quinine +T580 GO:0050909 30108 30113 taste +T581 SO:0000771 30114 30117 QTL +T582 PR:000036337 30126 30132 Tas2rs +T583 SO:0000771 30147 30150 QTL +T584 SO:0000771 30385 30388 QTL +T585 GO:0050909 30723 30728 taste +T586 SO:0000771 30838 30841 QTL +T587 SO:0001023 31007 31014 alleles +T588 SO:0001026 31244 31250 genome +T589 PR:000036337 31383 31389 Tas2rs +T590 GO:0050909 31474 31479 taste +T591 http://purl.obolibrary.org/obo/MONDO_0002182 31760 31783 Communication Disorders diff --git a/src/ontogpt/evaluation/craft/database/all/15938754.txt b/src/ontogpt/evaluation/craft/database/all/15938754.txt new file mode 100644 index 000000000..0ddb89237 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/15938754.txt @@ -0,0 +1,151 @@ +Haplotypes at the Tas2r locus on distal chromosome 6 vary with quinine taste sensitivity in inbred mice + +Abstract + +Background + +The detection of bitter-tasting compounds by the gustatory system is thought to alert animals to the presence of potentially toxic food. Some, if not all, bitter stimuli activate specific taste receptors, the T2Rs, which are expressed in subsets of taste receptor cells on the tongue and palate. However, there is evidence for both receptor-dependent and -independent transduction mechanisms for a number of bitter stimuli, including quinine hydrochloride (QHCl) and denatonium benzoate (DB). + +Results + +We used brief-access behavioral taste testing of BXD/Ty recombinant inbred (RI) mouse strains to map the major quantitative trait locus (QTL) for taste sensitivity to QHCl. This QTL is restricted to a ~5 Mb interval on chromosome 6 that includes 24 genes encoding T2Rs (Tas2rs). Tas2rs at this locus display in total 307 coding region single nucleotide polymorphisms (SNPs) between the two BXD/Ty RI parental strains, C57BL/6J (quinine-sensitive) and DBA/2J (quinine insensitive); approximately 50% of these mutations are silent. Individual RI lines contain exclusively either C57BL/6J or DBA/2J Tas2r alleles at this locus, and RI lines containing C57BL/6J Tas2r alleles are more sensitive to QHCl than are lines containing DBA/2J alleles. Thus, the entire Tas2r cluster comprises a large haplotype that correlates with quinine taster status. + +Conclusion + +These studies, the first using a taste-salient assay to map the major QTL for quinine taste, indicate that a T2R-dependent transduction cascade is responsible for the majority of strain variance in quinine taste sensitivity. Furthermore, the large number of polymorphisms within coding exons of the Tas2r cluster, coupled with evidence that inbred strains exhibit largely similar bitter taste phenotypes, suggest that T2R receptors are quite tolerant to variation. + +Background + +Animals use the gustatory system to provide information about food quality. For example, sweet-tasting foods may have a high caloric content and are preferred, while bitter-tasting foods often contain toxic substances, and are generally avoided. Two families of G protein-coupled receptors (GPCRs) expressed in subpopulations of taste receptor cells (TRCs) of the gustatory epithelium have been implicated in the detection and transduction of sweet, bitter and umami (i.e., glutamate) taste: T1Rs for sweet-and umami-tasting stimuli [1-8], and T2Rs for bitter-tasting compounds [9-11]. + +The genes that encode T2Rs, the Tas2rs, were first identified by database mining of mammalian genomes near chromosomal markers previously linked to differences in bitter taste sensitivity [9,11-18]. In mice, the majority of Tas2rs lie within a single cluster on distal chromosome 6. Thirty-three human Tas2rs (including 8 pseudogenes) and thirty-six mouse Tas2rs (including 3 pseudogenes in C57BL/6J mice) have been identified [9,11,19], and several of these respond to particular bitter stimuli in heterologous expression assays [10,20-23], or represent a strong candidate gene for a specific bitter taste quantitative trait [18,24,25]. + +Several quantitative trait loci (QTL) have been identified that influence two-bottle intake of bitter stimuli in the mouse, including loci for quinine (Qui) [12,16,26], cyclohexamide (Cyx) [13] and sucrose octaacetate (Soa) [14,15,17] sensitivity. Each of these QTL map to mouse distal chromosome 6 and are linked to the marker D6Mit13, which lies within a cluster of 24 intact Tas2rs in the C57BL/6 genome (e.g., [16,27,28]). However, the interpretation of these studies remains problematic for two reasons. First, the density of chromosomal markers and number of recombinant inbred (RI) strains used in these earlier studies did not permit the physical definition of the intervals containing each QTL. Second, these previous attempts to map bitter taste QTLs relied on behavioral assays that measured consumption, and were thus susceptible to contributions of post-ingestive effects such as toxicity. As we have shown previously, such effects can confound the quantification of bitter taste behaviors [29]. Therefore, the relevance and/or contribution of the aforementioned QTLs to bitter taste remain unclear. + +Furthermore, a number of physiological studies have suggested that the transduction of some amphiphilic bitter compounds, such as quinine and denatonium benzoate, may stimulate taste receptor cells independently of GPCRs (e.g. [30]). Quinine may directly activate G proteins, and both quinine and denatonium can block K+ channels [31-36] ; caffeine, another bitter-tasting substance, directly inhibits intracellular phosphodiesterase [33]. However, the relative contributions of T2R-dependent and T2R-independent mechanisms to the detection of these bitter stimuli are unknown. + +Here we use a taste-salient brief-access lick test [29,37] to measure taste sensitivities in C57BL/6J (B6), DBA/2J (D2) and BXD/Ty (BXD) recombinant inbred (RI) mice to two bitter stimuli, quinine hydrochloride (QHCl) and denatonium benzoate (DB). Using 17 BXD lines that were genotyped at 762 informative chromosomal markers, we mapped a major QTL for QHCl taste to a ~5 Mb interval on distal chromosome 6 that contains all 24 of the Tas2r genes in the distal cluster. We analyzed the sequence of each Tas2r allele in the parental strains (B6 and D2) and 29 RI lines. This analysis revealed that all 24 genes are polymorphic between the two strains, and that these 24 Tas2rs comprise a single haplotype that correlates with QHCl taste sensitivity. + +Results + +Taste testing + +Previous efforts to map QTL for bitter taste have utilized consumption tests that may be confounded by the contributions of post-ingestive effects [29]. We used a modified brief-access lick test, which minimizes the contribution of such effects [29,37] to determine whether B6 and D2 mice display differences in taste sensitivity to the taste stimuli QHCl and DB. After initially screening B6 and D2 mice to determine stimulus concentrations that were aversive but not saturating [47], we selected two ligand concentrations for each compound that best differentiated the two strains. Subsequent taste testing of BXD RI lines was restricted to these two concentrations (1 and 3 mM for both QHCl and DB). Avoidance by male and female B6 and D2 mice increased (as indicated by the decreased lick ratio) in a concentration-dependent manner for both compounds (Figure 1A; Table 1). There was a significant strain difference for both 1 and 3 mM QHCl (F[1,25] > 24.6; p < 0.0001). D2 mice displayed decreased aversion relative to B6 mice at both concentrations. On the other hand, the strains did not significantly differ in taste sensitivity to DB (Figure 1A). There were no significant effects of gender. + +Table 1 + +Mean lick ratios for B6, D2 and BXD mice. + +The number of individual mice tested for each strain (n) is listed in the second column. Subsequent columns show the mean lick rate to water during testing (± SEM), mean lick ratio for denatonium benzoate (DB), PROP (6-n-propylthiouracil; PR) and quinine hydrochloride (QH) at each of two concentrations (see Methods for details). + +Figure 1 + +Lick ratios (mean ± SE) for B6, D2 and BXD strains. (A) Mean lick ratios for B6 (filled circles) and D2 (open circles) mice at two concentrations of QHCl and DB. In all panels, a lower mean lick ratio indicates a greater aversion, and therefore greater taste sensitivity, to the stimulus. For panels B, C, and D, each BXD strain is represented by a different color, and listed in order from least sensitive to most sensitive to 1 mM QHCL. (B) Mean lick ratios for the six BXD strains that are most sensitive to QHCl in this assay. (C) Mean lick ratios for the five BXD strains that are least sensitive to QHCl in this assay. (D) Mean lick ratios for the six BXD strains intermediate in QHCl taste sensitivity to those in (B) and (C). Cutoffs for the three QHCl taster groups were arbitrarily set, as there was a continuity of the phenotype at 1 mM QHCl: sensitive strains exhibited a lick ratio for 1 mM QHCl of ≤ 0.3, intermediate strains from 0.31-0.6, and insensitive strains > 0.6. The absence of two distinct phenotypic classes suggests that sensitivity to QHCL is under polygenic control. + +We next tested mice from 17 BXD lines in the same manner. BXD mice also typically avoided both stimuli in a concentration dependent manner (Figures 1B–1D; Table 1). However, QHCl and DB taste sensitivity vary independently across these RI strains: some strains highly sensitive to QHCl are relatively insensitive to DB, and vice versa (Figures 1B–1D). + +QTL mapping + +Linkage analysis was conducted using Map Manager QTX (version 0.30[38]). No significant QTLs were identified for DB taste sensitivity, although several associations with markers on chromosomes 2,8 and 12 were "suggestive" (LRS > 9.4, genome-wide p = 0.65; see Additional File 1). A significant (LRS > 20.5; genome-wide p = 0.05) QTL for sensitivity to 1 mM QHCl was indicated on chromosome 6, with a second, suggestive (LRS > 11.4; genome-wide p = 0.65) QTL on chromosome 8 (Figure 2A); at 3 mM QHCl, both of these QTL were suggestive (LRS > 10.9) but did not reach genome-wide significance (Figure 2B). + +Figure 2 + +A major QTL for QHCl taste on mouse chromosome 6. (Top panel) The interval map (see Methods) shows a significant QTL on chromosome 6 (green) and a suggestive QTL on chromosome 8 (yellow) affecting taste responses to 1 mM QHCl. (Bottom panel) For 3 mM QHCl, both QTL were suggestive (yellow). The dashed line indicates genome-wide significance. + +The chromosome 6 QTL was linked to a single marker, D6Mit13 (Table 2, Figure 3). Adjacent proximal markers D6Mit254 and D6Mit194 are unlinked to the QHCl QTL, as is distal marker D6Mit374. Across the 17 RI lines tested there is at least one recombination event between D6Mit13 and either D6Mit254 (and D6Mit194, the physical position of which is not well defined) or D6Mit374. An additional proximal marker, D6Mit61, which lies between D6Mit194 and D6Mit13, was identified from genotypes of the BXD lines reported by the Jackson Laboratories. BXD/Ty-34 RI mice display a clear D2 phenotype for QHCl taste (Figure 1C) and D2 genotype for D6Mit13, but have a B6 genotype for D6Mit61 [39,40], indicating that D6Mit61 is unlinked to the QHCl QTL. Therefore, this QTL interval can be conservatively defined as that portion of mouse chromosome 6 that lies between D6Mit254 and D6Mit374, but is most likely restricted to the region between D6Mit61 and D6Mit374. + +Physical mapping of the single linked marker, D6Mit13, and the two closest unlinked markers, D6Mit61and D6Mit374, was performed in silico based on the May, 2004 build of the public B6 genome. Based on these marker positions, the size of the QHCl chromosome 6 QTL is less than 5.0 Mb (Figure 3). This region contains a number of known genes, all but eleven of which encode members of two large receptor families: natural killer cell lectin-like receptors, and T2R-type taste receptors. The Tas2r genes (which encode the T2Rs) are found clustered within a 1.2 Mb interval on either side of D6Mit13 (Figure 3, Figure 4). Because of their proximity to the linked marker, their demonstrated expression in taste receptor cells, and their role in the detection of at least some bitter-tasting compounds, we hypothesized that one or more of the 24 Tas2rs at this locus were responsible for the major QHCl taste sensitivity QTL. + +Figure 3 + +The QHCl QTL is linked to a single marker on chromosome 6. (A) As shown in the interval map for chromosome 6, the trait value (lick ratio for 1 mM QHCl) correlates strongly across BXD RI strains with the polymorphic marker D6Mit13 (bold). The dashed line indicates genome-wide significance. (B) The QHCl QTL (which lies between unlinked markers D6Mit61 and D6Mit 374) contains a cluster of putative bitter taste receptor genes, the Tas2rs (gray box). Physical positions of the polymorphic markers are given in Mb, and are based on the May, 2004 build of the B6 mouse genome. The physical position of D6Mit194 (*) is tentative. + +Figure 4 + +A map of the distal chromosome 6 Tas2r cluster. Twenty-four intact Tas2r genes map to distal chromosome 6 (black). The Tas2rs are found in two subclusters on either side of the polymorphic marker D6Mit13 (red) and two genes encoding proline-rich salivary proteins (Prp2 and Prh1; red). Map positions, in Mb, represent chromosome 6 positions in the May, 2004 assembly of the B6 genome. + +T2R alleles + +If one (or more) Tas2rs underlie the chromosome 6 QHCl taste sensitivity QTL, we would predict that one (or more) Tas2r genes would exhibit one of three likely characteristics: (1) A Tas2r allele is a pseudogene, or is deleted, in D2 (QHCL-insensitive), but not B6 (QHCl-sensitive), mice; (2) Missense mutations in the single coding exon of a D2 Tas2r allele impact protein functions such as ligand binding or receptor coupling to downstream signaling cascades; (3) Mutations in noncoding or regulatory regions of a D2 Tas2r allele affects expression of the protein product. Though we considered all three of these to be valid possibilities, we initially focused on the likelihood that deletion or mutation within the coding sequence of a single D2 Tas2r would correlate with the QHCl taste insensitivity phenotype. + +Twenty-four intact Tas2rs, along with three apparent Tas2r pseudogenes, have been identified in the distal chromosome 6 cluster of B6 mice [19] (Figure 4). We designed oligonucleotides to non-coding regions flanking the coding sequence of each intact Tas2r [see Additional file 2]. Using these oligos, we amplified each Tas2r coding sequence from D2 genomic DNA. PCR products were subcloned into cloning vectors and sequenced. Comparisons of the sequences of B6 and D2 orthologues revealed that only two of the twenty-four Tas2r alleles examined, Tas2r106 and Tas2r124, were identical across strains at the amino acid level (data not shown). A third, Tas2r120, could not be amplified from D2 genomic DNA (Figure 5) using either of two pairs of oligonucleotides (Additional file 2), suggesting that this Tas2r is deleted in D2 mice. Two D2 alleles, Tas2r103 and Tas2r117, contained numerous missense mutations and small deletions that create frame shifts and premature termination; these two genes may be pseudogenes in this strain. The remaining 19 Tas2rs contained between one and 16 missense mutations. All 24 Tas2rs examined have different alleles in B6 and D2 mice, and 307 single nucleotide polymorphisms are present within coding exons (data not shown). Although polymorphic residues between B6 and D2 Tas2rs are found in all regions of the receptors, 23% of the amino acid changes seen are within the first two extracellular loops of the T2Rs (data not shown). + +Figure 5 + +Allelic variation across strains for four Tas2rs. B6 and D2 alleles of four Tas2rs can be differentiated based on diagnostic restriction digests of amplified PCR products (Tas2r105, Tas2r116 and Tas2r131) or on the presence or absence of a PCR product (Tas2r120). In each of the 17 BXD strains tested, Tas2r genotype was always correlated with QHCl taster phenotype (blue = B6 taster phenotype, red = D2 taster phenotype). See additional file 1: Table 3 for restriction enzymes and oligonucleotides. + +The variability between orthologous receptors in these two inbred strains suggested that it might be possible to narrow the physical boundaries of the QHCl taste QTL by determining which Tas2r alleles are correlated with QHCl taste sensitivity. Therefore, we proceeded to screen genomic DNA from 29 available BXD RI lines, including the 17 that we had used in taste testing, for the Tas2r alleles they contained. In most cases, we were able to identify diagnostic restriction endonuclease digests that would allow us to quickly identify whether a particular Tas2r PCR product was amplified from a B6 or D2 allele. We did not analyze three genes (Tas2r104, Tas2r114 and Tas2r110) where no diagnostic restriction endonuclease could be identified. For Tas2r120, which is likely deleted in D2 mice, the absence of a PCR product was diagnostic of the D2 genotype for this gene. + +Surprisingly, we discovered that there have been no apparent recombination events within the distal chromosome 6 cluster during the generation of the BXD RI lines. For all RI lines tested, every Tas2r within an individual RI line originated from the same parental strain (Figures 5, 6). Furthermore, the genotype of each Tas2r gene always correlated with the QHCl taste phenotype (Figures 6, 7), suggesting that the entire Tas2r cluster is a single haplotype that varies with QHCl taster status. + +Figure 6 + +The Tas2r cluster is a single haplotype in BXD/Ty RI mice. The coding exon of each of 21 Tas2rs in the distal chromosome 6 cluster was amplified genomic DNA from 29 BXD/Ty RI strains. Each Tas2r within an individual BXD strain originated from the same parental strain (B6 allele = gray, D2 allele = white). The 17 BXD strains that were behaviorally tested in this study are indicated (*). + +Figure 7 + +Tas2r genotype correlates with QHCl taste phenotype. Mean lick ratios of B6, D2 and BXD strains reported in Figure 1 are grouped based on Tas2r haplotype (B6 haplotype = blue, D2 haplotype = red). B6 mice (blue line on left panel) are more sensitive to 1 mM and 3 mM QHCl than are D2 mice (red line on left panel) in brief access taste tests. Similarly, BXD strains with the B6 Tas2r haplotype (blue lines, right panel) are more sensitive to QHCl than are BXD strains with the D2 Tas2r haplotype (red lines, right panel). The BXD strains are listed in order from least to most sensitive to 1 mM QHCL. + +Discussion + +The gustatory system of mammals is thought to detect thousands of chemically-diverse bitter-tasting substances [41]. Although specific receptors, enzymes and channels have been implicated in the transduction of bitter stimuli, how interactions of bitter stimuli with taste receptor cells lead to cellular activation and signaling to the central nervous system is still poorly understood. We have found that a single QTL on distal chromosome 6 accounts for most of the variation in QHCL taste sensitivity between B6 and D2 mice. This QTL maps to the same chromosomal position as a previously identified QTL for quinine intake, Qui [16,28], indicating that taste is the major factor in regulating quinine aversion. This is an important distinction, as the consumption of bitter-tasting stimuli can be dependent on factors independent of taste, such as toxicity [29]. + +Using 17 RI lines and 762 chromosomal markers, we have restricted the quinine taste QTL to a < 5 Mb region on distal chromosome 6 that contains 24 Tas2r genes. At least 60 other genes also lie within this interval, including two genes that encode proline-rich salivary proteins, Prp2 and Prh1; these proteins appear to play no direct role in bitter taste [48]. Tas2rs are the most likely candidates for the QHCl quantitative trait gene(s) due to: (1) their expression in taste receptor cells and (2) genetic and functional evidence linking them to the detection of a number of bitter taste stimuli. As of yet there is no evidence for quinine activation of T2Rs from functional assays of these receptors in heterologous cells or membrane preparations, likely due to the lipophilic nature of quinine [23]. However, several physiological studies have suggested that quinine can directly activate G proteins or cationic conductances, or can block K+ channels in taste receptor cells [34-36]. While our data indicates that quinine taste is largely T2R-dependent, it is not exclusively so. For example, the BXD RI lines exhibited a range of quinine sensitivity, with several strains having similar sensitivities to that of B6, some strains with sensitivities similar to that of D2, and a third group with a more intermediate phenotype (Figures 1, 7). This observation is consistent with a polygenic basis for quinine taste [16,26]. Also, a suggestive QTL on chromosome 8 does not contain any Tas2r genes, but does contain a number of genes encoding ion channels, enzymes and members of other receptor families (our unpublished data). It will be interesting to determine whether this suggestive QTL is linked to quinine taste and, if so, whether it is specific for this single bitter stimulus or more broadly related to all bitter taste. + +Of the 29 BXD RI lines examined, there was no apparent recombination event within the chromosome 6 Tas2r cluster. While increasing the number of BXD RI lines or the number of markers used for genotyping them would facilitate the definition of smaller QTL intervals, in this case such an effort is unlikely to permit the identification of one or a few Tas2rs involved in quinine taste. For example, we examined six lines of AXB and BXA RIs with reported recombinations around D6Mit13; a small sampling of the Tas2rs in these RI lines again indicated no recombinations within the Tas2r cluster (data not shown). Behavioral genetic approaches have been invaluable for identifying genes involved in taste function, such as the Tas1r3 gene that encodes a receptor important for sweet and umami taste [42]. Positional cloning also permitted the identification of the Tas2r responsible for the majority of variance of phenylthiocarbamide (PTC) taste sensitivity in humans [18]. In both of these cases, however, the genes linked to saccharin or PTC taste were not tightly clustered with paralogues. For bitter taste, behavioral genetic approaches may be more useful for identifying genes encoding downstream signaling molecules or components of T2R-independent transduction mechanisms. For example, a QTL for PROP avoidance has been suggested on chromosome 7 [16], and we observe a suggestive QTL for quinine taste on chromosome 8 (Figure 2); in neither case are Tas2rs found at these loci (data not shown). + +It is somewhat puzzling that 22 of the 24 Tas2rs examined encode variant proteins in B6 and D2 mice even though these strains exhibit similar taste responses to bitter compounds such as DB or cyclohexamide [47]. Taken together, these observations suggest that Tas2rs are quite tolerant of variation, and that perhaps most of the differences observed do not affect domains important for ligand interactions or receptor-mediated signaling mechanisms. Interestingly, 23% of missense mutations in D2 Tas2rs affect the first two extracellular loops of the receptors. These two loops have been recently shown to affect the ligand response profiles of some T2Rs [23]. More systematic analyses of structure-function relationships between these T2R variants and an array of bitter stimuli are necessary to determine which changes may impact ligand binding, interactions with other proteins, or overall receptor structure. + +Such large numbers of nonsynonymous substitutions between orthologues is suggestive of adaptive selection. Analysis of sequence diversity of Tas2rs in humans, great apes and old world monkeys suggest that Tas2rs are subject to some degree of positive selection [43,44]. However, the fact that these two mouse strains, members of the same species, are so closely related makes this explanation problematic. It is possible that B6 and D2 mice, which have a similar origin in the early 20th century, inherited different Tas2r haplotypes present in wild mouse populations prior to inbreeding. Characterization of Tas2r sequences of several wild mouse species or subspecies, or in other inbred lines, would shed light on this issue. + +Conclusion + +In conclusion, we have found that sensitivity to the bitter-tasting substance quinine, as assayed by a taste specific brief-access test, is a polygenic trait in mice. However, the major mechanism for quinine taste transduction is likely dependent on one or more T2R receptors. Most Tas2r genes in the distal chromosome 6 cluster are polymorphic across inbred strains of mice, and this cluster forms a single haplotype that correlates with quinine taste sensitivity. The numerous differences in T2R protein sequence between these two mouse strains suggests that T2Rs are broadly tuned receptors quite tolerant to sequence variation. This tolerance may help to preserve the ability of T2R-expressing, bitter-sensitive taste cells to respond to a wide array of potentially toxic stimuli. + +Methods + +Mice and solutions + +A total of 188 adult male and female mice were behaviorally tested in these experiments: 16 C57BL/6J (B6; 9 males, 7 females), 12 DBA/2J (6 females, 6 males), and 90 BXD/Ty recombinant inbred mice (average = 5 / line; 64 males, 26 females) from 17 unique lines (1, 2, 5, 6, 11, 13, 14, 15, 20, 21, 24, 27, 29, 31, 32, 33, 34). All mice were either obtained from Jackson Laboratories (Bar Harbor, ME), or were bred from mating pairs at UTHSC. At time of testing, mice were individually housed in standard shoebox cages with woodchip bedding and ad libitum food (Teklad 8640 rodent diet). Taste stimuli used in this experiment were made from reagent-grade chemicals: Sucrose, denatonium benzoate, 6-n-propylthiouracil and quinine hydrochloride (Sigma Aldrich Corp.; St. Louis, MO). Concentrations of each solution were made fresh daily using distilled water, and all taste stimuli were presented at room temperature. All animal protocols were approved by the UTHSC Institutional Animal care and Use Committee. + +Brief-access tests + +All behavioral tests were conducted in the commercially available Davis MS-160 gustometer (DiLog Instruments, Inc., Tallahassee FL). Testing procedures were similar to those described earlier [29,37]. Briefly, after 24 hours of water deprivation, naïve mice are given a single 20-minute trial consisting of access to a single bottle of distilled water (sipper tube training). On day 2, mice could initiate up to sixteen 5 s trials with a single lick to one of four bottles containing distilled water (trial training). Testing occurred in sessions 3 and 4, with one test session per day per mouse. Trials were 5 s in length with an inter-trial interval of 10 s, and mice had up to 120 s to initiate a trial; if a trial was not initiated during this interval, the shutter closed for 10 s and the next trial was presented. Mice were tested with 2 concentrations each of 4 different taste stimuli [1 and 3 mM QHCl, 1 and 3 mM DB, 3 and 10 mM PROP (unpublished data), and 0.01 and 0.1 M sucrose]. Stimulus trials were presented in 3 blocks of 8 trials, for a total of 24 possible trials per test session. Each block consisted of each concentration of stimulus plus four presentations of distilled water in random order. Individual mice were also tested in random order. + +The dependent measure for each computed for each mouse was the lick ratio (average number of licks to stimulusx /average number of licks to water) where x is a given concentration of stimulus and the average number of licks to water is derived from the water trials during both test sessions. Lick ratio data for each stimulus were compiled for all individual mice, and means were prepared for each strain. B6 vs. D2 comparisons (Fig. 1A) were made using main effects ANOVA. Lick ratios for individual mice to sucrose were generally ~1.0 (data not shown), indicating that either concentration of this compound was licked at a similar rate to water by these thirsty animals. This stimulus was intended as a "neutral" stimulus, albeit one that has different sensory properties than water, and therefore not analyzed further. This was done to encourage sampling on "non-water" trials, as there is some evidence that mice detect distilled vs. adulterated water in brief-access taste tests based on olfactory clues; there is no evidence that mice can detect or distinguish among concentrations of a particular stimulus [37]. + +QTL mapping + +Linkage analysis was conducted on BXD mice using freely available software (Map Manager QTX [38]), and BXD genotype data shared by Robert W. Williams, University of Tennessee Health Science Center [45]. Simple interval mapping was conducted. This method evaluates the association between trait values (lick ratios) and expected genotype of a hypothetical quantitative trait locus (QTL) at multiple analysis points between each pair of adjacent marker loci. The significance of each potential association is measured by the likelihood ratio statistic (LRS; e.g. [46]). Permutation analysis (x2000) was used to determine genome-wide significance criteria for LRS scores. Significance was set at p < 0.05 and suggestive refers to p < 0.63. Additional markers used to refine the QTL on chromosome 6 were identified from the Jackson Laboratories online resources for the BXD RI strains [40]. + +Identification of T2R alleles + +Oligonucleotides were based on published mTas2r B6 or 129/SvJ cDNA sequences or on the public B6 genome. Entire coding regions plus ~50 kb of flanking sequence of each single-exon Tas2r was amplified from D2 or BXD RI genomic DNA (Jackson Laboratories, Bar Harbor, ME) by polymerase chain reaction (PCR) using a high-fidelity polymerase TaqPro Complete (Denville Scientific, South Plainfield, NJ). PCR products were subcloned into pGemT-Easy (Promega, Madison, WI) and sequenced at the University of Maryland School of Medicine Biopolymer Core. The sequences of D2 products were compared to B6 sequences available in Genbank (see Additional file 2), and polymorphisms identified. When possible, unique restriction sites were identified that differentiated B6 and D2 alleles, and the corresponding restriction endonucleases were used in diagnostic digests of Tas2r cDNAs amplified from genomic DNA of each BXD/Ty RI strain. For Tas2r120, the absence of a PCR product was considered diagnostic of the D2 allele. + +Authors' Contributions + +TN conducted the in silico analysis of the quinine taste QTL, analyzed the Tas2r genes, participated in the design of the study and drafted the manuscript. JB conducted the behavioral studies and QTL analysis. SM assisted with the in silico analysis of the quinine taste QTL and the Tas2rs, and with the QTL analysis. JB and SM conceived of the study, participated in its design, and edited the manuscript. All authors read and approved the final manuscript. Comments and requests should be addressed to JB or SM. + +Table 2 + +Linkage of a QHCl QTL to D6Mit13 on chromosome 6. + +Markers are listed from proximal (D6Mit150) to distal (SO8Gnf046.785), with physical position indicated in Mb. The physical position of D6Mit194 (italics) should be considered tentative. The LRS (likelihood ratio statistic) is listed for each locus, signifying the level of association of the trait (QHCl taste sensitivity) with each locus. Variance (Var) refers to the amount of the total trait variance explained by a QTL at this locus, as a percentage. Additive regression coefficients (Add) are listed for each association; in each case the coefficient is positive, indicating that D2 alleles increase the trait value (i.e. higher lick ratios). In simple marker regression analysis, all of these loci are associated with QHCl sensitivity at p < 0.001; only the association of sensitivity to 1 mM QHCl with D6Mit13 reaches genome-wide significance (asterisk). + +Supplementary Material + +Additional File 2 + +Table 4: Molecular biological methods for the analysis of Tas2rs. + +Click here for file + +Additional File 1 + +Table 3: Marker regression results for DB taste sensitivity. + +Click here for file + +Acknowledgements + +The authors thank Sandeep Raghow and Aldan Shank for technical assistance with the behavioral studies and Robert Lane for useful discussions. This study was supported by grants from the National Institute on Deafness and Other Communication Disorders (DC05786 to SM, DC04935 to JB), a Program Enrichment Fellowship from the University of Maryland, Baltimore (TN) and by the University of Tennessee Health Sciences Center (JB). diff --git a/src/ontogpt/evaluation/craft/database/all/16026622.ann b/src/ontogpt/evaluation/craft/database/all/16026622.ann new file mode 100644 index 000000000..b8a9f2c6d --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16026622.ann @@ -0,0 +1,597 @@ +T1 GO:0002507 35 49;60 69 development of ... tolerance +T2 UBERON:0002048 50 59 pulmonary +T3 CHEBI:36560 81 91 zinc oxide +T4 NCBITaxon:10088 95 99 mice +T5 NCBITaxon:1 123 134 Individuals +T6 UBERON:0002048 185 194 pulmonary +T7 CHEBI:64909 243 252 toxicants +T8 SO:0000704 287 294 genetic +T9 GO:0002507 337 351;362 371 development of ... tolerance +T10 UBERON:0002048 352 361 pulmonary +T11 CHEBI:36560 383 393 zinc oxide +T12 CHEBI:36560 395 398 ZnO +T13 NCBITaxon:10088 410 415 mouse +T14 CL:0000094 440 468 polymorphonuclear leukocytes +T15 CL:0000094 470 474 PMNs +T16 CL:0000235 477 488 macrophages +T17 SO:0000704 709 714 genes +T18 GO:0065007 720 727 control +T19 GO:0002507 732 746;757 766 development of ... tolerance +T20 UBERON:0002048 747 756 pulmonary +T21 CHEBI:36560 778 781 ZnO +T22 SO:0001026 793 799 Genome +T23 NCBITaxon:10088 850 855 mouse +T24 CL:0000094 891 895 PMNs +T25 CL:0000235 901 912 macrophages +T26 CHEBI:36560 975 978 ZnO +T27 SO:0001024 998 1007 haplotype +T28 SO:0000771 1071 1095 quantitative trait locus +T29 SO:0000771 1097 1100 QTL +T30 SO:0000771 1106 1109 QTL +T31 SO:0000704 1170 1175 genes +T32 SO:0000771 1204 1207 QTL +T33 SO:0000704 1248 1252 gene +T34 SO:0000771 1287 1311 quantitative trait locus +T35 SO:0000771 1313 1316 QTL +T36 SO:0000771 1357 1361 QTLs +T37 SO:0000771 1445 1449 QTLs +T38 CL:0000094 1462 1465 PMN +T39 CL:0000235 1470 1480 macrophage +T40 SO:0001024 1572 1582 haplotypes +T41 SO:0000771 1621 1625 QTLs +T42 PR:000001156 1660 1680 Toll-like receptor 5 +T43 PR:000001156 1682 1686 Tlr5 +T44 SO:0000704 1731 1735 gene +T45 SO:0000771 1759 1762 QTL +T46 PR:000001156 1809 1813 Tlr5 +T47 NCBITaxon:10088 1829 1833 mice +T48 CHEBI:36560 1882 1885 ZnO +T49 SO:0000704 1909 1916 Genetic +T50 GO:0002507 1961 1975;1986 1995 acquisition of ... tolerance +T51 UBERON:0002048 1976 1985 pulmonary +T52 CL:0000094 2012 2016 PMNs +T53 CL:0000235 2022 2033 macrophages +T54 CHEBI:36560 2044 2047 ZnO +T55 SO:0000704 2078 2083 genes +T56 SO:0000771 2112 2115 QTL +T57 PR:000001156 2187 2191 Tlr5 +T58 NCBITaxon:9606 2242 2248 humans +T59 SO:0000704 2308 2312 gene +T60 NCBITaxon:9606 2383 2389 person +T61 NCBITaxon:9606 2393 2399 person +T62 GO:0065007 2403 2413 regulating +T63 GO:0002507 2418 2432;2443 2452 development of ... tolerance +T64 UBERON:0002048 2433 2442 pulmonary +T65 CHEBI:64909 2464 2473 toxicants +T66 NCBITaxon:1 2490 2501 Individuals +T67 CHEBI:64909 2593 2602 toxicants +T68 UBERON:0002048 2667 2676 Pulmonary +T69 UBERON:0002048 2709 2713 lung +T70 CHEBI:36357 2772 2780 compound +T71 NCBITaxon:9606 2858 2863 human +T72 CHEBI:36560 2999 3009 zinc oxide +T73 CHEBI:36560 3011 3014 ZnO +T74 CHEBI:25812 3042 3047 ozone +T75 UBERON:0001004 3071 3082 respiratory +T76 NCBITaxon:1 3115 3125 individual +T77 UBERON:0002048 3165 3174 pulmonary +T78 SO:0000704 3321 3328 genetic +T79 SO:0000704 3344 3348 gene +T80 GO:0002507 3392 3406;3417 3426 development of ... tolerance +T81 UBERON:0002048 3407 3416 pulmonary +T82 NCBITaxon:9606 3430 3436 humans +T83 UBERON:0002048 3470 3479 pulmonary +T84 NCBITaxon:10088 3514 3519 mouse +T85 CL:0000094 3565 3593 polymorphonuclear leukocytes +T86 CL:0000094 3595 3599 PMNs +T87 CHEBI:36560 3670 3673 ZnO +T88 SO:0000704 3709 3714 genes +T89 GO:0065007 3715 3725 regulating +T90 GO:0002507 3730 3744;3755 3764 development of ... tolerance +T91 UBERON:0002048 3745 3754 pulmonary +T92 CHEBI:64909 3777 3785 toxicant +T93 SO:0000704 3812 3819 genetic +T94 GO:0002507 3839 3853;3864 3873 development of ... tolerance +T95 UBERON:0002048 3854 3863 pulmonary +T96 CHEBI:36560 3877 3880 ZnO +T97 CHEBI:25812 3897 3902 ozone +T98 NCBITaxon:10088 3948 3952 mice +T99 CL:0000094 4103 4107 PMNs +T100 CL:0000235 4113 4124 macrophages +T101 CHEBI:36560 4144 4147 ZnO +T102 NCBITaxon:10088 4174 4179 mouse +T103 SO:0001026 4241 4247 genome +T104 SO:0005858 4284 4312 regions of conserved synteny +T105 NCBITaxon:9606 4318 4324 humans +T106 NCBITaxon:33208 4344 4350 animal +T107 SO:0000704 4428 4433 genes +T108 GO:0065007 4434 4445 controlling +T109 UBERON:0002048 4446 4455 pulmonary +T110 CHEBI:64909 4477 4486 toxicants +T111 NCBITaxon:10088 4563 4567 mice +T112 SO:0000771 4603 4627 quantitative trait locus +T113 SO:0000771 4629 4632 QTL +T114 SO:0000704 4665 4670 genes +T115 GO:0065007 4676 4683 control +T116 UBERON:0002048 4710 4719 pulmonary +T117 NCBITaxon:10088 4834 4839 mouse +T118 SO:0000771 4869 4873 QTLs +T119 GO:0002507 4888 4902;4913 4922 development of ... tolerance +T120 UBERON:0002048 4903 4912 pulmonary +T121 CL:0000094 4939 4942 PMN +T122 CL:0000235 4948 4958 macrophage +T123 CHEBI:36560 4989 4992 ZnO +T124 NCBITaxon:10088 5027 5031 Mice +T125 NCBITaxon:10088 5088 5092 mice +T126 NCBITaxon:10088 5183 5187 mice +T127 NCBITaxon:33208 5256 5262 animal +T128 NCBITaxon:10088 5277 5281 mice +T129 CHEBI:15377 5450 5455 water +T130 NCBITaxon:9989 5480 5486 rodent +T131 CHEBI:33290 5487 5491 chow +T132 NCBITaxon:10088 5558 5562 mice +T133 NCBITaxon:33208 5633 5639 Animal +T134 NCBITaxon:33208 5781 5787 Animal +T135 CHEBI:36560 5800 5810 Zinc oxide +T136 NCBITaxon:10088 5855 5859 Mice +T137 CHEBI:36560 5876 5879 ZnO +T138 CHEBI:36560 5977 5980 ZnO +T139 CHEBI:36560 6051 6054 ZnO +T140 CHEBI:53251 6281 6304 polytetrafluoroethylene +T141 CHEBI:36560 6378 6381 ZnO +T142 GO:0002507 6647 6661;6672 6681 Development of ... tolerance +T143 UBERON:0002048 6662 6671 pulmonary +T144 NCBITaxon:33208 6776 6783 animals +T145 CHEBI:36560 6789 6792 ZnO +T146 NCBITaxon:10088 6809 6813 mice +T147 NCBITaxon:10088 6858 6862 mice +T148 NCBITaxon:10088 6916 6920 Mice +T149 UBERON:0001179 6945 6955 peritoneal +T150 CHEBI:650657 6970 6982 ketamine HCl +T151 CHEBI:7984 7055 7075 sodium pentobarbital +T152 UBERON:0001516 7149 7164 abdominal aorta +T153 UBERON:0002048 7182 7187 lungs +T154 NCBITaxon:10088 7196 7201 mouse +T155 CHEBI:29108 7285 7289 Ca2+ +T156 CHEBI:18420 7293 7297 Mg2+ +T157 SO:0001026 7569 7576 Genomic +T158 UBERON:0002113 7599 7605 kidney +T159 UBERON:0000479 7606 7612 tissue +T160 NCBITaxon:33208 7635 7641 animal +T161 SO:0001026 7683 7690 Genomic +T162 SO:0000207 7923 7928 SSLPs +T163 NCBITaxon:10088 7952 7957 mouse +T164 SO:0001026 7958 7964 genome +T165 SO:0000112 7987 7993 primer +T166 SO:0000207 8004 8009 SSLPs +T167 CHEBI:9754 8385 8389 Tris +T168 CHEBI:17883 8390 8393 HCl +T169 CHEBI:32588 8410 8413 KCL +T170 CHEBI:6636 8422 8427 MgCl2 +T171 SO:0000207 8540 8544 SSLP +T172 SO:0000112 8545 8551 primer +T173 CHEBI:60004 8572 8579 mixture +T174 SO:0001026 8603 8610 genomic +T175 NCBITaxon:271 8629 8632 Taq +T176 CHEBI:60004 8706 8714 mixtures +T177 SO:0000006 8959 8971 PCR products +T178 CHEBI:2511 8998 9005 agarose +T179 CHEBI:4883 9059 9075 ethidium bromide +T180 CHEBI:36560 9628 9631 ZnO +T181 NCBITaxon:10088 9660 9664 mice +T182 NCBITaxon:10088 9743 9747 mice +T183 SO:0001026 9784 9790 genome +T184 NCBITaxon:10088 9899 9904 mouse +T185 SO:0000771 10168 10171 QTL +T186 SO:0000207 10196 10200 SSLP +T187 SO:0000207 10245 10250 SSLPs +T188 SO:0001026 10664 10670 genome +T189 NCBITaxon:33208 10726 10733 animals +T190 CL:0000094 10763 10767 PMNs +T191 CL:0000235 10773 10784 macrophages +T192 SO:0000771 11099 11102 QTL +T193 SO:0000771 11185 11189 QTLs +T194 SO:0000207 11239 11244 SSLPs +T195 SO:0001024 11608 11617 Haplotype +T196 SO:0001024 11630 11639 haplotype +T197 SO:0000771 11756 11759 QTL +T198 SO:0000771 11764 11767 QTL +T199 SO:0001024 11922 11931 haplotype +T200 SO:0001024 11933 11943 Haplotypes +T201 SO:0000207 11962 11967 SSLPs +T202 NCBITaxon:10088 12110 12114 mice +T203 SO:0001024 12129 12138 haplotype +T204 SO:0000771 12147 12150 QTL +T205 SO:0000771 12154 12157 QTL +T206 NCBITaxon:10088 12232 12236 mice +T207 SO:0001024 12252 12262 haplotypes +T208 SO:0000771 12303 12307 QTLs +T209 SO:0000704 12424 12431 genetic +T210 GO:0002507 12450 12464;12475 12484 development of ... tolerance +T211 UBERON:0002048 12465 12474 pulmonary +T212 CL:0000094 12614 12617 PMN +T213 CL:0000235 12623 12633 macrophage +T214 NCBITaxon:10088 12738 12742 mice +T215 CL:0000094 12810 12814 PMNs +T216 CL:0000235 12823 12834 macrophages +T217 NCBITaxon:10088 12908 12912 mice +T218 CHEBI:36560 12967 12970 ZnO +T219 SO:0001026 13112 13118 genome +T220 NCBITaxon:10088 13195 13199 mice +T221 SO:0000771 13231 13235 QTLs +T222 GO:0002507 13252 13266;13277 13286 development of ... tolerance +T223 UBERON:0002048 13267 13276 pulmonary +T224 SO:0001026 13507 13513 genome +T225 SO:0000771 13613 13616 QTL +T226 SO:0000771 13696 13700 QTLs +T227 CL:0000094 13725 13728 PMN +T228 CL:0000235 13733 13743 macrophage +T229 SO:0001026 13793 13799 Genome +T230 SO:0000771 13814 13818 QTLs +T231 SO:0000207 14293 14298 SSLPs +T232 SO:0000771 14368 14371 QTL +T233 SO:0000771 14481 14484 QTL +T234 SO:0000771 14613 14616 QTL +T235 SO:0000771 14786 14789 QTL +T236 SO:0000207 15359 15364 SSLPs +T237 CL:0000094 15386 15389 PMN +T238 CL:0000235 15395 15405 macrophage +T239 SO:0000771 15429 15433 QTLs +T240 SO:0000771 15702 15706 QTLs +T241 CL:0000094 15719 15722 PMN +T242 CL:0000235 15727 15738 macrophages +T243 SO:0000771 15835 15839 QTLs +T244 SO:0000771 16263 16267 QTLs +T245 CL:0000094 16315 16318 PMN +T246 CL:0000235 16323 16333 macrophage +T247 SO:0001024 16688 16697 Haplotype +T248 NCBITaxon:10088 16738 16742 mice +T249 SO:0001024 16757 16766 haplotype +T250 NCBITaxon:10088 16835 16839 mice +T251 SO:0001024 16858 16867 haplotype +T252 SO:0000771 16915 16918 QTL +T253 SO:0000771 16939 16943 QTLs +T254 SO:0000207 17002 17006 SSLP +T255 NCBITaxon:33208 17011 17018 animals +T256 SO:0000207 17121 17125 SSLP +T257 NCBITaxon:10088 17198 17202 mice +T258 NCBITaxon:10088 17286 17290 mice +T259 SO:0001024 17308 17318 haplotypes +T260 NCBITaxon:10088 17381 17385 mice +T261 SO:0001024 17420 17430 haplotypes +T262 SO:0000207 17434 17439 SSLPs +T263 SO:0000771 17468 17472 QTLs +T264 NCBITaxon:10088 17488 17492 mice +T265 NCBITaxon:10088 17553 17557 mice +T266 NCBITaxon:10088 17640 17644 mice +T267 SO:0001024 17672 17681 haplotype +T268 SO:0001024 17746 17756 haplotypes +T269 SO:0001024 17777 17787 haplotypes +T270 SO:0000771 17872 17876 QTLs +T271 SO:0000771 17951 17955 QTLs +T272 NCBITaxon:10088 17960 17964 mice +T273 SO:0001024 17982 17991 haplotype +T274 NCBITaxon:10088 18219 18223 mice +T275 SO:0001024 18246 18255 haplotype +T276 SO:0000771 18270 18274 QTLs +T277 NCBITaxon:10088 18365 18369 mice +T278 SO:0000704 18515 18520 genes +T279 SO:0000771 18540 18544 QTLs +T280 SO:0000704 18556 18561 genes +T281 GO:0065007 18597 18608 controlling +T282 GO:0002507 18613 18637 development of tolerance +T283 CHEBI:36560 18649 18652 ZnO +T284 SO:0000704 18692 18697 genes +T285 SO:0000704 18811 18815 gene +T286 NCBITaxon:10088 18876 18881 mouse +T287 SO:0000673 18882 18892 transcript +T288 SO:0000704 18934 18939 genes +T289 UBERON:0002048 18965 18974 pulmonary +T290 CHEBI:36560 19019 19022 ZnO +T291 SO:0000704 19064 19069 genes +T292 UBERON:0002048 19095 19104 pulmonary +T293 CL:0000094 19122 19126 PMNs +T294 CL:0000235 19131 19142 macrophages +T295 CHEBI:36560 19162 19165 ZnO +T296 GO:0002507 19177 19191;19202 19211 Development of ... tolerance +T297 UBERON:0002048 19192 19201 pulmonary +T298 NCBITaxon:10088 19223 19227 mice +T299 PR:000001156 19243 19263 toll-like receptor 5 +T300 PR:000001156 19265 19269 Tlr5 +T301 SO:0000704 19299 19303 gene +T302 SO:0000771 19327 19330 QTL +T303 NCBITaxon:10088 19386 19391 mouse +T304 NCBITaxon:57486 19415 19431 M. m. molossinus +T305 PR:000001156 19473 19477 Tlr5 +T306 CHEBI:36560 19534 19537 ZnO +T307 PR:000001156 19568 19572 Tlr5 +T308 GO:0002507 19593 19607;19618 19627 development of ... tolerance +T309 UBERON:0002048 19608 19617 pulmonary +T310 CHEBI:36560 19722 19725 ZnO +T311 NCBITaxon:10088 19770 19774 mice +T312 NCBITaxon:10088 19969 19973 mice +T313 CHEBI:36560 20036 20039 ZnO +T314 NCBITaxon:10088 20095 20100 mouse +T315 CHEBI:36560 20119 20122 ZnO +T316 NCBITaxon:10088 20211 20215 mice +T317 GO:0002507 20474 20498 acquisition of tolerance +T318 CHEBI:64909 20510 20519 toxicants +T319 SO:0000704 20533 20540 genetic +T320 SO:0000704 20556 20560 gene +T321 GO:0002507 20604 20618;20629 20638 development of ... tolerance +T322 UBERON:0002048 20619 20628 pulmonary +T323 NCBITaxon:9606 20642 20648 humans +T324 SO:0000704 20687 20694 genetic +T325 NCBITaxon:10088 20717 20722 mouse +T326 UBERON:0002048 20732 20741 pulmonary +T327 CHEBI:36560 20764 20767 ZnO +T328 NCBITaxon:10088 20843 20848 mouse +T329 SO:0000704 20955 20962 genetic +T330 GO:0002507 20981 20995;21006 21015 development of ... tolerance +T331 UBERON:0002048 20996 21005 pulmonary +T332 SO:0000704 21040 21045 genes +T333 GO:0002507 21086 21110 acquisition of tolerance +T334 NCBITaxon:10088 21174 21178 mice +T335 SO:0000771 21228 21231 QTL +T336 SO:0000771 21388 21391 QTL +T337 SO:0000771 21490 21494 QTLs +T338 CHEBI:36560 21689 21692 ZnO +T339 SO:0000771 21831 21834 QTL +T340 NCBITaxon:10088 21909 21913 mice +T341 SO:0000771 21926 21929 QTL +T342 SO:0000771 22020 22024 QTLs +T343 NCBITaxon:10088 22134 22139 mouse +T344 SO:0000771 22167 22170 QTL +T345 NCBITaxon:10088 22242 22247 mouse +T346 SO:0000771 22265 22268 QTL +T347 NCBITaxon:10088 22475 22480 mouse +T348 SO:0000771 22524 22527 QTL +T349 SO:0000771 22622 22625 QTL +T350 SO:0000771 22635 22638 QTL +T351 NCBITaxon:10088 22715 22719 mice +T352 SO:0001024 22741 22750 haplotype +T353 NCBITaxon:10088 22766 22770 Mice +T354 SO:0001023 22785 22792 allelic +T355 SO:0000771 22810 22814 QTLs +T356 NCBITaxon:10088 23056 23060 mice +T357 SO:0001024 23071 23080 haplotype +T358 SO:0000771 23085 23089 QTLs +T359 SO:0000771 23219 23223 QTLs +T360 GO:0002507 23245 23259;23270 23279 development of ... tolerance +T361 UBERON:0002048 23260 23269 pulmonary +T362 GO:0065007 23336 23345 regulated +T363 SO:0000704 23371 23376 genes +T364 SO:0000704 23470 23475 genes +T365 SO:0000771 23499 23502 QTL +T366 GO:0065007 23552 23563 controlling +T367 GO:0002507 23568 23592 development of tolerance +T368 CHEBI:36560 23627 23630 ZnO +T369 PR:000001132 23670 23687 Duffy blood group +T370 UBERON:0000178 23676 23681 blood +T371 PR:000001132 23689 23692 Dfy +T372 GO:0065007 23712 23720 modulate +T373 CL:0009002 23814 23831 inflammatory cell +T374 UBERON:0001986 23919 23930 endothelium +T375 PR:000012289 23937 23961 ADP-ribosyltransferase 1 +T376 PR:000012289 23963 23969 Adprt1 +T377 http://purl.obolibrary.org/obo/MONDO_0005070 23975 23980 tumor +T378 PR:000002191 23975 24029 tumor necrosis factor (TNF) receptor-associated factor +T379 PR:000002291 24031 24036 Traf5 +T380 GO:0071159 24071 24085;24090 24093 nuclear factor ... -κB +T381 GO:0071159 24087 24089;24090 24093 NF ... -κB +T382 GO:0065007 24129 24139 regulation +T383 PR:000012289 24205 24211 ADPRT1 +T384 PR:000000046 24287 24315 transforming growth factor-β +T385 CHEBI:22907 24351 24360 bleomycin +T386 UBERON:0002048 24384 24388 lung +T387 NCBITaxon:10088 24425 24429 mice +T388 PR:000015073 24445 24469;24490 24498 solute carrier family 30 ... member 1 +T389 PR:000015073 24471 24487;24490 24498 zinc transporter ... member 1 +T390 PR:000015073 24500 24507 Slc30a1 +T391 GO:0005886 24539 24554 plasma membrane +T392 SO:0000704 24661 24666 genes +T393 SO:0000771 24726 24729 QTL +T394 CL:0000233 24821 24829 platelet +T395 GO:0030168 24821 24840 platelet-activating +T396 CHEBI:44811 24821 24847 platelet-activating factor +T397 CHEBI:64909 24945 24953 toxicant +T398 PR:000001393 24993 25004;25009 25011 Interleukin ... -6 +T399 PR:000001393 25006 25008;25009 25011 IL ... -6 +T400 CHEBI:36560 25044 25047 ZnO +T401 NCBITaxon:9606 25060 25066 humans +T402 CHEBI:36560 25137 25140 ZnO +T403 UBERON:0002048 25149 25153 lung +T404 UBERON:0002048 25187 25191 lung +T405 PR:000001393 25192 25196 IL-6 +T406 UBERON:0002048 25231 25240 pulmonary +T407 CHEBI:25812 25254 25259 ozone +T408 NCBITaxon:10114 25263 25267 rats +T409 PR:000015075 25282 25306;25326 25335 solute carrier family 30 ... members 2 +T410 PR:000015076 25282 25306;25326 25333;25340 25341 solute carrier family 30 ... members ... 3 +T411 PR:000015075 25308 25324;25326 25335 zinc transporter ... members 2 +T412 PR:000015076 25308 25324;25326 25333;25340 25341 zinc transporter ... members ... 3 +T413 PR:000015075 25343 25350 Slc30a2 +T414 PR:000015076 25355 25362 Slc30a3 +T415 NCBITaxon:33208 25474 25480 animal +T416 GO:0002507 25501 25515;25526 25535 development of ... tolerance +T417 UBERON:0002048 25516 25525 pulmonary +T418 GO:0065007 25573 25582 regulated +T419 SO:0000704 25629 25634 genes +T420 GO:0065007 25723 25733 regulation +T421 GO:0002507 25766 25790 development of tolerance +T422 SO:0000771 25808 25812 QTLs +T423 CL:0000094 25834 25838 PMNs +T424 CL:0000235 25843 25854 macrophages +T425 SO:0000704 25938 25943 genes +T426 PR:000001992 25955 25986 Chemokine (C-C motif) ligand 20 +T427 PR:000001992 25988 25993 Ccl20 +T428 PR:000001208 25996 26030 chemokine (C-X-C motif) receptor 4 +T429 PR:000001208 26032 26037 Cxcr4 +T430 PR:000001471 26044 26058 interleukin-10 +T431 PR:000001471 26060 26064 Il10 +T432 CL:0000094 26104 26107 PMN +T433 CL:0000094 26135 26139 PMNs +T434 UBERON:0000479 26158 26165 tissues +T435 GO:0065007 26169 26178 regulated +T436 GO:0006935 26182 26193 chemotactic +T437 CL:0000094 26276 26280 PMNs +T438 CL:0000094 26372 26376 PMNs +T439 PR:000001208 26453 26458 CXCR4 +T440 GO:0010467 26459 26469 expression +T441 NCBITaxon:9606 26491 26496 human +T442 CL:0000094 26497 26501 PMNs +T443 SO:0000704 26543 26547 gene +T444 GO:0010467 26543 26558 gene expression +T445 GO:0065007 26563 26570 control +T446 UBERON:0000479 26575 26581 tissue +T447 CL:0000094 26626 26630 PMNs +T448 UBERON:0002048 26640 26644 lung +T449 NCBITaxon:9606 26646 26651 Human +T450 CL:0000094 26652 26656 PMNs +T451 GO:0010467 26674 26681 express +T452 PR:000001992 26682 26687 CCL20 +T453 GO:0097511 26728 26737 dendritic +T454 CL:0000451 26728 26743 dendritic cells +T455 UBERON:0002405 26797 26803 immune +T456 GO:0006955 26797 26812 immune response +T457 PR:000001471 26863 26868 IL-10 +T458 CL:0000084 26888 26895 T cells +T459 CL:0000094 26920 26923 PMN +T460 GO:0010467 26948 26958 expression +T461 GO:0071159 26995 27000 NF-κB +T462 GO:0065007 27053 27061 modulate +T463 CHEBI:35224 27072 27080 effector +T464 http://purl.obolibrary.org/obo/MONDO_0005271 27110 27127 allergic response +T465 CL:0000235 27153 27163 macrophage +T466 UBERON:0007376 27179 27188 epidermal +T467 PR:000006928 27179 27202 epidermal growth factor +T468 PR:000004200 27227 27239 amphiregulin +T469 PR:000004200 27241 27245 Areg +T470 PR:000004837 27251 27263 betacellulin +T471 PR:000004837 27265 27268 Btc +T472 SO:0000704 27299 27304 genes +T473 SO:0000771 27327 27330 QTL +T474 GO:0000165 27449 27477 MAP kinase signaling cascade +T475 GO:0010467 27491 27501 expression +T476 GO:0046903 27525 27533 Secreted +T477 PR:000015561 27525 27550 Secreted phosphoprotein 1 +T478 PR:000015561 27552 27556 Spp1 +T479 PR:000015561 27572 27583 osteopontin +T480 CL:0000235 27646 27657 macrophages +T481 CL:0000235 27780 27790 macrophage +T482 GO:0048246 27780 27801 macrophage chemotaxis +T483 CL:0000094 27832 27836 PMNs +T484 CL:0000235 27841 27852 macrophages +T485 SO:0000704 27913 27918 genes +T486 PR:000001096 27957 27975 toll-like receptor +T487 PR:000001156 27977 27981 Tlr5 +T488 SO:0000704 27998 28002 gene +T489 SO:0000771 28031 28034 QTL +T490 PR:000001096 28081 28100 Toll-like receptors +T491 GO:0005622 28110 28124 intra-cellular +T492 GO:0035556 28110 28134 intra-cellular signaling +T493 CHEBI:35224 28186 28194 effector +T494 SO:0000704 28195 28200 genes +T495 PR:000001156 28207 28211 Tlr5 +T496 SO:0000704 28246 28250 gene +T497 UBERON:0002405 28258 28264 immune +T498 GO:0006955 28258 28273 immune response +T499 NCBITaxon:2 28309 28318 bacterial +T500 PR:000022655 28319 28328 flagellin +T501 NCBITaxon:10088 28372 28377 mouse +T502 PR:000001156 28440 28444 Tlr5 +T503 GO:0010467 28487 28497 expression +T504 PR:000001156 28524 28528 Tlr5 +T505 GO:0002507 28549 28573 development of tolerance +T506 CHEBI:36560 28596 28599 ZnO +T507 NCBITaxon:10088 28623 28627 mice +T508 PR:000001156 28650 28654 TLR5 +T509 GO:0010467 28660 28670 expression +T510 CHEBI:36560 28780 28783 ZnO +T511 NCBITaxon:10088 28863 28867 mice +T512 CHEBI:36560 28926 28929 ZnO +T513 PR:000001156 29014 29018 Tlr5 +T514 GO:0002507 29026 29050 development of tolerance +T515 PR:000001155 29119 29123 Tlr4 +T516 SO:0000704 29139 29143 gene +T517 NCBITaxon:10088 29201 29205 mice +T518 CHEBI:25812 29245 29250 ozone +T519 PR:000001096 29290 29308 toll-like receptor +T520 GO:0002224 29290 29318 toll-like receptor signaling +T521 GO:0065007 29343 29353 regulation +T522 CHEBI:64909 29365 29373 toxicant +T523 PR:000001156 29435 29439 Tlr5 +T524 GO:0034146 29435 29449 Tlr5 signaling +T525 GO:0065007 29454 29462 regulate +T526 GO:0002507 29467 29491 development of tolerance +T527 CHEBI:36560 29495 29498 ZnO +T528 NCBITaxon:2 29536 29545 bacterial +T529 PR:000022655 29546 29555 flagellin +T530 PR:000001155 29611 29615 Tlr4 +T531 PR:000001156 29620 29624 Tlr5 +T532 PR:000001096 29678 29696 toll-like receptor +T533 GO:0002224 29678 29706 toll-like receptor signaling +T534 CHEBI:64909 29737 29746 toxicants +T535 CHEBI:25812 29755 29760 ozone +T536 CHEBI:36560 29765 29768 ZnO +T537 PR:000001156 29843 29847 Tlr5 +T538 PR:000001155 29880 29884 Tlr4 +T539 PR:000007581 29920 29931 fibronectin +T540 CHEBI:16336 29941 29956 hyaluronic acid +T541 UBERON:0002048 29978 29982 lung +T542 UBERON:0002048 29996 30000 lung +T543 PR:000001155 30027 30031 Tlr4 +T544 PR:000001156 30078 30082 Tlr5 +T545 GO:0065007 30087 30095 regulate +T546 GO:0002507 30100 30124 development of tolerance +T547 CHEBI:36560 30128 30131 ZnO +T548 GO:0071159 30171 30176 NF-κB +T549 CHEBI:36560 30263 30266 ZnO +T550 PR:000001395 30290 30294 IL-8 +T551 PR:000001393 30299 30303 IL-6 +T552 GO:0071159 30325 30330 NF-κB +T553 SO:0000704 30341 30345 gene +T554 GO:0010467 30341 30356 gene expression +T555 PR:000001156 30373 30377 Tlr5 +T556 PR:000022655 30400 30409 flagellin +T557 PR:000001156 30466 30470 Tlr5 +T558 GO:0065007 30474 30484 regulatory +T559 SO:0005836 30474 30492 regulatory element +T560 GO:0071159 30505 30510 NF-κB +T561 GO:0065007 30528 30536 modulate +T562 CHEBI:36560 30576 30579 ZnO +T563 PR:000001156 30599 30603 Tlr5 +T564 GO:0002507 30652 30666;30671 30680 development of ... tolerance +T565 CHEBI:36560 30667 30670 ZnO +T566 GO:0045087 30720 30735 innate immunity +T567 UBERON:0002405 30777 30790 immune system +T568 CHEBI:59132 30821 30829 antigens +T569 CHEBI:64909 30896 30905 toxicants +T570 PR:000001096 30923 30941 toll-like receptor +T571 GO:0002224 30923 30951 toll-like receptor signaling +T572 UBERON:0002048 30970 30974 lung +T573 GO:0065007 30978 30986 regulate +T574 NCBITaxon:10088 31070 31075 mouse +T575 SO:0000771 31119 31122 QTL +T576 CHEBI:36560 31226 31229 ZnO +T577 SO:0000771 31242 31246 QTLs +T578 CL:0000094 31332 31336 PMNs +T579 CL:0000235 31366 31377 macrophages +T580 SO:0001024 31379 31388 Haplotype +T581 PR:000001156 31558 31562 Tlr5 +T582 SO:0000771 31601 31604 QTL +T583 PR:000001156 31651 31655 Tlr5 +T584 NCBITaxon:10088 31671 31675 mice +T585 CHEBI:36560 31741 31744 ZnO +T586 PR:000001156 31777 31781 Tlr5 +T587 GO:0002507 31789 31803;31814 31823 development of ... tolerance +T588 UBERON:0002048 31804 31813 pulmonary +T589 CHEBI:64909 31835 31844 toxicants +T590 SO:0000704 31883 31890 genetic +T591 GO:0002507 31935 31949;31960 31969 acquisition of ... tolerance +T592 UBERON:0002048 31950 31959 pulmonary +T593 CHEBI:64909 32000 32009 toxicants +T594 CHEBI:36560 32018 32021 ZnO +T595 SO:0000704 32047 32052 genes +T596 SO:0000771 32081 32084 QTL +T597 NCBITaxon:9606 32660 32666 people diff --git a/src/ontogpt/evaluation/craft/database/all/16026622.txt b/src/ontogpt/evaluation/craft/database/all/16026622.txt new file mode 100644 index 000000000..63779553a --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16026622.txt @@ -0,0 +1,167 @@ +Quantitative trait analysis of the development of pulmonary tolerance to inhaled zinc oxide in mice + +Abstract + +Background + +Individuals may develop tolerance to the induction of adverse pulmonary effects following repeated exposures to inhaled toxicants. Previously, we demonstrated that genetic background plays an important role in the development of pulmonary tolerance to inhaled zinc oxide (ZnO) in inbred mouse strains, as assessed by polymorphonuclear leukocytes (PMNs), macrophages, and total protein in bronchoalveolar lavage (BAL) phenotypes. The BALB/cByJ (CBy) and DBA/2J (D2) strains were identified as tolerant and non-tolerant, respectively. The present study was designed to identify candidate genes that control the development of pulmonary tolerance to inhaled ZnO. + +Methods + +Genome-wide linkage analyses were performed on a CByD2F2 mouse cohort phenotyped for BAL protein, PMNs, and macrophages following 5 consecutive days of exposure to 1.0 mg/m3 inhaled ZnO for 3 hours/day. A haplotype analysis was carried out to determine the contribution of each quantitative trait locus (QTL) and QTL combination to the overall BAL protein phenotype. Candidate genes were identified within each QTL interval using the positional candidate gene approach. + +Results + +A significant quantitative trait locus (QTL) on chromosome 1, as well as suggestive QTLs on chromosomes 4 and 5, for the BAL protein phenotype, was established. Suggestive QTLs for the BAL PMN and macrophage phenotypes were also identified on chromosomes 1 and 5, respectively. Analysis of specific haplotypes supports the combined effect of three QTLs in the overall protein phenotype. Toll-like receptor 5 (Tlr5) was identified as an interesting candidate gene within the significant QTL for BAL protein on chromosome 1. Wild-derived Tlr5-mutant MOLF/Ei mice were tolerant to BAL protein following repeated ZnO exposure. + +Conclusion + +Genetic background is an important influence in the acquisition of pulmonary tolerance to BAL protein, PMNs, and macrophages following ZnO exposure. Promising candidate genes exist within the identified QTL intervals that would be good targets for additional studies, including Tlr5. The implications of tolerance to health risks in humans are numerous, and this study furthers the understanding of gene-environment interactions that are likely to be important factors from person-to-person in regulating the development of pulmonary tolerance to inhaled toxicants. + +Introduction + +Individuals may develop tolerance to adverse health effects elicited from repeated exposure to inhaled toxicants in several different occupational and environmental situations. Pulmonary tolerance can be defined as the lung's ability to withstand the detrimental effects of a toxic compound following multiple exposures. There are several implications of tolerance to human health, having both advantages and disadvantages with respect to the development of harmful health effects. Clinical investigations of zinc oxide (ZnO)- [1], endotoxin- [2], and ozone- [3-6] induced adverse respiratory effects have demonstrated inter-individual variability in the capacity to develop pulmonary tolerance following inhalation exposure. Because these clinical studies are more tightly controlled than epidemiologic studies, they suggest that genetic background and gene-environment interactions contribute to the development of pulmonary tolerance in humans. + +We initially characterized the pulmonary tolerance phenotype in an outbred mouse model by assessing levels of BAL protein and polymorphonuclear leukocytes (PMNs) following single (1X) and 5 daily repeated (5X) exposures to inhaled ZnO to begin the identification of the genes regulating the development of pulmonary tolerance to repeated toxicant exposure [7]. Significant genetic variability in the development of pulmonary tolerance to ZnO, endotoxin, and ozone was established in several inbred strains of mice in a subsequent study [8]. Of the strains tested, the BALB/cByJ (CBy) strain was tolerant and the DBA/2J (D2) strain was non-tolerant to BAL protein, PMNs, and macrophages following repeated ZnO exposure. + +Because inbred mouse strains are virtually identical at all loci throughout their genome, and also share several chromosomal regions of conserved synteny with humans, they are an ideal animal model in which to investigate genotype-environment interactions and identify genes controlling pulmonary responses to inhaled toxicants where no a priori evidence for their location exists [9]. Inbred strains of mice have been successfully utilized in quantitative trait locus (QTL) analyses to identify candidate genes that control susceptibility to adverse pulmonary responses induced by a variety of inhaled gases [10-13] and particulates [14,15]. In the present study, a CByD2F2 mouse cohort was used to determine QTLs linked to the development of pulmonary tolerance to BAL protein, PMN, and macrophage phenotypes following repeated ZnO exposure. + +Materials and methods + +Mice + +Inbred BALB/cByJ (CBy), DBA/2J (D2) and CByD2F1/J (F1) mice (6–7 weeks of age) were purchased from The Jackson Laboratory (Bar Harbor, ME). CByD2F1/J mice were crossed to produce F2 (intercross) offspring in our laboratory animal facility. All mice were acclimated for at least 1 week before exposure, housed in a positive pressure environment with a 12-hour light/dark cycle starting at 6:00 a.m., and provided with water and standard laboratory rodent chow (Purina, Indianapolis, IN) ad libitum except during exposure. All mice were handled in accordance with the standards established by the U.S. Animal Welfare Acts set forth in National Institutes of Health guidelines, and by the New York University School of Medicine Division of Laboratory Animal Resources. + +Zinc oxide generation, characterization, and exposure + +Mice were exposed to ZnO (1.0 ± 0.2 mg/m3, mean ± SD) in stainless steel cages placed inside a 0.07 m3 Plexiglas chamber. ZnO fumes were generated in a furnace as previously described [7,16]. The ZnO particles had a mass median aerodynamic diameter of 0.3 μm and geometric standard deviation of 1.5. Samples of the chamber atmosphere were collected approximately every 40 minutes from the manifold of the exposure system with polytetrafluoroethylene filters (Type TX40HI20-WW, Pallflex Products Corp., Putnam, CT), and the ZnO concentration was determined gravimetrically using a microbalance (Model C-30, Cahn Instruments, Cerritos, CA). + +For linkage analyses, all F2 offspring (n = 299, 138 males and 161 females) were exposed at 7–12 weeks of age in a total of seven 5X exposure regimens. Development of pulmonary tolerance was assessed 24 hours after the fifth exposure, and exposure group sizes ranged from 36 to 45 animals. All ZnO exposures of F2 mice also contained at least two CBy, D2, and F1 mice for control purposes. + +Bronchoalveolar lavage (BAL) + +Mice were euthanized by intraperitoneal injections of ketamine HCl (100 mg/kg, Vetalar, Fort Dodge Laboratories, Inc., Fort Dodge, IA) and sodium pentobarbital (175 mg/kg, Sleepaway, Fort Dodge Laboratories, Inc.), and the posterior abdominal aorta was severed. The lungs of each mouse were lavaged two times with 1.2 ml of Dulbecco's phosphate buffered saline without Ca2+ or Mg2+ (pH 7.2–7.4, 37°C, Invitrogen, Carlsbad, CA). The collected BAL was immediately placed on ice (4°C) following recovery. Measurement of BAL protein, total cell counts, and differential cell counts were performed as previously described [8]. + +DNA isolation and genotyping + +Genomic DNA was isolated from kidney tissue of each phenotyped F2 animal and for CBy, D2, and F1 controls (Wizard Genomic DNA Purification Kit, Promega, Madison, WI). DNA concentration was determined using a Beckman DU-650 spectrophotometer, and each sample was diluted to 10 ng/μl for genotype analysis. PCRs were performed to genotype F2 offspring for SSLPs located throughout the mouse genome. Eighty-six unlabeled primer pairs for SSLPs that differed in length by at least 5% between the CBy and D2 progenitor strains were purchased from Research Genetics (ResGen/Invitrogen). PCR was performed in 20 μl reaction volumes in 96-well low profile plates (Fisherbrand, Fisher Scientific, Fairlawn, NJ) using a PTC-100 thermal cycler (MJ Research, Watertown, MA). The final concentration for each reaction was: 10 mM Tris-HCl (pH 8.3), 50 mM KCL, 2.5 mM MgCl2, 0.2 mM of each deoxynucleotide triphosphate (Promega), 1.1X Rediload (ResGen/Invitrogen), and 0.132 μM of each SSLP primer pair. This reaction mixture was added to 100 ng of genomic DNA and 0.45 U of Taq DNA polymerase (Roche Applied Science, Indianapolis, IN). Final reaction mixtures were initially denatured at 94°C for 3 min, followed by 36 amplification cycles (94°C for 30 seconds, 57°C for 45 seconds, and 72°C for 30 seconds + 1 second/cycle). A final extension step at 72°C for 7 min was followed by refrigeration (4°C). PCR products were differentiated on 3% agarose (Invitrogen) gels and all samples were visualized by ethidium bromide staining using a ChemiImager-4400 low light imaging system (Imgen Technologies, Alexandria, VA) and called by a single investigator. Any questionable calls in reading the genotype from the image were reviewed by a second investigator and if not resolved, that sample was rerun. + +Estimation of loci + +The number of independently segregating loci was calculated using the following formula by Wright [17]: n = (P2 - F1)2/4(|σ2F2 - σ2F1|), where n is an estimate of the number of independent loci; P2 and F1 are the mean BAL protein responses following 5X ZnO exposure in CBy and CByD2F1 mice, respectively; σ2F2 and σ2F1 are the computed variances of the F2 and CByD2F1 mice, respectively. + +Linkage analyses + +A genome scan was performed to identify associations between genotypes and the BAL protein phenotype using a CByD2F2 mouse cohort. All phenotypic data were natural log normalized to generate a normal distribution to meet normality assumptions of the Map Manager QT computer program. Interval analyses were then performed by fitting a regression equation for the effect of a theoretical QTL at the position of each SSLP and at 1-centimorgan (cM) intervals between SSLPs using free, additive, recessive, and dominant regression models. The regressions and significance of each genotype/phenotype association (or likelihood χ2 statistic) were calculated by Map Manager [18]. Permutation tests were performed on the phenotypic and genotypic data using Map Manager to generate empirical thresholds for significance following the methods of Churchill and Doerge [19,20]. + +For the initial genome scan, the 15 most tolerant and 15 most non-tolerant F2 animals with respect to BAL protein, PMNs, and macrophages (i.e., the phenotypic extremes) were used for selective genotyping [9,21]. Interval analyses were done as stated previously, and 10,000 permutations were performed to generate significant and suggestive likelihood χ2 statistic thresholds for the BAL protein phenotype. Following the identification of a suggestive QTL for BAL protein on chromosome 1, the entire F2 cohort was examined for additional QTLs. For the BAL protein phenotype, three additional SSLPs on chromosome 1 were analyzed, and a permutation test (10,000 permutations) was performed with only chromosome 1. This method was similar to that used in previous linkage studies with inhaled particles and gases that utilized Map Manager QT [10,14,22]. All likelihood χ2 statistic thresholds corresponded to those reported in the aforementioned linkage studies. + +Haplotype analysis + +A haplotype analysis was carried out similar to that done previously by Prows and Leikauf to determine the contribution of each QTL and QTL combination to the overall BAL protein phenotype [15]. This method quantifies any difference in mean BAL protein levels that are linked with a particular haplotype. Haplotypes for the following SSLPs were used for this analysis: D1Mit291 (101.5 cM), D4Mit254 (82.5 cM), and D5Mit193 (1.0 cM). Mean BAL protein concentrations for groups of F2 mice with the same haplotype at each QTL or QTL combinations were calculated and compared with the mean BAL protein of F2 mice with the other haplotypes to determine the contributions of these QTLs to the overall BAL protein phenotype. + +Results + +Phenotypes of the CByD2F2 cohort + +To further understand the role of genetic background in the development of pulmonary tolerance, an F2 (backcross) cohort derived from the CBy and D2 progenitors was phenotyped. The frequency distribution of the BAL protein, PMN, and macrophage phenotypic responses of the F2 cohort were within the ranges of similarly exposed CBy and D2 progenitor mice (Figure 1). + +Figure 1 + +Frequency distribution of the number of BAL PMNs (×104), macrophages (×104), and protein (μg/ml) in BALB/cByJ, DBA/2J, CByD2F1/J, and CByD2F2 mice following 5 consecutive days of exposure to 1.0 mg/m3 ZnO for 3 h/day. Vertical dashed lines represent approximate separation points between BALB/cByJ and DBA/2J phenotypes. + +Selective genotyping + +A genome-wide scan was performed using the 15 most tolerant and 15 most non-tolerant mice to initially identify possible QTLs influencing the development of pulmonary tolerance. Permutation tests on the BAL protein-extreme data set established a suggestive likelihood χ2 statistic threshold of 9.9 and a significant likelihood χ2 statistic threshold of 17.4. These values were consistent with the genome-wide probabilities projected by Lander and Kruglyak [23]. Interval mapping identified a suggestive QTL for the BAL protein phenotype on the distal end of chromosome 1 (Figure 2). No QTLs were identified for the PMN and macrophage phenotypes from selective genotyping. + +Figure 2 + +Genome-wide scan for QTLs associated with the BAL protein phenotype by selective genotyping of the CByD2F2 cohort. For each plot, the x-axis is the length of the chromosome in centimorgans (cM), and the y-axis is the likelihood χ2 statistic value as calculated by Map Manager. The upper and lower dashed lines represent significant (LRS = 17.4) and suggestive (LRS = 9.9) linkage thresholds, respectively. + +Genotyping of the entire F2 cohort + +The entire F2 cohort was genotyped with three additional SSLPs on distal chromosome 1 to further analyze the suggestive BAL protein QTL on chromosome 1 identified from selective genotyping. Interval mapping of the entire F2 cohort confirmed the QTL on chromosome 1 between 101.0 cM (D1Mit426) and 109.6 cM (D1Mit293) (Figure 3). The peak likelihood χ2 statistic value for this QTL exceeded the threshold value of 10.0 for significant linkage as determined by 10,000 permutations with all loci from chromosome 1 only. + +Figure 3 + +Plot of a significant QTL on chromosome 1 that is associated with the BAL protein phenotype from analysis of the entire CByD2F2 cohort. The x-axis is the length of the chromosome in centimorgans (cM), and the y-axis is the likelihood χ2 statistic value as calculated by Map Manager. The lower dashed line represents the suggestive linkage threshold (LRS = 3.8), the middle dashed line represents significant linkage threshold (LRS = 10.0), and the upper dashed line represents the highly significant linkage threshold (LRS = 18.8). + +The entire F2 cohort was further analyzed with the initial 86 SSLPs for the BAL protein, PMN, and macrophage phenotypes. Suggestive QTLs for the BAL protein phenotype that were not previously characterized by selective genotyping were identified on chromosome 4 between 53.6 cM (D4Mit146) and 82.5 cM (D4Mit254), and on chromosome 5 between 1.0 cM (D5Mit193) and 18.0 cM (D5Mit148) (Figure 4). Suggestive QTLs for the BAL PMN and macrophages were identified on chromosomes 1 and 5, respectively (Figure 5). + +Figure 4 + +Plots of suggestive QTLs on chromosomes 4 and 5 associated with the BAL protein phenotype from analysis of the entire CByD2F2 cohort. The x-axis is the length of the chromosome in centimorgans (cM), and the y-axis is the likelihood χ2 statistic as calculated by Map Manager. The upper and lower dashed lines in each plot represent significant (LRS = 15.8) and suggestive (LRS = 9.2) linkage thresholds, respectively. + +Figure 5 + +Plots of suggestive QTLs on chromosomes 1 and 5 associated with the BAL PMN and macrophage phenotypes, respectively, from analysis of the entire CByD2F2 cohort. The x-axis is the length of the chromosome in centimorgans (cM), and the y-axis is the likelihood χ2 statistic as calculated by Map Manager. The upper and lower dashed lines in each plot represent significant (LRS = 15.6) and suggestive (LRS = 9.2) linkage thresholds, respectively. + +Haplotype analysis + +Mean BAL protein levels of F2 mice with the same haplotype were calculated and compared with the mean BAL protein levels of F2 mice with the opposite haplotype in order to determine the contribution of each QTL and combinations of QTLs to the overall BAL protein phenotype (Figure 6). For each SSLP, F2 animals were genotyped as a homozygous CBy (CC), a homozygous D2 (DD) or heterozygous (H). For any individual SSLP, the greatest difference in mean BAL protein was found for D1Mit291. F2 mice that were DD at that locus had an average of 156 μg/ml more BAL protein than those mice that had CC or H haplotypes. + +Figure 6 + +Differences in mean BAL protein levels of CByD2F2 mice with tolerant versus non-tolerant haplotypes at SSLPs representing the identified QTLs. Open bars, F2 mice with CC or CD genotypes (represented as H). Filled bars, F2 mice with a DD genotype (represented as D). Number within each bar is the number of F2 mice with the given genotype or haplotype. Values are means ± SE. All comparisons of tolerant (open bars) haplotypes versus non-tolerant haplotypes (filled bars) were significant (P < 0.05, t test). + +The combinatorial effect of the QTLs on chromosomes 1, 4, and 5 were also examined. For any combination of two QTLs, F2 mice that had a DD-DD haplotype for markers on chromosomes 1 and 5 (D1Mit291 and D5Mit193) had an average of 310 μg/ml more BAL protein than those that were CC or H (CC/H) for those markers. The greatest difference in mean BAL protein levels were found in F2 mice that had a DD-CC/H-DD haplotype for the three QTLs on chromosomes 1, 4, and 5, (i.e., D1Mit291, D4Mit254, and D5Mit193), respectively. These mice had an average of 345 μg/ml more BAL protein than those that were CC/H at the markers across the three chromosomes. + +Identification of candidate genes + +Within all of the QTLs, candidate genes discovered with potential roles in controlling the development of tolerance to inhaled ZnO are presented in Tables 1 and 2. These genes were chosen as candidates from a thorough review of the existing literature and through the positional candidate gene approach, which combines knowledge of map position with the mouse transcript map [24]. + +Table 1 + +Positional candidate genes from linkage analysis of pulmonary tolerance to BAL protein following repeated ZnO exposure. + +Table 2 + +Positional candidate genes from linkage analysis of pulmonary tolerance to BAL PMNs and macrophages following repeated ZnO exposure. + +Development of pulmonary tolerance in MOLF/Ei mice + +We identified toll-like receptor 5 (Tlr5) as an interesting candidate gene within the significant QTL for BAL protein on chromosome 1. A wild-derived inbred mouse strain called MOLF/Ei (M. m. molossinus) which has non-conservative mutations in Tlr5 [25] was phenotyped for BAL protein following 1X and 5X ZnO exposure to determine whether Tlr5 may function in the development of pulmonary tolerance (Figure 7). MOLF/Ei BAL protein was significantly increased above control values following 1X ZnO exposure (395 ± 38 μg/ml). However, MOLF/Ei mice exhibited a tolerant phenotype, as 5X BAL protein values (215 ± 22 μg/ml) were significantly decreased below that of the 1X exposure group. + +Figure 7 + +BAL protein levels in wild-derived MOLF/Ei mice 24 h after single (1X) or repeated (5X) exposure to 1.0 mg/m3 ZnO or air for 3 h. Protein levels of BALB/cByJ and DBA/2J mouse strains following ZnO exposure are also shown for comparison purposes. Values are means ± SE (n = 4–5 MOLF/Ei mice/exposure group). * indicates significantly different from air-exposed MOLF/Ei controls, P < 0.05 [Student-Newman Keuls (SNK) test]. + indicates significantly different from 1X MOLF/Ei exposure group, P < 0.05 (SNK test). + +Discussion + +Clinical studies on the acquisition of tolerance to inhaled toxicants suggest that genetic background and gene-environment interactions contribute to the development of pulmonary tolerance in humans. We have previously determined that a genetic component exists in a mouse model of pulmonary tolerance to repeated ZnO exposure [8]. In the present study, we performed linkage analyses on an F2 mouse population derived from tolerant CBy and non-tolerant D2 strains to further ascertain the contribution of genetic background to the development of pulmonary tolerance, and identify candidate genes that may be important regulators in the acquisition of tolerance. + +Initial analysis using the most tolerant and non-tolerant F2 mice with respect to BAL protein generated a putative QTL (designated as the zinc-induced tolerance (ZIT1) locus) on the distal end of chromosome 1. Further assessment of the entire F2 cohort demonstrated that the QTL on chromosome 1 attained an LRS value for significant linkage, and also identified two suggestive QTLs located on chromosomes 4 and 5. Using a variation of the Wright equation [17], a minimum of three loci were estimated to be independently segregating with the BAL protein phenotype following 5X ZnO exposure, thus in agreement with the results of the linkage analysis. There are several approaches that can be pursued to focus in on the QTL intervals that were identified. For instance, increasing the number of F2 mice used in the QTL analysis is one alternative. The major disadvantage of this, however, is that segregating QTLs contribute a great deal of phenotypic "noise," making it problematic when determining whether or not a given mouse has inherited a particular QTL [9]. Thus, in order to separate the effects of multiple loci, congenic mouse strains for each QTL could be generated, which could then be used to breed multicongenic lines to examine the existence of any epistatic effects. Additionally, future studies could employ the use of a backcross (CByD2F1 × CBy) mouse population to expand the evaluation of the QTL effects to the overall BAL protein phenotype. + +To measure the contribution of each individual QTL and each QTL combination to the overall BAL protein phenotype, the protein levels for F2 mice with each particular haplotype were compared. Mice with opposite allelic combinations for QTLs on chromosomes 1 and 5 had a difference of 310 μg/ml BAL protein, which accounts for approximately one-third of the total difference in mean BAL protein between the parental CBy and D2 strains. Additionally, the mean BAL protein level of F2 mice with a DD haplotype for QTLs on chromosomes 1, 4, and 5 was over half that of the non-tolerant D2 parental strain. These analyses suggest that although three QTLs were identified, the development of pulmonary tolerance to BAL protein is a decidedly complex phenotype that is regulated by a number of different genes, some of which were likely not identified by linkage analysis using an F2 cohort. + +Candidate genes within the significant QTL on chromosome 1 (ZIT1) that could play a role in controlling the development of tolerance to BAL protein following repeated ZnO exposure are presented in Table 1. The Duffy blood group (Dfy) has been shown to modulate the intensity of inflammation following endotoxin exposure [26], and has a role in enhancing inflammatory cell recruitment to sites of inflammation by facilitating movement of chemokines across the endothelium [27]. ADP-ribosyltransferase 1 (Adprt1) and tumor necrosis factor (TNF) receptor-associated factor (Traf5) are functionally associated with nuclear factor (NF)-κB, a key transcription factor in the regulation of the inflammatory process [28,29]. Additionally, activation of ADPRT1 plays a role in endotoxin-induced BAL protein increases [30,31]. Activated transforming growth factor-β has been shown to be a mediator of bleomycin- and endotoxin-induced lung permeability (i.e., BAL protein) in mice [32]. Finally, solute carrier family 30 (zinc transporter), member 1 (Slc30a1) is a metal transporter on the plasma membrane that confers resistance to zinc and cadmium toxicity in vitro via an efflux mechanism [33,34]. + +Candidate genes for BAL protein tolerance identified within the suggestive QTL intervals on chromosomes 4 and 5 are also presented in Table 1. Notable candidates include platelet-activating factor receptor (Ptafr) and phospholipase A2 (Pla2) that have been implicated as potential mediators of toxicant-induced BAL protein increases [35-37]. Interleukin (IL)-6 protein was increased following ZnO exposure in humans [1,38], and was hypothesized to be an anti-inflammatory suppressor of ZnO-induced lung injury. Interestingly, increased lung IL-6 levels have been shown to mediate pulmonary tolerance to ozone in rats [39]. Lastly, solute carrier family 30 (zinc transporter) members 2 and 3 (Slc30a2 and Slc30a3) have been identified as zinc transporters that protect against zinc-induced toxicity in both cell culture and animal models [40,41]. The development of pulmonary tolerance to BAL protein in our model could be regulated by any number of the aforementioned candidate genes. These candidates will be investigated in future studies of transcriptional and protein regulation to determine their roles in the development of tolerance. + +The suggestive QTLs for tolerance to BAL PMNs and macrophages (on chromosomes 1 and 5, respectively) were also examined for positional candidate genes (Table 2). Chemokine (C-C motif) ligand 20 (Ccl20), chemokine (C-X-C motif) receptor 4 (Cxcr4), and interleukin-10 (Il10) were identified as candidates for BAL PMN tolerance. The movement of PMNs into inflammatory tissues is regulated by chemotactic factors (e.g., chemokines) that signal through numerous chemokine receptors [42]. PMNs are capable of producing several chemokines and proinflammatory cytokines, indicating that PMNs may be important in auto-direction of cell trafficking during inflammation. CXCR4 expression has been detected on human PMNs [43], and coordinated chemokine receptor gene expression may control the tissue-specific migration and activation status of PMNs into the lung. Human PMNs are also able to express CCL20 [44], which is able to recruit immature dendritic cells that play an important role in the initiation of the immune response as well as chronic inflammation [45-47]. Finally, IL-10 can be produced by T cells and is able to diminish PMN influx by inhibition of expression of proinflammatory chemokines [48], NF-κB (via I kappa kinase) [49], and TNF [50], as well as modulate cells and effector functions associated with an allergic response. With respect to the BAL macrophage phenotype, the epidermal growth factor receptor (EGFR) ligands amphiregulin (Areg) and betacellulin (Btc) were identified as candidate genes within the suggestive QTL on chromosome 5. Ligand-dependent activation of the EGFR by particles rich in metal content lead to activation of the MAP kinase signaling cascade and cytokine expression and secretion [51,52]. Secreted phosphoprotein 1 (Spp1, also known as osteopontin) was also identified, and it can act as a chemoattractant for macrophages [53,54]. Lastly, a host of chemokine (C-X-C motif) ligands were identified between 51 and 53 cM that could be involved in macrophage chemotaxis [55]. Again, tolerance to BAL PMNs and macrophages may be under the influence of any number of these candidate genes. + +In the present study, we identified toll-like receptor (Tlr5) as a candidate gene within the significant ZIT1 QTL on chromosome 1 for tolerance to BAL protein. Toll-like receptors activate intra-cellular signaling that culminates in the induction of a multitude of effector genes [56]. Tlr5 has been shown to be an important gene in the immune response to Gram-positive and Gram-negative bacterial flagellin [57,58]. We utilized a wild-derived inbred mouse strain called MOLF/Ei which has non-conservative mutations in Tlr5 that are associated with a lower level of expression [25] to determine whether Tlr5 plays a role in the development of tolerance to BAL protein in our ZnO model. Because MOLF/Ei mice have a lower level of TLR5 mRNA expression compared to other strains [25], it was unclear whether we would observe tolerance after a single exposure to ZnO. Although the MOLF/Ei strain has no "wild-type" control strain per se, MOLF/Ei mice were tolerant to increased BAL protein following repeated ZnO exposure when compared to the non-tolerant D2 strain. These data support a role for Tlr5 in the development of tolerance to BAL protein. Interestingly, Kleeberger and colleagues identified Tlr4 as a candidate gene in a study of susceptibility to increased BAL protein in mice following a single exposure to 0.3 ppm ozone for 72 hours [22], which also suggests toll-like receptor signaling may be important in the regulation of inhaled toxicant-induced changes in BAL protein. + +The mechanism through which Tlr5 signaling may regulate the development of tolerance to ZnO is unknown. While endotoxin [59] and bacterial flagellin [60] have been demonstrated as the primary ligands for Tlr4 and Tlr5, respectively, the ligand(s) that is responsible for toll-like receptor signaling following exposure to inhaled toxicants such as ozone and ZnO is unknown. Although there have been no studies on endogenous ligands for Tlr5, several endogenous ligands for Tlr4 have been identified. For example, fibronectin [61] and hyaluronic acid [62] are produced by lung cells during lung injury and are endogenous Tlr4 ligands. The downstream pathway through which Tlr5 may regulate the development of tolerance to ZnO-induced BAL protein is potentially via NF-κB, a transcription factor that is known to induce several cytokines involved in inhaled ZnO responsiveness such as IL-8 and IL-6 [1,63]. Furthermore, NF-κB-dependent gene expression is decreased in Tlr5-mediated tolerance to flagellin in vitro [64]. Thus, it is plausible that a mutation in Tlr5, a regulatory element upstream of NF-κB signaling, could modulate tolerance to BAL protein from repeated ZnO exposure. Finally, Tlr5 may function as a danger signal receptor in the development of ZnO tolerance, consistent with the "danger model" of innate immunity that explicates activation of the innate immune system by factors other than foreign antigens [65]. Whatever the case, much work is needed in understanding how toxicants function through toll-like receptor signaling mechanisms in the lung to regulate adverse responses such as BAL protein. + +In summary, linkage analysis of a large F2 mouse cohort identified significant linkage of a QTL (ZIT1) on chromosome 1 associated with tolerance to BAL protein following repeated exposure to inhaled ZnO. Suggestive QTLs were also identified on chromosomes 4 and 5 for BAL protein, on chromosome 1 for BAL PMNs, and on chromosome 5 for BAL macrophages. Haplotype analysis suggested that the combinatorial effects of these three loci contributed to the overall phenotype, which agrees with the calculated number of segregating loci. Tlr5 was identified within the significant QTL for BAL protein on chromosome 1. Wild-derived Tlr5-mutant MOLF/Ei mice were determined to be tolerant to BAL protein following repeated ZnO exposure, suggesting a role for Tlr5 in the development of pulmonary tolerance to inhaled toxicants. + +Conclusion + +These data substantiate genetic background as an important influence in the acquisition of pulmonary tolerance following exposure to inhaled toxicants such as ZnO, and promising candidate genes exist within the identified QTL intervals that would be good targets for additional studies on the pathogenesis of tolerance. + +Competing interests + +The author(s) declare that they have no competing interests. + +Authors' contributions + +SCW: Participated in the design and coordination of the study, performed the study, and drafted the manuscript. + +LCC: Participated in the design of the study and helped draft the manuscript. + +TG: Conceived the study, participated in the design and coordination of the study, and helped draft the manuscript. + +Acknowledgements + +The authors would like to thank the following people for their contributions to this study: Karen Galdanes, Margaret Krasinski, Kathy Baker, and Dr. Moon-shong Tang (New York University); Dr. George D. Leikauf (University of Cincinnati); Dr. Daniel R. Prows (Cincinnati Children's Hospital); Dr. Steven R. Kleeberger (National Institute of Environmental Health Sciences); Dr. Carrie L. Welch (Columbia University). This study was supported by USEPA Grant R-826244, USEPA Particulate Matter Center Grant R-827351, USEPA STAR Fellowship U-91578301, NIEHS Center Grant of Excellence ES-00260, and CDC/NIOSH (via Mt. Sinai School of Medicine, New York, NY) Grant T-42CCT210425-06-01. diff --git a/src/ontogpt/evaluation/craft/database/all/16027110.ann b/src/ontogpt/evaluation/craft/database/all/16027110.ann new file mode 100644 index 000000000..8bff80e1e --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16027110.ann @@ -0,0 +1,795 @@ +T1 NCBITaxon:10088 43 48 mouse +T2 GO:0005634 49 56 nuclear +T3 GO:0051168 49 63 nuclear export +T4 PR:000011535 103 106 TAP +T5 PR:Q9UBU9 107 112 hNXF1 +T6 GO:0006406 160 176;181 188 mRNA export from ... nucleus +T7 GO:0005634 181 188 nucleus +T8 SO:0000855 198 207 orthologs +T9 NCBITaxon:9606 266 272 humans +T10 NCBITaxon:33208 274 283 Metazoans +T11 SO:0000857 322 330 homology +T12 SO:0000417 335 341 domain +T13 PR:000011535 360 363 TAP +T14 PR:Q9UBU9 364 369 hNXF1 +T15 GO:0016071 405 420 mRNA metabolism +T16 NCBITaxon:9606 517 522 human +T17 PR:Q9GZY0 542 547 hNXF2 +T18 GO:0010467 564 573 expressed +T19 UBERON:0000955 581 586 brain +T20 UBERON:0000955 601 606 brain +T21 GO:0016071 624 639 mRNA metabolism +T22 SO:0000704 738 743 genes +T23 PR:000011536 745 749 Nxf2 +T24 PR:000011535 784 787 TAP +T25 PR:Q9UBU9 788 793 hNXF1 +T26 SO:0000855 796 804 ortholog +T27 PR:000011535 805 809 Nxf1 +T28 NCBITaxon:39107 823 829 murine +T29 SO:0000417 870 876 domain +T30 GO:0010467 945 954 expressed +T31 NCBITaxon:10088 962 967 mouse +T32 UBERON:0000955 968 973 brain +T33 GO:0007420 968 985 brain development +T34 PR:000011535 998 1001 TAP +T35 PR:Q9UBU9 1002 1007 hNXF1 +T36 GO:0005634 1043 1050 nucleus +T37 GO:0005635 1056 1072 nuclear envelope +T38 GO:0005737 1077 1086 cytoplasm +T39 GO:0005737 1171 1182 cytoplasmic +T40 PR:000011535 1348 1351 TAP +T41 PR:Q9UBU9 1352 1357 hNXF1 +T42 GO:0005737 1413 1422 cytoplasm +T43 PR:000011535 1439 1442 TAP +T44 PR:Q9UBU9 1443 1448 hNXF1 +T45 GO:0005634 1481 1488 nuclear +T46 GO:0006406 1481 1504 nuclear export of mRNAs +T47 SO:0000855 1520 1529 orthologs +T48 NCBITaxon:4932 1533 1557 Saccharomyces cerevisiae +T49 NCBITaxon:7227 1559 1582 Drosophila melanogaster +T50 NCBITaxon:6239 1587 1609 Caenorhabditis elegans +T51 NCBITaxon:33208 1677 1686 Metazoans +T52 PR:000011535 1748 1751 TAP +T53 PR:Q9UBU9 1752 1757 hNXF1 +T54 SO:0000855 1758 1767 orthologs +T55 GO:0005634 1805 1812 nuclear +T56 GO:0051168 1805 1819 nuclear export +T57 NCBITaxon:9606 1883 1889 humans +T58 SO:0000857 1908 1916 homology +T59 SO:0000417 1937 1943 domain +T60 PR:000011535 1999 2002 TAP +T61 PR:Q9UBU9 2003 2008 hNXF1 +T62 GO:0016071 2028 2043 mRNA metabolism +T63 NCBITaxon:9606 2057 2062 human +T64 PR:Q9GZY0 2063 2068 hNXF2 +T65 PR:Q9H4D5 2073 2078 hNXF3 +T66 NCBITaxon:7215 2165 2175 Drosophila +T67 PR:000011535 2187 2191 NXF1 +T68 SO:0000855 2192 2200 ortholog +T69 PR:Q9U1H9 2201 2207 dmNXF1 +T70 PR:Q9U1H9 2377 2383 dmNXF1 +T71 NCBITaxon:6239 2419 2428 C.elegans +T72 PR:000011535 2429 2433 NXF1 +T73 SO:0000853 2434 2441 homolog +T74 NCBITaxon:6239 2442 2444 Ce +T75 PR:Q9XVS7 2442 2449 Ce-NXF1 +T76 GO:0016246 2497 2501 RNAi +T77 NCBITaxon:6239 2515 2517 Ce +T78 PR:000011536 2518 2522 NXF2 +T79 GO:0065007 2608 2618 regulation +T80 PR:P34709 2622 2627 tra-2 +T81 NCBITaxon:9606 2683 2688 human +T82 PR:Q9H1B4 2689 2694 hNXF5 +T83 PR:000011535 2802 2805 TAP +T84 PR:Q9UBU9 2806 2811 hNXF1 +T85 SO:0000855 2812 2821 orthologs +T86 NCBITaxon:33208 2863 2871 metazoan +T87 NCBITaxon:species 2872 2879 species +T88 NCBITaxon:10088 2996 3001 mouse +T89 NCBITaxon:10088 3087 3092 mouse +T90 SO:0000704 3097 3102 genes +T91 PR:000011536 3104 3108 Nxf2 +T92 SO:0000704 3178 3182 Gene +T93 SO:0000112 3192 3199 primers +T94 NCBITaxon:10088 3259 3264 mouse +T95 SO:0000153 3265 3268 BAC +T96 NCBITaxon:9606 3290 3295 human +T97 PR:000011536 3296 3301 TAPX2 +T98 PR:Q9GZY0 3302 3307 hNXF2 +T99 NCBITaxon:10088 3388 3393 mouse +T100 UBERON:0000955 3394 3399 brain +T101 SO:0000112 3445 3452 primers +T102 SO:0000440 3532 3538 vector +T103 NCBITaxon:10088 3648 3653 mouse +T104 PR:000011536 3654 3658 Nxf2 +T105 SO:0000704 3668 3673 genes +T106 NCBITaxon:10088 3713 3718 mouse +T107 SO:0000153 3719 3722 BAC +T108 GO:0010467 3788 3798 Expression +T109 SO:0000155 3799 3807 plasmids +T110 SO:0000006 3993 4005 PCR products +T111 SO:0000155 4039 4046 plasmid +T112 SO:0001817 4065 4073 in-frame +T113 SO:0000155 4251 4258 plasmid +T114 SO:0000155 4692 4699 plasmid +T115 CHEBI:16856 4772 4783 glutathione +T116 SO:0000440 4916 4922 vector +T117 GO:0010467 4937 4947 expression +T118 NCBITaxon:562 4951 4967 Escherichia coli +T119 NCBITaxon:10088 4969 4974 Mouse +T120 PR:000011546 4975 4978 p15 +T121 PR:000011546 4979 4983 NXT1 +T122 GO:0010467 4984 4994 expression +T123 SO:0000155 4995 5002 plasmid +T124 NCBITaxon:9606 5067 5072 human +T125 GO:0010467 5079 5089 expression +T126 SO:0000155 5090 5097 plasmid +T127 GO:0009294 5146 5158 transfection +T128 NCBITaxon:9606 5175 5180 Human +T129 NCBITaxon:10088 5204 5209 mouse +T130 GO:0009294 5238 5249 transfected +T131 CHEBI:33893 5264 5271 reagent +T132 GO:0009294 5326 5339 transfections +T133 GO:0009294 5447 5459 transfection +T134 CHEBI:16842 5487 5499 formaldehyde +T135 GO:0009294 5562 5574 transfection +T136 CHEBI:52646 5605 5617 leptomycin B +T137 CHEBI:27666 5619 5632 actinomycin D +T138 CHEBI:23888 5788 5792 drug +T139 GO:0010467 5873 5883 expressing +T140 CHEBI:17698 6199 6214 Chloramphenicol +T141 GO:0009294 6346 6357 transfected +T142 GO:0019835 6392 6397 lysis +T143 CHEBI:9750 6411 6423 Triton X-100 +T144 CHEBI:9754 6435 6439 Tris +T145 CHEBI:17883 6440 6443 HCl +T146 CHEBI:53325 6515 6529 nitrocellulose +T147 UBERON:0001977 6555 6559 sera +T148 NCBITaxon:9606 6568 6573 human +T149 PR:000011536 6574 6579 TAPX2 +T150 PR:Q9GZY0 6580 6585 hNXF2 +T151 NCBITaxon:10088 6590 6595 mouse +T152 NCBITaxon:9986 6617 6624 rabbits +T153 SO:0000417 6658 6664 domain +T154 PR:000011536 6720 6725 TAPX2 +T155 CHEBI:60816 6773 6783 immunogens +T156 PR:000011536 6789 6794 TAPX2 +T157 PR:Q9GZY0 6795 6800 hNXF2 +T158 GO:0042571 6801 6811 antibodies +T159 PR:000011536 6854 6859 TAPX2 +T160 CHEBI:60816 6860 6869 immunogen +T161 NCBITaxon:9986 6909 6915 rabbit +T162 PR:000011536 6921 6926 TAPX2 +T163 PR:Q9GZY0 6927 6932 hNXF2 +T164 GO:0042571 6947 6955 antibody +T165 NCBITaxon:9793 7019 7025 donkey +T166 NCBITaxon:9986 7031 7037 rabbit +T167 NCBITaxon:3704 7038 7049 horseradish +T168 MOP:0000779 7061 7071 conjugated +T169 GO:0042571 7082 7090 antibody +T170 NCBITaxon:9606 7268 7273 human +T171 UBERON:0000479 7274 7281 tissues +T172 NCBITaxon:10088 7298 7303 mouse +T173 UBERON:0000955 7304 7309 brain +T174 GO:0007568 7310 7315 aging +T175 CL:0000558 7460 7472 Reticulocyte +T176 GO:0008152 7512 7525 metabolically +T177 GO:0006412 7559 7570 translation +T178 NCBITaxon:10760 7583 7585 T7 +T179 CL:0000558 7594 7606 Reticulocyte +T180 NCBITaxon:10760 7638 7640 T7 +T181 SO:0000167 7641 7649 promoter +T182 CL:0000558 7848 7860 reticulocyte +T183 NCBITaxon:562 7893 7899 E.coli +T184 CHEBI:16856 7954 7965 glutathione +T185 CHEBI:46756 8048 8053 HEPES +T186 CHEBI:32588 8069 8072 KCl +T187 CHEBI:9750 8095 8107 Triton X-100 +T188 CHEBI:26710 8134 8138 NaCl +T189 CHEBI:8984 8302 8305 SDS +T190 CHEBI:8984 8339 8342 SDS +T191 NCBITaxon:10088 8635 8640 mouse +T192 SO:0000704 8645 8650 genes +T193 PR:000011536 8749 8753 Nxf2 +T194 NCBITaxon:10088 8816 8821 mouse +T195 SO:0000704 8834 8839 genes +T196 SO:0000147 8959 8965 exonic +T197 NCBITaxon:10088 8984 8989 mouse +T198 PR:000011536 8990 8994 Nxf2 +T199 SO:0000704 9054 9059 genes +T200 NCBITaxon:10088 9148 9153 mouse +T201 SO:0000853 9154 9162 homologs +T202 NCBITaxon:9606 9166 9171 human +T203 PR:000011535 9172 9175 TAP +T204 PR:Q9UBU9 9176 9181 hNXF1 +T205 NCBITaxon:10088 9234 9239 mouse +T206 GO:0010467 9240 9249 expressed +T207 SO:0000345 9240 9263 expressed sequence tags +T208 SO:0000345 9265 9269 ESTs +T209 SO:0000857 9312 9320 homology +T210 NCBITaxon:10088 9328 9333 mouse +T211 SO:0000153 9334 9337 BAC +T212 NCBITaxon:9606 9362 9367 human +T213 PR:000011536 9368 9373 TAPX2 +T214 PR:Q9GZY0 9374 9379 hNXF2 +T215 PR:Q9GZY0 9393 9398 hNXF2 +T216 SO:0001060 9399 9406 isoform +T217 SO:0000317 9422 9432 cDNA clone +T218 PR:000011536 9441 9445 Nxf2 +T219 NCBITaxon:10088 9466 9471 mouse +T220 UBERON:0000955 9472 9477 brain +T221 SO:0000188 9511 9517 intron +T222 SO:0000153 9553 9556 BAC +T223 SO:0001026 9594 9601 genomic +T224 SO:0000147 9617 9621 exon +T225 SO:0000188 9626 9632 intron +T226 NCBITaxon:10088 9650 9655 mouse +T227 PR:000011536 9656 9660 Nxf2 +T228 SO:0000704 9661 9665 gene +T229 NCBITaxon:10088 9712 9717 mouse +T230 PR:000011536 9718 9722 Nxf2 +T231 SO:0000704 9723 9727 gene +T232 SO:0000195 9767 9779 coding exons +T233 SO:0001817 9788 9796 in-frame +T234 SO:0000147 9816 9821 exons +T235 SO:0000704 10139 10143 gene +T236 SO:0000153 10155 10158 BAC +T237 SO:0000345 10196 10199 EST +T238 SO:0000857 10200 10208 homology +T239 NCBITaxon:10088 10246 10251 mouse +T240 SO:0000147 10278 10284 exonic +T241 NCBITaxon:10088 10341 10346 mouse +T242 SO:0000704 10352 10356 gene +T243 SO:0000147 10369 10374 exons +T244 SO:0000317 10478 10488 cDNA clone +T245 SO:0000343 10503 10517 sequence match +T246 SO:0000147 10531 10537 exonic +T247 NCBITaxon:10088 10555 10560 mouse +T248 SO:0001026 10566 10573 genomic +T249 SO:0000857 10708 10716 homology +T250 SO:0001060 10749 10757 isoforms +T251 GO:0008380 11022 11028 splice +T252 SO:0000147 11046 11050 exon +T253 SO:0000704 11221 11226 genes +T254 NCBITaxon:10088 11234 11239 mouse +T255 PR:000011536 11240 11245 Nxf-2 +T256 SO:0000704 11319 11324 genes +T257 GO:0000805 11340 11352 X chromosome +T258 SO:0000704 11388 11392 gene +T259 PR:000011535 11416 11420 Nxf1 +T260 NCBITaxon:10088 11460 11465 mouse +T261 NCBITaxon:9606 11516 11521 human +T262 NCBITaxon:9606 11619 11625 humans +T263 SO:0000857 11652 11660 homology +T264 NCBITaxon:species 11684 11691 species +T265 NCBITaxon:10088 11711 11716 mouse +T266 PR:Q99JX7 11717 11722 mNXF1 +T267 PR:000011535 11747 11750 TAP +T268 PR:Q9UBU9 11751 11756 hNXF1 +T269 SO:0000855 11757 11766 orthologs +T270 NCBITaxon:8782 11773 11778 birds +T271 NCBITaxon:9606 11782 11788 humans +T272 NCBITaxon:10088 11839 11844 mouse +T273 NCBITaxon:9606 11930 11935 human +T274 NCBITaxon:10088 11997 12002 mouse +T275 PR:000011535 12019 12022 TAP +T276 PR:Q9UBU9 12023 12028 hNXF1 +T277 NCBITaxon:10088 12084 12089 mouse +T278 PR:Q99JX7 12090 12095 mNXF1 +T279 NCBITaxon:9606 12104 12109 human +T280 PR:000011535 12110 12113 TAP +T281 PR:Q9UBU9 12114 12119 hNXF1 +T282 PR:Q99JX7 12158 12163 mNXF1 +T283 SO:0000857 12197 12207 homologies +T284 SO:0000417 12327 12333 domain +T285 NCBITaxon:9606 12356 12361 human +T286 PR:000011535 12362 12365 TAP +T287 PR:Q9UBU9 12366 12371 hNXF1 +T288 SO:0000417 12432 12438 domain +T289 PR:000011533 12483 12487 NTF2 +T290 SO:0000417 12493 12499 domain +T291 PR:000011546 12529 12532 p15 +T292 PR:000011546 12533 12537 NXT1 +T293 SO:0000417 12573 12579 domain +T294 SO:0000409 12619 12633 binding region +T295 SO:0000417 12744 12750 domain +T296 GO:0010467 12822 12832 Expression +T297 UBERON:0000955 12857 12862 brain +T298 GO:0010467 12891 12901 expression +T299 UBERON:0001977 12923 12928 serum +T300 NCBITaxon:9986 12966 12973 rabbits +T301 UBERON:0001977 13017 13022 serum +T302 GO:0009294 13171 13182 transfected +T303 NCBITaxon:9606 13183 13188 human +T304 NCBITaxon:9606 13245 13250 human +T305 PR:000011535 13251 13254 TAP +T306 PR:Q9UBU9 13255 13260 hNXF1 +T307 NCBITaxon:10088 13264 13269 mouse +T308 NCBITaxon:10088 13433 13438 mouse +T309 UBERON:0000955 13493 13498 brain +T310 NCBITaxon:10088 13600 13605 mouse +T311 UBERON:0000955 13606 13611 brain +T312 UBERON:0000955 13707 13712 brain +T313 NCBITaxon:10088 13736 13741 mouse +T314 UBERON:0000922 13742 13748 embryo +T315 GO:0010467 13769 13779 expression +T316 GO:0007567 13818 13823 birth +T317 GO:0010467 13964 13974 expression +T318 GO:0042571 14024 14034 antibodies +T319 NCBITaxon:9986 14071 14077 rabbit +T320 UBERON:0001977 14082 14087 serum +T321 NCBITaxon:9606 14101 14106 human +T322 PR:000011536 14107 14112 TAPX2 +T323 SO:0001060 14132 14139 isoform +T324 PR:Q9GZY0 14143 14148 hNXF2 +T325 NCBITaxon:9606 14166 14171 human +T326 UBERON:0000479 14181 14187 tissue +T327 PR:000011536 14222 14227 TAPX2 +T328 PR:Q9GZY0 14228 14233 hNXF2 +T329 GO:0010467 14251 14260 expressed +T330 UBERON:0000955 14268 14273 brain +T331 UBERON:0000479 14322 14329 tissues +T332 UBERON:0000473 14348 14354 testis +T333 NCBITaxon:9606 14487 14492 human +T334 NCBITaxon:10088 14497 14502 mouse +T335 PR:000011536 14503 14507 NXF2 +T336 GO:0010467 14513 14522 expressed +T337 UBERON:0000955 14530 14535 brain +T338 UBERON:0000955 14561 14566 brain +T339 GO:0016071 14576 14591 mRNA metabolism +T340 GO:0009294 14717 14729 transfection +T341 NCBITaxon:9606 14733 14738 human +T342 SO:0000155 14755 14763 plasmids +T343 GO:0010467 14764 14774 expressing +T344 GO:0005634 14846 14853 nucleus +T345 GO:0005730 14880 14889 nucleolus +T346 GO:0034399 14910 14921 nuclear rim +T347 GO:0005737 14944 14953 cytoplasm +T348 NCBITaxon:9606 15008 15013 human +T349 PR:000011535 15014 15017 TAP +T350 PR:Q9UBU9 15018 15023 hNXF1 +T351 GO:0005737 15087 15096 cytoplasm +T352 NCBITaxon:9606 15206 15211 human +T353 NCBITaxon:10088 15236 15241 mouse +T354 CHEBI:52646 15438 15450 Leptomycin B +T355 PR:000017497 15477 15481 CRM1 +T356 CHEBI:27666 15523 15536 Actinomycin D +T357 GO:0006913 15634 15663 nucleocytoplasmic trafficking +T358 NCBITaxon:11676 15732 15737 HIV-1 +T359 GO:0005634 15790 15797 nuclear +T360 SO:0001528 15790 15817 nuclear localization signal +T361 GO:0005634 15851 15858 nuclear +T362 SO:0001528 15851 15878 nuclear localization signal +T363 SO:0001528 15880 15883 NLS +T364 GO:0034399 16037 16048 nuclear rim +T365 SO:0000417 16114 16120 domain +T366 GO:0005634 16176 16183 nucleus +T367 GO:0005737 16260 16271 cytoplasmic +T368 SO:0001528 16345 16348 NLS +T369 GO:0005634 16486 16493 nucleus +T370 GO:0005737 16684 16693 cytoplasm +T371 GO:0005634 16794 16801 nuclear +T372 GO:0051170 16794 16808 nuclear import +T373 GO:0005634 16913 16920 nuclear +T374 GO:0005737 16951 16962 cytoplasmic +T375 GO:0005634 17014 17021 nuclear +T376 GO:0051170 17014 17028 nuclear import +T377 GO:0005634 17057 17064 nuclear +T378 GO:0051170 17057 17071 nuclear import +T379 GO:0005737 17172 17181 cytoplasm +T380 GO:0005634 17255 17262 nuclear +T381 GO:0051170 17255 17269 nuclear import +T382 GO:0005634 17361 17368 nuclear +T383 SO:0001528 17431 17434 NLS +T384 GO:0005634 17633 17640 nuclear +T385 GO:0005737 17754 17765 cytoplasmic +T386 GO:0005634 17876 17883 nuclear +T387 SO:0001528 18037 18040 NLS +T388 GO:0005634 18156 18163 nuclear +T389 GO:0051170 18156 18170 nuclear import +T390 SO:0001528 18212 18215 NLS +T391 SO:0001528 18244 18247 NLS +T392 CHEBI:22695 18379 18384 basic +T393 SO:0001528 18429 18432 NLS +T394 CHEBI:22695 18543 18548 basic +T395 SO:0001528 18591 18594 NLS +T396 SO:0000417 18595 18601 domain +T397 SO:0001528 18649 18652 NLS +T398 GO:0005737 18669 18680 cytoplasmic +T399 SO:0001528 18776 18779 NLS +T400 SO:0001528 18869 18872 NLS +T401 SO:0001528 18937 18940 NLS +T402 SO:0001528 19004 19007 NLS +T403 SO:0001528 19104 19107 NLS +T404 SO:0001528 19217 19220 NLS +T405 CHEBI:22695 19256 19261 basic +T406 SO:0001528 19358 19361 NLS +T407 SO:0001528 19412 19415 NLS +T408 NCBITaxon:9606 19423 19428 human +T409 PR:000011535 19429 19432 TAP +T410 PR:000011535 19433 19437 NXF1 +T411 SO:0001528 19581 19584 NLS +T412 NCBITaxon:9606 19612 19617 human +T413 PR:000011535 19618 19621 TAP +T414 PR:Q9UBU9 19622 19627 hNXF1 +T415 SO:0001528 19648 19651 NLS +T416 CHEBI:22695 19671 19676 basic +T417 GO:0005635 19770 19786 nuclear envelope +T418 NCBITaxon:9606 19812 19817 human +T419 PR:000011535 19818 19821 TAP +T420 PR:Q9UBU9 19822 19827 hNXF1 +T421 GO:0005635 19869 19885 nuclear envelope +T422 GO:0005737 20012 20021 cytoplasm +T423 GO:0034399 20030 20047 nuclear periphery +T424 GO:0005634 20141 20148 nucleus +T425 GO:0034399 20173 20184 nuclear rim +T426 GO:0034399 20238 20249 nuclear rim +T427 CHEBI:27729 20412 20421 digitonin +T428 GO:0005635 20508 20524 nuclear envelope +T429 NCBITaxon:9606 20661 20666 human +T430 GO:0034399 20824 20835 nuclear rim +T431 GO:0005634 20939 20946 nucleus +T432 GO:0034399 20979 20990 nuclear rim +T433 GO:0034399 21121 21132 nuclear rim +T434 SO:0001528 21238 21241 NLS +T435 PR:000011535 21245 21248 TAP +T436 PR:Q9UBU9 21249 21254 hNXF1 +T437 GO:0034399 21322 21333 nuclear rim +T438 GO:0005654 21342 21353 nucleoplasm +T439 GO:0005643 21430 21450 nuclear pore complex +T440 GO:0005643 21452 21455 NPC +T441 GO:0005634 21473 21480 nuclear +T442 GO:0051170 21473 21487 nuclear import +T443 SO:0001528 21533 21536 NLS +T444 PR:000011535 21631 21634 TAP +T445 PR:Q9UBU9 21635 21640 hNXF1 +T446 SO:0000417 21740 21746 domain +T447 GO:0005737 21882 21893 cytoplasmic +T448 SO:0000417 22001 22008 domains +T449 PR:000011533 22076 22080 NTF2 +T450 SO:0000417 22098 22105 domains +T451 PR:000011535 22973 22976 TAP +T452 PR:Q9UBU9 22977 22982 hNXF1 +T453 SO:0000417 23004 23010 domain +T454 GO:0005634 23044 23051 nuclear +T455 GO:0051168 23044 23058 nuclear export +T456 CHEBI:23357 23059 23068 cofactors +T457 GO:1990124 23088 23102 mRNP complexes +T458 SO:0000155 23248 23255 plasmid +T459 SO:0000155 23264 23271 Plasmid +T460 PR:000003225 23356 23359 env +T461 SO:0000188 23360 23366 intron +T462 NCBITaxon:11676 23370 23375 HIV-1 +T463 SO:0000357 23377 23384 flanked +T464 GO:0008380 23390 23396 splice +T465 SO:0000673 23447 23458 transcripts +T466 GO:0005634 23479 23486 nucleus +T467 GO:0010467 23559 23569 expression +T468 NCBITaxon:11676 23600 23605 HIV-1 +T469 PR:000011535 23613 23616 TAP +T470 PR:Q9UBU9 23617 23622 hNXF1 +T471 PR:000011535 23705 23708 TAP +T472 PR:Q9UBU9 23709 23714 hNXF1 +T473 GO:0005634 23735 23742 nuclear +T474 GO:0051168 23735 23749 nuclear export +T475 SO:0000673 23757 23768 transcripts +T476 GO:0005634 23823 23830 nuclear +T477 GO:0006406 23823 23840;23855 23859 nuclear export of ... mRNA +T478 GO:0010467 23890 23899 expressed +T479 NCBITaxon:11676 23912 23917 HIV-1 +T480 PR:000011535 24051 24054 TAP +T481 PR:Q9UBU9 24055 24060 hNXF1 +T482 GO:0009294 24106 24118 transfection +T483 CHEBI:23357 24128 24136 cofactor +T484 NCBITaxon:9606 24142 24147 human +T485 PR:000011546 24148 24151 p15 +T486 PR:000011546 24152 24156 NXT1 +T487 PR:000011546 24258 24261 p15 +T488 PR:000011546 24262 24266 NXT1 +T489 GO:0009294 24322 24334 transfection +T490 GO:0010467 24398 24408 expression +T491 GO:0009294 24471 24483 transfection +T492 NCBITaxon:9606 24489 24494 human +T493 NCBITaxon:10088 24498 24503 mouse +T494 PR:000011546 24504 24507 p15 +T495 PR:000011546 24508 24512 NXT1 +T496 NCBITaxon:9606 24585 24590 human +T497 PR:000011535 24591 24594 TAP +T498 PR:Q9UBU9 24595 24600 hNXF1 +T499 PR:000011546 24633 24636 p15 +T500 PR:000011546 24637 24641 NXT1 +T501 GO:0005643 24737 24740 NPC +T502 PR:000011535 24754 24757 TAP +T503 PR:Q9UBU9 24758 24763 hNXF1 +T504 SO:0000409 24811 24824 binding sites +T505 PR:000011546 24847 24850 p15 +T506 PR:000011546 24851 24855 NXT1 +T507 PR:000011546 24906 24909 p15 +T508 PR:000011546 24910 24914 NXT1 +T509 PR:000011546 25005 25008 p15 +T510 PR:000011546 25009 25013 NXT1 +T511 GO:0005737 25084 25095 cytoplasmic +T512 NCBITaxon:10088 25136 25141 mouse +T513 GO:0016071 25193 25208 mRNA metabolism +T514 NCBITaxon:10088 25243 25248 mouse +T515 GO:0005634 25319 25326 nuclear +T516 GO:0051168 25319 25333 nuclear export +T517 PR:000013809 25377 25380 Y14 +T518 PR:000010086 25381 25386 MAGOH +T519 PR:000016944 25409 25415 U2AF35 +T520 PR:000011535 25444 25447 TAP +T521 PR:Q9UBU9 25448 25453 hNXF1 +T522 PR:000011535 25509 25512 TAP +T523 PR:Q9UBU9 25513 25518 hNXF1 +T524 NCBITaxon:2 25560 25571 bacterially +T525 SO:0000417 25633 25640 domains +T526 PR:000011535 25654 25657 TAP +T527 PR:Q9UBU9 25658 25663 hNXF1 +T528 GO:0008152 25744 25757 metabolically +T529 PR:000006358 25837 25841 DBP5 +T530 PR:000011535 25868 25871 TAP +T531 PR:Q9UBU9 25872 25877 hNXF1 +T532 PR:000010086 25909 25914 MAGOH +T533 PR:000014390 25916 25920 REF2 +T534 PR:000016944 25928 25934 U2AF35 +T535 PR:000011535 25957 25960 TAP +T536 PR:Q9UBU9 25961 25966 hNXF1 +T537 PR:000013809 26001 26004 Y14 +T538 PR:000006358 26009 26013 DBP5 +T539 PR:000010086 26063 26068 MAGOH +T540 PR:000014390 26070 26074 REF2 +T541 PR:000016944 26082 26088 U2AF35 +T542 PR:000011535 26092 26095 TAP +T543 PR:Q9UBU9 26096 26101 hNXF1 +T544 PR:000010086 26161 26166 MAGOH +T545 PR:000011535 26184 26187 TAP +T546 PR:Q9UBU9 26188 26193 hNXF1 +T547 PR:000013809 26216 26219 Y14 +T548 PR:000016313 26238 26242 REF1 +T549 PR:000013809 26250 26253 Y14 +T550 CL:0000558 26380 26392 reticulocyte +T551 PR:000016313 26402 26406 REF1 +T552 PR:000013809 26417 26420 Y14 +T553 GO:0006457 26429 26435 folded +T554 GO:0006412 26458 26471 translational +T555 PR:000011535 26544 26547 TAP +T556 PR:Q9UBU9 26548 26553 hNXF1 +T557 PR:000011535 26660 26663 TAP +T558 PR:Q9UBU9 26664 26669 hNXF1 +T559 GO:0006457 26726 26736;26754 26761 folding of ... protein +T560 PR:000011535 26920 26923 TAP +T561 PR:Q9UBU9 26924 26929 hNXF1 +T562 NCBITaxon:10088 27029 27034 mouse +T563 SO:0000704 27046 27051 genes +T564 NCBITaxon:40674 27097 27106 mammalian +T565 GO:0010467 27138 27147 expressed +T566 UBERON:0000955 27168 27173 brain +T567 NCBITaxon:9606 27187 27192 human +T568 PR:Q9H1B4 27193 27198 hNXF5 +T569 SO:0000704 27340 27344 gene +T570 NCBITaxon:10088 27355 27360 mouse +T571 NCBITaxon:species 27375 27382 species +T572 NCBITaxon:9606 27418 27423 human +T573 UBERON:0001016 27424 27436 neurological +T574 http://purl.obolibrary.org/obo/MONDO_0005071 27424 27444 neurological disease +T575 PR:000011535 27469 27473 Nxf1 +T576 SO:0000704 27482 27487 genes +T577 NCBITaxon:10088 27494 27499 mouse +T578 NCBITaxon:9606 27514 27519 human +T579 PR:Q9GZY0 27520 27525 hNXF2 +T580 GO:0010467 27530 27539 expressed +T581 UBERON:0000955 27547 27552 brain +T582 UBERON:0000955 27604 27609 brain +T583 GO:0010467 27619 27629 expression +T584 PR:Q9H1B4 27677 27682 hNXF5 +T585 PR:Q9GZY0 27690 27695 hNXF2 +T586 GO:0010467 27794 27804 expression +T587 GO:0008380 27863 27869 splice +T588 UBERON:0000479 27890 27897 tissues +T589 UBERON:0000473 27906 27912 testis +T590 UBERON:0000955 28026 28031 brain +T591 GO:0010467 28041 28051 expression +T592 GO:0042571 28106 28116 antibodies +T593 NCBITaxon:9606 28134 28139 human +T594 PR:000011536 28153 28158 TAPX2 +T595 PR:Q9GZY0 28159 28164 hNXF2 +T596 GO:0042571 28181 28191 antibodies +T597 GO:0010467 28215 28225 expression +T598 NCBITaxon:9606 28233 28238 human +T599 PR:000011536 28239 28244 TAPX2 +T600 PR:Q9GZY0 28245 28250 hNXF2 +T601 UBERON:0000955 28280 28285 brain +T602 NCBITaxon:10088 28312 28317 mouse +T603 GO:0010467 28324 28334 expression +T604 UBERON:0000955 28349 28354 brain +T605 GO:0065007 28375 28384 regulated +T606 UBERON:0000955 28409 28414 brain +T607 PR:000011535 28532 28535 TAP +T608 PR:Q9UBU9 28536 28541 hNXF1 +T609 GO:0005634 28663 28670 nucleus +T610 GO:0005635 28679 28695 nuclear envelope +T611 GO:0005634 28717 28724 nuclear +T612 GO:0005643 28742 28745 NPC +T613 PR:000011535 28806 28809 TAP +T614 PR:Q9UBU9 28810 28815 hNXF1 +T615 NCBITaxon:9606 28849 28854 human +T616 PR:000011535 28855 28858 TAP +T617 PR:Q9UBU9 28859 28864 hNXF1 +T618 GO:0008380 28905 28913 splicing +T619 CHEBI:23357 28921 28930 cofactors +T620 PR:000010086 28940 28945 MAGOH +T621 PR:000016944 28955 28961 U2AF35 +T622 GO:0005634 28992 28999 nuclear +T623 GO:0051168 28992 29006 nuclear export +T624 PR:000011535 29139 29142 TAP +T625 PR:Q9UBU9 29143 29148 hNXF1 +T626 CHEBI:23357 29156 29164 cofactor +T627 PR:000011546 29165 29168 p15 +T628 PR:000011535 29285 29288 TAP +T629 PR:Q9UBU9 29289 29294 hNXF1 +T630 GO:0005737 29326 29337 cytoplasmic +T631 GO:0005635 29342 29358 nuclear envelope +T632 PR:000011535 29390 29393 TAP +T633 PR:Q9UBU9 29394 29399 hNXF1 +T634 GO:0005654 29423 29434 nucleoplasm +T635 CHEBI:23357 29487 29495 cofactor +T636 PR:000011546 29496 29499 p15 +T637 PR:000011546 29500 29504 NXT1 +T638 GO:0005634 29516 29523 nuclear +T639 GO:0051168 29516 29530 nuclear export +T640 UBERON:0000955 29613 29618 brain +T641 PR:000011535 29672 29675 TAP +T642 PR:Q9UBU9 29676 29681 hNXF1 +T643 PR:000011535 29740 29743 TAP +T644 PR:Q9UBU9 29744 29749 hNXF1 +T645 PR:000011535 29840 29843 TAP +T646 PR:Q9UBU9 29844 29849 hNXF1 +T647 SO:0000673 29893 29904 transcripts +T648 UBERON:0000955 29912 29917 brain +T649 GO:0065007 29936 29946 regulation +T650 SO:0000673 30013 30024 transcripts +T651 GO:0005737 30145 30156 cytoplasmic +T652 GO:0005634 30202 30209 nucleus +T653 GO:0005634 30301 30308 nucleus +T654 SO:0000976 30351 30358 cryptic +T655 SO:0001528 30359 30362 NLS +T656 NCBITaxon:species 30442 30449 species +T657 GO:0005737 30494 30503 cytoplasm +T658 GO:0005634 30719 30726 nuclear +T659 GO:0051170 30719 30733 nuclear import +T660 GO:0005635 30738 30754 nuclear envelope +T661 GO:0005634 31060 31067 nuclear +T662 GO:0051170 31060 31073 nuclear entry +T663 GO:0005643 31078 31081 NPC +T664 GO:0005634 31154 31161 nuclear +T665 GO:0051168 31154 31168 nuclear export +T666 GO:0005634 31256 31263 nucleus +T667 SO:0000673 31377 31388 transcripts +T668 GO:0005737 31514 31523 cytoplasm +T669 SO:0000857 31590 31598 homology +T670 PR:000011535 31602 31605 TAP +T671 PR:Q9UBU9 31606 31611 hNXF1 +T672 SO:0000417 31623 31629 domain +T673 GO:0016071 31682 31697 mRNA metabolism +T674 GO:0006406 31771 31790 mRNA nuclear export +T675 GO:0005634 31776 31783 nuclear +T676 SO:0000155 32152 32160 plasmids +T677 NCBITaxon:10088 32541 32546 Mouse +T678 PR:000011536 32547 32551 Nxf2 +T679 SO:0000147 32566 32570 Exon +T680 SO:0000188 32571 32577 intron +T681 NCBITaxon:10088 32591 32596 mouse +T682 PR:000011536 32597 32601 Nxf2 +T683 SO:0000704 32611 32616 genes +T684 SO:0001026 32668 32675 genomic +T685 SO:0000147 32703 32708 Exons +T686 GO:0006413 32740 32764 translational initiation +T687 SO:0000318 32740 32770 translational initiation codon +T688 GO:0006415 32785 32808 translation termination +T689 SO:0000319 32785 32815 translation termination codons +T690 SO:0001817 32848 32856 in-frame +T691 SO:0000360 32861 32867 codons +T692 NCBITaxon:10116 32939 32941 rn +T693 NCBITaxon:10116 32943 32960 Rattus norvegicus +T694 NCBITaxon:93934 32962 32964 cj +T695 NCBITaxon:93934 32966 32983 Coturnix japonica +T696 NCBITaxon:7227 32985 32987 dm +T697 NCBITaxon:7227 32989 33012 Drosophila melanogaster +T698 NCBITaxon:4896 33014 33016 sp +T699 NCBITaxon:4896 33018 33043 Schizosaccharomyces pombe +T700 NCBITaxon:4932 33045 33047 sc +T701 NCBITaxon:4932 33049 33073 Saccharomyces cerevisiae +T702 NCBITaxon:10088 33079 33084 mouse +T703 NCBITaxon:9606 33154 33159 human +T704 PR:000011535 33160 33163 TAP +T705 PR:Q9UBU9 33164 33169 hNXF1 +T706 NCBITaxon:10088 33174 33179 mouse +T707 SO:0000417 33198 33213 protein domains +T708 SO:0000417 33271 33278 domains +T709 GO:0005634 33289 33296 nuclear +T710 NCBITaxon:9606 33313 33318 human +T711 PR:000011535 33319 33322 TAP +T712 PR:Q9UBU9 33323 33328 hNXF1 +T713 GO:0005737 33356 33367 cytoplasmic +T714 SO:0000417 33452 33458 domain +T715 PR:000011533 33486 33490 NTF2 +T716 GO:0005634 33492 33499 nuclear +T717 GO:0051169 33492 33509 nuclear transport +T718 PR:000011533 33518 33522 NTF2 +T719 SO:0000417 33529 33535 domain +T720 SO:0000417 33573 33579 domain +T721 GO:0010467 33601 33611 expression +T722 UBERON:0000955 33639 33644 brain +T723 UBERON:0000479 33645 33651 tissue +T724 GO:0009294 33698 33709 transfected +T725 GO:0010467 33793 33803 expression +T726 SO:0000155 33804 33812 plasmids +T727 NCBITaxon:9986 33835 33841 rabbit +T728 UBERON:0001977 33857 33862 serum +T729 NCBITaxon:10088 33893 33898 mouse +T730 UBERON:0000955 33899 33904 brain +T731 UBERON:0000479 33905 33911 tissue +T732 NCBITaxon:9986 33931 33937 rabbit +T733 UBERON:0001977 33953 33958 serum +T734 NCBITaxon:10088 33970 33975 mouse +T735 UBERON:0000955 33976 33981 brain +T736 GO:0007568 33982 33987 aging +T737 UBERON:0000955 34042 34047 brain +T738 NCBITaxon:9606 34124 34129 human +T739 UBERON:0000479 34130 34137 tissues +T740 UBERON:0001977 34165 34170 serum +T741 NCBITaxon:9606 34174 34179 human +T742 PR:000011536 34180 34185 TAPX2 +T743 PR:Q9GZY0 34186 34191 hNXF2 +T744 UBERON:0000479 34279 34286 tissues +T745 GO:0009294 34378 34389 transfected +T746 GO:0010467 34422 34432 expression +T747 SO:0000155 34433 34441 plasmids +T748 GO:0010467 34755 34765 expressing +T749 SO:0001528 34864 34867 NLS +T750 GO:0009294 34904 34915 transfected +T751 SO:0000155 34921 34929 plasmids +T752 GO:0005634 35117 35118 N +T753 GO:0005634 35120 35127 nucleus +T754 GO:0005737 35129 35130 C +T755 GO:0005737 35132 35141 cytoplasm +T756 SO:0001528 35167 35170 NLS +T757 SO:0001528 35313 35316 NLS +T758 GO:0005635 35430 35446 nuclear envelope +T759 GO:0009294 35464 35475 transfected +T760 SO:0000155 35481 35489 plasmids +T761 GO:0009294 35795 35807 transfection +T762 GO:0005634 35823 35824 N +T763 GO:0005634 35826 35833 nucleus +T764 GO:0005737 35835 35836 C +T765 GO:0005737 35838 35847 cytoplasm +T766 GO:0005634 35917 35923 nuclei +T767 NCBITaxon:9606 36049 36054 Human +T768 GO:0009294 36070 36081 transfected +T769 NCBITaxon:11676 36144 36149 HIV-1 +T770 GO:0010467 36154 36164 expression +T771 SO:0000155 36165 36172 plasmid +T772 SO:0000155 36232 36240 plasmids +T773 GO:0009294 36260 36272 transfection +T774 CHEBI:60004 36273 36281 mixtures +T775 GO:0010467 36296 36306 expression +T776 SO:0000155 36307 36315 plasmids +T777 NCBITaxon:9606 36330 36335 human +T778 PR:000011546 36336 36339 p15 +T779 PR:000011546 36340 36344 NXT1 +T780 PR:000011546 36348 36351 p15 +T781 PR:000011546 36352 36356 NXT1 +T782 CHEBI:17698 36455 36470 chloramphenicol +T783 MOP:0000030 36471 36482 acetylation +T784 GO:0010467 36509 36519 Expression +T785 GO:0009294 36529 36540 transfected +T786 GO:0010467 36552 36562 expression +T787 SO:0000155 36563 36570 plasmid +T788 PR:000011535 36708 36711 TAP +T789 PR:Q9UBU9 36712 36717 hNXF1 +T790 CHEBI:23357 36725 36734 cofactors +T791 NCBITaxon:2 36736 36747 Bacterially +T792 CHEBI:16856 36801 36812 glutathione +T793 CL:0000558 36863 36875 reticulocyte +T794 GO:0008152 36886 36899 metabolically +T795 CHEBI:8984 37017 37020 SDS diff --git a/src/ontogpt/evaluation/craft/database/all/16027110.txt b/src/ontogpt/evaluation/craft/database/all/16027110.txt new file mode 100644 index 000000000..2f1ab7d9d --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16027110.txt @@ -0,0 +1,143 @@ +Identification and characterization of the mouse nuclear export factor (Nxf) family members + +Abstract + +TAP/hNXF1 is a key factor that mediates general cellular mRNA export from the nucleus, and its orthologs are structurally and functionally conserved from yeast to humans. Metazoans encode additional proteins that share homology and domain organization with TAP/hNXF1, suggesting their participation in mRNA metabolism; however, the precise role(s) of these proteins is not well understood. Here, we found that the human mRNA export factor hNXF2 is specifically expressed in the brain, suggesting a brain-specific role in mRNA metabolism. To address the roles of additional NXF factors, we have identified and characterized the two Nxf genes, Nxf2 and Nxf7, which together with the TAP/hNXF1's ortholog Nxf1 comprise the murine Nxf family. Both mNXF2 and mNXF7 have a domain structure typical of the NXF family. We found that mNXF2 protein is expressed during mouse brain development. Similar to TAP/hNXF1, the mNXF2 protein is found in the nucleus, the nuclear envelope and cytoplasm, and is an active mRNA export receptor. In contrast, mNXF7 localizes exclusively to cytoplasmic granules and, despite its overall conserved sequence, lacks mRNA export activity. We concluded that mNXF2 is an active mRNA export receptor similar to the prototype TAP/hNXF1, whereas mNXF7 may have a more specialized role in the cytoplasm. + +INTRODUCTION + +TAP/hNXF1 is the key factor mediating the nuclear export of mRNAs (1–3), and its orthologs in Saccharomyces cerevisiae, Drosophila melanogaster and Caenorhabditis elegans were shown to be essential for general cellular mRNA export (4–6). Metazoans encode additional NXF-like proteins, which together with the TAP/hNXF1 orthologs comprise a family of proteins termed nuclear export factors (NXFs) that are evolutionarily conserved from yeast to humans. Besides sequence homology, the NXFs share the domain architecture, and therefore are thought, by analogy to TAP/hNXF1, to participate in mRNA metabolism (7). For the human hNXF2 and hNXF3 proteins, the mRNA export activity has been confirmed by mRNA export assays (6,8). In Drosophila, while the NXF1 ortholog dmNXF1 is essential (9), additional NXF proteins were shown to be nonessential for general mRNA export in cell culture, suggesting roles that are more specialized than that of dmNXF1 (10). We previously found that the C.elegans NXF1 homolog Ce-NXF1 is essential for mRNA export (4). Although the RNAi depletion of Ce-NXF2 was not lethal (5), this protein was recently implicated in the post-transcriptional regulation of tra-2 mRNA, which is required for female development (11). A human hNXF5 nullisomy was linked to mental retardation (12). Taken together, these observations suggest that while the TAP/hNXF1 orthologs are essential for general mRNA export in metazoan species, additional NXF family members have more specialized roles. To understand these roles, we studied the family of the mouse NXF proteins. Here, we describe the isolation and characterization of two additional mouse Nxf genes, Nxf2 and Nxf7. + +MATERIALS AND METHODS + +Isolation of cDNAs encoding mNXF2 + +Gene-specific primers were designed using a short region of identity between the mouse BAC clone RP23 65A22 and human TAPX2/hNXF2 cDNA (13). 5′ and 3′ rapid amplification of cDNA ends (RACEs) were performed on mouse brain Marathon-ready cDNA (Clontech) using outward primers complementary to this identity region. The RACE products were cloned into TOPO vector (Invitrogen), and the cDNAs were sequenced by BigDye™ Terminator Sequencing Kit (AB Applied Biosystems). The mouse Nxf2 and Nxf7 genes were identified and sequenced from the mouse BAC clones RP23 65A22 and BAC441N13, respectively. + +Recombinant DNA + +Expression plasmids for the green fluorescent protein (GFP)-tagged mNXF2 or mNXF7 proteins or their deletion mutants were generated by PCR amplification of corresponding cDNAs, and subsequent insertion of PCR products into the SacII and NheI sites in plasmid pCMV-GFPsg25 (14) in-frame with GFP. GFP-βGal-tagged mNXF2 and its mutants were generated by PCR amplification, followed by insertion of SacII- and XbaI-digested PCR fragment into SacII and NheI sites in plasmid pGFP-βgal (13), respectively. Histidine-tagged mNXF2 was constructed by PCR amplification of full-length mNXF2 coding sequence, then cloned into BssHII and XhoI sites in pCMV37M1-10D (15) replacing gag. Single or multiple point mutations, using 40–80Gβgal as a template, were generated by the QuickChange XL Site-Directed Mutagenesis kit (Stratagene) according to the manufacturer's protocol. The nucleotide sequences of each mutant plasmid were confirmed by automated BigDye fluorescence sequencing. To generate glutathione S-transferase (GST)-tagged mNXF2, the coding region for amino acids 1–400 of mNXF2 was PCR amplified and subcloned into the pGEX-2T vector (Promega) for expression in Escherichia coli. Mouse p15/NXT1 expression plasmid was generated based on GenBank accession no. NM_019761, and the human p15-1 expression plasmid was obtained from E. Izaurralde. + +Cell culture, transfection and microscopy + +Human HeLa and 293 cells and mouse NIH3T3 and PA317 cells were transfected with FuGene 6 reagent (Roche) according to the manufacturer's protocol. All transfections for subcellular localization analysis were performed in 35 mm glass-bottom plates. Approximately 24 h post-transfection cells were fixed with 3.7% formaldehyde in phosphate-buffered saline. For some experiments, 20 h post-transfection, fresh media containing 30 nM leptomycin B, actinomycin D (2 μg/ml) or 5,6-dichlororibofuranosylbenzimidazole (DRB, 30 μg/ml) were added, and the cells were incubated for 4 h prior to fixation. As control for the drug-induced relocalization experiments, the same treatments were performed on cells expressing GFP-tagged HIV Rev protein. Microscopic analysis of GFP fluorescence was performed as described previously (13). Three-dimensional reconstruction of fluorescent images from confocal Z-stacks was performed using the maximum intensity projection renderer implemented in Imaris software (Bitplane). + +Protein analysis + +Chloramphenicol acetyltransferase (CAT) assays and luciferase measurements were performed as described previously (13). For western blot analyses, transfected cells were extracted in 500 μl of lysis buffer (0.5% Triton X-100 and 100 mM Tris–HCl, pH 7.4), separated on denaturing polyacrylamide gels and blotted onto nitrocellulose membrane. Polyclonal antisera against human TAPX2/hNXF2 and mouse mNXF2 were raised in rabbits using purified cellulose-binding domain (CBD) fusion protein containing amino acids 102–372 of TAPX2, or GST fusion containing full-length mNXF2 as immunogens. The TAPX2/hNXF2 antibodies were affinity purified on immobilized CBD-TAPX2 immunogen. For western blots, after probing with rabbit anti-TAPX2/hNXF2 or anti-mNXF2 antibody (1:1000) in 5% nonfat dry milk, and subsequent incubation with donkey anti-rabbit horseradish peroxidase-conjugated secondary antibody, immunoreactive proteins were visualized by enhanced chemiluminescence (ECL plus Western Blotting Detection System, Amersham) and autography. Pre-made western blots of multiple human tissues (GenoTech) and ‘mouse brain aging’ blots (RNAWAY) were quality controlled by the manufacturers to ensure equal loading and transfer efficiency. + +In vitro protein binding assays + +Reticulocyte-produced proteins were synthesized and metabolically labeled in coupled transcription/translation system (TNT T7 Coupled Reticulocyte Lysate System, Promega), using T7 promoter-containing PCR fragments as templates, and were adjusted with unprogrammed extract to equal molar concentrations. These stocks were used in the binding reactions that contained equimolar amounts of reticulocyte-produced proteins and 1–2 μg of E.coli-produced GST-tagged proteins that were immobilized on glutathione–Sepharose beads (Amersham). The binding was performed in 200 μl RBB buffer (15 mM HEPES, pH 7.9, 50 mM KCl, 0.1 mM EDTA and 0.2% Triton X-100) supplemented with 200 mM NaCl. Following incubation for 15 min at room temperature, the beads were pelleted and washed three times with binding buffer. Bound proteins were eluted by boiling in SDS–PAGE sample buffer, separated by SDS–PAGE and detected using Phosphoimager. + +Biocomputing + +Database similarity searches, multiple sequence alignments and phylogenetic analyses were performed using the standard programs of Genetics Computer Group package, with default parameters. + +Nucleotide sequence accession + +The sequences of mouse Nxf genes and cDNAs were submitted previously to GenBank under the accession numbers AY017476, AF490577 for Nxf2, and AY260550, AY266683 for Nxf7. + +RESULTS + +Identification of mouse NXF-related genes + +As a first step to study the role of the NXF family of proteins, we have identified the complete cDNAs as well as the exonic structures of the mouse Nxf2 (GenBank accession numbers AY017476 and AF490577) and Nxf7 genes (GenBank accession numbers AY260550 and AY266683). To achieve the identification of the mouse homologs of human TAP/hNXF1-related proteins, database searches for NXF-related mouse expressed sequence tags (ESTs) or cDNAs were employed, which revealed a homology in the mouse BAC clone RP23 65A22 to the human TAPX2/hNXF2 cDNA (13), a hNXF2 isoform. A full-length cDNA clone, termed Nxf2, was obtained using mouse brain cDNA library as template and the intron sequences were identified from the BAC clone. By comparison of the cDNA and genomic sequences, the exon and intron structure of the mouse Nxf2 gene was determined (Figure 1A). We found that the mouse Nxf2 gene is >17 kb in length and consists of 21 coding exons with 20 in-frame AUGs present in 11 exons. Comparison of our mNXF2 protein sequence with those published by Wang et al. (16) (GenBank accession no. NM_031259) and Jun et al. (12) (GenBank accession no. NP_112549) shows 99% identity with a single E347D amino acid change. Database searches further revealed a partial cDNA sequence of an additional Nxf-related gene on another BAC clone. By PCR amplification based on EST homology, a full-length cDNA corresponding to mouse Nxf7 was obtained and its exonic structure was determined (Figure 1A). We found that the mouse Nxf7 gene contains 22 exons, spans >14 kb (GenBank accession no. AY266683) and encodes a predicted protein of 620 amino acids. Our cDNA clone has a perfect sequence match with all the exonic sequences in the mouse Nxf7 genomic DNA. The proteins encoded by Nxf-a1 (GenBank accession no. AJ305317) and Nxf-a2 (GenBank accession no. AJ305318) have a high sequence homology to mNXF7, although both protein isoforms lack 110 amino acids at the N-terminus as compared with mNXF7. Comparison of the mNXF7 protein with NXF-a1 and NXF-a2 reveals the following changes: L354P and L361P (NXF-a2); the replacement of 121-STF-123 with two valine residues (NXF-a1 and NXF-a2); NXF-a1 is a splice variant (lacking exon 10), which results in an internal deletion of 36 amino acids (nt 271–306) and removes the sequence between L271 and L306. + +Taken together, we identified and isolated two genes termed mouse Nxf-2 and Nxf-7, whose sequences we have previously submitted to GenBank. Both genes are located on X chromosome, suggesting that they arose from a gene amplification, whereas Nxf1 is located on chromosome 19. Thus, the mouse NXF family consists of three members, whereas the human NXF family comprises five (7). Figure 1B shows a dendrogram of NXF family proteins from yeast to humans, illustrating significant homology both across and within species. On this tree, the mouse mNXF1 clusters with the other TAP/hNXF1 orthologs, from birds to humans, as expected (7). Interestingly, the ‘additional’ mouse factors mNXF2 and mNXF7 are close together and further cluster with the ‘additional’ human factors. We then performed a more detailed comparison of the mouse NXF family with TAP/hNXF1 protein, a prototype NXF factor (Figure 1C). While the mouse mNXF1 and the human TAP/hNXF1 share 90% identity, the comparison of mNXF1 to mNXF2 and mNXF7 shows reduced homologies of 47 and 49% amino acid identity, respectively. Despite this, the mNXF2 and mNXF7 proteins are predicted to share the domain organization with the human TAP/hNXF1 protein (7), including a non-canonical RNP-type RNA-binding domain (RBD), the leucine-rich repeats (LRRs), the NTF2-like domain (mediating interactions with p15/NXT1) and the ubiquitin associated-like domain (UBA-like) that is part of nucleoporin-binding region. mNXF2 also has an insertion of 5 tandem 12 amino acid imperfect repeats located at the C-terminus of its LRR domain, as previously noted (12), but its role was not further investigated. + +Expression of mNXF2 protein in the brain + +To study the mNXF2 protein expression, we generated an antiserum specific against GST-tagged mNXF2 in rabbits. Western blot analysis showed that the antiserum recognized untagged, His-tagged, GFP-tagged or GFP-βGal-tagged mNXF2 protein (Figure 2A, left panel), whereas it did not react with proteins from untransfected human 293 cells. In addition, it did not cross-react with the human TAP/hNXF1 or mouse mNXF7 proteins (Figure 2A, right panel). The untagged mNXF2 protein migrated with an apparent molecular mass of ∼75 kDa. + +It has been previously reported that the mouse NXF-related mRNAs can be detected using RT–PCR in the brain (12). Using western immunoblot analysis, we found that the mNXF2 protein can also be detected in the mouse brain at different stages of development (Figure 2B). While the mNXF2 protein could be detected in a brain sample from an 18-week mouse embryo (E18), its level of expression is slightly increased at 1 week after birth and kept constant for ∼3 months and is still detectable, although at lower levels, after the age of 6 months. We were unable to address the expression of mNXF7 protein because of the lack of specific antibodies. + +Similarly, we used a monospecific rabbit antiserum generated to human TAPX2 that represents an isoform of hNXF2 (13), to probe a human multiple tissue blot. This analysis revealed that TAPX2/hNXF2 was specifically expressed in the brain (Figure 2C), but could not be detected in other tissues examined, such as testis, where mRNA was readily detectable (data not shown). Taken together, these results confirmed that NXF-related proteins, such as the human and mouse NXF2, are expressed in the brain, suggesting the roles in brain-specific mRNA metabolism. + +Distinct subcellular localization of mNXF2 and mNXF7 + +We next studied the subcellular localization of mNXF2 and mNXF7 upon transfection of human HeLa cells with plasmids expressing GFP-tagged fusion proteins. Figure 3 shows that mNXF2 localizes to the nucleus, but is excluded from the nucleolus, accumulates at the nuclear rim and is present in the cytoplasm. Its localization is similar to that observed for the human TAP/hNXF1 (13,17,18). In contrast, mNXF7 is localized exclusively to the cytoplasm (Figure 3), where it accumulates in granules. Similar localization patterns of mNXF2 and mNXF7 were found in human 293 cells as well as in mouse NIH3T3 and PA317 cells (data not shown); thus, the distinct subcellular localization is independent of the tested cell lines. The localization of both proteins was not affected in the presence of Leptomycin B, which excludes a role of CRM1 in protein export. Also, the presence of Actinomycin D or DRB did not alter the localization of mNXF2 and mNXF7 (data not shown), indicating that their nucleocytoplasmic trafficking is transcription-independent, while it affected the localization of HIV-1 Rev as expected (19). + +Identification of the active nuclear localization signal of mNXF2 + +We next determined the nuclear localization signal (NLS) of mNXF2 using GFP-tagged deletion mutants (Figure 4A). We found that mutant 1–264 which lacks 407 amino acids from the C-terminus (Figure 4A) lost the nuclear rim association as expected, because it lacks the conserved UBA-like domain (see Figure 1C). Mutant 1–80GFP still localized to the nucleus (Figure 4A, 1–80GFP), whereas further deletion to amino acid 70 resulted in cytoplasmic accumulation of the mutant protein (Figure 4A, 1–70GFP). This suggests a NLS located between amino acid 1 and 80 at the N-terminus. + +Since GFP is a small protein, a GFP-tagged small polypeptide may localize to the nucleus due to passive diffusion. To distinguish passive diffusion from active import, we used GFP-β-galactosidase (Gβgal) fusion protein as a tag (13). The Gβgal fusion protein is localized to the cytoplasm because the fusion protein has a higher molecular mass, and neither GFP nor βgal contains an active nuclear import signal (Figure 4B). In contrast, insertion of the N-terminal 80 residues of mNXF2 (1–80Gβgal) conferred nuclear localization on the otherwise cytoplasmic GβGal, demonstrating that mNXF2 contains an active nuclear import signal. We also tested this nuclear import signal using GST–GFP (13) as a tag (data not shown). The GST–GFP fusion protein is localized to the cytoplasm because GST can form a dimer, and neither GST nor GFP contains an active nuclear import signal. Fusion of GST–GFP to amino acids 2–80 or to amino acids 40–80 of mNXF2 resulted in nuclear localization (data not shown). + +We further mapped the minimal NLS within amino acids 1–80 of mNXF2 and examined the localization of several deletion mutants (Figure 4B). For the N-terminal deletion mutants, we found that deletion of amino acids 1–39 did not alter nuclear localization of the mutant protein (40–80Gβgal), whereas further deletion to residue 49 (49–80Gβgal) resulted in cytoplasmic accumulation of the fusion protein. Deletion of 10 amino acids from the C-terminus (1–70Gβgal) also abolished nuclear localization of the protein. These results demonstrate that amino acids 40–49 and amino acids 71–80 define the N- and C-terminal boundaries of the mNXF2 NLS. Taken together, these results demonstrate that amino acids 40–80 of mNXF2 are necessary and sufficient to promote nuclear import and this peptide constitutes the minimal NLS. + +Fine mapping of the mNXF2 NLS + +Inspection of the N-terminal region (40-QGRKRGVNY-48) and the C-terminal region (71-MKRRRERCSY-80) of mNXF2 revealed stretches of basic amino acids. To further study their role in NLS function, alanine substitutions were introduced within 40–80Gβgal (Figure 4C). In mutant M4, changes of three basic amino acids in the N-terminal part of the NLS domain (R42A, K43A, R44A) led to an impairment of the NLS and significant cytoplasmic accumulation of the mutant protein. Double alanine substitutions in the C-terminal part of the NLS as in mutants M10 (K72A, R73A), M6 (R73A, R74A), and M5 (R74A, R75A) resulted in loss of NLS function, whereas mutant M3 (E76A, R77A) has a fully functional NLS. Using single alanine substitutions, we further found that the NLS function is abrogated in M11 (K72A) and M7 (R73A) or significantly affect in M9 (R75A), but the NLS function is only slightly affected in M8 (R74A). Taken together, our results demonstrate that the mNXF2 core NLS consists of two short stretches of basic amino acids (amino acids 42–44 and amino acids 72–75), which are essential for maintaining full NLS activity. Similarly, we previously found that the NLS of the human TAP/NXF1 is bipartite consisting of a C-terminal region contributing (Figure 1C, dotted line) and an N-terminal region (solid line) being essential for NLS activity (13). In both the human TAP/hNXF1 and mNXF2, the core NLS is composed of few basic residues located toward the N-terminus of the proteins. + +Association of mNXF2 and mNXF7 with nuclear envelope + +As previously shown for human TAP/hNXF1, mNXF2-GFP protein also localized to the nuclear envelope (Figures 3 and 5A). Fusion of the C-terminal portion 545–671 to GFP-βgal led to the accumulation of the mutant protein in the cytoplasm and the nuclear periphery (Figure 5A, 545–671Gβgal), whereas fusion to GFP resulted in diffusion of the protein to the nucleus and accumulation at the nuclear rim. Deletion mutant 565–671GFP still accumulated at the nuclear rim, whereas further deletion to amino acids 581 abolished rim association (581–671GFP). Rim association of the mutants 545–671GFP and 565–671GFP was not affected by digitonin treatment prior to fixation (data not shown), indicating strong interactions with the nuclear envelope. Therefore, mNXF2 contains a signal for rim association at the C-terminus, which shows conservation in location and sequence with other human NXF proteins (7) (Figure 1C). + +Interestingly, we found that although GFP-tagged full-length mNXF7 or the N-terminal deletion 95–620GFP did not show apparent nuclear rim association, the deletion of the N-terminal 263 residues led to the accumulation of the protein in the nucleus and to its association with the nuclear rim (Figure 5B, 264–620GFP). Inspection of mNXF7 protein sequence confirmed the conservation of the C-terminal portion containing the nuclear rim association determinants (Figure 1C). By analogy, we previously reported that deletion of the N-terminal NLS of TAP/hNXF1 resulted in a mutant protein, which was found to accumulate at the nuclear rim and the nucleoplasm, suggesting that the ability of NXF to associate with mobile factors of the nuclear pore complex (NPC) facilitates its nuclear import even in the absence of the active N-terminal NLS (13,17,18). We conclude that although mNXF7 protein shares the key signals with the prototype TAP/hNXF1, other regions within the protein are responsible for its distinct localization. + +Mapping of mNXF7 domain responsible for granule formation + +A key property distinguishing mNXF7 from the other members of the NXF family is its localization to cytoplasmic granules. Figure 6A shows a series of N- and C-terminal deletion mutants of mNXF7 designed to identify the domains responsible for granule formation. Removal of part of the LRR, the NTF2 and the UBA-like domains as well as the N-terminal 95 residues did not abolish the localization to granules. The minimal region necessary and sufficient for granule formation spans amino acids 95–274, and further truncation to amino acids 110–210 resulted in uniform localization of the mutant (Figure 6). The localization of some mutants was further verified using 3D rendering, an approach that allows to better distinguish the granular and homogeneous localization patterns. As shown in Figure 6B, this analysis indeed led to better visualization of the granular pattern of the wild-type mNXF7 and its mutants encompassing amino acids 95–562, 1–274 and 95–274, whereas the mutant spanning amino acids 110–210 showed mostly homogeneous localization, confirming the granule phenotypes presented in Figure 6A. We note that the granule localization determinant in mNXF7 corresponds to part of TAP/hNXF1's ‘substrate-binding domain’ through which it interacts with nuclear export cofactors and assembles with mRNP complexes (2,7,20–22). + +mNXF2, but not mNXF7, can promote mRNA export + +To test whether mNXF2 or mNXF7 play a role in mRNA export, we used the CAT reporter plasmid pDM128. Plasmid pDM128 contains CAT coding sequence and the Rev response element located within the env intron of HIV-1, flanked by a splice donor and acceptor sites (23). In 293 cells, such transcripts are retained in the nucleus and, therefore, produce only background levels of CAT enzyme, whereas coexpression of export activators, such as HIV-1 Rev or TAP/hNXF1, leads to the stimulation of CAT production. We have previously demonstrated that TAP/hNXF1 indeed enhances the nuclear export of CAT transcripts (22); therefore, CAT production directly reflects the nuclear export of this reporter mRNA. + +As a positive control, we coexpressed pDM128 with HIV-1 Rev and found a strong (∼10-fold) activation of CAT production (Figure 7A), as expected (23). We further showed that the presence of TAP/hNXF1 resulted in an ∼2-fold activation, whereas cotransfection with its cofactor, the human p15/NXT1, led to ∼10-fold activation, in agreement with previous observations (7,24), whereas the presence of p15/NXT1 alone had no effect. Using this assay, we found that cotransfection of mNXF2 alone yielded a significant ∼5-fold activation of CAT expression, demonstrating that mNXF2 is a bonafide mRNA export factor. Cotransfection with human or mouse p15/NXT1 did not significantly affect mNXF2 activity. This is in contrast to the human TAP/hNXF1, which depends on the exogenous p15/NXT1 for maximal activity in this assay. We hypothesize that the mNXF2 has a higher affinity to the NPC similar to a TAP/hNXF1 mutant having a duplication of its nucleoporin-binding sites (25) or to endogenous p15/NXT1; therefore, it can act independently of exogenous p15/NXT1. In contrast, mNXF7 failed to activate CAT production both in the absence and presence of p15/NXT1 (Figure 7A), consistent with the lack of mRNA export function of this cytoplasmic protein. This finding suggests that the mouse NXF7 protein functions at post-export steps in the mRNA metabolism. Similar results were obtained in mouse PA317 cells (data not shown). + +To understand the mechanism of mNXF2's nuclear export activity, we studied its interactions with Y14/MAGOH, the REF proteins and U2AF35, which are known to bind to TAP/hNXF1 directly and are thought to facilitate the addition of TAP/hNXF1 to its export substrates (22,26–30). The bacterially produced recombinant proteins spanning the substrate-binding domains of mNXF2 and TAP/hNXF1 (amino acids 1–400) were immobilized and used in pull-down assays together with metabolically labeled binding factors. As a negative control, we used the mRNA export factor DBP5 (31), which does not bind TAP/hNXF1 directly. Figure 7B shows that MAGOH, REF2-II and U2AF35 bound readily to both TAP/hNXF1 and mNXF2, whereas the binding of Y14 and DBP5 was low or undetectable. The observed binding of MAGOH, REF2-II and U2AF35 to TAP/hNXF1 is consistent with previous findings (22,26–30). Since the MAGOH subunit binds to TAP/hNXF1 much more avidly than Y14 (28), the lack of REF1-II and Y14 binding confirmed that only strong interactions were revealed under our assay conditions. However, we cannot exclude that the reticulocyte-produced REF1-II and/or Y14 were misfolded or lacked proper post-translational modifications, and this possibility was not further addressed. Although TAP/hNXF1 and mNXF2 proteins were used at similar amounts, the overall binding efficiency of the tested proteins to TAP/hNXF1 was higher. This effect could be attributed to a better folding of this recombinant protein, and it was not further examined. In conclusion, this study shows that mNXF2 is an mRNA export factor sharing the localization and functional properties with TAP/hNXF1. + +DISCUSSION + +In this report, we present the identification and functional characterization of the mouse Nxf family genes and their corresponding proteins. Since some mammalian NXF factors are believed to be expressed specifically in the brain, and because human hNXF5 nullisomy is linked to mental retardation (12), we wished to understand the possible roles of such proteins. We have chosen to study the Nxf gene family in mouse, because this species is widely used as a model to study human neurological disease, yet possesses only two Nxf1-related genes. + +The mouse mNXF2 and the human hNXF2 are expressed in the brain + +Previously, mRNA analysis was used as evidence of brain-specific expression for the NXF factors, such as mNXF2, mNXF7, and hNXF5. Using hNXF2 as a model, we found that this approach can lead to gross overestimation of the predicted protein expression levels, because of high abundance of aberrant, non-coding splice products in certain tissues such as testis (A. Zolotukhin, S. Lindtner, S. Smulevitch and B.K. Felber, unpublished data). We therefore sought to verify the brain-specific expression of NXF family factors at the protein level and raised antibodies to mNXF2 and its human counterpart, TAPX2/hNXF2. By using these antibodies, we show here that the expression of the human TAPX2/hNXF2 protein is restricted to the brain (Figure 2C), and that the mouse mNXF2 expression levels in the brain are developmentally regulated (Figure 2B), suggesting brain-related function(s) of these proteins. + +mNXF2 is an active mRNA export receptor, with properties similar to those of TAP/hNXF1 + +We show here that mNXF2 behaves like a typical NXF factor, since it shows mRNA export activity, localizes mostly to the nucleus and the nuclear envelope, and contains active nuclear localization and NPC-association signals that are positioned similar to those of TAP/hNXF1. We also show that mNXF2 and the human TAP/hNXF1 share the ability to interact with mRNA splicing/export cofactors, such as MAGOH, REF and U2AF35, strongly suggesting a shared nuclear export mechanism. While this manuscript was under review, another group (32) has reported that mNXF2, but not mNXF7, binds in vitro to the TAP/hNXF1 export cofactor p15, further supporting our conclusions. The few differences we observed between mNXF2 and the prototype export factor, TAP/hNXF1, include (i) a more pronounced cytoplasmic and nuclear envelope localization of mNXF2, whereas TAP/hNXF1 is mostly found in the nucleoplasm (ii) mNXF2 shows a more relaxed requirement for the cofactor p15/NXT1 in the CAT nuclear export assays used. + +We believe that these small differences do not likely account for a brain-specific role of mNXF2 that is distinct from that of TAP/hNXF1. Rather, we propose that mNXF2 functions differently from TAP/hNXF1 due to its restricted substrate specificity. In this model, mNXF2, unlike the promiscuous TAP/hNXF1, only associates with a specific subset of transcripts in the brain, leading to their regulation during development. Further work is required to characterize such transcripts and the responsible determinants within mNXF2. + +mNXF7 is atypical of NXF family + +Unexpectedly, we found that mNXF7 is a cytoplasmic protein that is completely excluded from the nucleus. However, these localization data at steady-state do not rule out that mNXF7 can enter the nucleus, especially considering the presence of a cryptic NLS (see below). Independent of its fusion to detection tags (such as GFP and HA), species and cell lines, mNXF7 forms granules in the cytoplasm and is targeted to these granules via a region that spans amino acids 95–274. Interestingly, when devoid of this region, mNXF7 assumes the subcellular localization that is typical of a generic NXF factor, revealing nuclear import and nuclear envelope localization determinants. Apparently, these determinants are overridden in the wild-type mNXF7 protein by a potent granule localization signal. However, the conservation of active, NXF-like signals within mNXF7 points to their functional importance and suggests that the role of this protein may require nuclear entry and NPC binding. In agreement with its unusual localization, mNXF7 did not show nuclear export activity in CAT assays, probably due to its absence from or short dwelling time in the nucleus. We cannot exclude though, that mNXF7 may have an export activity that is strictly specific toward a subclass of transcripts and is not revealed using the cat mRNA reporter. In summary, we propose that mNXF7 plays a highly specialized role(s) in the cytoplasm that is distinct from that of other NXFs studied so far. Based on homology to TAP/hNXF1 and shared domain organization, it is likely that mNXF7 is engaged in mRNA metabolism. At present, we do not know whether such proposed activities include the mRNA nuclear export per se, or are restricted to post-export events. Understanding the precise biological role of mNXF7 and the underlying molecular mechanisms remains a goal for future studies. + +Acknowledgements + +We thank S. Lockett, J. Brenner, E. Cho and S. Costes for help with confocal microscopy, P. Aplan, G. Dreyfuss, E. Izaurralde and F. Stutz for their generous gifts of plasmids and T. Jones for editorial assistance. We are grateful to our summer students P. Sood and A. Witten, and our Werner H. Kirsten Student Intern program recipients D. Waddelow and C. Jodrie for their contributions. Funding to pay the Open Access publication charges for this article was provided by NCI. + +Conflict of interest statement. None declared. + +Figures and Tables + +Figure 1 + +Mouse Nxf2 and Nxf7. (A) Exon–intron structure of mouse Nxf2 and Nxf7 genes. The cDNAs were isolated and the sequences and the genomic structure were determined. Exons are shown in black boxes. ATG, translational initiation codon; TAA and TGA, translation termination codons. Asterisks indicate location of in-frame ATG codons. (B) Dendrogram illustrating the NXF family of proteins. Abbrevations: rn, Rattus norvegicus; cj, Coturnix japonica; dm, Drosophila melanogaster; sp, Schizosaccharomyces pombe; sc, Saccharomyces cerevisiae. The mouse family members are shown in bold. (C) Multiple sequence alignment of human TAP/hNXF1 and mouse NXF proteins. The protein domains based on Herold et al. (7) are indicated. The identified domains mediating nuclear localization of human TAP/hNXF1 and mNXF2, localization to cytoplasmic granules of mNXF7 and the rim association of mNXF2 are underlined. RBD, RNA-binding domain; LRR, leucine-rich repeat; NTF2, nuclear transport factor (NTF2)-like domain; UBA-like, ubiquitin associated-like domain. + +Figure 2 + +Specific expression of NXF-related proteins in brain tissue. (A) Western immunoblot analysis of 293 cells transfected with untagged and tagged (HIS, GFP and Gβgal) mNXF2 (left panel) and different NXF expression plasmids (right panel) using a rabbit anti-mNXF2 antiserum. (B) Western blot analysis of mouse brain tissue using monospecific rabbit anti-mNXF2 antiserum. Pre-made ‘mouse brain aging’ blots (RNAWAY) contained normalized amounts of whole brain lysates from fetus to 1-year-old as indicated. (C) Western blot analysis of human tissues using the monospecific antiserum to human TAPX2/hNXF2. Pre-made blots (GenoTech) contained normalized amounts of proteins from the indicated tissues. + +Figure 3 + +Distinct subcellular localization of mNXF2 and mNXF7 proteins. HeLa cells were transfected with GFP-tagged mNXF2 and mNXF7 expression plasmids, as indicated and the proteins were visualized in living cells. The images were obtained by fluorescent microscopy (Axiovert135TV, Zeiss) and by the use of a CCD camera and processed using IPLab Spectrum software (13). Similar results were obtained in numerous experiments and are typical of the vast majority of expressing cells in each individual experiment. Representative cells are shown. + +Figure 4 + +Identification of NLS of mNXF2. (A and B) HeLa cells were transfected with plasmids producing mNXF2 and N- and C-terminal deletions fused to GFP (A) or GFP-βgal (B) and the proteins were visualized as described in Figure 2. The localization of the proteins is indicated. N, nucleus; C, cytoplasm. (C) Fine mapping of the NLS. Alanine substitution in 40–80Gβgal generated a series of mutants and the mutant proteins are visualized. The protein sequence containing the NLS regions is shown and the critical residues are indicated in bold. + +Figure 5 + +Association of mNXF2 and mNXF7 with nuclear envelope. HeLa cells were transfected with plasmids producing the indicated GFP-tagged mNXF2 and mNXF7 deletion mutants and the images were analyzed as described in Figure 2. + +Figure 6 + +Identification of signal mediating granule localization of mNXF7. (A) A panel of N- and C-terminal deletion mutants of GFP-tagged mNXF7 deletion mutants was analyzed upon transfection of HeLa cells. N, nucleus; C, cytoplasm. Confocal images of GFP fluorescence in the mid-sections through the nuclei. (B) Three-dimensional rendering of GFP images from confocal Z-stacks. + +Figure 7 + +mNXF2 is an active mRNA export factor. (A) Human 293 cells were transfected with the cat reporter pDM128 either alone, in the presence of HIV-1 rev expression plasmid or in the presence of the indicated untagged NXF producing plasmids. As indicated, the transfection mixtures contained NXF expression plasmids together with human p15/NXT1 or p15/NXT1 alone. Two days later, the cells were harvested and the CAT production, measured as percentage of chloramphenicol acetylation, is shown by filled bars. Expression of the cotransfected luciferase expression plasmid was analyzed for each plate and the relative luciferase values are shown in open bars. A typical experiment is shown. (B) mNXF2 binds to TAP/hNXF1 export cofactors. Bacterially produced GST-tagged NXF proteins were immobilized on glutathione–Sepharose beads and used in pull-down assays with reticulocyte-produced, metabolically labeled factors (shown to the left). The bound (B) and 1:100 aliquots of the unbound (U) fractions were separated on SDS–PAGE and visualized on Phosphoimager. diff --git a/src/ontogpt/evaluation/craft/database/all/16098226.ann b/src/ontogpt/evaluation/craft/database/all/16098226.ann new file mode 100644 index 000000000..d67332d51 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16098226.ann @@ -0,0 +1,730 @@ +T1 PR:000012318 41 45 PAX6 +T2 PR:000008680 93 99 HOMER3 +T3 PR:000002325 101 106 DNCL1 +T4 PR:000016642 111 117 TRIM11 +T5 PR:000012318 145 149 PAX6 +T6 UBERON:0000970 208 214 ocular +T7 GO:0001654 208 214;232 243 ocular ... development +T8 UBERON:0001016 219 231 neurological +T9 GO:0007399 219 243 neurological development +T10 NCBITaxon:1 245 256 Individuals +T11 PR:000012318 309 313 PAX6 +T12 SO:0000704 314 318 gene +T13 http://purl.obolibrary.org/obo/MONDO_0016054 324 340;353 358 malformations of brain +T14 UBERON:0000970 345 348 eye +T15 UBERON:0000955 353 358 brain +T16 PR:000012318 402 406 PAX6 +T17 PR:000012318 498 502 PAX6 +T18 PR:000012318 616 620 PAX6 +T19 UBERON:0000955 697 702 brain +T20 GO:0010467 703 712 expressed +T21 PR:000012318 784 788 PAX6 +T22 SO:0000417 819 825 domain +T23 PR:000012318 839 843 PAX6 +T24 GO:0014069 886 907 post-synaptic density +T25 GO:0014069 909 912 PSD +T26 PR:000008680 922 928 HOMER3 +T27 GO:0030286 934 940 dynein +T28 PR:000002325 949 954 DNCL1 +T29 PR:000016642 989 995 TRIM11 +T30 PR:000012318 1014 1018 PAX6 +T31 UBERON:0000970 1069 1072 eye +T32 PR:000012318 1178 1182 PAX6 +T33 PR:000008680 1198 1204 HOMER3 +T34 PR:000002325 1206 1211 DNCL1 +T35 PR:000016642 1216 1222 TRIM11 +T36 PR:000012318 1259 1263 PAX6 +T37 PR:000008680 1269 1275 HOMER3 +T38 PR:000002325 1280 1285 DNCL1 +T39 GO:0045202 1310 1318 synaptic +T40 CL:0000540 1355 1363 neuronal +T41 PR:000012318 1445 1449 PAX6 +T42 PR:000012318 1538 1542 PAX6 +T43 PR:000037797 1570 1573 PAX +T44 PR:000037797 1575 1585 paired-box +T45 UBERON:0000970 1652 1658 ocular +T46 GO:0001654 1652 1658;1670 1681 ocular ... development +T47 GO:0007399 1663 1681 neural development +T48 NCBITaxon:9606 1717 1722 human +T49 PR:000012318 1723 1727 PAX6 +T50 SO:0000704 1728 1732 gene +T51 http://purl.obolibrary.org/obo/MONDO_0019172 1739 1747 aniridia +T52 http://purl.obolibrary.org/obo/MONDO_0019172 1749 1759;1764 1768 absence of iris +T53 UBERON:0001769 1764 1768 iris +T54 http://purl.obolibrary.org/obo/MONDO_0021140 1791 1801 congenital +T55 UBERON:0000970 1802 1805 eye +T56 UBERON:0001786 1848 1854 foveal +T57 http://purl.obolibrary.org/obo/MONDO_0044203 1848 1865 foveal hypoplasia +T58 UBERON:0000970 1870 1875 optic +T59 PR:000012318 1907 1911 PAX6 +T60 UBERON:0000970 1923 1926 eye +T61 http://purl.obolibrary.org/obo/MONDO_0005328 1923 1934 eye disease +T62 NCBITaxon:9606 1966 1969 man +T63 NCBITaxon:10088 1974 1979 mouse +T64 GO:0007608 2088 2097 olfactory +T65 UBERON:0000955 2119 2124 brain +T66 http://purl.obolibrary.org/obo/MONDO_0016054 2119 2138 brain malformations +T67 PR:000012318 2159 2163 PAX6 +T68 UBERON:0000955 2167 2172 brain +T69 GO:0007420 2167 2184 brain development +T70 NCBITaxon:10088 2231 2235 mice +T71 NCBITaxon:10114 2239 2243 rats +T72 UBERON:0000956 2276 2291 cerebral cortex +T73 GO:0021905 2297 2309;2314 2351 formation of ... prosencephalon-mesencephalon boundary +T74 UBERON:0001890 2314 2328 prosencephalon +T75 UBERON:0001891 2329 2342 mesencephalon +T76 GO:0030424 2357 2361 axon +T77 GO:0007411 2357 2370 axon guidance +T78 CL:0000540 2395 2402 neurons +T79 CL:0000125 2408 2412 glia +T80 CL:0000540 2421 2429 neuronal +T81 GO:0001764 2421 2439 neuronal migration +T82 UBERON:0002037 2447 2457 cerebellum +T83 PR:000012318 2513 2517 PAX6 +T84 UBERON:0000955 2521 2526 brain +T85 GO:0007420 2521 2538 brain development +T86 http://purl.obolibrary.org/obo/MONDO_0019172 2564 2572 aniridia +T87 UBERON:0000955 2610 2615 brain +T88 UBERON:0000935 2713 2732 anterior commissure +T89 UBERON:0011768 2791 2803 pineal gland +T90 UBERON:0001851 2805 2813 cortical +T91 http://purl.obolibrary.org/obo/MONDO_0000087 2814 2828 polymicrogyria +T92 UBERON:0002316 2830 2842 white matter +T93 UBERON:0002336 2858 2873 corpus callosum +T94 UBERON:0002020 2878 2889 grey matter +T95 UBERON:0002037 2905 2915 cerebellum +T96 GO:0007605 2993 3001 auditory +T97 PR:000037797 3049 3052 PAX +T98 SO:0000417 3117 3123 domain +T99 SO:0000417 3201 3207 domain +T100 PR:000012318 3209 3213 PAX6 +T101 SO:0000417 3301 3307 domain +T102 SO:0000417 3341 3347 domain +T103 PR:000012318 3401 3405 PAX6 +T104 PR:000012318 3469 3473 PAX6 +T105 GO:0065007 3491 3500 regulates +T106 SO:0000704 3524 3529 genes +T107 PR:000012315 3546 3550 Pax2 +T108 PR:000011157 3557 3561 Ngn2 +T109 PR:000012318 3591 3595 Pax6 +T110 SO:0000704 3596 3600 gene +T111 GO:0010467 3640 3650 expression +T112 UBERON:0000970 3666 3669 eye +T113 UBERON:0000955 3671 3676 brain +T114 UBERON:0000004 3678 3683 nasal +T115 UBERON:0002240 3696 3707 spinal cord +T116 UBERON:0001264 3712 3720 pancreas +T117 PR:000012318 3735 3739 PAX6 +T118 PR:000012318 3851 3855 PAX6 +T119 UBERON:0003714 3859 3873 neural tissues +T120 PR:000012318 3888 3892 Pax6 +T121 GO:0010467 3893 3903 expression +T122 UBERON:0000966 3968 3974 retina +T123 UBERON:0001950 3988 3997 neocortex +T124 PR:000012318 4003 4007 PAX6 +T125 GO:0030424 4048 4054 axonal +T126 UBERON:0000966 4079 4085 retina +T127 UBERON:0000955 4094 4099 brain +T128 UBERON:0001890 4120 4129 forebrain +T129 CL:0002319 4184 4195 neural cell +T130 UBERON:0000966 4237 4243 retina +T131 UBERON:0000956 4257 4272 cerebral cortex +T132 SO:0000704 4304 4309 genes +T133 PR:000011157 4318 4322 Ngn2 +T134 PR:000004362 4327 4332 Mash1 +T135 PR:000012318 4377 4381 PAX6 +T136 UBERON:0000966 4398 4404 retina +T137 UBERON:0000955 4413 4418 brain +T138 UBERON:0001016 4459 4471 neurological +T139 NCBITaxon:1 4486 4497 individuals +T140 PR:000012318 4503 4507 PAX6 +T141 GO:0065007 4741 4751 regulatory +T142 PR:000012318 4813 4817 PAX6 +T143 PR:000015426 4873 4877 SOX2 +T144 SO:0000165 4899 4907 enhancer +T145 SO:0000704 4936 4940 gene +T146 GO:0065007 4964 4973 modulates +T147 PR:000012318 4974 4978 PAX6 +T148 CL:0000540 4997 5005 neuronal +T149 SO:0000167 5062 5070 promoter +T150 GO:0010467 5095 5108;5133 5137 expression of ... gene +T151 UBERON:0001264 5114 5124 pancreatic +T152 SO:0000704 5133 5137 gene +T153 PR:000012318 5247 5251 PAX6 +T154 SO:0000417 5351 5357 domain +T155 PR:000012318 5405 5409 PAX6 +T156 UBERON:0000955 5438 5443 brain +T157 PR:000008680 5556 5562 HOMER3 +T158 PR:000002325 5564 5569 DNCL1 +T159 PR:000016642 5574 5580 TRIM11 +T160 PR:000012318 5606 5610 PAX6 +T161 PR:000012318 5678 5682 PAX6 +T162 PR:000012318 5734 5738 PAX6 +T163 PR:000012318 5853 5857 PAX6 +T164 PR:000012318 5916 5920 PAX6 +T165 SO:0000417 5925 5931 domain +T166 NCBITaxon:species 6105 6112 species +T167 NCBITaxon:8296 6121 6128 axolotl +T168 NCBITaxon:8296 6130 6149 Ambystoma mexicanum +T169 NCBITaxon:7625 6155 6165 sea urchin +T170 NCBITaxon:7656 6167 6188 Paracentrotus lividus +T171 PR:000012318 6238 6242 PAX6 +T172 PR:000012318 6319 6323 PAX6 +T173 NCBITaxon:species 6337 6344 species +T174 PR:000012318 6731 6735 PAX6 +T175 SO:0000417 6740 6746 domain +T176 SO:0000993 6888 6897 consensus +T177 SO:0000417 6946 6952 domain +T178 SO:0000417 7109 7115 domain +T179 SO:0000417 7254 7260 domain +T180 SO:0000319 7305 7315 stop codon +T181 PR:000012318 7924 7928 PAX6 +T182 SO:0000319 7964 7974 stop codon +T183 PR:P04386 8301 8305 GAL4 +T184 SO:0000417 8318 8324 domain +T185 NCBITaxon:10088 8347 8352 mouse +T186 UBERON:0000955 8353 8358 brain +T187 NCBITaxon:9606 8378 8383 human +T188 PR:000012318 8461 8465 PAX6 +T189 NCBITaxon:9606 8490 8493 man +T190 NCBITaxon:10088 8498 8503 mouse +T191 NCBITaxon:10088 8524 8529 mouse +T192 UBERON:0000955 8530 8535 brain +T193 SO:0000417 8667 8673 domain +T194 SO:0000417 8789 8795 domain +T195 SO:0000155 8837 8845 plasmids +T196 SO:0000667 8873 8880 inserts +T197 PR:000008680 8937 8943 Homer3 +T198 PR:000002325 8957 8962 Dncl1 +T199 GO:0030286 8964 8970 Dynein +T200 PR:000002325 8964 8996 Dynein cytoplasmic light chain 1 +T201 GO:0005737 8971 8982 cytoplasmic +T202 PR:000002325 9023 9026 Pin +T203 PR:000002325 9030 9034 Dlc8 +T204 PR:000016642 9040 9046 Trim11 +T205 PR:000016642 9048 9089 Tripartite motif protein family member 11 +T206 PR:000008680 9103 9109 Homer3 +T207 PR:000002325 9125 9130 Dncl1 +T208 PR:000008680 9192 9198 Homer3 +T209 PR:000002325 9211 9216 Dncl1 +T210 PR:000016642 9232 9238 Trim11 +T211 SO:0000417 9277 9283 domain +T212 SO:0000667 9301 9308 inserts +T213 SO:0001817 9314 9322 in-frame +T214 PR:P04386 9359 9363 GAL4 +T215 SO:0000417 9375 9381 domain +T216 PR:000008680 9455 9461 HOMER3 +T217 CL:0000540 9497 9505 neuronal +T218 GO:0014069 9506 9527 post-synaptic density +T219 GO:0014069 9529 9532 PSD +T220 PR:000002325 9549 9554 DNCL1 +T221 GO:0043234 9581 9598 protein complexes +T222 GO:0030286 9600 9606 dynein +T223 PR:000010865 9611 9620 myosin-Va +T224 GO:0005622 9652 9665 intracellular +T225 GO:0006886 9652 9689 intracellular trafficking of proteins +T226 GO:0051656 9666 9680;9694 9704 trafficking of ... organelles +T227 GO:0043226 9694 9704 organelles +T228 CL:0000540 9708 9715 neurons +T229 PR:000016642 9725 9731 TRIM11 +T230 SO:0001080 9834 9845 coiled coil +T231 SO:0000417 9846 9852 domain +T232 SO:0000417 9865 9871 domain +T233 PR:000012318 9951 9955 PAX6 +T234 PR:000008680 10018 10024 Homer3 +T235 PR:000002325 10026 10031 Dncl1 +T236 PR:000016642 10036 10042 Trim11 +T237 PR:000008680 10223 10229 Homer3 +T238 PR:000002325 10231 10236 Dncl1 +T239 PR:000016642 10238 10244 Trim11 +T240 PR:000012318 10249 10253 Pax6 +T241 PR:000004453 10269 10275 Atp5a1 +T242 PR:000004453 10287 10293 Atp5a1 +T243 GO:0010467 10324 10334 expression +T244 UBERON:0000479 10351 10358 tissues +T245 UBERON:0000955 10373 10378 brain +T246 PR:000008680 10388 10394 Homer3 +T247 PR:000002325 10396 10401 Dncl1 +T248 PR:000016642 10403 10409 Trim11 +T249 PR:000012318 10414 10418 Pax6 +T250 PR:000004453 10593 10599 Atp5a1 +T251 PR:000008680 10693 10699 Homer3 +T252 PR:000002325 10701 10706 Dncl1 +T253 PR:000016642 10708 10714 Trim11 +T254 PR:000012318 10719 10723 Pax6 +T255 NCBITaxon:10088 10731 10736 mouse +T256 UBERON:0000955 10737 10742 brain +T257 SO:0000112 10789 10796 primers +T258 PR:000012318 10810 10814 Pax6 +T259 SO:0000028 10820 10822 bp +T260 PR:000008680 10825 10831 Homer3 +T261 SO:0000028 10837 10839 bp +T262 PR:000002325 10847 10852 Dncl1 +T263 SO:0000028 10858 10860 bp +T264 PR:000016642 10866 10872 Trim11 +T265 SO:0000028 10878 10880 bp +T266 PR:000004453 10911 10917 Atp5a1 +T267 SO:0000028 10923 10925 bp +T268 SO:0000028 10942 10944 bp +T269 GO:0010467 10964 10973 expressed +T270 UBERON:0000955 10981 10986 brain +T271 NCBITaxon:10847 11033 11038 Φ×174 +T272 SO:0000028 11088 11090 bp +T273 SO:0000028 11099 11101 bp +T274 PR:000008680 11149 11155 Homer3 +T275 PR:000002325 11157 11162 Dncl1 +T276 PR:000016642 11167 11173 Trim11 +T277 SO:0000417 11367 11373 domain +T278 PR:000012318 11377 11381 PAX6 +T279 PR:000008680 11473 11479 HOMER3 +T280 PR:000002325 11484 11489 DNCL1 +T281 PR:000008680 11553 11559 HOMER3 +T282 PR:000002325 11561 11566 DNCL1 +T283 PR:000016642 11571 11577 TRIM11 +T284 SO:0000417 11612 11618 domain +T285 PR:000008680 11639 11645 HOMER3 +T286 PR:000002325 11650 11655 DNCL1 +T287 PR:000016642 11712 11718 TRIM11 +T288 SO:0000417 11768 11774 domain +T289 PR:000012318 11944 11948 PAX6 +T290 PR:000008680 11953 11959 HOMER3 +T291 PR:000002325 11961 11966 DNCL1 +T292 PR:000016642 11971 11977 TRIM11 +T293 PR:000002325 11983 11988 Dncl1 +T294 PR:000016642 11993 11999 Trim11 +T295 PR:000008680 12069 12075 Homer3 +T296 PR:000008680 12178 12184 Homer3 +T297 GO:0010467 12212 12222 expression +T298 PR:000008680 12327 12333 Homer3 +T299 PR:000012318 12371 12375 PAX6 +T300 SO:0000417 12380 12386 domain +T301 PR:000008680 12402 12408 HOMER3 +T302 PR:000016642 12449 12455 TRIM11 +T303 PR:000002325 12460 12465 DNCL1 +T304 SO:0000704 12489 12494 genes +T305 PR:000008680 12621 12627 HOMER3 +T306 PR:000002325 12631 12636 DNCL1 +T307 CHEBI:74498 12756 12776 5-fluoro-orotic acid +T308 PR:P03962 12830 12834 URA3 +T309 PR:P06633 12853 12857 HIS3 +T310 PR:000033987 12862 12866 LacZ +T311 PR:000008680 13138 13144 HOMER3 +T312 PR:000002325 13149 13154 DNCL1 +T313 PR:000016642 13290 13296 TRIM11 +T314 PR:000016642 13423 13429 TRIM11 +T315 SO:0000417 13456 13462 domain +T316 PR:000012318 13594 13598 PAX6 +T317 PR:000016642 13603 13609 TRIM11 +T318 PR:000033987 13622 13626 LacZ +T319 SO:0000704 13636 13640 gene +T320 PR:000012318 13781 13785 PAX6 +T321 SO:0000417 13790 13796 domain +T322 PR:000012318 13813 13817 PAX6 +T323 SO:0000417 13822 13828 domain +T324 PR:000012318 13869 13873 PAX6 +T325 SO:0000417 13878 13884 domain +T326 PR:000012318 13929 13933 PAX6 +T327 SO:0000417 13938 13944 domain +T328 PR:000012318 13987 13991 PAX6 +T329 SO:0000417 13996 14002 domain +T330 PR:000008680 14044 14050 HOMER3 +T331 PR:000008680 14055 14061 HOMER3 +T332 PR:000012318 14280 14284 PAX6 +T333 PR:000008680 14300 14306 HOMER3 +T334 PR:000002325 14308 14313 DNCL1 +T335 PR:000016642 14318 14324 TRIM11 +T336 PR:000012318 14509 14513 PAX6 +T337 SO:0000417 14518 14524 domain +T338 PR:000012318 14535 14539 PAX6 +T339 PR:000012318 14572 14576 PAX6 +T340 SO:0000417 14581 14587 domain +T341 PR:000012318 14633 14637 PAX6 +T342 SO:0000417 14642 14648 domain +T343 PR:000012318 14689 14693 PAX6 +T344 SO:0000417 14698 14704 domain +T345 PR:000012318 14747 14751 PAX6 +T346 SO:0000417 14756 14762 domain +T347 PR:000008680 14793 14799 HOMER3 +T348 PR:000008680 14804 14810 HOMER3 +T349 PR:000008680 14830 14836 HOMER3 +T350 PR:000008680 14851 14857 HOMER3 +T351 PR:000008680 14926 14932 HOMER3 +T352 PR:000002325 14934 14939 DNCL1 +T353 PR:000016642 14944 14950 TRIM11 +T354 PR:000012318 14969 14973 PAX6 +T355 SO:0000417 14978 14984 domain +T356 PR:000012318 15062 15066 PAX6 +T357 UBERON:0000970 15130 15136 ocular +T358 SO:0000360 15283 15288 codon +T359 PR:000012318 15292 15296 PAX6 +T360 http://purl.obolibrary.org/obo/MONDO_0019503 15380 15407 anterior segment dysgenesis +T361 UBERON:0001768 15413 15418 uveal +T362 http://purl.obolibrary.org/obo/MONDO_0002043 15419 15428 ectropion +T363 http://purl.obolibrary.org/obo/MONDO_0019172 15450 15458 aniridia +T364 UBERON:0001786 15463 15469 foveal +T365 http://purl.obolibrary.org/obo/MONDO_0044203 15463 15480 foveal hypoplasia +T366 http://purl.obolibrary.org/obo/MONDO_0019172 15539 15547 aniridia +T367 PR:000012318 15613 15617 PAX6 +T368 SO:0000319 15618 15628 stop codon +T369 GO:0006451 15655 15681 translational read-through +T370 SO:0000205 15691 15713 3' untranslated region +T371 GO:0006412 15696 15706 translated +T372 NCBITaxon:1 15847 15858 individuals +T373 GO:0050890 15939 15948 cognition +T374 UBERON:0002020 16004 16015 grey matter +T375 UBERON:0016525 16037 16049 frontal lobe +T376 UBERON:0001871 16051 16064 temporal lobe +T377 UBERON:0002037 16069 16079 cerebellum +T378 UBERON:0002316 16085 16097 white matter +T379 UBERON:0002336 16114 16129 corpus callosum +T380 http://purl.obolibrary.org/obo/MONDO_0019172 16202 16210 aniridia +T381 SO:0000319 16299 16309 stop codon +T382 SO:0000360 16329 16334 codon +T383 PR:000012318 16369 16373 PAX6 +T384 SO:0000205 16461 16483 3' untranslated region +T385 GO:0006412 16466 16476 translated +T386 UBERON:0000955 16551 16556 brain +T387 UBERON:0000935 16604 16623 anterior commissure +T388 UBERON:0001905 16625 16637 pineal gland +T389 GO:0007608 16642 16651 olfactory +T390 UBERON:0002264 16642 16657 olfactory bulbs +T391 http://purl.obolibrary.org/obo/MONDO_0000087 16690 16704 polymicrogyria +T392 http://purl.obolibrary.org/obo/MONDO_0005027 16730 16738 epilepsy +T393 PR:000008680 16905 16911 HOMER3 +T394 PR:000002325 16913 16918 DNCL1 +T395 PR:000016642 16923 16929 TRIM11 +T396 PR:000008680 17067 17073 HOMER3 +T397 PR:000002325 17078 17083 DNCL1 +T398 PR:000008680 17229 17235 HOMER3 +T399 PR:000002325 17240 17245 DNCL1 +T400 PR:000016642 17326 17332 TRIM11 +T401 PR:000016642 17384 17390 TRIM11 +T402 SO:0000417 17440 17446 domain +T403 PR:000012318 17566 17570 PAX6 +T404 SO:0000417 17579 17585 domain +T405 SO:0000417 17724 17730 domain +T406 PR:000008680 17783 17789 HOMER3 +T407 PR:000002325 17791 17796 DNCL1 +T408 PR:000016642 17801 17807 TRIM11 +T409 PR:000008680 17829 17835 HOMER3 +T410 PR:000002325 17840 17845 DNCL1 +T411 SO:0000417 17897 17903 domain +T412 PR:000016642 17910 17916 TRIM11 +T413 SO:0000417 17946 17952 domain +T414 PR:000008680 17970 17976 HOMER3 +T415 PR:000002325 17981 17986 DNCL1 +T416 PR:000016642 18043 18049 TRIM11 +T417 SO:0000417 18099 18105 domain +T418 SO:0000417 18140 18146 domain +T419 PR:000008680 18151 18157 HOMER3 +T420 PR:000002325 18159 18164 DNCL1 +T421 PR:000016642 18169 18175 TRIM11 +T422 PR:000008680 18262 18268 HOMER3 +T423 PR:000002325 18272 18277 DNCL1 +T424 PR:000012318 18395 18399 PAX6 +T425 PR:000008680 18400 18406 HOMER3 +T426 PR:000012318 18410 18414 PAX6 +T427 PR:000002325 18415 18420 DNCL1 +T428 PR:000008680 18441 18447 HOMER3 +T429 PR:000002325 18452 18457 DNCL1 +T430 SO:0000417 18486 18492 domain +T431 PR:000008680 18536 18542 HOMER3 +T432 GO:0014069 18559 18562 PSD +T433 CL:0000540 18566 18573 neurons +T434 CHEBI:29108 18704 18708 Ca2+ +T435 GO:0005622 18714 18727 intracellular +T436 GO:0031982 18728 18736 vesicles +T437 PR:000008680 18746 18752 HOMER3 +T438 GO:0010467 18821 18830 expressed +T439 UBERON:0000955 18838 18843 brain +T440 GO:0098794 18863 18876 post-synaptic +T441 GO:0014069 18969 18972 PSD +T442 GO:0030424 19050 19054 axon +T443 GO:0007411 19050 19063 axon guidance +T444 UBERON:0000955 19071 19076 brain +T445 GO:0007420 19071 19088 brain development +T446 PR:000002325 19096 19101 DNCL1 +T447 GO:0005622 19122 19135 intracellular +T448 GO:0046907 19122 19145 intracellular transport +T449 GO:1990351 19136 19163 transport protein complexes +T450 GO:0030286 19165 19171 dynein +T451 PR:000010865 19176 19185 myosin Va +T452 GO:0030286 19192 19198 Dynein +T453 PR:000010865 19203 19212 myosin Va +T454 GO:0005874 19233 19244 microtubule +T455 GO:0007018 19233 19250;19267 19275 microtubule-based ... movement +T456 GO:0015031 19267 19275;19289 19300 movement ... of proteins +T457 GO:0051650 19267 19275;19289 19291;19317 19325 movement ... of ... vesicles +T458 GO:0043226 19302 19312 organelles +T459 GO:0031982 19317 19325 vesicles +T460 CL:0000540 19329 19336 neurons +T461 PR:000010865 19346 19355 Myosin Va +T462 GO:0014069 19375 19378 PSD +T463 PR:000002325 19389 19394 DNCL1 +T464 GO:0014069 19417 19420 PSD +T465 PR:000006516 19440 19482 guanylate kinase domain-associated protein +T466 SO:0000417 19457 19463 domain +T467 CL:0000540 19492 19500 neuronal +T468 PR:000011326 19492 19522 neuronal nitric oxide synthase +T469 CHEBI:16480 19501 19513 nitric oxide +T470 PR:000016642 19530 19536 TRIM11 +T471 NCBITaxon:10088 19556 19561 mouse +T472 SO:0001080 19749 19760 coiled coil +T473 SO:0000417 19761 19767 domain +T474 SO:0000417 19788 19794 domain +T475 PR:000016642 19849 19855 TRIM11 +T476 PR:000008649 19871 19878 Humanin +T477 http://purl.obolibrary.org/obo/MONDO_0004975 19940 19959 Alzheimer's disease +T478 PR:000016642 19966 19972 TRIM11 +T479 PR:000008649 19980 19987 Humanin +T480 GO:0043161 20034 20075 ubiqutin-mediated proteasomal degradation +T481 GO:0000502 20052 20063 proteasomal +T482 PR:000012318 20269 20273 PAX6 +T483 PR:000008680 20279 20285 HOMER3 +T484 PR:000002325 20290 20295 DNCL1 +T485 GO:0045202 20337 20345 synaptic +T486 GO:0099536 20337 20356 synaptic signalling +T487 SO:0000704 20375 20379 gene +T488 GO:0010467 20375 20390 gene expression +T489 PR:000012318 20408 20412 PAX6 +T490 GO:0051235 20416 20427 sequestered +T491 GO:0014069 20435 20438 PSD +T492 PR:000008680 20453 20459 HOMER3 +T493 PR:000012318 20531 20535 PAX6 +T494 GO:0045202 20567 20575 synaptic +T495 PR:000002325 20615 20620 DNCL1 +T496 PR:000012318 20626 20630 PAX6 +T497 PR:000002325 20631 20636 DNCL1 +T498 GO:0032991 20637 20644 complex +T499 PR:000010865 20671 20680 myosin Va +T500 GO:0014069 20710 20713 PSD +T501 GO:0015629 20725 20743 actin cytoskeleton +T502 GO:0030286 20756 20762 dynein +T503 GO:0005874 20792 20803 microtubule +T504 GO:0005634 20837 20844 nucleus +T505 PR:000016642 20857 20863 TRIM11 +T506 GO:0030163 20881 20900 protein degradation +T507 PR:000012318 20929 20933 PAX6 +T508 GO:0014069 21014 21035 post-synaptic density +T509 PR:000002089 21044 21049 STAT3 +T510 CHEBI:33280 21073 21083 messengers +T511 GO:0045202 21096 21103 synapse +T512 GO:0005634 21112 21119 nucleus +T513 PR:000012318 21154 21158 PAX6 +T514 GO:0005634 21184 21191 nuclear +T515 NCBITaxon:10088 21219 21224 mouse +T516 NCBITaxon:6231 21236 21244 nematode +T517 SO:0001060 21252 21259 isoform +T518 SO:0000417 21282 21288 domain +T519 GO:0005634 21298 21305 nuclear +T520 GO:0005737 21310 21321 cytoplasmic +T521 NCBITaxon:10088 21379 21383 mice +T522 SO:0001060 21400 21407 isoform +T523 UBERON:0000955 21434 21439 brain +T524 CL:0000540 21486 21494 neurones +T525 GO:0014069 21543 21546 PSD +T526 PR:000008680 21654 21660 HOMER3 +T527 GO:0014069 21713 21716 PSD +T528 GO:0005737 21725 21734 cytoplasm +T529 PR:000002325 21741 21746 DNCL1 +T530 GO:0005737 21758 21769 cytoplasmic +T531 GO:0005634 21780 21787 nuclear +T532 PR:000016642 21829 21835 TRIM11 +T533 GO:0005634 21844 21851 nuclear +T534 GO:0005737 21856 21867 cytoplasmic +T535 GO:0005737 21884 21895 cytoplasmic +T536 PR:000012318 21896 21900 PAX6 +T537 GO:0005634 21959 21966 nuclear +T538 PR:000012318 21967 21971 PAX6 +T539 PR:000016642 21992 21998 TRIM11 +T540 PR:000002325 22003 22008 DNCL1 +T541 UBERON:0000479 22017 22023 tissue +T542 PR:000008680 22031 22037 HOMER3 +T543 GO:0010467 22038 22048 expression +T544 UBERON:0002370 22070 22076 thymus +T545 UBERON:0002048 22081 22085 lung +T546 UBERON:0000955 22120 22125 brain +T547 UBERON:0001890 22151 22160 forebrain +T548 UBERON:0002037 22178 22188 cerebellum +T549 PR:000002325 22195 22200 DNCL1 +T550 PR:000016642 22205 22211 TRIM11 +T551 GO:0010467 22227 22237 expression +T552 UBERON:0000955 22263 22268 brain +T553 GO:0010467 22287 22297 expression +T554 PR:000012318 22337 22341 PAX6 +T555 UBERON:0000479 22349 22355 tissue +T556 GO:0010467 22389 22399 expression +T557 PR:000012318 22403 22407 PAX6 +T558 PR:000008680 22409 22415 HOMER3 +T559 PR:000002325 22417 22422 DNCL1 +T560 PR:000016642 22427 22433 TRIM11 +T561 NCBITaxon:9606 22447 22452 human +T562 UBERON:0007023 22453 22458 adult +T563 UBERON:0000955 22459 22464 brain +T564 SO:0000417 22569 22575 domain +T565 PR:000008680 22580 22586 HOMER3 +T566 PR:000002325 22590 22595 DNCL1 +T567 PR:000012318 22672 22676 PAX6 +T568 PR:000012318 22804 22808 PAX6 +T569 PR:000008680 22853 22859 HOMER3 +T570 PR:000002325 22864 22869 DNCL1 +T571 PR:000008680 22967 22973 HOMER3 +T572 PR:000002325 22978 22983 DNCL1 +T573 GO:0006412 23055 23066 translation +T574 SO:0000205 23076 23098 3' untranslated region +T575 GO:0006412 23081 23091 translated +T576 PR:000016642 23383 23389 TRIM11 +T577 SO:0000417 23481 23487 domain +T578 PR:000016642 23542 23548 TRIM11 +T579 SO:0000417 23623 23629 domain +T580 http://purl.obolibrary.org/obo/MONDO_0019172 23688 23696 aniridia +T581 SO:0001587 23754 23781 premature termination codon +T582 PR:000012318 23791 23795 PAX6 +T583 SO:0000236 23796 23814 open reading frame +T584 SO:0001023 23829 23836 alleles +T585 GO:0000184 23938 23961 nonsense-mediated decay +T586 UBERON:0000955 23988 23993 brain +T587 http://purl.obolibrary.org/obo/MONDO_0019172 24031 24039 aniridia +T588 PR:000012318 24105 24109 PAX6 +T589 PR:000008680 24114 24120 HOMER3 +T590 PR:000002325 24122 24127 DNCL1 +T591 PR:000016642 24132 24138 TRIM11 +T592 http://purl.obolibrary.org/obo/MONDO_0000087 24205 24219 polymicrogyria +T593 UBERON:0000955 24425 24430 brain +T594 UBERON:0000966 24460 24466 retina +T595 PR:000012318 24493 24497 PAX6 +T596 UBERON:0000966 24562 24569 retinal +T597 NCBITaxon:1 24590 24601 individuals +T598 PR:000012318 24607 24611 PAX6 +T599 GO:0007399 24692 24710 neurodevelopmental +T600 PR:000012318 24737 24741 PAX6 +T601 PR:000008680 24757 24763 HOMER3 +T602 PR:000002325 24765 24770 DNCL1 +T603 PR:000016642 24775 24781 TRIM11 +T604 PR:000012318 24818 24822 PAX6 +T605 PR:000008680 24828 24834 HOMER3 +T606 PR:000002325 24839 24844 DNCL1 +T607 GO:0045202 24869 24877 synaptic +T608 GO:0099536 24869 24888 synaptic signalling +T609 GO:0065007 24903 24912 regulated +T610 SO:0000704 24924 24928 gene +T611 GO:0010467 24924 24939 gene expression +T612 CL:0000540 24943 24950 neurons +T613 PR:000012318 25019 25023 PAX6 +T614 SO:0000857 25343 25353 homologous +T615 NCBITaxon:9606 25375 25380 human +T616 PR:000012318 25381 25385 PAX6 +T617 SO:0000993 25480 25489 consensus +T618 NCBITaxon:9606 25586 25591 human +T619 PR:000012318 25592 25596 PAX6 +T620 NCBITaxon:9606 25653 25658 Human +T621 PR:000012318 25659 25663 PAX6 +T622 SO:0001023 25664 25671 Allelic +T623 PR:000012318 25767 25771 PAX6 +T624 GO:0010467 25802 25812 expression +T625 SO:0000440 25813 25819 vector +T626 PR:P04386 25907 25911 GAL4 +T627 SO:0000417 25924 25930 domain +T628 SO:0000417 25963 25969 domain +T629 PR:000012318 26010 26014 PAX6 +T630 SO:0000112 26025 26032 Primers +T631 SO:0001030 26045 26052 forward +T632 SO:0001031 26113 26114 R +T633 SO:0000112 26242 26249 Primers +T634 SO:0001030 26262 26263 F +T635 SO:0001031 26325 26326 R +T636 SO:0000417 26357 26363 domain +T637 MOP:0000780 26421 26428 cutting +T638 SO:0000061 26541 26558 restriction sites +T639 PR:000012318 26670 26674 PAX6 +T640 SO:0000417 26679 26685 domain +T641 SO:0000132 26806 26813;26818 26824 reverse ... primer +T642 GO:0001171 27066 27085 reverse transcribed +T643 SO:0000112 27146 27153 Primers +T644 SO:0001030 27166 27167 F +T645 SO:0001031 27212 27213 R +T646 MOP:0000780 27291 27294 cut +T647 NCBITaxon:10088 27431 27436 mouse +T648 UBERON:0000955 27437 27442 brain +T649 SO:0000440 27581 27587 vector +T650 PR:P04386 27632 27636 GAL4 +T651 SO:0000417 27648 27654 domain +T652 PR:P04386 27678 27682 GAL4 +T653 SO:0000704 27702 27707 genes +T654 PR:P06633 27709 27713 HIS3 +T655 PR:P03962 27715 27719 URA3 +T656 PR:000033987 27724 27728 lacZ +T657 PR:P04386 27824 27828 GAL4 +T658 SO:0000417 27841 27847 domain +T659 PR:P04386 27902 27906 GAL4 +T660 SO:0000417 27918 27924 domain +T661 PR:P04386 27954 27958 GAL4 +T662 PR:P06633 27969 27973 HIS3 +T663 PR:P03962 28033 28037 URA3 +T664 CHEBI:74498 28088 28107 5-fluro-orotic acid +T665 PR:P03962 28121 28125 URA3 +T666 PR:000033987 28178 28182 LacZ +T667 CHEBI:75055 28201 28206 X-gal +T668 GO:0009294 28532 28543 transformed +T669 SO:0000155 28579 28586 plasmid +T670 PR:P06633 28698 28702 HIS3 +T671 PR:P06633 28715 28719 HIS3 +T672 SO:0000155 28784 28791 plasmid +T673 PR:P06633 28814 28818 HIS3 +T674 PR:P03962 28819 28823 URA3 +T675 PR:000033987 28824 28828 LacZ +T676 SO:0000667 28852 28858 insert +T677 SO:0000667 28921 28927 insert +T678 GO:0009294 28995 29007 transforming +T679 PR:P06633 29164 29168 HIS3 +T680 PR:P03962 29170 29174 URA3 +T681 PR:000033987 29179 29183 LacZ +T682 PR:000008680 29230 29236 Homer3 +T683 SO:0000317 29265 29275 cDNA clone +T684 SO:0000345 29414 29417 EST +T685 PR:000008680 29608 29614 Homer3 +T686 SO:0000155 29615 29622 plasmid +T687 GO:0010467 29647 29657 expression +T688 PR:000012318 29800 29804 Pax6 +T689 PR:000008680 29806 29812 Homer3 +T690 PR:000002325 29814 29819 Dncl1 +T691 PR:000016642 29821 29827 Trim11 +T692 GO:0010467 29851 29860 expressed +T693 SO:0000704 29861 29866 genes +T694 PR:000004453 29877 29883 Atp5a1 +T695 SO:0000112 29893 29900 Primers +T696 SO:0000188 29937 29943 intron +T697 GO:0008380 29968 29975 spliced +T698 SO:0000112 29999 30005 Primer +T699 PR:000012318 30022 30026 Pax6 +T700 SO:0001030 30027 30028 F +T701 PR:000012318 30058 30062 Pax6 +T702 SO:0001031 30063 30064 R +T703 PR:000008680 30093 30099 Homer3 +T704 SO:0001030 30100 30101 F +T705 PR:000008680 30127 30133 Homer3 +T706 SO:0001031 30134 30135 R +T707 PR:000016642 30167 30173 Trim11 +T708 SO:0001030 30174 30175 F +T709 PR:000016642 30201 30207 Trim11 +T710 SO:0001031 30208 30209 R +T711 PR:000002325 30238 30243 Dncl1 +T712 SO:0001030 30244 30245 F +T713 PR:000002325 30273 30278 Dncl1 +T714 SO:0001031 30279 30280 R +T715 SO:0001030 30316 30317 F +T716 SO:0001031 30353 30354 R +T717 PR:000004453 30384 30390 Atp5a1 +T718 SO:0001030 30391 30392 F +T719 PR:000004453 30422 30428 Atp5a1 +T720 SO:0001031 30429 30430 R +T721 CHEBI:6636 30570 30575 MgCl2 +T722 SO:0000112 30588 30594 primer +T723 CHEBI:2511 30807 30814 agarose +T724 NCBITaxon:10847 30824 30829 ΦX174 +T725 SO:0000417 30881 30887 domain +T726 SO:0000417 30926 30932 domain +T727 GO:0014069 30934 30937 PSD +T728 GO:0014069 30939 30960 post-synaptic density +T729 PR:000008680 31445 31451 Homer3 +T730 GO:0007601 31496 31501 Sight diff --git a/src/ontogpt/evaluation/craft/database/all/16098226.txt b/src/ontogpt/evaluation/craft/database/all/16098226.txt new file mode 100644 index 000000000..2354b4cde --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16098226.txt @@ -0,0 +1,151 @@ +A screen for proteins that interact with PAX6: C-terminal mutations disrupt interaction with HOMER3, DNCL1 and TRIM11 + +Abstract + +Background + +The PAX6 protein is a transcriptional regulator with a key role in ocular and neurological development. Individuals with heterozygous loss-of-function mutations in the PAX6 gene have malformations of the eye and brain. Little is known about the interactions of PAX6 with other proteins, so we carried out a systematic screen for proteins that interact with PAX6. + +Results + +We used bioinformatics techniques to characterise a highly conserved peptide at the C-terminus of the PAX6 protein. Yeast two-hybrid library screens were then carried out to identify brain-expressed proteins that interact with the C-terminal peptide and with the entire PAX6 proline-serine-threonine-rich domain. Three novel PAX6-interacting proteins were identified: the post-synaptic density (PSD) protein HOMER3, the dynein subunit DNCL1, and the tripartite motif protein TRIM11. Three C-terminal PAX6 mutations, previously identified in patients with eye malformations, all reduced or abolished the interactions. + +Conclusion + +Our preliminary data suggest that PAX6 interacts with HOMER3, DNCL1 and TRIM11. We propose that the interaction of PAX6 with HOMER3 and DNCL1 is a mechanism by which synaptic activation could lead to changes in neuronal transcriptional activity, and that some of the neural anomalies in patients with PAX6 mutations could be explained by impaired protein-protein interactions. + +Background + +The PAX6 protein is a member of the PAX (paired-box) family of transcriptional regulators and is essential for normal ocular and neural development [1]. Heterozygous mutations of the human PAX6 gene cause aniridia (absence of the iris) and a range of other congenital eye malformations [2]. Neural defects such as foveal hypoplasia and optic nerve hypoplasia are common in PAX6-associated eye disease [3-5]. Homozygous mutations in man and mouse are lethal and result in severe developmental abnormalities including anophthalmia, severe reduction of the olfactory structures and gross brain malformations [2,6]. The roles of PAX6 in brain development have mainly been studied in homozygous mutant mice or rats and include arealisation of the cerebral cortex [7], formation of the prosencephalon-mesencephalon boundary [8], axon guidance [8], differentiation of neurons from glia [9] and neuronal migration in the cerebellum [10]. + +The discovery of multiple and diverse roles for PAX6 in brain development prompted MRI analyses of aniridia patients, and a range of distinctive brain anomalies were uncovered. The most common and striking of these was absence or hypoplasia of the anterior commissure [11]. Other defects included absence or hypoplasia of the pineal gland, cortical polymicrogyria, white matter changes in the corpus callosum and grey matter changes in the cerebellum [11-13]. Functional changes included hyposmia and abnormal inter-hemispheric auditory transfer [11,14]. + +The defining feature of all PAX proteins is the presence of a 128 amino acid DNA-binding paired domain that has been highly conserved over evolution [1]. In addition to the paired domain, PAX6 also contains a DNA-binding homeodomain and a proline, serine and threonine-rich (PST) domain at the C-terminus [1,6]. The PST domain, which encompasses the C-terminal 145 amino acids of PAX6, has been shown to act as a transcriptional activator [6]. The PAX6 protein directly regulates a wide range of target genes [1,2] including Pax2 [15], Ngn2 [16] and glucagon [17]. + +The Pax6 gene has a spatially and temporally complex expression pattern in the eye, brain, nasal structures, spinal cord and pancreas [1]. Although PAX6 is clearly involved in multiple developmental processes, common themes are now emerging concerning the role of PAX6 in neural tissues. Gradients of Pax6 expression are important for determining positional characteristics in the retina [18] and the neocortex [7]. PAX6 plays a role in development of specific axonal connections between the retina and the brain [18] and within the forebrain [8,19]. It is also involved in the differentiation of neural cell types from multipotent precursors in the retina [16] and the cerebral cortex [9] through activation of bHLH genes such as Ngn2 and Mash1. These studies provide a clear link between PAX6 function in the retina and the brain, and are of particular relevance to the neurological phenotypes of individuals with PAX6 mutations. + +It is becoming apparent that transcription factors do not act in isolation but are dependent on interactions with other proteins to carry out their function [20,21]. These interactions introduce more specificity into the regulatory function of a given transcription factor. To date only three PAX6 protein-protein interactions have been described: with SOX2 on the lens-specific enhancer element of the δ-crystallin gene [22]; with MDIA, which modulates PAX6 activity in early neuronal development [23], and with MAF proteins on the glucagon promoter, which causes increased expression of this pancreatic hormone gene [17]. + +Here we report the preliminary results of the first systematic screen for proteins that interact with PAX6. We used sequence alignment algorithms and secondary structure prediction programs to define a new domain of 32 amino acids at the C-terminal end of the PAX6 protein. We then screened a brain library with this peptide using the yeast two-hybrid technique and identified three novel interacting proteins, HOMER3, DNCL1 and TRIM11. The interaction between PAX6 and these proteins was disrupted by naturally occurring C-terminal PAX6 mutations. + +Results + +A highly conserved C-terminal PAX6 peptide + +We and others [31-33] noted that there is significant sequence conservation at the C-terminal end of the PAX6 protein. BLAST analysis of the amino acid sequence of the PAX6 PST domain (aa 278–422) revealed a highly conserved motif within the last 28 amino acids (beginning at the 'GLISP' motif, Figure 1a). Strong conservation was seen in distantly related species such as axolotl (Ambystoma mexicanum) and sea urchin (Paracentrotus lividus) (Figure 1a). + +Figure 1 + +Characterisation of the PAX6 C-terminal peptide. (a) CLUSTAL alignment of the terminal 42 amino acids of PAX6 from diverse species. (*) indicates invariant residues, (:) indicates highly similar substitutions and (.) indicates moderately similar substitutions. (b) Secondary structure analysis of the highly conserved terminal 28 amino acids predicts a single beta sheet (arrow) in the SVPVQ peptide. (c) When 4 residues are added in the N-terminal direction, 2 beta sheets are now predicted. + +We subjected the whole PAX6 PST domain to secondary structure analysis using JPRED, a program that uses a number of different protein structure prediction algorithms to generate a consensus secondary structure (Figure 1) [25,26]. The PST domain was largely devoid of predicted secondary structure except for the C-terminal region, which contained two predicted beta sheets within the highly conserved domain, one in the 'GLISP' motif and one in the 'SPVPQ' motif (identical to the pattern shown in Figure 1c). Initially we defined the C-terminal domain as running from the 'GLISP' motif up to the stop codon, since this region was most highly conserved and contained strongly predicted secondary structure elements. However when we performed secondary structure prediction analysis on this 28 amino acid peptide, the first beta sheet was lost (Figure 1b). Addition of another 4 amino acids (TTST) immediately before 'GLISP' caused recovery of the first beta sheet (Figure 1c). Although these 4 residues are not highly conserved (Figure 1a), they appear to be important for seeding the first beta sheet and therefore for secondary structure. Thus we define the C-terminal peptide as being the final 32 amino acids of PAX6, running from threonine 391 to the stop codon (top line of Figure 1c). + +Yeast two-hybrid screening + +We hypothesised that the C-terminal peptide might be involved in protein-protein interactions, and we tested this by screening a cDNA library using the yeast two-hybrid system with a construct (PAX6CTP) in which the 32 amino acid C-terminal peptide was fused to the yeast GAL4 DNA binding domain. We chose to screen a mouse brain cDNA library as no human libraries were available. Given the fact that the amino acid sequence of the PAX6 protein is identical in man and mouse, we reasoned that a mouse brain library would yield relevant interactors. For comparison we also carried out the screen using a construct containing the whole PST domain (PAX6PST). + +The C-terminal peptide screen gave 15 colonies that were positive with all three reporters and the PST domain screen gave 62 colonies. The interacting plasmids were isolated and the cDNA inserts sequenced. Three cDNAs were identified 3 or more times, Homer3 (NM_011984), Dncl1 (Dynein cytoplasmic light chain 1, NM_019682, also known as Pin or Dlc8) and Trim11 (Tripartite motif protein family member 11, NM_053168). Homer3 (6 clones) and Dncl1 (2 clones) were identified in the C-terminal peptide screen. Homer3 (7 clones), Dncl1 (2 clones) and Trim11 (6 clones) were identified in the PST domain screen. All cDNA inserts were in-frame with the coding region of the pPC86 GAL4 activation domain. None of the cDNAs was present in a list of known false positives [34]. + +HOMER3 is a member of the HOMER family of neuronal post-synaptic density (PSD) proteins [35]. DNCL1 is a subunit of two motor protein complexes, dynein and myosin-Va, both of which are involved in intracellular trafficking of proteins and organelles in neurons [36,37]. TRIM11 is a member of the tripartite motif protein family and contains a RING finger, a B-box zinc finger, a coiled coil domain and a B30.2 domain [38]. The possible significance of the interactions between these proteins and PAX6 is discussed below. + +Semi-quantitative PCR + +To check that the Homer3, Dncl1 and Trim11 clones were not identified multiple times solely because they are highly abundant in the library, we performed a semi-quantitative PCR assay. We compared the relative abundance of Homer3, Dncl1, Trim11 and Pax6 with Gapdh and Atp5a1. Gapdh and Atp5a1 both show strong constitutive expression in a variety of tissues including the brain [29,30]. Homer3, Dncl1, Trim11 and Pax6 were only amplified strongly after 35 cycles of PCR (Figure 2) and were therefore present at relatively low levels compared to Gapdh (amplified strongly after 25 cycles) and Atp5a1 (amplified strongly after 30 cycles; Figure 2). + +Figure 2 + +Semi-quantitative PCR analysis of Homer3, Dncl1, Trim11 and Pax6 in the mouse brain cDNA library. Library cDNA was amplified with primers specific for Pax6 (600 bp), Homer3 (485 bp band), Dncl1 (485 bp) and Trim11 (609 bp) for 20, 25, 30 or 35 cycles. Atp5a1 (415 bp) and Gapdh (450 bp), which are highly expressed in the brain, are included for comparison. M indicates the Φ×174 HaeIII DNA size marker; the positions of the 603 bp and 310 bp marker bands are indicated. + +We concluded that Homer3, Dncl1 and Trim11 clones were not highly abundant in the library. This is consistent with the idea that they were pulled out because the encoded proteins interact specifically with the C-terminal peptide or PST domain of PAX6. + +Yeast two-hybrid pairwise interactions + +By library screening we identified two proteins (HOMER3 and DNCL1) that interact with the C-terminal peptide and three proteins (HOMER3, DNCL1 and TRIM11) that interact with the whole PST domain. This suggests that HOMER3 and DNCL1 interact specifically with the C-terminal peptide while TRIM11 interacts with a more N-terminal part of the PST domain. We conducted pairwise tests between specific constructs to confirm the interactions identified in the library screen and to further investigate the interaction between PAX6 and HOMER3, DNCL1 and TRIM11. The Dncl1 and Trim11 clones that were pulled out of the library were full-length, but the Homer3 cDNAs lacked the N-terminal 70 amino acids. The missing coding region was inserted into the truncated Homer3 cDNA to give a full-length expression construct (see Methods). Pairwise interactions were carried out with both the full-length and truncated Homer3 clones. + +We confirmed that the whole PAX6 PST domain interacts with HOMER3 (full-length and truncated constructs), TRIM11 and DNCL1, as all three reporter genes were strongly activated in pairwise tests (Figure 3; Table 1). In contrast the interaction between the C-terminal peptide and HOMER3 or DNCL1 could not be confirmed with pairwise tests (Table 1). Occasionally, partial suppression of growth on plates containing 5-fluoro-orotic acid was observed, indicating low-level activation of the URA3 reporter; however HIS3 and LacZ activation were not observed. The reasons for this are not clear, although it may be that the pairwise tests were of sub-optimal sensitivity compared to the library screen. However we were able to confirm that the C-terminal peptide is important for the interaction with HOMER3 and DNCL1 because interaction with the PAX6PST-CT construct, which lacks the final 32 amino acids, was completely abolished (Figure 3, Table 1). TRIM11 interacted equally well with PAX6PST and PAX6PST-CT (Figure 3, Table 1). This is consistent with the library screens in which TRIM11 was isolated with the PST domain but not with the C-terminal peptide and supports the idea that the C-terminal peptide is not important for the interaction between PAX6 and TRIM11. + +Figure 3 + +LacZ reporter gene activation in pairwise tests. pPC86 constructs are shown across the top, and pDBLeu constructs are shown down the right hand side. PAX6PST, PAX6 PST domain; PAX6PST/Q422R, PAX6 PST domain with the Q422R mutation; PAX6PST/X423L, PAX6 PST domain with the X423L mutation, PAX6PST/1615del10, PAX6 PST domain with the 1615 del10 mutation; PAX6PST-CT, PAX6 PST domain minus the C-terminal peptide. 'Truncated HOMER3' is HOMER3 lacking the N-terminal 70 amino acids. Five control strains are shown for comparison (left). These range from non-interactor (A) to strong interactor (E). + +Table 1 + +Pairwise interaction tests between normal and mutant PAX6 constructs and HOMER3, DNCL1 and TRIM11. +++: strong interaction; ++: moderate interaction; +: weak interaction; (+) borderline interaction with one or two reporters activated at very low levels; 0: no interaction. PAX6PST, PAX6 PST domain; PAX6CTP, PAX6 C-terminal peptide; PAX6PST-CT, PAX6 PST domain minus the C-terminal peptide; PAX6PST/Q422R, PAX6 PST domain with the Q422R mutation; PAX6PST/X423L, PAX6 PST domain with the X423L mutation, PAX6PST/1615del, PAX6 PST domain with the 1615 del10 mutation. HOMER3-FL, HOMER3 full-length clone; HOMER3-Tr, truncated HOMER3 clone lacking the N-terminal 70 amino acids. + +Having confirmed that HOMER3, DNCL1 and TRIM11 interact with the PAX6 PST domain, we next investigated how the interactions were affected by three C-terminal PAX6 mutations that have been previously described in patients with ocular anomalies. The first mutation is a single nucleotide substitution 1627A>G that causes a glutamine to arginine amino acid substitution in the last codon of PAX6. This missense mutation (Q422R) has been reported in two patients, one affected by anterior segment dysgenesis with uveal ectropion and one with typical aniridia and foveal hypoplasia [27,33]. + +The second mutation (1615del10) was found in an aniridia family [28]. This frame-shifting deletion occurs just before the PAX6 stop codon and is predicted to cause translational read-through into the 3' untranslated region, generating a protein in which the last 5 amino acids of the C-terminal peptide are replaced by a 103 amino acid-extension. Affected individuals in this family showed unusual neurobehavioural traits including impaired social cognition and poor verbal inhibition [28]. MRI analysis revealed grey matter abnormalities in the frontal lobe, temporal lobe and cerebellum, and white matter deficits in the corpus callosum [13]. + +The third mutation 1629insT (X423L) has been reported in several aniridia patients [11,12,27,33]. Insertion of a single T nucleotide at position 1629 changes the stop codon (TAA) to a leucine codon (TTA) and generates a full length PAX6 protein with a C-terminal extension that extends for a further 35 amino acids into the 3' untranslated region. MRI analysis of six patients with this mutation revealed variable brain defects including absence or hypoplasia of the anterior commissure, pineal gland and olfactory bulbs [12]. Two patients had temporal polymicrogyria, one in association with epilepsy [12]. + +The three mutations were introduced into the PAX6PST construct, and pairwise tests were carried out to investigate the interaction of each mutant protein with HOMER3, DNCL1 and TRIM11. All three mutations had a clear effect on the interactions. The most subtle mutation (Q422R) caused a reduction in the interaction with HOMER3 and DNCL1 (Figure 3, Table 1). The C-terminal extension mutations X423L and1615del10 mutations both dramatically reduced or abolished the interaction with HOMER3 and DNCL1 (Figure 3, Table 1). + +None of the three mutations affected the interaction with TRIM11 which again is consistent with the hypothesis that TRIM11 interacts with a more N-terminal part of the PST domain. + +Discussion + +On the basis of secondary structure predictions and amino acid sequence conservation, we defined a novel PAX6 protein domain, which we have called the C-terminal peptide. We performed yeast two-hybrid library screens with the C-terminal peptide and the whole PST domain and we identified three novel interacting proteins, HOMER3, DNCL1 and TRIM11. In library screens, HOMER3 and DNCL1 interacted with the C-terminal peptide and the PST domain while TRIM11 interacted only with the PST domain, suggesting that HOMER3 and DNCL1 specifically interact with the C-terminal peptide while TRIM11 interacts with a more N-terminal part of the PST domain. The interactions between the PST domain and HOMER3, DNCL1 and TRIM11 were confirmed in pairwise tests. We were not able to confirm the interaction between HOMER3 or DNCL1 with the C-terminal peptide construct in pairwise tests, but we showed that the C-terminal peptide was important for PAX6/HOMER3 or PAX6/DNCL1 interaction because HOMER3 and DNCL1 did not interact with a PST domain construct lacking the C-terminal peptide. + +HOMER3 is found in the PSD of neurons and directly binds to type I metabotropic glutamate receptors, which act via phospholipase C to stimulate IP3-mediated release of Ca2+ from intracellular vesicles [35,39]. HOMER3 is a member of the HOMER family of proteins that are constitutively expressed in the brain and play a role in post-synaptic signalling and receptor trafficking by forming multivalent links with various receptors and PSD scaffolding proteins [35,39-41]. HOMER proteins have also been implicated in axon guidance during brain development [42]. + +DNCL1 is a subunit of two intracellular transport protein complexes, dynein and myosin Va [36]. Dynein and myosin Va are involved in the microtubule-based and actin-based movement respectively of proteins, organelles and vesicles in neurons [36,37]. Myosin Va is enriched in the PSD [43], and DNCL1 binds to a variety of PSD proteins including guanylate kinase domain-associated protein [44] and neuronal nitric oxide synthase [45]. + +TRIM11 is a member of the mouse tripartite motif protein family (also known as the RBCC family), and contains the three characteristic structural motifs of this protein family, a RING finger, a B-box zinc finger, and a coiled coil domain, as well as a B30.2 domain that is found in many but not all TRIM proteins [38]. TRIM11 interacts with Humanin, a protein that suppresses the neurotoxicity associated with Alzheimer's disease [46]. TRIM11 lowers Humanin levels by a mechanism that appears to involve ubiqutin-mediated proteasomal degradation [46]. + +At present our data must be considered preliminary because the interactions have not been confirmed by any other approach. However it is interesting to speculate that the interaction of PAX6 with HOMER3 and DNCL1 may be the basis of a mechanism by which synaptic signalling causes changes in gene expression. We propose that PAX6 is sequestered in the PSD by binding to HOMER3. Since receptor activation causes dissociation of HOMER proteins [35], PAX6 may be released as a result of synaptic activity, allowing it to interact with DNCL1. The PAX6/DNCL1 complex could then participate in myosin Va-mediated transport along the PSD-associated actin cytoskeleton followed by dynein-mediated transport along the microtubule network, eventually reaching the nucleus [36]. Since TRIM11 is implicated in protein degradation [46], it may play a role in PAX6 protein turnover. + +Precedents for association of transcription factors with the post-synaptic density include STAT3 and CREB, which act as messengers between the synapse and the nucleus [47,48]. Although the full-length PAX6 protein is predominantly nuclear, there is good evidence in mouse, quail and nematode for an isoform that lacks the paired domain, is both nuclear and cytoplasmic, and binds DNA through the homeodomain alone [49-51]. In mice the paired-less isoform is relatively abundant in brain [49]; however its subcellular localisation in neurones, and the possibility of an association with the PSD, remains to be investigated. + +Regarding the subcellular localisation of the putative interacting proteins, HOMER3 is predominantly found at the interface between the PSD and the cytoplasm [39], DNCL1 is chiefly cytoplasmic, although nuclear localisation has been reported [51], and TRIM11 is both nuclear and cytoplasmic [38]. Therefore cytoplasmic PAX6 could potentially interact with all three proteins, while nuclear PAX6 could interact with TRIM11 and DNCL1. At the tissue level, HOMER3 expression has been detected in thymus and lung but it has mainly been studied in brain where it is found in the forebrain, hippocampus and cerebellum [39]. DNCL1 and TRIM11 both have wide expression domains that include the brain [44,38]. Thus the expression of all three interactors overlaps with PAX6 at the tissue level [2,7,8,10]. We detected co-expression of PAX6, HOMER3, DNCL1 and TRIM11 by RT-PCR in human adult brain RNA (IH, L Harrison and A Brown, data not shown). + +We demonstrated that the interaction between the PST domain and HOMER3 or DNCL1 was impaired by three naturally occurring mutations that are located in the PAX6 C-terminal peptide. The Q422R mutation, which involves a glutamine to arginine substitution at the last amino acid position of PAX6, caused a reduction in the interaction with HOMER3 and DNCL1. The X423L and 1615del10 mutations severely reduced or completely abolished the interaction with HOMER3 and DNCL1. The predicted effect of the X423L and 1615del10 mutations is to cause translation into the 3' untranslated region, thus generating proteins with abnormal extensions that might be expected to disrupt the conformation of the C-terminal end of the protein. The 1615del10 mutation also removes the last 5 amino acids of the C-terminal peptide [28]. + +None of the mutations affected the interaction with TRIM11, suggesting that they do not alter the conformation of the more N-terminal part of the PST domain. All our data are consistent with the hypothesis that TRIM11 does not interact with the C-terminal peptide, but interacts with the PST domain between the homeodomain and the C-terminal peptide. + +Most aniridia patients are heterozygous for mutations that introduce a premature termination codon into the PAX6 open reading frame [2,52]. These alleles would be expected to encode truncated proteins or no protein at all if the mutant RNA is degraded by nonsense-mediated decay [52]. We propose that the brain anomalies that have been observed in aniridia patients may be partly explained by impaired interaction between PAX6 and HOMER3, DNCL1 and TRIM11. The neurobehavioural phenotype associated with 1615del10 and the polymicrogyria associated with X423L may result from a specific effect of these unusual C-terminal extension mutations. There is evidence that signalling and transport mechanisms that were initially characterized in the brain may also be conserved in the retina, suggesting that impaired PAX6 protein-protein interactions may also have implications for the retinal defects observed in individuals with PAX6 mutations [53,54]. + +Conclusion + +We have presented preliminary evidence that the neurodevelopmental transcriptional regulator PAX6 interacts with HOMER3, DNCL1 and TRIM11. We suggest that the interaction of PAX6 with HOMER3 and DNCL1 is a mechanism by which synaptic signalling could lead to regulated changes in gene expression in neurons. We also propose that some of the neural anomalies in patients with PAX6 mutations may be explained by impaired protein-protein interactions. + +Methods + +Bioinformatics techniques + +Sequence database searches were carried out using the BLAST program available through the Bioinformatics Applications at the Rosalind Franklin Centre for Genomics Research [24]. Protein sequences that were highly homologous to the C-terminus of human PAX6 were aligned using CLUSTAL [24]. Secondary structure prediction was performed using the JPRED consensus method [25,26]. + +Yeast two-hybrid constructs + +All cDNA and amino acid numbering is based on the human PAX6 cDNA and protein reference sequences available from the Human PAX6 Allelic Variant Database web site [27]. Standard PCR and subcloning techniques were used to make three PAX6 cDNA constructs in the pDBLeu expression vector (ProQuest Two-Hybrid System, Invitrogen), which generates a protein fused to the yeast GAL4 DNA binding domain. PAX6PST contains the whole PST domain (amino acids 278–422 of the full-length PAX6 protein). Primers were ST001 (forward) 5'-AAA AGT TCG ACT GCC AGC AAC ACA CCT AGT C-3' and ST005 (R) 5'-TTT TGC GGC TTT TTA CTG TAA TCT TGG CCA GTA TTG-3'. PAX6CTP contains the newly defined C-terminal peptide alone (391–422). Primers were ST004 (F) 5'-AAA AGT CGA CTA CCA CTT CAA CAG GAC TCA TT-3' and ST005 (R). PAX6PST-CT contains the PST domain minus the C-terminal peptide (278–390). This was made by cutting PAX6PST with NdeI and NotI to drop out the C-terminal peptide, and inserting a synthetic linker between the two restriction sites. All fragments generated by PCR or with linkers were sequenced to check that no errors had been introduced. + +A PAX6 PST domain construct containing the mutation 1627A>G (Q422R) was generated in the same way as the PAX6PST construct, but using the reverse PCR primer ST006 5'-TTT TGC GGC CGC TTT TTA CCG TAA TCT TGG CCA GTA TTG AG-3', which contains the mutant nucleotide substitution (underlined). + +cDNA sequences containing the mutations 1615del10 [28] and 1629insT (X423L) [12] were generated by PCR from reverse transcribed RNA (a gift from Dr K Williamson and Prof V van Heyningen). Primers were ST015 (F) 5'-CCC ACA TAT GCA GAC ACA C-3' and ST031 (R) 5'-TTG CGG CCG CAT CCA TCC AGT CTA CAT TGT TC-3'. The PAX6PST construct was cut with NdeI and NotI to release the normal C-terminal peptide, and the mutant sequence was inserted. + +Yeast two-hybrid library screens + +A mouse brain cDNA library (ProQuest, Invitrogen) was screened with the PAX6PST and PAX6CTP pDBLeu constructs. The library was constructed in the pPC86 vector, which produces proteins fused to the yeast GAL4 activation domain. The system uses three GAL4-activated reporter genes, HIS3, URA3 and lacZ, to identify positive interactions. Reporters are activated when the bait protein fused to the GAL4 DNA binding domain (pDBLeu) interacts with the prey protein fused to the GAL4 activation domain (pPC86), thus reconstituting GAL4 function. HIS3 activation allows growth on plates lacking histidine. Weak URA3 activation suppresses growth on plates containing 5-fluro-orotic acid while strong URA3 activation permits growth on plates lacking uracil. LacZ activation causes X-gal to turn blue in a beta-galactosidase assay. All assays were carried out in parallel with the five ProQuest control yeast strains A-E, which range from non-interactor (A) to strong interactor (E). + +All procedures were carried out according to the supplier's protocols. Briefly, chemically competent MaV203 yeast cells were co-transformed with the cDNA library and the bait plasmid pDBLeu PAX6CTP or pDBLeu PAX6PST. Transformants (5 × 107) were plated on medium lacking histidine to check for HIS3 activation. HIS3 positives were then assayed for all 3 reporters. The pPC86 prey plasmid was isolated from all HIS3/URA3/LacZ positives and the cDNA insert sequenced. BLAST searches were performed to identify the cDNA insert [24]. + +Pairwise interactions + +Specific interactions were tested by transforming competent MaV203 yeast cells with one bait construct (in pDBLeu) and one prey construct (in pPC86) and testing the resulting colonies for activation of the HIS3, URA3 and LacZ reporters as before. + +To create a full-length Homer3 clone for pairwise tests, a cDNA clone (IMAGE clone 3602414, accession number BE569374) containing the missing N-terminal 70 amino acids was identified by a BLAST search of the EST nucleotide sequence database and obtained from the Rosalind Franklin Centre for Genomics Research. The missing fragment was amplified from the IMAGE clone by PCR and inserted into the pPC86-Homer3 plasmid to create a full-length expression construct. + +Semi-quantitative PCR + +To check the relative representation of clones in the cDNA library, semi-quantitative PCR was performed on Pax6, Homer3, Dncl1, Trim11 and the constitutively expressed genes Gapdh and Atp5a1 [29,30]. Primers were designed to cross at least one intron, so that only correctly spliced clones were amplified. Primer sequences were: Pax6-F CAG CCA AAA TAG ATC TAC CTG; Pax6-R CGA TCA CAT GCT CTC TCC TT; Homer3-F CCC AGG TGG CTG TAG AGC; Homer3-R CTC TAC ACA GTG CAA AGC TCA G; Trim11-F GTG CAG GAT GTG AAG CTG; Trim11-R GCC TGC AGA TAG TCA TAG GG; Dncl1-F CAA AAA TGC AGA CAT GTC G; Dncl1-R CTA AGG GAG AAA AAA ATG GGG; Gapdh-F: CAT CAC CAT CTT CCA GGA GC; Gapdh-R: ATG ACC TTG CCC ACA GCC TT; Atp5a1-F: CAC ACG TGA GAT GTC CTC CA; Atp5a1-R: CAC AGA GAT TCG GGG ATA A. 10 ng library cDNA were amplified in a reaction containing 1xAmpliTaq polymerase buffer (Perkin Elmer), 1.5 mM MgCl2, 200μM each primer and 2.5 units of AmpliTaq polymerase (Perkin Elmer). PCR conditions were (95°C for 30 sec) × 1, (94°C for 30 sec, 55°C for 30 sec, 72°C for 30 sec) × 32 and (72°C for 2 min) × 1. Products were resolved on a 2.5% agarose gel with ΦX174/HaeIII size markers (Promega). + +Abbreviations + +PST domain, proline-, serine- and threonine-rich domain; PSD, post-synaptic density; PCR, polymerase chain reaction. + +Authors' contributions + +STC carried out the bioinformatic analyses and all experimental work. IMH conceived, designed and supervised the study, and obtained funding. The manuscript was prepared jointly by STC and IMH, who have both read and approved the final version. + +Acknowledgements + +We gratefully acknowledge Dr A Brown for technical advice on the yeast two-hybrid system and the Rosalind Franklin Centre for Genomics Research for supplying the Homer3 IMAGE clone. STC was supported by Fight for Sight and IMH was supported by a Career Development Award from the UK Medical Research Council. diff --git a/src/ontogpt/evaluation/craft/database/all/16103912.ann b/src/ontogpt/evaluation/craft/database/all/16103912.ann new file mode 100644 index 000000000..66b212b83 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16103912.ann @@ -0,0 +1,839 @@ +T1 PR:000017653 0 4 Fog2 +T2 UBERON:0001103 28 37 Diaphragm +T3 GO:0060539 28 37;47 58 Diaphragm ... Development +T4 UBERON:0002048 42 46 Lung +T5 GO:0030324 42 58 Lung Development +T6 NCBITaxon:10088 62 66 Mice +T7 NCBITaxon:9606 71 77 Humans +T8 http://purl.obolibrary.org/obo/MONDO_0005711 89 120 Congenital diaphragmatic hernia +T9 UBERON:0001103 100 113 diaphragmatic +T10 http://purl.obolibrary.org/obo/MONDO_0021140 131 141 congenital +T11 UBERON:0001103 142 155 diaphragmatic +T12 UBERON:0000922 331 338 embryos +T13 NCBITaxon:10088 352 356 mice +T14 CHEBI:23995 370 391 N-ethyl-N-nitrosourea +T15 UBERON:0002048 430 439 pulmonary +T16 UBERON:0001103 464 477 diaphragmatic +T17 GO:0060539 464 489 diaphragmatic development +T18 PR:000017653 491 495 Fog2 +T19 PR:000017653 497 502 Zfpm2 +T20 CHEBI:23995 554 575 N-ethyl-N-nitrosourea +T21 PR:000017653 616 620 Fog2 +T22 GO:0008380 648 654 splice +T23 SO:0000673 693 703 transcript +T24 NCBITaxon:9606 734 739 Human +T25 UBERON:0001103 759 772 diaphragmatic +T26 UBERON:0002048 784 793 pulmonary +T27 PR:000017653 837 841 FOG2 +T28 SO:0001587 904 924 premature stop codon +T29 GO:0016265 940 944 died +T30 UBERON:0000104 965 969 life +T31 UBERON:0002048 1000 1009 pulmonary +T32 UBERON:0001103 1052 1061 diaphragm +T33 PR:000017653 1123 1127 Fog2 +T34 UBERON:0001103 1151 1160 diaphragm +T35 UBERON:0002048 1165 1169 lung +T36 GO:0030324 1165 1181 lung development +T37 PR:000017653 1232 1236 FOG2 +T38 SO:0000704 1250 1254 gene +T39 http://purl.obolibrary.org/obo/MONDO_0002254 1292 1301 syndromic +T40 NCBITaxon:9606 1302 1307 human +T41 http://purl.obolibrary.org/obo/MONDO_0020419 1308 1318 congenital +T42 UBERON:0001103 1319 1332 diaphragmatic +T43 UBERON:0002048 1364 1373 pulmonary +T44 GO:0030324 1364 1385 pulmonary development +T45 http://purl.obolibrary.org/obo/MONDO_0005711 1430 1461 congenital diaphragmatic hernia +T46 UBERON:0001103 1441 1454 diaphragmatic +T47 UBERON:0002048 1484 1493 pulmonary +T48 GO:0030324 1484 1507 pulmonary developmental +T49 GO:0007567 1536 1541 Birth +T50 http://purl.obolibrary.org/obo/MONDO_0000839 1536 1549 Birth defects +T51 UBERON:0001103 1564 1573 diaphragm +T52 SO:0000704 1606 1613 genetic +T53 http://purl.obolibrary.org/obo/MONDO_0003847 1606 1623 genetic disorders +T54 http://purl.obolibrary.org/obo/MONDO_0009061 1632 1647 cystic fibrosis +T55 GO:0007567 1717 1721 born +T56 UBERON:0001103 1727 1740 diaphragmatic +T57 UBERON:0002048 1765 1770 lungs +T58 GO:0016265 1781 1784 die +T59 UBERON:0007221 1792 1806 newborn period +T60 UBERON:0002048 1875 1880 lungs +T61 UBERON:0001103 1917 1930 diaphragmatic +T62 SO:0000704 1977 1984 genetic +T63 GO:0030324 2014 2028;2038 2042 development of ... lung +T64 GO:0060539 2014 2028;2051 2060 development of ... diaphragm +T65 UBERON:0002048 2038 2042 lung +T66 UBERON:0001103 2051 2060 diaphragm +T67 NCBITaxon:10088 2099 2103 mice +T68 SO:0000704 2132 2139 genetic +T69 SO:0000704 2192 2196 gene +T70 PR:000017653 2197 2201 Fog2 +T71 PR:000017653 2203 2219 Friend of gata 2 +T72 UBERON:0001103 2238 2247 diaphragm +T73 GO:0060539 2238 2259 diaphragm development +T74 UBERON:0002048 2270 2275 lungs +T75 UBERON:0002048 2281 2286 lungs +T76 UBERON:0000101 2367 2376 lung lobe +T77 UBERON:0001103 2443 2456 diaphragmatic +T78 NCBITaxon:9606 2491 2496 human +T79 NCBITaxon:9606 2533 2538 human +T80 SO:0000704 2539 2543 gene +T81 PR:000017653 2544 2548 FOG2 +T82 GO:0016265 2553 2557 died +T83 UBERON:0000104 2575 2579 life +T84 UBERON:0001103 2618 2631 diaphragmatic +T85 UBERON:0002048 2650 2655 lungs +T86 PR:000017653 2692 2696 Fog2 +T87 UBERON:0001103 2719 2728 diaphragm +T88 GO:0060539 2719 2728;2738 2749 diaphragm ... development +T89 UBERON:0002048 2733 2737 lung +T90 GO:0030324 2733 2749 lung development +T91 NCBITaxon:10088 2753 2757 mice +T92 NCBITaxon:9606 2765 2771 humans +T93 http://purl.obolibrary.org/obo/MONDO_0002254 2811 2820 syndromic +T94 http://purl.obolibrary.org/obo/MONDO_0020419 2821 2831 congenital +T95 UBERON:0001103 2832 2845 diaphragmatic +T96 UBERON:0002048 2945 2949 lung +T97 UBERON:0001103 2958 2967 diaphragm +T98 http://purl.obolibrary.org/obo/MONDO_0020419 2984 2994 Congenital +T99 UBERON:0001103 2995 3008 diaphragmatic +T100 GO:0007567 3053 3058 birth +T101 http://purl.obolibrary.org/obo/MONDO_0000839 3053 3066 birth defects +T102 http://purl.obolibrary.org/obo/MONDO_0005711 3130 3161 congenital diaphragmatic hernia +T103 UBERON:0001103 3141 3154 diaphragmatic +T104 http://purl.obolibrary.org/obo/MONDO_0005711 3163 3166 CDH +T105 UBERON:0001103 3250 3263 diaphragmatic +T106 GO:0007567 3285 3290 birth +T107 UBERON:0001103 3292 3305 diaphragmatic +T108 UBERON:0001103 3318 3331 diaphragmatic +T109 UBERON:0001062 3430 3438 anatomic +T110 GO:0016265 3542 3548 mortem +T111 UBERON:0002048 3563 3572 Pulmonary +T112 UBERON:0001103 3606 3619 diaphragmatic +T113 UBERON:0001103 3723 3736 diaphragmatic +T114 UBERON:0002048 3749 3758 pulmonary +T115 UBERON:0002048 3836 3845 pulmonary +T116 http://purl.obolibrary.org/obo/MONDO_0005711 3906 3909 CDH +T117 GO:0016265 4081 4087 deaths +T118 GO:0007567 4097 4102 birth +T119 UBERON:0001004 4198 4209 respiratory +T120 http://purl.obolibrary.org/obo/MONDO_0021113 4198 4217 respiratory failure +T121 GO:0007567 4221 4226 birth +T122 UBERON:0002048 4346 4350 lung +T123 GO:0015671 4383 4394 oxygenation +T124 UBERON:0001004 4653 4664 respiratory +T125 GO:0050890 4680 4689 cognitive +T126 GO:0050905 4694 4704 neuromotor +T127 GO:0007605 4719 4726 hearing +T128 http://purl.obolibrary.org/obo/MONDO_0005365 4719 4731 hearing loss +T129 http://purl.obolibrary.org/obo/MONDO_0002254 4939 4948 syndromic +T130 UBERON:0001103 4949 4962 diaphragmatic +T131 UBERON:0002048 4975 4984 pulmonary +T132 NCBITaxon:9606 4999 5005 humans +T133 SO:0000704 5158 5165 genetic +T134 UBERON:0001103 5316 5329 diaphragmatic +T135 GO:0060539 5316 5341 diaphragmatic development +T136 http://purl.obolibrary.org/obo/MONDO_0005711 5449 5452 CDH +T137 NCBITaxon:9606 5533 5539 humans +T138 NCBITaxon:33208 5585 5591 animal +T139 NCBITaxon:9606 5602 5607 human +T140 GO:0007567 5608 5613 birth +T141 http://purl.obolibrary.org/obo/MONDO_0000839 5608 5621 birth defects +T142 SO:0000704 5639 5646 genetic +T143 NCBITaxon:33208 5670 5676 animal +T144 NCBITaxon:9606 5712 5717 human +T145 NCBITaxon:10088 5744 5748 mice +T146 CHEBI:25435 5775 5782 mutagen +T147 CHEBI:23995 5783 5804 N-ethyl-N-nitrosourea +T148 CHEBI:23995 5806 5809 ENU +T149 UBERON:0012101 5868 5884 perinatal period +T150 GO:0007567 5872 5877 natal +T151 NCBITaxon:10088 5933 5937 mice +T152 UBERON:0002048 5992 6001 pulmonary +T153 UBERON:0001103 6026 6039 diaphragmatic +T154 GO:0060539 6026 6039;6052 6063 diaphragmatic ... development +T155 UBERON:0000948 6044 6051 cardiac +T156 GO:0007507 6044 6063 cardiac development +T157 PR:000017653 6104 6108 Fog2 +T158 PR:000017653 6110 6115 Zfpm2 +T159 GO:0008380 6184 6190 splice +T160 SO:0000673 6229 6239 transcript +T161 SO:0000858 6312 6323 orthologous +T162 SO:0000704 6324 6328 gene +T163 NCBITaxon:9606 6332 6338 humans +T164 PR:000017653 6435 6439 FOG2 +T165 GO:0016265 6457 6461 died +T166 GO:0007567 6465 6470 birth +T167 UBERON:0001103 6478 6491 diaphragmatic +T168 UBERON:0002048 6510 6519 pulmonary +T169 NCBITaxon:9606 6609 6614 human +T170 UBERON:0002048 6677 6686 pulmonary +T171 http://purl.obolibrary.org/obo/MONDO_0000001 6745 6754 disorders +T172 UBERON:0002048 6795 6799 lung +T173 PR:000017653 6812 6816 Fog2 +T174 CHEBI:23995 6858 6861 ENU +T175 NCBITaxon:10088 6874 6878 mice +T176 UBERON:0000922 6882 6891 embryonic +T177 NCBITaxon:10088 6965 6969 mice +T178 UBERON:0000922 6997 7004 embryos +T179 UBERON:0002048 7043 7052 pulmonary +T180 UBERON:0001103 7075 7084 diaphragm +T181 UBERON:0002048 7123 7127 lung +T182 SO:0001024 7199 7208 haplotype +T183 NCBITaxon:10088 7371 7375 mice +T184 SO:0000357 7715 7723 flanking +T185 UBERON:0002048 7794 7803 pulmonary +T186 UBERON:0001103 7831 7840 diaphragm +T187 UBERON:0002048 7842 7851 Pulmonary +T188 NCBITaxon:10088 7890 7894 mice +T189 GO:0007567 7912 7917 birth +T190 NCBITaxon:10088 7964 7968 mice +T191 UBERON:0000104 7994 7998 life +T192 UBERON:0002048 8046 8050 lung +T193 NCBITaxon:10088 8098 8102 mice +T194 UBERON:0002048 8123 8127 lung +T195 UBERON:0002048 8180 8184 lung +T196 UBERON:0002048 8227 8232 lungs +T197 NCBITaxon:10088 8245 8249 mice +T198 UBERON:0009912 8276 8280 lobe +T199 UBERON:0009912 8353 8357 lobe +T200 UBERON:0001103 8371 8381 Diaphragms +T201 NCBITaxon:10088 8398 8402 mice +T202 CL:0002372 8470 8480 Myotubules +T203 CL:0002372 8553 8563 myotubules +T204 UBERON:0000309 8627 8637 body walls +T205 CL:0000187 8664 8677 muscle fibers +T206 UBERON:0006670 8685 8702 central tendonous +T207 UBERON:0001103 8725 8734 diaphragm +T208 CL:0002372 8736 8746 myotubules +T209 UBERON:0002385 8793 8806 muscle tissue +T210 UBERON:0000309 8846 8856 body walls +T211 NCBITaxon:10088 8896 8900 mice +T212 GO:0007567 8918 8923 birth +T213 UBERON:0000922 9048 9057 embryonic +T214 UBERON:0000995 9127 9134 uterine +T215 GO:0016265 9135 9141 demise +T216 UBERON:0000922 9208 9215 embryos +T217 UBERON:0000995 9267 9274 uterine +T218 GO:0016265 9275 9281 demise +T219 UBERON:0000479 9325 9331 tissue +T220 UBERON:0000922 9348 9355 embryos +T221 GO:0016265 9391 9397 demise +T222 GO:0016265 9569 9573 died +T223 UBERON:0000922 9643 9650 embryos +T224 GO:0016265 9674 9678 died +T225 UBERON:0000922 9701 9708 embryos +T226 UBERON:0000922 9767 9773 embryo +T227 UBERON:0001103 9786 9799 Diaphragmatic +T228 NCBITaxon:10088 9843 9847 mice +T229 UBERON:0002048 9867 9876 Pulmonary +T230 GO:0007567 9967 9972 birth +T231 UBERON:0000948 9999 10006 cardiac +T232 UBERON:0000948 10030 10036 hearts +T233 NCBITaxon:10088 10059 10063 mice +T234 UBERON:0002062 10148 10168 endocardial cushions +T235 http://purl.obolibrary.org/obo/MONDO_0018089 10172 10201 double-outlet right ventricle +T236 UBERON:0004145 10179 10185 outlet +T237 UBERON:0002080 10186 10201 right ventricle +T238 UBERON:0002087 10218 10240 atrioventricular canal +T239 UBERON:0002349 10246 10256 myocardium +T240 UBERON:0004125 10311 10324 compact layer +T241 NCBITaxon:10088 10373 10377 mice +T242 GO:0007567 10395 10400 birth +T243 UBERON:0000948 10410 10417 cardiac +T244 UBERON:0002087 10442 10464 atrioventricular-canal +T245 UBERON:0002094 10470 10488 ventricular septal +T246 http://purl.obolibrary.org/obo/MONDO_0002070 10470 10496 ventricular septal defects +T247 UBERON:0009149 10498 10511 ostium primum +T248 http://purl.obolibrary.org/obo/MONDO_0020437 10498 10533 ostium primum atrial septal defects +T249 UBERON:0002085 10512 10525 atrial septal +T250 UBERON:0002081 10548 10553 atria +T251 UBERON:0000948 10613 10620 cardiac +T252 UBERON:0000948 10653 10660 cardiac +T253 GO:0007507 10653 10672 cardiac development +T254 SO:0000996 10776 10791 predicted genes +T255 SO:0000704 10807 10812 genes +T256 PR:000017653 10824 10828 Fog2 +T257 PR:000017653 10852 10856 Fog2 +T258 UBERON:0000948 10862 10869 cardiac +T259 http://purl.obolibrary.org/obo/MONDO_0005453 10862 10877 cardiac defects +T260 NCBITaxon:10088 10934 10938 mice +T261 UBERON:0002087 10950 10972 atrioventricular canal +T262 http://purl.obolibrary.org/obo/MONDO_0020290 10950 10980 atrioventricular canal defects +T263 UBERON:0002349 10990 11000 myocardium +T264 UBERON:0005985 11013 11021 coronary +T265 UBERON:0002049 11022 11033 vasculature +T266 PR:000017653 11088 11092 Fog2 +T267 SO:0000673 11109 11120 transcripts +T268 UBERON:0000922 11161 11168 embryos +T269 SO:0000673 11207 11217 transcript +T270 SO:0000028 11273 11275 bp +T271 GO:0008380 11312 11318 splice +T272 SO:0000147 11354 11358 exon +T273 GO:0008380 11383 11391 splicing +T274 SO:0000028 11435 11437 bp +T275 SO:0000188 11441 11449 intronic +T276 SO:0000673 11475 11485 transcript +T277 SO:0000319 11504 11514 stop codon +T278 NCBITaxon:10088 11604 11608 mice +T279 PR:000017653 11629 11633 Fog2 +T280 SO:0001023 11643 11649 allele +T281 SO:0000704 11671 11675 gene +T282 NCBITaxon:10088 11712 11716 mice +T283 UBERON:0000922 11724 11733 embryonic +T284 PR:000017653 11812 11816 Fog2 +T285 NCBITaxon:10088 11848 11852 mice +T286 PR:000017653 11885 11889 Fog2 +T287 SO:0000673 11960 11970 transcript +T288 GO:0008380 11999 12005 splice +T289 SO:0000162 11999 12010 splice site +T290 UBERON:0002048 12022 12026 Lung +T291 GO:0030324 12022 12026;12041 12052 Lung ... Development +T292 UBERON:0001103 12031 12040 Diaphragm +T293 GO:0060539 12031 12052 Diaphragm Development +T294 PR:000017653 12060 12064 Fog2 +T295 NCBITaxon:10088 12072 12077 Mouse +T296 PR:000017653 12130 12134 Fog2 +T297 UBERON:0002048 12138 12147 pulmonary +T298 GO:0030324 12138 12147;12166 12177 pulmonary ... development +T299 UBERON:0001103 12152 12165 diaphragmatic +T300 GO:0060539 12152 12177 diaphragmatic development +T301 UBERON:0002048 12183 12192 pulmonary +T302 UBERON:0009912 12277 12281 lobe +T303 UBERON:0009912 12316 12320 lobe +T304 UBERON:0001103 12375 12388 diaphragmatic +T305 GO:0060539 12375 12400 diaphragmatic development +T306 UBERON:0002048 12446 12455 pulmonary +T307 PR:000017653 12553 12557 Fog2 +T308 GO:0010467 12561 12570 expressed +T309 UBERON:0004883 12588 12608 pulmonary mesenchyme +T310 GO:0001763 12630 12653 branching morphogenesis +T311 GO:0010467 12667 12677 expression +T312 UBERON:0001135 12699 12713 smooth muscles +T313 UBERON:0001005 12717 12724 airways +T314 UBERON:0002048 12729 12738 pulmonary +T315 UBERON:0000055 12739 12746 vessels +T316 UBERON:0002048 12790 12795 lungs +T317 UBERON:0001103 12853 12866 diaphragmatic +T318 UBERON:0002048 12914 12923 pulmonary +T319 UBERON:0001103 12963 12976 diaphragmatic +T320 UBERON:0002048 13010 13015 lungs +T321 PR:000017653 13036 13040 Fog2 +T322 NCBITaxon:10088 13044 13048 mice +T323 UBERON:0001103 13099 13112 diaphragmatic +T324 PR:000017653 13121 13125 Fog2 +T325 NCBITaxon:10088 13129 13133 mice +T326 UBERON:0002048 13252 13257 Lungs +T327 PR:000017653 13280 13284 Fog2 +T328 UBERON:0000922 13288 13295 embryos +T329 UBERON:0009912 13360 13364 lobe +T330 PR:000017653 13379 13383 Fog2 +T331 UBERON:0002048 13387 13391 lung +T332 UBERON:0009912 13454 13458 lobe +T333 UBERON:0002048 13486 13491 lungs +T334 UBERON:0002048 13612 13621 pulmonary +T335 PR:000017653 13636 13640 Fog2 +T336 NCBITaxon:10088 13648 13652 mice +T337 UBERON:0009912 13703 13708 lobes +T338 UBERON:0009912 13818 13823 lobes +T339 PR:000017653 13860 13864 Fog2 +T340 UBERON:0009912 13900 13905 lobar +T341 PR:000017653 13935 13939 Fog2 +T342 GO:0010467 13940 13950 expression +T343 UBERON:0000922 13975 13982 embryos +T344 UBERON:0009912 14010 14015 lobar +T345 PR:000017653 14051 14055 Fog2 +T346 GO:0010467 14056 14066 expression +T347 UBERON:0009912 14102 14107 lobar +T348 GO:0010467 14114 14124 Expression +T349 NCBITaxon:10088 14142 14146 mice +T350 PR:000033987 14158 14162 lacZ +T351 SO:0000704 14163 14167 gene +T352 PR:000017653 14190 14194 Fog2 +T353 NCBITaxon:10088 14242 14246 mice +T354 UBERON:0002048 14270 14275 lungs +T355 PR:000017653 14309 14313 Fog2 +T356 GO:0010467 14314 14324 expression +T357 UBERON:0003104 14332 14342 mesenchyme +T358 UBERON:0009912 14394 14398 lobe +T359 UBERON:0009912 14418 14423 lobes +T360 PR:000017653 14456 14460 Fog2 +T361 NCBITaxon:10088 14468 14472 mice +T362 GO:0010467 14495 14505 expression +T363 UBERON:0004883 14525 14545 pulmonary mesenchyme +T364 GO:0097617 14583 14596 hybridization +T365 UBERON:0000479 14600 14606 tissue +T366 UBERON:0001103 14618 14628 Diaphragms +T367 PR:000017653 14634 14638 Fog2 +T368 NCBITaxon:10088 14643 14647 mice +T369 UBERON:0001103 14756 14765 diaphragm +T370 CL:0000680 14808 14830 muscle precursor cells +T371 UBERON:0002101 14846 14851 limbs +T372 NCBITaxon:10088 14861 14865 Mice +T373 SO:0000704 14882 14887 genes +T374 GO:0065007 14918 14925 control +T375 UBERON:0001103 14968 14978 diaphragms +T376 CL:0000182 14991 15001 Hepatocyte +T377 PR:000008534 14991 15015 Hepatocyte growth factor +T378 PR:000008534 15016 15030 Scatter factor +T379 PR:000008534 15032 15035 HGF +T380 UBERON:0001103 15132 15141 diaphragm +T381 PR:000008534 15166 15169 HGF +T382 GO:0010467 15173 15182 expressed +T383 UBERON:0001062 15194 15202 anatomic +T384 NCBITaxon:10088 15221 15225 mice +T385 PR:000008534 15246 15249 HGF +T386 PR:000010335 15259 15264 c-Met +T387 UBERON:0009133 15291 15313 pleuroperitoneal folds +T388 UBERON:0009133 15315 15319 PPFs +T389 UBERON:0001103 15346 15356 diaphragms +T390 PR:000017653 15366 15370 Fog2 +T391 GO:0010467 15374 15383 expressed +T392 UBERON:0001103 15417 15426 diaphragm +T393 UBERON:0001103 15473 15482 diaphragm +T394 PR:000012316 15507 15511 Pax3 +T395 PR:000010875 15516 15520 MyoD +T396 UBERON:0009133 15642 15646 PPFs +T397 PR:000017653 15650 15654 Fog2 +T398 NCBITaxon:10088 15659 15663 mice +T399 GO:0010467 15699 15709 expression +T400 GO:0010467 15741 15751 expression +T401 PR:000008534 15755 15758 HGF +T402 UBERON:0001103 15815 15824 diaphragm +T403 PR:000017653 15849 15853 Fog2 +T404 NCBITaxon:10088 15861 15865 mice +T405 PR:000017653 15898 15902 Fog2 +T406 PR:000008534 15968 15971 HGF +T407 UBERON:0001103 15990 15999 diaphragm +T408 GO:0065007 16008 16018 regulation +T409 PR:000008534 16022 16025 HGF +T410 CL:0000680 16055 16076 muscle precursor cell +T411 GO:0016477 16072 16086 cell migration +T412 UBERON:0009133 16099 16102 PPF +T413 UBERON:0001103 16111 16120 diaphragm +T414 NCBITaxon:10088 16166 16170 mice +T415 PR:000017653 16173 16177 FOG2 +T416 UBERON:0001103 16205 16214 Diaphragm +T417 UBERON:0002048 16219 16223 Lung +T418 PR:000017653 16239 16243 FOG2 +T419 UBERON:0000479 16287 16293 tissue +T420 UBERON:0001062 16334 16342 anatomic +T421 UBERON:0001103 16356 16369 diaphragmatic +T422 http://purl.obolibrary.org/obo/MONDO_0005711 16572 16575 CDH +T423 UBERON:0001103 16590 16603 diaphragmatic +T424 UBERON:0001103 16630 16643 diaphragmatic +T425 http://purl.obolibrary.org/obo/MONDO_0006726 16630 16655 diaphragmatic eventration +T426 http://purl.obolibrary.org/obo/MONDO_0005711 16699 16702 CDH +T427 UBERON:0002048 16792 16801 Pulmonary +T428 UBERON:0002048 16832 16836 lung +T429 UBERON:0003215 16866 16874 alveolar +T430 PR:000017653 17066 17070 FOG2 +T431 UBERON:0001004 17155 17166 respiratory +T432 http://purl.obolibrary.org/obo/MONDO_0021113 17155 17174 respiratory failure +T433 GO:0007567 17178 17183 birth +T434 GO:0016265 17188 17192 died +T435 GO:0016265 17225 17231 mortem +T436 UBERON:0002048 17261 17265 lung +T437 UBERON:0000160 17286 17291 bowel +T438 UBERON:0001443 17299 17304 chest +T439 http://purl.obolibrary.org/obo/MONDO_0005711 17343 17346 CDH +T440 UBERON:0002048 17402 17411 pulmonary +T441 UBERON:0002048 17433 17437 lung +T442 UBERON:0002168 17537 17546 left lung +T443 UBERON:0002048 17552 17556 lung +T444 UBERON:0003215 17680 17688 alveolar +T445 UBERON:0002048 17746 17750 lung +T446 UBERON:0001103 17790 17803 diaphragmatic +T447 http://purl.obolibrary.org/obo/MONDO_0006726 17790 17815 diaphragmatic eventration +T448 UBERON:0000211 17877 17888 ligamentous +T449 UBERON:0001103 17906 17919 diaphragmatic +T450 UBERON:0001115 17943 17955;17960 17965 left lobe of ... liver +T451 UBERON:0002107 17997 18002 liver +T452 UBERON:0001103 18043 18052 diaphragm +T453 UBERON:0000948 18168 18173 heart +T454 UBERON:0003978 18230 18235 valve +T455 SO:0000147 18318 18322 exon +T456 SO:0000319 18378 18388 stop codon +T457 SO:0000417 18476 18483 domains +T458 UBERON:0007023 18568 18574 adults +T459 PR:000017653 18843 18847 FOG2 +T460 http://purl.obolibrary.org/obo/MONDO_0005711 18874 18906 Congenital diaphragmatic defects +T461 UBERON:0001103 18885 18898 diaphragmatic +T462 http://purl.obolibrary.org/obo/MONDO_0000001 18936 18945 disorders +T463 UBERON:0012101 19007 19023 perinatal period +T464 GO:0007567 19011 19016 natal +T465 UBERON:0001103 19050 19063 diaphragmatic +T466 UBERON:0001103 19396 19405 diaphragm +T467 GO:0007567 19478 19483 natal +T468 GO:0007567 19502 19507 natal +T469 UBERON:0001443 19508 19513 chest +T470 UBERON:0001103 19541 19554 diaphragmatic +T471 UBERON:0000916 19942 19951 abdominal +T472 UBERON:0002048 19982 19991 pulmonary +T473 UBERON:0001004 20007 20018 respiratory +T474 http://purl.obolibrary.org/obo/MONDO_0005711 20113 20147 congenital diaphragm abnormalities +T475 UBERON:0001103 20124 20133 diaphragm +T476 NCBITaxon:9606 20157 20162 Human +T477 PR:000017653 20163 20167 FOG2 +T478 UBERON:0001103 20235 20244 diaphragm +T479 http://purl.obolibrary.org/obo/MONDO_0005711 20353 20356 CDH +T480 PR:000017653 20426 20430 FOG2 +T481 PR:000017653 20516 20520 FOG2 +T482 GO:0016265 20532 20536 died +T483 http://purl.obolibrary.org/obo/MONDO_0000839 20551 20571 congenital anomalies +T484 http://purl.obolibrary.org/obo/MONDO_0005711 20582 20585 CDH +T485 SO:0000704 20616 20620 gene +T486 PR:000017653 20772 20776 FOG2 +T487 UBERON:0002048 20833 20842 pulmonary +T488 UBERON:0001103 20847 20860 diaphragmatic +T489 NCBITaxon:10088 20889 20894 mouse +T490 NCBITaxon:9606 20899 20904 human +T491 PR:000017653 20939 20943 FOG2 +T492 NCBITaxon:9606 20977 20982 human +T493 UBERON:0001103 20997 21010 diaphragmatic +T494 UBERON:0002048 21015 21024 pulmonary +T495 NCBITaxon:10088 21068 21072 mice +T496 PR:000017653 21109 21113 Fog2 +T497 SO:0000704 21212 21216 gene +T498 NCBITaxon:9606 21277 21283 humans +T499 NCBITaxon:10088 21289 21293 mice +T500 PR:000017653 21323 21327 Fog2 +T501 UBERON:0001103 21328 21341 diaphragmatic +T502 http://purl.obolibrary.org/obo/MONDO_0005711 21386 21389 CDH +T503 UBERON:0001103 21493 21502 diaphragm +T504 UBERON:0004290 21528 21540 dermomyotome +T505 UBERON:0005434 21544 21552 cervical +T506 UBERON:0002329 21553 21560 somites +T507 UBERON:0001103 21590 21599 diaphragm +T508 UBERON:0009133 21619 21622 PPF +T509 UBERON:0000479 21639 21645 tissue +T510 UBERON:0000309 21684 21693 body wall +T511 UBERON:0003283 21701 21721 esophageal mesentery +T512 UBERON:0004161 21751 21769 septum transversum +T513 UBERON:0009133 21804 21807 PPF +T514 GO:0008283 21827 21838 proliferate +T515 UBERON:0002228 21896 21902 costal +T516 UBERON:0000975 21904 21911 sternal +T517 UBERON:0002228 21912 21918 costal +T518 UBERON:0000978 21924 21930 crural +T519 UBERON:0001103 21957 21966 diaphragm +T520 UBERON:0009133 21986 21989 PPF +T521 GO:0060539 22037 22049;22054 22063 formation of ... diaphragm +T522 UBERON:0001103 22054 22063 diaphragm +T523 PR:000017653 22093 22097 Fog2 +T524 PR:000008534 22138 22141 HGF +T525 GO:0010467 22142 22152 expression +T526 CL:0000680 22181 22203 muscle precursor cells +T527 UBERON:0001103 22232 22241 diaphragm +T528 PR:000017653 22325 22329 Fog2 +T529 UBERON:0001103 22337 22346 diaphragm +T530 PR:000012316 22357 22361 Pax3 +T531 PR:000010875 22366 22370 MyoD +T532 GO:0010467 22371 22381 expression +T533 UBERON:0009133 22401 22404 PPF +T534 CL:0000680 22467 22488 muscle precursor cell +T535 GO:0016477 22484 22498 cell migration +T536 GO:0030154 22484 22488;22503 22518 cell ... differentiation +T537 UBERON:0009133 22557 22560 PPF +T538 CL:0000680 22586 22607 muscle precursor cell +T539 GO:0016477 22603 22617 cell migration +T540 UBERON:0009133 22630 22633 PPF +T541 UBERON:0001103 22653 22662 diaphragm +T542 PR:000017653 22664 22668 Fog2 +T543 PR:000007857 22712 22719 Gatas 1 +T544 PR:000007861 22712 22717;22720 22721 Gatas ... 6 +T545 PR:000011405 22770 22778 CoupTFII +T546 PR:000017653 22807 22811 Fog2 +T547 PR:000007859 22812 22817 Gata4 +T548 UBERON:0000948 22853 22860 cardiac +T549 GO:0007507 22853 22860;22873 22884 cardiac ... development +T550 UBERON:0000991 22865 22872 gonadal +T551 GO:0008406 22865 22884 gonadal development +T552 UBERON:0002048 22917 22921 lung +T553 UBERON:0001103 22926 22935 diaphragm +T554 UBERON:0002048 22983 22992 pulmonary +T555 UBERON:0001103 23066 23075 diaphragm +T556 UBERON:0002048 23084 23093 Pulmonary +T557 UBERON:0001103 23133 23146 diaphragmatic +T558 UBERON:0001062 23147 23154 anatomy +T559 UBERON:0001103 23239 23252 diaphragmatic +T560 UBERON:0001884 23307 23320 phrenic nerve +T561 http://purl.obolibrary.org/obo/MONDO_0005711 23371 23374 CDH +T562 NCBITaxon:9940 23430 23434 lamb +T563 UBERON:0002048 23482 23491 pulmonary +T564 GO:0030324 23482 23505 pulmonary developmental +T565 UBERON:0001103 23558 23571 diaphragmatic +T566 CHEBI:50905 23620 23631 teratogenic +T567 http://purl.obolibrary.org/obo/MONDO_0005711 23641 23644 CDH +T568 UBERON:0009912 23759 23764 lobar +T569 http://purl.obolibrary.org/obo/MONDO_0005711 23795 23798 CDH +T570 http://purl.obolibrary.org/obo/MONDO_0000001 23839 23847 disorder +T571 UBERON:0002048 23895 23904 pulmonary +T572 NCBITaxon:10088 23935 23939 mice +T573 PR:000017653 23962 23966 Fog2 +T574 UBERON:0002048 24008 24012 lung +T575 GO:0030324 24008 24024 lung development +T576 UBERON:0009912 24072 24076 lobe +T577 UBERON:0009912 24114 24118 lobe +T578 UBERON:0009912 24133 24138 lobar +T579 PR:000017653 24171 24175 Fog2 +T580 GO:0010467 24176 24186 expression +T581 UBERON:0009912 24208 24213 lobar +T582 PR:000017653 24229 24233 Fog2 +T583 GO:0010467 24234 24244 expression +T584 UBERON:0004883 24263 24283 pulmonary mesenchyme +T585 UBERON:0009912 24290 24295 lobar +T586 GO:0010467 24354 24363 expressed +T587 UBERON:0003104 24371 24381 mesenchyme +T588 UBERON:0009912 24411 24415 lobe +T589 UBERON:0009912 24444 24449 lobes +T590 UBERON:0009912 24499 24503 lobe +T591 UBERON:0009912 24518 24522 lobe +T592 PR:000017653 24547 24551 Fog2 +T593 UBERON:0009912 24609 24614 lobes +T594 PR:000017653 24649 24653 Fog2 +T595 PR:000017653 24695 24699 Fog2 +T596 UBERON:0002048 24700 24705 lungs +T597 PR:000017653 24797 24801 Fog2 +T598 UBERON:0002048 24802 24807 lungs +T599 UBERON:0009912 24868 24873 lobes +T600 UBERON:0002048 24923 24928 lungs +T601 PR:000017653 24995 24999 Fog2 +T602 NCBITaxon:10088 25007 25012 mouse +T603 UBERON:0001103 25046 25059 diaphragmatic +T604 UBERON:0002048 25088 25097 pulmonary +T605 SO:0000704 25161 25165 gene +T606 UBERON:0002048 25204 25208 lung +T607 GO:0030324 25204 25208;25223 25234 lung ... development +T608 UBERON:0001103 25213 25222 diaphragm +T609 GO:0060539 25213 25234 diaphragm development +T610 PR:000017653 25297 25301 Fog2 +T611 PR:000017653 25334 25338 Fog2 +T612 NCBITaxon:10088 25349 25354 mouse +T613 UBERON:0001103 25368 25377 diaphragm +T614 GO:0060539 25368 25377;25387 25398 diaphragm ... development +T615 UBERON:0002048 25382 25386 lung +T616 GO:0030324 25382 25398 lung development +T617 NCBITaxon:9606 25618 25623 human +T618 http://purl.obolibrary.org/obo/MONDO_0000001 25624 25631 disease +T619 NCBITaxon:33208 25818 25824 Animal +T620 NCBITaxon:33208 25860 25866 Animal +T621 SO:0000704 25929 25936 Genetic +T622 NCBITaxon:10088 25952 25957 mouse +T623 NCBITaxon:10088 26059 26063 mice +T624 SO:0000704 26073 26080 genetic +T625 NCBITaxon:10088 26167 26171 Mice +T626 PR:000017653 26200 26204 Fog2 +T627 SO:0000704 26218 26222 gene +T628 NCBITaxon:10088 26309 26313 mice +T629 GO:0007565 26322 26333 pregnancies +T630 UBERON:0000922 26376 26383 embryos +T631 UBERON:0000922 26385 26392 Embryos +T632 UBERON:0000922 26472 26479 embryos +T633 UBERON:0001103 26547 26556 diaphragm +T634 UBERON:0002048 26558 26563 lungs +T635 UBERON:0000948 26569 26574 heart +T636 UBERON:0002048 26594 26599 lungs +T637 UBERON:0007196 26604 26625 tracheobronchial tree +T638 UBERON:0001103 26658 26668 diaphragms +T639 UBERON:0000915 26694 26702 thoracic +T640 UBERON:0000479 26703 26709 tissue +T641 UBERON:0000922 26731 26738 embryos +T642 UBERON:0002048 26744 26748 lung +T643 UBERON:0002048 26766 26771 lungs +T644 UBERON:0000922 26798 26805 embryos +T645 CHEBI:33284 26952 26960 nutrient +T646 CHEBI:60004 26961 26968 mixture +T647 NCBITaxon:27592 27060 27066 bovine +T648 UBERON:0001977 27067 27072 serum +T649 CHEBI:17334 27110 27120 penicillin +T650 CHEBI:17076 27133 27145 streptomycin +T651 CHEBI:2682 27163 27177 amphotericin B +T652 UBERON:0002048 27179 27183 Lung +T653 CHEBI:16526 27230 27233 CO2 +T654 NCBITaxon:10088 27397 27401 mice +T655 PR:000033987 27415 27419 lacZ +T656 SO:0000704 27420 27424 gene +T657 PR:000017653 27439 27443 Fog2 +T658 SO:0000167 27444 27452 promoter +T659 NCBITaxon:33208 27498 27505 animals +T660 PR:000033987 27511 27515 lacZ +T661 SO:0000704 27516 27520 gene +T662 PR:000017653 27561 27565 Fog2 +T663 GO:0010467 27597 27607 expression +T664 SO:0001817 27628 27636 in frame +T665 PR:000017653 27675 27679 FOG2 +T666 PR:000017653 27693 27697 Fog2 +T667 PR:000033987 27698 27702 lacZ +T668 SO:0000243 27728 27732 ires +T669 SO:0005853 27738 27746 cassette +T670 SO:0001023 27768 27774 allele +T671 PR:000017653 27778 27782 Fog2 +T672 SO:0000704 27783 27787 gene +T673 PR:000017653 27793 27797 Fog2 +T674 PR:000033987 27798 27802 LacZ +T675 CL:0002322 27875 27883 ES cells +T676 UBERON:0000358 27984 27995 blastocysts +T677 PR:000017653 27997 28001 Fog2 +T678 PR:000033987 28002 28006 lacZ +T679 NCBITaxon:33208 28012 28019 animals +T680 PR:000033987 28074 28078 lacZ +T681 GO:0010467 28079 28089 Expression +T682 UBERON:0000922 28109 28118 embryonic +T683 UBERON:0002048 28119 28124 lungs +T684 CHEBI:75055 28184 28189 X-gal +T685 NCBITaxon:10088 28254 28259 mouse +T686 UBERON:0000915 28308 28316 thoracic +T687 UBERON:0005291 28317 28333 embryonic tissue +T688 SO:0000112 28366 28372 primer +T689 PR:000017653 28400 28404 Fog2 +T690 SO:0000704 28405 28409 gene +T691 SO:0000112 28449 28456 primers +T692 GO:0008380 28482 28489 spliced +T693 SO:0000842 28490 28499;28504 28508 region of ... gene +T694 SO:0000006 28617 28628 PCR product +T695 SO:0000440 28652 28658 vector +T696 SO:0000704 28755 28759 gene +T697 SO:0000112 28769 28776 primers +T698 SO:0000704 28827 28831 Gene +T699 GO:0097617 28885 28898 hybridization +T700 CHEBI:73702 28945 28948 wax +T701 GO:0097617 29002 29015 hybridization +T702 CHEBI:37983 29055 29058 35S +T703 SO:0000155 29112 29119 plasmid +T704 GO:0097617 29134 29144 hybridized +T705 UBERON:0000479 29148 29154 tissue +T706 GO:0005634 29165 29171 Nuclei +T707 NCBITaxon:9606 29279 29284 Human +T708 CHEBI:15882 29365 29371 phenol +T709 CHEBI:35255 29372 29382 chloroform +T710 UBERON:0000479 29419 29426 tissues +T711 SO:0000112 29451 29458 Primers +T712 PR:000017653 29484 29488 FOG2 +T713 SO:0000195 29489 29501 coding exons +T714 SO:0000028 29510 29512 bp +T715 SO:0000357 29516 29524 flanking +T716 SO:0000112 29628 29634 Primer +T717 SO:0000704 29722 29726 Gene +T718 UBERON:0000178 29805 29810 blood +T719 UBERON:0000178 29836 29841 blood +T720 SO:0000694 30043 30046 SNP +T721 SO:0000112 30226 30233 Primers +T722 NCBITaxon:10088 30246 30251 Mouse +T723 SO:0000673 30280 30290 Transcript +T724 PR:000017653 30294 30298 Fog2 +T725 NCBITaxon:10088 30312 30316 Mice +T726 NCBITaxon:9606 30379 30384 Human +T727 SO:0000112 30385 30392 Primers +T728 PR:000017653 30414 30418 FOG2 +T729 SO:0001026 30440 30447 Genomic +T730 UBERON:0001103 30663 30676 diaphragmatic +T731 UBERON:0002048 30681 30685 lung +T732 NCBITaxon:9606 30758 30763 Human +T733 PR:000017653 30976 30980 Fog2 +T734 NCBITaxon:10088 30984 30988 mice +T735 http://purl.obolibrary.org/obo/MONDO_0005711 31122 31125 CDH +T736 http://purl.obolibrary.org/obo/MONDO_0005711 31128 31159 congenital diaphragmatic hernia +T737 UBERON:0001103 31139 31152 diaphragmatic +T738 UBERON:0000922 31173 31182 embryonic +T739 CHEBI:23995 31197 31200 ENU +T740 CHEBI:23995 31203 31224 N-ethyl-N-nitrosourea +T741 PR:000008534 31226 31229 HGF +T742 CL:0000182 31232 31242 Hepatocyte +T743 PR:000008534 31232 31256 Hepatocyte growth factor +T744 PR:000008534 31257 31271 Scatter factor +T745 UBERON:0002048 31286 31290 lung +T746 UBERON:0009133 31292 31295 PPF +T747 UBERON:0009133 31298 31319 pleuroperitoneal fold +T748 UBERON:0002048 31349 31358 Pulmonary +T749 GO:0030324 31349 31358;31377 31388 Pulmonary ... Development +T750 UBERON:0001103 31363 31376 Diaphragmatic +T751 GO:0060539 31363 31388 Diaphragmatic Development +T752 NCBITaxon:10088 31400 31405 Mouse +T753 UBERON:0002048 31434 31438 lung +T754 UBERON:0009912 31486 31490 lobe +T755 UBERON:0009912 31536 31540 lobe +T756 UBERON:0001103 31608 31618 diaphragms +T757 UBERON:0001103 31728 31737 diaphragm +T758 UBERON:0001103 31763 31772 diaphragm +T759 CHEBI:23995 31803 31806 ENU +T760 PR:000017653 31831 31835 Fog2 +T761 GO:0008380 31841 31847 Splice +T762 SO:0000162 31841 31852 Splice Site +T763 SO:0000673 31896 31906 transcript +T764 NCBITaxon:10088 31910 31914 mice +T765 NCBITaxon:10088 31963 31968 mouse +T766 NCBITaxon:10088 32003 32008 mouse +T767 PR:000017653 32026 32030 Fog2 +T768 GO:0008380 32042 32048 splice +T769 SO:0000162 32042 32053 splice site +T770 SO:0000028 32095 32097 bp +T771 SO:0000188 32101 32109 intronic +T772 NCBITaxon:10088 32133 32138 mouse +T773 SO:0001587 32158 32178 premature stop codon +T774 PR:000017653 32226 32230 Fog2 +T775 GO:0010467 32234 32243 Expressed +T776 UBERON:0002048 32262 32266 Lung +T777 UBERON:0001103 32271 32280 Diaphragm +T778 PR:000017653 32282 32286 Fog2 +T779 GO:0010467 32290 32299 expressed +T780 UBERON:0004883 32315 32335 pulmonary mesenchyme +T781 UBERON:0003104 32362 32372 mesenchyme +T782 UBERON:0004242 32399 32408;32422 32435 bronchial ... smooth muscle +T783 UBERON:0001135 32437 32439 sm +T784 PR:000017653 32455 32459 Fog2 +T785 GO:0010467 32463 32472 expressed +T786 UBERON:0001103 32501 32510 diaphragm +T787 UBERON:0001103 32512 32515 Dia +T788 PR:000017653 32593 32597 Fog2 +T789 UBERON:0002048 32623 32627 Lung +T790 GO:0030324 32623 32639 Lung Development +T791 PR:000017653 32641 32645 Fog2 +T792 UBERON:0002048 32651 32656 lungs +T793 UBERON:0001103 32674 32687 diaphragmatic +T794 GO:0040007 32708 32713 grown +T795 UBERON:0009912 32741 32745 lobe +T796 UBERON:0009912 32769 32773 lobe +T797 UBERON:0002048 32824 32829 lungs +T798 PR:000017653 32842 32846 Fog2 +T799 GO:0010467 32847 32857 Expression +T800 UBERON:0000922 32861 32870 Embryonic +T801 UBERON:0002048 32882 32887 Lungs +T802 UBERON:0000922 32892 32901 embryonic +T803 UBERON:0002048 32913 32918 lungs +T804 PR:000017653 32920 32924 Fog2 +T805 GO:0010467 32940 32949 expressed +T806 UBERON:0009912 33012 33017 lobes +T807 GO:0010467 33070 33080 expression +T808 PR:000008534 33118 33121 HGF +T809 PR:000017653 33148 33152 Fog2 +T810 NCBITaxon:10088 33158 33162 Mice +T811 GO:0097617 33172 33185 hybridization +T812 PR:000008534 33189 33192 HGF +T813 PR:000017653 33220 33224 Fog2 +T814 UBERON:0000922 33232 33239 embryos +T815 GO:0010467 33263 33273 expression +T816 UBERON:0009133 33298 33301 PPF +T817 UBERON:0001103 33323 33332 diaphragm +T818 UBERON:0002107 33334 33336 Li +T819 UBERON:0002107 33338 33343 liver +T820 UBERON:0002048 33345 33347 Lu +T821 UBERON:0002048 33349 33353 lung +T822 PR:000017653 33366 33370 FOG2 +T823 UBERON:0001103 33398 33407 Diaphragm +T824 UBERON:0002048 33412 33416 Lung +T825 GO:0016265 33510 33514 died +T826 GO:0007567 33518 33523 birth +T827 UBERON:0002048 33536 33545 pulmonary +T828 UBERON:0001103 33578 33591 diaphragmatic +T829 http://purl.obolibrary.org/obo/MONDO_0006726 33578 33603 diaphragmatic eventration +T830 http://purl.obolibrary.org/obo/MONDO_0005711 33639 33642 CDH +T831 SO:0000417 33710 33717 domains +T832 CHEBI:33893 34070 34078 reagents +T833 PR:000017653 34216 34220 Fog2 +T834 UBERON:0001103 34244 34253 diaphragm +T835 GO:0060539 34244 34253;34263 34274 diaphragm ... development +T836 UBERON:0002048 34258 34262 lung +T837 GO:0030324 34258 34274 lung development +T838 NCBITaxon:10088 34278 34282 mice +T839 NCBITaxon:9606 34287 34293 humans diff --git a/src/ontogpt/evaluation/craft/database/all/16103912.txt b/src/ontogpt/evaluation/craft/database/all/16103912.txt new file mode 100644 index 000000000..ffcff4da2 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16103912.txt @@ -0,0 +1,199 @@ +Fog2 Is Required for Normal Diaphragm and Lung Development in Mice and Humans + +Abstract + +Congenital diaphragmatic hernia and other congenital diaphragmatic defects are associated with significant mortality and morbidity in neonates; however, the molecular basis of these developmental anomalies is unknown. In an analysis of E18.5 embryos derived from mice treated with N-ethyl-N-nitrosourea, we identified a mutation that causes pulmonary hypoplasia and abnormal diaphragmatic development. Fog2 (Zfpm2) maps within the recombinant interval carrying the N-ethyl-N-nitrosourea-induced mutation, and DNA sequencing of Fog2 identified a mutation in a splice donor site that generates an abnormal transcript encoding a truncated protein. Human autopsy cases with diaphragmatic defect and pulmonary hypoplasia were evaluated for mutations in FOG2. Sequence analysis revealed a de novo mutation resulting in a premature stop codon in a child who died on the first day of life secondary to severe bilateral pulmonary hypoplasia and an abnormally muscularized diaphragm. Using a phenotype-driven approach, we have established that Fog2 is required for normal diaphragm and lung development, a role that has not been previously appreciated. FOG2 is the first gene implicated in the pathogenesis of nonsyndromic human congenital diaphragmatic defects, and its necessity for pulmonary development validates the hypothesis that neonates with congenital diaphragmatic hernia may also have primary pulmonary developmental abnormalities. + +Synopsis + + + +Birth defects involving the diaphragm are as common and as serious as genetic disorders such as cystic fibrosis, yet the underlying causes of these defects are unknown. Most babies born with diaphragmatic defects have very small lungs, and many die in the newborn period with severe breathing difficulties. It is unknown whether the small lungs occur because these children have a diaphragmatic defect, or whether some patients might have a genetic abnormality that affects the development of both the lung and the diaphragm simultaneously. + +In a screen of fetal mice carrying chemically induced genetic mutations, the authors found that a mutation in the gene Fog2 (Friend of gata 2), causes abnormal diaphragm development and small lungs. The lungs have a primary developmental abnormality that includes the specific loss of one lung lobe. Based on this result, the authors studied children affected with diaphragmatic abnormalities, and identified one human baby with a serious mutation in the human gene FOG2 who died at five hours of life with severe breathing difficulties, a diaphragmatic defect, and small lungs. + +The authors have established that Fog2 is necessary for both diaphragm and lung development in mice and in humans. This is the first known cause of a nonsyndromic congenital diaphragmatic defect and establishes that some patients may have a primary developmental abnormality of both the lung and the diaphragm. + +Introduction + +Congenital diaphragmatic defects are a spectrum of relatively common birth defects. The Bochdalek or posterolateral hernias (often referred to as congenital diaphragmatic hernia [CDH]) occur in 1/3,000 live births [1], and although these are the most common type of diaphragmatic defect presenting at birth, diaphragmatic aplasia and diaphragmatic muscularization defects (eventrations) may have a similar clinical presentation. + +Making specific anatomic distinctions among these types of defects can be difficult without direct gross (intraoperative or postmortem) examination. Pulmonary hypoplasia associated with these diaphragmatic defects causes severe mortality and morbidity. The pathogenesis and developmental relationship between diaphragmatic defects and pulmonary hypoplasia is not understood. Although advances in the medical management of pulmonary hypoplasia may have decreased the mortality associated with CDH patients who survive to receive care at high-volume centers [2,3], the population-based mortality has been reported to be as great as 62%, and there are a large number of deaths prior to birth or to transfer to a tertiary care facility [4]. As these patients commonly present with severe respiratory failure at birth, therapy has been centered around developing better methods to provide ventilatory support while not producing further lung injury. Extracorporeal membrane oxygenation (ECMO) is used in some centers to provide an extended period of cardiopulmonary bypass [5,6], while other centers have success using other ventilatory support techniques [7]. The morbidity in those who survive is high, and many patients survive with chronic respiratory insufficiency, cognitive and neuromotor deficits, and hearing loss as a result of necessary intensive interventions and associated structural and irreversible developmental abnormalities [8–11]. + +To date, there have been no specific mutations found to be associated with nonsyndromic diaphragmatic defects and pulmonary hypoplasia in humans. The heritability of these defects is unclear, as the high morbidity and mortality limit the collection of multigenerational families for analysis. The genetic etiologies are likely to be complex and probably arise from different mutations in various parts of the molecular developmental pathways required for diaphragmatic development. Indeed, there are numerous reports implicating different chromosomal abnormalities in the pathogenesis of CDH [12,13]. Given the difficulty of studying lethal developmental abnormalities in humans, it is of great potential utility to develop animal models of human birth defects, as the specific genetic abnormalities found in animal models can then be investigated in human populations. + +We screened mice treated with the chemical mutagen N-ethyl-N-nitrosourea (ENU) for lines with developmental defects that present in the perinatal period [14]. From this screen, we identified a line of mice carrying a recessive mutation that results in primary pulmonary hypoplasia and abnormal diaphragmatic and cardiac development. Positional cloning analysis identified Fog2 (Zfpm2) as a likely candidate, and DNA sequencing revealed a mutation in a splice donor site that generates an abnormal transcript encoding a truncated protein. This result suggested that we examine the orthologous gene in humans with similar developmental defects, and we report the finding of a de novo nonsense mutation in FOG2 in a patient who died at birth with a diaphragmatic defect and severe pulmonary hypoplasia. This is the first reported mutation associated with these abnormalities in a human. We present additional data that provide direct evidence that pulmonary hypoplasia may be a primary component of this spectrum of disorders. + +Results + +Identification of the little lung Mutation in Fog2 + +We screened third-generation progeny of ENU-mutagenized mice at embryonic day 18.5 (E18.5) for abnormal developmental phenotypes [14]. One line of mice was found to have multiple embryos in independent litters that displayed pulmonary hypoplasia and a thin diaphragm. The mutation, which we called little lung (lil), was mapped to Chromosome 15 by utilizing a strategy of interval haplotype analysis (data not shown) [15]. For high-resolution mapping, F2 progeny from two crosses were analyzed. In 450 progeny of an intercross of F1 (A/J × FVB/N) lil/+ mice, the interval containing the mutation was defined by 19 recombinants between D15Mit220 and D15Mit154. Because of the lack of informative markers within this interval, an additional 39 F2 progeny from an A/J × C57BL/6J cross were tested. The identification of two recombinants established D15Mit85 as the proximal and D15Mit5 as the distal flanking markers. + +The lil phenotype was identified at E18.5 to have bilateral pulmonary hypoplasia and an abnormal diaphragm. Pulmonary hypoplasia was apparent in all mutant mice that survived to birth. In a comparison between wild-type and mutant mice found dead on day one of life, body weights were not different; however, the lung weights were significantly lower in the mutant mice. The average mutant lung weight was 9.6 ± 2.5 mg while the average wild-type lung weight was 26.4 ± 4.6 mg (p < 0.001). All lungs from mutant mice were lacking an accessory lobe on the right side and had underdevelopment of the anterior right middle lobe (Figure 1A). Diaphragms from mutant lil mice were intact, but muscularization was absent in the dorsal regions. Myotubules were present in a limited and abnormal distribution. More specifically, myotubules normally radiate in a mediolateral fashion to meet the lateral body walls, with a normal paucity of muscle fibers in the central tendonous region. In the mutant diaphragm, myotubules radiated in a dorsal–ventral orientation, and muscle tissue did not meet the entire surface of the body walls (Figure 1B). + +The number of lil mutant mice that survived to birth was less than 5% of total progeny, rather than the 25% expected for a recessive mutation. We evaluated litters at different embryonic time points to determine whether the reduced number was due to intra-uterine demise. Litters were collected at E12.5, 13.5, 15.5, 17.5, and 18.5, and embryos were genotyped and evaluated for evidence of intra-uterine demise, including growth retardation, pallor, and tissue friability. lil embryos had a progressively higher rate of demise between E13.5 and E15.5. At E12.5, 20 out of 87 (23%) were homozygous for the mutation, and all of these appeared viable. At E13.5, 13 of 49 (27%) were mutant and two had died. At E15.5, 22 out of 91 (24%) were mutant and the majority of mutant embryos (17 out of the 22) had died. By E18.5, nine of 29 embryos (31%) were homozygous for the mutation, of which only one embryo was viable. Diaphragmatic muscularization was abnormal in all mutant mice examined (n > 25). Pulmonary hypoplasia was observed in 100% of mutants evaluated for that phenotype between E11.5 and birth (n > 50). + +Examination of cardiac morphology showed that hearts from E15.5 lil mutant mice had a variety of developmental defects, including enlarged and abnormally developed endocardial cushions, a double-outlet right ventricle, and a complete atrioventricular canal. The myocardium was also poorly developed, with thinning of the outer compact layer and decreased vascularity. The cohort of mutant mice that survived to birth also had cardiac malformations including atrioventricular-canal-type ventricular septal defects, ostium primum atrial septal defects, and enlarged atria (data not shown). All mutants specifically evaluated for a cardiac phenotype (n = 10) had abnormal cardiac development. + +Examination of the 3-Mb region between D15Mit85 and D15Mit5 in DNA sequence databases revealed three predicted genes and four known genes, including Fog2. Targeted mutations of Fog2 have cardiac defects strikingly similar to those we identified in lil mutant mice, including atrioventricular canal defects, thinned myocardium, and absent coronary vasculature [16]. RT-PCR amplification of the proximal portion of Fog2 revealed longer transcripts in the lil mutant than in the wild-type embryos (Figure 2A). Sequencing of the mutant transcript revealed a point mutation (from thymine to cytosine) 2 bp after position 301, which is in the splice donor site at the end of the third exon. This mutation causes a splicing defect that results in the insertion of 85 bp of intronic sequence into the mutant transcript, and introduces a stop codon that generates a severely truncated protein product (Figure 2B). Heterozygous lil mutant mice were crossed with a Fog2+/− (null allele) mutant generated by gene targeting [16]. Doubly heterozygous mice had an embryonic lethal phenotype; this failure to complement proves that lil is a mutation in Fog2. The variable phenotype of lil mice (relative to that found for the Fog2 null mutant) is likely due to the generation of a low level of normal transcript despite the presence of the splice site mutation. + +Lung and Diaphragm Development in the Fog2 Mutant Mouse + +Experiments were conducted to evaluate the role of Fog2 in pulmonary and diaphragmatic development. The pulmonary phenotype is characterized by diffuse hypoplasia and specific loss of the accessory lobe and a portion of the right middle lobe. + +It is well established that abnormalities in either diaphragmatic development or fetal breathing can result in a secondary pulmonary hypoplasia, although loss of normal structure has never been documented in this context [17,18]. Fog2 is expressed diffusely in the pulmonary mesenchyme during the period of branching morphogenesis, while later expression is restricted to the smooth muscles of airways and pulmonary vessels (Figure 3). This, and the observation that lungs appeared small on transverse sections evaluated prior to diaphragmatic muscularization or function, suggests that the pulmonary hypoplasia occurs independently of the diaphragmatic defect. To test this hypothesis, lungs were dissected from Fog2−/− mice and littermate controls before the onset of fetal diaphragmatic motion. Fog2−/− mice were used for this experiment, as we wanted to avoid potential phenotypic variance from the lil hypomorphic mutation. Lungs dissected at E12 from Fog2−/− embryos were smaller in size and lacked the development of an accessory lobe. In 11 viable Fog2−/− lung culture explants, there was never development of an accessory lobe, and the weights of mutant lungs cultured for 5 d were significantly lower than those of littermate controls (Figure 4). These data demonstrate that the pulmonary hypoplasia in Fog2 mutant mice is a primary defect. + +Branching in the unaffected lobes appeared to be delayed by 6–12 h, but all mutants developed an intricate branching pattern in the unaffected lobes after culture for 5 d. + +Because the Fog2 phenotype is striking for specific lobar loss, the spatial pattern of Fog2 expression was evaluated in normal embryos during the period of early lobar establishment to determine whether Fog2 expression is specifically different at these lobar buds. Expression was evaluated in mice carrying a lacZ gene incorporated into the Fog2 locus (S. Tevosian, unpublished data). In nine mice examined at E11.5, all lungs showed a specific enhancement of Fog2 expression in the mesenchyme surrounding the accessory bud and the right middle lobe bud, which are the lobes that do not develop normally in Fog2 mutant mice (Figure 5). By E12.5, expression was diffuse in the pulmonary mesenchyme, as was seen previously with in situ hybridization on tissue sections. + +Diaphragms from Fog2 lil mice show an intact membrane with a defect in muscular patterning (see Figure 1B). The membranous portion of the diaphragm is populated by a migratory population of muscle precursor cells, much like the limbs [19,20]. Mice with defects in genes known to be important for the control of this process have intact but amuscular diaphragms [17,21,22]. Hepatocyte growth factor/Scatter factor (HGF) is one potential candidate responsible for the guidance of muscle precursors to the membranous diaphragm. It has been shown that HGF is expressed along this anatomic pathway [23], and mice with absence of the HGF receptor c-Met fail to form muscularized pleuroperitoneal folds (PPFs), and thus have amuscular diaphragms [24,25]. Fog2 is expressed diffusely in the early amuscular diaphragm at E11.5 as well as in the later muscularized diaphragm (see Figure 3C and 3D). Pax3 and MyoD, transcription factors required for appropriate migration and determination of myogenic precursors, were detected in the PPFs of Fog2 lil mice (data not shown). However, in situ expression analysis demonstrated that the expression of HGF in the region where this structure meets the membranous diaphragm was markedly reduced in Fog2 mutant mice (Figure 6). We hypothesize that Fog2 is required (either directly or indirectly) for the induction of HGF in the developing diaphragm, and dysregulation of HGF patterning along the path of muscle precursor cell migration between the PPF and the diaphragm accounts for the abnormal phenotype in these mice. + +FOG2 Mutation in a Patient with Diaphragm and Lung Abnormalities + +FOG2 sequence analysis was performed on autopsy tissue from 30 of 32 deceased children with an anatomic diagnosis of diaphragmatic defect evaluated at the Children's Hospital in Boston, Massachusetts, between 1993 and 2003. Autopsy reports were reviewed to determine the specific diagnoses. Of these 30 cases, 17 (57%) had Bochdalek CDH, two (7%) had diaphragmatic agenesis, seven (23%) had diaphragmatic eventration/muscularization defects (without Bochdalek CDH), and four (13%) had Bochdalek hernia of one hemidiaphragm and eventration of the other. Pulmonary hypoplasia was assessed using lung/body weight ratio and radial alveolar counts [26]. The material available for review included written reports and histologic slides in all cases and gross kodachromes in a subset of cases. + +One child carried a highly significant FOG2 sequence change. The patient was a full-term 3,500-g baby girl who developed severe respiratory failure at birth and died after 5 h of resuscitation. Antemortem radiographs showed opacified lung fields and possible bowel in the chest. The patient's clinical diagnosis was CDH. + +Review of autopsy material revealed severe bilateral pulmonary hypoplasia (combined lung weight, 11.1 g; expected for body length/weight, 46.8 ± 26.2 g; [27]), most markedly involving the left lung. The lung/body weight ratio was 0.0037 (expected > 0.010) [28]. There were a reduced number of bronchial generations, and the radial alveolar count was 2–3 (expected = 5) [29]. There were incomplete lung fissures bilaterally. A deep posterior diaphragmatic eventration was present on the left side. Additionally, two muscularized ligamentous bands resembling diaphragmatic remnants encircled the left lobe of the liver, creating an abnormal fissured liver contour. Away from the eventration, the diaphragm appeared well muscularized, measuring 0.3 cm in thickness. A complete autopsy revealed no other malformations; the heart was determined to be grossly normal and was donated for valve harvest. + +Sequence analysis revealed a cytosine to thymine heterozygous change in exon 4 that changes the 112th amino acid from arginine to a stop codon. This mutation produces a severely truncated peptide that does not contain zinc finger domains (Figure 7). This base change was not present in the analysis of DNA from 400 normal adults. To assess the likelihood that the mutation was causal for the developmental phenotype, we examined both parents. Paternity was confirmed, and sequence analysis revealed that the neither parent carried this mutation, proving that the patient had a de novo mutation in FOG2 (Figure 7). + + Discussion + +Congenital diaphragmatic defects are a heterogeneous group of disorders of unknown etiology. The defects that present in the pre- or perinatal period include Bochdalek hernia, diaphragmatic aplasia, and various degrees of muscularization defects or eventrations. Different types of defects occur in the same patients or in siblings, suggesting these represent variable expression of the same underlying pathogenesis [30,31]. Clinical differentiation between these defects may be very difficult, as the residual membranous diaphragm of a muscularization defect is thin and may not be easily visible on prenatal ultrasound or postnatal chest radiographs [32]. Although diaphragmatic muscularization defects were historically considered to be predictive of a good outcome, there have been inadequate population-based studies that include fetal or neonatal cases and autopsy diagnoses to make this conclusion definitive. In fact, the series of patients we report here and the published literature indicate that an eventration defect may be associated with displacement of abdominal contents and also with severe pulmonary hypoplasia and respiratory insufficiency [33,34]. + +Numerous chromosome abnormalities have been found in association with congenital diaphragm abnormalities [12,35]. Human FOG2 maps to Chromosome 8q23.1, and, importantly, several patients with diaphragm defects and rearrangements involving this locus have been reported. Specifically, there are three unrelated CDH patients with cytogenetically balanced translocations at or near the FOG2 locus [36,37]. Additionally, two patients with deletions apparently encompassing the FOG2 locus have died from multiple congenital anomalies including CDH [38–40]. Inactivation of this gene due to chromosomal rearrangement or deletion would result in a heterozygous null mutation similar to that found in the patient we report. + +Because the FOG2 mutation we report is de novo and the phenotypes of the pulmonary and diaphragmatic defects are similar between mouse and human, we suggest that this mutation in FOG2 is the first reported cause of a human developmental diaphragmatic and pulmonary defect. In contrast to the affected child, mice heterozygous for a null mutation of Fog2 appear normal. However, there is ample precedent for the observation that haploinsufficiency of a gene with developmental functions is much less well tolerated in humans than mice [41]. + +It is unclear how the Fog2 diaphragmatic defect relates to the more common Bochdalek CDH, as the pathogenic mechanisms for both are largely unknown. Muscle precursors destined to populate the diaphragm migrate from the lateral dermomyotome of cervical somites. Prior to migration onto the diaphragm, they populate the PPF, a wedge-shaped tissue that tapers medially from the lateral body wall to the esophageal mesentery and fuses ventrally with the septum transversum [42]. Muscle precursors reach the PPF by E11, where they proliferate, differentiate, and then migrate toward the dorsolateral costal, sternal costal, and crural regions of the developing diaphragm. Thus, a defect in PPF formation subsequently results in the abnormal formation of the diaphragm [43]. We have shown that the Fog2 mutant does have an abnormal pattern of HGF expression in the region through which muscle precursor cells migrate onto the developing diaphragm. This finding may account for the abnormally patterned muscle that develops in the Fog2 mutant diaphragm. Although Pax3 and MyoD expression is detected in the PPF, a detailed analysis of transcription factors responsible for muscle precursor cell migration and differentiation will need to be completed both in the PPF and along the pathway of muscle precursor cell migration between the PPF and the membranous diaphragm. Fog2 can interact with any of the Gata factors, Gatas 1–6, as well as other transcription factors such as CoupTFII [44,45]. It is known that a Fog2–Gata4 interaction is critical for normal cardiac and gonadal development, but interacting factors in the lung and diaphragm have not yet been determined. + +The severity of pulmonary hypoplasia in the patient we report was out of proportion to that of the diaphragm defect. Pulmonary hypoplasia is associated with abnormal diaphragmatic anatomy or function, and is known to occur as a secondary developmental defect in models of diaphragmatic dysfunction such as complete amuscularization [17] or phrenic nerve disruption [46]. It occurs in a surgical model of CDH in which a hernia is physically created in an in utero lamb [47,48]. However, the possibility that primary pulmonary developmental abnormalities occur with, rather than secondary to, diaphragmatic defects has been suggested by others based on a teratogenic model of CDH [49–51] and has long been suspected by clinicians who care for these patients. In addition, the high incidence of lobar abnormalities associated with CDH [52] supports the possibility that this disorder can be associated with a primary developmental pulmonary abnormality. + +Our analysis of mice carrying mutations of Fog2 proves that there is a primary defect in lung development that results in specific loss of the accessory lobe and partial loss of the right middle lobe. The specific lobar defects prompted us to evaluate Fog2 expression at the time of early lobar budding. While Fog2 expression is diffuse in the pulmonary mesenchyme after lobar structure is well established (E12.5), it is more focally expressed in the mesenchyme surrounding the right middle lobe and accessory buds as these lobes form. This matches the phenotype of right middle lobe and accessory lobe loss, and suggests that Fog2 has a specific patterning role in establishment of these lobes. It is less clear whether loss of Fog2 results in a global branching defect, as Fog2 lungs appear to have a slight developmental delay, which could result from many causes. Cultured Fog2 lungs do develop an intricate branching pattern in the unaffected lobes that appears similar in the pattern to wild-type lungs after 5 d in culture. + +In this report, we show that a mutation of Fog2 in the mouse causes the phenotype of abnormal diaphragmatic muscularization and primary pulmonary hypoplasia. We furthermore demonstrate that a mutation in this gene is associated with a lethal defect in lung and diaphragm development in a child. It is notable that, despite extensive analysis of Fog2 biology and the generation of a Fog2 knock-out mouse, its role in diaphragm and lung development was previously not recognized. It is only as a consequence of phenotype-driven analyses such as those we are pursuing that one has the opportunity to assay all of the potential molecular derangements that may result in human disease. + +Materials and Methods + + + +These investigations were conducted with approval of the institutional review board for Children's Hospital, Boston, and Brigham and Women's Hospital, Boston. Animal use was approved by the Center for Animal Resources and Comparative Medicine (Harvard Medical School). + +Genetic mapping of the mouse mutation lil. + +The lil mutation was identified as described in results. Wild-type FVB/N and C57BL/6J mice used for genetic crosses were obtained from the Jackson Laboratory (Bar Harbor, Maine, United States). Mice carrying a null mutation of Fog2 generated by gene targeting [16] were the generous gift of Dr. Stuart Orkin. + +Developmental analysis of mice. + +Timed pregnancies were set up for collection of E11.5–E17.5 embryos. Embryos were fixed, dehydrated, and embedded in paraffin prior to sectioning. In older embryos, a median sternotomy was performed under microscopic guidance, and diaphragm, lungs, and heart were examined. The lungs and tracheobronchial tree were removed and weighed. Whole diaphragms were isolated from fixed thoracic tissue from E15.5 and E17.5 embryos. For lung explant culture, lungs were dissected from fresh embryos at E11.5 and E12.5 and placed on porous 24-mm (0.4-μ) polyester membranes floated in wells containing 2 ml of Dulbecco's modified Eagle's medium, nutrient mixture F-12 (11039–021, Gibco, San Diego, California, United States), supplemented with 10% fetal bovine serum, 0.3 mg/ml L-glutamine, 100 units/ml penicillin, 100 mcg/ml streptomycin, and 0.25 mcg/ml amphotericin B. Lung explants were cultured at 37 °C in 95% air/5% CO2 for up to 5 d. They were photographed daily with a dissecting microscope (MZ12.5, Leica, Wetzlar, Germany) equipped with a Leica DC500 digital camera. + +Transgenic mice carrying the lacZ gene driven by the Fog2 promoter have been developed by S. Tevosian. In these animals, the lacZ gene is incorporated (“knocked-in”) into the Fog2 locus to allow β-galactosidase expression as a fusion protein in frame with the first 235 amino acids of the FOG2 protein. The Fog2-lacZ module is followed by an ires-eGFP cassette. This creates a null allele of Fog2 gene. The Fog2-LacZ-eGFP construct was linearized with KspI and electroporated into the CJ7 ES cells. The correctly targeted clone was selected by the Southern blot analysis and injected into C57BL/6J blastocysts. Fog2-lacZ-eGFP animals were maintained on the mixed C57BL/6J/129 background. lacZ Expression in whole dissected embryonic lungs was analyzed by staining for β-galactosidase activity with X-gal after fixation for 30 min. + +RT-PCR and sequence analysis in the mouse. + +RNA was extracted by standard techniques from thoracic embryonic tissue. RT-PCR was performed using six primer sets designed to cover the Fog2 gene. RT-PCR was repeated with radiolabeled primers to amplify an abnormally spliced region of the gene (Table S1), and the product was run on a denaturing sequencing gel according to standard techniques. The RT-PCR product was cloned into pCR2.1 vector using TOPO TA Cloning Kit (Invitrogen, Carlsbad, California, United States) and sequenced using gene-specific primers. Sequence analysis was done using Sequencher 4.1 (Gene Codes, Ann Arbor, Michigan, United States). + +In situ hybridization. + +After dehydration and embedding in paraffin wax, 10-μ sections were subjected to radioactive in situ hybridization as described [53]. Probes labeled with 35S were prepared by run-off transcription of linearized plasmid templates and hybridized to tissue sections. Nuclei were counterstained with Hoescht 33258, and signal was imaged using fluorescent and darkfield microscopy. + +Human DNA extraction and sequence analysis. + +DNA was isolated from paraffin blocks by phenol-chloroform extraction [54,55], and from frozen tissues by standard techniques. Primers were designed to amplify FOG2 coding exons plus 50 bp of flanking upstream and downstream sequence. PCR amplification and sequencing were performed by standard methods. Primer sequences used are listed in Table S2. Sequence analysis was done with Sequencher 4.1 (Gene Codes). + +DNA from the parents of one autopsy patient was extracted from fresh blood samples. A second set of blood samples was sent to an outside CLIA-certified laboratory for DNA extraction, PCR, sequencing, and analysis. Paternity testing was performed by the outside laboratory using a standard panel of markers. SNP genotyping was done using Harvard Partners Center for Genetics and Genomics genotyping core facility (Cambridge, Massachusetts, United States). + +Supporting Information + +Table S1 + +Primers for RT-PCR (Mouse): Amplification of Abnormal Transcript in Fog2 Mutant (lil) Mice + +(26 KB DOC) + +Click here for additional data file. + +Table S2 + +Human Primers for Amplification of FOG2 Coding Sequence from Genomic DNA + +(46 KB DOC) + +Click here for additional data file. + +Acknowledgements + +This manuscript is dedicated to Baby Lucy and her parents for the contribution that they have made to helping us to understand developmental diaphragmatic and lung defects. This work was funded by National Institute of Child Health and Human Development (NIHCD) grant HD36404 (DRB) and the Hearst Foundation (KGA). KGA also received salary support from NICHD grant T32HD040129–02. We would like to thank Stuart Orkin, M. D., for the generous gift of his Fog2−/− mice. We would also like to thank Raju Kucherlapati, Ph. D., and Birgit Funke, Ph. D., for providing control DNA samples. + +Abbreviations + +CDH - congenital diaphragmatic hernia + +E[number] - embryonic day [number] + +ENU - N-ethyl-N-nitrosourea + +HGF - Hepatocyte growth factor/Scatter factor + +lil - little lung + +PPF - pleuroperitoneal fold + +Figures + +Figure 1 + +Abnormal Pulmonary and Diaphragmatic Development in the lil Mouse + +(A) The mutant hypoplastic lung (right) lacks the development of the accessory lobe and the anterior portion of the right middle lobe (marked with arrows on the control sample on the left). + +(B) Whole diaphragms show a lack of normal muscularization in the posterolateral regions and the peripheral regions of the mutant diaphragm (control on left and lil diaphragm on the right). + +Figure 2 + +The ENU-Induced lil Mutation in Fog2 is a Splice Site Mutation + +(A) RT-PCR revealed a lengthened transcript in mice with the lil phenotype: the first band is a lil mouse, and the second band is a control mouse. + +(B) Sequencing Fog2 revealed a splice site mutation that causes the insertion of 85 bp of intronic sequence in the mutant mouse. This results in a premature stop codon prior to zinc finger transcription. + +Figure 3 + +Fog2 Is Expressed in the Developing Lung and Diaphragm + +Fog2 is expressed in the diffuse pulmonary mesenchyme at E13.5 (A) (arrow shows mesenchyme) and is restricted to the bronchial and vascular smooth muscle (sm) at E16.5 (B). Fog2 is expressed diffusely in the developing diaphragm (Dia) both prior to (E11.5) (C) and after muscularization (E13.5) (D). + +Figure 4 + +Fog2 Is Necessary for Primary Lung Development + +Fog2 null lungs removed prior to diaphragmatic muscularization and grown in vitro show no accessory lobe development. Accessory lobe is labeled with black arrow in control littermate lungs. + +Figure 5 + +Fog2 Expression in Embryonic Non-Mutant Lungs + +In embryonic non-mutant lungs, Fog2 is most highly expressed at the tips of the accessory (single arrows) and right middle lobes (double arrows) at E11.25 (A) and E11.75 (B), while expression is diffuse by E12.75 (C). + +Figure 6 + +HGF Patterning Is Abnormal in Fog2 Null Mice + +In situ hybridization of HGF in E12.5 wild-type (A) and Fog2−/− (B) embryos demonstrates decreased expression in the region where the PPF meets the membranous diaphragm. Li, liver; Lu, lung. + +Figure 7 + +FOG2 Mutation in a Patient with Diaphragm and Lung Abnormalities + +Sequencing revealed a de novo heterozygote nonsense mutation in a patient who died at birth with severe pulmonary hypoplasia and a posterior deep diaphragmatic eventration. She was clinically diagnosed with CDH. This nonsense mutation occurs prior to the functional zinc finger domains. + +Footnotes + +Competing interests. The authors have declared that no competing interests exist. + +Author contributions. KGA, JJG, and DRB conceived and designed the experiments. KGA, BJH, HH, SGT, LK, CR, RPB, and JAE performed the experiments. KGA, BJH, HH, LK, CR, JAE, JJG, and DRB analyzed the data. KGA, SOV, SGT, BRP, JAE, JJG, and DRB contributed reagents/materials/analysis tools. KGA and DRB wrote the paper. + +Citation: Ackerman KG, Herron BJ, Vargas SO, Huang H, Tevosian SG, et al. (2005) Fog2 is required for normal diaphragm and lung development in mice and humans. PLoS Genet 1(1): e10. diff --git a/src/ontogpt/evaluation/craft/database/all/16109169.ann b/src/ontogpt/evaluation/craft/database/all/16109169.ann new file mode 100644 index 000000000..8a08635de --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16109169.ann @@ -0,0 +1,1154 @@ +T1 http://purl.obolibrary.org/obo/MONDO_0007739 20 40 Huntington's disease +T2 SO:0000704 41 45 gene +T3 PR:000008840 47 50 Hdh +T4 UBERON:0004341 69 75 streak +T5 GO:0090009 69 85 streak formation +T6 NCBITaxon:10088 114 119 mouse +T7 UBERON:0000922 120 126 embryo +T8 PR:000008840 150 160 Huntingtin +T9 PR:000008840 166 168 HD +T10 SO:0000704 169 173 gene +T11 http://purl.obolibrary.org/obo/MONDO_0007739 228 248 Huntington's disease +T12 UBERON:0005292 265 287 extraembryonic tissues +T13 GO:0007369 299 311 gastrulation +T14 UBERON:0000922 385 391 embryo +T15 GO:0097617 455 468 hybridization +T16 UBERON:0000922 480 489 embryonic +T17 GO:0048598 480 489;505 518 embryonic ... morphogenesis +T18 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GO:0005737 3155 3166 cytoplasmic +T89 GO:0005634 3171 3178 nuclear +T90 PR:000008840 3226 3236 huntingtin +T91 GO:0009987 3269 3287 cellular processes +T92 GO:0036454 3319 3342 growth factor complexes +T93 SO:0000704 3346 3350 gene +T94 PR:000008840 3432 3442 huntingtin +T95 http://purl.obolibrary.org/obo/MONDO_0007739 3532 3534 HD +T96 PR:000008840 3546 3556 huntingtin +T97 PR:000008840 3672 3682 huntingtin +T98 PR:000008840 3733 3743 huntingtin +T99 NCBITaxon:10088 3762 3767 mouse +T100 NCBITaxon:10088 3789 3794 mouse +T101 SO:0000704 3798 3802 gene +T102 PR:000008840 3804 3807 Hdh +T103 PR:000008840 3824 3834 huntingtin +T104 NCBITaxon:10088 3903 3908 mouse +T105 UBERON:0000922 3909 3918 embryonic +T106 CL:0002322 3909 3929 embryonic stem cells +T107 CL:0000540 3934 3941 neurons +T108 PR:000008840 3952 3962 huntingtin +T109 PR:000008840 3981 3991 huntingtin +T110 NCBITaxon:1 4022 4030 organism +T111 NCBITaxon:40674 4042 4051 mammalian +T112 UBERON:0000922 4052 4061 embryonic +T113 GO:0009790 4052 4073 embryonic development +T114 PR:000008840 4103 4113 huntingtin +T115 GO:0007369 4153 4165 gastrulation +T116 PR:000008840 4193 4203 huntingtin +T117 GO:0022008 4231 4243 neurogenesis +T118 UBERON:0012101 4248 4257 perinatal +T119 GO:0007567 4252 4257 natal +T120 PR:000008840 4287 4297 huntingtin +T121 PR:000008840 4308 4311 Hdh +T122 PR:000008840 4317 4320 Hdh +T123 UBERON:0000922 4326 4333 embryos +T124 NCBITaxon:10088 4378 4383 mouse +T125 PR:000008840 4384 4386 HD +T126 SO:0000704 4387 4391 gene +T127 UBERON:0000922 4456 4463 embryos +T128 UBERON:0004341 4484 4500 primitive streak +T129 UBERON:0003062 4542 4546 node +T130 UBERON:0009746 4551 4561 head folds +T131 GO:0010467 4623 4633 expression +T132 PR:000008840 4637 4647 huntingtin +T133 UBERON:0005292 4656 4678 extraembryonic tissues +T134 GO:0007369 4704 4716 gastrulation +T135 PR:000008840 4744 4754 huntingtin +T136 UBERON:0000922 4793 4802 embryonic +T137 UBERON:0005292 4873 4895 Extraembryonic tissues +T138 CHEBI:33284 4924 4933 nutrients +T139 GO:0008595 4958 4991;5007 5009;5025 5031 anterior/posterior axis formation ... in ... embryo +T140 UBERON:0000922 5025 5031 embryo +T141 PR:000008840 5064 5074 huntingtin +T142 PR:000008840 5205 5215 huntingtin +T143 UBERON:0000922 5226 5233 embryos +T144 UBERON:0004877 5257 5274 visceral endoderm +T145 GO:0006826 5326 5340 iron transport +T146 GO:0006897 5410 5421 endocytosis +T147 PR:000008840 5441 5451 huntingtin +T148 UBERON:0000922 5462 5469 embryos +T149 UBERON:0000922 5473 5482 embryonic +T150 CL:0002322 5473 5493 embryonic stem cells +T151 PR:000008840 5523 5533 huntingtin +T152 GO:0005634 5555 5562 nucleus +T153 GO:0005634 5596 5603 nuclear +T154 PR:000008840 5671 5681 huntingtin +T155 UBERON:0005292 5727 5749 extraembryonic tissues +T156 UBERON:0000922 5767 5773 embryo +T157 GO:0010467 5843 5853 expression +T158 SO:0000704 5857 5862 genes +T159 UBERON:0000922 5885 5894 embryonic +T160 GO:0048598 5885 5894;5910 5923 embryonic ... 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PR:000008840 7361 7364 Hdh +T214 PR:000008840 7370 7373 Hdh +T215 UBERON:0004341 7440 7456 primitive streak +T216 UBERON:0009746 7485 7495 head folds +T217 UBERON:0003062 7499 7503 node +T218 GO:0097617 7536 7549 hybridization +T219 UBERON:0000923 7589 7600 germ layers +T220 UBERON:0005292 7605 7626 extraembryonic tissue +T221 PR:000008840 7641 7651 huntingtin +T222 UBERON:0000922 7662 7669 embryos +T223 PR:000012086 7672 7676 Otx2 +T224 GO:0010467 7687 7696 expressed +T225 UBERON:0002346 7713 7726 neuroectoderm +T226 UBERON:0004044 7731 7757 anterior visceral endoderm +T227 GO:0010467 7767 7776 expressed +T228 UBERON:0000922 7787 7794 embryos +T229 GO:0010467 7828 7838 expression +T230 PR:000008523 7874 7879 Hesx1 +T231 GO:0010467 7880 7890 expression +T232 UBERON:0000922 7919 7926 embryos +T233 GO:0010467 7933 7943 expression +T234 UBERON:0004044 7961 7964 AVE +T235 UBERON:0002346 7969 7982 neuroectoderm +T236 GO:0010467 8015 8025 expression +T237 GO:0001714 8090 8103;8117 8119;8138 8146;8153 8158 specification ... of ... endoderm ... cells +T238 GO:0048870 8108 8119;8153 8158 movement of ... cells +T239 UBERON:0004044 8120 8146 anterior visceral endoderm +T240 CL:0000223 8138 8146;8153 8158 endoderm ... cells +T241 UBERON:0004044 8148 8151 AVE +T242 UBERON:0002346 8196 8209 neuroectoderm +T243 UBERON:0000922 8235 8242 embryos +T244 UBERON:0004044 8255 8258 AVE +T245 UBERON:0002346 8285 8297 neurectoderm +T246 PR:000008840 8341 8351 huntingtin +T247 GO:0097617 8373 8386 hybridization +T248 PR:000012086 8399 8403 Otx2 +T249 PR:000008523 8414 8418 Hesx +T250 UBERON:0000922 8467 8474 embryos +T251 UBERON:0002346 8488 8501 neuroectoderm +T252 UBERON:0004044 8506 8532 anterior visceral endoderm +T253 UBERON:0004044 8534 8537 AVE +T254 PR:000008840 8559 8569 huntingtin +T255 UBERON:0000922 8580 8587 embryos +T256 UBERON:0002346 8602 8615 neuroectoderm +T257 GO:0010467 8616 8626 expression +T258 GO:0010467 8659 8669 expression +T259 PR:000008523 8673 8677 Hesx +T260 UBERON:0000922 8688 8695 embryos +T261 GO:0065007 8741 8748 control +T262 PR:000007603 8764 8769 Hnf3β +T263 GO:0010467 8770 8780 expression +T264 UBERON:0000925 8799 8807 endoderm +T265 UBERON:0004044 8860 8863 AVE +T266 UBERON:0000922 8900 8907 embryos +T267 UBERON:0000924 8960 8968 ectoderm +T268 UBERON:0000925 8973 8981 endoderm +T269 PR:000008840 9012 9015 Hdh +T270 PR:000008840 9021 9024 Hdh +T271 UBERON:0000922 9030 9037 embryos +T272 UBERON:0000922 9039 9046 Embryos +T273 UBERON:0000922 9148 9155 Embryos +T274 UBERON:0000925 9227 9235 endoderm +T275 GO:0007492 9227 9245 endoderm formation +T276 GO:0010467 9251 9261 expression +T277 PR:000007603 9265 9270 Hnf3β +T278 PR:000007603 9272 9277 FoxA2 +T279 UBERON:0000922 9303 9310 embryos +T280 UBERON:0000922 9338 9345 embryos +T281 PR:000007603 9347 9352 Hnf3β +T282 GO:0010467 9353 9363 expression +T283 UBERON:0003062 9383 9387 node +T284 UBERON:0004046 9392 9420 anterior definitive endoderm +T285 UBERON:0000922 9445 9452 embryos 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11446 embryos +T360 PR:000008840 11459 11469 Huntingtin +T361 GO:0090009 11486 11498;11508 11524 formation of ... primitive streak +T362 GO:0048339 11486 11498;11529 11546 formation of ... paraxial mesoderm +T363 UBERON:0004341 11508 11524 primitive streak +T364 UBERON:0003077 11529 11546 paraxial mesoderm +T365 GO:0097617 11579 11593 hybridizations +T366 UBERON:0000922 11602 11609 embryos +T367 PR:000016001 11616 11617 T +T368 PR:000016001 11619 11628 Brachyury +T369 GO:0010467 11639 11648 expressed +T370 UBERON:0004341 11656 11672 primitive streak +T371 UBERON:0003062 11674 11678 node +T372 UBERON:0003068 11680 11694 axial mesoderm +T373 PR:000007241 11699 11703 Evx1 +T374 GO:0010467 11713 11722 expressed +T375 UBERON:0004341 11730 11746 primitive streak +T376 UBERON:0004341 11778 11784 streak +T377 UBERON:0000922 11795 11802 embryos +T378 UBERON:0000922 11823 11830 embryos +T379 PR:000016001 11837 11838 T +T380 PR:000007241 11850 11854 Evx1 +T381 GO:0010467 11875 11884 expressed +T382 UBERON:0000922 11897 11906 embryonic +T383 PR:000017443 11915 11920 Wnt3A +T384 GO:0010467 11921 11931 expression +T385 UBERON:0000922 11953 11960 embryos +T386 GO:0010467 11999 12009 expression +T387 UBERON:0004341 12026 12032 streak +T388 UBERON:0000922 12061 12068 embryos +T389 UBERON:0003077 12086 12103 paraxial mesoderm +T390 PR:000016154 12112 12116 Tbx6 +T391 PR:000006523 12127 12131 Dll1 +T392 UBERON:0000922 12188 12195 embryos +T393 UBERON:0003077 12223 12240 paraxial mesoderm +T394 GO:0048339 12223 12251 paraxial mesoderm production +T395 PR:000008840 12270 12280 huntingtin +T396 UBERON:0000922 12282 12289 Embryos +T397 UBERON:0000922 12361 12368 Embryos +T398 UBERON:0004340 12490 12492 al +T399 UBERON:0004340 12495 12504 allantois +T400 UBERON:0000305 12506 12507 a +T401 UBERON:0000305 12510 12516 amnion +T402 UBERON:0003124 12518 12520 ch +T403 UBERON:0003124 12523 12530 chorion +T404 UBERON:0000922 12537 12546 embryonic +T405 UBERON:0003062 12547 12551 node +T406 UBERON:0005728 12556 12558 em +T407 UBERON:0005728 12561 12584 extraembryonic mesoderm +T408 UBERON:0003062 12586 12587 n +T409 UBERON:0003062 12590 12594 node +T410 UBERON:0004341 12596 12598 ps +T411 UBERON:0004341 12601 12617 primitive streak +T412 UBERON:0003062 12633 12637 node +T413 UBERON:0000922 12646 12653 embryos +T414 UBERON:0004341 12731 12747 primitive streak +T415 UBERON:0004341 12763 12769 streak +T416 UBERON:0003077 12780 12797 paraxial mesoderm +T417 UBERON:0003077 12821 12838 paraxial mesoderm +T418 GO:0048339 12821 12848 paraxial mesoderm formation +T419 UBERON:0000922 12873 12880 embryos +T420 UBERON:0000926 12904 12912 mesoderm +T421 PR:000017443 12944 12949 Wnt3A +T422 GO:0010467 12953 12962 expressed +T423 UBERON:0004341 12970 12986 primitive streak +T424 UBERON:0003077 13012 13029 paraxial mesoderm +T425 PR:000008840 13034 13044 huntingtin +T426 PR:000017443 13064 13069 Wnt3a +T427 UBERON:0004341 13097 13103 streak +T428 UBERON:0009746 13208 13218 head folds +T429 GO:0010467 13229 13239 expression +T430 PR:000017443 13243 13248 Wnt3a +T431 PR:000008840 13274 13277 Hdh +T432 PR:000008840 13283 13286 Hdh +T433 UBERON:0000922 13292 13299 embryos +T434 UBERON:0003077 13324 13341 paraxial mesoderm +T435 GO:0048339 13324 13353 paraxial mesoderm development +T436 GO:0010467 13373 13383 expression +T437 PR:000016154 13387 13391 Tbx6 +T438 UBERON:0000926 13399 13407 mesoderm +T439 UBERON:0004341 13423 13439 primitive streak +T440 UBERON:0000922 13450 13457 embryos +T441 UBERON:0000922 13523 13530 embryos +T442 GO:0010467 13544 13554 expression +T443 PR:000006523 13558 13562 Dll1 +T444 UBERON:0004341 13577 13583 streak +T445 UBERON:0003077 13669 13686 paraxial mesoderm +T446 UBERON:0004341 13751 13767 primitive streak +T447 GO:0090009 13751 13777 primitive streak formation +T448 UBERON:0003068 13812 13817;13831 13839 axial ... mesoderm +T449 GO:0048318 13812 13817;13831 13849 axial ... mesoderm formation +T450 UBERON:0003077 13822 13839 paraxial mesoderm +T451 GO:0048339 13822 13849 paraxial mesoderm formation +T452 GO:0010467 13931 13941 expression +T453 PR:000008840 13960 13970 huntingtin +T454 CHEBI:62488 14036 14055 signaling molecules +T455 PR:000000105 14096 14101 Nodal +T456 PR:000000046 14119 14123 Tgfβ +T457 GO:0046903 14134 14142 secreted +T458 CHEBI:36357 14143 14152 molecules +T459 GO:0090009 14173 14182;14199 14201;14206 14222 formation ... of ... primitive streak +T460 UBERON:0004341 14206 14222 primitive streak +T461 UBERON:0004044 14244 14247 AVE +T462 PR:000000105 14257 14262 Nodal +T463 GO:0010467 14275 14284 expressed +T464 UBERON:0002532 14300 14308 epiblast +T465 UBERON:0004877 14323 14340 visceral endoderm +T466 GO:0007566 14355 14367 implantation +T467 UBERON:0000922 14434 14440 embryo +T468 UBERON:0004341 14456 14472 primitive streak +T469 UBERON:0004877 14491 14508 visceral endoderm +T470 GO:0010467 14509 14519 expression +T471 PR:000000105 14558 14563 Nodal +T472 GO:0010467 14564 14574 expression +T473 UBERON:0003062 14596 14600 node +T474 PR:000000105 14602 14607 Nodal +T475 GO:0010467 14608 14618 expression +T476 PR:000008840 14635 14638 Hdh +T477 PR:000008840 14644 14647 Hdh +T478 UBERON:0000922 14653 14660 embryos +T479 PR:000000105 14682 14685 Ndl +T480 PR:000033987 14686 14691 lac Z +T481 SO:0001023 14692 14698 allele +T482 PR:000000105 14738 14743 nodal +T483 PR:000008840 14763 14766 Hdh +T484 PR:000008840 14772 14775 Hdh +T485 PR:000008840 14823 14826 Hdh +T486 PR:000008840 14832 14835 Hdh +T487 PR:000000105 14849 14852 Ndl +T488 UBERON:0000922 14854 14861 embryos +T489 PR:000008840 14876 14879 Hdh +T490 PR:000008840 14885 14888 Hdh +T491 UBERON:0000922 14894 14901 embryos +T492 UBERON:0000922 14945 14952 embryos +T493 PR:000000105 14989 14994 Nodal +T494 PR:000033987 14995 14999 LacZ +T495 GO:0010467 15000 15010 expression +T496 UBERON:0003062 15018 15022 node +T497 PR:000008840 15024 15027 Hdh +T498 PR:000008840 15033 15036 Hdh +T499 PR:000000105 15050 15053 Ndl +T500 UBERON:0000922 15055 15062 embryos +T501 GO:0010467 15063 15070 express +T502 PR:000000105 15071 15076 Nodal +T503 PR:000033987 15077 15081 LacZ +T504 UBERON:0000925 15097 15105 endoderm +T505 UBERON:0002532 15120 15128 epiblast +T506 PR:000000105 15237 15242 nodal +T507 UBERON:0003062 15300 15304 node +T508 SO:0000704 15360 15364 gene +T509 GO:0010467 15360 15375 gene expression +T510 PR:000008840 15379 15389 huntingtin +T511 UBERON:0000922 15400 15407 embryos +T512 CHEBI:75055 15409 15414 X-gal +T513 PR:000000105 15427 15432 Nodal +T514 PR:000033987 15433 15437 LacZ +T515 UBERON:0000922 15438 15445 embryos +T516 UBERON:0000925 15464 15472 endoderm +T517 UBERON:0003062 15482 15486 node +T518 UBERON:0000922 15497 15504 embryos +T519 UBERON:0000922 15540 15547 embryos +T520 GO:0010467 15565 15575 expression +T521 UBERON:0003062 15614 15618 node +T522 GO:0010467 15619 15629 expression +T523 PR:000000105 15633 15638 Nodal +T524 UBERON:0000922 15649 15656 embryos +T525 UBERON:0000922 15679 15686 embryos +T526 UBERON:0003062 15736 15740 node +T527 PR:000008840 15759 15769 huntingtin +T528 GO:0097617 15795 15808 hybridization +T529 UBERON:0000922 15821 15828 embryos +T530 PR:000007499 15842 15846 Fgf8 +T531 UBERON:0004341 15875 15881 streak +T532 UBERON:0004341 15933 15939 streak +T533 UBERON:0000922 15950 15957 embryos +T534 PR:000007499 15978 15982 Fgf8 +T535 GO:0010467 15998 16007 expressed +T536 UBERON:0000922 16018 16025 embryos +T537 GO:0010467 16043 16053 expression +T538 PR:000008278 16057 16060 Gsc +T539 UBERON:0000925 16079 16087 endoderm +T540 UBERON:0000033 16114 16118 head +T541 UBERON:0000922 16136 16143 embryos +T542 UBERON:0000119 16176 16187 cell layers +T543 UBERON:0000922 16198 16205 embryos +T544 UBERON:0000922 16241 16248 embryos +T545 GO:0010467 16274 16284 expression +T546 PR:000008278 16288 16291 Gsc +T547 UBERON:0000922 16321 16328 embryos +T548 UBERON:0000922 16370 16377 embryos +T549 UBERON:0000922 16383 16390 Embryos +T550 UBERON:0000922 16475 16482 Embryos +T551 PR:000007499 16515 16519 Fgf8 +T552 GO:0007369 16559 16571 gastrulation +T553 NCBITaxon:10088 16579 16584 mouse +T554 UBERON:0000922 16585 16591 embryo +T555 PR:000007499 16593 16597 Fgf8 +T556 GO:0016477 16614 16628 cell migration +T557 UBERON:0004341 16643 16659 primitive streak +T558 GO:0010467 16666 16675 Expressed +T559 UBERON:0004341 16690 16696 streak +T560 GO:0090009 16690 16706 streak formation +T561 UBERON:0002532 16724 16732 epiblast +T562 UBERON:0004877 16737 16754 visceral endoderm +T563 PR:000007499 16756 16760 Fgf8 +T564 UBERON:0004341 16782 16788 streak +T565 UBERON:0000926 16789 16797 mesoderm +T566 UBERON:0004341 16905 16911 streak +T567 PR:000008840 16928 16931 Hdh +T568 PR:000008840 16937 16940 Hdh +T569 UBERON:0000922 16946 16953 embryos +T570 PR:000007499 16955 16959 Fgf8 +T571 GO:0010467 16960 16970 expression +T572 GO:0010467 16983 16992 expressed +T573 UBERON:0004341 17024 17040 primitive streak +T574 UBERON:0000925 17064 17072 endoderm +T575 UBERON:0002532 17094 17102 epiblast +T576 UBERON:0004341 17125 17131 streak +T577 GO:0010467 17207 17217 expression +T578 PR:000012086 17237 17241 Otx2 +T579 PR:000007603 17243 17248 Hnf3β +T580 PR:000008523 17253 17258 Hesx1 +T581 UBERON:0000922 17298 17305 embryos +T582 UBERON:0004341 17334 17340 streak +T583 PR:000007499 17374 17378 Fgf8 +T584 UBERON:0000925 17400 17408 endoderm +T585 PR:000008840 17411 17414 Hdh +T586 PR:000008840 17420 17423 Hdh +T587 UBERON:0000922 17429 17436 embryos +T588 GO:0010467 17463 17473 expression +T589 PR:000008278 17477 17486 goosecoid +T590 PR:000008278 17488 17491 Gsc +T591 PR:000008278 17504 17507 Gsc +T592 GO:0010467 17521 17530 expressed +T593 UBERON:0004877 17538 17555 visceral endoderm +T594 UBERON:0004341 17597 17613 primitive streak +T595 GO:0007369 17633 17645 gastrulation +T596 UBERON:0004341 17654 17670 primitive streak +T597 PR:000008278 17690 17693 Gsc +T598 GO:0010467 17697 17706 expressed +T599 UBERON:0004341 17721 17727 streak +T600 UBERON:0003062 17733 17737 node +T601 UBERON:0003068 17747 17761 axial mesoderm +T602 UBERON:0003062 17792 17796 node +T603 PR:000008840 17842 17845 Hdh +T604 PR:000008840 17851 17854 Hdh +T605 UBERON:0000922 17860 17867 embryos +T606 PR:000008278 17884 17887 Gsc +T607 GO:0010467 17888 17898 expression +T608 UBERON:0000925 17926 17934 endoderm +T609 UBERON:0000922 17956 17962 embryo +T610 UBERON:0004364 18004 18022 ectoplacental cone +T611 PR:000007603 18086 18091 Hnf3β +T612 GO:0065007 18092 18102 regulation +T613 PR:000008278 18104 18107 Gsc +T614 UBERON:0004877 18152 18160;18176 18184 visceral ... endoderm +T615 UBERON:0005439 18165 18184 definitive endoderm +T616 PR:000008840 18198 18208 huntingtin +T617 PR:000008840 18278 18288 Huntingtin +T618 GO:0010467 18309 18319 expression +T619 UBERON:0000922 18328 18337 embryonic +T620 CHEBI:62488 18338 18357 signaling molecules +T621 UBERON:0000922 18388 18395 embryos +T622 PR:000008840 18409 18419 huntingtin +T623 UBERON:0005292 18444 18465 extraembryonic tissue +T624 UBERON:0005292 18512 18533 extraembryonic tissue +T625 UBERON:0000922 18568 18577 embryonic +T626 UBERON:0002532 18609 18617 epiblast +T627 UBERON:0000922 18650 18659 embryonic +T628 PR:000008840 18675 18685 huntingtin +T629 UBERON:0000922 18696 18703 embryos +T630 PR:000008654 18705 18709 Hnf4 +T631 GO:0010467 18736 18745 expressed +T632 UBERON:0008776 18753 18771 primitive endoderm +T633 UBERON:0000479 18788 18794 tissue +T634 UBERON:0004877 18838 18855 visceral endoderm +T635 GO:0046903 18856 18864 secreted +T636 PR:000003809 18881 18897 alphafetoprotein +T637 CHEBI:39015 18899 18914 apolipoproteins +T638 PR:000016261 18920 18931 transferrin +T639 PR:000008654 18949 18953 Hnf4 +T640 GO:0007369 18974 18986 gastrulation +T641 PR:000008654 19005 19009 Hnf4 +T642 GO:0010467 19013 19022 expressed +T643 UBERON:0004877 19039 19056 visceral endoderm +T644 CL:0000223 19048 19062 endoderm cells +T645 UBERON:0000922 19075 19084 embryonic +T646 UBERON:0000924 19085 19093 ectoderm +T647 PR:000008840 19123 19126 Hdh +T648 PR:000008840 19132 19135 Hdh +T649 UBERON:0000922 19141 19148 embryos +T650 UBERON:0008776 19173 19182;19196 19204 primitive ... endoderm +T651 UBERON:0004877 19187 19204 visceral endoderm +T652 PR:000008654 19222 19226 Hnf4 +T653 GO:0010467 19227 19237 expression +T654 UBERON:0000922 19296 19303 embryos +T655 UBERON:0000922 19326 19333 embryos +T656 PR:000014397 19356 19359 Pem +T657 GO:0010467 19384 19393 expressed +T658 UBERON:0004877 19406 19423 visceral endoderm +T659 UBERON:0004364 19428 19446 ectoplacental cone +T660 UBERON:0000922 19460 19467 embryos +T661 GO:0010467 19485 19494 expressed +T662 UBERON:0000479 19504 19511 tissues +T663 UBERON:0000922 19526 19533 embryos +T664 PR:000014397 19560 19563 Pem +T665 GO:0010467 19564 19574 expressing +T666 UBERON:0004877 19575 19592 visceral endoderm +T667 UBERON:0000922 19631 19638 embryos +T668 GO:0010467 19726 19736 expression +T669 UBERON:0000922 19745 19754 embryonic +T670 PR:000008840 19766 19776 huntingtin +T671 UBERON:0000922 19787 19794 embryos +T672 GO:0097617 19816 19829 hybridization +T673 UBERON:0005292 19865 19887 extraembryonic tissues +T674 GO:0010467 19911 19921 expression +T675 PR:000008840 19940 19950 huntingtin +T676 PR:000008654 19952 19956 Hnf4 +T677 GO:0010467 19958 19967 expressed +T678 UBERON:0004877 19975 19992 visceral endoderm +T679 UBERON:0000922 20012 20021 embryonic +T680 UBERON:0000924 20022 20030 ectoderm +T681 UBERON:0000922 20065 20072 embryos +T682 GO:0010467 20133 20143 expression +T683 PR:000014397 20147 20150 Pem +T684 SO:0000673 20151 20162 transcripts +T685 UBERON:0000922 20187 20194 embryos +T686 UBERON:0000922 20217 20224 embryos +T687 PR:000014397 20239 20242 Pem +T688 GO:0010467 20246 20255 expressed +T689 UBERON:0004877 20293 20310 visceral endoderm +T690 UBERON:0000922 20343 20350 embryos +T691 GO:0010467 20352 20362 Expression +T692 UBERON:0000922 20371 20380 embryonic +T693 CHEBI:62488 20381 20400 signaling molecules +T694 PR:000008840 20430 20440 huntingtin +T695 GO:0010467 20462 20472 expression +T696 PR:000000165 20476 20480 Bmp4 +T697 UBERON:0004366 20494 20517 extraembryonic ectoderm +T698 PR:000000287 20523 20529 Lefty1 +T699 PR:000006499 20534 20538 Dkk1 +T700 UBERON:0004044 20552 20555 AVE +T701 UBERON:0000922 20566 20573 embryos +T702 PR:000000165 20575 20579 Bmp4 +T703 UBERON:0004366 20620 20643 extraembryonic ectoderm +T704 UBERON:0000922 20654 20661 embryos +T705 UBERON:0000922 20679 20686 embryos +T706 CL:0000670 20692 20712 Primitive germ cells +T707 CL:0000670 20714 20718 PCGs +T708 UBERON:0000922 20774 20781 embryos +T709 PR:000000165 20802 20806 Bmp4 +T710 UBERON:0004366 20826 20849 extraembryonic ectoderm +T711 UBERON:0002532 20857 20865 epiblast +T712 PR:000000287 20877 20883 Lefty1 +T713 GO:0010467 20884 20894 expression +T714 UBERON:0000922 20926 20933 embryos +T715 UBERON:0000922 20960 20967 embryos +T716 GO:0010467 20999 21009 expression +T717 PR:000006499 21013 21017 Dkk1 +T718 UBERON:0004044 21025 21028 AVE +T719 UBERON:0000922 21039 21046 embryos +T720 GO:0010467 21073 21083 expression +T721 UBERON:0004044 21112 21115 AVE +T722 UBERON:0009746 21127 21137 head folds +T723 UBERON:0000922 21161 21168 embryos +T724 CHEBI:33284 21192 21200 nutrient +T725 UBERON:0000922 21241 21248 embryos +T726 NCBITaxon:10114 21271 21274 rat +T727 UBERON:0001977 21275 21280 serum +T728 UBERON:0002329 21290 21297 somites +T729 UBERON:0000948 21303 21308 heart +T730 UBERON:0009746 21330 21340 head folds +T731 PR:000008840 21387 21397 huntingtin +T732 UBERON:0000922 21408 21415 embryos +T733 UBERON:0009746 21460 21469 headfolds +T734 UBERON:0000948 21471 21476 heart +T735 UBERON:0002329 21480 21487 somites +T736 UBERON:0000922 21493 21500 Embryos +T737 UBERON:0000922 21576 21583 Embryos +T738 UBERON:0005292 21672 21694 extraembryonic tissues +T739 UBERON:0004044 21710 21736 anterior visceral endoderm +T740 UBERON:0004366 21741 21764 extraembryonic ectoderm +T741 UBERON:0002532 21821 21829 epiblast +T742 PR:000000165 21836 21840 Bmp4 +T743 CHEBI:62488 21846 21864 signaling molecule +T744 GO:0010467 21879 21888 expressed +T745 UBERON:0004366 21914 21937 extraembryonic ectoderm +T746 UBERON:0004366 21981 22004 extraembryonic ectoderm +T747 UBERON:0002532 22021 22029 epiblast +T748 GO:0065007 22063 22073 regulating +T749 GO:0090009 22078 22090;22104 22120 formation of ... primitive streak +T750 UBERON:0003062 22095 22099 node +T751 UBERON:0004341 22104 22120 primitive streak +T752 PR:000000165 22122 22126 Bmp4 +T753 UBERON:0000922 22158 22164 embryo +T754 PR:000008840 22221 22231 huntingtin +T755 PR:000000165 22233 22237 Bmp4 +T756 GO:0010467 22238 22248 expression +T757 PR:000008840 22279 22282 Hdh +T758 PR:000008840 22288 22291 Hdh +T759 UBERON:0004366 22297 22320 extraembryonic ectoderm +T760 GO:0010467 22333 22342 expressed +T761 UBERON:0004366 22358 22381 extraembryonic ectoderm +T762 PR:000000165 22430 22434 Bmp4 +T763 GO:0010467 22435 22445 expression +T764 UBERON:0004366 22488 22511 extraembryonic ectoderm +T765 UBERON:0000922 22537 22544 embryos +T766 PR:000000165 22555 22559 Bmp4 +T767 UBERON:0004366 22579 22602 extraembryonic ectoderm +T768 CL:0000670 22617 22638 primordial germ cells +T769 CL:0000670 22640 22644 PGCs +T770 PR:000000165 22661 22665 Bmp4 +T771 CL:0000670 22692 22696 PGCs +T772 UBERON:0004341 22782 22798 primitive streak +T773 UBERON:0000922 22850 22857 embryos +T774 CL:0000670 22905 22909 PCGs +T775 PR:000008840 22918 22921 Hdh +T776 PR:000008840 22927 22930 Hdh +T777 UBERON:0000922 22936 22943 embryos +T778 PR:000000165 22961 22965 Bmp4 +T779 PR:000008840 23008 23018 huntingtin +T780 UBERON:0004044 23037 23063 anterior visceral endoderm +T781 UBERON:0004044 23065 23068 AVE +T782 UBERON:0000922 23086 23095 embryonic +T783 PR:000000105 23162 23167 nodal +T784 PR:000006499 23181 23185 Dkk1 +T785 PR:O54908 23187 23193 mdkk-1 +T786 PR:000000287 23199 23205 Lefty1 +T787 GO:0010467 23224 23233 expressed +T788 UBERON:0004044 23241 23244 AVE +T789 UBERON:0000922 23312 23318 embryo +T790 PR:000000105 23334 23339 Nodal +T791 GO:0038092 23334 23339;23348 23357 Nodal ... signaling +T792 GO:0016055 23344 23357 Wnt signaling +T793 PR:000008840 23370 23373 Hdh +T794 PR:000008840 23379 23382 Hdh +T795 UBERON:0000922 23388 23395 embryos +T796 PR:000006499 23402 23406 Dkk1 +T797 PR:000000287 23423 23429 Lefty1 +T798 GO:0010467 23446 23455 expressed +T799 UBERON:0004044 23472 23475 AVE +T800 UBERON:0000922 23503 23510 embryos +T801 PR:000006499 23521 23526 Dkk-1 +T802 PR:000008840 23569 23572 Hdh +T803 PR:000008840 23578 23581 Hdh +T804 UBERON:0000922 23587 23594 embryos +T805 PR:000006499 23620 23625 Dkk-1 +T806 GO:0010467 23626 23636 expression +T807 GO:0010467 23709 23719 expression +T808 GO:0010467 23762 23772 expression +T809 PR:000000105 23776 23781 Nodal +T810 PR:000017443 23812 23817 Wnt3a +T811 GO:0010467 23818 23828 expression +T812 UBERON:0000922 23849 23856 embryos +T813 GO:0010467 23902 23912 expression +T814 PR:000000287 23924 23930 Lefty1 +T815 PR:000006499 23934 23938 Dkk1 +T816 UBERON:0004044 23956 23959 AVE +T817 UBERON:0002346 23974 23987 neuroectoderm +T818 UBERON:0009746 23999 24009 head folds +T819 PR:000008840 24025 24028 Hdh +T820 PR:000008840 24034 24037 Hdh +T821 UBERON:0000922 24043 24050 embryos +T822 UBERON:0000922 24091 24098 embryos +T823 UBERON:0009746 24133 24143 head folds +T824 UBERON:0000922 24145 24152 embryos +T825 UBERON:0000922 24258 24265 embryos +T826 UBERON:0009746 24287 24297 head folds +T827 UBERON:0002329 24299 24306 somites +T828 UBERON:0000948 24311 24317 hearts +T829 UBERON:0000922 24361 24368 embryos +T830 UBERON:0009746 24385 24394 headfolds +T831 UBERON:0000948 24396 24402 hearts +T832 UBERON:0002329 24406 24413 somites +T833 UBERON:0000922 24430 24437 embryos +T834 PR:000008840 24521 24531 huntingtin +T835 UBERON:0000922 24533 24540 embryos +T836 GO:0048513 24563 24576 organogenesis +T837 CHEBI:33284 24623 24631 nutrient +T838 UBERON:0000922 24688 24697 embryonic +T839 PR:000008840 24721 24731 huntingtin +T840 PR:000008840 24769 24779 huntingtin +T841 PR:000008840 24922 24932 huntingtin +T842 http://purl.obolibrary.org/obo/MONDO_0007739 24942 24944 HD +T843 PR:000008840 24988 24998 huntingtin +T844 UBERON:0005292 25017 25039 extraembryonic tissues +T845 GO:0007369 25051 25063 gastrulation +T846 SO:0000704 25121 25125 gene +T847 GO:0010467 25121 25136 gene expression +T848 PR:000008840 25140 25143 Hdh +T849 PR:000008840 25149 25152 Hdh +T850 UBERON:0000922 25158 25165 embryos +T851 PR:000008840 25200 25210 huntingtin +T852 UBERON:0004341 25243 25249 streak +T853 GO:0090009 25243 25259 streak formation +T854 GO:0048339 25279 25310 production of paraxial mesoderm +T855 UBERON:0003077 25293 25310 paraxial mesoderm +T856 PR:000008840 25352 25362 huntingtin +T857 GO:0010467 25422 25431 expressed +T858 SO:0000704 25432 25437 genes +T859 PR:000008840 25468 25478 huntingtin +T860 GO:0010467 25568 25577 expressed +T861 SO:0000704 25578 25583 genes +T862 GO:0010467 25611 25621 expression +T863 GO:0009948 25666 25699 anterior/posterior axis formation +T864 UBERON:0004044 25738 25741 AVE +T865 UBERON:0004341 25761 25777 primitive streak +T866 PR:000017443 25881 25886 Wnt3a +T867 CL:0000670 25891 25912 primordial germ cells +T868 UBERON:0000922 25956 25963 embryos +T869 GO:0010467 26009 26019 expression +T870 PR:000016001 26023 26024 T +T871 PR:000007241 26029 26033 Evx1 +T872 UBERON:0005728 26041 26064 extraembryonic mesoderm +T873 UBERON:0000922 26075 26082 embryos +T874 UBERON:0000922 26113 26120 embryos +T875 PR:000008840 26177 26187 huntingtin +T876 UBERON:0000922 26198 26205 embryos +T877 UBERON:0004341 26235 26241 streak +T878 UBERON:0005728 26261 26284 extraembryonic mesoderm +T879 SO:0000704 26339 26344 genes +T880 PR:000008840 26406 26416 huntingtin +T881 UBERON:0000922 26427 26434 embryos +T882 GO:0010467 26460 26470 expression +T883 PR:000007499 26474 26478 Fgf8 +T884 PR:000000105 26480 26485 Nodal +T885 PR:000008278 26490 26493 Gsc +T886 PR:000008840 26501 26511 huntingtin +T887 GO:0065007 26558 26568 regulation +T888 SO:0000704 26597 26602 genes +T889 GO:0065007 26669 26677 regulate +T890 GO:0010467 26682 26692 expression +T891 SO:0000704 26702 26707 genes +T892 PR:000008840 26730 26740 huntingtin +T893 UBERON:0005292 26748 26770 extraembryonic tissues +T894 UBERON:0000922 26817 26826 embryonic +T895 GO:0065007 26867 26877 regulation +T896 SO:0000704 26881 26885 gene +T897 GO:0010467 26881 26896 gene expression +T898 UBERON:0002532 26908 26916 epiblast +T899 PR:000008840 26937 26940 Hdh +T900 PR:000008840 26946 26949 Hdh +T901 UBERON:0000922 26955 26962 embryos +T902 UBERON:0000922 26969 26978 embryonic +T903 PR:000008840 26994 26997 Hdh +T904 PR:000008840 27003 27006 Hdh +T905 UBERON:0000922 27012 27019 embryos +T906 GO:0010467 27062 27072 expression +T907 PR:000008654 27076 27080 Hnf4 +T908 UBERON:0008776 27088 27106 primitive endoderm +T909 PR:000014397 27111 27114 Pem +T910 UBERON:0003124 27140 27147 chorion +T911 GO:0010467 27156 27166 expression +T912 PR:000000165 27199 27203 Bmp4 +T913 UBERON:0004366 27213 27236 extraembryonic ectoderm +T914 PR:000006499 27242 27246 Dkk1 +T915 PR:000000287 27251 27257 Lefty1 +T916 UBERON:0004044 27267 27270 AVE +T917 PR:000006499 27325 27330 Dkk-1 +T918 GO:0010467 27331 27341 expression +T919 PR:000008840 27345 27348 Hdh +T920 PR:000008840 27354 27357 Hdh +T921 UBERON:0000922 27363 27370 embryos +T922 GO:0016055 27412 27425 Wnt signaling +T923 UBERON:0000922 27455 27464 embryonic +T924 PR:000000165 27465 27469 Bmp4 +T925 PR:000008840 27514 27524 huntingtin +T926 CL:0000670 27546 27550 PCGs +T927 UBERON:0000922 27561 27568 embryos +T928 UBERON:0000922 27644 27653 embryonic +T929 PR:000000105 27672 27677 Nodal +T930 PR:000007499 27679 27683 Fgf8 +T931 PR:000008278 27688 27691 Gsc +T932 GO:0010467 27696 27705 expressed +T933 UBERON:0004877 27725 27742 visceral endoderm +T934 PR:000008840 27746 27749 Hdh +T935 PR:000008840 27755 27758 Hdh +T936 UBERON:0000922 27764 27771 embryos +T937 PR:000000105 27778 27783 Nodal +T938 PR:000007499 27788 27792 Fgf8 +T939 UBERON:0002532 27858 27866 epiblast +T940 GO:0065007 27880 27889 regulated +T941 GO:0007369 27897 27909 gastrulation +T942 GO:0010467 27925 27935 expression +T943 PR:000008278 27978 27987 goosecoid +T944 UBERON:0004877 27996 28013 visceral endoderm +T945 PR:000008840 28038 28041 Hdh +T946 PR:000008840 28047 28050 Hdh +T947 UBERON:0000922 28118 28127 embryonic +T948 PR:000008840 28200 28210 huntingtin +T949 CL:0000349 28223 28243 extraembryonic cells +T950 UBERON:0000922 28228 28237 embryonic +T951 UBERON:0000922 28254 28261 embryos +T952 PR:000008840 28264 28274 Huntingtin +T953 UBERON:0000922 28285 28292 embryos +T954 UBERON:0009746 28311 28320 headfolds +T955 GO:0048513 28337 28350 organogenesis +T956 CHEBI:33284 28376 28384 nutrient +T957 UBERON:0009746 28412 28420 headfold +T958 UBERON:0000922 28440 28447 embryos +T959 UBERON:0002346 28502 28514 neurectoderm +T960 UBERON:0004044 28540 28543 AVE +T961 UBERON:0000922 28558 28565 embryos +T962 GO:0010467 28566 28573 express +T963 PR:000012086 28590 28594 Otx2 +T964 PR:000006499 28596 28600 Ddk1 +T965 PR:000000287 28602 28608 Lefty1 +T966 PR:000008523 28613 28618 Hesx1 +T967 UBERON:0003062 28639 28643 node +T968 UBERON:0003062 28722 28726 node +T969 PR:000008840 28730 28740 huntingtin +T970 UBERON:0000922 28751 28758 embryos +T971 UBERON:0009746 28799 28808 headfolds +T972 UBERON:0003077 28836 28853 paraxial mesoderm +T973 PR:000008840 28857 28860 Hdh +T974 PR:000008840 28866 28869 Hdh +T975 UBERON:0000922 28875 28882 embryos +T976 UBERON:0009746 28909 28918 headfolds +T977 UBERON:0003077 28925 28942 paraxial mesoderm +T978 UBERON:0002346 28984 28997 neuroectoderm +T979 UBERON:0009746 29017 29026 headfolds +T980 UBERON:0009746 29069 29078 headfolds +T981 PR:000008840 29098 29108 huntingtin +T982 UBERON:0001017 29154 29157 CNS +T983 GO:0007417 29154 29169 CNS development +T984 PR:000008840 29250 29260 huntingtin +T985 PR:000008840 29281 29284 Hdh +T986 SO:0001023 29285 29291 allele +T987 UBERON:0000955 29310 29316 brains +T988 UBERON:0000922 29326 29335 embryonic +T989 GO:0009790 29326 29347 embryonic development +T990 PR:000008840 29389 29392 Hdh +T991 SO:0000704 29515 29519 gene +T992 PR:000006902 29527 29530 Eed +T993 UBERON:0004366 29532 29550 embryonic ectoderm +T994 PR:000006902 29532 29562 embryonic ectoderm development +T995 GO:0009790 29532 29541;29551 29562 embryonic ... development +T996 GO:0007398 29542 29562 ectoderm development +T997 PR:000008840 29604 29614 huntingtin +T998 PR:000006902 29622 29625 eed +T999 UBERON:0004341 29652 29658 streak +T1000 GO:0090009 29652 29670 streak development +T1001 UBERON:0009746 29680 29688 headfold +T1002 PR:000016001 29708 29709 T +T1003 PR:000007241 29711 29715 Evx1 +T1004 PR:000000105 29720 29725 Nodal +T1005 GO:0010467 29726 29736 expression +T1006 UBERON:0004341 29764 29780 primitive streak +T1007 UBERON:0000926 29781 29789 mesoderm +T1008 GO:0007498 29781 29800 mesoderm production +T1009 PR:000006902 29822 29825 Eed +T1010 UBERON:0000088 29862 29873 trophoblast +T1011 GO:0060819 29912 29936 imprinted X-inactivation +T1012 GO:0000805 29922 29923 X +T1013 SO:0001026 29941 29948 genomic +T1014 GO:0071514 29941 29959 genomic imprinting +T1015 PR:000008840 30027 30037 huntingtin +T1016 UBERON:0000922 30048 30055 embryos +T1017 PR:000008840 30134 30144 huntingtin +T1018 SO:0000417 30223 30229 domain +T1019 http://purl.obolibrary.org/obo/MONDO_0007739 30313 30315 HD +T1020 http://purl.obolibrary.org/obo/MONDO_0007739 30333 30335 HD +T1021 SO:0000704 30361 30368 genetic +T1022 PR:000008840 30379 30381 HD +T1023 NCBITaxon:10088 30395 30399 mice +T1024 CL:0000540 30468 30475 neurons +T1025 UBERON:0002435 30483 30491 striatum +T1026 PR:000008840 30511 30521 huntingtin +T1027 UBERON:0000922 30548 30557 embryonic +T1028 GO:0009790 30548 30569 embryonic development +T1029 http://purl.obolibrary.org/obo/MONDO_0007739 30598 30600 HD +T1030 PR:000008840 30628 30638 huntingtin +T1031 NCBITaxon:10088 30652 30657 mouse +T1032 PR:000008840 30675 30678 Hdh +T1033 SO:0001023 30679 30685 allele +T1034 PR:000008840 30704 30714 huntingtin +T1035 PR:000008840 30732 30742 huntingtin +T1036 UBERON:0000922 30754 30763 embryonic +T1037 http://purl.obolibrary.org/obo/MONDO_0007739 30812 30814 HD +T1038 PR:000008840 30865 30875 huntingtin +T1039 UBERON:0002435 30906 30914 striatal +T1040 PR:000008840 30971 30981 huntingtin +T1041 PR:000008840 31006 31016 huntingtin +T1042 SO:0000704 31041 31045 gene +T1043 PR:000013894 31077 31081 NRSF +T1044 PR:000013894 31082 31086 REST +T1045 PR:000004716 31096 31100 BDNF +T1046 GO:0010467 31101 31111 expression +T1047 PR:000008840 31140 31150 huntingtin +T1048 GO:0065007 31199 31209 regulation +T1049 SO:0000704 31213 31218 genes +T1050 GO:0010467 31232 31242 expression +T1051 PR:000008840 31348 31358 huntingtin +T1052 SO:0000704 31368 31372 gene +T1053 GO:0065007 31373 31383 regulation +T1054 PR:000008840 31425 31435 huntingtin +T1055 UBERON:0002532 31477 31485 epiblast +T1056 GO:0009790 31499 31512 embryogenesis +T1057 UBERON:0004341 31534 31540 streak +T1058 GO:0090009 31534 31540;31550 31559 streak ... formation +T1059 UBERON:0003062 31545 31549 node +T1060 UBERON:0004341 31561 31577 primitive streak +T1061 UBERON:0003077 31591 31608 paraxial mesoderm +T1062 GO:0048339 31591 31608;31623 31632 paraxial mesoderm ... formation +T1063 UBERON:0009746 31613 31622 head fold +T1064 GO:0010467 31687 31696 expressed +T1065 SO:0000704 31729 31734 genes +T1066 PR:000008840 31789 31799 huntingtin +T1067 UBERON:0000922 31810 31817 embryos +T1068 PR:000008840 31902 31912 huntingtin +T1069 UBERON:0000922 32003 32012 embryonic +T1070 UBERON:0004341 32045 32051 streak +T1071 UBERON:0000922 32082 32088 embryo +T1072 PR:000008840 32160 32170 huntingtin +T1073 UBERON:0002435 32191 32199 striatal +T1074 http://purl.obolibrary.org/obo/MONDO_0007739 32215 32217 HD +T1075 NCBITaxon:10088 32229 32233 Mice +T1076 PR:000008840 32254 32257 Hdh +T1077 NCBITaxon:10088 32263 32267 mice +T1078 NCBITaxon:10088 32337 32342 mouse +T1079 PR:000008840 32343 32345 HD +T1080 SO:0000704 32346 32350 gene +T1081 SO:0000853 32351 32360 homologue +T1082 NCBITaxon:33208 32508 32514 Animal +T1083 PR:000008840 32532 32535 Hdh +T1084 PR:000008840 32541 32544 Hdh +T1085 GO:0007565 32596 32607 pregnancies +T1086 GO:0007618 32613 32619 mating +T1087 PR:000008840 32623 32626 Hdh +T1088 PR:000008840 32632 32635 Hdh +T1089 UBERON:0010148 32706 32710 plug +T1090 UBERON:0000922 32733 32740 Embryos +T1091 UBERON:0000922 32825 32832 embryos +T1092 PR:000000105 32847 32852 Nodal +T1093 GO:0010467 32853 32863 expression +T1094 UBERON:0000922 32882 32889 embryos +T1095 GO:0007618 32895 32902 matings +T1096 PR:000008840 32906 32909 Hdh +T1097 PR:000008840 32915 32918 Hdh +T1098 PR:000000105 32929 32932 Ndl +T1099 GO:0097617 33035 33048 hybridization +T1100 UBERON:0000922 33094 33101 embryos +T1101 UBERON:0002450 33168 33175 decidua +T1102 GO:0097617 33272 33286 hybridizations +T1103 PR:000000105 33332 33337 Nodal +T1104 PR:000033987 33338 33342 lacZ +T1105 GO:0010467 33343 33353 expression +T1106 UBERON:0000922 33416 33423 embryos +T1107 UBERON:0000922 33459 33466 Embryos +T1108 CHEBI:17754 33487 33495 glycerol +T1109 PR:000008840 33528 33538 huntingtin +T1110 UBERON:0000922 33638 33645 embryos +T1111 UBERON:0000922 33689 33695 embryo +T1112 CL:0000670 33781 33802 Primordial Germ Cells +T1113 CL:0000670 33804 33808 PCGs +T1114 UBERON:0000922 33830 33837 embryos +T1115 CHEBI:9750 33918 33924 TX-100 +T1116 UBERON:0000922 33933 33940 Embryos +T1117 CHEBI:67100 33963 33975 Tris-Maleate +T1118 CHEBI:67100 33990 34002 Tris-Maleate +T1119 CHEBI:6636 34021 34026 MgCl2 +T1120 CHEBI:75958 34093 34101 solution +T1121 CHEBI:67100 34109 34121 Tris-Maleate +T1122 CHEBI:6636 34140 34145 MgCl2 +T1123 UBERON:0000922 34210 34217 embryos +T1124 CHEBI:9750 34246 34252 TX-100 +T1125 UBERON:0000922 34261 34267 embryo +T1126 UBERON:0000922 34277 34284 Embryos +T1127 UBERON:0000922 34328 34335 Embryos +T1128 NCBITaxon:10114 34422 34425 rat +T1129 UBERON:0001977 34426 34431 serum +T1130 CHEBI:16526 34505 34508 CO2 +T1131 UBERON:0000922 34510 34517 Embryos +T1132 UBERON:0004044 34587 34590 AVE +T1133 UBERON:0004044 34592 34618 anterior visceral endoderm +T1134 PR:000008840 34620 34622 HD +T1135 http://purl.obolibrary.org/obo/MONDO_0007739 34624 34644 Huntington's disease +T1136 SO:0000704 34645 34649 gene +T1137 http://purl.obolibrary.org/obo/MONDO_0007739 34651 34653 HD +T1138 http://purl.obolibrary.org/obo/MONDO_0007739 34655 34675 Huntington's disease +T1139 PR:000008840 34677 34680 Hdh +T1140 NCBITaxon:10088 34682 34687 mouse +T1141 PR:000008840 34688 34690 HD +T1142 SO:0000704 34691 34695 gene +T1143 SO:0000853 34696 34705 homologue +T1144 CL:0000670 34707 34711 PCGs +T1145 CL:0000670 34713 34734 primordial germ cells +T1146 GO:0097617 34815 34828 hybridization +T1147 GO:0042571 35266 35274 antibody +T1148 CHEBI:33893 35275 35283 reagents +T1149 NCBITaxon:10088 35317 35321 mice +T1150 NCBITaxon:33208 35506 35512 animal +T1151 http://purl.obolibrary.org/obo/MONDO_0007739 35752 35772 Huntington's disease +T1152 http://purl.obolibrary.org/obo/MONDO_0007739 35801 35821 Huntington's Disease +T1153 http://purl.obolibrary.org/obo/MONDO_0003847 35872 35890 Hereditary Disease +T1154 http://purl.obolibrary.org/obo/MONDO_0003847 35990 36008 Hereditary Disease diff --git a/src/ontogpt/evaluation/craft/database/all/16109169.txt b/src/ontogpt/evaluation/craft/database/all/16109169.txt new file mode 100644 index 000000000..945013167 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16109169.txt @@ -0,0 +1,129 @@ +Inactivation of the Huntington's disease gene (Hdh) impairs anterior streak formation and early patterning of the mouse embryo + +Abstract + +Background + +Huntingtin, the HD gene encoded protein mutated by polyglutamine expansion in Huntington's disease, is required in extraembryonic tissues for proper gastrulation, implicating its activities in nutrition or patterning of the developing embryo. To test these possibilities, we have used whole mount in situ hybridization to examine embryonic patterning and morphogenesis in homozygous Hdhex4/5 huntingtin deficient embryos. + +Results + +In the absence of huntingtin, expression of nutritive genes appears normal but E7.0–7.5 embryos exhibit a unique combination of patterning defects. Notable are a shortened primitive streak, absence of a proper node and diminished production of anterior streak derivatives. Reduced Wnt3a, Tbx6 and Dll1 expression signify decreased paraxial mesoderm and reduced Otx2 expression and lack of headfolds denote a failure of head development. In addition, genes initially broadly expressed are not properly restricted to the posterior, as evidenced by the ectopic expression of Nodal, Fgf8 and Gsc in the epiblast and T (Brachyury) and Evx1 in proximal mesoderm derivatives. Despite impaired posterior restriction and anterior streak deficits, overall anterior/posterior polarity is established. A single primitive streak forms and marker expression shows that the anterior epiblast and anterior visceral endoderm (AVE) are specified. + +Conclusion + +Huntingtin is essential in the early patterning of the embryo for formation of the anterior region of the primitive streak, and for down-regulation of a subset of dynamic growth and transcription factor genes. These findings provide fundamental starting points for identifying the novel cellular and molecular activities of huntingtin in the extraembryonic tissues that govern normal anterior streak development. This knowledge may prove to be important for understanding the mechanism by which the dominant polyglutamine expansion in huntingtin determines the loss of neurons in Huntington's disease. + +Background + +Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder that is caused by CAG repeats in the HD locus that extend a polyglutamine tract in a ubiquitous HEAT domain protein called huntingtin [1]. The molecular mechanism by which the new property that is conferred on huntingtin by the polyglutamine expansion leads to the hallmark loss of striatal neurons in HD is not known. However, polyglutamine expansions in unrelated proteins that target distinct neuronal cell populations cause distinct 'polyglutamine' neurodegenerative disorders. This observation strongly suggests that the striatal cell specificity of the polyglutamine expansion in the context of huntingtin must be determined by some aspect of huntingtin's structure, subcellular location or activities [2]. + +Huntingtin is postulated to function as a flexible ~350 kDa HEAT domain scaffold that may facilitate the assembly and possibly the subcellular location of large protein complexes [3-7]. Huntingtin's large number of diverse cytoplasmic and nuclear protein binding partners strongly suggest that huntingtin may participate in a variety of cellular processes that range from trafficking of growth factor complexes to gene transcription (reviewed in [5,8,9]. However, despite the potential importance of huntingtin's normal function to our understanding of how the dominant polyglutamine mutation causes HD pathology, huntingtin's precise molecular and cellular activities have not been defined. + +Therefore, we, and others, set out to discover huntingtin's essential activities by studying the effects of huntingtin deficiency in the mouse. Inactivation of the mouse HD gene (Hdh) has shown that huntingtin is not required for cell viability, as evidenced by the survival of mouse embryonic stem cells and neurons that lack huntingtin [10-12]. However, huntingtin is needed at the level of the organism for proper mammalian embryonic development [10,13,14]. Complete lack of huntingtin results in developmental arrest during gastrulation, while severe reduction of huntingtin levels results in abnormal neurogenesis and perinatal lethality [15]. + +Analysis of huntingtin deficient Hdhex4/5/Hdhex4/5 embryos reveals that homozygous inactivation of the mouse HD gene does not overtly affect development until E7.0. By E7.5, mutant embryos exhibit a shortened primitive streak, reduced size and, by morphology, lack a node and head folds. Mutants are rapidly resorbed by E8.0 [10]. Importantly, the expression of huntingtin only in extraembryonic tissues in chimeras rescues this gastrulation phenotype, suggesting that huntingtin is required only in cells of the extraembryonic lineage and acts in a cell non-autonomous manner at this stage [16]. + +Extraembryonic tissues are essential for supplying nutrients and signals that direct anterior/posterior axis formation and patterning in the developing embryo (reviewed in [17]), implicating huntingtin in either or both of these processes. Of these possibilities, the nutritive role has been more extensively investigated. However, huntingtin deficient embryos do not display obvious visceral endoderm defects, with the notable exception of compromised iron transport in later stage mutants, although iron uptake is undisturbed [16] and endocytosis is not impaired in huntingtin deficient embryos or embryonic stem cells [16,18]. + +By the same token, huntingtin shuttles through the nucleus, where it is required for proper nuclear localization of its transcription factor partners, suggesting that huntingtin may play a role in transcription cascades in extraembryonic tissues that pattern the embryo [18]. Therefore, we have examined this hypothesis, by monitoring the expression of genes that determine normal embryonic patterning and morphogenesis in Hdhex4/5/Hdhex4/5 huntingtin deficient embryos. Our results support and refine the hypothesis, indicating that huntingtin is required for proper mesoderm patterning and for normal regional restriction of the expression of a subset of growth and transcription factors. + +Results + +Huntingtin-deficient embryos exhibit abnormal streak progression and paraxial mesoderm production + +Since extraembryonic tissues supply nutrients to the developing embryo, we tested the possibility that huntingtin deficiency may perturb this function by performing RT-PCR analysis to examine the expression of a panel of 'nutritive' genes in E7.5 wild-type and Hdhex4/5/Hdhex4/5 huntingtin deficient embryos. Consistent with a previous report [16], no obvious differences were found in the expression of "nutritive" genes (Hnf4, Afp, Tfn, ApoAI, Apo-AIV, and ApoB) or genes involved in yolk sac hematopoiesis or vasculogenesis (Ttr, Rbp, Flt1, Flk1, Tal1, Rbtn2, GATA1) (data not shown), suggesting that huntingtin is not essential for the proper expression of genes required for the nutritive function of the extraembryonic tissues. + +To investigate huntingtin's developmental activities, we then analyzed the expression of genes which pattern the early embryo or mark morphogenic landmarks in wild-type and Hdhex4/5/Hdhex4/5 embryos by whole mount and section in situ hybridization. The dissections confirmed previous morphologic data at E7.0–7.5 that all Hdhex4/5/Hdhex4/5 homozygotes exhibit abnormal morphology, including shortened primitive streak and a lack of morphological head folds or node [10,13]. The results of in situ hybridization analysis also confirmed that all three germ layers and extraembryonic tissue are formed in huntingtin deficient embryos. + +Otx2, normally expressed in the anterior neuroectoderm and anterior visceral endoderm [19], is expressed in mutant embryos at E7.5 (Fig. 1A,B) although the expression domain appears reduced. Similarly, Hesx1 expression is grossly normal in mutant embryos, with expression localized to the AVE and neuroectoderm (Fig. 1C–F, [20]), although the expression domain also appears reduced. These results indicate appropriate specification and movement of anterior visceral endoderm (AVE) cells from the distal tip and suggest that neuroectoderm is induced in the mutant embryos. + +Figure 1 + +AVE displacement and anterior neurectoderm induction occur normally in the absence of huntingtin. Whole mount in situ hybridization analysis of Otx2 (A,B) and Hesx (C-F) in E7.5 normal (A,C,E) and mutant (B,D,F) embryos reveals that neuroectoderm and anterior visceral endoderm (AVE) develop normally in huntingtin deficient embryos, although the neuroectoderm expression domain is reduced. Asymmetrical expression of Hesx in mutant embryos (F) suggests that left-right transcriptional control is maintained. Hnf3β expression in the definitive endoderm extends around the distal tip and is reduced in the AVE (*) in both normal (G,I) and mutant embryos (H,J). Taken together, these results suggest normal ectoderm and endoderm induction and localization in Hdhex4/5/Hdhex4/5 embryos. Embryos are shown in lateral views, with anterior to the left in all pictures with the exception of E and F. Embryos are viewed from the anterior aspect in E and F. + +To examine definitive endoderm formation, the expression of Hnf3β (FoxA2) in mutant and wild-type embryos was analyzed. In wild-type embryos, Hnf3β expression is confined to the node and anterior definitive endoderm (Fig. 1G,I[21]). Mutant embryos exhibit Hnf3β-reactive definitive endoderm over the disorganized anterior streak region and proceeding rostrally around the distal tip (Fig. 1H,J). In both normal and mutant embryos, the AVE exhibits little Hnf3β expression. Therefore, huntingtin deficiency does not greatly affect Hnf3β regulation or the reorganization of the visceral endoderm. + +The lack of a morphological node and presence of a shortened streak, together with reduced neuroectoderm and lack of headfolds, suggest that anterior streak formation may be impaired in huntingtin deficient embryos. To investigate this possibility, we examined mesoderm formation in mutant embryos. Mesoderm is specified in the mutant embryos, as marked by the expression of T (Brachyury) and Evx1 (Fig. 2A–F). However, close inspection of the data reveals abnormal patterning within this tissue and its derivatives.T, normally expressed in the primitive streak, node and axial head process/notochord mesoderm [22], is detected in the shortened streak and axial mesoderm in Hdhex4/5/Hdhex4/5 embryos, extending rostrally from a region of weakly positive cells (Fig. 2A,B). T expression appears weaker, however, in the anterior streak, corresponding to cells that will give rise to axial mesoderm (Fig. 2D). T is also ectopically expressed in mutant extraembryonic mesoderm at the anterior embryonic junction and along the chorion (Fig. 2B,D). Similarly, Evx1, normally expressed in primitive streak mesoderm at E7.5 with highest levels in proximal cells [23], is expressed in the proximal shortened streak but is also aberrantly expressed throughout the extraembryonic mesoderm, allantois and chorion (Fig. 2E,F). Extraembryonic mesoderm, derived from the proximal streak, does not normally express T or Evx1 in wild-type embryos [22]. Therefore, the inappropriate expression of T and Evx1, the shortened primitive streak, and the absence of a morphological node, all suggest that the anterior primitive streak is deficient in the mutant embryos. + +Figure 2 + +Huntingtin is required for formation of anterior primitive streak and paraxial mesoderm. Whole mount and section insitu hybridizations of E7.5 embryos shows T (Brachyury) (A-D) is expressed in the primitive streak, node, axial mesoderm and Evx1 (E-F) is expressed in the primitive streak, most strongly in the proximal streak wild-type embryos. However, in mutant embryos, both T (B, D) and Evx1 (F) are ectopically expressed in the extraembryonic region. Wnt3A expression is reduced in mutant embryos (H), although the localization of its expression to the proximal streak is the same as in wild-type embryos (G). Analysis of paraxial mesoderm markers Tbx6 (I,J) and Dll1 (K,L), reveals that these markers are reduced in mutant embryos (J,L), suggesting impaired paraxial mesoderm production in the absence of huntingtin. Embryos in A-H are shown in a lateral view with anterior oriented to the left. Embryos in I-L are shown in a posterior view (I,K) or near posterior (J,L) view with proximal oriented toward the top. In (C,D), al = allantois, a = amnion, ch = chorion, ee = embryonic node(N), em = extraembryonic mesoderm, n = node, ps = primitive streak. Rather than a node, mutant embryos exhibit a region of disorganized cells (*) at the distal extent of the short primitive streak. + +The anterior streak generates paraxial mesoderm. Therefore we examined paraxial mesoderm formation in wild-type and mutant embryos, revealing deficits in mesoderm patterning. Starting at E.7.5, Wnt3A is expressed in the primitive streak in cells fated to become paraxial mesoderm. In huntingtin deficient mutants, Wnt3a is induced in the proximal streak (Fig. 2G,H), confirming stage appropriate posterior development, in contrast to the absence of anterior head folds. However, expression of Wnt3a is noticeably reduced in Hdhex4/5/Hdhex4/5 embryos, suggesting a defect in paraxial mesoderm development (Fig. 2H). Reduced expression of Tbx6 in the mesoderm lateral to the primitive streak in mutant embryos confirms this interpretation (Fig. 2I,J). Furthermore, in mutant embryos at E7.5, the expression of Dll1 in the distal streak region and in only a narrow swath of cells located laterally confirms the paucity of paraxial mesoderm (Fig. 2K,L, [24]). These results strongly suggest that anterior primitive streak formation is impaired, resulting in reduced axial and paraxial mesoderm formation and impaired neural development. + +Impaired regional restriction of growth factor expression in the absence of huntingtin + +To elucidate the apparent patterning deficits, we next analyzed signaling molecules that are required for early patterning. Nodal, a member of the Tgfβ family of secreted molecules is required for the formation and maintenance of the primitive streak and induction of the AVE [25-27]. Nodal is normally expressed throughout the epiblast and overlying visceral endoderm at early post implantation stages [28], but later becomes restricted to the posterior of the embryo to the site of primitive streak with asymmetrical visceral endoderm expression marking the left-right axis. By E7.5, Nodal expression is restricted to the node. Nodal expression was assessed in Hdhex4/5/Hdhex4/5 embryos heterozygous for the Ndl lac Z allele [28,29]. Notably, heterozygous loss of nodal does not alter the Hdhex4/5/Hdhex4/5 phenotype, as determined by morphology of Hdhex4/5/Hdhex4/5:Ndllacz/Ndl+ embryos compared with Hdhex4/5/Hdhex4/5 embryos (data not shown). In contrast to wild-type embryos, which exhibit tight restriction of Nodal.LacZ expression to the node, Hdhex4/5/Hdhex4/5:Ndllacz/Ndl+ embryos express Nodal.LacZ throughout the endoderm overlying the epiblast, with higher levels in the posterior in an asymmetric pattern (Fig. 3A–D). The lack of tight restriction of nodal signal is consistent with a failure to form an organized node structure. + +Figure 3 + +Impaired regional restriction of gene expression in huntingtin deficient embryos. X-gal staining of Nodal-LacZ embryos shows staining in endoderm near the node of normal embryos (A,C) but broad staining in mutant embryos (B, D), although expression is higher in the posterior. The tight node expression of Nodal in normal embryos (C) is lost in mutant embryos (D), consistent with the loss of a morphological node in the absence of huntingtin. Whole mount and in situ hybridization of E7.5 day embryos reveals that Fgf8 is detected in the proximal streak and is downregulated in cells migrating out of the streak in normal embryos (E,G). In contrast, Fgf8 remains highly expressed in mutant embryos (F,H). Transient expression of Gsc in the definitive endoderm overlying the prospective head region in normal embryos (I,K) is distinguished in other cell layers in normal embryos but remains unrestricted in mutant embryos (J,L). Earlier posterior expression of Gsc is also maintained in mutant embryos (J) while it is down-regulated in normal embryos (I). Embryos (A,B,E,F,G,H,I,K,L) are shown in a lateral view with anterior oriented to the left. Embryos (C,D) are in a posterior view. + +Fgf8 signaling is also essential for normal gastrulation in the mouse embryo. Fgf8 is required for cell migration away from the primitive streak [30]. Expressed just prior to streak formation in the posterior epiblast and visceral endoderm, Fgf8 is restricted to the streak mesoderm at E7.5 in a decreasing proximal-distal gradient and is downregulated in cells shortly after they exit the streak (Fig. 3E,G). In Hdhex4/5/Hdhex4/5 embryos, Fgf8 expression is strongly expressed in the posterior region in the primitive streak and ectopically in the endoderm overlying the entire epiblast (Fig. 3F,H). However, streak derivatives appear to migrate normally as evidenced by the proper anterior expression of markers such as Otx2, Hnf3β and Hesx1 anteriorly (Fig. 1). Therefore, mutant embryos exhibit normal migration of streak derivatives but display impaired Fgf8 repression in mutant endoderm. + +Hdhex4/5/Hdhex4/5 embryos also fail to restrict the expression of goosecoid (Gsc). Normally, Gsc is initially expressed in the visceral endoderm and proximal, posterior streak where the primitive streak will form prior to gastrulation. As the primitive streak forms and extends, Gsc is expressed in the distal streak, the node, and the axial mesoderm extending anteriorly from the node (Fig. 3I,K, [31,32]). However, in the mutant Hdhex4/5/Hdhex4/5 embryos, high levels of Gsc expression remain unrestricted in the endoderm overlying the entire embryo and ectopically in cells adjacent to the ectoplacental cone (Fig. 3J,L). These results suggest that, in contrast to proper Hnf3β regulation, Gsc remains inappropriately activated in mutant visceral and definitive endoderm, implicating huntingtin in the proper restriction of this homeodomain transcription factor. + +Huntingtin is not required for expression of extraembryonic signaling molecules + +Previous studies of chimeric embryos suggest that huntingtin is required only in the extraembryonic tissue for proper development [16]. Signals from the extraembryonic tissue are critical for the induction of embryonic signals and for patterning the epiblast. Consequently, we examined extraembryonic development in huntingtin deficient embryos. Hnf4 is a transcription factor expressed in the primitive endoderm as soon as this tissue becomes distinct and is a key regulator of visceral endoderm secreted factors such as alphafetoprotein, apolipoproteins, and transferrin. Inactivation of Hnf4 results in impaired gastrulation [33,34]. At E7.5, Hnf4 is expressed in the columnar visceral endoderm cells at the extraembryonic-ectoderm junction (Fig. 4A, [33]). In Hdhex4/5/Hdhex4/5 embryos, consistent with normal primitive and visceral endoderm differentiation, Hnf4 expression appears normal, although the signal is stronger in mutant embryos compared to wild-type embryos (Fig. 4B). Similarly, Pem, a transcription factor expressed in proximal visceral endoderm and ectoplacental cone in wild-type embryos at E7.5, also is expressed in these tissues in the mutant embryos (Fig. 4C,D[35]). However, Pem expressing visceral endoderm hangs over the anterior of the mutant embryos, revealing abnormal location despite grossly normal differentiation. + +Figure 4 + +Normal expression of extraembryonic markers in huntingtin deficient embryos. Whole mount in situ hybridization analysis at E7.5 of markers of the extraembryonic tissues reveals grossly normal expression in the absence of huntingtin. Hnf4, expressed in the visceral endoderm at the junction of embryonic-ectoderm junction (A), is normal in mutant embryos, although the signal is slightly higher (B). Similarly, the expression of Pem transcripts is maintained in mutant embryos (D) similar to normal embryos (C), although Pem is expressed in the abnormal lopsided overhang of visceral endoderm over the anterior of the mutant embryos. Expression of extraembryonic signaling molecules is unaffected by the loss of huntingtin, as evidenced by the expression of Bmp4 (E,F) in the extraembryonic ectoderm, and Lefty1 and Dkk1 (I-L) in the AVE in mutant embryos. Bmp4 is not localized, however, to a ring of extraembryonic ectoderm in mutant embryos (F) as in normal embryos (E). Primitive germ cells (PCGs) are induced normally in both wild-type (G) and mutant embryos (H), suggesting the Bmp4 signaling from the extraembryonic ectoderm to the epiblast is normal. Lefty1 expression appears disorganized in mutant embryos (I) compared to wild-type embryos (J). In contrast, the anterior expression of Dkk1 in the AVE in mutant embryos (L) matches the wild-type expression pattern (K). Despite normal AVE formation, head folds fail to form in mutant embryos, even when cultured in nutrient rich media for 24 hours. Wild-type E7.5 embryos, when cultured in 75% rat serum, develop somites (M), heart (white arrow, N) and head folds (blue arrow head, N) in culture. In contrast, huntingtin deficient embryos continue to live in culture but do not form headfolds, heart or somites (O). Embryos are shown in a lateral view (A-F, I-J) with anterior oriented to the left. Embryos in (G,H,K,L) are shown in an anterior view with proximal oriented up. + +Signals from the extraembryonic tissues, including the anterior visceral endoderm and extraembryonic ectoderm are required for proper formation and patterning of the epiblast [17]. Bmp4 is a signaling molecule that is first expressed uniformly throughout the extraembryonic ectoderm and subsequently is localized to a ring of extraembryonic ectoderm adjacent to the epiblast (Fig. 4E, [36]). A key factor in regulating the formation of the node and primitive streak, Bmp4 is required for patterning the embryo along the proximodistal axis [37-40]. In the absence of huntingtin, Bmp4 expression is properly maintained in the Hdhex4/5/Hdhex4/5 extraembryonic ectoderm but is also expressed throughout the extraembryonic ectoderm (Fig. 4F) in a pattern that is similar to early Bmp4 expression rather than being restricted to a ring of extraembryonic ectoderm as seen in the wild-type embryos To assess Bmp4 signaling from the extraembryonic ectoderm, we evaluated primordial germ cells (PGCs), which require Bmp4 for their induction [37]. PGCs can first be detected at E7.0 and subsequently underlie the posterior portion of the primitive streak. Whole mount staining of E7.5 mutant and wild-type embryos for alkaline phosphatase activity reveals that PCGs form in Hdhex4/5/Hdhex4/5 embryos, suggesting that Bmp4 signaling is functional in the absence of huntingtin (Fig. 4G,H). + +The anterior visceral endoderm (AVE) is also an extraembryonic source of signals that are critical for early patterning. Wnt and nodal antagonists, Dkk1 (mdkk-1) and Lefty1 respectively, are expressed in the AVE and are important in limiting the posteriorization of the anterior embryo by restricting Nodal and Wnt signaling [41-43]. In Hdhex4/5/Hdhex4/5 embryos, both Dkk1 (Fig. 4I,J) and Lefty1 (Fig. 4K,L) are expressed normally in the AVE as compared with wild-type embryos. However, Dkk-1 levels appear to be slightly increased in Hdhex4/5/Hdhex4/5 embryos, although the pattern of Dkk-1 expression remains unchanged and this increase may just reflect the same amount of expression in a smaller area. Therefore, the ectopic expression of Nodal (Fig. 3A–D) and the decreased Wnt3a expression (Fig. 2H) in mutant embryos do not appear to be result of changes in the expression pattern of Lefty1 or Dkk1. + +Despite normal AVE formation and neuroectoderm induction, head folds do not form in Hdhex4/5/Hdhex4/5 embryos. Therefore, to determine whether mutant embryos are inherently capable of forming head folds, embryos harvested at stage E7.5 were allowed to progress in rich culture medium in vitro for 24 hours. Wild-type embryos continued to develop head folds, somites and hearts (Fig. 4M,N). In contrast, mutant stage 7.5 embryos did not develop headfolds, hearts or somites, although these embryos continued to live (Fig. 4O). These results strongly suggest that in the absence of huntingtin, embryos are unable to undergo organogenesis, even if they continue to live past E7.5 in a nutrient rich environment. + +Discussion + +We have investigated the embryonic processes that require huntingtin in order to more precisely delineate huntingtin's essential molecular and cellular activities and to provide clues to the mechanism by which the dominant polyglutamine expansion mutation in huntingtin leads to HD pathogenesis. In pursuing the finding that huntingtin is needed only in extraembryonic tissues for normal gastrulation, our data fail to provide evidence of abnormal nutritive gene expression in Hdhex4/5/Hdhex4/5 embryos. Instead, our results reveal that huntingtin is required for normal anterior streak formation and the consequent production of paraxial mesoderm, with a previously unrecognized role for huntingtin in the proper extinction of transiently and/or dynamically expressed genes. + +Indeed, the hallmark of the huntingtin deficient molecular phenotype is the impaired down-regulation of a subset of dynamically expressed genes, after the proper onset of expression. This phenomenon does not reflect a lack of anterior/posterior axis formation, as evidenced by the formation of the AVE anteriorly and the primitive streak posteriorly. Nor can it be simply explained by delayed development, as stage-specific markers, such as Wnt3a and primordial germ cells, which are detectable at E7.0 in wild-type embryos, are induced appropriately. Furthermore, the expression of T and Evx1 in the extraembryonic mesoderm of mutant embryos is not a feature of wild-type embryos, even at earlier stages. This strongly suggests that in huntingtin deficient embryos, the migration of the distal streak derivatives to the extraembryonic mesoderm occurs normally but that the down-regulation of these genes is impaired. This impairment may also explain the failure of huntingtin deficient embryos to properly restrict the expression of Fgf8, Nodal and Gsc. Thus, huntingtin may play a direct role in the transcriptional regulation, or mRNA stability of these genes or it may act indirectly by intersecting with other pathways that regulate the expression of these genes. + +The requirement for huntingtin in the extraembryonic tissues had prompted us to test whether impaired extraembryonic signals might be responsible for the dysregulation of gene expression within the epiblast that is observed in Hdhex4/5/Hdhex4/5 embryos. Extraembryonic development in Hdhex4/5/Hdhex4/5 embryos is associated with mildly elevated levels expression of Hnf4 in the primitive endoderm and Pem in the lopsided anterior chorion but the expression of other known signals, such as Bmp4 from the extraembryonic ectoderm, and Dkk1 and Lefty1 from the AVE, appear to be normal, although the slight increase in Dkk-1 expression in Hdhex4/5/Hdhex4/5 embryos suggests that further investigation into Wnt signaling is warranted. Moreover, extraembryonic Bmp4 signaling is not impaired in the absence of huntingtin, as the induction of PCGs in mutant embryos is normal, implying proper transport and secretion of the appropriate extraembryonic signals. However, Nodal, Fgf8 and Gsc are expressed ectopically in the visceral endoderm of Hdhex4/5/Hdhex4/5 embryos. Both Nodal and Fgf8, important growth factors required for normal development of the epiblast, are tightly regulated during gastrulation. Therefore, misexpression of either or both of these factors, or of goosecoid, in the visceral endoderm could contribute to the Hdhex4/5/Hdhex4/5 mutant phenotype. In addition, it is possible that other extraembryonic signal(s) that we have not analyzed may also be affected by the lack of huntingtin activity in extraembryonic cells in mutant embryos. + +Huntingtin deficient embryos also fail to form headfolds, and to undergo organogenesis, even after culturing in nutrient rich media. The absence of headfold formation in these embryos does not appear to be a result of a failure to induce neurectoderm or a failure to form the AVE, since mutant embryos express markers such as Otx2, Ddk1, Lefty1 and Hesx1. In addition, since node formation is not required for neural induction [44-46], the failure to form a node in huntingtin deficient embryos is also unlikely to explain the lack of headfolds. The apparent reduction of paraxial mesoderm in Hdhex4/5/Hdhex4/5 embryos could explain the lack of headfolds since paraxial mesoderm is important for the full development of neuroectoderm, and consequently, headfolds. Alternatively, the inability to manifest headfolds could suggest that huntingtin is required at a very early stage for normal CNS development. This conclusion is consistent with the finding that severely reduced levels of huntingtin, from a hypomorphic Hdh allele, lead to abnormal brains later in embryonic development [15]. + +The cardinal features of complete Hdh inactivation that we observe are similar to the phenotypes that stem from the complete inactivation of the Polycomb group gene (Pc-g) Eed (embryonic ectoderm development). Indeed, complete deficiency for either huntingtin or the eed protein leads to abnormal streak development, lack of headfold formation, ectopic T, Evx1 and Nodal expression and disruption of anterior primitive streak mesoderm production [47]. Interestingly, Eed protein is also required for proper trophoblast development and normal maintenance of imprinted X-inactivation and genomic imprinting [47-49], suggesting that these activities warrant investigation in huntingtin deficient embryos. + +Thus, our observations provide unexpected starting-points in the search for huntingtin's precise molecular activities, which began with the discovery that this HEAT domain protein hosts the dominant polyglutamine property that is the fundamental basis of HD pathogenesis. In HD patients and in accurate genetic replicas, HD CAG knock-in mice, the dominant mutation specifically affects the major population of neurons in the striatum, without impairing huntingtin's essential activities in embryonic development [50-53]. Indeed, homozygous HD patients make no wild-type huntingtin, and, in the mouse, a single mutant Hdh allele's worth of mutant huntingtin can fully rescue huntingtin deficiency embryonic phenotypes [15,51]. The quest to understand the HD mechanism, therefore, is aimed at delineating the huntingtin activity that may explain the striatal cell specificity of the polyglutamine mutant version of huntingtin. One hypothesis is that huntingtin is normally involved in gene transcription, as proposed for NRSF/REST mediated BDNF expression [54]. Now, our finding that huntingtin can be absolutely necessary for the appropriate regulation of genes with dynamic expression patterns in vivo, provides a compelling reason to elucidate the cellular machinery that is necessary for huntingtin mediated gene regulation. + +Conclusion + +Our findings indicate that huntingtin is required for proper patterning of the epiblast during early embryogenesis, for proper anterior streak and node formation, primitive streak progression, paraxial mesoderm and head fold formation, as well as for the proper restriction of transiently expressed growth and transcription factor genes. Knowledge of the molecular basis of these changes in huntingtin deficient embryos should facilitate the identification of the cellular pathways that are dependent on huntingtin activities. These will be important for implicating candidates to be assessed in the extraembryonic signals that determine anterior streak progression in the developing embryo and in delineating the dominant activity of the polyglutamine tract in huntingtin that determines the striatal specificity of HD. + +Methods + +Mice and genotyping + +The Hdhex4/5 mice carrying a pGKneo insertion/replacement inactivating mutation in the mouse HD gene homologue have been described previously [10]. The experiments were conducted in accordance with an IACUC approved protocol, through the MGH Subcommittee on Animal Research. Mutant Hdhex4/5/Hdhex4/5 and normal littermates were obtained in timed pregnancies from mating of Hdhex4/5/Hdh+ heterozygotes, genotyped by PCR assay, as described [10]. The day of plug was taken to be E0.5. Embryos that were morphologically normal were pooled separately from morphologically mutant embryos for analysis. Nodal expression was determined in embryos from matings of Hdhex4/5/Hdh+; NdllacZ/Ndl+ compound heterozygotes genotyped by PCR assay as described in [29]. + +Whole mount and section in situ hybridization and β-gal staining + +After dissection in PBS, embryos were fixed overnight in 4% paraformaldehyde at 4°C. For sections, decidua fixed in 4% paraformaldehyde, were embedded in paraffin and sectioned at 7 microns. RNA in situ hybridizations were performed as described previously [55]. Nodal.lacZ expression was assessed by β-galactosidase staining as reported [29], on embryos post fixed in 4% paraformaldehyde. Embryos were mounted in 80% glycerol before being photographed. + +The huntingtin deficient phenotype is fully penetrant at each of the stages that were assessed [10]. Three to six embryos were evaluated for each marker, with every embryo exhibiting the same mutant phenotype in each case. + +Alkaline phosphatase staining of Primordial Germ Cells (PCGs) + +After dissections, embryos were fixed in 4% paraformaldehyde briefly and washed and stored in 1 × PBS/0.1% TX-100 at 4°C. Embryos were washed once with Tris-Maleate Buffer (25 mM Tris-Maleate, pH = 9.0, 0.8 mM MgCl2) and were subsequently incubated in alkaline phosphatase staining solution (25 mM Tris-Maleate, pH = 9.0, 0.8 mM MgCl2, 0.4 mg/ml alpha-naphthyl phosphate, 1 mg/ml Fast-Red). Stained embryos were washed in 1 × PBS/0.1% TX-100. + +Whole embryo culture + +Embryos were dissected at E7.5 and washed in DMEM. Embryos were then cultured individually in 1 ml of culture media (75% immediately centrifuged rat serum and 25% DMEM [56]) for 24 hours while rotating in a 37°C incubator in 5% CO2. Embryos were then fixed in 4% paraformaldehyde for analysis. + +Abbreviations + +AVE, anterior visceral endoderm; HD, Huntington's disease gene; HD, Huntington's disease; Hdh, mouse HD gene homologue; PCGs, primordial germ cells + +Authors' contributions + +JMW, TC, PH-M and MD performed whole mount and in situ hybridization assays. MEM and RC contributed to the conception of this study. JMW, TC, PH-M and MEM drafted the manuscript and RC contributed to its finalization. All authors read and approved the final manuscript. + +Acknowledgements + +We are grateful to Drs. A. Gossler, J. Darnell, Jr., J Rossant, G. Keller, S. Orkin, G. Martin, T. Yamaguchi, A. McMahon, R. Maas, K., Muneoka, A. Simeone, Hamada H. and C. Niehrs for the generous gifts of clones and antibody reagents and Dr. E. Robertson for NdllacZ mice. We would like to thank Kathy Molyneaux for her helpful suggestions and technical assistance. We also thank Vladimir Vrbanac, Janice Espinola and Edith Toral Lopez for assistance with animal husbandry. We also thank the members of the MacDonald lab for helpful discussions during the completion of this work. This work was supported by the NINDS grants NS32765 and NS16367, and grants from the Foundation for the Care and Cure of Huntington's disease and with the support of the Huntington's Disease Society of America Coalition for the Cure and the Hereditary Disease Foundation. Juliana M. Woda is the recipient of the Milton Wexler Postdoctoral Fellowship from the Hereditary Disease Foundation. diff --git a/src/ontogpt/evaluation/craft/database/all/16110338.ann b/src/ontogpt/evaluation/craft/database/all/16110338.ann new file mode 100644 index 000000000..12e7a8e7d --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16110338.ann @@ -0,0 +1,1565 @@ +T1 CL:0000210 9 22 Photoreceptor +T2 GO:0010467 23 33 Expressing +T3 CL:0000604 39 42 Rod +T4 CL:0000573 47 51 Cone +T5 SO:0000704 52 57 Genes +T6 NCBITaxon:10088 63 68 Mouse +T7 http://purl.obolibrary.org/obo/MONDO_0009989 78 102 Enhanced S-Cone Syndrome +T8 CL:0000573 89 93 Cone +T9 CL:0000604 114 117;127 141 Rod ... photoreceptors +T10 CL:0000573 122 141 cone photoreceptors +T11 GO:0007601 151 157 vision +T12 SO:0000704 286 290 gene +T13 GO:0010467 286 301 gene expression +T14 GO:0045202 330 338 synaptic +T15 CL:0000210 391 410 photoreceptor cells +T16 UBERON:0000966 418 425 retinal +T17 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GO:0005634 3479 3486 nuclear +T113 GO:0010467 3507 3516 expressed +T114 CL:0000604 3532 3536 rods +T115 NCBITaxon:9606 3551 3556 human +T116 SO:0000704 3557 3564 genetic +T117 SO:0000704 3614 3618 gene +T118 http://purl.obolibrary.org/obo/MONDO_0002254 3637 3645 syndrome +T119 CHEBI:26130 3672 3682 pigmentary +T120 UBERON:0000966 3683 3690 retinal +T121 http://purl.obolibrary.org/obo/MONDO_0004580 3683 3703 retinal degeneration +T122 http://purl.obolibrary.org/obo/MONDO_0009989 3747 3751 ESCS +T123 CL:0000604 3808 3825 rod photoreceptor +T124 CL:0000495 3984 3988;3996 4010 blue ... photoreceptors +T125 CL:0000573 3991 4010 cone photoreceptors +T126 CL:0000573 4014 4019 cones +T127 UBERON:0000966 4081 4087 retina +T128 NCBITaxon:9606 4095 4100 human +T129 http://purl.obolibrary.org/obo/MONDO_0009989 4114 4118 ESCS +T130 UBERON:0000966 4133 4140 retinal +T131 http://purl.obolibrary.org/obo/MONDO_0004580 4133 4153 retinal degeneration +T132 PR:000001245 4181 4190 rhodopsin +T133 PR:000001195 4239 4251 S-cone opsin +T134 CL:0000573 4241 4245 cone +T135 GO:0010467 4252 4262 expressing +T136 CL:0000573 4307 4312 cones +T137 http://purl.obolibrary.org/obo/MONDO_0000001 4497 4504 disease +T138 http://purl.obolibrary.org/obo/MONDO_0009989 4553 4557 ESCS +T139 UBERON:0000966 4574 4581 retinas +T140 UBERON:0001789 4612 4631 outer nuclear layer +T141 GO:0005634 4618 4625 nuclear +T142 UBERON:0001789 4633 4636 ONL +T143 CL:0000210 4644 4657 photoreceptor +T144 GO:0044297 4658 4669 cell bodies +T145 UBERON:0000966 4689 4696 retinal +T146 http://purl.obolibrary.org/obo/MONDO_0004580 4689 4709 retinal degeneration +T147 http://purl.obolibrary.org/obo/MONDO_0001941 4747 4756 blindness +T148 NCBITaxon:10088 4784 4789 mouse +T149 SO:0000855 4790 4798 ortholog +T150 PR:000011403 4802 4807 NR2E3 +T151 UBERON:0000966 4855 4862 retinal +T152 http://purl.obolibrary.org/obo/MONDO_0004580 4855 4875 retinal degeneration +T153 UBERON:0000966 4920 4927 retinal +T154 http://purl.obolibrary.org/obo/MONDO_0004580 4920 4940 retinal degeneration +T155 UBERON:0001789 4972 4975 ONL +T156 NCBITaxon:10088 5034 5039 mouse +T157 GO:1990603 5061 5076 dark adaptation +T158 PR:000001195 5258 5265 S-opsin +T159 UBERON:0000966 5292 5298 retina +T160 CL:0000573 5396 5400 cone +T161 SO:0000704 5401 5406 genes +T162 http://purl.obolibrary.org/obo/MONDO_0009989 5490 5494 ESCS +T163 CL:0000210 5557 5571 photoreceptors +T164 NCBITaxon:10088 5590 5595 mouse +T165 GO:0097617 5620 5633 hybridization +T166 PR:000001195 5687 5694 S-opsin +T167 CL:0000573 5746 5750 cone +T168 SO:0000704 5760 5765 genes +T169 CL:0000604 5773 5796 rod photoreceptor cells +T170 GO:0010467 5857 5864 express +T171 CL:0000604 5865 5868 rod +T172 CL:0000573 5873 5877 cone +T173 SO:0000704 5878 5883 genes +T174 UBERON:0000966 5895 5901 retina +T175 CL:0000604 5976 5980 rods +T176 CL:0000573 5985 5990 cones +T177 CL:0000573 6030 6034 Cone +T178 SO:0000704 6035 6040 Genes +T179 UBERON:0000966 6059 6065 Retina +T180 UBERON:0000966 6127 6134 retinas +T181 GO:0007567 6189 6194 natal +T182 UBERON:0000966 6361 6367 retina +T183 UBERON:0001016 6372 6386 nervous system +T184 SO:0000704 6439 6444 genes +T185 SO:0000704 6572 6576 gene +T186 CL:0000573 6718 6722 cone +T187 CL:0000573 6736 6740 cone +T188 SO:0000704 6750 6755 genes +T189 UBERON:0000966 6767 6773 retina +T190 GO:0007567 6793 6798 natal +T191 CL:0000573 6843 6847 cone +T192 CL:0000573 6860 6864 cone +T193 SO:0000704 6874 6879 genes +T194 SO:0000704 6935 6940 genes +T195 SO:0000704 6972 6977 genes +T196 GO:0007602 7006 7031 phototransduction cascade +T197 PR:000001119 7039 7045 opsins +T198 SO:0000704 7114 7119 genes +T199 CL:0000573 7135 7139 cone +T200 SO:0000704 7149 7154 genes +T201 SO:0000704 7240 7245 genes +T202 SO:0000704 7320 7325 genes +T203 UBERON:0000966 7374 7380 retina +T204 PR:000011403 7485 7490 Nr2e3 +T205 GO:0010467 7491 7501 expression +T206 GO:0097617 7533 7546 hybridization +T207 UBERON:0000922 7554 7563 embryonic +T208 UBERON:0007023 7632 7637 adult +T209 GO:0010467 7711 7721 expression +T210 SO:0000704 7733 7737 gene +T211 GO:0010467 7733 7748 gene expression +T212 SO:0000704 7878 7882 gene +T213 SO:0000704 7916 7920 gene +T214 SO:0000704 7946 7950 gene +T215 SO:0000704 8034 8038 gene +T216 GO:0010467 8055 8065 expression +T217 CL:0000573 8125 8129 Cone +T218 SO:0000704 8130 8134 Gene +T219 GO:0097617 8213 8226 hybridization +T220 CL:0000573 8265 8269 cone +T221 SO:0000704 8270 8275 genes +T222 UBERON:0000966 8316 8322 retina +T223 CL:0000573 8408 8412 cone +T224 SO:0000704 8422 8427 genes +T225 GO:0097617 8519 8532 hybridization +T226 CL:0000573 8578 8582 cone +T227 SO:0000704 8583 8587 gene +T228 SO:0000704 8664 8668 gene +T229 GO:0010467 8664 8679 gene expression +T230 UBERON:0001789 8695 8698 ONL +T231 SO:0000704 8716 8720 gene +T232 GO:0010467 8716 8731 gene expression +T233 CL:0000210 8739 8753 photoreceptors +T234 GO:0010467 8924 8934 expression +T235 CL:0000573 8979 8983 cone +T236 SO:0000704 8984 8988 gene +T237 GO:0010467 8984 8999 gene expression +T238 UBERON:0001773 9052 9059 scleral +T239 UBERON:0001789 9072 9075 ONL +T240 CL:0000573 9112 9116 cone +T241 SO:0000704 9117 9121 gene +T242 CHEBI:26710 9168 9172 salt +T243 GO:0010467 9203 9213 expression +T244 UBERON:0001789 9279 9282 ONL +T245 UBERON:0000966 9392 9398 retina +T246 GO:0010467 9515 9525 expression +T247 CL:0000573 9637 9642 cones +T248 GO:0044297 9677 9688 cell bodies +T249 UBERON:0001773 9696 9703 scleral +T250 UBERON:0001789 9716 9719 ONL +T251 UBERON:0000966 9761 9767 retina +T252 GO:0097617 9812 9825 hybridization +T253 GO:0097617 9853 9866 hybridization +T254 PR:000001195 9903 9915 S-cone opsin +T255 CL:0000573 9905 9909 cone +T256 SO:0000704 9916 9920 gene +T257 PR:000001195 9922 9928 Opn1sw +T258 PR:000001195 9987 9994 S-opsin +T259 GO:0010467 9999 10008 expressed +T260 UBERON:0000966 10020 10027 retinal +T261 CL:0009004 10020 10033 retinal cells +T262 PR:000001195 10056 10063 S-opsin +T263 CHEBI:51231 10092 10118 6-diamidino-2-phenylindole +T264 CHEBI:51231 10120 10124 DAPI +T265 PR:000001195 10209 10216 S-opsin +T266 UBERON:0000966 10264 10271 retinas +T267 PR:000001195 10283 10290 S-opsin +T268 CHEBI:51231 10319 10323 DAPI +T269 PR:000001195 10414 10421 S-opsin +T270 GO:0042571 10480 10488 antibody +T271 UBERON:0000479 10501 10507 tissue +T272 CL:0000573 10581 10586 cones +T273 NCBITaxon:10088 10594 10599 mouse +T274 UBERON:0000966 10600 10606 retina +T275 UBERON:0000966 10620 10627 retinal +T276 CL:0009004 10620 10633 retinal cells +T277 PR:000001195 10649 10656 S-opsin +T278 CL:0000573 10681 10686 cones +T279 UBERON:0000966 10714 10720 retina +T280 PR:000001195 10749 10756 S-opsin +T281 UBERON:0000966 10789 10795 retina +T282 UBERON:0000966 10860 10867 retinal +T283 CL:0009004 10860 10873 retinal cells +T284 PR:000001195 10878 10885 S-opsin +T285 CL:0000210 10952 10966 photoreceptors +T286 UBERON:0007023 11016 11021 adult +T287 NCBITaxon:10088 11022 11027 mouse +T288 UBERON:0000966 11028 11034 retina +T289 GO:0010467 11043 11050 express +T290 SO:0000704 11056 11060 gene +T291 PR:000001195 11109 11116 S-opsin +T292 GO:0010467 11117 11127 expressing +T293 GO:0010467 11136 11145 expressed +T294 CL:0000604 11146 11149 rod +T295 GO:0042571 11177 11185 antibody +T296 PR:000001195 11199 11206 S-opsin +T297 PR:000001245 11211 11220 rhodopsin +T298 GO:0010467 11284 11294 expression +T299 PR:000001195 11298 11305 S-opsin +T300 PR:000001245 11310 11319 rhodopsin +T301 CL:0000210 11327 11340 photoreceptor +T302 GO:0001750 11327 11355 photoreceptor outer segments +T303 PR:000001195 11417 11424 S-opsin +T304 GO:0010467 11425 11435 expressing +T305 UBERON:0000966 11453 11459 retina +T306 CL:0000573 11488 11493 cones +T307 CL:0000573 11502 11506 Cone +T308 SO:0000704 11516 11521 Genes +T309 CL:0000573 11579 11583 cone +T310 SO:0000704 11593 11598 genes +T311 SO:0000704 11666 11671 genes +T312 CL:0000573 11727 11731 cone +T313 GO:0010467 11741 11751 expression +T314 GO:0097617 11761 11774 hybridization +T315 SO:0000704 11822 11827 genes +T316 CL:0000573 11915 11919 cone +T317 CL:0000573 11932 11936 cone +T318 GO:0010467 11957 11967 expression +T319 UBERON:0000966 11985 11991 retina +T320 SO:0000704 12003 12008 genes +T321 UBERON:0000966 12086 12092 retina +T322 UBERON:0001773 12164 12171 scleral +T323 UBERON:0001789 12184 12187 ONL +T324 CL:0000573 12207 12211 cone +T325 GO:0010467 12232 12242 expression +T326 SO:0000704 12257 12262 genes +T327 UBERON:0000966 12299 12305 retina +T328 CL:0000573 12331 12335 cone +T329 SO:0000704 12345 12350 genes +T330 SO:0000673 12391 12402 transcripts +T331 CL:0000210 12410 12423 photoreceptor +T332 GO:0001917 12410 12437 photoreceptor inner segment +T333 PR:000004855 12445 12450 Bub1b +T334 PR:000016206 12455 12459 Tcta +T335 GO:0097617 12511 12524 hybridization +T336 UBERON:0001789 12586 12589 ONL +T337 GO:0001750 12617 12630 outer segment +T338 UBERON:0003926 12617 12636 outer segment layer +T339 SO:0000673 12665 12675 transcript +T340 CL:0000604 12714 12717 rod +T341 SO:0000704 12727 12732 genes +T342 PR:000001245 12740 12743 Rho +T343 PR:000012351 12757 12763 Pcdh21 +T344 PR:000013820 12765 12769 Rbp3 +T345 PR:000000686 12775 12780 Cnga1 +T346 CL:0000573 12827 12831 cone +T347 SO:0000704 12841 12846 genes +T348 CL:0000573 12889 12894 cones +T349 NCBITaxon:10088 12902 12907 mouse +T350 UBERON:0000966 12927 12933 retina +T351 SO:0000704 12948 12953 genes +T352 SO:0000673 12996 13006 transcript +T353 SO:0000704 13068 13073 genes +T354 CL:0000573 13086 13090 cone +T355 GO:0010467 13100 13110 expression +T356 UBERON:0000966 13128 13134 retina +T357 SO:0000704 13153 13158 genes +T358 UBERON:0000966 13187 13193 retina +T359 GO:0097617 13205 13218 hybridization +T360 SO:0000704 13230 13235 genes +T361 SO:0000704 13260 13265 genes +T362 GO:0010467 13279 13289 expression +T363 CL:0000573 13295 13299 cone +T364 SO:0000704 13333 13338 genes +T365 GO:0010467 13382 13391 expressed +T366 UBERON:0001789 13407 13410 ONL +T367 CL:0000573 13436 13441 cones +T368 CL:0000604 13450 13454 rods +T369 SO:0000704 13466 13470 gene +T370 SO:0000704 13511 13516 genes +T371 CL:0000573 13541 13545 cone +T372 UBERON:0000966 13572 13578 retina +T373 CL:0000573 13613 13617 cone +T374 SO:0000704 13683 13688 genes +T375 CL:0000573 13707 13711 cone +T376 SO:0000704 13721 13726 genes +T377 GO:0010467 13735 13744 expressed +T378 GO:0097617 13802 13815 hybridization +T379 CL:0000573 13849 13853 cone +T380 SO:0000704 13854 13859 genes +T381 SO:0000673 13995 14005 transcript +T382 GO:0001917 14026 14039 inner segment +T383 SO:0000704 14175 14180 genes +T384 CL:0000573 14241 14245 cone +T385 SO:0000704 14246 14251 genes +T386 CHEBI:17234 14282 14289 glucose +T387 GO:0006006 14282 14300 glucose metabolism +T388 PR:000013522 14302 14306 Pygm +T389 PR:000008038 14311 14315 Glo1 +T390 CHEBI:35366 14318 14328 fatty acid +T391 GO:0006631 14318 14339 fatty acid metabolism +T392 PR:000007037 14341 14347 Elovl2 +T393 GO:0006281 14350 14360 DNA repair +T394 PR:000015305 14362 14367 Smug1 +T395 GO:0007049 14370 14380 cell cycle +T396 GO:0007059 14381 14403 chromosome segregation +T397 PR:000004855 14405 14410 Bub1b +T398 PR:000016206 14429 14433 Tcta +T399 UBERON:0001986 14436 14447 endothelial +T400 PR:000006874 14457 14461 Ece1 +T401 GO:0005856 14464 14476 cytoskeletal +T402 PR:000007108 14487 14495 Ebp4.1l1 +T403 UBERON:0002280 14507 14514 otolith +T404 GO:0048840 14507 14524 otolith formation +T405 PR:000012076 14526 14531 Otop3 +T406 CL:0000573 14598 14602 cone +T407 SO:0000704 14612 14617 genes +T408 SO:0000704 14701 14705 gene +T409 GO:0010467 14701 14716 gene expression +T410 CL:0000210 14729 14742 photoreceptor +T411 GO:0010467 14789 14799 expression +T412 GO:0097617 14841 14854 hybridization +T413 UBERON:0001773 14875 14882 scleral +T414 UBERON:0000966 14897 14903 retina +T415 GO:0007567 14914 14919 natal +T416 PR:000008099 14958 14962 Gnb3 +T417 PR:000016321 14967 14972 Thrb2 +T418 CL:0000573 14999 15003 cone +T419 SO:0000704 15004 15009 genes +T420 GO:0010467 15037 15047 expression +T421 CL:0000573 15080 15084 cone +T422 SO:0000704 15085 15090 genes +T423 CL:0000210 15137 15150 photoreceptor +T424 GO:0010467 15162 15172 expression +T425 PR:000006874 15174 15178 Ece1 +T426 PR:000012076 15183 15188 Otop3 +T427 SO:0000704 15211 15216 genes +T428 GO:0097617 15316 15329 hybridization +T429 UBERON:0000113 15333 15345 adult stages +T430 GO:0010467 15371 15381 expression +T431 GO:0097617 15393 15406 hybridization +T432 CL:0000210 15457 15470 photoreceptor +T433 SO:0000704 15490 15495 genes +T434 UBERON:0000922 15510 15519 embryonic +T435 GO:0010467 15520 15530 expression +T436 SO:0000704 15555 15560 genes +T437 UBERON:0000922 15587 15596 embryonic +T438 GO:0097617 15605 15618 hybridization +T439 CL:0000210 15706 15719 photoreceptor +T440 GO:0010467 15720 15730 expression +T441 SO:0000704 15771 15776 genes +T442 CL:0000573 15789 15793 cone +T443 GO:0010467 15811 15820 expressed +T444 UBERON:0007023 15855 15860 adult +T445 PR:000001224 15863 15870 M-Opsin +T446 CHEBI:60311 15875 15890 Thyroid Hormone +T447 PR:000016321 15875 15902 Thyroid Hormone Receptor β2 +T448 CL:0000573 15945 15949 cone +T449 SO:0000704 15959 15964 genes +T450 GO:0010467 15994 16004 expression +T451 GO:0097617 16037 16050 hybridization +T452 PR:000001224 16070 16077 M-opsin +T453 PR:000001224 16079 16085 Opn1mw +T454 UBERON:0002046 16091 16098 thyroid +T455 CHEBI:60311 16091 16106 thyroid hormone +T456 PR:000016321 16091 16118 thyroid hormone receptor β2 +T457 PR:000016321 16120 16125 Thrb2 +T458 PR:000016321 16184 16189 Thrb2 +T459 GO:0010467 16221 16231 expression +T460 PR:000001224 16235 16242 M-opsin +T461 PR:000001195 16280 16287 S-opsin +T462 GO:0010467 16288 16298 expression +T463 NCBITaxon:10088 16326 16331 mouse +T464 UBERON:0000966 16332 16338 retina +T465 PR:000016321 16382 16387 Thrb2 +T466 PR:000001195 16394 16401 S-opsin +T467 PR:000016321 16435 16440 Thrb2 +T468 PR:000001195 16482 16489 S-opsin +T469 UBERON:0000966 16529 16535 retina +T470 SO:0000704 16574 16578 gene +T471 GO:0010467 16674 16684 expression +T472 PR:000016321 16709 16714 Thrb2 +T473 GO:0044297 16775 16786 cell bodies +T474 PR:000001224 16794 16801 M-opsin +T475 UBERON:0001789 16855 16858 ONL +T476 PR:000001224 17017 17024 M-opsin +T477 GO:0044297 17034 17045 cell bodies +T478 UBERON:0001773 17110 17117 scleral +T479 UBERON:0001789 17130 17133 ONL +T480 GO:0044297 17179 17190 cell bodies +T481 CL:0000573 17194 17213 cone photoreceptors +T482 NCBITaxon:10088 17221 17226 mouse +T483 UBERON:0001789 17265 17268 ONL +T484 UBERON:0001773 17307 17314 scleral +T485 UBERON:0001789 17327 17330 ONL +T486 UBERON:0000966 17386 17392 retina +T487 PR:000001224 17442 17449 M-opsin +T488 GO:0010467 17450 17460 expressing +T489 CL:0000306 17461 17465 cone +T490 GO:0044297 17466 17477 cell bodies +T491 UBERON:0001773 17485 17492 scleral +T492 UBERON:0001789 17505 17508 ONL +T493 CL:0000604 17511 17514 Rod +T494 SO:0000704 17515 17520 Genes +T495 CL:0000573 17604 17608 cone +T496 SO:0000704 17609 17613 gene +T497 GO:0010467 17609 17624 gene expression +T498 CL:0000604 17626 17629 rod +T499 SO:0000704 17639 17644 genes +T500 GO:0097617 17720 17733 hybridization +T501 CL:0000604 17755 17758 rod +T502 SO:0000704 17759 17764 genes +T503 GO:0010467 17800 17810 expression +T504 CL:0000604 17913 17916 rod +T505 SO:0000704 17917 17922 genes +T506 GO:0097617 17953 17966 hybridization +T507 CL:0000604 17996 17999 rod +T508 CL:0000210 18017 18030 photoreceptor +T509 SO:0000704 18031 18036 genes +T510 GO:0010467 18081 18091 expression +T511 SO:0000704 18108 18113 genes +T512 PR:000008353 18115 18121 gucy2e +T513 PR:000013965 18126 18130 Rgs9 +T514 GO:0010467 18159 18169 expression +T515 PR:000011403 18178 18183 Nr2e3 +T516 PR:000000686 18189 18194 Cnga1 +T517 CL:0000604 18239 18242 rod +T518 SO:0000704 18243 18247 gene +T519 GO:0010467 18243 18258 gene expression +T520 GO:0007567 18272 18277 natal +T521 PR:000001245 18348 18357 rhodopsin +T522 PR:000001245 18359 18362 Rho +T523 GO:0010467 18364 18374 expression +T524 PR:000001245 18566 18575 rhodopsin +T525 GO:0097617 18596 18609 hybridization +T526 CL:0000604 18630 18633 rod +T527 SO:0000704 18643 18648 genes +T528 PR:000001245 18669 18678 rhodopsin +T529 GO:0010467 18718 18728 expression +T530 PR:000001245 18823 18832 rhodopsin +T531 GO:0010467 18833 18843 expression +T532 SO:0000704 18856 18860 gene +T533 CL:0000604 19000 19003 rod +T534 CL:0000573 19014 19018 cone +T535 SO:0000704 19028 19033 genes +T536 GO:0010467 19040 19049 expressed +T537 CL:0000210 19069 19083 photoreceptors +T538 UBERON:0000966 19115 19122 Retinal +T539 CL:0011107 19148 19160 Müller Glial +T540 SO:0000704 19161 19165 Gene +T541 GO:0010467 19161 19176 Gene Expression +T542 CL:0000210 19205 19218 photoreceptor +T543 PR:000005904 19288 19291 Crx +T544 PR:000011432 19296 19299 Nrl +T545 PR:000011432 19382 19385 Nrl +T546 CL:0000604 19391 19394 rod +T547 CHEBI:22695 19405 19410 basic +T548 CL:0000604 19483 19486 rod +T549 SO:0000704 19496 19501 genes +T550 CL:0000573 19529 19533 cone +T551 SO:0000704 19543 19548 genes +T552 CL:0000604 19552 19556 rods +T553 PR:000011432 19563 19566 Nrl +T554 SO:0000704 19582 19593 genetically +T555 PR:000011403 19606 19611 Nr2e3 +T556 GO:0010467 19636 19646 expression +T557 PR:000005904 19653 19656 Crx +T558 GO:0010467 19692 19701 expressed +T559 CL:0000604 19710 19714 rods +T560 CL:0000573 19719 19724 cones +T561 GO:0010467 19749 19759 expression +T562 CL:0000604 19776 19779 rod +T563 CL:0000573 19785 19789 cone +T564 SO:0000704 19799 19804 genes +T565 PR:000011432 19846 19849 Nrl +T566 PR:000011403 19858 19863 Nr2e3 +T567 GO:0010467 19864 19874 expression +T568 PR:000005904 19891 19894 Crx +T569 PR:000011403 19947 19952 Nr2e3 +T570 GO:0097617 20035 20048 hybridization +T571 PR:000011403 20142 20147 Nr2e3 +T572 SO:0000417 20213 20220 domains +T573 SO:0001023 20277 20283 allele +T574 SO:0000673 20296 20306 transcript +T575 PR:000011403 20389 20394 Nr2e3 +T576 SO:0000704 20453 20457 gene +T577 SO:0000704 20492 20496 gene +T578 SO:0000704 20584 20588 gene +T579 GO:0010467 20616 20626 expression +T580 UBERON:0007023 20634 20639 adult +T581 UBERON:0000966 20651 20657 retina +T582 GO:0010467 20710 20720 expression +T583 UBERON:0001791 20728 20747 inner nuclear layer +T584 GO:0005634 20734 20741 nuclear +T585 UBERON:0001791 20749 20752 INL +T586 UBERON:0000966 20771 20777 retina +T587 SO:0000704 20784 20788 gene +T588 GO:0010467 20803 20813 expression +T589 UBERON:0001791 20854 20857 INL +T590 GO:0010467 20876 20886 expression +T591 UBERON:0001792 20894 20913 ganglion cell layer +T592 UBERON:0001792 20915 20918 GCL +T593 UBERON:0001773 20931 20938 scleral +T594 UBERON:0001789 20951 20954 ONL +T595 GO:0097617 20991 21004 hybridization +T596 CL:0011107 21044 21055 Müller glia +T597 CL:0000125 21071 21081 glial cell +T598 NCBITaxon:10088 21094 21099 mouse +T599 UBERON:0000966 21100 21106 retina +T600 GO:0010467 21152 21162 expression +T601 CL:0011107 21214 21225 Müller glia +T602 CL:0000210 21273 21287 Photoreceptors +T603 UBERON:0000966 21299 21305 Retina +T604 SO:0000704 21408 21412 gene +T605 GO:0010467 21408 21423 gene expression +T606 CL:0000210 21459 21472 photoreceptor +T607 GO:0044297 21473 21484 cell bodies +T608 GO:0044297 21521 21532 cell bodies +T609 GO:0001750 21576 21590 outer segments +T610 NCBITaxon:10088 21605 21610 mouse +T611 CL:0000604 21652 21655 rod +T612 CL:0000573 21660 21664 cone +T613 GO:0044297 21665 21671 somata +T614 GO:0001750 21726 21740 outer segments +T615 NCBITaxon:10088 21764 21769 mouse +T616 CL:0000306 21771 21775 cone +T617 GO:0044297 21776 21787 cell bodies +T618 UBERON:0001773 21810 21817 scleral +T619 UBERON:0001789 21832 21835 ONL +T620 CL:0000604 21871 21875 rods +T621 GO:0005720 21921 21944 nuclear heterochromatin +T622 CHEBI:10545 22055 22063 electron +T623 GO:0000791 22071 22082 euchromatin +T624 CL:0000604 22088 22092 rods +T625 GO:0043226 22134 22143 organelle +T626 GO:0005737 22149 22158 cytoplasm +T627 GO:0005634 22173 22179 nuclei +T628 GO:0005739 22206 22218 mitochondria +T629 CL:0000573 22299 22303 cone +T630 UBERON:0000966 22360 22366 retina +T631 GO:0044297 22401 22407 somata +T632 UBERON:0001789 22438 22441 ONL +T633 GO:0010467 22517 22527 expressing +T634 PR:000001195 22528 22540 S-cone opsin +T635 CL:0000573 22530 22534 cone +T636 GO:0005634 22648 22655 nuclear +T637 UBERON:0000966 22682 22689 retinal +T638 CL:0009004 22682 22695 retinal cells +T639 PR:000001195 22708 22720 S-cone opsin +T640 CL:0000573 22710 22714 cone +T641 GO:0097617 22749 22762 hybridization +T642 PR:000001245 22857 22866 rhodopsin +T643 CL:0000573 22931 22935 cone +T644 UBERON:0000966 22966 22972 retina +T645 CL:0000573 23008 23013 cones +T646 GO:0044297 23053 23064 cell bodies +T647 CL:0000306 23086 23090 cone +T648 GO:0044297 23091 23100 cell body +T649 CL:0000604 23116 23119 rod +T650 GO:0044297 23120 23124 soma +T651 GO:0000792 23190 23205 heterochromatin +T652 CHEBI:10545 23232 23240 electron +T653 GO:0000791 23247 23258 euchromatin +T654 GO:0048471 23275 23297 juxtanuclear cytoplasm +T655 GO:0043226 23306 23316 organelles +T656 UBERON:0001789 23360 23366;23375 23381 ONL of ... retina +T657 GO:0005634 23398 23405 nuclear +T658 CL:0000604 23425 23429 rods +T659 GO:0000792 23463 23478 heterochromatin +T660 CHEBI:10545 23494 23502 electron +T661 GO:0000791 23509 23520 euchromatin +T662 CL:0000573 23572 23577 cones +T663 GO:0000791 23590 23601 euchromatin +T664 CL:0000604 23665 23669 rods +T665 UBERON:0001789 23782 23786 ONLs +T666 PR:000001195 23825 23832 S-opsin +T667 CL:0000604 23999 24002 rod +T668 GO:0044297 24008 24019 cell bodies +T669 CL:0000604 24116 24119 rod +T670 GO:0044297 24125 24131 somata +T671 CL:0000604 24190 24193 rod +T672 GO:0044297 24194 24200 somata +T673 CL:0000604 24283 24287 rods +T674 GO:0044297 24410 24415 somal +T675 GO:0044297 24535 24540 somal +T676 CL:0000210 24656 24670 photoreceptors +T677 GO:0044297 24684 24689 somal +T678 GO:0005634 24727 24734 nuclear +T679 GO:0005739 24735 24747 mitochondria +T680 GO:0005634 24802 24809 nuclear +T681 GO:0043226 24810 24820 organelles +T682 CL:0000604 24857 24861 rods +T683 CL:0000573 24918 24923 cones +T684 UBERON:0000966 25024 25030 retina +T685 CL:0000604 25073 25077 rods +T686 CL:0000573 25082 25087 cones +T687 GO:0010467 25110 25120 expression +T688 CL:0000604 25129 25132 rod +T689 CL:0000573 25138 25142 cone +T690 SO:0000704 25152 25157 genes +T691 CL:0000604 25249 25252 rod +T692 PR:000011403 25284 25289 Nr2e3 +T693 CL:0000573 25306 25310 cone +T694 SO:0000704 25311 25316 genes +T695 GO:0006342 25317 25341 transcriptionally silent +T696 CL:0000604 25349 25353 rods +T697 CL:0000573 25390 25394 cone +T698 SO:0000704 25395 25399 gene +T699 UBERON:0000966 25431 25437 retina +T700 GO:0010467 25570 25580 expression +T701 CL:0000573 25584 25588 cone +T702 SO:0000704 25589 25594 genes +T703 UBERON:0001789 25632 25635 ONL +T704 GO:0010467 25670 25677 express +T705 CL:0000604 25682 25685 rod +T706 SO:0000704 25686 25691 genes +T707 SO:0000704 25738 25742 gene +T708 GO:0010467 25738 25753 gene expression +T709 CHEBI:10545 25770 25778 electron +T710 CL:0000604 25864 25868 rods +T711 CL:0000573 25873 25878 cones +T712 SO:0000704 25890 25895 genes +T713 UBERON:0001789 25978 25981 ONL +T714 SO:0000704 26024 26029 genes +T715 GO:0010467 26099 26109 expression +T716 PR:000001195 26113 26120 S-opsin +T717 GO:0010467 26121 26131 expressing +T718 CL:0000573 26132 26137 cones +T719 SO:0000704 26209 26214 genes +T720 UBERON:0000966 26233 26239 retina +T721 SO:0000704 26261 26266 genes +T722 UBERON:0001789 26354 26357 ONL +T723 PR:000001195 26507 26519 S-cone opsin +T724 CL:0000573 26509 26513 cone +T725 GO:0010467 26520 26530 expressing +T726 SO:0000704 26583 26588 genes +T727 GO:0010467 26605 26614 expressed +T728 PR:000001195 26618 26625 S-opsin +T729 GO:0010467 26626 26636 expressing +T730 CL:0009004 26662 26670;26679 26685 cells of ... retina +T731 UBERON:0000966 26679 26685 retina +T732 GO:0010467 26749 26759 expression +T733 SO:0000704 26783 26788 genes +T734 PR:000011432 26800 26803 Nrl +T735 PR:000011432 26847 26850 Nrl +T736 UBERON:0000966 26856 26863 retinal +T737 CL:0000604 26939 26943 rods +T738 PR:000001195 26949 26956 S-opsin +T739 GO:0010467 26957 26967 expressing +T740 CL:0000573 26968 26973 cones +T741 PR:000011432 27019 27022 Nrl +T742 GO:0016458 27061 27073;27088 27093 silencing of ... genes +T743 CL:0000573 27074 27078 cone +T744 SO:0000704 27088 27093 genes +T745 CL:0000604 27097 27101 rods +T746 PR:000011432 27110 27113 Nrl +T747 CL:0000573 27191 27195 cone +T748 SO:0000704 27205 27210 genes +T749 UBERON:0001789 27226 27229 ONL +T750 UBERON:0000966 27254 27260 retina +T751 CL:0000573 27350 27354 cone +T752 SO:0000704 27355 27359 gene +T753 GO:0010467 27355 27370 gene expression +T754 PR:000011403 27388 27393 Nr2e3 +T755 GO:0010467 27394 27404 expression +T756 PR:000011432 27406 27409 Nrl +T757 CL:0000573 27462 27466 cone +T758 SO:0000704 27467 27472 genes +T759 UBERON:0000966 27593 27599 retina +T760 UBERON:0001789 27676 27679 ONL +T761 CL:0000573 27715 27719 cone +T762 SO:0000704 27729 27734 genes +T763 PR:000001195 27746 27758 S-cone opsin +T764 CL:0000573 27748 27752 cone +T765 CL:0000573 27821 27826 cones +T766 GO:0044297 27850 27861 cell bodies +T767 PR:000001195 27899 27911 S-cone opsin +T768 CL:0000573 27901 27905 cone +T769 UBERON:0000966 28001 28007 retina +T770 GO:0042571 28034 28042 antibody +T771 CL:0000573 28190 28194 cone +T772 SO:0000704 28195 28200 genes +T773 PR:000001195 28216 28228 S-cone opsin +T774 CL:0000573 28218 28222 cone +T775 NCBITaxon:10088 28252 28257 mouse +T776 CL:0000604 28343 28346 rod +T777 SO:0000704 28356 28361 genes +T778 PR:000001245 28373 28382 rhodopsin +T779 GO:0097617 28433 28446 hybridization +T780 PR:000001245 28465 28474 rhodopsin +T781 GO:0010467 28475 28485 expression +T782 PR:000001245 28601 28610 rhodopsin +T783 GO:0097617 28772 28785 hybridization +T784 CL:0000604 28831 28834 rod +T785 SO:0000704 28844 28848 gene +T786 GO:0010467 28844 28859 gene expression +T787 CL:0000573 28985 28989 cone +T788 SO:0000704 28999 29004 genes +T789 SO:0000704 29094 29099 genes +T790 PR:000007037 29119 29125 Elovl2 +T791 PR:000007315 29130 29135 Fabp7 +T792 SO:0000704 29147 29152 genes +T793 PR:000007037 29260 29266 Elovl2 +T794 CL:0000573 29277 29281 cone +T795 GO:0010467 29302 29312 expression +T796 GO:0097617 29337 29350 hybridization +T797 PR:000007315 29363 29368 Fabp7 +T798 UBERON:0000966 29416 29422 retina +T799 PR:000007315 29494 29499 Fabp7 +T800 GO:0010467 29503 29512 expressed +T801 CL:0000681 29516 29527 radial glia +T802 CL:0002626 29532 29551 immature astrocytes +T803 UBERON:0000955 29559 29564 brain +T804 GO:0010467 29584 29594 expression +T805 UBERON:0001016 29620 29634 nervous system +T806 PR:000007315 29656 29661 Fabp7 +T807 CL:0011107 29681 29692 Müller glia +T808 UBERON:0000966 29704 29710 retina +T809 CL:0011107 29763 29775 Müller glial +T810 SO:0000704 29776 29780 gene +T811 SO:0000704 29840 29844 gene +T812 PR:000007315 29918 29923 Fabp7 +T813 PR:000011432 29966 29969 Nrl +T814 PR:000005904 29974 29977 Crx +T815 UBERON:0000966 29985 29992 retinas +T816 UBERON:0000966 30103 30109 retina +T817 CL:0000573 30140 30144 cone +T818 SO:0000704 30154 30158 gene +T819 NCBITaxon:7955 30262 30271 zebrafish +T820 SO:0000853 30272 30279 homolog +T821 PR:000011403 30283 30288 Nr2e3 +T822 GO:0010467 30309 30318 expressed +T823 CL:0000210 30322 30336 photoreceptors +T824 SO:0000853 30375 30382 homolog +T825 CL:0000210 30405 30418 photoreceptor +T826 GO:0010467 30430 30440 expression +T827 CL:0000604 30489 30492 rod +T828 GO:0010467 30513 30523 expression +T829 GO:0010467 30536 30546 expression +T830 CL:0000573 30550 30555 cones +T831 GO:0010467 30594 30604 expression +T832 CL:0000573 30608 30612 cone +T833 SO:0000704 30622 30626 gene +T834 PR:000011403 30732 30737 Nr2e3 +T835 UBERON:0000966 30764 30770 retina +T836 NCBITaxon:1 30799 30807 organism +T837 SO:0000409 30915 30927 binding site +T838 PR:000011403 30932 30937 Nr2e3 +T839 PR:000011403 31013 31018 Nr2e3 +T840 SO:0000704 31026 31031 genes +T841 SO:0000704 31038 31042 gene +T842 GO:0010467 31038 31053 gene expression +T843 UBERON:0000966 31091 31097 retina +T844 SO:0000704 31141 31145 gene +T845 GO:0065007 31146 31156 regulation +T846 NCBITaxon:10088 31160 31165 mouse +T847 CL:0000604 31166 31170 rods +T848 CL:0000573 31274 31278 cone +T849 SO:0000704 31279 31284 genes +T850 CL:0000604 31292 31296 rods +T851 PR:000011403 31298 31303 Nr2e3 +T852 PR:000011432 31315 31318 Nrl +T853 PR:000011432 31386 31389 Nrl +T854 CL:0000573 31497 31501 cone +T855 SO:0000704 31502 31507 genes +T856 PR:000011403 31545 31550 Nr2e3 +T857 GO:0065007 31610 31620 regulation +T858 SO:0000704 31628 31632 gene +T859 SO:0000704 31668 31673 genes +T860 PR:000011403 31675 31680 Nr2e3 +T861 SO:0000704 31784 31789 genes +T862 PR:000011403 31871 31876 Nr2e3 +T863 CL:0000573 31983 31987 cone +T864 SO:0000704 31997 32002 genes +T865 SO:0000704 32026 32031 genes +T866 SO:0000704 32083 32087 gene +T867 PR:000001224 32097 32104 M-opsin +T868 PR:000016321 32109 32114 Thrb2 +T869 PR:000016321 32119 32124 Thrb2 +T870 GO:0010467 32157 32167 expression +T871 PR:000001224 32171 32178 M-opsin +T872 PR:000001224 32214 32221 M-opsin +T873 PR:000016321 32282 32287 Thrb2 +T874 GO:0010467 32288 32298 expression +T875 CL:0000573 32440 32444 cone +T876 SO:0000704 32445 32450 genes +T877 PR:000011331 32458 32464 Notch1 +T878 UBERON:0000966 32468 32474 retina +T879 PR:000011331 32526 32532 Notch1 +T880 UBERON:0000966 32536 32543 retinas +T881 PR:000016321 32572 32577 Thrb2 +T882 SO:0000704 32618 32622 gene +T883 PR:000001224 32636 32643 M-opsin +T884 PR:000001224 32687 32694 M-opsin +T885 PR:000001195 32699 32706 S-opsin +T886 GO:0065007 32711 32721 controlled +T887 UBERON:0000966 32806 32813 retinas +T888 GO:0010467 32814 32821 express +T889 PR:000001195 32822 32829 S-opsin +T890 PR:000001224 32834 32841 M-opsin +T891 UBERON:0000966 32877 32884 retinas +T892 GO:0010467 32885 32892 express +T893 PR:000001195 32898 32905 S-opsin +T894 GO:0065007 32926 32937 controlling +T895 UBERON:0000966 33026 33033 retinas +T896 UBERON:0000966 33064 33071 retinas +T897 CHEBI:36357 33108 33117 molecules +T898 GO:0010467 33145 33155 expression +T899 PR:000001224 33159 33166 M-opsin +T900 PR:000001224 33263 33270 M-opsin +T901 GO:0010467 33271 33281 expression +T902 UBERON:0000966 33320 33326 retina +T903 PR:000001224 33369 33376 M-opsin +T904 SO:0000673 33377 33387 transcript +T905 UBERON:0000966 33417 33423 retina +T906 GO:0097617 33483 33496 hybridization +T907 PR:000001224 33518 33525 M-opsin +T908 GO:0010467 33526 33536 expressing +T909 UBERON:0001789 33582 33585 ONL +T910 PR:000001224 33640 33647 M-opsin +T911 SO:0000673 33648 33658 transcript +T912 CL:0000573 33739 33743 cone +T913 CL:0000573 33756 33760 cone +T914 SO:0000704 33770 33775 genes +T915 SO:0000704 33823 33828 genes +T916 PR:000013522 33830 33834 Pygm +T917 CHEBI:24384 33851 33859 glycogen +T918 GO:0005977 33851 33859;33868 33878 glycogen ... metabolism +T919 CHEBI:17234 33860 33867 glucose +T920 GO:0006006 33860 33878 glucose metabolism +T921 PR:000008038 33894 33898 Glo1 +T922 GO:0098754 33916 33930 detoxification +T923 CHEBI:17158 33934 33947 methylglyoxal +T924 GO:0006096 33964 33974 glycolysis +T925 SO:0000704 33989 33993 gene +T926 CHEBI:17234 34006 34013 glucose +T927 GO:0006006 34006 34024 glucose metabolism +T928 PR:000008609 34026 34038 hexokinase 2 +T929 PR:000008609 34040 34043 Hk2 +T930 GO:0010467 34107 34117 expression +T931 UBERON:0000966 34135 34141 retina +T932 GO:0010467 34162 34172 expression +T933 CL:0000573 34176 34181 cones +T934 CL:0000604 34190 34194 rods +T935 SO:0000704 34230 34234 gene +T936 CHEBI:17234 34247 34254 glucose +T937 GO:0006006 34247 34265 glucose metabolism +T938 PR:000007896 34267 34297 glucokinase regulatory protein +T939 GO:0065007 34279 34289 regulatory +T940 PR:000007896 34299 34303 Gckr +T941 GO:0097617 34403 34416 hybridization +T942 GO:0010467 34443 34453 expression +T943 PR:000007896 34457 34461 Gckr +T944 UBERON:0000966 34476 34482 retina +T945 CL:0000573 34513 34517 cone +T946 SO:0000704 34527 34531 gene +T947 SO:0000704 34574 34579 genes +T948 PR:000013522 34581 34585 Pygm +T949 PR:000008609 34590 34593 Hk2 +T950 UBERON:0001789 34635 34638 ONL +T951 SO:0000704 34667 34671 gene +T952 GO:0010467 34667 34682 gene expression +T953 CL:0000210 34748 34762 photoreceptors +T954 CHEBI:24384 34826 34834 glycogen +T955 GO:0005977 34826 34834;34847 34857 glycogen ... metabolism +T956 CHEBI:17234 34839 34846 glucose +T957 GO:0006006 34839 34857 glucose metabolism +T958 NCBITaxon:9443 34866 34873 primate +T959 CL:0000604 34874 34878 rods +T960 CL:0000573 34883 34888 cones +T961 PR:000013522 34961 34965 Pygm +T962 NCBITaxon:9606 34989 34994 human +T963 http://purl.obolibrary.org/obo/MONDO_0000001 34995 35002 disease +T964 SO:0000704 35022 35026 gene +T965 http://purl.obolibrary.org/obo/MONDO_0009293 35036 35053 McArdle's disease +T966 CHEBI:24384 35055 35063 glycogen +T967 http://purl.obolibrary.org/obo/MONDO_0009293 35055 35086 glycogen storage disease type V +T968 http://purl.obolibrary.org/obo/MONDO_0000866 35160 35173 myoglobinuria +T969 UBERON:0000966 35218 35225 retinal +T970 UBERON:0000966 35307 35313 retina +T971 PR:000001195 35397 35404 S-opsin +T972 GO:0010467 35405 35415 expressing +T973 CL:0000573 35416 35421 cones +T974 CL:0000604 35446 35450 rods +T975 CL:0000210 35463 35477 photoreceptors +T976 PR:000011403 35496 35501 Nr2e3 +T977 GO:0010467 35505 35514 expressed +T978 CL:0000604 35523 35527 rods +T979 SO:0000673 35537 35547 transcript +T980 GO:0007067 35575 35582 mitotic +T981 PR:000011403 35645 35650 Nr2e3 +T982 PR:000001195 35714 35720 S-cone +T983 CL:0000573 35716 35720 cone +T984 CL:0000210 35751 35765 photoreceptors +T985 UBERON:0000966 35788 35794 retina +T986 GO:0007067 35835 35842 mitotic +T987 CL:0000604 35878 35882 rods +T988 CL:0000604 35976 35979 rod +T989 CL:0000573 36080 36085 cones +T990 CL:0000604 36228 36231 rod +T991 PR:000011403 36251 36256 Nr2e3 +T992 CL:0000573 36275 36279 cone +T993 NCBITaxon:10114 36382 36385 rat +T994 CL:0000604 36442 36446 rods +T995 CL:0000604 36547 36550 rod +T996 PR:000001195 36658 36665 S-opsin +T997 UBERON:0000966 36692 36698 retina +T998 CL:0000604 36889 36892 rod +T999 UBERON:0000966 36949 36955 retina +T1000 GO:0007067 37053 37060 mitotic +T1001 CL:0000604 37061 37064 rod +T1002 NCBITaxon:10088 37083 37088 mouse +T1003 CL:0000573 37142 37146 cone +T1004 SO:0000704 37318 37322 gene +T1005 PR:000011403 37391 37396 Nr2e3 +T1006 GO:0065007 37448 37455 control +T1007 SO:0000704 37456 37460 gene +T1008 GO:0010467 37464 37473 expressed +T1009 CL:0000604 37477 37480 rod +T1010 GO:0042670 37567 37589;37592 37601 differentiation toward ... cone fate +T1011 CL:0000573 37592 37596 cone +T1012 CL:0000573 37732 37736 cone +T1013 NCBITaxon:9606 37780 37785 Human +T1014 http://purl.obolibrary.org/obo/MONDO_0009989 37800 37804 ESCS +T1015 UBERON:0000966 37860 37866 retina +T1016 UBERON:0000966 37980 37987 retinal +T1017 http://purl.obolibrary.org/obo/MONDO_0004580 37980 38000 retinal degeneration +T1018 UBERON:0000966 38019 38026 retinal +T1019 NCBITaxon:10088 38087 38091 mice +T1020 NCBITaxon:species 38215 38222 species +T1021 http://purl.obolibrary.org/obo/MONDO_0009989 38277 38281 ESCS +T1022 NCBITaxon:10088 38306 38311 mouse +T1023 http://purl.obolibrary.org/obo/MONDO_0009989 38372 38376 ESCS +T1024 CL:0000210 38425 38438 photoreceptor +T1025 GO:0010467 38548 38558 expressing +T1026 CL:0000604 38564 38567 rod +T1027 CL:0000573 38572 38576 cone +T1028 SO:0000704 38577 38582 genes +T1029 PR:000001245 38609 38618 rhodopsin +T1030 PR:000001195 38627 38639 S-cone opsin +T1031 CL:0000573 38629 38633 cone +T1032 NCBITaxon:9606 38694 38699 human +T1033 http://purl.obolibrary.org/obo/MONDO_0009989 38700 38704 ESCS +T1034 CL:0000604 38744 38747 rod +T1035 PR:000001245 38780 38789 rhodopsin +T1036 http://purl.obolibrary.org/obo/MONDO_0009989 38882 38886 ESCS +T1037 NCBITaxon:10088 38906 38910 mice +T1038 PR:000011432 38977 38980 Nrl +T1039 NCBITaxon:10088 38988 38993 mouse +T1040 CL:0000604 39044 39048 rods +T1041 CL:0000573 39059 39064 cones +T1042 NCBITaxon:9606 39147 39152 human +T1043 CL:0000210 39179 39197 photoreceptor cell +T1044 GO:0010467 39213 39222 expresses +T1045 PR:000001195 39223 39230 S-opsin +T1046 SO:0000704 39262 39266 gene +T1047 GO:0065007 39267 39277 regulatory +T1048 NCBITaxon:10088 39298 39302 mice +T1049 NCBITaxon:9606 39307 39313 humans +T1050 NCBITaxon:9606 39327 39332 human +T1051 PR:000011403 39333 39338 NR2E3 +T1052 PR:000001195 39348 39355 S-opsin +T1053 NCBITaxon:10088 39439 39444 mouse +T1054 GO:0010467 39463 39472 expressed +T1055 CL:0000210 39494 39513 photoreceptor cells +T1056 CL:0000573 39559 39564 cones +T1057 CL:0000210 39575 39589 photoreceptors +T1058 UBERON:0000966 39606 39612 retina +T1059 http://purl.obolibrary.org/obo/MONDO_0009989 39616 39620 ESCS +T1060 CL:0000604 39688 39692 rods +T1061 http://purl.obolibrary.org/obo/MONDO_0009989 39696 39700 ESCS +T1062 CL:0000573 39719 39724 cones +T1063 CL:0000210 39744 39758 photoreceptors +T1064 CL:0000604 39855 39858 rod +T1065 GO:0065007 39909 39919 regulatory +T1066 SO:0000704 39920 39924 gene +T1067 GO:0010467 39920 39935 gene expression +T1068 NCBITaxon:10088 39981 39986 mouse +T1069 NCBITaxon:9606 39995 40000 human +T1070 PR:000011403 40001 40006 NR2E3 +T1071 NCBITaxon:species 40021 40028 species +T1072 UBERON:0000966 40046 40053 retinal +T1073 http://purl.obolibrary.org/obo/MONDO_0004580 40046 40066 retinal degeneration +T1074 CL:0000210 40161 40179 photoreceptor cell +T1075 GO:0010467 40227 40237 expression +T1076 CL:0000604 40246 40249 rod +T1077 CL:0000573 40254 40258 cone +T1078 SO:0000704 40259 40264 genes +T1079 GO:0006915 40311 40320 apoptosis +T1080 NCBITaxon:10088 40356 40361 mouse +T1081 NCBITaxon:9606 40370 40375 human +T1082 PR:000011403 40376 40381 NR2E3 +T1083 UBERON:0000966 40433 40439 retina +T1084 CL:0000210 40581 40599 photoreceptor cell +T1085 UBERON:0000483 40666 40675 epithelia +T1086 GO:0065007 40719 40730 controlling +T1087 SO:0000704 40812 40816 gene +T1088 PR:P10040 40817 40823 crumbs +T1089 PR:000005848 40825 40829 CRB1 +T1090 UBERON:0001789 40891 40894 ONL +T1091 NCBITaxon:9606 40903 40909 humans +T1092 NCBITaxon:10088 40914 40918 mice +T1093 NCBITaxon:10088 40951 40955 mice +T1094 PR:000005848 41112 41116 CRB1 +T1095 PR:000011403 41144 41149 NR2E3 +T1096 http://purl.obolibrary.org/obo/MONDO_0004891 41161 41170 hyperopic +T1097 http://purl.obolibrary.org/obo/MONDO_0004892 41171 41188 refractive errors +T1098 UBERON:0000966 41246 41252 retina +T1099 NCBITaxon:10088 41289 41294 mouse +T1100 PR:P10040 41295 41301 crumbs +T1101 SO:0000855 41302 41310 ortholog +T1102 UBERON:0000966 41348 41354 retina +T1103 GO:0010467 41397 41407 expression +T1104 CL:0000573 41417 41422 cones +T1105 CL:0000604 41431 41435 rods +T1106 GO:0097617 41501 41514 hybridization +T1107 PR:P10040 41591 41597 crumbs +T1108 UBERON:0000966 41605 41611 retina +T1109 GO:0010467 41679 41689 expression +T1110 PR:P10040 41703 41709 crumbs +T1111 NCBITaxon:7215 41713 41723 Drosophila +T1112 UBERON:0000483 41788 41797 epithelia +T1113 GO:0005634 41826 41832 nuclei +T1114 CL:0000210 41840 41854 photoreceptors +T1115 UBERON:0000966 41892 41898 retina +T1116 GO:0010467 41961 41971 expressing +T1117 PR:000005848 41984 41988 Crb1 +T1118 CL:0000210 42096 42109 photoreceptor +T1119 CL:0000210 42161 42174 photoreceptor +T1120 CL:0000604 42207 42211 rods +T1121 NCBITaxon:species 42221 42228 species +T1122 CL:0000604 42302 42306 rods +T1123 CL:0000573 42311 42316 cones +T1124 GO:0010629 42537 42552;42561 42563;42570 42580 down-regulation ... of ... expression +T1125 PR:000011403 42564 42569 Nr2e3 +T1126 CL:0000604 42591 42595 rods +T1127 CL:0000210 42633 42651 photoreceptor cell +T1128 GO:0007601 42793 42799 vision +T1129 NCBITaxon:species 42825 42832 species +T1130 NCBITaxon:10088 42865 42869 mice +T1131 PR:000011403 42872 42877 Nr2e3 +T1132 NCBITaxon:10088 42888 42892 mice +T1133 NCBITaxon:10088 43035 43039 mice +T1134 UBERON:0000966 43083 43090 retinal +T1135 PR:000011403 43143 43148 Nr2e3 +T1136 NCBITaxon:10088 43156 43160 mice +T1137 CHEBI:33893 43178 43185 reagent +T1138 UBERON:0000966 43239 43246 retinal +T1139 NCBITaxon:10088 43294 43298 mice +T1140 UBERON:0000966 43374 43381 retinas +T1141 NCBITaxon:33208 43399 43406 animals +T1142 CHEBI:37987 43634 43637 Cy3 +T1143 CHEBI:37989 43641 43644 Cy5 +T1144 CHEBI:37987 43942 43945 Cy3 +T1145 CHEBI:37989 43969 43972 Cy5 +T1146 CHEBI:37958 44005 44009 dyes +T1147 GO:0097617 44042 44052 hybridized +T1148 SO:0000317 44108 44119 cDNA clones +T1149 UBERON:0001062 44145 44152 anatomy +T1150 SO:0000317 44275 44286 cDNA clones +T1151 GO:0097617 44336 44346 hybridized +T1152 GO:0097617 44375 44388 hybridization +T1153 CHEBI:37987 44945 44948 Cy3 +T1154 CHEBI:37989 44952 44955 Cy5 +T1155 CHEBI:37987 45453 45456 Cy3 +T1156 CHEBI:37989 45457 45460 Cy5 +T1157 SO:0000704 45782 45787 genes +T1158 UBERON:0000966 45925 45932 retinas +T1159 NCBITaxon:10088 45961 45966 mouse +T1160 SO:0001026 45967 45973 genome +T1161 GO:0097617 46077 46091 hybridizations +T1162 GO:0097617 46304 46317 Hybridization +T1163 SO:0000704 46699 46704 genes +T1164 SO:0000704 46859 46863 gene +T1165 SO:0000673 46901 46912 transcripts +T1166 SO:0000704 47061 47066 genes +T1167 SO:0000673 47108 47119 transcripts +T1168 SO:0000704 47145 47150 genes +T1169 SO:0000704 47330 47335 genes +T1170 SO:0000704 47396 47401 genes +T1171 GO:0097617 47597 47610 hybridization +T1172 GO:0097617 47629 47642 hybridization +T1173 UBERON:0000479 47729 47735 tissue +T1174 GO:0097617 47775 47789 hybridizations +T1175 CHEBI:42098 47901 47912 digoxygenin +T1176 CHEBI:7586 47971 47974 NBT +T1177 CHEBI:75508 47975 47979 BCIP +T1178 GO:0097617 48145 48158 hybridization +T1179 PR:000001195 48217 48224 S-opsin +T1180 CHEBI:42098 48225 48236 digoxygenin +T1181 GO:0097617 48276 48289 hybridization +T1182 PR:000001195 48416 48423 S-opsin +T1183 GO:0097617 48484 48497 hybridization +T1184 UBERON:0000966 48623 48630 retinas +T1185 GO:0042571 48660 48668 antibody +T1186 GO:0042571 48756 48766 antibodies +T1187 PR:000001195 48795 48805 blue opsin +T1188 NCBITaxon:9986 48816 48822 rabbit +T1189 NCBITaxon:10088 48906 48911 mouse +T1190 PR:000001245 48928 48937 rhodopsin +T1191 GO:0042571 48965 48975 antibodies +T1192 CHEBI:52302 48986 48989 Cy2 +T1193 CHEBI:37987 48994 48997 Cy3 +T1194 MOP:0000779 48998 49008 conjugated +T1195 NCBITaxon:9925 49009 49013 goat +T1196 NCBITaxon:9986 49019 49025 rabbit +T1197 NCBITaxon:10088 49035 49040 mouse +T1198 GO:0042571 49125 49133 antibody +T1199 CHEBI:51231 49144 49151 4′-DAPI +T1200 GO:0005634 49173 49179 nuclei +T1201 CHEBI:10545 49301 49309 Electron +T1202 NCBITaxon:33208 49484 49491 animals +T1203 UBERON:0001179 49525 49535 peritoneal +T1204 UBERON:0000970 49565 49569 eyes +T1205 UBERON:0000964 49593 49599 cornea +T1206 UBERON:0000970 49682 49685 eye +T1207 CHEBI:75958 49712 49720 solution +T1208 CHEBI:64276 49747 49761 glutaraldehyde +T1209 CHEBI:16223 49765 49775 cacodylate +T1210 CHEBI:48765 49790 49804 cacodylic acid +T1211 CHEBI:3312 49811 49827 calcium chloride +T1212 UBERON:0003072 49866 49872 eyecup +T1213 UBERON:0000970 49927 49930 eye +T1214 UBERON:0001091 49950 49956 dental +T1215 CHEBI:73702 49957 49960 wax +T1216 UBERON:0000966 50028 50035 retinas +T1217 UBERON:0000004 50089 50094 nasal +T1218 UBERON:0000966 50115 50122 retinas +T1219 UBERON:0001782 50137 50163 retinal pigment epithelium +T1220 CHEBI:26130 50145 50152 pigment +T1221 UBERON:0001773 50204 50210 sclera +T1222 UBERON:0000966 50238 50245 retinas +T1223 CHEBI:30059 50395 50417 potassium ferrocyanide +T1224 UBERON:0000966 50429 50436 retinas +T1225 CHEBI:16236 50693 50700 ethanol +T1226 CHEBI:16236 50727 50734 ethanol +T1227 CHEBI:16236 50772 50779 ethanol +T1228 UBERON:0000966 50791 50798 retinas +T1229 CHEBI:38685 50842 50857 propylene oxide +T1230 CHEBI:38909 51041 51054 sodium borate +T1231 UBERON:0000966 51108 51114 retina +T1232 UBERON:0000966 51163 51169 retina +T1233 PR:000001195 51210 51217 S-opsin +T1234 GO:0010467 51218 51228 expressing +T1235 CHEBI:10545 51307 51315 electron +T1236 CL:0000210 51515 51528 photoreceptor +T1237 UBERON:0001787 51515 51534 photoreceptor layer +T1238 CHEBI:10545 51579 51587 electron +T1239 CHEBI:10545 51625 51633 electron +T1240 UBERON:0000966 51728 51735 retinas +T1241 GO:0044297 51754 51765 cell bodies +T1242 CL:0000604 51773 51777 rods +T1243 CL:0000604 51783 51786 rod +T1244 UBERON:0001789 51827 51831 ONLs +T1245 UBERON:0001789 51918 51921 ONL +T1246 GO:0044297 52083 52094 cell bodies +T1247 GO:0044297 52142 52153 cell bodies +T1248 UBERON:0000970 52355 52358 eye +T1249 GO:0005886 52382 52396 cell membranes +T1250 CHEBI:10545 52488 52496 electron +T1251 CL:0000604 52770 52774 rods +T1252 UBERON:0000966 52792 52798 retina +T1253 GO:0005634 52817 52824 nuclear +T1254 GO:0005739 52825 52837 mitochondria +T1255 CL:0000604 52843 52847 rods +T1256 GO:0005634 52924 52931 nuclear +T1257 GO:0043226 52932 52942 organelles +T1258 CL:0000604 52980 52984 rods +T1259 GO:0005634 53009 53016 nuclear +T1260 GO:0043226 53017 53026 organelle +T1261 CL:0000604 53081 53085 rods +T1262 GO:0005634 53095 53102 nuclear +T1263 GO:0005739 53103 53115 mitochondria +T1264 GO:0044297 53186 53197 cell bodies +T1265 GO:0097617 53274 53287 hybridization +T1266 SO:0000704 53302 53307 genes +T1267 UBERON:0000966 53438 53444 retina +T1268 SO:0000704 53510 53514 gene +T1269 UBERON:0000966 53549 53556 retinas +T1270 GO:0007567 53624 53629 natal +T1271 SO:0000704 53667 53671 gene +T1272 SO:0000704 53686 53690 gene +T1273 UBERON:0000966 53728 53734 retina +T1274 NCBITaxon:33208 53755 53761 animal +T1275 UBERON:0000922 53788 53797 embryonic +T1276 UBERON:0000966 53817 53823 retina +T1277 UBERON:0000922 53847 53856 embryonic +T1278 GO:0097617 53888 53901 Hybridization +T1279 GO:0097617 54004 54017 Hybridization +T1280 GO:0097617 54121 54134 Hybridization +T1281 GO:0097617 54238 54251 Hybridization +T1282 GO:0097617 54355 54368 Hybridization +T1283 GO:0097617 54472 54485 Hybridization +T1284 SO:0000704 54497 54502 Genes +T1285 GO:0097617 54592 54605 Hybridization +T1286 SO:0000704 54617 54622 Genes +T1287 GO:0097617 54713 54726 Hybridization +T1288 PR:000001224 54739 54746 M-Opsin +T1289 PR:000016321 54751 54756 Thrb2 +T1290 PR:000001224 54772 54779 M-opsin +T1291 UBERON:0001789 54824 54827 ONL +T1292 UBERON:0001773 54871 54878 scleral +T1293 UBERON:0001789 54891 54894 ONL +T1294 PR:000001224 54939 54946 M-opsin +T1295 PR:000016321 54951 54956 Thrb2 +T1296 CL:0000573 55054 55058 Cone +T1297 CL:0000573 55072 55076 Cone +T1298 SO:0000704 55086 55091 Genes +T1299 GO:0097617 55146 55159 Hybridization +T1300 GO:0097617 55286 55299 hybridization +T1301 SO:0000704 55328 55332 gene +T1302 SO:0000842 55384 55393;55398 55402 region of ... gene +T1303 GO:0097617 55453 55466 hybridization +T1304 CL:0000604 55590 55593 Rod +T1305 SO:0000704 55594 55599 Genes +T1306 GO:0097617 55639 55652 Hybridization +T1307 GO:0097617 55700 55713 hybridization +T1308 SO:0000704 55729 55734 genes +T1309 CL:0000604 55754 55757 rod +T1310 UBERON:0000966 55796 55802 retina +T1311 GO:0097617 55882 55895 hybridization +T1312 SO:0000704 55924 55928 gene +T1313 SO:0000842 55980 55989;55994 55998 region of ... gene +T1314 GO:0097617 56049 56062 hybridization +T1315 GO:0097617 56108 56121 hybridization +T1316 CHEBI:37987 56592 56595 Cy3 +T1317 CHEBI:37989 56596 56599 Cy5 +T1318 CHEBI:37987 56699 56702 Cy3 +T1319 CHEBI:37989 56703 56706 Cy5 +T1320 SO:0000704 56963 56968 Genes +T1321 UBERON:0000966 56996 57002 Retina +T1322 SO:0000704 57041 57046 genes +T1323 PR:000011403 57180 57185 Nr2e3 +T1324 SO:0000673 57253 57263 transcript +T1325 SO:0000704 57374 57379 Genes +T1326 UBERON:0000966 57409 57415 Retina +T1327 SO:0000704 57454 57459 genes +T1328 PR:000011403 57595 57600 Nr2e3 +T1329 SO:0000673 57668 57678 transcript +T1330 CHEBI:10545 59492 59500 electron +T1331 CHEBI:33893 59581 59589 reagents +T1332 PR:000011432 59883 59886 Nrl +T1333 NCBITaxon:10088 59894 59898 mice +T1334 CHEBI:51231 59916 59920 DAPI +T1335 CHEBI:51231 59923 59949 6-diamidino-2-phenylindole +T1336 http://purl.obolibrary.org/obo/MONDO_0009989 60012 60016 ESCS +T1337 http://purl.obolibrary.org/obo/MONDO_0009989 60019 60043 enhanced S-cone syndrome +T1338 CL:0000573 60030 60034 cone +T1339 UBERON:0001792 60045 60048 GCL +T1340 UBERON:0001792 60051 60070 ganglion cell layer +T1341 UBERON:0001791 60072 60075 INL +T1342 UBERON:0001791 60078 60097 inner nuclear layer +T1343 GO:0005634 60084 60091 nuclear +T1344 UBERON:0001789 60099 60102 ONL +T1345 UBERON:0001789 60105 60124 outer nuclear layer +T1346 GO:0005634 60111 60118 nuclear +T1347 GO:0007567 60142 60147 natal +T1348 CL:0000573 60181 60185 Cone +T1349 CL:0000573 60199 60203 Cone +T1350 SO:0000704 60213 60218 Genes +T1351 GO:0097617 60273 60286 Hybridization +T1352 SO:0000704 60375 60380 genes +T1353 CL:0000573 60427 60431 cone +T1354 CL:0000573 60444 60448 cone +T1355 GO:0010467 60470 60480 expression +T1356 CL:0000573 60506 60510 cone +T1357 SO:0000704 60511 60516 genes +T1358 CL:0000573 60598 60602 cone +T1359 SO:0000704 60603 60608 genes +T1360 SO:0000704 60706 60711 genes +T1361 CL:0000573 60818 60822 cone +T1362 GO:0097617 60850 60863 hybridization +T1363 SO:0000704 60893 60898 genes +T1364 GO:0010467 60978 60988 expression +T1365 GO:0097617 61025 61039 hybridizations +T1366 SO:0000704 61125 61130 genes +T1367 GO:0097617 61231 61244 hybridization +T1368 GO:0097617 61320 61333 hybridization +T1369 SO:0000842 61378 61387;61392 61396 region of ... gene +T1370 GO:0007567 61537 61542 natal +T1371 NCBITaxon:10088 61684 61689 mouse +T1372 SO:0001026 61690 61696 genome +T1373 SO:0000704 61760 61764 gene +T1374 SO:0000704 61969 61973 gene +T1375 SO:0000704 62120 62124 gene +T1376 UBERON:0000966 62155 62161 retina +T1377 SO:0000704 62215 62220 Genes +T1378 GO:0097617 62430 62443 hybridization +T1379 UBERON:0000966 62470 62476 retina +T1380 GO:0010467 62568 62578 expression +T1381 GO:0010467 62630 62640 Expression +T1382 GO:0010467 62698 62708 expression +T1383 SO:0000704 62724 62728 gene +T1384 SO:0000704 62757 62762 genes +T1385 GO:0097617 62807 62820 hybridization +T1386 GO:0010467 62857 62867 expression +T1387 CL:0000573 62937 62941 cone +T1388 CL:0000604 62944 62947 rod +T1389 SO:0000704 62968 62972 gene +T1390 GO:0010467 62976 62985 expressed +T1391 CL:0000210 62993 63007 photoreceptors +T1392 CL:0000573 63033 63038 cones +T1393 CL:0000604 63044 63048 rods +T1394 CL:0000573 63051 63055 cone +T1395 CL:0000573 63092 63096 cone +T1396 CL:0000103 63117 63119 BP +T1397 CL:0000103 63121 63134 bipolar cells +T1398 CL:0000210 63146 63159 photoreceptor +T1399 GO:0010467 63160 63170 expression +T1400 GO:0001917 63180 63182 IS +T1401 GO:0001917 63184 63189;63195 63202 inner ... segment +T1402 CL:0011107 63217 63219 MG +T1403 CL:0011107 63221 63232 Müller glia +T1404 GO:0097617 63306 63319 hybridization +T1405 UBERON:0001782 63321 63324 RPE +T1406 UBERON:0001782 63326 63352 retinal pigment epithelium +T1407 CHEBI:26130 63334 63341 pigment +T1408 CL:0000573 63365 63369 Cone +T1409 CL:0000604 63374 63377 Rod +T1410 SO:0000704 63378 63382 Gene +T1411 GO:0010467 63378 63393 Gene Expression +T1412 CL:0000573 63498 63502 cone +T1413 SO:0000704 63503 63507 gene +T1414 UBERON:0000966 63539 63545 retina +T1415 CL:0000604 63613 63616 rod +T1416 SO:0000704 63626 63631 genes +T1417 GO:0010467 63726 63736 expression +T1418 CL:0000210 63754 63767 photoreceptor +T1419 SO:0000704 63890 63894 gene +T1420 PR:000001195 63938 63945 S-Opsin +T1421 GO:0097617 63971 63984 Hybridization +T1422 PR:000001195 63989 63996 S-Opsin +T1423 PR:000001245 63997 64006 Rhodopsin +T1424 GO:0042571 64007 64015 Antibody +T1425 UBERON:0000966 64039 64045 Retina +T1426 GO:0097617 64080 64093 hybridization +T1427 PR:000001195 64102 64109 S-opsin +T1428 UBERON:0000966 64148 64155 retinal +T1429 CL:0009004 64148 64161 retinal cells +T1430 CHEBI:51231 64175 64179 DAPI +T1431 UBERON:0001789 64228 64250;64265 64271 outer nuclear layer of ... retina +T1432 GO:0005634 64234 64241 nuclear +T1433 GO:0042571 64283 64291 antibody +T1434 PR:000001195 64296 64303 S-opsin +T1435 PR:000001245 64314 64323 rhodopsin +T1436 UBERON:0001773 64337 64344 scleral +T1437 UBERON:0001789 64357 64376 outer nuclear layer +T1438 GO:0005634 64363 64370 nuclear +T1439 CHEBI:51231 64384 64388 DAPI +T1440 GO:0001750 64477 64491 outer segments +T1441 PR:000001195 64515 64522 S-opsin +T1442 PR:000001245 64527 64536 rhodopsin +T1443 GO:0010467 64572 64582 Expression +T1444 CL:0000573 64609 64613 Cone +T1445 SO:0000704 64614 64619 Genes +T1446 UBERON:0001773 64720 64727 scleral +T1447 UBERON:0001789 64740 64759 outer nuclear layer +T1448 GO:0005634 64746 64753 nuclear +T1449 CL:0000573 64765 64769 cone +T1450 SO:0000704 64795 64800 genes +T1451 PR:000004855 64846 64851 Bub1b +T1452 PR:000016206 64856 64860 Tcta +T1453 SO:0000673 64866 64876 transcript +T1454 GO:0001917 64911 64927;64932 64946 inner segment of ... photoreceptors +T1455 CL:0000210 64932 64946 photoreceptors +T1456 UBERON:0000966 64948 64955 Retinas +T1457 UBERON:0001773 64983 64990 scleral +T1458 SO:0000704 65026 65031 Genes +T1459 CL:0000210 65066 65079 Photoreceptor +T1460 GO:0010467 65091 65101 Expression +T1461 PR:000008099 65103 65107 Gnb3 +T1462 PR:000016321 65112 65117 Thrb2 +T1463 CL:0000573 65152 65156 cone +T1464 SO:0000704 65157 65162 genes +T1465 UBERON:0001773 65189 65196 scleral +T1466 UBERON:0000922 65209 65218 embryonic +T1467 NCBITaxon:10088 65219 65224 mouse +T1468 UBERON:0000966 65225 65231 retina +T1469 CL:0000210 65270 65284 photoreceptors +T1470 PR:000006874 65286 65290 Ece1 +T1471 PR:000012076 65295 65300 Otop3 +T1472 CL:0000573 65315 65319 cone +T1473 SO:0000704 65320 65325 genes +T1474 CL:0000210 65406 65419 photoreceptor +T1475 GO:0010467 65431 65441 expression +T1476 PR:000001831 65443 65448 Prdm1 +T1477 SO:0000704 65486 65491 genes +T1478 SO:0000704 65533 65538 genes +T1479 PR:000001831 65576 65581 Prdm1 +T1480 GO:0010467 65608 65618 expression +T1481 UBERON:0007023 65630 65635 adult +T1482 UBERON:0000922 65711 65720 embryonic +T1483 UBERON:0000966 65721 65727 retina +T1484 CL:0000210 65745 65758 photoreceptor +T1485 UBERON:0000966 65794 65800 retina +T1486 PR:000008099 65808 65812 Gnb3 +T1487 PR:000001245 65858 65867 rhodopsin +T1488 GO:0010467 65868 65878 Expression +T1489 UBERON:0000966 65908 65914 Retina +T1490 PR:000001245 65958 65967 rhodopsin +T1491 UBERON:0000966 65986 65992 retina +T1492 CL:0000604 66022 66025 rod +T1493 SO:0000704 66035 66040 genes +T1494 GO:0010467 66068 66078 expression +T1495 PR:000012478 66136 66141 Pde6a +T1496 UBERON:0000966 66205 66211 Retina +T1497 CL:0000210 66246 66264 Photoreceptor Cell +T1498 PR:000001195 66299 66306 S-Opsin +T1499 CL:0000573 66316 66321 Cones +T1500 UBERON:0001789 66382 66401 outer nuclear layer +T1501 GO:0005634 66388 66395 nuclear +T1502 UBERON:0001773 66403 66410 scleral +T1503 GO:0005634 66519 66526 nuclear +T1504 CL:0000573 66539 66544 cones +T1505 GO:0044297 66641 66652 cell bodies +T1506 UBERON:0001789 66682 66701 outer nuclear layer +T1507 GO:0005634 66688 66695 nuclear +T1508 UBERON:0001789 66757 66776 outer nuclear layer +T1509 GO:0005634 66763 66770 nuclear +T1510 UBERON:0000966 66842 66848 retina +T1511 UBERON:0000966 66879 66885 retina +T1512 CL:0000573 66911 66921 cone cells +T1513 UBERON:0001789 67015 67034 outer nuclear layer +T1514 GO:0005634 67021 67028 nuclear +T1515 UBERON:0001773 67036 67043 scleral +T1516 GO:0097617 67072 67085 hybridization +T1517 PR:000001195 67090 67097 S-opsin +T1518 UBERON:0001773 67143 67150 scleral +T1519 UBERON:0001789 67163 67182 outer nuclear layer +T1520 GO:0005634 67169 67176 nuclear +T1521 UBERON:0000966 67216 67222 retina +T1522 PR:000001195 67243 67250 S-opsin +T1523 UBERON:0001789 67291 67310 outer nuclear layer +T1524 GO:0005634 67297 67304 nuclear +T1525 CL:0000573 67363 67373 cone cells +T1526 UBERON:0000966 67434 67441 retinas +T1527 CHEBI:10545 67545 67553 Electron +T1528 UBERON:0001789 67573 67592 outer nuclear layer +T1529 GO:0005634 67579 67586 nuclear +T1530 CL:0000604 67651 67654 rod +T1531 GO:0044297 67655 67666 cell bodies +T1532 GO:0044297 67693 67704 cell bodies +T1533 GO:0005634 67758 67765 nucleus +T1534 GO:0000792 67795 67810 heterochromatin +T1535 GO:0000792 67904 67919 heterochromatin +T1536 GO:0000791 67927 67938 euchromatin +T1537 GO:0005634 67953 67960 nuclear +T1538 GO:0005739 67961 67973 mitochondria +T1539 CL:0000573 68010 68014 cone +T1540 GO:0044297 68015 68024 cell body +T1541 CL:0000604 68057 68060 rod +T1542 GO:0000792 68075 68090 heterochromatin +T1543 GO:0005634 68109 68116 nuclear +T1544 GO:0005739 68117 68130 mitochondrion +T1545 CL:0000210 68172 68186 photoreceptors +T1546 CL:0000604 68259 68276 Rod Photoreceptor +T1547 GO:0065007 68293 68303 Regulatory +T1548 PR:000011432 68519 68522 Nrl +T1549 SO:0000704 68553 68558 genes +T1550 PR:000011403 68663 68668 Nr2e3 +T1551 GO:0065007 68727 68737 regulatory +T1552 CL:0000604 68828 68831 rod +T1553 SO:0000704 68841 68846 genes +T1554 PR:000011403 68850 68855 Nr2e3 +T1555 CL:0000210 68934 68947 photoreceptor +T1556 PR:000005904 68979 68982 Crx +T1557 CHEBI:33893 69231 69239 reagents +T1558 CL:0000210 69341 69354 photoreceptor +T1559 GO:0010467 69355 69365 expressing +T1560 CL:0000604 69371 69374 rod +T1561 CL:0000573 69379 69383 cone +T1562 SO:0000704 69384 69389 genes +T1563 NCBITaxon:10088 69395 69400 mouse +T1564 http://purl.obolibrary.org/obo/MONDO_0009989 69410 69434 enhanced s-cone syndrome +T1565 CL:0000573 69421 69425 cone diff --git a/src/ontogpt/evaluation/craft/database/all/16110338.txt b/src/ontogpt/evaluation/craft/database/all/16110338.txt new file mode 100644 index 000000000..f51e09580 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16110338.txt @@ -0,0 +1,461 @@ +A Hybrid Photoreceptor Expressing Both Rod and Cone Genes in a Mouse Model of Enhanced S-Cone Syndrome + +Abstract + +Rod and cone photoreceptors subserve vision under dim and bright light conditions, respectively. The differences in their function are thought to stem from their different gene expression patterns, morphologies, and synaptic connectivities. In this study, we have examined the photoreceptor cells of the retinal degeneration 7 (rd7) mutant mouse, a model for the human enhanced S-cone syndrome (ESCS). This mutant carries a spontaneous deletion in the mouse ortholog of NR2E3, an orphan nuclear receptor transcription factor mutated in ESCS. Employing microarray and in situ hybridization analysis we have found that the rd7 retina contains a modestly increased number of S-opsin–expressing cells that ultrastructurally appear to be normal cones. Strikingly, the majority of the photoreceptors in the rd7 retina represent a morphologically hybrid cell type that expresses both rod- and cone-specific genes. In addition, in situ hybridization screening of genes shown to be up-regulated in the rd7 mutant retina by microarray identified ten new cone-specific or cone-enriched genes with a wide range of biochemical functions, including two genes specifically involved in glucose/glycogen metabolism. We suggest that the abnormal electroretinograms, slow retinal degeneration, and retinal dysmorphology seen in humans with ESCS may, in part, be attributable to the aberrant function of a hybrid photoreceptor cell type similar to that identified in this study. The functional diversity of the novel cone-specific genes identified here indicates molecular differences between rods and cones extending far beyond those previously discovered. + +Synopsis + + + +Vision begins with light entering the eye. This light is projected onto the retina, a thin neural structure lining the inside of the eye. Photoreceptors, among the most important cell types in the retina, are the first to receive the incoming rays of light. In mammals, there are two types of photoreceptors: rods and cones. Rods are specialized for nighttime vision, and cones for daytime and color vision. In this study, the authors examined the photoreceptors of a mouse with a gene mutation that causes photoreceptors to develop abnormally. Humans with a similar mutation have a form of blindness called enhanced S-cone syndrome (ESCS). Surprisingly, the majority of photoreceptors in this mutant mouse were found to have features of both normal rods and cones. It is possible that the abnormal features of these photoreceptors predispose them to undergo premature death. If this model accurately reflects the situation in human patients with ESCS, it may provide an explanation for the loss of vision seen in this disease. This study also elucidated previously unknown molecular differences between normal rods and cones. This new knowledge may contribute to a better overall understanding of the mechanisms underlying night, day, and color vision. + +Introduction + +Enhanced S-cone syndrome (ESCS) is an unusual disease of photoreceptors that includes night blindness (suggestive of rod dysfunction), an abnormal electroretinogram (ERG) with a waveform that is nearly identical under both light and dark adaptation, and an increased sensitivity of the ERG to short-wavelength light [1,2]. The disease is caused by mutations in the orphan nuclear receptor transcription factor NR2E3 (also known as photoreceptor nuclear receptor), which is expressed exclusively in rods [3,4]. Recent human genetic studies have also demonstrated mutations in this gene in Goldmann-Favre syndrome and many cases of clumped pigmentary retinal degeneration [5]. + +The initial reports of patients with ESCS attributed the unusual ERG to an abnormally functioning rod photoreceptor system with persistent activity under light adaptation [6–8]. Subsequent studies, however, concluded that the ERG was due to supernumerary short-wavelength (“blue”) cone photoreceptors (S-cones) in these patients [1,2,9–11]. Histopathologic analysis of a retina from a human patient with ESCS and extensive retinal degeneration demonstrated an absence of rhodopsin-positive cells and an increase in the number of S-cone opsin-expressing cells. Nevertheless, the overall density of cones was only modestly increased in this patient (approximately 2-fold), suggesting that there might be additional factors that contribute to the very large, light-adapted ERG seen in this disease. In addition to the ERG findings, patients with ESCS have dysmorphic retinas with rosette formation in the outer nuclear layer (ONL) where photoreceptor cell bodies reside, and a slow retinal degeneration that can ultimately lead to complete blindness [12–14]. + +Mutations in the mouse ortholog of NR2E3 have been identified in the spontaneous mutant retinal degeneration 7 (rd7) [15]. This mutant demonstrates slow retinal degeneration and abnormal lamination of the ONL with rosette formation [15,16]. Curiously, the ERG of the mouse under both light and dark adaptation has been reported to be normal, showing progressive attenuation with time, presumably due to degenerative cell loss [15]. A prior study showed a 2- to 3-fold increase in the number S-opsin–positive cells in the rd7 retina compared to wild type [17]. In addition, two groups recently reported derepression of additional cone genes in the rd7 mutant [18,19]. + +In order to better understand the mechanistic basis of ESCS, we undertook a molecular and ultrastructural analysis of the photoreceptors of the rd7 mutant mouse. Microarray and in situ hybridization analyses revealed a modest increase in the number of S-opsin–positive cells and widespread derepression of many cone-specific genes within rod photoreceptor cells. Ultrastructural studies demonstrated that the cells that coexpress rod and cone genes in the rd7 retina represent a morphologically hybrid cell type, intermediate between normal rods and cones. + +Results + +Widespread Up-Regulation of Cone Genes in the rd7 Mutant Retina + +In an initial analysis of the rd7 mutant, homozygous mutant retinas were compared with wild-type controls at multiple postnatal time points using both cDNA and Affymetrix microarrays. The cDNA microarray used in this study contains approximately 12,000 different cDNAs largely derived from the retina and nervous system, and the Affymetrix microarray contains over 34,000 genes. Experiments at all timepoints were carried out in triplicate, and stringent criteria were applied in deciding whether a given gene was up- or down-regulated in the mutant (see Materials and Methods for details). + +These experiments demonstrated widespread up-regulation of cone-specific and cone-enriched genes in the rd7 retina, especially by postnatal day 14 (P14) and P21 (Figure 1). Most known cone-specific or cone-enriched genes were found to be up-regulated in the mutant (Figure 1, genes G1–G15). The majority of these genes represent components of the phototransduction cascade (e.g., opsins, transducins, and phosphodiesterase subunits). In addition to these genes, several novel cone-specific genes of unknown function recently identified in our lab were also up-regulated (Figure 1, genes G16, G17, G21, and G24; unpublished data). Finally, a wide range of other genes, most with no previously recognized role in the retina, were found to be up-regulated in the rd7 mutant (Figure 1, G26–G53; Tables S1 and S2; Figures S1–S7). + +Nr2e3 expression is first detectable by in situ hybridization around embryonic day 18 (E18); it then peaks around P6 and subsequently decreases to adult levels by P21 (unpublished data). In accordance with this time course of expression, almost no gene expression changes were found at P0, with progressively more changes at later timepoints (Figure 1). One exception to this statement is the gene RIKEN cDNA 4933409K07 (Figure 1, gene G47), which was the only gene shown to be up-regulated at all timepoints examined. Additional discussion of this gene and its unusual expression pattern will be presented below. + +Two Distinct Patterns of Cone Gene Derepression in rd7 + +In order to confirm these microarray results, an in situ hybridization analysis of the putative up-regulated cone genes was carried out in which the rd7 mutant retina was compared with age-matched, wild-type controls. We found that the majority of the cone-specific genes that were up-regulated in microarray experiments were derepressed when assessed by in situ hybridization (Figure 2). There were two major patterns of cone gene derepression. The more common pattern (type I) manifested itself as ectopic gene expression throughout the ONL, consistent with gene expression in all photoreceptors (Figure 2; upper left photomicrographs). Typical examples of this pattern of derepression are shown in Figure 2, and many more are available in Table S1. This pattern of expression contrasts sharply with the usual pattern of cone gene expression, which consists of scattered cells localized to the scleral edge of the ONL (Figure 2). + +The second category of cone gene derepression (type II) consisted of a patchy, salt-and-pepper pattern of ectopic expression in which individual positive cells were scattered throughout the ONL (Figure 2, upper right photomicrographs; Table S1). Although numerous positive cells were present in the rd7 retina (particularly in the ventral portion), there were clearly many interspersed cells that showed a complete absence of expression. In order to rule out the possibility that these scattered positive cells were simply the normal complement of cones that had failed to localize their cell bodies to the scleral edge of the ONL, the number of positive cells in the rd7 retina was quantitated by dissociated cell in situ hybridization. + +Dissociated cell in situ hybridization was performed using a probe for the S-cone opsin gene (Opn1sw), which shows type II derepression (Figures 2 and 3A–3C). S-opsin was expressed in 3.2% of retinal cells in the rd7 mutant (66 S-opsin–positive cells out of 2,056 6-diamidino-2-phenylindole [DAPI]-positive cells). This value is approximately 2-fold greater than the percentage of S-opsin–positive cells identified in wild-type control retinas, 1.65% (54 S-opsin-positive cells out of 3,271 DAPI-positive cells), and accords well with the previously reported value of 2- to 3-fold more S-opsin–positive cells in rd7 compared to wild type arrived at by antibody staining of tissue sections [17]. + +Previous studies have estimated that the total number of cones in the mouse retina is 2% of all retinal cells [20], and that S-opsin is largely repressed in cones in the dorsal third of the retina [21]. The estimate of 1.65% S-opsin–positive cells in the wild-type retina is in agreement with these data. The fact that only 3.2% of all retinal cells are S-opsin–positive in the rd7 mutant also confirms that the majority of the photoreceptors (which make up just over 70% of the cells in the adult mouse retina) do not express this gene. In order to assess whether these supernumerary S-opsin–expressing cells coexpressed rod-specific markers, a double antibody staining for S-opsin and rhodopsin was performed. This study showed mutually exclusive domains of expression of S-opsin and rhodopsin in the photoreceptor outer segments (Figure 3D–3F). This finding suggests that the supernumerary S-opsin–expressing cells in the rd7 retina may represent normal “blue” cones. + +Novel Cone-Specific Genes Are Derepressed in rd7 + +Given that the majority of known cone-specific genes showed marked derepression in the rd7 mutant, additional candidate genes up-regulated on microarray analysis were evaluated for cone-specific expression. In situ hybridization was performed on an additional 45 up-regulated genes, confirming that 21 of them were derepressed. Of these, at least ten showed a definite cone-specific or cone-enriched pattern of expression in the wild-type retina (Figure 1, genes G26–G35). Several examples are given in Figure 4. Note that in the wild-type retina, there is a relatively weak pattern of scattered positive cells at the scleral edge of the ONL, consistent with a cone-specific pattern of expression. All of these genes show marked derepression in the rd7 retina. A number of these novel cone-specific genes showed a striking localization of their transcripts to the photoreceptor inner segment (e.g., Bub1b and Tcta). This localization manifests in a section in situ hybridization as a dark band of staining just beyond the outer edge of the ONL immediately underlying the outer segment layer. Although such a pattern of transcript localization is commonly seen in many rod-specific genes (e.g., Rho in Figure 2; Pcdh21, Rbp3, and Cnga1 in Table S2), it is not easily appreciated in cone-specific genes, possibly due to the relative scarcity of cones in the mouse. In the rd7 mutant retina in which such genes are widely derepressed, such a pattern of transcript localization often becomes apparent. + +In addition to the ten genes that showed cone-specific expression in the wild-type retina, another 11 novel genes were derepressed in the rd7 retina by in situ hybridization (Figure 1, genes G36–G46). Some of these genes showed faint expression in a cone-like distribution (see Table S1, genes G36, G40, and G44), and one appeared to be expressed throughout the ONL but at greater levels in cones than in rods (Table S1, gene G37). The remainder of the up-regulated genes did not have detectable cone staining in the wild-type retina. Despite this apparent absence of cone staining, the pattern of derepression in rd7 suggests that these genes may also be novel cone-specific genes, albeit expressed at levels below the sensitivity threshold of our in situ hybridization assay. + +In most cases, the novel cone genes identified in this study appear to have a type I pattern of derepression. However, due to the weakness of the signal in some cases, or transcript localization to the inner segment in others, it was not always possible to determine with confidence which of the two patterns of derepression (if either) each of these genes displayed. In terms of functional categorization, the novel cone genes cover a broad range including glucose metabolism (Pygm and Glo1), fatty acid metabolism (Elovl2), DNA repair (Smug1), cell cycle/chromosome segregation (Bub1b), carcinogenesis (Tcta), endothelial biology (Ece1), cytoskeletal function (Ebp4.1l1), and even otolith formation (Otop3). + +A relatively frequent finding among both previously identified cone-specific genes, as well as in some of those identified in the present study, is the occurrence of gene expression in an early photoreceptor precursor pattern (Figure 5). This pattern of expression consists of positive staining by in situ hybridization specifically at the scleral border of the retina during prenatal timepoints (in the range of E13–E18). Gnb3 and Thrb2 are two examples of known cone genes with this early pattern of expression (Figure 5). Two of the 11 novel cone genes identified in this study also have this early photoreceptor pattern of expression (Ece1 and Otop3). Intriguingly, three genes shown to be up-regulated in rd7 on microarray, but that had either no detectable signal by in situ hybridization at adult stages or no apparent change in expression by in situ hybridization between wild type and rd7, also showed this early photoreceptor pattern (Figure 1, genes G48–G50). The embryonic expression pattern of two of these genes is shown in Figure 5 (the embryonic in situ hybridization for the third, G50, can be found in Table S1). Although the significance of such early photoreceptor expression is not known, it is possible that these genes may also be cone-specific but are expressed at undetectably low levels in the adult. + +M-Opsin and Thyroid Hormone Receptor β2 Are Unchanged in the rd7 Mutant + +Only two cone-specific genes failed to show any change in expression by either microarray or in situ hybridization in the rd7 mutant: M-opsin (Opn1mw) and thyroid hormone receptor β2 (Thrb2) (Figure S8). This result is particularly notable because Thrb2 is absolutely required for the expression of M-opsin [22]. Furthermore, the repression of S-opsin expression in the dorsal third of the mouse retina is thought to depend, at least in part, on Thrb2 since S-opsin shows dorsal derepression in the Thrb2 mutant [22]. Despite the derepression of S-opsin seen in the ventral portion of the rd7 retina, the normal dorsal repression of this gene is still present in this mutant (unpublished data). This finding is consistent with the normal expression pattern and function of Thrb2 in the rd7 mutant. + +One further finding to note is that the cell bodies of the M-opsin–positive cells appear to be scattered throughout the ONL in the rd7 mutant at P14 (Figure S8). Despite this fact, their overall number does not appear to be increased relative to wild-type. In addition, by P28, the M-opsin-positive cell bodies in rd7 appear to have relocated to their normal position at the scleral edge of the ONL (Figure S8). It is known that until P11, the cell bodies of cone photoreceptors in the mouse are normally dispersed throughout the ONL, only to relocate subsequently to the scleral edge of the ONL around P12 [23]. It is possible that in the rd7 mutant retina, there is a short delay in the relocation of the M-opsin–expressing cone cell bodies to the scleral edge of the ONL. + +Rod Genes Are Only Modestly and Temporarily Affected in rd7 + +In sharp contrast to changes in cone gene expression, rod-specific genes were much less severely affected in the rd7 mutant. Microarray and in situ hybridization analysis of numerous rod genes failed to reveal marked changes in expression levels at P14 and P21 (see Figure 2, lower left photomicrographs; Table S2). In addition to the three rod genes depicted in Figure 2, in situ hybridization analysis on an additional 19 rod-specific and pan-photoreceptor genes demonstrated only a very mild diminution of expression in two of these genes, gucy2e and Rgs9, at P14, and an increase in expression in two, Nr2e3, and Cnga1 (Table S2). + +Despite the minimal changes in rod gene expression at later postnatal timepoints, there was evidence of a significant delay in the onset of rhodopsin (Rho) expression in rd7 mutants relative to wild-type. Microarray analysis at P6 demonstrated five cDNA spots that were down-regulated in three out of three experiments. Of these spots, three corresponded to rhodopsin (Table S3). In situ hybridization analysis of several rod-specific genes at P6 revealed that rhodopsin alone showed a markedly lower level of expression compared to wild type (Figure 6; unpublished data). Despite this modest delay in the onset of rhodopsin expression, by P14 the gene had attained nearly normal levels in the rd7 mutant (see Figure 2, lower left photomicrographs). This latter finding suggests that all the rod- and many cone-specific genes are coexpressed in the majority of photoreceptors in the rd7 mutant. + +Changes in Retinal Transcription Factor and Müller Glial Gene Expression in rd7 + +Analysis of several photoreceptor transcription factors in the rd7 mutant indicated that the levels of Crx and Nrl are unaffected in the mutant at P14 (see Figure 2, lower right photomicrographs). Nrl is a rod-specific, basic leucine zipper transcription factor required for the activation of many rod-specific genes and the repression of most cone-specific genes in rods [24]. Nrl is known to be genetically upstream of Nr2e3 and is required for its expression [24]. Crx is a homeobox transcription factor expressed in both rods and cones and is required for the expression of a variety of rod- and cone-specific genes [25]. In contrast to what is seen in the Nrl mutant, Nr2e3 expression is unchanged in Crx mutant homozygotes (unpublished data). + +Strikingly, Nr2e3 itself was markedly up-regulated in the rd7 mutant both by microarray and in situ hybridization (see Figure 2, lower right photomicrographs; Table S2). The rd7 mutant carries a deletion in Nr2e3 that removes portions of both the DNA-binding and ligand-binding domains [15]. Although this deletion most likely creates a null allele, a residual transcript is clearly present and up-regulated in the rd7 mutant. This finding suggests that Nr2e3 is required for repression of its own transcription. + +One gene, RIKEN cDNA 4933409K07 (Figure 1, gene G47), was found to be up-regulated on microarray at all four timepoints examined. This gene showed a unique pattern of expression in the adult rd7 mutant retina. Whereas there was only a barely detectable hint of expression in the inner nuclear layer (INL) in the wild-type retina, this gene showed strong expression in the middle and vitreal thirds of the INL as well as patchy expression in the ganglion cell layer (GCL) and at the scleral edge of the ONL in rd7 (see Table S1). This in situ hybridization pattern is consistent with staining in Müller glia, the principal glial cell type of the mouse retina. One possible interpretation of this unusual expression pattern is that it represents an early reaction of Müller glia to injury in this mutant. + +The Majority of the Photoreceptors in the rd7 Retina Represent a Morphologically Hybrid Cell Type + +In order to assess the morphologic effects of the above gene expression changes, the ultrastructure of the photoreceptor cell bodies in the rd7 mutant was examined. The cell bodies were chosen for evaluation rather than the outer segments, since in the mouse, the ultrastructural differences between rod and cone somata are much greater than are the differences between the outer segments [26]. In the wild-type mouse, cone cell bodies are aligned along the scleral border of the ONL, and they are larger than those of rods. They have a smaller, more irregular mass of nuclear heterochromatin that is often broken up into multiple discrete clumps connected by thin threads. They also have more abundant electron-lucent euchromatin than rods. Lastly, they frequently have a patch of organelle-rich cytoplasm next to their nuclei, usually containing large mitochondria [26]. + +Analysis of toluidine blue-stained semi-thin sections revealed that such cone-like cells were present in greater abundance in the rd7 retina than in wild-type, and that their somata were scattered throughout the ONL (Figure 7). A comparison between the distribution of these cells and those expressing S-cone opsin strongly suggests that they represent the same cell population (compare Figure 7D and 7F). Analysis of the nuclear morphology of dissociated retinal cells stained for S-cone opsin by dissociated cell in situ hybridization confirmed that this is the case (unpublished data). These findings, along with the absence of rhodopsin staining in these cells (see Figure 3D–3F), suggest that these “cone-like” cells in the rd7 mutant retina may represent supernumerary normal cones with an abnormal localization of their cell bodies. + +In contrast to the cone cell body, the wild-type rod soma is small and nearly round. It has a single, large clump of dense heterochromatin, a thin rim of moderately electron-dense euchromatin, and very scant juxtanuclear cytoplasm without organelles [26,27]. The second cell population in the ONL of the rd7 retina has some of the nuclear features of normal rods, such as a single, dense mass of heterochromatin and moderately electron-dense euchromatin (Figure 7H); yet these cells also show features of cones. First, the euchromatin is, on average, more abundant in these cells than in wild-type rods (compare Figure 7G and 7H). In addition, comparison of the diagrammatic representation of the wild-type and rd7 ONLs suggests that the average area of the S-opsin–negative cells in rd7 is greater than in the wild-type (Figure 7C and 7D). In order to confirm this impression, we quantitated the area of 50 wild-type and 50 mutant rod-like cell bodies (see Materials and Methods for details). This experiment confirmed that the average area of the rod-like somata in rd7 is approximately 30% larger than that of wild-type rod somata (mean area in rd7 was 9.75 ± 1.36 (standard deviation) μm2, compared to wild-type rods, with 7.53 ± 0.72 μm2 ; n = 50; p = 7.6 × 10–16, Student's t-test). It is also notable that the standard deviation of the somal area is nearly twice as great in rd7 than in wild-type, confirming the subjective impression of greater variability in somal size and shape in the mutant compared to the wild-type (compare Figure 7C and 7D). + +Lastly, 38% (19/50) of the rd7 photoreceptors selected for somal area quantitation had prominent juxtanuclear mitochondria (red arrow in Figure 7H; unpublished data). Such juxtanuclear organelles are only very rarely seen in normal rods (1.5%; six out of 399 cells counted), but are common in cones (yellow arrow in Figure 7H). In conclusion, it is clear that this second cell population in the rd7 retina has morphological features of both normal rods and cones consistent with the coexpression of many rod- and cone-specific genes in these cells. + +Discussion + +In this paper we have determined that the primary role of the rod-specific transcription factor, Nr2e3, is to maintain cone genes transcriptionally silent within rods. We have identified two patterns of cone gene derepression in the rd7 mutant retina, in agreement with a previous report by Chen et al. [18]. The first pattern of derepression identified (type I) consists of ectopic expression of cone genes in the vast majority of cells in the ONL. These cells were also shown to coexpress all rod genes tested. Consistent with the hybrid pattern of gene expression in these cells, electron microscopic analysis revealed them to be morphologically intermediate between normal rods and cones. + +Although genes showing type I derepression demonstrated staining in the majority of cells in the ONL, two lines of evidence suggest that these genes are not completely derepressed in these cells when compared to their expression in S-opsin–expressing cones. First, close evaluation of the staining pattern of a number of type I genes in the rd7 mutant retina (e.g., see Table S1, genes G9, G19, and G24), reveals that, in addition to the background staining throughout the ONL, there is a more darkly staining subpopulation of cells scattered throughout this layer in a distribution corresponding to that of the supernumerary S-cone opsin-expressing cells. This pattern of staining suggests that these genes are more highly expressed in S-opsin expressing cells than in the hybrid cells of the rd7 retina. + +The second line of evidence derives from a comparison of the expression pattern of many type I genes in rd7 and Nrl−/− mutant backgrounds. As mentioned above, Nrl is a retinal transcription factor that, when mutated, results in en masse conversion of rods into S-opsin–expressing cones [24]. It can be inferred from this fact that Nrl is absolutely required for the normal silencing of cone-specific genes in rods. In the Nrl homozygous mutant, there is a stronger and more uniform derepression of many cone-specific genes throughout the ONL than is seen in the rd7 retina (unpublished data). This finding further suggests that, in addition to its repression of cone gene expression via induction of Nr2e3 expression, Nrl exerts an additional level of negative control over cone genes either directly or via a second, as yet uncharacterized, repressor. + +The second pattern of derepression seen in the rd7 retina (type II), is represented by a scattered population of cells throughout the ONL that shows derepression of several cone-specific genes, including S-cone opsin. By ultrastructural criteria, these cells appear to be normal cones, albeit with displaced cell bodies. Quantitation of these supernumerary S-cone opsin-positive cells indicates that they are approximately 2-fold more abundant than in normal retina, consistent with previous antibody studies [17]. + +Two recent studies have presented data that are consistent with many of the findings in our study [18,19]. Both studies showed that cone genes in addition to S-cone opsin are derepressed in the mouse rd7 mutant. In addition, Peng et al. [19] found by RT-PCR that the levels of several rod-specific genes, including rhodopsin, were modestly reduced in rd7 at P28. Our in situ hybridization data suggest that rhodopsin expression is markedly reduced at P6, but that it attains levels indistinguishable from wild-type by P14. Since the change in rhodopsin levels identified by Peng et al. were relatively small (an approximately 15% reduction), it is not surprising that such a difference was not detected by in situ hybridization. The overall finding of modest reductions in rod-specific gene expression is entirely in keeping with the results of the present study. + +In addition to demonstrating derepression of a range of known cone-specific genes in rd7 mutants, Chen et al. [18] showed up-regulation by Northern blot of two additional genes in the rd7 mutant, Elovl2 and Fabp7. These two genes were also found to be up-regulated in rd7 in the present study (see Figure 1; Table S1). Although we found Elovl2 to have a cone-enriched pattern of expression (see Figure 1), in situ hybridization analysis of Fabp7 failed to show a signal in wild-type or mutant retina (unpublished data). Nevertheless, previous studies have suggested that Fabp7 is expressed in radial glia and immature astrocytes in the brain [28–30]. Given the expression pattern elsewhere in the nervous system, it is possible that Fabp7 is up-regulated in Müller glia in the rd7 retina in response to injury in a manner akin to the novel Müller glial gene identified in this study, RIKEN cDNA 4933409K07 (Figure 1, gene G47). Indirect support for this idea is provided by the observation that Fabp7 is up-regulated by microarray analysis in Nrl and Crx mutant retinas as well (unpublished data), suggesting that this change may represent a generalized response to injury in the retina rather than derepression of a cone-enriched gene. + +The study by Chen et al. [18] made two further observations worthy of note. First, they identified a zebrafish homolog of Nr2e3 and showed it to be expressed in photoreceptors. Interestingly, they showed that this homolog appears to have a pan-photoreceptor pattern of expression early in development that later resolves into a rod-specific pattern of expression. This early expression in cones may represent a mechanism whereby the expression of cone-specific gene products is temporarily repressed. It will be important to determine the extent to which the function of Nr2e3 has been conserved in the retina of such a distantly related organism. Secondly, Chen et al. [18] used an in vitro oligonucleotide selection protocol to determine the preferred binding site for Nr2e3. This information will be very useful for future bioinformatic analyses of Nr2e3 target genes. + +The gene expression changes identified in the rd7 mutant retina in the present study suggest the scheme of gene regulation in mouse rods depicted in Figure 8. As this diagram implies, there appear to be at least two different repressors of cone genes within rods, Nr2e3 and either Nrl itself or an additional unknown transcription factor downstream of Nrl, here termed “transcription factor X.” In fact, it appears that the differences between type I and type II cone genes may simply depend on which repressor—Nr2e3 or transcription factor X—is primarily responsible for the regulation of the gene in question. In the case of type I genes, Nr2e3 is the primary repressor and transcription factor X is of secondary importance. In the case of type II genes, transcription factor X exerts the major repressive effect on transcription, and Nr2e3 plays a lesser, but still important role. + +In contrast to the marked derepression of the vast majority of cone-specific genes in the rd7 mutant, two genes stand out as being unaffected by the mutation: the gene encoding M-opsin and Thrb2. As Thrb2 is known to be required for the expression of M-opsin [22], the absence of supernumerary M-opsin–positive cells may simply be a consequence of the fact that Thrb2 expression is unchanged in the rd7 mutant. Further support for this idea has been provided by recent work in our lab showing widespread derepression of cone genes in the Notch1−/− retina (unpublished data). In contrast to the rd7 mutant, Notch1−/− retinas show marked derepression of Thrb2 and a corresponding derepression of the gene that encodes M-opsin. An additional observation suggesting that M-opsin and S-opsin are controlled by different mechanisms comes from in vitro experiments [31,32]. While explanted P3 retinas express S-opsin and M-opsin with normal kinetics, explanted P0 retinas express only S-opsin [32]. The factor(s) controlling these differences are unknown, but may be intrinsic, as cocultures of older and younger retinas, conditioned media from older retinas, and addition of a variety of small molecules were unable to promote the expression of M-opsin in the P0-initiated cultures [32]. + +In contrast to our findings, Peng et al. [19] reported that M-opsin expression is mildly increased in the rd7 mutant retina. It is possible that a subtle increase in M-opsin transcript levels does occur in the rd7 retina, and that this difference could not be detected by in situ hybridization. Since virtually all M-opsin–expressing cells are localized at the outer edge of the ONL by P28 in the rd7 mutant (Figure S8), any increase in M-opsin transcript in the mutant must have occurred in cells in that location. + +A variety of novel cone-specific or cone-enriched genes were characterized in this study. One of these genes, Pygm, is involved in glycogen/glucose metabolism, and a second, Glo1, is required for detoxification of methylglyoxal, a byproduct of glycolysis [33]. A third gene involved in glucose metabolism, hexokinase 2 (Hk2), is also derepressed in the rd7 mutant and shows a pattern of expression in the wild-type retina, suggesting greater expression in cones than in rods (see Figure 1; Table S1). A fourth gene involved in glucose metabolism, glucokinase regulatory protein (Gckr), was found to be increased in three out of three microarrays at P21 but was not tested by in situ hybridization (Table S4). The increased expression of Gckr in rd7 mutant retina suggests that it too may be a cone-enriched gene. A previous study found that two of these genes, Pygm and Hk2, have markedly elevated tag levels in an ONL-specific serial analysis of gene expression library consistent with their being highly enriched in wild-type photoreceptors [34]. Furthermore, prior studies have suggested differences in glycogen and glucose metabolism between primate rods and cones [35]. Our findings lend further support to this concept. Interestingly, Pygm has been implicated in human disease. Mutations in this gene underlie McArdle's disease (glycogen storage disease type V), the symptoms of which include exercise intolerance, muscle cramps, and myoglobinuria [36]. To our knowledge, no abnormalities of retinal function have been reported. + +One of the most curious findings in the rd7 mutant retina was the occurrence of two different types of changes: an increase in the number of S-opsin–expressing cones and a transformation of rods into hybrid photoreceptors. It is known that Nr2e3 is expressed only in rods, and the transcript is first detectable in postmitotic cells (J. Trimarchi and CLC, unpublished data). Assuming that Nr2e3 acts cell autonomously, we can conclude that the supernumerary S-cone–positive cells and the hybrid photoreceptors identified in the rd7 retina were redirected to these fates from postmitotic cells that were destined to become rods. This conclusion raises this question: Why does loss of a single transcription factor within rod precursors lead to two alternative fates—a hybrid cell type on the one hand and apparently normal S-cones on the other? There are at least two possible explanations for these differences. + +First, it is possible that there are two distinct types of rod precursor; loss of Nr2e3 in one leads to S-cone fate and in the other results in a hybrid cell type. In fact, there is experimental evidence from the rat to support the conclusion that early-born and late-born rods are intrinsically different [37]. One test of the hypothesis that there are two temporally distinct rod precursor populations would be to carry out birthdating experiments to determine whether the supernumerary S-opsin–positive cells in the rd7 retina derived exclusively from an early- or late-born population. Of course, if this were not the case, this experiment could not rule out the possibility that molecularly distinct populations of rod precursors are present simultaneously in the developing retina. + +An alternative explanation would be that there is only a single, homogeneous population of postmitotic rod precursors in the mouse, and a stochastic event triggers assumption of the S-cone fate in a small subpopulation of these cells in the rd7 mutant. Recent studies in a variety of experiment systems suggest that such a stochastic, all-or-none mechanism of gene activation is commonplace [38–44]. In this scenario, the absence of Nr2e3 would alter the probability that an unknown master control gene is expressed in rod precursors. Once this event takes place, it would initiate an irreversible program of differentiation toward S-cone fate, albeit at a relatively low frequency. In this way, a subset of cells from an initially homogeneous population would select the S-cone fate in an entirely probabilistic manner. + +Human patients with ESCS display three types of abnormality attributable to the retina: (1) an atypical ERG waveform that is preferentially sensitive to short-wavelength light, (2) slowly progressive retinal degeneration, and (3) abnormal retinal lamination with rosette formation [1,12,13]. The rd7 mutant mice also demonstrate the latter two defects, but have a normal ERG [15,45]. These similarities and differences between the two species help to explain the possible mechanistic basis of the ESCS. + +The fact that the rd7 mouse has a normal ERG strongly suggests that the aberrant ERG in ESCS is not attributable to the activity of a hybrid photoreceptor identical to that found in this study. Namely, the signal is unlikely to derive from a population of cells coexpressing both rod and cone genes but whose photopigment is rhodopsin and not S-cone opsin. This conclusion is consistent with the evidence from human ESCS patients indicating a markedly reduced rod system and a lack of measurable rhodopsin by reflection densitometry [1,2,10,11]. It is also unlikely that we would fail to detect an ESCS-like ERG signal in mice if it were present, as such a signal has been demonstrated in the Nrl mutant mouse, which has a near total transformation of all its rods into blue cones [24]. + +These findings, however, do not rule out the possibility that the abnormal human ERG derives from a hybrid photoreceptor cell type that also expresses S-opsin. It is possible that there are gene regulatory differences between mice and humans such that in human NR2E3 mutants, S-opsin shows a type I pattern of derepression rather than a type II as in seen in the rd7 mouse, and is therefore expressed in all of the hybrid photoreceptor cells. Alternatively, the ratio of supernumerary S-cones to hybrid photoreceptors produced in the retina of ESCS patients might be such that a higher percentage of the presumptive rods in ESCS patients become S-cones rather than hybrid photoreceptors. As discussed above, this ratio could depend either on the relative percentages of two distinct rod precursor populations or on stochastic effects on regulatory gene expression. + +In contrast to the ERG differences between mouse rd7 and human NR2E3 mutants, both species demonstrate slow retinal degeneration. It is possible that this degeneration is attributable to the abnormal function of the hybrid photoreceptor cell type characterized in the present study. The coexpression of both rod and cone genes in the same cell could predispose the cell to apoptosis. + +The final common feature between mouse rd7 and human NR2E3 mutants is the presence of an abnormally laminated retina with waviness and rosette formation in the ONL [12–15]. The cause of this abnormality is not known, but it is possibly related to defects in photoreceptor cell polarity in the rd7 mutant. Rosette formation and abnormally wavy epithelia are common sequelae of defects in pathways controlling cell polarity [46,47]. In particular, loss-of-function mutations in the polarity gene crumbs (CRB1) have been shown to cause morphological abnormalities of the ONL in both humans and mice, including rosette formation in mice very similar to that seen in the rd7 mutant [48,49]. Interestingly, Sharon et al. [5] have recently pointed out additional features shared by patients with CRB1 mutations and mutations in NR2E3, including hyperopic refractive errors and a distinctive pattern of clumped pigmentation in the retina. + +In the present study we found the mouse crumbs ortholog to be up-regulated in the rd7 mutant retina by microarray, consistent with its higher expression level in cones than in rods [50]. Although we were unable to confirm this finding by in situ hybridization due to the weakness of the signal, it is possible that the up-regulation of crumbs in the retina is the cause of the lamination defects seen in the rd7 mutant. Overexpression of wild-type crumbs in Drosophila has been shown to cause polarity defects leading to waviness of epithelia and even to misalignment of nuclei within photoreceptors analogous to what is seen in the rd7 retina [47,51]. Future experiments will address this question by overexpressing full-length Crb1 in a wild-type background. + +One further point worthy of note is the striking similarity between the hybrid photoreceptor identified in this study and a naturally occurring photoreceptor found in ground squirrels. The “rods” of this species have electrophysiologic, molecular, and ultrastructural features of both rods and cones [52–58]. Although these unusual findings have been difficult to interpret under the usual assumptions of “duplicity theory” [56], we would like to suggest that ground squirrels may have experienced a naturally occurring down-regulation or loss of Nr2e3 expression in their “rods” that transformed them into a hybrid photoreceptor cell type. The adaptive significance of such a change, if any, is unknown, and it may simply be due to relaxation of selective pressure for night vision in this strictly diurnal species. + +Materials and Methods + +Mutant mice. + +Nr2e3rd7 mutant mice were obtained from Jackson Laboratories (Bar Harbor, Maine, United States; stock #004643) and maintained on a C57BL/6 background. All control mice were C57BL/6. + +Microarray analysis. + +Total retinal RNA samples were isolated from P0, P6, P14, and P21 Nr2e3 mutant mice using the Trizol reagent (Gibco, San Diego, California. United States). Total retinal RNA samples from age-matched wild-type C57BL/6 mice were used as controls. Individual total RNA samples were derived from four retinas (pooled from two animals). All microarray experiments were performed in triplicate, in each case with separate RNA preparations. Microarray experiments with cDNAs were performed with the P0, P6, and P14 derived samples. Probes were labeled with either Cy3 or Cy5 using the Array 900 kit from Genisphere (Hatfield, Pennsylvania, United States) starting with 5 μg of total RNA according to the manufacturer's instructions. Wild-type control probes were compared to mutant on the same microarray. In two of the three replicates, the mutant probe was labeled with Cy3 and the wild type with Cy5, and in the third replicate the dyes were swapped. Labeled probe was hybridized to microarray slides spotted with approximately 11,500 cDNA clones from the brain molecular anatomy project library (kind gift of B. Soares, University of Iowa; see http://trans.nih.gov/bmap/index.htm for details) and 500 cDNA clones from our lab collection. Slides were printed and hybridized as described [59,60]. After hybridization and washing of slides according to the manufacturer's instructions (Genisphere), the slides were scanned on an Axon Instruments (Union City, California, United States) GenePix 4000 scanner and images were analyzed using the accompanying GenePix Pro software package. The complete raw cDNA microarray data set are available in Tables S6–S14. + +Two types of normalization were performed on the raw intensity scores derived from the GenePix Pro analysis. First, for a given experiment, the average intensity of all the spots in the weaker of the two channels (Cy3 or Cy5) was normalized to those in the stronger channel. Second, in a given set of experiments done in triplicate at a particular time point, the two experiments with the weaker average signal intensity over all spots were normalized to those in the third microarray with the strongest average signal intensity. All spots with signal levels equal to or below background were removed from the analysis. The resulting files contained on average about 6,000 spots. These files were then sorted according to Cy3/Cy5 signal intensity, and those spots with the 10% highest and 10% lowest intensity ratios (approximately 600 spots/experiment) were compared across the three experiments at a given time point using custom Perl scripts (available upon request from JCC). All spots which were present in the top 10% most up- or down-regulated genes in two out of three or three out of three experiments were recorded (the latter are listed in Table S3). + +Microarray analysis of the P21 retinas was performed on Affymetrix mouse genome 430 2.0 GeneChip array (Affymetrix, Santa Clara, California, United States). A total of six microarray hybridizations were performed: three with probes derived from mutant RNA and three from wild-type. Probes were synthesized starting with 10 μg of total RNA for each sample according to manufacturer's instructions (Affymetrix). Hybridization, washing, and scanning of the microarrays were all performed at the Bauer Center for Genomics Research at Harvard University according to manufacturer's instructions (Affymetrix). Initial data analysis was carried out using the GeneChip Operating System (GCOS) software from Affymetrix. Pairwise comparisons were made between individual mutant microarray results and controls. All genes were removed from the analysis for which “absent” calls were made by the software for both the wild-type and mutant samples being compared. The remaining gene lists contained approximately 26,000 transcripts. These lists were then sorted according to the mutant-to-wild-type “signal log ratio” in order to identify the most markedly up- and down-regulated genes. The top 500 most up- and down-regulated transcripts (approximately 2% of all genes in each case) from each of the three pairwise comparisons between mutant and wild-type were compared using custom Perl scripts (available upon request from JCC) to identify those genes that were present in two or three out of three lists. Those genes that were up- or down-regulated in three out of three experiments were recorded (Tables S4 and S5). The complete pairwise Affymetrix microarray datasets are available in Tables S15–S17. + +In Situ hybridization. + +Section in situ hybridization was performed as previously described [61] using 20-μm cryosections from OCT-embedded tissue or 4-μm paraffin sections. All in situ hybridizations were performed with the mutant and wild-type control sections on the same glass slide. Riboprobes labeled with digoxygenin-tagged UTP (Roche, Basel, Switzerland) were detected with NBT/BCIP (Sigma, St. Louis, Missouri, United States). The sources of the individual riboprobes used in this study are described in Tables S1 and S2. Dissociated cell in situ hybridization was performed as described previously [62] using the same S-opsin digoxygenin-labeled probe used for section in situ hybridization. All images were captured on a compound microscope (Eclipse e1000; Nikon, Tokyo, Japan) using a CCD camera (DXM1200F, Nikon). S-opsin positive cells were quantitated on dissociated cell in situ hybridization as previously described [62]. Twenty fields were quantitated in this manner at 200× magnification for both rd7 and wild-type retinas. + +Immunohistochemistry. + +For antibody staining, cryosections were prepared and stained as described previously [63]. Primary antibodies used were a polyclonal anti-blue opsin raised in rabbit (1:300; Chemicon International, Temecula, California, United States; AB5407) and a mouse monoclonal anti-rhodopsin (1:200; rho4D2). Secondary antibodies used were Cy2- or Cy3-conjugated goat anti-rabbit and anti-mouse (1:500; Jackson Immunologicals, West Grove, Pennsylvania, United States). Following antibody staining, 4′-DAPI was applied to stain nuclei (Sigma), and the sections were coverslipped and mounted in Gel/Mount (Biomeda, Foster City, California, United States). + +Electron microscopy. + +This protocol was adapted from one used by Raviola [64] with some modifications derived from Carter-Dawson and Lavail [26]. Four adult wild-type and four mutant animals were deeply anesthetized by intraperitoneal injection of Avertin and the eyes were then removed. The cornea was gently punctured with sharp forceps and excised with iridectomy scissors. The eye was then transferred to a solution of 2% paraformaldehyde/2% glutaraldehyde in cacodylate buffer (0.1 M cacodylic acid; 0.1% calcium chloride). The lens was gently removed and the eyecup allowed to fix for 2 h at room temperature. The fixed eye was then placed on dental wax and sectioned in the midline with a fresh razor blade (half of the retinas were sectioned along the D-V axis and half along the nasal-temporal axis). The retinas (and attached retinal pigment epithelium) were carefully dissected away from the sclera, which was discarded. + +The retinas were then rinsed four times for 15 min each with Sorenson's buffer (pH 7.4). They were then stained for 2 h at 4 °C with 1% osmium tetroxide in 1.5% potassium ferrocyanide. Next, the retinas were rinsed four times for 15 min in maleate buffer (pH 5.1) and then stained for 2 h at room temperature with 1% uranyl acetate in maleate buffer (pH 6.2). They were then washed four times for 15 min with maleate buffer (pH 5.1); once for 10 min with 70% ethanol; once for 10 min with 95% ethanol; and four times for 30 min with 100% ethanol. Next, the retinas were washed three times over one hour with propylene oxide and then embedded in TAAB 812 Resin (Marivac, Quebec, Canada) for 1–2 d in a 60 °C oven. Semi-thin sections were cut at a thickness of 0.5 μm and stained with 1% toluidine blue in 1% sodium borate buffer. Images of semi-thin sections from the mutant retina were taken within the ventral two-thirds of the retina where the majority of the supernumerary S-opsin–expressing cells reside. Wild-type images were taken in comparable regions. Sections for electron microscopy were cut at a thickness of 95 nm, placed on grids, and poststained with 2% uranyl acetate followed by 0.2% lead citrate. All sections were cut in a plane perpendicular to the plane of the photoreceptor layer. They were then visualized on a Jeol 1200EX electron microscope (Jeol, Tokyo, Japan). The electron microscopic images in Figure 7 derive from the ventral two-thirds of the wild-type and mutant retinas. + +The area of the cell bodies of the rods and “rod-like” cells in the wild-type and mutant ONLs, respectively, were quantitated in the following manner. First, ten fields within the ONL were chosen at random at 1,000× magnification and then photographed at 4,000× magnification for each of the two genotypes. Such images typically contained 35–45 cell bodies. In order to quantitate only the area of those cell bodies that were cut as near to the midline of the cell as possible (i.e., in order to obtain the maximal cross-sectional area) the five cells with the largest apparent area in each photograph were chosen by eye and the outline of the cell membranes were traced onto white paper. These tracings were scanned along with the size bar from the electron micrographs, and the areas of the resulting digital images were quantitated using Scion Image software (NIH Image, http://rsb.info.nih.gov/nih-image). A total of 50 cells of each genotype were quantitated in this manner. In addition, in order to evaluate the percentage of rods in the wild-type retina that had any juxtanuclear mitochondria, all rods within all ten images were counted as were the number of cells showing juxtanuclear organelles. A total of six out of 399 wild-type rods (1.5%) possessed a juxtanuclear organelle. This analysis permitted us to evaluate the wild-type rods for juxtanuclear mitochondria at multiple planes of section; however, serial sections of individual cell bodies were not performed. + +Supporting Information + +Figures S1–S7 show the in situ hybridization images of all genes discussed in the paper (see Tables S1 and S2). All paired images (which show the wild-type control on the left and the rd7 mutant retina on the right) are labeled in the lower left-hand corner with the gene symbol followed by the age of the retinas in question (P6, P14, P28, or adult). Unpaired images represent prenatal time points and are labeled with the gene symbol of the gene in question (“wt” indicates that the retina is from a wild-type animal) and a designation of the embryonic day from which the retina derives (e.g., e17.5 = embryonic day 17.5). + +Figure S1 + +In Situ Hybridization Images for G1–G13 in Table S1 + +(3.1 MB JPG) + +Click here for additional data file. + +Figure S2 + +In Situ Hybridization Images for G14–G25 in Table S1 + +(2.4 MB JPG) + +Click here for additional data file. + +Figure S3 + +In Situ Hybridization Images for G26–G35 in Table S1 + +(2.1 MB JPG) + +Click here for additional data file. + +Figure S4 + +In Situ Hybridization Images for G36–G46 in Table S1 + +(2.0 MB JPG) + +Click here for additional data file. + +Figure S5 + +In Situ Hybridization Images for G47–G53 in Table S1 + +(1.7 MB JPG) + +Click here for additional data file. + +Figure S6 + +In Situ Hybridization Images for Genes 1–11 in Table S2 + +(2.9 MB JPG) + +Click here for additional data file. + +Figure S7 + +In Situ Hybridization Images for Genes 12–22 in Table S2 + +(3.2 MB JPG) + +Click here for additional data file. + +Figure S8 + +In Situ Hybridization Results for M-Opsin and Thrb2 + +Note that the M-opsin–positive cells are scattered throughout the ONL at P14, but appear to have migrated to the scleral edge of the ONL by P28. There is no change in the number of M-opsin– or Thrb2-positive cells in the rd7 mutant. + +(2.1 MB PDF) + +Click here for additional data file. + +Table S1 + +Cone-Specific and Cone-Enriched Genes Evaluated in the rd7 Mutant by Microarray and In Situ Hybridization + +This table is a supplemental version of Figure 1. “Figure Number” indicates which figure (Figure S1–S5) contains the in situ hybridization images corresponding to the gene in question. “Lab Clone Information” indicates the region of the gene in question from which the probe used for in situ hybridization was derived. All abbreviations are as indicated in Figure 1. + +(26 KB XLS) + +Click here for additional data file. + +Table S2 + +Rod Genes Evaluated in the rd7 Mutant by In Situ Hybridization + +This table contains details about the in situ hybridization patterns of 22 genes (many of which are rod-specific) evaluated in the rd7 mutant retina. “Figure Number” indicates which figure (Figure S6 or S7) contains the in situ hybridization images corresponding to the gene in question. “Lab Clone Information” indicates the region of the gene in question from which the probe used for in situ hybridization was derived. The color coding of the in situ hybridization results under “In Situ Pattern” is as follows: dark green, markedly down-regulated; light green, mildly down-regulated; red, markedly up-regulated; orange, mildly up-regulated. + +(20 KB XLS) + +Click here for additional data file. + +Table S3 + +Summary of cDNA Microarray Results from P0, P6, and P14 + +The spots listed in this table represent those that were either up- or down-regulated in three out of three microarray experiments as described in Materials and Methods. The Cy3/Cy5 signal ratios are indicated for all three microarray experiments at each time point. Note that the Cy3/Cy5 ratios for “Microarray #3” are reversed relative to the other two, since the fluorescent tag used to label wild-type and mutant RNA was swapped as described in Materials and Methods. + +(22 KB XLS) + +Click here for additional data file. + +Table S4 + +Summary of Genes Up-Regulated in rd7 Mutant Retina at P21 by Affymetrix Microarray + +Only genes that were found to be up-regulated in three out of three microarray experiments (as described in Materials and Methods) are listed. “Nr2e3 signal” and “C57BL/6 signal” represent the average signal for that transcript in all three microarray experiments. + +(92 KB XLS) + +Click here for additional data file. + +Table S5 + +Summary of Genes Down-Regulated in rd7 Mutant Retina at P21 by Affymetrix Microarray + +Only genes that were found to be down-regulated in three out of three microarray experiments (as described in Materials and Methods) are listed. “Nr2e3 signal” and “C57BL/6 signal” represent the average signal for that transcript in all three microarray experiments. + +(58 KB XLS) + +Click here for additional data file. + +Table S6 + +Raw cDNA Microarray Data for rd7 versus Wild-Type Comparison at P0 (I) + +(5.6 MB XLS) + +Click here for additional data file. + +Table S7 + +Raw cDNA Microarray Data for rd7 versus Wild-Type Comparison at P0 (II) + +(5.6 MB XLS) + +Click here for additional data file. + +Table S8 + +Raw cDNA Microarray Data for rd7 versus Wild-Type Comparison at P0 (III) + +(5.6 MB XLS) + +Click here for additional data file. + +Table S9 + +Raw cDNA Microarray Data for rd7 versus Wild-Type Comparison at P6 (I) + +(5.6 MB XLS) + +Click here for additional data file. + +Table S10 + +Raw cDNA Microarray Data for rd7 versus Wild-Type Comparison at P6 (II) + +(5.6 MB XLS) + +Click here for additional data file. + +Table S11 + +Raw cDNA Microarray Data for rd7 versus Wild-Type Comparison at P6 (III) + +(5.6 MB XLS) + +Click here for additional data file. + +Table S12 + +Raw cDNA Microarray Data for rd7 versus Wild-Type Comparison at P14 (I) + +(5.5 MB XLS) + +Click here for additional data file. + +Table S13 + +Raw cDNA Microarray Data for rd7 versus Wild-Type Comparison at P14 (II) + +(5.6 MB XLS) + +Click here for additional data file. + +Table S14 + +Raw cDNA Microarray Data for rd7 versus Wild-Type Comparison at P14 (III) + +(5.6 MB XLS) + +Click here for additional data file. + +Table S15 + +Raw Affymetrix Microarray Data for rd7 versus Wild-Type Comparison at P21 (I) + +(18 MB XLS) + +Click here for additional data file. + +Table S16 + +Raw Affymetrix Microarray Data for rd7 versus Wild-Type Comparison at P21 (II) + +(18 MB XLS) + +Click here for additional data file. + +Table S17 + +Raw Affymetrix Microarray Data for rd7 versus Wild-Type Comparison at P21 (III) + +(18 MB XLS) + +Click here for additional data file. + +Acknowledgements + +We are grateful to E. Raviola and T. Reese for help with electron microscopy and to A. Jadhav and J. Trimarchi for access to unpublished data and reagents. Thanks to A. Jadhav, J. Trimarchi, D. Kim, and T. Cherry for helpful comments on the manuscript. This work was supported by the Howard Hughes Medical Institute and grants from the National Institutes of Health (EY014822 to JCC and EY009676 to CLC). Thanks to A. Swaroop for providing us with Nrl mutant mice. + +Abbreviations + +DAPI - 6-diamidino-2-phenylindole + +E[number] - embryonic day [number] + +ERG - electroretinogram + +ESCS - enhanced S-cone syndrome + +GCL - ganglion cell layer + +INL - inner nuclear layer + +ONL - outer nuclear layer + +P[number] - postnatal day [number] + +Figures + +Figure 1 + +Cone-Specific and Cone-Enriched Genes Evaluated in the rd7 Mutant by Microarray and In Situ Hybridization + +The color coding of text in the column “Gene Name” is as follows: light blue (G1–G15), genes previously reported in the literature to have cone-specific or cone-enriched patterns of expression; yellow (G16–G25), novel cone genes identified in an unrelated study (unpublished data); dark green (G26–G36), novel cone genes identified in the present study that were up-regulated in rd7; light green (G37–G46), additional genes found to be up-regulated in rd7 by microarray in the present study but that had either weak or inapparent cone-specific signal on in situ hybridization; white (G47–G53), additional genes up-regulated by microarray at two different timepoints but with either unusual expression patterns or nonconfirmatory in situ hybridizations. The column “ID” contains identifiers used in the present paper to refer to specific genes. “GenBank ID” contains the GenBank accession number of the clone used to make the probe for in situ hybridization. Within this column, “lab clone” indicates that the probe used for in situ hybridization derived from a clone in our laboratory. The region of the gene to which it corresponds is indicated in Table S1. Columns “P0” through “P21” contain the results of microarray experiments at the given postnatal dates. P0, P6, and P14 time points represent analyses on cDNA microarrays; the P21 time point represents data from an Affymetrix microarray (mouse genome 432 2.0). A red cell with a single up arrow indicates that the gene in question was up-regulated in three out of three microarrays at that time point (as described in Materials and Methods). Those cells labeled orange with a single up arrow and asterisk indicate that the gene in question was up-regulated in two out of three microarrays at that time point. The column “In Situ” lists the type of derepression seen for the gene in question in the rd7 mutant retina (type I and type II are described in the main text). Genes designated “unclassified” represent patterns of derepression that were difficult to classify as either type I or type II (see main text for more details). “Wild type” in this column indicates that the in situ hybridization pattern in the rd7 mutant retina was not different from the wild-type pattern; and “special” indicates a special pattern of expression discussed more fully in the main text. The column “Expression Pattern” contains a concise description of the wild-type expression pattern of the gene in question. In the case of genes for which no signal was obtained on in situ hybridization in the present study, the specified expression pattern derives from reports in the literature. Within this column, “cone > rod” indicates that the gene is expressed in all photoreceptors, but at higher levels in cones than rods; “cone?” indicates very weak staining in a cone-like distribution. + +BP, bipolar cells; EP, early photoreceptor expression pattern; IS, inner core segment localization; MG, Müller glia; N/A, not available on the microarray; NS, no signal detected on in situ hybridization; RPE, retinal pigment epithelium. + +Figure 2 + +Cone and Rod Gene Expression in the rd7 Mutant at P14 + +The upper sets of photomicrographs demonstrate examples of type I and type II cone gene derepression in the rd7 mutant retina as explained in the main text. The bottom left images show several rod-specific genes that are essentially unchanged in the rd7 background at P14. The bottom right images show the expression pattern of three photoreceptor transcription factors in the rd7 mutant. Abbreviations in the lower left hand corner of each pair of panels represent the gene symbols summarized in Figure 1. + +Figure 3 + +S-Opsin Dissociated Cell In Situ Hybridization and S-Opsin/Rhodopsin Antibody Staining on rd7 Mutant Retina + +(A–C) A dissociated cell in situ hybridization with an S-opsin probe (red) on dissociated rd7 mutant retinal cells stained with DAPI (blue). (C) shows the merged images. + +(D–F) The outer nuclear layer of an rd7 mutant retina stained by antibody for S-opsin (red) and rhodopsin (green). The scleral edge of the outer nuclear layer is up. DAPI staining is in blue. (F) shows the merged images. Insets are higher-power images of the outer segments showing non-overlap of S-opsin and rhodopsin staining in the mutant. + +Figure 4 + +Expression Patterns of Several Novel Cone Genes Up-Regulated in rd7 + +In the wild-type images (wt), note the scattered, weakly positive cells at the scleral edge of the outer nuclear layer in a cone distribution. All of the genes show marked up-regulation in the rd7 mutant. Bub1b and Tcta show transcript localization predominantly to the inner segment of the photoreceptors. Retinas are oriented such that the scleral edge is up. + +Figure 5 + +Some of the Genes Up-Regulated in rd7 Show an Early Photoreceptor Pattern of Expression + +Gnb3 and Thrb2 are both previously characterized cone genes that show staining at the scleral edge of the embryonic mouse retina in cells that will differentiate into photoreceptors. Ece1 and Otop3 are two novel cone genes identified in this study that were up-regulated in rd7 and also showed an early photoreceptor pattern of expression. Prdm1 and RIKEN cDNA 1300018I05 (Figure 1, genes G48 and G49, respectively) are two other genes that had either undetectable signal (Prdm1) or no apparent change in expression pattern in adult rd7 mutants (RIKEN cDNA 1300018I05), but which also showed staining in the embryonic retina in a presumptive photoreceptor pattern. All images are from E17.5 retina except Gnb3, which was from E16. + +Figure 6 + +The Onset of rhodopsin Expression Is Delayed in the rd7 Mutant Retina + +Note the nearly undetectable staining for rhodopsin in this P6 mutant retina (top right). The majority of rod-specific genes did not show this delay in expression onset, as indicated by the normal amount of staining for Pde6a in the mutant at P6 (bottom images). + +Figure 7 + +The rd7 Mutant Retina Contains a Morphologically Hybrid Photoreceptor Cell Type in Addition to Supernumerary S-Opsin–Positive Cones + +(A and B) Toluidine blue-stained semi-thin sections of the outer nuclear layer (scleral edge oriented up). + +(C and D) Hand-drawn diagrams of the cells in (A) and (B), respectively. Cells with the nuclear features of cones are highlighted in blue. Note that the number of such cells is greater in the mutant, and their cell bodies are scattered throughout the outer nuclear layer. In addition, the overall columnar architecture of the outer nuclear layer seen in the wild type is disrupted in this portion of the mutant retina. Other portions of the mutant retina with fewer supernumerary cone cells, however, retain the normal columnar appearance (unpublished data). + +(E and F) Images of the outer nuclear layer (scleral edge up) stained by in situ hybridization for S-opsin. Note the typical pattern of staining at the scleral edge of the outer nuclear layer in the wild type. The rd7 mutant retina shows supernumerary S-opsin–positive cells scattered throughout the outer nuclear layer in a distribution very similar to the supernumerary cone cells seen in (B). Since images (E) and (F) derive from different retinas than those depicted in (A) and (B), the location of the individual cells do not correspond. + +(G and H) Electron micrographs of the outer nuclear layer (10,000× magnification). Note the uniform distribution of rod cell bodies in the wild type (G). The cell bodies are nearly round and consist almost exclusively of a nucleus with a single, dense mass of heterochromatin. In the rd7 mutant (H), two types of cell are shown. The ovoid one with a lesser quantity of heterochromatin, paler euchromatin, and two juxtanuclear mitochondria (yellow arrow) represents a typical cone cell body. The adjacent cell with a more “rod-like” mass of heterochromatin and a single juxtanuclear mitochondrion (red arrow) represents one of the hybrid photoreceptors discussed more fully in the main text. + +Figure 8 + +A Partial View of the Rod Photoreceptor Transcriptional Regulatory Network + +Note that green lines indicate activation, and yellow and red lines indicate weak and strong repression, respectively. The dotted lines associated with a question mark indicate that it is not known whether Nrl directly represses the target genes in question or whether its repression is mediated by a downstream transcription factor (“X”). Note that Nr2e3 appears to negatively regulate its own transcription. The regulatory linkages depicted in this diagram are not necessarily direct. The weak activation of some rod-specific genes by Nr2e3 is omitted from this diagram for clarity. Also not shown is the role of other photoreceptor transcription factors, such as Crx. + +Footnotes + +Competing interests. The authors have declared that no competing interests exist. + +Author contributions. JCC and CLC conceived and designed the experiments. JCC performed the experiments. JCC and CLC analyzed the data. JCC contributed reagents/materials/analysis tools. JCC and CLC wrote the paper. + +Citation: Corbo JC, Cepko CL (2005) A hybrid photoreceptor expressing both rod and cone genes in a mouse model of enhanced s-cone syndrome. PLoS Genet 1(2): e11. diff --git a/src/ontogpt/evaluation/craft/database/all/16121255.ann b/src/ontogpt/evaluation/craft/database/all/16121255.ann new file mode 100644 index 000000000..26e6baeae --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16121255.ann @@ -0,0 +1,1060 @@ +T1 http://purl.obolibrary.org/obo/MONDO_0004782 0 18 Diabetes Insipidus +T2 NCBITaxon:10088 22 26 Mice +T3 PR:000004182 46 57 Aquaporin-2 +T4 http://purl.obolibrary.org/obo/MONDO_0021140 70 80 Congenital +T5 GO:0001822 81 92 nephrogenic +T6 http://purl.obolibrary.org/obo/MONDO_0016383 81 111 nephrogenic diabetes insipidus +T7 http://purl.obolibrary.org/obo/MONDO_0016383 113 116 NDI +T8 http://purl.obolibrary.org/obo/MONDO_0000001 123 130 disease +T9 UBERON:0002113 163 169 kidney +T10 UBERON:0001088 185 190 urine +T11 NCBITaxon:9606 219 224 Human +T12 GO:0001822 239 250 nephrogenic +T13 http://purl.obolibrary.org/obo/MONDO_0016383 239 269 nephrogenic diabetes insipidus +T14 PR:000001683 313 335 vasopressin receptor 2 +T15 PR:000001683 337 342 Avpr2 +T16 SO:0000704 344 348 gene +T17 CHEBI:15377 378 383 water +T18 PR:000004182 392 403 aquaporin-2 +T19 PR:000004182 405 409 Aqp2 +T20 SO:0000704 411 415 gene +T21 NCBITaxon:33208 494 500 animal +T22 SO:0000704 524 531 genetic +T23 CHEBI:23995 545 561 ethylnitrosourea +T24 NCBITaxon:10088 574 578 mice +T25 NCBITaxon:10088 635 640 mouse +T26 http://purl.obolibrary.org/obo/MONDO_0016383 650 653 NDI +T27 PR:000004182 685 689 Aqp2 +T28 SO:0000704 690 694 gene +T29 NCBITaxon:39107 724 730 murine +T30 http://purl.obolibrary.org/obo/MONDO_0016383 741 744 NDI +T31 NCBITaxon:10088 750 754 mice +T32 UBERON:0000113 766 775 adulthood +T33 NCBITaxon:9606 810 815 human +T34 http://purl.obolibrary.org/obo/MONDO_0000001 816 824 disorder +T35 UBERON:0002113 862 867 renal +T36 CL:1000497 862 872 renal cell +T37 PR:000004182 897 901 Aqp2 +T38 CHEBI:15377 957 962 water +T39 NCBITaxon:33208 981 988 animals +T40 PR:000004182 1042 1046 AQP2 +T41 GO:0001822 1088 1099 nephrogenic +T42 http://purl.obolibrary.org/obo/MONDO_0016383 1088 1118 nephrogenic diabetes insipidus +T43 NCBITaxon:1 1133 1141 organism +T44 UBERON:0002113 1165 1170 renal +T45 CL:1000497 1165 1175 renal cell +T46 PR:000004182 1215 1219 AQP2 +T47 GO:0005783 1246 1267 endoplasmic reticulum +T48 NCBITaxon:10088 1352 1357 mouse +T49 CHEBI:52217 1425 1440 pharmacological +T50 SO:0000704 1445 1449 gene +T51 http://purl.obolibrary.org/obo/MONDO_0016383 1464 1467 NDI +T52 GO:0001822 1482 1493 Nephrogenic +T53 http://purl.obolibrary.org/obo/MONDO_0016383 1482 1512 Nephrogenic diabetes insipidus +T54 http://purl.obolibrary.org/obo/MONDO_0016383 1514 1517 NDI +T55 http://purl.obolibrary.org/obo/MONDO_0000001 1524 1531 disease +T56 GO:0060073 1552 1561 urination +T57 UBERON:0001898 1588 1600 hypothalamus +T58 CHEBI:15377 1625 1630 water +T59 UBERON:0002113 1661 1667 kidney +T60 CHEBI:15377 1680 1685 water +T61 UBERON:0001088 1704 1709 urine +T62 CHEBI:62488 1715 1733 signaling molecule +T63 GO:0046903 1734 1742 secreted +T64 UBERON:0001898 1750 1762 hypothalamus +T65 GO:0009986 1835 1845;1853 1858 surface of ... cells +T66 UBERON:0002113 1846 1852 kidney +T67 CL:1000497 1846 1858 kidney cells +T68 PR:000001683 1860 1872 AVP receptor +T69 PR:000001683 1874 1879 AVPR2 +T70 UBERON:0002113 1913 1919 kidney +T71 CL:1000497 1913 1925 kidney cells +T72 PR:000004182 2019 2030 aquaporin 2 +T73 PR:000004182 2032 2036 AQP2 +T74 GO:0009986 2054 2064;2076 2080 surface of ... cell +T75 UBERON:0002113 2069 2075 kidney +T76 CL:1000497 2069 2080 kidney cell +T77 PR:000004182 2104 2108 AQP2 +T78 CHEBI:15377 2137 2142 water +T79 UBERON:0001088 2152 2159 urinary +T80 UBERON:0001088 2204 2209 urine +T81 CHEBI:15377 2225 2230 water +T82 http://purl.obolibrary.org/obo/MONDO_0021140 2232 2242 Congenital +T83 http://purl.obolibrary.org/obo/MONDO_0016383 2243 2246 NDI +T84 CHEBI:15377 2284 2289 water +T85 PR:000004182 2299 2303 AQP2 +T86 PR:000001683 2325 2330 AVPR2 +T87 NCBITaxon:33208 2409 2416 animals +T88 NCBITaxon:10088 2479 2484 mouse +T89 http://purl.obolibrary.org/obo/MONDO_0016383 2494 2497 NDI +T90 SO:0000704 2563 2568 genes +T91 http://purl.obolibrary.org/obo/MONDO_0000001 2597 2604 disease +T92 NCBITaxon:10088 2630 2635 mouse +T93 SO:0000704 2657 2664 genetic +T94 SO:0000704 2683 2690 genetic +T95 NCBITaxon:10088 2741 2746 mouse +T96 http://purl.obolibrary.org/obo/MONDO_0016383 2756 2759 NDI +T97 SO:0000704 2793 2800 genetic +T98 SO:0000704 2819 2824 genes +T99 NCBITaxon:33208 2851 2858 animals +T100 http://purl.obolibrary.org/obo/MONDO_0000001 2876 2883 disease +T101 SO:0000704 3003 3007 gene +T102 http://purl.obolibrary.org/obo/MONDO_0016383 3052 3055 NDI +T103 GO:0001822 3072 3083 Nephrogenic +T104 http://purl.obolibrary.org/obo/MONDO_0016383 3072 3102 Nephrogenic diabetes insipidus +T105 http://purl.obolibrary.org/obo/MONDO_0016383 3104 3107 NDI +T106 http://purl.obolibrary.org/obo/MONDO_0000001 3114 3121 disease +T107 GO:0060073 3149 3158 urination +T108 GO:0000805 3288 3289 X +T109 PR:000001683 3333 3338 Avpr2 +T110 SO:0000704 3339 3343 gene +T111 GO:0030849 3352 3361 autosomal +T112 PR:000004182 3385 3389 Aqp2 +T113 SO:0000704 3390 3394 gene +T114 PR:000004182 3400 3411 Aquaporin-2 +T115 PR:000004182 3413 3417 AQP2 +T116 CHEBI:15377 3470 3475 water +T117 GO:0010467 3500 3509 expressed +T118 UBERON:0001232 3513 3528 collecting-duct +T119 CL:1001431 3513 3544 collecting-duct principal cells +T120 UBERON:0002113 3552 3558 kidney +T121 GO:0006833 3600 3616 passage of water +T122 GO:0055085 3600 3607;3617 3624;3636 3644 passage ... through ... membrane +T123 CHEBI:15377 3611 3616 water +T124 GO:0005886 3629 3644 plasma membrane +T125 GO:0005886 3646 3648 PM +T126 CHEBI:15377 3728 3733 water +T127 GO:0070295 3728 3746;3758 3760;3765 3771 water reabsorption ... in ... kidney +T128 UBERON:0001088 3752 3757 urine +T129 UBERON:0002113 3765 3771 kidney +T130 GO:0051262 3848 3859 tetramerize +T131 GO:0005886 3892 3907 plasma membrane +T132 GO:0044459 3990 4000;4005 4007 regions of ... PM +T133 PR:000004182 4022 4026 AQP2 +T134 GO:0016324 4044 4068 apical membrane of cells +T135 UBERON:0001232 4085 4100 collecting duct +T136 PR:000004183 4128 4132 AQP3 +T137 GO:1990794 4161 4177 basolateral face +T138 PR:000004182 4212 4216 AQP2 +T139 GO:0005886 4257 4272 plasma membrane +T140 GO:0016324 4324 4330 apical +T141 GO:0005622 4331 4344 intracellular +T142 GO:0031982 4345 4353 vesicles +T143 CHEBI:15377 4391 4396 water +T144 PR:000004182 4407 4411 AQP2 +T145 GO:0016324 4432 4447 apical membrane +T146 CHEBI:15377 4460 4465 water +T147 PR:000001683 4537 4542 AVPR2 +T148 GO:1990794 4551 4570;4591 4596 basolateral face of ... cells +T149 UBERON:0001232 4575 4590 collecting duct +T150 CL:1001225 4575 4596 collecting duct cells +T151 GO:0005622 4619 4632 intracellular +T152 CHEBI:17489 4633 4637 cAMP +T153 PR:000004182 4682 4686 AQP2 +T154 CHEBI:17489 4704 4708 cAMP +T155 GO:0005886 4768 4783 plasma membrane +T156 PR:000004182 4804 4808 AQP2 +T157 PR:000004182 4879 4883 Aqp2 +T158 http://purl.obolibrary.org/obo/MONDO_0016383 4914 4917 NDI +T159 NCBITaxon:9606 4921 4927 humans +T160 PR:000004182 4946 4950 Aqp2 +T161 GO:0006457 5045 5051 folded +T162 CHEBI:15377 5052 5057 water +T163 CHEBI:17489 5106 5110 cAMP +T164 GO:0016323 5242 5262 basolateral membrane +T165 NCBITaxon:10088 5280 5285 mouse +T166 http://purl.obolibrary.org/obo/MONDO_0004782 5296 5314 diabetes insipidus +T167 NCBITaxon:9606 5374 5379 human +T168 http://purl.obolibrary.org/obo/MONDO_0016383 5380 5383 NDI +T169 NCBITaxon:10088 5385 5389 mice +T170 PR:000004182 5428 5432 Aqp2 +T171 PR:000001683 5437 5442 Avpr2 +T172 NCBITaxon:10088 5482 5487 mouse +T173 PR:000004182 5526 5530 Aqp2 +T174 SO:0000704 5531 5535 gene +T175 NCBITaxon:10088 5569 5573 mice +T176 GO:0016265 5574 5578 died +T177 GO:0007567 5596 5601 birth +T178 PR:000001683 5618 5623 AVPR2 +T179 GO:0016265 5649 5652 die +T180 GO:0007567 5681 5686 birth +T181 NCBITaxon:10088 5727 5732 mouse +T182 NCBITaxon:1 5759 5767 organism +T183 CHEBI:15377 5783 5788 water +T184 GO:0030104 5783 5800 water homeostasis +T185 SO:0000704 5856 5863 genetic +T186 NCBITaxon:10088 5874 5879 mouse +T187 PR:000004182 5888 5892 Aqp2 +T188 NCBITaxon:39107 5969 5975 murine +T189 GO:0001822 5995 6006 nephrogenic +T190 http://purl.obolibrary.org/obo/MONDO_0016383 5995 6009 nephrogenic DI +T191 PR:000004182 6055 6059 Aqp2 +T192 SO:0000704 6060 6064 gene +T193 SO:0001023 6071 6077 allele +T194 PR:000004182 6081 6085 Aqp2 +T195 NCBITaxon:10088 6115 6120 mouse +T196 http://purl.obolibrary.org/obo/MONDO_0016383 6130 6133 NDI +T197 UBERON:0000104 6168 6172 life +T198 PR:000004182 6215 6219 AQP2 +T199 UBERON:0001232 6267 6288 renal collecting-duct +T200 CL:1001225 6267 6294 renal collecting-duct cells +T201 CHEBI:4450 6342 6354 desmopressin +T202 NCBITaxon:9608 6414 6420 canine +T203 UBERON:0002113 6421 6427 kidney +T204 CL:1000497 6421 6427;6435 6439 kidney ... cell +T205 GO:0005783 6461 6482 endoplasmic reticulum +T206 GO:0030849 6592 6601 autosomal +T207 http://purl.obolibrary.org/obo/MONDO_0007451 6592 6601;6612 6615 autosomal NDI +T208 PR:000004182 6645 6649 AQP2 +T209 NCBITaxon:40674 6672 6678 mammal +T210 SO:0000704 6703 6710 genetic +T211 CHEBI:23995 6728 6744 ethylnitrosourea +T212 CHEBI:23995 6746 6749 ENU +T213 NCBITaxon:33208 6784 6790 animal +T214 GO:0008152 6850 6860 metabolism +T215 NCBITaxon:10088 6891 6895 mice +T216 GO:0060073 6901 6909 urinated +T217 GO:0042756 6914 6919 drank +T218 UBERON:0001977 6933 6938 Serum +T219 UBERON:0001088 6943 6948 urine +T220 UBERON:0001969 6970 6976 plasma +T221 CHEBI:17234 6977 6984 glucose +T222 CHEBI:17234 7021 7028 glucose +T223 UBERON:0001088 7036 7041 urine +T224 http://purl.obolibrary.org/obo/MONDO_0004782 7092 7110 diabetes insipidus +T225 http://purl.obolibrary.org/obo/MONDO_0000001 7117 7125 disorder +T226 NCBITaxon:10088 7135 7139 mice +T227 SO:0000704 7160 7165 genic +T228 GO:0030849 7167 7176 autosomal +T229 PR:000004182 7202 7206 Aqp2 +T230 SO:0000704 7219 7223 gene +T231 PR:000004182 7239 7243 Aqp2 +T232 NCBITaxon:10088 7270 7274 mice +T233 SO:1000022 7288 7306;7316 7328 thymine to guanine ... transversion +T234 SO:1000022 7308 7314;7316 7328 T to G ... transversion +T235 PR:000004182 7455 7459 AQP2 +T236 GO:0016020 7474 7482 membrane +T237 CHEBI:15377 7483 7488 water +T238 GO:0005576 7521 7534 extracellular +T239 GO:0016020 7553 7561 membrane +T240 SO:0000417 7571 7577 domain +T241 GO:0016020 7640 7648 membrane +T242 SO:0000417 7658 7665 domains +T243 NCBITaxon:7742 7686 7696 vertebrate +T244 NCBITaxon:species 7697 7704 species +T245 NCBITaxon:7742 7799 7809 vertebrate +T246 PR:000004182 7810 7814 AQP2 +T247 PR:000004182 7872 7876 Aqp2 +T248 NCBITaxon:10088 7888 7892 mice +T249 UBERON:0001088 7921 7926 urine +T250 UBERON:0001088 7976 7981 urine +T251 CHEBI:15377 8092 8097 water +T252 CHEBI:15377 8169 8174 water +T253 GO:0007631 8175 8181 intake +T254 NCBITaxon:10088 8198 8202 mice +T255 CHEBI:15377 8236 8241 water +T256 GO:0007631 8242 8248 intake +T257 UBERON:0001088 8343 8350 urinary +T258 CHEBI:15377 8362 8367 water +T259 GO:0007631 8368 8374 intake +T260 NCBITaxon:33208 8453 8460 animals +T261 http://purl.obolibrary.org/obo/MONDO_0004782 8470 8488 Diabetes insipidus +T262 UBERON:0001088 8535 8540 urine +T263 PR:000004182 8611 8615 Aqp2 +T264 NCBITaxon:10088 8627 8631 mice +T265 UBERON:0001088 8652 8657 urine +T266 UBERON:0001088 8677 8682 urine +T267 NCBITaxon:10088 8707 8711 mice +T268 NCBITaxon:10088 8773 8777 mice +T269 UBERON:0001088 8801 8806 urine +T270 GO:0065007 8834 8841 control +T271 UBERON:0001898 8849 8861 hypothalamus +T272 GO:0046903 8920 8928 secretes +T273 CHEBI:4450 8960 8994 1-deamino-8-D-arginine vasopressin +T274 CHEBI:4450 8996 9001 dDAVP +T275 CHEBI:4450 9015 9027 desmopressin +T276 PR:000001683 9053 9058 AVPR2 +T277 NCBITaxon:10088 9091 9095 mice +T278 CHEBI:4450 9097 9102 dDAVP +T279 UBERON:0001088 9135 9140 urine +T280 NCBITaxon:10088 9235 9239 mice +T281 UBERON:0001088 9258 9263 urine +T282 NCBITaxon:33208 9361 9368 animals +T283 UBERON:0001088 9410 9415 urine +T284 CHEBI:4450 9469 9474 dDAVP +T285 CHEBI:4450 9500 9505 dDAVP +T286 PR:000004182 9553 9557 AQP2 +T287 NCBITaxon:1 9617 9627 individual +T288 NCBITaxon:10088 9663 9668 mouse +T289 GO:0007618 9698 9705 matings +T290 NCBITaxon:33208 9718 9725 animals +T291 UBERON:0001088 9959 9964 urine +T292 CHEBI:15377 9980 9985 water +T293 GO:0007631 9986 9992 intake +T294 NCBITaxon:10088 10033 10037 mice +T295 UBERON:0002113 10054 10061 kidneys +T296 NCBITaxon:33208 10096 10103 animals +T297 NCBITaxon:10088 10160 10164 mice +T298 UBERON:0000104 10187 10195 lifespan +T299 NCBITaxon:33208 10205 10211 animal +T300 NCBITaxon:33208 10258 10265 animals +T301 PR:000004182 10282 10286 Aqp2 +T302 NCBITaxon:10088 10298 10302 mice +T303 http://purl.obolibrary.org/obo/MONDO_0005510 10322 10336 hydronephrosis +T304 UBERON:0002113 10455 10462 kidneys +T305 PR:000004182 10470 10474 Aqp2 +T306 NCBITaxon:10088 10486 10490 mice +T307 NCBITaxon:10088 10550 10554 mice +T308 http://purl.obolibrary.org/obo/MONDO_0005510 10585 10599 hydronephrosis +T309 PR:000004177 10639 10643 Aqp1 +T310 PR:000004183 10644 10648 Aqp3 +T311 NCBITaxon:10088 10659 10663 mice +T312 PR:000004182 10705 10709 Aqp2 +T313 NCBITaxon:10088 10721 10725 mice +T314 http://purl.obolibrary.org/obo/MONDO_0005510 10730 10744 hydronephrosis +T315 PR:000004182 10771 10775 Aqp2 +T316 NCBITaxon:10088 10787 10791 mice +T317 UBERON:0001224 10830 10842 renal pelvis +T318 UBERON:0000056 10872 10878 ureter +T319 UBERON:0006660 10918 10936 muscularis propria +T320 UBERON:0000365 11025 11035 urothelium +T321 UBERON:0005910 11046 11069 transitional epithelium +T322 UBERON:0002113 11121 11127 kidney +T323 UBERON:0002015 11145 11158 renal capsule +T324 UBERON:0001224 11162 11174 renal pelvis +T325 UBERON:0000074 11217 11226 glomeruli +T326 UBERON:0004134 11231 11239;11247 11254 proximal ... tubules +T327 UBERON:0004135 11240 11254 distal tubules +T328 PR:000004182 11365 11369 AQP2 +T329 CHEBI:37684 11666 11673 mannose +T330 PR:000004182 11691 11695 AQP2 +T331 UBERON:0002113 11778 11785 kidneys +T332 NCBITaxon:33208 11799 11806 animals +T333 PR:000004182 11808 11812 AQP2 +T334 NCBITaxon:33208 11825 11832 animals +T335 NCBITaxon:33208 12012 12019 animals +T336 MOP:0000780 12176 12183 cleaves +T337 CHEBI:37684 12184 12191 mannose +T338 CHEBI:16646 12197 12209 carbohydrate +T339 PR:000004182 12261 12265 AQP2 +T340 UBERON:0002113 12271 12278 kidneys +T341 NCBITaxon:33208 12318 12325 animals +T342 PR:000004182 12465 12469 Aqp2 +T343 NCBITaxon:10088 12481 12485 mice +T344 PR:000004182 12532 12536 Aqp2 +T345 NCBITaxon:10088 12548 12552 mice +T346 CHEBI:4450 12602 12607 dDAVP +T347 NCBITaxon:9606 12613 12619 humans +T348 SO:0001023 12631 12638 alleles +T349 PR:000004182 12642 12646 Aqp2 +T350 http://purl.obolibrary.org/obo/MONDO_0016383 12671 12674 NDI +T351 GO:0045177 12723 12729 apical +T352 GO:0009986 12730 12742 cell surface +T353 PR:000004182 12829 12833 Aqp2 +T354 NCBITaxon:9606 12857 12862 human +T355 http://purl.obolibrary.org/obo/MONDO_0000001 12863 12870 disease +T356 GO:0009294 12885 12896 transfected +T357 UBERON:0002113 12902 12908 kidney +T358 CL:1000497 12902 12913 kidney cell +T359 SO:0001023 12948 12955 alleles +T360 GO:0031982 13030 13038 vesicles +T361 GO:0005783 13072 13093 endoplasmic reticulum +T362 GO:0005783 13095 13097 ER +T363 NCBITaxon:10088 13104 13109 mouse +T364 http://purl.obolibrary.org/obo/MONDO_0016383 13119 13122 NDI +T365 NCBITaxon:33208 13189 13195 animal +T366 PR:000004182 13250 13254 AQP2 +T367 GO:0016324 13298 13304 apical +T368 GO:0044464 13305 13314;13331 13336 region of ... cells +T369 UBERON:0001232 13315 13330 collecting duct +T370 CL:1001225 13315 13336 collecting duct cells +T371 UBERON:0002113 13340 13347 kidneys +T372 NCBITaxon:10088 13361 13365 mice +T373 CHEBI:4450 13389 13394 dDAVP +T374 PR:000004182 13396 13400 AQP2 +T375 GO:0009986 13429 13441 cell surface +T376 UBERON:0002113 13470 13477 kidneys +T377 NCBITaxon:33208 13496 13503 animals +T378 PR:000004182 13514 13518 AQP2 +T379 PR:000004183 13613 13617 AQP3 +T380 GO:1990794 13657 13668 basolateral +T381 GO:0009986 13669 13676 surface +T382 CHEBI:4450 13701 13706 dDAVP +T383 PR:000004182 13720 13724 AQP2 +T384 GO:0009986 13760 13772 cell surface +T385 UBERON:0002113 13854 13861 kidneys +T386 NCBITaxon:10088 13885 13889 mice +T387 CHEBI:4450 13943 13948 dDAVP +T388 PR:000004182 14044 14048 AQP2 +T389 GO:0009294 14069 14081 transfection +T390 GO:0010467 14115 14125 expressing +T391 NCBITaxon:10088 14126 14131 mouse +T392 PR:000004182 14142 14146 AQP2 +T393 PR:000004182 14151 14155 AQP2 +T394 NCBITaxon:10088 14311 14315 mice +T395 GO:0010467 14408 14418 expressing +T396 PR:000004182 14419 14423 AQP2 +T397 GO:0009294 14516 14527 transfected +T398 PR:000004182 14564 14568 AQP2 +T399 GO:0016324 14646 14652 apical +T400 GO:0031982 14719 14728 vesicular +T401 PR:000004182 14751 14755 AQP2 +T402 GO:0005634 14813 14820 nuclear +T403 CHEBI:42471 14878 14887 forskolin +T404 CHEBI:17489 14891 14895 cAMP +T405 PR:000004182 14942 14946 AQP2 +T406 GO:0045177 14967 14973 apical +T407 GO:0009986 14974 14984;15000 15005 surface of ... cells +T408 GO:0005634 15060 15067 nuclear +T409 PR:000004182 15084 15088 AQP2 +T410 CHEBI:42471 15153 15162 forskolin +T411 GO:0005634 15215 15222 nuclear +T412 PR:000004182 15239 15243 AQP2 +T413 GO:0005783 15272 15274 ER +T414 PR:000004182 15319 15323 AQP2 +T415 GO:0005783 15347 15349 ER +T416 GO:0009294 15371 15382 transfected +T417 PR:000004182 15388 15392 Aqp2 +T418 PR:000004182 15409 15413 AQP2 +T419 GO:0005783 15421 15423 ER +T420 PR:000005005 15432 15440 calnexin +T421 PR:000005005 15472 15480 calnexin +T422 PR:000004182 15486 15490 AQP2 +T423 PR:000004182 15551 15555 AQP2 +T424 PR:000005005 15587 15595 calnexin +T425 GO:0005783 15648 15650 ER +T426 PR:000004182 15665 15669 AQP2 +T427 GO:0005783 15719 15721 ER +T428 GO:0005783 15729 15731 ER +T429 PR:000004182 15814 15818 AQP2 +T430 UBERON:0002113 15835 15842 kidneys +T431 NCBITaxon:33208 15861 15868 Animals +T432 PR:000004182 15890 15894 Aqp2 +T433 UBERON:0001088 15936 15941 urine +T434 UBERON:0001088 15956 15961 urine +T435 http://purl.obolibrary.org/obo/MONDO_0016383 16037 16040 NDI +T436 SO:0001023 16041 16047 allele +T437 PR:000004182 16049 16053 AQP2 +T438 CL:0000023 16105 16112 oocytes +T439 GO:0051260 16131 16147 homo-oligomerize +T440 UBERON:0002113 16179 16186 kidneys +T441 NCBITaxon:33208 16205 16212 animals +T442 PR:000004182 16262 16266 AQP2 +T443 UBERON:0001232 16322 16345 kidney collecting ducts +T444 PR:000004182 16351 16355 Aqp2 +T445 NCBITaxon:10088 16363 16367 mice +T446 PR:000004182 16432 16436 AQP2 +T447 GO:0045177 16468 16474 apical +T448 GO:0009986 16475 16487 cell surface +T449 CHEBI:4450 16493 16498 dDAVP +T450 PR:000004182 16884 16888 AQP2 +T451 GO:0009986 16896 16908 cell surface +T452 PR:000004182 16978 16982 AQP2 +T453 PR:000004182 17044 17048 AQP2 +T454 GO:0010467 17075 17084 expressed +T455 UBERON:0001232 17113 17129 collecting ducts +T456 PR:000004182 17135 17139 Aqp2 +T457 NCBITaxon:10088 17147 17151 mice +T458 PR:000004182 17245 17249 AQP2 +T459 GO:0009294 17354 17365 transfected +T460 GO:0010467 17408 17418 expressing +T461 PR:000004182 17429 17433 AQP2 +T462 GO:0009294 17451 17462 transfected +T463 GO:0010467 17472 17482 expression +T464 PR:000004182 17524 17528 AQP2 +T465 PR:000004182 17530 17534 AQP2 +T466 GO:0042571 17556 17566 Antibodies +T467 PR:000004182 17610 17614 AQP2 +T468 PR:000004182 17620 17624 AQP2 +T469 PR:000004182 17632 17636 AQP2 +T470 GO:0009294 17663 17674 transfected +T471 GO:0009294 17719 17730 transfected +T472 GO:0010467 17754 17764 expressing +T473 PR:000004182 17775 17779 AQP2 +T474 GO:0016020 17828 17837 membranes +T475 PR:000004182 17860 17864 AQP2 +T476 GO:0010467 17881 17890 expressed +T477 GO:0009294 18134 18145 transfected +T478 PR:000004182 18161 18165 AQP2 +T479 GO:0010467 18166 18176 expressing +T480 SO:0000440 18177 18183 vector +T481 SO:0000440 18198 18204 vector +T482 GO:0009294 18238 18249 transfected +T483 PR:000004182 18250 18254 AQP2 +T484 PR:000004182 18262 18266 AQP2 +T485 GO:0010467 18277 18287 expression +T486 PR:000004182 18300 18304 AQP2 +T487 GO:0045177 18326 18332 apical +T488 GO:0009986 18333 18340 surface +T489 CHEBI:42471 18346 18355 forskolin +T490 GO:0009294 18395 18406 transfected +T491 SO:0000440 18412 18418 vector +T492 PR:000004182 18479 18483 AQP2 +T493 PR:000004182 18516 18520 AQP2 +T494 GO:0010467 18537 18546 expressed +T495 GO:0009294 18560 18572 transfection +T496 SO:0000440 18578 18584 vector +T497 GO:0005737 18614 18625 cytoplasmic +T498 GO:0010467 18677 18686 expressed +T499 PR:000004182 18700 18704 AQP2 +T500 PR:000004182 18721 18725 AQP2 +T501 GO:0045177 18753 18759 apical +T502 GO:0009986 18760 18767 surface +T503 GO:0009294 18896 18908 transfection +T504 GO:0009294 18928 18939 transfected +T505 GO:0010467 18987 18997 expressing +T506 PR:000004182 19008 19012 AQP2 +T507 GO:0045177 19023 19029 apical +T508 CHEBI:42471 19035 19044 forskolin +T509 GO:0010467 19076 19086 expression +T510 PR:000004182 19116 19120 AQP2 +T511 PR:000004182 19139 19143 AQP2 +T512 PR:000004182 19195 19199 AQP2 +T513 PR:000004182 19227 19231 AQP2 +T514 GO:0045177 19253 19259 apical +T515 CHEBI:42471 19265 19274 forskolin +T516 PR:000004182 19334 19338 AQP2 +T517 GO:0045177 19377 19383 apical +T518 GO:0009986 19384 19391 surface +T519 PR:000004182 19424 19428 AQP2 +T520 GO:0045177 19456 19462 apical +T521 PR:000004182 19478 19482 AQP2 +T522 GO:0010467 19492 19501 expressed +T523 GO:0009294 19547 19558 transfected +T524 PR:000004182 19590 19594 AQP2 +T525 SO:0000440 19598 19604 vector +T526 MOP:0000093 19622 19634 biotinylated +T527 GO:0009986 19635 19642 surface +T528 CHEBI:42471 19658 19667 forskolin +T529 MOP:0000093 19702 19714 biotinylated +T530 PR:000004182 19737 19741 AQP2 +T531 GO:0010467 19755 19764 expressed +T532 MOP:0000093 19839 19851 biotinylated +T533 PR:000004182 19872 19876 AQP2 +T534 GO:0010467 19885 19894 expressed +T535 GO:0009986 19907 19914 surface +T536 MOP:0000093 19915 19927 biotinylated +T537 GO:0009986 19941 19953 cell surface +T538 CHEBI:15956 19981 19987 biotin +T539 PR:000004182 20017 20021 AQP2 +T540 GO:0009986 20050 20062 cell surface +T541 PR:000004182 20078 20082 AQP2 +T542 PR:000004182 20128 20132 Aqp2 +T543 NCBITaxon:10088 20143 20147 mice +T544 GO:0040007 20163 20167 grow +T545 GO:0000003 20172 20181 reproduce +T546 UBERON:0001088 20262 20267 urine +T547 UBERON:0001088 20290 20295 urine +T548 CHEBI:15377 20307 20312 water +T549 GO:0007631 20313 20319 intake +T550 NCBITaxon:10088 20348 20353 mouse +T551 http://purl.obolibrary.org/obo/MONDO_0016383 20363 20366 NDI +T552 NCBITaxon:9606 20394 20400 humans +T553 http://purl.obolibrary.org/obo/MONDO_0016383 20402 20405 NDI +T554 PR:000001683 20432 20437 Avpr2 +T555 PR:000004182 20441 20445 Aqp2 +T556 GO:0000805 20463 20464 X +T557 PR:000001683 20472 20477 Avpr2 +T558 SO:0000704 20478 20482 gene +T559 NCBITaxon:10088 20486 20490 mice +T560 http://purl.obolibrary.org/obo/MONDO_0016383 20504 20507 NDI +T561 UBERON:0007023 20596 20602 adults +T562 NCBITaxon:10088 20610 20614 mice +T563 GO:0016265 20615 20619 died +T564 GO:0007567 20635 20640 birth +T565 UBERON:0007023 20646 20651 adult +T566 UBERON:0001088 20713 20720 urinary +T567 CHEBI:15377 20732 20737 water +T568 GO:0007631 20738 20744 intake +T569 UBERON:0001088 20759 20764 urine +T570 NCBITaxon:10088 20793 20798 mouse +T571 PR:000004182 20799 20803 Aqp2 +T572 SO:0000704 20804 20808 gene +T573 NCBITaxon:9606 20848 20853 human +T574 http://purl.obolibrary.org/obo/MONDO_0000001 20854 20861 disease +T575 NCBITaxon:10088 20923 20927 mice +T576 UBERON:0001088 20942 20947 urine +T577 GO:0016265 20998 21003 death +T578 GO:0007567 21019 21024 birth +T579 PR:000004182 21037 21041 AQP2 +T580 UBERON:0001232 21123 21138 collecting duct +T581 NCBITaxon:10088 21221 21225 mice +T582 PR:000004182 21266 21270 Aqp2 +T583 SO:0000704 21287 21291 gene +T584 NCBITaxon:10088 21316 21320 mice +T585 PR:000004182 21347 21351 AQP2 +T586 CHEBI:15377 21382 21387 water +T587 GO:0006833 21382 21400 water transporting +T588 CHEBI:15377 21456 21461 water +T589 PR:000004182 21497 21501 AQP2 +T590 NCBITaxon:33208 21529 21536 animals +T591 CHEBI:4450 21565 21570 dDAVP +T592 CHEBI:4450 21581 21586 dDAVP +T593 UBERON:0001088 21598 21603 urine +T594 UBERON:0002113 21652 21658 kidney +T595 PR:000004182 21670 21674 AQP2 +T596 CHEBI:15377 21712 21717 water +T597 GO:0045177 21755 21761 apical +T598 GO:0009986 21762 21774 cell surface +T599 CHEBI:4450 21781 21786 dDAVP +T600 PR:000004182 21824 21828 AQP2 +T601 GO:0009986 21919 21931 cell surface +T602 GO:0009986 21937 21944 surface +T603 MOP:0000093 21945 21958 biotinylation +T604 GO:0009986 22026 22033 surface +T605 GO:0005783 22251 22253 ER +T606 CHEBI:37684 22350 22357 mannose +T607 GO:0005783 22615 22617 ER +T608 PR:000004182 22674 22678 AQP2 +T609 GO:0005783 22694 22696 ER +T610 PR:000005005 22705 22713 calnexin +T611 GO:0009294 22717 22728 transfected +T612 GO:0005783 22803 22805 ER +T613 GO:0005783 22837 22839 ER +T614 CHEBI:4450 22864 22869 dDAVP +T615 NCBITaxon:33208 22933 22940 animals +T616 GO:0015031 22950 22962;22984 22991 transport of ... protein +T617 GO:0005783 23003 23005 ER +T618 PR:000004182 23060 23064 AQP2 +T619 GO:0006457 23074 23081 folding +T620 CHEBI:15377 23130 23135 water +T621 GO:0006833 23130 23148 water transporting +T622 NCBITaxon:33208 23245 23252 animals +T623 GO:0005783 23289 23291 ER +T624 PR:000004182 23308 23312 AQP2 +T625 CHEBI:37684 23350 23357 mannose +T626 CHEBI:37684 23386 23393 mannose +T627 CHEBI:50699 23399 23414 oligosaccharide +T628 GO:0005783 23431 23433 ER +T629 GO:0005794 23478 23493 Golgi apparatus +T630 CHEBI:37684 23525 23532 mannose +T631 PR:000004182 23554 23558 AQP2 +T632 GO:0005783 23614 23616 ER +T633 CHEBI:50699 23633 23647 oligosaccharyl +T634 PR:000004182 23693 23697 AQP2 +T635 NCBITaxon:10088 23742 23746 mice +T636 NCBITaxon:10088 23797 23801 mice +T637 NCBITaxon:10088 23862 23866 mice +T638 UBERON:0002113 23968 23975 kidneys +T639 GO:0005783 24123 24125 ER +T640 PR:000004182 24180 24184 Aqp2 +T641 NCBITaxon:33208 24192 24199 animals +T642 SO:0001023 24343 24349 allele +T643 PR:000004182 24353 24357 Aqp2 +T644 GO:0010467 24401 24410 expressed +T645 PR:000004182 24553 24557 Aqp2 +T646 NCBITaxon:10088 24565 24569 mice +T647 PR:000004182 24591 24595 AQP2 +T648 PR:000004182 24630 24634 AQP2 +T649 GO:0010467 24659 24668 expressed +T650 PR:000004182 24693 24697 AQP2 +T651 GO:0009986 24718 24730 cell surface +T652 SO:0001023 24821 24827 allele +T653 PR:000004182 24838 24842 AQP2 +T654 http://purl.obolibrary.org/obo/MONDO_0016383 24853 24856 NDI +T655 PR:000004182 24890 24894 AQP2 +T656 PR:000004182 24918 24922 AQP2 +T657 SO:0001023 25037 25043 allele +T658 UBERON:0002113 25092 25099 kidneys +T659 PR:000004182 25114 25118 Aqp2 +T660 NCBITaxon:10088 25130 25134 mice +T661 GO:0010467 25157 25167 expressing +T662 UBERON:0001232 25168 25183 collecting duct +T663 CL:1001225 25168 25189 collecting duct cells +T664 CHEBI:15377 25206 25211 water +T665 GO:0016324 25262 25284 apical plasma membrane +T666 CHEBI:4450 25300 25305 dDAVP +T667 PR:000004182 25418 25422 Aqp2 +T668 GO:0009294 25434 25446 Transfection +T669 PR:000004182 25481 25485 Aqp2 +T670 NCBITaxon:9606 25523 25528 human +T671 SO:0001023 25529 25536 alleles +T672 GO:0005886 25636 25651 plasma membrane +T673 GO:0006457 25662 25669 folding +T674 GO:0005783 25688 25690 ER +T675 CHEBI:4450 25734 25739 dDAVP +T676 GO:0030849 25775 25784 autosomal +T677 http://purl.obolibrary.org/obo/MONDO_0007451 25775 25784;25795 25798 autosomal NDI +T678 NCBITaxon:9606 25877 25882 human +T679 http://purl.obolibrary.org/obo/MONDO_0016383 25883 25886 NDI +T680 NCBITaxon:10088 25893 25898 mouse +T681 http://purl.obolibrary.org/obo/MONDO_0016383 25908 25911 NDI +T682 PR:000004182 25924 25928 Aqp2 +T683 SO:0001023 25929 25935 allele +T684 SO:0000704 26010 26014 gene +T685 CHEBI:23995 26112 26115 ENU +T686 NCBITaxon:10088 26116 26120 mice +T687 CHEBI:23995 26135 26138 ENU +T688 NCBITaxon:10088 26159 26163 mice +T689 NCBITaxon:10088 26198 26202 Mice +T690 NCBITaxon:33208 26244 26251 animals +T691 NCBITaxon:33208 26359 26365 animal +T692 NCBITaxon:33208 26519 26525 Animal +T693 NCBITaxon:10088 26596 26601 mouse +T694 PR:000004182 26602 26606 AQP2 +T695 SO:0000155 26661 26668 plasmid +T696 GO:0006266 26693 26700 ligated +T697 SO:0000077 26955 26964 antisense +T698 PR:000004182 27047 27051 AQP2 +T699 PR:000004182 27069 27073 AQP2 +T700 SO:0000112 27120 27127 primers +T701 SO:0000319 27189 27199 stop codon +T702 PR:000004182 27203 27207 AQP2 +T703 PR:P23940 27248 27253 BamHI +T704 GO:0006266 27258 27265 ligated +T705 CHEBI:25435 27385 27394 mutagenic +T706 CHEBI:17334 27655 27665 penicillin +T707 CHEBI:17076 27684 27696 streptomycin +T708 CHEBI:16526 27712 27715 CO2 +T709 GO:0009294 27764 27775 transfected +T710 GO:0010467 27831 27841 expression +T711 PR:000004182 27875 27879 AQP2 +T712 PR:000004182 27881 27885 AQP2 +T713 SO:0000667 27899 27905 insert +T714 CHEBI:42768 27935 27939 G418 +T715 CHEBI:33282 28046 28056 antibiotic +T716 GO:0009294 28107 28120 transfections +T717 PR:000004182 28219 28223 Aqp2 +T718 NCBITaxon:10088 28242 28246 mice +T719 SO:0000147 28253 28258 exons +T720 PR:000004182 28262 28266 Aqp2 +T721 NCBITaxon:10088 28287 28292 mouse +T722 SO:0001026 28293 28300 genomic +T723 SO:0000147 28336 28340 exon +T724 SO:0000112 28367 28374 primers +T725 UBERON:0001088 28435 28440 Urine +T726 UBERON:0001088 28462 28467 urine +T727 UBERON:0007023 28510 28515 adult +T728 NCBITaxon:10088 28516 28520 mice +T729 GO:0008152 28532 28541 Metabolic +T730 UBERON:0001088 28615 28620 urine +T731 UBERON:0001088 28640 28645 Urine +T732 UBERON:0001088 28767 28772 Urine +T733 UBERON:0001179 28824 28834 peritoneal +T734 CHEBI:4450 28848 28853 dDAVP +T735 NCBITaxon:10088 28867 28871 Mice +T736 CHEBI:4450 28897 28902 dDAVP +T737 UBERON:0001088 28940 28945 Urine +T738 UBERON:0002113 29031 29037 Kidney +T739 GO:0016020 29038 29046 membrane +T740 NCBITaxon:10088 29067 29072 mouse +T741 UBERON:0002113 29073 29080 kidneys +T742 CHEBI:9754 29107 29111 Tris +T743 CHEBI:17992 29129 29136 sucrose +T744 GO:0016020 29391 29400 membranes +T745 UBERON:0002113 29516 29522 Kidney +T746 GO:0016020 29523 29531 membrane +T747 CHEBI:8984 29573 29576 SDS +T748 CHEBI:53325 29617 29631 nitrocellulose +T749 GO:0016020 29642 29651 Membranes +T750 CHEBI:9754 29686 29690 Tris +T751 CHEBI:53424 29718 29726 Tween 20 +T752 PR:000004182 29786 29790 AQP2 +T753 GO:0042571 29802 29810 antibody +T754 GO:0016020 29888 29897 Membranes +T755 MOP:0000779 29942 29952 conjugated +T756 NCBITaxon:9793 29953 29959 donkey +T757 NCBITaxon:9925 29965 29969 goat +T758 GO:0042571 29970 29978 antibody +T759 GO:0016020 29980 29989 Membranes +T760 CHEBI:33893 30054 30061 reagent +T761 UBERON:0002113 30148 30154 Kidney +T762 GO:0016020 30155 30164 membranes +T763 CHEBI:37586 30197 30213 sodium phosphate +T764 CHEBI:8984 30229 30232 SDS +T765 CHEBI:41218 30244 30261 β-mercaptoethanol +T766 CHEBI:33893 30465 30474 reactants +T767 MOP:0000093 30541 30554 biotinylation +T768 GO:0010467 30589 30599 expressing +T769 PR:000004182 30610 30614 AQP2 +T770 GO:0040007 30616 30621 grown +T771 GO:0009294 30644 30655 transfected +T772 PR:000004182 30677 30681 AQP2 +T773 PR:000004182 30689 30693 AQP2 +T774 SO:0000440 30704 30710 vector +T775 CHEBI:9754 30754 30758 Tris +T776 CHEBI:17992 30791 30798 sucrose +T777 GO:0016020 30887 30896 membranes +T778 CHEBI:9177 30951 30970 sodium deoxycholate +T779 GO:0016020 31022 31031 membranes +T780 GO:0016020 31082 31090 membrane +T781 GO:0016020 31115 31124 membranes +T782 UBERON:0001977 31215 31219 sera +T783 PR:000029158 31247 31256 protein A +T784 GO:0016020 31420 31428 membrane +T785 GO:0009986 31486 31498 Cell surface +T786 MOP:0000093 31499 31512 biotinylation +T787 PR:000004182 31563 31567 AQP2 +T788 GO:0009294 31579 31590 transfected +T789 GO:0010467 31614 31624 expressing +T790 PR:000004182 31635 31639 AQP2 +T791 SO:0000440 31667 31673 vector +T792 GO:0009294 31704 31716 transfection +T793 CHEBI:42471 31745 31754 forskolin +T794 CHEBI:15956 31856 31862 biotin +T795 CHEBI:9754 31950 31954 Tris +T796 GO:0016020 31998 32007 membranes +T797 GO:0016020 32070 32079 membranes +T798 MOP:0000093 32294 32306 biotinylated +T799 GO:0042571 32349 32357 antibody +T800 PR:000004182 32361 32365 AQP2 +T801 UBERON:0002113 32368 32374 Kidney +T802 NCBITaxon:10088 32404 32409 mouse +T803 UBERON:0002113 32410 32417 kidneys +T804 CHEBI:16842 32455 32463 formalin +T805 UBERON:0002113 32474 32481 Kidneys +T806 CHEBI:59132 32552 32559 antigen +T807 CHEBI:53258 32582 32596 sodium citrate +T808 PR:000004183 32670 32674 AQP3 +T809 PR:000004182 32688 32692 AQP2 +T810 NCBITaxon:9793 32724 32730 donkey +T811 UBERON:0001977 32731 32736 serum +T812 NCBITaxon:9925 32749 32753 goat +T813 PR:000004183 32759 32763 AQP3 +T814 GO:0042571 32764 32772 antibody +T815 CHEBI:52661 32865 32879 AlexaFluor 488 +T816 MOP:0000779 32880 32890 conjugated +T817 NCBITaxon:9793 32891 32897 donkey +T818 NCBITaxon:9925 32903 32907 goat +T819 GO:0042571 32908 32916 antibody +T820 NCBITaxon:9031 33011 33018 chicken +T821 UBERON:0001977 33019 33024 serum +T822 NCBITaxon:9986 33043 33049 rabbit +T823 PR:000004182 33055 33059 AQP2 +T824 GO:0042571 33060 33068 antibody +T825 CHEBI:51248 33152 33166 AlexaFluor 594 +T826 MOP:0000779 33167 33177 conjugated +T827 NCBITaxon:9031 33178 33185 chicken +T828 NCBITaxon:9986 33191 33197 rabbit +T829 GO:0042571 33198 33206 antibody +T830 CHEBI:51231 33265 33269 DAPI +T831 GO:0010467 33411 33421 expressing +T832 SO:0000440 33422 33428 vector +T833 PR:000004182 33446 33450 AQP2 +T834 PR:000004182 33455 33459 AQP2 +T835 GO:0010467 33497 33507 expressing +T836 GO:0040007 33530 33535 grown +T837 PR:000007581 33539 33550 fibronectin +T838 CHEBI:42471 33641 33650 forskolin +T839 CHEBI:17790 33676 33684 methanol +T840 CHEBI:9750 33754 33766 Triton X-100 +T841 PR:000004182 33806 33810 AQP2 +T842 GO:0043226 33815 33824 organelle +T843 GO:0005886 33848 33850 PM +T844 GO:0005783 33858 33860 ER +T845 PR:000004182 33862 33866 AQP2 +T846 NCBITaxon:9925 33886 33890 goat +T847 PR:000004182 33896 33900 AQP2 +T848 CHEBI:52661 33969 33983 AlexaFluor 488 +T849 MOP:0000779 33984 33994 conjugated +T850 NCBITaxon:9793 33995 34001 donkey +T851 NCBITaxon:9925 34007 34011 goat +T852 GO:0042571 34022 34030 antibody +T853 GO:0005886 34036 34038 PM +T854 GO:0005783 34043 34045 ER +T855 NCBITaxon:10088 34064 34069 mouse +T856 CHEBI:29101 34075 34078 Na+ +T857 CHEBI:29103 34079 34081 K+ +T858 NCBITaxon:9986 34141 34147 rabbit +T859 PR:000005005 34153 34161 calnexin +T860 GO:0042571 34224 34234 antibodies +T861 GO:0042571 34253 34263 antibodies +T862 CHEBI:37987 34265 34268 Cy3 +T863 MOP:0000779 34269 34279 conjugated +T864 NCBITaxon:9925 34280 34284 goat +T865 NCBITaxon:10088 34290 34295 mouse +T866 CHEBI:51248 34372 34386 AlexaFluor 594 +T867 MOP:0000779 34387 34397 conjugated +T868 NCBITaxon:9031 34398 34405 chicken +T869 NCBITaxon:9986 34411 34417 rabbit +T870 CHEBI:51231 34486 34490 DAPI +T871 PR:000004182 34567 34571 AQP2 +T872 GO:0042571 34593 34601 antibody +T873 UBERON:0002113 34623 34629 kidney +T874 PR:000004182 34674 34678 AQP2 +T875 PR:000004182 35281 35285 Aqp2 +T876 PR:000004182 35354 35358 AQP2 +T877 NCBITaxon:10088 35476 35480 mice +T878 NCBITaxon:10088 35537 35541 mice +T879 UBERON:0000479 35630 35636 tissue +T880 PR:000001683 35744 35749 AVPR2 +T881 PR:000001683 35752 35771 AVP type 2 receptor +T882 CHEBI:4450 35801 35806 dDAVP +T883 CHEBI:4450 35809 35843 1-deamino-8-D-arginine vasopressin +T884 GO:0005783 35845 35847 ER +T885 GO:0005783 35850 35871 endoplasmic reticulum +T886 NCBITaxon:9608 35892 35898 canine +T887 UBERON:0002113 35899 35905 kidney +T888 http://purl.obolibrary.org/obo/MONDO_0016383 35907 35910 NDI +T889 GO:0001822 35913 35924 nephrogenic +T890 http://purl.obolibrary.org/obo/MONDO_0016383 35913 35943 nephrogenic diabetes insipidus +T891 GO:0005886 35945 35947 PM +T892 GO:0005886 35950 35965 plasma membrane +T893 PR:000004182 35998 36002 Aqp2 +T894 NCBITaxon:10088 36036 36040 Mice +T895 PR:000004182 36072 36076 Aqp2 +T896 SO:0000360 36119 36124 codon +T897 NCBITaxon:10088 36227 36232 mouse +T898 PR:000004182 36233 36237 AQP2 +T899 PR:P41181 36318 36323 hAQP2 +T900 NCBITaxon:9606 36325 36330 human +T901 PR:000004182 36331 36335 AQP2 +T902 PR:Q02013 36337 36342 mAQP1 +T903 NCBITaxon:10088 36344 36349 mouse +T904 PR:000004177 36350 36354 AQP1 +T905 PR:P56402 36356 36361 mAQP2 +T906 NCBITaxon:10088 36363 36368 mouse +T907 PR:000004182 36369 36373 AQP2 +T908 PR:P34080 36375 36380 rAQP2 +T909 NCBITaxon:10114 36382 36385 rat +T910 PR:000004182 36386 36390 AQP2 +T911 NCBITaxon:8353 36399 36406 Xenopus +T912 PR:000004182 36407 36411 AQP2 +T913 UBERON:0001088 36418 36423 Urine +T914 CHEBI:15377 36444 36449 water +T915 GO:0007631 36450 36461 consumption +T916 NCBITaxon:10088 36476 36480 mice +T917 NCBITaxon:10088 36536 36540 mice +T918 NCBITaxon:10088 36678 36682 mice +T919 UBERON:0001088 36689 36694 Urine +T920 PR:000004182 36735 36739 Aqp2 +T921 CHEBI:4450 36836 36841 dDAVP +T922 NCBITaxon:10088 37042 37047 Mouse +T923 UBERON:0002113 37048 37055 Kidneys +T924 NCBITaxon:10088 37090 37095 mouse +T925 UBERON:0001224 37169 37181 renal pelvis +T926 UBERON:0008987 37218 37234 renal parenchyma +T927 UBERON:0002113 37289 37295 kidney +T928 UBERON:0004100 37300 37317 collecting system +T929 UBERON:0018707 37323 37330 bladder +T930 UBERON:0004538 37353 37364 Left kidney +T931 NCBITaxon:10088 37377 37382 mouse +T932 UBERON:0002113 37418 37424 kidney +T933 CHEBI:51686 37484 37495 Hematoxylin +T934 UBERON:0000056 37525 37531 ureter +T935 NCBITaxon:10088 37546 37551 mouse +T936 UBERON:0002113 37602 37608 kidney +T937 CHEBI:51686 37615 37626 Hematoxylin +T938 UBERON:0002113 37669 37675 kidney +T939 NCBITaxon:10088 37706 37711 mouse +T940 UBERON:0002113 37724 37730 kidney +T941 UBERON:0001224 37762 37774 renal pelvis +T942 UBERON:2001995 37796 37803 papilla +T943 UBERON:0001851 37834 37840 cortex +T944 UBERON:0000958 37845 37852 medulla +T945 PR:000004182 37888 37892 AQP2 +T946 NCBITaxon:10088 37898 37903 Mouse +T947 UBERON:0002113 37904 37911 Kidneys +T948 UBERON:0002113 37948 37954 kidney +T949 GO:0016020 37955 37964 membranes +T950 NCBITaxon:10088 37981 37985 mice +T951 PR:000004182 38011 38015 AQP2 +T952 UBERON:0002113 38044 38050 kidney +T953 GO:0016020 38051 38060 membranes +T954 NCBITaxon:10088 38075 38080 mouse +T955 NCBITaxon:10088 38119 38124 mouse +T956 UBERON:0002113 38143 38149 kidney +T957 GO:0016020 38150 38159 membranes +T958 CHEBI:37684 38247 38254 mannose +T959 CHEBI:37684 38258 38260 m. +T960 GO:0005783 38309 38311 ER +T961 PR:000004182 38369 38373 AQP2 +T962 NCBITaxon:10088 38420 38425 Mouse +T963 UBERON:0001232 38426 38449 Kidney Collecting Ducts +T964 UBERON:0001232 38499 38515 collecting ducts +T965 PR:000004182 38543 38547 AQP2 +T966 NCBITaxon:10088 38567 38572 mouse +T967 NCBITaxon:10088 38623 38627 Mice +T968 UBERON:0001179 38647 38659 peritoneally +T969 CHEBI:4450 38677 38682 dDAVP +T970 UBERON:0002113 38722 38729 kidneys +T971 UBERON:0002113 38731 38738 Kidneys +T972 PR:000004182 38771 38775 AQP2 +T973 GO:1990794 38790 38801 basolateral +T974 PR:000004183 38809 38813 AQP3 +T975 GO:0044444 38853 38860;38865 38874 area of ... cytoplasm +T976 PR:000004182 38914 38918 AQP2 +T977 GO:0016324 38955 38961 apical +T978 CHEBI:4450 38998 39003 dDAVP +T979 GO:0009294 39034 39045 transfected +T980 NCBITaxon:10088 39071 39076 mouse +T981 PR:000004182 39083 39087 AQP2 +T982 CHEBI:42471 39132 39141 forskolin +T983 PR:000004182 39237 39241 AQP2 +T984 GO:1990794 39269 39280 basolateral +T985 CHEBI:29101 39288 39291 Na+ +T986 CHEBI:29103 39292 39294 K+ +T987 GO:0005634 39333 39340 nuclear +T988 CHEBI:51231 39347 39351 DAPI +T989 PR:000004182 39449 39453 AQP2 +T990 GO:0009986 39479 39491 cell surface +T991 CHEBI:42471 39497 39506 forskolin +T992 GO:0005634 39536 39543 nuclear +T993 GO:0005783 39575 39577 ER +T994 PR:000004182 39601 39605 AQP2 +T995 GO:0010467 39631 39641 expressing +T996 PR:000004182 39642 39646 AQP2 +T997 GO:0040007 39657 39662 grown +T998 PR:000007581 39666 39677 fibronectin +T999 CHEBI:42471 39776 39785 forskolin +T1000 PR:000004182 39866 39870 AQP2 +T1001 PR:000005005 39883 39891 calnexin +T1002 GO:0005783 39902 39904 ER +T1003 PR:000004182 39941 39945 AQP2 +T1004 GO:0005783 39973 39994 endoplasmic reticulum +T1005 PR:000004182 40041 40045 AQP2 +T1006 NCBITaxon:10088 40075 40080 Mouse +T1007 UBERON:0001232 40081 40097 Collecting Ducts +T1008 GO:0009294 40107 40118 transfected +T1009 NCBITaxon:33208 40151 40158 animals +T1010 PR:000004182 40160 40164 AQP2 +T1011 CHEBI:4450 40191 40196 dDAVP +T1012 UBERON:0002113 40247 40253 kidney +T1013 PR:000004182 40271 40275 Aqp2 +T1014 NCBITaxon:10088 40283 40288 mouse +T1015 CHEBI:4450 40311 40316 dDAVP +T1016 UBERON:0002113 40318 40324 Kidney +T1017 PR:000004182 40370 40374 AQP2 +T1018 GO:1990794 40389 40400 basolateral +T1019 PR:000004183 40408 40412 AQP3 +T1020 PR:000004182 40448 40452 AQP2 +T1021 GO:0010467 40492 40502 expressing +T1022 PR:000004182 40513 40517 AQP2 +T1023 GO:0009294 40535 40546 transfected +T1024 PR:000004182 40573 40577 AQP2 +T1025 PR:000004182 40579 40583 AQP2 +T1026 GO:0016020 40617 40626 membranes +T1027 GO:0042571 40662 40670 antibody +T1028 GO:0016020 40678 40687 membranes +T1029 GO:0042571 40750 40758 antibody +T1030 PR:000004182 40767 40771 AQP2 +T1031 PR:000004182 40783 40787 AQP2 +T1032 PR:000004182 40828 40832 AQP2 +T1033 PR:000004182 40875 40879 AQP2 +T1034 PR:000004182 40913 40917 AQP2 +T1035 PR:000004182 40958 40962 AQP2 +T1036 GO:0010467 41005 41014 expressed +T1037 GO:0010467 41051 41061 expressing +T1038 SO:0000440 41062 41068 vector +T1039 SO:0000440 41076 41082 vector +T1040 PR:000004182 41110 41114 AQP2 +T1041 CHEBI:42471 41171 41180 forskolin +T1042 PR:000004182 41270 41274 AQP2 +T1043 GO:0009986 41293 41305 cell surface +T1044 GO:0010467 41317 41327 expressing +T1045 PR:000004182 41338 41342 AQP2 +T1046 PR:000004182 41344 41348 AQP2 +T1047 PR:000004182 41367 41371 AQP2 +T1048 GO:0010467 41382 41391 expressed +T1049 GO:0010467 41406 41416 expressing +T1050 PR:000004182 41427 41431 AQP2 +T1051 SO:0000440 41435 41441 vector +T1052 CHEBI:42471 41476 41485 forskolin +T1053 GO:0009986 41491 41503 cell surface +T1054 MOP:0000093 41504 41516 biotinylated +T1055 PR:000004182 41588 41592 AQP2 +T1056 PR:000004182 41605 41609 AQP2 +T1057 CHEBI:33893 41904 41912 reagents +T1058 http://purl.obolibrary.org/obo/MONDO_0004782 42028 42046 Diabetes insipidus +T1059 NCBITaxon:10088 42050 42054 mice +T1060 PR:000004182 42074 42085 aquaporin-2 diff --git a/src/ontogpt/evaluation/craft/database/all/16121255.txt b/src/ontogpt/evaluation/craft/database/all/16121255.txt new file mode 100644 index 000000000..05cdf71f8 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16121255.txt @@ -0,0 +1,213 @@ +Diabetes Insipidus in Mice with a Mutation in Aquaporin-2 + +Abstract + +Congenital nephrogenic diabetes insipidus (NDI) is a disease characterized by failure of the kidney to concentrate urine in response to vasopressin. Human kindreds with nephrogenic diabetes insipidus have been found to harbor mutations in the vasopressin receptor 2 (Avpr2) gene or the vasopressin-sensitive water channel aquaporin-2 (Aqp2) gene. Development of a treatment is rendered difficult due to the lack of a viable animal model. Through forward genetic screening of ethylnitrosourea-mutagenized mice, we report the identification and characterization of a mouse model of NDI, with an F204V mutation in the Aqp2 gene. Unlike previously attempted murine models of NDI, our mice survive to adulthood and more exactly recapitulate the human disorder. Previous in vitro experiments using renal cell lines suggest recessive Aqp2 mutations result in improper trafficking of the mutant water pore. Using these animals, we have directly proven this hypothesis of improper AQP2 translocation as the molecular defect in nephrogenic diabetes insipidus in the intact organism. Additionally, using a renal cell line we show that the mutated protein, AQP2-F204V, is retained in the endoplasmic reticulum and that this abnormal localization can be rescued by wild-type protein. This novel mouse model allows for further mechanistic studies as well as testing of pharmacological and gene therapies for NDI. + +Synopsis + + + +Nephrogenic diabetes insipidus (NDI) is a disease marked by excessive urination and thirst. Normally, the hypothalamus senses situations where water is limited and signals to the kidney to increase water reabsorption from urine. The signaling molecule secreted by the hypothalamus is arginine vasopressin (AVP), which binds to a specific protein on the surface of kidney cells, AVP receptor (AVPR2). AVP binding to its receptor on kidney cells begins a series of biochemical events that ultimately results in the insertion of a protein, aquaporin 2 (AQP2), into the outer surface of the kidney cell. As its name suggests, AQP2 facilitates the reuptake of water from the urinary space into the cell, thus concentrating the urine and conserving water. Congenital NDI is caused by mutations in either the water channel, AQP2, or in the receptor, AVPR2. While these mutations have been studied extensively in the lab, work in live animals has been very limited. This report describes the first viable mouse model of NDI. Previous models have been attempted by targeted mutation, i.e., genes known to be involved in the disease have been altered in the mouse, a so-called reverse genetic approach. Reverse genetic approaches have so far failed to produce a viable mouse model of NDI. Here the authors take a forward genetic approach in which genes are mutated at random and animals are screened for disease-like properties. As well as proving hypotheses that come from lab studies, this model opens the door to the testing of gene therapy or other therapies for treatment of NDI. + +Introduction + +Nephrogenic diabetes insipidus (NDI) is a disease characterized by excessive urination and thirst, despite normal production of the antidiuretic hormone arginine vasopressin (AVP) [1]. The inherited forms are either X-linked as a consequence of mutation of the Avpr2 gene [2], or autosomal due to mutation of the Aqp2 gene [3]. Aquaporin-2 (AQP2) is a pore-forming protein belonging to a family of water channels [4], and it is expressed in collecting-duct principal cells in the kidney [5]. Generally these proteins permit the passage of water through the plasma membrane (PM) of cells, several of which carry out this role specifically in the process of water reabsorption from urine in the kidney. It has been established that aquaporins, although functional as a monomer, tetramerize before their insertion into the plasma membrane [4,6]. Furthermore these proteins can also be differentially targeted to distinct regions of the PM; for example, AQP2 is routed to the apical membrane of cells surrounding the collecting duct, whereas other aquaporins (AQP3 or 4) are inserted into the basolateral face. Unlike all other family members, AQP2 is not constitutively inserted into the plasma membrane. Under basal conditions, the protein resides in subapical intracellular vesicles; however, under conditions requiring water retention AQP2 translocates to the apical membrane, permitting water reabsorption [7,8]. For this process to occur, AVP binds its receptor, AVPR2, on the basolateral face of the collecting duct cells, leading to a rise in intracellular cAMP, ultimately resulting in phosphorylation of AQP2 at serine 256 by cAMP-dependent protein kinase [9] and its redistribution to the plasma membrane. + +The importance of AQP2 redistribution has been highlighted by functional characterization of Aqp2 mutations resulting in severe NDI in humans [3,10]. Recessive Aqp2 mutations are generally thought to produce an abnormally localized and, in most instances, misfolded water pore that responds abnormally to an increase in cAMP [6,11]. Furthermore, dominant mutations have been described and found to misroute both the mutant and the wild-type protein to the basolateral membrane [6,12]. + +Several mouse models of diabetes insipidus have been generated [13–17]. In an attempt to recapitulate human NDI, mice have been generated with mutations in Aqp2 and Avpr2 [15,18]. Yang and colleagues created a mouse with a T126M knock-in mutation in the Aqp2 gene. Unexpectedly, homozygous mutant mice died within 6 d after birth. Interestingly, AVPR2-deficient male pups also die within the first week after birth. Together these models suggest that the mouse may be a highly sensitive organism with regard to water homeostasis, and is unable to survive with polyuria. + +In a forward genetic screen, a mouse with an Aqp2 mutation was identified. The purpose of this study was to characterize this murine model of recessive nephrogenic DI. We now report a novel F204V mutation in the Aqp2 gene. This allele of Aqp2 was found to cause the first mouse model of NDI to survive past the first week of life. Molecular analyses concluded that mutant AQP2 adopts a different subcellular localization in renal collecting-duct cells, and was resistant to translocation induced by desmopressin, an agonist of AVP. In vitro studies using the Madin-Darby canine kidney (MDCK) cell line demonstrated an endoplasmic reticulum pattern for the mutant protein, and apparent resistance to translocation. These data conclusively prove that autosomal recessive NDI is a consequence of improper AQP2 routing in the intact mammal. + +Results + +In a forward genetic screen that used ethylnitrosourea (ENU) to induce mutations in a founder animal whose offspring were then screened for abnormal whole body metabolism [19,20], we found a family of mice that urinated and drank excessively. Serum and urine analysis showed that plasma glucose levels were normal and there was no glucose in the urine (unpublished data). Hence, this was an example of diabetes insipidus. + +The disorder in these mice segregated in a monogenic, autosomal recessive manner, making Aqp2 a candidate gene. Sequencing of Aqp2 coding region of affected mice identified a thymine to guanine (T to G) transversion (Figure 1A), which is predicted to lead to a valine for phenylalanine substitution at amino acid 204 of the protein (F204V). + +AQP2 is a six-transmembrane water channel, and F204 lies near the extracellular face of the sixth membrane spanning domain, a region rich in hydrophobic amino acids. This and the other membrane-spanning domains are conserved among vertebrate species. The phenylalanine at position 204 is particularly well conserved (Figure 1B), not only among vertebrate AQP2 proteins, but also among others members of this family. + +Aqp2F204V/F204V mice have dramatically increased urine production, in some cases producing an amount of urine in 24 h that exceeds their body weight, compared to their heterozygous or wild-type littermates. Such loss of water would rapidly lead to dehydration were it not compensated by increased water intake. Indeed, mutant mice also dramatically increase their water intake (Figure 1C) compared to their heterozygous or wild-type littermates. This phenotype—increased urinary output and water intake—showed complete concordance with homozygosity of the F204V mutation in the 58 animals tested. + +Diabetes insipidus can be defined as an inability to concentrate urine where appropriate. Compared to wild-type or heterozygous littermates, Aqp2F204V/F204V mice produce very dilute urine (Figure 1D). Basal urine concentration in mutant mice is about 161 mOsm, compared to about 1,293 mOsm in wild-type mice (p < 0.001). Normally, urine concentration is under the control of the hypothalamus, which, in response to hypovolemia or hypernatremia [21], secretes AVP. The synthetic AVP analog, 1-deamino-8-D-arginine vasopressin (dDAVP; also called desmopressin), is a potent agonist of AVPR2. When administered to wild-type mice, dDAVP leads to a dramatic increase in urine concentration, from 1,293 to 5,885 mOsm (4.6-fold; Figure 1D). With similar treatment, mutant mice concentrate their urine to a lesser but still significant extent, from 161 to 470 mOsm (2.9-fold), indicating that these animals are not only unable to concentrate their urine properly but are also defective in their response to dDAVP. The smaller response to dDAVP indicates some residual activity of the mutant AQP2 channel, which must be sufficient to allow survival of the individual, in contrast to the T126M knock-in mouse [18]. + +Multiple heterozygous matings yielded 101 animals, which appeared at a ratio of 26:49:26, near the expected Mendelian wild type, heterozygote, and mutant frequencies, respectively, indicating that there is no reduced viability associated with this mutation. Other than the increased urine production and water intake, there was no overt phenotype in mutant mice, save distended kidneys, which appeared variably in adult animals (Figure 2A). Although not specifically measured, mutant mice seem to have a normal lifespan. The one animal that was followed lived to 18 mo, typical for animals in our colony. + +Aqp2F204V/F204V mice suffer from severe hydronephrosis (Figure 2A and 2B), presumably as a consequence of an inability to cope with the extreme polyuria. We found distended kidneys in all Aqp2F204V/F204V mice; however, the degree of inflation was variable in affected mice and worsened with age. Severe hydronephrosis has previously been observed in double Aqp1/Aqp3 knock-out mice [17], and appears at 6 wk. Even at 4 wk, Aqp2F204V/F204V mice had hydronephrosis. Histologic sections from Aqp2F204V/F204V mice demonstrated marked dilatation of the renal pelvis yet normal morphology of the ureter (Figure 2C and 2D). In particular, the muscularis propria was neither hypertrophied nor thinned. There was the normal festooned appearance of the urothelium, and this transitional epithelium was of normal thickness. There was thinning of the kidney as measured from renal capsule to renal pelvis. However, the morphologic features of the glomeruli and proximal/distal tubules were unremarkable (Figure 2D). + +As shown previously [18,22], immunoblotting revealed three different forms of AQP2, due to different degrees and forms of glycosylation (Figure 3A). Previous reports have demonstrated that nonglycosylated protein appears as a 29 kDa band, while complex glycosylated protein runs as a smear between 35 and 45 kDa. A short-lived intermediate form of 31 kDa representing core, high-mannose glycosylation of AQP2 is apparent from pulse-chase labeling experiments [22]. Compared to that from the kidneys of wild-type animals, AQP2 from mutant animals was reduced in both the high molecular weight, diffuse form and the lowest molecular weight form, but enriched in the intermediate molecular weight form (Figure 3A). Heterozygous animals showed intermediate amounts of all three forms. The nature of these glycosylated forms was revealed by digestion with endoglycosidase H, which specifically cleaves mannose-rich carbohydrate from the protein backbone. Treatment of endogenous AQP2 from kidneys of wild-type, heterozygous, and mutant animals specifically affected the intermediate molecular weight form (Figure 3B). The presence of some mature glycosylated proteins (35–45 kDa) in Aqp2F204V/F204V mice presumably permits their survival compared to Aqp2T126M/T126M mice, and is consistent with a diminished response to dDAVP. + +In humans, recessive alleles of Aqp2 are postulated to cause NDI because they do not properly translocate to the apical cell surface in response to AVP. This postulate comes solely from in vitro studies in which mutant Aqp2 cDNAs corresponding to human disease mutations are transfected into kidney cell lines. In general, such recessive alleles, when visualized immunocytochemically, fail to localize to AVP-responsive vesicles. Rather, they get trapped in the endoplasmic reticulum (ER). Our mouse model of NDI affords the first opportunity to test this hypothesis in a mature animal. As shown in Figure 4A (top row of photomicrographs), AQP2 (stained red) normally localized to the subapical region of collecting duct cells in kidneys of wild-type mice. Upon stimulation with dDAVP, AQP2 translocated to or near the cell surface (Figure 4A, second row). In kidneys taken from mutant animals, however, AQP2 was distributed randomly throughout the cell in the basal state (Figure 4A, third row), while AQP3 (green) appropriately localized to the basolateral surface [23]. Furthermore, upon dDAVP stimulation, AQP2-F204V failed to translocate to the cell surface (Figure 4A, bottom row). To confirm these findings, the staining was repeated in kidneys taken from two further mice for each class, wild-type or mutant, with or without dDAVP treatment, with identical results. + +To investigate the mechanism of defective translocation of AQP2-F204V, we turned to transfection of MDCK cells. Stable cell lines expressing mouse wild-type AQP2 and AQP2-F204V were established. Immunoblots of protein extracts from stable cell lines showed that MDCK cells recapitulate the glycosylation defect seen in mutant mice (unpublished data). The wild-type protein was again present in three different forms. Cells expressing AQP2-F204V lacked the 35–45 kDa form and were enriched in the core-glycosylated 31 kDa form. + +In transfected, unstimulated MDCK cells, wild-type AQP2 (stained green) appeared in a punctate pattern distributed throughout the subapical region (Figure 4B, left column photomicrographs), consistent with vesicular compartmentalization. AQP2-F204V, on the other hand, appeared in a punctate but perinuclear pattern (Figure 4B, third column). Upon stimulation with forskolin, a cAMP-dependent protein kinase activator, wild-type AQP2 translocated to the apical surface of polarized MDCK cells (Figure 4B, second column). Along the z-axis, the perinuclear distribution of AQP2-F204V was clearly seen, and this distribution is not altered by forskolin (Figure 4B, bottom row, two right columns). The perinuclear distribution of AQP2-F204V is consistent with an ER compartmentalization. To test the idea that AQP2-F204V localizes to the ER, we co-stained cells transfected with Aqp2F204V (cDNA) for AQP2 and an ER marker, calnexin (Figure 4C). Colocalization of calnexin with AQP2 was investigated directly, and it was found that 80% of all AQP2-F204V protein colocalized with calnexin. The remaining 20% appeared at the periphery of the ER, representing AQP2-F204V that had potentially progressed beyond the ER. This “ER escape” was consistent with the small proportion of mature, complex glycosylated, AQP2-F204V in mutant kidneys (see Figure 3A). + +Animals heterozygous for the Aqp2F204V mutation were not affected in their urine production or urine osmolality (see Figure 1C and 1D). It has also been shown that a recessive NDI allele, AQP2-R187C, does not interact with wild-type protein in oocytes [24], nor does it homo-oligomerize in MDCK cells [22]. Therefore, kidneys from heterozygous animals were examined for evidence of two populations of AQP2 protein. Surprisingly, immunohistochemical staining of kidney collecting ducts from Aqp2F204V/+ mice revealed a pattern remarkably similar to wild type (Figure 5A). AQP2 translocated completely to the apical cell surface upon dDAVP stimulation. This wild-type staining pattern may simply reflect the fact that decreasing the amount of mutant protein by half makes it undetectable by immunocytochemistry. Alternatively, the presence of wild-type protein may alter the localization of the mutant protein. Indeed, Hendriks et al. proposed a “piggy-back” mechanism to explain the transport of nonglycosylated subunits of AQP2 to the cell surface by glycosylated subunits [22]. It has also been shown that wild-type AQP2 protein can rescue a translocation-defective mutant protein, AQP2-P262L, when the two are coexpressed in MDCK cells [25]. + +In the collecting ducts from Aqp2F204V/+ mice, the wild type may rescue the mutant protein as suggested by the subcellular distribution of AQP2 protein. To test this idea, we first looked for an interaction between mutant and wild-type proteins in transfected MDCK cells (Figure 5B). MDCK cells stably expressing wild-type AQP2 were transiently transfected with GFP expression constructs encoding GFP-tagged wild-type AQP2, AQP2-F204V, or GFP alone. Antibodies against GFP coimmunoprecipitated wild-type AQP2 when AQP2-GFP or AQP2-F204V-GFP was transiently transfected, but not when GFP by itself was transiently transfected into MDCK cells stably expressing wild-type AQP2 (Figure 5B, upper blots). Western blot of total membranes showed that wild-type AQP2 is equivalently expressed in all three cases (Figure 5B, lower blots). + +If wild-type and mutant proteins are indeed interacting in the cell, is this interaction sufficient to rescue the localization of mutant protein? To answer this question, we used MDCK cells stably transfected with wild-type AQP2 expressing vector or with empty vector. On top of these, we transiently transfected AQP2-GFP or AQP2-F204V-GFP expression constructs. AQP2-GFP localized to the apical surface upon forskolin stimulation whether it was transiently transfected into vector-only cells (Figure 5C, upper left images) or into wild-type AQP2 cells (Figure 5C, upper right). AQP2-F204V-GFP, when expressed by transient transfection into vector only cells, showed a diffuse cytoplasmic distribution pattern (Figure 5C, lower left). When expressed in wild-type AQP2 cells, however, AQP2-F204V-GFP localized to the apical surface to varying degrees (Figure 5C, lower right images [i–iii]). The lower right images of Figure 5C shows three cells from a single transfection. The first is a nontransfected cell that shows the localization of the stably expressing wild-type AQP2, which is apical upon forskolin stimulation. The next two show expression of both the stable wild-type AQP2 and the transient AQP2-F204V-GFP. In cell (ii), localization of wild-type AQP2 was indistinguishable from AQP2-F204V-GFP; both were apical upon forskolin stimulation. Although the effect was subtle in cell (iii), AQP2-F204V-GFP was partly localized to the apical surface. Generally, the localization of AQP2-F204V-GFP was clearly more apical when wild-type AQP2 was also expressed. + +To confirm these results biochemically, we transfected the same cell lines (wild-type AQP2 or vector) with F204V-GFP, biotinylated surface proteins after forskolin stimulation, and precipitated the biotinylated proteins (Figure 5D). AQP2-F204V-GFP is expressed approximately equally in both cell lines (Figure 5D, total cells), but is biotinylated only when wild-type AQP2 is also expressed (Figure 5D, surface biotinylated). Since only cell surface proteins are accessible to biotin, these results indicate that AQP2-F204V is transported to the cell surface when wild-type AQP2 is present, but not on its own. + +Discussion + +Aqp2F204/F204V mice are viable and grow and reproduce normally. They are, however, severely defective in their ability to concentrate urine, leading to increased urine output and water intake, thus making them the first mouse model of NDI to survive to maturity. In humans, NDI is caused by mutations in Avpr2 or Aqp2. Knockout of the X-linked Avpr2 gene in mice [15] gave an NDI-like phenotype in male, hemizygous neonates, but the phenotype could not be assessed in adults as the mice died within 1 wk of birth. The adult heterozygous females showed a mild tendency toward increased urinary output and water intake and decreased urine osmolality. Knockout of the mouse Aqp2 gene has not been reported. A knock-in of a human disease-causing mutation (T126M), however, has been made [18]. These mice have a severe urine-concentrating defect resulting in dehydration and death within 1 wk of birth. Curiously, AQP2-T126M does localize properly in at least a subset of cells. The grossly abnormal collecting duct morphology makes it impossible to pinpoint the molecular defect in these knock-in mice. + +The T126M knock-in clearly shows that Aqp2 is an essential gene [18]. The fact that our mice survive shows either that AQP2-F204V possesses some residual water transporting ability or that there are AVP-independent pathways for water reabsorption. Residual activity of AQP2-F204V is likely, as mutant animals show some small response to dDAVP, although dDAVP-stimulated urine osmolality remains quite low. Immunostaining of kidney shows that AQP2-F204V does not efficiently transport water, because it fails to localize to the apical cell surface after dDAVP treatment. Some residual activity of AQP2 would imply that some small, undetectable portion of the mutant protein is getting to the cell surface. The surface biotinylation experiment (Figure 5D) suggests that no mutant protein gets to the surface, but this does not necessarily reflect the situation in vivo. While this small fraction of protein may not be detectable by immunofluorescence, Western blotting shows that some mutant protein does progress beyond the ER (35–45 kDa species in Figure 3A). Compared to wild-type, mutant protein is enriched in the high-mannose, core-glycosylated form (31 kDa) and deficient in nonglycosylated (29 kDa) and complex glycosylated (35–45 kDa) forms. The presence of a reduced but detectable amount of protein in the 35–45 kDa range indicates that mutant protein is transported out of the ER, but with greatly reduced efficiency. Colocalization of AQP2-F204V with the ER protein calnexin in transfected MDCK cells shows that, while most of the mutant protein is trapped in the ER, some does progress beyond the ER. Diminished response to dDAVP, diminished abundance of mature glycosylated protein in mutant animals, and the transport of a fraction of mutant protein beyond the ER in MDCK cells are all consistent with the notion that AQP2-F204V misfolding is limited and that it may retain some residual water transporting activity. Evidently this residual activity is sufficient for the viability and growth of mutant animals. + +Reduced efficiency in exiting the ER may explain why AQP2-F204V is enriched in the 31 kDa high-mannose glycosylated form. The high-mannose core oligosaccharide is added in the ER and is later modified and elaborated in the Golgi apparatus [26]. The increase in the high-mannose glycosylated form of AQP2-F204V may simply reflect its prolonged presence in the ER and exposure to oligosaccharyl transferase. + +While improper localization of AQP2 explains the phenotype of homozygous mutant mice, the complete lack of a phenotype in heterozygous mice is more difficult to explain. Physiologically, heterozygous mice have no symptoms (see Figure 1C), and they are indistinguishable from wild type on immunostaining of kidneys (Figure 5A). The presence of 50% of the normal amount of wild-type protein may explain the lack of symptoms, but it cannot explain the lack of any ER-retained mutant protein. Rather, the phenotype of the Aqp2F204V/+ animals suggests that the mutant protein is being rescued by the wild-type protein. Indeed, de Mattia et al. (26) have demonstrated that one recessive allele of Aqp2, P262L, does not properly translocate when expressed by itself in MDCK cells, but that in the presence of wild-type protein, it localizes normally. The same mechanism seems to apply in vivo with Aqp2F204V/+ mice. In support of this, AQP2-F204V can interact with wild-type AQP2 (Figure 5B), and when coexpressed with wild-type protein, AQP2-F204V can reach the cell surface (Figure 5C bottom right panel and 5D). Although it has been demonstrated that a recessive allele (encoding AQP2-R187C) of NDI fails to interact with wild-type AQP2 [6], here we show that AQP2-F204V does interact with the wild-type protein, presumably as part of heterotetramers, and represents a rescuable allele, both in vitro and in vivo. + +Immunostaining the kidneys of homozygous Aqp2F204V/F204V mice shows that the mutant-expressing collecting duct cells can not mediate water reabsorption, because it fails to insert into the apical plasma membrane in response to dDAVP. This is this first in vivo proof of a long-standing hypothesis that comes from in vitro studies with recessive Aqp2 mutations. Transfection into MDCK cells of any of several Aqp2 mutations corresponding to recessive human alleles shows abnormal subcellular localization [25], [27] and failure to appropriately translocate to the plasma membrane. Thus, misfolding, retention in the ER, and failure to translocate in response to dDAVP were proposed as the mechanism for autosomal recessive NDI. Here we not only prove this hypothesis but also establish a useful model for human NDI. This mouse model of NDI based on an Aqp2 allele that can be rescued provides the opportunity to test therapies, including gene therapy, that may promote proper subcellular localization. + +Materials and Methods + +Generation of ENU mice and housing. + +ENU mutagenized C57BL/6 mice were generated as described [19]. Mice were maintained by backcrossing affected animals to C57BL/6 and housed in the Genomics Institute of the Novartis Research Foundation Specific Pathogen Free animal facility (La Jolla, California, United States). All procedures were approved by the Genomics Institute of the Novartis Research Foundation Institutional Animal Care and Use Committee. + +Constructs. + +The complete coding sequence of mouse AQP2 from an IMAGE clone was digested from the pCMV⋅SPORT6 plasmid with EcoRI and NotI and ligated into pcDNA3.1 (Invitrogen, Carlsbad, California, United States). The F204V mutation was introduced by site-directed mutagenesis (Stratagene, La Jolla, California, United States), using the sense oligonucleotide 5′-GATGATCACTGGGTCGTCTGGATCGGACCCC-3′, and antisense oligonucleotide 5′-GGGGTCCGATCCAGACGACCCAGTGATCATC-3′. To generate GFP fusions of AQP2, the pCMV⋅SPORT6 AQP2 construct was used in a PCR reaction with the primers Sp6 and 5′-GACTGGATCCCGGCCTTGCTGCCGCGCGGCAG-3′ to remove the stop codon of AQP2. The product was digested with KpnI and BamHI and ligated into pEGFP-N2 (BD Biosciences, San Diego, California, United States). The F204V mutation was introduced using the same mutagenic oligonucleotides. + +Cell culture and generation of stable cell lines. + +MDCK cells (CCL-34; ATCC, Manassas, Virginia, United States) were cultured in DMEM (Sigma-Aldrich, St. Louis, Missouri, United States) supplemented with 10% FBS (Sigma-Aldrich), 100 U/ml of penicillin, and 100 μg/ml of streptomycin at 37 °C in 5% CO2. To generate stable MDCK cell lines, cells were transfected using Lipofectamine 2000 (Invitrogen) and the pcDNA3.1 expression constructs (containing wild-type AQP2, AQP2-F204V, or no insert) and selected with 900 μg/ml G418 (Sigma-Aldrich). Individual colonies were expanded 14 d later. For the duration of these experiments, the antibiotic was continually added to the media. Transient GFP transfections were carried out in subconfluent stable cells lines also using Lipofectamine 2000. + +Sequencing of Aqp2 and genotyping of mice. + +All exons of Aqp2 were amplified from mouse genomic DNA and sequenced. For genotyping, exon 4 was amplified using the primers 5′-TCAGAACTTGCCCACTAGCC-3′ and 5′-TGTAGAGGAGGGAACCGATG-3′. + +Urine measurements. + +Total urine output was measured by separately housing adult mice in Nalgene Metabolic Cages (Minimitter, Bend, Oregon, United States) for 2–3 d and collecting urine every 24 h period. Urine osmolalities were determined using an Osmometer (Osmette 5004; Precision Systems, Natick, Massachusetts, United States). Urine concentrating experiments were carried out by intraperitoneal injection of dDAVP (0.4 μg/kg). Mice were injected twice with dDAVP, once at time 0 and again at 30 min. Urine was collected at the start of the experiment and 30 min after the second injection. + +Kidney membrane preparation. + +Whole mouse kidneys were homogenized in 10 mM Tris (pH 7.4), 350 mM sucrose, and 5 mM EDTA containing protease inhibitors (Sigma-Aldrich, #P-8340) in a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 2,000 g for 10 min and the supernatant was subjected to ultracentrifugation at 100,000 g for 1 h at 4 °C. Pelleted membranes were resuspended in the same buffer, and protein concentration was determined by Bradford assay. + +Immunoblotting. + +Kidney membrane fractions (60 μg) were resolved on a 12% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane. Membranes were blocked in 5% nonfat milk in Tris-buffered saline with 0.05% Tween 20 (TBST), followed by an overnight incubation (at 4 °C) with AQP2 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, California, United States; #sc-9882). Membranes were washed in TBST then incubated with HRP-conjugated donkey anti-goat antibody. Membranes were washed further in TBST and bands were visualized using ECL reagent (Amersham Biosciences, Little Chalfont, United Kingdom). + +Endoglycosidase digestion. + +Kidney membranes (60 μg) were incubated in 50 mM sodium phosphate (pH 5.5), 0.1% SDS, and 50 mM β-mercaptoethanol, heated to 100 °C for 5 min, then cooled. Endoglycosidase H (0.01 units; Sigma-Aldrich) was added and incubated at 37 °C for 2 h. The reaction was stopped by boiling the samples in Laemmli buffer. Total reactants were immunoblotted as described above. + +Coimmunoprecipitation and biotinylation in MDCK cells. + +MDCK cells stably expressing wild-type AQP2 (grown on 10-cm plates) were transfected with pEGFP-wild-type AQP2, pEGFP-AQP2-F204V, or vector alone. The cells were homogenized in 10 mM Tris (pH 7.4), 1 mM EDTA, and 250 mM sucrose 40 h later. The clarified supernatant was centrifuged at 200,000 g for 30 min. Pelleted membranes were resuspended in the same buffer but containing 4% sodium deoxycholate and incubated at 37 °C for 1 h. From the dissolved membranes, a 30 μl sample was removed and used as the total membrane fraction. The remaining membranes were diluted with 600 μl of the homogenization buffer, and incubated with 1 μl of GFP antisera (Invitrogen, #46–0092) and protein A/G sepharose. Following overnight incubation, the precipitated proteins were washed in RIPA buffer and finally boiled in 50 μl of Laemmli buffer. Half of the total membrane and the IP fractions were processed for immunoblotting. + +Cell surface biotinylation was performed in a similar manner. However, pEGFP-AQP2-F204V, was transfected into MDCK cells stably expressing wild-type AQP2 and cells made stable with vector alone. Twenty-four hours post-transfection, cells were stimulated with forskolin, trypsinized, resuspended in 1 ml of PBS (2.5 × 106 cells/ml), and incubated with 0.5 mg of NHS-PEO4-biotin (Pierce Biotechnology) for 30 min at room temperature. Cells were washed once in 10 mM Tris (pH 8) and three times in PBS, after which membranes were purified and solubilized as described above. Solubilized membranes were incubated with 20 μl of immobilized streptavidin (Pierce Biotechnology) for 2 h at 4 °C. Finally the precipitated proteins were washed in RIPA buffer and boiled in 50 μl of Laemmli buffer. Total cells and the biotinylated precipitates were immunoblotting using an antibody to AQP2. + +Kidney immunohistochemistry. + +Whole mouse kidneys were fixed in 10% phosphate-buffered formalin for 24 h. Kidneys were embedded in paraffin, and 5-μm sections were prepared. Following antigen retrieval using 10 mM sodium citrate (pH 8) for 10 min at 98 °C, sections were sequentially probed, first for AQP3 and then for AQP2. Sections were incubated in 5% donkey serum and then in goat anti-AQP3 antibody (1:100; Santa Cruz Biotechnology; #sc-9885). Slides were washed with PBS and incubated with AlexaFluor 488-conjugated donkey anti-goat antibody (Molecular Probes, Eugene, Oregon, United States). The slides were subsequently blocked in 5% chicken serum, incubated with a rabbit anti-AQP2 antibody (1:250; USB, Cleveland, Ohio, United States; #A3000–06), which was detected with a AlexaFluor 594-conjugated chicken anti-rabbit antibody (1:500; Molecular Probes). The sections were stained with DAPI and mounted in Vectashield (Vector Labs, Burlingame, California, United States). + +Immunocytochemistry on MDCK cells. + +MDCK stable cell lines expressing vector alone, wild-type AQP2, or AQP2-F204V (and in some cases transiently expressing a GFP construct) were grown on fibronectin-coated coverslips until tight junctions formed. Cells were treated with or without 150 μM forskolin for 90 min, and fixed in methanol at −20 °C. Subsequently, cells were washed and permeabilized in 0.2% Triton X-100 for 5 min, and sequentially probed for AQP2 and organelle markers for either the PM or the ER. AQP2 was detected using goat anti-AQP2 (1:100; Santa Cruz Biotechnology; #sc-9882) and a 1:200 dilution of AlexaFluor 488-conjugated donkey anti-goat secondary antibody. The PM and ER were probed using mouse anti-Na+/K+-ATPase (Upstate, Waltham, Massachusetts, United States) or rabbit anti-calnexin (Stressgen Biotechnology, Victoria, British Columbia, Canada) antibodies and the secondary antibodies, Cy3-conjugated goat anti-mouse (1:200; Jackson ImmunoResearch, West Grove, Pennsylvania, United States) or AlexaFluor 594-conjugated chicken anti-rabbit (1:200) respectively. Cells were washed in PBS, counterstained with DAPI, and mounted in Vectashield. In experiments in which GFP fusions were used, AQP2 was probed using the antibody combination used for kidney immunohistochemistry in order to detect the AQP2 at 594 nm, to distinguish between the GFP fusion proteins. + +Confocal microscopy. + +Optical z-section images were collected on a BioRad (Hercules, California, United States) Rainbow Radiance 2100 Laser Scanning Confocal Microscope. Image stacks were flattened, or sectioned along the z-axis, then further processed using BioRad Laser Sharp 2000 software and Image J software (v. 1.32; National Institutes of Health). Colocalization was performed using the overlay coefficient of Image J software. + +Supporting Information + +Accession Numbers + +The GenBank (http://www.ncbi.nlm.nih.gov/) accession number of Aqp2 is NM_009699. The IMAGE (http://image.llnl.gov) accession number of AQP2 is 4222942. + +Acknowledgements + +We thank Debby Stradley for all genotyping, Lacey Kischassey for breeding and care of mice, Karina Ayala and Sandy Bohan for phenotyping the study mice, Miah Gilmore for performing endoglycosidase H experiments, James Watson for sectioning tissue, and Dr. William Kiossis for collecting confocal images. + +Abbreviations + +AQP[number] - aquaporin-[number] + +AVPR2 - AVP type 2 receptor + +AVP - arginine vasopressin + +dDAVP - 1-deamino-8-D-arginine vasopressin + +ER - endoplasmic reticulum + +MDCK - Madin-Darby canine kidney + +NDI - nephrogenic diabetes insipidus + +PM - plasma membrane + +Figures + +Figure 1 + +Analysis of Aqp2 Sequence and Phenotype in Mutant Mice + +(A) Chromatographic traces of Aqp2 F204V mutation. The box shows the mutated codon, TTC (Phe) to GTC (Val) at position 204. + +WT, wild type; Mut, mutant. + +(B) Amino acid conservation of mouse AQP2 (residues 194–214). The boxed residue indicates phenylalanine at position 204. + +hAQP2, human AQP2; mAQP1, mouse AQP1; mAQP2, mouse AQP2; rAQP2, rat AQP2; xAQP2, Xenopus AQP2. + +(C) Urine production (ml) and water consumption (ml) of 58 F2 mice over a 24-h period (both sexes, aged 10–22 wk). Mutant mice (black squares) exhibit overt polyuria and polydipsia compared to littermate wild-type (white triangles) and heterozygous (grey circles) mice. + +(D) Urine osmolality and concentrating ability in Aqp2 mutant and their littermates (10–22 wk, both sexes), before (white bars) and after (black bars) dDAVP treatment. Wild type (WT; n = 12); heterozygote (Het; n = 20); mutant (Mut; n = 9). Data represent averages ± standard error of the mean, **p < 0.01; ***p < 0.001. + +Figure 2 + +Anatomy and Histology of Mouse Kidneys + +(A) Gross anatomy of an affected mouse (8-mo-old male). This shows the enlargement and cystic dilatation of the renal pelvis. There is thinning of the overlying renal parenchyma imparting a translucent appearance to portions of the kidney and collecting system. The bladder is also dilated. + +(B) Left kidney from mutant mouse (right) shown in (A) compared to a kidney from an age-sex matched unaffected littermate (left). + +(C) Hematoxylin and eosin stained section of ureter from a mutant mouse, showing normal histology despite bloating of the kidney. + +(D) Hematoxylin and eosin stained histologic section of a kidney from a 4-wk-old female mutant mouse. The mutant kidney shows marked dilatation of the renal pelvis with blunting of the papilla. There is preservation of the cortex and medulla. + +Figure 3 + +Immunoblot Analyses of AQP2 from Mouse Kidneys + +(A) Western blot analyses of total kidney membranes from littermate mice. An intermediate form of AQP2 at 31 kDa was identified in kidney membranes from a mutant mouse (Mut) and partially in a heterozygous mouse (Het). + +(B) Total kidney membranes were subjected to endoglycosidase H treatment (Endo H) prior to Western blotting. High-mannose (h.m.) glycosylated proteins that have not exited the ER are sensitive to endoglycosidase H digestion. + +Figure 4 + +AQP2 Subcellular Localization and Translocation in Mouse Kidney Collecting Ducts and MDCK Cell Lines + +(A) Immunohistochemistry on collecting ducts in kidney sections from an AQP2-F204V mutant (Mut) mouse and an age-sex matched wild-type (WT) littermate. Mice were injected intraperitoneally with PBS (NT) or dDAVP before sacrificing and fixation of the kidneys. Kidneys sections were immunostained for AQP2 (red) and the basolateral marker AQP3 (green). The images were merged and an area of the cytoplasm was magnified (zoom). Note that mutant AQP2 is not properly localized to the subapical compartment, nor does it respond to dDAVP. + +(B) MDCK cell lines, stably transfected with constructs encoding mouse WT or AQP2-F204V, were treated with and without 150 μM forskolin for 90 min, after which cells were fixed, permeabilized, and subjected to immunocytochemistry. AQP2 is shown in green, and the basolateral marker Na+/K+-ATPase is shown in red, alongside the nuclear stain DAPI. The z-profile images were reconstructed from multiple z-sections, along the dotted line. Mutant AQP2 fails to localize to the cell surface upon forskolin stimulation. Rather, the perinuclear staining is consistent with an ER localization of mutant AQP2. + +(C) The MDCK cell line expressing AQP2-F204V was grown on fibronectin-coated coverslips until tight junctions formed, at which point the cells were treated with 150 μM forskolin for 90 min. Cells were fixed, permeabilized, and sequentially immunoblotted for AQP2 (green) and calnexin (red), an ER marker. The merged image shows that AQP2-F204V colocalizes with the endoplasmic reticulum marker. Scale bar refers to 10 μm. + +Figure 5 + +AQP2-F204V Rescue in Heterozygous Mouse Collecting Ducts and in Cotransfected MDCK Cells + +(A) In heterozygous animals, AQP2 localizes and responds to dDAVP normally. Immunohistochemistry was carried out on kidney sections from an Aqp2F204V/+ mouse, after injection with dDAVP. Kidney sections were sequentially immunostained for AQP2 (red) and the basolateral marker AQP3 (green). + +(B) Mutant and wild-type AQP2 physically interact. MDCK cells stably expressing wild-type AQP2 were transiently transfected with GFP tagged wild-type AQP2, AQP2-F204V, or GFP alone. Solubilized membranes were immunoprecipitated with a GFP antibody. Total membranes and immunoprecipitates (GFP-IP) were Western blotted using an antibody against AQP2 (arrow) or AQP2-GFP fusions (arrowhead). + +(C) Wild-type AQP2 rescues the localization defect of mutant AQP2. GFP fusions of either wild-type AQP2 (WT-GFP, top photomicrographs) or F204V AQP2 (F204V-GFP, bottom photomicrographs) were expressed in polarized MDCK stable cell lines expressing vector alone (vector, left photomicrographs) or AQP2-WT (right photomicrographs). Cells were stimulated with forskolin, processed for immunocytochemistry, and used to generate z-sectional images. + +(D) Mutant AQP2 is present at the cell surface in cells coexpressing wild-type AQP2 (AQP2-WT). GFP fused to AQP2-F204V was expressed in MDCK cells expressing wild-type AQP2 or vector alone. Cells were stimulated with forskolin, and cell surface biotinylated proteins were precipitated then analyzed for the presence of wild-type AQP2 (arrow) and AQP2-F204V (arrowhead) by Western blot. + +Footnotes + +Competing interests. The authors have declared that no competing interests exist. + +Author contributions. DJL and NG conceived and designed the experiments. DJL performed the experiments. DJL, FWH, and NG analyzed the data. DJL and LMT contributed reagents/materials/analysis tools. DJL and NG wrote the paper. + +Citation: Lloyd DJ, Hall FW, Tarantino LM, Gekakis N (2005) Diabetes insipidus in mice with a mutation in aquaporin-2. 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UBERON:0001348 8110 8119 brown fat +T193 UBERON:0002113 8121 8127 kidney +T194 UBERON:0000948 8129 8134 heart +T195 UBERON:0004288 8136 8144 skeletal +T196 UBERON:0002107 8157 8162 liver +T197 GO:0010467 8176 8186 expression +T198 UBERON:0000955 8194 8199 brain +T199 UBERON:0001347 8201 8210 white fat +T200 UBERON:0000473 8216 8222 testes +T201 GO:0008380 8291 8298 spliced +T202 SO:1001187 8291 8298;8305 8315 spliced ... transcript +T203 SO:0000673 8326 8337 transcripts +T204 NCBITaxon:10088 8391 8395 mice +T205 GO:0000184 8510 8537 nonsense mediated RNA decay +T206 NCBITaxon:10088 8614 8618 Mice +T207 SO:0000673 8626 8633 message +T208 CHEBI:33695 8626 8633 message +T209 UBERON:0000948 8656 8661 heart +T210 UBERON:0002107 8663 8668 liver +T211 UBERON:0001348 8670 8679 brown fat +T212 UBERON:0000955 8681 8686 brain +T213 UBERON:0002113 8688 8694 kidney +T214 UBERON:0001347 8712 8721 white fat +T215 UBERON:0000473 8726 8732 testes +T216 NCBITaxon:10088 8766 8770 mice +T217 GO:0010467 8784 8794 expression +T218 UBERON:0001348 8807 8816 brown fat +T219 UBERON:0002113 8818 8824 kidney +T220 UBERON:0000948 8826 8831 heart +T221 UBERON:0004288 8837 8845 skeletal +T222 NCBITaxon:10088 8862 8866 mice +T223 SO:0000673 8885 8892 message +T224 CHEBI:33695 8885 8892 message +T225 UBERON:0000479 8900 8907 tissues +T226 UBERON:0002107 8919 8924 Liver +T227 UBERON:0002107 8965 8970 liver +T228 UBERON:0001977 9002 9006 sera +T229 NCBITaxon:10088 9072 9076 mice +T230 NCBITaxon:10088 9097 9101 mice +T231 CHEBI:61905 9142 9162 short-chain acyl-CoA +T232 GO:0010467 9230 9240 expression +T233 NCBITaxon:10088 9285 9289 mice +T234 UBERON:0002107 9328 9333 Liver +T235 NCBITaxon:10088 9371 9375 Mice +T236 GO:0042571 9410 9418 antibody +T237 GO:0042571 9432 9440 antibody +T238 NCBITaxon:10088 9461 9465 mice +T239 UBERON:0002107 9608 9613 liver +T240 GO:0042571 9648 9656 antibody +T241 NCBITaxon:10088 9724 9728 mice +T242 GO:0042571 9788 9798 antibodies +T243 NCBITaxon:10088 9877 9882 mouse +T244 UBERON:0002107 9883 9888 liver +T245 CHEBI:10545 9911 9919 electron +T246 MOP:0000615 9911 9929 electron transport +T247 GO:0045251 9911 9942 electron transport flavoprotein +T248 CHEBI:5086 9930 9942 flavoprotein +T249 GO:0045251 9944 9947 ETF +T250 MOP:0000569 9949 9958 reduction +T251 CHEBI:15533 9970 9982 octanoyl-CoA +T252 CHEBI:15525 9994 10007 palmitoyl-CoA +T253 NCBITaxon:10088 10039 10043 mice +T254 MOP:0000590 10086 10099 dehydrogenate +T255 CHEBI:15533 10100 10112 octanoyl-CoA +T256 CHEBI:15525 10155 10168 palmitoyl-CoA +T257 NCBITaxon:10088 10189 10193 mice +T258 MOP:0000590 10223 10238 dehydrogenation +T259 CHEBI:15533 10242 10254 octanoyl-CoA +T260 CHEBI:15525 10259 10272 palmitoyl-CoA +T261 NCBITaxon:10088 10341 10345 mice +T262 http://purl.obolibrary.org/obo/MONDO_0008721 10408 10422 MCAD-Deficient +T263 NCBITaxon:10088 10423 10427 Mice +T264 GO:0016265 10682 10688 Deaths +T265 GO:0007567 10784 10788 born +T266 NCBITaxon:10088 10814 10818 mice +T267 NCBITaxon:10088 10840 10844 mice +T268 NCBITaxon:10088 10895 10899 mice +T269 GO:0016265 10937 10941 loss +T270 GO:0007618 11298 11305 matings +T271 GO:0007618 11329 11336 matings +T272 GO:0007618 11403 11410 matings +T273 http://purl.obolibrary.org/obo/MONDO_0008721 11496 11510 MCAD-deficient +T274 NCBITaxon:10088 11511 11515 mice +T275 NCBITaxon:10088 11570 11574 mice +T276 UBERON:0001977 11591 11596 serum +T277 CHEBI:17234 11597 11604 glucose +T278 UBERON:0001977 11618 11623 serum +T279 CHEBI:35366 11629 11639 fatty acid +T280 NCBITaxon:10088 11711 11715 mice +T281 NCBITaxon:10088 11769 11773 mice +T282 NCBITaxon:10088 11856 11860 mice +T283 UBERON:0001052 12022 12028 rectal +T284 NCBITaxon:10088 12052 12056 mice +T285 NCBITaxon:10088 12112 12116 mice +T286 UBERON:0001052 12126 12132 Rectal +T287 NCBITaxon:10088 12230 12234 mice +T288 NCBITaxon:10088 12292 12296 mice +T289 NCBITaxon:10088 12345 12349 mice +T290 UBERON:0001052 12403 12409 rectal +T291 CHEBI:64709 12435 12447 Organic Acid +T292 CHEBI:17387 12452 12465 Acylcarnitine +T293 UBERON:0001088 12476 12481 Urine +T294 CHEBI:64709 12482 12494 organic acid +T295 NCBITaxon:10088 12526 12530 mice +T296 CHEBI:64709 12544 12556 organic acid +T297 http://purl.obolibrary.org/obo/MONDO_0008721 12576 12590 MCAD-deficient +T298 NCBITaxon:9606 12591 12596 human +T299 NCBITaxon:10088 12651 12655 mice +T300 CHEBI:30832 12707 12713;12736 12741 adipic ... acids +T301 CHEBI:9300 12715 12722;12736 12741 suberic ... acids +T302 CHEBI:41865 12728 12741 sebacic acids +T303 CHEBI:64390 12746 12761 hexanoylglycine +T304 CHEBI:64709 12851 12864 organic acids +T305 CHEBI:30832 12876 12887 Adipic acid +T306 http://purl.obolibrary.org/obo/MONDO_0008721 12907 12922 MCAD deficiency +T307 CHEBI:20067 12942 12958 β-hydroxybutyric +T308 CHEBI:15344 12963 12974 acetoacetic +T309 http://purl.obolibrary.org/obo/MONDO_0008721 13035 13039 MCAD +T310 NCBITaxon:10088 13103 13107 mice +T311 UBERON:0001977 13153 13158 serum +T312 CHEBI:17126 13159 13168 carnitine +T313 NCBITaxon:10088 13212 13216 mice +T314 NCBITaxon:10088 13230 13234 mice +T315 UBERON:0001977 13267 13272 serum +T316 CHEBI:86063 13273 13290 decenoylcarnitine +T317 CHEBI:17387 13306 13319 acylcarnitine +T318 UBERON:0001970 13341 13345 Bile +T319 CHEBI:17387 13346 13359 acylcarnitine +T320 CHEBI:17387 13388 13401 acylcarnitine +T321 UBERON:0001977 13416 13421 serum +T322 CHEBI:17387 13448 13461 acylcarnitine +T323 NCBITaxon:10088 13486 13490 mice +T324 NCBITaxon:9606 13519 13524 human +T325 http://purl.obolibrary.org/obo/MONDO_0008721 13525 13539 MCAD-deficient +T326 NCBITaxon:9606 13562 13567 Human +T327 CHEBI:17387 13627 13641 acylcarnitines +T328 NCBITaxon:10088 13661 13665 mice +T329 CHEBI:17387 13704 13717 acylcarnitine +T330 NCBITaxon:9606 13721 13727 humans +T331 NCBITaxon:10088 13744 13749 mouse +T332 CHEBI:17387 13763 13776 acylcarnitine +T333 CHEBI:17387 13789 13802 Acylcarnitine +T334 UBERON:0001977 13817 13822 Serum +T335 CHEBI:17387 13823 13836 acylcarnitine +T336 NCBITaxon:10088 13877 13881 mice +T337 CHEBI:17387 13927 13940 acylcarnitine +T338 NCBITaxon:10088 13973 13977 mice +T339 UBERON:0001970 14131 14135 bile +T340 CHEBI:17387 14136 14150 acylcarnitines +T341 NCBITaxon:10088 14163 14167 mice +T342 UBERON:0001970 14265 14269 Bile +T343 CHEBI:17387 14270 14283 acylcarnitine +T344 NCBITaxon:10088 14306 14311 mouse +T345 NCBITaxon:9606 14326 14331 human +T346 http://purl.obolibrary.org/obo/MONDO_0008721 14345 14360 MCAD deficiency +T347 NCBITaxon:10088 14508 14512 mice +T348 GO:0031982 14557 14566 vesicular +T349 GO:0031982 14576 14585 vesicular +T350 UBERON:0002107 14586 14593 hepatic +T351 http://purl.obolibrary.org/obo/MONDO_0004790 14586 14603 hepatic steatosis +T352 NCBITaxon:10088 14626 14630 mice +T353 NCBITaxon:10088 14647 14651 mice +T354 NCBITaxon:10088 14747 14751 mice +T355 UBERON:0000062 14815 14821 organs +T356 NCBITaxon:10088 14856 14860 mice +T357 UBERON:0002107 14886 14893 hepatic +T358 http://purl.obolibrary.org/obo/MONDO_0004790 14886 14903 hepatic steatosis +T359 UBERON:0000948 14936 14943 cardiac +T360 NCBITaxon:10088 14972 14976 mice +T361 NCBITaxon:10088 15027 15031 Mice +T362 NCBITaxon:10088 15045 15049 mice +T363 UBERON:0002107 15069 15076 hepatic +T364 http://purl.obolibrary.org/obo/MONDO_0004790 15069 15086 hepatic steatosis +T365 UBERON:0002107 15110 15115 Liver +T366 http://purl.obolibrary.org/obo/MONDO_0004790 15151 15166 Hepatosteatosis +T367 NCBITaxon:10088 15178 15183 mouse +T368 UBERON:0002107 15225 15230 liver +T369 GO:0031982 15273 15282 vesicular +T370 GO:0031982 15292 15301 vesicular +T371 UBERON:0002107 15302 15309 hepatic +T372 http://purl.obolibrary.org/obo/MONDO_0004790 15302 15319 hepatic steatosis +T373 NCBITaxon:10088 15331 15335 mice +T374 http://purl.obolibrary.org/obo/MONDO_0004994 15356 15370 cardiomyopathy +T375 CL:0000187 15402 15409 myocyte +T376 NCBITaxon:10088 15447 15451 mice +T377 NCBITaxon:10088 15481 15486 mouse +T378 http://purl.obolibrary.org/obo/MONDO_0004994 15499 15513 cardiomyopathy +T379 CL:0000187 15545 15552 myocyte +T380 CL:0000187 15623 15631 myocytes +T381 GO:0016528 15690 15700 sarcoplasm +T382 GO:0030016 15717 15727 myofibrils +T383 CL:0000771 15749 15761 eosinophilic +T384 GO:0005634 15772 15778 Nuclei +T385 CL:0000187 15791 15799 myocytes +T386 GO:0031982 15822 15831 vesicular +T387 GO:0005730 15850 15858 nucleoli +T388 CL:0000187 15915 15923 myocytes +T389 UBERON:0004663 15956 15963;15968 15973 wall of ... aorta +T390 UBERON:0000948 15993 15998 heart +T391 UBERON:0000479 16048 16054 tissue +T392 CHEBI:26130 16131 16138 pigment +T393 UBERON:0001013 16224 16238 adipose tissue +T394 NCBITaxon:10088 16293 16298 mouse +T395 http://purl.obolibrary.org/obo/MONDO_0008721 16309 16324 MCAD deficiency +T396 NCBITaxon:9606 16411 16416 human +T397 http://purl.obolibrary.org/obo/MONDO_0008721 16427 16441 MCAD-deficient +T398 UBERON:0001969 16465 16471 plasma +T399 UBERON:0001088 16476 16481 urine +T400 CHEBI:25212 16482 16493 metabolites +T401 http://purl.obolibrary.org/obo/MONDO_0008721 16554 16568 MCAD-deficient +T402 UBERON:0001088 16614 16621 urinary +T403 CHEBI:64390 16622 16637 hexanoylglycine +T404 NCBITaxon:10088 16667 16671 mice +T405 CHEBI:17387 16673 16686 Acylcarnitine +T406 NCBITaxon:10088 16716 16721 mouse +T407 NCBITaxon:9606 16782 16787 human +T408 CHEBI:61910 16843 16867 very long-chain acyl-CoA +T409 NCBITaxon:10088 16896 16901 mouse +T410 CHEBI:22221 16934 16938 acyl +T411 NCBITaxon:9606 16965 16970 human +T412 CHEBI:22221 17021 17025 acyl +T413 GO:0045251 17044 17047 ETF +T414 MOP:0000569 17048 17057 reduction +T415 UBERON:0002107 17068 17073 liver +T416 NCBITaxon:10088 17129 17133 mice +T417 NCBITaxon:10088 17166 17170 mice +T418 MOP:0000590 17210 17223 dehydrogenate +T419 CHEBI:15346 17227 17230 CoA +T420 NCBITaxon:9606 17250 17255 human +T421 CHEBI:15346 17317 17320 CoA +T422 CHEBI:15525 17345 17358 palmitoyl-CoA +T423 CHEBI:59132 17402 17409 antigen +T424 NCBITaxon:10088 17430 17434 mice +T425 CHEBI:17984 17566 17574 acyl-CoA +T426 NCBITaxon:10088 17639 17643 mice +T427 http://purl.obolibrary.org/obo/MONDO_0008721 17753 17767 MCAD-deficient +T428 NCBITaxon:10088 17786 17790 mice +T429 GO:0016265 17875 17880 dying +T430 NCBITaxon:9606 17914 17919 Human +T431 http://purl.obolibrary.org/obo/MONDO_0004946 17942 17954 hypoglycemia +T432 UBERON:0000104 18070 18074 life +T433 UBERON:0001913 18217 18221 milk +T434 GO:0007631 18229 18237 ingested +T435 UBERON:0007023 18278 18283 adult +T436 NCBITaxon:10088 18292 18296 mice +T437 NCBITaxon:10088 18395 18399 mice +T438 UBERON:0001348 18442 18451 Brown fat +T439 GO:0031649 18485 18498 thermogenesis +T440 GO:0010467 18512 18521 expresses +T441 GO:0031982 18559 18568 vesicular +T442 GO:0031982 18578 18587 vesicular +T443 UBERON:0002107 18588 18595 hepatic +T444 http://purl.obolibrary.org/obo/MONDO_0004790 18588 18605 hepatic steatosis +T445 NCBITaxon:10088 18629 18633 mice +T446 NCBITaxon:9606 18694 18699 human +T447 http://purl.obolibrary.org/obo/MONDO_0008721 18700 18704 MCAD +T448 UBERON:0000948 18744 18751 cardiac +T449 NCBITaxon:10088 18771 18775 mice +T450 http://purl.obolibrary.org/obo/MONDO_0004994 18842 18856 cardiomyopathy +T451 CL:0000187 18873 18880 myocyte +T452 NCBITaxon:10088 18927 18931 mice +T453 NCBITaxon:9606 18957 18962 human +T454 http://purl.obolibrary.org/obo/MONDO_0008721 18963 18967 MCAD +T455 UBERON:0000948 18987 18994 cardiac +T456 http://purl.obolibrary.org/obo/MONDO_0007263 18987 19006 cardiac arrhythmias +T457 http://purl.obolibrary.org/obo/MONDO_0008721 19045 19059 MCAD-deficient +T458 http://purl.obolibrary.org/obo/MONDO_0004994 19094 19108 cardiomyopathy +T459 http://purl.obolibrary.org/obo/MONDO_0008723 19130 19146 VLCAD deficiency +T460 http://purl.obolibrary.org/obo/MONDO_0000001 19162 19171 disorders +T461 CHEBI:15904 19175 19189 long chain fat +T462 GO:0001676 19175 19200 long chain fat metabolism +T463 PR:000005835 19216 19221 CPT-1 +T464 PR:000005838 19216 19219;19226 19228 CPT ... -2 +T465 http://purl.obolibrary.org/obo/MONDO_0009705 19216 19221;19229 19241 CPT-1 deficiencies +T466 http://purl.obolibrary.org/obo/MONDO_0015515 19216 19219;19226 19241 CPT -2 deficiencies +T467 UBERON:0000948 19281 19288 cardiac +T468 NCBITaxon:10088 19305 19310 mouse +T469 NCBITaxon:10088 19369 19374 mouse +T470 UBERON:0002107 19398 19403 liver +T471 UBERON:0000948 19408 19415 cardiac +T472 NCBITaxon:10088 19433 19437 mice +T473 http://purl.obolibrary.org/obo/MONDO_0008721 19530 19545 MCAD deficiency +T474 NCBITaxon:9606 19549 19555 humans +T475 NCBITaxon:10088 19616 19621 mouse +T476 CHEBI:17984 19633 19641 acyl-CoA +T477 http://purl.obolibrary.org/obo/MONDO_0017714 19633 19668 acyl-CoA dehydrogenase deficiencies +T478 NCBITaxon:10088 19703 19707 mice +T479 NCBITaxon:10088 19750 19754 mice +T480 NCBITaxon:10088 19805 19810 mouse +T481 UBERON:0002107 19919 19924 liver +T482 UBERON:0000948 19926 19931 heart +T483 UBERON:0002113 19937 19943 kidney +T484 NCBITaxon:10088 19953 19957 mice +T485 GO:0016265 19980 19986 deaths +T486 GO:0007565 19991 20002 gestational +T487 NCBITaxon:10088 20090 20094 mice +T488 NCBITaxon:10088 20127 20132 mouse +T489 NCBITaxon:10088 20255 20259 mice +T490 NCBITaxon:33208 20287 20294 animals +T491 http://purl.obolibrary.org/obo/MONDO_0008721 20330 20344 MCAD-deficient +T492 NCBITaxon:10088 20345 20350 mouse +T493 GO:0005739 20396 20409 mitochondrial +T494 MOP:0000568 20412 20421 oxidation +T495 CHEBI:35366 20499 20510 fatty acids +T496 http://purl.obolibrary.org/obo/MONDO_0000001 20529 20537 diseases +T497 SO:0001644 20584 20600 targeting vector +T498 CHEBI:7507 20605 20613 neomycin +T499 SO:0005853 20625 20638 gene cassette +T500 SO:0000155 20697 20704 plasmid +T501 GO:0006266 20790 20798 ligation +T502 SO:0000440 20836 20842 vector +T503 SO:0000155 20913 20920 plasmid +T504 SO:0001026 20942 20949 genomic +T505 SO:0000147 20968 20973 exons +T506 SO:0000357 20991 20999 flanking +T507 SO:0000188 21000 21006 intron +T508 NCBITaxon:10088 21064 21069 mouse +T509 SO:0001026 21070 21077 genomic +T510 SO:0000440 21169 21175 vector +T511 PR:P23940 21194 21199 BamHI +T512 PR:P23940 21229 21234 BamHI +T513 SO:0001026 21241 21248 genomic +T514 SO:0000147 21269 21273 exon +T515 SO:0000357 21281 21289 flanking +T516 SO:0000188 21290 21296 intron +T517 SO:0000440 21321 21327 vector +T518 PR:P23940 21348 21353 BamHI +T519 SO:0001026 21360 21367 genomic +T520 GO:0006266 21433 21441 ligation +T521 SO:0000440 21551 21557 vector +T522 GO:0006266 21578 21586 ligation +T523 SO:0000147 21679 21683 exon +T524 http://purl.obolibrary.org/obo/MONDO_0008721 21722 21736 MCAD-deficient +T525 NCBITaxon:10088 21737 21741 mice +T526 SO:0000440 21764 21770 vector +T527 SO:0001026 21828 21835 genomic +T528 CL:0002322 21885 21893 ES cells +T529 NCBITaxon:10088 21941 21945 mice +T530 SO:0000440 21982 21988 vector +T531 SO:0000985 22052 22067 double stranded +T532 GO:0006302 22052 22080 double stranded–break repair +T533 SO:0001026 22193 22200 Genomic +T534 CHEBI:42768 22210 22214 G418 +T535 CL:0002322 22225 22227 ES +T536 SO:0000028 22311 22313 bp +T537 SO:0000147 22358 22362 exon +T538 SO:0000188 22369 22375 intron +T539 SO:0000440 22436 22442 vector +T540 GO:0097617 22463 22472 hybridize +T541 SO:0001026 22486 22493 genomic +T542 CL:0002322 22557 22564 ES cell +T543 UBERON:0000358 22599 22610 blastocysts +T544 NCBITaxon:10088 22632 22636 mice +T545 NCBITaxon:10088 22647 22651 mice +T546 NCBITaxon:10088 22698 22702 mice +T547 SO:0000704 22714 22718 gene +T548 NCBITaxon:10088 22728 22732 mice +T549 NCBITaxon:10088 22808 22812 mice +T550 NCBITaxon:10088 22814 22818 Mice +T551 NCBITaxon:10088 22837 22842 mouse +T552 NCBITaxon:10239 22899 22906 viruses +T553 NCBITaxon:2 22916 22925 bacterial +T554 UBERON:0001728 22938 22949 nasopharynx +T555 UBERON:0001153 22954 22959 cecum +T556 UBERON:0000062 23035 23041 organs +T557 UBERON:0000948 23091 23096 heart +T558 NCBITaxon:10088 23141 23145 mice +T559 CHEBI:30087 23175 23186 guanidinium +T560 CHEBI:18022 23187 23198 thiocyanate +T561 GO:0001171 23212 23233 Reverse transcription +T562 SO:0000147 23433 23437 exon +T563 SO:0000147 23444 23448 exon +T564 SO:0000440 23592 23598 vector +T565 GO:0010467 23693 23702 expressed +T566 NCBITaxon:10088 23720 23724 mice +T567 UBERON:0000948 23792 23797 heart +T568 UBERON:0002107 23799 23804 liver +T569 UBERON:0001348 23806 23815 brown fat +T570 UBERON:0000955 23817 23822 brain +T571 UBERON:0002113 23824 23830 kidney +T572 UBERON:0004288 23832 23840 skeletal +T573 UBERON:0001347 23849 23858 white fat +T574 UBERON:0000473 23864 23870 testes +T575 NCBITaxon:10088 23903 23907 mice +T576 CHEBI:2511 24097 24104 agarose +T577 CHEBI:16842 24105 24117 formaldehyde +T578 CHEBI:53325 24138 24152 nitrocellulose +T579 GO:0097617 24257 24267 hybridized +T580 CHEBI:37972 24273 24276 32P +T581 NCBITaxon:10088 24302 24307 mouse +T582 GO:0097617 24357 24371 Hybridizations +T583 CHEBI:16397 24443 24452 formamide +T584 CHEBI:34674 24458 24473 dextran sulfate +T585 CHEBI:33893 24489 24496 reagent +T586 CHEBI:8984 24501 24504 SDS +T587 CL:0000019 24517 24522 sperm +T588 GO:0097617 24542 24552 hybridized +T589 CHEBI:8984 24597 24600 SDS +T590 CHEBI:8984 24631 24634 SDS +T591 CHEBI:2511 24802 24809 agarose +T592 CHEBI:16842 24810 24822 formaldehyde +T593 CHEBI:4883 24844 24860 ethidium bromide +T594 NCBITaxon:10088 24973 24978 mouse +T595 UBERON:0000479 24979 24985 tissue +T596 UBERON:0002107 24987 24992 liver +T597 NCBITaxon:10088 25053 25057 mice +T598 UBERON:0000479 25137 25143 tissue +T599 GO:0019835 25164 25169 lysed +T600 CHEBI:78708 25197 25209 Nonidet P-40 +T601 CHEBI:9177 25216 25235 sodium deoxycholate +T602 CHEBI:8984 25244 25247 SDS +T603 CHEBI:17754 25258 25266 glycerol +T604 CHEBI:8102 25379 25407 phenylmethylsulfonylfluoride +T605 CHEBI:35607 25421 25441 sodium orthovanadate +T606 CHEBI:8984 25593 25596 SDS +T607 CHEBI:53325 25644 25658 nitrocellulose +T608 UBERON:0001913 25761 25765 milk +T609 CHEBI:53424 25805 25813 Tween-20 +T610 GO:0042571 25900 25908 antibody +T611 NCBITaxon:10088 25939 25944 mouse +T612 GO:0071735 25945 25948 IgG +T613 MOP:0000779 25953 25963 conjugated +T614 GO:0042571 25974 25982 antibody +T615 UBERON:0002107 26155 26160 Liver +T616 NCBITaxon:10088 26217 26221 mice +T617 UBERON:0002107 26223 26228 liver +T618 NCBITaxon:10088 26288 26292 mice +T619 GO:0045251 26346 26349 ETF +T620 MOP:0000569 26350 26359 reduction +T621 UBERON:0000479 26378 26384 tissue +T622 CHEBI:15533 26399 26411 octanoyl-CoA +T623 CHEBI:15525 26421 26434 palmitoyl-CoA +T624 NCBITaxon:10088 26547 26551 mice +T625 UBERON:0001977 26611 26616 serum +T626 CHEBI:17234 26617 26624 glucose +T627 CHEBI:35366 26631 26641 fatty acid +T628 CHEBI:64709 26643 26655 organic acid +T629 CHEBI:17126 26661 26670 carnitine +T630 CHEBI:17234 26703 26710 Glucose +T631 UBERON:0001977 26747 26751 sera +T632 CHEBI:35366 26884 26895 fatty acids +T633 CHEBI:33893 26964 26972 reagents +T634 UBERON:0001088 27173 27178 Urine +T635 CHEBI:64709 27179 27191 organic acid +T636 CHEBI:32936 27299 27310 tetracosane +T637 CHEBI:17387 27495 27508 Acylcarnitine +T638 UBERON:0001977 27521 27526 serum +T639 UBERON:0001970 27531 27535 bile +T640 NCBITaxon:10088 27629 27633 mice +T641 NCBITaxon:10088 27730 27735 mouse +T642 NCBITaxon:10088 27779 27784 mouse +T643 NCBITaxon:10088 27827 27831 mice +T644 NCBITaxon:10088 27878 27882 mice +T645 UBERON:0002113 27893 27899 Kidney +T646 UBERON:0002106 27901 27907 spleen +T647 UBERON:0001264 27909 27917 pancreas +T648 UBERON:0002107 27919 27924 liver +T649 UBERON:0000955 27926 27931 brain +T650 UBERON:0000948 27933 27938 heart +T651 UBERON:0000473 27940 27949 testicles +T652 UBERON:0000992 27951 27958 ovaries +T653 UBERON:0004288 27964 27972 skeletal +T654 CHEBI:16842 28020 28028 formalin +T655 CHEBI:51686 28111 28122 hematoxylin +T656 UBERON:0002107 28141 28146 liver +T657 NCBITaxon:10088 28300 28304 mice +T658 http://purl.obolibrary.org/obo/MONDO_0008721 28348 28355 disease +T659 UBERON:0000948 28376 28383 cardiac +T660 UBERON:0000948 28415 28422 cardiac +T661 NCBITaxon:10088 28697 28702 mouse +T662 SO:0000704 28703 28707 gene +T663 http://purl.obolibrary.org/obo/MONDO_0004992 28884 28890 Cancer +T664 NCBITaxon:33208 28930 28936 Animal +T665 UBERON:0002204 28979 28994 Musculoskeletal +T666 http://purl.obolibrary.org/obo/MONDO_0002081 28979 29002 Musculoskeletal Disease +T667 http://purl.obolibrary.org/obo/MONDO_0005578 29007 29016 Arthritis +T668 SO:0000704 29025 29029 Gene +T669 SO:0000028 29287 29289 bp +T670 SO:0000028 29292 29301 base pair +T671 UBERON:0000922 29308 29317 embryonic +T672 GO:0045251 29324 29327 ETF +T673 CHEBI:10545 29330 29338 electron +T674 MOP:0000615 29330 29348 electron transport +T675 GO:0045251 29330 29361 electron transport flavoprotein +T676 CHEBI:5086 29349 29361 flavoprotein +T677 CHEBI:61907 29370 29391 medium-chain acyl-CoA +T678 CHEBI:61905 29414 29434 short-chain acyl-CoA +T679 CHEBI:61910 29483 29507 very long-chain acyl-CoA +T680 CHEBI:33893 29848 29856 reagents +T681 CHEBI:61907 30011 30032 Medium-chain acyl-CoA +T682 http://purl.obolibrary.org/obo/MONDO_0008721 30011 30057 Medium-chain acyl-CoA dehydrogenase deficiency +T683 SO:0000704 30061 30065 gene +T684 NCBITaxon:10088 30075 30079 mice diff --git a/src/ontogpt/evaluation/craft/database/all/16121256.txt b/src/ontogpt/evaluation/craft/database/all/16121256.txt new file mode 100644 index 000000000..2ed5328f3 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16121256.txt @@ -0,0 +1,203 @@ +Medium-Chain Acyl-CoA Dehydrogenase Deficiency in Gene-Targeted Mice + +Abstract + +Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is the most common inherited disorder of mitochondrial fatty acid β-oxidation in humans. To better understand the pathogenesis of this disease, we developed a mouse model for MCAD deficiency (MCAD−/−) by gene targeting in embryonic stem (ES) cells. The MCAD−/− mice developed an organic aciduria and fatty liver, and showed profound cold intolerance at 4 °C with prior fasting. The sporadic cardiac lesions seen in MCAD−/− mice have not been reported in human MCAD patients. There was significant neonatal mortality of MCAD−/− pups demonstrating similarities to patterns of clinical episodes and mortality in MCAD-deficient patients. The MCAD-deficient mouse reproduced important aspects of human MCAD deficiency and is a valuable model for further analysis of the roles of fatty acid oxidation and pathogenesis of human diseases involving fatty acid oxidation. + +Synopsis + + + +Medium-chain acyl-CoA dehydrogenase (MCAD) deficiency is one of the most common inherited disorders of metabolism. This defect in fatty acid oxidation can lead to severe and sometimes fatal disease, especially in young children because they are unable to tolerate a fasting episode. Metabolic complications include very low blood glucose concentrations and generation of toxic by-products. This disorder can result in sudden infant death. Using a process known as gene targeting in mouse embryonic stem cells, the authors have developed a mouse model with the same enzyme deficiency. This mouse model of MCAD deficiency develops many of the same disease characteristics found in affected children. The MCAD-deficient mouse model shows a high rate of newborn loss, intolerance to cold, and the characteristic biochemical changes in the blood, tissues, and urine that are very similar to those found in the human disease counterpart. The MCAD-deficient mouse model will allow researchers to better understand disease mechanisms so that new preventive measures or therapies can be developed. + +Introduction + +Mitochondrial β-oxidation of fatty acids provides energy, especially during fasting conditions. Fatty acid oxidation occurs in mitochondria and consists of a repeating circuit of four sequential steps. There are four straight-chain acyl-CoA dehydrogenases involved in the initial step. Medium-chain acyl-CoA dehydrogenase (MCAD) (the mouse gene is Acadm, whereas the protein is MCAD), specifically, is responsible for catalyzing the dehydrogenation of medium-chain length (C6–C12) fatty acid thioesters [1]. Acadm is transcribed in the nucleus, translated in the cytosol, and translocated into the mitochondrial matrix [2–4]. Once inside the mitochondrial matrix, the MCAD monomers are assembled into homotetramers to gain enzymatic activity [4]. + +MCAD activity is essential for complete fatty acid oxidation. Inherited MCAD deficiency exists in humans as an autosomal recessive disorder. MCAD deficiency was first described in 1982—1983 [5–7] and has been described in numerous patients [1,8–11]. The carrier frequency in the Caucasian population has been estimated to be between 1 in 50 to 80 with an incidence of clinical disease expected at around 1 in 15,000 [1,9,12]. + +MCAD-deficient patients exhibit clinical episodes often associated with fasting. Patients manifest disease usually during the first two years of life. Symptoms include hypoketotic hypoglycemia and Reye-like episodes [1]. It is estimated that approximately 59% of patients presenting clinically between 15 to 26 mo of age die during their first clinical episode [1]. + +The pathogenesis of the wide range of metabolic disturbances in MCAD deficiency is poorly understood and certain aspects of patient management are controversial. An animal model for MCAD deficiency is essential to better understand the pathogenesis of MCAD deficiency and to develop better management regimens for human patients. To gain further insight into the mechanisms of this disease, we developed a mouse model of MCAD deficiency by gene targeting in embryonic stem (ES) cells (for reviews [13,14]). The mutant mice had many relevant features characteristic of the disease found in human MCAD-deficient patients, along with some unexpected findings. + +Results + +Gene Targeting and Generation of MCAD-Deficient Mice + +MCAD insertion vector (MCAD IV2) was designed to undergo gap repair of the 1.3-kb deleted region upon homologous recombination in 129P2 (129P2/OlaHsd) ES cells E14–1. Correct targeting of the MCAD locus resulted in a duplication of exons 8, 9, and 10 and integration of flanking plasmid and Neo sequences (Figure 1A). The insertion vector was designed to duplicate exon 8, 9, and 10 at the MCAD locus. Translation of the duplicated exon 8 region results in the formation of premature stop codons resulting in truncation of the MCAD monomer. Specifically, the first premature stop codon arises after translation of only seven amino acids from the duplicated exon 8. The resulting MCAD monomer is missing the C-terminal domain α-helixes that are responsible for making intersubunit contacts to generate the functional MCAD homotetramer. + +Figure 1 + +Strategy for Disruption of the Mouse Acadm Gene + +(A) The MCAD IV2 insertion targeting vector with a deleted 1.3-kb region encompassing exon 10 and flanking sequences. MCAD IV2 undergoes gap repair upon homologous recombination at the endogenous Acadm locus resulting in a duplication of exons 8, 9, and 10 at the disrupted allele. + +(B) Southern blot analysis of EcoRI-digested genomic DNA from ES cells screened by PCR. Probe A, a DNA fragment consisting of a portion of exon 10 that is not present in the targeting vector, hybridizes to an endogenous 3.1-kb fragment and, upon homologous recombination, to a 13.2-kb fragment. Lane 1 represents a wild-type ES cell line, and Lane 2 and 3 represent targeted ES cell lines. + +ES cell clones were screened by PCR (data not shown) and confirmed by Southern blot analysis. Southern blot analysis used an exon 10 probe (probe A), not present in the targeting vector, hybridized to a 13.2-kb band in addition to the 3.1-kb endogenous band indicating targeted insertion of the vector at the Acadm locus (Figure 1B). Correctly targeted ES cell clones were microinjected into B6 (C57BL/6NTac) blastocysts to generate chimeric mice. Chimeric mice were backcrossed to both 129P2 and B6 inbred mice to produce MCAD+/− and eventually MCAD−/− mice on a B6/129 mixed background. The studies described here were conducted exclusively on the B6/129 mixed background compared with littermate controls or B6/129 control groups maintained by intercrosses as were the mutants. Perpetuating this mutation as a congenic mutant line on the 129P2 background proved impractical. The 129P2 mice were poor breeders as wild-types, and when introduced, the Acadm mutation was nearly lost on this background because of the high rate of neonatal death. Because of the molecular structure of the targeted allele, it proved virtually impossible to distinguish all three potential genotypes. We could clearly detect the presence or absence of the targeted allele, however, whether a particular mouse was MCAD−/− or MCAD+/− could not be determined by Southern blot or PCR of genomic DNA. Ultimately MCAD−/− mice were ascertained by immunoblot analysis of offspring with subsequent perpetuation of MCAD−/− and MCAD+/+ mice as separate groups. + +RNA Analysis + +RT-PCR amplification from exon 7 to 11 from total heart RNA amplified the expected 600-base pair (bp) fragment in MCAD+/+ and MCAD+/− mice, and a 1.5-kb fragment in MCAD−/− mice (data not shown). Sequence analysis of the 1.5-kb fragment revealed that the amplified fragment consisted of exon 7 to exon 10 with 280 bp of pGEM plasmid sequence followed by exons 8–11. Some of the plasmid sequences, along with the pPGKNEOpA sequence, were deleted from this spliced mRNA. + +Northern blot analysis revealed Acadm was normally expressed in all tissues analyzed from MCAD+/+ mice with the most robust expression occurring in brown fat, kidney, heart, skeletal muscle, and liver with minimal expression in the brain, white fat, and testes (Figure 2). Interestingly, although RT-PCR amplified an incorrectly spliced Acadm transcript, no Acadm transcripts were detected by northern blot analysis from MCAD−/− mice. These results strongly suggest that the mutant RNA is unstable and degraded rapidly or, alternatively, undergoes nonsense mediated RNA decay. + +Figure 2 + +Northern Blot Analysis from MCAD−/− (n = 2) and MCAD+/+ (n = 2) Mice + +Acadm message was detected from the heart, liver, brown fat, brain, kidney, and muscle (and white fat and testes, data not shown) of only MCAD+/+ mice. Most robust expression occurred in brown fat, kidney, heart, and skeletal muscle. MCAD−/− mice had no detectable message in all tissues examined. + +Liver Enzyme Analyses + +Immunoblot analyses of liver homogenates with anti-MCAD antisera demonstrated that the 42 kDa MCAD monomer was present in MCAD+/+ mice, but not in MCAD−/− mice (Figure 3). As a control analysis, anti–short-chain acyl-CoA dehydrogenase (SCAD) antisera revealed no differences in levels of expression of SCAD protein between MCAD+/+ and MCAD−/− mice (Figure 3). + +Figure 3 + +Immunoblots of Liver Homogenates from MCAD+/+ and MCAD−/− Mice + +These were probed with anti-MCAD antibody or anti-SCAD antibody. Homozygous MCAD−/− mice had no detectable MCAD protein. MCAD protein is only detectable under the MCAD protein–spiked (positive control) lane. As a control analysis, liver homogenates probed with anti-SCAD antibody revealed that SCAD protein was present in both MCAD+/+ and MCAD−/− mice. No MCAD positive-control protein is detected by anti-SCAD antibodies (MCAD lane). mw, molecular weight standards. + +Enzyme activity was analyzed in mouse liver homogenates using the electron transport flavoprotein (ETF) reduction assay with octanoyl-CoA (C8:0) and palmitoyl-CoA (C16:0) as substrates. MCAD−/− mice had a significant reduction in ability to dehydrogenate octanoyl-CoA and a modest reduction in activity toward palmitoyl-CoA compared to MCAD+/+ mice (Table 1). Specifically, the dehydrogenation of octanoyl-CoA and palmitoyl-CoA substrates were reduced by 75% and by 30%, respectively, in MCAD−/− mice as compared to MCAD+/+ controls. + +Table 1 + +Characteristics of MCAD-Deficient Mice + +Values given are mean ± SD. + +aMCAD+/+ n = 5 and MCAD−/− n = 5. + +bMCAD+/+ n = 8 litters and MCAD−/− n = 10 litters. + +cMCAD+/+ n = 5 and MCAD−/− n = 6. + +dMCAD+/+ n = 4 and MCAD−/− n = 5. + +eExpressed as a ratio relative to the internal standard. + +Neonatal Deaths + +Significant neonatal mortality was noted in MCAD−/− pups. Although equal numbers of pups were born from MCAD+/+ and MCAD−/− mice, only 41% of MCAD−/− mice survived to weaning as compared to 98% of MCAD+/+ mice (Table 1). The mechanism of neonatal loss remains undetermined. The MCAD−/− pups are abandoned more frequently than MCAD+/+ pups for unknown reasons. They are likely more prone to hypothermia than MCAD+/+. Because of the difficulty of distinguishing the MCAD−/− mutants from the MCAD+/− heterozygous pups by molecular analysis due to the insertion mutation, we could only compare MCAD+/+ × MCAD+/+ matings with MCAD−/− × MCAD−/− matings. Thus, we were unable to evaluate the pedigrees from heterozygous matings. + +Fasting and Cold Intolerance + +In order to examine the stress effects of fasting on MCAD-deficient mice, they were fasted for 24 h prior to analysis. MCAD−/− mice displayed lower serum glucose and elevated serum free fatty acid levels although neither result was significant, as compared to MCAD+/+ mice (Table 1). + +To determine the effects of cold stress, mice were fasted for 18 h and placed in 4 °C environment for a 3-h period. The MCAD−/− mice were significantly compromised within this short period of cold stress, some severe enough to result in fatalities. After 1 h of the cold challenge, the average rectal temperature of MCAD−/− mice (n = 5) was 23.4 °C as compared with 35 °C for MCAD+/+ mice (n = 4). Rectal temperatures declined to unrecoverable temperatures of 16.7–19.2 °C in three of the five MCAD−/− mice. By the end of the 1.5-h mark, the two surviving MCAD−/− mice averaged 22.7 °C. In contrast, all four MCAD+/+ mice survived the 3-h cold stress, ending with an average rectal temperature of 33.6 °C. + +Organic Acid and Acylcarnitine Analysis + +Urine organic acid analysis revealed that MCAD−/− mice developed an organic acid profile similar to MCAD-deficient human patients. Specifically, when fasted for 18 h, MCAD−/− mice developed significantly elevated concentrations of adipic, suberic, and sebacic acids and hexanoylglycine as compared to MCAD+/+ controls, which showed trace to no detectable amounts of the same organic acids (Table 1). Adipic acid is not specific to MCAD deficiency. We also evaluated β-hydroxybutyric and acetoacetic concentrations and found no significant differences between MCAD genotypes (data not shown). + +Comparison of MCAD+/+ and MCAD−/− mice revealed no significant differences in total serum carnitine concentrations between MCAD+/+ and MCAD−/− mice, but MCAD−/− mice had a 5- to 6-fold elevation of serum decenoylcarnitine evident in the acylcarnitine profile (Figure 4A). Bile acylcarnitine analysis revealed a similar acylcarnitine pattern as in serum (Figure 4B). However, the acylcarnitine profiles of the MCAD−/− mice are different from those of human MCAD-deficient patients (Figure 4C). Human patients present with elevated levels of C6, C8, and C10:1 acylcarnitines, as did the mutant mice; however, the predominant peak was C8 acylcarnitine in humans, whereas in the mouse it was C10:1 acylcarnitine. + +Figure 4 + +Acylcarnitine Analyses + +(A) Serum acylcarnitine analysis of MCAD+/+ (n = 4) and MCAD−/− mice (n = 4) + +There are significant elevations in acylcarnitine species as indicated in MCAD−/− mice. Values shown are mean values ± standard deviation (SD). Asterisk indicates p < 0.002 and ‡ indicates p < 0.01. + +(B) There are significant elevations in bile acylcarnitines of the same mice shown in (A) as indicated. Values shown are mean values ± SD. Asterisk indicates p < 0.001. + +(C) Bile acylcarnitine profile of an MCAD−/− mouse compared to a human patient with MCAD deficiency. Internal standards are indicated by an asterisk. + +Histopathology + +Complete histopathologic examination of one group of mutant and MCAD+/+ control mice after a 24-h fast demonstrated diffuse microvesicular and macrovesicular hepatic steatosis in 6–8-wk-old MCAD−/− mice whereas MCAD+/+ mice had no histologic changes (Figure 5A and 5B). In another group of 4-wk-old MCAD+/+ and MCAD−/− mice fasted for 24-h, there were minimal to no abnormalities in all organs evaluated. Only the older MCAD−/− mice, therefore, demonstrated hepatic steatosis. In addition, we found sporadic cardiac lesions in multiple MCAD−/− mice. + +Figure 5 + +Histopathology of MCAD+/+ and MCAD−/− Mice + +(A) MCAD+/+ mice had no evidence of hepatic steatosis following a 24-h fast. Liver section with Oil-Red O stain. + +(B) Hepatosteatosis in MCAD−/− mouse following a 24-h fast. Oil-Red O stained liver sections revealed severe and diffuse microvesicular and macrovesicular hepatic steatosis in MCAD−/− mice. + +(C and D) Diffuse cardiomyopathy with chronic active multifocal myocyte degeneration and necrosis in MCAD−/− mice. + +In one example, an MCAD−/− mouse had diffuse cardiomyopathy with chronic active multifocal myocyte degeneration and necrosis (Figure 5C and 5D). Changes in degenerating myocytes included swelling, pale staining, and, in portions of the sarcoplasm, replacement of myofibrils with finely granular eosinophilic material. Nuclei of affected myocytes were large, pale, and vesicular and had prominent nucleoli. In the most severely affected areas, there was loss of myocytes accompanied by fibrosis. In the wall of the aorta at the base of the heart there was multifocal degeneration of the elastic tissue, accompanied by multifocal collections of globular translucent yellow-brown pigment interpreted to be ceroid/lipofuscin. Similar deposits were scattered within adjacent adipose tissue. + +Discussion + +Successfully targeting Acadm produced a mouse model for MCAD deficiency with features that mimic the clinical, biochemical, and pathologic phenotype found in human patients. MCAD-deficient patients have abnormal plasma and urine metabolites associated with the medium chain–length enzyme specificity. MCAD-deficient patients [15] often display a characteristic urinary hexanoylglycine peak, as was seen in MCAD−/− mice. Acylcarnitine analysis indicated, however, mouse MCAD is more active toward longer chain substrates than the human MCAD enzyme. This finding is similar to that seen with very long-chain acyl-CoA dehydrogenase (VLCAD) where mouse VLCAD is most active toward C16 acyl-substrates as compared to human VLCAD with the most enzymatic activity toward C14 acyl-substrates [16]. + +ETF reduction assays of liver homogenates were performed to characterize the MCAD−/− mice at the enzymatic level. MCAD−/− mice had a significantly reduced ability to dehydrogenate C8-CoA, as is the case in human patients where MCAD activity is reduced to near zero with C8-CoA . This was less so with palmitoyl-CoA (C16:0). Because there was clearly no MCAD antigen detected in MCAD−/− mice, the residual dehydrogenase activity measured with these two substrates must represent the activity of other chain length–specific acyl-CoA dehydrogenases. + +A high degree of neonatal mortality in MCAD−/− mice was a striking observation and appears to be analogous to the patterns of clinical episodes and mortality in MCAD-deficient patients. MCAD−/− mice exhibited significant neonatal mortality with approximately 60% of the MCAD−/− pups dying prior to weaning at 3 wk of age. Human patients present with hypoglycemia, hypoketonemia, and nonketotic organic aciduria precipitated by fasting, most frequently during the first 24 mo in life [1]. It is likely that neonatal MCAD−/− pups are manifesting sensitivity to fasting with decompensation in a short period of time if maternal milk is not ingested. In contrast, no mortality was noted in adult MCAD−/− mice unless challenged with cold stress and fasting. Under cold challenge conditions, however, MCAD−/− mice were unable to maintain body temperature. Brown fat is predominantly responsible for thermogenesis and normally expresses high levels of Acadm mRNA. + +The microvesicular and macrovesicular hepatic steatosis seen in fasted MCAD−/− mice is consistent with the primary pathological finding seen in human MCAD patients with fasting stress. Sporadic cardiac lesions in MCAD−/− mice, however, were an interesting and unexpected finding. The diffuse cardiomyopathy with multifocal myocyte degeneration and necrosis observed in MCAD−/− mice has not been reported in human MCAD patients, however, cardiac arrhythmias and dysfunction have been reported in MCAD-deficient patients [17, 18]. Interestingly, cardiomyopathy has been observed in VLCAD deficiency [19] and other disorders of long chain fat metabolism such as severe CPT-1 and -2 deficiencies [1]. Thus it is tempting to relate the cardiac problems in the mouse to the apparent broader range of substrate utilization of mouse MCAD. The inconsistent liver and cardiac lesions in these mice is analogous with the significant inter- and intrafamilial phenotypic heterogeneity seen in MCAD deficiency in humans [1,20]. + +In comparisons with our experiences with the other mouse models for acyl-CoA dehydrogenase deficiencies, the overall phenotype of MCAD−/− mice is less severe than that found in LCAD−/− mice, yet more pronounced than the VLCAD−/− or SCAD−/− mouse models [16,21,22]. All of these mutants are cold intolerant and display varying degrees of fatty changes in liver, heart, and kidney. LCAD−/− mice show more spontaneous deaths and gestational losses than the other deficiencies [21]. The significant neonatal mortality in MCAD−/− mice is distinctive from these other mouse models suggesting a greater degree of sensitivity to fasting intolerance. The phenotypes of both the VLCAD−/− and SCAD−/− mice are relatively mild if the animals are not cold stressed [16,22]. The MCAD-deficient mouse offers new insights into the pathogenesis of mitochondrial β-oxidation deficiencies and will provide a robust tool to better understand the role of fatty acids in other relevant diseases. + +Materials and Methods + +Construction of MCAD targeting vector. + +A neomycin resistance gene cassette [23] was subcloned into the SalI site of pGEM11Zf(+). The plasmid was digested with EcoRI and the overhangs were filled with Klenow enzyme. Subsequent ligation of the blunt ends recircularized the vector and destroyed the EcoRI site within the polylinker of the pGEMl1Zf(+) plasmid. Next, an 8-kb Acadm genomic fragment spanning exons 8, 9, and 10 and flanking intron sequences, originally obtained from a Lambda FIXII 129Sv mouse genomic library [24,25], was directionally cloned into the NotI and XhoI sites of pGEM11Zf(+). The vector was digested with BamHI and EcoRI to remove a 1.3-kb BamHI/EcoRI genomic fragment containing exon 10 and flanking intron sequences. The digested vector, without the 1.3-kb BamHI/EcoRI genomic fragment, was purified by gel purification and recircularized by ligation using three oligonucleotides: 5′-AATTGTCGACA-3′; 5′GATCGTCGACA-3′; and 5′-TCGATGTCGAC-3′. The recircularized vector, resulting from the ligation of the long arm to the short arm of homology, contained a unique SalI site where the 1.3-kb exon 10 region was deleted. + +Generation of MCAD-deficient mice. + +The Acadm insertion vector was linearized by SalI digestion, the site of the 1.3-kb genomic fragment deletion, and electroporated into E14–1 ES cells (a kind gift from R. Kuhn), derived from 129P2 mice. Correctly targeted Acadm insertion vector was designed to undergo gap repair of the 1.3-kb deletion upon double stranded–break repair [26] during homologous recombination. Southern blot analysis was conducted to confirm homologous recombination. Genomic DNA from G418 resistant ES colonies was digested with EcoRI, electrophoresed, blotted, and probed with an 850-bp probe (probe A) generated by PCR from Acadm exon 10 to intron 10. This DNA fragment is not present within Acadm insertion vector and was expected to hybridize to a 13.2-kb genomic DNA fragment upon homologous recombination. Correctly targeted ES cell clones were microinjected into B6 blastocysts to generate chimeric mice. Chimeric mice were subsequently backcrossed to B6 and 129P2 mice to produce gene-targeted mice AcadmtmUab1/+ (MCAD+/−) and eventually AcadmtmUab1/tm1Uab MCAD−/− (B6;129) mice. Mice were negative for mouse pathogens based on serological assays for ten different viruses, aerobic bacterial cultures of nasopharynx and cecum, examinations for endo- and ectoparasites, and histopathology of all major organs. + +RNA analysis. + +Total RNA was isolated from the heart of 30 day old MCAD+/+, MCAD+/−, and MCAD−/− mice by standard techniques using guanidinium thiocyanate method [27]. Reverse transcription was performed using random oligonucleotides as recommended by the manufacturer (Clontech, Mountain View, California, United States). PCR was subsequently performed using oligonucleotides specific to exon 7 and exon 11 of Acadm. PCR amplifications were performed as described above. PCR fragments were subsequently sequenced after subcloning into pGEM-T Easy vector (Promega, Madison, Wisconsin, United States). + +In order to determine the extent of Acadm mRNA expressed from the MCAD−/− mice, northern blot analysis was performed. Total RNA was isolated from heart, liver, brown fat, brain, kidney, skeletal muscle, white fat, and testes of 3-mo-old MCAD+/+ and MCAD−/− mice using the Ultraspec RNA Isolation Kit (BIOTEX Laboratories, Inc., Houston, Texas, United States) as per manufacturer's protocol. Ten μg of total RNA from each sample was loaded onto a 0.8% agarose-formaldehyde gel, transferred to nitrocellulose filter (Hybond N; GE Healthcare Amersham Biosciences Corp., Piscataway, New Jersey, United States), and hybridized with 32P-radiolabeled full-length mouse Acadm cDNA probe using standard procedures [28]. Hybridizations were performed under highly stringent conditions (42 °C in 2× SSC, 50% formamide, 10% dextran sulfate, 5× Denhardt's reagent, 1% SDS, and salmon sperm DNA) for 18 h. The hybridized filter was washed two times in 4× SSC, 0.1% SDS and two times in 2× SSC; 0.1% SDS at 55 °C for 1 h. The filter was exposed to autoradiographic film (Hyperfilm MP; GE Healthcare Amersham Biosciences, Piscataway, New Jersey, United States). Replicate agarose-formaldehyde gels were stained by ethidium bromide to verify equal RNA loading. + +Immunoblot analysis of MCAD protein. + +To evaluate the quantity of MCAD protein in mouse tissue, liver samples from 6–8-wk-old MCAD+/+ (n = 1) and MCAD−/− (n = 3) mice were immediately frozen in liquid nitrogen and stored at −80 °C. For analysis, tissue was homogenized and lysed in RIPA buffer (1× PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, and 1% SDS) with 10% glycerol and Complete Protease Inhibitor, (Roche Diagnostics Corporation, Indianapolis, Indiana, United States), 1 mM of phenylmethylsulfonylfluoride, and 1 mM of sodium orthovanadate. Lysates were quantified by Bradford BCA protein assay (Bio-Rad, Hercules, California, United States). Protein lysates were denatured, separated in 8% SDS-PAGE, and transferred overnight onto a 0.45 μm nitrocellulose membrane (Schleicher and Schuell, Keene, New Hampshire, United States). After blocking with 5% nonfat milk in phosphate-buffered saline with 0.1% Tween-20, the membrane was immunoblotted overnight at 4 °C with 1:500 dilution of an anti-MCAD antibody. Blots were incubated in anti-mouse IgG HRP-conjugated secondary antibody for 2–4 h at room temperature using standard procedures and developed by chemiluminiscence (Renaissance, NEN Lifesciences Products, Boston, Massachusetts, United States). + +Liver enzyme activity. + +In order to evaluate MCAD activity in mice, liver homogenates were prepared from MCAD+/+ (n = 5) and MCAD−/− mice (n = 5). The sensitive and highly specific anaerobic ETF reduction assay was used on tissue extracts with octanoyl-CoA (C8) and palmitoyl-CoA (C16) as substrate as previously described [29]. + +Fasting and cold challenge. + +Eight-wk-old MCAD+/+ and MCAD−/− mice were fasted for 18 h (cold tolerance experiments) or 24 h (serum glucose, free fatty acid, organic acid, and carnitine experiments) prior to analysis. Glucose concentration was measured in 10 μl sera using an Ektachem DT II system (Johnson and Johnson Clinical Diagnostics, Rochester, New York, United States). Total non-esterified fatty acids (NEFA) were measured by an enzymatic, colorimetric method (“NEFA-C” reagents, Wako Diagnostics, Richmond, Virginia, United States). The assay was modified to accommodate a reduced sample size (10 μl) and use of a microplate reader for measurement of optical density at 550 nm. Urine organic acid analyses were performed using gas chromatography-mass spectroscopy as previously described [22,30], except tetracosane (C24; Sigma, St. Louis, Missouri, United States) was used as the internal standard, and quantitative determinations were made based on comparison with synthetic calibration standards. Acylcarnitine analyses in serum and bile were conducted using electrospray tandem mass spectrometry [31,32]. + +Histopathology. + +Twelve mice were examined for gross and histologic abnormalities, including one male and one female MCAD−/− mouse 18-mo-old, one male and one female MCAD+/+ mouse 6-mo-old, two male and two female MCAD−/− mice 4-wk-old, and two male and two female MCAD+/+ mice 4-wk-old. Kidney, spleen, pancreas, liver, brain, heart, testicles, ovaries, and skeletal muscle were fixed by immersion in buffered 10% formalin, processed routinely for paraffin sectioning, sectioned at 5 μm, and stained with hematoxylin and eosin. Frozen liver sections were prepared using standard methods and sections were stained with Oil-red-O. Slides were examined without knowledge of genotype or age. Other mice were examined because of sporadic clinical disease. Those with visible cardiac enlargement were evaluated for cardiac lesions. + +Statistical analyses. + +Results between groups were tested for statistical significance using Student's t-test. A p < 0.05 was set as significant. + +Supporting Information + +Accession Number + +The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession number for the mouse gene Acadm is U07159. + +Acknowledgements + +We thank Sushama Varma for technical assistance. This research was supported by the University of Alabama at Birmingham (UAB) Comprehensive Cancer Center (Oligonucleotide and Transgenic Animal Shared Facilities) grant P30-CA13148, UAB Musculoskeletal Disease and Arthritis Center (Gene Targeting Core Facility) grant P60-AR20614, UAB Clinical Nutrition Research Center grant P30-DK-56336, and by National Institutes of Health grants R01-RR02599 (PAW), T32-RR-07003 (RJT), K01-RR00129 (RJT), RO1-DK45482 (JV), and DK54936 (JV). + +Abbreviations + +bp - base pair + +ES - embryonic stem + +ETF - electron transport flavoprotein + +MCAD - medium-chain acyl-CoA dehydrogenase + +SCAD - short-chain acyl-CoA dehydrogenase + +SD - standard deviation + +VLCAD - very long-chain acyl-CoA dehydrogenase + +Footnotes + +Competing interests. The authors have declared that no competing interests exist. + +Author contributions. RJT, JV, PR, DM, and PAW conceived and designed the experiments. RJT, DAH, LT, JV, PR, DM, JDS, and PAW performed the experiments. RJT, DAH, LT, JV, JDS, PR, DM, TRS, and PAW analyzed the data and contributed reagents/materials/analysis tools. RJT, JV, PR, DM, TRS, JDS, and PAW wrote the paper. + +Citation: Tolwani RJ, Hamm DA, Tian L, Sharer JD, Vockley J, et al. (2005) Medium-chain acyl-CoA dehydrogenase deficiency in gene-targeted mice. PLoS Genet 1(2): e23. diff --git a/src/ontogpt/evaluation/craft/database/all/16216087.ann b/src/ontogpt/evaluation/craft/database/all/16216087.ann new file mode 100644 index 000000000..9bb62dff8 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16216087.ann @@ -0,0 +1,1470 @@ +T1 GO:0007608 42 51 Olfactory +T2 GO:0007613 52 58 Memory +T3 GO:0065007 72 82 Controlled +T4 CHEBI:34018 93 97 AMPA +T5 SO:0000704 119 126 Genetic +T6 CHEBI:34018 144 192 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate +T7 GO:0007612 279 287 learning +T8 GO:0007613 293 299 memory +T9 CHEBI:29108 344 348 Ca2+ +T10 GO:0007608 369 378 olfactory +T11 PR:000008234 441 447 GluR-B +T12 GO:0010467 459 469 expression +T13 PR:000008234 482 488 GluR-B +T14 CHEBI:29108 519 523 Ca2+ +T15 NCBITaxon:10088 548 552 Mice +T16 UBERON:0001890 604 613 forebrain +T17 GO:0007608 631 640 olfactory +T18 GO:0007612 671 679 learning +T19 GO:0035106 694 714 operant conditioning +T20 GO:0007608 721 730 Olfactory +T21 GO:0007613 731 737 memory +T22 PR:000008234 775 781 GluR-B +T23 UBERON:0001890 795 804 forebrain +T24 NCBITaxon:1 847 858 individuals +T25 GO:0007608 898 907 olfactory +T26 GO:0007613 908 914 memory +T27 UBERON:0001851 934 940 cortex +T28 GO:0007613 955 961 memory +T29 PR:000008234 988 994 GluR-B +T30 GO:0010467 995 1005 expression +T31 UBERON:0004725 1020 1035 piriform cortex +T32 PR:000008234 1112 1118 GluR-B +T33 PR:000008234 1146 1152 GluR-B +T34 GO:0010467 1153 1163 expression +T35 PR:000008234 1220 1226 GluR-B +T36 GO:0010467 1227 1237 expression +T37 GO:0008355 1286 1304 olfactory learning +T38 GO:0007613 1326 1332 memory +T39 GO:0007608 1366 1380 sense of smell +T40 NCBITaxon:9989 1412 1419 rodents +T41 GO:0007616 1465 1477;1488 1494 long-lasting ... memory +T42 GO:0007608 1478 1487 olfactory +T43 GO:0007608 1598 1607 olfactory +T44 NCBITaxon:9989 1624 1631 rodents +T45 GO:0007608 1723 1732 olfactory +T46 GO:0007613 1733 1739 memory +T47 UBERON:0004725 1740 1755 piriform cortex +T48 GO:0007608 1786 1795 olfactory +T49 UBERON:0002264 1786 1800 olfactory bulb +T50 GO:0007608 1946 1955 olfactory +T51 GO:0007613 1956 1962 memory +T52 GO:0007608 2035 2044 olfactory +T53 GO:0007608 2138 2147 olfactory +T54 GO:0007613 2148 2154 memory +T55 CL:0000540 2172 2180 neuronal +T56 UBERON:0004725 2200 2215 piriform cortex +T57 GO:0045202 2310 2318 synaptic +T58 UBERON:0024914 2457 2466 circuitry +T59 GO:0007608 2474 2483 olfactory +T60 UBERON:0002264 2474 2488 olfactory bulb +T61 UBERON:0024914 2540 2548 circuits +T62 UBERON:0000966 2580 2586 retina +T63 UBERON:0001062 2723 2730 anatomy +T64 GO:0045202 2862 2870 synapses +T65 CL:0000527 2893 2907 output neurons +T66 CL:1001502 2909 2921 mitral cells +T67 CL:0000540 2944 2951 neurons +T68 CL:0000120 2953 2966 granule cells +T69 GO:0007608 2975 2984 olfactory +T70 UBERON:0002264 2975 2989 olfactory bulb +T71 GO:0045202 3009 3017 synapses +T72 CHEBI:16865 3068 3091 gamma-aminobutyric acid +T73 GO:0065007 3108 3118 controlled +T74 CHEBI:16865 3212 3235 gamma-aminobutyric acid +T75 GO:0014051 3212 3243 gamma-aminobutyric acid release +T76 CHEBI:29108 3247 3251 Ca2+ +T77 CL:0000540 3336 3344 neuronal +T78 UBERON:0024914 3336 3353 neuronal circuits +T79 GO:0007608 3397 3406 olfactory +T80 GO:0007613 3407 3413 memory +T81 CHEBI:34018 3483 3531 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate +T82 SO:0000704 3575 3582 genetic +T83 CHEBI:34018 3630 3678 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate +T84 CHEBI:34018 3680 3684 AMPA +T85 PR:000008234 3727 3733 GluR-B +T86 PR:000008233 3767 3773 GluR-A +T87 PR:000008236 3767 3772;3777 3778 GluR- ... D +T88 PR:000008233 3780 3785 GluR1 +T89 PR:000008234 3868 3874 GluR-B +T90 PR:000008234 3915 3921 GluR-B +T91 CHEBI:24870 4009 4012 ion +T92 CHEBI:29108 4082 4086 Ca2+ +T93 CHEBI:34018 4103 4107 AMPA +T94 CL:0000540 4130 4137 neurons +T95 PR:000008234 4160 4166 GluR-B +T96 GO:0006396 4325 4336 RNA editing +T97 PR:000008234 4340 4346 GluR-B +T98 SO:0000932 4347 4355 pre-mRNA +T99 PR:000008234 4396 4402 GluR-B +T100 GO:0010467 4422 4432 expression +T101 PR:000008234 4454 4460 GluR-B +T102 CHEBI:29108 4535 4539 Ca2+ +T103 CHEBI:34018 4556 4560 AMPA +T104 SO:0000704 4596 4600 gene +T105 NCBITaxon:10088 4610 4614 mice +T106 PR:000008234 4651 4657 GluR-B +T107 GO:0010467 4666 4676 expression +T108 PR:000008234 4680 4686 GluR-B +T109 GO:0007608 4697 4706 olfactory +T110 UBERON:0002264 4697 4711 olfactory bulb +T111 CL:1001502 4750 4762 mitral cells +T112 PR:000008234 4790 4796 GluR-B +T113 PR:000008234 4852 4858 GluR-B +T114 GO:0060076 4886 4905 excitatory synaptic +T115 GO:0007268 4897 4918 synaptic transmission +T116 SO:0000704 4926 4937 genetically +T117 CL:0000540 4948 4956 neuronal +T118 UBERON:0024914 5077 5085 circuits +T119 GO:0045202 5109 5117 synaptic +T120 CHEBI:29108 5136 5140 Ca2+ +T121 UBERON:0004725 5179 5194 piriform cortex +T122 GO:0007613 5213 5225 memorization +T123 UBERON:0002616 5284 5297 brain regions +T124 NCBITaxon:10088 5451 5455 mice +T125 PR:000008234 5468 5474 GluR-B +T126 NCBITaxon:10088 5675 5679 mice +T127 GO:0010467 5680 5690 expressing +T128 UBERON:0000955 5706 5711 brain +T129 PR:000008234 5739 5745 GluR-B +T130 GO:0016265 5812 5815 die +T131 GO:0010467 5929 5939 expression +T132 SO:0000346 5979 5982 lox +T133 GO:0010467 6012 6022 expression +T134 UBERON:0002616 6034 6045 brain areas +T135 SO:0000704 6049 6053 gene +T136 NCBITaxon:10088 6063 6067 mice +T137 PR:000008234 6077 6083 GluR-B +T138 SO:0001023 6084 6091 alleles +T139 SO:0000346 6102 6112 loxP sites +T140 GO:0010467 6177 6187 expression +T141 PR:000008234 6209 6215 GluR-B +T142 UBERON:0001890 6219 6228 forebrain +T143 UBERON:0000104 6255 6263 lifespan +T144 NCBITaxon:10088 6313 6317 Mice +T145 UBERON:0001890 6323 6332 forebrain +T146 PR:000008234 6342 6348 GluR-B +T147 UBERON:0000104 6404 6408 life +T148 GO:0007608 6504 6513 olfactory +T149 GO:0008355 6568 6586 olfactory learning +T150 GO:0007613 6608 6614 memory +T151 SO:0000902 6656 6666 transgenes +T152 GO:0010467 6697 6707 expression +T153 SO:0000704 6753 6764 genetically +T154 NCBITaxon:1 6775 6786 individuals +T155 GO:0010467 6827 6837 expression +T156 PR:000008234 6901 6907 GluR-B +T157 SO:0000704 6912 6916 gene +T158 SO:0000359 6927 6933 floxed +T159 PR:000008234 6934 6940 GluR-B +T160 SO:0001023 6941 6948 alleles +T161 NCBITaxon:10088 6979 6984 mouse +T162 GO:0010467 7010 7020 expression +T163 UBERON:0001890 7024 7033 forebrain +T164 PR:000008234 7050 7056 GluR-B +T165 GO:0007608 7067 7076 olfaction +T166 UBERON:0002616 7085 7098 brain regions +T167 PR:000008234 7168 7174 GluR-B +T168 GO:0007608 7178 7187 olfactory +T169 GO:0007613 7207 7213 memory +T170 UBERON:0002616 7242 7253 brain areas +T171 GO:0007608 7284 7293 olfactory +T172 PR:000008234 7335 7341 GluR-B +T173 UBERON:0004725 7368 7383 piriform cortex +T174 PR:000008234 7487 7493 GluR-B +T175 UBERON:0002616 7532 7545 brain regions +T176 GO:0008355 7549 7562 odor learning +T177 GO:0007613 7584 7590 memory +T178 PR:000008234 7602 7608 GluR-B +T179 GO:0010467 7612 7622 Expression +T180 UBERON:0001890 7630 7639 Forebrain +T181 GO:0008355 7650 7668 Olfactory Learning +T182 PR:000008234 7757 7763 GluR-B +T183 CHEBI:34018 7775 7779 AMPA +T184 GO:0007608 7792 7801 olfactory +T185 NCBITaxon:10088 7831 7835 mice +T186 GO:0010467 7841 7848 express +T187 PR:000008234 7861 7867 GluR-B +T188 PR:000008234 7927 7933 GluR-B +T189 NCBITaxon:10088 7965 7969 mice +T190 PR:000008234 8004 8010 GluR-B +T191 SO:0001023 8011 8017 allele +T192 SO:0000704 8021 8025 gene +T193 PR:000008234 8035 8041 GluR-B +T194 SO:0001023 8045 8051 allele +T195 SO:0000188 8065 8073 intronic +T196 PR:000008234 8096 8102 GluR-B +T197 SO:0000932 8103 8111 pre-mRNA +T198 SO:0000363 8153 8159;8167 8171 floxed ... gene +T199 GO:0008380 8196 8204 splicing +T200 SO:0000188 8221 8227 intron +T201 GO:0010467 8253 8263 expression +T202 PR:000008234 8271 8277 GluR-B +T203 SO:0001023 8281 8287 allele +T204 GO:0016458 8299 8306 silence +T205 PR:000008234 8322 8328 GluR-B +T206 SO:0001023 8332 8338 allele +T207 GO:0007567 8364 8369 natal +T208 UBERON:0001890 8370 8379 forebrain +T209 SO:0000902 8406 8415 transgene +T210 PR:000003199 8468 8475 αCaMKII +T211 SO:0000167 8476 8484 promoter +T212 UBERON:0001890 8538 8547 forebrain +T213 CL:0012001 8538 8555 forebrain neurons +T214 SO:0000188 8560 8568 intronic +T215 SO:0000704 8576 8580 gene +T216 PR:000008234 8604 8610 GluR-B +T217 SO:0001023 8615 8621 allele +T218 PR:000008234 8644 8650 GluR-B +T219 NCBITaxon:10088 8712 8716 mice +T220 GO:0010467 8717 8727 expressing +T221 GO:0007567 8732 8739 natally +T222 UBERON:0001890 8740 8749 forebrain +T223 PR:000008234 8777 8783 GluR-B +T224 CHEBI:29108 8797 8801 Ca2+ +T225 PR:000008234 8912 8918 GluR-B +T226 NCBITaxon:10088 8926 8930 mice +T227 NCBITaxon:10088 8951 8955 mice +T228 GO:0010467 8956 8966 expressing +T229 PR:000008234 8967 8973 GluR-B +T230 UBERON:0000104 8999 9007 lifespan +T231 PR:000008234 9181 9187 GluR-B +T232 NCBITaxon:10088 9195 9199 mice +T233 GO:0007608 9270 9279 olfactory +T234 CHEBI:15377 9320 9325 water +T235 NCBITaxon:10088 9335 9339 mice +T236 CHEBI:15377 9369 9374 water +T237 PR:000008234 9466 9472 GluR-B +T238 NCBITaxon:10088 9492 9496 mice +T239 PR:000008234 9659 9665 GluR-B +T240 NCBITaxon:10088 9673 9677 mice +T241 GO:0007612 9696 9704 learning +T242 GO:0007612 9875 9883 learning +T243 UBERON:0000033 10009 10013 head +T244 NCBITaxon:33208 10092 10099 animals +T245 UBERON:0000033 10111 10115 head +T246 UBERON:0000033 10226 10230 head +T247 PR:000008234 10543 10549 GluR-B +T248 NCBITaxon:10088 10557 10561 mice +T249 GO:0010467 10890 10900 expression +T250 CHEBI:29108 10904 10908 Ca2+ +T251 UBERON:0001890 10929 10938 forebrain +T252 GO:0007608 10960 10969 olfactory +T253 UBERON:0002264 10960 10974 olfactory bulb +T254 GO:0008355 10999 11012 odor learning +T255 GO:0007608 11026 11035 olfactory +T256 PR:000008234 11053 11059 GluR-B +T257 NCBITaxon:10088 11063 11067 Mice +T258 GO:0007608 11086 11095 Olfactory +T259 GO:0008355 11149 11167 olfactory learning +T260 CHEBI:29108 11232 11236 Ca2+ +T261 CHEBI:34018 11253 11257 AMPA +T262 PR:000008234 11315 11321 GluR-B +T263 NCBITaxon:10088 11325 11329 mice +T264 PR:000008234 11342 11348 GluR-B +T265 UBERON:0001890 11352 11361 forebrain +T266 UBERON:0001890 11403 11412 forebrain +T267 GO:0010467 11430 11440 expression +T268 SO:0000704 11462 11466 gene +T269 PR:000008234 11476 11482 GluR-B +T270 NCBITaxon:10088 11487 11491 mice +T271 PR:000008234 11510 11516 GluR-B +T272 SO:0001023 11517 11524 alleles +T273 SO:0000359 11528 11534 floxed +T274 SO:0000147 11535 11539 exon +T275 PR:000008234 11569 11575 GluR-B +T276 NCBITaxon:10088 11599 11603 mice +T277 PR:000008234 11723 11729 GluR-B +T278 UBERON:0001851 11814 11822 cortical +T279 GO:0007608 11834 11843 olfactory +T280 UBERON:0002264 11834 11848 olfactory bulb +T281 PR:000008234 11876 11882 GluR-B +T282 PR:000008234 11893 11899 GluR-B +T283 PR:000008234 11943 11949 GluR-B +T284 CHEBI:34018 11990 11994 AMPA +T285 PR:000008234 12033 12039 GluR-B +T286 PR:000008234 12040 12046 GluR-B +T287 CHEBI:29108 12105 12109 Ca2+ +T288 CHEBI:34018 12131 12135 AMPA +T289 PR:000008234 12174 12180 GluR-B +T290 PR:000008234 12277 12283 GluR-B +T291 NCBITaxon:10088 12295 12299 mice +T292 UBERON:0001890 12305 12314 forebrain +T293 PR:000008234 12324 12330 GluR-B +T294 UBERON:0000104 12368 12372 life +T295 UBERON:0000113 12451 12460 adulthood +T296 PR:000008234 12487 12493 GluR-B +T297 PR:000008234 12593 12599 GluR-B +T298 NCBITaxon:10088 12603 12607 mice +T299 NCBITaxon:10088 12795 12799 mice +T300 PR:000008234 12831 12837 GluR-B +T301 http://purl.obolibrary.org/obo/MONDO_0005618 12918 12925 anxiety +T302 PR:000008234 12929 12935 GluR-B +T303 NCBITaxon:10088 12939 12943 mice +T304 PR:000008234 12990 12996 GluR-B +T305 PR:000008234 13067 13073 GluR-B +T306 PR:000008234 13151 13157 GluR-B +T307 PR:000008234 13225 13231 GluR-B +T308 PR:000008234 13243 13249 GluR-B +T309 NCBITaxon:10088 13253 13257 mice +T310 PR:000008234 13431 13437 GluR-B +T311 PR:000008234 13438 13444 GluR-B +T312 CHEBI:29108 13482 13486 Ca2+ +T313 CHEBI:34018 13507 13511 AMPA +T314 GO:0008355 13544 13548;13568 13576 odor ... learning +T315 PR:000008234 13599 13605 GluR-B +T316 PR:000008234 13685 13691 GluR-B +T317 NCBITaxon:10088 13695 13699 mice +T318 GO:0008306 13751 13762;13782 13794 associative ... conditioning +T319 GO:0007608 13772 13781 olfactory +T320 GO:0007608 13859 13868 olfaction +T321 CHEBI:60004 13954 13962 mixtures +T322 GO:0046959 13974 13985 habituation +T323 PR:000008234 13987 13993 GluR-B +T324 NCBITaxon:10088 14009 14013 mice +T325 CHEBI:60004 14211 14219 mixtures +T326 CHEBI:23243 14223 14229 cineol +T327 CHEBI:4917 14234 14241 eugenol +T328 CHEBI:29019 14324 14334;14347 14351 pelargonic ... acid +T329 CHEBI:17418 14339 14351 valeric acid +T330 PR:000008234 14364 14370 GluR-B +T331 PR:000008234 14379 14385 GluR-B +T332 NCBITaxon:10088 14389 14393 mice +T333 GO:0007612 14415 14423 learning +T334 PR:000008234 14804 14810 GluR-B +T335 NCBITaxon:10088 14826 14830 mice +T336 CHEBI:60004 15039 15047 mixtures +T337 CHEBI:60004 15121 15128 mixture +T338 CHEBI:29019 15165 15168 Pel +T339 CHEBI:17418 15169 15172 Val +T340 PR:000008234 15477 15483 GluR-B +T341 NCBITaxon:10088 15487 15491 mice +T342 PR:000008234 15573 15579 GluR-B +T343 UBERON:0001890 15583 15592 forebrain +T344 GO:0008355 15628 15646 olfactory learning +T345 GO:0007608 15728 15737 olfaction +T346 PR:000008234 15766 15772 GluR-B +T347 NCBITaxon:10088 15776 15780 mice +T348 GO:0007608 15806 15815 olfactory +T349 GO:0007612 15846 15854 learning +T350 GO:0007608 16068 16077 olfactory +T351 GO:0007608 16137 16151 sense of smell +T352 PR:000008234 16313 16319 GluR-B +T353 PR:000008234 16331 16337 GluR-B +T354 NCBITaxon:10088 16341 16345 mice +T355 PR:000008234 16467 16473 GluR-B +T356 PR:000008234 16510 16516 GluR-B +T357 CHEBI:29108 16544 16548 Ca2+ +T358 CHEBI:29108 16602 16606 Ca2+ +T359 GO:0008355 16643 16661 olfactory learning +T360 GO:0007608 16683 16692 Olfactory +T361 GO:0007613 16693 16699 Memory +T362 PR:000008234 16750 16756 GluR-B +T363 NCBITaxon:10088 16760 16764 Mice +T364 CHEBI:29108 16808 16812 Ca2+ +T365 GO:0007608 16833 16842 olfactory +T366 CHEBI:34018 16893 16897 AMPA +T367 GO:0007608 16910 16919 olfactory +T368 GO:0007613 16920 16926 memory +T369 GO:0007608 16937 16946 olfactory +T370 GO:0007613 16947 16953 memory +T371 PR:000008234 16957 16963 GluR-B +T372 NCBITaxon:10088 16967 16971 mice +T373 CHEBI:29019 17129 17139;17155 17159 pelargonic ... acid +T374 CHEBI:17418 17147 17159 valeric acid +T375 NCBITaxon:10088 17302 17306 mice +T376 GO:0007613 17364 17370 memory +T377 PR:000008234 17413 17419 GluR-B +T378 NCBITaxon:10088 17423 17427 mice +T379 GO:0007608 17443 17452 olfactory +T380 GO:0007613 17453 17459 memory +T381 GO:0007612 17525 17533 learning +T382 PR:000008234 17546 17552 GluR-B +T383 GO:0007608 17596 17605 olfactory +T384 GO:0007613 17606 17612 memory +T385 GO:0007613 17766 17772 memory +T386 GO:0007613 17862 17868 memory +T387 GO:0007613 18031 18037 memory +T388 GO:0007613 18102 18108 memory +T389 GO:0007608 18206 18215 olfactory +T390 GO:0007613 18216 18222 memory +T391 GO:0007608 18254 18263 olfaction +T392 GO:0007612 18361 18369 learning +T393 GO:0007613 18438 18444 memory +T394 PR:000008234 18469 18475 GluR-B +T395 NCBITaxon:10088 18479 18483 mice +T396 NCBITaxon:33208 18521 18528 animals +T397 GO:0007608 18596 18605 olfactory +T398 GO:0007613 18606 18612 memory +T399 GO:0010467 18670 18680 expression +T400 UBERON:0001890 18684 18693 forebrain +T401 NCBITaxon:10088 18715 18719 mice +T402 NCBITaxon:10088 18772 18777 mouse +T403 GO:0010467 18842 18852 expression +T404 UBERON:0001890 18866 18875 forebrain +T405 NCBITaxon:10088 18915 18919 mice +T406 GO:0042571 19008 19016 antibody +T407 SO:0000902 19148 19157 transgene +T408 NCBITaxon:33208 19186 19193 animals +T409 SO:0000704 19237 19244 genetic +T410 GO:0007608 19342 19351 olfactory +T411 GO:0007613 19352 19358 memory +T412 GO:0010467 19465 19475 expression +T413 NCBITaxon:33208 19487 19494 animals +T414 GO:0007608 19516 19525 olfactory +T415 GO:0007613 19526 19532 memory +T416 UBERON:0002616 19567 19578 brain areas +T417 GO:0007608 19621 19630 Olfactory +T418 GO:0007613 19631 19637 Memory +T419 PR:000008234 19663 19669 GluR-B +T420 GO:0010467 19678 19688 Expression +T421 PR:000008234 19692 19698 GluR-B +T422 NCBITaxon:10088 19702 19706 Mice +T423 GO:0007608 19763 19772 olfactory +T424 GO:0007613 19773 19779 memory +T425 PR:000008234 19783 19789 GluR-B +T426 NCBITaxon:10088 19793 19797 mice +T427 PR:000008234 19834 19840 GluR-B +T428 PR:000008234 19851 19857 GluR-B +T429 NCBITaxon:10088 19861 19865 mice +T430 PR:000008234 19902 19908 GluR-B +T431 NCBITaxon:10088 19920 19924 mice +T432 GO:0007613 19940 19946 memory +T433 NCBITaxon:10088 19984 19988 mice +T434 GO:0007613 20005 20011 memory +T435 GO:0007613 20022 20028 memory +T436 PR:000008234 20069 20075 GluR-B +T437 UBERON:0001876 20100 20108 amygdala +T438 GO:0007608 20110 20119 olfactory +T439 UBERON:0002264 20110 20124 olfactory bulb +T440 UBERON:0004725 20130 20145 piriform cortex +T441 NCBITaxon:10088 20192 20196 mice +T442 GO:0007613 20218 20224 memory +T443 PR:000008234 20256 20262 GluR-B +T444 UBERON:0002616 20277 20288 brain areas +T445 PR:000008234 20387 20393 GluR-B +T446 GO:0007608 20406 20415 olfactory +T447 GO:0007613 20416 20422 memory +T448 GO:0007613 20428 20434 memory +T449 PR:000008234 20480 20486 GluR-B +T450 NCBITaxon:10088 20490 20494 mice +T451 NCBITaxon:33208 20522 20529 animals +T452 GO:0007613 20630 20636 memory +T453 PR:000008234 20669 20675 GluR-B +T454 GO:0007608 20732 20741 olfactory +T455 UBERON:0002264 20732 20747 olfactory bulbs +T456 UBERON:0001851 20749 20757 cortical +T457 NCBITaxon:10088 20790 20795 mouse +T458 PR:000008234 20801 20807 GluR-B +T459 NCBITaxon:33208 20903 20910 animals +T460 GO:0007612 21022 21030 learning +T461 GO:0007613 21159 21165 memory +T462 PR:000008234 21170 21176 GluR-B +T463 UBERON:0001851 21259 21267 cortical +T464 GO:0007608 21372 21381 olfactory +T465 UBERON:0002264 21372 21386 olfactory bulb +T466 PR:000008233 21429 21435 GluR-A +T467 PR:000008233 21554 21560 GluR-A +T468 NCBITaxon:10088 21636 21640 mice +T469 PR:000008234 21654 21660 GluR-B +T470 UBERON:0001890 21671 21680 forebrain +T471 GO:0007608 21704 21713 olfactory +T472 GO:0007613 21714 21720 memory +T473 PR:000008234 21767 21773 GluR-B +T474 UBERON:0001851 21804 21812 cortical +T475 GO:0008355 21829 21842 odor learning +T476 PR:000008234 21910 21916 GluR-B +T477 GO:0007608 21931 21940 olfactory +T478 UBERON:0002264 21931 21945 olfactory bulb +T479 UBERON:0001890 21956 21965 forebrain +T480 PR:000008234 21998 22004 GluR-B +T481 GO:0008355 22052 22065 odor learning +T482 PR:000008234 22155 22161 GluR-B +T483 GO:0007608 22245 22254 olfactory +T484 GO:0007613 22255 22261 memory +T485 GO:0008355 22267 22285 olfactory learning +T486 GO:0007608 22354 22363 Olfactory +T487 GO:0007613 22364 22370 Memory +T488 PR:000008234 22403 22409 GluR-B +T489 GO:0010467 22410 22420 Expression +T490 UBERON:0004725 22440 22455 Piriform Cortex +T491 PR:000008234 22459 22465 GluR-B +T492 NCBITaxon:10088 22469 22473 Mice +T493 PR:000008234 22499 22505 GluR-B +T494 NCBITaxon:10088 22519 22523 mice +T495 PR:000008234 22539 22545 GluR-B +T496 GO:0007608 22592 22601 olfactory +T497 UBERON:0001851 22603 22609 cortex +T498 GO:0007608 22641 22650 olfactory +T499 GO:0007613 22651 22657 memory +T500 GO:0007608 22663 22672 olfactory +T501 GO:0007613 22673 22679 memory +T502 PR:000008234 22719 22725 GluR-B +T503 GO:0007608 22729 22738 olfactory +T504 UBERON:0002894 22729 22745 olfactory cortex +T505 GO:0007612 22771 22779 learning +T506 PR:000008234 22853 22859 GluR-B +T507 GO:0007608 22867 22876 olfactory +T508 UBERON:0002264 22867 22881 olfactory bulb +T509 CHEBI:29108 22975 22979 Ca2+ +T510 GO:0010467 23001 23010 expressed +T511 PR:000008234 23083 23089 GluR-B +T512 UBERON:0004725 23122 23137 piriform cortex +T513 PR:000008234 23141 23147 GluR-B +T514 NCBITaxon:10088 23151 23155 mice +T515 PR:000008234 23255 23261 GluR-B +T516 UBERON:0004725 23293 23308 piriform cortex +T517 GO:0007613 23322 23328 memory +T518 NCBITaxon:10088 23383 23388 mouse +T519 PR:000008234 23428 23434 GluR-B +T520 SO:0000704 23450 23457 genetic +T521 PR:000008234 23472 23478 GluR-B +T522 NCBITaxon:10088 23482 23486 mice +T523 PR:000008234 23562 23568 GluR-B +T524 GO:0010467 23569 23579 expression +T525 CHEBI:27902 23599 23611 tetracycline +T526 GO:0065007 23612 23622 controlled +T527 GO:0065007 23693 23700 control +T528 PR:000003199 23715 23722 αCaMKII +T529 SO:0000167 23723 23731 promoter +T530 GO:0010467 23756 23766 expression +T531 GO:0010467 23839 23849 expression +T532 PR:000008234 23862 23868 GluR-B +T533 PR:000008234 23939 23945 GluR-B +T534 PR:000008234 24010 24016 GluR-B +T535 GO:0010467 24017 24027 expression +T536 UBERON:0000955 24031 24036 brain +T537 PR:000008234 24049 24055 GluR-B +T538 NCBITaxon:10088 24062 24066 mice +T539 GO:0010467 24076 24086 expression +T540 UBERON:0004725 24106 24121 piriform cortex +T541 UBERON:0001851 24131 24137 cortex +T542 UBERON:0001876 24139 24147 amygdala +T543 UBERON:0002435 24153 24161 striatum +T544 PR:000008234 24271 24277 GluR-B +T545 GO:0010467 24278 24288 expression +T546 PR:000008234 24313 24319 GluR-B +T547 NCBITaxon:10088 24326 24330 mice +T548 GO:0007608 24351 24360 Olfactory +T549 GO:0007613 24361 24367 memory +T550 PR:000008234 24385 24391 GluR-B +T551 PR:000008234 24409 24415 GluR-B +T552 PR:000008234 24423 24429 GluR-B +T553 NCBITaxon:10088 24434 24438 mice +T554 GO:0007613 24532 24538 Memory +T555 PR:000008234 24577 24583 GluR-B +T556 PR:000008234 24609 24615 GluR-B +T557 NCBITaxon:10088 24637 24641 mice +T558 GO:0007608 24721 24730 olfactory +T559 GO:0007613 24731 24737 memory +T560 PR:000008234 24741 24747 GluR-B +T561 NCBITaxon:10088 24754 24758 mice +T562 PR:000008234 24801 24807 GluR-B +T563 PR:000008234 24847 24853 GluR-B +T564 NCBITaxon:10088 24857 24861 mice +T565 GO:0007613 24873 24879 memory +T566 GO:0007613 24969 24975 memory +T567 GO:0007613 24995 25001 memory +T568 PR:000008234 25193 25199 GluR-B +T569 NCBITaxon:10088 25206 25210 mice +T570 GO:0007613 25232 25238 memory +T571 PR:000008234 25266 25272 GluR-B +T572 GO:0007613 25318 25324 memory +T573 GO:0007613 25350 25356 memory +T574 PR:000008234 25438 25444 GluR-B +T575 GO:0007613 25459 25465 memory +T576 GO:0007613 25541 25547 memory +T577 PR:000008234 25587 25593 GluR-B +T578 GO:0010467 25594 25604 expression +T579 UBERON:0004725 25624 25639 piriform cortex +T580 GO:0007613 25662 25668 memory +T581 GO:0010467 25787 25797 expression +T582 GO:0007613 25813 25819 memory +T583 GO:0007608 25854 25863 olfactory +T584 GO:0007613 25864 25870 memory +T585 PR:000008234 25888 25894 GluR-B +T586 NCBITaxon:33208 25898 25905 animals +T587 GO:0007608 25907 25916 olfactory +T588 GO:0007613 25917 25923 memory +T589 PR:000008234 25945 25951 GluR-B +T590 GO:0010467 25952 25962 expression +T591 UBERON:0001851 25966 25972 cortex +T592 UBERON:0001851 26017 26023 cortex +T593 GO:0007613 26066 26072 memory +T594 PR:000008234 26133 26139 GluR-B +T595 UBERON:0002616 26149 26162 brain regions +T596 PR:000008234 26179 26185 GluR-B +T597 NCBITaxon:33208 26192 26199 animals +T598 GO:0007608 26242 26251 olfactory +T599 GO:0007613 26252 26258 memory +T600 UBERON:0002616 26377 26388 brain areas +T601 GO:0010467 26417 26427 expression +T602 PR:000008234 26606 26612 GluR-B +T603 NCBITaxon:10088 26616 26620 mice +T604 PR:000008234 26719 26725 GluR-B +T605 CHEBI:29108 26902 26906 Ca2+ +T606 GO:0007608 26931 26940 olfactory +T607 UBERON:0002264 26931 26945 olfactory bulb +T608 PR:000008234 26964 26970 GluR-B +T609 GO:0010467 26971 26981 expression +T610 UBERON:0004725 26985 27000 piriform cortex +T611 PR:000008234 27036 27042 GluR-B +T612 GO:0010467 27043 27053 expression +T613 UBERON:0004725 27075 27090 piriform cortex +T614 PR:000008234 27114 27120 GluR-B +T615 GO:0007613 27159 27165 memory +T616 GO:0008355 27194 27203;27223 27231 olfactory ... learning +T617 GO:0008355 27311 27320;27337 27345 olfactory ... learning +T618 GO:0007613 27351 27357 memory +T619 SO:0000704 27371 27375 gene +T620 NCBITaxon:10088 27400 27404 mice +T621 GO:0010467 27426 27436 expression +T622 PR:000008234 27469 27475 GluR-B +T623 CHEBI:34018 27499 27503 AMPA +T624 PR:000003199 27516 27523 αCaMKII +T625 GO:0010467 27524 27534 expressing +T626 CL:0012001 27535 27545;27552 27561 neurons of ... forebrain +T627 NCBITaxon:10088 27546 27551 mouse +T628 UBERON:0001890 27552 27561 forebrain +T629 GO:0007608 27573 27582 olfactory +T630 UBERON:0002264 27573 27587 olfactory bulb +T631 CL:1001502 27573 27594;27607 27612 olfactory bulb mitral ... cells +T632 CL:0000120 27599 27612 granule cells +T633 GO:0008355 27623 27632;27652 27660 olfactory ... learning +T634 GO:0007608 27673 27682 olfactory +T635 GO:0007613 27683 27689 memory +T636 GO:0007608 27707 27716 olfactory +T637 GO:0035106 27755 27775 operant conditioning +T638 NCBITaxon:10088 27888 27892 mice +T639 GO:0007608 27919 27928 olfactory +T640 GO:0007613 27929 27935 memory +T641 GO:0010467 28057 28067 expression +T642 SO:0000359 28124 28136 loxP-flanked +T643 SO:0000704 28149 28153 gene +T644 SO:0001023 28163 28170 alleles +T645 PR:000008234 28202 28208 GluR-B +T646 PR:000008234 28267 28273 GluR-B +T647 CHEBI:29108 28297 28301 Ca2+ +T648 GO:0007613 28340 28346 memory +T649 GO:0008355 28348 28357;28377 28385 olfactory ... learning +T650 GO:0010467 28458 28468 expression +T651 SO:0000902 28533 28542 transgene +T652 PR:000008234 28569 28575 GluR-B +T653 UBERON:0004725 28593 28608 piriform cortex +T654 PR:000008234 28659 28665 GluR-B +T655 GO:0007613 28697 28703 memory +T656 GO:0007608 28745 28754 olfactory +T657 GO:0007608 28803 28812 olfactory +T658 CHEBI:29108 28873 28877 Ca2+ +T659 GO:0007608 28902 28911 olfactory +T660 UBERON:0002264 28902 28916 olfactory bulb +T661 GO:0007608 28926 28935 olfactory +T662 GO:0007613 28936 28942 memory +T663 SO:0000704 28965 28976 genetically +T664 PR:000008234 28985 28991 GluR-B +T665 UBERON:0000955 29011 29016 brain +T666 UBERON:0004725 29042 29057 piriform cortex +T667 GO:0007608 29060 29069 Olfactory +T668 NCBITaxon:10088 29101 29105 Mice +T669 UBERON:0001890 29111 29120 Forebrain +T670 PR:000008234 29130 29136 GluR-B +T671 PR:000008234 29149 29155 GluR-B +T672 GO:0010467 29159 29169 Expression +T673 UBERON:0001890 29176 29185 forebrain +T674 PR:000008234 29195 29201 GluR-B +T675 GO:0010467 29205 29215 expression +T676 PR:000008234 29220 29226 GluR-B +T677 GO:0008355 29254 29272 olfactory learning +T678 PR:000008234 29337 29343 GluR-B +T679 GO:0010467 29347 29357 expressing +T680 NCBITaxon:10088 29358 29362 mice +T681 PR:000008234 29442 29448 GluR-B +T682 UBERON:0001890 29470 29479 forebrain +T683 CHEBI:60004 29719 29727 mixtures +T684 GO:0007608 29788 29797 olfactory +T685 UBERON:0001890 29827 29836 forebrain +T686 PR:000008234 29846 29852 GluR-B +T687 NCBITaxon:10088 29862 29866 mice +T688 CHEBI:60004 29895 29903 mixtures +T689 GO:0007612 30046 30054 learning +T690 GO:0007608 30162 30171 olfactory +T691 GO:0007612 30226 30234 learning +T692 GO:0007613 30285 30291 memory +T693 GO:0010467 30321 30331 expression +T694 GO:0007608 30370 30387 olfactory sensory +T695 CL:0000207 30370 30395 olfactory sensory neurons +T696 GO:0007608 30457 30466 olfactory +T697 UBERON:0001997 30457 30477 olfactory epithelial +T698 SO:0000704 30508 30515 genetic +T699 UBERON:0002255 30534 30551 vomeronasal organ +T700 GO:0010467 30573 30583 expression +T701 GO:0007608 30663 30672 olfactory +T702 GO:0007608 30809 30818 olfactory +T703 PR:000008234 31056 31062 GluR-B +T704 GO:0007612 31130 31138 learning +T705 UBERON:0024914 31248 31256 circuits +T706 GO:0008355 31323 31327;31347 31355 Odor ... Learning +T707 CL:0012001 31403 31429 neurons of forebrain areas +T708 UBERON:0001890 31414 31423 forebrain +T709 GO:0007608 31444 31453 olfactory +T710 UBERON:0002264 31444 31458 olfactory bulb +T711 GO:0007608 31460 31469 olfactory +T712 UBERON:0002894 31460 31476 olfactory cortex +T713 UBERON:0001851 31488 31496 cortical +T714 GO:0008355 31540 31544;31564 31572 odor ... learning +T715 GO:0010467 31600 31610 expression +T716 PR:000008234 31617 31623 GluR-B +T717 PR:000008234 31627 31633 GluR-B +T718 NCBITaxon:10088 31637 31641 mice +T719 SO:0000704 31643 31650 genetic +T720 UBERON:0004725 31678 31693 piriform cortex +T721 GO:0010467 31729 31739 expression +T722 GO:0007608 31747 31756 olfactory +T723 UBERON:0002264 31747 31761 olfactory bulb +T724 GO:0007612 31805 31813 learning +T725 GO:0007608 31874 31883 olfactory +T726 UBERON:0002264 31874 31888 olfactory bulb +T727 GO:0008355 31892 31901;31921 31929 olfactory ... learning +T728 GO:0007612 32024 32032 learning +T729 GO:0007613 32056 32062 memory +T730 NCBITaxon:10088 32123 32127 mice +T731 UBERON:0004725 32133 32148 piriform cortex +T732 PR:000008234 32158 32164 GluR-B +T733 CHEBI:29108 32218 32222 Ca2+ +T734 SO:0000704 32238 32249 genetically +T735 CHEBI:34018 32259 32263 AMPA +T736 NCBITaxon:10088 32299 32303 mice +T737 GO:0010467 32304 32314 expressing +T738 PR:000008234 32333 32339 GluR-B +T739 GO:0008355 32359 32372 odor learning +T740 NCBITaxon:10088 32418 32422 mice +T741 PR:000008234 32437 32443 GluR-B +T742 NCBITaxon:10088 32453 32458 mouse +T743 CHEBI:29108 32466 32470 Ca2+ +T744 CHEBI:34018 32486 32490 AMPA +T745 CHEBI:34018 32552 32556 AMPA +T746 NCBITaxon:10088 32608 32612 mice +T747 GO:0010467 32617 32627 expressing +T748 PR:000008234 32628 32634 GluR-B +T749 GO:0045202 32663 32671 synaptic +T750 CHEBI:34018 32672 32676 AMPA +T751 NCBITaxon:10088 32735 32739 mice +T752 GO:0010467 32740 32750 expressing +T753 PR:000008234 32772 32778 GluR-B +T754 GO:0007268 32881 32896;32913 32921 transmission in ... synapses +T755 GO:0045202 32913 32921 synapses +T756 GO:0045202 33034 33042 synaptic +T757 PR:000008234 33069 33075 GluR-B +T758 GO:0010467 33079 33089 expression +T759 NCBITaxon:10088 33143 33148 mouse +T760 GO:0007612 33190 33198 learning +T761 PR:000008234 33237 33243 GluR-B +T762 GO:0045202 33304 33312 synapses +T763 CHEBI:29108 33366 33370 Ca2+ +T764 CHEBI:48828 33424 33428 Co2+ +T765 UBERON:0000955 33445 33450 brain +T766 CHEBI:48828 33489 33493 Co2+ +T767 CHEBI:29108 33505 33509 Ca2+ +T768 UBERON:0002313 33530 33551 hippocampal pyramidal +T769 CL:1001571 33530 33559 hippocampal pyramidal neurons +T770 NCBITaxon:10088 33563 33567 mice +T771 GO:0010467 33568 33578 expressing +T772 PR:000008234 33579 33585 GluR-B +T773 PR:000008234 33609 33615 GluR-B +T774 PR:000008234 33665 33671 GluR-B +T775 GO:0045202 33739 33746 synapse +T776 CL:1001502 33755 33761;33774 33779 mitral ... cells +T777 CL:0000120 33766 33779 granule cells +T778 GO:0007608 33787 33796 olfactory +T779 UBERON:0002264 33787 33801 olfactory bulb +T780 CHEBI:29108 33814 33818 Ca2+ +T781 PR:000008234 33952 33958 GluR-B +T782 PR:000008234 33979 33985 GluR-B +T783 GO:0007608 33996 34005 olfactory +T784 UBERON:0002264 33996 34010 olfactory bulb +T785 CL:0000540 34078 34085 neurons +T786 GO:0007608 34127 34136 olfactory +T787 PR:000008234 34220 34226 GluR-B +T788 PR:000008234 34268 34274 GluR-B +T789 NCBITaxon:10088 34280 34284 mice +T790 PR:000008234 34376 34382 GluR-B +T791 CHEBI:29108 34417 34421 Ca2+ +T792 PR:000008234 34466 34472 GluR-B +T793 SO:0001023 34476 34482 allele +T794 PR:000008234 34509 34515 GluR-B +T795 NCBITaxon:10088 34546 34550 mice +T796 PR:000008234 34581 34587 GluR-B +T797 SO:0001023 34589 34595 allele +T798 GO:0007608 34629 34638 olfactory +T799 UBERON:0002264 34629 34643 olfactory bulb +T800 GO:0008355 34864 34875;34884 34892 learning of ... odorants +T801 PR:000008234 35034 35040 GluR-B +T802 GO:0010467 35044 35054 expression +T803 PR:000008234 35060 35066 GluR-B +T804 NCBITaxon:10088 35094 35098 mice +T805 GO:0098793 35356 35360;35369 35377 pre- ... synaptic +T806 GO:0098794 35365 35377 postsynaptic +T807 GO:0005622 35378 35391 intracellular +T808 GO:0007608 35407 35416 Olfactory +T809 GO:0007613 35417 35423 Memory +T810 PR:000008234 35438 35444 GluR-B +T811 NCBITaxon:10088 35454 35458 Mice +T812 PR:000008234 35475 35481 GluR-B +T813 GO:0010467 35482 35492 Expression +T814 UBERON:0004725 35496 35511 Piriform Cortex +T815 GO:0007608 35539 35548 olfactory +T816 GO:0007613 35549 35555 memory +T817 PR:000008234 35559 35565 GluR-B +T818 NCBITaxon:10088 35569 35573 mice +T819 NCBITaxon:10088 35594 35598 mice +T820 GO:0007616 35746 35755;35766 35772 long-term ... memory +T821 GO:0007608 35756 35765 olfactory +T822 PR:000008234 35815 35821 GluR-B +T823 NCBITaxon:10088 35829 35833 mice +T824 GO:0007613 35835 35841 Memory +T825 PR:000008234 35845 35851 GluR-B +T826 NCBITaxon:10088 35855 35859 mice +T827 GO:0007613 35985 35991 memory +T828 GO:0007608 36093 36102 olfactory +T829 GO:0007613 36103 36109 memory +T830 GO:0007613 36276 36282 memory +T831 GO:0007612 36373 36381 learning +T832 GO:0007613 36459 36465 memory +T833 GO:0007613 36490 36496 memory +T834 GO:0007613 36571 36577 memory +T835 GO:0007613 36643 36649 memory +T836 NCBITaxon:10088 36668 36672 mice +T837 PR:000008234 36678 36684 GluR-B +T838 UBERON:0001890 36764 36773 forebrain +T839 PR:000008234 36795 36801 GluR-B +T840 GO:0007616 36832 36841;36852 36858 long-term ... memory +T841 GO:0007608 36842 36851 olfactory +T842 GO:0008355 36894 36898;36918 36926 odor ... learning +T843 GO:0007612 36973 36981 learning +T844 GO:0007613 36987 36993 memory +T845 GO:0010467 37095 37105 expression +T846 NCBITaxon:10088 37116 37120 mice +T847 PR:000008234 37142 37148 GluR-B +T848 NCBITaxon:10088 37182 37187 mouse +T849 GO:0008355 37272 37285 odor learning +T850 GO:0007608 37307 37316 olfactory +T851 GO:0007613 37317 37323 memory +T852 PR:000008234 37377 37383 GluR-B +T853 UBERON:0001851 37410 37418 cortical +T854 GO:0007608 37430 37439 olfactory +T855 GO:0007613 37440 37446 memory +T856 CL:0002608 37457 37468;37485 37492 hippocampal ... neurons +T857 UBERON:0001851 37476 37484 cortical +T858 CL:0010012 37476 37492 cortical neurons +T859 GO:0007613 37524 37536 memorization +T860 GO:0007612 37586 37594 learning +T861 PR:000008234 37640 37646 GluR-B +T862 PR:000008234 37686 37692 GluR-B +T863 GO:0007612 37885 37893 learning +T864 GO:0007613 37898 37904 memory +T865 UBERON:0001851 37962 37970 cortical +T866 GO:0007608 37995 38004 olfactory +T867 GO:0007613 38005 38011 memory +T868 PR:000008234 38022 38028 GluR-B +T869 GO:0007608 38043 38052 olfactory +T870 UBERON:0002264 38043 38057 olfactory bulb +T871 GO:0007613 38091 38097 memory +T872 GO:0010467 38122 38132 expression +T873 PR:000008234 38139 38145 GluR-B +T874 UBERON:0004725 38149 38164 piriform cortex +T875 GO:0007613 38196 38202 memory +T876 PR:000008234 38305 38311 GluR-B +T877 GO:0007608 38483 38492 olfactory +T878 GO:0007613 38493 38499 memory +T879 GO:0050890 38570 38579 cognitive +T880 UBERON:0002421 38643 38664 hippocampal formation +T881 NCBITaxon:10114 38668 38672 rats +T882 GO:0007616 38695 38704;38715 38721 long-term ... memory +T883 GO:0007608 38705 38714 olfactory +T884 GO:0007608 38745 38754 olfactory +T885 UBERON:0004725 38821 38836 piriform cortex +T886 GO:0007608 38890 38899 olfactory +T887 GO:0007613 38900 38906 memory +T888 UBERON:0004725 38964 38979 piriform cortex +T889 GO:0007613 39000 39006 memory +T890 GO:0007612 39026 39034 learning +T891 UBERON:0004725 39057 39072 piriform cortex +T892 GO:0007608 39118 39127 Olfactory +T893 GO:0007613 39128 39134 Memory +T894 GO:0007616 39187 39203 long-term memory +T895 PR:000008234 39237 39243 GluR-B +T896 PR:000008234 39276 39282 GluR-B +T897 UBERON:0003876 39347 39364 hippocampal field +T898 UBERON:0001876 39414 39422 amygdala +T899 PR:000008234 39440 39446 GluR-B +T900 CHEBI:29108 39606 39610 Ca2+ +T901 PR:000008234 39626 39632 GluR-B +T902 CHEBI:34018 39638 39642 AMPA +T903 GO:0098793 39740 39744;39753 39761 pre- ... synaptic +T904 GO:0098794 39749 39761 postsynaptic +T905 GO:0007613 39803 39809 memory +T906 GO:0007613 39909 39921 memorization +T907 PR:000008234 39941 39947 GluR-B +T908 NCBITaxon:10088 39951 39955 mice +T909 GO:0045202 40088 40096 synapses +T910 UBERON:0014548 40130 40143 CA1 pyramidal +T911 CL:0000598 40134 40149 pyramidal cells +T912 UBERON:0002616 40255 40266 brain areas +T913 GO:0007608 40320 40329 olfactory +T914 UBERON:0004725 40351 40366 piriform cortex +T915 GO:0007608 40370 40379 olfactory +T916 UBERON:0002264 40370 40384 olfactory bulb +T917 GO:0045202 40432 40440 synapses +T918 GO:0065007 40466 40475 regulated +T919 UBERON:0003883 40493 40508 hippocampal CA3 +T920 UBERON:0003881 40493 40504;40509 40512 hippocampal ... CA1 +T921 GO:0045202 40513 40521 synapses +T922 CHEBI:29108 40554 40558 Ca2+ +T923 CHEBI:29108 40583 40587 Ca2+ +T924 GO:0019722 40583 40597 Ca2+ signaling +T925 CHEBI:34018 40602 40606 AMPA +T926 GO:0007613 40675 40681 memory +T927 CHEBI:29108 40738 40742 Ca2+ +T928 GO:0007608 40791 40800 olfactory +T929 GO:0007613 40801 40807 memory +T930 PR:000008234 40862 40868 GluR-B +T931 UBERON:0002037 40896 40906 cerebellar +T932 PR:000008234 40969 40975 GluR-B +T933 SO:0000704 41086 41093 genetic +T934 PR:000008234 41192 41198 GluR-B +T935 PR:000008234 41213 41219 GluR-B +T936 GO:0010467 41223 41233 expression +T937 UBERON:0004725 41237 41252 piriform cortex +T938 SO:0000704 41319 41323 Gene +T939 GO:0010467 41319 41334 Gene Expression +T940 GO:0007612 41415 41423 learning +T941 GO:0007613 41444 41450 memory +T942 GO:0007613 41513 41519 memory +T943 PR:000008234 41527 41533 GluR-B +T944 NCBITaxon:33208 41543 41550 animals +T945 GO:0010467 41605 41615 expression +T946 PR:000008234 41669 41675 GluR-B +T947 GO:0007613 41732 41738 memory +T948 SO:0000771 41816 41839 quantitative trait loci +T949 SO:0000704 42009 42016 genetic +T950 SO:0000704 42179 42184 genic +T951 NCBITaxon:9989 42226 42233 rodents +T952 NCBITaxon:1 42247 42258 individuals +T953 SO:0000704 42376 42381 genes +T954 SO:0000704 42432 42437 genes +T955 SO:0000771 42572 42595 quantitative trait loci +T956 SO:0000704 42634 42639 genic +T957 SO:0000704 42722 42727 genes +T958 GO:0010467 42833 42843 expression +T959 SO:0000704 42929 42936 genetic +T960 SO:0000704 43197 43208 genetically +T961 PR:000008234 43370 43376 GluR-B +T962 CHEBI:29108 43404 43408 Ca2+ +T963 GO:0008355 43430 43439;43455 43463 olfactory ... learning +T964 GO:0007613 43469 43475 memory +T965 GO:0007608 43496 43505 olfactory +T966 UBERON:0002264 43496 43510 olfactory bulb +T967 UBERON:0004725 43515 43530 piriform cortex +T968 PR:000008234 43622 43628 GluR-B +T969 GO:0007613 43685 43691 memory +T970 GO:0007612 43723 43731 learning +T971 PR:000008234 43776 43782 GluR-B +T972 NCBITaxon:10088 43790 43794 mice +T973 UBERON:0004725 43800 43815 piriform cortex +T974 GO:0010467 43836 43846 expression +T975 PR:000008234 43850 43856 GluR-B +T976 SO:0000704 43954 43961 genetic +T977 UBERON:0024914 44051 44069 neural circuitries +T978 GO:0007608 44125 44134 olfactory +T979 UBERON:0000467 44145 44152 systems +T980 NCBITaxon:10088 44178 44183 Mouse +T981 PR:000008234 44241 44247 GluR-B +T982 NCBITaxon:10088 44255 44259 mice +T983 SO:0000704 44291 44298 genetic +T984 PR:000008234 44361 44367 GluR-B +T985 NCBITaxon:10088 44378 44382 mice +T986 PR:000008234 44384 44390 GluR-B +T987 NCBITaxon:10088 44396 44400 mice +T988 PR:000008234 44419 44425 GluR-B +T989 SO:0001023 44426 44432 allele +T990 SO:0000704 44439 44443 gene +T991 PR:000008234 44453 44459 GluR-B +T992 SO:0001023 44460 44466 allele +T993 SO:0000188 44480 44486 intron +T994 SO:0000704 44553 44557 gene +T995 SO:0000357 44558 44565 flanked +T996 SO:0000346 44569 44579 loxP sites +T997 SO:0000359 44582 44588 floxed +T998 PR:000008234 44592 44598 GluR-B +T999 NCBITaxon:10088 44603 44607 mice +T1000 SO:0000704 44619 44623 gene +T1001 PR:000008234 44633 44639 GluR-B +T1002 SO:0001023 44640 44647 alleles +T1003 SO:0000147 44657 44661 exon +T1004 SO:0000359 44668 44674 floxed +T1005 PR:000008234 44676 44682 GluR-B +T1006 NCBITaxon:10088 44686 44690 mice +T1007 SO:0000704 44707 44714 genetic +T1008 PR:000008234 44756 44762 GluR-B +T1009 NCBITaxon:10088 44767 44771 mice +T1010 NCBITaxon:10088 44785 44789 mice +T1011 NCBITaxon:10088 44834 44838 mice +T1012 GO:0045120 44934 44944 pronucleus +T1013 UBERON:0001890 44977 44986 forebrain +T1014 GO:0010467 44987 44997 expression +T1015 GO:0010467 45014 45024 expression +T1016 PR:000008234 45031 45037 GluR-B +T1017 NCBITaxon:10088 45043 45048 mouse +T1018 SO:0000155 45096 45103 plasmid +T1019 PR:000008234 45114 45120 GluR-B +T1020 GO:0045120 45143 45153 pronucleus +T1021 CL:0000023 45157 45164 oocytes +T1022 SO:0000155 45274 45281 Plasmid +T1023 PR:000008234 45292 45298 GluR-B +T1024 PR:000008233 45330 45336 GluR-A +T1025 PR:000008233 45355 45361 GluR-A +T1026 NCBITaxon:10114 45376 45379 rat +T1027 PR:000008234 45389 45395 GluR-B +T1028 PR:000008234 45411 45417 GluR-B +T1029 NCBITaxon:10088 45472 45476 mice +T1030 SO:0000902 45493 45502 transgene +T1031 UBERON:0001890 45507 45516 forebrain +T1032 GO:0010467 45542 45552 expression +T1033 NCBITaxon:10088 45581 45585 Mice +T1034 SO:0005853 45642 45650 cassette +T1035 PR:000008234 45658 45664 GluR-B +T1036 SO:0001023 45665 45671 allele +T1037 PR:000008234 45673 45679 GluR-B +T1038 SO:0000359 45710 45716 floxed +T1039 PR:000008234 45717 45723 GluR-B +T1040 PR:000008234 45725 45731 GluR-B +T1041 NCBITaxon:10088 45779 45783 mice +T1042 PR:000008234 45785 45791 GluR-B +T1043 SO:0000363 45844 45850;45858 45862 floxed ... gene +T1044 PR:000008234 45851 45857 GluR-B +T1045 CHEBI:27902 45885 45888 tet +T1046 SO:0000902 45909 45918 transgene +T1047 GO:0010467 45943 45953 expressing +T1048 SO:0000902 45964 45973 transgene +T1049 PR:000008234 46054 46060 GluR-B +T1050 NCBITaxon:10088 46064 46068 mice +T1051 PR:000008234 46126 46132 GluR-B +T1052 PR:000008234 46143 46149 GluR-B +T1053 NCBITaxon:10088 46186 46190 mice +T1054 PR:000008234 46270 46276 GluR-B +T1055 PR:000008234 46363 46369 GluR-B +T1056 PR:000008234 46380 46386 GluR-B +T1057 NCBITaxon:10088 46458 46462 Mice +T1058 NCBITaxon:10088 46487 46492 mouse +T1059 UBERON:0002415 46493 46497 tail +T1060 SO:0000112 46516 46523 primers +T1061 SO:0000028 46725 46734 basepairs +T1062 SO:0000028 46736 46738 bp +T1063 PR:000008234 46742 46748 GluR-B +T1064 SO:0000028 46891 46893 bp +T1065 SO:0000028 46910 46912 bp +T1066 SO:0000028 47019 47021 bp +T1067 SO:0000028 47038 47040 bp +T1068 SO:0000028 47146 47148 bp +T1069 SO:0000028 47249 47251 bp +T1070 SO:0001026 47278 47285 Genomic +T1071 NCBITaxon:10088 47295 47300 mouse +T1072 UBERON:0002415 47301 47305 tail +T1073 UBERON:0002107 47306 47311 liver +T1074 SO:0000028 47404 47406 bp +T1075 PR:000003199 47453 47460 αCaMKII +T1076 SO:0000167 47461 47469 promoter +T1077 PR:000008234 47657 47663 GluR-B +T1078 GO:0042571 47870 47880 antibodies +T1079 CHEBI:37926 47886 47890 FITC +T1080 MOP:0000779 47891 47898 coupled +T1081 MOP:0000779 47949 47956 coupled +T1082 NCBITaxon:9925 48022 48026 goat +T1083 NCBITaxon:9986 48032 48038 rabbit +T1084 GO:0042571 48039 48049 antibodies +T1085 GO:0007608 48061 48070 olfactory +T1086 UBERON:0001997 48061 48081 olfactory epithelium +T1087 GO:0042571 48168 48176 antibody +T1088 CHEBI:75055 48211 48216 X-gal +T1089 NCBITaxon:10088 48281 48286 Mouse +T1090 UBERON:0000955 48287 48293 brains +T1091 GO:0007608 48329 48338 olfactory +T1092 UBERON:0002264 48329 48343 olfactory bulb +T1093 UBERON:0001890 48359 48368 forebrain +T1094 GO:0042571 48468 48478 Antibodies +T1095 PR:000008234 48497 48503 GluR-B +T1096 PR:000003676 48535 48542 β-actin +T1097 NCBITaxon:9925 48648 48652 goat +T1098 NCBITaxon:9986 48658 48664 rabbit +T1099 NCBITaxon:9925 48669 48673 goat +T1100 NCBITaxon:10088 48679 48684 mouse +T1101 GO:0042571 48685 48695 antibodies +T1102 NCBITaxon:10088 48910 48914 mice +T1103 NCBITaxon:33208 49090 49096 animal +T1104 NCBITaxon:33208 49189 49196 animals +T1105 CHEBI:33290 49215 49219 food +T1106 CHEBI:15377 49229 49234 water +T1107 CHEBI:33290 49301 49305 food +T1108 CHEBI:15377 49330 49335 water +T1109 NCBITaxon:33208 49380 49386 animal +T1110 NCBITaxon:33208 49435 49441 animal +T1111 GO:0007608 49503 49512 olfactory +T1112 NCBITaxon:33208 49904 49911 animals +T1113 CHEBI:15377 50021 50026 water +T1114 CHEBI:15377 50105 50110 water +T1115 UBERON:0000033 50144 50148 Head +T1116 CHEBI:29019 50277 50292 pelargonic acid +T1117 CHEBI:17418 50294 50306 valeric acid +T1118 CHEBI:60004 50319 50327 mixtures +T1119 CHEBI:23243 50331 50337 cineol +T1120 CHEBI:4917 50342 50349 eugenol +T1121 CHEBI:46662 50403 50410 mineral +T1122 CHEBI:46662 50581 50588 mineral +T1123 GO:0046959 50647 50658 habituation +T1124 CHEBI:15377 50708 50713 water +T1125 NCBITaxon:33208 50736 50743 animals +T1126 GO:0035106 50772 50792 operant-conditioning +T1127 CHEBI:15377 50855 50860 water +T1128 UBERON:0000033 50941 50945 head +T1129 CHEBI:46662 51087 51094 mineral +T1130 NCBITaxon:33208 51104 51111 animals +T1131 GO:0007612 51112 51119 learned +T1132 NCBITaxon:10088 51212 51217 mouse +T1133 NCBITaxon:33208 51418 51424 animal +T1134 NCBITaxon:33208 51519 51525 animal +T1135 CHEBI:15377 51677 51682 water +T1136 NCBITaxon:33208 51735 51741 animal +T1137 NCBITaxon:33208 51911 51917 animal +T1138 NCBITaxon:33208 51956 51962 animal +T1139 NCBITaxon:33208 52210 52217 animals +T1140 CHEBI:15377 52664 52669 water +T1141 NCBITaxon:33208 52901 52907 animal +T1142 NCBITaxon:33208 53367 53373 animal +T1143 UBERON:0000033 53464 53468 head +T1144 GO:0046959 54357 54368 habituation +T1145 NCBITaxon:10088 54370 54374 mice +T1146 GO:0007613 54592 54598 memory +T1147 GO:0007612 54663 54671 learning +T1148 GO:0007612 54760 54768 learning +T1149 GO:0007612 54828 54836 learning +T1150 NCBITaxon:33208 54885 54892 animals +T1151 CHEBI:60004 54983 54991 mixtures +T1152 CHEBI:4917 54997 55004 eugenol +T1153 CHEBI:23243 55010 55016 cineol +T1154 CHEBI:4917 55026 55033 eugenol +T1155 CHEBI:23243 55039 55045 cineol +T1156 NCBITaxon:33208 55172 55179 animals +T1157 CHEBI:29019 55248 55263 pelargonic acid +T1158 CHEBI:17418 55271 55283 valeric acid +T1159 SO:0000704 55407 55414 genetic +T1160 NCBITaxon:9606 55575 55581 person +T1161 NCBITaxon:33208 55595 55602 animals +T1162 NCBITaxon:10088 55668 55672 mice +T1163 GO:0007613 55675 55681 Memory +T1164 GO:0007613 55705 55711 memory +T1165 CHEBI:29019 55766 55781 pelargonic acid +T1166 CHEBI:17418 55786 55798 valeric acid +T1167 GO:0007613 55800 55806 memory +T1168 GO:0007613 55969 55975 Memory +T1169 http://purl.obolibrary.org/obo/MONDO_0005027 56231 56240 epileptic +T1170 PR:000008234 56294 56299 GluRB +T1171 NCBITaxon:10088 56307 56311 mice +T1172 GO:0007612 56381 56389 Learning +T1173 GO:0007612 56539 56547 learning +T1174 GO:0007613 56864 56870 memory +T1175 PR:000008234 56890 56896 GluR-B +T1176 PR:000008234 56915 56921 GluR-B +T1177 NCBITaxon:10088 56934 56938 mice +T1178 NCBITaxon:33208 57028 57035 animals +T1179 PR:000008234 57076 57082 GluR-B +T1180 NCBITaxon:10088 57086 57090 mice +T1181 GO:0007613 57101 57107 memory +T1182 GO:0007613 57171 57177 memory +T1183 PR:000008234 57196 57202 GluR-B +T1184 GO:0007613 57209 57215 memory +T1185 UBERON:0001890 57313 57322 Forebrain +T1186 SO:0000704 57332 57336 Gene +T1187 PR:000003199 57398 57405 αCaMKII +T1188 SO:0000167 57406 57414 promoter +T1189 GO:0065007 57415 57422 control +T1190 GO:0010467 57446 57456 expression +T1191 PR:000033987 57477 57481 LacZ +T1192 NCBITaxon:10088 57505 57510 mouse +T1193 UBERON:0001890 57530 57539 forebrain +T1194 UBERON:0002298 57569 57578 brainstem +T1195 UBERON:0002037 57579 57589 cerebellum +T1196 NCBITaxon:10088 57602 57606 mice +T1197 GO:0010467 57662 57672 expression +T1198 CHEBI:75055 57729 57734 X-gal +T1199 UBERON:0001890 57849 57858 forebrain +T1200 UBERON:0001876 57886 57887 A +T1201 UBERON:0001876 57889 57897 amygdala +T1202 UBERON:0002037 57899 57901 Ce +T1203 UBERON:0002037 57903 57913 Cerebellum +T1204 UBERON:0001851 57915 57917 Cx +T1205 UBERON:0001851 57919 57925 cortex +T1206 UBERON:0001896 57943 57945 Me +T1207 UBERON:0001896 57947 57964 medulla oblongata +T1208 GO:0007608 57971 57980 Olfactory +T1209 UBERON:0002264 57971 57985 Olfactory bulb +T1210 NCBITaxon:10088 58007 58012 mouse +T1211 GO:0010467 58030 58040 expression +T1212 CHEBI:75055 58127 58132 X-gal +T1213 CL:0000120 58160 58173 granule cells +T1214 CL:0000120 58175 58177 GC +T1215 CL:1001502 58183 58195 mitral cells +T1216 CL:1001502 58197 58199 MC +T1217 PR:000008234 58360 58366 GluR-B +T1218 NCBITaxon:10088 58411 58415 Mice +T1219 GO:0007613 58605 58611 Memory +T1220 GO:0007613 58660 58666 Memory +T1221 PR:000008234 58749 58755 GluR-B +T1222 PR:000008234 58768 58774 GluR-B +T1223 NCBITaxon:33208 58787 58794 animals +T1224 GO:0007613 58819 58825 memory +T1225 GO:0007613 58868 58874 Memory +T1226 PR:000008234 58878 58884 GluR-B +T1227 NCBITaxon:33208 58888 58895 animals +T1228 GO:0007613 59176 59182 memory +T1229 GO:0007613 59281 59287 memory +T1230 NCBITaxon:33208 59332 59339 animals +T1231 PR:000008234 59347 59353 GluR-B +T1232 PR:000008234 59366 59372 GluR-B +T1233 GO:0007613 59467 59473 memory +T1234 CHEBI:60004 59528 59536 mixtures +T1235 NCBITaxon:33208 59568 59575 animals +T1236 GO:0007612 59622 59630 learning +T1237 GO:0007613 59712 59718 memory +T1238 GO:0010467 59843 59853 Expression +T1239 GO:0007608 59866 59875 Olfactory +T1240 UBERON:0001997 59866 59886 Olfactory Epithelium +T1241 NCBITaxon:10088 59897 59901 Mice +T1242 GO:0007608 59928 59937 olfactory +T1243 UBERON:0001997 59928 59948 olfactory epithelium +T1244 CHEBI:51240 60005 60021 propidium iodide +T1245 CL:0000120 60110 60123 granule cells +T1246 UBERON:0001885 60131 60144 dentate gyrus +T1247 GO:0005634 60157 60164 nuclear +T1248 NCBITaxon:10088 60191 60196 mouse +T1249 PR:000003199 60438 60445 αCaMKII +T1250 SO:0000167 60446 60454 promoter +T1251 PR:000008248 60481 60485 NR2C +T1252 SO:0000625 60486 60494 silencer +T1253 CHEBI:34018 61099 61103 AMPA +T1254 CHEBI:34018 61106 61154 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate +T1255 CHEBI:34018 61164 61212 α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate +T1256 SO:0000028 61223 61225 bp +T1257 SO:0000028 61228 61236 basepair +T1258 GO:0008355 61387 61400 Odor Learning +T1259 PR:000008234 61435 61441 GluR-B +T1260 NCBITaxon:10088 61449 61453 Mice +T1261 PR:000008234 61514 61520 GluR-B +T1262 SO:0000359 61540 61552 loxP-flanked +T1263 PR:000008234 61572 61578 GluR-B +T1264 SO:0000188 61594 61600 intron +T1265 GO:0010467 61640 61650 expression +T1266 PR:000008234 61687 61693 GluR-B +T1267 SO:0001023 61697 61703 allele +T1268 SO:0000147 61705 61709 Exon +T1269 GO:0016020 61727 61735 membrane +T1270 PR:000008234 61784 61790 GluR-B +T1271 SO:0000188 61804 61812 intronic +T1272 SO:0001023 61883 61889 allele +T1273 NCBITaxon:10088 61961 61966 mouse +T1274 NCBITaxon:10088 62054 62059 mouse +T1275 NCBITaxon:10088 62076 62081 mouse +T1276 UBERON:0000033 62110 62114 head +T1277 CHEBI:15377 62137 62142 water +T1278 NCBITaxon:10088 62191 62196 mouse +T1279 NCBITaxon:10088 62296 62301 mouse +T1280 NCBITaxon:33208 62460 62467 animals +T1281 PR:000008234 62473 62479 GluR-B +T1282 PR:000008234 62504 62510 GluR-B +T1283 PR:000008234 62672 62678 GluR-B +T1284 UBERON:0000033 62856 62860 head +T1285 NCBITaxon:10088 62878 62883 mouse +T1286 UBERON:0000033 63012 63016 head +T1287 UBERON:0000033 63059 63063 head +T1288 UBERON:0000033 63123 63127 head +T1289 GO:0008355 63404 63417 Odor Learning +T1290 GO:0007613 63459 63465 Memory +T1291 PR:000008234 63480 63486 GluR-B +T1292 NCBITaxon:10088 63490 63494 Mice +T1293 SO:0000359 63553 63565 loxP-flanked +T1294 SO:0000147 63566 63570 exon +T1295 PR:000008234 63581 63587 GluR-B +T1296 SO:0001023 63588 63595 alleles +T1297 PR:000008234 63613 63619 GluR-B +T1298 PR:000008234 63638 63643 GluRB +T1299 CHEBI:23243 63771 63774 Cin +T1300 CHEBI:4917 63780 63782 Eu +T1301 CHEBI:23243 63795 63798 Cin +T1302 CHEBI:4917 63804 63806 Eu +T1303 CHEBI:29019 63827 63830 Pel +T1304 CHEBI:17418 63841 63844 Val +T1305 PR:000008234 63846 63852 GluR-B +T1306 NCBITaxon:10088 63856 63860 mice +T1307 GO:0007612 63878 63886 learning +T1308 CHEBI:60004 64255 64262 mixture +T1309 NCBITaxon:10088 64343 64347 mice +T1310 PR:000008234 64363 64369 GluR-B +T1311 NCBITaxon:10088 64373 64377 mice +T1312 GO:0007608 64431 64440 Olfactory +T1313 GO:0007613 64441 64447 memory +T1314 PR:000008234 64506 64512 GluR-B +T1315 NCBITaxon:10088 64522 64526 mice +T1316 GO:0007608 64528 64537 Olfactory +T1317 GO:0007613 64538 64544 memory +T1318 CHEBI:29019 64622 64625 Pel +T1319 CHEBI:17418 64630 64633 Val +T1320 CHEBI:23243 64692 64695 Cin +T1321 CHEBI:23243 64697 64703 cineol +T1322 CHEBI:4917 64724 64726 Eu +T1323 CHEBI:4917 64728 64735 eugenol +T1324 CHEBI:29019 64737 64740 Pel +T1325 CHEBI:29019 64742 64757 pelargonic acid +T1326 CHEBI:17418 64759 64762 Val +T1327 CHEBI:17418 64764 64776 valeric acid +T1328 GO:0010467 64808 64818 Expression +T1329 NCBITaxon:10088 64822 64827 Mouse +T1330 GO:0010467 64858 64868 expression +T1331 UBERON:0001890 64872 64882 forebrains +T1332 NCBITaxon:10088 64902 64906 mice +T1333 GO:0007567 64973 64978 natal +T1334 CHEBI:75055 65048 65053 X-gal +T1335 UBERON:0000955 65089 65094 brain +T1336 SO:0001026 65169 65176 genomic +T1337 NCBITaxon:10088 65177 65182 mouse +T1338 NCBITaxon:10088 65202 65206 mice +T1339 GO:0010467 65232 65242 expression +T1340 SO:0000028 65295 65297 bp +T1341 SO:0000028 65335 65337 bp +T1342 SO:0001023 65339 65346 alleles +T1343 GO:0007608 65359 65368 Olfactory +T1344 GO:0007613 65369 65375 Memory +T1345 GO:0008355 65384 65397 Odor Learning +T1346 PR:000008234 65441 65447 GluR-B +T1347 UBERON:0001890 65474 65483 Forebrain +T1348 PR:000008234 65487 65493 GluR-B +T1349 NCBITaxon:10088 65497 65501 Mice +T1350 GO:0007608 65511 65520 olfactory +T1351 GO:0007613 65521 65527 memory +T1352 PR:000008234 65547 65553 GluR-B +T1353 NCBITaxon:10088 65597 65601 mice +T1354 NCBITaxon:10088 65739 65743 mice +T1355 PR:000008234 65858 65864 GluR-B +T1356 PR:000008234 65934 65940 GluR-B +T1357 PR:000008234 65968 65974 GluR-B +T1358 UBERON:0001876 66010 66018 amygdala +T1359 UBERON:0004725 66020 66035 piriform cortex +T1360 GO:0007608 66041 66050 olfactory +T1361 UBERON:0002264 66041 66055 olfactory bulb +T1362 PR:000008234 66084 66090 GluR-B +T1363 NCBITaxon:10088 66114 66119 mouse +T1364 UBERON:0000955 66120 66125 brain +T1365 UBERON:0001851 66190 66198 cortical +T1366 UBERON:0001890 66199 66208 forebrain +T1367 UBERON:0001890 66210 66212 FB +T1368 GO:0007608 66219 66228 olfactory +T1369 UBERON:0002264 66219 66233 olfactory bulb +T1370 UBERON:0002264 66235 66237 OB +T1371 PR:000008234 66276 66282 GluR-B +T1372 NCBITaxon:10088 66286 66290 mice +T1373 GO:0042571 66324 66334 antibodies +T1374 PR:000008234 66345 66351 GluR-B +T1375 PR:000003676 66356 66363 β-actin +T1376 PR:000008234 66471 66477 GluR-B +T1377 NCBITaxon:10088 66481 66485 mice +T1378 GO:0008355 66502 66515 odor learning +T1379 GO:0007608 66535 66544 olfactory +T1380 GO:0007613 66545 66551 memory +T1381 PR:000008234 66606 66612 GluR-B +T1382 UBERON:0001890 66651 66660 forebrain +T1383 GO:0007608 66666 66675 olfactory +T1384 UBERON:0002264 66666 66680 olfactory bulb +T1385 GO:0007613 66699 66705 Memory +T1386 CHEBI:60004 66841 66848 mixture +T1387 PR:000008234 66930 66936 GluR-B +T1388 GO:0007613 66945 66951 Memory +T1389 PR:000008234 66978 66984 GluR-B +T1390 UBERON:0001851 67041 67049 cortical +T1391 UBERON:0001890 67050 67059 forebrain +T1392 GO:0007608 67106 67115 olfactory +T1393 UBERON:0002264 67106 67120 olfactory bulb +T1394 GO:0007612 67158 67166 learning +T1395 CHEBI:60004 67217 67224 mixture +T1396 UBERON:0004725 67441 67456 Piriform Cortex +T1397 GO:0010467 67457 67467 Expression +T1398 PR:000008234 67485 67491 GluR-B +T1399 UBERON:0001890 67526 67535 forebrain +T1400 PR:000008234 67545 67551 GluR-B +T1401 GO:0010467 67596 67606 expression +T1402 PR:000008234 67613 67619 GluR-B +T1403 GO:0005634 67624 67631 nuclear +T1404 PR:000008234 67669 67675 GluR-B +T1405 NCBITaxon:10088 67679 67683 mice +T1406 PR:000008234 67710 67716 GluR-B +T1407 GO:0065007 67767 67777 controlled +T1408 PR:000008248 67797 67801 NR2C +T1409 SO:0000625 67802 67810 silencer +T1410 PR:000003199 67832 67839 αCaMKII +T1411 SO:0000167 67840 67848 promoter +T1412 UBERON:0000955 67892 67897 brain +T1413 NCBITaxon:10088 67910 67914 mice +T1414 SO:0000902 67933 67943 transgenes +T1415 CHEBI:75055 68001 68006 X-gal +T1416 CL:0002608 68048 68067 hippocampal neurons +T1417 UBERON:0001885 68076 68078 DG +T1418 CL:0000540 68084 68091 neurons +T1419 UBERON:0004725 68099 68114 piriform cortex +T1420 CL:0000540 68125 68132 neurons +T1421 PR:000008234 68141 68147 GluR-B +T1422 GO:0010467 68148 68158 expression +T1423 GO:0042571 68213 68221 antibody +T1424 PR:000008234 68292 68298 GluR-B +T1425 PR:000008234 68317 68323 GluR-B +T1426 NCBITaxon:10088 68362 68366 mice +T1427 PR:000008234 68436 68442 GluR-B +T1428 PR:000008234 68468 68474 GluR-B +T1429 PR:000008234 68506 68512 GluR-B +T1430 GO:0010467 68513 68523 Expression +T1431 UBERON:0001890 68543 68552 Forebrain +T1432 GO:0007613 68575 68581 Memory +T1433 PR:000008234 68593 68599 GluR-B +T1434 NCBITaxon:10088 68603 68607 Mice +T1435 GO:0007608 68613 68622 Olfactory +T1436 GO:0007613 68623 68629 memory +T1437 PR:000008234 68677 68683 GluR-B +T1438 NCBITaxon:10088 68702 68706 mice +T1439 PR:000008234 68730 68736 GluR-B +T1440 PR:000008234 68761 68767 GluR-B +T1441 PR:000008234 68791 68797 GluR-B +T1442 PR:000008234 68917 68923 GluR-B +T1443 PR:000008234 68965 68971 GluR-B +T1444 GO:0007613 69029 69035 memory +T1445 GO:0007613 69049 69055 Memory +T1446 PR:000008234 69218 69224 GluR-B +T1447 GO:0010467 69225 69235 expression +T1448 GO:0007613 69276 69282 memory +T1449 PR:000008234 69287 69293 GluR-B +T1450 UBERON:0004725 69304 69319 piriform cortex +T1451 GO:0007613 69344 69350 memory +T1452 GO:0007613 69486 69492 memory +T1453 PR:000008234 69508 69514 GluR-B +T1454 NCBITaxon:10088 69521 69525 mice +T1455 CHEBI:60004 69625 69632 mixture +T1456 PR:000008234 69658 69663 GluRB +T1457 PR:000008234 69679 69685 GluR-B +T1458 NCBITaxon:33208 69720 69727 animals +T1459 NCBITaxon:10088 69876 69881 Mouse +T1460 PR:000008234 69933 69939 GluR-B +T1461 PR:000008234 69944 69950 GluR-B +T1462 PR:000008234 69979 69985 GluR-B +T1463 PR:000008234 70038 70044 GluR-B +T1464 GO:0007608 70059 70068 Olfactory +T1465 PR:000008234 70137 70143 GluR-B +T1466 GO:0007608 70233 70242 olfactory +T1467 GO:0007608 70501 70510 olfactory +T1468 GO:0007613 70511 70517 memory +T1469 GO:0065007 70531 70541 controlled +T1470 CHEBI:34018 70552 70556 AMPA diff --git a/src/ontogpt/evaluation/craft/database/all/16216087.txt b/src/ontogpt/evaluation/craft/database/all/16216087.txt new file mode 100644 index 000000000..79ee23642 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16216087.txt @@ -0,0 +1,335 @@ +Enhanced Odor Discrimination and Impaired Olfactory Memory by Spatially Controlled Switch of AMPA Receptors + +Abstract + +Genetic perturbations of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) are widely used to dissect molecular mechanisms of sensory coding, learning, and memory. In this study, we investigated the role of Ca2+-permeable AMPARs in olfactory behavior. AMPAR modification was obtained by depletion of the GluR-B subunit or expression of unedited GluR-B(Q), both leading to increased Ca2+ permeability of AMPARs. Mice with this functional AMPAR switch, specifically in forebrain, showed enhanced olfactory discrimination and more rapid learning in a go/no-go operant conditioning task. Olfactory memory, however, was dramatically impaired. GluR-B depletion in forebrain was ectopically variable (“mosaic”) among individuals and strongly correlated with decreased olfactory memory in hippocampus and cortex. Accordingly, memory was rescued by transgenic GluR-B expression restricted to piriform cortex and hippocampus, while enhanced odor discrimination was independent of both GluR-B variability and transgenic GluR-B expression. Thus, correlated differences in behavior and levels of GluR-B expression allowed a mechanistic and spatial dissection of olfactory learning, discrimination, and memory capabilities. + +Introduction + +The sense of smell is of paramount importance for rodents [1], for which rapid odor discrimination and long-lasting olfactory memory permits responses to predator and prey critical for survival. Consequently, the behavioral analyses of olfactory capabilities in rodents are efficient, quantitative, and reproducible [2–4]. While in the formation and storage of olfactory memory piriform cortex [5–7], hippocampus [8,9], and olfactory bulb [10–12] are all implicated, the cellular correlates for these processes have not been clearly delineated. The contribution of the hippocampus to olfactory memory is presently controversial [2,13–18], but is deemed unlikely for simple olfactory discrimination tasks [9,19]. In fact, the most likely candidates for a cellular correlate of olfactory memory appear to be the neuronal connections in the piriform cortex due to the associational connectivity [5] and the expression of several forms of cellular and synaptic plasticity [7,20–23]. + +Concerning odor discrimination itself, cellular mechanisms for this process are often attributed to the inhibitory circuitry of the olfactory bulb ([24–30]; reviewed in [31–33]). Lateral inhibitory circuits were postulated, in analogy to retina [34,35], to mediate contrast enhancement [24], for which physiological recordings [24,36,37] and modeling data, based on the well-known anatomy [29], provide additional support. Such contrast enhancement may rest in large part on the particular properties of dendrodendritic synapses between the principal output neurons (mitral cells) and local inhibitory neurons (granule cells) of the olfactory bulb. In these distinct synapses, lateral and recurrent inhibition mediated by the gamma-aminobutyric acid-A system may be controlled by the activity of the closely appositioned glutamatergic part, perhaps triggering increased gamma-aminobutyric acid release by Ca2+ influx through glutamate-gated receptor channels ([38]; see also [39]). + +Given that neuronal circuits underlying odor discrimination, as well as olfactory memory, rely on properties of fast excitatory neurotransmission mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs), we sought to alter, by genetic means, the specific functional contribution of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) channels containing the dominant subunit GluR-B. Of the four AMPAR constituents, GluR-A to D (GluR1 to 4), which form tetrameric channels with different binary subunit combinations, GluR-B is contained in the majority of AMPARs. GluR-B is critically involved in the formation and trafficking of AMPARs, and dominates their ion conductance and gating properties [40–46]. Notably, the normally low Ca2+ permeability of AMPA channels in principal neurons is solely mediated by GluR-B, due to a unique arginine residue (R587) in the functionally critical glutamine/arginine (Q/R) site of the pore-forming segment M2 [44,47,48], resulting from RNA editing of GluR-B pre-mRNA ([49]; reviewed in [50]). Hence, either GluR-B deficiency, or the expression of Q/R site-unedited GluR-B with a glutamine residue at the critical channel site, leads to increased Ca2+ permeability of AMPA channels, as amply demonstrated in gene-targeted mice [51,53,58,60]. Thus, the absence of GluR-B, or the expression of GluR-B(Q) in the olfactory bulb, may generate increased inhibition in mitral cells. Moreover, the ablation of GluR-B, but also changes in the extent of Q/R site editing of GluR-B, can alter the strength of excitatory synaptic transmission in the genetically addressed neuronal populations [51,58,60], thus potentially shifting the balance of excitatory and inhibitory transmission in the affected circuits. Similarly, changes in synaptic plasticity due to Ca2+-permeable AMPARs [51,52,60], e.g., in piriform cortex, might alter odor memorization processes. Thus, alterations of AMPAR properties in these brain regions will allow investigation and possibly separation of mechanisms underlying these behavioral traits. + +Concerning this intended switch in AMPAR properties, mice lacking all GluR-B, however, show a widespread impairment in behavior, including lethargy, motor coordination problems, and deficits in exploratory activity [51], which preclude detailed behavioral analyses. Similarly, mice expressing (in the entire brain) a substantial part of the GluR-B population in the Q/R site-unedited form become seizure-prone and die prematurely [53]. Some of these problems can be partially overcome by use of spatially and temporally restricted expression systems [54–56], in particular the Cre-lox system, with Cre-recombinase expression in defined brain areas of gene-targeted mice carrying GluR-B alleles marked by loxP sites for Cre-mediated recombination [55,57]. Indeed, restricting the expression of Q/R site-unedited GluR-B to forebrain resulted in almost normal lifespan and an only weakly seizure-prone phenotype [58]. Mice with forebrain-specific GluR-B depletion appeared almost completely normal throughout life with no developmental abnormalities, thus permitting a detailed, quantitative investigation of olfactory behavior. + +To allow for the mechanistic separation of olfactory learning, discrimination, and memory, we exploited a well-known phenomenon of transgenes, which concerns heterogeneous expression among different founder lines and even among genetically identical individuals of a given line. Although such “mosaic” expression is usually undesired, here we took advantage of it by ablating GluR-B via gene-targeted, floxed GluR-B alleles with the help of a transgenic mouse line with variegated Cre expression in forebrain. By correlating GluR-B levels in olfaction-related brain regions with quantitative behavioral data, we investigated the dependence on GluR-B of olfactory discrimination and memory. Moreover, to delineate the brain areas involved in these distinctive olfactory processes we used transgenic “rescue” of GluR-B ablation, specifically in piriform cortex and hippocampus. + +These efforts allowed us to dissect, both spatially and mechanistically, the role of GluR-B-mediated AMPAR properties in selected brain regions in odor learning, discrimination, and memory. + +Results + +GluR-B(Q) Expression in the Forebrain Increases Olfactory Learning and Discrimination + +To explore the role of fast excitatory neurotransmission carried by GluR-B-containing AMPA channels in olfactory processes, we first analyzed mice that express part of the GluR-B population in a Q/R site-unedited form (Figure 1A; termed “GluR-BΔECS:FB”; see also [58]). These mice carry, in addition to a wild-type GluR-B allele, a gene-targeted GluR-Bneo allele in which the intronic sequence critical for GluR-B pre-mRNA editing at the Q/R site is replaced by a floxed TK-neo gene, which severely reduces splicing of the modified intron and hence attenuates the expression of the GluR-Bneo allele [60]. To unsilence the attenuated GluR-Bneo allele, specifically in the postnatal forebrain, we crossed in the TgCre4 transgene, which encodes Cre-recombinase and is driven by the αCaMKII promoter ([59]; “Camkcre4”). Thus, Cre-recombinase removes in forebrain neurons the intronic TK-neo gene, leading to the active GluR-BΔECS allele for Q/R site-unedited GluR-B(Q) subunits (Figures 1 and S1). As expected [53,60], also in mice expressing postnatally forebrain-specific Q/R site-unedited GluR-B(Q) subunits, Ca2+ permeability through AMPAR was increased and AMPAR currents showed rectification ([58] and unpublished data). GluR-BΔECS:FB mice had, in contrast to mice expressing GluR-B(Q) globally, a prolonged lifespan and no severe developmental alterations. They were however, still seizure-prone [58], and hence behavioral training was restricted to short periods of time. + +We trained six GluR-BΔECS:FB mice and six littermate controls on one odor pair in an automated go/no-go olfactory conditioning task [3,61]. In this task, water-deprived mice are trained to distinguish a water-rewarded odor (S+) and an unrewarded odor (S−) by their licking response (Figure 1B). Both GluR-BΔECS:FB and control mice acquired the “simple” task to discriminate between the monomolecular odors amylacetate and ethylbutyrate (percentage correct > 70% after 400 trials). Strikingly, GluR-BΔECS:FB mice showed more rapid learning and enhanced discrimination capabilities (Figure 1C, group effect: F(1,10) = 10.2, p < 0.01). This was confirmed by fitting linear trend lines to the initial part of the learning curve (slope difference: p < 0.05; see Materials and Methods). The training system employed allows for careful monitoring of head positions [3]. For rewarded trials (Figure 1D, green), already weakly trained animals kept their head in the sampling port (large tube in Figure 1B) during the entire 2-s trial, whereas for unrewarded trials the head is retracted quickly (Figure 1D, red). The difference of these two curves (Figure 1E, black) is a very sensitive assay for discrimination performance; the fitted maximum of this curve (Figure 1E, blue) is referred to as the “discrimination index” in the remainder of the paper and is again strongly improved for GluR-BΔECS:FB mice, compared with controls (Figure 1F; group effect: F(1,10) = 11.7, p < 0.01). This was not due to general motor performance, attention, or motivation changes, as both the intertrial interval (group effect: F(1,10) = 0.56, p > 0.4) and the overall licking frequency (group effect: F(1,10) = 0.72, p > 0.4) were unaffected. + +Thus, expression of Ca2+-permeable AMPARs in forebrain areas, including the olfactory bulb, resulted in more rapid odor learning and enhanced olfactory discrimination. + +GluR-BΔFB Mice Exhibit Increased Olfactory Discrimination Performance + +To ascertain if enhanced olfactory learning and odor discrimination may indeed correlate with the increased Ca2+ permeability of AMPA channels in the Q/R site-unedited form, we next analyzed GluR-BΔFB mice, which lack GluR-B in forebrain. This specific ablation was generated by forebrain-selective TgCre4 expression ([59] “Camkcre4”) in gene-targeted GluR-B2lox mice carrying, in both GluR-B alleles, a floxed exon 11 (Figure 2A). The specific GluR-B depletion in GluR-BΔFB mice can be monitored by immunohistochemistry (see below) and immunoblotting. In quantitative immunoblot analyses, we found GluR-B levels reduced to 28 ± 7%, 29 ± 8%, and 52 ± 9% (± SEM; n = 10) in the hippocampus, cortical areas, and olfactory bulb, respectively, relative to GluR-B levels in GluR-B2lox littermate controls. In the absence of GluR-B, the electrophysiological properties of AMPA channels become similar to those with GluR-B/GluR-B(Q) switch [51] showing strong rectification and increased Ca2+ permeability through AMPA channels (unpublished data). However, GluR-B depletion is not lethal and does not produce seizures. In addition, in contrast to the complete GluR-B knockouts, mice with forebrain-specific GluR-B depletion appeared normal throughout life with no developmental abnormalities, or difference in body size and weight in adulthood (wild-type: 31.0 g ± 1.2; GluR-BΔFB: 28.4 ± 0.9; each n = 10). Exploratory activity in an open field task was slightly increased in GluR-BΔFB mice (3,480 cm ± 180, n = 11), compared with wild-type littermates (2,512 cm ± 96, n = 12, p <0.01). Motor coordination measured in an accelerating rotarod was somewhat impaired in the mutant mice (wild-type: 156 s ± 37, n = 6; GluR-BΔFB: 37 s ± 10, n = 6; p < 0.05). Tests in the dark/light box revealed increased anxiety of GluR-BΔFB mice (latency of first exit: wild-type [17 s ± 3], GluR-BΔFB [97 s ± 42], p = 0.047; compartment changes: wild-type [17 s ± 3], GluR-BΔFB [7 ± 2], p < 0.05; time spent in lit compartment: wild-type [103 s ± 10], GluR-BΔFB [59 s ± 19], p = 0.051; each n = 6). Hence, unlike the complete GluR-B knockouts, GluR-BΔFB mice show only very minor changes in general activity and no sign of any major developmental disturbance, thus allowing detailed, quantitative behavioral investigations. + +If the GluR-B/GluR-B(Q) switch-induced alterations in the Ca2+ permeability of the AMPA channels are linked to enhanced odor discrimination and learning, the depletion of the GluR-B subunit should lead to a similar phenotypic readout. We therefore trained nine GluR-BΔFB mice and nine littermate controls in the same automated associative go/no-go olfactory conditioning task described above. To cover even small phenotypic changes in olfaction, we tested different odor pairs, “simple” monomolecular odors and “difficult” binary mixtures [3]. After habituation, GluR-BΔFB and control mice were trained to discriminate between the “simple” monomolecular odors amylacetate and ethylbutyrate, and subsequently additionally on a “difficult” discrimination task consisting of similar binary mixtures of cineol and eugenol; and finally again on a “simple” discrimination task with the monomolecular odors pelargonic and valeric acid. Similar to GluR-BΔECS:FB, GluR-BΔFB mice also showed enhanced learning and discrimination compared with controls (Figure 2B; group effect: F(1,16) = 6.55; p < 0.05). Increased discrimination performance is expected to show more pronounced effects for closely related odors because of the more challenging “difficult” discrimination task that is closer to the psychophysical limits of the system [3,4]. Consistent with this notion, differences between GluR-BΔFB and control mice were not only larger if the detailed sampling pattern and discrimination index were investigated (Figure 2C and 2D, group effect: F(1,16) = 29.5; p < 10−4), but in particular for the discrimination of binary mixtures with similar composition (group effect: F(1,16) = 27.8; p < 10−4 for the mixture; FAA EB(1,16) = 7.0; p = 0.02, and FPel Val(1,16) = 5.8; p = 0.03 for the “simple” discrimination tasks; 3-way ANOVA: F(6,96) = 2.9; p = 0.01). As activity, measured by the intertrial interval, was not significantly different between genotypes (F(1,16) = 3.1; p = 0.1), and analysis of the lick frequency showed a tendency to reduced motivation of GluR-BΔFB mice in this particular task (F(1,16) = 9.2; p < 0.01), we conclude that depletion of GluR-B in forebrain areas indeed resulted in increased olfactory learning and discrimination performances, rather than motivational alterations. To assess olfaction specificity, we trained ten GluR-BΔFB mice and ten controls in a nonolfactory hippocampus-dependent spatial learning task (elevated Y-maze). Performance in this task was not improved compared with controls (Figure S2); on the contrary, the acquisition of this task was slightly impaired, allowing the conclusion that the enhanced olfactory discrimination performance is likely to be specific to the sense of smell and possibly related to enhanced discrimination capability, rather than a general increase in alertness. Furthermore, enhancement in odor discrimination in both GluR-BΔECS:FB and GluR-BΔFB mice makes it likely that improvement in this task results from the common AMPAR property change mediated by the depletion of GluR-B and the lack of the Q/R site-edited GluR-B subunit, both resulting in Ca2+-permeable AMPARs. Thus, this suggests that increased Ca2+ influx via AMPARs leads to enhanced olfactory learning and discrimination. + +Olfactory Memory Is Significantly Decreased but Highly Variable in GluR-BΔFB Mice + +To capture the full extent of the role of Ca2+-permeable AMPARs in olfactory behavior, we next assessed the effects of altered AMPA channels on olfactory memory. To probe olfactory memory in GluR-BΔFB mice, six days after the end of the first training phase for odor discrimination (amylacetate versus ethylbutyrate), the training trials for the third odor pair (pelargonic versus valeric acid) were interleaved with unrewarded trials in which amylacetate or ethylbutyrate were again presented (black bar in Figure 2C). Whereas control mice reliably responded only to the previously rewarded odor (memory of 86 ± 8%, mean ± SD, n = 9, Figure 2E), GluR-BΔFB mice showed reduced olfactory memory (69 ± 16%, n = 9, p < 0.05, Mann-Whitney). Due to the more rapid learning observed in GluR-BΔFB, one could speculate that a decrease in olfactory memory might simply reflect increased extinction. However, extinction levels were low in general, no significant group-trial interaction could be found for the memory trials (2-way ANOVA, F(6,90) = 1.5, p > 0.1), and a restriction of the analysis to early memory trials displayed essentially the same pattern (Figure S3A). Thus, reduced performance in the probe trials is not due to increased extinction but reflects genuine memory impairment. Moreover, because the hippocampus-dependent spatial memory after-task acquisition in the Y-maze was not affected (Figure S2), we conclude that the observed olfactory memory deficit is rather specific for olfaction and does not readily generalize to other modalities. + +While the improved odor discrimination and learning behavior showed only little variability, the significantly impaired memory performance observed in GluR-BΔFB mice was highly variable among individual animals compared with control littermates (Figure 2E). This variability in olfactory memory was reflected in the level and extent of Cre-recombinase expression in forebrain of transgenic TgCre4 mice, as visualized by Cre-activity in the Cre-indicator mouse line R26R. We observed that onset and extent of Cre-recombinase expression in different forebrain regions varied among individual TgCre4 mice (Figure 3A), which could also be directly visualized by immunohistochemistry with a Cre-antibody (unpublished data). As this variability persisted after several backcrosses, and Southern blot analysis revealed no differences of transgene integration or number among animals (Figure 3B), it could not be attributed to genetic differences but rather to epigenetic mechanisms. + +Hence, we hypothesized that the variability in olfactory memory reflected the mosaicism observed in the transgenic TgCre4 line. The evaluation of regional differences in expression pattern of animals with robust and poor olfactory memory could then be applied to identify brain areas responsible for the observed phenotypes. + +Olfactory Memory Correlates with Residual GluR-B Protein Expression in GluR-BΔFB Mice + +Thus, to examine whether the pronounced variability of olfactory memory in GluR-BΔFB mice (Figure 2E) reflects variability of GluR-B levels in GluR-BΔFB mice, we analyzed the residual amount of GluR-B protein in mice with disparate memory performances (Figure 4A–C). Notably, mice with pronounced memory deficits (memory < 70%) showed essentially no detectable GluR-B protein in hippocampus, amygdala, olfactory bulb, and piriform cortex (n = 2, Figure 4B, and unpublished data), but mice with almost complete memory displayed substantial residual GluR-B levels in all brain areas investigated (n = 2, Figure 4B, and unpublished data). + +To quantify the relation between residual GluR-B protein and olfactory memory, the memory experiment was repeated with nine additional GluR-BΔFB mice and two GluR-B2lox control animals (indicated with shaded symbols in Figure 4A), resulting in the same mean, variability, and range of memory performance (control: 89 ± 10%; GluR-BΔFB: 63 ± 14%). Subsequently, protein was extracted from olfactory bulbs, cortical areas, and hippocampi from each mouse, and GluR-B protein was quantified (Figure 4C). The summarized correlations are depicted in Figure 4D (two animals were used for immunofluorescent analysis that yielded the same results as in the first experiment). Whereas no learning or discrimination-related parameter correlated with residual protein levels (Figure 4D, R2 < 0.3), a strong correlation between memory and GluR-B protein was observed in hippocampus (Figure 4D, R2 = 0.72, p < 0.003, n = 10) and cortical areas (Figure 4D, R2 = 0.62, p < 0.006, n = 10). Only a weakly significant correlation was found in the olfactory bulb (Figure 4D, R2 = 0.48, p = 0.03, n = 10). GluR-A levels were unchanged from wild-type, indicating that compensatory up-regulation of other AMPAR subunits is unlikely (GluR-A levels relative to control: 1.02 ± 0.05, mean ± SEM, n = 10). + +In summary, mice with reduced GluR-B levels in forebrain areas showed decreased olfactory memory, which correlated tightly with a reduction in GluR-B levels in the hippocampus and cortical areas. Enhanced odor learning and discrimination, on the other hand, was independent of residual GluR-B levels in the olfactory bulb and other forebrain areas, indicating that moderate GluR-B reductions are sufficient to saturate enhanced odor learning and discrimination. Thus, although both are mediated by alterations in the AMPAR subunit GluR-B, due to the qualitatively different dose-response curves, the phenotypes regarding olfactory memory, and olfactory learning/discrimination must be mechanistically distinct. + +Partial Rescue of Olfactory Memory Deficit by Selective Transgenic GluR-B Expression in Hippocampus and Piriform Cortex in GluR-BΔFB Mice + +The effect of selective GluR-B depletion in mice indicated that GluR-B-containing AMPARs in the hippocampus, and/or (olfactory) cortex are likely to be important for olfactory memory. The olfactory memory phenotype could be due to depletion of GluR-B in olfactory cortex or hippocampus; enhanced learning and discrimination capabilities might rather be evoked by AMPARs lacking GluR-B in the olfactory bulb. + +To obtain independent evidence for this spatial and mechanistic dissection of the roles of Ca2+-permeable AMPARs, we expressed by transgenic means N-terminally green fluorescent protein (GFP)-tagged GluR-B specifically in hippocampus and piriform cortex of GluR-BΔFB mice (Figure 5A and 5B). In accordance with the proposed region-dependence, we expected that additional GluR-B subunits in hippocampus and/or piriform cortex improve odor memory but do not alter odor discrimination performance. The mouse line employed for this purpose, termed GluR-BRescue, had the genetic background of GluR-BΔFB mice but additionally carried a bidirectional module for β-galactosidase and GFPGluR-B expression, responsive to the tetracycline-controlled transcriptional transactivator [62]. The transactivator was under the control of a modified αCaMKII-promoter fragment to obtain high expression selectivity (Figure 5A; see also Materials and Methods). The transgenic expression level of GFPGluR-B was 9.72% ± 1.25 (n = 3) in the hippocampus, compared with endogenous GluR-B (Figure 5C and 5D). Analysis of β-galactosidase activity and GFPGluR-B expression in brain sections of GluR-BRescue mice revealed expression in hippocampus and piriform cortex, whereas cortex, amygdala, and striatum only rarely showed any positive cells (Figure 5B). Importantly, both the spatial pattern and intensity of GFPGluR-B expression were constant among all GluR-BRescue mice analyzed (n = 11). + +Olfactory memory experiments with GluR-BRescue , and both GluR-BΔFB and GluR-B2lox mice as controls, were performed as described above (indicated with shaded symbols in Figure 6A). Memory was again highly reproducible in both GluR-BΔFB (66 ± 12%; n = 4) and GluR-B2lox (94 ± 2%; n = 3) mice, compared with experiments performed earlier (Figures 2E and 4A). Importantly, olfactory memory in GluR-BRescue mice was intermediate (75 ± 15%, n = 8), below GluR-B2lox control levels, but better than in GluR-BΔFB mice. Assessing memory under extinction-free condition, where each trial was rewarded, confirmed again that the memory deficit was a true memory deficit and not due to increased extinction (Figure S3B). Data from the experiments described in Figures 2 and 4 were combined to allow statistical comparison (Figure 6A and 6B). In summary, GluR-BRescue mice showed both enhanced memory performances compared with GluR-BΔFB (overall ANOVA: F(2,41) = 13.6, p < 10−4; memoryRescue = 75 ± 15%, n = 8; memoryΔFB = 66 ± 14%, n = 22; p < 0.05; Figure 6A), but were still impaired relative to GluR-B2lox controls (memory2lox = 88 ± 8%, n = 14; p < 0.005), consistent with a partial rescue of the memory deficit by circumscribed transgenic GFPGluR-B expression in hippocampus and piriform cortex. Notably, the partial memory is in numerical agreement with the predictions from the protein correlation and the measurement of transgenic protein expression (see predicted memory, blue line, in Figure 6B). In the olfactory memory experiments with GluR-BΔFB animals, olfactory memory linearly depended on GluR-B expression in cortex and hippocampus with a slope of 9.1 ± 2.5% (cortex) and 8.9 ± 2.0% (hippocampus) increase in memory per 10% increase in protein (Figure 4D). A 9.7% increase in GluR-B in these brain regions, as achieved by GluR-BRescue animals (Figure 5), is thus predicted to increase olfactory memory by approximately 9% throughout the heterogeneous population (blue line in Figure 6B). This confirms the role of these brain areas as inferred from the mosaic expression and protein correlation analysis described above (Figure 4). However, odor discrimination (measured by the discrimination index as in Figures 1, 2, and 4D) was as enhanced as in GluR-BΔFB mice (0.79 ± 0.05, mean ± SEM compared with 0.76 ± 0.02, p > 0.7, Figure 6C), and improved relative to GluR-B2lox controls (0.48 ± 0.06; Figure 6C; overall ANOVA: F(2,41) = 17.2, p < 10−5; post hoc Newman Keuls: p < 10−3), as expected if the enhanced discrimination phenotype is due to Ca2+-permeable AMPARs in the olfactory bulb and unaffected by GluR-B expression in piriform cortex or hippocampus. + +Hence, transgenic GluR-B expression, specifically in the piriform cortex and hippocampus in the GluR-B knockout background, rescues the odor memory deficit but leaves enhanced olfactory discrimination and learning unaltered. + +Discussion + +Here we present mechanistic and spatial dissections of olfactory discrimination, learning, and memory. We employed gene-targeted and transgenic mice with region-specific expression to demonstrate that a change in GluR-B-mediated properties of AMPA channels in αCaMKII-expressing neurons of mouse forebrain, including olfactory bulb mitral and granule cells, enhances olfactory discrimination and learning but impairs olfactory memory. These pertinent olfactory behaviors were assessed in a go/no-go operant conditioning task, which provides a quantitative, robust, and reproducible behavioral tool [3]. We observed among individual mice a striking variability in olfactory memory performance but not in odor discrimination. This variability could be traced to epigenetic variability in the transgenic expression of Cre-recombinase, which mediated recombination within loxP-flanked segments of gene-targeted alleles for the dominant AMPAR subunit GluR-B, and hence operated the switch in AMPAR properties toward GluR-B ablation and increased Ca2+ permeability. In contrast to variable memory, olfactory discrimination and learning performances appeared already saturated by even moderate extents of Cre expression, and hence moderate changes in AMPAR properties. The subsequent transgene-driven re-introduction of GluR-B, specifically in piriform cortex and hippocampus, reversed the Cre-induced loss of GluR-B and partially rescued the odor memory deficit, but left unaltered the enhanced olfactory discrimination. In a nutshell, we conclude that olfactory discrimination is enhanced by an increase in AMPAR-mediated Ca2+ permeability within the olfactory bulb, whereas olfactory memory becomes impaired upon genetically induced GluR-B ablation in higher brain centers, specifically in piriform cortex. + +Olfactory Discrimination Is Increased in Mice with Forebrain-Specific GluR-B Ablation or GluR-B(Q) Expression + +Both forebrain-specific GluR-B(Q) expression and GluR-B depletion led to increased olfactory learning and discrimination capabilities. This was rather pronounced for GluR-B(Q)-expressing mice, consistent with the overall stronger phenotypic consequences in comparison to GluR-B depletion, both when forebrain-selective ([58], and this study) or global [53,60]. + +A detailed analysis of the sampling pattern [3], and in particular the analysis of discrimination tasks that involved “simple” dissimilar monomolecular odor pairs and “difficult” binary mixtures, were necessary to fully capture the characteristics of the olfactory discrimination phenotype for forebrain-specific GluR-B-depleted mice. For closely related binary mixtures, discrimination improvements were largest, consistent with a specific alteration in odor discrimination, rather than a general enhancement of learning capabilities. This is further supported by the notion that no general improvement was observed in other nonolfactory behavioral tasks, e.g., hippocampus-dependent spatial learning tasks such as matching-to-place spatial reference memory tasks (Figure S2). As no Cre expression and activity was observed in the main olfactory sensory neurons at any developmental stage (Figure S4 and unpublished data), olfactory epithelial function was unaltered by the genetic modification. The vomeronasal organ showed very weak Cre expression (unpublished data), but a role of this structure concerning performance in the olfactory discrimination task is unlikely (for review, see [63]). The behavioral phenotype is thus likely to be associated with the processing of olfactory information rather than the detection of odors. Furthermore, increased performance even after long stretches of training and, in particular, increased sampling pattern differences (TB and ATS, unpublished data) supported the notion that GluR-B depletion resulted in enhanced odor discrimination capability. The learning phenotype might thus be a result of this enhanced discrimination capability or reflect additional changes in circuits underlying task acquisition. + +Putative Cellular Basis of Enhanced Odor Discrimination and Learning + +AMPAR properties were altered specifically in neurons of forebrain areas, most notably olfactory bulb, olfactory cortex, and other cortical areas and hippocampus, leading to enhanced odor discrimination and learning. Interestingly, transgenic expression of GFPGluR-B in GluR-BΔFB mice (genetic “rescue”), specifically in piriform cortex and hippocampus with no detectable expression in the olfactory bulb, did not alter enhanced discrimination and learning capabilities. This is consistent with a primary role of the olfactory bulb in olfactory discrimination and learning. Alternatively, transgenic protein levels might have been too low to alter discrimination and learning capabilities, although memory was clearly affected. This notion will be further tested in mice with piriform cortex-specific GluR-B ablation. + +A direct contribution to the phenotype by Ca2+ influx through genetically modified AMPA channels seems to be likely, since mice expressing Q/R site-unedited GluR-B showed even better odor learning and discrimination performance compared with mice with depleted GluR-B. In both mouse models Ca2+ influx through AMPA channels is increased, whereas the effect on the macroscopic AMPA conductance differs. AMPAR currents are reduced in mice not expressing GluR-B [51], possibly due to fewer synaptic AMPA channels and impaired AMPAR trafficking and recycling. In mice expressing the unedited form of GluR-B(Q), macroscopic conductance in whole-cell patches of CA1 pyramidals is increased [60], but excitatory transmission in CA3-to-CA1 cell synapses is somewhat reduced, in spite of a lower threshold for generating a population spike [58], indicating increased synaptic excitability by sustained GluR-B(Q) expression, in line with the seizure-prone phenotype. Yet, both mouse models yield enhanced discrimination and learning capability. The milder phenotype upon GluR-B depletion could be attributed to reduced AMPAR densities at synapses [40,41] and therefore probably to a lesser extent of Ca2+ influx. This is further supported by kainate-induced Co2+ uptake in acute brain slices, revealing considerably higher Co2+ uptake via Ca2+-permeable AMPARs in hippocampal pyramidal neurons of mice expressing GluR-B(Q), than those lacking GluR-B (DRS, RS, and PHS, unpublished data). + +Normally, GluR-B-containing AMPARs are prominently localized at the dendrodendritic synapse between mitral and granule cells in the olfactory bulb [64,65]. As Ca2+ influx through glutamate receptors is thought to contribute to lateral and recurrent inhibition ([38] see also [39]), the absence of GluR-B, or the presence of GluR-B(Q) in the olfactory bulb, is likely to result in increased inhibition between the principal neurons in this structure. Notably, the improved olfactory discrimination capabilities were apparent even upon relatively small reductions in GluR-B levels. It will be interesting to see if GluR-B+/neo mice also exhibit enhanced odor discrimination, which is indeed likely given that low levels of GluR-B(Q), and hence a small increase in Ca2+-permeable AMPARs, arise from the attenuated GluR-Bneo allele [60], and, moreover, that GluR-B levels are decreased in these mice due to the single copy of the GluR-B+ allele. + +In virtually all models of the olfactory bulb, lateral (and, in fewer models, also recurrent) inhibition plays a dominant role, either in establishing spatiotemporal dynamics [32,66–69], or in directly enhancing contrast and therefore simplifying discrimination and learning of similar odorants [24,28,29,36]. Increased inhibition will thus in general improve discriminability, consistent with the behavioral improvements observed with GluR-B(Q) expression, the GluR-B knockout, and the “rescue” mice. A direct test of this link would require the quantitative measurement of inhibition in the intact preparation, a task that might be feasible with further improved in vivo electrophysiological techniques, such as targeted recordings [70,71] or simultaneous pre- and postsynaptic intracellular measurements. + +Olfactory Memory Is Reduced in GluR-B Knockout Mice and Improved by GluR-B Expression in Piriform Cortex and Hippocampus + +To assess olfactory memory of GluR-BΔFB mice, after six days the mice were probed with unrewarded odor presentations that interleaved a simple discrimination task. Prolonged behavioral tasks such as the assessment of long-term olfactory memory were not performed with the seizure-prone GluR-BΔECS:FB mice. Memory in GluR-BΔFB mice, however, was dramatically impaired. This cannot be attributed to a general, unspecific deficit because, simultaneous to the memory trials, the normal, rewarded discrimination task was performed even better than by controls. Reduced olfactory memory can also not be simply attributed to increased extinction, as no significant trial-group interactions were observed and also, when restricted to the first unrewarded memory trials, a significant impairment was observed (Figure S3A). Additionally, investigating relearning of the first discrimination task revealed a significant correlation with the memory performance, showing a “memory” deficit under extinction-free conditions (Figure S3B). Finally, in other memory-related tasks, such as a hippocampus-dependent spatial reference memory task (Figure S2), mice with GluR-B depletion were not impaired after task acquisition. We therefore conclude that forebrain-specific ablation of GluR-B results in a specific loss of long-term olfactory memory but, at the same time, in enhanced odor discrimination and learning capabilities. + +To dissect the discrimination, learning, and memory phenotypes, and ultimately identify potential cellular correlates, we made use of the variegated Cre expression of TgCre4 mice. Therefore, residual GluR-B levels were determined from each mouse that had been tested in the behavioral experiments, and levels were correlated with odor learning, discrimination, and olfactory memory. The significant direct correlation between residual GluR-B levels in hippocampus and cortical areas with olfactory memory suggested hippocampal and/or cortical neurons as putative mediators for odor memorization and/or storage. No measure of discrimination and learning, on the other hand, correlated with residual GluR-B levels; even the smallest reduction in GluR-B levels (about 20%–60%) was sufficient to establish and saturate enhanced odor discrimination capabilities. Together, this allows the conclusion that distinct mechanisms mediate discrimination/learning and memory. This is furthermore consistent with a prominent role of cortical areas or hippocampus in olfactory memory, although GluR-B levels in the olfactory bulb were also weakly correlated with memory performance. Transgenic expression of GFPGluR-B in piriform cortex and hippocampus indeed rescued memory to an extent strikingly consistent with the transgenic protein levels (approximately 10% of wild-type GluR-B), confirming the reliability of the correlation analysis and the sensitivity of the behavioral assay. + +In general, the hippocampus is thought to be involved in only those olfactory memory tasks that involve temporal sequence analysis or require other higher cognitive features [2,13,72–74]. In particular, extensive lesions to the hippocampal formation in rats do not interfere with long-term olfactory memory in go/no-go successive olfactory discrimination tasks [72], such as the one described. This leaves piriform cortex as the most prominent candidate for the locus of the olfactory memory deficit described, consistent with the prevalent view of piriform cortex as an associational memory structure [75] and learning-associated changes in piriform cortex [7,20–23]. + +Potential Mechanisms of Specific Olfactory Memory Impairment + +What could be the cellular basis of the long-term memory deficit brought about by lack of GluR-B-containing AMPARs? For complete GluR-B knockouts, increased long-term plasticity (LTP) was reported in hippocampal field recordings [51]. In addition, in hippocampal and amygdala pathways lacking GluR-B, an AMPAR-dependent, N-methyl-D-aspartate (NMDA) receptor-independent form of LTP can be readily induced [51,52]. Mechanistically, this is likely to be due to Ca2+ influx through GluR-B-less AMPA channels leading to non-hebbian forms of plasticity. However, hebbian dependence on simultaneous pre- and postsynaptic activity is often a critical feature for memory storage (reviewed in e.g. [76]); hence, a non-hebbian form of plasticity should result in impaired memorization. + +Surprisingly, in GluR-BΔFB mice no LTP changes could be observed in field measurements in the hippocampus, nor could NMDA-independent LTP be induced in hippocampal synapses between Schaffer collaterals and CA1 pyramidal cells in presence of the NMDA antagonist APV (K. Jensen, O. Hvalby, personal communication). However, in other brain areas that are potentially important for the processing of olfactory information, such as piriform cortex or olfactory bulb, LTP measurements have not yet been performed; synapses of these pathways may be regulated differently than hippocampal CA3/CA1 synapses. + +Additionally, by changing the Ca2+ permeability of AMPARs, Ca2+ signaling via AMPA and colocalized NMDA channels might be disturbed, thereby impairing memory formation. Other forms of plasticity [77,78] induced by Ca2+-permeable AMPARs might play a prevalent role in olfactory memory. Alternatively, long-term stabilization might involve GluR-B phosphorylation similar to cerebellar long-term depression [79] and thus be selectively impaired by GluR-B depletion. Physiological experiments to assess these hypotheses will ideally make use of even more restricted genetic modifications with completely undisturbed input structures. One possibility would be Cre-mediated GluR-B depletion and GluR-B(Q) expression in piriform cortex as suggested above. + +Correlating Quantitative Behavior and Mosaic Gene Expression for Dissecting Phenotypes + +A critical step in discerning the discrimination and learning phenotypes from the memory phenotype was the observation of increased variability in the memory of the GluR-B knockout animals and, subsequently, the individual analyses of protein expression levels. We made use of the epigenetic variability in GluR-B excision that could be correlated to the variability in memory but showed no correlation to variability in discrimination. + +The analysis of quantitative trait loci (for review, see [80]), or classically correlating different behavioral phenotypes within a truly wild-type population [81–84], also attempts to find common or distinct genetic origins of different behavioral traits. These attempts undoubtedly contribute significantly to unraveling the molecular basis of behavior. Due to subtle and multigenic differences between different strains of rodents or different individuals in a wild-type population, however, they suffer from a rather low “signal-to-noise” ratio. Differences in individual genes are rather small compared with the vast number of genes involved. As a result, purely correlating behavioral traits within a wild-type population usually remains rather descriptive, whereas quantitative trait loci analysis is capable of revealing multigenic basis of behavioral traits but usually lacks the power to identify the individual genes themselves. + +Herein, we described a way to enhance the signal-to-noise ratio by making use of the mosaic expression often associated with transgenic approaches: combining the advantages of “classical” genetic manipulation—namely, that the target of the manipulation is well defined—with the possibility to analyze variability, might also in future provide novel ways to define molecular and cellular correlates of complex behavioral traits. + +In this study, we combined genetically induced manipulation of the AMPAR composition with quantitative behavioral and molecular analyses. We could thus provide evidence for opposing roles of specific GluR-B manipulation and increased Ca2+ influx via AMPARs in olfactory discrimination/learning, and memory, potentially in the olfactory bulb and piriform cortex, respectively. To achieve this, we made use of the epigenetic variability in the extent of GluR-B ablation, which correlated with the variability in odor memory, but not in discrimination and learning; this finding was subsequently confirmed in GluR-B rescue mice with piriform cortex-specific transgenic expression of GluR-B. Extending these principles of combining quantitative behavioral analyses with minimal and dosed genetic interference, together with further physiological analyses, will ultimately pinpoint the neural circuitries underlying related, but distinct, behavioral traits in olfactory and other systems. + +Materials and Methods + +Mouse lines + +The R26R line [85] was employed as Cre indicator. GluR-BΔECS:FB mice were of mixed C57Bl/6 and NMRI genetic background and generated from TgCre4 (“Camkcre4” in [59]) and GluR-B+/neo [60] mice. GluR-B+/neo mice carry a wild-type GluR-B allele and a gene-targeted GluR-B allele in which the intron 11 sequence critical for Q/R site editing is replaced by a TK-neo gene flanked by loxP sites (“floxed”). GluR-B2lox mice [86] carry gene-targeted GluR-B alleles in which exon 11 is floxed. GluR-BΔFB mice were of C57Bl/6 genetic background and generated from TgCre4 and GluR-B2lox mice. TgCN12-itTA mice were generated as described for TgCN10-itTA mice (see Materials and Methods in [58]), and represent another founder line obtained from the same pronucleus injection, with more widespread forebrain expression. For exogeneous expression of GFPGluR-B, the mouse line TgOCN1 was generated: an AseI fragment of plasmid pnlacZ/GFPGluR-B was injected into the pronucleus of oocytes obtained from DBA1/C57Bl/6 F1 hybrids. Positive founders were backcrossed into C57Bl/6 for further analysis. Plasmid pnlacZ/GFPGluR-B was constructed from pnlacZ/GFPGluR-A [87] by replacing GluR-A cDNA with the rat cDNA for GluR-B. Transgenic GFPGluR-B protein levels were measured in hippocampus of TgOCN1 mice also carrying a transgene for forebrain-specific homogeneous tTA expression [88]. + +Experimental groups + +Mice heterozygous for TgCre4 and heterozygous for the TK-neo cassette in the GluR-B allele (GluR-BΔECS:FB) or homozygous for the floxed GluR-B (GluR-BΔFB) were used in the experiments. The “rescue” mice (GluR-BRescue) were positive for TgCre4, homozygous for the floxed GluR-B gene, and positive for the tet-sensitive responder transgene TgOCN1 and for the itTA expressing activator transgene TgCN12-itTA. + +Control groups + +Littermate controls were used in all experiments. GluR-B+/+ mice were used as controls in the task described in Figure 1. GluR-B+/2lox and GluR-B2lox/2lox (both negative for TgCre4) mice were used as controls in experiments described in Figures 2, 4, and 6. For the GluR-BRescue experiments (Figure 6), controls positive for either TgCN12-itTA or TgOCN1 in a GluR-B+/2lox and GluR-B2lox/2lox (both negative for TgCre4) background were used. + +Genotyping + +Mice were selected by PCR of mouse-tail DNA with specific primers as described below. Indicated are the sequences and the approximate length of the amplified DNA fragments. TgCre4: rspCre1 (5′-ACCAGGTTCGTTCACTCATGG-3′) and rspCre2 (5′-AGGCTAAGTGCCTTCTCTACAC-3′), 200 basepairs (bp). + +GluR-Bneo: MH60 (5′-CACTCACAGCAATGAAGCAGGAC-3′), MH53a (5′-GAATGTTGATCATGTGTTTCCCTG-3′), and MH117 (5′-GTTCGAATTCGCCAATGACAAGACG-3′), wild-type: 500 bp and mutant: 400 bp. + +GluR-B2lox: VM12 (5′-GCGTAAGCCTGTGAAATACCTG-3′) and VM10 (5′-GTTGTCTAACAAGTTGTTGACC-3′), wild-type: 250 bp and mutant: 350 bp. + +TgOCN1: VM4 (5′-CTCCCAGACAACCATTACCTGTCC-3′) and GluR-B882BST (5′-CGAAGTATACTTAATTGTCGCTGTGTG-3′), 600 bp. + +TgCN12-itTA: htTA1 (5′-AGAGCAAAGTCATCAACTCTG-3′) and htTA2 (5′-GTGAGAGCCAGACTCACATTTCA-3′), 1,000 bp. + +Southern blot analysis + +Genomic DNA from mouse-tail/liver was digested with restriction enzyme BglII (NEB), and the Southern blot was done with a 320-bp probe (“integ”) obtained by PCR detecting the αCaMKII promoter. + +Histochemistry + +Histochemistry was performed as described previously [89], with the following exceptions: Coronal 70- to 100-μm vibratome slices were used for immunohistochemistry with GluR-B (1:60, polyclonal; Chemicon, Temecula, California, United States), GFP (1:8,000, polyclonal; MobiTech, Göttingen, Germany), and Cre (1:8,000, polyclonal; BAbCO, Berkeley, California, United States) primary antibodies, and FITC-coupled (1:200; Dianova, Hamburg, Germany) and peroxidase-coupled (1:600; Vector, Burlingame, California, United States) secondary goat anti-rabbit antibodies. + +The main olfactory epithelium was obtained via cryostat sectioning, and immunohistochemistry was performed (primary antibody Cre, 1:5,000, polyclonal; BAbCO). X-gal staining was performed as described [89]. + +Immunoblot analysis + +Mouse brains were removed, and the hippocampus, olfactory bulb, and remaining forebrain areas were isolated. Total protein was prepared, and immunoblots were performed as described [87]. Antibodies used were against GluR-B (1:800, monoclonal; Chemicon), β-actin (1:40,000, monoclonal; Sigma, St. Louis, Missouri, United States) as an internal standard, and secondary goat anti-rabbit and goat anti-mouse antibodies (Vector, 1:15,000). Immunoreactivity was detected with ECLplus (Amersham, Little Chalfont, United Kingdom), and immunoblots were scanned and quantitatively analyzed with ImageJ. + +Behavioral analysis: Subjects + +All mice were four to six weeks old at the beginning of the experiments. Subjects were maintained on a 12-h light-dark cycle in isolated cages in a temperature and humidity-controlled animal facility. All behavioral training was conducted during daytime. During the training period, animals were kept on free food but on a water-restriction schedule designed to keep them at > 85% of their free food body weight. Continuous water restriction was never longer than 12 h. All animal care and procedures were in accordance with the animal ethics guidelines of the Max-Planck Society. + +Apparatus + +All olfactory discrimination experiments were performed using three modified eight- channel olfactometers ([61], Knosys, Bethesda, Maryland, United States of America) operated by custom-written software in Igor (Wave Metrics, Lake Oswego, Oregon, United States of America) on Pentium I, II, and III PCs running Microsoft Windows 98. Great care was taken to counterbalance groups between setups. In brief, animals were presented with odor from one out of eight possible odor channels and rewarded with a 2- to 4-μl drop of water in a combined odor/reward port (Figure 1B), ensuring tight association of the water-reward with a presented odorant. Head insertion into the port was monitored by an IR beam and photodiode (Figure 1B). Odors used were n-amyl acetate, ethyl butyrate, pelargonic acid, valeric acid, and binary mixtures of cineol and eugenol. If not otherwise noted, odors were diluted to 1% in mineral oil (Fluka Chemie, Steinheim, Germany) and further diluted by airflow to a final concentration of approximately 0.15%. All dilutions in the text refer to the dilution in mineral oil. All chemicals were obtained from Fluka Chemie. + +Task habituation training + +Beginning 1–3 d after the start of the water restriction schedule, animals were trained using standard operant-conditioning procedures [3]. In a first pretraining step, each lick at the water delivery tube was rewarded. After 20 licks, a second stage was entered in which head insertion initiated a 2-s “odor” presentation during which a lick was rewarded. The “odorant” used in the pretraining was the carrier medium mineral oil. All animals learned this task within one day (2–3 sessions 30 min each). + +Structure of an individual trial + +The mouse initiates each trial by breaking a light barrier at the opening of the sampling port (see also [3]). This opens one of eight odor valves, and a diversion valve that diverts all air flow away from the animal for typically 500 ms. After the release of the diversion valve, the odor is accessible to the animal for 2,000 ms. If it continuously licks at the lick port during this time (once in at least three out of four 500-ms bins), it can receive a 2- to 4-μl water reward after the end of the 2,000-ms period. If the animal does not continuously lick, or if the presented odor was a designated nonrewarded odor, neither a reward is given nor any sort of punishment, to minimize stress for the animal. Trials are counted as correct if the animal licks continuously upon presentation of a rewarded odor or does not lick continuously with a nonrewarded odor. A second trial cannot be initiated unless an intertrial interval of at least 5 s has passed. This interval is sufficiently long so that animals typically retract quickly after the end of the trial. Odors are presented in a pseudo-randomized scheme (no more than two successive presentations of the same odor, equal numbers within each 20-trial block). No intrinsic preference toward any of the odors was observed but controlled for by counterbalancing. + +A total of 100–300 trials were performed each day, separated into 30- to 40-min stretches to ensure maximal motivation despite the mild water restriction scheme. Additionally, motivation was controlled by monitoring intertrial intervals and the response frequency [3]. + +Measurement of performance + +The simplest measure of performance is the fraction of trials in which the animal responds correctly—that is, responds with licking to the presentation of the S+ odor and does not lick with presentation of the S− odor. + +It was shown previously, however, that the detailed sampling pattern is a more sensitive measure of discrimination performance [3]. To avoid long (> 3-week) training periods, we chose not to measure discrimination times [3] but to analyze the average sampling behavior in total. Upon presentation of a rewarded odor, the animal usually continuously breaks the beam, whereas upon presentation of an unrewarded odor the head is quickly retracted. The difference in response to the rewarded and unrewarded odor is approximately sigmoidal (Figures 1D, 1E, and 2D) and yields a sensitive measure of the discrimination performance. From this difference or from a sigmoidal fit to the difference (Figure 1E), several measures of discrimination can be determined: the average difference, peak, or maximum, time of half maximum, and slope of the fitted sigmoid. Whereas for small trial numbers (< 200) the slope often is not well constrained, any of the other parameters yielded essentially the same results. The discrimination index plotted in Figures 1F, 2C, 4D, and 6C refers to the fitted maximum, generally ranging from zero to one, one indicating the best discrimination. Identical results were obtained with other measures of discrimination, such as the average sampling difference. + +Structure of training + +After habituation, mice were trained to discriminate 1% amylacetate from 1% ethylbutyrate for 500 trials. During the last 100 trials, the S+ odor was rewarded in only 50% of the cases to increase the resistance to extinction of the acquired memory. These trials were excluded for the statistical analysis of the learning curves. Inclusion did not alter the result of the ANOVA; however, linear fitting of the learning curve was not appropriate anymore as partial saturation of learning performance had already occurred. Subsequently, animals were trained for 500 trials on the “difficult” discrimination task [3] between the binary mixtures 0.6% eugenol/0.4% cineol and 0.4% eugenol/0.6% cineol. To allow comparison, the last 100 trials were altered as for the “simple” discrimination task above. After two days of rest, animals were finally trained on the “simple” discrimination task between 1% pelargonic acid and 1% valeric acid for another 600 trials. + +In all experiments, counterbalancing between both odors and setups was ensured within and between genetic groups, or results were compared with counterbalanced subgroups, which in every case yielded identical results. During the entire course of the experiment, the person handling the animals and operating the olfactometers was blind to the genotype of the mice. + +Memory measurement + +To assess memory, after 280 trials of training to discriminate between pelargonic acid and valeric acid, memory trials were interleaved for 120 trials; that is, within each block of 20 trials two unrewarded amylacetate and two unrewarded ethylbutyrate trials were included. Memory scores are given as the fraction of those unrewarded trials that were responded to “correctly” (licking response to the odor that was rewarded in the initial training session [S+], no response to the odor that was not rewarded initially [S−]). Due to the epileptic phenotype and the slightly increased mortality [58], GluRBΔECS:FB mice were trained only for the initial period of 400 trials. + +Statistics + +Learning curves for both correct performance (“percentage correct”) and the discrimination performance were analyzed by repeated measure ANOVA. Additionally, learning curves were assessed by linearly fitting of trend lines to the data with fixed offsets, leaving the slope as the only variable. In general, binning was 100 trials per block. To allow for the investigation of group/block interactions, the repeated measure ANOVA binning was reduced to 20 trials per block. To compare memory performance in the GluR-BRescue (n = 8) and GluR-BΔFB (n = 22) mice, due to the high variability, a bootstrap approach was employed. Subpopulations of eight animals were selected from the population of 22 GluR-BΔFB mice, and mean memory was determined. In only 343 out of 20,000 subpopulations, mean memory exceeded the mean GluR-BRescue memory of 74.99%, resulting in a p value of p = 343/20,000 = 0.017. + +Supporting Information + +Figure S1 + +Forebrain-Specific Gene Manipulation + +(A) Schematic diagrams depicting Cre under the αCaMKII promoter control [59] and Cre-dependent expression of β-galactosidase (LacZ) by the R26R indicator mouse [85]. + +(B) Coronal forebrain sections (left) and sagittal brainstem/cerebellum sections of mice at P42 positive for TgCre4 and R26R Cre-indicator. Cre expression visualized by enzymatic β-galactosidase activity (blue, X-gal, counterstain by eosin) of R26R and by anti-Cre immunostainings (brown, DAB [diaminobenzadine]) was restricted to forebrain regions. Scale bars: 1 mm. A, amygdala; Ce, Cerebellum; Cx, cortex; H, hippocampus; Me, medulla oblongata. + +(C) Olfactory bulb sections of the same mouse to visualize Cre expression by Cre immunoreactivity (left, DAB) and by enzymatic β-galactosidase activity (right, X-gal, counterstain by eosin) in granule cells (GC) and mitral cells (MC) as indicated by arrows. Scale bars: 400 μm (upper panel), 50 μm (lower panel). + +(3.4 MB TIF). + +Click here for additional data file. + +Figure S2 + +Performance of GluR-BΔFB (n = 10) and Littermate Control (n = 10) Mice in a Spatial Reference Task on the Elevated Y-Maze + +Details of the methodology are described in [90]. * indicates p < 0.05. + +(126 KB TIF). + +Click here for additional data file. + +Figure S3 + +Memory Deficit Is Not Due to Increased Extinction + +(A) Memory performance as a function of time for the experiment described in Figure 2E (nine GluR-BΔFB and nine GluR-B2lox control animals). Only four unrewarded “memory +” trials are binned for each data point. Memory of GluR-BΔFB animals was significantly reduced (F(1,33) = 17; p < 0.001). Whereas a weak overall time effect could be observed (F(2,66) = 3.6; p = 0.03), there was no genotype-time interaction effect (F(2,66) = 0.67; p = 0.5), indicating that there is no differential effect of putative extinction on memory performance. * indicates p-values for a Mann-Whitney U test (* < 0.05, ** < 0.01). + +(B) After the memory experiments from Figure 6A, the last set of animals (eight GluR-BRescue, four GluR-BΔFB, and three controls) was further trained on AA versus EB for 900 trials one week after the memory experiment. Subsequently, training continued on AA/EB mixtures for 1,200 trials. Finally, the animals were retrained on AA and EB for 100 trials. Relearning performance during the last retraining task is highly correlated to the original memory performance (R2 = 0.46, p = 0.006, n = 15). + +(264 KB TIF). + +Click here for additional data file. + +Figure S4 + +Absence of Cre Expression in the Main Olfactory Epithelium of TgCre4 Mice + +(A) Overview of the main olfactory epithelium. (B) Higher magnification of (A) showing all cells with propidium iodide (red) and absence of Cre-positive cells (green, anti-Cre). (C) Positive control showing granule cells of the dentate gyrus stained for nuclear Cre (green) from the same mouse. + +(3.7 MB TIF). + +Click here for additional data file. + +Acknowledgements + +The authors want to thank Troy Margrie and Bert Sakmann for support, encouragement, and discussion; Onyeka Nobuka for help with the construct OCN; Mark Mayford for the αCaMKII promoter; Bettina Suchanek for the NR2C silencer; Annette Herold, Juliana Kling, and Christiane Zacher for experimental and technical supports; Nixon Abraham for assistance with some behavioral experiments; and David Bannerman, Thomas Kuner, and Pavel Osten for critically reading early versions of the manuscript. This work was supported by a Deutsche Forschungsgemeinschaft grant (SFB 636) to RS, the Heidelberger Akademie der Wissenschaften, the Boeringer Ingelheim Fonds, and the Leopoldina Akademie der Wissenschaften to ATS, the BMBF, and the MPG. + +Competing interests. The authors have declared that no competing interests exist. + +Abbreviations + +AMPA - α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate + +AMPAR - α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor + +bp - basepair + +GFP - green fluorescent protein + +LTP - long-term plasticity + +NMDA - N-methyl-D-aspartate + +Q - glutamine + +R - arginine + +Figures and Tables + +Figure 1 + +Odor Learning and Discrimination Is Enhanced in GluR-BΔECS:FB Mice + +(A) Schematic diagram depicting Cre-mediated activation of GluR-B(Q) by removing the loxP-flanked TK-neo (TK-neo pA, GluR-Bneo) element in intron 11, which is acting as a suppressor in expression from the Q/R site editing deficient GluR-Bneo allele. Exon 10 and 11 encode membrane-spanning segments 1, 2, and 3 (M1, 2, 3) of the GluR-B subunit. The intronic element necessary for editing the Q/R site is shown for the wild-type allele (+). + +(B) Scheme of an individual trial. Breaking a light barrier, the mouse initiates a trial. An odor is presented, and (depending on the odor denotation and the mouse's response) the mouse is rewarded or retracts its head. A small (2- to 4-μl) water reward is given at the end of an S+ odor if the mouse continuously licks at the delivery tube during the 2-s trial. A trial is counted as correct if the mouse licks continuously upon presentation of a rewarded (S+) odor or does not lick continuously with a nonrewarded (S−) odor [3]. + +(C) Twelve experimentally naïve animals (six GluR-BΔECS:FB [orange] and six GluR-B+/+ littermate controls [black]) were trained on 1% AA versus 1% EB for two tasks of 200 trials each. Both groups acquired the task (> 70% correct); however, the GluR-BΔECS:FB were both quicker, and performed better overall, than the littermate controls (group effect: F(1,10 ) = 10.2; p < 0.01). AA, amylacetate; EB, ethylbutyrate. + +(D) Average head position for one mouse and 50 presentations of the S+ (green) and 50 presentations of the S− (red) odor. “1” indicates the breaking of the light beam (head in the sampling port [3]). Note the rapid head retraction for the S− odor. + +(E) Difference of the average head positions from (D) for S+ and S− odors. Blue line indicates sigmoidal fit. “Discrimination index” refers to the maximum of the fitted sigmoid. + +(F) As (C) but depicting the discrimination index as a function of trial number (group effect: F(1,10) = 1.7; p < 0.01). + +Figure 2 + +Odor Learning and Discrimination Is Enhanced, but Odor Memory Is Reduced in GluR-BΔFB Mice + +(A) Schematic diagram depicting Cre-mediated ablation of loxP-flanked exon 11 of the GluR-B alleles. + +(B and C) Nine GluR-BΔFB (red) and nine GluRB2lox littermate controls (black) were trained for successive odor discrimination tasks on 1% AA versus 1% EB (400 trials), 0.4% Cin/0.6% Eu versus 0.6% Cin/0.4% Eu (400 trials) and 1% Pel versus 1% Val. GluR-BΔFB mice showed increased learning/discrimination compared with controls, both using the performance as measured by percentage of correct trials ([B]; group effect: F1,16 = 6.55, p < 0.05) or the discrimination index (C), that is the maximal difference of the sampling pattern (see Materials and Methods and Figure 1D–1F; F1,16 = 29.5, p < 10−4). + +(D) Sampling difference for the last 100 trials of the mixture discrimination task (indicated with a black arrow in [C]) for all 18 individual mice. Note that the GluR-BΔFB mice show a consistently larger sampling difference. + +(E) Olfactory memory performance for nine littermate controls (black) and nine GluR-BΔFB (red) mice. Olfactory memory was tested at the time indicated by the black bar in (C) by interleaving the Pel and Val trials with unrewarded AA and EB trials. AA, amylacetate; Cin, cineol; EB, ethylbutyrate; Eu, eugenol; Pel, pelargonic acid; Val, valeric acid. + +Figure 3 + +Variability of Cre Expression of Mouse Line TgCre4 + +(A) Variable Cre expression in forebrains of three different mice positive for TgCre4 and R26R Cre indicator (see Figure S1) at postnatal day 12 pictured by the Cre-dependent β-galactosidase activity (blue, X-gal, counterstain by eosin) in coronal brain slices. Scale bar: 1.25 mm. + +(B) Southern blot analysis of BglII-digested genomic mouse DNA of four TgCre4 mice that differed in the Cre expression pattern. Southern probe detects the wild-type (4.5 kbp) and the transgenic (7, 5, 3, and 2 kbp) alleles. + +Figure 4 + +Olfactory Memory but not Odor Learning/Discrimination Is Correlated with Residual GluR-B Levels in Hippocampus and Forebrain of GluR-BΔFB Mice + +(A) The olfactory memory performance for 18 GluR-BΔFB (red) and 11 littermate control (black) mice is given as mean (thick lines ± SEM) and as individual performance in open circles and triangles. Arrows with numbers (#) indicate those mice used in experiments (B–C). Data were combined from Figure 2 (open symbols) and an additional experiment with nine GluR-BΔFB and two littermate controls (shaded symbols). + +(B and C) Residual GluR-B levels as detected by anti-GluR-B immunofluorescence in hippocampus, amygdala, piriform cortex, and olfactory bulb of one control (#1) and two GluR-BΔFB (#2 and #3) coronal mouse brain sections (B) and by immunoblot analysis from hippocampal (Hip), cortical forebrain (FB), and olfactory bulb (OB) protein extracts of control (#4) and GluR-BΔFB mice (#5, #6, #7, and #8) probed with antibodies detecting GluR-B and β-actin as an internal loading control (C). Scale bars: 200 μm (first panel), 100 μm (other panels). + +(D) From ten GluR-BΔFB mice, the individual odor learning/discrimination and olfactory memory performance was determined together with the relative GluR-B levels in immunoblots of hippocampal, forebrain, and olfactory bulb protein extracts. Memory performance (top panels) and discrimination capability (bottom panels; discrimination index is measured for the last 100 trials of the mixture discrimination task as indicated by the arrow in Figure 2C) were plotted against GluR-B levels. Memory was tightly correlated to GluR-B protein level in hippocampus (R2 = 0.72; p < 0.003) and cortical forebrain (R2 = 0.62; p < 0.006) and only weakly in the olfactory bulb (R2 = 0.48; p = 0.03). No measure of learning/discrimination (discrimination index for last 100 mixture trials [D], slopes of trend lines, average discrimination index, average sampling pattern differences, correct performance, etc. [not shown]) displayed any correlation (R2 < 0.3). + +Figure 5 + +Specific Hippocampus and Piriform Cortex Expression of Transgenic GFPGluR-B + +(A) Schematic diagrams depicting forebrain-specific GluR-B deletion as in Figure 2A and itTA-dependent expression of GFPGluR-B and nuclear-localized β-galactosidase (nLacZ) in GluR-BΔFB mice (termed GluR-BRescue). GFPGluR-B and nLacZ are both encoded by TgOCN1, and itTA is controlled by a fusion of the NR2C silencer element [91] and the αCaMKII promoter (termed TgCN12-itTA [92]). + +(B) In coronal brain sections of mice positive for both transgenes (TgCN12-itTA and TgOCN1) β-galactosidase activity (blue, X-gal, counterstain by eosin) is restricted to hippocampal neurons in CA1, DG, and neurons in the piriform cortex. The same neurons show GFPGluR-B expression when analyzed in immunohistochemical sections with an antibody against GFP. Scale bars: 500 μm. + +(C) Immunoblot detecting endogenous GluR-B and transgenic GFPGluR-B in the hippocampus of three different mice (#1, #2, #3). + +(D) Relative quantification from (C) of transgenic GFPGluR-B compared with endogenous GluR-B in the hippocampus. + +Figure 6 + +GluR-B Expression in Hippocampus and Forebrain Partially Rescues the Memory Deficit of GluR-BΔFB Mice + +(A) Olfactory memory experiments as in Figure 2 were performed with GluR-BRescue. Individual mice are indicated: control GluR-B2lox with black circles, GluR-BΔFB with red triangles, GluR-BRescue with green squares. Data were combined from Figure 3 and 4 (open symbols) and an additional experiment with four GluR-BΔFB, three littermate controls, and eight GluR-BRescue (shaded symbols). + +(B) Cumulative histogram of the memory performance. Memory performance is indicated as a stepped line. The sigmoidal fit is indicated as a continuous line. The predicted rescue on the basis of the extent of transgenic GFPGluR-B expression (Figure 5D) and the correlation between memory and GluR-B levels in piriform cortex (9.1 ± 2.5% increase in memory for 10% increase in protein, Figure 4D) is shown in blue. Note that the predicted rescue is in perfect numerical correspondence to the memory performance of GluR-BRescue mice. + +(C) The discrimination index was calculated as in Figures 1E, 2C, and 4D (last 100 trials of the mixture discrimination task) for GluRBRescue (n = 8), GluR-BΔFB (n = 22), and control (n = 14) animals. + +Footnotes + +Author contributions. DRS and ATS conceived and designed most of the experiments. The manuscript was written by DRS, PHS, RS, and ATS. Mouse lines were conceived and designed by DRS, PHS, RS (GluR-BΔFB, GluR-BΔECS:FB); DRS, PHS, RS, ATS (GluR-BRescue); JK, PHS, RS (TgCN12-itTA); and VM, PHS, RS (GluR-B2lox, TgOCN1). Olfactory behavioral experiments were done by TB and ATS; AM performed the GFPGluR-B Western blots; immunohistochemical experiments and other Western blots as well as the nonolfactory behavioral experiments were performed by DRS. + +¤ Current address: Department of Physiology, University College London, London, United Kingdom + +Citation: Shimshek DR, Bus T, Kim J, Mihaljevic A, Mack V, et al. (2005) Enhanced odor discrimination and impaired olfactory memory by spatially controlled switch of AMPA receptors. PLoS Biol 3(11): e354. diff --git a/src/ontogpt/evaluation/craft/database/all/16221973.ann b/src/ontogpt/evaluation/craft/database/all/16221973.ann new file mode 100644 index 000000000..bdb96f640 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16221973.ann @@ -0,0 +1,1322 @@ +T1 GO:0006952 35 42 defense +T2 NCBITaxon:10088 50 55 mouse +T3 PR:000010717 57 62 MTF-1 +T4 CHEBI:16856 84 95 glutathione +T5 PR:000010717 107 146 Metal-responsive transcription factor 1 +T6 PR:000010717 148 153 MTF-1 +T7 GO:0065007 155 164 regulates +T8 GO:0010467 165 175 expression +T9 SO:0000704 190 195 genes +T10 SO:0000165 328 336 enhancer +T11 SO:0000167 337 345 promoter +T12 UBERON:0000922 398 407 embryonic +T13 GO:0009790 398 407;414 425 embryonic ... development +T14 UBERON:0002107 408 413 liver +T15 GO:0001889 408 425 liver development +T16 PR:000010717 457 461 Mtf1 +T17 UBERON:0002107 469 474 liver +T18 GO:0007567 481 486 birth +T19 PR:000010717 524 528 Mtf1 +T20 NCBITaxon:10088 550 554 mice +T21 UBERON:0002107 618 623 liver +T22 SO:0000704 708 713 genes +T23 PR:000010717 727 732 MTF-1 +T24 SO:0000704 740 745 genes +T25 SO:0000167 769 777 promoter +T26 PR:000010717 794 799 MTF-1 +T27 GO:0010467 826 836 expression +T28 PR:000014662 840 865 selenoprotein W, muscle 1 +T29 SO:0000704 866 870 gene +T30 PR:000014662 872 877 Sepw1 +T31 CHEBI:16856 894 905 glutathione +T32 PR:000010717 969 974 MTF-1 +T33 MOP:0000568 982 991 oxidative +T34 PR:000010717 1022 1027 MTF-1 +T35 GO:0010467 1057 1067 expression +T36 PR:000011059 1071 1104 N-myc downstream regulated gene 1 +T37 GO:0065007 1088 1097 regulated +T38 SO:0000704 1098 1102 gene +T39 PR:000011059 1106 1111 Ndrg1 +T40 GO:0010467 1171 1180 expressed +T41 http://purl.obolibrary.org/obo/MONDO_0004992 1189 1196 cancers +T42 PR:000010717 1198 1203 MTF-1 +T43 PR:000005958 1248 1284 cysteine- and glycine-rich protein 1 +T44 SO:0000704 1285 1289 gene +T45 PR:000005958 1291 1296 Csrp1 +T46 GO:0005856 1322 1334 cytoskeletal +T47 PR:000010717 1362 1367 MTF-1 +T48 GO:0010467 1388 1398 expression +T49 PR:000015124 1402 1410 Slc39a10 +T50 PR:000010717 1469 1474 MTF-1 +T51 SO:0000704 1518 1523 genes +T52 GO:0006750 1540 1549;1567 1581 synthesis ... of glutathione +T53 CHEBI:16856 1570 1581 glutathione +T54 GO:0006968 1675 1683;1697 1704 cellular ... defense +T55 PR:000010717 1714 1719 MTF-1 +T56 SO:0000704 1735 1740 genes +T57 CHEBI:16856 1782 1793 glutathione +T58 PR:000014662 1823 1838 selenoprotein W +T59 NCBITaxon:1 1859 1868 organisms +T60 CHEBI:29105 2111 2115 Zn2+ +T61 CHEBI:29036 2117 2121 Cu2+ +T62 CHEBI:48775 2126 2130 Cd2+ +T63 GO:0042592 2234 2245 homeostasis +T64 GO:0098754 2250 2264 detoxification +T65 CHEBI:24870 2316 2320 ions +T66 NCBITaxon:10088 2335 2340 mouse +T67 SO:0000704 2373 2378 genes +T68 PR:000010925 2394 2397 Mt1 +T69 PR:000010706 2401 2404 Mt4 +T70 GO:0010467 2445 2455 expression +T71 PR:000010925 2459 2462 Mt1 +T72 PR:000010704 2467 2470 Mt2 +T73 PR:000010717 2486 2525 metal-responsive transcription factor 1 +T74 PR:000010717 2527 2532 MTF-1 +T75 SO:0000993 2652 2661 consensus +T76 SO:0000167 2706 2715 promoters +T77 SO:0000704 2726 2731 genes +T78 PR:000010717 2739 2744 MTF-1 +T79 SO:0000853 2776 2784 homologs +T80 NCBITaxon:10088 2816 2821 mouse +T81 NCBITaxon:9606 2828 2834 humans +T82 NCBITaxon:7215 2841 2851 Drosophila +T83 PR:000010717 2891 2896 MTF-1 +T84 NCBITaxon:10088 2938 2943 mouse +T85 PR:000010717 2983 2988 MTF-1 +T86 SO:0000704 3026 3031 genes +T87 MOP:0000568 3080 3089 oxidative +T88 PR:000015073 3179 3183 Znt1 +T89 GO:0005886 3204 3219 plasma membrane +T90 SO:0000704 3296 3300 gene +T91 UBERON:0001987 3305 3314 placental +T92 PR:000012605 3305 3328 placental growth factor +T93 PR:000012605 3330 3334 Plgf +T94 GO:0001525 3340 3350 angiogenic +T95 UBERON:0001986 3375 3386 endothelial +T96 http://purl.obolibrary.org/obo/MONDO_0005070 3454 3459 tumor +T97 PR:000010717 3494 3499 MTF-1 +T98 GO:0009790 3533 3546 embryogenesis +T99 PR:000010717 3571 3575 Mtf1 +T100 UBERON:0000922 3587 3596 embryonic +T101 GO:0007620 3627 3633 coitum +T102 CL:0000182 3641 3651 hepatocyte +T103 NCBITaxon:10088 3680 3684 mice +T104 SO:0000704 3746 3751 genes +T105 PR:000010925 3753 3756 Mt1 +T106 PR:000010704 3761 3764 Mt2 +T107 PR:000010717 3852 3857 MTF-1 +T108 SO:0000704 3865 3870 genes +T109 SO:0000346 3922 3926 loxP +T110 UBERON:0000922 3992 4001 embryonic +T111 PR:000010717 4035 4039 Mtf1 +T112 NCBITaxon:10088 4049 4053 mice +T113 PR:000010717 4122 4126 Mtf1 +T114 UBERON:0002107 4136 4141 liver +T115 GO:0007567 4148 4153 birth +T116 UBERON:0002107 4239 4244 liver +T117 PR:000010717 4254 4258 Mtf1 +T118 NCBITaxon:10088 4268 4273 mouse +T119 PR:000010717 4317 4322 MTF-1 +T120 SO:0000704 4330 4335 genes +T121 SO:0000704 4358 4363 genes +T122 UBERON:0007023 4371 4376 adult +T123 UBERON:0002107 4377 4382 liver +T124 SO:0000704 4403 4407 gene +T125 PR:000010717 4486 4490 Mtf1 +T126 NCBITaxon:10088 4512 4516 mice +T127 SO:0000704 4650 4655 genes +T128 SO:0000704 4690 4695 genes +T129 PR:000010717 4699 4704 MTF-1 +T130 PR:000010717 4719 4724 MTF-1 +T131 UBERON:0002107 4748 4753 liver +T132 GO:0010467 4754 4764 expression +T133 SO:0000704 4772 4776 gene +T134 PR:000014662 4781 4806 selenoprotein W, muscle 1 +T135 PR:000014662 4808 4813 Sepw1 +T136 GO:0010467 4846 4856 expression +T137 PR:000011059 4860 4893 N-myc downstream regulated gene 1 +T138 GO:0065007 4877 4886 regulated +T139 SO:0000704 4887 4891 gene +T140 PR:000011059 4895 4900 Ndrg1 +T141 SO:0000704 4910 4914 gene +T142 PR:000005958 4924 4960 cysteine- and glycine-rich protein 1 +T143 PR:000005958 4962 4967 Csrp1 +T144 PR:000010717 4983 4988 MTF-1 +T145 GO:0010467 5012 5022 expression +T146 PR:000015124 5026 5061 solute carrier family 39, member 10 +T147 SO:0000704 5062 5066 gene +T148 PR:000015124 5068 5076 Slc39a10 +T149 CHEBI:24870 5110 5113 ion +T150 PR:000010717 5133 5138 MTF-1 +T151 SO:0000704 5183 5188 genes +T152 CHEBI:16856 5201 5212 glutathione +T153 GO:0006749 5201 5223 glutathione metabolism +T154 GO:0006952 5283 5290 defense +T155 CHEBI:16856 5300 5311 glutathione +T156 PR:000010717 5332 5337 MTF-1 +T157 SO:0000704 5353 5358 genes +T158 PR:000010717 5426 5430 Mtf1 +T159 NCBITaxon:10088 5452 5456 mice +T160 UBERON:0002107 5461 5466 liver +T161 PR:000010717 5486 5490 Mtf1 +T162 NCBITaxon:10088 5512 5516 mice +T163 SO:0000040 5595 5609 genomic clones +T164 SO:0000147 5621 5626 exons +T165 PR:000010717 5637 5641 Mtf1 +T166 SO:0000704 5667 5671 gene +T167 SO:0001644 5672 5688 targeting vector +T168 CHEBI:7507 5742 5750 neomycin +T169 SO:0005853 5762 5770 cassette +T170 SO:0000357 5781 5788 flanked +T171 SO:0000346 5796 5806 loxP sites +T172 SO:0000147 5843 5847 exon +T173 PR:000010717 5853 5857 Mtf1 +T174 SO:0000346 5869 5878 loxP site +T175 SO:0000147 5915 5919 exon +T176 SO:0005853 5947 5955 cassette +T177 SO:0000147 5992 5996 exon +T178 CL:0002322 6004 6012 ES cells +T179 SO:0001644 6053 6069 targeting vector +T180 CHEBI:42768 6099 6103 G418 +T181 GO:0010467 6203 6213 expression +T182 SO:0005853 6263 6271 cassette +T183 NCBITaxon:10088 6277 6281 mice +T184 PR:000010717 6304 6308 Mtf1 +T185 SO:0000346 6308 6312 loxP +T186 SO:0001023 6313 6319 allele +T187 UBERON:0000358 6380 6391 blastocysts +T188 NCBITaxon:33208 6448 6455 animals +T189 PR:000010717 6457 6461 Mtf1 +T190 SO:0000346 6461 6465 loxP +T191 SO:0000346 6466 6470 loxP +T192 PR:000010788 6526 6528 Mx +T193 UBERON:0002107 6590 6595 liver +T194 PR:000010717 6605 6609 Mtf1 +T195 NCBITaxon:10088 6629 6633 mice +T196 SO:0000112 6676 6683 primers +T197 PR:000010717 6780 6784 Mtf1 +T198 SO:0000346 6784 6788 loxP +T199 SO:0001023 6802 6808 allele +T200 NCBITaxon:33208 6880 6886 Animal +T201 PR:000010717 6922 6926 Mtf1 +T202 SO:0000346 6926 6930 loxP +T203 SO:0000346 6931 6935 loxP +T204 NCBITaxon:10088 6936 6940 mice +T205 PR:000010788 6955 6957 Mx +T206 SO:0000902 6962 6971 transgene +T207 PR:000010717 6973 6977 Mtf1 +T208 SO:0000346 6977 6981 loxP +T209 SO:0000346 6982 6986 loxP +T210 PR:000010788 6987 6989 Mx +T211 PR:000010717 7002 7006 Mtf1 +T212 PR:000010788 7006 7008 Mx +T213 SO:0000902 7046 7055 transgene +T214 PR:000010717 7057 7061 Mtf1 +T215 SO:0000346 7061 7065 loxP +T216 SO:0000346 7066 7070 loxP +T217 PR:000010717 7079 7083 Mtf1 +T218 SO:0000346 7083 7087 loxP +T219 UBERON:0001179 7108 7118 peritoneal +T220 SO:0000985 7155 7170 double-stranded +T221 CHEBI:76777 7175 7187;7202 7206 polyinosinic ... acid +T222 CHEBI:76777 7208 7210 pI +T223 PR:000010717 7360 7365 MTF-1 +T224 SO:0000704 7373 7377 gene +T225 NCBITaxon:10088 7402 7406 mice +T226 CHEBI:76777 7419 7421 pI +T227 NCBITaxon:10088 7475 7479 mice +T228 CHEBI:76777 7511 7513 pI +T229 CHEBI:50292 7592 7597 CdSO4 +T230 CHEBI:50292 7604 7609 CdSO4 +T231 CHEBI:15377 7613 7616 H2O +T232 CHEBI:15377 7661 7664 H2O +T233 UBERON:0002107 7781 7786 liver +T234 UBERON:0000479 7787 7793 tissue +T235 CHEBI:76777 7797 7799 pI +T236 PR:000010717 7837 7841 Mtf1 +T237 PR:000010788 7841 7843 Mx +T238 PR:000010717 7852 7856 Mtf1 +T239 SO:0000346 7856 7860 loxP +T240 NCBITaxon:10088 7861 7865 mice +T241 SO:0000704 7980 7984 Gene +T242 GO:0010467 7980 7995 Gene expression +T243 NCBITaxon:10088 8076 8081 Mouse +T244 SO:0001026 8082 8088 Genome +T245 CHEBI:33893 8180 8188 reagents +T246 SO:0000985 8244 8259 Double-Stranded +T247 SO:0000673 8384 8394 Transcript +T248 PR:000010717 9013 9018 MTF-1 +T249 SO:0000704 9031 9036 genes +T250 SO:0000704 9038 9043 Genes +T251 GO:0010467 9081 9090 expressed +T252 GO:0010467 9136 9146 expression +T253 SO:0000704 9298 9302 gene +T254 NCBITaxon:10088 9342 9346 mice +T255 NCBITaxon:10088 9384 9388 mice +T256 SO:0000993 9456 9465 consensus +T257 SO:0000167 9490 9498 promoter +T258 SO:0000704 9548 9553 genes +T259 SO:0001026 9620 9626 Genome +T260 SO:0000112 9935 9942 primers +T261 PR:000005958 9944 9949 Csrp1 +T262 CHEBI:17368 10009 10020 hypoxanthin +T263 PR:000010717 10120 10124 Mtf1 +T264 PR:000011059 10190 10195 Ndrg1 +T265 PR:000014662 10255 10260 Sepw1 +T266 PR:000015124 10320 10328 Slc39a10 +T267 PR:000025471 10389 10400 S1 nuclease +T268 SO:0000673 10412 10423 transcripts +T269 PR:000025471 10425 10427 S1 +T270 PR:000025471 10439 10441 S1 +T271 PR:000025471 10643 10645 S1 +T272 CHEBI:37972 10683 10686 32P +T273 PR:000025471 10718 10720 S1 +T274 PR:000014662 10802 10807 Sepw1 +T275 PR:000025471 10808 10810 S1 +T276 UBERON:0002107 10983 10988 liver +T277 UBERON:0000479 10989 10995 tissue +T278 UBERON:0000479 11008 11014 Tissue +T279 CHEBI:33893 11034 11041 Reagent +T280 NCBITaxon:10088 11119 11124 mouse +T281 UBERON:0000479 11125 11131 tissue +T282 CHEBI:33893 11146 11153 reagent +T283 CHEBI:37972 11284 11287 32P +T284 SO:0000442 11300 11332 double-stranded oligonucleotides +T285 UBERON:0002107 11352 11357 liver +T286 GO:0097617 11625 11633 annealed +T287 PR:000005958 11662 11667 Csrp1 +T288 PR:000005958 11747 11752 Csrp1 +T289 PR:000005958 11829 11834 Csrp1 +T290 PR:000005958 11911 11916 Csrp1 +T291 PR:P04386 11993 11997 Gal4 +T292 NCBITaxon:10088 12078 12083 mouse +T293 PR:000010925 12084 12087 Mt1 +T294 SO:0000167 12088 12096 promoter +T295 SO:0000351 12197 12206;12221 12229 synthetic ... sequence +T296 SO:0000993 12211 12220 consensus +T297 PR:000011059 12323 12328 Ndrg1 +T298 PR:000011059 12401 12406 Ndrg1 +T299 PR:000011059 12479 12484 Ndrg1 +T300 PR:000014662 12588 12593 Sepw1 +T301 PR:000014662 12683 12688 Sepw1 +T302 PR:000015124 12767 12775 Slc39a10 +T303 PR:000015124 12861 12869 Slc39a10 +T304 PR:000000133 12946 12967 Specificity protein 1 +T305 PR:000000133 12969 12972 Sp1 +T306 UBERON:0000922 13074 13083 embryonic +T307 CL:0000057 13084 13095 fibroblasts +T308 PR:000010717 13130 13134 Mtf1 +T309 SO:0000346 13134 13138 loxP +T310 NCBITaxon:10088 13139 13144 mouse +T311 UBERON:0000922 13145 13151 embryo +T312 GO:0040007 13156 13161 grown +T313 NCBITaxon:27592 13198 13204 bovine +T314 UBERON:0001977 13205 13210 serum +T315 CHEBI:17334 13227 13237 penicillin +T316 CHEBI:17076 13238 13250 streptomycin +T317 CL:0000001 13316 13329 primary cells +T318 GO:0009294 13335 13346 transfected +T319 GO:0010467 13364 13374 expression +T320 SO:0000155 13375 13382 plasmid +T321 NCBITaxon:10633 13394 13409 simian virus 40 +T322 NCBITaxon:10633 13411 13415 SV40 +T323 CHEBI:59132 13425 13432 antigen +T324 NCBITaxon:10358 13447 13462 cytomegalovirus +T325 NCBITaxon:10358 13464 13467 CMV +T326 SO:0000167 13469 13477 promoter +T327 CHEBI:33893 13500 13507 reagent +T328 NCBITaxon:10088 13612 13617 mouse +T329 UBERON:0000922 13618 13627 embryonic +T330 CL:0002321 13618 13627;13639 13643 embryonic ... cell +T331 CL:0000057 13628 13643 fibroblast cell +T332 PR:000010717 13688 13692 Mtf1 +T333 SO:0000346 13692 13696 loxP +T334 SO:0001026 13734 13741 genomic +T335 CHEBI:59132 13763 13770 antigen +T336 GO:0009294 13838 13849 transfected +T337 CHEBI:77635 13857 13874 calcium phosphate +T338 GO:0010467 13906 13916 expression +T339 SO:0000155 13917 13924 plasmid +T340 NCBITaxon:10358 13959 13962 CMV +T341 SO:0000167 13963 13971 promoter +T342 GO:0010467 13989 13999 expression +T343 SO:0000155 14000 14007 plasmid +T344 CHEBI:7507 14016 14024 neomycin +T345 SO:0000704 14036 14040 gene +T346 GO:0065007 14051 14058 control +T347 SO:0000167 14069 14077 promoter +T348 GO:0009294 14086 14097 transfected +T349 CHEBI:42768 14147 14151 G418 +T350 GO:0040007 14220 14225 grown +T351 GO:0010467 14249 14259 expression +T352 SO:0000147 14295 14300 exons +T353 PR:000010717 14312 14316 Mtf1 +T354 PR:000010717 14402 14406 Mtf1 +T355 UBERON:0000479 14518 14524 tissue +T356 CHEBI:28714 14658 14688 l-buthionine-[S,R]-sulfoximine +T357 CHEBI:28714 14690 14693 BSO +T358 CHEBI:23888 14706 14710 drug +T359 CHEBI:16856 14725 14736 glutathione +T360 GO:0006750 14725 14746 glutathione synthesis +T361 CHEBI:35456 14800 14805 CdCl2 +T362 CHEBI:53233 14904 14907 MTT +T363 CHEBI:53233 14909 14969 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid +T364 GO:0008283 14977 14995 Cell Proliferation +T365 UBERON:0002107 15094 15099 liver +T366 PR:000010717 15109 15113 Mtf1 +T367 NCBITaxon:10088 15123 15128 mouse +T368 NCBITaxon:10088 15178 15182 mice +T369 PR:000010717 15213 15217 Mtf1 +T370 SO:0001023 15218 15224 allele +T371 PR:000010717 15225 15229 Mtf1 +T372 SO:0000346 15229 15233 loxP +T373 SO:0000147 15240 15245 exons +T374 SO:0000417 15312 15318 domain +T375 SO:0000357 15324 15331 flanked +T376 SO:0000346 15335 15345 loxP sites +T377 NCBITaxon:10088 15359 15363 Mice +T378 PR:000010717 15383 15387 Mtf1 +T379 SO:0000346 15387 15391 loxP +T380 SO:0001023 15392 15398 allele +T381 NCBITaxon:33208 15425 15432 animals +T382 PR:000010788 15472 15474 Mx +T383 GO:0010467 15499 15508 expressed +T384 GO:0065007 15532 15539 control +T385 NCBITaxon:10088 15547 15552 mouse +T386 PR:000010788 15553 15556 Mx1 +T387 SO:0000704 15557 15561 gene +T388 SO:0000167 15562 15570 promoter +T389 PR:000024938 15612 15628 interferon alpha +T390 PR:000024939 15612 15622;15632 15636 interferon ... beta +T391 SO:0000985 15651 15666 double-stranded +T392 CHEBI:76777 15671 15673 pI +T393 UBERON:0002107 15740 15745 liver +T394 UBERON:0000479 15770 15777 tissues +T395 UBERON:0002106 15799 15805 spleen +T396 UBERON:0000955 15815 15820 brain +T397 SO:0000147 15858 15863 exons +T398 GO:0006412 15917 15928 translation +T399 PR:000010717 15949 15954 MTF-1 +T400 PR:000010717 16015 16019 Mtf1 +T401 NCBITaxon:10088 16041 16045 mice +T402 PR:000010788 16060 16062 Mx +T403 SO:0000902 16067 16076 transgene +T404 PR:000010717 16078 16082 Mtf1 +T405 PR:000010788 16082 16084 Mx +T406 UBERON:0001179 16109 16119 peritoneal +T407 CHEBI:76777 16120 16122 pI +T408 CHEBI:76777 16157 16159 pI +T409 SO:0000902 16203 16212 transgene +T410 PR:000010717 16214 16218 Mtf1 +T411 SO:0000346 16218 16222 loxP +T412 PR:000010717 16327 16331 Mtf1 +T413 PR:000010788 16331 16333 Mx +T414 UBERON:0002107 16338 16344 livers +T415 SO:0000147 16382 16387 exons +T416 PR:000010717 16399 16403 Mtf1 +T417 NCBITaxon:33208 16413 16420 animals +T418 NCBITaxon:10088 16527 16531 mice +T419 PR:000010717 16586 16590 Mtf1 +T420 PR:000010717 16621 16626 MTF-1 +T421 PR:000010717 16669 16674 MTF-1 +T422 GO:0032993 16675 16694 protein–DNA complex +T423 UBERON:0002107 16715 16720 liver +T424 PR:000010717 16745 16749 Mtf1 +T425 SO:0000346 16749 16753 loxP +T426 NCBITaxon:10088 16762 16767 mouse +T427 PR:000010717 16828 16832 Mtf1 +T428 PR:000010788 16832 16834 Mx +T429 SO:0000147 16865 16870 exons +T430 PR:000010717 16882 16886 Mtf1 +T431 UBERON:0002107 16894 16899 liver +T432 PR:000010717 16903 16907 Mtf1 +T433 PR:000010788 16907 16909 Mx +T434 NCBITaxon:10088 16914 16918 mice +T435 UBERON:0002107 16956 16961 liver +T436 NCBITaxon:10088 16980 16984 mice +T437 PR:000010717 17047 17052 MTF-1 +T438 SO:0000704 17060 17064 gene +T439 PR:000010717 17099 17104 MTF-1 +T440 SO:0000704 17112 17117 genes +T441 UBERON:0002107 17135 17140 liver +T442 SO:0000673 17141 17151 transcript +T443 NCBITaxon:10088 17164 17168 mice +T444 PR:000010717 17197 17201 Mtf1 +T445 SO:0000704 17202 17206 gene +T446 SO:0000673 17327 17338 transcripts +T447 SO:0000985 17419 17434 double-stranded +T448 SO:0000412 17444 17465 restriction fragments +T449 PR:000010925 17582 17585 Mt1 +T450 PR:000010704 17590 17593 Mt2 +T451 SO:0000673 17626 17637 transcripts +T452 UBERON:0002107 17693 17699 livers +T453 PR:000010717 17727 17732 MTF-1 +T454 SO:0000704 17733 17737 gene +T455 PR:000010717 17796 17801 MTF-1 +T456 GO:0010467 17835 17845 expression +T457 SO:0000704 17865 17870 genes +T458 SO:0000704 17899 17903 gene +T459 GO:0010467 17899 17914 gene expression +T460 UBERON:0002107 17926 17932 livers +T461 NCBITaxon:10088 17956 17960 mice +T462 NCBITaxon:10088 17998 18003 Mouse +T463 SO:0001026 18004 18010 Genome +T464 UBERON:0002107 18076 18082 livers +T465 PR:000010717 18101 18105 Mtf1 +T466 PR:000010788 18105 18107 Mx +T467 PR:000010717 18116 18120 Mtf1 +T468 SO:0000346 18120 18124 loxP +T469 NCBITaxon:10088 18125 18129 mice +T470 GO:0010629 18160 18188 downregulation of expression +T471 PR:000010717 18205 18209 Mtf1 +T472 PR:000010788 18209 18211 Mx +T473 UBERON:0002107 18216 18222 livers +T474 SO:0000704 18296 18301 genes +T475 SO:0000704 18331 18336 genes +T476 SO:0000993 18366 18375 consensus +T477 SO:0000028 18418 18420 bp +T478 SO:0000704 18520 18525 genes +T479 PR:000010717 18585 18589 Mtf1 +T480 PR:000010788 18589 18591 Mx +T481 UBERON:0002107 18596 18602 livers +T482 SO:0000704 18632 18637 genes +T483 SO:0000993 18655 18664 consensus +T484 UBERON:0002107 18716 18722 livers +T485 PR:000010717 18742 18746 Mtf1 +T486 PR:000010788 18746 18748 Mx +T487 PR:000010717 18757 18761 Mtf1 +T488 SO:0000346 18761 18765 loxP +T489 NCBITaxon:10088 18766 18770 mice +T490 PR:000010717 18827 18831 Mtf1 +T491 PR:000010788 18831 18833 Mx +T492 UBERON:0002107 18838 18844 livers +T493 SO:0000704 18907 18912 genes +T494 SO:0000993 18956 18965 consensus +T495 SO:0000704 19062 19067 genes +T496 SO:0000704 19200 19205 genes +T497 SO:0000345 19207 19211 ESTs +T498 PR:000010717 19274 19278 Mtf1 +T499 PR:000010788 19278 19280 Mx +T500 PR:000010717 19289 19293 Mtf1 +T501 SO:0000346 19293 19297 loxP +T502 UBERON:0002107 19298 19304 livers +T503 GO:0010467 19326 19335 expressed +T504 PR:000010925 19379 19382 Mt1 +T505 PR:000010704 19387 19390 Mt2 +T506 PR:000010717 19407 19411 Mtf1 +T507 PR:000010788 19411 19413 Mx +T508 UBERON:0002107 19418 19424 livers +T509 PR:000010925 19505 19508 Mt1 +T510 NCBITaxon:33208 19525 19532 animals +T511 PR:000010717 19575 19580 MTF-1 +T512 SO:0000704 19588 19593 genes +T513 PR:000010925 19624 19627 Mt1 +T514 PR:000010704 19629 19632 Mt2 +T515 PR:000015073 19637 19641 Znt1 +T516 PR:000010717 19643 19648 MTF-1 +T517 PR:000010717 19809 19814 MTF-1 +T518 SO:0000409 19815 19828 binding sites +T519 SO:0000442 19857 19889 double-stranded oligonucleotides +T520 PR:000010717 19945 19950 MTF-1 +T521 PR:000010717 20053 20058 MTF-1 +T522 SO:0000704 20066 20070 gene +T523 SO:0000704 20103 20107 gene +T524 GO:0010467 20149 20159 expression +T525 PR:000014662 20163 20168 Sepw1 +T526 PR:000010717 20180 20185 MTF-1 +T527 PR:000014662 20187 20192 Sepw1 +T528 UBERON:0002107 20263 20269 livers +T529 PR:000010717 20301 20305 Mtf1 +T530 PR:000010788 20305 20307 Mx +T531 NCBITaxon:10088 20312 20316 mice +T532 PR:000014662 20337 20342 SEPW1 +T533 CHEBI:16856 20393 20404 glutathione +T534 PR:000014662 20465 20470 Sepw1 +T535 GO:0010467 20471 20481 expression +T536 UBERON:0002107 20485 20491 livers +T537 CHEBI:76777 20495 20497 pI +T538 PR:000010717 20535 20539 Mtf1 +T539 PR:000010788 20539 20541 Mx +T540 PR:000010717 20550 20554 Mtf1 +T541 SO:0000346 20554 20558 loxP +T542 NCBITaxon:10088 20559 20563 mice +T543 PR:000025471 20617 20619 S1 +T544 PR:000014662 20717 20722 Sepw1 +T545 UBERON:0002107 20753 20759 livers +T546 PR:000010717 20765 20769 Mtf1 +T547 SO:0000346 20769 20773 loxP +T548 NCBITaxon:10088 20774 20778 mice +T549 UBERON:0002107 20834 20840 livers +T550 PR:000010717 20872 20876 Mtf1 +T551 PR:000010788 20876 20878 Mx +T552 NCBITaxon:10088 20883 20887 mice +T553 PR:000010717 20905 20910 MTF-1 +T554 GO:0010467 20938 20948 expression +T555 PR:000014662 20952 20957 Sepw1 +T556 SO:0000993 20975 20984 consensus +T557 NCBITaxon:10088 21036 21041 mouse +T558 PR:000014662 21042 21047 Sepw1 +T559 SO:0000028 21190 21192 bp +T560 SO:0000028 21249 21251 bp +T561 PR:000010717 21274 21279 MTF-1 +T562 PR:000014662 21283 21288 Sepw1 +T563 UBERON:0002107 21349 21354 liver +T564 PR:000010717 21379 21383 Mtf1 +T565 SO:0000346 21383 21387 loxP +T566 NCBITaxon:10088 21396 21401 mouse +T567 CHEBI:76777 21481 21483 pI +T568 PR:000010717 21495 21499 Mtf1 +T569 PR:000010788 21499 21501 Mx +T570 NCBITaxon:10088 21506 21511 mouse +T571 PR:000010717 21583 21588 MTF-1 +T572 PR:000011059 21611 21616 Ndrg1 +T573 PR:000010717 21628 21633 MTF-1 +T574 PR:000011059 21635 21640 Ndrg1 +T575 UBERON:0002107 21691 21696 liver +T576 SO:0000673 21697 21708 transcripts +T577 PR:000010717 21730 21734 Mtf1 +T578 PR:000010788 21734 21736 Mx +T579 NCBITaxon:10088 21741 21745 mice +T580 PR:000010717 21776 21780 Mtf1 +T581 SO:0000346 21780 21784 loxP +T582 NCBITaxon:10088 21793 21797 mice +T583 PR:000011059 21812 21817 Ndrg1 +T584 CHEBI:36357 21912 21921 compounds +T585 GO:0010467 21932 21942 expression +T586 NCBITaxon:9989 21946 21952 rodent +T587 PR:000011059 21953 21958 Ndrg1 +T588 NCBITaxon:9606 21970 21975 human +T589 SO:0000855 21976 21984 ortholog +T590 PR:000011059 21999 22004 Ndrg1 +T591 PR:000010717 22086 22090 Mtf1 +T592 SO:0000346 22090 22094 loxP +T593 UBERON:0002107 22103 22109 livers +T594 PR:000011059 22131 22136 Ndrg1 +T595 GO:0010467 22137 22147 expression +T596 UBERON:0002107 22188 22194 livers +T597 PR:000010717 22200 22204 Mtf1 +T598 PR:000010788 22204 22206 Mx +T599 NCBITaxon:10088 22211 22215 mice +T600 GO:0010467 22271 22281 expression +T601 GO:0010467 22343 22353 expression +T602 PR:000011059 22357 22362 Ndrg1 +T603 PR:000010717 22374 22379 MTF-1 +T604 SO:0000993 22396 22405 consensus +T605 NCBITaxon:10088 22444 22449 mouse +T606 PR:000011059 22450 22455 Ndrg1 +T607 SO:0000028 22544 22546 bp +T608 SO:0000028 22603 22605 bp +T609 PR:000010717 22643 22648 MTF-1 +T610 GO:0032991 22878 22885 complex +T611 PR:000010717 22919 22924 MTF-1 +T612 GO:0032991 22925 22934 complexes +T613 UBERON:0002107 23000 23005 liver +T614 PR:000010717 23030 23034 Mtf1 +T615 SO:0000346 23034 23038 loxP +T616 NCBITaxon:10088 23039 23044 mouse +T617 NCBITaxon:10088 23113 23118 mouse +T618 PR:000010717 23127 23132 MTF-1 +T619 PR:000010717 23134 23138 Mtf1 +T620 PR:000010788 23138 23140 Mx +T621 PR:000005958 23168 23173 Csrp1 +T622 PR:000010717 23185 23190 MTF-1 +T623 PR:000005958 23192 23197 Csrp1 +T624 PR:000010717 23284 23288 Mtf1 +T625 PR:000010788 23288 23290 Mx +T626 NCBITaxon:10088 23295 23299 mice +T627 PR:000010717 23312 23316 Mtf1 +T628 SO:0000346 23316 23320 loxP +T629 NCBITaxon:10088 23321 23325 mice +T630 PR:000005958 23340 23345 CSRP1 +T631 GO:0005856 23455 23467 cytoskeletal +T632 PR:000005958 23561 23566 Csrp1 +T633 GO:0010467 23567 23577 expression +T634 PR:000010717 23593 23597 Mtf1 +T635 SO:0000346 23597 23601 loxP +T636 UBERON:0002107 23602 23608 livers +T637 UBERON:0002107 23695 23701 livers +T638 PR:000010717 23707 23711 Mtf1 +T639 PR:000010788 23711 23713 Mx +T640 NCBITaxon:10088 23718 23722 mice +T641 PR:000010717 23740 23745 MTF-1 +T642 PR:000005958 23783 23788 Csrp1 +T643 SO:0000993 23806 23815 consensus +T644 PR:000005958 23853 23858 Csrp1 +T645 SO:0000028 23906 23908 bp +T646 SO:0000028 23958 23960 bp +T647 PR:000010717 23994 23999 MTF-1 +T648 PR:000010717 24076 24080 Mtf1 +T649 SO:0000346 24080 24084 loxP +T650 UBERON:0002107 24085 24090 liver +T651 PR:000010717 24103 24107 Mtf1 +T652 PR:000010788 24107 24109 Mx +T653 UBERON:0002107 24114 24119 liver +T654 PR:000010717 24136 24141 MTF-1 +T655 PR:000010717 24175 24180 MTF-1 +T656 GO:0032991 24188 24195 complex +T657 PR:000010717 24210 24215 MTF-1 +T658 GO:0010467 24225 24235 expression +T659 PR:000015124 24239 24247 Slc39a10 +T660 PR:000015124 24249 24257 Slc39a10 +T661 UBERON:0002107 24329 24335 livers +T662 PR:000010717 24372 24376 Mtf1 +T663 PR:000010788 24376 24378 Mx +T664 NCBITaxon:10088 24383 24387 mice +T665 NCBITaxon:33208 24408 24415 animals +T666 CHEBI:24870 24518 24521 ion +T667 CHEBI:24870 24582 24585 ion +T668 GO:0005886 24604 24622 cellular membranes +T669 GO:0005737 24632 24641 cytoplasm +T670 GO:0010629 24722 24739;24749 24759 downregulation of ... expression +T671 PR:000015124 24740 24748 Slc39a10 +T672 UBERON:0002107 24763 24769 livers +T673 PR:000010717 24773 24777 Mtf1 +T674 SO:0000346 24777 24781 loxP +T675 NCBITaxon:10088 24782 24786 mice +T676 PR:000010717 24826 24830 Mtf1 +T677 PR:000010788 24830 24832 Mx +T678 NCBITaxon:10088 24837 24841 mice +T679 GO:0010467 24853 24863 expression +T680 PR:000015124 24943 24951 Slc39a10 +T681 GO:0010467 24952 24962 expression +T682 PR:000015124 25066 25074 Slc39a10 +T683 PR:000010717 25107 25111 Mtf1 +T684 PR:000010788 25111 25113 Mx +T685 PR:000010717 25122 25126 Mtf1 +T686 SO:0000346 25126 25130 loxP +T687 NCBITaxon:10088 25131 25135 mice +T688 PR:000010717 25163 25168 MTF-1 +T689 UBERON:0002107 25271 25277 livers +T690 PR:000010717 25391 25396 MTF-1 +T691 GO:0010467 25436 25446 expression +T692 PR:000015124 25450 25458 Slc39a10 +T693 SO:0000704 25518 25522 gene +T694 SO:0000993 25588 25597 consensus +T695 NCBITaxon:10088 25638 25643 mouse +T696 PR:000015124 25644 25652 Slc39a10 +T697 SO:0000028 25684 25686 bp +T698 SO:0000028 25732 25734 bp +T699 PR:000010717 25768 25773 MTF-1 +T700 UBERON:0002107 25818 25823 liver +T701 PR:000010717 25848 25852 Mtf1 +T702 SO:0000346 25852 25856 loxP +T703 PR:000010717 25873 25877 Mtf1 +T704 PR:000010788 25877 25879 Mx +T705 NCBITaxon:10088 25884 25889 mouse +T706 PR:000010717 25965 25970 MTF-1 +T707 SO:0000704 25983 25988 genes +T708 SO:0000704 26049 26054 genes +T709 PR:000010717 26080 26085 MTF-1 +T710 NCBITaxon:10088 26153 26157 mice +T711 NCBITaxon:10088 26192 26196 mice +T712 SO:0000704 26378 26383 genes +T713 SO:0000704 26448 26453 genes +T714 SO:0000704 26525 26530 genes +T715 GO:0006749 26547 26560;26577 26588 metabolism of ... glutathione +T716 CHEBI:16856 26577 26588 glutathione +T717 SO:0000704 26650 26655 genes +T718 PR:000007897 26669 26715 catalytic subunit of glutamate-cysteine ligase +T719 PR:000007897 26717 26721 Gclc +T720 GO:0006750 26767 26791 synthesis of glutathione +T721 CHEBI:16856 26780 26791 glutathione +T722 CHEBI:16856 26798 26809 glutathione +T723 PR:000008292 26798 26821 glutathione reductase 1 +T724 MOP:0000569 26833 26841 reducing +T725 MOP:0000568 26853 26861 oxidized +T726 CHEBI:17858 26853 26873 oxidized glutathione +T727 CHEBI:16856 26884 26895 glutathione +T728 PR:000030257 26884 26915 glutathione-S-transferase, mu 4 +T729 PR:000030257 26917 26922 Gstm4 +T730 CHEBI:16856 26950 26961 glutathione +T731 GO:0098754 26996 27010 detoxification +T732 PR:000007897 27118 27122 Gclc +T733 PR:000007897 27144 27200 heavy chain subunit of gamma-glutamylcysteine synthetase +T734 CHEBI:24195 27167 27189 gamma-glutamylcysteine +T735 PR:000007897 27202 27209 Ggcs-hc +T736 SO:0000704 27254 27258 gene +T737 PR:000010717 27262 27267 MTF-1 +T738 GO:0010467 27277 27287 expression +T739 PR:000007897 27307 27311 Gclc +T740 UBERON:0007023 27355 27360 adult +T741 NCBITaxon:10088 27361 27366 mouse +T742 UBERON:0002107 27367 27372 liver +T743 PR:000010717 27391 27396 MTF-1 +T744 CHEBI:16856 27426 27437 glutathione +T745 GO:0051716 27452 27460;27469 27477 cellular ... response +T746 NCBITaxon:10088 27479 27484 mouse +T747 UBERON:0000922 27485 27494 embryonic +T748 CL:0000057 27495 27506 fibroblasts +T749 PR:000010717 27535 27539 Mtf1 +T750 CHEBI:28714 27586 27589 BSO +T751 CHEBI:75599 27602 27640 inhibitor of glutamate-cysteine ligase +T752 CHEBI:78021 27716 27727 tetrazolium +T753 CHEBI:24866 27728 27732 salt +T754 CHEBI:53233 27733 27736 MTT +T755 CHEBI:28714 27778 27781 BSO +T756 CHEBI:28714 27878 27881 BSO +T757 PR:000010717 27974 27978 Mtf1 +T758 CHEBI:16856 28011 28022 glutathione +T759 PR:000010717 28047 28051 Mtf1 +T760 GO:0006952 28085 28092 defense +T761 CHEBI:16856 28109 28120 glutathione +T762 PR:000010717 28139 28144 MTF-1 +T763 SO:0000704 28160 28165 genes +T764 SO:0000704 28240 28245 genes +T765 CHEBI:16856 28261 28272 glutathione +T766 SO:0000704 28311 28316 genes +T767 SO:0000704 28367 28372 genes +T768 PR:000016871 28377 28400 thioredoxin reductase 1 +T769 PR:000016871 28402 28408 Txnrd1 +T770 MOP:0000569 28422 28430 reducing +T771 PR:000009274 28476 28531 KDEL endoplasmic reticulum protein retention receptor 2 +T772 GO:0005783 28481 28502 endoplasmic reticulum +T773 GO:0035437 28481 28520 endoplasmic reticulum protein retention +T774 PR:000009274 28533 28539 Kdelr2 +T775 GO:0005783 28558 28560 ER +T776 PR:000004621 28606 28634 Bcl2-associated athanogene 3 +T777 PR:000004621 28636 28640 Bag3 +T778 GO:0006915 28669 28678 apoptosis +T779 PR:000010717 28746 28750 Mtf1 +T780 UBERON:0002107 28758 28763 liver +T781 UBERON:0007023 28767 28772 adult +T782 CHEBI:76777 28774 28776 pI +T783 PR:000010717 28788 28792 Mtf1 +T784 PR:000010788 28792 28794 Mx +T785 NCBITaxon:10088 28799 28803 mice +T786 NCBITaxon:10088 28862 28866 mice +T787 PR:000010717 28912 28917 MTF-1 +T788 UBERON:0007023 28940 28945 adult +T789 UBERON:0002107 28946 28951 liver +T790 UBERON:0000922 28995 29004 embryonic +T791 GO:0009790 28995 29004;29011 29022 embryonic ... development +T792 UBERON:0002107 29005 29010 liver +T793 GO:0001889 29005 29022 liver development +T794 SO:0000704 29048 29052 gene +T795 GO:0010467 29048 29063 gene expression +T796 UBERON:0002107 29067 29073 livers +T797 PR:000010717 29102 29106 Mtf1 +T798 PR:000010788 29106 29108 Mx +T799 PR:000010717 29117 29121 Mtf1 +T800 SO:0000346 29121 29125 loxP +T801 NCBITaxon:10088 29126 29130 mice +T802 PR:000010717 29148 29153 MTF-1 +T803 SO:0000704 29161 29165 gene +T804 SO:0000673 29178 29189 Transcripts +T805 SO:0000704 29235 29240 genes +T806 PR:000010925 29241 29244 Mt1 +T807 PR:000010704 29249 29252 Mt2 +T808 PR:000010717 29308 29313 MTF-1 +T809 GO:0010467 29347 29357 expression +T810 SO:0000704 29416 29421 genes +T811 PR:000014662 29425 29430 Sepw1 +T812 PR:000014662 29464 29469 SEPW1 +T813 CHEBI:16856 29569 29580 glutathione +T814 GO:0010467 29620 29630 expression +T815 NCBITaxon:10088 29634 29639 mouse +T816 PR:000014662 29640 29645 Sepw1 +T817 CHEBI:16240 29673 29690 hydrogen peroxide +T818 CHEBI:16856 29739 29750 glutathione +T819 GO:0010467 29803 29813 expression +T820 SO:0000704 29828 29832 gene +T821 NCBITaxon:10114 29844 29847 rat +T822 PR:000014662 29848 29853 Sepw1 +T823 SO:0000167 29854 29862 promoter +T824 NCBITaxon:10114 29890 29893 rat +T825 CL:0000125 29894 29905 glial cells +T826 NCBITaxon:10114 30035 30038 rat +T827 PR:000014662 30039 30044 Sepw1 +T828 PR:000010717 30121 30126 MTF-1 +T829 GO:0010467 30162 30172 expression +T830 PR:000010717 30219 30224 MTF-1 +T831 GO:0010467 30252 30262 expression +T832 NCBITaxon:10088 30266 30271 mouse +T833 PR:000014662 30272 30277 Sepw1 +T834 PR:000011059 30346 30351 Ndrg1 +T835 PR:000010717 30373 30378 MTF-1 +T836 SO:0000704 30386 30390 gene +T837 PR:000011059 30401 30434 N-myc downstream regulated gene 1 +T838 GO:0065007 30418 30427 regulated +T839 SO:0000704 30428 30432 gene +T840 PR:000010812 30489 30494 N-myc +T841 GO:0010467 30509 30519 expression +T842 NCBITaxon:10088 30523 30528 mouse +T843 PR:000011059 30529 30534 Ndrg1 +T844 PR:000011059 30558 30563 Ndrg1 +T845 NCBITaxon:9606 30575 30580 human +T846 SO:0000855 30581 30589 ortholog +T847 CHEBI:50113 30664 30673 androgens +T848 CHEBI:36357 30682 30691 compounds +T849 GO:0010467 30763 30772 expressed +T850 NCBITaxon:9606 30776 30781 human +T851 http://purl.obolibrary.org/obo/MONDO_0004992 30782 30789 cancers +T852 UBERON:0000479 30798 30805 tissues +T853 UBERON:0002048 30815 30819 lung +T854 UBERON:0002107 30821 30826 liver +T855 UBERON:0000955 30828 30833 brain +T856 UBERON:0000310 30835 30841 breast +T857 UBERON:0002113 30843 30849 kidney +T858 PR:000011059 30874 30879 Ndrg1 +T859 NCBITaxon:9606 30899 30904 human +T860 SO:0000855 30905 30913 ortholog +T861 GO:0051716 31068 31072;31080 31088 cell ... response +T862 PR:000011059 31143 31148 Ndrg1 +T863 SO:0000704 31149 31153 gene +T864 GO:0010467 31149 31164 gene expression +T865 PR:000010717 31202 31207 MTF-1 +T866 PR:000005958 31264 31269 Csrp1 +T867 GO:0010467 31271 31281 expression +T868 PR:000010717 31329 31334 MTF-1 +T869 NCBITaxon:9606 31440 31445 human +T870 NCBITaxon:8782 31447 31452 avian +T871 NCBITaxon:9031 31457 31464 chicken +T872 PR:000005958 31465 31470 CSRP1 +T873 GO:0070161 31516 31532 adhesion plaques +T874 GO:0031941 31557 31574 filamentous actin +T875 GO:0070161 31599 31614 adhesion plaque +T876 PR:000018242 31623 31628 zyxin +T877 GO:0051764 31645 31664 actin-cross-linking +T878 GO:0005856 31750 31762 cytoskeletal +T879 CL:0000010 31794 31808 cultured cells +T880 GO:0044257 31846 31870;31879 31887 destruction of, cellular ... proteins +T881 GO:0015629 31896 31914 actin cytoskeleton +T882 NCBITaxon:10114 31957 31960 rat +T883 UBERON:0002113 31961 31967 kidney +T884 GO:0051017 32001 32015 actin-bundling +T885 PR:000017296 32024 32030 villin +T886 MOP:0000630 32098 32114 depolymerization +T887 GO:0007019 32098 32130 depolymerization of microtubules +T888 GO:0005874 32118 32130 microtubules +T889 PR:000005958 32151 32156 CSRP1 +T890 GO:0044430 32194 32215 cytoskeletal elements +T891 NCBITaxon:10088 32223 32228 mouse +T892 NCBITaxon:1 32276 32284 organism +T893 GO:0005856 32304 32316 cytoskeleton +T894 PR:000010717 32360 32365 MTF-1 +T895 GO:0010467 32391 32401 expression +T896 PR:000010717 32428 32433 MTF-1 +T897 PR:000015124 32467 32475 Slc39a10 +T898 GO:0010467 32522 32532 expression +T899 SO:0000704 32549 32554 genes +T900 PR:000010925 32560 32563 Mt1 +T901 PR:000010704 32565 32568 Mt2 +T902 PR:000015073 32574 32578 Znt1 +T903 PR:000015124 32587 32595 SLC39A10 +T904 NCBITaxon:10088 32609 32614 mouse +T905 CHEBI:24870 32670 32673 ion +T906 GO:0005622 32756 32769 intracellular +T907 GO:0005737 32770 32781 cytoplasmic +T908 CHEBI:24870 32788 32791 ion +T909 GO:0005576 32820 32833 extracellular +T910 GO:0006858 32820 32833;32852 32861 extracellular ... transport +T911 GO:0031982 32838 32847 vesicular +T912 GO:0016192 32838 32847;32852 32861 vesicular ... transport +T913 CHEBI:24870 32848 32851 ion +T914 GO:0006811 32848 32861 ion transport +T915 GO:0005737 32871 32880 cytoplasm +T916 PR:000015124 32994 33002 SLC39A10 +T917 PR:000010717 33139 33144 MTF-1 +T918 GO:0010467 33173 33183 expression +T919 NCBITaxon:10088 33211 33216 mouse +T920 PR:000015073 33217 33221 Znt1 +T921 SO:0000704 33222 33226 gene +T922 GO:0005622 33303 33316 intracellular +T923 GO:0005737 33317 33328 cytoplasmic +T924 GO:0005622 33378 33391 intracellular +T925 GO:0031982 33392 33400 vesicles +T926 GO:0019725 33500 33508;33514 33525 cellular ... homeostasis +T927 PR:000015124 33546 33554 SLC39A10 +T928 PR:000010717 33585 33590 MTF-1 +T929 GO:0065007 33597 33604 control +T930 GO:0010467 33605 33615 expression +T931 PR:000015073 33678 33682 Znt1 +T932 PR:000015124 33687 33695 Slc39a10 +T933 PR:000010717 33717 33722 MTF-1 +T934 PR:000015124 33778 33786 Slc39a10 +T935 SO:0000315 33888 33914 transcriptional start site +T936 SO:0000704 34023 34027 gene +T937 PR:P47043 34090 34125 zinc-responsive activator protein 1 +T938 PR:P47043 34127 34131 Zap1 +T939 SO:0000704 34148 34152 gene +T940 PR:Q12436 34154 34182 zinc-regulated transporter 2 +T941 GO:0065007 34159 34168 regulated +T942 PR:Q12436 34184 34188 ZRT2 +T943 PR:000015124 34223 34231 Slc39a10 +T944 GO:0010467 34232 34242 expression +T945 PR:000010717 34246 34251 MTF-1 +T946 SO:0000167 34300 34308 promoter +T947 PR:000010717 34333 34338 MTF-1 +T948 PR:000015124 34390 34398 Slc39a10 +T949 SO:0000673 34399 34410 transcripts +T950 SO:0000704 34493 34497 gene +T951 PR:000010717 34509 34514 MTF-1 +T952 NCBITaxon:10088 34520 34525 mouse +T953 UBERON:0000922 34526 34533 embryos +T954 PR:000010717 34550 34554 Mtf1 +T955 SO:0000704 34608 34613 genes +T956 PR:000003809 34635 34652 alpha-fetoprotein +T957 PR:000003809 34654 34657 Afp +T958 UBERON:0002107 34667 34672 liver +T959 SO:0000172 34703 34708 CCAAT +T960 PR:000005307 34703 34739 CCAAT/enhancer binding protein alpha +T961 SO:0000165 34709 34717 enhancer +T962 PR:000005307 34741 34746 Cebpa +T963 GO:0001889 34802 34815 hepatogenesis +T964 PR:000003809 34817 34820 Afp +T965 GO:0010467 34821 34831 expression +T966 GO:0007567 34849 34856 natally +T967 PR:000003918 34873 34880 albumin +T968 UBERON:0007023 34897 34902 adult +T969 PR:000010717 34903 34907 Mtf1 +T970 PR:000010788 34907 34909 Mx +T971 NCBITaxon:10088 34914 34918 mice +T972 PR:000010717 34927 34932 MTF-1 +T973 PR:000003809 34962 34965 Afp +T974 GO:0010467 34966 34976 expression +T975 PR:000005307 34978 34983 Cebpa +T976 GO:0010467 34987 34996 expressed +T977 UBERON:0007023 35004 35009 adult +T978 UBERON:0002107 35010 35015 liver +T979 UBERON:0000479 35036 35043 tissues +T980 GO:0010467 35106 35116 expression +T981 UBERON:0002107 35132 35138 livers +T982 UBERON:0007023 35144 35149 adult +T983 PR:000010717 35150 35154 Mtf1 +T984 SO:0000346 35154 35158 loxP +T985 PR:000010717 35163 35167 Mtf1 +T986 PR:000010788 35167 35169 Mx +T987 NCBITaxon:10088 35174 35178 mice +T988 PR:000010717 35208 35213 MTF-1 +T989 PR:000005307 35225 35230 Cebpa +T990 GO:0010467 35231 35241 expression +T991 UBERON:0000922 35254 35263 embryonal +T992 GO:0009790 35254 35275 embryonal development +T993 PR:000010717 35386 35391 MTF-1 +T994 SO:0000704 35498 35503 genes +T995 PR:000010717 35507 35512 MTF-1 +T996 UBERON:0007023 35520 35525 adult +T997 NCBITaxon:10088 35526 35531 mouse +T998 UBERON:0002107 35532 35537 liver +T999 PR:000014662 35554 35559 Sepw1 +T1000 PR:000010717 35561 35566 MTF-1 +T1001 GO:0010467 35597 35607 expression +T1002 NCBITaxon:10088 35630 35635 mouse +T1003 PR:000010717 35636 35641 MTF-1 +T1004 MOP:0000568 35645 35654 oxidative +T1005 PR:000010717 35685 35690 MTF-1 +T1006 GO:0010467 35726 35736 expression +T1007 PR:000011059 35740 35745 Ndrg1 +T1008 PR:000005958 35750 35755 Csrp1 +T1009 PR:000010717 35770 35775 MTF-1 +T1010 GO:0010467 35803 35813 expression +T1011 PR:000015124 35817 35825 Slc39a10 +T1012 SO:0000704 35884 35889 genes +T1013 PR:000010925 35895 35898 Mt1 +T1014 PR:000010704 35900 35903 Mt2 +T1015 PR:000015073 35907 35911 Znt1 +T1016 SO:0000704 36020 36024 gene +T1017 UBERON:0002107 36045 36050 liver +T1018 SO:0000704 36051 36055 gene +T1019 GO:0010467 36051 36066 gene expression +T1020 NCBITaxon:10088 36096 36100 mice +T1021 SO:0000704 36127 36132 genes +T1022 PR:000010717 36217 36222 MTF-1 +T1023 GO:1903409 36251 36288 production of reactive oxygen species +T1024 CHEBI:26523 36265 36288 reactive oxygen species +T1025 CL:0000010 36358 36372 cultured cells +T1026 NCBITaxon:33208 36376 36383 animals +T1027 MOP:0000569 36427 36434 reduced +T1028 CHEBI:16856 36427 36446 reduced glutathione +T1029 CHEBI:18059 36448 36453 lipid +T1030 MOP:0000568 36491 36500 Oxidative +T1031 GO:0019725 36542 36562 cellular homeostasis +T1032 GO:0010467 36593 36603 expression +T1033 SO:0000704 36607 36612 genes +T1034 GO:0006953 36622 36633 acute-phase +T1035 NCBITaxon:40674 36676 36683 mammals +T1036 UBERON:0002113 36720 36726 kidney +T1037 UBERON:0002107 36731 36736 liver +T1038 GO:0032991 36766 36773 complex +T1039 MOP:0000568 36876 36885 oxidative +T1040 CHEBI:16856 36934 36945 glutathione +T1041 GO:0006952 36980 36987 defense +T1042 CHEBI:16856 37019 37030 Glutathione +T1043 GO:0065003 37043 37052 complexes +T1044 CHEBI:26519 37080 37093 free radicals +T1045 CHEBI:26523 37104 37127 reactive oxygen species +T1046 CHEBI:16856 37202 37213 glutathione +T1047 MOP:0000568 37217 37225 oxidized +T1048 CHEBI:16856 37255 37266 glutathione +T1049 CHEBI:16856 37284 37295 glutathione +T1050 MOP:0000779 37323 37334 conjugation +T1051 CHEBI:16856 37363 37374 glutathione +T1052 PR:000007897 37421 37425 Gclc +T1053 PR:000030257 37435 37440 Gstm4 +T1054 CHEBI:16856 37468 37479 glutathione +T1055 GO:0051716 37487 37495;37504 37512 cellular ... response +T1056 NCBITaxon:10088 37577 37582 mouse +T1057 UBERON:0000922 37583 37592 embryonic +T1058 CL:0000057 37593 37604 fibroblasts +T1059 PR:000010717 37627 37631 Mtf1 +T1060 CHEBI:16856 37658 37669 glutathione +T1061 PR:000010717 37709 37714 MTF-1 +T1062 SO:0000704 37730 37735 genes +T1063 MOP:0000569 37747 37754 reduced +T1064 CHEBI:16856 37747 37766 reduced glutathione +T1065 GO:0006952 37797 37804 defense +T1066 GO:0006968 37869 37877;37891 37898 cellular ... defense +T1067 PR:000010717 37908 37913 MTF-1 +T1068 SO:0000704 37929 37934 genes +T1069 CHEBI:16856 37976 37987 glutathione +T1070 PR:000014662 38017 38022 Sepw1 +T1071 PR:000010717 38484 38488 Mtf1 +T1072 NCBITaxon:10088 38510 38514 mice +T1073 PR:000010788 38662 38664 Mx +T1074 NCBITaxon:10088 38669 38673 mice +T1075 PR:000010717 38999 39003 Mtf1 +T1076 UBERON:0007023 39007 39012 adult +T1077 NCBITaxon:10088 39013 39018 mouse +T1078 UBERON:0002107 39019 39024 liver +T1079 PR:000010717 39044 39048 Mtf1 +T1080 NCBITaxon:10088 39070 39074 mice +T1081 SO:0001023 39089 39095 allele +T1082 SO:0001023 39155 39161 allele +T1083 SO:0001644 39166 39182 targeting vector +T1084 CL:0002322 39186 39194 ES cells +T1085 CHEBI:7507 39211 39219 neomycin +T1086 SO:0005853 39220 39228 cassette +T1087 SO:0001023 39286 39292 allele +T1088 PR:000010717 39293 39297 Mtf1 +T1089 SO:0000346 39297 39301 loxP +T1090 SO:0000147 39340 39345 exons +T1091 PR:000010717 39355 39359 Mtf1 +T1092 SO:0000417 39439 39445 domain +T1093 SO:0000319 39474 39484 stop codon +T1094 SO:0000147 39497 39501 exon +T1095 SO:0000147 39505 39510 Exons +T1096 PR:000010717 39521 39525 Mtf1 +T1097 SO:0000346 39555 39565 loxP sites +T1098 SO:0005853 39607 39615 cassette +T1099 SO:0001644 39765 39781 targeting vector +T1100 UBERON:0002107 39806 39811 liver +T1101 CHEBI:76777 39821 39823 pI +T1102 PR:000010717 39840 39844 Mtf1 +T1103 PR:000010788 39844 39846 Mx +T1104 PR:000010717 39854 39858 Mtf1 +T1105 SO:0000346 39858 39862 loxP +T1106 NCBITaxon:10088 39863 39867 mice +T1107 SO:0000112 39878 39884 primer +T1108 SO:0000028 39917 39919 bp +T1109 SO:0000028 39928 39930 bp +T1110 SO:0000147 39970 39975 exons +T1111 UBERON:0002107 40013 40018 liver +T1112 CHEBI:76777 40040 40042 pI +T1113 PR:000010717 40059 40063 Mtf1 +T1114 PR:000010788 40063 40065 Mx +T1115 PR:000010717 40073 40077 Mtf1 +T1116 SO:0000346 40077 40081 loxP +T1117 NCBITaxon:10088 40082 40087 mouse +T1118 PR:000010717 40089 40094 MTF-1 +T1119 GO:0032993 40095 40114 protein–DNA complex +T1120 GO:0065004 40095 40124 protein–DNA complex formation +T1121 CHEBI:37972 40141 40144 32P +T1122 SO:0000993 40157 40166 consensus +T1123 PR:P04386 40280 40284 Gal4 +T1124 PR:000000133 40302 40305 Sp1 +T1125 CHEBI:37972 40322 40325 32P +T1126 PR:000000133 40334 40337 Sp1 +T1127 SO:0000993 40338 40347 consensus +T1128 PR:000014662 40411 40416 Sepw1 +T1129 GO:0010467 40423 40433 expression +T1130 PR:000010717 40445 40450 MTF-1 +T1131 PR:000014662 40485 40490 Sepw1 +T1132 UBERON:0002107 40508 40513 liver +T1133 CHEBI:76777 40523 40525 pI +T1134 PR:000010717 40542 40546 Mtf1 +T1135 PR:000010788 40546 40548 Mx +T1136 PR:000010717 40556 40560 Mtf1 +T1137 SO:0000346 40560 40564 loxP +T1138 NCBITaxon:10088 40565 40569 mice +T1139 NCBITaxon:33208 40575 40582 animals +T1140 CHEBI:50292 40677 40682 CdSO4 +T1141 PR:000025471 40814 40816 S1 +T1142 PR:000014662 40830 40835 Sepw1 +T1143 CHEBI:37972 40876 40879 32P +T1144 PR:000014662 40888 40893 Sepw1 +T1145 PR:000025471 40894 40896 S1 +T1146 CHEBI:37972 40906 40909 32P +T1147 PR:000025471 40918 40920 S1 +T1148 SO:0000993 40997 41006 consensus +T1149 SO:0000357 41044 41052 flanking +T1150 SO:0000028 41089 41091 bp +T1151 PR:000014662 41106 41111 Sepw1 +T1152 UBERON:0002107 41196 41201 liver +T1153 PR:000010717 41229 41233 Mtf1 +T1154 SO:0000346 41233 41237 loxP +T1155 CHEBI:76777 41243 41245 pI +T1156 PR:000010717 41263 41267 Mtf1 +T1157 PR:000010788 41267 41269 Mx +T1158 NCBITaxon:10088 41274 41279 mouse +T1159 PR:000010717 41300 41305 MTF-1 +T1160 GO:0032993 41306 41325 protein–DNA complex +T1161 GO:0065004 41306 41335 protein–DNA complex formation +T1162 CHEBI:37972 41352 41355 32P +T1163 PR:000014662 41364 41369 Sepw1 +T1164 PR:P04386 41504 41508 Gal4 +T1165 CHEBI:37972 41526 41529 32P +T1166 PR:000010717 41588 41593 MTF-1 +T1167 GO:0032991 41598 41605 complex +T1168 PR:000000133 41654 41657 Sp1 +T1169 CHEBI:37972 41663 41666 32P +T1170 PR:000000133 41675 41678 Sp1 +T1171 SO:0000993 41679 41688 consensus +T1172 PR:000011059 41778 41783 Ndrg1 +T1173 PR:000010717 41795 41800 MTF-1 +T1174 PR:000011059 41835 41840 Ndrg1 +T1175 UBERON:0002107 41858 41863 liver +T1176 CHEBI:76777 41873 41875 pI +T1177 PR:000010717 41892 41896 Mtf1 +T1178 PR:000010788 41896 41898 Mx +T1179 PR:000010717 41906 41910 Mtf1 +T1180 SO:0000346 41910 41914 loxP +T1181 NCBITaxon:10088 41915 41919 mice +T1182 NCBITaxon:33208 41925 41932 animals +T1183 CHEBI:50292 42027 42032 CdSO4 +T1184 SO:0000993 42173 42182 consensus +T1185 SO:0000357 42220 42228 flanking +T1186 SO:0000028 42265 42267 bp +T1187 PR:000011059 42282 42287 Ndrg1 +T1188 UBERON:0002107 42372 42377 liver +T1189 PR:000010717 42405 42409 Mtf1 +T1190 SO:0000346 42409 42413 loxP +T1191 CHEBI:76777 42419 42421 pI +T1192 PR:000010717 42439 42443 Mtf1 +T1193 PR:000010788 42443 42445 Mx +T1194 NCBITaxon:10088 42450 42455 mouse +T1195 PR:000010717 42476 42481 MTF-1 +T1196 GO:0032993 42482 42501 protein–DNA complex +T1197 GO:0065004 42482 42511 protein–DNA complex formation +T1198 CHEBI:37972 42528 42531 32P +T1199 PR:000011059 42540 42545 Ndrg1 +T1200 CHEBI:37972 42581 42584 32P +T1201 PR:P04386 42738 42742 Gal4 +T1202 CHEBI:37972 42760 42763 32P +T1203 PR:000010717 42822 42827 MTF-1 +T1204 GO:0032991 42832 42839 complex +T1205 PR:000000133 42841 42844 Sp1 +T1206 CHEBI:37972 42861 42864 32P +T1207 PR:000000133 42873 42876 Sp1 +T1208 SO:0000993 42877 42886 consensus +T1209 PR:000005958 42976 42981 Csrp1 +T1210 PR:000010717 42993 42998 MTF-1 +T1211 PR:000005958 43033 43038 Csrp1 +T1212 UBERON:0002107 43056 43061 liver +T1213 CHEBI:76777 43071 43073 pI +T1214 PR:000010717 43090 43094 Mtf1 +T1215 PR:000010788 43094 43096 Mx +T1216 PR:000010717 43104 43108 Mtf1 +T1217 SO:0000346 43108 43112 loxP +T1218 NCBITaxon:10088 43113 43117 mice +T1219 NCBITaxon:33208 43123 43130 animals +T1220 CHEBI:50292 43225 43230 CdSO4 +T1221 SO:0000993 43371 43380 consensus +T1222 SO:0000357 43418 43426 flanking +T1223 SO:0000028 43463 43465 bp +T1224 PR:000005958 43480 43485 Csrp1 +T1225 UBERON:0002107 43570 43575 liver +T1226 PR:000010717 43603 43607 Mtf1 +T1227 SO:0000346 43607 43611 loxP +T1228 CHEBI:76777 43617 43619 pI +T1229 PR:000010717 43637 43641 Mtf1 +T1230 PR:000010788 43641 43643 Mx +T1231 NCBITaxon:10088 43648 43653 mouse +T1232 PR:000010717 43674 43679 MTF-1 +T1233 GO:0032993 43680 43699 protein–DNA complex +T1234 GO:0065004 43680 43709 protein–DNA complex formation +T1235 CHEBI:37972 43726 43729 32P +T1236 PR:000005958 43738 43743 Csrp1 +T1237 PR:P04386 43890 43894 Gal4 +T1238 CHEBI:37972 43912 43915 32P +T1239 PR:000010717 43974 43979 MTF-1 +T1240 GO:0032991 43984 43991 complex +T1241 PR:000000133 43993 43996 Sp1 +T1242 CHEBI:37972 44013 44016 32P +T1243 PR:000000133 44025 44028 Sp1 +T1244 SO:0000993 44029 44038 consensus +T1245 PR:000010717 44108 44113 MTF-1 +T1246 GO:0010467 44130 44140 expression +T1247 PR:000015124 44144 44152 Slc39a10 +T1248 PR:000015124 44187 44195 Slc39a10 +T1249 UBERON:0002107 44213 44218 liver +T1250 CHEBI:76777 44228 44230 pI +T1251 PR:000010717 44247 44251 Mtf1 +T1252 PR:000010788 44251 44253 Mx +T1253 PR:000010717 44261 44265 Mtf1 +T1254 SO:0000346 44265 44269 loxP +T1255 NCBITaxon:10088 44270 44274 mice +T1256 NCBITaxon:33208 44280 44287 animals +T1257 CHEBI:50292 44382 44387 CdSO4 +T1258 SO:0000993 44528 44537 consensus +T1259 SO:0000357 44575 44583 flanking +T1260 SO:0000028 44620 44622 bp +T1261 PR:000015124 44637 44645 Slc39a10 +T1262 UBERON:0002107 44730 44735 liver +T1263 PR:000010717 44763 44767 Mtf1 +T1264 SO:0000346 44767 44771 loxP +T1265 CHEBI:76777 44777 44779 pI +T1266 PR:000010717 44797 44801 Mtf1 +T1267 PR:000010788 44801 44803 Mx +T1268 NCBITaxon:10088 44808 44813 mouse +T1269 PR:000010717 44834 44839 MTF-1 +T1270 GO:0032993 44840 44859 protein–DNA complex +T1271 GO:0065004 44840 44869 protein–DNA complex formation +T1272 CHEBI:37972 44886 44889 32P +T1273 PR:000015124 44898 44906 Slc39a10 +T1274 PR:P04386 45041 45045 Gal4 +T1275 CHEBI:37972 45063 45066 32P +T1276 PR:000010717 45125 45130 MTF-1 +T1277 GO:0032991 45135 45142 complex +T1278 PR:000000133 45144 45147 Sp1 +T1279 CHEBI:37972 45164 45167 32P +T1280 PR:000000133 45176 45179 Sp1 +T1281 SO:0000993 45180 45189 consensus +T1282 MOP:0000569 45270 45277 reduced +T1283 CHEBI:16856 45270 45289 reduced glutathione +T1284 PR:000010717 45311 45316 MTF-1 +T1285 CHEBI:53233 45403 45406 MTT +T1286 NCBITaxon:10088 45414 45419 Mouse +T1287 UBERON:0000922 45420 45429 embryonic +T1288 CL:0000057 45430 45441 fibroblasts +T1289 PR:000010717 45505 45509 Mtf1 +T1290 CHEBI:28714 45593 45596 BSO +T1291 CHEBI:35456 45647 45652 CdCl2 +T1292 UBERON:0002107 45850 45855 liver +T1293 SO:0000704 45856 45860 gene +T1294 GO:0010467 45856 45871 gene expression +T1295 CHEBI:76777 45876 45878 pI +T1296 PR:000010717 45890 45894 Mtf1 +T1297 PR:000010788 45894 45896 Mx +T1298 PR:000010717 45905 45909 Mtf1 +T1299 SO:0000346 45909 45913 loxP +T1300 NCBITaxon:10088 45914 45918 mice +T1301 NCBITaxon:33208 45974 45981 animals +T1302 CHEBI:50292 46076 46081 CdSO4 +T1303 GO:0010467 46121 46131 expression +T1304 SO:0000704 46148 46152 gene +T1305 NCBITaxon:33208 46186 46193 animals +T1306 PR:000010717 46243 46247 Mtf1 +T1307 SO:0000346 46247 46251 loxP +T1308 NCBITaxon:33208 46318 46325 animals +T1309 SO:0000993 46439 46448 consensus +T1310 SO:0000028 46487 46489 bp +T1311 SO:0000028 46589 46591 bp +T1312 UBERON:0002107 46779 46784 liver +T1313 SO:0000704 46785 46789 gene +T1314 GO:0010467 46785 46800 gene expression +T1315 NCBITaxon:10088 46831 46835 mice +T1316 NCBITaxon:33208 46891 46898 animals +T1317 CHEBI:50292 46993 46998 CdSO4 +T1318 GO:0010467 47038 47048 expression +T1319 SO:0000704 47065 47069 gene +T1320 NCBITaxon:33208 47103 47110 animals +T1321 PR:000010717 47160 47164 Mtf1 +T1322 SO:0000346 47164 47168 loxP diff --git a/src/ontogpt/evaluation/craft/database/all/16221973.txt b/src/ontogpt/evaluation/craft/database/all/16221973.txt new file mode 100644 index 000000000..ae67791c4 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16221973.txt @@ -0,0 +1,201 @@ +Two major branches of anti-cadmium defense in the mouse: MTF-1/metallothioneins and glutathione + +Abstract + +Metal-responsive transcription factor 1 (MTF-1) regulates expression of its target genes in response to various stress conditions, notably heavy metal load, via binding to metal response elements (MREs) in the respective enhancer/promoter regions. Furthermore, it serves a vital function in embryonic liver development. However, targeted deletion of Mtf1 in the liver after birth is no longer lethal. For this study, Mtf1 conditional knockout mice and control littermates were both mock- or cadmium-treated and liver-specific transcription was analyzed. Besides the well-characterized metallothionein genes, several new MTF-1 target genes with MRE motifs in the promoter region emerged. MTF-1 is required for the basal expression of selenoprotein W, muscle 1 gene (Sepw1) that encodes a glutathione-binding and putative antioxidant protein, supporting a role of MTF-1 in the oxidative stress response. Furthermore, MTF-1 mediates the cadmium-induced expression of N-myc downstream regulated gene 1 (Ndrg1), which is induced by several stress conditions and is overexpressed in many cancers. MTF-1 is also involved in the cadmium response of cysteine- and glycine-rich protein 1 gene (Csrp1), which is implicated in cytoskeletal organization. In contrast, MTF-1 represses the basal expression of Slc39a10, a putative zinc transporter. In a pathway independent of MTF-1, cadmium also induced the transcription of genes involved in the synthesis and regeneration of glutathione, a cadmium-binding antioxidant. These data provide strong evidence for two major branches of cellular anti-cadmium defense, one via MTF-1 and its target genes, notably metallothioneins, the other via glutathione, with an apparent overlap in selenoprotein W. + +INTRODUCTION + +All organisms have evolved mechanisms to cope with a variety of stress situations. One type of stress response is triggered by heavy metals, such as zinc, copper and cadmium (for convenience, the terms zinc, copper and cadmium are also used here to denote Zn2+, Cu2+ and Cd2+, respectively). Metallothioneins (MTs), small, cysteine-rich proteins, play an important role in metal homeostasis and detoxification due to their ability to bind different heavy metal ions (1–3). In the mouse, there are four metallothionein genes, designated as Mt1 to Mt4. Basal, as well as heavy metal-induced, expression of Mt1 and Mt2 is mediated by metal-responsive transcription factor 1 (MTF-1) (4–7). This zinc finger protein recognizes short cis-acting DNA sequences, termed metal response elements (MREs; core consensus sequence TGCRCNC), which are present in the promoters of target genes (8,9). MTF-1 is conserved in evolution, and homologs have been characterized in the mouse (10), humans (11), Drosophila (12–14) and fish (15,16). + +The role of MTF-1 has been studied most extensively in the mouse. Besides coping with heavy metal load, MTF-1 can also mediate the induction of Mt genes in response to other stress situations, such as oxidative stress (5,17) and hypoxia (18). In addition, it is required for the metalloregulation of Znt1, encoding the major plasma membrane-localized zinc efflux transporter (19), the hypoxic/anoxic induction of the gene for placental growth factor (Plgf), an angiogenic protein of the vascular endothelial growth factor (VEGF) family (20), and has recently been invoked in tumor development (21,22). Furthermore, MTF-1 has an essential function during embryogenesis: targeted disruption of Mtf1 results in embryonic lethality around 14 days post coitum due to hepatocyte necrosis (23). In contrast, mice with null mutations for the stress-inducible metallothionein genes (Mt1 and Mt2) are viable, though sensitive to cadmium (24,25), indicating that additional important MTF-1 target genes are involved in the lethal phenotype. With the Cre-loxP conditional knockout technique, it is possible to circumvent the embryonic lethal phenotype of conventional Mtf1 knockout mice. Previous experiments with this technique revealed that deletion of Mtf1 from the liver after birth is no longer lethal under non-stress conditions (26). + +For this study, an inducible, liver-specific Mtf1 knockout mouse line was generated to perform a search for MTF-1 target genes and cadmium-inducible genes in the adult liver. A number of target gene candidates emerged upon a transcriptome analysis of mock- and cadmium-treated Mtf1 conditional knockout mice and control littermates and several of these were confirmed by semiquantitative RT–PCR. Besides the stress-inducible metallothionein genes that were already known as target genes of MTF-1, we find that MTF-1 is important for basal liver expression of the gene for selenoprotein W, muscle 1 (Sepw1) as well as for cadmium-induced expression of N-myc downstream regulated gene 1 (Ndrg1) and the gene encoding cysteine- and glycine-rich protein 1 (Csrp1). In addition, MTF-1 appears to repress the expression of solute carrier family 39, member 10 gene (Slc39a10), which encodes a putative metal ion transporter. In an MTF-1-independent transcriptome response, several genes involved in glutathione metabolism are induced. Further studies confirmed a dual anti-cadmium defense, one via glutathione and another one via MTF-1 and its target genes, including metallothioneins. + +MATERIALS AND METHODS + +Generation of Mtf1 conditional knockout mice and liver-specific deletion + +Mtf1 conditional knockout mice were generated in collaboration with Dr Michael Leviten (San Carlos, CA). Two genomic clones containing exons 3 to 6 of Mtf1 were used to construct a gene targeting vector for homologous recombination (Supplementary Data). A neomycin resistance cassette (PGK-neo) flanked by two loxP sites was cloned into the SacI site 5′ of exon 3 of Mtf1, the third loxP site was cloned into the ScaI site 3′ of exon 4. A thymidine kinase (TK) cassette was inserted in the HpaI site 3′ of exon 6. 129 ES cells were electroporated with the linearized targeting vector, selected in the presence of G418 and FIAU, and screened for correct integration events by PCR and Southern blot analysis. Transient expression of Cre recombinase led to removal of the PGK-neo cassette, and mice carrying the modified Mtf1loxP allele were generated by injection of positive clones into C57Bl/6 blastocysts and subsequent crosses. Homozygous conditional knockout animals (Mtf1loxP/loxP) were crossed with the Cre recombinase transgenic line Mx-cre (a gift from Prof. Michel Aguet) to obtain an inducible, liver-specific Mtf1 knockout line. The mice were genotyped by PCR using the following primers (Microsynth): Cre recombinase: 5′-CTATCCAGCAACATTTGGGCCAGC-3′; 5′-CCAGGTTACGGATATAGTTCATGAC-3′, Mtf1loxP or wild-type allele: 5′-CACACCCAGTTTGTGTATGTCTTC-3′; 5′-CAGTCACAAGCAAATTACCAAACACTGCC-3′. + +Animal treatment + +At 8 weeks of age, male Mtf1loxP/loxP mice harboring the Mx-cre transgene (Mtf1loxP/loxP Mx-cre, abbr.: Mtf1Mx-cre) and control littermates without transgene (Mtf1loxP/loxP, abbr.: Mtf1loxP) received four intraperitoneal injections each of 300 µg synthetic double-stranded RNA polyinosinic–polycytidylic acid [pI–pC; Sigma; in a volume of 60 µl phosphate-buffered saline (PBS)] at 3 day intervals. Only in the case of DNA-binding studies with MRE sequences from MTF-1 target gene candidates, the control mice received no pI–pC injections. For experiments with metal treatment, mice received 2 days after the last pI–pC treatment a subcutaneous (s.c.) injection of either 20 µmol/kg body weight CdSO4 (2 mM CdSO4 in H2O; cadmium treatment) or 10 ml/kg body weight H2O (mock treatment) 6 h before sacrificing them. + +Microarray analysis and data processing + +Total RNA was isolated from liver tissue of pI–pC-induced, mock- or cadmium-treated Mtf1Mx-cre and Mtf1loxP mice (n = 3 per genotype and respective treatment; all male) essentially as described by Chomczynski and Sacchi (27). + +Gene expression analysis was performed in the Functional Genomics Center Zurich using GeneChip® Mouse Genome 430 2.0 Arrays (Affymetrix) according to the manufacturer's instructions and the following reagents and conditions. cDNA was synthesized with SuperScript™ Double-Stranded cDNA Synthesis Kit (Invitrogen), using 15 µg total RNA. In vitro transcription was performed with BioArray™ High Yield™ RNA Transcript Labeling Kit (Enzo) and 3.5 to 6 µg of each cDNA. Clean-up of both cDNA and cRNA samples was done using GeneChip® Sample Clean-up Module (Affymetrix). For the automated washing and staining in the Affymetrix fluidics station 450, the protocol EukGE-Ws2v4_450 was used. The probe arrays were scanned with the Affymetrix GS 3000 scanner. Raw data are available at ArrayExpress (, accession number E-MEXP-438). + +Data analysis was performed with GeneSpring 6.1 software (Silicon Genetics), applying a significance level P ≤ 0.05. Furthermore, multiple testing correction was used in addition to obtain cadmium-responsive, MTF-1-independent genes. Genes were considered to be differentially expressed if there was at least a 2-fold difference in expression levels of the compared experimental groups (genotype and respective treatment). The result was considered reliable if signal values for the respective gene were scored ‘present’ at least for all mice in one experimental group or for two mice in each of two groups. + +To screen for the presence of the MRE core consensus sequence TGCRCNC in the promoter region, the upstream sequences of the respective genes were obtained from the University of California Santa Cruz (UCSC) Genome Browser Database (; October/November 2004) (28). + +RT–PCR + +All RT–PCRs were performed with QIAGEN® OneStep RT–PCR Kit (QIAGEN) according to the manufacturer's instructions, using 150–200 ng DNase I-digested total RNA (RNA isolation see microarray analysis). The reactions were carried out using the following primers: Csrp1: 5′-TTCCGATGTGCCAAGTGTGGC-3′; 5′-AGTAGAGAGTGGACATTCAGC-3′, hypoxanthin-guanin-phosphoribosyltransferase (Hprt): 5′-GCTGGTGAAAAGGACCTCTCG-3′; 5′-CCACAGGACTAGGACACCTGC-3′, Mtf1: 5′-GTGACTTTTGAGACTGTACTGAGTG-3′; 5′-CATGCCAAGAAACATTGAAGGTG-3′, Ndrg1: 5′-AGATACACAACAACGTGGAGG-3′; 5′-TGTGCGAGCGGCTTCGGGGGC-3′, Sepw1: 5′-TAGAGGCAGGGTCCTGAAAGC-3′; 5′-ACACCTGGAAACATGGCTGCC-3′, Slc39a10: 5′-GCTGTGGCTGGTAGTAAAAGC-3′; 5′-GTGGCATGGGATGTAAACAGC-3′. + +S1 nuclease mapping of transcripts (S1 analysis) + +S1 analysis was performed as previously described (29), using 100 µg DNase I-digested total RNA (RNA isolation see microarray analysis). The gels were developed using PhosphorImager (Molecular Dynamics). S1 analysis was done with the following 32P-labeled oligonucleotides: Hprt S1: 5′-TCTTCAGTCTGATAAAATCTACAGTCATAGGAATGGATCTATCACTATTTCTATTCAGTGATTACATTAAAG-3′, Sepw1 S1: 5′-TTCAACCGGGAACACCTGGAAACATGGCTGCCTGTCTTCTTGAAGTCTTGAGGTGGAAAGGGAAAGCAAAGCAGGAGGGTTTTCCCACCC-3′. + +Electrophoretic mobility shift assay (EMSA) + +Protein was extracted from liver tissue with T-PER™ Tissue Protein Extraction Reagent (Pierce) according to the manufacturer's instructions, using a 1:10 ratio of mouse tissue (mg) to T-PER reagent (µl). + +EMSA was essentially performed as previously described (10). All binding reactions were carried out by incubating 2–5 fmol 32P-end-labeled double-stranded oligonucleotides with 100 to 130 µg liver protein extract. For competition experiments, 5 pmol of unlabeled competitor oligonucleotide was added to the binding reaction before addition of the extract. All EMSA gels were developed using PhosphorImager (Molecular Dynamics). The following oligonucleotides were annealed and used for the reactions: Csrp1 MRE1: 5′-GGAAACAAAACGGCGCGCACTCCGGCGC-3′; 5′-GGCTGCGC CGGAGTGCGCGCCGTTTTGT-3′, Csrp1 MRE2: 5′-TGTTGTGGTGCAGTGTGCAAAGCCTAC-3′; 5′-ACCAGTAGGCTTTGCACACTGCACCAC-3′, Csrp1 MRE3: 5′-GAGATCGCCATAGGGTGCAAAGAGAAG-3′; 5′-GTGACTTCTCTTTGCACCCTATGGCGA-3′, Csrp1 MRE4: 5′-TGTCTTATTCTGGAGTGCAAGTTAGTC-3′; 5′-AGGGGACTAACTTGCACTCCAGAATAA-3′, Gal4: 5′-TCCGGAGGACTGTCCTCCGG-3′; 5′-GCCGGAGGACAGTCCTCCGG-3′, MREd [MRE derived from mouse Mt1 promoter (10)]: 5′-CGAGGGAGCTCTGCACTCCGCCCGAAAAGTG-3′; 5′-TCGACACTTTTCGGGCGGAGTGCAGAGCTCCCTCGAGCT-3′, MRE-s [synthetic MRE consensus sequence (10)]: 5′-CGAGGGAGCTCTGCACACGGCCCGAAAAGTG-3′; 5′-TCGACACTTTTCGGGCCGTGTGCAGAGCTCCCTCGAGCT-3′, Ndrg1 MRE1: 5′-CAGCCCAGGCAGGGTGCAGCACGAG-3′; 5′-CCGCCTCGTGCTGCACCCTGCCTGG-3′, Ndrg1 MRE2: 5′-CACACGTTCGCTGCACACGCCGCGG-3′; 5′-GGGACCGCGGCGTGTGCAGCGAACG-3′, Ndrg1 MRE3,4: 5′-GGAGTCCTTATGCACACGCGCACGAGCGCGCACGGGCAC-3′; 5′-TGGTGTGCCCGTGCGCGCTCGTGCGCGTGTGCATAAGGAC-3′, Sepw1 MRE1: 5′-GAGGCAGTCGGCTGTGCGCACGGCCCCACGCTC-3′; 5′-CTCTGAGCGTGGGGCCGTGCGCACAGCCGACTGC-3′, Sepw1 MRE2: 5′-ATGGTTTTGGGGGTGCGCAGGGGGTCTG-3′; 5′-CGACAGACCCCCTGCGCACCCCCAAAAC-3′, Slc39a10 MRE1: 5′-GAATACACGACTGGGTGCAGCCGGGGTTTGG-3′; 5′-GGTACCAAACCCCGGCTGCACCCAGTCGTGTA-3′, Slc39a10 MRE2: 5′-GCGGAGAGGAGATGCACACGGCACTCG-3′; 5′-CACTCGAGTGCCGTGTGCATCTCCTCT-3′, Specificity protein 1 (Sp1) binding sequence (10): 5′-CGAGGCCCCGCCCAG-3′; 5′-TCGACTGGGCGGGGCCTCGAGct-3′. + +Cell culture + +Primary embryonic fibroblasts were isolated from a 12.5 day old Mtf1loxP mouse embryo and grown in DMEM supplemented with 10% fetal bovine serum (ICN), 100 U/ml penicillin–streptomycin (Gibco BRL) and 2 mM l-glutamine (Gibco BRL). 100 mm plates with primary cells were transfected with 10 µg of an expression plasmid coding for simian virus 40 (SV40) large T antigen driven by the cytomegalovirus (CMV) promoter, using lipofectamine™ reagent (Invitrogen) according to the manufacturer's instructions. Cell foci were isolated and the immortalized mouse embryonic fibroblast cell line ckoC was derived from one of them. The Mtf1loxP genotype of this line as well as the genomic integration of the T antigen were confirmed by PCR. 100 mm plates with these cells were further transfected by the calcium phosphate method (30) with 19.6 µg of an expression plasmid for Cre recombinase driven by the CMV promoter and 0.4 µg of an expression plasmid for the neomycin resistance gene under the control of the TK promoter. Stably transfected cells were selected in the presence of 0.4 µg/µl G418 (Calbiochem), isolated clones of resistant cells were harvested and grown independently, and the expression of Cre recombinase and excision of exons 3 and 4 of Mtf1 were analyzed by RT–PCR. The cell lines delC19, delC21 and delC23 with a deletion of Mtf1 were chosen for further experiments. + +Cytotoxicity assay + +Samples of 1 × 104 cells/well were plated in 96-well tissue culture plates and allowed to adhere for 24 h. The cells were then pre-incubated for 24 h in medium containing 0, 5, 10, 25 or 50 µM l-buthionine-[S,R]-sulfoximine (BSO) (Sigma), a drug that inhibits glutathione synthesis (31). Later, cells were exposed to 0, 5, 10 or 20 µM CdCl2 in the specified pre-incubation medium for an additional 24 h. Cytotoxicity was determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromid)-based Cell Proliferation Kit I (Roche) according to the manufacturer's instructions. + +RESULTS + +Generation of an inducible, liver-specific Mtf1 knockout mouse line + +Using a homologous recombination strategy, mice were obtained with a modified Mtf1 allele Mtf1loxP where exons 3 and 4, encoding four of the six zinc fingers of the DNA-binding domain, are flanked by loxP sites (Figure 1a). Mice homozygous for the Mtf1loxP allele were further crossed with animals of the Cre recombinase transgenic line Mx-cre. Cre recombinase is expressed in this line under the control of the mouse Mx1 gene promoter, which is inducible by administration of interferon alpha or beta, or synthetic double-stranded RNA pI–pC (32). Cre-mediated deletion was reported to be complete in the liver, while varying in other tissues, ranging from 94% in spleen to 8% in brain (32). After Cre-mediated deletion of exons 3 and 4, which results in a frameshift and premature translation stop, no functional MTF-1 protein can be produced. + +For induction of Cre recombinase, Mtf1 conditional knockout mice harboring the Mx-cre transgene (Mtf1Mx-cre) received four intraperitoneal pI–pC injections at 3 day intervals (pI–pC induction); control littermates without transgene (Mtf1loxP) received similar injections. Using RT–PCRs (Figure 1b), a shortened product was obtained with RNA from Mtf1Mx-cre livers, indicating a successful excision of exons 3 and 4 of Mtf1 in these animals. On close examination, a very faint band similar in size to full-length signal was also observed in those mice, probably due to a low amount of residual full-length Mtf1 mRNA. The level of functional MTF-1 protein was examined by EMSA (Figure 1c): MTF-1 protein–DNA complex was detectable with liver protein extract from an Mtf1loxP control mouse, but no functional MRE-binding protein was observed with an Mtf1Mx-cre sample. Thus, deletion of exons 3 and 4 of Mtf1 in the liver of Mtf1Mx-cre mice was virtually complete. All examined liver-specific knockout mice were viable under laboratory conditions and appeared normal. + +MTF-1 target gene search + +For the identification of MTF-1 target genes, we compared the liver transcript profiles of mice with and without functional Mtf1 gene that had been mock-treated or exposed to cadmium (n = 3 per genotype and respective treatment). + +In a first screen, the transcripts were analyzed with a differential display-based method, called amplification of double-stranded cDNA end restriction fragments (ADDER) (33). Thereby an overwhelming number of signals was obtained for the two stress-inducible metallothioneins (Mt1 and Mt2), due to the abundance of their transcripts both in mock-treated and especially in cadmium-treated livers that harbored a functional MTF-1 gene (data not shown). This result confirmed the importance of MTF-1 for both basal and metal-induced expression of metallothionein genes. + +In a second approach, the gene expression profile in livers of the above mentioned mice was compared by Affymetrix GeneChip® Mouse Genome 430 2.0 Arrays (Table 1). When analyzing the probe array data of livers from mock-treated Mtf1Mx-cre and Mtf1loxP mice, an at least 2-fold, reliable downregulation of expression was detected in Mtf1Mx-cre livers for 13 Affymetrix GeneChip® probe sets corresponding to 11 characterized genes (Table 1, a). Seven of these genes contain one or more MRE core consensus sequence TGCRCNC within a segment of 1000 bp upstream of the transcription start. For 26 probe sets corresponding to 24 different characterized genes, a 2-fold or higher, reliable upregulation was detected in Mtf1Mx-cre livers (Table 1, b); 17 of these 24 genes contain MRE core consensus sequences in the upstream region. The data set for livers of cadmium-treated Mtf1Mx-cre and Mtf1loxP mice revealed an at least 2-fold, reliable downregulation in Mtf1Mx-cre livers for 21 probe sets corresponding to 16 different characterized genes (Table 1, c); 10 of these contain MRE core consensus sequences in their upstream region. For 9 probe sets corresponding to 9 different characterized genes, an at least 2-fold, reliable upregulation was detected (Table 1, d); five of them contain MRE motifs. In addition to characterized genes, ESTs and RIKEN cDNA sequences were also found in the comparison of Mtf1Mx-cre and Mtf1loxP livers to be differentially expressed (Supplementary Table 1). Downregulation of Mt1 and Mt2 was detected in Mtf1Mx-cre livers for both conditions (though the level of significance for the downregulation of Mt1 in mock-treated animals was above 0.05; data not shown). + +For all MTF-1 target genes characterized so far, such as Mt1, Mt2 and Znt1, MTF-1 exerts its transcriptional activation activity via standard MRE sequences located proximal to the transcription start (4,5,8,18,19). Even a specific search for MTF-1 binding sites by selection from a pool of double-stranded oligonucleotides with random sequences yielded no new binding motif for MTF-1 in addition to the known MREs (34). Thus, an MRE sequence is to date the only indication for a direct MTF-1 target gene, and four MRE-containing target gene candidates were further analyzed. + +Basal expression of Sepw1 depends on MTF-1 + +Sepw1 was found in microarray analysis to be significantly downregulated in livers from cadmium- and mock-treated Mtf1Mx-cre mice (Table 1, a and c). SEPW1 is a selenocysteine-containing protein that binds glutathione (35) and is thought to act as an antioxidant in vivo (36). + +Sepw1 expression in livers of pI–pC-induced, mock- or cadmium-treated Mtf1Mx-cre and Mtf1loxP mice was further analyzed by semiquantitative RT–PCRs and S1 analysis (Figure 2a and b). In accordance with microarray data a slight, if any, upregulation of Sepw1 transcription was observed in livers from Mtf1loxP mice upon cadmium treatment. The basal level was reduced in livers from mock- and cadmium-treated Mtf1Mx-cre mice, indicating that MTF-1 is important for the basal expression of Sepw1. + +Three MRE core consensus sequences were found in the region upstream of the mouse Sepw1 transcription start (Figure 2c). Two of them in opposite orientation overlap almost completely proximal to the transcription start (MRE1, −40 bp), the third one is located further upstream (MRE2, −527 bp). Specific binding of MTF-1 to Sepw1 MRE1 but not MRE2 oligonucleotide was observed in EMSA with liver protein extract from an Mtf1loxP control mouse (Figure 2d). As a control, no binding to MRE1 was detected with extract from a pI–pC-induced Mtf1Mx-cre mouse, confirming that the bandshift was indeed dependent on the presence of MTF-1. + +Cadmium response of Ndrg1 depends on MTF-1 + +Ndrg1 was significantly downregulated in microarrays of liver transcripts from cadmium-treated Mtf1Mx-cre mice compared to similarly treated Mtf1loxP control mice (Table 1, c). Ndrg1 probably has some role in stress response since various stimuli, including hypoxia and nickel compounds, activate expression of rodent Ndrg1 and/or its human ortholog (37–40). + +The Ndrg1 microarray results were confirmed with semiquantitative RT–PCRs (Figure 3a): for Mtf1loxP control livers, a clear increase of Ndrg1 expression was observed after cadmium exposure; in livers from Mtf1Mx-cre mice, this cadmium response was not detectable, while basal expression was similar to controls. This indicates that cadmium-induced expression of Ndrg1 depends on MTF-1. + +Five MRE core consensus sequences are located upstream of the mouse Ndrg1 transcription start (Figure 3b). Four of them are clustered (MRE1 to MRE4, −138 to −332 bp), the fifth one is located farther upstream (MRE5, −883 bp). EMSA was performed to test whether MTF-1 is interacting with some or all of the four proximal MRE sequences (Figure 3c). Separate oligonucleotides were tested for MRE1 and MRE2, whereas one oligonucleotide spanning both sequences was used for MRE3 and MRE4 (MRE3,4). No complex was seen with MRE1, but specific MTF-1 complexes were observed for both the MRE2 and MRE3,4 oligonucleotides with liver protein extract from an Mtf1loxP mouse. As expected, no bandshift was observed with protein extract from a mouse lacking MTF-1 (Mtf1Mx-cre). + +Cadmium response of Csrp1 depends on MTF-1 + +Csrp1 was found in microarray analyses to be significantly downregulated in cadmium-treated Mtf1Mx-cre mice compared to Mtf1loxP mice (Table 1, c). CSRP1 is a member of the evolutionary conserved CRP family of proteins that have been implicated in myogenesis and cytoskeletal remodeling (41,42). + +Semiquantitative RT–PCRs confirmed the microarray results, namely, that Csrp1 expression is elevated in Mtf1loxP livers upon cadmium exposure (Figure 4a). In contrast, no cadmium response was detectable in livers from Mtf1Mx-cre mice, suggesting that MTF-1 is required for cadmium induction of Csrp1. + +Three MRE core consensus sequences were found upstream of the Csrp1 transcription start (MRE2 to MRE4, −56 to −366 bp), one was found immediately downstream (MRE1, +7 bp; Figure 4b). Specific binding of MTF-1 was observed with EMSA for MRE2 oligonucleotide and protein extract from an Mtf1loxP liver, but not an Mtf1Mx-cre liver extract lacking MTF-1, confirming the participation of MTF-1 in the complex (Figure 4c). + +MTF-1 inhibits expression of Slc39a10 + +Slc39a10 was detected in microarray analysis to be significantly upregulated in livers from both mock- and cadmium-treated Mtf1Mx-cre mice compared to control animals (Table 1, b and d). SLC39 proteins are members of the Zrt- and Irt-like protein (ZIP) family of metal ion transporters that transport, with no known exception, metal ion substrates across cellular membranes into the cytoplasm (43,44). + +In accordance with microarray data, semiquantitative RT–PCRs showed a downregulation of Slc39a10 expression in livers of Mtf1loxP mice upon cadmium exposure. In samples from Mtf1Mx-cre mice, the basal expression was significantly increased; cadmium treatment still resulted in a decrease of Slc39a10 expression (Figure 5a). It cannot be judged by this experiment whether the degree of cadmium-induced reduction of Slc39a10 transcription was identical for Mtf1Mx-cre and Mtf1loxP mice or lower in the absence of MTF-1. In microarray analysis, the degree of the downregulation was either comparable to the one in control livers or lower, depending on the considered Affymetrix GeneChip® probe set (data not shown). The results indicate that MTF-1 is involved in repression of the basal expression of Slc39a10. It might also participate in the cadmium response of this gene, but it is apparently not exclusively responsible. + +One MRE core consensus sequence was found just upstream of the mouse Slc39a10 transcription start (MRE1, −21 bp), another one directly downstream (MRE2, +17 bp; Figure 5b). Specific binding of MTF-1 was observed in EMSA analysis for MRE2 with liver protein extract from an Mtf1loxP but not from an Mtf1Mx-cre mouse, while no binding was detected with MRE1 (Figure 5c). + +Cadmium-responsive, MTF-1-independent genes + +Finally, we also identified a number of cadmium-responsive genes that were independent of MTF-1 presence, by comparing the probe array data of all cadmium-treated mice with the data of all mock-treated mice, irrespective of the genotype (Table 2). An at least 2-fold, reliable upregulation was observed after cadmium exposure for 31 probe sets corresponding to 21 different characterized genes (Table 2, a). For 2 probe sets corresponding to 2 characterized genes, an at least 2-fold downregulation was detected (Table 2, b). + +Several genes involved in the metabolism of the antioxidant glutathione were found to be upregulated by cadmium exposure, namely the genes encoding the catalytic subunit of glutamate-cysteine ligase (Gclc) that is the rate limiting enzyme in de novo synthesis of glutathione (45); glutathione reductase 1 (Gsr), the reducing enzyme for oxidized glutathione (45); and glutathione-S-transferase, mu 4 (Gstm4), which is a member of the glutathione-S-transferase supergene family of detoxification enzymes (45). In all of these cases, induction was confirmed by semiquantitative RT–PCRs (data not shown). Gclc, also referred to as heavy chain subunit of gamma-glutamylcysteine synthetase (Ggcs-hc), had been discussed previously as a target gene of MTF-1 (6). Our expression data indicate that Gclc is induced by cadmium but, at least in the adult mouse liver, not dependent on MTF-1. + +To analyze the role of the glutathione system in the cellular cadmium response, mouse embryonic fibroblasts with and without functional Mtf1 were treated with cadmium in combination with BSO, a specific inhibitor of glutamate-cysteine ligase (31), and cell viability was assessed by a colorimetric assay based on the tetrazolium salt MTT (Figure 6). Increasing concentrations of BSO or cadmium alone were to some extent cytotoxic for the examined cell lines. Treatment with both BSO and cadmium resulted in an enhanced lethality particularly for the cells without functional Mtf1, indicating that a depletion of glutathione together with a lack of Mtf1 impair an efficient anti-cadmium defense. Thus, adequate glutathione supply as well as MTF-1 and its target genes are essential for the survival of the cell under cadmium stress. + +Besides genes related to the glutathione pathway, several other stress-related genes were upregulated upon cadmium exposure, including genes for thioredoxin reductase 1 (Txnrd1), one of the reducing enzymes of the antioxidant thioredoxin (46); KDEL endoplasmic reticulum protein retention receptor 2 (Kdelr2) participating in ER stress response (47); and the anti-apoptotic Bcl2-associated athanogene 3 (Bag3) involved in stress-induced apoptosis (48). + +DISCUSSION + +In this study, a virtually complete deletion of Mtf1 in the liver of adult, pI–pC-induced Mtf1Mx-cre mice did not detectably affect the phenotype of the respective mice under non-stress conditions, confirming that MTF-1 is dispensable in the adult liver (26), in contrast to its essential role in embryonic liver development (23). + +The comparison of gene expression in livers of mock- or cadmium-treated Mtf1Mx-cre and Mtf1loxP mice revealed several MTF-1 target gene candidates. Transcripts of the two stress-responsive metallothionein genes Mt1 and Mt2 were severely reduced, in support of a crucial role of MTF-1 for both basal and metal-induced expression of metallothioneins (4,6). + +One of the newly found target genes is Sepw1. The exact molecular function of SEPW1 protein is unknown to date, but a role as antioxidant has been proposed due to its ability to bind glutathione (35). In accordance with this, ectopic expression of mouse Sepw1 renders cells resistant to hydrogen peroxide, and this resistance is dependent on it binding glutathione (36). Furthermore, Amantana et al. (49) showed that expression of a reporter gene fused to a rat Sepw1 promoter fragment can be induced in rat glial cells by copper and zinc, but not cadmium. This response was dependent on an overlapping-inverted MRE sequence located proximal to the rat Sepw1 transcription start (49), even though initial studies failed to demonstrate MTF-1 binding to that sequence (50). Our expression and DNA-binding studies strongly suggest that MTF-1 is important for the basal expression of mouse Sepw1 by binding to the corresponding overlapping-inverted MRE sequence. + +Ndrg1, another interesting MTF-1 target gene was named N-myc downstream regulated gene 1 following the discovery that the transcription factor N-myc represses the expression of mouse Ndrg1 (51). Transcription of Ndrg1 and/or its human ortholog is induced by different physiological and cell stress conditions, such as androgens, nickel compounds, DNA damage and hypoxia (37–40,52,53). In addition, the protein is overexpressed in human cancers of many tissues, such as lung, liver, brain, breast, kidney and skin (40). Although Ndrg1 and especially its human ortholog have been quite intensely studied, its function remains unclear; however, the induction by stimuli like nickel and hypoxia suggests an involvement in the cell stress response. Such a role is strongly endorsed by our finding that Ndrg1 gene expression is also induced by cadmium, and that MTF-1 plays a crucial role in this induction. + +In the case of Csrp1, expression analyses and DNA-binding studies indicate that MTF-1 is required for cadmium induction by binding to an MRE upstream of the transcription start. Studies with human, avian and chicken CSRP1 have shown that this protein is localized at adhesion plaques and in association with filamentous actin, and interacts with the adhesion plaque protein zyxin, as well as the actin-cross-linking protein alpha-actinin (54–57). The ability to bind these partners suggests a role in cytoskeletal organization (58). Exposure of cultured cells to cadmium causes a decrease in, and destruction of, cellular contact proteins and the actin cytoskeleton (59). In the proximal tubule cells of the rat kidney, a partial loss of actin and the actin-bundling protein villin is observed upon cadmium treatment, as well as the derangement and depolymerization of microtubules (60). Assuming that CSRP1 is important for the organization of cytoskeletal elements in the mouse, its upregulation by cadmium might protect the organism from damage of the cytoskeleton. Such a mechanism would expand the role of MTF-1 in stress response. + +Our expression studies also suggest that MTF-1 represses basal transcription of Slc39a10, in contrast to its role as activator for the expression of other target genes like Mt1, Mt2, and Znt1 (4,19). SLC39A10 is one of 14 mouse SLC39 members, which belong to the ZIP family of metal ion transporters (43,44). All members of the ZIP family characterized so far increase intracellular cytoplasmic metal ion concentrations by promoting extracellular and vesicular ion transport into the cytoplasm. ZIP proteins have been reported to be transporters of zinc, iron, manganese and/or cadmium (44,61–63). Although SLC39A10 is largely uncharacterized (44), it is referred to in several databases as putative zinc transporter. It has been previously shown that MTF-1 is important for both basal expression and metal induction of the mouse Znt1 gene (19). ZnT proteins represent a different family of transporters that reduce intracellular cytoplasmic zinc by promoting zinc efflux from cells or into intracellular vesicles. Thus, members of the ZnT and ZIP family with zinc as predominant substrate have opposite roles in cellular zinc homeostasis (43). Assuming that SLC39A10 is indeed a zinc transporter, MTF-1 would control expression of two zinc transporters with antagonistic functions, namely, Znt1 and Slc39a10. Specific binding of MTF-1 was observed for an MRE located just downstream of the Slc39a10 transcription start. In a simple model, such a binding could interfere with the accessibility of the transcriptional start site for RNA polymerase II and/or general transcription factors, thus preventing transcription initiation of the gene. Indeed, such a mechanism has been described in yeast for the zinc-responsive activator protein 1 (Zap1) and its target gene, zinc-regulated transporter 2 (ZRT2) (64). However, the inhibition of Slc39a10 expression by MTF-1 may well be more complex than a competition for promoter binding. Independent of MTF-1, cadmium treatment also leads to downregulation of Slc39a10 transcripts, suggesting that some other factor is mediating this response. + +A previous target gene search for MTF-1 with mouse embryos of conventional Mtf1 knockout phenotype revealed, besides metallothionein genes, the multifunctional alpha-fetoprotein (Afp) and the liver-enriched transcription factor CCAAT/enhancer binding protein alpha (Cebpa) as prime candidates (65). After an early onset during hepatogenesis, Afp expression is repressed postnatally and replaced by albumin (66). Thus, our adult Mtf1Mx-cre mice lacking MTF-1 were not suitable to analyze Afp expression. Cebpa is expressed in the adult liver as well as in other tissues (67), but the present microarray data revealed no significant expression differences in livers from adult Mtf1loxP and Mtf1Mx-cre mice (data not shown). Therefore, MTF-1 may affect Cebpa expression only during embryonal development, perhaps in combination with as yet unidentified factors. + +The present study confirms and extends the role of MTF-1 as an important stress response regulator. We have identified and preliminarily characterized four target genes of MTF-1 in the adult mouse liver: in the case of Sepw1, MTF-1 is required to maintain basal expression, supporting a role of mouse MTF-1 in oxidative stress response. In addition, MTF-1 contributes to the cadmium-induced expression of Ndrg1 and Csrp1. Furthermore, MTF-1 helps to repress the basal expression of Slc39a10, in contrast to its role as transcriptional activator for genes like Mt1, Mt2 or Znt1. Thus the same transcription factor apparently serves as an activator or repressor, depending on the target gene. + +The comparison of liver gene expression of cadmium- and mock-treated mice also revealed a number of genes that were responsive to cadmium exposure, independent of the presence or absence of MTF-1. Evidence suggests that the production of reactive oxygen species is a major effect of acute cadmium toxicity (68,69), and exposure of cultured cells or animals to cadmium is associated with depletion of reduced glutathione, lipid peroxidation and DNA damage (70–73). Oxidative stress and the subsequent restoration of cellular homeostasis have been shown to induce the expression of genes encoding acute-phase proteins and antioxidant enzymes (74). In mammals, cadmium tends to accumulate in the kidney and liver as a cadmium-metallothionein complex that has an extremely slow turnover (75,76). Furthermore, metallothioneins provide protection against oxidative stress (1,17). In addition to metallothioneins, glutathione was postulated as a first line of defense against cadmium toxicity (77). Glutathione efficiently complexes cadmium (78) and scavenges free radicals and other reactive oxygen species directly, and indirectly via enzymatic reactions (45). In such reactions, glutathione is oxidized and has to be regenerated by glutathione reductase. Also, glutathione-S-transferases mediate the conjugation of various electrophiles to glutathione. The observed cadmium-induced upregulation of Gclc, Gsr and Gstm4 supports the importance of glutathione in the cellular cadmium response. The enhanced sensitivity to cadmium toxicity that we found for mouse embryonic fibroblasts upon a combination of Mtf1 deletion and depletion of glutathione further corroborates the importance of MTF-1 and its target genes as well as reduced glutathione for an efficient anti-cadmium defense. + +Our data provide strong evidence for at least two branches of cellular anti-cadmium defense, one via MTF-1 and its target genes, notably metallothioneins, the other via glutathione, with an apparent overlap in Sepw1. + +SUPPLEMENTARY DATA + +Supplementary data are available at NAR online. + +Supplementary Material + +[Supplementary Material] + +Acknowledgements + +We are indebted to the Functional Genomics Center Zurich (Zurich, CH) for financial as well as technical support (thanks especially to Andrea Patrignani, Dr Ulrich Wagner, and Dr Hubert Rehrauer for their assistance in data generation and evaluation), and to Dr Michael Leviten (San Carlos, CA) for the help in generating Mtf1 conditional knockout mice. We also thank Prof. Ueli Schibler (Geneva, CH) for his advice on the ADDER technique, Prof. Michel Aguet (Epalinges-Lausanne, CH) for the gift of Mx-Cre mice, Dr George Hausmann and Dr Michael Fetchko for critical reading of the manuscript, and Fritz Ochsenbein for preparing the figures. Funding to pay the Open Access publication charges for this article was provided by the Kanton Zurich. + +Conflict of interest statement. None declared. + +Figures and Tables + +Figure 1 + +Deletion of Mtf1 in adult mouse liver. (a) Generation of Mtf1 conditional knockout mice. The targeted allele was obtained by homologous recombination of wild-type (wt) allele and targeting vector in ES cells. Removal of the neomycin cassette (NEO) by Cre recombinase led to the conditional knockout allele Mtf1loxP. Conditional Cre-mediated deletion of exons 3 and 4 (Mtf1Δ) results in loss of function via loss of an essential part of the DNA-binding domain and the generation of a new stop codon right after exon 2. Exons 3 to 7 of Mtf1 are indicated by grey boxes, loxP sites by black triangles. TK, thymidine kinase cassette. Restriction enzymes: San, SanDI; B, BbvCI; S, SrfI; H, HpaI. The HpaI site indicated by the crossed H was lost during the cloning procedure for the targeting vector. (b) RT–PCRs with total liver RNA from pI–pC-induced male Mtf1Mx-cre or Mtf1loxP mice. The used primer pair results in products of 589 bp and 218 bp with full-length mRNA and mRNA without exons 3 and 4, respectively. (c) EMSA with liver protein extract of a pI–pC-induced male Mtf1Mx-cre or Mtf1loxP mouse. MTF-1 protein–DNA complex formation was tested with 32P-labeled MRE consensus oligonucleotide MRE-s. Specificity of binding was verified with excess of unlabeled competitor MREd or unrelated Gal4 oligonucleotide; Sp1 bandshifts with 32P-labeled Sp1 consensus oligonucleotide were included as a loading control. + +Figure 2 + +Sepw1 basal expression depends on MTF-1. (a) Semiquantitative RT–PCRs for Sepw1 mRNA using total liver RNA from pI–pC-induced male Mtf1Mx-cre or Mtf1loxP mice. The animals had obtained either mock s.c. injections (−Cd) or s.c. injections with 20 µmol/kg body weight CdSO4 (+Cd) 6 h before sacrificing them. RT–PCRs for Hprt mRNA were used as internal control to adjust the amount of total RNA used. (b) S1 analysis for Sepw1 mRNA with RNA described in (a), using a 32P-labeled Sepw1 S1 probe. A 32P-labeled S1 probe for Hprt mRNA was used to adjust the amount of RNA used. (c) MRE core consensus sequences TGCRCNC (bold letters) and flanking sequences found in a region of 1000 bp upstream from Sepw1 transcription start; the position of each core sequence is indicated. (d) EMSA with liver protein extracts of a male Mtf1loxP or a pI–pC-induced, male Mtf1Mx-cre mouse, both mock-treated. MTF-1 protein–DNA complex formation was tested with 32P-labeled Sepw1 MRE1 or MRE2 oligonucleotide, respectively. Specificity of binding was verified with excess of unlabeled competitor MREd or unrelated Gal4 oligonucleotide. 32P-labeled MRE-s was included to indicate the position of an MTF-1-DNA complex; bandshifts for the common transcription factor Sp1 with 32P-labeled Sp1 consensus oligonucleotide were obtained as protein loading control. + +Figure 3 + +Cadmium response of Ndrg1 depends on MTF-1. (a) Semiquantitative RT–PCRs for Ndrg1 mRNA using total liver RNA from pI–pC-induced male Mtf1Mx-cre or Mtf1loxP mice. The animals had obtained either mock s.c. injections (−Cd) or s.c. injections with 20 µmol/kg body weight CdSO4 (+Cd) 6 h before sacrificing them. RT–PCRs for Hprt mRNA were used as internal control to adjust the amount of total RNA used. (b) MRE core consensus sequences TGCRCNC (bold letters) and flanking sequences found in a region of 1000 bp upstream from Ndrg1 transcription start; the position of each core sequence is indicated. (c) EMSA with liver protein extracts of a male Mtf1loxP or a pI–pC-induced, male Mtf1Mx-cre mouse, both mock-treated. MTF-1 protein–DNA complex formation was tested with 32P-labeled Ndrg1 MRE1 or MRE2 oligonucleotide, or a 32P-labeled oligonucleotide including both MRE3 and MRE4 (MRE3,4). Specificity of binding was verified with excess of unlabeled competitor MREd or unrelated Gal4 oligonucleotide. 32P-labeled MRE-s was included to indicate the position of an MTF-1-DNA complex; Sp1 bandshifts with 32P-labeled Sp1 consensus oligonucleotide were obtained as protein loading control. + +Figure 4 + +Cadmium response of Csrp1 depends on MTF-1. (a) Semiquantitative RT–PCRs for Csrp1 mRNA using total liver RNA from pI–pC-induced male Mtf1Mx-cre or Mtf1loxP mice. The animals had obtained either mock s.c. injections (−Cd) or s.c. injections with 20 µmol/kg body weight CdSO4 (+Cd) 6 h before sacrificing them. RT–PCRs for Hprt mRNA were used as internal control to adjust the amount of total RNA used. (b) MRE core consensus sequences TGCRCNC (bold letters) and flanking sequences found in a region of 1000 bp upstream from Csrp1 transcription start; the position of each core sequence is indicated. (c) EMSA with liver protein extracts of a male Mtf1loxP or a pI–pC-induced, male Mtf1Mx-cre mouse, both mock-treated. MTF-1 protein–DNA complex formation was tested with 32P-labeled Csrp1 MRE1, MRE2, MRE3 or MRE4 oligonucleotide, respectively. Specificity of binding was verified with excess of unlabeled competitor MREd or unrelated Gal4 oligonucleotide. 32P-labeled MRE-s was included to indicate the position of an MTF-1–DNA complex; Sp1 bandshifts with 32P-labeled Sp1 consensus oligonucleotide were obtained as protein loading control. + +Figure 5 + +MTF-1 represses basal expression of Slc39a10. (a) Semiquantitative RT–PCRs for Slc39a10 mRNA using total liver RNA from pI–pC-induced male Mtf1Mx-cre or Mtf1loxP mice. The animals had obtained either mock s.c. injections (−Cd) or s.c. injections with 20 µmol/kg body weight CdSO4 (+Cd) 6 h before sacrificing them. RT–PCRs for Hprt mRNA were used as internal control to adjust the amount of total RNA used. (b) MRE core consensus sequences TGCRCNC (bold letters) and flanking sequences found in a region of 1000 bp upstream from Slc39a10 transcription start; the position of each core sequence is indicated. (c) EMSA with liver protein extracts of a male Mtf1loxP or a pI–pC-induced, male Mtf1Mx-cre mouse, both mock-treated. MTF-1 protein–DNA complex formation was tested with 32P-labeled Slc39a10 MRE1 or MRE2 oligonucleotide, respectively. Specificity of binding was verified with excess of unlabeled competitor MREd or unrelated Gal4 oligonucleotide. 32P-labeled MRE-s was included to indicate the position of an MTF-1–DNA complex; Sp1 bandshifts with 32P-labeled Sp1 consensus oligonucleotide were obtained as protein loading control. + +Figure 6 + +Cells with reduced glutathione level that also lack MTF-1 are hypersensitive to cadmium. The viability of cells was assessed with the so-called MTT assay. Mouse embryonic fibroblasts with (ckoC) and without (delC19, delC21 and delC23) functional Mtf1 were compared. Cells were pre-incubated in medium containing 0, 5, 10, 25 or 50 µM BSO for 24 h and further exposed to 0, 5, 10 or 20 µM CdCl2 (Cd) in the specified pre-incubation medium for an additional 24 h. Results are expressed as mean values ± SD (n = 3) normalized to the respective value of untreated cells. + +Table 1 + +Comparison of liver gene expression for pI–pC-induced Mtf1Mx-cre and Mtf1loxP mice (up- or downregulation at least 2-fold, P ≤ 0.05) + +The animals had obtained either mock s.c. injections (−Cd) or s.c. injections with 20 µmol/kg body weight CdSO4 (+Cd) 6 h before sacrificing them. The expression values for each gene are given as mean value of three animals per group, normalized to the mean value of group Mtf1loxP −Cd (relative activity). Grey shading indicates the two groups of animals compared, relative activities for the other two groups are shown for a complete overview. The number of MRE core consensus sequences TGCRCNC in a region of 1000 bp upstream from the annotated transcription start is indicated. + +aOnly incomplete region up to −1000 bp from transcription start is available in database. + +bMean values of two independent Affymetrix probe sets. + +cMean value of four independent Affymetrix probe sets. + +Table 2 + +Comparison of liver gene expression for cadmium- and mock-treated mice (up- or downregulation at least 2-fold, P ≤ 0.05) + +The animals had obtained either mock s.c. injections (−Cd) or s.c. injections with 20 µmol/kg body weight CdSO4 (+Cd) 6 h before sacrificing them. The expression values for each gene are given as mean value of three animals per group, normalized to the mean value of group Mtf1loxP −Cd (relative activity). + +aMean values of three independent Affymetrix probe sets. + +bMean value of six independent Affymetrix probe sets. + +cMean value of two independent Affymetrix probe sets. diff --git a/src/ontogpt/evaluation/craft/database/all/16255782.ann b/src/ontogpt/evaluation/craft/database/all/16255782.ann new file mode 100644 index 000000000..626e3930a --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16255782.ann @@ -0,0 +1,640 @@ +T1 PR:000005311 0 5 CEBPG +T2 GO:0006281 59 69 DNA repair +T3 SO:0000704 70 75 genes +T4 UBERON:0002031 86 106 bronchial epithelial +T5 CL:0002328 86 112 bronchial epithelial cells +T6 NCBITaxon:1 124 135 individuals +T7 http://purl.obolibrary.org/obo/MONDO_0002806 141 163 bronchogenic carcinoma +T8 http://purl.obolibrary.org/obo/MONDO_0002806 229 251 bronchogenic carcinoma +T9 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12036 12047 individuals +T270 PR:000008210 12094 12098 GPX1 +T271 PR:000005311 12152 12157 CEBPG +T272 PR:000017504 12162 12167 XRCC1 +T273 http://purl.obolibrary.org/obo/MONDO_0002806 12175 12177 BC +T274 NCBITaxon:1 12178 12189 individuals +T275 http://purl.obolibrary.org/obo/MONDO_0002806 12193 12195 BC +T276 NCBITaxon:1 12196 12207 individuals +T277 SO:0000704 12257 12262 genes +T278 PR:000005311 12272 12277 CEBPG +T279 PR:000017504 12283 12288 XRCC1 +T280 PR:000007167 12290 12295 ERCC5 +T281 PR:000029950 12297 12302 GSTP1 +T282 PR:000015399 12304 12308 SOD1 +T283 PR:000008210 12312 12316 GPX1 +T284 http://purl.obolibrary.org/obo/MONDO_0002806 12390 12392 BC +T285 NCBITaxon:1 12393 12404 individuals +T286 http://purl.obolibrary.org/obo/MONDO_0002806 12406 12408 BC +T287 NCBITaxon:1 12409 12420 individuals +T288 http://purl.obolibrary.org/obo/MONDO_0002806 12453 12455 BC +T289 NCBITaxon:1 12456 12467 individuals +T290 PR:000005311 12529 12534 CEBPG +T291 GO:0010467 12572 12582 expression +T292 PR:000017504 12586 12591 XRCC1 +T293 PR:000007167 12599 12604 ERCC5 +T294 PR:000029950 12612 12617 GSTP1 +T295 PR:000015399 12625 12629 SOD1 +T296 PR:000008210 12641 12645 GPX1 +T297 PR:000006852 12653 12657 E2F1 +T298 http://purl.obolibrary.org/obo/MONDO_0002806 12745 12747 BC +T299 NCBITaxon:1 12748 12759 individuals +T300 http://purl.obolibrary.org/obo/MONDO_0002806 12764 12766 BC +T301 NCBITaxon:1 12767 12778 individuals +T302 PR:000006852 12812 12816 E2F1 +T303 PR:000007167 12851 12856 ERCC5 +T304 PR:000029950 12858 12863 GSTP1 +T305 PR:000015399 12868 12872 SOD1 +T306 http://purl.obolibrary.org/obo/MONDO_0002806 12900 12902 BC +T307 NCBITaxon:1 12903 12914 individuals +T308 PR:000006852 12916 12920 E2F1 +T309 PR:000029950 12941 12946 GSTP1 +T310 http://purl.obolibrary.org/obo/MONDO_0002806 12990 12992 BC +T311 NCBITaxon:1 12993 13004 individuals +T312 http://purl.obolibrary.org/obo/MONDO_0002806 13057 13059 BC +T313 NCBITaxon:1 13060 13071 individuals +T314 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transcript +T339 PR:000005311 13924 13929 CEBPG +T340 SO:0000704 13953 13958 genes +T341 http://purl.obolibrary.org/obo/MONDO_0002806 13970 13972 BC +T342 NCBITaxon:1 13973 13984 individuals +T343 PR:000005311 13989 13994 CEBPG +T344 SO:0000673 13995 14005 transcript +T345 SO:0000673 14084 14094 transcript +T346 GO:0006281 14140 14150 DNA repair +T347 SO:0000704 14151 14156 genes +T348 http://purl.obolibrary.org/obo/MONDO_0002806 14164 14166 BC +T349 NCBITaxon:1 14167 14178 individuals +T350 PR:000005311 14225 14230 CEBPG +T351 SO:0000704 14241 14246 genes +T352 http://purl.obolibrary.org/obo/MONDO_0002806 14250 14252 BC +T353 NCBITaxon:1 14253 14264 individuals +T354 GO:0006281 14336 14346 DNA repair +T355 SO:0000704 14347 14352 genes +T356 PR:000005311 14369 14374 CEBPG +T357 http://purl.obolibrary.org/obo/MONDO_0002806 14382 14384 BC +T358 NCBITaxon:1 14385 14396 individuals +T359 GO:0065007 14400 14409 regulated +T360 PR:000005311 14413 14418 CEBPG +T361 PR:000005311 14464 14469 CEBPG +T362 SO:0000704 14600 14605 genes +T363 SO:0000704 14667 14672 genes +T364 PR:000005308 14678 14683 CEBPB +T365 SO:0000409 14709 14725 recognition site +T366 PR:000005311 14729 14734 CEBPG +T367 SO:0000409 14751 14767 recognition site +T368 GO:0006281 14802 14812 DNA repair +T369 SO:0000704 14813 14818 genes +T370 PR:000005311 14896 14901 CEBPG +T371 GO:0006281 14923 14933 DNA repair +T372 SO:0000704 14934 14939 genes +T373 http://purl.obolibrary.org/obo/MONDO_0002806 14947 14949 BC +T374 NCBITaxon:1 14950 14961 individuals +T375 PR:000005311 14987 14992 CEBPG +T376 GO:0006281 15035 15045 DNA repair +T377 SO:0000704 15046 15051 genes +T378 GO:0065007 15055 15064 regulated +T379 SO:0000409 15133 15149 recognition site +T380 PR:000005311 15315 15320 CEBPG +T381 GO:0006281 15340 15350 DNA repair +T382 SO:0000704 15351 15356 genes +T383 http://purl.obolibrary.org/obo/MONDO_0002806 15360 15362 BC +T384 NCBITaxon:1 15363 15374 individuals +T385 http://purl.obolibrary.org/obo/MONDO_0002806 15397 15399 BC +T386 NCBITaxon:1 15400 15410 individual +T387 http://purl.obolibrary.org/obo/MONDO_0002806 15415 15417 BC +T388 NCBITaxon:1 15418 15428 individual +T389 SO:0000673 15656 15666 transcript +T390 PR:000005311 15687 15692 CEBPG +T391 PR:000017504 15694 15699 XRCC1 +T392 PR:000007167 15701 15706 ERCC5 +T393 PR:000029950 15708 15713 GSTP1 +T394 PR:000015399 15715 15719 SOD1 +T395 PR:000008210 15724 15728 GPX1 +T396 http://purl.obolibrary.org/obo/MONDO_0002806 15964 15966 BC +T397 NCBITaxon:1 15967 15978 individuals +T398 http://purl.obolibrary.org/obo/MONDO_0002806 15995 15997 BC +T399 NCBITaxon:1 15998 16009 individuals +T400 http://purl.obolibrary.org/obo/MONDO_0002806 16039 16041 BC +T401 http://purl.obolibrary.org/obo/MONDO_0004992 16101 16107 cancer +T402 NCBITaxon:1 16197 16208 individuals +T403 http://purl.obolibrary.org/obo/MONDO_0002806 16274 16276 BC +T404 SO:0000673 16307 16317 transcript +T405 PR:000005311 16339 16344 CEBPG +T406 SO:0000673 16357 16367 transcript +T407 GO:0006281 16416 16426 DNA repair +T408 SO:0000704 16427 16432 genes +T409 http://purl.obolibrary.org/obo/MONDO_0002806 16497 16499 BC +T410 PR:000005311 16592 16597 CEBPG +T411 GO:0006281 16663 16673 DNA repair +T412 SO:0000704 16674 16679 genes +T413 http://purl.obolibrary.org/obo/MONDO_0002806 16694 16696 BC +T414 NCBITaxon:1 16697 16708 individuals +T415 http://purl.obolibrary.org/obo/MONDO_0002806 16731 16733 BC +T416 NCBITaxon:1 16734 16745 individuals +T417 PR:000005311 16903 16908 CEBPG +T418 GO:0065007 16952 16962 regulation +T419 GO:0006281 16985 16995 DNA repair +T420 SO:0000704 16996 17001 genes +T421 NCBITaxon:1 17034 17044 individual +T422 GO:0065007 17062 17072 regulation +T423 SO:0000704 17090 17095 genes +T424 PR:000005311 17099 17104 CEBPG +T425 NCBITaxon:1 17141 17152 individuals +T426 http://purl.obolibrary.org/obo/MONDO_0002806 17174 17176 BC +T427 GO:0065007 17255 17265 regulation +T428 GO:0006281 17307 17317 DNA repair +T429 SO:0000704 17318 17323 genes +T430 http://purl.obolibrary.org/obo/MONDO_0002806 17380 17382 BC +T431 NCBITaxon:1 17383 17394 individuals +T432 NCBITaxon:1 17398 17409 individuals +T433 PR:000005311 17443 17448 CEBPG +T434 GO:0006281 17493 17503 DNA repair +T435 SO:0000704 17504 17509 genes +T436 PR:000005311 17512 17517 CEBPG +T437 GO:0046982 17622 17643 heterodimer formation +T438 PR:000005311 17705 17710 CEBPG +T439 UBERON:0000479 17774 17781 tissues +T440 GO:0065007 17850 17859 regulated +T441 SO:0000704 17860 17864 gene +T442 PR:000005311 17866 17871 CEBPG +T443 PR:000001393 17915 17919 IL-6 +T444 PR:000001395 17924 17928 IL-8 +T445 SO:0000167 17929 17938 promoters +T446 CL:0000236 17942 17948 B cell +T447 PR:000005307 18014 18019 CEBPA +T448 PR:000005308 18024 18029 CEBPB +T449 CL:0000057 18033 18043 fibroblast +T450 CL:0000236 18048 18054 B cell +T451 PR:000005311 18082 18087 CEBPG +T452 NCBITaxon:10088 18097 18101 mice +T453 PR:000005311 18121 18126 CEBPG +T454 UBERON:0002048 18141 18146 lungs +T455 PR:000005311 18168 18173 CEBPG +T456 NCBITaxon:10088 18186 18190 mice +T457 GO:0007567 18206 18211 birth +T458 GO:0016265 18225 18228 die +T459 http://purl.obolibrary.org/obo/MONDO_0004849 18283 18296 emphysematous +T460 UBERON:0002048 18297 18302 lungs +T461 NCBITaxon:9606 18312 18318 humans +T462 http://purl.obolibrary.org/obo/MONDO_0004849 18329 18338 emphysema +T463 http://purl.obolibrary.org/obo/MONDO_0004849 18436 18445 emphysema +T464 http://purl.obolibrary.org/obo/MONDO_0002806 18459 18461 BC +T465 PR:000005311 18558 18563 CEBPG +T466 GO:0065007 18567 18577 regulating +T467 GO:0006281 18598 18608 DNA repair +T468 SO:0000704 18609 18614 genes +T469 PR:000005311 18659 18664 CEBPG +T470 SO:0000704 18676 18680 gene +T471 SO:0000673 18681 18691 transcript +T472 PR:000006852 18776 18780 E2F1 +T473 GO:0006281 18798 18808 DNA repair +T474 SO:0000704 18825 18830 genes +T475 PR:000005311 18875 18880 CEBPG +T476 PR:000006852 18890 18894 E2F1 +T477 http://purl.obolibrary.org/obo/MONDO_0002806 18932 18934 BC +T478 NCBITaxon:1 18935 18946 individuals +T479 http://purl.obolibrary.org/obo/MONDO_0002806 18958 18960 BC +T480 NCBITaxon:1 18961 18972 individuals +T481 PR:000006852 19004 19008 E2F1 +T482 GO:0006281 19014 19024 DNA repair +T483 SO:0000704 19041 19046 genes +T484 http://purl.obolibrary.org/obo/MONDO_0002806 19050 19052 BC +T485 NCBITaxon:1 19053 19064 individuals +T486 GO:0065007 19109 19119 controlled +T487 http://purl.obolibrary.org/obo/MONDO_0002806 19192 19194 BC +T488 PR:000006852 19196 19200 E2F1 +T489 GO:0065007 19233 19241 regulate +T490 GO:0006281 19259 19269 DNA repair +T491 SO:0000704 19277 19282 genes +T492 NCBITaxon:9606 19322 19327 human +T493 CL:0000057 19328 19339 fibroblasts +T494 NCBITaxon:10088 19344 19349 mouse +T495 UBERON:0007376 19350 19359 epidermal +T496 CL:0000362 19350 19365 epidermal cells +T497 GO:0006281 19420 19430 DNA repair +T498 SO:0000704 19431 19435 gene +T499 PR:000006852 19465 19469 E2F1 +T500 GO:0006281 19490 19500 DNA repair +T501 GO:0006260 19517 19528 replicating +T502 http://purl.obolibrary.org/obo/MONDO_0002806 19694 19696 BC +T503 SO:0000704 20012 20016 gene +T504 UBERON:0002031 20034 20054 bronchial epithelium +T505 CHEBI:50903 20060 20071 carcinogens +T506 CHEBI:63248 20073 20081 oxidants +T507 GO:0065007 20666 20675 regulated +T508 SO:0000704 20704 20709 genes +T509 http://purl.obolibrary.org/obo/MONDO_0000001 20788 20795 disease +T510 SO:0000704 20860 20865 genes +T511 SO:0000673 20955 20965 transcript +T512 SO:0000704 21062 21067 genes +T513 http://purl.obolibrary.org/obo/MONDO_0002806 21093 21095 BC +T514 GO:0065007 21158 21168 regulation +T515 PR:000017504 21172 21177 XRCC1 +T516 PR:000007167 21179 21184 ERCC5 +T517 PR:000029950 21186 21191 GSTP1 +T518 PR:000015399 21193 21197 SOD1 +T519 PR:000008210 21203 21207 GPX1 +T520 PR:000005311 21211 21216 CEBPG +T521 UBERON:0000178 21326 21331 blood +T522 UBERON:0001567 21335 21341 buccal +T523 GO:0065007 21398 21408 regulation +T524 GO:0006281 21460 21470 DNA repair +T525 SO:0000704 21471 21476 genes +T526 NCBITaxon:1 21527 21538 individuals +T527 http://purl.obolibrary.org/obo/MONDO_0002806 21551 21553 BC +T528 NCBITaxon:1 21591 21602 individuals +T529 http://purl.obolibrary.org/obo/MONDO_0002806 21615 21617 BC +T530 SO:0000704 21752 21756 gene +T531 CHEBI:35222 21766 21776 inhibitors +T532 SO:0000673 21885 21895 transcript +T533 SO:0000673 22044 22054 transcript +T534 SO:0000673 22161 22171 transcript +T535 SO:0000673 22210 22220 transcript +T536 SO:0000704 22275 22280 genes +T537 SO:0000673 22302 22312 transcript +T538 SO:0000673 22350 22360 transcript +T539 SO:0000704 22405 22410 genes +T540 GO:0065007 22423 22432 regulated +T541 http://purl.obolibrary.org/obo/MONDO_0002806 22498 22500 BC +T542 NCBITaxon:1 22501 22512 individuals +T543 PR:000005311 22514 22519 CEBPG +T544 GO:0065007 22520 22529 regulates +T545 GO:0006281 22566 22576 DNA repair +T546 SO:0000704 22577 22582 genes +T547 http://purl.obolibrary.org/obo/MONDO_0002806 22623 22625 BC +T548 PR:000005311 22627 22632 CEBPG +T549 GO:0065007 22633 22643 regulation +T550 GO:0006281 22705 22715 DNA repair +T551 SO:0000704 22716 22721 genes +T552 SO:0000704 22822 22826 Gene +T553 CHEBI:33893 22880 22888 reagents +T554 http://purl.obolibrary.org/obo/MONDO_0004992 23778 23784 Cancer +T555 http://purl.obolibrary.org/obo/MONDO_0004992 23855 23861 Cancer +T556 CHEBI:33893 23887 23895 reagents +T557 SO:0000704 23927 23932 genes +T558 SO:0000704 23961 23965 Gene +T559 SO:0000704 24000 24004 Gene +T560 PR:000017504 24263 24268 XRCC1 +T561 PR:000007167 24270 24275 ERCC5 +T562 PR:000029950 24277 24282 GSTP1 +T563 PR:000015399 24284 24288 SOD1 +T564 PR:000008210 24293 24297 GPX1 +T565 SO:0000704 24426 24431 genes +T566 PR:000005308 24437 24442 CEBPB +T567 PR:000005311 24448 24453 CEBPG +T568 PR:000006852 24459 24463 E2F1 +T569 PR:000006854 24469 24473 E2F3 +T570 PR:000006857 24479 24483 E2F6 +T571 PR:000035849 24489 24493 EVI1 +T572 PR:000005311 24571 24576 CEBPG +T573 http://purl.obolibrary.org/obo/MONDO_0002806 24638 24640 BC +T574 NCBITaxon:1 24641 24652 individuals +T575 http://purl.obolibrary.org/obo/MONDO_0002806 24657 24659 BC +T576 NCBITaxon:1 24660 24671 individuals +T577 SO:0000704 24730 24734 gene +T578 PR:000005311 24889 24894 CEBPG +T579 PR:000017504 24900 24905 XRCC1 +T580 PR:000005311 24914 24919 CEBPG +T581 PR:000017504 24920 24925 XRCC1 +T582 http://purl.obolibrary.org/obo/MONDO_0002806 24982 24984 BC +T583 NCBITaxon:1 24985 24996 individuals +T584 http://purl.obolibrary.org/obo/MONDO_0002806 25002 25004 BC +T585 NCBITaxon:1 25005 25016 individuals +T586 http://purl.obolibrary.org/obo/MONDO_0002806 25118 25140 bronchogenic carcinoma +T587 NCBITaxon:1 25141 25151 individual +T588 http://purl.obolibrary.org/obo/MONDO_0002806 25221 25243 Bronchogenic carcinoma +T589 NCBITaxon:1 25244 25254 individual +T590 http://purl.obolibrary.org/obo/MONDO_0005233 25257 25283 Non-small cell lung cancer +T591 UBERON:0002048 25272 25276 lung +T592 http://purl.obolibrary.org/obo/MONDO_0005096 25287 25305 Squamous carcinoma +T593 http://purl.obolibrary.org/obo/MONDO_0008433 25309 25331 Small cell lung cancer +T594 UBERON:0002048 25320 25324 lung +T595 http://purl.obolibrary.org/obo/MONDO_0004647 25335 25352 Carcinoma-in-situ +T596 http://purl.obolibrary.org/obo/MONDO_0004970 25356 25370 Adenocarcinoma +T597 http://purl.obolibrary.org/obo/MONDO_0002806 25373 25392 Bronchogenic Cancer +T598 http://purl.obolibrary.org/obo/MONDO_0004993 25443 25452 carcinoma +T599 SO:0000112 25482 25488 primer +T600 SO:0000673 25530 25540 transcript +T601 SO:0000673 25643 25653 transcript +T602 SO:0000673 25693 25703 transcript +T603 SO:0000704 25728 25732 gene +T604 CHEBI:36357 25749 25758 molecules +T605 PR:000003676 25763 25770 β-actin +T606 CHEBI:36357 25771 25780 molecules +T607 GO:0010467 25842 25852 Expression +T608 SO:0000673 25954 25964 transcript +T609 SO:0000673 26035 26045 transcript +T610 GO:0010467 26132 26142 Expression +T611 SO:0000673 26241 26251 transcript +T612 GO:0006281 26337 26347 DNA repair +T613 SO:0000704 26348 26353 genes +T614 PR:000006852 26407 26411 E2F1 +T615 NCBITaxon:1 26454 26465 individuals +T616 http://purl.obolibrary.org/obo/MONDO_0002806 26474 26476 BC +T617 NCBITaxon:1 26477 26488 individuals +T618 http://purl.obolibrary.org/obo/MONDO_0002806 26496 26498 BC +T619 NCBITaxon:1 26499 26510 individuals +T620 CHEBI:36357 26543 26552 molecules +T621 PR:000006852 26677 26681 E2F1 +T622 http://purl.obolibrary.org/obo/MONDO_0002806 26732 26734 BC +T623 NCBITaxon:1 26735 26745 individual +T624 http://purl.obolibrary.org/obo/MONDO_0002806 26754 26756 BC +T625 NCBITaxon:1 26757 26768 individuals +T626 http://purl.obolibrary.org/obo/MONDO_0002806 26776 26778 BC +T627 NCBITaxon:1 26779 26790 individuals +T628 SO:0000673 26847 26857 transcript +T629 GO:0006281 26904 26914 DNA repair +T630 SO:0000704 26915 26919 gene +T631 SO:0000673 27024 27034 transcript +T632 GO:0006281 27143 27153 DNA repair +T633 SO:0000704 27154 27159 genes +T634 GO:0010467 27234 27244 expression +T635 SO:0000704 27253 27257 gene +T636 GO:0006281 27590 27600 DNA repair +T637 SO:0000704 27601 27606 genes +T638 PR:000006852 27622 27626 E2F1 +T639 http://purl.obolibrary.org/obo/MONDO_0002806 27651 27653 BC +T640 NCBITaxon:1 27654 27665 individuals diff --git a/src/ontogpt/evaluation/craft/database/all/16255782.txt b/src/ontogpt/evaluation/craft/database/all/16255782.txt new file mode 100644 index 000000000..324cf2867 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16255782.txt @@ -0,0 +1,157 @@ +CEBPG transcription factor correlates with antioxidant and DNA repair genes in normal bronchial epithelial cells but not in individuals with bronchogenic carcinoma + +Abstract + +Background + +Cigarette smoking is the primary cause of bronchogenic carcinoma (BC), yet only 10–15% of heavy smokers develop BC and it is likely that this variation in risk is, in part, genetically determined. We previously reported a set of antioxidant genes for which transcript abundance was lower in normal bronchial epithelial cells (NBEC) of BC individuals compared to non-BC individuals. In unpublished studies of the same NBEC samples, transcript abundance values for several DNA repair genes were correlated with these antioxidant genes. From these data, we hypothesized that antioxidant and DNA repair genes are co-regulated by one or more transcription factors and that inter-individual variation in expression and/or function of one or more of these transcription factors is responsible for inter-individual variation in risk for BC. + +Methods + +The putative transcription factor recognition sites common to six of the antioxidant genes were identified through in silico DNA sequence analysis. The transcript abundance values of these transcription factors (n = 6) and an expanded group of antioxidant and DNA repair genes (n = 16) were measured simultaneously by quantitative PCR in NBEC of 24 non-BC and 25 BC individuals. + +Results + +CEBPG transcription factor was significantly (p < 0.01) correlated with eight of the antioxidant or DNA repair genes in non-BC individuals but not in BC individuals. In BC individuals the correlation with CEBPG was significantly (p < 0.01) lower than that of non-BC individuals for four of the genes (XRCC1, ERCC5, GSTP1, and SOD1) and the difference was nearly significant for GPX1. The only other transcription factor correlated with any of these five target genes in non-BC individuals was E2F1. E2F1 was correlated with GSTP1 among non-BC individuals, but in contrast to CEBPG, there was no significant difference in this correlation in non-BC individuals compared to BC individuals. + +Conclusion + +We conclude that CEBPG is the transcription factor primarily responsible for regulating transcription of key antioxidant and DNA repair genes in non-BC individuals. Further, we conclude that the heavy smokers selected for development of BC are those who have sub-optimal regulation of antioxidant and DNA repair genes by CEBPG. + +Background + +BC is currently the leading cause of cancer-related death in the United States, causing 28% of all cancer deaths [1]. Although cigarette smoking is the primary risk factor, only 10–15% of heavy smokers (greater than 20 pack years) develop BC [1-3]. Antioxidant and DNA repair enzymes that provide protection from the effects of cigarette smoke are expressed in the progenitor cells for BC, normal bronchial epithelial cells (NBEC) [1]. Inherited inter-individual variation in the function of these genes plays a role in determining risk for BC [4-6]. Antioxidant enzymes protect NBEC from reactive oxygen species produced by interaction with and metabolism of xenobiotics such as pollution and cigarette smoke [4-7] as well as those produced by normal cellular metabolism. Reactive oxygen species cause many damaging reactions including denaturation of proteins, cross-linking of lipids and proteins and modification of nucleic acid bases, which can lead to cancer [7]. DNA repair enzymes repair the frequent damage to DNA caused by oxidant stress as well as other stresses, including bulky adducts derived from carcinogens in cigarette smoke [8]. + +We previously reported that an interactive transcript abundance index comprising antioxidant genes was lower in NBEC of BC individuals compared to non-BC individuals, suggesting that BC individuals are selected on the basis of poor antioxidant protection [9]. In that study, there was a tendency towards correlation in transcript abundance between several pairs of antioxidant or DNA repair genes in non-BC individuals, but not in BC individuals. Gene pairs included in that observation were GSTP1/GPX1, CAT/GPX3, and GPX3/SOD1. + +Correlation is one typical characteristic of co-regulated genes. Another is shared transcription factor recognition sites in the regulatory regions of those genes [10]. Based on the above findings, it was hypothesized first, that there is inter-individual variation in regulation of key antioxidant and DNA repair genes by one or more transcription factors and second, that individuals with sub-optimal regulation are selected for development of BC if they smoke cigarettes. To test these hypotheses, transcription factor recognition sites common to the regulatory regions of the above correlated gene pairs were identified through in silico DNA sequence analysis, and their transcript abundance measured simultaneously with an expanded group of ten antioxidant and six DNA repair genes. + +Methods + +NBEC sample procurement + +Brush biopsy samples of normal bronchial epithelium were obtained for research studies at the time of diagnostic bronchoscopy according to previously described methods [9,11]. Normal bronchial epithelium in the lung not involved with cancer was brushed prior to biopsy of the suspected cancerous area. Samples were collected in a manner satisfying all requirements of the Institutional Review Board for the Medical University of Ohio. Each BC diagnosis and subtype identification was determined by histopathological examination in the Department of Pathology at the Medical University of Ohio. NBEC samples from a total of 49 individuals, including 24 non-BC individuals and 25 BC individuals, were evaluated in this study. The biographical characteristics of these individuals are presented in Table 1. + +Transcript abundance measurement + +Total RNA samples extracted from NBEC were reverse transcribed using M-MLV reverse transcriptase and oligo dT primers as previously described [9,11]. Standardized RT (StaRT)-PCR was used for transcript abundance measurement in these studies. With StaRT-PCR, an internal standard for each gene within a standardized mixture of internal standards (SMIS) is included in each PCR reaction. After amplification, products were electrophoresed on an Agilent 2100 Bioanalyzer using DNA Chips with DNA 1000 Kit reagents for visualization according to the manufacturer's protocol (Agilent Technologies Deutschland GmbH, Waldbronn, Germany). + +The StaRT-PCR technology is licensed to Gene Express, Inc. (Toledo, OH). Many of the reagents are available commercially and were obtained through Gene Express, Inc. for this study. StaRT-PCR reagents for each of the measured genes that were not commercially available, including primers and SMIS, were prepared according to previously described methods [11,12]. Sequence information for the primers is provided in Table 2. + +Including an internal standard within a SMIS in each measurement controls for all known sources of variation during PCR, including inhibitors in samples, and generates virtually-multiplexed transcript abundance data that are directly comparable across multiple experiments and institutions [13]. The performance characteristics of StaRT-PCR are superior to other forms of commercially available quantitative PCR technology in the areas critical to this study. With respect to these studies, the key property of a quantitative PCR method is not whether the PCR products are measured kinetically or at endpoint, but rather whether there are internal standards in each measurement or not. The overall performance characteristics of StaRT-PCR, including extensive validation of the method in independent laboratories have been presented in several recent articles and chapters [13-15]. With respect to the genes measured in this study, for each gene the StaRT-PCR reagents had lower detection threshold of less than 10 molecules, linear dynamic range of more than six orders of magnitude (less than 10 to over 107 molecules), and signal-to-analyte response of 100%. In addition, the presence of an internal standard controls for inter-sample variation in presence of PCR inhibitors (which often are gene-specific) and ensures no false negatives (if the PCR fails the internal standard PCR product is not observed and there are no data to report). False positives are eliminated through use of a control PCR reaction with no cDNA in it. + +Statistical analysis + +More than 6,000 transcript abundance measurements were conducted in multiple experiments over two years to assess the six transcription factors and sixteen antioxidant and DNA repair genes in NBEC samples from 49 individuals (24 non-BC individuals and 25 BC individuals). + +Correlation of each of the six transcription factors with each of the antioxidant or DNA repair genes was determined by Pearson's correlation following logarithmic transformation. The transformation was necessary due to the wide biological variation in expression of each gene among the individuals. Significance level was defined as p < 0.01 following Bonferroni adjustment for multiple comparison, specifically comparison of each of six transcription factors to each of the antioxidant or DNA repair genes. Comparison for significant differences between pairs of correlation coefficients was done by Fisher's Z-transformation test [16]. + +Analysis of the relationship between virtually-multiplexed transcript abundance data for each gene with age was assessed by Pearson's correlation, with gender by t-test, and with smoking history by ANOVA followed by Duncan's test. + +Transcription factor recognition site analysis + +The El Dorado (Build 35) program from the Genomatix software package was used to locate the correlated genes within the genome and define 1101 base pairs of the promoter regions (1000 base pairs upstream of and 100 base pairs into the transcription start site) for each gene (Genomatix Software GmbH, Munich, Germany, [17]). The 1101 base pair sequences obtained from the El Dorado program then were used as the target sequences for putative transcription factor recognition site identification using the MatInspector Version 4.2 program, which yielded sites for 11 transcription factors (Genomatix Software GmbH, Munich, Germany, [17]). The parameters used were the standard (0.75) core similarity and the optimized matrix similarity [18]. StaRT-PCR reagents were optimized for ten of these transcription factors, including CEBPB, CEBPE, CEBPG, E2F1, E2F3, E2F4, E2F5, E2F6, EVI1, and PAX5. Four transcription factors were expressed at low and invariant levels among multiple NBEC samples and were therefore excluded from the study. The remaining six, CEBPB, CEBPG, E2F1, E2F3, E2F6, and EVI, were evaluated for correlation with an expanded group of ten antioxidant and six DNA repair genes. + +Results + +Virtually-multiplexed transcript abundance data were obtained for each gene in each of the 49 samples, except for E2F1 measurement in sample 147 (Table 3). A gene-specific inhibitor in sample 147 prevented amplification of E2F1. Neither the internal standard, nor the native cDNA PCR product was observed. The presence of gene-specific PCR inhibition was observable in some other samples as reduction in peak heights in internal standard PCR products relative to that expected for the number of internal standard molecules present at the beginning of the PCR reaction. However, in each such case, the PCR amplification was efficient enough to enable quantification. + +Bivariate analysis + +In non-BC individuals there was significant (p < 0.01) correlation between CEBPG and eight of the 16 antioxidant or DNA repair genes, specifically XRCC1, ERCC5, GSTP1, SOD1, GPX1, ERCC1, CAT and ERCC2 (Table 4). In contrast, in BC individuals samples CEBPG was not correlated with any of the antioxidant or DNA repair genes. These relationships were not observed with any of the other transcription factors studied. + +For XRCC1, ERCC5, GSTP1, and SOD1 the correlation with CEBPG was significantly lower in BC individuals compared to non-BC individuals and the difference was nearly significant for GPX1 (Fig. 1b). Scatter plots of the relationship between CEBPG and XRCC1 in non-BC individuals or BC individuals (Fig. 2a,b) are representative of the other four genes. Neither CEBPG, nor XRCC1, ERCC5, GSTP1, SOD1 or GPX1 was significantly correlated with age, gender, or smoking history in non-BC individuals, BC individuals, or the combined group. + +In non-BC individuals, based on the r2 values from Pearson's correlation analysis, CEBPG accounts for much of the variance in expression of XRCC1 (69%), ERCC5 (62%), GSTP1 (55%), SOD1 (44%), and GPX1 (52%). E2F1 accounts for some of the remaining variance. For example, when samples from all 49 non-BC individuals and BC individuals were assessed as a single group, E2F1 was significantly correlated with ERCC5, GSTP1 and SOD1 (Table 4). Further, in non-BC individuals, E2F1 was correlated with GSTP1 (Fig. 1c) and the correlation was lower in BC individuals. However, the difference in correlation between non-BC individuals and BC individuals was not significant. None of the other transcription factors were correlated with XRCC1, ERCC5, GSTP1, SOD1, or GPX1 (Fig. 1a,d,e,f). + +Comparison of gene expression with demographic characteristics + +E2F1 and GSTZ1 each were positively correlated with age. GSTM1-5 was the only gene with a difference in expression by gender. There was a difference in ERCC2 expression between former and never smokers. + +Discussion + +In this study, we tested two hypotheses. First, that there is inter-individual variation in regulation of key antioxidant and DNA repair genes by one or more transcription factors. Second, that individuals with sub-optimal regulation are selected for development of BC if they smoke cigarettes. + +These hypotheses are supported by the findings that a) there was large inter-individual variation in transcript levels of CEBPG and each of the target genes and in non-BC individuals, b) CEBPG transcript abundance values were significantly correlated by bivariate analysis with the transcript abundance values of four key antioxidant and DNA repair genes in non-BC individuals, and c) that there was no correlation between CEBPG and these genes in BC individuals. + +These results support the hypothesis that each of the antioxidant or DNA repair genes correlated with CEBPG in non-BC individuals is regulated by CEBPG. This is supported by the specificity of the CEBPG correlation. That is, there was lack of correlation between any of the other five transcription factors assessed and these target genes. Of particular note is the lack of correlation of the target genes with CEBPB, which binds to the same recognition site as CEBPG, and shares its recognition site within each of the antioxidant or DNA repair genes. However, there are alternative explanations for the observed correlation of CEBPG with antioxidant and DNA repair genes in non-BC individuals. One possibility is that CEBPG and each of the correlated antioxidant or DNA repair genes is regulated by a transcription factor that is as yet undiscovered, and/or has a recognition site that is not yet known and was not in the Genomatix software database. + +There also is more than one possible explanation for the observed lack of correlation between CEBPG and antioxidant or DNA repair genes in BC individuals. For example, the non-BC individual and BC individual groups are not perfectly matched with respect to age, gender or smoking history (Table 1) and each of these factors could contribute to the observed difference in correlation between groups. However, the lack of association of transcript abundance level for CEBPG, XRCC1, ERCC5, GSTP1, SOD1, or GPX1 with age, gender or smoking history argues against such an explanation. One way to examine this possibility is through additional, larger, more closely matched studies. Another possible explanation is that any differences in NBEC from BC individuals compared to non-BC individuals resulted from development of BC, instead of being a hereditary cause of increased risk for cancer. The best way to determine this will be to conduct a prospective study. In such a study, individuals matched for smoking history will be monitored for development of BC over time. The correlation of transcript abundance values for CEBPG relative to transcript abundance values for each of the antioxidant or DNA repair genes will be assessed. It is expected that the greatest incidence of BC will be among the heaviest smokers. Among the matched heaviest smokers, it is expected that CEBPG will be significantly correlated with each of the antioxidant or DNA repair genes among the non-BC individuals but not correlated in BC individuals. + +Thus, there are multiple possible explanations for the observed findings. However, based on the preponderance of data thus far available, we conclude that CEBPG is responsible for optimal transcriptional regulation of key antioxidant or DNA repair genes in NBEC and that there is inter-individual variation in the regulation of each of these genes by CEBPG. If this conclusion is correct, the individuals at greatest risk for BC will be those with the most extreme smoking history combined with sub-optimal regulation of the largest number of antioxidant and DNA repair genes. This, in turn, leads to increased representation among BC individuals of individuals with lack of correlation between CEBPG and each of the affected antioxidant and/or DNA repair genes. + +CEBPG is a truncated CEBP transcription factor [19] and possesses the sequences necessary for DNA binding and heterodimer formation, but lacks the sequences necessary for transactivation [20]. CEBPG forms heterodimers with other CEBP family members and in other tissues this leads to increased [21] or decreased [20] transcription of the regulated gene. CEBPG is known to have stimulatory effect on the IL-6 and IL-8 promoters in B cell lines [21], and can also act as a dominant negative regulator of CEBPA and CEBPB in fibroblast and B cell lines [20]. + +The data from CEBPG knockout mice support a role for CEBPG in protecting lungs from oxidant damage. CEBPG-/- knockout mice are healthy at birth but begin to die within 24 hours, and histological examination reveals emphysematous lungs [22]. In humans, risk for emphysema is associated with antioxidant capacity [23], and there is a strong correlation between risk for emphysema and risk for BC. + +However, it will be important to obtain direct experimental evidence in NBECs for the role of CEBPG in regulating the antioxidant and DNA repair genes included in this study. Correlation between CEBPG and target gene transcript levels may not be associated with correlation at the protein level. + +In this study, E2F1 correlation with DNA repair and antioxidant genes was less than the correlation observed with CEBPG, and the E2F1 correlation was observed in both non-BC individuals as well as BC individuals. The maintained correlation of E2F1 with DNA repair and antioxidant genes in BC individuals suggests that this function is more tightly controlled in the population and does not play a role in determination of risk for BC. E2F1 has previously been reported to regulate transcription of DNA repair enzyme genes in other cell types, including primary human fibroblasts and mouse epidermal cells [24,25]. Clearly this would have survival value since DNA repair gene up-regulation in response to E2F1 provides additional DNA repair when the DNA is replicating and is particularly vulnerable to damage. + +Epidemiologic assessment of the correlation between a particular variation in DNA sequence, or polymorphism, and risk for BC has been a dominant paradigm for many years. Thus far, these efforts have met with scant success [26]. A common limitation in design of such studies is that they involve assessment of a single polymorphism or occasionally, a few polymorphisms. Further, although the polymorphism assessed typically resides within a gene known to protect bronchial epithelium from carcinogens, oxidants, or DNA damage, the selection of the particular polymorphism for study is largely empiric, and not based on known functional properties. These are problems because multiple infrequent polymorphisms at different sites may all contribute to risk and unless the key polymorphisms can be identified through a functional test, a statistically valid assessment would require much larger study populations [27]. + +The findings of this study support a novel approach to identifying clinically useful biomarkers. According to the paradigm used in this study, a) a normal phenotype results from regulated transcription of a group of genes by one or more transcription factors, b) the corresponding risk-conferring or disease phenotype results from sub-optimal interaction among those same genes, and c) each phenotype is identifiable and distinguishable through virtually-multiplexed transcript abundance analysis. The data presented here support the utility of this paradigm in identifying genes associated with risk for BC. + +The next step will be to identify polymorphisms that affect regulation of XRCC1, ERCC5, GSTP1, SOD1, and GPX1 by CEBPG. Such polymorphisms should yield biomarkers suitable for more readily accessible samples, such as peripheral blood or buccal smears. A biomarker combining polymorphisms that affect regulation with those that affect function of antioxidant and DNA repair genes is likely to be the most accurate for identifying individuals at risk for BC. Biomarkers that accurately identify individuals at risk for BC will improve efficacy of chemoprevention and early detection clinical trials. + +The observed inter-sample variation in the presence of gene-specific inhibitors of PCR provides evidence supporting the need for inclusion of an internal standard in each quantitative PCR transcript abundance measurement. Including such internal standards in the form of standardized mixtures of internal standards improves the reproducibility of transcript abundance measurement and enables development of a standardized database comprising virtually-multiplexed transcript abundance data. Virtually-multiplexed transcript abundance data are highly suited to identification of genes that have correlated transcript abundance values. Correlation at the transcript abundance level is an important property of genes that are co-regulated at the transcription level. + +Conclusion + +We conclude that in non-BC individuals, CEBPG regulates transcription of key antioxidant or DNA repair genes in NBEC and that in smokers who develop BC, CEBPG regulation is sub-optimal for a sufficient number of antioxidant and/or DNA repair genes to cause increased risk. + +Competing interests + +JCW and ELC each have significant equity interest in Gene Express, Inc., which produces and markets StaRT-PCR™ reagents used in this study. + +Authors' contributions + +DNM participated in the design of the study, performed the TF identification, carried out TA measurements, coordinated and participated in the statistical analysis and drafted the manuscript. ELC contributed the preliminary data, participated in the design of the study and carried out TA measurements. SAK performed the statistical analysis for interpretation of data. DAH and YY consented patients and obtained the primary samples necessary for the study according to IRB regulations. JCW conceived of the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript. + +Pre-publication history + +The pre-publication history for this paper can be accessed here: + + + +Acknowledgements + +These studies and manuscript preparation were supported by grants from the National Cancer Institute (NCI), including CA85147 and CA 95806, and the George Isaac Cancer Research Fund. StaRT-PCR reagents for measurement of some of the genes were provided at no cost by Gene Express, Inc. Neither the NCI nor Gene Express, Inc. had any role in study design, collection, analysis, or interpretation of data, writing of the manuscript, or in the decision to submit the manuscript for publication. + +Figures and Tables + +Figure 1 + +Correlation of each transcription factor with XRCC1, ERCC5, GSTP1, SOD1, or GPX1. (a-f) Each panel presents the correlation coefficients (r values) for one transcription factor in relation to each of the five genes: (a) CEBPB, (b) CEBPG, (c) E2F1, (d) E2F3, (e) E2F6, (f) EVI1. The p value for each significant correlation is provided above the bar. For CEBPG, presented in panel b, the difference in r value between non-BC individuals and BC individuals was significant or nearly significant for each correlated gene, and the p value for each comparison is provided below the corresponding pair of bars. + +Figure 2 + +Scatter plot representation of bivariate correlation of CEBPG with XRCC1. (a, b) CEBPG/XRCC1 data from Figure 1b presented as scatter plots: (a) non-BC individuals, (b) BC individuals. + +Table 1 + +Demographic data of patients from whom the NBEC samples were obtained. + +1Pack years; 2Non-bronchogenic carcinoma individual; 3White; 4African-American; 5Non-smoker; 6Not available; 7Hispanic; 8Bronchogenic carcinoma individual; 9Non-small cell lung cancer; 10Squamous carcinoma; 11Small cell lung cancer; 12Carcinoma-in-situ; 13Adenocarcinoma;14Bronchogenic Cancer, histology not specified; 15Poorly differentiated carcinoma. + +Table 2 + +Sequence for each primer used for StaRT-PCR virtually-multiplexed transcript abundance measurement or for internal standard preparation (CT) [15]. + +Table 3 + +Virtually-multiplexed transcript abundance data. + +Virtually-multiplexed transcript abundance data for each gene (in the form of molecules/106 β-actin molecules) from all experiments were included in the same Standardized Expression Database (SED). These data are now directly comparable to previously published virtually-multiplexed transcript abundance data from this laboratory [15], or to Virtually-multiplexed transcript abundance data collected by others who use the NCI-funded (R24 CA 95806) Standardized Expression Measurement (SEM) Center. The data presented here represent more than 6,000 Virtually-multiplexed transcript abundance measurements conducted in multiple experiments. The sixteen antioxidant or DNA repair genes and each of the six transcription factors except for E2F1 were measured in each NBEC sample from 49 individuals (24 non-BC individuals and 25 BC individuals). + +1ND, Not detectable (When 60 molecules of Competitive Template (CT) were added to the reaction, CT was detected but native template was not.) + +2NA, Not available. E2F1 was measured in all of the samples except for one BC individual (24 non-BC individuals and 24 BC individuals). + +Table 4 + +Bivariate analysis of virtually-multiplexed transcript abundance data values for each antioxidant or DNA repair gene versus each transcription factor. + +The correlation of logarithmically transformed virtually-multiplexed transcript abundance data (Table 3) from each of the six transcription factors with each of the sixteen antioxidant or DNA repair genes was determined. The transformation was necessary due to the wide range of expression of each gene among the samples. The correlation coefficient (r value) and level of significance (p value) for each correlation are presented. Significance (p < 0.01) was determined using a two-tailed test following Bonferroni adjustment for six multiple comparisons (comparison of each of six transcription factors to each of the antioxidant or DNA repair genes). + +*values for E2F1 obtained in all but one BC individuals. diff --git a/src/ontogpt/evaluation/craft/database/all/16279840.ann b/src/ontogpt/evaluation/craft/database/all/16279840.ann new file mode 100644 index 000000000..60175b55a --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16279840.ann @@ -0,0 +1,1371 @@ +T1 http://purl.obolibrary.org/obo/MONDO_0019065 11 22 Amyloidosis +T2 GO:0034205 48 61 Aβ Production +T3 http://purl.obolibrary.org/obo/MONDO_0004975 87 104 Alzheimer Disease +T4 MOP:0000780 160 166 cleave +T5 PR:000004168 199 224 amyloid precursor protein +T6 PR:000004168 226 229 APP +T7 http://purl.obolibrary.org/obo/MONDO_0004975 318 335 Alzheimer disease +T8 GO:0034205 375 388 Aβ production +T9 UBERON:0000955 396 401 brain +T10 UBERON:0000479 528 534 tissue +T11 CHEBI:60425 550 557 amyloid +T12 NCBITaxon:33208 603 609 animal +T13 CHEBI:60425 620 627 amyloid +T14 NCBITaxon:10088 741 746 mouse +T15 SO:0000704 758 769 genetically +T16 GO:0034205 791 804 Aβ production +T17 CHEBI:35222 844 854 inhibitors +T18 NCBITaxon:10088 862 866 mice +T19 GO:0010467 871 878 express +T20 PR:000004168 886 889 APP +T21 SO:0000440 897 903 vector +T22 GO:0065007 916 925 regulated +T23 CHEBI:50845 929 940 doxycycline +T24 GO:0010467 978 988 expression +T25 PR:000004168 992 995 APP +T26 CHEBI:60425 1022 1029 amyloid +T27 CHEBI:50845 1054 1065 doxycycline +T28 PR:000004168 1101 1104 APP +T29 GO:0010467 1105 1115 expression +T30 GO:0034205 1148 1161 Aβ production +T31 NCBITaxon:10088 1195 1199 mice +T32 GO:0034205 1227 1239 Aβ synthesis +T33 CHEBI:60425 1288 1295 amyloid +T34 CHEBI:60425 1348 1355 amyloid +T35 NCBITaxon:10088 1460 1464 Mice +T36 PR:000004168 1474 1477 APP +T37 GO:0042983 1474 1487 APP synthesis +T38 CHEBI:60425 1580 1587 amyloid +T39 CHEBI:60425 1674 1681 amyloid +T40 NCBITaxon:10088 1704 1708 mice +T41 GO:0034205 1813 1826 Aβ production +T42 http://purl.obolibrary.org/obo/MONDO_0004975 1844 1861 Alzheimer disease +T43 CHEBI:60425 1967 1974 amyloid +T44 CHEBI:60425 2082 2089 amyloid +T45 http://purl.obolibrary.org/obo/MONDO_0004975 2239 2256 Alzheimer disease +T46 http://purl.obolibrary.org/obo/MONDO_0004975 2258 2260 AD +T47 SO:0000704 2311 2318 genetic +T48 http://purl.obolibrary.org/obo/MONDO_0004975 2347 2349 AD +T49 PR:000004168 2370 2395 amyloid precursor protein +T50 PR:000004168 2397 2400 APP +T51 PR:000013344 2433 2446 presenilins 1 +T52 PR:000013345 2433 2444;2451 2452 presenilins ... 2 +T53 GO:0032991 2509 2516 complex +T54 PR:000004168 2539 2542 APP +T55 GO:0034205 2589 2605 production of Aβ +T56 http://purl.obolibrary.org/obo/MONDO_0000001 2649 2656 disease +T57 http://purl.obolibrary.org/obo/MONDO_0001627 2771 2779 dementia +T58 GO:0042571 2821 2829 antibody +T59 NCBITaxon:9606 2900 2906 humans +T60 CHEBI:60425 2953 2960 amyloid +T61 CHEBI:60425 3143 3150 amyloid +T62 GO:0050890 3185 3194 cognitive +T63 GO:0042571 3270 3278 antibody +T64 GO:0042571 3341 3351 antibodies +T65 UBERON:0000955 3401 3406 brain +T66 http://purl.obolibrary.org/obo/MONDO_0004975 3555 3557 AD +T67 PR:000004168 3637 3640 APP +T68 PR:000004168 3670 3673 APP +T69 PR:000004615 3708 3731 β-APP cleaving enzyme 1 +T70 MOP:0000780 3714 3722 cleaving +T71 PR:000004615 3733 3738 BACE1 +T72 GO:0070765 3744 3755 γ-secretase +T73 MOP:0000780 3763 3769 cleave +T74 CHEBI:33837 3774 3785 holoprotein +T75 CHEBI:35222 3839 3849 inhibitors +T76 GO:0070765 3853 3864 γ-secretase +T77 CHEBI:36357 3909 3917 molecule +T78 CHEBI:35222 3918 3928 inhibitors +T79 PR:000004615 3932 3955 β-APP cleaving enzyme 1 +T80 MOP:0000780 3938 3946 cleaving +T81 NCBITaxon:9606 4050 4056 humans +T82 PR:000004615 4124 4147 β-APP cleaving enzyme 1 +T83 MOP:0000780 4130 4138 cleaving +T84 NCBITaxon:10088 4210 4215 mouse +T85 http://purl.obolibrary.org/obo/MONDO_0004975 4227 4229 AD +T86 UBERON:0000955 4314 4319 brain +T87 CHEBI:60425 4343 4350 amyloid +T88 GO:0034205 4365 4381 production of Aβ +T89 CHEBI:60425 4417 4424 amyloid +T90 NCBITaxon:33208 4476 4482 animal +T91 CHEBI:33893 4503 4511 reagents +T92 CHEBI:35222 4568 4578 inhibitors +T93 NCBITaxon:9606 4600 4605 human +T94 NCBITaxon:10088 4665 4670 mouse +T95 http://purl.obolibrary.org/obo/MONDO_0004975 4680 4689 Alzheimer +T96 CHEBI:60425 4695 4702 amyloid +T97 GO:0010467 4708 4717 expresses +T98 GO:0065007 4720 4732 controllable +T99 PR:000004168 4733 4736 APP +T100 SO:0000902 4737 4746 transgene +T101 GO:0065007 4806 4815 regulated +T102 CHEBI:27902 4830 4842 tetracycline +T103 CHEBI:33290 4859 4863 food +T104 CHEBI:15377 4867 4872 water +T105 GO:0010467 4893 4903 expression +T106 SO:0000440 4937 4944 vectors +T107 GO:0010467 4987 4997 expression +T108 CHEBI:27902 5034 5046 tetracycline +T109 CHEBI:52217 5108 5122 pharmaceutical +T110 CHEBI:35222 5123 5132 inhibitor +T111 GO:0034205 5136 5149 Aβ production +T112 CHEBI:60425 5182 5189 amyloid +T113 SO:0000902 5292 5301 Transgene +T114 CHEBI:27902 5329 5341 tetracycline +T115 NCBITaxon:10088 5362 5367 mouse +T116 NCBITaxon:9606 5368 5373 human +T117 SO:0000440 5406 5412 vector +T118 SO:0000167 5430 5445 promoter region +T119 PR:P04925 5453 5458 moPrP +T120 SO:0000440 5464 5470 vector +T121 CHEBI:27902 5517 5529 tetracycline +T122 SO:0000167 5541 5549 promoter +T123 GO:0006266 5630 5638 ligating +T124 NCBITaxon:10088 5639 5644 mouse +T125 PR:000004168 5645 5648 APP +T126 SO:0000417 5669 5675 domain +T127 NCBITaxon:10088 5677 5679 mo +T128 SO:0000440 5708 5714 vector +T129 CHEBI:27902 5740 5752 tetracycline +T130 SO:0000167 5764 5772 promoter +T131 SO:0000028 5774 5776 bp +T132 SO:0000112 5813 5820 primers +T133 PR:P23940 5841 5846 BamHI +T134 PR:P23940 5882 5887 BamHI +T135 PR:P23940 5934 5939 BamHI +T136 SO:0000167 5957 5965 promoter +T137 SO:0001030 5967 5974 forward +T138 SO:0001031 6031 6038 reverse +T139 PR:P04925 6107 6112 moPrP +T140 SO:0000188 6118 6124 intron +T141 SO:0000112 6147 6154 primers +T142 PR:P23940 6178 6183 BamHI +T143 SO:0000147 6206 6210 exon +T144 SO:0000147 6248 6252 exon +T145 SO:0001030 6256 6263 forward +T146 SO:0001031 6305 6312 reverse +T147 SO:0000188 6362 6368 intron +T148 SO:0000440 6407 6413 vector +T149 GO:0006266 6502 6509 ligated +T150 PR:P04925 6543 6548 moPrP +T151 SO:0000147 6565 6569 exon +T152 SO:0000147 6573 6577 exon +T153 SO:0000205 6585 6591 3′ UTR +T154 SO:0000440 6637 6643 vector +T155 SO:0000147 6659 6664 exons +T156 SO:0000188 6679 6685 intron +T157 SO:0000167 6693 6701 promoter +T158 SO:0000440 6708 6714 vector +T159 PR:P23940 6738 6743 BamHI +T160 SO:0000188 6767 6773 intron +T161 SO:0000112 6782 6788 primer +T162 GO:0006266 6794 6801 ligated +T163 PR:P23940 6816 6821 BamHI +T164 MOP:0000780 6822 6825 cut +T165 CHEBI:27902 6826 6838 tetracycline +T166 SO:0000167 6839 6847 promoter +T167 GO:0006266 6863 6871 ligation +T168 SO:0000440 6891 6897 vector +T169 CHEBI:27902 6911 6923 tetracycline +T170 SO:0000167 6924 6932 promoter +T171 SO:0000147 6958 6963 exons +T172 SO:0000188 6969 6975 intron +T173 SO:0000205 6994 7000 3′ UTR +T174 PR:P04925 7008 7013 moPrP +T175 SO:0000440 7019 7025 vector +T176 SO:0000440 7066 7072 vector +T177 NCBITaxon:10088 7156 7158 mo +T178 SO:0000112 7204 7210 primer +T179 SO:0000112 7306 7313 primers +T180 SO:0001030 7357 7364 forward +T181 SO:0001031 7422 7429 reverse +T182 SO:0001030 7470 7477 forward +T183 SO:0001031 7515 7522 reverse +T184 SO:0000006 7533 7545 PCR products +T185 GO:0006266 7551 7558 ligated +T186 NCBITaxon:10088 7623 7625 mo +T187 SO:0000440 7641 7647 vector +T188 PR:P04925 7720 7725 moPrP +T189 SO:0000440 7731 7737 vector +T190 GO:0045120 7777 7787 Pronuclear +T191 PR:P04925 7856 7861 moPrP +T192 NCBITaxon:10088 7867 7869 mo +T193 SO:0000440 7886 7892 vector +T194 SO:0000440 7980 7986 vector +T195 GO:0045120 8009 8019 pronucleus +T196 GO:0009566 8023 8033 fertilized +T197 CL:0000025 8034 8038 eggs +T198 GO:0007618 8064 8071 matings +T199 NCBITaxon:33208 8081 8088 animals +T200 SO:0000902 8127 8136 transgene +T201 PR:000001886 8172 8175 PrP +T202 PR:000001886 8178 8181 PrP +T203 SO:0000112 8185 8192 primers +T204 SO:0000902 8210 8219 Transgene +T205 NCBITaxon:33208 8251 8258 animals +T206 GO:0010467 8259 8269 expressing +T207 CHEBI:27902 8274 8286 tetracycline +T208 GO:0065007 8314 8321 control +T209 PR:000003199 8329 8358 calcium-calmodulin kinase IIα +T210 PR:000003199 8360 8367 CaMKIIα +T211 SO:0000167 8369 8377 promoter +T212 PR:000003199 8476 8482 Camk2a +T213 PR:000004168 8570 8573 APP +T214 PR:000004168 8605 8608 APP +T215 NCBITaxon:10088 8620 8624 mice +T216 CHEBI:33290 8645 8649 food +T217 CHEBI:15377 8654 8659 water +T218 NCBITaxon:33208 8672 8678 Animal +T219 NCBITaxon:33208 8797 8803 Animal +T220 CHEBI:50845 8830 8841 Doxycycline +T221 CHEBI:50845 8858 8869 Doxycycline +T222 CHEBI:50845 8871 8874 dox +T223 CHEBI:50845 8924 8927 dox +T224 CHEBI:33290 8939 8943 chow +T225 CHEBI:33290 8998 9002 chow +T226 CHEBI:33282 9026 9036 antibiotic +T227 GO:0007631 9057 9068 consumption +T228 NCBITaxon:10088 9080 9085 mouse +T229 NCBITaxon:33208 9121 9127 animal +T230 CHEBI:50845 9137 9140 dox +T231 NCBITaxon:33208 9167 9173 animal +T232 CHEBI:50845 9202 9205 dox +T233 CHEBI:33290 9236 9240 Chow +T234 GO:0017001 9283 9295;9300 9310 breakdown of ... antibiotic +T235 CHEBI:33282 9300 9310 antibiotic +T236 SO:0000902 9375 9384 transgene +T237 GO:0006277 9392 9408;9417 9420 amplification of ... DNA +T238 SO:0001026 9409 9416 genomic +T239 UBERON:0002415 9443 9447 tail +T240 UBERON:0002415 9456 9461 Tails +T241 CHEBI:32145 9513 9517 NaOH +T242 CHEBI:9754 9576 9580 Tris +T243 CHEBI:17883 9581 9584 HCl +T244 PR:000004168 9704 9707 APP +T245 SO:0000902 9716 9726 transgenes +T246 SO:0000112 9787 9794 primers +T247 PR:000004168 9796 9799 APP +T248 SO:0000121 9820 9834 forward primer +T249 PR:000004168 9868 9871 APP +T250 SO:0000132 9919 9933 reverse primer +T251 PR:000001886 9934 9937 PrP +T252 SO:0000205 9958 9964 3′ UTR +T253 SO:0000440 9972 9978 vector +T254 SO:0000902 10021 10030 transgene +T255 SO:0000112 10052 10058 primer +T256 SO:0001030 10113 10120 forward +T257 PR:P04483 10140 10144 Tn10 +T258 CHEBI:27902 10145 10157 tetracycline +T259 SO:0001031 10209 10216 reverse +T260 NCBITaxon:10298 10228 10232 HSV1 +T261 PR:P06492 10233 10237 VP16 +T262 SO:0000902 10292 10301 transgene +T263 SO:0000842 10331 10341;10371 10375 segment of ... gene +T264 PR:000001886 10357 10370 prion protein +T265 SO:0000121 10413 10427 forward primer +T266 PR:000001886 10429 10432 PrP +T267 NCBITaxon:10088 10454 10459 mouse +T268 PR:000001886 10460 10463 PrP +T269 SO:0000236 10464 10482 open reading frame +T270 SO:0000132 10522 10536 reverse primer +T271 PR:000001886 10538 10541 PrP +T272 SO:0000205 10562 10568 3′ UTR +T273 PR:000001886 10587 10590 PrP +T274 SO:0000704 10591 10595 gene +T275 SO:0000902 10604 10613 transgene +T276 SO:0000440 10614 10620 vector +T277 SO:0000028 10776 10778 bp +T278 PR:000001886 10807 10810 PrP +T279 SO:0000704 10811 10815 gene +T280 PR:000004168 10821 10824 APP +T281 SO:0000902 10825 10834 transgene +T282 SO:0000028 10869 10871 bp +T283 SO:0000028 10912 10914 bp +T284 UBERON:0001851 10953 10961 cortical +T285 UBERON:0001890 10971 10980 forebrain +T286 UBERON:0000479 10981 10987 tissue +T287 NCBITaxon:40674 11109 11118 Mammalian +T288 CHEBI:60004 11124 11132 cocktail +T289 UBERON:0000955 11459 11464 brain +T290 CHEBI:9754 11526 11530 Tris +T291 CHEBI:17883 11531 11534 HCl +T292 CHEBI:9754 11649 11653 Tris +T293 CHEBI:8984 11662 11684 sodium dodecyl sulfate +T294 CHEBI:8984 11691 11694 SDS +T295 CHEBI:9754 11763 11767 Tris +T296 CHEBI:46760 11768 11775 tricine +T297 CHEBI:8984 11776 11779 SDS +T298 CHEBI:53325 11900 11914 nitrocellulose +T299 UBERON:0001913 12055 12059 milk +T300 CHEBI:75958 12122 12130 solution +T301 GO:0042571 12157 12167 antibodies +T302 NCBITaxon:10088 12169 12174 mouse +T303 NCBITaxon:10088 12270 12275 mouse +T304 NCBITaxon:9986 12369 12375 rabbit +T305 CHEBI:18421 12392 12402 superoxide +T306 PR:000015399 12392 12414 superoxide dismutase 1 +T307 PR:P00441 12418 12423 hSOD1 +T308 NCBITaxon:9986 12461 12467 rabbit +T309 CHEBI:53424 12590 12598 Tween-20 +T310 NCBITaxon:9925 12631 12635 goat +T311 NCBITaxon:10088 12641 12646 mouse +T312 NCBITaxon:9925 12651 12655 goat +T313 NCBITaxon:9986 12661 12667 rabbit +T314 MOP:0000779 12672 12682 conjugated +T315 GO:0042571 12693 12701 antibody +T316 CHEBI:75958 12730 12738 solution +T317 CHEBI:53424 12789 12797 Tween-20 +T318 CHEBI:33893 12852 12859 reagent +T319 UBERON:0000955 13368 13373 brain +T320 UBERON:0002107 13375 13380 liver +T321 UBERON:0002113 13382 13388 kidney +T322 UBERON:0000948 13390 13395 heart +T323 UBERON:0002048 13397 13401 lung +T324 UBERON:0002106 13403 13409 spleen +T325 UBERON:0004288 13415 13423 skeletal +T326 CHEBI:53325 13476 13490 nitrocellulose +T327 MOP:0000779 13568 13574 linked +T328 SO:0000028 13611 13613 bp +T329 NCBITaxon:10088 13642 13644 mo +T330 GO:0097617 13666 13677 hybridizing +T331 CHEBI:37586 13723 13739 sodium phosphate +T332 CHEBI:8984 13759 13762 SDS +T333 CHEBI:37586 13850 13866 sodium phosphate +T334 CHEBI:8984 13886 13889 SDS +T335 CHEBI:37586 13951 13967 sodium phosphate +T336 CHEBI:8984 13987 13990 SDS +T337 CHEBI:60425 14078 14085 Amyloid +T338 NCBITaxon:10088 14097 14101 Mice +T339 CHEBI:25698 14121 14126 ether +T340 UBERON:0000955 14142 14148 brains +T341 CHEBI:50913 14225 14233 fixative +T342 UBERON:0000955 14243 14249 brains +T343 CHEBI:30879 14289 14297 alcohols +T344 CHEBI:31832 14330 14346 methylsalicylate +T345 CHEBI:27338 14595 14601 xylene +T346 CHEBI:30879 14606 14614 alcohols +T347 CHEBI:15377 14624 14629 water +T348 CHEBI:32130 14665 14679 silver nitrate +T349 CHEBI:75958 14680 14688 solution +T350 CHEBI:15377 14746 14751 water +T351 CHEBI:32130 14781 14795 silver nitrate +T352 CHEBI:75958 14796 14804 solution +T353 CHEBI:18219 14825 14843 ammonium hydroxide +T354 CHEBI:18219 14883 14896 ammonia water +T355 CHEBI:16842 14972 14984 formaldehyde +T356 CHEBI:15377 15006 15011 water +T357 CHEBI:48107 15035 15046 nitric acid +T358 CHEBI:30769 15061 15072 citric acid +T359 CHEBI:32130 15101 15115 silver nitrate +T360 CHEBI:75958 15116 15124 solution +T361 CHEBI:15377 15157 15162 water +T362 CHEBI:30879 15219 15227 alcohols +T363 CHEBI:27338 15232 15238 xylene +T364 CHEBI:27338 15312 15318 xylene +T365 CHEBI:30879 15323 15331 alcohols +T366 CHEBI:60425 15333 15340 amyloid +T367 CHEBI:15377 15520 15525 water +T368 CHEBI:75958 15585 15593 solution +T369 CHEBI:16995 15648 15659 oxalic acid +T370 CHEBI:75958 15660 15668 solution +T371 CHEBI:15377 15729 15736 aqueous +T372 CHEBI:75958 15756 15764 solution +T373 CHEBI:16236 15874 15881 ethanol +T374 CHEBI:15377 15905 15910 water +T375 CHEBI:15377 15948 15955 aqueous +T376 CL:0000125 16018 16023 glial +T377 PR:000007939 16018 16049 glial fibrillary acidic protein +T378 CHEBI:37527 16035 16041 acidic +T379 CHEBI:27338 16173 16179 xylene +T380 CHEBI:30879 16214 16222 alcohols +T381 CHEBI:15377 16232 16237 water +T382 CHEBI:16240 16305 16322 hydrogen peroxide +T383 CHEBI:17790 16326 16334 methanol +T384 CHEBI:15377 16374 16379 water +T385 NCBITaxon:9925 16475 16479 goat +T386 UBERON:0001977 16480 16485 serum +T387 CHEBI:9750 16495 16507 Triton-X 100 +T388 GO:0042571 16553 16561 antibody +T389 NCBITaxon:9986 16563 16569 rabbit +T390 GO:0042571 16597 16605 antibody +T391 NCBITaxon:9986 16675 16681 rabbit +T392 NCBITaxon:9986 16701 16707 rabbit +T393 CL:0000125 16713 16718 glial +T394 PR:000007939 16713 16744 glial fibrillary acidic protein +T395 CHEBI:37527 16730 16736 acidic +T396 PR:000007939 16746 16750 GFAP +T397 GO:0042571 16763 16773 antibodies +T398 NCBITaxon:9925 16857 16861 goat +T399 UBERON:0001977 16862 16867 serum +T400 GO:0042571 16950 16958 antibody +T401 NCBITaxon:9986 17048 17054 rabbit +T402 CHEBI:33893 17172 17180 reagents +T403 PR:000007939 17207 17211 GFAP +T404 GO:0042571 17320 17328 Antibody +T405 CHEBI:51686 17413 17424 hematoxylin +T406 UBERON:0001851 17465 17473 cortical +T407 CHEBI:8984 17562 17565 SDS +T408 CHEBI:8984 17644 17647 SDS +T409 GO:0042571 17898 17906 antibody +T410 CHEBI:75958 17956 17964 solution +T411 UBERON:0001913 17989 17993 milk +T412 CHEBI:53424 18069 18077 Tween-20 +T413 MOP:0000779 18123 18133 conjugated +T414 PR:000029158 18134 18143 protein A +T415 CHEBI:75958 18180 18188 solution +T416 CHEBI:53424 18251 18259 Tween-20 +T417 GO:0042571 18268 18276 antibody +T418 UBERON:0001851 18595 18603 cortical +T419 CHEBI:8984 18728 18731 SDS +T420 CHEBI:30751 18741 18752 formic acid +T421 CHEBI:30751 18754 18756 FA +T422 CHEBI:75958 19038 19046 solution +T423 CHEBI:8984 19067 19070 SDS +T424 CHEBI:30751 19115 19117 FA +T425 CHEBI:9754 19147 19151 Tris +T426 NCBITaxon:9606 19223 19228 Human +T427 NCBITaxon:10088 19419 19424 mouse +T428 NCBITaxon:9606 19427 19432 human +T429 UBERON:0001851 19646 19654 cortical +T430 UBERON:0000479 19655 19661 tissue +T431 PR:000003199 19724 19731 CaMKIIα +T432 NCBITaxon:10088 19762 19766 mice +T433 NCBITaxon:33208 19794 19801 Animals +T434 CHEBI:50845 19937 19940 dox +T435 NCBITaxon:33208 20039 20045 animal +T436 NCBITaxon:33208 20385 20391 animal +T437 GO:0010467 20509 20519 expression +T438 SO:0000902 20919 20928 transgene +T439 GO:0010467 20939 20946 express +T440 NCBITaxon:10088 20983 20985 mo +T441 CHEBI:27902 21036 21048 tetracycline +T442 SO:0000167 21060 21068 promoter +T443 PR:000004168 21089 21092 APP +T444 GO:0010467 21093 21103 expression +T445 NCBITaxon:10088 21145 21149 mice +T446 NCBITaxon:33208 21153 21160 animals +T447 GO:0065007 21181 21188 control +T448 PR:000003199 21196 21203 CaMKIIα +T449 SO:0000167 21204 21212 promoter +T450 NCBITaxon:10088 21299 21303 mice +T451 SO:0000902 21338 21347 transgene +T452 GO:0010467 21371 21381 expressing +T453 PR:000004168 21436 21439 APP +T454 CHEBI:60425 21474 21481 amyloid +T455 PR:000004168 21598 21601 APP +T456 PR:000004168 21716 21719 APP +T457 PR:000004168 21756 21759 APP +T458 NCBITaxon:10088 21760 21764 mice +T459 GO:0042571 21774 21782 antibody +T460 PR:000004168 21815 21818 APP +T461 PR:000004137 21824 21856 amyloid precursor-like protein 2 +T462 GO:0042571 21897 21905 antibody +T463 PR:000004168 21969 21972 APP +T464 NCBITaxon:10088 21973 21977 mice +T465 SO:0000902 22020 22029 transgene +T466 CHEBI:50845 22040 22043 dox +T467 SO:0000902 22188 22197 transgene +T468 CHEBI:50845 22259 22262 dox +T469 CHEBI:50845 22268 22271 dox +T470 NCBITaxon:10088 22339 22343 mice +T471 GO:0007567 22348 22352 born +T472 CHEBI:50845 22367 22370 dox +T473 CHEBI:50845 22404 22407 dox +T474 CHEBI:50845 22454 22457 dox +T475 CHEBI:33290 22496 22500 chow +T476 NCBITaxon:33208 22544 22551 Animals +T477 GO:0007567 22552 22556 born +T478 CHEBI:50845 22571 22574 dox +T479 PR:000004168 22598 22601 APP +T480 CHEBI:50845 22646 22649 dox +T481 PR:000004168 22672 22675 APP +T482 GO:0010467 22676 22686 expression +T483 CHEBI:50845 22732 22735 dox +T484 GO:0010467 22757 22767 expression +T485 NCBITaxon:10088 22797 22801 mice +T486 CHEBI:50845 22879 22882 dox +T487 PR:000004168 23016 23019 APP +T488 NCBITaxon:33208 23105 23112 animals +T489 NCBITaxon:10088 23176 23180 mice +T490 GO:0034205 23318 23331 Aβ production +T491 CHEBI:50845 23367 23370 dox +T492 UBERON:0001890 23465 23474 forebrain +T493 NCBITaxon:33208 23506 23513 animals +T494 NCBITaxon:10088 23535 23539 mice +T495 CHEBI:60425 23555 23562 amyloid +T496 UBERON:0000955 23645 23650 brain +T497 SO:0000902 23661 23670 transgene +T498 GO:0043043 23798 23815 peptide synthesis +T499 CHEBI:8984 23878 23881 SDS +T500 CHEBI:30751 23891 23893 FA +T501 NCBITaxon:9606 23964 23969 human +T502 SO:0000902 23970 23979 transgene +T503 NCBITaxon:10088 24015 24019 mice +T504 NCBITaxon:33208 24044 24051 animals +T505 GO:0007567 24073 24077 born +T506 CHEBI:50845 24092 24095 dox +T507 CHEBI:50845 24156 24159 dox +T508 CHEBI:33290 24160 24164 chow +T509 SO:0000902 24397 24406 transgene +T510 NCBITaxon:33208 24455 24462 animals +T511 GO:0007567 24463 24467 born +T512 CHEBI:50845 24482 24485 dox +T513 CHEBI:33282 24535 24545 antibiotic +T514 NCBITaxon:10088 24602 24606 mice +T515 CHEBI:50845 24660 24663 dox +T516 CHEBI:8984 24734 24737 SDS +T517 CHEBI:50845 24807 24810 dox +T518 CHEBI:30751 24843 24845 FA +T519 CHEBI:50845 24872 24875 dox +T520 NCBITaxon:33208 24921 24928 animals +T521 NCBITaxon:33208 25076 25083 animals +T522 GO:0007567 25094 25098 born +T523 CHEBI:50845 25113 25116 dox +T524 NCBITaxon:10088 25224 25228 mice +T525 NCBITaxon:10088 25277 25281 mice +T526 SO:0000902 25308 25317 transgene +T527 PR:000004168 25374 25377 APP +T528 NCBITaxon:10088 25378 25382 mice +T529 NCBITaxon:33208 25454 25461 animals +T530 CHEBI:50845 25584 25587 dox +T531 NCBITaxon:33208 25869 25876 animals +T532 PR:000003199 25930 25937 CaMKIIα +T533 NCBITaxon:10088 25960 25964 mice +T534 CHEBI:60425 25986 25993 amyloid +T535 CHEBI:60425 26027 26034 Amyloid +T536 NCBITaxon:10088 26056 26060 mice +T537 UBERON:0001890 26131 26140 forebrain +T538 UBERON:0001851 26156 26162 cortex +T539 GO:0007608 26177 26186 olfactory +T540 UBERON:0002264 26177 26191 olfactory bulb +T541 UBERON:0002435 26197 26205 striatum +T542 PR:000003199 26217 26224 CaMKIIα +T543 SO:0000167 26225 26233 promoter +T544 CHEBI:60425 26298 26305 amyloid +T545 UBERON:0001851 26356 26362 cortex +T546 UBERON:0002037 26420 26430 cerebellum +T547 UBERON:0002298 26434 26444 brain stem +T548 PR:000003199 26480 26487 CaMKIIα +T549 GO:0065007 26488 26498 controlled +T550 SO:0000902 26499 26508 transgene +T551 GO:0010467 26509 26519 expression +T552 NCBITaxon:9606 26560 26565 human +T553 http://purl.obolibrary.org/obo/MONDO_0000001 26566 26573 disease +T554 PR:000004168 26616 26619 APP +T555 NCBITaxon:10088 26620 26624 mice +T556 PR:000004168 26731 26734 APP +T557 NCBITaxon:10088 26746 26750 mice +T558 PR:000004168 26836 26839 APP +T559 NCBITaxon:10088 26840 26844 mice +T560 CHEBI:60425 26917 26924 amyloid +T561 UBERON:0001897 26954 26962 thalamus +T562 NCBITaxon:10088 26996 27000 mice +T563 GO:0010467 27001 27011 expressing +T564 PR:000004168 27019 27022 APP +T565 PR:000001843 27031 27036 Thy-1 +T566 SO:0000167 27037 27045 promoter +T567 CHEBI:60425 27063 27070 amyloid +T568 GO:0030424 27119 27125 axonal +T569 GO:0008088 27119 27135 axonal transport +T570 PR:000004168 27139 27142 APP +T571 UBERON:0001851 27166 27174 cortical +T572 CL:0010012 27166 27182 cortical neurons +T573 UBERON:0001897 27190 27198 thalamus +T574 NCBITaxon:10088 27246 27250 mice +T575 GO:0010467 27252 27262 expressing +T576 PR:000004168 27280 27283 APP +T577 SO:0000902 27284 27294 transgenes +T578 CHEBI:60425 27306 27313 amyloid +T579 NCBITaxon:10088 27341 27345 mice +T580 CHEBI:60425 27417 27424 amyloid +T581 NCBITaxon:33208 27484 27491 animals +T582 GO:0007567 27492 27496 born +T583 CHEBI:50845 27511 27514 dox +T584 NCBITaxon:33208 27516 27523 Animals +T585 GO:0010467 27541 27551 expressing +T586 CHEBI:50845 27582 27585 dox +T587 CHEBI:60425 27612 27619 amyloid +T588 SO:0000902 27684 27693 transgene +T589 GO:0010467 27694 27704 expression +T590 CHEBI:50845 27724 27727 dox +T591 CHEBI:60425 27777 27784 amyloid +T592 CHEBI:35222 27859 27869 inhibitors +T593 GO:0034205 27873 27886 Aβ production +T594 NCBITaxon:10088 27930 27934 mice +T595 PR:000003199 27936 27943 CaMKIIα +T596 PR:000004168 27950 27953 APP +T597 CHEBI:33290 27974 27978 food +T598 CHEBI:60425 28011 28018 amyloid +T599 UBERON:0000955 28062 28067 brain +T600 NCBITaxon:33208 28090 28097 animals +T601 CHEBI:33290 28124 28128 chow +T602 CHEBI:33290 28132 28136 food +T603 CHEBI:50845 28148 28151 dox +T604 NCBITaxon:33208 28236 28243 animals +T605 CHEBI:33290 28266 28270 chow +T606 CHEBI:50845 28492 28495 dox +T607 NCBITaxon:33208 28504 28511 animals +T608 SO:0000902 28571 28580 transgene +T609 NCBITaxon:10088 28624 28628 mice +T610 NCBITaxon:33208 28685 28692 animals +T611 NCBITaxon:33208 28735 28742 animals +T612 CHEBI:50845 28748 28751 dox +T613 PR:000004168 28812 28815 APP +T614 UBERON:0000479 29021 29027 Tissue +T615 NCBITaxon:33208 29047 29053 animal +T616 CHEBI:60425 29100 29107 amyloid +T617 CHEBI:60425 29262 29269 amyloid +T618 CHEBI:60425 29334 29341 amyloid +T619 CHEBI:60425 29377 29384 amyloid +T620 NCBITaxon:10088 29398 29402 mice +T621 NCBITaxon:33208 29667 29674 animals +T622 SO:0000902 29689 29698 transgene +T623 CHEBI:60425 29819 29826 amyloid +T624 CHEBI:60425 29963 29970 amyloid +T625 CHEBI:50845 30078 30081 dox +T626 PR:000004168 30116 30119 APP +T627 NCBITaxon:10088 30272 30276 mice +T628 CHEBI:50845 30294 30297 dox +T629 PR:000004168 30349 30352 APP +T630 NCBITaxon:10088 30353 30357 mice +T631 PR:000003199 30359 30366 CaMKIIα +T632 CHEBI:50845 30423 30426 dox +T633 NCBITaxon:10088 30467 30471 mice +T634 SO:0000902 30501 30510 transgene +T635 PR:000004168 30618 30621 APP +T636 SO:0000902 30637 30646 transgene +T637 NCBITaxon:33208 30674 30681 animals +T638 NCBITaxon:10088 30714 30718 mice +T639 CHEBI:60425 30736 30743 amyloid +T640 NCBITaxon:10088 30791 30795 mice +T641 SO:0000902 30838 30847 transgene +T642 GO:0010467 30848 30858 expression +T643 CHEBI:50845 31039 31042 dox +T644 NCBITaxon:33208 31051 31058 animals +T645 UBERON:0000955 31122 31128 brains +T646 NCBITaxon:10088 31136 31140 mice +T647 SO:0000902 31158 31167 transgene +T648 UBERON:0001851 31210 31218 cortical +T649 UBERON:0000479 31219 31225 tissue +T650 NCBITaxon:33208 31236 31242 animal +T651 GO:0007601 31487 31493 visual +T652 PR:000004168 31546 31549 APP +T653 NCBITaxon:10088 31550 31554 mice +T654 CHEBI:50845 31568 31571 dox +T655 NCBITaxon:10088 31721 31725 mice +T656 PR:000004168 31834 31837 APP +T657 NCBITaxon:10088 31838 31842 mice +T658 NCBITaxon:33208 31920 31927 animals +T659 SO:0000902 31981 31990 transgene +T660 UBERON:0000955 32072 32078 brains +T661 GO:0010467 32190 32200 expression +T662 UBERON:0001851 32217 32225 Cortical +T663 CHEBI:8984 32297 32300 SDS +T664 CHEBI:30751 32307 32309 FA +T665 SO:0000902 32334 32343 transgene +T666 NCBITaxon:9606 32383 32388 human +T667 NCBITaxon:33208 32417 32424 animals +T668 CHEBI:60425 32435 32442 amyloid +T669 CHEBI:8984 32521 32524 SDS +T670 CHEBI:30751 32529 32531 FA +T671 CHEBI:8984 32660 32663 SDS +T672 CHEBI:30751 32668 32670 FA +T673 UBERON:0000955 32792 32798 brains +T674 NCBITaxon:10088 32922 32926 mice +T675 NCBITaxon:33208 33267 33274 animals +T676 NCBITaxon:33208 33331 33338 animals +T677 GO:0043005 33358 33366 neuritic +T678 CL:0000125 33371 33376 glial +T679 SO:0000902 33492 33501 transgene +T680 GO:0043005 33576 33584 neuritic +T681 CL:0002627 33642 33662 activated astrocytes +T682 PR:000007939 33681 33685 GFAP +T683 NCBITaxon:33208 33717 33724 animals +T684 GO:0043005 33737 33745 Neuritic +T685 CL:0000125 33750 33755 glial +T686 NCBITaxon:10088 33806 33810 mice +T687 SO:0000902 33825 33834 transgene +T688 NCBITaxon:10088 33913 33917 mice +T689 GO:0050890 34093 34102 cognitive +T690 NCBITaxon:10088 34124 34128 mice +T691 GO:0050890 34155 34164 cognition +T692 GO:0050890 34238 34247 cognitive +T693 NCBITaxon:10088 34329 34333 mice +T694 PR:000004168 34343 34346 APP +T695 NCBITaxon:33208 34347 34354 animals +T696 GO:0036268 34441 34449 swimming +T697 NCBITaxon:10088 34477 34481 mice +T698 CHEBI:15377 34508 34513 water +T699 CHEBI:15377 34534 34539 water +T700 GO:0036268 34557 34561 swim +T701 NCBITaxon:33208 34688 34695 animals +T702 NCBITaxon:10088 34766 34770 mice +T703 NCBITaxon:33208 34908 34915 animals +T704 NCBITaxon:10088 34974 34978 mice +T705 GO:0010467 35030 35037 express +T706 CHEBI:60425 35180 35187 amyloid +T707 GO:0050890 35197 35206 cognitive +T708 PR:000004168 35306 35309 APP +T709 NCBITaxon:10088 35310 35314 mice +T710 GO:0050890 35364 35373 cognitive +T711 NCBITaxon:33208 35390 35397 animals +T712 GO:0050890 35447 35456 cognitive +T713 PR:000004168 35549 35552 APP +T714 NCBITaxon:10088 35553 35557 mice +T715 NCBITaxon:33208 35616 35623 animals +T716 GO:0040011 35729 35739 ambulation +T717 NCBITaxon:10088 35809 35813 mice +T718 GO:0007623 35977 35990 diurnal cycle +T719 PR:000004168 36090 36093 APP +T720 NCBITaxon:10088 36094 36098 mice +T721 SO:0000902 36223 36232 transgene +T722 PR:000004168 36357 36360 APP +T723 NCBITaxon:10088 36361 36365 mice +T724 CHEBI:50845 36369 36372 dox +T725 NCBITaxon:33208 36374 36381 Animals +T726 GO:0007567 36382 36386 born +T727 CHEBI:50845 36401 36404 dox +T728 CHEBI:50845 36500 36503 dox +T729 NCBITaxon:33208 36511 36518 animals +T730 GO:0007623 36566 36583 circadian rhythms +T731 NCBITaxon:10088 36690 36695 mouse +T732 http://purl.obolibrary.org/obo/MONDO_0004975 36706 36708 AD +T733 GO:0034205 36766 36779 Aβ production +T734 CHEBI:60425 36799 36806 amyloid +T735 NCBITaxon:10088 36842 36846 mice +T736 GO:0010467 36882 36889 express +T737 GO:0065007 36926 36933 control +T738 CHEBI:27902 36939 36951 tetracycline +T739 SO:0000167 36963 36971 promoter +T740 CHEBI:50845 37008 37011 dox +T741 GO:0010467 37182 37192 expression +T742 CHEBI:60425 37263 37270 amyloid +T743 NCBITaxon:10088 37295 37299 mice +T744 CHEBI:60425 37351 37358 Amyloid +T745 UBERON:0001851 37431 37437 cortex +T746 NCBITaxon:10088 37451 37455 mice +T747 PR:000004168 37539 37542 APP +T748 CHEBI:60425 37594 37601 amyloid +T749 NCBITaxon:33208 37649 37656 animals +T750 CHEBI:60425 37688 37695 amyloid +T751 CHEBI:35222 37794 37804 inhibitors +T752 CHEBI:60425 37878 37885 amyloid +T753 SO:0000902 37912 37921 transgene +T754 http://purl.obolibrary.org/obo/MONDO_0000001 38032 38039 disease +T755 PR:000008840 38085 38095 huntingtin +T756 GO:0005576 38155 38168 extracellular +T757 CHEBI:60425 38169 38176 amyloid +T758 CHEBI:52217 38207 38221 pharmaceutical +T759 GO:0070765 38222 38233 γ-secretase +T760 CHEBI:35222 38234 38244 inhibitors +T761 GO:1902003 38280 38307 regulation of Aβ production +T762 CHEBI:36357 38385 38394 compounds +T763 NCBITaxon:10088 38445 38449 mice +T764 PR:000004168 38627 38630 APP +T765 GO:0010467 38631 38641 expression +T766 NCBITaxon:10088 38656 38660 mice +T767 GO:0034205 38747 38760 Aβ production +T768 GO:0070765 38827 38838 γ-secretase +T769 CHEBI:35222 38839 38849 inhibitors +T770 CHEBI:52217 38887 38908 pharmaceutical agents +T771 GO:1902003 38946 38970 control of Aβ production +T772 CHEBI:36357 39072 39081 compounds +T773 CHEBI:60425 39112 39119 amyloid +T774 CHEBI:60425 39198 39205 amyloid +T775 SO:0000902 39249 39258 transgene +T776 NCBITaxon:33208 39302 39309 animals +T777 CHEBI:50845 39325 39328 dox +T778 CHEBI:60425 39382 39389 amyloid +T779 NCBITaxon:33208 39527 39534 animals +T780 CHEBI:50845 39548 39551 dox +T781 GO:0034205 39797 39810 Aβ production +T782 CHEBI:60425 39874 39881 amyloid +T783 UBERON:0000104 39925 39934 life span +T784 NCBITaxon:9606 40004 40010 humans +T785 UBERON:0000104 40079 40088 life span +T786 NCBITaxon:9989 40092 40098 rodent +T787 http://purl.obolibrary.org/obo/MONDO_0004975 40258 40260 AD +T788 GO:0034205 40300 40316 production of Aβ +T789 PR:000004168 40372 40375 APP +T790 GO:0010467 40376 40386 expression +T791 CHEBI:60425 40485 40492 amyloid +T792 NCBITaxon:10088 40591 40595 mice +T793 PR:000004168 40608 40611 APP +T794 NCBITaxon:9606 40798 40803 human +T795 http://purl.obolibrary.org/obo/MONDO_0000001 40804 40811 disease +T796 NCBITaxon:10088 40886 40890 mice +T797 SO:0000902 41041 41050 transgene +T798 GO:0010467 41051 41061 expression +T799 CHEBI:50845 41091 41094 dox +T800 NCBITaxon:10088 41115 41120 mouse +T801 NCBITaxon:10088 41208 41213 mouse +T802 NCBITaxon:9606 41235 41240 human +T803 CHEBI:60425 41365 41372 amyloid +T804 PR:000003199 41387 41394 CaMKIIα +T805 NCBITaxon:10088 41415 41419 mice +T806 CHEBI:50845 41430 41433 dox +T807 CHEBI:60425 41466 41473 amyloid +T808 SO:0000704 41530 41537 genetic +T809 GO:0034205 41565 41581 production of Aβ +T810 NCBITaxon:9606 41605 41611 humans +T811 PR:000004168 41759 41762 APP +T812 GO:0042983 41759 41762;41766 41775 APP ... synthetic +T813 GO:0034205 41763 41775 Aβ synthetic +T814 CHEBI:60425 41921 41928 amyloid +T815 CHEBI:60425 41978 41985 amyloid +T816 NCBITaxon:9606 41998 42003 human +T817 UBERON:0000955 42004 42009 brain +T818 NCBITaxon:10088 42067 42072 mouse +T819 UBERON:0000955 42073 42078 brain +T820 NCBITaxon:9606 42093 42098 human +T821 UBERON:0000955 42099 42104 brain +T822 NCBITaxon:10088 42151 42155 mice +T823 CHEBI:60425 42211 42218 amyloid +T824 CHEBI:60425 42254 42261 amyloid +T825 NCBITaxon:9606 42301 42306 human +T826 http://purl.obolibrary.org/obo/MONDO_0000001 42307 42314 disease +T827 NCBITaxon:10088 42327 42332 mouse +T828 CL:0000129 42351 42361 microglial +T829 GO:0006909 42362 42374 phagocytosis +T830 CL:0000129 42385 42394 microglia +T831 NCBITaxon:10088 42409 42414 mouse +T832 UBERON:0000479 42434 42440 tissue +T833 CHEBI:60425 42489 42496 amyloid +T834 CL:0000129 42530 42539 microglia +T835 CHEBI:60425 42552 42559 amyloid +T836 NCBITaxon:9606 42571 42576 human +T837 UBERON:0000955 42577 42582 brain +T838 GO:0010467 42635 42645 expression +T839 CL:0000129 42693 42702 microglia +T840 CHEBI:60425 42706 42713 amyloid +T841 GO:0008152 42714 42724 metabolism +T842 NCBITaxon:9606 42771 42776 human +T843 CHEBI:35472 42868 42891 anti-inflammatory drugs +T844 CL:0000129 42902 42912 microglial +T845 GO:0001774 42902 42923 microglial activation +T846 CHEBI:60425 42940 42947 amyloid +T847 PR:000004168 42956 42959 APP +T848 NCBITaxon:10088 42971 42975 mice +T849 NCBITaxon:10088 42999 43004 mouse +T850 CL:0000129 43005 43014 microglia +T851 CHEBI:60425 43051 43058 amyloid +T852 CHEBI:35472 43158 43181 anti-inflammatory drugs +T853 GO:0070765 43185 43196 γ-secretase +T854 MOP:0000780 43197 43205 cleavage +T855 CL:0000129 43240 43249 microglia +T856 NCBITaxon:9606 43262 43267 human +T857 NCBITaxon:10088 43286 43291 mouse +T858 CL:0000129 43340 43350 microglial +T859 CHEBI:60425 43413 43420 amyloid +T860 UBERON:0000955 43438 43444 brains +T861 http://purl.obolibrary.org/obo/MONDO_0004975 43462 43464 AD +T862 PR:000004168 43496 43499 APP +T863 NCBITaxon:10088 43500 43504 mice +T864 CL:0000129 43549 43558 microglia +T865 NCBITaxon:10088 43568 43573 mouse +T866 http://purl.obolibrary.org/obo/MONDO_0019065 43584 43595 amyloidosis +T867 CHEBI:60425 43663 43670 amyloid +T868 PR:000004168 43696 43699 APP +T869 NCBITaxon:10088 43700 43704 mice +T870 CHEBI:50845 43758 43761 dox +T871 CL:0000129 43813 43822 microglia +T872 NCBITaxon:10088 43838 43842 mice +T873 CHEBI:50845 43844 43847 Dox +T874 CHEBI:50694 43875 43886 minocycline +T875 CHEBI:35472 43899 43921 anti-inflammatory drug +T876 CHEBI:35222 43926 43935 inhibitor +T877 CL:0000129 43939 43949 microglial +T878 GO:0001774 43939 43960 microglial activation +T879 CHEBI:50845 43979 43982 dox +T880 CHEBI:67079 44064 44083 anti-inflammatories +T881 CHEBI:60425 44121 44128 amyloid +T882 CHEBI:50845 44136 44139 dox +T883 NCBITaxon:33208 44148 44155 animals +T884 CHEBI:50845 44215 44218 dox +T885 CHEBI:60425 44250 44257 amyloid +T886 CL:0000129 44313 44323 microglial +T887 NCBITaxon:10088 44359 44364 mouse +T888 http://purl.obolibrary.org/obo/MONDO_0004975 44365 44367 AD +T889 CHEBI:50845 44413 44416 dox +T890 CL:0000129 44426 44436 microglial +T891 CHEBI:60425 44517 44524 amyloid +T892 NCBITaxon:10088 44624 44629 mouse +T893 GO:0042571 44674 44684 antibodies +T894 UBERON:0000955 44712 44717 brain +T895 CHEBI:60425 44747 44754 amyloid +T896 GO:0042571 44821 44829 antibody +T897 CHEBI:60425 44850 44857 amyloid +T898 CHEBI:60425 44972 44979 amyloid +T899 NCBITaxon:11646 45081 45091 lentiviral +T900 PR:000001898 45104 45114 neprilysin +T901 GO:0042571 45249 45257 antibody +T902 CL:0002629 45296 45315 activated microglia +T903 CL:0000129 45492 45502 microglial +T904 GO:0042571 45519 45527 antibody +T905 CHEBI:59132 45528 45535 antigen +T906 GO:0032991 45536 45545 complexes +T907 PR:000004168 45550 45553 APP +T908 NCBITaxon:10088 45565 45570 mouse +T909 GO:0042571 45616 45624 antibody +T910 CL:0000129 45694 45703 microglia +T911 GO:0001774 45694 45714 microglia activation +T912 UBERON:0002405 45811 45824 immune system +T913 GO:0042571 45893 45901 antibody +T914 NCBITaxon:10239 45905 45910 viral +T915 GO:0001774 45941 45964 activation of microglia +T916 CL:0000129 45955 45964 microglia +T917 GO:0006909 46043 46055 phagocytosis +T918 PR:000001898 46110 46120 neprilysin +T919 GO:0042571 46136 46146 antibodies +T920 GO:0001774 46243 46266 activation of microglia +T921 CL:0000129 46257 46266 microglia +T922 GO:0010467 46286 46296 expression +T923 PR:000000046 46300 46304 TGFβ +T924 CHEBI:16412 46334 46352 lipopolysaccharide +T925 PR:000004168 46413 46416 APP +T926 NCBITaxon:10088 46428 46432 mice +T927 GO:0042571 46459 46467 antibody +T928 GO:0042571 46546 46554 antibody +T929 CHEBI:60425 46647 46654 amyloid +T930 CL:0000129 46759 46768 microglia +T931 NCBITaxon:10088 46772 46777 mouse +T932 NCBITaxon:9606 46858 46864 humans +T933 NCBITaxon:10088 46914 46918 mice +T934 GO:0010467 46924 46931 express +T935 NCBITaxon:9606 46938 46943 human +T936 PR:000010173 46944 46947 tau +T937 SO:0000440 46962 46968 vector +T938 PR:000004168 47000 47003 APP +T939 NCBITaxon:10088 47004 47008 mice +T940 PR:000010173 47038 47041 tau +T941 GO:0097418 47038 47065 tau neurofibrillary tangles +T942 CHEBI:60425 47072 47079 amyloid +T943 SO:0000902 47127 47136 transgene +T944 http://purl.obolibrary.org/obo/MONDO_0004975 47190 47192 AD +T945 NCBITaxon:10088 47297 47302 mouse +T946 http://purl.obolibrary.org/obo/MONDO_0007739 47313 47323;47339 47346 Huntington disease +T947 http://purl.obolibrary.org/obo/MONDO_0005429 47333 47346 prion disease +T948 CHEBI:50845 47423 47426 dox +T949 SO:0000902 47436 47445 transgene +T950 http://purl.obolibrary.org/obo/MONDO_0004975 47602 47604 AD +T951 http://purl.obolibrary.org/obo/MONDO_0005559 47641 47668 neurodegenerative disorders +T952 PR:000010173 47702 47705 tau +T953 GO:0005622 47791 47797;47807 47815 intra- ... cellular +T954 GO:0005576 47802 47815 extracellular +T955 GO:0034205 47916 47928;47933 47935 synthesis of ... Aβ +T956 GO:0050890 47961 47970 cognitive +T957 PR:000010173 48005 48008 tau +T958 NCBITaxon:10088 48009 48013 mice +T959 GO:0050890 48077 48086 cognitive +T960 SO:0000902 48109 48118 transgene +T961 GO:0050890 48174 48183 cognitive +T962 PR:000004168 48246 48249 APP +T963 NCBITaxon:10088 48250 48254 mice +T964 CHEBI:60425 48314 48321 amyloid +T965 PR:000004168 48351 48354 APP +T966 NCBITaxon:10088 48355 48359 mice +T967 NCBITaxon:10088 48515 48519 mice +T968 GO:0036268 48556 48564 swimming +T969 CHEBI:15377 48617 48622 water +T970 GO:0007612 48682 48690 learning +T971 GO:0007613 48695 48701 memory +T972 GO:0050890 48820 48829 cognitive +T973 GO:0010467 48928 48938 expression +T974 GO:0006508 48976 48987 proteolytic +T975 CHEBI:50845 49077 49080 dox +T976 NCBITaxon:10088 49088 49092 mice +T977 PR:000004168 49254 49257 APP +T978 GO:0042983 49254 49267 APP synthesis +T979 CL:0000540 49369 49377 neuronal +T980 SO:0000902 49397 49406 transgene +T981 GO:0010467 49407 49417 expression +T982 GO:0007567 49435 49440 natal +T983 GO:0034205 49573 49586 Aβ production +T984 NCBITaxon:10088 49647 49652 mouse +T985 http://purl.obolibrary.org/obo/MONDO_0004975 49662 49671 Alzheimer +T986 http://purl.obolibrary.org/obo/MONDO_0019065 49677 49688 amyloidosis +T987 GO:0034205 49730 49742 Aβ synthesis +T988 GO:0043005 49744 49752 neuritic +T989 GO:0034205 49881 49894 Aβ production +T990 GO:0006909 49933 49945 phagocytosis +T991 CHEBI:60425 49959 49966 amyloid +T992 http://purl.obolibrary.org/obo/MONDO_0004975 50017 50019 AD +T993 http://purl.obolibrary.org/obo/MONDO_0000001 50071 50078 disease +T994 CHEBI:35222 50090 50100 inhibitors +T995 CHEBI:60425 50183 50190 amyloid +T996 http://purl.obolibrary.org/obo/MONDO_0000001 50231 50238 disease +T997 CL:0000129 50268 50278 microglial +T998 GO:0001774 50268 50289 microglial activation +T999 NCBITaxon:9606 50293 50298 human +T1000 http://purl.obolibrary.org/obo/MONDO_0004975 50299 50301 AD +T1001 CHEBI:60425 50357 50364 amyloid +T1002 GO:0006909 50392 50404 phagocytosis +T1003 CHEBI:35222 50495 50505 inhibitors +T1004 NCBITaxon:9606 50559 50564 human +T1005 UBERON:0000955 50565 50570 brain +T1006 CHEBI:60425 50598 50605 amyloid +T1007 http://purl.obolibrary.org/obo/MONDO_0004975 50627 50629 AD +T1008 PR:000004168 50724 50727 APP +T1009 GO:0010467 50728 50738 Expression +T1010 CHEBI:50845 50758 50761 Dox +T1011 PR:000004168 50786 50789 APP +T1012 NCBITaxon:9606 50819 50824 human +T1013 GO:0042571 50834 50842 antibody +T1014 SO:0000902 50856 50865 transgene +T1015 PR:000004168 50886 50889 APP +T1016 NCBITaxon:33208 50929 50936 animals +T1017 SO:0000902 51016 51025 transgene +T1018 GO:0010467 51026 51036 expression +T1019 UBERON:0000955 51057 51062 brain +T1020 PR:000004168 51101 51104 APP +T1021 CHEBI:50845 51201 51204 dox +T1022 PR:000004168 51259 51262 APP +T1023 CHEBI:50845 51302 51305 dox +T1024 PR:000004168 51411 51414 APP +T1025 UBERON:0000955 51423 51428 Brain +T1026 UBERON:0000479 51482 51489 tissues +T1027 PR:000004168 51523 51526 APP +T1028 GO:0010467 51597 51607 expression +T1029 GO:0097617 51609 51622 Hybridization +T1030 UBERON:0000955 51643 51648 brain +T1031 UBERON:0000479 51698 51704 tissue +T1032 CHEBI:60425 51771 51778 Amyloid +T1033 UBERON:0001851 51796 51802 Cortex +T1034 CHEBI:60425 51839 51846 Amyloid +T1035 NCBITaxon:10088 51915 51919 mice +T1036 SO:0000902 52072 52081 transgene +T1037 CHEBI:60425 52161 52168 amyloid +T1038 UBERON:0001851 52186 52192 cortex +T1039 NCBITaxon:10088 52254 52258 mice +T1040 SO:0000902 52323 52332 transgene +T1041 CHEBI:50845 52338 52341 dox +T1042 NCBITaxon:33208 52437 52443 animal +T1043 CHEBI:60425 52448 52455 amyloid +T1044 PR:000004168 52494 52497 APP +T1045 NCBITaxon:33208 52523 52530 animals +T1046 PR:000004168 52788 52791 APP +T1047 NCBITaxon:10088 52792 52796 mice +T1048 SO:0000902 52884 52893 transgene +T1049 UBERON:0001870 52959 52973 frontal cortex +T1050 CHEBI:60425 53041 53048 amyloid +T1051 NCBITaxon:10088 53238 53242 mice +T1052 NCBITaxon:33208 53276 53283 animals +T1053 SO:0000902 53658 53667 Transgene +T1054 PR:000004168 53751 53754 APP +T1055 NCBITaxon:10088 53755 53759 Mice +T1056 PR:000003199 53761 53768 CaMKIIα +T1057 PR:000004168 53862 53865 APP +T1058 PR:000004168 53901 53904 APP +T1059 SO:0000366 53935 53951 integration site +T1060 UBERON:0001851 53963 53971 Cortical +T1061 CHEBI:50845 54011 54014 dox +T1062 NCBITaxon:10088 54041 54045 mice +T1063 PR:000004168 54081 54084 APP +T1064 NCBITaxon:9606 54094 54099 human +T1065 GO:0042571 54109 54117 antibody +T1066 SO:0000902 54134 54143 transgene +T1067 CHEBI:18421 54210 54220 superoxide +T1068 PR:000015399 54232 54236 SOD1 +T1069 PR:000004168 54357 54360 APP +T1070 CHEBI:50845 54429 54432 dox +T1071 UBERON:0001851 54679 54687 cortical +T1072 CHEBI:60425 54816 54823 amyloid +T1073 NCBITaxon:10088 54941 54945 mice +T1074 SO:0000902 55055 55064 transgene +T1075 NCBITaxon:10088 55107 55111 mice +T1076 CHEBI:50845 55125 55128 dox +T1077 NCBITaxon:33208 55154 55161 animals +T1078 NCBITaxon:10088 55294 55298 mice +T1079 CHEBI:60425 55396 55403 Amyloid +T1080 SO:0000902 55427 55436 Transgene +T1081 PR:000004168 55468 55471 APP +T1082 NCBITaxon:10088 55472 55476 Mice +T1083 CHEBI:60425 55478 55485 Amyloid +T1084 UBERON:0001851 55499 55507 cortical +T1085 PR:000004168 55600 55603 APP +T1086 NCBITaxon:10088 55604 55608 mice +T1087 PR:000004168 55704 55707 APP +T1088 GO:0010467 55708 55718 expression +T1089 CHEBI:50845 55804 55807 dox +T1090 http://purl.obolibrary.org/obo/MONDO_0004975 55971 55988 Alzheimer disease +T1091 http://purl.obolibrary.org/obo/MONDO_0004975 55990 55992 AD +T1092 CHEBI:60425 56106 56113 amyloid +T1093 UBERON:0000955 56131 56137 brains +T1094 NCBITaxon:1 56194 56205 individuals +T1095 PR:000004168 56248 56273 amyloid precursor protein +T1096 PR:000004168 56275 56278 APP +T1097 MOP:0000780 56283 56286 cut +T1098 PR:000004168 56390 56393 APP +T1099 GO:0034205 56441 56457 production of Aβ +T1100 UBERON:0000955 56473 56478 brain +T1101 CHEBI:60425 56510 56517 amyloid +T1102 CHEBI:23888 56602 56607 drugs +T1103 http://purl.obolibrary.org/obo/MONDO_0004975 56709 56711 AD +T1104 CHEBI:23888 56765 56770 drugs +T1105 NCBITaxon:33208 56863 56869 animal +T1106 http://purl.obolibrary.org/obo/MONDO_0000001 56884 56891 disease +T1107 CHEBI:23888 57018 57023 drugs +T1108 NCBITaxon:10088 57062 57066 mice +T1109 PR:000004168 57089 57092 APP +T1110 CHEBI:60425 57119 57126 amyloid +T1111 NCBITaxon:9606 57141 57146 human +T1112 http://purl.obolibrary.org/obo/MONDO_0004975 57161 57163 AD +T1113 NCBITaxon:10088 57179 57183 mice +T1114 NCBITaxon:10088 57191 57195 mice +T1115 SO:0000704 57220 57224 gene +T1116 PR:000004168 57266 57269 APP +T1117 GO:0007631 57273 57280 feeding +T1118 NCBITaxon:10088 57285 57289 mice +T1119 CHEBI:33290 57298 57302 food +T1120 PR:000004168 57316 57319 APP +T1121 NCBITaxon:10088 57329 57333 mice +T1122 CHEBI:23888 57388 57393 drugs +T1123 CHEBI:60425 57456 57463 amyloid +T1124 GO:0034205 57478 57491 Aβ production +T1125 GO:0034205 57533 57546 Aβ production +T1126 CHEBI:60425 57560 57567 amyloid +T1127 http://purl.obolibrary.org/obo/MONDO_0000001 57602 57609 disease +T1128 CHEBI:23888 57665 57670 drugs +T1129 http://purl.obolibrary.org/obo/MONDO_0000001 57695 57702 disease +T1130 UBERON:0000955 57720 57725 brain +T1131 CHEBI:60425 57785 57792 amyloid +T1132 GO:0034205 57981 57997 production of Aβ +T1133 http://purl.obolibrary.org/obo/MONDO_0004975 58033 58035 AD +T1134 CHEBI:60425 58093 58100 amyloid +T1135 NCBITaxon:10088 58129 58134 mouse +T1136 UBERON:0000955 58135 58140 brain +T1137 UBERON:0000955 58203 58208 brain +T1138 UBERON:0000955 58252 58257 brain +T1139 http://purl.obolibrary.org/obo/MONDO_0004975 58471 58488 Alzheimer disease +T1140 GO:0007568 58612 58617 Aging +T1141 http://purl.obolibrary.org/obo/MONDO_0004975 58642 58659 Alzheimer disease +T1142 http://purl.obolibrary.org/obo/MONDO_0004975 58694 58705 Alzheimer's +T1143 PR:000003199 58951 58958 CaMKIIα +T1144 NCBITaxon:10088 58963 58967 mice +T1145 NCBITaxon:33208 59253 59259 animal +T1146 GO:0042571 59422 59432 antibodies +T1147 GO:0042571 59516 59524 antibody +T1148 GO:0042571 59554 59562 antibody +T1149 http://purl.obolibrary.org/obo/MONDO_0004975 59621 59640 Alzheimer's Disease +T1150 http://purl.obolibrary.org/obo/MONDO_0005090 59702 59715 Schizophrenia +T1151 http://purl.obolibrary.org/obo/MONDO_0002050 59720 59730 Depression +T1152 http://purl.obolibrary.org/obo/MONDO_0004975 59802 59813 Alzheimer's +T1153 GO:0007568 59874 59879 Aging +T1154 UBERON:0001016 59999 60009 Neurologic +T1155 http://purl.obolibrary.org/obo/MONDO_0005071 59999 60017 Neurologic Disease +T1156 http://purl.obolibrary.org/obo/MONDO_0004992 60068 60074 Cancer +T1157 NCBITaxon:33208 60164 60170 animal +T1158 http://purl.obolibrary.org/obo/MONDO_0004975 60368 60370 AD +T1159 http://purl.obolibrary.org/obo/MONDO_0004975 60373 60390 Alzheimer disease +T1160 PR:000004168 60399 60416 amyloid precursor +T1161 PR:000004168 60431 60434 APP +T1162 PR:000004168 60437 60462 amyloid precursor protein +T1163 PR:000003199 60464 60471 CaMKIIα +T1164 PR:000003199 60474 60503 calcium-calmodulin kinase IIα +T1165 CHEBI:50845 60505 60508 dox +T1166 CHEBI:50845 60511 60522 doxycycline +T1167 CHEBI:30751 60524 60526 FA +T1168 CHEBI:30751 60529 60540 formic acid +T1169 PR:000007939 60542 60546 GFAP +T1170 CL:0000125 60549 60554 glial +T1171 PR:000007939 60549 60580 glial fibrillary acidic protein +T1172 CHEBI:37527 60566 60572 acidic +T1173 NCBITaxon:10088 60582 60584 mo +T1174 NCBITaxon:10088 60596 60601 mouse +T1175 PR:000004168 60602 60605 APP +T1176 SO:0000417 60626 60632 domain +T1177 CHEBI:8984 60667 60670 SDS +T1178 CHEBI:8984 60673 60695 sodium dodecyl sulfate +T1179 CHEBI:27902 60730 60742 tetracycline +T1180 GO:0010468 60789 60799;60815 60825 Control of ... Expression +T1181 PR:000004168 60811 60814 APP +T1182 CHEBI:50845 60829 60832 Dox +T1183 PR:000004168 60870 60873 APP +T1184 NCBITaxon:9606 60884 60889 human +T1185 GO:0042571 60904 60912 antibody +T1186 GO:0010467 60919 60929 expression +T1187 UBERON:0001890 60956 60965 forebrain +T1188 UBERON:0000479 60966 60972 tissue +T1189 NCBITaxon:33208 61013 61020 animals +T1190 CHEBI:50845 61062 61065 dox +T1191 NCBITaxon:33208 61087 61094 animals +T1192 SO:0000902 61115 61124 transgene +T1193 GO:0010467 61125 61135 expression +T1194 NCBITaxon:33208 61173 61180 animals +T1195 CHEBI:50845 61202 61205 dox +T1196 NCBITaxon:33208 61295 61302 animals +T1197 NCBITaxon:10088 61329 61333 mice +T1198 GO:0007567 61334 61338 born +T1199 CHEBI:50845 61353 61356 dox +T1200 GO:0042571 61398 61406 antibody +T1201 CHEBI:50845 61473 61476 dox +T1202 PR:000004168 61495 61498 APP +T1203 NCBITaxon:10088 61537 61541 mice +T1204 PR:000004168 61626 61629 APP +T1205 CHEBI:50845 61668 61671 dox +T1206 NCBITaxon:33208 61712 61719 animals +T1207 CHEBI:50845 61743 61746 dox +T1208 CHEBI:50845 61768 61771 dox +T1209 PR:000004168 61854 61857 APP +T1210 CHEBI:50845 61880 61883 dox +T1211 CHEBI:50845 61895 61898 dox +T1212 PR:000004168 62067 62070 APP +T1213 CHEBI:50845 62086 62089 dox +T1214 PR:000004168 62102 62105 APP +T1215 NCBITaxon:10088 62106 62110 mice +T1216 NCBITaxon:10088 62162 62166 mice +T1217 NCBITaxon:33208 62276 62283 animals +T1218 NCBITaxon:10088 62355 62359 mice +T1219 SO:0000902 62476 62485 Transgene +T1220 UBERON:0001851 62499 62507 Cortical +T1221 PR:000004168 62547 62550 APP +T1222 NCBITaxon:10088 62551 62555 mice +T1223 CHEBI:8984 62668 62671 SDS +T1224 CHEBI:30751 62681 62683 FA +T1225 NCBITaxon:9606 62696 62701 human +T1226 SO:0000902 62731 62740 transgene +T1227 CHEBI:50845 62893 62896 dox +T1228 NCBITaxon:33208 63034 63041 animals +T1229 NCBITaxon:33208 63089 63096 animals +T1230 NCBITaxon:33208 63147 63154 animals +T1231 CHEBI:60425 63201 63208 amyloid +T1232 CHEBI:8984 63249 63252 SDS +T1233 SO:0000902 63282 63291 transgene +T1234 CHEBI:8984 63379 63382 SDS +T1235 CHEBI:50845 63421 63424 dox +T1236 NCBITaxon:33208 63457 63464 animals +T1237 NCBITaxon:33208 63576 63583 animals +T1238 NCBITaxon:10088 63732 63736 mice +T1239 GO:0007567 63737 63741 born +T1240 CHEBI:50845 63756 63759 dox +T1241 CHEBI:30751 63861 63863 FA +T1242 NCBITaxon:33208 63959 63966 animals +T1243 CHEBI:30751 64146 64148 FA +T1244 CHEBI:30751 64246 64248 FA +T1245 CHEBI:50845 64304 64307 dox +T1246 CHEBI:50845 64348 64351 dox +T1247 SO:0000902 64501 64510 transgene +T1248 NCBITaxon:33208 64539 64546 animals +T1249 GO:0007567 64547 64551 born +T1250 CHEBI:50845 64566 64569 dox +T1251 NCBITaxon:33208 64614 64621 animals +T1252 SO:0000902 64761 64770 transgene +T1253 GO:0043043 64798 64810;64816 64824 synthesis of ... peptides +T1254 CHEBI:50845 64832 64835 dox +T1255 NCBITaxon:33208 65008 65015 animals +T1256 CHEBI:8984 65067 65070 SDS +T1257 CHEBI:30751 65075 65077 FA +T1258 NCBITaxon:10088 65158 65162 mice +T1259 SO:0000902 65243 65252 Transgene +T1260 NCBITaxon:10088 65274 65278 Mice +T1261 CHEBI:60425 65296 65303 Amyloid +T1262 UBERON:0001851 65319 65327 Cortical +T1263 NCBITaxon:33208 65361 65368 animals +T1264 NCBITaxon:9606 65447 65452 human +T1265 GO:0042571 65462 65470 antibody +T1266 SO:0000902 65487 65496 transgene +T1267 CHEBI:50845 65532 65535 dox +T1268 CHEBI:18421 65592 65602 superoxide +T1269 PR:000015399 65592 65614 superoxide dismutase 1 +T1270 PR:000015399 65616 65620 SOD1 +T1271 PR:000004168 65733 65736 APP +T1272 CHEBI:50845 65796 65799 dox +T1273 NCBITaxon:33208 65887 65894 animals +T1274 SO:0000902 66023 66032 transgene +T1275 CHEBI:60425 66092 66099 amyloid +T1276 CHEBI:60425 66231 66238 amyloid +T1277 NCBITaxon:33208 66345 66352 animals +T1278 CHEBI:50845 66363 66366 dox +T1279 CHEBI:50845 66431 66434 Dox +T1280 SO:0000902 66460 66469 transgene +T1281 PR:000004168 66503 66506 APP +T1282 NCBITaxon:10088 66507 66511 mice +T1283 NCBITaxon:10088 66599 66603 mice +T1284 CHEBI:50845 66626 66629 dox +T1285 PR:000004168 66683 66686 APP +T1286 GO:0042571 66718 66726 antibody +T1287 MOP:0000780 66761 66769 cleavage +T1288 CHEBI:18421 66856 66866 superoxide +T1289 PR:000015399 66856 66878 superoxide dismutase 1 +T1290 PR:000004168 67012 67015 APP +T1291 CHEBI:60425 67039 67046 Amyloid +T1292 UBERON:0001851 67094 67102 cortical +T1293 UBERON:0000479 67103 67109 tissue +T1294 CHEBI:50845 67115 67118 dox +T1295 PR:000004168 67143 67146 APP +T1296 NCBITaxon:10088 67147 67151 mice +T1297 NCBITaxon:10088 67607 67611 mice +T1298 SO:0000902 67731 67740 transgene +T1299 NCBITaxon:10088 67809 67813 mice +T1300 NCBITaxon:33208 67856 67863 animals +T1301 CHEBI:50845 67877 67880 dox +T1302 NCBITaxon:10088 68070 68074 mice +T1303 NCBITaxon:10088 68138 68142 mice +T1304 CHEBI:60425 68167 68174 Amyloid +T1305 NCBITaxon:10088 68222 68226 mice +T1306 CHEBI:60425 68354 68361 Amyloid +T1307 NCBITaxon:33208 68431 68438 animals +T1308 SO:0000902 68462 68471 transgene +T1309 NCBITaxon:10088 68483 68487 mice +T1310 CHEBI:50845 68522 68525 dox +T1311 CHEBI:50845 68542 68545 dox +T1312 NCBITaxon:33208 68566 68573 animals +T1313 CHEBI:60425 68612 68619 amyloid +T1314 NCBITaxon:10088 68732 68736 Mice +T1315 PR:000004168 68809 68812 APP +T1316 NCBITaxon:10088 68822 68826 mice +T1317 NCBITaxon:33208 68890 68897 animals +T1318 CHEBI:50845 68911 68914 dox +T1319 PR:000004168 68955 68958 APP +T1320 UBERON:0001851 69003 69011 Cortical +T1321 CHEBI:8984 69091 69094 SDS +T1322 CHEBI:30751 69104 69106 FA +T1323 NCBITaxon:9606 69119 69124 human +T1324 SO:0000902 69154 69163 transgene +T1325 UBERON:0000955 69263 69269 brains +T1326 NCBITaxon:10088 69288 69292 mice +T1327 CHEBI:30751 69315 69317 FA +T1328 CHEBI:8984 69322 69325 SDS +T1329 CHEBI:60425 69353 69360 amyloid +T1330 CHEBI:8984 69395 69398 SDS +T1331 CHEBI:30751 69404 69406 FA +T1332 NCBITaxon:10088 69449 69453 mice +T1333 NCBITaxon:10088 69507 69511 mice +T1334 CHEBI:8984 69582 69585 SDS +T1335 CHEBI:30751 69590 69592 FA +T1336 SO:0000902 69660 69669 transgene +T1337 NCBITaxon:10088 69792 69796 mice +T1338 NCBITaxon:10088 69889 69893 mice +T1339 PR:000004168 69933 69936 APP +T1340 NCBITaxon:10088 69963 69967 mice +T1341 CHEBI:50845 70138 70141 dox +T1342 NCBITaxon:10088 70150 70154 mice +T1343 NCBITaxon:10088 70194 70198 mice +T1344 NCBITaxon:10088 70296 70300 mice +T1345 NCBITaxon:10088 70712 70716 mice +T1346 NCBITaxon:10088 70780 70784 mice +T1347 GO:0043005 70815 70823 Neuritic +T1348 CL:0000125 70828 70833 Glial +T1349 SO:0000902 70868 70877 Transgene +T1350 GO:0043005 70902 70910 neurites +T1351 CL:0002627 70915 70935 activated astrocytes +T1352 PR:000004168 70977 70980 APP +T1353 NCBITaxon:10088 70981 70985 mice +T1354 GO:0043005 71029 71037 neurites +T1355 CL:0000127 71051 71061 astrocytes +T1356 NCBITaxon:10088 71158 71162 mice +T1357 SO:0000902 71239 71248 transgene +T1358 PR:000007939 71293 71297 GFAP +T1359 SO:0000902 71389 71398 Transgene +T1360 PR:000004168 71443 71446 APP +T1361 NCBITaxon:10088 71447 71451 Mice +T1362 GO:0040011 71475 71485 ambulation +T1363 NCBITaxon:10088 71547 71551 mice +T1364 NCBITaxon:10088 71694 71698 mice +T1365 CHEBI:50845 71702 71705 dox +T1366 CHEBI:50845 71796 71799 dox +T1367 PR:000004168 72034 72037 APP +T1368 NCBITaxon:33208 72038 72045 animals +T1369 http://purl.obolibrary.org/obo/MONDO_0019065 72222 72233 amyloidosis +T1370 GO:0034205 72259 72272 Aβ production +T1371 http://purl.obolibrary.org/obo/MONDO_0004975 72298 72315 Alzheimer disease diff --git a/src/ontogpt/evaluation/craft/database/all/16279840.txt b/src/ontogpt/evaluation/craft/database/all/16279840.txt new file mode 100644 index 000000000..9ab8abbc5 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16279840.txt @@ -0,0 +1,345 @@ +Persistent Amyloidosis following Suppression of Aβ Production in a Transgenic Model of Alzheimer Disease + +Abstract + +Background + +The proteases (secretases) that cleave amyloid-β (Aβ) peptide from the amyloid precursor protein (APP) have been the focus of considerable investigation in the development of treatments for Alzheimer disease. The prediction has been that reducing Aβ production in the brain, even after the onset of clinical symptoms and the development of associated pathology, will facilitate the repair of damaged tissue and removal of amyloid lesions. However, no long-term studies using animal models of amyloid pathology have yet been performed to test this hypothesis. + +Methods and Findings + +We have generated a transgenic mouse model that genetically mimics the arrest of Aβ production expected from treatment with secretase inhibitors. These mice overexpress mutant APP from a vector that can be regulated by doxycycline. Under normal conditions, high-level expression of APP quickly induces fulminant amyloid pathology. We show that doxycycline administration inhibits transgenic APP expression by greater than 95% and reduces Aβ production to levels found in nontransgenic mice. Suppression of transgenic Aβ synthesis in this model abruptly halts the progression of amyloid pathology. However, formation and disaggregation of amyloid deposits appear to be in disequilibrium as the plaques require far longer to disperse than to assemble. Mice in which APP synthesis was suppressed for as long as 6 mo after the formation of Aβ deposits retain a considerable amyloid load, with little sign of active clearance. + +Conclusion + +This study demonstrates that amyloid lesions in transgenic mice are highly stable structures in vivo that are slow to disaggregate. Our findings suggest that arresting Aβ production in patients with Alzheimer disease should halt the progression of pathology, but that early treatment may be imperative, as it appears that amyloid deposits, once formed, will require additional intervention to clear. + +Introduction + +Over a decade ago the amyloid cascade hypothesis predicted that increased levels of amyloid-β (Aβ) peptide lead to secondary pathologies that ultimately culminate in the onset of Alzheimer disease (AD) [1]. Early support for this hypothesis came from genetic studies linking early-onset AD to mutations in the amyloid precursor protein (APP), from which Aβ is derived, and presenilins 1 and 2, which are interchangeable components of a endoprotease complex that releases Aβ from APP (for review see [2,3]). If, as predicted, overproduction of Aβ initiates the cascade of events leading to disease, then therapeutic strategies that lower Aβ levels should either arrest or reverse the progression from peptide to dementia. + +Early evidence from clinical trials of antibody-mediated clearance, one of the first Aβ-lowering approaches tested in humans, suggested that treatments designed to reduce amyloid burden may indeed be beneficial. Although the trials were halted because of adverse effects in a subset of volunteers [4,5], further analysis of several patients found evidence that amyloid pathology, and to a lesser degree cognitive function, was improved in proportion to the patient's titer of Aβ-specific antibody [6,7]. While this approach is promising, constant exposure to antibodies that recognize an epitope highly enriched in the brain may have unexpected side effects that will limit its long-term use. + +An alternative approach that is being actively pursued for future treatment of AD seeks to lower Aβ levels by limiting its production from the precursor protein APP. Peptide Aβ is released from APP by the action of two enzymes, the β-APP cleaving enzyme 1 (BACE1) and γ-secretase, which cleave the holoprotein at the N- and C-termini of Aβ, respectively. Several inhibitors of γ-secretase have already been produced [8,9], and small molecule inhibitors of β-APP cleaving enzyme 1 are currently being developed [10,11]. The long-term effectiveness of this approach in either humans or model systems, however, has not been reported. Although loss of β-APP cleaving enzyme 1 function can prevent the development of plaques in transgenic mouse models for AD (F. Laird, H. Cai, P. C. Wong, personal communication), it is not known whether the brain can clear pre-existing amyloid deposits once production of Aβ has been suppressed. + +Clearly, the amyloid-lowering approach should be rigorously examined in animal models before these reagents are tested in patients. However, the chemical secretase inhibitors most likely to reach human trials are still in development. Therefore, we developed a mouse model of Alzheimer-type amyloid that expresses a controllable APP transgene. This system, commonly known as the tet-off system, can be regulated by analogs of tetracycline administered in food or water [12,13]. The strong expression levels produced with the tet-off vectors, combined with the ability to reduce this expression by several orders of magnitude with tetracycline [14], allowed for a stringent test of how a highly effective pharmaceutical inhibitor of Aβ production would impact the progression of amyloid pathology and whether reversal of these lesions might be possible following such treatment. + +Methods + +Transgene Construction + +We created a tetracycline-responsive chimeric mouse/human APP695Swedish/Indiana (swe/ind) vector by replacing the promoter region of the moPrP.XhoI vector (also known as pPrPpE1/E2,3sal [15]) with the tetracycline-responsive promoter of pTetSplice (Life Technologies, Rockville, Maryland, United States), and then ligating mouse APP with a humanized Aβ domain (mo/huAPP695) cDNA into the new vector. We began by cloning the tetracycline-responsive promoter (bp 6–481) from pTetSplice by PCR using primers that added external BamHI and NotI sites to the 5′ end and a BamHI site to the 3′ end, while destroying XhoI and BamHI sites within the promoter (forward: GCC GGA TCC GCG GCC GCC GTC GAG TTT ACC ACT CCC TAT C; reverse: GCC GGA TCC ACT CTA GAA GAT CCC CGG GTA CCG). We then isolated the moPrP.XhoI intron by amplification with primers that added an external BamHI site to the 5′ end of exon 1 and ran through the Asp718 site of exon 2 (forward: GCC GGA TCC GAT CAG CAG ACC GAT TCT GG; reverse: GCC GGT ACC ACT AGG AAG GCA GAA TGC). This 2-kb intron fragment was cloned into a TA cloning vector (Invitrogen, Carlsbad, California, United States), then excised by Asp718 digestion and ligated to the 6.8-kb Asp718 fragment of moPrP.XhoI containing exon 2, exon 3, the 3′ UTR, and pBluescript to generate an intermediate vector with all three exons and a central intron but no promoter. This vector was then opened at the BamHI site introduced by the intron cloning primer, and ligated to the 0.5-kb BamHI-cut tetracycline promoter fragment. This ligation generated a 9.3-kb vector encoding the tetracycline promoter from pTetSplice with two exons, one intron, and the original 3′ UTR of the moPrP.XhoI vector, all carried in the pBluescript cloning vector. + +We incorporated the Swedish (KM570/571NL) and Indiana (V617F) mutations into the mo/huAPP695 cDNA (in BS-KS) by PCR using a four-primer strategy: first, two partially overlapping products were generated in separate reactions using primers that encode the desired mutations (Swedish forward: GGA GAT CTC TGA AGT GAA TCT GGA TGC AGA ATT CCG/Indiana reverse: GGG TGA TGA AAA TCA CGG TTG C; Indiana forward: CAA CCG TGA TTT TCA TCA CCC TGG/M13 reverse). The two PCR products were ligated, digested with BglII and ApaI and cloned back into the original mo/huAPP695-BS-KS vector. Finally, the new APP695swe/ind was subcloned into the XhoI site of the moPrP-tetP vector from above to complete the construct. + +Pronuclear Injection, Screening of Founders, and Maintenance of the Lines + +The moPrP-tetP-mo/huAPP695swe/ind vector was linearized and the pBluescript domain excised by digestion with NotI. The purified vector was injected into the pronucleus of fertilized eggs from C57BL/6J × C3HeJ F1 matings. Founder animals were screened for the presence of the transgene by three-way PCR using the S36 and PrP-S/PrP-AS primers described below. Transgene-positive founders were bred to animals expressing the tetracycline transactivator (tTA) under control of the calcium-calmodulin kinase IIα (CaMKIIα) promoter obtained from Jackson Laboratory [16] (Bar Harbor, Maine, United States; stock # 3010; B6;CBA-TgN[Camk2a-tTA]1Mmay). The colony was thereafter maintained by crossing single transgenic tTA and APP offspring for each of the four APP lines. All mice were provided fresh food and water ad libitum. Animal protocols were approved by both the Johns Hopkins University and the California Institute of Technology Institutional Animal Care and Use Committees. + +Doxycycline Administration + +Doxycycline (dox) was administered through commercially available dox-containing chow (BioServ, Frenchtown, New Jersey, United States). The chow contained 200 mg/kg of antibiotic; based on estimated consumption of 5 g per mouse per day, the expected dose to each animal was 1 mg dox per day. The average 25-g animal therefore received 40 μg of dox per gram body weight per day. Chow was changed 1–2 times per week to prevent breakdown of the antibiotic. + +Genotyping + +Offspring were genotyped for the presence of each transgene by PCR amplification of genomic DNA extracted from a 5-mm tail biopsy. Tails were heated to 95 °C for 45 min in 250 μl of 50 mM NaOH, vortexed, then neutralized with an equal volume of 0.5 M Tris-HCl (pH 5.5). Debris was sedimented by centrifugation, and 3 μl of supernatant was used for amplification. + +Genotyping for APP and tTA transgenes was performed in the same PCR reaction, using five separate primers. APP was amplified using forward primer S36 located in the 3′ end of the APP cDNA (CCG AGA TCT CTG AAG TGA AGA TGG ATG) and reverse primer PrP-AS-J located in the 3′ UTR of the vector (CCA AGC CTA GAC CAC GAG AAT GC). The tTA transgene was detected using a primer set that amplified across its two subdomains with tet forward located within the Tn10 tetracycline repressor (CGC TGT GGG GCA TTT TAC TTT AG) and tet reverse within the HSV1 VP16 (CAT GTC CAG ATC GAA ATC GTC). All reactions, whether transgene-positive or not, amplified a segment of the endogenous prion protein gene as a control for DNA quality using a forward primer, PrP-S-J, specific to the mouse PrP open reading frame (GGG ACT ATG TGG ACT GAT GTC GG) and a reverse primer, PrP-AS-J, shared by the 3′ UTR of the endogenous PrP gene and the transgene vector. Amplification reactions were run for 37 cycles at 94 °C for 30 s, 64 °C for 1 min, and 72 °C for 1 min. All samples, transgenic and wild-type, gave a 750-bp product from the endogenous PrP gene. The APP transgene yielded an additional band at 400 bp; the tTA product fell in between at 480 bp. + +Immunoblotting/Quantitation + +Frozen cortical or whole forebrain tissue was homogenized by sonication in five volumes of phosphate-buffered saline (PBS) with 5 mM EDTA and protease inhibitors (Mammalian cell cocktail, Sigma, St. Louis, Missouri, United States), using a probe sonicator set to 50% output (TEKMAR, Cincinnati, Ohio, United States). After dilution with an equal volume of PBS/EDTA/protease inhibitor, the samples were centrifuged briefly and the supernatant used for analysis. Fifty micrograms (6E10 and CT15) or 5 μg (22C11) of brain homogenate was loaded per lane onto 7.5%, 10%–20%, or 4%–20% Tris-HCl PAGE gels (Bio-Rad Laboratories, Hercules, California, United States) and electrophoresed for several hours in 1× Tris-glycine–sodium dodecyl sulfate (1×TG-SDS) buffer (6E10 and 22C11; Amresco, Solon, Ohio, United States) or 1× Tris-tricine-SDS buffer (CT15; Invitrogen, Carlsbad, California, United States). Proteins were transferred overnight to 0.45-μm Optitran nitrocellulose (Schleicher and Schuell, Keene, New Hampshire, United States) in 1× TG buffer (Amresco). Blots were blocked in PBS containing 5% nonfat dry milk powder, and incubated for 3 h at room temperature in blocking solution with one of the following antibodies: mouse monoclonal 22C11 (kind gift of Konrad Beyreuther and Andreas Weidemann; [17]) diluted 1:1,000, mouse monoclonal 6E10 (Signet Laboratories, Dedham, Massachusetts, United States) diluted 1:2,500, rabbit polyclonal anti-superoxide dismutase 1 (m/hSOD1) [18] diluted 1:2,500 to 1:4,000, or rabbit polyclonal CT15 (kind gift of Ed Koo; [19]) diluted 1:1,000. Subsequently, the blots were washed with PBS containing 0.1% Tween-20, and then incubated with either goat anti-mouse– or goat anti-rabbit–HRP conjugated secondary antibody diluted 1:1,000 in blocking solution. After several additional rinses in PBS with 0.1% Tween-20, blots were developed with enhanced chemiluminescence reagent and imaged with the Bio-Rad Molecular Imager FX system. + +Staining intensity within each lane was quantified using the Quantity One image analysis software (Molecular Imager FX, Bio-Rad Laboratories). Background was calculated from across the image and subtracted from the entire file. The signal intensity for each band (corrected signal intensity × pixel number) was then calculated using the Volume report tool. + +Slot Blot mRNA Analysis + +Five micrograms per sample of total RNA extracted from fresh-frozen brain, liver, kidney, heart, lung, spleen, and skeletal muscle was vacuum-filtered through 0.45-μm Optitran nitrocellulose. After several washes through the manifold with 10× SSC, blots were UV-cross-linked and probed with a radiolabeled ∼350-bp BglII–XhoI cDNA fragment of mo/huAPP695 cDNA. After hybridizing overnight at 65 °C in 1% BSA/1 mM EDTA/0.5 M sodium phosphate buffer (pH 7.2)/7% SDS [20], the blots were washed twice at 65 °C for 30 min each in 0.1% BSA/1 mM EDTA/40 mM sodium phosphate buffer (pH 7.2)/5% SDS before two final 30-min washes at 65 °C with 1 mM EDTA/40 mM sodium phosphate buffer (pH 7.2)/1% SDS. Blots were wrapped wet and exposed to phosphorscreens overnight at room temperature. + +Amyloid Histology + +Mice were euthanized by ether inhalation and brains removed for immersion fixation in 4% paraformaldehyde/1× PBS. After 48 h in fixative at 4 °C, brains were transferred to PBS, dehydrated in alcohols, treated with cedarwood oil and methylsalicylate, and embedded in paraffin for sectioning. + +Hirano silver stain + +Silver impregnation histology was performed on 10-μm paraffin-embedded sections by Hirano's modification of the Bielschowsky method [21]. Briefly, sections were deparaffinized through xylene and alcohols into tap water before being placed into fresh 20% silver nitrate solution for 20 min. After being washed thoroughly with distilled water, slides were immersed in 20% silver nitrate solution titrated with fresh ammonium hydroxide. After 20 min, slides were washed with ammonia water before being individually developed with 100 μl of developer (20 ml of 37% formaldehyde, 100 ml of distilled water, 50 μl of concentrated nitric acid, and 0.5 g of citric acid) added to 50 ml of titrated silver nitrate solution. Slides were then rinsed in tap water, fixed in 5% sodium thiosulfate, and dehydrated through alcohols and xylene. + +Thioflavin-S staining + +Following deparaffinization of sections through xylene and alcohols, amyloid impregnation with thioflavin-S was performed according to the Guntern modification of the standard protocol. Slides holding 10-μm paraffin sections were washed twice in distilled water, then immersed for 5 min in a 0.25% potassium permanganate solution, followed by 5 min in a 1% potassium metabisulfate/1% oxalic acid solution. After this preparation, slides were placed into a filtered aqueous 0.02% thioflavin-S solution (Chroma-Gesellschaft Schmid, Kongen, Germany) for 8 min. Excess stain was removed by two brief rinses in 80% ethanol, then two in distilled water, after which slides were finished in aqueous mounting medium for florescence photomicrography. + +Ubiquitin, glial fibrillary acidic protein, and Aβ immunohistochemistry + +Prior to immunostaining, slides were deparaffinized by oven heating followed by immersion in xylene. After rehydration through graded alcohols into tap water, endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide in methanol. Slides were microwaved for 5–7 min in water, cooled for 5 min, then washed in TBS. Nonspecific staining was blocked for 1 h with 3% normal goat serum and 0.1% Triton-X 100 in TBS. Slides were then placed into primary antibody (rabbit anti-Aβ peptide polyclonal antibody, Zymed Laboratories, South San Francisco, California, United States; rabbit anti-ubiquitin and rabbit anti–glial fibrillary acidic protein (GFAP) polyclonal antibodies, Dako, Carpinteria, California, United States) diluted 1:500 in TBS with 2% normal goat serum and incubated overnight at room temperature. After being washed of excess primary antibody with several changes of TBS, slides were incubated with either the Vectastain Elite anti-rabbit secondary system (anti-Aβ; Vector Laboratories, Burlingame, California, United States) or peroxidase/anti-peroxidase reagents (anti-ubitquitin and anti-GFAP; Sternberger Monoclonals, Lutherville, Maryland, United States) according to the manufacturers' directions. Antibody binding was visualized with diaminobenzidene, and sections were counterstained with hematoxylin. + +Filter Trap Assay + +An aliquot of each cortical homogenate used for Western blotting above was partially solubilized by the addition of SDS to a final concentration of 1%. Serial 1:2 dilutions were made with 1× PBS/1% SDS, and 100 μl of each dilution was then vacuum-filtered through a pre-wet 0.22-μm cellulose acetate membrane (Schleicher and Schuell) [22]. Each well was washed several times with PBS, after which blots were incubated overnight with polyclonal anti-Aβ antibody (Zymed Laboratories) diluted 1:600 in a blocking solution of 1× TBS/5% nonfat dry milk powder. After washing the blots three times for 10 min each in 1× TBS/0.1% Tween-20, the membrane was incubated for 1 h with HRP-conjugated protein A (Sigma) diluted 1:5,000 in blocking solution. The membranes were again washed three times with 1× TBS/0.1% Tween-20, before antibody binding was detected with enhanced chemiluminescence (PerkinElmer, Boston, Massachusetts, United States). Digital images of each blot were captured with a Molecular Imager FX gel documentation system, and the intensity of Aβ staining was quantified using Quantity One image analysis software. + +Aβ ELISA + +An aliquot of cortical homogenate generated for Western analysis described above was subjected to a three-step sequential extraction using PBS, 2% SDS, and 70% formic acid (FA). At each step, the sample was sonicated in appropriate buffer and centrifuged at 100,000g for 30 min (1- to 1.5-mo samples) or 60 min (6- to 12-mo samples) at 4 °C as previously described [23–25]. The supernatant was removed for analysis, and the pellet was sonicated in the next solution in sequence. The 2% SDS extracts were diluted in EC buffer, and the FA extracts neutralized with 1M Tris-phosphate buffer (pH 11) then diluted with EC buffer prior to testing. Human Aβ was measured in each fraction using BAN50 for capture (epitope Aβ1–16) and BA27 and BC05 for detection (Aβ40 and Aβ42, respectively) (Takeda Chemical Industries, Osaka, Japan). Total Aβ (mouse + human; 1- to 1.5-mo samples only) was measured in each fraction using BNT77 for capture (epitope Aβ11–28) and BA27 and BC05 for detection. All values were calculated as picomoles per gram based on the initial weight of cortical tissue. + +Activity Monitoring + +Daily basal activity was studied in 28 CaMKIIα-tTA × tet-APPswe/ind line 107 mice between 4 and 5 mo of age. Animals were separated into individual cages immediately before the start of each experiment (n = 3–6 per genotype untreated, 2–5 per genotype dox-reared). The cages were placed inside activity-monitoring frames designed to count every time the animal passed through one of three photobeams spanning the width of the cage (San Diego Instruments, San Diego, California, United States). Experiments were started midway through the light phase of the day, and data were collected in 1-h bins for the following 48 h. Testing rooms were maintained on the same 13:11 h day:night cycle as the main animal housing areas and were closed to entry during the experiment. + +Statistical Analyses + +Statistical analyses of protein expression, ELISA data, and filter trap assays were performed by ANOVA with Tukey's honest significant difference post-hoc test applied to significant main effects or interactions (Statistica 6.0, StatSoft, Tulsa, Oklahoma, United States). In cases of positively skewed data distribution, log10(x + 0.5) transformation was applied to the raw data before submitting them to ANOVA. + +Results + +We used the tet-off transgene system to express a double mutant version of chimeric mo/huAPP695 (swe/ind KM570, 571NL, and V617F) from a tetracycline-responsive promoter [12,13]. Transgenic APP expression was activated by crossing the APPswe/ind mice to animals producing tTA under control of the CaMKIIα promoter [16]. After initial screening of founders, we identified four lines of tet-APPswe/ind mice that produced very high levels of transgene product in offspring coexpressing tTA (Figures 1 and Figure S1). Compared to a standard APP transgenic line used for previous amyloid studies by our laboratory (line C3–3; [15,26,27]), we estimated that the four controllable lines produce transgenic APP protein at 10- to 30-fold over endogenous levels (Figure S1). This estimate was confirmed by direct comparison of APP levels in nontransgenic and tet-off APP mice using an antibody that recognizes both endogenous APP (and amyloid precursor-like protein 2) and the transgenic protein (monoclonal antibody 22C11; Figure 1D). + +Importantly, all four new lines of tet-off APP mice showed nearly complete suppression of the transgene following dox treatment (Figures 1 and Figure S1). We focused on one of the four lines, line 107, to examine in more detail the time dependence and extent of transgene suppression following either acute or chronic treatment with dox. Two dox-treated groups were compared to two untreated groups: one group of mice was born and raised on dox, a second group was treated with dox for 2 wk starting at 1 mo of age (4 wk + 2 wk dox); two untreated groups kept on normal chow were harvested at either 4 or 6 wk of age. Animals born and raised on dox harbored no transgenic APP (Figure 1A). Following as little as 2 wk of dox treatment, transgenic APP expression was reduced by more than 95% compared to pre-dox levels. The residual expression remaining in acutely treated mice represents less than 4% of the transgenic protein produced in the absence of dox (Figure 1C), and likely results from slight leakage at the level of transcription (data not shown). Importantly, the total amount of APP (endogenous plus transgenic) and related APLPs in both acute and chronically treated animals was statistically indistinguishable from that in nontransgenic mice (Figure 1D; statistical analyses for experiments throughout the study are presented in the accompanying figure legends). + +To ensure that Aβ production was suppressed in concert with the dox-mediated inhibition of its precursor APPswe/ind, we measured Aβ40 and Aβ42 levels by ELISA in forebrain homogenates from young tet-off animals. At 1 mo of age, the mice lacked visible amyloid aggregates that might act as an intractable reservoir of peptide remaining in the brain after the transgene had been suppressed. To further ensure we could detect any such insoluble aggregates that might bias our measure of changes in peptide synthesis, we performed a sequential three-step extraction with PBS, 2% SDS, and 70% FA that would separate peptides by solubility. We compared the levels of human transgene-derived Aβ40 and Aβ42 in untreated mice at 4 and 6 wk of age to animals that had either been born and raised on dox or that had been left untreated for 4 wk and then placed on dox chow for 2 wk prior to harvest (the same groups described above for immunoblot analysis of APPswe/ind levels, line 107). Consistent with the reduction in full-length APPswe/ind synthesis shown by immunoblot (see Figure 1), we found that transgene-derived Aβ levels were completely suppressed in animals born and raised on dox, and were sharply reduced following acute (2 wk) antibiotic treatment. Compared to the levels in untreated 4-wk-old mice, PBS-soluble Aβ42 dropped by 95.2% following 2 wk of dox treatment and by 99.2% with chronic treatment (Figure 2A). Similarly, SDS-soluble Aβ42 decreased by 75.2% and 94.8% following 2-wk or lifelong dox treatment (Figure 2B). Only the FA fraction revealed a small dox-resistant pool of peptide in acutely treated animals that we believe represents stable predeposit aggregates that have already accumulated by 4 wk of age when treatment was begun (Figure 2C). Indeed, animals that were born and raised on dox did not harbor this reservoir of treatment-resistant peptide, with 96.3% less Aβ42 than untreated 4-wk-old mice. Measurement of total Aβ in chronically treated mice, including endogenous and transgene-derived peptide, demonstrated that Aβ levels in tet-off APP mice were reduced to the level of endogenous peptide found in nontransgenic animals (Figure 2D). Taken together with the immunoblotting data for full-length APPswe/ind, the ELISA measurements indicate that dox-mediated suppression of transgenic APPswe/ind synthesis leads to parallel reduction of Aβ levels. + +The ELISA data also confirmed that incorporation of the Swedish and Indiana mutations led to high levels of Aβ42, which we predicted would induce rapid plaque formation in untreated animals. Histological characterization of double transgenic (CaMKIIα-tTA × tet-APPswe/ind) mice revealed early-onset amyloid formation in all four new lines. Amyloid plaques were seen in mice as young as 8 wk of age (data not shown). Plaques were limited to the forebrain, including the cortex, hippocampus, olfactory bulb, and striatum, where the CaMKIIα promoter is known to be most active [16,28] (Figure S2). By 6 mo of age, amyloid burden became severe, covering large areas of the cortex and hippocampus (Figure S3). No lesions were seen in the cerebellum or brain stem even at late ages, consistent with CaMKIIα-controlled transgene expression. Unlike what is thought to occur in the human disease, the first visible plaques in the tet-off APP mice are fibrillar-cored deposits. We have noted the same early appearance of cored deposits in other lines of APP transgenic mice that harbor the Swedish mutation [27]. Diffuse plaques were apparent in 6-mo-old tTA/APP mice, and became relatively abundant by 9 mo of age. At older ages (9–12 mo) amyloid deposits were visible in the thalamus, which has also been observed in mice expressing mutant APP via the Thy-1 promoter. The presence of amyloid pathology in this region has been attributed to axonal transport of APP/Aβ to the terminals of cortical neurons in the thalamus [29]. Most importantly, only double transgenic mice, expressing both the tTA and APP transgenes, developed amyloid lesions. Single transgenic mice up to 15 mo of age showed no sign of pathology (Figure S3). Similarly, amyloid pathology can be completely prevented in double transgenic animals born and raised on dox. Animals from our highest expressing line (line 885) maintained on dox for up to 1 y harbored no amyloid pathology (data not shown), indicating that residual leakage of transgene expression in the presence of dox does not provide sufficient Aβ peptide to induce amyloid formation even over long periods. + +To mimic therapeutic intervention with inhibitors of Aβ production, we raised a group of 25 double transgenic mice (CaMKIIα-tTA × APP line 107) on normal food until 6 mo of age, when we knew amyloid formation was already well underway in the brain. At 6 mo, half of the animals were switched from normal chow to food containing dox at 200 mg/kg until they were sacrificed at 9 or 12 mo of age. The remaining control animals were kept on standard chow (untreated). In all, four cohorts were created: 6 mo untreated (n = 7), 9 mo untreated (n = 5), 6 mo + 3 mo treated (n = 8), and 6 mo + 6 mo treated (n = 5). Full suppression (>95%) of transgenic APPswe/ind levels in the dox-treated animals was confirmed by immunoblot (Figure 3). To ensure that the transgene could be suppressed as rapidly in 6-mo-old mice with fulminant pathology as it can in young, predeposit animals, we treated an additional set of 6-mo-old animals with dox for 1 wk prior to harvest. Importantly, both APPswe/ind and APP–C-terminal fragment levels were fully suppressed after only 1 wk of treatment, indicating that the in vivo half-life of APPswe/ind and its processed C-terminal fragments are relatively short (Figure 3D). + +Tissue sections from each animal in the four treatment groups were stained for amyloid pathology by Hirano silver, Campbell-Switzer silver, thioflavin-S, and Aβ immunohistochemistry. As expected, the 6 mo untreated cohort displayed moderate amyloid pathology, and the 9 mo untreated cohort progressed to a severe amyloid burden. In contrast, the extent of amyloid pathology in mice from the 6 mo + 3 mo treated or 6 mo + 6 mo treated cohorts closely resembled that of the 6 mo untreated cohort, despite the significant age difference between the treated and untreated groups (Figures 4 and Figure S3). Well-formed plaques remained in the treated animals after 6 mo of transgene suppression, even though as much time was given to clear the lesions as they had taken to form. Moreover, both types of amyloid, diffuse and fibrillar, remained intact throughout treatment. Using the Campbell-Switzer silver stain to distinguish different forms of amyloid, we found diffuse plaques were as persistent as cored deposits (Figure S4). It was nevertheless clear that dox-induced suppression of transgenic APP had completely halted the progression of pathology. + +To confirm that the arrest of plaques without any sign of clearance was not unique to the line 107 mice, we repeated the dox-suppression experiment in a second line of tet-off APP mice (CaMKIIα-tTA × tet-APPswe/ind line 18; n = 22). Again, long-term dox treatment was begun at 6 mo of age, and mice were harvested after 3 mo of transgene suppression (6 mo untreated, n = 8; 9 mo untreated, n = 6; 6 mo + 3 mo treated, n = 8). Immunoblotting for APP confirmed full transgene suppression in the treated animals (Figure S5). As in the line 107 mice described above, amyloid burden worsened substantially in the untreated mice between 6 and 9 mo of age. Suppression of transgene expression abruptly arrested progression of pathology (Figure S6), but again without any sign of reduction. Both silver- and thioflavin-S-positive plaques could still be found in each of the dox-treated animals. + +We biochemically measured the amount of aggregated Aβ in the brains of our mice before and after transgene suppression using filter trap analysis of cortical tissue from each animal. In this assay, serial dilutions of protein homogenate are passed through a cellulose acetate filter; particles larger than the pore size of the filter become trapped in the membrane and are revealed by immunoblotting [22]. Consistent with our visual analysis of the histological sections, line 107 tTA/APP mice treated with dox for 3 or 6 mo had the same amount of aggregated Aβ as when they started treatment at 6 mo of age (Figure 4A and 4B). In contrast, untreated 9-mo-old mice had almost twice as much aggregated Aβ as either of the treated groups. Filter trap analysis of line 18 tTA/APP mice yielded similar results: the increase in aggregated Aβ observed in untreated animals between 6 and 9 mo of age was completely arrested by transgene suppression (Figure S5C and S5D). + +We next used ELISA to measure total Aβ in the brains of each group to determine whether any change in the amount or solubility of peptide occurred while APPswe/ind expression was suppressed. Cortical homogenates were sequentially extracted to separate peptide into PBS-, SDS-, and FA-soluble fractions, then transgene-derived Aβ40 and Aβ42 were measured by human-specific ELISA [23]. In all animals harboring amyloid deposits, we found that the vast majority of Aβ (>99%) was extracted into the SDS and FA fractions (Figure 5A and 5B). Consistent with the filter trap results presented above, there were no significant differences in SDS- or FA-soluble Aβ between the 6 mo untreated cohort and either the 6 mo + 3 mo treated or 6 mo + 6 mo treated cohorts. However, brains of both 6 mo + 3 mo and 6 mo + 6 mo treated cohorts contained roughly twice as much PBS-soluble Aβ40 as untreated 6-mo-old mice (Figure 5C). Levels of Aβ42 showed a similar trend, but did not reach statistical significance. In fact, levels of PBS-soluble Aβ40 and Aβ42 in the 6 mo + 3 mo and 6 mo + 6 mo treated cohorts were most similar to that of the 9 mo untreated cohort, suggesting that age, as opposed to synthetic rate (which would be negligible in the treated animals), may determine the fraction of PBS-soluble Aβ in these animals. + +We also assessed neuritic and glial pathology surrounding the plaques to determine whether there were any changes in nearby tissue following long-term transgene suppression. Both Hirano silver stain and ubiquitin immunostaining showed neuritic pathology in all treatment groups (Figure 6). Similarly, activated astrocytes immunostained for GFAP were found near plaques in all animals (Figure 6). Neuritic and glial pathology were more severe in the older untreated mice. In contrast, transgene suppression prevented the growth of individual deposits apparent in untreated mice, and limited the surrounding pathology to what was already present when treatment began. + +An obvious question we sought to address was whether the deposition of Aβ diminished cognitive ability in untreated mice, and what might happen to cognition when the process was interrupted. Unfortunately, efforts to characterize cognitive behavior were compromised by severe hyperactivity in untreated double transgenic mice. The tTA/APP animals were often seen running in circles around the perimeter of their cages, and a similar swimming pattern was noted when the mice were tested in the Morris water maze. In the radial water maze, repetitive swim patterns were noted with no evidence of choice-motivated actions. Other studies have dealt with similar problems by excluding animals that do not show adequate attention to the task, retaining only those mice that meet certain performance criteria [30]. In our case, the penetrance of hyperactivity was close to 100%, leaving us with no testable animals. This phenotype has not affected previous lines of APPswe mice we have produced, such as lines E1–2 or C3–3, that express lower levels of transgenic protein. Indeed, in past studies where hyperactivity was not a factor, we established a clear relationship between amyloid load and cognitive ability [31]. However, in the current study, we feel that although the poor performance of the tTA/APP mice in the maze tests could technically be scored as cognitive impairment, the animals' severe hyperactivity made interpretation of the cognitive tasks impossible. + +In order to understand the nature and extent of hyperactivity in the tTA/APP mice, we quantified daily activity levels in double transgenic animals along with their single transgenic and nontransgenic siblings using four-beam frames designed to monitor ambulation within an enclosed cage. As shown in Figure 7, the double transgenic mice were up to 10-fold more active during the dark phase of the day–night cycle than any of the control groups. Activity levels appeared to follow a relatively normal diurnal cycle, decreasing substantially during the daylight hours. However, even during the light phase, the tTA/APP mice remained many-fold more active than normal controls. This behavior was partially, but not consistently, reversed by 1 mo of transgene suppression beginning at 4–5 mo of age (data not shown). In contrast, hyperactivity was completely abolished by rearing tTA/APP mice on dox. Animals born and raised on dox showed activity levels similar to the untreated controls (Figure 7C). Intriguingly, all of the dox-reared animals, both transgenic and wild-type, showed altered circadian rhythms with far less distinction between their day- and nighttime activity levels. + +Discussion + +We present a new mouse model for AD that was designed to test the consequences of inhibiting Aβ production after the onset of amyloid pathology. New lines of transgenic mice were developed for this study that express high levels of APPswe/ind under the control of a tetracycline-responsive promoter. We demonstrate that treatment with dox suppresses steady-state levels of both APPswe/ind and its C-terminal fragments, indicating that the mutant proteins have a relatively short half-life in vivo. Transgenic expression of APPswe/ind and consequent overproduction of Aβ42 cause early-onset amyloid deposition in untreated mice, in which deposits appear as early as 2 mo of age. Amyloid burden worsens significantly with age, and by 9 mo, the hippocampus and cortex of untreated mice are largely filled with aggregated peptide. We find that suppression of transgenic APP by more than 95% abruptly halts the progression of amyloid pathology. Importantly, this outcome occurs in animals already harboring considerable amyloid pathology, a situation similar to what might be expected in patients to be treated with secretase inhibitors. Somewhat unexpectedly, we observe no appreciable clearance of deposited amyloid even following periods of transgene suppression equal to the time taken for plaques to form. This latter finding indicates that compared to other disease-associated protein aggregates such as mutant huntingtin, which clear in less than 3 mo [32], the disaggregation of extracellular amyloid is relatively slow. + +Notably, pharmaceutical γ-secretase inhibitors published to date show less strict regulation of Aβ production following chronic administration than we have attained here. Two independent compounds tested in Tg2576 [33] and TgCRND8 [34] transgenic mice show no more than 85% suppression of Aβ40 levels, and where measured, even less suppression of Aβ42 (approximately 60%) [35,36]. Thus, even after accounting for the much higher APP expression levels in our mice than in the Tg2576 and TgCRND8 lines, we have achieved better absolute suppression of Aβ production with the tet-off system than is currently possible with published γ-secretase inhibitors. Since even the most advanced future pharmaceutical agents are unlikely to attain more complete control of Aβ production than achieved here, this system provides a salient test of therapeutic intervention with Aβ-lowering compounds. + +Although the progression of amyloid deposition was sharply arrested by this approach, we found that a substantial amyloid burden remained even after long periods of transgene suppression. We examined a small number of animals after 12 mo of dox treatment (beginning at 6 mo of age), and found that amyloid deposits were still relatively abundant. Longer-term treatments are now in progress. At the latest treatment interval analyzed by ELISA, animals administered dox for 6 mo showed elevated levels of PBS-soluble Aβ (see Figure 5) that could be interpreted as an indication that the plaques are slowly releasing peptide (or oligomeric Aβ) into the soluble pool and might eventually dissolve. Whether inhibiting Aβ production longer than 6 (or 12) mo may ultimately result in clearance of amyloid is under investigation; unfortunately, the life span of the model eventually limits this experiment. Therapeutics used in humans will have considerably more time to act than is possible within the life span of rodent models. Long-term treatments would certainly be possible and could be a key to effective therapy. Overall, however, we interpret our findings as evidence that AD therapies that significantly lower the production of Aβ (by either inhibiting secretase activity or inhibiting APP expression) may not quickly reverse preexisting pathology, but should effectively halt further deposition of amyloid. + +In interpreting our study, it should be remembered that the earliest plaques to appear in these mice, like other APP transgenics harboring the Swedish mutation [27], are predominantly fibrillar deposits, which may be less tractable than the diffuse aggregates thought to come first in the course of the human disease. However, our data suggest that once diffuse deposits are formed in these mice, they are no more easily cleared in our system than cored plaques (see Figure S4). An additional consideration we recognize is that a small amount of transgene expression continues in the presence of dox and that endogenous mouse Aβ continues to be produced. It is possible that the combined low levels of endogenous mouse Aβ and nonsuppressed human peptide are sufficient to maintain existing deposits. However, these low levels of peptide are not sufficient to induce new amyloid formation, as CaMKIIα-tTA × tetAPPswe/ind mice raised on dox for up to a year do not develop amyloid lesions (data not shown). It is also clear that in this genetic system, we have raised the production of Aβ to levels not found in humans to accelerate pathology into an experimentally feasible time frame. This system allowed us to create an approximately 20-fold differential between APP/Aβ synthetic rates before and after treatment, yet the in vivo equilibrium between aggregated and disaggregated states of Aβ still favored the maintenance of amyloid deposits. In our opinion, it seems unlikely that amyloid deposits in human brain would be inherently any less stable than those formed in mouse brain. However, the human brain may harbor clearance mechanisms not shared by mice that would allow more efficient removal of preexisting amyloid. + +One potential mechanism by which amyloid may be more efficiently removed in the human disease than in the mouse models is through microglial phagocytosis. Resident microglia in transgenic mouse models localize to tissue surrounding plaques but show little evidence of amyloid engulfment [37–40]. In contrast, microglia surrounding amyloid plaques in human brain show a much higher state of activation with greater expression of complement receptor [40]. Thus, the role of microglia in amyloid metabolism is minor in transgenic models compared to the human condition. Somewhat paradoxically, several studies further demonstrate that treatment with anti-inflammatory drugs to reduce microglial activation actually lowers amyloid load in APP transgenic mice, suggesting a role for mouse microglia in the formation and maintenance of amyloid aggregates [41–43]. However, this outcome may be alternatively explained by direct effects of many anti-inflammatory drugs on γ-secretase cleavage [44–47]. Nonetheless, the role of microglia in both the human condition and the mouse models is poorly understood, and differences in microglial reactivity between the two could lead to significantly faster amyloid clearance in the brains of patients with AD than we observe in the tet-off APP mice. + +Given the relatively minor role played by microglia in other mouse models of amyloidosis, we think it unlikely that these cells have influenced the rate of amyloid clearance in the tet-off APP mice. Even so, we considered the possibility that chronic dox treatment may have altered the activation state of microglia in our treated mice. Dox is structurally similar to minocycline, a reported anti-inflammatory drug and inhibitor of microglial activation [48]. However, if dox does have anti-inflammatory activity, then, based on previous studies with other anti-inflammatories, we would have expected to find less amyloid in the dox-treated animals. Clearly, that was not the case. While it is possible that dox acts in some other way to slow amyloid clearance, data from multiple studies demonstrate that microglial responses are normally weak in the mouse AD models [37–40], and thus it is doubtful that dox-mediated microglial inhibition affected the outcome of our study. + +The persistence and stability of amyloid deposits in our system is unexpected given the speed with which Aβ aggregates are cleared in other mouse models of therapeutic intervention. Anti-Aβ antibodies injected directly into the brain have been shown to eliminate amyloid deposits in as little as 1 wk after treatment [49–51]. Peripheral antibody injection decreases amyloid load more broadly, and although it does not appear to act as quickly as local injection, can significantly reduce amyloid load within 2 mo of initial treatment [52,53]. More recently, an alternative approach has shown that lentiviral transfer of neprilysin can also reduce the number of aggregates in the area of the injection site [54]. Careful study of the mechanism behind several of the antibody-mediated therapies has suggested that activated microglia play an important role in the removal of fibrillar plaques after immunization [50,52,55]. However, it has been noted that deletion of the Fc receptor (the primary receptor for microglial opsinization of antibody–antigen complexes) in APP transgenic mouse models has no impact on the effectiveness of antibody-mediated therapy [56,57]. It is, nevertheless, possible that lack of microglia activation is the major difference between the slow clearance described here, where no perturbation of the immune system is expected, and the rapid clearance described in studies involving antibody or viral injection. In isolation, mild activation of microglia by injection damage or opsinization may not be adequate to induce substantial phagocytosis, but when combined with an Aβ-lowering agent, such as neprilysin or Aβ-targeted antibodies, the two may work in concert to clear peptide deposits. Consistent with this hypothesis, strong activation of microglia through transgenic expression of TGFβ [58] or central injection of lipopolysaccharide [59,60] can by itself substantially reduce plaque burden in APP transgenic mice. But in the case of acute antibody- and/or injury-mediated activation, once the inflammation has passed, and the antibody and bound peptide have been cleared and degraded, the remaining Aβ quickly reaggregates and amyloid pathology is reestablished [49]. This finding reinforces the notion that without continued stimulation, microglia in mouse models do not maintain the same level of sustained activation that may occur in humans. + +SantaCruz et al. recently published a study of mice that express P301L human tau via a similar vector system [61]. As in our tet-off APP mice, SantaCruz et al. found that tau neurofibrillary tangles, like amyloid plaques, are not cleared efficiently following transgene suppression. The lack of clearance in both models of AD pathology comes as a stark contrast to the rapid removal of protein aggregates found in similar tet-off mouse models of Huntington [32] and prion disease [62]. In these cases, disrupting the input of new monomer to the system via dox-mediated transgene suppression led to relatively rapid clearance of protein aggregates. By contrast, our study and that of SantaCruz et al. suggest that protein aggregates in AD may be more tenacious than in other neurodegenerative disorders. Perhaps once aggregated, Aβ and tau are either inherently more stable than other protein aggregates or more resistant to intra- and extracellular clearance mechanisms. + +One question we were not able to address in this study is whether abrogating synthesis of new Aβ halts the progression of cognitive decline. Studies from the tet-off tau mice suggest that protein clearance may in fact not be required for cognitive improvement following transgene suppression [61]. At present, because of unexpected noncognitive behavioral abnormalities, it is not clear whether the tet-off APP mice can be used to address the same question in the context of amyloid pathology. Both lines of tTA/APP mice we studied here display extreme hyperactivity visible as cage circling and quantified by activity monitoring (see Figure 7). Many of the double transgenic mice showed similar circular patterns of swimming near the edge of the tank when tested in the Morris water maze. Expression of this phenotype makes standard tests of learning and memory uninterpretable. Hyperactivity nonspecifically inhibits choice-driven changes in movement, the key element behind all cognitive behavioral paradigms. We are currently working to determine whether hyperactivity correlates with expression of the APPswe/ind holoprotein or its proteolytic derivatives. Preliminary studies suggest that hyperactivity does not appear quickly when dox-reared mice are shifted to nonmedicated diets (J. L. J., unpublished data). These data may indicate that the neuroactive culprit is not immediately present after transgenic APP synthesis is initiated, but requires additional time to develop. Alternatively, hyperactivity may be caused by neuronal alterations due to transgene expression during early postnatal development. Further experiments are needed to distinguish between these possibilities. + +In summary, we demonstrate that abrogating Aβ production halts the progression of pathologic changes in a transgenic mouse model of Alzheimer-type amyloidosis. However, despite dramatic reductions in Aβ synthesis, neuritic plaques are stable structures in vivo that do not quickly disaggregate. It is possible that a combination of therapies to limit Aβ production, increase Aβ degradation, and enhance phagocytosis of deposited amyloid may be required to reverse damage associated with AD. However, if started early enough in the course of disease, secretase inhibitors alone could provide substantial benefit in slowing pathogenic processes linked to amyloid deposition. Even at later stages in the disease, the presence of substantial microglial activation in human AD [40] suggests that simply slowing the formation of new amyloid deposits may allow ongoing phagocytosis to diminish preexisting lesions. However, the development of safe and effective secretase inhibitors will ultimately be required to determine whether the human brain has the capacity to repair amyloid-associated damage of AD once the progression of pathology is arrested. + +Supporting Information + +Figure S1 + +Transgenic APP Expression and Suppression by Dox in the Four New Tet-Off APP Lines + +Western blotting with human-specific antibody 6E10 reveals transgene-derived full-length APP in cortical homogenates from untreated animals (left lanes of each panel). The new lines produce exceptionally high levels of transgene expression; an equal amount of brain homogenate from a standard transgenic APP line is shown for comparison (extreme left lane, Standard Tg line C3–3; [15,63]). After 1 mo of dox treatment, transgenic protein in all four new tet-off APP lines is reduced to residual levels (+ dox; right lanes of each panel). + +(474 KB TIF). + +Click here for additional data file. + +Figure S2 + +Transgenic APP mRNA Is Brain-Specific + +A slot blot of mRNA harvested from various tissues in three of the four new tet-off APP lines and a nontransgenic control was used to examine transgenic mRNA expression. Hybridization is seen only in the brain; no signal above background is seen in any other tissue. + +(781 KB PSD). + +Click here for additional data file. + +Figure S3 + +Amyloid Pathology in the Cortex Reiterates That in the Hippocampus + +Amyloid histology was performed on sections from line 107 double transgenic mice by Hirano silver stain (top row), thioflavin-S (middle row), and Aβ immunohistochemistry (bottom row) to examine the persistence of pathology following transgene suppression. As in the hippocampus (see Figure 4 and text), the progression of amyloid pathology in the cortex worsens substantially between 6 and 9 mo of age in untreated mice. This progression is completely prevented by suppression of the transgene with dox. For comparison, normal neurohistology is shown in an age-matched single transgenic (tTA only) animal. No amyloid pathology has been detected in either APP or tTA single transgenic animals up to 15 mo of age. + +(4.8 MB PSD). + +Click here for additional data file. + +Figure S4 + +Diffuse Deposits Do Not Disperse During Aβ Suppression + +Campbell–Switzer silver stain was used to differentiate cored (brown) from diffuse (black) deposits in line 107 tTA/APP mice. This stain demonstrates that both types of deposit persist throughout long periods of transgene suppression. The lower panels, showing low-power images (10×) of frontal cortex from each condition, reveal little change in the extent of diffuse amyloid following up to 6 mo of Aβ suppression. High-power images (40×) in the upper panels show that the diffuse halo surrounding individual cored deposits remains relatively unchanged in treated mice. Untreated tTA single transgenic animals are shown as a negative control. Protocol for the Campbell-Switzer Alzheimer's Method was kindly shared by Robert Switzer, III (NeuroScience Associates, Knoxville, Tennessee, United States), and can be downloaded at http://www.nsalabs.com/Documents/publications/campbell-switzer_protocol.htm [64,65]. + +(923 KB JPG). + +Click here for additional data file. + +Figure S5 + +Chronic Transgene Suppression and Arrest of Aβ Aggregate Formation in an Independent Line of Tet-Off APP Mice (CaMKIIα-tTA × tet-APPswe/ind Line 18) + +(A) The experiment presented in the text for line 107 tet-off APP was repeated with a second tet-off APP line (line 18) to control for integration site artifacts. Cortical homogenates from untreated control and dox-treated double transgenic mice were immunoblotted for full-length APP with the human-specific antibody 6E10 to confirm transgene suppression at the time of harvest. Immunostaining for endogenous superoxide dismutase (SOD1) was included as a loading control. + +(B) Quantitation of signal intensity from the Western blot in (A) shows transgenic APP levels in line 18 are suppressed by more than 98% following 3 mo of dox treatment (significant effect of group ANOVA F2,8 = 1559.7, p < 0.001). This level of suppression was equal to or better than that attained in line 107 (see Figure 3B). + +(C) Serial dilution filter trap assay was used to quantify aggregated Aβ in cortical homogenates. + +(D) Quantitation of signal intensity in the linear range of the dilution series shown in (C). Consistent with the amyloid histology shown in Figure S5, aggregate formation was significantly increased between 6 and 9 mo of age in untreated mice (significant effect of group ANOVA F2,18 = 12.14, p < 0.001). Aggregate formation was completely arrested by transgene suppression, and is identical in 9-mo-old mice treated with dox for 3 mo as in untreated animals harvested when treatment began (p > 0.5, Tukey post-hoc test). *, p < 0.05; **, p < 0.005; ***, p < 0.001 versus 9-mo-old untreated mice, Tukey post-hoc test. + +(962 KB TIF). + +Click here for additional data file. + +Figure S6 + +Arrest of Amyloid Progression by Chronic Transgene Suppression in Line 18 Tet-Off APP Mice + +Amyloid histology in cortical (first and third rows) and hippocampal (second and fourth rows) sections from untreated tTA/APP mice shows a dramatic progression of pathology between 6 and 9 mo of age. Suppression of transgenic APP expression arrests this progression, although without any sign of plaque clearance (6 mo + 3 mo dox). Hirano silver stain (top panels); thioflavin-S (bottom panels). + +(5.8 MB PSD). + +Click here for additional data file. + +Patient Summary + +Background + +Patients with Alzheimer disease (AD) have elevated levels of a small protein called amyloid-β peptide that sticks together to form what are known as amyloid plaques in their brains. This peptide is normally made at low levels in healthy individuals, and is made when a larger protein called amyloid precursor protein (APP) is cut down in size. New treatments are now being developed that will decrease the amount of Aβ produced from APP. However, it is not clear whether lowering the production of Aβ will allow the brain to heal itself by clearing the amyloid plaques. The answer to this question may be important for deciding when Aβ-lowering drugs should be started, and may also determine how effective they are in reversing the mental symptoms of AD. + +What Did the Researchers Do and Find? + +Because new drugs designed to lower Aβ levels are still in development, they are not available for testing in animal models of the disease. Instead, basic questions about the effectiveness of this type of treatment must be answered using systems that mimic how the drugs work. To do this, the authors created mice that produce too much APP and that develop the same amyloid lesions as do human patients with AD. Unlike normal mice, these mice also carried a “switch” gene that allowed the researchers to turn off APP by feeding the mice special food. Turning off APP in these mice had the same effect as treating them with Aβ-lowering drugs, and so the researchers were able to ask what happened to the amyloid plaques after Aβ production was shut down. They showed that lowering Aβ production prevents the amyloid lesions from getting worse as the disease progresses. This means that treatment with Aβ-lowering drugs may be able to stop the disease from filling the brain with plaques. However, the researchers also found that the amyloid lesions that had formed before treatment was started remained intact throughout the experiment. + +What Do These Findings Mean? + +These results indicate that treatments designed to lower the production of Aβ may be an important part of future AD treatment, as this approach seems to prevents additional amyloid plaques from forming in the mouse brain. However, by itself, this strategy may not be able to rid the brain of plaques that have already formed in the brain before treatment is started. The findings suggest that early treatment may be important for this approach to succeed. + +Where Can I Get More Information Online? + +MedlinePlus has several Web pages of information on Alzheimer disease: + +http://www.nlm.nih.gov/medlineplus/alzheimersdisease.html + +The ADEAR Center of the US Government's National Institute on Aging also has information on Alzheimer disease: + +http://www.alzheimers.org/ + +The Alzheimer's Association Web site contains information on both caregiving and research: + +http://www.alz.org + +Acknowledgements + +We thank Patrick Tremblay for helpful advice on the tet system at a critical time in the project, and Mark Mayford for sharing the CaMKIIα-tTA mice through Jackson Laboratory. We also thank Fraser Moss for saving several immunoblots with last-minute shipments, Andy Groves for many thoughtful discussions, Neil Segil for generously sharing his laboratory and equipment, Beth Olson, Natasha Bouey, and Yolanda Jackson for outstanding animal care, Debbie Swing for expert microinjection, and Dave Fromholt for genotyping and dissection. We gratefully acknowledge Takeda Chemical Industries for providing antibodies BAN50, BA27, and BC05, Konrad Beyreuther and Andreas Weidemann for providing 22C11 antibody, and Ed Koo for sharing CT15 antibody. This work was supported by grants from the Johns Hopkins Alzheimer's Disease Research Center (JLJ), the National Alliance for Research on Schizophrenia and Depression (Young Investigator Award [JLJ]), the Rose Hills Foundation (JLJ), the Alzheimer's Association (Zenith Award [DRB]), the National Institute of Aging (K01 AG26144–01 [JLJ], P50 AGO5146–20 [DRB], R01 AG006656–16 [SGY], and P01 AG015453 [SGY]), the National Institute of Neurologic Disease and Stoke (R01 NS 047225 [DRB]), and the National Cancer Institute (NAJ and NGC). The funding agencies generously provided for research supplies, animal care, and salary support; the funders of this work had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. + +Abbreviations + +Aβ - amyloid-β + +AD - Alzheimer disease + +APLP - amyloid precursor–like protein + +APP - amyloid precursor protein + +CaMKIIα - calcium-calmodulin kinase IIα + +dox - doxycycline + +FA - formic acid + +GFAP - glial fibrillary acidic protein + +mo/huAPP695 - mouse APP with a humanized Aβ domain + +PBS - phosphate-buffered saline + +SDS - sodium dodecyl sulfate + +swe/ind - Swedish/Indiana + +tTA - tetracycline transactivator + +Figures and Tables + +Figure 1 + +Control of Transgenic APP Expression by Dox + +(A) Western blotting for transgenic APP using the human-specific 6E10 antibody shows expression of full-length protein in forebrain tissue from young predeposit double transgenic animals (line 107) and its suppression following dox treatment. Untreated animals show high levels of transgene expression; protein levels drop dramatically in animals acutely treated with dox for 2 wk. A faint, but detectable band of full-length protein remains in acutely treated animals that can be eliminated in mice born and raised on dox. + +(B) Immunoblotting with the N-terminal antibody 22C11 to detect both transgenic and endogenous protein shows that dox treatment reduces APP/APLP to levels found in nontransgenic mice. + +(C) Measurement of signal intensity from the Western blot in (A) shows transgenic APP levels are decreased more than 95% by dox in both acutely and chronically treated animals (97.2% for 4 wk + 2 wk dox, 98.0% for reared on dox versus 4 wk untreated; ANOVA effect of treatment group F4,15 = 85.55, p < 0.001). APP levels in 4 wk + 2 wk dox, reared on dox, and nontransgenic (NTg) were not significantly different (p > 0.9, Tukey post-hoc test). + +(D) Measurement of signal intensity from the Western blot in (B) shows total APP/APLP levels in dox-treated tTA/APP mice are significantly lower than in 4-wk-old untreated mice (ANOVA effect of treatment group F4,15 = 84.41, p < 0.001) and indistinguishable from those of nontransgenic animals (p > 0.9, Tukey post-hoc test). *, p < 0.001 versus untreated 4-wk-old mice, Tukey post-hoc test applied to significant effect of group ANOVA. + +Figure 2 + +Aβ Levels Are Dramatically Reduced by Transgene Suppression + +Cortical homogenates from young, predeposit tTA/APP mice used for Western blot in Figure 1 (line 107) were fractionated by sequential multi-step extraction with PBS, 2% SDS, and 70% FA followed by human-specific Aβ ELISA to measure transgene-derived peptide in each fraction. Aβ40 is shown in white, Aβ42 in black. + +(A) PBS-soluble Aβ levels are substantially reduced by both acute and chronic dox treatment (ANOVA, effect of treatment group F4,24 = 137.10 and 386.01, p < 0.001, for Aβ40 and Aβ42, respectively). Aβ levels in treated animals are indistinguishable from nontransgenic (NTg) animals (p > 0.3, Tukey post-hoc test). + +(B) In the young animals tested here prior to the formation of visible amyloid deposits, most Aβ is extracted into the SDS fraction (84% and 76% of all transgene-derived Aβ40 and Aβ42, respectively). As in the PBS-soluble fraction, Aβ levels in the SDS fraction are significantly lowered by dox treatment compared to untreated animals (ANOVA effect of group F4,24 = 197.57 and 163.48, p < 0.001, for Aβ40 and Aβ42, respectively). Acutely treated animals retained a small (although significant) amount of residual peptide (p < 0.001 compared to nontransgeinc, Tukey post-hoc test), whereas Aβ levels in mice born and raised on dox were reduced to levels indistinguishable from nontransgenic (p > 0.8, Tukey post-hoc test). + +(C) The FA-soluble fraction already contains a small but significant pool of aggregated Aβ42 in untreated animals by 4 wk of age (p < 0.05 versus nontransgenic; Tukey post-hoc test applied to significant effect of group ANOVA F4,24 = 17.11, p < 0.001). By 6 wk of age, the amount of Aβ in the FA fraction is increased significantly preceding the appearance of visible deposits 2 wk later. The FA pool is the only peptide fraction not lowered by acute dox treatment (4 wk untreated = 4 wk + 2 wk dox, p > 0.9, Tukey post-hoc test), consistent with poor turnover of aggregated Aβ species. + +(D) Measurements of total Aβ, including both endogenous and transgene-derived peptides, show that animals born and raised on dox harbor Aβ levels identical to nontransgenic animals (p > 0.9, Tukey post-hoc test, effect of group ANOVA F4,24 = 39.13 and 35.29, p < 0.001, for Aβ40 and Aβ42, respectively). Whereas chronic transgene suppression fully prevents synthesis of both peptides, acute dox treatment fully suppresses Aβ40 levels (p > 0.8, Tukey post-hoc test), but leaves a small amount of nonsuppressed Aβ42. The residual Aβ42 observed in acutely treated young animals derives from uncleared aggregates extracted in the SDS and FA fractions. *, p < 0.05; **, p < 0.005; ***, p < 0.001 versus 4-wk-old untreated mice, Tukey post-hoc applied to significant effect of group ANOVA. + +Figure 3 + +Robust Transgene Suppression in Older Mice with Preexisting Amyloid Pathology + +(A) Cortical homogenates from 6- to 12-mo-old animals used for pathology studies described below (line 107) were immunoblotted with human-specific antibody 6E10 to examine transgene suppression following 3 or 6 mo of dox treatment. The blot was co-immunostained for endogenous superoxide dismutase 1 (SOD1) as a control for loading. + +(B) Quantitation of signal intensity from the Western blot shown in (A). Transgenic APP levels are significantly suppressed following 3 or 6 mo of dox treatment (96.9% and 97.6%, respectively). *, p < 0.001 compared to 6-mo-old untreated animals, Tukey post-hoc test applied to significant effect of group ANOVA F3,12 = 107.22, p < 0.001. These data demonstrate that strong transgene suppression is attained both before and after the onset of amyloid pathology (see Figure 1 for predeposit experiments). + +(C) Experimental design. To examine the effects of chronic Aβ suppression on amyloid pathology after the onset of deposition, we compared untreated controls harvested at 6 and 9 mo of age to animals placed on dox at 6 mo of age and harvested after 3 or 6 mo of treatment. + +(D) Dox treatment leads to rapid transgene suppression even in 6-mo-old tTA/APP mice. Immunostaining with 6E10 shows APPswe/ind levels are dramatically reduced in 6-mo-old mice treated for 1 wk with dox (upper panel). A separate blot was immunostained for APP C-terminal fragments with CT15 antibody to show that the precursors to Aβ cleavage are decreased in parallel with the full-length protein (middle panel). Costaining for superoxide dismutase 1 was used as an internal control for loading (lower panel, taken from bottom half of 6E10 blot). + +Figure 4 + +Suppression of Transgenic APP Arrests Progression of Amyloid Pathology + +(A) Aggregated Aβ was quantified in cortical tissue from dox-treated and control tTA/APP mice (line 107) using a filter trap assay. Serial dilutions of protein homogenate were passed through a cellulose acetate filter; protein aggregates larger than the pore size were trapped and immunostained for Aβ. + +(B) Quantitation of signal intensity in the linear range of each filter trap dilution series (arrow in [A]) was used to compare aggregate load across treatment groups. Aggregated Aβ increased significantly between 6 and 9 mo of age in untreated mice (significant effect of group ANOVA F3,18 = 7.85, p < 0.002). This progression of pathology was completely prevented by transgene suppression. The amount of aggregated Aβ was identical in untreated mice at 6 mo of age to that in 9- or 12-mo-old animals treated with dox (p > 0.9, Tukey post-hoc test). Single transgenic tTA samples were included as negative controls and showed no signal above background. *, p < 0.01; **, p < 0.005 versus 9-mo-old untreated mice, Tukey post-hoc test; ***, p < 0.001 versus 9-mo-old untreated mice, Student's t-test. + +(C) Amyloid pathology in the hippocampus of representative mice from each treatment group: Hirano silver stain (top row), thioflavin-S (middle row), and Aβ immunohistochemistry (bottom row). Amyloid burden increases dramatically between 6 and 9 mo of age in untreated animals, but remains stable in transgene-suppressed mice over the same period (6 mo + 3 mo dox and 6 mo + 6 mo dox). Single transgenic animals (tTA only shown here) show no sign of amyloid pathology at any age tested. + +Figure 5 + +Aβ ELISA Confirms Arrest of Progression without Clearance of Peptide in Mice with Preexisting Aggregates + +Aβ levels in untreated 6- and 9-mo-old tTA/APP line 107 mice (shown in Figure 4) were compared to those in 9- and 12-mo-old animals treated with dox from the age of 6 mo. Single transgenic APP samples were included as negative controls. Cortical homogenates were fractionated by sequential multi-step extraction with PBS, 2% SDS, and 70% FA followed by human-specific Aβ ELISA to measure transgene-derived peptide in each fraction. Aβ40 is shown in white, Aβ42 in black. + +(A and B) Most Aβ in the brains of plaque-bearing mice is extracted into the FA and SDS fractions. Consistent with amyloid burden (Figures 4 and Figure S3), SDS- and FA-extracted Aβ levels in untreated 9-mo-old mice were significantly higher than in untreated 6-mo-old mice (Tukey post-hoc test applied to significant effect of group ANOVA for SDS and FA fractions F3,18 = 4.72–12.92, p < 0.02). In contrast, 3 or 6 mo of transgene suppression held Aβ at levels equivalent to those harbored when treatment was started (p > 0.2 compared to 6 mo untreated mice, Tukey post-hoc test). *, p < 0.05; **, p < 0.005; ***, p < 0.001 versus 9-mo-old untreated mice, Tukey post-hoc test. Significance for APP versus 9-mo-old untreated mice is based on Student's t-test. + +(C) The PBS fraction represents less than 0.1% of total Aβ (note the change in y-axis from [A] and [B]), but only here do Aβ levels in the dox-treated mice differ from those in younger untreated mice. Although both peptides appear elevated in the treated groups compared to the untreated 6-mo-old mice, only Aβ40 reaches statistical significance (p < 0.05, Tukey post-hoc test applied to significant effect of group ANOVA for Aβ40 F3,18 = 4.60, p < 0.02). A similar trend was seen for Aβ42, where ANOVA yielded a significant effect of group for PBS-soluble Aβ42 (F3,18 = 3.75, p < 0.03), however this was due only to differences between the untreated 6- and 9-mo-old groups. •, p < 0.05 versus 6-mo-old untreated mice, Tukey post-hoc test; •••, p < 0.001 versus 6-mo-old untreated mice, Student's t-test. + +Figure 6 + +Neuritic and Glial Pathology Are Unchanged following Transgene Suppression + +Dystrophic neurites and activated astrocytes surround most compact plaques in tet-off APP mice (line 107). Dark-stained, ubiquitin-filled neurites and reactive astrocytes form a halo around cored, fibrillar deposits by 6 mo of age that worsens with time in untreated mice. Both plaque-associated pathologies are arrested, although not reversed, by transgene suppression. Hirano silver stain (top row); GFAP immunohistochemistry (middle row); ubiquitin immunohistochemistry (bottom row). + +Figure 7 + +Transgene Suppression Attenuates Hyperactivity in tTA/APP Mice + +(A) A 48-h measure of ambulation records extreme hyperactivity in untreated double transgenic mice compared to single transgenic and nontransgenic controls (line 107). This phenotype is completely eliminated by rearing the double transgenic mice on dox. + +(B) The same data shown in (A) are replotted to magnify data from untreated control and dox-treated groups. + +(C and D) Activity levels in the combined control groups of (A) and (B) are here separated by genotype. None of the single transgenic or nontransgenic control groups display the hyperactivity present in untreated tTA/APP animals. Again, note the y-axes have been enlarged for detail compared to (A). + +Footnotes + +Citation: Jankowsky JL, Slunt HH, Gonzales V, Savonenko AV, Wen JC, et al. (2005) Persistent amyloidosis following suppression of Aβ production in a transgenic model of Alzheimer disease. 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+T290 PR:000009279 10458 10463 Sam68 +T291 NCBITaxon:10088 10484 10488 Mice +T292 PR:000027234 10517 10520 Src +T293 NCBITaxon:33208 10526 10533 animals +T294 GO:0008152 10557 10567 metabolism +T295 PR:000009279 10592 10597 Sam68 +T296 PR:000027234 10616 10619 Src +T297 PR:000009279 10647 10652 Sam68 +T298 NCBITaxon:10088 10653 10657 mice +T299 UBERON:0004288 10662 10670 skeletal +T300 PR:000009279 10697 10702 Sam68 +T301 PR:000009279 10710 10715 Sam68 +T302 NCBITaxon:10088 10719 10723 mice +T303 UBERON:0004288 10770 10778 skeletal +T304 NCBITaxon:10088 10929 10933 mice +T305 NCBITaxon:10088 10965 10969 mice +T306 PR:000009279 11038 11043 Sam68 +T307 NCBITaxon:10088 11047 11051 mice +T308 PR:000009279 11070 11075 Sam68 +T309 NCBITaxon:10088 11079 11083 mice +T310 UBERON:0011137 11128 11133;11151 11159 axial ... skeleton +T311 UBERON:0002091 11138 11159 appendicular skeleton +T312 UBERON:0001851 11367 11375 cortical +T313 UBERON:0001438 11412 11423 metaphyseal +T314 UBERON:0000981 11454 11460 femora +T315 PR:000009279 11477 11482 Sam68 +T316 NCBITaxon:10088 11486 11490 mice +T317 NCBITaxon:10088 11517 11521 mice +T318 PR:000009279 11558 11563 Sam68 +T319 NCBITaxon:10088 11567 11571 mice +T320 UBERON:0002483 11595 11610 trabecular bone +T321 UBERON:0004621 11654 11676 fifth lumbar vertebrae +T322 PR:000009279 11697 11702 Sam68 +T323 NCBITaxon:10088 11706 11710 mice +T324 PR:000009279 11726 11731 Sam68 +T325 NCBITaxon:10088 11735 11739 mice +T326 CHEBI:46662 11776 11783 mineral +T327 NCBITaxon:10088 11831 11836 mouse +T328 PR:000009279 11915 11920 Sam68 +T329 PR:000009279 11928 11933 Sam68 +T330 NCBITaxon:10088 11937 11941 mice +T331 PR:000009279 12018 12023 Sam68 +T332 NCBITaxon:10088 12027 12031 mice +T333 UBERON:0000981 12096 12101 femur +T334 UBERON:0002412 12106 12114 vertebra +T335 PR:000009279 12122 12127 Sam68 +T336 NCBITaxon:10088 12131 12135 mice +T337 CHEBI:46662 12142 12149 mineral +T338 NCBITaxon:10088 12212 12216 mice +T339 UBERON:0000981 12274 12281 femoral +T340 UBERON:0002412 12286 12295 vertebral +T341 PR:000009279 12323 12328 Sam68 +T342 PR:000009279 12366 12371 Sam68 +T343 NCBITaxon:10088 12375 12379 mice +T344 UBERON:0011137 12423 12428;12446 12454 axial ... skeleton +T345 UBERON:0002091 12433 12454 appendicular skeleton +T346 PR:000009279 12689 12694 Sam68 +T347 NCBITaxon:10088 12698 12702 Mice +T348 UBERON:0004288 12778 12787 skeletons +T349 NCBITaxon:10088 12804 12808 mice +T350 NCBITaxon:9606 12872 12878 humans +T351 NCBITaxon:1 12902 12912 individual +T352 http://purl.obolibrary.org/obo/MONDO_0005315 12916 12924 fracture +T353 UBERON:0000479 13034 13040 tissue +T354 PR:000009279 13093 13098 Sam68 +T355 NCBITaxon:10088 13102 13106 mice +T356 NCBITaxon:10088 13148 13152 mice +T357 PR:000009279 13201 13206 Sam68 +T358 NCBITaxon:10088 13210 13214 mice +T359 PR:000009279 13530 13535 Sam68 +T360 NCBITaxon:10088 13539 13543 mice +T361 NCBITaxon:10088 13733 13737 mice +T362 UBERON:0000440 13802 13812 trabeculae +T363 UBERON:0000440 13853 13863 trabeculae +T364 PR:000009279 13885 13890 Sam68 +T365 NCBITaxon:10088 13894 13898 mice +T366 NCBITaxon:10088 13940 13944 mice +T367 GO:0046849 13947 13962 Bone Remodeling +T368 PR:000009279 13984 13989 Sam68 +T369 NCBITaxon:10088 13993 13997 Mice +T370 PR:000009279 14082 14087 Sam68 +T371 NCBITaxon:10088 14091 14095 mice +T372 UBERON:0000981 14140 14146 femora +T373 UBERON:0000979 14151 14156 tibia +T374 CL:0000062 14169 14179 osteoblast +T375 CL:0000092 14184 14194 osteoclast +T376 CL:0000062 14335 14346 osteoblasts +T377 PR:000001937 14351 14373 tartrate-resistant ALP +T378 PR:000001937 14375 14410 tartrate-resistant acid phosphatase +T379 CHEBI:37527 14394 14398 acid +T380 PR:000001937 14412 14416 TRAP +T381 CL:0000092 14455 14466 osteoclasts +T382 PR:000009279 14503 14508 Sam68 +T383 PR:000009279 14535 14540 Sam68 +T384 NCBITaxon:10088 14563 14567 mice +T385 PR:000001937 14597 14601 TRAP +T386 PR:000009279 14650 14655 Sam68 +T387 NCBITaxon:10088 14659 14663 mice +T388 PR:000009279 14722 14727 Sam68 +T389 NCBITaxon:10088 14731 14735 mice +T390 CL:0000062 14778 14788 osteoblast +T391 CL:0000092 14793 14803 osteoclast +T392 PR:000009279 14833 14838 Sam68 +T393 NCBITaxon:10088 14842 14846 mice +T394 CL:0000092 14918 14928 osteoclast +T395 CL:0000062 14934 14944 osteoblast +T396 http://purl.obolibrary.org/obo/MONDO_0000001 14980 14987 disease +T397 UBERON:0002495 15034 15044 long bones +T398 UBERON:0008883 15152 15159 osteoid +T399 CHEBI:46662 15173 15180 mineral +T400 PR:000009279 15250 15255 Sam68 +T401 NCBITaxon:10088 15259 15263 mice +T402 PR:000009279 15290 15295 Sam68 +T403 NCBITaxon:10088 15299 15303 mice +T404 UBERON:0002371 15369 15375 marrow +T405 CL:0000062 15413 15424 osteoblasts +T406 CL:0000092 15438 15449 osteoclasts +T407 UBERON:0000479 15463 15469 tissue +T408 PR:000009279 15548 15553 Sam68 +T409 NCBITaxon:10088 15557 15561 mice +T410 UBERON:0002371 15615 15621 marrow +T411 NCBITaxon:10088 15652 15656 mice +T412 CL:0000062 15822 15833 osteoblasts +T413 CL:0000092 15837 15848 osteoclasts +T414 NCBITaxon:10088 15878 15882 mice +T415 UBERON:0001977 15917 15922 serum +T416 PR:000009279 16077 16082 Sam68 +T417 NCBITaxon:10088 16086 16090 mice +T418 UBERON:0001977 16109 16114 Serum +T419 CHEBI:50114 16115 16123 estrogen +T420 NCBITaxon:10088 16172 16176 mice +T421 PR:000001393 16230 16243 interleukin-6 +T422 GO:0065007 16300 16310 regulating +T423 UBERON:0001977 16331 16336 serum +T424 PR:000009279 16360 16365 Sam68 +T425 PR:000009279 16373 16378 Sam68 +T426 NCBITaxon:10088 16382 16386 mice +T427 PR:000009279 16405 16410 Sam68 +T428 NCBITaxon:10088 16414 16418 mice +T429 PR:000009279 16469 16474 Sam68 +T430 NCBITaxon:10088 16478 16482 mice +T431 PR:000009279 16493 16498 Sam68 +T432 NCBITaxon:10088 16502 16506 mice +T433 PR:000009279 16604 16609 Sam68 +T434 NCBITaxon:10088 16613 16617 mice +T435 CHEBI:50114 16651 16659 estrogen +T436 CL:0000062 16744 16754 Osteoblast +T437 CL:0000092 16764 16774 Osteoclast +T438 PR:000009279 16799 16804 Sam68 +T439 NCBITaxon:10088 16808 16812 Mice +T440 UBERON:0007023 16858 16863 adult +T441 CL:0000062 16913 16923 osteoblast +T442 CL:0000092 16927 16937 osteoclast +T443 PR:000009279 17049 17054 Sam68 +T444 NCBITaxon:10088 17058 17062 mice +T445 PR:000009279 17137 17142 Sam68 +T446 CL:0000062 17146 17157 osteoblasts +T447 CL:0000092 17162 17173 osteoclasts +T448 UBERON:0007195 17214 17233 bone marrow stromal +T449 CL:0010001 17214 17239 bone marrow stromal cells +T450 PR:000009279 17266 17271 Sam68 +T451 NCBITaxon:10088 17275 17279 mice +T452 CL:0000062 17310 17320 osteoblast +T453 PR:000009279 17491 17496 Sam68 +T454 NCBITaxon:10088 17500 17504 mice +T455 CL:0000057 17537 17547 fibroblast +T456 CL:0000057 17574 17575 F +T457 CL:0000062 17665 17675 osteoblast +T458 PR:000009279 17754 17759 Sam68 +T459 PR:000009279 17767 17772 Sam68 +T460 NCBITaxon:10088 17776 17780 mice +T461 PR:000003263 17791 17806 type I collagen +T462 PR:000009279 17861 17866 Sam68 +T463 NCBITaxon:10088 17870 17874 mice +T464 CL:0000092 17906 17917 osteoclasts +T465 UBERON:0002495 17944 17954 long bones +T466 PR:000009279 17958 17963 Sam68 +T467 PR:000009279 17971 17976 Sam68 +T468 NCBITaxon:10088 17980 17984 mice +T469 PR:000001937 18010 18014 TRAP +T470 UBERON:0001751 18082 18088 dentin +T471 CHEBI:10545 18123 18131 electron +T472 CL:0000092 18188 18199 osteoclasts +T473 PR:000009279 18233 18238 Sam68 +T474 NCBITaxon:10088 18242 18246 mice +T475 PR:000027234 18263 18266 Src +T476 NCBITaxon:10088 18272 18276 mice +T477 PR:000009279 18299 18304 Sam68 +T478 CL:0000136 18314 18323 Adipocyte +T479 CL:0000062 18353 18363 Osteoblast +T480 UBERON:0007195 18408 18427 bone marrow stromal +T481 CL:0010001 18408 18433 bone marrow stromal cells +T482 CL:0000062 18452 18463 osteoblasts +T483 CL:0000136 18468 18478 adipocytes +T484 CL:0000136 18568 18577 adipocyte +T485 PR:000009279 18615 18620 Sam68 +T486 UBERON:0007195 18641 18660 bone marrow stromal +T487 CL:0010001 18641 18666 bone marrow stromal cells +T488 GO:0001503 18694 18704 osteogenic +T489 GO:0060612 18716 18726 adipogenic +T490 PR:000009279 18763 18768 Sam68 +T491 GO:0065007 18786 18794 regulate +T492 GO:0065007 18848 18855 control +T493 UBERON:0000013 18889 18915 sympathetic nervous system +T494 PR:000009279 18992 18997 Sam68 +T495 GO:0045595 19005 19018;19029 19044 regulation of ... differentiation +T496 CL:0000136 19019 19028 adipocyte +T497 PR:000009279 19089 19094 Sam68 +T498 PR:000009279 19102 19107 Sam68 +T499 UBERON:0000922 19111 19118 embryos +T500 PR:000009279 19132 19137 Sam68 +T501 CL:0000136 19171 19181 adipocytes +T502 CHEBI:8228 19225 19237 pioglitazone +T503 GO:0060612 19248 19260 adipogenesis +T504 GO:0060612 19331 19343 adipogenesis +T505 GO:0060612 19345 19357 Adipogenesis +T506 PR:000009279 19421 19426 Sam68 +T507 PR:000009279 19484 19489 Sam68 +T508 CL:0000136 19493 19502 adipocyte +T509 GO:0010467 19535 19545 expression +T510 PR:000013058 19589 19594 PPARγ +T511 PR:000009365 19599 19603 KLF5 +T512 PR:000009279 19620 19625 Sam68 +T513 PR:000009279 19663 19668 Sam68 +T514 GO:0060612 19703 19715 adipogenesis +T515 PR:000009279 19734 19739 Sam68 +T516 PR:000009279 19814 19819 Sam68 +T517 NCBITaxon:10088 19823 19827 mice +T518 PR:000009279 19902 19907 Sam68 +T519 GO:0065007 19908 19917 modulates +T520 GO:0030154 19922 19940;19953 19958 differentiation of ... cells +T521 UBERON:0003104 19941 19952 mesenchymal +T522 CL:0000134 19941 19958 mesenchymal cells +T523 UBERON:0000922 20037 20046 embryonic +T524 CL:0002321 20037 20046;20085 20090 embryonic ... cells +T525 UBERON:0003104 20047 20058 mesenchymal +T526 CL:0000134 20047 20058;20085 20090 mesenchymal ... cells +T527 PR:000000164 20118 20123 BMP-2 +T528 CL:0000062 20132 20142 osteoblast +T529 GO:0010467 20214 20224 expression +T530 PR:000030444 20232 20243 osteocalcin +T531 PR:000030444 20245 20248 OCN +T532 SO:0000704 20250 20254 gene +T533 GO:0009294 20299 20310 transfected +T534 SO:0000313 20381 20388 hairpin +T535 PR:000009279 20397 20402 Sam68 +T536 CHEBI:17939 20432 20441 puromycin +T537 GO:0010467 20447 20457 expression +T538 PR:000009279 20461 20466 Sam68 +T539 PR:000003676 20542 20549 β-actin +T540 CL:0000062 20584 20594 Osteoblast +T541 GO:0010467 20635 20645 expressing +T542 PR:000000164 20698 20703 BMP-2 +T543 GO:0010467 20733 20743 expression +T544 PR:000030444 20747 20750 OCN +T545 PR:000003676 20755 20762 β-actin +T546 GO:0010467 20825 20835 expressing +T547 CL:0000062 20870 20880 osteoblast +T548 GO:0010467 20939 20949 expression +T549 PR:000030444 20953 20956 OCN +T550 PR:000009279 21037 21042 Sam68 +T551 UBERON:0007195 21046 21065 bone marrow stromal +T552 CL:0010001 21046 21071 bone marrow stromal cells +T553 SO:0002031 21112 21129 short hairpin RNA +T554 SO:0002031 21131 21136 shRNA +T555 NCBITaxon:10088 21210 21214 mice +T556 UBERON:0002371 21242 21248 marrow +T557 PR:000009279 21331 21336 Sam68 +T558 PR:000009279 21344 21349 Sam68 +T559 NCBITaxon:10088 21353 21357 mice +T560 UBERON:0000479 21420 21426 tissue +T561 UBERON:0002371 21450 21456 marrow +T562 CL:0000136 21457 21467 adipocytes +T563 CHEBI:51217 21514 21526 fluorochrome +T564 PR:000009279 21595 21600 Sam68 +T565 NCBITaxon:10088 21604 21608 mice +T566 UBERON:0002483 21641 21656 trabecular bone +T567 UBERON:0002371 21722 21728 marrow +T568 CHEBI:51217 21768 21780 fluorochrome +T569 UBERON:0001474 21848 21853 bones +T570 PR:000009279 21870 21875 Sam68 +T571 NCBITaxon:10088 21879 21883 mice +T572 NCBITaxon:10088 21929 21933 mice +T573 PR:000009279 21982 21987 Sam68 +T574 GO:0065007 21988 21997 regulates +T575 GO:0030154 22002 22020;22045 22050 differentiation of ... cells +T576 UBERON:0002371 22021 22032 bone marrow +T577 CL:0002540 22021 22050 bone marrow mesenchymal cells +T578 UBERON:0003104 22033 22044 mesenchymal +T579 CL:0000136 22062 22071 adipocyte +T580 CL:0000062 22100 22110 osteoblast +T581 GO:0007568 22130 22135 aging +T582 PR:000009279 22218 22223 Sam68 +T583 GO:0065007 22230 22238 modulate +T584 UBERON:0002371 22239 22250 bone marrow +T585 CL:0002540 22239 22273 bone marrow mesenchymal stem cells +T586 UBERON:0003104 22251 22262 mesenchymal +T587 PR:000009279 22281 22286 Sam68 +T588 NCBITaxon:10088 22290 22294 mice +T589 PR:000009279 22400 22405 Sam68 +T590 NCBITaxon:10088 22409 22413 mice +T591 PR:000009279 22465 22470 Sam68 +T592 NCBITaxon:40674 22570 22577 mammals +T593 PR:000009279 22589 22594 Sam68 +T594 NCBITaxon:33208 22598 22605 animals +T595 GO:0007568 22637 22642 aging +T596 GO:0030154 22648 22666;22684 22689 differentiation of ... cells +T597 UBERON:0002371 22667 22678 bone marrow +T598 CL:0002092 22667 22678;22684 22689 bone marrow ... cells +T599 CL:0000034 22679 22689 stem cells +T600 PR:000009279 22704 22709 Sam68 +T601 NCBITaxon:10088 22713 22717 mice +T602 UBERON:0000922 22722 22731 embryonic +T603 CL:0002321 22722 22731;22770 22775 embryonic ... cells +T604 UBERON:0003104 22732 22743 mesenchymal +T605 CL:0000134 22732 22743;22770 22775 mesenchymal ... cells +T606 PR:000009279 22799 22804 Sam68 +T607 SO:0002031 22805 22810 shRNA +T608 CL:0000062 22841 22851 osteoblast +T609 PR:000009279 22913 22918 Sam68 +T610 CL:0000062 22928 22938 osteoblast +T611 PR:000009279 23006 23011 Sam68 +T612 NCBITaxon:33208 23015 23022 animals +T613 CL:0000136 23065 23074 adipocyte +T614 PR:000009279 23105 23110 Sam68 +T615 PR:000009279 23171 23176 Sam68 +T616 GO:0065007 23183 23191 regulate +T617 GO:0060612 23212 23222 adipogenic +T618 GO:0001503 23227 23237 osteogenic +T619 UBERON:0002371 23261 23272 bone marrow +T620 UBERON:0003104 23273 23284 mesenchymal +T621 PR:000009279 23287 23292 Sam68 +T622 GO:0010467 23301 23311 expression +T623 NCBITaxon:10088 23351 23356 mouse +T624 UBERON:0000922 23357 23363 embryo +T625 PR:000009279 23418 23423 Sam68 +T626 GO:0010467 23442 23451 expressed +T627 PR:000009279 23469 23474 Sam68 +T628 UBERON:0000955 23491 23496 brain +T629 UBERON:0000948 23498 23503 heart +T630 UBERON:0000160 23509 23518 intestine +T631 UBERON:0002107 23545 23550 liver +T632 UBERON:0002113 23562 23568 kidney +T633 UBERON:0000323 23591 23609 late-stage embryos +T634 GO:0010467 23616 23626 expression +T635 PR:000009279 23686 23691 Sam68 +T636 CL:0000540 23695 23702 neurons +T637 UBERON:0000160 23734 23744 intestinal +T638 PR:000013434 23734 23748 intestinal SIK +T639 UBERON:0000310 23749 23755 breast +T640 http://purl.obolibrary.org/obo/MONDO_0021100 23749 23761 breast tumor +T641 PR:000013434 23749 23768 breast tumor kinase +T642 GO:0010467 23779 23789 expression +T643 PR:000009279 23793 23798 Sam68 +T644 GO:0007568 23818 23823 aging +T645 UBERON:0007195 23824 23843 bone marrow stromal +T646 CL:0010001 23824 23849 bone marrow stromal cells +T647 GO:0007568 23856 23868 senescencing +T648 PR:000009279 23917 23922 Sam68 +T649 CL:0000138 23926 23938 chondrocytes +T650 CL:0000062 23940 23951 osteoblasts +T651 CL:0000092 23956 23967 osteoclasts +T652 UBERON:0004288 24045 24053 skeletal +T653 GO:0001501 24045 24065 skeletal development +T654 PR:000009279 24072 24077 Sam68 +T655 NCBITaxon:10088 24081 24085 mice +T656 SO:0000417 24164 24170 domain +T657 PR:000009279 24218 24223 Sam68 +T658 NCBITaxon:10088 24227 24231 mice +T659 UBERON:0000113 24244 24253 adulthood +T660 PR:000009279 24324 24329 Sam68 +T661 PR:000009280 24417 24422 SLM-1 +T662 PR:000009281 24427 24432 SLM-2 +T663 PR:000009279 24454 24459 Sam68 +T664 UBERON:0000922 24483 24492 embryonic +T665 GO:0009790 24483 24504 embryonic development +T666 PR:000009279 24533 24538 Sam68 +T667 PR:000009279 24555 24560 Sam68 +T668 PR:000009279 24591 24596 Sam68 +T669 UBERON:0007023 24711 24716 Adult +T670 PR:000009279 24717 24722 Sam68 +T671 NCBITaxon:10088 24726 24730 mice +T672 UBERON:0000104 24745 24754 life span +T673 PR:000009279 24787 24792 Sam68 +T674 http://purl.obolibrary.org/obo/MONDO_0005047 24807 24814 sterile +T675 PR:000009279 24823 24828 Sam68 +T676 PR:000007598 24944 24948 FosB +T677 NCBITaxon:10088 24952 24956 mice +T678 UBERON:0001977 24963 24968 Serum +T679 CHEBI:50114 24979 24987 estrogen +T680 GO:0007568 25001 25006 aging +T681 PR:000009279 25007 25012 Sam68 +T682 PR:000009279 25082 25087 Sam68 +T683 PR:000009279 25109 25114 Sam68 +T684 http://purl.obolibrary.org/obo/MONDO_0011122 25135 25140 obese +T685 GO:0007568 25257 25262 aging +T686 http://purl.obolibrary.org/obo/MONDO_0021124 25428 25444 female sterility +T687 UBERON:0004288 25456 25464 skeletal +T688 PR:000009279 25491 25496 Sam68 +T689 PR:000009279 25504 25509 Sam68 +T690 NCBITaxon:10088 25513 25517 mice +T691 PR:000009279 25562 25567 Sam68 +T692 NCBITaxon:10088 25571 25575 mice +T693 CL:0000062 25674 25684 osteoblast +T694 CL:0000092 25689 25699 osteoclast +T695 PR:000009279 25737 25742 Sam68 +T696 GO:0065007 25743 25753 regulatory +T697 PR:000027234 25763 25766 Src +T698 http://purl.obolibrary.org/obo/MONDO_0017198 25771 25784 osteopetrosis +T699 PR:000009279 25838 25843 Sam68 +T700 CL:0000092 25847 25858 osteoclasts +T701 PR:000027234 25872 25875 Src +T702 GO:0046849 25920 25935 bone remodeling +T703 PR:000027234 25941 25944 Src +T704 NCBITaxon:10088 25948 25952 mice +T705 GO:0016265 25953 25957 died +T706 http://purl.obolibrary.org/obo/MONDO_0017198 25985 25998 osteopetrotic +T707 CL:0000092 26057 26067 osteoclast +T708 CL:0000092 26115 26126 osteoclasts +T709 PR:000009279 26142 26147 Sam68 +T710 PR:000009279 26155 26160 Sam68 +T711 NCBITaxon:10088 26164 26168 mice +T712 UBERON:0001751 26180 26186 dentin +T713 PR:000009279 26247 26252 Sam68 +T714 CL:0000092 26256 26267 osteoclasts +T715 PR:000009279 26290 26295 Sam68 +T716 CL:0000092 26299 26310 osteoclasts +T717 GO:0008152 26407 26417 metabolism +T718 PR:000009279 26434 26439 Sam68 +T719 NCBITaxon:10088 26443 26447 mice +T720 PR:000009279 26506 26511 Sam68 +T721 NCBITaxon:10088 26515 26519 mice +T722 GO:0045453 26552 26567 bone resorption +T723 GO:0045453 26640 26655 bone resorption +T724 CL:0000092 26677 26687 osteoclast +T725 PR:000009279 26776 26781 Sam68 +T726 NCBITaxon:10088 26785 26789 mice +T727 PR:000009279 26883 26888 Sam68 +T728 NCBITaxon:10088 26892 26896 mice +T729 CL:0000092 26897 26907 osteoclast +T730 UBERON:0000104 26946 26950 life +T731 GO:0030154 27040 27058;27087 27092 differentiation of ... cells +T732 CL:0000001 27059 27066;27087 27092 primary ... cells +T733 UBERON:0007195 27067 27086 bone marrow stromal +T734 CL:0010001 27067 27092 bone marrow stromal cells +T735 PR:000009279 27109 27114 Sam68 +T736 PR:000009279 27122 27127 Sam68 +T737 NCBITaxon:10088 27131 27135 mice +T738 CL:0000062 27146 27156 osteoblast +T739 GO:0001503 27177 27187 osteogenic +T740 PR:000009279 27240 27245 Sam68 +T741 NCBITaxon:10088 27249 27253 mice +T742 CL:0000001 27331 27338;27359 27364 primary ... cells +T743 UBERON:0007195 27339 27358 bone marrow stromal +T744 CL:0010001 27339 27364 bone marrow stromal cells +T745 PR:000009279 27375 27380 Sam68 +T746 NCBITaxon:10088 27384 27388 mice +T747 UBERON:0000922 27426 27435 embryonic +T748 CL:0002321 27426 27435;27474 27479 embryonic ... cells +T749 UBERON:0003104 27436 27447 mesenchymal +T750 CL:0000134 27436 27447;27474 27479 mesenchymal ... cells +T751 PR:000009279 27503 27508 Sam68 +T752 SO:0002031 27509 27514 shRNA +T753 CL:0000062 27541 27552 osteoblasts +T754 PR:000000164 27558 27562 BMP2 +T755 PR:000009279 27587 27592 Sam68 +T756 NCBITaxon:10088 27596 27600 mice +T757 UBERON:0002371 27622 27633 bone marrow +T758 PR:000009279 27661 27666 Sam68 +T759 NCBITaxon:10088 27671 27675 mice +T760 CL:0000136 27690 27699 adipocyte +T761 PR:000009279 27731 27736 Sam68 +T762 GO:0065007 27737 27746 regulates +T763 CL:0000136 27752 27761 adipocyte +T764 CL:0000062 27766 27776 osteoblast +T765 PR:000009279 27818 27823 Sam68 +T766 GO:0065007 27860 27868 regulate +T767 UBERON:0003104 27869 27880 mesenchymal +T768 CL:0000134 27869 27885 mesenchymal cell +T769 GO:0030154 27881 27901 cell differentiation +T770 GO:0065007 27973 27982 regulates +T771 CL:0000062 28018 28028 osteoblast +T772 PR:000027234 28052 28055 Src +T773 NCBITaxon:10088 28059 28063 mice +T774 PR:000009279 28133 28138 Sam68 +T775 NCBITaxon:10088 28142 28146 mice +T776 GO:0065007 28172 28181 regulated +T777 PR:000027234 28185 28188 Src +T778 GO:0065007 28219 28228 regulates +T779 GO:0045453 28229 28244 bone resorption +T780 UBERON:0000013 28275 28301 sympathetic nervous system +T781 CL:0000062 28318 28329 osteoblasts +T782 PR:000002107 28410 28415 RANKL +T783 CL:0000092 28432 28443 osteoclasts +T784 PR:000009279 28503 28508 Sam68 +T785 NCBITaxon:10088 28512 28516 mice +T786 NCBITaxon:10088 28542 28546 mice +T787 PR:000009279 28686 28691 Sam68 +T788 NCBITaxon:10088 28695 28699 mice +T789 UBERON:0001977 28775 28780 serum +T790 PR:000009279 28784 28789 Sam68 +T791 NCBITaxon:10088 28793 28797 mice +T792 PR:000009279 28827 28832 Sam68 +T793 GO:0065007 28840 28850 regulating +T794 GO:0008152 28856 28866 metabolism +T795 PR:000009279 28911 28916 Sam68 +T796 GO:0045453 28964 28979 bone resorption +T797 UBERON:0000013 28988 29014 sympathetic nervous system +T798 PR:000009279 29038 29043 Sam68 +T799 CL:0000062 29051 29061 osteoblast +T800 CL:0000136 29075 29084 adipocyte +T801 PR:000009279 29158 29163 Sam68 +T802 GO:0008152 29172 29182 metabolism +T803 UBERON:0002371 29187 29198 bone marrow +T804 CL:0002540 29187 29220 bone marrow mesenchymal stem cell +T805 UBERON:0003104 29199 29210 mesenchymal +T806 GO:0030154 29216 29236 cell differentiation +T807 PR:000009279 29269 29274 Sam68 +T808 NCBITaxon:10088 29278 29282 mice +T809 CHEBI:35222 29294 29304 inhibitors +T810 PR:000009279 29308 29313 Sam68 +T811 PR:000009279 29394 29399 Sam68 +T812 GO:0010467 29400 29410 expression +T813 NCBITaxon:9606 29450 29456 humans +T814 UBERON:0002371 29489 29495 marrow +T815 CL:0000136 29496 29505 adipocyte +T816 http://purl.obolibrary.org/obo/MONDO_0005298 29523 29535 osteoporosis +T817 NCBITaxon:33208 29570 29576 animal +T818 GO:0007568 29592 29597 aging +T819 UBERON:0000922 29781 29790 embryonic +T820 NCBITaxon:10088 29791 29795 mice +T821 GO:0007565 29820 29828 pregnant +T822 PR:000009279 30142 30147 Sam68 +T823 GO:0042571 30148 30156 antibody +T824 CHEBI:51686 30209 30220 hematoxylin +T825 UBERON:0000922 30223 30228 Adult +T826 NCBITaxon:10088 30229 30233 mice +T827 UBERON:0001179 30253 30263 peritoneal +T828 CHEBI:51903 30286 30293 calcein +T829 CHEBI:27902 30317 30329 tetracycline +T830 UBERON:0000981 30477 30482 femur +T831 UBERON:0000979 30487 30492 tibia +T832 CHEBI:34840 30534 30537 MMA +T833 CHEBI:60004 30544 30551 mixture +T834 CHEBI:34840 30559 30562 MMA +T835 CHEBI:34288 30571 30589 glycolmethacrylate +T836 CHEBI:34288 30591 30594 GMA +T837 CHEBI:34840 30627 30630 MMA +T838 UBERON:0000479 30640 30647 tissues +T839 CHEBI:34840 30754 30757 MMA +T840 CHEBI:34288 30758 30761 GMA +T841 PR:000001937 30788 30792 TRAP +T842 CHEBI:46662 31227 31234 Mineral +T843 NCBITaxon:10088 31265 31269 mice +T844 PR:000009279 31302 31307 sam68 +T845 SO:0000704 31308 31312 gene +T846 PR:000009279 31352 31357 Sam68 +T847 SO:0000147 31358 31363 exons +T848 SO:0001026 31399 31406 genomic +T849 PR:000009279 31433 31438 Sam68 +T850 PR:000009279 31470 31475 Sam68 +T851 SO:0001026 31476 31483 genomic +T852 SO:0000147 31520 31524 exon +T853 SO:0000852 31531 31543 part of exon +T854 SO:0000852 31565 31577 part of exon +T855 SO:0000147 31584 31588 exon +T856 SO:0000112 31805 31811 primer +T857 SO:0001644 32182 32198 targeting vector +T858 PR:000009279 32204 32209 Sam68 +T859 SO:0000147 32219 32223 exon +T860 SO:0000852 32230 32242 part of exon +T861 CHEBI:7507 32252 32260 neomycin +T862 SO:0005853 32271 32284 gene cassette +T863 SO:0000155 32383 32390 plasmid +T864 UBERON:0000922 32416 32425 embryonic +T865 CL:0002322 32416 32430;32436 32441 embryonic stem ... cells +T866 CL:0002322 32432 32434;32436 32441 ES ... cells +T867 CL:0002322 32463 32465 ES +T868 PR:000009279 32539 32544 Sam68 +T869 SO:0001023 32552 32558 allele +T870 CL:0002322 32605 32613 ES cells +T871 UBERON:0000358 32652 32663 blastocysts +T872 NCBITaxon:33208 32715 32722 animals +T873 UBERON:0010166 32749 32753 coat +T874 GO:0007618 32765 32770 mated +T875 NCBITaxon:10088 32783 32787 mice +T876 NCBITaxon:10088 32833 32837 mice +T877 NCBITaxon:10088 32881 32885 mice +T878 NCBITaxon:10088 32916 32920 mice +T879 NCBITaxon:10088 33022 33026 mice +T880 NCBITaxon:10088 33125 33130 mouse +T881 NCBITaxon:33208 33247 33253 Animal +T882 SO:0001026 33260 33267 Genomic +T883 UBERON:0002415 33290 33294 tail +T884 SO:0001026 33342 33349 genomic +T885 PR:000009279 33580 33585 sam68 +T886 SO:0001023 33586 33592 allele +T887 SO:0001026 33611 33618 genomic +T888 PR:000009279 33744 33749 sam68 +T889 SO:0001023 33759 33765 allele +T890 SO:0001026 33784 33791 genomic +T891 UBERON:0001977 33923 33928 serum +T892 PR:000009279 33945 33950 Sam68 +T893 PR:000009279 33958 33963 Sam68 +T894 NCBITaxon:10088 33967 33971 mice +T895 NCBITaxon:10088 34062 34066 Mice +T896 CHEBI:38867 34102 34112 anesthetic +T897 NCBITaxon:10088 34437 34441 mice +T898 UBERON:0000981 34558 34563 femur +T899 UBERON:0004620 34568 34590 fourth lumbar vertebra +T900 UBERON:0000479 34613 34620 tissues +T901 UBERON:0004377 34672 34689 distal metaphysis +T902 UBERON:0001977 35032 35037 Serum +T903 NCBITaxon:9989 35092 35098 Rodent +T904 UBERON:0001977 35238 35243 serum +T905 CHEBI:16469 35294 35307 17β estradiol +T906 PR:000001393 35418 35431 interleukin-6 +T907 CL:0000062 35472 35482 osteoblast +T908 CL:0000092 35487 35497 osteoclast +T909 UBERON:0002371 35509 35520 Bone marrow +T910 UBERON:0000979 35542 35547 tibia +T911 UBERON:0000981 35552 35558 femora +T912 PR:000009279 35582 35587 Sam68 +T913 PR:000009279 35595 35600 Sam68 +T914 NCBITaxon:10088 35604 35608 mice +T915 UBERON:0003891 35619 35626 stromal +T916 CL:0000499 35619 35632 stromal cells +T917 CL:0000062 35889 35899 osteoblast +T918 SO:0000704 35908 35912 gene +T919 GO:0010467 35908 35923 gene expression +T920 CL:0000092 35965 35976 Osteoclasts +T921 UBERON:0002495 36008 36018 long bones +T922 PR:000009279 36031 36036 Sam68 +T923 PR:000009279 36044 36049 Sam68 +T924 NCBITaxon:10088 36053 36057 mice +T925 UBERON:0001751 36164 36170 dentin +T926 PR:000001937 36329 36333 TRAP +T927 UBERON:0001751 36354 36360 Dentin +T928 CHEBI:18219 36422 36440 ammonium hydroxide +T929 CHEBI:10545 36520 36528 electron +T930 CHEBI:10545 36548 36556 Electron +T931 NCBITaxon:10088 36898 36903 mouse +T932 UBERON:0000922 36904 36913 embryonic +T933 CL:0000057 36914 36924 fibroblast +T934 CL:0000136 36942 36951 adipocyte +T935 UBERON:0000922 37007 37014 embryos +T936 CL:0000136 37097 37106 adipocyte +T937 CL:0000010 37191 37205 Cultured cells +T938 GO:0060612 37291 37303 adipogenesis +T939 PR:000009279 37307 37312 Sam68 +T940 CHEBI:33893 37365 37372 reagent +T941 GO:0001171 37486 37505 reverse-transcribed +T942 SO:0000112 37632 37639 primers +T943 GO:0060612 37662 37672 adipogenic +T944 PR:000009365 37690 37694 KLF5 +T945 PR:000013058 37759 37764 PPARγ +T946 PR:000003676 37842 37849 β-Actin +T947 PR:000009279 37924 37929 Sam68 +T948 PR:000009279 38010 38015 Sam68 +T949 SO:0002031 38063 38068 shRNA +T950 SO:0000985 38078 38084 duplex +T951 NCBITaxon:10088 38186 38191 mouse +T952 PR:000009279 38192 38197 Sam68 +T953 GO:0009294 38363 38376 Transfections +T954 SO:0000440 38464 38470 vector +T955 GO:0009294 38486 38498 transfection +T956 CHEBI:17939 38540 38549 puromycin +T957 PR:000009279 38605 38610 Sam68 +T958 GO:0042571 38617 38625 antibody +T959 GO:0001503 38628 38638 Osteogenic +T960 GO:0001503 38689 38699 osteogenic +T961 UBERON:0000479 38766 38772 tissue +T962 PR:000000164 38904 38909 BMP-2 +T963 PR:000030444 39011 39022 osteocalcin +T964 PR:000003676 39024 39031 β-actin +T965 SO:0000704 39043 39047 gene +T966 GO:0010467 39043 39058 gene expression +T967 PR:000000164 39084 39089 BMP-2 +T968 PR:000009279 39179 39184 Sam68 +T969 NCBITaxon:10088 39196 39201 Mouse +T970 CL:0000062 39202 39213 Osteoblasts +T971 CL:0000092 39218 39229 Osteoclasts +T972 SO:0000704 39295 39299 Gene +T973 GO:0010467 39295 39310 Gene Expression +T974 CL:0000010 39322 39330;39339 39344 Cultured ... Cells +T975 UBERON:0003891 39331 39338 Stromal +T976 CL:0000499 39331 39344 Stromal Cells +T977 PR:000009279 39387 39392 Sam68 +T978 PR:000009279 39400 39405 Sam68 +T979 NCBITaxon:10088 39409 39413 Mice +T980 UBERON:0001977 39479 39484 Serum +T981 NCBITaxon:10088 39541 39545 Mice +T982 UBERON:0001758 39675 39686 Periodontal +T983 CHEBI:46662 40166 40173 mineral +T984 CHEBI:46662 40194 40201 mineral +T985 PR:000000164 40211 40216 BMP-2 +T986 GO:0060349 40219 40237 bone morphogenetic +T987 PR:000000164 40219 40247 bone morphogenetic protein-2 +T988 NCBITaxon:10088 40302 40307 mouse +T989 UBERON:0000922 40308 40314 embryo +T990 CL:0000057 40315 40325 fibroblast +T991 PR:000013058 40327 40332 PPARγ +T992 GO:0005777 40335 40345 peroxisome +T993 PR:000013058 40335 40379 peroxisome proliferator–activated receptor γ +T994 SO:0002031 40381 40386 shRNA +T995 SO:0002031 40389 40406 short hairpin RNA +T996 PR:000001937 40408 40412 TRAP +T997 PR:000001937 40415 40450 tartrate-resistant acid phosphatase +T998 CHEBI:37527 40434 40438 acid +T999 PR:000009279 40518 40523 Sam68 +T1000 UBERON:0000922 40527 40536 Embryonic +T1001 NCBITaxon:10088 40537 40541 Mice +T1002 UBERON:0000922 40547 40556 Embryonic +T1003 NCBITaxon:10088 40557 40561 mice +T1004 GO:0007565 40580 40588 pregnant +T1005 UBERON:0000922 40681 40687 embryo +T1006 PR:000009279 40724 40729 Sam68 +T1007 GO:0042571 40730 40738 antibody +T1008 UBERON:0000922 40832 40841 Embryonic +T1009 UBERON:0000479 40847 40854 tissues +T1010 UBERON:0000955 40864 40869 brain +T1011 UBERON:0000948 40871 40876 heart +T1012 UBERON:0001555 40881 40884 gut +T1013 CHEBI:51686 40903 40914 hematoxylin +T1014 PR:000009279 40950 40955 Sam68 +T1015 GO:0042571 40956 40964 antibody +T1016 PR:000009279 41039 41044 Sam68 +T1017 CL:0000138 41074 41086 chondrocytes +T1018 UBERON:0001706 41094 41106 nasal septum +T1019 UBERON:0002412 41135 41143 vertebra +T1020 UBERON:0004384 41169 41186 femoral epiphysis +T1021 UBERON:0004769 41215 41225 diaphyseal +T1022 CL:0000062 41226 41237 osteoblasts +T1023 CHEBI:51686 41285 41296 hematoxylin +T1024 GO:0042571 41336 41344 antibody +T1025 PR:000009279 41407 41412 Sam68 +T1026 GO:0005634 41444 41460 nucleus of cells +T1027 UBERON:0000479 41477 41484 tissues +T1028 GO:0005737 41524 41533 cytoplasm +T1029 UBERON:0000922 41665 41672 embryos +T1030 PR:000009279 41699 41704 Sam68 +T1031 NCBITaxon:10088 41715 41719 Mice +T1032 SO:0001026 41729 41736 genomic +T1033 PR:000009279 41781 41786 sam68 +T1034 SO:0001023 41787 41794 alleles +T1035 PR:000009279 42014 42019 sam68 +T1036 SO:0001023 42020 42027 alleles +T1037 SO:0001023 42042 42048 allele +T1038 SO:0000147 42058 42062 exon +T1039 SO:0000852 42069 42081 part of exon +T1040 PR:000009279 42087 42092 sam68 +T1041 SO:0005853 42113 42121 cassette +T1042 SO:0001026 42154 42161 genomic +T1043 NCBITaxon:10088 42229 42233 mice +T1044 SO:0001023 42311 42318 alleles +T1045 PR:000009279 42366 42371 Sam68 +T1046 GO:0010467 42372 42382 expression +T1047 NCBITaxon:9986 42498 42504 rabbit +T1048 UBERON:0001977 42505 42510 serum +T1049 PR:000009279 42517 42522 Sam68 +T1050 GO:0042571 42527 42535 antibody +T1051 GO:0042571 42549 42557 antibody +T1052 CHEBI:60816 42583 42594 immunogenic +T1053 NCBITaxon:10088 42643 42648 mouse +T1054 PR:000009279 42649 42654 Sam68 +T1055 PR:000009279 42661 42666 Sam68 +T1056 GO:0042571 42673 42681 antibody +T1057 PR:000009279 42731 42736 Sam68 +T1058 GO:0042571 42753 42763 antibodies +T1059 UBERON:0000981 42897 42902 Femur +T1060 PR:000009279 42920 42925 Sam68 +T1061 PR:000009279 42933 42938 Sam68 +T1062 NCBITaxon:10088 42942 42946 Mice +T1063 NCBITaxon:10088 42952 42956 Mice +T1064 CHEBI:38867 42985 42995 anesthetic +T1065 UBERON:0000981 43058 43064 femora +T1066 UBERON:0000981 43178 43183 femur +T1067 PR:000009279 43187 43192 Sam68 +T1068 PR:000009279 43206 43211 Sam68 +T1069 NCBITaxon:10088 43221 43225 mice +T1070 UBERON:0001851 43300 43308 cortical +T1071 UBERON:0000981 43382 43387 femur +T1072 NCBITaxon:10088 43395 43399 mice +T1073 NCBITaxon:10088 43412 43416 mice +T1074 UBERON:0001474 43423 43428 Bones +T1075 UBERON:0000479 43457 43463 tissue +T1076 UBERON:0000981 43745 43750 femur +T1077 PR:000009279 43754 43759 Sam68 +T1078 PR:000009279 43773 43778 Sam68 +T1079 NCBITaxon:10088 43788 43792 mice +T1080 UBERON:0002483 43843 43858 trabecular bone +T1081 UBERON:0001851 43874 43882 cortical +T1082 UBERON:0000981 43921 43926 femur +T1083 NCBITaxon:10088 43947 43951 mice +T1084 NCBITaxon:33208 44042 44049 animals +T1085 UBERON:0002483 44101 44116 Trabecular Bone +T1086 UBERON:0000479 44163 44169 tissue +T1087 UBERON:0000981 44240 44245 femur +T1088 UBERON:0004620 44250 44272 fourth lumbar vertebra +T1089 NCBITaxon:10088 44289 44293 mice +T1090 PR:000009279 44469 44474 Sam68 +T1091 NCBITaxon:10088 44478 44482 mice +T1092 PR:000009279 44514 44519 Sam68 +T1093 NCBITaxon:10088 44523 44527 mice +T1094 PR:000009279 44572 44577 Sam68 +T1095 NCBITaxon:10088 44581 44585 mice +T1096 PR:000009279 44617 44622 Sam68 +T1097 NCBITaxon:10088 44626 44630 mice +T1098 UBERON:0000440 44675 44685 trabeculae +T1099 PR:000009279 44768 44773 Sam68 +T1100 PR:000009279 44804 44809 Sam68 +T1101 PR:000009279 44843 44848 Sam68 +T1102 PR:000009279 44877 44882 Sam68 +T1103 PR:000009279 44915 44920 Sam68 +T1104 PR:000009279 45014 45019 Sam68 +T1105 PR:000009279 45027 45032 Sam68 +T1106 NCBITaxon:10088 45036 45040 Mice +T1107 UBERON:0000979 45054 45059 tibia +T1108 CL:0000062 45166 45177 osteoblasts +T1109 PR:000001937 45185 45189 TRAP +T1110 CL:0000092 45222 45233 osteoclasts +T1111 PR:000009279 45281 45286 Sam68 +T1112 PR:000009279 45313 45318 Sam68 +T1113 PR:000009279 45350 45355 Sam68 +T1114 NCBITaxon:10088 45369 45373 mice +T1115 PR:000009279 45401 45406 Sam68 +T1116 NCBITaxon:10088 45410 45414 mice +T1117 NCBITaxon:33208 45580 45587 animals +T1118 PR:000009279 45620 45625 Sam68 +T1119 PR:000009279 45633 45638 Sam68 +T1120 CL:0000062 45642 45653 Osteoblasts +T1121 CL:0000092 45658 45669 Osteoclasts +T1122 UBERON:0007195 45671 45685 Marrow stromal +T1123 CL:0010001 45671 45691 Marrow stromal cells +T1124 UBERON:0002495 45715 45725 long bones +T1125 NCBITaxon:10088 45738 45742 mice +T1126 CL:0000062 45788 45798 osteoblast +T1127 CHEBI:32130 45929 45943 silver nitrate +T1128 PR:000009279 45987 45992 Sam68 +T1129 CL:0000092 46176 46187 osteoclasts +T1130 UBERON:0002495 46219 46229 long bones +T1131 NCBITaxon:10088 46242 46246 mice +T1132 UBERON:0001751 46284 46290 dentin +T1133 CL:0000092 46346 46357 Osteoclasts +T1134 CL:0002242 46377 46387;46402 46408 cells with ... nuclei +T1135 GO:0005634 46402 46408 nuclei +T1136 PR:000001937 46435 46439 TRAP +T1137 UBERON:0001751 46479 46485 dentin +T1138 PR:000001937 46604 46608 TRAP +T1139 GO:0060612 46676 46688 Adipogenesis +T1140 PR:000009279 46701 46706 Sam68 +T1141 NCBITaxon:10088 46710 46715 Mouse +T1142 UBERON:0000922 46716 46725 Embryonic +T1143 CL:0000057 46726 46737 Fibroblasts +T1144 NCBITaxon:10088 46763 46768 mouse +T1145 UBERON:0000922 46769 46776 embryos +T1146 UBERON:0000922 46780 46789 embryonic +T1147 PR:000009279 46826 46831 Sam68 +T1148 PR:000009279 46839 46844 Sam68 +T1149 CL:0000136 46899 46908 Adipocyte +T1150 CHEBI:8228 47009 47021 pioglitazone +T1151 CL:0000136 47135 47145 adipocytes +T1152 CHEBI:2511 47385 47392 agarose +T1153 CHEBI:4883 47411 47427 ethidium bromide +T1154 GO:0010467 47433 47443 expression +T1155 GO:0060612 47447 47457 adipogenic +T1156 PR:000005308 47466 47472 C/EBPβ +T1157 PR:000005309 47474 47480 C/EBPδ +T1158 PR:000013056 47482 47487 PPARα +T1159 PR:000009365 47493 47497 KLF5 +T1160 GO:0010467 47526 47536 expression +T1161 PR:000009279 47559 47564 Sam68 +T1162 PR:000003676 47566 47573 β-actin +T1163 GO:0001503 47606 47616 Osteogenic +T1164 UBERON:0000922 47650 47659 Embryonic +T1165 CL:0002321 47650 47664 Embryonic Cell +T1166 PR:000009279 47693 47698 Sam68 +T1167 GO:0009294 47720 47731 transfected +T1168 SO:0000440 47746 47752 vector +T1169 SO:0000440 47773 47779 vector +T1170 SO:0002031 47794 47799 shRNA +T1171 PR:000009279 47801 47806 Sam68 +T1172 SO:0002031 47807 47812 shRNA +T1173 CHEBI:17939 47833 47842 puromycin +T1174 PR:000009279 47882 47887 Sam68 +T1175 PR:000009279 47922 47927 Sam68 +T1176 PR:000009279 47977 47982 Sam68 +T1177 GO:0042571 47989 47997 antibody +T1178 PR:000003676 48007 48014 β-actin +T1179 GO:0042571 48015 48025 antibodies +T1180 GO:0001503 48052 48062 Osteogenic +T1181 PR:000000164 48130 48135 BMP-2 +T1182 GO:0001503 48184 48194 osteogenic +T1183 GO:0010467 48227 48237 expression +T1184 CL:0000062 48246 48256 osteoblast +T1185 PR:000030444 48265 48276 osteocalcin +T1186 PR:000030444 48278 48281 OCN +T1187 PR:000003676 48325 48332 β-actin +T1188 CHEBI:2511 48390 48397 agarose +T1189 CHEBI:4883 48415 48431 ethidium bromide +T1190 PR:000009279 48448 48453 Sam68 +T1191 NCBITaxon:10088 48457 48461 Mice +T1192 GO:0048539 48485 48499;48506 48517 Development of ... Bone Marrow +T1193 UBERON:0002371 48506 48517 Bone Marrow +T1194 UBERON:0000479 48693 48699 tissue +T1195 UBERON:0002371 48712 48718 marrow +T1196 CL:0000136 48719 48729 adipocytes +T1197 PR:000009279 48812 48817 Sam68 +T1198 UBERON:0002371 48891 48897 marrow +T1199 CL:0000136 48898 48908 adipocytes +T1200 CHEBI:51217 48968 48980 fluorochrome +T1201 NCBITaxon:33208 49098 49105 animals +T1202 PR:000009279 49128 49133 Sam68 +T1203 CHEBI:46662 49186 49193 Mineral +T1204 NCBITaxon:10088 49232 49236 Mice +T1205 UBERON:0002495 49277 49287 Long Bones +T1206 NCBITaxon:10088 49313 49317 Mice +T1207 NCBITaxon:10088 49620 49624 mice +T1208 PR:000009279 49882 49887 Sam68 +T1209 NCBITaxon:10088 49917 49921 mice diff --git a/src/ontogpt/evaluation/craft/database/all/16362077.txt b/src/ontogpt/evaluation/craft/database/all/16362077.txt new file mode 100644 index 000000000..fc069d482 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16362077.txt @@ -0,0 +1,269 @@ +Ablation of the Sam68 RNA Binding Protein Protects Mice from Age-Related Bone Loss + +Abstract + +The Src substrate associated in mitosis of 68 kDa (Sam68) is a KH-type RNA binding protein that has been shown to regulate several aspects of RNA metabolism; however, its physiologic role has remained elusive. Herein we report the generation of Sam68-null mice by homologous recombination. Aged Sam68−/− mice preserved their bone mass, in sharp contrast with 12-month-old wild-type littermates in which bone mass was decreased up to approximately 75%. In fact, the bone volume of the 12-month-old Sam68−/− mice was virtually indistinguishable from that of 4-month-old wild-type or Sam68−/− mice. Sam68−/− bone marrow stromal cells had a differentiation advantage for the osteogenic pathway. Moreover, the knockdown of Sam68 using short hairpin RNA in the embryonic mesenchymal multipotential progenitor C3H10T1/2 cells resulted in more pronounced expression of the mature osteoblast marker osteocalcin when differentiation was induced with bone morphogenetic protein-2. Cultures of mouse embryo fibroblasts generated from Sam68+/+ and Sam68−/− littermates were induced to differentiate into adipocytes with culture medium containing pioglitazone and the Sam68−/− mouse embryo fibroblasts shown to have impaired adipocyte differentiation. Furthermore, in vivo it was shown that sections of bone from 12-month-old Sam68−/− mice had few marrow adipocytes compared with their age-matched wild-type littermate controls, which exhibited fatty bone marrow. Our findings identify endogenous Sam68 as a positive regulator of adipocyte differentiation and a negative regulator of osteoblast differentiation, which is consistent with Sam68 being a modulator of bone marrow mesenchymal cell differentiation, and hence bone metabolism, in aged mice. + +Synopsis + +Osteoporosis is a debilitating bone disease that is characterized by reduced bone mass and microarchitectural damage, which result in increased bone fragility and susceptibility to fracture. Peak bone mass, which is achieved by the age of 30 in humans, has been identified as a major determinant of resistance or susceptibility to osteoporosis. The authors generated mice deficient for the Sam68 RNA binding protein, a protein of unknown physiologic function. The mice develop normally and are protected against bone loss during aging. Age-related bone loss has long been associated with an increase in marrow adipocytes, which are derived from the same mesenchymal lineage as osteoblasts in bone marrow. The authors showed that Sam68 regulates the differentiation of this mesenchymal lineage, such that in its absence, osteoblasts continued to be generated in aging bone, leading to preservation of bone mass. This study identifies a physiologic role for Sam68 as a modulator of the bone marrow stem cell niche and hence of bone metabolism. The data identify Sam68 as a potential therapeutic target for the prevention and treatment of age-related bone loss. + +Introduction + +During skeletal development, the anabolic activity of osteoblasts [1] is favored over the catabolic activity of osteoclasts [2], which results in a net gain in bone mass. At skeletal maturity, bone mass is maintained through the balanced activity of osteoblasts and osteoclasts during the remodeling cycle. During skeletal aging, there is a shift in the balance that favors osteoclast over osteoblast activity, which results in net bone loss [3]. The amount and rate at which bone is gained during development and lost during aging are determined in large part by genetics [4–6] but also by physical activity and by alterations in the availability and response of bone cells to circulating hormones [7–9] and locally derived growth factors [10,11]. Whereas genetic-based studies have provided novel insights into the pathways that regulate bone development [12–14], relatively little is known about the etiology of age-related bone loss. Increased bone resorption in elderly men and women is associated with a reduction in bone mass and an increase in circulating levels of bone biomarkers [15]. These changes have been attributed primarily to nutritional deficits resulting in alterations in the parathyroid hormone–vitamin D axis [16], to gonadal hormone deficiency [17], to leptin levels and the sympathetic nervous system [8,18–20], and to alterations in bone cell apoptosis [21]. Bone loss in the elderly has also been attributed to alterations in the response of bone marrow stromal cells to their microenvironment that favors differentiation down the adipocyte lineage rather than the osteoblast lineage [22]. + +Aging has long been associated with an increase in marrow fat, where the generation of adipocytes is favored over osteoblasts [23]. Osteoblasts and adipocytes are derived from a common mesenchymal precursor cell present in bone marrow, and the factors that control this age-induced switch toward adipogenic differentiation is not well understood [24]. While several transcriptional regulatory proteins have been associated with cell fate determination of bone marrow mesenchymal cells, including peroxisome proliferator–activated receptor γ (PPARγ) and KLF5 [25–30], the role of RNA binding proteins in this process remains unknown. RNA binding proteins of the KH type are known regulators of cellular differentiation. For example, expression defects in the KH domain proteins NOVA and FMRP are known to cause paraneoplastic neurologic disorders [31] and the fragile X syndrome, respectively, in humans [32]. The phenotype of the quaking viable mice suggests a role for the QUAKING RNA binding protein in oligodendrocytes and myelination [33]. Indeed, the ectopic expression of the QKI-6/7 isoforms in vivo led to the formation of glial cells rather than neurons from neural progenitor, demonstrating its role in cell fate determination [34]. The loss of GLD-1 protein in Caenorhabditis elegans prevents the appearance of stem cells [35], and the absence of the KH domain protein HOW in Drosophila prevents muscle differentiation and results in flies with held-out-wings [36,37]. The Src substrate associated in mitosis of 68 kDa (Sam68) is also a member of the family of KH domain RNA binding proteins [38]; however, its physiologic role has remained undefined. + +Sam68 was identified as an SH3 and SH2 domain interacting protein for Src family kinases and is also a known substrate of Src kinases [39–42] and of the breast tumor kinase [43]. Sam68 has been shown to facilitate the export of unspliced HIV RNA [44] and to regulate pre-mRNA processing [45]. In the present paper, we report the generation of Sam68−/− mice and analysis of their skeletal phenotype. Our data indicate that the absence of Sam68 confers resistance to age-related bone loss in mice such that old Sam68 have a higher bone mass than their wild-type littermates. We provide evidence that Sam68 regulates the differentiation of bone marrow stromal cells by showing that cells isolated from Sam68−/− animals had enhanced osteogenic activity and decreased adipogenic activity than those harvested from wild-type littermates. Furthermore, Sam68−/− mouse embryo fibroblasts (MEFs) were impaired in their capacity to differentiate into adipocytes, consistent with Sam68 being a regulator of bone marrow mesenchymal cell differentiation. These results also characterize a new animal model to study bone metabolism, regeneration, and repair during aging. + +Results + +Sam68 Is Expressed in the Developing Skeleton of Embryonic Mice + +The Sam68 mRNA is known to be widely expressed [38], whereas its pattern of protein expression in vivo remains to be defined. Sections of paraffin-embedded wild-type E14.5 and E16.5 embryonic mice were immunostained with the well-characterized AD1 anti-Sam68 antibody, raised in rabbits against an immunizing peptide that corresponds to amino acids 330 to 348 of the mouse Sam68 protein [46]. Sam68 immunoreactivity was observed in the skeleton and soft tissues of developing wild-type mice at E14.5 and E16.5 (Figure 1). Intense staining was seen in the nucleus of cells in the developing brain, heart, and small intestine (Figure 1B), as well as in chondrocytes in the nasal septum and the glandular tissue adjacent to the nasal cartilage (Figure 1C, panels A–D), in the vertebra and intervertebral discs (panels E–H) and in the epiphysis (panels I–K) and metaphysis (panel L) of long bones. Nuclear staining was observed in proliferating chondrocytes and hypertrophic chondrocytes (Figure 1C, panel H), in osteoblasts (panel L; Dataset S1), and in osteoclasts (Dataset S1). No staining was observed with preimmune serum or when the antiserum was preadsorbed with the immunizing peptide (Figure 1C, panels B, F, and J). Taken together, these data demonstrate that Sam68 protein is selectively expressed in the developing mouse embryo, with particularly elevated expression in cartilage and bone. + +Sam68 is Not Essential for Mouse Development + +To define the physiologic role of Sam68, we generated Sam68-deficient mice by targeted disruption of exons 4 and 5 of the sam68 gene, which encode the functional region of the KH-type RNA binding domain (Figure 2A). The integrity of the targeted allele was verified by Southern blot analysis (Figure 2B) and by PCR of genomic DNA (unpublished data). Sam68 transcripts encoded by exons 1 to 5 were absent as evidenced by RT-PCR (Dataset S2), and Sam68−/− mice were devoid of Sam68 protein, as visualized by immunoblotting with anti-Sam68 AD1 and SC-333 antibodies or control normal rabbit serum or anti-Sam68 AD1 preabsorbed with peptide (Figure 2C). SC-333 is a rabbit anti-Sam68 antibody that was raised against the C-terminal 20 amino acids of Sam68 [47]. These data confirmed the generation of a mouse deficient in Sam68. The genotypes of offspring from heterozygote intercrosses exhibited a Mendelian segregation at E18.5 (Table 1). Despite the lack of visible deformity, many of the Sam68−/− pups died at birth of unknown causes. Sam68+/- mice were phenotypically normal and Sam68−/− pups that survived the perinatal period invariably lived to old age. Despite evidence that Sam68 mRNA is widely expressed and phosphorylated in mitosis [41,42], the Sam68−/− mice did not develop tumors and showed no immunologic or other major illnesses. Sam68−/− mice did, however, have difficulty breeding due to male infertility and the females rarely provided adequate care to their young. + +Sam68 Deficiency Protects Mice from Age-Related Bone Loss + +Src null animals are known to have bone metabolism defects [48], and since Sam68 is a substrate of Src, we decided to analyze the Sam68 mice for skeletal abnormalities. Cohorts of Sam68+/+ and Sam68−/− mice were euthanized at 4 and 12 months of age for skeletal phenotyping. To minimize differences in the bone phenotype that might arise secondary to gender or weight differences, we selected age-matched female mice for these analyses. The female mice demonstrated similar increases in body weight, although 4-month-old Sam68−/− mice weighed less than Sam68+/+ mice, and similar changes in bone lengths in the axial and appendicular skeleton between 4 and 12 months of age (Table 2). Faxitron radiography (Faxitron X-ray Corporation, Wheeling, Illinois, United States) (Figure 3A) and micro–computed tomography (CT) (Figure 3B) revealed significant cortical thinning (arrow) and a reduction in metaphyseal bone (asterisk) in the distal femora of 12-month-old Sam68+/+ mice compared with 4-month-old mice of either genotype and 12-month-old Sam68−/− mice. Similar reductions in trabecular bone were shown by Faxitron and micro-CT in the fifth lumbar vertebrae of the 12-month-old Sam68+/+ mice but not in the Sam68−/− mice (unpublished data). Total body bone mineral content (BMC), quantified with a Lunar PIXImus mouse densitometer (GE-Lunar, Madison, Wisconsin, United States), increased in both Sam68+/+ and Sam68−/− mice between 4 months and 12 months of age, but only reached significance in the Sam68−/− mice (Table 2). Similarly, a greater increase in BMC was seen in the femur and vertebra in the Sam68−/− mice. Bone mineral density (BMD) remained constant or decreased in the wild type mice but increased significantly in the total body and in the femoral and vertebral regions of interest in the Sam68−/− (Table 2). These data showed that Sam68−/− mice continued to thrive and accrue bone in the axial and appendicular skeleton for longer than 12 months. This was in contrast to the situation in age-matched, littermate controls in which a significant amount of bone was lost over the same timeframe. + +Three-Dimensional Architecture of Bone Is Preserved in Aged Sam68−/− Mice + +Bone loss and compromised architecture are characteristic features of the skeletons of aged C57BL/6 mice and resemble the clinical features of age-related bone loss in humans that can predispose an individual to fracture. Quantification of the micro-CT data shown in Figure 3B confirmed the reduction in bone volume compared with tissue volume (BV/TV; Figure 4A, left) in the 12-month-old Sam68+/+ mice (hatched bars) compared with 4-month-old mice of both genotypes (solid bars) and 12-month-old Sam68−/− mice (stippled bars). This was associated with a significant increase (p < 0.01; denoted by the asterisks) in the structure model index, which measures the ratio of plate-like to rod-like structures (Figure 4A, right). A quantifiable increase in the mean trabecular separation (Tb.Sp) (unpublished data) in 12-month-old Sam68+/+ mice was due to an increase in the percentage of spaces falling in the range of 350 to 700 μm (Figure 4B, red hatched line). Trabecular thickness remained constant among the different groups of mice (unpublished data). In effect, this meant that there were fewer trabeculae, rather than equivalent numbers of thin trabeculae, in the 12-month-old Sam68+/+ mice compared with any of the other groups of mice. + +Bone Remodeling Is Preserved in Aged Sam68−/− Mice + +To further define the mechanisms involved in the preservation of bone mass in aged Sam68−/− mice, we prepared sections from plastic-embedded femora and tibia to evaluate osteoblast and osteoclast activity (Figure 5). In situ enzyme histochemical staining for alkaline phosphatase (ALP; brown stain) activity was used as a biomarker for osteoblasts and tartrate-resistant ALP (tartrate-resistant acid phosphatase [TRAP]; red stain) activity as a marker for osteoclasts. Little difference was seen between Sam68+/+ (Figure 5A and 5B) and Sam68−/− (Figure 5C and 5D) mice at 4 months of age. ALP- and TRAP-positive cells were reduced in the 12-month-old Sam68+/+ mice (Figure 5E and 5F) and remained unchanged in 12-month-old Sam68−/− mice (Figure 5G and 5H). The reduction in both osteoblast and osteoclast activity in the 12-month-old Sam68+/+ mice argued against bone being lost primarily due to a relative increase in osteoclast over osteoblast activity, as seen in high turnover disease [49]. + +Histomorphometric analyses [50] of the long bones (Table 3) corroborated the radiologic evidence of age-related bone loss. Bone volume (BV/TV), newly formed osteoid (OV/TV), and mineral apposition rate (MAR) were all significantly reduced in 12-month-old Sam68+/+ mice compared with 4-month-old Sam68+/+ mice. These reductions were associated with a significant increase in marrow fat (FV/TV) and decreased numbers of osteoblasts (nOB/TV) and osteoclasts (nOC/TV) per tissue volume (Table 3). These results were in contrast to those of the 12-month-old Sam68−/− mice in which all histomorphometric parameters, including marrow fat, resembled those of young mice of either genotype (Table 3). When cells were expressed as a function of the bone perimeter (nOB/BP, nOC/BP), there were no statistical differences and the ratio of osteoblasts to osteoclasts was similar in all groups of mice (Table 3). + +Circulating levels of serum C-telopeptide (sCTX; Roche Diagnostics, Mannheim, Germany) and ALP showed little difference among the groups, except for a small decrease in 12-month-old Sam68−/− mice and (Dataset S3). Serum estrogen was significantly decreased in all 12-month-old mice regardless of genotype with a reciprocal increase in interleukin-6 levels (Dataset S3). Given the involvement of leptin in regulating body and bone mass, serum leptin was measured in Sam68+/+ and Sam68−/− mice. Twelve-month-old Sam68−/− mice had significantly lower levels than young and old Sam68+/+ mice and young Sam68−/− mice (Dataset S3). Taken together, these observations suggested that preservation of bone mass in the Sam68−/− mice was not due primarily to altered estrogen status but could have been influenced by differences in circulating leptin levels. + +Osteoblast, but Not Osteoclast, Activity Is Altered in Sam68−/− Mice Ex Vivo + +Maintenance of bone mass during the adult remodeling cycle is dependent on the coupling of osteoblast to osteoclast activity, such that there is no net gain or loss of bone [49]. To further explore the age-related advantage of Sam68−/− mice with respect to bone preservation, we examined the functional activity of Sam68−/− osteoblasts and osteoclasts ex vivo (Figure 6A and 6B). Cultures of bone marrow stromal cells harvested from 4-week-old Sam68−/− mice and maintained for 18 days in osteoblast differentiation medium demonstrated more intense staining for ALP at 6 days (unpublished data) and 18 days (Figure 6A), and more mineralized nodules at 18 days, than the Sam68+/+ mice, even though similar amounts of fibroblast colony-forming units (CFU-F) were observed (Figure 6A and unpublished data). RT-PCR analysis of molecular markers of osteoblast differentiation (Dataset S2) revealed similar increases over time in RNA from Sam68+/+ and Sam68−/− mice, although type I collagen appeared to be up-regulated at both timepoints in the Sam68−/− mice. Short-term cultures of mature osteoclasts released from the crushed long bones of Sam68+/+ and Sam68−/− mice stained equally well for TRAP and excavated approximately equal numbers of pits of equal size in dentin slices, as visualized by scanning electron microscopy (Figure 6B). These findings suggest that the osteoclasts may not be the primary defect in Sam68−/− mice, as they are in Src null mice [48]. + +The Absence of Sam68 Prevents Adipocyte Differentiation and Promotes Osteoblast Differentiation + +It is well recognized that bone marrow stromal cells give rise to both osteoblasts and adipocytes and that age-related bone loss is accompanied by an increase in differentiation down the adipocyte lineage [22]. Therefore, the loss of Sam68 could influence the bone marrow stromal cells to differentiate along the osteogenic versus the adipogenic pathway. Alternatively, the loss of Sam68 could indirectly regulate bone mass as a general disturbance of neuroendocrine control as was shown when leptin and the sympathetic nervous system axis were shown to negatively regulate bone mass [8]. To confirm a role for Sam68 in the regulation of adipocyte differentiation, we isolated primary MEFs from 14.5-day-old Sam68+/+ and Sam68−/− embryos. The primary Sam68−/− MEFs were differentiated into adipocytes in vitro in culture medium containing 5 μM pioglitazone to induce adipogenesis. Cells at days 0, 4, 6, and 12 were stained with Oil red O to monitor adipogenesis. Adipogenesis was more pronounced in the wild-type MEFs cultures than in the Sam68−/− MEFs, consistent with the positive role of endogenous Sam68 in adipocyte differentiation (Figure 7). The expression of key transcription factors including the PPARγ and KLF5 was impaired in Sam68−/− differentiated MEFs compared with Sam68+/+ MEFs, consistent with impaired adipogenesis in the absence of Sam68 (Figure 7). These data, together with data confirming a lean phenotype in Sam68−/− mice (N. Torabi and S. Richard, unpublished data), support the hypothesis that Sam68 modulates the differentiation of mesenchymal cells. + +To further examine this phenotype in a cell autonomous system, we chose the embryonic mesenchymal multipotential progenitor cells C3H10T1/2. The addition of BMP-2 induced osteoblast differentiation, as evidenced by an approximately 400-fold increase in expression of the osteocalcin (OCN) gene [51]. Populations of C3H10T1/2 cells stably transfected with either pSuper-retro (control) and pSuper-retro harboring a short hairpin against Sam68 (Sam68sh) were selected with puromycin. The expression of Sam68 was reduced by approximately 80% as evidenced by immunoblot analyses using β-actin as a loading control (Figure 8A). Osteoblast differentiation was induced in cultures expressing pSuper-retro or pSuper-retro Sam68sh by addition of BMP-2 to the culture medium. . The expression of OCN and β-actin mRNAs was examined by semiquantitative RT-PCR and the Sam68sh-expressing cells displayed a more pronounced osteoblast phenotype compared with control cells, as assessed by the expression of OCN (Figure 8B). + +Given the apparent enhancement of mineralized nodule formation by Sam68−/− bone marrow stromal cells ex vivo and the phenotype observed with short hairpin RNA (shRNA)-treated C3H10T1/2, we stained sections of bone from 4- and 12-month-old mice for evidence of changes in marrow adiposity. Figure 9 shows sections of undecalcified bone from 4- and 12-month-old Sam68+/+ and Sam68−/− mice stained with von Kossa and toluidine blue to show mineralized tissue (Figure 9A, black) and marrow adipocytes (Figure 9B, white) and left unstained to show fluorochrome labeling of the mineralization fronts (Figure 9C). Twelve-month-old Sam68+/+ mice showed a noticeable decrease in trabecular bone (Figure 9A), which was associated with a significant increase in marrow adiposity (Figure 9B) and with the two fluorochrome labels superimposed upon one another (Figure 9C). In contrast, the bones of 12-month-old Sam68−/− mice appeared similar to those of the 4-month-old mice of either genotype. These data demonstrate that Sam68 regulates the differentiation of bone marrow mesenchymal cells to promote adipocyte differentiation and inhibit osteoblast differentiation in aging bone. + +Discussion + +The present study provides evidence that a physiologic role of Sam68 is to modulate bone marrow mesenchymal stem cells. Young Sam68−/− mice developed normally and contained similar bone mass compared with wild-type littermates. Aged (12 months) Sam68−/− mice displayed a high bone mass phenotype compared with Sam68+/+ littermates. The wild-type littermates underwent age-related bone loss that occurs naturally in mammals, while the Sam68−/− animals preserved their bone mass with aging. The differentiation of bone marrow stem cells isolated from Sam68−/− mice and embryonic mesenchymal multipotential progenitor cells C3H10T1/2 treated with Sam68 shRNA resulted in a more pronounced osteoblast differentiation. These findings demonstrate that the loss of Sam68 enhances osteoblast differentiation. The converse was also true, as MEFs isolated from Sam68−/− animals were impaired in their ability to undergo adipocyte differentiation compared with Sam68+/+ MEFs. These findings suggest that a physiologic role for Sam68 is to regulate the balance between adipogenic and osteogenic differentiation of the bone marrow mesenchymal. + +Sam68 protein expression was observed throughout the developing mouse embryo, in keeping with previous reports that identified the Sam68 mRNA to be widely expressed [38]. We observe Sam68 staining in the brain, heart, and intestine (see Figure 1) as well as liver, skin, and kidney (unpublished data) of late-stage embryos. This expression pattern is consistent with previous work that has observed Sam68 in neurons [52] and as a substrate of the intestinal SIK/breast tumor kinase [43]. The expression of Sam68 was not altered in aging bone marrow stromal cells or in senescencing WI-38 cells (unpublished data). The presence of Sam68 in chondrocytes, osteoblasts and osteoclasts in developing cartilage and bone predicted a pivotal role for the protein in skeletal development. + +The Sam68−/− mice were generated using a traditional targeting approach where the functional KH domain was deleted and approximately one third of the Sam68−/− mice survived to adulthood with no apparent defects. One explanation for this phenomenon is that Sam68 function is sub-served by one or more of its family members during development such as SLM-1 and SLM-2 [47]. Alternatively, Sam68 is not required during embryonic development. However, two thirds of the Sam68−/−, but not the Sam68+/- pups, were killed by their Sam68+/- mothers. These findings suggest that the mothers are able to detect a subtle defect/difference that we cannot. Adult Sam68−/− mice live a normal life span of approximately two years. The Sam68−/− males were sterile and the Sam68−/− females provided inadequate care to their young, but the pups were not scattered and neglected as observed with FosB−/− mice [53]. Serum levels of estrogen decreased in aging Sam68−/− females as expected; however, the leptin levels decreased in aged Sam68−/− females. The aged Sam68−/− females were not obese and actually weighed less than the littermate controls (see Table 2). Moreover, their appetite was not altered with aging (N. Torabi and S. Richard, unpublished data), suggesting that the observed leptin reduction was not recreating ob/ob-like phenotypes related to weight, appetite and female sterility [54]. + +The skeletal phenotyping of cohorts of Sam68+/+ and Sam68−/− mice showed that bone mass was preserved in aged Sam68−/− mice. Traditional histology and histomorphometry suggested that the mechanism involved preservation of osteoblast and osteoclast activity. The documented role of the Sam68 regulatory protein, Src, in osteopetrosis led us to investigate the morphology and activity of Sam68−/− osteoclasts ex vivo. The Src tyrosine kinase was shown to play a role in bone remodeling when Src−/− mice died at 6 months of age with an osteopetrotic phenotype [48] and the defect was attributed to defective osteoclast function [55−59]. We therefore cultured mature osteoclasts harvested from Sam68+/+ and Sam68−/− mice ex vivo on dentin slices to quantify their resorptive capacity. The fact that Sam68−/− osteoclasts looked and acted like Sam68+/+ osteoclasts ex vivo and in vivo made it unlikely that this was the primary source of the difference in bone metabolism in 12-month-old Sam68−/− mice. The fact that the CTX levels were lower in young and old Sam68−/− mice suggested that there is reduced bone resorption compared with wild-type littermate controls. However, this reduction in bone resorption occurred with normal osteoclast activity, as assessed by in vitro culturing. These observations are consistent with the Sam68−/− mice having a youth-like bone phenotype. However, it is still possible, that a mild impairment in Sam68−/− mice osteoclast function may manifest itself later in life in overall accumulation of bone and this will require further detailed studies. + +Ex vivo differentiation of primary bone marrow stromal cells, harvested from Sam68+/+ and Sam68−/− mice, down the osteoblast lineage revealed an osteogenic advantage in the cultures of cells derived from the Sam68−/− mice. It will be important to demonstrate that a similar effect is observed using primary bone marrow stromal cells from aged Sam68−/− mice. Similar findings were observed when embryonic mesenchymal multipotential progenitor cells C3H10T1/2 treated with Sam68 shRNA, were differentiated into osteoblasts with BMP2. Our findings that aged Sam68−/− mice do not develop fatty bone marrow and that MEFs derived from Sam68 −/− mice have impaired adipocyte differentiation, indicate that Sam68 regulates both adipocyte and osteoblast differentiation. These findings identify Sam68 as the first RNA binding protein to regulate mesenchymal cell differentiation and the challenge will be to identify the specific RNA targets that it regulates during this process. The fact that osteoblast function is altered in Src−/− mice [60,61] raises the possibility that preservation of bone mass in the Sam68−/− mice could be linked with and regulated by Src. + +The pathway by which leptin regulates bone resorption was identified to involve the sympathetic nervous system relaying to the osteoblasts via the β-adrenergic pathway leading to the release of growth factors including RANKL that causes the osteoclasts to thrive [8,18–20]. The lowering of leptin levels in aged Sam68−/− mice is consistent with these mice having a high bone mass compared with their aged littermates. These data would suggest that the leptin-sympathetic pathway is unaltered in Sam68−/− mice and that the lowering of leptin may explain the lower levels of CTX in the serum of Sam68−/− mice. These findings suggest that Sam68 may be regulating bone metabolism at two different levels: (1) the absence of Sam68 results in lower leptin levels that may reduce bone resorption via the sympathetic nervous system and (2) the absence of Sam68 favors osteoblast, rather than adipocyte, differentiation. + +In conclusion, our data define a physiologic role for Sam68 in bone metabolism and bone marrow mesenchymal stem cell differentiation. The bone phenotype observed in Sam68−/− mice imply that inhibitors of Sam68 could prevent age-related bone loss. Furthermore, the results also suggest that Sam68 expression levels, hypomorphism, and mutations in humans may influence susceptibility to marrow adipocyte accumulation and osteoporosis. Our findings also identify a new animal model to study aging bone loss. + +Materials and Methods + +Histologic, immunohistochemical, and histomorphometric analyses. + +All analyses were performed essentially as described previously [62,63]. Briefly, embryonic mice were removed from timed pregnant dams at E14.5 and E16.5 and fixed intact for 36 h in 4% paraformaldehyde, rinsed thoroughly in PBS, and processed for paraffin embedding. Serial 4-μm sections were cut on a modified Leica RM 2155 rotary microtome (Leica Microsystems, Richmond Hill, Ontario, Canada), stained with a 1:600 dilution of the AD1 anti-Sam68 antibody [46] and counterstained with either methyl green or hematoxylin. + +Adult mice were given an intraperitoneal injection of 30 mg/kg calcein at 7 days and 30 mg/kg tetracycline at 2 days prior to sacrifice to label actively mineralizing surfaces. After overnight fixation in 4% paraformaldehyde and rinsing in PBS, the left femur and tibia were embedded in polymethylmethacrylate (MMA) or a mixture of 50% MMA and 50% glycolmethacrylate (GMA). Serial 4- to 6-μm sections of MMA-embedded tissues were left unstained or stained with von Kossa and toluidine blue or with toluidine blue alone, while 4-μm MMA-GMA sections were stained for TRAP and ALP activity. Images were captured using a Leica DMR microscope (Leica Microsystems) equipped with a Retiga 1300 camera (Qimaging, Burnaby, British Columbia, Canada) and the primary histomorphometric data obtained using Bioquant Nova Prime image analysis software (Bioquant Image Analysis Corp, Nashville, Tennessee, United States). Nomenclature and abbreviations conform to those recommended by the American Society for Bone and Mineral Research [50]. + +Generation of mice with targeted disruption of the sam68 gene. + +A λ bacteriophage clone encompassing Sam68 exons 3 to 9 was isolated from a 129/SvJ genomic library using full-length Sam68 cDNA as a probe. XbaI-digested Sam68 genomic DNA fragments of 4 kb (encompassing exon 4 and part of exon 5) and 3kb (spanning part of exon 5 and exon 6) were subcloned in Bluescript SK resulting in pBS4 and pBS3, respectively. A DNA fragment was amplified from pBS4 with the following oligonucleotides (5′-AAT GTC TAG AAA CAA CTC ATA TAC AGA C-3′) and the universal primer. The XbaI-digested 1-kb DNA fragment was subcloned in the XbaI site of pPNT (a gift from Andrew Karaplis, McGill University, Montréal, Quebec, Canada). The 3-kb fragment from pBS3 was amplified by PCR with (5′-GGG ATG CGG CCG CTC TAG AAT TGT CCT ACT TGA ACG G-3') and (5′-CGG TGG CGG CCG CTG TCG ACC TGA GTA ACA TTT CTT A-3′) and subcloned in the NotI site of pPNT. The targeting vector pPNT-Sam68 replaces exon 4 and part of exon 5 with a neomycin-resistant gene cassette. An SalI site was introduced at the 3′ end of the 3-kb DNA fragment and was used to linearize the plasmid for electroporation into embryonic stem (ES) cells. Approximately 1,000 ES colonies were screened and two clones were identified that contained the Sam68 mutant allele, as determined by Southern blotting. Targeted ES cells were injected into 3.5-day-old BALB/c blastocysts and were transferred into CD-1 foster mothers, and animals classified as chimeras by coat color were mated with BALB/c mice. Germ line transmission was achieved and the mice were maintained in C57BL/6 background. The mice used for this study represent mice that were backcrossed in C57BL/6 between three and eight generations; in addition, we maintained the mice in the 129/SvJ strain and observed a similar phenotype. + +Genotyping and immunoblot analyses. + +All mouse procedures were performed in accordance with McGill University guidelines, which are set by the Canadian Council on Animal Care. Genomic DNA was isolated from tail biopsies and analyzed by Southern blotting and genomic PCR analysis. The DNA fragment utilized as the probe for the Southern blotting analysis was amplified with the following two oligonucleotides (5′-AAG CCT TTA CTG GTT GTG T-3′) and (5′-CTT GAA ACG CAC CGT AGG CT-3′). The wild-type sam68 allele was identified by genomic PCR using the following oligonucleotides 5′-AAA TCC TAA CCC TCC TCA GTC AG-3′ and 5′-GAT ATG ATG GAT GAT ATC TGT CAG-3′. The sam68-targeted allele was identified by genomic PCR using the following oligonucleotides 5′-CTT GGG TGG AGA GGC TAT TCG-3′ and 5′-GTC GGG CAT GCG CGC CTT GAG C-3′. + +Radiology and serum biochemistry on Sam68+/+ and Sam68−/− mice. + +Radiography, BMD, and micro-CT were performed essentially as described previously [63]. Mice were administered a lethal dose of anesthetic at the indicated times, exsanguinated, and imaged using a Faxitron MX20 equipped with an FPX-2 Imaging system (Dalsa Medoptics, Waterloo, Ontario, Canada). Body fat, BMC, and BMD were evaluated using a Lunar PixiMUS 1.46 (GE-Lunar, Madison, Wisconsin, United States). Morphometric parameters were determined on anesthetized mice at the time of sacrifice by direct measurement or from the Faxitron radiograph. + +Micro-CT was performed on the left femur and fourth lumbar vertebra after removal of soft tissues and overnight fixation in 4% paraformaldehyde. The distal metaphysis was scanned with a Skyscan 1072 micro-CT instrument (Skyscan, Antwerp, Belgium). Image acquisition was performed at 100 kV and 98 μA, with a 0.9° rotation between frames. The two-dimensional images were used to generate three-dimensional reconstructions to obtain quantitative data with the 3D Creator software supplied with the instrument. + +Serum biochemistry (ALP, Ca, PO4, Mg) was determined at the Rodent Diagnostics Lab (McGill University, Montréal, Quebec, Canada) using routine automated techniques. Commercial assays were used to determine serum levels of CTX (RatLaps ELISA, Nordic Bioscience), 17β estradiol (IBL Immuno-Biological, Hamburg, Germany), leptin (R & D Systems, Minneapolis, Minnesota, United States), and interleukin-6 (R & D Systems). + +Ex vivo assessment of osteoblast and osteoclast activity. + +Bone marrow was flushed from the tibia and femora of juvenile 4-week-old Sam68+/+ and Sam68−/− mice to obtain stromal cells that were maintained in differentiation medium for 6 or 18 days as described [63]. Cultures were stained in situ for ALP activity and with von Kossa stain to detect mineralized nodules. Total RNA was harvested from parallel cultures for RT-PCR analysis of osteoblast-related gene expression over time as described previously [63]. + +Osteoclasts were isolated from the crushed long bones of juvenile Sam68+/+ and Sam68−/− mice and used for quantitative studies as described [64]. Briefly, cells were plated on glass coverslips or on dentin slices in 24-well cluster plates for assessment of cell number and pit number, respectively. Cover slips were immersed in 4% paraformaldehyde and stained for TRAP activity after 2 h. Dentin slices were left for 28 h before removing the cells with 1 M ammonium hydroxide and air drying before coating with sputter Au-Pd and examination with scanning electron microscopy (McGill Electron Microscopy Centre). Light microscope images were captured using a Leica DMR microscope equipped with a Retiga 1300 camera. Quantitative analyses were performed using Adobe Photoshop and the data presented as the mean ± SD of two or three independent experiments. Statistical comparisons were made using the Student's t test. + +Preparation of mouse embryonic fibroblast and induction of adipocyte differentiation. + +MEFs were prepared from 14.5-day-old embryos. Only early-passage MEFs were used for the experimental studies, and induction of adipocyte differentiation was carried out according to the methods previously described [30]. Cultured cells were stained with Oil Red O as described [29]. + +RNA preparation and RT-PCR to assess adipogenesis in Sam68−/− MEFs. + +Total cellular RNA was prepared by TRIzol reagent according to the manufacturer's protocol (Invitrogen, Carlsbad, California, United States). Total RNA (1 μg) was reverse-transcribed, and cDNA samples were subjected to PCR. RT-PCR was normalized by the transcriptional level of GAPDH. The following 5′ and 3′ primers were used to evaluate adipogenic differentiation: KLF5: 5'-AGA CAA GCT GAG ATG CTG C-3′, 5′-GGC AAA CCT CCA GTC GC-3′; PPARγ: 5′-GTG CGA TCA AAG TAG AAC CTG C-3′, 5′-CCT ATC ATA AAT AAG CTT CAA TCG-3′; β-Actin: 5′-TAG GCG GAC TGT TAC TGA GC-3′, 5′-AGC CTT CAT ACA TCA AGT TGG-3′; and Sam68: 5′- GTG GAG ACC CCA AAT ATG CCC A-3′ and 5′-AAA CTG CTC CTG ACA GAT ATC A-3′. + +Sam68 down-regulation using Sam68sh. + +To generate an shRNA, 64-base duplex DNA nucleotides were purchased from Invitrogen. This fragment comprised 19 base residues specific to mouse Sam68 (GATCCCC AAGATGACGAGGAGAATTATTCAAGAGA TAATTCTCCTCGTCATCTT TTTTTGGAAA). This fragment was cloned into pSUPER retro (OligoEngine, Seattle, Washington, United States). Transfections were performed using LipofectAMINE Plus (Invitrogen) with cloned DNA fragment or empty vector. At 48 h after transfection, populations were selected for 2.5 μg/ml puromycin and the knockdown observed by immunoblotting with anti-Sam68 (AD1) antibody. + +Osteogenic differentiation analysis of C3HT101/2 cells. + +For osteogenic differentiation, C3HT101/2 (ATCC) cells were incubated in 48-well tissue culture plates at 1.25 × 104 cells/cm2 . After 24-h incubation, the culture media were changed to fresh media containing 300 ng/ml BMP-2 (Sigma, St. Louis, Missouri, United States). Total cellular RNA was prepared as described above, and osteocalcin, β-actin, and GAPDH gene expression at indicated times after BMP-2 treatment was quantified by RT-PCR. + +Supporting Information + +Dataset S1 + +Localization of Sam68 in Primary Mouse Osteoblasts and Osteoclasts + +(124 KB PPT) + +Click here for additional data file. + +Dataset S2 + +Gene Expression Profile of Cultured Stromal Cells at Days 6 and 18 of Culture Isolated from Sam68+/+ and Sam68−/− Mice + +(204 KB PPT) + +Click here for additional data file. + +Dataset S3 + +Serum Biochemistry and Bone Biomarkers in 4- and 12-Month-Old Mice + +(46 KB DOC) + +Click here for additional data file. + +Acknowledgements + +We acknowledge Jean-Sebastien Binette (Centre for Bone and Periodontal Research) and Jacinthe Sirois (Québec Transgenic Research Network) for excellent technical assistance. We are grateful to Erique Lukong, Daniel Larocque, and Mark Bedford for helpful discussions. This work was supported by grant MT13377 to SR and grant MOP-13419 to JEH from the Canadian Institutes of Health Research (CIHR). SR holds an Investigator Award from the CIHR and JEH is a Chercheur Boursier, Senior of the FRSQ. + +Abbreviations + +ALP - alkaline phosphatase + +BMC - bone mineral content + +BMD - bone mineral density + +BMP-2 - bone morphogenetic protein-2 + +CT - computed tomography + +CTX - C-telopeptide + +MEF - mouse embryo fibroblast + +PPARγ - peroxisome proliferator–activated receptor γ + +shRNA - short hairpin RNA + +TRAP - tartrate-resistant acid phosphatase + +Figures and Tables + +Figure 1 + +Immunohistochemical Localization of Sam68 in Embryonic Mice + +(A) Embryonic mice were removed from pregnant dams at E14.5 and E16.5, fixed in 4% paraformaldehyde, and embedded in paraffin. The entire embryo was immunostained with the AD1 anti-Sam68 antibody and counterstained with methyl green, and the image was captured at ×1.2 magnification. + +(B) Embryonic soft tissues from the brain, heart and gut were stained with hematoxylin (left) and immunostained with anti-Sam68 antibody (right), and images were captured at ×20 magnification. + +(C) Intense anti-Sam68 immunoreactivity was seen in chondrocytes in the nasal septum (panels A–D), in developing vertebra (panels E–H), and in the femoral epiphysis (panels I–K), as well as in diaphyseal osteoblasts (panel L). Adjacent sections were stained with hematoxylin and eosin (panels A, E, and I) or with antibody preadsorbed with the immunizing peptide (panels B, F, and J). Sam68 was localized primarily in the nucleus of cells in a variety of tissues but was also found occasionally in the cytoplasm. Magnification at source ×20, except for panels D, H, and L, which were ×40. Staining patterns are representative of three to five embryos. + +Figure 2 + +Generation of Sam68-Deficient Mice + +(A) The genomic organizations of the wild-type and targeted sam68 alleles after homologous recombination are depicted. The location of the DNA fragment used as a probe for the Southern blot analysis is shown, as well as the sizes of the two BglII fragments detected for wild-type and targeted sam68 alleles. The targeted allele replaces exon 4 and part of exon 5 of sam68 with a PGK-neomycin cassette. + +(B) Southern-blot analysis of genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. DNA fragments corresponding to wild-type (4.5 kb) and the targeted (5.5 kb) alleles are illustrated. + +(C) Western blot analysis of Sam68 expression. Protein extracts from wild-type, heterozygous, and homozygous cells subjected to immunoblot analyses using normal rabbit serum, anti-Sam68 AD1 antibody, the peptide antibody AD1 preabsorbed with the immunogenic peptide corresponding to amino acids 330–348 of mouse Sam68, anti-Sam68 Sc333 antibody that recognizes the C-terminal 20 amino acids of Sam68, and anti-actin antibodies as loading control. The migration of the molecular mass markers is known on the left in kDa. + +Figure 3 + +Radiologic Assessment of the Femur of Young and Old Sam68+/+ and Sam68−/− Mice + +(A) Mice were given a lethal dose of anesthetic at the indicated times, and contact radiographs of the distal femora were obtained on a Faxitron MX20 equipped with an FPX-2 Imaging system. Representative radiographs of the distal femur of Sam68+/+ (+/+) and Sam68−/− (−/−) mice revealed comparable radiopacity at 4 months (left). At 12 months (right), cortical thinning (arrow) and radiolucency (asterisk) were apparent in the distal femur of +/+ mice but not −/− mice. + +(B) Bones were dissected free of soft tissue and fixed overnight in 4% paraformaldehyde before scanning on a Skyscan 1072 static instrument equipped with 3D Creator analytical software. Representative three-dimensional re-constructions and two-dimensional cross-sectional scans demonstrated similar architecture in the distal femur of Sam68+/+ (+/+) and Sam68−/− (−/−) mice. In keeping with the results from Faxitron x-ray, trabecular bone (asterisk) and cortical thickness (arrow) were reduced in the femur of 12-month-old +/+ mice compared with all other groups. The images are representative of those from five to seven animals in each group. + +Figure 4 + +Quantitative Micro-CT of Trabecular Bone Composition and Architecture + +(A) Bone volume/tissue volume (BV/TV) and structure model index (SMI) were calculated on the femur and fourth lumbar vertebra of six or seven mice in each group using 3D Creator software supplied with the Skyscan instrument. Results expressed as the mean ± SD showed significant differences (p < 0.01) between 4-month-old Sam68+/+ mice (solid black) and 12-month-old Sam68+/+ mice (hatched black) but not between 4-month-old Sam68−/− mice (solid white) and 12-month-old Sam68−/− mice (stippled white). + +(B) The distance between trabeculae was reflected in a shift to the right of the distribution curves for 12-month-old Sam68+/+. Solid black = 4-month-old Sam68+/+; hatched black = 12-month-old Sam68+/+; solid red = 4-month-old Sam68−/−; stippled red = 12-month-old Sam68−/−. The asterisks denote p < 0.01. + +Figure 5 + +Histologic Analysis of Undecalcified Bone from Sam68+/+ and Sam68−/− Mice + +Sections of tibia fixed in 4% paraformaldehyde and embedded in plastic were stained for ALP (A–C, E–G) activity to identify osteoblasts or for TRAP (B–D, F–H) activity to identify osteoclasts. Staining patterns were similar in 4-month-old Sam68+/+ (A and B), 4-month-old Sam68−/− (C and D), and 12-month-old Sam68−/− (G and H) mice compared with 12-month-old Sam68+/+ mice (E and F). Magnification at source, left panels ×10 and right panels ×40. Micrographs are representative of those taken from five to seven sections in each group of animals. + +Figure 6 + +Ex Vivo Activity of Sam68+/+ and Sam68−/− Osteoblasts and Osteoclasts + +Marrow stromal cells were isolated from the long bones of juvenile mice and maintained under conditions that promote osteoblast differentiation. + +(A) Cultures were fixed in 4% paraformaldehyde after 6 or 18 days and stained in situ for ALP activity and with silver nitrate (von Kossa) to detect mineralized nodules. Sam68−/− cultures stained more intensely for ALP at early and late time points and produced significantly more mineralized nodules after 18 days. Asterisks represent p < 0.01. + +(B) Primary osteoclasts were isolated from the crushed long bones of the same mice and plated on glass coverslips or on dentin slices to quantify numbers and activity, respectively. Osteoclasts were identified as cells with three or more nuclei that stained positive for TRAP activity (upper) and excavated pits in dentin slices, as demonstrated by SEM (lower, bar = 20 μm). No statistical differences were observed either in the number of TRAP-positive cells or in their resorptive activity. + +Figure 7 + +Ex Vivo Adipogenesis Analysis of Sam68−/− Mouse Embryonic Fibroblasts + +MEFs were isolated from mouse embryos at embryonic day 14.5. Equal number of MEFs from Sam68+/+ and Sam68−/− was plated on glass cover slips in 24 well-plates. Adipocyte differentiation was carried out at indicated times by the addition of complete media containing the pioglitazone. + +(A) Cultures were fixed in 4% paraformaldehyde and stained with Oil Red O to detect the fat droplets stored in adipocytes and photographed (top). The cell images were magnified ×10 and ×20 as indicated. + +(B) RT-PCR was carried out on total cellular RNA isolated after differentiation of the MEFs for day 0, 2, 4, 6, and 12. The DNA fragments were visualized on agarose gels stained with ethidium bromide. The expression of adipogenic markers C/EBPβ, C/EBPδ, PPARα, and KLF5 was examined as well as the expression of controls including Sam68, β-actin, and GAPDH. + +Figure 8 + +Enhanced Osteogenic Differentiation of the C3HT101/2 Embryonic Cell Line Depleted of Endogenous Sam68 + +(A) C3HT101/2 cells transfected with an empty vector (pSuper-retro) or a vector containing an shRNA (Sam68 shRNA) were selected with puromycin, and knockdown populations depleted of Sam68 were identified. The reduction in Sam68 protein was analyzed by immunoblotting with anti-Sam68 (AD1) antibody and anti–β-actin antibodies as loading controls. + +(B) Osteogenic differentiation was carried out with conditioned medium containing BMP-2 for the indicated times. To assess the level of osteogenic differentiation in these cells, expression of late osteoblast marker, osteocalcin (OCN), was analyzed by RT-PCR and compared with β-actin and GAPDH controls. The DNA fragments were visualized by agarose gel stained with ethidium bromide. + +Figure 9 + +Old Sam68−/− Mice Are Protected from the Development of Fatty Bone Marrow + +Sections of undecalcified bone were stained with von Kossa and toluidine blue and images captured at original magnifications of ×2 (A), ×40 (B and C) to evaluate mineralized tissue (A, black), marrow adipocytes (B, white), and the mineralization fronts (C, yellow and green). The 12-month-old Sam68+/+ bone demonstrated a significant reduction in bone (A) and increase in marrow adipocytes (B) and a decrease in the distance between two consecutive fluorochrome labels (C). Magnification at source was ×40. Micrographs are representative of four to six screened in each group of animals. + +Table 1 + +Progeny of Sam68 Heterozygote Breeding + +Table 2 + +Morphology and Bone Mineral Density in 4- and 12-Month-Old Female Mice + +Table 3 + +Histomorphometric Analysis of Long Bones from 4- and 12-Month-Old Mice + +Footnotes + +Competing interests. The authors have declared that no competing interests exist. + +Author contributions. SR and JEH conceived and designed the experiments. SR, NT, GVF, GAT, TC, GV, MM, PC, AFR, SK, WL, AL, and YJG performed the experiments. SR and JEH analyzed the data. MLT generated the mice. SR and JEH wrote the paper. + +A previous version of this article appeared as an Early Online Release on November 11, 2005 (DOI: 10.1371/journal.pgen.0010074.eor). + +Citation: Richard S, Torabi N, Franco GV, Tremblay GA, Chen T, et al. (2005) Ablation of the Sam68 RNA binding protein protects mice from age-related bone loss. PLoS Genet 1(6): e74. diff --git a/src/ontogpt/evaluation/craft/database/all/16410827.ann b/src/ontogpt/evaluation/craft/database/all/16410827.ann new file mode 100644 index 000000000..feb4f3cbc --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16410827.ann @@ -0,0 +1,1429 @@ +T1 PR:000001921 17 21 JAG1 +T2 NCBITaxon:40674 76 85 Mammalian +T3 UBERON:0001846 86 95 Inner Ear +T4 NCBITaxon:40674 110 117 mammals +T5 UBERON:0001846 155 164 inner ear +T6 GO:0007605 183 190 hearing +T7 CL:0000855 247 257 hair cells +T8 CL:0000630 309 325 supporting cells +T9 GO:0065007 418 424 govern +T10 GO:0007423 429 443;450 464 development of ... sensory organs +T11 UBERON:0000020 450 464 sensory organs +T12 GO:0007219 466 481 Notch signaling +T13 GO:0030154 510 528;534 539 differentiation of ... cells +T14 GO:0030154 510 528;555 560 differentiation of ... cells +T15 CL:0000855 529 539 hair cells +T16 CL:0000630 544 560 supporting cells +T17 GO:0046331 574 592 lateral inhibition +T18 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UBERON:0001844 3275 3282 cochlea +T95 UBERON:0001840 3314 3333 semicircular canals +T96 UBERON:0001349 3345 3354 vestibule +T97 UBERON:0001860 3382 3400 endolymphatic duct +T98 UBERON:0002223 3382 3395;3405 3408 endolymphatic ... sac +T99 UBERON:0001860 3436 3454 endolymphatic duct +T100 UBERON:0002223 3436 3449;3459 3462 endolymphatic ... sac +T101 UBERON:0001690 3491 3494 ear +T102 UBERON:0000020 3507 3521 sensory organs +T103 CL:0000855 3535 3553 sensory hair cells +T104 CL:0000630 3575 3591 supporting cells +T105 UBERON:0000020 3633 3647 sensory organs +T106 UBERON:0004721 3649 3656 cristae +T107 UBERON:0001840 3686 3704 semicircular canal +T108 UBERON:0000054 3706 3713 maculae +T109 UBERON:0001349 3741 3750 vestibule +T110 UBERON:0002227 3760 3774 organ of Corti +T111 UBERON:0001855 3792 3805 cochlear duct +T112 UBERON:0000020 3816 3829 sensory organ +T113 UBERON:0002227 3835 3849 organ of Corti +T114 GO:0007605 3867 3874 hearing +T115 UBERON:0000062 3891 3897 organs +T116 NCBITaxon:40674 3943 3950 mammals +T117 SO:0000704 4008 4015 genetic +T118 GO:0031099 4036 4046 regenerate +T119 GO:0007605 4068 4075 hearing +T120 http://purl.obolibrary.org/obo/MONDO_0005365 4068 4075;4091 4101 hearing impairment +T121 UBERON:0001690 4220 4223 ear +T122 UBERON:0000020 4349 4362 sensory organ +T123 NCBITaxon:7742 4466 4476 vertebrate +T124 UBERON:0001846 4477 4486 inner ear +T125 NCBITaxon:7215 4521 4531 Drosophila +T126 UBERON:0000020 4532 4543 sense organ +T127 GO:0007423 4532 4555 sense organ development +T128 NCBITaxon:7215 4583 4593 Drosophila +T129 UBERON:0000020 4603 4614 sense organ +T130 GO:0007423 4603 4626 sense organ development +T131 GO:0046331 4628 4646 lateral inhibition +T132 GO:0007219 4659 4674 Notch signaling +T133 UBERON:0000020 4719 4732 sensory organ +T134 UBERON:0000020 4790 4803 sensory organ +T135 NCBITaxon:7742 4829 4839 vertebrate +T136 UBERON:0001690 4840 4843 ear +T137 GO:0046331 4845 4863 lateral inhibition +T138 GO:0007219 4876 4891 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exon +T265 PR:000001921 8450 8454 Jag1 +T266 SO:0000704 8455 8459 gene +T267 SO:0000346 8465 8475 loxP sites +T268 SO:0000417 8496 8502 domain +T269 SO:0000842 8640 8649;8654 8658 region of ... gene +T270 SO:0001023 8723 8729 allele +T271 PR:000001921 8774 8778 Jag1 +T272 SO:0000359 8778 8782 flox +T273 NCBITaxon:10088 8785 8789 mice +T274 NCBITaxon:10088 8793 8797 mice +T275 GO:0010467 8798 8808 expressing +T276 GO:0065007 8854 8861 control +T277 PR:000018212 8869 8872 Zp3 +T278 SO:0000167 8873 8881 promoter +T279 PR:000018212 8883 8886 Zp3 +T280 NCBITaxon:10088 8891 8895 mice +T281 PR:000018212 8902 8905 Zp3 +T282 NCBITaxon:10088 8910 8915 mouse +T283 GO:0010467 8941 8948 express +T284 GO:0040007 8972 8979 growing +T285 CL:0000023 8980 8986 oocyte +T286 GO:0007127 9018 9040 first meiotic division +T287 PR:000018212 9054 9057 Zp3 +T288 PR:000001921 9065 9069 Jag1 +T289 SO:0000359 9069 9073 flox +T290 NCBITaxon:10088 9115 9119 mice +T291 SO:0000359 9194 9200 floxed +T292 SO:0001023 9201 9207 allele +T293 PR:000001921 9220 9224 Jag1 +T294 PR:000001921 9273 9277 Jag1 +T295 NCBITaxon:10088 9284 9288 mice +T296 PR:000001921 9312 9316 Jag1 +T297 PR:000001921 9321 9325 Jag1 +T298 PR:000001921 9401 9405 Jag1 +T299 PR:000001921 9410 9414 Jag1 +T300 UBERON:0000922 9426 9433 embryos +T301 UBERON:0000922 9499 9506 embryos +T302 PR:000001921 9533 9537 Jag1 +T303 SO:0001023 9543 9549 allele +T304 PR:000001921 9551 9555 Jag1 +T305 PR:000001921 9584 9588 Jag1 +T306 PR:000001921 9593 9597 Jag1 +T307 UBERON:0000922 9609 9616 embryos +T308 UBERON:0001040 9627 9635 yolk sac +T309 UBERON:0002406 9722 9737 pericardial sac +T310 PR:000001921 9758 9762 Jag1 +T311 PR:000001921 9767 9771 Jag1 +T312 UBERON:0000922 9783 9790 embryos +T313 UBERON:0000922 9901 9908 embryos +T314 SO:0000112 9915 9922 primers +T315 SO:0000359 9937 9943 floxed +T316 SO:0000147 9944 9948 exon +T317 PR:000001921 10063 10067 Jag1 +T318 SO:0000359 10067 10071 flox +T319 SO:0001023 10072 10078 allele +T320 PR:000001921 10102 10106 Jag1 +T321 SO:0001023 10114 10120 allele +T322 SO:0001023 10163 10169 Allele +T323 PR:000001921 10177 10181 Jag1 +T324 SO:0000704 10182 10186 Gene +T325 PR:000001921 10246 10250 Jag1 +T326 SO:0000359 10250 10254 flox +T327 NCBITaxon:10088 10255 10259 mice +T328 SO:0001644 10265 10281 targeting vector +T329 SO:0000346 10305 10315 loxP sites +T330 SO:0000147 10353 10357 exon +T331 SO:0000417 10369 10375 domain +T332 SO:0000147 10385 10389 exon +T333 SO:0000147 10405 10409 exon +T334 CHEBI:7507 10418 10426 neomycin +T335 SO:0005853 10438 10446 cassette +T336 SO:0000357 10476 10483 flanked +T337 SO:0000350 10487 10496 FRT sites +T338 GO:0010467 10569 10579 expressing +T339 NCBITaxon:10088 10580 10584 mice +T340 http://purl.obolibrary.org/obo/MONDO_0005504 10588 10598 diphtheria +T341 CHEBI:27026 10599 10604 toxin +T342 SO:0000704 10605 10609 gene +T343 SO:0000112 10757 10763 Primer +T344 http://purl.obolibrary.org/obo/MONDO_0005504 10901 10911 diphtheria +T345 CHEBI:27026 10912 10917 toxin +T346 CL:0002322 10993 11001 ES cells +T347 CL:0002322 11142 11150 ES cells +T348 PR:000001921 11230 11234 Jag1 +T349 PR:000001921 11239 11243 Jag1 +T350 UBERON:0000922 11248 11255 Embryos +T351 PR:000001921 11319 11323 JAG1 +T352 UBERON:0000922 11350 11357 embryos +T353 UBERON:0001981 11390 11403 blood vessels +T354 PR:000001921 11433 11437 Jag1 +T355 PR:000001921 11442 11446 Jag1 +T356 UBERON:0001040 11451 11460 yolk sacs +T357 PR:000001921 11482 11486 Jag1 +T358 UBERON:0000922 11527 11534 embryos +T359 PR:000001921 11567 11571 Jag1 +T360 PR:000001921 11576 11580 Jag1 +T361 UBERON:0000922 11585 11591 embryo +T362 UBERON:0002406 11638 11653 pericardial sac +T363 UBERON:0004535 11724 11738 cardiovascular +T364 SO:0000112 11769 11776 primers +T365 SO:0000147 11787 11791 exon +T366 PR:000018212 11841 11844 ZP3 +T367 UBERON:0000922 11857 11864 embryos +T368 SO:0000028 11894 11896 bp +T369 SO:0001023 11927 11933 allele +T370 SO:0000028 11967 11969 bp +T371 SO:0001023 12001 12007 allele +T372 SO:0000147 12030 12034 exon +T373 PR:000001921 12055 12059 Jag1 +T374 UBERON:0001690 12080 12083 Ear +T375 PR:000001921 12096 12100 JAG1 +T376 UBERON:0001846 12121 12130 inner ear +T377 PR:000001921 12143 12147 Jag1 +T378 SO:0000359 12147 12151 flox +T379 PR:000001921 12152 12156 Jag1 +T380 SO:0000359 12156 12160 flox +T381 NCBITaxon:10088 12161 12165 mice +T382 NCBITaxon:10088 12171 12175 mice +T383 PR:000007623 12204 12209 Foxg1 +T384 SO:0001023 12214 12220 allele +T385 NCBITaxon:10088 12228 12232 mice +T386 GO:0010467 12233 12240 express +T387 UBERON:0003051 12272 12279 otocyst +T388 UBERON:0001890 12292 12301 forebrain +T389 UBERON:0000970 12303 12306 eye +T390 UBERON:0001041 12312 12319 foregut +T391 PR:000001921 12334 12338 Jag1 +T392 SO:0001023 12343 12349 allele +T393 PR:000007623 12384 12389 Foxg1 +T394 PR:000001921 12397 12401 Jag1 +T395 PR:000001921 12406 12410 Jag1 +T396 SO:0000359 12410 12414 flox +T397 PR:000001921 12437 12441 Jag1 +T398 UBERON:0001846 12492 12501 inner ear +T399 PR:000001921 12564 12568 Jag1 +T400 UBERON:0000922 12573 12580 embryos +T401 UBERON:0001844 12597 12605 cochleae +T402 GO:0097617 12632 12645 hybridization +T403 SO:0000147 12699 12703 exon +T404 GO:0010467 12744 12754 expression +T405 UBERON:0003051 12762 12769 otocyst +T406 UBERON:0001844 12836 12844 cochlear +T407 GO:0010467 12845 12855 expression +T408 GO:0010467 12882 12892 expression +T409 CL:0000057 13020 13030 Fibroblast +T410 PR:000001448 13020 13055 Fibroblast growth factor receptor 1 +T411 PR:000001448 13057 13062 Fgfr1 +T412 SO:0000359 13064 13070 floxed +T413 SO:0001023 13071 13077 allele +T414 PR:000007623 13088 13093 Foxg1 +T415 PR:000001921 13127 13131 Jag1 +T416 SO:0000359 13131 13135 flox +T417 SO:0001023 13136 13142 allele +T418 UBERON:0001846 13156 13165 inner ear +T419 GO:0048839 13156 13177 inner ear development +T420 PR:000001921 13202 13206 Jag1 +T421 PR:000007623 13230 13235 Foxg1 +T422 UBERON:0000922 13285 13292 embryos +T423 GO:0097617 13327 13340 hybridization +T424 PR:000001921 13349 13353 Jag1 +T425 SO:0000147 13354 13358 exon +T426 PR:000001921 13407 13411 Jag1 +T427 UBERON:0003051 13426 13433 otocyst +T428 UBERON:0000970 13455 13458 eye +T429 GO:0010467 13514 13523 expressed +T430 PR:000001921 13528 13532 Jag1 +T431 GO:0010467 13633 13643 expression +T432 UBERON:0002240 13651 13662 spinal cord +T433 UBERON:0009201 13667 13679 nephric duct +T434 GO:0010467 13711 13721 expression +T435 PR:000007623 13747 13752 Foxg1 +T436 NCBITaxon:10088 13757 13761 mice +T437 GO:0010467 13772 13782 expression +T438 PR:000001921 13824 13828 Jag1 +T439 UBERON:0000922 13833 13840 embryos +T440 GO:0010467 13896 13906 expression +T441 PR:000001921 13929 13933 Jag1 +T442 UBERON:0000922 13938 13945 embryos +T443 UBERON:0003051 13959 13966 otocyst +T444 UBERON:0000970 13975 13978 eye +T445 GO:0010467 14022 14032 expression +T446 PR:000007623 14079 14084 Foxg1 +T447 GO:0010467 14089 14099 expression +T448 GO:0097617 14120 14133 hybridization +T449 UBERON:0001844 14143 14151 cochleae +T450 PR:000001921 14166 14170 Jag1 +T451 GO:0010467 14171 14181 expression +T452 PR:000001921 14203 14207 Jag1 +T453 UBERON:0001844 14212 14220 cochleae +T454 GO:0010467 14232 14242 expression +T455 UBERON:0001846 14305 14314 Inner Ear +T456 PR:000001921 14318 14322 Jag1 +T457 PR:000001921 14369 14373 Jag1 +T458 UBERON:0001846 14378 14388 inner ears +T459 UBERON:0001846 14410 14420 inner ears +T460 PR:000001921 14556 14560 Jag1 +T461 UBERON:0001846 14565 14575 inner ears +T462 UBERON:0001840 14652 14671 semicircular canals +T463 UBERON:0001841 14732 14740;14753 14772 anterior ... semicircular canals +T464 UBERON:0001843 14745 14772 lateral semicircular canals +T465 UBERON:0001853 14791 14798 utricle +T466 UBERON:0001854 14819 14826 saccule +T467 UBERON:0001844 14850 14857 cochlea +T468 UBERON:0001846 14905 14914 inner ear +T469 GO:0048880 14944 14961 sensory formation +T470 UBERON:0001860 14977 14995 endolymphatic duct +T471 UBERON:0002223 14977 14990;15000 15003 endolymphatic ... sac +T472 UBERON:2005411 15012 15023 common crus +T473 UBERON:0001846 15068 15077 Inner Ear +T474 PR:000001921 15095 15099 Jag1 +T475 PR:000001921 15107 15111 Jag1 +T476 UBERON:0000922 15116 15123 Embryos +T477 UBERON:0001846 15131 15141 inner ears +T478 UBERON:0001846 15221 15230 inner ear +T479 UBERON:0004043 15306 15313 ampulla +T480 UBERON:0001841 15315 15318 asc +T481 UBERON:0001841 15320 15347 anterior semicircular canal +T482 UBERON:0001855 15349 15351 cd +T483 UBERON:0001855 15353 15365 cochlea duct +T484 UBERON:0001860 15367 15369 ed +T485 UBERON:0001860 15371 15389 endolymphatic duct +T486 UBERON:0004043 15403 15410 ampulla +T487 UBERON:0001843 15412 15415 lsc +T488 UBERON:0001843 15417 15443 lateral semicircular canal +T489 UBERON:0004043 15459 15466 ampulla +T490 UBERON:0001842 15468 15471 psc +T491 UBERON:0001842 15473 15501 posterior semicircular canal +T492 UBERON:0001854 15503 15506 sac +T493 UBERON:0001854 15508 15515 saccule +T494 UBERON:0001853 15517 15519 ut +T495 UBERON:0001853 15521 15528 utricle +T496 PR:000001921 15535 15539 Jag1 +T497 UBERON:0001846 15553 15563 inner ears +T498 PR:000001921 15572 15576 Jag1 +T499 PR:000007623 15586 15591 Foxg1 +T500 PR:000001921 15599 15603 Jag1 +T501 SO:0000359 15603 15607 flox +T502 UBERON:0001842 15629 15658 posterior semicircular canals +T503 UBERON:0004043 15671 15679 ampullae +T504 UBERON:0004043 15735 15743 ampullae +T505 PR:000001921 15855 15859 Jag1 +T506 NCBITaxon:33208 15864 15871 animals +T507 UBERON:0004043 15886 15894 ampullae +T508 UBERON:0001840 15906 15924 semicircular canal +T509 GO:0060872 15906 15936 semicircular canal development +T510 UBERON:0001841 15977 15991 anterior canal +T511 UBERON:0001843 16032 16045 lateral canal +T512 UBERON:0001853 16059 16066 utricle +T513 UBERON:0001854 16071 16078 saccule +T514 UBERON:0001844 16099 16107 cochleae +T515 PR:000001921 16176 16180 Jag1 +T516 UBERON:0001846 16192 16202 Inner Ears +T517 PR:000001921 16214 16218 Jag1 +T518 SO:0000704 16219 16223 gene +T519 GO:0010467 16227 16236 expressed +T520 UBERON:0001690 16265 16268 ear +T521 PR:000001921 16323 16327 Jag1 +T522 UBERON:0001846 16339 16349 inner ears +T523 UBERON:0001690 16394 16397 ear +T524 UBERON:0000020 16413 16427 sensory organs +T525 UBERON:0001690 16468 16471 ear +T526 UBERON:0002227 16501 16515 organ of Corti +T527 UBERON:0000020 16521 16534 sensory organ +T528 UBERON:0001844 16542 16549 cochlea +T529 CHEBI:10545 16572 16580 electron +T530 CL:0000855 16629 16639 hair cells +T531 UBERON:0002227 16651 16665 organ of Corti +T532 GO:0007049 16682 16692 cell cycle +T533 CL:0000855 16766 16775 hair cell +T534 PR:000001921 16827 16831 Jag1 +T535 UBERON:0001844 16843 16851 cochleae +T536 UBERON:0001844 16912 16919 cochlea +T537 CL:0000855 16930 16939 hair cell +T538 GO:0048468 16935 16949 cell formation +T539 UBERON:0002227 17007 17021 organ of Corti +T540 CL:0000855 17023 17033 hair cells +T541 CL:0000589 17126 17131;17142 17152 inner ... hair cells +T542 CL:0000601 17136 17152 outer hair cells +T543 CL:0000855 17181 17191 hair cells +T544 UBERON:0002227 17227 17241 organ of Corti +T545 CL:0000855 17273 17283 hair cells +T546 CL:0000855 17410 17420 hair cells +T547 CL:0000855 17467 17477 hair cells +T548 CL:0000855 17509 17518 Hair Cell +T549 UBERON:0001844 17545 17552 Cochlea +T550 CHEBI:10545 17563 17571 electron +T551 CL:0000855 17624 17633 hair cell +T552 GO:0048468 17629 17644 cell production +T553 UBERON:0001844 17669 17676 cochlea +T554 PR:000001921 17680 17684 Jag1 +T555 UBERON:0000922 17689 17696 embryos +T556 UBERON:0002819 17728 17734;17745 17753 apical ... cochlear +T557 UBERON:0014626 17739 17753 basal cochlear +T558 CL:0000855 17878 17888 hair cells +T559 UBERON:0014626 17896 17903;17917 17924 base of ... cochlea +T560 PR:000001921 17908 17912 Jag1 +T561 CL:0000855 18057 18067 hair cells +T562 CL:0000589 18150 18155;18166 18176 inner ... hair cells +T563 CL:0000601 18160 18176 outer hair cells +T564 CL:0000855 18209 18219 hair cells +T565 CL:0000855 18306 18310;18326 18330 Hair ... Cell +T566 CL:0000630 18315 18330 Supporting Cell +T567 PR:000001921 18343 18347 Jag1 +T568 UBERON:0001846 18352 18362 Inner Ears +T569 PR:000001921 18430 18434 Jag1 +T570 UBERON:0001844 18446 18454 cochleae +T571 CL:0000855 18495 18504 hair cell +T572 CL:0000630 18509 18524 supporting cell +T573 UBERON:0001690 18549 18552 ear +T574 UBERON:0001844 18572 18579 cochlea +T575 GO:0042571 18592 18600 antibody +T576 PR:000010869 18609 18614 MYO7A +T577 CL:0000855 18628 18638 hair cells +T578 GO:0042571 18646 18654 antibody +T579 PR:000014402 18663 18669 S100A1 +T580 CL:0000589 18679 18695 inner hair cells +T581 CL:0000635 18697 18722 Deiter's supporting cells +T582 CL:0005015 18728 18761 inner phalangeal supporting cells +T583 UBERON:0003221 18734 18744 phalangeal +T584 CL:0000589 18812 18828 inner hair cells +T585 CL:0000601 18830 18846 outer hair cells +T586 CL:0000630 18857 18872 supporting cell +T587 UBERON:0002819 18951 18958;18963 18970 apex of ... cochlea +T588 CL:0000589 18972 18988 inner hair cells +T589 CL:0000630 19063 19079 supporting cells +T590 CL:0005015 19085 19107 inner phalangeal cells +T591 UBERON:0003221 19091 19101 phalangeal +T592 CL:0000601 19128 19144 Outer hair cells +T593 CL:0000630 19166 19182 supporting cells +T594 CL:0000635 19188 19202 Deiter's cells +T595 UBERON:0001844 19267 19274 cochlea +T596 CL:0000589 19281 19286;19310 19320 inner ... hair cells +T597 CL:0000601 19304 19320 outer hair cells +T598 UBERON:0005972 19412 19427 tunnel of Corti +T599 CL:0000589 19479 19495 inner hair cells +T600 CL:0000601 19521 19536 outer hair cell +T601 CL:0000635 19563 19588 Deiter's supporting cells +T602 CL:0000855 19612 19622 hair cells +T603 CL:0000630 19627 19643 supporting cells +T604 UBERON:0001844 19689 19696 cochlea +T605 CL:0000855 19721 19725;19741 19745 Hair ... Cell +T606 CL:0000630 19730 19745 Supporting Cell +T607 PR:000001921 19809 19813 Jag1 +T608 UBERON:0001846 19818 19827 Inner Ear +T609 PR:000010869 19868 19879 myosin VIIA +T610 CL:0000855 19890 19900 hair cells +T611 PR:000014402 19906 19911 S100a +T612 CL:0000589 19920 19936 inner hair cells +T613 CL:0000635 19938 19952 Dieter's cells +T614 CL:0005015 19958 19980 inner phalangeal cells +T615 UBERON:0003221 19964 19974 phalangeal +T616 CL:0000855 20006 20010;20026 20030 hair ... cell +T617 CL:0000630 20015 20030 supporting cell +T618 GO:0048468 20026 20041 cell production +T619 PR:000001921 20066 20070 Jag1 +T620 UBERON:0001846 20075 20085 inner ears +T621 UBERON:0001844 20138 20145 cochlea +T622 CL:0000855 20166 20175 hair cell +T623 PR:000001921 20230 20234 Jag1 +T624 UBERON:0001844 20239 20246 cochlea +T625 UBERON:0000483 20335 20345 epithelial +T626 CL:0000589 20353 20357 IHCs +T627 CL:0000589 20359 20375 inner hair cells +T628 UBERON:0000483 20410 20420 epithelial +T629 CL:0000601 20428 20432 OHCs +T630 CL:0000601 20434 20450 outer hair cells +T631 CL:0000630 20470 20473 SCs +T632 CL:0000630 20475 20491 supporting cells +T633 CL:0000855 20532 20536;20552 20556 hair ... cell +T634 CL:0000630 20541 20556 supporting cell +T635 GO:0048468 20552 20567 cell production +T636 UBERON:0004681 20575 20592 vestibular system +T637 UBERON:0002214 20598 20614 utricular macula +T638 CL:0000855 20648 20658 hair cells +T639 UBERON:0001854 20673 20680 saccule +T640 CL:0000855 20698 20702;20718 20722 hair ... cell +T641 CL:0000630 20707 20722 supporting cell +T642 GO:0048468 20718 20733 cell production +T643 UBERON:0000062 20760 20765 organ +T644 UBERON:0006585 20837 20862 vestibular sensory organs +T645 PR:000001921 20866 20870 Jag1 +T646 UBERON:0001846 20882 20892 inner ears +T647 UBERON:0001840 20937 20955 semicircular canal +T648 GO:0060872 20937 20955;20968 20977 semicircular canal ... formation +T649 UBERON:0004043 20960 20967 ampulla +T650 UBERON:0004721 21029 21035 crista +T651 PR:000001921 21051 21055 Jag1 +T652 UBERON:0002214 21060 21076 utricular macula +T653 CL:0000855 21127 21137 hair cells +T654 UBERON:0001854 21169 21176 saccule +T655 UBERON:0000054 21185 21191 macula +T656 PR:000001921 21225 21229 Jag1 +T657 UBERON:0001846 21234 21244 inner ears +T658 CL:0000855 21258 21267 Hair cell +T659 GO:0030154 21263 21283 cell differentiation +T660 UBERON:0001854 21336 21344 saccular +T661 UBERON:0000020 21511 21525 sensory organs +T662 UBERON:0001846 21537 21546 inner ear +T663 PR:000001921 21582 21586 Jag1 +T664 UBERON:0001846 21591 21601 inner ears +T665 UBERON:0000020 21617 21631 sensory organs +T666 UBERON:0004721 21645 21652 cristae +T667 PR:000001921 21697 21701 JAG1 +T668 CL:0000855 21741 21750 hair cell +T669 PR:000001921 21765 21769 Jag1 +T670 UBERON:0001844 21774 21782 cochleae +T671 CL:0000855 21805 21814 hair cell +T672 GO:0048468 21810 21824 cell formation +T673 CL:0000855 21871 21880 hair cell +T674 CL:0000855 21950 21959 hair cell +T675 GO:0032420 21960 21971 stereocilia +T676 CL:0000855 22009 22018 hair cell +T677 GO:0048468 22014 22028 cell formation +T678 UBERON:0001844 22112 22120 cochleae +T679 CL:0000855 22136 22145 hair cell +T680 GO:0030154 22141 22161 cell differentiation +T681 CL:0000589 22229 22234;22245 22255 inner ... hair cells +T682 CL:0000601 22239 22255 outer hair cells +T683 CL:0000589 22329 22345 inner hair cells +T684 CL:0000855 22445 22454 hair cell +T685 GO:0030154 22450 22470 cell differentiation +T686 PR:000001921 22524 22528 Jag1 +T687 UBERON:0001844 22533 22541 cochleae +T688 CL:0000855 22606 22616 hair cells +T689 CL:0000855 22686 22696 hair cells +T690 PR:000001921 22764 22768 Jag1 +T691 CL:0000855 22797 22806 hair cell +T692 GO:0048468 22802 22816 cell formation +T693 GO:0030154 22802 22806;22829 22844 cell ... differentiation +T694 PR:000001921 22885 22889 Jag1 +T695 UBERON:0001844 22894 22902 cochleae +T696 CL:0000855 23060 23070 hair cells +T697 UBERON:0001844 23094 23102 cochleae +T698 PR:000001921 23172 23176 Jag1 +T699 UBERON:0001844 23181 23188 Cochlea +T700 GO:0048468 23242 23254;23263 23268 Formation of ... Cells +T701 UBERON:0001844 23333 23340 cochlea +T702 UBERON:0001844 23395 23402 cochlea +T703 UBERON:0000483 23417 23427 epithelial +T704 UBERON:0001844 23482 23489 cochlea +T705 UBERON:0001844 23586 23593 cochlea +T706 CL:0000855 23629 23639 hair cells +T707 PR:000001921 23668 23672 Jag1 +T708 UBERON:0001844 23677 23684 cochlea +T709 CL:0000855 23820 23830 hair cells +T710 CL:0000855 23974 23984 hair cells +T711 PR:000001921 24177 24181 Jag1 +T712 UBERON:0001846 24186 24196 Inner Ears +T713 PR:000001921 24219 24223 JAG1 +T714 GO:0048880 24248 24267 sensory development +T715 PR:000003090 24329 24336 p27kip1 +T716 PR:000015426 24341 24345 SOX2 +T717 GO:0010467 24366 24376 expression +T718 PR:000001921 24408 24412 Jag1 +T719 UBERON:0001844 24424 24432 cochleae +T720 CL:0000855 24471 24481 hair cells +T721 CL:0000630 24486 24502 supporting cells +T722 UBERON:0002227 24510 24524 organ of Corti +T723 CL:0000855 24566 24576 hair cells +T724 UBERON:0001844 24637 24644 cochlea +T725 PR:000003090 24651 24658 p27kip1 +T726 GO:0007049 24662 24672 cell-cycle +T727 CHEBI:35222 24673 24682 inhibitor +T728 UBERON:0001844 24704 24712 cochlear +T729 GO:0007049 24745 24755 cell cycle +T730 UBERON:0001844 24826 24833 cochlea +T731 PR:000003090 24843 24850 p27kip1 +T732 GO:0010467 24864 24873 expressed +T733 UBERON:0001844 24906 24913 cochlea +T734 CL:0000855 24921 24931 hair cells +T735 CL:0000630 24936 24952 supporting cells +T736 GO:0007049 24962 24972 cell cycle +T737 PR:000015643 25060 25063 SRY +T738 PR:000015426 25093 25097 SOX2 +T739 UBERON:0001846 25161 25170 inner ear +T740 PR:000001921 25288 25292 JAG1 +T741 GO:0010467 25301 25311 expression +T742 PR:000001921 25334 25338 Jag1 +T743 UBERON:0001844 25343 25351 cochleae +T744 PR:000001921 25382 25386 JAG1 +T745 GO:0010467 25395 25404 expressed +T746 PR:000003090 25449 25456 p27kip1 +T747 GO:0010467 25457 25467 expression +T748 GO:0010467 25494 25503 expressed +T749 UBERON:0001844 25594 25601 cochlea +T750 UBERON:0000062 25614 25619 organ +T751 PR:000015426 25653 25657 SOX2 +T752 PR:000003090 25701 25708 p27kip1 +T753 PR:000015426 25772 25776 SOX2 +T754 GO:0010467 25777 25787 expression +T755 PR:000003090 25824 25831 p27kip1 +T756 UBERON:0000062 25866 25871 organ +T757 PR:000001921 25897 25901 JAG1 +T758 PR:000001921 25932 25936 JAG1 +T759 GO:0010467 25945 25954 expressed +T760 PR:000003090 25999 26006 p27kip1 +T761 PR:000015426 26011 26015 SOX2 +T762 GO:0010467 26016 26026 expression +T763 UBERON:0001844 26066 26073 cochlea +T764 UBERON:0001690 26154 26158 ears +T765 GO:0010467 26178 26188 expression +T766 PR:000001921 26319 26323 Jag1 +T767 UBERON:0001844 26328 26335 cochlea +T768 PR:000001921 26357 26361 JAG1 +T769 GO:0010467 26365 26374 Expressed +T770 PR:000001921 26439 26443 Jag1 +T771 UBERON:0001846 26448 26458 Inner Ears +T772 PR:000015426 26533 26537 Sox2 +T773 PR:000003090 26542 26549 p27Kip1 +T774 PR:000001921 26571 26575 JAG1 +T775 UBERON:0001844 26618 26625 cochlea +T776 PR:000001921 26641 26645 JAG1 +T777 PR:000003090 26680 26687 p27Kip1 +T778 PR:000015426 26726 26730 SOX2 +T779 PR:000003090 26764 26771 p27Kip1 +T780 PR:000015426 26797 26801 SOX2 +T781 PR:000003090 26806 26813 p27Kip1 +T782 PR:000001921 26840 26844 Jag1 +T783 UBERON:0001844 26849 26856 cochlea +T784 GO:0010467 26891 26901 expression +T785 UBERON:0000483 26948 26958 epithelial +T786 UBERON:0000483 26978 26988 epithelial +T787 PR:000001921 27022 27026 JAG1 +T788 GO:0010467 27035 27044 expressed +T789 UBERON:0001844 27077 27084 cochlea +T790 PR:000001921 27138 27142 JAG1 +T791 PR:000015426 27157 27161 SOX2 +T792 PR:000003090 27176 27183 p27Kip1 +T793 GO:0010467 27191 27200 expressed +T794 UBERON:0001846 27208 27217 inner ear +T795 PR:000001921 27286 27290 Jag1 +T796 UBERON:0001844 27295 27303 cochleae +T797 PR:000001921 27340 27344 JAG1 +T798 PR:000015426 27348 27352 SOX2 +T799 UBERON:0001844 27437 27444 cochlea +T800 PR:000001921 27493 27497 JAG1 +T801 GO:0010467 27498 27508 expression +T802 PR:000015426 27530 27534 SOX2 +T803 PR:000001921 27578 27582 JAG1 +T804 GO:0010467 27596 27605 expressed +T805 GO:0010467 27719 27729 expression +T806 PR:000001921 27733 27737 JAG1 +T807 PR:000015426 27742 27746 SOX2 +T808 PR:000001921 27790 27794 Jag1 +T809 UBERON:0001844 27799 27806 cochlea +T810 PR:000015426 27808 27812 SOX2 +T811 PR:000001921 27950 27954 JAG1 +T812 GO:0010467 27958 27967 expressed +T813 UBERON:0001844 28004 28011 cochlea +T814 PR:000001921 28057 28061 JAG1 +T815 GO:0048880 28072 28089 sensory formation +T816 GO:0007049 28112 28122 cell cycle +T817 GO:0030154 28132 28150;28164 28169 differentiation of ... cells +T818 GO:0030154 28132 28150;28196 28201 differentiation of ... cells +T819 CL:0000855 28151 28169 sensory hair cells +T820 CL:0000630 28185 28201 supporting cells +T821 PR:000001921 28223 28227 JAG1 +T822 GO:0010467 28231 28240 Expressed +T823 PR:000015426 28274 28278 SOX2 +T824 GO:0010467 28279 28289 Expression +T825 PR:000001921 28330 28334 Jag1 +T826 UBERON:0001844 28339 28347 Cochleae +T827 UBERON:0000922 28400 28406 embryo +T828 GO:0042571 28447 28457 antibodies +T829 PR:000001921 28473 28477 JAG1 +T830 PR:000015426 28481 28485 SOX2 +T831 PR:000001921 28528 28532 JAG1 +T832 PR:000015426 28537 28541 SOX2 +T833 UBERON:0014626 28549 28556;28561 28568 base of ... cochlea +T834 PR:000015426 28660 28664 SOX2 +T835 GO:0010467 28672 28681 expressed +T836 PR:000001921 28711 28715 Jag1 +T837 UBERON:0001844 28720 28727 cochlea +T838 GO:0010467 28752 28762 expression +T839 UBERON:0001981 28780 28782 bv +T840 UBERON:0001981 28784 28796 blood vessel +T841 UBERON:0001855 28798 28800 cd +T842 UBERON:0001855 28802 28815 cochlear duct +T843 PR:000001921 28835 28839 JAG1 +T844 PR:000015426 28844 28848 SOX2 +T845 GO:0010467 28849 28859 expression +T846 UBERON:0001846 28893 28902 inner ear +T847 PR:000001921 28925 28929 Jag1 +T848 UBERON:0000922 28941 28948 embryos +T849 PR:000001921 28950 28954 JAG1 +T850 PR:000015426 28959 28963 SOX2 +T851 GO:0010467 28994 29004 expression +T852 UBERON:0000020 29060 29074 sensory organs +T853 UBERON:0001690 29108 29111 ear +T854 GO:0010467 29133 29143 expression +T855 UBERON:0004721 29210 29217 cristae +T856 PR:000001921 29225 29229 JAG1 +T857 GO:0010467 29230 29240 expression +T858 GO:0010467 29283 29293 expression +T859 PR:000015426 29310 29314 SOX2 +T860 GO:0010467 29315 29325 expression +T861 PR:000001921 29392 29396 JAG1 +T862 UBERON:0004721 29533 29540 cristae +T863 PR:000015426 29571 29575 SOX2 +T864 GO:0010467 29585 29594 expressed +T865 PR:000001921 29609 29613 Jag1 +T866 UBERON:0000054 29629 29644 sensory patches +T867 PR:000015426 29646 29650 SOX2 +T868 GO:0010467 29651 29661 expression +T869 PR:000001921 29758 29762 Jag1 +T870 UBERON:0001854 29767 29774 saccule +T871 PR:000015426 29799 29803 SOX2 +T872 GO:0010467 29804 29814 expression +T873 UBERON:0002212 29882 29897 saccular macula +T874 PR:000015426 29912 29916 SOX2 +T875 GO:0010467 29917 29927 expression +T876 UBERON:0001853 29935 29942 utricle +T877 GO:0010467 29965 29975 expression +T878 UBERON:0002214 30095 30111 utricular macula +T879 PR:000001921 30115 30119 Jag1 +T880 UBERON:0001846 30124 30134 inner ears +T881 PR:000015426 30149 30153 SOX2 +T882 GO:0010467 30154 30164 expression +T883 PR:000001921 30172 30176 Jag1 +T884 UBERON:0004721 30181 30188 cristae +T885 UBERON:0004043 30213 30221 ampullae +T886 UBERON:0004721 30363 30370 cristae +T887 UBERON:0004043 30375 30383 ampullae +T888 PR:000001921 30422 30426 JAG1 +T889 PR:000015426 30431 30435 SOX2 +T890 UBERON:0001349 30471 30480 Vestibule +T891 PR:000015426 30486 30490 SOX2 +T892 GO:0010467 30491 30501 Expression +T893 GO:0048880 30527 30544 Sensory Formation +T894 PR:000001921 30552 30556 Jag1 +T895 UBERON:0001349 30561 30570 Vestibule +T896 PR:000001921 30640 30644 JAG1 +T897 PR:000015426 30648 30652 SOX2 +T898 GO:0010467 30653 30663 expression +T899 UBERON:0001846 30701 30711 inner ears +T900 PR:000001921 30753 30757 Jag1 +T901 UBERON:0001846 30762 30771 inner ear +T902 PR:000015426 30786 30790 SOX2 +T903 GO:0010467 30791 30801 expression +T904 UBERON:0004721 30843 30850 cristae +T905 UBERON:0004043 30855 30863 ampullae +T906 PR:000001921 30883 30887 Jag1 +T907 UBERON:0001846 30892 30901 inner ear +T908 UBERON:0004721 30916 30923 cristae +T909 UBERON:0004721 30937 30944 cristae +T910 UBERON:0004721 30960 30967 cristae +T911 UBERON:0002212 30969 30972 sac +T912 UBERON:0002212 30974 30989 saccular macula +T913 UBERON:0002214 30991 30993 ut +T914 UBERON:0002214 30995 31011 utricular macula +T915 GO:0007219 31052 31067 Notch signaling +T916 PR:000001921 31085 31089 JAG1 +T917 UBERON:0001690 31179 31182 ear +T918 GO:0010467 31197 31207 expression +T919 PR:000001921 31211 31215 JAG1 +T920 PR:000015426 31257 31261 SOX2 +T921 PR:000003090 31266 31273 p27kip1 +T922 PR:000001921 31294 31298 JAG1 +T923 UBERON:0001690 31335 31338 ear +T924 UBERON:0002227 31405 31419 organ of Corti +T925 GO:0007049 31468 31478 cell cycle +T926 CL:0000855 31510 31520 hair cells +T927 CL:0000630 31525 31541 supporting cells +T928 PR:000015426 31548 31552 SOX2 +T929 PR:000003090 31557 31564 p27kip1 +T930 PR:000001921 31626 31630 Jag1 +T931 UBERON:0001846 31635 31644 inner ear +T932 PR:000001921 31665 31669 JAG1 +T933 UBERON:0001846 31739 31748 inner ear +T934 CL:0000855 31775 31784 Hair Cell +T935 GO:0048468 31780 31794 Cell Formation +T936 PR:000001921 31798 31802 Jag1 +T937 UBERON:0001846 31807 31817 Inner Ears +T938 PR:000001921 31976 31980 Jag1 +T939 PR:000001921 32011 32015 Jag1 +T940 UBERON:0004681 32020 32037 vestibular system +T941 UBERON:0004721 32043 32050 cristae +T942 CL:0000855 32103 32113 hair cells +T943 UBERON:0002214 32136 32153 utricular maculae +T944 UBERON:0002212 32172 32188 saccular maculae +T945 CL:0000855 32221 32230 hair cell +T946 GO:0048468 32226 32240 cell formation +T947 UBERON:0000062 32276 32281 organ +T948 PR:000001921 32303 32307 Jag1 +T949 UBERON:0001844 32312 32319 cochlea +T950 CL:0000855 32321 32330 hair cell +T951 GO:0030154 32326 32346 cell differentiation +T952 UBERON:0001844 32446 32453 cochlea +T953 CL:0000589 32459 32475 inner hair cells +T954 UBERON:0001844 32606 32613 cochlea +T955 CL:0000855 32626 32636 hair cells +T956 CL:0000601 32721 32737 outer hair cells +T957 PR:000014402 32803 32809 S100A1 +T958 CL:0000635 32818 32832 Dieter's cells +T959 UBERON:0001844 32884 32891 cochlea +T960 CL:0000855 32901 32911 hair cells +T961 CL:0000630 32916 32932 supporting cells +T962 CL:0000855 32963 32973 hair cells +T963 UBERON:0001844 33008 33015 cochlea +T964 UBERON:0001844 33100 33107 cochlea +T965 NCBITaxon:10088 33196 33201 mouse +T966 SO:0000704 33213 33218 genes +T967 UBERON:0001690 33291 33294 ear +T968 SO:0001023 33328 33334 allele +T969 SO:0001023 33363 33369 allele +T970 PR:000001448 33377 33382 Fgfr1 +T971 SO:0000704 33383 33387 gene +T972 CL:0000855 33409 33419 hair cells +T973 UBERON:0001844 33439 33446 cochlea +T974 PR:000001921 33468 33472 Jag1 +T975 PR:000001448 33509 33514 Fgfr1 +T976 CL:0000589 33552 33568 inner hair cells +T977 CL:0000601 33626 33642 outer hair cells +T978 PR:000001921 33655 33659 Jag1 +T979 PR:000001448 33675 33680 Fgfr1 +T980 UBERON:0001844 33715 33722 cochlea +T981 NCBITaxon:10088 33732 33737 mouse +T982 SO:0001023 33760 33766 allele +T983 PR:000015426 33774 33778 Sox2 +T984 SO:0000704 33779 33783 gene +T985 PR:000015426 33803 33807 Sox2 +T986 CL:0000855 33839 33849 hair cells +T987 UBERON:0001844 33879 33886 cochlea +T988 UBERON:0001844 33939 33946 cochlea +T989 PR:000001921 33973 33977 Jag1 +T990 PR:000015426 33993 33997 SOX2 +T991 UBERON:0001844 34024 34032 cochlear +T992 CL:0000589 34097 34113 inner hair cells +T993 UBERON:0001844 34144 34152 cochleae +T994 CL:0000589 34181 34197 inner hair cells +T995 CL:0000589 34339 34344;34363 34373 inner ... hair cells +T996 CL:0000601 34357 34373 outer hair cells +T997 GO:0007049 34489 34499 cell cycle +T998 UBERON:0001844 34667 34674 cochlea +T999 CL:0000589 34698 34714 inner hair cells +T1000 PR:000001921 34727 34731 Jag1 +T1001 CL:0000589 34891 34906 inner hair cell +T1002 PR:000001921 34998 35002 Jag1 +T1003 UBERON:0001844 35007 35014 cochlea +T1004 NCBITaxon:10088 35034 35039 mouse +T1005 GO:0060026 35089 35109 convergent extension +T1006 UBERON:0000479 35180 35186 tissue +T1007 GO:0008283 35222 35235 proliferation +T1008 CL:0000855 35268 35278 hair cells +T1009 UBERON:0001844 35364 35371 cochlea +T1010 PR:000001921 35482 35486 JAG1 +T1011 PR:000003090 35596 35603 p27kip1 +T1012 GO:0008283 35639 35655;35670 35675 proliferation of ... cells +T1013 UBERON:0001844 35683 35690 cochlea +T1014 GO:0051301 35711 35724 cell division +T1015 CL:0000589 35805 35821 inner hair cells +T1016 PR:000001921 35933 35937 Jag1 +T1017 UBERON:0001846 35942 35952 inner ears +T1018 GO:0007219 35962 35977 Notch signaling +T1019 CL:0000057 35979 35989 fibroblast +T1020 GO:0008543 35979 36013 fibroblast growth factor signaling +T1021 PR:000015426 36044 36048 SOX2 +T1022 UBERON:0001846 36152 36161 inner ear +T1023 UBERON:0001690 36199 36202 Ear +T1024 PR:000003090 36258 36265 p27Kip1 +T1025 PR:000015426 36270 36274 SOX2 +T1026 PR:000001921 36335 36339 Jag1 +T1027 UBERON:0001846 36344 36354 inner ears +T1028 PR:000001921 36453 36457 JAG1 +T1029 UBERON:0001690 36492 36495 ear +T1030 GO:0043583 36492 36507 ear development +T1031 PR:000006523 36591 36595 DLL1 +T1032 PR:000009194 36600 36604 JAG2 +T1033 GO:0007219 36724 36739 Notch signaling +T1034 UBERON:0001846 36778 36787 inner ear +T1035 UBERON:0000467 36810 36817 systems +T1036 UBERON:0001016 36833 36847 nervous system +T1037 UBERON:0001277 36873 36894 intestinal epithelium +T1038 GO:0007219 36926 36941 Notch signaling +T1039 NCBITaxon:40674 37020 37029 mammalian +T1040 UBERON:0001016 37030 37044 nervous system +T1041 GO:0007219 37076 37091 Notch signaling +T1042 GO:0007219 37299 37314 Notch signaling +T1043 CL:0000034 37353 37362 stem cell +T1044 GO:0007219 37413 37428 Notch signaling +T1045 CL:0000681 37438 37450 radial glial +T1046 UBERON:0001017 37528 37550 central nervous system +T1047 UBERON:0001016 37601 37615 nervous system +T1048 GO:0007219 37617 37632 Notch signaling +T1049 PR:000001921 37637 37641 JAG1 +T1050 UBERON:0001846 37709 37718 inner ear +T1051 UBERON:0001016 37740 37754 nervous system +T1052 PR:000001921 37831 37835 JAG1 +T1053 PR:000001921 37976 37980 JAG1 +T1054 GO:0010467 38047 38057 expressing +T1055 PR:000011331 38097 38103 Notch1 +T1056 UBERON:0000054 38155 38170 sensory patches +T1057 GO:0007219 38211 38226 Notch signaling +T1058 UBERON:0001690 38407 38410 ear +T1059 GO:0010467 38518 38528 expressing +T1060 PR:000002198 38550 38559 β-catenin +T1061 GO:0016055 38601 38622 Wnt signaling pathway +T1062 NCBITaxon:9031 38631 38638 chicken +T1063 UBERON:0001846 38639 38648 inner ear +T1064 PR:000011331 38665 38671 Notch1 +T1065 UBERON:0001690 38766 38769 ear +T1066 GO:0010467 38827 38837 expression +T1067 PR:000002198 38841 38850 β-catenin +T1068 UBERON:0001844 38907 38915 cochlear +T1069 GO:0016055 38948 38961 Wnt signaling +T1070 GO:0065007 38962 38969 governs +T1071 NCBITaxon:7215 39069 39079 Drosophila +T1072 PR:P09615 39112 39120 Wingless +T1073 CHEBI:62488 39152 39171 signaling molecules +T1074 NCBITaxon:7742 39260 39271 vertebrates +T1075 PR:000000034 39292 39318 Bone morphogenetic protein +T1076 GO:0030509 39292 39318;39325 39334 Bone morphogenetic protein ... signaling +T1077 PR:000000034 39320 39323 BMP +T1078 GO:0030509 39320 39323;39325 39334 BMP ... signaling +T1079 GO:0048880 39361 39378 sensory formation +T1080 UBERON:0004721 39409 39416 cristae +T1081 PR:000000165 39421 39425 BMP4 +T1082 NCBITaxon:10088 39453 39458 mouse +T1083 UBERON:0004721 39459 39466 cristae +T1084 NCBITaxon:9031 39523 39530 chicken +T1085 PR:000000034 39556 39559 BMP +T1086 GO:0030509 39556 39569 BMP signaling +T1087 GO:0048880 39605 39624 sensory development +T1088 GO:0010467 39682 39692 expression +T1089 PR:000001921 39754 39758 JAG1 +T1090 UBERON:0001690 39841 39844 ear +T1091 PR:000000034 39950 39953 BMP +T1092 UBERON:0000020 39973 39987 sensory organs +T1093 GO:0048880 40037 40054 Sensory Formation +T1094 PR:000001921 40071 40075 Jag1 +T1095 UBERON:0001846 40080 40090 Inner Ears +T1096 PR:000001921 40135 40139 JAG1 +T1097 GO:0048880 40202 40219 sensory formation +T1098 PR:000001921 40229 40233 Jag1 +T1099 UBERON:0001846 40238 40248 inner ears +T1100 PR:000001921 40327 40331 JAG1 +T1101 GO:0010467 40428 40438 expression +T1102 PR:000001921 40450 40454 JAG1 +T1103 UBERON:0001690 40462 40465 ear +T1104 PR:000006523 40489 40493 Dll1 +T1105 PR:000009194 40498 40502 Jag2 +T1106 SO:0000704 40503 40508 genes +T1107 GO:0010467 40513 40522 expressed +T1108 CL:0000855 40534 40544 hair cells +T1109 GO:0007049 40565 40575 cell cycle +T1110 CL:0000855 40627 40636 hair cell +T1111 GO:0010467 40637 40647 expression +T1112 GO:0010467 40669 40682;40692 40696 expression of ... gene +T1113 PR:000006523 40687 40691 Dll1 +T1114 SO:0000704 40692 40696 gene +T1115 UBERON:0003051 40733 40740 otocyst +T1116 PR:000001921 40811 40815 JAG1 +T1117 GO:0010467 40849 40859 expression +T1118 GO:0014019 40916 40928;40933 40944 formation of ... neuroblasts +T1119 CL:0000031 40933 40944 neuroblasts +T1120 GO:0060232 40950 40960 delaminate +T1121 UBERON:0003249 40970 40985 otic epithelium +T1122 CL:0000540 41019 41026 neurons +T1123 GO:0060384 41037 41046 innervate +T1124 CL:0000855 41051 41061 hair cells +T1125 NCBITaxon:7955 41088 41097 zebrafish +T1126 CL:0000031 41111 41121 neuroblast +T1127 GO:0014019 41111 41131 neuroblast formation +T1128 GO:0007219 41141 41155;41164 41173 Notch-mediated ... signaling +T1129 NCBITaxon:40674 41191 41198 mammals +T1130 PR:000006523 41265 41269 Dll1 +T1131 SO:0000704 41270 41274 gene +T1132 PR:000006523 41357 41361 DLL1 +T1133 GO:0010467 41362 41372 expression +T1134 PR:000001921 41452 41456 JAG1 +T1135 GO:0010467 41457 41467 expression +T1136 PR:000001921 41499 41503 Jag1 +T1137 UBERON:0001846 41508 41518 Inner Ears +T1138 GO:0048880 41550 41567 sensory formation +T1139 PR:000001921 41575 41579 Jag1 +T1140 UBERON:0001846 41584 41594 inner ears +T1141 UBERON:0001846 41607 41617 inner ears +T1142 UBERON:0001840 41671 41690 semicircular canals +T1143 UBERON:0001841 41750 41758;41771 41777 anterior ... canals +T1144 UBERON:0001843 41763 41777 lateral canals +T1145 UBERON:0004043 41802 41810 ampullae +T1146 UBERON:0001853 41828 41835 utricle +T1147 UBERON:0001844 41855 41862 cochlea +T1148 GO:0010467 42016 42025 expressed +T1149 UBERON:0004721 42041 42048 cristae +T1150 UBERON:0001840 42057 42075 semicircular canal +T1151 GO:0060872 42057 42085 semicircular canal formation +T1152 PR:000000164 42111 42115 BMP2 +T1153 UBERON:0004721 42140 42147 cristae +T1154 UBERON:0000025 42193 42198 canal +T1155 GO:0035295 42193 42208 canal formation +T1156 NCBITaxon:10088 42233 42238 mouse +T1157 SO:0000704 42269 42274 genes +T1158 GO:0048880 42287 42304 sensory formation +T1159 UBERON:0001846 42334 42344 inner ears +T1160 PR:000015426 42376 42380 Sox2 +T1161 SO:0000704 42381 42385 gene +T1162 PR:000001921 42448 42452 Jag1 +T1163 UBERON:0001846 42470 42480 inner ears +T1164 UBERON:0000922 42484 42491 embryos +T1165 SO:0001023 42528 42535 alleles +T1166 PR:000015426 42539 42543 Sox2 +T1167 PR:000015426 42545 42549 Sox2 +T1168 PR:000015426 42561 42565 Sox2 +T1169 UBERON:0000025 42591 42596 canal +T1170 GO:0035295 42591 42606 canal formation +T1171 UBERON:0001853 42616 42625 utricular +T1172 UBERON:0001854 42630 42638 saccular +T1173 UBERON:0001844 42678 42686 cochleae +T1174 PR:000007480 42706 42711 FGF10 +T1175 NCBITaxon:10088 42712 42717 mouse +T1176 UBERON:0004721 42750 42757 cristae +T1177 UBERON:0000025 42794 42799 canal +T1178 UBERON:0000025 42837 42842 canal +T1179 UBERON:0001844 42855 42863 cochlear +T1180 GO:0090102 42855 42873 cochlear formation +T1181 GO:0048513 42918 42932;42937 42942 development of ... organ +T1182 UBERON:0002227 42937 42951 organ of Corti +T1183 UBERON:0001844 42958 42965 cochlea +T1184 GO:0048880 43022 43039 sensory formation +T1185 UBERON:0001844 43062 43070 cochlear +T1186 GO:0048880 43105 43122 sensory formation +T1187 GO:0060026 43151 43171 convergent extension +T1188 SO:0000704 43195 43200 genes +T1189 UBERON:0001844 43224 43231 cochlea +T1190 UBERON:0001844 43300 43307 cochlea +T1191 GO:0060026 43342 43362 convergent extension +T1192 UBERON:0001844 43414 43421 cochlea +T1193 PR:000001921 43434 43438 Jag1 +T1194 GO:0060026 43496 43516 convergent extension +T1195 PR:000001921 43622 43626 Jag1 +T1196 SO:0000704 43627 43631 gene +T1197 UBERON:0001846 43685 43694 inner ear +T1198 PR:000001921 43758 43762 JAG1 +T1199 GO:0007219 43772 43787 Notch signaling +T1200 GO:0008543 43873 43886 FGF signaling +T1201 PR:000015426 43891 43895 SOX2 +T1202 GO:0010467 43896 43906 expression +T1203 GO:0031099 43987 43999 regeneration +T1204 GO:0007605 44050 44057 hearing +T1205 http://purl.obolibrary.org/obo/MONDO_0005365 44050 44062;44078 44087 hearing loss disorders +T1206 http://purl.obolibrary.org/obo/MONDO_0002643 44067 44087 vestibular disorders +T1207 PR:000001921 44138 44142 Jag1 +T1208 SO:0001023 44150 44156 allele +T1209 PR:000001921 44176 44180 Jag1 +T1210 SO:0001023 44188 44194 allele +T1211 NCBITaxon:2 44196 44205 bacterial +T1212 SO:0000153 44196 44227 bacterial artificial chromosome +T1213 PR:000001921 44250 44254 Jag1 +T1214 SO:0001026 44255 44262 genomic +T1215 NCBITaxon:10088 44314 44319 mouse +T1216 NCBITaxon:2 44320 44329 bacterial +T1217 SO:0000153 44320 44351 bacterial artificial chromosome +T1218 GO:0097617 44405 44418 hybridization +T1219 NCBITaxon:10088 44431 44436 mouse +T1220 PR:000001921 44437 44441 Jag1 +T1221 SO:0001644 44500 44516 targeting vector +T1222 SO:0000147 44553 44557 exon +T1223 SO:0000440 44638 44644 vector +T1224 SO:0000346 44662 44666 loxP +T1225 SO:0000350 44667 44670 FRT +T1226 SO:0000350 44678 44681 FRT +T1227 SO:0005853 44682 44690 cassette +T1228 SO:0000147 44730 44734 exon +T1229 SO:0000346 44765 44769 loxP +T1230 SO:0000350 44770 44773 FRT +T1231 SO:0000350 44781 44784 FRT +T1232 SO:0005853 44785 44793 cassette +T1233 SO:0000147 44878 44882 exon +T1234 SO:0000440 44959 44965 vector +T1235 SO:0000346 44990 44999 loxP site +T1236 SO:0000440 45102 45108 vector +T1237 http://purl.obolibrary.org/obo/MONDO_0005504 45122 45132 diphtheria +T1238 CHEBI:27026 45133 45138 toxin +T1239 SO:0000704 45139 45143 gene +T1240 SO:0000346 45205 45209 loxP +T1241 SO:0000350 45210 45213 FRT +T1242 SO:0000350 45221 45224 FRT +T1243 SO:0000440 45291 45297 vector +T1244 PR:000001921 45354 45358 Jag1 +T1245 SO:0001644 45366 45382 targeting vector +T1246 PR:000001921 45414 45418 Jag1 +T1247 SO:0000359 45418 45422 flox +T1248 NCBITaxon:10088 45423 45427 mice +T1249 PR:000001921 45434 45438 Jag1 +T1250 UBERON:0000922 45519 45528 embryonic +T1251 CL:0002322 45519 45533;45539 45544 embryonic stem ... cells +T1252 CL:0002322 45535 45537;45539 45544 ES ... cells +T1253 CL:0002322 45589 45596 ES cell +T1254 SO:0000112 45651 45657 primer +T1255 SO:0000346 45922 45931 loxP site +T1256 SO:0000346 46068 46077 loxP site +T1257 SO:0000112 46113 46120 primers +T1258 SO:0000357 46126 46133 flanked +T1259 SO:0000346 46138 46147 loxP site +T1260 UBERON:0000358 46252 46263 blastocysts +T1261 NCBITaxon:10088 46278 46282 mice +T1262 NCBITaxon:10088 46312 46316 mice +T1263 GO:0007618 46322 46327 mated +T1264 PR:000001921 46402 46406 Jag1 +T1265 SO:0001023 46414 46420 allele +T1266 PR:000001921 46434 46438 Jag1 +T1267 SO:0000112 46455 46462 primers +T1268 PR:000001921 46464 46468 Jag1 +T1269 NCBITaxon:10088 46478 46482 mice +T1270 PR:000001921 46522 46526 Jag1 +T1271 PR:000001921 46534 46538 Jag1 +T1272 PR:000001921 46568 46572 Jag1 +T1273 PR:000001921 46580 46584 Jag1 +T1274 NCBITaxon:10088 46592 46596 mice +T1275 CHEBI:7507 46646 46654 neomycin +T1276 SO:0005853 46666 46674 cassette +T1277 PR:000001921 46709 46713 Jag1 +T1278 GO:0010467 46714 46724 expression +T1279 SO:0005853 46794 46802 cassette +T1280 SO:0000361 46808 46819 FRT-flanked +T1281 SO:0005853 46827 46835 cassette +T1282 GO:0007618 46851 46857 mating +T1283 PR:000001921 46858 46862 Jag1 +T1284 NCBITaxon:10088 46870 46874 mice +T1285 GO:0010467 46895 46905 expressing +T1286 NCBITaxon:10088 46906 46910 mice +T1287 SO:0000359 47009 47013 flox +T1288 NCBITaxon:10088 47014 47018 mice +T1289 PR:000001921 47025 47029 Jag1 +T1290 PR:000001921 47041 47045 Jag1 +T1291 SO:0000359 47045 47049 flox +T1292 NCBITaxon:10088 47050 47054 mice +T1293 SO:0001023 47190 47196 allele +T1294 PR:000001921 47226 47230 Jag1 +T1295 SO:0001023 47243 47249 allele +T1296 PR:000001921 47251 47255 Jag1 +T1297 PR:000001921 47284 47288 Jag1 +T1298 SO:0001023 47289 47295 allele +T1299 PR:000001921 47350 47354 Jag1 +T1300 SO:0000359 47354 47358 flox +T1301 PR:000001921 47362 47366 Jag1 +T1302 SO:0001023 47374 47381 alleles +T1303 PR:000001921 47386 47390 Jag1 +T1304 SO:0001023 47395 47401 allele +T1305 NCBITaxon:10088 47404 47409 Mouse +T1306 PR:000007623 47437 47442 Foxg1 +T1307 NCBITaxon:10088 47447 47451 mice +T1308 PR:000018212 47536 47539 ZP3 +T1309 NCBITaxon:10088 47544 47548 mice +T1310 PR:000007623 47686 47691 Foxg1 +T1311 SO:0001023 47696 47702 allele +T1312 PR:000001921 47734 47738 Jag1 +T1313 SO:0001023 47744 47750 allele +T1314 PR:000001921 47752 47756 Jag1 +T1315 PR:000001921 47816 47820 Jag1 +T1316 SO:0000359 47820 47824 flox +T1317 PR:000001921 47825 47829 Jag1 +T1318 SO:0000359 47829 47833 flox +T1319 NCBITaxon:10088 47887 47891 Mice +T1320 PR:000007623 47909 47914 Foxg1 +T1321 PR:000001921 47922 47926 Jag1 +T1322 PR:000001921 47931 47935 Jag1 +T1323 SO:0000359 47935 47939 flox +T1324 PR:000007623 47944 47949 Foxg1 +T1325 PR:000001921 47957 47961 Jag1 +T1326 PR:000001921 47966 47970 Jag1 +T1327 PR:000001921 48027 48031 Jag1 +T1328 NCBITaxon:10088 48036 48040 mice +T1329 PR:000001921 48074 48078 Jag1 +T1330 SO:0000359 48078 48082 flox +T1331 NCBITaxon:10088 48083 48087 mice +T1332 SO:0000112 48093 48100 primers +T1333 SO:0001030 48147 48154 forward +T1334 SO:0001031 48198 48205 reverse +T1335 SO:0000112 48214 48221 primers +T1336 SO:0000357 48222 48227 flank +T1337 SO:0000346 48235 48244 LoxP site +T1338 SO:0000112 48292 48298 primer +T1339 SO:0000346 48318 48327 LoxP site +T1340 SO:0000132 48398 48412 reverse primer +T1341 PR:000007623 48434 48439 Foxg1 +T1342 PR:000018212 48448 48451 ZP3 +T1343 SO:0001023 48456 48463 alleles +T1344 SO:0000112 48478 48485 primers +T1345 SO:0001030 48531 48538 forward +T1346 SO:0001031 48578 48585 reverse +T1347 SO:0000112 48597 48604 primers +T1348 SO:0001030 48674 48681 forward +T1349 SO:0001031 48722 48729 reverse +T1350 SO:0000132 48744 48758 reverse primer +T1351 SO:0001023 48829 48835 allele +T1352 PR:000001921 48861 48865 Jag1 +T1353 PR:000001921 48875 48879 Jag1 +T1354 SO:0000359 48879 48883 flox +T1355 CHEBI:10545 48957 48965 electron +T1356 PR:000001921 49003 49007 Jag1 +T1357 UBERON:0001846 49012 49022 inner ears +T1358 UBERON:0001846 49105 49115 Inner ears +T1359 CHEBI:9549 49202 49220 thiocarbohydrazide +T1360 CHEBI:10545 49288 49296 electron +T1361 UBERON:0008816 49403 49418 embryonic heads +T1362 UBERON:0000033 49489 49494 heads +T1363 CHEBI:73702 49521 49524 wax +T1364 GO:0042571 49595 49605 Antibodies +T1365 PR:000010869 49625 49630 Myo7a +T1366 PR:000003090 49683 49690 p27kip1 +T1367 PR:000015426 49766 49770 SOX2 +T1368 PR:000001921 49842 49846 JAG1 +T1369 PR:000014402 49936 49942 S100A1 +T1370 GO:0042571 49984 49994 antibodies +T1371 CHEBI:59132 50004 50011 antigen +T1372 CHEBI:30769 50085 50096 citric acid +T1373 GO:0042571 50108 50118 antibodies +T1374 CHEBI:52661 50136 50151 Alexa-Fluor 488 +T1375 CHEBI:51760 50136 50147;50155 50158 Alexa-Fluor ... 546 +T1376 NCBITaxon:9925 50159 50163 goat +T1377 NCBITaxon:10088 50169 50174 mouse +T1378 NCBITaxon:9986 50178 50184 rabbit +T1379 CHEBI:51231 50312 50316 DAPI +T1380 NCBITaxon:3850 50419 50440 Griffonia simplifonia +T1381 GO:0097617 50512 50525 hybridization +T1382 PR:000001921 50549 50553 Jag1 +T1383 SO:0001023 50565 50571 allele +T1384 SO:0001817 50583 50591 in-frame +T1385 SO:0000417 50612 50618 domain +T1386 SO:0000359 50671 50677 floxed +T1387 PR:000001921 50692 50696 Jag1 +T1388 SO:0000359 50696 50700 flox +T1389 SO:0001023 50701 50707 allele +T1390 GO:0097617 50719 50732 hybridization +T1391 SO:0000028 50776 50778 bp +T1392 SO:0000147 50800 50804 exon +T1393 SO:0000112 50808 50815 primers +T1394 SO:0000440 50937 50943 vector +T1395 UBERON:0000922 50974 50981 embryos +T1396 UBERON:0000922 50991 50998 embryos +T1397 GO:0097617 51052 51065 hybridization +T1398 UBERON:0001844 51115 51123 cochlear +T1399 UBERON:0001846 51133 51143 inner ears +T1400 UBERON:0000033 51168 51172 head +T1401 UBERON:0006612 51248 51253 shell +T1402 UBERON:0002282 51258 51263 stria +T1403 UBERON:0001844 51286 51294 cochleae +T1404 CHEBI:17790 51330 51338 methanol +T1405 GO:0097617 51348 51361 hybridization +T1406 GO:0097617 51425 51438 hybridization +T1407 PR:000001921 51545 51549 Jag1 +T1408 PR:000001921 51554 51558 Jag1 +T1409 UBERON:0000922 51563 51570 embryos +T1410 NCBITaxon:11866 51738 51741 AMV +T1411 SO:0000112 51823 51829 primer +T1412 SO:0000112 51909 51916 primers +T1413 SO:0000357 51922 51929 flanked +T1414 SO:0000147 51930 51934 exon +T1415 CHEBI:33893 52090 52098 reagents +T1416 UBERON:0000358 52293 52303 blastocyst +T1417 PR:000000034 52498 52501 BMP +T1418 GO:0060349 52504 52522 bone morphogenetic +T1419 PR:000000034 52504 52530 bone morphogenetic protein +T1420 PR:P10041 52566 52571 Delta +T1421 PR:P10041 52584 52589 Delta +T1422 PR:P18168 52590 52597 Serrate +T1423 UBERON:0000922 52616 52625 embryonic +T1424 UBERON:0000922 52645 52654 embryonic +T1425 CL:0000057 52667 52677 fibroblast +T1426 CHEBI:10545 52722 52730 electron +T1427 PR:000001921 53188 53192 JAG1 +T1428 NCBITaxon:40674 53247 53256 mammalian +T1429 UBERON:0001846 53257 53266 inner ear diff --git a/src/ontogpt/evaluation/craft/database/all/16410827.txt b/src/ontogpt/evaluation/craft/database/all/16410827.txt new file mode 100644 index 000000000..28213f561 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16410827.txt @@ -0,0 +1,227 @@ +The Notch Ligand JAG1 Is Required for Sensory Progenitor Development in the Mammalian Inner Ear + +Abstract + +In mammals, six separate sensory regions in the inner ear are essential for hearing and balance function. Each sensory region is made up of hair cells, which are the sensory cells, and their associated supporting cells, both arising from a common progenitor. Little is known about the molecular mechanisms that govern the development of these sensory organs. Notch signaling plays a pivotal role in the differentiation of hair cells and supporting cells by mediating lateral inhibition via the ligands Delta-like 1 and Jagged (JAG) 2. However, another Notch ligand, JAG1, is expressed early in the sensory patches prior to cell differentiation, indicating that there may be an earlier role for Notch signaling in sensory development in the ear. Here, using conditional gene targeting, we show that the Jag1 gene is required for the normal development of all six sensory organs within the inner ear. Cristae are completely lacking in Jag1-conditional knockout (cko) mutant inner ears, whereas the cochlea and utricle show partial sensory development. The saccular macula is present but malformed. Using SOX2 and p27kip1 as molecular markers of the prosensory domain, we show that JAG1 is initially expressed in all the prosensory regions of the ear, but becomes down-regulated in the nascent organ of Corti by embryonic day 14.5, when the cells exit the cell cycle and differentiate. We also show that both SOX2 and p27kip1 are down-regulated in Jag1-cko inner ears. Taken together, these data demonstrate that JAG1 is expressed early in the prosensory domains of both the cochlear and vestibular regions, and is required to maintain the normal expression levels of both SOX2 and p27kip1. These data demonstrate that JAG1-mediated Notch signaling is essential during early development for establishing the prosensory regions of the inner ear. + +Synopsis + +Deafness and adult-onset hearing loss are significant health problems. In most cases, deafness or vestibular dysfunction results when the sensory cells in the inner ear, known as hair cells, degenerate due to environmental or genetic causes. In the mammalian inner ear, the hair cells and their associated supporting cells can be found in six different patches that have particular functions related to hearing or balance. Unfortunately, unlike in birds or fish, mammalian hair cells show little ability to regenerate, resulting in a permanent hearing or balance disorder when damaged. Here, the authors show that a protein called JAG1, a ligand in the Notch signaling pathway, is required for the normal development of all six sensory regions in the mammalian inner ear. In ears that lacked JAG1, some of the sensory patches were missing completely, whereas others were small and lacked particular cell types. The authors showed that JAG1 is required by the sensory precursors, progenitor cells that give rise to both the hair cells and the supporting cells. By understanding how the sensory areas develop normally, it is hoped that molecular tools can be developed that will aid sensory regeneration in the mammalian inner ear. + +Introduction + +The mammalian inner ear is a complex structure consisting of a coiled cochlea, three orthogonally positioned semicircular canals, a central vestibule, and a dorsally projecting endolymphatic duct and sac. With the exception of the endolymphatic duct and sac, the different parts of the ear all contain sensory organs populated by sensory hair cells and their associated supporting cells. There are three different categories of sensory organs: cristae, located at the base of each semicircular canal; maculae, housed within the central vestibule; and the organ of Corti, which lines the cochlear duct. Only one sensory organ, the organ of Corti, is required for hearing; the other five organs are important for balance. Unfortunately, in mammals, if these regions are damaged due to an environmental or genetic insult, they cannot regenerate, leaving a permanent hearing and/or balance impairment. + +Although some progress has been made in understanding how the individual cell types within the sensory areas of the ear are formed [1,2], little is known about the molecular mechanisms that establish the prosensory lineage and how the different sensory organ types are formed. Interestingly, the molecular mechanisms that underlie sensory differentiation in the vertebrate inner ear demonstrate strong parallels with Drosophila sense organ development [3–5]. For example, during Drosophila external sense organ development, lateral inhibition mediated by Notch signaling is required to restrict the adoption of the sensory organ precursor cell fate, which then gives rise to the entire sensory organ [6–8]. Similarly, in the vertebrate ear, lateral inhibition mediated by Notch signaling appears to be important for restricting the number of cells that can adopt the hair cell fate [9–15]. Lineage analysis has also shown that, at least in the chicken, hair cells and supporting cells arise from a common progenitor [16], consistent with an equipotent epithelium that undergoes lateral signaling to specify cell fates. Unlike in Drosophila, which has a single Notch receptor and two ligands (Delta and Serrate/Jagged), in mammals Notch signaling pathway components include four receptors (Notch 1–4) and five ligands (Delta-like [DLL] 1, 3, and 4, and Jagged [JAG] 1 and 2; for reviews, see [8,17–19]). In the mouse, both DLL1 and JAG2 are expressed in nascent hair cells [10,20] and act synergistically during lateral inhibition [15]. Both DLL1 and JAG2 appear to signal through the NOTCH1 receptor [15]. In Drosophila, prosensory regions that undergo lateral inhibition are first delineated by expression of members of the atonal or acheate-scute family of basic helix-loop-helix transcription factors. Further parallels with Drosophila have arisen with the finding that an atonal homolog, the Math1 gene, is required for hair cell differentiation [21–23]. However, the situation is not entirely similar to Drosophila, as MATH1 does not appear to be required to establish the prosensory regions [22,24]. Instead, it has turned out that another type of transcription factor, the HMG-box factor SOX2, is required for sensory organ formation in the inner ear [25]. This finding does not demonstrate direct parallels with Drosophila sense organ formation, since the Drosophila SOX2 homologs SoxNeuro and Dicheate have no known role in peripheral sense organ formation. Instead, both genes play a role in the formation of neural progenitor cells in the central nervous system [26,27]. Consistent with a prosensory role, SOX2 marks sensory progenitors early in development and acts upstream of the Math1 gene during sensory organ formation in the ear [25]. Interestingly, one of the Notch ligands, JAG1, is expressed early in the prosensory regions of the ear [4,20], indicating that Notch signaling also may play a role in early sensory organ formation. Consistent with this finding, mice heterozygous for N-ethyl-N-nitrosourea-induced point mutations in the Jag1 gene show mild sensory organ defects in the ear [28,29]. Unfortunately, embryos homozygous for available mutant alleles of the Jag1 gene do not survive past embryonic day (E) 11.5 due to vascular defects, precluding an analysis of their inner ears. To circumvent this early lethality, we have created a conditional allele of the Jag1 gene using Cre/loxP technology. Using the Foxg1-Cre mouse line to express Cre recombinase in the early otocyst [30,31], we have disrupted JAG1 function in the ear and show that sensory formation in the inner ear is severely attenuated in these mutants. Analysis of the patterns of hair and supporting cell formation in the Jag1-conditional knockout (cko) inner ears suggests that fewer progenitors form in Jag1-cko inner ears. This result is confirmed by analysis of the prosensory markers SOX2 and p27kip1, which are down-regulated as early as E12.5, indicating that the Jag1 gene acts early during sensory progenitor formation. These data demonstrate an early role for Notch signaling in establishing the sensory progenitors of the inner ear. + +Results + +Creation of a Conditional Allele of the Jag1 Gene + +We created an allele for conditional inactivation of JAG1 function by flanking the Delta-Serrate-Lag2 (DSL) domain-encoding exon (exon 4) of the Jag1 gene with loxP sites (Figure 1). The DSL domain has been shown to be the region of the DLL and JAG proteins that interacts with Notch family receptors [32,33]; therefore, removing this region of the gene should create a nonfunctional protein. To demonstrate that this allele encodes a nonfunctional protein, we crossed Jag1flox/+ mice to mice expressing Cre recombinase in the female germline under control of the Zp3 promoter (Zp3-Cre mice); the Zp3-Cre mouse strain has been shown to express Cre recombinase in the growing oocyte prior to the completion of the first meiotic division [34]. Female Zp3-Cre/+; Jag1flox/+ offspring were then crossed to male B6 mice to produce offspring that were heterozygous for the deleted region of the floxed allele (designated Jag1del2/+; see Materials and Methods). Heterozygous Jag1del2/+ mice were intercrossed, and Jag1del2/Jag1del2 homozygous offspring were analyzed for defects between E9.5 and E11.5. Jag1del2/Jag1del2 mutant embryos exhibited the same vascular phenotype we described previously in embryos homozygous for a targeted Jag1 null allele, Jag1del1 [35]. Specifically, the Jag1del2/Jag1del2 mutant embryos exhibited yolk sac vascular remodeling defects and cranial hemorrhaging, and often exhibited an enlarged pericardial sac (Figure 2A–2D). All Jag1del2/Jag1del2 mutant embryos were necrotic by E11.5, and most showed vascular defects by E10.5. RT-PCR of cDNA synthesized from the mutant embryos using primers that span the floxed exon 4 demonstrated that this region was deleted, as expected (Figure 2E). These data demonstrate that deletion of the Jag1flox allele yields a nonfunctional Jag1 mutant allele. + +Figure 1 + +Construction of a Conditional Allele of the Jag1 Gene + +(A) Schematic diagram showing the strategy for generating Jag1flox mice. The targeting vector was designed to insert loxP sites (black arrowheads) on either side of exon 4, the DSL domain-encoding exon (white area in exon 4). The neomycin resistance cassette (for positive selection) was flanked by FRT sites (gray arrowheads) so that it could later be removed by crossing to FLPe-expressing mice. A diphtheria toxin gene was included for negative selection. Dotted lines depict the recombination events that occur when either the FLPe or Cre recombinases are present. Primer positions for genotyping are shown as small black arrows (a, DSLF; b, J1LoxR1; c, J1FlpF1; see Materials and Methods for sequences). DT, diphtheria toxin; R, EcoR1; X, Xba1. + +(B) Southern blot analysis of EcoRI-digested DNA from ES cells using the external probe shown in (A). Left lane shows the wild-type band (12.3 kb), and the center and right lanes show correctly targeted ES cells that have both a wild-type band and a smaller mutant band (9.4 kb). + +Figure 2 + +Jag1del2/Jag1del2 Embryos Exhibit Vascular Defects and Lethality Consistent with Loss of JAG1 Function + +(A and B) E10.5 embryos demonstrating the loss of large blood vessels (white arrows in [A]) in the Jag1del2/Jag1del2 yolk sacs (B) similar to other Jag1 loss-of-function mutants. (C and D) E11 embryos demonstrating a small, necrotic Jag1del2/Jag1del2 embryo (D). White arrow in (D) indicates an enlarged pericardial sac (enlarged, inset), which is frequently observed in mutants exhibiting cardiovascular defects. RT-PCR results using primers that span exon 4 using RNA extracted from E10.5 control (+) and ZP3-Cre deleted embryos (−) (E). The upper band (541 bp) indicates that the wild-type allele is present. The lower bands (286 bp) indicates the Jag1del2 mutant allele that does not contain exon 4. + +Inactivation of Jag1 Function within the Ear + +To disrupt JAG1 function within the inner ear, we crossed Jag1flox/Jag1flox mice with mice doubly heterozygous for the Foxg1-Cre allele (these mice express Cre recombinase throughout the otocyst, as well as forebrain, eye, and foregut) [30] and the Jag1del1 allele [35]. Offspring with the genotype Foxg1-Cre/+; Jag1del1/Jag1flox (hereafter designated Jag1-cko) survived through E18.5 and were analyzed for inner ear defects. We examined the patterns of Cre-mediated excision in Jag1-cko embryos at E10.5 and in cochleae at E16.5–E18.5 by in situ hybridization using a probe that specifically detected the deleted exon 4 (Figure 3). These results showed that expression in the otocyst was weak or absent by E10.5 (Figure 3B). In addition, analysis of cochlear expression at later stages showed no expression at E16.5 (Figure 3F) and E18.5 (unpublished data). These data indicate that, as previously shown for conditional deletion of a Fibroblast growth factor receptor 1 (Fgfr1) floxed allele [31], the Foxg1-Cre line efficiently deletes the Jag1flox allele early during inner ear development. + +Figure 3 + +Conditional Jag1 Inactivation Using the Foxg1-Cre Line + +(A–D) Low- and high-power views of E10 embryos processed for whole-mount in situ hybridization using a Jag1 exon 4-specific probe. White arrows (A) point to the Jag1 signal in the otocyst (left arrow) and the eye (right arrow), two structures where Cre recombinase is expressed. In Jag1-cko mutants at E10, this signal is either absent or extremely weak. Black arrows (A and B) point to expression in the spinal cord and nephric duct, regions where Cre recombinase expression has not been reported in Foxg1-Cre mice. However, expression is consistently weaker in these areas in Jag1-cko embryos, indicating that there may be low levels of widespread expression of Cre recombinase in Jag1-cko embryos. In (D), the otocyst and the eye are outlined by a dotted line. Very little expression is observed in these regions, consistent with Foxg1-Cre expression. + +(E and F) In situ hybridization of E16.5 cochleae demonstrating Jag1 expression in wild-type (E) and Jag1-cko cochleae (F), where expression is entirely absent. Scale bars = 500 μm. + +Malformation of the Inner Ear in Jag1-cko Mutants + +To examine the morphology of the Jag1-cko inner ears, paintfilling of the inner ears of mutants and controls was performed at E15.5 (Figure 4). Results of this analysis showed a severe disruption in the structure of the Jag1-cko inner ears compared to their littermate controls (Figure 4C and 4D). Specifically, the semicircular canals were largely absent, with the exception of a portion of the anterior and lateral semicircular canals. In addition, the utricle appeared small, the saccule was misshapen, and the cochlea was undercoiled. In contrast, the parts of the inner ear that are not associated with sensory formation, including the endolymphatic duct and sac and the common crus, appeared relatively unaffected. + +Figure 4 + +Inner Ear Dysmorphology in Jag1+/− and Jag1-cko Embryos + +E15.5 inner ears that have been paintfilled to display their overall morphology. + +(A) Wild-type inner ear showing normal morphology. Structures are labeled as follows: aa, anterior ampulla; asc, anterior semicircular canal; cd, cochlea duct; ed, endolymphatic duct; la, lateral ampulla; lsc, lateral semicircular canal; pa, posterior ampulla; psc, posterior semicircular canal; sac, saccule; ut, utricle. + +(B) Jag1 heterozygote inner ears (either Jag1del1/+ or Foxg1-Cre/+; Jag1flox/+) display truncated posterior semicircular canals and missing ampullae (asterisk). Arrows point to the anterior and posterior ampullae, which are small compared to the wild-type control (A). + +(C and D) A much more severe phenotype is observed in Jag1-cko animals. There are no ampullae and little semicircular canal development; an asterisk indicates a remnant of the anterior canal. In (D), there is also a remnant of the lateral canal (arrow). The utricle and saccule are smaller and the cochleae are shorter and undercoiled. Scale bar = 500μm. + +Sensory Defects in Jag1-cko Mutant Inner Ears + +Since the Jag1 gene is expressed in the sensory areas of the ear, and because the structural malformations observed in Jag1-cko mutant inner ears appeared to primarily affect regions of the ear that contained sensory organs, we examined the sensory regions of the ear for defects. We examined the organ of Corti, the sensory organ of the cochlea, at E18.5 by scanning electron microscopy (SEM) (Figure 5). By this stage, all hair cells within the organ of Corti have exited the cell cycle, and most are well-differentiated although not fully mature [36]. Severe hair cell patterning defects were apparent by SEM within the Jag1-cko mutant cochleae. This phenotype was most striking in the basal turns of the cochlea, where no hair cell formation was observed (Figure 5D). In the midbasal regions of the organ of Corti, hair cells formed in patches, within which there was no clear formation of rows or distinction between inner and outer hair cells (Figure 5F). More apically, hair cells appeared more continuous along the organ of Corti (Figure 5H). However, although hair cells were present in the apical region, their numbers were clearly reduced; rather than the normal, perfectly ordered four rows of hair cells, there were only two rows of loosely arranged hair cells of indistinct type. + +Figure 5 + +Hair Cell Patterning Defects in the Cochlea + +Scanning electron micrographs demonstrating the different patterns of hair cell production along the length of the cochlea in Jag1-cko embryos. + +(A–D) Low-power views of the apical and basal cochlear turns. The boxed-in area along the base in (A) and (B) is shown at higher magnification in (C) and (D). Note the absence of hair cells in the base of the Jag1-cko cochlea, except for a small patch of cells in the more apical portion (arrow). Scale bars = 500 μm. + +(E and F) In the midbasal region, more hair cells are observed, but they are arranged in patches, with no clear distinction between inner and outer hair cells. + +(G and H) In the apical turn, hair cells are continuous but generally arranged in only two rows. Scale bar = 100 μm. + +Abnormal Hair and Supporting Cell Patterns in Jag1-cko Inner Ears + +To determine which sensory cell types were differentiating in the Jag1-cko mutant cochleae, specific markers were used to identify hair cell and supporting cell subtypes throughout the ear (Figure 6). In the cochlea, we used an antibody against MYO7A to label all hair cells and an antibody against S100A1 to label inner hair cells, Deiter's supporting cells, and inner phalangeal supporting cells [24]. When both markers were used in combination, inner hair cells, outer hair cells, and some supporting cell types could be distinguished (Figure 6A–6F). This analysis showed that in the apex of the cochlea, inner hair cells were present and usually formed as doublets (Figure 6B). Their associated supporting cells, the inner phalangeal cells, were also present. Outer hair cells and their associated supporting cells, the Deiter's cells, were not present in this region. In the middle portions of the cochlea, both inner and occasionally outer hair cells were present, although their patterning was clearly abnormal (Figure 6D). In addition, the tunnel of Corti was not apparent, and there were often doublets of inner hair cells and increased numbers of outer hair cell rows without accompanying Deiter's supporting cells. As shown by SEM, both hair cells and supporting cells were absent in the very basal regions of the cochlea (Figure 6F). + +Figure 6 + +Hair and Supporting Cell Markers Demonstrate Sensory Areas Are Reduced or Absent in the Jag1-cko Inner Ear + +Immunocytochemistry using two markers, myosin VIIA (red; all hair cells) and S100a (green; inner hair cells, Dieter's cells, and inner phalangeal cells) demonstrate patterns of hair and supporting cell production at E18.5 in control and Jag1-cko inner ears. + +(A–F) Sections through the indicated turns of the cochlea. Note the different hair cell patterns in the apical, middle and basal turns of the Jag1-cko cochlea. Normal morphology is shown (A) along with labeled structures, as follows: GER, greater epithelial ridge; IHCs, inner hair cells (color-coded yellow); LER, lesser epithelial ridge; OHCs, outer hair cells (color-coded red); SCs, supporting cells (color-coded green). + +(G–J) Patterns of hair and supporting cell production in the vestibular system. The utricular macula is extremely small with very few hair cells (H) while the saccule (J) shows robust hair and supporting cell production although the shape of the organ is smaller and malformed. + +Using the same markers we also examined the vestibular sensory organs in Jag1-cko mutant inner ears (Figure 6G–6J). Consistent with the lack of semicircular canal and ampulla formation observed by paintfilling, there was no evidence of crista formation. The Jag1-cko utricular macula was extremely small with very few differentiating hair cells (Figure 6H). Surprisingly, the saccule and its macula were only mildly affected in the Jag1-cko inner ears (Figure 6J). Hair cell differentiation appeared relatively unaffected, although the entire saccular structure was shaped differently than in the controls, a feature that was also observed in the paintfilled specimens (see Figure 4C and 4D). These data show that all sensory organs within the inner ear are affected to varying degrees in Jag1-cko inner ears. However, some sensory organs, such as the cristae, appear to be more sensitive to the loss of JAG1 function. + +To examine whether aberrant hair cell patterning in Jag1-cko cochleae was due to defects in hair cell formation or in subsequent differentiation, we examined hair cell patterning at an earlier stage (E16.5). Using a lectin that binds to hair cell stereocilia, we examined whether the patterns of hair cell formation at E16.5 looked similar to the patterns at E18.5 (Figure 7). At E16.5 in wild-type cochleae, a gradient of hair cell differentiation was evident (Figure 7A, 7C, 7E, and 7G); in the basal regions both inner and outer hair cells could be recognized (unpublished data), while in the middle regions only inner hair cells were clearly detected by most markers (Figure 7A and 7C). In the more apical regions, little to no hair cell differentiation had taken place by this stage (Figure 7E and 7G). In Jag1-cko cochleae, the patterns looked similar to those at E18.5, with patches of hair cells in the midbasal regions (Figure 7B and 7D) and a complete absence of hair cells in the very basal regions (Figure 7B). These data suggest that the Jag1-cko mutants have defects in hair cell formation rather than differentiation. In addition, the apical regions in the Jag1-cko cochleae did not appear more differentiated than the controls (Figure 7E–7H), arguing against precocious differentiation as an explanation for the reduced numbers of hair cells observed in the mutant cochleae. + +Figure 7 + +Early Analysis of the Patterns of Differentiation in the Jag1-cko Cochlea Indicates the Defects Are Caused by a Failure in the Formation of Sensory Cells and Not Subsequent Degeneration + +Lectin staining of whole-mount cochlea at E16.5. + +(A) Normal patterning in wild-type control cochlea. GER, greater epithelial ridge. + +(B) Both the basal and middle portions of the cochlea are shown, although because it is much longer in the control (A), the very basal portion of the cochlea has been removed. Note the lack of hair cells in the basal portion of the Jag1-cko cochlea. + +(C–H) Boxed-in areas of (A) and (B) are shown at higher magnification in (C) and (D). Arrows in (D) indicate the abnormal patches of hair cells also observed at E18.5. Similarly, the boxed-in regions of (E) and (F) are shown at higher magnification in (G) and (H), demonstrating the few hair cells that are just beginning to differentiate in this region in both the control and the mutant (arrowheads). Scale bars = 500 μm for the corresponding panels. + +Disrupted Prosensory Development in Jag1-cko Inner Ears + +To determine how the JAG1 ligand functions during sensory development, we used several markers of the prosensory domain, including p27kip1 and SOX2, and examined their expression patterns in both wild-type and Jag1-cko mutant cochleae (Figure 8). At E14.5, the majority of hair cells and supporting cells in the organ of Corti have completed their final division, and hair cells are beginning to differentiate in the basal portions of the cochlea [36]. p27kip1, a cell-cycle inhibitor, is required for the cochlear sensory progenitors to exit the cell cycle on time, and is an established marker of the prosensory domain in the cochlea [22,37]. p27kip1 begins to be expressed in a discrete domain within the cochlea as the hair cells and supporting cells exit the cell cycle around E13.5 to E14.5 (Figure 8A, 8B, 8D, and 8E). Recently it has been shown that the SRY-related transcription factor SOX2 is required for establishment of the prosensory regions in the inner ear [25]. Using fluorescence immunocytochemistry double labeling, we examined the relationship between these markers and JAG1 protein expression in both wild-type and Jag1-cko cochleae. As previously reported [22], JAG1 was not expressed within the prosensory domain as assessed by p27kip1 expression at E14.5, but instead was expressed immediately adjacent (possibly with some slight overlap) in the inner (neural) portion of cochlea (Kölliker's organ; Figure 8A and 8D). In contrast, SOX2 did show a largely overlapping domain with p27kip1 (Figure 8B and 8E), as originally described [25]. However, the SOX2 expression domain was slightly larger than the p27kip1 domain, extending into Kölliker's organ and overlapping with the JAG1 domain. Despite the fact that JAG1 was not expressed within the prosensory domain at E14.5, both p27kip1 and SOX2 expression was absent in the basal regions of the cochlea (Figure 8C), indicating that prosensory formation is already disrupted in these ears. In the apex, weak expression of both markers was observed (Figure 8F), consistent with the fact that some sensory differentiation occurs in this region of the Jag1-cko cochlea. + +Figure 8 + +At E14.5 JAG1 Is Expressed Directly adjacent to the Prosensory Domain That Is Disrupted in Jag1-cko Inner Ears + +Immunocytochemistry at E14.5 using two markers of the prosensory domain, Sox2 and p27Kip1, in combination with JAG1 in both the basal and apical turns of the cochlea. Note that the JAG1 domain (red) does not overlap the p27Kip1 domain (green) (A and D), whereas the SOX2 domain does largely overlap with p27Kip1 (yellow) (B and E). Both SOX2 and p27Kip1 are down-regulated in the Jag1-cko cochlea (C and F), although there is weak expression of both markers in the apex (F). GER, greater epithelial ridge; LER, lesser epithelial ridge. + +In order to determine if JAG1 is ever expressed in the prosensory region of the cochlea, we examined an earlier age (E12.5) and compared the JAG1 domain to the SOX2 domain (since p27Kip1 is not expressed in the inner ear prior to E13.5 to E14.5). Adjacent sections from both wild-type and Jag1-cko cochleae were immunostained to detect either JAG1 or SOX2 protein (Figure 9). This analysis showed that in the basal regions of the wild-type cochlea, where sensory progenitors were still dividing, JAG1 expression did overlap with the SOX2 domain (Figure 9A and 9B), indicating that JAG1 is initially expressed within the prosensory domain. However, in the apical regions, where the sensory precursors have ceased dividing, expression of JAG1 and SOX2 did not overlap (Figure 9D and 9E). In the Jag1-cko cochlea, SOX2 was absent from the basal regions and significantly down-regulated in the apical regions (Figure 9C and 9F). These data demonstrate that JAG1 is expressed within the prosensory domain of the cochlea at early stages, and that, in the absence of JAG1 function, sensory formation is disrupted prior to cell cycle exit and differentiation of sensory hair cells and nonsensory supporting cells. + +Figure 9 + +At E12.5 JAG1 Is Expressed within the Prosensory Domain and SOX2 Expression Is Down-Regulated within This Domain in Jag1-cko Cochleae + +(A, B, D, and E) Alternate sections from a control embryo processed for immunocytochemistry using antibodies against either JAG1 or SOX2. Note the similar domain occupied by both JAG1 and SOX2 in the base of the cochlea (A and B; brackets). In the apical region, the two proteins are not colocalized (D and E). SOX2 is not expressed in the basal portions of the Jag1-cko cochlea (C) and shows only weak expression in the apex (F). bv, blood vessel; cd, cochlear duct. + +We also compared JAG1 and SOX2 expression in the vestibular regions of the inner ear in both wild-type and Jag1-cko mutant embryos. JAG1 and SOX2 exhibited largely overlapping expression domains that corresponded to the locations of the five sensory organs in the vestibular portion of the ear (Figure 10). The two expression domains only differed significantly in the anterior and posterior cristae, where JAG1 expression had a negative patch in the middle of its expression domain, whereas SOX2 expression did not show this same patch (Figure 10A, 10B, 10G, and 10H). The JAG1-negative region may correspond to the eminentia cruciatum, a nonsensory region present in the middle of both the anterior and posterior cristae, although it is not clear why SOX2 would be expressed there. In the Jag1-cko vestibular sensory patches, SOX2 expression was consistent with the patterns of sensory differentiation observed at E18.5. For example, the Jag1-cko saccule displayed fairly normal SOX2 expression (Figure 10C), consistent with the almost normal development of the saccular macula. In contrast, SOX2 expression in the utricle was very weak and the expression domain was much smaller than in controls (Figure 10F), consistent with the severe disruption of differentiation of the utricular macula in Jag1-cko inner ears. There was no SOX2 expression in the Jag1-cko cristae, and in fact the entire ampullae appeared to be missing or severely disrupted even at this early stage (Figure 10C and 10I; dotted line regions), consistent with the lack of cristae and ampullae observed at later stages. + +Figure 10 + +JAG1 and SOX2 Mark the Prosensory Regions of the Vestibule, and SOX2 Expression Correlates with Impaired Sensory Formation in the Jag1-cko Vestibule + +(A and B, D and E, G and H) Alternate sections demonstrating either JAG1 or SOX2 expression in the vestibular regions of control inner ears. + +(C, F, I) Similar sections through the Jag1-cko inner ear demonstrating SOX2 expression. Dotted lines indicate regions where the cristae and ampullae are missing in the Jag1-cko inner ear. ac, anterior cristae; lc, lateral cristae; pc, posterior cristae; sac, saccular macula; ut, utricular macula. + +Discussion + +We have demonstrated that Notch signaling, mediated by the JAG1 ligand, is required early in development for the formation of the sensory regions of the ear. By comparing expression of JAG1 to two markers of the prosensory domain, SOX2 and p27kip1, we have shown that JAG1 marks all prosensory regions of the ear from early time points (E12.5), but becomes down-regulated in the organ of Corti by E14.5, when the sensory progenitors exit the cell cycle and begin differentiating into hair cells and supporting cells. Both SOX2 and p27kip1 are down-regulated in the affected prosensory regions of the Jag1-cko inner ear, demonstrating that JAG1 is necessary for the development of early sensory progenitors in the inner ear. + +Distinctive Patterns of Hair Cell Formation in Jag1-cko Inner Ears Suggest Progenitor Cell Numbers Are Reduced + +One intriguing result from our studies was that the six sensory regions were not equally affected by the loss of Jag1 function. For example, in the Jag1-cko vestibular system, the cristae were lacking altogether, and only a small number of hair cells differentiated in the utricular maculae. In contrast, the saccular maculae exhibited little disturbance in hair cell formation, although the overall shape of the organ was abnormal. In the Jag1-cko cochlea, hair cell differentiation patterns varied based on their apical or basal location. For example, in the apical regions of the cochlea only inner hair cells formed, and these were often arranged in multiple rows rather than the normal single row. In the middle and midbasal turns of the cochlea, patches of hair cells with nonsensory intervening regions were frequently observed. Within these patches, outer hair cells were sometimes present, although the patterning was abnormal and S100A1-labeled Dieter's cells were not present. In the very basal regions of the cochlea, neither hair cells nor supporting cells were present. + +The patches of hair cells found in the basal regions of the cochlea and the differential effect of the mutation on the basal and apical portions of the cochlea were particularly interesting, as similar defects have been found in at least two other mouse mutants of genes known to play a role in the generation of the sensory precursors of the ear. For example, both a hypomorphic allele and a conditionally deleted allele of the Fgfr1 gene exhibited patches of hair cells in portions of the cochlea [31]. Similar to the Jag1-cko phenotype, these patches in the Fgfr1 conditional mutants contained mostly inner hair cells that were often arranged in multiple rows, with very few outer hair cells. Unlike the Jag1-cko phenotype, Fgfr1 function was required only in the cochlea. Another mouse mutant, a hypomorphic allele of the Sox2 gene (yellow submarine; Sox2ysb), also displayed patches of hair cells in the basal portions of the cochlea and a milder phenotype in the apical regions of the cochlea [25]. More similar to the Jag1-cko phenotype, SOX2 was required for both the cochlear and vestibular sensory regions [25]. The finding that primarily inner hair cells differentiate in these mutant cochleae may be due to the fact that inner hair cells are the first to differentiate [10,37], suggesting that if there are reduced numbers of progenitor cells, they would likely differentiate as inner rather than outer hair cells. Similarly, the milder phenotype in the apical regions of these mutants may be due to the fact that cells exit the cell cycle earliest in the apex [36]. This may mean that, if there are reduced numbers of progenitor cells, they would reside in the apical rather than the basal portions of the cochlea. + +The multiple rows of inner hair cells observed in Jag1-cko and other mutants could be explained by a number of different scenarios. One possibility is that the multiple rows are not a result of actual increases in inner hair cell numbers, but rather are caused by defects in their eventual arrangement due to the shorter Jag1-cko cochlea. Recent studies of mouse mutants with defects in planar cell polarity and convergent extension (a term referring to the intercalation of cells, leading to growth of tissue in one dimension in the absence of proliferation) indicate that multiple rows of hair cells can be obtained in this way and are frequently observed in the apical regions of the cochlea [38–41]. An alternative possibility is that the multiple rows are a result of a second, later function of the JAG1 ligand, distinct from its prosensory role described here. A third possibility is that the down-regulation of p27kip1, a protein that inhibits continued proliferation of the precursor cells in the cochlea, leads to continued cell division of the remaining sensory progenitors, ultimately resulting in excess numbers of inner hair cells in the regions where they form. Taken together, these data suggest that sensory progenitors are reduced in the Jag1-cko inner ears and that Notch signaling, fibroblast growth factor signaling, and the transcription factor SOX2 all act in either common or parallel pathways involved in the production of sensory progenitors in the inner ear. + +A Prosensory Role for Notch in the Ear + +An examination of early prosensory markers, including p27Kip1 and SOX2, demonstrated that prosensory establishment is disrupted in Jag1-cko inner ears, consistent with the suggestion that progenitors are reduced in these mutants. Our data show that JAG1 plays an early prosensory role in ear development, quite unlike the role played later during development by the other Notch ligands, DLL1 and JAG2, which are involved in lateral signaling and differentiation [10,15]. These data are consistent with an early role for Notch signaling in progenitor cell maintenance in the inner ear. In a number of other systems, including the nervous system and more recently in the intestinal epithelium, it has been demonstrated that Notch signaling is involved in maintaining cells in an undifferentiated state [42–46]. In the mammalian nervous system it has been shown that loss of Notch signaling leads to premature differentiation and a reduction in the progenitor pool [42]. Consistent with these findings, in vitro studies have demonstrated that the frequency of neurosphere production was reduced in Notch signaling mutants [47,48], indicating a loss of stem cell potential. Moreover, studies have also shown that Notch signaling promotes radial glial identity, a cell type that has been shown to act as a progenitor cell in the central nervous system [49–52]. Our results suggest that, similar to the nervous system, Notch signaling via JAG1 is important for sensory precursor formation or maintenance in the inner ear. However, unlike the nervous system, we see no evidence for precocious differentiation, suggesting instead that JAG1 may affect the specification, survival, or proliferative capacity of the sensory precursors. + +Recent evidence from the chick indicates that JAG1 may be important for the initial sensory specification events. By expressing a constitutively active form of Notch (Notch1-ICD), Daudet et al. [14] demonstrated that ectopic sensory patches could be induced, indicating that early Notch signaling may be important for the induction of sensory areas, and not just for their maintenance. However, it should be noted that ectopic sensory areas formed only in certain areas of the ear, indicating that some sensory competence is required for this effect. A similar result was obtained by overexpressing an activated form of β-catenin, an essential component of the canonical Wnt signaling pathway, in the chicken inner ear [53]. As in the Notch1-ICD studies, ectopic sensory regions were obtained, but again, only in certain regions of the ear. However, unlike the Notch gain-of-function studies, overexpression of β-catenin also led to a change in sensory region character (i.e., cochlear to vestibular), indicating that Wnt signaling governs not only whether a sensory region will form but also the type of sensory region that will form. In Drosophila, interactions between Notch and Wingless, a member of the Wnt family of signaling molecules, are well established [54,55], and evidence of an interaction has begun accumulating in vertebrates as well [45,56,57]. Bone morphogenetic protein (BMP) signaling may also be important for sensory formation, particularly for the sensory cristae, as BMP4 has been shown to mark the mouse cristae from very early in development [58]. Experiments in the chicken have shown that blocking BMP signaling sometimes leads to disturbances in sensory development [59]. Taken together, these data indicate that, based on expression patterns, previous studies, and the evidence presented here, JAG1 is the ligand responsible for the prosensory function of the Notch pathway in the ear. Furthermore, the Notch pathway likely interacts with other signaling pathways such as the Wnt, FGF, and BMP pathways to create sensory organs of the proper size, organization and character. + +Sensory Formation Still Occurs in Jag1-cko Inner Ears + +One somewhat puzzling question is that, if JAG1 is important for sensory progenitor development, why does any sensory formation occur in Jag1-cko inner ears? One possibility is that another Notch ligand is compensating for the loss of JAG1 function. This explanation seems unlikely since none of the other Notch ligands shows a similar expression pattern to JAG1 in the ear. For example, both the Dll1 and Jag2 genes are expressed in nascent hair cells after they exit the cell cycle and begin differentiating. However, in addition to hair cell expression, there is also early expression of the Dll1 gene in the anteroventral portion of the otocyst at about E10.5 [4,20], that likely overlaps with at least part of the JAG1 domain (see Figure 2) [60]. This expression domain has previously been thought to be related to the formation of the neuroblasts that delaminate from the otic epithelium and later differentiate into the neurons that will innervate the hair cells [4]. It has been shown in zebrafish that correct neuroblast formation requires Notch-mediated lateral signaling [9]; however, in mammals it has not been shown definitively that this is the role that the Dll1 gene plays at early stages. This leaves open the possibility that this early domain of DLL1 expression may be at least partially involved in prosensory specification, similar to the JAG1 expression domain. + +Nonsensory Defects in Jag1-cko Inner Ears + +In addition to the defects in sensory formation in the Jag1-cko inner ears, the mutant inner ears also exhibited nonsensory defects. Specifically, the semicircular canals were largely absent, with the exception of portions of the anterior and lateral canals. In addition, all three ampullae were absent, the utricle was small, and the cochlea was undercoiled. Based on recent studies, it is likely that these defects are secondary to the sensory defects. For example, it has been shown that FGFs expressed in the sensory cristae promote semicircular canal formation through up-regulation of BMP2 [61]. Thus, loss of the cristae would be expected to have a severe affect on canal formation. Emerging evidence from mouse mutants has demonstrated that genes involved in sensory formation result in severely malformed inner ears. For example, mutations in the Sox2 gene lead to malformations very similar to those described here in Jag1-cko mutants. The inner ears of embryos homozygous for two different mutant alleles of Sox2, Sox2lcc/lcc and Sox2ysb/ysb, showed disrupted canal formation; smaller utricular and saccular compartments; and thinner, undercoiled cochleae [25]. In addition, FGF10 mouse knockouts also showed disrupted cristae development associated with loss of canal structures [62]. However, unlike the canal structures, cochlear formation does not appear to be strictly dependent on development of the organ of Corti, as a cochlea, albeit short and thin, will form in the absence of any sensory formation [25]. However, normal cochlear length appears to be dependent on sensory formation, at least partially through convergent extension. Recently, a number of genes have been found in the cochlea that lead to defects in planar cell polarity as well as a shortened cochlea, presumably because of defects in convergent extension [38,41]. Therefore it is likely that the shortened cochlea observed in Jag1-cko mutants is at least partially a result of failure of convergent extension caused by a reduction in the number of sensory precursors. + +The data presented here demonstrate that the Jag1 gene is required for sensory precursor development in the inner ear. Further studies are required to establish the exact role that JAG1-mediated Notch signaling plays in early sensory progenitors, and also its relationship to the roles played by FGF signaling and SOX2 expression. Understanding how the sensory precursors form is an important prerequisite for regeneration studies that may provide molecular tools to treat hearing loss and vestibular disorders [63]. + +Materials and Methods + +Construction of the Jag1floxneo allele. + +To construct the Jag1floxneo allele, bacterial artificial chromosome clones containing the Jag1 genomic locus were isolated from a RPCI-22 (129S6/SvEvTac) mouse bacterial artificial chromosome library (filters obtained from Research Genetics) by hybridization to a 1.8-kb mouse Jag1 cDNA probe. To make the shorter 5′ homology region of the targeting vector, a 2.2-kb KpnI fragment upstream of exon 4 was isolated, blunt-ended, and subcloned into the SmaI site of a modified pBS vector that contained a loxP-FRT-PGKneo-FRT cassette. A 1.5-kb KpnI fragment that contained exon 4 was also subcloned into the loxP-FRT-PGKneo-FRT cassette. To construct the longer 3′ homology region, a 3.5-kb KpnI-SmaI fragment containing exon 5 was blunt-ended and subcloned into the EcoRV site of another modified pBS vector that contained a single loxP site. A 3.5-kb SmaI-SalI fragment from this construct was then cloned into the SmaI-XhoI site of a pKO 905 vector containing a diphtheria toxin gene for negative selection. A 5.7-kb SalI-NotI fragment from the loxP-FRT-PGKneo-FRT construct was then cloned into the SalI-NotI sites of the pKO 905 vector containing the 3′ homology region to generate the final Jag1floxneo targeting vector (see Figure 1). + +Generation of Jag1flox mice. + +The Jag1floxneo targeting construct was linearized with Not1 and electroporated into CJ7 embryonic stem (ES) cells, as described previously [64]. DNA from 288 ES cell clones was screened by PCR using an internal/external primer set, and positive clones were then confirmed by Southern blot by probing EcoRI-digested DNA with an external 1.7-kb Stu1-EcoRI fragment located 3′ to the targeting construct (see Figure 1). This probe also detected partial recombination events in which the distal loxP site was lost; in these cases a slightly larger fragment (11 kb rather than 9.3 kb) was obtained (see Figure 1A). The presence of the distal loxP site was further confirmed by PCR using primers that flanked the loxP site (DSLF and J1LoxR1; see below for sequences). Correctly targeted clones were injected into C57BL/6J (B6) blastocysts, and chimeric mice were obtained. Chimeric male mice were mated to B6 females and the agouti progeny were assayed for the presence of the Jag1floxneo allele by PCR using Jag1floxneo specific primers. Jag1floxneo/+ mice were intercrossed to create homozygous Jag1floxneo/Jag1floxneo offspring. Homozygous Jag1floxneo/Jag1floxneo mice appeared normal and healthy, suggesting that the neomycin resistance cassette (PGKneo) did not adversely affect Jag1 expression. To control for any possible effects from the presence of the PGKneo cassette, the FRT-flanked PGKneo cassette was deleted by mating Jag1floxneo mice to FLPe-recombinase expressing mice (Gt[ROSA]26Sor tm1(FLP1)Dym; Jackson Laboratory, Bar Harbor, Maine, United States) to produce Jag1flox mice. Both Jag1floxneo and Jag1flox mice were used in these experiments, and no differences in the resulting phenotype were observed. To differentiate the deleted form of this allele from our previously reported Jag1 null mutant allele (Jag1del1) [35], we designate the Jag1 allele generated by Cre recombinase-mediated deletion of the Jag1flox or Jag1floxneo alleles the Jag1del2 allele. + +Mouse husbandry and genotyping. + +Foxg1-Cre mice ([30]; gift of Rob Burgess) were maintained on an outbred Swiss Webster background. ZP3-Cre mice ([34]; gift of Mimi de Vries and Barbara Knowles) were maintained on a B6 background. Typically, males that were heterozygous for both a Foxg1-Cre allele and our previously constructed Jag1 null allele (Jag1del1) [35] (maintained on a B6 background), were crossed to Jag1flox/Jag1flox females that were maintained on a B6/129 background. Mice of the genotypes Foxg1-Cre/+; Jag1del1/Jag1flox and Foxg1-Cre/+; Jag1del1/Jag1floxneo were used interchangeably, and are designated as Jag1-cko mice in this report. + +To genotype the Jag1flox mice, the primers used were: DSLF, 5′-TCAGGCATGATAAACCCTAGC-3′ (forward) and J1LoxR1, 5′-CTACATACAGCATCTACATGC-3′ (reverse); these primers flank the 5′ LoxP site. To genotype for CRE-mediated recombination, a primer upstream of the 3′ LoxP site was used: J1FlpF1, 5′-CAGGTTGAGTGCTGACTTAG-3′, along with the J1LoxR1 reverse primer. To genotype for the Foxg1-Cre and ZP3-Cre alleles, Cre-specific primers were used: Cre1, 5′-TGATGAGGTTCGCAAGAACC-3′ (forward) and Cre2, 5′-CCATGAGTGAACGAACCTGG-3′ (reverse). Jag1del1 primers were as follows for the mutant: JGKO1, 5′-TCTCACTCAGGCATGATAAACC-3′ (forward) and SOL1, 5′-TGGATGTGGAATGTGTGCGAG-3′ (reverse). A different reverse primer, JGKO2, 5′-TAACGGGGACTCCGG ACAGGG-3′ was used to detect the wild-type allele. Littermates (wild type, Jag1del1/+ or Jag1flox/+) were used as controls for all experiments. + +Paintfilling and scanning electron microscopy. + +The paintfilling of the Jag1-cko inner ears was performed at E15.5. The technique was performed as previously described [28]. Inner ears were prepared for SEM as described previously using a version of the osmium tetroxide-thiocarbohydrazide method [65]. Specimens were examined with a Hitachi 3000N scanning electron microscope (Hitachi, Tokyo, Japan). + +Immunohistochemistry and lectin staining. + +For immunohistochemistry, embryonic heads were bisected and fixed for 1–2 h in 4% paraformaldehyde in PBS. Half heads were embedded in paraffin wax, and immunocytochemistry was performed on standard 7-micron sections. Antibodies used included anti-Myo7a (1:1,000; gift of A. EL-Amraoui and C. Petit), anti-p27kip1 (1:100; Neomarkers, Stratech, Soham, Cambridgeshire, United Kingdom), anti-SOX2 (1:2,000; Chemicon, Temecula, California, United States; AB5603), anti-JAG1 (1:100; Santa Cruz Biotechnology, Santa Cruz, California, United States; H-114) and anti-S100A1 (1:50; Dako, Glostrup, Denmark). For all antibodies used, an antigen retrieval step was performed by boiling the sections for 10 min in 10 mM citric acid. Secondary antibodies used were either Alexa-Fluor 488 or 546 goat anti-mouse or rabbit (1:400; Invitrogen, Carlsbad, California, United States). Slides were coverslipped in Vectashield HardSet Mounting Medium with DAPI (Vector Laboratories, Burlingame, California, United States). Lectin staining was performed using the Griffonia simplifonia I lectin (Vector Laboratories) essentially as described [15]. + +In situ hybridization and RT-PCR. + +Since the Jag1del2 mutant allele creates an in-frame deletion of the DSL domain, an in situ probe was designed for detection of the floxed region of the Jag1flox allele by in situ hybridization. The probe was created by amplifying a 433-bp product encompassing exon 4 (primers: J1-420F, 5′-CGACCGTAATCGCATCGTAC-3′ and J1-853R, 5′-ATGCACTTGTCGCAGTACAG-3′) and subcloning the product into the PCR II vector (Invitrogen). For whole-mount embryos in situ, embryos were fixed overnight in 4% paraformaldehyde. In situ hybridization was performed essentially as described [66]. For cochlear in situ, inner ears were dissected from the head and fixed overnight in 4% paraformaldehyde. After washing in PBS, the bony shell and stria were removed from the cochleae and the samples were dehydrated in methanol. In situ hybridization was performed as described [67], with the exception of the posthybridization washes, which were done according to [68]. For RT-PCR, total RNA was extracted from the E10.5 control and Jag1del2/Jag1del2 embryos, using an RNAeasy kit (Qiagen, Valencia, California, United States) and following the manufacturer's instructions. First-strand cDNA synthesis was performed using the AMV reverse transcriptase (Promega, Madison, Wisconsin, United States) with specific primer J1-961R (5′-AGTCCCACAGTAATTCAGATC-3′). Products were amplified from cDNA using primers that flanked exon 4 (J1-420F, 5′-CGACCGTAATCGCATCGTAC-3′ and J1-961R). + +Acknowledgements + +We thank Drs. A. El-Amraoui, C. Petit, R. Burgess, M. de Vries, and B. Knowles for reagents; Peter Finger of the Jackson Laboratory Histopathology and Microscopy Services for help with the SEM processing; and the Jackson Laboratory Cell Biology and Microinjection Core Facility for the blastocyst injections. This work was supported by grants from the National Institutes of Health to AEK (DC05865) and TG (NS036437 and DK066387), and from the Jackson Laboratory (CA034196). + +Abbreviations + +BMP - bone morphogenetic protein + +cko - conditional knockout + +DLL - Delta-like + +DSL - Delta-Serrate-Lag2 + +E[number] - embryonic day [number] + +ES - embryonic stem + +FGF - fibroblast growth factor + +JAG - Jagged + +SEM - scanning electron microscopy + +Footnotes + +Author contributions. AEK and TG conceived and designed experiments. AEK and JX performed experiments. All authors analyzed the data. AEK wrote the paper. + +Competing interests. The authors have declared that no competing interests exist. + +A previous version of this article appeared as an Early Online Release on December 1, 2005 (DOI: 10.1371/journal.pgen.0020004.eor). + +Citation: Kiernan AE, Xu J, Gridley T (2006) The Notch ligand JAG1 is required for sensory progenitor development in the mammalian inner ear. PLoS Genet 2(1): e4. diff --git a/src/ontogpt/evaluation/craft/database/all/16433929.ann b/src/ontogpt/evaluation/craft/database/all/16433929.ann new file mode 100644 index 000000000..6ec5d62ec --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16433929.ann @@ -0,0 +1,840 @@ +T1 GO:0007566 29 41 implantation +T2 UBERON:0000922 42 51 embryonic +T3 GO:0009790 42 63 embryonic development +T4 NCBITaxon:10088 67 71 mice +T5 GO:0010467 136 145 expressed +T6 GO:0016477 170 188 cellular migration +T7 GO:0007049 190 200 cell cycle +T8 GO:0008219 241 251 cell death +T9 PR:000005009 337 342 Capn1 +T10 PR:000005015 358 363 Capn2 +T11 GO:0065007 390 400 regulatory +T12 PR:000005022 420 425 Capn4 +T13 NCBITaxon:10088 445 450 mouse +T14 PR:000005022 451 456 Capn4 +T15 SO:0000704 457 461 gene +T16 UBERON:0000922 527 536 embryonic +T17 NCBITaxon:40674 623 632 mammalian +T18 GO:0009790 633 646 embryogenesis +T19 PR:000005009 666 671 Capn1 +T20 SO:0000704 672 676 gene +T21 NCBITaxon:10088 702 706 mice +T22 GO:0016265 832 837 death +T23 PR:000005022 841 846 Capn4 +T24 NCBITaxon:10088 850 854 mice +T25 NCBITaxon:10088 968 973 mouse +T26 PR:000005015 974 979 Capn2 +T27 SO:0000704 980 984 gene +T28 UBERON:0000922 988 997 embryonic +T29 CL:0002322 988 1009 embryonic stems cells +T30 SO:0001023 1038 1044 allele +T31 NCBITaxon:10088 1057 1062 mouse +T32 NCBITaxon:33208 1098 1105 animals +T33 GO:0007566 1160 1169 implanted +T34 UBERON:0000922 1170 1177 embryos +T35 GO:0007566 1247 1259 implantation +T36 UBERON:0000922 1267 1274 embryos +T37 UBERON:0000085 1296 1302 morula +T38 UBERON:0000358 1318 1328 blastocyts +T39 PR:000005015 1395 1400 Capn2 +T40 SO:0000704 1401 1405 gene +T41 GO:0007566 1421 1433 implantation +T42 UBERON:0000922 1434 1443 embryonic +T43 UBERON:0000085 1466 1472 morula +T44 UBERON:0000358 1477 1487 blastocyst +T45 GO:0009790 1598 1612;1640 1646 development of ... embryo +T46 GO:0007566 1620 1632 implantation +T47 NCBITaxon:39107 1633 1639 murine +T48 UBERON:0000922 1640 1646 embryo +T49 CHEBI:29108 1680 1684 Ca2+ +T50 PR:000005015 1761 1769 capain-2 +T51 SO:0000704 1802 1806 gene +T52 SO:0000704 1828 1833 genes +T53 NCBITaxon:40674 1837 1844 mammals +T54 PR:000005009 1948 1953 Capn1 +T55 PR:000005009 1955 1961 μ-80 k +T56 PR:000005015 1967 1972 Capn2 +T57 PR:000005015 1974 1980 m-80 k +T58 SO:0000704 1982 1987 genes +T59 GO:0065007 2039 2049 regulatory +T60 PR:000005022 2073 2078 Capn4 +T61 SO:0000704 2079 2083 gene +T62 PR:000005009 2089 2095;2107 2115 μ-80 k ... subunits +T63 PR:000005015 2100 2115 m-80 k subunits +T64 GO:0010467 2300 2310 expression +T65 NCBITaxon:40674 2343 2352 mammalian +T66 UBERON:0000479 2353 2360 tissues +T67 SO:0001060 2384 2391 isoform +T68 CHEBI:29108 2476 2480 Ca2+ +T69 CHEBI:29108 2532 2536 Ca2+ +T70 CHEBI:29108 2567 2571 Ca2+ +T71 GO:0005737 2772 2783 cytoplasmic +T72 CHEBI:29108 2789 2793 Ca2+ +T73 GO:0065007 2902 2912 regulation +T74 GO:0065007 2977 2987 regulating +T75 CL:0000540 3232 3240 neuronal +T76 GO:0007049 3275 3285 cell cycle +T77 GO:0016020 3368 3376 membrane +T78 GO:0005856 3411 3423 cytoskeleton +T79 GO:0016477 3428 3442 cell migration +T80 GO:0010941 3455 3479 regulation of cell death +T81 GO:0006915 3502 3511 apoptosis +T82 GO:0008219 3637 3647 cell death +T83 CHEBI:82824 3716 3738 inhibitors of calpains +T84 GO:0010467 3850 3860 expression +T85 PR:000005060 3864 3875 calpastatin +T86 CHEBI:82824 3900 3912;3922 3929 inhibitor of ... calpain +T87 SO:0001060 4004 4011 isoform +T88 SO:0001060 4067 4074 isoform +T89 UBERON:0007376 4123 4132 epidermal +T90 PR:000006928 4123 4146 epidermal growth factor +T91 PR:000006928 4148 4151 EGF +T92 GO:0048870 4161 4174 cell motility +T93 PR:000006065 4211 4241 interferon-inducible protein 9 +T94 GO:0051546 4250 4276 migration of keratinocytes +T95 CL:0000312 4263 4276 keratinocytes +T96 CHEBI:82824 4300 4317 calpain inhibitor +T97 CHEBI:29917 4347 4352 thiol +T98 GO:0008283 4442 4455 proliferation +T99 GO:0010467 4493 4503 expression +T100 SO:0000704 4611 4615 gene +T101 NCBITaxon:10088 4628 4632 mice +T102 SO:0001060 4759 4766 isoform +T103 PR:000005022 4812 4817 Capn4 +T104 PR:000005022 4925 4930 Capn4 +T105 NCBITaxon:39107 4934 4940 murine +T106 UBERON:0000922 4941 4948 embryos +T107 GO:0016265 4949 4953 died +T108 GO:0007565 4980 4989 gestation +T109 UBERON:0000922 5064 5071 embryos +T110 PR:000005022 5078 5083 Capn4 +T111 NCBITaxon:39107 5087 5093 murine +T112 UBERON:0000922 5094 5103 embryonic +T113 CL:0000057 5104 5115 fibroblasts +T114 UBERON:0000922 5152 5159 embryos +T115 GO:0005925 5390 5405 focal adhesions +T116 PR:000005022 5436 5441 Capn4 +T117 SO:0000704 5495 5499 gene +T118 UBERON:0000922 5524 5533 embryonic +T119 GO:0007566 5565 5577 implantation +T120 UBERON:0000922 5613 5622 embryonic +T121 PR:000005022 5667 5672 Capn4 +T122 NCBITaxon:10088 5676 5680 mice +T123 GO:0007565 5817 5826 gestation +T124 PR:000005022 5854 5859 Capn4 +T125 NCBITaxon:10088 5863 5867 mice +T126 PR:000005009 5921 5926 Capn1 +T127 PR:000005009 5941 5968 μ-calpain catalytic subunit +T128 NCBITaxon:10088 6025 6029 mice +T129 CL:0000233 6075 6084 platelets +T130 PR:000005022 6155 6160 Capn4 +T131 NCBITaxon:10088 6166 6170 mice +T132 GO:0016265 6171 6174 die +T133 GO:0009790 6182 6195 embryogenesis +T134 PR:000005009 6306 6311 Capn1 +T135 NCBITaxon:10088 6322 6326 mice +T136 GO:0009790 6437 6450 embryogenesis +T137 PR:000005015 6606 6611 Capn2 +T138 SO:0000704 6612 6616 gene +T139 PR:000005015 6630 6644 m-80 k subunit +T140 NCBITaxon:10088 6648 6652 mice +T141 PR:000005015 6674 6679 Capn2 +T142 UBERON:0000922 6685 6692 embryos +T143 GO:0016265 6693 6697 died +T144 GO:0007566 6711 6723 implantation +T145 GO:0009790 6784 6797 embryogenesis +T146 GO:0010467 6852 6861 expressed +T147 UBERON:0000922 6865 6874 embryonic +T148 CL:0002322 6865 6879;6885 6890 embryonic stem ... cells +T149 CL:0002322 6881 6883;6885 6890 ES ... cells +T150 GO:0007565 6942 6951 gestation +T151 GO:0009790 7061 7074 embryogenesis +T152 PR:000005015 7120 7125 Capn2 +T153 CL:0002322 7135 7142 ES cell +T154 PR:000005015 7167 7172 Capn2 +T155 CL:0002322 7176 7183 ES cell +T156 CHEBI:23888 7252 7256 drug +T157 PR:000005015 7300 7305 Capn2 +T158 GO:0097617 7350 7363 hybridization +T159 GO:0097617 7443 7453 hybridized +T160 PR:P23940 7466 7471 BamHI +T161 SO:0001023 7498 7504 allele +T162 PR:P23940 7516 7521 BamHI +T163 SO:0001023 7545 7551 allele +T164 SO:0001026 7570 7577 genomic +T165 SO:0005853 7854 7862 cassette +T166 PR:P23940 7890 7895 BamHI +T167 PR:000005015 7908 7913 Capn2 +T168 CL:0002322 7917 7925 ES cells +T169 SO:0001644 7947 7963 targeting vector +T170 PR:000005015 7993 7998 Capn2 +T171 SO:0000028 8106 8108 bp +T172 SO:0000028 8121 8123 bp +T173 SO:0001023 8148 8154 allele +T174 SO:0000028 8166 8168 bp +T175 CL:0002322 8278 8285 ES cell +T176 PR:000005015 8324 8329 Capn2 +T177 PR:P23940 8409 8414 BamHI +T178 SO:0001026 8424 8431 genomic +T179 CL:0002322 8451 8459 ES cells +T180 GO:0097617 8464 8474 hybridized +T181 CHEBI:42098 8482 8485 DIG +T182 SO:0000028 8498 8500 bp +T183 PR:P23940 8501 8506 BamHI +T184 PR:P43870 8507 8514 HindIII +T185 SO:0001644 8577 8593 targeting vector +T186 PR:P23940 8615 8620 BamHI +T187 SO:0001023 8661 8667 allele +T188 SO:0001023 8736 8742 allele +T189 SO:0000028 8908 8910 bp +T190 SO:0001023 8938 8944 allele +T191 SO:0000028 8956 8958 bp +T192 SO:0001023 8983 8989 allele +T193 SO:0000112 9026 9032 primer +T194 SO:0000188 9044 9050 intron +T195 SO:0001644 9083 9099 targeting vector +T196 SO:0001023 9114 9120 allele +T197 SO:0000077 9130 9139 antisense +T198 SO:0000112 9140 9147 primers +T199 SO:0001023 9186 9192 allele +T200 SO:0000077 9201 9210 antisense +T201 SO:0000112 9211 9217 primer +T202 SO:0000147 9229 9233 exon +T203 SO:0000077 9300 9309 antisense +T204 SO:0000112 9310 9316 primer +T205 SO:0005853 9332 9340 cassette +T206 SO:0001023 9392 9398 allele +T207 SO:0001023 9432 9438 allele +T208 NCBITaxon:10088 9557 9561 mice +T209 PR:000005015 9602 9607 Capn2 +T210 SO:0001023 9608 9614 allele +T211 NCBITaxon:10088 9636 9640 mice +T212 UBERON:0000085 9658 9664 morula +T213 PR:000005015 9699 9704 Capn2 +T214 PR:000005015 9759 9764 Capn2 +T215 PR:000005015 9840 9845 Capn2 +T216 NCBITaxon:33208 9849 9856 animals +T217 UBERON:0001062 9907 9914 anatomy +T218 GO:0000003 9916 9928 reproduction +T219 UBERON:0000104 9933 9942 life span +T220 PR:000005015 10001 10006 Capn2 +T221 UBERON:0012101 10076 10085 perinatal +T222 GO:0007567 10080 10085 natal +T223 GO:0016265 10086 10091 death +T224 PR:000005015 10100 10105 Capn2 +T225 PR:000005015 10155 10160 Capn2 +T226 NCBITaxon:33208 10164 10171 animals +T227 GO:0016265 10172 10180 perished +T228 UBERON:0000922 10202 10211 embryonic +T229 GO:0009790 10202 10223 embryonic development +T230 UBERON:0000922 10255 10264 embryonic +T231 GO:0016265 10265 10270 death +T232 GO:0007566 10290 10302 implantation +T233 UBERON:0000922 10310 10317 embryos +T234 PR:000005015 10395 10400 Capn2 +T235 UBERON:0000922 10404 10411 embryos +T236 UBERON:0000922 10442 10448 embryo +T237 PR:000005015 10509 10514 Capn2 +T238 UBERON:0000922 10518 10525 embryos +T239 GO:0016265 10535 10540 dying +T240 GO:0007566 10550 10562 implantation +T241 UBERON:0000922 10564 10571 Embryos +T242 UBERON:0000993 10599 10607 oviducts +T243 GO:0007565 10611 10619 pregnant +T244 GO:0007566 10742 10754 implantation +T245 UBERON:0000922 10755 10762 embryos +T246 PR:000005015 10768 10773 Capn2 +T247 PR:000005015 10813 10818 Capn2 +T248 UBERON:0000922 10822 10829 embryos +T249 UBERON:0019252 10851 10863 8-cell stage +T250 UBERON:0000358 10957 10967 blastocyst +T251 UBERON:0000922 10975 10982 embryos +T252 PR:000005015 10988 10993 Capn2 +T253 PR:000005015 11014 11019 Capn2 +T254 UBERON:0000922 11030 11037 embryos +T255 UBERON:0019252 11055 11067 8-cell stage +T256 UBERON:0019248 11155 11168 early embryos +T257 SO:0000673 11247 11257 transcript +T258 PR:000005015 11306 11311 Capn2 +T259 UBERON:0000922 11315 11322 embryos +T260 UBERON:0000085 11341 11347 morula +T261 PR:000005015 11413 11418 Capn2 +T262 NCBITaxon:10088 11430 11434 mice +T263 GO:0007566 11511 11523 implantation +T264 UBERON:0000922 11524 11531 embryos +T265 SO:0000704 11568 11575 genetic +T266 GO:0007566 11647 11659 implantation +T267 UBERON:0000922 11660 11667 embryos +T268 SO:0000028 11715 11717 bp +T269 SO:0001023 11746 11752 allele +T270 SO:0000028 11763 11765 bp +T271 SO:0001023 11790 11796 allele +T272 SO:0001644 11858 11874 targeting vector +T273 SO:0000028 11901 11903 bp +T274 SO:0000112 12029 12036 primers +T275 SO:0000077 12112 12121 antisense +T276 SO:0000112 12122 12129 primers +T277 SO:0001023 12135 12141 allele +T278 SO:0000112 12156 12163 primers +T279 SO:0000147 12258 12263 Exons +T280 SO:0005853 12322 12330 cassette +T281 SO:0001023 12544 12550 allele +T282 SO:0005853 12583 12591 cassette +T283 SO:0001023 12606 12612 allele +T284 GO:0007566 12643 12655 implantation +T285 UBERON:0000922 12656 12663 embryos +T286 UBERON:0000922 12708 12715 embryos +T287 GO:0007566 12725 12737 implantation +T288 PR:000005015 12764 12769 Capn2 +T289 NCBITaxon:10088 12773 12777 mice +T290 GO:0007618 12783 12788 mated +T291 GO:0009566 12805 12818 fertilization +T292 UBERON:0010148 12852 12865 vaginal plugs +T293 UBERON:0000358 12867 12877 Blastocyst +T294 UBERON:0019252 12888 12902 8-cell embryos +T295 UBERON:0000993 12932 12940 oviducts +T296 SO:0001023 13061 13068 alleles +T297 SO:0001644 13165 13181 targeting vector +T298 SO:0001023 13206 13213 alleles +T299 SO:0000028 13243 13245 bp +T300 SO:0001023 13264 13270 allele +T301 SO:0000028 13276 13278 bp +T302 SO:0001023 13294 13300 allele +T303 SO:0000028 13310 13312 bp +T304 UBERON:0000358 13389 13399 blastocyst +T305 UBERON:0000922 13406 13413 embryos +T306 UBERON:0000922 13415 13422 Embryos +T307 PR:000005015 13447 13452 Capn2 +T308 UBERON:0000922 13464 13471 embryos +T309 PR:000005015 13487 13492 Capn2 +T310 SO:0000028 13531 13533 bp +T311 UBERON:0019252 13585 13599 8-cell embryos +T312 UBERON:0000922 13610 13617 Embryos +T313 PR:000005015 13642 13647 Capn2 +T314 UBERON:0000922 13657 13663 embryo +T315 PR:000005015 13671 13676 Capn2 +T316 SO:0000028 13714 13716 bp +T317 GO:0007566 13820 13832 implantation +T318 GO:0007566 13842 13854 implantation +T319 UBERON:0000922 13855 13862 embryos +T320 PR:000005015 13909 13914 Capn2 +T321 PR:000005015 13921 13926 Capn2 +T322 NCBITaxon:33208 13929 13936 animals +T323 NCBITaxon:33208 14056 14063 animals +T324 PR:000005015 14116 14121 Capn2 +T325 PR:000005015 14149 14154 Capn2 +T326 NCBITaxon:33208 14158 14165 animals +T327 PR:000005015 14194 14199 Capn2 +T328 PR:000005015 14206 14211 Capn2 +T329 NCBITaxon:33208 14215 14222 animals +T330 PR:000005015 14464 14469 Capn2 +T331 NCBITaxon:33208 14473 14480 animals +T332 GO:0007566 14511 14523 implantation +T333 GO:0007566 14532 14544 implantation +T334 UBERON:0000922 14545 14552 embryos +T335 NCBITaxon:33208 14625 14632 animals +T336 PR:000005022 14665 14670 Capn4 +T337 PR:000005015 14758 14763 Capn2 +T338 PR:000005015 14771 14776 Capn2 +T339 NCBITaxon:33208 14780 14787 animals +T340 PR:000005015 14843 14848 Capn2 +T341 NCBITaxon:33208 14852 14859 animals +T342 PR:000005015 14896 14901 Capn2 +T343 SO:0001023 14950 14956 allele +T344 PR:000005015 15058 15063 Capn2 +T345 PR:000005015 15070 15075 Capn2 +T346 NCBITaxon:33208 15079 15086 animals +T347 PR:000005015 15205 15210 Capn2 +T348 SO:0001023 15230 15236 allele +T349 PR:000005015 15300 15305 Capn2 +T350 SO:0001023 15325 15331 allele +T351 GO:0048468 15365 15378;15391 15396 generation of ... cells +T352 PR:000005015 15379 15384 Capn2 +T353 CL:0002322 15388 15396 ES cells +T354 PR:000005015 15398 15403 Capn2 +T355 CL:0002322 15407 15415 ES cells +T356 CHEBI:42768 15478 15482 G418 +T357 SO:0000704 15534 15538 gene +T358 GO:0035822 15534 15549 gene conversion +T359 CL:0002322 15607 15615 ES cells +T360 PR:000005022 15656 15661 Capn4 +T361 CL:0002322 15665 15673 ES cells +T362 PR:000005015 15706 15711 Capn2 +T363 CL:0002322 15715 15723 ES cells +T364 CHEBI:23888 15756 15760 drug +T365 PR:000005015 15804 15809 Capn2 +T366 CL:0002322 15813 15821 ES cells +T367 PR:000005015 15854 15859 Capn2 +T368 UBERON:0000922 15863 15870 embryos +T369 UBERON:0019252 15882 15894 8-cell stage +T370 CL:0002322 15999 16006 ES cell +T371 CHEBI:35222 16232 16242 inhibitors +T372 SO:0001060 16441 16449 isoforms +T373 GO:0065007 16475 16484 regulated +T374 SO:0000704 16527 16531 Gene +T375 NCBITaxon:10088 16545 16549 mice +T376 SO:0001060 16638 16646 isoforms +T377 PR:000005022 16683 16688 Capn4 +T378 SO:0000704 16689 16693 gene +T379 GO:0065007 16714 16724 regulatory +T380 UBERON:0000922 16804 16813 embryonic +T381 PR:000005022 16827 16832 Capn4 +T382 NCBITaxon:10088 16842 16846 mice +T383 UBERON:0000922 17082 17091 embryonic +T384 GO:0009790 17082 17103 embryonic development +T385 SO:0001060 17113 17121 isoforms +T386 SO:0001060 17250 17257 isoform +T387 PR:000005009 17340 17345 Capn1 +T388 NCBITaxon:10088 17349 17353 mice +T389 PR:000005009 17370 17397 μ-calpain catalytic subunit +T390 CL:0000233 17434 17442 platelet +T391 GO:0070527 17434 17454 platelet aggregation +T392 GO:0009790 17618 17631 embryogenesis +T393 SO:0001060 17648 17655 isoform +T394 UBERON:0000922 17692 17701 embryonic +T395 PR:000005015 17734 17739 Capn2 +T396 NCBITaxon:10088 17743 17747 mice +T397 PR:000005015 17764 17791 m-calpain catalytic subunit +T398 GO:0016265 17793 17796 die +T399 GO:0007566 17807 17819 implantation +T400 GO:0009790 17932 17945 embryogenesis +T401 PR:000005022 17982 17987 Capn4 +T402 SO:0000704 17988 17992 gene +T403 PR:000005022 18117 18122 Capn4 +T404 GO:0007566 18150 18162 implantation +T405 UBERON:0000922 18181 18188 embryos +T406 PR:000005022 18229 18234 Capn4 +T407 UBERON:0000922 18238 18245 embryos +T408 GO:0007565 18319 18328 gestation +T409 PR:000005022 18479 18484 Capn4 +T410 PR:000005022 18580 18601 calpain small subunit +T411 SO:0005853 18630 18638 cassette +T412 SO:0000147 18658 18662 exon +T413 GO:0010467 18895 18904 expressed +T414 PR:000005015 18914 18937 m-calpain large subunit +T415 NCBITaxon:562 18941 18948 E. coli +T416 PR:000005022 18972 18977 Capn4 +T417 SO:0000147 19077 19082 exons +T418 PR:000005022 19190 19195 Capn4 +T419 SO:0001023 19269 19275 allele +T420 SO:0001023 19334 19340 allele +T421 SO:0000704 19455 19459 gene +T422 PR:000010445 19569 19591 mixed lineage leukemia +T423 http://purl.obolibrary.org/obo/MONDO_0005059 19583 19591 leukemia +T424 PR:000010445 19593 19596 Mll +T425 SO:0000704 19598 19602 gene +T426 UBERON:0000922 19642 19649 embryos +T427 GO:0016265 19650 19658 perished +T428 GO:0009790 19666 19679 embryogenesis +T429 SO:0001023 19866 19873 alleles +T430 PR:000005022 19920 19925 Capn4 +T431 SO:0000673 19972 19983 transcripts +T432 PR:000005022 20030 20035 Capn4 +T433 SO:0001023 20036 20042 allele +T434 SO:0001023 20049 20055 allele +T435 SO:0000147 20153 20157 exon +T436 SO:0000976 20186 20193 cryptic +T437 GO:0008380 20194 20200 splice +T438 SO:0000162 20194 20206 splice sites +T439 SO:0000167 20218 20226 promoter +T440 SO:0000673 20248 20259 transcripts +T441 GO:0010467 20434 20444 expression +T442 GO:0010467 20550 20559 expressed +T443 NCBITaxon:562 20563 20570 E. coli +T444 NCBITaxon:40674 20604 20613 mammalian +T445 NCBITaxon:2759 20767 20777 eukaryotic +T446 CL:0000255 20767 20783 eukaryotic cells +T447 GO:0010467 20944 20953 expressed +T448 NCBITaxon:562 20970 20977 E. coli +T449 NCBITaxon:40674 20984 20993 mammalian +T450 PR:000005022 21047 21052 Capn4 +T451 SO:0000704 21108 21115 genetic +T452 NCBITaxon:10088 21222 21226 mice +T453 SO:0001060 21345 21353 isoforms +T454 MOP:0000780 21521 21527 cleave +T455 CHEBI:29108 21717 21721 Ca2+ +T456 SO:0001060 21775 21783 isoforms +T457 GO:0065007 21803 21812 regulated +T458 SO:0000704 21853 21857 gene +T459 NCBITaxon:10088 21881 21885 mice +T460 GO:0009790 21965 21978 embryogenesis +T461 PR:000005015 21998 22003 Capn2 +T462 NCBITaxon:39107 22009 22015 murine +T463 UBERON:0000922 22016 22023 embryos +T464 GO:0016265 22024 22027 die +T465 GO:0007566 22037 22049 implantation +T466 PR:000005009 22080 22103 μ-calpain large subunit +T467 SO:0000704 22104 22108 gene +T468 PR:000005009 22110 22115 Capn1 +T469 NCBITaxon:10088 22149 22153 mice +T470 PR:000005009 22208 22213 Capn1 +T471 CL:0000233 22251 22259 platelet +T472 GO:0006468 22308 22326;22335 22343 phosphorylation of ... proteins +T473 CL:0000233 22356 22364 platelet +T474 GO:0030168 22356 22375 platelet activation +T475 NCBITaxon:40674 22430 22439 mammalian +T476 CL:0000233 22587 22596 Platelets +T477 CL:0000232 22601 22613 erythrocytes +T478 CL:0000233 22777 22786 platelets +T479 CL:0000232 22791 22803 erythrocytes +T480 PR:000005009 22918 22923 Capn1 +T481 NCBITaxon:33208 22927 22934 animals +T482 PR:000005015 23094 23099 Capn2 +T483 UBERON:0000922 23103 23110 embryos +T484 UBERON:0000922 23165 23174 embryonic +T485 GO:0009790 23165 23186 embryonic development +T486 UBERON:0019252 23198 23210 8-cell stage +T487 GO:0007566 23325 23337 implantation +T488 PR:000005015 23351 23356 Capn2 +T489 UBERON:0000922 23360 23367 embryos +T490 UBERON:0000922 23420 23427 embryos +T491 UBERON:0019252 23446 23458 8-cell stage +T492 GO:0007566 23579 23591 implantation +T493 UBERON:0000922 23592 23599 embryos +T494 PR:000005015 23613 23618 Capn2 +T495 GO:0007566 23649 23661 implantation +T496 SO:0000704 23687 23691 gene +T497 UBERON:0000922 23796 23805 embryonic +T498 UBERON:0000922 23885 23892 embryos +T499 UBERON:0000085 23931 23937 morula +T500 UBERON:0000922 23977 23986 embryonic +T501 SO:0000704 24044 24048 gene +T502 UBERON:0019248 24123 24135 early embryo +T503 UBERON:0000922 24183 24190 embryos +T504 CL:0002322 24231 24239 ES cells +T505 PR:000005015 24267 24272 Capn2 +T506 PR:000005015 24412 24417 Capn2 +T507 UBERON:0000922 24421 24428 embryos +T508 UBERON:0019252 24436 24448 8-cell stage +T509 GO:0051301 24546 24560 cell divisions +T510 GO:0008283 24708 24726 cell proliferation +T511 GO:0000070 24793 24830 chromosome segregation during mitosis +T512 GO:0045132 24793 24815;24847 24861 chromosome segregation ... during meiosis +T513 PR:000005022 24902 24907 Capn4 +T514 UBERON:0000922 24949 24958 embryonic +T515 GO:0009790 24949 24970 embryonic development +T516 PR:000005022 25041 25046 Capn4 +T517 PR:000005022 25243 25248 Capn4 +T518 GO:0008283 25289 25307 cell proliferation +T519 GO:0051318 25412 25420 G1 stage +T520 GO:0007049 25428 25438 cell cycle +T521 GO:0007049 25502 25512 cell cycle +T522 PR:000003035 25534 25537 p53 +T523 PR:000000118 25539 25543 p107 +T524 PR:000005121 25545 25554 cyclin D1 +T525 PR:000003090 25560 25567 p27kip1 +T526 CL:0000019 25611 25616 Sperm +T527 CL:0000023 25632 25638 oocyte +T528 GO:0005737 25658 25669 cytoplasmic +T529 GO:0001669 25697 25705 acrosome +T530 GO:0007340 25697 25714 acrosome reaction +T531 NCBITaxon:9989 25774 25780 rodent +T532 CL:0000019 25781 25786 sperm +T533 CL:0000023 25796 25803 oocytes +T534 GO:0001669 25842 25850 acrosome +T535 GO:0007340 25842 25859 acrosome reaction +T536 GO:0005938 25923 25931 cortical +T537 GO:0016020 25932 25940 membrane +T538 CL:0000023 25944 25951 oocytes +T539 GO:0060473 25997 26013 cortical granule +T540 CL:0000023 26094 26100 oocyte +T541 GO:0007126 26101 26108 meiotic +T542 GO:0072687 26101 26116 meiotic spindle +T543 GO:0009566 26123 26136 fertilization +T544 GO:0007059 26167 26189 chromosome segregation +T545 GO:0007059 26220 26242 chromosome segregation +T546 UBERON:0019248 26277 26292 early embryonic +T547 GO:0009790 26283 26304 embryonic development +T548 PR:000005022 26339 26344 Capn4 +T549 CL:0002322 26348 26350;26359 26364 ES ... cells +T550 SO:0001023 26398 26404 allele +T551 PR:000005022 26736 26741 Capn4 +T552 SO:0001023 26742 26748 allele +T553 UBERON:0019248 26940 26955 early embryonic +T554 CL:0000007 26940 26961 early embryonic cells +T555 SO:0000704 26996 27000 gene +T556 GO:0035822 26996 27011 gene conversion +T557 PR:000005015 27015 27020 Capn2 +T558 PR:000005015 27027 27032 Capn2 +T559 CL:0002322 27036 27044 ES cells +T560 CHEBI:42768 27094 27098 G418 +T561 CL:0002322 27199 27207 ES cells +T562 GO:0010467 27234 27241 express +T563 PR:000005015 27246 27260 m-80 k subunit +T564 PR:000005015 27268 27273 Capn2 +T565 SO:0000902 27286 27295 transgene +T566 SO:0000704 27305 27309 gene +T567 GO:0035822 27305 27320 gene conversion +T568 SO:0001023 27414 27420 allele +T569 GO:0010467 27470 27480 expression +T570 SO:0000902 27495 27504 transgene +T571 PR:000005015 27673 27678 Capn2 +T572 PR:000005015 27693 27698 Capn2 +T573 NCBITaxon:10088 27828 27832 mice +T574 PR:000005022 27972 27977 Capn4 +T575 NCBITaxon:33208 28159 28166 animals +T576 GO:0010467 28350 28360 expression +T577 SO:0001060 28566 28574 isoforms +T578 NCBITaxon:39107 28667 28673 murine +T579 GO:0009790 28674 28687 embryogenesis +T580 GO:0007566 28713 28725 implantation +T581 SO:0001060 28892 28900 isoforms +T582 GO:0007566 28982 28994 implantation +T583 GO:0009790 28995 29008 embryogenesis +T584 UBERON:0007023 29016 29021 adult +T585 NCBITaxon:10088 29022 29026 mice +T586 SO:0000704 29101 29105 gene +T587 NCBITaxon:10088 29166 29171 mouse +T588 PR:000005015 29172 29177 Capn2 +T589 NCBITaxon:10088 29189 29194 mouse +T590 PR:000005015 29195 29200 Capn2 +T591 SO:0000704 29201 29205 gene +T592 PR:000005015 29241 29264 m-calpain large subunit +T593 PR:000005015 29266 29272 m-80 k +T594 SO:0000147 29293 29298 exons +T595 SO:0000317 29339 29349 cDNA clone +T596 NCBITaxon:10088 29376 29381 mouse +T597 PR:000005015 29382 29396 m-80 k subunit +T598 SO:0000319 29560 29570 stop codon +T599 SO:0000204 29604 29610 3'-UTR +T600 SO:0000551 29618 29630 polyA signal +T601 NCBITaxon:10088 29697 29702 mouse +T602 SO:0001026 29703 29710 genomic +T603 SO:0000754 29722 29723 λ +T604 SO:0000040 29745 29759 genomic clones +T605 SO:0000028 29790 29792 bp +T606 PR:P23940 29811 29816 BamHI +T607 SO:0001014 29817 29831 site in intron +T608 PR:P23940 29839 29844 BamHI +T609 SO:0001014 29845 29859 site in intron +T610 SO:0000730 30085 30089 gaps +T611 PR:000005015 30245 30250 Capn2 +T612 SO:0001644 30251 30267 targeting vector +T613 SO:0000028 30321 30323 bp +T614 PR:P23940 30324 30329 BamHI +T615 PR:P43870 30330 30337 HindIII +T616 SO:0000147 30359 30363 exon +T617 PR:000005015 30373 30378 Capn2 +T618 SO:0000704 30379 30383 gene +T619 SO:0005853 30402 30410 cassette +T620 SO:0000147 30423 30427 Exon +T621 PR:P43870 30612 30619 HindIII +T622 PR:P23940 30620 30625 BamHI +T623 SO:0000147 30647 30652 exons +T624 SO:0000440 30695 30701 vector +T625 SO:0005853 30726 30734 cassette +T626 PR:P23940 30761 30766 BamHI +T627 PR:P23940 30835 30840 BamHI +T628 SO:0001023 30860 30866 allele +T629 PR:P23940 30907 30912 BamHI +T630 SO:0005853 30947 30955 cassette +T631 SO:0001023 31018 31025 alleles +T632 PR:P43870 31109 31116 HindIII +T633 SO:0000188 31147 31153 intron +T634 SO:0000188 31159 31165 intron +T635 SO:0005853 31225 31234 cassettes +T636 NCBITaxon:39107 31288 31294 murine +T637 PR:000005015 31295 31300 Capn2 +T638 SO:0000704 31301 31305 gene +T639 NCBITaxon:39107 31311 31317 murine +T640 PR:000005015 31318 31323 Capn2 +T641 SO:0000704 31324 31328 gene +T642 PR:000005015 31343 31357 m-80 k subunit +T643 CL:0002322 31376 31384 ES cells +T644 SO:0001023 31446 31452 allele +T645 SO:0001644 31464 31480 targeting vector +T646 SO:0001023 31506 31512 allele +T647 SO:0001023 31550 31556 allele +T648 SO:0005853 31568 31576 cassette +T649 SO:0001026 31595 31602 genomic +T650 SO:0000147 31623 31627 exon +T651 SO:0100019 31665 31676 active site +T652 SO:0001644 31712 31728 targeting vector +T653 SO:0005853 31742 31750 cassette +T654 SO:0000357 31754 31761 flanked +T655 PR:000005015 31775 31780 Capn2 +T656 PR:P23940 31952 31957 BamHI +T657 SO:0001023 31986 31992 allele +T658 PR:P23940 32006 32011 BamHI +T659 SO:0001023 32037 32043 allele +T660 SO:0000147 32045 32050 Exons +T661 SO:0000147 32103 32107 exon +T662 SO:0000112 32293 32300 primers +T663 CL:0002322 32337 32344 ES cell +T664 GO:0009294 32354 32366 transfection +T665 CL:0002322 32410 32418 ES cells +T666 CHEBI:5291 32443 32450 gelatin +T667 NCBITaxon:10088 32487 32492 mouse +T668 UBERON:0000922 32493 32502 embryonic +T669 CL:0000057 32503 32514 fibroblasts +T670 CHEBI:16526 32532 32535 CO2 +T671 CL:0002322 32539 32546 ES cell +T672 CHEBI:17234 32566 32573 glucose +T673 NCBITaxon:27592 32603 32609 bovine +T674 UBERON:0001977 32610 32615 serum +T675 CHEBI:50144 32672 32687 sodium pyruvate +T676 CHEBI:41218 32696 32713 2-mercaptoethanol +T677 CHEBI:17334 32724 32734 penicillin +T678 CHEBI:17076 32746 32758 streptomycin +T679 CHEBI:2682 32769 32781 amphotericin +T680 NCBITaxon:27592 32826 32832 bovine +T681 UBERON:0001977 32833 32838 serum +T682 CL:0002322 32921 32928 ES cell +T683 CHEBI:5291 32937 32944 Gelatin +T684 UBERON:0000479 33034 33040 tissue +T685 CHEBI:33893 33049 33057 reagents +T686 CL:0002322 33164 33172 ES cells +T687 CHEBI:5291 33217 33224 gelatin +T688 GO:0009294 33243 33254 transformed +T689 CHEBI:42768 33305 33309 G418 +T690 CHEBI:465284 33332 33343 ganciclovir +T691 CHEBI:23888 33375 33379 Drug +T692 CHEBI:5291 33422 33429 gelatin +T693 NCBITaxon:10088 33534 33538 mice +T694 PR:000005015 33540 33545 Capn2 +T695 CL:0002322 33549 33557 ES cells +T696 UBERON:0019252 33589 33603 8-cell embryos +T697 GO:0007618 33623 33630 matings +T698 UBERON:0000358 33679 33690 blastocysts +T699 NCBITaxon:33208 33748 33755 animals +T700 GO:0007567 33777 33782 birth +T701 UBERON:0000970 33792 33795 eye +T702 UBERON:0010166 33847 33851 coat +T703 SO:0000704 34040 34047 genetic +T704 NCBITaxon:10088 34060 34065 Mouse +T705 NCBITaxon:33208 34116 34122 Animal +T706 NCBITaxon:33208 34193 34199 Animal +T707 PR:000005015 34325 34330 Capn2 +T708 CHEBI:42098 34382 34393 digoxigenin +T709 CHEBI:42098 34395 34398 DIG +T710 PR:P23940 34475 34480 BamHI +T711 SO:0001026 34490 34497 genomic +T712 GO:0097617 34506 34516 hybridized +T713 CHEBI:42098 34524 34527 DIG +T714 SO:0000028 34540 34542 bp +T715 SO:0000147 34543 34547 exon +T716 PR:P23940 34561 34566 BamHI +T717 PR:P43870 34567 34574 HindIII +T718 SO:0000028 34660 34662 bp +T719 SO:0005853 34699 34707 cassette +T720 SO:0001026 34864 34871 genomic +T721 SO:0000112 34914 34921 primers +T722 CL:0002322 35002 35010 ES cells +T723 GO:0007566 35033 35045 implantation +T724 UBERON:0000922 35046 35053 embryos +T725 SO:0000028 35088 35090 bp +T726 SO:0001023 35116 35122 allele +T727 SO:0000028 35135 35137 bp +T728 SO:0001023 35160 35166 allele +T729 SO:0000188 35220 35226 intron +T730 SO:0000112 35236 35242 primer +T731 SO:0000077 35300 35309 antisense +T732 SO:0000112 35310 35317 primers +T733 GO:0097617 35324 35334 hybridized +T734 SO:0000147 35356 35360 exon +T735 GO:0097617 35548 35557 annealing +T736 SO:0000112 35667 35674 primers +T737 PR:000005015 35696 35701 Capn2 +T738 SO:0000704 35738 35745 genetic +T739 GO:0007566 35772 35784 implantation +T740 UBERON:0000922 35785 35792 embryos +T741 UBERON:0000922 35889 35896 embryos +T742 SO:0001023 36065 36071 allele +T743 SO:0001023 36131 36137 allele +T744 SO:0001023 36208 36214 allele +T745 SO:0000188 36277 36283 intron +T746 SO:0000112 36292 36298 primer +T747 SO:0000077 36306 36315 antisense +T748 SO:0000112 36316 36322 primer +T749 SO:0000188 36334 36340 intron +T750 SO:0000188 36441 36447 intron +T751 SO:0000112 36456 36462 primer +T752 SO:0000077 36470 36479 antisense +T753 SO:0000112 36480 36486 primer +T754 SO:0000147 36490 36494 exon +T755 SO:0001023 36537 36543 allele +T756 SO:0000112 36581 36587 primer +T757 SO:0000188 36610 36616 intron +T758 SO:0000112 36625 36631 primer +T759 SO:0000077 36639 36648 antisense +T760 SO:0000112 36649 36655 primer +T761 SO:0005853 36671 36679 cassette +T762 SO:0000112 36692 36698 primer +T763 SO:0000188 36732 36738 intron +T764 SO:0000112 36747 36753 primer +T765 SO:0000077 36761 36770 antisense +T766 SO:0000112 36771 36777 primer +T767 SO:0000112 36900 36907 primers +T768 SO:0001026 37046 37053 genomic +T769 SO:0001644 37089 37105 targeting vector +T770 SO:0001023 37157 37164 alleles +T771 SO:0000112 37176 37182 primer +T772 SO:0000188 37225 37231 intron +T773 SO:0000112 37240 37246 primer +T774 SO:0000188 37254 37260 intron +T775 SO:0000077 37263 37272 antisense +T776 SO:0000112 37273 37279 primer +T777 SO:0000112 37292 37298 primer +T778 SO:0000147 37317 37321 exon +T779 SO:0000112 37330 37336 primer +T780 SO:0000188 37350 37356 intron +T781 SO:0000077 37359 37368 antisense +T782 SO:0000112 37369 37375 primer +T783 SO:0000028 37405 37407 bp +T784 SO:0000028 37433 37435 bp +T785 SO:0000028 37467 37469 bp +T786 GO:0097617 37664 37673 annealing +T787 GO:0007566 37779 37791 implantation +T788 UBERON:0000922 37792 37799 embryos +T789 GO:0009566 37809 37822 fertilization +T790 GO:0007620 37856 37866 copulation +T791 UBERON:0010148 37856 37872 copulation plugs +T792 GO:0007566 37920 37932 implantation +T793 UBERON:0000922 37933 37940 embryos +T794 UBERON:0000995 37973 37978 uteri +T795 GO:0007565 37986 37994 pregnant +T796 UBERON:0000993 38036 38044 oviducts +T797 CL:0002322 38050 38057 ES cell +T798 UBERON:0000922 38075 38082 embryos +T799 GO:0019835 38137 38142 lysis +T800 CHEBI:9754 38157 38161 Tris +T801 CHEBI:17883 38162 38165 HCl +T802 CHEBI:9750 38172 38184 Triton X-100 +T803 PR:000006928 38350 38353 EGF +T804 UBERON:0007376 38355 38364 Epidermal +T805 PR:000006928 38355 38378 Epidermal growth factor +T806 UBERON:0000922 38384 38393 Embryonic +T807 NCBITaxon:10088 38405 38410 Mouse +T808 UBERON:0000922 38411 38420 embryonic +T809 CL:0000057 38421 38431 fibroblast +T810 SO:0000028 38464 38466 bp +T811 SO:0000028 38468 38477 base pair +T812 PR:000005015 38521 38526 Capn2 +T813 CL:0002322 38575 38577 ES +T814 NCBITaxon:33208 38693 38700 animals +T815 NCBITaxon:10088 38746 38751 mouse +T816 PR:000005015 39068 39073 Capn2 +T817 SO:0001026 39098 39105 Genomic +T818 NCBITaxon:10088 39129 39134 mouse +T819 UBERON:0002415 39135 39139 tail +T820 PR:P23940 39255 39260 BamHI +T821 SO:0001023 39289 39295 allele +T822 NCBITaxon:33208 39316 39323 animals +T823 PR:P23940 39339 39344 BamHI +T824 SO:0001023 39370 39376 allele +T825 PR:000005015 39417 39422 Capn2 +T826 GO:0007566 39474 39486 implantation +T827 UBERON:0000922 39487 39494 embryos +T828 UBERON:0000922 39505 39511 embryo +T829 PR:000005015 39567 39572 Capn2 +T830 UBERON:0000922 39576 39583 embryos +T831 GO:0016265 39584 39592 perished +T832 GO:0007566 39602 39614 implantation +T833 CL:0002322 39697 39704 ES cell +T834 NCBITaxon:10088 39740 39745 mouse +T835 SO:0001026 39746 39753 genomic +T836 SO:0000440 39815 39821 vector +T837 PR:000005015 39842 39847 Capn2 +T838 SO:0000440 39852 39858 vector +T839 UBERON:0000948 40123 40128 Heart +T840 http://purl.obolibrary.org/obo/MONDO_0005098 40133 40139 Stroke diff --git a/src/ontogpt/evaluation/craft/database/all/16433929.txt b/src/ontogpt/evaluation/craft/database/all/16433929.txt new file mode 100644 index 000000000..6ae448d78 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16433929.txt @@ -0,0 +1,153 @@ +m-Calpain is required for preimplantation embryonic development in mice + +Abstract + +Background + +μ-calpain and m-calpain are ubiquitously expressed proteases implicated in cellular migration, cell cycle progression, degenerative processes and cell death. These heterodimeric enzymes are composed of distinct catalytic subunits, encoded by Capn1 (μ-calpain) or Capn2 (m-calpain), and a common regulatory subunit encoded by Capn4. Disruption of the mouse Capn4 gene abolished both μ-calpain and m-calpain activity, and resulted in embryonic lethality, thereby suggesting essential roles for one or both of these enzymes during mammalian embryogenesis. Disruption of the Capn1 gene produced viable, fertile mice implying that either m-calpain could compensate for the loss of μ-calpain, or that the loss of m-calpain was responsible for death of Capn4-/- mice. + +Results + +To distinguish between the alternatives described above, we deleted an essential coding region in the mouse Capn2 gene in embryonic stems cells and transmitted this mutant allele through the mouse germline. Breeding of heterozygous animals failed to produce homozygous mutant live offspring or implanted embryos. A nested PCR genotyping protocol was established, and homozygous preimplantation mutant embryos were detected at the morula but not at the blastocyts stage. + +Conclusion + +We conclude that homozygous disruption of the Capn2 gene results in pre-implantation embryonic lethality between the morula and blastocyst stage. This establishes that μ-calpain and m-calpain have distinct functions, and that m-calpain is vital for development of the preimplantation murine embryo. + +Background + +The two ubiquitous Ca2+-dependent, cysteine proteases known as μ-calpain (calpain-1) and m-calpain (capain-2), are the founding members of a gene family comprising 13 genes in mammals [1-3]. Both are heterodimeric enzymes consisting of distinct 80 kDa catalytic subunits, encoded by the Capn1 (μ-80 k) and Capn2 (m-80 k) genes, respectively, that associate with a common 28 kDa regulatory subunit encoded by the Capn4 gene. The μ-80 k and m-80 k subunits share 62% amino acid sequence identity, and are very similar in terms of structure, protein chemistry, and in vitro substrate specificity. Despite these similarities, the differential expression patterns of μ- and m-calpain in mammalian tissues suggest they have some isoform specific and distinct functions. The μ and m designations derive from the levels of Ca2+ required in vitro for optimal activation; 10–50 μM Ca2+ for μ-calpain and 0.3–0.35 mM Ca2+ for m-calpain. It is generally assumed that μ- and m-calpain maintain their differential sensitivities to calcium in vivo, although this has not yet been strictly demonstrated. Furthermore, since the cytoplasmic free Ca2+ concentration is typically less than 1 μM, it is also assumed that other in vivo factors must contribute to regulation of these enzymes [3]. + +Without apriori knowledge of the factors regulating calpain activity or their relevant substrates, elucidation of biological functions for calpains presents a challenge. Research on calpains has linked them with a wide variety of functions including muscle growth, development, degeneration (3), neuronal growth and neurodegeneration [4], cell cycle progression [5,6], signal cascades triggered by integrins and growth factors [7], membrane protrusion [8], remodeling of the cytoskeleton and cell migration [9-15], and regulation of cell death via both necrosis and apoptosis [16-22]. To date, the literature suggests a complex interplay between caspases and calpains [23,24] and impact of calpain on cell death pathway components [25]. The lack of highly specific cell-permeable inhibitors of calpains contributes to the challenge of investigating and defining calpain functions in these processes. Although over-expression of calpastatin, the endogenous protein inhibitor of μ- and m-calpain provides an important approach for these efforts, it will not distinguish isoform specific functions [24,26,27]. Some work has suggested isoform specific roles, such as a role for m-calpain in epidermal growth factor (EGF)-induced cell motility [28,29] and a role for μ-calpain in interferon-inducible protein 9-induced migration of keratinocytes [28]. A cell permeable calpain inhibitor (which likely inhibits other thiol-proteases as well) has been used to select cells lacking μ-calpain which display reduced proliferation rates [30]. Interestingly, m-calpain expression persisted in these cells, suggesting a possible requirement of m-calpain for cell survival [30]. + +Targeted gene deletion in mice provides a powerful approach to determining the physiological roles of μ- and m-calpain and the opportunity to approach their isoform specific functions. Initial studies targeted Capn4 based on the prediction that loss of this calpain subunit would abolish activity of both μ- and m-calpain. Capn4-/- murine embryos died between days 10 and 11 of gestation, and there was no detectable μ- or m-calpain activity in these or younger embryos [31]. Capn4-/- murine embryonic fibroblasts (MEFs) could be cultured from these embryos, although they also lacked calpain activity as assessed by casein zymography or by the formation of characteristic spectrin breakdown products, and they displayed migration defects consistent with a role for calpain in release of focal adhesions [9]. An independently derived Capn4 knockout, involving a more extensive deletion of the gene, resulted in an earlier embryonic lethality, apparently at a pre-implantation stage [32]. The different times of embryonic lethality suggested that the first reported Capn4-/- mice [31] were targeted with a hypomorphic mutation, which retained some small level of calpain activity, allowing for their survival to mid-gestation, while the second reported Capn4-/- mice [32] represented a true null mutation. Disruption of Capn1, encoding the μ-calpain catalytic subunit, was subsequently reported to result in fertile, viable mice with some mild defects in the μ-calpain rich platelets relating to their aggregation and clot retraction [33]. The fact that Capn4 null mice die during embryogenesis indicates that at least one of the ubiquitous calpains is essential for development to term. The viability of Capn1-deficient mice does not however distinguish between two possibilities: either that m-calpain is specifically required during embryogenesis, or that either form of calpain alone is sufficient and can compensate for the absence of the other. To resolve this question, we have now knocked out the Capn2 gene encoding the m-80 k subunit in mice. We report here that Capn2 null embryos died prior to the implantation stage, indicating that m-calpain is indispensable for early embryogenesis. This role cannot be fulfilled by μ-calpain, which is expressed in embryonic stem (ES) cells [31] and is assumed to be present at this stage of gestation. This demonstrates unequivocally that m-calpain and μ-calpain have distinct physiological roles during early embryogenesis. + +Results + +Isolation and characterization of Capn2 targeted ES cell clones + +Two independent Capn2+/- ES cell lines, designated ES27 and ES36, were isolated from a screen of 305 drug-resistant clones. Correct targeting of the Capn2 locus was established both by Southern blot hybridization and PCR analysis. A probe located outside the short (upstream) arm of homology hybridized to a 3.5-kb BamHI fragment of the wild-type allele and 5.3-kb BamHI fragment of the mutant allele as predicted from genomic maps (Figure 2A). The same probe also detected the expected 7.2-kb wild-type and 6.4-kb mutant BglII fragments, 4.9-kb wild-type and 5.7-kb mutant NcoI fragments, as well as 7.2-kb wild-type and 4.9-kb mutant BglII/AgeI fragments (not shown). A probe derived from the PGK-Neo cassette recognized only the 5.3-kb BamHI fragment in Capn2+/- ES cells, suggesting that the targeting vector had integrated solely at the Capn2 locus (not shown). A PCR screening method was also established that generated a wild-type product of 2,749 bp and a 2,711 bp product from the mutant allele. The 2,711 bp product was only evident in the two targeted cell lines (Figure 2B). + +Figure 2 + +Characterization of targeted ES cell lines. (A) Targeted disruption of the Capn2 locus was detected initially by Southern blotting. Membranes were blotted with BamHI-digested genomic DNA extracted from ES cells and hybridized with a DIG-labeled 823 bp BamHI/HindIII fragment located immediately upstream of the short arm of the targeting vector (Figure 1). A 3.5-kb BamHI fragment corresponding to the wild-type allele was present in all cells, whereas a 5.3-kb fragment from the mutant allele was detected in two targeted cell lines, designated ES27 and ES36. (B) PCR genotyping was carried out with two separate reactions designed to amplify either a 2,748 bp segment from the wild-type allele or a 2,711 bp segment from the mutant allele. Both reactions used a common sense primer located in intron 4, outside the short arm of the targeting vector, and distinct allele-specific antisense primers. The reaction to detect the wild-type allele used an antisense primer located in exon 7 while the amplification of the mutant sequence was done with an antisense primer in the PGK-Neo cassette. The results confirm the presence of the wild-type allele in all cells, whereas the mutant allele signal was observed only in the two targeted clones. (M) denotes the molecular weight marker. + +Generation of chimeric mice and germline transmission of the mutant Capn2 allele + +Eight chimeric male mice were produced in morula aggregation experiments using the Capn2+/- ES27 cell line. Two of these males transmitted the Capn2+/- ES27 genotype through the germline into the F1 generation. Heterozygous Capn2+/- animals appeared normal, with no obvious defects in gross anatomy, reproduction, or life span. + +Out of 199 weanlings from heterozygous intercrosses, no Capn2-/- progeny were detected (Table 2). We did not observe high rates of perinatal death, and no Capn2-/- stillborns were observed. This suggested that Capn2-/- animals perished at some stage during embryonic development. In an attempt to determine if embryonic death occurred at a post-implantation stage, embryos were harvested for genotyping at different times between E10.5 and E18.5. No Capn2-/- embryos were observed and no signs of embryo resorption were detected (Table 2). This indicated that the Capn2-/- embryos might be dying prior to implantation. Embryos were then flushed from the oviducts of pregnant females at E2.5 or E3.5, and genotyped by means of a nested PCR strategy (Figure 4). Two of 90 successfully genotyped pre-implantation embryos were Capn2-/-, (Table 2; Figure 5). Both of these Capn2-/- embryos were isolated at the 8-cell stage and did not display any obvious morphological defects. None of the 46 successfully genotyped blastocyst-staged embryos were Capn2-/-. The scarcity of Capn2-deficient embryos surviving to the 8-cell stage suggested that the loss of m-calpain activity must fatally compromise the viability of early embryos. Furthermore, it is possible that persistence of some maternally derived mRNA transcript or protein might have allowed a small number of Capn2-/- embryos to survive to the morula-stage. + +Table 2 + +Genotype distribution of offspring derived from Capn2 transgenic mice. + +* ND, not determined + +Figure 4 + +Nested PCR strategy for genotyping of pre-implantation embryos. Due to the scarcity of extractable genetic material, a nested PCR strategy was developed in order to genotype pre-implantation embryos. Separate reactions were used to amplify a 429 bp fragment from the wild-type allele and a 389 bp segment from the mutant allele, both spanning the 3' end of the short (upstream) arm of the targeting vector. In both reactions, a 213 bp sequence located within the short arm was co-amplified with the 'diagnostic' products as an internal control. The same sense primers were used to amplify 'diagnostic' sequences in both reactions, whereas the antisense primers were allele-specific. The primers, represented by triangles, are depicted in two (nested) sets for each of the three reactions. Exons are represented by open, vertical rectangles, the PGK-Neo cassette by an open, horizontal rectangle, while thin vertical lines denote the boundaries of the short arm and the 5' end of the long (downstream) arm. A grey, horizontal rectangle delineates the segment of the wild-type allele that is replaced by the PGK-Neo cassette in the mutant allele. + +Figure 5 + +Genotyping of pre-implantation embryos. A nested PCR strategy was used to genotype embryos prior to implantation as described in Figure 4. Capn2+/- mice were mated and the date of fertilization established by the appearance of vaginal plugs. Blastocyst (E3.5) or 8-cell embryos (E2.5) were flushed from the oviducts and then digested with proteinase K. In separate reactions segments found exclusively in either the wild-type or mutant alleles were co-amplified with an internal control sequence, located in the short (upstream) arm of the targeting vector, which is found in both alleles. The final products were 429 bp for the wild-type allele, 389 bp for the mutant allele, and 213 bp for the internal control. (A) A representative example of the genotyping of blastocyst stage embryos. Embryos #1, 2, 4, 5, and 6 were Capn2+/- whereas embryos #3 and #7 were Capn2+/+, denoted by the absence of the 389 bp mutant signal. (B) An example of the genotyping of 8-cell embryos is shown. Embryos #1, 2, 3, 5, and 6 were Capn2+/- while embryo #4 was Capn2-/-, marked by the absence of the 429 bp wild-type signal. (M) denotes the molecular weight marker. + +The genotyping results for weanlings, post-implantation, and pre-implantation embryos are shown in Table 2. Curiously, the ratio of Capn2+/+ to Capn2+/-animals from inbred heterozygous intercrosses was substantially less than the predicted 1:2 Mendelian ratio. In a group of 199 animals derived from heterozygote breeding (33 litters), 23 Capn2+/+ (11.6%) and 176 (88.4%) Capn2+/- animals were observed. The ratio of Capn2+/+ to Capn2+/- animals among males (14% to 82%) or females (13% to 90%) was essentially the same is it was for the combined population, and there were an average of six pups per litter, which is normal for this background strain. A larger than expected proportion Capn2+/- animals was also evident in both post-implantation and pre-implantation embryos (Table 2). Interestingly, a similar over-representation of heterozygous animals was also reported in one of the Capn4 transgenic lines, though the genotype skewing was not as extreme [32]. Crosses between Capn2+/+ and Capn2+/- animals also resulted in a greater than expected proportion of Capn2+/- animals (Table 2). An even higher degree of Capn2+/- over-representation was seen when the mutant allele came the mother (73%) compared to when it came from the father (59%). In these crosses the ratios of Capn2+/+ to Capn2+/- animals among males or females compared well with the ratio in the combined populations; 77% of males and 69% of females were Capn2+/- when the mutant allele came from the mother, and 62% of males and 55% of females were Capn2+/- when the mutant allele came from the father. + +Attempted generation of Capn2-/- ES cells + +Capn2+/- ES cells were subjected to clonal selection in the presence of 2 mg/mL G418 in attempts to generate homozygous mutant cells by gene conversion. This procedure has been extensively applied to targeted ES cells and was readily achieved in the case of Capn4+/- ES cells [31]. In this case, however, no Capn2-/- ES cells were isolated in screens of 126 drug-resistant clones. The inability to isolate Capn2-/- ES cells, in concert with the absence of Capn2-/- embryos beyond the 8-cell stage, suggested that m-calpain activity might be essential for cell viability or the establishment of viable ES cell clones. + +Discussion + +Although calpain activity was first identified four decades ago, a clear understanding of the substrates and functions of the enzymes has remained elusive. In large part, this has been due to the lack of inhibitors capable of specifically down-regulating the calpains without affecting other proteases. In the past decade, the story has been further complicated by the discovery of a number of previously unknown isoforms which may be differently regulated and have different substrate specificity. Gene targeting in mice has provided a powerful approach to examine the physiologic roles of individual calpain isoforms. This was first used to disrupt the Capn4 gene, encoding the small regulatory subunit common to both μ- and m-calpain. Two independent laboratories observed embryonic lethality in Capn4 knockout mice, albeit at different stages of development [31,32]. These observations supported the hypothesis that the small subunit is required for both μ- and m-calpain, and furthermore suggested four possibilities regarding their requirement for embryonic development: 1) both isoforms were required; 2) μ-calpain was required; 3) m-calpain was required, or 4) μ- and m-calpain are redundant, and one or the other isoform was required. These options were narrowed down by the subsequent observation that Capn1-/- mice, which lack the μ-calpain catalytic subunit, were healthy and fertile, although platelet aggregation and clot retraction defects were observed [33]. At that point, we were left with the last two possibilities that either m-calpain was specifically required during embryogenesis, or that either isoform alone was sufficient for sustaining embryonic viability. + +We report here that Capn2-/- mice, which lack the m-calpain catalytic subunit, die at the preimplantation stage of development. This observation allows us to now conclude that m-calpain is specifically required during embryogenesis. Since homozygous disruption of the Capn4 gene was also expected to abolish m-calpain activity, this result is in agreement with the phenotype presented by one of the two Capn4 targeted lines in which preimplantation lethality of null embryos was also observed [32]. The survival of Capn4-/- embryos from the original targeted line reported by Arthur and colleagues to mid-gestation is more difficult to reconcile [31]. In retrospect, it seems likely that the latter line represents a hypomorphic state, rather than a true null. The Capn4 targeting strategy employed by Arthur and colleagues involved disrupting the C-terminus of the calpain small subunit by insertion of the PGK-Neo cassette into the middle of exon 9, which caused truncation of the protein [31]. This strategy was based upon previous structure/function studies showing that excision of the C-terminal 25 amino acid residues of the small subunit abolished calpain activity when co-expressed with the m-calpain large subunit in E. coli [34]. In contrast, the Capn4 targeting strategy employed by Zimmerman and colleagues involved a much more extensive deletion of exons 4 through 8 [32]. It now seems probable that the difference in the time of lethality of these two targeted Capn4 lines can be explained by different extents of disruption. The Zimmerman allele probably represents a true null genotype while the Arthur allele is likely a hypomorphic mutation. Alternate targeting strategies have been shown to yield different phenotypes in gene disruption studies. For example, three different targeting strategies were independently used to disrupt the mixed lineage leukemia (Mll) gene. In all three studies, homozygous null embryos perished during embryogenesis, but at different stages (E0.5, E10.5, E14.5) [35]. The variation in phenotype was attributed to the differences in degree of function of the truncated proteins produced from the mutant alleles. A similar effect might be at work in the two Capn4 transgenic lines. Efforts were made to detect transcripts or calpain activities derived from the Arthur Capn4 allele. This allele gave rise to multiple mRNA species, detectable by RT-PCR, reading through from the first half of exon 9 to at least two different cryptic splice sites in the PGK promoter sequence [31]. These transcripts could give rise to defective calpain small subunits with 10–30 inappropriate C-terminal acids, which might be sufficient to support a low level of calpain activity. However, expression of calpains with these modified small subunits did not give rise to any detectable calpain activity when expressed in E. coli, although their functionality in mammalian cells has yet to be determined (J.S. Elce, unpublished work) It has also been suggested that calpain large subunits alone might provide some activity in eukaryotic cells, although the Zimmerman et al. knockout appears to exclude that possibility, and no calpain activity was observed in our hands when calpain large subunits were expressed alone either in E. coli or in mammalian cells [36]. The different timing of lethality in the Capn4 knockouts might also be a consequence of the different genetic backgrounds of the two transgenic lines, which has been observed to influence the phenotype of transgenic mice on a number of occasions [37]. + +One of the enduring questions in calpain research has been whether the two ubiquitous isoforms, μ- and m-calpain, possess distinct in vivo roles. The two enzymes share 62% sequence identity and are very similar in their structure and biochemistry. Notably, they cleave essentially the same set of substrates in vitro, suggesting that they have the potential to carry out the same functions in vivo. On the other hand, since they require different amounts of Ca2+ for in vitro activation, it is possible that the two isoforms are differentially regulated inside cells. It is now clear, from the gene targeting work done in mice, that μ- and m-calpain have some distinct physiological roles, at least during embryogenesis. As noted, whereas Capn2 null murine embryos die prior to implantation, homozygous disruption of the μ-calpain large subunit gene, Capn1, did not affect the viability of mice [33]. The principal phenotype observed as a result of Capn1 deficiency involved a disturbance in platelet function, possibly at the level of the tyrosine phosphorylation of certain proteins involved in platelet activation. Both μ- and m-calpain activities are present in most mammalian cells, although the published data, owing to weaknesses in the available methodology, do not provide reliable estimates of their relative amounts. Platelets and erythrocytes contain abundant μ-calpain activity, while m-calpain activity is barely detectable. Compensation for μ-calpain deficiency by m-calpain is therefore less likely in platelets and erythrocytes than in other cell types. As a result, it is not possible to determine whether the absence of marked phenotype in Capn1-/- animals is due to a compensatory affect by the remaining m-calpain activity, or whether the functions of μ-calpain are simply not essential. In contrast, lethality in Capn2-/- embryos demonstrates that m-calpain activity is essential for embryonic development beyond the 8-cell stage. It follows that at least some functions of μ-calpain and m-calpain are distinct. + +The underlying cause of the preimplantation lethality in Capn2-/- embryos has not yet been clarified. The two homozygous null embryos identified at the 8-cell stage did not present any obvious morphological defects. However, the fact that only two out of 90 successfully genotyped pre-implantation embryos proved to be Capn2-/- is in itself revealing. Preimplantation lethality resulting from gene knockouts can typically be attributed to two general causes. In some cases, defects are incurred in the embryonic differentiation program which can often be observed morphologically [38]. Null embryos of this type often survive beyond the morula stage, and a Mendelian distribution of embryonic genotypes is usually noted. In other cases, however, the gene disruption is thought to compromise fatally the viability of cells in the early embryo [3,39-43]. In these instances, only a few null embryos are ever observed and homozygous mutant ES cells could not be isolated. The Capn2 knockout fits into the latter category. If it is true that m-calpain is essential for some aspect of cell viability, the survival of a few Capn2-/- embryos to the 8-cell stage is most likely due to the persistence of some maternal m-calpain mRNA and/or protein through 2–3 cell divisions. + +The mechanistic reasons for lethality in the absence of m-calpain are still unclear. There have been several reports of m-calpain involvement in cell proliferation in certain circumstances, including reports of its involvement in chromosome segregation during mitosis [44] as well as during meiosis [45]. Defects in migration, reported in Capn4-/- cells [9], could contribute to failed embryonic development. The association between calpain and cell viability has been noted in Capn4-/- MEFs and other cell lines, although the reported work frequently did not distinguish between μ- and m-calpain, and did not show that calpain was strictly essential [5,6]. In both CHO cells and Capn4-/- MEFs, calpain was shown to influence cell proliferation, but only at very low cell densities [5]. Calpain has also been associated with progression through the G1 stage of the cell cycle [6]. Furthermore, some of the proteins known to be involved in cell cycle progression, such as p53, p107, cyclin D1, and p27kip1 are reputed to be calpain substrates [3]. + +Sperm binding to the oocyte leads to increased cytoplasmic calcium which triggers the acrosome reaction [46]. Both μ- and m-calpain have recently been detected in rodent sperm [47] and oocytes [45]. m-calpain was implicated in the acrosome reaction [47] which correlated with a translocation of m-calpain to the cortical membrane in oocytes where it might participate in the release of cortical granule contents required to prevent polyspermy [45]. m-calpain also relocalized to the oocyte meiotic spindle after fertilization, were it could be involved in chromosome segregation [45]. Polyspermy or defective chromosome segregation would both have lethal effects on early embryonic development. + +It should also be stressed that Capn4-/- ES and MEF cells from the presumptive hypomorphic allele can be maintained in culture despite an apparent lack of calpain activity, as assessed by casein zymography or by the appearance of characteristic spectrin breakdown products. It is conceivable, as discussed earlier, that a trace amount of calpain activity, beneath levels detectable by these methods, is retained from this mutant Capn4 allele, and it is sufficient for maintaining the viability of the cells. Furthermore, calpain-independent mechanisms for protecting cell viability might exist in these cell lines that are absent in early embryonic cells. + +The repeated failure to achieve gene conversion of Capn2+/- to Capn2-/- ES cells by selection of clones in high concentrations of G418 suggests that the homozygous mutant state somehow compromises cell viability or clonal selection of ES cells. Efforts are under way to express the m-80 k subunit from a Capn2 cDNA rescue transgene prior to gene conversion in order to preemptively rescue m-calpain activity before the remaining endogenous wild-type allele is lost. However, difficulty in achieving stable expression of the rescue transgene has thus far hampered these attempts. + +One curiosity which arose during the genotyping of progeny from heterozygous interbreeding was the highly non-Mendelian ratio of Capn2+/+ (11.6%) to Capn2+/- (88.4%) of weanlings. At present, there is no obvious explanation for this result. Crosses between wild-type and heterozygous mice also produced progeny with a greater than expected proportion of heterozygous offspring. Interestingly, heterozygous crosses involving the Capn4 transgenic line generated by Zimmerman and colleagues yielded progeny with a similar, if less extreme, skewing in favor of the heterozygous genotype. Out of a total of 80 genotyped animals, 22.5% were wild-type and 77.5% were heterozygous, with no homozygous null progeny observed [32]. These observations suggest a developmental advantage associated with reduced calpain expression. Perhaps future studies will reveal a mechanistic basis for this. + +Conclusion + +The work presented here has clarified two important questions regarding the physiological roles of the two ubiquitous calpain isoforms, μ- and m-calpain. Firstly, it was determined that m-calpain plays an indispensable role in murine embryogenesis, possibly related to pre-implantation development. Furthermore, this function cannot be carried out by μ-calpain despite the apparent in vitro similarities of μ- and m-calpain, demonstrating that the two isoforms clearly have some distinct roles in vivo. The functions of m-calpain during post-implantation embryogenesis and in adult mice remain to be elucidated and will have to be addressed using a conditional gene targeting strategy. + +Methods + +Cloning and sequencing of the mouse Capn2 locus + +The mouse Capn2 gene encodes the 700 amino acids of the m-calpain large subunit (m-80 k) and consists of 21 exons extending over 50-kb on chromosome 1. A cDNA clone encoding a portion of the mouse m-80 k subunit was purchased (dbEST Id, 807416, Image:606689, Image Consortium, LLNL). It was found to contain 2.8-kb of sequence from position 247 of the coding sequence to the stop codon at position 2,101, including the 3'-UTR to the polyA signal. A fragment of this cDNA was subcloned and used to screen a 129Sv mouse genomic library in λ-Dash II. Overlapping genomic clones were obtained covering 15,354 bp, extending from a BamHI site in intron 3 to a BamHI site in intron 15. These clones were sequenced completely on both strands and the data were submitted to GenBank (accession no. AF497625). The submitted sequences agreed precisely with the public databases, and also filled in several small gaps corresponding to short repetitive sequences which could only be firmly established by repeated sequencing in non-standard conditions. + +Construction of the Capn2 targeting vector + +A targeting construct was designed to replace a 785 bp BamHI-HindIII fragment, containing exon 7 of the Capn2 gene, with the PGK-Neo cassette (Figure 1). Exon 7 encodes 24 amino acids in the active-site region, including Asn286, one of the catalytic triad residues. The short (upstream) arm of the targeting construct was provided by a 2.7-kb HindIII-BamHI fragment, containing exons 5 and 6, which was inserted into the pPNT vector upstream of the PGK-Neo cassette [48]. During cloning, the BamHI site at the 3' end of the short arm was abolished. The loss of this BamHI site in the mutant allele, coupled with the introduction of a new BamHI site at the 3' end of the PGK-Neo cassette, provided a basis for distinguishing the wild-type and mutant alleles by Southern blotting. The long (downstream) arm of homology was provided by a 7-kb HindIII-KpnI fragment, extending from intron 7 to intron 12, inserted between the PGK-Neo and thymidine kinase (tk) cassettes. + +Figure 1 + +Targeting strategy for disruption of the murine Capn2 gene. The murine Capn2 gene, encoding the m-80 k subunit, was disrupted in ES cells by homologous recombination. The structures of the wild-type allele (top), the targeting vector (middle), and the mutant allele (bottom) are depicted. In the mutant allele, a PGK-Neo cassette replaces a 0.8-kb genomic fragment containing exon 7 (grey rectangle) which encodes the active site asparagine residue (Asn286) In the targeting vector, the PGK-Neo cassette is flanked by 2.7-kb of Capn2 homologous sequence in the upstream (short) arm and 7.9-kb of homology in the downstream (long) arm. A probe located immediately outside of the short arm detects a 3.5-kb BamHI fragment from the wild-type allele and a 5.3-kb BamHI fragment from the mutant allele. Exons are depicted as open vertical rectangles except for exon 7 which is represented by a solid vertical rectangle. The probe used in most Southern blot analyses is shown as a solid, horizontal rectangle, while triangles mark the positions of PCR primers also used for genotyping purposes. + +ES cell culture, transfection and selection of targeted clones + +Mouse R1 ES cells [49] were maintained on gelatin-coated plates with feeder layers of mouse embryonic fibroblasts at 37°C under 5% CO2 in ES cell medium (DMEM [high glucose] supplemented with 15% fetal bovine serum, 0.1 mM non-essential amino acids, 2 mM glutamine, 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 100 U/ml penicillin, 100 μg/ml streptomycin, 25 μg/ml amphotericin, and 1,000 U/ml of ESGro [Chemicon]). Fetal bovine serum from HyClone Laboratories Inc (Logan, Utah) was tested for its ability to support ES cell growth. Gelatin was from Sigma-Aldrich Canada (Oakville, Ontario). Unless otherwise specified, all other tissue culture reagents were from Gibco-BRL. + +The targeting construct was linearized by NotI digestion and electroporated into R1 ES cells. Cells were plated without feeder layers on gelatin-coated plates and transformed clones were selected in the presence of 200 μg/ml G418 (Gibco- BRL) and 2 μM ganciclovir (Syntex, Inc.) for eight days. Drug-resistant clones were picked, expanded on gelatin-coated plates, and genotyped by Southern blotting and PCR analysis (see below). + +Generation of targeted mice + +Capn2+/- ES cells were aggregated overnight with 8-cell embryos recovered from CD1 matings, as previously described [31]. On the next day, blastocysts were transferred to pseudopregnant CD1 females. Chimeric animals were identifiable at birth by black eye pigmentation and subsequently by patches of agouti coat colour. Chimeric males were bred with CD1 females to identify those males capable of germline transmission. These were then bred with 129SvJ females to establish the mutation in an inbred genetic background. Mouse protocols were approved by the Queen's University Animal Care Committee according to the guidelines of the Canadian Council on Animal Care. + +Genotyping methods + +Several Southern blot and PCR strategies were exploited in order to determine the genotype of the Capn2 locus. Southern blotting was carried out using the digoxigenin (DIG) non-radioactive system (Roche). In most cases, membranes were blotted with BamHI-digested genomic DNA and hybridized with a DIG-labeled 823 bp exon 4-containing BamHI-HindIII fragment located immediately upstream of the short arm of homology (Figure 1). A 681 bp PstI-XbaI fragment from the PGK-Neo cassette was also used to probe Southern blots in order to verify a single integration event in targeted clones. + +Genotyping was also carried out by PCR analysis of genomic DNA. The sequences of all oligonucleotide primers are listed in Table 1. A single-step PCR strategy was sufficient for genotyping ES cells or biopsies from post-implantation embryos and weanlings (Figure 1). A 2,748 bp segment of the wild-type allele and a 2,711 bp segment of the mutant allele were amplified in separate reactions using a common (intron 4) sense primer, located outside the short arm of homology, and distinct antisense primers which hybridized to either wild-type (exon 7) or mutant (PGK-Neo) sequence (Table I). The thermocycling parameters included a five minute initial denaturation step at 95°C, 30 cycles of one minute denaturation at 95°C, one minute annealing at 56°C, and one minute extension at 72°C, with a ten minute final extension step. + +Table 1 + +Oligonucleotide primers used to genotype the Capn2 locus + +Due to the limited amount of genetic material available in pre-implantation embryos, a nested PCR strategy was developed to yield reliable genotyping information (Figure 4). Whole embryos were first digested in 20 μL of proteinase K buffer (see below). The lysate was then divided in two, with half (10 μL) being used in the amplification of the wild-type allele and the remaining 10 μL in the amplification of the mutant allele. + +The first reaction in the nested PCR amplification of the wild-type allele was carried out in a final reaction volume of 50 μL, using an intron 6 sense primer and an antisense primer located in intron 7. Two μL of the first reaction were used as template in the second PCR amplification using another intron 6 sense primer and an antisense primer in exon 7. The nested amplification of the mutant allele was carried out similarly. The first primer pair consisted of the intron 6 sense primer and an antisense primer in the PGK-Neo cassette. The nested primer pair was comprised of the second intron 6 sense primer and an antisense primer also located within the PGK-Neo sequence. It should be noted that amplification of both sequences involved the same sense primers in both steps of the nested PCR strategy. In addition, the two sets of reactions included a common internal control designed to amplify a genomic region within the short arm of the targeting vector that is preserved in both the wild-type and mutant alleles. The first primer pair of the control PCR was made up of an intron 4 sense primer and an intron 5 antisense primer. The second primer pair comprised an exon 5 sense primer and a nested intron 5 antisense primer. The final products were 213 bp for the control PCR, 429 bp for the wild-type PCR, and 389 bp for the mutant PCR. All reactions were carried out using identical PCR conditions entailing an initial five minute denaturation at 95°C, 35 cycles of one minute denaturation at 95°C, one minute annealing at 56°C, and one minute extension at 72°C, with a final extension step of ten minutes. + +Isolation of pre-implantation embryos + +Time of fertilization was determined by observation of copulation plugs, and noon of that day was defined as E0.5. Pre-implantation embryos were obtained by dissecting the uteri out of pregnant females at E2.5 or E3.5 and flushing the oviducts with ES cell medium. Isolated embryos were then digested for five hours at 55°C in 20 μL of lysis buffer (50 mM Tris-HCl, 0.5% Triton X-100, 200 μL/mL proteinase K, pH 8.0), followed by ten minutes at 95°C to inactivate the proteinase K. Lysates were then used for PCR genotyping. + +List of abbreviations + +EGF: Epidermal growth factor + +ES: Embryonic stem + +MEF: Mouse embryonic fibroblast + +kDa: kilodalton + +kb: kilobase + +bp: base pair + +Authors' contributions + +DEC generated the Capn2 targeting construct. PD and PAG carried out the ES electroporation and selection of targeted clones. KW performed aggregation chimeras to establish germline chimeric animals. TDV established and maintained the knockout mouse colony and performed genotyping analysis. PD, JSCA, JSE, DEC and PAG conceived the study and helped draft the manuscript. All authors read and approved the final manuscript. + +Figure 3 + +Genotyping of weanlings from heterozygote intercrosses. A representative example of the Southern blot genotyping of progeny from a Capn2+/- intercross is shown. Genomic DNA was extracted from mouse tail biopsies taken from three-week old weanlings and genotyped by Southern blotting as described in Figure 1. A 3.5-kb BamHI fragment from the wild-type allele was detected in all animals while a 5.3-kb BamHI fragment from the mutant allele was observed in a subset of progeny. No Capn2-/- offspring were detected among weanlings or post-implantation embryos, nor were embryo resorption sites observed. These results indicate that Capn2-/- embryos perished at a pre-implantation stage. + +Acknowledgements + +We would like to thank Andras Nagy for providing the R1 ES cell line, Janet Rossant for the 129SvJ mouse genomic DNA library, and Ralph Zirngibl for modification of the pPNT vector. Initial cloning of Capn2 and vector construction was undertaken by DEC as a sabbatical visitor at Queen's University with support from the National Science Foundation (MCB9723636) and the University of Maine. + +This work was supported by grants from the Canadian Institutes of Health Research and The Heart and Stroke Foundation of Canada. 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SO:0000704 5303 5307 gene +T112 SO:0000704 5409 5416 genetic +T113 http://purl.obolibrary.org/obo/MONDO_0011122 5433 5440 obesity +T114 GO:0010468 5460 5473;5480 5495 regulation of ... gene expression +T115 UBERON:0002107 5474 5479 liver +T116 SO:0000704 5480 5484 gene +T117 CHEBI:39015 5586 5600 apolipoprotein +T118 PR:000004155 5586 5602 apolipoprotein E +T119 PR:000004155 5609 5613 ApoE +T120 PR:000004155 5638 5642 ApoE +T121 GO:0008152 5742 5751 metabolic +T122 http://purl.obolibrary.org/obo/MONDO_0004955 5742 5760 metabolic syndrome +T123 NCBITaxon:33208 5788 5795 animals +T124 SO:0000771 5866 5870 QTLs +T125 SO:0000704 5908 5912 gene +T126 GO:0010467 5908 5923 gene expression +T127 SO:0000771 5924 5928 QTLs +T128 SO:0000704 6027 6034 genetic +T129 GO:0065007 6035 6045 regulation +T130 UBERON:0000991 6053 6060 gonadal +T131 SO:0000704 6092 6097 genes +T132 SO:0000771 6135 6139 QTLs +T133 UBERON:0000991 6156 6163 Gonadal +T134 PR:000004155 6200 6204 ApoE +T135 PR:000004155 6216 6220 ApoE +T136 PR:000004155 6247 6251 ApoE +T137 UBERON:0000991 6311 6318 Gonadal +T138 PR:000004155 6410 6414 ApoE +T139 PR:000004155 6444 6448 ApoE +T140 NCBITaxon:10088 6452 6456 mice +T141 UBERON:0000991 6458 6465 Gonadal +T142 UBERON:0003916 6483 6490 fat pad +T143 NCBITaxon:33208 6528 6535 animals +T144 UBERON:0000991 6695 6702 gonadal +T145 UBERON:0000991 6860 6867 gonadal +T146 NCBITaxon:10088 6897 6901 mice +T147 SO:0000694 6958 6989 single nucleotide polymorphisms +T148 SO:0000694 6991 6995 SNPs +T149 GO:0030849 7013 7022 autosomes +T150 SO:0000771 7024 7027 QTL +T151 SO:0000771 7075 7079 QTLs +T152 UBERON:0000991 7130 7137 gonadal +T153 SO:0000771 7669 7672 QTL +T154 SO:0000704 7724 7728 gene +T155 GO:0010467 7724 7739 gene expression +T156 SO:0000771 7876 7879 QTL +T157 SO:0000771 8069 8073 QTLs +T158 SO:0000771 8086 8089 QTL +T159 SO:0000771 8286 8289 QTL +T160 SO:0000704 8298 8302 gene +T161 GO:0010467 8298 8313 gene expression +T162 SO:0000771 8367 8370 QTL +T163 SO:0001026 8540 8546 genome +T164 SO:0000771 8563 8566 QTL +T165 SO:0000771 8858 8861 QTL +T166 UBERON:0000991 9217 9224 gonadal +T167 UBERON:0000991 9755 9762 gonadal +T168 SO:0001026 9798 9804 genome +T169 NCBITaxon:33208 10082 10089 animals +T170 NCBITaxon:33208 10364 10371 animals +T171 SO:0000771 10412 10415 QTL +T172 UBERON:0000991 10721 10728 gonadal +T173 UBERON:0000991 11264 11271 gonadal +T174 NCBITaxon:10088 11451 11455 mice +T175 GO:0065007 11600 11610 regulation +T176 UBERON:0000991 11635 11642 gonadal +T177 UBERON:0002107 11673 11678 Liver +T178 UBERON:0002107 11686 11692 Livers +T179 NCBITaxon:33208 11705 11712 animals +T180 NCBITaxon:10088 11894 11899 mouse +T181 SO:0000673 11900 11911 transcripts +T182 SO:0000673 11924 11934 transcript +T183 NCBITaxon:10088 12118 12122 mice +T184 SO:0000673 12296 12307 transcripts +T185 GO:0010467 12328 12337 expressed +T186 NCBITaxon:33208 12366 12373 animals +T187 SO:0000704 12439 12444 genes +T188 GO:0010467 12491 12501 expression +T189 SO:0000673 12632 12643 transcripts +T190 NCBITaxon:10088 12663 12667 mice +T191 UBERON:0002107 12774 12779 liver +T192 SO:0000704 12780 12784 gene +T193 GO:0010467 12780 12795 gene expression +T194 GO:0065007 12830 12841 controlling +T195 SO:0000673 12865 12876 transcripts +T196 GO:0010467 12878 12888 expression +T197 GO:0010467 12920 12930 expression +T198 SO:0000673 12952 12963 transcripts +T199 SO:0000771 13207 13211 QTLs +T200 SO:0000771 13318 13322 QTLs +T201 SO:0001026 13432 13438 genome +T202 SO:0000771 13527 13530 QTL +T203 SO:0000771 13584 13587 QTL +T204 SO:0000771 13648 13651 QTL +T205 PR:000004155 13863 13867 ApoE +T206 SO:0000673 14074 14085 transcripts +T207 SO:0000704 14206 14210 gene +T208 GO:0065007 14238 14247 regulated +T209 SO:0000704 14283 14287 gene +T210 SO:0001026 14546 14552 genome +T211 SO:0000704 15783 15787 gene +T212 GO:0010467 15790 15800 expression +T213 UBERON:0002107 15908 15913 liver +T214 SO:0000704 15989 15996 genetic +T215 GO:0010468 15997 16026 regulation of gene expression +T216 SO:0000704 16011 16015 gene +T217 SO:0000704 16053 16058 Genes +T218 UBERON:0000991 16067 16074 Gonadal +T219 UBERON:0002107 16126 16131 liver +T220 SO:0000704 16132 16136 gene +T221 GO:0010467 16132 16147 gene expression +T222 SO:0000704 16156 16163 genetic +T223 GO:0065007 16164 16174 regulation +T224 UBERON:0000991 16178 16185 gonadal +T225 SO:0000704 16316 16321 genes +T226 SO:0000704 16509 16514 genes +T227 SO:0000704 16536 16541 genes +T228 SO:0000704 16664 16669 Genes +T229 UBERON:0000991 16707 16714 Gonadal +T230 UBERON:0000991 16875 16882 gonadal +T231 SO:0000673 16902 16912 transcript +T232 SO:0000704 16957 16961 gene +T233 SO:0000704 16976 16980 gene +T234 SO:0000704 17001 17005 gene +T235 SO:0000704 17069 17073 gene +T236 SO:0000704 17432 17437 genes +T237 UBERON:0000991 17473 17480 gonadal +T238 SO:0000704 17500 17505 genes +T239 SO:0000704 17539 17543 gene +T240 SO:0000704 17614 17621 genetic +T241 GO:0065007 17622 17632 regulation +T242 SO:0000704 17671 17676 genes +T243 UBERON:0000991 17693 17700 gonadal +T244 SO:0000704 17749 17754 genes +T245 UBERON:0000991 17771 17778 gonadal +T246 SO:0000704 17890 17895 genes +T247 SO:0000704 17993 17998 genes +T248 SO:0000704 18069 18074 Genes +T249 UBERON:0000991 18118 18125 gonadal +T250 SO:0000704 18224 18229 genes +T251 SO:0000704 18270 18277 genetic +T252 SO:0000704 18296 18300 gene +T253 SO:0000704 18362 18367 genes +T254 SO:0000704 18443 18448 genes +T255 UBERON:0000062 18517 18522 organ +T256 GO:0065007 18523 18533 regulation +T257 SO:0000704 18582 18589 genetic +T258 GO:0065007 18590 18600 regulation +T259 UBERON:0002107 18633 18638 liver +T260 UBERON:0002107 18670 18675 liver +T261 UBERON:0002107 18713 18718 liver +T262 UBERON:0000479 18778 18784 tissue +T263 SO:0000704 18787 18792 Genes +T264 UBERON:0000991 18832 18839 gonadal +T265 SO:0001026 19009 19015 genome +T266 SO:0000704 19101 19105 gene +T267 GO:0065007 19184 19193 regulated +T268 SO:0000704 19211 19215 gene +T269 SO:0000704 19307 19312 genes +T270 GO:0065007 19360 19369 regulated +T271 UBERON:0000479 19382 19389 tissues +T272 UBERON:0000062 19394 19400 organs +T273 UBERON:0002107 19470 19475 liver +T274 SO:0000704 19476 19480 gene +T275 GO:0010467 19476 19491 gene expression +T276 UBERON:0002107 19596 19601 liver +T277 SO:0000704 19602 19606 gene +T278 GO:0010467 19602 19617 gene expression +T279 SO:0000704 19686 19691 genes +T280 GO:0065007 19720 19730 controlled +T281 UBERON:0000479 19740 19747 tissues +T282 SO:0001026 19869 19875 genome +T283 SO:0001026 20029 20035 genome +T284 UBERON:0002107 20093 20098 liver +T285 SO:0001026 20247 20253 genome +T286 SO:0000673 20322 20333 transcripts +T287 UBERON:0000991 20354 20361 gonadal +T288 SO:0000704 20525 20532 genetic +T289 GO:0065007 20533 20543 regulation +T290 UBERON:0002107 20608 20613 liver +T291 SO:0000704 20614 20618 gene +T292 GO:0010467 20614 20629 gene expression +T293 SO:0000704 20731 20735 gene +T294 GO:0010467 20731 20746 gene expression +T295 SO:0000704 20854 20859 genes +T296 UBERON:0000991 20876 20883 gonadal +T297 GO:0065007 20897 20906 regulated +T298 SO:0000704 20970 20974 gene +T299 GO:0010467 20970 20985 gene expression +T300 GO:0065007 20993 21003 controlled +T301 SO:0000673 21131 21142 transcripts +T302 SO:0000704 21226 21233 genetic +T303 GO:0010468 21234 21263 regulation of gene expression +T304 SO:0000704 21248 21252 gene +T305 UBERON:0000991 21481 21488 gonadal +T306 SO:0000704 21584 21591 genetic +T307 GO:0065007 21592 21602 regulation +T308 UBERON:0002107 21606 21611 liver +T309 SO:0000704 21612 21617 genes +T310 UBERON:0000062 21702 21707 organ +T311 NCBITaxon:10088 21790 21795 mouse +T312 SO:0000704 21829 21836 genetic +T313 GO:0065007 21837 21847 regulation +T314 GO:0008152 21896 21905 metabolic +T315 http://purl.obolibrary.org/obo/MONDO_0004955 21896 21914 metabolic syndrome +T316 SO:0001026 22015 22021 genome +T317 SO:0000704 22027 22031 gene +T318 GO:0010467 22027 22042 gene expression +T319 NCBITaxon:33208 22147 22154 animals +T320 SO:0000704 22282 22289 genetic +T321 GO:0065007 22290 22300 regulation +T322 GO:0010468 22331 22344;22369 22384 Regulation of ... Gene Expression +T323 SO:0000704 22369 22373 Gene +T324 GO:0065007 22464 22471 control +T325 http://purl.obolibrary.org/obo/MONDO_0011122 22475 22482 obesity +T326 SO:0000771 22559 22563 QTLs +T327 SO:0000771 22786 22789 QTL +T328 SO:0000771 22987 22990 QTL +T329 SO:0001026 23023 23029 genome +T330 SO:0000771 23132 23136 QTLs +T331 SO:0000771 23241 23245 QTLs +T332 UBERON:0000991 23312 23319 gonadal +T333 SO:0000771 23544 23548 QTLs +T334 http://purl.obolibrary.org/obo/MONDO_0011122 23565 23572 obesity +T335 UBERON:0000991 23574 23581 gonadal +T336 UBERON:0007808 23591 23604 abdominal fat +T337 SO:0000771 23773 23776 QTL +T338 SO:0001024 23945 23954 haplotype +T339 SO:0000704 24053 24060 genetic +T340 GO:0065007 24061 24071 regulation +T341 http://purl.obolibrary.org/obo/MONDO_0021187 24115 24129 hyperlipidemic +T342 PR:000004155 24147 24151 ApoE +T343 NCBITaxon:9606 24276 24281 human +T344 http://purl.obolibrary.org/obo/MONDO_0000001 24287 24294 disease +T345 UBERON:0000991 24415 24422 gonadal +T346 SO:0000704 24449 24456 genetic +T347 GO:0065007 24457 24467 regulation +T348 SO:0000704 24514 24519 genes +T349 SO:0001026 24589 24595 genome +T350 GO:0010467 24601 24611 expression +T351 UBERON:0000062 24700 24706 organs +T352 SO:0000704 24756 24761 genes +T353 SO:0000673 24820 24830 transcript +T354 SO:0000704 24877 24884 genetic +T355 GO:0065007 24885 24895 regulation +T356 SO:0000704 24920 24924 gene +T357 GO:0010467 24920 24935 gene expression +T358 SO:0000704 25043 25048 genes +T359 SO:0001026 25083 25089 genome +T360 SO:0000704 25166 25171 genes +T361 GO:0010468 25610 25639 regulation of gene expression +T362 SO:0000704 25624 25628 gene +T363 SO:0000704 25679 25683 gene +T364 SO:0000704 25711 25718 genetic +T365 GO:0065007 25719 25729 regulation +T366 SO:0000673 25738 25749 transcripts +T367 SO:0000704 25893 25900 genetic +T368 GO:0010468 25901 25930 regulation of gene expression +T369 SO:0000704 25915 25919 gene +T370 CHEBI:50112 25964 25975 sex hormone +T371 NCBITaxon:10088 26182 26187 mouse +T372 SO:0001026 26188 26194 genome +T373 UBERON:0000991 26255 26262 gonadal +T374 UBERON:0000991 26354 26361 gonadal +T375 SO:0000704 26722 26727 genes +T376 SO:0000704 26791 26796 genes +T377 SO:0000673 26824 26834 transcript +T378 GO:0010467 26835 26845 expression +T379 SO:0000704 26958 26963 genes +T380 SO:0000704 27059 27064 genes +T381 SO:0000704 27148 27152 gene +T382 GO:0006412 27169 27182 translational +T383 GO:0065007 27247 27256 regulated +T384 UBERON:0000062 27409 27414 organ +T385 SO:0000704 27424 27428 gene +T386 GO:0010467 27424 27439 gene expression +T387 SO:0000704 27507 27511 gene +T388 UBERON:0000479 27519 27525 tissue +T389 UBERON:0000479 27545 27551 tissue +T390 GO:0065007 27562 27569 control +T391 UBERON:0001013 27632 27646 adipose tissue +T392 GO:0065007 27662 27672 controlled +T393 UBERON:0000479 27685 27692 tissues +T394 SO:0000704 27733 27738 genes +T395 SO:0000704 27781 27786 Genes +T396 UBERON:0000991 27803 27810 Gonadal +T397 UBERON:0000479 27831 27837 Tissue +T398 GO:0065007 27847 27857 Regulation +T399 SO:0000704 27897 27902 genes +T400 SO:0000704 27969 27974 genes +T401 SO:0000673 28071 28082 transcripts +T402 UBERON:0000991 28087 28094 gonadal +T403 SO:0000771 28113 28117 QTLs +T404 SO:0000704 28182 28187 genes +T405 UBERON:0000991 28235 28242 gonadal +T406 SO:0000704 28345 28350 genes +T407 SO:0000704 28472 28477 genes +T408 SO:0000704 28534 28538 gene +T409 GO:0010467 28534 28549 gene expression +T410 SO:0000704 29007 29012 genes +T411 UBERON:0000991 29029 29036 gonadal +T412 SO:0000673 29311 29322 transcripts +T413 UBERON:0000991 29369 29376 gonadal +T414 SO:0000704 29474 29479 genes +T415 GO:0065007 29539 29549 regulatory +T416 SO:0000704 29550 29555 genes +T417 SO:0000704 29624 29629 genes +T418 SO:0000704 29666 29670 gene +T419 UBERON:0000991 29739 29746 gonadal +T420 GO:0008152 29771 29780 metabolic +T421 SO:0000704 29891 29896 genes +T422 GO:0065007 29961 29971 controlled +T423 UBERON:0000479 29979 29985 tissue +T424 SO:0000167 30123 30131 promoter +T425 SO:0000704 30144 30149 genes +T426 SO:0000704 30212 30216 gene +T427 GO:0010467 30212 30227 gene expression +T428 GO:0065007 30347 30357 regulation +T429 SO:0001026 30417 30423 genome +T430 GO:0010467 30429 30439 expression +T431 SO:0001026 30512 30519 genomic +T432 SO:0000704 30764 30771 genetic +T433 GO:0065007 30772 30782 regulation +T434 SO:0000704 31030 31034 gene +T435 GO:0010467 31030 31045 gene expression +T436 SO:0000704 31301 31308 genetic +T437 SO:0001026 31313 31320 genomic +T438 NCBITaxon:9606 31417 31422 human +T439 GO:0008152 31423 31432 metabolic +T440 http://purl.obolibrary.org/obo/MONDO_0004955 31423 31441 metabolic syndrome +T441 NCBITaxon:10088 31453 31457 mice +T442 SO:0000704 31610 31617 genetic +T443 GO:0065007 31618 31628 regulation +T444 UBERON:0000991 31683 31690 gonadal +T445 UBERON:0002107 31790 31795 liver +T446 SO:0000704 31898 31902 gene +T447 GO:0010467 31898 31913 gene expression +T448 SO:0000704 32043 32048 genes +T449 UBERON:0002107 32102 32107 liver +T450 SO:0000704 32108 32113 genes +T451 UBERON:0000479 32169 32175 tissue +T452 SO:0000704 32197 32204 genetic +T453 GO:0065007 32205 32215 regulation +T454 SO:0000704 32351 32358 genetic +T455 GO:0065007 32359 32369 regulation +T456 NCBITaxon:33208 32425 32432 Animals +T457 UBERON:0000479 32437 32443 tissue +T458 PR:000004155 32466 32470 ApoE +T459 PR:000004155 32478 32482 ApoE +T460 PR:000004155 32570 32574 ApoE +T461 PR:000004155 32583 32587 ApoE +T462 PR:000004155 32626 32630 ApoE +T463 NCBITaxon:10088 32665 32669 mice +T464 PR:000004155 32726 32730 ApoE +T465 PR:000004155 32742 32746 ApoE +T466 NCBITaxon:10088 32758 32762 mice +T467 NCBITaxon:10088 32806 32810 mice +T468 NCBITaxon:10088 32827 32831 mice +T469 NCBITaxon:10088 32874 32878 mice +T470 GO:0007631 32884 32887 fed +T471 CHEBI:33290 32895 32899 Chow +T472 CHEBI:16113 33006 33017 cholesterol +T473 NCBITaxon:10088 33029 33033 Mice +T474 GO:0016265 33070 33075 death +T475 UBERON:0002107 33077 33083 livers +T476 CHEBI:17997 33138 33140 N2 +T477 UBERON:0003428 33146 33162 gonadal fat pads +T478 GO:0097617 33227 33240 hybridization +T479 GO:0010467 33246 33256 expression +T480 GO:0097617 33294 33308 hybridizations +T481 NCBITaxon:10088 33572 33577 mouse +T482 NCBITaxon:10088 33661 33666 Mouse +T483 UBERON:0002107 33667 33673 livers +T484 CHEBI:33893 33728 33735 reagent +T485 GO:0001171 33858 33877 reverse transcribed +T486 CHEBI:37987 33902 33905 Cy3 +T487 CHEBI:37989 33909 33912 Cy5 +T488 CHEBI:51217 33913 33925 fluorochrome +T489 CHEBI:37987 33936 33939 Cy3 +T490 CHEBI:37989 33943 33946 Cy5 +T491 GO:0097617 33969 33979 hybridized +T492 GO:0097617 34048 34061 hybridization +T493 SO:0000704 34371 34375 Gene +T494 GO:0010467 34371 34386 Gene expression +T495 NCBITaxon:10088 34452 34456 mice +T496 SO:0000704 34679 34683 gene +T497 GO:0010467 34716 34725 expressed +T498 SO:0001026 34935 34942 Genomic +T499 UBERON:0002113 34965 34971 kidney +T500 CHEBI:15882 34975 34981 phenol +T501 CHEBI:35255 34982 34992 chloroform +T502 SO:0000694 35064 35068 SNPs +T503 GO:0030849 35161 35170 autosomes +T504 SO:0000694 35208 35212 SNPs +T505 SO:0000704 35449 35453 gene +T506 GO:0010467 35449 35464 gene expression +T507 SO:0000771 35600 35604 QTLs +T508 UBERON:0002107 35873 35878 liver +T509 SO:0000673 35879 35890 transcripts +T510 SO:0001026 35901 35907 genome +T511 SO:0000771 36383 36386 QTL +T512 SO:0000771 36674 36677 QTL +T513 SO:0000771 36740 36743 QTL +T514 SO:0000771 37191 37195 QTLs +T515 NCBITaxon:33208 37266 37273 animals +T516 SO:0000771 37369 37373 QTLs +T517 SO:0000771 37516 37520 QTLs +T518 SO:0000771 37776 37779 QTL +T519 SO:0001026 37931 37937 genome +T520 SO:0000771 38345 38348 QTL +T521 SO:0000704 39217 39222 genes +T522 SO:0000704 39261 39266 genes +T523 GO:0010467 39279 39288 expressed +T524 UBERON:0002107 39296 39301 liver +T525 SO:0000704 39320 39324 gene +T526 SO:0000704 39484 39488 gene +T527 GO:0010467 39484 39499 gene expression +T528 SO:0000704 39573 39578 genes +T529 SO:0000771 39687 39690 QTL +T530 SO:0000771 39797 39800 QTL +T531 SO:0000704 39832 39836 gene +T532 SO:0000771 40038 40041 QTL +T533 SO:0000704 40061 40065 gene +T534 GO:0010467 40061 40076 gene expression +T535 SO:0000771 40107 40110 QTL +T536 SO:0000704 40536 40540 gene +T537 GO:0010467 40536 40551 gene expression +T538 SO:0000771 40607 40611 QTLs +T539 SO:0000704 40646 40651 genes +T540 SO:0000771 40715 40719 QTLs +T541 SO:0000771 40837 40841 QTLs +T542 GO:0010467 41488 41498 expression +T543 SO:0000771 41710 41713 QTL +T544 UBERON:0000991 41954 41961 gonadal +T545 SO:0000673 41981 41991 transcript +T546 SO:0000704 42292 42296 gene +T547 SO:0000704 42670 42675 Genes +T548 UBERON:0000991 42696 42703 gonadal +T549 SO:0000704 42806 42811 genes +T550 UBERON:0002107 42953 42958 liver +T551 SO:0001026 43001 43007 genome +T552 SO:0001026 43295 43301 genome +T553 UBERON:0000991 43425 43432 gonadal +T554 SO:0000673 43676 43687 Transcripts +T555 UBERON:0000991 43718 43725 Gonadal +T556 PR:000004155 44067 44071 ApoE +T557 PR:000004155 44121 44125 ApoE +T558 CHEBI:39015 44131 44145 apolipoprotein +T559 PR:000004155 44131 44147 apolipoprotein E +T560 SO:0000771 44170 44173 QTL +T561 SO:0000704 44182 44186 gene +T562 GO:0010467 44182 44197 gene expression +T563 SO:0000771 44198 44201 QTL +T564 SO:0000771 44284 44287 QTL +T565 SO:0000771 44290 44314 quantitative trait locus +T566 SO:0000694 44357 44360 SNP +T567 SO:0000694 44363 44393 single nucleotide polymorphism +T568 SO:0001026 44425 44431 Genome +T569 UBERON:0000991 44441 44448 Gonadal +T570 NCBITaxon:33208 44463 44470 Animals +T571 SO:0000694 44527 44531 SNPs +T572 SO:0001026 44677 44683 genome +T573 SO:0000771 44733 44736 QTL +T574 SO:0001026 44982 44988 Genome +T575 UBERON:0000991 44999 45006 gonadal +T576 NCBITaxon:10088 45044 45049 mouse +T577 NCBITaxon:33208 45093 45100 animals +T578 SO:0000771 45367 45370 QTL +T579 SO:0000771 45692 45695 QTL +T580 SO:0000771 45776 45780 QTLs +T581 UBERON:0002107 45953 45958 Liver +T582 SO:0000704 45959 45963 Gene +T583 GO:0010467 45959 45974 Gene Expression +T584 GO:0010467 46014 46023 expressed +T585 SO:0000704 46024 46029 genes +T586 UBERON:0002107 46033 46038 liver +T587 NCBITaxon:33208 46067 46074 animals +T588 SO:0000673 46092 46102 transcript +T589 GO:0010467 46120 46130 Expression +T590 UBERON:0002107 46289 46294 Liver +T591 UBERON:0002107 46338 46343 liver +T592 SO:0001026 46361 46367 genome +T593 UBERON:0002107 46451 46456 liver +T594 SO:0000673 46457 46468 transcripts +T595 SO:0000673 46641 46652 transcripts +T596 SO:0001026 47065 47071 genome +T597 SO:0001026 47115 47121 genome +T598 SO:0001026 47395 47401 genome +T599 SO:0000673 47532 47543 Transcripts +T600 UBERON:0000991 47574 47581 Gonadal +T601 SO:0000704 47631 47635 gene +T602 SO:0000673 47657 47668 transcripts +T603 UBERON:0000991 47673 47680 gonadal +T604 SO:0000673 47712 47723 transcripts +T605 SO:0000704 47813 47818 genes +T606 UBERON:0000991 47849 47856 gonadal +T607 SO:0000704 47883 47888 genes +T608 UBERON:0000991 47993 48000 gonadal +T609 SO:0001026 48021 48027 genome +T610 SO:0001026 48065 48072 genomic +T611 UBERON:0000991 48116 48123 gonadal +T612 SO:0001026 48156 48162 genome +T613 UBERON:0000991 48507 48514 gonadal +T614 UBERON:0000991 48611 48618 Gonadal +T615 SO:0000704 48687 48691 gene +T616 GO:0010467 48687 48702 gene expression +T617 SO:0000673 48801 48811 transcript +T618 UBERON:0000991 49073 49080 gonadal +T619 SO:0000704 49298 49302 gene +T620 GO:0010467 49298 49313 gene expression +T621 PR:000004155 49488 49492 ApoE +T622 UBERON:0000991 49522 49529 Gonadal +T623 UBERON:0002107 49568 49573 Liver +T624 SO:0000704 49604 49609 Genes +T625 UBERON:0000991 49630 49637 Gonadal +T626 SO:0000673 49662 49673 Transcripts +T627 UBERON:0000991 49745 49752 Gonadal +T628 SO:0000704 49783 49788 Genes +T629 CHEBI:33893 49999 50007 reagents +T630 UBERON:0000948 50416 50421 Heart +T631 SO:0000704 50626 50633 Genetic +T632 SO:0001026 50638 50645 genomic diff --git a/src/ontogpt/evaluation/craft/database/all/16462940.txt b/src/ontogpt/evaluation/craft/database/all/16462940.txt new file mode 100644 index 000000000..253983ae2 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16462940.txt @@ -0,0 +1,273 @@ +Genetic and Genomic Analysis of a Fat Mass Trait with Complex Inheritance Reveals Marked Sex Specificity + +Abstract + +The integration of expression profiling with linkage analysis has increasingly been used to identify genes underlying complex phenotypes. The effects of gender on the regulation of many physiological traits are well documented; however, “genetical genomic” analyses have not yet addressed the degree to which their conclusions are affected by sex. We constructed and densely genotyped a large F2 intercross derived from the inbred mouse strains C57BL/6J and C3H/HeJ on an apolipoprotein E null (ApoE−/−) background. This BXH.ApoE−/− population recapitulates several “metabolic syndrome” phenotypes. The cross consists of 334 animals of both sexes, allowing us to specifically test for the dependence of linkage on sex. We detected several thousand liver gene expression quantitative trait loci, a significant proportion of which are sex-biased. We used these analyses to dissect the genetics of gonadal fat mass, a complex trait with sex-specific regulation. We present evidence for a remarkably high degree of sex-dependence on both the cis and trans regulation of gene expression. We demonstrate how these analyses can be applied to the study of the genetics underlying gonadal fat mass, a complex trait showing significantly female-biased heritability. These data have implications on the potential effects of sex on the genetic regulation of other complex traits. + +Synopsis + +Although their genomes are nearly identical, the males and females of a species exhibit striking differences in many traits, including complex traits such as obesity. This study combines genetic and genomic tools to identify in parallel quantitative trait loci (QTLs) for a measure of gonadal fat mass and for expression of transcripts in the liver. The results are used to explore the relationship between genetic variation, sexual differentiation, and obesity in the mouse model. Using over 300 intercross progeny of two inbred mouse strains, five loci in the genome were found to be highly correlated with abdominal fat mass. Four of the five loci exhibited opposite effects on obesity in the two sexes, a phenomenon known as sexual antagonism. To identify candidate genes that may be involved in obesity through their expression in the liver, global gene expression analysis was employed using microarrays. Many of these expression QTLs also show sex-specific effects on transcription. A hotspot for trans-acting QTLs regulating the expression of transcripts whose abundance is correlated with gonadal fat mass was identified on Chromosome 19. This region of the genome colocalizes with a clinical QTL for gonadal fat mass, suggesting that it harbors a good candidate gene for obesity. + +Introduction + +Females and males share nearly identical genetic information, but vary widely with respect to disease susceptibility [1,2]. Apart from the obvious gender-specific diseases such as cervical or prostate cancer, sex influences susceptibility to nearly all highly prevalent diseases that affect both women and men, including atherosclerosis and diabetes and their precursor conditions of hyperlipidemia, obesity, and insulin resistance. These are multifactorial in their pathogenesis, encompassing environmental and behavioral aspects, as well as significant genetic determination, on which these other factors interact. The genetic component is multigenic, with the heritability embedded in the genetic variation intrinsic to our population. Although the sex differences in disease susceptibility are recognized, the interplay between sex and gene expression that is at the basis of these differences is not well understood. Females and males inherit (on average) the same genes that may be risk factors, but their expression and effect on disease risk varies significantly. An understanding and recognition of the significance of specific disease-associated polymorphisms/mutations in the context of sex is therefore of critical clinical importance. + +We have utilized the mouse as a model system to study the genetics of metabolic and vascular diseases [3–5]. Rather than focus initially on natural or induced mutants of single genes, we utilize the complex endogenous genetic variation between strains in genetic crosses to identify causative genetic loci and ultimately the underlying variations responsible for trait differences [6]. This design more closely reflects the situation faced when studying human populations. The genetic composition of each individual mouse is restricted to that of the two parental strains and is defined at every locus across the genome. The application of quantitative trait locus (QTL) analysis allows the identification of those chromosomal regions that contain a genetic variation that influences trait expression (for a comprehensive review on QTLs see [7]). We [5,8], and others [9–17], have recently extended the power of this approach by incorporating genome-wide gene expression array analysis, which allows us to model the “genetics of gene expression” using similar methods. An immediate extension of this approach is toward dissecting the genetic regulation of complex phenotypes, which would greatly improve the progression from candidate locus to candidate gene. + +Here we report the application of this integrated approach to study the significance of sex on the genetic determinants of obesity and the associated regulation of liver gene expression in an F2 intercross derived from the inbred strains C57BL/6J (B6) and C3H/HeJ (C3H) on an apolipoprotein E null (ApoE−/−) background. The BXH.ApoE−/− population was designed to recapitulate several of the phenotypes associated with the so-called metabolic syndrome. The cross consists of 334 animals of both sexes, allowing us to specifically test for the dependence of QTLs on sex. We detected several thousand gene expression QTLs (eQTLs), a significant proportion of which were sex-biased. We used these analyses to dissect the genetic regulation of the gonadal fat mass trait and to identify genes associated with the trait. + +Results + +QTLs Associated with Gonadal Fat Mass + +Characteristics of the B6.ApoE−/− and C3H.ApoE−/− parents and the F2 BXH.ApoE−/− generation on a Western diet are summarized in Table 1. Gonadal fat mass differed significantly between the sexes in F2 (p < 10−4) and in the parental C3H.ApoE−/− (p < 0.05), but not in B6.ApoE−/− mice. Gonadal fat mass was the fat pad collection that represented the most animals and the most accurate collections and was thus chosen for further analysis. Broad sense heritability (h2) calculated as (σ2Total − σ2Parental)/σ2Total for the gonadal fat mass trait was 54% for females and 36% for males, which is in close agreement with previous reports [18,19] and demonstrates significant heritability of gonadal fat mass. + +A total of 334 F2 mice were genotyped at an average 1.5 cM density using 1,032 single nucleotide polymorphisms (SNPs) spanning all 19 autosomes. QTL analysis for several clinical traits (clinical QTLs [cQTLs]), including the unadjusted raw values for gonadal fat mass, was performed using a single marker regression approach (justified by the high-density of markers, making interval mapping unnecessary). In order to test specifically for sex effects of linkage, we included additive, dominant, sex, sex-additive, and sex-dominant parameters in our calculations (see Materials and Methods). A stepwise regression procedure was used to determine whether the addition of the final two terms significantly improved the linear regression model, conditional on realizing a significant additive QTL effect. We performed permutation analyses over all gene expression traits, estimating false discovery rates (FDRs) at different logarithm of odds (LOD) score thresholds and assessing the overall rate of QTL detection. From these analyses we constructed receiver operating characteristic (ROC)-like curves to demonstrate that our straightforward method has significantly increased power to detect QTLs compared to QTL mapping methods that do not incorporate sex and genotype–sex interactions (Figure S1). It is clear from the ROC curves that the sex and sex-interaction terms add significantly to the detection of QTL for the gene expression traits. Using previously described conventions [20], QTL models without the final two interaction terms (sex*add and sex*dom) have a suggestive threshold of 3.0 (p < 1 × 10−3) and a significant threshold of 4.3 (p < 5 × 10−5, genome-wide p < 0.05). QTL models incorporating only the sex*add interaction term in addition to the additive terms have one extra degree of freedom that leads to a corresponding increase in the LOD score thresholds to 3.5 (suggestive) and 4.9 (significant) for the 0.001 and 0.00005 p-value thresholds, respectively. QTL models incorporating both sex*add and sex*dom interaction terms possess two extra degrees of freedom, with a corresponding increase in LOD score thresholds to 4.0 (suggestive) and 5.4 (significant) for the 0.001 and 0.00005 p-value thresholds, respectively. + +One suggestive (Chromosome 1) and four significant (Chromosomes 3, 5, 11, and 19) cQTLs for the gonadal fat mass trait were identified (Figure 1A; Table 2). Four out of the five cQTLs showed statistically significant better fits with the full model incorporating the interaction terms sex*add and sex*dom, compared to the model including only the additive terms. Interestingly, the cQTL over Chromosome 11 did not improve, suggesting that the additional terms did not contribute to improved detection of this locus. Table 2 summarizes the position and LOD score of maximal linkage for each cQTL. While the focus of this study was the gonadal fat mass trait, it is noted that a genome scan for the adiposity trait resulted in cQTLs at the same locations, with very similar LOD scores and sex dependence (unpublished data). + +Results from the various regression models used to determine linkage for the Chromosome 5 cQTL are depicted in Figure 1B. Analysis of all animals with and without sex as a covariate failed to demonstrate evidence of linkage on Chromosome 5. When females were analyzed alone, a suggestive LOD score of 3.7 was realized (p = 2 × 10−4); males analyzed alone did not demonstrate evidence for linkage. However, using all 334 animals and adding the interaction terms to the QTL model significantly improved sensitivity, and a cQTL with a maximum LOD of 7.56 (p = 1.7 × 10−6) was realized. + +Given the improved detection of four of the five cQTLs when sex-additive and sex-dominant interaction terms were considered, we hypothesized that the main genotype effect of these cQTLs on the gonadal fat mass trait would differ between the sexes (Figure 2). Indeed, cQTLs located on Chromosomes 1, 3, and 5 showed opposing effects on fat mass, or sex antagonism. The effect of the cQTL on Chromosome 11 was in the same direction in both males and females, but was sex-biased toward a larger effect in females (R2 = 0.091 in females versus R2 = 0.046 in males), confirming the minimal sex specificity of this cQTL. The cQTL on Chromosome 19 showed a sex-specific effect in females, with no effect in males. + +Overall, all five cQTLs for gonadal fat mass were biased toward a larger effect in females. Assuming purely additive effects of each genotype, these cQTLs account for approximately 42% of the variation in female F2 mice and 13% in males, consistent with the narrow sense heritability estimates for this trait and again demonstrating significant differences in the regulation and heritability of the gonadal fat trait between the sexes. + +Liver eQTLs + +Livers from 312 F2 animals (156 female, 156 male) were profiled using oligonucleotide microarrays manufactured by Agilent Technologies (Palo Alto, California, United States), which included probes for 23,574 mouse transcripts. Individual transcript intensities were corrected for experimental variation and normalized and are reported as the mean log10 ratio (mlratio) of an individual experiment relative to a pool composed of 150 mice randomly selected from the F2 population [21]. Each measurement was fitted to an error model and assigned a significance measurement (type I error). A heat map of the 2,320 transcripts most differentially expressed (p < 0.05 in 10% or more of animals) relative to the pool is depicted in Figure 3. This selection of genes was not biased on a priori known differential expression between the sexes, linkage, or correlation with a clinical phenotype. This is noteworthy because hierarchical clustering of these transcripts against the 312 F2 mice shows an almost perfect clustering into male and female subgroups, emphasizing striking effects of sex on liver gene expression levels and suggesting that sex is controlling more variance in these transcripts' expression than any other parameter. + +The expression values of the 23,574 transcripts were treated as quantitative traits and fitted to the same linear regression models used to compute LOD scores for clinical traits (eQTLs). The FDR at each threshold was determined by permuting the data 100 times and taking the mean number of QTLs detected over all of the permuted datasets at a given threshold, and dividing this count by the number of QTLs detected at the same threshold in the observed data. At the threshold for significant linkage (p < 5 × 10−5, genome-wide p < 0.05, based on a single trait), the FDR was estimated at 3.4% for the standard QTL model not accounting for any sex terms, 3.1% for the QTL model accounting for additive sex effects, and 3.2% for the QTL model accounting for additive sex effects and allowing for the sex interaction terms to enter the model. A list of all detected suggestive (p < 1 × 10−3) and significant eQTLs (p < 5 × 10−5) detected in the BXH.ApoE−/− intercross is provided in Table S1. + +Characteristics of the eQTLs at different significance levels are summarized in Table 3 and shown graphically in Figure 4. We detected 6,676 eQTLs representing 4,998 transcripts at the 5 × 10−5 significant level. Of these, 2,118 eQTLs were located within 20 Mb (roughly 10 cM) of the corresponding gene, likely representing eQTLs regulated by cis-acting variation within the gene itself. Of the 6,676 significant eQTLs, 1,166 (17%) demonstrated a sex bias and were subsequently significantly improved with the addition of the sex-additive and sex-dominant terms. + +The distribution of all 6,676 significant eQTLs (p < 5 × 10−5) across the genome in 2 cM bins is shown in Figure 4A. Evidence for eQTL hotspots is clear on Chromosomes 1, 2, 4, 5, 6, and 7 where significant fractions of the 6,676 eQTLs colocalize within 2 cM regions on each chromosome. Approximately 67% of eQTLs at this threshold are trans, and these eQTL hotspots consist primarily of trans-acting effects on transcriptional variation. Distribution of the 1,166 eQTLs with significant sex effects, of which 852 (73%) are trans, showed enrichment on Chromosome 5 at approximately 49 cM (Figure 4B) as assessed using the Fisher exact test (p = 8.7 × 10−25 after Bonferroni correction). At this locus there were 250 eQTLs, 140 of which exhibited genotype–sex interactions. + +At increasing thresholds for linkage, a higher fraction of detected eQTLs were cis-acting (Figure 4C). The increased proportion of cis-eQTLs with increasing LOD score thresholds has been reported before [5,15] and confirms what is likely to be our increased power to detect first-order cis-acting variations affecting transcription. The proportion of eQTLs with significant sex effects remained relatively constant at all thresholds (Figure 4C). Furthermore, the majority of these sex-specific eQTLs (73%) are acting in trans on a given gene's expression (Figure 4D), and similar proportions of sex-specific eQTLs (26%) are cis compared to the proportion of all liver cis-eQTLs (32%). These data demonstrate the profound effects of sex on the genetic regulation of gene expression. + +Cis-eQTLs Are Candidate Genes for the Gonadal Fat Mass Trait + +Given the marked effects of sex on liver gene expression and the genetic regulation of gonadal fat mass, we reasoned that cis-eQTLs with significant “sex-additive” and “sex-dominant” interactions would be potential candidate genes for our trait. Of the 2,118 significant cis-eQTLs, 304 (14%) were improved by the sex-interaction terms. Cis-eQTLs overlapping the confidence interval for the fat mass cQTL are candidate genes for the trait. Those genes with significant sex interactions receive increased consideration as potential candidates (Tables 4 and 5). + +Thousands of Genes Show a Sex-Specific Correlation with Gonadal Fat Mass + +For each of the 23,574 oligonucleotides represented on the array, we computed a linear regression analysis to test for association between the trait “gonadal fat mass” and each transcript abundance measure, incorporating the terms “gene,” “sex,” and “gene-by-sex,” where the “gene-by-sex” parameter tests for sex-specific correlation between a gene and the trait. As before, a stepwise regression procedure was used to determine if the addition of the interaction term significantly improved the model fit (see Materials and Methods). Multiple testing was addressed by controlling for the FDR. Distribution of the p-values obtained from these 23,574 correlations is shown in Figure 5A. At FDR = 0.01, 4,613 genes were significantly correlated with gonadal fat mass. Of these genes, 4,524 (98%) showed significant “gene-by-sex” effects, supporting the high degree of sex specificity in the genetic regulation of this trait. A complete list of all genes correlated with gonadal fat mass is provided in Table S2. + +Of the 4,613 genes correlated with gonadal fat mass, 1,130 generate 1,478 significant eQTLs (Figure 5B). The colocalization of eQTLs for these correlated genes with the cQTL for the fat mass trait provides useful implications for the possible role of these genes. Whether the eQTLs are cis or trans determines what that role may be. Genes that show significant correlation with the gonadal fat mass trait and that have cis-eQTLs coincident with the fat mass cQTLs are potential candidate genes for the trait (i.e., they may contain a genetic variation in that gene that is the cause of the trait cQTL). Table 5 summarizes the genes that possess these properties for each cQTL, increasing evidence for these genes as potential candidates. As addressed below, given the complex multiorgan regulation of adipose tissue mass, it is unlikely that the genetic regulation of all five loci resides in the liver. However, some may involve the liver, and even for those that do not, the liver transcriptional variation may reflect that of the relevant tissue. + +Genes that show significant correlation with gonadal fat mass and have trans-eQTLs coincident with the fat mass cQTL cannot be candidates directly responsible for the trait, as they are physically located elsewhere in the genome. However, they are potentially involved in the pathway(s) leading from the causative gene to the expression of the fat mass trait (i.e., their transcription is closely regulated by the causative gene at the locus). All of the five fat mass cQTLs have colocalizing trans-eQTLs for correlated genes. However, for a trait such as fat mass that is regulated by multiple tissues and organs, it is unlikely that all five fat mass cQTLs are primarily driven by liver gene expression. As an approach to this problem, we hypothesized that those cQTLs that are most closely associated with liver gene expression would show an overrepresentation of colocalized eQTL for correlated genes, while those loci primarily controlled by other tissues would not have shown this pattern. + +To assess this, we first determined the distribution of these 1,478 eQTLs across the genome in 2-cM bins as shown in Figure 5C. In order to see if there exist any hotspots for these eQTLs, we tested eQTLs with p < 0.001 for enrichment along the genome in overlapping 2-cM bins against the distribution of all liver eQTLs (Figure 4A) using a Fisher exact test. Figure 5D shows the significance of enrichment reported as −log10 of the enrichment p-value across the genome. One locus on Chromosome 19 was significantly enriched for eQTLs of transcripts correlated with the gonadal fat mass trait. As anticipated, there was an overlap of a correlated eQTL hotspot and a fat mass cQTL, specifically Chromosome 19 at 40 cM. This suggests that the genetic regulation of fat mass for the Chromosome 19 locus is more closely tied to liver gene expression than are the other four fat mass cQTLs. + +The effect of the trans-eQTLs at the Chromosome 19 locus on gene expression is summarized in Figure 6. Twenty-nine trans-eQTLs colocalize to Chromosome 9 at 40 cM, suggesting that 29 genes correlated with gonadal fat mass are regulated in trans by a polymorphism at this position. The proportion of gene expression levels controlled by this locus (approximated as the coefficients of determination R2) differs between males and females for the majority of the transcripts (as in Figure 6A), and for Chromosome 19 (Figure 6B), females demonstrated greater genetic regulation of gene expression than males. This substantial female bias is significantly higher than would be expected to arise by chance for the Chromosome 19 locus (p < 0.001 by χ2). This locus corresponds to one of the four sex-biased cQTLs for gonadal fat mass reported in this study, and the significant sex specificity of both the cis and trans genetic regulation of liver genes correlated with fat mass supports the functional significance of this locus in this organ. + +Discussion + +In this study, we described a large, densely mapped, segregating F2 mouse population designed to study the genetic regulation of several traits associated with the so-called metabolic syndrome. Several groups, including ours, have reported the advantage of combining traditional genetics with genome-wide gene expression analysis for the dissection of complex traits. This study improved on past models by including over 300 animals (three times the size of previous studies) of both sexes, allowing for the incorporation of sex-specific effects on underlying genetic regulation. + +Significant Sex Bias in the Regulation of Both Complex Traits and Gene Expression + +Given the known dichotomy between females and males in the susceptibility and control of obesity, this study was designed to sufficiently power the detection of significant QTLs for this and other traits with sex-dependent effects. Note, however, that these effects can extend to traits without overall mean differences between the sexes. Previous studies have described the advantages of performing QTL analysis both with and without sex as an interactive covariate [22–25]. Analyzing the sexes separately is suboptimal since it reduces sample size in both groups, thus reducing power to detect main QTL effects, as demonstrated by our genome scan of Chromosome 5 (Figure 1B). Furthermore, separate analyses would not allow for the detection of QTLs that have opposing, or sex-antagonistic, effects in females and males and would hinder the detection of QTLs specific to one sex. + +Accordingly, we detected five cQTLs for the gonadal fat mass trait on Chromosomes 1, 3, 5, 11, and 19. The detection of all five cQTLs was “driven” by the larger effect in females, with significant improvement by the incorporation of sex*additive and sex*dominant parameters. QTLs associated with obesity, gonadal fat, and abdominal fat have been reported before overlapping with cQTLs on Chromosomes 1 [26–28], 5 [26,29], and 11 [19,29] reported here, whereas the cQTL on Chromosome 3 represents a novel QTL for this trait. The Chromosome 19 cQTL for fat mass was recently reported by us [5] in the BXD intercross F2 progeny from the strains B6 and DBA (which shares the same haplotype at this region as the C3H strain used in this study). Interestingly, significant heritability and genetic regulation was seen in this F2 population despite the hyperlipidemic, proinflammatory ApoE−/− background and the high-fat Western diet. This background possesses several advantages, such as allowing the modeling of human-like disease states. The predominantly female-driven effects of the five cQTLs likely reflect the significant effect of differential gonadal hormone secretions on the genetic regulation of this complex trait. + +The identification of genes underlying cQTLs remains a challenge. The widespread availability of genome-wide expression analysis has begun to address this by providing a snapshot of transcription in relevant organs and thus providing initial information for which genes can differentiate a given trait. Furthermore, by treating transcript levels as quantitative traits, we can map the genetic regulation underlying differential gene expression (eQTLs). Those eQTLs that have cis-acting variations affecting their transcription are potential candidate genes for the trait. At a single trait, genome-wide significance level of 0.05, we detected 6,676 eQTLs representing 4,998 genes, of which 2,118 were cis-acting. At increased thresholds, the proportion of cis-eQTLs increased, which is in good agreement with previous studies [5,15] and likely reflects the increased power to detect cis-acting variations affecting transcription. Of all 6,676 significant eQTLs, 1,166 possessed significant sex interactions. Of these, 304 were cis and 852 were trans, suggesting that only a minority of the sex-specific effects on the regulation of gene expression occur through polymorphisms within the gene itself. Rather, underlying genetic regulation of most transcripts is the result of interactions between trans loci and sex-specific factors (e.g., hormones). As with cQTLs, sex bias in the predominantly trans genetic regulation of gene expression is likely secondary to different sex hormone profiles. + +Recently, using a similar dataset, our group demonstrated that significant cis-eQTLs (p < 5 × 10−5) largely represent true positives [30] and are enriched for highly polymorphic regions over the mouse genome. The cis-eQTLs presented in Table 5 overlap with one of the gonadal fat mass cQTLs and should be considered potential candidates. Given the sex effects in the gonadal fat mass cQTLs, we reasoned that the cis-eQTLs with significant sex*additive and sex*dominant effects should receive priority consideration. The use of eQTLs to dissect cQTLs is a method still in its infancy, with uncertain efficacy and applicability. Nevertheless, application of this analysis to this dataset provides some tantalizingly attractive candidate genes. + +One shortcoming of this approach, however, is that candidate genes are limited to those whose transcript expression levels vary in association with a nearby polymorphism that differs between the parental strains—in other words, genes with significant and detectable cis-eQTLs. However, it is not strictly necessary for candidate genes to have evidence of such linkage: polymorphisms underlying a trait cQTL can affect gene function or posttranslational modifications. Nevertheless, several phenotypes are known to be regulated, at least partly, at the level of transcription or mRNA stability, which is exactly what our methods are designed to detect. A separate problem is that organ-specific gene expression differences may preclude one from detecting the relevant causative gene if the tissue arrayed is not the tissue where the control is exerted. This is particularly relevant for a trait such as adipose tissue mass, which is controlled by multiple tissues. We propose that analysis of correlated genes can provide guidance as discussed below. + +Genes Correlated with Gonadal Fat Mass Illustrate Tissue-Specific Regulation of the Trait + +In an effort to identify genes associated with the fat mass trait, but not necessarily candidate genes underlying the trait cQTLs, we fitted linear models to assess the degree of association between transcripts and gonadal fat mass. As with QTLs, sex-specific correlations were modeled. At an FDR of 1%, 4,613 genes were found to be significantly correlated with gonadal fat mass, of which 4,254 (98%) showed sex-biased correlation. As indicated in Tables 4 and 5, several genes with detectable cis-eQTLs are also significantly correlated with the trait and are even further prioritized as candidate genes. + +Thus far, studies that have examined the “genetics of gene expression” are in good agreement regarding the increased power to detect cis-eQTLs relative to trans [5,9,15,16,31]. It is unclear at this time, however, what exactly is the significance of trans-eQTLs and the nature of the underlying polymorphisms associated with them. Furthermore, the eQTL hotspots reported in this and previous studies [5,9,10] largely represent trans-eQTLs. This localization suggests some functional significance to these regions. Of the 4,613 genes correlated with gonadal fat mass, 1,130 generate 1,478 significant eQTLs, of which 1,023 (69%) are trans-acting. These eQTLs are significantly enriched at one locus (Chromosome 19). Interestingly, this hotspot was coincident with a cQTL associated with fat mass reported in this study. Since these transcripts represent those significantly correlated with gonadal fat mass, the localization of their eQTLs to these regions strongly supports the notion that the genes with trans-eQTLs represent downstream targets of candidate regulatory genes located at the position of significant linkage. This means that the genes may be causal but downstream of the gene responsible for the cQTLs, or they may be reacting to the increased gonadal fat mass and associated metabolic changes. These data also suggest that identifying such loci that show overrepresentation of highly correlated genes is a means to identify which of the trait cQTLs are more likely controlled by the tissue arrayed. As expected, the Chromosome 19 locus was enriched for trans-eQTLs with substantially greater effects in females. Functional and promoter analysis of genes with a common trans-eQTL may prove enlightening. Furthermore, gene expression network construction and analysis may be improved by the incorporation of experimentally demonstrated cis versus trans regulation. + +Conclusion + +The integration of traditional genetics with genome-wide expression analysis was first proposed by Jansen and Nap 4 y ago [32]. Advances in genomic technology and bioinformatic resources since then have vastly improved the applicability of these methods to the dissection of complex traits. Taking into account sex-specific effects will similarly improve the sensitivity to detect underlying genetic regulation, especially for phenotypes known to be affected by sex. Furthermore, network analyses are increasingly being applied to complex phenotypes [11,33]. Regardless of which variables are used in the construction of these networks, whether they measure gene expression or protein interactions, accounting for sex specificity, hormonal status, or construction of different networks for females and males would likely more accurately represent the complexity associated with these phenotypes. + +We reported here on the initial genetic and genomic analysis of an F2 intercross population designed to recapitulate several traits associated with human metabolic syndrome. Using 334 mice of both sexes genotyped at high density, this is the largest study of its kind to date designed, and it is strongly powered to detect subtle effects of genetic regulation and sex specificity. We identified five cQTLs for the gonadal fat mass trait, all with greater effects in females. We also detected several thousand significant liver eQTLs, a significant fraction of which are sex-biased, demonstrating how meaningful effects of sex on gene expression extend beyond overall mean differences. We demonstrated the application of linkage and correlation methods to identify candidate genes. Finally, we showed that localization of a subset of liver genes linked in trans to a cQTL region can identify relative tissue contributions to the genetic regulation of a complex trait. We anticipate that the application of these and similar methods would significantly improve the elucidation of the genetic regulation underlying complex phenotypes. + +Materials and Methods + +Animals and tissue collection. + +C57BL/6J ApoE−/− (B6.ApoE−/−) were purchased from Jackson Laboratory (Bar Harbor, Maine, United States). C3H/HeJ ApoE−/− (C3H.ApoE−/−) were generated by backcrossing B6.ApoE−/− to C3H for ten generations. F1 mice were generated from reciprocal intercrossing between B6.ApoE−/− and C3H.ApoE−/−, and F2 mice were subsequently bred by intercrossing F1 mice. A total of 334 mice (169 female, 165 male) were produced. All mice were fed Purina Chow containing 4% fat until 8 wk of age and then transferred to a “Western” diet containing 42% fat and 0.15% cholesterol for 16 wk. Mice were sacrificed at 24 wk of age. At death, livers were immediately collected and flash-frozen in liquid N2, and gonadal fat pads were extracted and weighed. + +RNA sample preparation, microarray hybridization, and expression analysis. + +RNA preparation and array hybridizations were performed at Rosetta Inpharmatics (Seattle, Washington, United States). The custom ink-jet microarrays used in this study (Agilent Technologies, previously described [21,34]) contain 2,186 control probes and 23,574 noncontrol oligonucleotides extracted from mouse UniGene clusters and combined with RefSeq sequences and RIKEN full-length clones. + +Mouse livers were homogenized and total RNA extracted using TRIzol reagent (Invitrogen, Carlsbad, California, United States) according to manufacturer's protocol. Three micrograms of total RNA was reverse transcribed and labeled with either Cy3 or Cy5 fluorochrome. Purified Cy3 or Cy5 complementary RNA was hybridized to at least two microarray slides with fluor reversal for 24 h in a hybridization chamber, washed, and scanned using a laser confocal scanner. Arrays were quantified on the basis of spot intensity relative to background, adjusted for experimental variation between arrays using average intensity over multiple channels, and fitted to an error model to determine significance (type I error). Gene expression is reported as the mlratio relative to the pool derived from 150 mice randomly selected from the F2 population. For subsequent analyses, mlratio data are assumed to be normally distributed, a valid assumption as previously demonstrated [21,30]. The error model used to assess whether a given gene is significantly differentially expressed in a single sample relative to a pool comprised of a randomly selected subset of 150 samples has been extensively described and tested in a number of publications [35,36]. + +Genotyping and linkage statistics. + +Genomic DNA was isolated from kidney by phenol-chloroform extraction. An examination of existing databases identified over 1,300 SNPs that showed variation between the B6 and C3H strains, and a complete linkage map for all 19 autosomes was constructed using 1,032 of these SNPs at an average density of 1.5 cM. Genotyping was conducted by ParAllele (South San Francisco, California, United States) using the molecular-inversion probe (MIB) multiplex technique [37]. Testing for linkage of both clinical traits and gene expression (using mlratio) was conducted using a linear model. Consider a phenotype denoted by y. The linear model that relates variation in y to QTLs and other covariates (e.g., sex) is given by the general form + +where μ is the trait mean, X′ a vector of covariates, β being the associated vector of regression coefficients, and e the residual error. Linkage was computed for over 20 clinical traits as well as 23,574 liver transcripts. Standard genome scans calculate linkage by comparing the linear model + +to the null model + +where β1 and β2 are the regression coefficients of the additive and dominant parameters, respectively. The LOD score represents the difference in the log10 of the likelihood of the above two equations, where the individual model likelihoods are maximized with respect to the model parameters, given the marker genotype and phenotype data. If a trait y differs on average between the two sexes but the QTL has the same effect in both males and females, we can model this interaction by including sex as an additive covariate in the above models, resulting in the new model + +which is then compared to the null model + +where β3 is the regression coefficient of the sex parameter. The effect of a QTL may be dependent on the state of a covariate; for instance, a QTL may have an effect specific to one sex, or may have opposite effects in the two sexes. This interaction can be modeled using a full model, which accounts for all additive covariates, as well as interactions between the covariates: + +which is compared to the above null model (Equation 5). + +The full model (Equation 6) allows us to model all heritable and sex-specific interactions in a single equation and maximally powers us to detect significant QTLs when the sex and sex-interaction terms are significant, given all 334 animals are included in the analysis. Furthermore, using the full model obviates the need for modeling QTLs in one sex only, a procedure that could decrease our power by halving the sample size, rendering it impossible to detect interactions between QTLs and sex. However, because the full model contains two more parameters than the model that treats sex as an additive covariate (Equation 4), the LOD score threshold for significant linkage is higher (using the convention of Lander and Kruglyak [20]), with QTL-specific significance levels of 2 × 10−3 and 5 × 10−5 equivalent to LOD scores of 4.0 (suggestive linkage) and 5.4 (significant linkage, equivalent to genome-wide p < 0.05), respectively. As a result, if there are no significant sex-additive or sex-dominant interactions, the full model will actually reduce power to detect linkage. To minimize the loss in power of fitting the full model when the sex-interaction terms are not significant, we employed a model selection procedure that introduces sex-interaction terms only if they add significantly to the overall QTL model. + +The model selection procedure makes use of forward stepwise regression techniques to determine whether it is beneficial to include the sex-interaction terms, conditional on realizing a significant additive effect (p < 0.001). That is, the data are fitted to Equation 4 for a given marker, and if the add term is significant at the 0.001 significance level, then we attempt to add the sex*add term into the model. The sex*add term is retained in the model if the Bayesian information criterion (BIC) for this model is smaller than the BIC for Equation 4. If sex*add is included in the model as a result of this procedure, then we again use BIC to consider including the sex*dom term in the model. + +To determine which of the four models (Equations 2, 4, 6, and the model selection) is optimal, we empirically estimated the FDR for each model over a set of 3,000 genes randomly selected from the set of all genes detected as expressed in the liver samples. For each gene and for each marker we fitted each of the four models to the data. We performed this same analysis on ten separate permutation sets in which each of the 3,000 gene expression trait vectors was permuted such that the correlation structure among the genes was preserved. The FDR for a given LOD score threshold was then computed as the ratio of the mean number of QTL detected in the permuted datasets (the mean taken over the ten permuted datasets) and the total number of QTL detected in the observed 3,000-gene dataset. We then generated ROC-like curves by varying the LOD score threshold, resulting in a range of FDRs (from 0% to more than 50%). To simplify this type of summary, we considered no more than one QTL per chromosome per gene expression trait by considering only the QTL with the max LOD score on each chromosome for each trait. The ROC curves for each of the four models are shown in Figure S1, with the black curve corresponding to Equation 2, the blue curve corresponding to Equation 4, the red curve corresponding to Equation 6, and the green curve corresponding to the model selection procedure. + +It is clear from the ROC curves that the sex and sex-interaction terms are significant in the gene expression dataset. For example, at an FDR of 5% we note that 800 QTLs were detected in the set of 3,000 genes for model 1, while 968 (21% increase) and 1,159 (45% increase) QTLs were detected for Equations 4 and 6, respectively. These results demonstrate significantly increased power to detect QTLs when sex is taken into account. We further note that the stepwise selection procedure captures more information than Equation 4, indicating a significant interaction signature in this dataset and also demonstrating that this simple statistical procedure is capable of identifying significant interaction events even at conservative FDR thresholds. Finally, it is of particular note that Equation 4 performed better than Equation 6, the model that incorporated the interaction terms at all times. That is, despite there being a significant interaction signature, the signature was not large enough to justify including interaction terms for every expression trait and at every marker tested. This fact motivated the need to employ the forward regression procedure, and these results further motivate the need to explore sex effects by employing even more sophisticated QTL detection methods, such as that recently described by Yi et al. [38]. + +Additional statistics. + +For each 23,574 oligonucleotides represented on the array, we computed a linear regression analysis to test for a correlation between the trait “gonadal fat mass” and each transcript. Similar to our method of calculating linkage, we employed a stepwise linear regression procedure using equations of the form + +or + +compared to the null model + +where β0 is the intercept, and β1, β2, and β3 represent the regression coefficients of their respective terms. As before, the parameter “sex*gene” is only retained if it significantly improves the fit of the model. The p-value threshold for significant correlation is calculated by an F test, which compares the appropriate model (Equation 7 or 8) to the null model (Equation 9). As before, multiple testing was addressed with use of the FDR, ranking the p-values obtained from the above F tests and setting α = 0.01. + +Genes correlated with the gonadal fat mass trait generated several significant eQTLs. In order to determine if eQTLs generated by these genes were enriched in any locus or if they were distributed randomly, we compared the distribution of these eQTLs against the distribution of all liver eQTLs in overlapping 6-cM bins across the genome using the Fisher exact test. This test is based on exact probabilities from a specific distribution (hypergeometric distribution). p-Values obtained from this test were corrected for multiple comparisons using a simple Bonferonni correction (given that we performed 772 tests across the genome, 0.05/772). Loci with p < 6.5 × 10−5 by Fisher exact test were considered significantly enriched for eQTLs correlated with gonadal fat mass. + +Supporting Information + +Figure S1 + +ROC Curves for Each of the Four Models + +(21 KB DOC) + +Click here for additional data file. + +Table S1 + +Suggestive and Significant eQTLs + +(3.7 MB XLS) + +Click here for additional data file. + +Table S2 + +Transcripts Significantly Correlated with Gonadal Fat Mass + +(716 KB XLS) + +Click here for additional data file. + +Acknowledgements + +The authors wish to thank Steve Horvath, Desmond Smith, Xia Yang, Sudheer Doss, Anatole Ghazalpour, Pek Lum, and John Lamb for valuable discussions and insights. We thank Leslie Ingram-Drake for work on data preparation. Initial work on construction of the BXH.ApoE−/− cross was done by Weibin Shi. + +Abbreviations + +ApoE−/− - apolipoprotein E null + +cQTL - clinical QTL + +eQTL - gene expression QTL + +FDR - false discovery rate + +LOD - logarithm of odds + +mlratio - mean log10 ratio + +QTL - quantitative trait locus + +ROC - receiver operating characteristic + +SNP - single nucleotide polymorphism + +Figures and Tables + +Figure 1 + +Genome Scan for Gonadal Fat Mass + +(A) Animals were genotyped at an average 1.5 cM density using 1,032 SNPs polymorphic between the parental strains. LOD scores computed using sex as an additive covariate (black) failed to detect significant linkage. A genome scan accounting for interactions between sex and QTL (red) showed evidence for suggestive linkage on Chromosome 1 and significant linkage on Chromosomes 3, 5, 11, and 19. Dashed and solid lines are thresholds for suggestive (p < 1 × 10−3) and significant linkage (p < 5 × 10−5), respectively. + +(B) Genome scans for gonadal fat mass using different models over mouse Chromosome 5. Scans for fat mass using all animals with (black) and without (green) sex as an additive covariate failed to detect significant linkage. Females analyzed alone (magenta) showed evidence for suggestive linkage (p < 2 × 10−4). When both sexes were analyzed to account for sex effects (red), a significant QTL was realized (p < 10−6). + +For clarity, only the model incorporating both the “sex*add” and “sex*dom” terms is shown in red, although additional models incorporating the terms separately were also computed. + +Figure 2 + +Effect of Genotype on Fat Mass + +Homozygous B6 (BB), C3H (CC), or heterozygous (BC) genotype at all five QTL positions, separated by sex, are shown. The underlying genotypic effects of the QTLs on fat mass differ between the sexes. Coefficients of determination (R2) are shown along with associated ANOVAs *p < 0.05, **p < 0.01, ***p < 0.001. + +Figure 3 + +Heat Map of Liver Gene Expression + +Over 2,300 of the most differentially expressed genes in liver hierarchically clustered by animals (x-axis) against transcript levels (y-axis). Expression is reported as mlratio of individual experiment against a common pool. Red is over- and green underrepresented relative to pool. + +Figure 4 + +Properties of All Liver eQTLs + +(A) Distribution of all significant liver eQTLs across the genome in 2-cM bins. A total of 6,676 significant eQTLs were realized, representing 4,998 liver transcripts. Hotspots of nonrandom eQTL colocalization are clearly evident. + +(B) Distribution of eQTLs with significant sex-specific effects. A total of 1,166 eQTLs representing 1,044 transcripts show an eQTL hotspot on Chromosome 5. + +(C) Properties of eQTLs at increasing significance levels. As the threshold for significant linkage increases (p-value decreases, or LOD score increases), the proportion of cis-eQTLs (black) increases. The fraction of all eQTLs with sex effects (red) and cis-eQTLs with sex effects (blue) remains relatively constant at increasing thresholds. The dashed line indicates the genome-wide significance threshold (p < 5 × 10−5; genome-wide p < 0.05). + +(D) Properties of sex-specific eQTLs at increasing significance levels. For eQTLs with significant sex effects, as with all eQTLs, the proportion of cis-eQTLs (black) increases and trans (blue) decreases as the threshold for significance increases. At the genome-wide threshold for significance (dashed line), over 70% of eQTLs with significant sex effects are trans. + +Figure 5 + +Properties of Transcripts Significantly Correlated with Gonadal Fat Mass + +(A) Distribution of p-values for trait–gene correlations between transcripts and gonadal fat mass. At FDR = 0.01, 4,613 transcripts are significantly correlated with the trait. + +(B) Number of eQTLs generated by the 4,613 genes significantly correlated with gonadal fat mass. Of these, 1,130 genes possessed at least one significant eQTL. + +(C) Distribution of 1,478 eQTLs significantly correlated with gonadal fat mass across the genome in 2-cM bins. + +(D) Identification of genomic regions enriched for eQTLs correlated with gonadal fat mass. The x-axis represents genome position in 2-cM bins, and the y-axis represents the −log10 Fisher exact test p-value for enrichment of eQTLs in overlapping 6-cM bins. The dashed line corresponds to p = 0.05 after correction for multiple comparisons. One significantly enriched region on Chromosome 19 is shown. The Chromosome 19 (40-cM) hotspot is coincident with a cQTL for gonadal fat mass and is highlighted in red. + +Figure 6 + +The Effects of Sex on Trans-eQTL Correlated with Gonadal Fat Mass + +(A) Example of the effect of genotype at a trans locus on gene expression. Presence of homozygous B6 (BB), C3H (CC), or heterozygous (BC) genotype at a trans locus affects transcript MMT00016118 levels (reported as mlratio) in a sex-specific manner, with effects detectable only in females. Coefficients of determination (R2, or proportion variance explained) are shown along with associated ANOVA p-values. Several trans-eQTLs correlated with gonadal fat mass localize to regions overlapping with cQTLs for this trait, specifically, to Chromosome 19, 40 cM. + +(B) For Chromosome 19, the vast majority of these correlated trans-eQTLs are biased toward larger effects on gene expression in females (red lines). The effect of any given trans-eQTL is approximated as R2 determined in a manner similar to that depicted in (A). + +Table 1 + +Characteristics of the BXH.ApoE−/− Cross + +Table 2 + +cQTLs for Gonadal Fat Mass + +Table 3 + +Characteristics of Liver eQTLs + +Table 4 + +Properties of Genes Coincident with the Gonadal Fat Mass cQTL + +Table 5 + +Transcripts Coincident with cQTLs with Significant Sex Effects and Correlated with Gonadal Fat Mass Are Strong Candidate Genes for the Trait + +Footnotes + +Author contributions. SW, TAD, and AJL conceived and designed the experiments. SW, NY, and EES performed the experiments. SW, NY, HW, and TAD analyzed the data. EES and HW contributed reagents/materials/analysis tools. HW designed script and code to analyze data. SW, NY, EES, TAD, and AJL wrote the paper. + +Funding. Work was supported in part by National Institutes of Health grants HL-30568 and HL-28481 to AJL, Public Health Services Training Grant HD07228–24 to SW, the Iris Cantor-UCLA Women's Health Center, UCLA National Center for Excellence in Women's Health Grant to TAD, and by an American Heart Association Medical Student Research Grant to NY. + +Competing interests. The authors have declared that no competing interests exist. + +Citation: Wang S, Yehya N, Schadt EE, Wang H, Drake TA, et al. (2006) Genetic and genomic analysis of a fat mass trait with complex inheritance reveals marked sex specificity. 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IV +T300 PR:000015906 13229 13232;13234 13236 Syt ... IV +T301 NCBITaxon:10088 13247 13251 mice +T302 NCBITaxon:10088 13304 13308 mice +T303 NCBITaxon:10088 13380 13384 mice +T304 PR:000015906 13512 13518 Syt IV +T305 GO:0061743 13567 13581 motor learning +T306 PR:000003710 13628 13634 ADAM11 +T307 NCBITaxon:10088 13645 13649 mice +T308 GO:0061743 13695 13709 motor learning +T309 GO:0061743 13775 13789 motor learning +T310 PR:000003710 13793 13799 ADAM11 +T311 NCBITaxon:10088 13810 13814 mice +T312 GO:0007612 13908 13913 learn +T313 GO:0061743 13936 13950 motor learning +T314 PR:000003710 13962 13968 ADAM11 +T315 NCBITaxon:10088 13979 13983 mice +T316 UBERON:0002037 14021 14031 cerebellar +T317 GO:0007612 14042 14050 learning +T318 GO:0008306 14068 14077;14085 14097 classical ... conditioning +T319 UBERON:0001711 14078 14084 eyelid +T320 PR:000003710 14177 14183 ADAM11 +T321 NCBITaxon:10088 14194 14198 mice +T322 PR:000003710 14251 14257 ADAM11 +T323 NCBITaxon:10088 14268 14272 mice +T324 UBERON:0002616 14473 14484 brain sites +T325 UBERON:0002037 14513 14523 cerebellum +T326 GO:0007612 14577 14585 learning +T327 PR:000003710 14626 14632 ADAM11 +T328 NCBITaxon:10088 14643 14647 mice +T329 GO:0097617 14657 14670 hybridisation +T330 PR:000003710 14693 14699 Adam11 +T331 SO:0000704 14700 14704 gene +T332 GO:0010467 14700 14715 gene expression +T333 UBERON:0002929 14723 14732 pyramidal +T334 CL:0000598 14723 14738 pyramidal cells +T335 UBERON:0003881 14742 14745;14750 14756 CA1 ... fields +T336 UBERON:0003883 14746 14756 CA3 fields +T337 CL:0000120 14761 14774 granule cells +T338 UBERON:0001885 14782 14795 dentate gyrus +T339 CL:0000120 14822 14836 granular cells +T340 UBERON:0002037 14844 14854 cerebellum +T341 GO:0007612 14951 14959 learning +T342 UBERON:0002037 15061 15071 cerebellum +T343 PR:000003710 15093 15099 ADAM11 +T344 NCBITaxon:10088 15110 15114 mice +T345 PR:000003710 15203 15209 ADAM11 +T346 CL:0000540 15226 15232 neuron +T347 CL:0000540 15233 15239 neuron +T348 CL:0000540 15243 15249 neuron +T349 CL:0000125 15250 15260 glial cell +T350 CHEBI:36357 15369 15378 molecules +T351 PR:000001023 15388 15417 neural cell adhesion molecule +T352 CHEBI:36357 15409 15417 molecule +T353 PR:000001023 15419 15423 NCAM +T354 UBERON:0001016 15478 15492 nervous system +T355 GO:0007399 15478 15504 nervous system development +T356 GO:0045202 15556 15564 synaptic +T357 UBERON:0007023 15583 15588 adult +T358 UBERON:0001016 15589 15603 nervous system +T359 CL:0000540 15621 15627 neuron +T360 GO:0060291 15756 15778 long-term potentiation +T361 GO:0060291 15780 15783 LTP +T362 GO:0045202 15820 15828 synaptic +T363 GO:0007612 15857 15865 learning +T364 GO:0007613 15870 15876 memory +T365 NCBITaxon:10088 15914 15918 mice +T366 PR:000008246 15950 15954;15964 15972 NR2A ... subunits +T367 PR:000008248 15959 15972 NR2C subunits +T368 GO:0098794 16058 16070 postsynaptic +T369 UBERON:0002037 16091 16101 cerebellar +T370 CL:0001031 16091 16115 cerebellar granule cells +T371 GO:0045202 16182 16190 synaptic +T372 GO:0007268 16182 16203 synaptic transmission +T373 UBERON:0002037 16242 16252 cerebellum +T374 PR:000003710 16256 16262 ADAM11 +T375 NCBITaxon:10088 16273 16277 mice +T376 PR:000003710 16330 16336 ADAM11 +T377 GO:0045202 16393 16401 synaptic +T378 GO:0045202 16439 16447 synaptic +T379 GO:0065007 16448 16458 regulation +T380 PR:000003710 16461 16467 ADAM11 +T381 NCBITaxon:10088 16478 16482 mice +T382 http://purl.obolibrary.org/obo/MONDO_0000437 16529 16535 ataxia +T383 GO:0007612 16579 16587 learning +T384 NCBITaxon:10088 16630 16634 mice +T385 PR:000003718 16647 16653 Adam22 +T386 SO:0000704 16654 16658 gene +T387 http://purl.obolibrary.org/obo/MONDO_0000437 16673 16679 ataxia +T388 UBERON:0001021 16721 16738 peripheral nerves +T389 GO:0016265 16744 16748 died +T390 NCBITaxon:10088 16770 16774 Mice +T391 PR:000003719 16787 16793 Adam23 +T392 SO:0000704 16794 16798 gene +T393 http://purl.obolibrary.org/obo/MONDO_0000437 16813 16819 ataxia +T394 GO:0016265 16835 16839 died +T395 PR:000003710 16889 16895 ADAM11 +T396 PR:000003718 16897 16903 ADAM22 +T397 PR:000003719 16908 16914 ADAM23 +T398 UBERON:0001016 16956 16970 nervous system +T399 SO:0001026 16974 16980 genome +T400 SO:0000855 17006 17015 orthologs +T401 PR:000003710 17019 17025 ADAM11 +T402 PR:000003718 17027 17033 ADAM22 +T403 PR:000003719 17038 17044 ADAM23 +T404 SO:0000704 17045 17050 genes +T405 NCBITaxon:7742 17063 17074 vertebrates +T406 NCBITaxon:40674 17083 17090 mammals +T407 NCBITaxon:8292 17102 17112 amphibians +T408 UBERON:0001016 17278 17292 nervous system +T409 NCBITaxon:1 17303 17312 organisms +T410 PR:000003710 17327 17333 ADAM11 +T411 NCBITaxon:10088 17344 17348 mice +T412 http://purl.obolibrary.org/obo/MONDO_0000437 17385 17391 ataxia +T413 UBERON:0001062 17540 17550 anatomical +T414 GO:0007612 17612 17620 learning +T415 UBERON:0002037 17625 17635 cerebellum +T416 PR:000003710 17668 17674 ADAM11 +T417 NCBITaxon:10088 17685 17689 mice +T418 CHEBI:15377 17722 17727 water +T419 SO:0000704 17761 17772 genetically +T420 NCBITaxon:10088 17782 17787 mouse +T421 UBERON:0001016 17910 17924 nervous system +T422 PR:000003710 17955 17961 Adam11 +T423 SO:0000704 17962 17966 gene +T424 SO:0001644 17967 17983 targeting vector +T425 PR:P23940 17997 18002 BamHI +T426 SO:0000147 18025 18030 exons +T427 PR:000003710 18045 18051 Adam11 +T428 SO:0000704 18052 18056 gene +T429 SO:0001026 18084 18091 genomic +T430 CHEBI:60004 18143 18146 mix +T431 NCBITaxon:10088 18215 18220 mouse +T432 PR:000003710 18221 18227 Adam11 +T433 SO:0000704 18228 18232 gene +T434 SO:0005853 18255 18263 cassette +T435 SO:0000147 18269 18274 exons +T436 PR:000003710 18288 18294 Adam11 +T437 SO:0000704 18295 18299 gene +T438 SO:0001026 18354 18361 genomic +T439 SO:0005853 18427 18435 cassette +T440 SO:0005853 18459 18467 cassette +T441 PR:000003710 18547 18553 Adam11 +T442 NCBITaxon:10088 18564 18568 mice +T443 SO:0001644 18585 18601 targeting vector +T444 UBERON:0000922 18630 18639 embryonic +T445 CL:0002322 18630 18644;18650 18655 embryonic stem ... cells +T446 CL:0002322 18646 18648;18650 18655 ES ... cells +T447 SO:0000112 18727 18734 primers +T448 SO:0005853 18793 18801 cassette +T449 SO:0001644 18858 18874 targeting vector +T450 CL:0002322 19027 19029 ES +T451 GO:0009566 19056 19066 fertilised +T452 NCBITaxon:10088 19071 19076 mouse +T453 CL:0000025 19077 19081 eggs +T454 UBERON:0007236 19089 19105 eight-cell stage +T455 GO:0007618 19140 19145 mated +T456 NCBITaxon:10088 19257 19261 mice +T457 NCBITaxon:10088 19306 19310 mice +T458 NCBITaxon:10088 19337 19342 Mouse +T459 SO:0001026 19343 19350 genomic +T460 UBERON:0002107 19410 19415 liver +T461 PR:P23940 19434 19439 BamHI +T462 SO:0001026 19449 19456 genomic +T463 CHEBI:37972 19482 19485 32P +T464 PR:P23940 19527 19532 BamHI +T465 SO:0001026 19541 19548 genomic +T466 SO:0000188 19569 19575 intron +T467 PR:000003710 19618 19624 ADAM11 +T468 NCBITaxon:10088 19646 19650 mice +T469 UBERON:0002037 19708 19718 cerebellum +T470 NCBITaxon:10088 19753 19758 mouse +T471 GO:0019835 19795 19805 cell lysis +T472 CHEBI:9754 19820 19824 Tris +T473 CHEBI:17883 19825 19828 HCl +T474 CHEBI:26710 19845 19849 NaCl +T475 CHEBI:63016 19854 19859 NP-40 +T476 CHEBI:60004 19880 19888 cocktail +T477 CHEBI:8984 20141 20144 SDS +T478 CHEBI:53250 20172 20176 PVDF +T479 GO:0042571 20231 20239 antibody +T480 PR:000003710 20252 20258 ADAM11 +T481 GO:0042571 20302 20312 antibodies +T482 NCBITaxon:3704 20334 20345 horseradish +T483 GO:0042571 20373 20381 antibody +T484 NCBITaxon:33208 20490 20496 animal +T485 NCBITaxon:33208 20549 20555 animal +T486 NCBITaxon:33208 20598 20604 Animal +T487 NCBITaxon:33208 20667 20673 Animal +T488 NCBITaxon:33208 20708 20714 Animal +T489 PR:000003710 20786 20792 ADAM11 +T490 NCBITaxon:10088 20808 20812 mice +T491 NCBITaxon:10088 21038 21043 mouse +T492 GO:0040011 21084 21093 Locomotor +T493 NCBITaxon:10088 21154 21158 mice +T494 CHEBI:15377 21328 21333 Water +T495 GO:0007612 21353 21361 learning +T496 CHEBI:15377 21407 21412 water +T497 NCBITaxon:10088 21440 21444 mice +T498 CHEBI:15377 21707 21712 water +T499 NCBITaxon:10088 21787 21792 mouse +T500 GO:0036268 21808 21812 swim +T501 NCBITaxon:33208 22056 22062 animal +T502 NCBITaxon:10088 22268 22273 mouse +T503 NCBITaxon:10088 22388 22393 mouse +T504 GO:0036268 22460 22464 swim +T505 GO:0036268 22477 22481 Swim +T506 NCBITaxon:10088 22683 22688 mouse +T507 NCBITaxon:10088 22738 22743 mouse +T508 GO:0036268 22759 22763 swim +T509 NCBITaxon:33208 22928 22934 animal +T510 NCBITaxon:10088 23230 23234 mice +T511 NCBITaxon:33208 23498 23504 animal +T512 NCBITaxon:10088 23570 23575 mouse +T513 NCBITaxon:10088 23655 23660 mouse +T514 UBERON:0002415 23764 23768 tail +T515 NCBITaxon:10088 23844 23849 mouse +T516 UBERON:0002415 24350 24354 tail +T517 NCBITaxon:10088 24478 24483 mouse +T518 NCBITaxon:10088 24492 24496 mice +T519 CHEBI:15377 24969 24974 water +T520 UBERON:0000479 25186 25193 tissues +T521 UBERON:0000955 25208 25213 brain +T522 UBERON:0002240 25215 25226 spinal cord +T523 UBERON:0000948 25228 25233 heart +T524 UBERON:0002048 25235 25239 lung +T525 UBERON:0002107 25241 25246 liver +T526 UBERON:0002113 25248 25254 kidney +T527 UBERON:0002106 25256 25262 spleen +T528 UBERON:0000473 25267 25273 testis +T529 NCBITaxon:10088 25279 25283 mice +T530 CHEBI:16842 25360 25368 formalin +T531 CHEBI:51686 25448 25459 hematoxylin +T532 SO:0000155 25557 25564 plasmid +T533 GO:0042571 25579 25587 antibody +T534 NCBITaxon:10088 25715 25719 mice +T535 NCBITaxon:10088 25824 25828 mice +T536 PR:000003710 26293 26299 Adam11 +T537 SO:0000417 26310 26316 Domain +T538 PR:000003710 26330 26336 ADAM11 +T539 SO:0001062 26346 26349 PRO +T540 SO:0001062 26351 26361 proprotein +T541 PR:000006928 26415 26418 EGF +T542 PR:000006928 26438 26441 EGF +T543 SO:0001077 26455 26457 TM +T544 SO:0001077 26459 26472 transmembrane +T545 GO:0016020 26464 26472 membrane +T546 GO:0005737 26480 26491 cytoplasmic +T547 SO:0000417 26492 26498 domain +T548 PR:000003710 26500 26506 Adam11 +T549 SO:0000704 26507 26511 gene +T550 SO:0000319 26548 26565 termination codon +T551 SO:0005853 26579 26587 cassette +T552 SO:0000147 26593 26598 exons +T553 SO:0005853 26616 26624 cassette +T554 SO:0001644 26643 26659 targeting vector +T555 PR:P23940 26757 26762 BamHI +T556 SO:0001023 26793 26799 allele +T557 SO:0001023 26819 26825 allele +T558 SO:0000112 26860 26867 primers +T559 CL:0002322 26929 26931 ES +T560 NCBITaxon:10088 26970 26975 mouse +T561 SO:0001026 26976 26983 genomic +T562 SO:0001023 27034 27040 allele +T563 SO:0001023 27059 27065 allele +T564 PR:000003710 27191 27197 ADAM11 +T565 GO:0010467 27198 27208 expression +T566 NCBITaxon:10088 27216 27221 mouse +T567 UBERON:0002037 27222 27232 cerebellum +T568 PR:000003710 27245 27251 ADAM11 +T569 UBERON:0002037 27280 27290 cerebellum +T570 PR:000003710 27312 27318 ADAM11 +T571 GO:0042571 27330 27338 antibody +T572 NCBITaxon:10088 27386 27391 mouse +T573 PR:000003710 27392 27398 ADAM11 +T574 GO:0010467 27407 27416 expressed +T575 PR:000003710 27470 27476 ADAM11 +T576 UBERON:0002037 27606 27616 Cerebellum +T577 CHEBI:15377 27674 27679 water +T578 PR:000003710 27693 27699 ADAM11 +T579 NCBITaxon:10088 27710 27714 mice +T580 NCBITaxon:10088 27850 27854 mice +T581 CHEBI:15377 27969 27974 water +T582 NCBITaxon:10088 28046 28050 mice +T583 GO:0036268 28159 28167 Swimming +T584 PR:000003710 28367 28373 ADAM11 +T585 NCBITaxon:10088 28384 28388 mice +T586 NCBITaxon:10088 28515 28519 mice +T587 PR:000003710 28604 28610 ADAM11 +T588 NCBITaxon:10088 28621 28625 mice +T589 PR:000003710 28713 28719 ADAM11 +T590 NCBITaxon:10088 28730 28734 mice +T591 PR:000003710 28909 28915 ADAM11 +T592 NCBITaxon:10088 28926 28930 mice diff --git a/src/ontogpt/evaluation/craft/database/all/16504143.txt b/src/ontogpt/evaluation/craft/database/all/16504143.txt new file mode 100644 index 000000000..d59187daa --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16504143.txt @@ -0,0 +1,145 @@ +Deficits in spatial learning and motor coordination in ADAM11-deficient mice + +Abstract + +Background + +ADAM11 is a member of the ADAM gene family and is mainly expressed in the nervous system. It is thought to be an adhesion molecule, since it has a disintegrin-like domain related to cell-cell or cell-matrix interactions. To elucidate the physiological functions of ADAM11, we generated ADAM11-deficient mice by means of gene targeting. + +Results + +ADAM11-deficient mice were apparently normal, and survived more than one year with no major histological abnormalities in the brain or spinal cord. Because ADAM11 is highly expressed in the hippocampus and cerebellum, we have examined ADAM11 mutant mice for learning using visual and hidden water maze tasks, and their motor coordination using a rotating rod task. Our results showed that their visual water maze task results are normal, but the hidden water maze and rotating rod task skills are impaired in ADAM11-deficient mice. + +Conclusion + +Our results indicate that ADAM11 mutation does not affect cell migration and differentiation during development, but affects learning and motor coordination. Thus, ADAM11 might play an important signalling or structural role as a cell adhesion molecule at the synapse, and may thus participate in synaptic regulation underlying behavioural changes. + +Background + +The ADAM (A Disintegrin And Metalloprotease) family comprises membrane-spanning multi-domain proteins containing a metalloproteinase-like domain and a disintegrin-like domain. Approximately half of the ADAMs are catalytically active metalloproteases that shed a broad range of substrates, such as cytokines, growth factors, receptors, adhesion molecules [1,2]. Recent gene-disruption studies in mice have disclosed a physiological role of each ADAM. For example, ADAM10, ADAM17 and ADAM19 are critical for normal development by providing growth signals at the correct times and places [3-6]. In contrast, the functions of ADAM8, ADAM9, ADAM12 and ADAM15 are not essential for embryogenesis, suggesting a possible functional redundancy with other protease [7-10]. The remaining half, which are non-protease-ADAMs, are thought to be adhesion molecules. More than ten ADAMs have been shown to support integrin-mediated cell adhesion in vitro [11]. More recently, the finding has been reported that integrin-mediated cell migration of tissue culture cells can be controlled by distinct ADAMs [12]. In C. elegans, unc-71 gene coding a non-protease-ADAM protein have been shown to be involved in cell migration events in vivo [13]. However, the physiological roles of mammalian non-protease-ADAMs are poorly understood in vivo. Genetic deletion studies in mice have shown that both Adam2-null and Adam3-null male mutants are infertile and their spermatozoa lack egg-binding abilities [14-16]. The precise mechanism is under investigation [17]. + +We have reported the findings of ADAM11, ADAM22 and ADAM23 genes and their restricted expression in the human and murine nervous systems [18,19]. These ADAMs lack a catalytic motif critical for the metalloproteinase activity, suggesting that they are not proteases. We assume that these ADAMs might contribute to cell migration, differentiation and survival or synaptic regulation by binding to integrins, cell-surface proteins or intracellular signalling molecules [20,21]. We have reported severe ataxia, seizures, and death before weaning, accompanied with hypomyelination of the peripheral nervous system in Adam22-null mutant mice [22]. It has been reported that the disruption of Adam23 gene in the mouse results in premature death associated with ataxia and tremor [23]. Is spite of such extreme phenotypes, no histological defects in the brain has been observed in either Adam22-null nor Adam23-null mice. + +ADAM11 is the closest paralog of ADAM22 and ADAM23, and its orthologs have been found in vertebrates, but not in invertebrates. It has been reported that the Xenopus ADAM11 ortholog, xMDC11a, has an expression pattern associated with neural development, with a proposed role in cell migration [24]; and it has been reported in analyses using Northern blot and in situ hybridisation methods that the murine Adam11 gene is expressed in both the developing and adult nervous system [19,25]. These findings led us to hypothesise that ADAM11 is an integrin binder that plays an important role in the nervous system, as do ADAM22 and ADAM23. To determine the physiological functions of ADAM11, we generated and analysed Adam11 gene-targeted mice. + +Results + +Generation of ADAM11-deficient mice + +Mice carrying a targeted mutation in their Adam11 gene were generated by homologous recombination (Fig. 1A). Correct targeting events were confirmed by Southern blot analysis (Fig. 1B). As a result of this procedure, since a termination codon was introduced in exon 5 in the pro-protein domain, only the truncated form of the ADAM11 protein would be synthesised from this targeted allele. Because pro-protein domains are always removed in mature functional ADAM-proteins by furin-like proteases and are thus thought to be non-functional, we concluded that this truncated form of ADAM11 protein would have no function [26]. Absence of mature ADAM11 protein in homozygous mutants was confirmed by Western blot analysis using a specific antibody that recognises the ADAM11 protein (Fig. 1C). Analysis by Western blot showed that heterozygous mutants produced approximately half of the wild-type levels of ADAM11 protein. + +ADAM11-deficient mice are viable and appear normal + +Mating of mice heterozygous for the ADAM11 mutation yielded a near-Mendelian distribution of genotypes in the offspring and the ratio of females and males was the same in all groups (data not shown). For the present experiments, ADAM11-deficient male mice (backcrossed generations into C57BL/6NCrj, n = 14) were used in all behavioural analyses and anatomical-histological studies. Homozygous ADAM11-deficient mice were viable and appeared normal, indicating that the Adam11 gene is not essential to development and survival. The gain in body weight of (-/-) mice was comparable to that of (+/+) and (+/-) mice at least until 24 weeks of age (data not shown). There was no significant difference in locomotor activity among (+/+), (+/-) and (-/-) genotypes (data not shown). No apparent anatomical-histological change was observed in the spinal cord, heart, lung, liver, kidney, spleen, testis or ovary (data not shown). The brain of (-/-) mice, including the hippocampus (Fig. 1D) and cerebellum (Fig. 1E) appeared normal after HE staining when (+/+), (+/-) and (-/-) mice were compared. Since Adam11 is predominantly expressed in the hippocampus and cerebellum, we applied hippocampus- or cerebellum-dependent behavioural analyses. + +Spatial learning in ADAM11-deficient mice + +To study the functions of ADAM11 in the hippocampus, we examined the spatial learning ability of ADAM11-deficient mice using the Morris Water Maze task [27]. First, mice were trained in a hidden platform task, in which mice searched for a submerged platform to escape from the water. Adam11 (-/-) mice were found to take a significantly longer time to locate the hidden platform than (+/+) and (+/-) mice (Fig. 2A). Nonetheless, the ability of (+/+), (+/-) and (-/-) mice to find the hidden platform improved over test sessions (Fig. 2A). Thus, (+/+), (+/-) and (-/-) mice were able to learn the location of the hidden platform during the course of the trials, although the capacity of (-/-) mice to find the platform was lower than that of (+/+) and (+/-) mice. We further performed a probe test, in which the platform was removed from the pool after completion of the hidden platform task, and the trained mice were allowed to swim freely for 60 sec. The time spent in the target quadrant of (-/-) is significantly lower than that of (+/+) mice (Fig. 2B). Adam11 (+/-) mice appeared to spend a shorter time in the target quadrant, but there was no significant difference between (+/+) and (+/-) mice (Fig. 2B). These results indicate a spatial learning impairment in (-/-) mice. The swimming distance of (+/+), (+/-) and (-/-) mice, however, was similar during the probe trial (Fig. 2C). In the visible platform task, the performance of mice with (+/+), (+/-) and (-/-) genotypes improved during the course of the test sessions, as indicated by a progressive decrease in the latency for locating the platform (Fig. 2D), suggesting that (-/-) mice learn this visible platform task just as well as (+/+) and (+/-) mice. These results also indicated that (-/-) mice have normal visible and motor function and the motivation needed in the water maze task. + +Motor function of ADAM11-deficient mice + +To study the functions of ADAM11 in the cerebellum, we examined the motor function of ADAM11-deficient mice using a rotating rod task [28]. In the rotating rod test, the retention time of (+/-) and (-/-) mice on the rod was not significantly different from that of (+/+) mice at 0 rpm (stationary) [+/-: F (1, 80) = 1.67, p = 0.21; -/-: F (1, 88) = 0.002, p = 0.96] (Fig. 3A). In (+/-) mice, the tendency of decreased retention times was seen at 5, 10 and 15 rpm, but there was no significant difference between (+/+) and (+/-) mice [5 rpm: F (1, 20) = 1.27, p = 0.27; 10 rpm: F (1, 20) = 1.07, p = 0.31; 15 rpm: F (1, 20) = 2.716, p = 0.11] (Figs. 3B, 3C and 3D). However, (-/-) mice fell more quickly from the rod than (+/+) mice at 5, 10 and 15 rpm [5 rpm: F (1, 27.3) = 17.51, p = 0.0002; 10 rpm: F (1, 22) = 11.05, p = 0.003; 15 rpm: F (1, 22) = 19.22, p = 0.0002] (Figs. 3B, 3C and 3D). The retention time was not significantly improved in (-/-) mice across trials of training at 5, 10 and 15 rpm [5 rpm: F (3, 15.3) = 2.05, p = 0.15; 10 rpm: F (7, 11) = 2.61, p = 0.07; 15 rpm: F (19, 26.6) = 1.57, p = 0.14] (Figs. 3B, 3C and 3D). In a traction test, (-/-) mice showed a high grip strength similar to that of (+/+) and (+/-) mice (Fig. 4). The hanging score and time of (-/-) mice were also similar to those of (+/+) and (+/-) mice (Figs. 5A and 5B). A footprint test did not reveal any abnormalities in walking patterns, including stride length and step width, as a function of genotypes (Figs. 6A and 6B). + +Discussion + +In the present study, we examined the function of ADAM11 by generating mice that lacked the Adam11 gene. It has been reported that the Adam11 gene is expressed in the brain, heart, liver and testis, as manifested in Northern blot analysis [19]. Interestingly, ADAM11-deficient mice appeared normal and survived more than one year without apparent histological abnormality, including the brain, heart, liver and testis. The expression patterns of the Adam11 gene in the brain have been studied using in situ hybridisation [25]. This study has shown high expression of ADAM11 mRNA in the hippocampus and cerebellum. We therefore focused on examining the hippocampus- and cerebellum-dependent behavioural tests to study the functions of ADAM11 in the nervous system. + +It is widely accepted that the hippocampus is involved in the performance of the hidden platform version of the water maze test, since lesions of the hippocampal pathways lead to a severe impairment of learning in this task [29]. In the hidden platform task, ADAM11-deficient mice needed more time to reach the platform than wild-type mice. Spatial learning was also assessed using a probe trial, in which the platform was removed from the pool on the day following nine days of training. The wild-type mice spent significantly more time searching for the platform in the target quadrant than the other quadrants. However, ADAM11-deficient mice did not search selectively in the target quadrant. On the other hand, in the visible platform version, in which the motor ability and visual acuity of the mouse are tested, ADAM11-defieient mice were normal. These results indicated that ADAM11-deficient mice showed a spatial learning impairment. + +The cerebellum is in charge of the smooth coordination of somatic motor activity, regulation of muscle tone, and mechanisms that influence and maintain equilibrium [30]. It is widely accepted that the cerebellum is involved in the performance of the rotating rod task, since mice with structural abnormalities in the cerebellum [31] or with disruptions in genes enriched in the cerebellum [32] exhibit performance deficits in this task. In the rotating rod test in this study, although the retention time of ADAM11-deficient mice on the rod was not different from wild-type mice at 0 rpm (stationary), ADAM11-deficient mice fell more quickly from the rod than wild-type mice at 5, 10 and 15 rpm. These results indicate that ADAM11-deficient mice have a deficit in motor coordination. On the other hand, in the visual platform version of the water maze task, motor ability for swimming in ADAM11-deficient mice was normal. These results suggested that there might be a different mechanism of motor function between swimming and the rotating rod task. It has been also reported that the cerebellum is involved in another important distinct function: learning associated with component movement (motor learning) [33]. The retention time of synaptotagmin (Syt) IV-deficient mice on the rod was significantly shorter than wild-type mice, and they improved it approximately to the same rates as did wild-type mice during rotating rod performance [34]. These data suggest the basis for the performance deficit on the rotating rod task in the Syt IV mutants is in motor coordination rather than in motor learning. The results in the present study showed that ADAM11-deficient mice slightly, though not significantly, improved motor learning ability during rotating rod performance. However, the deficit of motor learning in ADAM11-deficient mice was not clear in this study. Because they could not ride on the rotating rod, they might not learn this task. To examine motor learning ability in ADAM11-deficient mice in detail, we will need to use other cerebellar-dependent learning tasks, such as a classical eyelid conditioning test. There was no impairment in the grip strength and wire suspension test in ADAM11-deficient mice. Spontaneous motor activity and walking patterns of ADAM11-deficient mice were also found to be normal, suggesting that the dysfunction found in the rotating rod test was not derived from muscle weakness or other peripheral disturbances. + +These results suggest that limited brain sites, mainly the hippocampus and cerebellum, might contribute to the dominant phenotype, spatial learning impairment and motor discoordination in ADAM11-deficient mice. In situ hybridisation analysis has detected Adam11 gene expression in the pyramidal cells of CA1–CA3 fields and granule cells of the dentate gyrus in the hippocampus and in granular cells in the cerebellum [25]. It is unlikely that morphological changes during development led to impairment of spatial learning and motor coordination, and morphological alterations in the cytoarchitecture of the hippocampus and cerebellum were not observed in ADAM11-deficient mice after histological investigation using HE staining. However, because it is thought that ADAM11 plays a role in neuron-neuron or neuron-glial cell interactions, a more precise morphological investigation will be needed. It has been reported that adhesion molecules, such as neural cell adhesion molecule (NCAM), are not only implicated in cell interactions during nervous system development, but are also recognised as important mediators of synaptic plasticity in the adult nervous system [35]. Changes in neuron excitability in the hippocampus, such as decreases in the post-burst after hyperpolarization (AHP), have been thought to affect long-term potentiation (LTP) [36], an experimental model of the synaptic changes thought to underlie learning and memory [37]. It has also been reported that mice lacking NMDA receptor for both NR2A and NR2C subunits showed motor dysfunction and complete loss of both spontaneous and evoked excitatory postsynaptic currents (EPSCs) in cerebellar granule cells [38]. Therefore, electrophyological studies to enable analysis of synaptic transmission and plasticity in the hippocampus and cerebellum of ADAM11-deficient mice will be needed. These studies might clarify whether ADAM11 plays an important signalling or structural role at the synaptic level and whether it participates in synaptic regulation. + +ADAM11-deficient mice remained alive for more than one year without ataxia or tremor, but showed a deficit in spatial learning and motor dysfunction. On the other hand, mice lacking the Adam22 gene showed severe ataxia, exhibited marked hypomyelination of the peripheral nerves, and died before weaning [22]. Mice lacking the Adam23 gene showed severe ataxia and tremor and died before weaning [23]. These findings suggest that ADAM11, ADAM22 and ADAM23 have separate and important roles in the nervous system. A genome database search revealed orthologs of ADAM11, ADAM22 and ADAM23 genes to exist in vertebrates such as mammals, fish, and amphibians, but not in invertebrates. Although the precise molecular functions of these ADAMs are still unclear, further investigations will provide clues to understanding the nervous system of higher organisms. + +Conclusion + +ADAM11-deficient mice survived more than one year without ataxia or tremor, showed no abnormalities in body weight gain, spontaneous motor activity, muscle strength, or walking patterns, and exhibited no apparent anatomical-histological changes. However, hippocampus-dependent spatial learning and cerebellum-dependent motor coordination in ADAM11-deficient mice were impaired when tested using water maze and rotating rod tasks. Our genetically modified mouse strain has the potential to be a useful and sensitive tools for detailed investigation of ADAM proteins' functions in the nervous system. + +Methods + +Construction of an Adam11 gene-targeting vector + +The 13.7-kb BamHI DNA fragment covering exons 2 – 20 of the Adam11 gene was amplified from C57BL/6 genomic DNA by the PCR using Expand Hi-Fidelity polymerase mix (Roche Diagnostics KK, Tokyo, Japan). To generate a mutation in the mouse Adam11 gene, we inserted a PGKneo cassette into exons 5 – 7 of the Adam11 gene. The final targeting construct consisted of a 10.1-kb genomic DNA fragment that was interrupted by the insertion of the PGKneo cassette and contained a MC1/TK cassette as a negative selection marker against random integration [39]. + +Generation of Adam11 deficient mice + +The linearised targeting vector was electroporated into TT2 embryonic stem (ES) cells [40]. Homologous recombinants were selected by PCR using the following primers, AGN1: 5'-TCGTGCTTTACGGTATCGCCGCTCCCGATT-3' in the PGKneo cassette, and SGN033: 5'-GGACCCGGAAGACATTTGCACGGT-3' outside the targeting vector. Homologous recombined DNA was efficiently amplified by 35 cycles of the following amplification steps (94°C-30 sec and 68°C-5 min). Correctly targeted ES clones were injected into fertilised ICR mouse eggs at the eight-cell stage. The resulting male chimeras were mated with C57BL/6NCrj females (Charles River Japan, Tokyo, Japan), and resulting heterozygous (+/-) male and female mice were interbred to generate homozygous (-/-) mice. + +Southern blot analysis + +Mouse genomic DNA used for Southern blot analysis was extracted from the liver of each genotype. BamHI-digested genomic DNA was probed with the [32P]-labelled BE2K probe, which was a 2.0-kb BamHI – EcoRI genomic fragment located in intron 2. + +Western blot analysis + +The absence of ADAM11 protein in the (-/-) mice was confirmed by Western blot analysis. To be brief, the cerebellum was isolated from a six month-old mouse of each genotype and homogenised in cell lysis buffer (50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1% NP-40, protease inhibitor cocktail [Roche Diagnostics]). After removal of cell debris by centrifugation, the glycosylated proteins in the supernatant were concentrated by the affinity chromatography using Con A Sepharose 4B (Amersham Biosciences Corp.). Each sample was separated on 10% SDS-PAGE, and transferred to a PVDF membrane. The blot was then incubated with monoclonal antibody against the ADAM11 (U. S. Patent 5,631,351) at 1 μg/ml. Bound antibodies were visualised with horseradish peroxidase-labelled second antibody and an ECL-plus chemiluminescence detection system (Amersham Biosciences Corp.). + +Behavioural analysis + +All animal procedures conformed to the Japanese regulations on animal care and use, following the Guideline for Animal Experimentation of the Japanese Association for Laboratory of Animal Science, and were approved by the Animal Care and Use Committee of Eisai Co., Ltd. For the present experiments, ADAM11-deficient male mice (backcrossed generations into C57BL/6NCrj, n = 14) were used in all behavioural analyses (22–24 weeks of age). All of the behavioural studies were conducted between 10:00 and 16:00 by a well-trained experimenter blind to the mouse genotypes. + +Spontaneous motor activity + +Locomotor activity in a novel environment was measured by introducing mice (+/+, +/- and -/-; n = 8, 8 and 8, respectively) for 90 min in a Plexiglas box (30 × 20 × 13 cm) using a VERSAMAX equipment and software (Accuscan, Columbus, OH, USA). + +Water maze task + +Spatial learning was assessed by three variants of the Morris water maze task [27] adapted for mice. Hidden platform task: The pool was divided into four quadrants with four starting locations, called north, east, south and west, at equal distances to the rim. A circular, transparent acrylic platform (diameter 8 cm) was submerged 1 cm below the surface of the water in the centre of the southeast quadrant for each trial of this task. Each mouse was allowed to swim for 60 sec and the time required to reach the platform (escape latency) was recorded in each trial. In total this task consisted of four trials per day over 9 days (1 min inter-trial intervals). Each trial was initiated by randomly placing an animal in one of the four starting locations. Probe trial: A single probe trial was carried out after the hidden platform task had been completed. In this trial, the platform was removed and the movement of each mouse was monitored using a computer-based video tracking system (BTA-2; Muromachi Kikai Co., Ltd., Tokyo, Japan). Each mouse was placed into the northwest quadrant of the pool and allowed to swim for 60 sec. Swim path length and the time spent in the trained (southeast) quadrant were calculated. Visible platform task: In this task, the circular platform was made visible by attaching a black board to it and the mouse was required to locate the visible platform. The mouse was allowed to swim for 60 sec and this task consisted of four trials per day for three consecutive days (1 min inter-trial intervals). Each trial was initiated by randomly placing an animal in one of the four starting locations. + +Rotating rod test + +Motor coordination was assessed with a rotating rod apparatus (KN-75, Natsume Seisakujo, Tokyo, Japan), which consisted of a plastic rod (3 cm diameter, 8 cm long) with a gritted surface flanked by two large discs (40 cm diameter). The mice were first placed on the stationary rod for 4 successive trials, followed by 4 trials at a rotation speed of 5 rpm, 8 trials at 10 rpm, and 20 trials at 15 rpm in that order. Latency until a fall occurred was monitored for 120 sec. Intra-trial intervals for each animal were more than 20 min. + +Traction test + +The grip strength of each mouse was measured with a traction apparatus (FU-1, Muromachi Kikai Co., Ltd.). Each mouse was made to grasp the attached bar (2 mm diameter) with the forepaws and was slowly pulled back by the tail. The maximum tension before release was recorded. + +Wire suspension test + +A mouse was placed on a stainless bar (50 cm length, 2 mm diameter, elevated up to 37 cm from a surface) at a point midway between the supports and observed for 30 sec in four separate trials. The amount of time spent hanging was recorded and scored according to the following system [41]: 0, fell off; 1, hung onto the bar with two forepaws; 2, in addition to 1, attempted to climb onto the bar; 3, hung onto the bar with two forepaws and one or both hind paws; 4, hung onto the bar with all four paws with tail wrapped around the bar; 5, escaped to one of the supports. + +Footprint test + +Black ink was applied to the hind paws of each mouse and the mice were placed in a narrow alley (9 × 25 × 10 cm) on white paper. Stride length and step width were calculated. + +Statistics + +Data are expressed as the means ± SEM. Statistical analysis was conducted using the software package SAS 8.1 (SAS Institute Japan Ltd., Tokyo, Japan). Statistical significance was determined by analysis of variance (ANOVA) followed by the Dunnett-type multiple comparison test, and p values of less than 0.05 were considered to be significant in the water maze task. Behavioural data in the rotating rod test were analysed by one- or two-way ANOVA with repeated measures, and p values of less than 0.05 were considered to be significant. + +Histopathology + +Sections of tissues including the brain, spinal cord, heart, lung, liver, kidney, spleen and testis from mice (+/+, +/- and -/-; n = 7, 7 and 7, respectively) were fixed in 10% buffered formalin and embedded in paraffin. Each paraffin section of thick 4 μm was stained with hematoxylin and eosin (HE). + +Authors' contributions + +KS is leading this project, and performed cDNA cloning, plasmid construction, antibody production, biochemical study, and wrote the manuscript. ET is also leading this project, performed the generation of knockout mice and behavioural analyses, and wrote the manuscript. TO and KY contributed to the generation of knockout mice. TN and JK supervised the project. All authors read and approved the manuscript. + +Acknowledgements + +We are grateful to Isao Tanaka, Masayuki Okada, Mitsuhiro Ino, Norimasa Miyamoto, Kappei Tsukahara, Hiroyuki Kato, Hiroo Ogura, Tatsuto Fukushima, Yoshiaki Furuya, Jiro Sonoda (Eisai Co., Ltd.), Paul Frankland (University of Toronto), and Hachiro Sugimoto (Kyoto University) for fruitful discussions and advice. + +Figures and Tables + +Figure 1 + +Targeted mutation of Adam11. (A) Top; Domain structure of ADAM11 protein. PRO, proprotein; MP, metalloprotease-like; DIS, disintegrin-like; CR/EGF, Cysteine-rich and EGF-like repeat; TM, transmembrane; Cyto, cytoplasmic domain. Adam11 gene was disrupted by the insertion of a termination codon and a PGKneo cassette into exons 5 – 7. An MC1/TK cassette at the end of the targeting vector allowed for negative selection. The BE2K-probe used for Southern blot analysis, and the expected BamHI "B" fragments of the targeted allele (TG) and wild-type allele (WT) are indicated by arrows. The primers, AGN1 and 033 were used for the selection of the recombinant ES clones. (B) Southern blot analysis of mouse genomic DNA. The expected DNA fragments for the wild-type allele (WT) and targeted allele (TG) are 13.7-kb and 10.3-kb, respectively. +/+, wild-type; +/-, heterozygote; -/-, homozygote. (C) Western blot analysis of ADAM11 expression in the mouse cerebellum. Absence of ADAM11 protein in the (-/-) mutant cerebellum was shown using anti-ADAM11 monoclonal antibody. "P" indicates the positive control, HA-tagged mouse ADAM11 protein expressed in HeLa cells. White circle indicates mature-form of ADAM11 protein and black circle shows immature-form, which was not processed. (D) Hippocampus sections with HE stain. Scar = 30 μm. (E) Cerebellum sections with HE stain. Scar = 200 μm. + +Figure 2 + +Morris water maze task of ADAM11-deficient mice. (A) Escape latencies (sec) in the hidden platform task over nine testing days. Asterisk: significant (p < 0.05) difference from (+/+) mice. (B) The probe test after the acquisition trials of the hidden platform task. Times spent in each quadrant of the water pool are shown. Asterisk: significant (p < 0.05) difference from (+/+) mice. TQ, training quadrant; AR, adjacent right quadrant; AL, adjacent left quadrant; OP, opposite quadrant. (C) Swimming distances (cm) during the probe test. (D) Escape latencies (sec) in the visible platform task over three testing days. +/+, +/- and -/-; n = 11, 14 and 12, respectively. + +Figure 3 + +Retention time of ADAM11-deficient mice on the rotating rod at speeds of 0 rpm (stationary), 5, 10 and 15 rpm. Asterisk: significant (p < 0.05) difference from (+/+) mice. +/+, +/- and -/-; n = 11, 14 and 12, respectively. + +Figure 4 + +Traction test (g) of ADAM11-deficient mice. +/+, +/- and -/-; n = 12, 10 and 12, respectively. + +Figure 5 + +Wire suspension test of ADAM11-deficient mice. (A) Score in the wire suspension test. (B) Retention time (sec) in the wire suspension test. +/+, +/- and -/-; n = 12, 10 and 12, respectively. + +Figure 6 + +Footprint test of ADAM11-deficient mice. (A) Stride length (cm) in the footprint test. (B) Step width (cm) in the footprint test. +/+, +/- and -/-; n = 12, 10 and 12, respectively. diff --git a/src/ontogpt/evaluation/craft/database/all/16504174.ann b/src/ontogpt/evaluation/craft/database/all/16504174.ann new file mode 100644 index 000000000..8386b56fd --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16504174.ann @@ -0,0 +1,621 @@ +T1 NCBITaxon:10088 46 51 mouse +T2 SO:0000704 52 56 gene +T3 PR:000006847 66 70 ESG1 +T4 PR:000006847 72 76 PH34 +T5 PR:000006847 77 82 ECAT2 +T6 PR:000006847 83 88 DPPA5 +T7 UBERON:0000922 113 122 Embryonic +T8 CL:0002322 113 132 Embryonic stem cell +T9 PR:000006847 113 146;153 154 Embryonic stem cell-specific gene ... 1 +T10 SO:0000704 142 146 gene +T11 PR:000006847 148 151;153 154 ESG ... 1 +T12 SO:0000417 175 181 domain +T13 GO:0010467 218 227 expressed +T14 UBERON:0019248 231 244 early embryos +T15 CL:0000586 246 256 germ cells +T16 UBERON:0000922 262 271 embryonic +T17 CL:0002322 262 276;282 287 embryonic stem ... cells +T18 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PR:000006847 9454 9458 ESG1 +T290 SO:0000704 9459 9463 gene +T291 SO:0000409 9537 9554 Recognition sites +T292 SO:0000704 9640 9644 gene +T293 http://purl.obolibrary.org/obo/MONDO_0005504 9654 9664 diphtheria +T294 CHEBI:27026 9665 9670 toxin +T295 SO:0001644 9713 9730 targeting vectors +T296 CL:0002322 9842 9850 ES cells +T297 CL:0002322 9865 9873 ES cells +T298 CL:0002322 9887 9895 ES cells +T299 PR:000006847 9902 9906 ESG1 +T300 CL:0002322 9912 9920 ES cells +T301 CL:0002322 10026 10034 ES cells +T302 PR:000006847 10041 10045 ESG1 +T303 CL:0002322 10051 10059 ES cells +T304 CL:0002322 10097 10105 ES cells +T305 CHEBI:4883 10219 10235 ethidium bromide +T306 GO:0005840 10248 10257 ribosomal +T307 CL:0002322 10315 10323 ES cells +T308 SO:0000440 10349 10355 vector +T309 CL:0002322 10379 10387 ES cells +T310 CHEBI:42768 10396 10400 G418 +T311 PR:000006847 10451 10455 ESG1 +T312 PR:000006847 10531 10535 ESG1 +T313 SO:0000440 10588 10594 vector +T314 SO:0000440 10617 10623 vector +T315 SO:0000440 10706 10712 vector +T316 PR:000006847 10754 10758 ESG1 +T317 CL:0002322 10762 10770 ES cells +T318 GO:0008283 10829 10841 proliferated +T319 PR:000006847 10935 10939 ESG1 +T320 http://purl.obolibrary.org/obo/MONDO_0005059 10994 11002 leukemia +T321 PR:000009799 10994 11020 leukemia inhibitory factor +T322 CHEBI:26536 11036 11049 retinoic acid +T323 UBERON:0000180 11101 11107 flanks +T324 NCBITaxon:10088 11116 11120 mice +T325 http://purl.obolibrary.org/obo/MONDO_0002601 11143 11152 teratomas +T326 http://purl.obolibrary.org/obo/MONDO_0005070 11154 11160 tumors +T327 UBERON:0000923 11196 11207 germ layers +T328 PR:000006847 11249 11253 ESG1 +T329 GO:0017145 11277 11289 self-renewal +T330 CL:0002322 11321 11329 ES cells +T331 PR:000006847 11354 11358 ESG1 +T332 CL:0002322 11364 11372 ES cells +T333 PR:000006847 11395 11399 ESG1 +T334 CL:0002322 11405 11412 ES cell +T335 GO:0040007 11422 11427 grown +T336 PR:000006847 11484 11488 ESG1 +T337 CL:0002322 11523 11531 ES cells +T338 http://purl.obolibrary.org/obo/MONDO_0002601 11785 11793 teratoma +T339 PR:000006847 11807 11811 ESG1 +T340 CL:0002322 11817 11825 ES cells +T341 CHEBI:51686 11827 11838 hematoxylin +T342 SO:0000704 11875 11879 gene +T343 GO:0010467 11875 11890 gene expression +T344 PR:000006847 11903 11907 ESG1 +T345 CL:0002322 11911 11919 ES cells +T346 SO:0000704 11985 11990 genes +T347 CL:0002322 12017 12025 ES cells +T348 PR:000006847 12027 12031 ESG1 +T349 SO:0000704 12054 12058 gene +T350 PR:000006847 12093 12097 ESG1 +T351 CL:0002322 12101 12109 ES cells +T352 GO:0010467 12127 12137 expression +T353 CL:0002322 12141 12148 ES cell +T354 SO:0000704 12156 12161 genes +T355 PR:000010968 12171 12176 Nanog +T356 PR:000013044 12181 12185 Oct3 +T357 PR:000006847 12203 12207 ESG1 +T358 CL:0002322 12211 12219 ES cells +T359 PR:000013044 12241 12245 Oct3 +T360 GO:0010467 12248 12258 expression +T361 SO:0000704 12318 12322 gene +T362 GO:0010467 12318 12333 gene expression +T363 CL:0002322 12372 12380 ES cells +T364 PR:000006847 12385 12389 ESG1 +T365 CL:0002322 12393 12401 ES cells +T366 SO:0000704 12411 12416 genes +T367 PR:000006847 12464 12468 ESG1 +T368 PR:000009450 12489 12493 Krt1 +T369 PR:000014397 12497 12500 Pem +T370 PR:000003172 12502 12506 Ctgf +T371 PR:000013428 12508 12513 Ptgs2 +T372 PR:000000216 12524 12529 Inhba +T373 SO:0000704 12537 12542 genes +T374 GO:0065007 12552 12561 regulated +T375 PR:000006847 12588 12592 ESG1 +T376 PR:000006847 12600 12604 ESG1 +T377 SO:0000417 12636 12642 domain +T378 SO:0000704 12675 12680 genes +T379 SO:0000704 12751 12755 Gene +T380 GO:0010467 12751 12766 Gene expression +T381 PR:000006847 12779 12783 ESG1 +T382 CL:0002322 12789 12797 ES cells +T383 CL:0002322 12852 12860 ES cells +T384 PR:000006847 12865 12869 ESG1 +T385 CL:0002322 12875 12883 ES cells +T386 CHEBI:37987 12902 12905 Cy3 +T387 CHEBI:37989 12910 12913 Cy5 +T388 GO:0097617 12942 12952 hybridized +T389 NCBITaxon:10088 12964 12969 Mouse +T390 PR:000006847 13091 13095 ESG1 +T391 PR:000006847 13103 13107 ESG1 +T392 GO:0010467 13135 13145 expression +T393 PR:000006847 13149 13153 ESG1 +T394 PR:000013044 13155 13159 Oct3 +T395 PR:000005257 13166 13170 CDK4 +T396 PR:000006847 13205 13209 ESG1 +T397 NCBITaxon:10088 13219 13223 mice +T398 PR:000006847 13243 13247 ESG1 +T399 CL:0002322 13251 13258 ES cell +T400 UBERON:0000358 13275 13286 blastocysts +T401 NCBITaxon:10088 13297 13301 mice +T402 PR:000006847 13368 13372 ESG1 +T403 NCBITaxon:10088 13376 13380 mice +T404 PR:000006847 13423 13427 ESG1 +T405 PR:000006847 13439 13443 ESG1 +T406 PR:000006847 13469 13473 ESG1 +T407 NCBITaxon:10088 13477 13481 mice +T408 NCBITaxon:33208 13489 13496 animals +T409 UBERON:0000473 13600 13606 testis +T410 UBERON:0000992 13611 13616 ovary +T411 PR:000006847 13696 13700 ESG1 +T412 NCBITaxon:10088 13725 13730 mouse +T413 CL:0000586 13747 13756 germ cell +T414 GO:0007281 13747 13766 germ cell formation +T415 CL:0002322 13787 13795 ES cells +T416 UBERON:0000358 13801 13812 blastocysts +T417 PR:000006847 13841 13845 ESG1 +T418 PR:000006847 13859 13863 ESG1 +T419 CL:0002322 13889 13896 ES cell +T420 PR:000006847 13932 13936 ESG1 +T421 PR:000006847 13947 13951 ESG1 +T422 CL:0002322 13957 13965 ES cells +T423 GO:0008283 13998 14011 proliferation +T424 PR:000006847 14062 14066 ESG1 +T425 CL:0002322 14085 14093 ES cells +T426 PR:000006847 14146 14150 ESG1 +T427 NCBITaxon:10088 14170 14175 mouse +T428 SO:0000704 14176 14180 gene +T429 CL:0002322 14243 14251 ES cells +T430 GO:0010467 14270 14280 expression +T431 UBERON:0019248 14284 14297 early embryos +T432 CL:0000586 14299 14309 germ cells +T433 PR:000006847 14361 14365 ESG1 +T434 NCBITaxon:10088 14385 14390 mouse +T435 CL:0000586 14404 14413 germ cell +T436 GO:0007281 14404 14423 germ cell formation +T437 CL:0002322 14429 14436 ES cell +T438 GO:0017145 14432 14449 cell self-renewal +T439 SO:0000153 14492 14495 BAC +T440 NCBITaxon:10088 14518 14523 mouse +T441 PR:000006847 14524 14528 ESG1 +T442 SO:0000704 14529 14533 gene +T443 NCBITaxon:2 14547 14556 bacterial +T444 SO:0000153 14547 14578 bacterial artificial chromosome +T445 SO:0000153 14580 14583 BAC +T446 NCBITaxon:10088 14603 14608 mouse +T447 PR:000006847 14609 14613 ESG1 +T448 SO:0000704 14614 14618 gene +T449 NCBITaxon:10088 14656 14661 mouse +T450 SO:0000153 14662 14665 BAC +T451 PR:000006847 14714 14718 pH34 +T452 PR:000006847 14757 14761 pH34 +T453 SO:0000112 14796 14803 primers +T454 SO:0000153 14884 14887 BAC +T455 PR:000006847 14923 14927 pH34 +T456 PR:000006847 14973 14977 pH34 +T457 PR:000006847 15012 15016 pH34 +T458 PR:000006847 15062 15066 pH34 +T459 PR:000006847 15109 15113 pH34 +T460 GO:0097617 15161 15174 Hybridization +T461 PR:000006847 15230 15234 ESG1 +T462 SO:0000704 15235 15239 gene +T463 SO:0000336 15243 15254 pseudogenes +T464 NCBITaxon:10088 15295 15300 mouse +T465 PR:000006847 15301 15305 ESG1 +T466 SO:0000704 15306 15310 gene +T467 SO:0001417 15319 15337 3' flanking region +T468 SO:0000028 15359 15361 bp +T469 SO:0000440 15398 15404 vector +T470 PR:P43870 15419 15426 HindIII +T471 SO:0000440 15464 15470 vector +T472 PR:000006847 15543 15547 ESG1 +T473 SO:0000336 15548 15558 pseudogene +T474 SO:0001417 15567 15585 3' flanking region +T475 SO:0000028 15593 15595 bp +T476 PR:P23940 15650 15655 BamHI +T477 SO:0000440 15684 15690 vector +T478 SO:0001416 15772 15791 5' flanking regions +T479 PR:000006847 15799 15803 ESG1 +T480 SO:0000704 15804 15808 gene +T481 SO:0000336 15825 15836 pseudogenes +T482 PR:000006847 15886 15890 pH34 +T483 PR:000006847 15938 15942 pH34 +T484 SO:0000112 15948 15955 primers +T485 SO:0000028 16035 16037 bp +T486 SO:0001416 16218 16236 5' flanking region +T487 PR:000006847 16244 16248 ESG1 +T488 SO:0000704 16249 16253 gene +T489 SO:0000028 16261 16263 bp +T490 SO:0000357 16396 16404 flanking +T491 SO:0000336 16409 16419 pseudogene +T492 PR:000006847 16438 16442 ESG1 +T493 SO:0001644 16443 16460 targeting vectors +T494 PR:000006847 16485 16489 ESG1 +T495 SO:0000147 16490 16495 exons +T496 SO:0001644 16505 16522 targeting vectors +T497 SO:0000243 16544 16548 IRES +T498 SO:0005853 16555 16563 cassette +T499 SO:0000243 16575 16579 IRES +T500 SO:0005853 16585 16593 cassette +T501 SO:0000167 16597 16605 promoter +T502 SO:0000028 16652 16654 bp +T503 PR:000006847 16689 16693 pH34 +T504 PR:000006847 16747 16751 pH34 +T505 SO:0000112 16805 16812 primers +T506 SO:0000028 16831 16833 bp +T507 PR:000006847 16859 16863 pH34 +T508 PR:000006847 16917 16921 pH34 +T509 SO:0000112 16971 16978 primers +T510 SO:0000243 16984 16988 IRES +T511 SO:0000243 16998 17002 IRES +T512 SO:0005853 17008 17017 cassettes +T513 GO:0006266 17023 17030 ligated +T514 http://purl.obolibrary.org/obo/MONDO_0005504 17069 17079 diphtheria +T515 CHEBI:27026 17080 17085 toxin +T516 SO:0005853 17088 17096 cassette +T517 SO:0001644 17172 17189 targeting vectors +T518 CL:0002322 17229 17237 ES cells +T519 SO:0000704 17251 17255 Gene +T520 GO:0009294 17273 17284 Transfected +T521 CHEBI:42768 17320 17324 G418 +T522 CHEBI:16976 17338 17350 hygromycin B +T523 SO:0001026 17366 17373 Genomic +T524 CHEBI:42768 17383 17387 G418 +T525 CHEBI:16976 17392 17404 hygromycin B +T526 CL:0002322 17543 17551 ES cells +T527 SO:0001026 17552 17559 genomic +T528 GO:0019835 17593 17603 Cell Lysis +T529 CHEBI:75958 17604 17612 Solution +T530 SO:0001026 17662 17669 genomic +T531 CHEBI:2511 17730 17737 agarose +T532 CHEBI:25614 17763 17768 nylon +T533 SO:0000028 17803 17805 bp +T534 SO:0000112 17925 17932 primers +T535 SO:0000028 17972 17974 bp +T536 SO:0000028 18012 18014 bp +T537 SO:0000028 18052 18054 bp +T538 SO:0001026 18081 18088 Genomic +T539 SO:0000028 18160 18162 bp +T540 SO:0000112 18290 18297 primers +T541 GO:0097617 18309 18319 hybridized +T542 SO:0000028 18332 18334 bp +T543 SO:0000028 18374 18376 bp +T544 SO:0000028 18415 18417 bp +T545 PR:000006847 18464 18468 ESG1 +T546 GO:0042571 18480 18490 antibodies +T547 PR:000006847 18515 18519 Esg1 +T548 PR:000006847 18550 18554 pH34 +T549 PR:000006847 18607 18611 pH34 +T550 SO:0000112 18660 18667 primers +T551 PR:000006847 18688 18692 pH34 +T552 SO:0000006 18708 18719 PCR product +T553 PR:000006847 18768 18772 pH34 +T554 GO:0010467 18871 18881 expression +T555 SO:0000440 18882 18888 vector +T556 PR:000006847 18897 18901 pH34 +T557 NCBITaxon:562 18915 18922 E. coli +T558 GO:0006412 18949 18967 protein production +T559 PR:000006847 19038 19042 ESG1 +T560 CHEBI:44557 19065 19086 nitrilotriacetic acid +T561 CHEBI:2511 19087 19094 agarose +T562 CHEBI:16199 19155 19159 urea +T563 CHEBI:16199 19188 19192 urea +T564 NCBITaxon:9986 19256 19263 rabbits +T565 PR:000006847 19281 19285 ESG1 +T566 GO:0042571 19297 19307 antibodies +T567 CL:0002322 19345 19352 ES cell +T568 CHEBI:8984 19419 19441 sodium dodecyl sulfate +T569 CHEBI:8984 19443 19446 SDS +T570 CHEBI:53325 19491 19505 nitrocellulose +T571 PR:000006847 19564 19568 ESG1 +T572 PR:000013044 19592 19596 Oct3 +T573 PR:000005257 19639 19643 CDK4 +T574 GO:0042571 19714 19724 antibodies +T575 NCBITaxon:3704 19726 19737 Horseradish +T576 MOP:0000779 19749 19759 conjugated +T577 NCBITaxon:9986 19765 19771 rabbit +T578 NCBITaxon:10088 19781 19786 mouse +T579 GO:0019814 19787 19802 immunoglobulins +T580 GO:0042571 19848 19856 antibody +T581 GO:0042571 19886 19894 antibody +T582 PR:000006847 19968 19972 ESG1 +T583 CL:0002322 19983 19991 ES cells +T584 UBERON:0000358 19997 20008 blastocysts +T585 PR:000006847 20010 20014 Esg1 +T586 PR:000006847 20020 20024 ESG1 +T587 NCBITaxon:10088 20042 20046 mice +T588 CHEBI:41774 20066 20075 Tamoxifen +T589 CHEBI:6716 20088 20100 Depo-provera +T590 GO:0007565 20143 20152 pregnancy +T591 UBERON:0000922 20171 20178 embryos +T592 UBERON:0000995 20215 20221 uterus +T593 NCBITaxon:27592 20346 20352 Bovine +T594 UBERON:0001977 20353 20358 Serum +T595 CHEBI:17334 20456 20466 Penicillin +T596 CHEBI:17076 20467 20479 Streptomysin +T597 CHEBI:41218 20506 20523 2-mercaptoethanol +T598 PR:000027795 20641 20648 trypsin +T599 PR:000027795 20793 20800 trypsin +T600 CL:0000001 20899 20906;20910 20914 primary ... cell +T601 CL:0002322 20907 20914 ES cell +T602 UBERON:0000119 21004 21015 cell layers +T603 CL:0002322 21129 21137 ES cells +T604 PR:000006847 21142 21146 ESG1 +T605 CL:0002322 21150 21158 ES cells +T606 CHEBI:37987 21176 21179 Cy3 +T607 CHEBI:37989 21184 21187 Cy5 +T608 GO:0097617 21220 21230 hybridized +T609 NCBITaxon:10088 21236 21241 Mouse +T610 GO:0097617 21390 21403 Hybridization +T611 PR:000006847 21576 21580 ESG1 +T612 NCBITaxon:10088 21590 21594 mice +T613 PR:000006847 21647 21651 ESG1 +T614 SO:0000704 21652 21656 gene +T615 SO:0000336 21661 21672 pseudogenes +T616 SO:0001644 21693 21709 targeting vector +T617 NCBITaxon:10088 21726 21731 mouse +T618 UBERON:0000922 21732 21738 embryo +T619 CL:0002322 21775 21782 ES cell +T620 SO:0000440 22260 22267 vectors +T621 CL:0002322 22324 22332 ES cells diff --git a/src/ontogpt/evaluation/craft/database/all/16504174.txt b/src/ontogpt/evaluation/craft/database/all/16504174.txt new file mode 100644 index 000000000..fe1c171c8 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16504174.txt @@ -0,0 +1,121 @@ +Identification and targeted disruption of the mouse gene encoding ESG1 (PH34/ECAT2/DPPA5) + +Abstract + +Background + +Embryonic stem cell-specific gene (ESG) 1, which encodes a KH-domain containing protein, is specifically expressed in early embryos, germ cells, and embryonic stem (ES) cells. Previous studies identified genomic clones containing the mouse ESG1 gene and five pseudogenes. However, their chromosomal localizations or physiological functions have not been determined. + +Results + +A Blast search of mouse genomic databases failed to locate the ESG1 gene. We identified several bacterial artificial clones containing the mouse ESG1 gene and an additional ESG1-like sequence with a similar gene structure from chromosome 9. The ESG1-like sequence contained a multiple critical mutations, indicating that it was a duplicated pseudogene. The 5' flanking region of the ESG1 gene, but not that of the pseudogene, exhibited strong enhancer and promoter activity in undifferentiated ES cells by luciferase reporter assay. To study the physiological functions of the ESG1 gene, we replaced this sequence in ES cells with a β-geo cassette by homologous recombination. Despite specific expression in early embryos and germ cells, ESG1-/- mice developed normally and were fertile. We also generated ESG1-/- ES cells both by a second independent homologous recombination and directly from blastocysts derived from heterozygous intercrosses. Northern blot and western blot analyses confirmed the absence of ESG1 in these cells. These ES cells demonstrated normal morphology, proliferation, and differentiation. + +Conclusion + +The mouse ESG1 gene, together with a duplicated pseudogene, is located on chromosome 9. Despite its specific expression in pluripotent cells and germ cells, ESG1 is dispensable for self-renewal of ES cells and establishment of germcells. + +Background + +Embryonic stem (ES) cells were first derived from the blastocysts of mice in 1981 [1,2] and humans in 1998 [3]. ES cells have two important properties: theability to maintain pluripotency, which is the ability to differentiate into a wide variety of cells, and rapid proliferation. These characteristics make mouse ES cells an essential component of gene targeting technology. These qualitiesalso make human ES cells attractive sources for cell transplantation therapy to treat various diseases, including spinal cord injuries and juvenile diabetes. The molecular mechanisms underlying the pluripotency and rapid proliferation of ES cells are currently a major focus of the field of stem cell biology [4-6]. + +To identify molecules essential in ES cells for these properties, several groups have utilized transcriptome analyses to identify genes specifically expressed in ES cells and early embryos. These analyses, including DNA microarray analyses [7] and expressed sequence tag analyses [8-12], identified several common transcripts, including ESG1 that was also designated dppa5 or ECAT2. + +ESG1 was originally identified as a transcript Ph34 that was down-regulated by retinoic acid in embryonic carcinoma cells [13]. The expression of this gene was confined in mice to early embryos and germ cells [14]. It is also expressed in pluripotent cells, including ES cells, embryonic germ cells, and multipotent germline stem cells [15]. ESG1 encodes a polypeptide of 118 amino acids that contains a single KH domain, which is an RNA-binding domain [16]. It remains unclear, however, if ESG1 functions as an RNA-binding protein or the roles it plays in ES cells and mice. + +Previous genomic library screening by identified genomic clones containing the mouse ESG1 gene and seven pseudogenes [17]. Two of these pseudogenes exhibit a similar exon-intron structure as the ESG1 gene, indicating their generation by gene duplication. The five remaining pseudogenes did not contain any introns, indicating that these were generated by retrotransposition of ESG1 transcripts. The chromosomal localizations of the mouse ESG1 gene and pseudogenes, however, have not been reported. + +In this study, we determined the structure of the mouse gene encoding this protein and related pseudogenes. We also performed gene targeting to determine the physiological function of ESG1. + +Results and discussion + +Chromosomal localization and structures of mouse ESG1 gene and psedogenes + +To determine the chromosomal localizations of the mouse ESG1 gene and pseudogenes, we performed a Blast analysis of the mouse genomic database with the ESG1 cDNA sequence as a query. We identified several putative pseudogenes without introns on chromosomes 1, 5, 11, 12, 14, 16, 17, and X (Figure 1). In addition, two intron-less pseudogenes were identified in DNA fragments for which the chromosomal localization remained unmapped. While these pseudodgenes have significant homology to ESG1 cDNA, they could not produce functional proteins, because of critical mutations. This result suggests that there are a larger number of intron-less pseudogenes than previously anticipated. Existence of multiple retropseudogenes is a hallmark of pluripotent cell-specific genes [18]. + +Figure 1 + +ESG1pseudogenes identified by a Blast search of mouse genomic databases. Substitution mutations are indicated by black lines. Intron-like gap sequences are indicated with triangles. Chromosomal localizations are shown on the right. + +On chromosome 9, we identified a DNA fragment similar to the ESG1 gene that included two putative introns. These putative first and second exons, however, contained (4) multiple mutations of the ESG1 cDNA sequence. The putative third exon was identical to that of the previously reported ESG1 gene. Also on chromosome 9, we identified another DNA fragment that was similar, but not identical, to the third exon of the ESG1 gene. These findings suggest that these ESG1-like sequences on chromosome 9 have not been correctly assembled. + +To obtain DNA fragments containing the ESG1 gene, we screened the bacterial artificial chromosome (BAC) DNA pool by PCR using primer pairs that would only amplify the real gene, not the pseudogenes. We obtained two independent, but overlapping BAC clones. Southern blot analyses and sequencing demonstrated that these clones contained a sequence exhibiting complete identity with ESG1 cDNA that was interrupted by two putative introns (Figure 2A). The two intron sequences begin with GT and terminate with AG, fulfilling the GT-AG rule of exon-intron junctions [19]. The 5' flanking region of this DNA fragment exhibited strong promoter/enhancer activity by luciferase reporter assays in undifferentiated ES cells, but not in somatic cells (Figure 3). The same fragment showed much weaker activity after induction of differentiation by retinoic acid. We concluded that this sequence is the ESG1 gene. + +Figure 2 + +BAC clones containing the ESG1 gene and a duplication pseudogene (PS). A) Localization of the gene and PS on chromosome 9. B) Sequence comparison of the gene and PS revealed that these are homologous from eight base pairs upstream of the first exon to 675 bp downstream of the third exon. Substitution mutations are indicated by black lines. The insertion is indicated by an open triangle. + +Figure 3 + +Promoter/enhancer activity of the ESG1 gene and pseudogene. DNA fragments of ~6 kbp isolated from the 5' flanking regions of the gene and PS were transferred into luciferase reporter plasmids. We introduced the reporter genes into undifferentiated ES cells (open columns), retinoic acid-treated ES cells (grey columns), and NIH3T3 cells (closed columns). Data represent the averages and standard deviations of three independent experiments. + +We also found that the two BAC clones contained another ESG1-like sequence (Figure 2A). The two sequences, separated by a 68 kbp intergenic sequence, were oriented in opposite directions. The ESG1-like sequence exhibited greater than 95% identity to the exons and introns of the ESG1 gene. This sequence, however, contained critical nucleotide substitutions in all of the exons and one nucleotide insertion in exon 2 (Figure 2B). Although 675 base pairs of the 3' flanking regions were conserved between the ESG1 gene and the pseudogene, only five base pairs of the 5' flanking region were identical. This 5' flanking region (~6 kbp) did not possess any promoter/enhancer activity in luciferase reporter assays (Figure 3). It is thus unlikely that this sequence is transcribed or translated into a functional protein. This sequence likely represents a duplication pseudogene. Bierbaum previously reported the existence of two pseudogenes with similar exon-intron organization as the ESG1 gene [17]. We could not determine which of these two pseudogenes corresponds to the one we identified or the location of the remaining pseudogene. + +Targeted disruption of the mouse ESG1 gene + +To study the function of ESG1, we deleted the gene by homologous recombination in mouse ES cells. We replaced the three exons with either a fusion of the neomycin-resistance and β-galactosidase genes (β-geo) or the hygromycin resistant gene (HygR) using two targeting vectors (Figure 4A) introduced into RF8 ES cells by electroporation. We obtained eight ES cell clones with correct homologous recombination of the β-geo targeting vector, which was confirmed by Southern blot analysis (Figure 4B). We obtained only one clone with correct homologous recombination of the HygR targeting vector. + +Figure 4 + +Targeted disruption of the mouse ESG1 gene. A) Targeting strategy. Homologous regions are indicated by thick lines. Recognition sites of PstI (P) and SpeI (S), which were used for Southern blot analyses, are shown. The gene encoding diphtheria toxin A (DTA) was inserted at the 3' end of the targeting vectors to facilitate negative selection. B) Southern blot analyses confirming homologous recombination. WT, wild-type ES cells; β, β-geo +/- ES cells; H, HygR +/- ES cells; -/-, ESG1-null ES cells. Numbers indicate clone numbers. C) Northern blot (upper) and western blot (lower) analyses of wild-type ES cells (WT), ESG1-null ES cells (-/-, three clones) and heterozygous ES cells (+/-). Northern blot was performed as previously described [20]. To confirm the loading of equal amounts of RNA, ethidium bromide staining of ribosomal RNA is also shown (middle). + +To obtain homozygous mutant ES cells, we introduced the β-geo vector into HygR heterozygous ES cells. Of 105 G418-resistant colonies tested, 49 were homozygous for ESG1 deletion. Northern blot and western blot analyses confirmed the absence of ESG1 in these cells (Figure 4C). In 29 clones, the β-geo vector had replaced the HygR vector, such that the cells remained heterozygous. In the remaining 27 clones, the β-geo vector was integrated at non-homologous sites. + +ESG1-/- ES cells exhibited normal morphology (Figure 5A). These cells also proliferated at a speed comparable to that of the control (heterozygous and wild-type) cells (Figure 5B). ESG1-/- cells differentiated normally after the removal of leukemia inhibitory factor (Figure 5B) or retinoic acid treatment (not shown). When transplanted into hind flanks of nude mice, these cells produced teratomas, tumors containing components of all three germ layers (Figure 5C). These results indicate that ESG1 is dispensable for the self-renewal properties and pluripotency of ES cells. + +Figure 5 + +Analyses of ESG1-null ES cells. A) The morphology of ESG1-null ES cell colonies grown on STO feeder cells. B) Growth curve of wild-type (WT), ESG1-null (-/-) and heterozygous (+/-) ES cells. Each clone (1 × 104 cells/well) was plated in 24-well plates. Cell numbers were determined with a Coulter counter at 2, 4, and 6 days. Data of +/- and -/- cells are shown as averages and standard deviations of three independent clones. C) A section of teratoma derived from ESG1-null ES cells (hematoxylin & eosin staining). + +We examined the gene expression profiles of ESG1-/- ES cells using oligonucleotide-based DNA microarrays representing ~20,000 genes. In comparison to control ES cells, ESG1 was identified as the gene reduced to the greatest extent in ESG1-/- ES cells (Figure 6A). The expression of ES cell marker genes, such as Nanog and Oct3/4, was normal in ESG1-/- ES cells. We confirmed normal Oct3/4 expression at protein levels by Western blot (Figure 6B). The overall gene expression profiles were similar between control ES cells and ESG1-/- ES cells. Several genes exhibited a greater than two-fold reduction in ESG1-/- cells, including Krt1-8, Pem, Ctgf, Ptgs2, Igf2 and Inhba. These genes might be regulated directly or indirectly by ESG1. Since ESG1 contains a KH-type RNA-binding domain, it may stabilize mRNA of these genes. Further studied are required to clarify this possibility. + +Figure 6 + +Gene expression analyses of ESG1-null ES cells. A) DNA microarray analyses. Total RNA from wild-type ES cells and ESG1-null ES cells were labeled with Cy3 and Cy5, respectively. Samples were hybridized to Agilent Mouse Developmental Microarrays. The averages of two independent clones are shown. B) Western blot analyses. Cell lysates from ESG1+/- and ESG1-/- cells were examined for expression of ESG1, Oct3/4 and CDK4 with immunoblotting. + +To generate ESG1-knockout mice, we injected β-geo-ESG1+/- ES cell clones into the blastocysts of C57BL6 mice. We obtained germline transmission from three clones. We obtained ESG1-/- mice at the Mendelian ratios (36 wild-type, 69 ESG1+/-, and 45 ESG1-/-) from intercrosses of ESG1+/- mice. These animals exhibited normal development, gross appearance, and fertility (not shown). Histological examination of testis and ovary could not identify any abnormalities (not shown). These data demonstrated that ESG1 is dispensable for both mouse development and germ cell formation. + +We also generated ES cells from blastocysts obtained by intercrosses of ESG1+/- males and ESG1-/- females. Of the eight ES cell lines established, two clones were ESG1-/-. These ESG1-null ES cells demonstrated normal morphology, proliferation, and differentiation (not shown), confirming that ESG1 is dispensable in ES cells. + +Conclusion + +To analyze the physiological roles of ESG1, we identified the mouse gene on chromosome 9 and deleted it by homologous recombination in ES cells. Despite specific expression in early embryos, germ cells, and pluripotent cells, our data demonstrated that ESG1 is dispensable for mouse development, germ cell formation, and ES cell self-renewal. + +Methods + +Identification and analyses of BAC clones containing the mouse ESG1 gene + +To identify bacterial artificial chromosome (BAC) clones containing mouse ESG1 gene, we performed PCR-based screening of mouse BAC library DNA pools (Research Genetics) using the pH34-u38 (5'-GAAGTCTGGTTCCTTGGCAGG-3') and pH34-L394 (5'-ACTCGATACACTGGCCTAGC-3') primers. Following restriction enzyme digestion, we performed Southern blot analyses of BAC clones as described [20] using the pH34-U258 (5'-CTCGAGTGTACAGTCAAGTGGTTGCTGGGA-3'), pH34-U65 (5'-GTGACCCTCGTGACCCGTAA-3'), pH34-intron1L (5'-CTGCGTGAGAGAAACACCAAACAGGC-3'), pH34-L545 (5'-TGTGAATGGGAAGGTTACCACTCT-3') and pH34-SCL1 (5'-GCCCTCTTCTGGTTTGTCTCGAAAT-3') probes. Hybridization with these probes revealed bands containing either the ESG1 gene or pseudogenes. + +To sequence the region containing the mouse ESG1 gene and the 3' flanking region, we subcloned a ~15 kbp XhoI-SalI fragment into the pZERO-2 vector (Invitrogen). HindIII- or EcoRI-digested fragments of this vector were then cloned into pBluescript KS(-) for sequencing. To sequence the ESG1 pseudogene and the 3' flanking region, an 8 kbp NotI/XhoI fragment was cloned into pBluescript KS(-). BamHI- or PstI- fragments of this vector were also cloned into pBluescript KS(-). To identify the sequence containing the 5' flanking regions of the ESG1 gene and the related pseudogenes, we used a TOPO walker kit (Invitrogen) with the pH34-T2L (5'-ACTAGTCGCAGCAGGGATCCAGGAATATCT-3') and pH34-L394 primers. The resulting sequence was cloned into pCR2.1 (Invitrogen). We obtained a ~6 kbp band from the NsiI-digensted library; XbaI-, SpeI-, EcoRI-, and PstI-digested fragments of this band were cloned into pBluescript KS(-) for sequencing. This fragment contained the 5' flanking region of the ESG1 gene. A ~3 kbp fragment, obtained from the SacI-digested library, was cloned into pCR2.1 for sequencing. This fragment was contained the 5' region flanking the pseudogene. + +Construction of ESG1 targeting vectors + +We replaced all of the ESG1 exons with two targeting vectors containing either an IRES-β-geo cassette [21] or an IRES-HygR cassette by promoter trap selection. We amplified the 5' arm (1.8 kbp) using KOD plus (TOYOBO) with the pH34-targetpair5-U (5'-CCGCGGAAAGTCAAGAGATTGGGTGG-3') and pH34-targetpair5-L (5'-GCGGCCGCCTTTACGGGTCACGAGGGTCAC-3') primers. The 3' arm (5.8 kbp) was amplified using the pH34-targetpair3-U (5'-TGTGGCCAGTGTTTGGTTCTGGCGGG-3') and pH34-targetpair3-L (5'-CTCGAGGACTCGCCATTCTAGCCAAG-3') primers. The IRES β-geo or IRES HygR cassettes were ligated in between the two PCR fragments. The diphtheria toxin A cassette was placed downstream of the 3' arm. After linearization with SacII, these targeting vectors were electroporated into 2.0 × 107 RF8 ES cells [22] using a Gene pulser (BIORAD). Transfected cells were selected with 250 μg/mL G418 or 100 μg/mL hygromycin B, respectively. Genomic DNA from G418- or hygromycin B-resistant colonies was screened for homologous recombination by Southern blotting. + +Southern blot screening for homologous recombination + +ES cells genomic DNA was extracted with PUREGENE™ Cell Lysis Solution (Gentra systems). For 5' Southern blot analysis, genomic DNA was first digested with PstI, then separated on an 0.8% agarose gel and transferred to a nylon membrane as described [20]. A 560 bp 5' probe was amplified using the ESG1S5 (5'- GATGGTGGTGGTGACTCAGAG -3') and ESG1AS5 as (5'- CCTCCATTGCCTCTATATCAG -3') primers. The probe specifically labeled an 18 kbp band from the wild-type locus, a 15 kbp band from the β-geo locus, and a 12 kbp band from the HygR locus. Genomic DNA was also digested with SpeI for 3' Southern blot analysis. A 1,010 bp 3' probe was amplified with the pH34U-8000 (5'- CCAACCAGCCAGAGTTTCAGTTAT -3') and pH34L-9000 (5'-GATAAGCTGCTGCCAAAAGACAAG -3') primers. The probe hybridized to an 11.5 kbp band from the wild-type locus, a 12.5 kbp band from the β-geo locus, and a 9.5 kbp band from the HygR locus. + +Generation of anti-ESG1 polyclonal antibodies + +The coding sequence of Esg1 was amplified by PCR with the pH34-gw-s (5'- AAAAAGCAGGCTGGATGATGGTGACCCTCGTGA-3') and pH34-gw-as (5'- AGAAAGCTGGGTCTGCATCCAGGTCGGAGACA-3') primers. To construct pDONR-pH34, the resulting PCR product was subcloned into pDONR201 (Invitrogen). pDONR-pH34 was interacted with pDEST17 (Invitrogen) by LR recombination. After introduction of the resulting expression vector pDEST17-pH34 into BL21-AI E. coli (Invitrogen), recombinant protein production was induced according to the manufacture's protocol. Histidine-tagged ESG1 was purified using Ni-nitrilotriacetic acid agarose (Qiagen) under denaturing conditions in the presence of 8 M urea. After dialysis against 6 M urea, the recombinant proteins were injected into New Zealand White rabbits to generate anti-ESG1 polyclonal antibodies. + +Western blot + +After preparation of ES cell extracts with M-Per (Pierce), cellular proteins were separated on sodium dodecyl sulfate (SDS)-14% polyacrylamide gels and transferred to nitrocellulose membranes (Millipore). Membranes were incubated with anti-ESG1 (1/500 dilution), anti-Oct3/4 (1/500; Santa Cruz Biotechnology), anti-CDK4 (1/200; Santa Cruz Biotechnology), and anti-GFP (1/1000; MBL) primary antibodies. Horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulins (1/5000; Cell Signaling) were used to detect antibody binding. We visualized bound antibody with an ECL Western Blotting Detection System (Amersham). + +Derivation of ESG1-deficient ES cells from blastocysts + +Esg1+/-or ESG1-/- mutant female mice were injected with Tamoxifen (10 μg) and Depo-provera (1 mg) subcutaneously on the third day of pregnancy. Four days later, embryos in diapause were flushed out of the uterus and cultured on STO feeder cells in four-well plates in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 20% Fetal Bovine Serum (Hyclone), 0.1 mM Non-Essential Amino Acids (Invitrogen), 2 mM L-glutamine (Invitrogen), 50 U/ml Penicillin-Streptomysin (Invitrogen), and 0.11 mM 2-mercaptoethanol (Invitrogen). After six days, the central mass of each explant was harvested, rinsed in PBS, and placed in a drop of trypsin for a few minutes. The cell mass was collected with a finely drawn-out Pasteur pipette preloaded with medium, ensuring minimal carryover of the trypsin. The cells were gently transfered into a fresh well with 20% FBS-containing medium. The resulting primary ES cell colonies were individually passaged into wells of four-well plates containing STO feeder cell layers. Thereafter, cells were expanded by trypsinization of the entire culture. + +Microarrays + +Total RNA from wild-type ES cells and ESG1-/- ES cells was labeled with Cy3 and Cy5, respectively. The samples were hybridized to a Mouse Development Microarray (Algilent) according to the manufacturer's protocol. Arrays were scanned with a G2565BA Microarray Scanner System (Agilent). Hybridization was repeated with two independent clones. Data were analyzed with GeneSprings software (Silico Genetics). + +Authors' contributions + +HA carried out the phenotypic studies of ESG1 knockout mice. KI determined the chromosomal localizations of the ESG1 gene and pseudogenes and constructed the targeting vector. TI carried out mouse embryo manipulation. MM and MN carried out ES cell culture. SY conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript. + +Acknowledgements + +We thank Chihiro Takigawa, Junko Iida, Masako Shirasaka, Yumi Ohuchi, and Megumi Narita for their technical and administrative assistance. We are grateful to Drs. Minoru Ko and Azim Surani for sharing helpful data prior to publication, Dr. Hitoshi Niwa for generously providing various vectors, and Dr. Robert Farese Jr. for his kind gift of the RF8 ES cells. This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Uehara Memorial Foundation, the Naito Foundation, the Sumitomo Research Foundation, the Mitsubishi Foundation. S.Y. was supported by a Toray Science and Technology Grant (to S.Y.). This work was also supported in part by a Grant-in-Aid for 21st Century COE (Center of Excellence) Research from the Ministry of Education, Culture, Sports, Science, and Technology. diff --git a/src/ontogpt/evaluation/craft/database/all/16507151.ann b/src/ontogpt/evaluation/craft/database/all/16507151.ann new file mode 100644 index 000000000..df1d43f58 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16507151.ann @@ -0,0 +1,340 @@ +T1 http://purl.obolibrary.org/obo/MONDO_0005578 35 44 arthritis +T2 NCBITaxon:10088 77 81 mice +T3 http://purl.obolibrary.org/obo/MONDO_0005578 110 119 arthritis +T4 NCBITaxon:10088 123 127 mice +T5 UBERON:0002405 165 171 immune +T6 http://purl.obolibrary.org/obo/MONDO_0008383 219 239 rheumatoid arthritis +T7 http://purl.obolibrary.org/obo/MONDO_0005578 264 273 arthritis +T8 SO:0000704 291 296 genic +T9 http://purl.obolibrary.org/obo/MONDO_0000001 297 304 disease +T10 http://purl.obolibrary.org/obo/MONDO_0000001 357 364 disease +T11 GO:0065007 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17882 17891 arthritis +T307 PR:000003266 17893 17896 CII +T308 PR:000003266 17899 17915 collagen type II +T309 GO:0042611 17946 17949 MHC +T310 GO:0042611 17952 17984 major histocompatibility complex +T311 MOP:0000635 18003 18017 chain reaction +T312 SO:0000771 18019 18022 QTL +T313 SO:0000771 18025 18049 quantitative trait locus +T314 http://purl.obolibrary.org/obo/MONDO_0008383 18051 18053 RA +T315 http://purl.obolibrary.org/obo/MONDO_0008383 18056 18076 rheumatoid arthritis +T316 http://purl.obolibrary.org/obo/MONDO_0005578 18696 18705 arthritis +T317 NCBITaxon:10088 18758 18762 mice +T318 SO:0000771 18804 18827 quantitative trait loci +T319 SO:0000771 18829 18832 QTL +T320 http://purl.obolibrary.org/obo/MONDO_0005578 18855 18864 arthritis +T321 SO:0000771 18900 18903 QTL +T322 PR:000003266 18908 18924 collagen type II +T323 PR:000003266 18926 18929 CII +T324 GO:0042571 18931 18939 antibody +T325 http://purl.obolibrary.org/obo/MONDO_0005578 19179 19188 arthritis +T326 PR:000003266 19198 19214 collagen type II +T327 PR:000003266 19221 19224 CII +T328 NCBITaxon:10088 19267 19271 mice +T329 http://purl.obolibrary.org/obo/MONDO_0005578 19352 19361 arthritic +T330 NCBITaxon:10088 19362 19366 mice +T331 http://purl.obolibrary.org/obo/MONDO_0005578 19485 19494 arthritic +T332 NCBITaxon:10088 19495 19499 mice +T333 NCBITaxon:10088 19562 19566 mice +T334 NCBITaxon:10088 19687 19691 mice +T335 http://purl.obolibrary.org/obo/MONDO_0005578 19751 19760 arthritic +T336 NCBITaxon:10088 19761 19765 mice +T337 http://purl.obolibrary.org/obo/MONDO_0005578 19875 19884 arthritic +T338 NCBITaxon:10088 19885 19889 mice +T339 NCBITaxon:10088 19997 20001 mice +T340 http://purl.obolibrary.org/obo/MONDO_0005578 20070 20079 arthritis diff --git a/src/ontogpt/evaluation/craft/database/all/16507151.txt b/src/ontogpt/evaluation/craft/database/all/16507151.txt new file mode 100644 index 000000000..efa7ac817 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16507151.txt @@ -0,0 +1,125 @@ +Identification of collagen-induced arthritis loci in aged multiparous female mice + +Abstract + +Collagen-induced arthritis in mice is one of the most commonly used autoimmune experimental models, with many similarities to rheumatoid arthritis. Since collagen-induced arthritis is a complex polygenic disease there is a need for identification of several major disease-controlling genes. Because rheumatoid arthritis particularly affects aged women, we have in the present study identified new genetic regions critical for collagen-induced arthritis by studying aged female mice of a cross between NFR/N and B10.Q (H-2q haplotype). The mice in the present study had different reproductive histories, which did not significantly affect the onset, incidence or severity of the disease. A total of 200 female mice were used in a total genome-wide screening with 125 microsatellite markers. We found one new significant quantitative trait locus affecting the arthritis incidence, severity and day of onset on chromosome 11 (denoted Cia40), which colocalizes with a locus controlling pregnancy failure. Furthermore, a quantitative trait locus of suggestive significance associated with the incidence, severity and day of onset was identified on chromosome 1. Finally, a suggestively significant quantitative trait locus associated with collagen type II antibody titers was identified on chromosome 13. This study indicates that several gene loci control arthritis in aged multiparous females, and that at least one of these loci coincides with pregnancy failure. + +Introduction + +The similarities between rheumatoid arthritis (RA) in man and collagen-induced arthritis (CIA) in mice are well established, although differences in the disease pattern exist [1-3]. Characteristic of RA is the fact that women of reproductive age are more susceptible to the disease than men [4]. It has also been reported that 75% of female patients (23 out of 31 females) with RA exhibit clinical remission during the course of pregnancy [5]. On the other hand, it has been suggested that the onset of RA is postponed by parity. The risk of onset is reduced during pregnancy, while in the first year postpartum the chance of onset is increased [6,7]. The results of a recent study of RA in women, however, indicate that pregnancy history does not increase the incidence of the disease later in life [8]. + +Adequate animal models are useful tools for the identification of genes involved in complex human diseases. CIA in mice shares both immunological and pathological characteristics with human RA and is one of the most used models for the identification of genes and mechanisms involved in arthritis. The incidence of CIA is sex dependent, like the incidence in RA, although different species and different variants of the disease could lead to both male and female predominance. Male mice are more often affected than females. Gender differences in CIA susceptibility are dependent on many factors, including genetic, hormonal and behavioral influences [9]. However, isolated factors are remarkably consistent between RA and the different animal models [3,9]. Pregnancy in mice, like pregnancy in women, normally causes remission of arthritis [5,10], while exacerbation often occurs postpartum [7,10,11]. Pregnancy-induced remission of CIA in mice appears to be caused by the increase in estrogen levels, while the postpartum exacerbation can be explained by the dramatic drop in estrogen [10], possibly together with increased prolactin levels [11]. To what extent the pregnancy history (parity) affects the incidence of CIA later in life has, to our knowledge, not previously been studied. Importantly, RA occurs predominantly in women, many of which have had several children, and it is therefore appropriate to also mimic this situation using animal models. + +In the present paper we have analyzed female mice that previously had undergone a reproductive study (Liljander M, Sällström MA, Andersson S, Wernhoff P, Andersson Å, Holmdahl R, Mattsson R, unpublished data). A total of 200 female mice (ten months of age) of the N2 backcross between NFR/N and C57BL/B10.Q (B10.Q) were used to analyze the genetic control of CIA in aged females with a multiparous history. + +Materials and methods + +Mice + +Inbred NFR/N mice were originally obtained from the National Institute of Health (Bethesda, MD, USA) and the B10.Q mice were originally bought from The Jackson Laboratory (Bar Harbor, ME, USA). (B10Q × NFR/N)F1 hybrids and (B10.Q × NFR/N) × B10.Q N2 mice were bred in individual ventilated cages in the BMC Barrier animal facility and at the animal house of the Department of Pathology, Lund University, Sweden. The animals were fed ad libitum with standard rodent chow (LAB FOR R36, irradiated breeding food for rats and mice; Lactamin AB, Stockholm, Sweden) and water in a photoperiod of 12 hours:12 hours light:dark. The mice used in the present study had clean health monitoring protocols according to the Federation of European Laboratory Animal Sciences Association recommendations. Ethical permissions were M125-04 (embryo transfer) and M290-03 (reproduction and arthritis). + +Experimental design + +A total of 200 female mice (approximately ten months old) of N2 backcross (B10.Q × NFR/N) × B10.Q were used. The mice had earlier undergone a reproductive study where each animal had four mating opportunities. First, the female mice were allowed to mate twice with B10.RIII males (allogeneic mating by means of MHC (major histocompatibility complex) and finally to mate twice with B10.Q males (syngeneic mating by means of MHC). After four possible pregnancies CIA was induced to the female mice. + +Induction and evaluation of CIA + +To induce CIA, the mice were immunized subcutaneously at the base of the tail with 100 μg rat collagen type II (CII) emulsified in 0.1 M acetic acid combined with an equal amount of complete Freud's adjuvant (Difco Laboratories, Detroit, MI, USA). A booster injection containing 50 μg CII emulsified in 0.1 M acetic acid combined with an equal amount of Freud's incomplete adjuvant (Difco Laboratories) was given after 30 days. The clinical scoring of arthritis commenced 25 days after the first immunization. Animals were examined for clinical signs of CIA three times per week and were graded on a 12-point scale. The arthritis index was assigned to each mouse using the following criteria: 0 = no visible sign of arthritis, 1 = redness and swelling in a single joint, 2 = inflammation in multiple joints, and 3 = severe inflammation in the entire paw and/or anklebone. Each paw was given the scores 0–3, with the index being the sum of all four paws. The severity trait is the maximum score observed in each individual female. The onset is the number of days calculated from the first immunization to the first clinical signs of arthritis excluding unaffected animals. + +Microsatellite genotyping and linkage analysis + +Tail biopsies were collected from all N2 females and the F0 generation. DNA was isolated according to a previously described protocol [12]. After screening of parental DNA with approximately 450 mouse fluorescence-labeled microsatellite markers (INTERACTIVA, Ulm, Germany), 125 informative markers were selected covering the genome. Two hundred and thirty-seven N2 mice were genotyped with markers covering all chromosomes except for the Y chromosome. PCR amplification for the markers was performed in a final volume of 10 μl in a 96-well V-bottom microtiter plate using 20 ng DNA, 10 mM KCl, 20 mM Tris–HCl, 10 mM (NH4)2SO4, 2 mM MgCl2, 0.1% Triton X-100, pH 8.8 (New England BioLabs Inc., Ipswich, MA, USA), 3 μM (10 pmol) each primer, 2 mM dNTPs (Advanced Biotechnologies, Epsom, Surrey, UK) and 0.25 U Taq DNA Polymerase (New England BioLabs Inc.). The following program was used to amplify the DNA: denaturation at 95°C for 3 minutes, annealing at 56°C for 45 seconds, polymerization at 72°C for 1 minute, 30 cycles of 95°C for 30 seconds, 56°C for 45 seconds and 72°C for 1 minute, and a final extension step of 7 minutes at 72°C. The PCR products were analyzed on a MegaBACE™ 1000 (Amersham Pharmacia Biotech Inc Piscataway, NJ, USA) according to the manufacturer's protocol. Data were analyzed with Genetic Profiler 1.1 (Amersham Pharmacia Biotech Inc). + +Map Manager QTXb20 free software [13] was used to perform linkage analysis and the permutation test. Ninety percent of the mouse genome was within a 20 cM intermarker distance. The marker map was generated using the Kosambis map function and 1,000 permutations were performed for every phenotype (P < 0.05). The permutation tests were carried out to establish empirical significance thresholds for the interactions. A threshold equal to or above the 37th percentile (P = 0.63) was considered suggestively significant, and the level for the significant threshold was set to the 95th percentile (P = 0.05). Interval mapping was made with a 2 cM increase under the additive regression model in order to calculate the test statistics. + +Enzyme-linked immunosorbent assay + +The adult female mice were sacrificed at 15 months of age and sera were collected. Anti-CII antibody titers in sera were analyzed by a sandwich ELISA technique [14]. In brief, CII (10 μg/ml) was coupled to immunosorbent plates overnight at 4°C. Bovine serum albumin (Sigma Chemical St Louis, MO, USA) was used for blocking, and thereafter different dilutions of control sera (purified mouse anti-CII antibodies), test sera, and positive and negative controls were added. The presence of CII-specific IgG was visualized by means of peroxidase-conjugated goat antimouse IgG. + +Statistical analysis + +Statistical comparison between the different experimental groups was performed using the Mann-Whitney U test or Student's unpaired t test. + +Results + +NFR/N female mice show delayed onset and lower incidence of CIA compared with B10.Q females + +The onset of the disease was significantly delayed in NFR/N mice, which resulted in lower incidence in the initial phase of the disease (Table 1 and Figure 1). The appearance of the arthritis was slightly different between the two strains, NFR/N mice showing a higher frequency of swelling of the entire paw than B10.Q mice. Later in the disease course the differences in CIA frequency and severity between the strains were less obvious. However, the anti-CII titers 100 days after primary immunization (and 70 days after the booster) differed significantly between NFR/N and B10.Q female mice (see Table 1). + +Pregnancy history does not significantly affect incidence or severity of CIA in female mice of mixed genetic background + +Table 2 presents the results of arthritic incidence, arthritis onset and maximum arthritic score for the female mice of the N2 generation, (NFR/N × B10.Q) × B10.Q, grouped according to the number of pregnancies they had experienced prior to immunization with CII. There was no significant difference in the incidence or severity of arthritis depending on the number of pregnancies that had been passed prior to the induction of the disease. We observed a trend towards lower severity in multiparous mice compared with mice with a history of zero to two pregnancies. The onset of diseases, however, was earlier in mice that had passed more than one pregnancy. + +There was no difference in the development of the disease between female mice that have only been pregnant with MHC mismatched males (B10.RIII) and female mice that only have been pregnant with MHC matched males (B10.Q). + +New loci associated with CIA in identified old female mice + +The genetic linkage analysis revealed one significant quantitative trait locus (QTL) located at 64–70 cM on chromosome 11 (Table 3 and Figure 2). This locus (denoted Cia40) gave significant LOD scores for the traits incidence (LOD = 4.1) and 'day of onset' (LOD = 4.0), and a suggestive LOD score for the trait 'arthritis score' (LOD = 3.3). Another QTL of suggestive significance for the incidence of disease was identified around 27 cM on chromosome 1 (Figure 2). Finally, a suggestive QTL for the trait anti-CII antibody titer was identified on chromosome 13 (Figure 2). + +Discussion + +Although carrying the same CIA-susceptible MHC region, the NFR/N and B10.Q mice are different in several respects. The NFR/N mouse, which is an inbred NMRI mouse of the H-2q haplotype, is larger in size than the B10.Q mouse and is also known for its extraordinarily good breeding properties. The mice used in the present study, (NFR/N × B10.Q) × B10.Q females, first underwent a genetic study of reproduction, in which a number of loci associated with breeding performance were identified (Liljander M, Sällström MA, Andersson S, Wernhoff P, Andersson Å, Holmdahl R, Mattsson R; unpublished data). Since the parental strains differ in susceptibility to CIA, in spite of both having the MHC class II A beta genes [15], we wanted to test the susceptibility to inflammatory disease in mice from this cross. In fact, this situation gave an opportunity to study the genetic control of arthritis in aged multiparous females, a common situation in human RA. This susceptibility was important to investigate for various reasons. The fact that the mice differed in their reproductive history made it possible to analyze whether this would significantly affect the incidence or severity of CIA. Second, almost all the linkage analyses performed for detection of CIA-associated loci in mice have been carried out with male mice. Another reason is that the use of old multiparous mice mimicked the RA situation (older women is the major risk group). Finally, the NFR/N strain had not previously been used in linkage analyses for detection of CIA-associated loci, which opened (up) the possibility of detecting new polymorphic genes of importance in this disease. + +The first conclusion from the present paper is that multiparity does not negatively influence the incidence or severity of CIA induced later in life (Table 2). This is in agreement with results from a recent study of RA in women, which similarly indicates that previous experience of pregnancy does not negatively affect the incidence or severity of RA later in life [8]. It is worth noting that the situation is quite different if pregnancy is entered during ongoing arthritis. This will normally lead to a temporary remission of the disease in both mice and humans, followed by an exacerbation phase after delivery [7,10,11]. + +The second conclusion from this study is that loci of possible importance for CIA have been detected, two associated with arthritis susceptibility (chromosomes 1 and 11) and one associated with anti-CII titers (chromosome 13). Some arthritis loci have previously been identified on chromosome 1: Cia15 at 8 cM [16], Cia20 at 44 cM [16] and Cia9 at 92 cM [17]. However, none of these loci appears to be close to the locus found on chromosome 1 in the present study. The newly identified Cia40 locus includes the Ctla4 (CD152) gene, which is a strong candidate associated with spontaneous diabetes identified in crosses between C57Bl and nonobese diabetic strains [18]. Analyses of CIA in crosses of the same backgrounds, however, did not identify a linkage to this locus, suggesting that the polymorphism underlying Cia40 differs from the genes associated with diabetes [18]. Interestingly, Bauer and colleagues previously identified a locus (Cia28) associated with anti-CII antibody production at 53 cM on chromosome 13 [19], which is approximately at the same position as where we find the linkage for this trait in the present study. It is therefore most likely that the QTL we have identified on chromosome 13 is the same as Cia28. + +Since the possible new locus of potential interest on chromosome 1 was of suggestive significance, and since the locus identified on chromosome 13 probably is Cia28, we are now paying special attention to the significant QTL for CIA incidence detected at 60–70 cM on chromosome 11 (denoted Cia40). No other CIA genes have previously been typed in this region, but the central part of chromosome 11 is known to contain a number of inflammation loci, such as Eae22, Eae6b, Eae23 and Eae7 [20-22]. One QTL for proteoglycan-induced arthritis, which was female specific for disease onset, have also been found on chromosome 11. Although this locus (Pgia28) is not located in the vicinity of Cia40, it is worth noting [23]. + +The mouse chromosome 11 contains several genes that are highly conserved with human chromosome 17. Linkages for human RA have been found in this particular region [24]. Another interesting locus is Cia5 on the homolog rat chromosome 10, which regulates the severity of CIA and pristane-induced arthritis [25]. + +Interestingly, from studies with the same cross, we have previously detected a significant QTL denoted Pregq2 for the trait 'pregnancy frequency' in the very same region as Cia40 (peak at 64–70 cM on chromosome 11) (Liljander M, Sällström MA, Andersson S, Wernhoff P, Andersson Å, Holmdahl R, Mattsson R, unpublished data). This means that this region contains genes affecting the CIA incidence in multiparous mice in addition to the rate of successful deliveries in female mice. The 'pregnancy rate' in mice is defined as successful pregnancies per detected vaginal plug, a phenotype associated with early pregnancy failure, which in turn possibly could have an inflammatory cause. We cannot exclude that the same gene(s) are affecting both these traits. + +Conclusion + +Our results show that multiparity does not negatively influence the incidence or severity of CIA induced later in life. Furthermore, two new loci linked to CIA susceptibility were detected on chromosome 11 (Cia40) and on chromosome 1. We detected on chromosome 13 a locus associated with anti-CII titers, which probably is the same as the recently reported Cia28 [19]. + +Abbreviations + +CIA = collagen-induced arthritis; CII = collagen type II; LOD = logarithm of the odds; MHC = major histocompatibility complex; PCR = polymerase chain reaction; QTL = quantitative trait locus; RA = rheumatoid arthritis. + +Competing interests + +The authors declare that they have no competing interest. + +Authors' contributions + +ML is responsible for genotyping, phenotyping, analyses and, together with RM, for interpretation and for writing the manuscript. M-AS and SA have contributed to collection of phenotype data. RM, ÅA, RH and ML designed the study. All authors read and approved the final manuscript. + +Acknowledgements + +This study was supported by Österlund's fund, Crafoord's fund, the Gustav V 80 Year Foundation, the Royal Physiographic Society in Lund and by Swegene. + +Figures and Tables + +Figure 1 + +The accumulated incidence of arthritis in the parental strains (NFR/N and B10.Q) of female mice. + +Figure 2 + +Chromosomal locations of the quantitative trait loci (QTL) for collagen-induced arthritis incidence on chromosomes 1 and 11. QTL for collagen type II (CII) antibody titers are illustrated on chromosome 13. *Suggestive level threshold (P = 0.63) according to the permutation test (n = 1,000). ** Significance level threshold (P = 0.05) according to the permutation test (n = 1,000). + +Table 1 + +Severity of arthritis and anti-collagen type II (anti-CII) titers in (NFR/N × B10.Q) × B10.Q female mice and parental strains + +aMedian value on the days of onset when calculated in all arthritic mice in the group on day 100 (maximum–minimum values in the series). + +bMean ± standard error of the maximum score from all arthritic mice in the respective group. + +cSignificantly higher than in B10.Q mice (P = 0.02 in Student's unpaired t test). + +Table 2 + +Incidence, onset and maximum score in (NFR/N × B10.Q) × B10.Q female mice + +aMedian value on the days of onset when calculated in all arthritic mice in the group (maximum–minimum values in the series). + +bMean (± standard error) of the maximum score from all arthritic mice in the respective group on day 100. + +Table 3 + +Distribution of genotypes between affected and unaffected N2 mice at different loci linked to clinical phenotypes of collagen-induced arthritis + +*Suggestive significance, **significant. diff --git a/src/ontogpt/evaluation/craft/database/all/16517939.ann b/src/ontogpt/evaluation/craft/database/all/16517939.ann new file mode 100644 index 000000000..d123e97de --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16517939.ann @@ -0,0 +1,809 @@ +T1 SO:0001060 3 10 isoform +T2 PR:000017718 14 20 ZBP-89 +T3 UBERON:0001155 37 42 colon +T4 http://purl.obolibrary.org/obo/MONDO_0005292 46 53 colitis +T5 GO:0000380 65 85 Alternative splicing +T6 GO:0010467 94 104 expression +T7 SO:0001060 137 145 isoforms +T8 PR:000017718 224 230 ZBP-89 +T9 GO:0000380 274 295 alternatively spliced +T10 SO:0000704 296 301 genes +T11 NCBITaxon:9606 319 324 human +T12 PR:000017718 325 331 ZBP-89 +T13 GO:0008380 332 338 splice +T14 PR:000017718 348 354 ZBP-89 +T15 PR:000044061 429 437 ZBP-89FL +T16 PR:000017718 440 446 ZBP-89 +T17 GO:0010467 461 470 expressed +T18 PR:000044061 480 488 ZBP-89FL +T19 UBERON:0005409 500 516 gastrointestinal +T20 UBERON:0000479 532 539 tissues +T21 PR:000017718 552 558 ZBP-89 +T22 GO:0010467 573 582 expressed +T23 PR:000017718 629 635 ZBP-89 +T24 NCBITaxon:10088 647 651 mice +T25 SO:0000147 665 669 exon +T26 PR:000017718 716 722 ZBP-89 +T27 NCBITaxon:10088 725 729 mice +T28 GO:0010467 731 741 expressing +T29 PR:000017718 747 753 ZBP-89 +T30 http://purl.obolibrary.org/obo/MONDO_0005292 864 871 colitis +T31 PR:000017718 890 896 ZBP-89 +T32 PR:000044061 911 919 ZBP-89FL +T33 GO:0010467 943 953 expression +T34 SO:0001060 971 978 isoform +T35 UBERON:0005409 988 1004 gastrointestinal +T36 GO:0042592 1005 1016 homeostasis +T37 GO:0000380 1033 1062 Alternative pre-mRNA splicing +T38 SO:0000932 1045 1053 pre-mRNA +T39 SO:0000167 1076 1084 promoter +T40 SO:0000704 1128 1135 genetic +T41 NCBITaxon:9606 1150 1156 humans +T42 NCBITaxon:10088 1165 1169 mice +T43 SO:0001026 1175 1181 Genome +T44 NCBITaxon:9606 1226 1231 human +T45 SO:0000704 1232 1237 genes +T46 GO:0010467 1238 1245 express +T47 GO:0000380 1246 1264 alternative splice +T48 http://purl.obolibrary.org/obo/MONDO_0005070 1310 1319 neoplasia +T49 http://purl.obolibrary.org/obo/MONDO_0000001 1329 1336 disease +T50 SO:0001060 1377 1385 isoforms +T51 PR:000003035 1393 1396 p53 +T52 SO:0000704 1397 1401 gene +T53 PR:000016567 1420 1423 p63 +T54 PR:000016568 1432 1435 p73 +T55 http://purl.obolibrary.org/obo/MONDO_0005070 1454 1459 tumor +T56 GO:0010467 1523 1532 expressed +T57 http://purl.obolibrary.org/obo/MONDO_0005070 1536 1542 tumors +T58 PR:000017718 1545 1551 ZBP-89 +T59 PR:000017718 1553 1559 ZNF148 +T60 PR:000017718 1561 1567 Zfp148 +T61 PR:000017718 1569 1575 BFCOL1 +T62 GO:0065007 1682 1691 regulates +T63 SO:0000167 1736 1744 promoter +T64 http://purl.obolibrary.org/obo/MONDO_0005070 1843 1848 tumor +T65 PR:000003035 1860 1863 p53 +T66 SO:0000417 1888 1894 domain +T67 PR:000043452 1924 1931 histone +T68 CHEBI:15358 1924 1931 histone +T69 PR:000007102 1983 1987 p300 +T70 PR:000017718 2021 2027 ZBP-89 +T71 PR:000003035 2065 2068 p53 +T72 GO:0006915 2098 2107 apoptosis +T73 PR:000003035 2118 2121 p53 +T74 NCBITaxon:10088 2150 2154 Mice +T75 PR:000017718 2177 2183 ZBP-89 +T76 PR:000017718 2185 2191 Zfp148 +T77 http://purl.obolibrary.org/obo/MONDO_0005047 2197 2204 sterile +T78 GO:0007283 2223 2238 spermatogenesis +T79 UBERON:0000922 2258 2267 embryonic +T80 CL:0002322 2258 2278 embryonic stem cells +T81 PR:000017718 2298 2304 ZBP-89 +T82 SO:0001023 2305 2311 allele +T83 PR:000003035 2328 2331 p53 +T84 PR:000017718 2365 2371 ZBP-89 +T85 GO:0032991 2380 2387 complex +T86 PR:000007102 2393 2397 p300 +T87 CHEBI:17968 2405 2413 butyrate +T88 PR:000005274 2427 2434 p21waf1 +T89 NCBITaxon:9606 2438 2443 human +T90 UBERON:0012652 2444 2454 colorectal +T91 PR:000017718 2494 2500 ZBP-89 +T92 CHEBI:37527 2513 2519 acidic +T93 SO:0000417 2520 2526 domain +T94 PR:000007102 2548 2552 p300 +T95 GO:0010467 2582 2592 expression +T96 SO:0000417 2616 2622 domain +T97 CHEBI:17968 2674 2682 butyrate +T98 PR:000005274 2696 2703 p21waf1 +T99 CHEBI:37527 2714 2720 acidic +T100 SO:0000417 2721 2727 domain +T101 PR:000017718 2731 2737 ZBP-89 +T102 SO:0000147 2767 2771 exon +T103 NCBITaxon:9606 2781 2786 human +T104 NCBITaxon:10088 2791 2796 mouse +T105 SO:0000704 2797 2802 genes +T106 PR:000017718 2833 2839 ZBP-89 +T107 GO:0010467 2845 2855 expression +T108 PR:000007102 2903 2907 p300 +T109 SO:0000417 2920 2926 domain +T110 PR:000017718 2950 2956 ZBP-89 +T111 NCBITaxon:10088 2962 2967 mouse +T112 UBERON:0000922 2981 2990 embryonic +T113 NCBITaxon:10088 3019 3024 mouse +T114 PR:000017718 3044 3050 ZBP-89 +T115 SO:0001060 3053 3060 isoform +T116 NCBITaxon:10088 3079 3083 mice +T117 http://purl.obolibrary.org/obo/MONDO_0005292 3197 3204 colitis +T118 GO:0010467 3227 3237 expression +T119 SO:0001060 3251 3258 isoform +T120 UBERON:0005409 3275 3291 gastrointestinal +T121 GO:0042592 3292 3303 homeostasis +T122 PR:000017718 3427 3433 ZBP-89 +T123 SO:0001026 3434 3441 genomic +T124 NCBITaxon:9606 3451 3463 Homo sapiens +T125 SO:0000153 3497 3500 BAC +T126 NCBITaxon:10090 3525 3537 Mus musculus +T127 SO:0000149 3552 3558 contig +T128 NCBITaxon:9598 3573 3588 Pan troglodytes +T129 SO:0001026 3602 3609 genomic +T130 NCBITaxon:9606 3631 3636 human +T131 PR:000017718 3637 3643 ZBP-89 +T132 SO:0000147 3655 3659 exon +T133 SO:0001026 3663 3670 genomic +T134 NCBITaxon:9598 3768 3781 P.troglodytes +T135 SO:0001026 3782 3789 genomic +T136 SO:0000857 3799 3809 homologous +T137 NCBITaxon:9606 3813 3818 human +T138 SO:0000147 3819 3823 exon +T139 NCBITaxon:9606 3882 3887 human +T140 PR:000017718 3889 3895 ZNF148 +T141 NCBITaxon:9596 3898 3903 chimp +T142 NCBITaxon:10088 3908 3913 mouse +T143 PR:000017718 3915 3921 Zfp148 +T144 PR:000017718 3923 3929 ZBP-89 +T145 NCBITaxon:9606 3938 3943 human +T146 SO:0000040 3944 3957 genomic clone +T147 SO:0000149 3958 3964 contig +T148 PR:000017718 3982 3988 ZBP-89 +T149 NCBITaxon:2 4026 4035 Bacterial +T150 SO:0000153 4026 4057 Bacterial artificial chromosome +T151 SO:0000153 4059 4062 BAC +T152 SO:0001421 4106 4128 intron/exon boundaries +T153 PR:000017718 4142 4148 ZNF148 +T154 SO:0000147 4156 4160 Exon +T155 SO:0000704 4182 4186 gene +T156 SO:0000153 4238 4241 BAC +T157 NCBITaxon:10710 4246 4266 bacteriophage lambda +T158 SO:0000149 4273 4279 contig +T159 NCBITaxon:10088 4293 4298 mouse +T160 PR:000017718 4299 4305 Zfp148 +T161 SO:0001026 4357 4364 genomic +T162 PR:000017718 4385 4391 Zfp148 +T163 SO:0000112 4399 4406 Primers +T164 SO:0001030 4414 4415 F +T165 SO:0001031 4455 4456 R +T166 NCBITaxon:9596 4518 4523 chimp +T167 NCBITaxon:9598 4525 4538 P.troglodytes +T168 SO:0001026 4540 4547 genomic +T169 SO:0000147 4563 4567 exon +T170 SO:0000730 4622 4625 gap +T171 NCBITaxon:9596 4642 4647 Chimp +T172 SO:0001026 4648 4655 genomic +T173 NCBITaxon:9443 4693 4700 Primate +T174 CL:0000010 4859 4873 cultured cells +T175 CHEBI:33893 4887 4894 reagent +T176 UBERON:0001155 4967 4972 colon +T177 http://purl.obolibrary.org/obo/MONDO_0002032 4967 4979 colon cancer +T178 UBERON:0000344 4991 4998 mucosal +T179 GO:0001171 5104 5125 reverse transcription +T180 NCBITaxon:9606 5186 5191 human +T181 SO:0000147 5192 5196 exon +T182 NCBITaxon:9606 5264 5269 Human +T183 SO:0000112 5270 5276 primer +T184 SO:0001030 5306 5307 F +T185 SO:0001031 5338 5339 R +T186 SO:0001030 5371 5372 F +T187 SO:0001031 5396 5397 R +T188 NCBITaxon:10088 5419 5424 Mouse +T189 SO:0000112 5425 5431 primer +T190 SO:0001030 5466 5467 F +T191 SO:0001030 5500 5501 F +T192 SO:0001031 5530 5531 R +T193 SO:0001031 5563 5564 R +T194 SO:0001030 5599 5600 F +T195 SO:0001031 5629 5630 R +T196 SO:0000006 5655 5667 PCR products +T197 NCBITaxon:9606 5779 5784 human +T198 SO:0000147 5785 5789 exon +T199 SO:0000147 5812 5816 exon +T200 SO:0000704 5899 5903 gene +T201 SO:0000112 5913 5919 primer +T202 SO:0000006 6013 6025 PCR products +T203 SO:0000112 6050 6057 primers +T204 SO:0001644 6176 6192 Targeting vector +T205 SO:0000440 6209 6215 vector +T206 PR:000017718 6234 6240 ZBP-89 +T207 SO:0000147 6241 6245 exon +T208 SO:0005853 6263 6271 cassette +T209 NCBITaxon:10088 6370 6375 mouse +T210 SO:0000153 6376 6379 BAC +T211 SO:0000112 6403 6410 primers +T212 SO:0001030 6433 6434 F +T213 SO:0001031 6490 6491 R +T214 SO:0001030 6560 6561 F +T215 SO:0001031 6612 6613 R +T216 SO:0000147 6705 6709 exon +T217 SO:0000188 6716 6722 intron +T218 SO:0000188 6804 6810 intron +T219 CHEBI:7507 6818 6826 neomycin +T220 SO:0005853 6827 6835 cassette +T221 SO:0000141 6849 6876 transcriptional stop signal +T222 GO:0048468 6952 6965;6978 6983 Generation of ... cells +T223 CL:0002322 6975 6983 ES cells +T224 NCBITaxon:10088 6994 6998 mice +T225 UBERON:0000922 7019 7028 embryonic +T226 CL:0002322 7019 7033;7039 7044 embryonic stem ... cells +T227 CL:0002322 7035 7037;7039 7044 ES ... cells +T228 SO:0001644 7061 7077 targeting vector +T229 UBERON:0000358 7100 7111 blastocysts +T230 CL:0002322 7126 7134 ES cells +T231 NCBITaxon:33208 7191 7197 Animal +T232 SO:0001026 7213 7220 Genomic +T233 CL:0002322 7268 7270 ES +T234 SO:0000112 7349 7355 Primer +T235 NCBITaxon:10088 7575 7580 mouse +T236 UBERON:0002106 7581 7587 spleen +T237 UBERON:0000479 7600 7606 tissue +T238 CHEBI:33893 7626 7633 reagent +T239 NCBITaxon:9606 7815 7820 human +T240 NCBITaxon:40674 7870 7879 mammalian +T241 CHEBI:33893 7899 7906 reagent +T242 CHEBI:28619 8162 8172 acrylamide +T243 CHEBI:53250 8216 8220 PVDF +T244 PR:000017718 8261 8267 ZBP-89 +T245 UBERON:0001977 8272 8277 serum +T246 http://purl.obolibrary.org/obo/MONDO_0005292 8339 8346 colitis +T247 http://purl.obolibrary.org/obo/MONDO_0005292 8352 8359 colitis +T248 PR:000017718 8389 8395 ZBP-89 +T249 NCBITaxon:10088 8421 8425 mice +T250 GO:0042756 8455 8463 drinking +T251 CHEBI:15377 8464 8469 water +T252 NCBITaxon:10088 8507 8511 mice +T253 GO:0042756 8536 8544 drinking +T254 CHEBI:15377 8545 8550 water +T255 CHEBI:51686 8611 8622 Hematoxylin +T256 CHEBI:51686 8634 8635 H +T257 UBERON:0001155 8647 8652 colon +T258 http://purl.obolibrary.org/obo/MONDO_0005292 8726 8733 colitis +T259 UBERON:0001983 8806 8811 crypt +T260 CL:0000738 8987 8996 leukocyte +T261 GO:0045123 8987 9009 leukocyte infiltration +T262 UBERON:0000344 9014 9021 mucosal +T263 http://purl.obolibrary.org/obo/MONDO_0005292 9396 9403 colitis +T264 NCBITaxon:33208 9424 9431 Animals +T265 GO:0016265 9437 9441 died +T266 http://purl.obolibrary.org/obo/MONDO_0005292 9470 9477 colitis +T267 NCBITaxon:1 9520 9531 Individuals +T268 NCBITaxon:9606 9709 9714 Human +T269 UBERON:0001276 9715 9733 gastric epithelial +T270 CL:0002178 9715 9739 gastric epithelial cells +T271 NCBITaxon:9606 9760 9765 human +T272 UBERON:0001155 9766 9771 colon +T273 http://purl.obolibrary.org/obo/MONDO_0002032 9766 9778 colon cancer +T274 NCBITaxon:9606 9820 9825 human +T275 http://purl.obolibrary.org/obo/MONDO_0005059 9833 9841 leukemia +T276 CHEBI:16526 9902 9905 CO2 +T277 CHEBI:18208 9968 9980 penicillin G +T278 CHEBI:17076 9996 10008 streptomycin +T279 NCBITaxon:9606 10050 10055 human +T280 PR:000017718 10056 10062 ZBP-89 +T281 SO:0001060 10065 10072 isoform +T282 SO:0001060 10090 10098 isoforms +T283 PR:000017718 10102 10108 ZBP-89 +T284 NCBITaxon:9606 10132 10137 human +T285 PR:000017718 10138 10144 ZNF148 +T286 SO:0001026 10145 10152 genomic +T287 SO:0000147 10217 10221 exon +T288 SO:0000147 10249 10253 exon +T289 SO:0000147 10306 10310 exon +T290 GO:0010467 10317 10326 expressed +T291 SO:0000147 10340 10344 Exon +T292 GO:0010467 10365 10374 expressed +T293 SO:0000147 10380 10384 exon +T294 SO:0000673 10399 10406 message +T295 CHEBI:33695 10399 10406 message +T296 UBERON:0001155 10415 10420 colon +T297 http://purl.obolibrary.org/obo/MONDO_0002032 10415 10427 colon cancer +T298 UBERON:0000479 10475 10481 tissue +T299 UBERON:0001155 10494 10499 colon +T300 UBERON:0001155 10504 10509 colon +T301 http://purl.obolibrary.org/obo/MONDO_0002271 10504 10524 colon adenocarcinoma +T302 GO:0010467 10558 10567 expressed +T303 PR:000017718 10644 10650 ZBP-89 +T304 SO:0001060 10700 10707 isoform +T305 SO:0000147 10709 10713 exon +T306 SO:0000147 10763 10767 exon +T307 SO:0000147 10798 10803 exons +T308 SO:0000147 10836 10841 exons +T309 SO:0000121 10884 10899 forward primers +T310 SO:0000147 10905 10910 exons +T311 SO:0000147 10949 10953 exon +T312 SO:0000077 10957 10966 antisense +T313 SO:0000112 10967 10974 primers +T314 SO:0000167 11007 11015 promoter +T315 GO:0065007 11016 11025 regulates +T316 SO:0000147 11026 11030 exon +T317 GO:0010467 11034 11044 expression +T318 SO:0000147 11182 11186 exon +T319 SO:0000147 11221 11225 exon +T320 GO:0008380 11233 11240 spliced +T321 SO:0000147 11244 11248 exon +T322 SO:0000717 11279 11292 reading frame +T323 SO:0000147 11325 11329 exon +T324 SO:0000147 11346 11350 Exon +T325 SO:0000203 11391 11413 untranslated sequences +T326 GO:0006412 11393 11403 translated +T327 SO:0000147 11428 11432 exon +T328 SO:0000167 11474 11482 promoter +T329 SO:0000147 11501 11505 exon +T330 GO:0010467 11525 11535 expression +T331 PR:000017718 11569 11575 ZBP-89 +T332 SO:0001060 11576 11583 isoform +T333 PR:000017718 11585 11591 ZBP-89 +T334 SO:0000318 11615 11631 initiation codon +T335 SO:0001060 11692 11699 isoform +T336 CHEBI:37527 11710 11716 acidic +T337 SO:0000417 11717 11723 domain +T338 PR:000007102 11728 11732 p300 +T339 PR:000044061 11779 11787 ZBP-89FL +T340 UBERON:0001043 11858 11867 esophagus +T341 UBERON:0000945 11869 11876 stomach +T342 UBERON:0001155 11878 11883 colon +T343 CL:0000084 11895 11902 T-cells +T344 SO:0000147 11924 11928 exon +T345 SO:0000167 11944 11952 promoter +T346 NCBITaxon:9606 11988 11993 human +T347 PR:000017718 11994 12000 ZBP-89 +T348 PR:000017718 12058 12064 ZBP-89 +T349 PR:000044061 12140 12148 ZBP-89FL +T350 CHEBI:8984 12263 12266 SDS +T351 PR:000005997 12332 12336 CTCF +T352 PR:000017493 12346 12349 XPA +T353 SO:0001060 12404 12412 isoforms +T354 CHEBI:37527 12498 12504 acidic +T355 SO:0000417 12505 12511 domain +T356 PR:000017718 12515 12521 ZBP-89 +T357 PR:000044061 12575 12583 ZBP-89FL +T358 PR:000017718 12711 12717 ZBP-89 +T359 UBERON:0001977 12722 12727 serum +T360 CHEBI:22695 12870 12875 basic +T361 PR:000017718 12884 12890 ZBP-89 +T362 PR:000017718 12895 12901 ZBP-89 +T363 SO:0000167 12916 12924 promoter +T364 NCBITaxon:9604 12952 12960 hominids +T365 NCBITaxon:9606 12975 12980 human +T366 PR:000017718 12982 12988 ZNF148 +T367 NCBITaxon:10088 12994 12999 mouse +T368 PR:000017718 13001 13007 Zfp148 +T369 PR:000017718 13009 13015 ZBP-89 +T370 SO:0001026 13044 13051 genomic +T371 SO:0000147 13071 13075 exon +T372 NCBITaxon:10088 13092 13096 mice +T373 NCBITaxon:40674 13112 13119 mammals +T374 SO:0000028 13171 13173 bp +T375 NCBITaxon:9596 13185 13195 chimpanzee +T376 NCBITaxon:9598 13197 13210 P.troglodytes +T377 SO:0000857 13261 13269 homology +T378 NCBITaxon:9606 13273 13278 human +T379 SO:0000147 13279 13283 exon +T380 SO:0000730 13304 13307 gap +T381 SO:0001026 13331 13338 genomic +T382 SO:0000730 13371 13374 gap +T383 SO:0001026 13381 13388 genomic +T384 GO:0006277 13389 13406 DNA amplification +T385 NCBITaxon:9596 13426 13431 chimp +T386 SO:0000028 13476 13478 bp +T387 NCBITaxon:9606 13479 13484 human +T388 SO:0000147 13485 13489 exon +T389 PR:000017718 13518 13524 ZBP-89 +T390 SO:0000167 13539 13547 promoter +T391 NCBITaxon:9604 13575 13583 hominids +T392 NCBITaxon:10088 13599 13604 mouse +T393 PR:000017718 13614 13620 ZBP-89 +T394 GO:0010467 13623 13633 expression +T395 NCBITaxon:10088 13641 13645 mice +T396 SO:0000147 13651 13655 exon +T397 GO:0010467 13681 13688 express +T398 PR:000017718 13691 13697 ZBP-89 +T399 SO:0001060 13700 13707 isoform +T400 NCBITaxon:10088 13725 13730 mouse +T401 SO:0000147 13731 13735 exon +T402 SO:0000417 13738 13744 domain +T403 SO:0000147 13819 13823 exon +T404 SO:0000188 13830 13836 intron +T405 SO:0000188 13905 13911 intron +T406 CL:0002322 13962 13964 ES +T407 UBERON:0000358 14110 14121 blastocysts +T408 SO:0000147 14152 14156 exon +T409 UBERON:0000922 14524 14531 embryos +T410 GO:0016265 14532 14535 die +T411 UBERON:0012101 14548 14559 perinatally +T412 GO:0007567 14552 14559 natally +T413 PR:000017718 14869 14875 ZBP-89 +T414 PR:000017718 14877 14883 Zfp148 +T415 SO:0001023 14885 14891 allele +T416 CL:0000586 14910 14919 germ cell +T417 GO:0007281 14910 14931 germ cell development +T418 GO:0000380 14972 14992 Alternative splicing +T419 SO:0000147 14996 15000 exon +T420 SO:0000147 15006 15010 exon +T421 NCBITaxon:10088 15028 15032 mice +T422 SO:0000147 15061 15065 exon +T423 PR:000017718 15080 15086 ZBP-89 +T424 GO:0010467 15087 15097 expression +T425 SO:0000147 15154 15158 exon +T426 NCBITaxon:33208 15170 15177 animals +T427 SO:0000147 15199 15203 exon +T428 SO:0000147 15280 15285 exons +T429 GO:0010467 15291 15300 expressed +T430 SO:0000112 15314 15321 primers +T431 SO:0000147 15331 15335 exon +T432 SO:0000028 15354 15356 bp +T433 SO:0000028 15387 15389 bp +T434 SO:0001023 15407 15414 alleles +T435 SO:0000028 15473 15475 bp +T436 SO:0000147 15489 15493 exon +T437 SO:0000147 15517 15521 exon +T438 GO:0008380 15528 15535 spliced +T439 SO:0000147 15548 15552 exon +T440 SO:0005853 15586 15594 cassette +T441 SO:0000147 15662 15666 exon +T442 SO:0000318 15681 15697 initiation codon +T443 SO:0000673 15710 15717 message +T444 CHEBI:33695 15710 15717 message +T445 SO:0000717 15754 15767 reading frame +T446 SO:0000704 15769 15773 Gene +T447 SO:0000318 15839 15855 initiation codon +T448 NCBITaxon:9606 15925 15930 human +T449 PR:000017718 15931 15937 ZBP-89 +T450 NCBITaxon:10088 15962 15967 Mouse +T451 PR:000017718 15968 15974 ZBP-89 +T452 GO:0010467 15985 15995 expression +T453 PR:000044061 16046 16054 ZBP-89FL +T454 PR:000017718 16059 16065 ZBP-89 +T455 GO:0010467 16081 16090 expressed +T456 SO:0001023 16097 16103 allele +T457 NCBITaxon:9606 16254 16259 human +T458 SO:0001060 16260 16268 isoforms +T459 SO:0001060 16374 16382 isoforms +T460 SO:0001060 16445 16452 isoform +T461 UBERON:0002106 16505 16511 spleen +T462 UBERON:0000479 16512 16518 tissue +T463 GO:0010467 16649 16659 expression +T464 PR:000017718 16695 16701 ZBP-89 +T465 NCBITaxon:10088 16721 16726 mouse +T466 SO:0001026 16727 16733 genome +T467 NCBITaxon:10088 16745 16750 mouse +T468 PR:000017718 16816 16822 ZBP-89 +T469 GO:0010467 16825 16835 expression +T470 PR:000017718 16850 16856 ZBP-89 +T471 NCBITaxon:10088 16862 16866 mice +T472 PR:000017718 16947 16953 ZBP-89 +T473 NCBITaxon:10088 16959 16963 mice +T474 PR:000044061 17028 17036 ZBP-89FL +T475 PR:000044061 17028 17032;17037 17039 ZBP- ... FL +T476 PR:000044061 17076 17084 ZBP-89FL +T477 PR:000017718 17180 17186 ZBP-89 +T478 NCBITaxon:10088 17204 17208 mice +T479 NCBITaxon:10088 17267 17271 mice +T480 PR:000017718 17318 17324 ZBP-89 +T481 NCBITaxon:10088 17330 17334 mice +T482 UBERON:0012101 17351 17360 perinatal +T483 GO:0007567 17355 17360 natal +T484 PR:000044061 17595 17603 ZBP-89FL +T485 PR:000044061 17595 17599;17604 17606 ZBP- ... FL +T486 PR:000044061 17614 17622 ZBP-89FL +T487 PR:000017718 17635 17641 ZBP-89 +T488 NCBITaxon:10088 17647 17651 mice +T489 UBERON:0000062 17671 17676 organ +T490 UBERON:0000479 17681 17687 tissue +T491 PR:000017718 17733 17739 ZBP-89 +T492 NCBITaxon:10088 17745 17749 mice +T493 UBERON:0001155 17786 17791 colon +T494 CL:0000542 17842 17853 lymphocytic +T495 UBERON:0001155 17921 17926 colon +T496 GO:0010467 17988 17997 expressed +T497 SO:0001023 18004 18010 allele +T498 GO:0010467 18081 18091 expression +T499 PR:000044061 18095 18103 ZBP-89FL +T500 PR:000017718 18108 18114 ZBP-89 +T501 SO:0001060 18117 18125 isoforms +T502 NCBITaxon:10088 18155 18159 mice +T503 GO:0010467 18222 18232 expression +T504 PR:000017718 18240 18246 ZBP-89 +T505 SO:0001060 18249 18256 isoform +T506 NCBITaxon:10088 18271 18275 mice +T507 PR:000017718 18325 18331 ZBP-89 +T508 http://purl.obolibrary.org/obo/MONDO_0005292 18346 18353 colitis +T509 PR:000017718 18425 18431 ZBP-89 +T510 CHEBI:17968 18478 18486 butyrate +T511 PR:000005274 18500 18507 p21waf1 +T512 CHEBI:17968 18564 18572 butyrate +T513 PR:000017718 18616 18622 ZBP-89 +T514 NCBITaxon:40674 18630 18639 mammalian +T515 UBERON:0001555 18640 18662 gastrointestinal tract +T516 CHEBI:17968 18669 18677 Butyrate +T517 UBERON:0001155 18689 18696 colonic +T518 http://purl.obolibrary.org/obo/MONDO_0005292 18780 18787 colitis +T519 PR:000017718 18803 18809 ZBP-89 +T520 NCBITaxon:10088 18815 18819 mice +T521 CHEBI:51686 18908 18919 hematoxylin +T522 http://purl.obolibrary.org/obo/MONDO_0005292 18984 18991 colitis +T523 PR:000017718 19015 19021 ZBP-89 +T524 SO:0000704 19024 19028 gene +T525 PR:000044061 19058 19066 ZBP-89FL +T526 PR:000044061 19058 19062;19067 19069 ZBP- ... FL +T527 NCBITaxon:10088 19070 19074 mice +T528 CL:0000542 19110 19121 lymphocytic +T529 UBERON:0000344 19150 19157 mucosal +T530 PR:000044061 19165 19173 ZBP-89FL +T531 NCBITaxon:10088 19177 19181 mice +T532 UBERON:0001983 19255 19260 crypt +T533 UBERON:0000344 19292 19299 mucosal +T534 PR:000017718 19307 19313 ZBP-89 +T535 NCBITaxon:10088 19319 19323 mice +T536 UBERON:0001983 19371 19376 crypt +T537 UBERON:0000344 19435 19442 mucosal +T538 PR:000017718 19454 19460 ZBP-89 +T539 SO:0000704 19463 19467 gene +T540 UBERON:0005409 19519 19535 gastrointestinal +T541 PR:000017718 19573 19579 ZBP-89 +T542 NCBITaxon:10088 19585 19589 mice +T543 GO:0016265 19590 19594 died +T544 http://purl.obolibrary.org/obo/MONDO_0005292 19698 19705 colitis +T545 PR:000017718 19739 19745 ZBP-89 +T546 SO:0000704 19748 19752 gene +T547 GO:0010467 19807 19817 expression +T548 PR:000017718 19821 19827 ZBP-89 +T549 http://purl.obolibrary.org/obo/MONDO_0005292 19877 19884 colitis +T550 NCBITaxon:9606 19959 19964 human +T551 PR:000017718 19974 19980 ZBP-89 +T552 GO:0008380 19981 19987 splice +T553 PR:000017718 19997 20003 ZBP-89 +T554 SO:0000167 20042 20050 promoter +T555 SO:0000147 20084 20088 exon +T556 PR:000017718 20111 20117 ZBP-89 +T557 SO:0000417 20152 20158 domain +T558 PR:000017718 20189 20195 ZBP-89 +T559 PR:000017718 20245 20251 ZBP-89 +T560 SO:0001060 20252 20259 isoform +T561 SO:0000704 20310 20314 gene +T562 NCBITaxon:10088 20338 20342 mice +T563 NCBITaxon:9606 20351 20357 humans +T564 NCBITaxon:9606 20377 20382 human +T565 SO:0000704 20383 20388 genes +T566 GO:0000380 20393 20414 alternatively spliced +T567 NCBITaxon:10088 20464 20469 mouse +T568 SO:0001026 20495 20502 genomic +T569 PR:000017718 20531 20537 ZBP-89 +T570 GO:0008380 20540 20548 splicing +T571 NCBITaxon:9606 20586 20592 humans +T572 NCBITaxon:9596 20597 20603 chimps +T573 GO:0065007 20648 20658 regulating +T574 PR:000017718 20659 20665 ZBP-89 +T575 NCBITaxon:9604 20692 20700 hominids +T576 GO:0010467 20720 20730 expression +T577 SO:0000704 20741 20748 genetic +T578 GO:0065007 20767 20777 regulatory +T579 http://purl.obolibrary.org/obo/MONDO_0004992 20817 20823 cancer +T580 http://purl.obolibrary.org/obo/MONDO_0000001 20845 20852 disease +T581 NCBITaxon:9606 20876 20881 human +T582 PR:000017718 20882 20888 ZBP-89 +T583 UBERON:0000317 20926 20940 colonic mucosa +T584 SO:0000417 21017 21023 domain +T585 PR:000017718 21040 21046 ZBP-89 +T586 CHEBI:17968 21058 21066 butyrate +T587 PR:000005274 21091 21098 p21waf1 +T588 PR:000007102 21119 21123 p300 +T589 SO:0000417 21175 21181 domain +T590 NCBITaxon:10088 21231 21236 mouse +T591 GO:0010467 21280 21289 expressed +T592 PR:000007102 21316 21320 p300 +T593 SO:0000417 21333 21339 domain +T594 UBERON:0000104 21382 21390 lifespan +T595 NCBITaxon:1 21441 21449 organism +T596 UBERON:0000062 21495 21501 organs +T597 UBERON:0001155 21566 21571 colon +T598 NCBITaxon:10088 21657 21661 mice +T599 PR:000017718 21685 21691 ZBP-89 +T600 GO:0010467 21694 21704 expressing +T601 NCBITaxon:10088 21705 21709 mice +T602 http://purl.obolibrary.org/obo/MONDO_0005292 21721 21728 colitis +T603 NCBITaxon:33208 21765 21772 animals +T604 GO:0010467 21847 21857 expression +T605 PR:000044061 21861 21869 ZBP-89FL +T606 http://purl.obolibrary.org/obo/MONDO_0005292 21895 21902 colitis +T607 SO:0000704 21908 21912 gene +T608 http://purl.obolibrary.org/obo/MONDO_0005292 21934 21941 colitis +T609 PR:000017718 21983 21989 ZBP-89 +T610 SO:0001060 21992 21999 isoform +T611 PR:000044061 22047 22055 ZBP-89FL +T612 SO:0001060 22128 22136 isoforms +T613 PR:000003035 22148 22151 p53 +T614 SO:0000704 22152 22156 gene +T615 PR:000017718 22208 22214 ZBP-89 +T616 PR:000017718 22269 22275 Zfp148 +T617 SO:0000417 22324 22339 protein domains +T618 PR:000017718 22373 22379 ZBP-89 +T619 NCBITaxon:10088 22381 22385 Mice +T620 PR:000017718 22410 22416 ZBP-89 +T621 SO:0001023 22417 22423 allele +T622 CL:0000015 22456 22470 male germ cell +T623 GO:0007281 22461 22482 germ cell development +T624 PR:000003035 22504 22507 p53 +T625 GO:0009790 22518 22531 embryogenesis +T626 PR:000017718 22580 22586 ZBP-89 +T627 PR:000003035 22604 22607 p53 +T628 SO:0000704 22636 22640 gene +T629 PR:000017718 22673 22679 ZBP-89 +T630 GO:0010467 22682 22692 expression +T631 UBERON:0000922 22712 22721 embryonic +T632 GO:0007567 22730 22735 natal +T633 SO:0001429 22802 22813;22827 22833 DNA-binding ... region +T634 PR:000017718 22837 22843 ZBP-89 +T635 PR:000003035 22874 22877 p53 +T636 SO:0000417 22893 22899 domain +T637 PR:000017718 22919 22925 ZBP-89 +T638 SO:0001060 22928 22935 isoform +T639 SO:0000417 22957 22963 domain +T640 PR:000007102 22984 22988 p300 +T641 SO:0000417 23001 23007 domain +T642 PR:000003035 23028 23031 p53 +T643 UBERON:0000922 23042 23051 embryonic +T644 GO:0009790 23042 23063 embryonic development +T645 GO:0007567 23072 23077 natal +T646 UBERON:0005409 23116 23132 gastrointestinal +T647 NCBITaxon:33208 23252 23258 Animal +T648 NCBITaxon:33208 23421 23427 Animal +T649 UBERON:0000104 23643 23647 Life +T650 http://purl.obolibrary.org/obo/MONDO_0004992 23873 23879 Cancer +T651 UBERON:0005409 23948 23964 Gastrointestinal +T652 SO:0000147 24457 24461 exon +T653 NCBITaxon:9606 24473 24478 human +T654 PR:000017718 24479 24485 ZBP-89 +T655 PR:000017718 24487 24493 ZNF148 +T656 NCBITaxon:9606 24510 24515 human +T657 PR:000017718 24516 24522 ZBP-89 +T658 PR:000017718 24524 24530 ZNF148 +T659 SO:0000198 24587 24599;24627 24632 untranslated ... exons +T660 GO:0006412 24589 24599 translated +T661 SO:0000195 24614 24620;24627 24632 coding ... exons +T662 SO:0000147 24658 24662 exon +T663 SO:0000147 24727 24731 exon +T664 SO:0000121 24768 24775;24803 24810 forward ... primers +T665 SO:0000121 24777 24778;24803 24810 F ... primers +T666 SO:0000132 24784 24791;24803 24810 reverse ... primers +T667 SO:0000132 24793 24794;24803 24810 R ... primers +T668 GO:0010467 24820 24830 expression +T669 PR:000044061 24843 24852 FL ZBP-89 +T670 SO:0000147 24861 24865 exon +T671 SO:0000188 24887 24893 intron +T672 PR:000044061 24943 24954;24977 24983 full-length ... ZBP-89 +T673 PR:000044061 24956 24958;24977 24983 FL ... ZBP-89 +T674 GO:0010467 24989 24999 expression +T675 CL:0000010 25060 25073 cultured cell +T676 SO:0000112 25086 25093 primers +T677 SO:0001030 25097 25098 F +T678 SO:0001031 25101 25102 R +T679 SO:0000028 25108 25110 bp +T680 SO:0000112 25121 25128 primers +T681 SO:0001030 25132 25133 F +T682 SO:0001031 25136 25137 R +T683 SO:0000028 25143 25145 bp +T684 SO:0000112 25179 25186 primers +T685 SO:0000028 25192 25194 bp +T686 NCBITaxon:9606 25266 25271 human +T687 UBERON:0001155 25272 25277 colon +T688 UBERON:0001155 25314 25319 colon +T689 UBERON:0001155 25324 25329 colon +T690 http://purl.obolibrary.org/obo/MONDO_0002271 25324 25344 colon adenocarcinoma +T691 UBERON:0000479 25345 25351 tissue +T692 SO:0000673 25365 25372 message +T693 CHEBI:33695 25365 25372 message +T694 GO:0010467 25376 25385 expressed +T695 SO:0000167 25391 25399 promoter +T696 SO:0000167 25403 25406 Pro +T697 PR:000017718 25427 25433 ZBP-89 +T698 GO:0010467 25442 25451 expressed +T699 SO:0000167 25457 25465 promoter +T700 SO:0000167 25469 25472 Pro +T701 PR:000017718 25522 25528 ZBP-89 +T702 SO:0000673 25529 25539 transcript +T703 PR:000017718 25569 25575 ZBP-89 +T704 SO:0000147 25597 25601 exon +T705 SO:0000147 25672 25676 exon +T706 SO:0000147 25709 25713 exon +T707 GO:0006412 25718 25728 translated +T708 PR:000044061 25753 25761 ZBP-89FL +T709 SO:0001215 25789 25793;25796 25811 exon ... coding sequence +T710 SO:0000147 25891 25895 exon +T711 SO:0001060 26078 26086 isoforms +T712 PR:000044061 26110 26118 ZBP-89FL +T713 CHEBI:37527 26131 26137 acidic +T714 PR:000007102 26142 26146 p300 +T715 SO:0000417 26159 26165 domain +T716 PR:000017718 26192 26198 ZBP-89 +T717 PR:000017718 26206 26212 ZBP-89 +T718 SO:0001060 26215 26222 isoform +T719 SO:0000417 26262 26269 domains +T720 PR:000044061 26338 26346 ZBP-89FL +T721 PR:000017718 26368 26374 ZBP-89 +T722 CHEBI:37527 26525 26531 acidic +T723 PR:000017718 26593 26599 ZBP-89 +T724 SO:0000147 26600 26604 exon +T725 NCBITaxon:10088 26610 26614 mice +T726 SO:0000147 26635 26639 exon +T727 CHEBI:7507 26649 26657 Neomycin +T728 SO:0005853 26669 26677 cassette +T729 CL:0002322 26715 26723 ES cells +T730 SO:0000147 26782 26786 exon +T731 SO:0000318 26800 26816 initiation codon +T732 CHEBI:37527 26818 26824 acidic +T733 SO:0000417 26825 26831 domain +T734 PR:000007102 26854 26858 p300 +T735 SO:0000417 26871 26877 domain +T736 CHEBI:22695 26891 26896 basic +T737 SO:0000417 26897 26904 domains +T738 GO:0006412 26954 26964 translated +T739 PR:000017718 26980 26986 Zfp148 +T740 SO:0001026 26987 26994 genomic +T741 SO:0000188 27002 27008 Intron +T742 UBERON:0002415 27068 27072 Tail +T743 SO:0000188 27100 27103 Int +T744 SO:0001030 27106 27107 F +T745 SO:0000188 27108 27111 Int +T746 SO:0001031 27114 27115 R +T747 SO:0000188 27129 27132 Int +T748 SO:0001030 27135 27136 F +T749 SO:0001031 27141 27142 R +T750 SO:0001023 27197 27204 alleles +T751 NCBITaxon:10088 27314 27319 mouse +T752 PR:000017718 27320 27326 ZBP-89 +T753 NCBITaxon:10088 27414 27418 mice +T754 UBERON:0002106 27420 27426 Spleen +T755 GO:0001171 27446 27465 reverse-transcribed +T756 SO:0000112 27485 27492 primers +T757 SO:0000147 27514 27519 exons +T758 GO:0010467 27552 27561 expressed +T759 PR:000000133 27596 27599 Sp1 +T760 SO:0000006 27714 27726 PCR products +T761 PR:000017718 27867 27873 ZBP-89 +T762 SO:0000043 27882 27902 processed pseudogene +T763 SO:0000147 27998 28002 exon +T764 GO:0008380 28024 28032 splicing +T765 SO:0000147 28036 28040 exon +T766 SO:0000147 28046 28050 exon +T767 SO:0005853 28072 28080 cassette +T768 SO:0000717 28096 28103 reading +T769 SO:0000318 28139 28155 initiation codon +T770 NCBITaxon:9606 28228 28233 human +T771 PR:000017718 28234 28240 ZBP-89 +T772 UBERON:0002106 28293 28299 spleen +T773 NCBITaxon:10088 28345 28349 mice +T774 NCBITaxon:9606 28408 28413 human +T775 NCBITaxon:10088 28491 28495 mice +T776 PR:000044061 28523 28531 ZBP-89FL +T777 PR:000044061 28523 28527;28532 28534 ZBP- ... FL +T778 PR:000044061 28563 28571 ZBP-89FL +T779 PR:000017718 28608 28614 ZBP-89 +T780 NCBITaxon:10088 28697 28701 mice +T781 PR:000017718 28777 28783 ZBP-89 +T782 GO:0016265 28953 28958 death +T783 PR:000044061 29001 29009 ZBP-89FL +T784 PR:000017718 29014 29020 ZBP-89 +T785 UBERON:0001155 29042 29047 colon +T786 http://purl.obolibrary.org/obo/MONDO_0005292 29092 29099 colitis +T787 PR:000017718 29103 29109 ZBP-89 +T788 NCBITaxon:10088 29112 29116 mice +T789 CHEBI:51686 29139 29140 H +T790 PR:000017718 29186 29192 ZBP-89 +T791 NCBITaxon:10088 29202 29206 mice +T792 CL:0000542 29227 29238 lymphocytes +T793 UBERON:0000344 29248 29255 mucosal +T794 GO:0010467 29303 29313 expression +T795 UBERON:0005409 29335 29351 gastrointestinal +T796 UBERON:0001988 29374 29379 fecal +T797 UBERON:0000178 29387 29392 blood +T798 PR:000017718 29447 29453 ZBP-89 +T799 PR:000017718 29456 29462 ZBP-89 +T800 NCBITaxon:10088 29465 29469 mice +T801 GO:0016265 29470 29473 die +T802 PR:000044061 29503 29511 ZBP-89FL +T803 PR:000044061 29512 29520 ZBP-89FL +T804 PR:000044061 29525 29533 ZBP-89FL +T805 PR:000017718 29534 29540 ZBP-89 +T806 NCBITaxon:10088 29543 29547 mice +T807 http://purl.obolibrary.org/obo/MONDO_0005292 29582 29589 Colitis +T808 NCBITaxon:10088 29619 29623 mice +T809 NCBITaxon:10088 29668 29672 mice diff --git a/src/ontogpt/evaluation/craft/database/all/16517939.txt b/src/ontogpt/evaluation/craft/database/all/16517939.txt new file mode 100644 index 000000000..f88bcb16c --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16517939.txt @@ -0,0 +1,127 @@ +An isoform of ZBP-89 predisposes the colon to colitis + +Abstract + +Alternative splicing enables expression of functionally diverse protein isoforms. The structural and functional complexity of zinc-finger transcription factor ZBP-89 suggests that it may be among the class of alternatively spliced genes. We identified a human ZBP-89 splice isoform (ZBP-89ΔN), which lacks amino terminal residues 1–127 of the full-length protein (ZBP-89FL). ZBP-89ΔN mRNA was co-expressed with its ZBP-89FL cognate in gastrointestinal cell lines and tissues. Similarly, ZBP-89ΔN protein was expressed. To define its function in vivo, we generated ZBP-89ΔN knock-in mice by targeting exon 4 that encodes the amino terminus. Homozygous ZBP-89ΔN mice, expressing only ZBP-89ΔN protein, experienced growth delay, reduced viability and increased susceptibility to dextran sodium sulfate colitis. We conclude that ZBP-89ΔN antagonizes ZBP-89FL function and that over-expression of the truncated isoform disrupts gastrointestinal homeostasis. + +INTRODUCTION + +Alternative pre-mRNA splicing and multiple promoter usage are common mechanisms for increasing genetic complexity in humans (1) and mice (2). Genome-wide analyses indicate that the majority of human genes express alternative splice isoforms (3) and some variants contribute to neoplasia or other disease processes (4,5). For example, truncated isoforms of the p53 gene family, including p63 (6) and p73 (7–9), oppose the tumor suppressor activity of their full-length cognates and are over-expressed in tumors. + +ZBP-89 (ZNF148; Zfp148; BFCOL1), a Krüppel-type zinc-finger protein (10), is both structurally (11) and functionally complex (12–14). It regulates diverse biological functions through direct promoter binding (10,14) and through multiple protein–protein interactions. It interacts directly with the tumor suppressor p53 through its zinc-finger domain (13) and indirectly with the histone acetyltransferase and transcriptional co-activator p300 through its amino terminus (12). ZBP-89 induces cell growth arrest through a p53-dependent mechanism (13) and apoptosis through a p53-independent mechanism (12). Mice haploinsufficient for ZBP-89 (Zfp148) are sterile owing to aberrant spermatogenesis (15). In addition, embryonic stem cells harboring a single ZBP-89 allele fail to exhibit p53 phosphorylation at Ser 15 (15). + +ZBP-89 forms a complex with p300 during butyrate induction of p21waf1 in human colorectal cell lines (12). The amino terminus of ZBP-89 contains an acidic domain that is required for p300-mediated induction, since an expression construct lacking this domain (Δ amino acids 1–113) loses its ability to enhance butyrate induction of p21waf1 (12). The acidic domain of ZBP-89 is contained entirely within exon 4 of the human and mouse genes (11). Given the heterogeneous ZBP-89 mRNA expression pattern (10), the functional importance of the p300 interaction domain, and the fact that the ZBP-89 null mouse is likely an embryonic lethal (15), we generated a mouse homozygous for the ZBP-89ΔN isoform. Homozygous ΔNter mice experienced growth delay, reduced viability and increased susceptibility to Dextran sodium sulfate (DSS) induced colitis, suggesting that over-expression of the ΔNter isoform disrupts normal gastrointestinal homeostasis. + +MATERIALS AND METHODS + +Database deposition + +National Center for Biotechnology Information (NCBI) reference sequences for ZBP-89 genomic loci are Homo sapiens chromosome 3 locus NC_000003 and BAC RP11-775J23 (AC108688); Mus musculus chromosome 16 contig NT_039624 and Pan troglodytes chromosome 3 genomic locus NW_104931. The human ZBP-89ΔN mRNA and exon 4B genomic sequences reported here are found in GenBank as entries DQ090088 and DQ090089, respectively. The P.troglodytes genomic sequence homologous to human exon 4B is deposited in Genbank DQ144540. + +Organization of the human (ZNF148), chimp and mouse (Zfp148) ZBP-89 loci + +A human genomic clone contig encompassing the ZBP-89 locus was previously described (14). Bacterial artificial chromosome (BAC) clones were used as templates to sequence intron/exon boundaries spanning the ZNF148 locus. Exon 4B was identified by gene prediction sequence analysis (16,17). Similarly, a BAC and bacteriophage lambda clone contig spanning the mouse Zfp148 locus was assembled and sequenced to determine the genomic organization of the Zfp148 locus. Primers Ptr-4B-F: 5′-TTCACCTCCCTGTCCTGTTC-3′ and Ptr-4B-R: 5′-TATCTGTCCCGTTTGCCTG-3′ were used to amplify and sequence chimp (P.troglodytes) genomic DNA containing exon 4B, after BLAST analysis (18) had revealed a sequence gap in this region. Chimp genomic DNA was obtained from the Integrated Primate Biomaterials and Information Resource through the Coriell Institute for Medical Research (Camden, NJ). + +RT–PCR analysis + +Total cellular RNA was isolated from cultured cells using TRIzol reagent (Invitrogen, Carlsbad, CA). Archived whole cell RNA samples from paired colon cancer and normal mucosal samples were also analyzed. Two-step RT–PCR was performed using SuperScript III RT (Invitrogen), however reverse transcription was performed at 55°C in order to obtain adequate yields of human exon 4B-containing cDNA. RT reactions were primed with random nonamers. Human primer sequences (5′ to 3′) were 4A-F: ATGAACATTGACGACAAACTGGAAG; 5-R: TTCCATATGATATTTTTGTATGAAT; 4B-F: TAGGGATGGTCAGCACTG; 6-R: GTTCTTTTGTGCCTTTCC. Mouse primer sequences (5′ to 3′) included Ex2-F: GCGGATAGAAGAGAAGAATCAGTGG; Ex4-F: CATTGACGACAAACTGGAAGG; Ex4-R: ACTTCGATCTTGAAGTACTGACTC; Ex5-R: CAGGAGAGCGTTGTTTCCG; Ex3/5fusion-F: CAGCCTCAGATAAGTGTA and Ex9-R: TTGTGGCATCTGGTGAAG. RT–PCR products were purified and subjected to DNA sequence analysis (University of Michigan DNA Sequencing Core). + +5′-RACE of human exon 4B + +The 5′ end of the exon 4B variant was identified using the GeneRacer™ system (Invitrogen, Carlsbad, CA), gene specific primer (5′ to 3′) 4B211-Rev: CAGTGCTGACCATCCCTATC CTACTTG and 2 µg of Jurkat whole cell RNA. Nested PCR products, generated with adaptor primers included with the GeneRacer™ system, were cloned using the TOPO-TA system (Invitrogen, Carlsbad, CA), and sequenced. + +Targeting vector + +We constructed vector pΔEx-4 to replace ZBP-89 exon 4 with a PGK-Neo cassette by homologous recombination. High fidelity long-range PCR was used to amplify targeting arms from mouse BAC clone pBmZBP-89, using primers (5′ to 3′): (Kpn)Int3-F, GATAGGTACCGCATTGGATGGCACAAGTGACTGAGAGG with (Xho)Int3-R, CTCGAGCCCGGGCTTAAGTATAACTGCCTAGAAAG for the left arm and (Apa)Int4-F, CTCGAGGGGCCCGTAAGTACTAAACTAGAAATG with (Apa)Int4-R, CTCGAGGGGCCCAAGAGCCTTGCTGACTCATAG for the right arm. The left targeting arm, encompassing exon 3 and intron 3, was 8 kb in length. The right targeting arm consisted of the proximal 2 kb of intron 4. The neomycin cassette, including a transcriptional stop signal, was isolated from pPNT (19), generously provided by Dr Richard Mulligan. + +Generation of targeted ES cells and ΔNter mice + +Electroporation of embryonic stem (ES) cells with the pΔEx-4 targeting vector and microinjection of blastocysts with targeted ES cells were performed by the University of Michigan Transgenic Animal Model Core (). Genomic PCR was utilized to genotype targeted 129 Sv/J ES clones, chimeric founders, and progeny resulting after germline transmission. Primer sequences (5′ to 3′) were Int3-F1, GGAGTATTCTCTGTCCGTT ATG; Int4-326R, GCAAGAACTACACAGAGAAACCAC and R506Neo, TGAGGAA GAGGAGAACAGCG. + +Two-dimensional western blot analysis + +Whole cell protein extracts were prepared from mouse spleen using T-PER tissue protein extraction reagent (Pierce, Rockford, IL), supplemented with Complete Mini protease inhibitors (Roche, Indianapolis, IN) according the manufacturers' recommendations. Whole cell protein extracts from human Jurkat cells were similarly prepared using M-PER mammalian protein extraction reagent (Pierce, Rockford, IL). Isoelectric focusing (IEF) was performed with 7 cm ZOOM® Strip ph3-10L (linear) immobilized pH gradient gels (Invitrogen, Carlsbad, CA), as recommended by the manufacturer. The focused proteins were then separated on NuPAGE® 4-12% acrylamide gels (Invitrogen). Electroblot transfer to PVDF membrane and immunoblot procedures with ZBP-89 antiserum are as previously described (10,12). + +Dextran sodium sulfate colitis + +DSS colitis was induced in 6–9 month old ZBP-89ΔN and littermate control mice by the addition of 4% DSS to drinking water for a period of 5 days (20). Treated mice were returned to normal drinking water for 2 days prior to necropsy for histopathological scoring. Hematoxylin and eosin (H&E) stained colon sections were prepared by the Swiss roll method (21) and were scored for colitis index by a modification of a previously described method (22). Briefly, crypt damage was scored from 0 to 3 as none, basal only, moderate damage and complete erosion, respectively. Inflammation was scored from 0 to 3 as none, minor, moderate and severe leukocyte infiltration. Submucosal edema was scored from 0 to 3 as none, minor, moderate and severe, respectively. Similarly, hemorrhage was scored from 0 to 3 as none, minor, moderate and severe. Each parameter was multiplied by an extent factor (1–3), <10%, up to 25%, 25 to 50% and >50%, respectively. Samples with transmural involvement received an additional four points. Therefore, the maximum possible colitis index score was 40. Animals that died during treatment because of colitis injury were also given the maximum score. Individuals scoring the samples were blinded to the genotype. + +Cell culture + +The following cell lines were obtained from ATCC (Manassas, VA) and maintained on the recommended growth media: Human gastric epithelial cells Kato III and MKN45; human colon cancer cells Colo 320DM, CaCo-2 and HCT116; and human Jurkat leukemia cells. Cells were cultured in a humidified atmosphere of 5% CO2 and 95% air at 37°C. All culture media were supplemented with penicillin G (100 U/ml) and streptomycin (100 µg/ml). + +RESULTS + +Identification of human ZBP-89ΔN isoform + +To determine if isoforms of ZBP-89 exist, we screened the human ZNF148 genomic locus in silico (16,17). After localizing candidate alternative exon 4B within 4 kb upstream of exon 5 (Figure 1A), we used RT–PCR analysis to show that exon 4B is expressed (Figure 1B). Exon 4B mRNA (ΔN) was co-expressed with exon 4A-containing message (FL) in colon cancer cells (ColoDM2, CaCo2, HCT116), and in primary tissue from normal colon and colon adenocarcinoma. Both forms also were abundantly expressed in Jurkat cells (data not shown). This suggested that at least two forms of ZBP-89 exist. + +To better understand the function of the isoform, exon 4B-containing cDNA was sequenced. In addition to exon 4B, the variant mRNA included exons 5 and 6 (Figure 2A), as well as exons 7–9 (not shown). In contrast, RT–PCR with forward primers from exons 1-4A failed to generate products with exon 4B antisense primers, suggesting that an independent promoter regulates exon 4B expression. This was confirmed by 5′-rapid amplification of cDNA ends (RACE), which showed that transcription was initiated immediately upstream of exon 4B. The cDNA sequence showed that exon 4B was spliced to exon 5 resulting in an alternative reading frame relative to the cDNA encoded by exon 4A (Figure 2A). Exon 4B was 329 nt in length and composed of untranslated sequences when fused to exon 5. These data predicted that alternative promoter usage upstream of exon 4B resulted in the expression of an amino-terminally truncated ZBP-89 isoform, ZBP-89ΔN, with an alternative initiation codon corresponding to M128 of full-length mRNA (Figure 2B). This isoform lacks the acidic domain and p300-interaction region (12) found in full-length (ZBP-89FL) protein (10). Identical results were obtained with cDNA derived from esophagus, stomach, colon and Jurkat T-cells, suggesting that the exon 4B alternative promoter mechanism is common. + +Detection of human ZBP-89ΔN protein + +We found that the electrophoretic mobility of ZBP-89ΔN protein, despite its shorter length, overlapped with the mobility of its ZBP-89FL cognate (Figure 2C). Both forms, isolated from Jurkat cells, migrated at 100 kDa on a 4–20% gradient gel. Similar SDS–PAGE anomalies have been reported with other proteins, including CTCF (23) and XPA (24). An alternative approach to separate the protein isoforms was suggested by comparing their predicted (25) isoelectric points (pI). Loss of the acidic domain in ZBP-89ΔN protein predicted a pI of 7.8, compared to 6.0 for ZBP-89FL. This difference could be revealed by two-dimensional (2D) gel electrophoresis, followed by western blot analysis (Figure 2C). ZBP-89 antiserum detected two protein species, with apparent electrophoretic mobilities of 100 kDa, but with a pI difference of ∼1.5, confirming that the more basic form is ZBP-89ΔN. + +ZBP-89ΔN alternative promoter mechanism is restricted to hominids + +Although the human (ZNF148) and mouse (Zfp148) ZBP-89 loci share many features of genomic organization (11), exon 4B is absent in mice and most other mammals (data not shown). In contrast, we identified a 133 bp segment of chimpanzee (P.troglodytes) chromosome 3 sequence with striking DNA sequence homology to human exon 4B, but there was a gap in the existing online genomic sequence. Sequencing across the gap after genomic DNA amplification, we found that the chimp shares 99% DNA sequence identity to the 329 bp human exon 4B. This suggested that the ZBP-89ΔN alternative promoter mechanism is restricted to hominids. + +Generating a mouse model of ZBP-89ΔN expression + +Since mice lack exon 4B, and therefore do not express a ZBP-89ΔN isoform, we targeted the mouse exon 4 domain by homologous recombination (Figure 3A). The left targeting arm contained exon 3 and intron 3, and the right targeting arm contained 1.8 kb of the 5′ margin of intron 4. Homologous recombination occurred in 4 of 1200 ES clones identified by PCR (Figure 3B). Of the four homologous recombinants, three demonstrated stable karyotypes and were therefore injected into blastocysts. Germline transmission of the exon 4-targeted locus resulted in two founder lines, 4E10 and 8C6, while a third founder, 9C6, failed to transmit the recombinant locus. Germline transmission was obtained first in the 4E10 lineage. Near-Mendelian ratios were observed at the age of weaning (3 weeks) in offspring of heterozygous intercrosses (Figure 3C). The data suggested that ∼20% of Δexon4 homozygous embryos die in utero or perinatally, however the difference between predicted and observed ΔExon4/ΔExon4 yields was not statistically significant (χ2-square analysis; P > 0.05). A similar pattern was observed in the 8C6 lineage. These results also suggest that the mutant locus was at least partially functional, since heterozygosity for a null ZBP-89 (Zfp148) allele results in failed germ cell development and therefore is not transmitted (15). + +Alternative splicing of exon 3 to exon 5 in recombinant mice + +To determine the effect of exon 4 deletion on ZBP-89 expression, we analyzed cDNA derived from normal, heterozygous and exon 4-targeted animals (Figure 4A). Whereas exon 4 sequences were absent from recombinant mRNA, both upstream and downstream exons were expressed. RT–PCR with primers spanning exon 4 generated a 690 bp cDNA from wild type and a 339 bp cDNA from mutant alleles. The size difference between WT and targeted cDNA was 351 bp, the size of exon 4. This suggested that exon 3 was spliced directly to exon 5, excluding the intervening Neo cassette, and this was confirmed by DNA sequencing (Figure 4B). Deletion of exon 4 removed the initiation codon found in FL message and also resulted in an alternative reading frame. Gene feature sequence analysis (16,17) predicted that the alternative initiation codon corresponded to M128 of the FL protein (Figure 4B), analogous to the human ZBP-89ΔN variant (Figure 2B). + +Mouse ZBP-89ΔN protein expression + +2D western blot analysis (Figure 4C) showed that ZBP-89FL and ZBP-89ΔN proteins are expressed in an allele-dependent manner. The two forms have similar electrophoretic mobilities of 100 kDa, but are readily separated by their pI differences, similar to the human isoforms (Figure 2C). As mentioned above, similar electrophoretic anomalies have been observed with other protein isoforms (23,24). The immunoblot data also suggest that the ΔN protein isoform may be present at higher levels than the FL form in spleen tissue, however mRNA levels appear comparable, suggesting a possible difference in protein stability. Collectively, the mRNA and protein expression data confirmed that we knocked the ZBP-89ΔN variant into the mouse genome. Thus, the mouse model offers the advantage of assessing the biological effect of ZBP-89ΔN expression exclusively. + +ZBP-89ΔN/ΔN mice experience growth delay and reduced viability + +At weaning, by age 3 weeks, male ZBP-89ΔN/ΔN mice weighed an average of 5.9 ± 0.5 g (Figure 5A), 34% smaller than ZBP-89FL/FL (12.1 ± 0.7 g) and 49% smaller than ZBP-89FL/ΔN heterozygous littermates (15.1 ± 0.8 g). From ages 4–8 weeks, the size differential between ZBP-89ΔN/ΔN and control mice was diminished. A similar effect was observed with female mice (data not shown). As shown above (Figure 3C), ZBP-89ΔN/ΔN mice experienced 20% perinatal mortality as determined by genotype ratios at weaning. Reduced viability persisted and became statistically significant (Figure 5B), with 50% mortality at 48 weeks and 69% mortality by 104 weeks. The 2 year survival ratios were 20:20 ZBP-89FL/FL, 39:40 ZBP-89FL/ΔN and 5:16 ZBP-89ΔN/ΔN mice. Histopathological organ and tissue surveys revealed no obvious abnormalities in ZBP-89ΔN/ΔN mice, with the possible exception of the colon, where we noted a trend toward slightly increased lymphocytic infiltrates (data not shown). Semi-quantitative RT–PCR analysis of colon mRNA levels (Figure 5C), suggested that FL and ΔN forms were expressed in an allele-dependent manner. Collectively, these data demonstrated that balanced expression of ZBP-89FL and ZBP-89ΔN isoforms, as observed in heterozygous mice, supports normal growth and viability. In contrast, exclusive expression of the ZBP-89ΔN isoform in transgenic mice resulted in growth delay and reduced viability. + +ZBP-89ΔN confers DSS colitis susceptibility + +We previously showed that the amino terminal region of ZBP-89 (amino acids 1–111) is required to potentiate butyrate induction of p21waf1, suggesting that the amino terminal region mediates the butyrate-dependent anti-proliferative activities of ZBP-89 in the mammalian gastrointestinal tract (12). Butyrate suppresses colonic inflammation when induced by DSS (26). Since DSS accentuates a tendency to develop colitis, we challenged ZBP-89ΔN/ΔN mice acutely with 4% DSS for 5 days, as previously described (20). Histopathogical analysis (hematoxylin and eosin stained sections) demonstrated the presence of severe colitis that correlated with a ZBP-89ΔN gene dosage effect (Figure 6A–C). ZBP-89FL/FL mice (Figure 6A) had localized areas of lymphocytic infiltration and minimal submucosal edema. ZBP-89FL/ΔN mice (Figure 6B) exhibited more extensive infiltration, with displaced normal crypt architecture in addition to submucosal edema. ZBP-89ΔN/ΔN mice (Figure 6C) had areas with complete erosion of crypt architecture, grossly visible hemorrhage and extensive submucosal edema. The ZBP-89ΔN gene dosage effect also correlated with the duration of gastrointestinal bleeding (Figure 6D). Moreover, only ZBP-89ΔN/ΔN mice died during DSS treatment, with 50% mortality by the conclusion of the 7 day regimen (Figure 6E). Composite colitis scoring similarly paralleled the ZBP-89ΔN gene dosage (Figure 6F). These data suggest that increased expression of ZBP-89ΔN is associated with increased susceptibility to colitis. + +DISCUSSION + +Through structural and functional analyses, we identified a human-specific ZBP-89 splice isoform, ZBP-89ΔN, which was generated by alternative promoter usage upstream of an alternative exon 4B. As a consequence, ZBP-89ΔN protein lacks a transcriptional domain (12) found in its full-length ZBP-89 cognate. This is the first characterization of a ZBP-89 isoform and is consistent with previous observations that gene complexity is lower in mice than in humans. As many as 59% of human genes are alternatively spliced (27), while the highest estimate to date for the mouse is 33% (28). Comparative genomic analysis suggested that the ZBP-89ΔN splicing mechanism has been conserved between humans and chimps, indicating that this specific mechanism of regulating ZBP-89 function is restricted to hominids. + +Alternative mRNA expression increases genetic diversity through regulatory mechanisms that also are implicated in cancer (1,3–5,29) and other disease processes (30,31). The human ZBP-89ΔN variant described here renders the colonic mucosa more susceptible to injury. Our previous studies showed that the N-terminal domain is required for ZBP-89 to mediate butyrate-dependent activation of p21waf1 by cooperating with p300 (12). This result demonstrated that the N terminal domain is functionally important in vitro. Generating a mouse model in which only the truncated form was expressed revealed that loss of the p300-interacting domain results in delayed growth and a shortened lifespan. Although the overall effect on the health of the organism was impressive, an initial survey of several organs did not reveal obvious abnormalities, with the exception of the colon, which appeared to exhibit slightly more inflammatory infiltrates than the wild type mice. The propensity of the ZBP-89ΔN expressing mice to develop colitis was subsequently uncovered when the animals were challenged with DSS. Therefore, we concluded from these studies that expression of ZBP-89FL tends to protect against colitis. The gene dosage dependence of colitis susceptibility further suggests that the ZBP-89ΔN isoform functions as a dominant negative antagonist of ZBP-89FL. Similar antagonistic interactions have been reported between FL and ΔN isoforms within the p53 gene family, including ΔNp63 (29) and ΔNp73 (7–9). + +The ZBP-89ΔN knock-in model reported here, along with an earlier Zfp148+/− model (15), help to elaborate how individual protein domains mediate the in vivo functions of ZBP-89. Mice heterozygous for a null ZBP-89 allele demonstrate complete failure of male germ cell development and are defective in p53-dependent embryogenesis (15). This finding suggests that the ability of ZBP-89 to interact with p53 is exquisitely sensitive to gene dosage. In contrast, homozygous ZBP-89ΔN expression is compatible with embryonic and postnatal survival, albeit at reduced levels. We previously showed that the DNA-binding, zinc-finger region of ZBP-89 mediates its interaction with p53 (13), and this domain is retained in the ZBP-89ΔN isoform. Therefore, the Nter domain, which includes the p300 interaction domain, is dispensable for p53-dependent embryonic development and postnatal survival, but is essential for normal gastrointestinal function. + +Acknowledgements + +The authors gratefully acknowledge the expertise of the University of Michigan Transgenic Animal Model Core, especially Thom Saunders, Linda Samuelson and Elizabeth Hughes. The authors also thank members of the DNA Sequencing Core and the Unit for Laboratory Animal Medicine at the University of Michigan, and Kathy McClinchey for assistance with histology. Proteomics data were provided by the Michigan Proteome Consortium () which is supported in part by funds from the Michigan Life Sciences Corridor. Specifically, valuable assistance with 2D gel electrophoresis was provided by Jennifer Callahan. This research is supported in part by the National Institutes of Health through the University of Michigan's Cancer Center Support Grant (5 P30 CA46592) and the University of Michigan Gastrointestinal Peptide Research Center (DK-34533). Art Tessier and Gail Kelsey provided administrative support. Stacey Ehrenberg provided technical assistance. This work was supported by grants from the National Institutes of Health to D.J.L. (R21 DK65004-01) and J.L.M. (RO1-DK55732). Funding to pay the Open Access publication charges for this article was provided by NIH grant RO1-DK55732 (J.L.M.). + +Conflict of interest statement. None declared. + +Figures and Tables + +Figure 1 + +Identification of a novel exon within the human ZBP-89 (ZNF148) locus. (A) The human ZBP-89 (ZNF148) locus spans 142 kb (upper panel) and encompasses three untranslated (1–3) and six coding (4–9) exons. A potential alternative exon 4B (shaded box) was identified at a position 4.2 kb upstream of exon 5. Arrows indicate the locations of forward (F) and reverse (R) RT–PCR primers used for expression analysis of FL ZBP-89 and the exon 4B variant. Selected intron sizes (kb) are indicated. (B) RT–PCR analysis of full-length (FL) and variant (ΔN) ZBP-89 mRNA expression, using actin (Ac) as control. The cDNAs were generated with cultured cell lines using primers 4A-F/5-R (726 bp product); primers 4B-F/6-R (432 bp product) and (Ac), Actin control primers (400 bp product). Whole cell RNA was isolated from ColoDM2, CaCo 2, and HCT116 human colon cell lines as well as paired normal colon and colon adenocarcinoma tissue. Full-length message is expressed from promoter 1 (Pro 1) and variant (ΔN) ZBP-89 mRNA is expressed from promoter 2 (Pro 2), as described in Figure 2. + +Figure 2 + +Variant ZBP-89 transcript encodes a truncated protein, ZBP-89ΔN. (A) The 5′ end of exon 4B-variant cDNA, determined by 5′-RACE, is shown. Underlined italics, exon 4B-encoded; lower case italics, exon 5 untranslated (frameshift relative to ZBP-89FL cDNA); upper case italics, exon 5 coding sequence commencing at M128 relative to FL protein; upper case underlined, beginning of exon 6-encoded. The remainder of the cDNA and amino acid sequences are identical to the corresponding segments of FL cDNA and protein. (B) Comparison of FL (upper) and ΔN (lower) protein isoforms. The amino terminus of ZBP-89FL includes an acidic and p300-interaction domain (PID), which is absent in ZBP-89ΔN. The ZBP-89ΔN isoform retains the DNA binding and C-terminal domains. (C) 2D western blot analysis of Jurkat whole cell protein extract. ZBP-89FL (solid triangle) and ZBP-89ΔN (open triangle) peptides share an electrohoretic mobility of ∼100 kDa and are separated on the basis of their distinct isoelectric points (pI). The acidic end of the focusing gel is at the left. + +Figure 3 + +Targeting ZBP-89 exon 4 in mice. (A) Replacement of exon 4 with a Neomycin resistance cassette (NEO) by homologous recombination in ES cells. Top panel: bracket shows location of features encoded by exon 4, including initiation codon, acidic domain (black rectangle) and p300 interaction domain. Open boxes, basic domains; hatched boxes, zinc-fingers; stippled region, untranslated. Middle panel; Zfp148 genomic locus. Intron sizes (kb) are shown. Lower panel; targeting strategy. (B) Tail biopsy DNA genotyping. WT (Int 3 F/Int 4 R) and Δexon4 (Int 3 F/Neo R) amplimers. (C) Near-Mendelian inheritance of ΔExon-4 alleles. The differences between predicted and observed values were not significant. + +Figure 4 + +ΔExon4 locus encodes mouse ZBP-89ΔN. (A) RT–PCR analysis was used to determine the variant mRNA structure in recombinant mice. Spleen whole-cell RNA was reverse-transcribed and amplified with primers within the indicated exons. RT–PCR of another ubiquitously expressed zinc-finger transcription factor, Sp1, was used for comparison and showed that the RNA samples were of uniform quality. Control experiments showed that PCR products were RT-dependent (data not shown), indicating that they were indeed derived from mRNA rather than possibly resulting from amplification of ZBP-89 related processed pseudogene sequences, such as ΨBERF1 (32). (B) DNA sequencing of recombinant cDNA showed that deletion of exon 4 resulted in direct splicing of exon 3 to exon 5, excluding the Neo cassette. The resulting reading frameshift predicts an alternative initiation codon corresponding to M128 of FL protein, similar to the naturally occurring human ZBP-89ΔN variant. (C) 2D immunoblot analysis of whole-cell spleen protein extracts from FL/FL, FL/ΔN and ΔN/ΔN mice, using conditions identical to those used for analysis of human samples (Figure 2C). + +Figure 5 + +Growth delay and decreased survival in ΔNter mice. (A) Growth curve for male ZBP-89FL/FL (gray dashed line, n = 20), ZBP-89FL/ΔN (black dotted line, n = 36), and ZBP-89ΔN/ΔN (black solid line, n = 16) offspring. A similar pattern was seen with female mice (not shown). ** P < 0.01; * P < 0.05. A trend toward lower body weights in ZBP-89ΔN/ΔN persisted after 12 weeks, but was no longer statistically significant (data not shown). (B) Kaplan–Meier analysis of survival interval to the onset of morbidity or death. (C) Semi-quantitative RT–PCR analysis of ZBP-89FL and ZBP-89ΔN mRNA levels in the colon. + +Figure 6 + +Increased susceptibility to DSS colitis in ZBP-89ΔN mice. (A–C) Representative H&E stains of normal (A), heterozygous (B) and ZBP-89ΔN/ΔN (C) mice. Inf = infiltrating lymphocytes; SE = submucosal edema; TM = transmural inflammation. (D) ΔNter expression accelerates onset of gastrointestinal bleeding, measured by fecal occult blood screening. (E) Mortality during DSS treatment: 50% of ZBP-89ΔN/ZBP-89ΔN mice die during 4% DSS treatment; all ZBP-89FL/ZBP-89FL and ZBP-89FL/ZBP-89ΔN mice survived (six in each group). (F) Colitis index scoring of DSS-treated mice, as described in Materials and Methods; six mice in each genotype cohort. diff --git a/src/ontogpt/evaluation/craft/database/all/16539743.ann b/src/ontogpt/evaluation/craft/database/all/16539743.ann new file mode 100644 index 000000000..ac8f30b76 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16539743.ann @@ -0,0 +1,1340 @@ +T1 PR:000014441 32 36 mr-s +T2 SO:0000417 50 56 domain +T3 GO:0010467 80 89 expressed +T4 UBERON:0000966 93 100 retinal +T5 CL:0010009 93 120 retinal photoreceptor cells +T6 http://purl.obolibrary.org/obo/MONDO_0005047 144 151 Sterile +T7 SO:0000417 170 177 domains +T8 SO:0000417 261 268 modules +T9 SO:0000417 275 281 domain +T10 SO:0000417 483 489 domain +T11 GO:0010467 533 542 expressed +T12 UBERON:0000966 546 553 retinal +T13 CL:0010009 546 568 retinal photoreceptors +T14 UBERON:0001905 577 589 pineal gland +T15 NCBITaxon:10088 608 613 mouse +T16 PR:000014441 614 618 mr-s +T17 PR:000014441 620 652 major retinal SAM domain protein +T18 UBERON:0000966 626 633 retinal +T19 SO:0000417 638 644 domain +T20 PR:000014441 665 669 mr-s +T21 NCBITaxon:7955 703 712 zebrafish +T22 NCBITaxon:9606 721 726 human +T23 NCBITaxon:1 728 737 organisms +T24 CL:0000210 769 782 photoreceptor +T25 GO:0042461 769 794 photoreceptor development +T26 SO:0000417 865 871 domain +T27 PR:000014441 875 879 mr-s +T28 NCBITaxon:10088 909 914 mouse +T29 SO:0000855 933 941 ortholog +T30 PR:Q64028 943 947 Mph1 +T31 PR:000012633 948 953 Rae28 +T32 CHEBI:36357 987 995 molecule +T33 GO:0000785 1008 1017 chromatin +T34 GO:0016568 1008 1031 chromatin modifications +T35 PR:000014441 1077 1081 mr-s +T36 GO:0065007 1110 1120 regulating +T37 SO:0000704 1121 1125 gene +T38 GO:0010467 1121 1136 gene expression +T39 CL:0000210 1140 1153 photoreceptor +T40 GO:0042461 1140 1165 photoreceptor development +T41 PR:000014441 1167 1171 mr-s +T42 GO:0010467 1190 1199 expressed +T43 CL:0000210 1207 1221 photoreceptors +T44 GO:0007567 1229 1234 natal +T45 CL:0000210 1256 1270 photoreceptors +T46 UBERON:0007023 1316 1321 adult +T47 UBERON:0001905 1322 1334 pineal gland +T48 PR:000014441 1353 1357 mr-s +T49 GO:0065007 1370 1379 regulated +T50 CL:0000573 1387 1391 cone +T51 CL:0000604 1392 1395 rod +T52 PR:000005904 1416 1419 Crx +T53 PR:000014441 1463 1467 mr-s +T54 SO:0000417 1515 1521 domain +T55 PR:000014441 1559 1563 mr-s +T56 GO:0005634 1596 1603 nucleus +T57 PR:000014441 1610 1614 mr-s +T58 GO:0010467 1622 1631 expressed +T59 PR:000014441 1700 1704 mr-s +T60 PR:P04386 1722 1726 GAL4 +T61 SO:0000417 1739 1745 domain +T62 PR:000014441 1832 1836 mr-s +T63 SO:0000417 1892 1898 domain +T64 SO:0000704 1964 1968 gene +T65 PR:000014441 1970 1974 mr-s +T66 GO:0010467 1999 2008 expressed +T67 UBERON:0000966 2012 2019 retinal +T68 CL:0010009 2012 2034 retinal photoreceptors +T69 UBERON:0001905 2039 2051 pineal gland +T70 GO:0010467 2066 2076 expression +T71 PR:000014441 2127 2131 mr-s +T72 CL:0000210 2179 2198 photoreceptor cells +T73 CL:0000652 2206 2218 pinealocytes +T74 UBERON:0001905 2226 2238 pineal gland +T75 GO:0060041 2260 2274;2289 2295 development of ... retina +T76 NCBITaxon:40674 2279 2288 mammalian +T77 UBERON:0000966 2289 2295 retina +T78 CL:0000048 2355 2366;2386 2391 multipotent ... cells +T79 UBERON:0000966 2367 2374 retinal +T80 CL:0002672 2367 2391 retinal progenitor cells +T81 CL:0000210 2417 2431 photoreceptors +T82 UBERON:0000966 2473 2479 retina +T83 NCBITaxon:7742 2484 2495 vertebrates +T84 CL:0000210 2522 2536 photoreceptors +T85 CL:0000604 2538 2542 rods +T86 CL:0000573 2547 2552 cones +T87 CL:0000604 2554 2558 Rods +T88 CL:0000573 2591 2596 cones +T89 GO:0007601 2652 2658 vision +T90 GO:0007602 2660 2677 Phototransduction +T91 CHEBI:30212 2732 2738 photon +T92 GO:0016037 2769 2785 capture of light +T93 CHEBI:16066 2791 2805 11-cis-retinal +T94 CHEBI:23240 2809 2820 chromophore +T95 PR:000001119 2834 2839 opsin +T96 PR:000001245 2850 2859 rhodopsin +T97 CL:0000604 2863 2867 rods +T98 CL:0000573 2872 2876 cone +T99 PR:000001119 2877 2883 opsins +T100 CL:0000573 2887 2892 cones +T101 GO:0007602 2922 2939 phototransduction +T102 GO:0005886 2991 3001 membranous +T103 GO:0001750 3017 3030 outer segment +T104 GO:0001750 3036 3049 outer segment +T105 SO:0000704 3138 3145 genetic +T106 NCBITaxon:9989 3168 3174 rodent +T107 UBERON:0000966 3175 3181 retina +T108 CL:0000604 3277 3294 Rod photoreceptor +T109 GO:0046548 3277 3305 Rod photoreceptor generation +T110 GO:0007567 3331 3336 birth +T111 CL:0000573 3338 3357 Cone photoreceptors +T112 UBERON:0000045 3359 3367 ganglion +T113 CL:0000740 3359 3373 ganglion cells +T114 CL:0000745 3375 3391 horizontal cells +T115 CL:0000561 3396 3410 amacrine cells +T116 CL:0011107 3440 3451 Müller glia +T117 CL:0000103 3456 3469 bipolar cells +T118 GO:0000785 3666 3675 chromatin +T119 GO:0016568 3666 3688 chromatin modification +T120 CL:0000210 3759 3772 photoreceptor +T121 GO:0042461 3759 3784 photoreceptor development +T122 SO:0000704 3816 3821 genes +T123 PR:000012086 3823 3827 Otx2 +T124 PR:000005904 3832 3835 Crx +T125 GO:0043703 3867 3890;3920 3937 cell fate determination ... of photoreceptors +T126 CL:0000210 3923 3937 photoreceptors +T127 PR:000012086 3965 3969 Otx2 +T128 CL:0000210 3996 4015 photoreceptor cells +T129 CL:0000561 4033 4041 amacrine +T130 CL:0000540 4047 4054 neurons +T131 PR:000005904 4060 4063 Crx +T132 PR:000012086 4088 4092 Otx2 +T133 GO:0065007 4094 4102 controls +T134 CL:0000210 4132 4150 photoreceptor cell +T135 SO:0000704 4160 4165 genes +T136 GO:0001750 4204 4218 outer segments +T137 GO:0045202 4220 4228 synaptic +T138 GO:0007602 4244 4261 phototransduction +T139 PR:000005904 4279 4282 Crx +T140 SO:0000673 4283 4294 transcripts +T141 GO:0010467 4307 4316 expressed +T142 CL:0000210 4331 4345 photoreceptors +T143 UBERON:0000922 4349 4358 embryonic +T144 NCBITaxon:10088 4383 4388 mouse +T145 PR:000005904 4418 4421 Crx +T146 CL:0000210 4475 4489 photoreceptors +T147 GO:0007567 4497 4502 natal +T148 CL:0000210 4515 4534 Photoreceptor cells +T149 PR:000005904 4542 4545 Crx +T150 NCBITaxon:10088 4560 4564 mice +T151 CL:0000210 4602 4615 photoreceptor +T152 CHEBI:36357 4616 4625 molecules +T153 GO:0007601 4636 4642 visual +T154 CHEBI:26130 4643 4651 pigments +T155 CL:0000210 4672 4685 photoreceptor +T156 GO:0001750 4672 4700 photoreceptor outer segments +T157 GO:0045202 4707 4715 synaptic +T158 NCBITaxon:9606 4759 4764 human +T159 PR:000005904 4765 4768 CRX +T160 CL:0000210 4829 4842 photoreceptor +T161 http://purl.obolibrary.org/obo/MONDO_0000001 4843 4851 diseases +T162 GO:0030849 4853 4862 autosomal +T163 CL:0000573 4872 4876 cone +T164 http://purl.obolibrary.org/obo/MONDO_0007362 4872 4892 cone-rod dystrophy 2 +T165 CL:0000604 4877 4880 rod +T166 GO:0030849 4894 4903 autosomal +T167 http://purl.obolibrary.org/obo/MONDO_0019200 4918 4938 retinitis pigmentosa +T168 http://purl.obolibrary.org/obo/MONDO_0018998 4944 4972 Leber's congenital amaurosis +T169 http://purl.obolibrary.org/obo/MONDO_0018998 4974 4977 LCA +T170 PR:000012086 4994 4998 Otx2 +T171 PR:000005904 5003 5006 Crx +T172 GO:0065007 5007 5014 control +T173 CL:0000210 5023 5036 photoreceptor +T174 GO:0042461 5023 5048 photoreceptor development +T175 PR:000016321 5085 5089 TRβ2 +T176 PR:000011432 5091 5094 Nrl +T177 PR:000011403 5100 5105 Nr2e3 +T178 GO:0065007 5106 5114 regulate +T179 CL:0000210 5136 5154 photoreceptor cell +T180 SO:0000417 5175 5182 domains +T181 SO:0000417 5219 5226 domains +T182 SO:0000417 5311 5318 modules +T183 SO:0000417 5333 5339 domain +T184 PR:000016568 5504 5507 p73 +T185 http://purl.obolibrary.org/obo/MONDO_0005070 5508 5513 tumor +T186 PR:Q23972 5555 5560 Smaug +T187 CHEBI:18035 5567 5581 diacylglycerol +T188 GO:0007618 5605 5611 mating +T189 SO:0000704 5752 5756 gene +T190 GO:0016458 5752 5766 gene silencing +T191 NCBITaxon:40674 5798 5805 mammals +T192 SO:0000704 5847 5851 gene +T193 GO:0065007 5852 5862 regulation +T194 GO:0016458 5905 5917;5935 5940 silencing of ... genes +T195 SO:0000704 5935 5940 genes +T196 GO:0000785 5949 5958 chromatin +T197 GO:0016568 5949 5972 chromatin modifications +T198 GO:0035102 5986 6015 polycomb repressive complex 1 +T199 GO:0035102 6017 6021 PRC1 +T200 SO:0000417 6043 6049 domain +T201 GO:0035102 6073 6085 PRC1 complex +T202 SO:0001114 6103 6110 helical +T203 CHEBI:33839 6125 6133 polymers +T204 SO:0000417 6150 6156 domain +T205 CHEBI:33839 6169 6178 polymeric +T206 GO:0000785 6230 6239 chromatin +T207 SO:0000704 6300 6305 genes +T208 SO:0000704 6345 6349 gene +T209 PR:000014441 6351 6355 mr-s +T210 SO:0000417 6377 6383 domain +T211 SO:0000417 6412 6418 domain +T212 PR:000014441 6422 6426 mr-s +T213 NCBITaxon:10088 6465 6470 mouse +T214 SO:0000855 6471 6479 ortholog +T215 PR:Q64028 6481 6485 MPH1 +T216 PR:000012633 6486 6491 Rae28 +T217 PR:000014441 6493 6497 mr-s +T218 GO:0010467 6515 6524 expressed +T219 UBERON:0000966 6528 6535 retinal +T220 CL:0010009 6528 6550 retinal photoreceptors +T221 UBERON:0007023 6599 6604 adult +T222 UBERON:0001905 6605 6617 pineal gland +T223 GO:0010467 6623 6633 expression +T224 PR:000014441 6637 6641 mr-s +T225 GO:0065007 6654 6663 regulated +T226 PR:000005904 6667 6670 Crx +T227 PR:000014441 6682 6686 mr-s +T228 GO:0005634 6707 6714 nucleus +T229 SO:0000417 6754 6760 domain +T230 PR:000014441 6799 6803 mr-s +T231 PR:P04386 6821 6825 GAL4 +T232 SO:0000417 6838 6844 domain +T233 PR:000014441 6915 6919 mr-s +T234 GO:0017053 6947 6980 transcriptional repressor complex +T235 UBERON:0000966 6984 6991 retinal +T236 CL:0010009 6984 7005 retinal photoreceptor +T237 GO:0042461 6992 7017 photoreceptor development +T238 NCBITaxon:10088 7040 7045 mouse +T239 PR:000014441 7046 7050 mr-s +T240 NCBITaxon:10088 7079 7084 mouse +T241 SO:0000704 7085 7090 genes +T242 GO:0010467 7106 7115 expressed +T243 UBERON:0000966 7134 7140 retina +T244 SO:0000345 7284 7287 EST +T245 NCBITaxon:10088 7330 7335 mouse +T246 UBERON:0000966 7336 7343 retinal +T247 SO:0000417 7434 7440 domain +T248 NCBITaxon:10088 7519 7524 mouse +T249 NCBITaxon:10088 7552 7557 mouse +T250 UBERON:0000966 7564 7571 retinal +T251 SO:0000317 7609 7619 cDNA clone +T252 SO:0000704 7673 7677 gene +T253 SO:0000417 7693 7699 domain +T254 PR:000014441 7751 7755 mr-s +T255 PR:000014441 7757 7789 major retinal SAM domain protein +T256 UBERON:0000966 7763 7770 retinal +T257 SO:0000417 7775 7781 domain +T258 GO:0006413 7815 7837 translation initiation +T259 SO:0000318 7815 7843 translation initiation codon +T260 SO:0000236 7867 7885 open reading frame +T261 SO:0000417 7897 7903 domain +T262 SO:0000318 7910 7925 initiation site +T263 SO:0000993 7950 7959 consensus +T264 SO:0000319 8066 8076 stop codon +T265 PR:000014441 8094 8098 mr-s +T266 SO:0000417 8172 8178 domain +T267 PR:000014441 8182 8186 mr-s +T268 SO:0000857 8230 8238 homology +T269 SO:0000417 8248 8255 domains +T270 PR:000007130 8259 8264 EphB2 +T271 PR:000007124 8266 8271 EphA4 +T272 PR:Q64028 8273 8277 MPH1 +T273 PR:000007229 8279 8282 TEL +T274 PR:Q23972 8287 8292 Smaug +T275 SO:0000417 8338 8344 domain +T276 PR:000014441 8348 8352 mr-s +T277 PR:Q64028 8388 8392 Mph1 +T278 PR:000012633 8393 8398 Rae28 +T279 NCBITaxon:10088 8402 8407 mouse +T280 SO:0000853 8408 8415 homolog +T281 NCBITaxon:10088 8433 8438 Mouse +T282 PR:000014441 8439 8443 mr-s +T283 NCBITaxon:10114 8468 8471 rat +T284 NCBITaxon:9606 8473 8478 human +T285 NCBITaxon:7955 8490 8499 zebrafish +T286 NCBITaxon:10088 8551 8556 mouse +T287 PR:000014441 8557 8561 mr-s +T288 SO:0000417 8595 8602 domains +T289 NCBITaxon:10114 8606 8609 rat +T290 NCBITaxon:9606 8611 8616 human +T291 NCBITaxon:7955 8628 8637 zebrafish +T292 PR:000014441 8638 8642 mr-s +T293 SO:0000417 8728 8734 domain +T294 NCBITaxon:10088 8738 8743 mouse +T295 PR:000014441 8744 8748 mr-s +T296 NCBITaxon:10088 8791 8796 mouse +T297 NCBITaxon:9606 8801 8806 human +T298 PR:000014441 8807 8811 mr-s +T299 SO:0000704 8812 8817 genes +T300 NCBITaxon:10088 8851 8856 mouse +T301 NCBITaxon:9606 8861 8866 human +T302 SO:0001026 8867 8873 genome +T303 NCBITaxon:10088 8906 8911 Mouse +T304 PR:000014441 8912 8916 mr-s +T305 NCBITaxon:9606 8950 8955 human +T306 PR:000014441 8956 8960 MR-S +T307 http://purl.obolibrary.org/obo/MONDO_0018998 8994 8997 LCA +T308 http://purl.obolibrary.org/obo/MONDO_0001941 9046 9055 blindness +T309 NCBITaxon:9606 9057 9062 Human +T310 PR:000014441 9063 9067 MR-S +T311 http://purl.obolibrary.org/obo/MONDO_0018998 9148 9151 LCA +T312 PR:000014441 9169 9173 mr-s +T313 PR:000014441 9215 9219 mr-s +T314 SO:0000417 9288 9294 domain +T315 GO:0005634 9344 9351 nuclear +T316 SO:0001528 9344 9371 nuclear localization signal +T317 GO:0043631 9408 9423 polyadenylation +T318 SO:0000551 9408 9442 polyadenylation termination signal +T319 SO:0000417 9465 9471 domain +T320 SO:0000417 9490 9496 domain +T321 SO:0001117 9527 9540 alpha helices +T322 SO:0000417 9782 9788 domain +T323 NCBITaxon:10088 10017 10022 mouse +T324 NCBITaxon:10114 10024 10027 rat +T325 NCBITaxon:9606 10029 10034 human +T326 NCBITaxon:7955 10046 10055 zebrafish +T327 PR:000014441 10056 10060 mr-s +T328 SO:0000417 10105 10112 domains +T329 NCBITaxon:10088 10167 10172 mouse +T330 NCBITaxon:10088 10228 10233 mouse +T331 GO:0010467 10266 10276 Expression +T332 PR:000014441 10280 10284 mr-s +T333 UBERON:0000966 10303 10309 retina +T334 UBERON:0001905 10318 10330 pineal gland +T335 PR:000014441 10347 10351 mr-s +T336 GO:0010467 10352 10362 expression +T337 GO:0097617 10403 10416 hybridization +T338 NCBITaxon:10088 10420 10425 mouse +T339 UBERON:0000922 10426 10433 embryos +T340 GO:0097617 10438 10451 hybridization +T341 PR:000014441 10505 10509 mr-s +T342 GO:0010467 10559 10569 expression +T343 PR:000014441 10577 10581 mr-s +T344 SO:0000704 10582 10586 gene +T345 UBERON:0000966 10605 10611 retina +T346 GO:0097617 10631 10644 hybridization +T347 UBERON:0000966 10711 10717 retina +T348 CL:0000031 10800 10812 neuroblastic +T349 CL:0000210 10840 10853 photoreceptor +T350 UBERON:0001787 10840 10859 photoreceptor layer +T351 PR:000014441 10895 10899 mr-s +T352 SO:0000673 10900 10910 transcript +T353 CL:0000210 10950 10963 photoreceptor +T354 UBERON:0001787 10950 10969 photoreceptor layer +T355 PR:000014441 10988 10992 mr-s +T356 GO:0010467 11005 11015 expression +T357 CL:0000210 11023 11036 photoreceptor +T358 UBERON:0001787 11023 11042 photoreceptor layer +T359 GO:0010467 11111 11121 expressing +T360 PR:000001245 11122 11131 rhodopsin +T361 GO:0007602 11142 11159 phototransduction +T362 SO:0000704 11160 11165 genes +T363 PR:000014441 11217 11221 mr-s +T364 UBERON:0001789 11280 11299 outer nuclear layer +T365 GO:0005634 11286 11293 nuclear +T366 UBERON:0001789 11301 11304 ONL +T367 GO:0010467 11330 11340 expression +T368 PR:000014441 11344 11348 mr-s +T369 CL:0000210 11377 11391 photoreceptors +T370 UBERON:0007023 11399 11404 adult +T371 UBERON:0000966 11405 11411 retina +T372 PR:000014441 11434 11438 mr-s +T373 GO:0010467 11439 11449 expression +T374 NCBITaxon:10088 11464 11469 mouse +T375 UBERON:0000966 11470 11476 retina +T376 UBERON:0001905 11481 11493 pineal gland +T377 PR:000014441 11501 11505 mr-s +T378 GO:0010467 11506 11516 expression +T379 GO:0060041 11524 11538;11549 11555 development of ... retina +T380 NCBITaxon:10088 11543 11548 mouse +T381 UBERON:0000966 11549 11555 retina +T382 GO:0097617 11569 11582 hybridization +T383 PR:000014441 11593 11597 mr-s +T384 PR:000014441 11713 11717 mr-s +T385 UBERON:0001781 11747 11755;11760 11766 layer of ... retina +T386 UBERON:0007023 11814 11819 adult +T387 UBERON:0000966 11820 11826 retina +T388 UBERON:0000966 11870 11876 retina +T389 PR:000014441 11940 11944 mr-s +T390 GO:0010467 11945 11955 expression +T391 UBERON:0007023 11959 11964 adult +T392 NCBITaxon:10088 11965 11970 mouse +T393 UBERON:0000062 11971 11977 organs +T394 PR:000014441 12010 12014 mr-s +T395 SO:0000673 12015 12025 transcript +T396 UBERON:0000922 12086 12092 embryo +T397 UBERON:0000468 12097 12107 whole body +T398 UBERON:0000970 12124 12127 eye +T399 UBERON:0000966 12133 12139 retina +T400 UBERON:0001905 12144 12156 pineal gland +T401 UBERON:0000955 12161 12166 brain +T402 UBERON:0002107 12171 12176 liver +T403 UBERON:0007023 12178 12183 adult +T404 UBERON:0000966 12184 12190 retina +T405 UBERON:0007023 12192 12197 adult +T406 UBERON:0001905 12198 12210 pineal gland +T407 UBERON:0007023 12212 12217 adult +T408 UBERON:0000955 12218 12223 brain +T409 UBERON:0007023 12228 12233 adult +T410 UBERON:0002107 12234 12239 liver +T411 UBERON:0001782 12255 12258 RPE +T412 UBERON:0001782 12260 12286 retinal pigment epithelium +T413 CHEBI:26130 12268 12275 pigment +T414 UBERON:0003902 12288 12290 NR +T415 UBERON:0003902 12292 12305 neural retina +T416 CL:0000031 12312 12324 neuroblastic +T417 UBERON:0001792 12332 12335 GCL +T418 CL:0000740 12337 12350 ganglion cell +T419 UBERON:0001792 12337 12356 ganglion cell layer +T420 UBERON:0001789 12358 12361 ONL +T421 UBERON:0001789 12363 12382 outer nuclear layer +T422 GO:0005634 12369 12376 nuclear +T423 UBERON:0001791 12384 12387 INL +T424 UBERON:0001791 12389 12408 inner nuclear layer +T425 GO:0005634 12395 12402 nuclear +T426 UBERON:0000479 12428 12434 tissue +T427 PR:000014441 12450 12454 mr-s +T428 GO:0010467 12455 12465 expression +T429 GO:0010467 12471 12481 expression +T430 PR:000014441 12489 12493 mr-s +T431 SO:0000704 12494 12498 gene +T432 UBERON:0007023 12510 12515 adult +T433 UBERON:0000479 12516 12523 tissues +T434 GO:0097617 12549 12562 hybridization +T435 UBERON:0000966 12591 12598 retinal +T436 UBERON:0000966 12695 12701 retina +T437 GO:0000380 12799 12818 alternative spliced +T438 SO:1001187 12799 12830 alternative spliced transcripts +T439 PR:000014441 12869 12873 mr-s +T440 UBERON:0007023 12909 12914 adult +T441 UBERON:0000479 12915 12922 tissues +T442 UBERON:0000479 12955 12962 tissues +T443 GO:0010467 12970 12977 express +T444 PR:000014441 12978 12982 mr-s +T445 UBERON:0000966 13031 13037 retina +T446 CL:0000210 13076 13089 photoreceptor +T447 SO:0000704 13099 13104 genes +T448 GO:0010467 13114 13123 expressed +T449 UBERON:0001905 13131 13143 pineal gland +T450 GO:0010467 13166 13176 expression +T451 PR:000014441 13180 13184 mr-s +T452 SO:0000673 13185 13196 transcripts +T453 UBERON:0000922 13210 13216 embryo +T454 UBERON:0000468 13218 13228 whole body +T455 UBERON:0000966 13230 13236 retina +T456 UBERON:0001905 13238 13250 pineal gland +T457 UBERON:0000955 13252 13257 brain +T458 UBERON:0002107 13259 13264 liver +T459 UBERON:0000062 13275 13281 organs +T460 SO:0000997 13357 13366;13406 13415 fragments ... for genes +T461 SO:0000028 13374 13376 bp +T462 SO:0000028 13385 13387 bp +T463 SO:0000112 13393 13399 primer +T464 NCBITaxon:10088 13425 13430 mouse +T465 PR:000014441 13431 13435 mr-s +T466 UBERON:0000922 13474 13480 embryo +T467 UBERON:0000468 13488 13498 whole body +T468 UBERON:0000970 13515 13518 eye +T469 PR:000014441 13524 13528 mr-s +T470 PR:000014441 13580 13584 mr-s +T471 GO:0010467 13588 13597 expressed +T472 UBERON:0007023 13612 13617 adult +T473 UBERON:0001905 13618 13630 pineal gland +T474 UBERON:0007023 13646 13651 adult +T475 UBERON:0000955 13652 13657 brain +T476 UBERON:0002107 13659 13664 liver +T477 UBERON:0000062 13683 13689 organs +T478 PR:000014441 13720 13724 mr-s +T479 PR:000014441 13793 13797 mr-s +T480 GO:0010467 13815 13824 expressed +T481 CL:0000210 13839 13853 photoreceptors +T482 UBERON:0001905 13862 13874 pineal gland +T483 GO:0065007 13877 13887 Regulation +T484 PR:000014441 13891 13895 mr-s +T485 PR:000005904 13913 13916 Crx +T486 PR:000012086 13960 13964 Otx2 +T487 PR:000005904 13969 13972 Crx +T488 UBERON:0000966 14007 14014 retinal +T489 CL:0010009 14007 14028 retinal photoreceptor +T490 GO:0042461 14015 14040 photoreceptor development +T491 PR:000012086 14064 14068 Otx2 +T492 PR:000005904 14073 14076 Crx +T493 GO:0010467 14081 14090 expressed +T494 CL:0000210 14105 14124 photoreceptor cells +T495 UBERON:0000922 14132 14141 embryonic +T496 UBERON:0000966 14142 14148 retina +T497 PR:000005904 14155 14158 Crx +T498 GO:0010467 14169 14178 expressed +T499 GO:0007567 14190 14195 natal +T500 CL:0000210 14196 14210 photoreceptors +T501 PR:000014441 14228 14232 mr-s +T502 GO:0065007 14240 14249 regulated +T503 PR:000005904 14253 14256 Crx +T504 PR:000005904 14274 14277 Crx +T505 GO:0065007 14278 14287 regulates +T506 PR:000014441 14288 14292 mr-s +T507 GO:0097617 14329 14342 hybridization +T508 PR:000014441 14346 14350 mr-s +T509 PR:000005904 14373 14376 Crx +T510 UBERON:0000966 14383 14390 retinas +T511 PR:000005904 14412 14415 Crx +T512 UBERON:0000966 14419 14425 retina +T513 PR:000014441 14431 14435 mr-s +T514 SO:0000673 14436 14446 transcript +T515 PR:000005904 14503 14506 Crx +T516 PR:000014441 14543 14547 mr-s +T517 PR:000014441 14596 14600 mr-s +T518 GO:0065007 14609 14618 regulated +T519 PR:000005904 14622 14625 Crx +T520 UBERON:0001905 14633 14645 pineal gland +T521 PR:000014441 14709 14713 mr-s +T522 PR:000005904 14735 14738 Crx +T523 UBERON:0001905 14742 14754 pineal gland +T524 PR:000014441 14792 14796 mr-s +T525 PR:000005904 14850 14853 Crx +T526 UBERON:0001905 14857 14869 pineal gland +T527 PR:000014441 14912 14916 mr-s +T528 GO:0065007 14934 14943 regulated +T529 PR:000005904 14947 14950 Crx +T530 CL:0000210 14969 14983 photoreceptors +T531 UBERON:0001905 14988 15000 pineal gland +T532 PR:000014441 15034 15038 mr-s +T533 GO:0065007 15042 15051 regulated +T534 PR:000005904 15055 15058 Crx +T535 GO:0097617 15075 15088 hybridization +T536 NCBITaxon:10088 15107 15112 mouse +T537 PR:000014441 15113 15117 mr-s +T538 PR:000005904 15157 15160 Crx +T539 UBERON:0000966 15164 15171 retinas +T540 PR:000014441 15183 15187 mr-s +T541 GO:0010467 15188 15198 expression +T542 PR:000005904 15230 15233 Crx +T543 UBERON:0000966 15237 15243 retina +T544 UBERON:0001905 15321 15334 pineal glands +T545 PR:000005904 15355 15358 Crx +T546 NCBITaxon:10088 15362 15367 mouse +T547 SO:0000006 15400 15412 PCR products +T548 SO:0000112 15430 15436 primer +T549 PR:000014441 15456 15460 mr-s +T550 CHEBI:15377 15492 15497 Water +T551 PR:000005904 15542 15545 Crx +T552 PR:000014441 15561 15565 mr-s +T553 SO:0000155 15590 15598 plasmids +T554 PR:000005904 15656 15659 Crx +T555 SO:0000409 15660 15673 binding sites +T556 GO:0009294 15748 15759 transfected +T557 PR:000005904 15765 15768 Crx +T558 PR:000012086 15770 15774 Otx2 +T559 PR:000011432 15776 15779 Nrl +T560 PR:000005904 15785 15788 Crx +T561 PR:000011432 15789 15792 Nrl +T562 GO:0009294 15898 15909 transfected +T563 PR:000005904 15919 15922 Crx +T564 GO:0010467 15923 15933 expression +T565 SO:0000440 15934 15940 vector +T566 PR:000005904 15942 15945 Crx +T567 SO:0000440 15961 15967 vector +T568 PR:000005904 15969 15972 Crx +T569 PR:000005904 16072 16075 Crx +T570 GO:0065007 16076 16085 regulates +T571 PR:000014441 16086 16090 mr-s +T572 SO:0001668 16176 16200 proximal promoter region +T573 PR:000014441 16204 16208 mr-s +T574 SO:0000704 16231 16235 gene +T575 PR:000005904 16288 16291 Crx +T576 PR:000012086 16293 16297 Otx2 +T577 PR:000011432 16299 16302 Nrl +T578 GO:0010467 16303 16313 expression +T579 SO:0000440 16314 16321 vectors +T580 PR:000014441 16363 16367 mr-s +T581 PR:000005904 16401 16404 Crx +T582 SO:0000993 16413 16422 consensus +T583 PR:000005904 16518 16521 Crx +T584 PR:000012086 16525 16529 Otx2 +T585 GO:0010467 16530 16540 expression +T586 SO:0000440 16541 16547 vector +T587 PR:000011432 16654 16657 Nrl +T588 GO:0010467 16658 16668 expression +T589 SO:0000440 16669 16675 vector +T590 PR:000005904 16760 16763 Crx +T591 PR:000011432 16782 16785 Nrl +T592 PR:000001245 16795 16804 rhodopsin +T593 SO:0000167 16805 16813 promoter +T594 SO:0000704 16837 16841 gene +T595 SO:0000704 16910 16914 gene +T596 GO:0010467 16910 16925 gene expression +T597 PR:000005904 16956 16959 Crx +T598 PR:000011432 16964 16967 Nrl +T599 GO:0010467 16968 16978 expression +T600 SO:0000440 16979 16986 vectors +T601 PR:000005904 17053 17056 Crx +T602 GO:0010467 17062 17072 expression +T603 SO:0000440 17073 17079 vector +T604 UBERON:0000966 17160 17167 retinal +T605 UBERON:0001905 17168 17174 pineal +T606 PR:000012086 17262 17266 Otx2 +T607 SO:0000994 17311 17320 consensus +T608 PR:000005904 17324 17327 Crx +T609 PR:000014441 17349 17353 mr-s +T610 GO:0010467 17354 17364 expression +T611 GO:0010467 17393 17403 expression +T612 PR:000014441 17415 17419 mr-s +T613 PR:000005904 17444 17447 Crx +T614 SO:0000673 17466 17477 transcripts +T615 PR:000012086 17481 17485 Otx2 +T616 CL:0000210 17513 17526 photoreceptor +T617 UBERON:0001787 17513 17532 photoreceptor layer +T618 UBERON:0000922 17536 17545 embryonic +T619 PR:000014441 17583 17587 mr-s +T620 GO:0065007 17614 17623 regulated +T621 PR:000005904 17634 17637 Crx +T622 SO:0000440 17669 17676 vectors +T623 PR:000005904 17725 17728 Crx +T624 SO:0000409 17729 17742 binding sites +T625 PR:000005904 17796 17799 Crx +T626 GO:0010467 17800 17810 expression +T627 SO:0000440 17811 17817 vector +T628 PR:000005904 17932 17935 Crx +T629 GO:0009294 18035 18046 transfected +T630 PR:000005904 18080 18083 Crx +T631 PR:000005904 18132 18135 Crx +T632 SO:0000409 18136 18149 binding sites +T633 SO:0000028 18153 18155 bp +T634 SO:0000028 18164 18166 bp +T635 SO:0000315 18185 18214 transcription initiation site +T636 GO:0065007 18242 18252 regulation +T637 PR:000014441 18256 18260 mr-s +T638 PR:000005904 18278 18281 Crx +T639 PR:000014441 18304 18308 mr-s +T640 SO:0000417 18322 18329 domains +T641 SO:0000417 18383 18390 modules +T642 SO:0000417 18413 18420 domains +T643 SO:0000417 18449 18455 domain +T644 SO:0000417 18506 18512 domain +T645 SO:0000417 18577 18583 domain +T646 PR:000014441 18591 18595 mr-s +T647 SO:0000417 18655 18661 module +T648 PR:000014441 18721 18725 mr-s +T649 NCBITaxon:10088 18838 18843 mouse +T650 UBERON:0000966 18850 18857 retinal +T651 PR:P04386 18882 18886 GAL4 +T652 SO:0000417 18898 18904 domain +T653 SO:0000417 18993 18999 domain +T654 PR:000014441 19003 19007 mr-s +T655 PR:000014441 19054 19058 mr-s +T656 SO:0000417 19095 19101 domain +T657 PR:000014441 19171 19175 mr-s +T658 PR:000014441 19244 19248 mr-s +T659 SO:0000417 19276 19282 domain +T660 PR:P04386 19317 19321 GAL4 +T661 PR:000014441 19397 19401 mr-s +T662 PR:P04386 19417 19421 GAL4 +T663 SO:0000417 19449 19455 domain +T664 GO:0009294 19513 19524 transformed +T665 SO:0000902 19551 19560 transgene +T666 PR:P04386 19566 19570 GAL4 +T667 SO:0000409 19571 19584 binding sites +T668 PR:000033987 19601 19605 lacZ +T669 SO:0000704 19606 19610 gene +T670 PR:000014441 19642 19646 mr-s +T671 PR:000033987 19670 19674 lacZ +T672 GO:0010467 19675 19685 expression +T673 PR:000014441 19707 19711 mr-s +T674 PR:000014441 19797 19801 mr-s +T675 SO:0000417 19832 19838 domain +T676 PR:000033987 19878 19882 lacZ +T677 PR:000014441 19937 19941 mr-s +T678 PR:000033987 19978 19982 lacZ +T679 PR:000014441 20033 20037 mr-s +T680 PR:000014441 20087 20091 mr-s +T681 PR:000033987 20117 20121 lacZ +T682 PR:000033987 20200 20204 lacZ +T683 GO:0010467 20205 20215 expression +T684 PR:000014441 20290 20294 mr-s +T685 PR:P04386 20327 20331 GAL4 +T686 PR:000014441 20385 20389 mr-s +T687 SO:0000417 20471 20478 domains +T688 GO:0005634 20576 20583 nucleus +T689 PR:P04386 20595 20599 GAL4 +T690 SO:0000409 20600 20613 binding sites +T691 SO:0000417 20638 20644 domain +T692 PR:P04386 20801 20805 GAL4 +T693 PR:000014441 20829 20833 mr-s +T694 PR:000014441 20940 20944 mr-s +T695 SO:0000417 20992 20999 domains +T696 PR:000014441 21030 21034 mr-s +T697 PR:P04386 21069 21073 GAL4 +T698 SO:0000417 21123 21130 domains +T699 PR:000033987 21151 21155 LacZ +T700 GO:0010467 21156 21166 expression +T701 SO:0000417 21361 21367 domain +T702 SO:0000417 21384 21390 domain +T703 PR:000014441 21410 21414 mr-s +T704 PR:000014441 21441 21445 mr-s +T705 PR:000014441 21495 21499 mr-s +T706 PR:000014441 21562 21566 mr-s +T707 NCBITaxon:40674 21578 21587 mammalian +T708 GO:0009294 21667 21679 transfection +T709 PR:000014441 21715 21719 mr-s +T710 PR:000014441 21758 21762 mr-s +T711 PR:000014841 21824 21838 Sonic hedgehog +T712 PR:000014841 21840 21843 Shh +T713 PR:P04386 21915 21919 GAL4 +T714 PR:000014441 21949 21953 mr-s +T715 PR:000014441 22019 22023 mr-s +T716 PR:000014441 22030 22033 mrs +T717 SO:0000417 22093 22099 domain +T718 GO:0009294 22207 22219 transfection +T719 PR:000014441 22269 22273 mr-s +T720 GO:0009294 22358 22369 transfected +T721 PR:000014441 22415 22418 mrs +T722 PR:000014441 22556 22560 mr-s +T723 NCBITaxon:40674 22591 22600 mammalian +T724 SO:0000440 22840 22846 vector +T725 GO:0009294 22902 22913 transfected +T726 GO:0042571 22983 22991 antibody +T727 GO:0042571 23016 23024 antibody +T728 PR:000014441 23113 23117 mr-s +T729 GO:0009294 23175 23186 transfected +T730 GO:0042571 23248 23256 antibody +T731 GO:0042571 23283 23291 antibody +T732 PR:000014441 23321 23325 mr-s +T733 SO:0000417 23373 23379 domain +T734 SO:0000417 23435 23441 domain +T735 PR:000014441 23445 23449 mr-s +T736 SO:0000417 23530 23536 domain +T737 SO:0000417 23669 23675 domain +T738 PR:Q9VHA0 23696 23714 Sex comb on midleg +T739 PR:Q9VHA0 23716 23719 Scm +T740 PR:000014441 23890 23894 mr-s +T741 PR:000014441 24067 24070 mrs +T742 PR:P04386 24128 24132 GAL4 +T743 PR:000014441 24158 24162 mr-s +T744 SO:0000417 24212 24218 domain +T745 SO:0000417 24273 24279 domain +T746 PR:000014441 24314 24318 mr-s +T747 NCBITaxon:40674 24330 24339 mammalian +T748 GO:0005634 24360 24367 nuclear +T749 SO:0001528 24360 24387 nuclear localization signal +T750 PR:000014441 24413 24417 mr-s +T751 PR:000014441 24466 24470 mr-s +T752 GO:0005634 24496 24503 nucleus +T753 PR:000014441 24550 24554 mr-s +T754 NCBITaxon:40674 24558 24567 mammalian +T755 PR:000014441 24614 24618 mr-s +T756 SO:0000155 24619 24626 plasmid +T757 PR:000014441 24683 24687 mr-s +T758 GO:0005634 24720 24730;24739 24744 nucleus of ... cells +T759 PR:000014441 24802 24806 mr-s +T760 PR:000014441 24828 24832 mr-s +T761 CHEBI:51231 24859 24863 DAPI +T762 PR:000014441 24904 24908 mr-s +T763 GO:0005634 24949 24956 nucleus +T764 NCBITaxon:40674 24960 24969 mammalian +T765 PR:000014441 25006 25010 mr-s +T766 GO:0065007 25050 25060 regulation +T767 PR:000007229 25078 25081 TEL +T768 PR:000014441 25122 25126 mr-s +T769 NCBITaxon:40674 25130 25139 mammalian +T770 PR:000014441 25176 25180 mr-s +T771 GO:0009294 25185 25196 transfected +T772 GO:0042571 25238 25246 antibody +T773 GO:0042571 25317 25325 antibody +T774 CHEBI:51231 25331 25335 DAPI +T775 PR:P04386 25386 25390 GAL4 +T776 PR:000014441 25391 25395 mr-s +T777 MOP:0000629 25607 25621 polymerization +T778 GO:0000785 25741 25750 chromatin +T779 PR:000014441 25766 25770 mr-s +T780 SO:0000417 25821 25827 domain +T781 PR:000014441 25873 25877 mr-s +T782 CHEBI:35224 25930 25938 effector +T783 SO:0000155 25939 25947 plasmids +T784 GO:0010467 25955 25962 express +T785 PR:000014441 25994 25998 mr-s +T786 PR:P04386 26012 26016 GAL4 +T787 SO:0000417 26029 26035 domain +T788 PR:000014441 26083 26087 mr-s +T789 PR:P04386 26097 26101 GAL4 +T790 SO:0000417 26114 26120 domain +T791 PR:000014441 26126 26129 mrs +T792 SO:0000167 26157 26165 promoter +T793 SO:0000155 26166 26173 plasmid +T794 PR:P04386 26182 26186 GAL4 +T795 SO:0000409 26187 26200 binding sites +T796 SO:0000155 26249 26256 plasmid +T797 GO:0009294 26264 26275 transfected +T798 PR:000014441 26281 26284 mrs +T799 PR:P04386 26380 26384 GAL4 +T800 SO:0000417 26397 26403 domain +T801 SO:0000155 26464 26471 plasmid +T802 PR:000014441 26516 26520 mr-s +T803 PR:P04386 26529 26533 GAL4 +T804 SO:0000155 26573 26580 plasmid +T805 PR:000014441 26678 26682 mr-s +T806 PR:P04386 26775 26779 GAL4 +T807 SO:0000704 26900 26904 gene +T808 GO:0010467 26900 26915 gene expression +T809 PR:000014441 26983 26986 mrs +T810 SO:0000417 27067 27073 domain +T811 PR:000014441 27128 27132 mr-s +T812 PR:000014441 27319 27322 mrs +T813 PR:000014441 27423 27427 mr-s +T814 PR:000014441 27617 27621 mr-s +T815 PR:P04386 27755 27759 GAL4 +T816 SO:0000155 27906 27913 plasmid +T817 GO:0009294 28021 28032 transfected +T818 SO:0000155 28052 28059 plasmid +T819 PR:000014441 28124 28128 mr-s +T820 UBERON:0000966 28141 28148 retinal +T821 NCBITaxon:9606 28196 28201 human +T822 http://purl.obolibrary.org/obo/MONDO_0008380 28206 28220 retinoblastoma +T823 PR:000014441 28259 28262 mrs +T824 http://purl.obolibrary.org/obo/MONDO_0008380 28317 28331 retinoblastoma +T825 GO:0009294 28506 28518 transfection +T826 GO:0005622 28584 28597 intracellular +T827 http://purl.obolibrary.org/obo/MONDO_0008380 28626 28640 retinoblastoma +T828 CL:0000210 28698 28712 photoreceptors +T829 PR:000014441 28809 28813 mr-s +T830 PR:000014441 28951 28955 mr-s +T831 CL:0000210 29011 29025 photoreceptors +T832 PR:000014441 29079 29083 mr-s +T833 PR:000014441 29155 29159 mr-s +T834 SO:0000417 29172 29178 domain +T835 NCBITaxon:10088 29261 29266 mouse +T836 PR:000014441 29267 29271 mr-s +T837 NCBITaxon:species 29298 29305 species +T838 NCBITaxon:10114 29383 29386 rat +T839 NCBITaxon:9606 29388 29393 human +T840 NCBITaxon:7955 29405 29414 zebrafish +T841 NCBITaxon:10088 29493 29498 mouse +T842 PR:000014441 29499 29503 mr-s +T843 SO:0000417 29546 29552 domain +T844 CL:0000210 29556 29569 photoreceptor +T845 GO:0042461 29556 29581 photoreceptor development +T846 SO:0000704 29723 29727 gene +T847 PR:000014441 29789 29793 mr-s +T848 PR:P04386 29803 29807 GAL4 +T849 SO:0000417 29820 29826 domain +T850 SO:0000155 29978 29985 plasmid +T851 GO:0009294 29993 30004 transfected +T852 CHEBI:35224 30029 30037 effector +T853 SO:0000155 30038 30046 plasmids +T854 GO:0010467 30047 30057 expressing +T855 PR:P04386 30092 30096 GAL4 +T856 PR:000014441 30134 30137 mrs +T857 SO:0000155 30154 30162 plasmids +T858 GO:0009294 30168 30179 transfected +T859 SO:0000155 30216 30223 plasmid +T860 SO:0000440 30277 30283 vector +T861 GO:0009294 30395 30406 transfected +T862 SO:0000155 30452 30459 plasmid +T863 PR:000014441 30554 30557 mrs +T864 GO:0009294 30648 30659 transfected +T865 SO:0000155 30686 30693 plasmid +T866 PR:000014441 30758 30761 mrs +T867 GO:0009294 30776 30787 transfected +T868 http://purl.obolibrary.org/obo/MONDO_0008380 30797 30811 retinoblastoma +T869 SO:0000155 30854 30861 plasmid +T870 NCBITaxon:10088 31099 31104 mouse +T871 NCBITaxon:10114 31106 31109 rat +T872 NCBITaxon:9606 31111 31116 human +T873 NCBITaxon:7955 31128 31137 zebrafish +T874 PR:000014441 31138 31142 mr-s +T875 PR:000014441 31260 31263 mrs +T876 PR:000014441 31341 31345 mr-s +T877 SO:0000417 31401 31407 domain +T878 SO:0000704 31520 31524 gene +T879 PR:000014441 31526 31530 mr-s +T880 GO:0010467 31555 31564 expressed +T881 UBERON:0000966 31568 31575 retinal +T882 CL:0010009 31568 31590 retinal photoreceptors +T883 UBERON:0001905 31599 31611 pineal gland +T884 PR:000014441 31625 31629 mr-s +T885 GO:0010467 31630 31640 expression +T886 UBERON:0000966 31659 31665 retina +T887 GO:0010467 31685 31695 expression +T888 PR:000005904 31742 31745 Crx +T889 PR:000001245 31747 31756 rhodopsin +T890 CL:0000210 31767 31780 photoreceptor +T891 SO:0000704 31781 31786 genes +T892 UBERON:0001790 31816 31837 outer plexiform layer +T893 UBERON:0001781 31868 31883 layer of retina +T894 UBERON:0001789 31911 31914 ONL +T895 UBERON:0001791 31919 31922 INL +T896 CL:0000210 31942 31956 photoreceptors +T897 GO:0001750 32012 32025 outer segment +T898 PR:000014441 32058 32062 mr-s +T899 CHEBI:36357 32072 32080 molecule +T900 GO:0042461 32093 32122 development of photoreceptors +T901 CL:0000210 32108 32122 photoreceptors +T902 PR:000012086 32153 32157 Otx2 +T903 PR:000005904 32162 32165 Crx +T904 CL:0000210 32190 32203 photoreceptor +T905 GO:0042461 32190 32215 photoreceptor development +T906 PR:000012086 32225 32229 Otx2 +T907 GO:0065007 32239 32248 regulates +T908 PR:000005904 32249 32252 Crx +T909 GO:0097617 32282 32295 hybridization +T910 PR:000014441 32339 32343 mr-s +T911 PR:000005904 32358 32361 Crx +T912 UBERON:0000966 32365 32371 retina +T913 UBERON:0001905 32376 32388 pineal gland +T914 PR:000012086 32442 32446 Otx2 +T915 PR:000005904 32451 32454 Crx +T916 PR:000014441 32500 32504 mr-s +T917 NCBITaxon:40674 32508 32517 mammalian +T918 UBERON:0000966 32528 32535 retinal +T919 CL:0010009 32528 32555 retinal photoreceptor cells +T920 PR:000012086 32561 32565 Otx2 +T921 SO:0000673 32566 32577 transcripts +T922 GO:0010467 32593 32602 expressed +T923 PR:000005904 32623 32626 Crx +T924 SO:0000673 32627 32638 transcripts +T925 PR:000014441 32717 32721 mr-s +T926 GO:0065007 32748 32757 regulated +T927 PR:000005904 32761 32764 Crx +T928 PR:000011432 32788 32791 Nrl +T929 CL:0000210 32795 32808 photoreceptor +T930 GO:0010467 32854 32863 expressed +T931 CL:0000210 32867 32881 photoreceptors +T932 GO:0007567 32893 32898 natal +T933 PR:000014441 32942 32946 mr-s +T934 PR:000011432 33009 33012 Nrl +T935 NCBITaxon:10088 33016 33021 mouse +T936 GO:0010467 33060 33070 expression +T937 PR:000011432 33097 33100 Nrl +T938 UBERON:0000966 33104 33111 retinas +T939 PR:000014441 33154 33158 mr-s +T940 GO:0010467 33198 33207 expressed +T941 SO:0000704 33208 33213 genes +T942 PR:000011432 33221 33224 Nrl +T943 UBERON:0000966 33228 33234 retina +T944 SO:0000417 33277 33283 domain +T945 SO:0000417 33317 33323 module +T946 SO:0000417 33333 33339 domain +T947 PR:000014441 33347 33351 mr-s +T948 PR:000007229 33397 33400 TEL +T949 SO:0000417 33412 33419 domains +T950 SO:0001114 33431 33438 helical +T951 CHEBI:33839 33453 33462 polymeric +T952 GO:0000785 33517 33526 chromatin +T953 PR:000014441 33582 33586 mr-s +T954 PR:000014441 33646 33650 mr-s +T955 SO:0000417 33778 33784 domain +T956 PR:000014441 33788 33792 mr-s +T957 PR:000014441 33822 33826 mr-s +T958 SO:0000417 33859 33865 domain +T959 SO:0000417 33940 33946 domain +T960 PR:000014441 33950 33954 mr-s +T961 PR:000014441 33978 33982 mr-s +T962 SO:0000417 34026 34032 domain +T963 NCBITaxon:40674 34036 34045 mammalian +T964 SO:0000417 34107 34113 domain +T965 PR:000014441 34117 34121 mr-s +T966 CHEBI:33839 34130 34139 polymeric +T967 SO:0000417 34205 34211 domain +T968 PR:000014441 34215 34219 mr-s +T969 SO:0000417 34234 34240 domain +T970 CHEBI:36357 34252 34261 molecules +T971 PR:000014441 34276 34280 mr-s +T972 CHEBI:33839 34303 34310 polymer +T973 SO:0000704 34323 34327 gene +T974 GO:0016458 34323 34337 gene silencing +T975 GO:0032991 34362 34371 complexes +T976 GO:0000785 34382 34391 chromatin +T977 PR:000007229 34413 34416 TEL +T978 SO:0000417 34530 34536 domain +T979 CHEBI:33839 34616 34623 polymer +T980 PR:000014441 34715 34719 mr-s +T981 GO:0010467 34746 34756 expression +T982 PR:000014441 34797 34801 mr-s +T983 CHEBI:33839 34810 34817 polymer +T984 PR:000007229 34876 34879 TEL +T985 SO:0000417 34921 34927 domain +T986 SO:0000417 34944 34950 domain +T987 SO:0000417 34992 34998 domain +T988 PR:000007229 35005 35008 TEL +T989 CHEBI:33839 35035 35042 polymer +T990 GO:0051259 35066 35081 oligomerization +T991 SO:0000417 35093 35099 domain +T992 PR:000007229 35116 35119 TEL +T993 SO:0000704 35356 35360 gene +T994 PR:P05084 35405 35414 Hunchback +T995 SO:0000417 35445 35451 domain +T996 MOP:0000629 35517 35531 polymerization +T997 PR:000014441 35544 35548 mr-s +T998 PR:000014441 35684 35688 mr-s +T999 PR:000014441 35816 35820 mr-s +T1000 PR:P04386 35834 35838 GAL4 +T1001 SO:0000417 35851 35857 domain +T1002 PR:000014441 35863 35866 mrs +T1003 PR:000014441 35941 35945 mr-s +T1004 GO:0032991 35972 35981 complexes +T1005 SO:0000417 36003 36009 domain +T1006 PR:000014441 36089 36093 mr-s +T1007 SO:0000417 36110 36116 domain +T1008 MOP:0000629 36179 36193 Polymerization +T1009 SO:0000417 36205 36211 domain +T1010 PR:000007229 36296 36299 TEL +T1011 NCBITaxon:9606 36328 36333 human +T1012 PR:000009640 36334 36365 lethal(3) malignant brain tumor +T1013 http://purl.obolibrary.org/obo/MONDO_0001657 36344 36365 malignant brain tumor +T1014 UBERON:0000955 36354 36359 brain +T1015 PR:000009640 36369 36376 L(3)MBT +T1016 SO:0000417 36413 36419 domain +T1017 NCBITaxon:9606 36513 36514 H +T1018 PR:000009640 36515 36522 L(3)MBT +T1019 SO:0000417 36587 36593 domain +T1020 PR:000014441 36657 36661 mr-s +T1021 PR:000014441 36894 36898 mr-s +T1022 SO:0000417 37004 37010 domain +T1023 SO:0000155 37060 37067 plasmid +T1024 PR:000014441 37198 37202 mr-s +T1025 PR:000014441 37264 37268 mr-s +T1026 NCBITaxon:7955 37274 37283 zebrafish +T1027 NCBITaxon:9606 37292 37297 human +T1028 GO:0031519 37380 37393 PcG complexes +T1029 GO:0035102 37395 37399 PRC1 +T1030 GO:0035098 37404 37408 PRC2 +T1031 GO:0035098 37432 37436 PRC2 +T1032 GO:0016458 37470 37479 silencing +T1033 PR:000043452 37493 37500 histone +T1034 CHEBI:15358 37493 37500 histone +T1035 PR:000043452 37526 37533 histone +T1036 CHEBI:15358 37526 37533 histone +T1037 CHEBI:15358 37574 37581 histone +T1038 PR:000027594 37574 37584 histone H3 +T1039 GO:0016458 37611 37619 silenced +T1040 GO:0035328 37611 37629 silenced chromatin +T1041 GO:0035102 37631 37635 PRC1 +T1042 CHEBI:15358 37666 37673 histone +T1043 PR:000027594 37666 37676 histone H3 +T1044 GO:0035098 37699 37703 PRC2 +T1045 SO:0000704 37736 37740 gene +T1046 GO:0035102 37761 37765 PRC1 +T1047 GO:0000785 37773 37782 chromatin +T1048 GO:0006338 37773 37793 chromatin remodeling +T1049 GO:0016514 37825 37840 SWI-SNF complex +T1050 GO:0035098 37892 37896 PRC2 +T1051 GO:0035102 37966 37970 PRC1 +T1052 PR:000014441 38075 38078 mrs +T1053 CHEBI:46024 38141 38155 trichostatin A +T1054 PR:000014441 38259 38263 mr-s +T1055 GO:0035102 38316 38328 PRC1 complex +T1056 PR:000014441 38402 38406 mr-s +T1057 GO:0010467 38509 38519 expression +T1058 SO:0000704 38534 38539 genes +T1059 UBERON:0000966 38547 38554 retinal +T1060 CL:0010009 38547 38568 retinal photoreceptor +T1061 GO:0042461 38555 38580 photoreceptor development +T1062 PR:000014441 38637 38641 mr-s +T1063 PR:000014441 38738 38742 mr-s +T1064 GO:0097617 38773 38786 hybridization +T1065 PR:000014441 38811 38815 mr-s +T1066 GO:0010467 38816 38826 expression +T1067 UBERON:0000966 38846 38853 retinal +T1068 CL:0010009 38846 38868 retinal photoreceptors +T1069 SO:0000704 38934 38939 genes +T1070 PR:000014441 38943 38947 mr-s +T1071 CL:0000210 38961 38974 photoreceptor +T1072 SO:0000704 38975 38980 genes +T1073 PR:000014441 38996 39000 mr-s +T1074 GO:0010467 39018 39028 expression +T1075 CL:0000210 39036 39049 photoreceptor +T1076 SO:0000704 39050 39055 genes +T1077 CL:0000604 39059 39062;39072 39086 rod ... photoreceptors +T1078 CL:0000573 39067 39086 cone photoreceptors +T1079 PR:000014441 39126 39130 mr-s +T1080 GO:0043703 39146 39172;39177 39191 cell fate determination of ... photoreceptors +T1081 GO:0042671 39146 39172;39199 39218 cell fate determination of ... cone photoreceptors +T1082 CL:0000604 39173 39191 rod photoreceptors +T1083 CL:0000573 39199 39218 cone photoreceptors +T1084 CL:0000573 39226 39245 cone photoreceptors +T1085 UBERON:0019248 39266 39281 early embryonic +T1086 NCBITaxon:10088 39292 39297 mouse +T1087 GO:0060041 39298 39311 retinogenesis +T1088 CL:0000604 39313 39331 rod photoreceptors +T1089 UBERON:0007220 39358 39372;39393 39399 late embryonic ... period +T1090 GO:0007567 39387 39392 natal +T1091 GO:0010467 39410 39420 expression +T1092 PR:000014441 39432 39436 mr-s +T1093 PR:000014441 39454 39458 mr-s +T1094 GO:0010467 39462 39471 expressed +T1095 CL:0000604 39475 39493 rod photoreceptors +T1096 CL:0000573 39505 39524 cone photoreceptors +T1097 PR:000011403 39536 39541 Nr2e3 +T1098 CL:0000573 39621 39639 cone photoreceptor +T1099 SO:0000704 39649 39654 genes +T1100 PR:000014441 39694 39698 mr-s +T1101 NCBITaxon:10088 39712 39716 mice +T1102 PR:000014441 39746 39750 mr-s +T1103 NCBITaxon:10088 39807 39812 mouse +T1104 PR:000014441 39813 39817 mr-s +T1105 GO:0010467 39842 39851 expressed +T1106 UBERON:0000966 39855 39862 retinal +T1107 CL:0010009 39855 39877 retinal photoreceptors +T1108 UBERON:0001905 39886 39898 pineal gland +T1109 PR:000014441 39900 39904 mr-s +T1110 NCBITaxon:7955 39938 39947 zebrafish +T1111 NCBITaxon:9606 39959 39964 human +T1112 PR:000014441 39999 40003 mr-s +T1113 CL:0000210 40007 40020 photoreceptor +T1114 GO:0042461 40007 40032 photoreceptor development +T1115 PR:000014441 40064 40068 mr-s +T1116 GO:0005634 40094 40101 nucleus +T1117 SO:0000417 40149 40155 domain +T1118 PR:000014441 40167 40171 mr-s +T1119 PR:P04386 40189 40193 GAL4 +T1120 PR:000014441 40267 40271 mr-s +T1121 PR:000014441 40345 40349 mr-s +T1122 CHEBI:36357 40371 40379 molecule +T1123 GO:0042461 40405 40419;40428 40442 development of ... photoreceptors +T1124 GO:0021982 40405 40419;40447 40459 development of ... pineal gland +T1125 UBERON:0000966 40420 40427 retinal +T1126 CL:0010009 40420 40442 retinal photoreceptors +T1127 UBERON:0001905 40447 40459 pineal gland +T1128 NCBITaxon:10088 40484 40489 mouse +T1129 PR:000014441 40490 40494 mr-s +T1130 NCBITaxon:10088 40596 40601 mouse +T1131 SO:0000704 40602 40607 genes +T1132 GO:0010467 40608 40617 expressed +T1133 UBERON:0000966 40640 40646 retina +T1134 NCBITaxon:10088 40710 40715 mouse +T1135 UBERON:0000966 40716 40723 retinal +T1136 NCBITaxon:10090 40761 40764 Mm. +T1137 SO:0000857 40784 40792 homology +T1138 SO:0000704 40822 40827 genes +T1139 SO:0000028 40835 40837 bp +T1140 NCBITaxon:10088 40925 40930 mouse +T1141 UBERON:0000966 40934 40941 retinal +T1142 GO:0097617 41015 41028 hybridization +T1143 GO:0097617 41042 41055 hybridization +T1144 NCBITaxon:10088 41059 41064 mouse +T1145 UBERON:0000966 41071 41078 retinal +T1146 NCBITaxon:10088 41116 41121 mouse +T1147 PR:000014441 41201 41205 mr-s +T1148 PR:000014441 41366 41370 mr-s +T1149 SO:0000704 41371 41375 gene +T1150 GO:0097617 41470 41483 hybridization +T1151 SO:0000028 41493 41495 bp +T1152 PR:000014441 41513 41517 mr-s +T1153 GO:0097617 41570 41583 hybridization +T1154 GO:0097617 41593 41606 hybridization +T1155 GO:0009294 41669 41681 transfection +T1156 NCBITaxon:27592 41794 41800 bovine +T1157 UBERON:0001977 41801 41806 serum +T1158 CHEBI:17334 41826 41836 penicillin +T1159 CHEBI:17076 41851 41863 streptomycin +T1160 GO:0009294 41875 41887 transfection +T1161 CHEBI:77635 41927 41944 calcium phosphate +T1162 GO:0009294 41963 41975 transfection +T1163 CHEBI:33893 41976 41983 reagent +T1164 http://purl.obolibrary.org/obo/MONDO_0008380 41997 42011 retinoblastoma +T1165 CHEBI:32139 42123 42141 sodium bicarbonate +T1166 GO:0009294 42162 42174 transfection +T1167 UBERON:0000479 42270 42277 tissues +T1168 UBERON:0007023 42281 42286 adult +T1169 NCBITaxon:10088 42287 42291 mice +T1170 CHEBI:2511 42369 42376 agarose +T1171 CHEBI:16842 42377 42389 formaldehyde +T1172 CHEBI:25614 42415 42420 nylon +T1173 SO:0000028 42464 42466 bp +T1174 SO:0000417 42498 42504 domain +T1175 PR:000014441 42508 42512 mr-s +T1176 GO:0097617 42537 42550 hybridization +T1177 GO:0097617 42552 42565 Hybridization +T1178 CHEBI:24866 42712 42718 saline +T1179 CHEBI:8984 42732 42754 sodium dodecyl sulfate +T1180 CHEBI:8984 42756 42759 SDS +T1181 UBERON:0000479 42813 42819 tissue +T1182 GO:0001171 42858 42877 reverse transcribed +T1183 SO:0000112 42919 42926 primers +T1184 SO:0000188 42939 42946 introns +T1185 PR:000014441 42965 42969 mr-s +T1186 SO:0000028 43074 43076 bp +T1187 SO:0000112 43137 43143 Primer +T1188 NCBITaxon:10088 43154 43159 mouse +T1189 SO:0000028 43252 43254 bp +T1190 PR:P04386 43354 43358 GAL4 +T1191 PR:P04386 43431 43435 GAL4 +T1192 PR:000014441 43534 43538 mr-s +T1193 SO:0000440 43555 43561 vector +T1194 NCBITaxon:10088 43597 43602 mouse +T1195 UBERON:0000966 43609 43616 retinal +T1196 SO:0000440 43637 43643 vector +T1197 SO:0000155 43770 43777 Plasmid +T1198 SO:0000155 43822 43829 Plasmid +T1199 GO:0040007 43934 43939 grown +T1200 CHEBI:75055 44024 44029 X-gal +T1201 CHEBI:75055 44047 44052 X-gal +T1202 PR:000014441 44125 44129 mr-s +T1203 PR:000014441 44232 44236 mr-s +T1204 SO:0000440 44260 44267 vectors +T1205 CHEBI:75055 44347 44352 X-gal +T1206 PR:000014441 44488 44492 mr-s +T1207 PR:000014441 44511 44514 mrs +T1208 GO:0010467 44689 44699 expression +T1209 SO:0000440 44700 44706 vector +T1210 PR:000014441 44847 44851 mr-s +T1211 GO:0009294 44856 44867 transfected +T1212 SO:0000155 44895 44902 plasmid +T1213 CHEBI:77635 44924 44941 calcium phosphate +T1214 GO:0009294 44976 44988 transfection +T1215 CHEBI:9754 45048 45052 Tris +T1216 CHEBI:17883 45053 45056 HCl +T1217 CHEBI:6636 45083 45088 MgCl2 +T1218 CHEBI:26710 45097 45101 NaCl +T1219 CHEBI:8102 45108 45137 phenylmethylsulfonyl fluoride +T1220 CHEBI:78708 45149 45161 Nonidet P-40 +T1221 CHEBI:17754 45167 45175 glycerol +T1222 CHEBI:60004 45215 45223 cocktail +T1223 GO:0042571 45315 45323 antibody +T1224 CHEBI:9754 45623 45627 Tris +T1225 CHEBI:17883 45628 45631 HCl +T1226 CHEBI:26710 45649 45653 NaCl +T1227 CHEBI:6636 45661 45666 MgCl2 +T1228 CHEBI:8984 45708 45711 SDS +T1229 CHEBI:8984 45730 45733 SDS +T1230 CHEBI:9754 45740 45744 Tris +T1231 CHEBI:18320 45761 45764 DTT +T1232 CHEBI:17754 45769 45777 glycerol +T1233 CHEBI:8682 45785 45795 pyronine Y +T1234 CHEBI:8984 45836 45858 sodium dodecyl sulfate +T1235 CHEBI:8984 45895 45898 SDS +T1236 CHEBI:53325 45928 45942 nitrocellulose +T1237 NCBITaxon:9986 46027 46033 rabbit +T1238 GO:0042571 46053 46061 antibody +T1239 NCBITaxon:3704 46112 46123 horseradish +T1240 MOP:0000779 46136 46146 conjugated +T1241 NCBITaxon:9925 46147 46151 goat +T1242 NCBITaxon:9986 46157 46163 rabbit +T1243 GO:0071735 46164 46167 IgG +T1244 GO:0009294 46266 46277 transfected +T1245 SO:0000155 46282 46289 plasmid +T1246 PR:000014441 46321 46325 mr-s +T1247 GO:0009294 46406 46418 transfection +T1248 CHEBI:9750 46590 46602 Triton X-100 +T1249 NCBITaxon:27592 46675 46681 bovine +T1250 UBERON:0001977 46682 46687 serum +T1251 PR:000003918 46682 46695 serum albumin +T1252 GO:0042571 46766 46774 antibody +T1253 CHEBI:37987 46905 46908 Cy3 +T1254 MOP:0000779 46909 46919 conjugated +T1255 NCBITaxon:9925 46920 46924 goat +T1256 GO:0071735 46925 46928 IgG +T1257 NCBITaxon:9986 46937 46943 rabbit +T1258 GO:0071735 46944 46947 IgG +T1259 SO:0000167 47215 47223 promoter +T1260 PR:000014441 47234 47238 mr-s +T1261 SO:0000155 47266 47273 plasmid +T1262 SO:0001026 47316 47323 genomic +T1263 SO:0000997 47324 47335;47341 47345 fragment of ... gene +T1264 PR:000014441 47336 47340 mr-s +T1265 SO:0000155 47378 47385 plasmid +T1266 GO:0010467 47401 47411 expression +T1267 SO:0000440 47412 47419 vectors +T1268 PR:000005904 47463 47466 Crx +T1269 PR:000012086 47468 47472 Otx2 +T1270 PR:000011432 47474 47477 Nrl +T1271 SO:0000704 47478 47483 genes +T1272 GO:0010467 47496 47506 expression +T1273 SO:0000440 47507 47513 vector +T1274 SO:0000440 47577 47583 vector +T1275 PR:000005904 47589 47592 Crx +T1276 SO:0000409 47593 47605 binding site +T1277 SO:0000028 47619 47621 bp +T1278 SO:0000440 47693 47699 vector +T1279 PR:000005904 47705 47708 Crx +T1280 SO:0000409 47709 47721 binding site +T1281 SO:0000028 47734 47736 bp +T1282 SO:0000440 47807 47813 vector +T1283 PR:000005904 47819 47822 Crx +T1284 SO:0000409 47823 47835 binding site +T1285 SO:0000028 47847 47849 bp +T1286 SO:0000440 47922 47928 vector +T1287 PR:000005904 47943 47946 Crx +T1288 SO:0000409 47947 47960 binding sites +T1289 SO:0000155 48004 48011 plasmid +T1290 PR:P04386 48047 48051 GAL4 +T1291 NCBITaxon:10633 48112 48116 SV40 +T1292 SO:0000167 48125 48133 promoter +T1293 PR:000014441 48230 48234 mr-s +T1294 CHEBI:35224 48253 48261 effector +T1295 SO:0000155 48262 48270 plasmids +T1296 SO:0000440 48355 48361 vector +T1297 PR:P04386 48440 48444 GAL4 +T1298 SO:0000417 48457 48463 domain +T1299 GO:0009294 48494 48505 transfected +T1300 SO:0000155 48525 48532 plasmid +T1301 GO:0010467 48553 48563 expression +T1302 SO:0000440 48564 48570 vector +T1303 GO:0009294 48622 48634 transfection +T1304 CHEBI:33893 48635 48642 reagent +T1305 GO:0009294 48688 48700 transfection +T1306 PR:000005904 48726 48729 Crx +T1307 CL:0000573 48731 48735 cone +T1308 PR:000005904 48731 48748 cone-rod homeobox +T1309 CL:0000604 48736 48739 rod +T1310 PR:000012086 48750 48754 Otx2 +T1311 PR:000012086 48756 48788 orthodenticle-related homeobox 2 +T1312 PR:000016321 48790 48794 TRβ2 +T1313 UBERON:0002046 48796 48803 thyroid +T1314 CHEBI:60311 48796 48811 thyroid hormone +T1315 PR:000016321 48796 48827 thyroid hormone receptor beta 2 +T1316 PR:000011432 48829 48832 Nrl +T1317 UBERON:0003902 48834 48847 neural retina +T1318 PR:000011432 48834 48862 neural retina leucine zipper +T1319 PR:000011403 48864 48869 Nr2e3 +T1320 GO:0005634 48871 48878 nuclear +T1321 PR:000011403 48871 48916 nuclear receptor subfamily 2 group E member 3 +T1322 SO:0000345 48918 48921 EST +T1323 GO:0010467 48923 48932 expressed +T1324 SO:0000345 48923 48945 expressed sequence tag +T1325 PR:000007130 48947 48952 EphB2 +T1326 PR:000007130 48954 48972 ephrin receptor B2 +T1327 PR:000007124 48974 48979 EphA4 +T1328 PR:000007124 48981 48999 ephrin receptor A4 +T1329 PR:000007229 49001 49004 TEL +T1330 PR:000007229 49006 49032 translocation ETS leukemia +T1331 http://purl.obolibrary.org/obo/MONDO_0005059 49024 49032 leukemia +T1332 UBERON:0000966 49182 49189 retinal +T1333 UBERON:0000479 49190 49197 tissues +T1334 PR:000005904 49234 49237 Crx +T1335 NCBITaxon:10088 49245 49249 mice +T1336 http://purl.obolibrary.org/obo/MONDO_0008380 49291 49305 retinoblastoma +T1337 SO:0000155 49555 49562 plasmid +T1338 UBERON:0000922 49809 49818 Embryonic +T1339 UBERON:0000955 49894 49899 Brain +T1340 UBERON:0000104 49957 49961 Life diff --git a/src/ontogpt/evaluation/craft/database/all/16539743.txt b/src/ontogpt/evaluation/craft/database/all/16539743.txt new file mode 100644 index 000000000..f9d85b16f --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16539743.txt @@ -0,0 +1,161 @@ +Cloning and characterization of mr-s, a novel SAM domain protein, predominantly expressed in retinal photoreceptor cells + +Abstract + +Background + +Sterile alpha motif (SAM) domains are ~70 residues long and have been reported as common protein-protein interaction modules. This domain is found in a large number of proteins, including Polycomb group (PcG) proteins and ETS family transcription factors. In this work, we report the cloning and functional characterization of a novel SAM domain-containing protein, which is predominantly expressed in retinal photoreceptors and the pineal gland and is designated mouse mr-s (major retinal SAM domain protein). + +Results + +mr-s is evolutionarily conserved from zebrafish through human, organisms through which the mechanism of photoreceptor development is also highly conserved. Phylogenetic analysis suggests that the SAM domain of mr-s is most closely related to a mouse polyhomeotic (ph) ortholog, Mph1/Rae28, which is known as an epigenetic molecule involved in chromatin modifications. These findings provide the possibility that mr-s may play a critical role by regulating gene expression in photoreceptor development. mr-s is preferentially expressed in the photoreceptors at postnatal day 3–6 (P3-6), when photoreceptors undergo terminal differentiation, and in the adult pineal gland. Transcription of mr-s is directly regulated by the cone-rod homeodomain protein Crx. Immunoprecipitation assay showed that the mr-s protein self-associates mainly through the SAM domain-containing region as well as ph. The mr-s protein localizes mainly in the nucleus, when mr-s is overexpressed in HEK293T cells. Moreover, in the luciferase assays, we found that mr-s protein fused to GAL4 DNA-binding domain functions as a transcriptional repressor. We revealed that the repression activity of mr-s is not due to a homophilic interaction through its SAM domain but to the C-terminal region. + +Conclusion + +We identified a novel gene, mr-s, which is predominantly expressed in retinal photoreceptors and pineal gland. Based on its expression pattern and biochemical analysis, we predict that mr-s may function as a transcriptional repressor in photoreceptor cells and in pinealocytes of the pineal gland. + +Background + +In the development of the mammalian retina, a diverse range of cell types is generated from a pool of multipotent retinal progenitor cells. Among these cell types, photoreceptors account for over 70% of all cells in the retina. In vertebrates, there are two classes of photoreceptors, rods and cones. Rods are sensors of dim light, while cones function in bright light and are responsible for color vision. Phototransduction, a series of signal amplifications detecting a single photon of light, is initiated by the capture of light with 11-cis-retinal, a chromophore bound by the opsin proteins: rhodopsin in rods and cone opsins in cones. The proteins that carry out phototransduction are located in an elaborate and highly specialized membranous structure, the outer segment. The outer segment appears to be relatively fragile, degenerating in response to many environmental and/or genetic perturbations. In the rodent retina, the production of specific cell types during development progresses in a general order [1,2]. Rod photoreceptor generation peaks around the time of birth. Cone photoreceptors, ganglion cells, horizontal cells and amacrine cells are generated earlier, while Müller glia and bipolar cells are generated later. This production of different cell types at different times appears to derive from differences in the intrinsic properties of progenitor cells involved in the transcription or chromatin modification. Recent studies identified several important transcription factors of photoreceptor development [3-7]. Two Otx family homeobox genes, Otx2 and Crx, play essential roles in early cell fate determination and terminal differentiation of photoreceptors [3,8,9]. In the absence of Otx2 function, differentiating photoreceptor cells are converted to amacrine-like neurons [9]. Crx, a downstream target of Otx2, controls the transcription of various photoreceptor cell-specific genes and is essential for the formation of outer segments, synaptic terminals, and phototransduction pathways [8,10]. Crx transcripts begin to be expressed in developing photoreceptors at embryonic day 12.5 (E12.5) in the mouse and a strong upregulation of Crx transcription is apparent across the differentiating photoreceptors at postnatal day 6 (P6). Photoreceptor cells in the Crx knockout (KO) mice exhibit a dramatic reduction of many photoreceptor molecules including visual pigments and develop neither photoreceptor outer segments nor a synaptic terminus [8,10]. In addition, mutations of human CRX have been demonstrated to be associated with three types of photoreceptor diseases: autosomal dominant cone-rod dystrophy 2, autosomal dominant-type retinitis pigmentosa, and Leber's congenital amaurosis (LCA) [11-14]. While Otx2 and Crx control general photoreceptor development, three other transcription factors, TRβ2, Nrl, and Nr2e3 regulate the specification of photoreceptor cell types [4,5,7]. + +SAM domains (also known as Pointed, HLH, or SPM domains) are ~70 residues long and have been reported as common protein-protein interaction modules [15-17]. This domain is found in a large number of proteins, including Polycomb group (PcG) proteins [18], serine threonine kinases [19], Eph family receptor tyrosine kinases [20], the p73 tumor suppressor [21], the RNA-binding protein Smaug [22], diacylglycerol kinases [23,24], yeast mating type signaling proteins [19,25] and ETS family transcription factors [26,27]. The PcG proteins are transcriptional repressors that maintain gene silencing during development [28-30]. In mammals, PcG proteins are also implicated in Hox gene regulation. Their biological activity lies in stable silencing of specific sets of genes through chromatin modifications. A member of polycomb repressive complex 1 (PRC1), ph, contains a SAM domain at the C-terminal, and PRC1 complex is known to form helical, head-to-tail polymers through its SAM domain [31]. These polymeric structures mediate the formation of a higher order chromatin structure and possibly stabilize the repressed state of Hox genes. + +In this study, we identified a novel gene, mr-s, which encodes a SAM domain-containing protein. The SAM domain of mr-s is most closely related to that of ph mouse ortholog, MPH1/Rae28. mr-s is predominantly expressed in retinal photoreceptors when they undergo terminal differentiation, and adult pineal gland. The expression of mr-s is directly regulated by Crx. Moreover, mr-s is localized in the nucleus and can self-associate through its SAM domain-containing region. We also found that mr-s protein fused to GAL4 DNA-binding domain functions as a transcriptional repressor. These findings suggest that mr-s functions as a member of a transcriptional repressor complex in retinal photoreceptor development. + +Results + +Cloning of mouse mr-s + +In order to identify novel mouse genes preferentially expressed in the developing retina, we screened the National Institute for Biotechnology Information (NCBI) database, UniGene, using Digital Differential Display (DDD) and found EST fragments which are frequently present in mouse retinal cDNA libraries. We found that one clone in these cDNAs encodes a protein containing a SAM domain related to that of polyhomeotic protein. A PCR fragment corresponding to this mouse clone was used to screen a mouse P0-P3 retinal cDNA library to obtain a full-length cDNA clone. Sequence analysis showed that this cDNA was a novel gene encoding a SAM domain-containing protein. We referred to this protein as mr-s (major retinal SAM domain protein). As shown in Fig. 1A, a translation initiation codon is present in the same open reading frame as the SAM domain. This initiation site shows similarity to the consensus sequence proposed by Kozak [32] including the presence of the highly conserved purine at position -3. The stop codon of the predicted mr-s protein is also indicated in Fig. 1A. The amino acid sequence of the SAM domain of mr-s protein (Fig. 1A, boxed sequence) exhibits homology with SAM domains of EphB2, EphA4, MPH1, TEL and Smaug (Fig. 1B). By phylogenetic analysis, the SAM domain of mr-s is most closely related to that of Mph1/Rae28, a mouse homolog of ph (Fig. 1C). Mouse mr-s protein is conserved in rat, human, chick and zebrafish, which display 91%, 70%, 36% and 26% identity with mouse mr-s, respectively (Fig. 1D). The SAM domains of rat, human, chick and zebrafish mr-s protein are highly conserved and display 96%, 90%, 76% and 72% identity with the SAM domain of mouse mr-s protein. The chromosomal localizations of mouse and human mr-s genes were determined by searching the mouse and human genome databases (NCBI), respectively. Mouse mr-s is mapped to chromosome 4E2, and human MR-S is mapped to chromosome 1p36.33. LCA is the most common cause of inherited childhood blindness. Human MR-S maps in the vicinity region of the LCA9, recently identified as a new locus for LCA [33]. + +Figure 1 + +mr-s nucleotide and amino acid sequences. (A) mr-s nucleotide and amino acids sequences. Boxed amino acids are the SAM domain sequence and the dashed box indicates a putative nuclear localization signal. The underline indicates a putative polyadenylation termination signal. (B) Alignment of SAM domain sequences for SAM domain-containing proteins. The five alpha helices are marked H1-H5. Conserved amino acid residues are shown with a dark shadow and functionally similar residues are shown with a light shadow. The sites that were targeted for mutagenesis are indicated by arrows. (C) Phylogenetic tree of SAM domain-containing proteins. Amino acid sequences were analyzed by the neighbor-joining method in MacVector 7.2. Branch lengths reflect the mean number of substitutions per site. (D) Schematic comparison of the amino acid sequences for mouse, rat, human, chick and zebrafish mr-s proteins. The percent similarity of the SAM domains and other regions to the corresponding regions of the mouse protein is shown. Overall sequence similarity with the mouse protein is shown on the right. + +Expression of mr-s in the developing retina and the pineal gland + +To investigate mr-s expression, we first performed whole mount in situ hybridization in mouse embryos. No hybridization signal was detected at E9.5, E10.5 and E11.5 with an mr-s probe (data not shown). We then investigated the expression of the mr-s gene in the developing retina by section in situ hybridization (Fig. 1A–G). No significant signal was detected in the developing retina at E13 (Fig. 2A). A weak signal was initially detected in the outer aspect of the neuroblastic layer (NBL), a presumptive photoreceptor layer at E18 (Fig. 2B, arrow). At P3, an mr-s transcript was clearly detected in the developing photoreceptor layer (Fig. 2E). At P6, mr-s showed peak expression in the photoreceptor layer (Fig. 2F). This pattern correlates with the rapid increase in cells expressing rhodopsin and other phototransduction genes between P6-P8 [34-37]. At P9, the intensity of the mr-s signal was significantly reduced but was localized to the outer nuclear layer (ONL) (data not shown). Faint expression of mr-s mRNA was observed in mature photoreceptors in the adult retina (Fig. 2G). + +Figure 2 + +mr-s expression in developing mouse retina and pineal gland. (A-G) mr-s expression during development of the mouse retina. The in situ hybridization signal of mr-s was not detected at E13 (A). The signal (arrow) was first detected in the outer aspect of NBL at E18 (B). A strong mr-s signal was detected in outer layer of the retina at P3-P6, and then the signal decreased in the adult retina (E-G). Control with the sense probe in E18 retina is shown (C). Scale bar, 100 μm. (H) Northern blot analysis of mr-s expression in adult mouse organs. The arrow corresponds to 2.2kb mr-s transcript. (I) RT-PCR analysis of total RNAs extracted from E13 whole embryo, P0 whole body (except for the eye), P7 retina, P7 pineal gland, P7 brain, P7 liver, adult retina, adult pineal gland, adult brain and adult liver, respectively. RPE, retinal pigment epithelium; NR, neural retina; NBL, neuroblastic layer; GCL, ganglion cell layer; ONL, outer nuclear layer; INL, inner nuclear layer. + +To determine the tissue specificity of mr-s expression, the expression of the mr-s gene in various adult tissues was examined by Northern hybridization (Fig. 2H). As a control, P7 retinal RNA was used. Four bands corresponding to 7.2-kb, 4.0-kb, 2.5-kb and 2.2-kb were detected in P7 retina. The 2.2-kb band corresponds to the cDNA characterized in this study. The larger bands, possibly alternative spliced transcripts, have not yet been characterized. The mr-s probe did not detect a band in the adult tissues examined, indicating that these tissues do not express mr-s at a level comparable to that of the developing retina. + +Previous reports revealed that many photoreceptor-specific genes are also expressed in the pineal gland [38]. We examined the expression of mr-s transcripts in the whole embryo, whole body, retina, pineal gland, brain, liver and other organs at various stages by RT-PCR (Fig. 2I and data not shown). We amplified PCR fragments of 292 bp and 452 bp with primer pairs for genes encoding mouse mr-s and G3PDH, respectively. In E13 whole embryo and P0 whole body (except for the eye), no mr-s signal was detected. As expected, we observed that mr-s is expressed in the P7 and adult pineal gland. In the P7 and adult brain, liver and several other organs, the RT-PCR amplified band of mr-s was not detected (Fig. 2I and data not shown). Our data showed that mr-s is predominantly expressed in developing photoreceptors and the pineal gland. + +Regulation of mr-s transcription by Crx homeodomain protein + +Transcription factors Otx2 and Crx are known to be key regulators of retinal photoreceptor development [3,8,9]. Although both Otx2 and Crx are expressed in developing photoreceptor cells in the embryonic retina, only Crx is highly expressed in the postnatal photoreceptors, suggesting that mr-s may be regulated by Crx. To test whether Crx regulates mr-s transcription, we performed in situ hybridization of mr-s mRNA on wild-type and Crx KO P5 retinas (Fig. 3A, B). In the Crx KO retina, the mr-s transcript was dramatically reduced (Fig. 3B). This indicates that Crx is essential for transactivation of mr-s. Moreover, to test whether the transcription of mr-s is also regulated by Crx in the pineal gland, RT-PCR analysis was used to compare transcriptional levels of mr-s in P28 wild-type and Crx KO pineal gland (Fig. 3C). The results revealed that mr-s transcription was significantly downregulated in the Crx KO pineal gland. Taken together, these data indicate that mr-s transcription is regulated by Crx in the developing photoreceptors and pineal gland. + +Figure 3 + +The transcription of mr-s is regulated by Crx. (A, B) In situ hybridization using a probe for mouse mr-s was performed on the wild-type (A) and Crx KO retinas (B) at P5. mr-s expression was drastically reduced in the Crx KO retina (B). Scale bar, 100 μm. (C) RT-PCR analysis of total RNAs extracted from the pineal glands of P5 wild-type and Crx KO mouse. The upper and lower lanes show PCR products amplified by the primer pairs specific for mr-s and G3PDH cDNAs, respectively. Water was used for control RT-PCR reaction. (D-F) Crx transactivates mr-s transcription. Reporter plasmids for the luciferase assay are shown. Blue boxes represent Crx binding sites (D). Relative activity of the luciferase is indicated when Pro1.2k was co-transfected with Crx, Otx2, Nrl, and Crx+Nrl, respectively (E). Fold activation is indicated when Pro1.2k, mut1259, mut198, mut72 and mut all were co-transfected with the Crx expression vector (Crx+) or the empty vector (Crx-) into HEK293T cells (F). Error bars represent standard error of mean. + +To further examine whether Crx regulates mr-s transcription directly or not, we next performed a luciferase assay using the 1.2-kb proximal promoter region of mr-s fused to a luciferase gene as a luciferase reporter (Fig. 3D, Pro1.2k) and the Crx, Otx2, Nrl expression vectors, respectively. This 1.2-kb region of the mr-s upstream sequence contains three Crx binding consensus sequences. As shown in Fig. 3E, the luciferase activity was significantly upregulated when the Crx or Otx2 expression vector was co-introduced with Pro1.2k into HEK293T cells, while the luciferase activity was not altered when the Nrl expression vector was co-introduced. A previous report suggested that the transcriptional activity of Crx is augmented with Nrl when the rhodopsin promoter was used as a reporter gene [6]. On the other hand, our present data showed that the luciferase gene expression was not upregulated when both Crx and Nrl expression vectors were co-introduced with Pro1.2k compared to the activity when the Crx only expression vector was introduced. This may be due to cell type differences because a cell type of retinal/pineal origin was not used in our luciferase assay. In addition, our present data showed that Otx2, which is reported to have the same binding consensus as Crx, also transactivated mr-s expression. As shown in Fig. 2A–F, the expression pattern of mr-s correlates with that of Crx. In contrast, the transcripts of Otx2 are mainly detected in the photoreceptor layer at embryonic stages. Therefore, we concluded that mr-s transcription is directly regulated mainly by Crx. + +We also constructed reporter vectors in which mutations were introduced at the three Crx binding sites (Fig. 3D, mut1259, mut198, mut72, mut all). Then the Crx expression vector was co-introduced with mut1259, mut198, mut72 and mut all, respectively (Fig. 3F). The transactivaton activity by Crx was clearly reduced when mut198 or mut72 was co-introduced. On the other hand, when mut1259 was co-transfected, the transactivation activity by Crx was not altered. These results suggest that the Crx binding sites 72-bp and 198-bp upstream from the transcription initiation site are crucial for the direct regulation of mr-s transcription by Crx. + +Self-association of mr-s protein + +SAM domains are known to function as protein-protein interaction modules [15-17]. Although SAM domains can bind to various non-SAM domain-containing proteins, many homo-SAM and hetero-SAM domain interactions have been reported. To investigate whether the SAM domain of the mr-s protein can also function as a protein-protein interaction module, we performed yeast two-hybrid screening using full-length mr-s protein as the bait. Using this bait, we screened the transcriptional activator fusion protein library in which mouse P0-P3 retinal cDNAs were fused to the GAL4 activation domain. The most frequent positive clones (5 out of 28) were cDNA fragments containing the SAM domain of mr-s (Fig. 4A). This result strongly suggests that mr-s protein self-associates through SAM domain-containing regions. We then directly tested this self-association of mr-s protein in yeast. We fused full-length or truncated portions of the mr-s protein to the DNA-binding domain of the yeast transcription factor GAL4 to make bait constructs. We fused full-length or truncated portions of the mr-s protein to the GAL4 transcriptional activation domain to make prey constructs (Fig. 4B). These constructs were transformed into yeast that contain a transgene with GAL4 binding sites upstream of the lacZ gene. We found that the full-length mr-s bait construct induced lacZ expression with the full-length mr-s prey construct (Fig. 4B, full × full). The N-terminus 400 amino acid (aa) stretch of mr-s, which does not contain a SAM domain, does weakly activate transcription of lacZ (Fig. 4B, full × N). The N-terminus 400 aa stretch of mr-s was able to induce transcription of lacZ weakly with the same N-terminus 400 aa stretch of mr-s (Fig. 4B, N × N). Although the N-terminus 400 aa mr-s protein weakly activates lacZ transcription with the same N-terminus portion, a much stronger activation of lacZ expression was observed with a C-terminus portion encoding the 391–542 aa stretch of mr-s (Fig. 4B, full × C, C × C). Our GAL4 assay indicated that the signal when the full-length mr-s was present in both the bait and prey contexts was weaker than when isolated SAM domains were used. This may simply reflect the tendency for the small fusion proteins to enter the yeast nucleus and occupy GAL4 binding sites. Alternatively, the SAM domain may be less accessible for interaction in the full-length protein context as previously reported [39]. + +Figure 4 + +Summary of yeast two-hybrid screening and GAL4 assay. (A) Full-length mr-s as a bait used in the yeast two-hybrid screening and positive clones are indicated. Note that all of five mr-s clones identified in the screening contain SAM domains. (B) Schematic diagram of the mr-s fusion proteins used in the yeast GAL4 assay. Black boxes represent the position of SAM domains. Relative levels of LacZ expression are shown on the right, respectively. Note; ++ indicates an intense blue signal visible after 12hr of incubation at 37°C, + indicates a blue signal visible after 24hr of incubation. BD, binding domain; AD, activation domain; full, full-length mr-s; N, N-terminal portion of mr-s (amino acids 1 to 400); C, C-terminal portion of mr-s (amino acids 391 to 542). + +To confirm self-association of the mr-s protein in mammalian cells, we next performed co-immunoprecipitation studies in HEK293T cells by co-transfection of HA-tagged full-length/truncated mr-s and Flag-tagged full-length/truncated mr-s (Fig. 5A). As a negative control, we constructed Flag-tagged Sonic hedgehog (Shh) (lane 2 and 7). In accordance with the result of the yeast two-hybrid GAL4 assay, HA-tagged full-length mr-s (full-HA) was co-immunoprecipitated with Flag-tagged full-length mr-s (Flag-mrs) and the Flag-tagged C-terminus portion containing the SAM domain (Flag-SAM), respectively (Fig 5B, lane3 and lane5). We also found a weak co-immunoprecipitation band in co-transfection of full-HA and Flag-tagged N-terminus portion of mr-s (Flag-ΔSAM, Fig. 5B, lane 4). When ΔSAM-HA and Flag-tagged deletion mutants were co-transfected, ΔSAM-HA was co-immunoprecipitated with Flag-mrs and Flag-ΔSAM (Fig. 5B, lane 8 and lane 9), while ΔSAM-HA was not co-immunoprecipitated with Flag-SAM (Fig. 5B, lane 10). + +Figure 5 + +The mr-s protein can self-associate in mammalian cells. (A) Schematic drawing of the constructs used for immunoprecipitation assay. HA-tagged or Flag-tagged full-length (amino acids 1 to 542), ΔSAM (amino acids 1 to 400) and SAM (amino acids 401 to 542) regions were inserted into pcDNA3 vector, respectively. (B) The constructs indicated above were transfected into HEK293T cells. Each lane was co-immunoprecipitated by anti-Flag antibody and detected by anti-HA antibody. Input protein lysates are shown in the lower panels. (C) Flag-tagged two site-directed mr-s mutants, Flag-W404A and Flag-G453A were generated and co-transfected with full-HA. Each lane was co-immunoprecipitated by anti-HA antibody and detected by anti-Flag antibody. + +To investigate whether the mr-s protein self-associates mainly through the SAM domain, two site-directed mutations were generated in the SAM domain of mr-s (Fig. 1B, arrows). These mutations alter residues that are conserved in the SAM domain of ph and previous report indicates that these mutations of ph-SAM cause significant reduction in binding activity to the other SAM domain-containing protein, Sex comb on midleg (Scm) (41). Based on this result, we introduced two types of site-directed mutations, which correspond to the mutations introduced in ph protein, into Flag-tagged full-length mr-s (Flag-W404A and Flag-G453A). We found that Flag-W404A binding activity was significantly reduced and Flag-G453A binding activity was also slightly reduced compared to Flag-mrs (Fig. 5C). These results, together with yeast two-hybrid GAL4 assay, indicate that the mr-s protein self-associates strongly through its SAM domain and weakly through the N-terminus portion lacking SAM domain. + +The subcellular localization of mr-s protein in mammalian cells + +The putative nuclear localization signal at the N-terminus of the mr-s protein (Fig. 1A, dashed box) suggests that the mr-s protein localizes in the nucleus. To determine the subcellular localization of mr-s in mammalian cells, we introduced an HA-tagged full-length mr-s plasmid into HEK293T cells. Confocal microscopy showed that the mr-s protein localized mainly in the nucleus of HEK293T cells (Fig. 6A, B). To confirm the precise localization of the mr-s protein, full-length mr-s was co-immunostained with DAPI (Fig. 6C–E). These data showed that the mr-s protein localizes preferentially to the nucleus in mammalian cells, supporting the idea that the mr-s protein is involved in transcriptional regulation as are ph and/or TEL. + +Figure 6 + +Subcellular localization of mr-s in mammalian cells. (A, B) HA-tagged full-length mr-s was transfected into HEK293T (B) and detected by anti-HA antibody (A). Scale bar, 20 μm. (C-E) HEK293T cells immunostained with anti-HA antibody (C), DAPI (D), and merged image (E). Scale bar, 50 μm. + +The GAL4-mr-s fusion protein functions as transcriptional repressor + +A member of PcG proteins, ph, does not contain an obvious sequence-specific DNA binding motif (16). Ph functions as a transcriptional repressor through its polymerization and protein-protein interaction with other sequence-specific transcriptional repressors, which can form a higher order chromatin structure. The mr-s protein also does not have an obvious DNA-binding domain. To characterize the biochemical activity of mr-s, we next performed a luciferase assay. We generated effector plasmids, which express various deletion constructs of mr-s fused to the GAL4 DNA-binding domain (Fig. 7A). We first confirmed that full-length mr-s fused to GAL4 DNA binding domain (DBD-mrs) had no effect on the pGL3 promoter plasmid lacking GAL4 binding sites (data not shown). When the 5xGAL4-pGL3 reporter plasmid was co-transfected, DBD-mrs repressed luciferase activity by about 90% in a dose-dependent manner (Fig. 7B). As a control, GAL4 DNA-binding domain (DBD) had no significant effect on the 5xGAL4-pGL3 reporter plasmid. In addition, we confirmed that full-length mr-s without GAL4 DBD had no effect on the same reporter plasmid (data not shown). We next analyzed deletion constructs in which the N-terminus 400 aa stretch of mr-s (amino acids 1 to 400) or the C-terminus portion (amino acids 391 to 542) were fused to the GAL4 DBD (Fig. 7A, DBD-N, DBD-C). While DBD-N had no repressive effect on this reporter activity, DBD-C repressed luciferase gene expression by about 65%. This result suggested to us the possibility that DBD-mrs exerts transcriptional repressive activity via self-association through its SAM domain. To investigate whether the homophilic association of mr-s is required for transcriptional repression, two site-directed mutants, DBD-W404A and DBD-G453A, either of which may reduce self-binding ability, were analyzed (Fig. 7C). Compared to DBD-mrs, DBD-W404A and DBD-G453A had repression activity of 72% and 87%, respectively. While the ability of mr-s self-association partially correlates with transcriptional repressive activity, these mutants do not compromise the ability to repress transcription critically. To determine the regions of mr-s involved in transcriptional repression more precisely, the C-terminus portion was divided into two regions and each was fused to the GAL4 DBD (Fig. 7A, DBD-tail, DBD-SAM). As a consequence, DBD-SAM (amino acids 384 to 462) did not have a repressive effect on the 5xGAL4-pGL3 reporter plasmid. On the other hand, luciferase activity was repressed by 55% when DBD-tail (amino acids 459 to 542) was co-transfected with this reporter plasmid (Fig. 7D). To assess the transcriptional repressive activity of mr-s in cells of retinal origin, we performed similar experiments using human Y79 retinoblastoma cells. The results indicated that DBD-mrs also reduced luciferase activity significantly in Y79 retinoblastoma cells (Fig. 7E). However, luciferase activity was repressed by about 30% in Y79 cells, while it was repressed by 90% in HEK293T cells. This might be due to the difference in transfection efficiency between these cell lines. Another possibility is that intracellular environment in Y79 cells, a retinoblastoma cell line, is insufficient for recapitulating developing photoreceptors. In this study, we did not address the question whether or not our in vitro data reflect native mr-s transcriptional repression in vivo. However, these in vitro experiments using HEK293T and Y79 cells strongly support our hypothesis that mr-s functions as a transcriptional repressor in developing photoreceptors. Our results suggested that the C-terminal region of mr-s (amino acids 463 to 542) is required for transcriptional repression of mr-s and the SAM domain appears to be dispensable for this repressive activity. This C-terminal region of mouse mr-s is highly conserved among species (Fig. 7F). The sequence identity of the region was 93%, 73%, 41% and 40% for rat, human, chick and zebrafish, respectively (Fig. 1D). This strongly suggests that the C-terminal region of mouse mr-s functions as a transcriptional repressive domain in photoreceptor development. However, this region does not contain characteristic amino acid motifs and the mechanism through which the region achieves and/or maintains gene repression remains to be clarified in the future. + +Figure 7 + +mr-s fused to GAL4 DNA binding domain functions as a transcriptional repressor in HEK293T cells. (A) Schematic drawing of the constructs used for the luciferase assay. 5xGAL4-pGL3 reporter plasmid was co-transfected into HEK293T cells with effector plasmids expressing various deletion mutants fused to GAL4-DBD. (B) Various amounts of DBD, DBD-mrs, DBD-N or DBD-C plasmids were transfected with 0.1 μg of 5xGAL4-pGL3 reporter plasmid. The reporter activity in the presence of the pcDNA3 vector (pcDNA3) was designated as 1. Error bars represent standard error of mean. (C) DBD-W404A and DBD-G453A were co-transfected into HEK293T cells with 5xGAL4-pGL3 reporter plasmid. Fold repression was calculated as the fold decrease in luciferase activity compared with DBD-mrs. Error bars represent standard deviation. (D) Various amounts of DBD-tail or DBD-SAM were transfected with 5xGAL4-pGL3 reporter plasmid. Error bars represent standard error of mean. (E) pcDNA3 or DBD-mrs (5 μg) was co-transfected into Y79 retinoblastoma cells with 0.5 μg of 5xGAL4-pGL3 reporter plasmid. The reporter activity in the presence of pcDNA3 was designated as 1. Error bars represent standard deviation. Asterisk marks statistically significant difference (Student's t test: p < 0.03). (F) Alignment of the C-terminal regions for mouse, rat, human, chick and zebrafish mr-s proteins. Conserved amino acid residues are shown with a dark shadow. + +Taken together, our findings suggest that DBD-mrs functions as a transcriptional repressor and that the repression activity of mr-s is not due to a homophilic interaction through its SAM domain but to the C-terminal region (amino acids 463 to 542). + +Discussion + +In the present study, we identified a novel gene, mr-s, which is predominantly expressed in retinal photoreceptors and the pineal gland. The peak of mr-s expression in the developing retina is around P6. This expression pattern correlates with the rapid increase of Crx, rhodopsin and other photoreceptor genes around P6-P8. Around P6, the outer plexiform layer becomes visible and the outer layer of retina separates into two layers, ONL and INL. At the same time, photoreceptors begin to undergo terminal differentiation, forming the outer segment. We therefore hypothesized that mr-s is a key molecule in the late development of photoreceptors. + +We previously reported that Otx2 and Crx have a critical role in photoreceptor development and that Otx2 directly regulates Crx transcription [3,9]. In situ hybridization and RT-PCR showed significant reduction of mr-s signal in the Crx KO retina and pineal gland. Furthermore, the luciferase assay demonstrated that Otx2 and Crx may directly upregulate the transcription of mr-s in mammalian cells. In retinal photoreceptor cells, the Otx2 transcripts are not highly expressed at P6-P9, while the Crx transcripts are strongly detected around P6. Therefore, our results strongly suggest that mr-s transcription is directly regulated by Crx. In the present study, Nrl, a photoreceptor-specific transcription factor that is highly expressed in photoreceptors at the postnatal stage, did not affect the transcription of mr-s. This finding is actually consistent with the analysis of the Nrl KO mouse which was recently reported [40]. The expression profiles of wild-type and Nrl KO retinas at P2, P10 and 2 months were analyzed and mr-s was not included in 161 differentially expressed genes in the Nrl KO retina. + +Previous reports suggested that the SAM domain is a protein-protein interaction module. The SAM domain of the mr-s protein is closely related to that of ph and TEL, whose SAM domains can form a helical, head-to-tail polymeric structure and mediate the formation of a higher order chromatin structure. To characterize the biochemical function of mr-s, we performed yeast two-hybrid screening using full-length mr-s as the bait. As a result, the most frequent positive clones (5/28) in the screening were the cDNA fragments containing the SAM domain of mr-s. This strongly suggests that mr-s self-associates through its SAM domain. An immunoprecipitation assay, using two site-directed mutants of the SAM domain of mr-s, demonstrated that the mr-s protein can self-associate through its SAM domain in mammalian cells. While we did not address the question whether the SAM domain of mr-s forms a polymeric structure in the present study, the phylogenetic analysis of SAM domain of mr-s and other SAM domain-containing molecules suggests that mr-s can form head-to-tail polymer and mediate gene silencing by spreading repressive complexes along the chromatin similar to ph and/or TEL. Although our results in the immunoprecipitation assay demonstrated that the N-terminal constructs lacking a SAM domain still interact with each other, these results do not fit into the head-to-tail polymer model. We cannot exclude the possibility that the resulting protein-protein interaction of mr-s is an artifact of the overexpression conditions. The issue of whether or not mr-s forms a polymer awaits future analysis. + +A previous report indicated that TEL contains a sequence-specific DNA binding domain, namely the ETS domain, and binds to specific sites via its ETS domain [41]. TEL could serve to nucleate a polymer, which would spread by oligomerization of the SAM domain. In contrast to TEL, ph does not contain an obvious sequence-specific DNA binding motif (16). Therefore, its initial binding to the template may require protein-protein interactions with other sequence-specific transcriptional repressors. The segmentation gene-encoding transcriptional repressors such as Hunchback have a role in recruiting SAM domain-containing PcG proteins, which can spread along the template via polymerization [42]. Since mr-s does not contain obvious DNA binding motifs, we suppose that there is a sequence-specific transcription factor(s) which interacts with mr-s. However, we did not find any transcription factors in the present yeast two-hybrid screening. + +We also found that full-length mr-s fused to the GAL4 DNA binding domain (DBD-mrs) functions as a transcriptional repressor. This may support the idea that mr-s is involved in repressive complexes similar to other SAM domain-containing proteins. Our results, however, showed that the self-association of mr-s through its SAM domain is not essential for the transcriptional repressive activity. Polymerization of the SAM domain has been previously reported to be essential for the repressive functions of ph and TEL [43,44]. On the other hand, human lethal(3) malignant brain tumor (H-L(3)MBT) protein, which also contains a SAM domain at the C-terminus, was reported as a transcriptional repressor and the repressor activity of H-L(3)MBT required mainly the presence of the MBT repeats but not the SAM domain [45]. To determine the transcriptional repressor region of the mr-s protein, we performed a luciferase assay using site-directed mutants (DBD-W404A and DBD-G453A). The result showed that the reduced binding ability of self-association partially compromises the transcriptional repressive activity of mr-s (Fig. 7D). However, the repressive effect was more significant when DBD-tail, which does not contain SAM domain, was co-introduced with the 5xGAL4-pGL3 reporter plasmid. Therefore, we conclude that a region from amino acids 463 to 542 is mainly responsible for the repressor activity in the case of mr-s. Evolutionary conservation of the C-terminal 80 aa region of mr-s from zebrafish through human may underlie the functional importance of this region. + +Two distinct multiprotein PcG complexes, PRC1 and PRC2, have been identified. PRC2 is involved in the initiation of silencing and contains histone deacetylases (HDACs) and histone methyltransferases, which can methylate histone H3 lysine 9 and 27, marks of silenced chromatin. PRC1, including ph, recognizes the histone H3 lysine 27 mark set by PRC2 and maintains a stable state of gene repression in which PRC1 blocks chromatin remodeling by the trithorax group-related SWI-SNF complex [46,47]. Therefore, the mechanism of repression by PRC2 is thought to be HDAC-dependent while the mechanism of repression by PRC1 appears to be HDAC-independent. Our luciferase assay also showed that transcriptional repression of DBD-mrs was not affected by the addition of various concentrations of trichostatin A, a potent HDAC inhibitor (data not shown). Therefore, we speculate that the mechanism of repression of mr-s may be HDAC-independent and more similar to that of PRC1 complex. + +In the present study, the biochemical experiments demonstrate that the mr-s protein functions as a transcriptional repressor and possibly down-regulates the spatial and temporal expression of the target genes during retinal photoreceptor development. Our data also revealed that the repressive activity of mr-s is mainly due to the C-terminal region (amino acids 463 to 542). However, downstream targets of mr-s still remain unclear. In situ hybridization showed that the peak of mr-s expression is around P6, when retinal photoreceptors undergo terminal differentiation. We hypothesize that the target genes of mr-s might be non-photoreceptor genes. In this case, mr-s may suppress the expression of non-photoreceptor genes in rod and cone photoreceptors. There may be another possibility that mr-s is involved in cell fate determination of rod photoreceptors versus cone photoreceptors. While cone photoreceptors are born during the early embryonic stages of mouse retinogenesis, rod photoreceptors are born primarily in the late embryonic and early postnatal period [48]. The expression pattern of mr-s may suggest that mr-s is expressed in rod photoreceptors but not in cone photoreceptors as well as Nr2e3, which is known as a transcriptional repressor and is thought to down-regulate cone photoreceptor-specific genes. To clarify the biological function of mr-s, analysis of mice with targeted disruptions of mr-s will be very important. + +Conclusion + +Here we identified mouse mr-s, which is predominantly expressed in retinal photoreceptors and the pineal gland. mr-s is evolutionarily conserved from zebrafish through to human, suggesting a significant role of mr-s in photoreceptor development. Our present data suggest that mr-s protein localizes in the nucleus and can self-associated mainly through the SAM domain. Moreover, mr-s protein fused to GAL4 DBD functions as a transcriptional repressor. The repressive activity of mr-s is due to its C-terminal region (amino acid 463 to 542). Taken together, mr-s is a novel repressor molecule possibly involved in the development of retinal photoreceptors and pineal gland. + +Methods + +Isolation of mouse mr-s cDNA + +We used the bioinformatics method Digital Differential Display (NCBI, UniGene) to screen novel mouse genes expressed preferentially in the retina. Some of the clusters in the UniGene database were mainly from mouse retinal cDNAs. One clone in these clusters (#Mm. 246385) has a weak homology with the polyhomeotic family genes. A 735-bp cDNA fragment of this clone, encoding amino acids 140–384 was amplified by RT-PCR from mouse P0 retinal cDNA. This fragment was used as the probe for library screening, in situ hybridization and Northern hybridization. A mouse P0-P3 retinal cDNA library was screened using this mouse cDNA fragment. Positive bacteriophage clones were isolated and the full-length mr-s fragment was inserted into pBluescriptII (Stratagene). DNA sequencing was performed on both strands by the cycle sequencing method. The nucleotide sequence for mr-s gene has been deposited in the GenBank database under GenBank Accession Number #AY458844. + +In situ hybridization + +The 735-bp cDNA fragment of mr-s amplified by RT-PCR was used as a probe for in situ hybridization. In situ hybridization was performed as described previously [49]. + +Cell culture and transfection + +HEK293T cells were maintained at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Sigma), 100 IU/ml penicillin and 100 μg/ml streptomycin. Transient transfection of HEK293T cells was carried out using calcium phosphate method or Fugene6 transfection reagent (Roche). Y79 retinoblastoma cells were maintained in Iscove's modified Dulbecco's medium with 4 mM L-glutamine adjusted to contain 1.5 g/L sodium bicarbonate, 20% FBS. Transient transfection was carried out using TransIt LT1 (Mirus). + +Northern-blot analysis + +RNA was extracted from the tissues of adult mice using Trizol (Invitrogen). A 5 μg of total RNA was electrophoresed in a 1.0% agarose-formaldehyde gel and transferred to a nylon membrane (Zeta-Probe GT, Bio-Rad). The 735-bp cDNA fragment encoding the SAM domain of mr-s was used as a probe for hybridization. Hybridization was performed according to the manufacturer's protocol. Washes with increasing stringency were performed, the last being at 50°C in 0.1× standard saline citrate/0.1% sodium dodecyl sulfate (SDS). + +RT-PCR analysis + +Total RNA was isolated from each tissue using Trizol. A 1 μg of total RNA was reverse transcribed using SuperscriptII (Invitrogen). RT-PCR primers, which span introns, for detection of mr-s cDNA were 5'-TGTCCAGCCCAGCCAACCCAAGGAGACGACA-3' and 5'-TGTGGTCTCCTCATCAGTGAAGA-3'. Product size was 292 bp (positions 965–1256 of Genbank Accession Number #AY458844). Primer pairs for mouse G3PDH were 5'-ACCACAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTGTTGCTGTA- 3' which amplified a 452-bp product (positions 587–1038 of Genbank Accession Number #BC85275). + +Yeast two-hybrid screening and GAL4 assay + +We carried out yeast two-hybrid experiments using the MATCHMAKER GAL4 two-hybrid system 3 (BD Bioscience) as recommended by the manufacturer. We cloned the full-length mr-s into the pGBKT7 vector and used it to screen a library of mouse P0-P3 retinal cDNAs in the pGADT7 vector. Transformants that conferred growth were picked, isolated and re-introduced with the bait into AH109 to confirm interaction. Plasmid DNA was isolated from yeast using RPM Yeast Plasmid Isolation Kit (Qbiogene). In order to confirm the protein interactions, single colonies were picked and grown individually in synthetic complete media lacking leucine, tryptophan and containing X-gal. After 4–5 days, X-gal positive clones were selected and analyzed. To test self-interaction of mr-s, we subcloned the full-length, N-terminus (amino acids 1–400) and C-terminus (amino acids 391–542) of mr-s into pGBKT7 and pGADT7 vectors. We assessed interactions by scoring blue color on plates of medium containing X-gal. + +Immunoprecipitation assay + +For immunoprecipitation assay, 4× hemagglutinin (HA) or 3× Flag tagged cDNA fragment encoding full-length mr-s (full-HA and Flag-mrs), a cDNA fragment encoding amino acids 1–400 (ΔSAM-HA and Flag-ΔSAM), and a cDNA fragment encoding amino acids 400–542 (Flag-SAM) were subcloned into the pcDNA3 (Invitrogen) expression vector. We also constructed two site-directed mutants, Flag-W404A and Flag-G453A, using PCR. Each of these mutations was also introduced into Flag-mr-s. We transfected HEK293T cells with 5 μg of plasmid DNA per 6 cm dish by calcium phosphate method. Approximately 48 hr after transfection, cells were harvested in immunoprecipitation buffer (50 mM Tris-HCl [pH 7.5], 1 mM EDTA, 2 mM MgCl2, 150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride [Wako], 1% Nonidet P-40, 10% glycerol) in the presence of protease inhibitor cocktail tablets (Roche). For each reaction, 1 mg of cell lysate was mixed with 0.5 μg of anti-Flag antibody (SIGMA, F3165) and 15 μl of protein G-Sepharose (Amersham) on a rotating wheel at 4°C for 2 hrs. Protein concentration was determined by the BCA protein assay system (Pierce). The beads were then washed three times with immunoprecipitation buffer and followed by three times with wash buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 20 mM MgCl2). Proteins were then boiled for 5 min in SDS sample buffer (1% SDS, 1 mM Tris [pH 6.8], 40 mM DTT, 4% glycerol, 0.01% pyronine Y). The supernatants were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the nitrocellulose membrane (Trans-Blot Transfer Medium, Bio-Rad). Western blotting was performed with rabbit polyclonal anti-HA antibody (Santa Cruz, #sc-805). Signals were detected with horseradish peroxidase- conjugated goat anti-rabbit IgG and ECL plus Western Blotting Detection System (Amersham). + +Subcellular localization analysis + +We transfected the plasmid encoding HA-tagged full-length mr-s into HEK293T cells, seeded cells on coverslips coated with collagen 48 hr after transfection, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 20 min, and washed with PBS. Then we permeabilized cells in PBS containing 0.1% Triton X-100 for 15 min, washed again with PBS, and incubated in PBS containing 0.1% bovine serum albumin (BSA) for 30 min at room temperature. We incubated cells with anti-HA antibody overnight at 4°C (1:200 in PBS containing 0.2% BSA). The following day, we washed cells three times with PBS and incubated with a Cy3-conjugated goat IgG against rabbit IgG (1:400 in PBS, Jackson ImmunoReseach Laboratories) for 30 min. We rinsed cells three times with PBS, followed by observation using a confocal microscope FV300 (Olympus) equipped with a 60× objective lens. + +Luciferase assay + +For transcriptional analysis of the 1.2-kb promoter region of mr-s, we constructed a reporter plasmid (pro1.2k) by subcloning a 1.2-kb upstream genomic fragment of mr-s gene into a pGL3 luciferase reporter plasmid (Promega). The expression vectors were constructed by subcloning full-length Crx, Otx2, Nrl genes into a pMIK expression vector (a gift from Dr. K. Maruyama), respectively. For the "mut1259" vector, the Crx binding site at the -1259 bp position was mutated by replacing GGATTA with AGATCT. For the "mut198" vector, the Crx binding site at the -198 bp position was mutated by replacing TAATCC with GAATTC. For the "mut72" vector, the Crx binding site at the -72 bp position was mutated by replacing GGATTA with GAATTC. For the "mut all" vector, all of three Crx binding sites were mutated. 5xGAL4-pGL3 Control reporter plasmid, which contains five copies of the GAL4 DNA recognition sequence positioned immediately upstream of SV40 minimal promoter, was kindly gifted from Dr. T. Noguchi and used for analysis of the transcriptional activity of mr-s [50]. To generate effector plasmids, various deletion fragments were produced by PCR reaction and subcloned into pGBKT7 vector, respectively. These fragments were digested with the sequence, which encodes GAL4 DNA binding domain, and inserted into pcDNA3. We transfected 0.1 μg of reporter plasmid DNA and 2 μg of the expression vector DNA per 6 cm dish into HEK293T cells using Fugene6 transfection reagent. We analyzed luciferase activity 48 hr after transfection. + +List of abbreviations + +Crx, cone-rod homeobox; Otx2, orthodenticle-related homeobox 2; TRβ2, thyroid hormone receptor beta 2; Nrl, neural retina leucine zipper; Nr2e3, nuclear receptor subfamily 2 group E member 3; EST, expressed sequence tag; EphB2, ephrin receptor B2; EphA4, ephrin receptor A4; TEL, translocation ETS leukemia. + +Authors' contributions + +TI and KT performed most of the experiments, interpreted the data and wrote the first draft of the manuscript. AF prepared retinal tissues and their sections of wild-type and Crx mutant mice. CK performed luciferase assay using Y79 retinoblastoma cells. YT, MA and TF have made substantial contributions to conception and design of the experiments and drafting of the manuscript. All authors read and approved the final manuscript. + +Acknowledgements + +We thank T. Noguchi for 5xGAL4-pGL3 reporter plasmid; A. Nishida for discussion and critical reading of the manuscript; A. Yoshimura for advice on confocal microscopy; A. Tani, Y. Kambara, M. Murai, Y. Hirao and H. Yoshii for technical assistance. This work was supported by Precursory Research for Embryonic Science and Technology (PRESTO), Dynamics of Development Systems Molecular Brain Science, Grant-in Aid for Scientific Research (B), Senri Life Science Foundation, and Uehara Foundation. diff --git a/src/ontogpt/evaluation/craft/database/all/16579849.ann b/src/ontogpt/evaluation/craft/database/all/16579849.ann new file mode 100644 index 000000000..9e5c2048f --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16579849.ann @@ -0,0 +1,951 @@ +T1 NCBITaxon:39107 35 41 murine +T2 http://purl.obolibrary.org/obo/MONDO_0017051 52 59;77 102 classic maple syrup urine disease +T3 http://purl.obolibrary.org/obo/MONDO_0017052 64 102 intermediate maple syrup urine disease +T4 NCBITaxon:4022 77 82 maple +T5 UBERON:0001088 89 94 urine +T6 NCBITaxon:4022 126 131 Maple +T7 http://purl.obolibrary.org/obo/MONDO_0009563 126 151 Maple Syrup Urine Disease +T8 UBERON:0001088 138 143 Urine +T9 http://purl.obolibrary.org/obo/MONDO_0009563 153 157 MSUD +T10 http://purl.obolibrary.org/obo/MONDO_0019052 165 191 inborn error of metabolism +T11 GO:0008152 181 191 metabolism +T12 CHEBI:25754 233 242 keto acid +T13 http://purl.obolibrary.org/obo/MONDO_0009563 258 262 MSUD +T14 http://purl.obolibrary.org/obo/MONDO_0000001 423 430 disease +T15 NCBITaxon:33208 508 514 animal +T16 NCBITaxon:39107 544 550 murine +T17 http://purl.obolibrary.org/obo/MONDO_0017051 560 572 classic MSUD +T18 SO:0000704 582 586 gene +T19 UBERON:0000922 601 610 embryonic +T20 CL:0002322 601 620 embryonic stem cell +T21 NCBITaxon:10088 646 651 mouse +T22 PR:000006300 682 692 E2 subunit +T23 SO:0000704 693 697 gene +T24 CHEBI:25754 716 725 keto acid +T25 NCBITaxon:39107 753 759 murine +T26 http://purl.obolibrary.org/obo/MONDO_0017052 769 786 intermediate MSUD +T27 GO:0010467 821 828 express +T28 NCBITaxon:9606 831 836 human +T29 PR:000006300 837 839 E2 +T30 NCBITaxon:10088 873 877 Mice +T31 NCBITaxon:33208 953 959 animal +T32 PR:000006300 996 1006 E2 subunit 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http://purl.obolibrary.org/obo/MONDO_0009563 4438 4442 MSUD +T118 CHEBI:22918 4514 4518 BCAA +T119 http://purl.obolibrary.org/obo/MONDO_0000001 4559 4566 disease +T120 GO:0008152 4629 4638 metabolic +T121 http://purl.obolibrary.org/obo/MONDO_0005550 4661 4670 infection +T122 http://purl.obolibrary.org/obo/MONDO_0009563 4783 4787 MSUD +T123 UBERON:0002107 4894 4899 liver +T124 GO:0006520 4903 4924 amino acid metabolism +T125 NCBITaxon:9606 4971 4976 human +T126 UBERON:0002107 4977 4982 liver +T127 http://purl.obolibrary.org/obo/MONDO_0009563 5007 5011 MSUD +T128 UBERON:0002107 5042 5047 liver +T129 UBERON:0002107 5105 5110 liver +T130 CHEBI:35705 5242 5259 immunosuppressant +T131 CHEBI:23888 5260 5265 drugs +T132 UBERON:0002107 5367 5372 liver +T133 UBERON:0002107 5419 5425 livers +T134 http://purl.obolibrary.org/obo/MONDO_0000001 5500 5507 disease +T135 http://purl.obolibrary.org/obo/MONDO_0009563 5577 5581 MSUD +T136 http://purl.obolibrary.org/obo/MONDO_0000001 5637 5644 disease +T137 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http://purl.obolibrary.org/obo/MONDO_0009563 6421 6425 MSUD +T158 NCBITaxon:10088 6493 6498 mouse +T159 NCBITaxon:9606 6517 6522 human +T160 http://purl.obolibrary.org/obo/MONDO_0009563 6523 6527 MSUD +T161 SO:0000704 6595 6606 genetically +T162 NCBITaxon:39107 6618 6624 murine +T163 http://purl.obolibrary.org/obo/MONDO_0009563 6635 6639 MSUD +T164 NCBITaxon:9606 6718 6723 human +T165 http://purl.obolibrary.org/obo/MONDO_0000001 6724 6731 disease +T166 PR:000006300 6795 6814 E2 subunit of BCKDH +T167 UBERON:0000922 6846 6855 embryonic +T168 CL:0002322 6846 6860;6866 6871 embryonic stem ... cells +T169 CL:0002322 6862 6864;6866 6871 ES ... cells +T170 http://purl.obolibrary.org/obo/MONDO_0017052 6886 6903 intermediate MSUD +T171 PR:000006300 6952 6954 E2 +T172 SO:0000704 6955 6959 gene +T173 NCBITaxon:39107 7037 7043 murine +T174 http://purl.obolibrary.org/obo/MONDO_0009563 7054 7058 MSUD +T175 SO:0000704 7143 7147 gene +T176 CL:0000034 7151 7160 stem cell +T177 http://purl.obolibrary.org/obo/MONDO_0009563 7191 7195 MSUD +T178 NCBITaxon:33208 7229 7236 animals +T179 NCBITaxon:33208 7312 7318 Animal +T180 SO:0000704 7344 7348 Gene +T181 SO:0001026 7372 7379 genomic +T182 SO:0000852 7432 7448 portion of Exons +T183 PR:000006300 7464 7466 E2 +T184 SO:0000704 7467 7471 gene +T185 SO:0000357 7476 7484 flanking +T186 NCBITaxon:10678 7509 7517 P1 phage +T187 SO:0001026 7531 7537 Genome +T188 NCBITaxon:10088 7576 7581 Mouse +T189 CL:0002322 7582 7584 ES +T190 SO:0000704 7633 7637 gene +T191 SO:0001644 7638 7654 targeting vector +T192 SO:0000852 7731 7746 portion of Exon +T193 SO:0000147 7760 7764 Exon +T194 SO:0000704 7790 7794 gene +T195 SO:0000440 7823 7829 vector +T196 CL:0002322 7912 7920 ES cells +T197 CL:0002322 7970 7978 ES cells +T198 CHEBI:42768 7998 8002 G418 +T199 UBERON:0000104 8015 8019 Life +T200 CHEBI:465284 8061 8072 gancyclovir +T201 SO:0000704 8154 8158 gene +T202 SO:0001026 8212 8219 genomic +T203 GO:0097617 8236 8246 hybridized +T204 SO:0000147 8255 8259 Exon +T205 SO:0000704 8302 8306 gene +T206 CL:0002322 8347 8355 ES cells +T207 UBERON:0000358 8384 8395 blastocysts +T208 NCBITaxon:10088 8416 8420 mice +T209 NCBITaxon:10088 8599 8603 mice +T210 NCBITaxon:10088 8687 8691 mice +T211 NCBITaxon:33208 8730 8737 animals +T212 SO:0000704 8782 8789 genetic +T213 PR:000006300 8813 8815 E2 +T214 SO:0000704 8816 8820 gene +T215 NCBITaxon:10088 8830 8835 mouse +T216 SO:0000704 8851 8855 Gene +T217 PR:000006300 8898 8900 E2 +T218 NCBITaxon:10088 8910 8915 mouse +T219 CL:0002322 8916 8924 ES cells +T220 SO:0000852 9010 9022 site in Exon +T221 SO:0000188 9044 9050 intron +T222 PR:000006300 9068 9070 E2 +T223 SO:0000704 9071 9075 gene +T224 SO:0000412 9100 9120 restriction fragment +T225 GO:0097617 9126 9136 hybridizes +T226 SO:0000147 9144 9148 Exon +T227 PR:000006300 9188 9190 E2 +T228 SO:0000412 9220 9240 restriction fragment +T229 GO:0097617 9246 9256 hybridizes +T230 SO:0001644 9338 9354 targeting vector +T231 SO:0001644 9385 9401 targeting vector +T232 SO:0001026 9446 9453 genomic +T233 CL:0002322 9494 9501 ES cell +T234 CL:0002322 9537 9544 ES cell +T235 NCBITaxon:10088 9632 9636 mice +T236 GO:0097617 9651 9661 hybridized +T237 SO:0000147 9670 9674 Exon +T238 UBERON:0002107 9741 9746 liver +T239 PR:000006300 9779 9781 E2 +T240 GO:0007567 9801 9806 natal +T241 NCBITaxon:10088 9813 9818 mouse +T242 PR:000006300 9851 9853 E2 +T243 PR:000006300 9863 9865 E2 +T244 GO:0042571 9875 9883 antibody +T245 GO:0005634 9898 9905 nuclear +T246 PR:000006300 9949 9951 E2 +T247 NCBITaxon:10088 10002 10007 mouse +T248 NCBITaxon:10088 10087 10092 mouse +T249 UBERON:0000922 10093 10102 embryonic +T250 CL:0000057 10103 10114 fibroblasts +T251 PR:000006300 10167 10169 E2 +T252 GO:0005739 10234 10246 mitochondria +T253 NCBITaxon:10088 10309 10313 mice +T254 PR:000006300 10398 10400 E2 +T255 SO:0000902 10407 10416 transgene +T256 CHEBI:27902 10430 10442 tetracycline +T257 NCBITaxon:10359 10454 10458 hCMV +T258 SO:0000167 10462 10470 promoter +T259 CHEBI:27902 10490 10502 tetracycline +T260 NCBITaxon:10359 10542 10546 hCMV +T261 SO:0000167 10547 10555 promoter +T262 SO:0000188 10615 10621 intron +T263 SO:0000673 10653 10660 message +T264 CHEBI:33695 10653 10660 message +T265 GO:0010467 10675 10685 expression +T266 SO:0000993 10703 10712 consensus +T267 SO:0000318 10729 10745 initiation codon +T268 GO:0006412 10758 10769 translation +T269 NCBITaxon:9606 10780 10785 human +T270 PR:000006300 10786 10788 E2 +T271 PR:000000084 10863 10868 c-myc +T272 NCBITaxon:10633 10937 10941 SV40 +T273 GO:0043631 10947 10962 polyadenylation +T274 SO:0000610 10947 10971 polyadenylation sequence +T275 GO:0006412 11013 11024 translation +T276 PR:000006300 11043 11045 E2 +T277 SO:0000902 11046 11055 transgene +T278 SO:0000440 11074 11080 vector +T279 PR:P23940 11125 11130 BamHI +T280 NCBITaxon:10088 11187 11192 mouse +T281 UBERON:0000922 11193 11200 embryos +T282 SO:0001026 11315 11322 Genomic +T283 UBERON:0002415 11336 11340 tail +T284 NCBITaxon:10088 11344 11348 mice +T285 GO:0097617 11423 11436 hybridization +T286 SO:0000028 11448 11450 bp +T287 NCBITaxon:10633 11474 11478 SV40 +T288 PR:000006300 11498 11500 E2 +T289 SO:0000902 11501 11510 transgene +T290 http://purl.obolibrary.org/obo/MONDO_0017052 11527 11544 intermediate MSUD +T291 NCBITaxon:39107 11545 11551 murine +T292 PR:000006300 11575 11577 E2 +T293 GO:0007618 11609 11614 mated +T294 NCBITaxon:10088 11632 11636 mice +T295 PR:000006300 11673 11675 E2 +T296 PR:000005308 11693 11696 LAP +T297 SO:0000902 11701 11710 transgene +T298 NCBITaxon:33208 11833 11840 animals +T299 SO:0000902 11873 11883 transgenes +T300 NCBITaxon:10088 11931 11935 mice +T301 NCBITaxon:33208 11979 11986 animals +T302 SO:0000902 12052 12062 transgenes +T303 NCBITaxon:33208 12240 12247 animals +T304 SO:0000902 12262 12272 transgenes +T305 UBERON:0007221 12298 12313 neonatal period +T306 NCBITaxon:10088 12332 12336 mice +T307 SO:0001026 12379 12386 Genomic +T308 UBERON:0002415 12406 12410 tail +T309 CHEBI:2511 12492 12499 agarose +T310 CHEBI:25614 12532 12537 nylon +T311 NCBITaxon:10088 12608 12613 mouse +T312 UBERON:0000922 12614 12623 embryonic +T313 CL:0000057 12624 12635 fibroblasts +T314 UBERON:0000922 12655 12664 embryonic +T315 CL:0000057 12707 12718 Fibroblasts +T316 CHEBI:9750 12848 12859 Triton X100 +T317 NCBITaxon:9925 13007 13011 goat +T318 GO:0071735 13012 13015 IgG +T319 PR:000006300 13116 13118 E2 +T320 UBERON:0001977 13132 13137 serum +T321 GO:0042571 13248 13256 antibody +T322 UBERON:0002107 13388 13394 livers +T323 CHEBI:30362 13549 13559 isopentane +T324 UBERON:0000178 13708 13713 Blood +T325 UBERON:0001825 13750 13755 sinus +T326 UBERON:0003481 13759 13768 tail vein +T327 NCBITaxon:10088 13772 13776 mice +T328 UBERON:0000178 13826 13831 blood +T329 GO:0007567 13859 13864 natal +T330 CHEBI:22918 13894 13898 BCAA +T331 UBERON:0000178 13924 13929 blood +T332 UBERON:0002107 14040 14046 Livers +T333 UBERON:0002107 14138 14144 livers +T334 CHEBI:17992 14195 14202 sucrose +T335 CHEBI:9754 14210 14214 Tris +T336 CHEBI:17883 14215 14218 HC1 +T337 UBERON:0002107 14228 14233 Liver +T338 UBERON:0000479 14375 14381 tissue +T339 UBERON:0002107 14442 14447 liver +T340 UBERON:0000479 14448 14454 tissue +T341 CHEBI:16526 14548 14551 CO2 +T342 CHEBI:17865 14557 14563;14572 14583 α-keto ... isocaproate +T343 CHEBI:36927 14567 14570 14C +T344 CHEBI:60004 14636 14643 mixture +T345 CHEBI:17865 14724 14741 α-ketoisocaproate +T346 CHEBI:15346 14751 14756 CoASH +T347 CHEBI:9532 14766 14787 thiamin pyrophosphate +T348 CHEBI:15846 14794 14798 NAD+ +T349 CHEBI:18320 14805 14819 dithiothreitol +T350 CHEBI:18420 14826 14830 Mg2+ +T351 CHEBI:17865 14861 14867;14876 14887 α-keto ... isocaproate +T352 CHEBI:36927 14871 14874 14C +T353 UBERON:0002107 14904 14909 liver +T354 CHEBI:16526 14985 14988 CO2 +T355 CHEBI:24651 15004 15013 hydroxide +T356 CHEBI:31264 15017 15024 Hyamine +T357 UBERON:0002107 15169 15174 liver +T358 NCBITaxon:10088 15258 15262 mice +T359 CHEBI:8984 15332 15335 SDS +T360 CHEBI:53250 15394 15398 PVDF +T361 PR:000006300 15477 15479 E2 +T362 NCBITaxon:9986 15505 15511 rabbit +T363 PR:000006300 15512 15514 E2 +T364 UBERON:0001977 15519 15523 sera +T365 NCBITaxon:10088 15554 15559 mouse +T366 NCBITaxon:9606 15573 15578 human +T367 PR:000006300 15588 15599 E2 subunits +T368 NCBITaxon:9986 15688 15694 rabbit +T369 PR:000000084 15700 15705 c-myc +T370 GO:0042571 15710 15718 antibody +T371 GO:0042571 15805 15813 antibody +T372 PR:000003676 15818 15825 β-actin +T373 NCBITaxon:9925 15905 15909 goat +T374 NCBITaxon:9986 15915 15921 rabbit +T375 GO:0042571 15932 15940 antibody +T376 MOP:0000779 15941 15951 conjugated +T377 NCBITaxon:3704 15955 15966 horseradish +T378 CHEBI:33893 16095 16102 reagent +T379 CHEBI:22918 16184 16188 BCAA +T380 PR:000006300 16384 16386 E2 +T381 SO:0000704 16387 16391 gene +T382 NCBITaxon:10088 16401 16405 mice +T383 PR:000006300 16417 16419 E2 +T384 NCBITaxon:10088 16429 16433 mice +T385 SO:0000704 16443 16447 gene +T386 NCBITaxon:10088 16461 16466 mouse +T387 CL:0002322 16467 16475 ES cells +T388 PR:000006300 16491 16493 E2 +T389 SO:0000704 16494 16498 gene +T390 PR:000006300 16540 16542 E2 +T391 SO:0000704 16543 16547 gene +T392 SO:0000704 16579 16583 gene +T393 SO:0001026 16649 16656 genomic +T394 SO:0000852 16683 16695 part of Exon +T395 SO:0000147 16709 16713 Exon +T396 CL:0002322 16767 16774 ES cell +T397 SO:0000193 16864 16905 restriction fragment length polymorphisms +T398 SO:0000704 16920 16924 gene +T399 PR:000006300 16942 16944 E2 +T400 PR:000006300 16989 16991 E2 +T401 SO:0000147 16992 16996 Exon +T402 GO:0097617 17061 17071 hybridizes +T403 SO:0000412 17094 17114 restriction fragment +T404 SO:0001023 17134 17140 allele +T405 CL:0002322 17160 17167 ES cell +T406 CL:0002322 17196 17204 ES cells +T407 GO:0097617 17222 17232 hybridizes +T408 SO:0000412 17250 17270 restriction fragment +T409 CL:0002322 17389 17397 ES cells +T410 UBERON:0000358 17422 17433 blastocysts +T411 NCBITaxon:10088 17454 17458 mice +T412 NCBITaxon:10088 17492 17496 mice +T413 SO:0001023 17546 17552 allele +T414 NCBITaxon:10088 17567 17571 mice +T415 NCBITaxon:33208 17655 17662 animals +T416 NCBITaxon:10088 17664 17668 Mice +T417 SO:0000147 17715 17719 Exon +T418 GO:0097617 17737 17747 hybridized +T419 SO:0000412 17770 17790 restriction fragment +T420 NCBITaxon:10088 17798 17802 mice +T421 NCBITaxon:10088 17834 17838 mice +T422 NCBITaxon:10088 17878 17882 mice +T423 NCBITaxon:10088 17895 17899 Mice +T424 PR:000006300 17919 17921 E2 +T425 GO:0007567 17936 17940 born +T426 GO:0007618 18018 18025 matings +T427 NCBITaxon:10088 18058 18062 mice +T428 NCBITaxon:33208 18134 18141 animals +T429 PR:000006300 18224 18226 E2 +T430 SO:0000704 18227 18231 gene +T431 UBERON:0000922 18259 18268 embryonic +T432 GO:0009790 18259 18280 embryonic development +T433 NCBITaxon:10088 18326 18330 mice +T434 GO:0016265 18331 18335 died +T435 UBERON:0012101 18343 18359 perinatal period +T436 GO:0007567 18347 18352 natal +T437 GO:0007567 18383 18388 birth +T438 GO:0001967 18504 18510 suckle +T439 GO:0007567 18538 18543 natal +T440 UBERON:0001004 18665 18676 respiratory +T441 GO:0016265 18718 18722 died +T442 GO:0007567 18742 18747 birth +T443 GO:0007567 18804 18809 natal +T444 PR:000006300 18881 18883 E2 +T445 NCBITaxon:10088 18894 18898 mice +T446 http://purl.obolibrary.org/obo/MONDO_0017051 18916 18928 classic MSUD +T447 SO:0000704 18954 18958 gene +T448 PR:000006300 18990 18992 E2 +T449 SO:0001023 18998 19004 allele +T450 UBERON:0002107 19038 19043 liver +T451 GO:0007567 19063 19068 natal +T452 NCBITaxon:10088 19075 19080 mouse +T453 GO:0007618 19110 19116 mating +T454 NCBITaxon:10088 19173 19177 mice +T455 NCBITaxon:10088 19266 19271 mouse +T456 UBERON:0002107 19272 19278 livers +T457 NCBITaxon:10088 19314 19318 mice +T458 NCBITaxon:10088 19414 19418 mice +T459 NCBITaxon:9606 19451 19457 humans +T460 http://purl.obolibrary.org/obo/MONDO_0017051 19463 19475 classic MSUD +T461 http://purl.obolibrary.org/obo/MONDO_0017051 19528 19540 classic MSUD +T462 NCBITaxon:39107 19541 19547 murine +T463 UBERON:0002107 19583 19588 liver +T464 NCBITaxon:10088 19675 19679 mice +T465 UBERON:0002107 19730 19735 liver +T466 UBERON:0002107 19797 19802 liver +T467 CHEBI:22918 19813 19817 BCAA +T468 UBERON:0000178 19836 19841 blood +T469 NCBITaxon:10088 19845 19849 mice +T470 CHEBI:22918 19857 19861 BCAA +T471 CHEBI:22918 19922 19926 BCAA +T472 UBERON:0000178 19945 19950 blood +T473 NCBITaxon:10088 19960 19964 mice +T474 CHEBI:22918 20036 20040 BCAA +T475 UBERON:0000178 20055 20060 blood +T476 NCBITaxon:10088 20064 20068 mice +T477 NCBITaxon:10088 20115 20119 mice +T478 NCBITaxon:10088 20144 20148 mice +T479 NCBITaxon:10088 20198 20202 mice +T480 PR:000006300 20350 20352 E2 +T481 GO:0042571 20362 20370 antibody +T482 PR:000006300 20391 20393 E2 +T483 NCBITaxon:10088 20409 20413 mice +T484 PR:000006300 20460 20462 E2 +T485 UBERON:0002107 20487 20492 liver +T486 UBERON:0000922 20497 20506 embryonic +T487 CL:0000057 20507 20518 fibroblasts +T488 NCBITaxon:10088 20525 20529 mice +T489 PR:000006300 20566 20568 E2 +T490 UBERON:0000479 20602 20609 tissues +T491 NCBITaxon:10088 20617 20621 mice +T492 PR:000006300 20635 20637 E2 +T493 NCBITaxon:10088 20647 20651 mice +T494 UBERON:0000178 20684 20689 blood +T495 UBERON:0001088 20706 20711 urine +T496 CHEBI:22918 20739 20743 BCAA +T497 CHEBI:22918 20897 20901 BCAA +T498 http://purl.obolibrary.org/obo/MONDO_0009563 20988 20992 MSUD +T499 NCBITaxon:10088 20993 20997 mice +T500 GO:0009081 21004 21022 metabolism of BCAA +T501 CHEBI:22918 21018 21022 BCAA +T502 GO:0006523 21042 21062 synthesis of alanine +T503 GO:0006537 21042 21054;21064 21073 synthesis of ... glutamate +T504 GO:0006542 21042 21054;21079 21088 synthesis of ... glutamine +T505 GO:0009081 21115 21133 metabolism of BCAA +T506 CHEBI:22918 21129 21133 BCAA +T507 http://purl.obolibrary.org/obo/MONDO_0009563 21137 21141 MSUD +T508 NCBITaxon:10088 21142 21146 mice +T509 UBERON:0000178 21233 21238 blood +T510 NCBITaxon:10088 21359 21363 mice +T511 NCBITaxon:10088 21414 21418 mice +T512 NCBITaxon:10088 21463 21467 mice +T513 NCBITaxon:10088 21500 21504 mice +T514 UBERON:0000178 21556 21561 blood +T515 UBERON:0000178 21595 21600 blood +T516 CHEBI:22918 21601 21605 BCAA +T517 http://purl.obolibrary.org/obo/MONDO_0009563 21640 21644 MSUD +T518 http://purl.obolibrary.org/obo/MONDO_0009563 21665 21669 MSUD +T519 CHEBI:22918 21711 21715 BCAA +T520 http://purl.obolibrary.org/obo/MONDO_0009563 21790 21794 MSUD +T521 CHEBI:22918 21800 21804 BCAA +T522 CHEBI:22918 21877 21881 BCAA +T523 UBERON:0000178 22006 22011 blood +T524 http://purl.obolibrary.org/obo/MONDO_0009563 22092 22096 MSUD +T525 UBERON:0000178 22139 22144 blood +T526 http://purl.obolibrary.org/obo/MONDO_0017051 22170 22182 classic MSUD +T527 GO:0007567 22297 22302 birth +T528 PR:000006300 22404 22406 E2 +T529 NCBITaxon:10088 22416 22420 mice +T530 PR:000006300 22452 22454 E2 +T531 CHEBI:22918 22510 22514 BCAA +T532 UBERON:0000178 22522 22527 blood +T533 UBERON:0001088 22532 22537 urine +T534 GO:0008152 22545 22554 metabolic +T535 NCBITaxon:9606 22669 22675 humans +T536 http://purl.obolibrary.org/obo/MONDO_0017051 22685 22705 classic form of MSUD +T537 PR:000006300 22713 22715 E2 +T538 NCBITaxon:10088 22725 22729 mice +T539 http://purl.obolibrary.org/obo/MONDO_0017051 22744 22756 classic MSUD +T540 NCBITaxon:10088 22768 22772 mice +T541 GO:0010467 22773 22783 expressing +T542 NCBITaxon:9606 22786 22791 human +T543 PR:000006300 22792 22794 E2 +T544 SO:0000902 22795 22804 transgene +T545 http://purl.obolibrary.org/obo/MONDO_0009563 22829 22833 MSUD +T546 http://purl.obolibrary.org/obo/MONDO_0017052 22846 22863 intermediate MSUD +T547 NCBITaxon:39107 22877 22883 murine +T548 http://purl.obolibrary.org/obo/MONDO_0017052 22897 22930 intermediate variant form of MSUD +T549 CHEBI:22918 22996 23000 BCAA +T550 http://purl.obolibrary.org/obo/MONDO_0017051 23043 23055 classic MSUD +T551 NCBITaxon:10088 23056 23061 mouse +T552 GO:0010467 23130 23137 express +T553 NCBITaxon:9606 23138 23143 human +T554 PR:000006300 23144 23146 E2 +T555 UBERON:0002107 23150 23155 liver +T556 PR:000006300 23163 23165 E2 +T557 PR:000005308 23228 23231 LAP +T558 SO:0000902 23236 23245 transgene +T559 PR:000006300 23256 23258 E2 +T560 SO:0000902 23259 23268 transgene +T561 PR:000005308 23284 23287 LAP +T562 SO:0000902 23292 23301 transgene +T563 UBERON:0002107 23330 23335 liver +T564 GO:0010467 23345 23355 expression +T565 CHEBI:27902 23363 23375 tetracycline +T566 GO:0065007 23376 23386 controlled +T567 GO:0010467 23448 23458 expression +T568 SO:0000167 23462 23471 promoters +T569 PR:000006300 23533 23535 E2 +T570 SO:0000902 23536 23545 transgene +T571 GO:0010467 23562 23569 express +T572 NCBITaxon:9606 23572 23577 human +T573 PR:000006300 23578 23580 E2 +T574 SO:0000167 23616 23624 promoter +T575 NCBITaxon:10088 23672 23677 mouse +T576 NCBITaxon:10088 23750 23754 mice +T577 GO:0010467 23760 23767 express +T578 NCBITaxon:9606 23768 23773 human +T579 PR:000006300 23774 23776 E2 +T580 PR:000005308 23778 23781 LAP +T581 NCBITaxon:10088 23797 23801 mice +T582 NCBITaxon:10088 23845 23849 mice +T583 GO:0010467 23850 23857 express +T584 CHEBI:27902 23862 23874 tetracycline +T585 GO:0065007 23875 23885 controlled +T586 UBERON:0002107 23916 23921 liver +T587 PR:000005308 23931 23934 LAP +T588 SO:0000167 23935 23943 promoter +T589 PR:000006300 23953 23955 E2 +T590 SO:0000902 23956 23965 transgene +T591 CHEBI:27902 23979 23991 tetracycline +T592 SO:0000167 24030 24038 promoter +T593 SO:0000188 24052 24058 intron +T594 NCBITaxon:9606 24076 24081 human +T595 PR:000006300 24082 24084 E2 +T596 PR:000000084 24112 24117 c-myc +T597 NCBITaxon:10633 24135 24139 SV40 +T598 GO:0043631 24148 24163 polyadenylation +T599 SO:0000610 24148 24172 polyadenylation sequence +T600 NCBITaxon:10088 24236 24240 mice +T601 PR:000006300 24270 24272 E2 +T602 UBERON:0002107 24284 24289 liver +T603 http://purl.obolibrary.org/obo/MONDO_0017052 24305 24322 intermediate MSUD +T604 NCBITaxon:10088 24323 24327 mice +T605 NCBITaxon:9606 24353 24358 human +T606 PR:000006300 24359 24361 E2 +T607 NCBITaxon:10088 24395 24399 mice +T608 NCBITaxon:33208 24455 24462 animals +T609 NCBITaxon:10088 24503 24508 mouse +T610 PR:000006300 24509 24511 E2 +T611 NCBITaxon:33208 24535 24542 animals +T612 PR:000000084 24562 24567 c-myc +T613 GO:0042571 24572 24580 antibody +T614 SO:0000902 24611 24620 transgene +T615 PR:000000084 24630 24635 c-myc +T616 NCBITaxon:9606 24644 24649 human +T617 PR:000006300 24650 24652 E2 +T618 NCBITaxon:10088 24667 24671 mice +T619 UBERON:0000955 24718 24723 brain +T620 UBERON:0002113 24729 24735 kidney +T621 SO:0000902 24787 24796 transgene +T622 PR:000006300 24805 24807 E2 +T623 UBERON:0000479 24817 24824 tissues +T624 PR:000003676 24859 24864 actin +T625 GO:0042571 24865 24873 antibody +T626 PR:000005308 24938 24941 LAP +T627 NCBITaxon:10088 24946 24950 mice +T628 GO:0045120 25025 25035 Pronuclear +T629 NCBITaxon:10088 25082 25086 mice +T630 PR:000006300 25109 25111 E2 +T631 SO:0000902 25112 25121 transgene +T632 SO:0000902 25174 25183 transgene +T633 SO:0000902 25363 25372 transgene +T634 NCBITaxon:33208 25456 25463 animals +T635 SO:0000902 25477 25486 transgene +T636 SO:0000366 25487 25502 insertion sites +T637 SO:0000902 25710 25719 transgene +T638 UBERON:0002415 25773 25777 tail +T639 PR:000006300 25816 25818 E2 +T640 NCBITaxon:10088 25819 25823 mice +T641 NCBITaxon:10088 25889 25893 mice +T642 PR:000005308 25921 25924 LAP +T643 SO:0000902 25929 25938 transgene +T644 PR:000006300 25969 25971 E2 +T645 NCBITaxon:10088 26032 26036 mice +T646 PR:000005308 26063 26066 LAP +T647 SO:0000902 26071 26080 transgene +T648 PR:000006300 26090 26092 E2 +T649 SO:0000902 26093 26102 transgene +T650 PR:000006300 26112 26114 E2 +T651 SO:0000902 26170 26180 transgenes +T652 NCBITaxon:33208 26335 26342 animals +T653 PR:000006300 26386 26388 E2 +T654 SO:0000902 26420 26430 transgenes +T655 NCBITaxon:10088 26472 26476 mice +T656 NCBITaxon:10088 26653 26657 mice +T657 SO:0000902 26784 26793 transgene +T658 PR:000006300 26874 26876 E2 +T659 NCBITaxon:33208 27018 27025 animals +T660 http://purl.obolibrary.org/obo/MONDO_0017052 27109 27126 intermediate MSUD +T661 NCBITaxon:10088 27127 27133 murine +T662 NCBITaxon:10088 27219 27223 mice +T663 PR:000006300 27348 27350 E2 +T664 PR:000005308 27377 27380 LAP +T665 PR:000006300 27393 27395 E2 +T666 SO:0000902 27396 27406 transgenes +T667 NCBITaxon:33208 27516 27523 animals +T668 CHEBI:22918 27620 27624 BCAA +T669 UBERON:0000178 27639 27644 blood +T670 NCBITaxon:10088 27648 27652 mice +T671 CHEBI:22918 27695 27699 BCAA +T672 CHEBI:22918 27754 27758 BCAA +T673 UBERON:0002107 27986 27991 liver +T674 NCBITaxon:10088 28024 28028 mice +T675 NCBITaxon:10088 28083 28087 mice +T676 NCBITaxon:10088 28118 28122 mice +T677 UBERON:0000178 28335 28340 blood +T678 CHEBI:22918 28391 28395 BCAA +T679 CHEBI:22918 28412 28416 BCAA +T680 NCBITaxon:10088 28436 28440 mice +T681 CHEBI:22918 28557 28561 BCAA +T682 NCBITaxon:10088 28596 28600 mice +T683 UBERON:0000178 28666 28671 Blood +T684 NCBITaxon:10088 28739 28743 mice +T685 NCBITaxon:10088 28775 28779 mice +T686 UBERON:0000178 28845 28850 blood +T687 http://purl.obolibrary.org/obo/MONDO_0017052 28876 28893 intermediate MSUD +T688 NCBITaxon:10088 28995 28999 mice +T689 PR:000005308 29234 29237 LAP +T690 NCBITaxon:10088 29242 29246 mice +T691 GO:0010467 29271 29281 expression +T692 NCBITaxon:9606 29296 29301 human +T693 PR:000006300 29302 29304 E2 +T694 UBERON:0002107 29339 29344 liver +T695 GO:0010467 29354 29364 expression +T696 GO:0006412 29397 29410;29420 29427 production of ... protein +T697 NCBITaxon:9606 29411 29416 human +T698 PR:000006300 29417 29419 E2 +T699 UBERON:0002107 29431 29436 liver +T700 UBERON:0002107 29499 29504 liver +T701 UBERON:0002107 29601 29606 liver +T702 NCBITaxon:9606 29653 29658 human +T703 PR:000006300 29659 29661 E2 +T704 NCBITaxon:10088 29703 29707 mice +T705 NCBITaxon:10088 29735 29739 mice +T706 NCBITaxon:10088 29769 29773 mice +T707 NCBITaxon:9606 29788 29793 human +T708 PR:000006300 29794 29796 E2 +T709 NCBITaxon:10088 29838 29843 mouse +T710 PR:000006300 29844 29846 E2 +T711 UBERON:0002107 29868 29873 liver +T712 NCBITaxon:10088 29899 29903 mice +T713 PR:000006300 29956 29958 E2 +T714 SO:0000902 30033 30042 transgene +T715 PR:000006300 30051 30053 E2 +T716 PR:000000084 30116 30121 c-myc +T717 SO:0000902 30157 30166 transgene +T718 NCBITaxon:9606 30175 30180 human +T719 PR:000006300 30181 30183 E2 +T720 CHEBI:15361 30353 30361 pyruvate +T721 GO:0045254 30353 30383 pyruvate dehydrogenase complex +T722 NCBITaxon:562 30496 30503 E. coli +T723 GO:0065003 30535 30551 subunit assembly +T724 NCBITaxon:9606 30583 30588 human +T725 PR:000006300 30589 30591 E2 +T726 GO:0032991 30622 30631 complexed +T727 NCBITaxon:10088 30637 30642 mouse +T728 PR:000006506 30650 30661 E3 subunits +T729 NCBITaxon:9606 30663 30668 Human +T730 PR:000006300 30669 30671 E2 +T731 NCBITaxon:10088 30696 30701 mouse +T732 PR:000006300 30702 30704 E2 +T733 GO:0010467 30776 30786 expression +T734 PR:000006300 30794 30796 E2 +T735 SO:0000902 30797 30806 transgene +T736 UBERON:0000955 30810 30815 brain +T737 UBERON:0002113 30817 30823 kidney +T738 GO:0010467 30882 30892 expression +T739 PR:000006300 30896 30898 E2 +T740 UBERON:0000479 30922 30929 tissues +T741 NCBITaxon:10088 30958 30962 mice +T742 NCBITaxon:33208 31082 31089 animals +T743 NCBITaxon:10088 31160 31164 mice +T744 NCBITaxon:10088 31199 31203 mice +T745 NCBITaxon:10088 31369 31373 mice +T746 GO:0016265 31427 31431 died +T747 NCBITaxon:10088 31488 31492 mice +T748 CHEBI:22918 31497 31501 BCAA +T749 GO:0010467 31580 31589 expressed +T750 UBERON:0002107 31635 31640 liver +T751 http://purl.obolibrary.org/obo/MONDO_0009563 31733 31737 MSUD +T752 http://purl.obolibrary.org/obo/MONDO_0000001 31781 31788 disease +T753 NCBITaxon:10088 31813 31817 mice +T754 http://purl.obolibrary.org/obo/MONDO_0017052 31855 31872 intermediate MSUD +T755 http://purl.obolibrary.org/obo/MONDO_0009563 31945 31949 MSUD +T756 NCBITaxon:33208 31993 31999 animal +T757 http://purl.obolibrary.org/obo/MONDO_0000001 32013 32020 disease +T758 SO:0000704 32129 32140 genetically +T759 NCBITaxon:10088 32152 32157 mouse +T760 http://purl.obolibrary.org/obo/MONDO_0009563 32205 32209 MSUD +T761 http://purl.obolibrary.org/obo/MONDO_0000001 32266 32273 disease +T762 http://purl.obolibrary.org/obo/MONDO_0017051 32289 32301 classic MSUD +T763 SO:0000704 32317 32321 gene +T764 PR:000006300 32338 32357 E2 subunit of BCKDH +T765 NCBITaxon:33208 32391 32398 animals +T766 NCBITaxon:9606 32424 32430 humans +T767 http://purl.obolibrary.org/obo/MONDO_0017051 32436 32448 classic MSUD +T768 NCBITaxon:10088 32459 32463 mice +T769 GO:0007567 32469 32473 born +T770 GO:0007567 32523 32528 birth +T771 GO:0007567 32546 32551 birth +T772 GO:0001967 32566 32574 suckling +T773 UBERON:0000178 32576 32581 blood +T774 CHEBI:22918 32592 32596 BCAA +T775 GO:0009081 32735 32753 metabolism of BCAA +T776 CHEBI:22918 32749 32753 BCAA +T777 UBERON:0002107 32806 32812 livers +T778 NCBITaxon:10088 32836 32841 mouse +T779 GO:0008152 32902 32913 metabolized +T780 CHEBI:22918 32914 32918 BCAA +T781 PR:000006300 32935 32937 E2 +T782 UBERON:0002107 32960 32965 liver +T783 CL:0000057 32970 32981 fibroblasts +T784 NCBITaxon:10088 33040 33045 mouse +T785 http://purl.obolibrary.org/obo/MONDO_0017051 33086 33098 classic MSUD +T786 UBERON:0001016 33132 33142 neurologic +T787 UBERON:0001004 33219 33230 respiratory +T788 UBERON:0001016 33255 33265 neurologic +T789 http://purl.obolibrary.org/obo/MONDO_0009563 33420 33424 MSUD +T790 GO:0016265 33505 33509 died +T791 GO:0007567 33529 33534 birth +T792 CHEBI:22918 33597 33601 BCAA +T793 UBERON:0000955 33638 33643 brain +T794 http://purl.obolibrary.org/obo/MONDO_0006684 33638 33649 brain edema +T795 http://purl.obolibrary.org/obo/MONDO_0009563 33700 33704 MSUD +T796 http://purl.obolibrary.org/obo/MONDO_0017051 33722 33734 classic MSUD +T797 CHEBI:22918 33809 33813 BCAA +T798 NCBITaxon:10088 33949 33954 mouse +T799 http://purl.obolibrary.org/obo/MONDO_0017051 33988 34000 classic MSUD +T800 NCBITaxon:9606 34043 34048 human +T801 http://purl.obolibrary.org/obo/MONDO_0000001 34049 34056 disease +T802 http://purl.obolibrary.org/obo/MONDO_0017052 34131 34148 intermediate MSUD +T803 GO:0010467 34183 34190 express +T804 NCBITaxon:9606 34191 34196 human +T805 PR:000006300 34197 34199 E2 +T806 UBERON:0002107 34207 34212 liver +T807 PR:000006300 34216 34218 E2 +T808 NCBITaxon:10088 34228 34232 mice +T809 PR:000006300 34254 34256 E2 +T810 SO:0000902 34275 34284 transgene +T811 PR:000006300 34293 34295 E2 +T812 GO:0016265 34296 34299 die +T813 UBERON:0007221 34317 34332 neonatal period +T814 GO:0010467 34347 34357 expression +T815 NCBITaxon:9606 34363 34368 human +T816 PR:000006300 34369 34371 E2 +T817 SO:0000902 34372 34381 transgene +T818 UBERON:0002107 34389 34394 liver +T819 NCBITaxon:10088 34413 34417 mice +T820 NCBITaxon:10088 34479 34483 mice +T821 UBERON:0000113 34496 34505 adulthood +T822 NCBITaxon:10088 34547 34552 mouse +T823 UBERON:0002107 34602 34607 liver +T824 CHEBI:22918 34668 34672 BCAA +T825 UBERON:0000178 34683 34688 blood +T826 NCBITaxon:10088 34705 34709 mice +T827 http://purl.obolibrary.org/obo/MONDO_0017052 34777 34794 intermediate MSUD +T828 NCBITaxon:9606 34795 34800 human +T829 CHEBI:22918 34847 34851 BCAA +T830 NCBITaxon:10088 34961 34965 mice +T831 NCBITaxon:9606 34973 34978 human +T832 SO:0000704 34995 35006 genetically +T833 NCBITaxon:10088 35018 35022 mice +T834 NCBITaxon:33208 35048 35054 animal +T835 http://purl.obolibrary.org/obo/MONDO_0017052 35068 35093 intermediate form of MSUD +T836 http://purl.obolibrary.org/obo/MONDO_0017052 35148 35165 intermediate MSUD +T837 CHEBI:27902 35189 35201 tetracycline +T838 GO:0065007 35202 35211 regulated +T839 SO:0000704 35212 35216 gene +T840 NCBITaxon:10088 35352 35356 mice +T841 http://purl.obolibrary.org/obo/MONDO_0017052 35365 35382 intermediate MSUD +T842 SO:0000902 35503 35512 transgene +T843 NCBITaxon:9606 35567 35572 human +T844 PR:000006300 35573 35575 E2 +T845 GO:0010467 35576 35586 expression +T846 SO:0005853 35587 35595 cassette +T847 CHEBI:27902 35610 35622 tetracycline +T848 CHEBI:50845 35633 35644 doxycycline +T849 CHEBI:50845 35756 35767 doxycycline +T850 SO:0000366 35807 35823 integration site +T851 SO:0000902 35831 35841 transgenes +T852 GO:0065007 35865 35874 regulated +T853 GO:0010467 35875 35885 expression +T854 GO:0065007 35985 35994 regulated +T855 CHEBI:23995 36104 36107 ENU +T856 NCBITaxon:10088 36108 36113 mouse +T857 NCBITaxon:9606 36125 36130 human +T858 http://purl.obolibrary.org/obo/MONDO_0009563 36131 36135 MSUD +T859 GO:0005739 36177 36190 mitochondrial +T860 PR:000004683 36177 36195 mitochondrial BCAT +T861 SO:0000704 36196 36200 gene +T862 UBERON:0002107 36233 36238 liver +T863 NCBITaxon:10088 36258 36263 mouse +T864 UBERON:0000178 36291 36296 blood +T865 CHEBI:22918 36297 36301 BCAA +T866 http://purl.obolibrary.org/obo/MONDO_0009563 36395 36399 MSUD +T867 http://purl.obolibrary.org/obo/MONDO_0009563 36497 36501 MSUD +T868 http://purl.obolibrary.org/obo/MONDO_0000001 36538 36545 disease +T869 NCBITaxon:10088 36586 36591 mouse +T870 NCBITaxon:9913 36601 36604 cow +T871 http://purl.obolibrary.org/obo/MONDO_0009563 36614 36618 MSUD +T872 NCBITaxon:33208 36725 36731 animal +T873 http://purl.obolibrary.org/obo/MONDO_0009563 36792 36796 MSUD +T874 NCBITaxon:9913 36797 36800 cow +T875 http://purl.obolibrary.org/obo/MONDO_0009563 36811 36815 MSUD +T876 NCBITaxon:9606 36816 36822 humans +T877 NCBITaxon:39107 36884 36890 murine +T878 NCBITaxon:9606 36967 36972 human +T879 http://purl.obolibrary.org/obo/MONDO_0009563 36973 36977 MSUD +T880 http://purl.obolibrary.org/obo/MONDO_0009563 37030 37034 MSUD +T881 NCBITaxon:10088 37035 37040 mouse +T882 SO:0000704 37084 37088 gene +T883 CL:0000182 37129 37140 hepatocytes +T884 UBERON:0000922 37152 37161 embryonic +T885 CL:0002322 37152 37172 embryonic stem cells +T886 SO:0000704 37192 37196 gene +T887 SO:0000704 37227 37231 gene +T888 NCBITaxon:9606 37243 37249 humans +T889 NCBITaxon:33208 37296 37302 animal +T890 SO:0000704 37313 37320 genetic +T891 http://purl.obolibrary.org/obo/MONDO_0003847 37313 37329 genetic diseases +T892 SO:0000704 37370 37374 gene +T893 NCBITaxon:33208 37415 37421 animal +T894 NCBITaxon:33208 37556 37562 animal +T895 NCBITaxon:9606 37660 37666 humans +T896 http://purl.obolibrary.org/obo/MONDO_0000001 37821 37828 disease +T897 NCBITaxon:9606 37882 37887 human +T898 UBERON:0000479 37957 37963 tissue +T899 http://purl.obolibrary.org/obo/MONDO_0017052 37983 38000 intermediate MSUD +T900 CHEBI:26948 38060 38067 thiamin +T901 CHEBI:26948 38071 38078 thiamin +T902 http://purl.obolibrary.org/obo/MONDO_0009563 38098 38102 MSUD +T903 CHEBI:26948 38211 38218 thiamin +T904 http://purl.obolibrary.org/obo/MONDO_0009563 38292 38296 MSUD +T905 CHEBI:26948 38327 38334 thiamin +T906 PR:000006300 38403 38405 E2 +T907 GO:0010467 38406 38416 expressing +T908 SO:0001023 38417 38423 allele +T909 http://purl.obolibrary.org/obo/MONDO_0017052 38471 38488 intermediate MSUD +T910 NCBITaxon:10088 38489 38494 mouse +T911 CHEBI:26948 38534 38541 thiamin +T912 UBERON:0000178 38577 38582 blood +T913 NCBITaxon:33208 38627 38633 animal +T914 GO:0032991 38700 38709 complexes +T915 http://purl.obolibrary.org/obo/MONDO_0017052 38729 38746 intermediate MSUD +T916 http://purl.obolibrary.org/obo/MONDO_0009563 38887 38891 MSUD +T917 GO:0005739 38964 38977 mitochondrial +T918 http://purl.obolibrary.org/obo/MONDO_0044970 38964 38987 mitochondrial disorders +T919 SO:0000704 39115 39126 genetically +T920 NCBITaxon:10088 39138 39143 mouse +T921 http://purl.obolibrary.org/obo/MONDO_0017051 39154 39161;39186 39190 classic MSUD +T922 http://purl.obolibrary.org/obo/MONDO_0017052 39173 39190 intermediate MSUD +T923 NCBITaxon:33208 39198 39205 animals +T924 http://purl.obolibrary.org/obo/MONDO_0009563 39272 39276 MSUD +T925 SO:0000704 39342 39346 gene +T926 GO:0008152 39397 39406 metabolic +T927 http://purl.obolibrary.org/obo/MONDO_0005066 39397 39414 metabolic disease +T928 http://purl.obolibrary.org/obo/MONDO_0009563 39460 39464 MSUD +T929 NCBITaxon:4022 39466 39471 Maple +T930 http://purl.obolibrary.org/obo/MONDO_0009563 39466 39491 Maple Syrup Urine Disease +T931 UBERON:0001088 39478 39483 Urine +T932 CHEBI:25754 39515 39523 ketoacid +T933 CHEBI:22918 39539 39543 BCAA +T934 CHEBI:22918 39545 39571 branched chain amino acids +T935 CHEBI:23995 39573 39576 ENU +T936 CHEBI:23995 39578 39599 N-ethyl-N-nitrosourea +T937 CL:0002322 39640 39647 ES cell +T938 UBERON:0000922 39649 39658 embryonic +T939 CL:0002322 39649 39668 embryonic stem cell +T940 CHEBI:27902 39675 39687 tetracycline +T941 NCBITaxon:10088 39713 39718 mouse +T942 UBERON:0000922 39719 39728 embryonic +T943 CL:0000057 39729 39739 fibroblast +T944 NCBITaxon:10088 39879 39884 mouse +T945 http://purl.obolibrary.org/obo/MONDO_0017052 40018 40035 intermediate MSUD +T946 NCBITaxon:10088 40078 40083 mouse +T947 PR:000006300 40666 40668 E2 +T948 UBERON:0001977 40673 40678 serum +T949 GO:0008152 40800 40809 Metabolic +T950 http://purl.obolibrary.org/obo/MONDO_0005066 40800 40817 Metabolic Disease +T951 http://purl.obolibrary.org/obo/MONDO_0009563 40832 40836 MSUD diff --git a/src/ontogpt/evaluation/craft/database/all/16579849.txt b/src/ontogpt/evaluation/craft/database/all/16579849.txt new file mode 100644 index 000000000..aa9f05e9d --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16579849.txt @@ -0,0 +1,189 @@ +Production and characterization of murine models of classic and intermediate maple syrup urine disease + +Abstract + +Background + +Maple Syrup Urine Disease (MSUD) is an inborn error of metabolism caused by a deficiency of branched-chain keto acid dehydrogenase. MSUD has several clinical phenotypes depending on the degree of enzyme deficiency. Current treatments are not satisfactory and require new approaches to combat this disease. A major hurdle in developing new treatments has been the lack of a suitable animal model. + +Methods + +To create a murine model of classic MSUD, we used gene targeting and embryonic stem cell technologies to create a mouse line that lacked a functional E2 subunit gene of branched-chain keto acid dehydrogenase. To create a murine model of intermediate MSUD, we used transgenic technology to express a human E2 cDNA on the knockout background. Mice of both models were characterized at the molecular, biochemical, and whole animal levels. + +Results + +By disrupting the E2 subunit gene of branched-chain keto acid dehydrogenase, we created a gene knockout mouse model of classic MSUD. The homozygous knockout mice lacked branched-chain keto acid dehydrogenase activity, E2 immunoreactivity, and had a 3-fold increase in circulating branched-chain amino acids. These metabolic derangements resulted in neonatal lethality. Transgenic expression of a human E2 cDNA in the liver of the E2 knockout animals produced a model of intermediate MSUD. Branched-chain keto acid dehydrogenase activity was 5–6% of normal and was sufficient to allow survival, but was insufficient to normalize circulating branched-chain amino acids levels, which were intermediate between wildtype and the classic MSUD mouse model. + +Conclusion + +These mice represent important animal models that closely approximate the phenotype of humans with the classic and intermediate forms of MSUD. These animals provide useful models to further characterize the pathogenesis of MSUD, as well as models to test novel therapeutic strategies, such as gene and cellular therapies, to treat this devastating metabolic disease. + +Background + +Maple Syrup Urine Disease (MSUD) is a genetic disorder caused by a deficiency of branched-chain keto acid dehydrogenase (BCKDH), a mitochondrial multienzyme complex responsible for the oxidative decarboxylation of branched-chain keto acids derived from branched-chain amino acids (BCAA), leucine, isoleucine and valine (for review, see: [1]). + +Patients with MSUD, depending on the mutation, show variable degrees of enzyme deficiency leading to several different clinical phenotypes [1]. Approximately 75% of MSUD patients have the classic form of the disease with BCKDH activity in the range of 0–2% of normal [1]. These patients show markedly elevated levels of BCAA in blood and other body fluids [1]. Besides the classic form, there are other variants of the disease. Patients with the intermediate form of the disease show BCKDH activity in the range of 3–30% of normal. In such patients the onset of the disease is delayed, but there are persistently elevated levels of BCAA [1]. Patients with the intermittent form of MSUD show BCKDH activity in the range of 5–20% and during the asymptomatic phase the blood BCAA levels are normal [1]. The overall incidence of MSUD in the general population is 1:185,000 [1], and in certain population groups, such as Mennonites of Pennsylvania, the incidence is estimated to be as high as 1:176 [2]. + +The BCKDH complex, the deficient enzyme in MSUD, consists of three catalytic proteins, a decarboxylase (E1), a dihydrolipoyl transacylase (E2), and a dihydrolipoyl dehydrogenase (E3). The E1 component is a heterotetramer composed of two α and two β subunits [3]. The E1 and E2 components are specific to BCKDH, whereas E3 is also used by pyruvate and α-ketoglutarate dehydrogenase complexes [3] and the glycine cleavage system [4]. BCKDH is also associated with two regulatory proteins, a specific kinase and a phosphatase which regulate the activity of this enzyme through a phosphorylation (inactivation) and dephosphorylation (activation) cycle of the E1 α subunit [5,6]. Mutations in the genes of the E1 and E2 subunits of BCKDH have been described, however, the majority of MSUD mutations identified thus far are in the E2 subunit [1,7]. To date, cases of MSUD have not been associated with defects in the regulatory kinase and phosphatase [1]. + +Current management of MSUD patients relies on a strict lifelong dietary restriction of protein or BCAA [1,8]. Such a dietary management of the disease, however, is not entirely satisfactory especially in times of metabolic decompensation due to infection, injuries and other stressors. In spite of dietary intervention, there is significant mortality associated with MSUD and there is a high incidence of mental retardation in survivors [9]. + +Because of the central role of the liver in amino acid metabolism and moderate/high levels of BCKDH activity in human liver [10-12], a few cases of MSUD have recently been treated by liver transplantation [13-16]. While the short-term outcome of liver transplantation is encouraging, long-term effects of this approach are not known. However, these patients are now required to take immunosuppressant drugs for the rest of their lives, often with undesirable side effects. Moreover, the cost associated with liver transplantation and the availability of donor livers are additional limiting factors for the practicality of treatment of this disease. + +Because of the current unsatisfactory options for the treatment of MSUD, there is a need for improved therapies to combat this disease. An obstacle to developing novel treatments for MSUD has been the lack of a suitable animal model to perform necessary preclinical studies. Although a Hereford calf model with MSUD has been described [17-19], this model is neither readily available nor practical to perform preclinical studies. Furthermore, comparison of this animal model with human MSUD has shown some differences in the pathology of the disease [1], making this animal a less desirable model for the human disease. + +Recently, a N-ethyl-N-nitrosourea (ENU)-induced mutant mouse that phenotypically resembles human MSUD has been described [20]. However, the mutation in this model disrupts a splice site in the mitochondrial branched-chain aminotransferase (BCAT) gene, not in BCKDH, the deficient enzyme in MSUD. Because the mutation is not in BCKDH, the validity of this mutant mouse line for modeling human MSUD is questionable. + +The objective of the present study was to create genetically engineered murine models of MSUD that mimic the pathology of the classic and intermediate variant forms of the human disease. The classic model was created by targeted inactivation of the E2 subunit of BCKDH by homologous recombination in embryonic stem (ES) cells. The model of intermediate MSUD was created by partial transgenic rescue of the E2 gene knockout. This report describes the generation and characterization of these murine models of MSUD. These models will allow for the development of novel treatment approaches, such as gene or stem cell therapies, to ultimately cure MSUD. + +Methods + +All studies involving animals were reviewed and approved by the University of Pittsburgh's Institutional Animal Care and Use Committee. + +Gene knockout production + +A genomic DNA subclone of Strain 129/SvJ DNA that contained a portion of Exons 4 and 5 of the E2 gene and flanking DNA was obtained from a P1 phage library from Genome Systems, Inc., (St. Louis, MO; '3-Hit Mouse ES Library'). A positive/negative replacement type gene targeting vector [21] was created by replacing a 1.67 kb EcoRV-Smal fragment that included a portion of Exon 4 and all of Exon 5 with the PGKneo marker gene (See Fig. 1A) from the pPNT vector [22]. The targeting construct was linearized with NotI and electroporated into R1 ES cells [23] under conditions described previously [24]. ES cells were selected with G418 (300 μg/ml; Life Technologies Inc., Gaithersburg, MD) and gancyclovir (2 μM; gift of Syntex, Palo Alto, CA). Doubly resistant clones were screened for gene targeting by Southern blot analysis of BglI digested genomic DNA. Blots were hybridized with an Exon 6 specific probe that was external to the gene targeting construct. Correctly targeted ES cells were injected into C57BL/6J blastocysts to produce chimeric mice using standard procedures. Heterozygous offspring from germline competent chimeras were intercrossed to produce wild type (+/+), heterozygous (+/-) and homozygous knockout (-/-) mice. At all generations, +/- breeding pairs were used. Results presented here are from mice derived from the F2+ generations. All animals were of a mixed C57BL/6J × Strain 129Sv/SvJ genetic background. + +Figure 1 + +E2 gene knockout mouse production. A, Gene targeting strategy used for targeting the E2 locus in mouse ES cells. The targeting construct was designed to delete 1.67 kb of sequence between an EcoRV site in Exon 4 and a Smal site in intron 5. The wild type E2 gene contains an ~16 kb BglI restriction fragment that hybridizes to the Exon 6 specific probe. A correctly targeted E2 locus harbors an ~11 kb BglI restriction fragment that hybridizes to the same probe. Note that the probe will not detect random integration of the targeting vector because it is external to the targeting vector. B, Southern blot analysis of Bgll digested genomic DNA derived from the parental wild type ES cell line (R1), a heterozygous targeted ES cell line (362), and from wild type (+/+), heterozygous (+/-) and homozygous knockout (-/-) mice. The blot was hybridized with an Exon 6 specific probe. C, Immunohistochemical analysis of fresh frozen liver sections from control (+/+) and E2 knockout (-/-) postnatal day 1 mouse pups. Sections were stained for E2 using an E2 specific antibody (green) and a nuclear stain (blue). Note the complete absence of E2 immunoreactivity in the section from the knockout mouse. D, Similar results were observed upon immunohistochemical analysis of primary mouse embryonic fibroblasts (MEFs). Note that the readily detectable signal for E2 in the control cells was present in a pattern characteristic of mitochondria, the subcellular location of BCKDH. + +Production of transgenic mice + +Standard molecular techniques were used to assemble the transgenic construct, pTRE-E2. This transgene contains the tetracycline responsive hCMV*-1 promoter [consisting of the tetracycline responsive element (TRE) and a minimal hCMV promoter] [25] from pTRE2 (Clontech Inc., Mt. View, CA), a chimeric intron from pCI (Promega) to increase message stability and expression [26,27], a Kozak consensus sequence at the initiation codon to optimize translation [28], the human E2 cDNA which has been modified to contain a 4× alanine linker followed by a c-myc epitope tag at the carboxy terminus to facilitate detection, and an SV40 late polyadenylation sequence from pCI for enhanced mRNA stability and translation. + +The 2.48 kb TRE-E2 transgene was purified from vector sequences following digestion with XhoI and BamHI and injected into C57BL/6J or C57BL/6J × Strain 129SvEv mouse embryos at the transgenic core facilities of the University of Pittsburgh and the University of Cincinnati, respectively. Genomic DNA from the tail of mice was screened by Southern blot analysis following digestion with EcoRI and hybridization to an ~400 bp probe derived from the SV40 portion of the TRE-E2 transgene. + +Production of intermediate MSUD murine model + +The various TRE-E2 transgenic lines produced were mated independently to mice that were heterozygous for both the E2 knockout and the LAP-tTA transgene [Tg(tTALap)Bjd/J; Stock 3272; The Jackson Laboratory, Bar Harbor, ME; NMRI × FVB × C57BL/6J background]. Interbreeding of animals that were heterozygous for both transgenes and the knockout resulted in the production of mice with a variety of genotypes including some animals that were homozygous for the knockout and were positive for both transgenes (we refer to this genotype as the "rescue" genotype). If our strategy for rescuing the neonatal lethal phenotype of the knockout were successful, then those homozygous knockout animals that had both transgenes would survive beyond the neonatal period. + +Genotyping + +All mice were genotyped by Southern blot analysis. Genomic DNAs prepared from tail snips were digested with an appropriate restriction enzyme, size fractionated by agarose gel electrophoresis, blotted to nylon, and probed using standard procedures. + +Immunohistochemistry + +Primary mouse embryonic fibroblasts were prepared from embryonic day ~16.5–18.5 fetuses as described [29]. Fibroblasts were passed onto glass, fixed in 2% paraformaldehyde in PBS for 10 minutes, permeabilized in 2% paraformaldehyde containing 0.1% Triton X100 for 10 minutes and washed three times in PBS containing 0.5% BSA and 0.15% glycine, pH 7.4 (Buffer A). Following a 30 min incubation with purified goat IgG (50 (μg/ml) at 25°C and three additional washes with Buffer A, cells were incubated for 60 min with E2-specific antiserum [30] at 1 μg/ml followed by three washes in Buffer A and 60 minute incubation in fluorescently labeled second antibody (1–2 μg/ml). The cells were then washed six times (5 min/wash) in Buffer A and then mounted in gelvatol (Monsanto, St Louis). When livers from newborn pups were examined, fixation was by immersion in 2% paraformaldehyde followed by cryoprotection and shock freezing in liquid nitrogen cooled isopentane and sectioning (5 microns). Otherwise processing was as for the cells above (without the fixation and permabilization steps). + +Amino acid analysis + +Blood was collected from the retroorbital sinus or tail vein of mice and spotted on a filter paper routinely used for blood amino acid analysis for prenatal screening. Concentrations of BCAA and other amino acids in blood were determined by tandem mass spectrometry (Pediatrix Screening, Bridgeville, PA). + +Assay of BCKDH activity + +Livers were removed, frozen in liquid nitrogen, and stored at -80°C. At the time of enzyme assay, livers were thawed, and homogenized (1:9, w/v) in 0.25 M sucrose, 10 mM Tris-HC1, pH 7.4. Liver homogenates were centrifuged at 600 × g for 10 min at 4°C and the supernatant fraction was saved to determine the BCKDH activity. The use of tissue homogenates was necessitated by the limited availability of liver tissue, particularly from newborn pups. BCKDH activity was determined by measuring the release of 14CO2 from α-keto [1-14C] isocaproate as described previously [31]. The complete reaction mixture contained (final volume 1 ml) 30 mM potassium phosphate buffer, pH 7.4, 0.20 mM α-ketoisocaproate, 0.40 mM CoASH, 0.40 mM thiamin pyrophosphate, 2 mM NAD+, 2 mM dithiothreitol, 5 mM Mg2+, approximately 250,000 DPM of α-keto [1-14C] isocaproate, and 0.10 ml of liver homogenate (2–3 mg protein). Assays were carried out for 15 min at 37°C, 14CO2 was trapped in hydroxide of Hyamine, and radioactivity was determined by liquid scintillation spectrometry. + +Western blot analysis + +Protein extracts were isolated from homogenized liver (freshly harvested and flash frozen) of wildtype, Line 525A, and Line A transgenic mice. Protein (25 μg per sample) was analyzed by electrophoresis on a 10% SDS-PAGE Ready Gel (Bio-Rad, Hercules, CA) and transferred to PVDF membrane (Sequi-Blot; Bio-Rad) via electroblotting. All blots were probed for E2 protein using polyclonal rabbit E2 antisera (1:5,000), which detects both mouse (~47 kD) and human (~54 kD) E2 subunits ([30]; gift from Dr. Susan Hutson, Wake Forest University). Blots were re-probed with a rabbit anti-c-myc tag antibody (1:10,000; abCam, Cambridge, MA; cat.# ab9106-100). Blots were also re-probed with an antibody for β-actin (43 kD; 1:10,000; abCam; cat.# ab8227-50) to allow for loading comparisons. A goat anti-rabbit secondary antibody conjugated to horseradish peroxidase (1:10,000; Novus, Littleton, CO; cat.# NB730-H) was used for detection using the Western Lightning chemiluminescence reagent (Perkin Elmer, Boston, MA) and exposed to X-ray film. + +Statistical analyses + +All BCAA and BCKDH enzyme activity data are presented as the mean +/- the standard error of the mean (S.E.M.). Differences between genotypes were compared by Student's t test[32]. + +Results + +Production of E2 gene knockout mice + +To create E2 knockout mice, we used gene targeting in mouse ES cells to disrupt the E2 gene. The overall strategy for disrupting the E2 gene is illustrated in Fig. 1A. The gene targeting construct was designed to replace a 1.67 kb EcoRV/Smal genomic DNA fragment encompassing part of Exon 4 and all of Exon 5 with the PGKneo selectable marker cassette. Of 522 ES cell clones screened for targeting by Southern blot analysis, 29 (5%) displayed the predicted restriction fragment length polymorphisms indicative of gene targeting at the E2 locus. As illustrated in Fig. 1A and 1B, an E2 Exon 6 specific probe, which is external to the targeting construct, hybridizes to only a ~16 kb BglI restriction fragment from the wild type allele in the parental R1 ES cell line. In correctly targeted ES cells, this probe also hybridizes to a ~11 kb BglI restriction fragment. Targeting was confirmed with several additional restriction enzymes and probes (data not shown). + +Correctly targeted ES cells were microinjected into blastocysts to produce chimeric mice. Chimerics were bred to C57BL/6J mice. Following germline transmission of the targeted allele, heterozygous mice were interbred to produce wild type (+/+), heterozygous (+/-) and homozygous (-/-) animals. Mice were genotyped by Southern blot analysis. The Exon 6 specific probe hybridized to only a ~16 kb BglI restriction fragment in +/+ mice, a ~11 kb BglI fragment in -/- mice, and to both of these fragments in +/- mice (Fig. 1B). + +Mice homozygous for the E2 mutation were born at the expected frequency. Genotype analysis of pups derived from +/- by +/- matings revealed that +/+, +/-, and -/- mice were present at nearly the expected 1:2:1 frequency. Of the initial 60 animals genotyped, 19 (32%) were +/+, 27 (45%) were +/-, and 14 (23%) were -/-. Thus, the E2 gene was dispensable for normal embryonic development. However, as expected, nearly all homozygous mice died in the perinatal period. Immediately following birth, homozygous pups were indistinguishable from their +/+ and +/- littermates; they were vigorous, active and able to suckle. By mid to late day on postnatal day one, most -/- pups became moribund and were readily identifiable as they were lethargic, pale, and exhibited gasping respiratory movements. With few exceptions, -/- pups died within 72 hours of birth. We have observed one rare -/- pup that survived to postnatal day 13. The reason for the prolonged survival of this pup is unknown. + +E2 deficient mice accurately model classic MSUD + +To demonstrate that the gene targeting event created a true E2 null allele, we determined BCKDH activity in liver homogenates of postnatal day 1 mouse pups derived from +/- by +/- mating pairs. As shown in Figure 2A, the BCKDH activity in +/+ mice was readily detectable. In marked contrast, BCKDH activity was completely absent in -/- mouse livers. As expected, homogenates from +/- mice had approximately half the activity of their +/+ littermates (Figure 2A). The results from -/- mice are similar to that observed in humans with classic MSUD [1]. + +Figure 2 + +Biochemical characterization of the classic MSUD murine model. A, BCKDH enzyme activity in liver of newborn wild type control (+/+), heterozygous (+/-), and homozygous (-/-) knockout mice. Enzyme activity was significantly reduced in +/- liver compared to +/+, and was below the level of detection in -/- liver. B, Total BCAA concentrations in blood of mice. Total BCAA represent the sum of leucine, isoleucine, and valine. Total BCAA concentrations in blood from -/- mice were significantly elevated compared to +/+ and +/-. C, Ratio of total BCAA to alanine in blood of mice. This ratio was significantly elevated in -/- mice compared to +/+ and +/- mice. The numbers on the bars indicates the number of mice analyzed. *, Significantly different from +/+ (P < 0.001); **, significantly different from +/+ and +/- (P < 0.001). + +Immunohistochemistry with an E2 specific antibody was used to examine E2 protein in the mice. As shown in Figure 1C and 1D, immunoreactive E2 protein was abundant in liver and embryonic fibroblasts of+/+ mice. In marked contrast, immunoreactive E2 protein was absent in these same tissues of -/- mice. + +Homozygous E2 knockout mice had a nearly 3-fold increase in blood (Figure 2B) and urine (data not shown) levels of BCAA (sum of leucine, isoleucine, and valine) as compared to their +/+ littermates. Because amino acids were analyzed by tandem mass spectrometry, the sum of BCAA shown in Figure 2B also may include alloisoleucine that may have been produced in -/- MSUD mice. + +The metabolism of BCAA is linked with the synthesis of alanine, glutamate, and glutamine [33]. Because of impaired metabolism of BCAA in MSUD mice, and to further characterize the abnormal biochemistry in this model, we analyzed the blood levels of the alanine, glutamate, and glutamine. As shown in Table 1, the levels of all three amino acids in homozygous mice were markedly lower than the levels in +/+ or +/- mice. The levels of these amino acids in the +/- mice were comparable to those in +/+ mice (Table 1). Because of the abnormal decrease in the blood alanine level and marked rise in blood BCAA levels that are characteristic of MSUD, a recent report on MSUD patients has suggested that the ratio of BCAA/alanine provides a more sensitive measure of the abnormal biochemistry of MSUD than BCAA level alone [8]. Therefore, we also expressed the amino acid results as BCAA/alanine ratio. As shown in Figure 2C, this ratio in -/- pups was more than 6-fold higher than +/+ or +/- littermates. These blood amino acid results are consistent with the concentrations seen in patients with MSUD [1,8,15]. + +Table 1 + +Summary of additional blood amino acid levels in the classic MSUD model and control littermates as determined by tandem mass spectrometry. All samples were collected on the day of birth. All values are mean +/- SEM. + +*: Significantly different from +/+ and +/- (P < 0.001). + +In summary, E2 knockout mice lack BCKDH enzymatic activity, E2 immunoreactivity, and have markedly elevated levels of BCAA in the blood and urine. These metabolic derangements ultimately result in neonatal lethality. These phenotypes are remarkably similar to that observed in humans with the classic form of MSUD. Thus, E2 knockout mice closely model classic MSUD. + +Knockout mice expressing a human E2 transgene model a variant form of MSUD that mimics intermediate MSUD + +To create a murine model of the intermediate variant form of MSUD, we used a transgenic strategy to rescue the severe elevation of BCAA and neonatal lethality that occurs in the classic MSUD mouse model. Our strategy was composed of a two part transgenic system to express human E2 in liver on the E2 knockout background. This bi-transgenic system consisted of a LAP-tTA transgene and a TRE-E2 transgene (Fig. 3A). The LAP-tTA transgene [34] directs high levels of liver specific expression of the tetracycline-controlled transactivator (tTA), a transcription factor that stimulates expression of promoters that harbor a transactivator response element (TRE). The TRE-E2 transgene was designed to express a human E2 cDNA from a TRE containing minimal promoter upon stimulation by tTA. + +Figure 3 + +Transgenic mouse production and characterization. A, Transgenic strategy used to produce mice that express human E2. LAP-tTA transgenic mice have been previously described [34]. These mice express the tetracycline-controlled transactivator (tTA) from the liver specific LAP promoter. The TRE-E2 transgene contains the tetracycline response element (TRE) as part of the promoter, a synthetic intron (thin line), the human E2 cDNA, an alanine spacer, a c-myc epitope tag, and SV40 derived polyadenylation sequence. This construct was used to create several lines of transgenic mice. B, Western blot analysis of E2 protein in liver of control and intermediate MSUD mice. Note that the amount of human E2 protein (predicted MW ~54 Kd) in mice from Lines A and 525 A was variable but in many of the animals the amount was similar to the amount of mouse E2 (MW=~47 Kd) in control animals. Re-probing with a c-myc tag antibody confirmed the presence of the transgene derived, c-myc tagged, human E2 in transgenic mice but not in controls. Western blot analysis of brain (C), kidney (D), and muscle (E) revealed negligible amounts of transgene derived E2 in those tissues. All blots were re-probed with an actin antibody to allow amount of protein loaded in each lane to be compared. + +LAP-tTA mice were previously produced and characterized by the Bujard laboratory [34]. Pronuclear microinjection was used to produce transgenic mice that harbored the TRE-E2 transgene. From injections at the University of Pittsburgh, 2 transgene positive founders were identified that led to the generation of 3 different transgenic lines (Lines A, B, & D). From the injections at the University of Cincinnati, we obtained 8 transgene positive founders. Subsequent breeding of these founders revealed that many of the animals had multiple transgene insertion sites that segregated. We established a total of 15 different transgenic lines from these founders. Limited resources allowed us to only focus on a total of 9 of these lines. These lines differed substantially in transgene copy number as compared by Southern blot analysis of tail DNA (data not shown). + +Transgenic TRE-E2 mice from each line (either founders or F1 offspring) were crossed to mice that were positive for the LAP-tTA transgene and were heterozygous for the E2 knockout. Ultimately, breeding pairs were used in which the mice were heterozygous for the LAP-tTA transgene, the TRE-E2 transgene, and the E2 knockout. To efficiently screen for the ability of the transgenes to rescue the knockout from neonatal lethality, we genotyped litters at weaning. This breeding strategy is expected to result in a theoretical maximum of animals with the rescue genotype (i.e., homozygous E2 knockout and positive for both transgenes) of 14%. Fig. 4A shows the percentage of mice alive at weaning with the rescue genotype from each transgenic line tested. Considerable variability was observed between lines. The line with the highest percentage of rescue mice at weaning was line 525 A. Approximately 10% of weaned, surviving pups from this line had the rescue genotype. Thus, the 525A transgene appears to be highly effective at rescuing the neonatal lethal phenotype of the E2 knockout. In contrast, line 520B completely failed to rescue the neonatal lethal phenotype. All other lines tested produced surviving rescue animals at a frequency between 1 and 5% of weaned pups. + +Figure 4 + +Characterization of the intermediate MSUD murine model. A, Survival analysis of transgenic rescue lines. Presented are percentages of mice alive at weaning from each transgenic line tested that had the rescue genotype (i.e., homozygous for knockout of endogenous E2 and positive for both the LAP-tTA and TRE-E2 transgenes). Also plotted is the theoretical maximum frequency at which this genotype is expected in this population of animals. The numbers on the bars indicates the numbers of observations for each line. B, Ratio of total BCAA to alanine in blood of mice from controls and Lines A and 525A. Total BCAA represent the sum of leucine, isoleucine, and valine. BCAA/alanine values were significantly greater for Lines A and 525A compared to controls at all ages tested. The numbers on the bars indicates the numbers of samples analyzed. *, P ≤ 0.01; **, P < 0.005. C, BCKDH enzyme activity in liver of control and Lines A and 525A mice. *, P ≤ 0.001; **, P ≤ 0.0001. D, Survival curves for mice from Lines A and 525A. Rescue mice alive at weaning were monitored until they were moribund and subsequently sacrificed or were found dead in their cages. + +Lines A and 525A were selected for detailed characterization. Fig. 4B shows the results of blood amino acid analysis presented as a ratio of total BCAA to alanine. The BCAA/alanine values for mice from both lines were significantly elevated compared to controls for all of the time points analyzed. Note that the BCAA/alanine values for the transgenic mice are intermediate between controls and knockouts (see Figure 2C). Blood levels of alanine, glutamate, and glutamine were reduced in Line A mice, as was glutamate in Line 525A mice, compared to controls (Table 2). + +Table 2 + +Summary of additional blood amino acid levels in the intermediate MSUD model and littermate controls as determined by tandem mass spectrometry. Samples were collected from mice that were 4–6 weeks of age. All values are mean +/- SEM. + +*: Significantly different from control (P < 0.05). + +**: Significantly different from control (P < 0.01). + +***: Significantly different from Line 525A (P < 0.05). + +Because the LAP-tTA mice that were used to drive expression of transgenic human E2 have been demonstrated to produce liver specific expression [34], BCKDH enzyme activity and production of human E2 protein in liver of Lines A and 525A was examined. BCKDH enzymatic activity in liver from Lines A and 525A was ~6 and 5%, respectively, of the enzymatic activity present in control liver (Fig. 4C). As shown in Fig. 3B, the amount of human E2 (predicted MW~54 Kd) in these transgenic mice was quite variable between mice. In some of these transgenic mice, the level of human E2 was approximately equal to the amount of mouse E2 (~47 Kd) produced in liver of nontransgenic control mice. The observations that these near normal amounts of E2 protein result in only ~5–6% of normal BCKDH enzyme activity suggest that transgene derived E2 is functioning at a suboptimal level. It is probable that the c-myc tag at the carboxy terminus of the transgene derived human E2 interfered with enzymatic activity. This interpretation is consistent with previous studies which have revealed that the carboxy terminus of an analogous subunit of the pyruvate dehydrogenase complex is essential for subunit interactions [35] and insertion of a Hisx6 tag on the carboxy terminus of an analogous E. coli subunit interfered with normal subunit assembly [36]. It is also possible that human E2 was not fully functional when complexed with mouse E1 and E3 subunits. Human E2 shares ~88% identity to mouse E2 at the amino acid level. We also used western blot analysis to analyze expression of the E2 transgene in brain, kidney and muscle. As shown in Figure 3C, D and 3E respectively, expression of E2 is negligible in those tissues. + +Long-term survival of the mice from these two transgenic lines that survived beyond weaning is plotted in Fig. 4D. Although survival data for control animals was not collected, it is readily apparent that survival of the rescue mice was compromised. By 16 weeks, all mice of Line A were moribund and humanely sacrificed, or were found dead in their cage. Survival of Line 525A appeared to be somewhat better. At 20 weeks of age, ~12% of mice (2 of 16) were still alive. These two rare survivors died at 40 and 60 weeks of age. + +In summary, these surviving mice had BCAA/alanine ratios that were intermediate between controls and knockouts and they expressed ~5–6% of normal BCKDH enzyme activity in the liver. These phenotypic observations are remarkably similar to the clinical phenotype observed in MSUD patients with the intermediate form of the disease [1]. Thus, these rescue mice represent a very useful model of the intermediate MSUD phenotype. + +Discussion + +A major hurdle in developing new treatments for MSUD has been the lack of a practical, accurate animal model of the disease. This hurdle has now been overcome. In this report, we describe the development and characterization of two genetically engineered mouse models that are phenotypically very similar to MSUD patients with the classic and intermediate forms of the disease. + +Our model of classic MSUD was created by gene knockout of the E2 subunit of BCKDH. The phenotype of these knockout animals is strikingly similar to humans with classic MSUD. Knockout mice were born at the expected frequency and appeared normal at birth. Within a day of birth and following suckling, blood levels of BCAA were markedly elevated. Concomitantly, levels of the amino acids alanine, glutamate, and glutamine, whose synthesis is linked with normal metabolism of BCAA, were decreased (Table 1). The activity of BCKDH in livers of homozygous knockout mouse pups was undetectable, accounting for the accumulation of unmetabolized BCAA. Immunoreactive E2 protein was absent in liver and fibroblasts of homozygous pups. The phenotypic behavior of homozygous mouse pups resembles symptoms seen in newborn classic MSUD patients. These include signs of neurologic dysfunction such as seizures, stupor, lethargy, loss of motor activity, and respiratory difficulties [1]. These neurologic symptoms may result from reduced levels of glutamate, glutamine, alanine, and other similar neuroactive amino acids, which are considered the culprit for MSUD encephalopathies in patients [1,37]. Finally, nearly all of the homozygous pups died within 72 hours of birth. This neonatal lethality is likely due to the accumulation of BCAA to neurotoxic levels, ketoacidosis, brain edema, dehydration, and malnutrition as observed in the MSUD calf [17] and in classic MSUD patients [1]. Heterozygous knockouts were normal and had normal levels of BCAA despite having approximately half of BCKDH enzymatic activity. From the characterization studies completed thus far, the null mutation mouse accurately represents a model of classic MSUD and appears to be a faithful model of the human disease with respect to several biochemical phenotypes [1]. + +To create a model of intermediate MSUD, we used a transgenic strategy to express human E2 in the liver of E2 knockout mice. As mentioned above, E2 knockouts without transgene derived E2 die during the early neonatal period. We show that expression of a human E2 transgene in the liver of these knockout mice is able to rescue the neonatal lethality. Many of the rescue mice survived to adulthood. It is interesting to note that in these mouse lines only ~5–6% of normal BCKDH activity in the liver was sufficient to allow survival. We also demonstrated that BCAA levels in blood of these rescue mice were intermediate between controls and knockouts. The hallmarks of intermediate MSUD human patients are persistently increased levels of BCAA and BCKDH activity in the range of 3–30% of normal [1]. Because of the phenotypic similarities of the rescue mice to the human patients, these genetically engineered mice represent a useful small animal model of the intermediate form of MSUD. + +The transgenic approach that was used to create the intermediate MSUD model was based on the tetracycline regulated gene switch system that has been used with great success in other studies, for example [34]. The strategy behind our approach was to create mice with an intermediate MSUD phenotype that could be converted to the classic phenotype at the investigator's discretion by turning off the rescuing transgene. However, we were unable to consistently turn off the human E2 expression cassette by the potent tetracycline analogue, doxycycline in either of the two transgenic lines tested (data not shown). The reason these two lines were unresponsive to doxycycline is unknown. It is conceivable that the integration site of the transgenes was not permissive for regulated expression. Screening of additional lines may be needed to find lines that allow for survival and can also be regulated. + +The models described in this communication are important advances over the previously described models. An ENU mouse resembling human MSUD was previously created by disrupting the mitochondrial BCAT gene [20]. The BCKDH activity in the liver and muscle of this mouse was normal even though the blood BCAA levels were markedly elevated. While this is an interesting model, it is not a true model of MSUD because the mutation is not in BCKDH and the levels of BCKDH are normal. Furthermore, no case of MSUD has been described attributing this disease to BCAT deficiency. In addition to this mouse model, a cow model of MSUD has also been previously described [17-19]. However, due to practical constraints imposed by such a large animal model and due to the observation of differences between the MSUD cow model and MSUD humans [1], this model is also of limited utility. In contrast, the murine models described in this report are more valid and appropriate for modeling human MSUD. + +The most significant opportunity presented by the MSUD mouse models is to test novel treatments such as gene [38-41] and cell based therapies (e.g., hepatocytes [42,43] or embryonic stem cells [44]). Relevant to gene therapy, recent problems with gene therapy in humans highlight the importance and critical need of animal models of genetic diseases for preclinical studies. The testing of gene and cell based therapies on appropriate animal model systems provides a preliminary method of establishing not only the efficacy, but also short- and long-term safety. Success with animal studies is expected to advance such therapeutic approaches and could pave the way for studies in humans. In addition to testing various treatments, these models should also be very useful for investigating the underlying pathophysiologic consequences of the disease. Such studies are very difficult/impossible to do in human patients for obvious ethical reasons, and often must rely on autopsy tissue. Additionally, the intermediate MSUD model may also be useful for studies to test the effect of thiamin. A thiamin-responsive form of MSUD has been described [1]. The phenotype of these patients is heterogeneous and treatment with a wide range of thiamin doses has produced limited success [1]. Recent cell culture studies with MSUD cells have suggested that the thiamin-responsive phenotype is dependent upon the presence of at least one E2 expressing allele [45,46]. In light of these newer findings, our intermediate MSUD mouse provides a model to test the effect of thiamin with respect to BCKDH activity and blood amino acid levels at the level of the whole animal. Because of recent interest in structural analysis of multienzyme complexes [35,36,45,46], the intermediate MSUD model may provide a valuable resource for studies of structural biology. Lastly, the information and knowledge gained from studies with the MSUD models described here will also be applicable and transferable to other mitochondrial disorders due to defects in multisubunit enzymes. + +Conclusion + +In summary, this report describes the development and characterization of genetically engineered mouse models of classic as well as intermediate MSUD. These animals provide useful models to further characterize the pathogenesis of MSUD, as well as models to test novel therapeutic strategies, such as gene and cellular therapies, to treat this devastating metabolic disease. + +Abbreviations + +The abbreviations used are: MSUD, Maple Syrup Urine Disease; BCKDH, branched chain ketoacid dehydrogenase; BCAA, branched chain amino acids; ENU, N-ethyl-N-nitrosourea; BCAT, branched chain aminotransferase; ES cell, embryonic stem cell; TRE, tetracycline responsive element; MEF, mouse embryonic fibroblast. + +Competing interests + +The author(s) declare that they have no competing interests. + +Authors' contributions + +GEH designed and produced the mouse models, coordinated experiments, interpreted data, and helped draft the manuscript. KS conducted much of the characterization of the intermediate MSUD model. CF assisted with production of the mouse models, data collection, analysis, and interpretation. SW directed the immunohistochemistry experiments. HSP conceived of the knockout model, interpreted data and drafted the manuscript. All authors contributed to composing and editing the manuscript and have read and approved the final version. + +Pre-publication history + +The pre-publication history for this paper can be accessed here: + + + +Acknowledgements + +The authors would like to thank Frank Kist, Jodi Dagget, Edward Mallick, Brian Sloat, and Judith Rodda for expert technical assistance, and Dr. Susan Hutson for the gift of E2 antiserum. This work was supported by National Institutes of Health Grants DK51960, DK57386, DK57956, AA10422, the Scott C. Foster Metabolic Disease Fund, and the MSUD Family Support Group. diff --git a/src/ontogpt/evaluation/craft/database/all/16611361.ann b/src/ontogpt/evaluation/craft/database/all/16611361.ann new file mode 100644 index 000000000..23b385aa9 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16611361.ann @@ -0,0 +1,570 @@ +T1 NCBITaxon:10088 14 18 mice +T2 SO:0001023 61 67 allele +T3 PR:000007861 71 76 Gata6 +T4 PR:000007861 125 130 GATA6 +T5 GO:0048513 174 188;197 203 development of ... organs +T6 UBERON:0000062 197 203 organs +T7 UBERON:0002107 218 223 liver +T8 UBERON:0001555 225 247 gastrointestinal tract +T9 UBERON:0000948 252 257 heart +T10 PR:000007861 300 305 GATA6 +T11 GO:0048513 313 326 organogenesis +T12 PR:000007861 354 359 Gata6 +T13 NCBITaxon:10088 363 367 mice +T14 GO:0007369 391 403 gastrulation +T15 UBERON:0008945 422 445 extraembryonic endoderm +T16 NCBITaxon:10088 486 491 mouse +T17 SO:0001023 539 545 allele +T18 PR:000007861 549 554 Gata6 +T19 SO:0000346 565 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+T48 GO:0065007 1312 1322 regulating +T49 UBERON:0000922 1323 1332 embryonic +T50 GO:0009790 1323 1344 embryonic development +T51 NCBITaxon:40674 1368 1377 mammalian +T52 NCBITaxon:10088 1407 1412 mouse +T53 PR:000007861 1413 1418 Gata6 +T54 SO:0000704 1419 1423 gene +T55 SO:0000417 1504 1511 domains +T56 PR:000007859 1625 1630 GATA4 +T57 GO:0048513 1706 1720;1729 1735 development of ... organs +T58 UBERON:0000062 1729 1735 organs +T59 UBERON:0000948 1750 1755 heart +T60 UBERON:0002048 1757 1761 lung +T61 UBERON:0001555 1763 1785 gastrointestinal tract +T62 UBERON:0002107 1790 1795 liver +T63 GO:0009790 1803 1817;1829 1836 Development of ... embryos +T64 PR:000007861 1818 1823 GATA6 +T65 UBERON:0000922 1829 1836 embryos +T66 GO:0007369 1852 1864 gastrulation +T67 UBERON:0008945 1896 1919 extraembryonic endoderm +T68 UBERON:0019248 1941 1956 early embryonic +T69 PR:000007861 2003 2008 GATA6 +T70 UBERON:0000922 2014 2021 embryos +T71 UBERON:0008945 2039 2053;2063 2071 extraembryonic ... endoderm +T72 UBERON:0004877 2054 2071 visceral endoderm +T73 UBERON:0000922 2089 2095 embryo +T74 UBERON:0000922 2117 2124 embryos +T75 PR:000007861 2195 2200 Gata6 +T76 CL:0002322 2203 2210 ES cell +T77 UBERON:0000922 2219 2226 embryos +T78 GO:0001889 2251 2264 hepatogenesis +T79 PR:000007861 2299 2304 GATA6 +T80 UBERON:0000922 2379 2386 embryos +T81 PR:000007861 2392 2397 Gata6 +T82 CL:0002322 2400 2408 ES cells +T83 UBERON:0000922 2423 2429 embryo +T84 PR:000007861 2502 2507 GATA6 +T85 GO:0009790 2521 2534 embryogenesis +T86 PR:000007861 2594 2599 GATA6 +T87 GO:0065007 2646 2657 controlling +T88 NCBITaxon:10088 2751 2755 mice +T89 SO:0001023 2786 2792 allele +T90 PR:000007861 2796 2801 Gata6 +T91 PR:000007861 2853 2858 GATA6 +T92 PR:000007861 2944 2949 GATA6 +T93 SO:0000357 2972 2977 flank +T94 PR:000007861 2978 2983 Gata6 +T95 SO:0000147 2984 2988 exon +T96 SO:0000346 3052 3065 loxP elements +T97 SO:0000147 3079 3083 exon +T98 PR:000007861 3112 3117 GATA6 +T99 SO:0000346 3225 3238 loxP elements +T100 SO:0001644 3301 3317 targeting vector +T101 SO:0000346 3330 3342 loxP element +T102 SO:0000147 3359 3364 exons +T103 SO:0005853 3389 3397 cassette +T104 SO:0000346 3411 3423 loxP element +T105 CHEBI:7507 3450 3458 neomycin +T106 SO:0000704 3484 3488 gene +T107 CHEBI:42768 3520 3524 G418 +T108 SO:0000147 3549 3554 exons +T109 SO:0000350 3574 3583 FRT sites +T110 SO:0000357 3584 3591 flanked +T111 SO:0000704 3600 3604 gene +T112 PR:P03870 3642 3645 Flp +T113 SO:0000061 3669 3708 restriction endonuclease cleavage sites +T114 MOP:0000780 3694 3702 cleavage +T115 PR:000007861 3756 3761 Gata6 +T116 SO:0001644 3762 3778 targeting vector +T117 PR:000007861 3829 3834 Gata6 +T118 SO:0001023 3835 3842 alleles +T119 SO:0001644 3874 3890 targeting vector +T120 CL:0002322 3914 3922 ES cells +T121 CHEBI:42768 3951 3955 G418 +T122 CL:0002322 3966 3973 ES cell +T123 PR:000007861 4029 4034 Gata6 +T124 CL:0002322 4117 4119;4145 4149 ES ... cell +T125 PR:000007861 4121 4126 Gata6 +T126 SO:0000346 4126 4130 loxP +T127 CL:0002322 4179 4186 ES cell +T128 SO:0001026 4187 4194 genomic +T129 SO:0001644 4273 4289 targeting vector +T130 PR:000007861 4330 4335 Gata6 +T131 SO:0000346 4570 4579 loxP site +T132 SO:0005853 4706 4714 cassette +T133 PR:000007861 4724 4729 Gata6 +T134 CL:0002322 4761 4768 ES cell +T135 NCBITaxon:10088 4852 4856 mice +T136 UBERON:0000085 4860 4866 morula +T137 NCBITaxon:33208 4900 4907 animals +T138 GO:0007618 4918 4923 mated +T139 NCBITaxon:10088 4934 4938 mice +T140 PR:000007861 4983 4988 Gata6 +T141 SO:0000346 4988 4992 loxP +T142 SO:0001023 5004 5010 allele +T143 UBERON:0002415 5054 5058 tail +T144 NCBITaxon:10088 5074 5078 Mice +T145 PR:000007861 5097 5102 Gata6 +T146 SO:0000346 5102 5106 loxP +T147 SO:0001023 5120 5126 allele +T148 NCBITaxon:10088 5186 5190 mice +T149 SO:0001023 5221 5227 allele +T150 SO:0005853 5275 5283 cassette +T151 PR:000007861 5294 5299 GATA6 +T152 UBERON:0000922 5325 5334 embryonic +T153 PR:000007861 5361 5366 Gata6 +T154 UBERON:0000922 5370 5377 embryos +T155 NCBITaxon:10088 5412 5417 mouse +T156 PR:000007861 5424 5429 Gata6 +T157 SO:0000346 5429 5433 loxP +T158 SO:0005853 5457 5465 cassette +T159 SO:0000350 5504 5513 FRT sites +T160 GO:0007618 5549 5555 mating +T161 PR:000007861 5556 5561 Gata6 +T162 SO:0000346 5561 5565 loxP +T163 NCBITaxon:10088 5579 5583 mice +T164 NCBITaxon:10088 5600 5605 mouse +T165 PR:P03870 5645 5648 Flp +T166 GO:0010467 5671 5680 expressed +T167 NCBITaxon:9606 5690 5695 human +T168 PR:000003676 5696 5706 beta actin +T169 SO:0000704 5707 5711 gene +T170 SO:0000167 5712 5720 promoter +T171 SO:0005853 5755 5763 cassette +T172 PR:000007861 5840 5845 Gata6 +T173 SO:0000346 5845 5849 loxP +T174 NCBITaxon:10088 5852 5856 mice +T175 PR:000007861 5892 5897 Gata6 +T176 SO:0000346 5897 5901 loxP +T177 SO:0000346 5902 5906 loxP +T178 PR:000007861 5993 5998 Gata6 +T179 SO:0001023 6016 6022 allele +T180 PR:000007861 6059 6064 Gata6 +T181 SO:0001026 6065 6072 genomic +T182 SO:0001644 6087 6103 targeting vector +T183 SO:0000147 6109 6114 exons +T184 SO:0000112 6201 6208 primers +T185 SO:0000346 6229 6233;6269 6274 loxP ... sites +T186 SO:0000350 6257 6260;6269 6274 FRT ... sites +T187 SO:0005853 6287 6296 cassettes +T188 CHEBI:7507 6306 6314 neomycin +T189 http://purl.obolibrary.org/obo/MONDO_0005504 6344 6354 diphtheria +T190 CHEBI:27026 6355 6360 toxin +T191 SO:0000412 6433 6467 restriction endonuclease fragments +T192 SO:0001026 6532 6539 genomic +T193 CL:0002322 6558 6566 ES cells +T194 NCBITaxon:10088 6592 6597 mouse +T195 UBERON:0002415 6598 6603 tails +T196 CL:0002322 6641 6648 ES cell +T197 SO:0000346 6691 6695 loxP +T198 SO:0001023 6707 6713 allele +T199 NCBITaxon:10088 6741 6745 Mice +T200 PR:000007861 6770 6775 Gata6 +T201 SO:0001023 6776 6782 allele +T202 CL:0002322 6809 6817 ES cells +T203 SO:0000147 6836 6840 Exon +T204 PR:000007861 6846 6851 Gata6 +T205 PR:000007861 6865 6870 Gata6 +T206 SO:0005853 6886 6894 cassette +T207 PR:000007861 6922 6927 Gata6 +T208 SO:0000147 6928 6932 exon +T209 SO:0000357 6935 6942 flanked +T210 SO:0000346 6946 6959 loxP elements +T211 PR:000007861 6961 6966 Gata6 +T212 SO:0000346 6966 6970 loxP +T213 PR:000007861 6985 6990 Gata6 +T214 SO:0000346 6990 6994 loxP +T215 NCBITaxon:10088 7006 7010 mice +T216 NCBITaxon:10088 7025 7029 mice +T217 GO:0010467 7030 7040 expressing +T218 PR:P03870 7070 7073 Flp +T219 SO:0000412 7123 7144 restriction fragments +T220 NCBITaxon:10088 7275 7279 mice +T221 UBERON:0000922 7284 7291 embryos +T222 GO:0006277 7320 7336;7345 7348 amplification of ... DNA +T223 SO:0001026 7337 7344 genomic +T224 SO:0000112 7350 7357 Primers +T225 PR:000007861 7404 7409 Gata6 +T226 SO:0000028 7425 7427 bp +T227 SO:0000346 7434 7438 loxP +T228 SO:0000028 7453 7455 bp +T229 PR:000007861 7461 7466 Gata6 +T230 SO:0000028 7485 7487 bp +T231 SO:0001023 7489 7496 alleles +T232 SO:0000028 7527 7529 bp +T233 PR:P03870 7532 7535 flp +T234 PR:P03870 7537 7540 Flp +T235 SO:0001030 7541 7542 F +T236 SO:0001031 7543 7544 R +T237 SO:0000028 7550 7552 bp +T238 SO:0000028 7577 7579 bp +T239 SO:0000902 7581 7591 transgenes +T240 PR:000007861 7631 7636 GATA6 +T241 GO:0010467 7665 7675 expression +T242 PR:000007861 7720 7725 Gata6 +T243 SO:0000704 7726 7730 gene +T244 UBERON:0001555 7738 7760 gastrointestinal tract +T245 GO:0010467 7782 7791 expressed +T246 UBERON:0000483 7799 7809 epithelium +T247 GO:0065007 7832 7843 controlling +T248 UBERON:0001555 7844 7847 gut +T249 GO:0048565 7844 7847;7862 7873 gut ... development +T250 PR:000007861 7886 7891 Gata6 +T251 SO:0000346 7891 7895 loxP +T252 PR:000017296 7897 7903 Villin +T253 NCBITaxon:10088 7908 7912 mice +T254 PR:000007861 7922 7927 Gata6 +T255 SO:0000346 7927 7931 loxP +T256 SO:0000346 7932 7936 loxP +T257 NCBITaxon:10088 7937 7941 mice +T258 NCBITaxon:10088 7958 7962 mice +T259 PR:000017296 7967 7970 Vil +T260 GO:0010467 7996 8006 expression +T261 UBERON:0000483 8027 8037 epithelial +T262 CL:0002254 8027 8046;8051 8066 epithelial cells of ... small intestine +T263 UBERON:0002108 8051 8066 small intestine +T264 PR:000017296 8074 8080 Villin +T265 PR:000017296 8082 8086 Vil1 +T266 SO:0000167 8088 8096 promoter +T267 NCBITaxon:10088 8135 8139 mice +T268 UBERON:0001902 8177 8190;8195 8210 epithelium of ... small intestine +T269 PR:000007861 8228 8233 Gata6 +T270 SO:0000346 8233 8237 loxP +T271 PR:000017296 8239 8245 Villin +T272 GO:0007618 8261 8266 mated +T273 PR:000007861 8272 8277 Gata6 +T274 SO:0000346 8277 8281 loxP +T275 SO:0000346 8282 8286 loxP +T276 PR:000007861 8305 8310 Gata6 +T277 SO:0000346 8310 8314 loxP +T278 PR:000017296 8316 8322 Villin +T279 PR:000007861 8339 8344 Gata6 +T280 SO:0000346 8344 8348 loxP +T281 SO:0000346 8349 8353 loxP +T282 PR:000017296 8353 8359 Villin +T283 SO:0000346 8494 8504 loxP sites +T284 NCBITaxon:10088 8546 8550 mice +T285 PR:000007861 8605 8610 Gata6 +T286 SO:0000346 8610 8614 loxP +T287 PR:000017296 8616 8622 Villin +T288 PR:000007861 8701 8706 Gata6 +T289 SO:0000346 8706 8710 loxP +T290 SO:0000346 8711 8715 loxP +T291 PR:000017296 8715 8721 Villin +T292 NCBITaxon:10088 8744 8748 mice +T293 PR:000007861 8816 8821 GATA6 +T294 UBERON:0000160 8829 8838 intestine +T295 PR:000007861 8886 8891 Gata6 +T296 SO:0000346 8891 8895 loxP +T297 PR:000017296 8897 8903 Villin +T298 PR:000007861 8979 8984 Gata6 +T299 SO:0000346 8984 8988 loxP +T300 SO:0000346 8989 8993 loxP +T301 PR:000017296 8993 8999 Villin +T302 UBERON:0002415 9023 9027 tail +T303 PR:000007861 9065 9070 Gata6 +T304 SO:0000112 9071 9078 primers +T305 PR:000007861 9169 9174 Gata6 +T306 SO:0000346 9175 9188 loxP elements +T307 PR:000007861 9228 9233 Gata6 +T308 UBERON:0000160 9242 9252 intestines +T309 NCBITaxon:10088 9292 9296 mice +T310 PR:000007861 9326 9331 Gata6 +T311 SO:0000346 9331 9335 loxP +T312 PR:000017296 9337 9343 Villin +T313 PR:000007861 9373 9378 Gata6 +T314 UBERON:0000160 9413 9422 intestine +T315 UBERON:0000160 9434 9443 intestine +T316 PR:000007861 9470 9475 Gata6 +T317 SO:0000346 9475 9479 loxP +T318 SO:0000346 9480 9484 loxP +T319 PR:000017296 9484 9490 Villin +T320 PR:000007861 9520 9525 Gata6 +T321 SO:0000346 9525 9529 loxP +T322 SO:0000346 9530 9534 loxP +T323 PR:000017296 9534 9540 Villin +T324 NCBITaxon:10088 9545 9549 mice +T325 GO:0010467 9576 9583 express +T326 PR:000007861 9584 9589 Gata6 +T327 GO:0010467 9648 9658 expression +T328 PR:000007861 9662 9667 GATA6 +T329 UBERON:0000160 9683 9693 intestines +T330 PR:000007861 9697 9702 Gata6 +T331 SO:0000346 9702 9706 loxP +T332 SO:0000346 9707 9711 loxP +T333 PR:000017296 9711 9717 Villin +T334 PR:000007861 9754 9759 Gata6 +T335 SO:0000346 9759 9763 loxP +T336 PR:000017296 9765 9771 Villin +T337 PR:000007861 9801 9806 GATA6 +T338 GO:0005634 9835 9842 nuclear +T339 UBERON:0000483 9858 9868 epithelial +T340 CL:0002563 9858 9877;9886 9896 epithelial cells of ... intestines +T341 UBERON:0000160 9886 9896 intestines +T342 PR:000007861 9907 9912 Gata6 +T343 SO:0000346 9912 9916 loxP +T344 SO:0000346 9917 9921 loxP +T345 PR:000017296 9922 9928 Villin +T346 NCBITaxon:10088 9933 9937 mice +T347 UBERON:0000160 9987 9997 intestinal +T348 PR:000007861 9998 10003 GATA6 +T349 PR:000007861 10056 10061 Gata6 +T350 SO:0000704 10062 10066 gene +T351 PR:000007861 10101 10106 Gata6 +T352 SO:0000346 10106 10110 loxP +T353 SO:0000346 10111 10115 loxP +T354 NCBITaxon:10088 10116 10120 mice +T355 GO:0010467 10143 10153 expression +T356 PR:000007861 10185 10190 Gata6 +T357 UBERON:0000160 10226 10235 intestine +T358 PR:000007861 10239 10244 Gata6 +T359 SO:0000346 10244 10248 loxP +T360 SO:0000346 10249 10253 loxP +T361 NCBITaxon:10088 10254 10258 mice +T362 GO:0010467 10262 10272 expression +T363 PR:000017296 10289 10295 Villin +T364 SO:0000167 10296 10304 promoter +T365 PR:000007861 10336 10341 Gata6 +T366 UBERON:0000160 10363 10373 intestines +T367 PR:000007861 10401 10406 Gata6 +T368 SO:0000346 10406 10410 loxP +T369 PR:000017296 10412 10418 Villin +T370 PR:000007861 10451 10456 Gata6 +T371 SO:0000346 10456 10460 loxP +T372 SO:0000346 10461 10465 loxP +T373 PR:000017296 10465 10471 Villin +T374 NCBITaxon:10088 10488 10492 mice +T375 PR:000012987 10517 10523 Polr2a +T376 PR:000007861 10648 10653 GATA6 +T377 GO:0005634 10682 10689 nuclear +T378 UBERON:0000483 10701 10711 epithelial +T379 CL:0002563 10701 10717;10727 10729;10738 10748 epithelial cells ... of ... intestines +T380 UBERON:0000160 10738 10748 intestines +T381 PR:000007861 10796 10801 Gata6 +T382 SO:0000346 10801 10805 loxP +T383 SO:0000346 10806 10810 loxP +T384 PR:000017296 10810 10816 Villin +T385 UBERON:0000160 10821 10831 intestines +T386 PR:000007861 10868 10873 Gata6 +T387 SO:0000346 10873 10877 loxP +T388 SO:0000346 10878 10882 loxP +T389 NCBITaxon:10088 10883 10887 mice +T390 PR:000007861 10923 10928 GATA6 +T391 UBERON:0000922 10975 10982 embryos +T392 SO:0001023 11010 11016 allele +T393 PR:000007861 11018 11023 Gata6 +T394 PR:000007861 11086 11091 Gata6 +T395 UBERON:0000922 11095 11102 embryos +T396 PR:000007861 11110 11115 Gata6 +T397 NCBITaxon:10088 11121 11125 mice +T398 GO:0007618 11143 11149 mating +T399 PR:000007861 11150 11155 Gata6 +T400 SO:0000346 11155 11159 loxP +T401 NCBITaxon:10088 11173 11177 mice +T402 NCBITaxon:10088 11206 11211 mouse +T403 NCBITaxon:10088 11259 11264 mouse +T404 GO:0010467 11265 11274 expresses +T405 UBERON:0000479 11305 11312 tissues +T406 GO:0007566 11337 11349 implantation +T407 UBERON:0000922 11350 11357 embryos +T408 SO:0000346 11421 11431 loxP sites +T409 CL:0000586 11435 11445 germ cells +T410 PR:000007861 11452 11457 Gata6 +T411 UBERON:0002415 11530 11534 tail +T412 GO:0097617 11576 11586 hybridized +T413 PR:000007861 11599 11604 Gata6 +T414 PR:000007861 11681 11686 Gata6 +T415 SO:0000346 11686 11690 loxP +T416 SO:0001023 11702 11708 allele +T417 PR:000007861 11753 11758 Gata6 +T418 SO:0001023 11762 11768 allele +T419 PR:000007861 11770 11775 Gata6 +T420 GO:0007618 11791 11796 mated +T421 UBERON:0000922 11827 11834 embryos +T422 SO:0001026 11902 11909 genomic +T423 UBERON:0000922 11921 11928 embryos +T424 PR:000007861 11948 11953 Gata6 +T425 PR:000007861 11969 11974 Gata6 +T426 UBERON:0002450 12032 12040 decidual +T427 PR:000007861 12136 12141 Gata6 +T428 UBERON:0000922 12149 12156 embryos +T429 PR:000007861 12240 12245 GATA6 +T430 GO:0010467 12266 12276 expression +T431 GO:0007566 12291 12303 implantation +T432 UBERON:0000922 12304 12311 embryos +T433 PR:000007861 12330 12335 Gata6 +T434 SO:0000346 12335 12339 loxP +T435 SO:0000346 12340 12344 loxP +T436 NCBITaxon:10088 12345 12349 mice +T437 PR:000007861 12363 12368 Gata6 +T438 NCBITaxon:10088 12382 12386 mice +T439 SO:0000902 12428 12437 transgene +T440 UBERON:0000922 12465 12472 embryos +T441 PR:000007861 12551 12556 Gata6 +T442 SO:0000346 12556 12560 loxP +T443 PR:000007861 12567 12572 Gata6 +T444 SO:0000346 12572 12576 loxP +T445 PR:000007861 12589 12594 Gata6 +T446 SO:0000346 12594 12598 loxP +T447 UBERON:0000922 12608 12615 embryos +T448 PR:000007861 12620 12625 Gata6 +T449 SO:0000346 12625 12629 loxP +T450 UBERON:0000922 12641 12648 embryos +T451 UBERON:0002450 12693 12701 decidual +T452 UBERON:0000922 12744 12753 Embryonic +T453 PR:000007861 12788 12793 GATA6 +T454 NCBITaxon:10088 12873 12877 mice +T455 PR:000007861 12887 12892 GATA6 +T456 GO:0010467 12936 12946 expression +T457 NCBITaxon:10088 12996 13001 mouse +T458 PR:000007861 13050 13055 GATA6 +T459 GO:0048513 13063 13076 organogenesis +T460 UBERON:0007023 13114 13119 adult +T461 NCBITaxon:10088 13120 13124 mice +T462 SO:0000155 13136 13143 Plasmid +T463 NCBITaxon:2 13160 13169 bacterial +T464 SO:0000153 13160 13191 bacterial artificial chromosome +T465 SO:0000153 13193 13196 BAC +T466 PR:000007861 13227 13232 Gata6 +T467 SO:0001026 13233 13240 genomic +T468 SO:0000155 13332 13339 Plasmid +T469 PR:000007861 13348 13353 GATA6 +T470 SO:0001026 13400 13407 genomic +T471 PR:000007861 13428 13433 Gata6 +T472 SO:0000147 13434 13439 exons +T473 SO:0000440 13479 13485 vector +T474 http://purl.obolibrary.org/obo/MONDO_0005504 13517 13527 diphtheria +T475 CHEBI:27026 13528 13533 toxin +T476 GO:0010467 13539 13549 expression +T477 SO:0005853 13550 13558 cassette +T478 SO:0000155 13613 13620 plasmid +T479 CL:0002322 13624 13632 ES cells +T480 SO:0005853 13636 13644 cassette +T481 SO:0000346 13656 13660 loxP +T482 SO:0000346 13665 13669 loxP +T483 SO:0000155 13700 13707 plasmid +T484 SO:0000112 13768 13775 primers +T485 SO:0000028 13993 13995 bp +T486 PR:000007861 14013 14018 Gata6 +T487 SO:0000188 14019 14025 intron +T488 PR:000007861 14076 14081 Gata6 +T489 SO:0000188 14082 14088 intron +T490 PR:000007861 14111 14116 GATA6 +T491 NCBITaxon:562 14148 14154 E.coli +T492 SO:0005853 14222 14230 cassette +T493 SO:0000346 14262 14271 loxP site +T494 GO:0010467 14275 14285 expression +T495 SO:0000346 14312 14316 loxP +T496 SO:0005853 14328 14336 cassette +T497 SO:0000112 14376 14383 primers +T498 SO:0000028 14598 14600 bp +T499 SO:0000028 14619 14621 bp +T500 PR:000007861 14650 14655 Gata6 +T501 SO:0000188 14656 14662 intron +T502 SO:0005853 14685 14693 cassette +T503 PR:000007861 14714 14719 Gata6 +T504 SO:0000188 14720 14726 intron +T505 NCBITaxon:562 14770 14776 E.coli +T506 SO:0001644 14800 14816 targeting vector +T507 CL:0002322 14829 14836 ES cell +T508 NCBITaxon:33208 14851 14858 animals +T509 SO:0000987 14860 14866 Linear +T510 SO:0001644 14867 14883 targeting vector +T511 CL:0002322 14915 14923 ES cells +T512 CHEBI:42768 14998 15007 Geneticin +T513 NCBITaxon:10088 15072 15076 mice +T514 GO:0098743 15095 15109;15113 15118 aggregation of ... cells +T515 CL:0002322 15110 15118 ES cells +T516 UBERON:0000085 15129 15136 morulae +T517 SO:0001023 15183 15189 allele +T518 NCBITaxon:10088 15250 15254 mice +T519 PR:000007861 15256 15261 Gata6 +T520 SO:0000346 15261 15265 loxP +T521 SO:0000346 15266 15270 loxP +T522 NCBITaxon:10088 15271 15275 mice +T523 PR:000007861 15302 15307 Gata6 +T524 SO:0000346 15307 15311 loxp +T525 NCBITaxon:10088 15325 15329 mice +T526 NCBITaxon:10088 15361 15365 mice +T527 SO:0005853 15410 15418 cassette +T528 PR:P03870 15422 15425 Flp +T529 SO:0000902 15478 15487 transgene +T530 PR:000007861 15515 15520 Gata6 +T531 SO:0000346 15520 15524 loxP +T532 SO:0000346 15525 15529 loxP +T533 NCBITaxon:10088 15530 15534 mice +T534 NCBITaxon:10088 15545 15549 mice +T535 PR:000007861 15551 15556 Gata6 +T536 NCBITaxon:10088 15562 15566 mice +T537 GO:0007618 15585 15591 mating +T538 PR:000007861 15592 15597 Gata6 +T539 SO:0000346 15597 15601 loxp +T540 NCBITaxon:33208 15615 15622 animals +T541 NCBITaxon:10088 15670 15674 mice +T542 SO:0000346 15738 15751 loxP elements +T543 SO:0000902 15782 15791 transgene +T544 PR:000007861 15816 15821 Gata6 +T545 NCBITaxon:10088 15830 15834 mice +T546 NCBITaxon:10088 15844 15848 mice +T547 NCBITaxon:33208 15904 15911 animals +T548 SO:0000112 16111 16117 primer +T549 PR:000007861 16125 16130 Gata6 +T550 PR:000007861 16184 16189 Gata6 +T551 PR:P03870 16255 16258 Flp +T552 SO:0001030 16259 16260 F +T553 SO:0001031 16261 16262 R +T554 SO:0000112 16514 16520 primer +T555 PR:000007861 16528 16533 Gata6 +T556 SO:0001030 16589 16590 F +T557 SO:0001031 16591 16592 R +T558 CHEBI:59132 16709 16716 antigen +T559 PR:000007861 16788 16793 GATA6 +T560 GO:0042571 16794 16802 antibody +T561 CHEBI:7507 16862 16870 neomycin +T562 SO:0001644 16936 16952 targeting vector +T563 NCBITaxon:10088 17002 17006 mice +T564 SO:0000155 17373 17381 plasmids +T565 NCBITaxon:2 17418 17427 bacterial +T566 GO:0007565 17460 17468 Pregnant +T567 UBERON:0001977 17474 17479 serum +T568 GO:0030728 17514 17523 ovulation +T569 SO:0000155 17720 17728 plasmids +T570 UBERON:0004535 17792 17806 cardiovascular diff --git a/src/ontogpt/evaluation/craft/database/all/16611361.txt b/src/ontogpt/evaluation/craft/database/all/16611361.txt new file mode 100644 index 000000000..d403ebdf2 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16611361.txt @@ -0,0 +1,65 @@ +Generation of mice harbouring a conditional loss-of-function allele of Gata6 + +Abstract + +The zinc finger transcription factor GATA6 is believed to have important roles in the development of several organs including the liver, gastrointestinal tract and heart. However, analyses of the contribution of GATA6 toward organogenesis have been hampered because Gata6-/- mice fail to develop beyond gastrulation due to defects in extraembryonic endoderm function. We have therefore generated a mouse line harbouring a conditional loss-of-function allele of Gata6 using Cre/loxP technology. + +LoxP elements were introduced into introns flanking exon 2 of the Gata6 gene by homologous recombination in ES cells. Mice containing this altered allele were bred to homozygosity and were found to be viable and fertile. To assess the functional integrity of the loxP sites and to confirm that we had generated a Gata6 loss-of-function allele, we bred Gata6 'floxed' mice to EIIa-Cre mice in which Cre is ubiquitously expressed, and to Villin-Cre mice that express Cre in the epithelial cells of the intestine. We conclude that we have generated a line of mice in which GATA6 activity can be ablated in a cell type specific manner by expression of Cre recombinase. This line of mice can be used to establish the role of GATA6 in regulating embryonic development and various aspects of mammalian physiology. + +Background + +The mouse Gata6 gene encodes a 45 kD protein containing two highly conserved zinc-finger DNA binding domains with a Cys-X2-Cys-X17-Cys-X2-Cys motif that directs binding to the nucleotide sequence element (A/T)GATA(A/G) 1. GATA4, 5 and 6 make up a subset of GATA factors that have been implicated in the development of several organs including the heart, lung, gastrointestinal tract and liver [1-4]. Development of GATA6 null embryos arrests during gastrulation as a consequence of defects in extraembryonic endoderm function [2,5]. This early embryonic lethality can be rescued by complementing the GATA6 null embryos with a wild type extraembryonic visceral endoderm using tetraploid embryo complementation, and embryos derived by this process can survive until E10.5 [3]. Analyses of such Gata6-/-ES cell-derived embryos has revealed defects in hepatogenesis, which supports the proposal that GATA6 is an important developmental regulator. Although the ability to generate embryos from Gata6-/-ES cells by tetraploid embryo complementation has provided important insight into the contribution of GATA6 during early embryogenesis, this approach is not compatible with studying the role of GATA6 at later stages in development or its role in controlling differentiation of specific cell types. In the current report, we describe the generation of mice containing a conditional null allele of Gata6 that can be used for cell type-specific removal of GATA6 by Cre-mediated recombination. + +Results and discussion + +To ensure the elimination of GATA6 activity, we chose to flank Gata6 exon 2 (based on nomenclature described by Brewer et al [6,7]) with loxP elements because this exon encodes the majority of the GATA6 protein (Fig. 1A). We decided to use a recombineering approach to facilitate the accurate placement of the loxP elements following the procedure described by Lee et al [8]. The final targeting vector contained a loxP element located between exons 2 and 3. In addition, a cassette containing a loxP element lying immediately 5' to a neomycin phosphotransferase (neo) gene, which conferred resistance to G418, was introduced between exons 1 and 2 (Fig. 1A). FRT sites flanked the neo gene, in order to allow removal of neo by Flp recombinase. Two novel restriction endonuclease cleavage sites (EcoRI and PacI) were also introduced into the Gata6 targeting vector to allow the identification of correctly modified Gata6 alleles by Southern blot analyses. The targeting vector was introduced into R1 ES cells [9] by electroporation, and G418-resistant ES cell clones were tested for homologous recombination at the Gata6 locus by Southern blot analysis. Fig. 1B shows an example of a correctly targeted ES (Gata6loxP(FRTneoFRT)/+) cell line. Following digestion of ES cell genomic DNA with EcoRI, a probe that lies 3' to the short arm of homology used in the targeting vector identified an expected 8.1 kb wild type Gata6 DNA fragment, while correctly targeted cells contain an additional 3.2 kb fragment due to the introduction of a new EcoRI site. When NdeI digested DNA was probed with a DNA fragment lying 5' to the expected position of the introduced loxP site, it identified the predicted 13.2 kb wild type fragment and a novel 15.2 kb fragment, which resulted from introducing the neo cassette into the Gata6 locus. Five correctly targeted ES cell clones were recovered and three of these cell lines were used to generate chimeric mice by morula aggregation [10]. These chimeric animals were then mated with CD-1 mice and successful germline transmission of the Gata6loxP(FRTneoFRT) allele was confirmed by Southern blot analysis of tail DNA (Fig. 1B). Mice carrying a single Gata6loxP(FRTneoFRT)/+ allele were viable and fertile. However, we were unable to obtain mice that were homozygous for this allele, which suggested that the inclusion of the neo cassette disrupted GATA6 function and resulted in embryonic lethality as observed for Gata6-/- embryos [2,5]. We therefore established a mouse line, Gata6loxP/+, that lacked the neo cassette by inducing recombination between the FRT sites in vivo [11]. This was achieved by mating Gata6loxP(FRTneoFRT)/+ mice to a transgenic mouse, B6;SJL-Tg(ACTFLPe)9205Dym/J, in which Flp recombinase is widely expressed from the human beta actin gene promoter [11]. Correct excision of the neo cassette was confirmed in offspring by Southern blot and PCR analyses (Fig. 1B & C). Gata6loxP/+ mice were finally interbred to generate Gata6loxP/loxPmice, which are healthy, fertile and have reached maturity. + +Figure 1 + +Generation of a Gata6 conditional null allele. (A) Schematic showing a map of the Gata6 genomic locus and the targeting vector with exons represented by open boxes. The relative position of Southern blot probes (lines), PCR primers (small arrowheads), loxP (large arrowheads) and FRT (ovals) sites, as well as cassettes encoding neomycin phosphotransferase (neo) and diphtheria toxin (DT), are included. Sizes of relevant EcoRI (e), NdeI (n), and PacI (p) restriction endonuclease fragments are shown in kilobase pairs (kb). (B) Southern blot analysis of genomic DNA isolated from ES cells (lanes 1, 2, 8 and 9) or mouse tails (lanes 3–7 and 10). An example of an ES cell line containing a correctly targeted Gata6loxP(FRTneoFRT) allele is shown in lanes 2 and 8. Mice harbouring the modified Gata6 allele were generated from these ES cells (lanes 3 and 10). Exon 2 of Gata6 was deleted (Gata6del) or the neo cassette alone was deleted, leaving Gata6 exon 2 flanked by loxP elements (Gata6loxP), by breeding Gata6loxP(FRTneoFRT) mice to transgenic mice expressing either Cre (lane 6 and 7) or Flp (lane 4) recombinases, respectively. The size of restriction fragments identified by 5' and 3' probes (Fig. 1A) was deduced from their position relative to standard DNA fragments. (C) The genotypes of mice and embryos were also determined by PCR amplification of genomic DNA. Primers were designed that differentiated between the Gata6+ (gt4F/4R; 159 bp), GataloxP (gt4F/4R; 250 bp) and Gata6del (k/o2F/2R; 568 bp) alleles, as well as neo (Neo A/B; 225 bp), flp (Flp F/R; 750 bp), and cre (Cre 1/2; 300 bp) transgenes. + +We next addressed whether removal of GATA6 could be induced in vivo by expression of Cre recombinase. We chose to disrupt the Gata6 gene in the gastrointestinal tract because it is highly expressed in the epithelium and may have roles in controlling gut physiology or development. To produce Gata6loxP/+Villin-Cre mice, we bred Gata6loxP/loxP mice with transgenic mice (Tg(Vil-cre)997Gum) in which Cre expression was directed to the epithelial cells of the small intestine by the Villin (Vil1) promoter [12]. Using this transgenic strain of mice, Cre activity can be detected in the epithelium of the small intestine from E14.5 [12]. Gata6loxP/+Villin-Cre males were mated with Gata6loxP/loxP females to obtain Gata6loxP/+Villin-Cre control and Gata6loxP/loxPVillin-Cre experimental offspring. In previous experiments, we observed that the efficiency through which Cre mediates recombination between loxP sites varies between different transgenic male mice [13]. We noted a similar situation when using various Gata6loxP/+Villin-Cre males. In the majority of cases (4/5 stud males) we were unable to obtain Gata6loxP/loxPVillin-Cre offspring (n = 95 mice genotyped), and this lethality associated with presumptive loss of GATA6 in the intestine is currently under investigation. However, one Gata6loxP/+Villin-Cre male did produce offspring (12/90) whose genotype was determined to be Gata6loxP/loxPVillin-Cre by PCR analysis of tail DNA. We, therefore, used RT-PCR with Gata6 primers corresponding to nucleotide sequences predicted to be deleted after recombination between Gata6 loxP elements, to compare the steady-state levels of Gata6 mRNA in intestines isolated from control and experimental mice generated by this particular Gata6loxP/+Villin-Cre male. Fig. 2A shows that Gata6 mRNA could be detected in control intestine but not in intestine isolated from a subset of Gata6loxP/loxPVillin-Cre offspring. The remaining Gata6loxP/loxPVillin-Cre mice were found to continue to express Gata6 mRNA at varying levels (data not shown). We also examined expression of GATA6 protein in the intestines of Gata6loxP/loxPVillin-Cre offspring derived from the same Gata6loxP/+Villin-Cre male. Fig. 2B shows that GATA6 was detected as an abundant nuclear protein in the epithelial cells of control intestines. However, Gata6loxP/loxP Villin-Cre mice displayed a marked reduction in the abundance of intestinal GATA6 (Fig. 2B). From these results, we conclude that the Gata6 gene can be conditionally disrupted in Gata6loxP/loxP mice by cell-type specific expression of Cre recombinase. + +Figure 2 + +Gata6 can be successfully ablated in the intestine of Gata6loxP/loxP mice by expression of Cre from the Villin promoter. (A) The steady-state level of Gata6 mRNA was compared in intestines isolated from four control Gata6loxP/+Villin-Cre (lanes 2–5) or experimental Gata6loxP/loxPVillin-Cre (lanes 6–9) mice. Amplification of Pol2 (Polr2a) mRNA showed that similar levels of starting material were utilized in each reaction. (B) Immunohistochemistry showing that GATA6 was detected as an abundant nuclear protein in epithelial cells (arrows) of control intestines, but was detected at greatly reduced levels in Gata6loxP/loxPVillin-Cre intestines. + +Cre-mediated deletion of exon2 in Gata6loxP/loxP mice was predicted to result in loss of GATA6 function. If this were true, we expected that embryos homozygous for the deleted allele (Gata6del) should undergo developmental arrest, as was described for Gata6-/- embryos [2,5]. Gata6del/+ mice were produced by mating Gata6loxP(FRTneoFRT)/+ mice with an EIIa-Cre transgenic mouse (B6.FVB-Tg(EIIa-cre)C5379Lmgd/J). The EIIa-Cre mouse expresses Cre recombinase in nearly all tissues, including those of pre-implantation embryos, and has been used previously to mediate recombination between loxP sites in germ cells [14]. Gata6+/del progeny were identified by Southern blot analyses of NdeI digested tail DNA. As shown in Fig. 1B, Southern blots hybridized with the 5' Gata6 probe (Fig. 1A) revealed the conversion of a 15.2 kb NdeI fragment from the Gata6loxP(FRTneoFRT) allele to an expected 12 kb NdeI fragment from the Gata6del allele. Gata6+/del were then mated inter se, and the genotype of embryos collected between E8.5 and E11.5 was determined by PCR analysis of genomic DNA. Of 77 embryos recovered, 27 were Gata6+/+ and 50 were Gata6+/del (Table 1). In addition, 23 partially resorbed empty decidual masses were recovered, which we believe likely resulted from the early developmental arrest of Gata6del/del embryos. We finally determined whether the developmental lethality associated with loss of GATA6 could be induced by expression of Cre in pre-implantation embryos. To achieve this, Gata6loxP/loxP mice were bred to Gata6+/delEIIa-Cre mice, which are heterozygous for the EIIa-Cre transgene. The genotype of resulting embryos ranging from E8.5 to E11.5 was again determined by PCR. While we recovered 14 Gata6loxP/+, 18 Gata6loxP/del, and 16 Gata6loxP/+EIIaCre embryos, no Gata6loxP/delEIIaCre embryos were identified although 13 empty resorbing decidual masses were observed (Table 1). + +Table 1 + +Embryonic lethality associated with loss of GATA6 function + +Conclusion + +In summary, we conclude that we have generated a line of mice in which GATA6 transcriptional activity can be ablated by expression of Cre. We believe that the availability of this mouse will be useful to elucidate the contribution of GATA6 during organogenesis as well as its physiological role in adult mice. + +Methods + +Plasmid construction + +A bacterial artificial chromosome (BAC # RP23-410I12) that contained Gata6 genomic DNA was obtained from BACPAC Resources, Children Hospital Research Institute, Oakland, CA. Plasmid pNeb-DT-GATA6 was generated by cloning a 12.2 kb EcoRV/PacI genomic fragment containing Gata6 exons 1 and 2 into the PacI/HincII site of a vector, pNEB193-DT, which contained a diphtheria toxin (DT) expression cassette to enrich against random integration of the targeting plasmid in ES cells. A cassette containing loxP-neo-loxP was amplified by PCR from the plasmid pSV-LNL (modified from Zhang et al)[15] using the following primers: Gata6ET1:GCTTGCTGTTTGAGTCTACCCCATTTCTGCCTGTTTCTTGACATCCCTTCGAATTCTGGTACCGGCGCGCCTAGTCGAC, Gata6ET2:ATCCATTATTGTCAATGTCTAAAGATGGAATTGTCTCTGCACAAGCTATCTTCTCAACTCGAGCCCTTAATTAACCGGT. These oligonucleotides contained 55 bp of sequence from Gata6 intron 2 (underlined). This amplicon was introduced into Gata6 intron 2 sequence in pNEB-DT-GATA6 by homologous recombination in E.coli following the procedure described by Lee et al [8] The loxPneoloxP cassette was then converted to a single loxP site by expression of Cre recombinase [8]. A loxP(FRTneoFRT) cassette was then amplified from pSV-LFNF using primers Gata6ET3:CACGCTGGTGGTTGTAAGGCGGTTTGTGTTTAAGGTGTGCGGTTGGCCTGGACGTGTGGTACCGGCGCGCCTAGTCGAC, Gata6ET4:GAAAAAGTTACCTAGCCCAGAGAAAGTGAGATGCCAGGAAAGGCATAAGGATATCAACTCGAGCCCTTAATTAACCGGT. These oligonucleotides contain 56 bp (Gata6ET3) and 52 bp (Gata6ET4) of sequence from Gata6 intron 1, respectively. This cassette was introduced into Gata6 intron 1, again using homologous recombination in E.coli. to generate the final targeting vector (Fig. 1A). + +ES cell targeting and animals + +Linear targeting vector (100μg) was introduced into R1 ES cells by electroporation, and the genotype of colonies resistant to 350μg/ml of Geneticin (Gibco BRL) was determined by Southern blot (Fig. 1B). Chimeric mice were generated by aggregation of ES cells with CD-1 morulae as described previously [10] and the modified allele was passed through the germline by breeding chimeras to CD1 mice. Gata6loxP/loxP mice were produced by breeding Gata6loxp(FRTneoFRT)/+ mice to B6;SJL-Tg(ACTFLPe)9205Dym/J mice [16] (Jackson Labs) to delete the FRTneoFRT cassette by Flp-mediated recombination in the germline. The ACTFLPe transgene was removed by breeding F1 Gata6loxP/loxP mice into CD-1 mice. Gata6+/del mice were generated by mating Gata6loxp(FRTneoFRT)/+ animals with B6.FVB-Tg(EIIa-cre)C5379Lmgd/J transgenic mice [17] (Jackson Labs) to allow Cre-mediate recombination between loxP elements in the germline. The EIIa-Cre transgene was removed by breeding Gata6+/del F1 mice with CD1 mice. The MCW IACUC committee approved all procedures using animals. + +Southern blot, PCR and RT-PCR + +Southern blot analyses were performed using standard conditions with probes indicated in Fig. 1. Genotypes were determined by PCR using the following oligonucleotide primer pairs: Gata6 gt4F/4R, GTGGTTGTAAGGCGGTTTGT, ACGCGAGCTCCAGAAAAAGT; Gata6 k/o2F/2R, AGTCTCCCTGTCATTCTTCCTGCTC, TGATCAAACCTGGGTCTACACTCCTA; Flp F/R, GGTCCAACTGCAGCCCAAGCTTCC, GTGGATCGATCCTACCCCTTGCG [16]; Cre 1/2, GTTCGCAAGAACCTGATGGACA, CTAGAGCCTGTTTTGCACGTTC [18]; Neo A/B, GCCAACGCTATGTCCTGATAGCGGT, AGCCGGTCTTGTCGATCAGGATGAT. RT-PCR was performed as described previously [19] with the following primer pairs: Gata6 9/10; AGTTTTCCGGCAGAGCAGTA, AGTCAAGGCCATCCACTGTC, Pol2 F/R; CTGATGCGGGTGCTGAGTGAGAAGG, GCGGTTGACCCCATGACGAGTG. + +Immunohistochemistry + +Immunohistochemistry was performed using antigen retrieval in citrate buffer as described previously [13] using an anti-GATA6 antibody (AF1700 R&D Systems,1/1000 dilution). + +Abbreviations + +Neo, neomycin phosphotransferase + +Authors' contributions + +C.P.S. generated the targeting vector and carried out analyses of conditional knockout mice, as well as contributed to experimental design and draft of the manuscript. J. L. generated aggregation chimeras. S.A.D. conceived of the study, contributed to experimental design and interpretation of results, and coordinated the project and writing of the manuscript. + +Acknowledgements + +We would like to thank Drs. Robert Burgess and Francis Stewart for providing plasmids and Dr. Neal Copeland for providing bacterial strains used in recombineering. Pregnant mare serum gonadotrophin (PMSG) used in superovulation was obtained from Dr. A.F. Parlow at the National Hormone and Peptide Program (Torrance, CA). We are also grateful to Dr. Michele Battle for critically evaluating the manuscript and for providing plasmids. This work was supported by an NIH training grant from the MCW cardiovascular research centre to C.P.S and NIH grants to S.A.D. diff --git a/src/ontogpt/evaluation/craft/database/all/16628246.ann b/src/ontogpt/evaluation/craft/database/all/16628246.ann new file mode 100644 index 000000000..a8adc86f7 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16628246.ann @@ -0,0 +1,1735 @@ +T1 PR:000004503 8 12 Atrx +T2 UBERON:0000088 21 32 Trophoblast +T3 GO:0000805 64 65 X +T4 GO:0009048 64 78 X-Inactivation +T5 UBERON:0005292 82 104 Extraembryonic Tissues +T6 PR:000004503 116 120 ATRX +T7 GO:0000805 127 128 X +T8 PR:P22082 151 155 SNF2 +T9 GO:0065007 202 210 regulate +T10 SO:0000704 211 215 gene +T11 GO:0010467 211 226 gene expression +T12 GO:0000785 240 249 chromatin +T13 PR:000004503 279 283 ATRX +T14 NCBITaxon:9606 316 321 human +T15 SO:0000704 322 329 genetic +T16 http://purl.obolibrary.org/obo/MONDO_0003847 322 337 genetic disease +T17 PR:000004503 413 417 ATRX +T18 http://purl.obolibrary.org/obo/MONDO_0010519 473 478;528 536 ATR-X syndrome +T19 GO:0000805 477 478 X +T20 http://purl.obolibrary.org/obo/MONDO_0010519 480 526;528 536 alpha thalassemia mental retardation, X-linked syndrome +T21 GO:0000805 518 519 X +T22 SO:0000853 571 578 homolog +T23 NCBITaxon:10088 582 586 mice +T24 PR:000004503 588 592 Atrx +T25 PR:000004503 650 654 Atrx +T26 GO:0010467 673 682 expressed +T27 UBERON:0000922 693 700 embryos +T28 PR:000004503 710 714 Atrx +T29 GO:0007566 715 724 implanted +T30 GO:0007369 729 740 gastrulated +T31 GO:0007620 790 796 coitus +T32 UBERON:0000922 838 847 embryonic +T33 UBERON:0000088 848 859 trophoblast +T34 UBERON:0000922 931 937 embryo +T35 NCBITaxon:10088 954 958 mice +T36 SO:0001023 988 994 allele +T37 GO:0000805 1034 1046 X chromosome +T38 UBERON:0005292 1074 1096 extraembryonic tissues +T39 UBERON:0001987 1163 1171 placenta +T40 GO:0060819 1216 1240 imprinted X-inactivation +T41 GO:0000805 1226 1227 X +T42 UBERON:0000479 1250 1257 tissues +T43 PR:000004503 1343 1347 Atrx +T44 NCBITaxon:39107 1374 1380 murine +T45 UBERON:0000088 1381 1392 trophoblast +T46 GO:0060819 1431 1466 imprinted X chromosome inactivation +T47 GO:0000805 1441 1453 X chromosome +T48 PR:000004503 1479 1483 ATRX +T49 GO:0000785 1556 1565 chromatin +T50 GO:0005634 1619 1626 nucleus +T51 PR:000004503 1648 1652 ATRX +T52 SO:0000704 1677 1681 gene +T53 GO:0010467 1677 1692 gene expression +T54 PR:000004503 1711 1715 ATRX +T55 SO:0000704 1716 1720 gene +T56 GO:0000803 1753 1767 sex chromosome +T57 GO:0000805 1769 1770 X +T58 NCBITaxon:9606 1805 1810 human +T59 http://purl.obolibrary.org/obo/MONDO_0000001 1811 1818 disease +T60 http://purl.obolibrary.org/obo/MONDO_0010519 1820 1834 ATR-X syndrome +T61 GO:0000805 1824 1825 X +T62 GO:0000805 1894 1895 X +T63 GO:0000806 1898 1899 Y +T64 PR:000004503 2023 2027 ATRX +T65 SO:0000704 2117 2121 gene +T66 NCBITaxon:10088 2125 2129 mice +T67 PR:000004503 2131 2135 Atrx +T68 NCBITaxon:10088 2160 2164 mice +T69 GO:0000805 2166 2167 X +T70 GO:0000806 2170 2171 Y +T71 GO:0016265 2207 2211 died +T72 UBERON:0001987 2270 2278 placenta +T73 UBERON:0001987 2287 2295 placenta +T74 NCBITaxon:10088 2304 2308 mice +T75 GO:0000805 2333 2345 X chromosome +T76 GO:0000805 2432 2444 X chromosome +T77 PR:000004503 2482 2486 Atrx +T78 UBERON:0001987 2576 2584 placenta +T79 PR:000004503 2710 2714 Atrx +T80 SO:0000704 2715 2719 gene +T81 GO:0000805 2791 2803 X-chromosome +T82 GO:0071514 2804 2814 imprinting +T83 http://purl.obolibrary.org/obo/MONDO_0010519 2831 2845 ATR-X syndrome +T84 GO:0000805 2835 2836 X +T85 GO:0000805 2882 2883 X +T86 http://purl.obolibrary.org/obo/MONDO_0000839 2954 2978 congenital abnormalities +T87 http://purl.obolibrary.org/obo/MONDO_0011399 3029 3043 α-thalassaemia +T88 GO:0010467 3063 3073 expression +T89 CHEBI:5386 3099 3105 globin +T90 SO:0000704 3106 3111 genes +T91 PR:000008457 3150 3158 β-globin +T92 CHEBI:5386 3152 3158 globin +T93 GO:0016234 3184 3200 inclusion bodies +T94 CL:0000232 3216 3231 red blood cells +T95 UBERON:0000178 3220 3225 blood +T96 GO:0016234 3285 3295 inclusions +T97 GO:0000805 3364 3376 X-chromosome +T98 GO:0009048 3364 3389 X-chromosome inactivation +T99 SO:0001023 3492 3498 allele +T100 UBERON:0000479 3515 3522 tissues +T101 GO:0000805 3548 3549 X +T102 GO:0009048 3548 3562 X-inactivation +T103 http://purl.obolibrary.org/obo/MONDO_0010519 3631 3645 ATR-X syndrome +T104 GO:0000805 3635 3636 X +T105 SO:0000704 3674 3678 gene +T106 PR:000004503 3680 3684 ATRX +T107 SO:0000147 3704 3709 exons +T108 SO:0001026 3729 3736 genomic +T109 SO:0000704 3772 3776 gene +T110 SO:0001060 3806 3814 isoforms +T111 PR:000004503 3854 3858 ATRX +T112 SO:0000673 3902 3912 transcript +T113 SO:0001060 3966 3973 isoform +T114 SO:0000673 4017 4027 transcript +T115 SO:0000188 4062 4068 intron +T116 GO:0008380 4084 4091 spliced +T117 SO:0000185 4101 4119 primary transcript +T118 SO:0000188 4139 4147 intronic +T119 SO:0000551 4148 4162 poly(A) signal +T120 NCBITaxon:10088 4180 4185 mouse +T121 SO:0000853 4186 4193 homolog +T122 PR:000004503 4201 4205 ATRX +T123 SO:0000704 4206 4210 gene +T124 PR:000004503 4212 4216 Atrx +T125 GO:0000805 4242 4254 X chromosome +T126 PR:000004503 4292 4296 Atrx +T127 SO:0001060 4340 4348 isoforms +T128 http://purl.obolibrary.org/obo/MONDO_0000001 4357 4364 Disease +T129 SO:0000842 4413 4423;4428 4432 regions of ... gene +T130 SO:0000417 4457 4463 domain +T131 PR:P22082 4470 4474 SNF2 +T132 SO:0000417 4487 4493 domain +T133 GO:0000785 4588 4597 chromatin +T134 GO:0000785 4634 4643 chromatin +T135 GO:0006338 4634 4655 chromatin-remodelling +T136 http://purl.obolibrary.org/obo/MONDO_0000001 4686 4693 disease +T137 SO:0000417 4757 4764 domains +T138 PR:000004503 4766 4770 ATRX +T139 GO:0000785 4797 4806 chromatin +T140 PR:P05205 4835 4838 HP1 +T141 GO:0000792 4842 4857 heterochromatin +T142 CL:0000836 4882 4895 promyelocytic +T143 GO:0016605 4882 4919 promyelocytic leukemia nuclear bodies +T144 PR:000006283 4944 4948 Daxx +T145 PR:000004503 4982 4986 ATRX +T146 GO:0006306 5015 5030 DNA methylation +T147 PR:000004503 5060 5064 ATRX +T148 SO:0000704 5074 5078 gene +T149 GO:0010467 5074 5089 gene expression +T150 UBERON:0000178 5144 5158 haematological +T151 http://purl.obolibrary.org/obo/MONDO_0010519 5180 5185 ATR-X +T152 GO:0000805 5184 5185 X +T153 PR:P22082 5219 5223 SWI2 +T154 PR:P22082 5224 5228 SNF2 +T155 GO:0000785 5229 5238 chromatin +T156 GO:0006338 5229 5250 chromatin-remodelling +T157 PR:000004503 5260 5264 ATRX +T158 GO:0065007 5274 5283 regulates +T159 SO:0000704 5326 5331 genes +T160 CHEBI:5386 5406 5412 globin +T161 SO:0000704 5413 5418 genes +T162 GO:0065007 5473 5483 regulation +T163 PR:000004503 5487 5491 ATRX +T164 PR:000004503 5549 5553 ATRX +T165 NCBITaxon:40674 5569 5578 mammalian +T166 NCBITaxon:10088 5639 5644 mouse +T167 SO:0001023 5695 5701 allele +T168 PR:000004503 5709 5713 Atrx +T169 SO:0000704 5714 5718 gene +T170 NCBITaxon:10088 5722 5727 mouse +T171 UBERON:0000922 5728 5737 embryonic +T172 CL:0002322 5728 5742;5748 5753 embryonic stem ... cells +T173 CL:0002322 5744 5746;5748 5753 ES ... cells +T174 GO:0010467 5810 5820 expression +T175 PR:000004503 5840 5844 Atrx +T176 CL:0002322 5856 5864 ES cells +T177 NCBITaxon:10088 5872 5877 mouse +T178 UBERON:0000922 5878 5885 embryos +T179 GO:0048468 5897 5910;5914 5919 Generation of ... Cells +T180 CL:0002322 5911 5919 ES Cells +T181 PR:000004503 5940 5944 Atrx +T182 NCBITaxon:9606 5955 5960 human +T183 SO:0000704 5961 5965 gene +T184 NCBITaxon:10088 5971 5976 mouse +T185 PR:000004503 5977 5981 Atrx +T186 SO:0000704 5982 5986 gene +T187 GO:0000805 5995 5996 X +T188 PR:000004503 6049 6053 Atrx +T189 SO:0001023 6054 6060 allele +T190 CL:0002322 6069 6077 ES cells +T191 CL:0002322 6216 6224 ES cells +T192 SO:0000440 6245 6252 vectors +T193 SO:0000147 6266 6270 exon +T194 PR:000004503 6281 6285 Atrx +T195 SO:0000704 6286 6290 gene +T196 SO:0000147 6292 6296 Exon +T197 PR:P22082 6362 6366 SNF2 +T198 SO:0000417 6372 6378 domain +T199 PR:000004503 6382 6386 Atrx +T200 PR:P22082 6448 6452 SNF2 +T201 GO:0016514 6495 6502 SWI/SNF +T202 SO:0000704 6513 6517 gene +T203 GO:0010467 6513 6528 gene expression +T204 SO:0000440 6585 6592 vectors +T205 PR:000004503 6608 6612 Atrx +T206 CL:0002322 6641 6648 ES cell +T207 SO:0000147 6795 6799 exon +T208 SO:0000147 6848 6852 exon +T209 SO:0000357 6865 6872 flanked +T210 SO:0000346 6876 6898 loxP recognition sites +T211 PR:000004503 6924 6928 Atrx +T212 SO:0000359 6928 6932 flox +T213 SO:0001023 6933 6939 allele +T214 SO:0001023 6972 6978 allele +T215 SO:0000359 6995 7007 loxP-flanked +T216 SO:0005853 7017 7025 cassette +T217 SO:0000188 7029 7035 intron +T218 PR:000004503 7125 7129 Atrx +T219 SO:0000359 7129 7133 flox +T220 GO:0010467 7154 7161 express +T221 PR:000004503 7179 7183 Atrx +T222 SO:0001060 7216 7223 isoform +T223 CL:0002322 7259 7267 ES cells +T224 PR:000004503 7273 7277 Atrx +T225 SO:0000359 7277 7281 flox +T226 GO:0009294 7324 7335 transfected +T227 GO:0010467 7359 7369 expression +T228 SO:0000155 7370 7377 plasmid +T229 SO:0000243 7390 7394 IRES +T230 SO:0001023 7441 7447 allele +T231 PR:000004503 7449 7453 Atrx +T232 SO:0000147 7489 7493 exon +T233 SO:0005853 7510 7518 cassette +T234 PR:000004503 7619 7623 Atrx +T235 SO:0000359 7623 7627 flox +T236 SO:0001023 7628 7634 allele +T237 PR:000004503 7752 7756 Atrx +T238 SO:0000673 7757 7767 transcript +T239 PR:000004503 7811 7815 Atrx +T240 SO:0000673 7936 7946 transcript +T241 SO:0001060 7981 7988 isoform +T242 SO:0000673 7994 8004 transcript +T243 SO:0000188 8035 8041 intron +T244 SO:0000147 8083 8087 exon +T245 SO:0001060 8155 8162 isoform +T246 SO:0000417 8192 8198 domain +T247 GO:0016514 8211 8218 SWI/SNF +T248 CL:0002322 8371 8379 ES cells +T249 PR:000004503 8410 8414 Atrx +T250 PR:000004503 8461 8465 Atrx +T251 CL:0002322 8470 8478 ES Cells +T252 PR:000004503 8480 8484 Atrx +T253 CL:0002322 8489 8497 ES cells +T254 GO:0040007 8555 8562 growing +T255 PR:000004503 8568 8572 Atrx +T256 CL:0002322 8574 8576 ES +T257 PR:000004503 8693 8697 Atrx +T258 CL:0002322 8701 8708 ES cell +T259 PR:000004503 8729 8733 Atrx +T260 PR:000004503 8739 8743 Atrx +T261 CL:0002322 8748 8755 ES cell +T262 PR:000004503 8805 8809 Atrx +T263 PR:000004503 8830 8834 Atrx +T264 PR:000004503 8843 8847 Atrx +T265 SO:0000359 8847 8851 flox +T266 SO:0001023 8852 8858 allele +T267 PR:000004503 8864 8868 Atrx +T268 PR:000004503 8885 8889 Atrx +T269 SO:0001023 8897 8903 allele +T270 CL:0002322 8905 8913 ES cells +T271 SO:0001023 9055 9062 alleles +T272 PR:000004503 9169 9173 Atrx +T273 SO:0001023 9181 9187 allele +T274 PR:000004503 9217 9221 Atrx +T275 CL:0002322 9224 9232 ES cells +T276 PR:000004503 9255 9259 Atrx +T277 SO:0000359 9259 9263 flox +T278 SO:0001023 9264 9270 allele +T279 PR:000004503 9352 9356 Atrx +T280 SO:0001023 9357 9364 alleles +T281 PR:000004503 9366 9370 Atrx +T282 PR:000004503 9377 9381 Atrx +T283 SO:0000359 9381 9385 flox +T284 PR:000004503 9479 9483 Atrx +T285 CL:0002322 9488 9496 ES cells +T286 PR:000004503 9641 9645 Atrx +T287 CL:0002322 9681 9688 ES cell +T288 GO:0007049 9724 9734 cell-cycle +T289 PR:000004503 9760 9764 Atrx +T290 GO:0007049 9782 9792 cell cycle +T291 CHEBI:472552 9809 9826 bromodeoxyuridine +T292 CHEBI:472552 9828 9832 BrdU +T293 CL:0002322 9841 9849 ES cells +T294 PR:000004503 9901 9905 Atrx +T295 CL:0002322 9910 9917 ES cell +T296 GO:0007049 9937 9947 cell cycle +T297 CL:0002322 9988 9996 ES cells +T298 PR:000004503 10018 10022 Atrx +T299 SO:0001023 10023 10029 allele +T300 PR:000004503 10031 10035 Atrx +T301 PR:000004503 10041 10045 Atrx +T302 SO:0000359 10045 10049 flox +T303 GO:0007067 10089 10096 mitotic +T304 CL:0002322 10159 10167 ES cells +T305 CHEBI:15358 10195 10202 histone +T306 PR:000027594 10195 10205 histone H3 +T307 GO:0007067 10228 10235 mitosis +T308 GO:0007049 10282 10292 cell-cycle +T309 GO:0007067 10359 10366 mitotic +T310 PR:000004503 10385 10389 Atrx +T311 CL:0002322 10394 10396 ES +T312 PR:000004503 10490 10494 Atrx +T313 CL:0002322 10499 10507 ES cells +T314 GO:0043065 10522 10548 up-regulation of apoptosis +T315 PR:000004078 10572 10581 Annexin V +T316 CL:0000445 10627 10642 apoptotic cells +T317 PR:000004503 10704 10708 Atrx +T318 CL:0002322 10746 10754 ES cells +T319 PR:000004503 10763 10767 Atrx +T320 GO:0007049 10793 10803 cell cycle +T321 GO:0008219 10838 10848 cell death +T322 PR:000004503 10917 10921 Atrx +T323 CL:0002322 10926 10934 ES cells +T324 CL:0002322 11137 11145 ES cells +T325 http://purl.obolibrary.org/obo/MONDO_0000001 11241 11248 disease +T326 NCBITaxon:9606 11274 11279 human +T327 PR:000004503 11280 11284 ATRX +T328 SO:0000704 11285 11289 gene +T329 GO:0006306 11336 11351 DNA methylation +T330 NCBITaxon:9606 11395 11400 human +T331 SO:0001026 11401 11407 genome +T332 GO:0005840 11453 11462 ribosomal +T333 http://purl.obolibrary.org/obo/MONDO_0010519 11529 11534 ATR-X +T334 GO:0000805 11533 11534 X +T335 NCBITaxon:1 11563 11574 individuals +T336 NCBITaxon:10088 11705 11710 mouse +T337 PR:000004503 11727 11731 Atrx +T338 CL:0002322 11736 11744 ES cells +T339 UBERON:0014374 11754 11769 embryoid bodies +T340 CL:0002322 11782 11790 ES cells +T341 UBERON:0014374 11795 11810 embryoid bodies +T342 PR:000004503 11832 11836 Atrx +T343 SO:0001023 11837 11843 allele +T344 PR:000004503 11845 11849 Atrx +T345 PR:000004503 11855 11859 Atrx +T346 SO:0000359 11859 11863 flox +T347 PR:000004503 11947 11951 Atrx +T348 CL:0002322 11961 11969 ES cells +T349 NCBITaxon:9606 12030 12035 human +T350 http://purl.obolibrary.org/obo/MONDO_0010519 12100 12105 ATR-X +T351 GO:0000805 12104 12105 X +T352 NCBITaxon:10088 12122 12127 mouse +T353 CL:0002322 12169 12176 ES cell +T354 GO:0000792 12193 12208 heterochromatic +T355 NCBITaxon:11632 12320 12330 retroviral +T356 CL:0002322 12582 12590 ES cells +T357 PR:000004503 12660 12664 Atrx +T358 PR:000004503 12773 12777 ATRX +T359 GO:0006306 12790 12805 DNA methylation +T360 NCBITaxon:9606 12818 12823 human +T361 NCBITaxon:10088 12852 12857 mouse +T362 UBERON:0019248 12866 12881 Early Embryonic +T363 PR:000004503 12895 12899 Atrx +T364 NCBITaxon:10088 12909 12913 Mice +T365 PR:000004503 12946 12950 Atrx +T366 NCBITaxon:10088 12966 12971 mouse +T367 NCBITaxon:10088 13019 13023 mice +T368 PR:000004503 13036 13040 Atrx +T369 SO:0000359 13040 13044 flox +T370 SO:0001023 13045 13051 allele +T371 PR:000004503 13080 13084 Atrx +T372 SO:0000359 13084 13088 flox +T373 CL:0002322 13089 13096 ES cell +T374 UBERON:0000358 13158 13169 blastocysts +T375 NCBITaxon:10088 13191 13195 mice +T376 PR:000004503 13290 13294 Atrx +T377 SO:0000359 13294 13298 flox +T378 PR:000004503 13328 13332 Atrx +T379 SO:0000359 13335 13339 flox +T380 SO:0000359 13349 13355 floxed +T381 SO:0001023 13356 13362 allele +T382 PR:000004503 13417 13421 Atrx +T383 SO:0000359 13421 13425 flox +T384 SO:0000359 13426 13430 flox +T385 PR:000004503 13497 13501 Atrx +T386 SO:0000359 13501 13505 flox +T387 SO:0001023 13506 13512 allele +T388 PR:000004503 13597 13601 Atrx +T389 SO:0000359 13601 13605 flox +T390 SO:0001023 13606 13612 allele +T391 PR:000004503 13650 13654 Atrx +T392 NCBITaxon:10088 13659 13663 mice +T393 PR:000004503 13699 13703 Atrx +T394 SO:0000359 13703 13707 flox +T395 NCBITaxon:10088 13708 13712 mice +T396 NCBITaxon:10088 13731 13735 mice +T397 SO:0000902 13748 13757 transgene +T398 GO:0010467 13790 13799 expressed +T399 GO:0065007 13810 13817 control +T400 GO:0065007 13825 13835 regulatory +T401 SO:0005836 13825 13844 regulatory elements +T402 NCBITaxon:10088 13852 13857 mouse +T403 PR:000007857 13858 13864 GATA-1 +T404 SO:0000704 13865 13869 gene +T405 PR:000007857 13871 13876 GATA1 +T406 GO:0010467 13899 13909 expression +T407 PR:000007857 13917 13922 GATA1 +T408 SO:0000902 13927 13936 transgene +T409 GO:0009790 13972 13985 embryogenesis +T410 PR:000007857 14032 14037 GATA1 +T411 GO:0010467 14042 14052 expression +T412 GO:0010467 14141 14150 expressed +T413 SO:0000359 14187 14199 loxP-flanked +T414 SO:0000141 14200 14213;14230 14249 transcription ... termination signals +T415 GO:0006415 14218 14241 translation termination +T416 SO:0000319 14218 14249 translation termination signals +T417 PR:000007857 14274 14279 GATA1 +T418 SO:0000902 14284 14293 transgene +T419 UBERON:0000085 14328 14334 morula +T420 GO:0007620 14370 14376 coitus +T421 PR:000004503 14410 14414 Atrx +T422 NCBITaxon:10088 14419 14423 mice +T423 SO:0000359 14438 14444 floxed +T424 PR:000004503 14454 14458 Atrx +T425 SO:0000359 14461 14465 flox +T426 GO:0007618 14472 14477 mated +T427 PR:000007857 14494 14499 GATA1 +T428 PR:000004503 14522 14526 Atrx +T429 PR:000007857 14531 14536 GATA1 +T430 PR:000004503 14549 14553 Atrx +T431 PR:000004503 14565 14569 Atrx +T432 PR:000007857 14576 14581 GATA1 +T433 GO:0007567 14608 14613 birth +T434 PR:000004503 14646 14650 Atrx +T435 UBERON:0000922 14662 14671 embryonic +T436 NCBITaxon:9606 14718 14723 human +T437 http://purl.obolibrary.org/obo/MONDO_0010519 14724 14729 ATR-X +T438 GO:0000805 14728 14729 X +T439 UBERON:0000113 14758 14767 adulthood +T440 UBERON:0000922 14786 14793 Embryos +T441 UBERON:0001040 14886 14894 yolk sac +T442 UBERON:0000922 14904 14910 embryo +T443 PR:000004503 14940 14944 Atrx +T444 PR:000004503 15109 15113 Atrx +T445 PR:000004503 15182 15186 Atrx +T446 PR:000004503 15247 15251 Atrx +T447 UBERON:0000922 15266 15275 embryonic +T448 NCBITaxon:10088 15289 15293 mice +T449 PR:000004503 15344 15348 Atrx +T450 UBERON:0000922 15353 15360 embryos +T451 GO:0016265 15370 15375 death +T452 UBERON:0000922 15377 15384 embryos +T453 UBERON:0002450 15442 15450 deciduas +T454 CHEBI:51686 15503 15515 haematoxylin +T455 PR:000004503 15544 15548 ATRX +T456 GO:0042571 15549 15557 antibody +T457 PR:000004503 15617 15621 Atrx +T458 GO:0010467 15633 15642 expressed +T459 UBERON:0000922 15664 15671 embryos +T460 GO:0010467 15685 15695 Expression +T461 UBERON:0000922 15715 15724 embryonic +T462 UBERON:0003124 15752 15759 chorion +T463 GO:0010467 15804 15814 expression +T464 UBERON:0004364 15836 15854 ectoplacental cone +T465 UBERON:0002450 15883 15891 decidual +T466 UBERON:0000479 15892 15898 tissue +T467 PR:000004503 15937 15941 Atrx +T468 GO:0010467 15942 15952 expression +T469 UBERON:0000088 15956 15967 trophoblast +T470 CL:0002488 15956 15979 trophoblast giant cells +T471 CL:0002488 15981 15985 TGCs +T472 UBERON:0004369 16003 16022 Reichert's membrane +T473 GO:0005634 16053 16059 nuclei +T474 CL:0002488 16069 16073 TGCs +T475 GO:0005634 16087 16094 nuclear +T476 PR:000004503 16110 16114 Atrx +T477 GO:0005721 16130 16161 pericentromeric heterochromatin +T478 UBERON:0005291 16255 16272 embryonic tissues +T479 GO:0010467 16295 16305 expression +T480 UBERON:0002450 16325 16333 decidual +T481 UBERON:0000479 16334 16340 tissue +T482 GO:0042571 16390 16398 antibody +T483 PR:000004503 16461 16465 Atrx +T484 UBERON:0000922 16470 16477 embryos +T485 UBERON:0000922 16586 16593 embryos +T486 UBERON:0002050 16694 16714 embryonic structures +T487 PR:000004503 16718 16722 Atrx +T488 UBERON:0004716 16727 16738 conceptuses +T489 UBERON:0000305 16768 16774 amnion +T490 UBERON:0003124 16779 16786 chorion +T491 UBERON:0000301 16816 16824;16857 16865 amniotic ... cavities +T492 UBERON:0003888 16826 16837;16857 16865 exocoelomic ... cavities +T493 UBERON:0008851 16843 16865 ectoplacental cavities +T494 UBERON:0000922 16906 16915 embryonic +T495 UBERON:0000923 16916 16927 germ layers +T496 UBERON:0000922 16956 16963 embryos +T497 UBERON:0002450 16987 16995 deciduas +T498 UBERON:0004716 17037 17048 conceptuses +T499 UBERON:0001040 17095 17103 yolk sac +T500 UBERON:0000922 17204 17210 embryo +T501 UBERON:0004716 17230 17241 conceptuses +T502 UBERON:0009746 17287 17296 head fold +T503 GO:0010467 17321 17331 expression +T504 UBERON:0000926 17345 17353 mesoderm +T505 PR:000016001 17361 17370 brachyury +T506 PR:000016001 17372 17373 T +T507 UBERON:0004341 17400 17416 primitive streak +T508 UBERON:0002328 17430 17439 notochord +T509 GO:0097617 17463 17476 hybridisation +T510 PR:000004503 17514 17518 Atrx +T511 UBERON:0000922 17523 17530 embryos +T512 GO:0007369 17535 17546 gastrulated +T513 PR:000004503 17596 17600 Atrx +T514 UBERON:0000922 17605 17612 embryos +T515 GO:0006915 17639 17648 apoptosis +T516 UBERON:0000922 17700 17707 embryos +T517 CL:0000445 17783 17798 apoptotic cells +T518 UBERON:0000922 17834 17841 embryos +T519 PR:000004503 17846 17850 Atrx +T520 UBERON:0000922 17855 17862 embryos +T521 PR:000004503 18003 18007 Atrx +T522 CL:0002322 18012 18020 ES cells +T523 PR:000004503 18069 18073 Atrx +T524 UBERON:0000922 18078 18085 embryos +T525 PR:000004503 18285 18289 Atrx +T526 UBERON:0000922 18294 18301 embryos +T527 GO:0008283 18314 18327 proliferation +T528 UBERON:0000922 18400 18406 embryo +T529 GO:0007067 18424 18431 mitosis +T530 CHEBI:15358 18462 18469 histone +T531 PR:000027594 18462 18472 histone H3 +T532 GO:0007067 18505 18512 mitotic +T533 UBERON:0000922 18541 18548 embryos +T534 GO:0007067 18568 18575 mitotic +T535 UBERON:0000922 18603 18610 embryos +T536 PR:000004503 18732 18736 Atrx +T537 UBERON:0000922 18741 18748 embryos +T538 GO:0006915 18863 18872 apoptosis +T539 PR:000004503 18920 18924 Atrx +T540 CL:0002322 18929 18937 ES cells +T541 PR:000004503 18970 18974 Atrx +T542 UBERON:0000922 18979 18986 embryos +T543 GO:0007067 18992 18999 mitotic +T544 CL:0002322 19013 19020 ES cell +T545 GO:0042571 19059 19067 antibody +T546 GO:0007067 19136 19143 mitotic +T547 UBERON:0000922 19163 19170 embryos +T548 PR:000004503 19240 19244 Atrx +T549 UBERON:0000088 19337 19348 trophoblast +T550 UBERON:0004345 19363 19376 Trophectoderm +T551 PR:000004503 19388 19392 Atrx +T552 UBERON:0000922 19397 19404 Embryos +T553 UBERON:0000922 19441 19448 embryos +T554 UBERON:0000922 19535 19541 embryo +T555 UBERON:0005292 19554 19576 extraembryonic tissues +T556 PR:000004503 19580 19584 Atrx +T557 UBERON:0004716 19589 19600 conceptuses +T558 UBERON:0000922 19636 19643 embryos +T559 UBERON:0002450 19662 19670 deciduas +T560 UBERON:0004345 19688 19707 trophectoderm layer +T561 PR:000004503 19741 19745 Atrx +T562 UBERON:0000922 19750 19757 embryos +T563 UBERON:0004364 19812 19830 ectoplacental cone +T564 UBERON:0002450 19891 19899 deciduas +T565 PR:000004503 19934 19938 Atrx +T566 UBERON:0000922 19943 19950 embryos +T567 GO:0010467 19991 20001 expression +T568 UBERON:0001987 20005 20014 placental +T569 CHEBI:81588 20005 20023 placental lactogen +T570 PR:000013543 20005 20025 placental lactogen-1 +T571 CHEBI:81588 20027 20029 Pl +T572 PR:000013543 20027 20031 Pl-1 +T573 CL:0002488 20072 20076 TGCs +T574 CHEBI:81588 20092 20094 Pl +T575 PR:000013543 20092 20096 Pl-1 +T576 GO:0010467 20097 20107 expressing +T577 UBERON:0002450 20130 20138 decidual +T578 UBERON:0000060 20139 20143 wall +T579 UBERON:0000922 20165 20171 embryo +T580 UBERON:0000088 20207 20218 trophoblast +T581 CL:0000351 20207 20224 trophoblast cells +T582 GO:0007566 20242 20254 implantation +T583 CHEBI:81588 20298 20300 Pl +T584 PR:000013543 20298 20302 Pl-1 +T585 GO:0010467 20303 20313 expressing +T586 UBERON:0002450 20340 20348 decidual +T587 GO:0007566 20349 20361 implantation +T588 PR:000004503 20379 20383 Atrx +T589 UBERON:0000922 20388 20395 embryos +T590 UBERON:0000922 20565 20572 embryos +T591 UBERON:0002450 20576 20584 deciduas +T592 CHEBI:81588 20598 20600 Pl +T593 PR:000013543 20598 20602 Pl-1 +T594 GO:0042571 20603 20611 antibody +T595 CL:0002488 20627 20630 TGC +T596 PR:000004503 20660 20664 Atrx +T597 PR:000004503 20705 20709 Atrx +T598 GO:0010467 20720 20729 expressed +T599 CL:0002488 20733 20744 giant cells +T600 UBERON:0000922 20775 20782 embryos +T601 UBERON:0000088 20824 20835 trophoblast +T602 CL:0002498 20879 20893 secondary TGCs +T603 UBERON:0004364 20933 20951 ectoplacental cone +T604 UBERON:0006280 20984 21003 polar trophectoderm +T605 UBERON:0000087 21018 21033 inner cell mass +T606 UBERON:0000358 21041 21051 blastocyst +T607 CL:0002497 21088 21100 primary TGCs +T608 UBERON:0006265 21140 21159 mural trophectoderm +T609 UBERON:0000358 21167 21177 blastocyst +T610 UBERON:0000358 21180 21191 blastocysts +T611 PR:000004503 21223 21227 Atrx +T612 SO:0000359 21230 21234 flox +T613 PR:000007857 21247 21252 GATA1 +T614 PR:000004503 21286 21290 Atrx +T615 PR:000007857 21295 21300 GATA1 +T616 UBERON:0000088 21376 21387 trophoblast +T617 UBERON:0000358 21411 21421 blastocyst +T618 UBERON:0000088 21469 21480 trophoblast +T619 PR:000004503 21500 21504 Atrx +T620 UBERON:0000358 21529 21539 blastocyst +T621 UBERON:0000358 21569 21580 blastocysts +T622 UBERON:0000086 21598 21612 zona pellucida +T623 UBERON:0000088 21630 21641 trophoblast +T624 CL:0000351 21630 21647 trophoblast cells +T625 UBERON:0000087 21671 21686 inner cell mass +T626 UBERON:0000088 21789 21800 trophoblast +T627 PR:000004503 21839 21843 Atrx +T628 UBERON:0000358 21848 21859 blastocysts +T629 PR:000004503 21861 21865 Atrx +T630 UBERON:0000358 21884 21895 blastocysts +T631 PR:000004503 21907 21911 Atrx +T632 SO:0001023 21914 21920 allele +T633 PR:000004503 21922 21926 Atrx +T634 PR:000004503 21940 21944 Atrx +T635 PR:000004503 21960 21964 Atrx +T636 CL:0002498 22061 22081 secondary giant cell +T637 PR:000004503 22140 22144 Atrx +T638 UBERON:0004716 22149 22160 conceptuses +T639 GO:0007566 22161 22168 implant +T640 GO:0007369 22197 22209 gastrulation +T641 PR:000004503 22259 22263 Atrx +T642 UBERON:0000088 22314 22325 trophoblast +T643 UBERON:0000922 22403 22409 embryo +T644 UBERON:0004716 22427 22438 conceptuses +T645 GO:0016265 22485 22488 die +T646 UBERON:0000088 22589 22600 trophoblast +T647 GO:0071514 22615 22624 Imprinted +T648 PR:000004503 22666 22670 Atrx +T649 SO:0001023 22673 22679 Allele +T650 UBERON:0005292 22683 22705 Extraembryonic Tissues +T651 NCBITaxon:10088 22724 22728 Mice +T652 NCBITaxon:10088 22737 22741 mice +T653 PR:000004503 22754 22758 Atrx +T654 SO:0001023 22763 22769 allele +T655 PR:000004503 22771 22775 Atrx +T656 PR:000007857 22783 22788 GATA1 +T657 GO:0007567 22849 22854 birth +T658 PR:000004503 22968 22972 Atrx +T659 PR:000007857 22978 22983 GATA1 +T660 UBERON:0000922 23048 23055 embryos +T661 GO:0016265 23056 23060 died +T662 UBERON:0007023 23081 23086 adult +T663 NCBITaxon:10088 23102 23106 mice +T664 GO:0000003 23204 23213 reproduce +T665 PR:000004503 23223 23227 Atrx +T666 UBERON:0000922 23250 23257 embryos +T667 PR:000004503 23284 23288 Atrx +T668 SO:0001023 23291 23297 allele +T669 PR:000004503 23332 23336 Atrx +T670 SO:0001023 23341 23347 allele +T671 NCBITaxon:10088 23379 23384 mouse +T672 GO:0000805 23386 23398 X chromosome +T673 GO:0009048 23386 23411 X chromosome inactivation +T674 GO:0071514 23435 23445 imprinting +T675 UBERON:0004345 23453 23466 trophectoderm +T676 UBERON:0008776 23471 23489 primitive endoderm +T677 UBERON:0005292 23521 23543 extraembryonic tissues +T678 GO:0060817 23558 23573;23578 23599 inactivation of ... paternal X chromosome +T679 GO:0060817 23558 23573;23601 23603 inactivation of ... Xp +T680 GO:0000805 23587 23599 X chromosome +T681 UBERON:0000479 23627 23634 tissues +T682 UBERON:0000922 23642 23648 embryo +T683 UBERON:0000087 23674 23689 inner cell mass +T684 UBERON:0000358 23697 23707 blastocyst +T685 GO:0000805 23709 23710 X +T686 GO:0009048 23709 23723 X-inactivation +T687 UBERON:0000922 23758 23767 embryonic +T688 PR:000004503 23783 23787 Atrx +T689 GO:0060819 23819 23843 imprinted X-inactivation +T690 GO:0000805 23829 23830 X +T691 GO:0016458 23875 23884 silencing +T692 PR:000004503 23911 23915 Atrx +T693 SO:0001023 23918 23924 allele +T694 PR:000004503 23943 23947 Atrx +T695 SO:0001023 23952 23958 allele +T696 GO:0000805 23982 23983 X +T697 UBERON:0005292 24012 24034 extraembryonic tissues +T698 PR:000004503 24056 24060 Atrx +T699 PR:000004503 24094 24098 Atrx +T700 UBERON:0000922 24111 24120 embryonic +T701 PR:000004503 24146 24150 Atrx +T702 GO:0000806 24155 24156 Y +T703 GO:0071514 24213 24222 imprinted +T704 PR:000004503 24262 24266 Atrx +T705 SO:0001023 24269 24275 allele +T706 UBERON:0000922 24288 24297 embryonic +T707 UBERON:0000922 24344 24351 embryos +T708 PR:000004503 24400 24404 Atrx +T709 SO:0000359 24404 24408 flox +T710 SO:0000359 24409 24413 flox +T711 PR:000007857 24437 24442 GATA1 +T712 PR:000004503 24465 24469 Atrx +T713 GO:0000806 24472 24473 Y +T714 PR:000007857 24474 24479 GATA1 +T715 PR:000004503 24527 24531 Atrx +T716 PR:000004503 24543 24547 Atrx +T717 GO:0000806 24552 24553 Y +T718 PR:000007857 24554 24559 GATA1 +T719 PR:000004503 24589 24593 Atrx +T720 PR:000007857 24601 24606 GATA1 +T721 PR:000004503 24646 24650 Atrx +T722 SO:0001023 24653 24659 allele +T723 UBERON:0000922 24685 24692 Embryos +T724 UBERON:0002450 24717 24725 deciduas +T725 PR:000004503 24783 24787 ATRX +T726 GO:0042571 24788 24796 antibody +T727 UBERON:0000922 24843 24849 embryo +T728 PR:000004503 24897 24901 Atrx +T729 GO:0010467 24902 24912 expression +T730 UBERON:0002532 24947 24955 epiblast +T731 UBERON:0000922 24957 24963 embryo +T732 UBERON:0000922 25000 25007 embryos +T733 UBERON:0002532 25038 25046 epiblast +T734 UBERON:0000922 25072 25079 embryos +T735 PR:000004503 25126 25130 Atrx +T736 PR:000004503 25160 25164 Atrx +T737 SO:0001023 25169 25175 allele +T738 PR:000004503 25208 25212 Atrx +T739 PR:000004503 25242 25246 Atrx +T740 SO:0001023 25249 25255 allele +T741 PR:000004503 25305 25309 Atrx +T742 SO:0000704 25310 25314 gene +T743 GO:0000805 25344 25345 X +T744 GO:0009048 25344 25358 X-inactivation +T745 UBERON:0002532 25366 25374 epiblast +T746 PR:000004503 25394 25398 Atrx +T747 GO:0010467 25399 25409 expression +T748 UBERON:0005292 25440 25462 extraembryonic tissues +T749 UBERON:0000922 25504 25513 embryonic +T750 UBERON:0003374 25522 25540 chorionic ectoderm +T751 PR:000004503 25554 25558 Atrx +T752 GO:0010467 25559 25569 expression +T753 CL:0000221 25602 25610;25625 25633 cells of ... ectoderm +T754 UBERON:0003374 25615 25633 chorionic ectoderm +T755 PR:000004503 25635 25639 Atrx +T756 GO:0010467 25640 25650 expression +T757 UBERON:0000478 25686 25711 extraembryonic structures +T758 CL:0002488 25723 25727 TGCs +T759 GO:0016458 25760 25769 silencing +T760 PR:000004503 25789 25793 Atrx +T761 SO:0001023 25796 25802 allele +T762 UBERON:0003374 25828 25846 chorionic ectoderm +T763 GO:0000805 25912 25913 X +T764 GO:0009048 25912 25926 X-inactivation +T765 UBERON:0002532 25954 25962 epiblast +T766 PR:000004503 25968 25972 Atrx +T767 SO:0001023 25975 25981 allele +T768 GO:0060819 26034 26058 imprinted X-inactivation +T769 GO:0000805 26044 26045 X +T770 UBERON:0000922 26071 26080 embryonic +T771 PR:000004503 26163 26167 Atrx +T772 NCBITaxon:10088 26179 26184 mouse +T773 PR:000004503 26267 26271 Atrx +T774 CL:0002322 26289 26297 ES cells +T775 UBERON:0014374 26302 26317 embryoid bodies +T776 NCBITaxon:10088 26342 26347 mouse +T777 UBERON:0000922 26348 26355 embryos +T778 PR:000004503 26358 26362 Atrx +T779 CL:0002322 26366 26374 ES Cells +T780 PR:000004503 26376 26380 Atrx +T781 CL:0002322 26385 26393 ES cells +T782 PR:000004503 26558 26562 Atrx +T783 GO:0010467 26573 26582 expressed +T784 CL:0002322 26586 26594 ES cells +T785 PR:000004503 26632 26636 Atrx +T786 PR:000004503 26706 26710 Atrx +T787 SO:0001023 26711 26717 allele +T788 PR:000004503 26771 26775 Atrx +T789 CL:0002322 26780 26788 ES cells +T790 GO:0006915 26839 26848 apoptosis +T791 CL:0002322 26886 26894 ES cells +T792 PR:000004503 26903 26907 Atrx +T793 PR:000004503 26939 26943 Atrx +T794 NCBITaxon:10088 26956 26961 mouse +T795 UBERON:0000922 26962 26969 embryos +T796 PR:000004503 27023 27027 Atrx +T797 UBERON:0000922 27108 27117 embryonic +T798 UBERON:0001851 27118 27124 cortex +T799 GO:0007567 27133 27138 natal +T800 PR:000004503 27157 27161 Atrx +T801 GO:0010467 27162 27172 expression +T802 NCBITaxon:10088 27203 27208 mouse +T803 UBERON:0001890 27209 27218 forebrain +T804 PR:000004503 27229 27233 Atrx +T805 SO:0000359 27233 27237 flox +T806 SO:0001023 27238 27244 allele +T807 NCBITaxon:9606 27270 27275 human +T808 PR:000004503 27276 27280 ATRX +T809 GO:0032991 27322 27329 complex +T810 PR:000006283 27335 27339 Daxx +T811 GO:0042981 27409 27432 regulation of apoptosis +T812 NCBITaxon:10088 27477 27482 mouse +T813 PR:000004503 27483 27487 Atrx +T814 PR:000006283 27488 27492 Daxx +T815 GO:0032991 27493 27500 complex +T816 PR:000004503 27521 27525 Atrx +T817 CL:0002322 27620 27628 ES cells +T818 GO:0006915 27663 27672 apoptosis +T819 PR:000004503 27730 27734 Atrx +T820 GO:0006915 27789 27798 apoptosis +T821 PR:000004503 27815 27819 Atrx +T822 UBERON:0001890 27827 27836 forebrain +T823 PR:000004503 27867 27871 Atrx +T824 PR:000004503 27929 27933 Atrx +T825 NCBITaxon:10088 27956 27961 Mouse +T826 UBERON:0000088 27962 27973 Trophoblast +T827 PR:000004503 27988 27992 Atrx +T828 NCBITaxon:10088 28002 28006 mice +T829 UBERON:0000922 28026 28033 embryos +T830 GO:0016265 28034 28037 die +T831 GO:0016265 28064 28069 death +T832 PR:000004503 28071 28075 Atrx +T833 UBERON:0000922 28080 28087 embryos +T834 GO:0007067 28115 28122 mitotic +T835 UBERON:0000922 28178 28187 embryonic +T836 UBERON:0004716 28207 28216 conceptus +T837 PR:000004503 28254 28258 Atrx +T838 UBERON:0000922 28263 28270 embryos +T839 UBERON:0000088 28315 28326 trophoblast +T840 CL:0002488 28371 28375 TGCs +T841 UBERON:0004716 28392 28401 conceptus +T842 UBERON:0004364 28437 28455 ectoplacental cone +T843 CL:0000415 28476 28483;28490 28494 diploid ... cell +T844 CL:0002488 28484 28494 giant cell +T845 CL:0002488 28512 28516 TGCs +T846 GO:0007067 28548 28555 mitotic +T847 UBERON:0000483 28586 28596 epithelial +T848 UBERON:0001987 28627 28635 placenta +T849 UBERON:0002450 28685 28692 decidua +T850 UBERON:0000995 28779 28786 uterine +T851 UBERON:0000479 28787 28793 tissue +T852 UBERON:0002450 28845 28852 decidua +T853 GO:0007566 28859 28871 implantation +T854 GO:0046903 28879 28888 secreting +T855 GO:0065007 28903 28911 regulate +T856 PR:000004503 28950 28954 Atrx +T857 UBERON:0000922 28959 28966 embryos +T858 GO:0007566 28977 28984 implant +T859 CL:0002488 29044 29047 TGC +T860 UBERON:0000922 29084 29093 Embryonic +T861 NCBITaxon:10088 29107 29111 mice +T862 PR:000004503 29130 29134 Atrx +T863 NCBITaxon:9606 29215 29220 human +T864 http://purl.obolibrary.org/obo/MONDO_0010519 29221 29235 ATR-X syndrome +T865 GO:0000805 29225 29226 X +T866 PR:000004503 29269 29273 Atrx +T867 UBERON:0000088 29281 29292 trophoblast +T868 NCBITaxon:10088 29308 29312 mice +T869 PR:000004503 29322 29326 ATRX +T870 NCBITaxon:9606 29362 29367 human +T871 UBERON:0000088 29368 29379 trophoblast +T872 GO:0007567 29393 29398 birth +T873 http://purl.obolibrary.org/obo/MONDO_0010519 29421 29435 ATR-X syndrome +T874 GO:0000805 29425 29426 X +T875 NCBITaxon:10088 29535 29539 mice +T876 PR:000004503 29552 29556 Atrx +T877 PR:000004503 29635 29639 Atrx +T878 http://purl.obolibrary.org/obo/MONDO_0000001 29678 29685 disease +T879 NCBITaxon:9606 29721 29726 human +T880 http://purl.obolibrary.org/obo/MONDO_0010519 29727 29732 ATR-X +T881 GO:0000805 29731 29732 X +T882 SO:0001023 29778 29785 alleles +T883 PR:000004503 29804 29808 ATRX +T884 PR:000004503 29934 29938 ATRX +T885 NCBITaxon:10376 29977 29995 Epstein-Barr virus +T886 GO:0009294 29996 30007 transformed +T887 CL:0000542 30008 30019 lymphocytes +T888 NCBITaxon:9606 30027 30032 human +T889 PR:000004503 30143 30147 ATRX +T890 SO:0001023 30148 30154 allele +T891 NCBITaxon:9606 30161 30166 human +T892 NCBITaxon:10088 30210 30215 mouse +T893 PR:000004503 30241 30245 ATRX +T894 NCBITaxon:9606 30275 30280 human +T895 PR:000004503 30351 30355 Atrx +T896 NCBITaxon:39107 30363 30369 murine +T897 UBERON:0004345 30370 30383 trophectoderm +T898 PR:000004503 30432 30436 Atrx +T899 UBERON:0000922 30441 30448 embryos +T900 PR:000004503 30496 30500 Atrx +T901 UBERON:0000479 30534 30541 tissues +T902 UBERON:0000922 30549 30555 embryo +T903 PR:000004503 30585 30589 Atrx +T904 GO:0010467 30600 30609 expressed +T905 UBERON:0000922 30643 30649 embryo +T906 PR:000004503 30696 30700 Atrx +T907 UBERON:0000479 30766 30773 tissues +T908 UBERON:0000922 30827 30834 embryos +T909 UBERON:0005292 30862 30884 extraembryonic tissues +T910 PR:000004503 30923 30927 Atrx +T911 NCBITaxon:10088 30941 30946 mouse +T912 PR:000004503 31035 31039 Atrx +T913 SO:0000359 31039 31043 flox +T914 SO:0001023 31044 31050 allele +T915 UBERON:0000479 31089 31095 tissue +T916 SO:0000902 31109 31119 transgenes +T917 PR:000004503 31196 31200 Atrx +T918 CL:0000540 31208 31216 neuronal +T919 UBERON:0007023 31236 31241 adult +T920 NCBITaxon:10088 31242 31246 mice +T921 PR:000004503 31275 31279 Atrx +T922 NCBITaxon:10088 31316 31321 mouse +T923 PR:000004503 31387 31391 Atrx +T924 UBERON:0000479 31423 31430 tissues +T925 NCBITaxon:10088 31449 31453 mice +T926 UBERON:0000479 31461 31468 tissues +T927 PR:000004503 31500 31504 Atrx +T928 PR:000004503 31518 31522 Atrx +T929 GO:0000805 31560 31561 X +T930 GO:0009048 31560 31574 X-inactivation +T931 PR:000004503 31612 31616 Atrx +T932 NCBITaxon:10088 31644 31649 mouse +T933 SO:0000704 31650 31655 genes +T934 GO:0007566 31702 31714 implantation +T935 UBERON:0000088 31740 31751 trophoblast +T936 UBERON:0001987 31755 31764 placental +T937 PR:000004503 31876 31880 Atrx +T938 UBERON:0000088 31884 31895 trophoblast +T939 PR:000004503 31909 31913 Atrx +T940 UBERON:0000922 31921 31928 embryos +T941 UBERON:0000922 31951 31958 embryos +T942 GO:0048866 32007 32023;32036 32046 specification of ... stem cells +T943 UBERON:0000088 32024 32035 trophoblast +T944 CL:0000351 32024 32035;32041 32046 trophoblast ... cells +T945 CL:0000034 32036 32046 stem cells +T946 PR:000005296 32056 32060 Cdx2 +T947 CL:0000034 32068 32077 stem cell +T948 GO:0019827 32068 32089 stem cell maintenance +T949 GO:0072089 32068 32077;32094 32107 stem cell ... proliferation +T950 PR:000003463 32117 32122 Eomes +T951 PR:000005296 32125 32129 Cdx2 +T952 UBERON:0000922 32137 32144 embryos +T953 GO:0007566 32153 32160 implant +T954 GO:0016265 32165 32168 die +T955 PR:000003463 32205 32210 Eomes +T956 UBERON:0000358 32218 32229 blastocysts +T957 GO:0007566 32230 32237 implant +T958 UBERON:0000995 32247 32253 uterus +T959 GO:0007566 32277 32289 implantation +T960 UBERON:0002050 32316 32325;32344 32354 embryonic ... structures +T961 UBERON:0000478 32329 32354 extraembryonic structures +T962 PR:000004503 32374 32378 Atrx +T963 UBERON:0000922 32386 32393 embryos +T964 GO:0007566 32394 32401 implant +T965 UBERON:0002050 32439 32459 embryonic structures +T966 PR:000004503 32476 32480 Atrx +T967 NCBITaxon:10088 32533 32537 mice +T968 CHEBI:22695 32558 32563 basic +T969 SO:0001114 32564 32569 helix +T970 SO:0001114 32575 32580 helix +T971 PR:000008439 32602 32607 Hand1 +T972 PR:000008439 32609 32614 Hand1 +T973 UBERON:0000922 32680 32689 embryonic +T974 PR:000004503 32713 32717 Atrx +T975 UBERON:0000922 32725 32732 embryos +T976 PR:000008439 32746 32751 Hand1 +T977 UBERON:0004364 32790 32808 ectoplacental cone +T978 CL:0002488 32824 32828 TGCs +T979 PR:000004503 32843 32847 Atrx +T980 PR:000008439 32883 32888 Hand1 +T981 UBERON:0004716 32896 32907 conceptuses +T982 PR:000004503 32948 32952 Atrx +T983 PR:000008439 32968 32973 Hand1 +T984 CL:0002498 32995 33015 secondary giant cell +T985 GO:0048468 33011 33025 cell formation +T986 UBERON:0000088 33039 33050 trophoblast +T987 UBERON:0000358 33067 33078 blastocysts +T988 PR:000008439 33096 33101 Hand1 +T989 CL:0002498 33146 33160 secondary TGCs +T990 UBERON:0000088 33181 33192 trophoblast +T991 CL:0000351 33181 33198 trophoblast cells +T992 UBERON:0004364 33234 33252 ectoplacental cone +T993 PR:000004503 33290 33294 Atrx +T994 PR:000008439 33300 33305 Hand1 +T995 PR:000004503 33348 33352 Atrx +T996 GO:0000785 33402 33411 chromatin +T997 PR:000004503 33467 33471 Atrx +T998 PR:000008439 33497 33502 Hand1 +T999 GO:0010467 33503 33513 expression +T1000 PR:000008439 33626 33631 Hand1 +T1001 UBERON:0000955 33663 33668 brain +T1002 PR:000004503 33678 33682 Atrx +T1003 NCBITaxon:10088 33692 33696 mice +T1004 CL:0000540 33752 33759 neurons +T1005 CL:0002498 33770 33784 secondary TGCs +T1006 PR:000004503 33811 33815 Atrx +T1007 UBERON:0000479 33892 33899 tissues +T1008 NCBITaxon:10088 33918 33923 mouse +T1009 PR:000004503 33967 33971 Atrx +T1010 GO:0010467 33990 34000 expression +T1011 UBERON:0000479 34009 34015 tissue +T1012 SO:0000704 34025 34030 genes +T1013 CHEBI:5386 34096 34102 globin +T1014 SO:0000704 34103 34108 genes +T1015 GO:0065007 34156 34166 regulation +T1016 NCBITaxon:9606 34170 34175 human +T1017 PR:000004503 34176 34180 ATRX +T1018 GO:0010467 34198 34207 expressed +T1019 CL:0000764 34264 34273 erythroid +T1020 PR:000004503 34284 34288 Atrx +T1021 GO:0060819 34297 34321 Imprinted X-Inactivation +T1022 GO:0000805 34307 34308 X +T1023 UBERON:0005292 34325 34347 Extraembryonic Tissues +T1024 NCBITaxon:10088 34366 34370 Mice +T1025 UBERON:0000922 34440 34447 embryos +T1026 PR:000004503 34472 34476 Atrx +T1027 SO:0001023 34479 34485 allele +T1028 GO:0060819 34519 34543 imprinted X-inactivation +T1029 GO:0000805 34529 34530 X +T1030 GO:0016458 34562 34570 silences +T1031 UBERON:0000922 34601 34610 embryonic +T1032 NCBITaxon:39107 34633 34639 murine +T1033 UBERON:0000479 34640 34647 tissues +T1034 GO:0016458 34654 34663 Silencing +T1035 PR:000004503 34671 34675 Atrx +T1036 SO:0001023 34678 34684 allele +T1037 PR:000004503 34728 34732 Atrx +T1038 UBERON:0005292 34740 34762 extraembryonic tissues +T1039 PR:000004503 34816 34820 Atrx +T1040 SO:0001023 34828 34834 allele +T1041 PR:000004503 34877 34881 Atrx +T1042 GO:0000003 34931 34940 reproduce +T1043 GO:0016458 34973 34980 silence +T1044 PR:000007899 35004 35008 Atrx +T1045 SO:0001023 35011 35017 allele +T1046 UBERON:0005292 35025 35047 extraembryonic tissues +T1047 PR:000007899 35111 35115 Atrx +T1048 PR:000004503 35131 35135 Atrx +T1049 UBERON:0000088 35194 35205 trophoblast +T1050 NCBITaxon:10088 35252 35257 mouse +T1051 PR:000004503 35276 35280 Atrx +T1052 NCBITaxon:39107 35366 35372 murine +T1053 GO:0000805 35373 35374 X +T1054 SO:0000704 35382 35387 genes +T1055 UBERON:0000922 35422 35431 embryonic +T1056 PR:000006498 35485 35493 dyskerin +T1057 PR:000006498 35495 35499 Dkc1 +T1058 CHEBI:14314 35502 35521 glucose 6-phosphate +T1059 PR:000005430 35548 35561 choroideremia +T1060 PR:000005430 35563 35566 Chm +T1061 SO:0000704 35568 35573 genes +T1062 UBERON:0000922 35580 35589 embryonic +T1063 UBERON:0000922 35613 35620 embryos +T1064 UBERON:0000922 35649 35658 embryonic +T1065 UBERON:0000479 35667 35674 tissues +T1066 NCBITaxon:10088 35691 35695 mice +T1067 SO:0000704 35724 35729 genes +T1068 GO:0000805 35758 35770 X chromosome +T1069 GO:0016265 35776 35779 die +T1070 PR:000004503 35875 35879 Atrx +T1071 SO:0000704 35887 35892 genes +T1072 UBERON:0005292 36007 36029 extraembryonic tissues +T1073 GO:0010467 36033 36040 express +T1074 GO:0000805 36069 36081 X chromosome +T1075 GO:0010467 36093 36103 expression +T1076 PR:000004503 36120 36124 Atrx +T1077 SO:0001023 36127 36133 allele +T1078 UBERON:0005292 36155 36177 extraembryonic tissues +T1079 PR:000004503 36185 36189 Atrx +T1080 GO:0000805 36249 36250 X +T1081 SO:0000704 36258 36263 genes +T1082 GO:0016458 36265 36277;36287 36291 silencing of ... gene +T1083 PR:000004503 36282 36286 Atrx +T1084 SO:0000704 36287 36291 gene +T1085 PR:000004503 36368 36372 Atrx +T1086 UBERON:0005292 36418 36440 extraembryonic tissues +T1087 PR:000004503 36498 36502 Atrx +T1088 SO:0001023 36516 36522 allele +T1089 GO:0016458 36537 36545 silenced +T1090 NCBITaxon:10088 36558 36563 mouse +T1091 UBERON:0000088 36564 36575 trophoblast +T1092 CL:0000351 36564 36575;36581 36585 trophoblast ... cell +T1093 CL:0000034 36576 36585 stem cell +T1094 PR:000004503 36613 36617 Atrx +T1095 GO:0060819 36643 36667 imprinted X-inactivation +T1096 GO:0000805 36653 36654 X +T1097 UBERON:0005292 36675 36697 extraembryonic tissues +T1098 GO:0010467 36730 36740 expression +T1099 PR:000004503 36758 36762 Atrx +T1100 SO:0001023 36765 36771 allele +T1101 PR:000004503 36825 36829 Atrx +T1102 SO:0001023 36834 36840 allele +T1103 GO:0060819 36950 36974 imprinted X-inactivation +T1104 GO:0000805 36960 36961 X +T1105 NCBITaxon:10088 37015 37020 mouse +T1106 UBERON:0005292 37021 37043 extraembryonic tissues +T1107 GO:0000805 37130 37142 X chromosome +T1108 GO:0010467 37353 37363 expression +T1109 GO:0000805 37390 37391 X +T1110 PR:000033987 37399 37403 lacZ +T1111 SO:0000902 37425 37435 transgenes +T1112 GO:0016458 37449 37457 silenced +T1113 CL:0000349 37486 37506 extraembryonic cells +T1114 UBERON:0000922 37491 37500 embryonic +T1115 CL:0000349 37562 37582 extraembryonic cells +T1116 UBERON:0000922 37567 37576 embryonic +T1117 GO:0006260 37636 37647 replication +T1118 UBERON:0000922 37757 37764 embryos +T1119 GO:0071514 37873 37882 imprinted +T1120 UBERON:0000922 37897 37906 embryonic +T1121 GO:0016458 37930 37939 silencing +T1122 GO:0030154 37958 37962;37973 37988 cell ... differentiation +T1123 GO:0071514 38082 38092 imprinting +T1124 SO:0000704 38141 38152 genetically +T1125 UBERON:0000922 38163 38170 embryos +T1126 SO:0001023 38273 38280 alleles +T1127 SO:0000704 38284 38289 genes +T1128 UBERON:0000922 38316 38325 embryonic +T1129 PR:000004503 38576 38580 Atrx +T1130 GO:0016265 38617 38622 dying +T1131 GO:0060817 38720 38735;38740 38750 inactivation of ... paternal X +T1132 GO:0000805 38749 38750 X +T1133 PR:000004503 38804 38808 Atrx +T1134 SO:0000704 38809 38813 gene +T1135 PR:000004503 38887 38891 Atrx +T1136 SO:0001023 38906 38912 allele +T1137 GO:0016458 38956 38965 silencing +T1138 SO:0000704 38997 39002 genes +T1139 GO:0000805 39019 39031 X chromosome +T1140 GO:0060817 39082 39097;39109 39122 inactivation of ... Xp chromosome +T1141 UBERON:0000922 39130 39139 embryonic +T1142 SO:0001023 39159 39166 allelic +T1143 GO:0010467 39167 39177 expression +T1144 GO:0000805 39181 39182 X +T1145 SO:0000704 39190 39195 genes +T1146 UBERON:0000088 39203 39214 trophoblast +T1147 http://purl.obolibrary.org/obo/MONDO_0010519 39231 39245 ATR-X syndrome +T1148 GO:0000805 39235 39236 X +T1149 NCBITaxon:9606 39259 39264 human +T1150 SO:0000704 39265 39272 genetic +T1151 http://purl.obolibrary.org/obo/MONDO_0003847 39265 39280 genetic disease +T1152 GO:0000785 39318 39327 chromatin +T1153 GO:0006338 39318 39339 chromatin remodelling +T1154 PR:000004503 39378 39382 ATRX +T1155 SO:0000704 39394 39398 gene +T1156 GO:0010467 39394 39409 gene expression +T1157 http://purl.obolibrary.org/obo/MONDO_0010519 39533 39547 ATR-X syndrome +T1158 GO:0000805 39537 39538 X +T1159 PR:000004503 39666 39670 Atrx +T1160 NCBITaxon:10088 39689 39694 mouse +T1161 UBERON:0001890 39695 39704 forebrain +T1162 PR:000004503 39719 39723 Atrx +T1163 SO:0000359 39723 39727 flox +T1164 SO:0001023 39728 39734 allele +T1165 PR:000004503 39762 39766 Atrx +T1166 CL:0000540 39819 39826 neurons +T1167 PR:000004503 39856 39860 Atrx +T1168 UBERON:0000479 39870 39877 tissues +T1169 NCBITaxon:33208 39915 39921 animal +T1170 PR:000004503 39945 39949 Atrx +T1171 SO:0000704 39950 39954 gene +T1172 UBERON:0000922 39971 39980 embryonic +T1173 PR:000004503 40031 40035 Atrx +T1174 NCBITaxon:39107 40060 40066 murine +T1175 UBERON:0000088 40067 40078 trophoblast +T1176 PR:000004503 40093 40097 Atrx +T1177 GO:0060819 40116 40151 imprinted X-chromosome inactivation +T1178 GO:0000805 40126 40138 X-chromosome +T1179 UBERON:0005292 40159 40181 extraembryonic tissues +T1180 NCBITaxon:10088 40205 40209 mice +T1181 GO:0048468 40235 40248;40252 40257 Generation of ... cells +T1182 CL:0002322 40249 40257 ES cells +T1183 PR:000004503 40270 40274 Atrx +T1184 SO:0000359 40274 40278 flox +T1185 SO:0001023 40279 40285 allele +T1186 SO:0001644 40301 40317 targeting vector +T1187 SO:0000346 40348 40357 loxP site +T1188 SO:0000188 40365 40371 intron +T1189 SO:0000359 40381 40393 loxP-flanked +T1190 SO:0005853 40413 40421 cassette +T1191 SO:0000188 40425 40431 intron +T1192 PR:000004503 40442 40446 Atrx +T1193 SO:0000704 40447 40451 gene +T1194 SO:0000155 40535 40542 plasmid +T1195 CL:0002322 40592 40600 ES cells +T1196 CHEBI:42768 40628 40632 G418 +T1197 CHEBI:465284 40637 40648 ganciclovir +T1198 GO:0097617 40751 40764 hybridisation +T1199 SO:0000112 40797 40804 primers +T1200 GO:0097617 40995 41008 hybridisation +T1201 SO:0000188 41034 41040 intron +T1202 SO:0000006 41047 41058 PCR product +T1203 SO:0000112 41074 41081 primers +T1204 SO:0000346 41121 41130 loxP site +T1205 SO:0000188 41138 41144 intron +T1206 GO:0035825 41177 41189 crossed-over +T1207 SO:0000112 41230 41237 primers +T1208 PR:000004503 41304 41308 Atrx +T1209 CL:0002322 41313 41321 ES cells +T1210 UBERON:0014374 41326 41341 embryoid bodies +T1211 CL:0002322 41344 41351 ES cell +T1212 PR:000004503 41371 41375 Atrx +T1213 SO:0000359 41375 41379 flox +T1214 SO:0001023 41380 41386 allele +T1215 GO:0009294 41420 41431 transfected +T1216 MOP:0000780 41448 41451 cut +T1217 GO:0010467 41456 41466 expression +T1218 SO:0000155 41467 41474 plasmid +T1219 SO:0000243 41487 41491 IRES +T1220 GO:0009294 41513 41525 transfection +T1221 CHEBI:42768 41603 41607 G418 +T1222 SO:0005853 41785 41793 cassette +T1223 GO:0097617 41825 41838 hybridisation +T1224 SO:0000188 41848 41854 intron +T1225 CHEBI:33893 42009 42016 Reagent +T1226 GO:0097617 42083 42093 hybridised +T1227 SO:0000147 42119 42123 exon +T1228 PR:000004503 42134 42138 Atrx +T1229 SO:0000704 42139 42143 gene +T1230 SO:0000112 42160 42167 primers +T1231 GO:0097617 42238 42248 hybridised +T1232 PR:000003676 42256 42263 β-actin +T1233 PR:000004503 42361 42365 Atrx +T1234 NCBITaxon:10088 42439 42444 mouse +T1235 PR:000004503 42461 42465 ATRX +T1236 GO:0042571 42466 42474 antibody +T1237 GO:0007049 42497 42507 cell cycle +T1238 GO:0006915 42512 42521 apoptosis +T1239 CL:0002322 42597 42604 ES cell +T1240 UBERON:0014374 42620 42635 embryoid bodies +T1241 SO:0001026 42714 42721 Genomic +T1242 NCBITaxon:9606 42886 42891 human +T1243 NCBITaxon:10088 42922 42927 mouse +T1244 SO:0000028 43229 43231 bp +T1245 SO:0000006 43232 43243 PCR product +T1246 SO:0000112 43245 43252 primers +T1247 NCBITaxon:10088 43306 43311 mouse +T1248 PR:000004372 43312 43318 agouti +T1249 SO:0000704 43319 43323 gene +T1250 SO:0000006 43384 43395 PCR product +T1251 SO:0000359 43513 43519 floxed +T1252 NCBITaxon:10088 43520 43524 mice +T1253 NCBITaxon:10088 43537 43541 mice +T1254 PR:000004503 43553 43557 Atrx +T1255 SO:0000359 43557 43561 flox +T1256 CL:0002322 43562 43569 ES cell +T1257 UBERON:0000358 43604 43615 blastocysts +T1258 GO:0007618 43719 43724 mated +T1259 PR:000004503 43793 43797 Atrx +T1260 SO:0000359 43797 43801 flox +T1261 SO:0001023 43802 43808 allele +T1262 UBERON:0002415 43863 43867 tail +T1263 SO:0000188 43882 43888 intron +T1264 PR:000004503 43951 43955 Atrx +T1265 SO:0000359 43955 43959 flox +T1266 NCBITaxon:10088 43960 43964 mice +T1267 PR:000007857 43983 43988 GATA1 +T1268 NCBITaxon:10088 44004 44008 mice +T1269 SO:0001023 44047 44054 alleles +T1270 GO:0097617 44179 44192 hybridisation +T1271 UBERON:0002450 44220 44228 decidual +T1272 UBERON:0000479 44273 44279 tissue +T1273 UBERON:0000922 44342 44349 embryos +T1274 CHEBI:16236 44413 44420 ethanol +T1275 CHEBI:27338 44432 44438 xylene +T1276 NCBITaxon:9986 44703 44709 rabbit +T1277 GO:0042571 44721 44731 antibodies +T1278 PR:000004503 44740 44744 ATRX +T1279 UBERON:0001987 44780 44789 Placental +T1280 CHEBI:81588 44780 44798 Placental lactogen +T1281 PR:000013543 44780 44800 Placental lactogen-I +T1282 CHEBI:32958 44875 44882 phospho +T1283 CHEBI:15358 44891 44898 histone +T1284 PR:000027594 44891 44901 histone H3 +T1285 CHEBI:51686 45029 45041 haematoxylin +T1286 CL:0000445 45105 45120 apoptotic cells +T1287 GO:0008219 45148 45158 cell death +T1288 CHEBI:51231 45271 45275 DAPI +T1289 SO:0000440 45277 45283 Vector +T1290 GO:0097617 45400 45414 hybridisations +T1291 UBERON:0000922 45441 45448 embryos +T1292 UBERON:0005292 45483 45505 extraembryonic tissues +T1293 PR:000016001 45515 45524 brachyury +T1294 PR:000016001 45526 45527 T +T1295 UBERON:0002450 45560 45568 decidual +T1296 GO:0007566 45569 45581 implantation +T1297 UBERON:0000922 45599 45606 embryos +T1298 UBERON:0001987 45642 45651 placental +T1299 CHEBI:81588 45642 45660 placental lactogen +T1300 PR:000013543 45642 45662 placental lactogen-1 +T1301 CHEBI:81588 45664 45666 Pl +T1302 PR:000013543 45664 45668 Pl-1 +T1303 UBERON:0000358 45712 45722 Blastocyst +T1304 GO:0030728 45749 45757 ovulated +T1305 NCBITaxon:10088 45778 45782 mice +T1306 PR:000004503 45784 45788 Atrx +T1307 SO:0000359 45791 45795 flox +T1308 GO:0007618 45802 45807 mated +T1309 PR:000007857 45822 45827 GATA1 +T1310 UBERON:0000358 45857 45868 blastocysts +T1311 UBERON:0002247 45887 45900 uterine horns +T1312 UBERON:0000358 45947 45958 blastocysts +T1313 UBERON:0000479 45986 45992 tissue +T1314 PR:000004503 46180 46184 Atrx +T1315 GO:0007049 46305 46315 Cell Cycle +T1316 PR:000004503 46328 46332 Atrx +T1317 CL:0002322 46355 46363 ES Cells +T1318 CHEBI:472552 46401 46405 BrdU +T1319 CL:0002322 46413 46421 ES cells +T1320 PR:000004503 46449 46453 Atrx +T1321 PR:000004503 46459 46463 Atrx +T1322 SO:0000359 46463 46467 flox +T1323 PR:000004503 46478 46482 Atrx +T1324 PR:000004503 46491 46495 Atrx +T1325 SO:0001023 46496 46503 alleles +T1326 CHEBI:472552 46512 46516 BrdU +T1327 CHEBI:37926 46517 46521 FITC +T1328 CHEBI:51240 46558 46574 propidium iodide +T1329 GO:0051318 46635 46637;46727 46736;46741 46751 G1 ... phases of ... cell cycle +T1330 CHEBI:472552 46646 46650 BrdU +T1331 GO:0051320 46667 46668;46727 46736;46741 46751 S ... phases of ... cell cycle +T1332 CHEBI:472552 46670 46674 BrdU +T1333 GO:0000086 46695 46699;46727 46736;46741 46751 G2/M ... phases of ... cell cycle +T1334 CHEBI:472552 46707 46711 BrdU +T1335 GO:0051318 46845 46847;46861 46878 G1 ... cell-cycle phases +T1336 GO:0051320 46849 46850;46861 46878 S ... cell-cycle phases +T1337 GO:0000086 46856 46878 G2/M cell-cycle phases +T1338 CL:0002322 46918 46926 ES cells +T1339 PR:000004503 46954 46958 Atrx +T1340 PR:000004503 46964 46968 Atrx +T1341 SO:0000359 46968 46972 flox +T1342 PR:000004503 46983 46987 Atrx +T1343 PR:000004503 46996 47000 Atrx +T1344 SO:0001023 47001 47008 alleles +T1345 GO:0007067 47025 47032 mitosis +T1346 CHEBI:15358 47063 47070 histone +T1347 PR:000027594 47063 47073 histone H3 +T1348 CHEBI:37926 47075 47079 FITC +T1349 CHEBI:51240 47116 47132 propidium iodide +T1350 GO:0007067 47173 47180 mitotic +T1351 CL:0002322 47266 47273 ES cell +T1352 GO:0006915 47393 47402 Apoptosis +T1353 PR:000004503 47406 47410 Atrx +T1354 PR:000004503 47424 47428 Atrx +T1355 CL:0002322 47433 47441 ES Cells +T1356 CL:0002322 47460 47468 ES cells +T1357 PR:000004503 47487 47491 Atrx +T1358 SO:0001023 47492 47499 alleles +T1359 PR:000004078 47531 47540 Annexin V +T1360 CHEBI:37926 47542 47546 FITC +T1361 CHEBI:51240 47579 47595 propidium iodide +T1362 GO:0006306 47854 47869 DNA Methylation +T1363 NCBITaxon:10088 47873 47878 Mouse +T1364 PR:000004503 47902 47906 Atrx +T1365 CL:0002322 47911 47919 ES Cells +T1366 PR:000004503 47930 47934 Atrx +T1367 PR:000004503 47963 47967 Atrx +T1368 PR:000004503 47973 47977 Atrx +T1369 SO:0000359 47977 47981 flox +T1370 SO:0001023 47982 47988 allele +T1371 PR:000004503 47993 47997 Atrx +T1372 PR:000004503 48015 48019 Atrx +T1373 SO:0001023 48027 48033 allele +T1374 CL:0002322 48035 48043 ES cells +T1375 GO:0010424 48071 48086 CpG-methylation +T1376 GO:0097617 48248 48258 hybridised +T1377 NCBITaxon:10088 48288 48293 mouse +T1378 GO:0010424 48392 48407 CpG-methylation +T1379 PR:000004503 48460 48464 Atrx +T1380 CL:0002322 48469 48477 ES cells +T1381 CL:0002322 48535 48543 ES cells +T1382 PR:000006606 48563 48568 Dnmt1 +T1383 PR:000006606 48570 48575 Dnmt1 +T1384 PR:000006607 48592 48598 Dnmt3a +T1385 PR:000006608 48603 48609 Dnmt3b +T1386 GO:0010424 48715 48730 CpG-methylation +T1387 GO:0071514 48878 48887 Imprinted +T1388 UBERON:0000922 48927 48933 Embryo +T1389 PR:000004503 48985 48989 Atrx +T1390 PR:000004503 49018 49022 Atrx +T1391 GO:0000806 49025 49026 Y +T1392 UBERON:0000922 49033 49040 embryos +T1393 UBERON:0002450 49065 49073 deciduas +T1394 PR:000004503 49141 49145 Atrx +T1395 SO:0001023 49148 49154 allele +T1396 PR:000004503 49183 49187 Atrx +T1397 SO:0001023 49192 49198 allele +T1398 PR:000004503 49277 49281 ATRX +T1399 GO:0042571 49282 49290 antibody +T1400 UBERON:0005062 49344 49355 neural fold +T1401 UBERON:0005062 49364 49366 nf +T1402 UBERON:0000922 49369 49375 embryo +T1403 PR:000004503 49405 49409 Atrx +T1404 UBERON:0000922 49418 49424 embryo +T1405 UBERON:0000479 49431 49437 tissue +T1406 PR:000004503 49474 49478 Atrx +T1407 PR:000004503 49492 49496 Atrx +T1408 GO:0000805 49526 49527 X +T1409 GO:0009048 49526 49540 X-inactivation +T1410 CHEBI:51686 49579 49591 haematoxylin +T1411 UBERON:0000922 49642 49648 embryo +T1412 UBERON:0000922 49683 49692 embryonic +T1413 UBERON:0003374 49701 49719 chorionic ectoderm +T1414 UBERON:0003374 49721 49723 ce +T1415 UBERON:0004877 49729 49746 visceral endoderm +T1416 UBERON:0004877 49748 49750 ve +T1417 UBERON:0000479 49757 49764 tissues +T1418 GO:0060819 49778 49802 imprinted X-inactivation +T1419 GO:0000805 49788 49789 X +T1420 PR:000004503 49813 49817 Atrx +T1421 GO:0010467 49828 49837 expressed +T1422 UBERON:0004877 49845 49862 visceral endoderm +T1423 GO:0010467 49918 49928 expression +T1424 PR:000004503 49932 49936 Atrx +T1425 UBERON:0003374 49956 49974 chorionic ectoderm +T1426 PR:000004503 50015 50019 Atrx +T1427 SO:0001023 50022 50028 allele +T1428 UBERON:0000479 50062 50068 tissue +T1429 SO:0000112 50220 50227 Primers +T1430 NCBITaxon:9606 50354 50357 Man +T1431 http://purl.obolibrary.org/obo/MONDO_0010519 50435 50449 ATR-X syndrome +T1432 GO:0000805 50439 50440 X +T1433 NCBITaxon:9606 50525 50530 human +T1434 PR:000004503 50531 50535 ATRX +T1435 NCBITaxon:10088 50551 50556 mouse +T1436 PR:000004503 50557 50561 Atrx +T1437 PR:000007857 50882 50887 GATA1 +T1438 NCBITaxon:10088 50903 50907 mice +T1439 CL:0002322 51018 51026 ES cells +T1440 SO:0000155 51064 51071 plasmid +T1441 GO:0097617 51195 51208 hybridisation +T1442 UBERON:0000922 51222 51228 embryo +T1443 CHEBI:472552 51257 51261 BrdU +T1444 CHEBI:472552 51264 51281 bromodeoxyuridine +T1445 GO:0007620 51298 51304 coitus +T1446 CL:0002322 51306 51314 ES cells +T1447 UBERON:0000922 51317 51326 embryonic +T1448 CL:0002322 51317 51337 embryonic stem cells +T1449 GO:0005840 51379 51388 ribosomal +T1450 CL:0002488 51394 51397 TGC +T1451 UBERON:0000088 51400 51411 trophoblast +T1452 CL:0002488 51400 51422 trophoblast giant cell +T1453 GO:0097617 51497 51510 hybridisation +T1454 PR:000004503 51574 51578 ATRX +T1455 SO:0001060 51579 51587 Isoforms +T1456 NCBITaxon:9606 51613 51618 human +T1457 PR:000004503 51619 51623 ATRX +T1458 SO:0000147 51657 51662 exons +T1459 SO:0000188 51668 51675 introns +T1460 GO:0000380 51698 51718 alternative splicing +T1461 SO:0000147 51722 51727 exons +T1462 PR:000004503 51779 51783 ATRX +T1463 SO:0001060 51792 51800 isoforms +T1464 PR:000004503 51814 51818 ATRX +T1465 SO:0000236 51856 51874 open reading frame +T1466 SO:0000417 51930 51936 domain +T1467 GO:0016514 51951 51958 SWI/SNF +T1468 PR:000004503 52005 52009 ATRX +T1469 SO:0001060 52039 52046 isoform +T1470 GO:0008380 52122 52128 splice +T1471 SO:0000188 52129 52135 intron +T1472 SO:0000188 52157 52165 intronic +T1473 SO:0000551 52166 52180 poly(A) signal +T1474 SO:0000188 52186 52192 intron +T1475 GO:0042571 52326 52336 antibodies +T1476 PR:000004503 52438 52442 Atrx +T1477 CL:0002322 52454 52462 ES Cells +T1478 SO:0000147 52502 52506 exon +T1479 PR:000004503 52517 52521 Atrx +T1480 SO:0000704 52522 52526 gene +T1481 SO:0001023 52561 52567 allele +T1482 PR:000004503 52569 52573 Atrx +T1483 SO:0000147 52603 52607 exon +T1484 SO:0001644 52631 52647 targeting vector +T1485 SO:0001023 52665 52671 allele +T1486 PR:000004503 52673 52677 Atrx +T1487 SO:0000359 52677 52681 flox +T1488 SO:0000346 52728 52745 loxP target sites +T1489 PR:000004503 52930 52934 Atrx +T1490 SO:0000359 52934 52938 flox +T1491 SO:0001023 52939 52945 allele +T1492 SO:0001023 52986 52992 allele +T1493 PR:000004503 52994 52998 Atrx +T1494 SO:0000147 53060 53064 exon +T1495 SO:0005853 53095 53103 cassette +T1496 GO:0000805 53205 53217 X chromosome +T1497 CL:0002322 53377 53385 ES cells +T1498 CL:0002322 53404 53411 ES cell +T1499 PR:000004503 53431 53435 Atrx +T1500 SO:0000359 53435 53439 flox +T1501 SO:0001023 53440 53446 allele +T1502 GO:0097617 53465 53475 hybridised +T1503 PR:000004503 53567 53571 Atrx +T1504 SO:0001023 53574 53580 allele +T1505 CL:0002322 53758 53766 ES cells +T1506 CL:0002322 53785 53792 ES cell +T1507 PR:000004503 53812 53816 Atrx +T1508 SO:0000359 53816 53820 flox +T1509 SO:0001023 53821 53827 allele +T1510 GO:0097617 53934 53944 hybridised +T1511 SO:0000188 53954 53960 intron +T1512 PR:000004503 54015 54019 Atrx +T1513 PR:000004503 54029 54033 Atrx +T1514 SO:0000359 54033 54037 flox +T1515 PR:000004503 54049 54053 Atrx +T1516 CL:0002322 54120 54128 ES cells +T1517 GO:0097617 54160 54170 hybridised +T1518 SO:0000147 54193 54197 exon +T1519 PR:000004503 54208 54212 Atrx +T1520 SO:0000704 54213 54217 gene +T1521 PR:000003676 54251 54258 β-actin +T1522 SO:0000673 54308 54319 transcripts +T1523 PR:000004503 54348 54352 Atrx +T1524 SO:0001060 54386 54394 isoforms +T1525 PR:000004503 54511 54515 ATRX +T1526 GO:0042571 54527 54535 antibody +T1527 NCBITaxon:9606 54575 54580 human +T1528 PR:000004503 54581 54585 ATRX +T1529 PR:000004503 54644 54648 Atrx +T1530 SO:0001060 54649 54657 isoforms +T1531 PR:000004503 54718 54722 Atrx +T1532 CL:0002322 54727 54735 ES Cells +T1533 CL:0002322 54793 54801 ES cells +T1534 PR:000004503 54820 54824 Atrx +T1535 SO:0001023 54825 54832 alleles +T1536 PR:000004503 54995 54999 Atrx +T1537 SO:0001023 55000 55007 alleles +T1538 GO:0097617 55058 55068 hybridised +T1539 SO:0001023 55146 55153 alleles +T1540 NCBITaxon:10088 55226 55231 mouse +T1541 SO:0000704 55272 55277 genes +T1542 PR:P23940 55334 55338 BamH +T1543 PR:P23940 55349 55350 B +T1544 PR:000004503 55644 55648 Atrx +T1545 PR:000004503 55659 55663 Atrx +T1546 PR:000004503 55684 55688 Atrx +T1547 PR:000004503 55694 55698 Atrx +T1548 SO:0000359 55698 55702 flox +T1549 SO:0001023 55703 55709 allele +T1550 PR:000004503 55714 55718 Atrx +T1551 PR:000004503 55736 55740 Atrx +T1552 SO:0001023 55748 55754 allele +T1553 CL:0002322 55756 55764 ES cells +T1554 UBERON:0014374 55773 55788 embryoid bodies +T1555 MOP:0000780 55933 55936 cut +T1556 MOP:0000780 56171 56174 cut +T1557 MOP:0000780 56181 56184 cut +T1558 PR:000004503 56290 56294 Atrx +T1559 PR:000004503 56308 56312 Atrx +T1560 PR:000007857 56386 56391 GATA1 +T1561 GO:0010467 56396 56406 Expression +T1562 PR:000004503 56429 56433 Atrx +T1563 SO:0001023 56434 56441 Alleles +T1564 PR:000007857 56447 56452 GATA1 +T1565 UBERON:0000922 56548 56555 embryos +T1566 UBERON:0000085 56592 56598 morula +T1567 CHEBI:75055 56623 56628 X-gal +T1568 SO:0001023 56693 56699 allele +T1569 UBERON:0000922 56729 56736 embryos +T1570 SO:0001023 56751 56758 alleles +T1571 PR:000004503 56808 56812 Atrx +T1572 SO:0001023 56813 56820 alleles +T1573 UBERON:0000922 56824 56831 embryos +T1574 SO:0000112 56838 56845 primers +T1575 SO:0000147 56855 56859 exon +T1576 SO:0000147 56876 56880 exon +T1577 SO:0000006 56927 56939 PCR products +T1578 SO:0001023 56959 56966 alleles +T1579 PR:000004503 56991 56995 Atrx +T1580 PR:000004503 57059 57063 Atrx +T1581 SO:0001023 57071 57077 allele +T1582 PR:000004503 57155 57159 Atrx +T1583 SO:0000006 57190 57203;57206 57218 products from ... PCR reaction +T1584 SO:0000112 57220 57227 primers +T1585 UBERON:0000922 57251 57258 embryos +T1586 SO:0000028 57294 57296 bp +T1587 SO:0000006 57297 57308 PCR product +T1588 NCBITaxon:10088 57329 57334 mouse +T1589 GO:0000806 57335 57347 Y chromosome +T1590 PR:000004503 57400 57404 Atrx +T1591 UBERON:0000922 57409 57416 Embryos +T1592 PR:000004503 57475 57479 Atrx +T1593 UBERON:0000922 57492 57499 embryos +T1594 UBERON:0002450 57520 57528 deciduas +T1595 CHEBI:51686 57548 57560 haematoxylin +T1596 PR:000004503 57581 57585 ATRX +T1597 GO:0042571 57586 57594 antibody +T1598 UBERON:0000305 57804 57805 a +T1599 UBERON:0000305 57807 57813 amnion +T1600 UBERON:0000301 57815 57817 ac +T1601 UBERON:0000301 57819 57834 amniotic cavity +T1602 UBERON:0003124 57836 57837 c +T1603 UBERON:0003124 57839 57846 chorion +T1604 UBERON:0002532 57848 57849 e +T1605 UBERON:0002532 57851 57859 epiblast +T1606 UBERON:0008851 57861 57863 ec +T1607 UBERON:0008851 57865 57885 ectoplacental cavity +T1608 UBERON:0003888 57887 57890 ecc +T1609 UBERON:0003888 57892 57910 exocoelomic cavity +T1610 UBERON:0004364 57912 57914 ep +T1611 UBERON:0004364 57916 57934 ectoplacental cone +T1612 UBERON:0002346 57936 57938 ne +T1613 UBERON:0002346 57940 57955 neural ectoderm +T1614 UBERON:0004369 57957 57959 rm +T1615 UBERON:0004369 57961 57980 Reichert's membrane +T1616 CL:0002488 57982 57985 tgc +T1617 UBERON:0000088 57987 57998 trophoblast +T1618 CL:0002488 57987 58009 trophoblast giant cell +T1619 PR:000016001 58029 58038 brachyury +T1620 PR:000016001 58040 58041 T +T1621 GO:0010467 58043 58053 expression +T1622 PR:000004503 58057 58061 Atrx +T1623 UBERON:0000922 58074 58080 embryo +T1624 UBERON:0009746 58082 58091 head fold +T1625 UBERON:0001040 58195 58203 yolk sac +T1626 UBERON:0009746 58205 58207 hf +T1627 UBERON:0009746 58209 58218 head fold +T1628 UBERON:0002328 58220 58221 n +T1629 UBERON:0002328 58232 58241 notochord +T1630 UBERON:0004341 58243 58245 ps +T1631 UBERON:0004341 58247 58263 primitive streak +T1632 GO:0006915 58288 58297 Apoptosis +T1633 GO:0007067 58302 58309 Mitosis +T1634 PR:000004503 58313 58317 Atrx +T1635 UBERON:0000922 58322 58329 Embryos +T1636 PR:000004503 58369 58373 Atrx +T1637 UBERON:0000922 58386 58393 embryos +T1638 UBERON:0002450 58414 58422 deciduas +T1639 CL:0000445 58457 58472 apoptotic cells +T1640 CHEBI:31624 58487 58498 fluorescein +T1641 CHEBI:51231 58539 58543 DAPI +T1642 PR:000004503 58584 58588 Atrx +T1643 UBERON:0000922 58601 58608 embryos +T1644 GO:0042571 58630 58638 antibody +T1645 GO:0007067 58651 58658 mitosis +T1646 CHEBI:15358 58689 58696 histone +T1647 PR:000027594 58689 58699 histone H3 +T1648 CHEBI:51686 58735 58747 haematoxylin +T1649 PR:000004503 58798 58802 Atrx +T1650 UBERON:0000922 58811 58817 embryo +T1651 PR:000004503 58877 58881 ATRX +T1652 GO:0042571 58882 58890 antibody +T1653 UBERON:0004345 58945 58958 Trophectoderm +T1654 UBERON:0000922 58978 58985 Embryos +T1655 UBERON:0000922 58999 59006 embryos +T1656 UBERON:0002450 59039 59047 decidual +T1657 UBERON:0000479 59048 59054 tissue +T1658 UBERON:0000922 59176 59183 embryos +T1659 UBERON:0002329 59250 59256 somite +T1660 UBERON:0000088 59287 59298 trophoblast +T1661 UBERON:0000088 59300 59301 t +T1662 UBERON:0000088 59313 59324 trophoblast +T1663 UBERON:0009746 59370 59378 headfold +T1664 UBERON:0002329 59382 59388 somite +T1665 UBERON:0002329 59406 59412 somite +T1666 UBERON:0000088 59489 59500 trophoblast +T1667 UBERON:0000922 59534 59543 embryonic +T1668 UBERON:0000922 59568 59574 embryo +T1669 UBERON:0004364 59615 59633 ectoplacental cone +T1670 UBERON:0004364 59635 59638 epc +T1671 GO:0010467 59688 59698 expression +T1672 CHEBI:81588 59702 59704 Pl +T1673 PR:000013543 59702 59706 Pl-1 +T1674 CL:0002488 59720 59724 TGCs +T1675 GO:0007566 59733 59745 implantation +T1676 UBERON:0002450 59763 59771 deciduas +T1677 PR:000004503 59810 59814 Atrx +T1678 PR:000004503 59824 59828 Atrx +T1679 PR:000004503 59834 59838 Atrx +T1680 GO:0000806 59846 59847 Y +T1681 UBERON:0000922 59849 59856 embryos +T1682 UBERON:0000922 59924 59931 embryos +T1683 CL:0002488 59933 59937 TGCs +T1684 CHEBI:81588 59955 59957 Pl +T1685 PR:000013543 59955 59959 Pl-1 +T1686 PR:000004503 60000 60004 Atrx +T1687 UBERON:0000922 60017 60024 embryos +T1688 UBERON:0002450 60045 60053 deciduas +T1689 CHEBI:81588 60081 60083 Pl +T1690 PR:000013543 60081 60085 Pl-1 +T1691 GO:0042571 60086 60094 antibody +T1692 PR:000004503 60123 60127 Atrx +T1693 UBERON:0000922 60136 60142 embryo +T1694 PR:000004503 60202 60206 ATRX +T1695 GO:0042571 60207 60215 antibody +T1696 UBERON:0000358 60280 60290 blastocyst +T1697 UBERON:0000088 60321 60332 trophoblast +T1698 UBERON:0000087 60353 60368 inner cell mass +T1699 UBERON:0000087 60370 60373 icm +T1700 PR:000004503 60410 60414 Atrx +T1701 UBERON:0000358 60439 60449 blastocyst +T1702 GO:0071514 60507 60516 Imprinted +T1703 PR:000004503 60558 60562 Atrx +T1704 SO:0001023 60565 60571 Allele +T1705 PR:000004503 60624 60628 Atrx +T1706 GO:0000806 60631 60632 Y +T1707 PR:000004503 60654 60658 Atrx +T1708 UBERON:0000922 60675 60682 embryos +T1709 UBERON:0002450 60703 60711 deciduas +T1710 PR:000004503 60740 60744 ATRX +T1711 GO:0042571 60745 60753 antibody +T1712 UBERON:0000922 60878 60885 embryos +T1713 UBERON:0000305 60908 60909 a +T1714 UBERON:0000305 60911 60917 amnion +T1715 UBERON:0003124 60919 60920 c +T1716 UBERON:0003124 60922 60929 chorion +T1717 UBERON:0002532 60931 60932 e +T1718 UBERON:0002532 60934 60942 epiblast +T1719 UBERON:0004364 60944 60946 ep +T1720 UBERON:0004364 60948 60966 ectoplacental cone +T1721 UBERON:0002532 61014 61022 epiblast +T1722 UBERON:0000922 61125 61134 embryonic +T1723 UBERON:0003374 61143 61161 chorionic ectoderm +T1724 UBERON:0003374 61200 61202 ce +T1725 UBERON:0003374 61204 61222 chorionic ectoderm +T1726 UBERON:0003124 61228 61237 chorionic +T1727 UBERON:0000926 61238 61246 mesoderm +T1728 PR:000004503 61274 61278 Atrx +T1729 GO:0007618 61298 61305 Matings +T1730 CHEBI:33893 61516 61524 reagents +T1731 PR:000004503 61863 61867 Atrx +T1732 UBERON:0000088 61876 61887 trophoblast +T1733 GO:0000805 61919 61920 X +T1734 GO:0009048 61919 61933 X-inactivation +T1735 UBERON:0005292 61937 61959 extraembryonic tissues diff --git a/src/ontogpt/evaluation/craft/database/all/16628246.txt b/src/ontogpt/evaluation/craft/database/all/16628246.txt new file mode 100644 index 000000000..661f1f867 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16628246.txt @@ -0,0 +1,297 @@ +Loss of Atrx Affects Trophoblast Development and the Pattern of X-Inactivation in Extraembryonic Tissues + +Abstract + +ATRX is an X-encoded member of the SNF2 family of ATPase/helicase proteins thought to regulate gene expression by modifying chromatin at target loci. Mutations in ATRX provided the first example of a human genetic disease associated with defects in such proteins. To better understand the role of ATRX in development and the associated abnormalities in the ATR-X (alpha thalassemia mental retardation, X-linked) syndrome, we conditionally inactivated the homolog in mice, Atrx, at the 8- to 16-cell stage of development. The protein, Atrx, was ubiquitously expressed, and male embryos null for Atrx implanted and gastrulated normally but did not survive beyond 9.5 days postcoitus due to a defect in formation of the extraembryonic trophoblast, one of the first terminally differentiated lineages in the developing embryo. Carrier female mice that inherit a maternal null allele should be affected, since the paternal X chromosome is normally inactivated in extraembryonic tissues. Surprisingly, however, some carrier females established a normal placenta and appeared to escape the usual pattern of imprinted X-inactivation in these tissues. Together these findings demonstrate an unexpected, specific, and essential role for Atrx in the development of the murine trophoblast and present an example of escape from imprinted X chromosome inactivation. + +Synopsis + +ATRX belongs to a class of proteins that may modify how DNA is packaged into chromatin, altering the accessibility of other proteins in the nucleus to DNA. In this way, ATRX is thought to influence gene expression. Mutations in the ATRX gene, which is located on the female sex chromosome (X), provided the first example of a human disease (ATR-X syndrome) associated with defects in such proteins. Affected males (XMUTY) have multiple developmental abnormalities in a wide variety of systems. Currently, it is not understood how proteins like ATRX influence cell biology. To address this question, the authors deleted the version of the gene in mice, Atrx. Although affected male mice (XMUTY) started to develop normally, they died early in development because they failed to form a normal placenta. In the placenta, female mice normally inactivate the X chromosome that they inherit from their fathers (Xp), so if females inherit from their mother an X chromosome (Xm) that bears the abnormal copy of Atrx (XmMUTXp), one would predict that, like affected males, they would fail to form a normal placenta. The authors unexpectedly found this not to be so. They showed, instead, that in such females the normal, paternally derived Atrx gene is active. This study has therefore demonstrated an important facet of X-chromosome imprinting. + +Introduction + +ATR-X syndrome is a severe, nonprogressive form of X-linked mental retardation that is frequently associated with multiple congenital abnormalities [1]. It is usually associated with a mild form of α-thalassaemia, caused by reduced expression of structurally intact α-globin genes, and characterised by the presence of β-globin tetramers (haemoglobin H inclusion bodies) in peripheral red blood cells. Carrier females occasionally manifest haemoglobin H inclusions, but are otherwise intellectually and physically normal. Studies of X-chromosome inactivation in carrier females have demonstrated preferential inactivation of the chromosome bearing the abnormal allele in a variety of tissues [2], and this skewing of X-inactivation is thought to explain the mild phenotype observed in carriers. + +The ATR-X syndrome is caused by mutations in a gene (ATRX) that comprises 36 exons spanning 300 kb of genomic DNA at Chromosome Xq13.3 [3]. This gene encodes two dominant protein isoforms (Figure 1). As well as the full-length ATRX protein of ~280 kDa, which is encoded by a transcript of ~10 kb, we recently demonstrated that a truncated isoform called ATRXt (~200 kDa) is produced from a transcript of around 7 kb, which arises when intron 11 fails to be spliced from the primary transcript and an alternative intronic poly(A) signal is used [4]. The mouse homolog of the ATRX gene, Atrx, is also situated on the X chromosome, and also gives rise to full-length (Atrx, ~280 kDa) and truncated (Atrxt, ~200 kDa) isoforms [4,5]. + +Disease-causing missense mutations are clustered in two regions of the gene: a PHD-like zinc finger domain and a SNF2-like ATPase domain (Figure 1) [6]. The former motif is thought to be involved in protein-protein interactions in chromatin [7], and the latter is a feature of chromatin-remodelling proteins, and the presence of disease-causing mutations indicates the functional importance of these domains. ATRX has been shown to remodel chromatin [8]. It also interacts with HP1 at heterochromatin [9] and is recruited to promyelocytic leukemia nuclear bodies via an interaction with Daxx [10]. Furthermore, disruption of ATRX leads to diverse changes in DNA methylation [11]. Nevertheless, the role ATRX plays in gene expression remains unclear. + +The consistent core of clinical and haematological features observed in ATR-X patients suggests that, like the SWI2/SNF2 chromatin-remodelling protein, ATRX probably regulates transcription of a discrete set of target genes. However, although there are clearly others to be found, at present the α-globin genes remain the only confirmed targets for transcriptional regulation by ATRX. Little is currently known about the precise role of the ATRX protein during mammalian development. To investigate the role of this protein during mouse development, we generated a conditionally deleted allele of the Atrx gene in mouse embryonic stem (ES) cells, and used these cells to examine the effect of ablating expression of the full-length Atrx protein in ES cells and in mouse embryos. + +Results + +Generation of ES Cells Lacking Full-Length Atrx + +Like the human gene, the mouse Atrx gene is also X-linked, such that a direct disruption of the single Atrx allele in male ES cells would immediately give rise to the null state. No targeted clones were recovered after attempted homologous recombination in male E14TG2a ES cells using two different vectors that removed exon 18 of the Atrx gene. Exon 18 encodes the first of the seven motifs composing the conserved SNF2-like domain of Atrx (Figure 1); mutation of the corresponding motif of the yeast SNF2 protein has been shown to severely impair SWI/SNF-dependent gene expression [12]. The failure to recover targeted clones with these vectors suggested that Atrx may be important for normal ES cell growth and expansion and that direct targeting of the single locus may not be possible. We therefore adopted a conditional strategy for targeting exon 18 (Figure 2) and recovered two clones in which exon 18 has been flanked by loxP recognition sites for the Cre recombinase (Atrxflox allele in Figure 2A) (Figure 2B). This allele also contains a loxP-flanked MC1-neor cassette in intron 17 (Figure 2A). Northern and Western blot analyses (Figure 2D and 2E) confirmed that the Atrxflox clones continued to express both full-length Atrx protein and the truncated Atrxt isoform. + +To generate the full deletion in ES cells, the Atrxflox clones (1/F12 and 1/G11) were transiently transfected with a Cre-recombinase expression plasmid (pCAGGS-Cre-IRESpuro), and subclones were recovered bearing an allele (AtrxΔ18Δneo in Figure 2A) in which both exon 18 and the neor cassette had been deleted by the Cre recombinase (resulting from the recombination event labelled “C” in the Atrxflox allele shown in Figure 2A) (Figure 2C). Northern and Western blot analyses (Figure 2D and 2E) revealed that the full-length Atrx transcript and protein is completely abolished in the AtrxΔ18Δneo recombinant clones, suggesting that deletion of this region has a highly destabilising effect on the full-length transcript. As expected, the truncated Atrxt isoform, the transcript of which is terminated within intron 11 [4], is unaffected by the deletion of exon 18 (Figure 2E). While the function of Atrxt is not yet clear, this isoform, which contains the PHD-like domain but not the SWI/SNF motifs (Figure 1), is unlikely to be functionally equivalent to the full-length protein. Thus, a conditional knockout strategy allowed the isolation of ES cells that are null for full-length Atrx. + +Perturbed Growth and Methylation Defects in Atrxnull ES Cells + +Atrxnull ES cells could be maintained in culture but were generally slower growing than Atrx+ ES clones, and appeared to undergo higher rates of spontaneous differentiation. We investigated directly the effect of Atrx on ES cell growth by comparing Atrx+ and Atrxnull ES cell clones in competition cultures. Equal numbers of Atrx+ (bearing either an AtrxWT or an Atrxflox allele) and Atrxnull (bearing an AtrxΔ18Δneo allele) ES cells were inoculated into cultures and the mixed cultures were passaged (1:3 split) every 2 d for 8–10 d. The relative abundance of the different alleles in the culture at each time point was analysed by Southern blotting (Figure 3A). The clone containing the AtrxΔ18Δneo allele was rapidly outgrown by both AtrxWT ES cells and cells bearing the Atrxflox allele. In a control competition experiment between different clones bearing functional Atrx alleles (AtrxWT and Atrxflox), both clones continued to be equally represented after 8 d of cocultivation. Thus, although Atrxnull ES cells could be recovered and maintained in culture by a conditional targeting approach, these cocultivation experiments suggested that the absence of Atrx does negatively impact upon normal ES cell growth. + +To investigate a possible cell-cycle defect in the absence of Atrx, we analysed the cell cycle distribution of bromodeoxyuridine (BrdU)-pulsed ES cells by flow cytometry (Figure S1A). Surprisingly, both Atrxnull ES cell clones exhibited a cell cycle profile that was indistinguishable from ES cells bearing a functional Atrx allele (AtrxWT or Atrxflox). We also specifically quantitated the mitotic index within each population by flow cytometry after staining ES cells for phosphorylated (Ser10) histone H3, a specific marker of mitosis (Figure S1B) [13]. Consistent with the normal cell-cycle profile observed above, there was no depletion in the size of the mitotic population in the Atrxnull ES clones, despite their slow growth. Finally, we investigated whether the growth defect in the Atrxnull ES cells was due to an up-regulation of apoptosis by staining cells with Annexin V (Figure S2) and found that the proportion of apoptotic cells was not significantly affected by the absence of full-length Atrx. Thus, the growth defect observed in ES cells lacking Atrx is not due to a specific cell cycle block or significant induction of cell death. While the cause of the proliferative delay is not yet clear, since Atrxnull ES cells appear to undergo higher rates of spontaneous differentiation (unpublished data), it seems likely that the observed growth defect reflects the spontaneous transition from fast-cycling, undifferentiated ES cells into more slowly cycling, differentiated cell types in these cultures. + +It has been shown that disease-causing mutations in the human ATRX gene give rise to changes in the normal pattern of DNA methylation at several repetitive sequences within the human genome [11]. Notably, the transcribed region of the ribosomal DNA (rDNA) repeat was found to be significantly hypomethylated in ATR-X patients relative to normal individuals. Using methylation-sensitive restriction enzymes, we also observed significant hypomethylation at several sites tested within the mouse rDNA repeats in Atrxnull ES cells and 12-d embryoid bodies relative to ES cells and embryoid bodies bearing a functional Atrx allele (AtrxWT or Atrxflox) (Figure 3B and 3C). The observation that rDNA is hypomethylated in the absence of Atrx, even in ES cells, is consistent with the finding that hypomethylation of the human rDNA repeats is detectable from an early developmental stage in ATR-X patients. Other mouse repetitive sequence elements surveyed in ES cell DNA include the heterochromatic major satellite (assayed with MaeII) and minor satellite (assayed with HpaII) repeats, as well as interspersed retroviral repeats of the intracisternal A particle (IAP) type and the Line 1 and Sine B1 families (all assayed with HpaII). These repeats were found to be moderately (Line 1 and Sine B1) or highly (IAP, major satellite, minor satellite) methylated in wild-type ES cells, and this methylation was not detectably perturbed by the absence of Atrx (Figure S3 and unpublished data). Taken together, these data indicate that the subtle interplay between the ATRX protein and DNA methylation observed in human patients is also present in mouse cells. + +Early Embryonic Lethality in Atrxnull Male Mice + +To investigate the role of the Atrx protein during mouse development, we initially established lines of mice bearing the Atrxflox allele. Two independently targeted Atrxflox ES cell clones with normal male karyotype were injected into C57BL/6 blastocysts to produce chimaeric mice, which were then used to obtain germline transmission. Intercrosses between males hemizygous (Atrxflox/Y) and females heterozygous (AtrxWT/flox) for the floxed allele were also carried out to generate homozygous females (Atrxflox/flox). Males hemizygous and females heterozygous or homozygous for the Atrxflox allele were viable, appeared healthy, and bred normally, suggesting that, as expected, the Atrxflox allele was functionally normal. To generate Atrxnull mice by Cre-mediated recombination, the Atrxflox mice were crossed with mice harboring a transgene in which the Cre recombinase is expressed under the control of the regulatory elements of the mouse GATA-1 gene (GATA1-cre) [14]. Widespread expression of the GATA1-cre transgene has been demonstrated during early embryogenesis [14]. We more accurately defined the onset of GATA1-cre expression using a ROSA26 reporter strain, in which a β-galactosidase/neor fusion reporter gene is expressed only after Cre-mediated excision of loxP-flanked transcription and translation termination signals [14]. We found that the GATA1-cre transgene was already active at the 16-cell morula stage of development (0.5 days postcoitus [dpc]) (Figure 4A). + +To generate Atrxnull mice, heterozygous floxed females (AtrxWT/flox) were mated with homozygous GATA1-cre transgenic males (AtrxWT/Y;GATA1-cre+/+). No Atrxnull males (Atrxnull/Y;GATA1-cre+/−) were recovered at birth, indicating that the absence of Atrx results in embryonic lethality. This finding was unexpected, since human ATR-X patients clearly survive to adulthood (see Discussion). Embryos were dissected at 7.5, 8.5, and 9.5 dpc and genotyped by PCR analysis of DNA extracted from yolk sac or total embryo (Figure 4B and Protocol S1). Atrxnull males were present at expected mendelian ratios (~25%) at both 7.5 dpc and 8.5 dpc (Table 1). However, by 9.5 dpc, depletion was observed both in the number of Atrxnull males (7%) and in the total number of males recovered (31%). No Atrxnull males were recovered after 9.5 dpc. Thus the absence of Atrx gives rise to embryonic lethality in mice before 9.5 dpc. + +To investigate the morphology of Atrxnull embryos prior to death, embryos from the above crosses were initially dissected in their deciduas at 7.5 dpc, and paraffin sections were stained with haematoxylin (Figure 5A) or with an anti-ATRX antibody (Figure 5B–5E). Immunohistochemical staining revealed that Atrx was widely expressed in wild-type 7.5 dpc embryos (Figure 5B). Expression was highest in the embryonic region (Figure 5C) and the chorion (Figure 5D). Detectable but lower levels of expression were observed in the ectoplacental cone (Figure 5D) and surrounding decidual tissue. We also observed very high levels of Atrx expression in trophoblast giant cells (TGCs) surrounding the Reichert's membrane (Figure 5E). Within the large nuclei of these TGCs, the typical nuclear association of Atrx with blocks of pericentromeric heterochromatin [15] was clearly observable. Only background staining was seen in the corresponding Atrxnull embryonic tissues (Figure 5B–5D), while expression in the surrounding decidual tissue (of maternal origin) was normal and served as an antibody staining control (unpublished data). Morphologically, 7.5 dpc Atrxnull embryos were dramatically reduced in size and appeared developmentally retarded relative to stage-matched wild-type embryos (Figure 5A and 5B). However, despite their reduced size, the general morphology and organisation of embryonic structures in Atrxnull conceptuses appeared grossly normal. The amnion and chorion were clearly present and the amniotic, exocoelomic, and ectoplacental cavities were distinguishable, as were all three embryonic germ layers (Figure 5A–5C). At 8.5 dpc, embryos were dissected free of deciduas, and observed in whole mount. Individual conceptuses were genotyped by PCR using DNA isolated from yolk sac as described in Protocol S1. Consistent with observations at 7.5 dpc, the general morphology of the embryo proper of Atrxnull conceptuses also appeared grossly normal at 8.5 dpc. The head fold had clearly formed, and expression of the early mesoderm marker brachyury (T) [16] was detected in the primitive streak and emerging notochord by whole-mount in situ hybridisation (WMISH) (Figure 5F), indicating that Atrxnull embryos had gastrulated. + +To investigate whether the reduced size of the Atrxnull embryos was due to an increase in apoptosis, we analysed sections of paraffin-embedded 7.5 dpc embryos by TdT-mediated dUTP nick end labeling (TUNEL) assay (Figure 6A). Very few apoptotic cells were detected in wild-type 7.5 dpc embryos. In Atrxnull embryos, a slight increase in the apoptotic population was evident. However, consistent with our observation of a grossly normal apoptotic index in Atrxnull ES cells (Figure S2), the apoptotic response observed in Atrxnull embryos was also not uniform, but was restricted to a low number of scattered TUNEL-positive cells. Since this small apoptotic response is unlikely to account fully for the dramatic size deficit observed in Atrxnull embryos, a possible proliferation defect was also investigated by immunohistochemical staining of 7.5 dpc embryo sections for the mitosis marker phosphorylated (Ser10) histone H3 [13]. Relative to the very high mitotic index observed in wild-type embryos, the proportion of mitotic cells observed in Atrxnull embryos at 7.5 dpc was dramatically reduced (Figure 6B). Taken together, these results suggest that the size deficit observed in Atrxnull embryos prior to lethality largely reflects a proliferative defect, with a minor but indirect contribution from increased apoptosis. Although a growth defect was also observed in Atrxnull ES cells (Figure 3A), in contrast to the Atrxnull embryos, the mitotic index of the ES cell population (as measured with the same antibody) was not depleted (Figure S1B). These observations suggest that the mitotic defect observed in embryos is unlikely to be a direct, cell-autonomous effect of the absence of Atrx, and is more likely to be a secondary effect resulting from the failure to develop a normal trophoblast (see below). + +Trophectoderm Failure in Atrxnull Embryos + +Whole-mount observation of 8.5 dpc embryos revealed that, in contrast to the basically normal although delayed morphology of the embryo itself, the extraembryonic tissues of Atrxnull conceptuses appeared highly disorganised. When embryos were removed from deciduas, the surrounding trophectoderm layer appeared dramatically reduced in Atrxnull embryos relative to wild-type littermates, and the underlying ectoplacental cone appeared reduced and abnormally shaped (Figure 7A). Vacated deciduas surrounding 8.5 dpc wild-type and Atrxnull embryos were bisected and analysed by WMISH for expression of placental lactogen-1 (Pl-1), a marker of terminally differentiated TGCs. The number of Pl-1-expressing cells attached to the decidual wall after removal of the embryo is an indication of the density of trophoblast cells surrounding each implantation site [17]. We found that the population of Pl-1-expressing cells was depleted in the decidual implantation sites containing Atrxnull embryos relative to those containing wild-type littermates (Figure 7B); this was also apparent at 7.5 dpc, as determined by immunohistochemical staining of paraffin sections of embryos in deciduas with an anti-Pl-1 antibody (Figure 7C). A TGC deficiency in the absence of Atrx is consistent with the observation that Atrx is highly expressed in giant cells surrounding wild-type 7.5 dpc embryos (Figure 5E). + +To investigate whether the trophoblast defect was restricted to the production of secondary TGCs (produced by diploid precursors in the ectoplacental cone and derived originally from the polar trophectoderm overlying the inner cell mass of the blastocyst) or also affected the production of primary TGCs (resulting from differentiation of the mural trophectoderm of the blastocyst), blastocysts (3.5 dpc) from crosses between AtrxWT/flox females and GATA1-Cre homozygous transgenic males (AtrxWT/Y;GATA1-Cre+/+) were cultured in vitro for 5 d to monitor outgrowth of the primary trophoblast. After 5 d, individual blastocyst cultures were scored for the extent of primary trophoblast outgrowth, and the Atrx genotype and sex of the blastocyst were determined by PCR. Most blastocysts hatched from the zona pellucida within 24 h, and trophoblast cells spreading out from the inner cell mass could usually be detected within 48 h of culture. No difference was observed in the rate or extent of trophoblast outgrowth over 5 d of culture between Atrxnull blastocysts (Atrxnull/Y, n = 6) and blastocysts bearing an AtrxWT allele (AtrxWT/WT, n = 6; AtrxWT/null, n = 6; AtrxWT/Y, n = 6) (examples shown in Figure 7D), suggesting that the defect specifically involves the secondary giant cell compartment. This is consistent with the observation that Atrxnull conceptuses implant successfully and survive to gastrulation. Taken together, these data suggest that loss of Atrx results in a defect in formation of the secondary trophoblast that is apparent from 7.5 dpc. Despite initiating normal organisation in the embryo proper, Atrxnull conceptuses exhibit a proliferative defect by 7.5 dpc and die by around 9.5 dpc, probably due to a nutritional deficit resulting from failure to develop a normal trophoblast. + +Escape from Imprinted Inactivation of the Paternally Inherited AtrxWT Allele in Extraembryonic Tissues of Carrier Female Mice + +Female mice carrying an Atrxnull allele (AtrxWT/null;GATA1-cre+/−) were detected at 9.5 dpc (Table 1) and recovered at birth (unpublished data), although at both time points the number of carrier females was lower than that of wild-type (AtrxWT/WT;GATA1-cre+/−) females, suggesting that a proportion of carrier female embryos died in utero. Surviving adult carrier female mice were not phenotypically normal and exhibited mild behavioural abnormalities, although some could reproduce. For all AtrxWT/null carrier female embryos presented in Table 1, the AtrxWT allele was paternally derived, while the Atrxnull allele was maternally derived. In the mouse, X chromosome inactivation is subject to parental imprinting in the trophectoderm and primitive endoderm lineages that give rise to the extraembryonic tissues, resulting in inactivation of the paternal X chromosome (Xp) [18]. In contrast, in tissues of the embryo proper (derived from the inner cell mass of the blastocyst) X-inactivation is random [19]. Thus, in the extraembryonic compartment of AtrxWT/null carrier females, normal imprinted X-inactivation would be expected to result in silencing of the paternally derived AtrxWT allele, leaving only the Atrxnull allele on the active maternal X (Xm) and thereby render the extraembryonic tissues null for full-length Atrx protein. However, the absence of Atrx in the extraembryonic compartment is lethal in Atrxnull/Y males. This suggested the possibility of an escape from imprinted inactivation of the paternally derived AtrxWT allele in the extraembryonic compartment of a proportion of carrier female embryos. + +To investigate further, we crossed homozygous Atrxflox/flox females and homozygous GATA1-cre transgenic males (AtrxWT/Y;GATA1-cre+/+), which would be expected to yield only Atrxnull males (Atrxnull/Y;GATA1-cre+/−) and carrier females (AtrxWT/null;GATA1-cre+/−). In these carrier females, the AtrxWT allele is paternally inherited. Embryos were dissected in their deciduas at 7.5 dpc, and paraffin sections were stained with anti-ATRX antibody, along with sections from a wild-type 7.5 dpc embryo for comparison (Figure 8). As described above, Atrx expression was detected in every cell in the epiblast (embryo proper) region of wild-type 7.5 dpc embryos (Figure 8B). In contrast, the epiblast region of carrier female embryos was composed of a mosaic of small clusters of Atrx-positive cells (in which the Atrxnull allele on Xm had been inactivated) and Atrx-negative cells (in which the AtrxWT allele on Xp had been inactivated), indicating that the Atrx gene was subject to normal random X-inactivation in the epiblast. Remarkably, clear Atrx expression could also be detected in the extraembryonic tissues of carrier females, as shown in the extraembryonic-derived chorionic ectoderm (Figure 8C). Atrx expression could be detected in almost all cells of the chorionic ectoderm. Atrx expression was also clearly detected in other extraembryonic structures, including TGCs (unpublished data). Escape from silencing of an Xp-inherited AtrxWT allele was also observed in the chorionic ectoderm of carrier females at 8.5 dpc (Figure S4). Thus, although random X-inactivation occurs normally within the epiblast, the AtrxWT allele (inherited on the Xp chromosome) escaped the normal imprinted X-inactivation in the extraembryonic compartment of some carrier females. + +Discussion + +We investigated the role of the Atrx protein in mouse development. By using a conditional knockout approach, we ablated the full-length Atrx protein first in ES cells and embryoid bodies, and then in developing mouse embryos. + +Atrx in ES Cells + +Atrxnull ES cells could not be recovered by direct targeting and were eventually generated by adopting a conditional targeting approach. This is consistent with our observation that Atrx is highly expressed in ES cells, and that the absence of full-length Atrx imparts a growth disadvantage relative to cells bearing a functional Atrx allele. At present, the cause of the proliferative delay in Atrxnull ES cells is not known. Interestingly, we demonstrated that apoptosis is not significantly up-regulated in ES cells lacking Atrx and is only mildly elevated in Atrxnull 7.5 dpc mouse embryos. In contrast, it was recently shown that the loss of Atrx markedly increased the apoptotic population in the differentiating cells of the embryonic cortex and postnatal hippocampus, when Atrx expression was ablated in the developing mouse forebrain using the Atrxflox allele described here [20]. The human ATRX protein has been shown to associate in a complex with Daxx [8], a protein that has been implicated in multiple pathways for the regulation of apoptosis [21]. It is possible that disruption of the mouse Atrx-Daxx complex (by ablation of the Atrx protein) could have triggered a universal proapoptotic response. However, our observations in ES cells demonstrate that the induction of apoptosis is not an automatic response triggered by the removal of Atrx in all cell types, and suggest that the inappropriate apoptosis observed in the Atrx-mutant forebrain may reflect a requirement for Atrx during terminal differentiation. + +An Unexpected Role for Atrx in Development of the Mouse Trophoblast + +We show that Atrxnull male mice are not viable and embryos die by around 9.5 dpc. Before death, Atrxnull embryos exhibit a markedly reduced mitotic index, suggesting a proliferative defect. Although the embryonic compartment of the conceptus appears initially normal, by 7.5 dpc Atrxnull embryos display abnormalities in development of the trophoblast, including a depletion in the population of TGCs surrounding the conceptus and a reduction in the size of the ectoplacental cone, which contains the diploid giant cell precursors [22]. TGCs are highly differentiated, postmitotic cells that ultimately form an epithelial layer at the periphery of the placenta, which interfaces with the maternal cells of the decidua [23]. These highly invasive cells are important for mediating initial invasion of the uterine tissue, but are also involved in remodelling the maternal decidua after implantation and in secreting hormones that regulate fetal and maternal growth [24]. Since Atrxnull embryos appear to implant normally, lethality is likely to arise due to a failure of TGC function later during development. + +Embryonic lethality in mice in the absence of Atrx was a surprising finding, as there had been no suggestion of foetal loss in the human ATR-X syndrome. It is possible that the role of Atrx in the trophoblast is specific to mice and that ATRX has no role or is redundant in the human trophoblast. Indeed, the birth weight of babies with ATR-X syndrome is usually normal. An alternative explanation for the unexpectedly severe phenotype we observed in mice is that the AtrxΔ18Δneo deletion generated by Cre recombination completely ablates full-length Atrx protein (Figure 2E). In contrast, all disease-causing mutations characterised in human ATR-X pedigrees appear to give rise to hypomorphic alleles. Some full-length ATRX protein is detected in cases predicted to have truncating mutations (RJG, unpublished data), and residual ATPase activity in ATRX immunoprecipitates can be detected in Epstein-Barr virus-transformed lymphocytes of all human patients analysed to date (A. Argentaro and M. Mitson, unpublished data). The failure to observe a truly null ATRX allele among human patients strongly suggests that, as in the mouse, the complete absence of ATRX protein is incompatible with human fetal survival. + +While this study has revealed an unexpected role for Atrx in the murine trophectoderm, as a result of the early lethality observed in Atrxnull embryos it is not possible to rule out other roles for Atrx at later developmental stages in tissues of the embryo proper. Indeed, we show that Atrx is highly expressed throughout the entire developing embryo at 7.5 dpc (Figure 5B), and it is likely that Atrx function will turn out to be important for other differentiating tissues. Tetraploid aggregation experiments (in which mutant embryos are rescued with wild-type extraembryonic tissues) might shed more light on the role of Atrx during later mouse development, but these issues can be more subtly dissected by combining the conditional Atrxflox allele that we have generated with different tissue-specific Cre transgenes. As mentioned above, this approach has already revealed a critical role for Atrx during neuronal differentiation in adult mice [20]. Further evidence that Atrx is also required at later stages of mouse development is provided by the observed dramatic skewing against Atrx-negative cells in some somatic tissues of carrier female mice, whose tissues initially comprise a mosaic of Atrx-positive and Atrx-negative cells as a result of random X-inactivation (M. Muers, personal communication). + +Atrx joins an expanding list of mouse genes for which targeted disruption results in peri-implantation lethality as a result of trophoblast or placental abnormalities (reviewed in [25]). Comparison with other phenotypes might provide some insight into the role of Atrx in trophoblast development. Atrx-mutant embryos progress further than embryos nullizygous for factors involved in the initial specification of trophoblast stem cells (such as Cdx2) or in stem cell maintenance and proliferation (such as Eomes). Cdx2-mutant embryos fail to implant and die between 3.5 and 5.5 dpc [26], while Eomes-mutant blastocysts implant into the uterus, but arrest soon after implantation without forming organised embryonic or extraembryonic structures [27]. In contrast, Atrx-mutant embryos implant successfully and establish organised embryonic structures by 7.5 dpc. The Atrx-mutant phenotype closely resembles that observed in mice nullizygous for the basic helix-loop-helix transcription factor Hand1. Hand1-mutant conceptuses arrest at around 7.5 dpc and display a normal embryonic compartment, but, like Atrx-mutant embryos, ablation of Hand1 causes a reduction in the size of the ectoplacental cone and density of TGCs [28]. As with Atrx mutants, only arrested or resorbed Hand1-mutant conceptuses were recovered after 8.5 dpc. Also like Atrx, disruption of Hand1 specifically affects secondary giant cell formation, and primary trophoblast outgrowths from blastocysts appeared normal. Hand1 is required for terminal differentiation of secondary TGCs, and in its absence trophoblast cells arrest at a precursor stage in the ectoplacental cone [17,28]. Given the similarity of the Atrx- and Hand1-mutant phenotypes and the likelihood that Atrx acts as a transcriptional regulator by modifying chromatin structure, it will be of interest to determine whether Atrx is itself a regulator of Hand1 expression, or alternatively whether it acts as a co-regulator of one or more of the downstream transcriptional targets of Hand1. It is noteworthy that, in the brain-specific Atrx knockout mice, the defect was observed in terminally differentiating neurons [20]. The secondary TGCs affected in the universal Atrx knockout reported here represent one of the first terminally differentiated tissues in the developing mouse, and this may point to the requirement for Atrx in the high-level expression of some tissue-specific genes during the final stages of differentiation. Interestingly, the α-globin genes, the only confirmed transcriptional targets of regulation by human ATRX, are also highly expressed specifically during terminal differentiation within the erythroid lineage. + +Atrx Escapes Imprinted X-Inactivation in Extraembryonic Tissues of Carrier Female Mice + +Another surprising finding of this study is that, in carrier female embryos, a paternally inherited AtrxWT allele appears to escape the process of imprinted X-inactivation, which ordinarily silences the Xp chromosome in the extraembryonic compartment of female murine tissues [18]. Silencing of the AtrxWT allele on Xp should render these females null for Atrx in the extraembryonic tissues, since the normally active Xm chromosome carries the AtrxΔ18Δneo allele. Although not phenotypically normal, some Atrx carrier females developed to term and went on to reproduce. Thus, the failure to correctly silence the paternally derived AtrxWT allele in the extraembryonic tissues of carrier females is consistent with our observations that in Atrxnull males, the Atrx protein plays an essential role in the development of the trophoblast and is necessary for survival in utero in the mouse. + +The survival of Atrx carrier females contrasts with the phenotypes seen in carriers of mutations of other murine X-linked genes known to be essential in the extraembryonic compartment. For example, targeted disruption of the dyskerin (Dkc1), glucose 6-phosphate dehydrogenase (G6PD), and choroideremia (Chm) genes cause embryonic lethality in null male embryos through defects of the extraembryonic-derived tissues [29–31]. Female mice carrying mutations of these genes on the maternally inherited X chromosome also die in utero, whereas females that inherit the mutation on the Xp chromosome survive. Thus, unlike Atrx, these genes and/or their effects on cell growth are unable to circumvent the processes that ultimately cause all cells in the extraembryonic tissues to express only the maternally derived X chromosome. How might expression of the paternal AtrxWT allele be maintained in the extraembryonic tissues of the Atrx carrier females? + +One possibility is that, like some other X-linked genes, silencing of the Atrx gene on Xp is incomplete, such that there is always a low-level, leaky output of Atrx from a normally inactivated Xp chromosome in extraembryonic tissues. However, it was recently demonstrated that the paternal Atrx (called Xnp) allele is completely silenced in a normal mouse trophoblast stem cell line [32], suggesting that Atrx does not normally escape imprinted X-inactivation in the extraembryonic tissues of wild-type females. Thus, the expression of the Xp-linked AtrxWT allele that we observed is unique to female carriers of the Atrxnull allele. + +Perhaps a more likely explanation for this phenomenon stems from experimental observations suggesting that imprinted X-inactivation is not imposed on all precursors of the mouse extraembryonic tissues: A subpopulation of cells may escape this process and make a random “choice” of which X chromosome will be inactivated. On average, 50% of the cells in this randomly inactivating subpopulation would be expected to maintain an active Xp chromosome. In support of this hypothesis, it has been demonstrated that expression of paternally transmitted X-linked lacZ [33,34] and GFP [35] transgenes failed to be silenced in a small subpopulation of extraembryonic cells. Further, it has been shown that in a subpopulation of extraembryonic cells, it is the Xm rather than the Xp that undergoes late replication, a molecular correlate of the inactive state [18,36]. Although initially small and quickly diluted in normal embryos, the cellular subpopulation that inactivates the Xm chromosome could rapidly expand to replace the normally imprinted cells in extraembryonic lineages if the normal silencing of Xp compromises cell growth or differentiation. Interestingly, it has been suggested that the size of the population that initially escapes imprinting may range widely (from 0% to 30%), even between genetically identical embryos [37], and this may account for the variable phenotype observed among females bearing Xm-linked mutant alleles of genes essential for normal extraembryonic development [38]. Put simply, carrier females bearing a small initial population of escaping cells would be more severely affected than those bearing a larger population. This could explain why we have observed significant phenotypic variation among Atrx carrier females, with some carriers dying in utero by 9.5 dpc (Table 1) and others developing to term. + +Another possible mechanism is that inactivation of the paternal X proceeds normally in all cells, but subsequently the Atrx gene within individual cells is reactivated. Alternatively, in the absence of Atrx, the paternal allele may partially escape the normal process of silencing. In both of these cases, other genes on the paternal X chromosome must be inactivated and remain so, since blocking inactivation of the entire Xp chromosome causes embryonic lethality due to biallelic expression of X-linked genes in the trophoblast [39]. + +Summary + +ATR-X syndrome is the first human genetic disease known to be caused by mutations in a chromatin remodelling factor. At present we do not know how ATRX influences gene expression or what effect it has on cell behaviour. Nevertheless, we have previously noted that none of the natural mutations causing ATR-X syndrome are nulls, which suggests that it plays a critical role in normal development. Results of conditional inactivation of Atrx in the developing mouse forebrain, based on the Atrxflox allele described here, shows that Atrx exerts a major effect on terminally differentiating neurons. Conditional inactivation of Atrx in other tissues is underway. Here we have shown that animal-wide disruption of the Atrx gene causes a severe embryonic-lethal phenotype, revealing an essential role for Atrx in the formation of the murine trophoblast. In addition, Atrx appears to escape imprinted X-chromosome inactivation in the extraembryonic tissues of some carrier female mice. + +Materials and Methods + +Generation of ES cells bearing the Atrxflox allele. + +Briefly, the targeting vector (shown in Figure 2A) places a loxP site within intron 18 and a loxP-flanked MC1neopA selection cassette in intron 17 of the Atrx gene. A detailed description of the targeting construct is provided in [20]. Linearised plasmid (150 μg) was electroporated into 1 × 108 E14Tg2a ES cells, and colonies resistant to G418 and ganciclovir were isolated. Homologous targeting events were identified by Southern blot of EcoRI-digested DNA and hybridisation with a 5′ probe (generated with primers PPS1.20 and PPS1.27) and a 3′ probe (a 0.9-kb HaeIII fragment) as shown in Figure 2A and 2B. DNA from correctly targeted clones was also digested with SacI and analysed by Southern blot and hybridisation with a probe from within intron 17 (a PCR product generated with primers PPS1.15 and Xnp46) to confirm that the loxP site within intron 18 had been included within the crossed-over region (Figure 2A and 2C). Sequences of primers are shown in Table S1. + +Cre-recombination and characterisation of Atrxnull ES cells and embryoid bodies. + +ES cell clones bearing the Atrxflox allele (1 × 107 cells) were transiently transfected with 50 μg of uncut Cre expression plasmid (pCAGGS-Cre-IRESpuro) [40]. Following transfection, cells were plated at a range of clonal densities in complete medium without G418, and isolated subclones were picked after 7 d. Subclones were expanded and analysed for the presence of a recombinant locus initially by PCR, to detect deletion of the MC1neopA cassette, and then by Southern blot and hybridisation with the intron 17 probe described above (Figure 2A and 2C). Northern blots were carried out according to standard techniques using 20 μg of total RNA isolated using TRI Reagent (Sigma-Aldrich, St. Louis, Missouri, United States). The blot was hybridised with a probe from within exon 10 of the Atrx gene (generated with primers Mxnp4 and Mxnp28 [Table S1]). After it was stripped, the membrane was hybridised with a β-actin cDNA probe (Clontech, Palo Alto, California, United States). Protein extraction and detection of Atrx by Western blotting was performed as described previously [4], using the mouse monoclonal anti-ATRX antibody 23C [15]. Analyses of cell cycle and apoptosis are described in Protocol S1. Methylation of rDNA was analysed in DNA from ES cell clones or from embryoid bodies recovered after 7 d of in vitro differentiation as described previously [41]. Genomic DNA was digested with methylation-sensitive restriction enzymes as described and analysed by Southern blotting. The RIB3 and RIB4 probes (which were amplified from human DNA, but cross-react with the mouse rDNA repeat) have been described previously [11]. Oligonucleotide probes to detect Line 1 and Sine B1 repeats have been described previously [42]. The minor satellite probe was a 27-mer oligonucleotide (mCENT2). The major satellite probe was a 27-mer oligonucleotide (DG27). The IAP probe was an ~400 bp PCR product (primers 14A and 13K) amplified from an IAP inserted into the mouse agouti gene [43] and was a gift from Peter Warnecke and Tim Bestor. The PCR product included the entire 5′ LTR of the IAP. All oligonucleotide sequences are shown in Table S1. + +Generation of chimeras, floxed mice, and mutant mice. + +Targeted Atrxflox ES cell clones were injected into C57BL/6 blastocysts and transferred into 2.5 dpc pseudopregnant recipients by standard techniques. Resulting chimeras were mated with C57BL/6 to establish germline transmission. Offspring with the Atrxflox allele were identified by Southern blotting of SacI-digested tail DNA using the intron 17 probe as shown in Figure 2A and 2C. For Cre-recombination, Atrxflox mice were crossed with GATA1-Cre transgenic mice as described in the text. Recombinant alleles were detected by Southern blotting as described above or by PCR as described in Protocol S1. + +Immunohistochemistry, in situ hybridisation, and TUNEL assay. + +7.5 dpc decidual swellings were dissected away from maternal tissue and fixed in 4% paraformaldehyde/PBS overnight at 4 °C. After embryos were washed thoroughly in PBS, they were dehydrated through an ethanol series and xylene, embedded in paraffin, and sectioned at 5 μm. Sections were processed for immunohistochemistry using the ABC Staining System (Santa Cruz Biotechnology, Santa Cruz, California, United States) according to the manufacturer's instructions. Sections were stained with rabbit polyclonal antibodies against ATRX (H-300, Santa Cruz Biotechnology), Placental lactogen-I (AB1288, Chemicon International, Temecula, California, United States) and phospho (Ser10)-histone H3 (06–570, Upstate Biotechnology, Waltham, Massachusetts, United States). Where appropriate, adjacent sections were stained with haematoxylin. In some cases, adjacent sections were also analysed to detect apoptotic cells by TUNEL using the in situ cell death detection kit (Roche, Basel, Switzerland). After labelling, these slides were mounted in Vectashield containing DAPI (Vector Laboratories, Burlingame, California, United States) and visualised by fluorescence microscopy. Whole-mount in situ hybridisations were performed on 8.5 dpc embryos (dissected away from maternal and extraembryonic tissues) using a brachyury (T) riboprobe [16] and on bisected decidual implantation sites from which embryos (8.5 dpc) had been removed using a placental lactogen-1 (Pl-1) riboprobe (see Protocol S1 for details). + +Blastocyst outgrowth cultures. + +Superovulated heterozygous female mice (AtrxWT/flox) were mated to homozygous GATA1-cre+/+ transgenic males, and blastocysts were flushed from uterine horns with M2 medium (Sigma) at 3.5 dpc. Individual blastocysts were cultured in multiwell tissue cultures plates as described previously [44]. Cultures were inspected and photographed daily and the extent of outgrowth scored. After 7 d, cultures were harvested and DNA extracted. The Atrx genotype and sex of each culture was determined by PCR as described in Protocol S1. + +Supporting Information + +Figure S1 + +Cell Cycle Analysis of Atrx-Positive and Atrxnull ES Cells + +(A) Representative FACS profiles of BrdU-pulsed ES cells bearing either functional (AtrxWT or Atrxflox) or null (AtrxΔ18Δneo) Atrx alleles showing BrdU-FITC (y-axis, logarithmic scale) against propidium iodide (x-axis, linear scale). The gated populations show cells in G1 (PIlow, BrdU-negative) (R1), S (BrdU-positive) (R2), and G2/M (PIhi, BrdU-negative) (R3) phases of the cell cycle. Below is shown the quantitation of gated populations, indicating the percentage of cells in G1, S, and G2/M cell-cycle phases in each culture. + +(B) FACS profiles of ES cells bearing either functional (AtrxWT or Atrxflox) or null (AtrxΔ18Δneo) Atrx alleles stained for the mitosis marker phosphorylated (Ser10) histone H3 (FITC, y-axis, logarithmic scale) against propidium iodide (x-axis, linear scale). The size of the mitotic population (phosphoH3S10-positive, PIhi)(gate R3) is indicated for each profile. The ES cell clone analysed in each trace is indicated. + +(3.2 MB PDF) + +Click here for additional data file. + +Figure S2 + +Analysis of Apoptosis in Atrx-Positive and Atrxnull ES Cells + +FACS analysis of ES cells bearing different Atrx alleles as shown, after costaining for Annexin V (FITC, x-axis, logarithmic scale) and propidium iodide (y-axis, logarithmic scale). The size of the early apoptotic (Annexin-positive, PIlow) and late apoptotic or necrotic (Annexin-positive, PIhi) populations is indicated for each genotype. + +(223 KB PDF) + +Click here for additional data file. + +Figure S3 + +Normal DNA Methylation at Mouse Repetitive Elements in Atrxnull ES Cells + +DNA from Atrx-positive (bearing either an AtrxWT or Atrxflox allele) or Atrxnull (bearing the AtrxΔ18Δneo allele) ES cells was digested with either a CpG-methylation-sensitive enzyme HpaII (H) or its methylation-insensitive isoschizomer MspI (M) as indicated, and digested DNA was analysed by Southern blotting. Membranes were hybridised with probes specific for the mouse Line 1 (A), Sine B1 (B), minor satellite (C), and IAP (D) repeat elements. No significant loss of CpG-methylation was observed at any of these repetitive elements in Atrxnull ES cells. As a positive control for loss of methylation, DNA from ES cells lacking either the Dnmt1 (Dnmt1−/−) or both the Dnmt3a and Dnmt3b (Dnmt3a3b−/−) DNA methyltransferases was included in the analysis of Line 1 and Sine B1 repeats. Loss of CpG-methylation was clearly observed at these repetitive elements in these cell lines. + +(1.8 MB PDF) + +Click here for additional data file. + +Figure S4 + +Escape from Imprinted Inactivation in 8.5 dpc Carrier Female Embryo + +A cross was carried out between a carrier female (AtrxWT/null) and wild-type male (AtrxWT/Y), and embryos were dissected in their deciduas at 8.5 dpc. Any carrier female progeny of this cross will carry an AtrxWT allele on the Xp chromosome and an Atrxnull allele on the Xm chromosome. Transverse paraffin sections were stained with the anti-ATRX antibody (H-300). + +(A) High-magnification image (400×) of the neural fold region (nf) (embryo proper) of a carrier female (AtrxWT/null) embryo. This tissue is clearly comprised of a mosaic of Atrx-positive and Atrx-negative cells due to random X-inactivation. This section was counterstained with haematoxylin. + +(B) High-magnification image (400×) of the same embryo depicted in (A), showing the extraembryonic derived-chorionic ectoderm (ce) and visceral endoderm (ve), two tissues that undergo imprinted X-inactivation. Although Atrx is poorly expressed in the visceral endoderm (even in wild-type females [unpublished data]), strong expression of Atrx can be seen in the chorionic ectoderm, indicating that the paternally-derived AtrxWT allele had escaped inactivation in this tissue. + +(1.7 MB PDF) + +Click here for additional data file. + +Protocol S1 + +Supplementary Methods + +(42 KB DOC) + +Click here for additional data file. + +Table S1 + +Primers Used in This Study + +(20 KB DOC) + +Click here for additional data file. + +Accession Numbers + +The Online Mendelian Inheritance in Man (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM) accession number for ATR-X syndrome is 301040. The GeneID (www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=gene) for human ATRX is 546 and for mouse Atrx is 22589. The GenBank (http://www.ncbi.nlm.nih.gov/) accession number for the minor satellite probe mCENT2 is X14470 (nucleotides 75–101), for the major satellite probe DG27 is M25032 (nucleotides 146–172), and for the IAP probe is L33247. + +Acknowledgements + +We would like to thank Stu Orkin for providing access to the GATA1-Cre transgenic mice, Gillian Morriss-Kay for advice, Peter Warnecke and Tim Bestor for the IAP probe, En Li for the Dnmt knockout ES cells, Frank Talamantes for the pRSV-mPL-1 plasmid, Ann Atzberger for assistance with flow cytometry, and Deb Bogani and Terry Hacker for assistance with whole-mount in situ hybridisation analyses and embryo sectioning. + +Abbreviations + +BrdU - bromodeoxyuridine + +dpc - days postcoitus + +ES cells - embryonic stem cells + +IAP - intracisternal A particle + +rDNA - ribosomal DNA + +TGC - trophoblast giant cell + +TUNEL - TdT-mediated dUTP nick end labeling + +WMISH - whole-mount in situ hybridisation + +Figures and Tables + +Figure 1 + +Schematic Representation of the ATRX Isoforms + +Shown at the top is the human ATRX cDNA. The boxes represent the 36 exons. The introns are not to scale. The alternative splicing of exons 6 and 7 is indicated. Shown underneath are the two ATRX protein isoforms. Full-length ATRX (~280 kDa) is encoded by the largest open reading frame. The positions of the principal features (the PHD-like domain and the seven SWI/SNF-like motifs) are indicated. Above full-length ATRX is shown the truncated ATRXt isoform (apparent molecular weight of ~200 kDa) that arises through the failure to splice intron 11 and the use of an intronic poly(A) signal. The intron-encoded region of ATRXt is indicated as a filled grey box. Locations of recombinant proteins (A2, FXNP5, and H-300) used to generate antibodies are shown. The scale bar represents 200 amino acids. + +Figure 2 + +Cre-Mediated Ablation of Full-Length Atrx Protein in ES Cells + +(A) Strategy for targeted deletion of exon 18 of the Atrx gene. The top line shows the wild-type allele (AtrxWT) at the region surrounding exon 18. Below is shown the targeting vector and the targeted allele (Atrxflox) resulting from homologous recombination. The loxP target sites of the Cre recombinase are shown as black triangles, and the three possible recombination events that can be mediated by the Cre recombinase are indicated (labelled A, B, and C in the Atrxflox allele). At bottom is shown the Cre-recombined allele (AtrxΔ18Δneo) (resulting from recombination event C) in which both exon 18 and the MC1neopA selection cassette have been deleted. EcoRI (labelled E) and SacI (labelled S) sites present on the targeted 129 strain X chromosome are indicated. Black bars indicate the positions of the probes used in Southern blots. + +(B) Southern blot analysis of EcoRI-digested DNA from either wild-type ES cells (E14) or targeted ES cell clones bearing the Atrxflox allele (1/F12 and 1/G11) hybridised with either the 20/27 (left blot) or Hae0.9 (right blot) probes. The EcoRI fragment of the AtrxWT allele (18.5 kb) has been replaced with the expected fragments of 11.2 kb (20/27 probe) or 8.5 kb (Hae0.9 probe) + +(C) Southern blot analysis of SacI-digested DNA from either wild-type ES cells (E14) or targeted ES cell clones bearing the Atrxflox allele (1/F12 and 1/G11) or Cre-recombinant clones derived from these (1/F12B1F12 and 1/G11D5). The membrane was hybridised with the intron 17 probe indicated in (A). The expected bands of 6.2 (AtrxWT), 5.0 (Atrxflox), and 2.8 (AtrxΔ18Δneo) kb were observed. + +(D) Northern blot analysis of RNA from ES cells shown in (C). The membrane was hybridised first to a probe from exon 10 of the Atrx gene (top blot) and subsequently to a β-actin cDNA probe as loading control (bottom blot). The transcripts responsible for full-length Atrx (~10 kb) and the truncated Atrxt isoforms (~7 kb) are indicated. + +(E) Western blot analysis of whole-cell extracts from the clones shown in (C) using an anti-ATRX monoclonal antibody (23C, raised against peptide A2 of the human ATRX protein shown in Figure 1). The full-length and truncated Atrx isoforms are indicated. + +Figure 3 + +Growth and Methylation Defects in Atrxnull ES Cells + +(A) Cultures were inoculated with equivalent numbers of ES cells bearing different Atrx alleles as indicated, and were serially passaged. After the indicated days of coculture, DNA extracted from a sample of cells was analysed by Southern blot to detect the Atrx alleles. DNA was digested with SpeI, and the membrane was hybridised with the 20/27 probe shown in Figure 2A. The expected sizes of the different alleles are indicated. + +(B) Schematic diagram of the transcribed portion of the mouse rDNA repeat with the 18S, 5.8S, and 28S genes indicated. The positions of the limit-digesting enzymes BamH (labelled B) and EcoRI (labelled E) and the probes (RIB3 and RIB4) used in the Southern blots shown in (C) are indicated. Below are shown the locations of the methylation-sensitive enzymes (SmaI, PvuI, and MluI) whose methylation status has been analysed in the Southern blots shown in (C). + +(C) DNA from Atrx-positive (Atrx+, bearing either an AtrxWT or Atrxflox allele) or Atrxnull (bearing the AtrxΔ18Δneo allele) ES cells and 7-d embryoid bodies were digested with the enzymes shown and analysed by Southern blotting using the probes indicated. Arrows indicate the fully methylated copies (cut by only the limit-digesting enzyme). Phosphorimager quantitation of the blots are shown below. The y-axis shows the percentage of copies that are undigested by the methylation-sensitive enzyme as a percentage of the total signal from cut and uncut rDNA. Mean values are indicated by horizontal lines, and the significance of the differences between the Atrx-positive and Atrxnull populations are shown for each enzyme. + +Figure 4 + +Timing of Onset of GATA1-Cre Expression and PCR Genotyping of Atrx Alleles + +(A) GATA1-cre+/+ transgenic males were crossed to females of the ROSA26 reporter strain (ROSA26+/−), and embryos were recovered at 0.5 dpc (~16-cell morula stage) and stained with X-gal. Cre-mediated activation of the ROSA26 β-galactosidase reporter allele was detected in all cells in embryos in which both alleles are coinherited. + +(B) Top gel: PCR genotyping of Atrx alleles in embryos using primers PPS1.15 (exon 17) and Mxnp30 (exon 20) as described in Protocol S1. The sizes of PCR products from the different alleles are indicated. Both the AtrxΔ18 (resulting from recombination event B in Figure 2A) and the AtrxΔ18Δneo allele (resulting from recombination event C in Figure 2A) are null for full-length Atrx protein. The bottom gel shows products from a PCR reaction (primers DG52/DG53) used to sex embryos as described in Protocol S1. A 450-bp PCR product is amplified from a mouse Y chromosome-specific satellite repeat. + +Figure 5 + +Morphology of Atrxnull Embryos at 7.5 dpc and 8.5 dpc + +Paraffin sections of wild-type or Atrxnull 7.5 dpc embryos (dissected in their deciduas) were stained with haematoxylin (A) or with an anti-ATRX antibody (H-300, Figure 1) (B–E). Photomicrographs C–E show higher magnification images (200×) of the stained sections shown in (B) (40×). Scale bars represent 200 μm (40× magnification) or 40 μm (200× magnification). a, amnion; ac, amniotic cavity; c, chorion; e, epiblast; ec, ectoplacental cavity; ecc, exocoelomic cavity; ep, ectoplacental cone; ne, neural ectoderm; rm, Reichert's membrane; tgc, trophoblast giant cell. + +(F) Detection of brachyury (T) expression in Atrxnull 8.5 dpc embryo (head fold stage) by WMISH. The genotype was determined by PCR (as shown in Protocol S1) using DNA extracted from yolk sac. hf, head fold; n, emerging notochord; ps, primitive streak. + +Figure 6 + +Analysis of Apoptosis and Mitosis in Atrxnull Embryos + +(A) Paraffin sections of wild-type or Atrxnull 7.5 dpc embryos (dissected in their deciduas) were analysed by TUNEL assay and apoptotic cells labelled with fluorescein-dUTP. Sections were counterstained with DAPI. + +(B) Paraffin sections of wild-type or Atrxnull 7.5 dpc embryos were stained with an antibody against the mitosis marker phosphorylated (Ser10) histone H3. Sections were counterstained with haematoxylin. For both (A) and (B), the presence or absence of Atrx in each embryo was determined by staining adjacent sections with the anti-ATRX antibody (H-300) as in Figure 5 (unpublished data). + +Figure 7 + +Trophectoderm Defect in Atrxnull Embryos + +(A) 8.5 dpc embryos were dissected from surrounding decidual tissue and observed in whole mount. The genotype of each (indicated above) was determined by PCR using DNA extracted from whole embryos after photography. In the left image, the wild-type female (three-somite stage, left) is surrounded by trophoblast (t) while the trophoblast component surrounding the Atrxnull males (at headfold/presomite [middle] and two-somite stages [right], respectively) is severely depleted. In the right image, the trophoblast has been dissected away from the embryonic region of the wild-type embryo, to reveal the small, abnormally shaped ectoplacental cone (epc) of the mutant littermates. + +(B) WMISH to detect expression of Pl-1 (a marker of TGCs) at the implantation sites in vacated deciduas that had contained 8.5 dpc wild-type (AtrxWT/WT) or Atrxnull (AtrxΔ18Δneo/Y) embryos. The genotype was determined by PCR using DNA extracted from whole embryos. TGCs are stained with Pl-1. + +(C) Paraffin sections of wild-type or Atrxnull 7.5 dpc embryos (dissected in their deciduas) were stained with an anti-Pl-1 antibody. The presence or absence of Atrx in each embryo was determined by staining adjacent sections with the anti-ATRX antibody (H-300) as in Figure 5 (unpublished data). + +(D) Examples of 5-d blastocyst outgrowth cultures. Extensive trophoblast outgrowing from the inner cell mass (icm) was observed in all genotypes. The Atrx genotype and sex of the blastocyst indicated were determined by PCR. + +Figure 8 + +Escape from Imprinted Inactivation of the Paternally Inherited AtrxWT Allele in Carrier Females + +Paraffin sections of wild-type (AtrxWT/Y) and carrier female (AtrxWT/null) 7.5 dpc embryos (dissected in their deciduas) were stained with the anti-ATRX antibody (H-300). Scale bars represent 200 μm (40× magnification) or 20 μm (400× magnification). + +(A) Stained sections showing whole embryos at 40× magnification. a, amnion; c, chorion; e, epiblast; ep, ectoplacental cone. + +(B) Higher-magnification image (400×) of the epiblast regions of the stained sections shown in (A). + +(C) Higher-magnification image (400×) showing the extraembryonic derived-chorionic ectoderm of the stained sections shown in (A). ce, chorionic ectoderm; cm, chorionic mesoderm. + +Table 1 + +Distribution of Atrx Genotypes in Timed Matings + +Footnotes + +Author contributions. DG, AJHS, WGW, DRH, and RJG conceived and designed the experiments. DG, JAS, RA, and RJG performed the experiments. DG, RA, WGW, DRH, and RJG analysed the data. LD contributed reagents/materials/analysis tools. LD provided technical assistance. DG wrote the paper. + +Competing interests. The authors have declared that no competing interests exist. + +Funding. This work was supported by the Medical Research Council of the United Kingdom. + +Citation: Garrick D, Sharpe JA, Arkell R, Dobbie L, Smith AJH, et al. (2006) Loss of Atrx affects trophoblast development and the pattern of X-inactivation in extraembryonic tissues. PLoS Genet 2(4): e58. DOI: 10.1371/journal.pgen.0020058 diff --git a/src/ontogpt/evaluation/craft/database/all/16670015.ann b/src/ontogpt/evaluation/craft/database/all/16670015.ann new file mode 100644 index 000000000..48ebbeabd --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16670015.ann @@ -0,0 +1,664 @@ +T1 SO:0001026 0 6 Genome +T2 http://purl.obolibrary.org/obo/MONDO_0011122 36 43 obesity +T3 SO:0000771 44 47 QTL +T4 NCBITaxon:10088 54 59 mouse +T5 SO:0000771 159 182 quantitative trait loci +T6 SO:0000771 184 187 QTL +T7 UBERON:0008979 221 228 carcass +T8 NCBITaxon:10088 271 276 mouse +T9 NCBITaxon:10090 290 293 MMU +T10 SO:0000771 362 365 QTL +T11 SO:0000771 452 455 QTL +T12 SO:0001023 456 463 alleles +T13 SO:0000704 530 537 genetic +T14 http://purl.obolibrary.org/obo/MONDO_0011122 842 849 obesity +T15 SO:0000771 850 853 QTL +T16 SO:0000771 1028 1031 QTL +T17 http://purl.obolibrary.org/obo/MONDO_0011122 1183 1190 obesity +T18 SO:0000771 1293 1296 QTL +T19 SO:0001026 1478 1485 genomic 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SO:0000771 24634 24637 QTL +T282 CHEBI:16236 24666 24673 ethanol +T283 SO:0000357 24708 24716 flanking +T284 SO:0000771 24717 24720 QTL +T285 SO:0001026 24737 24743 genome +T286 SO:0000771 24806 24809 QTL +T287 SO:0001026 24832 24838 genome +T288 SO:0000704 24919 24926 genetic +T289 SO:0000704 25037 25044 genetic +T290 http://purl.obolibrary.org/obo/MONDO_0011122 25255 25262 obesity +T291 NCBITaxon:10088 25297 25302 mouse +T292 SO:0001026 25436 25443 genomic +T293 SO:0001023 25616 25623 alleles +T294 http://purl.obolibrary.org/obo/MONDO_0011122 25736 25743 obesity +T295 SO:0000771 25744 25747 QTL +T296 SO:0000771 25757 25760 QTL +T297 SO:0000771 25854 25857 QTL +T298 SO:0000704 26018 26023 genes +T299 http://purl.obolibrary.org/obo/MONDO_0011122 26045 26052 obesity +T300 SO:0000771 26222 26225 QTL +T301 http://purl.obolibrary.org/obo/MONDO_0011122 26322 26329 obesity +T302 SO:0000771 26401 26404 QTL +T303 SO:0000771 26488 26491 QTL +T304 GO:0010467 26511 26521 expression +T305 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UBERON:0008979 31014 31021 carcass +T378 SO:0000704 31064 31068 gene +T379 PR:000001672 31096 31101 Sstr5 +T380 CHEBI:35222 31128 31137 inhibitor +T381 PR:000001672 31148 31153 Sstr5 +T382 UBERON:0001264 31231 31241 pancreatic +T383 CL:0000169 31231 31251 pancreatic beta cell +T384 PR:000001672 31273 31278 Sstr5 +T385 PR:000045358 31303 31310 insulin +T386 CHEBI:17234 31325 31332 glucose +T387 GO:0042593 31325 31344 glucose homeostasis +T388 SO:0000704 31390 31395 genes +T389 SO:0000694 31625 31628 SNP +T390 SO:0000704 31756 31760 gene +T391 GO:0010467 31756 31771 gene expression +T392 SO:0000694 31791 31794 SNP +T393 SO:0000694 31818 31821 SNP +T394 SO:0000028 31833 31835 bp +T395 SO:0000704 31845 31850 genes +T396 SO:0000704 32067 32071 gene +T397 GO:0010467 32067 32082 gene expression +T398 GO:0010467 32114 32124 expression +T399 SO:0000704 32158 32163 genes +T400 SO:0001816 32303 32316 nonsynonomous +T401 SO:0000771 32350 32353 QTL +T402 SO:0000704 32477 32482 genes +T403 SO:0000704 32659 32664 genes +T404 PR:000017012 32666 32670 Ubr1 +T405 PR:000001968 32672 32678 Ptpns1 +T406 PR:000017005 32680 32688 Ubce7ip5 +T407 PR:000010491 32693 32697 Mmp9 +T408 PR:000015393 32779 32784 Socs2 +T409 GO:0043161 32797 32845 ubiquitination dependent proteasomal degradation +T410 GO:0000502 32822 32833 proteasomal +T411 GO:0060396 32857 32869 Gh signaling +T412 PR:000017012 32876 32880 Ubr1 +T413 PR:000017005 32885 32893 Ubce7ip5 +T414 GO:0016567 32915 32937 protein ubiquitination +T415 PR:000017012 32942 32946 Ubr1 +T416 NCBITaxon:10088 32956 32960 mice +T417 UBERON:0001013 33026 33040 adipose tissue +T418 PR:000001968 33050 33056 Ptpns1 +T419 NCBITaxon:10088 33066 33070 mice +T420 PR:000010491 33121 33125 Mmp9 +T421 NCBITaxon:10088 33135 33139 mice +T422 SO:0000704 33220 33225 genes +T423 SO:0000704 33288 33293 genes +T424 SO:0001026 33456 33462 genome +T425 SO:0000771 33468 33471 QTL +T426 http://purl.obolibrary.org/obo/MONDO_0011122 33490 33497 obesity +T427 UBERON:0008979 33502 33509 carcass +T428 SO:0000771 33596 33599 QTL +T429 SO:0000771 33731 33734 QTL +T430 SO:0000704 33847 33854 genetic +T431 http://purl.obolibrary.org/obo/MONDO_0011122 33882 33889 obesity +T432 NCBITaxon:10088 33901 33906 Mouse +T433 NCBITaxon:10088 33955 33959 mice +T434 NCBITaxon:10088 34175 34179 mice +T435 NCBITaxon:10088 34258 34262 Mice +T436 NCBITaxon:33208 34530 34536 Animal +T437 UBERON:0002415 34613 34617 tail +T438 CHEBI:63016 34702 34706 NP40 +T439 CHEBI:53424 34722 34730 Tween 20 +T440 CHEBI:15377 34829 34832 H2O +T441 NCBITaxon:10088 35071 35075 mice +T442 SO:0000112 35110 35116 primer +T443 SO:0000112 35139 35145 primer +T444 SO:0001030 35154 35155 F +T445 SO:0001031 35184 35185 R +T446 SO:0000028 35249 35251 bp +T447 NCBITaxon:10088 35278 35282 mice +T448 SO:0001030 35307 35308 F +T449 SO:0001031 35342 35343 R +T450 SO:0000028 35383 35385 bp +T451 PR:000005845 35386 35391 Cradd +T452 NCBITaxon:10088 35450 35454 mice +T453 GO:0097617 35469 35478 annealing +T454 CHEBI:6636 35508 35513 MgCl2 +T455 NCBITaxon:10088 35739 35743 mice +T456 SO:0000028 35847 35849 bp +T457 SO:0001026 35978 35984 genome +T458 SO:0000771 36039 36042 QTL +T459 SO:0001026 36133 36139 genome +T460 SO:0001023 36192 36199 alleles +T461 NCBITaxon:10088 36687 36691 mice +T462 GO:0007618 36702 36707 mated +T463 GO:0007618 37037 37043 mating +T464 SO:0000771 37509 37512 QTL +T465 SO:0000771 37746 37749 QTL +T466 SO:0000771 37795 37798 QTL +T467 SO:0001023 37867 37874 alleles +T468 SO:0000771 37894 37897 QTL +T469 SO:0000771 38004 38007 QTL +T470 SO:0001023 38343 38350 alleles +T471 SO:0001026 38383 38389 genome +T472 SO:0001026 38418 38424 genome +T473 SO:0000771 38534 38537 QTL +T474 NCBITaxon:10088 38648 38652 mice +T475 GO:0007618 38762 38768 mating +T476 SO:0000704 39093 39100 genetic +T477 SO:0001023 39171 39178 alleles +T478 SO:0001023 39239 39246 alleles +T479 NCBITaxon:10088 39613 39617 mice +T480 NCBITaxon:10088 39644 39648 Mice +T481 SO:0001023 39703 39710 alleles +T482 GO:0007618 39746 39751 mated +T483 GO:0007618 39865 39871 mating +T484 SO:0001023 40062 40069 alleles +T485 NCBITaxon:10088 40393 40397 mice +T486 NCBITaxon:10088 40611 40615 Mice +T487 CHEBI:33290 40647 40651 Feed +T488 CHEBI:15377 40707 40712 water +T489 NCBITaxon:10088 40738 40742 Mice +T490 NCBITaxon:10088 40830 40834 mice +T491 CHEBI:6015 40859 40869 isoflurane +T492 UBERON:0000004 40874 40879 nasal +T493 UBERON:0001245 40880 40884 anal +T494 UBERON:0000004 40901 40906 nasal +T495 UBERON:0002415 40907 40911 tail +T496 UBERON:0002415 40950 40954 Tail +T497 NCBITaxon:10088 41006 41010 mice +T498 UBERON:0012283 41067 41082 Femoral fat pad +T499 UBERON:0012283 41084 41087 FFP +T500 UBERON:0003428 41090 41105 gonadal fat pad +T501 UBERON:0003428 41107 41110 GFP +T502 UBERON:0015143 41113 41131 mesenteric fat pad +T503 UBERON:0015143 41133 41136 MFP +T504 UBERON:0010411 41142 41165 retroperitoneal fat pad +T505 UBERON:0010411 41167 41170 RFP +T506 UBERON:0008979 41286 41295 carcasses +T507 UBERON:0003916 41381 41388 fat pad +T508 UBERON:0008979 41409 41416 carcass +T509 UBERON:0001555 41429 41445;41451 41456 gastrointestinal ... tract +T510 UBERON:0005409 41447 41449 GI +T511 UBERON:0008979 41486 41495 carcasses +T512 UBERON:0008979 41543 41550 carcass +T513 UBERON:0008979 41565 41574 Carcasses +T514 UBERON:0008979 41688 41697 carcasses +T515 CHEBI:15377 41735 41740 water +T516 CHEBI:25698 41841 41846 ether +T517 CHEBI:15347 41871 41878 acetone +T518 UBERON:0008979 41928 41935 Carcass +T519 UBERON:0008979 42022 42029 Carcass +T520 UBERON:0008979 42091 42098 carcass +T521 SO:0000704 42982 42987 genes +T522 SO:0000704 42989 42994 Genes +T523 SO:0001026 43107 43114 genomic +T524 SO:0000704 43151 43155 gene +T525 GO:0060396 43176 43188 Gh signaling +T526 SO:0000704 43189 43193 Gene +T527 SO:0000704 43298 43303 genes +T528 SO:0000704 43351 43356 genes +T529 SO:0000771 43372 43375 QTL +T530 SO:0000704 43418 43422 gene +T531 SO:0000204 43490 43492;43500 43520 5' ... untranslated regions +T532 SO:0000205 43497 43520 3' untranslated regions +T533 GO:0006412 43502 43512 translated +T534 SO:0000704 43549 43553 gene +T535 UBERON:0000955 43689 43694 brain +T536 UBERON:0002107 43696 43701 liver +T537 UBERON:0002106 43703 43709 spleen +T538 UBERON:0002048 43711 43715 lung +T539 UBERON:0000473 43720 43726 testis +T540 UBERON:0007023 43748 43753 adult +T541 NCBITaxon:10088 43764 43769 mouse +T542 SO:0000112 43857 43863 primer +T543 SO:0000704 43878 43882 gene +T544 CHEBI:75958 44124 44132 solution +T545 CHEBI:32599 44141 44146 MgSO4 +T546 SO:0000112 44173 44179 primer +T547 NCBITaxon:271 44202 44205 Taq +T548 CHEBI:2511 44470 44477 agarose +T549 CHEBI:4883 44505 44509 EtBr +T550 CHEBI:15377 44585 44588 H2O +T551 CHEBI:6636 44683 44688 MgCl2 +T552 SO:0000112 44715 44721 primer +T553 NCBITaxon:271 44735 44738 Taq +T554 CHEBI:2511 44906 44913 agarose +T555 CHEBI:4883 44941 44945 EtBr +T556 CHEBI:2511 45083 45090 agarose +T557 CHEBI:4883 45117 45121 EtBr +T558 SO:0000006 45200 45211 PCR product +T559 SO:0000112 45233 45239 primer +T560 SO:0000704 45475 45479 gene +T561 SO:0001026 45541 45547 genome +T562 SO:0000150 45728 45733 reads +T563 SO:0000149 45763 45770 contigs +T564 SO:0000704 45793 45797 gene +T565 SO:0000704 46846 46851 genes +T566 SO:0000112 46906 46913 primers +T567 SO:0000704 46972 46977 genes +T568 NCBITaxon:10088 47045 47050 mouse +T569 NCBITaxon:10088 47223 47228 mouse +T570 NCBITaxon:33208 47323 47329 Animal +T571 UBERON:0008979 47363 47370 carcass +T572 SO:0001026 47700 47706 genome +T573 http://purl.obolibrary.org/obo/MONDO_0011122 47723 47730 obesity +T574 SO:0000771 47731 47734 QTL +T575 GO:0007618 47760 47767 matings +T576 GO:0007618 47799 47806 matings +T577 GO:0007618 48154 48161 matings +T578 SO:0001026 48318 48324 Genome +T579 SO:0000771 48368 48371 QTL +T580 SO:0000028 48528 48530 bp +T581 NCBITaxon:10088 48750 48754 mice +T582 SO:0000771 48856 48859 QTL +T583 UBERON:0002415 48965 48969 tail +T584 UBERON:0000004 48982 48987 nasal +T585 UBERON:0001245 48988 48992 anal +T586 UBERON:0003916 49031 49039 fat pads +T587 GO:0060396 49671 49685;49691 49708 growth hormone ... signaling pathway +T588 GO:0060396 49687 49689;49691 49708 Gh ... signaling pathway +T589 SO:0000704 49710 49715 Genes +T590 GO:0060396 49755 49767 Gh signaling +T591 SO:0000704 49831 49836 Genes +T592 NCBITaxon:10090 49883 49886 MMU +T593 SO:0001026 49887 49894 genomic +T594 SO:0000704 49913 49917 gene +T595 SO:0000704 49953 49958 genes +T596 SO:0000704 50056 50061 Genes +T597 SO:0000704 50118 50123 genes +T598 SO:0000771 50136 50139 QTL +T599 SO:0001026 50290 50296 genome +T600 SO:0000771 50302 50305 QTL +T601 http://purl.obolibrary.org/obo/MONDO_0011122 50327 50334 obesity +T602 SO:0000771 50336 50339 QTL +T603 SO:0000771 50341 50365 quantitative trait locus +T604 NCBITaxon:10090 50367 50370 MMU +T605 NCBITaxon:10088 50372 50377 mouse +T606 SO:0000771 50414 50417 QTL +T607 SO:0000771 50561 50564 QTL +T608 SO:0000028 50587 50589 bp +T609 SO:0001026 50642 50648 genome +T610 SO:0000028 50814 50816 bp +T611 NCBITaxon:10088 50830 50834 mice +T612 SO:0000028 50858 50860 bp +T613 NCBITaxon:10088 50872 50876 mice +T614 SO:0001026 51223 51230 genomic +T615 SO:0001023 51260 51267 alleles +T616 UBERON:0000004 51372 51377 nasal +T617 UBERON:0001245 51378 51382 anal +T618 UBERON:0002415 51402 51406 tail +T619 UBERON:0003428 51415 51418 GFP +T620 UBERON:0003428 51420 51435 gonadal fat pad +T621 UBERON:0012283 51437 51440 FFP +T622 UBERON:0012283 51442 51457 femoral fat pad +T623 UBERON:0015143 51459 51462 MFP +T624 UBERON:0015143 51464 51482 mesenteric fat pad +T625 UBERON:0010411 51484 51487 RFP +T626 UBERON:0010411 51489 51512 retroperitoneal fat pad +T627 UBERON:0003916 51540 51548 fat pads +T628 UBERON:0000004 52199 52204 nasal +T629 UBERON:0001245 52205 52209 anal +T630 UBERON:0003916 52233 52240 fat pad +T631 UBERON:0000004 52712 52717 nasal +T632 UBERON:0001245 52718 52722 anal +T633 UBERON:0002415 52742 52746 tail +T634 UBERON:0003428 53014 53017 GFP +T635 UBERON:0003428 53019 53034 gonadal fat pad +T636 UBERON:0012283 53036 53039 FFP +T637 UBERON:0012283 53041 53056 femoral fat pad +T638 UBERON:0015143 53058 53061 MFP +T639 UBERON:0015143 53063 53081 mesenteric fat pad +T640 UBERON:0010411 53083 53086 RFP +T641 UBERON:0010411 53088 53111 retroperitoneal fat pad +T642 UBERON:0003916 53139 53147 fat pads +T643 UBERON:0008979 53455 53462 Carcass +T644 CHEBI:15377 53521 53524 H2O +T645 UBERON:0008979 53526 53533 carcass +T646 CHEBI:15377 53534 53539 water +T647 CHEBI:15377 53542 53545 H2O +T648 CHEBI:15377 53547 53552 water +T649 UBERON:0008979 53569 53576 carcass +T650 UBERON:0008979 53590 53597 carcass +T651 UBERON:0008979 53629 53636 carcass +T652 UBERON:0008979 53650 53657 carcass +T653 UBERON:0008979 53689 53696 carcass +T654 UBERON:0008979 53711 53718 carcass +T655 UBERON:0008979 53759 53766 carcass +T656 SO:0001816 53979 53992 Nonsynonymous +T657 SO:0000694 53993 53996 SNP +T658 SO:0000704 54037 54042 genes +T659 NCBITaxon:10090 54101 54104 MMU +T660 NCBITaxon:10088 54106 54111 mouse +T661 SO:0001023 54299 54305 allele +T662 SO:0001023 54339 54345 allele +T663 SO:0001023 54366 54372 allele +T664 SO:0001023 54418 54424 allele diff --git a/src/ontogpt/evaluation/craft/database/all/16670015.txt b/src/ontogpt/evaluation/craft/database/all/16670015.txt new file mode 100644 index 000000000..10ea4e077 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16670015.txt @@ -0,0 +1,301 @@ +Genome-wide isolation of growth and obesity QTL using mouse speed congenic strains + +Abstract + +Background + +High growth (hg) modifier and background independent quantitative trait loci (QTL) affecting growth, adiposity and carcass composition were previously identified on mouse chromosomes (MMU) 1, 2, 5, 8, 9, 11 and 17. To confirm and further characterize each QTL, two panels of speed congenic strains were developed by introgressing CAST/EiJ (CAST) QTL alleles onto either mutant C57Bl/6J-hg/hg (HG) or wild type C57Bl/6J (B6) genetic backgrounds. + +Results + +The first speed congenic panel was developed by introgressing four overlapping donor regions spanning MMU2 in its entirety onto both HG and B6 backgrounds, for a total of eight strains. Phenotypic characterization of the MMU2 panel confirmed the segregation of multiple growth and obesity QTL and strongly suggested that a subset of these loci modify the effects of the hg deletion. The second panel consisted of individual donor regions on an HG background for each QTL on MMU1, 5, 8, 9, 11 and 17. Of the six developed strains, five were successfully characterized and displayed significant differences in growth and/or obesity as compared to controls. All five displayed phenotypes similar to those originally attributed to each QTL, however, novel phenotypes were unmasked in several of the strains including sex-specific effects. + +Conclusion + +The speed congenic strains developed herein constitute an invaluable genomic resource and provide the foundation to identify the specific nature of genetic variation influencing growth and obesity. + +Background + +The use of mouse models has provided valuable insight into the etiology of monogenic syndromes caused by single gene mutations. However, such models do not mimic the genetic complexity of disease traits commonly seen in the human population. Complex traits, such as polygenic growth and obesity are influenced by the small to moderate direct effects of quantitative trait loci (QTL), epistasis between QTL alleles, environmental perturbations and QTL-environment interactions. + +To date numerous mouse growth and obesity QTL have been localized [1,2], however, little progress has been made in determining the specific nature of the underlying genetic variants. One resource used to fine map QTL are congenic strains which are designed to convert a complex polygenic trait into one that is mono- or oligogenic. This is accomplished by eliminating segregating genetic variation outside the locus of interest and reducing the environmental variation influencing a trait by characterizing large numbers of genetically identical mice. The Complex Trait Consortium (CTC) considers congenic analysis an excellent method to confirm and subsequently fine map QTL [3]. + +Traditionally, congenic strains are developed by introgressing QTL alleles from a donor strain, whose boundaries are defined by genetic markers, on the genetic background of a recipient strain via 10 backcrosses [4]. While technically straightforward this is a time consuming endeavor taking over three years to construct a single strain. The speed congenic approach is an alternative to this lengthy process and can reduce the number of required backcrosses from 10 to five [5,6]. This strategy uses marker-assisted selection to identify male mice inheriting fewer donor alleles, than expected on average, during each backcross. Numerous traditionally developed and speed congenics have been used to successfully isolate mouse QTL for a wide array of traits, including growth and obesity [7-13]. + +The C57Bl/6J-hg/hg (HG) mouse is a model of systemic overgrowth resulting from a spontaneous deletion on MMU10, which eliminates expression of the Socs2 (suppressor of cytokine signaling 2) gene [14-16]. The role of Socs2 in the HG phenotype was confirmed by an independently engineered Socs2-/- knockout mouse which shared a number of phenotypes in common with HG, including gigantism [17]. + +QTL which alter the phenotypic effects of another locus are referred to as modifier QTL [18]. Modifier QTL have been mapped for numerous traits and in these studies the modified locus is typically a known gene containing a spontaneously arisen or engineered mutation with major phenotypic effects, such as hg [11,19-21]. Epistasis forms the basis of these interactions, implying that the known gene and its modifiers are members of the same biochemical or metabolic pathway. Different genetic backgrounds have been shown to modify the growth-enhancing effects of hg [15,22]. Since the primary function of Socs2 is to negatively regulate growth hormone (Gh) [23], it is likely these background effects are the result of polymorphism influencing interactions between members of the Gh signaling pathway. Thus, identification of hg modifiers has the potential to uncover novel members of metabolically important pathways or previously unknown convergences between pathways. + +As an initial step, growth and carcass composition QTL were identified in a cross between CAST/EiJ (CAST) and HG [24]. In the F2, mice homozygous for the hg deletion (hg/hg) and wild type (+/+) were assayed and hg modifier QTL were defined as those either absent in +/+ mice and segregating in hg/hg or loci with altered gene action dependent on background. Four hg modifier QTL (Wg2 on MMU2, Carfhg2 on MMU9, Carp2 on MMU11 and Feml3 on MMU17) were identified, along with 12 additional QTL (Q1Ucd1 on MMU1 (which only reached a suggestive level of statistical significance), Wg1, Carp1, Cara1 and Feml1 on MMU2, Carfhg1 on MMU5, Wg3 on MMU8, Feml2 on MMU9, Wg4 and Cara2 on MMU11 and Carp3 and Cara3 on MMU17) whose expression was independent of the hg locus. + +In the current study we have developed speed congenic strains to isolate all the aforementioned QTL. Single strains on an HG background were created for each chromosomal region outside of MMU2, while a comprehensive panel of overlapping strains with identical donor regions on both B6 and HG backgrounds were developed for MMU2. The MMU2 panel was developed to confirm the presence of multiple QTL and test for QTL-hg interactions. Additionally, based on the knowledge of potential QTL-hg interactions, genes involved in Gh signaling, whose genomic location overlapped hg modifier QTL on MMU2, 9, 11 and 17, were sequenced. + +Results + +Speed congenic strain development + +Two speed congenic panels were created, the first comprehensively dissected MMU2 while the second isolated QTL on MMU1, 5, 8, 9, 11 and 17 (Table 1). The MMU2 panel consisted of eight congenic strains developed by introgressing four overlapping donor regions onto both B6 and HG genetic backgrounds (Table 1). Single donor regions bred onto an HG background were created to isolate the remaining QTL on MMU1, 5, 8, 9, 11 and 17. Implementation of a speed congenic approach using marker-assisted breeding with 79 genome-wide microsatellite markers accelerated production of all strains [5,6] (Additional File 1 and Figure 1). Strain abbreviations and genomic region isolated by each strain are listed in Table 1. In addition to both congenic panels, two control strains homozygous B6 (B6.CASTC; B6C) or HG (HG.CASTC; HGC) for all genome-wide markers genotyped were developed from the same cross (see Methods) and served as the basis for strain comparisons. After stabilizing each congenic, 12 of 14 were phenotypically characterized for growth and adiposity. The HG2D and HG5 strains were not characterized due to reproductive problems. The recombinant end points for all strains were refined using microsatellite markers flanking each donor region (Additional File 2). + +MMU2 speed congenic strain characterization + +Due to the overlapping nature of the MMU2 congenics, five distinct chromosomal regions (Regions I–V) were queried for the presence of QTL (Figure 2). As a separate analysis we also tested for interactions between each donor region and the two distinct genetic backgrounds. The following sections describe the phenotypes for each B6.CAST and HG.CAST speed congenic strain as well as the results of the interaction analysis. + +B6.CAST MMU2 speed congenic strains + +The B62D strain exhibited the largest (P < 0.0001) decreases in body weight of any B6.CAST congenic (Additional File 3). Both sexes had reductions in weight at 2 (2WK), 3 (3WK), 6 (6WK) and 9 (9WK) weeks of age compared to control B6C mice (Figure 3). Despite large decreases in body weight, no differences in growth rates (G26, weight gain from 2 to 6 weeks and G29, weight gain from 2 to 9 weeks) were observed (Additional File 3). Therefore, the B62D unique region (Region V) harbors either an early-growth QTL or maternal genotype effect which produces a distinct decrease in body weight prior to 2WK. These effects are evident in the growth curves for both sexes of each strain (Figure 3). Additionally, B62D mice displayed significantly shorter tail lengths despite no difference in naso-anal length (NA) (Table 2). + +In contrast, B62M mice displayed small decreases in body weights and growth (male 9WK was significant, female 6WK and 9WK were suggestive at P < 0.05) and significant decreases in NA (male P = 0.0152) and tail length (Additional File 3 and Table 2). The decrease in NA in B62M mice is surprising since the 2M donor region is entirely nested within the 2D donor region and B62D mice showed no difference in NA (Figure 2). These data indicate the presence of three QTL with the 2D unique region. Two of which are disjoined by the B62M and B62D strains and additively decrease body weight and tail length, but not NA. The third QTL, located within the B62D unique region (Region V), increases NA canceling the effects of the B62M QTL (Region IV) decreasing NA. + +B62PM females had decreases in all growth-related traits except 2WK and 3WK, while no differences were seen in males (Additional File 3). Similarly, only B62P females and not males showed significant decreases in 9WK, G26 and G29 (Additional File 3), indicating these two strains share a female specific growth QTL (Region II). + +In the original genome scan body fat percentage as determined by chemical compositional analysis was not linked to markers on MMU2 [24]. However, since numerous obesity QTL have been found on MMU2 [2], some of which were discovered in B6 by CAST crosses [9,25], we chose to measure dissected fat pad weights as a more sensitive measure of adiposity. + +Table 2 lists the weights of gonadal (GFP), femoral (FFP), mesenteric (MFP) and retroperitoneal (RFP) fat pads, along with total fat pad weight (sum of the four fat pads), adiposity index (AI = TF/weight at sacrifice (WSAC)) and body mass index (BMI = WSAC/NA2 *100) for each MMU2 congenic strain. Similar to large differences in body weight the B62D strain displayed highly significant decreases in adiposity. B62D was the only strain where both sexes had significant decreases in TF, AI and BMI. Interestingly, no difference in adiposity was seen in B62M mice. This discordance indicates the leanness promoting QTL is located in the B62D unique region (Region V). Only minor differences in various fat pad weights were observed for the B62P and B62PM strains (Table 2). + +HG.CAST MMU2 speed congenic panel + +HG2P and HG2PM mice of both sexes displayed significant decreases in post-weaning body weights and growth rates (Additional Table 3). In addition, the HG2M strain displayed highly significant decreases in length, similar to B62M mice (Table 2). These results indicate QTL within Region II shared between the HG2P and HG2PM strains decrease body size, weight gain and NA (only in females), while a QTL in the HG2M region significantly decreases tail length. + +Similar to growth traits a general decrease in GFP, MFP, RFP, TF and AI were seen in HG2P and HG2PM mice (Region II) (Table 2). These differences in adiposity are likely pleiotropic effects of the growth QTL shared between these two strains. Surprisingly, HG2M mice displayed striking sex differences in adiposity. Males exhibited an increase in AI (P = 0.0256) with a decrease in BMI (P = 0.0462) and the exact opposite was seen in females (AI, P < 0.0001; BMI, P = 0.0425) (Table 2). Therefore, the obesity QTL in the HG2M unique region (Region IV) is profoundly impacted by the presence of hg. + +Confirmation of QTL-hg interactions + +An important feature of our experimental design was the ability to test for interactions between QTL in each MMU2 donor region and genotype at the HG locus, since identical donor regions were introgressed on two genetic backgrounds, B6 (+/+) and HG (hg/hg). Significant interactions between donor region and HG genotype were viewed as strong evidence that hg modifier QTL reside within that unique genomic region. + +In the original linkage analysis the mode of gene action and peak location of Wg2 were dependent on the presence of hg [24] (Table 1). In the present study, only homozygous congenic mice were characterized, thus, the overdominant effects of Wg2 were not tested. However, since its original peak location differed dependent on background we hypothesized that Wg2 represents a set of linked QTL between 74.9 and 181.8 Mbp within the 2M and 2D donor regions (Region III–V), some of which interact with hg. Following this logic we expected one or both of these strains would exhibit donor region by HG genotype interactions. Unfortunately, we were unable to characterize HG2D mice. However, as noted above, strong sex-specific effects were seen in HG2M mice. Significant 2M donor region by HG genotype by sex three-way interactions for AI (P = 0.0004; Figure 4) and TF were identified (Table 3). The basis of these interactions was a decrease in HG2M female and an increase in HG2M male adiposity, with no differences in fat accumulation across B62M sexes. Significant 2PM donor region by HG genotype two-way and 2PM donor region by HG genotype by sex three-way interactions were also seen for TF and AI (Table 3). In addition, significant 2P donor region × HG genotype interactions were observed for all traits listed, although some traits did reached significance at the critical P < 0.0071 (Table 3). The basis for each interaction was a decrease in phenotype in HG2P compared to HGC and no difference between B62P and B6C. Together these data confirm that MMU2 QTL modify the effects of the hg deletion. + +Speed congenic strains for QTL on MMU1, 8, 9, 11 and 17 + +HG1 + +Q1Ucd1 affecting G26 was the least significant of any QTL identified in the original intercross and only reached a suggestive level of significance (LOD = 2.46, P < 0.05) [26]. In accordance, HG1 mice had small differences in all growth and length traits. NA in both sexes and 2WK, 3WK, 6WK and tail length in females was significant at a critical P < 0.005 (Table 4). The only difference in adiposity was a significant decrease in male MFP (Table 5). The successful capture and confirmation of the small effect Q1Ucd1-w26 QTL adds support to pursuing QTL identified at suggestive levels of statistical confidence from genome scans. + +HG8 + +Markers on MMU8 were linked with G29 in the original intercross [24]. This QTL was small, only accounting for 4.3% of the phenotypic variance. Interestingly, in HG8 mice no differences in growth were detected; however, they were significantly leaner than controls (Table 5). In males a general decrease in fat pad mass, TF and AI was detected; however, only MFP was significant at a critical P < 0.005. Male AI was nearly significant at a nominal P = 0.0082. Larger decreases in fat mass were seen in HG8 females, with significant decreases detected for GFP, MFP and AI. Additionally, RFP and TF were nearly significant with nominal P values of 0.0208 and 0.0153, respectively. HG8 mice represent a lean congenic model in which CAST alleles protect against fat accumulation. + +HG9 + +CAST alleles in the proximal region of MMU9 were previously found to be linked with an increase in body fat percentage and a decrease in femur length [24]. The effects appeared to be the result of two linked QTL, Carfhg2 at 10 cM and Feml2 at 20 cM, instead of one pleiotropically impacting both traits (Table 1). In support of this, the increase in fat was dependent on hg; however, differences in femur length were not [24]. + +In confirmation of Carfhg2 all measures of adiposity in HG9 mice were increased relative to control mice; except MFP in both sexes and BMI in males (Table 5). In males, GFP, RFP, FFP and AI were increased by 29.0%, 51.2%, 42.9% and 30.0%, respectively. In females these increases were much larger. The same measurements were increased by 68.8%, 99.4%, 69.7% and 57.3%, relative to HGC females. A 2.9% and 3.6% reduction in male and female NA, respectively, was also detected. This was expected since NA was previously found to be an excellent indicator of femur length (R2 = 0.88) in HG mice [27] and as noted above this region contains the Feml2 QTL [24]. These data confirm the effects of Carfhg2 on adiposity and Feml2 on length and indicate the isolated effects of Carfhg2 are more significant than originally observed. + +HG11 + +In the original intercross CAST alleles near 50 cM on MMU11 were linked with a reduction in G29, carcass ash (ASH) and carcass protein (PROT) [24]. Therefore, we measured each carcass component using chemical compositional analysis in the HGC, HG11 and HG17 strains. Originally, Carp2 was dependent on the presence of hg and no significant sex effects were detected [24]. In contrast to those results, HG11 mice displayed significant differences in growth and obesity between the sexes. In general, males demonstrated a 5% increase and females a 5% decrease in all growth-related traits (Table 4). Most of these differences, however, were not significant at a critical P < 0.005, although almost all were at a nominal P < 0.05. Following the same trend, males displayed an increase in tail length and a small suggestive increase in NA (P = 0.06), while females displayed significant decreases in NA. In support of the sex-specific QTL effects strain by sex interactions (P < 0.04) were seen for the following growth traits: 3WK, 6WK, 9WK, G26, G29 and NA (data not shown). The basis of each interaction was an increase in the males and a decrease in females. + +In our analysis of the HGC, HG11 and HG17 strains, concordant results were observed measuring adiposity using chemical lipid extraction and weighing fat pad mass. The phenotypic correlation between carcass fat (FAT) as a percent of the empty carcass weight (ECW; weight of total body (including organs) minus the head and gastrointestinal tract) (%FAT) determined by chemical analysis and AI, using data from all three lines (HGC, HG11 and HG17), was 0.92. These data suggest that individual fat pad dissection is an excellent proxy for measuring whole body adiposity and provides a much more sensitive technique to measure fat accumulation in specific body regions. + +HG11 females displayed slight increases in all fat pads (except MFP) and AI, although, only the 16.5% increase in AI was significant (Table 5). In contrast, HG11 males did not show a difference in fat mass leading to a significant strain by sex interaction (P = 0.008) for AI (data not shown). Identical to the results obtained using fat pad weights; female HG11 carcasses displayed an increase in %FAT (Table 6). + +HG11 male carcasses had higher levels carcass ash (ASH) as a percent of ECW (%ASH) and female carcasses displayed lower levels of H2O and ASH (Table 6). Strains by sex interactions (P < 0.005) were identified for H2O, %H2O, %FAT, ASH and PROT (data not shown). If the aggregate phenotype in HG11 is due to a single locus, its function may be to disrupt energy partitioning by decreasing the deposition of lean tissue and increasing lipid accumulation in females, while having the opposite action in males. However, it may also be due to distinct sex-specific QTL with opposing action. + +HG17 + +In the original intercross CAST alleles at MMU17 markers were associated with decreases in femur length, ASH and PROT [24] and similar results were seen in HG17 mice. Interestingly, both sexes were heavier at 2WK (males, P = 0.0045; females, P = 0.0342), but lighter at 9WK, leading to substantially lower G26 and G29 (Table 4). In general growth differences ranged between 5% and 15% lower in congenic mice. + +Both sexes had significant reductions in length traits relative to control mice. NA was reduced by 2.7% (P = 0.0186) and 3.4% in males and females, respectively (Table 4). In addition, tail was reduced by approximately 6% in both sexes (Table 4). + +In general, HG17 mice displayed increases in all fat pads (except MFP), TF and AI (Table 5). However, none of these differences were significant at critical P < 0.005. In addition, HG17 male carcasses possessed lower levels of H2O and ASH, while female carcasses contained lower levels of % H2O, ASH, %ASH and %PROT (Table 6). + +MMU2, 9, 11 and 17 hg modifier candidate gene sequencing + +The HG phenotype is due to the deletion of Socs2, therefore we reasoned that QTL on MMU2, 9, 11 and 17 interacting with hg possibly represent variation within genes participating in various aspects of Gh function. To select candidate genes for sequencing we identified genes known to be or potentially involved in Gh signaling, are responsive to Gh or that propagate downstream Gh functions. Forty-four hg modifier candidate genes were identified from primary literature, reviews and book chapters and coordinated using the GenMAPP (Gene MicroArray Pathway Profiler) pathway building software (Figure 5). The coding region of each gene was sequenced from the CAST strain and compared to the publicly available B6 sequence to identify polymorphisms (Additional File 4). A total of 94.492 kbp was sequenced (75.378 kbp CDS and 19.114 Kbp 5' and 3' untranslated region (UTR)), representing 25,083 amino acids. Comparison with the public B6 assembly (May 2004 University of California, Santa Cruz (UCSC [28]) mm5 genome assembly, National Center for Biotechnology Information (NCBI) Build 33) identified 307 polymorphisms between CAST and B6. Of these, 295 were single nucleotide polymorphisms (SNP) and 12 were insertions or deletions in CAST (Additional File 4). All 12 insertions or deletions were located within 5' and 3' UTRs. Fifty-six nonsynonomous SNP (nsSNP) were identified in 14 different genes (Additional File 4). PolyPhen [29] and SIFT [30] are software programs designed to identify nsSNP which potentially alter protein function by evaluating evolutionary conservation at specific amino acid residues using a multiple sequence alignment of protein sequences homologous to the query. When applied to our data set PolyPhen, SIFT or both programs predicted that 15 of the 56 nsSNP in 9 different genes would possibly alter protein function (Table 7). + +Discussion + +Speed congenic strains provide a powerful approach to confirm and physically confine QTL within intervals defined by molecular markers. In the current study, approximately 20% of the CAST genome harboring all previously detected growth and carcass composition QTL was isolated on an HG or B6 background through the development of 14 speed congenic strains. Two distinct speed congenic panels were developed, the first provided a comprehensive isolation of all MMU2 QTL between the B6, HG and CAST strains and the second targeted all QTL outside of MMU2. Each successfully characterized strain exhibited phenotypic differences relative to control mice. These strains represent important resources and provide the genetic resource to positionally clone numerous quantitative trait genes. + +One criticism of the speed congenic approach is the potential for QTL to reside among unwanted donor alleles not eliminated during backcrossing. In this case differences between the congenic and control strains would be due in part or whole to these contaminating alleles. We took several precautions to reduce the probability of this occurrence. First, our control strains were developed from mice undergoing the same selection as all of the other congenics. Therefore, it is possible that any unwanted QTL or mutations arising during congenic construction are shared between all strains. More importantly, we have knowledge of all large previously detected growth and obesity QTL in the current cross [24]. Using this information we increased the density of markers in each QTL region (MMU1, 2, 5, 8, 9, 11 and 17; Table 1 and Additional File 1), ensuring the absence of CAST alleles at each of these QTL. This approach, termed "QTL-Marker-Assisted Counter Selection" or QMACS, has been previously used to characterize QTL for hypnotic sensitivity to ethanol [31]. In that study, only markers flanking QTL were typed, not genome-wide markers. In contrast, we selected not only against known QTL, we also screened for genome wide heterozygosity increasing the probability that effects observed are due to genetic variation within each donor region. + +Although great effort was put forth to eliminate non-donor region direct genetic effects, other factors such as maternal genotype (maternal genotype for each congenic versus control dams differed) and environmental effects could confound our results. Maternal genotype effects on growth and obesity have been observed in a number of mouse crosses [8,32-35] and their existence in the current cross cannot be discounted. However, our congenics provide the ideal foundation genomic resource to test for the influence of any of these possible effects. Future fine mapping experiments can be designed to randomize the influences of any contaminating donor alleles and environmental differences, as well as test for maternal genotype effects. + +MMU2 is a hotspot for growth and obesity QTL. Over 30 QTL have been identified in various experiments [1,2]. Several previously reported or novel MMU2 QTL have been isolated and characterized using congenic strains [7,9-11,36]. Our findings are no different and indicate that MMU2 is highly complex with regards to genes affecting growth and obesity. The overlapping nature of our MMU2 strains allowed us to parse the chromosome into five regions (Regions I–V) (Figure 2). The data support the presence of at least one QTL in each of the five regions (Figure 2). Each of the five pleiotropically impact both growth and obesity, although to varying degrees. + +In addition to the large number of MMU2 QTL, the presence of hg adds complexity by either eliciting interactions with the same QTL or by inducing the expression of novel QTL. The 2P unique region (Region I) contains an hg modifier with large effects on growth and smaller effects on obesity (Table 3). In contrast, interactions between 2PM/2M QTL and hg primarily affect fat deposition (Table 3). As illustrated in Figure 4 the 2M donor region exhibits strong sex effects on the rate of lipid storage, dependent on background. In control mice, HGC females have a higher AI than males, while the opposite is seen in B6C mice. Interestingly, the 2M hg modifier QTL abolished the hg induced sexual dimorphism in adiposity (Figure 4). Although these results provide insight into the nature of hg modifier QTL, it should be noted, that the actual number and precise location of loci is still unclear. + +In addition to MMU2, three other congenics (HG9, HG11 and HG17) captured hg modifier QTL. In classical terms, they are QTL which modify the expressivity of growth and obesity in HG mice [18]. These QTL are novel since they represent epistasis between hg and the modifier gene. QTL-hg epistasis implies that Socs2 and the hg modifiers are in the same biological pathway. Therefore, these QTL are likely due to polymorphism in genes interacting with Gh, responsive to Gh or which in some way modulate Gh function. This information will significantly aid QTL cloning by providing another filter to screen candidates. Cloning these QTL has major implications to improve our understanding of Gh and its regulation of growth and adiposity and in the administration of human Gh therapeutics. + +HG1 mice displayed differences only in growth traits. Originally, Q1Ucd1 had small effects and these results illustrate the power of congenic strain analysis to isolate small effect QTL, although it is likely this QTL represents the lower boundary of detection using only 20–30 congenic mice. The most notable candidate genes located within the HG1 interval are the signal transducer and activator of transcription 1 (Stat1) and Stat4. + +Two of the congenics had major alterations in the deposition of adipose tissue, HG8 and HG9. The HG8 donor region promotes leanness. Congenic females displayed a reduction of over 25% in AI. In contrast, the HG9 strain is an obese mouse model. The strain is quite novel for two reasons. First, the effect size is large; AI was 57% higher in HG9 females and 30% higher in males. Secondly, it is dependent on hg for its expression. The HG9 strain represents a major epistasis-based obese mouse model and promises to aid in the understanding of obesity and specifically the modulation of adipose tissue deposition by Gh. Studies are currently underway to identify the causative mutation and to characterize the effects of age and diet on obesity in this strain as well as testing for other physiological consequences such as alterations in food intake and insulin sensitivity. + +The HG11 strain is of particular interest for a number of reasons. First, HG11 congenic mice demonstrated significant strain by sex interactions for a number of traits. Males were generally larger, faster growing and longer and the converse was seen in females. The confounding effects of sex are likely the reason for the discrepancies between the congenic and genome scan results, where both sexes were analyzed together. Secondly, Carp2 was found to interact with hg and MMU11 is saturated with genes involved in the central Gh intracellular signaling pathway, such as Gh, Stat5b and Stat5a. Thirdly, MMU11 growth QTL overlapping HG11 have been identified in a number of crosses using different mouse strains [37-41]. Given the well documented sexually dimorphic nature of Gh secretion and Gh induced gene expression [42,43], it is probable that the underlying mutation in the HG11 congenic may reside in a gene enhancing Gh induced sex-specific effects. The structural Gh gene itself and Stat5b are excellent candidates. The potential role of Gh would include polymorphism that alters protein function in the absence of Socs2 or that causes transcriptional deregulation of Gh. Additionally, functional variation in Stat5b may explain the sex-specific phenotypes in HG11 mice since it is the primary transcription factor responsible for Gh induced sex-specific liver gene expression [44]. Future studies aimed at identifying the HG11 QTG, will certainly include a thorough characterization of Gh and Stat5b sequence and expression patterns in congenic mice. + +The hg modifier QTL located within the HG17 strain had large effects on growth, body length and carcass components. The most intriguing candidate gene located in the congenic is Sstr5. Somatostatin is a potent inhibitor of Gh and Sstr5 is one of five receptors which mediate these effects [45,46]. Ubiquitous and pancreatic beta cell specific knockout of Sstr5 leads to alterations in insulin secretion and glucose homeostasis [47,48]. + +We sequenced candidate hg modifier genes to complement the characterization of the speed congenics on MMU2, 9, 11 and 17. A limited number of studies have identified variation within CAST coding sequence; so sequencing candidates gave us the opportunity to estimate the SNP frequency in coding sequence relative to B6. This information will be vital to future fine mapping studies, which will include gene expression analysis. The high SNP frequency (0.312%; 295 SNP in 94.492 kbp) in CAST genes may lead to a higher rate of false positives using DNA microarrays or quantitative real-time PCR assays, most of which are based on B6 sequence. Therefore, these sequence data can be used to guide the development of gene expression assays to confirm differential expression for candidates and suggests that genes identified by downstream experiments should also be sequenced. + +More importantly, sequencing hg modifier candidates allowed us to identify nonsynonomous polymorphism, which may underlie QTL. SIFT and/or PolyPhen predicted alterations in protein function for 15 of the 56 total nsSNP (27%) in nine of the 44 total genes (20%) (Table 7). It has been shown that predicting function based on evolutionary conservation using programs such as SIFT and PolyPhen is very accurate [49]. Four of the nine genes (Ubr1, Ptpns1, Ubce7ip5 and Mmp9; all of which are on MMU2) are of particular interest. It has been suggested the Socs2 may rely on ubiquitination dependent proteasomal degradation to inhibit Gh signaling [23]. Ubr1 and Ubce7ip5 are both involved in protein ubiquitination and Ubr1 knockout mice show an 20% decrease in body weight partly due to a reduction in adipose tissue [50,51]. Ptpns1 knockout mice displayed a 10% reduction in body weight [52] and Mmp9 knockout mice show a reduction in size and drastically reduced bone length [53]. All of these genes are excellent functional and positional hg modifier candidate genes and this information can be incorporated into future fine mapping studies. + +Conclusion + +The speed congenic strains developed herein confirm previously identified genome-wide QTL affecting growth, obesity and carcass composition. Our novel congenic development approach on MMU2 provided confirmation of QTL-hg interactions, which will significantly aid in future fine mapping experiments. The identification of the molecular mediators of QTL-hg epistasis as well as the QTG for the remaining congenics will provide essential information on the molecular genetic architecture of growth and obesity. + +Methods + +Mouse strains and husbandry + +CAST/EiJ (stock #000928) mice were purchased from the Jackson Laboratory. The hg mutation was originally identified in a selected outcross line [22] and has been introgressed onto a B6 background via nine backcrosses to create the HG strain. HG mice used in this experiment were from the 17th or later generation of inbreeding. Mice were housed in polycarbonate cages under controlled conditions of temperature (21°C ± 2°C), humidity (40–70%) and lighting (14 h light, 10 h dark, lights on at 7 AM), and managed according to the guidelines of the American Association for Accreditation of Laboratory Animal Care (AAALAC). + +Genotyping + +DNA for genotyping was isolated from 1.0–2.0 mm tail clips by digesting with Proteinase K (Fisher) at 55°C in a buffer composed of 0.45% NP40 (Sigma), 0.45% Tween 20 (Fisher) and 1X PCR buffer (Promega). The product of this digestion was diluted (1:10) in sterile H2O and used for genotyping without further purification. Microsatellite genotyping was performed using standard PCR and gel electrophoresis protocols. Reaction conditions for each marker are listed in Additional File 1 and 2. + +MMU2 congenic mice were genotyped for hg using a two primer genotyping assay. One primer set (HG-F, ctcctgtctgggctgtgag and HG-R, caaaggcagaagtggggtaa) spanned the hg deletion producing a 447 bp product in hg/hg and +/hg mice. The other set (CRADD3a.F, gtccatcagcattcctgaaa and CRADD3.R, tgtccagcaacagcattgtc) amplified a 232 bp Cradd amplicon (located within the hg deletion) in +/+ and +/hg mice [54]. The PCR annealing temperature was 55°C and the MgCl2 concentration was 1.5 mM. + +Development of B6.CAST and HG.CAST MMU2 speed congenic strains + +All speed congenic strains were developed starting with an initial cross between a CAST male and HG females (Figure 1) [5,6]. Male F1 mice were then backcrossed to HG females. All agouti (the dominant nonagouti (a) locus is located at 154.8 Mbp on MMU2) N2 males were genotyped for 79 microsatellite markers (Additional File 1). These markers were evenly spaced across the genome, except in regions previously identified as harboring QTL [1], which were more densely screened. The "best" N2 agouti male with the lowest level of genome-wide unwanted heterozygosity while maintaining CAST alleles for all MMU2 markers was selected for breeding. This selection scheme was used at each generation until a N4 male was identified as homozygous HG for all markers typed outside MMU2. After an additional backcross to HG females, recombinant males were identified providing the foundation for the four overlapping donor regions. Selected recombinant males were then backcrossed to both B6 and HG females to create strains, which were B6 (+/+) or HG (hg/hg) and heterozygous congenic. These mice were intermated to produce homozygous founders for each strain. This novel breeding scheme created four identical founder congenics on two backgrounds B6 (+/+) and HG (hg/hg), which formed the basis for our examination of interactions caused by the presence of the hg deletion. MMU2 speed congenic strains were maintained through brother-sister mating. Once each congenic was stabilized, 20 additional microsatellite markers were used to refine the position of each congenic recombinant end point (Additional File 2). + +Development of HG.CAST speed congenic strains for MMU1, 5, 8, 9, 11 and 17 + +All black N2 males from the first two crosses described above were genotyped for 12 markers (D1Mit432, -480, D5Mit353, -311, D9Mit60, -262, D11Mit5, -67, D8Mit234, -211 and D17Mit28 and -142), two spanning each of the six QTL harboring regions (MMU1, 5, 8, 9, 11 and 17; Table 1 and Additional File 1). Markers were selected to capture, at a minimum, the 2-LOD support interval. Two N2 males were selected to propagate the N3 generation; one heterozygous for QTL on MMU1 and 9 and the other heterozygous for QTL on MMU5, 8, 11 and 17 (Figure 1). Both males were homozygous for HG alleles at all other known QTL. These males were backcrossed to HG females and two of the resulting N3 males inheriting the same sets of QTL as their sire were selected for breeding. These males were subsequently backcrossed to HG females and three N4 males were identified heterozygous for the following regions: 1) MMU1 and 9; 2) MMU5 and 11; 3) MMU8, 11 and 17 (Figure 1). Starting at N4 and continuing through N6, the "best" male with the lowest percent of unwanted donor alleles was selected after performing a genome scan using the remaining 67 genome-wide markers (79 total markers minus the 12 markers genotyped in the first two backcrosses spanning the know QTL intervals). At N5 a distinct strain was created for each of the six individual donor regions and heterozygous mice were intermated (Figure 1). Homozygous HG.CAST speed congenic strains were maintained through brother-sister mating. Once each congenic was stabilized, 19 additional microsatellite markers were used to refine the position of each congenic recombinant end point (Additional File 2). + +Development of B6.CASTC and HG.CASTC control strains + +HG is a strain in which the hg deletion has been introgressed onto a B6 background, therefore the only genetic differences between the strains would be the hg locus, tightly linked alleles from the outbred strain on which hg arose and contaminating alleles remaining after the nine backcrosses and fixed during inbreeding. Instead of using parental B6 and HG strains as controls for phenotypic comparisons with each speed congenic, we choose to develop independent control strains originating from the same cross as the congenic panels. Separate B6.CAST control (B6C) and HG.CAST control (HGC) strains were developed using mice from the MMU2 experiment. Mice from the last backcross inheriting only B6 or HG MMU2 alleles at markers spanning MMU2 were intermated to serve as the basis for each control. Both control strains were subsequently maintained through brother-sister mating. + +The control strains were coisogenic with the parental B6 or HG strain with the exception of mutations that arose during congenic construction and a small percentage of contaminating donor alleles missed after 6 backcrosses. Therefore, since the congenics and controls were developed through the same selection scheme and possibly share common contaminating regions, the B6C and HGC strains are the most ideal control to compare each congenic. + +Phenotypic characterization + +Trait data were collected on approximately 40 mice (20 of each sex) from each congenic and control strain. To eliminate parity and reduce litter size effects only progeny from uniparous dams were characterized and all litters were standardized to 5–7 pups/litter. Mice were weaned at 3 weeks of age. Feed (Purina 5008; 23.5% protein, 6.5% fat, 3.3 Kcal/g) and water were offered ad libitum. Mice were weighed to the nearest 0.1 g at 2WK, 3WK, 6WK, and 9WK of age. At 9WK ( ± 5 days) mice were anesthetized under isoflurane and nasal-anal length (NA) and nasal-tail (NT) were measured to the nearest mm. Tail length was calculated as NA minus NT. Anesthetized mice were then sacrificed by decapitation and exsanguinated. Femoral fat pad (FFP), gonadal fat pad (GFP), mesenteric fat pad (MFP) and retroperitoneal fat pad (RFP) were removed and weighed to the nearest mg. + +Chemical compositional analysis was performed for HGC, HG11 and HG17 carcasses as previously described with slight modifications [24]. Briefly, after weighing each fat pad was returned to the carcass. The entire gastrointestinal (GI) tract was subsequently removed and carcasses were again weighed. This represented the empty carcass weight (ECW). Carcasses were labeled and secured in two layers of cheesecloth (Fisher) and frozen at -20°C until analysis. At this time, carcasses were freeze-dried for seven days and water content was determined by subtracting the freeze-dried weight from ECW. FAT was then extracted with ether for 7 days, followed by acetone for an additional 7 days in a Soxhlet apparatus. Carcass ash (ASH) was determined measuring the remains after a 16 hour incineration at 575°C. Carcass protein (PROT) was calculated as the remaining portion after carcass fat (FAT) and ASH were subtracted from ECW. + +Statistical analysis + +The MEANS and UNIVARIATE procedures of SAS were used to generate descriptive statistics and test normality assumptions for each trait [55]. All data were then analyzed using the GLM procedure of SAS [55] with a linear model that included the fixed effects of strain, sex and strain by sex interaction; dam's weight at breeding by strain was used as a covariate. A second linear model was used to test for strain by hg genotype (+/+ or hg/hg) interactions. This model included the fixed effects of donor region, sex and HG genotype and all possible two and three-way interactions. Choosing a nominal P value of 0.05 and applying the Bonferroni correction for multiple comparisons established significant differences in the ANOVA's. The critical P values used are indicated in each table. + +Identification of candidate genes + +Genes were identified by manual data mining of primary literature, reviews and book chapters. To organize and collate genomic and functional information for each gene we created a custom Gh signaling Gene Map Annotator and Pathway Profiler (GenMAPP) pathway [56] (Figure 5). Visualization and color-coding of genes using GenMAPP aided the selection of candidate genes mapping within QTL regions on MMU2, 9, 11 and 17. + +Candidate gene sequencing + +PCR amplicons covering the coding sequence and partial 5' and 3' untranslated regions for each selected candidate gene were amplified, purified and sequenced from the CAST strain using protocols outlined in [57] with slight modifications. Total RNA from brain, liver, spleen, lung and testis was isolated from an adult CAST male mouse using Trizol (Ambion). cDNA was produced from total RNA using standard procedures. PCR primer sets for each gene were designed using Primer3 [58] (Additional File 5). The initial amplification was performed in 10 μl PCR reactions using the Invitrogen Platinum TaqPCRx amplification system (Invitrogen). The reactions contained 1X PCR buffer, 1X Enhancer solution, 1.5 mM MgSO4, 0.17 mM dNTPs, 1 μM each primer, 0.1 unit of Platinum Taq (Invitrogen) and approximately 25 ng of cDNA. Reactions were incubated for 5 min at 95°C, then cycled for 45 s at 95°C, 45 s at 55°C, 1 min at 72°C for 35 cycles with a final 72°C extension for 10 min on a MJ Research PTC-200. The products were visualized on 1.5% agarose gels containing 0.06 μg/ml EtBr. Bands of the correct size were excised and incubated in 100 μl of sterile H2O at 80°C for 10 min to elute DNA. Reamplification reactions consisted of 1X PCR buffer, 1.5 mM MgCl2, 0.17 mM dNTPs, 1 μM each primer, 0.1 unit of Taq (Promega) and 10 μl of eluate in a total volume of 50 μl. The same cycling parameters were used for reamplification reactions. Products were visualized on 1% 0.5X TBE agarose gels containing 0.06 μg/ml EtBr, excised and purfied using PCR purification columns (Promega). To quantity each fragment 1 μl of each purified product was run on a 1.0% agarose gel containing 0.06 μg/ml EtBr, along with a DNA mass ladder (Invitrogen). In a 96 well plate, 15 ng of each PCR product was added to 5 pM of primer for sequencing. The College of Agriculture and Environmental Sciences (CAES) Genomics Facility at the University of California, Davis, performed bidirectional sequencing of each amplicon. + +Sequence analysis + +B6 mRNA sequences for each gene were downloaded from the May 2004 (mm5) UCSC (NCBI Build 33) genome assembly [28]. These sequences were imported along with all CAST sequence traces into the SeqMan sequence assembly program (DNASTAR) and manually curated for quality. Poor quality reads were resequenced. Individual contigs were created for each gene and polymorphisms were detected and curated by manual inspection. + +Authors' contributions + +CRF and JFM conceived the study. CRF developed and characterized each speed congenic strain, assisted with phenotypic data analysis and drafted the manuscript. PMC assisted with experimental design and phenotypic data analysis. JFM assisted in the evaluation of experimental results and the manuscript and provided coordination of the project. All authors read and approved the final manuscript. + +Supplementary Material + +Additional File 1 + +Table of microsatellite markers used in the construction of B6.CAST and HG.CAST speed congenic strains + +Click here for file + +Additional File 2 + +Table of microsatellite markers used to position the recombinant ends of each speed congenic strain + +Click here for file + +Additional File 3 + +Table of body weight and growth rate phenotypes of B6.CAST and HG.CAST MMU2 speed congenic strains + +Click here for file + +Additional File 4 + +Table of results of sequence comparisons between CAST and B6 for MMU2 hg modifier candidate genes + +Click here for file + +Additional File 5 + +Table of PCR primers used to sequence MMU2, 9, 11 and 17 hg modifier candidate genes + +Click here for file + +Acknowledgements + +We thank Vince De Vera for mouse care and Alma Islas-Trejo, Kerri Morimoto, Gonzalo Rincon, Ricardo Verdugo, Karina Guevara, James Chitwood, Lee Nguyen, Galen Williams and Emily Farber for assistance with mouse phenotyping. We also thank Scott Taylor and Dr. Ed DePeters for assistance and use of the UCD Animal Science Nutrition Laboratory for carcass composition analysis. This work was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension Service, grant number 2005–35205–15453. CRF was supported by the Austin Eugene Lyons fellowship. + +Figures and Tables + +Figure 1 + +Outline of the speed congenic approach used to capture genome-wide growth and obesity QTL. Details of the congenic matings are outlined in "Methods". The matings used to construct the MMU2 speed congenic panel are listed on the left of the figure, along with the expected and observed % heterozygosity at each generation. % heterozygosity is defined as the number of markers heterozygous B6 (or HG)/CAST as a percent of the total number of markers typed (N = 79). The right side of the illustration lists the matings used to construct the MMU1, 5, 8, 9, 11 and 17 speed congenic strains, along with the expected and observed % heterozygosity at each generation. + +Figure 2 + +Genome-wide speed congenic strains and summary of QTL effects. White bars indicate the boundaries of CAST donor regions and hatched bars indicate intervals of unknown genotype. The minimum physical intervals (Mbp) of each donor region are listed in Table 1. To provide a general summary of the results, MMU2 is divided into five chromosomal regions (I, II, III, IV and V). Phenotypic differences for male and female (+/+ and hg/hg) mice relative to the appropriate controls are listed. For congenics outside of MMU2, a general summary of QTL effects is listed to the right of each donor region. Abbreviations: WT, body weight; GR, growth rate; T, tail length; NA, nasal-anal body length; TF, total weight of four fat pads; AI, adiposity index and BMI, body mass index. + +Figure 3 + +Growth curves for MMU2 B6.CAST and HG.CAST speed congenic strains. Body weight LSMEANS ± SEM are plotted as a function of time (weeks). Top plot, growth curves in male B6 (+/+) and HG (hg/hg) speed congenic strains; bottom plot, growth curves in female B6 (+/+) and HG (hg/hg) speed congenic strains. + +Figure 4 + +Significant three-way donor region by HG genotype by sex interaction for AI in the 2M donor region. AI plot illustrates the effect of the hg deletion on altering fat deposition between the sexes. The interaction is significant at P = 0.0004. + +Figure 5 + +GenMAPP growth hormone (Gh) signaling pathway. Genes known to be or potentially involved in Gh signaling were mined from primary literature, reviews and book chapters. Genes are organized broadly based on functionality. MMU genomic position for each gene is to the right of each symbol and genes located on MMU2, 9, 11 and 17 are highlighted in yellow, purple, orange and green, respectively. Genes located on MMUX are labeled in white. Forty-four of the genes overlapping QTL intervals were selected for sequencing in the CAST strain. + +Table 1 + +B6.CAST and HG.CAST speed congenic strains developed to isolate and characterize genome-wide QTL affecting growth and obesity + +QTL, quantitative trait locus; MMU, mouse chromosome; LOD, log of the odds + +a QTL MGI [59] nomenclature. Q1Ucd1-w26 never received an official MGI name, because it only reached a suggestive level of significance; hg modifier QTL are in bold [24]. + +b Mbp position according to the August 2005 mm7 UCSC [28] genome assembly (NCBI Build 35), peak LOD from [24]. + +c In the linkage analysis performed in [24], the peak location for Wg2, Carp1, Cara1 and Feml1 was 59–63 cM (115–120 Mbp) in hg/hg F2 mice and 78–83 cM (135–150 Mbp) in +/+ F2 mice. + +d Full congenic name consists of three parts: 1. B6.CAST, indicates the recipient strain is B6 and the donor strain is CAST; HG.CAST, indicates the recipient strain is HG and the donor strain is CAST; 2. markers defining the minimal congenic interval; 3. the number of backcrosses used for speed congenic development. + +e Represents the minimum genomic region spanned by CAST donor alleles. + +Table 2 + +Body length and adiposity phenotypes of B6.CAST and HG.CAST MMU2 speed congenic strains + +NA, nasal-anal body length; Tail, tail length; GFP, gonadal fat pad; FFP, femoral fat pad; MFP, mesenteric fat pad; RFP, retroperitoneal fat pad; TF, summed weight of four fat pads; AI, adiposity index (TF/body weight*100) and BMI, body mass index (body weight at sacrifice/NA2 *100). Values are expressed as LSMEANS ± SEM. LSMEANS in bold are significantly different than the respective control after Bonferroni correction (B6 (critical P < 0.00625) or HG (P < 0.00833)) within each sex. + +Table 3 + +Significance levels for the main effects, two-way and three way interactions of MMU2 speed congenic donor region (DR), HG genotype (HG) (+/+ or hg/hg) and sex for selected traits + +6WK, weight at 6 weeks of age; 9WK, weight at 9 weeks of age; G26, weight gain from 2 to 6 weeks of age; G29, weight gain from 2 to 9 weeks of age; NA, nasal-anal body length; TF, total fat pad weight and AI, adiposity index. Significant effects after a Bonferroni correction (critical P < 0.0071) are in bold. + +a The distal (2D) donor region was not analyzed. + +Table 4 + +Body size, growth rate and length phenotypes of HG.CAST speed congenic strains + +2WK, weight at 2 weeks of age; 3WK, weight at 3 weeks of age; 6WK, weight at 6 weeks of age; 9WK, weight at 9 weeks of age; G26, weight gain from 2 to 6 weeks of age; G29, weight gain from 2 to 9 weeks of age; NA, nasal-anal body length; Tail, tail length. Values are expressed as least square means (LSMEANS) ± SEM. LSMEANS in bold are significantly different than the control (HGC) (critical P < 0.005) after Bonferroni correction within each sex. + +Table 5 + +Adiposity phenotypes of HG.CAST speed congenic strains + +GFP, gonadal fat pad; FFP, femoral fat pad; MFP, mesenteric fat pad; RFP, retroperitoneal fat pad; TF, summed weight of four fat pads; AI, adiposity index (TF/body weight*100) and BMI, body mass index (body weight at sacrifice/NA2 *100). Values are expressed as least square means (LSMEANS) ± SEM. LSMEANS in bold are significantly different than the control (HGC) (critical P < 0.005) after Bonferroni correction within each sex. + +Table 6 + +Carcass composition phenotypes of HG.CAST speed congenic strains + +H2O, carcass water; %H2O, water as a percent of carcass weight; FAT, carcass fat; %FAT, fat as a percent of carcass weight; ASH, carcass ash; %ASH, ash as a percent of carcass weight; PROT, carcass protein, %PROT, protein as a percent of carcass weight. Values are expressed as least square means (LSMEANS) ± SEM. LSMEANS in bold are significantly different than the control (HGC) (critical P < 0.0125) after Bonferroni correction within each sex. + +Table 7 + +Nonsynonymous SNP in MMU2, 9 and 17 hg modifier candidate genes predicted by SIFT and Polyphen to alter protein function + +MMU, mouse chromosome; AA, amino acid; PBD, probably damaging, PSD, possibly damaging; B, Benign; T, tolerated and D, deleterious. + +a Amino acid change, first residue listed corresponds to the CAST allele and second corresponds to the B6 allele. + +b Indicates which allele was designated as "mutant", i.e. B6/CAST, B6 allele is mutant and CAST is wild type. diff --git a/src/ontogpt/evaluation/craft/database/all/16700629.ann b/src/ontogpt/evaluation/craft/database/all/16700629.ann new file mode 100644 index 000000000..0f68c978e --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16700629.ann @@ -0,0 +1,1402 @@ +T1 PR:000007500 0 4 Fgf9 +T2 PR:000017444 9 13 Wnt4 +T3 GO:0065007 45 53 Regulate +T4 NCBITaxon:40674 54 63 Mammalian +T5 GO:0007530 64 81 Sex Determination +T6 SO:0000704 97 102 genes +T7 UBERON:0000023 127 131 wing +T8 PR:P09615 127 135 wingless +T9 NCBITaxon:11757 144 148 MMTV +T10 SO:0000366 149 165 integration site +T11 CL:0000057 176 186 fibroblast +T12 GO:0009653 235 248 morphogenesis +T13 UBERON:0007688 278 284 fields +T14 NCBITaxon:10088 321 326 mouse +T15 PR:000007500 328 332 Fgf9 +T16 PR:000017444 337 341 Wnt4 +T17 GO:0010467 346 355 expressed +T18 UBERON:0000991 359 365 gonads +T19 GO:0007530 389 406 sex determination +T20 PR:000007500 416 420 Fgf9 +T21 PR:000017444 463 467 Wnt4 +T22 UBERON:0000473 487 493 testis +T23 GO:0008584 487 505 testis development +T24 UBERON:0000991 512 518 gonads +T25 GO:0030238 576 596 male sex-determining +T26 SO:0000704 597 601 gene +T27 PR:000015643 603 606 Sry +T28 CL:0000057 681 691 fibroblast +T29 PR:000007500 681 707 fibroblast growth factor 9 +T30 PR:000007500 709 713 FGF9 +T31 PR:000017444 719 723 WNT4 +T32 GO:0065007 751 759 regulate +T33 GO:0007530 760 777 sex determination +T34 NCBITaxon:10088 786 791 mouse +T35 UBERON:0000991 795 800 gonad +T36 PR:000015643 802 805 Sry +T37 PR:000015435 853 857 Sox9 +T38 PR:000007500 862 866 Fgf9 +T39 PR:000007500 887 891 Fgf9 +T40 PR:000017444 906 910 Wnt4 +T41 UBERON:0000473 928 934 testis +T42 PR:000017444 966 970 Wnt4 +T43 UBERON:0000991 977 983 gonads +T44 PR:000007500 1013 1017 Fgf9 +T45 PR:000015435 1022 1026 Sox9 +T46 PR:000015643 1045 1048 Sry +T47 UBERON:0000991 1090 1095 gonad +T48 GO:0065007 1099 1109 controlled +T49 PR:000007500 1132 1136 Fgf9 +T50 PR:000017444 1141 1145 Wnt4 +T51 GO:0030238 1163 1183 male sex-determining +T52 PR:000015643 1191 1194 Sry +T53 NCBITaxon:40674 1210 1217 mammals +T54 GO:0007530 1299 1316 sex determination +T55 NCBITaxon:7742 1326 1337 vertebrates +T56 GO:0048513 1455 1469;1502 1508 development of ... organs +T57 GO:0000003 1489 1501 reproductive +T58 UBERON:0003133 1489 1508 reproductive organs +T59 NCBITaxon:33208 1535 1541 animal +T60 NCBITaxon:species 1542 1549 species +T61 UBERON:0000473 1555 1561 testis +T62 UBERON:0000992 1566 1571 ovary +T63 UBERON:0000922 1641 1650 embryonic +T64 UBERON:0000991 1651 1656 gonad +T65 GO:0008406 1694 1708;1713 1718 development of ... gonad +T66 UBERON:0000991 1713 1718 gonad +T67 NCBITaxon:species 1743 1750 species +T68 UBERON:0000992 1821 1826 ovary +T69 UBERON:0000473 1830 1836 testis +T70 NCBITaxon:species 1881 1888 species +T71 UBERON:0000922 1894 1903 embryonic +T72 UBERON:0000991 1904 1909 gonad +T73 CL:0000586 1946 1956 germ cells +T74 CL:0002371 1961 1974 somatic cells +T75 UBERON:0000479 1981 1987 tissue +T76 UBERON:0000992 2086 2093 ovarian +T77 UBERON:0000473 2097 2107 testicular +T78 UBERON:0001048 2168 2178 primordium +T79 SO:0000704 2238 2243 genes +T80 PR:000011390 2307 2311 Dax1 +T81 PR:000011390 2313 2418 dosage-sensitive sex reversal-congenital adrenal hypoplasia critical region on the X chromosome protein 1 +T82 http://purl.obolibrary.org/obo/MONDO_0016241 2343 2372 congenital adrenal hypoplasia +T83 UBERON:0002369 2354 2361 adrenal +T84 GO:0000805 2396 2408 X chromosome +T85 PR:000015435 2421 2425 Sox9 +T86 PR:000015435 2427 2445 Sry-like HMG box 9 +T87 PR:000007500 2448 2452 Fgf9 +T88 CL:0000057 2454 2464 fibroblast +T89 PR:000007500 2454 2480 fibroblast growth factor 9 +T90 PR:000017444 2487 2491 Wnt4 +T91 UBERON:0000023 2493 2497 wing +T92 PR:000017444 2493 2533 wingless-related MMTV integration site 4 +T93 NCBITaxon:11757 2510 2514 MMTV +T94 SO:0000366 2515 2531 integration site +T95 GO:0010467 2550 2559 expressed +T96 UBERON:0000991 2593 2599 gonads +T97 NCBITaxon:40674 2632 2641 mammalian +T98 GO:0007530 2642 2659 sex determination +T99 UBERON:0000062 2689 2694 organ +T100 GO:0048513 2689 2706 organ development +T101 UBERON:0000992 2710 2717 ovarian +T102 UBERON:0000473 2732 2738 testis +T103 SO:0000704 2751 2755 gene +T104 UBERON:0000473 2798 2804 testis +T105 GO:0008584 2798 2816 testis development +T106 CL:0000630 2867 2882 supporting cell +T107 GO:0009653 2922 2935 morphogenetic +T108 GO:0008283 3054 3067;3086 3094 proliferation ... of cells +T109 GO:0016477 3076 3094 migration of cells +T110 UBERON:0005297 3106 3117 testis cord +T111 CL:0000216 3173 3185 Sertoli cell +T112 GO:0030154 3181 3201 cell differentiation +T113 PR:000015643 3204 3207 Sry +T114 GO:0000806 3211 3223 Y chromosome +T115 SO:0000704 3231 3235 gene +T116 GO:0007530 3252 3267 sex-determining +T117 SO:0000704 3268 3272 gene +T118 NCBITaxon:40674 3276 3283 mammals +T119 PR:000015643 3311 3314 Sry +T120 GO:0010467 3315 3325 expression +T121 UBERON:0000922 3332 3339 embryos +T122 UBERON:0000922 3350 3357 embryos +T123 PR:000015643 3381 3384 Sry +T124 UBERON:0000991 3398 3403 gonad +T125 UBERON:0000992 3414 3421 ovarian +T126 SO:0000704 3447 3454 Genetic +T127 NCBITaxon:10088 3478 3482 mice +T128 GO:0010467 3493 3503 expression +T129 SO:0000902 3527 3537 transgenes +T130 PR:000015643 3560 3563 Sry +T131 GO:0010467 3564 3574 expression +T132 CL:0000630 3621 3636 supporting cell +T133 GO:0010467 3646 3656 Expression +T134 PR:000015643 3660 3663 Sry +T135 UBERON:0000473 3725 3731 testis +T136 CL:0000216 3741 3754 Sertoli cells +T137 CL:0000477 3770 3784 follicle cells +T138 UBERON:0000992 3816 3821 ovary +T139 CL:0000216 3851 3863 Sertoli cell +T140 UBERON:0000991 3911 3916 gonad +T141 CL:0000216 3989 4002 Sertoli cells +T142 UBERON:0000473 4028 4034 testis +T143 UBERON:0000992 4111 4116 ovary +T144 PR:000015643 4147 4150 Sry +T145 GO:0010467 4151 4161 expression +T146 GO:0010467 4170 4180 expression +T147 SO:0000704 4199 4204 genes +T148 UBERON:0000991 4212 4217 gonad +T149 SO:0000704 4246 4250 gene +T150 PR:000015643 4265 4268 Sry +T151 CL:0000216 4314 4326 Sertoli cell +T152 SO:0000704 4351 4355 gene +T153 GO:0010467 4356 4365 expressed +T154 UBERON:0000479 4374 4381 tissues +T155 UBERON:0000922 4400 4406 embryo +T156 PR:000015435 4408 4412 Sox9 +T157 PR:000015435 4428 4432 Sox9 +T158 GO:0010467 4433 4443 expression +T159 UBERON:0000991 4454 4459 gonad +T160 PR:000015435 4535 4539 Sox9 +T161 UBERON:0000991 4550 4555 gonad +T162 UBERON:0000473 4565 4571 testis +T163 GO:0008584 4565 4583 testis development +T164 PR:000015435 4621 4625 Sox9 +T165 GO:0007530 4650 4667 sex determination +T166 PR:000015643 4676 4679 Sry +T167 NCBITaxon:40674 4702 4709 mammals +T168 GO:0010467 4711 4721 expression +T169 PR:000015435 4725 4729 Sox9 +T170 UBERON:0000991 4762 4767 gonad +T171 NCBITaxon:species 4776 4783 species +T172 NCBITaxon:40674 4788 4795 mammals +T173 PR:000015435 4797 4801 Sox9 +T174 PR:000015643 4836 4839 Sry +T175 GO:0010467 4840 4850 expression +T176 PR:000015643 4882 4885 Sry +T177 GO:0010467 4886 4896 expressing +T178 UBERON:0000473 4950 4956 testis +T179 CL:0000216 4993 5006 Sertoli cells +T180 GO:0010467 5012 5019 express +T181 PR:000015435 5020 5024 Sox9 +T182 GO:0010467 5060 5069 expressed +T183 PR:000015643 5070 5073 Sry +T184 PR:000015435 5110 5114 Sox9 +T185 PR:000015643 5146 5149 Sry +T186 PR:000007500 5210 5214 Fgf9 +T187 PR:000003273 5219 5224 Igf1r +T188 PR:000009065 5225 5228 Irr +T189 PR:000003273 5233 5270 insulin-like growth factor 1 receptor +T190 PR:000009065 5271 5304 insulin receptor-related receptor +T191 PR:000045358 5305 5312 insulin +T192 PR:000015435 5343 5347 Sox9 +T193 GO:0010467 5348 5358 expression +T194 GO:0005576 5421 5434 extracellular +T195 GO:0007538 5485 5510 primary sex determination +T196 NCBITaxon:10088 5513 5517 Mice +T197 PR:000007500 5552 5556 Fgf9 +T198 UBERON:0000473 5621 5627 testis +T199 GO:0009987 5637 5652 cellular events +T200 GO:0008283 5664 5682 cell proliferation +T201 UBERON:0000080 5684 5695 mesonephric +T202 GO:0016477 5696 5710 cell migration +T203 UBERON:0005297 5712 5723 testis cord +T204 GO:0030154 5743 5761;5770 5775 differentiation of ... cells +T205 CL:0000216 5762 5775 Sertoli cells +T206 PR:000007500 5785 5789 Fgf9 +T207 UBERON:0000991 5832 5838 gonads +T208 GO:0010467 5853 5862 expressed +T209 UBERON:0000991 5866 5872 gonads +T210 PR:000015643 5920 5923 Sry +T211 GO:0010467 5927 5936 expressed +T212 GO:0010467 5962 5972 expression +T213 PR:000017444 5976 5980 Wnt4 +T214 UBERON:0000991 6016 6022 gonads +T215 UBERON:0000991 6070 6076 gonads +T216 PR:000017444 6101 6105 Wnt4 +T217 UBERON:0000473 6138 6148 testicular +T218 CL:0000216 6196 6209 Sertoli cells +T219 UBERON:0000473 6234 6244 testicular +T220 CL:0000216 6284 6291 Sertoli +T221 PR:000017444 6330 6334 Wnt4 +T222 UBERON:0000991 6341 6347 gonads +T223 GO:0010467 6358 6368 expression +T224 CL:0000216 6372 6384 Sertoli cell +T225 PR:000017444 6497 6501 Wnt4 +T226 GO:0007538 6522 6547 primary sex determination +T227 UBERON:0000991 6555 6560 gonad +T228 SO:0000704 6612 6619 genetic +T229 PR:000015643 6636 6639 Sry +T230 PR:000015435 6641 6645 Sox9 +T231 PR:000007500 6647 6651 Fgf9 +T232 PR:000017444 6657 6661 Wnt4 +T233 GO:0065007 6669 6679 regulatory +T234 GO:0065007 6693 6700 governs +T235 UBERON:0000991 6705 6712 gonadal +T236 UBERON:0007688 6713 6718 field +T237 PR:000007500 6745 6749 Fgf9 +T238 UBERON:0000991 6774 6780 gonads +T239 GO:0010467 6801 6811 expression +T240 PR:000015643 6815 6818 SRY +T241 PR:000015435 6851 6855 SOX9 +T242 PR:000015435 6866 6870 SOX9 +T243 GO:0010467 6871 6881 expression +T244 PR:000007500 6907 6911 Fgf9 +T245 UBERON:0000991 6922 6928 gonads +T246 UBERON:0000473 6934 6940 testis +T247 PR:000007500 6994 6998 FGF9 +T248 UBERON:0000992 7013 7018 ovary +T249 SO:0000704 7029 7033 gene +T250 PR:000017444 7035 7039 Wnt4 +T251 PR:000007500 7061 7065 FGF9 +T252 CL:0000216 7109 7116 Sertoli +T253 GO:0046903 7140 7147 secrete +T254 PR:000007500 7148 7152 FGF9 +T255 PR:000017444 7208 7212 Wnt4 +T256 PR:000015435 7253 7257 SOX9 +T257 GO:0010467 7258 7268 expression +T258 UBERON:0000991 7302 7307 gonad +T259 PR:000017444 7309 7313 WNT4 +T260 GO:0046661 7334 7346 male pathway +T261 GO:0010467 7361 7371 expression +T262 PR:000015435 7375 7379 SOX9 +T263 PR:000007500 7384 7388 FGF9 +T264 GO:0046661 7409 7421 male pathway +T265 SO:0000704 7422 7427 genes +T266 PR:000017444 7457 7461 Wnt4 +T267 UBERON:0000991 7468 7474 gonads +T268 GO:0000806 7497 7498 Y +T269 SO:0000704 7506 7510 gene +T270 PR:000015643 7511 7514 Sry +T271 SO:0000704 7530 7537 genetic +T272 UBERON:0000991 7607 7612 gonad +T273 GO:0065007 7616 7626 controlled +T274 PR:000007500 7668 7672 FGF9 +T275 PR:000017444 7677 7681 WNT4 +T276 UBERON:0000991 7689 7696 gonadal +T277 UBERON:0007688 7697 7702 field +T278 GO:0009653 7768 7781 morphogenesis +T279 GO:0030154 7787 7811 cellular differentiation +T280 GO:0010467 7841 7851 Expression +T281 PR:000007500 7855 7859 FGF9 +T282 GO:0042571 7917 7925 antibody +T283 PR:000007500 7938 7942 FGF9 +T284 GO:0010467 7956 7966 expression +T285 UBERON:0000991 7981 7986 gonad +T286 GO:0008406 7981 7998 gonad development +T287 PR:000007500 8000 8004 FGF9 +T288 UBERON:0000991 8053 8058 gonad +T289 PR:000007500 8115 8119 FGF9 +T290 UBERON:0000991 8144 8150 gonads +T291 UBERON:0000991 8196 8202 gonads +T292 UBERON:0005297 8231 8243 testis cords +T293 GO:0010467 8263 8273 expression +T294 UBERON:0000991 8300 8306 gonads +T295 PR:000007500 8335 8339 FGF9 +T296 GO:0010467 8340 8350 expression +T297 UBERON:0005297 8358 8370 testis cords +T298 CL:0000216 8388 8401 Sertoli cells +T299 CL:0000586 8420 8430 germ cells +T300 CL:0000586 8444 8453 germ cell +T301 GO:0005886 8449 8462 cell membrane +T302 CL:0000233 8470 8478 platelet +T303 PR:000001904 8470 8513 platelet/endothelial cell adhesion molecule +T304 UBERON:0001986 8479 8490 endothelial +T305 CL:0000115 8479 8495 endothelial cell +T306 CHEBI:23367 8505 8513 molecule +T307 PR:000001904 8515 8520 PECAM +T308 CL:0000300 8579 8586 gametic +T309 UBERON:0000991 8587 8593 gonads +T310 PR:000002065 8599 8602 Kit +T311 UBERON:0000922 8629 8636 embryos +T312 UBERON:0005297 8652 8664 testis cords +T313 CL:0000216 8676 8689 Sertoli cells +T314 PR:000007500 8696 8700 FGF9 +T315 UBERON:0005297 8736 8748 testis cords +T316 PR:000002065 8755 8758 Kit +T317 UBERON:0000991 8763 8769 gonads +T318 CL:0000586 8831 8841 germ cells +T319 PR:000007500 8874 8878 FGF9 +T320 GO:0010467 8882 8891 expressed +T321 CL:0000216 8895 8908 Sertoli cells +T322 GO:0010467 8923 8933 expression +T323 CL:0000586 8970 8980 germ cells +T324 PR:000007500 8994 8998 FGF9 +T325 GO:0010467 8999 9009 expression +T326 UBERON:0000991 9040 9046 gonads +T327 UBERON:0000991 9098 9104 gonads +T328 UBERON:0000473 9108 9114 testis +T329 PR:000015643 9143 9146 Sry +T330 GO:0010467 9147 9157 Expression +T331 PR:000007500 9187 9191 Fgf9 +T332 UBERON:0000991 9195 9201 Gonads +T333 GO:0010467 9213 9223 expression +T334 PR:000007500 9227 9231 Fgf9 +T335 UBERON:0000991 9247 9253 gonads +T336 PR:000007500 9285 9289 Fgf9 +T337 PR:000015643 9318 9321 Sry +T338 GO:0007618 9359 9364 mated +T339 PR:000007500 9365 9369 Fgf9 +T340 NCBITaxon:10088 9373 9377 mice +T341 SO:0000902 9468 9477 transgene +T342 PR:000015643 9492 9495 Sry +T343 SO:0000167 9496 9504 promoter +T344 PR:000015643 9505 9508 Sry +T345 SO:0000902 9537 9546 transgene +T346 PR:000015643 9584 9587 Sry +T347 GO:0010467 9588 9598 expression +T348 GO:0010467 9608 9618 expression +T349 PR:000007500 9656 9660 Fgf9 +T350 UBERON:0000991 9667 9673 gonads +T351 PR:000007500 9688 9692 Fgf9 +T352 GO:0065007 9768 9778 regulation +T353 PR:000015643 9782 9785 Sry +T354 PR:000007500 9804 9808 Fgf9 +T355 PR:000015643 9874 9877 Sry +T356 GO:0010467 9878 9888 expression +T357 PR:000015643 9931 9934 SRY +T358 GO:0051170 9935 9946;9951 9958 import into ... nucleus +T359 GO:0005634 9951 9958 nucleus +T360 SO:0000902 9969 9978 transgene +T361 PR:000015643 9980 9983 Sry +T362 GO:0005622 10011 10024 intracellular +T363 PR:000015643 10045 10048 SRY +T364 PR:000015643 10088 10091 SRY +T365 GO:0065007 10092 10102 regulation +T366 PR:000007500 10112 10116 Fgf9 +T367 NCBITaxon:10088 10120 10124 mice +T368 PR:000015643 10138 10141 Sry +T369 NCBITaxon:10088 10151 10156 mouse +T370 PR:000000020 10173 10176 Myc +T371 PR:000015643 10184 10187 Sry +T372 SO:0000902 10188 10197 transgene +T373 PR:000015643 10199 10202 Sry +T374 PR:000000020 10202 10205 Myc +T375 GO:0005622 10242 10255 intracellular +T376 PR:000015643 10256 10259 SRY +T377 GO:0010467 10260 10270 expression +T378 GO:0042571 10294 10302 antibody +T379 PR:000000084 10311 10316 c-MYC +T380 GO:0010467 10322 10332 expression +T381 GO:0005634 10337 10344 nuclear +T382 PR:000015643 10361 10364 SRY +T383 PR:000000020 10364 10367 MYC +T384 PR:000015643 10371 10374 Sry +T385 PR:000000020 10374 10377 Myc +T386 PR:000007500 10379 10383 Fgf9 +T387 UBERON:0000991 10387 10393 gonads +T388 PR:000015643 10492 10495 Sry +T389 PR:000015643 10544 10547 Sry +T390 GO:0010467 10548 10558 expression +T391 PR:000007500 10579 10583 Fgf9 +T392 PR:000007500 10596 10600 Fgf9 +T393 PR:000015643 10653 10656 Sry +T394 GO:0065007 10660 10668 regulate +T395 UBERON:0000473 10669 10675 testis +T396 GO:0008584 10669 10687 testis development +T397 PR:000007500 10690 10694 FGF9 +T398 PR:000015435 10711 10715 SOX9 +T399 GO:0010467 10716 10726 Expression +T400 PR:000015435 10769 10773 SOX9 +T401 GO:0010467 10774 10784 expression +T402 PR:000007500 10800 10804 Fgf9 +T403 UBERON:0000991 10811 10817 gonads +T404 UBERON:0000473 10851 10857 testes +T405 PR:000015435 10885 10889 SOX9 +T406 GO:0010467 10890 10900 expression +T407 CL:0000216 10951 10958 Sertoli +T408 SO:0000704 11007 11014 genetic +T409 PR:000007500 11035 11039 FGF9 +T410 PR:000015435 11044 11048 SOX9 +T411 PR:000015435 11060 11064 Sox9 +T412 GO:0010467 11075 11084 expressed +T413 UBERON:0005294 11098 11112 genital ridges +T414 UBERON:0000922 11131 11138 embryos +T415 PR:000015643 11175 11178 Sry +T416 GO:0010467 11179 11189 expression +T417 UBERON:0000991 11213 11219 gonads +T418 PR:000007500 11257 11261 Fgf9 +T419 PR:000015643 11308 11311 Sry +T420 PR:000007500 11337 11341 Fgf9 +T421 GO:0010628 11362 11378;11384 11394 up-regulation of ... expression +T422 PR:000015435 11379 11383 Sox9 +T423 CL:0000001 11396 11408 Primary cell +T424 UBERON:0000991 11421 11426 gonad +T425 PR:000015435 11463 11467 Sox9 +T426 PR:000007500 11492 11496 FGF9 +T427 CL:0001034 11502 11515 in vitro cell +T428 UBERON:0000991 11559 11565 gonads +T429 UBERON:0000080 11574 11585 mesonephroi +T430 GO:0031012 11603 11623 extracellular matrix +T431 PR:000007500 11679 11683 FGF9 +T432 PR:000015435 11726 11730 SOX9 +T433 GO:0010467 11731 11741 expression +T434 GO:0042571 11762 11770 antibody +T435 PR:000015435 11783 11787 SOX9 +T436 PR:000007500 11832 11836 FGF9 +T437 PR:000007500 11937 11941 FGF9 +T438 PR:000015435 12004 12008 SOX9 +T439 PR:000015435 12054 12058 SOX9 +T440 UBERON:0000991 12100 12106 gonads +T441 PR:000007500 12131 12135 FGF9 +T442 UBERON:0000991 12216 12223 gonadal +T443 UBERON:0000991 12237 12243 gonads +T444 PR:000007500 12290 12294 FGF9 +T445 UBERON:0000991 12366 12371 gonad +T446 PR:000007500 12388 12392 FGF9 +T447 GO:0031012 12443 12463 extracellular matrix +T448 UBERON:0000991 12474 12479 gonad +T449 PR:000007500 12509 12513 FGF9 +T450 UBERON:0000991 12534 12539 gonad +T451 PR:000007500 12571 12575 FGF9 +T452 PR:000015435 12647 12651 SOX9 +T453 GO:0010467 12652 12662 expression +T454 UBERON:0000991 12724 12729 gonad +T455 PR:000007500 12750 12754 FGF9 +T456 PR:000007500 12834 12838 FGF9 +T457 PR:000015435 12860 12864 SOX9 +T458 GO:0010467 12865 12875 expression +T459 UBERON:0000991 12882 12889 gonadal +T460 PR:000007500 12939 12943 Fgf9 +T461 PR:000015435 12948 12952 Sox9 +T462 PR:000007500 12955 12959 Fgf9 +T463 PR:000015435 12988 12992 SOX9 +T464 GO:0010467 12993 13003 Expression +T465 UBERON:0000991 13010 13016 Gonads +T466 PR:000007500 13041 13045 Fgf9 +T467 PR:000015435 13084 13088 Sox9 +T468 PR:000015435 13110 13114 SOX9 +T469 GO:0010467 13115 13125 expression +T470 PR:000007500 13146 13150 Fgf9 +T471 PR:000007500 13158 13162 Fgf9 +T472 UBERON:0000991 13169 13175 gonads +T473 UBERON:0000991 13249 13255 gonads +T474 PR:000015435 13269 13273 SOX9 +T475 UBERON:0000991 13321 13326 gonad +T476 GO:0005634 13374 13381 nuclear +T477 PR:000015435 13382 13386 SOX9 +T478 UBERON:0001851 13427 13433 cortex +T479 UBERON:0000991 13478 13483 gonad +T480 PR:000015643 13535 13538 Sry +T481 PR:000015435 13543 13547 Sox9 +T482 GO:0010467 13548 13558 expression +T483 PR:000007500 13614 13618 Fgf9 +T484 UBERON:0000991 13625 13631 gonads +T485 CL:0002371 13652 13665 Somatic cells +T486 PR:000007500 13673 13677 Fgf9 +T487 UBERON:0000991 13681 13687 gonads +T488 PR:000015435 13706 13710 SOX9 +T489 GO:0010467 13781 13791 expression +T490 PR:000015435 13813 13817 SOX9 +T491 PR:000007500 13840 13844 Fgf9 +T492 UBERON:0000991 13858 13864 gonads +T493 PR:000007500 13896 13900 Fgf9 +T494 UBERON:0000991 13907 13913 gonads +T495 PR:000015435 13915 13919 SOX9 +T496 CL:0000216 13962 13969 Sertoli +T497 UBERON:0005297 14022 14033 testis cord +T498 PR:000007500 14092 14096 Fgf9 +T499 PR:000015435 14138 14142 Sox9 +T500 PR:000015435 14184 14188 Sox9 +T501 GO:0010467 14189 14199 expression +T502 CL:0000216 14203 14210 Sertoli +T503 PR:000015435 14229 14233 Sox9 +T504 PR:000007500 14250 14254 Fgf9 +T505 UBERON:0000991 14275 14281 Gonads +T506 SO:0000704 14347 14352 genes +T507 PR:000015643 14358 14361 Sry +T508 PR:000007500 14364 14368 Fgf9 +T509 PR:000015435 14371 14375 Sox9 +T510 GO:0010467 14377 14387 expression +T511 PR:000007500 14391 14395 Fgf9 +T512 UBERON:0000991 14418 14424 gonads +T513 PR:000015435 14443 14447 Sox9 +T514 PR:000015643 14489 14492 Sry +T515 PR:000015435 14495 14499 Sox9 +T516 PR:000007500 14502 14506 Fgf9 +T517 GO:0010467 14508 14518 expression +T518 PR:000007500 14522 14526 Fgf9 +T519 UBERON:0000991 14561 14567 gonads +T520 PR:000015435 14586 14590 Sox9 +T521 PR:000007500 14604 14608 Fgf9 +T522 GO:0010467 14609 14619 expression +T523 PR:000015435 14623 14627 Sox9 +T524 PR:000015435 14647 14651 Sox9 +T525 UBERON:0000991 14659 14665 gonads +T526 NCBITaxon:10088 14688 14692 mice +T527 SO:0000359 14728 14732 flox +T528 SO:0001023 14734 14740 allele +T529 PR:000015435 14744 14748 Sox9 +T530 PR:000015435 14750 14754 Sox9 +T531 SO:0000359 14754 14758 flox +T532 SO:0000359 14759 14763 flox +T533 NCBITaxon:10088 14770 14774 mice +T534 SO:0000902 14806 14816 transgenes +T535 PR:000013249 14818 14822 Prm1 +T536 PR:000018212 14839 14842 Zp3 +T537 PR:000015435 14870 14874 Sox9 +T538 UBERON:0000922 14887 14894 embryos +T539 GO:0016265 14895 14898 die +T540 UBERON:0004535 14925 14939 cardiovascular +T541 PR:000015435 15009 15013 Sox9 +T542 UBERON:0000991 15026 15032 gonads +T543 PR:000015435 15119 15123 Sox9 +T544 UBERON:0000991 15131 15137 gonads +T545 GO:0097617 15260 15273 hybridization +T546 GO:0010467 15294 15304 expression +T547 PR:000007500 15308 15312 Fgf9 +T548 PR:000015435 15357 15361 Sox9 +T549 UBERON:0000991 15365 15371 gonads +T550 PR:000015643 15403 15406 Sry +T551 GO:0010467 15407 15417 expression +T552 PR:000007500 15508 15512 Fgf9 +T553 GO:0010467 15513 15523 expression +T554 UBERON:0000991 15540 15546 gonads +T555 GO:0010467 15567 15577 expression +T556 PR:000015435 15581 15585 Sox9 +T557 GO:0010467 15621 15631 expression +T558 PR:000015643 15635 15638 Sry +T559 GO:0065007 15660 15668 regulate +T560 PR:000007500 15669 15673 Fgf9 +T561 PR:000015435 15692 15696 Sox9 +T562 PR:000015435 15726 15730 Sox9 +T563 PR:000007500 15748 15752 Fgf9 +T564 GO:0010467 15753 15763 expression +T565 PR:000007500 15769 15773 Fgf9 +T566 PR:000015435 15796 15800 Sox9 +T567 GO:0010467 15801 15811 expression +T568 SO:0000704 15871 15876 genes +T569 UBERON:0000991 15883 15889 gonads +T570 PR:000007500 15897 15901 Fgf9 +T571 UBERON:0000991 15909 15915 Gonads +T572 PR:000015435 15917 15921 Sox9 +T573 UBERON:0000991 15928 15934 Gonads +T574 GO:0008283 15951 15969 Cell Proliferation +T575 GO:0008283 16011 16029 cell proliferation +T576 PR:000007500 16046 16050 Fgf9 +T577 UBERON:0000991 16057 16063 gonads +T578 PR:000015435 16077 16081 Sox9 +T579 PR:000007500 16114 16118 Fgf9 +T580 GO:0010467 16119 16129 expression +T581 GO:0008283 16153 16171 cell proliferation +T582 UBERON:0000991 16178 16184 gonads +T583 PR:000015435 16221 16225 Sox9 +T584 GO:0008283 16239 16252 proliferation +T585 PR:000015435 16256 16260 Sox9 +T586 UBERON:0000991 16264 16270 gonads +T587 GO:0007067 16291 16298 mitotic +T588 CHEBI:15358 16327 16334 histone +T589 PR:000027594 16327 16337 histone H3 +T590 GO:0008283 16339 16352 Proliferating +T591 UBERON:0000991 16440 16446 gonads +T592 GO:0008283 16469 16487 cell proliferation +T593 PR:000015435 16506 16510 Sox9 +T594 SO:0000359 16510 16514 flox +T595 PR:000015435 16562 16566 Sox9 +T596 UBERON:0000991 16573 16579 gonads +T597 GO:0008283 16580 16593 proliferation +T598 UBERON:0000991 16624 16630 gonads +T599 PR:000015435 16724 16728 Sox9 +T600 PR:000007500 16733 16737 Fgf9 +T601 SO:0000704 16793 16798 genes +T602 CL:0002371 16833 16846 somatic cells +T603 CL:0000216 16858 16870 Sertoli cell +T604 UBERON:0000991 16889 16895 gonads +T605 GO:0046661 16902 16914 Male Pathway +T606 PR:000007500 16929 16933 Fgf9 +T607 CL:0000216 16937 16944 Sertoli +T608 PR:000015435 16980 16984 Sox9 +T609 GO:0010467 16998 17007 expressed +T610 PR:000007500 17011 17015 Fgf9 +T611 UBERON:0000991 17019 17025 gonads +T612 SO:0000704 17057 17062 genes +T613 GO:0046661 17070 17082 male pathway +T614 CL:0000216 17126 17138 Sertoli cell +T615 GO:0030154 17134 17154 cell differentiation +T616 PR:000000073 17156 17178 anti-Mullerian hormone +T617 PR:000000073 17180 17183 Amh +T618 PR:000006459 17194 17209 Desert hedgehog +T619 PR:000006459 17211 17214 Dhh +T620 PR:000007500 17224 17228 Fgf9 +T621 UBERON:0000991 17235 17241 gonads +T622 GO:0097617 17268 17281 hybridization +T623 PR:000006459 17283 17286 Dhh +T624 GO:0010467 17297 17306 expressed +T625 UBERON:0000991 17340 17346 gonads +T626 PR:000007500 17386 17390 Fgf9 +T627 UBERON:0000991 17397 17403 gonads +T628 UBERON:0000080 17414 17425 mesonephric +T629 GO:0010467 17426 17436 expression +T630 PR:000000073 17476 17479 Amh +T631 PR:000015435 17525 17529 SOX9 +T632 PR:000007500 17598 17602 Fgf9 +T633 UBERON:0000991 17609 17615 gonads +T634 PR:000000073 17666 17669 Amh +T635 GO:0010467 17699 17709 expression +T636 PR:000015435 17713 17717 SOX9 +T637 PR:000007500 17721 17725 Fgf9 +T638 UBERON:0000991 17729 17735 gonads +T639 PR:000000073 17775 17778 Amh +T640 PR:000006459 17832 17835 Dhh +T641 CL:0000216 17859 17866 Sertoli +T642 CL:0000216 17921 17933 Sertoli cell +T643 PR:000007500 17977 17981 Fgf9 +T644 PR:000015643 18006 18009 Sry +T645 PR:000015435 18014 18018 Sox9 +T646 GO:0010467 18019 18029 expression +T647 PR:000007500 18033 18037 Fgf9 +T648 UBERON:0000991 18041 18047 gonads +T649 PR:000015435 18089 18093 SOX9 +T650 GO:0010467 18094 18104 expression +T651 PR:000007500 18111 18115 Fgf9 +T652 UBERON:0000991 18119 18125 gonads +T653 CL:0000216 18176 18183 Sertoli +T654 GO:0008219 18297 18307 cell death +T655 PR:000007500 18329 18333 Fgf9 +T656 UBERON:0000991 18337 18343 gonads +T657 PR:000015435 18378 18382 SOX9 +T658 GO:0010467 18383 18393 expressing +T659 PR:000026878 18442 18458 active caspase-3 +T660 CL:0000445 18463 18477 apoptotic cell +T661 CL:0000445 18486 18501 Apoptotic cells +T662 PR:000007500 18523 18527 Fgf9 +T663 UBERON:0000991 18531 18537 gonads +T664 PR:000007500 18586 18590 Fgf9 +T665 GO:0008219 18628 18638 cell death +T666 UBERON:0000083 18642 18661 mesonephric tubules +T667 UBERON:0003074 18642 18653;18666 18671 mesonephric ... ducts +T668 PR:000007500 18689 18693 Fgf9 +T669 GO:0010467 18694 18704 expression +T670 PR:000015435 18763 18767 SOX9 +T671 GO:0010467 18768 18778 expression +T672 PR:000007500 18782 18786 Fgf9 +T673 UBERON:0000991 18793 18799 gonads +T674 GO:0008219 18818 18828 cell death +T675 PR:000007500 18854 18858 FGF9 +T676 PR:000015435 18859 18863 SOX9 +T677 GO:0065007 18877 18887 regulation +T678 GO:0046661 18930 18942 male pathway +T679 PR:000007500 18946 18950 Fgf9 +T680 CL:0000216 18954 18961 Sertoli +T681 CL:0000630 19011 19027 supporting cells +T682 GO:0046661 19033 19037;19048 19063 male ... differentiation +T683 GO:0046660 19041 19063 female differentiation +T684 GO:0010467 19081 19091 expression +T685 PR:000017444 19095 19099 Wnt4 +T686 UBERON:0000992 19104 19109 ovary +T687 SO:0000704 19120 19124 gene +T688 PR:000017444 19138 19142 Wnt4 +T689 PR:000007500 19166 19170 Fgf9 +T690 PR:000007500 19188 19192 Fgf9 +T691 UBERON:0000991 19196 19201 gonad +T692 PR:000007500 19253 19257 Fgf9 +T693 PR:000017444 19298 19302 Wnt4 +T694 UBERON:0000991 19325 19331 gonads +T695 PR:000007500 19362 19366 Fgf9 +T696 PR:000017444 19371 19375 Wnt4 +T697 PR:000017444 19431 19435 Wnt4 +T698 PR:000007500 19447 19451 Fgf9 +T699 UBERON:0000991 19458 19464 gonads +T700 SO:0000704 19475 19482 genetic +T701 PR:000007500 19515 19519 Fgf9 +T702 PR:000017444 19524 19528 Wnt4 +T703 PR:000015643 19538 19541 SRY +T704 PR:000015435 19546 19550 SOX9 +T705 GO:0010467 19565 19574 expressed +T706 PR:000007500 19578 19582 Fgf9 +T707 UBERON:0000991 19589 19595 gonads +T708 PR:000017444 19653 19657 Wnt4 +T709 PR:000007500 19685 19689 FGF9 +T710 GO:0010467 19710 19720 expression +T711 PR:000017444 19724 19728 Wnt4 +T712 UBERON:0000991 19749 19754 gonad +T713 UBERON:0000080 19755 19766 mesonephros +T714 PR:000007500 19791 19795 FGF9 +T715 PR:000017444 19818 19822 Wnt4 +T716 GO:0010467 19823 19833 expression +T717 GO:0097617 19857 19870 hybridization +T718 UBERON:0000991 19888 19894 gonads +T719 PR:000007500 19910 19914 FGF9 +T720 GO:0010467 19937 19947 expression +T721 PR:000017444 19951 19955 Wnt4 +T722 PR:000007500 20003 20007 Fgf9 +T723 PR:000015643 20021 20024 Sry +T724 PR:000015435 20028 20032 Sox9 +T725 PR:000017444 20061 20065 Wnt4 +T726 UBERON:0000991 20082 20088 gonads +T727 PR:000007500 20111 20115 FGF9 +T728 PR:000017444 20120 20124 WNT4 +T729 PR:000017444 20183 20187 Wnt4 +T730 UBERON:0000991 20208 20213 gonad +T731 PR:000007500 20274 20278 FGF9 +T732 PR:000017444 20309 20313 Wnt4 +T733 PR:000017444 20321 20325 Wnt4 +T734 UBERON:0000991 20329 20335 gonads +T735 PR:000007500 20376 20380 FGF9 +T736 PR:000015435 20404 20408 SOX9 +T737 GO:0010467 20409 20419 expression +T738 PR:000007500 20450 20454 FGF9 +T739 PR:000015435 20463 20467 SOX9 +T740 PR:000017444 20488 20492 Wnt4 +T741 UBERON:0000991 20496 20502 gonads +T742 PR:000017444 20515 20519 Wnt4 +T743 UBERON:0000991 20526 20532 gonads +T744 PR:000017444 20598 20602 WNT4 +T745 PR:000007500 20607 20611 FGF9 +T746 PR:000007500 20764 20768 Fgf9 +T747 PR:000017444 20802 20806 Wnt4 +T748 GO:0042571 20832 20840 antibody +T749 PR:000007500 20849 20853 FGF9 +T750 PR:000007500 20869 20873 FGF9 +T751 GO:0010467 20878 20887 expressed +T752 PR:000017444 20891 20895 Wnt4 +T753 UBERON:0000991 20902 20908 gonads +T754 PR:000017444 20920 20924 Wnt4 +T755 PR:000007500 20987 20991 FGF9 +T756 PR:000017444 21022 21026 WNT4 +T757 UBERON:0000991 21033 21039 gonads +T758 PR:000007500 21065 21069 FGF9 +T759 PR:000015435 21095 21099 Sox9 +T760 PR:000017444 21122 21126 Wnt4 +T761 UBERON:0000991 21130 21136 gonads +T762 GO:0010467 21155 21165 expression +T763 PR:000015435 21169 21173 SOX9 +T764 PR:000017444 21197 21201 Wnt4 +T765 UBERON:0000991 21205 21211 gonads +T766 GO:0042571 21231 21239 antibody +T767 PR:000015435 21248 21252 SOX9 +T768 GO:0010467 21267 21277 expression +T769 PR:000017444 21308 21312 Wnt4 +T770 UBERON:0000991 21319 21325 gonads +T771 GO:0097617 21495 21508 hybridization +T772 PR:000015435 21530 21534 Sox9 +T773 SO:0000673 21535 21546 transcripts +T774 PR:000017444 21559 21563 Wnt4 +T775 UBERON:0000991 21570 21576 gonads +T776 PR:000017444 21590 21594 Wnt4 +T777 UBERON:0000991 21601 21607 gonads +T778 UBERON:0000991 21656 21662 gonads +T779 CL:0000216 21696 21708 Sertoli cell +T780 GO:0030154 21704 21724 cell differentiation +T781 UBERON:0005297 21729 21740 testis cord +T782 PR:000015435 21818 21822 SOX9 +T783 PR:000015643 21861 21864 Sry +T784 PR:000017444 21908 21912 Wnt4 +T785 PR:000007500 21931 21935 FGF9 +T786 GO:0007530 21968 21985 sex determination +T787 UBERON:0000991 22130 22135 gonad +T788 CL:0000630 22157 22172 supporting cell +T789 CL:0000477 22208 22222 follicle cells +T790 CL:0000216 22226 22239 Sertoli cells +T791 PR:000007500 22244 22248 Fgf9 +T792 UBERON:0000991 22255 22261 gonads +T793 CL:0000216 22293 22300 Sertoli +T794 PR:000007500 22332 22336 Fgf9 +T795 PR:000015435 22358 22362 Sox9 +T796 GO:0010467 22363 22373 expression +T797 GO:0046661 22399 22412 male pathways +T798 CL:0000216 22426 22439 Sertoli cells +T799 PR:000007500 22446 22450 Fgf9 +T800 UBERON:0000991 22454 22460 gonads +T801 GO:0008219 22475 22485 cell death +T802 CL:0000630 22518 22533 supporting cell +T803 PR:000015435 22542 22546 SOX9 +T804 GO:0010467 22547 22557 expression +T805 PR:000007500 22630 22634 FGF9 +T806 PR:000017444 22636 22640 WNT4 +T807 GO:0065007 22665 22671 govern +T808 CL:0002371 22672 22684 somatic cell +T809 UBERON:0000991 22697 22704 gonadal +T810 UBERON:0007688 22705 22710 field +T811 NCBITaxon:7215 22717 22727 Drosophila +T812 UBERON:3011048 22728 22735 genital +T813 UBERON:0007688 22751 22756 field +T814 UBERON:3011048 22890 22897 genital +T815 GO:0065007 22933 22940 control +T816 PR:P23023 22944 22954 double sex +T817 PR:P23023 22956 22959 dsx +T818 GO:0007530 22987 23004 sex determination +T819 SO:0000704 23058 23065 genetic +T820 GO:0065007 23183 23191 regulate +T821 GO:0017015 23270 23283;23298 23339 regulation of ... transforming growth factor beta signaling +T822 GO:0030111 23270 23287;23330 23339 regulation of WNT ... signaling +T823 GO:0040036 23270 23283;23289 23292;23330 23339 regulation of ... FGF ... signaling +T824 PR:000000046 23298 23329 transforming growth factor beta +T825 GO:0065007 23355 23363 regulate +T826 GO:0030154 23376 23396 cell differentiation +T827 GO:0009653 23402 23415 morphogenesis +T828 GO:0065007 23450 23460 regulation +T829 UBERON:0000991 23464 23469 gonad +T830 GO:0008406 23464 23483 gonad organogenesis +T831 NCBITaxon:7742 23487 23498 vertebrates +T832 GO:0007530 23587 23602 sex-determining +T833 GO:0030238 23624 23640;23645 23649;23660 23667 establishment of ... male ... program +T834 GO:0030237 23624 23640;23653 23667 establishment of ... female program +T835 GO:0030154 23804 23824 cell differentiation +T836 GO:0009653 23829 23842 morphogenesis +T837 UBERON:0000991 23850 23855 gonad +T838 NCBITaxon:40674 23936 23945 mammalian +T839 UBERON:0000991 23946 23951 gonad +T840 CL:0000216 24039 24046 Sertoli +T841 GO:0038001 24196 24205 paracrine +T842 GO:0060009 24239 24269 establishment of Sertoli cells +T843 CL:0000216 24256 24269 Sertoli cells +T844 PR:000007500 24318 24322 FGF9 +T845 PR:000015435 24334 24338 SOX9 +T846 PR:000007500 24411 24415 FGF9 +T847 UBERON:0000991 24458 24463 gonad +T848 PR:000017444 24497 24501 Wnt4 +T849 PR:000007500 24534 24538 FGF9 +T850 CL:0000216 24610 24617 Sertoli +T851 PR:000015435 24643 24647 SOX9 +T852 PR:000015643 24675 24678 Sry +T853 PR:000015435 24705 24709 SOX9 +T854 PR:000007500 24728 24732 Fgf9 +T855 PR:000007500 24750 24754 FGF9 +T856 PR:000007500 24796 24800 FGF9 +T857 GO:0008283 24820 24838 cell proliferation +T858 CL:0000216 24865 24872 Sertoli +T859 GO:0046661 24926 24938 male pathway +T860 UBERON:0000991 25029 25035 gonads +T861 CL:0000216 25048 25061 Sertoli cells +T862 PR:000007500 25070 25074 FGF9 +T863 GO:0008283 25084 25097 proliferation +T864 CL:0000216 25101 25108 Sertoli +T865 PR:000007500 25170 25174 FGF9 +T866 GO:0038001 25194 25203 paracrine +T867 PR:000017444 25286 25290 WNT4 +T868 GO:0008283 25334 25352 cell proliferation +T869 GO:0030238 25382 25398;25403 25415 establishment of ... male pathway +T870 CL:0000216 25426 25433 Sertoli +T871 GO:0010467 25508 25518 expression +T872 PR:000007500 25522 25526 Fgf9 +T873 UBERON:0004208 25536 25559 nephrogenous mesenchyme +T874 UBERON:0000991 25584 25591 gonadal +T875 SO:0000704 25599 25603 gene +T876 GO:0010467 25599 25614 gene expression +T877 PR:000007500 25628 25632 Fgf9 +T878 UBERON:0000991 25636 25643 gonadal +T879 GO:0008283 25644 25662 cell proliferation +T880 NCBITaxon:species 25670 25677 species +T881 PR:000015435 25707 25711 SOX9 +T882 PR:000017444 25722 25726 WNT4 +T883 GO:0010467 25739 25749 expression +T884 PR:000007500 25798 25802 FGF9 +T885 UBERON:0000991 25817 25822 gonad +T886 GO:0010467 25854 25864 expression +T887 PR:000017444 25868 25872 Wnt4 +T888 PR:000017444 25893 25897 Wnt4 +T889 PR:000015435 25927 25931 SOX9 +T890 PR:000015435 25936 25940 SOX9 +T891 PR:000015643 26011 26014 SRY +T892 PR:000015435 26019 26023 SOX9 +T893 GO:0010467 26038 26047 expressed +T894 PR:000007500 26051 26055 Fgf9 +T895 UBERON:0000991 26062 26068 gonads +T896 PR:000017444 26070 26074 Wnt4 +T897 PR:000007500 26115 26119 Fgf9 +T898 PR:000007500 26170 26174 FGF9 +T899 PR:000017444 26201 26205 Wnt4 +T900 PR:000017444 26314 26318 Wnt4 +T901 GO:0005622 26384 26397 intracellular +T902 PR:000015435 26423 26427 SOX9 +T903 GO:0010467 26428 26438 expression +T904 CL:0000138 26452 26463 chondrocyte +T905 GO:0005622 26506 26519 intracellular +T906 PR:000017444 26661 26665 Wnt4 +T907 GO:0046661 26682 26694 male pathway +T908 PR:000017444 26722 26726 Wnt4 +T909 PR:000015435 26760 26764 SOX9 +T910 PR:000007500 26769 26773 FGF9 +T911 UBERON:0000991 26780 26786 gonads +T912 PR:000015643 26793 26796 Sry +T913 GO:0046661 26828 26840 male pathway +T914 PR:000017444 26892 26896 Wnt4 +T915 PR:000007500 26901 26905 Fgf9 +T916 NCBITaxon:7742 26956 26966 vertebrate +T917 GO:0007530 26967 26984 sex-determination +T918 PR:000015643 27002 27005 Sry +T919 GO:0007530 27017 27032 sex determining +T920 PR:000015435 27067 27071 Sox9 +T921 UBERON:0000473 27103 27109 testis +T922 GO:0008584 27103 27121 testis development +T923 PR:000015435 27198 27202 Sox9 +T924 GO:0010467 27209 27218 expressed +T925 UBERON:0000991 27229 27234 gonad +T926 GO:0010467 27260 27270 expression +T927 PR:000015435 27278 27282 Sox9 +T928 GO:0010467 27283 27293 expression +T929 GO:0065007 27344 27354 regulatory +T930 SO:0005836 27344 27364 regulatory sequences +T931 PR:000017444 27432 27436 Wnt4 +T932 PR:000015435 27446 27450 SOX9 +T933 GO:0010467 27451 27461 expression +T934 PR:000017444 27518 27522 Wnt4 +T935 UBERON:0000991 27529 27534 gonad +T936 PR:000007500 27611 27615 FGF9 +T937 PR:000015435 27616 27620 SOX9 +T938 GO:0010467 27621 27631 expressing +T939 PR:000015643 27762 27765 Sry +T940 PR:000015435 27793 27797 SOX9 +T941 GO:0010467 27798 27808 expression +T942 GO:0031012 27847 27867 extracellular matrix +T943 PR:000015435 27992 27996 SOX9 +T944 GO:0010467 27997 28007 expression +T945 GO:0010467 28058 28067 expressed +T946 UBERON:0000991 28078 28083 gonad +T947 PR:000017444 28158 28162 Wnt4 +T948 PR:000017444 28193 28197 WNT4 +T949 GO:0046661 28244 28256 male pathway +T950 GO:0010628 28281 28297;28303 28313 up-regulation of ... expression +T951 PR:000015435 28298 28302 SOX9 +T952 NCBITaxon:9606 28360 28365 human +T953 PR:000017444 28395 28399 WNT4 +T954 GO:0010467 28544 28551 express +T955 PR:000017444 28552 28556 Wnt4 +T956 UBERON:0000991 28563 28569 gonads +T957 CL:0000216 28642 28654 Sertoli cell +T958 GO:0030154 28650 28670 cell differentiation +T959 PR:000017444 28697 28701 WNT4 +T960 NCBITaxon:10088 28767 28771 mice +T961 GO:0010467 28799 28808 expressed +T962 PR:000017444 28940 28944 Wnt4 +T963 GO:0065007 29034 29042 controls +T964 GO:0007530 29043 29060 sex determination +T965 PR:000015643 29086 29089 Sry +T966 NCBITaxon:40674 29111 29120 mammalian +T967 UBERON:0000991 29233 29238 gonad +T968 UBERON:0000473 29247 29257 testicular +T969 UBERON:0000992 29262 29269 ovarian +T970 NCBITaxon:7742 29279 29290 vertebrates +T971 SO:0000704 29307 29314 genetic +T972 GO:0046661 29370 29382 male pathway +T973 NCBITaxon:40674 29435 29444 mammalian +T974 UBERON:0000991 29445 29450 gonad +T975 PR:000007500 29536 29540 Fgf9 +T976 GO:0010467 29542 29551 expressed +T977 UBERON:0011997 29561 29569 coelomic +T978 PR:000017444 29583 29587 Wnt4 +T979 GO:0010467 29589 29598 expressed +T980 UBERON:0000080 29608 29619 mesonephric +T981 NCBITaxon:40674 29642 29651 mammalian +T982 UBERON:0000991 29655 29661 gonads +T983 PR:000015643 29676 29679 Sry +T984 GO:0010467 29680 29690 expression +T985 GO:0046661 29705 29717 male pathway +T986 PR:000015435 29735 29739 Sox9 +T987 PR:000015435 29741 29745 SOX9 +T988 PR:000007500 29759 29763 Fgf9 +T989 PR:000015435 29783 29787 Sox9 +T990 PR:000007500 29788 29792 Fgf9 +T991 GO:0030238 29828 29841;29846 29858 commitment to ... male pathway +T992 UBERON:0000991 29866 29872 gonads +T993 UBERON:0000991 29886 29892 gonads +T994 PR:000015643 29901 29904 Sry +T995 PR:000015435 29906 29910 Sox9 +T996 PR:000007500 29915 29919 Fgf9 +T997 PR:000015435 29925 29929 SOX9 +T998 PR:000007500 29930 29934 FGF9 +T999 PR:000017444 29977 29981 WNT4 +T1000 GO:0065007 29988 29995 control +T1001 UBERON:0000991 30003 30010 gonadal +T1002 UBERON:0007688 30011 30016 field +T1003 PR:000015435 30057 30061 Sox9 +T1004 PR:000007500 30066 30070 Fgf9 +T1005 GO:0030237 30099 30112;30117 30131 commitment to ... female pathway +T1006 PR:000007500 30207 30211 FGF9 +T1007 PR:000017444 30216 30220 WNT4 +T1008 GO:0065007 30350 30360 regulating +T1009 GO:0010467 30361 30371 expression +T1010 UBERON:0000473 30379 30385 testis +T1011 PR:000015435 30405 30409 SOX9 +T1012 NCBITaxon:33208 30435 30442 Animals +T1013 PR:000007500 30464 30468 Fgf9 +T1014 PR:000007500 30563 30567 Fgf9 +T1015 PR:000015643 30582 30585 Sry +T1016 NCBITaxon:10088 30591 30595 mice +T1017 PR:000007500 30754 30758 Fgf9 +T1018 PR:000007500 30836 30840 Fgf9 +T1019 PR:000015643 30884 30887 Sry +T1020 PR:000000020 30887 30890 Myc +T1021 NCBITaxon:10088 30891 30895 mice +T1022 PR:000017444 30937 30941 Wnt4 +T1023 UBERON:0000922 30980 30987 embryos +T1024 GO:0000806 31012 31024 Y chromosome +T1025 SO:0000112 31034 31041 primers +T1026 NCBITaxon:10088 31088 31092 Mice +T1027 PR:000015435 31112 31116 Sox9 +T1028 SO:0000704 31167 31171 gene +T1029 GO:0097617 31216 31229 hybridization +T1030 GO:0097617 31264 31277 hybridization +T1031 GO:0097617 31384 31397 hybridization +T1032 GO:0097617 31466 31479 hybridization +T1033 PR:000000073 31486 31489 Amh +T1034 PR:000006459 31496 31499 Dhh +T1035 PR:000017444 31506 31510 Wnt4 +T1036 PR:000007500 31520 31524 Fgf9 +T1037 CHEBI:42098 31531 31542 Digoxigenin +T1038 GO:0042571 31626 31636 Antibodies +T1039 NCBITaxon:10088 31683 31688 mouse +T1040 PR:000010812 31705 31710 N-MYC +T1041 NCBITaxon:9986 31786 31792 rabbit +T1042 PR:000015435 31798 31802 SOX9 +T1043 NCBITaxon:10114 31833 31836 rat +T1044 PR:000001904 31842 31847 PECAM +T1045 NCBITaxon:9986 31907 31913 rabbit +T1046 PR:000002312 31919 31928 caspase-3 +T1047 NCBITaxon:9986 32004 32010 rabbit +T1048 CHEBI:15358 32031 32038 histone +T1049 PR:000027594 32031 32041 histone H3 +T1050 GO:0042571 32067 32075 Antibody +T1051 CHEBI:51217 32103 32114 fluorophore +T1052 MOP:0000779 32115 32125 conjugated +T1053 GO:0042571 32136 32146 antibodies +T1054 PR:000007500 32233 32237 FGF9 +T1055 UBERON:0000991 32254 32260 gonads +T1056 NCBITaxon:9986 32299 32305 rabbit +T1057 NCBITaxon:10088 32311 32316 mouse +T1058 PR:000007500 32317 32321 FGF9 +T1059 NCBITaxon:9986 32352 32358 rabbit +T1060 GO:0071735 32359 32362 IgG +T1061 MOP:0000779 32363 32373 conjugated +T1062 GO:0042571 32400 32408 antibody +T1063 CHEBI:37987 32460 32463 Cy3 +T1064 CHEBI:51217 32464 32475 fluorophore +T1065 GO:0042571 32532 32540 antibody +T1066 PR:000007500 32556 32560 FGF9 +T1067 UBERON:0000992 32573 32578 ovary +T1068 PR:000007500 32582 32586 Fgf9 +T1069 UBERON:0000991 32602 32608 gonads +T1070 CL:0000001 32705 32712;32721 32725 Primary ... cell +T1071 UBERON:0000991 32713 32720 gonadal +T1072 UBERON:0000922 32745 32752 embryos +T1073 NCBITaxon:10088 32777 32781 mice +T1074 UBERON:0000305 32818 32825 amnions +T1075 UBERON:0005294 32851 32865 genital ridges +T1076 UBERON:0000991 32885 32891 gonads +T1077 UBERON:0000080 32912 32923 mesonephroi +T1078 UBERON:0000991 32929 32935 gonads +T1079 PR:000027795 32979 32986 trypsin +T1080 GO:0031012 33166 33186 extracellular matrix +T1081 NCBITaxon:27592 33242 33248 bovine +T1082 UBERON:0001977 33249 33254 serum +T1083 CHEBI:33282 33262 33273 antibiotics +T1084 CHEBI:35718 33274 33286 antimycotics +T1085 CHEBI:16526 33300 33303 CO2 +T1086 PR:000007500 33335 33339 FGF9 +T1087 PR:000015435 33527 33531 SOX9 +T1088 GO:0005634 33572 33579 nuclear +T1089 UBERON:0000991 33643 33648 Gonad +T1090 UBERON:0000991 33667 33672 Gonad +T1091 UBERON:0000080 33673 33684 mesonephros +T1092 CHEBI:16526 33772 33775 CO2 +T1093 PR:000007500 33799 33803 FGF9 +T1094 PR:000007500 33824 33828 FGF9 +T1095 PR:000007500 33891 33895 FGF9 +T1096 UBERON:0000991 33937 33942 gonad +T1097 CHEBI:28304 33959 33966 heparin +T1098 CHEBI:2511 33967 33974 agarose +T1099 PR:000007500 34052 34056 FGF9 +T1100 PR:000015435 34178 34182 Sox9 +T1101 PR:000017444 34202 34206 Wnt4 +T1102 UBERON:0000991 34213 34219 Gonads +T1103 GO:0097617 34241 34254 hybridization +T1104 PR:000015435 34259 34263 Sox9 +T1105 PR:000015435 34273 34277 Sox9 +T1106 GO:0010467 34278 34288 expression +T1107 PR:000017444 34301 34305 Wnt4 +T1108 PR:000017444 34325 34329 Wnt4 +T1109 UBERON:0000991 34336 34342 gonads +T1110 PR:000015643 34419 34422 Sry +T1111 PR:000015435 34594 34598 Sox9 +T1112 UBERON:0004122 34618 34628 urogenital +T1113 NCBITaxon:33208 34714 34721 animals +T1114 GO:0007620 34837 34843 coitum +T1115 CL:0000057 34894 34904 fibroblast +T1116 PR:000001904 34920 34925 PECAM +T1117 CL:0000233 34928 34936 platelet +T1118 PR:000001904 34928 34971 platelet/endothelial cell adhesion molecule +T1119 UBERON:0001986 34937 34948 endothelial +T1120 CL:0000115 34937 34953 endothelial cell +T1121 CHEBI:36357 34963 34971 molecule +T1122 PR:000015643 34979 34982 SRY +T1123 PR:000015643 34997 35000 SRY +T1124 GO:0007530 35003 35018 sex-determining +T1125 PR:000015643 35003 35034 sex-determining region of the Y +T1126 GO:0000806 35033 35034 Y +T1127 UBERON:0000023 35042 35046 wing +T1128 PR:P09615 35042 35050 wingless +T1129 NCBITaxon:11757 35059 35063 MMTV +T1130 SO:0000366 35064 35080 integration site +T1131 GO:0010467 35137 35147 Expression +T1132 PR:000007500 35151 35155 FGF9 +T1133 UBERON:0000922 35159 35168 Embryonic +T1134 UBERON:0000991 35169 35175 Gonads +T1135 PR:000007500 35196 35200 FGF9 +T1136 UBERON:0000991 35238 35243 gonad +T1137 GO:0008406 35238 35255 gonad development +T1138 PR:000007500 35257 35261 FGF9 +T1139 UBERON:0000991 35284 35290 gonads +T1140 PR:000007500 35423 35427 Fgf9 +T1141 UBERON:0000991 35431 35437 gonads +T1142 PR:000002065 35523 35526 Kit +T1143 UBERON:0000991 35534 35540 gonads +T1144 PR:000007500 35596 35600 FGF9 +T1145 UBERON:0005297 35617 35629 Testis cords +T1146 CL:0000586 35659 35669 germ cells +T1147 PR:000002065 35676 35679 Kit +T1148 UBERON:0000991 35691 35697 gonads +T1149 GO:0010467 35728 35738 Expression +T1150 PR:000007500 35742 35746 FGF9 +T1151 UBERON:0000991 35772 35778 gonads +T1152 CL:0000216 35785 35798 Sertoli cells +T1153 UBERON:0005297 35839 35844 cords +T1154 UBERON:0000991 35909 35914 gonad +T1155 UBERON:0000080 35919 35930 mesonephroi +T1156 PR:000001904 35932 35937 PECAM +T1157 CL:0000586 35952 35962 germ cells +T1158 CL:0000071 35967 35993 vascular endothelial cells +T1159 UBERON:0001986 35976 35987 endothelial +T1160 UBERON:0000991 36044 36045 g +T1161 UBERON:0000991 36047 36052 gonad +T1162 UBERON:0000080 36054 36055 m +T1163 UBERON:0000080 36057 36068 mesonephroi +T1164 PR:000015643 36107 36110 Sry +T1165 PR:000007500 36112 36116 Fgf9 +T1166 PR:000015435 36122 36126 Sox9 +T1167 PR:000015643 36134 36137 Sry +T1168 GO:0010467 36138 36148 expression +T1169 PR:000007500 36169 36173 Fgf9 +T1170 PR:000007500 36175 36179 Fgf9 +T1171 PR:000007500 36191 36195 Fgf9 +T1172 UBERON:0000991 36206 36212 gonads +T1173 GO:0010467 36225 36235 expressing +T1174 PR:000015643 36257 36260 Sry +T1175 SO:0000167 36261 36269 promoter +T1176 CL:0000398 36271 36286 polygonal cells +T1177 CL:0000081 36297 36308 Blood cells +T1178 PR:000007500 36363 36367 Fgf9 +T1179 PR:000007500 36379 36383 Fgf9 +T1180 UBERON:0000991 36394 36400 gonads +T1181 GO:0010467 36413 36423 expressing +T1182 PR:000015643 36424 36427 SRY +T1183 PR:000000020 36427 36430 MYC +T1184 GO:0005634 36470 36477 nuclear +T1185 PR:000015643 36525 36528 SRY +T1186 PR:000000020 36528 36531 MYC +T1187 PR:000001904 36533 36538 PECAM +T1188 UBERON:0001986 36552 36563 endothelial +T1189 CL:0000115 36552 36563;36573 36578 endothelial ... cells +T1190 CL:0000586 36568 36578 germ cells +T1191 PR:000007500 36625 36629 FGF9 +T1192 PR:000015435 36646 36650 SOX9 +T1193 GO:0010467 36651 36661 expression +T1194 UBERON:0000991 36668 36674 gonads +T1195 PR:000015435 36694 36698 SOX9 +T1196 UBERON:0000991 36730 36737 gonadal +T1197 PR:000007500 36799 36803 FGF9 +T1198 PR:000015435 36822 36826 SOX9 +T1199 GO:0010467 36827 36837 expression +T1200 GO:0005634 36899 36906 nuclear +T1201 PR:000015435 36947 36951 SOX9 +T1202 UBERON:0000991 36961 36966 gonad +T1203 PR:000007500 36998 37002 FGF9 +T1204 PR:000015435 37017 37021 SOX9 +T1205 GO:0010467 37025 37034 expressed +T1206 UBERON:0000991 37041 37047 gonads +T1207 PR:000007500 37069 37073 FGF9 +T1208 UBERON:0000991 37121 37127 gonads +T1209 PR:000001904 37205 37210 PECAM +T1210 UBERON:0001986 37224 37235 endothelial +T1211 CL:0000115 37224 37235;37245 37250 endothelial ... cells +T1212 CL:0000586 37240 37250 germ cells +T1213 PR:000007500 37333 37337 Fgf9 +T1214 PR:000015435 37342 37346 Sox9 +T1215 PR:000015435 37372 37376 SOX9 +T1216 PR:000007500 37386 37390 Fgf9 +T1217 PR:000007500 37398 37402 Fgf9 +T1218 UBERON:0000991 37409 37415 gonads +T1219 PR:000007500 37427 37431 Fgf9 +T1220 PR:000015435 37463 37467 SOX9 +T1221 PR:000015435 37490 37494 SOX9 +T1222 CL:0000216 37498 37505 Sertoli +T1223 PR:000007500 37540 37544 Fgf9 +T1224 UBERON:0000991 37548 37554 gonads +T1225 PR:000015435 37632 37636 SOX9 +T1226 UBERON:0000991 37674 37680 gonads +T1227 GO:0097617 37771 37784 hybridization +T1228 PR:000015643 37789 37792 Sry +T1229 PR:000007500 37797 37801 Fgf9 +T1230 PR:000015435 37805 37809 Sox9 +T1231 SO:0000359 37809 37813 flox +T1232 PR:000015435 37820 37824 Sox9 +T1233 UBERON:0000991 37831 37837 gonads +T1234 PR:000015435 37849 37853 Sox9 +T1235 PR:000007500 37870 37874 Fgf9 +T1236 GO:0010467 37875 37885 expression +T1237 PR:000015643 37887 37890 Sry +T1238 GO:0010467 37891 37901 expression +T1239 PR:000015435 37922 37926 Sox9 +T1240 SO:0000359 37926 37930 flox +T1241 PR:000015435 37937 37941 Sox9 +T1242 UBERON:0000991 37945 37951 gonads +T1243 PR:000007500 37983 37987 Fgf9 +T1244 GO:0010467 37988 37998 expression +T1245 PR:000015435 38034 38038 Sox9 +T1246 UBERON:0000991 38042 38048 gonads +T1247 GO:0008283 38093 38111 cell proliferation +T1248 PR:000015435 38115 38119 Sox9 +T1249 PR:000015435 38130 38134 Sox9 +T1250 SO:0000359 38134 38138 flox +T1251 UBERON:0000991 38141 38147 gonads +T1252 CHEBI:15358 38200 38207 histone +T1253 PR:000027594 38200 38210 histone H3 +T1254 GO:0008283 38224 38237 proliferation +T1255 UBERON:0000991 38245 38250 gonad +T1256 PR:000015435 38292 38296 Sox9 +T1257 GO:0008283 38338 38351 proliferation +T1258 UBERON:0001851 38395 38403 cortical +T1259 UBERON:0000991 38419 38424 gonad +T1260 PR:000015435 38486 38490 Sox9 +T1261 SO:0000359 38490 38494 flox +T1262 UBERON:0000991 38497 38503 gonads +T1263 UBERON:0000991 38540 38545 gonad +T1264 UBERON:0000991 38565 38571 gonads +T1265 PR:000001904 38591 38596 PECAM +T1266 CL:0000216 38662 38674 Sertoli Cell +T1267 GO:0010467 38698 38708 Expression +T1268 GO:0046661 38712 38716;38727 38734 Male ... Pathway +T1269 GO:0046660 38720 38734 Female Pathway +T1270 SO:0000704 38735 38740 Genes +T1271 GO:0097617 38768 38781 hybridization +T1272 SO:0000704 38786 38791 genes +T1273 GO:0046661 38799 38811 male pathway +T1274 PR:000015435 38826 38830 Sox9 +T1275 PR:000006459 38832 38835 Dhh +T1276 PR:000000073 38841 38844 Amh +T1277 PR:000006459 38846 38849 Dhh +T1278 GO:0010467 38850 38860 expression +T1279 PR:000007500 38880 38884 Fgf9 +T1280 UBERON:0000991 38888 38894 gonads +T1281 PR:000000073 38922 38925 Amh +T1282 GO:0010467 38926 38936 expression +T1283 PR:000007500 38963 38967 Fgf9 +T1284 UBERON:0000991 38971 38977 gonads +T1285 GO:0008219 39024 39034 cell death +T1286 PR:000007500 39038 39042 Fgf9 +T1287 UBERON:0000991 39049 39055 gonads +T1288 PR:000026878 39083 39099 active caspase-3 +T1289 GO:0006915 39120 39129 apoptosis +T1290 PR:000007500 39148 39152 Fgf9 +T1291 UBERON:0000991 39156 39162 gonads +T1292 UBERON:0000991 39192 39198 gonads +T1293 CL:0000445 39209 39224 apoptotic cells +T1294 UBERON:0000083 39246 39265 mesonephric tubules +T1295 UBERON:0000083 39267 39268 m +T1296 UBERON:0000991 39284 39290 gonads +T1297 UBERON:0000080 39360 39371 mesonephros +T1298 UBERON:0000991 39376 39381 gonad +T1299 PR:000001904 39384 39389 PECAM +T1300 GO:0097617 39426 39439 hybridization +T1301 PR:000017444 39444 39448 Wnt4 +T1302 UBERON:0000992 39453 39458 ovary +T1303 PR:000017444 39467 39471 Wnt4 +T1304 GO:0010467 39475 39484 expressed +T1305 PR:000007500 39488 39492 Fgf9 +T1306 UBERON:0000991 39499 39505 gonads +T1307 PR:000007500 39549 39553 Fgf9 +T1308 UBERON:0000991 39631 39632 g +T1309 UBERON:0000991 39634 39639 gonad +T1310 UBERON:0000080 39641 39642 m +T1311 UBERON:0000080 39644 39655 mesonephros +T1312 PR:000007500 39694 39698 Fgf9 +T1313 PR:000017444 39703 39707 Wnt4 +T1314 PR:000017444 39715 39719 Wnt4 +T1315 GO:0097617 39740 39753 hybridization +T1316 UBERON:0000991 39757 39762 gonad +T1317 PR:000007500 39790 39794 FGF9 +T1318 UBERON:0000991 39798 39803 gonad +T1319 GO:0010629 39828 39846;39852 39862 down-regulation of ... expression +T1320 PR:000017444 39847 39851 Wnt4 +T1321 UBERON:0000991 39878 39884 gonads +T1322 PR:000007500 39931 39935 FGF9 +T1323 PR:000017444 39977 39981 Wnt4 +T1324 PR:000007500 39989 39993 FGF9 +T1325 PR:000015435 40004 40008 SOX9 +T1326 UBERON:0000991 40015 40021 gonads +T1327 PR:000015435 40041 40045 SOX9 +T1328 PR:000007500 40075 40079 FGF9 +T1329 PR:000015435 40093 40097 SOX9 +T1330 GO:0010467 40098 40108 expression +T1331 PR:000017444 40125 40129 Wnt4 +T1332 UBERON:0000991 40136 40142 gonads +T1333 PR:000017444 40159 40163 Wnt4 +T1334 UBERON:0000991 40170 40176 gonads +T1335 PR:000001904 40182 40187 PECAM +T1336 GO:0010467 40247 40257 Expression +T1337 PR:000015435 40275 40279 SOX9 +T1338 PR:000007500 40284 40288 FGF9 +T1339 PR:000017444 40296 40300 Wnt4 +T1340 UBERON:0000991 40304 40310 Gonads +T1341 PR:000007500 40318 40322 FGF9 +T1342 PR:000007500 40355 40359 FGF9 +T1343 GO:0010467 40363 40372 expressed +T1344 PR:000017444 40379 40383 Wnt4 +T1345 UBERON:0000991 40387 40393 gonads +T1346 PR:000015435 40460 40464 SOX9 +T1347 PR:000015435 40497 40501 SOX9 +T1348 PR:000017444 40536 40540 Wnt4 +T1349 UBERON:0000991 40544 40550 gonads +T1350 PR:000015435 40552 40556 SOX9 +T1351 GO:0010467 40557 40567 expression +T1352 PR:000017444 40583 40587 Wnt4 +T1353 UBERON:0000991 40594 40600 gonads +T1354 UBERON:0000991 40671 40676 gonad +T1355 PR:000015435 40693 40697 SOX9 +T1356 PR:000017444 40728 40732 Wnt4 +T1357 UBERON:0000991 40736 40742 gonads +T1358 PR:000017444 40752 40756 Wnt4 +T1359 UBERON:0000991 40763 40769 gonads +T1360 UBERON:0011997 40799 40807 coelomic +T1361 UBERON:0000055 40808 40814 vessel +T1362 PR:000017444 40821 40825 Wnt4 +T1363 UBERON:0000991 40829 40835 gonads +T1364 PR:000001904 40869 40874 PECAM +T1365 GO:0065007 40943 40951 Regulate +T1366 GO:0007530 40952 40969 Sex Determination +T1367 UBERON:0000991 40989 40994 Gonad +T1368 UBERON:0000991 41014 41020 gonads +T1369 UBERON:0002329 41043 41049 somite +T1370 PR:000007500 41058 41062 Fgf9 +T1371 SO:0000673 41063 41074 transcripts +T1372 UBERON:0000991 41112 41117 gonad +T1373 PR:000017444 41145 41149 Wnt4 +T1374 SO:0000673 41150 41161 transcripts +T1375 UBERON:0000991 41184 41189 gonad +T1376 UBERON:0000080 41190 41201 mesonephric +T1377 UBERON:0000991 41266 41271 gonad +T1378 SO:0000704 41319 41326 genetic +T1379 GO:0046661 41365 41377 male pathway +T1380 NCBITaxon:40674 41429 41436 mammals +T1381 PR:000015435 41489 41493 Sox9 +T1382 PR:000015435 41495 41499 Sox9 +T1383 PR:000007500 41513 41517 Fgf9 +T1384 PR:000007500 41523 41527 Fgf9 +T1385 PR:000015435 41538 41542 Sox9 +T1386 UBERON:0000991 41587 41593 gonads +T1387 PR:000007500 41634 41638 FGF9 +T1388 PR:000017444 41643 41647 WNT4 +T1389 PR:000007500 41679 41683 FGF9 +T1390 GO:0046661 41710 41722 male pathway +T1391 PR:000015435 41785 41789 SOX9 +T1392 PR:000007500 41794 41798 FGF9 +T1393 UBERON:0000991 41812 41818 gonads +T1394 PR:000017444 41821 41825 WNT4 +T1395 PR:000007500 41833 41837 Fgf9 +T1396 GO:0046660 41854 41868 female pathway +T1397 CHEBI:33893 42086 42094 reagents +T1398 PR:000007500 42258 42262 Fgf9 +T1399 PR:000017444 42267 42271 Wnt4 +T1400 GO:0065007 42303 42311 regulate +T1401 NCBITaxon:40674 42312 42321 mammalian +T1402 GO:0007530 42322 42339 sex determination diff --git a/src/ontogpt/evaluation/craft/database/all/16700629.txt b/src/ontogpt/evaluation/craft/database/all/16700629.txt new file mode 100644 index 000000000..b3184637d --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16700629.txt @@ -0,0 +1,201 @@ +Fgf9 and Wnt4 Act as Antagonistic Signals to Regulate Mammalian Sex Determination + +Abstract + +The genes encoding members of the wingless-related MMTV integration site (WNT) and fibroblast growth factor (FGF) families coordinate growth, morphogenesis, and differentiation in many fields of cells during development. In the mouse, Fgf9 and Wnt4 are expressed in gonads of both sexes prior to sex determination. Loss of Fgf9 leads to XY sex reversal, whereas loss of Wnt4 results in partial testis development in XX gonads. However, the relationship between these signals and the male sex-determining gene, Sry, was unknown. We show through gain- and loss-of-function experiments that fibroblast growth factor 9 (FGF9) and WNT4 act as opposing signals to regulate sex determination. In the mouse XY gonad, Sry normally initiates a feed-forward loop between Sox9 and Fgf9, which up-regulates Fgf9 and represses Wnt4 to establish the testis pathway. Surprisingly, loss of Wnt4 in XX gonads is sufficient to up-regulate Fgf9 and Sox9 in the absence of Sry. These data suggest that the fate of the gonad is controlled by antagonism between Fgf9 and Wnt4. The role of the male sex-determining switch—Sry in the case of mammals—is to tip the balance between these underlying patterning signals. In principle, sex determination in other vertebrates may operate through any switch that introduces an imbalance between these two signaling pathways. + +Introduction + +The development of sexually dimorphic reproductive organs is a common feature among animal species. The testis and ovary represent two divergent pathways of development from the bipotential embryonic gonad. The switch that initiates divergent development of the gonad is highly diverse among species; however, the underlying mechanisms that lead to the establishment of ovary or testis pathways are likely to be conserved. In all species, the embryonic gonad is made up of a mixed population of germ cells and somatic cells. This tissue is remarkable in that all of its cells are believed to be bipotential, and can differentiate into ovarian or testicular lineages [1,2]. Consistent with the idea that cells in this primordium are poised between two developmental pathways, some of the genes that are involved in establishing sexual dimorphism, including Dax1 (dosage-sensitive sex reversal-congenital adrenal hypoplasia critical region on the X chromosome protein 1), Sox9 (Sry-like HMG box 9), Fgf9 (fibroblast growth factor 9), and Wnt4 (wingless-related MMTV integration site 4), are initially expressed in similar patterns in XX and XY gonads [3–8]. The conventional view of mammalian sex determination is that the basic pathway of organ development is ovarian, and that the testis-determining gene operates by diverting this program toward testis development by simultaneously influencing the fate of the key supporting cell lineage and initiating a male-specific morphogenetic program. All of the experimental evidence suggests that these two processes are closely interwoven. For example, both proliferation [9] and migration of cells to trigger testis cord formation [10,11] appear to be closely integrated with Sertoli cell differentiation. + +Sry, a Y chromosome-linked gene, is the primary sex-determining gene in mammals [12–14]. In the absence of Sry expression—in XX embryos, or in XY embryos carrying a deletion of Sry—cells in the gonad follow an ovarian differentiation pathway. Genetic evidence from chimeric mice [15], and expression studies using reporter transgenes [2,16], indicate that Sry expression is required only in precursors of the somatic supporting cell lineage. Expression of Sry in these bipotential cells leads to their differentiation as testis-specific Sertoli cells rather than as follicle cells, the parallel cell type of the ovary [2]. It is believed that the Sertoli cell is the first cell type to differentiate in the gonad [17]. There is substantial evidence that a critical threshold number of Sertoli cells is required to establish testis differentiation [9,15,18–20]. In cases where this threshold is not reached, ovary differentiation ensues. + +Once Sry expression begins, expression patterns of other genes in the gonad begin to diverge. The first gene downstream of Sry known to show male-specific up-regulation in Sertoli cell precursors is a related gene expressed in many tissues in the developing embryo, Sox9. Disruption of Sox9 expression in the XY gonad causes male-to-female sex reversal [21,22], whereas increasing the dose of Sox9 in the XX gonad leads to testis development [23–25]. These studies indicate that Sox9 plays a central role in sex determination. Unlike Sry, which is specific to mammals, expression of Sox9 is known to be conserved in the gonad of many species. In mammals, Sox9 is up-regulated immediately after Sry expression initiates. Experiments tracing Sry-expressing cells using a stable reporter demonstrated that once testis differentiation is established, all Sertoli cells that express Sox9 are descendants of cells that have expressed Sry [16], suggesting that activation of Sox9 is a cell-autonomous effect of Sry. However, mutations in several signaling pathways including Fgf9 and Igf1r/Irr/Ir (insulin-like growth factor 1 receptor/insulin receptor-related receptor/insulin receptor) resulted in loss of Sox9 expression and partial or complete sex reversal [26,27], suggesting that extracellular signaling pathways play a significant role during primary sex determination. + +Mice homozygous for a null mutation in Fgf9 display male-to-female sex reversal caused by disruption of all testis-specific cellular events, including cell proliferation, mesonephric cell migration, testis cord formation, and the differentiation of Sertoli cells [26,27]. Fgf9, like many of the founding signals in the gonads, is initially expressed in gonads of both sexes, but becomes male-specific after Sry is expressed. In a reciprocal manner, expression of Wnt4, which is also initially common to gonads of both sexes, becomes female-specific [8]. XX gonads with a null mutation in Wnt4 display some obvious aspects of testicular differentiation [28]. Based on the theory that Sertoli cells initiate all downstream testicular differentiation, this might imply that Sertoli differentiation had been initiated in Wnt4−/− XX gonads. However, expression of Sertoli cell markers was not previously detected in these mutants during fetal stages [8,29], leading to the conclusion that Wnt4 was not involved in primary sex determination in the gonad. + +To integrate these findings, we investigated the genetic relationship of Sry, Sox9, Fgf9, and Wnt4 in the regulatory network that governs the gonadal field. We show that the loss of Fgf9 in homozygous mutant XY gonads does not affect the expression of SRY or the initial up-regulation of SOX9; however, SOX9 expression is not maintained in the Fgf9−/− mutant gonads, and testis differentiation is aborted. We also demonstrate that FGF9 represses the ovary-promoting gene, Wnt4. We hypothesize that FGF9 functions in a feed-forward loop to expand Sertoli precursor cells, which secrete FGF9, to a critical threshold number sufficient to suppress Wnt4. This directly or indirectly stabilizes SOX9 expression and secures the male fate of the gonad. WNT4 seems to oppose the male pathway by repressing expression of SOX9 and FGF9. Surprisingly, both male pathway genes are transiently activated in Wnt4−/− XX gonads in the absence of the Y-linked gene Sry. Based on this genetic and in vitro data, we suggest that the plasticity of the bipotential gonad is controlled by mutually antagonistic signals between FGF9 and WNT4 in the gonadal field. These signals coordinate sexually dimorphic patterns of growth, morphogenesis, and cellular differentiation. + +Results + +Early Bipotential Expression of FGF9 Resolves to an XY-Specific Pattern by 12.5 dpc + +Using an antibody specific to FGF9, we examined expression during normal gonad development. FGF9 protein was distributed throughout the 11.5 dpc gonad in both sexes (Figure 1A and 1B). However, by 12.5 dpc, FGF9 was detected only in XY gonads in two domains: in cells near the surface of gonads and in cells located within testis cords. This sex-specific expression pattern was maintained in gonads at 13.5 dpc (Figure 1C–1F). FGF9 expression within testis cords was localized to Sertoli cells and excluded from germ cells based on the germ cell membrane marker platelet/endothelial cell adhesion molecule (PECAM) (Figure 1D and 1F). To confirm this result, we examined agametic gonads from KitW/Wv compound heterozygous embryos, which develop testis cords containing Sertoli cells only. FGF9 was detectable at normal levels in testis cords in XY KitW/Wv gonads, where alkaline phosphatase staining verified the absence of germ cells (Figure 1G–1J), indicating that FGF9 is expressed by Sertoli cells, and that its expression is not dependent on the presence of germ cells. In summary, FGF9 expression was present in both XX and XY gonads at bipotential stages, and became restricted to XY gonads as testis differentiation proceeded. + +Sry Expression Is Normal in Homozygous Null Fgf9 XY Gonads + +The early expression of Fgf9 in bipotential gonads raised the question of whether Fgf9 is an upstream regulator of Sry. To investigate this possibility, we mated Fgf9+/− mice with a transgenic reporter line that carries an enhanced green fluorescent protein (EGFP) transgene driven by the Sry promoter Sry-EGFP. At early stages, this transgene represents the pattern of endogenous Sry expression [2]. The expression of the EGFP reporter was detected in Fgf9−/− XY gonads comparable to Fgf9+/− littermate controls (Figure 2A and 2B), suggesting that transcriptional regulation of Sry is independent of Fgf9. Sex reversal is caused not only by the loss of normal levels of Sry expression [30,31], but also by mutations disrupting SRY import into the nucleus [32]. The transgene, Sry-EGFP, does not reflect the intracellular distribution of the SRY protein. To investigate this aspect of SRY regulation, we bred Fgf9+/− mice with another Sry reporter mouse line carrying a Myc-tagged Sry transgene, SryMyc, which recapitulates the endogenous intracellular SRY expression pattern [16]. Using an antibody against c-MYC, the expression and nuclear localization of SRYMYC in SryMyc; Fgf9−/− gonads was indistinguishable from littermate controls (Figure 2C and 2D). These data using two different Sry transgenic reporter lines provide evidence that Sry expression is not dependent on Fgf9. Therefore, Fgf9 signaling must act in parallel and/or downstream of Sry to regulate testis development. + +FGF9 Can Up-Regulate SOX9 Expression + +In our previous study we did not observe SOX9 expression at 12.5 dpc in Fgf9−/− XY gonads that fail to develop into normal testes [26]. However, the loss of SOX9 expression at 12.5 dpc could be a consequence of the loss of Sertoli differentiation rather than a reflection of the genetic interaction between FGF9 and SOX9. Normally, Sox9 is weakly expressed in wild-type genital ridges of both XX and XY embryos at 10.5 dpc and, after the onset of Sry expression, is up-regulated in XY gonads [5,33,16]. As results indicated that Fgf9 functioned downstream of, or in parallel with Sry, we investigated whether Fgf9 was involved in the up-regulation of Sox9 expression. Primary cell culture and gonad culture systems were used to assess Sox9 activation by exogenous FGF9. For in vitro cell culture, cells were isolated from 11.5 dpc gonads free of mesonephroi, and cultured on extracellular matrix-coated coverslips with or without addition of purified FGF9 in culture media. After culture for 24 h, SOX9 expression was monitored by an antibody specific to SOX9 in XX cells and control XY cells. Exogenous FGF9 increased cell number in XX and XY cell cultures compared with cells in a duplicate culture without FGF9 treatment (unpublished data), and caused the up-regulation of SOX9 in XX cells (Figure 2E–2H). Up-regulation of SOX9 had not previously been seen in whole XX gonads cultured with exogenous FGF9 [9]. To explain the difference between experimental results from dissociated XX gonadal cells and XX gonads, we reasoned that the local concentrations of FGF9 might not be high enough to override blocking signals in the intact XX gonad, or that active FGF9 was not efficiently localized or presented in the extracellular matrix of the XX gonad. To test the local effect of FGF9, we modified the XX gonad culture by stably immobilizing FGF9, or BSA as a control, on beads (Figure 2I–2K). Under these conditions, SOX9 expression was up-regulated locally in cells near the surface of the XX gonad in contact with the FGF9 bead (Figure 2K). Taken together, these in vitro data demonstrate that ectopic FGF9 signaling can induce SOX9 expression in XX gonadal cells, suggesting a positive interaction between Fgf9 and Sox9. + +Fgf9 Is Required for Maintaining SOX9 Expression in XY Gonads + +To investigate whether Fgf9 is essential for the up-regulation of Sox9 in vivo, we assessed SOX9 expression in loss-of-function Fgf9+/− and Fgf9−/− XY gonads at 11.5–12.5 dpc (Figure 3A–3F). In wild-type and heterozygous mutant XY gonads at 11.5 dpc, SOX9 was detected in a small number of cells in the gonad (Figure 3A). Over the next 6 h of development, nuclear SOX9 accumulated rapidly in cells toward the cortex and the anterior and posterior poles of the gonad, replicating patterns previously reported for both Sry and Sox9 expression [2,16,34–36]. This unique pattern was also observed in Fgf9−/− XY gonads (Figure 3D and 3E). Somatic cells within Fgf9−/− gonads were positive for SOX9 at 11.5 dpc, the earliest stages examined, demonstrating that initial expression and up-regulation of SOX9 were not disrupted in Fgf9−/− mutant XY gonads prior to 12.0 dpc. Notably, in Fgf9−/− XY gonads, SOX9 was no longer detectable by 12.5 dpc, and Sertoli precursor cells never began to organize into normal testis cord structures (Figure 3F). These data indicate that although Fgf9 is not required for the up-regulation of Sox9 in vivo, it is indispensable to maintain Sox9 expression in Sertoli precursor cells. + +Sox9 Is Required for Fgf9 Up-Regulation in XY Gonads + +We hypothesized that if the linear relationship among the three genes were Sry → Fgf9 → Sox9, expression of Fgf9 would be normal in XY gonads in the absence of Sox9. Alternatively, if the relationship were Sry → Sox9 → Fgf9, expression of Fgf9 should be reduced or absent in XY gonads in the absence of Sox9. We examined Fgf9 expression in Sox9 homozygous mutant (Sox9Δ/Δ) XY gonads generated by crossing mice homozygous for a conditional null (flox) allele of Sox9 (Sox9flox/flox) with mice carrying germline-specific Cre transgenes, Prm1-Cre in male and Zp3-Cre in female [22,37]. The Sox9 null mutant embryos die after 11.5 dpc because of cardiovascular defects [37]. Chaboissier et al. [22] successfully cultured 11.5 dpc Sox9 null mutant gonads in vitro and detected male and female markers after 2–3 d of culture, suggesting that Sox9 mutant gonads are viable and developmentally competent at 11.5 dpc—the time point at which we collected samples to perform mRNA in situ hybridization (Figure 3G–3J). The expression of Fgf9 was significantly decreased or absent in XY Sox9Δ/Δ gonads at 11.5 dpc (Figure 3J), while Sry expression was similar to wild-type (Figure 3G and 3H), as previously reported [20], suggesting that Fgf9 expression in wild-type XY gonads is dependent on the expression of Sox9. These findings also indicate that expression of Sry is not sufficient to regulate Fgf9 in the absence of Sox9. Therefore, we conclude that Sox9 is essential for Fgf9 expression, and Fgf9, in return, maintains Sox9 expression, generating a positive feed-forward loop between these two genes in XY gonads. + +Like Fgf9 Mutant Gonads, Sox9Δ/Δ XY Gonads Show Defects in Cell Proliferation + +We previously reported that XY-specific cell proliferation is defective in Fgf9−/− XY gonads [7]. Because Sox9 acts as a positive regulator of Fgf9 expression, we questioned whether cell proliferation in XY gonads was also compromised by the loss of Sox9. We examined proliferation in Sox9Δ/Δ gonads at 11.5 dpc using a mitotic cell marker, phosphorylated histone H3. Proliferating cells were more abundant and concentrated in a domain near the surface of wild-type XY gonads, and this XY-specific cell proliferation was evident in XY Sox9flox/Δ littermate controls (Figure 3K). However, in Sox9Δ/Δ XY gonads proliferation was reduced and similar to XX gonads (Figure 3L–3O). This result supports the idea that there is a mutual interdependence between Sox9 and Fgf9 generating a positive feed-forward loop, and that both genes are required for the expansion of somatic cells, including Sertoli cell precursors, in XY gonads. + +The Male Pathway Is Aborted in Fgf9−/− Sertoli Precursors + +Based on the fact that Sox9 is initially expressed in Fgf9−/− gonads, we investigated whether other genes in the male pathway are activated. We examined two markers for Sertoli cell differentiation, anti-Mullerian hormone (Amh) [38] and Desert hedgehog (Dhh) [39] in Fgf9−/− XY gonads using whole-mount in situ hybridization. Dhh, which is expressed in XY wild-type and heterozygous gonads beginning at 11.5 dpc, was absent from Fgf9−/− XY gonads, although mesonephric expression was still detected (Figure 4A and 4B). Amh, which is a direct transcriptional target of SOX9 activated after 11.5 dpc [38,40], was detected at reduced levels in Fgf9−/− XY gonads at 12.5 dpc (Figure 4C–4F). The residual level of Amh suggested that the transient expression of SOX9 in Fgf9−/− gonads at 11.5 dpc was sufficient to activate Amh, a direct downstream target. However, the absence of Dhh indicated that not all Sertoli pathways are initiated. + +The initial specification of Sertoli cell precursors was not affected by the loss of Fgf9, as evidenced by normal Sry and Sox9 expression in Fgf9−/− gonads at 11.5 dpc (Figures 2 and 3D). However, SOX9 expression in XY Fgf9−/− gonads rapidly disappeared (Figure 3E and 3F), and other Sertoli markers were absent or severely reduced (Figure 4A–4F). To investigate the possibility that this loss was due to cell death, we immunostained XY Fgf9−/− gonads at 12.0 dpc—a time point at which SOX9-expressing cells were declining in numbers (Figure 3E)—for active caspase-3, an apoptotic cell marker. Apoptotic cells were not observed in Fgf9−/− gonads or in littermate controls at 12.0 dpc, although Fgf9−/− samples showed somewhat increased cell death in mesonephric tubules and ducts, another site of Fgf9 expression (Figure 4G and 4H). These data suggested that the loss of SOX9 expression in Fgf9−/− XY gonads was not caused by cell death but by the disruption of FGF9/SOX9 feed-forward regulation. To determine whether the aborting of the male pathway in Fgf9−/− Sertoli precursors was associated with the transition of supporting cells from male to female differentiation, we investigated expression of Wnt4, an ovary-promoting gene. At 12.5 dpc Wnt4 was up-regulated in XY Fgf9−/− but not in XY Fgf9+/− gonad controls (Figure 4I–4K). This result suggests that Fgf9 is necessary for the down-regulation of Wnt4 in differentiating XY gonads at/after bipotential stages. + +Fgf9 and Wnt4 Antagonize Each Other + +Our finding that high levels of Wnt4 persist in Fgf9−/− XY gonads implies a genetic antagonism specifically between Fgf9 and Wnt4, as both SRY and SOX9 are initially expressed in Fgf9−/− XY gonads at 11.5 dpc, yet this is not sufficient to down-regulate Wnt4. To test whether exogenous FGF9 could down-regulate expression of Wnt4, we cultured the XX gonad/mesonephros complex with or without FGF9 protein, and examined Wnt4 expression by whole-mount in situ hybridization. Treatment of XX gonads with exogenous FGF9 suppressed the normal expression of Wnt4 (Figure 5A–5C), supporting the hypothesis that Fgf9, rather than Sry or Sox9, functions to down-regulate Wnt4 in wild-type XY gonads. + +We reasoned that if FGF9 and WNT4 do act as opposing signals, then reduction in the dose of Wnt4 might render the XX gonad more susceptible to the male-promoting effects of exogenous FGF9. To test this possibility, XX Wnt4+/− and Wnt4+/+ gonads were cultured in medium with or without FGF9, and were examined for SOX9 expression (Figure 5D–5G). We found that FGF9 induced SOX9 up-regulation in XX Wnt4+/− gonads, but not in Wnt4+/+ XX gonads (Figure 5F and 5G). These results demonstrate antagonism between WNT4 and FGF9 under in vitro gain-of-function conditions. To test antagonism between these factors under loss-of-function conditions in vivo, we investigated whether Fgf9 is derepressed in the absence of Wnt4 (Figure 6A–6C). Using an antibody against FGF9, we found that FGF9 was expressed in Wnt4−/− XX gonads but not in Wnt4+/− XX controls (Figure 6B and 6C). This result suggested that FGF9 is normally down-regulated by WNT4 in XX gonads. + +Given our finding that FGF9, a positive regulator of Sox9, is derepressed in XX Wnt4−/− gonads, we asked whether expression of SOX9 might also occur in XX Wnt4−/− gonads (Figure 6D–6L). An antibody against SOX9 revealed that expression was initially up-regulated in Wnt4−/− XX gonads at 11.5 dpc (Figure 6F and 6I), although it was rapidly down-regulated by 12.0 dpc and absent at 12.5 dpc (Figure 6I and 6L). This finding was confirmed by mRNA in situ hybridization, which also detected Sox9 transcripts in 11.5 dpc Wnt4−/− XX gonads (Figure S1). Wnt4−/− XX gonads do not increase in size comparable to normal XY gonads (Figure 6C, 6F, 6I, and 6L), and Sertoli cell differentiation and testis cord formation do not occur. Nevertheless, it is noteworthy that up-regulation of SOX9 occurs in this case in the absence of Sry, by eliminating the antagonistic effect of Wnt4 and up-regulating FGF9, supporting our hypothesis that sex determination occurs by tipping the balance between these two opposing signals. + +Discussion + +Many studies support the view that cells in the undifferentiated gonad are bipotential; the supporting cell precursor lineage can develop into follicle cells or Sertoli cells. In Fgf9−/− XY gonads, cells initially embark on the Sertoli pathway, but in the absence of Fgf9 can neither maintain Sox9 expression nor establish downstream male pathways. The loss of Sertoli cells in XY Fgf9−/− gonads is not due to cell death, but instead to a transition of supporting cell fate as SOX9 expression is lost. We suggest that in the absence of the antagonizing activity of FGF9, WNT4 signals predominate and govern somatic cell fate in the gonadal field. + +The Drosophila genital disk is also a field of cells that normally follows one of two sexually dimorphic fates. For many years it was believed that the fate of each cell in the genital disk was under the cell-autonomous control of double sex (dsx), the key regulator of the sex determination pathway. However, mosaic studies have shown that the genetic sex of the cells in the anterior/posterior organizers of the disk, not the sex of the majority of cells in the disk, regulate the sexually dimorphic fate of the disk. This occurs through the sex-specific regulation of WNT, FGF, and transforming growth factor beta signaling, which in turn regulate the growth, cell differentiation, and morphogenesis of the disk [41,42]. Sex-specific regulation of gonad organogenesis in vertebrates may occur in a similar manner, where some cells are cell-autonomously responsive to the sex-determining switch; however, the establishment of the male or female program occurs through the non-cell-autonomous activity of classic signaling pathways that act in an antagonistic manner and coordinate growth, cell differentiation and morphogenesis in the gonad. + +The interplay between cell-autonomous and non-cell-autonomous pathways in the mammalian gonad is not well understood. In XX↔XY chimera experiments, XX cells can be recruited to the Sertoli lineage, indicating that non-cell-autonomous signaling mechanisms operate under these conditions [15]. Other more recent studies have suggested that paracrine signals could be involved in the establishment of Sertoli cells [43–46]. The current study reveals that ectopic FGF9 can induce SOX9 under conditions in which XX cells are dissociated (Figure 2F), when an FGF9-coated bead is directly applied to the XX gonad (Figure 2K), or when the dose of Wnt4 is reduced (Figure 5G). Whether FGF9 normally acts non-cell-autonomously in vivo to recruit XY cells to the Sertoli lineage by up-regulating SOX9 is not clear. We show that Sry can initially up-regulate SOX9 in the absence of Fgf9, suggesting that FGF9 is not necessary for this step. However, FGF9 may act to trigger cell proliferation, increasing the number of Sertoli precursors above a threshold needed to stabilize the male pathway, consistent with threshold requirements deduced from earlier studies using XX↔XY chimeric gonads [15]. Since Sertoli cells produce FGF9, loss of proliferation of Sertoli precursors may result in a reduction of the overall level of FGF9, and/or other male paracrine signals, below a critical threshold level required to antagonize the influence of WNT4. This model is appealing, because it links cell proliferation, believed to be required for establishment of the male pathway [9], with Sertoli fate determination. A recent study by Yoshioka et al. [47] showed that misexpression of Fgf9 in chick nephrogenous mesenchyme led to the expansion of gonadal marker gene expression, implicating Fgf9 in gonadal cell proliferation across species. + +It has been suggested that SOX9 represses WNT4 based on misexpression studies [48]. Here we show that the addition of FGF9 protein to XX gonad explant cultures repressed the expression of Wnt4. Down-regulation of Wnt4 is unlikely to occur through SOX9, as SOX9 is not up-regulated in this situation [7]. Furthermore, although both SRY and SOX9 are initially expressed in Fgf9−/− XY gonads, Wnt4 is not down-regulated in the absence of Fgf9 (Figure 4K). These findings support the idea that FGF9 acts as the antagonist of Wnt4. Antagonism of WNT signals may be a multistep process involving both the transcriptional down-regulation of Wnt4 observed in this study and the destabilization of downstream Wnt intracellular pathways that antagonize SOX9 expression, as shown in chondrocyte differentiation [49], or that compete for intracellular signal transducers as has been reported in other systems [50,51]. Future work will address these possibilities. + +In support of the idea that Wnt4 antagonizes the male pathway, we found that the loss of Wnt4 caused the up-regulation of both SOX9 and FGF9 in XX gonads where Sry is absent. It appears that the male pathway can be initiated by disrupting the balance between Wnt4 and Fgf9, a finding that has strong implications for other vertebrate sex-determination systems in which Sry is not the sex determining factor. However, up-regulation of Sox9 is not sufficient to establish testis development in this mutant, as occurs in Odsex and other gain-of-function mutants where Sox9 is misexpressed in the XX gonad [24,25]. In those two misexpression cases, Sox9 expression may have been artificially sustained by exogenous regulatory sequences that bypass the fine dosage balance in this signaling network. + +In Wnt4 mutants, SOX9 expression is not maintained. In light of the observation that the Wnt4−/− XX gonad does not increase significantly in size (Figure 6), it is possible that the FGF9/SOX9-expressing population did not reach a critical threshold. Alternatively (or in addition), another male-specific factor normally dependent on Sry may be required to sustain SOX9 expression, possibly FGF-binding proteins in the extracellular matrix or FGF receptors. It is equally plausible that there are other female-specific factors that antagonize the establishment of SOX9 expression. It has been observed that several other WNTs are expressed in the XX gonad [52], and these or other factors may partially compensate for the loss of Wnt4. + +These findings suggest that WNT4 signaling normally acts as a repressor of the male pathway by interfering with the up-regulation of SOX9 expression. One report of a duplication of the region of human Chromosome 1, which includes WNT4, led to an intersex phenotype [53]. However, the report constitutes only circumstantial evidence. Such a role is not supported by efforts to misexpress Wnt4 in XY gonads, which have led to very mild phenotypes with no evidence for defects in Sertoli cell differentiation [54]. It is possible that WNT4 protein did not function as an active signal in these transgenic mice, either because it was not expressed in the right cells, at the right time, or at the right level. Consistent with our data and the partially sex-reversed phenotype of Wnt4−/− XX mutants, other WNTs or additional female factors may be required. + +The switch that controls sex determination is biologically diverse. Sry is not present in nonmammalian systems; however, antagonistic signaling between FGFs and WNTs may be the conserved mechanism that balances the gonad between testicular and ovarian fates in vertebrates. In theory, any genetic or environmental switch may tip the balance toward the male pathway. Based on our findings we propose that cells in the mammalian gonad are balanced between two competing cell fates by counterbalanced signaling pathways, Fgf9, expressed near the coelomic surface, and Wnt4, expressed near the mesonephric border (Figure 7). In mammalian XY gonads, the onset of Sry expression initiates the male pathway by up-regulating Sox9. SOX9 up-regulates Fgf9, which initiates a Sox9/Fgf9 feed-forward loop that accelerates commitment to the male pathway. In XX gonads or XY mutant gonads lacking Sry, Sox9, or Fgf9, the SOX9/FGF9 feed-forward loop is not established, and WNT4 gains control of the gonadal field. This results in the down-regulation of Sox9 and Fgf9, tilting the balance toward commitment to the female pathway. Further experiments will be required to define the molecular mechanism of FGF9 and WNT4 action. However, our in vivo and in vitro data strongly support the antagonistic relationship of these two signaling pathways in regulating expression of the testis-determining factor SOX9. + +Materials and Methods + +Animals and genotyping. + +The Fgf9 mutation was maintained on a C57BL/6 (B6) background that leads to sex reversal in 100% of XY Fgf9−/− offspring. Sry-EGFP mice, a kind gift from K. Albrecht and E. Eicher, were initially on a mixed B6/129 and were backcrossed to B6 for five generations. Offspring were then crossed to Fgf9+/− and intercrossed and backcrossed to B6 in alternating generations. All XY Fgf9−/− offspring showed complete sex reversal. SryMyc mice were maintained on a CBA background, and Wnt4 on a mixed 129/SVJ background. Mutant embryos were sexed by PCR using Y chromosome-specific primers and were genotyped as described [2,16,26,55]. Mice homozygous for the Sox9 deletion were generated using a germline-specific gene deletion system as described [20]. + +In situ hybridization and immunocytochemistry. + +In situ hybridization was performed on paraformaldehyde-fixed/OCT embedded cryosections, as described [56]. Whole-mount in situ hybridization was performed as previously described [57]. Probes used for in situ hybridization were: Amh [58], Dhh [20], Wnt4 [8], and Fgf9 [59]. Digoxigenin-labeled probes were prepared according to the Boehringer-Mannheim-Roche protocol. + +Antibodies used in whole-mount immunocytochemistry were: mouse monoclonal anti-N-MYC (Cell Signaling Technology, Beverly, Massachusetts, United States; 1:100), rabbit anti-SOX9 (gift of F. Poulat; 1:1,000), rat anti-PECAM (Pharmingen, San Diego, California, United States; 1:500), rabbit anti-caspase-3 fragment (BD Bioscience, San Diego, California, United States; 1:100), and rabbit anti-phosphorylated histone H3 (Cell Signaling; 1:250). Antibody binding was detected using fluorophore-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, Pennsylvania, United States) as recommended. For FGF9 immunostaining, gonads were prepared as frozen sections, and rabbit anti-mouse FGF9 (Cell Science; 1:50) and anti-rabbit IgG conjugated with peroxidase secondary antibody were used, followed by amplification with tyramide-Cy3 fluorophore (Molecular Probes, Eugene, Oregon, United States). This antibody did not detect FGF9 in 12.5 dpc ovary or Fgf9−/− null mutant gonads. Immunostained samples were mounted in DABCO and imaged on a Zeiss LSM420 confocal microscope. + +Primary gonadal cell culture. + +11.5 dpc embryos were collected from CD1 mice, and sex was determined by staining amnions as described [15]. Whole genital ridges were dissected and gonads were separated from mesonephroi. The gonads were treated with collagenase (0.025%) and trypsin (0.025%) in HAT buffer at 37 °C for 10 min. After the digestion, cells were mechanically dissociated by pipetting, washed in DMEM, plated on 10-mm diameter coverslips coated with extracellular matrix (Sigma), and were cultured in DMEM containing 5% fetal bovine serum and 1× antibiotics/antimycotics at 37 °C, 5% CO2. In one of duplicate cultures, FGF9 (R&D Systems, Minneapolis, Minnesota, United States) was added to the final concentration of 50 ng/ml in the culture medium. After 36 h of culture, cells were fixed and immunostained for SOX9. Syto13 (Molecular Probes) was used for nuclear counterstaining, according to the manufacturer's instruction. + +Gonad explant culture. + +Gonad/mesonephros complexes were dissected at 11.5 dpc and cultured on agar blocks for 36 h at 37 °C, 5% CO2 as described [60]. For FGF9 treatment, 50 ng/ml FGF9 (R&D Systems) was added directly to the culture medium, or an FGF9-loaded bead was placed on the surface of gonad. To coat beads, heparin-agarose beads (Sigma, St. Louis, Missouri, United States) were incubated in 50 μg/ml FGF9 for 2 h, and washed five times in culture medium. + +Supporting Information + +Figure S1 + +Confirmation that Transcription of Sox9 Is Up-Regulated in Wnt4−/− XX Gonads + +Whole-mount in situ hybridization for Sox9 detected Sox9 expression in 11.5 dpc Wnt4+/− XY controls and Wnt4−/− XX gonads. + +(428 KB PPT) + +Click here for additional data file. + +Acknowledgements + +The Sry-EGFP transgenics were a kind gift from Eva M. Eicher. We thank Serge Nef for freely providing information from his microarray screen, Hao Chang for generating some of the Sox9 mutant and control urogenital ridges, members of the Capel lab for discussion, and Iordan Batchvarov for help with animals. + +Competing interests. The authors have declared that no competing interests exist. + +Abbreviations + +dpc - day post coitum + +EGFP - enhanced green fluorescent protein + +FGF - fibroblast growth factor + +PECAM - platelet/endothelial cell adhesion molecule + +SOX - SRY-like HMG box + +SRY - sex-determining region of the Y + +WNT - wingless-related MMTV integration site + +Figures and Tables + +Figure 1 + +Stage- and Cell-Specific Expression of FGF9 in Embryonic Gonads + +(A–F) Detection of FGF9 protein (red) at different stages of gonad development. FGF9 is up-regulated in XY gonads at 11.5 (B), 12.5 (D), and 13.5 dpc (F) while it is down-regulated in XX after 11.5 dpc (A, C, and E). No signal was detected in XY Fgf9−/− gonads (unpublished data). + +(G–J) Serial sections of wild-type XX and compound heterozygous KitW/Wv XY gonads stained for alkaline phosphatase (purple; G and I) and FGF9 (red; H and J). Testis cords are formed in the absence of germ cells in XY KitW/Wv mutant gonads at 12.5 dpc (arrowhead in J). Expression of FGF9 is present in the mutant gonads where Sertoli cells are the only remaining cell type in the cords (J). Semitransparent dotted line indicates the boundary between gonad and mesonephroi. PECAM (green) marks germ cells and vascular endothelial cells (C–F, H, and J). The scale bars represent 25 μm. + +g, gonad; m, mesonephroi. + +Figure 2 + +Epistatic Relationship of Sry, Fgf9, and Sox9 + +(A–D) Sry expression is not dependent on Fgf9. Fgf9+/− (A) and Fgf9−/− (B) XY gonads at 11.5 dpc expressing GFP (green) from the Sry promoter (polygonal cells, arrows). Blood cells show background fluorescence (doughnut-shaped cells). Fgf9+/− (C) and Fgf9−/− (D) XY gonads at 11.5 dpc expressing SRYMYC protein (red, arrowheads). Inset shows nuclear counterstain (green, Syto13) colocalizing with SRYMYC. PECAM (blue) marks endothelial and germ cells. Scale bars represent 25 μm. + +(E–K) Exogenous FGF9 can up-regulate SOX9 expression in XX gonads. Immunostaining of SOX9 (green) in primary cultures of gonadal cells. XX cells (E) and XY cells (G) cultured with exogenous FGF9 show induction of SOX9 expression (F and H, respectively). Cells were counterstained using the nuclear marker, Syto13 (red). Immunostaining of SOX9 (red) in gonad explants cultured with BSA- or FGF9-coated beads. SOX9 is expressed in XY gonads and cells contacting FGF9-coated beads (dotted circle labeled “F”) in XX gonads (I and K) but not in XX cells contacting BSA-coated control beads (“B”) (J). PECAM (blue) marks endothelial and germ cells. Scale bars (I–K) represent 50 μm. + +Figure 3 + +Interdependent Relationship between Fgf9 and Sox9 + +(A–F) Immunostaining of SOX9 (red) in Fgf9+/− and Fgf9−/− XY gonads shows that Fgf9 is required for maintenance of SOX9. The up-regulation of SOX9 in Sertoli precursor cells appears normal in Fgf9−/− gonads at 11.5 dpc (D) compared with heterozygous littermate controls (A). However, SOX9 is detected in fewer cells in mutant gonads at 12.0 dpc (B and E), and is lost by 12.5 dpc (C and F). + +(G–J) mRNA whole-mount in situ hybridization for Sry and Fgf9 in Sox9flox/Δ and Sox9Δ/Δ XY gonads shows that Sox9 is required for Fgf9 expression. Sry expression is detected in both Sox9flox/Δ and Sox9Δ/Δ gonads at 11.5 dpc (G and H), whereas Fgf9 expression is markedly decreased or absent in Sox9Δ/Δ gonads at 11.5 dpc (I and J). + +(K–O) Comparison of cell proliferation in Sox9Δ/Δ versus Sox9flox/Δ gonads at 11.5 dpc using immunostaining for phosphorylated histone H3. XY-specific proliferation at the gonad surface (K) is reduced in the absence of Sox9 (L). Bar graph (O) shows quantitation of proliferation obtained by counting positive cells in the cortical region of each gonad (right brace) and normalizing to the number obtained from XY Sox9flox/Δ gonads. n = 30, with five sections of each gonad and three pairs of gonads for each genotype. PECAM, green (A–F and K–N). The scale bars represent 25 μm. + +Figure 4 + +Sertoli Cell Precursors Switch from Expression of Male to Female Pathway Genes + +(A–F) Whole-mount in situ hybridization for genes in the male pathway downstream of Sox9, Dhh, and Amh. Dhh expression is disrupted in XY Fgf9−/− gonads (g) at 11.5 dpc (A and B). Amh expression is severely reduced in XY Fgf9−/− gonads at 12.5 dpc (E and F). + +(G and H) Analysis of cell death in Fgf9−/− XY gonads using an apoptotic marker, active caspase-3 (red). No increased apoptosis is observed in XY Fgf9−/− gonads (g) compared with control XY gonads, although apoptotic cells are increased around mesonephric tubules (m) of the mutant gonads (arrow in H). Semitransparent dotted line indicates boundary between mesonephros and gonad. (PECAM, green). + +(I–K) Whole-mount in situ hybridization for Wnt4, an ovary marker. Wnt4 is expressed in Fgf9−/− XY gonads at 12.5 dpc (K) similar to the level in XX Fgf9+/− controls (I) but not in XY controls (J). The scale bars represent 50 μm. + +g, gonad; m, mesonephros. + +Figure 5 + +Mutual Antagonism between Fgf9 and Wnt4 + +(A–C) Wnt4 whole-mount in situ hybridization on gonad cultures. Adding exogenous FGF9 in gonad cultures results in the down-regulation of Wnt4 expression in cultured XX gonads (C). Controls (A and B) were cultured without FGF9 peptide. + +(D–G) Reduction in the dose of Wnt4 allows FGF9 to induce SOX9 in XX gonads. Immunostaining of SOX9 (red) shows that addition of FGF9 up-regulates SOX9 expression in heterozygous Wnt4+/− XX gonads (G), but not in Wnt4+/+ XX gonads (F). PECAM, green. The scale bars represent 50 μm. + +Figure 6 + +Ectopic Expression of Male Factors, SOX9 and FGF9, in XX Wnt4−/− Gonads + +(A–C) FGF9 (red) immunostaining shows that FGF9 is expressed in XX Wnt4−/− gonads at 12.5 dpc (C) relative to littermate controls (A and B). + +(D–L) SOX9 (red) immunostaining shows that SOX9 is transiently up-regulated in XX Wnt4−/− gonads. SOX9 expression is detected in Wnt4−/− XX gonads at 11.5–12.0 dpc (F and I), albeit at reduced levels compared with XY gonad controls (E-H). SOX9 is not detected in control XX Wnt4+/− gonads (D-J) or Wnt4−/− XX gonads at 12.5 dpc (L). The ectopic coelomic vessel in XX Wnt4−/− gonads [28] is indicated by arrowheads. PECAM, green. The scale bars represent 50 μm. + +Figure 7 + +Opposing Signals Regulate Sex Determination in the Bipotential Gonad + +In both XX and XY gonads at 11.25 dpc (15 tail-somite stage), Fgf9 transcripts (white arrows) are detected near the gonad surface (A and B), whereas Wnt4 transcripts are detected near the gonad mesonephric boundary (C and D). We propose a model in which the fate of the gonad is balanced between these competing signals. A genetic or environmental switch initiates the male pathway by creating an imbalance between these signals. In mammals, this imbalance occurs through the up-regulation of Sox9. Sox9 up-regulates Fgf9, and Fgf9 maintains Sox9, forming a positive feed-forward loop in XY gonads. In this situation, the balance between FGF9 and WNT4 signals is shifted in favor of FGF9, and the dominance of the male pathway is established. In the absence of a feed-forward loop between SOX9 and FGF9 (e.g., in XX gonads), WNT4 blocks Fgf9, initiating the female pathway. + +Footnotes + +Author contributions. YK, AK, and BC conceived and designed the experiments. YK, AK, LD, and JB performed the experiments. YK, RLB, and BC analyzed the data. YK, RS, MCC, FP, RRB, RLB, and BC contributed reagents/materials/analysis tools. YK and BC wrote the paper with significant input from RLB. + +Citation: Kim Y, Kobayashi A, Sekido R, DiNapoli L, Brennan J, et al. (2006) Fgf9 and Wnt4 act as antagonistic signals to regulate mammalian sex determination. PLoS Biol 4(6): e187. DOI: 10.1371/journal.pbio.0040187 + +Funding. This work was funded by grants from the National Institutes of Health to BC (HD39963), and to RRB (HD30284). diff --git a/src/ontogpt/evaluation/craft/database/all/16787536.ann b/src/ontogpt/evaluation/craft/database/all/16787536.ann new file mode 100644 index 000000000..68cdc0759 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16787536.ann @@ -0,0 +1,1022 @@ +T1 PR:000006043 23 30 cubilin +T2 UBERON:0000925 102 110 endoderm +T3 UBERON:0002329 118 124 somite +T4 PR:000006043 158 165 Cubilin +T5 GO:0019898 171 190 peripheral membrane +T6 GO:0016021 223 240 integral membrane +T7 PR:000009921 250 257 megalin +T8 UBERON:0000305 262 268 amnion +T9 PR:000003996 262 272 amnionless +T10 GO:0006897 291 302 endocytosis +T11 UBERON:0000483 317 326 epithelia +T12 UBERON:0008945 339 353;363 371 extraembryonic ... endoderm +T13 UBERON:0004877 354 371 visceral endoderm +T14 UBERON:0004877 373 375 VE +T15 SO:0000704 422 429 genetic +T16 PR:000006043 442 449 cubilin +T17 NCBITaxon:10088 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UBERON:0002116 1924 1929 ileum +T67 UBERON:0003257 1934 1942;1967 1975 yolk sac ... endoderm +T68 UBERON:0008945 1943 1957;1967 1975 extraembryonic ... endoderm +T69 UBERON:0004877 1958 1975 visceral endoderm +T70 UBERON:0004877 1977 1979 VE +T71 PR:000006043 2018 2025 cubilin +T72 CHEBI:30411 2067 2078 vitamin B12 +T73 CHEBI:30411 2079 2088 cobalamin +T74 CHEBI:30411 2090 2093 Cbl +T75 UBERON:0000160 2127 2137 intestinal +T76 GO:0050892 2127 2148 intestinal absorption +T77 CHEBI:30411 2152 2155 Cbl +T78 PR:000006043 2163 2170 Cubilin +T79 GO:0006897 2196 2205 endocytic +T80 CHEBI:39015 2219 2233 apolipoprotein +T81 PR:000004140 2219 2237 apolipoprotein A-I +T82 PR:000004140 2239 2245 apoA-I +T83 UBERON:0002113 2310 2316 kidney +T84 UBERON:0004877 2321 2323 VE +T85 PR:000006043 2337 2344 cubilin +T86 PR:000016261 2370 2381 transferrin +T87 GO:0019814 2383 2397 immunoglobulin +T88 CHEBI:27300 2412 2421 vitamin D +T89 PR:000007883 2412 2437 vitamin D-binding protein +T90 PR:000009771 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UBERON:0003061 12519 12531 blood island +T346 UBERON:0000922 12618 12625 embryos +T347 UBERON:0000922 12732 12739 embryos +T348 UBERON:0000922 12765 12772 embryos +T349 UBERON:0000948 12796 12801 heart +T350 UBERON:0009746 12813 12823 head folds +T351 UBERON:0010190 12861 12881 paired dorsal aortae +T352 UBERON:0002329 12911 12918 somites +T353 UBERON:0000922 12931 12938 embryos +T354 UBERON:0000922 13028 13035 embryos +T355 UBERON:0002329 13054 13061 somites +T356 PR:000006043 13126 13133 cubilin +T357 UBERON:0000922 13290 13296 embryo +T358 UBERON:0002329 13328 13335 somites +T359 UBERON:0002329 13358 13365 somites +T360 UBERON:0003077 13430 13447 paraxial mesoderm +T361 PR:000006043 13523 13530 Cubilin +T362 UBERON:0000922 13549 13556 embryos +T363 UBERON:0002329 13595 13602 somites +T364 UBERON:0000922 13632 13638 embryo +T365 UBERON:0005062 13666 13668 NF +T366 UBERON:0005062 13670 13682 neural folds +T367 UBERON:0004340 13684 13685 A +T368 UBERON:0004340 13687 13696 allantois +T369 UBERON:0000948 13698 13699 H +T370 UBERON:0000948 13701 13706 heart +T371 UBERON:0001040 13708 13710 YS +T372 UBERON:0001040 13712 13720 yolk sac +T373 UBERON:0000922 13778 13785 embryos +T374 UBERON:0000922 14014 14021 embryos +T375 UBERON:0001040 14099 14107 yolk sac +T376 UBERON:0001981 14108 14121 blood vessels +T377 UBERON:0001981 14170 14182 blood vessel +T378 GO:0001568 14170 14192 blood vessel formation +T379 UBERON:0000922 14194 14201 embryos +T380 GO:0007618 14220 14227 matings +T381 PR:000001904 14257 14264 PECAM-1 +T382 UBERON:0001040 14289 14297 yolk sac +T383 UBERON:0001981 14298 14310 blood vessel +T384 GO:0001568 14298 14320 blood vessel formation +T385 UBERON:0001040 14430 14438 yolk sac +T386 UBERON:0000055 14439 14446 vessels +T387 UBERON:0000922 14464 14470 embryo +T388 UBERON:0000055 14521 14528 vessels +T389 UBERON:0001040 14575 14583 yolk sac +T390 UBERON:0001981 14680 14693 blood vessels +T391 UBERON:0000922 14760 14766 embryo +T392 UBERON:0001040 14767 14775 yolk sac +T393 UBERON:0000922 14803 14812 embryonic +T394 UBERON:0001040 14813 14821 yolk sac +T395 PR:000001904 14823 14830 PECAM-1 +T396 UBERON:0003909 14862 14872 sinusoidal +T397 UBERON:0000055 14878 14885 vessels +T398 UBERON:0003061 14943 14955 blood island +T399 CHEBI:51686 14984 14985 H +T400 UBERON:0001040 15026 15034 Yolk sac +T401 UBERON:0001981 15035 15048 blood vessels +T402 PR:000006043 15060 15067 cubilin +T403 PR:000001904 15109 15116 PECAM-1 +T404 UBERON:0002049 15125 15136 vasculature +T405 UBERON:0000922 15161 15167 embryo +T406 UBERON:0000922 15198 15204 embryo +T407 UBERON:0000922 15260 15266 embryo +T408 UBERON:0000922 15295 15301 embryo +T409 UBERON:0001040 15379 15387 yolk sac +T410 UBERON:0002049 15388 15399 vasculature +T411 UBERON:0001040 15445 15453 yolk sac +T412 UBERON:0001040 15455 15457 YS +T413 UBERON:0001040 15459 15467 yolk sac +T414 UBERON:0004340 15469 15470 A +T415 UBERON:0004340 15472 15481 allantois +T416 UBERON:0005062 15483 15485 NF +T417 UBERON:0005062 15487 15499 neural folds +T418 UBERON:0000948 15501 15502 H +T419 UBERON:0000948 15504 15509 heart +T420 PR:000001904 15573 15580 PECAM-1 +T421 UBERON:0001040 15589 15597 yolk sac +T422 UBERON:0002049 15598 15609 vasculature +T423 UBERON:0000922 15664 15671 embryos +T424 UBERON:0001981 15702 15715 blood vessels +T425 UBERON:0000922 15730 15737 embryos +T426 UBERON:0000922 15790 15797 embryos +T427 UBERON:0001040 15822 15830 yolk sac +T428 UBERON:0001981 15831 15844 blood vessels +T429 UBERON:0001040 15956 15964 yolk sac +T430 UBERON:0004340 16000 16009 allantoic +T431 UBERON:0002049 16010 16021 vasculature +T432 UBERON:0001981 16086 16098 blood vessel +T433 UBERON:0000922 16130 16136 embryo +T434 UBERON:0010260 16147 16170 allantoic blood vessels +T435 GO:0009653 16324 16337 morphogenesis +T436 UBERON:0004340 16345 16354 allantoic +T437 UBERON:0002049 16355 16366 vasculature +T438 UBERON:0005335 16417 16432 chorioallantoic +T439 UBERON:0001987 16433 16442 placental +T440 UBERON:0000922 16469 16478 embryonic +T441 UBERON:0004877 16483 16500 visceral endoderm +T442 PR:000006043 16504 16511 cubilin +T443 PR:000006043 16538 16545 cubilin +T444 GO:0045177 16573 16579 apical +T445 GO:0009986 16580 16587 surface +T446 UBERON:0004877 16604 16606 VE +T447 CL:0000076 16669 16683 squamous cells +T448 UBERON:0005439 16695 16713 embryonic endoderm +T449 UBERON:0005439 16715 16717 EE +T450 PR:000006043 16776 16783 cubilin +T451 UBERON:0005439 16804 16824 embryonic endodermal +T452 UBERON:0000479 16825 16832 tissues +T453 UBERON:0000922 16870 16877 embryos +T454 UBERON:0000925 16899 16909 endodermal +T455 UBERON:0005439 16960 16979 definitive endoderm +T456 UBERON:0000922 17002 17009 embryos +T457 UBERON:0007603 17061 17091 stratified cuboidal epithelium +T458 UBERON:0000485 17120 17146 simple columnar epithelium +T459 UBERON:0000483 17196 17205 epithelia +T460 GO:0045177 17210 17216 apical +T461 GO:0042995 17217 17226 processes +T462 GO:0005902 17246 17256 microvilli +T463 GO:0005929 17260 17265 cilia +T464 GO:0009986 17280 17287 surface +T465 UBERON:0005439 17325 17327 EE +T466 PR:000006043 17373 17380 cubilin +T467 GO:0060429 17394 17406;17410 17420 formation of ... epithelium +T468 UBERON:0000483 17410 17420 epithelium +T469 UBERON:0005439 17468 17487 definitive endoderm +T470 UBERON:0005439 17625 17644 definitive endoderm +T471 PR:000006043 17657 17664 Cubilin +T472 UBERON:0000483 17698 17708 epithelial +T473 UBERON:0005439 17723 17741 embryonic endoderm +T474 CHEBI:51686 17753 17754 H +T475 UBERON:0000922 17834 17841 embryos +T476 UBERON:0000922 17843 17850 Embryos +T477 UBERON:0003061 17947 17949 BI +T478 UBERON:0003061 17951 17963 blood island +T479 UBERON:0002329 17965 17966 S +T480 UBERON:0002329 17968 17974 somite +T481 UBERON:0005439 17976 17978 EE +T482 UBERON:0005439 17980 18009 definitive embryonic endoderm +T483 UBERON:0004877 18011 18013 VE +T484 UBERON:0001040 18015 18023 yolk sac +T485 UBERON:0004877 18024 18041 visceral endoderm +T486 UBERON:0000483 18071 18081 epithelium +T487 UBERON:0005439 18105 18107 EE +T488 UBERON:0004877 18151 18153 VE +T489 UBERON:0000922 18178 18185 embryos +T490 UBERON:0004877 18270 18272 VE +T491 UBERON:0000922 18283 18290 embryos +T492 UBERON:0004877 18296 18298 VE +T493 PR:000006043 18302 18309 cubilin +T494 UBERON:0000486 18324 18345 stratified epithelium +T495 UBERON:0004877 18409 18411 VE +T496 GO:0045177 18440 18446 apical +T497 GO:0005773 18447 18455 vacuoles +T498 UBERON:0004877 18483 18485 VE +T499 CL:0000223 18559 18567;18585 18593 cells of ... endoderm +T500 UBERON:0005439 18575 18593 embryonic endoderm +T501 UBERON:0005439 18664 18666 EE +T502 UBERON:0004877 18671 18673 VE +T503 UBERON:0000483 18674 18683 epithelia +T504 UBERON:0004877 18726 18728 VE +T505 UBERON:0000483 18736 18746 epithelium +T506 UBERON:0005439 18770 18772 EE +T507 GO:0010467 18834 18844 expression +T508 SO:0005853 18860 18868 cassette +T509 PR:000006043 18887 18894 cubilin +T510 SO:0000704 18895 18899 gene +T511 PR:000009921 18905 18912 megalin +T512 GO:0010467 18913 18923 expression +T513 UBERON:0000922 18942 18949 embryos +T514 UBERON:0004877 19024 19026 VE +T515 UBERON:0005439 19040 19042 EE +T516 PR:000009921 19119 19126 megalin +T517 GO:0010467 19145 19154 expressed +T518 GO:0045177 19162 19168 apical +T519 GO:0009986 19169 19177 surfaces +T520 UBERON:0004877 19192 19194 VE +T521 PR:000006043 19210 19217 cubilin +T522 GO:0009986 19244 19256 cell surface +T523 PR:000009921 19272 19279 megalin +T524 PR:000009921 19322 19329 megalin +T525 GO:0010467 19330 19340 expression +T526 UBERON:0003257 19344 19352;19362 19370 yolk sac ... endoderm +T527 UBERON:0004877 19353 19370 visceral endoderm +T528 UBERON:0005439 19362 19373;19381 19388 endoderm of ... embryos +T529 PR:000006043 19394 19401 cubilin +T530 SO:0000704 19402 19406 gene +T531 PR:000006043 19466 19473 cubilin +T532 GO:0010467 19480 19490 expression +T533 CHEBI:51686 19509 19510 H +T534 UBERON:0000922 19553 19559 embryo +T535 UBERON:0003061 19667 19680 blood islands +T536 GO:0010467 19685 19695 expressing +T537 PR:000009921 19736 19743 megalin +T538 UBERON:0000922 19767 19773 embryo +T539 UBERON:0000922 19775 19782 Embryos +T540 UBERON:0000922 19879 19885 embryo +T541 UBERON:0004877 19957 19959 VE +T542 UBERON:0003257 19961 19969;19979 19987 yolk sac ... endoderm +T543 UBERON:0004877 19970 19987 visceral endoderm +T544 UBERON:0003061 19989 19991 BI +T545 UBERON:0003061 19993 20005 blood island +T546 UBERON:0000305 20007 20009 Am +T547 UBERON:0000305 20011 20017 amnion +T548 UBERON:0004877 20059 20061 VE +T549 UBERON:0005439 20075 20077 EE +T550 UBERON:0004877 20084 20101 visceral endoderm +T551 UBERON:0005439 20093 20104;20116 20123 endoderm of ... embryos +T552 UBERON:0004877 20194 20196 VE +T553 PR:000006043 20286 20293 cubilin +T554 UBERON:0004877 20304 20306 VE +T555 UBERON:0000922 20403 20410 embryos +T556 CHEBI:52029 20416 20419 DiI +T557 CHEBI:52029 20454 20457 DiI +T558 UBERON:0004877 20474 20476 VE +T559 CHEBI:52029 20484 20487 DiI +T560 UBERON:0000922 20503 20510 embryos +T561 UBERON:0004877 20538 20540 VE +T562 UBERON:0003257 20559 20578 yolk sac endodermal +T563 GO:0010467 20589 20599 expressing +T564 PR:000006043 20600 20607 cubilin +T565 GO:0010467 20624 20634 expression +T566 PR:000006043 20642 20649 cubilin +T567 SO:0000704 20650 20654 gene +T568 CHEBI:52029 20708 20711 DiI +T569 UBERON:0004877 20728 20730 VE +T570 UBERON:0000922 20745 20752 embryos +T571 UBERON:0004877 20795 20797 VE +T572 PR:000006043 20817 20824 cubilin +T573 UBERON:0003257 20901 20909;20919 20927 yolk sac ... endoderm +T574 UBERON:0004877 20910 20927 visceral endoderm +T575 UBERON:0005439 20919 20930;20949 20956 endoderm of ... embryos +T576 PR:000006043 20931 20938 cubilin +T577 UBERON:0000922 21015 21021 embryo +T578 CHEBI:52029 21084 21087 DiI +T579 UBERON:0004877 21147 21149 VE +T580 UBERON:0000922 21171 21178 embryos +T581 UBERON:0004877 21186 21188 VE +T582 UBERON:0004877 21190 21207 visceral endoderm +T583 UBERON:0005439 21209 21211 EE +T584 UBERON:0005439 21213 21231 embryonic endoderm +T585 PR:000006043 21263 21270 cubilin +T586 GO:0032991 21279 21286 complex +T587 PR:000003996 21292 21295 AMN +T588 GO:0032991 21315 21322 complex +T589 PR:000003996 21330 21333 AMN +T590 PR:000006043 21338 21345 cubilin +T591 GO:0010467 21353 21362 expressed +T592 UBERON:0000483 21377 21387 epithelial +T593 UBERON:0004877 21402 21404 VE +T594 PR:000003996 21420 21423 AMN +T595 PR:000006043 21462 21469 cubilin +T596 GO:0016020 21486 21494 membrane +T597 GO:0045177 21508 21514 apical +T598 PR:000003996 21576 21579 AMN +T599 PR:000006043 21633 21640 cubilin +T600 PR:000006043 21667 21674 cubilin +T601 UBERON:0000922 21680 21687 embryos +T602 PR:000003996 21689 21692 AMN +T603 UBERON:0000922 21703 21710 embryos +T604 UBERON:0000922 21815 21821 embryo +T605 SO:0000704 21848 21855 genetic +T606 UBERON:0000305 21889 21895 amnion +T607 UBERON:0000926 21905 21913 mesoderm +T608 GO:0007498 21905 21923 mesoderm formation +T609 PR:000006043 21931 21938 cubilin +T610 PR:000003996 21943 21946 AMN +T611 UBERON:0000922 21954 21961 embryos +T612 UBERON:0004340 22006 22015 allantois +T613 UBERON:0003061 22017 22030 blood islands +T614 UBERON:0001040 22032 22040 yolk sac +T615 UBERON:0001981 22041 22054 blood vessels +T616 UBERON:0000948 22061 22066 heart +T617 UBERON:0002329 22103 22110 somites +T618 UBERON:0003077 22190 22207 paraxial mesoderm +T619 UBERON:0000925 22282 22290 endoderm +T620 PR:000006043 22332 22339 cubilin +T621 UBERON:0004877 22350 22352 VE +T622 PR:000003996 22411 22414 AMN +T623 UBERON:0004877 22425 22427 VE +T624 UBERON:0005439 22432 22434 EE +T625 UBERON:0002532 22456 22464 epiblast +T626 UBERON:0000922 22489 22496 embryos +T627 UBERON:0004877 22538 22540 VE +T628 UBERON:0005439 22545 22564 definitive endoderm +T629 PR:000006043 22581 22588 cubilin +T630 PR:000006043 22630 22637 cubilin +T631 GO:0060429 22641 22650;22670 22672;22679 22688 formation ... of ... epithelia +T632 UBERON:0000483 22679 22688 epithelia +T633 PR:000003996 22761 22764 AMN +T634 PR:000006043 22821 22828 cubilin +T635 PR:000003996 22833 22836 AMN +T636 UBERON:0005439 22903 22922 definitive endoderm +T637 GO:0007369 22943 22955 gastrulation +T638 UBERON:0002532 22978 22986 epiblast +T639 CL:0000352 22978 22992 epiblast cells +T640 UBERON:0004341 23005 23021 primitive streak +T641 NCBITaxon:10088 23078 23083 mouse +T642 PR:000003996 23084 23087 amn +T643 PR:000006043 23092 23099 cubilin +T644 SO:0000704 23131 23136 genes +T645 UBERON:0004877 23163 23165 VE +T646 UBERON:0000922 23177 23186 embryonic +T647 GO:0065007 23201 23208 control +T648 GO:0048339 23213 23243 formation of paraxial mesoderm +T649 UBERON:0003077 23226 23243 paraxial mesoderm +T650 PR:000003996 23249 23252 AMN +T651 PR:000006043 23257 23264 cubilin +T652 GO:0006897 23333 23342 endocytic +T653 GO:0032991 23365 23372 complex +T654 GO:0045177 23381 23387 apical +T655 GO:0010467 23388 23398 expression +T656 UBERON:0004877 23402 23404 VE +T657 CHEBI:33284 23483 23492 nutrients +T658 CHEBI:33284 23537 23546 nutrients +T659 UBERON:0004877 23570 23572 VE +T660 PR:000006043 23655 23662 cubilin +T661 PR:000016261 23687 23698 transferrin +T662 GO:0019814 23700 23714 immunoglobulin +T663 CHEBI:27300 23729 23738 vitamin D +T664 PR:000007883 23729 23754 vitamin D-binding protein +T665 PR:000009771 23767 23777 galectin-3 +T666 CL:0000158 23779 23789 Clara cell +T667 PR:000014505 23779 23807 Clara cell secretory protein +T668 GO:0046903 23790 23799 secretory +T669 PR:000004140 23809 23815 apoA-I +T670 PR:000006043 23855 23862 cubilin +T671 GO:0032991 23881 23888 complex +T672 CHEBI:39015 23919 23934 apolipoproteins +T673 CHEBI:16247 23936 23949 phospholipids +T674 CHEBI:16113 23951 23962 cholesterol +T675 CHEBI:24020 23967 23989 lipid soluble vitamins +T676 CHEBI:50211 23998 24005 retinol +T677 CHEBI:26536 24029 24042 retinoic acid +T678 CHEBI:15035 24065 24078 retinaldehyde +T679 PR:000003921 24065 24094 retinaldehyde dehydrogenase-2 +T680 PR:000003921 24096 24102 Raldh2 +T681 SO:0000704 24113 24120 genetic +T682 NCBITaxon:10088 24135 24140 mouse +T683 PR:000003921 24141 24147 Raldh2 +T684 UBERON:0019248 24157 24169 early embryo +T685 UBERON:0002329 24217 24224 somites +T686 UBERON:0001040 24226 24234 yolk sac +T687 UBERON:0000948 24262 24267 heart +T688 UBERON:0000922 24285 24292 embryos +T689 PR:000006043 24343 24350 cubilin +T690 PR:000003996 24355 24358 amn +T691 PR:000006043 24439 24446 cubilin +T692 UBERON:0000305 24451 24457 amnion +T693 PR:000003996 24451 24461 amnionless +T694 PR:000009921 24469 24476 megalin +T695 NCBITaxon:10088 24487 24491 mice +T696 GO:0016265 24492 24495 die +T697 UBERON:0012101 24496 24507 perinatally +T698 GO:0007567 24500 24507 natally +T699 GO:0048853 24529 24545;24550 24559 morphogenesis of ... forebrain +T700 GO:0060425 24529 24545;24587 24591 morphogenesis of ... lung +T701 GO:0060993 24529 24545;24596 24602 morphogenesis of ... kidney +T702 UBERON:0001890 24550 24559 forebrain +T703 http://purl.obolibrary.org/obo/MONDO_0016296 24567 24584 holoprosencephaly +T704 UBERON:0002048 24587 24591 lung +T705 UBERON:0002113 24596 24602 kidney +T706 PR:000009921 24641 24648 megalin +T707 PR:000006043 24673 24680 cubilin +T708 GO:0010467 24699 24708 expressed +T709 PR:000006043 24714 24721 cubilin +T710 UBERON:0019248 24797 24809 early embryo +T711 NCBITaxon:10088 24849 24853 mice +T712 PR:000006259 24867 24877 disabled-2 +T713 PR:000006259 24879 24883 dab2 +T714 PR:000009921 24900 24907 megalin +T715 PR:000006043 24958 24965 cubilin +T716 UBERON:0004877 25005 25007 VE +T717 GO:0045177 25020 25026 apical +T718 GO:0031982 25027 25035 vesicles +T719 PR:000006259 25051 25055 dab2 +T720 PR:000006043 25105 25112 cubilin +T721 GO:0007369 25138 25150 gastrulation +T722 PR:000006259 25152 25156 Dab2 +T723 PR:000006043 25196 25203 cubilin +T724 GO:0006897 25204 25215 endocytosis +T725 UBERON:0004877 25219 25221 VE +T726 PR:000006259 25253 25257 dab2 +T727 UBERON:0000922 25263 25270 embryos +T728 GO:0006897 25306 25315 endocytic +T729 GO:0010467 25326 25335 expressed +T730 UBERON:0004877 25343 25345 VE +T731 PR:000009921 25356 25363 megalin +T732 PR:000006043 25365 25372 cubilin +T733 UBERON:0000305 25385 25391 amnion +T734 PR:000003996 25385 25395 amnionless +T735 PR:000006259 25413 25417 dab2 +T736 CHEBI:16113 25483 25494 cholesterol +T737 PR:000006043 25563 25570 cubilin +T738 PR:000006043 25599 25606 cubilin +T739 UBERON:0004877 25618 25620 VE +T740 CHEBI:47775 25649 25675 HDL-associated cholesterol +T741 PR:000004140 25680 25686 apoA-I +T742 NCBITaxon:10088 25712 25717 mouse +T743 UBERON:0000922 25718 25725 embryos +T744 UBERON:0000922 25811 25820 embryonic +T745 PR:000006043 25870 25877 cubilin +T746 UBERON:0004877 25903 25905 VE +T747 UBERON:0004877 25931 25933 VE +T748 GO:0010467 25934 25943 expresses +T749 PR:000004140 25944 25950 apoA-I +T750 PR:000004145 25982 25986 apoB +T751 CHEBI:16113 26011 26022 cholesterol +T752 UBERON:0004877 26079 26081 VE +T753 PR:000006043 26092 26099 cubilin +T754 UBERON:0000922 26182 26188 embryo +T755 GO:0005791 26290 26317 rough endoplasmic reticulum +T756 GO:0046903 26322 26331 secretory +T757 GO:0099503 26322 26340 secretory vesicles +T758 NCBITaxon:10088 26356 26361 mouse +T759 UBERON:0001040 26362 26370 yolk sac +T760 UBERON:0000925 26421 26429 endoderm +T761 PR:000006043 26459 26466 cubilin +T762 PR:000003996 26471 26474 AMN +T763 NCBITaxon:10088 26489 26493 mice +T764 UBERON:0000922 26503 26512 embryonic +T765 NCBITaxon:9606 26537 26542 human +T766 NCBITaxon:9608 26547 26553 canine +T767 PR:000003996 26554 26557 AMN +T768 GO:0045177 26578 26584 apical +T769 CHEBI:30411 26637 26648 vitamin B12 +T770 GO:0032991 26663 26670 complex +T771 UBERON:0000922 26703 26712 embryonic +T772 GO:0009790 26703 26724 embryonic development +T773 NCBITaxon:species 26806 26813 species +T774 UBERON:0001040 26829 26837 yolk sac +T775 NCBITaxon:9989 26850 26857 rodents +T776 NCBITaxon:9615 26859 26863 dogs +T777 NCBITaxon:9606 26868 26874 humans +T778 SO:0000147 26981 26986 exons +T779 NCBITaxon:9606 26994 26999 human +T780 PR:000003996 27000 27003 AMN +T781 SO:0000673 27084 27095 transcripts +T782 SO:0000315 27140 27167 transcriptional start sites +T783 GO:0006413 27184 27206 translation initiation +T784 SO:0000323 27184 27212 translation initiation sites +T785 PR:000003996 27230 27233 AMN +T786 GO:0006415 27263 27289 termination of translation +T787 GO:0010467 27315 27322 express +T788 PR:000003996 27338 27341 AMN +T789 PR:000003996 27440 27443 AMN +T790 PR:000000010 27475 27482 chordin +T791 SO:0000417 27488 27494 module +T792 GO:0016020 27501 27509 membrane +T793 SO:0000417 27510 27516 domain +T794 GO:0005737 27521 27532 cytoplasmic +T795 SO:0000417 27533 27539 domain +T796 CHEBI:30411 27711 27722 vitamin B12 +T797 UBERON:0000922 27787 27796 embryonic +T798 GO:0009790 27787 27808 embryonic development +T799 PR:000006043 27884 27891 cubilin +T800 PR:000006043 27994 28001 cubilin +T801 GO:0045177 28009 28015 apical +T802 GO:0009986 28016 28027;28039 28044 surfaces of ... cells +T803 UBERON:0000483 28028 28038 epithelial +T804 CL:0000066 28028 28044 epithelial cells +T805 NCBITaxon:9608 28072 28078 canine +T806 PR:000003996 28079 28082 AMN +T807 SO:0000704 28083 28087 gene +T808 PR:000006043 28101 28108 cubilin +T809 GO:0005622 28109 28122 intracellular +T810 GO:0046907 28109 28134 intracellular trafficking +T811 UBERON:0000922 28162 28171 embryonic +T812 GO:0009790 28162 28183 embryonic development +T813 NCBITaxon:9608 28218 28224 canine +T814 PR:000003996 28225 28228 AMN +T815 GO:0010467 28237 28244 express +T816 PR:000003996 28257 28260 AMN +T817 NCBITaxon:9606 28303 28309 humans +T818 GO:0045177 28405 28411 apical +T819 GO:0010467 28412 28422 expression +T820 GO:0032991 28436 28443 complex +T821 PR:000006043 28540 28547 cubilin +T822 NCBITaxon:10088 28551 28556 mouse +T823 GO:0009790 28557 28570 embryogenesis +T824 GO:0007492 28591 28612 formation of endoderm +T825 UBERON:0000925 28604 28612 endoderm +T826 UBERON:0002329 28617 28624 somites +T827 PR:000006043 28672 28679 cubilin +T828 UBERON:0000922 28707 28713 embryo +T829 UBERON:0004877 28738 28740 VE +T830 SO:0001644 28752 28768 Targeting vector +T831 NCBITaxon:10088 28801 28805 mice +T832 SO:0000112 28839 28846 primers +T833 NCBITaxon:10114 28977 28980 rat +T834 PR:000006043 28981 28988 cubilin +T835 NCBITaxon:9606 29008 29013 human +T836 PR:000006043 29014 29021 cubilin +T837 UBERON:0007023 29071 29076 adult +T838 NCBITaxon:10088 29077 29082 mouse +T839 UBERON:0002113 29083 29089 kidney +T840 SO:0000028 29327 29329 bp +T841 NCBITaxon:10114 29408 29411 rat +T842 NCBITaxon:10088 29432 29437 mouse +T843 PR:000006043 29438 29445 cubilin +T844 NCBITaxon:10088 29494 29499 mouse +T845 SO:0001026 29500 29507 genomic +T846 SO:0001026 29517 29523 Genome +T847 SO:0000153 29545 29548 BAC +T848 SO:0000153 29609 29612 BAC +T849 SO:0000188 29653 29659 intron +T850 SO:0000147 29660 29664 exon +T851 SO:0000842 29688 29698;29711 29715 portion of ... gene +T852 PR:000006043 29703 29710 cubilin +T853 SO:0000147 29749 29754 exons +T854 SO:0000153 29809 29812 BAC +T855 NCBITaxon:10088 29850 29855 mouse +T856 SO:0001026 29869 29876 genomic +T857 SO:0000149 29877 29883 contig +T858 PR:000006043 29912 29919 cubilin +T859 SO:0001644 29937 29953 targeting vector +T860 SO:0000147 30010 30015 exons +T861 NCBITaxon:10088 30031 30036 mouse +T862 PR:000006043 30037 30044 cubilin +T863 SO:0000704 30045 30049 gene +T864 PR:000006043 30122 30129 cubilin +T865 SO:0000147 30130 30134 exon +T866 SO:0000346 30247 30251 loxP +T867 SO:0000359 30252 30258 floxed +T868 SO:0005853 30260 30268 cassette +T869 SO:0005853 30353 30361 cassette +T870 SO:0001644 30420 30436 targeting vector +T871 NCBITaxon:39107 30494 30500 murine +T872 UBERON:0000922 30515 30524 embryonic +T873 CL:0002322 30515 30529;30535 30540 embryonic stem ... cells +T874 CL:0002322 30531 30533;30535 30540 ES ... cells +T875 CHEBI:42768 30585 30589 G418 +T876 CL:0002322 30814 30816 ES +T877 UBERON:0000358 30851 30862 blastocysts +T878 NCBITaxon:10088 30927 30931 mice +T879 NCBITaxon:10088 31050 31054 mice +T880 PR:000006043 31087 31094 cubilin +T881 SO:0001023 31095 31101 allele +T882 NCBITaxon:10088 31103 31107 Mice +T883 SO:0000704 31168 31175 genetic +T884 UBERON:0000922 31269 31276 embryos +T885 GO:0007620 31306 31312 coitum +T886 UBERON:0000922 31351 31357 embryo +T887 UBERON:0000922 31379 31386 embryos +T888 UBERON:0001040 31454 31462 yolk sac +T889 SO:0000112 31468 31474 primer +T890 PR:000006043 31541 31548 cubilin +T891 SO:0001023 31549 31555 allele +T892 UBERON:0002415 31580 31584 tail +T893 SO:0001026 31590 31597 genomic +T894 PR:000006043 31623 31630 cubilin +T895 SO:0000112 31644 31650 primer +T896 SO:0000077 31731 31740 antisense +T897 SO:0000112 31748 31754 primer +T898 SO:0000028 32000 32002 bp +T899 PR:000006043 32027 32034 cubilin +T900 SO:0001023 32035 32041 allele +T901 SO:0000112 32098 32104 primer +T902 SO:0000077 32176 32185 antisense +T903 SO:0000112 32193 32199 primer +T904 SO:0000028 32434 32436 bp +T905 UBERON:0000922 32443 32450 embryos +T906 GO:0007618 32480 32487 matings +T907 UBERON:0000479 32532 32538 tissue +T908 PR:000006043 32642 32649 cubilin +T909 PR:000006043 32656 32663 cubilin +T910 UBERON:0000922 32753 32760 embryos +T911 UBERON:0000922 32838 32845 embryos +T912 UBERON:0000922 32939 32946 Embryos +T913 GO:0007565 32967 32975 pregnant +T914 PR:000006043 33000 33007 cubilin +T915 GO:0007618 33022 33029 matings +T916 CHEBI:51686 33080 33091 Hematoxylin +T917 CHEBI:51686 33103 33104 H +T918 UBERON:0000922 33138 33144 embryo +T919 PR:000001904 33243 33249 PECAM1 +T920 PR:000001904 33250 33254 CD31 +T921 GO:0042571 33299 33309 Antibodies +T922 PR:000001904 33313 33318 PECAM +T923 PR:000009921 33399 33406 megalin +T924 NCBITaxon:9986 33463 33469 rabbit +T925 GO:0042571 33470 33480 antibodies +T926 NCBITaxon:9986 33528 33534 rabbit +T927 PR:000009921 33535 33542 megalin +T928 GO:0005737 33543 33554 cytoplasmic +T929 SO:0000417 33555 33561 domain +T930 GO:0042571 33562 33572 antibodies +T931 MOP:0000779 33614 33624 conjugated +T932 GO:0042571 33635 33645 antibodies +T933 UBERON:0000479 33720 33726 tissue +T934 UBERON:0000922 33948 33955 embryos +T935 GO:0007618 33974 33981 matings +T936 PR:000006043 34117 34124 cubilin +T937 SO:0000673 34125 34135 transcript +T938 SO:0000112 34192 34198 primer +T939 SO:0000673 34232 34242 transcript +T940 SO:0000112 34280 34286 primer +T941 SO:0000121 34309 34323 forward primer +T942 SO:0000147 34372 34376 exon +T943 SO:0000132 34405 34419 reverse primer +T944 SO:0000147 34466 34470 exon +T945 SO:0000028 34663 34665 bp +T946 SO:0000112 34674 34680 primer +T947 SO:0000121 34703 34717 forward primer +T948 SO:0000147 34766 34770 exon +T949 SO:0000132 34800 34814 reverse primer +T950 SO:0000147 34865 34869 exon +T951 SO:0000028 35063 35065 bp +T952 SO:0000673 35083 35093 transcript +T953 SO:0000121 35127 35141 forward primer +T954 SO:0000132 35209 35223 reverse primer +T955 SO:0000028 35458 35460 bp +T956 PR:000003676 35472 35479 b-actin +T957 SO:0000673 35480 35490 transcript +T958 SO:0000121 35500 35514 forward primer +T959 SO:0000132 35585 35599 reverse primer +T960 SO:0000028 35841 35843 bp +T961 SO:0000673 35860 35870 transcript +T962 SO:0000121 35880 35894 forward primer +T963 SO:0000132 35964 35978 reverse primer +T964 SO:0000028 36222 36224 bp +T965 UBERON:0000922 36239 36248 embryonic +T966 NCBITaxon:9606 36281 36286 Human +T967 CHEBI:52029 36287 36290 DiI +T968 CHEBI:52029 36304 36307 DiI +T969 CHEBI:52029 36373 36376 DiI +T970 PR:000004155 36397 36401 apoE +T971 CHEBI:28304 36416 36423 heparin +T972 CHEBI:26710 36490 36494 NaCl +T973 CHEBI:9754 36502 36506 Tris +T974 CHEBI:52029 36652 36655 DiI +T975 UBERON:0007318 36764 36778 saphenous vein +T976 GO:0007565 36782 36790 pregnant +T977 NCBITaxon:10088 36804 36808 mice +T978 UBERON:0000922 36892 36899 embryos +T979 UBERON:0008800 36922 36939 parietal endoderm +T980 UBERON:0004877 36993 36995 VE +T981 UBERON:0004877 36997 37014 visceral endoderm +T982 UBERON:0005439 37016 37018 EE +T983 UBERON:0005439 37020 37038 embryonic endoderm +T984 UBERON:0000305 37040 37042 Am +T985 UBERON:0000305 37044 37050 amnion +T986 CHEBI:52029 37083 37086 DiI +T987 CHEBI:52029 37092 37095 DiI +T988 PR:000004140 37109 37115 apoA-I +T989 CHEBI:39015 37117 37131 apolipoprotein +T990 PR:000004140 37117 37135 apolipoprotein A-I +T991 PR:000003996 37137 37140 AMN +T992 UBERON:0000305 37142 37148 amnion +T993 PR:000003996 37142 37152 amnionless +T994 CHEBI:30411 37154 37157 Cbl +T995 CHEBI:30411 37159 37168 cobalamin +T996 GO:0007620 37184 37190 coitum +T997 CHEBI:51686 37192 37193 H +T998 CHEBI:51686 37197 37208 Hematoxylin +T999 SO:0001644 37310 37326 targeting vector +T1000 NCBITaxon:10088 37361 37365 mice +T1001 UBERON:0000922 37505 37511 embryo +T1002 UBERON:0000922 37612 37618 embryo +T1003 UBERON:0000922 37662 37668 embryo +T1004 NCBITaxon:10088 37725 37730 mouse +T1005 PR:000006043 37731 37738 cubilin +T1006 SO:0000153 37789 37792 BAC +T1007 SO:0001644 37814 37830 targeting vector +T1008 PR:000006043 37844 37851 cubilin +T1009 SO:0001644 37911 37927 targeting vector +T1010 SO:0000440 37948 37954 vector +T1011 CL:0002322 37994 38002 ES cells +T1012 UBERON:0000358 38004 38014 blastocyst +T1013 GO:0007566 38004 38014;38029 38041 blastocyst ... implantation +T1014 UBERON:0000922 38106 38113 embryos +T1015 PR:000006043 38358 38365 cubilin +T1016 SO:0000315 38366 38396 transcription initiation sites +T1017 SO:0000204 38505 38511 5' UTR +T1018 SO:0000147 38515 38519 exon +T1019 SO:0000704 38962 38966 Gene +T1020 NCBITaxon:10088 38990 38995 Mouse +T1021 PR:000006043 39044 39051 cubilin +T1022 NCBITaxon:10088 39061 39066 mouse diff --git a/src/ontogpt/evaluation/craft/database/all/16787536.txt b/src/ontogpt/evaluation/craft/database/all/16787536.txt new file mode 100644 index 000000000..810244515 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16787536.txt @@ -0,0 +1,169 @@ +Targeted disruption of cubilin reveals essential developmental roles in the structure and function of endoderm and in somite formation + +Abstract + +Background + +Cubilin is a peripheral membrane protein that interacts with the integral membrane proteins megalin and amnionless to mediate ligand endocytosis by absorptive epithelia such as the extraembryonic visceral endoderm (VE). + +Results + +Here we report the effects of the genetic deletion of cubilin on mouse embryonic development. Cubilin gene deletion is homozygous embryonic lethal with death occurring between 7.5–13.5 days post coitum (dpc). Cubilin-deficient embryos display developmental retardation and do not advance morphologically beyond the gross appearance of wild-type 8–8.5 dpc embryos. While mesodermal structures such as the allantois and the heart are formed in cubilin mutants, other mesoderm-derived tissues are anomalous or absent. Yolk sac blood islands are formed in cubilin mutants but are unusually large, and the yolk sac blood vessels fail to undergo remodeling. Furthermore, somite formation does not occur in cubilin mutants. Morphological abnormalities of endoderm occur in cubilin mutants and include a stratified epithelium in place of the normally simple columnar VE epithelium and a stratified cuboidal epithelium in place of the normally simple squamous epithelium of the definitive endoderm. Cubilin-deficient VE is also functionally defective, unable to mediate uptake of maternally derived high-density lipoprotein (HDL). + +Conclusion + +In summary, cubilin is required for embryonic development and is essential for the formation of somites, definitive endoderm and VE and for the absorptive function of VE including the process of maternal-embryo transport of HDL. + +Background + +Cubilin is a 460-kDa peripheral membrane protein expressed by a number of absorptive epithelial cells including those of the renal proximal convoluted tubule, ileum and yolk sac extraembryonic visceral endoderm (VE) [1]. The first described function of cubilin was as the receptor for intrinsic factor-vitamin B12/cobalamin (Cbl), serving a critical role in the intestinal absorption of Cbl [2,3]. Cubilin was later shown to be an endocytic receptor for apolipoprotein A-I (apoA-I)/high density lipoprotein (HDL), mediating uptake of HDL in the kidney and VE [4,5]. Other cubilin ligands include albumin, transferrin, immunoglobulin light chains, vitamin D-binding protein, myoglobin, galectin-3 and Clara cell secretory protein [6]. + +Three cell surface integral membrane proteins have been shown to interact with cubilin. The first identified was megalin, an endocytic receptor belonging to the low density lipoprotein receptor (LDLR) family [7,8]. Megalin functions together with cubilin to mediate endocytosis of apoA-I/HDL, presumably facilitating endocytosis of the cubilin-apoA-I/HDL complex. The cation-independent mannose 6-phosphate/insulin-like growth factor II-receptor (CIMPR) is another endocytic receptor that binds to cubilin [9], although the functional significance of its interaction with cubilin remains to be established. The ~48-kDa type I transmembrane protein, amnionless (AMN), is the most recent integral membrane protein found to interact with cubilin [10]. AMN is essential for efficient transport of cubilin to the apical cell surface as well as for membrane anchoring of cubilin [11,12], + +Given the fact that cubilin is expressed by trophectoderm and VE [13,14], it is believed to play an important role in maternal-embryonic transport of nutrients. Several additional pieces of evidence support this hypothesis. First, cubilin has been shown to mediate VE uptake of holoparticle HDL, HDL-associated cholesterol and apolipoprotein A-I [8,14]. Furthermore, the work of Sahali et al. [15] demonstrated that cubilin monoclonal antibodies infused into circulation of pregnant rats (9 dpc), bound to VE cubilin and induced a spectrum of developmental abnormalities and embryonic resorption within 24–48 hours of infusion. The abnormalities included retarded embryonic growth, craniofacial defects involving the eye, ear and neural tube, hydrocephaly and telencephalic hypoplasia. Similarly, growth retardation and morphological anomalies were obtained when rat embryos (10 dpc) were cultured ex utero in the presence of cubilin antibodies [16]. Here we characterize the consequences of targeted deletion of the mouse cubilin gene on embryonic development. + +Results + +Generation of cubilin-/- mice + +To generate mice with targeted disruption of the cubilin gene (and concomitant knock-in of the EGFP reporter gene) we cloned and fully mapped the exon-intron structure of the 5' portion of the murine cubilin gene. A mouse cubilin gene-targeting vector was designed to create a null mutation through deletion of exons 1–6 (Fig. 1). After electroporation of the construct and G418 positive selection and FIAU negative selection, ten ES clones (out of 132 screened) were identified that had the desired recombination based on Southern analysis (using both upstream and downstream flanking probes) (Fig. 1). Targeted ES clones were injected into C57BL/6J blastocysts and the blastocysts were transferred to foster mothers to obtain chimeric mice. Two male chimeras were obtained from the first targeted ES cell line tested and found to be germ line competent through the generation of heterozygous offspring. As shown in Figure 1C, Southern analysis confirmed that offspring from one of these mice contained the properly targeted cubilin allele. + +Figure 1 + +Targeted deletion of the mouse cubilin gene. A, is a diagram of the structural and functional domains of cubilin. Ligand binding regions indicated in the diagram are based on published studies [12, 33, 34]. B, is a diagram of the gene targeting strategy showing the organization of cubilin gene exons (vertical lines in uppermost model and rectangles in expanded region shown below), the wild-type and targeted alleles and the location of 5' and 3' DNA probes used for Southern blot analysis. White portions of boxed exons depict untranslated sequences. The promoter-less EGFP (containing Kozak and ATG sequences) and loxP-floxed neoR cassettes were inserted into exon 1, 33 bp upstream of the cubilin ATG and 16 bp downstream from the proximal-most transcription initiation site, resulting in a replacement of the majority of exon 1. Homologous recombination between the targeting construct and the cubilin locus results in deletion of exon 1 coding sequence and all of exons 2–6. C, Southern analysis of HindIII/HhaI and BglII digested DNA from representative wild-type (WT) and targeted (-/+) ES cell clones using 5' and 3' probes (left and right panels, respectively). The wild-type allele yields a 7 kb band and the knockout allele yields a 5.5 kb band when genomic DNA is digested with HindIII and HhaI and hybridized with the 5' probe. Additionally, the wild-type allele yields an 11.3 kb band and the knockout allele yields a 7 kb band when genomic DNA is digested with BglII and hybridized with the 3' probe. The results show that the correct recombinant allele is present in the selected ES cell clone. D, Southern analysis of BglII digested tail DNA from a wild-type (WT) and a correctly targeted heterozygous (-/+) mouse. + +The cubilin exon 1–6 deletion leads to embryonic lethality + +Genotypic analysis was performed on 220 offspring from heterozygous intercross matings. As a result, 100 wild-type, 120 heterozygous and no homozygous offspring were detected (Table 1). These non-Mendelian ratios indicate that lethality occurs in embryos homozygous for the targeted cubilin gene deletion. Furthermore, since the frequency of heterozygotes at 4 weeks was much lower than expected (1:1 versus the expected 2:1 heterozygous:wild-type ratio), embryonic lethality appeared to be occurring in some heterozygotes. To substantiate this possibility we performed genotypic analysis of 181 4-wk offspring from wild-type × heterozygote matings. As a result, 109 wild-type and 72 heterozygous offspring were detected. Based on a chi-square test of these data and a resulting p value = 0.0074, the data are consistent with the hypothesis that haploinsufficiency of cubilin causes embryonic lethality, although with incomplete penetrance. + +Table I + +Genotype frequency of progeny from heterozygote (cubilin exon1–6+/-) matings + +Asterisk indicates that the chi-square test was based on a 1:2 ratio of wild-type to heterozygote offspring. + +Retrograde genotypic analysis was performed on embryos from heterozygous intercross matings (Table 1). At 7.5 dpc homozygous embryos were obtained at a frequency consistent with a normal Mendelian ratio. From 8.5–10.5 dpc, homozygous embryos were detected, but not at a frequency in accordance with Mendelian expectations (Table 1). After 13.5 dpc, no homozygous embryos were detected. Together, these findings indicate that homozygous embryos die over a range of developmental stages between 7.5 to 13.5 dpc. Consistent with this conclusion was the relatively high number of embryo resorptions observed after 7.5 dpc (Table 1). + +To establish that embryos homozygous for the targeted cubilin gene deletion of exons 1–6 were indeed cubilin null, RT-PCR analysis was performed using two primer pairs designed to amplify separate 5' and 3' regions of the cubilin transcript. No cubilin mRNA was detectable by either primer pair using RNA isolated from homozygous 8.5 dpc embryos (Fig. 2). Furthermore, immunohistological analysis confirmed that there was no cubilin detectable in paraffin embedded sections of homozygous 8.5 dpc embryos (data not shown). + +Figure 2 + +Embryos homozygous for targeted deletion of cubilin do not express cubilin transcripts. Shown are RT-PCR analyses of the expression of cubilin transcripts in RNA from heterozygous (+/-) and null (-/-) 8.5 dpc embryos. In A, a primer pair corresponding to a 5' region of the cubilin transcript was used to assess cubilin transcript expression. In B, a primer pair corresponding to a 3' region of the cubilin transcript was used to assess cubilin transcript expression. + +Developmental retardation and mesodermal defects of homozygous embryos + +Morphological analysis of 8–8.5 dpc embryos revealed that homozygous embryos were considerably smaller than wild-type or heterozygous littermates (Fig. 3). Mutant 8–8.5 dpc embryos were morphologically similar to wild-type 7.5 dpc embryos. However, rather than exhibiting a normal cylindrical shape, mutant 8–8.5 embryos appeared acorn shaped owing to a relatively smaller embryonic component as compared to extraembryonic component. + +Figure 3 + +Cubilin deficient 8–8.5 dpc embryos display growth retardation with formation of some mesodermal structures, but not somites. Shown are wild-type (A), heterozygous (B) and homozygous 8–8.5 dpc embryos (C-E), derived from a heterozygous intercross mating. Embryos in A and B have been dissected to lie in a planar configuration where as embryos in C-D are intact. Shown in F and G are H&E stained sections of 8.5 dpc wild-type (F) and mutant (G) embryos. Mutant 8.5 dpc embryos possess an allantois and amnion, but lack somites. A, allantois; Am, amnion; S, somite. Bars in A-G = 100 μm. + +Histological examination of null 8–8.5 dpc embryos revealed that all mutant embryos underwent gastrulation, but did not form somites (Fig. 3F and 3G). In contrast, 8–8.5 dpc wild-type littermates had formed 3–9 somites. Other mesodermal structures did form in the 8–8.5 dpc mutants, albeit with some variability. For example, an allantois was present in 5 of 7 mutant 8–8.5 dpc embryos examined. Most mutant embryos (5 of 7) also had an amnion and chorion, tissues having mesoderm components. While blood islands formed in the yolk sacs of all 8–8.5 dpc mutants they appeared unusually large, extending into the exocoelomic cavity to a greater extent than wild-type blood islands (Fig. 4). Such blood islands appeared to have a larger than normal number of mesodermal cells. Additionally, blood island hematopoietic development appeared to be retarded as evidenced by the failure of the cells to exhibit the characteristic rounded hematopoietic cell morphology (Fig. 4). + +Figure 4 + +Enlargement of blood islands in cubilin mutants. Shown are H&E stained sections of an 8–8.5 dpc wild-type (A) and mutant (B) embryo. C and D show high magnification views of blood islands from embryos shown in A and B. Embryos in panels A and B are oriented such that anterior is to the right and posterior is to the left. BI, blood island. Bars in A and B = 100 μm. Bars in C and D = 25 μm. + +By 9.5 dpc, surviving homozygous embryos had not undergone axial rotation but displayed an overall morphological appearance of wild-type 8–8.5-dpc embryos (Fig. 5). Mutant 9.5 dpc embryos had formed a primitive heart and neural head folds (4 of 4 nulls examined) (Fig. 5) and paired dorsal aortae (data not shown), but lacked somites. Homozygous embryos surviving beyond 9.5 dpc (i.e., 11.5 dpc) were also grossly similar to wild-type 8.0 dpc embryos, but still lacked somites (data not shown). Together these findings indicate that lack of cubilin results in developmental retardation that initiates prior to 8.0 dpc, and a failure to achieve developmental milestones appropriate for a wild-type 8.0 dpc embryo, most notably, failure to form somites. The complete lack of somites at 9.5 dpc suggests that the basis is due to some defect of the paraxial mesoderm rather than merely being a consequence of retarded development. + +Figure 5 + +Cubilin-deficient 9.5 dpc embryos are developmentally retarded and lack somites. A shows a wild-type 9.5 dpc embryo and B a mutant littermate. NF, neural folds; A, allantois; H, heart; YS, yolk sac. Bars in A and B = 200 μm. + +It is important to note that embryos confirmed by genotyping to be heterozygous were indistinguishable morphologically from wild-type littermates over a range of stages examined extending from 7–11.5 dpc (Fig. 3B). This indicates that the lethality of heterozygous embryos, apparent from genotypic analysis, is occurring beyond 11.5 dpc. + +Failure of yolk sac blood vessels to undergo remodeling + +To assess the effects on blood vessel formation, embryos from heterozygous matings were immunolabeled with anti-PECAM-1. As shown in Figure 6A, yolk sac blood vessel formation in 9.5 dpc mutants appeared retarded as compared to wild-type littermates (Fig. 6B). Normally by 9.5 dpc the yolk sac vessels of the wild-type embryo have undergone remodeling to form larger diameter vessels (Fig. 6B, inset). By contrast, the pattern of yolk sac vascular development in 9.5 dpc mutants appeared as an interconnected network of small diameter blood vessels (Fig. 6A), a pattern similar to that seen in an 8.5 dpc wild-type embryo yolk sac. In some areas of the extraembryonic yolk sac, PECAM-1 labeling often showed enlarged sinusoidal-like vessels (data not shown), which was consistent with the enlarged blood island-like structures observed in H&E stained sections (Fig. 4). + +Figure 6 + +Yolk sac blood vessels of 9.5 dpc cubilin mutants fail to undergo remodeling. Anti-PECAM-1-labeled vasculature in a 9.5 dpc homozygous embryo (A), a 9.5 dpc wild-type (WT) embryo (littermate to that shown in A) (B), 8.5 dpc wild-type embryo (C) and 11.5 dpc homozygous embryo (D). The inset panel in B shows a higher magnification view of the remodeled yolk sac vasculature in the boxed region of the 9.5 dpc wild-type yolk sac. YS, yolk sac; A, allantois; NF, neural folds; H, heart. Bars in A and B = 500 μm. Bars in C and D = 200 μm. + +When the PECAM-1-labeled yolk sac vasculature of 11.5 dpc mutants was compared to that of wild-type embryos it was again evident that the blood vessels of homozygous embryos had not progressed beyond that of 8.5 dpc wild-type embryos. As shown in Figure 6D, yolk sac blood vessels of an 11.5 dpc mutant had not undergone the remodeling that normally occurs after 8.5 dpc. + +In addition to the yolk sac vascular remodeling anomalies, the allantoic vasculature of 11.5 dpc mutants was aberrant. Instead of a branched central blood vessel normally present in an 8.5 dpc embryo [17], the allantoic blood vessels of 11.5 dpc mutants were bulbous and lacked branching (Fig. 6D). Since this abnormality was not observed in 9.5 dpc mutants, it can be concluded that dysmorphogenesis of the allantoic vasculature occurs after 9.5 dpc and perhaps contributes to a chorioallantoic placental defect. + +Abnormalities of embryonic and visceral endoderm in cubilin mutants + +Considering that cubilin is normally present on the apical surface of the columnar VE (from 6.0 dpc to at least 9.5 dpc) and on a population of the squamous cells within the embryonic endoderm (EE) (from 7.3–8.0 dpc) [13], we next evaluated the impact of cubilin deficiency on these embryonic endodermal tissues. Examination of homozygous 8–8.5 dpc embryos revealed a number of endodermal anomalies (Fig. 7). In place of a normal squamous definitive endoderm (Fig. 7C), the mutant embryos had both cuboidal and columnar cells arranged in a stratified cuboidal epithelium (Fig. 7D, arrow) as well as simple columnar epithelium (not shown). Additionally, mutant cells in these epithelia had apical processes that may be either microvilli or cilia. Such luminal surface structures are normally not found on EE cells. Based on the findings, the absence of cubilin inhibits the formation of an epithelium morphologically comparable to that of a normal definitive endoderm at this stage, however, it remains to be determined whether this is a result of a defect in the specification and differentiation of the definitive endoderm. + +Figure 7 + +Cubilin mutants display anomalies in the epithelial morphology of embryonic endoderm. Shown are H&E stained sections of wild-type (A, C and E) and mutant (B, D and F) 8–8.5 dpc embryos. Embryos in panels A and B are oriented such that anterior is to the right and posterior is to the left. BI, blood island; S, somite; EE, definitive embryonic endoderm; VE, yolk sac visceral endoderm, Asterisk points to aberrant epithelium in place of the normal EE. Bars in A-F = 100 μm. + +Examination of the VE of homozygous 8–8.5 dpc embryos revealed it to also be morphologically abnormal. In contrast to the simple columnar VE of normal embryos, the VE of cubilin mutants was a stratified epithelium, comprised of cuboidal and columnar cells. Furthermore, mutant VE cells also lacked the large apical vacuoles that are characteristic of VE cells (Fig. 7E and 7F). Given the morphological similarities between the cells of mutant embryonic endoderm there was not a discernable demarcation between the normally distinct EE and VE epithelia. However, a clear demarcation between the VE and an epithelium that would normally be EE was evident upon immunohistological analysis of GFP reporter expression (from the EGFP cassette inserted into the cubilin gene) and megalin expression in mutant 8.0 dpc embryos (Fig. 8). This indicated that although being morphologically similar, the VE and presumed EE of mutants were distinct at the molecular level. Furthermore, the fact that megalin was appropriately expressed on the apical surfaces of the mutant VE indicated that cubilin is not required to direct cell surface trafficking of megalin. + +Figure 8 + +Green fluorescent protein and megalin expression in yolk sac visceral endoderm of mutant embryos. The cubilin gene targeting strategy (see Fig. 1) created a GFP reporter for cubilin locus expression. Shown in A is an H&E stained section of a homozygous 8.0 dpc embryo. Shown in B is anti-GFP fluorescence of a serial section to the one shown in A. Note that cells within the blood islands are expressing the GFP reporter. Shown in C is an anti-megalin stained 8.0 dpc mutant embryo. Embryos in panels A-B are oriented such that anterior is to the left and posterior is to the right. The embryo in C is oriented with anterior to the right and posterior to the left. VE, yolk sac visceral endoderm; BI, blood island; Am, amnion; Asterisk, points to the boundary of the VE and presumed EE. + +The visceral endoderm of homozygous embryos does not mediate uptake of maternal-derived HDL + +A normal function of VE is to mediate uptake of maternal-derived HDL [8]. We therefore evaluated the capacity of cubilin-deficient VE to mediate uptake of maternal-derived HDL. As shown in Figure 9, the VE of heterozygous 8.0 dpc embryos from DiI-HDL-infused mothers showed strong DiI labeling within VE cells. DiI label in these embryos was present exclusively in VE which is the only yolk sac endodermal component expressing cubilin as evidenced by expression of the cubilin gene reporter, EGFP. By contrast, there was no detectable DiI labeling of the VE of homozygous embryos (Fig. 9D–F). These findings indicate that VE uptake of HDL is a cubilin dependent process. + +Figure 9 + +Maternally derived HDL is not taken up by the yolk sac visceral endoderm of cubilin-deficient embryos. Shown are confocal micrographs of a heterozygous 8.0 dpc embryo (A-C) and a homozygous littermate (D-F) isolated 1 hour after DiI-HDL was infused into the mother. The distal portion of the VE was removed from all embryos shown. VE, visceral endoderm; EE, embryonic endoderm. + +Discussion + +Considering that cubilin forms a complex with AMN (forming the cubam complex), that AMN and cubilin are co-expressed in absorptive epithelial including the VE [11], and that AMN is required to mediate key aspects of cubilin function (e.g., membrane association, apical sorting) [11,12], it is not surprising that the phenotype of AMN-deficient mutants [18] closely resembles that of the cubilin nulls described. Like the cubilin null embryos, AMN-deficient embryos display arrested development, with most mutants not advancing morphologically beyond a normal 8–8.5 dpc embryo. On a mixed 129Sv/C57BL/6 genetic background, both mutants form an amnion. General mesoderm formation in the cubilin and AMN mutant embryos appears normal in that both mutants form an allantois, blood islands, yolk sac blood vessels and a heart. However, both mutants fail to form somites, indicating that somitogenesis is in some manner defective (e.g., insufficient paraxial mesoderm or defective condensation and/or segmentation). Both mutants also display endoderm defects as evidenced by the inability of cubilin-deficient VE to facilitate uptake of maternal HDL and the inability of AMN-deficient VE and EE to support wild-type epiblast development in chimeric embryos [18]. The morphological abnormalities of VE and definitive endoderm observed in the cubilin mutants also highlight the importance of cubilin in formation and/or maintenance of these epithelia. Such morphological abnormalities were not reported in the phenotype of AMN-deficient mutants. It remains to be established whether cubilin and AMN are playing roles in the specification and differentiation of the definitive endoderm, which forms during gastrulation by the recruitment of epiblast cells through the primitive streak [19]. + +The close similarities between the phenotypes of mouse amn and cubilin mutants also suggest that both genes act co-operatively in the VE to support embryonic growth and to control the formation of paraxial mesoderm. How AMN and cubilin mechanistically mediate these processes remains to be resolved. The endocytic function of the cubam complex and its apical expression in VE supports the possibility that it functions in the transport of vital maternal nutrients/factors. The spectrum of maternally derived nutrients/factors transported by VE cubam is potentially quite broad considering the multi-ligand binding capacity of cubilin which includes albumin, transferrin, immunoglobulin light chains, vitamin D-binding protein, myoglobin, galectin-3, Clara cell secretory protein, apoA-I and HDL [6]. Furthermore, at least one cubilin ligand, HDL, is a complex of multiple factors including apolipoproteins, phospholipids, cholesterol and lipid soluble vitamins such as retinol, which is converted to retinoic acid through the action of retinaldehyde dehydrogenase-2 (Raldh2). Indeed, genetic deficiency of mouse Raldh2 leads to early embryo lethality (~10.5 dpc), absence or reduction in somites, yolk sac vascular defects, enlarged heart and a failure of embryos to undergo axial rotation [20,21], all similar to cubilin and amn mutants. + +By contrast to the developmental anomalies and early lethality of the cubilin and amnionless nulls, megalin-deficient mice die perinatally and display abnormal morphogenesis of the forebrain (e.g., holoprosencephaly), lung and kidney [22]. Thus, despite the evidence that megalin functions together with cubilin and that it is co-expressed with cubilin in the VE [13], any joint function that these two proteins may have in the early embryo is evidently not vital. Interestingly, mice deficient in disabled-2 (dab2), an adaptor of megalin, display several of the abnormalities observed in cubilin nulls including disorganization of the VE and loss of apical vesicles [23]. However, dab2-/- mutants arrest earlier in development than do cubilin nulls and do not undergo gastrulation. Dab2 has also been shown to be required for cubilin endocytosis in VE [24]. The earlier lethality of dab2 null embryos may be an indication that numerous endocytic receptors expressed in the VE including megalin, cubilin and perhaps amnionless are dependent on dab2. + +The importance of maternal derived lipoproteins as a source of cholesterol for normal development is well established [25]. Through the use of cubilin antagonists, the ability of cubilin to mediate VE uptake of holoparticle HDL, HDL-associated cholesterol and apoA-I has been demonstrated in mouse embryos cultured in vitro [8,14]. Our findings provide in vivo evidence that early (8.0 dpc) embryonic uptake of HDL from the maternal circulation is a cubilin-dependent process of the VE. Given the fact that the VE expresses apoA-I and the major LDL constituent, apoB [26], it is likely that cholesterol and other maternal HDL-derived constituents taken up by VE cells via cubilin are incorporated into new HDL and LDL particles and subsequently delivered to the embryo. In support of this, there is evidence that nascent lipoprotein particles have been localized to the rough endoplasmic reticulum and secretory vesicles of the 9.5 dpc mouse yolk sac and seen in the pericellular space underlying the endoderm [27]. + +Despite the fact that cubilin and AMN deficiency in mice leads to embryonic lethality, mutations in human and canine AMN that cause impaired apical targeting [28] and function (i.e., malabsorption of vitamin B12) of the cubam complex, nevertheless have no effect on embryonic development. One explanation for this apparent paradox may relate to the fact that there are species differences in yolk sac function in rodents, dogs and humans. Another explanation comes from the studies of Tanner et al. [29] who have shown that mutations affecting exons 1–4 of human AMN (OMIM 261100, megaloblastic anemia, MGA1) do not prevent the production variant transcripts and polypeptides resulting from alternative transcriptional start sites and alternative translation initiation sites. Indeed, the two AMN mutants that cause premature termination of translation (14ΔG and 208-2A→G) also express an alternative AMN polypeptide of 40 kDa and several minor species of 44, 42 and 38 kDa [29]. All of the alternative AMN polypeptides would contain the chordin-like module, transmembrane domain and cytoplasmic domain, but would lack various lengths of the amino terminal portion of the full-length protein. While these alternative polypeptides apparently are missing regions required for vitamin B12 uptake, they evidently confer enough normal function to sustain embryonic development. How these alternative polypeptides function, particularly with respect to cubilin, remains to be established. One possibility is that they retain the ability to mediate trafficking of cubilin to the apical surfaces of epithelial cells. However, mutations of the canine AMN gene that disrupt cubilin intracellular trafficking have no apparent effect on embryonic development [28]. It is not known whether the canine AMN mutants express alternative AMN polypeptides similar to those observed in humans. If so, it is possible that the polypeptides possess some activity that overcomes the need for apical expression of the cubam complex to mediate normal development. + +Conclusion + +The present study reveals an indispensable role for cubilin in mouse embryogenesis particularly in the formation of endoderm and somites. The findings also highlight the importance of cubilin in the process of maternal-embryo transport of HDL by the VE. + +Methods + +Targeting vector design and generation of mutant mice + +Degenerate deoxyoligonucleotide primers 5'-CTICACCARCCICGIATG-3' and 5'-CCRTTRRATYTCRCAYTC-3' were synthesized based on cDNA sequences conserved in the 5' region of both rat cubilin (GI: 24475743) and human cubilin (GI: 3929528). Preparation of template cDNA from adult mouse kidney total RNA and PCR amplification were done using methods described previously [8]. Cycling parameters for PCR amplification were: 94°C for 5 min and then 30 cycles of 94°C for 1 min, 46°C for 1 min and 72°C for 1.5 min. The expected ~820 bp product was sequenced and found to have 93% identity with a 5' portion of the rat cDNA sequence. + +The mouse cubilin cDNA was used as a probe to screen a 129-strain mouse genomic library (Genome Systems, Inc.) and a BAC clone (24267) was isolated. DNA sequencing of ~20 kb of the BAC clone was performed to characterize the intron-exon organization of the 5' portion of the cubilin gene, including the region containing exons 1–10. A search of GenBank showed that the sequence of BAC clone 24267 was contained within the mouse chromosome 2 genomic contig GI:82796355. + +The 3' arm of cubilin replacement-type targeting vector was constructed from a 5.4-kb EcoRI fragment containing exons 7 and 8 of the mouse cubilin gene. The 5' arm was constructed from a 4.3 kb ApaI-NaeI fragment containing cubilin exon 1 disrupted by insertion of a enhanced green fluorescent protein (EGFP)-N1 (Clontech, Mountain View, CA), neoR (loxP floxed) cassette 33 nucleotides 5' to the first ATG (see Additional file 1). A tk negative selection cassette was placed at the 5' end of this construct. The resulting targeting vector was linearized by NotI digestion and electroporated into murine 129/Sv-strain embryonic stem (ES) cells. After electroporation of the construct and G418 positive selection and 1-(2-deoxy-2-fluoro-8-d-arabinofuranosyl)-5-iodouracil (FIAU) negative selection, clones having the desired homologous recombination were identified by Southern analysis (Fig. 1C). One of the targeted ES clones was injected into C57BL/6J blastocysts that were then transferred to foster mothers to obtain chimeric mice. Two male chimeras were obtained that were germ line competent. Southern analysis confirmed that offspring from these mice contained the properly targeted cubilin allele. Mice used in this study were maintained on a mixed 129Sv/C57BL/6 genetic background. + +Genotypes of progeny from heterozygote intercrosses were determined by PCR. For embryos between 7.5 and 8.5 days postcoitum (dpc) DNA was isolated from the whole embryo and used in PCR. For embryos between 9.5 and 14.5 dpc, DNA for genotyping was isolated from the yolk sac. Two primer pairs were used for PCR-based genotyping. To detect the wild-type cubilin allele, PCR was performed with tail clip genomic DNA preparations using a cubilin sense strand primer, 5'-GCCAAGTAGACCAGGCTGAC-3' (residues 10422223–10422242 in GI: 82796355, and an antisense strand primer, 5'-GCTTCTGAGCCCAGTGAAAC-3' (residues 10422576–10422595 in GI: 82796355). Cycling parameters for PCR amplification were: 98°C for 5 min and then 40 cycles of 98°C for 0.5 min, 55°C for 1 min and 72°C for 1 min. The expected amplicon size is 373 bp. To detect the targeted cubilin allele, PCR reactions were performed with an EGFP sense strand primer, 5'-CCTGAAGTTCATCTGCACCA-3' (residues 810–829 in GI:1377911), and EGFP antisense strand primer 5'-TGCTCAGGTAGTGGTTGTCG-3' (residues 1288–1269 in GI:1377911). Cycling parameters for PCR amplification were: 98°C for 5 min and then 40 cycles of 98°C for 0.5 min, 55°C for 1 min and 72°C for 1 min. The expected amplicon size is 478 bp. + +For embryos from intercross heterozygous matings that were paraffin embedded within maternal tissue, the assignment of 'mutant' was based on two criteria: 1) by the absence of immunologically detectable cubilin (anti-cubilin T-16 from Santa Cruz Biotechnology, Santa Cruz, CA) in sections of the paraffin embedded embryos; and 2) the distinct mutant morphological phenotype corresponding to that of embryos confirmed by genotypic analysis to be homozygous nulls. + +Immunohistochemistry and histology + +Embryos were harvested from pregnant females following timed cubilin+/- intercross matings, and fixed for 40 min in 4% paraformaldehyde/PBS. Hematoxylin and eosin (H&E) staining of paraffin embedded embryo sections (7 μm) was performed using standard techniques. Whole-mount immunohistochemistry for the PECAM1/CD31 was performed as described previously [30]. Antibodies to PECAM (clone Mec-13.3) were purchased from BD PharMingen (San Diego, CA). For GFP and megalin immunohistological staining, sections were stained with rabbit antibodies to GFP purchased from Abcam (Cambridge, MA) or rabbit megalin cytoplasmic domain antibodies described previously [13]. Fluorescently conjugated secondary antibodies were purchased from Jackson ImmunoResearch Labs (West Grove, PA). Stained tissue sections were analyzed using a Leica DMR research-grade microscope equipped with Leica objectives and a SPOT-RT camera (Diagnostic Instruments, Sterling Heights, MI). + +RT-PCR analysis + +Total RNA was isolated from 8.5 dpc embryos from heterozygous matings using Trizol (Invitrogen). Template cDNA was prepared from 1 mg RNA using Superscript II Reverse Transcriptase (Invitrogen). To assess cubilin transcript levels two sets of PCR reactions were performed using a primer pair targeting the 5' end of the transcript and one targeting the 3' end. The 5' primer pair consisted of the forward primer 5'-ATGATGATGACCTTGGCGAATG-3' (residues 275–296 (exon 2) in GI: 94365995) and the reverse primer 5'-GCAGCCAAAGGGTGTTCCAG-3' (residues 587–606 (exon 6) in GI: 94365995). Cycling parameters for PCR amplification were: 98°C for 5 min and then 40 cycles of 98°C for 0.5 min, 58°C for 1 min and 72°C for 1 min. The expected amplicon size is 332 bp. The 3' primer pair consisted of the forward primer 5'-TCTCATACACCAACTACCCC-3' (residues 9221–9240 (exon 58) in GI: 94365995) and the reverse primer 5'-AGCAGTCTTGTGAGGGCAGC-3' (residues 10041–10060 (exon 62) in GI: 94365995). Cycling parameters for PCR amplification were: 98°C for 5 min and then 40 cycles of 98°C for 0.5 min, 58°C for 1 min and 72°C for 1 min. The expected amplicon size is 840 bp. To assess GAPDH transcript levels PCR was performed using a forward primer 5'-CGGTGTGAACGGATTTGGC-3' (residues 70–88 in GI: 47607489) and the reverse primer 5'-GCAGTGATGGCATGGACTGT-3' (residues 581–600 in GI: 47607489). Cycling parameters for PCR amplification were: 98°C for 5 min and then 40 cycles of 98°C for 0.5 min, 54°C for 1 min and 72°C for 1 min. The expected amplicon size is 531 bp. To assess b-actin transcript levels a forward primer 5'-CGGGACCTGACAGACTACCTC-3' (residues 627–647 in GI: 6671508) and the reverse primer 5'-AACCGCTCGTTGCCAATA-3' (residues 827–844 in GI: 6671508) were used. Cycling parameters for PCR amplification were: 98°C for 5 min and then 40 cycles of 98°C for 0.5 min, 55°C for 1 min and 72°C for 1 min. The expected amplicon size is 218 bp. To assess EGFP transcript levels a forward primer 5'-ACGTAAACGGCCACAAGTTC-3' (residues 743–762 in GI: 1377911) and the reverse primer 5'-AAGTCGTGCTGCTTCATGTG-3' (residues 910–929 in GI: 1377911) were used. Cycling parameters for PCR amplification were: 98°C for 5 min and then 40 cycles of 98°C for 0.5 min, 58°C for 1 min and 72°C for 1 min. The expected amplicon size is 187 bp. + +Analysis of embryonic uptake of maternal derived HDL + +Human DiI-labeled HDL (DiI-HDL) was purchased from Biomedical Technologies (Stoughton, MA). DiI-HDL was depleted of apoE-HDL and other heparin-binding particles according to Oram [31], dialyzed against 150 mM NaCl, 50 mM Tris pH 7.4 (TBS) containing 0.3 mM EDTA and filter-sterilized. Lipoprotein concentration was determined by BCA protein assay (Pierce, Rockford, IL). DiI-HDL was diluted into PBS to a final concentration of 0.265 mg protein/ml and 75–125 μl was infused into the saphenous vein of pregnant heterozygous mice at 8.0 dpc according to the procedure of Hem et al. [32]. One hour after infusion, embryos were isolated free of parietal endoderm and analyzed by confocal microscopy. + +Abbreviations + +VE, visceral endoderm; EE, embryonic endoderm; Am, amnion; HDL, high density lipoprotein; DiI-HDL, DiI-labeled HDL; apoA-I, apolipoprotein A-I; AMN, amnionless; Cbl, cobalamin; dpc, days postcoitum; H&E, Hematoxylin and eosin; EGFP, enhanced green fluorescent protein. + +Authors' contributions + +B. T. S. generated the targeting vector, carried out analyses of knockout mice, contributed to experimental design and helped draft the manuscript. J. C. M. carried out morphological and genotypic analyses of the null embryo phenotype, maternal HDL transport experiments and writing of the manuscript. P. A. F. assisted with embryo isolation, immunohistological analysis and embryo imaging. J. L. B. performed degenerate amplification of mouse cubilin 5' cDNA sequences, isolated and characterized the BAC used to generate the targeting vector and directed cubilin 5' RACE analysis. M. A. C. assisted with the design of the targeting vector. D. D. S. performed vector electroporation, selection of targeted ES cells, blastocyst injection and implantation. C. J. D. assisted with the phenotypic characterization of null embryos and writing of the manuscript. W. S. A. conceived of the study, contributed to experimental design and interpretation of results, and coordinated the project and writing of the manuscript. + +Supplementary Material + +Additional File 1 + +Mapping of cubilin transcription initiation sites. The file contains findings from 5' RACE experiments used to position the EGFP reporter sequence within the 5' UTR of exon 1 of the reporter knock-in/KO targeting construct. + +Click here for file + +Acknowledgements + +This work was supported by NIH grants HL061873 and DE14347 (WSA), HL57375 (CJD) and CA109958 (DDS) as well as DOD contract 4400122478 (DDS). Brian Smith was a recipient of support from NIH postdoctoral training grant T32 HL007710. Jason Mussell was a recipient of support from NIH postdoctoral training grant T32 HL07260. We also acknowledge the MUSC Gene Targeting and Knockout Mouse Core for assistance with the development of the cubilin knockout mouse. diff --git a/src/ontogpt/evaluation/craft/database/all/16800892.ann b/src/ontogpt/evaluation/craft/database/all/16800892.ann new file mode 100644 index 000000000..61b01821e --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16800892.ann @@ -0,0 +1,1099 @@ +T1 SO:0000704 12 16 gene +T2 GO:0010467 12 27 gene expression +T3 NCBITaxon:10088 35 40 mouse +T4 GO:0030431 49 57 Sleeping +T5 SO:0000704 65 69 gene +T6 SO:0000101 75 85 transposon +T7 SO:0000101 149 170 transposable elements +T8 NCBITaxon:1 222 231 organisms +T9 NCBITaxon:562 237 244 E. coli +T10 NCBITaxon:7215 248 258 Drosophila +T11 NCBITaxon:10088 283 288 mouse +T12 GO:0030431 314 322 Sleeping +T13 SO:0000101 335 345 transposon +T14 CHEBI:25435 416 423 mutagen +T15 NCBITaxon:10088 431 436 mouse +T16 SO:0000101 450 460 transposon +T17 SO:0000440 461 467 vector +T18 CHEBI:36357 533 542 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NCBITaxon:8015 2453 2466 salmonid fish +T77 GO:0030431 2486 2494 Sleeping +T78 SO:0000101 2507 2517 transposon +T79 SO:0000101 2578 2599 transposable elements +T80 NCBITaxon:7742 2632 2642 vertebrate +T81 GO:0009294 2643 2657 transformation +T82 CHEBI:25435 2692 2699 mutagen +T83 SO:0000101 2744 2754 transposon +T84 NCBITaxon:10088 2779 2784 mouse +T85 NCBITaxon:10088 2940 2945 mouse +T86 NCBITaxon:10088 2998 3003 mouse +T87 SO:0000704 3004 3009 genes +T88 SO:0000704 3093 3100 genetic +T89 SO:0000704 3149 3156 genetic +T90 NCBITaxon:7215 3169 3179 Drosophila +T91 GO:0065007 3196 3204 regulate +T92 SO:0000704 3205 3210 genes +T93 UBERON:0000479 3216 3222 tissue +T94 SO:0000704 3307 3312 genes +T95 GO:0065007 3339 3346 control +T96 GO:0010467 3351 3361 expression +T97 NCBITaxon:10088 3378 3383 mouse +T98 SO:0000704 3384 3388 gene +T99 UBERON:0000479 3428 3435 tissues +T100 GO:0010468 3511 3537 control of gene expression +T101 SO:0000704 3522 3526 gene +T102 SO:0000704 3592 3596 gene 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+T131 NCBITaxon:40674 4678 4687 mammalian +T132 CHEBI:25435 4736 4745 mutagenic +T133 SO:0000704 4756 4760 gene +T134 SO:0000101 4766 4776 transposon +T135 SO:0000440 4777 4783 vector +T136 NCBITaxon:10088 4807 4812 mouse +T137 SO:0000704 4813 4817 gene +T138 GO:0010467 4822 4829 express +T139 CHEBI:27902 4834 4837 Tet +T140 UBERON:0000479 4886 4892 tissue +T141 GO:0065007 4909 4918 regulated +T142 SO:0000704 4932 4936 gene +T143 SO:0000704 4958 4963 genes +T144 GO:0010467 4981 4991 expression +T145 GO:0010467 5073 5083 expression +T146 SO:0000902 5087 5097 transgenes +T147 GO:0065007 5103 5112 regulable +T148 GO:0030431 5206 5214 Sleeping +T149 SO:0000101 5222 5232 transposon +T150 NCBITaxon:10088 5243 5248 mouse +T151 SO:0000101 5291 5301 transposon +T152 SO:0000704 5441 5445 gene +T153 SO:0000101 5468 5478 transposon +T154 SO:0000704 5485 5489 gene +T155 SO:0000440 5499 5505 vector +T156 NCBITaxon:7955 5543 5552 zebrafish +T157 UBERON:0000922 5553 5560 embryos +T158 SO:0000704 5571 5575 gene +T159 SO:0000440 5585 5591 vector +T160 UBERON:0000479 5622 5629 tissues +T161 NCBITaxon:7955 5633 5642 zebrafish +T162 GO:0065007 5662 5671 regulated +T163 SO:0000902 5676 5685 transgene +T164 SO:0000704 5750 5754 gene +T165 PR:000003676 5791 5798 β-actin +T166 GO:0008380 5799 5805 splice +T167 NCBITaxon:12104 5820 5846 encephalomyocarditis virus +T168 NCBITaxon:12104 5848 5852 EMCV +T169 SO:0000243 5854 5883 internal ribosomal entry site +T170 GO:0005840 5863 5872 ribosomal +T171 SO:0000243 5885 5889 IRES +T172 CHEBI:27902 5896 5899 Tet +T173 NCBITaxon:10633 5962 5966 SV40 +T174 GO:0043631 5972 5987 polyadenylation +T175 SO:0000551 5972 5994 polyadenylation signal +T176 SO:0000319 6232 6242 stop codon +T177 SO:0000717 6261 6274 reading frame +T178 SO:0000243 6296 6300 IRES +T179 SO:0000101 6317 6327 transposon +T180 SO:0000440 6328 6334 vector +T181 GO:0008380 6384 6390 splice +T182 NCBITaxon:10088 6409 6414 mouse +T183 CHEBI:17368 6415 6427 hypoxanthine +T184 SO:0000704 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translation initiation +T212 SO:0000236 7245 7263 open reading frame +T213 SO:0000440 7318 7324 vector +T214 SO:0000704 7346 7350 gene +T215 SO:0000704 7410 7414 gene +T216 GO:0008380 7456 7464 splicing +T217 GO:0008380 7479 7485 splice +T218 GO:0010467 7509 7516 express +T219 NCBITaxon:10088 7548 7552 mice +T220 SO:0000704 7592 7596 gene +T221 SO:0000704 7642 7646 gene +T222 GO:0010467 7642 7657 gene expression +T223 CHEBI:42768 7677 7681 G418 +T224 SO:0005853 7693 7701 cassette +T225 SO:0000101 7733 7743 transposon +T226 SO:0000440 7744 7750 vector +T227 GO:0065007 7842 7851 regulated +T228 SO:0000902 7863 7872 transgene +T229 GO:0009294 7882 7894 transfection +T230 SO:0000101 7920 7930 transposon +T231 SO:0000755 7931 7945 vector plasmid +T232 SO:0000704 7995 7999 gene +T233 SO:0001026 8049 8055 genome +T234 SO:0005853 8067 8075 cassette +T235 CHEBI:42768 8084 8088 G418 +T236 GO:0010467 8128 8135 express +T237 SO:0000101 8140 8150 transposon +T238 SO:0000440 8151 8157 vector +T239 SO:0000704 8167 8171 gene +T240 SO:0005853 8181 8189 cassette +T241 GO:0008380 8204 8212 splicing +T242 GO:0010467 8217 8224 express +T243 SO:0000101 8241 8251 transposon +T244 GO:0010467 8264 8273 expressed +T245 SO:0000704 8274 8278 gene +T246 CHEBI:42768 8371 8375 G418 +T247 SO:0000101 8418 8428 transposon +T248 CHEBI:42768 8453 8457 G418 +T249 SO:0005853 8469 8477 cassette +T250 GO:0008380 8491 8497 splice +T251 SO:0000243 8507 8511 IRES +T252 SO:0005853 8516 8524 cassette +T253 GO:0010467 8554 8564 expression +T254 GO:0065007 8753 8762 regulable +T255 CHEBI:23888 8784 8788 drug +T256 CHEBI:27902 8789 8801 tetracycline +T257 CHEBI:50845 8816 8827 doxycycline +T258 CHEBI:50845 8878 8889 doxycycline +T259 CHEBI:50845 8970 8981 doxycycline +T260 SO:0000704 9209 9213 gene +T261 GO:0008380 9251 9259 splicing +T262 SO:0000147 9277 9281 exon +T263 GO:0010467 9288 9297 expressed +T264 SO:0000704 9298 9302 gene +T265 SO:0000112 9366 9373 primers +T266 SO:0000704 9390 9394 gene +T267 SO:0000704 9506 9510 gene +T268 SO:0000147 9579 9583 exon +T269 SO:0000704 9591 9595 gene +T270 GO:0008380 9599 9606 spliced +T271 SO:0000704 9616 9620 gene +T272 CHEBI:36357 9706 9714 molecule +T273 GO:0010467 9722 9731 expressed +T274 SO:0000704 9741 9745 gene +T275 SO:0000440 9755 9761 vector +T276 SO:0000704 9790 9794 gene +T277 GO:0065007 9813 9822 regulated +T278 SO:0000902 9823 9832 transgene +T279 CHEBI:50845 9892 9903 doxycycline +T280 NCBITaxon:10088 9937 9942 mouse +T281 GO:0065007 9956 9965 regulated +T282 SO:0000704 9966 9970 gene +T283 NCBITaxon:10088 10093 10097 mice +T284 SO:0000101 10148 10158 transposon +T285 SO:0000440 10159 10166 vectors +T286 GO:0045120 10179 10189 pronuclear +T287 SO:0000902 10300 10309 transgene +T288 NCBITaxon:10088 10344 10349 mouse +T289 SO:0001026 10350 10356 genome +T290 SO:0000902 10389 10399 transgenes +T291 NCBITaxon:10088 10405 10410 mouse +T292 SO:0000101 10428 10438 transposon +T293 SO:0000902 10439 10448 transgene +T294 SO:0000704 10588 10592 gene +T295 SO:0000101 10602 10613 transposons +T296 SO:0000101 10742 10753 transposons +T297 SO:0000101 10918 10928 transposon +T298 SO:0000101 11024 11035 transposons +T299 GO:0010467 11116 11126 expressing +T300 NCBITaxon:10088 11179 11183 mice +T301 NCBITaxon:33208 11213 11220 animals +T302 SO:0000704 11232 11236 gene +T303 UBERON:0002415 11298 11302 tail +T304 NCBITaxon:10088 11337 11341 mice +T305 SO:0000101 11413 11423 transposon +T306 CL:0000300 11439 11445 gamete +T307 NCBITaxon:10088 11488 11492 mice +T308 SO:0000101 11566 11576 transposon +T309 SO:0000101 11699 11709 transposon +T310 SO:0000101 11913 11923 transposon +T311 SO:0000101 11984 11995 transposons +T312 NCBITaxon:10088 12010 12014 mice +T313 SO:0000101 12024 12035 transposons +T314 SO:0001026 12131 12137 genome +T315 SO:0000704 12312 12316 gene +T316 SO:0000101 12429 12440 transposons +T317 GO:0007276 12484 12497 gametogenesis +T318 GO:0018995 12499 12503 host +T319 GO:0006281 12504 12514 DNA repair +T320 SO:0000028 12577 12587 base pairs +T321 GO:0006281 12594 12603 repairing +T322 SO:0000985 12608 12621 double-strand +T323 NCBITaxon:10088 12722 12726 mice +T324 SO:0000101 12753 12763 transposon +T325 SO:0000902 12805 12814 transgene +T326 SO:0000357 12950 12955 flank +T327 UBERON:0000922 13097 13106 embryonic +T328 NCBITaxon:10088 13157 13162 mouse +T329 PR:000015018 13163 13171 Slc25a22 +T330 SO:0000704 13172 13176 gene +T331 SO:0000704 13207 13211 gene +T332 NCBITaxon:10088 13313 13317 mice +T333 NCBITaxon:10088 13344 13348 mice +T334 SO:0000346 13480 13490 loxP sites +T335 SO:0000440 13511 13517 vector +T336 SO:0000357 13529 13537 flanking +T337 CHEBI:25435 13542 13551 mutagenic +T338 SO:0000704 13567 13571 gene +T339 SO:0000440 13577 13583 vector +T340 UBERON:0000479 13615 13622 tissues +T341 SO:0000704 13807 13811 gene +T342 NCBITaxon:10088 13833 13838 mouse +T343 SO:0000704 13839 13843 gene +T344 GO:0010467 13864 13874 expression +T345 UBERON:0000479 13899 13905 tissue +T346 NCBITaxon:10088 14179 14183 mice +T347 SO:0000902 14239 14248 transgene +T348 NCBITaxon:10088 14355 14359 mice +T349 SO:0000101 14580 14590 transposon +T350 CHEBI:25435 14726 14735 mutagenic +T351 GO:0030431 14747 14755 Sleeping +T352 SO:0000704 14772 14776 gene +T353 SO:0000101 14870 14880 transposon +T354 NCBITaxon:10088 14917 14921 mice +T355 SO:0000101 14946 14956 transposon +T356 SO:0001026 14957 14964 genomic +T357 NCBITaxon:10088 15096 15100 mice +T358 NCBITaxon:10088 15163 15168 mouse +T359 SO:0001026 15169 15175 genome +T360 NCBITaxon:10088 15293 15298 mouse +T361 SO:0000101 15565 15576 transposons +T362 SO:0001026 15687 15693 genome +T363 SO:0000704 15726 15731 genes +T364 SO:0000112 15745 15751 primer +T365 SO:0000101 15818 15828 transposon +T366 SO:0000704 15898 15902 gene +T367 SO:0000440 15912 15918 vector +T368 NCBITaxon:10088 15951 15956 mouse +T369 SO:0000704 15957 15961 gene +T370 NCBITaxon:10088 16017 16022 mouse +T371 SO:0000704 16023 16028 genes +T372 SO:0000704 16106 16110 gene +T373 SO:0000188 16172 16178 intron +T374 PR:000004915 16186 16207 carbonic anhydrase-12 +T375 SO:0000704 16208 16212 gene +T376 PR:000004915 16214 16219 Car12 +T377 SO:0000188 16254 16260 intron +T378 SO:0000996 16270 16279;16286 16290 predicted ... gene +T379 GO:0010467 16356 16366 expression +T380 NCBITaxon:10088 16378 16383 mouse +T381 PR:000004915 16384 16389 Car12 +T382 UBERON:0000479 16467 16474 tissues +T383 SO:0000112 16481 16488 primers +T384 SO:0000147 16492 16497 exons +T385 SO:0000704 16514 16518 gene +T386 GO:0008380 16527 16535 splicing +T387 SO:0000243 16550 16554 IRES +T388 SO:0000147 16592 16596 exon +T389 PR:000004915 16606 16611 Car12 +T390 PR:000004915 16672 16677 Car12 +T391 PR:000004915 16682 16687 Car12 +T392 SO:0000243 16688 16692 IRES +T393 SO:0000079 16697 16720 bicistronic transcripts +T394 SO:0000673 16839 16849 transcript +T395 UBERON:0000479 16858 16865 tissues +T396 GO:0010467 16891 16901 expression +T397 PR:000004915 16913 16918 Car12 +T398 SO:0000673 16939 16949 transcript +T399 GO:0010467 16971 16981 expression +T400 UBERON:0000479 17005 17011 tissue +T401 SO:0000673 17046 17056 transcript +T402 UBERON:0000948 17080 17085 heart +T403 UBERON:0000948 17119 17124 heart +T404 PR:000004915 17228 17233 Car12 +T405 GO:0010467 17237 17246 expressed +T406 UBERON:0000948 17254 17259 heart +T407 PR:000003676 17289 17296 β-actin +T408 GO:0008380 17297 17303 splice +T409 SO:0000704 17361 17365 gene +T410 SO:0000147 17398 17402 exon +T411 GO:0010467 17536 17546 expression +T412 PR:000004915 17561 17566 Car12 +T413 SO:0000673 17589 17600 transcripts +T414 PR:000004915 17650 17655 Car12 +T415 UBERON:0002113 17668 17674 kidney +T416 UBERON:0000955 17679 17684 brain +T417 GO:0010467 17732 17742 Expression +T418 NCBITaxon:10088 17802 17807 mouse +T419 UBERON:0000955 17808 17813 brain +T420 PR:000003676 17927 17934 β-actin +T421 GO:0008380 17935 17941 splice +T422 PR:000004915 18007 18012 Car12 +T423 NCBITaxon:10088 18020 18024 mice +T424 GO:0010467 18175 18185 expression +T425 PR:000004915 18213 18219 CA-XII +T426 NCBITaxon:10088 18271 18276 mouse +T427 UBERON:0002113 18277 18283 kidney +T428 UBERON:0001155 18285 18290 colon +T429 UBERON:0000473 18296 18302 testes +T430 PR:000004915 18313 18319 CA-XII +T431 PR:000004915 18407 18413 CA-XII +T432 GO:0010467 18414 18424 expression +T433 UBERON:0002113 18445 18451 kidney +T434 UBERON:0002113 18482 18488 kidney +T435 GO:0042571 18490 18498 Antibody +T436 UBERON:0002113 18592 18598 kidney +T437 GO:0042571 18618 18626 antibody +T438 UBERON:0000479 18688 18694 Tissue +T439 SO:0000167 18724 18732 promoter +T440 GO:0065007 18733 18742 regulated +T441 SO:0000902 18743 18752 transgene +T442 SO:0000704 18793 18797 gene +T443 SO:0000440 18842 18849 vectors +T444 SO:0000167 18869 18877 promoter +T445 GO:0065007 18878 18887 regulated +T446 SO:0000902 18888 18897 transgene +T447 UBERON:0000479 18903 18909 tissue +T448 NCBITaxon:10088 18968 18972 mice +T449 GO:0065007 18995 19004 regulated +T450 SO:0000902 19016 19025 transgene +T451 NCBITaxon:10088 19060 19064 mice +T452 UBERON:0000479 19106 19112 tissue +T453 GO:0010467 19126 19136 expression +T454 CHEBI:50845 19142 19153 doxycycline +T455 UBERON:0000062 19220 19225 organ +T456 NCBITaxon:33208 19285 19292 animals +T457 UBERON:0000479 19332 19339 tissues +T458 SO:0000704 19456 19460 gene +T459 PR:000004915 19462 19467 Car12 +T460 GO:0010467 19476 19485 expressed +T461 UBERON:0002107 19493 19498 liver +T462 UBERON:0000479 19512 19519 tissues +T463 SO:0000101 19648 19659 transposons +T464 SO:0000902 19702 19712 transgenes +T465 GO:0010467 19757 19764 express +T466 GO:0010467 19812 19822 expression +T467 SO:0000101 19990 20011 transposable elements +T468 SO:0000101 20155 20166 transposons +T469 SO:0000101 20187 20197 transposon +T470 SO:0000704 20244 20248 gene +T471 NCBITaxon:10088 20305 20309 mice +T472 UBERON:0000479 20339 20345 tissue +T473 UBERON:0000955 20466 20471 brain +T474 NCBITaxon:10088 20520 20524 mice +T475 UBERON:0000479 20540 20547 tissues +T476 NCBITaxon:10088 20598 20602 mice +T477 UBERON:0000955 20667 20672 brain +T478 GO:0010467 20673 20683 expression +T479 GO:0008380 20726 20734 splicing +T480 GO:0008380 20755 20761 splice +T481 UBERON:0000479 20791 20797 tissue +T482 GO:0010467 20807 20817 expression +T483 GO:0010467 20905 20915 expression +T484 CHEBI:25078 21039 21048 luciferin +T485 UBERON:0000479 21119 21125 tissue +T486 SO:0000704 21165 21169 gene +T487 NCBITaxon:10088 21213 21217 mice +T488 UBERON:0000026 21261 21272 extremities +T489 UBERON:0000004 21274 21278 nose +T490 UBERON:0001690 21280 21284 ears +T491 UBERON:0002415 21290 21295 tails +T492 UBERON:0002398 21357 21362 hands +T493 UBERON:0000479 21388 21394 tissue +T494 NCBITaxon:10088 21510 21514 mice +T495 GO:0010467 21532 21542 expression +T496 UBERON:0000062 21580 21585 organ +T497 UBERON:0000062 21817 21822 organ +T498 GO:0010467 21860 21870 expression +T499 GO:0016020 21882 21890 Membrane +T500 PR:000010921 21882 21933 Membrane-spanning 4-domains, subfamily A, member 6C +T501 SO:0000417 21902 21909 domains +T502 PR:000010921 21935 21941 Ms4a6c +T503 PR:000002042 21965 21980 Integrin beta 3 +T504 PR:000002042 21982 21987 Itgb3 +T505 NCBITaxon:10088 22026 22031 mouse +T506 UBERON:0000948 22032 22037 heart +T507 UBERON:0019248 22041 22056 early embryonic +T508 GO:0010467 22104 22114 expression +T509 SO:0000704 22124 22128 gene +T510 SO:0000704 22163 22168 genes +T511 GO:0010467 22177 22186 expressed +T512 UBERON:0007023 22190 22195 adult +T513 UBERON:0000479 22196 22203 tissues +T514 GO:0010467 22252 22262 expression +T515 NCBITaxon:10088 22330 22334 mice +T516 NCBITaxon:10088 22336 22341 mouse +T517 NCBITaxon:10088 22392 22397 mouse +T518 UBERON:0001062 22484 22494 anatomical +T519 UBERON:0000975 22524 22531 sternum +T520 UBERON:0002106 22533 22539 spleen +T521 UBERON:0000981 22541 22547 femurs +T522 UBERON:0002412 22552 22561 vertebrae +T523 UBERON:0002371 22629 22640 bone marrow +T524 GO:0030097 22645 22658 hematopoietic +T525 CL:0000988 22645 22664 hematopoietic cells +T526 PR:000031436 22670 22675 MacF1 +T527 SO:0000704 22676 22680 gene +T528 SO:0000147 22699 22704 exons +T529 GO:0010467 22733 22742 expressed +T530 SO:0001060 22755 22763 isoforms +T531 NCBITaxon:10088 22771 22776 mouse +T532 UBERON:0002048 22810 22814 lung +T533 SO:0000112 22827 22834 primers +T534 SO:0000147 22838 22843 exons +T535 SO:0000357 22849 22854 flank +T536 PR:000031436 22889 22894 MacF1 +T537 SO:0000673 22895 22906 transcripts +T538 GO:0008380 22947 22953 splice +T539 GO:0010467 22995 23004 expressed +T540 UBERON:0000479 23016 23023 tissues +T541 PR:000031436 23029 23034 MacF1 +T542 GO:0010467 23051 23060 expressed +T543 UBERON:0002371 23068 23079 bone marrow +T544 NCBITaxon:10088 23095 23100 mouse +T545 PR:000031436 23149 23154 MacF1 +T546 GO:0010467 23174 23184 expression +T547 SO:0000704 23192 23196 gene +T548 UBERON:0002371 23204 23215 bone marrow +T549 GO:0030097 23220 23233 hematopoietic +T550 CL:0000988 23220 23239 hematopoietic cells +T551 PR:000031436 23292 23297 MacF1 +T552 SO:0000243 23298 23302 IRES +T553 SO:0000673 23307 23317 transcript +T554 UBERON:0002371 23323 23334 bone marrow +T555 NCBITaxon:10088 23357 23361 mice +T556 GO:0008380 23410 23417 spliced +T557 NCBITaxon:10088 23511 23516 mouse +T558 PR:000031436 23517 23522 MacF1 +T559 SO:0000704 23523 23527 gene +T560 NCBITaxon:10088 23581 23586 mouse +T561 NCBITaxon:10088 23672 23676 mice +T562 NCBITaxon:10088 23835 23839 mice +T563 SO:0000101 23904 23914 transposon +T564 NCBITaxon:10088 23929 23934 mouse +T565 PR:000031436 23950 23955 MacF1 +T566 NCBITaxon:10088 23970 23975 mouse +T567 PR:000031436 24148 24153 MacF1 +T568 SO:0000704 24189 24200 genetically +T569 SO:0000101 24266 24277 transposons +T570 UBERON:0002371 24483 24494 bone marrow +T571 UBERON:0000479 24556 24562 tissue +T572 SO:0000704 24572 24576 gene +T573 GO:0010467 24586 24596 expression +T574 NCBITaxon:10088 24657 24661 mice +T575 NCBITaxon:10088 24681 24685 mice +T576 UBERON:0000479 24878 24884 tissue +T577 NCBITaxon:10088 25012 25016 mice +T578 CHEBI:30212 25123 25130 photons +T579 SO:0000581 25257 25260 cap +T580 GO:0006412 25273 25284 translation +T581 CHEBI:36357 25296 25304 molecule +T582 SO:0000243 25314 25318 IRES +T583 NCBITaxon:10088 25327 25332 mouse +T584 SO:0000704 25425 25429 gene +T585 GO:0010467 25425 25440 gene expression +T586 CHEBI:27902 25444 25456 tetracycline +T587 CHEBI:50845 25472 25483 doxycycline +T588 GO:0065007 25527 25534 control +T589 SO:0000704 25535 25539 gene +T590 CHEBI:50845 25591 25602 doxycycline +T591 GO:0042756 25610 25618 drinking +T592 CHEBI:15377 25619 25624 water +T593 NCBITaxon:10088 25657 25661 mice +T594 NCBITaxon:33208 25725 25732 animals +T595 SO:0000101 25896 25906 transposon +T596 SO:0000704 25912 25916 gene +T597 SO:0000440 25922 25928 vector +T598 GO:0010467 25934 25943 expresses +T599 CHEBI:27902 25948 25951 Tet +T600 SO:0000704 26014 26019 genes +T601 SO:0000704 26046 26050 gene +T602 CL:0000010 26089 26103 cultured cells +T603 SO:0000704 26137 26142 genes +T604 GO:0008380 26153 26161 splicing +T605 SO:0000704 26171 26176 genes +T606 GO:0010467 26180 26187 express +T607 GO:0065007 26254 26263 regulated +T608 SO:0000902 26264 26273 transgene +T609 CHEBI:50845 26279 26290 doxycycline +T610 SO:0000704 26331 26335 gene +T611 SO:0000101 26345 26355 transposon +T612 NCBITaxon:10088 26372 26377 mouse +T613 SO:0000704 26378 26383 genes +T614 SO:0000440 26402 26408 vector +T615 GO:0010467 26443 26453 expression +T616 NCBITaxon:10088 26459 26464 mouse +T617 SO:0000704 26465 26469 gene +T618 UBERON:0000479 26492 26498 tissue +T619 GO:0010467 26521 26531 expression +T620 SO:0000167 26541 26549 promoter +T621 GO:0065007 26550 26559 regulated +T622 SO:0000902 26560 26569 transgene +T623 CHEBI:50845 26575 26586 doxycycline +T624 SO:0000440 26635 26641 vector +T625 NCBITaxon:10088 26763 26767 mice +T626 GO:0010467 26773 26780 express +T627 GO:0065007 26804 26814 controlled +T628 SO:0000101 26838 26848 transposon +T629 NCBITaxon:10088 26875 26880 mouse +T630 CL:0000300 26970 26976 gamete +T631 SO:0000101 27013 27023 transposon +T632 SO:0001026 27142 27148 genome +T633 SO:0001026 27154 27161 genomic +T634 SO:0000101 27178 27188 transposon +T635 SO:0001026 27339 27345 genome +T636 GO:0031507 27615 27637 heterochromatinization +T637 SO:0000101 27694 27704 transposon +T638 SO:0000704 27711 27715 gene +T639 SO:0000440 27721 27728 vectors +T640 GO:0010467 27765 27775 expression +T641 NCBITaxon:10088 27779 27784 mouse +T642 SO:0000704 27785 27790 genes +T643 SO:0000440 27807 27813 vector +T644 GO:0010467 27840 27850 expression +T645 NCBITaxon:10088 27856 27861 mouse +T646 SO:0000704 27862 27866 gene +T647 NCBITaxon:10088 27930 27935 mouse +T648 PR:000004915 27936 27941 Car12 +T649 SO:0000704 27942 27946 gene +T650 SO:0000704 27956 27960 gene +T651 SO:0000704 27991 27995 gene +T652 GO:0010467 27991 28006 gene expression +T653 NCBITaxon:10088 28163 28168 mouse +T654 SO:0000704 28169 28173 gene +T655 SO:0000440 28239 28246 vectors +T656 GO:0008380 28248 28254 Splice +T657 SO:0000704 28288 28293 genes +T658 GO:0043631 28318 28333 polyadenylation +T659 SO:0000551 28318 28341 polyadenylation signals +T660 CL:0002322 28400 28407 ES cell +T661 SO:0000155 28408 28415 plasmid +T662 NCBITaxon:11632 28421 28431 retroviral +T663 SO:0000704 28438 28442 gene +T664 SO:0000440 28548 28555 vectors +T665 NCBITaxon:10088 28569 28574 mouse +T666 SO:0000704 28575 28580 genes +T667 SO:0000440 28601 28608 vectors +T668 SO:0000704 28636 28641 genes +T669 CHEBI:27902 28685 28697 tetracycline +T670 GO:0065007 28698 28708 controlled +T671 GO:0010467 28763 28772 expressed +T672 SO:0000243 28782 28786 IRES +T673 SO:0000704 28794 28798 gene +T674 SO:0000704 28870 28874 gene +T675 SO:0000440 28880 28886 vector +T676 GO:0010467 28925 28935 expression +T677 SO:0000243 28971 28975 IRES +T678 SO:0000243 29007 29011 IRES +T679 NCBITaxon:39107 29029 29035 murine +T680 PR:000011251 29036 29039 Gtx +T681 SO:0000704 29052 29056 gene +T682 SO:0000243 29082 29086 IRES +T683 GO:0010467 29131 29141 expression +T684 GO:0010467 29188 29198 expression +T685 PR:000031436 29271 29276 MacF1 +T686 SO:0000704 29277 29281 gene +T687 UBERON:0002371 29328 29339 bone marrow +T688 GO:0030097 29345 29358 hematopoietic +T689 CL:0000988 29345 29364 hematopoietic cells +T690 GO:0010467 29383 29393 expression +T691 SO:0000831 29394 29403;29409 29413 domain of ... gene +T692 SO:0000704 29480 29484 gene +T693 GO:0008380 29493 29500 spliced +T694 SO:0000704 29510 29514 gene +T695 SO:0000101 29773 29784 transposons +T696 GO:0010467 29821 29831 expression +T697 PR:000031436 30030 30035 MacF1 +T698 SO:0001026 30081 30088 genomic +T699 NCBITaxon:10088 30177 30181 mice +T700 SO:0000704 30257 30261 gene +T701 NCBITaxon:10088 30364 30368 mice +T702 UBERON:0000479 30374 30380 tissue +T703 GO:0010467 30394 30404 expression +T704 SO:0000101 30585 30596 transposons +T705 NCBITaxon:10088 30641 30645 mice +T706 SO:0000101 30772 30783 transposons +T707 SO:0000155 30864 30871 plasmid +T708 SO:0000704 30886 30890 gene +T709 SO:0000101 30900 30910 transposon +T710 NCBITaxon:10088 30985 30990 mouse +T711 UBERON:0000922 30991 30998 embryos +T712 UBERON:0000922 31016 31022 embryo +T713 GO:0006412 31048 31058 translated +T714 NCBITaxon:10088 31106 31111 mouse +T715 SO:0001026 31112 31118 genome +T716 GO:0045120 31176 31186 pronuclear +T717 NCBITaxon:10088 31247 31251 mice +T718 SO:0001026 31300 31306 genome +T719 SO:0000018 31321 31335 linkage groups +T720 PR:P04386 31503 31507 GAL4 +T721 SO:0000165 31512 31520 enhancer +T722 NCBITaxon:7147 31546 31549 fly +T723 GO:0010467 31551 31561 expression +T724 SO:0000704 31610 31615 genes +T725 GO:0010467 31644 31654 expression +T726 UBERON:0000479 31677 31683 tissue +T727 GO:0010467 31697 31707 expression +T728 NCBITaxon:10088 31758 31763 mouse +T729 GO:0010467 31764 31774 expressing +T730 NCBITaxon:7049 31775 31782 firefly +T731 GO:0065007 31804 31811 control +T732 CHEBI:27902 31819 31822 tet +T733 SO:0000165 31832 31840 enhancer +T734 SO:0000167 31841 31849 promoter +T735 SO:0000704 31880 31884 gene +T736 SO:0000704 32029 32033 gene +T737 GO:0010467 32067 32077 expression +T738 UBERON:0000479 32094 32100 tissue +T739 NCBITaxon:10088 32163 32168 mouse +T740 GO:0065007 32191 32200 regulated +T741 SO:0000902 32201 32211 transgenes +T742 GO:0010467 32258 32268 expression +T743 SO:0000704 32331 32335 gene +T744 SO:0000101 32345 32355 transposon +T745 UBERON:0000479 32424 32430 tissue +T746 NCBITaxon:10088 32481 32486 mouse +T747 SO:0000704 32512 32516 gene +T748 SO:0000440 32526 32532 vector +T749 NCBITaxon:10088 32538 32543 mouse +T750 SO:0000704 32544 32549 genes +T751 GO:0010467 32586 32596 expression +T752 NCBITaxon:10088 32643 32648 mouse +T753 SO:0000704 32649 32656 genetic +T754 UBERON:0000479 32666 32672 Tissue +T755 SO:0000167 32682 32691 promoters +T756 GO:0065007 32696 32703 control +T757 GO:0010467 32708 32718 expression +T758 CHEBI:27902 32765 32777 tetracycline +T759 GO:0065007 32778 32788 controlled +T760 NCBITaxon:33208 32819 32826 animals +T761 UBERON:0000479 32843 32849 tissue +T762 GO:0010468 32872 32898 control of gene expression +T763 SO:0000704 32883 32887 gene +T764 NCBITaxon:10088 32906 32911 mouse +T765 UBERON:0000479 32912 32918 tissue +T766 SO:0000704 32991 32995 gene +T767 SO:0000101 33080 33090 transposon +T768 SO:0000440 33091 33097 vector +T769 NCBITaxon:10088 33208 33213 mouse +T770 SO:0001026 33214 33220 genome +T771 SO:0000704 33247 33251 gene +T772 GO:0010467 33289 33299 expression +T773 SO:0000902 33329 33338 transgene +T774 GO:0065007 33372 33381 regulated +T775 SO:0000704 33414 33418 gene +T776 GO:0010467 33512 33522 expression +T777 SO:0000902 33526 33536 transgenes +T778 SO:0000704 33556 33560 gene +T779 SO:0000101 33570 33580 transposon +T780 SO:0000440 33581 33588 vectors +T781 SO:0000112 33594 33600 primer +T782 SO:0000704 33676 33680 gene +T783 SO:0000440 33690 33697 vectors +T784 SO:0000147 33788 33792 exon +T785 PR:000003676 33808 33815 β-actin +T786 SO:0000147 33816 33820 exon +T787 SO:0000319 33830 33841 stop codons +T788 SO:0000717 33850 33863 reading frame +T789 GO:0097617 33880 33889 annealing +T790 GO:0006266 33967 33975 ligation +T791 NCBITaxon:10665 33981 33983 T4 +T792 PR:P43870 34243 34250 HindIII +T793 PR:P43870 34267 34274 HindIII +T794 PR:P43870 34362 34369 HindIII +T795 SO:0000155 34616 34623 plasmid +T796 SO:0000112 34629 34636 primers +T797 SO:0000028 34670 34672 bp +T798 SO:0001261 34673 34680 overlap +T799 GO:0009294 34720 34734 transformation +T800 NCBITaxon:562 34736 34743 E. coli +T801 GO:0006281 34744 34751 repairs +T802 SO:0000155 34756 34763 plasmid +T803 SO:0001261 34799 34816 overlapped region +T804 SO:0000155 34835 34842 plasmid +T805 SO:0000346 34863 34872 loxP site +T806 GO:0097617 34885 34894 annealing +T807 SO:0000028 34942 34944 bp +T808 SO:0000346 34945 34958 loxP sequence +T809 SO:0000346 35109 35122 loxP sequence +T810 SO:0000346 35143 35147 loxP +T811 SO:0000346 35187 35191 loxP +T812 SO:0000101 35236 35246 transposon +T813 SO:0000440 35247 35253 vector +T814 SO:0000346 35319 35323 loxP +T815 GO:0008380 35383 35389 splice +T816 GO:0097617 35449 35457 annealed +T817 SO:0000346 35503 35507 loxP +T818 SO:0000346 35530 35534 loxp +T819 SO:0000028 35632 35634 bp +T820 SO:0000141 35635 35671 transcriptional termination sequence +T821 NCBITaxon:9606 35681 35686 human +T822 SO:0000704 35695 35699 gene +T823 SO:0000346 35804 35808 loxp +T824 SO:0000346 35835 35839 loxp +T825 SO:0000755 35920 35935 plasmid vectors +T826 GO:0043631 35955 35970 polyadenylation +T827 SO:0000551 35955 35978 polyadenylation signals +T828 SO:0000704 36072 36076 gene +T829 SO:0000346 36101 36110 loxP site +T830 GO:0097617 36123 36132 annealing +T831 GO:0006266 36159 36167 ligation +T832 SO:0000346 36202 36206 loxp +T833 SO:0000704 36318 36322 gene +T834 GO:0009294 36404 36415 transfected +T835 SO:0000155 36425 36432 plasmid +T836 CHEBI:33893 36516 36523 reagent +T837 CHEBI:17939 36554 36563 puromycin +T838 GO:0010467 36733 36743 expression +T839 GO:0009294 36779 36790 transfected +T840 GO:0009294 36908 36919 transfected +T841 CHEBI:42768 37008 37012 G418 +T842 GO:0040007 37105 37110 grown +T843 GO:0019835 37189 37194 Lysis +T844 CHEBI:33893 37195 37202 Reagent +T845 GO:0019835 37271 37276 lysis +T846 CHEBI:50845 37554 37565 Doxycycline +T847 SO:0000112 37759 37765 primer +T848 CL:0000010 37840 37854 cultured cells +T849 NCBITaxon:10088 37858 37863 mouse +T850 UBERON:0000479 37864 37871 tissues +T851 CL:0000010 38162 38175 cultured cell +T852 NCBITaxon:10088 38179 38184 mouse +T853 UBERON:0000479 38185 38191 tissue +T854 SO:0000112 38250 38257 primers +T855 CHEBI:18320 38296 38310 dithiothreitol +T856 CHEBI:60004 38361 38368 mixture +T857 CHEBI:15377 38557 38562 water +T858 SO:0000112 38683 38690 primers +T859 CHEBI:6636 38768 38773 MgCl2 +T860 NCBITaxon:271 38789 38792 Taq +T861 SO:0000112 39181 39187 primer +T862 NCBITaxon:10088 39250 39254 mice +T863 GO:0045120 39258 39268 pronuclear +T864 GO:0097617 39300 39313 hybridization +T865 SO:0000028 39397 39399 bp +T866 CHEBI:16236 39454 39461 ethanol +T867 CHEBI:9754 39506 39510 Tris +T868 CHEBI:17996 39511 39513 Cl +T869 GO:0045120 39540 39550 pronuclear +T870 NCBITaxon:10088 39586 39590 mice +T871 SO:0000155 39729 39736 plasmid +T872 SO:0000815 39779 39787 minigene +T873 UBERON:0010166 39853 39857 coat +T874 NCBITaxon:33208 39869 39876 animals +T875 SO:0000101 39894 39904 transposon +T876 SO:0000815 39916 39924 minigene +T877 SO:0000366 39936 39950 insertion site +T878 GO:0010467 39952 39962 Expression +T879 NCBITaxon:10088 40090 40095 mouse +T880 UBERON:0002106 40119 40126 splenic +T881 CL:0000542 40127 40138 lymphocytes +T882 GO:0006412 40186 40197 translation +T883 SO:0000155 40223 40230 plasmid +T884 SO:0000153 40240 40243 BAC +T885 PR:P23940 40375 40380 BamHI +T886 SO:0001026 40390 40397 genomic +T887 SO:0000028 40457 40459 bp +T888 SO:0000236 40491 40509 open reading frame +T889 PR:P23940 40522 40527 BamHI +T890 SO:0000028 40616 40618 bp +T891 SO:0000028 40627 40629 bp +T892 SO:0000101 40658 40669 Transposons +T893 SO:0001026 40705 40712 genomic +T894 SO:0001026 40837 40844 genomic +T895 PR:P23940 40845 40850 BamHI +T896 PR:000004915 40893 40899 CA-XII +T897 GO:0010467 40900 40910 expression +T898 UBERON:0002113 40918 40924 kidney +T899 PR:000004915 40964 40970 CA-XII +T900 GO:0042571 40971 40979 antibody +T901 CHEBI:8984 41071 41074 SDS +T902 CHEBI:53250 41112 41116 PVDF +T903 PR:000004915 41159 41164 CAXII +T904 GO:0042571 41173 41181 antibody +T905 NCBITaxon:3704 41216 41227 horseradish +T906 MOP:0000779 41239 41249 conjugated +T907 NCBITaxon:9986 41255 41261 rabbit +T908 GO:0071735 41262 41265 IgG +T909 CHEBI:9754 41314 41318 Tris +T910 CHEBI:17883 41319 41322 HCl +T911 CHEBI:26710 41339 41343 NaCl +T912 CHEBI:53424 41351 41359 Tween 20 +T913 UBERON:0001913 41386 41390 milk +T914 GO:0010467 41447 41457 expression +T915 CHEBI:50845 41459 41470 doxycycline +T916 NCBITaxon:10088 41493 41497 mice +T917 CHEBI:25078 41561 41570 luciferin +T918 UBERON:0000062 41636 41641 organ +T919 NCBITaxon:10088 41652 41656 mice +T920 UBERON:0000062 41684 41690 organs +T921 GO:0019835 41743 41748 Lysis +T922 CHEBI:33893 41749 41756 Reagent +T923 GO:0042756 41823 41831 Drinking +T924 CHEBI:15377 41832 41837 water +T925 CHEBI:50845 41868 41879 Doxycycline +T926 CHEBI:32112 41889 41906 sodium saccharine +T927 SO:0000440 42005 42011 vector +T928 SO:0000704 42050 42054 gene +T929 SO:0000902 42080 42090 transgenes +T930 NCBITaxon:10088 42136 42140 mice +T931 GO:0010467 42282 42292 expression +T932 SO:0000704 42415 42419 gene +T933 NCBITaxon:10088 42454 42458 mice +T934 SO:0000704 42488 42492 gene +T935 SO:0000704 42791 42795 gene +T936 GO:0008380 42811 42819 Splicing +T937 SO:0000704 42830 42835 genes +T938 SO:0000704 42845 42849 gene +T939 GO:0008380 42961 42969 splicing +T940 SO:0000611 42986 42998 branch point +T941 PR:000003676 43011 43018 β-actin +T942 GO:0008380 43019 43025 splice +T943 SO:0000704 43075 43079 gene +T944 SO:0000147 43123 43127 exon +T945 SO:0000704 43144 43149 genes +T946 SO:0001026 43328 43335 genomic +T947 NCBITaxon:10088 43414 43418 mice +T948 SO:0000996 43555 43570 predicted genes +T949 NCBITaxon:10088 43817 43821 mice +T950 NCBITaxon:10088 43902 43907 mouse +T951 NCBITaxon:10088 43951 43956 mouse +T952 NCBITaxon:10088 44050 44054 mice +T953 SO:0000112 44220 44226 Primer +T954 PR:000004915 44374 44379 CAXII +T955 GO:0042571 44380 44388 antibody +T956 SO:0000101 44459 44469 Transposon +T957 SO:0000704 44705 44709 Gene +T958 SO:0000440 44719 44725 vector +T959 SO:0000440 44788 44795 vectors +T960 SO:0000704 44838 44843 genes +T961 SO:0000704 44898 44903 genes +T962 SO:0000704 44947 44951 gene +T963 PR:P23940 44957 44962 BamHI +T964 GO:0010467 44993 45003 expression +T965 SO:0000101 45035 45045 transposon +T966 SO:0000704 45052 45056 gene +T967 SO:0000440 45062 45068 vector +T968 SO:0000704 45084 45088 gene +T969 GO:0008380 45143 45151 splicing +T970 SO:0000243 45169 45173 IRES +T971 SO:0000716 45200 45216 bicistronic mRNA +T972 NCBITaxon:10633 45249 45253 SV40 +T973 GO:0043631 45259 45274 polyadenylation +T974 SO:0000553 45259 45279 polyadenylation site +T975 SO:0000716 45285 45301 bicistronic mRNA +T976 SO:0000581 45309 45312 cap +T977 GO:0006412 45325 45336 translation +T978 CHEBI:36357 45348 45356 molecule +T979 SO:0000147 45404 45409 exons +T980 GO:0065007 45428 45437 regulated +T981 SO:0000101 45500 45510 transposon +T982 SO:0000440 45511 45517 vector +T983 GO:0009294 45528 45540 transfection +T984 SO:0000155 45548 45555 plasmid +T985 GO:0010467 45658 45668 expression +T986 SO:0000704 45747 45751 gene +T987 CHEBI:50845 45845 45856 doxycycline +T988 GO:0010467 45908 45918 expression +T989 SO:0000101 45952 45962 transposon +T990 SO:0000101 46012 46022 transposon +T991 NCBITaxon:10088 46054 46059 mouse +T992 PR:000015018 46060 46068 Slc25a22 +T993 SO:0000704 46069 46073 gene +T994 NCBITaxon:33208 46125 46132 Animals +T995 SO:0000101 46158 46168 transposon +T996 SO:0000101 46425 46435 transposon +T997 SO:0000704 46519 46523 gene +T998 NCBITaxon:10088 46572 46577 mouse +T999 PR:000004915 46578 46583 Car12 +T1000 SO:0000704 46584 46588 gene +T1001 NCBITaxon:10088 46619 46624 mouse +T1002 SO:0000704 46644 46648 gene +T1003 SO:0000704 46650 46655 Genes +T1004 SO:0000147 46741 46746 exons +T1005 PR:000004915 46849 46854 Car12 +T1006 PR:000004915 46865 46870 Car12 +T1007 SO:0000243 46871 46875 IRES +T1008 UBERON:0000479 46946 46953 tissues +T1009 UBERON:0002113 46955 46957 Kd +T1010 UBERON:0002113 46958 46964 kidney +T1011 UBERON:0000955 46966 46968 Br +T1012 UBERON:0000955 46969 46974 brain +T1013 UBERON:0002107 46976 46978 Lv +T1014 UBERON:0002107 46979 46984 liver +T1015 UBERON:0000473 46986 46988 Te +T1016 UBERON:0000473 46989 46995 testes +T1017 UBERON:0001013 46997 46999 Ad +T1018 UBERON:0001013 47000 47007 adipose +T1019 UBERON:0001155 47009 47011 Co +T1020 UBERON:0001155 47012 47017 colon +T1021 UBERON:0002108 47018 47020 SI +T1022 UBERON:0002108 47021 47036 small intestine +T1023 UBERON:0000992 47038 47040 Ov +T1024 UBERON:0000992 47041 47046 ovary +T1025 UBERON:0000948 47059 47061 Ht +T1026 UBERON:0000948 47062 47067 heart +T1027 SO:0000704 47090 47094 gene +T1028 UBERON:0000479 47109 47116 tissues +T1029 PR:000004915 47255 47261 CA-XII +T1030 GO:0010467 47262 47272 expression +T1031 PR:000004915 47370 47376 CA-XII +T1032 GO:0042571 47377 47385 antibody +T1033 SO:0001060 47397 47405 isoforms +T1034 SO:0000704 47469 47473 gene +T1035 UBERON:0000479 47490 47496 tissue +T1036 CHEBI:27902 47522 47525 tet +T1037 SO:0000902 47537 47546 transgene +T1038 NCBITaxon:10088 47586 47591 mouse +T1039 SO:0000704 47592 47597 genes +T1040 SO:0000704 47641 47646 genes +T1041 SO:0000147 47716 47721 exons +T1042 SO:0000243 47784 47788 IRES +T1043 SO:0005853 47802 47810 cassette +T1044 UBERON:0000479 47865 47872 tissues +T1045 NCBITaxon:10088 47965 47969 mice +T1046 SO:0000704 48001 48005 gene +T1047 CHEBI:27902 48030 48033 tet +T1048 SO:0000902 48045 48054 transgene +T1049 NCBITaxon:10088 48140 48145 mouse +T1050 GO:0010467 48282 48292 expression +T1051 CHEBI:30212 48308 48315 photons +T1052 PR:000031436 48350 48355 MacF1 +T1053 GO:0010467 48356 48366 expression +T1054 UBERON:0007023 48379 48384 adult +T1055 UBERON:0000479 48385 48392 tissues +T1056 CL:0002322 48442 48444 ES +T1057 UBERON:0000922 48445 48454 embryonic +T1058 CL:0002322 48445 48465 embryonic stem cells +T1059 UBERON:0002107 48467 48469 Lv +T1060 UBERON:0002107 48470 48475 liver +T1061 UBERON:0002113 48477 48479 Kd +T1062 UBERON:0002113 48480 48486 kidney +T1063 UBERON:0000955 48488 48490 Br +T1064 UBERON:0000955 48491 48496 brain +T1065 UBERON:0002048 48498 48500 Lu +T1066 UBERON:0002048 48501 48505 lung +T1067 UBERON:0000948 48507 48509 Ht +T1068 UBERON:0000948 48510 48515 heart +T1069 UBERON:0002106 48517 48519 Sp +T1070 UBERON:0002106 48520 48526 spleen +T1071 UBERON:0000945 48528 48530 St +T1072 UBERON:0000945 48531 48538 stomach +T1073 UBERON:0002371 48540 48542 BM +T1074 UBERON:0002371 48543 48554 bone marrow +T1075 UBERON:0002108 48556 48558 SI +T1076 UBERON:0002108 48559 48574 small intestine +T1077 UBERON:0001155 48576 48578 Co +T1078 UBERON:0001155 48579 48584 colon +T1079 UBERON:0001013 48586 48588 Ad +T1080 UBERON:0001013 48589 48596 adipose +T1081 UBERON:0000992 48609 48611 Ov +T1082 UBERON:0000992 48612 48617 ovary +T1083 UBERON:0000473 48621 48627 testes +T1084 UBERON:0002370 48629 48631 Th +T1085 UBERON:0002370 48632 48638 thymus +T1086 NCBITaxon:10088 48663 48667 mice +T1087 PR:P23940 48758 48763 BamHI +T1088 SO:0000028 48781 48791 base pairs +T1089 SO:0000112 48850 48856 primer +T1090 NCBITaxon:10088 49019 49024 mouse +T1091 NCBITaxon:10088 49030 49034 Mice +T1092 CHEBI:30212 49123 49130 photons +T1093 CHEBI:50845 49236 49247 doxycycline +T1094 NCBITaxon:10088 49260 49264 mice +T1095 SO:0000704 49299 49303 gene +T1096 SO:0000101 49313 49324 transposons +T1097 NCBITaxon:10088 49350 49354 mice +T1098 SO:0000101 49359 49369 Transposon +T1099 NCBITaxon:10088 49627 49631 mice diff --git a/src/ontogpt/evaluation/craft/database/all/16800892.txt b/src/ontogpt/evaluation/craft/database/all/16800892.txt new file mode 100644 index 000000000..2d9ac0663 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16800892.txt @@ -0,0 +1,185 @@ +Conditional gene expression in the mouse using a Sleeping Beauty gene-trap transposon + +Abstract + +Background + +Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB) transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern. + +Results + +Here we report the systematic development and testing of a transposon-based gene-trap system incorporating the doxycycline-repressible Tet-Off (tTA) system that is capable of activating the expression of genes under control of a Tet response element (TRE) promoter. We demonstrate that the gene trap system is fully functional in vitro by introducing the "gene-trap tTA" vector into human cells by transposition and identifying clones that activate expression of a TRE-luciferase transgene in a doxycycline-dependent manner. In transgenic mice, we mobilize gene-trap tTA vectors, discover parameters that can affect germline mobilization rates, and identify candidate gene insertions to demonstrate the in vivo functionality of the vector system. We further demonstrate that the gene-trap can act as a reporter of endogenous gene expression and it can be coupled with bioluminescent imaging to identify genes with tissue-specific expression patterns. + +Conclusion + +Akin to the GAL4/UAS system used in the fly, we have made progress developing a tool for mutating and revealing the expression of mouse genes by generating the tTA transactivator in the presence of a secondary TRE-regulated reporter molecule. A vector like the gene-trap tTA could provide a means for both annotating mouse genes and creating a resource of mice that express a regulable transcription factor in temporally- and tissue-specific patterns for conditional gene expression studies. These mice would be a valuable resource to the mouse genetics community for purpose of dissecting mammalian gene function. + +Background + +Derived from ancient salmonid fish sequences [1], the Sleeping Beauty (SB) transposon is a member of the Tc1/mariner superfamily of cut-and-paste transposable elements [2] and has been developed as a vertebrate transformation tool [3] and germline insertional mutagen [4,5]. We and others have shown that the SB transposon is highly active in the mouse germline and can generate heritable loss-of-function mutations that lead to detectable phenotypes [4-7]. + +Data accumulated from spontaneous and engineered mouse mutations suggests that a significant percentage of mouse genes are essential for early development. This has necessitated the creation of various genetic tools for conditional loss- or gain-of-function genetic studies. In Drosophila, the ability to regulate genes in a tissue- or temporally-specific manner has become one method to study the function of these genes. Likewise, the ability to control the expression of an essential mouse gene is one way to discover its function in tissues that are formed after lethal phenotypes are manifest. Temporal and spatial control of gene expression in mutant backgrounds can be used to determine when a gene product is required during a developmental process [8-10]. With this in mind, and taking lessons from the Drosophila literature on GAL4/UAS-based P-element transposon systems [11], we designed a SB transposon-based gene trap system that allows us to annotate the expression pattern and function of mouse genes. + +Previous studies using the SB transposon system for generating insertional mutations in the germline of mice used various gene- and polyA-trap transposon vectors [4,5]. These vectors were designed to truncate an endogenous mRNA, incorporate coding sequences for a visible reporter like β-Galactosidase or green fluorescent protein (GFP), and report the expression patterns of trapped genes. If a transcriptional transactivator, similar to GAL4, were used in place of a visible reporter molecule, a small amount of the molecule could lead to high levels of a secondary reporter transgene under control of a responsive promoter. Analogous to the GAL4/UAS system, the tetracycline-controlled transactivator (tTA) and Tet-response element promoter (TRE) were developed for mammalian systems [12]. + +Here we report the creation of a mutagenic SB-based, gene-trap transposon vector that can insert into a mouse gene and express the Tet-off transcriptional transactivator (tTA) in its tissue- and temporally-regulated manner. The "gene-trap tTA" can mutate genes and record their expression patterns for functional annotation as well as create a mechanism for driving the expression of transgenes in a regulable manner in vivo. We also report several general parameters that impact the application of the Sleeping Beauty transposon system to mouse functional genomics including analysis of transposon mobilization rates in several different transgenic lines and the rescue of a mutant phenotype by remobilization. + +Results + +Creation of the gene-trap tTA system + +A SB transposon-based gene-trap tTA vector was first designed for and tested in zebrafish embryos [13]. The gene-trap tTA vector was shown function in somatic tissues of zebrafish and activate a TRE-regulated GFP transgene. T2/GT2/tTA and T2/GT3/tTA (Fig. 1A) are similar to the earlier gene-trap tTA (GT/tTA) and encode a carp β-actin splice acceptor, the encephalomyocarditis virus (EMCV) internal ribosomal entry site (IRES), the Tet-Off® (tTA) coding sequence (Clontech, Palo Alto, CA), and the SV40 late polyadenylation signal. Modifications are described in the Materials and Methods and include an upgrade to the 'T2' versions of the SB inverted terminal repeat sequences (ITR), demonstrated to increase transposition activity in vitro [14], and the addition of stop codon sequences in each reading frame just upstream of the IRES. The T2/GT3/tTA transposon vector (Fig. 1A) was additionally modified to include a splice acceptor from the mouse hypoxanthine phosphoribosyltransferase gene (HPRT) and a V5-epitope tag on the opposite strand of the transposon. This allows the gene trap to disrupt gene expression upon insertion into a mouse gene in either orientation. If open reading frame one were conserved after splicing from the endogenous exon, the V5-epitope would be added to the truncated peptide. + +Fig. 1B shows the method by which the gene-trap tTA can disrupt a mouse gene. When the gene-trap transposon lands in the intron of a gene, it will intercept splicing from an upstream exon and incorporate the IRES and tTA elements to form a bicistronic messenger RNA. Translation of the endogenous gene peptide is truncated at one of the stop codons incorporated just upstream of the IRES. The IRES element allows cap-independent translation initiation from the second open reading frame to produce tTA from the same mRNA. For the T2/GT2/tTA vector, an insertion into a gene in the opposite orientation is not predicted to mutate the gene, however, the T2/GT3/tTA would interrupt splicing with the HPRT splice acceptor, but will not express tTA. + +Before making transgenic mice, we validated the functionality of the gene-trap tTA as a faithful activator of reporter gene expression in vitro. First, a G418-resistance cassette was cloned into the T2/GT2/tTA transposon vector to generate T2/GT2/tTA/SVNeo (Fig. 1C). We then generated a HeLa cell line harboring a TRE-regulated luciferase transgene. Upon co-transfection of the pT2/GT2/tTA/SVNeo transposon vector plasmid and a source of SB transposase, pCMV-SB [1], the gene-trap tTA transposes at random into the HeLa cell genome. The SVNeo cassette confers G418 resistance to cells that integrate and express the transposon vector, but the gene-trap tTA cassette can intercept splicing and express tTA only if the transposon lands in an expressed gene in the correct orientation. Fig. 1D shows the luciferase activation in sixty-two individual G418-resistant clone extracts. As a control, a transposon that contained the same G418 resistance cassette, but not the splice acceptor-IRES-tTA cassette, did not activate luciferase expression (Fig. 1D, white bars). As expected, only a subset of T2/GT2/tTA/SVNeo-transgenic clones, 17 out of 50, showed activation of TRE-luciferase above background levels. + +The activity of tTA is regulable by administering the drug tetracycline or its analog doxycycline [15]. We tested this sensitivity by administering doxycycline to three of the TRE-luciferase activated clones from Fig. 1D. Fig. 1E shows the doxycycline-dependent repression of TRE-luciferase activation in each clone. Luciferase activity was reduced approximately two orders of magnitude in clones, 43 and 44, and about one order of magnitude in clone 39. Finally, to assure that gene trap tTA activation was dependent on splicing from an upstream exon of an expressed gene, 5' rapid amplification of cDNA ends (RACE) was performed with primers specific to the gene trap on mRNA extracts prepared from several clones. Additional file 1 shows the sequence tags, chromosomes and gene identification obtained for seven clones. In each case, an upstream exon of the gene is spliced into the gene-trap tTA at the predicted position (underlined). These data demonstrate that the tTA molecule can be expressed from the gene-trap tTA vector in the context of a trapped gene to activate a TRE-regulated transgene and that this activation is repressible by the addition of doxycycline. With the prospect of generating mouse strains with regulated gene-trap tTA activation, we set out to test the system in vivo. + +Parameters affecting germline mobilization rates + +Transgenic mice were generated with the T2/GT2/tTA and T2/GT3/tTA transposon vectors by standard pronuclear injection (see Materials and Methods). A typical result of this protocol is the head-to-tail concatenation of transgene units before integrating into the mouse genome to create a multi-copy array of transgenes in a mouse chromosome. Each transposon transgene concatemer, thus, serves as a donor site containing multiple substrates for mobilization by the SB transposase. To determine the number of gene-trap tTA transposons in each transgenic line, we performed Southern blot analysis using a restriction digest scheme that resolves the concatemerized transposons to a single band and estimated the number of copies based on its intensity (see Materials and Methods). Table 1 shows ten independent transgenic founder lines with transposon copy numbers ranging from approximately three to sixty copies. To test for mobilization of the transposons in the germline, each transgenic line was crossed to the CAGGS-SB10 transposase-expressing strain [16]. The resulting doubly transgenic "seed" mice were outcrossed to wild type animals to observe gene trap mobilization in the germline. Southern blot analysis on tail biopsy DNA from offspring of seed mice was used, as previously described [4,16], to observe the number of new transposon insertions per gamete. We averaged the insertion data from seed mice from each line to calculate the likelihood (rate per copy number) that a transposon could be mobilized from their respective concatemers (Table 1). The data demonstrates that lines with fewer copies of the transposon substrate show the lowest mobilization rates. This, however, is not a strict correlation. This is evident in comparing lines 6632 and 6657, where the former has fewer copies, but shows a higher per-copy-transposon mobilization rate of the latter. + +The inability to mobilize transposons in transgenic mice with few transposons in the concatemer is consistent with our efforts to re-mobilize a single copy insertion in the genome. We analyzed the mobilization of two independently generated single copy insertions. Insertion 01-0032 was generated and described in an earlier report [4] and 02A-0016 is a gene-trap tTA insertion identified in an offspring of a T2/GT2/tTA seed male (line 4583) as described below. When SB transposons are excised from a chromosome locus during gametogenesis, host DNA repair machinery typically creates a "footprint" of a few or several base pairs while repairing the double-strand-break gap [17]. We mobilized the insertions 01-0032 and 02A-0016 by intercrossing doubly transgenic mice that harbor a single-copy transposon insertion and the CAGGS-SB10 transposase transgene (Fig. 2A). We detected transposition in about one out of every 100 offspring for either insertion by PCR amplifying the sequences that flank the donor site and sequencing to detect a footprint (Fig. 2B). Notably, remobilization of insertion 01-0032, which causes an early recessive embryonic lethal phenotype as a result of disruption of the mouse Slc25a22 gene [4], restores activity of the gene and rescues the recessive lethality associated with this insertion as heterozygous 01-0032/footprint mice are phenotypically normal mice. The inability to efficiently remobilize a single copy insertion has been reported elsewhere [6]. For this reason, we incorporated loxP sites into the T2/GT3/tTA vector (Fig. 1A), flanking the mutagenic portion of the gene-trap vector. Cre-mediated recombination in tissues and in the germline have been demonstrated and are significantly higher than the 1% mobilization rate we can achieve by remobilizing single copy insertions [18-20]. Thus, a T2/GT3/tTA gene trap disruption of a mouse gene might be rescued by expression of Cre recombinase in a tissue- or germline-specific manner. + +Table 1 also demonstrates that germline mobilization rates are lower in the female germline when compared to their male siblings (dashed boxes). Finally, the solid box shows the difference in transposition rates between two sibling male seed mice, one hemizygous and one homozygous for the transposase transgene. While the SB10+/+ seed male did not show increased transposition over other independently generated seed mice for the 4563 line, there was an increase over its sibling SB10+/- seed male, suggesting that we may be able to increase transposition rates by increasing the transposase dosage in future studies. The analysis of several transposon-transgenic lines has thus revealed parameters that affect transposition rates, and suggest careful line selection will be required for mutagenic programs. + +Sleeping Beauty-mediated gene insertion in vivo + +T2/GT2/tTA line 4563 and T2/GT3/tTA line 6660 were useful to identify new transposon insertions in the offspring of seed mice. Techniques for cloning transposon-genomic DNA junction sequences have been described [1,4,21]. The chromosomal positions of thirty transposition events in offspring of seed mice from line 4563 were identified by querying the ENSEMBL online mouse genome database using the BLAST function. The T2/GT3/tTA line 6660 was used in a study of mutations induced specifically on mouse chromosome 11 and the insertion data from this line will be reported elsewhere (A.M.G., manuscript in preparation). Additional file 2 and additional file 3 show the data accumulated from the thirty T2/GT2/tTA events. Consistent with previous reports, the T2/GT2/tTA transposons exhibit local hopping, where a significant percentage of new insertions cluster in a particular region of the genome, and the insertions can land in genes [4,5]. Three-primer PCR [3] can readily be used to genotype carriers for any specific transposon insertion. Because we were interested in testing the function of the gene-trap tTA vector in the context of an endogenous mouse gene, carriers of insertions 02A-0001 and 02A-0002, both in mouse genes, (Fig. 3A) were propagated for further study. + +Molecular characterization of gene-trap tTA function + +Insertion number 02A-0001 is in the first intron of the carbonic anhydrase-12 gene (Car12), while 02A-0002 is inserted into intron 8 of the predicted novel gene ENSMUSG00000066992 (Fig. 3A). The top panel in Fig. 3B shows the expression pattern of mouse Car12 by reverse-transcriptase PCR (RT-PCR) on RNA extracts from several wild type tissues using primers in exons 1 and 5. If the gene trap is splicing properly, the IRES and tTA sequences should be fused to exon 1 of the Car12 mRNA. The second panel shows RT-PCR detection of endogenous Car12 and Car12-IRES-tTA bicistronic transcripts in RNA samples taken from a heterozygous carrier of insertion 02A-0001. Nested PCR was necessary to detect the fusion transcript in some tissues. Comparing the wild type expression pattern of Car12 mRNA and the fusion transcript, it is apparent that expression of the trapped mRNA is tissue specific as predicted. The fusion transcript can be detected in the heart of the carrier, but not wildtype heart RNA extracts. We attribute this to the extra sensitivity of the nested PCR and suspect that endogenous Car12 is expressed in the heart at a low level. + +If the carp β-actin splice acceptor is efficient, it should truncate the endogenous gene mRNA after the nearest upstream exon. Total RNA extracts from wild type, and heterozygous or homozygous carriers of both insertions 02A-0001 and 02A-0002 were probed for expression of endogenous Car12 or ENSMUSG00000066992 transcripts respectively. Fig. 3C shows complete ablation of Car12 mRNA in the kidney and brain of a homozygous carrier of insertion 02A-0001. Expression of ENSMUSG00000066992, detectable by RT-PCR exclusively in mouse brain (data not shown), was not ablated in homozygous carriers of insertion 02-0002, however, suggesting that the carp β-actin splice acceptor does not work efficiently in each case. + +Interestingly, Car12 mutant mice demonstrate reduced fitness, as an intercross of 02A-0001 carriers results in non-mendelian inheritance of the homozygous class (data not shown). The expression of the carbonic anhydrase (CA-XII) has been previously localized to the membranes of mouse kidney, colon, and testes [22]. The CA-XII peptide has been previously reported to be 46 kDA [23]. Fig. 3C shows the reduction of CA-XII expression in the heterozygous kidney and absence in the homozygous kidney. Antibody cross-reaction with a ~ 30-kDa peptide is consistent with cross-reactions previously seen in kidney extracts with this antibody [23], and serves as a loading control (arrowhead, Fig. 3C). + +Tissue-specific activation of a TRE promoter-regulated transgene in vivo + +The final in vivo test for the gene-trap tTA system is to determine whether the vectors can activate a TRE promoter-regulated transgene in a tissue-specific manner. We obtained the Tg(tetL)1Bjd/J strain of mice, transgenic for a TRE-regulated luciferase transgene, from Jackson Laboratories. These mice have been previously shown to respond to tissue-specific tTA expression in a doxycycline-dependent manner [24]. Luciferase activity assays from individual organ extracts from a 02A-0001; Tg(tetL)1Bjd/J doubly transgenic animals showed activation of luciferase in all tissues tested when compared to singly transgenic controls (data not shown). This result was surprising because the trapped gene, Car12, is not expressed in the liver (among other tissues) by RT-PCR (Fig. 3B). Insertion 02A-0001 is the result of local transposition, and thus is linked to the original concatemer of transposons. We reasoned that these linked T2/GT2/tTA transgenes present in carriers of this insertion might express tTA, therefore resulting ubiquitous luciferase expression when crossed to the Tg(tetL)1Bjd/J strain. To circumvent this issue, we focused on insertions that were not the result of local hopping and would segregate from other transposable elements. Insertions 02A-0002, 03A-0184, 03A-0217, and 03A-0241 (Figs. 3A and 4A) are the result of non-local transposition of T2/GT2/tTA or T2/GT3/tTA transposons in their respective transposon transgenic strains. Carriers of each of these gene insertions were crossed to the Tg(tetL)1Bjd/J strain of mice with the hope of visualizing tissue-specific, tTA-dependent transactivation of luciferase. As predicted, Fig. 4B shows weak activation of luciferase in the brain of a 02A-0002; Tg(tetL)1Bjd/J doubly transgenic mice, but not other tissues. This pattern was reproduced in three out of four mice. As shown above, this insertion does not completely disrupt the brain expression of ENSMUSG00000066992 (Fig. 3C), but some splicing into the T2/GT2/tTA splice acceptor must occur to allow tissue-specific expression of the tTA. + +Hasan, et al. (2001) demonstrated that non-invasive imaging of luciferase expression of a similar tTA-activated strain using an intensified charge-coupled device camera system after subcutaneous injection of luciferin substrate [25]. We used the Xenogen IVIS™100 Imaging System to see if tissue-specific patterns could be detected in gene-trap tTA; Tg(tetL)1Bjd/J doubly transgenic mice. Light emissions are often observed in the extremities, nose, ears, and tails of control Tg(tetL)1Bjd/J in a tTA-independent manner in our hands (Fig. 5A). No additional tissue-specific signals were initially detected in 02A-0001; Tg(tetL)1Bjd/J or 02A-0002; Tg(tetL)1Bjd/J doubly transgenic mice, suggesting that expression of luciferase levels detected in the organ extracts were not sufficient to be detected by the imager. Likewise, when crossed to the Tg(tetL)1Bjd/J strain, insertions 03A-0184 and 03A-0217 (Fig. 4A) showed no activation of luciferase by imaging or by luminometer readings of organ extracts (data not shown). While the expression pattern of Membrane-spanning 4-domains, subfamily A, member 6C (Ms4a6c) is not characterized, Integrin beta 3 (Itgb3) has been localized to the developing mouse heart in early embryonic stages [26]. It is possible that we missed the expression of these gene-trap tTA insertions because these genes are not expressed in adult tissues. + +In contrast, a striking pattern of luciferase expression was captured by imaging 03A-0241; Tg(tetL)1Bjd/J doubly transgenic mice (mouse 936) when compared to singly 03A-0241 transgenic (mouse 933) or Tg(tetL)1Bjd/J controls (Fig. 5A). Light emission patterns were detected from anatomical origins corresponding to the sternum, spleen, femurs and vertebrae, presumably reflecting luciferase activation in a component of the bone marrow and hematopoietic cells. The MacF1 gene has more than 100 exons and has been reported to be expressed in multiple isoforms in the mouse with high levels produced in the lung [27]. Using primers in exons that flank the 03A-0241 insertion to amplify MacF1 transcripts, Fig. 5B demonstrates that at least two splice variants of different sizes are variably expressed in several tissues, but MacF1 is not normally expressed in the bone marrow of a wild-type mouse. This suggests that the 03A-0241 insertion into MacF1 may cause abnormal expression of the gene in the bone marrow and hematopoietic cells. However, multiple attempts to amplify the chimeric MacF1-IRES-tTA transcript from bone marrow extracts from carrier mice by RT-PCR were unsuccessful in identifying this spliced transcript. + +To determine whether this pattern was linked only to insertion 03A-0241 and the mouse MacF1 gene, Southern blots and genotyping PCR were performed on mouse 936 (and siblings 931–935) and his offspring (mice 949–976) (Fig. 5C). Surprisingly, mice that inherited insertion 03A-0241 by PCR showed linkage to the concatemer donor site by Southern blot (Fig. 5A, arrowhead). This was unexpected because these mice were generated from T2/GT3/tTA line 6660 (Table 1), which has a transposon donor site on mouse chromosome 11. MacF1 is located on mouse chromosome 4 at 57.4 cM, thus the Southern data suggests that a large portion of the chromosome-11 concatemer had translocated by some mechanism to chromosome-4. Thus, the MacF1 insertion on chromosome 4 is still genetically linked to a translocated donor concatemer of multiple T2/GT3/tTA transposons in addition to other linked single-copy insertions (Fig. 5C, arrows). Repeated attempts to identify these other linked chromosome-4 insertions by our cloning techniques and 5'RACE for trapped sequences in bone marrow extracts were unsuccessful in identifying the origin of this tissue-specific gene-trap tTA expression. Nevertheless, the pattern seen in 03A-0241; Tg(tetL)1Bjd/J mice is unique to these mice, as it is not seen in carriers of other T2/GT2/tTA or T2/GT3/tTA insertions (with or without associated concatemers) when crossed to the Tg(tetL)1Bjd/J strain (above and data not shown). + +The tissue-specific luciferase pattern in carriers of this insertion could be reproducibly transmitted through the germline to offspring (mice 962–964 and 971–973, Fig. 5D). The upper range of luciferase activation from each offsrpring, measured in photons/second/cm2, ranged approximately a thousand-fold (scale bars). We attribute these differences to differences in the levels of cap-independent translation of the tTA molecule from the IRES in each mouse. Finally, other groups have demonstrated in vivo sensitivity of tTA-dependent activation of gene expression to tetracycline, or its analog doxycycline [24,28,29]. We determined whether we could control gene-trap tTA activation of luciferase by administering doxycycline to the drinking water of the 03A-0241; Tg(tetL)1Bjd/J mice. Fig. 5E demonstrates absence of luciferase signal in the same animals from Fig. 5D, after six days of treatment, suggesting complete repression of the tTA-dependent luciferase activation. + +Discussion + +We examined the utility of a SB transposon-base gene trap vector that expresses the Tet-off transcriptional transactivator in the patterns of trapped genes in vitro and in vivo. The gene-trap tTA demonstrated full utility in cultured cells, having the ability to insert in genes, trap the splicing of those genes to express the tTA transcription factor which, in turn, could activate a TRE-regulated transgene in a doxycycline-dependent manner. Likewise, analysis of gene-trap tTA transposon insertions into mouse genes revealed that the vector system can completely disrupt the expression of a mouse gene in some cases and can tissue-specifically activate expression of a TRE promoter-regulated transgene in a doxycycline-dependent manner in vivo. While ultimately this vector system can function as designed, several observations suggest we need a more optimal approach for creating a resource of mice that express tTA in developmentally-controlled patterns. + +Germline SB transposon mobilization rates in the mouse germline are variable and ranged from less than one in twenty to nearly three events per gamete. An evaluation of several different transposon-transgenic lines has given us insight into the potential the SB system to perform regional mutagenesis throughout the genome. The genomic location of the transposon donor site, along with copy number, impacts the germline mobilization rate from any concatemer (see Table 1). This suggests that not all parts of the genome are equally accessible to SB transposition. Understanding the mechanisms behind this "mobilization position effect" will likely require an increased understanding about molecular regulators of SB transposition, but may include a well known director of position effect, heterochromatinization [30]. + +As demonstrated in previous studies and here, SB transposon-based gene trap vectors can function in vivo to disrupt the expression of mouse genes. The T2/GT2/tTA vector can completely ablate the expression of a mouse gene as demonstrated by the null mutation that was generated in the mouse Car12 gene. Not all gene insertions completely disrupt gene expression however, as in the case of insertion 02A-0002 into ENSMUSG00000066992. While hypomorphic mutations may be valuable to discover the function of an essential mouse gene, this has lead to ideas on how to improve the efficacy of future vectors. Splice acceptors derived from different genes, termination sequences (polyadenylation signals) and other functional sequences have been used in several ES cell plasmid- and retroviral-based gene-trapping studies with varying efficiencies [31-33]. Drawing data from other groups will lead to improved vectors for trapping mouse genes and perhaps lead to vectors to trap certain classes of genes [34]. + +The activation of luciferase by the tetracycline-controlled transactivator varied rougly 1000-fold (Fig. 5D) when expressed from the IRES in the gene-trap tTA. Once again, improvements to the functional components of the gene trap vector may lead to more reliable and greater expression of the transactivator. Alternative IRES elements like the 9-nucleotide IRES element from the murine Gtx homeodomain gene [35], or eliminating the IRES entirely, may allow for more consistent tTA expression. + +The brilliant pattern of in vivo luciferase expression seen if Fig. 5 was initially thought to originate from insertion in the MacF1 gene. Published reports suggested that neither the bone marrow, nor hematopoietic cells are in the normal expression domain of this gene. Upon further molecular characterization, we discovered that this gene was not spliced into the gene-trap tTA, and by Southern blot, found it was tightly linked to other insertions as well as the concatemer. It seems more likely that the pattern of tTA-dependent luciferase activation seen in carriers of the 03A-0241 insertion comes from one of these linked transposons. Efforts to find the source of this expression by 5'RACE were unfortunately unsuccessful. We propose that in the case of insertion 03A-0241, the concatemer translocated to chromosome 4 first, then a single copy of T2/GT3/tTA hopped locally into MacF1. We have observed similar and other types of genomic rearrangements associated with transposition from the chromosome-11 donor site in other mice (A.M.G., manuscript in preparation). These observations would suggest that gene-trap tTA mobilization in the germline may not be the most efficient means of generating a resource of mice with tissue-specific tTA expression patterns. It is not clear, however, that these large donor site insertions are not rare events that could be tolerated. Independent insertions, unlinked to the concatemer or other transposons, are frequently recovered. By pre-selecting mice that do not inherit the concatemer by Southern blot or PCR analysis, a screen would largely avoid the complications of linked transposons. + +An alternative and relatively facile approach would be to inject a linearized plasmid harboring the gene-trap tTA transposon, along with in vitro-transcribed transposase messenger RNA, into one-cell mouse embryos. In the one-cell embryo, the transposase mRNA is translated and transposase catalyzes integration into the mouse genome at rates that are two to three-fold higher than standard pronuclear injection of naked DNA [3]. This method produces transgenic mice with insertions distributed randomly across the genome, and multiple linkage groups are frequently obtained from a single injection [3]. This would be another method to avoid the complications of linked insertions and concatemers. + +Finally, using the GAL4/UAS enhancer trap systems used in the fly, expression-based screening has been useful for identifying genes with similar or overlapping expression patterns [36] and for tissue-specific overexpression studies [37]. As we have shown here, a transgenic mouse expressing firefly luciferase under the control of the tet-response enhancer/promoter (TRE) can be coupled with the gene-trap tTA system to identify in vivo patterns with an intensified CCD camera system. In a screen, this type of imaging could be used to identify gene trap insertions that lead to tTA expression in a particular tissue or developmental stage, even in utero [38,39]. Alternatively, mouse strains for other TRE-regulated transgenes have been created, allowing for tTA-dependent expression of β-galactosidase [40] or GFP [41]. If the efficiency of the gene-trap tTA transposon can be improved, these strains could be used by any lab to identify tissue-specific patterns. + +Conclusion + +As a tool for the mouse community, introducing a gene-trap tTA vector into mouse genes with different spatial and temporal expression patterns would create a valuable tool for the mouse genetic toolbox. Tissue-specific promoters can control the expression of transactivators like tTA and rtTA (reverse tetracycline-controlled transactivator) in transgenic animals [42-44]. Having tissue-specific and temporal control of gene expression in any mouse tissue at any developmental stage could allow for the dissection of biological gene functions that were previously masked by a phenotype such as lethality. An SB-based transposon vector is well suited to accomplish this task by introducing precisely integrated functional reporter units into the mouse genome. In addition, an improved gene-trap tTA could provide more reliable expression of the transactivator than a transgene construct since transcription is regulated in the context of an endogenous gene and is thus less likely to be subject to the position effect variegation that can hinder the expression of transgenes. + +Methods + +Cloning gene-trap tTA transposon vectors + +All primer/oligo sequences for PCR and cloning are provided in Additional file 4. All gene-trap tTA vectors are descendent of pGT/tTA, originally described in Clark et al . 2004 [13]. An artificial exon (based on carp β-actin exon 2), with stop codons in each reading frame, was created by annealing four oligos (AMG049-AMG052), filling in the gaps with Klenow DNA polymerase, ligation with T4 DNA ligase, PCR amplifying with AMG049 and AMG051, and subcloning the AgeI fragment into the partially-digested SmaI-AgeI fragment of pGT/tTA to create pGT2/tTA. Subcloning the exonuclease-treated EcoRV-PstI fragment of pGT2/tTA into the Klenow-treated blunt HindIII fragment of pT2/HindIII [14] generated pT2/GT2/tTA. pT2/GT2/SVNeo was created by subcloning the Klenow-treated HindIII fragment of pT2/SVneo [14] into the EcoRV fragment of pT2/GT2/tTA. + +The construction of pT2/GT3/tTA was a multi-step process to add additional features to the GT2 version. First, an NheI site was added to pT2/GT2/tTA by PCR amplifying the entire plasmid with primers AG023 and AG024, containing a 12-bp overlap containing the NheI site sequence, and transformation. E. coli repairs the plasmid by homologous recombination in the overlapped region, resulting in the plasmid pT2/GT2/tTA/NheI. A loxP site was made by annealing two oligos (AG025 and AG026) containing the 34-bp loxP sequence with overhanging 4-base cohesive ends to NheI and cloned into the NheI site of pT2/GT2/tTA/NheI, destroying the NheI site on one side, and adding the loxP sequence to make pT2/GT2/tTA/loxP. The NheI-SpeI fragment of pT2/GT2/tTA/loxP was subcloned into the XbaI fragment of the transposon vector pT2/HB, which has a multiple cloning site, to create pT2/GT2/tTA/loxP-2. 70-mer oligos (SA/V5-1 and SA/V5-2) containing the HPRT splice acceptor and V5 epitope tag with EagI compatible ends were annealed and cloned into the EagI site of pT2/GT2/tTA/loxP-2 to make pT2/GT2/tTA/loxp/SA-V5. In the process, one side of the EagI site was destroyed, leaving a single EagI site. A 32-bp transcriptional termination sequence from the human GASTRIN gene was created by overlapping oligos (Termin-1 and Termin-2) and cloned into this EagI site of pT2/GT2/tTA/loxp/SA-V5 to make pT2/GT2/tTA/loxp/SA-V5/Termin. This sequence was shown to enhance transcriptional termination in plasmid vectors when downstream of polyadenylation signals [45], and was included to increase the chances of mRNA truncation, and thus mutation, in the gene trap. Finally, a second loxP site was made by annealing the same oligos above and ligation into the SpeI site of pT2/GT2/tTA/loxp/SA-V5/Termin to make the final product, pT2/GT3/tTA. + +Generation of Tre-Luciferase/Puro cell line and in vitro gene trap testing + +pTRE-Luc was kindly provided by Dr. Perry Hackett's lab and was co-transfected with the plasmid pKO Select-Puro (Stratagene, La Jolla, CA) into HeLa cells using Mirus TransIT-LT1 reagent (Promega, Madison, WI). After puromycin resistant clones were isolated, ten clones were tested for inducibility by pTet-off (Clontech, Palo Alto, CA). One clone TLP-10 showed a four-log increase in luciferase expression over background in the presence of transfected pTet-off (data not shown), and was subsequently used for the in vitro studies presented here. + +pT2/GT2/tTA/SVNeo was transfected into the TLP-10 cell line along with pCMV-SB [1], and clones were selected in 800 μg/mL G418. pT2/SVNeo [14] was used as a control. Fifty of these clones and twelve control clones were grown to confluency on 60 mm plates and cells harvested with Promega's Cell Culture Lysis Reagent for luciferase assays. Luciferase assays were performed on 20 μL of lysis extract using 100 μL of Promega's Luciferase Assay Substrate and a Lumat LB 9507 luminometer (Berthold, Bundoora, Australia) with a 15 second measuring time. Relative light unit (RLU) measurements were normalized to total protein concentration as determined by Bradford assay. Doxycycline (Sigma cat. #D-9891) was added to a final concentration of 2 mM to repress tTA-induction of TRE-Luciferase in clones 39, 43, and 44. + +RT-PCR and 5' rapid amplification of cDNA ends (RACE) + +All primer/oligo sequences for PCR are provided in Additional file 4. Total RNA from cultured cells or mouse tissues was extracted with Trizol® (Invitrogen, Carlsbad, CA). RT-PCR was performed using the one-step RobusT™ I RT-PCR Kit (Finnzymes, Espoo, Finland) according to the manufacturer's protocol. A template-switching reaction with Superscript III (Invitrogen) was used to make template for 5'RACE on cultured cell or mouse tissue RNA extract. 250 ng total RNA was mixed with 1 μM each of primers GT2-RT3 and RACE-1, along with 0.01 M dithiothreitol, 500 μM dNTPs, and 1 unit of Superscript III. The mixture was cycled six times at 52°C for 10', 50°C for 15', and then gradually cooled from 50°C to 37°C over two minutes before a final extension for 90" at 37°C. Template was diluted ten-fold in water and 2 μL was used for primary 5'RACE PCR. Briefly, the template was amplified in a 50 μL PCR reaction supplemented with primers GT2-RT3 (0.4 μM), KJC-002 (0.04 μM) and KJC-003 (0.1 μM), 200 μM dNTPS, 2 mM MgCl2, and 1 unit of Taq DNA polymerase (CLP, San Diego, CA). The PCR machine was programmed for touchdown PCR at 95°C for 3', 10 cycles of 95°C for 30", 65°C for 30" (-0.5°C per cycle), 70°C for 2', and then 25 cycles of 95°C for 30", 60°C for 30", and 70°C for 2'. The primary RACE reaction was diluted 1:50 and 2 μL used in a nested PCR under the same exact conditions, except supplemented with 0.5 μM of each primer GT2-RT2 and KJC-004. + +Generation of T2/GT2/tTA and T2/GT3/tTA mice by pronuclear injection, fluorescent in situ hybridization, and Southern blotting + +The 3379-bp FspI -SapI fragment of pT2/GT2/tTA or the 3738-bp FspI -SapI fragment of pT2/GT3/tTA were gel-purified, ethanol precipitated twice, and resuspended in 5 mM Tris-Cl (pH 7.5), 0.1 mM EDTA for pronuclear injection into the FVB/N strain of mice (Charles River Laboratories, Wilmington, MA). The pT2/GT3/tTA fragment was co-injected with the similarly-prepared HinP1I fragment of the plasmid pTYBS [Overbeek, 1991 #211], a tyrosinase minigene construct kindly provided by Dr. Paul Overbeek, in an attempt to coat-color mark animals that inherit the transposon/tyrosinase minigene transgenic insertion site. Expression of the tyrosinase, for unknown reasons, was not evident in any of the founders (data not shown). FISH on T2/GT3/tTA transgenic mouse lines was performed on splenic lymphocytes using standard techniques, and performing nick translation to label the pT2/GT3/tTA plasmid or whole BAC DNA from the Wellcome Trust Sanger Institute [46] were used as a probe. Standard Southern blotting techniques were used to analyze BamHI-digested genomic DNA from founders and subsequent generations using the 842-bp NcoI -SphI fragment of the tTA open reading frame as a probe. BamHI restricts each copy in any T2/GT2/tTA or T2/GT3/tTA concatemer to a single band at 2124-bp or 2236-bp respectively (see Fig. 1A). Transposons mobilized from the concatemer to a genomic site are detected as bands of variable size which are larger than the concatemer band, their size determined by the nearest genomic BamHI site (see Fig. 5C). + +Western blotting for CA-XII expression + +Whole kidney extracts were probed with a polyclonal CA-XII antibody kindly provided by the laboratory of Dr. William Sly. 50 μg total protein was run on a 10% SDS-PAGE (Invitrogen) and transferred to PVDF and probed with a 1:1000 dilution of anti-CAXII primary antibody followed by a 1:10000 dilution of horseradish peroxidase-conjugated Anti-rabbit IgG secondary (Amersham Biosciences) in TBST (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 % Tween 20) supplemented with 1% dry milk. + +In vivo luciferase imaging and analysis of luciferase expression, doxycycline treatment + +Transgenic mice were imaged as described in Wilber, et al . (2005) [47], using luciferin from Xenogen Corporation (Alameda, CA). For luciferase assays on organ extracts, mice were sacrificed, and whole organs dissected and homogenized in Promega's Cell Culture Lysis Reagent. Extracts were analyzed as described above for the in vitro work. Drinking water was supplemented with 5 mg/mL Doxycycline and 0.1% sodium saccharine for 6 days to suppress tTA-induced activation. + +Authors' contributions + +AMG performed the updated vector construction, in vitro testing of the gene-trap tTA, preparation of transgenes, breeding and characterization of transgenic mice, 5'RACE, RT-PCR, western blotting, Southern blotting, and aided the in vivo imaging studies. AW performed the in vivo imaging for luciferase expression. CMC bred and characterized 'rescue by remobilization' for insertion 01-0032. PDL performed PCR-based methods to identify gene-trap tTA insertions in transgenic mice. KJC originally designed the gene-trap tTA system and was instrumental to upgrading its design and functionality. PBH, RSM, and DAL are not only the mentors of the above authors, but were involved in the design and execution of this manuscript. + +Supplementary Material + +Additional File 1 + +T2/GT2/tTA/SVNeo clone 5'RACE sequence and gene identification Splicing events of genes into the gene-trap tTA were identified by 5'RACE PCR and sequencing. For seven clones, the partial sequence tag demonstrates splicing into the proper branch point of the carp β-actin splice acceptor, allowing identification of the trapped gene by the sequence of the adjoined endogenous exon sequence. These genes and their functions are described according to the ENSEMBL May 17, 2005 freeze of the NCBI m34 build. + +Click here for file + +Additional File 2 + +Germline T2/GT2/tTA insertions The genomic positions of thirty T2/GT2/tTA insertions identified in the offspring of seed mice from line 4563 (Table 1) are reported. Shown are the chromosome and position, along with the identification and description of known or predicted genes potentially disrupted for each insertion. This data is based on the ENSEMBL May17, 2005 freeze of the NCBI m34 build. + +Click here for file + +Additional File 3 + +Distribution of thirty T2/GT2/tTA insertions Cloning insertions from offspring of seed mice from transgenic line 4563 (Table 1) reveals two local hopping intervals, one on mouse chromosome 1 near 45.8 Mb, and a second on mouse chromosome-9 around 66.5 Mb. By Southern blot, it was later determined that the 4563 line of mice originally obtained and segregated two independent concatemer integrations during the initial transgenesis (data not shown) + +Click here for file + +Additional File 4 + +Primer sequences + +Click here for file + +Acknowledgements + +The authors would like to thank Dr. Abdul Waheed (Saint Louis University) for providing the anti-CAXII antibody in addition to all members of the Arnold and Mabel Beckman Center for Transposon Research for continuous dedication to stimulating discussion. The authors were supported by N.I.D.A. grant R01-DA014764 (AMG, DAL), N.I.H. grant T32 HD007480 (AMG), and N.I.G.M.S. grant T32 GM08347 (AW). + +Figures and Tables + +Figure 1 + +Gene-trap tTA vector design and in vitro testing A). The T2/GT2/tTA and T2/GT3/tTA vectors. The 'GT2' version is capable of mutating genes in one orientation while the 'GT3' version can mutate genes in both orientations upon insertion into a gene. (B- BamHI sites ) B) Compared to normal expression (left), when the T2/GT2/tTA SB transposon-based gene-trap vector inserts into a gene in the direction of transcription (right), endogenous splicing incorporates the IRES and tTA sequences and the bicistronic mRNA is prematurely truncated at the SV40 late polyadenylation site. The bicistronic mRNA allows cap-independent translation of the tTA molecule in addition to any peptide encoded by upstream exons. C) A stable, TRE-regulated luciferase cell line was created to test the T2/GT2/tTA/SVNeo transposon vector. After co-transfection with a plasmid source of transposase, G418R clones were individually expanded for analysis. D) Individual luciferase expression levels of G418R clone cell extracts from twelve control (pT2/SVNeo) and fifty gene-trap tTA clones from C. E) Incubation of three clones from C in media supplemented with 2 mM doxycycline results in 10- to 100-fold reduction of luciferase expression. + +Figure 2 + +Phenotypic rescue by transposon mobilization A). Breeding scheme to remobilize a transposon insertion, 01-0032, out of the mouse Slc25a22 gene as previously reported by Carlson, et al . (2003). Animals for both the single-copy transposon insertion 01-0032 and the CAGGS-SB10 transposase were intercrossed. B) Germline single-copy remobilization rates of the independently generated insertions 01-0032 and 02A-0016 (Additional file 2) were detected by analyzing the donor site for evidence of a transposon footprint by sequencing (data not shown). + +Figure 3 + +Molecular characterization of gene-trap tTAfunction A). Insertions 02A-0001 in the mouse Car12 gene and insertion 02A-0002 in the mouse ENSMUSG00000066992 gene. Genes are shown in the orientation of transcription from left to right, vertical lines are exons, along with the position and orientation of the T2/GT2/tTA insertion (arrows). B) RT-PCR detection of Car12 (top) and Car12-IRES-tTA chimeric (bottom) transcription in wild type and 02A-0001 carrier tissues. Kd-kidney, Br-brain, Lv-liver, Te-testes, Ad-adipose, Co-colon,SI-small intestine, Ov-ovary, Mu-muscle, Ht-heart C) RT-PCR analysis of gene disruption in tissues from wild type (+/+), heterozygous (+/ins), and homozygous (ins/ins) carriers of insertions 02A-0001 and 02A-0002. C) Western analysis of CA-XII expression in a wild type, heterozygous and homozygous carrier of insertion 02A-0001. Cross-reaction of the CA-XII antibody with other isoforms serves as a loading control (arrowhead). + +Figure 4 + +T2/GT3/tTA gene disruptions and tissue-specific activation of a tet-responsive transgene in vivo A). T2/GT3/tTA insertions into mouse genes not linked to the original donor site. The genes are shown in the orientation of transcription with vertical lines as exons (from left to right) with the position and orientation of the IRES-tTA trapping cassette (arrow). B) Luciferase assays performed on individual tissues from four independent 02A-0002; Tg(tetL)1Bjd/J doubly transgenic and control Tg(tetL)1Bjd/J mice. + +Figure 5 + +In vivo imaging of gene-trap tTAactivation of a tet-responsive transgene A). Luciferase emission pattern seen in a 03A-0241; Tg(tetL)1Bjd/J doubly transgenic mouse (936) as imaged by an intensified charge-coupled device (CCD) camera. Ventral and dorsal aspects are shown. The intensity of luciferase expression is compared in photons/second/cm2. B) RT-PCR analysis of MacF1 expression in multiple adult tissues, Gapdh was used as a control for sample quality. ES-embryonic stem cells, Lv-liver, Kd-kidney, Br-brain, Lu-lung, Ht-heart, Sp-spleen, St-stomach, BM-bone marrow, SI-small intestine, Co-colon, Ad-adipose, Mu-muscle, Ov-ovary, b-testes, Th-thymus C) Southern analysis of mice from A and B (see Materials and Methods). The donor site concatemer appears as an intense BamHI fragment at 2236 base pairs (arrowhead). The detection of insertion 03A-0241 by three-primer PCR is shown below each lane. Three bands marked by arrows segregate with insertion 03A-0241 genotyping and the concatemer.D) Pattern inheritance by offspring of mouse 936. Mice were imaged and scaled to different ranges of intensity that ranged from 1x104 to 1×108 photons/second/cm2 (scale bars). E) Repression of in vivo luciferase activation after six days of treatment with doxycycline in the same mice from D. + +Table 1 + +Mobilization of gene-trap tTA transposons in the germlines of seed mice. + +a Transposon copy number for each transgenic line was determined by Southern blot (see Materials and Methods) + +b The number of single-copy insertions as determined by the presence of new bands on a Southern blot after mobilization by transposase in the germline of seed mice + +c Reported is the average hits/gemete divided by the copy number diff --git a/src/ontogpt/evaluation/craft/database/all/16870721.ann b/src/ontogpt/evaluation/craft/database/all/16870721.ann new file mode 100644 index 000000000..d1362f45e --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16870721.ann @@ -0,0 +1,536 @@ +T1 SO:0000704 13 17 gene +T2 CL:0002322 39 41;54 59 ES ... cells +T3 CL:0002371 46 59 somatic cells +T4 PR:000003035 159 162 p53 +T5 GO:0065007 163 173 regulation +T6 PR:000003035 204 207 p53 +T7 CL:0002322 248 256 ES cells +T8 NCBITaxon:10088 276 280 mice +T9 PR:000003035 385 388 p53 +T10 SO:0005853 442 450 Cassette +T11 CL:0002322 519 527 ES cells +T12 NCBITaxon:10088 567 571 mice +T13 CL:0002371 637 650 Somatic cells +T14 PR:000003035 734 737 p53 +T15 CL:0000057 779 790 fibroblasts +T16 SO:0000948 890 898;931 936 inverted ... sites +T17 CL:0002322 1015 1023 ES cells +T18 NCBITaxon:10088 1042 1047 mouse +T19 PR:000003035 1145 1148 p53 +T20 CL:0000010 1227 1241 cultured cells +T21 GO:0009294 1264 1276 transfection +T22 SO:0000155 1280 1288 plasmids +T23 GO:0010467 1289 1299 expressing +T24 PR:000003035 1308 1311 p53 +T25 PR:000003035 1360 1363 p53 +T26 GO:0065007 1367 1376 regulated +T27 PR:000003035 1473 1476 p53 +T28 UBERON:0000922 1517 1526 embryonic +T29 CL:0002322 1517 1531;1537 1542 embryonic stem ... cells +T30 CL:0002322 1533 1535;1537 1542 ES ... cells +T31 NCBITaxon:10088 1562 1566 mice +T32 SO:0001026 1632 1639 genomic +T33 GO:0010467 1652 1661 expressed +T34 SO:0000167 1682 1690 promoter +T35 GO:0010467 1715 1725 expression +T36 PR:000003035 1831 1834 p53 +T37 GO:0009294 1855 1867 transfection +T38 PR:000003035 2021 2024 p53 +T39 GO:0065007 2025 2035 regulation +T40 CL:0002322 2081 2089 ES cells +T41 NCBITaxon:10088 2109 2113 mice +T42 CL:0002322 2233 2241 ES cells +T43 SO:0000155 2375 2383 plasmids +T44 NCBITaxon:10088 2476 2480 mice +T45 NCBITaxon:10088 2511 2516 mouse +T46 PR:000003035 2767 2770 p53 +T47 SO:0000704 2780 2785 genes +T48 CL:0002322 2842 2850 ES cells +T49 SO:0000704 2916 2920 gene +T50 CL:0000057 2934 2945 fibroblasts +T51 SO:0000646 2990 2996 siRNAs +T52 CL:0000057 3000 3011 fibroblasts +T53 SO:0000704 3060 3064 gene +T54 NCBITaxon:33208 3079 3086 animals +T55 http://purl.obolibrary.org/obo/MONDO_0000001 3115 3122 disease +T56 GO:0010467 3208 3218 expression +T57 SO:0001023 3219 3226 alleles +T58 CL:0000057 3257 3268 fibroblasts +T59 SO:0001023 3342 3349 alleles +T60 GO:0030849 3399 3408 autosomal +T61 PR:000003035 3495 3498 p53 +T62 CL:0002322 3513 3521 ES cells +T63 CL:0000057 3526 3537 fibroblasts +T64 SO:0005853 3560 3568 Cassette +T65 SO:0000704 3673 3677 gene +T66 SO:0005853 3723 3731 cassette +T67 SO:0000357 3732 3739 flanked +T68 SO:0000346 3774 3784 loxP sites +T69 SO:0000359 3819 3825 floxed +T70 SO:0005853 3889 3897 cassette +T71 SO:0000359 3911 3917 floxed +T72 SO:0001023 3925 3931 allele +T73 SO:0000704 3985 3989 gene +T74 NCBITaxon:10088 4098 4102 mice +T75 SO:0005853 4133 4142 cassettes +T76 SO:0000346 4166 4176 loxP sites +T77 SO:0000346 4250 4260 loxP sites +T78 SO:0005853 4434 4442 cassette +T79 GO:0010467 4555 4565 expression +T80 SO:0000704 4609 4613 gene +T81 SO:0000704 4668 4672 gene +T82 SO:0000948 4744 4752;4771 4776 inverted ... sites +T83 SO:0000346 4766 4776 loxP sites +T84 GO:0010467 4855 4865 expression +T85 SO:0005853 4866 4874 cassette +T86 SO:0001023 5005 5011 allele +T87 CL:0002371 5080 5092 somatic cell +T88 NCBITaxon:10088 5118 5122 mice +T89 CL:0002322 5144 5152 ES cells +T90 CHEBI:24753 5363 5373 Hygromycin +T91 NCBITaxon:10088 5474 5479 mouse +T92 http://purl.obolibrary.org/obo/MONDO_0005047 5480 5489 sterility +T93 CL:0002322 5534 5542 ES cells +T94 SO:0000948 5615 5623;5642 5647 inverted ... sites +T95 SO:0000346 5637 5647 loxP sites +T96 CL:0002322 5726 5734 ES cells +T97 NCBITaxon:10088 5753 5758 mouse +T98 PR:000003035 5887 5890 p53 +T99 CL:0002322 5900 5908 ES cells +T100 NCBITaxon:10088 5928 5932 mice +T101 CL:0002371 5987 6000 Somatic cells +T102 PR:000003035 6184 6187 p53 +T103 SO:0000155 6218 6225 plasmid +T104 SO:0000155 6338 6345 plasmid +T105 SO:0000346 6376 6386 loxP sites +T106 SO:0000346 6459 6463 loxP +T107 SO:0000346 6473 6477 loxP +T108 SO:0000346 6569 6573 loxP +T109 SO:0000346 6689 6693 loxP +T110 SO:0000346 6767 6777 loxP sites +T111 SO:0000155 6920 6927 plasmid +T112 SO:0000346 6993 7003 LoxP sites +T113 SO:0000155 7016 7023 plasmid +T114 SO:0000155 7061 7068 plasmid +T115 SO:0000704 7181 7185 gene +T116 SO:0000704 7256 7260 gene +T117 SO:0000155 7273 7280 plasmid +T118 PR:000003035 7335 7340 Trp53 +T119 SO:0000155 7424 7431 plasmid +T120 SO:0000155 7506 7513 plasmid +T121 SO:0000147 7525 7530 exons +T122 PR:000003035 7539 7542 p53 +T123 SO:0000155 7607 7614 plasmid +T124 SO:0000346 7703 7713 loxP sites +T125 SO:0000155 7810 7817 plasmid +T126 PR:000003035 7874 7877 p53 +T127 SO:0000704 7878 7882 gene +T128 SO:0000704 7895 7899 gene +T129 GO:0006266 7943 7951 ligation +T130 PR:P43870 7987 7994 HindIII +T131 PR:P43870 8007 8014 HindIII +T132 PR:000003035 8035 8040 Trp53 +T133 SO:0000704 8102 8106 gene +T134 SO:0000155 8113 8120 plasmid +T135 SO:0000155 8169 8176 plasmid +T136 PR:000003035 8241 8244 p53 +T137 SO:0000155 8361 8368 plasmid +T138 PR:P23940 8421 8426 BamHI +T139 SO:0000188 8446 8452 intron +T140 PR:000003035 8458 8461 p53 +T141 SO:0000155 8488 8495 plasmid +T142 SO:0000155 8538 8546 plasmids +T143 SO:0000155 8624 8631 plasmid +T144 SO:0000155 8643 8650 Plasmid +T145 SO:0000112 8754 8761 primers +T146 PR:000003035 8793 8796 p53 +T147 SO:0001421 8819 8840 exon–intron junctions +T148 SO:0000346 8877 8887 loxP sites +T149 PR:000003035 8922 8925 p53 +T150 PR:000003035 8933 8936 p53 +T151 SO:0000155 8942 8950 plasmids +T152 PR:000003035 8962 8965 p53 +T153 PR:P43870 9013 9020 HindIII +T154 PR:000003035 9037 9040 p53 +T155 SO:0000852 9065 9077 part of exon +T156 SO:0000704 9112 9116 gene +T157 PR:P43870 9145 9152 HindIII +T158 SO:0000831 9207 9214;9223 9227 part of ... gene +T159 PR:000003035 9219 9222 p53 +T160 SO:0000112 9273 9280 primers +T161 SO:0000319 9390 9400 stop codon +T162 PR:P23940 9418 9423 BamHI +T163 SO:0000112 9440 9447 primers +T164 SO:0000155 9609 9616 plasmid +T165 PR:P23940 9640 9645 BamHI +T166 SO:0001817 9667 9675 in frame +T167 PR:P23940 9697 9703 Bam HI +T168 SO:0000155 9722 9729 plasmid +T169 PR:000003035 9787 9790 p53 +T170 SO:0000155 9858 9865 plasmid +T171 PR:P43870 9881 9888 HindIII +T172 PR:000003035 9926 9929 p53 +T173 PR:000003035 10001 10004 p53 +T174 PR:000003035 10076 10079 p53 +T175 SO:0000155 10092 10099 plasmid +T176 PR:000003035 10123 10126 p53 +T177 SO:0000112 10242 10249 primers +T178 SO:0000704 10301 10305 gene +T179 SO:0000155 10332 10339 plasmid +T180 PR:000003035 10409 10412 p53 +T181 SO:0000704 10413 10417 gene +T182 PR:000003035 10485 10488 p53 +T183 PR:000003035 10495 10498 p53 +T184 SO:0000155 10504 10512 plasmids +T185 PR:000003035 10587 10590 p53 +T186 SO:0000155 10605 10613 plasmids +T187 PR:000003035 10659 10662 p53 +T188 SO:0000188 10663 10669 intron +T189 PR:000003035 10718 10721 p53 +T190 SO:0000188 10722 10728 intron +T191 PR:000003035 10748 10751 p53 +T192 SO:0000155 10757 10764 plasmid +T193 PR:000003035 10852 10855 p53 +T194 SO:0001023 10856 10862 allele +T195 PR:000003035 11002 11005 p53 +T196 SO:0001023 11009 11015 allele +T197 UBERON:0000922 11114 11121 embryos +T198 CHEBI:33282 11192 11203 antibiotics +T199 CL:0002322 11215 11223 ES cells +T200 GO:0040007 11229 11234 grown +T201 CHEBI:27504 11314 11325 mitomycin C +T202 CHEBI:17939 11420 11429 puromycin +T203 CHEBI:465284 11451 11462 ganciclovir +T204 CL:0002322 11520 11528 ES cells +T205 CHEBI:17939 11599 11608 puromycin +T206 UBERON:0000358 11695 11706 blastocysts +T207 CL:0002322 11765 11773 ES cells +T208 PR:000003035 11794 11797 p53 +T209 CL:0002322 11804 11812 ES cells +T210 GO:0040007 11818 11823 grown +T211 CHEBI:17939 11832 11841 puromycin +T212 NCBITaxon:10358 11878 11881 CMV +T213 SO:0000155 11886 11893 plasmid +T214 GO:0040007 12113 12118 grown +T215 PR:000003035 12271 12274 p53 +T216 GO:0040007 12297 12302 grown +T217 CHEBI:17939 12311 12320 puromycin +T218 GO:0040007 12428 12433 grown +T219 CHEBI:465284 12505 12516 ganciclovir +T220 GO:0040007 12636 12641 grown +T221 CHEBI:28748 12772 12782 adriamycin +T222 GO:0019835 12789 12794 lysed +T223 CHEBI:9754 12839 12843 Tris +T224 CHEBI:26710 12872 12876 NaCl +T225 CHEBI:78708 12883 12895 Nonidet P-40 +T226 CHEBI:8102 12902 12906 PMSF +T227 CHEBI:35607 12913 12928 sodium vanadate +T228 CHEBI:28741 12936 12939 NaF +T229 CHEBI:8984 13215 13218 SDS +T230 CHEBI:53250 13270 13296 poly(vinylidene difluoride +T231 UBERON:0001913 13366 13370 milk +T232 CHEBI:9754 13387 13391 Tris +T233 CHEBI:26710 13407 13411 NaCl +T234 CHEBI:53424 13417 13425 Tween-20 +T235 GO:0042571 13483 13493 antibodies +T236 PR:000003035 13502 13505 p53 +T237 GO:0042571 13555 13565 antibodies +T238 MOP:0000779 13590 13600 conjugated +T239 NCBITaxon:9925 13601 13605 goat +T240 NCBITaxon:10088 13611 13616 mouse +T241 GO:0071735 13617 13620 IgG +T242 NCBITaxon:9986 13630 13636 rabbit +T243 GO:0071735 13637 13640 IgG +T244 CHEBI:472552 13939 13943 BrdU +T245 CHEBI:16236 13966 13973 ethanol +T246 CHEBI:37926 13995 13999 FITC +T247 CHEBI:472552 14005 14009 BrdU +T248 CHEBI:51240 14014 14030 propidium iodide +T249 SO:0000359 14261 14267 floxed +T250 SO:0001023 14268 14274 allele +T251 CL:0002322 14278 14286 ES cells +T252 CL:0002322 14357 14364 ES cell +T253 CHEBI:17939 14467 14476 puromycin +T254 CL:0002322 14594 14602 ES cells +T255 PR:000003035 14643 14646 p53 +T256 PR:000003035 14661 14664 p53 +T257 PR:000003035 14688 14691 p53 +T258 CL:0002322 14946 14953 ES cell +T259 PR:000003035 15306 15309 p53 +T260 CL:0002322 15315 15317 ES +T261 GO:0042571 15363 15371 antibody +T262 PR:000003035 15380 15383 p53 +T263 GO:0010467 15398 15405 express +T264 PR:000003035 15495 15498 p53 +T265 PR:000003035 15518 15521 p53 +T266 PR:000003035 15556 15559 p53 +T267 PR:000003035 15604 15607 p53 +T268 PR:000003035 15609 15612 p53 +T269 PR:000003035 15714 15717 p53 +T270 PR:000003035 15724 15727 p53 +T271 CHEBI:28748 15750 15760 adriamycin +T272 PR:000003035 15801 15804 p53 +T273 UBERON:0000358 15836 15847 blastocysts +T274 GO:0007565 15874 15882 pregnant +T275 GO:0007565 15933 15944 pregnancies +T276 PR:000003035 15987 15990 p53 +T277 GO:0065007 16002 16011 regulated +T278 CL:0002322 16032 16034;16047 16052 ES ... cells +T279 CL:0002371 16039 16052 somatic cells +T280 CL:0002322 16054 16062 ES cells +T281 PR:000003035 16087 16090 p53 +T282 PR:000003035 16111 16114 p53 +T283 CL:0002371 16154 16167 somatic cells +T284 PR:000003035 16215 16218 p53 +T285 NCBITaxon:10088 16242 16247 mouse +T286 GO:0009790 16248 16261 embryogenesis +T287 GO:0007565 16318 16329 pregnancies +T288 PR:000003035 16368 16371 p53 +T289 CL:0002322 16382 16390 ES cells +T290 UBERON:0000358 16405 16416 blastocysts +T291 UBERON:0000922 16445 16454 embryonic +T292 GO:0009790 16445 16466 embryonic development +T293 PR:000003035 16515 16518 p53 +T294 PR:000003035 16598 16601 p53 +T295 PR:000003035 16627 16630 p53 +T296 SO:0000417 16644 16650 domain +T297 PR:000003035 16670 16673 p53 +T298 PR:000003035 16686 16689 p53 +T299 SO:0000417 16764 16770 domain +T300 UBERON:0000922 16913 16922 embryonic +T301 GO:0009790 16913 16934 embryonic development +T302 PR:000003035 16948 16951 p53 +T303 SO:0000155 16969 16976 plasmid +T304 PR:000003035 17090 17093 p53 +T305 CL:0002322 17101 17109 ES cells +T306 GO:0010467 17175 17185 expression +T307 PR:000003035 17194 17197 p53 +T308 PR:000003035 17231 17234 p53 +T309 PR:000003035 17244 17247 p53 +T310 PR:000003035 17302 17305 p53 +T311 CL:0002322 17313 17321 ES cells +T312 NCBITaxon:10088 17346 17350 mice +T313 SO:0001023 17377 17383 allele +T314 PR:000003035 17410 17413 p53 +T315 CL:0002322 17421 17428 ES cell +T316 UBERON:0000358 17455 17466 blastocysts +T317 NCBITaxon:10088 17494 17498 mice +T318 GO:0007618 17556 17562 mating +T319 NCBITaxon:10088 17584 17588 mice +T320 CL:0002322 17668 17676 ES cells +T321 PR:000003035 17844 17847 p53 +T322 SO:0001023 17848 17854 allele +T323 CL:0002371 17858 17871 somatic cells +T324 PR:000003035 17930 17933 p53 +T325 PR:000003035 17941 17944 p53 +T326 GO:0007618 17995 18001 mating +T327 PR:000003035 18002 18005 p53 +T328 PR:000003035 18026 18029 p53 +T329 NCBITaxon:10088 18033 18037 mice +T330 PR:000003035 18096 18099 p53 +T331 PR:000003035 18148 18151 p53 +T332 CL:0002371 18161 18174 somatic cells +T333 PR:000003035 18227 18230 p53 +T334 GO:0010467 18253 18263 expression +T335 SO:0000155 18264 18271 plasmid +T336 PR:000003035 18280 18283 p53 +T337 SO:0000155 18287 18294 plasmid +T338 CHEBI:465284 18331 18342 ganciclovir +T339 SO:0001023 18384 18390 allele +T340 PR:000003035 18435 18438 p53 +T341 CL:0002322 18445 18453 ES cells +T342 PR:000003035 18466 18469 p53 +T343 GO:0010467 18476 18485 expressed +T344 PR:000003035 18543 18546 p53 +T345 CL:0002322 18613 18621 ES cells +T346 PR:000003035 18674 18677 p53 +T347 CL:0002322 18703 18711 ES cells +T348 GO:0008283 18784 18795 proliferate +T349 PR:000003035 18823 18826 p53 +T350 PR:000003035 18867 18870 p53 +T351 GO:0010467 18918 18928 expression +T352 SO:0000155 18929 18936 plasmid +T353 GO:0008283 19116 19127 proliferate +T354 CHEBI:465284 19158 19169 ganciclovir +T355 PR:000003035 19217 19220 p53 +T356 GO:0010467 19279 19289 expression +T357 SO:0000155 19290 19297 plasmid +T358 UBERON:0007379 19427 19430 egg +T359 PR:000003035 19610 19613 p53 +T360 GO:0010467 19617 19627 expressing +T361 GO:0016265 19643 19646 die +T362 PR:000003035 19664 19667 p53 +T363 PR:000003035 19708 19711 p53 +T364 PR:000003035 19732 19735 p53 +T365 PR:000003035 19752 19755 p53 +T366 GO:0010467 19761 19771 expressing +T367 CL:0002322 19850 19857 ES cell +T368 GO:0010467 19871 19880 expressed +T369 PR:000003035 19883 19886 p53 +T370 PR:000003035 19926 19929 p53 +T371 PR:000003035 19961 19964 p53 +T372 PR:000003035 20083 20086 p53 +T373 GO:0010467 20105 20112 express +T374 PR:000003035 20125 20128 p53 +T375 PR:000003035 20185 20188 p53 +T376 SO:0000417 20379 20385 domain +T377 PR:000003035 20407 20410 p53 +T378 GO:0051726 20425 20443 cell cycle control +T379 PR:000003035 20515 20518 p53 +T380 GO:0065007 20519 20529 regulation +T381 SO:0001023 20687 20693 allele +T382 CL:0002322 20710 20718 ES cells +T383 NCBITaxon:10088 20731 20735 mice +T384 SO:0000948 20853 20861;20880 20885 inverted ... sites +T385 SO:0000346 20875 20885 loxP sites +T386 CL:0002322 20964 20972 ES cells +T387 CL:0002371 20983 20996 somatic cells +T388 SO:0001023 21027 21033 allele +T389 SO:0000704 21041 21045 gene +T390 NCBITaxon:40674 21151 21160 mammalian +T391 SO:0000704 21161 21166 genes +T392 GO:0065007 21205 21212 control +T393 UBERON:0000479 21237 21244 tissues +T394 CL:0000057 21295 21312 fibroblastic cell +T395 SO:0000704 21373 21378 genes +T396 PR:000003035 21380 21383 p53 +T397 PR:000000084 21385 21390 c-myc +T398 GO:0071159 21392 21397 NF-KB +T399 SO:0000704 21470 21474 gene +T400 SO:0000704 21520 21524 gene +T401 http://purl.obolibrary.org/obo/MONDO_0004992 21782 21788 Cancer +T402 SO:0000704 22038 22042 gene +T403 SO:0000147 22073 22078 exons +T404 SO:0000346 22153 22157 loxP +T405 SO:0005853 22221 22229 cassette +T406 SO:0000346 22275 22279 loxP +T407 CL:0002322 22320 22328 ES cells +T408 GO:0009294 22376 22388 transfecting +T409 GO:0010467 22395 22405 expression +T410 SO:0000155 22406 22413 plasmid +T411 SO:0000155 22432 22439 plasmid +T412 SO:0000359 22447 22453 floxed +T413 SO:0000147 22485 22489 exon +T414 NCBITaxon:10088 22512 22517 mouse +T415 SO:0005853 22652 22660 cassette +T416 NCBITaxon:10088 22720 22724 mice +T417 SO:0005853 22761 22769 cassette +T418 CL:0002371 22888 22901 somatic cells +T419 NCBITaxon:10088 23037 23042 mouse +T420 NCBITaxon:10088 23048 23052 mice +T421 CL:0002371 23080 23093 somatic cells +T422 SO:0001023 23134 23140 allele +T423 NCBITaxon:10088 23182 23187 mouse +T424 UBERON:0000922 23188 23197 embryonic +T425 CL:0000057 23198 23209 fibroblasts +T426 CL:0002322 23399 23407 ES cells +T427 PR:000003035 23415 23418 p53 +T428 PR:000003035 23441 23444 p53 +T429 SO:0000704 23445 23449 gene +T430 SO:0000203 23544 23548 UTRs +T431 SO:0000346 23777 23781 loxP +T432 PR:000003035 23805 23808 p53 +T433 SO:0000147 23809 23814 exons +T434 SO:0000346 23926 23930 loxP +T435 http://purl.obolibrary.org/obo/MONDO_0005504 23987 23996 diphteria +T436 CHEBI:27026 23999 24004 toxin +T437 SO:0000704 24011 24015 gene +T438 GO:0035825 24087 24101 crossing-overs +T439 SO:0000112 24192 24199 primers +T440 CL:0002322 24330 24338 ES cells +T441 PR:000003035 24356 24359 p53 +T442 SO:0000155 24363 24370 plasmid +T443 SO:0000155 24385 24392 plasmid +T444 PR:000003035 24447 24450 p53 +T445 SO:0000357 24471 24478 flanked +T446 GO:0010467 24532 24542 expression +T447 SO:0000155 24543 24550 plasmid +T448 SO:0000112 24600 24607 primers +T449 SO:0000155 25166 25173 plasmid +T450 GO:0042571 25323 25331 antibody +T451 PR:000003035 25335 25338 p53 +T452 PR:000003035 25406 25409 p53 +T453 CL:0002322 25415 25417 ES +T454 CHEBI:28748 25461 25471 adriamycin +T455 CHEBI:28748 25473 25476 ADR +T456 PR:000003035 25537 25540 p53 +T457 GO:0010467 25592 25601 expressed +T458 PR:000003035 25668 25671 p53 +T459 SO:0000155 25677 25684 plasmid +T460 PR:000003035 25690 25693 p53 +T461 PR:000003035 25777 25780 p53 +T462 SO:0000147 25829 25833 exon +T463 SO:0000112 25964 25971 primers +T464 GO:0010467 26115 26124 expressed +T465 PR:000003035 26125 26128 p53 +T466 PR:000003035 26135 26138 p53 +T467 PR:000003035 26239 26242 p53 +T468 PR:000003035 26279 26282 p53 +T469 GO:0007618 26348 26354 mating +T470 PR:000003035 26357 26360 p53 +T471 NCBITaxon:10088 26384 26389 mouse +T472 SO:0000112 26416 26423 primers +T473 PR:000003035 26459 26462 p53 +T474 PR:000003035 26471 26474 p53 +T475 PR:000003035 26509 26512 p53 +T476 NCBITaxon:10088 26520 26525 mouse +T477 PR:000003035 26610 26613 p53 +T478 PR:000003035 26632 26635 p53 +T479 CL:0002322 26642 26650 ES cells +T480 UBERON:0000358 26670 26681 blastocysts +T481 NCBITaxon:10088 26703 26707 mice +T482 GO:0007618 26735 26740 mated +T483 PR:000003035 26746 26749 p53 +T484 NCBITaxon:10088 26753 26757 mice +T485 SO:0000112 26830 26837 primers +T486 SO:0001023 26902 26908 allele +T487 PR:000003035 26959 26962 p53 +T488 SO:0000112 27066 27073 primers +T489 PR:000003035 27149 27152 p53 +T490 NCBITaxon:10088 27156 27160 mice +T491 SO:0000112 27199 27206 Primers +T492 SO:0000028 27229 27231 bp +T493 SO:0001023 27258 27264 allele +T494 SO:0000112 27272 27279 primers +T495 SO:0000028 27315 27317 bp +T496 SO:0001023 27356 27362 allele +T497 PR:000003035 27374 27377 p53 +T498 PR:000003035 27405 27408 p53 +T499 PR:000003035 27476 27479 p53 +T500 SO:0000155 27483 27490 plasmid +T501 GO:0005634 27548 27555 nuclear +T502 PR:000003035 27570 27573 p53 +T503 GO:0010467 27612 27622 expression +T504 SO:0000155 27623 27630 plasmid +T505 PR:000003035 27639 27642 p53 +T506 SO:0000155 27655 27662 plasmid +T507 PR:000003035 27897 27900 p53 +T508 SO:0000155 27906 27913 plasmid +T509 PR:000003035 27915 27918 p53 +T510 GO:0010467 27957 27967 expression +T511 SO:0000155 27968 27975 plasmid +T512 PR:000003035 27984 27987 p53 +T513 SO:0000155 27993 28000 plasmid +T514 CHEBI:465284 28021 28032 ganciclovir +T515 SO:0000112 28043 28050 primers +T516 CHEBI:465284 28091 28102 ganciclovir +T517 SO:0000112 28222 28229 primers +T518 PR:000003035 28310 28313 p53 +T519 PR:000003035 28477 28480 p53 +T520 CHEBI:28748 28504 28507 ADR +T521 PR:000003035 28534 28537 p53 +T522 PR:000003035 28565 28568 p53 +T523 GO:0051726 28583 28601 cell cycle control +T524 PR:000003035 28616 28619 p53 +T525 PR:000003035 28630 28633 p53 +T526 PR:000003035 28842 28845 p53 +T527 PR:000003035 28869 28872 p53 +T528 CL:0002322 28921 28929 ES cells +T529 PR:000003035 28941 28944 p53 +T530 PR:000003035 28953 28956 p53 +T531 PR:000003035 29062 29065 p53 +T532 PR:000003035 29122 29125 p53 +T533 PR:000003035 29193 29196 p53 +T534 CL:0000057 29264 29275 fibroblasts +T535 PR:000003035 29376 29379 p53 +T536 SO:0001023 29380 29387 alleles diff --git a/src/ontogpt/evaluation/craft/database/all/16870721.txt b/src/ontogpt/evaluation/craft/database/all/16870721.txt new file mode 100644 index 000000000..79544b40e --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16870721.txt @@ -0,0 +1,107 @@ +RMCE-ASAP: a gene targeting method for ES and somatic cells to accelerate phenotype analyses + +Abstract + +In recent years, tremendous insight has been gained on p53 regulation by targeting mutations at the p53 locus using homologous recombination in ES cells to generate mutant mice. Although informative, this approach is inefficient, slow and expensive. To facilitate targeting at the p53 locus, we developed an improved Recombinase-Mediated Cassette Exchange (RMCE) method. Our approach enables efficient targeting in ES cells to facilitate the production of mutant mice. But more importantly, the approach was Adapted for targeting in Somatic cells to Accelerate Phenotyping (RMCE-ASAP). We provide proof-of-concept for this at the p53 locus, by showing efficient targeting in fibroblasts, and rapid phenotypic read-out of a recessive mutation after a single exchange. RMCE-ASAP combines inverted heterologous recombinase target sites, a positive/negative selection marker that preserves the germline capacity of ES cells, and the power of mouse genetics. These general principles should make RMCE-ASAP applicable to any locus. + +INTRODUCTION + +p53 is one of the most highly analyzed proteins for the past 25 years. Studies in cultured cells, often relying on the transfection of plasmids expressing various p53 mutants, have established models to explain how p53 is regulated. In recent years, some of these models were tested in vivo by targeting subtle mutations at the p53 locus using homologous recombination in embryonic stem (ES) cells to generate mutant mice. The strength of this approach is that mutations are tested in a genomic setting and expressed from the endogenous promoter, ensuring physiological expression levels and correct spatio-temporal profiles. As significant differences between phenotypes from targeted p53 mutants in vivo and transfection data were observed [e.g. Refs (1–5)], more targeted mutations need to be generated and analyzed in multiple tissues to formulate more accurate models of p53 regulation. + +However, using homologous recombination in ES cells to generate mutant mice is an inefficient, slow and expensive method because (i) homologous recombination typically occurs at low frequency in ES cells, requiring sophisticated selection schemes and screening of hundreds of clones to identify the desired mutant; (ii) large (15–20 kb) plasmids, often difficult to clone, are required to increase targeting efficiency and (iii) breeding mice to homozygosity and housing a mouse colony generate further delays and costs. Such limitations make the repeated targeting of a locus a technically daunting and economically impractical task. + +Improvements in current technologies are needed to enable such analyses to be applied to the p53 or other genes. Developing methods to increase targeting efficiency in ES cells is clearly an important goal. In addition, efficient methods for gene targeting in fibroblasts could expedite phenotypic analyses. Indeed, siRNAs in fibroblasts often provide a faster read-out than equivalent gene knock-outs in animals (6). However, modeling most disease-associated mutations requires generating subtle mutations, not knock-outs or reduced expression alleles. Targeting point mutations in fibroblasts by homologous recombination is extremely inefficient, and targeting both alleles is required to reveal the phenotype of recessive autosomal mutations. + +Here we report an approach that enables highly efficient targeting at the p53 locus in both ES cells and fibroblasts. Recombinase-Mediated Cassette Exchange (RMCE) approaches were developed to improve targeting efficiency using a two-step process: the gene of interest is first replaced by a selection cassette flanked by recombinase target sites (e.g. loxP sites for Cre recombinase, to create a ‘floxed’ locus). Then, Cre-mediated recombination in the presence of a cassette containing a floxed mutant allele removes the resident sequence and inserts the mutant gene (7). Previously, technical difficulties have prevented RMCE from being applied routinely to generate mutant mice. For example, exchanges using cassettes with directly repeated loxP sites were inefficient because excisions dominated the intended exchanges (8). loxP sites with different sequences were generated to overcome this problem, but these sites also underwent intramolecular recombination, making RMCE efficient only if the replacement cassette contained a marker enabling selection of the desired recombinant (7,9–12). However, interference resulting from expression of the selection marker and the endogenous gene (13) necessitates strategies to remove the selectable gene. Together, previous studies indicate that an optimal RMCE requires (i) inverted heterologous loxP sites diverging by at least 2 nt to maximize the efficiency of exchange and (ii) an expression cassette enabling both positive selection to identify the initial recombinant and then negative selection to obtain a ‘marker-free’ mutant allele (14). + +Most RMCE experiments have been performed at random sites in somatic cell lines. Only a few mutant mice generated by RMCE in ES cells have been reported, but the RMCE systematically introduced a selectable marker (15–17), or, when tested without an incoming marker, proved inefficient (12). A recent report disclosed an additional problem: the Hygromycin–Thymidine Kinase fusion gene used most frequently for positive/negative selection in RMCE, leads to mouse sterility, so that exchanges can only be performed in ES cells (16). + +The RMCE strategy presented here relies on the integrated use of inverted heterologous loxP sites, a positive/negative selection marker that preserves the germline capacity of ES cells, and the power of mouse genetics to expedite phenotypic analyses. We show that our approach enables efficient targeting of marker-free mutations at the p53 locus in ES cells to generate mutant mice, but more importantly, it is Adapted for targeting in Somatic cells to Accelerate Phenotyping (ASAP). Because it relies on very general principles, RMCE-ASAP could be applied to any locus of interest. + +MATERIALS AND METHODS + +Targeting construct for a p53 RMCE-ready locus + +Details for plasmid construction are available upon request owing to space limitations mandating a brief outline. We started from a plasmid L3-1L containing heterologous loxP sites (L3 is the mutant loxP257 recently described (14), 1L is an inverted WT loxP). The WT loxP and loxP257 differ in their spacer sequences: the spacer sequence is 5′-ATGTATGC-3′ for WT loxP and 5′-AAGTCTCC-3′ for loxP257. The three mutations in the loxP257 spacer sequence prevent it to recombine with WT loxP, ensuring efficient RMCE in several cell lines: accurate RMCE with these loxP sites occurred with an average frequency of 81% at two loci in CHO cells and an average frequency of 69% at four loci in Hela cells (14). The L3-1L plasmid was first modified to include a ClaI and a FseI site between the LoxP sites, leading to plasmid L3-CF-1L. We next modified a puroΔTK plasmid (YTC37, a kind gift from A. Bradley) by using oligonucleotides to destroy a NotI site downstream of the puroΔTK gene and introduce a NotI site upstream, and a FseI site downstream of the gene (leading to plasmid CN-PuroΔTK-F). Next, a PmlI-MfeI 6.3 kb fragment from Trp53 was subcloned in a modified pBluescript KSII+ (pBS, Stratagene), and the resulting plasmid was digested with SwaI to introduce an EagI site, leading to p53PmlEag, a plasmid containing exons 2–11 of p53. We then inserted a 5.5 kb ClaI-EagI fragment from p53PmlEag in plasmid CN-PuroΔTK-F digested by ClaI and NotI, and inserted the resulting fragment between the loxP sites of L3-CF-1L by ClaI and FseI digestion, leading to L3-p53PmlEagPuroΔTK-1L. We next engineered a plasmid containing the region for 3′-homology downstream of the p53 gene and the DTA gene in two steps: (i) we performed a three-way ligation between a modified pBS digested by HindIII and NotI, a HindIII-EcoRI fragment from Trp53 for 3′homology and an EcoRI-NotI fragment containing the DTA gene, from plasmid pgkdtabpa (kind gift of P. Soriano), leading to plasmid 3′ + DTA and (ii) because the Bsu36I-EcoRI region downstream of p53 contains repetitive sequences (F. Toledo and G. M. Wahl, unpublished data), we later deleted this region, to obtain plasmid 3′ + DTA. The 5′ homology consists of a 3.4 kb-long BamHI-PmlI fragment from intron 1 of p53 cloned in a modified pBS (plasmid p5′). Finally, appropriate fragments from plasmids p5′, L3-p53PmlEagPuroΔTK-1L, and 3′ + DTA were assembled in a modified pSP72 plasmid (Promega). Plasmid Flox, the resulting targeting construct, was verified by restriction analysis, then sequenced using 30 primers chosen to precisely verify all p53 coding sequences, all exon–intron junctions and the sequences at and around the loxP sites. + +Exchange constructs: making the p53GFP and p53ΔPGFP plasmids + +To make a p53-GFP fusion protein, we first subcloned a SacII-HindIII fragment of the p53 locus (corresponding to part of exon 10 to sequences downstream of the gene) into pBS, then mutated the HindIII site into a FseI site. We next mutated the C-terminal part of the p53 gene in two rounds of PCR mutagenesis, first with primers 5′-GGGCCTGACTCAGACGGATCCCCTCTGCATCCCGTC-3′ and 5′-GACGGGATGCAGAGGGGATCCGTCTGAGTCAGGCCC-3′, which removed the stop codon and introduced a BamHI site, then with primers 5′-GACGGATCCCCTCTGAATTCCGTCCCCATCACCA-3′ and 5′-TGGTGATGGGGACGGAATACAGAGGGGATCCGTC-3′, which introduced an EcoRI site. We verified the sequence from the mutated plasmid, then digested it with BamHI and EcoRI, to insert in frame GFP sequences from a Bam HI-EcoRI fragment of plasmid phr-GFP-1 (Stratagene). We verified the sequence of this p53-GFP fusion fragment, then swapped it in the L3-p53PmlEagPuroΔTK-1L plasmid (see above) by HindIII and FseI digestion, resulting in the p53GFP exchange construct, the sequence which was verified before use. The p53ΔPGFP exchange construct was engineered by combining sequences from the p53GFP exchange plasmid and sequences from the p53ΔP targeting construct described recently (5). Its sequence was also verified before use. + +Sequences and use of PCR primers + +a: 5′-CCCCGGCCCTCACCCTCATCTTCG-3′, from the PuΔTK gene, assays targeting of Flox plasmid; b: 5′-AACAAAACAAAACAGCAGCAACAA-3′, from sequences downstream of the p53 gene and outside Flox sequences, assays targeting of Flox and RMCE with p53GFP or p53ΔPGFP plasmids; c: 5′-TGAAGAGCAAGGGCGTGGTGAAGGA-3′, from GFP sequences, assays RMCE with p53GFP orp53ΔPGFP plasmids; d: 5′-CAAAAAATGGAAGGAAATCAGGAACTAA-3′, from p53 intron 3, and e: 5′-TCTAGACAGAGAAAAAAGAGGCATT-3′, from p53 intron 4, assay RMCE with p53ΔPGFP plasmid; f: 5′-ATGGGAGGCTGCCAGTCCTAACCC and g: 5′-GTGTTTCATTAGTTCCCCACCTTGAC-3′ amplify the WT p53 allele according to Taconic's procedures, h: 5′-TTTACGGAGCCCTGGCGCTCGATGT-3′ and i: 5′-GTGGGAGGGACAAAAGTTCGAGGCC-3′ amplify the Neo marker in the p53 KO allele according to Taconic's procedures. + +Cell culture conditions + +Primary MEFs, isolated from 13.5 day embryos, were cultured in DMEM with 15% FBS, 100 mM BME, 2 mM l-glutamine and antibiotics. 129/SvJae ES cells were grown in the same medium supplemented with 1000 U/ml ESGRO (Chemicon), on a layer of mitomycin C-treated SNLPuro-7/4 feeders (kind gift of A. Bradley). Selections were performed with 2 μg/ml puromycin, 0.2 μM FIAU or 2 μM ganciclovir. + +Targeting/genotyping of the RMCE-ready locus + +29/SvJae ES cells were electroporated with the Flox construct linearized with PmeI, and puromycin resistant clones were analyzed as described (Figure 2). Two clones were injected into blastocysts and transmitted through the germline. + +Performing RMCE in ES cells + +A total of 8 × 105 p53RMCE/+ ES cells were grown without puromycin for 12 h, electroporated with 15 μg CMV-Cre plasmid (pOG231) and 200 μg of the exchange construct, and plated in T25 flasks at 105 cells per flask. FIAU was added to the medium 3–4 days after electroporation. Individual clones, picked 10 days after electroporation, were grown in 96-well plates and expanded to generate duplicate plates for freezing and DNA analysis by PCR and Southern. + +Performing RMCE in MEFs + +A total of 106 p53RMCE/− MEFs cells were grown without puromycin for 12 h, electroporated with 3 μg pOG231 and 30 μg exchange construct, and plated in a single 10 cm-dish, grown for 3 days then split in several dishes at 105 cells per dish. FIAU or ganciclovir was added to the medium 4 days after electroporation, for 3–4 days. Clones, picked 10 days after electroporation, were grown in 24-well plates and expanded for freezing and DNA analysis. + +Western-blots + +Cells, untreated or treated for 24 h with 0.5 μg/ml adriamycin, were lysed on the dish in a buffer consisting of 50 mM Tris (pH 8.0), 5 mM EDTA, 150 mM NaCl, 0.5% Nonidet P-40, 1 mM PMSF, 1 mM sodium vanadate, 10 mM NaF and Complete Mini Protease Inhibitors (Roche Diagnostics) at 4°C for 30 min. Lysates were scraped, then spun at 6000× g at 4°C for 10 min. Protein concentration in the supernatant was determined using the Bio-Rad DC protein assay. Lysates were separated on single percentage SDS/PAGE gels, then electrophoretically transferred to poly(vinylidene difluoride), using standard procedures.Blots were incubated in 5% non-fat dried milk in TBST (0.02 M Tris, pH 7.6/0.35 M NaCl/0.1% Tween-20) for 1 h at room temperature before probing with primary antibodies against p53 (CM-5, Novacastra) and -actin (Sigma). Secondary antibodies used include peroxidase-conjugated goat anti-mouse IgG and anti-rabbit IgG (Pierce). Probed blots were incubated with Pierce Supersignal West Pico chemiluminescent substrate and exposed to X-ray films. + +Flow cytometry + +Log phase cells were irradiated at RT with a 60 Co γ-irradiator at doses of 6 or 12 Gy and incubated for 24 h. Cells were then pulse-labeled for 1 h with BrdU (10 μM), fixed in 70% ethanol, double-stained with FITC anti-BrdU and propidium iodide, then sorted by using a Becton Dickinson FACScan machine. Data were analyzed using Becton Dickinson Cellquest Pro. + +RESULTS AND DISCUSSION + +The rationale for RMCE-ASAP is detailed in Figure 1. The first step requires generating a floxed allele in ES cells that will serve as the substrate for subsequent exchanges (RMCE-ready ES cell, Figure 1). The targeting strategy is detailed in Figure 2. The frequency of targeting was 4% (12/300 puromycin-resistant clones, analyzed by Southern blot and long-range PCR, Figure 2). + +We next tested the efficiency of RMCE in ES cells, using a replacement construct encoding p53 fused to GFP (p53GFP) to enable tracking p53 in individual live cells. Importantly however, GFP fluorescence was not used to screen cells with targeted events, as we wanted to develop a general method to isolate marker-free recombinants. The exchange strategy is detailed in Figure 3A. We picked 65 ES cell clones resistant to 1-(-2-deoxy-2-fluoro-1-b-d-arabino-furanosyl)-5-iodouracil (FIAU) due to TK loss and analyzed their DNA by PCR and Southern blot. Strikingly, 54 proper recombinants were identified, indicating very high RMCE efficiency (83%). RMCE also proved to be precise, as no aberrant bands were detected in PCR and Southern blots (Figure 3A). p53+/GFP ES clones were analyzed by western blot with an antibody against p53, and found to express an additional band at the expected size (ca. 80 kDa). Surprisingly, the fusion of GFP to p53 apparently altered p53 stability: steady-state levels of p53GFP were much higher than those of wild-type p53 (p53WT) in unstressed cells, and did not vary significantly after DNA damage, so that the levels for both p53WT and p53GFP were similar after adriamycin treatment (Figure 3A). + +Six independent p53+/GFP clones were injected into blastocysts and transferred to pseudo-pregnant females using standard procedures. Strikingly, no pregnancies were obtained. It has been shown that the p53 pathway is regulated very differently in ES and somatic cells: ES cells contain relatively high p53 levels and lack the p53-mediated DNA damage responses found in somatic cells (18). This, together with the observation that p53 levels decrease during mouse embryogenesis (19), suggested an explanation for the observed lack of pregnancies: we speculate that the high levels of p53GFP in the ES cells injected into blastocysts might have prevented normal embryonic development once these cells began to differentiate and the p53 pathway became functional. + +To test this possibility, we performed RMCE with a p53 fusion gene in which the p53 proline-rich domain (PRD) was deleted (p53ΔP), and the p53ΔP was fused to GFP. We used this mutant because deleting the proline-rich domain decreases stability and compromises DNA-damage responses in vivo (5). According to our hypothesis, this hypomorphic mutant should not prevent embryonic development. RMCE with a p53ΔPGFP replacement plasmid was again very efficient and western blots revealed an additional band of the predicted molecular weight only in p53+/ΔPGFP ES cells (Figure 3B). As expected, the PRD deletion correlated with lower expression levels: p53ΔPGFP was much less abundant than p53WT in all p53+/ΔPGFP clones (Figure 3B). We next determined whether p53+/ΔPGFP ES cells could generate chimeric mice and transmit the modified allele through the germline. Two p53+/ΔPGFP ES cell clones were injected into blastocysts and highly chimeric (>80%) mice were obtained. Heterozygote pups were recovered from the mating the chimeras with WT mice (Figure 3C). These data demonstrate that marker-free RMCE is very efficient in ES cells and allows germline transmission of a targeted mutation (see Figure 1, path A). + +We next determined whether the RMCE approach could be used to target mutations at the p53 allele in somatic cells (Figure 1, path B). We first verified that the RMCE-ready p53 locus (p53RMCE) could be transmitted through the germline by mating p53RMCE/+ chimeras with p53+/− mice (20) (Figure 4). Importantly, this allowed us to generate p53RMCE/− MEFs, which were used to test RMCE at the p53 locus in somatic cells. We first attempted RMCE in MEFs by electroporating p53RMCE/− MEFs with a Cre-expression plasmid and the p53GFP plasmid, followed by selection with FIAU or ganciclovir. Strikingly, no clones with an exchanged allele were identified (data not shown). RMCE with p53GFP in ES cells showed that p53GFP is expressed at high levels (Figure 3A), and as mentioned before, the p53 pathway that can be activated in MEFs is not readily activated in ES cells (18). The results above suggest that high levels of p53GFP could be tolerated by ES cells but toxic to MEFs, so that MEFs in which an RMCE had occurred failed to proliferate. To test this possibility, p53RMCE/− MEFs were electroporated with the p53GFP replacement construct with or without a Cre-expression plasmid, then analyzed by fluorescence microscopy 48 h after electroporation. The experiment was done without selection to enable observation of cells under conditions where a failure to proliferate would not derive from FIAU or ganciclovir toxicity but rather solely from the effects of p53GFP. We observed a few fluorescent cells only when the Cre expression plasmid was co-electroporated, suggesting that such cells resulted from RMCE. Importantly, the rare fluorescent cells had a flat, ‘fried-egg’ appearance typical of senescent cells (Figure 5A), and when plates were observed 5 days later, the cells had detached. Altogether, the results suggest that RMCE can give rise to p53GFP-expressing MEFs, but they die rapidly owing to p53GFP toxicity. + +We also performed RMCE in p53RMCE/− MEFs with the p53ΔPGFP construct. p53ΔPGFP-expressing MEFs were viable, recovered with an efficiency of ∼40%, and, as expected from ES cell experiments, expressed a p53ΔPGFP protein at much lower levels than p53WT (Figure 5B). Unlike WT MEFs, p53ΔP/ΔP MEFs are unable to arrest cycling after irradiation (5). Likewise, we found that irradiation doses that arrested p53RMCE/− MEFs (which express a wild-type p53 from the RMCE-ready locus, see Figure 2) did not arrest p53ΔPGFP/− MEFs (Figure 5C). These data show that a single RMCE-ASAP reaction in heterozygous MEFs enables detection of a recessive phenotype. The results confirm that deleting the proline rich domain leads to less active p53 with impaired cell cycle control, and also indicate that a GFP C-terminal fusion can dramatically alter p53 regulation. A summary of our results is presented in Table 1. + +These data report the development and implementation of an improved RMCE approach that enables efficient allele modification in ES cells to generate mice and in heterozygous MEFs to accelerate phenotypic analyses. The success of RMCE-ASAP relied on the integrated use of inverted heterologous loxP sites, a positive/negative selection marker that preserves the germline capacity of ES cells, and, for somatic cells, the existence of a knock-out allele of the gene of interest. These characteristics should make RMCE-ASAP a robust and general technology for analysis of mammalian genes under conditions that preserve normal control mechanisms in different tissues. In addition, RMCE-ASAP could be used to generate fibroblastic cell lines tailored for the repeated targeting of widely studied genes (p53, c-myc, NF-KB, etc.). + +Acknowledgements + +The authors thank A. Bradley for the PuroΔTK gene and SNLPuro-7/4 cells, P Soriano for the DTA gene, G. Campbell for technical assistance and E. T. Wong for helpful discussions. The work was supported by NIH grants CA100845 and CA061449 to G.M.W. F.T. was supported in part by the Institut Pasteur and a fellowship from Association pour la Recherche sur le Cancer. Funding to pay the Open Access publication charges for this article was provided by the Institut Pasteur. + +Conflict of interest statement. None declared. + +Figures and Tables + +Figure 1 + +Rationale for a RMCE-ASAP. Using homologous recombination, the gene of interest (GOI, open boxes: exons), is targeted with a construct introducing upstream of coding regions one loxP (blue arrowhead) and downstream, a positive/negative selection cassette (red box) and a second inverted heterologous loxP (purple arrowhead) to create RMCE-ready ES cells. An exchange is performed in these cells by co-transfecting a Cre expression plasmid and a marker-free plasmid with a floxed mutant GOI (green box: mutated exon), to produce a mutant mouse (path A). Importantly RMCE-ASAP incorporates two major improvements over classical RMCE (path B): (i) the positive/negative selection cassette does not prevent germline transmission, so that RMCE-ready mice can be obtained; (ii) the selection cassette does not replace, but rather lies downstream of the GOI. This is a crucial requirement for accelerated phenotyping in somatic cells, as maintaining a functional GOI ensures that the RMCE-ready locus still behaves like a WT locus. Hence, after breeding the RMCE-ready mouse with mice heterozygotes for the GOI, somatic cells with an RMCE-ready locus and a WT or KO allele can be recovered [e.g. RMCE/+ and RMCE/− mouse embryonic fibroblasts (MEFs)]. Such cells, phenotypically similar to +/+ and +/− cells, can then be used for phenotypic analyses of dominant or recessive mutations after a single exchange. + +Figure 2 + +Generating ES cells with a p53 RMCE-ready locus. The p53 gene is contained in a 17 kb-long EcoRI (RI) fragment (black boxes: coding sequences, white boxes: UTRs). The Flox targeting construct (below), the sequence which was verified before use (Materials and Methods), contains (i) a 3.4 kb-long 5′ homology region; (ii) 0.2 kb upstream of coding sequences, an EcoRI site and L3, a mutant loxP [loxP257, (14)]; (iii) p53 exons; (iv) 0.4 kb downstream, a puroΔTK fusion gene (puDTK) for positive/negative selection (21) and an inverted WT loxP (1L); (v) a 1.2 kb-long 3′ homology region and (vi) the diphteria α-toxin (DTA) gene for targeting enrichment. The recombinants resulting from the depicted crossing-overs are identified by a 6.5 kb band in Southern blot with probe A and a 3 kb band by PCR with primers a and b. A representative Southern, and PCR of one positive (β) and two negative clones, are shown. + +Figure 3 + +Performing RMCE in ES cells. (A) RMCE with a p53GFP plasmid. The exchange plasmid, the sequence which was verified before use, contains p53GFP coding sequences flanked by L3 and 1L sites. It was electroporated with a Cre expression plasmid. FIAU-resistant clones were analyzed by PCR with primers b and c and Southern blot with probe B. Both approaches led to identical results and identified 54/65 RMCE recombinants. Representative clones (P–Z) are shown (left), analyzed by PCR (top) and Southern (bottom): all clones but Q and T are positive with both assays. All positive clones produced a band of the expected size by PCR, indicating correct recombination at 1L, and displayed only the expected 12 and 5 kb bands by Southern, indicating correct recombination at L3. The absence of bands of aberrant size in Southerns also indicated that the exchange plasmid was neither rearranged nor inserted at ectopic sites. Thus RMCE was efficient and accurate. Recombinant clones were analyzed by western blot with an antibody to p53. In the representative western (right), cells from two independent p53+/GFP ES clones were left untreated or treated with adriamycin (ADR) at 0.5 μg/ml for 24 h, and protein extracts were prepared. p53GFP migrated at the expected size of 80 kDa and was expressed at unexpectedly high levels regardless of stress. (B) RMCE with a p53ΔPGFP plasmid. The p53ΔPGFP exchange construct (which sequence was verified before use) differed from the p53GFP construct only in that it contains a mutated exon 4 (4*) encoding a PRD deletion. RMCE was again very efficient, with 10/12 FIAU-resistant clones producing a 3 kb band by PCR with primers b and c. A western analysis of four FIAU-resistant clones is shown below (with low/high exposures: Lo X/Hi X). As expected, all except clone x expressed p53ΔPGFP. p53ΔPGFP migrated at the expected size of 75 kDa and accumulated after stress, but at lower levels than p53WT. (C) Germline transmission of the p53ΔPGFP mutation. DNA of seven littermates (U42–U48), obtained from mating a p53ΔPGFP chimera with a WT mouse, was analyzed by PCR with primers d and e (see B), with DNA from WT, p53+/ΔP and p53ΔP/ΔP MEFs (5) as controls. U45, a p53+/ΔPGFP mouse, demonstrated transmission of the mutation. + +Figure 4 + +Germline transmission of the p53 RMCE-ready locus. p53RMCE/+ ES cells were injected into blastocysts to generate chimeric mice. Chimeras (>80%) were then mated with p53+/− mice (Taconic) and MEFs were prepared. MEFs were first genotyped by PCR with primers a and b (see Figure 2) to detect the PuroΔTK marker of the RMCE allele (top). This revealed germline transmission of the p53 RMCE-ready locus in MEFs 1, 2 and 6. Each of these three MEF clones was further analyzed (bottom) with primers f and g (left lanes) and h and i (right lanes), routinely used to genotype p53+/− mice (sequences in Materials and Methods). Primers f and g amplify a 320 bp product from a WT or RMCE allele, while primers h and i specifically amplify a 150 bp product from the Neo marker in the KO allele. MEF 1 are p53RMCE/+ and MEFs 2 and 6 are p53RMCE/− cells. + +Figure 5 + +Performing RMCE in MEFs. (A) RMCE with the p53GFP plasmid leads to the transient observation of cells with intense nuclear fluorescence. p53RMCE/− MEFs, electroporated with a Cre expression plasmid and the p53GFP exchange plasmid, were analyzed 48 h later by fluorescence microscopy. A typical field (left to right: fluorescence, phase contrast, merged) with a fluorescent cell (arrow) is shown. The fluorescent cell is enlarged (extreme right). (B) RMCE with the p53ΔPGFP plasmid. p53RMCE/− MEFs, electroporated with a Cre expression plasmid and the p53ΔPGFP plasmid, were selected with ganciclovir. PCR with primers d and e (Figure 3B) indicated that 9/22 ganciclovir-resistant clones integrated the ΔP mutation [top row, a representative analysis of 10 clones (Q–Z) is shown]. PCR with primers b and c next verified that the detected PRD deletions resulted from RMCE at the p53 locus, not random integration (middle row, as expected clones R, S, T and W are positive, but not Q). Western analysis of positive clones (bottom row) showed that p53ΔPGFP accumulated after ADR, but at lower levels than p53WT. (C) Phenotypic assay of p53ΔPGFP: loss of cell cycle control. Asynchronous p53RMCE/− and p53ΔPGFP/− MEFs left untreated, or irradiated with doses of 6 or 12 Gy, were analyzed (top shows a typical experiment; bottom plots results from ≥4 independent experiments and ≥3 independent MEFs). Note that the p53RMCE locus encodes a WT p53. + +Table 1 + +Summary of targeting experiments + +In ES cells, targeting p53ΔPGFP or p53GFP by RMCE was ∼20 times more efficient than targeting Flox by homologous recombination. In MEFs, due to p53GFP toxicity, only the targeting efficiency of RMCE with p53ΔPGFP could be evaluated. Importantly, a phenotypic read-out of the p53ΔPGFP mutation in MEFs, if it had been performed after targeting in fibroblasts by homologous recombination, would have required two rounds of inefficient targeting to target both p53 alleles. diff --git a/src/ontogpt/evaluation/craft/database/all/16968134.ann b/src/ontogpt/evaluation/craft/database/all/16968134.ann new file mode 100644 index 000000000..8392c805f --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16968134.ann @@ -0,0 +1,1457 @@ +T1 GO:0005921 14 26 Gap Junction +T2 PR:000025844 56 62 ephrin +T3 http://purl.obolibrary.org/obo/MONDO_0010570 84 110 Craniofrontonasal Syndrome +T4 GO:0000805 135 136 X +T5 PR:000006921 144 153 ephrin-B1 +T6 NCBITaxon:9606 157 163 humans +T7 http://purl.obolibrary.org/obo/MONDO_0010570 170 196 craniofrontonasal syndrome +T8 http://purl.obolibrary.org/obo/MONDO_0010570 198 202 CFNS +T9 http://purl.obolibrary.org/obo/MONDO_0000001 207 214 disease +T10 PR:000006921 281 290 ephrin-B1 +T11 GO:0000805 330 331 X +T12 GO:0009048 330 344 X-inactivation +T13 PR:000006921 366 375 ephrin-B1 +T14 NCBITaxon:10088 379 383 mice +T15 PR:000006921 425 434 ephrin-B1 +T16 GO:0010467 435 445 expression +T17 PR:000006921 525 534 ephrin-B1 +T18 NCBITaxon:10088 538 542 mice +T19 UBERON:0004339 551 560 calvarial +T20 UBERON:0002342 596 608 neural crest +T21 UBERON:0004339 679 688 calvarial +T22 GO:0001503 728 738 osteogenic +T23 GO:0005921 764 776 gap junction +T24 PR:000025844 821 827 ephrin +T25 PR:000006921 848 857 ephrin-B1 +T26 PR:000008373 873 883 connexin43 +T27 GO:0065007 888 897 regulates +T28 SO:0000704 937 944 genetic +T29 UBERON:0004339 984 993 calvarial +T30 PR:000006921 1014 1023 ephrin-B1 +T31 UBERON:0000922 1027 1034 embryos +T32 PR:000025844 1077 1084 ephrins +T33 GO:0065007 1088 1098 regulating +T34 http://purl.obolibrary.org/obo/MONDO_0010570 1160 1164 CFNS +T35 GO:0065007 1194 1204 regulation +T36 UBERON:0000479 1224 1231 tissues +T37 NCBITaxon:33208 1387 1393 animal +T38 PR:000025844 1434 1441 ephrins +T39 GO:0065007 1442 1450 regulate +T40 UBERON:0000479 1517 1523 tissue +T41 PR:000025844 1661 1668 ephrins +T42 PR:000006921 1824 1833 ephrin-B1 +T43 NCBITaxon:10088 1854 1858 mice +T44 PR:000006921 1911 1920 ephrin-B1 +T45 SO:0000704 1921 1925 gene +T46 NCBITaxon:9606 1929 1934 human +T47 http://purl.obolibrary.org/obo/MONDO_0010570 1935 1961 craniofrontonasal syndrome +T48 http://purl.obolibrary.org/obo/MONDO_0010570 1963 1967 CFNS +T49 GO:0000805 2008 2009 X +T50 GO:0009048 2008 2022 X-inactivation +T51 GO:0000805 2024 2025 X +T52 PR:000006921 2033 2042 ephrin-B1 +T53 GO:0010467 2043 2053 expression +T54 PR:000006921 2067 2076 ephrin-B1 +T55 NCBITaxon:10088 2080 2084 mice +T56 PR:000006921 2089 2098 ephrin-B1 +T57 PR:000006921 2112 2121 ephrin-B1 +T58 http://purl.obolibrary.org/obo/MONDO_0021003 2188 2199 polydactyly +T59 PR:000006921 2236 2245 ephrin-B1 +T60 NCBITaxon:33208 2251 2258 animals +T61 http://purl.obolibrary.org/obo/MONDO_0010570 2277 2281 CFNS +T62 GO:0000805 2288 2289 X +T63 http://purl.obolibrary.org/obo/MONDO_0000425 2288 2296;2311 2319 X-linked disorder +T64 UBERON:0004766 2408 2415;2426 2431 cranial ... bones +T65 UBERON:0001681 2420 2431 nasal bones +T66 http://purl.obolibrary.org/obo/MONDO_0015469 2437 2453 craniosynostosis +T67 UBERON:0002489 2479 2494 coronal sutures +T68 http://purl.obolibrary.org/obo/MONDO_0021003 2542 2553 polydactyly +T69 http://purl.obolibrary.org/obo/MONDO_0021002 2558 2568 syndactyly +T70 PR:000025844 2699 2706 ephrins +T71 PR:000025844 2777 2784 ephrins +T72 PR:000025844 2939 2945 ephrin +T73 GO:0065007 2966 2976 regulation +T74 GO:0015629 3030 3048 actin cytoskeleton +T75 PR:000025844 3099 3106 ephrins +T76 GO:0065007 3107 3115 regulate +T77 GO:0005921 3116 3128 gap junction +T78 NCBITaxon:7955 3173 3182 zebrafish +T79 GO:0010467 3199 3209 expression +T80 PR:000025844 3231 3238 ephrins +T81 NCBITaxon:33208 3242 3248 animal +T82 CL:0000548 3242 3248;3253 3258 animal ... cells +T83 CL:0000676 3249 3258 cap cells +T84 GO:0005921 3334 3347 Gap junctions +T85 GO:0016020 3366 3374 membrane +T86 CHEBI:36357 3444 3453 molecules +T87 NCBITaxon:7742 3489 3499 vertebrate +T88 UBERON:0001892 3616 3627 rhombomeric +T89 UBERON:0001892 3666 3677 rhombomeres +T90 GO:0005921 3801 3815 gap junctional +T91 NCBITaxon:9606 3855 3860 human +T92 http://purl.obolibrary.org/obo/MONDO_0000001 3861 3869 diseases +T93 UBERON:0004288 3916 3924 skeletal +T94 GO:0001501 3916 3936 skeletal development +T95 PR:000008373 3959 3969 connexin43 +T96 PR:000008373 3971 3975 Cx43 +T97 UBERON:0004288 3997 4005 skeletal +T98 NCBITaxon:9606 4022 4028 humans +T99 NCBITaxon:10088 4033 4037 mice +T100 PR:000006921 4136 4145 ephrin-B1 +T101 NCBITaxon:10088 4159 4163 mice +T102 PR:000006921 4194 4203 ephrin-B1 +T103 UBERON:0004339 4223 4232 calvarial +T104 GO:0030154 4261 4279;4293 4298 differentiation of ... cells +T105 UBERON:0002342 4280 4292 neural crest +T106 PR:000025844 4360 4366 ephrin +T107 PR:000006921 4387 4396 ephrin-B1 +T108 PR:000008373 4423 4427 Cx43 +T109 GO:0010467 4493 4503 expression +T110 PR:000008373 4507 4511 Cx43 +T111 UBERON:0004339 4534 4543 calvarial +T112 PR:000006921 4564 4573 ephrin-B1 +T113 GO:0065007 4611 4621 regulation +T114 PR:000025844 4677 4683 ephrin +T115 PR:000006921 4748 4757 ephrin-B1 +T116 PR:000025844 4847 4853 ephrin +T117 UBERON:0000479 4890 4896 tissue +T118 http://purl.obolibrary.org/obo/MONDO_0010570 4987 4991 CFNS +T119 NCBITaxon:9606 4995 5001 humans +T120 PR:000025844 5039 5046 ephrins +T121 PR:000006921 5093 5102 ephrin-B1 +T122 PR:000006921 5166 5175 ephrin-B1 +T123 http://purl.obolibrary.org/obo/MONDO_0021003 5197 5208 polydactyly +T124 PR:000025844 5241 5247 ephrin +T125 PR:000006921 5263 5272 ephrin-B1 +T126 http://purl.obolibrary.org/obo/MONDO_0010570 5299 5303 CFNS +T127 NCBITaxon:9606 5304 5309 human +T128 PR:000006921 5399 5408 ephrin-B1 +T129 NCBITaxon:10088 5429 5433 mice +T130 UBERON:0000479 5479 5486 tissues +T131 PR:000006921 5556 5565 ephrin-B1 +T132 UBERON:0005744 5621 5628 foramen +T133 UBERON:0000209 5642 5655 frontal bones +T134 UBERON:0000209 5735 5748 frontal bones +T135 UBERON:0000210 5755 5768 parietal bone +T136 PR:000006921 5804 5813 Ephrin-B1 +T137 PR:000006921 5853 5862 ephrin-B1 +T138 UBERON:0001474 5932 5937 bones +T139 UBERON:0000209 5977 5989 frontal bone +T140 PR:000006921 6065 6074 ephrin-B1 +T141 UBERON:0000209 6152 6165 frontal bones +T142 PR:000006921 6172 6181 Ephrin-B1 +T143 UBERON:0006378 6234 6243 vibrissae +T144 PR:000006921 6300 6309 ephrin-B1 +T145 PR:000006921 6319 6328 ephrin-B1 +T146 SO:0000346 6330 6333 lox +T147 http://purl.obolibrary.org/obo/MONDO_0010570 6479 6483 CFNS +T148 PR:000025844 6550 6557 ephrins +T149 PR:000025844 6642 6648 ephrin +T150 GO:0048013 6642 6658 ephrin signaling +T151 UBERON:0004339 6673 6682 calvarial +T152 PR:000006921 6697 6706 ephrin-B1 +T153 PR:000006922 6742 6751 ephrin-B2 +T154 SO:0000704 6771 6775 gene +T155 PR:000006922 6867 6876 ephrin-B2 +T156 GO:0010467 6880 6889 expressed +T157 NCBITaxon:10088 6941 6946 mouse +T158 PR:000006922 6985 6994 ephrin-B2 +T159 SO:0000704 6995 6999 gene +T160 CHEBI:15358 7054 7061 histone +T161 PR:000036509 7054 7064 histone 2B +T162 PR:000036509 7066 7069 H2B +T163 GO:0065007 7121 7128 control +T164 PR:000006922 7136 7145 ephrin-B2 +T165 SO:0000167 7146 7154 promoter +T166 PR:000006922 7196 7205 ephrin-B2 +T167 PR:000006921 7210 7219 ephrin-B1 +T168 NCBITaxon:33208 7233 7240 animals +T169 GO:0007618 7246 7251 mated +T170 PR:000006921 7264 7273 ephrin-B1 +T171 PR:000006922 7274 7283 ephrin-B2 +T172 NCBITaxon:33208 7304 7311 animals +T173 UBERON:0004288 7313 7321 Skeleton +T174 PR:000006922 7370 7379 ephrin-B2 +T175 PR:000006921 7386 7395 ephrin-B1 +T176 UBERON:0004339 7423 7432 calvarial +T177 UBERON:0000209 7504 7517 frontal bones +T178 PR:000006921 7532 7541 ephrin-B1 +T179 PR:000006922 7545 7554 ephrin-B2 +T180 UBERON:0000922 7560 7567 embryos +T181 UBERON:0002489 7588 7602 coronal suture +T182 PR:000006921 7635 7644 ephrin-B1 +T183 UBERON:0002489 7694 7709 coronal sutures +T184 PR:000006921 7713 7722 ephrin-B1 +T185 PR:000006922 7726 7735 ephrin-B2 +T186 UBERON:0000922 7741 7748 embryos +T187 UBERON:4200215 7814 7820 suture +T188 UBERON:0003104 7821 7831 mesenchyme +T189 UBERON:0004288 7861 7869 skeleton +T190 UBERON:0000922 7894 7901 embryos +T191 PR:000006921 7944 7953 ephrin-B1 +T192 PR:000006922 7957 7966 ephrin-B2 +T193 UBERON:0000922 7972 7979 embryos +T194 PR:000006921 8004 8013 ephrin-B1 +T195 UBERON:0000922 8017 8024 embryos +T196 UBERON:0002489 8087 8101 coronal suture +T197 PR:000006921 8129 8138 ephrin-B1 +T198 PR:000006922 8142 8151 ephrin-B2 +T199 UBERON:0004339 8213 8222 calvarial +T200 PR:000006921 8321 8330 ephrin-B1 +T201 UBERON:0004339 8378 8387 calvarial +T202 UBERON:0001474 8388 8393 bones +T203 PR:000006922 8418 8427 ephrin-B2 +T204 PR:000006921 8434 8443 ephrin-B1 +T205 UBERON:0002489 8510 8524 coronal suture +T206 UBERON:0004339 8554 8563 calvarial +T207 PR:000006921 8578 8587 ephrin-B1 +T208 UBERON:0000922 8593 8600 embryos +T209 UBERON:0004339 8630 8639 calvarial +T210 UBERON:0000922 8659 8666 embryos +T211 PR:000006922 8697 8706 ephrin-B2 +T212 PR:000006921 8774 8783 ephrin-B1 +T213 UBERON:0000922 8787 8794 Embryos +T214 UBERON:0004339 8835 8844 calvarial +T215 PR:000006921 8868 8877 ephrin-B1 +T216 GO:0010467 8909 8919 expression +T217 PR:000006921 8931 8940 ephrin-B1 +T218 PR:000006922 8945 8954 ephrin-B2 +T219 PR:000007130 8984 8989 EphB2 +T220 PR:000007131 8994 8999 EphB3 +T221 http://purl.obolibrary.org/obo/MONDO_0021003 9083 9094 polydactyly +T222 GO:0060021 9116 9128;9133 9139 formation of ... palate +T223 PR:000006921 9189 9198 ephrin-B1 +T224 GO:0010467 9210 9220 expression +T225 PR:000006921 9224 9233 ephrin-B1 +T226 PR:000007130 9235 9240 EphB2 +T227 PR:000007131 9246 9251 EphB3 +T228 UBERON:0000209 9286 9299 frontal bones +T229 PR:000006921 9313 9322 Ephrin-B1 +T230 PR:000007131 9327 9332 EphB3 +T231 GO:0010467 9337 9346 expressed +T232 PR:000007130 9375 9380 EphB2 +T233 GO:0010467 9381 9391 expression +T234 PR:000006921 9443 9452 ephrin-B1 +T235 GO:0010467 9470 9479 expressed +T236 UBERON:0002360 9487 9502 meningeal layer +T237 UBERON:0002342 9522 9534 neural crest +T238 UBERON:0004339 9605 9614 calvarial +T239 PR:000006921 9637 9646 ephrin-B1 +T240 GO:0010467 9650 9659 expressed +T241 UBERON:0005253 9675 9690 head mesenchyme +T242 UBERON:0006378 9709 9718 vibrissae +T243 UBERON:0000922 9737 9744 embryos +T244 PR:000006921 9776 9785 ephrin-B1 +T245 UBERON:0000922 9789 9796 embryos +T246 GO:0010467 9798 9808 expression +T247 PR:000006921 9812 9821 ephrin-B1 +T248 UBERON:0003104 9860 9870 mesenchyme +T249 UBERON:0001893 9882 9895 telencephalon +T250 UBERON:0006378 9939 9948 vibrissae +T251 PR:000006922 9973 9982 ephrin-B2 +T252 PR:000007130 9987 9992 EphB2 +T253 GO:0010467 10001 10011 expression +T254 PR:000006921 10041 10050 ephrin-B1 +T255 GO:0010467 10073 10083 Expression +T256 PR:000006922 10087 10096 ephrin-B2 +T257 PR:000006921 10123 10132 ephrin-B1 +T258 PR:000007130 10157 10162 EphB2 +T259 GO:0010467 10163 10173 expression +T260 GO:0010467 10247 10257 expression +T261 PR:000006921 10261 10270 ephrin-B1 +T262 PR:000006921 10274 10283 ephrin-B1 +T263 UBERON:0004347 10287 10296 limb buds +T264 PR:000006921 10322 10331 ephrin-B1 +T265 PR:000006921 10398 10407 ephrin-B1 +T266 GO:0000805 10440 10441 X +T267 GO:0009048 10440 10454 X-inactivation +T268 GO:0010467 10478 10488 expression +T269 PR:000006921 10492 10501 ephrin-B1 +T270 UBERON:0004347 10509 10517 limb bud +T271 http://purl.obolibrary.org/obo/MONDO_0021003 10536 10547 polydactyly +T272 PR:000006921 10578 10587 ephrin-B1 +T273 UBERON:0004339 10636 10645 calvarial +T274 PR:000006921 10669 10678 ephrin-B1 +T275 GO:0010467 10727 10737 expression +T276 PR:000006921 10741 10750 ephrin-B1 +T277 PR:000007130 10755 10760 EphB2 +T278 UBERON:0000209 10780 10792 frontal bone +T279 PR:000006921 10829 10838 ephrin-B1 +T280 PR:000006921 10852 10861 ephrin-B1 +T281 UBERON:0003104 10897 10907 mesenchyme +T282 UBERON:0000209 10914 10926 Frontal Bone +T283 GO:0001503 10956 10966 Osteogenic +T284 PR:000006921 11047 11056 ephrin-B1 +T285 PR:000006921 11113 11122 ephrin-B1 +T286 PR:000006921 11136 11145 ephrin-B1 +T287 GO:0009888 11191 11205;11211 11217 development of ... tissue +T288 UBERON:0000479 11211 11217 tissue +T289 PR:000006921 11263 11272 ephrin-B1 +T290 UBERON:0000209 11301 11313 frontal bone +T291 PR:000006921 11351 11360 ephrin-B1 +T292 PR:000006922 11365 11374 ephrin-B2 +T293 GO:0065007 11375 11382 control +T294 GO:0016477 11383 11395;11402 11407 migration of ... cells +T295 UBERON:0000209 11505 11517 frontal bone +T296 SO:0001023 11565 11572 alleles +T297 PR:000006921 11667 11676 ephrin-B1 +T298 NCBITaxon:33208 11712 11719 animals +T299 UBERON:0000209 11801 11813 frontal bone +T300 GO:0008283 11841 11854 proliferation +T301 UBERON:0000209 11916 11929 frontal bones +T302 UBERON:0004339 11982 11991 calvarial +T303 PR:000006921 12012 12021 ephrin-B1 +T304 UBERON:0000922 12025 12032 embryos +T305 CL:0000062 12065 12077 osteoblastic +T306 CL:0000062 12159 12171 osteoblastic +T307 UBERON:0000209 12214 12227 frontal bones +T308 PR:000006921 12262 12271 ephrin-B1 +T309 PR:000006922 12275 12284 ephrin-B2 +T310 PR:000006921 12294 12303 ephrin-B1 +T311 PR:000006921 12414 12423 ephrin-B1 +T312 PR:000006921 12432 12441 ephrin-B1 +T313 UBERON:0000922 12445 12452 embryos +T314 PR:000006921 12607 12616 ephrin-B1 +T315 UBERON:0000922 12620 12627 embryos +T316 UBERON:0000209 12696 12708 frontal bone +T317 PR:000006921 12731 12740 ephrin-B1 +T318 UBERON:0000922 12754 12761 embryos +T319 UBERON:0004339 12813 12822 calvarial +T320 PR:000006921 12843 12852 ephrin-B1 +T321 GO:0001755 12888 12897;12910 12917 migration ... of NCCs +T322 GO:0001503 12994 13004 osteogenic +T323 UBERON:0003104 13005 13015 mesenchyme +T324 PR:000006921 13069 13078 ephrin-B1 +T325 GO:0001503 13118 13128 osteogenic +T326 UBERON:0003104 13129 13140 mesenchymal +T327 CL:0000134 13129 13146 mesenchymal cells +T328 PR:000006921 13172 13181 ephrin-B1 +T329 UBERON:0000922 13185 13192 embryos +T330 GO:0001503 13283 13293 osteogenic +T331 PR:000006921 13348 13357 ephrin-B1 +T332 UBERON:0000922 13361 13368 embryos +T333 CL:0007010 13489 13510 osteoprogenitor cells +T334 GO:0010467 13539 13549 expression +T335 PR:000006921 13553 13562 ephrin-B1 +T336 GO:0010467 13677 13687 expression +T337 PR:000006921 13691 13700 ephrin-B1 +T338 PR:000006921 13755 13764 ephrin-B1 +T339 GO:0065007 13774 13782 regulate +T340 GO:0001503 13816 13826 Osteogenic +T341 PR:000006921 13846 13855 ephrin-B1 +T342 UBERON:0000922 13859 13866 Embryos +T343 PR:000008373 13892 13896 Cx43 +T344 GO:0065007 13993 14001 regulate +T345 GO:0001503 14002 14012 osteogenic +T346 PR:000001443 14040 14050 N-cadherin +T347 PR:000008373 14055 14059 Cx43 +T348 GO:0010467 14060 14070 expression +T349 GO:0000187 14083 14101 activation of MAPK +T350 PR:000008373 14121 14125 Cx43 +T351 PR:000006921 14200 14209 ephrin-B1 +T352 UBERON:0000922 14223 14230 embryos +T353 PR:000008373 14265 14269 Cx43 +T354 GO:0005921 14305 14319 gap junctional +T355 GO:0005737 14347 14358 cytoplasmic +T356 PR:000008373 14359 14363 Cx43 +T357 GO:0009986 14412 14424 cell surface +T358 GO:0005921 14499 14513 gap junctional +T359 GO:0005911 14525 14545 cell–cell interfaces +T360 UBERON:0003104 14562 14573 mesenchymal +T361 CL:0000134 14562 14579 mesenchymal cells +T362 UBERON:0000922 14604 14611 embryos +T363 GO:0010467 14639 14648 expressed +T364 PR:000008373 14665 14669 Cx43 +T365 GO:0005921 14675 14689 gap junctional +T366 UBERON:0003104 14793 14804 mesenchymal +T367 CL:0000134 14793 14810 mesenchymal cells +T368 PR:000006921 14825 14834 ephrin-B1 +T369 UBERON:0000922 14848 14855 embryos +T370 PR:000008373 14877 14881 Cx43 +T371 GO:0005921 14898 14912 gap junctional +T372 PR:000008373 15019 15023 Cx43 +T373 PR:000006921 15072 15081 ephrin-B1 +T374 UBERON:0000922 15095 15102 embryos +T375 GO:0010467 15149 15159 expression +T376 PR:000008373 15163 15167 Cx43 +T377 PR:000006921 15205 15214 ephrin-B1 +T378 UBERON:0000922 15218 15225 embryos +T379 PR:000008373 15280 15284 Cx43 +T380 PR:000006921 15305 15314 ephrin-B1 +T381 UBERON:0000922 15322 15329 embryos +T382 UBERON:0000922 15424 15431 embryos +T383 PR:000008373 15444 15448 Cx43 +T384 UBERON:0000209 15490 15502 frontal bone +T385 PR:000006921 15529 15538 ephrin-B1 +T386 UBERON:0000922 15542 15548 embryo +T387 UBERON:0000209 15574 15586 frontal bone +T388 PR:000008373 15663 15667 Cx43 +T389 PR:000006921 15725 15734 ephrin-B1 +T390 UBERON:0000922 15740 15747 embryos +T391 UBERON:0004339 15760 15769 calvarial +T392 GO:0001503 15792 15802 osteogenic +T393 PR:000006921 15837 15846 ephrin-B1 +T394 PR:000008373 15903 15907 Cx43 +T395 PR:000008373 15950 15954 Cx43 +T396 PR:000006921 15998 16007 ephrin-B1 +T397 UBERON:0000922 16051 16058 embryos +T398 PR:000006921 16100 16109 ephrin-B1 +T399 PR:000006921 16123 16132 ephrin-B1 +T400 UBERON:0000922 16171 16178 embryos +T401 PR:000006921 16192 16201 ephrin-B1 +T402 UBERON:0000922 16207 16216 embryonic +T403 CL:0002322 16207 16221;16227 16232 embryonic stem ... cells +T404 CL:0002322 16223 16225;16227 16232 ES ... cells +T405 UBERON:0000358 16253 16264 blastocysts +T406 GO:0010467 16271 16278 express +T407 CHEBI:75055 16332 16337 X-gal +T408 UBERON:0000922 16361 16368 embryos +T409 PR:000008373 16417 16421 Cx43 +T410 GO:0042571 16422 16430 antibody +T411 GO:0005921 16432 16446 Gap junctional +T412 PR:000008373 16447 16451 Cx43 +T413 PR:000006921 16509 16518 ephrin-B1 +T414 GO:0005921 16552 16566 gap junctional +T415 PR:000008373 16567 16571 Cx43 +T416 PR:000006921 16606 16615 ephrin-B1 +T417 PR:000006921 16712 16721 ephrin-B1 +T418 PR:000006921 16731 16740 ephrin-B1 +T419 PR:000006921 16771 16780 Ephrin-B1 +T420 PR:000008373 16797 16801 Cx43 +T421 GO:0065007 16806 16815 Regulates +T422 PR:000007130 16867 16881 EphB2 receptor +T423 PR:000006921 16902 16911 ephrin-B1 +T424 PR:000006921 16932 16941 ephrin-B1 +T425 UBERON:0000922 16945 16952 embryos +T426 PR:000025844 16980 16986 ephrin +T427 PR:000008373 17051 17055 Cx43 +T428 PR:000006921 17059 17068 ephrin-B1 +T429 PR:000006921 17078 17087 ephrin-B1 +T430 GO:0048013 17163 17183 Eph/ephrin signaling +T431 PR:000025844 17167 17173 ephrin +T432 GO:0048013 17201 17221 Eph/ephrin signaling +T433 PR:000025844 17205 17211 ephrin +T434 GO:0065007 17228 17236 regulate +T435 PR:000006921 17324 17333 ephrin-B1 +T436 PR:000007130 17338 17344 Eph-B2 +T437 GO:0010467 17410 17420 expressing +T438 PR:000006921 17421 17430 ephrin-B1 +T439 GO:0010467 17490 17500 expressing +T440 PR:000007130 17515 17521 Eph-B2 +T441 PR:000006921 17609 17618 ephrin-B1 +T442 GO:0010467 17619 17629 expressing +T443 GO:0010467 17647 17657 expressing +T444 PR:000007130 17673 17679 Eph-B2 +T445 GO:0010467 17789 17796 express +T446 PR:000006921 17802 17811 ephrin-B1 +T447 PR:000007130 17816 17822 Eph-B2 +T448 CL:0000057 17907 17918 fibroblasts +T449 CL:0000057 17954 17965 fibroblasts +T450 GO:0010467 17966 17976 expressing +T451 PR:000006921 17977 17986 ephrin-B1 +T452 CHEBI:37958 18006 18009 dye +T453 CHEBI:37958 18183 18186 dye +T454 CL:0000057 18293 18304 fibroblasts +T455 GO:0010467 18305 18315 expressing +T456 PR:000007130 18316 18322 Eph-B2 +T457 CHEBI:37958 18501 18504 dye +T458 CHEBI:37958 18624 18627 dye +T459 PR:000006921 18724 18733 ephrin-B1 +T460 PR:000007130 18758 18763 EphB2 +T461 GO:0010467 18766 18776 expressing +T462 PR:000007130 18835 18840 EphB2 +T463 PR:000006921 18845 18854 ephrin-B1 +T464 PR:000025844 18945 18952 ephrins +T465 PR:000008373 18970 18974 Cx43 +T466 GO:0065007 18992 19000 regulate +T467 PR:000006921 19043 19052 ephrin-B1 +T468 PR:000008373 19057 19061 Cx43 +T469 PR:000008373 19121 19125 Cx43 +T470 PR:000006921 19130 19139 ephrin-B1 +T471 CL:0000001 19165 19172;19185 19190 primary ... cells +T472 UBERON:0003104 19173 19184 mesenchymal +T473 CL:0000134 19173 19190 mesenchymal cells +T474 PR:000025844 19239 19245 ephrin +T475 PR:000008373 19260 19264 Cx43 +T476 PR:000006921 19301 19310 ephrin-B1 +T477 GO:0009294 19327 19338 transfected +T478 GO:0010467 19340 19350 Expression +T479 PR:000006921 19354 19363 ephrin-B1 +T480 PR:000008373 19368 19372 Cx43 +T481 GO:0005911 19441 19465 interfaces between cells +T482 GO:0010467 19466 19476 expressing +T483 PR:000006921 19477 19486 ephrin-B1 +T484 PR:000008373 19511 19515 Cx43 +T485 PR:000007130 19565 19570 EphB2 +T486 PR:000007130 19593 19607 EphB2 receptor +T487 PR:000006921 19610 19619 ephrin-B1 +T488 PR:000008373 19677 19681 Cx43 +T489 PR:000006921 19795 19804 ephrin-B1 +T490 PR:000008373 19809 19813 Cx43 +T491 PR:000025844 19897 19903 ephrin +T492 PR:000006921 19933 19942 ephrin-B1 +T493 PR:000008373 19947 19951 Cx43 +T494 GO:0010467 20021 20031 expressing +T495 PR:000006921 20032 20041 ephrin-B1 +T496 GO:0005576 20091 20104 extracellular +T497 SO:0000417 20105 20111 domain +T498 PR:000007130 20115 20130 Eph-B2 receptor +T499 NCBITaxon:9606 20159 20164 human +T500 GO:0071735 20165 20168 IgG +T501 PR:000007130 20170 20175 EphB2 +T502 PR:000006921 20193 20202 ephrin-B1 +T503 PR:000008373 20204 20208 Cx43 +T504 PR:000006921 20274 20283 ephrin-B1 +T505 PR:000006921 20324 20333 ephrin-B1 +T506 PR:000008373 20373 20377 Cx43 +T507 GO:0042571 20378 20386 antibody +T508 PR:000006921 20429 20438 ephrin-B1 +T509 PR:000008373 20443 20447 Cx43 +T510 GO:0065007 20475 20485 Regulation +T511 PR:000025844 20503 20509 ephrin +T512 SO:0000417 20548 20554 domain +T513 PR:000006921 20558 20567 ephrin-B1 +T514 PR:000008373 20602 20606 Cx43 +T515 GO:0005576 20679 20692 extracellular +T516 SO:0000417 20693 20699 domain +T517 PR:000006921 20703 20712 ephrin-B1 +T518 NCBITaxon:9606 20741 20746 human +T519 GO:0071735 20747 20750 IgG +T520 PR:000006921 20752 20760 ephrinB1 +T521 PR:000008373 20766 20770 Cx43 +T522 GO:0005622 20825 20838 intracellular +T523 SO:0000417 20839 20845 domain +T524 PR:000006921 20849 20858 ephrin-B1 +T525 PR:000008373 20896 20900 Cx43 +T526 SO:0000417 20953 20959 domain +T527 PR:000006921 20963 20972 ephrin-B1 +T528 PR:000008373 21012 21016 Cx43 +T529 PR:000008373 21024 21028 Cx43 +T530 CHEBI:36357 21076 21085 molecules +T531 PR:000008373 21131 21135 Cx43 +T532 GO:0005622 21155 21168 intracellular +T533 SO:0000417 21169 21175 domain +T534 PR:000006921 21179 21188 ephrin-B1 +T535 PR:000006921 21220 21229 ephrin-B1 +T536 PR:000006921 21285 21294 ephrin-B1 +T537 PR:000006921 21340 21349 ephrin-B1 +T538 PR:000008373 21367 21371 Cx43 +T539 GO:0009294 21425 21436 transfected +T540 PR:000006921 21459 21468 ephrin-B1 +T541 PR:000006921 21472 21481 ephrin-B1 +T542 GO:0010467 21565 21574 expressed +T543 GO:0010467 21613 21623 expression +T544 PR:000006921 21627 21636 ephrin-B1 +T545 PR:000008373 21685 21689 Cx43 +T546 CHEBI:8984 21732 21735 SDS +T547 GO:0065007 21761 21769 regulate +T548 PR:000008373 21787 21791 Cx43 +T549 GO:0010467 21839 21849 expressing +T550 PR:000006921 21857 21866 ephrin-B1 +T551 PR:000006921 21880 21889 ephrin-B1 +T552 PR:000008373 22007 22011 Cx43 +T553 PR:000006921 22050 22059 ephrin-B1 +T554 PR:000008373 22111 22115 Cx43 +T555 PR:000006921 22136 22145 ephrin-B1 +T556 SO:0000417 22217 22223 domain +T557 PR:000006921 22227 22236 ephrin-B1 +T558 PR:000008373 22278 22282 Cx43 +T559 PR:000006921 22293 22302 ephrin-B1 +T560 PR:000006921 22321 22330 ephrin-B1 +T561 PR:000008373 22379 22383 Cx43 +T562 PR:000008373 22409 22413 Cx43 +T563 PR:000008373 22449 22453 Cx43 +T564 PR:000008373 22479 22483 Cx43 +T565 GO:0005737 22509 22520 cytoplasmic +T566 PR:000006921 22545 22554 ephrin-B1 +T567 PR:000008373 22577 22581 Cx43 +T568 PR:000007130 22606 22611 EphB2 +T569 PR:000006921 22625 22634 ephrin-B1 +T570 GO:0009986 22658 22670 cell surface +T571 PR:000006921 22736 22745 ephrin-B1 +T572 PR:000008373 22754 22758 Cx43 +T573 PR:000007130 22783 22788 EphB2 +T574 UBERON:0004339 22844 22853 calvarial +T575 PR:000006921 22880 22889 ephrin-B1 +T576 UBERON:0000922 22903 22910 embryos +T577 UBERON:0004288 22927 22935 skeletal +T578 PR:000006921 22958 22967 ephrin-B1 +T579 UBERON:0000922 23001 23008 embryos +T580 UBERON:0004339 23040 23049 calvarial +T581 UBERON:0001474 23050 23055 bones +T582 CL:0002322 23125 23133 ES cells +T583 http://purl.obolibrary.org/obo/MONDO_0021003 23168 23179 polydactyly +T584 UBERON:0000975 23242 23249 sternum +T585 PR:000006921 23316 23325 ephrin-B1 +T586 UBERON:0004339 23418 23427 calvarial +T587 http://purl.obolibrary.org/obo/MONDO_0021003 23432 23443 polydactyly +T588 UBERON:0000922 23471 23478 embryos +T589 PR:000006921 23524 23533 ephrin-B1 +T590 CL:0002322 23538 23546 ES cells +T591 PR:000006921 23582 23591 ephrin-B1 +T592 PR:000008373 23628 23632 Cx43 +T593 UBERON:0000922 23664 23671 embryos +T594 CL:0002322 23692 23700 ES cells +T595 PR:000006921 23714 23723 ephrin-B1 +T596 SO:0001023 23728 23734 allele +T597 PR:000006921 23739 23748 ephrin-B1 +T598 CL:0002322 23754 23762 ES cells +T599 UBERON:0000358 23798 23809 blastocysts +T600 CHEBI:75055 23846 23851 X-gal +T601 UBERON:0000922 23875 23882 embryos +T602 PR:000006921 23904 23913 ephrin-B1 +T603 GO:0010467 23932 23942 expressing +T604 PR:000006921 23943 23952 ephrin-B1 +T605 SO:0000417 24092 24098 domain +T606 UBERON:0004339 24170 24179 calvarial +T607 PR:000006921 24216 24225 ephrin-B1 +T608 PR:000008373 24250 24254 Cx43 +T609 GO:0065007 24312 24322 regulation +T610 PR:000006921 24388 24397 ephrin-B1 +T611 PR:000006921 24411 24420 ephrin-B1 +T612 GO:0009294 24477 24488 transfected +T613 PR:000008373 24544 24548 Cx43 +T614 PR:000008373 24555 24559 Cx43 +T615 GO:0009294 24630 24641 transfected +T616 PR:000008373 24649 24653 Cx43 +T617 GO:0010467 24658 24668 expressing +T618 GO:0009294 24693 24704 transfected +T619 GO:0010467 24845 24855 expression +T620 PR:000008373 24859 24863 Cx43 +T621 UBERON:0004339 24886 24895 Calvarial +T622 UBERON:0004339 25036 25045 calvarial +T623 GO:0065007 25064 25074 regulation +T624 PR:000006921 25085 25094 ephrin-B1 +T625 UBERON:0000922 25108 25115 embryos +T626 SO:0000704 25132 25139 genetic +T627 NCBITaxon:10088 25159 25163 Mice +T628 NCBITaxon:10358 25175 25178 CMV +T629 PR:000008373 25179 25183 Cx43 +T630 SO:0000902 25184 25193 transgene +T631 GO:0010467 25230 25240 expression +T632 PR:000008373 25244 25248 Cx43 +T633 PR:000006921 25267 25276 ephrin-B1 +T634 NCBITaxon:10088 25280 25284 mice +T635 PR:000008373 25349 25353 Cx43 +T636 UBERON:0000209 25369 25382 frontal bones +T637 UBERON:0000922 25386 25393 embryos +T638 SO:0000902 25407 25416 transgene +T639 UBERON:0004288 25437 25445 Skeleton +T640 UBERON:0004288 25501 25509 skeletal +T641 PR:000006921 25545 25554 ephrin-B1 +T642 PR:000006921 25600 25609 ephrin-B1 +T643 SO:0000704 25655 25662 genetic +T644 GO:0010467 25716 25726 expression +T645 PR:000008373 25730 25734 Cx43 +T646 UBERON:0004339 25756 25765 calvarial +T647 PR:000006921 25788 25797 ephrin-B1 +T648 UBERON:0005744 25828 25835 foramen +T649 UBERON:0000209 25853 25866 frontal bones +T650 GO:0010467 25956 25966 expression +T651 PR:000008373 25970 25974 Cx43 +T652 UBERON:0000209 26027 26040 frontal bones +T653 UBERON:0005744 26059 26066 foramen +T654 PR:000006921 26093 26102 ephrin-B1 +T655 NCBITaxon:10358 26124 26127 CMV +T656 PR:000008373 26128 26132 Cx43 +T657 SO:0000902 26133 26142 transgene +T658 PR:000006921 26155 26164 ephrin-B1 +T659 SO:0000902 26185 26194 transgene +T660 UBERON:0005744 26233 26240 foramen +T661 PR:000006921 26258 26267 ephrin-B1 +T662 NCBITaxon:10358 26297 26300 CMV +T663 PR:000008373 26301 26305 Cx43 +T664 SO:0000902 26306 26315 transgene +T665 UBERON:0000209 26357 26369 frontal bone +T666 SO:0000902 26401 26410 transgene +T667 GO:0010467 26483 26493 expression +T668 PR:000008373 26497 26501 Cx43 +T669 UBERON:0004288 26521 26529 skeletal +T670 UBERON:0000975 26582 26589 sternum +T671 PR:000006921 26669 26678 ephrin-B1 +T672 GO:0065007 26714 26724 regulation +T673 UBERON:0004339 26753 26762 calvarial +T674 PR:000006921 26783 26792 ephrin-B1 +T675 PR:000006921 26828 26837 ephrin-B1 +T676 NCBITaxon:10088 26841 26845 Mice +T677 http://purl.obolibrary.org/obo/MONDO_0010570 26861 26865 CFNS +T678 NCBITaxon:9606 26914 26920 humans +T679 PR:000006921 26943 26952 ephrin-B1 +T680 NCBITaxon:10088 26956 26960 mice +T681 UBERON:0004339 27001 27012 skull vault +T682 NCBITaxon:9606 27014 27019 Human +T683 http://purl.obolibrary.org/obo/MONDO_0010570 27020 27024 CFNS +T684 PR:000006921 27060 27069 ephrin-B1 +T685 SO:0000704 27070 27074 gene +T686 UBERON:0000970 27125 27131 ocular +T687 http://purl.obolibrary.org/obo/MONDO_0007778 27132 27145 hypertelorism +T688 UBERON:0001456 27167 27171 face +T689 UBERON:0008200 27191 27199 forehead +T690 UBERON:0000004 27208 27212 nose +T691 UBERON:0003128 27215 27222 cranium +T692 http://purl.obolibrary.org/obo/MONDO_0015469 27245 27261 craniosynostosis +T693 GO:0007567 27292 27297 birth +T694 PR:000006921 27299 27308 ephrin-B1 +T695 NCBITaxon:10088 27312 27316 mice +T696 GO:0001503 27340 27352 ossification +T697 UBERON:0004339 27356 27365 calvarial +T698 UBERON:0001474 27366 27371 bones +T699 UBERON:0005744 27393 27400 foramen +T700 UBERON:0003128 27412 27419 cranium +T701 http://purl.obolibrary.org/obo/MONDO_0000110 27466 27476 bifid nose +T702 UBERON:0000004 27472 27476 nose +T703 NCBITaxon:9606 27492 27497 human +T704 http://purl.obolibrary.org/obo/MONDO_0000001 27498 27505 disease +T705 PR:000006922 27528 27537 ephrin-B2 +T706 UBERON:0005744 27584 27591 foramen +T707 UBERON:0002489 27611 27625 coronal suture +T708 PR:000006921 27639 27648 ephrin-B1 +T709 PR:000006921 27685 27694 ephrin-B1 +T710 UBERON:0000922 27700 27707 embryos +T711 PR:000006921 27750 27759 ephrin-B1 +T712 PR:000006922 27818 27827 ephrin-B2 +T713 PR:000006922 27867 27876 ephrin-B2 +T714 UBERON:0004339 27880 27889 calvarial +T715 UBERON:0019248 27935 27950 early embryonic +T716 PR:000006922 27964 27973 ephrin-B2 +T717 UBERON:0000922 27979 27986 embryos +T718 NCBITaxon:10088 28015 28019 mice +T719 http://purl.obolibrary.org/obo/MONDO_0015469 28036 28052 craniosynostosis +T720 UBERON:0002489 28060 28075 coronal sutures +T721 NCBITaxon:9606 28084 28090 humans +T722 UBERON:0004339 28117 28126 calvarial +T723 UBERON:0005744 28127 28135 foramina +T724 http://purl.obolibrary.org/obo/MONDO_0015469 28140 28156 craniosynostosis +T725 SO:0000704 28161 28172 genetically +T726 PR:000010687 28273 28277 Msx2 +T727 PR:000027825 28347 28352 Twist +T728 http://purl.obolibrary.org/obo/MONDO_0015469 28365 28381 craniosynostosis +T729 UBERON:0005744 28397 28405 foramina +T730 GO:0065007 28438 28446 regulate +T731 UBERON:0000209 28466 28478 frontal bone +T732 UBERON:0002489 28558 28572 coronal suture +T733 PR:000006921 28576 28585 ephrin-B1 +T734 PR:000006922 28589 28598 ephrin-B2 +T735 UBERON:0000922 28604 28611 embryos +T736 UBERON:4200215 28660 28666 suture +T737 NCBITaxon:33208 28736 28743 animals +T738 GO:0016265 28744 28747 die +T739 GO:0007567 28751 28756 birth +T740 SO:0000704 28802 28809 genetic +T741 UBERON:0002489 28865 28879 coronal suture +T742 PR:000006921 28898 28907 ephrin-B1 +T743 NCBITaxon:10088 28921 28925 mice +T744 NCBITaxon:9606 28930 28936 humans +T745 SO:0000704 28953 28960 genetic +T746 GO:0010467 28976 28986 expression +T747 GO:0010467 29040 29050 expression +T748 PR:000006921 29054 29063 ephrin-B1 +T749 GO:0065007 29064 29072 controls +T750 UBERON:4200215 29073 29079 suture +T751 UBERON:0003104 29133 29143 mesenchyme +T752 PR:000006921 29175 29184 ephrin-B1 +T753 NCBITaxon:33208 29190 29197 animals +T754 UBERON:0004339 29217 29226 calvarial +T755 UBERON:0001474 29227 29232 bones +T756 PR:000006921 29261 29270 ephrin-B1 +T757 UBERON:0004339 29307 29316 calvarial +T758 UBERON:4200215 29344 29350 suture +T759 PR:000006921 29395 29404 ephrin-B1 +T760 GO:0010467 29408 29417 expressed +T761 UBERON:0002360 29425 29440 meningeal layer +T762 UBERON:4200215 29468 29474 suture +T763 UBERON:0000210 29505 29518 parietal bone +T764 PR:000006921 29541 29550 ephrin-B1 +T765 UBERON:0000922 29554 29561 embryos +T766 UBERON:4200215 29596 29602 suture +T767 UBERON:0000210 29607 29620 parietal bone +T768 PR:000006921 29666 29675 ephrin-B1 +T769 http://purl.obolibrary.org/obo/MONDO_0010570 29705 29709 CFNS +T770 SO:0000704 29752 29756 gene +T771 SO:0001587 29833 29853 premature stop codon +T772 PR:000006921 29904 29913 ephrin-B1 +T773 SO:0000704 30037 30041 gene +T774 PR:000006921 30109 30118 ephrin-B1 +T775 UBERON:0000922 30149 30156 embryos +T776 SO:0000417 30204 30210 domain +T777 PR:000006921 30214 30223 ephrin-B1 +T778 PR:000006921 30288 30297 ephrin-B1 +T779 UBERON:0000922 30303 30310 embryos +T780 http://purl.obolibrary.org/obo/MONDO_0016064 30321 30333 cleft palate +T781 UBERON:0004339 30355 30364 calvarial +T782 http://purl.obolibrary.org/obo/MONDO_0021003 30376 30387 polydactyly +T783 http://purl.obolibrary.org/obo/MONDO_0010570 30440 30444 CFNS +T784 CHEBI:35224 30526 30534 effector +T785 CHEBI:36357 30535 30544 molecules +T786 SO:0000417 30568 30574 domain +T787 GO:0001503 30587 30597 Osteogenic +T788 PR:000025844 30719 30725 ephrin +T789 PR:000006921 30740 30749 ephrin-B1 +T790 UBERON:0000209 30869 30882 frontal bones +T791 http://purl.obolibrary.org/obo/MONDO_0021003 30908 30919 polydactyly +T792 PR:000025844 30978 30984 ephrin +T793 PR:000008373 31035 31039 Cx43 +T794 PR:000025844 31069 31075 ephrin +T795 PR:000008373 31119 31123 Cx43 +T796 GO:0001503 31150 31160 osteogenic +T797 GO:0030154 31161 31179;31188 31193 differentiation of ... cells +T798 CL:0000001 31180 31193 primary cells +T799 PR:000006921 31208 31217 ephrin-B1 +T800 UBERON:0000922 31231 31238 embryos +T801 GO:0010467 31267 31277 expression +T802 PR:000008373 31281 31285 Cx43 +T803 UBERON:0004339 31308 31317 calvarial +T804 PR:000006921 31340 31349 ephrin-B1 +T805 PR:000008373 31452 31456 Cx43 +T806 GO:0001503 31460 31470 osteogenic +T807 SO:0000704 31517 31524 genetic +T808 GO:0065007 31559 31569 regulation +T809 UBERON:0002544 31602 31607 digit +T810 PR:000006921 31664 31673 ephrin-B1 +T811 NCBITaxon:10088 31738 31742 Mice +T812 PR:000008373 31757 31761 Cx43 +T813 GO:0001503 31778 31790 ossification +T814 UBERON:0004339 31798 31807 calvarial +T815 UBERON:0001474 31808 31813 bones +T816 PR:000008373 31886 31890 GJA1 +T817 SO:0000704 31896 31900 gene +T818 PR:000008373 31912 31916 Cx43 +T819 NCBITaxon:9606 31921 31927 humans +T820 http://purl.obolibrary.org/obo/MONDO_0008111 31948 31975 oculodentodigital dysplasia +T821 http://purl.obolibrary.org/obo/MONDO_0008111 31977 31981 ODDD +T822 http://purl.obolibrary.org/obo/MONDO_0002254 31986 31994 syndrome +T823 UBERON:0002544 32059 32064 digit +T824 CHEBI:23995 32084 32087 ENU +T825 NCBITaxon:10088 32098 32102 mice +T826 NCBITaxon:10088 32133 32138 mouse +T827 http://purl.obolibrary.org/obo/MONDO_0008111 32194 32198 ODDD +T828 NCBITaxon:10088 32243 32247 mice +T829 PR:000008373 32268 32272 GJA1 +T830 GO:0005921 32325 32337 gap junction +T831 GO:0016264 32325 32346 gap junction assembly +T832 GO:0048013 32436 32456 Eph/ephrin signaling +T833 PR:000025844 32440 32446 ephrin +T834 GO:0001503 32479 32489 osteogenic +T835 SO:0000417 32590 32596 domain +T836 PR:000006921 32600 32609 ephrin-B1 +T837 GO:0005737 32625 32636 cytoplasmic +T838 GO:0048013 32671 32691 Eph/ephrin signaling +T839 PR:000025844 32675 32681 ephrin +T840 PR:000002997 32693 32711 Src family kinases +T841 GO:0001503 32768 32778 osteogenic +T842 GO:0000187 32964 32979 MAPK activation +T843 PR:000006921 32983 32992 ephrin-B1 +T844 PR:000006921 33040 33049 ephrin-B1 +T845 PR:000008373 33054 33058 Cx43 +T846 GO:0032991 33066 33073 complex +T847 PR:000006921 33122 33131 ephrin-B1 +T848 PR:000008373 33136 33140 Cx43 +T849 GO:0006897 33193 33204 endocytosis +T850 PR:000025844 33241 33248 ephrins +T851 GO:0032991 33249 33258 complexes +T852 GO:0006897 33311 33322 endocytosis +T853 GO:0065007 33352 33362 regulation +T854 GO:0009986 33383 33395 cell surface +T855 GO:0005886 33431 33446 plasma membrane +T856 PR:000008373 33521 33525 Cx43 +T857 PR:000025844 33560 33566 ephrin +T858 GO:0032991 33567 33576 complexes +T859 GO:0006897 33612 33623 endocytosis +T860 GO:0065007 33640 33649 regulated +T861 GO:0048013 33723 33743 Eph/ephrin signaling +T862 PR:000025844 33727 33733 ephrin +T863 GO:0065007 33744 33753 regulates +T864 GO:0072583 33754 33783 clathrin-mediated endocytosis +T865 PR:000015882 33815 33829 Synaptojanin 1 +T866 PR:000008373 33864 33868 Cx43 +T867 GO:0065007 33905 33914 regulated +T868 PR:000006921 33950 33959 ephrin-B1 +T869 PR:000008373 34006 34010 Cx43 +T870 PR:000006921 34019 34028 ephrin-B1 +T871 PR:000008373 34081 34085 Cx43 +T872 PR:000006921 34127 34136 ephrin-B1 +T873 PR:000008373 34141 34145 Cx43 +T874 GO:0009986 34230 34242 cell surface +T875 GO:0005737 34253 34262 cytoplasm +T876 GO:0065007 34265 34275 Regulation +T877 PR:000008373 34329 34333 Cx43 +T878 GO:0005911 34350 34368;34388 34393 interfaces between ... cells +T879 PR:000006921 34369 34378 ephrin-B1 +T880 PR:000025844 34441 34447 ephrin +T881 PR:000025844 34484 34490 ephrin +T882 PR:000025844 34491 34497 ephrin +T883 GO:0010467 34557 34567 expression +T884 PR:000008373 34571 34575 Cx43 +T885 GO:0065007 34622 34632 regulation +T886 PR:000025844 34659 34665 ephrin +T887 GO:0065007 34805 34815 regulation +T888 GO:0005911 34834 34852 cell–cell contacts +T889 GO:0042571 34969 34979 antibodies +T890 NCBITaxon:8353 35050 35057 Xenopus +T891 GO:0010467 35068 35078 expression +T892 PR:000006921 35082 35091 ephrin-B1 +T893 CL:0000353 35152 35163 blastomeres +T894 GO:0010467 35194 35204 expressing +T895 PR:000008373 35205 35209 Cx43 +T896 GO:0065007 35290 35300 regulation +T897 PR:000025844 35354 35360 ephrin +T898 PR:000006921 35469 35478 ephrin-B1 +T899 PR:000006921 35573 35582 ephrin-B1 +T900 GO:0065007 35586 35596 regulating +T901 PR:000007124 35677 35682 EphA4 +T902 PR:000025844 35724 35731 ephrins +T903 http://purl.obolibrary.org/obo/MONDO_0010570 35769 35773 CFNS +T904 GO:0005576 35813 35826 extracellular +T905 SO:0000417 35827 35833 domain +T906 PR:000006921 35837 35846 ephrin-B1 +T907 PR:000025844 35885 35891 ephrin +T908 PR:000006921 36070 36079 ephrin-B1 +T909 PR:000006921 36083 36092 ephrin-B1 +T910 PR:000025844 36153 36159 ephrin +T911 UBERON:0000922 36163 36169 embryo +T912 NCBITaxon:10088 36207 36211 mice +T913 PR:000025844 36252 36258 ephrin +T914 GO:0010467 36259 36269 expressing +T915 PR:000006921 36374 36383 ephrin-B1 +T916 PR:000025844 36463 36469 ephrin +T917 GO:0006897 36516 36527 endocytosis +T918 PR:000008373 36531 36535 Cx43 +T919 UBERON:0001892 36736 36747 rhombomeric +T920 PR:000025844 36796 36802 ephrin +T921 GO:0010467 36832 36842 expression +T922 PR:000008373 36846 36850 Cx43 +T923 UBERON:0001017 36887 36909 central nervous system +T924 NCBITaxon:10088 36929 36933 mice +T925 GO:0065007 37022 37032 regulating +T926 GO:0015629 37037 37055 actin cytoskeleton +T927 PR:000025844 37075 37082 ephrins +T928 GO:0065007 37114 37124 regulating +T929 GO:0005921 37125 37139 gap junctional +T930 GO:0065007 37181 37191 regulation +T931 PR:000006921 37235 37244 ephrin-B1 +T932 NCBITaxon:1 37258 37269 individuals +T933 PR:000025844 37320 37327 ephrins +T934 UBERON:0000479 37372 37378 tissue +T935 UBERON:0000922 37397 37406 embryonic +T936 GO:0009790 37397 37418 embryonic development +T937 NCBITaxon:10088 37444 37448 Mice +T938 PR:000006921 37451 37460 Ephrin-B1 +T939 NCBITaxon:10088 37468 37472 mice +T940 NCBITaxon:10358 37477 37480 CMV +T941 PR:000008373 37481 37485 Cx43 +T942 NCBITaxon:10088 37497 37501 mice +T943 PR:000006922 37544 37553 ephrin-B2 +T944 SO:0001023 37557 37563 allele +T945 SO:0001037 37607 37615 cassette +T946 SO:0000852 37628 37635;37646 37650 site in ... exon +T947 PR:000006922 37654 37663 ephrin-B2 +T948 SO:0000318 37705 37716 start codon +T949 PR:000006922 37724 37733 ephrin-B2 +T950 SO:0000704 37734 37738 gene +T951 CL:0002322 37796 37804 ES cells +T952 NCBITaxon:10088 37834 37838 Mice +T953 SO:0000112 37947 37954 primers +T954 SO:0001030 37960 37961 F +T955 SO:0001031 37999 38000 R +T956 NCBITaxon:10088 38034 38038 Mice +T957 NCBITaxon:33208 38169 38175 Animal +T958 CL:0002322 38276 38284 ES cells +T959 PR:000006921 38298 38307 ephrin-B1 +T960 SO:0000112 38377 38383 primer +T961 SO:0001023 38407 38413 allele +T962 PR:000006921 38456 38465 ephrin-B1 +T963 CL:0002322 38471 38479 ES cells +T964 CL:0002322 38503 38511 ES cells +T965 SO:0001023 38537 38543 allele +T966 PR:000006921 38547 38556 ephrin-B1 +T967 PR:000006921 38558 38567 ephrin-B1 +T968 SO:0000346 38567 38570 lox +T969 GO:0010467 38606 38616 expression +T970 SO:0000440 38617 38623 vector +T971 CL:0002322 38625 38632 ES cell +T972 PR:000006921 38703 38712 ephrin-B1 +T973 CL:0002322 38729 38737 ES cells +T974 UBERON:0000358 38757 38768 blastocysts +T975 CHEBI:75055 38898 38903 X-gal +T976 UBERON:0004288 38917 38925 skeletal +T977 CHEBI:75055 38961 38966 X-gal +T978 UBERON:0004288 38980 38988 skeletal +T979 NCBITaxon:10088 39071 39075 mice +T980 PR:000008373 39093 39097 Cx43 +T981 SO:0000902 39098 39107 transgene +T982 UBERON:0005744 39131 39138 foramen +T983 UBERON:0000209 39152 39165 frontal bones +T984 UBERON:0005744 39293 39300 foramen +T985 SO:0000902 39384 39393 transgene +T986 SO:0000902 39539 39548 transgene +T987 GO:0097617 39656 39669 hybridization +T988 GO:0097617 39688 39701 hybridization +T989 UBERON:0000922 39758 39765 embryos +T990 GO:0097617 39816 39829 hybridization +T991 PR:000006921 39919 39928 ephrin-B1 +T992 PR:000007130 39930 39935 EphB2 +T993 PR:000007131 39941 39946 EphB3 +T994 UBERON:0000209 40007 40019 frontal bone +T995 UBERON:0003104 40020 40030 mesenchyme +T996 UBERON:0000922 40056 40063 embryos +T997 PR:000027795 40086 40093 trypsin +T998 PR:000027795 40133 40140 Trypsin +T999 CHEBI:7586 40580 40583 NBT +T1000 CHEBI:75508 40584 40588 BCIP +T1001 UBERON:0002539 40931 40947 branchial arches +T1002 UBERON:0000922 40956 40963 embryos +T1003 GO:0009294 41092 41103 transfected +T1004 GO:0010467 41109 41119 expression +T1005 SO:0000440 41120 41127 vectors +T1006 PR:000006921 41132 41141 ephrin-B1 +T1007 PR:000007130 41143 41158 Eph-B2 receptor +T1008 SO:0000440 41182 41188 vector +T1009 GO:0010467 41256 41266 expressing +T1010 PR:000006921 41267 41276 ephrin-B1 +T1011 GO:0009294 41529 41540 transfected +T1012 GO:0010467 41563 41573 expressing +T1013 PR:000006921 41574 41583 ephrin-B1 +T1014 PR:000007130 41605 41611 Eph-B2 +T1015 GO:0005921 41644 41657 gap junctions +T1016 CHEBI:37958 41838 41841 dye +T1017 CHEBI:37958 41897 41900 dye +T1018 CHEBI:37958 41944 41947 dye +T1019 GO:0007601 41992 41998 visual +T1020 CHEBI:63016 42191 42195 NP40 +T1021 GO:0019835 42196 42201 lysis +T1022 CHEBI:46756 42216 42221 Hepes +T1023 CHEBI:26710 42239 42243 NaCl +T1024 CHEBI:17754 42249 42257 glycerol +T1025 CHEBI:6636 42266 42271 MgCl2 +T1026 CHEBI:28741 42290 42293 NaF +T1027 CHEBI:9754 42353 42357 Tris +T1028 CHEBI:26710 42375 42379 NaCl +T1029 PR:000007130 42440 42445 EphB2 +T1030 PR:000008373 42457 42461 Cx43 +T1031 GO:0042571 42473 42481 antibody +T1032 PR:000029158 42556 42564 ProteinA +T1033 GO:0032991 42585 42594 complexes +T1034 CHEBI:8984 42612 42615 SDS +T1035 GO:0042571 42641 42651 antibodies +T1036 PR:000006921 42653 42662 ephrin-B1 +T1037 PR:000008373 42742 42746 Cx43 +T1038 PR:000008373 42826 42830 Cx43 +T1039 PR:000006921 42834 42843 ephrin-B1 +T1040 GO:0019835 42864 42869 lysis +T1041 GO:0009294 43007 43018 transfected +T1042 GO:0010467 43027 43037 expression +T1043 SO:0000440 43038 43044 vector +T1044 PR:000006921 43049 43058 ephrin-B1 +T1045 GO:0009294 43078 43090 transfection +T1046 PR:000007130 43152 43157 EphB2 +T1047 CHEBI:9750 43239 43245 Tx-100 +T1048 CHEBI:60004 43293 43296 mix +T1049 PR:000007130 43300 43305 EphB2 +T1050 PR:000008373 43330 43334 Cx43 +T1051 GO:0042571 43346 43354 antibody +T1052 PR:000001443 43358 43368 N-cadherin +T1053 GO:0042571 43380 43388 antibody +T1054 CL:0000001 43438 43451 primary cells +T1055 PR:000006921 43453 43462 ephrin-B1 +T1056 NCBITaxon:10114 43492 43495 rat +T1057 GO:0042571 43507 43515 antibody +T1058 CHEBI:37926 43522 43526 FITC +T1059 CHEBI:37987 43532 43535 Cy3 +T1060 MOP:0000779 43536 43546 conjugated +T1061 GO:0042571 43557 43567 antibodies +T1062 GO:0009294 43704 43715 transfected +T1063 PR:000008373 43739 43743 Cx43 +T1064 GO:0010467 43744 43754 expression +T1065 GO:0009294 43790 43802 transfection +T1066 GO:0009294 43939 43951 transfection +T1067 PR:000008373 43960 43964 Cx43 +T1068 GO:0042571 43965 43973 antibody +T1069 CHEBI:59132 44096 44104 antigens +T1070 UBERON:0000479 44106 44112 Tissue +T1071 CHEBI:75958 44147 44155 solution +T1072 NCBITaxon:9796 44164 44169 horse +T1073 UBERON:0001977 44170 44175 serum +T1074 PR:000008373 44236 44240 Cx43 +T1075 GO:0042571 44241 44249 antibody +T1076 CHEBI:75958 44269 44277 solution +T1077 CHEBI:37987 44308 44311 Cy3 +T1078 MOP:0000779 44312 44322 conjugated +T1079 GO:0042571 44333 44341 antibody +T1080 CHEBI:75958 44393 44401 solution +T1081 PR:000008373 44418 44422 Cx43 +T1082 PR:000008373 44587 44591 Cx43 +T1083 UBERON:0006378 44897 44906 Vibrissae +T1084 PR:000006921 44926 44935 ephrin-B1 +T1085 UBERON:0000922 44939 44946 Embryos +T1086 UBERON:0000922 44964 44971 embryos +T1087 CHEBI:51686 44990 44991 H +T1088 UBERON:0006378 45007 45015 whiskers +T1089 PR:000006921 45024 45033 ephrin-B1 +T1090 PR:000006921 45062 45071 ephrin-B1 +T1091 CHEBI:75055 45101 45106 X-gal +T1092 PR:000006921 45125 45134 ephrin-B1 +T1093 SO:0000346 45136 45139 lox +T1094 UBERON:0000922 45140 45146 embryo +T1095 PR:000017435 45169 45173 Wnt1 +T1096 SO:0001023 45178 45185 alleles +T1097 UBERON:0006378 45209 45218 vibrissae +T1098 PR:000006921 45343 45352 ephrin-B1 +T1099 UBERON:0006378 45413 45422 vibrissae +T1100 GO:0097617 45452 45465 hybridization +T1101 UBERON:0000922 45505 45511 embryo +T1102 PR:000006921 45529 45538 ephrin-B1 +T1103 PR:000007130 45547 45552 EphB2 +T1104 GO:0010467 45565 45575 expression +T1105 SO:0000704 45585 45590 genes +T1106 UBERON:0003104 45598 45608 mesenchyme +T1107 UBERON:0006378 45616 45625 vibrissae +T1108 UBERON:0004339 45709 45718 Calvarial +T1109 GO:0001503 45741 45751 Osteogenic +T1110 UBERON:0004288 45773 45781 Skeleton +T1111 UBERON:0000033 45804 45809 heads +T1112 UBERON:0000922 45832 45839 embryos +T1113 PR:000006922 45841 45850 ephrin-B2 +T1114 PR:000006921 45889 45898 ephrin-B1 +T1115 PR:000006921 45939 45948 ephrin-B1 +T1116 PR:000006922 45952 45961 ephrin-B2 +T1117 UBERON:4200215 46032 46038 suture +T1118 UBERON:0003104 46039 46049 mesenchyme +T1119 PR:000006921 46053 46062 ephrin-B1 +T1120 PR:000006922 46063 46072 ephrin-B2 +T1121 UBERON:0005744 46126 46133 foramen +T1122 UBERON:0000209 46146 46159 frontal bones +T1123 PR:000006921 46178 46187 ephrin-B1 +T1124 UBERON:0000922 46191 46198 embryos +T1125 UBERON:0001474 46218 46223 bones +T1126 UBERON:0000922 46311 46318 embryos +T1127 PR:000006921 46340 46349 ephrin-B1 +T1128 PR:000006921 46372 46381 ephrin-B1 +T1129 PR:000006922 46385 46394 ephrin-B2 +T1130 UBERON:0000922 46468 46474 embryo +T1131 PR:000006921 46623 46632 ephrin-B1 +T1132 PR:000006922 46636 46645 ephrin-B2 +T1133 UBERON:0000209 46730 46742 frontal bone +T1134 PR:000006921 46841 46850 Ephrin-B1 +T1135 PR:000008373 46855 46859 Cx43 +T1136 UBERON:0000209 46913 46926 Frontal Bones +T1137 PR:000006921 46930 46939 ephrin-B1 +T1138 UBERON:0000922 46943 46950 Embryos +T1139 PR:000006921 46956 46965 Ephrin-B1 +T1140 CL:0000001 47016 47023;47036 47041 primary ... cells +T1141 UBERON:0003104 47024 47035 mesenchymal +T1142 CL:0000134 47024 47041 mesenchymal cells +T1143 GO:0010467 47106 47116 Expression +T1144 PR:000006921 47120 47129 ephrin-B1 +T1145 CL:0000001 47183 47196 primary cells +T1146 PR:000008373 47298 47302 Cx43 +T1147 UBERON:0003104 47333 47344 mesenchymal +T1148 CL:0000134 47333 47350 mesenchymal cells +T1149 PR:000008373 47428 47432 Cx43 +T1150 UBERON:0000922 47496 47503 embryos +T1151 PR:000006921 47505 47514 ephrin-B1 +T1152 PR:000006921 47535 47544 ephrin-B1 +T1153 UBERON:0000922 47548 47555 embryos +T1154 PR:000008373 47640 47644 Cx43 +T1155 UBERON:0000209 47703 47715 frontal bone +T1156 PR:000006921 47719 47728 ephrin-B1 +T1157 UBERON:0000922 47732 47738 embryo +T1158 UBERON:0000922 47777 47783 embryo +T1159 PR:000008373 47911 47915 Cx43 +T1160 UBERON:0000479 47924 47930 tissue +T1161 PR:000006921 47996 48005 ephrin-B1 +T1162 UBERON:0000922 48019 48026 Embryos +T1163 UBERON:0004339 48042 48051 Calvarial +T1164 UBERON:0004288 48065 48073 Skeletal +T1165 UBERON:0000033 48096 48101 heads +T1166 PR:000006921 48113 48122 ephrin-B1 +T1167 UBERON:0000922 48136 48143 embryos +T1168 CL:0002322 48196 48204 ES cells +T1169 UBERON:0002415 48225 48229 tail +T1170 UBERON:0004288 48244 48252 Skeletal +T1171 PR:000006921 48290 48299 ephrin-B1 +T1172 PR:000006921 48312 48321 ephrin-B1 +T1173 UBERON:0000922 48366 48373 embryos +T1174 UBERON:0000975 48382 48389 sternum +T1175 PR:000006921 48413 48422 ephrin-B1 +T1176 UBERON:0000922 48428 48435 embryos +T1177 NCBITaxon:10358 48544 48547 CMV +T1178 PR:000008373 48548 48552 Cx43 +T1179 NCBITaxon:10088 48553 48557 mice +T1180 PR:000007130 48593 48599 Eph-B2 +T1181 GO:0010467 48600 48610 expression +T1182 PR:000007130 48647 48652 EphB2 +T1183 PR:000008373 48681 48685 Cx43 +T1184 GO:0042571 48686 48694 antibody +T1185 PR:000008373 48703 48707 Cx43 +T1186 GO:0010467 48708 48718 expression +T1187 NCBITaxon:10114 48760 48763 rat +T1188 GO:0042571 48775 48783 antibody +T1189 http://purl.obolibrary.org/obo/MONDO_0010570 48986 48990 CFNS +T1190 http://purl.obolibrary.org/obo/MONDO_0010570 48993 49019 craniofrontonasal syndrome +T1191 PR:000008373 49021 49025 Cx43 +T1192 PR:000008373 49028 49038 connexin43 +T1193 UBERON:0000922 49045 49054 embryonic +T1194 GO:0005921 49100 49112 gap junction +T1195 UBERON:0002342 49150 49162 neural crest +T1196 PR:000006921 49199 49208 ephrin-B1 +T1197 UBERON:0000922 49212 49219 Embryos +T1198 UBERON:0004288 49260 49268 Skeleton +T1199 UBERON:0000033 49291 49296 heads +T1200 UBERON:0000922 49315 49322 embryos +T1201 UBERON:0001474 49330 49335 Bones +T1202 CHEBI:16866 49353 49365 Alizarin Red +T1203 UBERON:0005744 49423 49430 foramen +T1204 UBERON:0000209 49440 49453 frontal bones +T1205 PR:000006921 49469 49478 ephrin-B1 +T1206 PR:000006921 49518 49527 ephrin-B1 +T1207 UBERON:0000922 49562 49569 embryos +T1208 PR:000006921 49579 49588 ephrin-B1 +T1209 UBERON:0000209 49648 49661 frontal bones +T1210 UBERON:0000209 49705 49718 frontal bones +T1211 UBERON:0000209 49746 49759 frontal bones +T1212 UBERON:0004288 49772 49780 Skeleton +T1213 UBERON:0000033 49803 49808 heads +T1214 UBERON:0000922 49820 49827 embryos +T1215 UBERON:0000922 49843 49850 embryos +T1216 PR:000006921 49867 49876 ephrin-B1 +T1217 PR:000006922 49880 49889 ephrin-B2 +T1218 UBERON:0000922 49895 49902 embryos +T1219 UBERON:0002489 49925 49939 coronal suture +T1220 PR:000006921 49974 49983 ephrin-B1 +T1221 UBERON:0002489 50067 50082 coronal sutures +T1222 PR:000006921 50086 50095 ephrin-B1 +T1223 PR:000006922 50099 50108 ephrin-B2 +T1224 UBERON:0000922 50114 50121 embryos +T1225 UBERON:4200215 50153 50159 suture +T1226 UBERON:0003104 50160 50170 mesenchyme +T1227 UBERON:0000210 50197 50205;50218 50222 parietal ... bone +T1228 UBERON:0000209 50210 50222 frontal bone +T1229 UBERON:0002489 50242 50256 coronal suture +T1230 PR:000006921 50268 50277 ephrin-B1 +T1231 PR:000006922 50281 50290 ephrin-B2 +T1232 UBERON:0000922 50296 50303 embryos +T1233 UBERON:0002489 50367 50381 Coronal suture +T1234 UBERON:0000209 50386 50399 frontal bones +T1235 PR:000006922 50421 50430 ephrin-B2 +T1236 PR:000006921 50468 50477 ephrin-B1 +T1237 PR:000006922 50481 50490 ephrin-B2 +T1238 UBERON:0000209 50508 50510 fb +T1239 UBERON:0000209 50512 50524 frontal bone +T1240 UBERON:0000210 50526 50528 pb +T1241 UBERON:0000210 50530 50543 parietal bone +T1242 GO:0010467 50556 50566 Expression +T1243 PR:000006921 50570 50579 ephrin-B1 +T1244 PR:000007130 50584 50589 EphB2 +T1245 PR:000006921 50605 50614 ephrin-B1 +T1246 UBERON:0000922 50618 50625 Embryos +T1247 GO:0097617 50639 50652 hybridization +T1248 UBERON:0000209 50656 50663 frontal +T1249 UBERON:0000922 50692 50698 embryo +T1250 PR:000006921 50716 50725 ephrin-B1 +T1251 PR:000007130 50731 50736 EphB2 +T1252 PR:000007131 50745 50750 EphB3 +T1253 PR:000006921 50756 50765 Ephrin-B1 +T1254 GO:0010467 50769 50778 expressed +T1255 UBERON:0000209 50805 50817 frontal bone +T1256 UBERON:0002360 50861 50876 meningeal layer +T1257 GO:0010467 50891 50901 Expression +T1258 PR:000007130 50905 50910 EphB2 +T1259 PR:000007131 50956 50961 EphB3 +T1260 GO:0010467 50965 50974 expressed +T1261 UBERON:0000209 51001 51013 frontal bone +T1262 GO:0097617 51028 51041 hybridization +T1263 PR:000006921 51076 51085 ephrin-B1 +T1264 UBERON:0000922 51089 51096 embryos +T1265 PR:000006921 51133 51142 ephrin-B1 +T1266 PR:000006922 51156 51165 ephrin-B2 +T1267 PR:000007130 51182 51187 EphB2 +T1268 PR:000006921 51206 51215 ephrin-B1 +T1269 PR:000007130 51220 51225 EphB2 +T1270 UBERON:0001893 51246 51259 telencephalon +T1271 UBERON:0003104 51281 51291 mesenchyme +T1272 PR:000006921 51295 51304 ephrin-B1 +T1273 UBERON:0000922 51308 51315 embryos +T1274 GO:0010467 51335 51345 expression +T1275 PR:000006922 51349 51358 ephrin-B2 +T1276 UBERON:0004339 51385 51394 Calvarial +T1277 UBERON:0005744 51395 51402 Foramen +T1278 GO:0001503 51428 51438 Osteogenic +T1279 UBERON:0000922 51466 51473 embryos +T1280 PR:000017435 51496 51500 Wnt1 +T1281 SO:0001023 51505 51512 alleles +T1282 CHEBI:75055 51532 51537 X-gal +T1283 GO:0001503 51583 51593 osteogenic +T1284 PR:000006921 51639 51648 ephrin-B1 +T1285 PR:000006921 51720 51729 ephrin-B1 +T1286 PR:000006921 51742 51751 ephrin-B1 +T1287 UBERON:0000922 51759 51766 embryos +T1288 GO:0001503 51810 51820 osteogenic +T1289 UBERON:0000922 51874 51881 embryos +T1290 CL:0000001 52040 52047;52060 52065 Primary ... cells +T1291 UBERON:0003104 52048 52059 mesenchymal +T1292 CL:0000134 52048 52065 mesenchymal cells +T1293 UBERON:0004339 52097 52105 calvaria +T1294 PR:000006921 52115 52124 ephrin-B1 +T1295 PR:000006921 52151 52160 ephrin-B1 +T1296 GO:0001503 52328 52338 Osteogenic +T1297 PR:000008373 52396 52400 Cx43 +T1298 CL:0000001 52406 52413;52426 52431 Primary ... cells +T1299 UBERON:0003104 52414 52425 mesenchymal +T1300 CL:0000134 52414 52431 mesenchymal cells +T1301 UBERON:0004339 52458 52466 calvaria +T1302 PR:000006921 52495 52504 ephrin-B1 +T1303 UBERON:0000922 52508 52515 embryos +T1304 PR:000008373 52600 52604 Cx43 +T1305 PR:000008373 52648 52652 Cx43 +T1306 UBERON:0000922 52725 52732 embryos +T1307 PR:000006921 52764 52773 ephrin-B1 +T1308 UBERON:0000922 52777 52784 embryos +T1309 PR:000008373 52804 52808 Cx43 +T1310 UBERON:0004347 52834 52842 limb bud +T1311 CHEBI:75055 52858 52863 X-gal +T1312 UBERON:0000922 52881 52887 embryo +T1313 PR:000006921 52910 52919 ephrin-B1 +T1314 UBERON:0000358 52950 52960 blastocyst +T1315 GO:0005921 52962 52976 Gap junctional +T1316 PR:000008373 52977 52981 Cx43 +T1317 PR:000006921 53071 53080 ephrin-B1 +T1318 PR:000006921 53242 53251 ephrin-B1 +T1319 PR:000008373 53258 53262 Cx43 +T1320 PR:000008373 53317 53321 Cx43 +T1321 PR:000008373 53395 53399 Cx43 +T1322 PR:000008373 53460 53464 Cx43 +T1323 PR:000025844 53531 53537 ephrin +T1324 GO:0005921 53559 53571 Gap Junction +T1325 GO:0010467 53612 53622 expressing +T1326 PR:000006921 53623 53632 ephrin-B1 +T1327 GO:0010467 53717 53727 expressing +T1328 GO:0010467 53735 53745 expressing +T1329 PR:000007130 53777 53792 Eph-B2 receptor +T1330 CHEBI:37958 53806 53809 dye +T1331 CHEBI:37958 53888 53891 dye +T1332 PR:000007130 53943 53949 Eph-B2 +T1333 GO:0010467 53955 53964 expressed +T1334 GO:0009294 54053 54064 transfected +T1335 SO:0000155 54087 54094 plasmid +T1336 GO:0010467 54115 54125 expression +T1337 PR:000006921 54140 54149 ephrin-B1 +T1338 PR:000007130 54157 54163 Eph-B2 +T1339 CHEBI:37958 54233 54236 dye +T1340 CHEBI:37958 54319 54322 dye +T1341 PR:000025844 54418 54424 ephrin +T1342 CHEBI:37958 54483 54486 dye +T1343 CHEBI:37958 54589 54592 dye +T1344 CHEBI:37958 54628 54631 dye +T1345 CHEBI:37958 54724 54727 dye +T1346 PR:000006921 54884 54893 Ephrin-B1 +T1347 PR:000008373 54898 54902 Cx43 +T1348 UBERON:0003104 54957 54968 mesenchymal +T1349 PR:000008373 54989 54993 Cx43 +T1350 PR:000006921 55016 55025 ephrin-B1 +T1351 GO:0009294 55083 55094 transfected +T1352 PR:000006921 55100 55109 ephrin-B1 +T1353 PR:000008373 55169 55173 Cx43 +T1354 PR:000006921 55190 55199 ephrin-B1 +T1355 PR:000006921 55255 55264 ephrin-B1 +T1356 PR:000008373 55292 55296 Cx43 +T1357 GO:0005911 55312 55330;55349 55354 interfaces between ... cells +T1358 PR:000025844 55331 55337 ephrin +T1359 GO:0010467 55338 55348 expressing +T1360 PR:000006921 55377 55386 ephrin-B1 +T1361 PR:000007130 55406 55411 EphB2 +T1362 PR:000006921 55440 55449 ephrin-B1 +T1363 PR:000008373 55458 55462 Cx43 +T1364 PR:000006921 55491 55500 ephrin-B1 +T1365 PR:000006921 55563 55572 Ephrin-B1 +T1366 PR:000007130 55624 55629 EphB2 +T1367 PR:000008373 55650 55654 Cx43 +T1368 GO:0032991 55671 55678 complex +T1369 GO:0010467 55748 55758 expressing +T1370 PR:000006921 55759 55768 ephrin-B1 +T1371 PR:000006921 55789 55797 ephrinB1 +T1372 PR:000008373 55822 55826 Cx43 +T1373 GO:0032991 55843 55852 complexes +T1374 PR:000008373 55886 55890 Cx43 +T1375 GO:0010467 55933 55943 expressing +T1376 PR:000006921 55944 55953 ephrin-B1 +T1377 PR:000006921 55974 55983 ephrin-B1 +T1378 GO:0065007 56112 56122 Regulation +T1379 GO:0009294 56195 56206 transfected +T1380 PR:000006921 56229 56238 ephrin-B1 +T1381 PR:000006921 56245 56254 ephrin-B1 +T1382 SO:0000155 56280 56287 plasmid +T1383 PR:000007130 56329 56334 EphB2 +T1384 PR:000008373 56359 56363 Cx43 +T1385 GO:0032991 56380 56387 complex +T1386 PR:000029158 56418 56423 ProtA +T1387 PR:000029158 56467 56476 Protein A +T1388 PR:000007130 56491 56496 EphB2 +T1389 PR:000008373 56611 56615 Cx43 +T1390 PR:000008373 56663 56667 Cx43 +T1391 GO:0009294 56729 56740 transfected +T1392 PR:000006921 56746 56755 ephrin-B1 +T1393 PR:000007130 56777 56782 EphB2 +T1394 PR:000008373 56815 56819 Cx43 +T1395 PR:000006921 56828 56837 ephrin-B1 +T1396 UBERON:0004347 56972 56981 limb buds +T1397 UBERON:0000922 57002 57009 embryos +T1398 PR:000006921 57044 57053 ephrin-B1 +T1399 CL:0002322 57059 57067 ES cells +T1400 PR:000006921 57081 57090 ephrin-B1 +T1401 CL:0002322 57095 57103 ES cells +T1402 UBERON:0000922 57125 57132 embryos +T1403 CHEBI:75055 57138 57143 X-gal +T1404 GO:0005634 57226 57233 Nuclear +T1405 GO:0010467 57313 57323 expressing +T1406 PR:000006921 57324 57333 ephrin-B1 +T1407 GO:0010467 57395 57405 expressing +T1408 PR:000008373 57406 57410 Cx43 +T1409 GO:0010467 57502 57512 expressing +T1410 GO:0010467 57543 57553 expressing +T1411 UBERON:0004339 57662 57671 Calvarial +T1412 PR:000006921 57682 57691 ephrin-B1 +T1413 NCBITaxon:10088 57695 57699 Mice +T1414 GO:0010467 57704 57714 expressing +T1415 PR:000008373 57715 57719 Cx43 +T1416 UBERON:0004288 57721 57729 Skeleton +T1417 PR:000006921 57751 57760 ephrin-B1 +T1418 PR:000006921 57777 57786 ephrin-B1 +T1419 PR:000006921 57808 57817 ephrin-B1 +T1420 NCBITaxon:10358 57841 57844 CMV +T1421 PR:000008373 57845 57849 Cx43 +T1422 SO:0000902 57850 57859 transgene +T1423 UBERON:0005744 57891 57898 foramen +T1424 UBERON:0000209 57916 57929 frontal bones +T1425 PR:000006921 57936 57945 ephrin-B1 +T1426 PR:000006921 57957 57959 B1 +T1427 PR:000006921 57971 57980 ephrin-B1 +T1428 NCBITaxon:10358 58004 58007 CMV +T1429 PR:000008373 58008 58012 Cx43 +T1430 SO:0000902 58013 58022 transgene +T1431 NCBITaxon:10358 58024 58027 CMV +T1432 PR:000008373 58028 58032 Cx43 +T1433 PR:000006921 58033 58035 B1 +T1434 SO:0000902 58090 58099 transgene +T1435 UBERON:0000922 58163 58170 embryos +T1436 PR:000006921 58299 58308 ephrin-B1 +T1437 GO:0005921 58389 58402 gap junctions +T1438 GO:0005912 58407 58425 adherens junctions +T1439 PR:000006921 58427 58436 Ephrin-B1 +T1440 PR:000008373 58455 58459 Cx43 +T1441 GO:0065007 58486 58496 regulation +T1442 GO:0005921 58500 58513 gap junctions +T1443 GO:0005912 58518 58536 adherens junctions +T1444 UBERON:0000922 58620 58626 embryo +T1445 UBERON:0004339 58682 58691 calvarial +T1446 UBERON:0001474 58692 58697 bones +T1447 GO:0010467 58709 58716 express +T1448 PR:000006921 58717 58726 ephrin-B1 +T1449 CHEBI:33280 58770 58780 messengers +T1450 PR:000025844 58838 58844 ephrin +T1451 PR:000006921 58859 58868 ephrin-B1 +T1452 UBERON:0000922 58872 58879 embryos +T1453 PR:000025844 58885 58891 ephrin +T1454 GO:0006897 58949 58960 endocytosis +T1455 PR:000008373 58964 58968 Cx43 +T1456 NCBITaxon:9606 59541 59546 Human +T1457 UBERON:0001091 59718 59724 Dental diff --git a/src/ontogpt/evaluation/craft/database/all/16968134.txt b/src/ontogpt/evaluation/craft/database/all/16968134.txt new file mode 100644 index 000000000..977fe896e --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/16968134.txt @@ -0,0 +1,293 @@ +Inhibition of Gap Junction Communication at Ectopic Eph/ephrin Boundaries Underlies Craniofrontonasal Syndrome + +Abstract + +Mutations in X-linked ephrin-B1 in humans cause craniofrontonasal syndrome (CFNS), a disease that affects female patients more severely than males. Sorting of ephrin-B1–positive and –negative cells following X-inactivation has been observed in ephrin-B1+/− mice; however, the mechanisms by which mosaic ephrin-B1 expression leads to cell sorting and phenotypic defects remain unknown. Here we show that ephrin-B1+/− mice exhibit calvarial defects, a phenotype autonomous to neural crest cells that correlates with cell sorting. We have traced the causes of calvarial defects to impaired differentiation of osteogenic precursors. We show that gap junction communication (GJC) is inhibited at ectopic ephrin boundaries and that ephrin-B1 interacts with connexin43 and regulates its distribution. Moreover, we provide genetic evidence that GJC is implicated in the calvarial defects observed in ephrin-B1+/− embryos. Our results uncover a novel role for Eph/ephrins in regulating GJC in vivo and suggest that the pleiotropic defects seen in CFNS patients are due to improper regulation of GJC in affected tissues. + +Introduction + +Physical segregation, or sorting, of different cell populations during development is essential for the proper spatial organization of the animal body. Eph receptor tyrosine kinases and ephrins regulate many developmental processes [1–3], and play an important role in tissue patterning by restricting cell intermingling and establishing developmental boundaries [4–6]. + +A dramatic example of the role of Eph and ephrins in cell sorting, as well as the importance of proper cell sorting during development, was recently provided by the analysis of the phenotypes exhibited by ephrin-B1 heterozygous female mice [7,8] and by the identification of mutations in the ephrin-B1 gene in human craniofrontonasal syndrome (CFNS) patients [9,10]. As a result of random X-inactivation, X-linked ephrin-B1 expression is mosaic in ephrin-B1+/− mice and ephrin-B1–positive and ephrin-B1–negative cells segregate from one another. This correlates with a polydactyly phenotype that is never observed in ephrin-B1 null animals [7,8]. Similarly, CFNS is an X-linked developmental disorder characterized by a number of craniofacial defects including abnormal development of the cranial and nasal bones, and craniosynostosis (premature fusion of the coronal sutures), as well as extracranial anomalies (including polydactyly and syndactyly), affecting mainly female patients [11]. + +Despite years of intensive studies, the molecular mechanisms by which Eph receptors and ephrins influence cell sorting are still poorly understood. Eph receptors and ephrins function as an unusual receptor/ligand pair in which both receptor and ligand are capable of activating a signaling cascade. One prominent outcome of Eph/ephrin interactions is the regulation of cell–substrate adhesion and reorganization of the actin cytoskeleton. It has also been reported that Eph receptors and ephrins regulate gap junction communication (GJC) [6]. Indeed, studies in zebrafish have shown that expression of Eph receptors and ephrins in animal cap cells was sufficient to block GJC at the boundary between both cell populations. Gap junctions are intercellular membrane channels that mediate cell coupling by allowing the passage of small molecules directly from cell to cell. During vertebrate development, regions of GJC coincide with developmental compartments [12,13]. For instance, GJC is reduced at inter-rhombomeric boundaries, as compared to GJC in the rhombomeres themselves. GJC is involved in various developmental processes and mutations in connexins, the structural proteins forming gap junctional pores, have been linked to a number of human diseases [14]. Notably, GJC plays an important role in skeletal development [15] and mutations in connexin43 (Cx43) lead to cranial and skeletal defects both in humans and mice [16,17]. + +In this report, we have investigated the underlying cause of the phenotypes observed in ephrin-B1 heterozygous mice. We show that cell sorting in ephrin-B1+/− females induces calvarial defects due to the impaired differentiation of neural crest cells (NCCs). We provide evidence that GJC is inhibited at ectopic ephrin boundaries and that ephrin-B1 physically interacts with Cx43 and influences its distribution. In addition, we report that overexpression of Cx43 partially rescues the calvarial defects observed in ephrin-B1 heterozygotes. Finally, we show that regulation of GJC correlates with cell sorting in response to Eph/ephrin interaction. From these results we conclude that mosaic loss of ephrin-B1 exerts a dominant effect during development involving perturbation of GJC at ectopic Eph/ephrin boundaries and leading to defective tissue differentiation. These observations extend our understanding of the mechanisms underlying CFNS in humans and of the role of Eph receptors and ephrins in vivo. + +Results + +Craniofacial Phenotypes in ephrin-B1 Heterozygous Females + +We and others have shown previously that ephrin-B1+/− females exhibit a polydactyly phenotype that is never seen in ephrin-B1Y/− males or ephrin-B1−/− females [7,8]. Because CFNS human female patients exhibit numerous craniofacial defects, we undertook a closer analysis of ephrin-B1 heterozygous female mice that revealed several defects in NCC-derived tissues that were not seen in hemizygous males or homozygous females. At P1, ephrin-B1+/− heterozygous females (n = 17) presented an opening (foramen) between the frontal bones and jagged bone fronts (Figure 1Ab), indicative of abnormal development of the frontal bones. (The parietal bone was affected at lower penetrance). Ephrin-B1−/− homozygous null females (n = 3) and ephrin-B1Y/− hemizygous males (n = 13) exhibited a normal development of these bones (Figure 1Aa and unpublished data). The frontal bone phenotype was recapitulated in heterozygous females carrying a deletion of ephrin-B1 specifically in NCCs (n = 3) (Figure 1Ac), consistent with the NCC origin of frontal bones [18]. Ephrin-B1+/− females also exhibited abnormal alignment of the vibrissae buds which was recapitulated by conditional deletion of ephrin-B1 in NCCs (ephrin-B1+/lox;Wnt1Cre+), indicating that the defect is autonomous to this lineage (Figure S1). + +It has been proposed by others that the mild manifestations of CFNS in male carriers might be due to compensatory mechanisms by other ephrins [10]. To test this hypothesis, we asked whether further alteration in the dosage of ephrin signaling would lead to calvarial phenotypes in ephrin-B1 null mutants. We chose to focus on ephrin-B2 since loss of this gene has been shown previously to affect migration of a sub-population of cranial NCCs [19] and ephrin-B2 is expressed in the craniofacial area (Figure 2) We generated a mouse line harboring a null mutation in the ephrin-B2 gene by placing a cDNA coding for a fusion protein between histone 2B (H2B) and the green fluorescent protein (GFP) under the control of the ephrin-B2 promoter (A. Davy, P. Soriano, unpublished data). ephrin-B2 and ephrin-B1 heterozygous animals were mated to generate ephrin-B1/ephrin-B2 double heterozygous animals. Skeleton preparations showed that removal of one copy of ephrin-B2 in an ephrin-B1+/− background worsened the calvarial phenotype (Figure 1B). Indeed, in addition to a larger gap between the frontal bones (Figure S2A), ephrin-B1+/−/ephrin-B2+/GFP embryos (n = 5) exhibited a coronal suture defect that was not observed in ephrin-B1 single mutants, males or females. Bone fronts at coronal sutures in ephrin-B1+/−/ephrin-B2+/GFP embryos did not overlap, and ectopic bone was frequently observed in the suture mesenchyme (Figure 1Bc). Examination of skeleton preparations from E15.5 embryos indicated that although bone formation in ephrin-B1+/−/ephrin-B2+/GFP embryos proceeded comparably to ephrin-B1+/− embryos, bone fronts seemed unable to extend toward each other at the coronal suture (Figure 1Bf). Importantly, ephrin-B1Y/−/ephrin-B2+/GFP double hemi/heterozygous males (n = 3) exhibited normal calvarial development (Figure 1Bd and unpublished data). Altogether, these results show that mosaic loss of ephrin-B1 exerts a dominant effect on the development of calvarial bones. Removal of one copy of ephrin-B2 in an ephrin-B1+/− background was sufficient to uncover additional defects at the coronal suture; however, it did not lead to calvarial phenotypes in ephrin-B1 null embryos, suggesting that the lack of calvarial phenotype in these embryos is not due to compensation by ephrin-B2. + +Defects in NCC-Derived Structures Correlate with Cell-Sorting in ephrin-B1+/− Embryos + +To better understand the basis for the calvarial phenotypes observed in ephrin-B1 heterozygotes, we analyzed the expression pattern of ephrin-B1 and ephrin-B2, and their cognate receptors EphB2 and EphB3 at various stages of development. These receptors have been implicated both in the polydactyly phenotype and in the formation of the palate [20], a NCC-derived structure that also requires ephrin-B1. At E14.5, expression of ephrin-B1, EphB2, and EphB3 can be detected in the developing frontal bones (Figure 2A). Ephrin-B1 and EphB3 are expressed throughout the bone whereas EphB2 expression appears to be restricted ventrally. Interestingly, ephrin-B1 is also strongly expressed in the meningeal layer which derives from neural crest cells [18]. At E12.5, a stage that corresponds to the early stages of calvarial bone differentiation, ephrin-B1 is expressed throughout the head mesenchyme as well as in the vibrissae buds in wild-type embryos (Figure 2Ba and Figure S1). In ephrin-B1+/− embryos, expression of ephrin-B1 is patchy throughout the craniofacial mesenchyme and in the telencephalon, and highlights the defective formation of vibrissae buds (Figure 2Bb). Both ephrin-B2 and EphB2 exhibit expression patterns that are similar to ephrin-B1 (Figure 2Bc and 2Be). Expression of ephrin-B2, however, is unchanged in ephrin-B1+/− (Figure 2Bd) whereas EphB2 expression appears patchy (Figure 2Bf). It has been reported previously that patchy expression of ephrin-B1 in ephrin-B1+/− limb buds reflects sorting between ephrin-B1–positive and –negative cell populations that are generated in the ephrin-B1 heterozygous females via random X-inactivation and that this abnormal expression of ephrin-B1 in the limb bud correlates with a polydactyly phenotype that is observed in ephrin-B1+/− females [7,8]. Our data demonstrate that the calvarial phenotypes observed in ephrin-B1 heterozygous females correlate with an abnormal expression of ephrin-B1 and EphB2 in the presumptive frontal bone, likely due to cell sorting between ephrin-B1–positive and ephrin-B1–negative cells in the craniofacial mesenchyme. + +The Frontal Bone Defect Is Caused by Abnormal Osteogenic Differentiation + +To uncover the nature of the dominant effect of mosaic loss of ephrin-B1, we reasoned that understanding why sorting-out between ephrin-B1–positive and ephrin-B1–negative cells has such consequences for the development of this tissue would shed light on the dominant function of ephrin-B1. NCCs are the source of the frontal bone osteoprogenitor population, and both ephrin-B1 and ephrin-B2 control migration of these cells, raising the possibility that improper migration of NCC progenitors could be responsible for the frontal bone phenotype. Using a combination of Wnt1Cre/R26R alleles to specifically label NCCs, we found no difference in the size of the progenitor pool between ephrin-B1 heterozygous females and wild-type animals (Figure 3A), indicating that defective migration is not the likely cause for the frontal bone phenotype. In addition, no proliferation or cell survival defects were detected on sections of mutant frontal bones (unpublished data). + +To test whether the defects in calvarial bone development in ephrin-B1+/− embryos correlated with perturbation of osteoblastic differentiation, we used alkaline phosphatase (AP) activity as a marker of early osteoblastic differentiation. At E16.5, AP staining of frontal bones showed delayed differentiation in ephrin-B1+/−/ephrin-B2+/GFP and ephrin-B1+/− (Figure S2B and unpublished data). At E12.5, similar levels of AP activity could be detected in wild-type, ephrin-B1Y/−, and ephrin-B1+/− embryos (Figure 3B), indicating that defective bone growth was not due to delayed onset of differentiation in heterozygous mutants. However, unlike wild-type and ephrin-B1Y/− embryos which showed continuous AP activity, AP staining of the presumptive frontal bone appeared irregular in ephrin-B1 heterozygous embryos (Figure 3Bc). These observations indicate that the calvarial defects observed in ephrin-B1+/− mutants are not due to abnormal migration or survival of NCCs, but instead might be due to a defective differentiation of the presumptive osteogenic mesenchyme. + +To confirm the differentiation defects observed in ephrin-B1 heterozygotes, we isolated presumptive osteogenic mesenchymal cells from E14.5 wild-type and ephrin-B1+/− embryos, and evaluated their ability to differentiate in vitro. Using AP activity as a marker for osteogenic differentiation, we observed that cells isolated from ephrin-B1+/− embryos were consistently less prone to differentiate in vitro (Figure 3C), indicating that these defects are autonomous to the osteoprogenitor cells. In these primary cultures, expression of ephrin-B1 was detected in a punctate pattern in both AP-positive as well as AP-negative cells (Figure S3A), indicating that expression of ephrin-B1 and AP do not strictly correlate, and suggesting that ephrin-B1 does not regulate AP activity directly. + +Defective Osteogenic Differentiation in ephrin-B1+/− Embryos Correlates with Abnormal Cx43 Distribution + +Using this in vitro system, we tested a number of markers that have been shown to regulate osteogenic differentiation, including N-cadherin and Cx43 expression, as well as activation of MAPK. Among these, only Cx43 distribution showed prevalent changes between cultures from wild-type and ephrin-B1 heterozygous embryos (Figure 4A and unpublished data). Cx43 is a structural protein that forms gap junctional pores (connexons). Whereas cytoplasmic Cx43 shows a diffuse staining by immunofluorescence, cell surface connexons appear as bright dots because they aggregate to form functional gap junctional plaques at cell–cell interfaces. In cultures of mesenchymal cells isolated from wild-type embryos, the differentiating cells expressed a high level of Cx43, and gap junctional plaques were readily visible between these cells (Figure 4Aa and 4Ab). On the contrary, in cultures of mesenchymal cells isolated from ephrin-B1 heterozygous embryos, the distribution of Cx43 was altered and gap junctional plaques were not detected (Figure 4Ac and 4Ad). Western blot analysis indicated that the overall level of Cx43 was unchanged in primary cultures isolated from ephrin-B1 heterozygous embryos, indicating that the distribution but not the expression of Cx43 was altered in primary cultures from ephrin-B1+/− embryos (Figure S3B). + +We next tested whether distribution of Cx43 was also altered in ephrin-B1 mosaic embryos. For this purpose, we co-stained paraffin sections from control and heterozygous mutant E12.5 embryos with AP and Cx43. However, even though AP staining of the frontal bone was very irregular in the ephrin-B1+/− embryo, compared to the control frontal bone (Figure S3Ca and S3Cc), we were unable to detect significant differences in Cx43 staining on these sections (Figure S3Cb and S3Cd). Since ephrin-B1 null embryos do not show calvarial phenotype or abnormal osteogenic differentiation, we reasoned that ephrin-B1 itself might not be required for proper localization of Cx43, but rather that abnormal distribution of Cx43 might be seen only at the boundary between ephrin-B1–positive and –negative cells in the mosaic embryos. To be able to detect boundaries between ephrin-B1–positive and ephrin-B1–negative cells, we generated chimeric embryos by injecting ephrin-B1 null embryonic stem (ES) cells in wild-type ROSA26 blastocysts which express β-galactosidase constitutively. Paraffin sections of X-gal–stained E11.5 chimeric embryos were processed for immunofluorescence using the Cx43 antibody. Gap junctional Cx43 was readily detected between wild-type cells and between ephrin-B1 null cells (Figure 4B). However, gap junctional Cx43 was almost never observed between ephrin-B1–positive and –negative cells. We concluded that the number of junctional pores is diminished at ephrin-B1–positive/ephrin-B1–negative boundaries in vivo. + +Ephrin-B1 Associates with Cx43 and Regulates GJC + +Compagni et al. have shown that the levels of EphB2 receptor are up-regulated in ephrin-B1–negative domains in ephrin-B1+/− embryos, thus creating ectopic Eph/ephrin boundaries [7]. We therefore reasoned that decreased junctional Cx43 at ephrin-B1–positive/ephrin-B1–negative boundaries in vivo could in fact indicate an inhibition of GJC by Eph/ephrin signaling. To test whether Eph/ephrin signaling could regulate GJC, we used calcein-AM as a marker of GJC in vitro. We found that interaction between ephrin-B1 and Eph-B2 resulted in inhibition of GJC in vitro (Figure 5). NIH 3T3 cells expressing ephrin-B1 showed reduced transfer of calcein-AM when plated on cells expressing low levels of Eph-B2 (Figure 5Ab), as compared to control cells (Figure 5Aa). More dramatically, plating of ephrin-B1–expressing cells over cells expressing high levels of Eph-B2 resulted in a complete inhibition of GJC (Figure 5Ac). Similar results were obtained using primary NCCs that express both ephrin-B1 and Eph-B2 at low levels. Although primary NCCs were able to establish strong GJC with control fibroblasts (Figure 5Ba and 5Bd), plating onto fibroblasts expressing ephrin-B1 markedly decreased dye transfer, even though the majority of the cells were able to spread normally (Figure 5Bb and 5Be). The fact that some cells spread normally, but were unable to transfer the dye (Figure 5Bf), indicates that inhibition of GJC is not due to cell repulsion. Plating of primary NCCs onto fibroblasts expressing Eph-B2 resulted in a moderate reduction in GJC (Figure 5Bc). We quantified GJC by two means: first, we evaluated the number of donor cells that had spread and were able to transfer the dye (Figure 5Ca). Second, we counted the number of receiving cells for each donor cell that had spread and transferred the dye (Figure 5Cb). Both measurements indicate that GJC is diminished when primary NCCs are plated on ephrin-B1– (and to a lower extent EphB2–) expressing cells. These results demonstrate that interaction between EphB2 and ephrin-B1 impairs establishment of GJC. + +To better understand the molecular mechanisms by which Eph/ephrins might impinge on Cx43 distribution and regulate GJC, we analyzed the distribution of both ephrin-B1 and Cx43 in cell culture. Co-immunofluorescence studies showed that Cx43 and ephrin-B1 partially co-localize in primary mesenchymal cells (Figure 6A). We then analyzed the effect of Eph/ephrin engagement on Cx43 distribution in cell lines in which ephrin-B1 was transiently transfected. Expression of ephrin-B1 and Cx43 was partially overlapping in untreated NIH 3T3 cells, especially at interfaces between cells expressing ephrin-B1, which exhibited strong Cx43 staining (Figure 6Ba–c). Following engagement by EphB2-Fc (a soluble form of EphB2 receptor), ephrin-B1 was found in clusters that partially overlapped with the Cx43 punctate staining (Figure 6Bd–f). Similar results were obtained using MDCK cells. These results demonstrate that ephrin-B1 and Cx43 partially co-localize at the subcellular level both in absence and presence of Eph/ephrin interaction. To test whether ephrin-B1 and Cx43 physically interact, we performed a pull-down assay in NIH 3T3 cells expressing ephrin-B1. We used a recombinant protein consisting of the extracellular domain of Eph-B2 receptor fused to the Fc fragment of human IgG (EphB2-Fc) to pull down ephrin-B1. Cx43 was detected in the pull down, indicating that it interacts with ephrin-B1 (Figure 6Ca). In a converse experiment, ephrin-B1 was co-immunoprecipitated with an anti-Cx43 antibody (Figure 6Cb). These results indicate that ephrin-B1 and Cx43 interact with each other. + +Regulation of GJC Underlies ephrin-Induced Cell Sorting + +To identify the domain of ephrin-B1 required for the interaction with Cx43, we performed a pull down using a recombinant protein consisting of the extracellular domain of ephrin-B1 fused to the Fc fragment of human IgG (ephrinB1-Fc). Cx43 was not detected in the pull down indicating that the intracellular domain of ephrin-B1 is required for the interaction with Cx43 (Figure 6Cb). We next asked whether the PDZ-binding domain of ephrin-B1 was necessary for the interaction with Cx43, since Cx43 has been shown to interact with PDZ-containing molecules, and the data presented above suggested that Cx43 interacts with the intracellular domain of ephrin-B1. We generated a mutant form of ephrin-B1 that shows reduced binding to PDZ-containing proteins (ephrin-B1ΔPDZ [8]). The ability of this mutant form of ephrin-B1 to interact with Cx43 was tested using NIH 3T3 cells that were transiently transfected with either wild-type ephrin-B1 or ephrin-B1ΔPDZ. Western-blot analysis of whole cell lysates indicated that both proteins were expressed, albeit at different levels, and that expression of ephrin-B1 did not influence the phosphorylation status of Cx43 (detected by differences in mobility on a SDS-PAGE), which is known to regulate GJC (Figure 7A). Cx43 could be detected in the pull downs from cells expressing either ephrin-B1 wild type or ephrin-B1ΔPDZ, however, the relative abundance of phosphorylated versus unphosphorylated band was changed. More phosphorylated Cx43 (slower mobility) was observed in the ephrin-B1 wild-type pull down, whereas more unphosphorylated Cx43 was detected in the ephrin-B1ΔPDZ pull down (Figure 7A). These results indicate that the PDZ binding domain of ephrin-B1 is not required for its interaction with Cx43; however, ephrin-B1ΔPDZ and wild-type ephrin-B1 interact preferentially with different forms of Cx43. + +Because phosphorylated Cx43 is thought to represent junctional Cx43 whereas unphosphorylated Cx43 represents the inactive, cytoplasmic pool, we tested whether ephrin-B1ΔPDZ co-localized with Cx43 following engagement by EphB2-Fc. Although ephrin-B1ΔPDZ was present at the cell surface and localized in clusters, we did not observe co-localization of ephrin-B1ΔPDZ and Cx43 following engagement by EphB2-Fc (Figure 7B). To test the effect of this mutation on calvarial development, we generated ephrin-B1ΔPDZ chimeric embryos. Examination of skeletal preparations of E18.5 ephrin-B1ΔPDZ chimeras revealed that these embryos did not exhibit defects in the calvarial bones, even in chimeras exhibiting a high degree of contribution of mutant ES cells (Figure S4), nor did they present polydactyly (unpublished data). However, subtle but consistent defects in sternum development, a phenotype that is associated with complete loss of ephrin-B1 [7,8], were observed in almost all of the chimeras (Figure S4), indicating that the lack of calvarial and polydactyly phenotypes in the chimeric embryos is not due to an ineffective contribution of ephrin-B1ΔPDZ ES cells to bone. To test the effect of the ephrin-B1ΔPDZ mutation on the distribution of Cx43 in vivo, we generated chimeric embryos by injecting mutant ES cells carrying the ephrin-B1ΔPDZ allele (or ephrin-B1 null ES cells as a control) in wild-type ROSA 26 blastocysts. Unexpectedly, paraffin sections of X-gal–stained E11.5 chimeric embryos revealed that unlike ephrin-B1 null cells, cells expressing ephrin-B1ΔPDZ do not sort-out from wild-type cells (Figure 7C). These results indicate that mosaic loss of reverse signaling through the PDZ binding domain is not sufficient to drive cell sorting and does not lead to defective calvarial bone development. + +The inability of ephrin-B1ΔPDZ to co-localize with Cx43 upon engagement and to drive cell sorting suggested that regulation of GJC itself may play a role in the sorting-out process between ephrin-B1–positive and ephrin-B1–negative cells. To test this hypothesis, we transiently transfected HEK293T cells (that have very low levels of endogenous Cx43) with Cx43 and allowed them to sort out following trypsinization. Unlike control transfected cells, Cx43-overexpressing cells segregated from untransfected cells and were consistently found in clusters (Figure 7Da), indicating that the establishment of GJC does indeed promote cell sorting. + +Overexpression of Cx43 Partially Rescues the Calvarial Phenotype + +Because the lack of cell sorting in the experiments described above precluded the establishment of a functional link between the calvarial phenotype and the regulation of GJC in ephrin-B1 heterozygote embryos, we performed a genetic rescue experiment. Mice carrying a CMV-Cx43 transgene that allows for the generalized overexpression of Cx43 [21] were bred to ephrin-B1−/− mice. Western-blot analysis showed a modest increase in the level of Cx43 in presumptive frontal bones of embryos carrying the transgene (unpublished data). Skeleton preparations of P1 offspring indicated that all of the skeletal defects previously observed in the ephrin-B1 mutants, including the phenotype specific to ephrin-B1 heterozygotes, were also found in this mixed genetic background (unpublished data). To assess whether overexpression of Cx43 had an effect on the calvarial phenotype observed in ephrin-B1+/− mutants, we quantified the foramen area between the frontal bones in newborn pups from all genotypes (see the Materials and Methods section). Although overexpression of Cx43 had no significant impact on the development of the frontal bones in male pups, the foramen area was decreased in the ephrin-B1+/− pups carrying the CMV-Cx43 transgene compared to ephrin-B1+/− pups without the transgene (Figure 8). Whereas the difference in foramen area between the ephrin-B1+/− pups with and without the CMV-Cx43 transgene was significant, the degree of rescue of frontal bone development in presence of the transgene was variable from sample to sample (unpublished data). Importantly, overexpression of Cx43 did not change the skeletal defects that are independent of cell sorting (i.e., sternum defects) (unpublished data). These results establish a functional link between ephrin-B1 and GJC, and suggest that improper regulation of GJC is implicated in the calvarial defects observed in ephrin-B1 heterozygous females. + +Discussion + +ephrin-B1+/− Mice as a Model for CFNS + +In this study we have shown that, analogous to humans, heterozygous loss of ephrin-B1 in mice results in defective development of the skull vault. Human CFNS patients carrying mutations in the ephrin-B1 gene exhibit a range of craniofacial defects including ocular hypertelorism, malformation of the face (in particular the forehead and the nose), cranium bifidum occultum, and craniosynostosis [22]. Our study shows that at birth, ephrin-B1+/− mice exhibit a delay in the ossification of calvarial bones leading to a frontal foramen similar to cranium bifidum occultum, and in many instances, to a bifid nose, mimicking the human disease. Removing one copy of ephrin-B2 resulted in increased severity of the frontal foramen, and an additional coronal suture defect in an ephrin-B1+/− background, but had no effect in ephrin-B1 null embryos, suggesting that the lack of phenotype in ephrin-B1 hemizygous males is not due to functional compensation by ephrin-B2. The effect of removing both copies of ephrin-B2 on calvarial development could not be analyzed due to the early embryonic lethality of ephrin-B2 null embryos. + +Interestingly, our mutant mice did not exhibit craniosynostosis of the coronal sutures seen in humans. The processes leading to calvarial foramina and craniosynostosis are genetically linked, as evidenced by loss of function and gain of function mutations in the transcription factor Msx2, respectively. Moreover, heterozygosity for the transcription factor Twist can lead to craniosynostosis, as well as to foramina, and both transcription factors regulate differentiation of frontal bone osteoprogenitors (Ishii et al., 2003). Ectopic bone growth observed within the coronal suture of ephrin-B1+/−/ephrin-B2+/GFP embryos might be prescient of a premature fusion of the suture, but this could not be analyzed at later stages, since 100% of these animals die at birth (A. Davy, P. Soriano, unpublished data). The genetic interaction suggests, however, that the discrepancy in coronal suture phenotype between ephrin-B1 heterozygous mice and humans could be due to genetic modifiers. Our expression analysis does not support a model in which wild-type expression of ephrin-B1 controls suture formation by establishing boundaries in craniofacial mesenchyme. In fact, the observation that ephrin-B1 null animals have no defects in calvarial bones argues that the function of ephrin-B1 is not normally required for either calvarial bone development or proper suture formation. On the other hand, the fact that ephrin-B1 is expressed in the meningeal layer could account for both the suture as well as the low-penetrance parietal bone phenotype observed in ephrin-B1+/− embryos because this layer is involved in suture and parietal bone formation [18,23]. + +Most of the mutations in ephrin-B1 that have been identified in CFNS patients are located in the 5′ end of the gene and are consistent with loss-of-function mutations, either by introducing a premature stop codon, or by presumably interfering with the binding of ephrin-B1 to Eph receptors [9,10]. However, it was reported more recently that some patients harbored mutations in the 3′ end of the gene, which might affect specifically the reverse signaling activity of ephrin-B1 [24]. Our data using chimeric embryos demonstrate that a mutation in the PDZ binding domain of ephrin-B1, which is known to phenocopy some of the phenotypes observed in ephrin-B1 null embryos including cleft palate [8], does not induce calvarial defects or polydactyly. These results indicate that the mutations found in CFNS patients might impinge on protein stability, protein localization, or binding of effector molecules independent of the PDZ domain. + +Defective Osteogenic Differentiation and Inhibition of GJC + +Our results are consistent with a model in which inhibition of GJC at ectopic Eph/ephrin boundaries in ephrin-B1+/− females results in an abnormal differentiation of osteoprogenitors, thereby leading to the defective development of frontal bones and also, presumably, to polydactyly. Consistent with a previous report [6], we found that Eph/ephrin interaction inhibits GJC. In addition, junctional Cx43 was decreased at ectopic Eph/ephrin boundaries in vivo, and reduced junctional Cx43 correlated with decreased osteogenic differentiation of primary cells isolated from ephrin-B1 heterozygous embryos. Finally, we found that overexpression of Cx43 partially rescued the calvarial phenotype observed in ephrin-B1 heterozygote females. These results are consistent with other reports documenting the role of GJC and Cx43 in osteogenic differentiation [25–27]. In addition, several genetic studies have shown that defective regulation of GJC affects craniofacial and digit development, indicating that the structures affected in ephrin-B1 heterozygous females are highly sensitive to alteration in GJC. Mice deficient for Cx43 exhibit delayed ossification of the calvarial bones and craniofacial abnormalities [28], but more importantly, mutations in GJA1 (the gene coding for Cx43) in humans are responsible for oculodentodigital dysplasia (ODDD), a syndrome that is characterized by defective craniofacial development and digit formation [16]. An ENU screen in mice recently uncovered a dominant mouse mutation that exhibits many of the classic features of ODDD, and positional cloning revealed that these mice carry a mutation in GJA1 that acts in a dominant-negative fashion to disrupt gap junction assembly and function [17]. On the basis of our results, we can not rule out the possibility that Eph/ephrin signaling might also impinge on osteogenic differentiation directly by activating signal transduction cascades (that would not involve the PDZ domain of ephrin-B1). However, the cytoplasmic kinases that are known targets of Eph/ephrin signaling (Src family kinases and MAPKs) have been shown to be positive regulators of osteogenic differentiation, which is not consistent with the inhibition of differentiation that we observe in vivo and in vitro. Moreover, we have not been able to detect a change in the level of MAPK activation in ephrin-B1+/− cultures (unpublished data). + +We found that ephrin-B1 and Cx43 form a complex and that following engagement by Eph receptors, ephrin-B1 and Cx43 co-localize in clusters that could be indicative of endocytosis. Recent reports have shown that Eph/ephrins complexes are internalized following interaction [29–31], and endocytosis is also a well-known mode of regulation of connexons at the cell surface [32]. In both cases, pieces of the plasma membrane from neighboring cells are internalized. It is therefore conceivable that Cx43 might be co-internalized with Eph/ephrin complexes. However, it is also possible that endocytosis of connexons is regulated via a signal transduction cascade, since it has been shown recently that Eph/ephrin signaling regulates clathrin-mediated endocytosis by tyrosine phosphorylation of Synaptojanin 1 [33]. Tyrosine phosphorylation of Cx43 is also a mechanism by which GJC is regulated. In our pull-down assay, wild-type ephrin-B1 interacted preferentially with phosphorylated Cx43 whereas ephrin-B1ΔPDZ interacted preferentially with unphosphorylated Cx43, suggesting that the interaction between ephrin-B1 and Cx43 might not be direct, and that these proteins might interact differently when at the cell surface or in the cytoplasm. + +Regulation of GJC and Cell Sorting + +We observed that junctional Cx43 was enriched at interfaces between ephrin-B1–positive cells, suggesting that while GJC is inhibited at Eph/ephrin interfaces, it might be promoted at ephrin/ephrin interfaces. This observation, as well as the fact that overexpression of Cx43 leads to cell sorting, supports the idea that regulation of GJC contributes to Eph/ephrin-induced cell sorting. Although GJC has not been formally linked to cell sorting previously, there is ample evidence in the literature that regulation of GJC influences cell–cell contacts. Indeed, it has been shown in various experimental settings that inhibiting GJC, either through the use of blocking antibodies or dominant negative constructs, induces loss of adhesion [34–37]. In Xenopus, both overexpression of ephrin-B1 and dominant-negative connexin result in de-adhesion of the blastomeres [36,38]. Sorting of cells overexpressing Cx43 has also been reported previously in PC12 cells [39]. We therefore propose that regulation of GJC contributes to cell sorting downstream of Eph/ephrin interaction (Figure 9A). + +A question that remains unanswered is whether or not the cell sorting observed in ephrin-B1 heterozygotes is fully dependent on Eph receptors. Interestingly, an Eph-independent role for ephrin-B1 in regulating tight junctions has recently been reported [40], and it was recently shown that EphA4 can induce cell sorting independently of ephrins [41,42]. However, the fact that some CFNS patients harbor point mutations in the extracellular domain of ephrin-B1 that presumably have an effect on Eph/ephrin interaction argues for the involvement of Eph receptors in the sorting process. + +On the basis of our data, we propose a model that explains the dominant effect of mosaic loss of ephrin-B1 in ephrin-B1 heterozygous females, and that revisits the function of Eph/ephrin in embryo patterning (Figure 9B). In wild-type mice and within a developmental compartment, ephrin-expressing cells establish GJC which stabilizes cell–cell interactions and creates a communication compartment. In ephrin-B1 heterozygotes as well as at a developmental boundary, GJC is inhibited between ephrin-positive and Eph-positive cells, possibly via endocytosis of Cx43, which prevents stable cell–cell interactions and leads to the formation of distinct compartments. Inhibition of GJC at developmental boundaries has been shown in a variety of models, including inter-rhombomeric boundaries [12,43], which also happen to be Eph/ephrin boundaries. In addition, overexpression of Cx43 has recently been shown to rescue a central nervous system boundary defect in mice [44]. + +In conclusion, our work demonstrates that in addition to their prominent role in regulating the actin cytoskeleton, Eph receptors and ephrins also play an important role in regulating gap junctional communication and suggests that improper regulation of GJC leads to the phenotypes observed in ephrin-B1 heterozygous individuals. Together, these functions make Eph receptors and ephrins potent regulators of boundary formation and tissue patterning during embryonic development. + +Materials and Methods + +Mice. + +Ephrin-B1 mutant mice and CMV-Cx43 transgenic mice have been described elsewhere [8,22]. The ephrin-B2GFP allele was generated by inserting the H2BGFP cDNA cassette at the MluI site in the first exon of ephrin-B2. The MluI-XbaI fragment encompassing the start codon of the ephrin-B2 gene was replaced with H2BGFP. The mutation was introduced in ES cells by homologous recombination. Mice were maintained in a 129S4/C57Bl6J mixed background. Genotyping was done by PCR using the following sets of primers: GFP-F: 5′-GCAAGAAGGCGGTGACTAAGGCGC-3′; GFP-R: 5′-GGCCGCCGCCAGTGCTTGAGGTCG-3′. Mice were housed in microisolator racks in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care, and experimentation was reviewed by the Hutchinson Center Institutional Review Committee. The ES cells carrying the ephrin-B1ΔPDZ mutation have been described previously [8]. The sequence of the primer specific to the mutant allele was: 5′-GCCATGCTGGGCCTTCACT-3′. To obtain ephrin-B1 null ES cells, one clone of targeted ES cells carrying the conditional allele of ephrin-B1 (ephrin-B1lox) was electroporated with a PGK-Cre expression vector. ES cell clones were isolated and screened by Southern-blot for the recombined ephrin-B1 locus. Targeted ES cells were injected into blastocysts obtained from crosses between F1 (129S4/C57Bl6J) ROSA26-βgal homozygous males and either wild-type C57BL6J/CBAJ or MF1 females. + +X-gal staining and skeletal preparations. + +Procedures used for X-gal staining and skeletal preparations have been described in detail elsewhere [8]. For the experiment with mice carrying the CMV-Cx43 transgene, quantification of the foramen area between frontal bones was performed using Photoshop (Adobe, San Jose, California, United States) to record the number of pixels corresponding to the foramen. The mean values for females with (97,901 pixels) and without (117,391 pixels) the transgene were then normalized to the mean value obtained for males (91,824 pixels) (no significant difference was found between males with or without the transgene, n = 10). Statistical significance was calculated using an unpaired t-test with Welch correction. + +In situ hybridization. + +Section in situ hybridization experiments were performed on frontal sections of E14.5 embryos as described previously [45]. Whole-mount in situ hybridization was performed according to a protocol described elsewhere [46]. Probe sequences used for ephrin-B1, EphB2, and EphB3 are available upon request. + +Primary cultures. + +Presumptive frontal bone mesenchyme was dissected from E14.5 embryos and incubated with 1% trypsin/0.5% DNAse in PBS for 10 min at 37 °C. Trypsin was inactivated by addition of complete medium (DMEM containing 15% FCS) and cells were dissociated by trituration with a glass Pasteur pipette. Cells were pelleted by centrifugation and washed twice in complete medium. Cells were plated at high density in a 24-well plate and kept in culture in complete medium. After 3 d, cells were fixed in 2% PFA and rinsed three times in NTMT. AP activity was detected by incubating fixed cells with NBT/BCIP (Roche, Basel, Switzerland). Some cultures were further processed for immunofluorescence as described below. Quantification of AP activity was performed on digital images using the Image J software (http://rsb.info.nih.gov/ij). The data presented are representative of five independent experiments. + +Primary NCCs were isolated from dissected branchial arches of E9.5 embryos as described above, except cells were cultured in F12 medium supplemented with 10% FCS. + +GJC assays. + +NIH 3T3 cells were stably transfected with expression vectors for ephrin-B1, Eph-B2 receptor, or the pcDNA3 control vector. Recipient cells were plated at high density. Donor cells (NIH 3T3 expressing ephrin-B1 or primary NCCs) were incubated with calcein-AM (Molecular Probes, Eugene, Oregon, United States) for 20 min at 37 °C. Calcein-AM loaded cells (donors) were extensively washed in PBS, trypsinized, and dropped onto confluent monolayers of NIH 3T3 cells transfected with either pcDNA3 or expressing ephrin-B1 or various levels of Eph-B2. Transfer of calcein-AM through gap junctions was assessed after 3 h incubation at 37 °C. GJC establishment was quantified by two means for each conditions: (1) the percentage of cells that were spread and had transferred the dye versus cells that were spread but did not transfer the dye; and (2) the number of cells receiving the dye for each donor. These data were acquired by visual assessment either directly on the inverted microscope or from digital images. Statistical significance was calculated using a Student t-test. + +Western blot analysis. + +Cells were scraped in 1% NP40 lysis buffer (50 mM Hepes [pH 7.5], 150 mM NaCl, 10% glycerol, 1.5 mM MgCl2, 1mM EGTA, 100 mM NaF), except for the experiment presented in Figure 7A (100 mM Tris [pH 7.4], 150 mM NaCl, 1mM EDTA). Protein lysates were incubated with either 5-μg EphB2-Fc or 4-μg Cx43 monoclonal antibody (generous gift from P. Lampe) for 4 h at 4 °C and subsequently with 20-μl ProteinA-Sepharose. Affinity complexes were analyzed by SDS-PAGE using the following antibodies: ephrin-B1 (A20 or C18, Santa Cruz Biotechnology, Santa Cruz, California, United States), Cx43 (Sigma, St. Louis, Missouri, United States). We have noted that the binding of Cx43 to ephrin-B1 is sensitive to the lysis buffer used: the interaction is lost in RIPA buffer. + +Immunofluorescence. + +NIH 3T3 cells were plated on glass coverslips and transiently transfected with an expression vector for ephrin-B1. Forty hours after transfection, cells were either fixed in 2% PFA or incubated with 4-μg/ml EphB2-Fc for 30 min at 37 °C and then fixed in PFA. Cells were permeabilized with 0.1% Tx-100 for 3 min, rinsed in PBS, and incubated with a mix of EphB2-Fc (4 μg/ml) and either Cx43 monoclonal antibody or N-cadherin monoclonal antibody (Zymed, Carlsbad, California, United States). In primary cells, ephrin-B1 was detected using the 25H11 rat monoclonal antibody [47]. FITC- and Cy3-conjugated secondary antibodies were from Jackson ImmunoResearch (West Grove, Pennsylvania, United States). For the sorting experiments, HEK293T cells were transiently transfected with either DsRed or a Cx43 expression construct. Twenty-four hours after transfection, cells were trypsinized into a single-cell suspension and replated onto glass coverslips. Immunofluorescence was performed at 48 h post-transfection using a Cx43 antibody (Sigma). + +For immunofluorescence on sections, paraffin sections were rehydrated and subjected to a citrate boil to reveal antigens. Tissue sections were blocked in Blocking solution (PBS/5% horse serum) 1 h at room temperature and incubated overnight at 4 °C in Cx43 antibody (1/100 in Blocking solution [Sigma]). Incubation with the Cy3-conjugated secondary antibody was for 1 h at room temperature (1/250 in Blocking solution). The number of Cx43 dots was counted on digital images acquired on a Delta Vision Deconvolution microscope (Applied Precision Inc., Issaquah, Washington, United States). The number of Cx43 dots was normalized to the number of junctions: for each section, we evaluated the number of wild-type/knock-out (KO) junctions (15–20) and counted a similar number of wild-type/wild-type and KO/KO junctions on each side of the wild-type/KO boundary. + +Supporting Information + +Figure S1 + +Patterning of the Vibrissae Buds Is Altered in ephrin-B1+/− Embryos + +(A) Whole E14.5 embryos were stained with H&E to highlight whiskers buds of ephrin-B1Y/− hemizygous males (a) and ephrin-B1+/− heterozygous females (b). X-gal staining of E14.5 ephrin-B1+/lox embryo carrying the R26R and Wnt1-Cre alleles (c). The patterning of vibrissae buds is abnormal in heterozygote but not homozygote mutants. This defect is autonomous to the NCC lineage since eliminating ephrin-B1 specifically in this lineage is sufficient to phenocopy the vibrissae bud defect (c). + +(B) In situ hybridization on frontal sections of E14.5 wild-type embryo using probes for ephrin-B1 (a) and EphB2 (b) showing expression of these genes in the mesenchyme around vibrissae buds (arrowheads). + +(3.0 MB PPT) + +Click here for additional data file. + +Figure S2 + +Calvarial Defects and Decreased Osteogenic Differentiation + +(A) Skeleton preparations of whole heads from E17.5 littermate embryos, ephrin-B2+/GFP single heterozygous mutants (a), ephrin-B1+/− single heterozygous females (b), and ephrin-B1+/−/ephrin-B2+/GFP double heterozygous females (c). Ectopic bone can be seen in the suture mesenchyme of ephrin-B1/ephrin-B2 double heterozygous females (arrowhead in c) and the foramen between the frontal bones is larger than in ephrin-B1+/− embryos. An outline of the bones is presented for better visualization (d–f). + +(B) AP staining on cryosections of E15.5 embryos. A wild-type (a), an ephrin-B1 null male (b), and an ephrin-B1+/−/ephrin-B2+/GFP double heterozygous female (c) are shown. Bone front in the control embryo (a) and (b) is closer to the midline than bone front in the heterozygous littermate (c). In addition, the thickness of the bone is decreased in the ephrin-B1+/−/ephrin-B2+/GFP double heterozygous female. Both observations show that differentiation of the frontal bone is decreased in the heterozygote. + +(2.2 MB PPT) + +Click here for additional data file. + +Figure S3 + +Ephrin-B1 and Cx43 Distribution in Primary Cultures and Differentiating Frontal Bones of ephrin-B1+/− Embryos + +(A) Ephrin-B1 was detected by immunofluorescence in cultures of primary mesenchymal cells (a) and (c) that were also stained for AP activity (b) and (d). Expression of ephrin-B1 was readily detected as a punctate staining on these primary cells, both in AP-positive (a) and (b) and in AP-negative cells (c) and (d). + +(B) Western blot analysis of Cx43 levels in primary cultures of mesenchymal cells isolated from littermates of various genotypes. No difference in the overall Cx43 levels was detected. + +(C) Paraffin sections from E12.5 control embryos (ephrin-B1Y/−) (a) and (b) and ephrin-B1+/− embryos (c) and (d) were stained for AP activity (a) and (c) and subsequently processed for Cx43 immunostaining (b) and (d). AP staining in the developing frontal bone of ephrin-B1+/− embryo is more irregular than in the control embryo, consistent with the results presented in Figure 3. However, no significant difference can be seen in the overall staining for Cx43 in this tissue. + +(878 KB PPT) + +Click here for additional data file. + +Figure S4 + +ephrin-B1ΔPDZ Chimeric Embryos Do Not Exhibit Calvarial Defects + +(A) Skeletal preparations of whole heads from E18.5 ephrin-B1ΔPDZ chimeric embryos showing low (a) and high (b) contribution of mutant ES cells, assessed by PCR on tail DNA (c). + +(B) Skeletal preparations of E18.5 wild-type (a), ephrin-B1−/− (b), and ephrin-B1ΔPDZ chimeras (c) and (d) show that chimeric embryos exhibit sternum defects reminiscent of ephrin-B1 null embryos. + +(1.1 MB PPT) + +Click here for additional data file. + +Acknowledgements + +We thank Cecilia Lo for sharing the CMV-Cx43 mice; Mark Henkemeyer for providing the Eph-B2 expression construct and the in situ probe for EphB2; Paul Lampe for sharing his Cx43 antibody and the Cx43 expression construct; Wieland Huttner for the 25H11 rat monoclonal antibody [47]; Philip Corrin, Jason Frazier, and Marc Grenley for excellent technical assistance; and our laboratory colleagues for critical reading of the manuscript. + +Abbreviations + +AP - alkaline phosphatase + +CFNS - craniofrontonasal syndrome + +Cx43 - connexin43 + +ES - embryonic stem + +GFP - green fluorescent protein + +GJC - gap junction communication + +KO - knock-out + +NCC - neural crest cell + +Figures and Tables + +Figure 1 + +ephrin-B1+/− Embryos Exhibit Defects in NCC Derivatives + +(A) Skeleton preparations of whole heads from E18.5 mutant embryos (a–c). Bones are stained with Alizarin Red while cartilage is stained with Alcian Blue. An opening (foramen) between frontal bones is observed in ephrin-B1+/− heterozygous females (b) but not in ephrin-B1−/− homozygous females (a). Mutant embryos in which ephrin-B1 is specifically deleted in NCC also exhibit defects of the frontal bones (c). Arrowheads show the bone front of the frontal bones. Schematic drawings of the frontal bones (d–f). + +(B) Skeleton preparations of whole heads from E18.5 embryos (a–c) or E15.5 embryos (d–f) show that ephrin-B1+/−/ephrin-B2+/GFP embryos present an additional coronal suture defect (c) and (f) as compared to ephrin-B1+/− single heterozygous females (b) and (e). At E18.5, bone fronts never overlap at coronal sutures of ephrin-B1+/−/ephrin-B2+/GFP embryos, and ectopic bone forms in the suture mesenchyme (arrow in [c]). At E15.5, parietal and frontal bone fronts forming the coronal suture (arrow) of ephrin-B1+/−/ephrin-B2+/GFP embryos (f) are further apart than in control littermates (d) and (e). Coronal suture and frontal bones are normal in single ephrin-B2+/GFP heterozygous mutants (a) and in ephrin-B1Y/−/ephrin-B2+/GFP males (d). + +fb, frontal bone; pb, parietal bone. + +Figure 2 + +Expression of ephrin-B1 and EphB2 Is Abnormal in ephrin-B1+/− Embryos + +(A) In situ hybridization on frontal sections of E14.5 wild-type embryo using probes for ephrin-B1 (a), EphB2 (b), or EphB3 (c). Ephrin-B1 is expressed throughout the developing frontal bone (marked by dotted lines) as well as in the meningeal layer (arrowheads). Expression of EphB2 is restricted ventrally (arrowheads) whereas EphB3 is expressed throughout the developing frontal bone. + +(B) In situ hybridization of wild-type (a), (c), and (e) or ephrin-B1+/− embryos (b), (d), and (f) using a probe for ephrin-B1 (a) and (b), ephrin-B2 (c) and (d) and EphB2 (e) and (f). Both ephrin-B1 and EphB2 show sorting in the telencephalon and the craniofacial mesenchyme of ephrin-B1+/− embryos (arrowheads) while expression of ephrin-B2 is unaffected. + +Figure 3 + +Calvarial Foramen Correlates with Impaired Osteogenic Differentiation + +(A) E11.5 embryos carrying the R26R and Wnt1-Cre alleles were processed for X-gal staining to label NCCs. No difference in the osteogenic precursor population (arrow) was detected in ephrin-B1+/− females (b) as compared to wild type (a). + +(B) E12.5 wild-type (a), ephrin-B1Y/− (b), and ephrin-B1+/− (c) embryos were stained for AP activity. The onset of osteogenic differentiation does not seem affected in the mutant embryos, but AP staining pattern is irregular in the heterozygote mutant as compared to wild-type and homozygote mutant, with areas devoid of staining (arrows). + +(C) Primary mesenchymal cells were isolated from presumptive calvaria of E14.5 ephrin-B1Y/− hemizygous male (a) or ephrin-B1+/− heterozygous female (b) and plated at high density. AP activity was evaluated after 3 d in culture. Digital quantification of AP activity (c). + +Figure 4 + +Decreased Osteogenic Differentiation Correlates with Abnormal Distribution of Cx43 + +(A) Primary mesenchymal cells isolated from presumptive calvaria of wild-type (a) and (b) or ephrin-B1+/− embryos (c) and (d) were stained for AP activity (a) and (c) and subsequently processed for Cx43 immunofluorescence (b) and (d). Junctional Cx43 evidenced by bright dots is readily detected in cultures from wild-type embryos (WT), but not in cultures from ephrin-B1+/− embryos. + +(B) Detection of Cx43 by immunofluorescence in limb bud sections of an X-gal–stained chimeric embryo obtained by injecting ephrin-B1 null cells into a ROSA26-βgal blastocyst. Gap junctional Cx43 appears as bright dots (a). The boundary between wild-type cells (dark cells in [c]) and ephrin-B1 null cells (white cells in [c]) is indicated by red arrows. A schematic representation of the results is shown (b). Grey cells are wild type and white cells are ephrin-B1 null. Cx43 dots are in red, whereas yellow in (b) marks the only Cx43 dot that might be between a wild-type and a null cell. Quantification of Cx43 dots in multiple sections show a reduction in the number of Cx43 dots between wild-type and null cells (WT-KO) (d). + +Figure 5 + +Eph/ephrin Interaction Inhibits Gap Junction Communication + +(A) NIH 3T3 cells stably expressing ephrin-B1 were loaded with calcein-AM and dropped onto monolayers of NIH 3T3 cells either not expressing (a) or expressing variable levels (b) and (c) of Eph-B2 receptor. Transfer of dye to neighboring cells (arrowheads) was evaluated by fluorescence after 3 h. No dye transfer (arrows) was observed when high levels of Eph-B2 were expressed. + +(B) Primary NCCs were loaded with Calcein-AM and dropped onto NIH 3T3 cells that were transfected either with a control plasmid (pcDNA3 [a]), or an expression construct for ephrin-B1 (b) or Eph-B2 (c). In the control situation, the majority of cells transferred the dye (arrowheads). The boxed area is shown at higher magnification in (d). Transfer of dye is visualized by faint staining of cells surrounding the very bright donor cell. Following Eph/ephrin interaction, many of the donor cells did not transfer the dye (arrows). Boxed areas in (b) are shown at higher magnification in (e) and (f). Cells transferring the dye (f) and cells not transferring the dye (e) are able to spread. + +(C) The percentage of cells that had spread and showed transfer of dye was evaluated after 3 hours (a). In addition, the number of receiving cells for each donor cell was counted (b). + +*p < 0.05 compared to control. + +Figure 6 + +Ephrin-B1 and Cx43 Co-localize and Interact with Each Other + +(A) Primary mesenchymal were co-stained for Cx43 ([a], red in [c]) and ephrin-B1 ([b], green in [c]). + +(B) NIH 3T3 cells were transiently transfected with ephrin-B1 and co-immunofluorescence studies were performed to detect Cx43 (a) and (d) and ephrin-B1 (b) and (e) either without (a–c) or with engagement of ephrin-B1 (d–f). In untreated cells, Cx43 is enriched at interfaces between ephrin expressing cells and co-localizes with ephrin-B1 (c). Engagement by EphB2-Fc results in clustering of ephrin-B1 (e) and Cx43 partially co-localizes with ephrin-B1 clusters (f). Insets present higher magnification views. + +(C) Ephrin-B1 was affinity precipitated from NIH 3T3 cells using EphB2-Fc. The presence of Cx43 in the affinity complex was assessed by Western-blot (a). Protein lysates from NIH 3T3 cells expressing ephrin-B1 were incubated with ephrinB1-Fc, and the presence of Cx43 in the affinity complexes was assessed by Western blot (b) Cx43 was immunoprecipitated from NIH 3T3 cells expressing ephrin-B1 and the presence of ephrin-B1 in the immunocomplexes was detected by Western blot (right panel). + +IP, immunoprecipitation; Wcl, whole cell lysate. + +Figure 7 + +Regulation of GJC Correlates with Cell Sorting + +(A) NIH 3T3 cells were transiently transfected with either wild-type ephrin-B1 (B1), ephrin-B1ΔPDZ (ΔPDZ), or a control plasmid (−). Protein lysates were incubated with EphB2-Fc, and the presence of Cx43 in the affinity complex was assessed by Western blot. ProtA indicates a sample that was incubated with Protein A in absence of EphB2-Fc. Whole cell lysates (a) and affinity precipitations (b) were analyzed by Western blot. + +np, non phosphorylated Cx43; p1, p2, two different forms of phosphorylated Cx43; wcl, whole cell lysate. + +(B) NIH 3T3 cells were transiently transfected with ephrin-B1ΔPDZ and treated with EphB2-Fc. Subcellular localization of Cx43 (a) and ephrin-B1ΔPDZ (b) was analyzed by immunofluorescence. No co-localization was observed between these two proteins (c). + +(C) Paraffin sections of limb buds from E11.5 chimeric embryos obtained from injection of either ephrin-B1 null ES cells (K/O) (a) or ephrin-B1ΔPDZ ES cells (ΔPDZ) (b). Chimeric embryos were X-gal stained, processed for histology, and paraffin sections were counter-stained with Nuclear Fast Red. The wild-type cells are blue whereas the mutant cells are red. Cells expressing ephrin-B1ΔPDZ do not sort from wild-type cells. + +(D) HEK293T cells overexpressing Cx43 were detected by immunofluorescence as a tightly packed cluster of cells (a) whereas cells expressing DsRed are scattered among non-expressing cells (b). (c) and (d) show the visible image for the same field of cells. + +Figure 8 + +Partial Rescue of the Calvarial Defect in ephrin-B1+/− Mice Overexpressing Cx43 + +Skeleton preparations from an ephrin-B1Y/− male (a), an ephrin-B1+/− female (b) and an ephrin-B1+/− female carrying the CMV-Cx43 transgene (c). Quantification (d) of the foramen area between the frontal bones of an ephrin-B1+/− female (B1+/−) and an ephrin-B1+/− female carrying the CMV-Cx43 transgene (CMV-Cx43;B1+/−). Mean values for the females with and without the transgene were normalized to the value obtained for males. The number of embryos for each genotype is indicated. Error bars represent standard error of the mean values. + +*p = 0.0224. + +Figure 9 + +Mosaic Loss of ephrin-B1 and Establishment of Developmental Boundaries + +(A) Potential cross-talk between gap junctions and adherens junctions. Ephrin-B1 co-localizes with Cx43 in junctional plaques. Co-regulation of gap junctions and adherens junctions has been extensively studied. + +(B) Establishment of communication compartments for embryo patterning. Within a compartment and in the developing calvarial bones, all cells express ephrin-B1 and are coupled via GJC, exchanging second messengers (black dots). At a developmental boundary and at ectopic ephrin boundaries in ephrin-B1+/− embryos, Eph/ephrin interaction leads to inhibition of GJC, possibly through endocytosis of Cx43, concomitant with a loss of stable cell–cell interactions between the two cell types. Sorting between these cells and inhibition of GJC concur to establish distinct developmental compartments. + +Footnotes + +Competing interests. The authors have declared that no competing interests exist. + +Author contributions. AD and PS conceived and designed the experiments. AD and JOB performed the experiments. AD, JOB, and PS analyzed the data. AD and PS wrote the paper. + +Funding. This work was supported by grants HD24875 and HD25326 from the National Institute of Child Health and Human Development to PS. AD is a Canadian Institute of Health Research postdoctoral fellow. JOB is the recipient of a F32 postdoctoral fellowship from the National Institute of Dental and Craniofacial Research (DE17506). diff --git a/src/ontogpt/evaluation/craft/database/all/17002498.ann b/src/ontogpt/evaluation/craft/database/all/17002498.ann new file mode 100644 index 000000000..22476ed48 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17002498.ann @@ -0,0 +1,1800 @@ +T1 http://purl.obolibrary.org/obo/MONDO_0004972 0 11 Adenomatous +T2 PR:000004122 0 26 Adenomatous Polyposis Coli +T3 PR:000004122 28 31 APC +T4 GO:0043588 56 75 Development of Skin +T5 GO:0048538 56 70;80 86 Development of ... Thymus +T6 UBERON:0002370 80 86 Thymus +T7 http://purl.obolibrary.org/obo/MONDO_0005070 102 107 tumor +T8 SO:0000704 119 123 gene +T9 PR:000004122 124 127 Apc +T10 http://purl.obolibrary.org/obo/MONDO_0004972 129 140 adenomatous +T11 PR:000004122 129 155 adenomatous polyposis coli +T12 GO:0016055 176 197 Wnt signaling pathway +T13 NCBITaxon:10088 271 275 mice +T14 PR:000004122 280 283 Apc +T15 http://purl.obolibrary.org/obo/MONDO_0005070 291 296 tumor +T16 UBERON:0000922 348 357 embryonic +T17 PR:000004122 395 398 Apc +T18 SO:0000359 429 435 floxed +T19 SO:0001023 436 442 allele +T20 NCBITaxon:10088 450 454 mice +T21 GO:0007618 460 465 mated +T22 GO:0065007 515 522 control +T23 NCBITaxon:9606 530 535 human +T24 PR:000009454 536 546 Keratin 14 +T25 PR:000009454 548 551 K14 +T26 SO:0000167 553 561 promoter +T27 CL:0002187 582 606 basal cells of epidermis +T28 CL:0000079 588 596;617 637 cells of ... stratified epithelia +T29 UBERON:0007376 597 606 epidermis +T30 UBERON:0000486 617 637 stratified epithelia +T31 NCBITaxon:10088 639 643 Mice +T32 SO:0000359 663 669 floxed +T33 SO:0001023 670 676 allele +T34 PR:000009454 697 700 K14 +T35 SO:0000902 705 714 transgene +T36 GO:0016265 754 758 died +T37 PR:000009454 834 837 K14 +T38 PR:000004122 851 854 Apc +T39 UBERON:0000924 896 908 ectodermally +T40 UBERON:0006914 917 935 squamous epithelia +T41 UBERON:0002073 947 961 hair follicles +T42 UBERON:0001091 963 968 teeth +T43 UBERON:0002424 974 978;991 1000 oral ... epithelia +T44 UBERON:0001772 983 1000 corneal epithelia +T45 UBERON:0000483 1059 1069 epithelial +T46 UBERON:0000479 1078 1085 tissues +T47 UBERON:0002370 1101 1107 thymus +T48 UBERON:0002073 1132 1146 hair follicles +T49 UBERON:0004529 1157 1167 appendages +T50 UBERON:0002370 1183 1189 thymic +T51 PR:000009454 1207 1210 K14 +T52 PR:000004122 1216 1219 Apc +T53 NCBITaxon:10088 1227 1231 mice +T54 PR:000004122 1244 1247 Apc +T55 SO:0000704 1248 1252 gene +T56 UBERON:0000922 1267 1276 embryonic +T57 CL:0002321 1267 1282 embryonic cells +T58 UBERON:0000483 1294 1304 epithelial +T59 CL:0000066 1294 1309 epithelial cell +T60 UBERON:0000062 1319 1325 organs +T61 UBERON:0000483 1339 1349 epithelial +T62 UBERON:0003104 1350 1361 mesenchymal +T63 http://purl.obolibrary.org/obo/MONDO_0021055 1423 1453 familial adenomatous polyposis +T64 http://purl.obolibrary.org/obo/MONDO_0021055 1455 1458 FAP +T65 http://purl.obolibrary.org/obo/MONDO_0019336 1477 1495 Gardner's syndrome +T66 UBERON:0012652 1522 1532 colorectal +T67 http://purl.obolibrary.org/obo/MONDO_0021392 1522 1539 colorectal polyps +T68 http://purl.obolibrary.org/obo/MONDO_0003847 1549 1566 heritable disease +T69 SO:0000704 1587 1594 genetic +T70 http://purl.obolibrary.org/obo/MONDO_0005070 1611 1616 tumor +T71 SO:0000704 1628 1632 gene +T72 PR:000004122 1633 1636 APC +T73 http://purl.obolibrary.org/obo/MONDO_0004972 1638 1649 adenomatous +T74 PR:000004122 1638 1664 adenomatous polyposis coli +T75 NCBITaxon:1 1673 1684 individuals +T76 UBERON:0001155 1703 1710 colonic +T77 http://purl.obolibrary.org/obo/MONDO_0021140 1737 1747 congenital +T78 UBERON:0001782 1767 1793 retinal pigment epithelium +T79 CHEBI:26130 1775 1782 pigment +T80 http://purl.obolibrary.org/obo/MONDO_0007608 1795 1809 desmoid tumors +T81 UBERON:0007376 1811 1821 epidermoid +T82 http://purl.obolibrary.org/obo/MONDO_0007547 1811 1827 epidermoid cysts +T83 http://purl.obolibrary.org/obo/MONDO_0005381 1829 1841;1869 1874 disorders of bones +T84 UBERON:0002397 1846 1855 maxillary +T85 UBERON:0004288 1860 1868 skeletal +T86 UBERON:0001474 1869 1874 bones +T87 UBERON:0001091 1880 1886 dental +T88 PR:000004122 1931 1934 APC +T89 UBERON:0000062 1954 1960 organs +T90 PR:000004122 1988 1991 Apc +T91 UBERON:0000062 2014 2020 organs +T92 UBERON:0000160 2032 2041 intestine +T93 PR:000004122 2056 2059 Apc +T94 NCBITaxon:10088 2067 2071 mice +T95 SO:0000704 2106 2110 gene +T96 NCBITaxon:10088 2150 2154 mice +T97 GO:0007618 2160 2165 mated +T98 PR:000009454 2171 2174 K14 +T99 PR:000009454 2176 2186 Keratin 14 +T100 NCBITaxon:10088 2192 2196 mice +T101 GO:0010467 2202 2209 express +T102 UBERON:0004529 2242 2252 appendages +T103 NCBITaxon:10088 2288 2292 mice +T104 PR:000004122 2308 2311 Apc +T105 PR:000009454 2315 2318 K14 +T106 SO:0000902 2323 2332 transgene +T107 GO:0010467 2333 2343 expressing +T108 UBERON:0000479 2344 2351 tissues +T109 GO:0016265 2392 2396 died +T110 NCBITaxon:10088 2426 2430 mice +T111 UBERON:0000483 2500 2510 epithelial +T112 UBERON:0000479 2519 2526 tissues +T113 UBERON:0001091 2538 2543 teeth +T114 UBERON:0002370 2548 2554 thymus +T115 PR:000004122 2597 2600 Apc +T116 GO:0048513 2604 2618;2624 2630 development of ... organs +T117 UBERON:0000062 2624 2630 organs +T118 PR:000004122 2705 2708 APC +T119 http://purl.obolibrary.org/obo/MONDO_0004972 2735 2746 Adenomatous +T120 PR:000004122 2735 2761 Adenomatous polyposis coli +T121 PR:000004122 2763 2766 APC +T122 GO:0016055 2787 2808 Wnt signaling pathway +T123 GO:0065007 2846 2854 regulate +T124 PR:000002198 2869 2878 β-catenin +T125 PR:000002198 2895 2904 β-catenin +T126 GO:0065007 2905 2915 regulation +T127 NCBITaxon:9606 2935 2940 human +T128 http://purl.obolibrary.org/obo/MONDO_0005070 2941 2947 tumors +T129 PR:000004122 2961 2964 APC +T130 GO:0005829 3005 3014 cytosolic +T131 PR:000002198 3015 3024 β-catenin +T132 GO:0051170 3056 3068;3073 3080 migration to ... nucleus +T133 GO:0005634 3073 3080 nucleus +T134 PR:000004122 3142 3145 APC +T135 PR:000004527 3216 3222 axin-2 +T136 PR:000004527 3224 3229 AXIN2 +T137 PR:000009235 3232 3243 plakoglobin +T138 PR:000009235 3245 3248 JUP +T139 PR:000004257 3251 3255 Asef +T140 PR:000004257 3257 3264 ARHGEF4 +T141 PR:000009322 3267 3307 kinesin superfamily–associated protein 3 +T142 PR:000009322 3309 3315 KIFAP3 +T143 PR:000010170 3318 3321 EB1 +T144 PR:000010170 3323 3329 MAPRE1 +T145 GO:0005874 3332 3344 microtubules +T146 NCBITaxon:9606 3354 3359 human +T147 SO:0000853 3360 3367 homolog +T148 PR:000006511 3360 3393 homolog of Drosophila discs large +T149 NCBITaxon:7215 3371 3381 Drosophila +T150 PR:000006511 3395 3399 DLG1 +T151 PR:000004122 3434 3437 APC +T152 GO:0065007 3454 3462 regulate +T153 GO:0005856 3522 3534 cytoskeletal +T154 GO:0065007 3549 3559 regulation +T155 PR:000009235 3563 3574 plakoglobin +T156 GO:0051726 3583 3596;3601 3611 regulation of ... cell cycle +T157 GO:0042981 3583 3596;3616 3625 regulation of ... apoptosis +T158 GO:0098722 3642 3671 asymmetric stem cell division +T159 CL:0000034 3653 3662 stem cell +T160 GO:2000114 3677 3705 control of cell polarization +T161 PR:000004122 3714 3717 APC +T162 http://purl.obolibrary.org/obo/MONDO_0005070 3723 3728 tumor +T163 SO:0000704 3740 3744 gene +T164 PR:000004122 3767 3770 APC +T165 http://purl.obolibrary.org/obo/MONDO_0005401 3809 3818;3823 3828 tumors of colon +T166 http://purl.obolibrary.org/obo/MONDO_0002165 3809 3818;3833 3839 tumors of rectum +T167 UBERON:0001155 3823 3828 colon +T168 UBERON:0001052 3833 3839 rectum +T169 GO:0030849 3841 3850 Autosomal +T170 PR:000004122 3882 3885 APC +T171 http://purl.obolibrary.org/obo/MONDO_0021055 3892 3921 familial adenomatous polypois +T172 http://purl.obolibrary.org/obo/MONDO_0021055 3923 3926 FAP +T173 http://purl.obolibrary.org/obo/MONDO_0019336 3945 3961 Gardner syndrome +T174 http://purl.obolibrary.org/obo/MONDO_0021055 3963 3966 FAP +T175 http://purl.obolibrary.org/obo/MONDO_0004972 4009 4020 adenomatous +T176 UBERON:0012652 4021 4031 colorectal +T177 http://purl.obolibrary.org/obo/MONDO_0021392 4021 4038 colorectal polyps +T178 UBERON:0012652 4081 4091 colorectal +T179 http://purl.obolibrary.org/obo/MONDO_0005575 4081 4098 colorectal cancer +T180 UBERON:0000104 4134 4138 life +T181 UBERON:0012652 4161 4171 colorectal +T182 http://purl.obolibrary.org/obo/MONDO_0005335 4161 4180 colorectal neoplams +T183 NCBITaxon:1 4188 4199 individuals +T184 UBERON:0001155 4217 4224 colonic +T185 UBERON:0001555 4257 4279 gastrointestinal tract +T186 http://purl.obolibrary.org/obo/MONDO_0024292 4257 4286 gastrointestinal tract polyps +T187 http://purl.obolibrary.org/obo/MONDO_0021140 4288 4298 congenital +T188 UBERON:0001782 4318 4344 retinal pigment epithelium +T189 CHEBI:26130 4326 4333 pigment +T190 http://purl.obolibrary.org/obo/MONDO_0007608 4346 4360 desmoid tumors +T191 http://purl.obolibrary.org/obo/MONDO_0005381 4362 4374;4402 4407 disorders of bones +T192 UBERON:0002397 4379 4388 maxillary +T193 UBERON:0004288 4393 4401 skeletal +T194 UBERON:0001474 4402 4407 bones +T195 UBERON:0001091 4413 4419 dental +T196 PR:000004122 4468 4471 APC +T197 SO:0000704 4472 4476 gene +T198 UBERON:0000467 4496 4509 organ systems +T199 PR:000004122 4533 4536 APC +T200 NCBITaxon:9606 4558 4563 human +T201 UBERON:0012652 4564 4574 colorectal +T202 http://purl.obolibrary.org/obo/MONDO_0005575 4564 4581 colorectal cancer +T203 UBERON:0000479 4621 4627 tissue +T204 GO:0030111 4705 4732 regulation of Wnt signaling +T205 UBERON:0000922 4736 4745 embryonic +T206 GO:0009880 4736 4763 embryonic pattern formation +T207 GO:0009887 4768 4784;4790 4796 morphogenesis of ... organs +T208 UBERON:0000062 4790 4796 organs +T209 PR:000004122 4827 4830 APC +T210 UBERON:0001155 4858 4865 colonic +T211 UBERON:0000479 4866 4873 tissues +T212 NCBITaxon:9606 4936 4942 humans +T213 PR:000004122 4983 4986 Apc +T214 NCBITaxon:10088 5016 5020 mice +T215 PR:000004122 5042 5045 Apc +T216 SO:0000704 5064 5075 genetically +T217 NCBITaxon:10088 5085 5090 mouse +T218 PR:000004122 5103 5106 Apc +T219 UBERON:0005409 5191 5207 gastrointestinal +T220 http://purl.obolibrary.org/obo/MONDO_0021223 5191 5207;5218 5223 gastrointestinal tumor +T221 NCBITaxon:10088 5257 5262 Mouse +T222 UBERON:0000922 5263 5270 embryos +T223 SO:0000704 5299 5306 genetic +T224 GO:0016265 5320 5323 die +T225 GO:0009790 5331 5344 embryogenesis +T226 GO:0007369 5391 5403 gastrulation +T227 PR:000004122 5460 5463 Apc +T228 UBERON:0000479 5498 5505 tissues +T229 NCBITaxon:10088 5523 5528 mouse +T230 SO:0001023 5574 5580 allele +T231 GO:0007618 5585 5589 mate +T232 NCBITaxon:10088 5600 5605 mouse +T233 SO:0001023 5666 5672 allele +T234 PR:000004122 5723 5726 Apc +T235 UBERON:0000105 5740 5754 stages of life +T236 NCBITaxon:10088 5784 5788 mice +T237 SO:0001023 5836 5842 allele +T238 PR:000004122 5846 5849 Apc +T239 PR:000004122 5851 5854 Apc +T240 NCBITaxon:10088 5866 5870 mice +T241 GO:0007618 5876 5881 mated +T242 GO:0065007 5931 5938 control +T243 NCBITaxon:9606 5946 5951 human +T244 PR:000009454 5952 5962 Keratin 14 +T245 PR:000009454 5964 5967 K14 +T246 SO:0000167 5969 5977 promoter +T247 CL:0002187 5998 6022 basal cells of epidermis +T248 CL:0000079 6004 6012;6033 6053 cells of ... stratified epithelia +T249 UBERON:0007376 6013 6022 epidermis +T250 UBERON:0000486 6033 6053 stratified epithelia +T251 PR:000009454 6075 6078 K14 +T252 SO:0000167 6079 6087 promoter +T253 PR:000004122 6103 6106 Apc +T254 GO:0048513 6128 6142;6151 6157 development of ... organs +T255 UBERON:0000062 6151 6157 organs +T256 UBERON:0000483 6181 6191 epithelial +T257 UBERON:0003104 6192 6203 mesenchymal +T258 UBERON:0002073 6228 6241 hair follicle +T259 UBERON:0001091 6243 6248 teeth +T260 UBERON:0002370 6254 6260 thymus +T261 GO:0016265 6287 6292 death +T262 NCBITaxon:10088 6296 6300 mice +T263 PR:000004122 6316 6319 Apc +T264 GO:0001709 6344 6372 determinations of cell fates +T265 UBERON:0000922 6384 6393 embryonic +T266 GO:0009790 6384 6405 embryonic development +T267 UBERON:0000479 6433 6439 tissue +T268 GO:0065007 6449 6459 regulation +T269 PR:000002198 6463 6472 β-catenin +T270 UBERON:0004529 6497 6507 appendages +T271 UBERON:0002370 6520 6526 thymus +T272 NCBITaxon:10088 6575 6579 Mice +T273 PR:000004122 6608 6611 Apc +T274 GO:0043588 6615 6634 development of skin +T275 UBERON:0004529 6643 6653 appendages +T276 SO:0000346 6671 6675 loxP +T277 PR:000004122 6730 6733 Apc +T278 SO:0000704 6734 6738 gene +T279 NCBITaxon:10088 6742 6746 mice +T280 UBERON:0000922 6763 6772 embryonic +T281 CL:0002322 6763 6777;6783 6788 embryonic stem ... cells +T282 CL:0002322 6779 6781;6783 6788 ES ... cells +T283 NCBITaxon:10088 6793 6797 mice +T284 PR:000004122 6810 6813 Apc +T285 SO:0001023 6814 6820 allele +T286 SO:0000346 6846 6856 loxP sites +T287 SO:0000357 6857 6865 flanking +T288 PR:000004122 6866 6869 Apc +T289 SO:0000147 6870 6874 exon +T290 PR:P03870 6892 6895 FLP +T291 SO:0000350 6892 6914;6921 6926 FLP recognition target ... sites +T292 SO:0000350 6916 6919;6921 6926 FRT ... sites +T293 SO:0000357 6927 6935 flanking +T294 CHEBI:7507 6940 6948 neomycin +T295 SO:0005853 6959 6967 cassette +T296 PR:000004122 7006 7009 Apc +T297 SO:0001023 7014 7020 allele +T298 CHEBI:7507 7028 7036 neomycin +T299 SO:0005853 7037 7045 cassette +T300 CHEBI:7507 7054 7062 neomycin +T301 SO:0005853 7063 7071 cassette +T302 PR:000004122 7128 7131 Apc +T303 SO:0000188 7135 7141 intron +T304 SO:0000704 7163 7167 gene +T305 SO:0000346 7173 7177;7186 7191 loxP ... sites +T306 SO:0000350 7182 7191 FRT sites +T307 NCBITaxon:10088 7251 7256 mouse +T308 CL:0002322 7332 7334 ES +T309 PR:000004122 7403 7406 Apc +T310 NCBITaxon:10088 7413 7417 mice +T311 PR:000004122 7461 7464 Apc +T312 NCBITaxon:10088 7470 7474 mice +T313 PR:000004122 7512 7515 Apc +T314 PR:000004122 7529 7532 Apc +T315 SO:0001023 7537 7543 allele +T316 CHEBI:7507 7565 7573 neomycin +T317 SO:0005853 7574 7582 cassette +T318 SO:0000346 7605 7615 loxP sites +T319 SO:0000188 7623 7630 introns +T320 SO:0000357 7631 7639 flanking +T321 SO:0000147 7640 7644 exon +T322 SO:0000147 7682 7686 exon +T323 NCBITaxon:10088 7693 7697 mice +T324 PR:000004122 7713 7716 Apc +T325 NCBITaxon:10088 7722 7726 mice +T326 PR:000004122 7803 7806 Apc +T327 PR:000004122 7819 7822 Apc +T328 SO:0001023 7841 7847 allele +T329 PR:000004122 7849 7852 Apc +T330 SO:0000147 7864 7868 exon +T331 SO:0000673 7889 7899 transcript +T332 SO:0000147 7913 7917 exon +T333 SO:0000717 7954 7967 reading frame +T334 SO:0001587 7984 8017 premature chain termination codon +T335 PR:000004122 8155 8158 Apc +T336 UBERON:0002415 8195 8199 tail +T337 PR:000004122 8230 8233 Apc +T338 PR:000004122 8242 8245 Apc +T339 PR:000004122 8304 8307 Apc +T340 SO:0001023 8308 8314 allele +T341 NCBITaxon:10088 8328 8332 Mice +T342 PR:000004122 8359 8362 Apc +T343 UBERON:0000104 8420 8428 lifespan +T344 SO:0000147 8483 8487 exon +T345 PR:000004122 8549 8552 Apc +T346 SO:0000704 8553 8557 gene +T347 PR:000004122 8567 8570 Apc +T348 NCBITaxon:10088 8577 8581 mice +T349 UBERON:0001052 8657 8663 rectal +T350 http://purl.obolibrary.org/obo/MONDO_0002280 8677 8683 anemia +T351 PR:000004122 8749 8752 Apc +T352 NCBITaxon:10088 8771 8776 mouse +T353 PR:000004122 8790 8793 Apc +T354 NCBITaxon:10088 8800 8804 mice +T355 UBERON:0000160 8842 8852 intestinal +T356 http://purl.obolibrary.org/obo/MONDO_0021118 8842 8859 intestinal tumors +T357 GO:0016265 8881 8886 death +T358 PR:000004122 8926 8929 Apc +T359 http://purl.obolibrary.org/obo/MONDO_0005070 8963 8968 tumor +T360 UBERON:0000160 8997 9007 intestinal +T361 http://purl.obolibrary.org/obo/MONDO_0021118 8997 9014 intestinal tumors +T362 PR:000004122 9020 9023 Apc +T363 NCBITaxon:10088 9030 9034 mice +T364 GO:0006412 9065 9076 translation +T365 PR:000004122 9118 9121 Apc +T366 PR:000004122 9206 9209 Apc +T367 SO:0001023 9229 9236 allelic +T368 SO:0001023 9254 9260 allele +T369 NCBITaxon:10088 9311 9315 mice +T370 PR:000004122 9320 9323 Apc +T371 GO:0001967 9389 9394 nurse +T372 http://purl.obolibrary.org/obo/MONDO_0005070 9427 9432 tumor +T373 PR:000004122 9442 9445 Apc +T374 NCBITaxon:10088 9451 9455 mice +T375 PR:000004122 9486 9489 Apc +T376 PR:000004122 9586 9589 Apc +T377 SO:0001023 9593 9599 allele +T378 NCBITaxon:10088 9607 9611 mice +T379 NCBITaxon:10088 9636 9640 mice +T380 PR:000004122 9645 9648 Apc +T381 SO:0001023 9652 9658 allele +T382 PR:000004122 9776 9779 Apc +T383 SO:0001023 9783 9789 allele +T384 SO:0001023 9819 9825 allele +T385 PR:000004122 9842 9845 Apc +T386 PR:000004122 9865 9868 Apc +T387 NCBITaxon:10088 9880 9885 mouse +T388 PR:000004122 9901 9904 Apc +T389 GO:0007567 9939 9943 born +T390 SO:0000346 10000 10010 loxP sites +T391 SO:0000188 10014 10021 introns +T392 SO:0000357 10022 10030 flanking +T393 SO:0000147 10031 10035 exon +T394 PR:000004122 10085 10088 Apc +T395 SO:0000704 10089 10093 gene +T396 PR:000009454 10096 10099 K14 +T397 PR:000004122 10115 10118 Apc +T398 PR:000004122 10206 10209 Apc +T399 GO:0010467 10221 10231 expressing +T400 PR:000009454 10232 10235 K14 +T401 CL:0002322 10252 10259 ES cell +T402 PR:000004122 10273 10276 Apc +T403 NCBITaxon:10088 10282 10286 mice +T404 PR:000009454 10292 10295 K14 +T405 NCBITaxon:10088 10312 10316 mice +T406 PR:000009454 10345 10348 K14 +T407 PR:000004122 10354 10357 Apc +T408 NCBITaxon:10088 10363 10367 mice +T409 PR:000009454 10412 10415 K14 +T410 PR:000004122 10421 10424 Apc +T411 PR:000004122 10452 10455 Apc +T412 CL:0000023 10512 10519 oocytes +T413 PR:000009454 10523 10526 K14 +T414 NCBITaxon:10088 10558 10562 mice +T415 NCBITaxon:10088 10620 10624 mice +T416 PR:000009454 10759 10762 K14 +T417 SO:0000167 10763 10771 promoter +T418 UBERON:0007376 10791 10800 epidermal +T419 CL:0000362 10791 10805 epidermal cell +T420 SO:0000167 10806 10814 promoter +T421 GO:0010467 10830 10840 expression +T422 GO:0007067 10848 10859 mitotically +T423 CL:0000362 10867 10875;10880 10889 cells of ... epidermis +T424 UBERON:0007376 10880 10889 epidermis +T425 UBERON:0004529 10898 10908 appendages +T426 UBERON:0000924 10963 10981 embryonic ectoderm +T427 UBERON:0000924 11013 11023 ectodermal +T428 UBERON:0000922 11033 11042 embryonic +T429 GO:0010467 11074 11084 expression +T430 PR:000009454 11088 11091 K14 +T431 UBERON:0003846 11109 11126 thymic epithelial +T432 CL:0002293 11109 11132 thymic epithelial cells +T433 CL:0002293 11134 11138 TECs +T434 UBERON:0002124 11147 11157;11165 11171 medulla of ... thymus +T435 PR:000009454 11272 11275 K14 +T436 PR:000004122 11281 11284 Apc +T437 PR:000004122 11294 11297 Apc +T438 NCBITaxon:10088 11305 11309 mice +T439 NCBITaxon:10088 11322 11326 mice +T440 PR:000009454 11343 11346 K14 +T441 PR:000004122 11352 11355 Apc +T442 GO:0007567 11391 11395 born +T443 GO:0007567 11622 11627 birth +T444 GO:0007567 11715 11719 born +T445 PR:000009454 11778 11781 K14 +T446 PR:000004122 11787 11790 Apc +T447 GO:0007567 11845 11849 born +T448 GO:0001967 11907 11913 nursed +T449 UBERON:0001913 11938 11942 milk +T450 UBERON:0000945 11952 11960 stomachs +T451 GO:0007567 11993 11998 birth +T452 GO:0007567 12045 12050 natal +T453 GO:0016265 12190 12194 died +T454 UBERON:0000922 12286 12293 embryos +T455 CHEBI:37958 12310 12313 dye +T456 UBERON:0007376 12338 12347 epidermal +T457 CHEBI:37958 12498 12501 dye +T458 UBERON:0007376 12523 12532 epidermal +T459 UBERON:0000922 12574 12583 embryonic +T460 UBERON:0008200 12743 12752 foreheads +T461 UBERON:0000033 12799 12803 head +T462 UBERON:0002415 12807 12811 tail +T463 UBERON:0001691 12819 12832 external ears +T464 UBERON:0001757 12836 12842 pinnae +T465 CHEBI:26130 12876 12885 pigmented +T466 GO:0007567 13003 13008 birth +T467 GO:0040007 13043 13047 grew +T468 UBERON:0010509 13074 13085 pelage hair +T469 UBERON:0001037 13158 13162 hair +T470 UBERON:0010166 13253 13265 coat of hair +T471 UBERON:0003451 13296 13310 lower incisors +T472 NCBITaxon:33208 13408 13415 Animals +T473 UBERON:0006378 13502 13511 vibrissae +T474 UBERON:0010509 13530 13542 pelage hairs +T475 UBERON:0001690 13630 13634 ears +T476 UBERON:0001711 13636 13643 eyelids +T477 UBERON:0008200 13645 13653 forehead +T478 UBERON:0000004 13655 13659 nose +T479 NCBITaxon:33208 13740 13747 animals +T480 UBERON:0000970 13766 13770 eyes +T481 UBERON:0001091 14070 14075 tooth +T482 UBERON:0001098 14089 14097 incisors +T483 UBERON:0003655 14101 14107 molars +T484 UBERON:0000945 14119 14127 stomachs +T485 CHEBI:33290 14169 14173 food +T486 GO:0007631 14288 14294 ingest +T487 CHEBI:33290 14301 14305 food +T488 UBERON:0001037 14372 14376 hair +T489 GO:0016265 14600 14605 death +T490 GO:0007567 14640 14644 born +T491 GO:0016265 14655 14659 died +T492 PR:000004122 14836 14839 Apc +T493 SO:0000704 14874 14881 genetic +T494 UBERON:0000062 14964 14970 organs +T495 UBERON:0002370 15001 15006 thymi +T496 UBERON:0002370 15218 15223 thymi +T497 NCBITaxon:10088 15242 15246 mice +T498 UBERON:0000479 15288 15294 tissue +T499 NCBITaxon:10088 15322 15326 mice +T500 UBERON:0004288 15354 15362 skeletal +T501 UBERON:0004288 15390 15398 skeletal +T502 NCBITaxon:10088 15420 15424 mice +T503 CHEBI:16866 15438 15450 Alizarin red +T504 NCBITaxon:10088 15500 15504 mice +T505 UBERON:0001684 15512 15527 mandibular bone +T506 NCBITaxon:10088 15560 15564 mice +T507 UBERON:0003450 15601 15619 maxillary incisors +T508 UBERON:0003666 15601 15610;15624 15630 maxillary ... molars +T509 UBERON:0004288 15671 15679 skeletal +T510 UBERON:0000479 15710 15716 Tissue +T511 GO:0010467 15726 15736 Expression +T512 PR:000004122 15754 15757 Apc +T513 SO:0000673 15758 15769 Transcripts +T514 PR:000009454 15810 15813 K14 +T515 PR:000004122 15882 15885 Apc +T516 PR:000004122 15887 15890 Apc +T517 SO:0001023 15896 15903 alleles +T518 SO:0001026 15905 15912 Genomic +T519 UBERON:0002107 15936 15941 liver +T520 UBERON:0002370 15943 15949 thymus +T521 PR:000009454 15991 15994 K14 +T522 PR:000004122 16000 16003 Apc +T523 PR:000009454 16012 16015 K14 +T524 PR:000004122 16021 16024 Apc +T525 PR:000004122 16031 16034 Apc +T526 PR:000004122 16047 16050 Apc +T527 SO:0001026 16071 16078 genomic +T528 UBERON:0000479 16094 16101 tissues +T529 PR:000004122 16118 16121 Apc +T530 SO:0001023 16126 16132 allele +T531 SO:0000028 16138 16140 bp +T532 UBERON:0002370 16186 16192 thymus +T533 PR:000009454 16200 16203 K14 +T534 NCBITaxon:10088 16217 16221 mice +T535 PR:000004122 16246 16249 Apc +T536 SO:0001023 16250 16256 allele +T537 UBERON:0002370 16264 16270 thymus +T538 PR:000009454 16274 16277 K14 +T539 PR:000004122 16283 16286 Apc +T540 NCBITaxon:10088 16292 16296 mice +T541 NCBITaxon:33208 16363 16369 animal +T542 UBERON:0000479 16379 16386 tissues +T543 UBERON:0002107 16472 16477 liver +T544 PR:000009454 16481 16484 K14 +T545 PR:000009454 16515 16518 K14 +T546 NCBITaxon:10088 16532 16537 mouse +T547 UBERON:0000479 16538 16545 tissues +T548 UBERON:0000479 16623 16629 tissue +T549 NCBITaxon:10088 16653 16657 mice +T550 PR:000009454 16673 16676 K14 +T551 PR:000004122 16695 16698 Apc +T552 SO:0000673 16699 16710 transcripts +T553 SO:0000112 16745 16752 primers +T554 SO:0000147 16762 16766 exon +T555 UBERON:0000479 16830 16836 tissue +T556 SO:0000006 16874 16885 PCR product +T557 SO:0000028 16891 16893 bp +T558 PR:000004122 16914 16917 Apc +T559 PR:000004122 16919 16922 Apc +T560 SO:0001023 16928 16934 allele +T561 UBERON:0000479 16947 16954 tissues +T562 GO:0010467 16992 17001 expressed +T563 PR:000009454 17009 17012 K14 +T564 NCBITaxon:10088 17026 17030 mice +T565 PR:000009454 17085 17088 K14 +T566 NCBITaxon:10088 17102 17107 mouse +T567 UBERON:0000479 17108 17115 tissues +T568 UBERON:0002107 17131 17136 liver +T569 PR:000009454 17140 17143 K14 +T570 NCBITaxon:10088 17157 17161 mice +T571 SO:0001023 17203 17209 allele +T572 SO:0000028 17215 17217 bp +T573 UBERON:0000479 17330 17336 tissue +T574 PR:000009454 17381 17384 K14 +T575 PR:000004122 17396 17399 Apc +T576 UBERON:0002073 17422 17436 Hair Follicles +T577 UBERON:0007376 17452 17461 Epidermis +T578 UBERON:0001037 17512 17516 hair +T579 UBERON:0002073 17628 17641 hair follicle +T580 UBERON:0007376 17648 17657 epidermal +T581 UBERON:0004529 17658 17667 appendage +T582 UBERON:0001037 17759 17763 hair +T583 UBERON:0005942 17777 17794 outer root sheath +T584 UBERON:0005942 17796 17799 ORS +T585 UBERON:0002025 17853 17867;17872 17881 basal layer of ... epidermis +T586 UBERON:0002073 17907 17920 hair follicle +T587 UBERON:0005941 17956 17973 inner root sheath +T588 UBERON:0001037 18005 18009 hair +T589 UBERON:0002073 18046 18059 hair follicle +T590 UBERON:0005932 18079 18097 hair follicle bulb +T591 GO:0008283 18122 18135 proliferating +T592 UBERON:0005941 18207 18224 inner root sheath +T593 UBERON:0002074 18233 18243 hair shaft +T594 GO:0042640 18268 18283;18288 18298 anagen phase of ... hair cycle +T595 UBERON:0001037 18288 18292 hair +T596 UBERON:0002073 18312 18326 hair follicles +T597 NCBITaxon:10088 18352 18356 mice +T598 GO:0040007 18357 18361 grew +T599 UBERON:0002190 18378 18394 subcutaneous fat +T600 NCBITaxon:10088 18689 18693 mice +T601 UBERON:0010166 18714 18718 coat +T602 UBERON:0007376 18971 18980 epidermis +T603 UBERON:0002073 19080 19094 hair follicles +T604 UBERON:0010166 19299 19303 coat +T605 NCBITaxon:10088 19320 19324 mice +T606 PR:000004122 19327 19330 Apc +T607 PR:000002198 19349 19358 β-catenin +T608 GO:0016055 19381 19394 Wnt signaling +T609 GO:0010467 19424 19434 expression +T610 PR:000002198 19438 19447 β-catenin +T611 UBERON:0000479 19464 19471 tissues +T612 PR:000002198 19493 19502 β-catenin +T613 GO:0005912 19520 19545 adherens junction complex +T614 UBERON:0005942 19564 19585 ORS of hair follicles +T615 UBERON:0002025 19590 19614 basal layer of epidermis +T616 PR:000009454 19622 19625 K14 +T617 GO:0010467 19626 19636 expression +T618 GO:0010467 19686 19696 expression +T619 PR:000009450 19700 19702 K1 +T620 PR:000009167 19704 19714 involucrin +T621 PR:000009882 19720 19728 loricrin +T622 UBERON:0002026 19742 19749;19763 19782 spinous ... layers of epidermis +T623 UBERON:0002069 19754 19782 granular layers of epidermis +T624 UBERON:0007376 19818 19827 epidermis +T625 GO:0010467 19864 19874 expression +T626 PR:000009454 19878 19881 K14 +T627 PR:000009450 19883 19885 K1 +T628 PR:000009167 19887 19897 involucrin +T629 PR:000009882 19903 19911 loricrin +T630 NCBITaxon:10088 19955 19959 mice +T631 GO:0010467 20086 20096 expression +T632 GO:0010467 20127 20136 expressed +T633 UBERON:0005942 20177 20183;20188 20201 ORS of ... hair follicle +T634 UBERON:0007376 20217 20226 epidermis +T635 UBERON:0002073 20306 20320 hair follicles +T636 UBERON:0005942 20490 20493 ORS +T637 PR:000009454 20542 20545 K14 +T638 PR:000009481 20549 20551 K5 +T639 GO:0008283 20589 20602 proliferating +T640 UBERON:0002025 20637 20661 basal layer of epidermis +T641 UBERON:0005932 20680 20699 hair follicle bulbs +T642 CHEBI:472552 20752 20756 BrdU +T643 PR:000010425 20774 20778 Ki67 +T644 GO:0010467 20779 20789 expression +T645 UBERON:0002073 20849 20862 hair follicle +T646 UBERON:0005942 20927 20933;20947 20961 ORS of ... hair follicles +T647 UBERON:0005932 21020 21038 hair follicle bulb +T648 GO:0008283 21050 21063 proliferating +T649 GO:0008283 21129 21142 proliferation +T650 UBERON:0005932 21254 21273 hair follicle bulbs +T651 GO:0016020 21423 21431 membrane +T652 GO:0005829 21492 21501 cytosolic +T653 PR:000002198 21502 21511 β-catenin +T654 PR:000002198 21591 21600 β-catenin +T655 GO:0010467 21601 21611 expressing +T656 GO:0008283 21645 21658 proliferating +T657 GO:0005829 21777 21786 cytosolic +T658 PR:000002198 21787 21796 β-catenin +T659 PR:000009454 21836 21839 K14 +T660 PR:000009450 21849 21851 K1 +T661 UBERON:0002025 21861 21876 basal epidermis +T662 PR:000009454 21880 21883 K14 +T663 CL:0000646 21905 21910;21915 21920 basal ... cells +T664 UBERON:0005942 21911 21914 ORS +T665 CL:0002561 21911 21920 ORS cells +T666 GO:0008283 21944 21957 proliferating +T667 UBERON:0002073 21997 22010 hair follicle +T668 GO:0031069 21997 22024 hair follicle morphogenesis +T669 GO:0010467 22059 22069 expression +T670 PR:000014841 22081 22095 Sonic hedgehog +T671 PR:000014841 22097 22100 Shh +T672 GO:0010467 22112 22121 expressed +T673 UBERON:0005932 22125 22135 hair bulbs +T674 UBERON:0000922 22139 22148 embryonic +T675 UBERON:0002073 22179 22192 hair follicle +T676 GO:0031069 22179 22206 hair follicle morphogenesis +T677 UBERON:0000922 22246 22255 embryonic +T678 GO:0010467 22285 22295 expression +T679 PR:000014841 22299 22302 Shh +T680 UBERON:0007376 22318 22327 epidermis +T681 UBERON:0000922 22365 22372 embryos +T682 NCBITaxon:10088 22452 22457 mouse +T683 UBERON:0002073 22458 22472 hair follicles +T684 UBERON:0000922 22552 22559 embryos +T685 PR:000014841 22689 22692 Shh +T686 GO:0010467 22693 22703 expression +T687 PR:000014841 22748 22751 Shh +T688 UBERON:0005086 22860 22873 hair placodes +T689 UBERON:0002073 22887 22900 hair follicle +T690 GO:0031069 22887 22914 hair follicle morphogenesis +T691 GO:0097617 22959 22972 hybridization +T692 UBERON:0000922 22995 23002 embryos +T693 PR:000002198 23007 23016 β-catenin +T694 GO:0010467 23042 23052 expression +T695 PR:000002198 23064 23073 β-catenin +T696 UBERON:0000922 23077 23084 embryos +T697 UBERON:0005086 23141 23154 hair placodes +T698 UBERON:0000922 23169 23178 embryonic +T699 UBERON:0005086 23280 23293 hair placodes +T700 UBERON:0000922 23319 23328 embryonic +T701 UBERON:0005086 23357 23370 hair placodes +T702 UBERON:0002101 23420 23425 limbs +T703 UBERON:0000922 23485 23492 embryos +T704 UBERON:0013623 23559 23567 footpads +T705 UBERON:0005086 23575 23588 hair placodes +T706 UBERON:0005086 23674 23687 hair placodes +T707 UBERON:0013623 23709 23717 footpads +T708 UBERON:0005086 23751 23764 hair placodes +T709 PR:000004122 23787 23790 Apc +T710 SO:0000704 23791 23795 gene +T711 UBERON:0000922 23942 23951 embryonic +T712 GO:0048598 23942 23951;23966 23979 embryonic ... morphogenesis +T713 UBERON:0002073 23952 23965 hair follicle +T714 GO:0031069 23952 23979 hair follicle morphogenesis +T715 UBERON:0002073 24039 24052 hair follicle +T716 GO:0031069 24039 24066 hair follicle morphogenesis +T717 GO:0007567 24074 24079 natal +T718 PR:000009454 24105 24108 K14 +T719 PR:000004122 24128 24131 Apc +T720 UBERON:0007376 24141 24150 Epidermal +T721 UBERON:0004529 24151 24161 Appendages +T722 UBERON:0002073 24189 24203 hair follicles +T723 PR:000009454 24205 24208 K14 +T724 PR:000004122 24228 24231 Apc +T725 UBERON:0007376 24271 24280 epidermal +T726 UBERON:0004529 24281 24291 appendages +T727 UBERON:0000483 24307 24317 epithelial +T728 UBERON:0003104 24318 24329 mesenchymal +T729 UBERON:0001091 24395 24401 dental +T730 UBERON:0001091 24428 24433 Tooth +T731 UBERON:0000924 24507 24519 ectodermally +T732 UBERON:0002424 24528 24543 oral epithelium +T733 UBERON:0007213 24564 24603 cranial neural crest–derived mesenchyme +T734 UBERON:0001091 24618 24623 tooth +T735 UBERON:0001091 24650 24655 tooth +T736 UBERON:0000167 24716 24729 oral cavities +T737 UBERON:0008281 24765 24775 tooth buds +T738 UBERON:0001091 24809 24814 teeth +T739 GO:0040007 24835 24839 grow +T740 UBERON:0001913 24879 24883 milk +T741 CHEBI:33290 24893 24897 food +T742 GO:0010467 24916 24926 expression +T743 PR:000009454 24939 24942 K14 +T744 PR:000002198 24947 24956 β-catenin +T745 GO:0016020 24985 24993 membrane +T746 GO:0010467 25000 25010 expression +T747 PR:000002198 25014 25023 β-catenin +T748 PR:000009454 25040 25043 K14 +T749 GO:0010467 25044 25054 expressing +T750 UBERON:0002424 25055 25070 oral epithelium +T751 CL:0000059 25075 25086 ameloblasts +T752 NCBITaxon:10088 25097 25101 mice +T753 PR:000009454 25127 25130 K14 +T754 GO:0010467 25131 25141 expressing +T755 GO:0005829 25167 25176 cytosolic +T756 GO:0005634 25177 25184 nuclear +T757 PR:000002198 25185 25194 β-catenin +T758 UBERON:0008281 25284 25294 tooth buds +T759 NCBITaxon:10088 25309 25313 mice +T760 PR:000014841 25352 25355 Shh +T761 GO:0010467 25356 25366 expression +T762 UBERON:0007115 25383 25396 primary teeth +T763 PR:000004122 25424 25427 Apc +T764 UBERON:0006914 25457 25475 squamous epithelia +T765 UBERON:0001772 25466 25485 epithelia of cornea +T766 UBERON:0004809 25466 25478;25493 25501;25517 25523 epithelia of ... salivary ... glands +T767 UBERON:0004694 25466 25478;25507 25523 epithelia of ... Hardarian glands +T768 UBERON:0010047 25487 25491;25517 25523 oral ... glands +T769 UBERON:0002073 25567 25580 hair follicle +T770 UBERON:0002073 25606 25619 hair follicle +T771 GO:0031069 25606 25633 hair follicle morphogenesis +T772 UBERON:0000483 25661 25670 epithelia +T773 PR:000009454 25673 25676 K14 +T774 PR:000004122 25688 25691 Apc +T775 UBERON:0002370 25721 25727 thymic +T776 NCBITaxon:10088 25728 25732 Mice +T777 UBERON:0002370 25734 25740 Thymus +T778 UBERON:0000062 25747 25752 organ +T779 PR:000009454 25780 25783 K14 +T780 GO:0010467 25784 25794 expression +T781 UBERON:0000062 25836 25841 organ +T782 CL:0000893 25846 25855 thymocyte +T783 CL:0002364 25906 25920 TECs of cortex +T784 CL:0002365 25906 25913;25925 25932 TECs of ... medulla +T785 UBERON:0001851 25914 25920 cortex +T786 UBERON:0000958 25925 25932 medulla +T787 UBERON:0001851 25976 25984 Cortical +T788 CL:0002364 25976 25984;25999 26002 Cortical ... TEC +T789 UBERON:0000958 25989 25998 medullary +T790 CL:0002365 25989 26002 medullary TEC +T791 GO:0010467 26045 26055 expression +T792 PR:000009495 26081 26083 K8 +T793 PR:000009458 26085 26088 K18 +T794 PR:000009481 26090 26092 K5 +T795 PR:000009454 26098 26101 K14 +T796 UBERON:0002370 26115 26121 thymus +T797 UBERON:0000062 26146 26151 organ +T798 UBERON:0009911 26158 26164 lobule +T799 CL:0002293 26198 26201 TEC +T800 UBERON:0001851 26225 26231 cortex +T801 UBERON:0000958 26245 26252 medulla +T802 UBERON:0002370 26317 26323 thymus +T803 CHEBI:51686 26405 26406 H +T804 UBERON:0002370 26421 26427 thymus +T805 UBERON:0001851 26433 26439 cortex +T806 UBERON:0001744 26460 26475 lymphoid tissue +T807 UBERON:0003891 26518 26524 stroma +T808 UBERON:0000958 26532 26539 medulla +T809 CL:0000542 26573 26584 lymphocytes +T810 UBERON:0001851 26594 26600 cortex +T811 UBERON:0000958 26606 26613 medulla +T812 UBERON:0001851 26644 26650 cortex +T813 NCBITaxon:10088 26662 26666 mice +T814 UBERON:0002370 26672 26678 thymus +T815 UBERON:0007023 26712 26717 adult +T816 GO:0060033 26726 26735 regresses +T817 NCBITaxon:10088 26779 26783 mice +T818 CL:0000893 26825 26835 thymocytes +T819 GO:0007067 26841 26852 mitotically +T820 UBERON:0001851 26867 26873 cortex +T821 CHEBI:472552 26891 26895 BrdU +T822 UBERON:0002370 26955 26961 thymus +T823 NCBITaxon:10088 26977 26981 mice +T824 PR:000009454 27020 27023 K14 +T825 GO:0010467 27036 27046 expression +T826 CL:0002293 27087 27091 TECs +T827 UBERON:0000958 27105 27114 medullary +T828 CL:0000312 27133 27146 keratinocytes +T829 UBERON:0003987 27150 27170 Hassall's corpuscles +T830 GO:0005737 27192 27203 cytoplasmic +T831 PR:000002198 27217 27226 β-catenin +T832 UBERON:0000958 27252 27261 medullary +T833 UBERON:0000483 27262 27272 epithelial +T834 CL:0000066 27262 27278 epithelial cells +T835 PR:000009454 27307 27310 K14 +T836 GO:0010467 27311 27321 expression +T837 PR:000009495 27344 27346 K8 +T838 UBERON:0000483 27363 27373 epithelial +T839 CL:0000066 27363 27379 epithelial cells +T840 UBERON:0000958 27392 27399 medulla +T841 UBERON:0001851 27404 27410 cortex +T842 PR:000009450 27424 27426 K1 +T843 NCBITaxon:10088 27462 27466 mice +T844 NCBITaxon:10088 27486 27490 mice +T845 CL:0000312 27525 27538 keratinocytes +T846 UBERON:0003987 27550 27570 Hassall's corpuscles +T847 UBERON:0002370 27619 27625 thymus +T848 UBERON:0002370 27692 27698 thymus +T849 UBERON:0009911 27715 27722 lobules +T850 NCBITaxon:10088 27740 27744 mice +T851 UBERON:0002370 27760 27766 thymus +T852 UBERON:0002370 28004 28010 thymus +T853 UBERON:0002370 28146 28152 thymus +T854 UBERON:0002370 28274 28280 thymus +T855 UBERON:0000483 28379 28389 epithelial +T856 UBERON:0002370 28406 28412 thymus +T857 CL:0000893 28477 28487 thymocytes +T858 UBERON:0001851 28503 28509 cortex +T859 CHEBI:51686 28577 28578 H +T860 UBERON:0001851 28618 28624 cortex +T861 UBERON:0000958 28639 28646 medulla +T862 GO:0005634 28691 28698 nuclear +T863 PR:000002198 28699 28708 β-catenin +T864 UBERON:0000958 28742 28751 medullary +T865 PR:000002198 28773 28782 β-catenin +T866 PR:000009454 28860 28863 K14 +T867 UBERON:0000958 28898 28905 medulla +T868 PR:000009495 28931 28933 K8 +T869 UBERON:0002370 28984 28990 thymus +T870 UBERON:0003846 29004 29021 thymic epithelial +T871 CL:0000542 29077 29088 lymphocytes +T872 CL:0000893 29205 29215 thymocytes +T873 UBERON:0002370 29248 29254 thymus +T874 UBERON:0000483 29264 29274 epithelial +T875 CL:0000066 29264 29280 epithelial cells +T876 UBERON:0002370 29373 29378 thymi +T877 UBERON:0002370 29401 29407 thymus +T878 PR:000009454 29425 29428 K14 +T879 GO:0010467 29429 29439 expression +T880 PR:000009495 29461 29463 K8 +T881 GO:0010467 29464 29474 expression +T882 CL:0000646 29515 29526 basal cells +T883 CL:0002293 29544 29548 TECs +T884 GO:0005634 29627 29634 nuclear +T885 GO:0005737 29639 29650 cytoplasmic +T886 PR:000002198 29651 29660 β-catenin +T887 GO:0005634 29687 29694 nuclear +T888 PR:000002198 29707 29716 β-catenin +T889 UBERON:0002370 29760 29766 thymus +T890 PR:000009454 29798 29801 K14 +T891 PR:000002198 29806 29815 β-catenin +T892 UBERON:0002370 29902 29908 thymus +T893 UBERON:0003987 29940 29959 Hassall's corpuscle +T894 PR:000009454 29995 29998 K14 +T895 PR:000009495 30004 30006 K8 +T896 GO:0010467 30007 30017 expressing +T897 GO:0031424 30018 30030 keratinizing +T898 CL:0000311 30018 30030;30042 30047 keratinizing ... cells +T899 UBERON:0000483 30031 30041 epithelial +T900 CL:0000066 30031 30047 epithelial cells +T901 CL:0000775 30123 30134 neutrophils +T902 CL:0000235 30139 30150 macrophages +T903 UBERON:0002370 30168 30174 thymus +T904 NCBITaxon:10088 30336 30340 mice +T905 PR:000009450 30355 30357 K1 +T906 PR:000009167 30362 30372 involucrin +T907 UBERON:0003987 30407 30427 Hassall's corpuscles +T908 UBERON:0002370 30458 30463 thymi +T909 NCBITaxon:10088 30486 30490 mice +T910 CL:0000893 30494 30504 thymocytes +T911 CHEBI:472552 30522 30526 BrdU +T912 GO:0031424 30571 30583 keratinizing +T913 CL:0000311 30571 30583;30595 30600 keratinizing ... cells +T914 UBERON:0000483 30584 30594 epithelial +T915 CL:0000066 30584 30600 epithelial cells +T916 GO:0010467 30682 30692 expression +T917 PR:000002198 30696 30705 β-catenin +T918 UBERON:0000483 30732 30742 epithelial +T919 CL:0000066 30732 30748 epithelial cells +T920 GO:0005634 30796 30803 nuclear +T921 PR:000002198 30804 30813 β-catenin +T922 NCBITaxon:10088 30861 30865 mice +T923 GO:0005634 30876 30883 nuclear +T924 PR:000002198 30900 30909 β-catenin +T925 PR:000009454 30931 30934 K14 +T926 CL:0000646 30984 30995 basal cells +T927 NCBITaxon:10088 31010 31014 mice +T928 GO:0010467 31047 31057 expression +T929 UBERON:0002370 31080 31086 thymus +T930 PR:000002198 31139 31148 β-catenin +T931 GO:0010467 31149 31159 expression +T932 PR:000009495 31220 31222 K8 +T933 PR:000009454 31223 31226 K14 +T934 GO:0010467 31227 31237 expression +T935 UBERON:0002370 31317 31323 thymus +T936 PR:000004122 31375 31378 Apc +T937 PR:000002198 31411 31420 β-catenin +T938 PR:000009454 31424 31427 K14 +T939 GO:0010467 31428 31438 expressing +T940 CL:0002293 31439 31443 TECs +T941 GO:0008283 31467 31480 proliferation +T942 GO:0030216 31485 31517 differentiation to keratinocytes +T943 CL:0000312 31504 31517 keratinocytes +T944 UBERON:0000958 31583 31592 medullary +T945 CL:0002365 31583 31592;31605 31609 medullary ... TECs +T946 UBERON:0001851 31596 31604 cortical +T947 CL:0002364 31596 31609 cortical TECs +T948 CL:0002293 31626 31629 TEC +T949 CL:0000893 31676 31686 thymocytes +T950 NCBITaxon:10088 31710 31714 mice +T951 UBERON:0002370 31723 31729 thymic +T952 PR:000004122 31745 31748 Apc +T953 GO:0016055 31770 31791 Wnt signaling pathway +T954 NCBITaxon:9606 31848 31853 Human +T955 PR:000004122 31876 31879 APC +T956 http://purl.obolibrary.org/obo/MONDO_0021055 31886 31889 FAP +T957 http://purl.obolibrary.org/obo/MONDO_0004972 31935 31946 adenomatous +T958 UBERON:0012652 31947 31957 colorectal +T959 http://purl.obolibrary.org/obo/MONDO_0021392 31947 31964 colorectal polyps +T960 UBERON:0012652 32007 32017 colorectal +T961 http://purl.obolibrary.org/obo/MONDO_0005575 32007 32024 colorectal cancer +T962 UBERON:0000104 32060 32064 life +T963 http://purl.obolibrary.org/obo/MONDO_0021055 32095 32098 FAP +T964 http://purl.obolibrary.org/obo/MONDO_0019336 32116 32134 Gardner's syndrome +T965 UBERON:0012652 32169 32179 colorectal +T966 http://purl.obolibrary.org/obo/MONDO_0021392 32169 32186 colorectal polyps +T967 NCBITaxon:1 32194 32205 individuals +T968 UBERON:0001155 32223 32230 colonic +T969 UBERON:0004908 32257 32285 upper gastrointestinal tract +T970 http://purl.obolibrary.org/obo/MONDO_0024292 32263 32292 gastrointestinal tract polyps +T971 http://purl.obolibrary.org/obo/MONDO_0021140 32294 32304 congenital +T972 UBERON:0001782 32324 32350 retinal pigment epithelium +T973 CHEBI:26130 32332 32339 pigment +T974 http://purl.obolibrary.org/obo/MONDO_0007608 32352 32366 desmoid tumors +T975 http://purl.obolibrary.org/obo/MONDO_0005381 32368 32380;32408 32413 disorders of bones +T976 UBERON:0002397 32385 32394 maxillary +T977 UBERON:0004288 32399 32407 skeletal +T978 UBERON:0001474 32408 32413 bones +T979 UBERON:0001091 32419 32425 dental +T980 NCBITaxon:10088 32477 32481 mice +T981 PR:000004122 32486 32489 Apc +T982 http://purl.obolibrary.org/obo/MONDO_0004972 32498 32509 adenomatous +T983 http://purl.obolibrary.org/obo/MONDO_0005079 32510 32516 polyps +T984 UBERON:0002108 32534 32549 small intestine +T985 UBERON:0000922 32566 32573 embryos +T986 GO:0016265 32574 32577 die +T987 GO:0007369 32585 32597 gastrulation +T988 PR:000004122 32641 32644 Apc +T989 UBERON:0000479 32653 32660 tissues +T990 UBERON:0001555 32672 32694 gastrointestinal tract +T991 UBERON:0000104 32702 32706 life +T992 NCBITaxon:33208 32710 32717 animals +T993 UBERON:0000922 32740 32749 embryonic +T994 PR:000004122 32776 32779 Apc +T995 NCBITaxon:10088 32808 32813 mouse +T996 SO:0001023 32844 32850 allele +T997 PR:000004122 32854 32857 Apc +T998 PR:000004122 32859 32862 Apc +T999 SO:0000147 32876 32880 exon +T1000 PR:000004122 32891 32894 Apc +T1001 SO:0000357 32898 32905 flanked +T1002 SO:0000346 32909 32923 loxP sequences +T1003 NCBITaxon:10088 32940 32944 mice +T1004 SO:0001023 32965 32971 allele +T1005 NCBITaxon:10088 33019 33023 mice +T1006 PR:000004122 33059 33062 Apc +T1007 UBERON:0000479 33068 33074 tissue +T1008 PR:000004122 33168 33171 Apc +T1009 SO:0000704 33172 33176 gene +T1010 GO:0010467 33191 33198 express +T1011 PR:000009454 33199 33202 K14 +T1012 NCBITaxon:10088 33215 33219 mice +T1013 PR:000004122 33248 33251 Apc +T1014 SO:0001023 33255 33261 allele +T1015 PR:000009454 33276 33279 K14 +T1016 SO:0000902 33284 33293 transgene +T1017 NCBITaxon:10088 33301 33305 mice +T1018 GO:0016265 33327 33331 died +T1019 UBERON:0001037 33368 33372 hair +T1020 UBERON:0001091 33374 33379 tooth +T1021 UBERON:0002370 33385 33391 thymus +T1022 PR:000004122 33405 33408 Apc +T1023 UBERON:0002073 33413 33426 Hair Follicle +T1024 GO:0031069 33413 33440 Hair Follicle Morphogenesis +T1025 UBERON:0002073 33460 33473 hair follicle +T1026 GO:0001942 33460 33485 hair follicle development +T1027 UBERON:0000483 33563 33573 epithelium +T1028 CHEBI:36357 33662 33671 molecules +T1029 GO:0016055 33769 33790 Wnt signaling pathway +T1030 UBERON:0002067 33798 33804 dermis +T1031 UBERON:0000483 33889 33899 epithelial +T1032 PR:000002198 33900 33909 β-catenin +T1033 GO:0010467 33924 33934 expression +T1034 PR:000002198 33995 34004 β-catenin +T1035 GO:0010467 34016 34026 expression +T1036 PR:000009751 34030 34034 Lef1 +T1037 NCBITaxon:10088 34070 34075 mouse +T1038 PR:000002198 34131 34140 β-catenin +T1039 PR:000009751 34160 34164 Lef1 +T1040 GO:0010467 34184 34194 expression +T1041 CHEBI:35222 34202 34211 inhibitor +T1042 PR:000006499 34212 34216 Dkk1 +T1043 PR:000002198 34244 34253 β-catenin +T1044 UBERON:0007376 34257 34266 epidermis +T1045 UBERON:0010419 34287 34296 vibrissae +T1046 UBERON:0010509 34306 34312 pelage +T1047 NCBITaxon:10088 34326 34330 mice +T1048 PR:000009454 34357 34360 K14 +T1049 PR:000004122 34368 34371 Apc +T1050 UBERON:0000924 34380 34398 embryonic ectoderm +T1051 UBERON:0005086 34467 34479 hair placode +T1052 GO:0009653 34504 34517 morphogenesis +T1053 UBERON:0008281 34589 34599 tooth buds +T1054 UBERON:0000483 34670 34680 epithelial +T1055 PR:000002198 34681 34690 β-catenin +T1056 PR:000004122 34750 34753 Apc +T1057 GO:0048866 34757 34773;34795 34805 specification of ... stem cells +T1058 GO:0001715 34757 34773;34784 34794;34800 34805 specification of ... ectodermal ... cells +T1059 UBERON:0000924 34774 34794 embryonic ectodermal +T1060 CL:0002322 34774 34783;34795 34805 embryonic ... stem cells +T1061 CL:0000221 34784 34794;34800 34805 ectodermal ... cells +T1062 UBERON:0002073 34819 34832 hair follicle +T1063 PR:000004122 34852 34855 Apc +T1064 PR:000002198 34878 34887 β-catenin +T1065 PR:000004122 34897 34900 Apc +T1066 GO:0010467 34934 34944 expression +T1067 PR:000002198 34948 34957 β-catenin +T1068 UBERON:0000924 34961 34971 ectodermal +T1069 CL:0000221 34961 34977 ectodermal cells +T1070 UBERON:0005086 34989 35001 hair placode +T1071 GO:0060789 34989 35011 hair placode formation +T1072 UBERON:0000922 35070 35079 embryonic +T1073 UBERON:0007376 35080 35089 epidermis +T1074 UBERON:0013623 35105 35113 footpads +T1075 UBERON:0001037 35130 35134 hair +T1076 NCBITaxon:10088 35250 35254 mice +T1077 PR:000009454 35284 35287 K14 +T1078 NCBITaxon:10088 35308 35312 mice +T1079 GO:0010467 35313 35323 expressing +T1080 PR:000002198 35331 35340 β-catenin +T1081 GO:0065007 35351 35358 control +T1082 PR:000009454 35362 35365 K14 +T1083 SO:0000167 35366 35374 promoter +T1084 PR:000009454 35384 35387 K14 +T1085 PR:000009751 35388 35392 Lef1 +T1086 NCBITaxon:10088 35404 35408 mice +T1087 NCBITaxon:10088 35440 35444 mice +T1088 PR:000009454 35479 35482 K14 +T1089 PR:000009751 35483 35487 Lef1 +T1090 NCBITaxon:10088 35488 35492 mice +T1091 PR:000009454 35502 35505 K14 +T1092 NCBITaxon:10088 35526 35530 mice +T1093 NCBITaxon:10088 35559 35563 mice +T1094 PR:000009454 35624 35627 K14 +T1095 PR:000009454 35643 35646 K14 +T1096 NCBITaxon:10088 35667 35671 mice +T1097 NCBITaxon:10088 35696 35700 mice +T1098 UBERON:0010166 35726 35735 hair coat +T1099 UBERON:0001037 35777 35782 hairs +T1100 PR:000009454 35812 35815 K14 +T1101 PR:000009751 35816 35820 Lef1 +T1102 NCBITaxon:10088 35821 35825 mice +T1103 PR:000009454 35838 35841 K14 +T1104 NCBITaxon:10088 35851 35855 mice +T1105 UBERON:0000922 35857 35866 embryonic +T1106 GO:0048598 35857 35866;35881 35894 embryonic ... morphogenesis +T1107 UBERON:0002073 35867 35880 hair follicle +T1108 GO:0031069 35867 35894 hair follicle morphogenesis +T1109 GO:0007567 35950 35957 natally +T1110 PR:000009454 36081 36084 K14 +T1111 SO:0000167 36085 36093 promoter +T1112 GO:0010467 36126 36136 expression +T1113 GO:0010467 36228 36238 expression +T1114 SO:0000167 36242 36250 promoter +T1115 SO:0000902 36251 36260 transgene +T1116 NCBITaxon:10088 36285 36289 mice +T1117 SO:0000366 36351 36367 integration site +T1118 SO:0000902 36375 36384 transgene +T1119 NCBITaxon:10088 36392 36397 mouse +T1120 SO:0001026 36398 36404 genome +T1121 SO:0000366 36423 36446 location of integration +T1122 SO:0000902 36494 36503 transgene +T1123 GO:0009790 36591 36604 embryogenesis +T1124 GO:0010467 36641 36651 expression +T1125 PR:000009454 36659 36662 K14 +T1126 SO:0000902 36672 36681 transgene +T1127 SO:0000167 36708 36716 promoter +T1128 PR:000009454 36783 36786 K14 +T1129 NCBITaxon:10088 36802 36806 mice +T1130 PR:000009454 36835 36838 K14 +T1131 PR:000004122 36870 36873 Apc +T1132 SO:0000704 36874 36878 gene +T1133 UBERON:0002073 36946 36959 hair follicle +T1134 GO:0031069 36946 36972 hair follicle morphgenesis +T1135 GO:0010467 37056 37066 expression +T1136 SO:0000902 37070 37079 transgene +T1137 CL:0002187 37099 37113;37118 37127 basal cells of ... epidermis +T1138 CL:0002561 37105 37113;37136 37157 cells of ... ORS of hair follicles +T1139 UBERON:0007376 37118 37127 epidermis +T1140 UBERON:0005942 37136 37157 ORS of hair follicles +T1141 PR:000009454 37169 37172 K14 +T1142 SO:0000167 37173 37181 promoter +T1143 GO:0010467 37197 37207 expression +T1144 SO:0000902 37211 37220 transgene +T1145 PR:000009454 37255 37258 K14 +T1146 GO:0010467 37259 37269 expressing +T1147 PR:000009454 37329 37332 K14 +T1148 PR:000004122 37358 37361 Apc +T1149 PR:000004122 37378 37381 Apc +T1150 PR:000009454 37403 37406 K14 +T1151 SO:0000167 37407 37415 promoter +T1152 UBERON:0000119 37451 37462 cell layers +T1153 PR:000009454 37485 37488 K14 +T1154 SO:0000167 37489 37497 promoter +T1155 UBERON:0010402 37548 37568 suprabasal epidermis +T1156 UBERON:0000483 37584 37594 epithelium +T1157 UBERON:0002073 37602 37615 hair follicle +T1158 PR:000002198 37634 37643 β-catenin +T1159 PR:000002198 37771 37780 β-catenin +T1160 GO:0010467 37781 37791 expression +T1161 PR:000009450 37802 37804 K1 +T1162 GO:0007567 37849 37854 natal +T1163 PR:000004122 37911 37914 Apc +T1164 PR:000002198 37946 37955 β-catenin +T1165 PR:000009454 37974 37977 K14 +T1166 GO:0010467 37978 37988 expressing +T1167 PR:000002198 38011 38020 β-catenin +T1168 GO:0010467 38021 38031 expression +T1169 GO:0001709 38048 38071 cell fate determination +T1170 CL:0000646 38095 38106 basal cells +T1171 PR:000004122 38191 38194 Apc +T1172 PR:000002198 38254 38263 β-catenin +T1173 NCBITaxon:10088 38358 38362 mice +T1174 NCBITaxon:10088 38396 38400 mice +T1175 UBERON:0002073 38474 38487 hair follicle +T1176 GO:0031069 38474 38501 hair follicle morphogenesis +T1177 PR:000009454 38503 38506 K14 +T1178 PR:000004122 38522 38525 Apc +T1179 UBERON:0008281 38555 38565 tooth buds +T1180 UBERON:0002073 38577 38591 hair follicles +T1181 UBERON:0000483 38658 38668 epithelium +T1182 UBERON:0003104 38673 38683 mesenchyme +T1183 UBERON:0008281 38729 38739 tooth buds +T1184 NCBITaxon:33208 38749 38756 animals +T1185 GO:0010467 38760 38770 expressing +T1186 PR:000009751 38771 38775 Lef1 +T1187 GO:0007567 38822 38827 birth +T1188 PR:000004122 38854 38857 Apc +T1189 UBERON:0000922 38888 38897 embryonic +T1190 GO:0009790 38888 38897;38904 38915 embryonic ... development +T1191 UBERON:0001091 38898 38903 tooth +T1192 PR:000014841 38947 38950 Shh +T1193 GO:0010467 38951 38961 expression +T1194 UBERON:0000922 38971 38980 embryonic +T1195 UBERON:0002424 38981 38996 oral epithelium +T1196 UBERON:0008281 39059 39069 tooth buds +T1197 UBERON:0001091 39124 39129 teeth +T1198 NCBITaxon:10088 39164 39168 mice +T1199 UBERON:0001091 39178 39183 tooth +T1200 UBERON:0001091 39209 39214 tooth +T1201 NCBITaxon:10088 39248 39252 mice +T1202 NCBITaxon:10088 39297 39301 mice +T1203 GO:0007567 39310 39317 natally +T1204 NCBITaxon:10088 39345 39349 mice +T1205 UBERON:0000062 39427 39433 organs +T1206 NCBITaxon:10088 39440 39444 mice +T1207 CHEBI:33290 39587 39591 food +T1208 UBERON:0000945 39601 39608 stomach +T1209 GO:0016265 39647 39651 died +T1210 UBERON:0001091 39779 39784 teeth +T1211 GO:0001967 39864 39870 nursed +T1212 UBERON:0001091 39915 39920 teeth +T1213 GO:0016265 39949 39954 death +T1214 UBERON:0001091 40010 40015 teeth +T1215 UBERON:0001911 40020 40034 mammary glands +T1216 NCBITaxon:10088 40057 40061 mice +T1217 PR:000009751 40075 40079 Lef1 +T1218 GO:0010467 40096 40106 expressing +T1219 PR:000006499 40107 40111 Dkk1 +T1220 UBERON:0008281 40230 40239 tooth bud +T1221 UBERON:0001091 40279 40284 teeth +T1222 NCBITaxon:10088 40289 40293 mice +T1223 UBERON:0000104 40328 40332 life +T1224 PR:000002198 40392 40401 β-catenin +T1225 UBERON:0001091 40428 40433 tooth +T1226 PR:000004122 40461 40464 Apc +T1227 UBERON:0002370 40469 40475 Thymus +T1228 GO:0048538 40469 40489 Thymus Organogenesis +T1229 PR:000009454 40523 40526 K14 +T1230 PR:000004122 40542 40545 Apc +T1231 UBERON:0002370 40566 40572 thymus +T1232 CL:0000893 40697 40706 thymocyte +T1233 NCBITaxon:10088 40733 40737 mice +T1234 UBERON:0002370 40739 40745 thymic +T1235 UBERON:0007023 40790 40795 adult +T1236 UBERON:0002370 40796 40802 thymus +T1237 PR:000009454 40803 40806 K14 +T1238 GO:0010467 40807 40817 expression +T1239 PR:000009481 40841 40843 K5 +T1240 UBERON:0000958 40860 40869 medullary +T1241 CL:0002365 40860 40874 medullary TECs +T1242 PR:000009481 40904 40906 K5 +T1243 CL:0002293 40908 40912 TECs +T1244 UBERON:0001851 40920 40926 cortex +T1245 UBERON:0001851 40937 40944 cortico +T1246 UBERON:0000958 40945 40954 medullary +T1247 PR:000009454 41008 41011 K14 +T1248 CL:0002293 41013 41017 TECs +T1249 GO:0010467 41027 41034 express +T1250 PR:000009495 41035 41037 K8 +T1251 UBERON:0000958 41069 41078 medullary +T1252 PR:000009495 41097 41099 K8 +T1253 PR:000009454 41100 41103 K14 +T1254 PR:000009495 41127 41129 K8 +T1255 PR:000009454 41130 41133 K14 +T1256 UBERON:0002370 41210 41216 thymus +T1257 PR:000009454 41217 41220 K14 +T1258 GO:0010467 41221 41231 expression +T1259 UBERON:0000958 41259 41268 medullary +T1260 CL:0002365 41259 41273 medullary TECs +T1261 PR:000009495 41295 41297 K8 +T1262 GO:0010467 41298 41308 expression +T1263 CL:0002293 41334 41338 TECs +T1264 GO:0010467 41414 41423 expressed +T1265 CL:0002293 41436 41440 TECs +T1266 UBERON:0002370 41592 41598 thymus +T1267 UBERON:0002370 41685 41691 thymus +T1268 UBERON:0002370 41704 41709 thymi +T1269 PR:000009454 41773 41776 K14 +T1270 UBERON:0002370 41826 41832 thymus +T1271 GO:0005634 41882 41889 nuclear +T1272 PR:000002198 41890 41899 β-catenin +T1273 GO:0010467 41930 41940 expressing +T1274 PR:000009454 41941 41944 K14 +T1275 UBERON:0003846 41960 41977 thymic epithelial +T1276 UBERON:0002370 42025 42031 thymus +T1277 UBERON:0000958 42093 42102 medullary +T1278 CL:0002365 42093 42102;42116 42120 medullary ... TECs +T1279 UBERON:0001851 42107 42115 cortical +T1280 CL:0002364 42107 42120 cortical TECs +T1281 CL:0000893 42125 42134 thymocyte +T1282 PR:000009454 42203 42206 K14 +T1283 PR:000004122 42214 42217 Apc +T1284 GO:0005634 42244 42251 nuclear +T1285 PR:000002198 42252 42261 β-catenin +T1286 PR:000009495 42266 42268 K8 +T1287 PR:000009454 42269 42272 K14 +T1288 UBERON:0000483 42290 42300 epithelial +T1289 CL:0000066 42290 42306 epithelial cells +T1290 GO:0008283 42354 42367 proliferation +T1291 CL:0002293 42409 42412 TEC +T1292 PR:000002198 42437 42446 β-catenin +T1293 GO:0010467 42447 42457 expression +T1294 UBERON:0000483 42496 42506 epithelial +T1295 CL:0000066 42496 42512 epithelial cells +T1296 GO:0005634 42520 42527 nuclear +T1297 PR:000009495 42545 42547 K8 +T1298 PR:000009454 42548 42551 K14 +T1299 UBERON:0000483 42602 42612 epithelial +T1300 CL:0000066 42602 42618 epithelial cells +T1301 CL:0000775 42689 42700 neutrophils +T1302 CL:0000235 42705 42716 macrophages +T1303 UBERON:0000483 42729 42739 epithelial +T1304 CL:0000066 42729 42745 epithelial cells +T1305 CL:0000893 42775 42785 thymocytes +T1306 PR:000004122 42861 42864 Apc +T1307 CL:0002293 42868 42872 TECs +T1308 GO:0005634 42894 42901 nuclear +T1309 PR:000002198 42918 42927 β-catenin +T1310 CL:0000312 42988 43001 keratinocytes +T1311 GO:0010467 43007 43016 expressed +T1312 PR:000009454 43022 43025 K14 +T1313 PR:000009495 43030 43032 K8 +T1314 CL:0000646 43049 43060 basal cells +T1315 CL:0002293 43085 43088 TEC +T1316 CL:0000312 43141 43154 keratinocytes +T1317 UBERON:0003846 43178 43195 thymic epithelial +T1318 CL:0000775 43302 43313 neutrophils +T1319 CL:0000235 43318 43329 macrophages +T1320 UBERON:0002370 43348 43354 thymus +T1321 UBERON:0000483 43371 43381 epithelial +T1322 UBERON:0002370 43436 43442 thymus +T1323 CL:0000893 43472 43482 thymocytes +T1324 PR:000009454 43484 43487 K14 +T1325 PR:000004122 43503 43506 Apc +T1326 GO:0010467 43535 43545 expression +T1327 PR:000002198 43549 43558 β-catenin +T1328 CL:0002293 43562 43566 TECs +T1329 UBERON:0000483 43608 43618 epithelial +T1330 CL:0000066 43608 43623 epithelial cell +T1331 UBERON:0001851 43676 43684 cortical +T1332 CL:0002364 43676 43684;43698 43702 cortical ... TECs +T1333 UBERON:0000958 43688 43697 medullary +T1334 CL:0002365 43688 43702 medullary TECs +T1335 CL:0000893 43734 43743 thymocyte +T1336 CL:0000893 43808 43818 thymocytes +T1337 UBERON:0002370 43833 43839 thymus +T1338 GO:0010467 43877 43887 expression +T1339 CL:0002293 43940 43943 TEC +T1340 PR:000004122 43968 43971 Apc +T1341 UBERON:0002370 43984 43990 thymic +T1342 GO:0048538 43984 44002 thymic development +T1343 CL:0000893 44028 44037 thymocyte +T1344 PR:000004122 44055 44058 Apc +T1345 PR:000004122 44093 44096 Apc +T1346 NCBITaxon:10088 44109 44113 mice +T1347 NCBITaxon:10088 44136 44140 mice +T1348 PR:000009454 44156 44159 K14 +T1349 CL:0002293 44167 44170 TEC +T1350 PR:000004122 44188 44191 Apc +T1351 UBERON:0002370 44232 44238 thymus +T1352 GO:0048538 44232 44250 thymus development +T1353 CL:0000893 44263 44273 thymocytes +T1354 CL:0002293 44286 44290 TECs +T1355 UBERON:0001091 44316 44322 dental +T1356 UBERON:0001091 44373 44378 teeth +T1357 NCBITaxon:10088 44419 44423 mice +T1358 http://purl.obolibrary.org/obo/MONDO_0019336 44462 44480 Gardner's syndrome +T1359 PR:000004122 44494 44497 APC +T1360 UBERON:0001091 44539 44545 odonto +T1361 UBERON:0000165 44546 44560 stomatological +T1362 NCBITaxon:10088 44776 44781 mouse +T1363 PR:000004122 44827 44830 Apc +T1364 UBERON:0000467 44852 44865 organ systems +T1365 NCBITaxon:9606 44927 44932 human +T1366 PR:000004122 44986 44989 Apc +T1367 PR:000009454 44993 44996 K14 +T1368 GO:0010467 44997 45007 expressing +T1369 UBERON:0000922 45008 45017 embryonic +T1370 CL:0002321 45008 45023 embryonic cells +T1371 GO:0009653 45040 45053 morphogenesis +T1372 UBERON:0004529 45070 45080 appendages +T1373 UBERON:0002073 45092 45106 hair follicles +T1374 UBERON:0001091 45111 45116 teeth +T1375 UBERON:0002370 45131 45137 thymus +T1376 GO:0048538 45131 45150 thymus organogensis +T1377 SO:0000704 45172 45179 genetic +T1378 GO:0010467 45194 45204 expression +T1379 PR:000004122 45208 45211 Apc +T1380 GO:0065007 45229 45239 regulation +T1381 UBERON:0000062 45270 45276 organs +T1382 UBERON:0000483 45290 45300 epithelial +T1383 UBERON:0003104 45301 45312 mesenchymal +T1384 SO:0001644 45367 45384 targeting vectors +T1385 PR:000004122 45401 45404 Apc +T1386 SO:0000153 45424 45427 BAC +T1387 PR:000004122 45480 45483 Apc +T1388 SO:0000204 45499 45505 5′ UTR +T1389 SO:0000147 45510 45514 exon +T1390 SO:0000153 45614 45617 BAC +T1391 SO:0001026 45664 45671 genomic +T1392 PR:000004122 45694 45697 Apc +T1393 SO:0000147 45698 45703 exons +T1394 SO:0000440 45745 45751 vector +T1395 SO:0000346 45764 45773 loxP site +T1396 SO:0000188 45794 45800 intron +T1397 SO:0000346 45816 45820 loxP +T1398 SO:0000350 45821 45824 FRT +T1399 SO:0000350 45833 45836 FRT +T1400 SO:0005853 45847 45855 cassette +T1401 PR:000004122 45877 45880 Apc +T1402 SO:0000704 45881 45885 gene +T1403 GO:0048468 45900 45913;45924 45929 Generation of ... cells +T1404 PR:000004122 45914 45917 Apc +T1405 CL:0002322 45921 45929 ES cells +T1406 NCBITaxon:10088 45934 45938 mice +T1407 SO:0001644 45956 45973 targeting vectors +T1408 CL:0002322 46013 46021 ES cells +T1409 CHEBI:42768 46053 46057 G418 +T1410 CL:0002322 46068 46070 ES +T1411 SO:0000704 46106 46110 gene +T1412 SO:0000346 46280 46289 loxP site +T1413 SO:0005853 46308 46316 cassette +T1414 CL:0002322 46322 46324 ES +T1415 PR:000004122 46345 46348 Apc +T1416 UBERON:0000358 46396 46407 blastocysts +T1417 NCBITaxon:10088 46430 46434 mice +T1418 CL:0002322 46455 46462 ES cell +T1419 SO:0001026 46555 46562 genomic +T1420 UBERON:0002415 46593 46597 tail +T1421 NCBITaxon:10088 46753 46757 mice +T1422 NCBITaxon:10088 46774 46778 mice +T1423 SO:0005853 46793 46801 cassette +T1424 SO:0000350 46833 46836 FRT +T1425 NCBITaxon:10088 46872 46876 mice +T1426 PR:000004122 46892 46895 Apc +T1427 SO:0001023 46899 46905 allele +T1428 PR:000004122 46907 46910 Apc +T1429 NCBITaxon:10088 46927 46931 mice +T1430 NCBITaxon:10088 46977 46981 mice +T1431 PR:000004122 47059 47062 Apc +T1432 NCBITaxon:10088 47067 47071 mice +T1433 PR:000004122 47074 47077 Apc +T1434 NCBITaxon:10088 47094 47098 mice +T1435 NCBITaxon:10088 47129 47133 mice +T1436 NCBITaxon:10088 47155 47159 mice +T1437 GO:0010467 47165 47172 express +T1438 UBERON:0019248 47180 47192 early embryo +T1439 PR:000004122 47215 47218 Apc +T1440 SO:0001023 47219 47225 allele +T1441 PR:000004122 47310 47313 Apc +T1442 NCBITaxon:10088 47320 47324 mice +T1443 PR:000009454 47393 47396 K14 +T1444 PR:000004122 47402 47405 Apc +T1445 NCBITaxon:10088 47413 47417 mice +T1446 NCBITaxon:10088 47424 47428 mice +T1447 PR:000004122 47479 47482 Apc +T1448 NCBITaxon:10088 47499 47503 mice +T1449 PR:000009454 47560 47563 K14 +T1450 NCBITaxon:10088 47579 47583 mice +T1451 PR:000009454 47602 47605 K14 +T1452 PR:000004122 47611 47614 Apc +T1453 NCBITaxon:10088 47625 47629 mice +T1454 PR:000004122 47668 47671 Apc +T1455 PR:000004122 47727 47730 Apc +T1456 PR:000009454 47767 47770 K14 +T1457 NCBITaxon:10088 47780 47784 mice +T1458 NCBITaxon:10088 47846 47850 mice +T1459 SO:0001026 47853 47860 Genomic +T1460 UBERON:0010162 47870 47877;47884 47889 tips of ... tails +T1461 NCBITaxon:10088 47878 47883 mouse +T1462 SO:0000112 47973 47980 primers +T1463 PR:000004122 47982 47985 Apc +T1464 PR:000004122 48023 48026 Apc +T1465 SO:0000028 48078 48080 bp +T1466 SO:0000028 48089 48091 bp +T1467 PR:000004122 48136 48139 Apc +T1468 SO:0001023 48143 48149 allele +T1469 PR:000004122 48207 48210 Apc +T1470 SO:0001023 48211 48217 allele +T1471 PR:000004122 48219 48222 Apc +T1472 SO:0000112 48232 48238 primer +T1473 PR:000004122 48239 48242 Apc +T1474 PR:000004122 48296 48299 Apc +T1475 SO:0000028 48324 48326 bp +T1476 SO:0000112 48336 48343 Primers +T1477 SO:0000902 48442 48451 transgene +T1478 SO:0000028 48486 48488 bp +T1479 CHEBI:9754 48562 48566 Tris +T1480 CHEBI:17883 48567 48570 HCl +T1481 CHEBI:32588 48587 48590 KCl +T1482 CHEBI:6636 48599 48604 MgCl2 +T1483 SO:0000112 48633 48639 primer +T1484 UBERON:0000922 49000 49007 embryos +T1485 CHEBI:75055 49049 49054 X-gal +T1486 CHEBI:60004 49064 49067 mix +T1487 CHEBI:37586 49086 49091 NaPO4 +T1488 CHEBI:6636 49100 49105 MgCl2 +T1489 CHEBI:30060 49112 49121 K3Fe(CN)6 +T1490 CHEBI:30059 49128 49137 K4Fe(CN)6 +T1491 CHEBI:75055 49151 49156 X-gal +T1492 UBERON:0007376 49223 49232 epidermal +T1493 CHEBI:75958 49246 49254 solution +T1494 UBERON:0007376 49266 49275 epidermis +T1495 UBERON:0000922 49384 49391 embryos +T1496 UBERON:0004288 49525 49533 Skeletal +T1497 CHEBI:32035 49683 49702 potassium hydroxide +T1498 CHEBI:32035 49704 49707 KOH +T1499 CHEBI:75958 49759 49767 solution +T1500 CHEBI:32035 49774 49777 KOH +T1501 CHEBI:16866 49809 49821 alizarin red +T1502 CHEBI:17754 49928 49936 glycerin +T1503 UBERON:0000479 49993 49999 tissue +T1504 UBERON:0000479 50034 50040 tissue +T1505 SO:0001023 50083 50089 allele +T1506 UBERON:0000479 50162 50169 tissues +T1507 UBERON:0000479 50276 50282 tissue +T1508 CHEBI:33893 50305 50312 reagent +T1509 GO:0010467 50331 50341 expression +T1510 PR:000004122 50369 50372 Apc +T1511 SO:0001023 50373 50380 alleles +T1512 UBERON:0002107 50568 50573 liver +T1513 UBERON:0002370 50578 50584 thymus +T1514 GO:0001171 50608 50627 reverse-transcribed +T1515 SO:0000112 50634 50641 primers +T1516 PR:000004122 50642 50645 Apc +T1517 PR:000004122 50679 50682 Apc +T1518 SO:0000028 50730 50732 bp +T1519 SO:0000028 50741 50743 bp +T1520 PR:000004122 50772 50775 Apc +T1521 SO:0001023 50779 50786 alleles +T1522 PR:000004122 50791 50794 Apc +T1523 SO:0001023 50799 50805 allele +T1524 CHEBI:472552 50822 50826 BrdU +T1525 NCBITaxon:10088 50838 50842 Mice +T1526 UBERON:0001179 50862 50874 peritoneally +T1527 CHEBI:472552 50917 50921 BrdU +T1528 GO:0016265 50964 50969 death +T1529 UBERON:0000479 50971 50977 Tissue +T1530 NCBITaxon:10088 51093 51097 mice +T1531 CHEBI:16526 51167 51170 CO2 +T1532 NCBITaxon:10088 51183 51187 Mice +T1533 CHEBI:33893 51300 51307 reagent +T1534 CHEBI:75958 51406 51414 solution +T1535 NCBITaxon:10088 51474 51478 mice +T1536 UBERON:0000479 51530 51537 tissues +T1537 UBERON:0000468 51607 51617 whole body +T1538 CHEBI:75958 51639 51647 solution +T1539 NCBITaxon:9989 51684 51690 Rodent +T1540 UBERON:0000479 51815 51821 tissue +T1541 CHEBI:27338 51853 51859 xylene +T1542 CHEBI:30879 51873 51880 alcohol +T1543 CHEBI:16240 51939 51943 H2O2 +T1544 CHEBI:17790 51947 51955 methanol +T1545 CHEBI:15377 51993 51998 water +T1546 CHEBI:59132 52007 52014 antigen +T1547 GO:0042571 52170 52180 antibodies +T1548 GO:0042571 52212 52222 Antibodies +T1549 PR:000002198 52233 52242 β-catenin +T1550 GO:0009293 52247 52259 Transduction +T1551 PR:000009450 52304 52314 keratins 1 +T1552 PR:000009481 52304 52312;52316 52317 keratins ... 5 +T1553 PR:000009454 52304 52312;52322 52324 keratins ... 14 +T1554 PR:000009167 52326 52336 involucrin +T1555 PR:000009882 52338 52346 loricrin +T1556 PR:000009495 52395 52404 keratin 8 +T1557 PR:000010425 52441 52445 Ki67 +T1558 CHEBI:472552 52511 52515 BrdU +T1559 MOP:0000093 52525 52537 Biotinylated +T1560 GO:0042571 52548 52558 antibodies +T1561 NCBITaxon:9793 52560 52566 donkey +T1562 NCBITaxon:9986 52572 52578 rabbit +T1563 NCBITaxon:9925 52583 52587 goat +T1564 NCBITaxon:10088 52593 52598 mouse +T1565 GO:0071735 52599 52602 IgG +T1566 CHEBI:51686 52830 52841 hematoxylin +T1567 GO:0097617 52852 52865 hybridization +T1568 NCBITaxon:10114 52886 52889 rat +T1569 PR:000014841 52890 52893 Shh +T1570 PR:000002198 52931 52940 β-catenin +T1571 CHEBI:42098 53024 53027 DIG +T1572 CHEBI:75958 53084 53092 solution +T1573 UBERON:0000160 53147 53157 Intestinal +T1574 http://purl.obolibrary.org/obo/MONDO_0021118 53147 53167 Intestinal Neoplasia +T1575 PR:000004122 53171 53174 Apc +T1576 NCBITaxon:10088 53179 53183 Mice +T1577 UBERON:0002108 53220 53235 small intestine +T1578 NCBITaxon:10088 53251 53256 mouse +T1579 UBERON:0002108 53331 53346 small intestine +T1580 CHEBI:51686 53363 53364 H +T1581 http://purl.obolibrary.org/obo/MONDO_0004972 53448 53455 adenoma +T1582 NCBITaxon:10088 53588 53592 Mice +T1583 UBERON:0000922 53631 53638 embryos +T1584 UBERON:0000922 53674 53681 embryos +T1585 CHEBI:37958 53703 53706 dye +T1586 UBERON:0001757 53794 53801 pinnnae +T1587 UBERON:0008200 53825 53833 forehead +T1588 UBERON:0000922 53844 53850 embryo +T1589 GO:0040007 53914 53918 grow +T1590 UBERON:0002370 54036 54041 Thymi +T1591 UBERON:0002370 54060 54066 thymus +T1592 UBERON:0002370 54085 54091 thymus +T1593 PR:000009495 54106 54108 K8 +T1594 PR:000009450 54129 54131 K1 +T1595 PR:000009167 54156 54166 involucrin +T1596 PR:000009167 54268 54278 involucrin +T1597 UBERON:0002370 54298 54304 thymus +T1598 UBERON:0002370 54352 54358 thymus +T1599 SO:0000704 54509 54514 genes +T1600 SO:0000704 54519 54523 gene +T1601 PR:000004122 54561 54564 Apc +T1602 PR:000002198 54578 54587 β-catenin +T1603 PR:000004527 54601 54607 AXIN 2 +T1604 PR:000009235 54621 54632 plakoglobin +T1605 PR:000004257 54646 54650 Asef +T1606 PR:000009322 54664 54704 kinesin superfamily–associated protein 3 +T1607 PR:000010170 54718 54721 EB1 +T1608 NCBITaxon:9606 54735 54740 human +T1609 SO:0000853 54741 54748 homolog +T1610 PR:000006511 54741 54774 homolog of Drosophila Discs large +T1611 NCBITaxon:7215 54752 54762 Drosophila +T1612 PR:000009751 54785 54789 Lef1 +T1613 PR:000006499 54803 54807 Dkk1 +T1614 PR:000009450 54821 54823 K1 +T1615 PR:000009481 54837 54839 K5 +T1616 PR:000009495 54869 54871 K8 +T1617 PR:000009454 54885 54888 K14 +T1618 PR:000009167 54902 54912 involucrin +T1619 PR:000009882 54926 54934 loricrin +T1620 PR:000014841 54952 54955 Shh +T1621 PR:000004122 54985 54988 APC +T1622 http://purl.obolibrary.org/obo/MONDO_0004972 54991 55002 adenomatous +T1623 PR:000004122 54991 55017 adenomatous polyposis coli +T1624 UBERON:0000922 55051 55060 embryonic +T1625 UBERON:0000922 55071 55080 embryonic +T1626 http://purl.obolibrary.org/obo/MONDO_0021055 55087 55090 FAP +T1627 http://purl.obolibrary.org/obo/MONDO_0021055 55093 55123 familial adenomatous polyposis +T1628 SO:0000350 55125 55128 FRT +T1629 PR:P03870 55131 55134 FLP +T1630 SO:0000350 55131 55153 FLP recognition target +T1631 GO:0097617 55169 55182 hybridization +T1632 PR:000009454 55184 55187 K14 +T1633 PR:000009454 55190 55200 keratin 14 +T1634 UBERON:0005942 55202 55205 ORS +T1635 UBERON:0005942 55208 55225 outer root sheath +T1636 GO:0007567 55235 55240 natal +T1637 CL:0002293 55246 55249 TEC +T1638 UBERON:0003846 55252 55269 thymic epithelial +T1639 CL:0002293 55252 55274 thymic epithelial cell +T1640 PR:000004122 55336 55339 Apc +T1641 SO:0001023 55340 55346 Allele +T1642 SO:0000147 55373 55378 exons +T1643 NCBITaxon:10088 55396 55401 mouse +T1644 PR:000004122 55402 55405 Apc +T1645 SO:0000704 55406 55410 gene +T1646 SO:0001644 55416 55432 targeting vector +T1647 SO:0001023 55464 55470 allele +T1648 SO:0000346 55478 55488 LoxP sites +T1649 SO:0000147 55505 55509 exon +T1650 CHEBI:7507 55522 55530 neomycin +T1651 SO:0005853 55531 55539 cassette +T1652 SO:0000188 55560 55566 intron +T1653 SO:0005853 55604 55612 cassette +T1654 SO:0000357 55616 55626 sandwiched +T1655 SO:0000350 55632 55641 FRT sites +T1656 GO:0010467 55684 55694 expressing +T1657 NCBITaxon:10088 55695 55699 mice +T1658 SO:0000112 55718 55725 primers +T1659 SO:0000147 55921 55925 exon +T1660 PR:000004122 55963 55966 Apc +T1661 SO:0001026 56073 56080 genomic +T1662 UBERON:0002415 56081 56085 tail +T1663 NCBITaxon:10088 56107 56111 mice +T1664 PR:000004122 56123 56126 Apc +T1665 NCBITaxon:10088 56127 56132 mouse +T1666 PR:000004122 56140 56143 Apc +T1667 PR:000004122 56149 56152 Apc +T1668 GO:0097617 56159 56169 hybridized +T1669 SO:0000028 56179 56181 bp +T1670 UBERON:0002415 56189 56193 Tail +T1671 SO:0001026 56194 56201 genomic +T1672 PR:000004122 56211 56214 Apc +T1673 NCBITaxon:10088 56222 56226 mice +T1674 CL:0002322 56251 56253 ES +T1675 PR:000004122 56288 56291 Apc +T1676 SO:0001023 56296 56302 allele +T1677 SO:0001023 56338 56344 allele +T1678 SO:0001026 56354 56361 genomic +T1679 PR:000004122 56375 56378 Apc +T1680 NCBITaxon:10088 56383 56388 mouse +T1681 PR:000004122 56414 56417 Apc +T1682 SO:0001023 56422 56428 allele +T1683 PR:000004122 56479 56482 Apc +T1684 NCBITaxon:10088 56488 56492 mice +T1685 PR:000004122 56520 56523 Apc +T1686 NCBITaxon:10088 56531 56535 mice +T1687 PR:000004122 56564 56567 Apc +T1688 NCBITaxon:10088 56574 56578 mice +T1689 PR:000004122 56672 56675 Apc +T1690 SO:0001023 56680 56686 allele +T1691 PR:000004122 56788 56791 Apc +T1692 NCBITaxon:10088 56795 56799 mice +T1693 GO:0007567 56879 56884 natal +T1694 PR:000009454 56917 56920 K14 +T1695 PR:000004122 56926 56929 Apc +T1696 NCBITaxon:10088 56944 56948 Mice +T1697 NCBITaxon:33208 56950 56957 Animals +T1698 PR:000004122 57028 57031 Apc +T1699 SO:0001023 57032 57038 allele +T1700 PR:000009454 57095 57098 K14 +T1701 PR:000004122 57104 57107 Apc +T1702 PR:000009454 57140 57143 K14 +T1703 PR:000004122 57166 57169 Apc +T1704 SO:0001023 57170 57176 allele +T1705 NCBITaxon:10088 57219 57223 mice +T1706 NCBITaxon:10088 57319 57324 mouse +T1707 UBERON:0010166 57377 57386 hair coat +T1708 UBERON:0001690 57400 57404 ears +T1709 UBERON:0006378 57413 57422 Vibrissae +T1710 NCBITaxon:10088 57482 57487 mouse +T1711 UBERON:0001098 57535 57543 incisors +T1712 NCBITaxon:10088 57577 57582 mouse +T1713 UBERON:0008200 57621 57629 forehead +T1714 UBERON:0001690 57664 57668 ears +T1715 UBERON:0002370 57912 57918 thymus +T1716 NCBITaxon:10088 57927 57931 mice +T1717 UBERON:0002370 57944 57950 thymus +T1718 UBERON:0004288 58069 58077 Skeletal +T1719 UBERON:0001098 58171 58178;58197 58202 incisor ... teeth +T1720 UBERON:0001098 58180 58181;58197 58202 I ... teeth +T1721 UBERON:0003655 58187 58192;58197 58202 molar ... teeth +T1722 UBERON:0003655 58194 58195;58197 58202 M ... teeth +T1723 UBERON:0000479 58215 58221 Tissue +T1724 GO:0010467 58245 58255 Expression +T1725 PR:000004122 58267 58270 Apc +T1726 SO:0001023 58271 58278 Alleles +T1727 UBERON:0000479 58284 58290 Tissue +T1728 SO:0001026 58321 58328 genomic +T1729 UBERON:0002370 58363 58369 thymus +T1730 UBERON:0002370 58371 58372 T +T1731 UBERON:0002107 58383 58388 liver +T1732 UBERON:0002107 58390 58391 L +T1733 NCBITaxon:10088 58396 58400 mice +T1734 PR:000009454 58414 58417 K14 +T1735 PR:000004122 58451 58454 Apc +T1736 SO:0001023 58459 58465 allele +T1737 UBERON:0000479 58486 58492 tissue +T1738 GO:0010467 58502 58512 expression +T1739 PR:000004122 58530 58533 Apc +T1740 SO:0000673 58534 58545 transcripts +T1741 SO:0000112 58584 58591 primers +T1742 UBERON:0002370 58647 58653 thymus +T1743 UBERON:0002107 58662 58667 liver +T1744 NCBITaxon:10088 58671 58675 mice +T1745 PR:000009454 58689 58692 K14 +T1746 SO:0000673 58702 58713 transcripts +T1747 SO:0000028 58739 58741 bp +T1748 SO:0000028 58760 58762 bp +T1749 PR:000004122 58764 58767 Apc +T1750 SO:0001023 58768 58775 alleles +T1751 UBERON:0001091 58848 58853 Teeth +T1752 UBERON:0000167 58918 58929 oral cavity +T1753 UBERON:0000167 58948 58959 oral cavity +T1754 CHEBI:51686 58974 58975 H +T1755 PR:000010425 59010 59014 Ki67 +T1756 PR:000002198 59023 59032 β-catenin +T1757 PR:000009454 59047 59050 K14 +T1758 GO:0009653 59100 59113 morphogenesis +T1759 GO:0001942 59132 59144;59178 59192 formation of ... hair follicles +T1760 UBERON:0002073 59178 59192 hair follicles +T1761 GO:0008283 59253 59266 proliferation +T1762 UBERON:0005932 59291 59300 hair bulb +T1763 UBERON:0000167 59445 59456 oral cavity +T1764 GO:0005829 59514 59523 cytosolic +T1765 PR:000002198 59540 59549 β-catenin +T1766 GO:0010467 59694 59704 Expression +T1767 PR:000014841 59708 59711 Shh +T1768 PR:000002198 59716 59725 β-catenin +T1769 SO:0000673 59726 59737 Transcripts +T1770 PR:000004122 59749 59752 Apc +T1771 PR:000009454 59773 59776 K14 +T1772 PR:000004122 59782 59785 Apc +T1773 UBERON:0000922 59794 59803 Embryonic +T1774 PR:000014841 59833 59836 Shh +T1775 UBERON:0000483 59971 59981 epithelium +T1776 UBERON:0003104 59986 59996 mesenchyme +T1777 PR:000002198 60050 60059 β-catenin +T1778 UBERON:0000922 60098 60105 embryos +T1779 UBERON:0005086 60139 60152 hair placodes +T1780 PR:000009454 60182 60185 K14 +T1781 PR:000004122 60193 60196 Apc +T1782 UBERON:0005086 60260 60273 hair placodes +T1783 UBERON:0001037 60286 60290 hair +T1784 UBERON:0013623 60295 60304 foot pads +T1785 UBERON:0002370 60407 60413 Thymus +T1786 UBERON:0002370 60431 60437 thymus +T1787 UBERON:0002370 60460 60466 thymus +T1788 UBERON:0002370 60491 60497 thymus +T1789 UBERON:0002370 60516 60522 thymus +T1790 CHEBI:51686 60537 60538 H +T1791 CHEBI:472552 60569 60573 BrdU +T1792 PR:000002198 60585 60594 β-catenin +T1793 PR:000009454 60613 60616 K14 +T1794 CL:0000893 60653 60663 thymocytes +T1795 UBERON:0002123 60703 60712;60723 60729 cortex of ... thymus +T1796 UBERON:0002370 60796 60802 thymus +T1797 GO:0007618 60919 60926 Matings +T1798 CHEBI:33893 61420 61428 reagents +T1799 http://purl.obolibrary.org/obo/MONDO_0004992 61547 61553 Cancer +T1800 UBERON:0001091 61675 61681 Dental diff --git a/src/ontogpt/evaluation/craft/database/all/17002498.txt b/src/ontogpt/evaluation/craft/database/all/17002498.txt new file mode 100644 index 000000000..9b62c178e --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17002498.txt @@ -0,0 +1,285 @@ +Adenomatous Polyposis Coli (APC) Is Required for Normal Development of Skin and Thymus + +Abstract + +The tumor suppressor gene Apc (adenomatous polyposis coli) is a member of the Wnt signaling pathway that is involved in development and tumorigenesis. Heterozygous knockout mice for Apc have a tumor predisposition phenotype and homozygosity leads to embryonic lethality. To understand the role of Apc in development we generated a floxed allele. These mice were mated with a strain carrying Cre recombinase under the control of the human Keratin 14 (K14) promoter, which is active in basal cells of epidermis and other stratified epithelia. Mice homozygous for the floxed allele that also carry the K14-cre transgene were viable but had stunted growth and died before weaning. Histological and immunochemical examinations revealed that K14-cre–mediated Apc loss resulted in aberrant growth in many ectodermally derived squamous epithelia, including hair follicles, teeth, and oral and corneal epithelia. In addition, squamous metaplasia was observed in various epithelial-derived tissues, including the thymus. The aberrant growth of hair follicles and other appendages as well as the thymic abnormalities in K14-cre; ApcCKO/CKO mice suggest the Apc gene is crucial in embryonic cells to specify epithelial cell fates in organs that require epithelial–mesenchymal interactions for their development. + +Synopsis + +Patients with familial adenomatous polyposis (FAP) and its variant, Gardner's syndrome, will develop hundreds of colorectal polyps. It is a heritable disease that is linked to a genetic mutation in the tumor suppressor gene APC (adenomatous polyposis coli). These individuals also develop extracolonic symptoms, among which are congenital hypertrophy of the retinal pigment epithelium, desmoid tumors, epidermoid cysts, disorders of the maxillary and skeletal bones, and dental abnormalities, suggesting the importance of APC functions in these organs. To understand the role of Apc in development and in organs other than intestine, we generated Apc mutant mice that can conditionally delete the gene when exposed to Cre recombinase. These mice were mated with K14 (Keratin 14)–cre mice that express Cre recombinase in skin and its appendages. The authors found that the mutant mice that have lost Apc in K14-cre transgene–expressing tissues were viable, but had stunted growth and died before weaning. These mutant mice showed developmental abnormalities not only in skin but also in many epithelial-derived tissues, including teeth and thymus. This work demonstrates the importance of Apc in development of many organs, and provides new insights into diagnosis and management of patients with APC mutations. + +Introduction + +Adenomatous polyposis coli (APC) is a member of the Wnt signaling pathway and one of its known functions is to regulate the levels of β-catenin. Alterations in β-catenin regulation are very common in human tumors [1]. Loss of APC is associated with stabilization of the cytosolic β-catenin that ultimately results in its migration to the nucleus and activating a cascade of events leading to tumorigenesis. APC also interacts with a multitude of other cellular proteins, including axin-2 (AXIN2), plakoglobin (JUP), Asef (ARHGEF4), kinesin superfamily–associated protein 3 (KIFAP3), EB1 (MAPRE1), microtubules, and the human homolog of Drosophila discs large (DLG1). These interactions suggest that APC can potentially regulate many cellular functions, including intercellular adhesion, cytoskeletal organization, regulation of plakoglobin levels, regulation of the cell cycle and apoptosis, orientation of asymmetric stem cell division, and control of cell polarization [2,3]. + +APC is a tumor suppressor gene. Somatic mutations in APC are frequently found in many sporadic tumors of the colon and rectum. Autosomal dominant germline mutations in APC cause familial adenomatous polypois (FAP) and its variant, Gardner syndrome. FAP patients are characterized by hundreds of adenomatous colorectal polyps, with an almost inevitable progression to colorectal cancer in the third and fourth decades of life [4,5]. In addition to colorectal neoplams, these individuals can develop extracolonic symptoms, among which are upper gastrointestinal tract polyps, congenital hypertrophy of the retinal pigment epithelium, desmoid tumors, disorders of the maxillary and skeletal bones, and dental abnormalities [6], suggesting the importance of APC gene functions in these organ systems. + +Although the role of APC in the initiation of human colorectal cancer is well established, its role in other tissue and developmental processes are not well understood. Given the importance of regulation of Wnt signaling in embryonic pattern formation and morphogenesis of many organs, mechanistic understanding of APC in development and in extracolonic tissues becomes critical to better assess potential adverse events in humans. One approach to understand the role of Apc in development is to develop mice with an inactivating Apc mutation. Several genetically modified mouse strains for Apc have been described [7–10]. Most of these models, in the heterozygous state, show a gastrointestinal and other tumor predisposition phenotype [7–10]. Mouse embryos that are homozygous for the genetic modification die during embryogenesis, and some of the models do not survive beyond gastrulation [8,11]. An alternate approach to understand the role of Apc in development and/or in specific tissues is to generate a mouse strain that carries a conditionally modified allele and mate it with a mouse strain that facilitates the modification of the conditional allele in specific cell lineages. + +To assess the role of Apc in different stages of life systematically, we generated mice containing a conditional knockout (CKO) mutant allele of Apc (ApcCKO). These mice were mated with a strain carrying Cre recombinase under the control of the human Keratin 14 (K14) promoter, which is active in basal cells of epidermis and other stratified epithelia. We report here that K14 promoter-driven loss of Apc resulted in aberrant development of several organs that require inductive epithelial–mesenchymal interactions, including hair follicle, teeth, and thymus, and resulted in neonatal death in mice. We found that Apc plays a crucial role in determinations of cell fates during the embryonic development, possibly via temporal and tissue-specific regulation of β-catenin levels in the skin, its appendages, and in the thymus. + +Results + +Generation of the ApcCKO and ApcΔ580 Mice + +To investigate the role of Apc in development of skin and its appendages, we used the Cre/loxP technology to introduce a conditional mutation of the Apc gene in mice. We constructed embryonic stem (ES) cells and mice carrying an Apc allele harboring both a pair of loxP sites flanking Apc exon 14 and a pair of FLP recognition target (FRT) sites flanking PGK-neomycin selection cassette by recombineering [12,13] (Figure 1A, ApcCKON allele, N for neomycin cassette). A PGK-neomycin cassette was inserted in the same transcriptional orientation as Apc in intron 14 of the endogenous gene. The loxP and FRT sites were used to aid unidirectional recombination [12,13]. Two mouse lines containing the same modification were generated from two independent ES clones to ensure that these two lines behave in the same way. These ApcCKON/+ mice were crossed with FLPe-deleter to generate ApcCKO/+ mice that were heterozygous for the final Apc conditional (ApcCKO) allele that removed the PGK-neomycin cassette and contains only the loxP sites in the introns flanking exon 14. To assess the effect of deleting exon 14 in mice, both lines of ApcCKO/+ mice were crossed with the Cre-deleter to generate the germline knockout line of Apc, designated ApcΔ580/+. The mutant allele (ApcΔ580) lacks exon 14 (Figure 1A). The transcript from loss of exon 14 results in a shift in the normal reading frame, resulting in a premature chain termination codon which, if utilized, would result in a truncated polypeptide that is 605 aa in length, of which the first 580 aa correspond to the normal Apc protein. + +Southern blot analysis of tail DNA from F1 offspring of both ApcCKON and ApcΔ580 lines confirmed the germline transmission of modified Apc allele (Figure 1B). Mice that are heterozygous for ApcΔ580 mutation are viable but have a significantly reduced lifespan (Figure 1C). These results suggested that deletion of exon 14 indeed results in either loss or abnormal function of the Apc gene product. ApcΔ580/+ mice have median survival of 5 mo of age (Figure 1C), with progressive signs of rectal bleeding and anemia. Similar to the results reported with an independently generated ApcΔ580/+ conditional mouse strain [14], ApcΔ580/+ mice had more than 100 (120 ± 37, n = 11) intestinal tumors at the time of their death (Figure S1). Inactivation of wild-type Apc is an important prerequisite for tumor development. We analyzed 30 intestinal tumors from ApcΔ580/+ mice by in vitro transcription and translation assay, but none of them showed truncated Apc products (unpublished data), indicating that the most likely mechanism of wild-type Apc inactivation is by allelic loss. The mutant allele had to be maintained and transmitted through male mice, as ApcΔ580/+ females were frequently not healthy enough to successfully nurse their own pups because of their tumor burden. + +ApcCKO/+ mice were intercrossed to generate ApcCKO/CKO offspring. Approximately one-quarter of the offspring (17 of 81) were homozygous for the ApcCKO allele. These mice as well as heterozygous mice for ApcCKO allele are normal, showing no differences in their survival to the wild-type littermates (Figure 1C). We tested whether our ApcCKO allele can compliment the wild-type allele by crossing the ApcCKO/CKO female with ApcΔ580/+ male mouse. The resultant ApcCKO/Δ580 offspring were viable and born in the Mendelian ratio, suggesting that the presence of loxP sites in introns flanking exon 14 have no adverse effect on the function of the Apc gene. + +K14-Driven Loss of Apc Results in Severe Growth Retardation and Early Lethality + +To introduce the mutation of Apc into cells expressing K14, we crossed WW6 ES cell–derived [15] ApcCKO/+ mice with K14-cre recombinase mice in FVB background [16]. The K14-cre; ApcCKO/+ mice were normal in appearance and were fertile. K14-cre; ApcCKO/+ males were crossed to ApcCKO/CKO females to avoid the potential deleter effect in oocytes of K14-cre–positive females [17]. The mice were intercrossed thereafter for maintenance; hence, the mice used for analysis were in a mixed background of FVB, 129/S, and C57BL/6 in similar proportions, with minimal contribution of SJ. + +The K14 promoter is a commonly used epidermal cell promoter because of its expression by the mitotically active cells of the epidermis and its appendages in mature skin [18], but most notably it is active in embryonic ectoderm as early as the single layered ectodermal stage of embryonic day (E) 9.5 [19]. A restricted expression of K14 is also found in thymic epithelial cells (TECs) in the medulla of normal thymus [20]. + +We genotyped a total of 458 pups (8–10 d old) from 67 litters resulting from crosses between K14-cre; ApcCKO/+ and ApcCKO/CKO mice. The mutant mice of the genotype K14-cre; ApcCKO/CKO (hereafter, KA mutant) were born, but the observed frequency of KA mutants was much less than expected (78 of 458 [17.0%]; p < 0.0005 Chi-square analysis, Table 1). To assess the basis for the neonatal lethality of KA mutants, we monitored three litters from birth to weaning by measuring the body weight of each pup every day. A total of 25 pups were born from three litters, of which 7 (28%) were confirmed to be K14-cre; ApcCKO/CKO by genotyping, indicating that KA mutants were born in the expected Mendelian ratio. The KA mutant pups were nursed normally, and there was milk in their stomachs during the first 2 or 3 d after birth, but they failed to thrive (Figure 2). By postnatal day (P) 8–10, at the time of genotyping, many KA mutant pups were considerably smaller than their littermates (Figure 2B–2F) and some have died at or prior to this age. None of KA mutants survived to weaning age. + +The ability of whole embryos to exclude blue dye was used to examine the epidermal barrier, normally acquired beginning at E16 and complete by E18.5 [21]. Analyses of E17.5–E18.5 KA mutants showed that they were able to exclude blue dye, indicating that the epidermal barrier was intact (Figure S2). At these embryonic ages, there were no differences in size between the mutants and their littermates, but the mutants showed a patch of “birthmark” or dark pigmentation on their foreheads and a dark median line that ran caudally from head to tail. Their external ears or pinnae were shriveled in appearance and pigmented compared to those of littermates. + +External characteristics of KA mutants that were evident at E18.5 persisted after birth and became more prominent as they grew (Figure 2A–2F). Growth of pelage hair was generally delayed in the mutants. At around P8, the KA mutants were hairless and had wrinkled skin while their phenotypically normal littermates had a smooth thin coat of hair (Figure 2B). At this age, two lower incisors start to erupt in normal littermates and these were absent in the KA mutants (Figure 2C and 2D). Animals also tended to be smaller and around P10–P12 displayed abnormally short and misshapen vibrissae and short, shaggy pelage hairs (Figure 2C and 2D). Development of thick ridges in their skin, particularly around the ears, eyelids, forehead, nose, and paws, became noticeable (Figure 2E). These regions looked scaly, and these animals hardly kept their eyes open. In contrast to the normal littermates that consistently increased their body weight with age, surviving KA mutants started to lose weight from P10 onwards; by P16–P17 they were all lethargic, and none of them survived to weaning (Figure 2E and 2F). At the time of autopsy all the mutants were toothless, without incisors or molars, and their stomachs were consistently small and had no solid food, unlike their age-matched littermates, suggesting that the observed weight loss could be the result of failure to ingest solid food (Figure 2F). Interestingly, changes in body weights and timing of hair growth varied considerably among mutant pups even if they were from the same litter, whereas those of phenotypically normal littermates tended to be similar. This difference was also reflected in the variation in timing of death in mutants: some mutant pups were born alive but died within a day or two, some survived close to the weaning age. This variability of the mutant phenotypes suggests possible variation in the timing and efficiency of cre-mediated Apc deletion. It is possible that the genetic background has a role to play in this variability. + +Gross examination of internal organs also showed that the mutants' thymi were consistently inconspicuous and were very small for their age, whereas those of their littermates were very prominent in size (Figure 2G). This difference was evident as early as P3. Quite frequently mutant thymi in P12–P17 mutant mice also contained black deposits within the tissue (unpublished data). Mutant mice were also examined for any skeletal abnormalities by preparing skeletal specimens of P16–P17 mice stained with Alizarin red. No differences between the normal and KA mutant mice in the mandibular bone can be detected, but the mutant mice lacked or had underdeveloped set of maxillary incisors and molars (Figure 2H). We detected no other major skeletal abnormalities. + +Genotype- and Tissue-Specific Expression of the Truncated Apc Transcripts + +To assess the molecular effects of the K14-cre–mediated recombination, we screened for the presence of deleted Apc (ApcΔ580) alleles. Genomic DNA was extracted from liver, thymus, and skin from all 4 possible genotypes: K14-cre; ApcCKO/CKO, K14-cre; ApcCKO/+, ApcCKO/CKO, and ApcCKO/+. Genotyping on genomic DNA from these tissues showed that the ApcΔ580 allele (500-bp product) was detected only from the skin and thymus of the K14-cre–positive mice. The presence of mutant Apc allele in the thymus of K14-cre; ApcCKO/+ mice was consistently much less than the DNA from the skin of the same animal or other tissues from the KA mutants. In addition, this product was not detected at all in either the liver of K14-cre–positive or in any of the K14-cre–negative mouse tissues samples, establishing that Cre-mediated recombination has taken place in the tissue-specific manner in the mice that inherited K14-cre (Figure 3A). + +Apc transcripts were also analyzed by RT-PCR with primers spanning exon 14 (Figure 1A) using total RNA isolated from the corresponding tissue samples. We detected the expected RT-PCR product (313 bp) from the truncated Apc (ApcΔ580) allele only in the tissues where Cre recombinase is known to be expressed in the K14-cre–positive mice. However, this product was not detected in either the K14-cre–negative mouse tissues samples or the liver of K14-cre–positive mice, and only the product from the wild-type allele (528 bp) was detected from these RNA samples, further confirming that Cre-mediated recombination has taken place in the tissue- and genotype-specific manner (Figure 3B). + +K14-cre–Driven Apc Loss Induced Aberrant Hair Follicles throughout the Epidermis + +To understand the basis for delayed and abnormal hair development in the KA mutants, we conducted a histological and immunohistochemical examination (Figure 4). The hair follicle is an epidermal appendage that consists of an upper permanent portion, and a lower cycling portion that produces the hair [22,23]. The outer root sheath (ORS) is contiguous with and biochemically similar to the basal layer of the epidermis. The inner layers of the hair follicle include three concentric layers of inner root sheath and three concentric layers of hair-producing cells. At the base of the hair follicle is the germinative hair follicle bulb, which contains rapidly proliferating “matrix” cells that differentiate to populate all of the layers of the inner root sheath and the hair shaft itself [22]. During the anagen phase of the hair cycle (until P15), hair follicles of phenotypically normal mice grew deeply into the subcutaneous fat and were uniformly spaced and aligned in parallel arrays at a specific angle relative to the skin surface (Figure 4A). In contrast, KA mutant follicles were irregularly spaced and often seen as disoriented and clamped invaginations at P3 that became even more remarkable at P12 when the mutant mice were covered by fur coat (Figure 4F). Bulbs were often bent in addition to being irregularly angled to one another and their sizes and locations were often variable. Clusters of multiple invaginations or dysplastic follicular structures were frequently observed throughout the epidermis, whereas other regions showed gaps with no follicles. Serial sectioning indicated that some of the hair follicles in the P12 mutant skin were not properly formed or shorter than normal. Taken together, these features could account for the apparently delayed, followed by outgrowth, of the short and shaggy-looking fur coat of these mutant mice. + +Apc is a regulator of β-catenin that is important for Wnt signaling. We examined the patterns of expression of β-catenin in the affected tissues. In the normal skin, β-catenin, a member of the adherens junction complex, was found in the ORS of hair follicles and basal layer of epidermis, where K14 expression is also observed (Figure 4C and 4D), whereas the expression of K1, involucrin, and loricrin (markers for spinous and granular layers of epidermis) was only observed in the nonbasal epidermis (unpublished data). The patterns of expression of K14, K1, involucrin, and loricrin, in skin from mutant and normal littermate mice at P3–P17, showed no significant differences in the terminal differentiation (Figure 4A–4D, 4F–4I). Similarly, the pattern of expression of K6, which is normally only expressed in the suprabasal or inner layer of the ORS of the hair follicle but not in the epidermis (Figure 4E), did not change. Due to the abnormal and disorganized structure of hair follicles themselves, K6 localization highlighted the histological abnormality (Figure 4J). Yet as in the normal skin, K6 was principally seen only in the suprabasal layer of the ORS that did not colocalize with the basal markers, K14 or K5 (Figure 4I and 4J). + +In normal skin, proliferating cells were detected in either the basal layer of epidermis or in germinative hair follicle bulbs at the base (Figure 4B). In the mutant skin, either BrdU incorporation or Ki67 expression was observed not only in cells in bulbs at the base of the hair follicle but also in bulb-like structures that were budding out from the ORS of the existing hair follicles (Figure 4G and 4G′). Each budding tip was becoming like a hair follicle bulb containing proliferating cells. Hence, despite the abnormal histology in the mutant skin, proliferation seems to be confined to bulb-like structures as in the normal skin (Figure 4G and 4G′). The exact locations of hair follicle bulbs were not as easy to define for some mutant follicles due to their disorganized structures. Interestingly, in the mutant skin, in addition to diffuse membrane-bound localization as in the normal skin, cells with strong cytosolic β-catenin localization were also observed frequently (Figure 4H and 4H′). These elevated β-catenin–expressing cells were usually surrounded by proliferating cells, forming bulb-like structures. Comparison of immunochemically stained serial sections showed that these intense cytosolic β-catenin stainings were usually found in either K14-positive K1-negative basal epidermis or K14-positive K6-negative basal ORS cells, and are surrounded by proliferating cells. + +To determine the initiation of hair follicle morphogenesis in these mutants, we examined the expression pattern of Sonic hedgehog (Shh), a factor expressed in hair bulbs in embryonic skin (Figure 5). The aberrant hair follicle morphogenesis is evident as early as E14.5 in mutant embryonic skin, by multiple apolarized expression of Shh throughout the epidermis (Figure 5B), whereas that of control embryos was well polarized and regularly spaced (Figure 5A). With development, control mouse hair follicles invaginate downward in a polarized manner (Figure 5C), whereas those of mutant embryos were completely irregular and apolarized (Figure 5D). It was also noted that the size of each “budding” follicle, as detected by Shh expression, was variable (Figure 5D). The intensity of Shh staining was generally stronger in mutant skin than in the normal skin. The aberrant initiation of multiple hair placodes during early hair follicle morphogenesis was also evident by the whole-mount in situ hybridization (ISH) of E15.5 mutant embryos for β-catenin (Figure 5F and 5F′). The expression pattern of β-catenin in embryos clearly demonstrated the formation of regular arrays of hair placodes in the normal embryonic skin (Figure 5E and 5E′), but such regular patterning was lost, and often tightly clustered abnormal hair placodes were initiated in mutant embryonic skin (Figure 5F′). Aberrant hair placodes were also evident throughout the skin surface of limbs in E15.5 mutants (Figure 5F), whereas those of the control embryos had not yet formed (Figure 5E). Most interestingly, in the mutant footpads, where hair placodes do not normally form (Figure 5G), we also found ectopic irregularly sized and spaced hair placodes, indicating that the footpads still have the potential to form hair placodes in the absence of the Apc gene (Figure 5H). + +These results collectively suggest that the terminal differentiation does take place normally in the mutant skin, but initiation of embryonic hair follicle morphogenesis is severely disrupted, accompanied by a continuous ectopic hair follicle morphogenesis in postnatal mutant skin. + +Effects of K14-cre–Driven Loss of Apc in Other Epidermal Appendages + +Similar to the biology of hair follicles, K14-cre–driven loss of Apc also affected the development of other epidermal appendages that depend on epithelial–mesenchymal interactions for their formation. The most striking of these was dental dysplasia (Figure 4K–4R). Tooth development is normally initiated between E11 and E12 by invagination of ectodermally derived oral epithelium into the underlying cranial neural crest–derived mesenchyme, generating a tooth germ. Despite the grossly toothless phenotype of KA mutants, histological analysis of their oral cavities revealed the formation of multiple tooth buds at each location. These aberrant teeth obviously failed to grow out during the dietary transition from milk to solid food. Analogous to the expression patterns of K14 and β-catenin in the normal skin, diffuse membrane-bound expression of β-catenin was detected in K14-expressing oral epithelium and ameloblasts of normal mice. In mutants, some of the K14-expressing cells also showed strong cytosolic/nuclear β-catenin staining, as observed in the mutant skin (Figure 4Q, 4Q′, and 4R). Initiation of ectopic tooth buds in the mutant mice was evident at E15.5 by extra dots of Shh expression adjacent to the primary teeth (data not shown). + +Loss of Apc also leads to hyperplasia in squamous epithelia of cornea, oral, salivary, and Hardarian glands (unpublished data). Squamous metaplasia to hair follicle–like structures, ectopic hair follicle morphogenesis was also observed in these epithelia. + +K14-cre–Driven Apc Loss Results in Hypoplastic/Athymic Mice + +Thymus is an organ that is also known to have K14 expression [16]. It represents the primary lymphoid organ for thymocyte development and selection. Distinct population of TECs of cortex and medulla mediates both of these critical functions. Cortical and medullary TEC subsets are characterized by differential expression of four keratin species: K8, K18, K5, and K14. + +The normal thymus is a lobulated lymphoid organ, each lobule clearly showing the two distinct TEC compartments, an outer cortex and an inner medulla (Figure 6). There were no major differences in the histology of thymus between the ages P3 to P17 in phenotypically normal littermates. As shown in the H&E staining of thymus, the cortex was formed of dense lymphoid tissue that lacks nodules (Figure 6A). Since the stroma of the medulla is less heavily infiltrated with lymphocytes than the cortex, the medulla stained more lightly than the cortex. In normal mice, the thymus retains its size until the young adult age and regresses thereafter by atrophy. In the normal young mice we examined (P3–P17), it is evident that thymocytes were mitotically active in the cortex as determined by BrdU immunostaining (Figure 6B). Immunohistochemistry of normal thymus from P3 to P17 mice showed a similar staining pattern for K14 in that its expression was restricted to a small population of TECs in the inner medullary region and in the keratinocytes in Hassall's corpuscles (Figure 6D). Diffuse cytoplasmic staining for β-catenin was also detected in the medullary epithelial cells (Figure 6C). In contrast to K14 expression, diffuse staining for K8 was observed in epithelial cells both in the medulla and cortex (Figure S3). K1 staining was not detected in young mice at P3 but in older mice it was detected in differentiated keratinocytes in some of Hassall's corpuscles (Figure S3). + +The histological abnormalities of thymus were evident as early as P3 in KA mutants (Figure 6E and 6H). The thymus was made of two lobules as in the normal mice but the mutant thymus was significantly smaller in size than that of the age-matched controls (Figure 2G). Interestingly, variations in the phenotypic severity of the mutant pups at P3 were prominently reflected in the extent of histological abnormalities of thymus. A P3 KA mutant pup that showed milder phenotype with a comparable body weight to its normal littermates (Figure 2A, M2) showed milder thymus abnormalities (Figure 6E) compared to its more severe mutant littermate (Figure 2A, M1; Figure 6H). The milder P3 mutant thymus was already much smaller in size compared to those of normal littermates (data not shown) but two epithelial compartments of thymus were histologically still distinguishable, with colonization of thymocytes evident in the cortex. However, there were small populations of lightly stained cells by H&E extending from the edge of the outer cortex towards inner medulla (Figure 6E), and these cells showed intense nuclear β-catenin staining whereas the rest of the medullary cells showed diffuse β-catenin staining pattern similar to that of the control (Figure 6F). Localization of K14 was limited to a few cells in the medulla and some overlapped with K8 localization (Figure 6G). + +In the other P3 mutant thymus the distinct thymic epithelial compartments have been lost completely, and only a few lymphocytes were remaining at the edges and some in the middle (Figure 6H). Proliferative activities were no longer observed in thymocytes as prominently as in the normal thymus, but the epithelial cells seemed to be forming concentric structures (Figure 6I). Unlike in the normal or mild mutant thymi, the severe P3 mutant thymus showed extensive K14 expression that overlapped with K8 expression (Figure 6K). These cells were more like basal cells of the skin than TECs and were adjacent to the most immature looking cells that were showing strong nuclear and cytoplasmic β-catenin staining (Figure 6J). The nuclear staining of β-catenin was not observed in the normal age-matched thymus (Figure 6C). Most notably, the K14 and β-catenin staining patterns were mutually exclusive (Figure 6J and 6K). + +At P10–P13, the mutant thymus consisted of numerous enlarged Hassall's corpuscle–like structures, made of arrays of K14- and K8-expressing keratinizing epithelial cells surrounding large keratin deposits (Figure 6L and 6O). There were numerous neutrophils and macrophages infiltrating the thymus in response to these keratins; hence, these structures could be called pyogranuloma. Varying degrees of differentiation-specific markers depending on the age of mice, in this case K1 and involucrin that are normally present only in Hassall's corpuscles, were also detected in mutant thymi (Figure S3). In these mice no thymocytes were detectable. BrdU incorporation was only observed in very few keratinizing epithelial cells, looking somewhat similar to the pattern of mature skin (Figure 6M). The diffuse expression of β-catenin was also present in these epithelial cells, and at this age fewer cells were positive for nuclear β-catenin staining (Figure 6N). As in the younger mutant mice, however, nuclear localization of β-catenin was only observed in K14-negative cells that looked like undifferentiated basal cells. In older P17 mice, the histopathology and keratin expression pattern of the mutant thymus was similar to that of P13 except for the fact that β-catenin expression became increasingly diffuse and appeared to colocalize with K8/K14 expression (data not shown). This coincided with fewer immature cells in the older mutant thymus. + +Collectively, these results suggest that loss of Apc and consequent stabilization of β-catenin in K14-expressing TECs lead to their aberrant proliferation and differentiation to keratinocytes, causing massive squamous metaplasia, rather than to form either medullary or cortical TECs. Loss of proper TEC compartments consequently resulted in loss of thymocytes for maturation and the mice to be “athymic.” + +Discussion + +Apc is implicated in the Wnt signaling pathway that is involved both in development and tumorigenesis. Human germline mutations in APC cause FAP [4,5], which is characterized by hundreds of adenomatous colorectal polyps, with an almost inevitable progression to colorectal cancer in the third and fourth decades of life. The phenotypical features of FAP and its variant, Gardner's syndrome, can be very variable. As well as colorectal polyps, these individuals can develop extracolonic symptoms, among which are upper gastrointestinal tract polyps, congenital hypertrophy of the retinal pigment epithelium, desmoid tumors, disorders of the maxillary and skeletal bones, and dental abnormalities [6]. While the heterozygous knockout mice for Apc develop adenomatous polyps predominantly in small intestine, the homozygous embryos die before gastrulation. To gain more insights into the effects of Apc loss in tissues other than gastrointestinal tract during life of animals and to circumvent the embryonic lethality associated with Apc nullizygosity, we created a mouse strain carrying a conditional allele of Apc (ApcCKO) in which exon 14 of the Apc is flanked by loxP sequences. The homozygous mice for the conditional allele are viable and indistinguishable to the normal mice, allowing us to study the roles of Apc in a tissue- and temporal-specific manner. + +To assess the phenotypic consequences of inactivation of the Apc gene in cells that express K14, we created mice that are homozygous for the ApcCKO allele and contain a K14-cre transgene. These mice failed to thrive and died before weaning. They also exhibited hair, tooth, and thymus phenotypes. + +Apc and Hair Follicle Morphogenesis + +Current models of hair follicle development suggest that the establishment of a regular array of placodes in the surface epithelium in response to the first dermal message is achieved through the competing activities of molecules that promote or repress placode fate [24]. There is accumulating evidence that activation of the Wnt signaling pathway in the dermis may be involved in establishing the first dermal signal. Experimental activation of epithelial β-catenin signaling (by expression of N-terminal–truncated, constitutively stabilized forms of β-catenin or ectopic expression of Lef1) induces ectopic follicles in both mouse and chick skin [25–27]. Conversely, down-regulation of β-catenin signaling (through Lef1 knock-out, ectopic expression of Wnt inhibitor Dkk1 or conditional deletion of β-catenin in epidermis) results in loss of vibrissae and some pelage follicles in mice [28–30]. Our finding that K14-driven Apc loss in embryonic ectoderm resulted in irregularly spaced and often tightly clustered abnormal hair placode initiation and follicle morphogenesis (Figures 4F, 5B, 5D, and 5F) as well as in the development of multiple tooth buds (Figure 4O and 4P), is in line with the effects seen in activation of epithelial β-catenin signaling during placode formation, indicating the role of Apc in specification of embryonic ectodermal stem cells to produce a hair follicle. Given the role of Apc in down-regulation of β-catenin, loss of Apc would inevitably lead to altered expression of β-catenin in ectodermal cells during the hair placode formation, giving rise to aberrant follicular growth throughout the embryonic epidermis, including the footpads, where normally hairless (Figure 5B, 5D, 5F, and 5H). Of interest are the phenotypic similarities and differences between our KA mutant mice and the previously described K14-ΔN87βcat transgenic mice expressing stable β-catenin under the control of K14 promoter [25] and K14-Lef1 transgenic mice [27]. Surprisingly, our mutant mice shared more similarities with the K14-Lef1 mice than the K14-ΔN87βcat transgenic mice. For example, the KA mutant mice displayed the disoriented and short, curly whiskers seen in K14-Lef1but not in K14-ΔN87βcat transgenic mice. In addition, KA mutant mice displayed a disorganized hair coat beginning with the emergence of neonatal hairs, similar to that observed in K14-Lef1 mice, whereas in K14-ΔN87βcat mice, embryonic hair follicle morphogenesis was unaffected and other skin changes only emerged postnatally. The reason for this difference could be attributed to one or all of three possible contributing factors. (1) Although the K14 promoter itself is known to initiate the expression at E9.5 and elevated dramatically by E13.5–E14.5 [19,31], many factors could influence the expression of promoter/transgene construct in transgenic mice. One of the most important considerations has to do with the integration site of the transgene in the mouse genome. Depending on the location of integration, the transcriptional activity of even the same transgene could vary considerably. It is possible that the apparent lack of ΔN87βcat function in embryogenesis could be due to the delayed or weak expression of the K14-ΔN87βcat transgene compared to the intrinsic promoter. In contrast, Cre-mediated recombination is seen in E13.5 skin of K14-cre transgenic mice used in this study [16] and K14-cre–mediated truncation of the Apc gene is likely to have occurred by then, in time for the second wave of hair follicle morphgenesis. (2) Another important fact is that in the aforementioned transgenic model the overexpression of transgene is confined to the basal cells of the epidermis and the ORS of hair follicles, where the K14 promoter is active. The expression of transgene is hence transient and stops once K14-expressing cells terminally differentiate. In contrast, in our model, K14-cre–mediated deletion of Apc would result in Apc mutation not only in K14 promoter active cells, but remain so in all cell layers that derived from the K14 promoter active progenitors. These derivatives include the suprabasal epidermis and the entire epithelium of the hair follicle. Stabilization of β-catenin in a broader population of cells could account for some of the phenotypic differences. However, we did not detect any elevated β-catenin expression in either K1- or K6-positive differentiated cells in postnatal mutant skin. This indicates that despite the absence of Apc and potential stabilization of β-catenin in derivatives of K14-expressing progenitors, elevated β-catenin expression, and subsequent cell fate determination may only take place in basal cells. (3) We cannot exclude the possibility that given the multifunctional properties of Apc, disruption of its functions other than down-regulation of β-catenin may also have contributed to the observed overt phenotype. + +While some features of our mutant mice were similar to these transgenic mice, other phenotypic aspects were largely distinct. In addition to aberrant hair follicle morphogenesis, K14-driven loss of Apc caused formation of multiple tooth buds that, like hair follicles, were known to develop through inductive interactions between the epithelium and mesenchyme. This observation was similar to the ectopic tooth buds found in animals misexpressing Lef1 [27], but more severe and was also present at birth, indicating the effect of Apc loss during the initiation of embryonic tooth development, which was evident by aberrant Shh expression in E15.5 embryonic oral epithelium (unpublished data). It should be noted that although multiple tooth buds were histologically visible (Figure 4O and 4P), these teeth never broke out and the KA mutant mice appeared toothless. This unusual severe tooth defect is unique to these mutant mice. In addition, neither of the two transgenic mice was postnatally lethal as in the KA mutant mice. We did not find any obvious histopathological abnormalities in the internal organs of KA mice that could contribute to the lethality. However, the fact that all the mutants had lower weight (Figure 2F) with hardly any evidence of solid food in their stomach indicates that the mutants might have died of starvation. Dermal fat was reduced in the mutant skin, possibly as a consequence of poor nutrition caused by the absence of teeth. Since the weight loss in KA mutants started from P8–P10 while pups were still nursed by their mothers, starvation due to lack of teeth cannot be the sole cause of death, but is likely to be a contributing factor. Absence of teeth and mammary glands have been observed in mice deficient in Lef1 and ectopically expressing Dkk1 [29,30] but their absence was due to the block in development before the bud stage. Hence, neither loss nor excess of tooth bud formation allows proper development of teeth for mice to have a healthy diet and normal life. Mechanistic studies to understand how increased levels of β-catenin leads to altered skin and tooth phenotypes are under way. + +Apc and Thymus Organogenesis + +In this study, we observed that K14-driven loss of Apc resulted in a small thymus with severe squamous metaplasia leading to the formation of numerous pyogranuloma and loss of proper meshwork structure for thymocyte maturation, rendering the mice athymic. Previous studies have shown that in normal adult thymus K14 expression is found together with K5 in the stellate medullary TECs, but not in association with K5+ TECs in the cortex or at the cortico-medullary junction. In addition, it has been demonstrated that K14+ TECs do not coexpress K8; hence, there are two distinct medullary subsets, namely a K8−K14+ stellate subset and a K8+K14− globular subset [32]. In agreement with the previous results, in P3 normal thymus K14 expression was restricted to stellate medullary TECs (Figure 6D), whereas K8 expression was found throughout the TECs (unpublished data). We could not clarify whether these two keratins were coexpressed in the same TECs without double-staining. There were individual differences among mutants and these were prominently reflected in the histological abnormalities of the thymus at P3, but as the older surviving mutants all showed the same histopathologies of the thymus, the mutant thymi eventually seem to result in the same fate. It is unclear when K14-cre induction actually takes place in the mutant thymus, but as the population of cells showing a strong nuclear β-catenin staining as well as the cells expressing K14 were small and thymic epithelial compartments still existed in a mild P3 mutant thymus (Figure 6E and 6F), it seems that initial differentiation to medullary and cortical TECs and thymocyte colonization have already taken place prior to the major effects of K14-driven Apc loss. However, cells with nuclear β-catenin and K8+K14+ double-positive epithelial cells increased subsequently, associated with active proliferation in the latter group of cells and loss of TEC compartments. With age, β-catenin expression pattern became more diffuse and fewer epithelial cells showed nuclear localization but K8+K14+ cells remained, forming concentric structures of epithelial cells filled with hard keratin deposits and infiltrated with vast number of neutrophils and macrophages. Only a few epithelial cells were dividing and hardly any thymocytes were present by this time. These results suggest that with the deletion of Apc in TECs, stabililization and nuclear localization of β-catenin took place and subsequently these cells differentiated into keratinocytes that expressed both K14 and K8, similar to the basal cells of the skin, instead of TEC subsets. The expansion and differentiation of these keratinocytes lead to loss of proper thymic epithelial compartments and in turn produced and deposited the hard keratins that consequently caused vast amount of neutrophils and macrophages to infiltrate the thymus. These aberrant epithelial structures eventually have overtaken the whole of the thymus and driven out the colonized thymocytes. K14-driven loss of Apc and subsequent constitutive expression of β-catenin in TECs have therefore misdirected them to wrong epithelial cell fate, not allowing proper differentiation to either cortical or medullary TECs, which is essential for normal thymocyte development. This is not only evident from the lack of dividing thymocytes in the mutant thymus by P13, but also by the differential expression pattern of keratins, which were more skin-like than TEC-like. The importance of Apc function in thymic development has been demonstrated by thymocyte-specific loss of Apc by crossing a different strain of Apc conditional mice and LckCre transgenic mice [33]. Here, by K14-driven TEC-specific loss of Apc, we have demonstrated its importance in thymus development not only in thymocytes but also in TECs. + +It is of interest that dental abnormalities, such as supernumerary and impacted teeth similar to those observed in our mutant mice, are frequently seen in patients with Gardner's syndrome, carriers of APC germline mutation [6]. The importance of odonto-stomatological examinations should hence be pointed out as a means of reaching a presumptive diagnosis, whose confirmation is vital to the patient. Further characterization of the mechanism of such developmental defects using our mouse model should provide important insights into Apc function in multiple organ systems and to give better insights into potential adverse events in human subjects. + +In conclusion, we have shown that loss of Apc in K14-expressing embryonic cells causes aberrant morphogenesis in various skin appendages, including hair follicles and teeth, and abnormal thymus organogensis. Our results provide genetic evidence that expression of Apc is essential for regulation of proper cell fates in these organs that require epithelial–mesenchymal interactions. + +Materials and Methods + +Construction of targeting vectors. + +To target the Apc locus, we obtained BAC clone RP23-233F17 that contains all the sequence of Apc except for the 5′ UTR and exon 1. Using a recombineering-based method for generating conditional mutations [12,13], subcloning of BAC and further modifications were conducted. The genomic sequence encompassing Apc exons 11 to 15 was first subcloned into pBR322 vector, and then a loxP site was introduced into intron 13 followed by loxP-FRT-PGKneor-FRT selection cassette into intron14 of the Apc gene (Figure 1A). + +Generation of ApcCKO ES cells and mice. + +The linearized targeting vectors were electroporated into 129/S-derived ES cells, WW6 cells [15]. All candidate G418-resistant ES clones were screened by long-range gene-specific PCR using Expand Long Template PCR System (Roche, Indianapolis, Indiana, United States), followed by sequencing to validate the correct insertion of the single loxP site and the selection cassette. Two ES clones with correct ApcCKON/+ modification were injected into C57BL/6J blastocysts, after which chimeric mice with high levels of ES cell contribution were backcrossed to C57BL/6J females to produce heterozygous F1 offspring. The genomic DNA samples obtained from the tail of F1 offspring were subsequently analyzed by Southern blot analysis to further confirm the correct homologous recombination. By crossing the heterozygous mice to FLPe deleter mice [34], PGKneor cassette was deleted in the germline by FRT-mediated recombination to generate mice with the final ApcCKO allele. ApcCKO heterozygous mice were crossed together to generate homozygous mice, and the homozygous offspring were interbred for maintenance. + +Generation of ApcΔ580 mice. + +ApcCKO heterozygote mice were crossed with Cre deleter mice, EIIA-cre transgenic mice that express Cre in early embryo [35], to knockout the Apc allele in the germline, consequently creating a knockout strain (Figure 1A). The resultant ApcΔ580/+ mice were maintained by backcrossing to C57BL/6J females. + +Generation of K14-cre; ApcCKO/CKO mice. + +The mice analyzed in this study were generated by crossing ApcCKO heterozygote mice of the F1 generation (C57BL/6J × 129/S background) with K14-cre transgenic mice (FVB background). K14-cre; ApcCKO/+ male mice thus generated were then crossed with ApcCKO/CKO females to generate homozygous and heterozygous ApcCKO offspring either with or without K14-cre. The mice were intercrossed thereafter for maintenance. + +Genotyping of mice. + +Genomic DNA from tips of mouse tails obtained at ~10 d of age was genotyped in a single PCR reaction with the following primers: Apc-Int13F2 (GAGAAACCCTGTCTCGAAAAAA) and Apc-Int13R2 (AGTGCTGTTTCTATGAGTCAAC), resulting in 320-bp and 430-bp products from the wild-type and conditional ApcCKO allele, respectively. For the detection of Cre-mediated deleted Apc allele, ApcΔ580, the primer Apc-Int14R4 (TTGGCAGACTGTGTATATAAGC) in combination with Apc-Int13F2 resulted in 500-bp product. Primers Cre-F1 (TCCAATTTACTGACCGTACACC) and Cre-R1 (CCGACGATGAAGCATGTTTAG) were used for detection of cre transgene in the germline, resulting in 300-bp products. Amplification was performed in a 25-μl volume containing 15 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.2 mM dNTP, 0.2μM of each primer, and 1.25 U of AmpliTaq Gold (Applied Biosystems, Foster City, California, United States). The reactions were heated for 10 min at 94 °C to heat-activate the enzyme followed by 30 PCR cycles at 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 45 s, followed by the final extension for 5 min at 72 °C. + +Skin permeability assay. + +Unfixed and freshly isolated E18.5 embryos were rinsed in PBS and then submerged in X-gal reaction mix at pH 4.5 (100 mM NaPO4, 1.3 mM MgCl2, 3 mM K3Fe(CN)6, 3 mM K4Fe(CN)6, and 1 mg/ml X-gal) at room temperature overnight [36]. At this pH in the absence of epidermal barrier, the solution penetrates epidermis and an endogenous β-galactosidase–like activity catalyzes production of a blue precipitate. After staining, embryos were washed twice in PBS, fixed overnight at 4 °C in 4% paraformaldehyde, and were photographed using a 35 mm Nikon digital camera. + +Skeletal preparations. + +The skinned and eviscerated bodies of the oldest surviving KA mutants (P16–P17) and their age-matched littermates were placed into 1% potassium hydroxide (KOH) for 5 d. The bodies were transferred into a fresh solution of 1% KOH containing a few drops of 0.5% alizarin red S (Sigma, St. Louis, Missouri, United States) and left for another 5 d. The stained bodies were stored in glycerin and viewed under a dissection microscope. + +Analysis for tissue-specific recombination. + +Genotype/tissue-specific recombination of the conditional allele was examined by both PCR and RT-PCR. DNA samples extracted from various tissues collected at the time of autopsy were examined by genotyping PCR as described. RNA extracted from various tissue homogenates in Trizol reagent were examined for expression of wild-type and truncated Apc alleles by SuperScript One-Step RT-PCR with Platinum Taq (Invitrogen, Carlsbad, California, United States), following manufacturer's protocol. Approximately 200 ng of total RNA from either skin, liver, or thymus from each genotype was reverse-transcribed using primers Apc-F546 (TGAGGAATTTGTCTTGGCGAG) and Apc-R721 (GCACTTCCCATGGCAATCATT), resulting in 528-bp and 313-bp products from the wild-type/ApcCKO alleles and ApcΔ580 allele, respectively. + +BrdU labeling. + +Mice were injected intraperitoneally with approximately 50 μg/g body weight of BrdU (Sigma) dissolved in PBS 2 h before their death. Tissue samples were fixed in Bouin's and processed as described below. + +Histological and immunochemical analysis. + +Mutant mice and age-matching littermates were humanely killed at various ages by CO2 inhalation. Mice were skinned and pieces of skin were either snap-frozen in liquid nitrogen or immediately homogenized in Trizol reagent and stored at −80 °C until molecular analysis, or fixed flat on a piece of paper towel in Bouin's solution for histological and immunohistochemical examinations. The mice were then dissected for gross examination, various tissues were similarly collected for future molecular analyses, and then the whole body was fixed in Bouin's solution. The samples were then submitted to Rodent Histopathological Core for processing and histopathological examinations. + +For immunohistochemistry, 5-μm paraffin-embedded tissue sections were deparafinized in xylene, followed by alcohol rehydration. After quenching endogenous peroxidases in 3% H2O2 in methanol, the slides were rinsed in distilled water, and an antigen retrieval step was carried out in a microwave oven for a total of 10 min in preheated citrate buffer (pH 6.0). The slides were then incubated with primary antibodies at room temperature overnight. Antibodies used were β-catenin (BD Transduction Lab, San Diego, California, United States), keratins 1, 5, 6, 14, involucrin, loricrin (Covance, Berkeley, California, United States), keratin 8 (Abcam, Cambridge, United Kingdom), Ki67 (Vector Laboratories, Burlingame, California, United States) and BrdU (Roche). Biotinylated secondary antibodies (donkey anti-rabbit and goat anti-mouse IgG, 1:250; Jackson ImmunoResearch, West Grove, Pennsylvania, United States), followed by the Vectastain Elite ABC kit (Vector Laboratories) were used for detection. The slides were stained with DAB and counterstained with Mayer's hematoxylin. + +In situ hybridization. + +Section ISH using rat Shh probe [37] and whole-mount ISH using β-catenin probe [38] were performed as previously described [39,40]. Riboprobes labeled with DIG were detected with BM purple AP substrate precipitation solution (Roche). + +Supporting Information + +Figure S1 + +Multiple Intestinal Neoplasia in ApcΔ580 Mice + +(A) A representative appearance of small intestine of 4-month-old mouse. Scale bar, 1.5 mm. + +(B) Histological analysis of longitudinal section of small intestine shown in (A) by H&E staining. Scale bar, 100 μm. + +(C) The typical histopathological appearance of an adenoma shown in (B). Scale bar, 20μm. + +(621 KB PPT) + +Click here for additional data file. + +Figure S2 + +Skin Permeability Assay and Neonatal Mice + +(A) Skin permeability assay of E18.5 embryos. Both normal (N) and KA mutant (M) embryos can exclude the blue dye at this age, showing no defects in skin barrier function. Note slight pigmentations in pinnnae and “birthmark” in the forehead of mutant embryo (B), which are present at P1 (C), and become prominent as they grow. + +(749 KB PPT) + +Click here for additional data file. + +Figure S3 + +Immunochemical Examination of P13 Normal and Mutant Thymi + +(A–E) P13 normal thymus. (F–K) P13 mutant thymus. Stained with K8 (A), (D), (F), (I), K1 (B), (E), (G), (J), and involucrin (C), (H), (K). Scale bars, 100 μm for (A–C), (F–H), (J–K) and 20 μm for (D–E), (I). Note the lack of involucrin staining in normal thymus but varying degree of positive cells in mutant thymus. + +(6.4 MB PPT) + +Click here for additional data file. + +Accession Numbers + +The GenBank ((http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the genes and gene products discussed in this paper are Apc (NM_007462), β-catenin (NM_007614), AXIN 2 (NM_004655), plakoglobin (NM_002230), Asef (NM_015320), kinesin superfamily–associated protein 3 (NM_014970), EB1 (NM_012325), human homolog of Drosophila Discs large (U13897), Lef1 (NM_010703), Dkk1 (NM_010051), K1 (NM_008473), K5 (NM_027011), K6 (NM_008476), K8 (NM_031170), K14 (NM_016958), involucrin (NM_008412), loricrin (NM_008508), and Shh (NM_009170). + +Abbreviations + +APC - adenomatous polyposis coli + +CKO - conditional knockout + +E - embryonic day + +ES - embryonic stem + +FAP - familial adenomatous polyposis + +FRT - FLP recognition target + +ISH - in situ hybridization + +K14 - keratin 14 + +ORS - outer root sheath + +P - postnatal day + +TEC - thymic epithelial cell + +Figures and Tables + +Figure 1 + +Generation of the Conditional Apc Allele + +(A) Schematic diagram of exons 14 and 15 of the mouse Apc gene, the targeting vector, and the resulting conditional allele with 2 LoxP sites sandwiching the exon 14. The PGK-neomycin cassette was inserted within intron 14 by recombineering technique. This cassette is sandwiched by 2 FRT sites that could be removed by crossing to FLPe-expressing mice. Positions of PCR primers used for genotyping PCR (F2, R2, R4) and RT-PCR (F546 and R721) are indicated. Positions of probe used for Southern blot analysis with NdeI sites are also shown. Upon Cre-mediated recombination, exon 14 is removed and leads to truncated Apc protein, of which the first 580 aa correspond to the normal. + +(B) Southern blot analysis of NdeI-digested genomic tail DNA isolated from F1 mice of various Apc mouse lines (ApcCKON, ApcΔ580), hybridized to a 600-bp probe. Tail genomic DNA from ApcCKON F1 mice derived from a modified ES clone showed a 12-kb band for the ApcCKON allele and a 10-kb band for the wild-type allele, whereas genomic DNA from the ApcΔ580 mouse was heterozygous for the ApcΔ580 allele (9.2-kb band). + +(C) Kaplan-Meier survival plot of ApcCKO/+ mice (thin solid line, n = 39), ApcCKO/CKO mice (thin dotted line, n = 57), ApcΔ580/+ mice (solid line, n = 51), and wild-type littermates (broken line, n = 21). Heterozygosity of the ApcΔ580 allele led to a significantly shortened survival (p < 0.0001), whereas those of heterozygous and homozygous ApcCKO mice had no significant difference to that of wild-type littermates. + +Figure 2 + +Postnatal Mortality and Stunted Growth in K14-cre; ApcCKO/CKO Mutant Mice + +Animals whose genotype is either heterozygous or homozygous for the wild-type Apc allele are referred to as normal (N); those whose genotype are K14-cre; ApcCKO/CKO and show the presence of K14-cre–recombined mutant Apc allele are called mutant (M). + +(A) Two P3 mutant mice, M1 and M2, and their normal littermates, showing size variation among mutants. + +(B) P8 mutant mouse (right) and a normal littermate. Note sparseness of hair coat and abnormal ears. + +(C–D) Vibrissae of whisker pads are short and oddly angled in a P12 mutant mouse (C), relative to control (D). Note the lack of incisors in the mutant. + +(E) A P17 mutant mouse (right) with its littermate. Its bare forehead, dorsal median line, and abnormal ears are evident. + +(F) Growth curve of mutants and normal littermates. Mutants exhibit stunted growth, which became more prominent as they aged, and weigh significantly less than littermates from P8 (p < 0.05). + +(G) Comparison of mutant and normal thymus from P3 mice. The mutant thymus (left) is dramatically smaller for its age compared to the normal littermate (right). The scale bar equals 1 mm. + +(H) Skeletal preparations of normal (left) and mutant (right), showing differences in development of both incisor (I) and molar (M) teeth. + +Figure 3 + +Tissue-Specific Detection and Expression of Deleted Apc Alleles + +(A) Tissue-specific genotyping PCR. Only genomic DNA samples from the skin (S) and thymus (T), but not liver (L) of mice positive for K14-cre show the presence of deleted ApcΔ580 allele. + +(B) Genotype- and tissue-specific expression of the truncated Apc transcripts. A representative gel of RT-PCR using primers F546 and R721, showing that only RNA from the skin and thymus but not liver of mice positive for K14-cre have transcripts from both wild-type (528 bp) and deleted (313 bp) Apc alleles. + +Figure 4 + +Histological and Immunochemical Examination of P12 Skin and Teeth + +(A–E) P12 normal skin. (F–J) P12 mutant skin. (K–N) P12 normal oral cavity. (O–R) P12 mutant oral cavity. Stained with H&E for histology (A, F, K–L, O–P), Ki67 (B, G), β-catenin (C, H, M, Q), K14 (D, I, N, R), and K6 (E, J). Aberrant follicular morphogenesis, characterized by formation of irregularly spaced, nonpolarized hair follicles, in mutant skin is evident. Despite the abnormal histology, proliferation seems to be confined to hair bulb-like structures (arrows in [G], inset [G′] at higher magnification), but in mutant skin (arrows in [H], inset [H′] at higher magnification) and oral cavity (arrows in insets [Q′] at higher magnification) elevated cytosolic localization of β-catenin is detected in some cells. Scale bars: 50 μm for (A–F), (H–J); 250 μm for (K) and (O); 100 μm for (G), (L–N), (P–R); 20 μm for (Q′). + +Figure 5 + +Expression of Shh and β-catenin Transcripts in Normal (ApcCKO/CKO) and Mutant (K14-cre; ApcCKO/CKO) Embryonic Skin + +(A–D) Section ISH with Shh probe in E14.5 normal (A), E14.5 mutant (B), E16.5 normal (C), and E16.5 mutant (D) skin. Broken lines indicate the interface between epithelium and mesenchyme. Scale bars: 50 μm. Whole mount in situ detection of β-catenin in E15.5 normal (E, G), mutant (F, H) embryos. Aberrant initiation of multiple hair placodes is evident at E14.5. Loss of K14-driven Apc loss caused aberrant pattern formation (F′) and formed ectopic hair placodes in normally hairless foot pads (H, arrows) which are absent in normal (G). + +Figure 6 + +Histological and Immunochemical Examination of Thymus + +(A–D) P3 normal thymus. (E–G) Mild P3 mutant thymus. (H–K) Severe P3 mutant thymus. (L–O) P13 mutant thymus. Stained with H&E for histology (A, E, H, L), BrdU (B, I, M), β-catenin (C, F, J, N), and K14 (D, G, K, O). (B) Actively dividing thymocytes are visible at the superficial edge of cortex of normal P3 thymus. Note the progression of histological abnormalities in the mutant thymus from mild P3, severe P3 to P13 (A, E, H, L). Scale bars, 20 μm. + +Table 1 + +Genotype Distribution of Progeny from the Matings + +Footnotes + +Competing interests. The authors have declared that no competing interests exist. + +A previous version of this article appeared as an Early Online Release on July 28, 2006 (DOI: 10.1371/journal.pgen.0020146.eor). + +Author contributions. M. Kuraguchi and R. Kucherlapati conceived and designed the experiments. M. Kuraguchi, X. Wang, R. Bronson, R. Rothenberg, N. Ohene-Baah, J. Lund, and M. Kucherlapati performed the experiments. M. Kuraguchi analyzed the data. R. Maas contributed reagents/materials/analysis tools. M. Kuraguchi wrote the paper. + +Funding. This work was supported by grants from the National Cancer Institute (CA-084301), the National Institute of Environmental Health Sciences (ES-11040), and the National Institute of Dental and Craniofacial Research (DE-11697, DE-16140). diff --git a/src/ontogpt/evaluation/craft/database/all/17020410.ann b/src/ontogpt/evaluation/craft/database/all/17020410.ann new file mode 100644 index 000000000..72f22132f --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17020410.ann @@ -0,0 +1,918 @@ +T1 http://purl.obolibrary.org/obo/MONDO_0020732 10 18 Progeria +T2 http://purl.obolibrary.org/obo/MONDO_0018053 22 41 Trichothiodystrophy +T3 PR:000007164 63 66 Xpd +T4 SO:0001023 67 74 Alleles +T5 SO:0001023 160 167 alleles +T6 SO:0000704 180 184 gene +T7 NCBITaxon:9606 199 204 human +T8 http://purl.obolibrary.org/obo/MONDO_0000001 215 222 disease +T9 http://purl.obolibrary.org/obo/MONDO_0000001 248 255 disease +T10 SO:0001023 378 385 allelic +T11 SO:0000704 429 436 genetic +T12 SO:0001023 499 506 alleles +T13 PR:000007164 565 568 XPD +T14 http://purl.obolibrary.org/obo/MONDO_0000001 579 588 disorders +T15 NCBITaxon:10088 614 619 mouse +T16 GO:0006281 691 701 DNA repair +T17 http://purl.obolibrary.org/obo/MONDO_0004992 798 804 cancer +T18 http://purl.obolibrary.org/obo/MONDO_0020732 845 853 progeria +T19 SO:0001023 880 887 allelic +T20 NCBITaxon:1 899 909 organismal +T21 PR:000007164 962 965 Xpd +T22 SO:0001023 966 973 alleles +T23 PR:000007164 1037 1040 Xpd +T24 SO:0001023 1041 1048 alleles +T25 http://purl.obolibrary.org/obo/MONDO_0000001 1076 1083 disease +T26 http://purl.obolibrary.org/obo/MONDO_0000001 1170 1177 disease +T27 SO:0001023 1186 1192 allele +T28 UBERON:0000479 1230 1236 tissue +T29 PR:000007164 1268 1271 Xpd +T30 SO:0001023 1272 1278 allele +T31 SO:0001023 1304 1311 allelic +T32 NCBITaxon:40674 1389 1396 mammals +T33 SO:0001023 1461 1468 alleles +T34 PR:000007164 1472 1475 XPD +T35 http://purl.obolibrary.org/obo/MONDO_0000001 1476 1485 disorders +T36 SO:0001023 1543 1550 alleles +T37 NCBITaxon:40674 1615 1622 mammals +T38 SO:0001023 1644 1651 allelic +T39 SO:0001023 1724 1731 alleles +T40 NCBITaxon:1 1827 1836 organisms +T41 NCBITaxon:9606 1899 1904 human +T42 http://purl.obolibrary.org/obo/MONDO_0000001 1905 1912 disease +T43 SO:0001023 1952 1959 allelic +T44 GO:0008152 2069 2078 metabolic +T45 http://purl.obolibrary.org/obo/MONDO_0005066 2069 2088 metabolic disorders +T46 CHEBI:15539 2123 2135 propinyl-CoA +T47 CHEBI:57472 2153 2170 argininosuccinate +T48 CHEBI:53025 2182 2203 galactose-1-phosphate +T49 CHEBI:16625 2233 2250 methylmalonyl CoA +T50 NCBITaxon:1 2291 2302 individuals +T51 SO:0001023 2333 2340 alleles +T52 SO:0000704 2353 2357 gene +T53 SO:0001023 2405 2411 allele +T54 SO:0000704 2413 2420 genetic +T55 SO:0001023 2452 2459 alleles +T56 SO:0001023 2484 2491 allelic +T57 SO:0001023 2563 2570 allelic +T58 http://purl.obolibrary.org/obo/MONDO_0000001 2613 2620 disease +T59 SO:0001023 2638 2645 allelic +T60 SO:0001023 2749 2756 allelic +T61 SO:0001023 2784 2791 allelic +T62 NCBITaxon:9606 2803 2808 human +T63 http://purl.obolibrary.org/obo/MONDO_0000001 2809 2816 disease +T64 SO:0000704 2904 2911 genetic +T65 PR:000007164 2925 2928 XPD +T66 GO:0005675 2975 3016 basal transcription/DNA repair factor IIH +T67 GO:0006281 2995 3005 DNA repair +T68 GO:0005675 3018 3023 TFIIH +T69 GO:0032991 3057 3064 complex +T70 GO:0006281 3152 3169 DNA damage repair +T71 GO:0006289 3178 3204 nucleotide excision repair +T72 GO:0006289 3206 3209 NER +T73 PR:000007164 3241 3244 XPD +T74 GO:0005675 3268 3273 TFIIH +T75 http://purl.obolibrary.org/obo/MONDO_0015938 3317 3338 multisystem disorders +T76 UBERON:0000467 3322 3328 system +T77 http://purl.obolibrary.org/obo/MONDO_0019600 3349 3370 xeroderma pigmentosum +T78 http://purl.obolibrary.org/obo/MONDO_0019600 3372 3374 XP +T79 http://purl.obolibrary.org/obo/MONDO_0019600 3377 3379 XP +T80 http://purl.obolibrary.org/obo/MONDO_0016006 3394 3411 Cockayne syndrome +T81 http://purl.obolibrary.org/obo/MONDO_0016006 3413 3415 CS +T82 http://purl.obolibrary.org/obo/MONDO_0018053 3422 3441 trichothiodystrophy +T83 http://purl.obolibrary.org/obo/MONDO_0018053 3443 3446 TTD +T84 http://purl.obolibrary.org/obo/MONDO_0019600 3456 3458 XP +T85 http://purl.obolibrary.org/obo/MONDO_0002898 3550 3561 skin cancer +T86 http://purl.obolibrary.org/obo/MONDO_0016006 3658 3660 CS +T87 http://purl.obolibrary.org/obo/MONDO_0018053 3665 3668 TTD +T88 http://purl.obolibrary.org/obo/MONDO_0015333 3703 3722 progeroid disorders +T89 GO:0007567 3757 3762 natal +T90 http://purl.obolibrary.org/obo/MONDO_0004992 3862 3868 cancer +T91 http://purl.obolibrary.org/obo/MONDO_0018053 3907 3910 TTD +T92 UBERON:0001037 3967 3971 hair +T93 UBERON:0001705 3976 3981 nails +T94 http://purl.obolibrary.org/obo/MONDO_0000001 4091 4099 disorder +T95 http://purl.obolibrary.org/obo/MONDO_0004992 4109 4115 cancer +T96 http://purl.obolibrary.org/obo/MONDO_0019600 4134 4136 XP +T97 GO:0007399 4155 4173 neurodevelopmental +T98 http://purl.obolibrary.org/obo/MONDO_0016006 4191 4193 CS +T99 http://purl.obolibrary.org/obo/MONDO_0016354 4195 4199 XPCS +T100 PR:000007164 4253 4256 XPD +T101 http://purl.obolibrary.org/obo/MONDO_0000001 4300 4307 disease +T102 PR:000007164 4325 4328 XPD +T103 http://purl.obolibrary.org/obo/MONDO_0018053 4339 4342 TTD +T104 PR:000007164 4347 4350 XPD +T105 http://purl.obolibrary.org/obo/MONDO_0019600 4361 4363 XP +T106 http://purl.obolibrary.org/obo/MONDO_0002254 4419 4428 syndromes +T107 SO:0001023 4430 4437 Alleles +T108 http://purl.obolibrary.org/obo/MONDO_0000001 4474 4482 disorder +T109 SO:0001023 4512 4519 alleles +T110 SO:0001023 4543 4550 alleles +T111 NCBITaxon:4896 4590 4621 Schizosaccharomyces pombe yeast +T112 PR:000007164 4657 4660 XPD +T113 SO:0000853 4661 4670 homologue +T114 PR:P26659 4671 4676 rad15 +T115 SO:0001023 4772 4779 alleles +T116 SO:0001023 4853 4860 allelic +T117 PR:000007164 4874 4877 XPD +T118 http://purl.obolibrary.org/obo/MONDO_0000001 4878 4885 disease +T119 http://purl.obolibrary.org/obo/MONDO_0019600 4934 4936 XP +T120 http://purl.obolibrary.org/obo/MONDO_0000001 4984 4991 disease +T121 http://purl.obolibrary.org/obo/MONDO_0019600 5033 5035 XP +T122 http://purl.obolibrary.org/obo/MONDO_0018053 5040 5043 TTD +T123 SO:0001023 5090 5097 allelic +T124 http://purl.obolibrary.org/obo/MONDO_0018053 5196 5199 TTD +T125 NCBITaxon:10088 5200 5205 mouse +T126 PR:000007164 5213 5216 XPD +T127 NCBITaxon:9606 5244 5249 human +T128 http://purl.obolibrary.org/obo/MONDO_0002254 5250 5258 syndrome +T129 PR:000007164 5319 5322 Xpd +T130 SO:0001023 5323 5330 alleles +T131 http://purl.obolibrary.org/obo/MONDO_0018053 5403 5406 TTD +T132 GO:0007568 5438 5444 ageing +T133 GO:0006281 5475 5485 DNA repair +T134 PR:000007164 5583 5586 Xpd +T135 PR:000007164 5627 5630 Xpd +T136 SO:0001023 5640 5646 allele +T137 PR:000007164 5706 5709 XPD +T138 http://purl.obolibrary.org/obo/MONDO_0016354 5729 5733 XPCS +T139 GO:0010467 5769 5779 expression +T140 SO:0001023 5798 5804 allele +T141 UBERON:0000922 5826 5835 embryonic +T142 CL:0002322 5826 5846 embryonic stem cells +T143 GO:0010467 5879 5889 expression +T144 UBERON:0000473 6012 6018 testis +T145 NCBITaxon:33208 6035 6042 animals +T146 PR:000007164 6107 6110 XPD +T147 PR:000007164 6120 6123 XPD +T148 GO:0010467 6130 6139 expressed +T149 SO:0001023 6154 6160 allele +T150 NCBITaxon:10088 6267 6271 mice +T151 UBERON:0000922 6323 6332 embryonic +T152 UBERON:0000922 6358 6365 embryos +T153 SO:0001023 6415 6421 allele +T154 http://purl.obolibrary.org/obo/MONDO_0016354 6500 6504 XPCS +T155 SO:0001023 6505 6511 allele +T156 GO:0010467 6547 6557 expression +T157 SO:0000704 6599 6603 gene +T158 PR:000007164 6662 6665 Xpd +T159 PR:000007164 6676 6679 Xpd +T160 UBERON:0000104 6717 6721 life +T161 GO:0009790 6752 6765 embryogenesis +T162 PR:000007164 6830 6833 Xpd +T163 PR:000007164 6852 6855 XPD +T164 http://purl.obolibrary.org/obo/MONDO_0019600 6887 6889 XP +T165 NCBITaxon:9606 6917 6923 humans +T166 GO:0010467 6944 6953 expressed +T167 SO:0001023 7008 7014 allele +T168 SO:0000205 7129 7134 3′UTR +T169 SO:0000704 7158 7162 gene +T170 PR:000007164 7192 7195 Xpd +T171 PR:000007164 7234 7237 Xpd +T172 PR:000007164 7248 7251 XPD +T173 NCBITaxon:33208 7264 7271 animals +T174 NCBITaxon:10088 7306 7311 Mouse +T175 PR:000007164 7312 7315 Xpd +T176 SO:0000704 7316 7320 Gene +T177 SO:0001026 7358 7365 genomic +T178 SO:0001250 7388 7403 restriction map +T179 NCBITaxon:10088 7427 7432 mouse +T180 PR:000007164 7433 7436 Xpd +T181 PR:000007164 7454 7457 Xpd +T182 SO:0001023 7458 7464 allele +T183 SO:0001215 7489 7512 coding regions of exons +T184 SO:0000205 7531 7536 3′UTR +T185 GO:0006415 7586 7604 translational stop +T186 SO:0000319 7586 7610 translational stop codon +T187 SO:0000551 7612 7617 PolyA +T188 GO:0043631 7632 7647 polyadenylation +T189 SO:0000551 7632 7654 polyadenylation signal +T190 PR:000007164 7664 7667 Xpd +T191 SO:0001023 7680 7686 allele +T192 SO:0000028 7696 7705 base pair +T193 SO:0000028 7707 7709 bp +T194 NCBITaxon:9606 7711 7716 human +T195 PR:000007164 7717 7720 XPD +T196 SO:0000147 7744 7748 exon +T197 NCBITaxon:9031 7848 7855 Chicken +T198 PR:000008457 7856 7864 β-globin +T199 CHEBI:5386 7858 7864 globin +T200 SO:0000147 7865 7870 exons +T201 SO:0000205 7893 7898 3′UTR +T202 PR:000008457 7978 7986 β-globin +T203 CHEBI:5386 7980 7986 globin +T204 GO:0043631 7987 8002 polyadenylation +T205 SO:0000551 7987 8009 polyadenylation signal +T206 SO:0000551 8011 8017 PolyA* +T207 PR:000007164 8028 8031 Xpd +T208 PR:000007164 8039 8042 Xpd +T209 SO:0001023 8057 8064 alleles +T210 SO:0000147 8150 8155 exons +T211 SO:0000061 8274 8291 Restriction sites +T212 PR:P23940 8293 8294 B +T213 PR:P23940 8296 8301 BamHI +T214 PR:P43870 8322 8323 H +T215 PR:P43870 8325 8332 HindIII +T216 SO:0001026 8400 8407 genomic +T217 PR:000007164 8421 8424 Xpd +T218 PR:000007164 8438 8441 Xpd +T219 UBERON:0000922 8460 8469 embryonic +T220 CL:0002322 8460 8479 embryonic stem cell +T221 GO:0097617 8487 8497 hybridised +T222 SO:0001023 8540 8546 allele +T223 PR:000007164 8606 8609 Xpd +T224 PR:000007164 8617 8620 Xpd +T225 SO:0001023 8626 8633 alleles +T226 SO:0001023 8694 8701 alleles +T227 SO:0000112 8715 8722 primers +T228 SO:0000028 8782 8784 bp +T229 SO:0000028 8793 8795 bp +T230 GO:0010467 8841 8851 expression +T231 SO:0001023 8896 8903 alleles +T232 UBERON:0000922 8907 8916 embryonic +T233 CL:0002322 8907 8926 embryonic stem cell +T234 SO:0000112 8940 8947 primers +T235 GO:0097617 8952 8963 hybridising +T236 SO:0000028 9043 9045 bp +T237 UBERON:0000473 9111 9117 testis +T238 PR:000007164 9139 9142 Xpd +T239 PR:000007164 9164 9167 Xpd +T240 PR:000007164 9217 9220 Xpd +T241 NCBITaxon:10088 9230 9234 mice +T242 GO:0097617 9249 9262 Hybridisation +T243 NCBITaxon:10088 9277 9282 mouse +T244 PR:000007164 9283 9286 Xpd +T245 PR:000007164 9343 9346 Xpd +T246 PR:000007164 9357 9360 Xpd +T247 SO:0001023 9364 9371 alleles +T248 CHEBI:4883 9390 9406 ethidium bromide +T249 CHEBI:4883 9408 9412 EtBr +T250 PR:000007164 9504 9507 Xpd +T251 PR:000007164 9516 9519 Xpd +T252 PR:000007164 9558 9561 Xpd +T253 UBERON:0000922 9571 9578 Embryos +T254 SO:0001023 9601 9607 Allele +T255 UBERON:0001037 9653 9657 Hair +T256 http://purl.obolibrary.org/obo/MONDO_0018053 9670 9673 TTD +T257 SO:0001023 9727 9733 allele +T258 NCBITaxon:1 9764 9774 organismal +T259 PR:000007164 9801 9804 Xpd +T260 SO:0001023 9810 9816 allele +T261 PR:000007164 9831 9834 Xpd +T262 SO:0001023 9838 9844 allele +T263 NCBITaxon:33208 9888 9895 animals +T264 NCBITaxon:10088 9923 9927 mice +T265 PR:000007164 9946 9949 Xpd +T266 SO:0001023 9959 9965 allele +T267 PR:000007164 9967 9970 Xpd +T268 PR:000007164 10001 10004 Xpd +T269 NCBITaxon:10088 10014 10018 mice +T270 GO:0007567 10024 10028 born +T271 GO:0010467 10068 10078 Expression +T272 PR:000007164 10088 10091 Xpd +T273 SO:0001023 10097 10103 allele +T274 UBERON:0000473 10128 10134 testis +T275 NCBITaxon:33208 10160 10167 animals +T276 GO:0010467 10177 10187 expression +T277 PR:000007164 10197 10200 Xpd +T278 SO:0001023 10204 10210 allele +T279 GO:0042571 10295 10305 antibodies +T280 PR:000007164 10371 10374 XPD +T281 PR:000007164 10477 10480 XPD +T282 SO:0001023 10508 10515 alleles +T283 GO:0010467 10549 10559 expression +T284 PR:000007164 10583 10586 Xpd +T285 SO:0001023 10592 10598 allele +T286 PR:000007164 10620 10623 Xpd +T287 http://purl.obolibrary.org/obo/MONDO_0000001 10638 10645 disease +T288 PR:000007164 10680 10683 Xpd +T289 NCBITaxon:33208 10693 10700 animals +T290 UBERON:0001037 10732 10736 hair +T291 NCBITaxon:10088 10804 10808 mice +T292 PR:000007164 10847 10850 Xpd +T293 PR:000007164 10863 10866 Xpd +T294 NCBITaxon:10088 10874 10878 mice +T295 UBERON:0001037 10903 10907 hair +T296 http://purl.obolibrary.org/obo/MONDO_0004907 10903 10912 hair loss +T297 UBERON:0001037 10926 10930 hair +T298 GO:0042633 10926 10936 hair cycle +T299 UBERON:0001037 10949 10953 hair +T300 http://purl.obolibrary.org/obo/MONDO_0004907 10949 10958 hair loss +T301 UBERON:0000104 10997 11002 lives +T302 PR:000007164 11031 11034 Xpd +T303 NCBITaxon:10088 11044 11048 mice +T304 UBERON:0001037 11064 11068 hair +T305 http://purl.obolibrary.org/obo/MONDO_0004907 11064 11073 hair loss +T306 UBERON:0001037 11096 11100 hair +T307 GO:0042633 11096 11106 hair cycle +T308 UBERON:0001137 11131 11135 back +T309 CHEBI:10545 11158 11166 electron +T310 PR:000007164 11190 11193 Xpd +T311 UBERON:0001037 11203 11207 hair +T312 http://purl.obolibrary.org/obo/MONDO_0018053 11251 11254 TTD +T313 UBERON:0001037 11284 11289 hairs +T314 UBERON:0001037 11401 11405 hair +T315 PR:000007164 11413 11416 Xpd +T316 NCBITaxon:10088 11426 11430 mice +T317 PR:000007164 11465 11468 Xpd +T318 NCBITaxon:33208 11476 11483 animals +T319 PR:000007164 11547 11550 Xpd +T320 PR:000007164 11656 11659 Xpd +T321 NCBITaxon:10088 11667 11671 mice +T322 http://purl.obolibrary.org/obo/MONDO_0018053 11707 11710 TTD +T323 UBERON:0000178 11722 11727 Blood +T324 PR:000007164 11783 11786 Xpd +T325 NCBITaxon:10088 11796 11800 Mice +T326 PR:000007164 11841 11844 Xpd +T327 PR:000007164 11875 11878 Xpd +T328 NCBITaxon:10088 11896 11900 mice +T329 UBERON:0001037 11932 11936 hair +T330 http://purl.obolibrary.org/obo/MONDO_0004907 11932 11941 hair loss +T331 PR:000007164 11985 11988 Xpd +T332 PR:000007164 11997 12000 Xpd +T333 NCBITaxon:10088 12018 12022 mice +T334 http://purl.obolibrary.org/obo/MONDO_0018053 12024 12027 TTD +T335 UBERON:0007376 12059 12068 epidermis +T336 UBERON:0001821 12158 12173 sebacious gland +T337 UBERON:0007376 12241 12250 epidermis +T338 PR:000007164 12254 12257 Xpd +T339 NCBITaxon:10088 12274 12278 mice +T340 UBERON:0001037 12325 12329 hair +T341 PR:000007164 12339 12342 Xpd +T342 PR:000007164 12355 12358 Xpd +T343 NCBITaxon:10088 12368 12372 mice +T344 PR:000007164 12463 12466 Xpd +T345 PR:000007164 12478 12481 Xpd +T346 NCBITaxon:10088 12491 12495 mice +T347 UBERON:0000178 12579 12584 blood +T348 PR:000007164 12588 12591 Xpd +T349 PR:000007164 12603 12606 Xpd +T350 NCBITaxon:10088 12616 12620 mice +T351 PR:000007164 12752 12755 Xpd +T352 PR:000007164 12767 12770 Xpd +T353 NCBITaxon:10088 12780 12784 mice +T354 http://purl.obolibrary.org/obo/MONDO_0018053 12954 12957 TTD +T355 UBERON:0007376 12974 12983 epidermis +T356 UBERON:0000119 13025 13033;13048 13053 layer of ... cells +T357 GO:0005634 13038 13047 nucleated +T358 CL:0002242 13038 13053 nucleated cells +T359 GO:0070268 13100 13109 cornified +T360 UBERON:0002027 13100 13115 cornified layer +T361 UBERON:0001821 13152 13167 sebacious gland +T362 UBERON:0001037 13214 13218 hair +T363 PR:000007164 13248 13251 Xpd +T364 NCBITaxon:10088 13261 13265 mice +T365 http://purl.obolibrary.org/obo/MONDO_0002280 13358 13365 anaemia +T366 http://purl.obolibrary.org/obo/MONDO_0018053 13415 13418 TTD +T367 PR:000007164 13431 13434 Xpd +T368 NCBITaxon:10088 13442 13446 mice +T369 PR:000007164 13505 13508 Xpd +T370 NCBITaxon:10088 13518 13522 mice +T371 http://purl.obolibrary.org/obo/MONDO_0020732 13554 13563 Progeroid +T372 http://purl.obolibrary.org/obo/MONDO_0018053 13576 13579 TTD +T373 NCBITaxon:10088 13580 13584 Mice +T374 PR:000007164 13606 13609 Xpd +T375 SO:0001023 13610 13617 Alleles +T376 http://purl.obolibrary.org/obo/MONDO_0018053 13641 13644 TTD +T377 http://purl.obolibrary.org/obo/MONDO_0016354 13646 13650 XPCS +T378 http://purl.obolibrary.org/obo/MONDO_0016006 13656 13658 CS +T379 http://purl.obolibrary.org/obo/MONDO_0019600 13668 13670 XP +T380 NCBITaxon:10088 13694 13699 mouse +T381 http://purl.obolibrary.org/obo/MONDO_0020732 13733 13742 progeroid +T382 GO:0007568 13785 13791 ageing +T383 NCBITaxon:10088 13836 13840 mice +T384 PR:000007164 13861 13864 Xpd +T385 NCBITaxon:33208 13872 13879 animals +T386 CHEBI:46662 13898 13905 mineral +T387 http://purl.obolibrary.org/obo/MONDO_0005298 13953 13965 osteoporosis +T388 UBERON:0002415 13993 13997 tail +T389 UBERON:0002412 13998 14007 vertebrae +T390 PR:000007164 14035 14038 Xpd +T391 NCBITaxon:10088 14048 14052 mice +T392 PR:000007164 14137 14140 Xpd +T393 NCBITaxon:10088 14148 14152 mice +T394 NCBITaxon:33208 14188 14195 animals +T395 PR:000007164 14249 14252 Xpd +T396 NCBITaxon:10088 14262 14266 mice +T397 http://purl.obolibrary.org/obo/MONDO_0018053 14344 14347 TTD +T398 PR:000007164 14462 14465 Xpd +T399 NCBITaxon:10088 14475 14479 mice +T400 UBERON:0000104 14513 14522 life span +T401 PR:000007164 14574 14577 Xpd +T402 NCBITaxon:10088 14585 14589 mice +T403 http://purl.obolibrary.org/obo/MONDO_0018053 14622 14625 TTD +T404 http://purl.obolibrary.org/obo/MONDO_0020732 14647 14656 Progeroid +T405 PR:000007164 14691 14694 Xpd +T406 NCBITaxon:10088 14705 14709 Mice +T407 PR:000007164 14766 14769 Xpd +T408 PR:000007164 14795 14798 Xpd +T409 NCBITaxon:10088 14806 14810 mice +T410 UBERON:0002190 14847 14863 subcutaneous fat +T411 PR:000007164 14872 14875 Xpd +T412 NCBITaxon:10088 14883 14888 mouse +T413 PR:000007164 14933 14936 Xpd +T414 NCBITaxon:10088 14946 14950 mice +T415 PR:000007164 14991 14994 Xpd +T416 PR:000007164 15009 15012 Xpd +T417 NCBITaxon:10088 15020 15024 mice +T418 GO:0007568 15026 15032 Ageing +T419 PR:000007164 15033 15036 Xpd +T420 NCBITaxon:10088 15044 15048 mice +T421 UBERON:0001130 15084 15097 spinal column +T422 CHEBI:46662 15121 15128 mineral +T423 UBERON:0002415 15172 15176 tail +T424 UBERON:0002412 15177 15186 vertebrae +T425 UBERON:0002355 15204 15210 pelvis +T426 PR:000007164 15278 15281 Xpd +T427 NCBITaxon:10088 15294 15299 mouse +T428 CHEBI:46662 15338 15345 mineral +T429 UBERON:0002415 15357 15361 tail +T430 UBERON:0002412 15362 15371 vertebrae +T431 PR:000007164 15404 15407 Xpd +T432 PR:000007164 15430 15433 Xpd +T433 NCBITaxon:10088 15449 15453 mice +T434 PR:000007164 15524 15527 Xpd +T435 PR:000007164 15657 15660 Xpd +T436 NCBITaxon:10088 15668 15672 mice +T437 PR:000007164 15709 15712 Xpd +T438 NCBITaxon:10088 15724 15728 mice +T439 PR:000007164 15769 15772 Xpd +T440 PR:000007164 15803 15806 Xpd +T441 NCBITaxon:10088 15816 15820 mice +T442 PR:000007164 15931 15934 Xpd +T443 SO:0001023 15937 15944 allelic +T444 NCBITaxon:10088 15956 15960 Mice +T445 PR:000007164 16015 16018 Xpd +T446 SO:0001023 16024 16030 allele +T447 PR:000007164 16100 16103 Xpd +T448 SO:0001023 16107 16113 allele +T449 PR:000007164 16150 16153 Xpd +T450 NCBITaxon:10088 16161 16165 mice +T451 NCBITaxon:33208 16209 16216 animals +T452 PR:000007164 16233 16236 Xpd +T453 SO:0001023 16243 16249 allele +T454 PR:000007164 16273 16276 Xpd +T455 SO:0001023 16281 16287 allele +T456 UBERON:0001037 16325 16329 hair +T457 http://purl.obolibrary.org/obo/MONDO_0004907 16325 16334 hair loss +T458 GO:0007568 16499 16505 ageing +T459 UBERON:0000104 16643 16652 life span +T460 SO:0001023 16721 16728 alleles +T461 http://purl.obolibrary.org/obo/MONDO_0018053 16828 16831 TTD +T462 SO:0001023 16900 16907 allelic +T463 GO:0071897 16982 16995 DNA synthesis +T464 GO:0032774 17039 17052 RNA synthesis +T465 GO:0006289 17121 17124 NER +T466 SO:0001026 17145 17151 genome +T467 GO:0006289 17152 17155 NER +T468 GO:0006283 17160 17185 transcription-coupled NER +T469 GO:0006289 17197 17200 NER +T470 http://purl.obolibrary.org/obo/MONDO_0018053 17370 17373 TTD +T471 PR:000007164 17411 17414 Xpd +T472 PR:000007164 17426 17429 Xpd +T473 NCBITaxon:33208 17436 17443 animals +T474 PR:000007164 17468 17471 Xpd +T475 CL:0000057 17591 17602 fibroblasts +T476 NCBITaxon:1 17678 17686 organism +T477 PR:000007164 17688 17691 Xpd +T478 PR:000007164 17823 17826 Xpd +T479 PR:000007164 17901 17904 Xpd +T480 SO:0001023 17910 17916 allele +T481 PR:000007164 17920 17923 Xpd +T482 PR:000007164 17991 17994 Xpd +T483 SO:0001023 17998 18004 allele +T484 UBERON:0000922 18029 18038 embryonic +T485 PR:000007164 18130 18133 Xpd +T486 PR:000007164 18142 18145 Xpd +T487 SO:0001023 18149 18156 alleles +T488 PR:000007164 18178 18181 XPD +T489 PR:000007164 18185 18188 XPD +T490 PR:000007164 18210 18213 XPD +T491 PR:000007164 18219 18222 XPD +T492 NCBITaxon:9606 18229 18234 human +T493 PR:000007164 18327 18330 Xpd +T494 PR:000007164 18341 18344 XPD +T495 NCBITaxon:10088 18357 18362 mouse +T496 PR:000007164 18422 18425 Xpd +T497 PR:000007164 18440 18443 Xpd +T498 SO:0001023 18521 18527 allele +T499 GO:0006289 18611 18614 NER +T500 PR:000007164 18639 18642 Xpd +T501 PR:000007164 18672 18675 Xpd +T502 GO:0005675 18747 18752 TFIIH +T503 http://purl.obolibrary.org/obo/MONDO_0018053 18804 18807 TTD +T504 PR:000007164 18809 18812 XPD +T505 http://purl.obolibrary.org/obo/MONDO_0016354 18820 18824 XPCS +T506 PR:000007164 18826 18829 XPD +T507 http://purl.obolibrary.org/obo/MONDO_0019600 18840 18842 XP +T508 PR:000007164 18844 18847 XPD +T509 GO:0006289 18890 18893 NER +T510 GO:0005675 18939 18954 TFIIH complexes +T511 SO:0001023 19020 19027 allelic +T512 GO:0006281 19133 19139 repair +T513 http://purl.obolibrary.org/obo/MONDO_0018053 19160 19163 TTD +T514 http://purl.obolibrary.org/obo/MONDO_0020732 19164 19173 progeroid +T515 NCBITaxon:33208 19188 19194 animal +T516 GO:0005675 19214 19219 TFIIH +T517 PR:000007164 19248 19251 XPD +T518 http://purl.obolibrary.org/obo/MONDO_0000001 19263 19270 Disease +T519 PR:000007164 19348 19351 Xpd +T520 PR:000007164 19370 19373 Xpd +T521 PR:000007164 19383 19386 Xpd +T522 SO:0001023 19390 19397 alleles +T523 PR:000007164 19479 19482 Xpd +T524 GO:0010467 19527 19536 expressed +T525 SO:0001023 19544 19550 allele +T526 PR:000007164 19552 19555 Xpd +T527 SO:0001026 19801 19807 genome +T528 GO:0006281 19808 19814 repair +T529 PR:000007164 19847 19850 Xpd +T530 PR:000007164 19867 19870 Xpd +T531 PR:000007164 19886 19889 Xpd +T532 PR:000007164 19908 19911 Xpd +T533 PR:000007164 20034 20037 Xpd +T534 PR:000007164 20092 20095 Xpd +T535 GO:0006283 20129 20157 transcription-coupled repair +T536 PR:000007164 20211 20214 Xpd +T537 PR:000007164 20231 20234 Xpd +T538 PR:000007164 20250 20253 Xpd +T539 PR:000007164 20272 20275 Xpd +T540 MOP:0000780 20352 20360 Incision +T541 GO:0005675 20406 20421 TFIIH complexes +T542 GO:0006289 20441 20444 NER +T543 GO:0005675 20515 20521 TFIIHs +T544 PR:000007164 20534 20537 XPD +T545 PR:000007165 20539 20542 XPB +T546 PR:000008317 20544 20547 p62 +T547 PR:000008320 20549 20552 p52 +T548 PR:000008318 20558 20561 p44 +T549 PR:000008319 20568 20571 p34 +T550 PR:000005265 20573 20577 cdk7 +T551 PR:000005130 20579 20587 cyclin H +T552 PR:000010496 20589 20593 Mat1 +T553 PR:000008321 20599 20601 p8 +T554 PR:000007167 20631 20634 XPG +T555 PR:000007166 20636 20639 XPF +T556 PR:000007163 20640 20645 ERCC1 +T557 PR:000017494 20647 20650 XPC +T558 PR:000013670 20651 20657 hHR23B +T559 GO:0005662 20659 20662 RPA +T560 GO:0006289 20694 20697 NER +T561 GO:0005675 20917 20930 TFIIH complex +T562 PR:000007164 21008 21011 Xpd +T563 GO:0005675 21040 21045 TFIIH +T564 PR:000007164 21060 21063 Xpd +T565 PR:000007164 21083 21086 Xpd +T566 PR:000007164 21120 21123 Xpd +T567 PR:000007164 21137 21140 Xpd +T568 PR:000008317 21195 21215 p62 subunit of TFIIH +T569 GO:0005675 21210 21215 TFIIH +T570 PR:000007164 21376 21379 Xpd +T571 PR:000007164 21421 21424 Xpd +T572 PR:000007164 21506 21509 Xpd +T573 PR:000007164 21599 21602 Xpd +T574 GO:0005634 21700 21706 nuclei +T575 PR:000007164 22048 22051 Xpd +T576 PR:000007164 22061 22064 Xpd +T577 SO:0001023 22068 22075 alleles +T578 http://purl.obolibrary.org/obo/MONDO_0018053 22120 22123 TTD +T579 GO:0005675 22155 22160 TFIIH +T580 PR:000007164 22193 22196 Xpd +T581 PR:000007164 22211 22214 Xpd +T582 GO:0005675 22333 22338 TFIIH +T583 CL:0000057 22357 22368 fibroblasts +T584 http://purl.obolibrary.org/obo/MONDO_0018053 22388 22391 TTD +T585 GO:0010467 22464 22474 expression +T586 PR:000007164 22492 22495 Xpd +T587 SO:0001023 22499 22505 allele +T588 SO:0001023 22525 22531 allele +T589 GO:0005675 22545 22550 TFIIH +T590 NCBITaxon:10088 22597 22602 mouse +T591 PR:000007164 22603 22606 Xpd +T592 CL:0000057 22614 22625 fibroblasts +T593 NCBITaxon:9606 22681 22686 human +T594 http://purl.obolibrary.org/obo/MONDO_0018053 22701 22704 TTD +T595 SO:0000704 22729 22733 gene +T596 PR:000007164 22818 22821 Xpd +T597 GO:0010467 22860 22870 expression +T598 PR:000007164 22891 22894 Xpd +T599 PR:000007164 22908 22911 Xpd +T600 SO:0001023 22915 22921 allele +T601 GO:0005675 22942 22947 TFIIH +T602 PR:000007164 22974 22977 Xpd +T603 http://purl.obolibrary.org/obo/MONDO_0018053 23137 23140 TTD +T604 http://purl.obolibrary.org/obo/MONDO_0020732 23141 23150 progeroid +T605 GO:0005675 23204 23209 TFIIH +T606 GO:0010467 23343 23353 expression +T607 PR:000007164 23401 23404 XPD +T608 http://purl.obolibrary.org/obo/MONDO_0018053 23426 23429 TTD +T609 UBERON:0001037 23430 23434 hair +T610 PR:000007164 23465 23468 Xpd +T611 NCBITaxon:33208 23479 23486 animals +T612 http://purl.obolibrary.org/obo/MONDO_0018053 23501 23504 TTD +T613 UBERON:0001037 23505 23509 hair +T614 PR:000007164 23552 23555 Xpd +T615 GO:0010467 23561 23571 expression +T616 PR:000007164 23581 23584 Xpd +T617 NCBITaxon:33208 23593 23600 animals +T618 UBERON:0001037 23608 23612 hair +T619 GO:0010467 23638 23648 expression +T620 PR:000007164 23672 23675 Xpd +T621 SO:0001023 23680 23686 allele +T622 GO:0010467 23738 23748 expression +T623 SO:0001023 23778 23785 alleles +T624 UBERON:0001037 23840 23844 hair +T625 PR:000007164 23899 23902 Xpd +T626 SO:0001023 23903 23910 alleles +T627 GO:0005675 23952 23957 TFIIH +T628 NCBITaxon:10088 23970 23974 mice +T629 SO:0001023 24039 24046 allelic +T630 SO:0001023 24149 24156 allelic +T631 SO:0001023 24202 24209 alleles +T632 SO:0000704 24365 24372 genetic +T633 SO:0000704 24395 24406 genetically +T634 NCBITaxon:40674 24415 24424 mammalian +T635 PR:000007164 24577 24580 Xpd +T636 SO:0001023 24581 24588 alleles +T637 SO:0001023 24606 24613 allelic +T638 SO:0001023 24669 24675 allele +T639 NCBITaxon:9606 24814 24819 human +T640 http://purl.obolibrary.org/obo/MONDO_0000001 24830 24837 disease +T641 PR:000007164 24853 24856 Xpd +T642 SO:0001023 24857 24864 alleles +T643 SO:0001023 24975 24982 alleles +T644 SO:0001023 25035 25042 alleles +T645 NCBITaxon:1 25222 25231 organisms +T646 NCBITaxon:9606 25242 25248 humans +T647 NCBITaxon:33208 25303 25309 animal +T648 PR:000007164 25315 25318 Xpd +T649 SO:0001023 25319 25326 allelic +T650 UBERON:0000479 25463 25469 tissue +T651 SO:0001023 25535 25542 allelic +T652 SO:0001023 25565 25572 allelic +T653 SO:0001023 25601 25608 alleles +T654 SO:0001023 25752 25759 allelic +T655 PR:000007164 25798 25801 Xpd +T656 UBERON:0001037 25846 25850 hair +T657 http://purl.obolibrary.org/obo/MONDO_0020732 25864 25872 progeria +T658 GO:0005675 25875 25880 TFIIH +T659 GO:0006281 25902 25908 Repair +T660 PR:000007164 25924 25927 XPD +T661 http://purl.obolibrary.org/obo/MONDO_0000001 25928 25935 Disease +T662 PR:000007164 25990 25993 Xpd +T663 PR:000007164 26022 26025 Xpd +T664 PR:000007164 26035 26038 Xpd +T665 SO:0001023 26042 26049 alleles +T666 NCBITaxon:1 26119 26127 organism +T667 UBERON:0000922 26152 26161 embryonic +T668 GO:0030154 26185 26203;26231 26236 differentiation of ... cells +T669 GO:0090601 26204 26215 enucleating +T670 CL:0000225 26204 26215;26231 26236 enucleating ... cells +T671 UBERON:0000178 26225 26230 blood +T672 CL:0000081 26225 26236 blood cells +T673 UBERON:0000358 26245 26255 blastocyst +T674 PR:000007164 26297 26300 Xpd +T675 PR:000007164 26304 26307 Xpd +T676 PR:000007164 26318 26321 Xpd +T677 SO:0001023 26325 26332 alleles +T678 UBERON:0000104 26412 26416 life +T679 PR:000007164 26421 26424 Xpd +T680 PR:000007164 26439 26442 Xpd +T681 NCBITaxon:10088 26473 26477 mice +T682 UBERON:0000922 26479 26488 embryonic +T683 PR:000007164 26524 26527 Xpd +T684 SO:0001023 26531 26537 allele +T685 UBERON:0000922 26547 26556 embryonic +T686 PR:000007164 26593 26596 Xpd +T687 NCBITaxon:10088 26614 26618 mice +T688 PR:000007164 26624 26627 Xpd +T689 SO:0001023 26631 26637 allele +T690 SO:0001023 26713 26720 alleles +T691 PR:000007164 26722 26725 Xpd +T692 PR:000007164 26729 26732 Xpd +T693 PR:000007164 26743 26746 Xpd +T694 http://purl.obolibrary.org/obo/MONDO_0018053 26805 26808 TTD +T695 http://purl.obolibrary.org/obo/MONDO_0002280 26832 26839 anaemic +T696 GO:0005675 26977 26982 TFIIH +T697 GO:0030154 27003 27021;27050 27055 differentiation of ... cells +T698 UBERON:0002074 27028 27038 hair-shaft +T699 CL:0002559 27028 27038;27050 27055 hair-shaft ... cells +T700 UBERON:0000178 27044 27049 blood +T701 CL:0000081 27044 27055 blood cells +T702 NCBITaxon:10088 27090 27094 mice +T703 PR:000007164 27119 27122 Xpd +T704 PR:000007164 27132 27135 Xpd +T705 SO:0001023 27139 27146 alleles +T706 PR:000007164 27170 27173 Xpd +T707 http://purl.obolibrary.org/obo/MONDO_0002280 27200 27207 anaemic +T708 PR:000007164 27262 27265 Xpd +T709 SO:0001023 27269 27275 allele +T710 UBERON:0000922 27350 27359 embryonic +T711 UBERON:0001037 27421 27425 hair +T712 UBERON:0000178 27431 27436 blood +T713 UBERON:0019248 27534 27549 early embryonic +T714 GO:0009790 27540 27561 embryonic development +T715 PR:000007164 27563 27566 Xpd +T716 PR:000007164 27591 27594 Xpd +T717 PR:000007164 27604 27607 Xpd +T718 SO:0001023 27611 27618 alleles +T719 GO:0043588 27641 27660 ontogenesis of skin +T720 GO:0048468 27641 27655;27684 27689 ontogenesis of ... cells +T721 UBERON:0002074 27662 27672 hair-shaft +T722 CL:0002559 27662 27672;27684 27689 hair-shaft ... cells +T723 UBERON:0000178 27678 27683 blood +T724 CL:0000081 27678 27689 blood cells +T725 GO:0006281 27738 27744 repair +T726 PR:000007164 27769 27772 Xpd +T727 SO:0001023 27778 27784 allele +T728 PR:000007164 27836 27839 Xpd +T729 SO:0001023 27843 27849 allele +T730 PR:000007164 27857 27860 Xpd +T731 SO:0001023 27865 27872 alleles +T732 GO:0010467 27888 27898 expression +T733 SO:0001023 27937 27944 allelic +T734 PR:000007164 28064 28067 Xpd +T735 GO:0006281 28117 28123 repair +T736 http://purl.obolibrary.org/obo/MONDO_0018053 28150 28153 TTD +T737 http://purl.obolibrary.org/obo/MONDO_0020732 28154 28163 progeroid +T738 SO:0001023 28188 28195 allelic +T739 PR:000007164 28216 28219 XPD +T740 SO:0001023 28245 28252 allelic +T741 PR:000007164 28300 28303 XPD +T742 SO:0001023 28319 28326 allelic +T743 GO:0032991 28369 28379 multimeric +T744 SO:0000417 28414 28421 domains +T745 http://purl.obolibrary.org/obo/MONDO_0000001 28482 28489 disease +T746 PR:000007164 28512 28515 XPD +T747 SO:0000417 28527 28534 domains +T748 PR:000007164 28581 28584 XPD +T749 GO:0005675 28618 28623 TFIIH +T750 GO:0005675 28725 28730 TFIIH +T751 PR:000007164 28743 28746 XPD +T752 PR:000007165 28751 28754 XPB +T753 GO:0006289 28770 28773 NER +T754 GO:0005675 28816 28821 TFIIH +T755 GO:0006289 28897 28900 NER +T756 PR:000007164 28924 28927 XPD +T757 GO:0005675 28972 28977 TFIIH +T758 SO:0001023 29033 29040 allelic +T759 PR:000007164 29084 29087 XPD +T760 CHEBI:36357 29088 29097 molecules +T761 GO:0005675 29109 29122 TFIIH complex +T762 GO:0005675 29138 29153 TFIIH complexes +T763 PR:000007164 29175 29178 XPD +T764 CHEBI:36357 29179 29188 molecules +T765 SO:0001026 29247 29253 genome +T766 GO:0006283 29265 29296;29329 29339 transcription-coupled repair of ... DNA damage +T767 SO:0001023 29346 29353 allelic +T768 PR:000007164 29367 29370 XPD +T769 http://purl.obolibrary.org/obo/MONDO_0000001 29371 29380 Disorders +T770 SO:0001023 29432 29439 alleles +T771 SO:0001023 29555 29562 alleles +T772 NCBITaxon:4896 29601 29609 S. pombe +T773 PR:000007164 29638 29641 XPD +T774 SO:0000853 29642 29651 homologue +T775 PR:P26659 29652 29657 rad15 +T776 SO:0001023 29708 29715 alleles +T777 http://purl.obolibrary.org/obo/MONDO_0000001 29819 29826 disease +T778 NCBITaxon:9606 29860 29866 humans +T779 NCBITaxon:10088 29882 29887 mouse +T780 http://purl.obolibrary.org/obo/MONDO_0019600 29968 29970 XP +T781 SO:0001023 30060 30067 allelic +T782 PR:000007164 30080 30083 XPD +T783 http://purl.obolibrary.org/obo/MONDO_0000001 30084 30093 disorders +T784 PR:000007164 30136 30139 XPD +T785 PR:000007164 30166 30169 XPD +T786 PR:000007164 30178 30181 XPD +T787 SO:0001023 30196 30203 alleles +T788 GO:0016265 30208 30212 died +T789 http://purl.obolibrary.org/obo/MONDO_0000001 30220 30227 disease +T790 PR:000007164 30273 30276 XPD +T791 PR:000007164 30308 30311 XPD +T792 PR:000007164 30320 30323 XPD +T793 SO:0001023 30338 30345 alleles +T794 PR:000007164 30379 30382 XPD +T795 http://purl.obolibrary.org/obo/MONDO_0000001 30428 30435 disease +T796 http://purl.obolibrary.org/obo/MONDO_0019600 30616 30618 XP +T797 http://purl.obolibrary.org/obo/MONDO_0018053 30623 30626 TTD +T798 http://purl.obolibrary.org/obo/MONDO_0002254 30635 30643 syndrome +T799 PR:000007164 30664 30667 Xpd +T800 PR:000007164 30681 30684 Xpd +T801 NCBITaxon:10088 30692 30696 mice +T802 UBERON:0001037 30772 30776 hair +T803 SO:0001023 30870 30876 allele +T804 PR:000007164 30913 30916 XPD +T805 PR:000007164 30949 30952 XPD +T806 SO:0001023 30967 30973 allele +T807 NCBITaxon:4896 31019 31027 S. pombe +T808 PR:P26659 31028 31033 rad15 +T809 SO:0001023 31086 31092 allele +T810 SO:0001023 31215 31222 alleles +T811 SO:0001023 31274 31281 allelic +T812 http://purl.obolibrary.org/obo/MONDO_0019600 31329 31331 XP +T813 PR:000007164 31404 31407 XPD +T814 http://purl.obolibrary.org/obo/MONDO_0000001 31408 31417 Disorders +T815 SO:0001023 31448 31455 allelic +T816 SO:0001023 31516 31522 allele +T817 SO:0001023 31555 31561 allele +T818 PR:000007164 31669 31672 XPD +T819 http://purl.obolibrary.org/obo/MONDO_0000001 31700 31708 disorder +T820 SO:0001023 31725 31732 allelic +T821 SO:0001023 31785 31792 alleles +T822 SO:0001023 31902 31908 allele +T823 http://purl.obolibrary.org/obo/MONDO_0000001 31936 31943 disease +T824 SO:0001023 32055 32061 allele +T825 SO:0000417 32125 32132 domains +T826 SO:0001023 32167 32174 Alleles +T827 NCBITaxon:40674 32209 32216 Mammals +T828 NCBITaxon:9606 32221 32227 humans +T829 SO:0001023 32257 32264 allelic +T830 SO:0001023 32286 32293 allelic +T831 SO:0001023 32341 32348 allelic +T832 SO:0001023 32395 32402 alleles +T833 CHEBI:30860 32473 32486 methylmalonic +T834 http://purl.obolibrary.org/obo/MONDO_0002012 32473 32496 methylmalonic acidaemia +T835 http://purl.obolibrary.org/obo/MONDO_0000001 32523 32530 disease +T836 http://purl.obolibrary.org/obo/MONDO_0020732 32627 32636 progeroid +T837 NCBITaxon:33208 32720 32727 animals +T838 SO:0001023 32736 32743 allelic +T839 NCBITaxon:9606 32774 32779 human +T840 http://purl.obolibrary.org/obo/MONDO_0000001 32780 32787 disease +T841 SO:0001023 32821 32828 alleles +T842 PR:000001044 32852 32856 CTRF +T843 SO:0000704 32857 32861 gene +T844 GO:0030849 32887 32896 autosomal +T845 http://purl.obolibrary.org/obo/MONDO_0006025 32887 32915 autosomal recessive disorder +T846 http://purl.obolibrary.org/obo/MONDO_0009061 32916 32931 cystic fibrosis +T847 SO:0001023 32990 32997 allelic +T848 SO:0001023 33036 33043 allelic +T849 SO:0000694 33094 33125 single nucleotide polymorphisms +T850 SO:0001026 33126 33132 genome +T851 SO:0001023 33178 33185 allelic +T852 GO:0030849 33301 33310 autosomal +T853 http://purl.obolibrary.org/obo/MONDO_0006025 33301 33328 autosomal recessive disease +T854 NCBITaxon:40674 33363 33370 mammals +T855 SO:0001023 33500 33506 allele +T856 SO:0001023 33614 33621 alleles +T857 GO:0010467 33682 33692 expression +T858 NCBITaxon:10088 33804 33808 mice +T859 PR:000007164 33825 33828 Xpd +T860 PR:000007164 33833 33836 XPD +T861 PR:000007164 33847 33850 Xpd +T862 NCBITaxon:10088 33857 33861 mice +T863 PR:000007164 33970 33973 Xpd +T864 PR:000007164 33983 33986 Xpd +T865 SO:0001023 33991 33998 alleles +T866 NCBITaxon:10088 34099 34103 mice +T867 NCBITaxon:10088 34108 34113 mouse +T868 UBERON:0000922 34114 34123 embryonic +T869 CL:0000057 34124 34135 fibroblasts +T870 CHEBI:51686 34185 34197 Haematoxylin +T871 UBERON:0000178 34321 34326 Blood +T872 NCBITaxon:33208 34354 34360 Animal +T873 UBERON:0000178 34361 34366 Blood +T874 CHEBI:46662 34460 34467 mineral +T875 NCBITaxon:10088 34513 34517 Mice +T876 NCBITaxon:10088 34630 34634 mice +T877 SO:0000704 34690 34697 genetic +T878 NCBITaxon:1 34716 34725 organisms +T879 NCBITaxon:33208 34734 34740 animal +T880 GO:0005675 34868 34873 TFIIH +T881 MOP:0000780 34874 34882 incision +T882 PR:000007164 35077 35080 Xpd +T883 PR:000007164 35093 35096 Xpd +T884 PR:000007164 35114 35117 Xpd +T885 PR:000007164 35199 35202 Xpd +T886 MOP:0000780 35405 35413 incision +T887 GO:0005675 35451 35456 TFIIH +T888 PR:000008317 35618 35632;35637 35642 p62 subunit of ... TFIIH +T889 GO:0005675 35637 35642 TFIIH +T890 NCBITaxon:10088 35703 35708 mouse +T891 UBERON:0000922 35709 35718 embryonic +T892 CL:0000057 35719 35730 fibroblasts +T893 PR:000007164 35800 35803 Xpd +T894 CHEBI:33893 36439 36447 reagents +T895 http://purl.obolibrary.org/obo/MONDO_0000001 36663 36671 Diseases +T896 http://purl.obolibrary.org/obo/MONDO_0004992 36886 36892 Cancer +T897 http://purl.obolibrary.org/obo/MONDO_0004992 36953 36959 Cancer +T898 SO:0000028 37002 37004 bp +T899 SO:0000028 37007 37016 base pair +T900 http://purl.obolibrary.org/obo/MONDO_0016006 37018 37020 CS +T901 http://purl.obolibrary.org/obo/MONDO_0016006 37023 37040 Cockayne syndrome +T902 UBERON:0000922 37054 37063 embryonic +T903 GO:0006289 37093 37096 NER +T904 GO:0006289 37099 37125 nucleotide excision repair +T905 GO:0005675 37161 37166 TFIIH +T906 GO:0005675 37169 37210 basal transcription/DNA repair factor IIH +T907 GO:0006281 37189 37199 DNA repair +T908 http://purl.obolibrary.org/obo/MONDO_0018053 37212 37215 TTD +T909 http://purl.obolibrary.org/obo/MONDO_0018053 37218 37237 trichothiodystrophy +T910 GO:0032774 37260 37273 RNA synthesis +T911 GO:0071897 37326 37339 DNA synthesis +T912 http://purl.obolibrary.org/obo/MONDO_0019600 37387 37389 XP +T913 http://purl.obolibrary.org/obo/MONDO_0019600 37392 37413 xeroderma pigmentosum +T914 http://purl.obolibrary.org/obo/MONDO_0016354 37415 37419 XPCS +T915 http://purl.obolibrary.org/obo/MONDO_0019600 37422 37443 xeroderma pigmentosum +T916 http://purl.obolibrary.org/obo/MONDO_0016006 37458 37475 Cockayne syndrome +T917 http://purl.obolibrary.org/obo/MONDO_0019600 37497 37518 xeroderma pigmentosum +T918 http://purl.obolibrary.org/obo/MONDO_0018053 37523 37542 trichothiodystrophy diff --git a/src/ontogpt/evaluation/craft/database/all/17020410.txt b/src/ontogpt/evaluation/craft/database/all/17020410.txt new file mode 100644 index 000000000..1a79e271c --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17020410.txt @@ -0,0 +1,211 @@ +Rescue of Progeria in Trichothiodystrophy by Homozygous Lethal Xpd Alleles + +Abstract + +Although compound heterozygosity, or the presence of two different mutant alleles of the same gene, is common in human recessive disease, its potential to impact disease outcome has not been well documented. This is most likely because of the inherent difficulty in distinguishing specific biallelic effects from differences in environment or genetic background. We addressed the potential of different recessive alleles to contribute to the enigmatic pleiotropy associated with XPD recessive disorders in compound heterozygous mouse models. Alterations in this essential helicase, with functions in both DNA repair and basal transcription, result in diverse pathologies ranging from elevated UV sensitivity and cancer predisposition to accelerated segmental progeria. We report a variety of biallelic effects on organismal phenotype attributable to combinations of recessive Xpd alleles, including the following: (i) the ability of homozygous lethal Xpd alleles to ameliorate a variety of disease symptoms when their essential basal transcription function is supplied by a different disease-causing allele, (ii) differential developmental and tissue-specific functions of distinct Xpd allele products, and (iii) interallelic complementation, a phenomenon rarely reported at clinically relevant loci in mammals. Our data suggest a re-evaluation of the contribution of “null” alleles to XPD disorders and highlight the potential of combinations of recessive alleles to affect both normal and pathological phenotypic plasticity in mammals. + +Introduction + +Interallelic complementation is defined as the ability of two differentially mutated alleles to function better together than either can on its own. Despite its near universality in lower organisms [1], its potential to contribute to clinical heterogeneity in human disease is seldom considered. Evidence of interallelic complementation at clinically relevant loci is limited to biochemical and cell-based studies of a handful of metabolic disorders with defects in enzymes including propinyl-CoA carboxylase [2], argininosuccinate lyase [3], galactose-1-phosphate uridylyltransferase [4], and methylmalonyl CoA mutase [5]. + +Compound heterozygotes are individuals carrying two different mutant alleles of the same gene. In the absence of a dominant (wild-type [wt]) allele, genetic interactions between recessive alleles (referred to here as “biallelic” effects) could result in different phenotypic outcomes including interallelic complementation. Although amelioration of disease symptoms by interallelic complementation would create an ascertainment bias in the clinic, the lack of evidence concerning interallelic complementation or other biallelic effects in human disease is likely caused by the difficulty in distinguishing such effects from environment and genetic background. + +XPD encodes one of the two helicase components of basal transcription/DNA repair factor IIH (TFIIH), a ten-subunit, multifunctional complex that is essential for multiple processes, including basal transcription initiation and DNA damage repair via the nucleotide excision repair (NER) pathway [6,7]. Alterations in XPD resulting in defective TFIIH function are associated with UV-sensitive, multisystem disorders including xeroderma pigmentosum (XP), XP combined with Cockayne syndrome (CS), and trichothiodystrophy (TTD) [8–10]. XP is marked by sun-induced pigmentation anomalies and a greater than 1,000-fold elevation in skin cancer risk. Severe cases can also present with growth retardation and primary neurodegeneration [11]. CS and TTD, on the other hand, are segmental progeroid disorders characterised by progressive post-natal growth failure and primary demyelination resulting in severe neurodysfunction, but without a clear cancer predisposition [12–15]. Patients with TTD additionally display hallmark sulphur-deficient brittle hair and nails and scaling skin [13], resulting from a basal transcription defect in specific cell types [16,17]. A related disorder with the cancer predisposition of XP combined with the neurodevelopmental complications of CS (XPCS), although rare, has also been described [18]. + +Many XPD mutations are associated with an exclusive disease phenotype (e.g., XPDR722W with TTD and XPDR683W with XP) and are thus viewed as causative of the corresponding syndromes. Alleles not associated exclusively with one disorder are considered “likely null” alleles [19,20]. Some of these alleles fail to support viability in a haploid Schizosaccharomyces pombe yeast strain with a null mutation in the XPD homologue rad15 and are thus considered devoid of significant biological activity [19]. This classification of alleles as either causative or null currently defines what we refer to as a “monoallelic” paradigm of XPD disease. However, the identification in recent years of XP complementation group D patients with atypical disease presentation, including symptoms of both XP and TTD [8], casts doubt on the ability of such a monoallelic paradigm to explain clinical heterogeneity in compound heterozygotes. + +Previously, we generated a TTD mouse model (XPDR722W) that phenocopies the human syndrome [15,21]. Here we report the generation of additional mutant Xpd alleles that fail to support viability on their own but nevertheless ameliorate TTD-associated premature segmental ageing, cutaneous features, cellular DNA repair capacity, and UV survival when present in a compound heterozygote state. + +Results + +Generation of Xpd Compound Heterozygotes + +We generated an Xpd knock-in allele with a point mutation encoding a single amino acid change (XPDG602D) found in the XPCS patient XPCS2 (Figure 1A–1C). mRNA expression from the targeted allele could be detected in embryonic stem cells by RT-PCR (Figure 1D), although expression was reduced approximately 5-fold relative to wt mRNA transcript levels as determined by Northern blotting of RNA from the testis of heterozygous animals (Figure 1E). Because patient XPCS2 was a hemizygote with mutant XPD protein (XPDG602D) expressed from a single allele, the corresponding mutation was expected to be viable in the homozygous state. However, homozygous mutant mice were not observed, neither amongst live births nor embryonic day 13.5 (E13.5) or E3.5 embryos (Table 1). The corresponding hypomorphic, mutant allele was thus designated as homozygous lethal (†XPCS). Homozygous lethality of the XPCS allele is likely due to reduced levels of expression of this essential protein as a result of gene targeting (Figure 1A) rather than to the mutation itself. Xpd ablation (XpdKO/KO) is similarly incompatible with life beyond the earliest stages of embryogenesis [22]. Consistent with this interpretation, a different targeted Xpd mutation encoding XPDR683W, which is associated with XP in the homozygous state in humans, was similarly underexpressed and lethal in the homozygous state (designated as †XP allele) (Figure 1A–1C; Table 1; unpublished data). Also, a different targeting approach leading to the use of the native 3′UTR and removal of the neo gene resulted in normalisation of XpdXPCS mRNA levels and viable homozygous XpdXPCS/XPCS (XPDG602D/G602D) animals [23]. + +Figure 1 + +Targeting of the Mouse Xpd Gene + +(A) Schematic representation of the genomic structure and partial restriction map of the wt and targeted mouse Xpd loci. For the wt Xpd allele, shaded boxes represent coding regions of exons 12 and 19–23; the 3′UTR is represented by an open box. TGA indicates the translational stop codon; PolyA indicates the polyadenylation signal. For the XpdTTD targeted allele, the 194–base pair (bp) human XPD cDNA fragment fused to exon 22 is indicated as a striped box including the TTD (R722W) mutation indicated by a vertical arrow. Chicken β-globin exons 2 and 3 including the 3′UTR are indicated as black boxes with corresponding Roman numerals followed by the β-globin polyadenylation signal (PolyA*). For the Xpd†XP and Xpd†XPCS targeted alleles, vertical arrows indicate XPCS (G602D-encoding) and XP (R683W-encoding) mutations in exons 19 and 22, respectively. The unique 3′ probe located outside the targeting construct is marked by a thick black line. Restriction sites: B, BamHI; C, ClaI; E, EcoRI; H, HindIII; Hp, HpaI; Sf, SfiI. + +(B) Southern blot analysis of EcoRI-digested genomic DNA from wt, Xpd†XPCS/wt, and Xpd†XP/wt recombinant embryonic stem cell clones hybridised with the 3′ probe depicted in (A). The wt allele yields a 6.5-kilobase (kb) fragment, whereas both targeted Xpd†XP and Xpd†XPCS alleles yield a 5.1-kb fragment. + +(C) Genotyping of wt and targeted alleles by PCR using primers F2, R1, and mR as indicated in (A) yields fragments of 399 bp and 468 bp, respectively. + +(D) RT-PCR detection of mRNA expression originating from the targeted †XP and †XPCS alleles in embryonic stem cell clones using primers F1 (hybridising outside the targeting construct) and mR as indicated in (A) results in a 1,416-bp fragment. + +(E) Northern blot analysis of total RNA isolated from testis of homozygous wt and XpdTTD/TTD, heterozygous Xpd†XPCS/wt and XpdTTD/wt, and compound heterozygous Xpd†XPCS/TTD mice as indicated. Hybridisation with a 1.4-kb mouse Xpd cDNA probe detects mRNAs of 4, 3.3, and 2.7 kb from wt, Xpd†XPCS, and XpdTTD alleles, respectively. An ethidium bromide (EtBr)–stained gel showing the amount of total RNA loaded is shown below. + +Table 1 + +Frequency of Xpd†XP/†XP, Xpd†XPCS/†XPCS, and Compound Heterozygous Xpd†XP/†XPCS Embryos and Offspring + +“Null” Allele Can Alleviate Developmental Delay, Skin, and Hair Features of TTD + +To test the potential of a homozygous lethal “null” allele to nevertheless contribute to organismal phenotype, we combined an Xpd†XPCS allele with a viable XpdTTD allele by crossing the corresponding heterozygous animals. Similar to hemizygous TTD mice carrying one true Xpd knockout allele (XpdTTD/KO), compound heterozygous XpdTTD/†XPCS mice were born at the expected Mendelian frequencies. Expression from the Xpd†XPCS allele was also reduced in the testis of compound heterozygous animals, whereas expression from the XpdTTD allele was increased relative to wt by ~5-fold (Figure 1E). Because of a lack of available antibodies and the inability to distinguish amongst various mutant forms of XPD differing only by single amino acid substitutions, we were unable to ascertain the relative amount of XPD protein from the different alleles. + +Despite reduced levels of mRNA expression, the homozygous lethal Xpd†XPCS allele ameliorated multiple XpdTTD-associated disease symptoms in compound heterozygous XpdTTD/†XPCS animals including the hallmark brittle hair and cutaneous features fully penetrant in homo- and hemizygous TTD mice (Figure 2A–2C). In marked contrast to XpdTTD/TTD (and XpdTTD/KO) mice, which display complete hair loss in the first hair cycle and partial hair loss in subsequent cycles throughout their lives [21], compound heterozygous XpdTTD/†XPCS mice displayed some hair loss only during the first hair cycle and only locally at the back (Figure 2A). Scanning electron microscope analysis of XpdTTD/†XPCS hair revealed an almost normal appearance, with TTD-like features such as broken hairs found only at very low frequency (unpublished data). Amino acid analysis confirmed that cysteine levels in the hair of the XpdTTD/†XPCS mice were significantly higher than in XpdTTD/TTD animals, but remained below the wt level (Figure 2C). TTD hemizygotes (XpdTTD/KO) do not display significant differences in cutaneous features and longevity relative to homozygous XpdTTD/TTD mice [21]. + +Figure 2 + +Partial Rescue of TTD Cutaneous, Blood, and Developmental Phenotypes in Compound Heterozygous XpdTTD/†XPCS Mice + +(A) Photographs of 5-mo-old homozygous XpdTTD/TTD, compound heterozygous XpdTTD/†XPCS, and wt mice. Insets: images of first-round hair loss. + +(B) Histological analysis of the skin of XpdTTD/TTD, XpdTTD/†XPCS, and wt mice. TTD-associated acanthosis (thicker epidermis, indicated by solid vertical line), pronounced granular layer (indicated by arrows), and sebacious gland hyperplasia (indicated by dotted vertical line) were absent in the epidermis of XpdTTD/†XPCS and wt mice. Magnification 400×. + +(C) Cysteine content of hair from wt, XpdTTD/TTD, and XpdTTD/†XPCS mice. The p-value indicates significant differences between mutants and wt, as well as between XpdTTD/TTD and XpdTTD/†XPCS mice. Error bars indicate standard error of the mean (SEM). + +(D) Hematocrit values from blood of XpdTTD/TTD and XpdTTD/†XPCS mice. The p-values indicate the significance of the difference relative to wt. Error bars indicate SEM. + +(E) Body weights of developing XpdTTD/TTD and XpdTTD/†XPCS mice after weaning plotted as a percentage of the weight of age-matched control wt and heterozygote (hz) littermates (set at 100%). Error bars indicate SEM. + +Other prominent TTD features in the epidermis, including acanthosis (thickening of the layer of the nucleated cells), hyperkeratosis (prominent thickening of the cornified layer), and pronounced granular layer and sebacious gland hyperplasia (causing greasy appearance of the hair), were absent in the skin of XpdTTD/†XPCS mice, as established by blind microscopic examination of skin sections (Figure 2B). Furthermore, anaemia and developmental delay present in patients with TTD [24] and in XpdTTD/TTD mice [15] were both partially rescued in compound heterozygous XpdTTD/†XPCS mice (Figure 2D and 2E). + +Rescue of Progeroid Features in TTD Mice by Homozygous Lethal Xpd Alleles + +Because patients with TTD, XPCS, and CS (but not XP) and the corresponding mouse models share similar accelerated progeroid symptoms [12,13,15,23], we next addressed ageing-related parameters in compound heterozygous mice (Figure 3). Whereas XpdTTD/TTD animals show reduced bone mineral density as an indication of the early onset of osteoporosis before ~14 mo of age [15], tail vertebrae from compound heterozygous XpdTTD/†XPCS mice were comparable to wt even at 20 mo of age (Figure 3B and 3C). Furthermore, whereas XpdTTD/TTD mice developed kyphosis earlier than wt animals (onset ~3 mo versus 12–20 mo), compound heterozygous XpdTTD/†XPCS mice did not (Figure 3B). Overall appearance and body weight curves revealed that TTD-associated age-related premature cachexia and lack of general fitness were fully rescued in compound heterozygous XpdTTD/†XPCS mice (Figure 3A and 3D). Finally, the life span of compound heterozygotes was extended relative to XpdTTD/TTD mice (Table 2). + +Figure 3 + +Rescue of TTD-Associated Segmental Progeroid Features in Compound Heterozygous Xpd TTD/†XPCS Mice + +(A) Photographs of 20-mo-old wt, compound heterozygous XpdTTD/†XPCS, and homozygous XpdTTD/TTD mice. Note the extreme cachexia (lack of subcutaneous fat) in the XpdTTD/TTD mouse and the absence of this phenotype in wt and XpdTTD/†XPCS mice. + +(B) Radiographs of 20-mo-old male wt, XpdTTD/†XPCS, and XpdTTD/TTD mice. Ageing XpdTTD/TTD mice develop kyphosis (curvature of the spinal column) and reduction of bone mineral density as shown in the 6–8 segment of the tail vertebrae counted from the pelvis (see close-up at right). Note the absence of these features in the XpdTTD / † XPCS mouse. + +(C) Quantification of relative bone mineral density of tail vertebrae from 20-mo-old male wt (n = 3), XpdTTD/†XPCS (n = 4), and XpdTTD/TTD (n = 3) mice. The p-values indicate the significance of the difference relative to XpdTTD/TTD. Error bars indicate SEM. + +(D) Body weight curves as a function of time. Note that the age-dependent cachexia observed in XpdTTD/TTD mice was rescued in both male and female XpdTTD / †XPCS mice. Significant differences between wt and XpdTTD/TTD but not between wt and XpdTTD/†XPCS mice were observed at 9 and 18 mo of age as indicated by asterisks. Error bars indicate SEM. + +Table 2 + +Pleiotropic Xpd Biallelic Effects in Mice and Cells + +To determine whether the homozygous lethal Xpd†XPCS allele was unique in its ability to ameliorate symptoms associated with the XpdTTD allele, we generated compound heterozygous XpdTTD/†XP mice by crossing the corresponding heterozygous animals. Similar to the Xpd †XPCS allele, the homozygous lethal Xpd †XP allele rescued cutaneous symptoms including hair loss (except locally during the first round; unpublished data), reduced cysteine content (cysteine index 9.3 ± 0.9 standard deviation [87% of wt], p = 0.01 versus TTD), ageing-associated premature cachexia (males and females were 36.1 ± 6.4 g [93% of wt] and 39.2 ± 3.2 g [116% of wt], respectively), and reduced life span (Table 2). Taken together, these data indicate that two independent alleles, which on their own are unable to support viability (Table 1), were nonetheless able to ameliorate TTD-associated phenotypes in vivo (Table 2). + +Molecular Mechanisms of Biallelic Effects + +We next turned to UV-based cellular assays including unscheduled DNA synthesis after UV irradiation (UV-UDS), recovery of RNA synthesis after UV irradiation (UV-RRS), and UV survival, which report on the NER subpathways (global genome NER and transcription-coupled NER) and total NER, respectively. In none of these assays was the response to UV improved in compound heterozygotes relative to TTD homozygotes (Figure 4A–4C). However, unlike the in vivo TTD phenotypes described above, in which XpdTTD/TTD and XpdTTD/KO animals were indistinguishable, XpdTTD dosage effects were observed in UV survival, UV-UDS, and UV-RRS, indicating that cellular parameters as measured in fibroblasts here do not always correlate with the phenotype at the level of the intact organism. XpdTTD/KO hemizygous cells were thus used as the baseline on which to compare the activity of compound heterozygous cells. Relative to XpdTTD/KO hemizygote cells, UV survival was improved by the homozygous lethal Xpd†XPCS allele in XpdTTD/†XPCS compound heterozygous cells and to a lesser degree by the Xpd†XP allele (Figure 4A). Because of embryonic and cellular lethality, we were unable to test UV survival associated exclusively with the Xpd†XPCS or Xpd†XP alleles. However, homozygous XPDXP (XPDR683W) and hemizygous XPDXPCS (XPDG602D) human cells are known to be highly sensitive to UV [19,25], as are cells from a homozygous viable XpdXPCS/XPCS (XPDG602D/G602D) mouse model (Figure 4A, dotted line) [23]. Thus, the survival of XpdTTD/†XPCS (and XpdTTD/†XP) cells likely represents a level of UV resistance that neither mutant allele can impart on its own (Table 2). Significant effects of compound heterozygosity on NER subpathways relative to XpdTTD/KO cells were observed in XpdTTD/†XP cells but only for UV-UDS activity. Finally, none of the mutant TFIIH combinations (carrying alterations associated with TTD [XPDR722W], XPCS [XPDG602D], or XP [XPDR683W]) exhibited synergism in an in vitro NER reaction reconstituted with different mutant TFIIH complexes (Figure 4D). Taken together, these data are consistent with interallelic complementation of UV sensitivity in cells but underscore the lack of any correlation between UV-related repair characteristics and TTD progeroid phenotypes in animal models. + +Figure 4 + +TFIIH Functions and Mechanisms of XPD-Associated Disease Pleiotropy + +(A) Cellular survival after UV irradiation. Rescue of hemizygous XpdTTD/KO survival by Xpd†XPCS and Xpd†XP alleles is illustrated by arrows marked A and B, respectively. UV survival of homozygous XpdXPCS/XPCS cells (asterisk) from the normally expressed viable allele (XpdXPCS) is depicted by a dotted line. Survival curves represent an average of four independent experiments; 1–2 cell lines per genotype were included in each experiment. Error bars indicate SEM between experiments. + +(B) UV-UDS, a measure of global genome repair. Number of experiments: n = 15 (XpdTTD/TTD), n = 6 (XpdTTD/KO), n = 4 (XpdTTD/†XPCS), n = 2 (XpdTTD/†XP); 1–2 cell lines per genotype were included in each experiment. The asterisk indicates significant difference with XpdTTD/TTD; crosses indicate significant differences with XpdTTD/KO. + +(C) UV-RRS, a measure of transcription-coupled repair of UV-induced lesions. Number of experiments: n = 7 (XpdTTD/TTD), n = 2 (XpdTTD/KO), n = 4 (XpdTTD/†XPCS), n = 2 (XpdTTD/†XP); 1–2 cell lines per genotype were included in each experiment. + +(D) Incision/excision activity of combinations of altered TFIIH complexes in a reconstituted NER reaction. Equal amounts of single or mixed populations of recombinant TFIIHs (containing XPD, XPB, p62, p52, His-p44, Flag-p34, cdk7, cyclin H, Mat1, and p8) were mixed with recombinant XPG, XPF/ERCC1, XPC/hHR23B, RPA, and a radiolabelled synthetic NER substrate. The excision products (26–34 nucleotides in length) were visualised at nucleotide resolution on a denaturing polyacrylamide gel as indicated . Note the weak activity corresponding to each single and combined TFIIH complex (lanes 3–8) relative to the wt (lane 1) and negative controls (lane 2). + +(E) Xpd dose-dependent reduction of TFIIH in homozygous XpdTTD/TTD, hemizygous XpdTTD/KO, and compound heterozygous XpdTTD/†XPCS and XpdTTD/†XP cells by comparative immunofluorescence of the p62 subunit of TFIIH. Roman numerals represent different microscopic slides and Arabic numerals different cell lines labelled as follows: (I) wt cells (1) labelled with 2-μm beads, XpdTTD/TTD cells (2) with 0.79-μm beads, and XpdTTD/KO cells (3) with no beads; (II) wt cells (1) labelled with 0.79-μm beads and XpdTTD/†XPCS cells (4) with no beads; and (III) wt cells (1) labelled with 0.79-μm beads and XpdTTD/†XP cells (5) with no beads. + +(F) Quantification of immunofluorescent signal from at least 50 nuclei per cell line and 2–6 experiments per genotype. Bars representing cells analysed on the same microscopic slide are depicted side by side, with wt set at 100%. The p-value indicates minimum significant difference between wt and the indicated cell lines analysed on the same microscopic slide within one experiment. + +Next we asked whether the Xpd†XPCS and Xpd†XP alleles, despite decreased mRNA levels, ameliorated TTD symptoms by increasing overall TFIIH levels in compound heterozygous XpdTTD/ †XPCS and XpdTTD/ †XP cells. Previously, using comparative immunohistochemistry, we and others have shown an up to 70% reduction of TFIIH levels in primary fibroblasts from patients with TTD compared with wt controls due to reduced stability [16,17]. Despite overexpression of mRNA from the XpdTTD allele relative to the wt allele (Figure 1E), TFIIH protein levels were reduced by 50% in primary mouse XpdTTD/TTD fibroblasts (Figure 4E and 4F), thereby mimicking the situation in human patients with TTD. In accordance with the gene dosage, a further reduction of up to 70% of the wt level was observed in hemizygous XpdTTD/KO cells. Consistent with low mRNA expression levels, neither the Xpd†XPCS nor the Xpd†XP allele was able to restore TFIIH abundance to wt levels in XpdTTD compound heterozygote cells (Figure 4E and 4F). Thus, the improved UV survival observed in compound heterozygote cells (Figure 4A) and likely the rescue of TTD progeroid symptoms (Figure 3) were not due to normalisation of TFIIH levels, suggesting a qualitative rather than a quantitative effect on these phenotypes in vivo. + +In contrast, the level of XPCS mRNA expression did affect the ability of the encoded protein (XPDG602D) to restore the TTD hair phenotype to normal. Notably, XpdTTD/ †XPCS animals had a partial TTD hair phenotype, correlating with low levels of Xpd†XPCS expression, whereas XpdTTD/XPCS animals had wt hair, correlating with normal expression levels from the viable XpdXPCS allele (Table 2 and unpublished data). Thus, the range of expression levels from these two mutant alleles affected their ability to complement some phenotypes (hair). An overview of the functional relationships between Xpd alleles, phenotypes, and the presumed underlying TFIIH function in mice and cells is presented in Table 2. + +Discussion + +Dissection of Biallelic Effects from other Determinants of Phenotype + +Although phenotypic consequences, referred to here as biallelic effects, resulting from two different mutant alleles in compound heterozygote patients have been postulated, such effects have historically been difficult to distinguish from the influence of environment and genetic background. We used a genetically defined mammalian model system under controlled environmental conditions to reveal phenotypic effects attributable specifically to combinations of differentially mutated Xpd alleles. + +The observed biallelic effects were of three general types. In the first, the allele associated in a homozygous state with a phenotype closer to wt singularly determined the phenotypic outcome, a phenomenon widely known in human recessive disease. Because these Xpd alleles functioned at or near wt levels with respect to a particular function, we call these effects “dominant”. Such alleles can also be referred to as “separation of function” alleles, because they allow dissection of the roles of multifunctional proteins in specific phenotypes. + +Secondly, highlighting the potential relevance of current findings to all diploid organisms including humans was the observation that in one compound heterozygous animal, the Xpd allelic relationship could shift from Adominant|arecessive to Arecessive|adominant with respect to different phenotypes in a time-dependent and tissue-specific manner (see below and Table 2). + +In the third type of biallelic effect, known as interallelic complementation, two mutant alleles produced a phenotype closer to wt than either could alone in a homo- or hemizygous state. As summarised in Table 2, examples of all types of biallelic effects were observed in a variety of Xpd-associated phenotypes, ranging from brittle hair to segmental progeria. + +TFIIH in Transcription and Repair: Mechanisms of XPD Disease Pleiotropy + +We observed differences in the ability of XpdTTD versus homozygous lethal Xpd†XPCS and Xpd†XP alleles to function in two transcription-related phenotypes separated in the organism by both time and space: embryonic lethality and terminal differentiation of enucleating skin and blood cells. The preblastocyst-stage homozygous lethality shared by the XpdKO, Xpd†XPCS, and Xpd†XP alleles most likely reflects a defect in basal transcription that is incompatible with life. In XpdTTD/ †XPCS and XpdTTD/ †XP compound heterozygous mice, embryonic lethality was fully rescued by the XpdTTD allele. Because embryonic lethality was also fully rescued in XpdTTD/KO hemizygous mice, the XpdTTD allele can be considered as wt and thus dominant to each of the homozygous lethal alleles (XpdKO, Xpd†XPCS, and Xpd†XP) with respect to this particular phenotype (Table 2). + +TTD-specific cutaneous and anaemic features, on the other hand, are thought to result from a specific kind of transcriptional insufficiency caused by depletion of unstable TFIIH during the terminal differentiation of skin, hair-shaft, and blood cells [16,24]. In compound heterozygous mice, both homozygous lethal Xpd†XPCS and Xpd†XP alleles were able to alleviate XpdTTD-specific cutaneous and anaemic features and can thus be defined as dominant over the XpdTTD allele with respect to these phenotypes. We conclude that the defects leading to embryonic lethality and aberrant terminal differentiation of the skin, hair, and blood represent two qualitatively and/or quantitatively different transcriptional deficiencies. During early embryonic development, XpdTTD is dominant over the Xpd†XPCS and Xpd†XP alleles, whereas later in the ontogenesis of skin, hair-shaft, and blood cells, the situation is reversed. + +In its role in the repair of UV photolesions, the Xpd†XPCS allele imparted a clear UV survival benefit over a single XpdTTD allele or two XpdXPCS alleles independent of expression levels, which is consistent with interallelic complementation. However, the observation that no other cellular or biochemical UV-related parameters were improved in XpdTTD/ †XPCS argues against complementation of this repair activity in the rescue of TTD progeroid symptoms in vivo. + +Interallelic Complementation and XPD Function + +What does interallelic complementation tell us about the mechanism of XPD function? Interallelic complementation is most often observed in multimeric proteins with multiple functional domains. Unfortunately, the structure–function relationship between disease-causing mutations and XPD functional domains, including detailed structural information on XPD or even its stoichiometry within TFIIH, remains unknown. However, based on the ability of cell extracts that are defective in two different TFIIH components (XPD and XPB) to complement NER activity in vitro [26], it is likely that TFIIH (or its components) can either multimerise or exchange at least during the NER reaction. Furthermore, XPD is known to be a “loosely bound” subunit of TFIIH [27]. We thus envisage the molecular mechanism of interallelic complementation to involve the exchange of XPD molecules within the TFIIH complex or turnover of TFIIH complexes containing different XPD molecules at the site of DNA damage during the course of the global genome as well as transcription-coupled repair of either UV-induced or endogenous DNA damage. + +A Biallelic Paradigm for XPD Disorders + +Recently, proteins originating from presumed null alleles were biochemically characterised as inactive in basal transcription [27], providing an explanation as to why these alleles failed to rescue lethality in haploid S. pombe with a null mutation in the XPD homologue rad15 [19]. Our data suggest that certain presumed null alleles, although unable on their own to support basal transcription, may in fact have a substantial impact on disease outcome in compound heterozygous humans, as they do in mouse models. + +Clinical evidence in support of this hypothesis comes from a number of XP complementation group D patients that do not fit within the framework of the current monoallelic paradigm of XPD disorders (Figure 5). In contrast to two hemizygous XPDXPCS patients carrying the XPDG47R- or XPDR666W-encoding alleles who died of the disease before 2 y of age, two compound heterozygous XPDXPCS patients carrying the same XPDG47R- or XPDR666W-encoding alleles in addition to the presumed null XPDL461V+del716−730 both had considerably milder disease symptoms and survived more than ten times longer (A. Lehmann, personal communication) (Figure 5). Compound heterozygosity is also associated with the recently reported combination XP and TTD (XPTTD) syndrome [8]. Similar to the XpdTTD/†XPCS and XpdTTD/†XP mice described here, both patients with XPTTD described so far had intermediate hair cysteine values. Furthermore, XPTTD patient XP38BR carried a “causative” TTD mutation in one allele and a novel point mutation encoding XPDL485P in the other. Although the XPDL485P-encoding allele fails to complement viability in the haploid S. pombe rad15 deletion strain and is thus interpretable as a null allele [8], we nonetheless suggest that the combined XPTTD phenotype in this patient involves phenotypic contributions from both alleles. Taken together, these data suggest a shift to a biallelic paradigm for compound heterozygous patients in XP complementation group D. + +Figure 5 + +Genotype–Phenotype Relationships in XPD Disorders + +According to the current monoallelic hypothesis, phenotype is determined solely by the causative allele product. If a second, different allele is present, it is considered a functional null. There is a lack of any correlation between the site of the XPD mutation and the resulting disorder. We propose a biallelic hypothesis for compound heterozygotes in which both alleles can contribute to the phenotype. Examples of compound heterozygous patients in which a second, presumed null allele is likely to contribute to disease outcome are provided above in comparison to corresponding homo- or hemizygous patients with the same causative allele. Numbers in the schematic of the protein indicate the helicase domains. + +Potential of Combined Recessive Alleles to Affect Phenotypic Diversity in Mammals + +In humans, the clinical relevance of biallelic effects such as interallelic complementation remains unknown. Although interallelic complementation between two endogenous mutant alleles has been described in cells from a compound heterozygous patient with methylmalonic acidaemia, no observable effects on disease outcome were noted in the patient [28]. Thus, to the best of our knowledge, the amelioration of progeroid features observed here is the first in vivo demonstration in compound heterozygous animals of interallelic complementation relevant to a human disease. Keeping in mind that the ~1,200 alleles known to exist for the CTRF gene implicated in the common autosomal recessive disorder cystic fibrosis alone [29] can theoretically result in ~700,000 different allelic combinations, the potential number of allelic combinations of different recessive mutations and single nucleotide polymorphisms genome-wide is currently incalculable. We suggest biallelic effects as a previously underestimated yet important variable in considering genotype–phenotype relationships from autosomal recessive disease to normal phenotypic diversity in mammals. Extension of the above concept implies that recessive mutations can enter evolutionary selection in F1 provided that the second allele carries a different recessive alteration. Finally, our data highlight the potential of clinically relevant alleles previously designated as null, with little or no detectable expression or activity, to nonetheless contribute to phenotype. + +Materials and Methods + +Derivation and analysis of mutant mice. + +Generation of XpdTTD (XPDR722W) and XpdTTD/KO mice has been described previously [21,22]. A detailed description of the generation of targeting constructs for Xpd†XPCS and Xpd †XP alleles carrying mutations encoding the G602D and R683W alterations will be provided upon request. Chimeric mice and mouse embryonic fibroblasts were generated according to standard procedures. Haematoxylin and eosin staining was performed according to standard procedures. Amino acid analysis was conducted as described in [21]. Blood values were analysed using Animal Blood Counter Vet (ABX Diagnostix, Montpellier, France). Radiographs were taken, and relative bone mineral density was calculated as described in [15]. Mice used in this study were in a 129Ola/C57BL6 mixed background unless noted differently. All experiments involving mice were judged and approved by the national committee for genetic identification of organisms and the animal ethical committee, and were conducted according to national and international guidelines. + +UV sensitivity, UV-UDS, UV-RRS, and TFIIH incision/excision activity. + +UV survival, UV-UDS, and UV-RRS assays were performed as described previously [21,30]. For UV-RRS, average values from the representative experiment containing two wt, three XpdTTD/TTD, two XpdTTD/XPCS, and one XpdTTD/XP cell line are presented. The ~48% UV-UDS value presented in this study for XpdTTD/TTD cells differs from our previously published data of 25% UV-UDS [21], possibly because of the high variability intrinsic to the assay or routine variations in the cell culture conditions. For the incision/excision activity assay, recombinant TFIIH was prepared and assayed as described previously [27]. + +Comparative immunofluorescence. + +Latex bead labelling and comparative immunofluorescence analysis of the p62 subunit of the TFIIH was performed as described previously [16,17] using primary mouse embryonic fibroblasts at passages 2–5. Two or more cell lines per genotype (except for the XpdTTD/†XP cells, in which only one cell line was used in repeated experiments) were used, and experiments were repeated 2–6 times per genotype. + +Acknowledgements + +We are very grateful to Steven Bergink, Koos Jaspers, and Bjorn Schumacher for thoughtful discussions and critical reading of the manuscript and to Ruud Koppenol and Tom de Vries for photography. + +Author contributions. JOA, JJ, JHJH, GTJvdH, and JRM conceived and designed the experiments. JOA, JJ, JdW, FC, DH, MvdV, WT, JH, WJvL, and JRM performed the experiments. JOA, JJ, JdW, FC, DH, MvdV, JH, HBT, WJvL, JME, JHJH, and JRM analyzed the data. JdB and GTJvdH contributed reagents/materials/analysis tools. JOA, JHJH, and JRM wrote the paper. + +Funding. This research was supported by the Netherlands Organization for Scientific Research (NWO) through the foundation of the Research Institute for Diseases of the Elderly, as well as grants from the National Institutes of Health (1PO1 AG17242–02), National Institute of Environmental Health Sciences (1UO1 ES011044), European Commission (QRTL-1999–02002), and the Dutch Cancer Society (EUR 99–2004). JRM was a fellow of the Damon Runyon Cancer Research Fund (DRG 1677). + +Abbreviations + +bp - base pair + +CS - Cockayne syndrome + +E[number] - embryonic day [number] + +kb - kilobase + +NER - nucleotide excision repair + +SEM - standard error of the mean + +TFIIH - basal transcription/DNA repair factor IIH + +TTD - trichothiodystrophy + +UV-RRS - recovery of RNA synthesis after ultraviolet irradiation + +UV-UDS - unscheduled DNA synthesis after ultraviolet irradiation + +wt - wild-type + +XP - xeroderma pigmentosum + +XPCS - xeroderma pigmentosum combined with Cockayne syndrome + +XPTTD - combination xeroderma pigmentosum and trichothiodystrophy + +Footnotes + +Competing interests. The authors have declared that no competing interests exist. + +¤a Current address: Institute of Biotechnology, University of Helsinki, Helsinki, Finland + +¤b Current address: Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California, United States of America + +¤c Current address: Institute for Biomedical Technology, University of Twente, Bilthoven, Netherlands diff --git a/src/ontogpt/evaluation/craft/database/all/17022820.ann b/src/ontogpt/evaluation/craft/database/all/17022820.ann new file mode 100644 index 000000000..1846b634e --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17022820.ann @@ -0,0 +1,826 @@ +T1 SO:0000704 0 7 Genetic +T2 GO:0007599 42 52 hemostasis +T3 http://purl.obolibrary.org/obo/MONDO_0000831 57 67 thrombosis +T4 GO:0007596 57 67 thrombosis +T5 http://purl.obolibrary.org/obo/MONDO_0000831 91 101 Thrombosis +T6 GO:0007596 91 101 Thrombosis +T7 UBERON:0004535 144 158 cardiovascular +T8 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NCBITaxon:10088 12281 12285 mice +T284 CHEBI:30808 12291 12296 FeCl3 +T285 UBERON:0005396 12297 12311 cartoid artery +T286 UBERON:0001637 12352 12360 arterial +T287 http://purl.obolibrary.org/obo/MONDO_0000831 12361 12371 thrombosis +T288 GO:0007596 12361 12371 thrombosis +T289 UBERON:0010210 12411 12419 thrombus +T290 http://purl.obolibrary.org/obo/MONDO_0000831 12411 12419 thrombus +T291 NCBITaxon:10088 12438 12443 mouse +T292 UBERON:0001637 12496 12504 arterial +T293 UBERON:0010210 12505 12513 thrombus +T294 http://purl.obolibrary.org/obo/MONDO_0000831 12505 12513 thrombus +T295 GO:0007599 12505 12523 thrombus occlusion +T296 NCBITaxon:10088 12566 12570 mice +T297 NCBITaxon:10088 12614 12618 mice +T298 NCBITaxon:10088 12651 12655 mice +T299 CHEBI:30808 12670 12675 FeCl3 +T300 NCBITaxon:10088 12705 12709 mice +T301 NCBITaxon:10088 12766 12770 mice +T302 UBERON:0000178 12978 12983 blood +T303 CHEBI:30808 12995 13000 FeCl3 +T304 NCBITaxon:10088 13085 13089 mice +T305 NCBITaxon:10088 13119 13123 mice +T306 UBERON:0000178 13128 13133 blood +T307 UBERON:0005396 13340 13348 carotids +T308 NCBITaxon:10088 13479 13483 mice +T309 NCBITaxon:10088 13496 13500 mice +T310 UBERON:0010210 13551 13559 thrombus +T311 http://purl.obolibrary.org/obo/MONDO_0000831 13551 13559 thrombus +T312 NCBITaxon:10088 13618 13622 mice +T313 NCBITaxon:10088 13698 13702 mice +T314 NCBITaxon:10088 13723 13727 mice +T315 UBERON:0001637 13740 13748 Arterial +T316 http://purl.obolibrary.org/obo/MONDO_0020673 13740 13758 Arterial occlusion +T317 NCBITaxon:10088 13778 13782 mice +T318 CHEBI:30808 13887 13902 ferric chloride +T319 UBERON:0005396 13922 13929 carotid +T320 NCBITaxon:10088 13943 13947 mice +T321 NCBITaxon:10088 13962 13966 mice +T322 UBERON:0001637 13977 13985 Arterial +T323 http://purl.obolibrary.org/obo/MONDO_0020673 13977 13995 Arterial occlusion +T324 NCBITaxon:10088 14022 14026 mice +T325 NCBITaxon:10088 14107 14111 mice +T326 NCBITaxon:10088 14191 14195 mice +T327 GO:0007599 14232 14242 hemostatic +T328 UBERON:0002415 14289 14293 tail +T329 UBERON:0002415 14332 14336 tail +T330 NCBITaxon:10088 14470 14475 mouse +T331 NCBITaxon:10088 14553 14557 mice +T332 NCBITaxon:10088 14573 14577 mice +T333 NCBITaxon:10088 14674 14678 mice +T334 GO:0007599 14705 14715 hemostasis +T335 NCBITaxon:10088 14723 14727 mice +T336 http://purl.obolibrary.org/obo/MONDO_0000831 14732 14742 thrombosis +T337 GO:0007596 14732 14742 thrombosis +T338 PR:000012867 14744 14747 Plg +T339 PR:000014702 14755 14758 PAI +T340 NCBITaxon:10088 14762 14766 mice +T341 NCBITaxon:10088 14796 14800 mice +T342 CL:0000233 14815 14823 platelet +T343 GO:0070527 14815 14835 platelet aggregation +T344 CL:0000233 14879 14887 platelet +T345 NCBITaxon:10088 14911 14915 mice +T346 NCBITaxon:10088 14996 15000 mice +T347 PR:000012867 15076 15079 Plg +T348 NCBITaxon:10088 15083 15087 mice +T349 NCBITaxon:10088 15103 15107 mice +T350 PR:000014702 15156 15161 PAI-1 +T351 NCBITaxon:10088 15165 15169 mice +T352 NCBITaxon:10088 15185 15189 mice +T353 NCBITaxon:10088 15216 15220 mice +T354 CL:0000233 15228 15236 platelet +T355 NCBITaxon:10088 15347 15351 mice +T356 CL:0000233 15366 15374 platelet +T357 GO:0070527 15366 15386 platelet aggregation +T358 PR:000012867 15483 15486 Plg +T359 PR:000014702 15494 15499 PAI-1 +T360 NCBITaxon:10088 15503 15507 mice +T361 PR:000012867 15665 15668 Plg +T362 PR:000014702 15733 15738 PAI-1 +T363 NCBITaxon:10088 15912 15916 mice +T364 NCBITaxon:10088 15953 15957 mice +T365 UBERON:0010210 16006 16014 thrombus +T366 http://purl.obolibrary.org/obo/MONDO_0000831 16006 16014 thrombus +T367 NCBITaxon:10088 16207 16211 mice +T368 NCBITaxon:10088 16224 16228 mice +T369 NCBITaxon:10088 16301 16305 mice +T370 NCBITaxon:10088 16318 16322 mice +T371 GO:0042730 16360 16372 fibrinolytic +T372 PR:000012867 16408 16411 Plg +T373 PR:000014702 16419 16424 PAI-1 +T374 NCBITaxon:10088 16428 16432 mice +T375 PR:000012867 16500 16503 Plg +T376 NCBITaxon:10088 16507 16511 mice +T377 PR:000014702 16544 16549 PAI-1 +T378 NCBITaxon:10088 16553 16557 mice +T379 NCBITaxon:10088 16655 16659 mice +T380 PR:000012867 16672 16675 Plg +T381 PR:000014702 16751 16756 PAI-1 +T382 GO:0065007 16879 16889 regulation +T383 PR:000012867 16897 16900 Plg +T384 UBERON:0000178 16928 16933 blood +T385 GO:0007596 16928 16945 blood coagulation +T386 NCBITaxon:10088 16965 16969 mice +T387 GO:0050817 17002 17013 coagulation +T388 GO:0007598 17193 17222 extrinsic coagulation pathway +T389 PR:000007301 17238 17248 Factor VII +T390 UBERON:0000479 17252 17258 tissue +T391 PR:000001940 17252 17265 tissue factor +T392 GO:0002542 17321 17346 activation of Factors XII +T393 PR:000007296 17335 17346 Factors XII +T394 PR:000007295 17335 17342;17348 17350 Factors ... XI +T395 PR:000007303 17335 17342;17352 17354 Factors ... IX +T396 PR:000007302 17335 17342;17359 17363 Factors ... VIII +T397 CHEBI:9574 17461 17469 thrombin +T398 CHEBI:5054 17485 17491 fibrin +T399 UBERON:0010210 17485 17496 fibrin clot +T400 GO:0050817 17492 17506 clot formation +T401 NCBITaxon:10088 17552 17556 mice +T402 NCBITaxon:10088 17603 17608 mouse +T403 GO:0050817 17659 17670 coagulation +T404 GO:0050817 17733 17744 Coagulation +T405 PR:000012867 17749 17760 Plasminogen +T406 NCBITaxon:10088 17800 17805 mouse +T407 PR:000012867 17875 17878 Plg +T408 PR:000014702 17883 17888 PAI-1 +T409 NCBITaxon:10088 17911 17915 mice +T410 PR:000012867 17917 17920 Plg +T411 NCBITaxon:10088 17961 17965 mice +T412 NCBITaxon:10088 17977 17981 mice +T413 GO:0042730 17996 18008 fibrinolytic +T414 PR:000014702 18019 18024 PAI-1 +T415 PR:000014706 18039 18053 α2-anitplasmin +T416 NCBITaxon:10088 18085 18089 mice +T417 NCBITaxon:10088 18114 18118 mice +T418 GO:0005577 18200 18210 fibrinogen +T419 NCBITaxon:10088 18237 18241 mice +T420 PR:000012867 18304 18307 Plg +T421 NCBITaxon:10088 18321 18325 mice +T422 UBERON:0001969 18327 18333 plasma +T423 NCBITaxon:10088 18358 18362 mice +T424 MOP:0000569 18403 18410 reduced +T425 MOP:0000569 18416 18423 reduced +T426 PR:000012867 18501 18504 Plg +T427 CHEBI:8984 18596 18599 SDS +T428 MOP:0000569 18611 18619 reducing +T429 UBERON:0001969 18634 18640 plasma +T430 NCBITaxon:10088 18676 18680 mice +T431 NCBITaxon:10088 18761 18765 mice +T432 NCBITaxon:10088 18782 18786 mice +T433 UBERON:0001969 18850 18856 plasma +T434 PR:000012867 18895 18898 Plg +T435 UBERON:0001969 18902 18908 plasma +T436 NCBITaxon:10088 18922 18926 mice +T437 UBERON:0001969 18943 18949 plasma +T438 NCBITaxon:10088 18962 18966 mice +T439 PR:000012867 19112 19115 Plg +T440 NCBITaxon:10088 19127 19132 mouse +T441 UBERON:0002415 19143 19147 Tail +T442 UBERON:0002415 19178 19183 tails +T443 NCBITaxon:10088 19191 19195 mice +T444 UBERON:0002415 19319 19323 tail +T445 NCBITaxon:10088 19344 19348 mice +T446 CHEBI:51686 19367 19378 Hematoxylin +T447 UBERON:0002073 19510 19524 hair follicles +T448 UBERON:0001821 19526 19542 sebaceous glands +T449 CHEBI:51686 19572 19583 Hematoxylin +T450 UBERON:0002415 19636 19641 tails +T451 CL:0000233 19724 19732 platelet +T452 UBERON:0010210 19771 19779 thrombus +T453 http://purl.obolibrary.org/obo/MONDO_0000831 19771 19779 thrombus +T454 GO:0007596 19771 19789 thrombus formation +T455 UBERON:0002415 19811 19815 tail +T456 NCBITaxon:10088 19947 19951 mice +T457 UBERON:0000055 19976 19983 vessels +T458 UBERON:0001013 19987 20001 adipose tissue +T459 NCBITaxon:10088 20013 20017 mice +T460 NCBITaxon:10088 20055 20059 mice +T461 UBERON:0002415 20089 20093 Tail +T462 UBERON:0002415 20119 20124 Tails +T463 CHEBI:51686 20184 20195 Hematoxylin +T464 CHEBI:51686 20271 20282 hematoxylin +T465 NCBITaxon:10088 20309 20313 mice +T466 NCBITaxon:10088 20396 20400 mice +T467 UBERON:0002415 20534 20538 tail +T468 UBERON:0002415 20613 20617 tail +T469 NCBITaxon:10088 20640 20645 mouse +T470 NCBITaxon:10088 20680 20684 mice +T471 NCBITaxon:10088 20764 20768 mice +T472 UBERON:0002415 20792 20796 Tail +T473 SO:0000771 20846 20849 QTL +T474 UBERON:0002415 20883 20887 tail +T475 GO:0007599 20940 20950 hemostasis +T476 http://purl.obolibrary.org/obo/MONDO_0000831 20955 20965 thrombosis +T477 GO:0007596 20955 20965 thrombosis +T478 NCBITaxon:10088 21044 21048 mice +T479 NCBITaxon:10088 21213 21217 mice +T480 NCBITaxon:10088 21385 21389 mice +T481 NCBITaxon:10088 21409 21413 mice +T482 NCBITaxon:10088 21551 21555 mice +T483 NCBITaxon:10088 21597 21601 mice +T484 NCBITaxon:10088 21743 21747 mice +T485 NCBITaxon:10088 21858 21862 mice +T486 NCBITaxon:10088 21920 21924 mice +T487 NCBITaxon:10088 22103 22107 mice +T488 NCBITaxon:10088 22144 22148 mice +T489 NCBITaxon:10088 22434 22438 mice +T490 NCBITaxon:10088 22465 22469 mice +T491 NCBITaxon:10088 22493 22497 mice +T492 NCBITaxon:10088 22618 22622 mice +T493 NCBITaxon:10088 22666 22670 mice +T494 NCBITaxon:10088 23290 23294 mice +T495 NCBITaxon:10088 23363 23367 mice +T496 NCBITaxon:10088 23435 23439 mice +T497 NCBITaxon:10088 23476 23480 mice +T498 PR:000014702 23702 23707 PAI-1 +T499 CHEBI:59132 23708 23715 antigen +T500 PR:000012867 23748 23751 Plg +T501 PR:000014702 23788 23793 PAI-1 +T502 PR:000014706 23804 23818 α2-antiplasmin +T503 NCBITaxon:10088 23871 23875 mice +T504 PR:000012867 23890 23893 Plg +T505 CHEBI:59132 23894 23901 antigen +T506 GO:0042730 23903 23915 fibrinolytic +T507 PR:000014706 23926 23940 α2-antiplasmin +T508 PR:000014702 23942 23947 PAI-1 +T509 GO:0005577 23961 23971 fibrinogen +T510 NCBITaxon:10088 23992 23996 mice +T511 NCBITaxon:10088 24008 24012 mice +T512 PR:000012867 24018 24021 Plg +T513 CHEBI:59132 24022 24029 antigen +T514 NCBITaxon:10088 24117 24121 mice +T515 PR:000012867 24157 24160 Plg +T516 CHEBI:59132 24161 24168 antigen +T517 NCBITaxon:10088 24202 24206 mice +T518 GO:0042730 24208 24220 Fibrinolytic +T519 NCBITaxon:10088 24298 24302 mice +T520 PR:000014706 24304 24318 α2-antiplasmin +T521 NCBITaxon:10088 24367 24371 mice +T522 NCBITaxon:10088 24502 24506 mice +T523 NCBITaxon:10088 24526 24530 mice +T524 PR:000014702 24533 24538 PAI-1 +T525 CHEBI:59132 24539 24546 antigen +T526 PR:000014702 24788 24793 PAI-1 +T527 CHEBI:59132 24794 24801 antigen +T528 NCBITaxon:10088 24822 24826 mice +T529 PR:000014702 24840 24845 PAI-1 +T530 CHEBI:59132 24846 24853 antigen +T531 PR:000014702 25070 25075 PAI-1 +T532 NCBITaxon:10088 25137 25141 mice +T533 PR:000014702 25196 25201 PAI-1 +T534 PR:000014702 25275 25280 PAI-1 +T535 CHEBI:59132 25281 25288 antigen +T536 NCBITaxon:10088 25364 25368 mice +T537 NCBITaxon:10088 25399 25403 mice +T538 PR:000014702 25416 25421 PAI-1 +T539 UBERON:0001969 25455 25461 Plasma +T540 PR:000014702 25462 25467 PAI-1 +T541 CHEBI:59132 25468 25475 antigen +T542 UBERON:0001969 25497 25503 Plasma +T543 PR:000014702 25504 25509 PAI-1 +T544 NCBITaxon:10088 25553 25557 mice +T545 NCBITaxon:10088 25626 25630 mice +T546 NCBITaxon:10088 25698 25702 mice +T547 NCBITaxon:10088 25739 25743 mice +T548 UBERON:0001637 25961 25969 arterial +T549 UBERON:0010210 25970 25978 thrombus +T550 http://purl.obolibrary.org/obo/MONDO_0000831 25970 25978 thrombus +T551 GO:0007596 25970 25988 thrombus formation +T552 UBERON:0002415 25994 25998 tail +T553 UBERON:0001637 26133 26141 arterial +T554 http://purl.obolibrary.org/obo/MONDO_0020673 26133 26151 arterial occlusion +T555 CHEBI:30808 26162 26167 FeCl3 +T556 NCBITaxon:10088 26246 26250 mice +T557 NCBITaxon:10088 26348 26352 mice +T558 NCBITaxon:10088 26450 26454 mice +T559 NCBITaxon:10088 26474 26478 mice +T560 NCBITaxon:10088 26524 26528 mice +T561 NCBITaxon:10088 26589 26593 mice +T562 UBERON:0001637 26619 26627 arterial +T563 http://purl.obolibrary.org/obo/MONDO_0020673 26619 26637 arterial occlusion +T564 SO:0000704 26683 26688 genes +T565 UBERON:0001637 26728 26736 arterial +T566 http://purl.obolibrary.org/obo/MONDO_0020673 26728 26746 arterial occlusion +T567 UBERON:0001637 26764 26772 Arterial +T568 http://purl.obolibrary.org/obo/MONDO_0020673 26764 26782 Arterial occlusion +T569 NCBITaxon:10088 26857 26861 mice +T570 NCBITaxon:10088 26931 26935 mice +T571 NCBITaxon:10088 27003 27007 mice +T572 NCBITaxon:10088 27044 27048 mice +T573 http://purl.obolibrary.org/obo/MONDO_0000831 27271 27281 thrombotic +T574 GO:0007596 27271 27281 thrombotic +T575 NCBITaxon:10088 27340 27344 mice +T576 http://purl.obolibrary.org/obo/MONDO_0011122 27408 27415 obesity +T577 http://purl.obolibrary.org/obo/MONDO_0005311 27430 27445 atherosclerosis +T578 UBERON:0000055 27495 27501 vessel +T579 UBERON:0001637 27514 27522 Arterial +T580 http://purl.obolibrary.org/obo/MONDO_0020673 27514 27532 Arterial occlusion +T581 UBERON:0002415 27539 27543 tail +T582 http://purl.obolibrary.org/obo/MONDO_0000831 27615 27625 thrombotic +T583 GO:0007596 27615 27625 thrombotic +T584 SO:0000704 27703 27707 gene +T585 NCBITaxon:10088 27717 27721 mice +T586 PR:000012867 27729 27732 Plg +T587 GO:0042730 27754 27766 fibrinolytic +T588 NCBITaxon:10088 27809 27813 mice +T589 CL:0000233 27829 27837 platelet +T590 http://purl.obolibrary.org/obo/MONDO_0000831 27885 27895 thrombotic +T591 GO:0007596 27885 27895 thrombotic +T592 GO:0050817 28011 28022 coagulation +T593 CL:0000233 28026 28034 platelet +T594 SO:0000704 28106 28111 genes +T595 NCBITaxon:10088 28181 28185 Mice +T596 PR:000014702 28348 28353 PAI-1 +T597 CHEBI:59132 28354 28361 antigen +T598 NCBITaxon:10088 28435 28439 mice +T599 UBERON:0001637 28574 28582 Arterial +T600 http://purl.obolibrary.org/obo/MONDO_0020673 28574 28592 Arterial occlusion +T601 CHEBI:30808 28735 28740 FeCl3 +T602 http://purl.obolibrary.org/obo/MONDO_0000831 28778 28788 thrombosis +T603 GO:0007596 28778 28788 thrombosis +T604 NCBITaxon:10088 28792 28796 mice +T605 SO:0000704 28828 28835 genetic +T606 CHEBI:52217 28840 28855 pharmacological +T607 http://purl.obolibrary.org/obo/MONDO_0004450 28938 28950;28955 28969 occlusion of carotid artery +T608 UBERON:0005396 28955 28969 carotid artery +T609 CHEBI:30808 28976 28981 FeCl3 +T610 CHEBI:30808 28996 29001 FeCl3 +T611 UBERON:0010210 29013 29021 thrombus +T612 http://purl.obolibrary.org/obo/MONDO_0000831 29013 29021 thrombus +T613 GO:0007596 29013 29031 thrombus formation +T614 GO:0007599 29013 29021;29036 29045 thrombus ... occlusion +T615 NCBITaxon:10088 29082 29086 mice +T616 NCBITaxon:10088 29106 29110 mice +T617 NCBITaxon:10114 29200 29204 rats +T618 UBERON:0000178 29273 29278 blood +T619 UBERON:0005396 29473 29481 carotids +T620 UBERON:0001637 29485 29493 arterial +T621 http://purl.obolibrary.org/obo/MONDO_0020673 29485 29503 arterial occlusion +T622 NCBITaxon:10088 29528 29532 mice +T623 UBERON:0010210 29610 29618 thrombus +T624 http://purl.obolibrary.org/obo/MONDO_0000831 29610 29618 thrombus +T625 NCBITaxon:10088 29633 29637 mice +T626 UBERON:0000178 29654 29659 blood +T627 NCBITaxon:10088 29735 29739 mice +T628 CL:0000233 29759 29767 platelet +T629 GO:0050817 29782 29793 coagulation +T630 NCBITaxon:10088 29838 29842 mice +T631 PR:000012867 29868 29871 Plg +T632 UBERON:0010210 29886 29894 thrombus +T633 http://purl.obolibrary.org/obo/MONDO_0000831 29886 29894 thrombus +T634 GO:0007596 29886 29904 thrombus formation +T635 PR:000012867 29929 29932 Plg +T636 NCBITaxon:10088 29936 29940 mice +T637 PR:000014702 29963 29966 PAI +T638 NCBITaxon:10088 29970 29974 mice +T639 UBERON:0010210 30048 30052 clot +T640 GO:0065007 30064 30072 modulate +T641 UBERON:0010210 30105 30113 thrombus +T642 http://purl.obolibrary.org/obo/MONDO_0000831 30105 30113 thrombus +T643 GO:0007596 30105 30123 thrombus formation +T644 UBERON:0002415 30130 30134 tail +T645 NCBITaxon:10088 30179 30183 mice +T646 GO:0010467 30230 30240 expression +T647 CL:0000233 30244 30252 platelet +T648 GO:0050817 30257 30268 coagulation +T649 NCBITaxon:10088 30281 30285 mice +T650 NCBITaxon:10088 30328 30332 mice +T651 NCBITaxon:10088 30359 30363 mice +T652 PR:000017364 30411 30433 von Willebrand antigen +T653 CHEBI:59132 30426 30433 antigen +T654 NCBITaxon:10088 30462 30466 mice +T655 SO:0000704 30472 30476 gene +T656 GO:0050817 30494 30505 coagulation +T657 UBERON:0002415 30619 30623 tail +T658 PR:000007302 30627 30632 FVIII +T659 PR:000007303 30637 30640 FIX +T660 NCBITaxon:10088 30651 30655 mice +T661 SO:0000704 30749 30756 genetic +T662 UBERON:0010210 30771 30779 thrombus +T663 http://purl.obolibrary.org/obo/MONDO_0000831 30771 30779 thrombus +T664 GO:0007596 30771 30789 thrombus formation +T665 NCBITaxon:10088 30805 30809 Mice +T666 CL:0000233 30860 30868 platelet +T667 CL:0000233 30879 30887 platelet +T668 CHEBI:17089 30888 30900 glycoprotein +T669 PR:000001664 30914 30943 protease-activated receptor-4 +T670 UBERON:0002415 30965 30969 tail +T671 CL:0000233 31020 31028 platelet +T672 GO:0070527 31020 31040 platelet aggregation +T673 PR:000012867 31125 31128 Plg +T674 PR:000012826 31136 31139 uPA +T675 NCBITaxon:10088 31143 31147 mice +T676 PR:000012867 31162 31165 Plg +T677 PR:000012826 31170 31173 uPA +T678 CL:0000233 31200 31208 platelet +T679 UBERON:0010210 31236 31244 thrombus +T680 http://purl.obolibrary.org/obo/MONDO_0000831 31236 31244 thrombus +T681 GO:0007596 31236 31254 thrombus formation +T682 NCBITaxon:10088 31325 31330 mouse +T683 SO:0000704 31348 31355 genetic +T684 GO:0050817 31375 31386 coagulation +T685 UBERON:0010210 31425 31433 thrombus +T686 http://purl.obolibrary.org/obo/MONDO_0000831 31425 31433 thrombus +T687 UBERON:0002415 31443 31447 tail +T688 NCBITaxon:10088 31468 31472 mice +T689 NCBITaxon:10088 31558 31562 mice +T690 PR:000012867 31578 31581 Plg +T691 NCBITaxon:10088 31585 31589 mice +T692 PR:000014702 31619 31624 PAI-1 +T693 PR:000012826 31663 31666 uPA +T694 NCBITaxon:10088 31670 31674 mice +T695 PR:000012867 31721 31724 Plg +T696 NCBITaxon:10088 31728 31732 mice +T697 PR:000012867 31874 31877 Plg +T698 NCBITaxon:10088 31895 31899 mice +T699 PR:000012825 31964 31967 tPA +T700 PR:000012826 31972 31975 uPA +T701 PR:000014702 31980 31985 PAI-1 +T702 NCBITaxon:10088 31989 31993 mice +T703 NCBITaxon:10088 32009 32013 mice +T704 UBERON:0002415 32050 32054 tail +T705 PR:000012867 32216 32219 Plg +T706 NCBITaxon:10088 32223 32227 mice +T707 PR:000012867 32262 32265 Plg +T708 NCBITaxon:10088 32269 32273 mice +T709 SO:0000704 32433 32444 genetically +T710 UBERON:0002415 32485 32489 tail +T711 SO:0000704 32509 32520 genetically +T712 UBERON:0000178 32540 32545 blood +T713 NCBITaxon:10088 32586 32590 mice +T714 UBERON:0002415 32605 32609 tail +T715 NCBITaxon:10088 32699 32703 mice +T716 UBERON:0000178 32735 32740 blood +T717 NCBITaxon:10088 32786 32790 mice +T718 UBERON:0000178 32792 32797 Blood +T719 PR:000012867 32827 32830 Plg +T720 NCBITaxon:10088 32834 32838 mice +T721 NCBITaxon:10088 32913 32917 mice +T722 UBERON:0000178 32931 32936 blood +T723 PR:000012826 32983 32986 uPA +T724 NCBITaxon:10088 33013 33017 mice +T725 UBERON:0000178 33036 33041 blood +T726 UBERON:0000178 33091 33096 blood +T727 UBERON:0002415 33110 33114 tail +T728 UBERON:0002415 33174 33178 tail +T729 UBERON:0002415 33233 33237 tail +T730 NCBITaxon:10088 33249 33253 mice +T731 GO:0032963 33291 33313 metabolism of collagen +T732 GO:0019538 33291 33304;33338 33346 metabolism of ... proteins +T733 GO:0031012 33317 33337 extracellular matrix +T734 NCBITaxon:10088 33428 33432 mice +T735 SO:0000704 33473 33478 genes +T736 PR:000012867 33483 33486 Plg +T737 PR:000014702 33488 33493 PAI-1 +T738 PR:000014706 33499 33513 α2-antiplasmin +T739 SO:0000771 33561 33564 QTL +T740 NCBITaxon:10088 33664 33668 mice +T741 UBERON:0001637 33796 33804 arterial +T742 http://purl.obolibrary.org/obo/MONDO_0020673 33796 33814 arterial occlusion +T743 NCBITaxon:10088 33831 33835 mice +T744 NCBITaxon:10088 33861 33865 mice +T745 NCBITaxon:10088 33875 33879 mice +T746 PR:000012867 33945 33948 Plg +T747 PR:000014702 33950 33955 PAI-1 +T748 PR:000014706 33961 33975 α2-antiplasmin +T749 SO:0001023 33981 33988 allelic +T750 NCBITaxon:10088 34002 34007 mouse +T751 PR:000012867 34008 34011 Plg +T752 SO:0000704 34012 34016 gene +T753 NCBITaxon:10088 34051 34055 mice +T754 PR:000012867 34062 34065 Plg +T755 NCBITaxon:10088 34101 34105 mice +T756 NCBITaxon:10088 34121 34125 mice +T757 PR:000012867 34169 34172 Plg +T758 NCBITaxon:10088 34348 34352 mice +T759 PR:000012867 34393 34396 Plg +T760 SO:0000704 34397 34401 gene +T761 NCBITaxon:10088 34526 34530 mice +T762 SO:0000771 34552 34555 QTL +T763 PR:000012867 34563 34566 Plg +T764 SO:0000704 34578 34583 genes +T765 GO:0031012 34608 34614 matrix +T766 SO:0000704 34676 34681 genes +T767 SO:0000028 34687 34696 base pair +T768 PR:000014702 34719 34724 PAI-1 +T769 SO:0000704 34725 34729 gene +T770 PR:000014706 34737 34751 α2-antiplasmin +T771 SO:0000704 34752 34756 gene +T772 NCBITaxon:10088 34771 34775 mice +T773 GO:0065007 34815 34825 regulatory +T774 SO:0000704 34826 34831 genes +T775 SO:0000704 34922 34927 genes +T776 PR:000012867 34950 34953 Plg +T777 NCBITaxon:10088 35074 35078 mice +T778 UBERON:0001637 35095 35103 arterial +T779 http://purl.obolibrary.org/obo/MONDO_0000831 35104 35114 thrombosis +T780 GO:0007596 35104 35124 thrombosis formation +T781 CHEBI:30808 35130 35135 FeCl3 +T782 UBERON:0002415 35180 35184 tail +T783 PR:000012867 35201 35212 plasminogen +T784 UBERON:0000479 35227 35233 tissue +T785 UBERON:0000055 35238 35244 vessel +T786 NCBITaxon:10088 35293 35297 mice +T787 SO:0000771 35323 35326 QTL +T788 SO:0000704 35352 35357 genes +T789 PR:000012867 35394 35397 Plg +T790 GO:0065007 35410 35418 modulate +T791 UBERON:0001637 35419 35427 arterial +T792 http://purl.obolibrary.org/obo/MONDO_0020673 35419 35437 arterial occlusion +T793 NCBITaxon:10088 35523 35527 mice +T794 SO:0000704 35557 35564 genetic +T795 http://purl.obolibrary.org/obo/MONDO_0000831 35581 35591 thrombosis +T796 GO:0007596 35581 35591 thrombosis +T797 GO:0007599 35596 35607 haemostasis +T798 SO:0000704 35672 35677 genes +T799 SO:0000704 35706 35711 genes +T800 UBERON:0001637 35750 35758 arterial +T801 http://purl.obolibrary.org/obo/MONDO_0000831 35759 35769 thrombosis +T802 GO:0007596 35759 35769 thrombosis +T803 UBERON:0002415 35771 35775 tail +T804 UBERON:0000055 35800 35806 vessel +T805 GO:0031012 35807 35827 extracellular matrix +T806 SO:0000704 35986 35993 genetic +T807 http://purl.obolibrary.org/obo/MONDO_0000831 36010 36020 thrombotic +T808 GO:0007596 36010 36020 thrombotic +T809 PR:000012867 36177 36180 Plg +T810 PR:000012867 36183 36194 plasminogen +T811 PR:000014702 36196 36201 PAI-1 +T812 PR:000014702 36204 36237 plasminogen activator inhibitor-1 +T813 CHEBI:35222 36226 36235 inhibitor +T814 PR:000012825 36239 36242 tPA +T815 UBERON:0000479 36245 36251 tissue +T816 PR:000012825 36245 36273 tissue plasminogen activator +T817 PR:000012826 36275 36278 uPA +T818 PR:000012826 36281 36312 urokinase plasminogen activator +T819 SO:0000771 36348 36351 QTL +T820 SO:0000771 36354 36378 quantitative trait locus +T821 UBERON:0002415 36612 36616 tail +T822 UBERON:0002415 36739 36743 tail +T823 UBERON:0002415 36780 36784 tail +T824 NCBITaxon:10088 36828 36833 mouse +T825 UBERON:0000178 37373 37378 blood +T826 PR:000012867 37426 37437 plasminogen diff --git a/src/ontogpt/evaluation/craft/database/all/17022820.txt b/src/ontogpt/evaluation/craft/database/all/17022820.txt new file mode 100644 index 000000000..5739f79b8 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17022820.txt @@ -0,0 +1,199 @@ +Genetic background determines response to hemostasis and thrombosis + +Abstract + +Background + +Thrombosis is the fatal and disabling consequence of cardiovascular diseases, the leading cause of mortality and morbidity in Western countries. Two inbred mouse strains, C57BL/6J and A/J, have marked differences in susceptibility to obesity, atherosclerosis, and vessel remodeling. However, it is unclear how these diverse genetic backgrounds influence pathways known to regulate thrombosis and hemostasis. The objective of this study was to evaluate thrombosis and hemostasis in these two inbred strains and determine the phenotypic response of A/J chromosomes in the C57BL/6J background. + +Methods + +A/J and C57Bl/6J mice were evaluated for differences in thrombosis and hemostasis. A thrombus was induced in the carotid artery by application of the exposed carotid to ferric chloride and blood flow measured until the vessel occluded. Bleeding and rebleeding times, as surrogate markers for thrombosis and hemostasis, were determined after clipping the tail and placing in warm saline. Twenty-one chromosome substitution strains, A/J chromosomes in a C57BL/6J background, were screened for response to the tail bleeding assay. + +Results + +Thrombus occlusion time was markedly decreased in the A/J mice compared to C57BL/6J mice. Tail bleeding time was similar in the two strains, but rebleeding time was markedly increased in the A/J mice compared to C57BL/6J mice. Coagulation times and tail morphology were similar, but tail collagen content was higher in A/J than C57BL/6J mice. Three chromosome substitution strains, B6-Chr5A/J, B6-Chr11A/J, and B6-Chr17A/J, were identified with increased rebleeding time, a phenotype similar to A/J mice. Mice heterosomic for chromosomes 5 or 17 had rebleeding times similar to C57BL/6J mice, but when these two chromosome substitution strains, B6-Chr5A/J and B6-Chr17A/J, were crossed, the A/J phenotype was restored in these doubly heterosomic progeny. + +Conclusion + +These results indicate that susceptibility to arterial thrombosis and haemostasis is remarkably different in C57BL/and A/J mice. Three A/J chromosome substitution strains were identified that expressed a phenotype similar to A/J for rebleeding, the C57Bl/6J background could modify the A/J phenotype, and the combination of two A/J QTL could restore the phenotype. The diverse genetic backgrounds and differences in response to vascular injury induced thrombosis and the tail bleeding assay, suggest the potential for identifying novel genetic determinants of thrombotic risk. + +Background + +Family history [1] is the strongest risk factor for cardiovascular diseases (CVD). While a number of genetic mutations have been identified, these account for only a small percentage of the CVD in human populations. Thrombus formation on fissured atherosclerotic plaques is the precipitating event in the transition from a stable or subclinical atherosclerotic disease and leads to acute myocardial infarction, ischemic stroke or peripheral arterial occlusion. Arterial and venous thrombosis are complex responses and are influenced by multiple genetic and environmental factors [2-5]. Polymorphisms and mutations in coagulation factors, fibrinolytic factors, platelet surface receptors, methylenetetrahydrofalate reductase, endothelial nitric oxide synthase, and antioxidant enzymes have been implicated as genetic determinants of susceptibility to thrombosis [6,7]. Great strides have been made in the diagnosis and treatment of thrombosis in the last decade. However, strategies to prevent thrombosis have lagged far behind due, in part, to the contribution of multiple, and as yet undefined, genetic factors that lead to thrombotic risk. Moreover, it remains unclear how genetic background influences the function of molecules and pathways known to regulate thrombus formation and lysis and, thereby, contributes to the risk of thrombotic disease. + +Many of the available inbred mouse strains, such as C57BL/6J (B6) and A/J, have been classified as resistant or susceptible to particular complex diseases (Table 1). These two strains have diverse responses to diet-induced obesity [8] (B6 susceptible; A/J resistant), diet-induced atherosclerosis (B6 susceptible; A/J resistant) [9], arterial ligation-induced neointimal hyperplasia (B6 resistant; A/J resistant), and ligation induced vessel remodeling (B6 resistant; A/J susceptible) [10]. These conditions are predisposing to thrombosis per se, but these mice have not been systematically evaluated for thrombosis. Clot formation and lysis, which ultimately determine thrombosis, requires platelet aggregation, coagulation, and fibrinolytic functions. Interactions among these pathways may occur and additional factors may modulate these processes. Identification of novel modulators and factors of these pathways can be identified by associating the phenotype with a location(s) on a specific chromosome, a quantitative trait locus (QTL). + +Table 1 + +Identification of mice susceptible or resistant to obesity, atherosclerosis, and vascular injury + +R: resistant; S: susceptible. Reference provided by number in parenthesis. Lepob, Plg-/-, and PAI-F1-/- mice were in a B6 background. + +A panel of chromosome substitution strains (CSS) was generated [11] by a "marker-assisted" breeding program where the progeny of a B6 × A/J cross were successively backcrossed to the B6 mice. Genetic markers were used to identify homozygosity in the background (B6) and the individual A/J chromosome. These strains have one chromosome from A/J mice in a B6 background. This allows the identification of a trait in one or more CSS and implies that at least one QTL resides on this chromosome. Use of this panel requires fewer mice to determine the QTL than does a genome-wide scan. Another advantage is the ability for detection of QTL in the presence of other QTL. In addition, initial screens with the CSS simplify the fine-mapping of QTL. Several studies of this CSS panel, B6 Chr1-19, X, YA/J, have been reported for behavior [12], weight gain [13], sterols [13], and plasma amino acids levels [13,14], and have identified many more QTL than studies of the same traits using a genome-wide scan. + +The purpose of this study was to evaluate the two inbred strains of mice for prothrombotic risk, utilizing a ferric chloride vascular injury model, tail bleeding assay, and measures of coagulation and fibrinolysis. The results of this study indicate that the two inbred strains, B6 and A/J mice, have diverse prothrombotic phenotypes, unrelated to coagulation or platelet aggregation. In addition, in the CSS, expression of the A/J phenotypes, rebleeding time and arterial occlusion, was modified in the B6 background, and suggested that interactions occurred among the A/J QTL. Thus, it should be possible to identify independent genetic determinants of susceptibility to pathological haemostasis and thrombosis. + +Methods + +Mice + +The inbred strains, B6 (#000664), Lepob (#000632), A/J (#000646) and gene-targeted plasminogen (Plg) activator inhibitor deficient mice (PAI-1-/-) (#002507) [15] mice in a B6 background were obtained from Jackson Laboratory (Bar Harbor, Maine) at 6 wks-of-age. The Plg-/- mice were generated as previously described [16] and maintained in the B6 background, generated by crossing mice from the original mixed (B6:129) background for eight generations with B6 mice. The Plg-/- mice do not reproduce well and Plg heterozygous pairs were used for breeding. Genotypes (Plg+/+, Plg+/-, Plg-/-) of the offspring were determined by a PCR assay at 3–4 weeks-of-age from an ear punch [17]. Breeding pairs of the CSS mice were transferred from Dr. Nadeau's laboratory. All mice were housed and bred in the Biological Resource Unit at the Cleveland Clinic Foundation (CCF). Mice were housed in sterilized isolator cages, maintained on a 14 hr light/10 hr dark cycle, and were provided sterilized food and water ad libitum. Mice were fed a standard autoclavable laboratory diet consisting of: 23% protein, 4.5% fat, 6% fiber, (Purina, St. Louis, MO), and tested between 7 and 9 wks-of-age. All animal experiments were performed in accordance with a protocol approved by the Institutional Animal Care and Research Advisory Committee at CCF. + +Vascular injury + +To induce thrombosis formation in the carotid artery, a ferric chloride (FeCl3) model of vessel injury [18,19] was employed. Mice were anesthetized with ketamine/xylazine (80 mg/kg, 5 mg/kg), a midline cervical incision was made and the left common carotid artery isolated by blunt dissection. The flow probe (Transonic Systems, model 0.5PSB) was placed under the artery and when a stable baseline was reached, a 0.5 × 2 mm strip of filter paper saturated with 10% FeCl3 solution was applied to the surface of the carotid artery for 3 min. Occlusion time was determined from the addition of the FeCl3 solution to the occlusion of the artery (minimum blood flow). The flow probe was in place from the establishment of the baseline until several min after the stable occlusion had been reached or stopped at 30 min if it had not occluded. Blood flow was recorded every 10 sec (Transonic Systems, model TS420). There was no difference in body weight among the mice that were tested in the vascular injury model. + +Bleeding and rebleeding assay + +For the tail bleeding assay, mice were anesthetized with ketamine/xylazine (80 mg/kg, 5 mg/kg), the tail prewarmed for 5 min in 10 mL of saline at 37°C in a water bath. The tail was lifted from the saline and a 5 mm tail segment amputated and immediately returned to the saline. Bleeding time was measured as the time between the start of bleeding to cessation of bleeding. With the tail remaining in the same saline solution, the rebleeding time was measured from the time between bleeding cessation and the start of the second bleeding. + +Coagulation, Plg, α2-antiplasmin, fibrinogen, fibrinolytic and PAI-1 assays + +Mice were anesthetized with Isoflurane (Abbott), bled from the orbital sinus into uncoated capillary tubes, and a drop of blood immediately placed on a reagent strip (Synbiotics) for prothrombin time (PT) or activated partial thromboplastin time (aPTT) determinations and inserted into the precalibrated Coagulation Analyzer (SCA2000, Synbiotics). PAI-1 activity was determined according to the method of Chandler et al [20]. PAI-1 antigen concentration was determined by ELISA [21] using sheep-anti mouse PAI-1 (American Diagnostica) as the capture antibody and mouse PAI-1 (American Diagnostica) as the standard (0–3.5 ng/mL). Plg was determined with a chromogenic assay [22] and the fibrinolytic activity determined by fibrin degradation [23]. For the negative controls in these assays, plasma from PAI-1 or Plg deficient mice was used. Functionally active mouse α2-antiplasmin was determined with a plasmin capture assay and detection with a mouse antibody (Molecular Innovations, Southfield, MI), and fibrinogen with an ELISA assay with antibodies that recognize mouse fibrinogen (Hyphen BioMed, France). + +Histochemical and immunohistochemistry analysis + +The frozen tails or injured carotids in OCT were sectioned at 5 μm thickness and stained with Masson's Trichrome (HT 15, Sigma Diagnostics). For quantitative analysis of collagen area in the tail sections, four sections from 2–3 sites were measured using computer assisted image analysis (Image-Pro Plus, Cybernetics). For the area determination of the injured carotid lumen, 3–4 sections from different sites approximately 100 μm apart were analyzed and averaged for each mouse. + +Statistics + +Data are presented as mean ± SEM. The statistical analysis for comparisons between A/J and B6 mice and between wild-type (WT) and Plg-/- littermates was compared with a two-tailed t-test. Differences among B6, Lepob, PAI-1-/- mice were determined by a one-way ANOVA and a Newman-Keuls post-test. A value of P < 0.05 was considered significant. The statistical analysis for comparisons between B6 mice and CSS were determined with two-tail t-tests and the level of the significant P-values (see figure legends) determined with a Bonferroni correction to account for multihypothesis testing [13]. + +Results + +Marked differences in arterial thrombus formation in B6 and A/J mice + +The FeCl3 cartoid artery injury model [24], a well-characterized arterial thrombosis model, was used to induce an occlusive thrombus in the B6 and A/J mouse strains under investigation. A marked difference in arterial thrombus occlusion time was found between the B6 and the A/J mice (Figure 1A). The occlusion time in the A/J mice was 2-fold less than for the B6 mice. A 5% dose of FeCl3 was tested in the A/J and B6 mice and the occlusion time was lower in the A/J than the B6 mice, the same results as with the 10% dose. The occlusion time (min for both B6 (15 ± 3, n = 3) and A/J (8 ± 1, n = 3) at 5% were longer than at 10% for B6 (10 ± 1, n = 17 and A/J (5 ± 2, n = 14). The curves of blood flow after FeCl3 application indicated a pattern with a gradual decrease before occlusion for the B6 mice (Figure 1B), but for the A/J mice the blood flow decreased abruptly to zero (Figure 1C), demonstrating a marked difference from the B6 strain. The mean occlusion times were significantly different between the two strains (Figure 1A). The size of the carotids in the two strains was noticeably different. The area (mm2) of the lumen (0.066 ± 0.007, n = 6) was significantly less in the A/J mice than for B6 mice (0.128 ± 0.007, n = 6). However, the ratio of the thrombus to lumen was similar for A/J (0.63 ± 0.03) compared to B6 mice (0.67 ± 0.09). Calculated sheer stress rates [25] were 14% less in the A/J mice when compared to B6 mice. + +Figure 1 + +Arterial occlusion time in B6 and A/J mice. Panels A and B are representative occlusion curves from each genotype. Red arrows indicate duration of ferric chloride application to the carotid. Panel A: B6 mice, Panel B: A/J mice. Panel C: Arterial occlusion time, mean ± SEM of 14–17 mice per genotype. Symbol indicates a significant difference (**P < 0.01) between B6 mice determined with a t-test. + +Marked differences in rebleeding time in B6 and A/J mice + +The bleeding time, an indicator of hemostatic activity, was determined after prewarming the tail, clipping a 5 mm segment, placing the tail in warm saline and measuring the time for the bleeding to stop. We found this procedure was consistent and reproducible in different mouse strains and was not gender dependent. Bleeding time was not different in A/J mice compared to B6 mice (Figure 2A). In order to determine if specific pathways were altered in the B6 and A/J strains, mice with known alterations in hemostasis, Lepob mice, or thrombosis, Plg-/- and PAI-/- mice, were also tested. The Lepob mice have impaired platelet aggregation [26] and were evaluated as a reference for platelet function. In the Lepob mice (Figure 2A) there was nearly a 10-fold increase in bleeding time compared to B6 mice. Bleeding time was also significantly (P = 0.0001) increased by 66% in the Plg-/- mice compared to WT mice littermates. Bleeding time was not different in PAI-1-/- mice compared to B6 mice. In contrast to the Lepob mice with a platelet defect and 10-fold increase in bleeding time, there was no difference in the bleeding time between B6 and A/J mice and suggested platelet aggregation is not altered in these two strains. + +Figure 2 + +Bleeding and rebleeding time in B6, A/J, Lepob, Plg-/- and PAI-1-/- mice. Panel A, B: Bleeding time (seconds) and Panel C, D: Rebleeding time (seconds). To determine statistical differences, values from B6 and A/J and from WT and Plg-/- littermates were compared with a t-test, and values from B6, PAI-1-/- and Lepob were compared by a one-way ANOVA and a Newman-Keuls post-test. Symbols indicate a significant difference (*P < 0.05, **P < 0.01, ***P < 0.001) between B6 or WT mice. Values are the mean ± SEM of 10–34 mice per genotype. + +Rebleeding time, an indicator of thrombus stability, was measured as the time between the cessation of bleeding and the start of the second bleeding (Figure 2B). Rebleeding times were significantly higher in A/J (2.6-fold, P < 0.001) mice than the B6 mice. In contrast, the rebleeding time was nearly 8.5-fold less in the Lepob mice than the B6 mice. To determine how alterations in the fibrinolytic system may affect rebleeding time, Plg-/- and PAI-1-/- mice were also tested (Figure 2B). Rebleeding time was not different in Plg-/- mice compared to WT littermates, but PAI-1-/- mice had a significantly (P < 0.05) reduced rebleeding time, which was 1.8-fold lower than for the B6 mice. Although a Plg deficiency did not result in a prolonged rebleeding time as anticipated, a PAI-1 deficiency resulted in a shortened time. The markedly increased rebleeding time in the A/J may suggest differences in the regulation of the Plg network. + +No difference in blood coagulation between B6 and A/J mice + +To determine if differences in coagulation were contributing factors to the bleeding and rebleeding results, prothrombin time (PT) and activated partial thromboplastin time (aPTT) were measured. PT is a measurement of the extrinsic coagulation pathway (activation of Factor VII by tissue factor) while aPTT is a measurement of the intrinsic pathway (activation of Factors XII, XI, IX and VIII). The products of both pathways activate Factor X, which initiates the common pathway leading to thrombin generation and fibrin clot formation. PT and aPTT were measured in the B6 and A/J mice and values were not different between the two mouse strains (Table 2). These results suggest that the coagulation pathway is not different in the two inbred strains. + +Table 2 + +Coagulation and Plasminogen System Parameters in B6 and A/J inbred mouse strains + +Values are the mean ± SEM. *P < 0.05, **P < 0.01. n = 5–8. + +Plg and PAI-1 higher in A/J than B6 mice + +Plg concentration was 34% higher in the A/J mice than in B6 mice. In addition, fibrinolytic activity, PAI-1 activity, and α2-anitplasmin were also increased in the A/J mice when compared to the B6 mice (Table 2). The functional consequences of these differences are not clear, since fibrinogen is also higher in the A/J mice. To determine whether there were protein structure changes of Plg from the A/J mice, plasma from the B6 and the A/J mice were subjected to electrophoresis under reduced, non-reduced, and non-denaturing conditions. No difference in migration for immunostained Plg under these conditions was observed (data not shown). The density of the Western blot from SDS-PAGE under reducing conditions of plasma of varying volumes from B6 and A/J mice was compared and the density of the bands was 56% higher in the A/J than the B6 mice (six individual mice were tested on at least two occasions) from the same amount of plasma (Fig. 1B). Thus, the concentration of Plg in plasma from the A/J mice was higher than plasma from the B6 mice as determined by two methods and no difference in migration after electrophoresis was observed, suggesting no marked changes in the structure of Plg in the two mouse strains. + +Tail morphology in B6 and A/J + +The tails of the mice were examined for differences that might account for the variations in the bleeding and rebleeding values. Sections of the tail from the B6 and A/J mice were stained with Hematoxylin/Eosin or Masson's Trichrome and representative sections are shown in Figure 3. No obvious qualitative differences were observed in hair follicles, sebaceous glands, or center bone/cartilage in Hematoxylin/Eosin (Figure 3A) stained sections. Sections of the tails were stained with Masson's Trichrome (Figure 3B) that detects collagen, the major platelet adhesive substratum for initiation of thrombus formation. Collagen content (% tail area) was quantified (Figure 3C) from 3–4 sections from 2–3 areas/mouse. The amount of collagen was greater in the A/J than the B6 mice (P < 0.05). Collagen in vessels in adipose tissue in the A/J mice is also increased compared to the B6 mice (data not shown). + +Figure 3 + +Tail morphology and collagen. Tails were excised, embedded in OCT, sectioned, and stained with Hematoxylin/Eosin or Masson's Trichrome. Panel A: Representative sections stained with hematoxylin and eosin from B6 and A/J mice. Panel B: Representative sections stained with Masson's Trichrome from B6 and A/J mice. The blue color identifies the collagen. Panel C: The percentage of the collagen area (excluding center bone/cartilage) of the total tail area of each section was determined in 3–4 sections at 2–3 sites for each tail and averaged for each mouse. Values are the mean ± SEM of 4–8 mice per genotype. Symbol indicates a significant difference (*P < 0.05) between B6 mice determined by t-test. + +Tail bleeding assay identifies three chromosomes with QTL for rebleeding in CSS + +Using the tail bleeding/rebleeding assay as a surrogate marker for hemostasis and thrombosis, the CSS were screened. The bleeding times (Figure 4A) for the B6 and the A/J mice were not different, but 6six of the strains, B6-Chr5, 6, 8, 14, 15, and YA/J, had lower (P < 0.002) bleeding times (49–79 seconds) compared to the value for the B6 mice (121 sec). The rebleeding time (Figure 4B), determined as the time between bleeding cessation and initiation of the second bleeding, was markedly increased in the A/J mice compared to the B6 mice and two strains, B6-Chr11A/J (CSS-11) and B6-Chr17A/J (CSS-17), had significantly (P < 0.002) longer rebleeding times compared to the B6 mice that were similar to values from the A/J mice. Another strain, B6-Chr5A/J (CSS-5), approached significance at P < 0.008. The rebleeding time was stopped at 600 sec, and the percentage of mice with this value determined for the three strains: CSS-5–60%, CSS-11–60% and CSS-A17–46%. The percentage of B6 mice with a rebleeding time of 600 sec was 7% and for the A/J mice the percentage was 85%. + +Figure 4 + +Bleeding and rebleeding time in the CSS. Panel A: Bleeding Time (seconds). Panel B: Rebleeding Time (seconds). Bars are the mean ± SEM of 7–28 mice. Statistical differences between B6 mice and CSS were determined with a t-test, and a Bonferroni correction was made to determine significance value (P < 0.002). Symbols indicate a significant difference between B6 values. *P < 0.002. + +Rebleeding time in the parent strains and CSS were compared to values from the progeny of mice that were crossed with B6 mice to produce heterosomic mice for the CSS-5, (B6xCSS-5)F1 and CSS-17 (B6xCSS-17)F1 strains (Figure 5). The rebleeding values for CSS-5F1 and CSS-17F1 mice were similar to the B6 rather than the A/J mice, suggesting the A/J trait is recessive. Bleeding and rebleeding in the progeny of the cross, B6-Chr5A/J × B6-Chr17A/J (CSS-5 × CSS-17) resulted in the recovery of the A/J phenotype in the progeny of these double heterosomic strains. Thus, despite the heterosomic chromosomes of 5 and 17 in the progeny of the CSS-5 × CSS-17, the phenotype was now similar to A/J. This suggested that traits on the A/J chromosomes were interacting to produce the phenotype. + +Figure 5 + +Bleeding and rebleeding time in the CSS backcrosses. Panel A: Bleeding Time (seconds) and Panel B: Rebleeding Time (seconds) were tested in heterosomic mice, (B6xCSS-5)F1 (5F1) and (B6xCSS-17)F1 (17F1) and doubly heterosomic mice for chromosomes 5 and 17 (5 × 17). Bars are the mean ± SEM of 5–28 mice. Statistical differences between B6 mice and the CSS were determined with a t-test, and a Bonferroni correction was made to determine significance value (P < 0.01). Symbols indicate a significant difference between B6 values. *P < 0.01. + +Interactions of CSS for PAI-1 antigen and activity + +Components of the Plg system reside on chromosomes CSS-5 (PAI-1), CSS-11 (α2-antiplasmin), and CSS-17 (Plg). As reported in Table 2, the A/J mice had increased Plg antigen, fibrinolytic activity, α2-antiplasmin, PAI-1 activity and fibrinogen when compared to B6 mice. In CSS-17 mice, the Plg antigen was also significantly (P = 0.015) increased (152 ± 6, n = 12) when compared to the B6 mice, but for CSS-5 and CSS-5 × 17, the Plg antigen was similar to values for the B6 mice. Fibrinolytic activity was not different in the CSS-5 or CSS-17 strains compared to the B6 mice. α2-antiplasmin was significantly (P = < 0.01) reduced in CSS-5 mice (142 ± 4 μg/mL, n = 5), significantly (P < 0.05) higher in the CSS-11 (201 ± 6, n = 8), but not different in CSS-17 or CSS-5 × 17 mice compared to the B6 mice. + +PAI-1 antigen and activity were determined in the CSS with increased rebleeding times, CSS-5, CSS-11, and CSS-17 (Figure 6), the heterosomic, CSS-5F1, CSS-17F1, and double hetersomic, CSS-5 × 17. CSS-5, CSS-17, CSS-5F1, and CSS-17F1 generally had reduced PAI-1 antigen when compared to B6 mice. The reduced PAI-1 antigen was a dominant trait due to the fact that the values were similar in the homosomic and heterosomic CSS-5 and CSS-17. The CSS-5 × CSS-17 double heterosomic strains had values similar to the B6 and A/J parent strains. PAI-1 activity was significantly reduced in the CSS-5 and CSS-17F1 mice, but again in the CSS-5 × CSS-17 strain the value for PAI-1 activity was restored to the value of the B6 and A/J parent strains. The PAI-1 antigen (5.4 ng/mL ± 0.5, n = 7) and activity (170 U/mL ± 20, n = 6) in the CSS-11 mice was not different than for B6 mice. + +Figure 6 + +PAI-1 in the CSS backcrosses. Panel A: Plasma PAI-1 antigen (ng/mL) and Panel B: Plasma PAI-1 activity (U/mL) were tested in heterosomic mice, (B6xCSS-5)F1 (5F1) and (B6xCSS-17)F1 (17F1) and doubly heterosomic mice for chromosomes 5 and 17 (5 × 17). Bars are the mean ± SEM of 5–18 mice. Statistical differences between B6 mice and CSS were determined with a t-test, and a Bonferroni correction was made to determine significance value (P < 0.01). Symbols indicate a significant difference between B6 values. *P < 0.01. + +Interactions of CSS for arterial thrombus formation + +The tail bleeding assay was rapid and facilitated the screening of the 21 CSS strains, and subsequently the identified strains were tested for arterial occlusion after the FeCl3 injury (Figure 7). The CSS-5 and CSS-17 had occlusion times similar to the B6 mice, but the doubly heterosomic CSS-5 × CSS-17 progeny had occlusion times higher than either the B6 mice or the heterosomic CSS-5F1 and CSS-17F1 or the A/J strains. In double heterosomic CSS-5 × CSS-17 mice, only one or three mice tested had occluded by 30 min; the other two mice did not occlude by 30 min. While only a small number of CSS mice have been tested for the arterial occlusion, the results suggest that interaction of the genes on chromosome 5 and 17 also determines arterial occlusion time. + +Figure 7 + +Arterial occlusion time in the CSS backcrosses. Occlusion time was determined in heterosomic mice, (B6xCSS-5)F1 (5F1) and (B6xCSS-17)F1 (17F1), and doubly heterosomic mice for chromosomes 5 and 17 (5 × 17). Bars are the mean ± SEM of 5–18 mice. Statistical differences between B6 mice and CSS were determined with a t-test, and a Bonferroni correction was made to determine significance value (P < 0.02). Symbols indicate a significant difference between B6 values. *P < 0.0001. + +Discussion + +In this study, thrombotic risk was systematically assessed in two inbred strains of mice that have marked differences in susceptibility to diet-induced obesity, diet-induced atherosclerosis, and ligation-induced neointimal hyperplasia and vessel remodeling. Arterial occlusion time, tail bleeding and rebleeding time were evaluated as potential predictors of thrombotic response. Assessments of the two inbred strains were compared to values from gene targeted mice of the Plg network with altered fibrinolytic responses, as well as in leptin deficient mice with a reduced platelet function. Marked differences were found in the thrombotic response among the two inbred strains, B6 and A/J, and the observed differences were not correlated with change in coagulation or platelet function. Screening the CSS identified three chromosomes that harbored genes which contributed to the A/J phenotype of increased rebleeding time. Mice homosomic for these chromosomes or doubly heterosomic for two of the chromosomes, 5 and 17, were required to express the A/J phenotype, elevated rebleeding time. PAI-1 antigen and activity were decreased in both CSS-5 and CSS-17 and the heterosomic mice, CSS-5F1 and CSS-17F1, but not in the CSS-11 strain. Values were restored in the doubly heterosomic for two of chromosomes, 5 and 17. Arterial occlusion time was similar to B6 in the CSS-5 and CSS-17 homozygous strains, but increased in doubly heterosomic for two of chromosomes, 5 and 17. + +The FeCl3-induced model of vascular injury and thrombosis in mice is now widely used to evaluate genetic and pharmacological interventions [24]. The two inbred strains had marked differences in the time for occlusion of the carotid artery after FeCl3 injury. After FeCl3 treatment, thrombus formation and occlusion was remarkably shortened in the A/J mice compared to the B6 mice. These results have not previously been reported. A recent study [25] of sheer stress in rats, found that the magnitude of changes in sheer stress with increased blood flow varied with the different strains. Further investigation, beyond the scope of this study would be necessary to determine the contribution in the differences in size and sheer stress of the carotids to arterial occlusion rates in the B6 and A/J mice. In preliminary studies, we have noted differences in the composition of the thrombus in B6 and A/J mice. The pattern of blood flow cessation for the two inbred strains was different than for the Lepob mice [26] with impaired platelet function, and coagulation time was similar in the two strains. In the mice with deficiencies of the Plg network [27], thrombus formation time was reduced in the Plg-/- mice, but increased in the PAI-/- mice, suggesting that alterations in plasmin activity that affect the rate of clot lysis, can modulate the events leading to occlusive thrombus formation. + +The tail-bleeding assay has been used extensively in mice to assess the impact of deficiencies and over expression of platelet and coagulation proteins in mice. Sweeny et al [28] screened 25 strains of mice and found only the RIIS/J mice to have increased bleeding time due to reduced von Willebrand antigen. Broze et al [29] evaluated mice with gene deletions of the coagulation pathway and found that while bleeding time was not increased, rebleeding persisted despite electrocautery of the tail in FVIII and FIX deficient mice. This observation raised the possibility that rebleeding time may be a sensitive reporter of genetic influences on thrombus formation and stability. Mice that are deficient in factors required for normal platelet function, platelet glycoprotein Ib [30], and protease-activated receptor-4, [31] have increased tail bleeding time. The leptin deficiency with reduced platelet aggregation clearly shows a marked increase in bleeding time and the increased bleeding time in Plg-/- and uPA-/- mice suggests that Plg and uPA may exert an influence on platelet response to promote normal thrombus formation. Overall, the marked increase in rebleeding times observed in certain mouse strains suggests genetic factors other than coagulation may play a role in the stability of a thrombus. + +In the tail bleeding assay, A/J mice did not have changes in bleeding time, but rebleeding time was higher compared to B6 mice. In our study, Plg-/- mice had increased bleeding time, PAI-1-/- had decreased rebleeding time, and uPA-/- mice had an increased bleeding time similar to the Plg-/- mice (J. Hoover-Plow, A. Shchurin, and E. Hart, unpublished results). Increased bleeding or rebleeding times have not been reported in any of the Plg network targeted mice. Matsuno et al [32] reported no difference in bleeding time for tPA-/-, uPA-/-, PAI-1-/- mice compared to WT mice, and the assay was similar, but the tail clip segment was considerably shorter and bleeding times reduced compared to the times reported in our study. Bleeding time has not been previously reported for Plg-/- mice. We have found similar values for Plg-/- mice in a 50%B6:50%129 background (J. Hoover-Plow, A. Shchurin, and E. Hart, unpublished results). The results of this study suggest that not only is bleeding time genetically determined by background, but also that tail rebleeding time is genetically determined. + +While blood pressure could conceivably be higher in mice with elevated tail bleeding time, several studies suggest that this is not the case. Studies of A/J [33-36] mice have reported either decreased blood pressure or no difference compared to the B6 mice. Blood pressure was measured in the Plg-/- mice (D. Hellard, J. Hoover-Plow, and E. Plow, unpublished results), and these mice have reduced blood pressure when compared to WT littermates, and uPA-/- [37] and Lepob [38,39] mice also have reduced blood pressure, but there is no correspondence between blood pressure and tail bleeding or rebleeding times. Morphometric analysis of the tail indicated a difference in the collagen content of the tail in the A/J mice. Differences in the structure and/or metabolism of collagen or extracellular matrix proteins may contribute to the increased rebleeding and reduced occlusion time in the A/J mice and warrant further investigation. + +The genes for Plg, PAI-1, and α2-antiplasmin reside on the three chromosomes identified for QTL for rebleeding time and may or may not be coincidental to the observed phenotypes found in the A/J mice and CSS. It is not likely that merely the concentration of these components explains the increased rebleeding time and reduced arterial occlusion time in the A/J mice. When compared to the B6 mice, the A/J mice and the three strains with elevated rebleeding time had variable Plg, PAI-1, and α2-antiplasmin. Two allelic forms of the mouse Plg gene have been reported for B6 and A/J mice [40]. Plg concentration was increased in A/J mice compared to B6 mice. Although we did not detect differences in Plg structure, this would not exclude possible functional differences. A congenic strain (specific for a homosomic region from A/J and the remaining Chr and background is from B6 mice) of A/J chromosome 17 that includes the Plg gene was tested in the bleeding assay. Values for bleeding and rebleeding in this congenic strain were similar to values from B6 mice, indicating that the QTL is not Plg. There are genes for other proteases and matrix proteins that reside on chromosome 17 in addition to unknown genes. DNA base pair sequence data for the PAI-1 gene or the α2-antiplasmin gene in B6 and A/J mice have reported no differences [51], but regulatory genes upstream of the coding region may be important. Our results suggest that novel or unknown genes may interact with the Plg system components to modify their function. + +In summary, this study reports marked differences in two inbred strains of mice, B6 and A/J, in arterial thrombosis formation in a FeCl3 vascular injury model, rebleeding time in a tail bleeding assay, plasminogen function, and tissue and vessel collagen deposition. Three chromosomes from A/J mice were identified that had QTL for rebleeding time. The genes may interact with components in the Plg network and modulate arterial occlusion time. + +Conclusion + +The marked independent differences demonstrated in the B6 and A/J mice can be exploited to identify genetic determinants of thrombosis and haemostasis. Our results of the CSS screening suggest that novel or unknown genes can be used to identify the genes responsible for the traits related to arterial thrombosis, tail bleeding/rebleeding and vessel extracellular matrix. Identification of differences in the parent strains, such as those described in this study, and screening of the CSS is a first-step in the discovery of new genetic determinants of thrombotic risk. + +Abbreviations + +B6 = C57BL/6J + +CSS = chromosome substitution strains + +CSS-# = B6-Chr #A/J + +CSS-#F1 = (B6 × CSS-#)F1 + +WT = wild-type + +-/- = deficient + +Plg = plasminogen + +PAI-1 = plasminogen activator inhibitor-1 + +tPA = tissue plasminogen activator + +uPA = urokinase plasminogen activator + +SEM = standard error of the mean + +QTL = quantitative trait locus + +Competing interests + +The author(s) declare that they have no competing interests. + +Authors' contributions + +JHP conceived and designed the study, coordinated the experiments and drafted the manuscript. AS developed and performed the tail bleeding assay and participated in drafting the manuscript. EH carried out the vascular injury model and analyses and the tail bleeding assay, participated in the tail collagen analyses, and coordination of the mouse breeding. JS participated in the analysis of the vascular injury model results and collagen analyses. JHN and AHE provided the CSS breeding pairs, JHN participated in discussions of the results, and JBS and JHN developed the CSS. All the authors have read and approved the final manuscript. + +Pre-publication history + +The pre-publication history for this paper can be accessed here: + + + +Acknowledgements + +The authors thank Dr. A. Vasanji, Shiyang Wang, and Jenna Saraniti for assistance with image analyses, David Hellard for performing the blood pressure measurements, Dr. Y.Shchurina for the plasminogen analyses, Drs. Lindsey Burrage and Jonathan Smith for helpful discussions, and Robin Lewis for assistance with manuscript preparation. This study was supported by grants from NIH, HL17964, HL65204, HL078701 (JHP), T32 HL07914 (AS), and RR12305 (JHN). diff --git a/src/ontogpt/evaluation/craft/database/all/17029558.ann b/src/ontogpt/evaluation/craft/database/all/17029558.ann new file mode 100644 index 000000000..6a1f6cef6 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17029558.ann @@ -0,0 +1,994 @@ +T1 PR:000035849 16 20 Evi1 +T2 NCBITaxon:10088 36 40 Mice +T3 http://purl.obolibrary.org/obo/MONDO_0005441 66 78 Otitis Media +T4 http://purl.obolibrary.org/obo/MONDO_0005441 90 102 Otitis media +T5 http://purl.obolibrary.org/obo/MONDO_0005441 104 106 OM +T6 http://purl.obolibrary.org/obo/MONDO_0005441 109 124;129 139 inflammation of middle ear +T7 UBERON:0001756 129 139 middle ear +T8 GO:0007605 174 181 hearing +T9 http://purl.obolibrary.org/obo/MONDO_0005365 174 192 hearing impairment +T10 NCBITaxon:9606 324 329 human +T11 NCBITaxon:10088 345 350 mouse +T12 SO:0000704 386 393 genetic +T13 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11344 11350 bullae +T250 http://purl.obolibrary.org/obo/MONDO_0005441 11373 11375 OM +T251 NCBITaxon:10088 11385 11389 mice +T252 NCBITaxon:10088 11418 11422 mice +T253 UBERON:0008959 11427 11433 bullae +T254 NCBITaxon:10088 11467 11471 mice +T255 UBERON:0008959 11472 11478 bullae +T256 UBERON:0001756 11576 11586 middle ear +T257 NCBITaxon:10088 11609 11613 mice +T258 UBERON:0008959 11615 11621 bullae +T259 http://purl.obolibrary.org/obo/MONDO_0005441 11766 11768 OM +T260 NCBITaxon:10088 11795 11799 mice +T261 UBERON:0008959 11800 11806 bullae +T262 NCBITaxon:10088 11911 11915 mice +T263 UBERON:0001690 11953 11957 ears +T264 NCBITaxon:10088 11973 11977 mice +T265 UBERON:0008959 11984 11990 bullae +T266 UBERON:0008959 12043 12049 bullae +T267 UBERON:0008959 12109 12115 bullae +T268 http://purl.obolibrary.org/obo/MONDO_0005441 12242 12244 OM +T269 GO:0007567 12256 12261 natal +T270 UBERON:0007023 12293 12298 adult +T271 NCBITaxon:10088 12331 12335 mice +T272 NCBITaxon:10088 12418 12422 mice +T273 NCBITaxon:10088 12450 12454 mice +T274 UBERON:0000922 12499 12508 embryonic +T275 UBERON:0002384 12509 12526 connective tissue +T276 UBERON:0004114 12549 12552 MEC +T277 UBERON:0004114 12612 12615 MEC +T278 NCBITaxon:10088 12659 12663 mice +T279 NCBITaxon:10088 12683 12687 mice +T280 http://purl.obolibrary.org/obo/MONDO_0005441 12698 12700 OM +T281 NCBITaxon:10088 12732 12736 mice +T282 UBERON:0007780 12753 12760 exudate +T283 UBERON:0004114 12773 12777 MECs +T284 NCBITaxon:10088 12795 12799 mice +T285 http://purl.obolibrary.org/obo/MONDO_0005441 12815 12817 OM +T286 UBERON:0007794 12822 12828 serous +T287 UBERON:0001690 12846 12849 ear +T288 UBERON:0001690 12897 12900 ear +T289 UBERON:0004114 12944 12947 MEC +T290 http://purl.obolibrary.org/obo/MONDO_0005079 12982 12988 polyps +T291 UBERON:0000922 13032 13041 embryonic +T292 UBERON:0002384 13042 13059 connective tissue +T293 UBERON:0000483 13085 13095 epithelial +T294 UBERON:0008959 13096 13102 bullae +T295 UBERON:0007794 13115 13127 serous fluid +T296 UBERON:0003891 13129 13136 stromal +T297 UBERON:0001982 13158 13169 capillaries +T298 UBERON:0001473 13175 13185 lymphatics +T299 CL:0000775 13225 13246 neutrophil leukocytes +T300 UBERON:0000483 13252 13262 epithelial +T301 CL:0000646 13292 13303 basal cells +T302 GO:0005929 13307 13315 ciliated +T303 CL:0000064 13307 13315;13325 13330 ciliated ... cells +T304 http://purl.obolibrary.org/obo/MONDO_0005441 13376 13378 OM +T305 NCBITaxon:10088 13419 13423 mice +T306 CL:0000775 13480 13501 neutrophil leucocytes +T307 CL:0000775 13526 13546 neutrophil leukocyte +T308 UBERON:0004114 13547 13550 MEC +T309 NCBITaxon:10088 13596 13600 mice +T310 http://purl.obolibrary.org/obo/MONDO_0005441 13620 13622 OM +T311 http://purl.obolibrary.org/obo/MONDO_0005441 13649 13651 OM +T312 UBERON:0000065 13705 13722 respiratory tract +T313 http://purl.obolibrary.org/obo/MONDO_0003014 13759 13767 rhinitis +T314 http://purl.obolibrary.org/obo/MONDO_0001040 13784 13799 nasopharyngitis +T315 http://purl.obolibrary.org/obo/MONDO_0004553 13821 13831 alveolitis +T316 UBERON:0005169 13832 13844 interstitial +T317 http://purl.obolibrary.org/obo/MONDO_0005249 13845 13854 pneumonia +T318 CL:0000771 13860 13881 eosinophil leukocytes +T319 NCBITaxon:2 13961 13969 bacteria +T320 NCBITaxon:10239 13973 13978 viral +T321 UBERON:0000160 14041 14050 intestine +T322 UBERON:0002107 14052 14057 liver +T323 UBERON:0001264 14059 14067 pancreas +T324 UBERON:0002113 14069 14075 kidney +T325 UBERON:0000948 14077 14082 heart +T326 UBERON:0002370 14084 14090 thymus +T327 UBERON:0002106 14096 14102 spleen +T328 UBERON:0001756 14128 14138 Middle Ear +T329 UBERON:0000004 14143 14147 Nose +T330 NCBITaxon:10088 14178 14182 Mice +T331 GO:0007567 14219 14224 natal +T332 NCBITaxon:10088 14225 14229 mice +T333 UBERON:0004114 14306 14309 MEC +T334 UBERON:0007780 14329 14336 exudate +T335 UBERON:0001756 14357 14367 middle ear +T336 UBERON:0001678 14368 14381 temporal bone +T337 UBERON:0001756 14451 14461 middle ear +T338 CL:0000775 14497 14517 neutrophil leukocyte +T339 CL:0000775 14534 14544 neutrophil +T340 UBERON:0007780 14550 14558 exudates +T341 UBERON:0004114 14566 14569 MEC +T342 UBERON:0001756 14586 14596 middle ear +T343 UBERON:0004114 14645 14648 MEC +T344 http://purl.obolibrary.org/obo/MONDO_0005079 14684 14689 polyp +T345 UBERON:0000922 14717 14726 embryonic +T346 UBERON:0001756 14727 14737 middle ear +T347 UBERON:0002384 14738 14755 connective tissue +T348 UBERON:0002364 14757 14774 tympanic membrane +T349 UBERON:0004114 14791 14794 MEC +T350 GO:0005929 14804 14812 ciliated +T351 CL:0000064 14804 14812;14822 14827 ciliated ... cells +T352 UBERON:0004114 14855 14858 MEC +T353 CL:0000646 14868 14879 basal cells +T354 http://purl.obolibrary.org/obo/MONDO_0003014 14920 14928 rhinitis +T355 UBERON:0001707 14930 14942 nasal cavity +T356 UBERON:0007780 14960 14967 exudate +T357 UBERON:0001706 14969 14981 nasal septum +T358 UBERON:0000004 14996 15001 nasal +T359 UBERON:0000344 15002 15008 mucosa +T360 UBERON:0007023 15036 15041 adult +T361 UBERON:0001756 15056 15066 middle ear +T362 http://purl.obolibrary.org/obo/MONDO_0001920 15072 15094 chronic suppurative OM +T363 http://purl.obolibrary.org/obo/MONDO_0005079 15124 15130 polyps +T364 GO:0005929 15169 15177 ciliated +T365 CL:0000067 15169 15194 ciliated epithelial cells +T366 UBERON:0000483 15178 15188 epithelial +T367 UBERON:0003891 15238 15244 stroma +T368 UBERON:0002364 15285 15302 tympanic membrane +T369 UBERON:0001352 15304 15319 outer ear canal +T370 UBERON:0002364 15354 15371 tympanic membrane +T371 UBERON:0001352 15373 15388 outer ear canal +T372 UBERON:0007780 15395 15396 E +T373 UBERON:0007780 15398 15405 exudate +T374 UBERON:0001707 15427 15429 NC +T375 UBERON:0001707 15431 15443 nasal cavity +T376 UBERON:0000004 15449 15454 nasal +T377 UBERON:0000344 15455 15461 mucosa +T378 UBERON:0001706 15463 15465 NS +T379 UBERON:0001706 15467 15479 nasal septum +T380 UBERON:0001352 15481 15484 OEC +T381 UBERON:0001352 15486 15501 outer ear canal +T382 UBERON:0001678 15503 15505 TB +T383 UBERON:0001678 15507 15520 temporal bone +T384 UBERON:0002364 15522 15524 TM +T385 UBERON:0002364 15526 15543 tympanic membrane +T386 UBERON:0002048 15572 15576 Lung +T387 UBERON:0001756 15581 15591 Middle Ear +T388 UBERON:0007780 15592 15599 Exudate +T389 NCBITaxon:10088 15609 15613 Mice +T390 GO:0007567 15628 15633 natal +T391 UBERON:0002048 15640 15644 lung +T392 UBERON:0002186 15671 15682 bronchiolar +T393 CL:0000771 15720 15741 eosinophil leukocytes +T394 UBERON:0002186 15755 15765 bronchiole +T395 UBERON:0002012 15767 15783 pulmonary artery +T396 CL:0000771 15800 15812 eosiniphilic +T397 http://purl.obolibrary.org/obo/MONDO_0004553 15813 15823 alveolitis +T398 UBERON:0004893 15850 15865 alveolar septae +T399 CL:0000771 15888 15898 eosinophil +T400 GO:0007567 15930 15935 natal +T401 UBERON:0004114 15942 15945 MEC +T402 UBERON:0002186 15993 15994 B +T403 UBERON:0002186 15996 16006 bronchiole +T404 UBERON:0002012 16008 16010 PA +T405 UBERON:0002012 16012 16028 pulmonary artery +T406 GO:0007567 16038 16043 natal +T407 http://purl.obolibrary.org/obo/MONDO_0005441 16044 16046 OM +T408 NCBITaxon:10088 16073 16077 mice +T409 http://purl.obolibrary.org/obo/MONDO_0005441 16118 16120 OM +T410 UBERON:0007023 16140 16146 adults +T411 http://purl.obolibrary.org/obo/MONDO_0005441 16176 16178 OM +T412 NCBITaxon:10088 16208 16212 mice +T413 UBERON:0007023 16247 16252 adult +T414 NCBITaxon:10088 16259 16263 mice +T415 http://purl.obolibrary.org/obo/MONDO_0003014 16296 16304 rhinitis +T416 NCBITaxon:10088 16337 16341 mice +T417 CL:0000771 16367 16379 eosinophilic +T418 http://purl.obolibrary.org/obo/MONDO_0005749 16367 16389 eosinophilic pneumonia +T419 http://purl.obolibrary.org/obo/MONDO_0005441 16466 16468 OM +T420 UBERON:0007780 16469 16476 exudate +T421 UBERON:0001728 16528 16542 nasopharyngeal +T422 http://purl.obolibrary.org/obo/MONDO_0005441 16616 16618 OM +T423 NCBITaxon:10088 16651 16655 mice +T424 GO:0008283 16694 16710;16718 16723 proliferation of ... cells +T425 UBERON:0000912 16711 16717 mucous +T426 CL:0000319 16711 16723 mucous cells +T427 CHEBI:29149 16727 16740 periodic acid +T428 UBERON:0000912 16757 16762 mucus +T429 UBERON:0004114 16770 16773 MEC +T430 UBERON:0007023 16776 16781 Adult +T431 NCBITaxon:10088 16788 16792 mice +T432 http://purl.obolibrary.org/obo/MONDO_0001920 16819 16841 chronic suppurative OM +T433 UBERON:0001756 16851 16861 middle ear +T434 CL:0000775 16938 16959 neutrophil leukocytes +T435 CL:0000235 16970 16981 macrophages +T436 NCBITaxon:2 16983 16991 bacteria +T437 CL:0000235 17064 17075 macrophages +T438 CHEBI:16113 17077 17088 cholesterol +T439 UBERON:0002364 17185 17192 eardrum +T440 CHEBI:29149 17248 17261 periodic acid +T441 UBERON:0000912 17278 17283 mucus +T442 http://purl.obolibrary.org/obo/MONDO_0005079 17368 17374 polyps +T443 GO:0005929 17433 17441 ciliated +T444 CL:0000064 17433 17441;17451 17456 ciliated ... cells +T445 UBERON:0000912 17471 17477 mucous +T446 CL:0000319 17471 17483 mucous cells +T447 UBERON:0003891 17516 17522 stroma +T448 CL:0000775 17557 17577 neutrophil leukocyte +T449 GO:0072672 17557 17590 neutrophil leukocyte infiltration +T450 CL:0000097 17602 17612 mast cells +T451 UBERON:0002393 17682 17697 eustachian tube +T452 UBERON:0007780 17724 17732 exudates +T453 UBERON:0000483 17742 17752 epithelial +T454 UBERON:0000912 17789 17795 mucous +T455 CL:0000319 17789 17801 mucous cells +T456 UBERON:0000483 17812 17822 epithelial +T457 CL:0000738 17823 17833 leukocytes +T458 UBERON:0002364 17861 17868 eardrum +T459 http://purl.obolibrary.org/obo/MONDO_0001443 17870 17886 tympanosclerosis +T460 http://purl.obolibrary.org/obo/MONDO_0005441 17918 17920 OM +T461 UBERON:0002364 17966 17973 eardrum +T462 http://purl.obolibrary.org/obo/MONDO_0005441 18062 18064 OM +T463 NCBITaxon:10088 18098 18102 mice +T464 UBERON:0000922 18169 18175 embryo +T465 NCBITaxon:10088 18253 18257 Mice +T466 UBERON:0001004 18428 18439 respiratory +T467 CHEBI:16134 18458 18465 ammonia +T468 http://purl.obolibrary.org/obo/MONDO_0005441 18496 18498 OM +T469 NCBITaxon:10088 18508 18512 mice +T470 http://purl.obolibrary.org/obo/MONDO_0003014 18582 18590 rhinitis +T471 CL:0009002 18639 18657 inflammatory cells +T472 UBERON:0004114 18701 18704 MEC +T473 NCBITaxon:10088 18749 18753 mice +T474 http://purl.obolibrary.org/obo/MONDO_0005441 18768 18770 OM +T475 http://purl.obolibrary.org/obo/MONDO_0005441 18787 18789 OM +T476 http://purl.obolibrary.org/obo/MONDO_0005441 18819 18821 OM +T477 NCBITaxon:10088 18849 18853 mice +T478 http://purl.obolibrary.org/obo/MONDO_0005441 18865 18867 OM +T479 http://purl.obolibrary.org/obo/MONDO_0005441 18910 18912 OM +T480 NCBITaxon:10088 18952 18956 mice +T481 NCBITaxon:10088 18999 19004 mouse +T482 NCBITaxon:10088 19025 19029 mice +T483 UBERON:0000062 19119 19124 organ +T484 UBERON:0001756 19150 19160 middle ear +T485 UBERON:0007023 19164 19169 adult +T486 NCBITaxon:10088 19176 19180 mice +T487 http://purl.obolibrary.org/obo/MONDO_0005550 19233 19243 infections +T488 UBERON:0002048 19267 19271 lung +T489 UBERON:0004122 19273 19283;19305 19312 urogenital ... systems +T490 UBERON:0005409 19288 19312 gastrointestinal systems +T491 UBERON:0002405 19337 19343 immune +T492 NCBITaxon:10088 19415 19419 mice +T493 http://purl.obolibrary.org/obo/MONDO_0005441 19455 19457 OM +T494 NCBITaxon:10088 19516 19520 mice +T495 http://purl.obolibrary.org/obo/MONDO_0005441 19533 19535 OM +T496 GO:0007567 19547 19552 natal +T497 UBERON:0001557 19571 19594 upper respiratory tract +T498 http://purl.obolibrary.org/obo/MONDO_0004867 19571 19602 upper respiratory tract disease +T499 NCBITaxon:10088 19640 19644 mice +T500 NCBITaxon:10088 19696 19700 mice +T501 http://purl.obolibrary.org/obo/MONDO_0005441 19702 19704 OM +T502 NCBITaxon:10088 19794 19798 Mice +T503 GO:0042571 19845 19853 antibody +T504 NCBITaxon:10088 19895 19899 mice +T505 CL:0000084 19921 19922 T +T506 NCBITaxon:54986 19952 19966 keyhole limpet +T507 CL:0000084 19983 19984 T +T508 NCBITaxon:1313 20015 20027 pneumococcal +T509 CHEBI:18154 20028 20042 polysaccharide +T510 CHEBI:59132 20051 20059 antigens +T511 UBERON:0000178 20089 20094 blood +T512 CL:0000775 20095 20106 neutrophils +T513 GO:0009986 20125 20137 cell surface +T514 GO:0034688 20155 20160 Mac-1 +T515 GO:0034688 20155 20160 Mac-1 +T516 UBERON:0000178 20269 20274 blood +T517 CL:0000775 20275 20286 neutrophils +T518 NCBITaxon:10088 20310 20314 mice +T519 CL:0000775 20445 20455 neutrophil +T520 NCBITaxon:10088 20480 20484 mice +T521 NCBITaxon:10088 20513 20517 mice +T522 CL:0000094 20574 20586 Granulocytes +T523 UBERON:0000178 20590 20595 Blood +T524 NCBITaxon:10088 20619 20623 Mice +T525 GO:0010467 20625 20635 Expression +T526 PR:000035849 20639 20643 Evi1 +T527 NCBITaxon:10088 20667 20671 Mice +T528 GO:0010467 20705 20715 expression +T529 PR:000035849 20719 20723 Evi1 +T530 UBERON:0005291 20732 20741;20753 20760 embryonic ... tissues +T531 UBERON:0000468 20742 20752 whole-body +T532 GO:0007567 20801 20806 birth +T533 GO:0007567 20816 20821 natal +T534 UBERON:0001756 20822 20832 middle ear +T535 UBERON:0000479 20833 20839 tissue +T536 GO:0010467 20936 20946 expression +T537 NCBITaxon:10088 20970 20974 mice +T538 GO:0010467 21044 21054 expression +T539 GO:0007567 21085 21090 birth +T540 GO:0007567 21138 21143 natal +T541 UBERON:0000033 21144 21148 head +T542 UBERON:0000479 21149 21156 tissues +T543 PR:000035849 21175 21179 Evi1 +T544 GO:0010467 21183 21192 expressed +T545 GO:0005634 21196 21205;21214 21219 nuclei of ... cells +T546 CL:0000763 21206 21219 myeloid cells +T547 UBERON:0002371 21223 21234 bone marrow +T548 CL:0000775 21236 21257 neutrophil leukocytes +T549 CL:0000057 21259 21270 fibroblasts +T550 CL:0000646 21276 21298 basal epithelial cells +T551 UBERON:0000483 21282 21292 epithelial +T552 UBERON:0001756 21315 21325 middle ear +T553 GO:0010467 21366 21376 expression +T554 NCBITaxon:10088 21412 21416 mice +T555 PR:000035849 21429 21433 Evi1 +T556 NCBITaxon:10088 21489 21493 Mice +T557 http://purl.obolibrary.org/obo/MONDO_0005441 21505 21507 OM +T558 CL:0000763 21528 21541 myeloid cells +T559 UBERON:0002371 21554 21565 bone marrow +T560 CL:0000138 21577 21589 chondrocytes +T561 CL:0000137 21594 21604 osteocytes +T562 CL:0000775 21671 21692 neutrophil leukocytes +T563 CL:0000057 21694 21705 fibroblasts +T564 CL:0000646 21711 21732 basal epithelial cell +T565 UBERON:0000483 21717 21727 epithelial +T566 GO:0005634 21728 21739 cell nuclei +T567 UBERON:0002371 21785 21796 bone marrow +T568 CL:0000646 21844 21845 B +T569 CL:0000646 21847 21868 basal epithelial cell +T570 UBERON:0000483 21853 21863 epithelial +T571 GO:0005634 21864 21875 cell nuclei +T572 CL:0000138 21877 21878 C +T573 CL:0000138 21880 21892 chrondrocyte +T574 CL:0000057 21894 21895 F +T575 CL:0000057 21897 21907 fibroblast +T576 CL:0000057 21909 21911 MC +T577 CL:0000763 21913 21925 myeloid cell +T578 CL:0000775 21927 21928 N +T579 CL:0000775 21930 21950 neutrophil leukocyte +T580 CL:0000137 21952 21953 O +T581 CL:0000137 21955 21964 osteocyte +T582 http://purl.obolibrary.org/obo/MONDO_0005441 21966 21968 OM +T583 PR:000035849 21986 21990 Evi1 +T584 NCBITaxon:10088 22000 22004 Mice +T585 http://purl.obolibrary.org/obo/MONDO_0005441 22006 22008 OM +T586 PR:000035849 22039 22043 Evi1 +T587 NCBITaxon:10088 22053 22057 mice +T588 PR:000035849 22074 22078 Evi1 +T589 NCBITaxon:10088 22088 22092 mice +T590 http://purl.obolibrary.org/obo/MONDO_0005441 22105 22107 OM +T591 http://purl.obolibrary.org/obo/MONDO_0005441 22152 22154 OM +T592 http://purl.obolibrary.org/obo/MONDO_0005441 22215 22217 OM +T593 PR:000035849 22221 22225 Evi1 +T594 NCBITaxon:10088 22235 22239 mice +T595 UBERON:0002544 22276 22282 digits +T596 NCBITaxon:10088 22304 22308 mice +T597 SO:0000704 22391 22398 genetic +T598 PR:000035849 22413 22417 Evi1 +T599 NCBITaxon:10088 22427 22431 mice +T600 NCBITaxon:10088 22463 22467 mice +T601 PR:000035849 22514 22518 Evi1 +T602 SO:0001060 22541 22548 isoform +T603 PR:000035849 22711 22715 Evi1 +T604 http://purl.obolibrary.org/obo/MONDO_0001920 22768 22790 chronic suppurative OM +T605 SO:0000704 22812 22819 Genetic +T606 NCBITaxon:10088 22928 22932 mice +T607 http://purl.obolibrary.org/obo/MONDO_0005441 22977 22979 OM +T608 UBERON:0001728 23040 23054 nasopharyngeal +T609 NCBITaxon:1279 23055 23069 Staphylococcus +T610 NCBITaxon:1301 23074 23087 Streptococcus +T611 NCBITaxon:species 23088 23092 spp. +T612 http://purl.obolibrary.org/obo/MONDO_0005441 23107 23109 OM +T613 UBERON:0000467 23145 23153 systemic +T614 http://purl.obolibrary.org/obo/MONDO_0005550 23185 23195 infections +T615 CL:0000084 23227 23228 T +T616 CL:0000084 23243 23244 T +T617 UBERON:0002405 23257 23263 immune +T618 GO:0006955 23257 23273 immune responses +T619 NCBITaxon:10088 23283 23287 mice +T620 UBERON:0002405 23312 23318 immune +T621 http://purl.obolibrary.org/obo/MONDO_0005441 23352 23354 OM +T622 PR:000035849 23361 23365 Evi1 +T623 SO:0000366 23409 23416;23428 23439 site of ... integration +T624 NCBITaxon:11632 23417 23427 retroviral +T625 CL:0000763 23469 23476 myeloid +T626 http://purl.obolibrary.org/obo/MONDO_0005170 23469 23483 myeloid tumors +T627 NCBITaxon:10088 23496 23501 mouse +T628 PR:000035849 23571 23575 Evi1 +T629 CL:0000763 23624 23631 myeloid +T630 http://purl.obolibrary.org/obo/MONDO_0004643 23624 23641 myeloid leukemias +T631 http://purl.obolibrary.org/obo/MONDO_0018881 23646 23670 myelodysplastic syndrome +T632 NCBITaxon:9606 23674 23680 humans +T633 GO:0005634 23716 23723 nuclear +T634 SO:0000417 23775 23782 domains +T635 SO:0000417 23867 23873 domain +T636 SO:0000993 23913 23922 consensus +T637 CHEBI:37527 23977 23983 acidic +T638 SO:0000417 23984 23991 domains +T639 SO:0000167 24091 24099 promoter +T640 PR:000035849 24134 24138 Evi1 +T641 PR:000000046 24191 24196 TGF-β +T642 GO:0007179 24191 24214 TGF-β signaling pathway +T643 PR:000000365 24241 24246 Smad3 +T644 SO:0000417 24281 24287 domain +T645 GO:0060284 24325 24352 control of cell development +T646 GO:0042127 24325 24340;24357 24370 control of cell ... proliferation +T647 PR:000035849 24436 24440 Evi1 +T648 GO:0065007 24464 24471 control +T649 PR:000007597 24475 24480 c-fos +T650 PR:000009232 24489 24493 AP-1 +T651 SO:0000417 24563 24569 domain +T652 CHEBI:33708 24948 24955 residue +T653 SO:0001117 25012 25023 alpha helix +T654 SO:0000417 25149 25155 domain +T655 SO:0000417 25241 25247 domain +T656 PR:000000046 25268 25273 TGF-β +T657 GO:0007179 25268 25283 TGF-β signaling +T658 http://purl.obolibrary.org/obo/MONDO_0005441 25317 25319 OM +T659 PR:000035849 25352 25356 Evi1 +T660 NCBITaxon:10088 25366 25370 mice +T661 PR:000035849 25385 25389 Evi1 +T662 SO:0001023 25397 25403 allele +T663 SO:0001060 25418 25425 isoform +T664 SO:0001023 25435 25441 allele +T665 PR:000035849 25458 25462 Evi1 +T666 SO:0000673 25463 25473 transcript +T667 SO:0001060 25497 25504 isoform +T668 PR:000035849 25537 25541 Evi1 +T669 CL:0002322 25577 25579;25583 25588 ES ... cells +T670 GO:0007618 25617 25622 mated +T671 NCBITaxon:10088 25631 25635 mice +T672 SO:0001060 25675 25682 isoform +T673 SO:0000704 25702 25709 genetic +T674 PR:000035849 25730 25734 Evi1 +T675 NCBITaxon:10088 25744 25748 mice +T676 NCBITaxon:10088 25759 25763 mice +T677 http://purl.obolibrary.org/obo/MONDO_0005441 25825 25827 OM +T678 PR:000035849 26016 26020 Evi1 +T679 http://purl.obolibrary.org/obo/MONDO_0005441 26059 26061 OM +T680 GO:0010467 26107 26116 expressed +T681 UBERON:0001756 26160 26170 middle ear +T682 PR:000035849 26219 26223 Evi1 +T683 GO:0010467 26227 26236 expressed +T684 CL:0000775 26240 26261 neutrophil leukocytes +T685 http://purl.obolibrary.org/obo/MONDO_0005441 26269 26271 OM +T686 PR:000035849 26285 26289 Evi1 +T687 GO:0030097 26325 26338 hematopoietic +T688 CL:0000763 26374 26381 myeloid +T689 http://purl.obolibrary.org/obo/MONDO_0004643 26374 26390 myeloid leukemia +T690 SO:0000704 26420 26424 gene +T691 PR:000035849 26429 26433 Evi1 +T692 CL:0000775 26437 26458 neutrophil leukocytes +T693 CHEBI:25450 26466 26487 inositol triphosphate +T694 PR:000009159 26466 26503 inositol triphosphate type 2 receptor +T695 SO:0000704 26504 26508 gene +T696 PR:000009159 26510 26515 Itpr2 +T697 GO:0065007 26549 26559 regulation +T698 CL:0000775 26563 26583 neutrophil leukocyte +T699 CHEBI:53490 26599 26612 F-met-leu-phe +T700 NCBITaxon:2 26647 26656 bacterial +T701 CL:0000775 26705 26716 neutrophils +T702 http://purl.obolibrary.org/obo/MONDO_0005441 26742 26744 OM +T703 http://purl.obolibrary.org/obo/MONDO_0001920 26768 26790 chronic suppurative OM +T704 UBERON:0001756 26857 26867 middle ear +T705 NCBITaxon:10088 26877 26881 mice +T706 CL:0000775 26927 26938 Neutrophils +T707 GO:0030097 26968 26981 hematopoietic +T708 UBERON:0012429 26968 26989 hematopoietic tissues +T709 NCBITaxon:10088 27034 27038 mice +T710 CL:0000775 27076 27086 neutrophil +T711 NCBITaxon:10088 27108 27112 mice +T712 CL:0000776 27127 27147 immature neutrophils +T713 NCBITaxon:10088 27228 27232 mice +T714 http://purl.obolibrary.org/obo/MONDO_0005441 27254 27256 OM +T715 NCBITaxon:10088 27273 27277 mice +T716 http://purl.obolibrary.org/obo/MONDO_0001920 27299 27321 chronic suppurative OM +T717 CL:0000776 27323 27331;27343 27353 immature ... neutrophil +T718 CL:0000096 27336 27353 mature neutrophil +T719 NCBITaxon:10088 27408 27412 mice +T720 PR:000000046 27478 27482 TGFβ +T721 GO:0010467 27505 27515 expression +T722 http://purl.obolibrary.org/obo/MONDO_0005441 27590 27592 OM +T723 PR:000035849 27609 27613 Evi1 +T724 PR:000000046 27636 27640 TGFβ +T725 GO:0007179 27636 27640;27646 27664 TGFβ ... signaling pathways +T726 GO:0060395 27641 27664 SMAD signaling pathways +T727 PR:000035849 27715 27719 Evi1 +T728 GO:0010467 27749 27759 expression +T729 http://purl.obolibrary.org/obo/MONDO_0005441 27799 27801 OM +T730 NCBITaxon:727 27824 27846 Haemophilus influenzae +T731 NCBITaxon:2 27863 27872 bacterial +T732 NCBITaxon:9606 27894 27899 human +T733 http://purl.obolibrary.org/obo/MONDO_0005441 27900 27902 OM +T734 PR:000000046 27914 27918 Tgfβ +T735 GO:0007179 27914 27927;27936 27945 Tgfβ receptor ... signaling +T736 PR:000000365 27928 27933 Smad3 +T737 PR:000001153 27965 27969 TLR2 +T738 PR:000001740 27970 27975 MyD88 +T739 PR:000003158 27976 27980 TAK1 +T740 PR:000001776 27985 27989 IKKβ +T741 PR:000003293 27992 27996 IκBα +T742 GO:0051092 28007 28026 activation of NF-κB +T743 GO:0071159 28021 28026 NF-κB +T744 PR:000010764 28060 28070 MUC2 mucin +T745 PR:000035849 28091 28095 Evi1 +T746 PR:000000046 28160 28164 TGFβ +T747 GO:0010628 28185 28200;28206 28216 upregulation of ... expression +T748 PR:000010764 28201 28205 MUC2 +T749 http://purl.obolibrary.org/obo/MONDO_0005441 28285 28287 OM +T750 PR:000031343 28349 28361 MUC5AC mucin +T751 PR:000001153 28395 28399 TLR2 +T752 PR:000001740 28400 28405 MyD88 +T753 PR:000003106 28416 28419 p38 +T754 PR:000000046 28461 28465 TGFβ +T755 GO:0007179 28461 28465;28471 28480 TGFβ ... signaling +T756 GO:0060395 28466 28480 SMAD signaling +T757 PR:000003106 28522 28525 p38 +T758 PR:000006736 28547 28565 MAPK phosphatase-1 +T759 PR:000031343 28582 28594 MUC5AC mucin +T760 PR:000035849 28633 28637 Evi1 +T761 PR:000000046 28672 28676 TGFβ +T762 PR:000031343 28740 28752 MUC5AC mucin +T763 GO:0006952 28791 28798 defense +T764 http://purl.obolibrary.org/obo/MONDO_0005975 28802 28816 suppurative OM +T765 http://purl.obolibrary.org/obo/MONDO_0005441 28953 28955 OM +T766 GO:0065007 29069 29079 regulation +T767 GO:0010467 29089 29099 expression +T768 http://purl.obolibrary.org/obo/MONDO_0005441 29147 29149 OM +T769 PR:000035849 29187 29191 Evi1 +T770 http://purl.obolibrary.org/obo/MONDO_0005441 29213 29215 OM +T771 SO:0000417 29280 29286 domain +T772 SO:0000704 29326 29331 genes +T773 PR:000007789 29332 29339 Gadd45g +T774 PR:000007858 29341 29346 Gata2 +T775 PR:000017653 29348 29353 Zfpm2 +T776 PR:000017653 29354 29358 Fog2 +T777 PR:000014906 29360 29364 Skil +T778 PR:P12757-1 29366 29370 SnoN +T779 PR:000009365 29373 29377 Klf5 +T780 PR:000009365 29379 29384 BTEB2 +T781 PR:000000094 29387 29390 Dcn +T782 PR:000010133 29396 29403 Map3k14 +T783 PR:000035849 29430 29434 Evi1 +T784 GO:0065007 29435 29445 regulation +T785 PR:000001153 29470 29474 TLR2 +T786 PR:000001740 29475 29480 MyD88 +T787 PR:000003158 29481 29485 TAK1 +T788 PR:000001776 29490 29494 IKKβ +T789 PR:000003293 29497 29501 IκBα +T790 GO:0071159 29526 29531 NF-κB +T791 PR:000010764 29564 29574 MUC2 mucin +T792 GO:0070498 29642 29673 cytokine IL-1 signaling pathway +T793 PR:000001091 29651 29655 IL-1 +T794 PR:000001740 29681 29686 MyD88 +T795 PR:000001782 29687 29691 IRAK +T796 PR:000002292 29692 29697 TRAF6 +T797 PR:000003158 29698 29702 TAK1 +T798 GO:0033256 29711 29720 NF-κB/IκB +T799 PR:000003292 29717 29720 IκB +T800 SO:0000704 29782 29789 genetic +T801 http://purl.obolibrary.org/obo/MONDO_0003847 29782 29797 genetic disease +T802 http://purl.obolibrary.org/obo/MONDO_0005441 29807 29809 OM +T803 NCBITaxon:9606 29878 29883 human +T804 http://purl.obolibrary.org/obo/MONDO_0021166 29899 29919 Inflammatory disease +T805 UBERON:0001756 29941 29951 middle ear +T806 UBERON:0002405 29986 29992 immune +T807 http://purl.obolibrary.org/obo/MONDO_0005441 30005 30007 OM +T808 GO:0007567 30040 30045 natal +T809 http://purl.obolibrary.org/obo/MONDO_0001920 30068 30090 chronic suppurative OM +T810 PR:000035849 30240 30244 Evi1 +T811 http://purl.obolibrary.org/obo/MONDO_0005441 30299 30301 OM +T812 PR:000035849 30342 30346 Evi1 +T813 SO:0000704 30356 30360 gene +T814 GO:0065007 30361 30371 regulation +T815 http://purl.obolibrary.org/obo/MONDO_0005441 30401 30403 OM +T816 PR:000035849 30425 30429 Evi1 +T817 SO:0000704 30430 30434 gene +T818 SO:0000704 30523 30530 genetic +T819 http://purl.obolibrary.org/obo/MONDO_0005441 30559 30561 OM +T820 NCBITaxon:9606 30569 30574 human +T821 NCBITaxon:10088 30611 30615 Mice +T822 NCBITaxon:10088 30653 30658 mouse +T823 CHEBI:23995 30718 30721 ENU +T824 NCBITaxon:10088 30786 30790 mice +T825 GO:0007618 30812 30817 mated +T826 GO:0060004 31011 31017 reflex +T827 NCBITaxon:10088 31236 31240 mice +T828 NCBITaxon:10088 31249 31253 mice +T829 NCBITaxon:10088 31315 31319 mice +T830 NCBITaxon:10239 31383 31388 viral +T831 NCBITaxon:11138 31396 31399 MHV +T832 NCBITaxon:10508 31450 31460 Adenovirus +T833 NCBITaxon:12124 31469 31473 TMEV +T834 UBERON:0001004 31501 31512 respiratory +T835 NCBITaxon:51027 31553 31561 pinworms +T836 UBERON:0001004 31583 31594 respiratory +T837 NCBITaxon:758 31604 31629 Pasteurella pneumotropica +T838 UBERON:0001004 31694 31705 respiratory +T839 http://purl.obolibrary.org/obo/MONDO_0005087 31694 31713 respiratory disease +T840 http://purl.obolibrary.org/obo/MONDO_0005441 31721 31723 OM +T841 NCBITaxon:10088 31731 31736 mouse +T842 http://purl.obolibrary.org/obo/MONDO_0005249 31745 31754 pneumonia +T843 NCBITaxon:11263 31745 31760 pneumonia virus +T844 NCBITaxon:11191 31762 31774 Sendai virus +T845 NCBITaxon:2107 31776 31795 Mycoplasma pulmonis +T846 NCBITaxon:1313 31797 31821 Streptococcus pneumoniae +T847 NCBITaxon:287 31827 31849 Pseudomonas aeruginosa +T848 NCBITaxon:2 31929 31937 bacteria +T849 UBERON:0001728 31956 31967 nasopharynx +T850 NCBITaxon:10088 31980 31984 mice +T851 NCBITaxon:1279 31993 32007 Staphylococcus +T852 NCBITaxon:species 32008 32012 spp. +T853 NCBITaxon:1280 32014 32035 Staphylococcus aureus +T854 NCBITaxon:1301 32054 32066 streptococci +T855 NCBITaxon:1301 32078 32091 Streptococcus +T856 NCBITaxon:species 32092 32095 spp +T857 NCBITaxon:10088 32103 32107 mice +T858 UBERON:0001728 32225 32239 nasopharyngeal +T859 SO:0000704 32265 32272 Genetic +T860 CHEBI:23995 32318 32321 ENU +T861 NCBITaxon:10088 32330 32335 mouse +T862 GO:0060004 32443 32449 reflex +T863 NCBITaxon:1 32495 32506 individuals +T864 SO:0001026 32513 32519 genome +T865 SO:0000704 32580 32587 genetic +T866 SO:0000704 32865 32872 genetic +T867 PR:000035849 32987 32991 Evi1 +T868 CL:0002322 33028 33036 ES cells +T869 GO:0007618 33053 33060 matings +T870 NCBITaxon:10088 33069 33073 mice +T871 PR:000035849 33080 33084 Evi1 +T872 NCBITaxon:10088 33094 33098 mice +T873 GO:0007618 33104 33109 mated +T874 NCBITaxon:10088 33121 33125 mice +T875 NCBITaxon:10088 33151 33155 mice +T876 PR:000035849 33209 33213 Evi1 +T877 NCBITaxon:10088 33223 33227 mice +T878 GO:0007618 33238 33243 mated +T879 NCBITaxon:10088 33255 33259 mice +T880 UBERON:0001756 33461 33471 middle ear +T881 http://purl.obolibrary.org/obo/MONDO_0003276 33461 33479 middle ear disease +T882 GO:0007567 33535 33540 natal +T883 NCBITaxon:10088 33541 33545 mice +T884 GO:0007567 33581 33586 natal +T885 UBERON:0007023 33662 33667 adult +T886 NCBITaxon:10088 33668 33672 mice +T887 NCBITaxon:10088 33821 33825 mice +T888 http://purl.obolibrary.org/obo/MONDO_0005441 33918 33920 OM +T889 NCBITaxon:10088 33959 33963 mice +T890 UBERON:0000922 33985 33991 embryo +T891 PR:000035849 34273 34277 Evi1 +T892 NCBITaxon:10088 34287 34291 mice +T893 http://purl.obolibrary.org/obo/MONDO_0005441 34354 34356 OM +T894 UBERON:0000479 34359 34366 Tissues +T895 CHEBI:16842 34410 34418 formalin +T896 CHEBI:73702 34450 34453 wax +T897 UBERON:0000033 34455 34460 Heads +T898 UBERON:0001474 34465 34470 bones +T899 UBERON:0001756 34608 34619 middle ears +T900 CHEBI:51686 34638 34650 Haemotoxylin +T901 CHEBI:51686 34662 34663 H +T902 NCBITaxon:10088 34688 34692 mice +T903 http://purl.obolibrary.org/obo/MONDO_0005441 34698 34700 OM +T904 UBERON:0001762 34729 34745 nasal turbinates +T905 UBERON:0001729 34776 34786 oropharynx +T906 UBERON:0002048 34788 34793 lungs +T907 UBERON:0002107 34795 34801 livers +T908 UBERON:0002113 34803 34810 kidneys +T909 UBERON:0000948 34812 34817 heart +T910 UBERON:0002106 34819 34825 spleen +T911 UBERON:0000160 34827 34837 intestines +T912 UBERON:0001264 34839 34847 pancreas +T913 UBERON:0002107 34853 34859 livers +T914 UBERON:0001756 34900 34911 middle ears +T915 UBERON:0006333 34913 34919 snouts +T916 UBERON:0002048 34925 34930 lungs +T917 http://purl.obolibrary.org/obo/MONDO_0005441 34936 34938 OM +T918 NCBITaxon:2 34963 34971 bacteria +T919 CL:0000771 35008 35029 eosinophil leukocytes +T920 http://purl.obolibrary.org/obo/MONDO_0005550 35075 35085 infections +T921 NCBITaxon:10088 35190 35194 mice +T922 UBERON:0000468 35226 35236 whole-body +T923 UBERON:0000479 35263 35270 tissues +T924 NCBITaxon:10088 35366 35370 mice +T925 UBERON:0000033 35465 35470 heads +T926 UBERON:0000955 35492 35498 brains +T927 UBERON:0001684 35503 35512 mandibles +T928 UBERON:0003129 35535 35540 skull +T929 UBERON:0000033 35606 35610 head +T930 UBERON:0000922 35864 35871 embryos +T931 UBERON:0007023 35929 35934 adult +T932 UBERON:0000033 35935 35940 heads +T933 NCBITaxon:10088 35986 35990 mice +T934 CHEBI:73702 36027 36030 wax +T935 CHEBI:27338 36064 36070 xylene +T936 CHEBI:16236 36112 36119 ethanol +T937 CHEBI:75958 36120 36129 solutions +T938 CHEBI:16240 36183 36200 hydrogen peroxide +T939 CHEBI:17824 36204 36215 isopropanol +T940 CHEBI:15956 36479 36485 biotin +T941 CHEBI:15956 36504 36510 Biotin +T942 CHEBI:75958 36548 36556 solution +T943 NCBITaxon:9793 36564 36570 donkey +T944 UBERON:0001977 36571 36576 serum +T945 NCBITaxon:9925 36605 36609 Goat +T946 GO:0042571 36621 36629 antibody +T947 NCBITaxon:9606 36669 36674 human +T948 PR:000035849 36675 36679 EVI1 +T949 MOP:0000093 36787 36799 Biotinylated +T950 NCBITaxon:9793 36800 36806 donkey +T951 NCBITaxon:9925 36812 36816 goat +T952 GO:0042571 36817 36825 antibody +T953 PR:000035849 36921 36926 EVI-1 +T954 NCBITaxon:9793 36980 36986 donkey +T955 UBERON:0001977 36987 36992 serum +T956 CHEBI:51686 37056 37068 Haematoxylin +T957 UBERON:0004288 37071 37079 Skeletal +T958 UBERON:0000922 37095 37102 Embryos +T959 CHEBI:16236 37121 37128 ethanol +T960 CHEBI:16866 37178 37190 alizarin red +T961 NCBITaxon:10088 37263 37267 mice +T962 CL:0000084 37274 37275 T +T963 UBERON:0002405 37297 37310 immune system +T964 NCBITaxon:54986 37345 37359 keyhole limpet +T965 CL:0000084 37415 37416 T +T966 NCBITaxon:1313 37454 37466 pneumococcal +T967 CHEBI:18154 37467 37481 polysaccharide +T968 NCBITaxon:10088 37539 37543 mice +T969 UBERON:0000178 37595 37600 blood +T970 CHEBI:28304 37626 37633 heparin +T971 UBERON:0004711 37649 37661 jugular vein +T972 NCBITaxon:10088 37679 37683 mice +T973 CHEBI:29745 37689 37700 barbiturate +T974 NCBITaxon:10088 37722 37726 mice +T975 CL:0000094 37824 37836 granulocytes +T976 CHEBI:37926 37899 37903 FITC +T977 GO:0034688 37912 37917 Mac-1 +T978 GO:0034688 37912 37917 Mac-1 +T979 GO:0042571 38036 38044 antibody +T980 CL:0000094 38056 38067 granulocyte +T981 CL:0000084 38115 38116 T +T982 CL:0000084 38131 38132 T +T983 UBERON:0002405 38158 38164 Immune +T984 NCBITaxon:10088 38197 38201 Mice +T985 GO:0007567 38529 38534 birth +T986 GO:0007620 38551 38557 coitum +T987 CHEBI:23995 38559 38562 ENU +T988 CHEBI:23995 38565 38586 N-ethyl-N-nitrosourea +T989 UBERON:0004114 38588 38591 MEC +T990 UBERON:0004114 38594 38611 middle ear cavity +T991 NCBITaxon:727 38632 38654 Haemophilus influenzae +T992 http://purl.obolibrary.org/obo/MONDO_0005441 38656 38658 OM +T993 http://purl.obolibrary.org/obo/MONDO_0005441 38661 38673 otitis media +T994 CHEBI:33893 39081 39089 reagents diff --git a/src/ontogpt/evaluation/craft/database/all/17029558.txt b/src/ontogpt/evaluation/craft/database/all/17029558.txt new file mode 100644 index 000000000..3994b343c --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17029558.txt @@ -0,0 +1,179 @@ +Mutation at the Evi1 Locus in Junbo Mice Causes Susceptibility to Otitis Media + +Abstract + +Otitis media (OM), inflammation of the middle ear, remains the most common cause of hearing impairment in children. It is also the most common cause of surgery in children in the developed world. There is evidence from studies of the human population and mouse models that there is a significant genetic component predisposing to OM, yet nothing is known about the underlying genetic pathways involved in humans. We identified an N-ethyl-N-nitrosourea-induced dominant mouse mutant Junbo with hearing loss due to chronic suppurative OM and otorrhea. This develops from acute OM that arises spontaneously in the postnatal period, with the age of onset and early severity dependent on the microbiological status of the mice and their air quality. We have identified the causal mutation, a missense change in the C-terminal zinc finger region of the transcription factor Evi1. This protein is expressed in middle ear basal epithelial cells, fibroblasts, and neutrophil leukocytes at postnatal day 13 and 21 when inflammatory changes are underway. The identification and characterization of the Junbo mutant elaborates a novel role for Evi1 in mammalian disease and implicates a new pathway in genetic predisposition to OM. + +Synopsis + +Otitis media (OM), inflammation of the middle ear, is the most common cause of deafness in children. Although acute episodes of OM in children are associated with middle ear infections, in a substantial portion of cases recurrent episodes of OM, or a chronic suppurative OM, will develop. There is evidence from genetic studies of human families that there is a significant genetic component contributing to the development of recurrent and chronic forms of OM. However, the genes involved have not been identified. The authors have identified and characterized mouse mutants that demonstrate chronic OM as a route to identifying genes involved with OM. This study describes one mutant, Junbo, which shares many features with human OM. Junbo develops an acute OM following birth that subsequently develops into a chronic suppurative form of OM. Junbo carries a mutation in the transcription factor gene, Evi1. Evi1 is expressed in a variety of cell types in the middle ear lining when inflammatory changes are underway. The identification of the Junbo mutation implicates a new gene involved in predisposition to OM. + +Introduction + +Otitis media (OM), inflammation of the middle ear, remains the most common cause of hearing impairment in children [1,2]. Acute episodes of OM in infants and children are most often associated with middle ear infections involving the pathogens Streptococcus pneumoniae and Haemophilus influenzae [3]. Prolonged stimulation of the inflammatory response, along with poor mucociliary clearance, can also lead to the persistence of middle ear fluid giving rise to the clinical presentation of otitis media with effusion [2]. In a substantial portion of children, recurrent episodes of OM or a chronic suppurative OM will develop. The high prevalence of the disease, coupled with its recurrent and chronic nature, accounts for the large number of tympanostomies undertaken in affected children. OM is still the most common cause of surgery in children in the developed world. There is still considerable debate over the etiology of OM and the underlying pathological mechanisms [2]. However, risk factors for OM include craniofacial abnormalities, impaired mucocilliary function, and the presence of an inflammatory stimulus, such as bacteria. There is evidence from studies of the human population and mouse models that there is a significant genetic component predisposing to recurrent or chronic OM [4–7], yet little is known about the underlying genetic pathways involved. While several inbred strains are predisposed to the development of OM, their genetic analysis and utility is compounded by the complex genetic bases and the low penetrance of the phenotype [6]. In addition, there are several mouse mutants that demonstrate an OM phenotype, but the OM develops as part of a complex syndrome with a wide spectrum of phenotypes [6]. It will be important to identify and characterize the genes underlying highly penetrant mouse mutants that develop OM in the absence of other diverse pathology and represent appropriate models for OM in the human population. + +Large-scale phenotype-driven mouse ENU (N-ethyl-N-nitrosourea) mutagenesis programs provide a rich source of novel mutant phenotypes that are the basis for systematic efforts to identify the genetic basis for diverse disease states [8–10]. One such screen at MRC Harwell, recovered a large number of mutant phenotypes representing ENU-induced mutations at a number of novel loci in the mammalian genome [9]. Mouse models have and continue to play an important role in studying the genetic causes of hearing impairment. A number of mutations have been cloned and have provided us with several profound insights into the critical proteins involved with the development and function of the auditory apparatus at the level of both the middle and inner ears [11,12]. Nevertheless, it is clear that we do not possess mouse mutants for all loci and pathways potentially involved in hearing impairment. + +We report the characterization of a new mutant, Junbo, identified in the Harwell mutagenesis program as having a deafness phenotype. Junbo is a model of OM that shares many features with the human condition. Acute OM arises spontaneously in the postnatal period and develops into chronic suppurative OM with otorrhea. The underlying molecular basis of this phenotype has been identified as a mutation in the Evi1 transcription factor causing a nonconservative Asn763Ile change in the second of the two zinc-finger domains in this protein. The Junbo mouse highlights a new role for the transcription factor Evi1 and provides the first evidence for the genetic pathways involved in the etiology of this complex childhood disease. + +Results + +Identification, Mapping, and Cloning of the Junbo Mutation + +An ENU mutagenesis screen [9] identified a new dominant mutant, Junbo (Jbo), with hearing loss. Preliminary phenotyping using a click-box test (see Materials and Methods) of an age-matched cohort derived from the founder mouse indicated that mice demonstrated a hearing loss at ~40 d after birth (DAB). We used a pooling strategy employing DNA from affected mice and genome-wide fluorescent simple sequence length polymorphism-based screening [9,13] to provide an initial map position for the Jbo mutation on Chromosome 3. This map position was further refined using additional affected animals to an approximately 1.5-Mb region delineated by the Eif5a2 locus and microsatellite marker D3Mit178. Direct sequence analysis of coding regions within this interval identified an A2288T transversion in the Evi1 locus, causing a nonconservative Asn763Ile change in the second of the two zinc-finger domains in this protein (Figure 1A and 1B). No other sequence changes were identified in any other coding sequences within the minimal nonrecombinant region containing the mutation. + +Figure 1 + +The Evi1 Gene Is Mutated in Junbo Mice + +(a) Sequence analysis of the Evi1 locus in BALB/c, C3H/HeN, Jbo/+ adult, and Jbo/Jbo embryonic DNA. An A2318T transversion is detected in Jbo/+ and Jbo/Jbo mutants that is not present in either parental substrains. + +(b) Schematic of the EVI1 peptide. Ten zinc finger motifs are clustered into two DNA-binding domains, ZF1 and ZF2. EVI1 contains a proline-rich repressor domain between the two sets of zinc fingers and a highly acidic domain at the C-terminus. Expanded peptide sequence across the ninth zinc finger motif shows the high degree of conservation of this region between orthologous proteins from different species: Mus musculus (P14404), Rattus norvegicus (ENSRNOG00000012645), Homo sapien (Q03112), Danio rerio (ENSDARP00000008993), Fugu rubrides (ENSDARP00000008993), Drosophila melanogaster (CG31753), Caenorhabditis elegans (R53.3a). Contact residues are highlighted in yellow, the position of the Junbo mutation is highlighted in red. + +(c) Extra digits are seen on the forelimbs of both heterozygote and homozygote mice E18.5 (white arrows). In heterozygotes, (Jbo/+, middle panel) an extra digit is observed on either forelimb. The homozygotes, (Jbo/Jbo, right panel) have extra digits on both forelimbs. The anterior digit is often reduced in size in the homozygote limbs (red arrow). Wild-type mice, left panel. + +A knockout allele of Evi1 has been produced, Evi1tm1Mmor [14]. Evi1tm1Mmor mice carry a targeted deletion resulting in an isoform-specific null for the longest Evi1 transcripts, while the Δ324 (shorter) isoform [15,16] remains unaltered. This isoform lacks the final two zinc-finger motifs from the first (N-terminal) DNA-binding domain and consequently is predicted to exhibit altered binding abilities. Characterization of heterozygote mice carrying this targeted mutant failed to uncover any phenotype, while homozygotes show prenatal lethality from presumptive cardiac failure [14]. In addition, these embryos displayed widespread hypocellularity, most markedly of the cortical mesenchyme, retarded development of the first and second branchial arches, and severely reduced vasculature of the yolk sac. To confirm that the A2318T alteration identified in Evi1 is responsible for the Junbo phenotype, we undertook a complementation screen between Jbo/+ and Evi1tm1Mmor/+ animals. 81 live births were produced from crosses of Jbo/+ and Evi1tm1Mmor/+ mice and all were genotyped. None of the progeny coinherited the Evi1tm1Mmor and the Jbo mutations. However, the other expected genotypes were recovered—Jbo/+ (38%), Evi1tm1Mmor/+ (28%), +/+ (33%)—confirming the allelism of the knockout and Junbo mutant. We established intercrosses between Jbo/+ animals using in vitro fertilization and implantation into wild-type females as Jbo/+ females undergo repeated spontaneous abortion. At 10.5 days postcoitum (dpc), 17% of homozygote mice had small hind limbs, a large pericardial sac, and a malformed forebrain, features demonstrated by the knockout [14]. However, many homozygotes were scored as normal in appearance at this and later stages and proceeded to die between E18.5 and birth, though homozygotes were of comparable body size to wild-type littermates. It appears that though some homozygote embryos may die at 10.5 dpc, others continue to develop unhindered when carried by a wild-type mother. The only other phenotypic feature detected in Jbo/+ mice was an extra digit on one forelimb. Jbo/Jbo mice have extra digits on both forelimbs (see Figure 1C). + +Pathology Phenotyping of the Junbo Mutant + +To determine the cause of the deafness phenotype in Junbo, we performed an initial examination of deaf adult animals and embryonic skeletal preparations (15.5 dpc–18.5 dpc). This revealed no gross morphological defects of the inner ear and ossicular chain or ossification of the temporal bone and tympanic ring in Jbo/+ animals. Dissection of the bullae of seven Jbo/+ mice and four +/+ mice (>130 DAB) revealed that the middle ear cavity (MEC) of mutant mice was filled with exudate, indicative of OM. X-ray analysis revealed that there was no consistent abnormality of bullae shape associated with OM in Jbo/+ mice. Examination of seven Jbo/+ mice (14 bullae, >180 DAB) revealed that 6 Jbo/+ mice bullae had a normal shape and demonstrated radio opacity, indicating the presence of an effusion in the middle ear, whereas in six Jbo/+ mice, bullae were misshapen and also had an effusion present (demonstrated by radio opacity). Although X-ray imaging is a less sensitive means of diagnosing OM than histology, two Jbo/+ mice bullae did not demonstrate a radio opacity reflecting the possibility of resolution of a small number of Junbo mice at advanced age (see below). Control ears (two wild-type mice, four bullae) were clear of effusion and presented with a normal bullae shape. We concluded that the presence of abnormally shaped bullae in the Junbo mutant is not necessary for the development of an effusion. We therefore proceeded to study the pathology of the OM in the postnatal period, at weaning, and in the adult, first in conventionally housed mice of relatively low microbiological status and then in specific pathogen-free (SPF) mice. + +In conventionally housed mice we found no evidence of inflammation of the embryonic connective tissue or exudation into the MEC in Jbo/+ or wild-type litter mates at 5 DAB. By 13 DAB the MEC is more fully formed and 100% of the Jbo/+ mice and ~33% wild-type mice had acute OM (Figure 2A–2D). However, Jbo/+ mice had suppurative exudate in both the MECs, while wild-type mice had unilateral OM, or serous exudation in one ear and suppurative exudation in the contralateral ear. Typical acute inflammatory changes in the MEC mucoperiosteum included edematous polyps (Figure 2E, which may represent unresorbed embryonic connective tissue [17]) and/or bulging sub-epithelial bullae filled with serous fluid, stromal edema, widely patent capillaries, and lymphatics, with variable numbers of infiltrating neutrophil leukocytes. The epithelial covering was formed by small basal cells or ciliated columnar cells (Figure 2F and 2G). A second, milder type of OM occurred in a further ~27% of wild-type mice consisting of only focal mucoperiosteal aggregations of neutrophil leucocytes with or without a light neutrophil leukocyte MEC exudation (unpublished data); ~40% wild-type mice had no evidence of OM at this age. Importantly, OM appeared at this stage as part of a more generalized respiratory tract inflammation comprising suppurative rhinitis (Figure 2H) and nasopharyngitis, and multifocal mild alveolitis/interstitial pneumonia with eosinophil leukocytes in perivascular cuffs (Figure 3A and 3B) but without evidence of intralesional bacteria or viral inclusions. There were no significant histological lesions in intestine, liver, pancreas, kidney, heart, thymus, and spleen. + +Figure 2 + +Histology of Middle Ear and Nose in Wild-Type and Junbo Mutant Mice + +Images (a–h) are from 13-d-old postnatal mice and are given with their original magnification (a) Jbo/+ dorsal section of MEC partly filled with exudate ×40, (b) +/+ normal middle ear temporal bone covered with thin mucoperiosteum (arrowheads) ×400, (c) +/+ inflamed middle ear with thickened mucoperiosteum with neutrophil leukocyte infiltrates and neutrophil-rich exudates in the MEC ×400, (d) Jbo/+ middle ear with more severe suppurative exudation into the MEC ×400, (e) Jbo/+ inflamed edematous polyp (arrowhead) of un-resorbed embryonic middle ear connective tissue, tympanic membrane ×100, (f) Jbo/+ MEC lined by ciliated columnar cells (arrowhead) ×600 (g) Jbo/+ MEC lined by basal cells (arrowhead) ×600, (h) Jbo/+ suppurative rhinitis: nasal cavity with suppurative exudate, nasal septum with inflamed nasal mucosa ×200. + +Images (i–l) are of adult (180-d) Jbo/+ middle ear with chronic suppurative OM; changes include (i) fibrous polyps (arrowheads) ×200, (j) hyperplasia of ciliated epithelial cells (arrowhead) and fibrosis of mucoperiosteum stroma ×400, and (k) fibrous thickening of the tympanic membrane, outer ear canal ×400 compared with (l) normal +/+ tympanic membrane, outer ear canal ×400. E, exudate; MP, mucoperiosteum; NC, nasal cavity; NM, nasal mucosa; NS, nasal septum; OEC, outer ear canal; TB, temporal bone; TM, tympanic membrane + +Figure 3 + +Histology of the Lung and Middle Ear Exudate in Junbo Mice + +(a) 13-d postnatal Jbo/+ lung with perivascular and peribronchiolar cuffs containing Sirius red positive eosinophil leukocytes (arrowhead), bronchiole, pulmonary artery ×400, (b) focal eosiniphilic alveolitis (arrowhead) and thickened alveolar septae (open arrowhead) with eosinophil-rich infiltrates. (c) 21-d postnatal Jbo/+ MEC pus with colonies of Gram positive cocci ×600. B, bronchiole; PA, pulmonary artery + +Any postnatal OM had resolved in wild-type mice by weaning (0% incidence at 21 DAB) and OM was exceptional in adults (3% incidence). In contrast, OM was present in 100% of Jbo/+ mice at weaning and occurred in 94% of adult Jbo/+ mice 29 DAB to >180 DAB. Suppurative rhinitis was still present in some Jbo/+ mice examined, but in two the eosinophilic pneumonia was absent or minimal. Large numbers of Gram positive cocci were present in OM exudate at day 21 in ~70% of cases (Figure 3C), suggesting nasopharyngeal staphylococcal and streptococcal flora may play a role in progression of OM, if not its initiation in Jbo/+ mice. There was no evidence of significant proliferation of mucous cells or periodic acid-Schiff-positive mucus in the MEC. + +Adult Jbo/+ mice >29 DAB develop bilateral chronic suppurative OM. Chronic middle ear effusions contained variable numbers and proportions of viable and necrotic neutrophil leukocytes and foamy macrophages; bacteria were infrequently present and there were small numbers of multinucleate macrophages, cholesterol clefts, and small amounts of birefringent foreign body (perhaps secondary to perforation of the eardrum). The effusions did not contain significant amounts of periodic acid-Schiff-positive mucus. Chronic changes in inflamed thickened mucoperiosteum include formation of multiple polyps (Figure 2I) covered by low cuboidal cells or hyperplastic ciliated columnar cells and scattered mucous cells (Figure 2J). The mucoperiosteal stroma has variable degrees of fibrosis, neutrophil leukocyte infiltration, scattered mast cells, lymphoplasmacytic infiltrates, and occasional lymphoid nodules. The eustachian tube is patent and can contain exudates, and the epithelial lining can have elevated numbers of mucous cells and intra-epithelial leukocytes. Fibrous thickening of the eardrum (tympanosclerosis) is common (Figure 2K and 2L). OM was often associated with perforation of the eardrum, possibly providing drainage to account for apparent resolution of a few cases: (6%) of OM in the oldest age group of Jbo/+ mice (>180 DAB). + +The Junbo colony has subsequently been re-derived by embryo transfer into new high-health status facilities (Mary Lyon Centre, Harwell). Mice are housed in individually ventilated cages, and screening shows all FELASA-listed pathogens (see Materials and Methods) have been excluded; 75 cage air changes/h reduce respiratory irritants such as ammonia to <3 ppm. In SPF conditions, OM in Jbo/+ mice is relatively milder at early time points and is not associated with rhinitis. Typically at 13 DAB there are small numbers of inflammatory cells in the mucoperiosteum and sometimes, light MEC effusion. By 20–22 DAB and 28 DAB, 4% Jbo/+ mice had bilateral OM, 54% unilateral OM, and 42% had very mild or no OM. However, by 54 DAB, Jbo/+ mice (100%) had OM, but in 10% of cases this was unilateral. OM does not occur in pre-weaned wild-type mice and in only 3% (a single case in a 22-DAB mouse) of older wild-type mice. + +Comprehensive pathology phenotyping failed to show a consistent pattern of significant organ pathology outside of the middle ear in adult Jbo/+ mice. Specifically there is no evidence of opportunistic infections in sites such as skin, lung, urogenital, or gastrointestinal systems that might be a sign of immune deficiency (see below). + +In summary, the microbiological status of the mice and/or air quality affect onset of OM, and under “dirty” conventional conditions even wild-type mice can develop OM in the postnatal period as part of upper respiratory tract disease; however, this resolves in wild-type mice by weaning. In conventionally housed and SPF Jbo/+ mice, OM emerges as a chronic condition. + +Immunology and FACS Analysis of SPF Wild-Type and Junbo Mice + +There were no significant differences in the antibody responses of Jbo/+ and wild-type control mice to immunization with T-dependent (IgG1 and IgG2a to keyhole limpet hemocyanin) and T-independent (IgGM and IgG3 to pneumococcal polysaccharide type 3) antigens (Table S1). FACS analysis of blood neutrophils identified by the cell surface markers Gr-1 and Mac-1 did not reveal significant differences between levels of immature, mature cell forms, and total circulating blood neutrophils in Jbo/+ and wild-type mice at either 20–22 DAB or 49–58 DAB. However, there was a significantly lower (p = 0.003) ratio of immature forms in the circulating neutrophil pool in 20–22 DAB Jbo/+ mice, but not in 54–58 DAB Jbo/+ mice (Table 1). + +Table 1 + +FACS Analysis of the Proportion of Granulocytes in Blood of Wild-Type and Junbo Mice + +Expression of Evi1 in Wild-Type and Junbo Mice + +We proceeded to investigate the expression of Evi1 in both embryonic whole-body tissues (E9.5 and at eight intervals through to birth) and postnatal middle ear tissue (13 DAB, 21 DAB) in order to relate the underlying mutation to the Junbo phenotype. We examined expression in wild-type and Jbo/+ mice, but at no time point did we identify any significant differences in expression patterns, up to and including birth, from those described [14,18]. However, in postnatal head tissues, we now find that Evi1 is expressed in nuclei of myeloid cells in bone marrow, neutrophil leukocytes, fibroblasts, and basal epithelial cells in the inflamed middle ear lining (Figure 4), and that patterns of expression are similar in Jbo/+ and wild-type mice. + +Figure 4 + +Evi1 Protein Immunostaining in 13-d-Old Jbo/+ and Wild-Type Mice with Acute OM + +(a) Jbo/+ positive myeloid cells in temporal bone marrow ×600. Note chondrocytes and osteocytes are also strongly positive. (b) Jbo/+ mucoperiosteum has positive neutrophil leukocytes, fibroblasts, and basal epithelial cell nuclei ×600. (c) +/+ similar pattern of staining in bone marrow ×600 and (d) +/+ inflamed mucoperiosteum ×600. B, basal epithelial cell nuclei; C, chrondrocyte; F, fibroblast; MC, myeloid cell; N, neutrophil leukocyte; O, osteocyte + +OM Phenotype of the Evi1tm1Mmor/+ Mice + +OM is not a prominent feature in Evi1tm1Mmor/+ mice. Only one of 12 Evi1tm1Mmor/+ mice >68 DAB had OM, and in this single case it was unilateral. OM was not present in 15 wild-type littermates. The absence of OM in Evi1tm1Mmor/+ mice, coupled with the presence of extra digits in Jbo/+ and Jbo/Jbo mice, suggests that the Junbo mutation may have gain-of-function effects. However, the genetic background of Evi1tm1Mmor/+ mice differs from Jbo/+ and Jbo/Jbo mice (see Materials and Methods). In addition, the Evi1tm1Mmor mutation is an isoform- specific null. It is therefore difficult to reach definitive conclusions as to the nature of the Junbo mutation (see Discussion). + +Discussion + +A mutation in the Evi1 transcription factor underlies the development of a chronic suppurative OM in the Junbo mutant. Genetic mechanisms appear to interact with microbiological status and environmental conditions, such that SPF Jbo/+ mice with improved air quality have milder early OM. Gnotobiotic studies are planned to investigate the role of nasopharyngeal Staphylococcus and Streptococcus spp. commensals in OM pathogenesis. The absence of multi-systemic inflammation and opportunistic infections at other sites, and the normal T-dependent and T-independent immune responses in Jbo/+ mice, argue against an overt immune deficiency being responsible for OM. + +The Evi1 locus was initially identified as a common site of retroviral integration underlying susceptibility to myeloid tumors in the AKXD mouse recombinant inbred strain [19,20]. Transcriptional activation of the Evi1 locus by translocations and inversions leads to myeloid leukemias and myelodysplastic syndrome in humans [21]. This locus encodes a 145-kDa nuclear transcription factor with two distinct zinc-finger domains composed of seven and three zinc-finger motifs, respectively [22]. Each zinc-finger domain has been shown to bind specific target consensus DNA sequences, and additional proline-rich and highly acidic domains located within the protein have been demonstrated to be capable of repressing or activating target promoter activities, respectively [21–24]. Evi1 has also been shown to be capable of repressing the TGF-β signaling pathway through direct binding of Smad3 mediated by the first zinc-finger domain, suggesting a functional role in the control of cell development and proliferation [25]. In addition, cell line assays have demonstrated a role for Evi1 in the transcriptional control of c-fos and the AP-1 proliferative pathway through interactions of the second zinc-finger domain [26]. The Junbo mutation results in a nonconservative Asn763Ile change in the second of these three zinc-fingers. Previous in vitro site-directed mutagenesis studies of the contact residues within these three zinc-fingers uncovered a complete loss of DNA-binding ability following any residue change [23]. The Junbo Asn763Ile alteration is within three amino acids of a contact residue, and the mutated amino acid contributes to the putative alpha helix of the Cys2His2 structure. While it seems likely that the Junbo mutation will disturb the function of the second zinc-finger domain, it is unclear whether or not there are effects on the role of the first zinc-finger domain that is involved in TGF-β signaling. + +It is interesting to note that OM is not part of the phenotype of Evi1tm1Mmor/+ mice. However, the Evi1tm1Mmor allele results in an isoform-specific allele for the longest Evi1 transcript while the Δ324 shorter isoform is unaffected. In addition, the Evi1tm1Mmor knockout was established in ES-D3 cells with chimaeras subsequently mated to CF-1 mice [14]. Both the presence of the shorter isoform and the dissimilar genetic backgrounds between Evi1tm1Mmor/+ mice and Jbo/+ mice may contribute to the differences in the expressivity of the OM phenotype. Alternatively, the Junbo mutation may lead to gain-of-function effects. However, it is not possible at this stage to distinguish between these possibilities. + +A mutation in the Evi1 transcription factor may give rise to OM by more than one mechanism, given that it is expressed in a number of different cell types in the middle ear. One mechanism arises from our observation that Evi1 is expressed in neutrophil leukocytes during OM development. Evi1 has multiple functions relating to hematopoietic differentiation and development of myeloid leukemia [27–30]. One putative target gene for Evi1 in neutrophil leukocytes is the inositol triphosphate type 2 receptor gene (Itpr2) that is required for functional regulation of neutrophil leukocyte maturation via F-met-leu-phe receptor signaling in response to bacterial proteins [31]. Our FACS analysis of circulating neutrophils at two time points, when OM first appears and when chronic suppurative OM is well established, indicates that localized inflammation in the middle ear in Jbo/+ mice does not result in significant neutrophilia. Neutrophils are apparently released from hematopoietic tissues at comparable levels in Jbo/+ and wild-type mice and there was no detectable block in neutrophil development in Jbo/+ mice. The ratio of immature neutrophils in the circulating pool was no higher; indeed, it is reduced in recently weaned mice when ~60% have acute OM. In older Jbo/+ mice with fully penetrant chronic suppurative OM, immature and mature neutrophil ratios are not significantly different from wild-type mice. + +A variety of in vitro studies have highlighted the role of the TGFβ/SMAD pathway on mucin expression and thus underlined the potential importance of this signaling pathway in OM [32]. A loss of Evi1 function could affect TGFβ/SMAD signaling pathways. There are two in vitro studies that suggest that Evi1 mutations could affect mucin expression that might underlie predisoposition to OM. Firstly, nontypeable Haemophilus influenzae (NTHi), a known bacterial pathogen involved in human OM, activates Tgfβ receptor-Smad3/4 signaling that together with TLR2-MyD88-TAK1-NIK-IKKβ/γ-IκBα-dependent activation of NF-κB is known to mediate NTHi-induced MUC2 mucin transcription [33]. Evi1 loss-of-function mutations might lead to a de-repression of the TGFβ/SMAD pathway and an upregulation of MUC2 expression leading to an enhancement of effusive processes as a contributor to OM. Alternatively, it has also been shown that NTHi upregulates MUC5AC mucin production via activation of the TLR2-MyD88-dependant p38 pathway [34]. However, the activation of TGFβ/SMAD signaling by NTHi also leads to down-regulation of p38 activity by inducing MAPK phosphatase-1 and suppressing MUC5AC mucin induction. Thus, in this case loss of Evi1 function and de-repression of the TGFβ/SMAD pathway would presumably lead to increased suppression of MUC5AC mucin induction that may reduce mucociliary defense in suppurative OM. Importantly, the identification of the Junbo mutation now provides in vivo evidence to support the role of these signaling pathways in OM. It will be interesting to explore further the molecular phenotype of the Junbo mutant and to examine whether disregulation of mucin expression is a contributing factor in the development of OM. + +Additional levels of complexity of Evi1 function relevant to OM pathogenesis may result from the first (N-terminal) zinc-finger domain binding to a number of putative target genes Gadd45g, Gata2, Zfpm2/Fog2, Skil (SnoN), Klf5 (BTEB2), Dcn, and Map3k14 (Nik) [35]. For instance, Evi1 regulation of Nik could act in the TLR2-MyD88-TAK1-NIK-IKKβ/γ-IκBα-dependent activation of NF-κB pathway to mediate NTHi-induced MUC2 mucin transcription as described above; and also in the pro inflammatory cytokine IL-1 signaling pathway IL-1R1-MyD88-IRAK-TRAF6-TAK1-NIK-IKK-NF-κB/IκB [36]. + +In conclusion, the Junbo mutant provides an important genetic disease model of OM; particularly because it shares important features with the chronic human condition [7]. Inflammatory disease is restricted to the middle ear and is not a consequence of overt immune deficiency. OM arises spontaneously in the postnatal period, develops into chronic suppurative OM with otorrhea, with the early severity and age of onset dependent on microbiological status and/or air quality. We have shown that a mutation at the Evi1 locus underlies the susceptibility and persistence of OM. Our observations underline the role of Evi1 in mucin gene regulation as a possible contributor to OM. In this regard, the Evi1 gene and associated pathway members can be considered important candidates for examining the genetic basis for susceptibility to OM in the human population. + +Materials and Methods + +Mice and deafness screening. + +The founder mouse carrying the Junbo mutation was generated in a large-scale ENU mutagenesis program at Harwell, United Kingdom [9]. Male BALB/c mice were mutagenized and mated to normal C3H/HeN females, and the offspring were screened for a variety of defects, including deafness and vestibular dysfunction. The Jbo founder was identified because of a lack of a Preyer reflex when presented with a calibrated 20 kHz 90 dB SPL tone burst. For analysis of the phenotype, the colony was maintained on a C3H/HeN background in accordance with Home Office regulations. + +Microbiological status of the mice. + +Junbo mice were originally derived in a conventional facility. Sentinel mice from this colony were seropositive for the FELASA- [37] listed viral agents MHV (judged by histology to be enteropathic strains), Adenovirus II, and TMEV, none of which are primary respiratory pathogens [38]. Intestinal flagellates, pinworms, and the opportunist respiratory pathogen Pasteurella pneumotropica were also common isolates. A number of pathogens known to cause respiratory disease and/or OM in the mouse such as pneumonia virus, Sendai virus, Mycoplasma pulmonis, Streptococcus pneumoniae, and Pseudomonas aeruginosa have not been found over many years in the sentinel screens. Non-FELASA-listed bacteria isolated from the nasopharynx of sentinel mice include Staphylococcus spp., Staphylococcus aureus, Alpha-haemolytic streptococci, and other Streptococcus spp. Junbo mice are now housed in a high- health-status SPF unit in which all FELASA-listed pathogens have been excluded. Non-FELASA nasopharyngeal flora remains the same. + +Genetic crosses and mapping. + +The founder C3HeNBALB/cENUF1-Jbo/+ mouse was maintained by repeated backcrossing to C3H/HeN. Mutant progeny were identified by the lack of a Preyer reflex. 41 DNAs were initially pooled from affected individuals and a genome scan was carried out as described [9,13]. A high-resolution genetic map was constructed using 242 affected progeny N4 and N5 backcross progeny using published microsatellite markers D3Mit90, D3Mit328, D3Mit92, D3Mit203, D3Mit55, D3Mit178, D3Mit151, D3Mit273, D3Mit180, D3Mit239, D3Mit21, D3Mit224, D3Mit182, D3Mit119, D3Mit241, and D3Mit22. The genetic map across the Jbo locus was constructed by minimizing the number of recombination events across the region. + +The Evi1tm1Mmor mutation was generated in D3 ES cells with subsequent matings to CF-1 mice [14]. Evi1tm1Mmor/+ mice were mated to C3H/HeN mice before crossing to Jbo/+ mice for complementation testing. For phenotypic analysis Evi1tm1Mmor/+ mice were also mated to C3H/HeN mice and F1 heterozygous mutant progeny along with wild-type littermate controls aged to appropriate time points. + +Pathology, histology, and X-Ray analysis. + +The onset and time course of development of the middle ear disease was examined by histology in conventionally housed postnatal mice 4–5 DAB (five Jbo/+, two +/+); postnatal 13 DAB (13 Jbo/+, 16 +/+); at weaning 21 DAB (seven Jbo/+, 17 +/+); and in adult mice 29 DAB (five Jbo/+, 11 +/+), 44 DAB (six Jbo/+, eight +/+), 180 DAB (seven Jbo/+, nine +/+), and 180–360 DAB (six Jbo/+, three +/+). Representative mice from one to three litters were examined at each time point. In a second study to assess the OM phenotype under SPF conditions, Junbo mice were sampled from an embryo re-derived colony that was housed in individually ventilated cages. The cohorts were as follows: 5 DAB (four Jbo/+, nine +/+), 13 DAB (11 Jbo/+, 19 +/+), 20–22 DAB, 28 DAB (24 Jbo/+, 17 +/+), 49–58 DAB (20 Jbo/+, 18 +/+), and 85–115 DAB (19 Jbo/+). 12 conventionally housed 68-DAB Evi1tm1Mmor/+ mice and 15 same-aged wild-type littermates were also examined for OM. + +Tissues were fixed 24–48 h in 10% neutral buffered formalin (NBF) and embedded in paraffin wax. Heads and bones were decalcified 24–48 h with Immunocal (Decal Corporation, Tallman, New York, United States). 4-μm dorsal plane sections of decalcified middle ears were stained with Haemotoxylin and Eosin (H & E). In 13- and 21-DAB mice with OM, transverse sections of the nasal turbinates, dorsal plane sections of the oropharynx, lungs, livers, kidneys, heart, spleen, intestines, pancreas, and livers were examined by histology. Sections of middle ears, snouts, and lungs from OM cases were examined for bacteria using Gram stain and Sirius red for eosinophil leukocytes. + +To assess the possibility of opportunistic infections at other body sites, a general pathology screen was performed on four 28-DAB and seven 56-DAB SPF Jbo/+ mice. In addition, a more extensive whole-body pathology analysis of 25+ tissues following EMPReSS necropsy SOPs [39] was performed on four female and three male 180-DAB Jbo/+ mice; five female and four male wild-type litter mates were used as controls. + +For X-ray analysis, heads were skinned and the brains and mandibles removed, and then the skull was fixed in 10% NBF. After the dorsoventral view was taken, the head was bisected midsagittally and lateromedial views taken. Images were taken on a MX-20 Faxitron X-ray machine (Faxitron X-ray Corporation, Wheeling, Illinois, United States) at 26 kV and 0.3 mA with an exposure time of 3 s. + +Immunohistochemistry. + +Whole embryos (E9.5, E10.5, E11.5, E12.5, E13.5, E16.5, and E18.5) and adult heads (13 DAB and 21 DAB) from wild-type and Jbo/+ mice were used for immunolabelling. 3-μm wax sections were de-paraffinised in xylene substitute and rehydrated through graded ethanol solutions. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in isopropanol. The sections were microwaved in 10 mM citrate buffer (pH6.0) and rinsed with phosphate-buffered saline at room temperature. The immunostaining was performed using a Dako (Glostrup, Denmark) autostainer at room temperature. To inhibit the non-specific endogenous biotin staining the Dako Biotin Blocking System was used. A blocking solution of 10% donkey serum (Serotec) was used for 1 h. Goat polyclonal antibody raised against the carboxy terminus of human EVI1 was used in concentration 1:100 (Santa Cruz Biotechnology, Santa Cruz, California, United States) for 1 h. Biotinylated donkey anti-goat antibody (Santa Cruz Biotechnology) and ChemMate Detection Kit (Dako) were used to develop the specific EVI-1 signals. Negative control sections were incubated in donkey serum and processed identically. The slides were counterstained with Haematoxylin. + +Skeletal preparations. + +Embryos were fixed in 95% ethanol and processed through a standard alcian blue and alizarin red bone/cartilage staining procedure. + +Immunology and FACS analysis in SPF mice. + +The T-dependent arm of the immune system was assessed by immunization with keyhole limpet hemocycanin and measurement of IgG1 and IgG2a, and the T-independent arm by immunization with pneumococcal polysaccharide type 3 and measurement of IgGM and IgG3 in 42–56-DAB SPF mice (ten Jbo/+, ten +/+) [40]. For FACS analysis whole blood was collected in lithium heparin tubes from the jugular vein after overdosing mice with barbiturate-administered IP. SPF mice were assessed at 20–22 DAB (17 Jbo/+, 20 +/+) and 49–58 DAB (14 Jbo/+, 14 +/+). FACS analysis of granulocytes was performed after labeling cells with R-PE-labeled Gr-1 and FITC-labeled Mac-1 markers. The Wilcoxon sum-of-ranks test was used to test for statistical differences between Jbo/+ and +/+ post-bleed antibody titers and granulocyte parameters. + +Supporting Information + +Table S1 + +T-Dependent and T-Independent Responses in Immune- Challenged Wild-Type and Junbo Mice + +(35 KB DOC) + +Click here for additional data file. + +Acknowledgements + +We thank Dr. Emma Coghill for advice on using the Amera software and Stuart Townsend for assistance with FACS analysis. We also thank the Histology and Pathology teams at Harwell, United Kingdom for processing of specimens. + +Abbreviations + +DAB - days after birth + +dpc - days postcoitum + +ENU - N-ethyl-N-nitrosourea + +MEC - middle ear cavity + +NTHi - nontypeable Haemophilus influenzae + +OM - otitis media + +SPF - specific pathogen-free + +Footnotes + +Competing interests. Part of this work was funded by GlaxoSmith-Kline. + +Author contributions. NP, REHH, HT, ND, AJH, MTC, and SDMB conceived and designed the experiments. NP, REHH, HT, HTT, DB, SM, ZL, FM, MF, PG, AMW, SP, IB, TAH, and MTC performed the experiments. NP, REHH, HT, HTT, DB, SM, ZL, FM, ND, and MTC analyzed the data. MF, PG, AMW, and SP contributed reagents/materials/analysis tools. NP, REHH, MTC, and SDMB wrote the paper. + +Funding. This work was supported by the Medical Research Council, United Kingdom. Hilda Tateossian is supported by the Eumorphia program (European Commission contract QPG2-CT-2002–00930). diff --git a/src/ontogpt/evaluation/craft/database/all/17069463.ann b/src/ontogpt/evaluation/craft/database/all/17069463.ann new file mode 100644 index 000000000..f2ae17a58 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17069463.ann @@ -0,0 +1,1424 @@ +T1 PR:000013712 0 6 RanBP2 +T2 GO:0065007 7 16 Modulates +T3 PR:000005770 17 22 Cox11 +T4 PR:000008608 27 39 Hexokinase I +T5 PR:000013712 77 83 RanBP2 +T6 CHEBI:17234 103 110 Glucose +T7 GO:0006006 103 121 Glucose Metabolism +T8 PR:000013712 137 158 Ran-binding protein 2 +T9 PR:000013712 160 166 RanBP2 +T10 SO:0000417 308 315 modules +T11 PR:000013712 319 325 RanBP2 +T12 PR:000013712 397 403 RanBP2 +T13 SO:0000704 412 419 genetic +T14 PR:000013712 449 455 RanBP2 +T15 NCBITaxon:33208 467 473 animal +T16 PR:000013712 556 562 RanBP2 +T17 PR:000013712 597 603 RanBP2 +T18 NCBITaxon:10088 609 614 mouse +T19 PR:000013712 622 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1438 RanBP2 +T49 PR:000008608 1491 1494 HKI +T50 UBERON:0001017 1529 1551 central nervous system +T51 PR:000013712 1560 1566 RanBP2 +T52 NCBITaxon:10088 1570 1574 mice +T53 CHEBI:17234 1617 1624 glucose +T54 GO:0006007 1617 1635 glucose catabolism +T55 CHEBI:17234 1658 1665 glucose +T56 GO:0046323 1658 1672 glucose uptake +T57 GO:0006094 1677 1692 gluconeogenesis +T58 CL:0000210 1782 1794;1814 1821 photosensory ... neurons +T59 PR:000013712 1830 1836 RanBP2 +T60 CL:0000540 1887 1895 neuronal +T61 PR:000008608 1896 1899 HKI +T62 CHEBI:17234 1901 1908 glucose +T63 GO:0006007 1901 1919 glucose catabolism +T64 GO:0097009 1921 1939 energy homeostasis +T65 GO:0008152 1957 1966 metabolic +T66 GO:0007568 1968 1973 aging +T67 http://purl.obolibrary.org/obo/MONDO_0000001 1974 1983 disorders +T68 http://purl.obolibrary.org/obo/MONDO_0005244 1995 2007 neuropathies +T69 PR:000013712 2024 2045 Ran-binding protein 2 +T70 PR:000013712 2047 2053 RanBP2 +T71 SO:0000417 2087 2094 domains +T72 SO:0000417 2168 2175 domains +T73 PR:000013712 2179 2185 RanBP2 +T74 SO:0000417 2246 2252 domain +T75 PR:000013712 2307 2313 RanBP2 +T76 GO:0006412 2344 2357;2369 2377 production of ... proteins +T77 GO:0051223 2379 2409 control of protein trafficking +T78 GO:0005634 2422 2429 nuclear +T79 GO:0005829 2434 2441 cytosol +T80 GO:0065007 2460 2467 control +T81 GO:0051301 2498 2511 cell division +T82 SO:0000704 2524 2531 genetic +T83 PR:000013712 2591 2597 RanBP2 +T84 PR:000013712 2638 2644 RanBP2 +T85 NCBITaxon:33208 2656 2662 animal +T86 GO:0005739 2736 2749 mitochondrial +T87 PR:000013712 2772 2778 RanBP2 +T88 PR:000005770 2780 2785 Cox11 +T89 PR:000008608 2790 2807 hexokinase type I +T90 PR:000008608 2809 2812 HKI +T91 PR:000013712 2871 2877 RanBP2 +T92 PR:000005770 2892 2897 Cox11 +T93 PR:000008608 2899 2902 HKI +T94 PR:000013712 2958 2964 RanBP2 +T95 PR:000008608 3000 3003 HKI +T96 PR:000005770 3008 3013 Cox11 +T97 PR:000013712 3015 3021 RanBP2 +T98 PR:000005770 3026 3031 Cox11 +T99 GO:0065007 3043 3051 modulate +T100 PR:000008608 3052 3055 HKI +T101 PR:000013712 3104 3110 RanBP2 +T102 NCBITaxon:10088 3116 3121 mouse +T103 GO:0006007 3165 3185 breakdown of glucose +T104 CHEBI:17234 3178 3185 glucose +T105 PR:000008608 3219 3222 HKI +T106 UBERON:0001017 3257 3279 central nervous system +T107 GO:0007601 3293 3299 visual +T108 PR:000013712 3352 3358 RanBP2 +T109 GO:0008152 3379 3398 metabolic processes +T110 http://purl.obolibrary.org/obo/MONDO_0000001 3410 3417 disease +T111 PR:000013712 3445 3451 RanBP2 +T112 PR:000013712 3452 3458 Nup358 +T113 NCBITaxon:7742 3471 3481 vertebrate +T114 SO:0000417 3557 3564 domains +T115 PR:000013712 3589 3595 RanBP2 +T116 PR:000013712 3624 3630 RanBP2 +T117 GO:0006913 3634 3663 nucleocytoplasmic trafficking +T118 GO:0006412 3671 3689 protein biogenesis +T119 GO:0090307 3701 3713;3718 3733 formation of ... mitotic spindle +T120 GO:0072686 3718 3733 mitotic spindle +T121 GO:0005635 3751 3767 nuclear envelope +T122 GO:0005635 3800 3816 nuclear envelope +T123 GO:0051081 3800 3826 nuclear envelope breakdown +T124 GO:0000776 3832 3843 kinetochore +T125 GO:0051382 3832 3853 kinetochore formation +T126 GO:0007067 3882 3889 mitotic +T127 PR:000013712 3935 3941 RanBP2 +T128 PR:000013712 4011 4017 RanBP2 +T129 UBERON:0000479 4120 4126 tissue +T130 PR:000013712 4153 4159 RanBP2 +T131 PR:000013712 4227 4233 RanBP2 +T132 PR:000013708 4252 4255 Ran +T133 SO:0000417 4264 4271 domains +T134 PR:000013712 4284 4290 RanBP2 +T135 GO:0005634 4310 4317 nuclear +T136 GO:0051170 4310 4324 nuclear import +T137 GO:0042571 4362 4372 antibodies +T138 PR:000013712 4381 4387 RanBP2 +T139 GO:0005634 4400 4407 nuclear +T140 GO:0051170 4400 4414 nuclear import +T141 NCBITaxon:8353 4479 4486 Xenopus +T142 CL:0000023 4487 4494 oocytes +T143 SO:0000417 4548 4555 domains +T144 PR:000013712 4573 4579 RanBP2 +T145 PR:000001244 4643 4646;4653 4658 red ... opsin +T146 PR:000001224 4647 4658 green opsin +T147 GO:0010467 4660 4669 expressed +T148 CL:0000210 4673 4693 photosensory neurons +T149 PR:000001119 4707 4712 opsin +T150 PR:000013712 4778 4784 RanBP2 +T151 GO:0005874 4819 4830 microtubule +T152 PR:000009317 4867 4872 KIF5B +T153 PR:000009318 4877 4882 KIF5C +T154 UBERON:0001017 4910 4932 central nervous system +T155 UBERON:0001017 4934 4937 CNS +T156 SO:0000417 5041 5047 domain +T157 PR:000013712 5051 5057 RanBP2 +T158 PR:000013712 5114 5120 RanBP2 +T159 SO:0000417 5156 5162 domain +T160 SO:0000417 5228 5235 domains +T161 PR:000013712 5239 5245 RanBP2 +T162 GO:0008541 5296 5302;5307 5317 cap of ... proteasome +T163 PR:000015829 5331 5337 SUMO-1 +T164 MOP:0000779 5338 5349 conjugating +T165 PR:000016977 5358 5362 Ubc9 +T166 GO:0005634 5378 5385 nuclear +T167 GO:0051168 5378 5392 nuclear export +T168 PR:000017497 5403 5407 CRM1 +T169 PR:000017497 5408 5418 exportin-1 +T170 PR:000013712 5439 5445 RanBP2 +T171 PR:000015829 5479 5484 SUMO1 +T172 PR:000013712 5543 5549 RanBP2 +T173 PR:000015829 5559 5565 SUMO-1 +T174 PR:000015829 5607 5613 SUMO-1 +T175 GO:0005829 5623 5632 cytosolic +T176 PR:000013717 5651 5657 RanGAP +T177 GO:1990876 5666 5683;5688 5708 cytosolic face of ... nuclear pore complex +T178 GO:1990876 5666 5683;5710 5713 cytosolic face of ... NPC +T179 PR:000013712 5730 5736 RanBP2 +T180 GO:0005739 5783 5795 mitochondria +T181 CL:0000210 5838 5858 photosensory neurons +T182 PR:000013708 5871 5880 RanGTPase +T183 GO:0005737 5903 5914 cytoplasmic +T184 GO:0005737 5963 5974 cytoplasmic +T185 GO:0005643 5998 6011 nuclear pores +T186 UBERON:0001017 6065 6068 CNS +T187 PR:000013712 6090 6096 RanBP2 +T188 http://purl.obolibrary.org/obo/MONDO_0005244 6143 6155 neuropathies +T189 PR:000012285 6157 6163 Parkin +T190 GO:0010467 6180 6189 expressed +T191 PR:000016977 6213 6217 Ubc9 +T192 SO:0000417 6230 6236 domain +T193 PR:000013712 6240 6246 RanBP2 +T194 GO:0016567 6271 6285 ubiquitination +T195 PR:000013712 6305 6311 RanBP2 +T196 SO:0000417 6358 6364 domain +T197 PR:000013712 6368 6374 RanBP2 +T198 GO:0008541 6388 6391 cap +T199 GO:0000502 6408 6418 proteasome +T200 PR:000013709 6456 6477 Ran-binding protein 1 +T201 PR:000013709 6479 6485 RanBP1 +T202 PR:P41920 6488 6493 Yrb1p +T203 SO:0000857 6507 6515 homology +T204 PR:000013708 6523 6526 Ran +T205 SO:0000417 6535 6542 domains +T206 PR:000013712 6546 6552 RanBP2 +T207 GO:0007049 6575 6585 cell-cycle +T208 GO:0065007 6586 6595 regulated +T209 GO:0030163 6596 6615 protein degradation +T210 PR:000012285 6622 6628 Parkin +T211 PR:000013712 6635 6641 RanBP2 +T212 PR:000012285 6704 6710 parkin +T213 http://purl.obolibrary.org/obo/MONDO_0017279 6720 6731;6761 6773 early onset Parkinsonism +T214 GO:0030849 6732 6741 autosomal +T215 http://purl.obolibrary.org/obo/MONDO_0000828 6752 6773 juvenile Parkinsonism +T216 CHEBI:18243 6790 6802 dopaminergic +T217 CL:0000700 6790 6811 dopaminergic neuronal +T218 PR:000012285 6847 6853 parkin +T219 PR:000012285 6879 6885 parkin +T220 NCBITaxon:10088 6889 6893 mice +T221 GO:0005739 6902 6915 mitochondrial +T222 PR:000012285 6978 6984 parkin +T223 GO:0032473 7002 7021;7026 7038 cytoplasmic face of ... mitochondria +T224 GO:0005739 7126 7139 mitochondrial +T225 PR:000013712 7236 7242 RanBP2 +T226 PR:000012285 7247 7253 parkin +T227 PR:000013712 7330 7336 RanBP2 +T228 PR:000013712 7361 7367 RanBP2 +T229 NCBITaxon:33208 7371 7377 animal +T230 PR:000013712 7491 7497 RanBP2 +T231 PR:000005770 7499 7504 Cox11 +T232 PR:000008608 7509 7526 hexokinase type I +T233 PR:000008608 7528 7531 HKI +T234 SO:0000910 7566 7572 orphan +T235 SO:0000417 7573 7579 domain +T236 PR:000013712 7583 7589 RanBP2 +T237 SO:0000417 7608 7614 domain +T238 PR:000013712 7671 7677 RanBP2 +T239 PR:000008608 7681 7684 HKI +T240 NCBITaxon:33208 7686 7692 animal +T241 CHEBI:17234 7709 7716 glucose +T242 GO:0006006 7709 7716;7724 7734 glucose ... metabolism +T243 PR:000013712 7756 7762 RanBP2 +T244 PR:000005770 7778 7783 Cox11 +T245 PR:000008608 7788 7791 HKI +T246 PR:000013712 7803 7809 RanBP2 +T247 SO:0000910 7837 7843 orphan +T248 SO:0000417 7844 7850 domain +T249 UBERON:0000955 7949 7954 Brain +T250 UBERON:0000966 7959 7965 retina +T251 PR:000005770 8016 8021 Cox11 +T252 SO:0000417 8058 8064 domain +T253 PR:000005770 8085 8090 Cox11 +T254 PR:000002199 8127 8139 cytochrome c +T255 CHEBI:18070 8127 8139 cytochrome c +T256 PR:Q6P8I6 8221 8227 mCox11 +T257 PR:000013712 8235 8241 RanBP2 +T258 PR:000005770 8368 8373 Cox11 +T259 UBERON:0000966 8416 8423 retinal +T260 CHEBI:16856 8438 8449 glutathione +T261 CHEBI:8984 8488 8510 sodium dodecyl sulfate +T262 SO:0001060 8527 8534 isoform +T263 PR:000005770 8538 8543 Cox11 +T264 PR:000005770 8623 8628 Cox11 +T265 GO:0005739 8655 8668 mitochondrial +T266 GO:0019867 8692 8706 outer membrane +T267 PR:000008608 8727 8730 HKI +T268 PR:000013712 8815 8821 RanBP2 +T269 PR:000008608 8871 8874 HKI +T270 SO:0001060 8875 8882 isoform +T271 PR:000008609 8892 8896 HKII +T272 PR:000008610 8898 8903 HKIII +T273 PR:000013712 8953 8959 RanBP2 +T274 PR:000005770 8999 9004 Cox11 +T275 PR:000008608 9006 9009 HKI +T276 PR:000013712 9027 9033 RanBP2 +T277 UBERON:0000966 9054 9061 retinal +T278 GO:0042571 9078 9088 antibodies +T279 PR:000013712 9107 9113 RanBP2 +T280 PR:000013712 9135 9141 RanBP2 +T281 PR:000008608 9158 9161 HKI +T282 UBERON:0000479 9243 9250 tissues +T283 PR:000013712 9270 9276 RanBP2 +T284 PR:000013712 9360 9366 RanBP2 +T285 PR:000005770 9371 9376 Cox11 +T286 GO:0006457 9428 9435 folding +T287 PR:000005770 9447 9452 Cox11 +T288 PR:000005770 9525 9530 Cox11 +T289 CHEBI:16199 9577 9581 urea +T290 PR:000005770 9608 9613 Cox11 +T291 PR:000005770 9652 9657 Cox11 +T292 PR:000005770 9788 9793 Cox11 +T293 PR:000005770 9827 9832 Cox11 +T294 GO:0010467 9847 9857 expression +T295 CHEBI:23414 9877 9882 CuSO4 +T296 CHEBI:26348 9886 9902 prosthetic group +T297 PR:000005770 9920 9925 Cox11 +T298 PR:000013712 9994 10000 RanBP2 +T299 CHEBI:23414 10025 10030 CuSO4 +T300 PR:000013712 10065 10071 RanBP2 +T301 PR:000005770 10087 10092 Cox11 +T302 PR:000008608 10097 10100 HKI +T303 PR:000013712 10127 10133 RanBP2 +T304 SO:0000417 10164 10171 domains +T305 PR:000013712 10194 10200 RanBP2 +T306 NCBITaxon:39107 10243 10249 murine +T307 PR:000005770 10260 10265 Cox11 +T308 PR:000005770 10277 10282 Cox11 +T309 SO:0000417 10301 10308 domains +T310 GO:0005739 10373 10386 mitochondrial +T311 MOP:0000780 10387 10395 cleavage +T312 GO:0016020 10405 10413 membrane +T313 SO:0001077 10405 10429 membrane-spanning domain +T314 PR:000005770 10512 10517 Cox11 +T315 PR:000005770 10524 10529 Cox11 +T316 PR:000013712 10633 10639 RanBP2 +T317 PR:000005770 10644 10649 Cox11 +T318 PR:000005770 10690 10695 Cox11 +T319 PR:000005770 10771 10776 Cox11 +T320 GO:0005829 10802 10811 cytosolic +T321 PR:000005770 10824 10829 Cox11 +T322 GO:0005758 10886 10913 mitochondrial intermembrane +T323 SO:0001075 10900 10920 intermembrane domain +T324 PR:000005770 10924 10929 Cox11 +T325 PR:000005770 10931 10936 Cox11 +T326 SO:0000417 11084 11090 domain +T327 PR:000013712 11094 11100 RanBP2 +T328 PR:000013712 11301 11307 RanBP2 +T329 SO:0000417 11331 11337 domain +T330 UBERON:0000966 11342 11349 retinal +T331 SO:0000417 11395 11401 domain +T332 PR:000013712 11405 11411 RanBP2 +T333 PR:000005770 11428 11433 Cox11 +T334 PR:000008608 11458 11461 HKI +T335 PR:000013712 11516 11522 RanBP2 +T336 GO:0042571 11528 11538 antibodies +T337 PR:000013712 11581 11587 RanBP2 +T338 GO:0032991 11596 11603 complex +T339 PR:000008608 11617 11620 HKI +T340 PR:000005770 11659 11664 Cox11 +T341 GO:0042571 11750 11760 antibodies +T342 PR:000013712 11773 11779 RanBP2 +T343 SO:0000417 11780 11787 domains +T344 PR:000008608 11868 11871 HKI +T345 GO:0042571 11877 11887 antibodies +T346 PR:000013712 11896 11902 RanBP2 +T347 PR:000005770 12100 12105 Cox11 +T348 CHEBI:16199 12200 12204 urea +T349 GO:0006457 12206 12213 Folding +T350 PR:000005770 12245 12250 Cox11 +T351 PR:000013712 12305 12311 RanBP2 +T352 PR:000005770 12378 12383 Cox11 +T353 GO:0010467 12384 12393 expressed +T354 CHEBI:23414 12451 12456 CuSO4 +T355 SO:0001060 12469 12476 isoform +T356 PR:000013712 12544 12550 RanBP2 +T357 SO:0000417 12569 12575 domain +T358 SO:0000417 12596 12602 domain +T359 PR:000013708 12612 12615 Ran +T360 SO:0000417 12624 12631 domains +T361 SO:0000417 12662 12668 domain +T362 PR:000009317 12684 12689 KIF5B +T363 PR:000009318 12690 12695 KIF5C +T364 SO:0000417 12705 12711 domain +T365 SO:0000417 12735 12741 domain +T366 SO:0000417 12763 12769 domain +T367 SO:0000417 12787 12793 domain +T368 PR:000005770 12796 12802 Cox 11 +T369 PR:000008608 12812 12815 HKI +T370 PR:000013712 12839 12845 RanBP2 +T371 PR:000005770 12873 12878 Cox11 +T372 PR:000008608 12884 12887 HKI +T373 PR:000005770 12925 12930 Cox11 +T374 PR:000008608 12935 12938 HKI +T375 PR:000013712 12954 12960 RanBP2 +T376 GO:0065007 12961 12970 modulates +T377 PR:000008608 12997 13000 HKI +T378 PR:000005770 13063 13068 Cox11 +T379 PR:000008608 13093 13096 HKI +T380 PR:000005770 13129 13134 Cox11 +T381 PR:000008608 13153 13156 HKI +T382 PR:000005770 13221 13226 Cox11 +T383 PR:000008608 13228 13231 HKI +T384 PR:000005770 13276 13281 Cox11 +T385 CHEBI:35222 13318 13327 inhibitor +T386 PR:000008608 13331 13334 HKI +T387 PR:000008608 13360 13363 HKI +T388 CHEBI:17234 13368 13375 glucose +T389 PR:000013712 13432 13438 RanBP2 +T390 PR:000005770 13513 13518 Cox11 +T391 CHEBI:17234 13548 13555 glucose +T392 SO:0000417 13634 13640 domain +T393 PR:000005770 13683 13688 Cox11 +T394 PR:000008608 13692 13695 HKI +T395 PR:000008608 13859 13862 HKI +T396 PR:000005770 13890 13895 Cox11 +T397 PR:000008608 13964 13967 HKI +T398 PR:000008608 14054 14057 HKI +T399 PR:000005770 14125 14130 Cox11 +T400 PR:000013712 14135 14141 RanBP2 +T401 PR:000008608 14145 14148 HKI +T402 CHEBI:17234 14196 14203 glucose +T403 PR:000008608 14207 14210 HKI +T404 PR:000005770 14268 14273 Cox11 +T405 PR:000008608 14340 14343 HKI +T406 PR:000005770 14388 14393 Cox11 +T407 PR:000008608 14409 14412 HKI +T408 PR:000005770 14463 14468 Cox11 +T409 CHEBI:17234 14532 14539 glucose +T410 PR:000005770 14596 14601 Cox11 +T411 PR:000005770 14709 14714 Cox11 +T412 CHEBI:35222 14743 14752 inhibitor +T413 PR:000008608 14756 14759 HKI +T414 PR:000008608 14784 14787 HKI +T415 CHEBI:17234 14810 14817 glucose +T416 PR:000008608 14824 14827 HKI +T417 CHEBI:17234 14892 14899 glucose +T418 PR:000005770 14910 14915 Cox11 +T419 PR:000013712 14990 14996 RanBP2 +T420 PR:000008608 15023 15026 HKI +T421 PR:000005770 15039 15044 Cox11 +T422 PR:000013712 15081 15087 RanBP2 +T423 PR:000008608 15091 15094 HKI +T424 PR:000005770 15133 15138 Cox11 +T425 CHEBI:17234 15207 15214 glucose +T426 PR:000013712 15261 15267 RanBP2 +T427 PR:000013712 15312 15318 RanBP2 +T428 PR:000008608 15334 15337 HKI +T429 CHEBI:17234 15385 15392 glucose +T430 PR:000013712 15395 15401 RanBP2 +T431 PR:000008608 15419 15422 HK1 +T432 PR:000005770 15424 15429 Cox11 +T433 UBERON:0000966 15449 15455 Retina +T434 CL:0000540 15469 15476 Neurons +T435 GO:0005643 15525 15529 NPCs +T436 PR:000013712 15561 15567 RanBP2 +T437 GO:0005739 15604 15616 mitochondria +T438 CL:0010009 15659 15671;15688 15698;15703 15709 photosensory ... neurons of ... retina +T439 CL:0000210 15673 15686 photoreceptor +T440 UBERON:0000966 15703 15709 retina +T441 PR:000013712 15770 15776 RanBP2 +T442 PR:000013712 15839 15845 RanBP2 +T443 PR:000005770 15847 15852 Cox11 +T444 PR:000008608 15854 15857 HKI +T445 CL:0002608 15884 15903 hippocampal neurons +T446 UBERON:0000956 15920 15935 cerebral cortex +T447 CL:0010012 15920 15943 cerebral cortex neurons +T448 GO:0005739 15971 15983 mitochondria +T449 CL:0010009 16018 16041;16046 16052 photosensory neurons of ... retina +T450 UBERON:0000966 16046 16052 retina +T451 CL:0000125 16093 16097 glia +T452 CL:0000540 16102 16108 neuron +T453 UBERON:0000955 16127 16132 brain +T454 GO:0042571 16176 16186 antibodies +T455 GO:0005739 16245 16257 mitochondria +T456 PR:000013712 16301 16307 RanBP2 +T457 CL:0000540 16401 16408 neurons +T458 UBERON:0000956 16414 16429 cerebral cortex +T459 PR:000008608 16467 16470 HKI +T460 PR:000013712 16482 16488 RanBP2 +T461 PR:000013712 16553 16559 RanBP2 +T462 PR:000008608 16564 16567 HKI +T463 GO:0010467 16579 16588 expressed +T464 CL:0002608 16613 16632 hippocampal neurons +T465 PR:000008608 16638 16641 HKI +T466 GO:0010467 16642 16652 expression +T467 PR:000013712 16675 16681 RanBP2 +T468 UBERON:0001851 16711 16719 cortical +T469 CL:0010012 16711 16727 cortical neurons +T470 CL:0000099 16736 16748 interneurons +T471 NCBITaxon:27592 16786 16792 bovine +T472 UBERON:0000966 16793 16800 retinal +T473 GO:0005634 16837 16844 nuclear +T474 CL:0000210 16854 16875 photoreceptor neurons +T475 GO:0001917 16886 16891;16914 16921 inner ... segment +T476 PR:000013712 16981 16987 RanBP2 +T477 PR:000005770 17008 17013 Cox11 +T478 PR:000008608 17019 17022 HKI +T479 PR:000005770 17031 17036 Cox11 +T480 PR:000013712 17110 17116 RanBP2 +T481 PR:000005770 17130 17135 Cox11 +T482 GO:0005739 17143 17155 mitochondria +T483 CL:0000210 17186 17200 photoreceptors +T484 PR:000013712 17227 17233 RanBP2 +T485 PR:000005770 17238 17243 Cox11 +T486 PR:000008608 17273 17276 HKI +T487 PR:000005770 17297 17302 Cox11 +T488 UBERON:0001893 17397 17405 cerebral +T489 CL:0000540 17406 17413 neurons +T490 CL:0000125 17418 17429 glial cells +T491 PR:000008608 17467 17470 HKI +T492 PR:000005770 17475 17480 Cox11 +T493 PR:000013712 17516 17522 RanBP2 +T494 PR:000008608 17552 17555 HKI +T495 UBERON:0001789 17638 17641 ONL +T496 UBERON:0001789 17643 17662 outer nuclear layer +T497 GO:0005634 17649 17656 nuclear +T498 PR:000013712 17687 17693 RanBP2 +T499 PR:000008608 17721 17724 HKI +T500 PR:000008608 17756 17759 HKI +T501 PR:000013712 17832 17838 RanBP2 +T502 PR:000008608 17843 17846 HKI +T503 NCBITaxon:39107 17883 17889 murine +T504 UBERON:0000922 17890 17899 embryonic +T505 CL:0002322 17890 17904;17912 17916 embryonic stem ... cell +T506 PR:000013712 17938 17944 RanBP2 +T507 SO:0000704 17963 17967 gene +T508 PR:000013712 18118 18124 RanBP2 +T509 PR:000013712 18128 18134 RanBP2 +T510 PR:000013712 18138 18144 RanBP2 +T511 PR:000013712 18242 18248 RanBP2 +T512 NCBITaxon:10088 18252 18256 mice +T513 UBERON:0000922 18262 18275 embryonically +T514 UBERON:0000922 18290 18297 embryos +T515 GO:0010467 18310 18320 expression +T516 PR:000013712 18324 18330 RanBP2 +T517 UBERON:0004128 18338 18351 optic vesicle +T518 UBERON:0000922 18379 18385 embryo +T519 PR:000013712 18516 18522 RanBP2 +T520 SO:0000704 18523 18527 gene +T521 GO:0010467 18531 18540 expressed +T522 UBERON:0000966 18555 18562 retinal +T523 CL:0000540 18563 18570 neurons +T524 GO:0010467 18576 18586 expression +T525 UBERON:0000045 18590 18598 ganglion +T526 CL:0000740 18590 18607;18618 18624 ganglion cells of ... retina +T527 UBERON:0007023 18612 18617 adult +T528 UBERON:0000966 18618 18624 retina +T529 NCBITaxon:39107 18706 18712 Murine +T530 PR:000013712 18713 18719 RanBP2 +T531 SO:0000704 18720 18724 Gene +T532 SO:0001026 18745 18752 genomic +T533 PR:000013712 18763 18769 RanBP2 +T534 SO:0000879 18817 18828 bicistronic +T535 SO:0000440 18838 18844 vector +T536 SO:0000147 18853 18857 exon +T537 SO:0000079 18871 18893 bicistronic transcript +T538 GO:0065007 18922 18932 regulation +T539 PR:000013712 18936 18942 RanBP2 +T540 GO:0008380 18949 18957 splicing +T541 PR:000013712 18961 18967 RanBP2 +T542 SO:0000147 18986 18990 exon +T543 SO:0000704 19039 19044 genes +T544 NCBITaxon:9606 19066 19071 human +T545 UBERON:0001987 19072 19081 placental +T546 PR:000003969 19072 19101 placental alkaline phophatase +T547 PR:000003969 19103 19107 PLAP +T548 GO:0006412 19126 19136 translated +T549 SO:0000243 19147 19175 internal ribosome entry site +T550 GO:0005840 19156 19164 ribosome +T551 GO:0010467 19215 19225 expression +T552 GO:0044297 19255 19266 cell bodies +T553 GO:0010467 19274 19284 expression +T554 GO:0030424 19318 19334 axonal processes +T555 SO:0000147 19402 19406 exon +T556 PR:000013712 19450 19456 RanBP2 +T557 SO:0001026 19493 19500 genomic +T558 UBERON:0002415 19508 19513 tails +T559 NCBITaxon:10088 19520 19524 mice +T560 PR:P43870 19562 19569 HindIII +T561 SO:0001417 19603 19605;19640 19656 3′ ... flanking regions +T562 SO:0001416 19623 19625;19640 19656 5′ ... flanking regions +T563 SO:0001414 19664 19684 insertion breakpoint +T564 SO:0000156 19694 19700 cosmid +T565 PR:000013712 19716 19722 RanBP2 +T566 SO:0000704 19723 19727 gene +T567 SO:0000147 19734 19738 exon +T568 UBERON:0000922 19822 19828 embryo +T569 PR:000003969 19833 19837 PLAP +T570 PR:000003969 19885 19889 PLAP +T571 GO:0010467 19902 19911 expressed +T572 UBERON:0002329 19919 19926 somites +T573 UBERON:0002101 19928 19933 limbs +T574 UBERON:0001017 19939 19942 CNS +T575 PR:000003969 19949 19953 PLAP +T576 GO:0010467 19980 19990 expression +T577 UBERON:0004128 20020 20033 optic vesicle +T578 CHEBI:75055 20043 20048 X-gal +T579 PR:000003969 20087 20091 PLAP +T580 UBERON:0000966 20101 20108 retinal +T581 PR:000013712 20131 20137 RanBP2 +T582 NCBITaxon:10088 20141 20146 mouse +T583 UBERON:0001781 20236 20248 neuroretinal +T584 GO:0001917 20260 20273 inner segment +T585 CL:0000210 20289 20303 photoreceptors +T586 GO:0010467 20330 20340 expression +T587 UBERON:0000045 20344 20352 ganglion +T588 CL:0000740 20344 20358 ganglion cells +T589 PR:000003969 20360 20364 PLAP +T590 GO:0010467 20365 20375 expression +T591 GO:0045202 20410 20418 synaptic +T592 GO:0001750 20430 20461 outer segment of photoreceptors +T593 CL:0000210 20447 20461 photoreceptors +T594 CL:0000740 20467 20469 GC +T595 UBERON:0000045 20471 20479 ganglion +T596 CL:0000740 20471 20484 ganglion cell +T597 PR:000003969 20486 20490 PLAP +T598 NCBITaxon:9606 20492 20497 human +T599 UBERON:0001987 20498 20507 placental +T600 PR:000003969 20498 20527 placental alkaline phophatase +T601 CL:0000604 20534 20537 rod +T602 GO:0001750 20538 20551 outer segment +T603 CL:0000604 20558 20561 rod +T604 GO:0001917 20562 20575 inner segment +T605 UBERON:0001789 20577 20580 ONL +T606 UBERON:0001789 20582 20601 outer nuclear layer +T607 GO:0005634 20588 20595 nuclear +T608 GO:0045202 20625 20633 synaptic +T609 UBERON:0001791 20642 20645 INL +T610 UBERON:0001791 20647 20666 inner nuclear layer +T611 GO:0005634 20653 20660 nuclear +T612 GO:0045202 20690 20698 synaptic +T613 UBERON:0001792 20707 20709 GC +T614 CL:0000740 20711 20724 ganglion cell +T615 UBERON:0001792 20711 20730 ganglion cell layer +T616 PR:000013712 20772 20778 RanBP2 +T617 PR:000005770 20784 20789 Cox11 +T618 PR:000008608 20794 20797 HKI +T619 GO:0065007 20835 20845 modulation +T620 PR:000008608 20849 20852 HKI +T621 PR:000013712 20875 20881 RanBP2 +T622 PR:000005770 20886 20891 Cox11 +T623 PR:000008608 20929 20932 HKI +T624 GO:0006096 20971 20981 glycolysis +T625 PR:000013712 21001 21007 RanBP2 +T626 NCBITaxon:10088 21011 21015 mice +T627 PR:000008608 21042 21045 HKI +T628 PR:000005770 21047 21052 Cox11 +T629 GO:0097009 21058 21076 energy homeostasis +T630 SO:0001023 21082 21089 allelic +T631 GO:0010467 21090 21100 expression +T632 PR:000013712 21104 21110 RanBP2 +T633 GO:0005643 21141 21145 NPCs +T634 CL:0002608 21172 21191 hippocampal neurons +T635 GO:0010467 21254 21264 expression +T636 PR:000013712 21268 21274 RanBP2 +T637 UBERON:0001017 21299 21302 CNS +T638 UBERON:0000955 21304 21309 brain +T639 UBERON:0000966 21314 21320 retina +T640 PR:000013712 21452 21458 RanBP2 +T641 NCBITaxon:10088 21469 21474 mouse +T642 PR:000013712 21504 21510 RanBP2 +T643 UBERON:0000966 21533 21539 retina +T644 UBERON:0000955 21541 21546 brain +T645 PR:000013712 21567 21573 RanBP2 +T646 NCBITaxon:10088 21576 21580 mice +T647 PR:000011508 21648 21654 Nup153 +T648 PR:000011521 21659 21664 Nup62 +T649 PR:000005770 21681 21686 Cox11 +T650 PR:000008608 21798 21801 HKI +T651 UBERON:0001017 21872 21875 CNS +T652 PR:000008608 21883 21886 HKI +T653 UBERON:0004288 21929 21937 skeletal +T654 UBERON:0002106 21946 21952 spleen +T655 UBERON:0002107 21958 21963 liver +T656 PR:000008608 21985 21988 HKI +T657 PR:000008608 22062 22065 HKI +T658 SO:0001060 22091 22098 isoform +T659 GO:0010467 22099 22108 expressed +T660 UBERON:0001017 22116 22119 CNS +T661 PR:000008608 22153 22156 HKI +T662 PR:000013712 22161 22167 RanBP2 +T663 UBERON:0001017 22294 22297 CNS +T664 UBERON:0000955 22299 22304 brain +T665 UBERON:0000966 22309 22315 retina +T666 CL:0000540 22333 22341 neuronal +T667 UBERON:0000479 22342 22349 tissues +T668 PR:000008608 22417 22420 HKI +T669 PR:000005770 22433 22438 Cox11 +T670 GO:0005739 22460 22472 mitochondria +T671 CL:0000604 22528 22552 rod photosensory neurons +T672 PR:000008608 22608 22611 HKI +T673 UBERON:0000966 22632 22638 retina +T674 GO:0045202 22678 22686 synaptic +T675 PR:000013712 22745 22751 RanBP2 +T676 PR:000008608 22773 22776 HKI +T677 GO:0005643 22830 22834 NPCs +T678 CL:0002608 22850 22869 hippocampal neurons +T679 NCBITaxon:10088 22912 22916 mice +T680 GO:0005643 22982 22986 NPCs +T681 GO:0005643 22992 22995 NPC +T682 GO:0005635 23008 23024 nuclear envelope +T683 PR:000013712 23043 23049 RanBP2 +T684 PR:000013712 23057 23063 RanBP2 +T685 NCBITaxon:10088 23067 23071 mice +T686 PR:000013712 23100 23106 RanBP2 +T687 PR:000011508 23107 23113 Nup153 +T688 PR:000011521 23114 23119 Nup62 +T689 PR:000008608 23131 23134 HKI +T690 PR:000005770 23150 23155 Cox11 +T691 GO:0042571 23156 23166 antibodies +T692 UBERON:0000966 23170 23177 retinal +T693 NCBITaxon:10088 23233 23237 mice +T694 PR:000013712 23256 23262 RanBP2 +T695 PR:000013712 23267 23273 RanBP2 +T696 NCBITaxon:10088 23277 23281 mice +T697 GO:0010467 23309 23319 expression +T698 PR:000013712 23330 23336 RanBP2 +T699 PR:000008608 23341 23344 HKI +T700 GO:0010467 23419 23429 expression +T701 PR:000013712 23440 23446 RanBP2 +T702 PR:000005770 23448 23453 Cox11 +T703 PR:000008608 23455 23458 HKI +T704 PR:000013712 23493 23499 RanBP2 +T705 PR:000013712 23507 23513 RanBP2 +T706 NCBITaxon:10088 23516 23520 mice +T707 PR:000013712 23559 23565 RanBP2 +T708 PR:000008608 23570 23573 HKI +T709 NCBITaxon:10088 23590 23594 mice +T710 PR:000008608 23614 23617 HKI +T711 UBERON:0000955 23636 23641 brain +T712 CL:0000540 23663 23671 neuronal +T713 UBERON:0000479 23672 23679 tissues +T714 UBERON:0002106 23696 23702 spleen +T715 UBERON:0002107 23708 23713 liver +T716 UBERON:0001017 23759 23762 CNS +T717 UBERON:0000479 23763 23770 tissues +T718 UBERON:0000955 23772 23777 brain +T719 UBERON:0000966 23782 23788 retina +T720 CL:0000540 23805 23813 neuronal +T721 UBERON:0000479 23814 23821 tissues +T722 UBERON:0002106 23836 23842 spleen +T723 GO:0008152 23846 23855 Metabolic +T724 PR:000013712 23901 23907 RanBP2 +T725 PR:000013712 23936 23942 RanBP2 +T726 NCBITaxon:10088 23946 23950 mice +T727 PR:000013712 24010 24016 RanBP2 +T728 NCBITaxon:10088 24020 24024 mice +T729 PR:000013712 24071 24077 RanBP2 +T730 NCBITaxon:10088 24081 24085 mice +T731 NCBITaxon:10088 24148 24152 mice +T732 PR:000013712 24179 24185 RanBP2 +T733 NCBITaxon:10088 24196 24200 mice +T734 SO:0000704 24266 24273 genetic +T735 CHEBI:33290 24325 24329 Food +T736 GO:0007631 24325 24341 Food consumption +T737 PR:000013712 24423 24429 RanBP2 +T738 NCBITaxon:10088 24433 24437 Mice +T739 NCBITaxon:10088 24514 24518 mice +T740 PR:000013712 24520 24526 RanBP2 +T741 NCBITaxon:10088 24530 24534 mice +T742 PR:000013712 24677 24683 RanBP2 +T743 NCBITaxon:10088 24687 24691 mice +T744 NCBITaxon:10088 24735 24739 mice +T745 PR:000013712 24796 24802 RanBP2 +T746 NCBITaxon:10088 24805 24809 mice +T747 SO:0000704 24818 24825 genetic +T748 PR:000013712 24877 24883 RanBP2 +T749 PR:000013712 24891 24897 RanBP2 +T750 NCBITaxon:10088 24901 24905 mice +T751 SO:0000704 24960 24967 genetic +T752 PR:000013712 24985 24991 RanBP2 +T753 PR:000013712 24999 25005 RanBP2 +T754 NCBITaxon:10088 25015 25019 mice +T755 CHEBI:33290 25045 25049 food +T756 GO:0007631 25045 25061 food consumption +T757 NCBITaxon:10088 25063 25067 Mice +T758 GO:0007567 25126 25131 birth +T759 PR:000008608 25142 25145 HKI +T760 UBERON:0001017 25153 25156 CNS +T761 UBERON:0000955 25158 25163 brain +T762 UBERON:0000966 25168 25174 retina +T763 GO:0010467 25203 25213 expression +T764 CHEBI:17234 25233 25240 glucose +T765 UBERON:0001017 25260 25263 CNS +T766 CHEBI:17234 25283 25290 glucose +T767 UBERON:0001017 25336 25339 CNS +T768 UBERON:0001017 25369 25372 CNS +T769 CHEBI:17234 25379 25386 glucose +T770 UBERON:0001017 25449 25452 CNS +T771 UBERON:0001017 25482 25485 CNS +T772 CHEBI:17234 25525 25532 glucose +T773 PR:000013712 25597 25603 RanBP2 +T774 GO:0006094 25643 25652;25665 25675 formation ... of glucose +T775 GO:0046323 25658 25675 uptake of glucose +T776 CHEBI:17234 25668 25675 glucose +T777 NCBITaxon:10088 25737 25741 mice +T778 CHEBI:33290 25761 25765 chow +T779 PR:000013712 25800 25806 RanBP2 +T780 NCBITaxon:10088 25809 25813 mice +T781 CHEBI:17234 25883 25890 glucose +T782 PR:000013712 25976 25982 RanBP2 +T783 NCBITaxon:10088 25986 25990 mice +T784 CHEBI:17234 26014 26021 glucose +T785 PR:000013712 26061 26067 RanBP2 +T786 NCBITaxon:10088 26071 26075 mice +T787 CHEBI:17234 26125 26132 Glucose +T788 PR:000045358 26184 26191 insulin +T789 CHEBI:17234 26201 26208 glucose +T790 GO:0046323 26201 26215 glucose uptake +T791 PR:000013712 26253 26259 RanBP2 +T792 GO:0006094 26282 26297 gluconeogenesis +T793 GO:0006094 26348 26358;26373 26383 production ... of glucose +T794 CHEBI:17234 26376 26383 glucose +T795 GO:0006094 26424 26437 gluconeogenic +T796 CHEBI:15361 26459 26467 pyruvate +T797 CHEBI:15361 26469 26477 pyruvate +T798 CHEBI:17234 26534 26541 glucose +T799 GO:0006094 26534 26552 glucose production +T800 PR:000013712 26556 26562 RanBP2 +T801 NCBITaxon:10088 26566 26570 mice +T802 PR:000013712 26619 26625 RanBP2 +T803 GO:0006094 26647 26662 gluconeogenesis +T804 CHEBI:17234 26686 26693 glucose +T805 GO:0006094 26686 26704 glucose production +T806 CHEBI:17234 26738 26745 glucose +T807 PR:000013712 26781 26787 RanBP2 +T808 PR:000013712 26799 26805 RanBP2 +T809 NCBITaxon:10088 26809 26813 mice +T810 CHEBI:17234 26855 26862 glucose +T811 GO:0006007 26855 26872 glucose breakdown +T812 GO:0008152 26885 26894 Metabolic +T813 PR:000013712 26909 26915 RanBP2 +T814 NCBITaxon:10088 26926 26930 Mice +T815 PR:000013712 26969 26975 RanBP2 +T816 NCBITaxon:10088 26979 26983 mice +T817 CHEBI:17234 27004 27011 glucose +T818 CHEBI:17234 27033 27040 glucose +T819 PR:000013712 27108 27114 RanBP2 +T820 NCBITaxon:10088 27118 27122 mice +T821 CHEBI:17234 27160 27167 glucose +T822 CHEBI:17234 27189 27196 glucose +T823 PR:000013712 27257 27263 RanBP2 +T824 PR:000013712 27271 27277 RanBP2 +T825 NCBITaxon:10088 27281 27285 mice +T826 PR:000045358 27308 27315 insulin +T827 CHEBI:17234 27325 27332 glucose +T828 GO:0046323 27325 27339 glucose uptake +T829 PR:000045358 27354 27361 insulin +T830 CHEBI:15361 27391 27399 Pyruvate +T831 CHEBI:17234 27436 27443 glucose +T832 CHEBI:17234 27458 27465 glucose +T833 PR:000013712 27491 27497 RanBP2 +T834 PR:000013712 27505 27511 RanBP2 +T835 NCBITaxon:10088 27515 27519 mice +T836 PR:000013712 27552 27558 RanBP2 +T837 CL:0000006 27613 27623;27651 27658 Receptoral ... Neurons +T838 UBERON:0000966 27643 27650 Retinal +T839 GO:0010467 27686 27696 expression +T840 PR:000013712 27700 27706 RanBP2 +T841 PR:000008608 27711 27714 HKI +T842 UBERON:0000966 27718 27725 retinal +T843 CL:0000540 27726 27733 neurons +T844 CL:0000540 27770 27778 neuronal +T845 UBERON:0000966 27779 27785 retina +T846 UBERON:0000955 27791 27796 brain +T847 CHEBI:17234 27801 27808 glucose +T848 GO:0008152 27891 27900 metabolic +T849 http://purl.obolibrary.org/obo/MONDO_0005066 27891 27910 metabolic disorders +T850 http://purl.obolibrary.org/obo/MONDO_0005015 27920 27928 diabetes +T851 UBERON:0000966 27933 27940 retinal +T852 http://purl.obolibrary.org/obo/MONDO_0005266 27957 27977 diabetic retinopathy +T853 PR:000013712 28021 28027 RanBP2 +T854 PR:000008608 28029 28032 HKI +T855 CL:0000604 28096 28099;28110 28123 rod ... photoreceptor +T856 CL:0000573 28104 28108;28110 28123 cone ... photoreceptor +T857 UBERON:0000966 28141 28148 retinal +T858 CL:0000540 28149 28156 neurons +T859 PR:000013712 28160 28166 RanBP2 +T860 PR:000013712 28177 28183 RanBP2 +T861 NCBITaxon:10088 28187 28191 mice +T862 GO:1990603 28197 28205;28221 28230 scotopic ... responses +T863 GO:1990603 28207 28219;28221 28230 dark-adapted ... responses +T864 CL:0000604 28247 28264 rod photoreceptor +T865 CL:0000604 28311 28314 rod +T866 CL:0000573 28319 28323 cone +T867 PR:000013712 28392 28398 RanBP2 +T868 NCBITaxon:10088 28402 28406 mice +T869 GO:0036367 28443 28451;28468 28477 photopic ... responses +T870 GO:0036367 28453 28466;28468 28477 light-adapted ... responses +T871 CL:0000573 28492 28511 cone photoreceptors +T872 CL:0000210 28537 28557 photosensory neurons +T873 NCBITaxon:10088 28565 28570 mouse +T874 UBERON:0000966 28571 28577 retina +T875 GO:1990603 28746 28764 scotopic responses +T876 CL:0000006 28876 28894 receptoral neurons +T877 CL:0000540 28938 28944 neuron +T878 CL:0000210 29081 29094 photoreceptor +T879 CL:0000540 29124 29130 neuron +T880 CL:0000210 29162 29176 photoreceptors +T881 GO:0036367 29278 29292 light response +T882 CL:0000210 29301 29315 photoreceptors +T883 CL:0000540 29335 29342 neurons +T884 CHEBI:38867 29344 29355 Anesthetics +T885 CHEBI:6121 29370 29378 ketamine +T886 CHEBI:17234 29413 29420 glucose +T887 NCBITaxon:10088 29424 29428 mice +T888 PR:000013712 29567 29573 RanBP2 +T889 PR:000013712 29581 29587 RanBP2 +T890 CHEBI:17234 29618 29625 glucose +T891 CHEBI:17234 29715 29722 glucose +T892 CHEBI:17234 29802 29809 Glucose +T893 PR:000013712 29988 29994 RanBP2 +T894 PR:000013712 30001 30007 RanBP2 +T895 NCBITaxon:10088 30018 30022 Mice +T896 CL:0000210 30031 30044 Photoreceptor +T897 CL:0000540 30062 30068 Neuron +T898 GO:1990603 30115 30123;30139 30148 Scotopic ... responses +T899 GO:1990603 30125 30137;30139 30148 dark-adapted ... responses +T900 PR:000013712 30154 30160 RanBP2 +T901 NCBITaxon:10088 30164 30168 mice +T902 PR:000013712 30289 30295 RanBP2 +T903 NCBITaxon:10088 30299 30303 mice +T904 CL:0000604 30370 30396 rod photoreceptor neuronal +T905 CL:0000604 30477 30480 rod +T906 CL:0000573 30485 30489 cone +T907 GO:0036367 30505 30513;30558 30567 Photopic ... responses +T908 GO:0036367 30515 30528;30558 30567 light-adapted ... responses +T909 CL:0000573 30530 30548 cone photoreceptor +T910 PR:000013712 30571 30577 RanBP2 +T911 NCBITaxon:10088 30581 30585 mice +T912 PR:000013712 30691 30697 RanBP2 +T913 NCBITaxon:10088 30701 30705 mice +T914 GO:1990603 30733 30741 scotopic +T915 PR:000013712 30765 30771 RanBP2 +T916 PR:000013712 30794 30800 RanBP2 +T917 NCBITaxon:10088 30821 30825 mice +T918 CL:0000540 30854 30860 neuron +T919 CL:0000210 30967 30980 photoreceptor +T920 PR:000013712 30995 31001 RanBP2 +T921 PR:000013712 31009 31015 RanBP2 +T922 NCBITaxon:10088 31019 31023 mice +T923 PR:000013712 31084 31090 RanBP2 +T924 NCBITaxon:10088 31094 31098 mice +T925 PR:000013712 31444 31450 RanBP2 +T926 GO:0065007 31479 31489 modulating +T927 CHEBI:17234 31490 31497 glucose +T928 GO:0042593 31490 31497;31509 31520 glucose ... homeostasis +T929 GO:0097009 31502 31520 energy homeostasis +T930 PR:000005770 31544 31548 Cox1 +T931 PR:000008608 31553 31556 HKI +T932 PR:000013712 31604 31610 RanBP2 +T933 PR:000013712 31640 31646 RanBP2 +T934 GO:0006457 31682 31689 folding +T935 PR:000005770 31707 31712 Cox11 +T936 PR:000008608 31739 31742 HKI +T937 PR:000005770 31755 31760 Cox11 +T938 PR:000008608 31803 31806 HKI +T939 CHEBI:36357 31817 31826 molecules +T940 PR:000005770 31830 31835 Cox11 +T941 PR:000005770 31859 31864 Cox11 +T942 CHEBI:36357 31921 31929 molecule +T943 PR:000008608 31933 31936 HKI +T944 PR:000005770 31957 31962 Cox11 +T945 GO:0051235 31963 31973 sequesters +T946 PR:000008608 31974 31977 HKI +T947 PR:000008608 31992 31995 HKI +T948 SO:0100019 32032 32043 active site +T949 PR:000008608 32089 32092 HKI +T950 PR:000008608 32160 32163 HKI +T951 SO:0100019 32210 32221 active site +T952 PR:000008608 32225 32228 HKI +T953 CHEBI:17234 32236 32243 glucose +T954 PR:000005770 32291 32296 Cox11 +T955 PR:000008608 32302 32305 HKI +T956 PR:000013712 32323 32329 RanBP2 +T957 PR:000008608 32411 32414 HKI +T958 PR:000013712 32469 32475 RanBP2 +T959 PR:000008608 32499 32502 HKI +T960 PR:000005770 32506 32511 Cox11 +T961 PR:000005770 32596 32601 Cox11 +T962 PR:000008608 32626 32629 HKI +T963 PR:000013712 32633 32639 RanBP2 +T964 PR:000013712 32650 32656 RanBP2 +T965 CHEBI:35225 32678 32684 buffer +T966 PR:000008608 32691 32694 HK1 +T967 PR:000005770 32699 32704 Cox11 +T968 PR:000013712 32741 32747 RanBP2 +T969 PR:000013712 32770 32776 RanBP2 +T970 NCBITaxon:10088 32780 32784 mice +T971 GO:0051235 32815 32828 sequestration +T972 PR:000008608 32832 32835 HKI +T973 CL:0000210 32868 32888 photosensory neurons +T974 PR:000008608 32934 32937 HKI +T975 CL:0000540 32972 32979 neurons +T976 PR:000008608 33058 33061 HKI +T977 PR:000013712 33088 33094 RanBP2 +T978 PR:000008608 33162 33165 HKI +T979 PR:000008608 33204 33207 HKI +T980 GO:0005739 33235 33248 mitochondrial +T981 GO:0005643 33253 33256 NPC +T982 PR:000013712 33339 33345 RanBP2 +T983 PR:000005770 33393 33398 Cox11 +T984 PR:000008608 33400 33403 HK1 +T985 PR:000013712 33409 33415 RanBP2 +T986 PR:000005770 33472 33477 Cox11 +T987 PR:000008608 33483 33486 HKI +T988 PR:000008608 33538 33541 HKI +T989 PR:000013712 33547 33553 RanBP2 +T990 PR:000008608 33574 33577 HKI +T991 PR:000008608 33619 33622 HKI +T992 GO:0008152 33720 33730 metabolize +T993 CHEBI:17234 33731 33738 glucose +T994 PR:000013712 33742 33748 RanBP2 +T995 NCBITaxon:10088 33752 33756 mice +T996 UBERON:0000966 33823 33829 retina +T997 UBERON:0000966 33895 33902 retinal +T998 CL:0000540 33903 33910 neurons +T999 SO:0000417 33932 33939 domains +T1000 PR:000013712 33943 33949 RanBP2 +T1001 PR:000001244 34068 34071;34078 34083 red ... opsin +T1002 PR:000001224 34072 34083 green opsin +T1003 SO:0000417 34118 34125 domains +T1004 PR:000013708 34161 34164 Ran +T1005 SO:0000417 34173 34180 domains +T1006 PR:000013712 34184 34190 RanBP2 +T1007 PR:000013708 34251 34260 RanGTPase +T1008 PR:000013708 34280 34283 Ran +T1009 PR:000013712 34366 34372 RanBP2 +T1010 GO:0006457 34445 34452 folding +T1011 PR:000005770 34464 34469 Cox11 +T1012 PR:000008608 34485 34488 HKI +T1013 PR:000013712 34584 34590 RanBP2 +T1014 PR:000013712 34674 34680 RanBP2 +T1015 GO:0008541 34714 34720;34725 34735 cap of ... proteasome +T1016 PR:000012285 34820 34826 parkin +T1017 PR:000015829 34844 34850 SUMO-1 +T1018 MOP:0000779 34851 34862 conjugating +T1019 PR:000016977 34872 34876 Ubc9 +T1020 PR:000008608 34923 34926 HKI +T1021 GO:0000502 34934 34944 proteasome +T1022 GO:0010498 34934 34965 proteasome-mediated proteolysis +T1023 GO:0065007 34970 34980 modulation +T1024 PR:000012285 35102 35108 parkin +T1025 PR:000008608 35133 35136 HKI +T1026 CHEBI:18243 35140 35152 dopaminergic +T1027 CL:0000700 35140 35160 dopaminergic neurons +T1028 PR:000012285 35168 35174 parkin +T1029 GO:0065007 35191 35199 modulate +T1030 PR:000013712 35200 35206 RanBP2 +T1031 PR:000012285 35226 35232 parkin +T1032 GO:0006110 35357 35370;35375 35393 regulation of ... glycolytic pathway +T1033 UBERON:0001017 35416 35419 CNS +T1034 GO:0006096 35427 35437 glycolysis +T1035 GO:0000502 35491 35501 proteasome +T1036 GO:0065007 35540 35550 modulating +T1037 SO:0000704 35644 35651 genetic +T1038 PR:000013712 35665 35671 RanBP2 +T1039 NCBITaxon:10088 35674 35678 mice +T1040 SO:0000704 35690 35697 genetic +T1041 PR:000013712 35742 35748 RanBP2 +T1042 GO:0065007 35755 35765 regulation +T1043 CHEBI:17234 35785 35792 glucose +T1044 GO:0042593 35785 35792;35800 35811 glucose ... homeostasis +T1045 GO:0097009 35793 35811 energy homeostasis +T1046 SO:0000771 35938 35962 quantitative trait locus +T1047 PR:000013712 35982 35988 RanBP2 +T1048 http://purl.obolibrary.org/obo/MONDO_0005015 36021 36029 diabetes +T1049 UBERON:0000479 36077 36084 tissues +T1050 PR:000013712 36104 36110 RanBP2 +T1051 PR:000008608 36115 36118 HKI +T1052 CHEBI:29101 36187 36190 Na+ +T1053 CHEBI:29103 36191 36193 K+ +T1054 UBERON:0001017 36326 36329 CNS +T1055 UBERON:0001017 36388 36391 CNS +T1056 UBERON:0000966 36487 36494 retinal +T1057 CL:0000540 36495 36502 neurons +T1058 PR:000008608 36537 36540 HKI +T1059 GO:0005622 36553 36566 intracellular +T1060 http://purl.obolibrary.org/obo/MONDO_0002909 36567 36580 hyperglycemia +T1061 UBERON:0001017 36588 36591 CNS +T1062 CHEBI:30911 36601 36609 sorbitol +T1063 CHEBI:29101 36678 36681 Na+ +T1064 CHEBI:29103 36682 36684 K+ +T1065 PR:000008608 36776 36779 HKI +T1066 GO:0005622 36861 36874 intracellular +T1067 http://purl.obolibrary.org/obo/MONDO_0002909 36875 36888 hyperglycemia +T1068 GO:0065007 36926 36934 modulate +T1069 CL:0000540 36974 36982 neuronal +T1070 PR:000013712 37110 37116 RanBP2 +T1071 GO:0097009 37230 37248 energy homeostasis +T1072 GO:0043335 37267 37276 unfolding +T1073 GO:0008541 37325 37331;37336 37346 cap of ... proteasome +T1074 GO:0065007 37354 37363 modulated +T1075 PR:000013712 37378 37384 RanBP2 +T1076 PR:000008608 37422 37425 HKI +T1077 GO:0005634 37488 37495 nuclear +T1078 GO:0051170 37488 37502 nuclear import +T1079 PR:000013708 37533 37536 Ran +T1080 SO:0000417 37545 37552 domains +T1081 PR:000013712 37556 37562 RanBP2 +T1082 GO:0005634 37588 37595 nuclear +T1083 GO:0051169 37588 37609 nuclear translocation +T1084 CHEBI:17138 37649 37675 glyceraldehyde-3-phosphate +T1085 CHEBI:23888 37764 37768 drug +T1086 CHEBI:50217 37770 37784 R-(-)-deprenyl +T1087 http://purl.obolibrary.org/obo/MONDO_0005180 37840 37857 Parkinson disease +T1088 PR:000013712 37874 37880 RanBP2 +T1089 SO:0000704 37931 37936 genes +T1090 SO:0000704 38007 38014 genetic +T1091 UBERON:0001017 38048 38051 CNS +T1092 http://purl.obolibrary.org/obo/MONDO_0005180 38073 38082 Parkinson +T1093 http://purl.obolibrary.org/obo/MONDO_0005015 38084 38092 diabetes +T1094 PR:000045358 38098 38105 insulin +T1095 http://purl.obolibrary.org/obo/MONDO_0005244 38128 38140 neuropathies +T1096 http://purl.obolibrary.org/obo/MONDO_0005559 38145 38171 neurodegenerative diseases +T1097 GO:0007568 38189 38194 aging +T1098 PR:000013712 38231 38237 RanBP2 +T1099 NCBITaxon:10088 38238 38243 mouse +T1100 SO:0000704 38273 38280 genetic +T1101 GO:0008152 38387 38406 metabolic processes +T1102 PR:000013712 38482 38488 RanBP2 +T1103 GO:0008152 38517 38526 Metabolic +T1104 CL:0000540 38531 38539 Neuronal +T1105 PR:000013712 38550 38556 RanBP2 +T1106 PR:000005770 38572 38577 Cox11 +T1107 PR:000008608 38582 38585 HKI +T1108 PR:000013712 38657 38663 RanBP2 +T1109 PR:000008608 38691 38694 HKI +T1110 PR:000005770 38698 38703 Cox11 +T1111 PR:000013712 38748 38754 RanBP2 +T1112 GO:0006096 38797 38815 glycolytic pathway +T1113 GO:0006754 38820 38837 production of ATP +T1114 GO:0006096 38843 38861 glycolytic pathway +T1115 CHEBI:29101 38899 38902 Na+ +T1116 CHEBI:29103 38903 38905 K+ +T1117 GO:0001917 38959 38964;38975 38982 inner ... segment +T1118 GO:0001750 38969 38982 outer segment +T1119 CL:0000210 38999 39019 photosensory neurons +T1120 PR:000013712 39055 39061 RanBP2 +T1121 PR:000013712 39095 39101 RanBP2 +T1122 PR:000008608 39103 39106 HKI +T1123 PR:000005770 39112 39117 Cox11 +T1124 PR:000008608 39197 39200 HKI +T1125 GO:0006754 39219 39233 ATP production +T1126 CL:0000540 39280 39287 neurons +T1127 CL:0000006 39332 39342;39362 39369 receptoral ... neurons +T1128 PR:000008608 39423 39426 HKI +T1129 PR:000008608 39463 39466 HKI +T1130 GO:0005622 39475 39488 intracellular +T1131 http://purl.obolibrary.org/obo/MONDO_0002909 39489 39502 hyperglycemia +T1132 CHEBI:29101 39562 39565 Na+ +T1133 CHEBI:29103 39566 39568 K+ +T1134 PR:000013712 39641 39647 RanBP2 +T1135 CL:0000604 39703 39706 rod +T1136 GO:0001917 39707 39720 inner segment +T1137 CL:0000604 39727 39730 rod +T1138 GO:0001750 39731 39744 outer segment +T1139 PR:000013712 39756 39762 RanBP2 +T1140 PR:000011526 39807 39812 Nup96 +T1141 NCBITaxon:1 39879 39888 organisms +T1142 GO:0010467 39956 39966 expression +T1143 NCBITaxon:33208 39994 40000 animal +T1144 UBERON:0000479 40038 40044 tissue +T1145 SO:0000646 40196 40217 short interfering RNA +T1146 PR:000013712 40240 40246 RanBP2 +T1147 PR:000011506 40270 40276 Nup107 +T1148 GO:0031080 40270 40291 Nup107-Nup160 complex +T1149 PR:000011510 40277 40283 Nup160 +T1150 GO:0071850 40335 40349 mitotic arrest +T1151 GO:0005643 40368 40380 nuclear pore +T1152 GO:0051292 40368 40389 nuclear pore assembly +T1153 PR:000013712 40462 40468 RanBP2 +T1154 PR:000011526 40485 40490 Nup96 +T1155 UBERON:0001017 40528 40531 CNS +T1156 GO:0008152 40562 40572 metabolism +T1157 UBERON:0002405 40596 40609 immune system +T1158 PR:000024939 40643 40655 interferon-β +T1159 GO:0065007 40656 40665 regulated +T1160 NCBITaxon:10239 40707 40712 viral +T1161 http://purl.obolibrary.org/obo/MONDO_0005108 40707 40722 viral infection +T1162 SO:0000704 40968 40975 genetic +T1163 NCBITaxon:33208 40986 40992 animal +T1164 SO:0000704 41077 41084 genetic +T1165 NCBITaxon:9606 41170 41175 human +T1166 http://purl.obolibrary.org/obo/MONDO_0000001 41176 41184 diseases +T1167 NCBITaxon:9606 41260 41265 human +T1168 PR:000013712 41266 41272 RanBP2 +T1169 SO:0001817 41305 41313 in-frame +T1170 PR:P04386 41323 41327 GAL4 +T1171 SO:0000417 41340 41346 domain +T1172 SO:0000440 41355 41361 vector +T1173 CHEBI:6104 41372 41381 kanamycin +T1174 PR:P04386 41403 41407 GAL4 +T1175 SO:0000440 41413 41419 vector +T1176 PR:P04386 41451 41455 GAL4 +T1177 SO:0000440 41456 41462 vector +T1178 GO:0007618 41567 41573 mating +T1179 NCBITaxon:39107 41579 41585 murine +T1180 UBERON:0000922 41601 41607 embryo +T1181 UBERON:0000955 41612 41617 brain +T1182 GO:0009294 41757 41771 transformation +T1183 NCBITaxon:27592 41798 41804 bovine +T1184 UBERON:0000966 41805 41812 retinal +T1185 SO:0001817 41880 41888 in-frame +T1186 UBERON:0000922 41933 41942 embryonic +T1187 UBERON:0007023 41947 41952 adult +T1188 UBERON:0000955 41953 41958 brain +T1189 PR:000005770 42046 42051 Cox11 +T1190 PR:000005770 42074 42079 Cox11 +T1191 SO:0000155 42639 42646 plasmid +T1192 SO:0000112 42713 42720 primers +T1193 SO:0000417 42729 42736 domains +T1194 SO:0000006 42782 42794 PCR products +T1195 PR:P04386 42842 42846 GAL4 +T1196 SO:0000440 42847 42853 vector +T1197 SO:0000440 42883 42890 vectors +T1198 NCBITaxon:9606 42938 42943 human +T1199 PR:000013712 42944 42950 RanBP2 +T1200 CHEBI:1418 43000 43005 CHAPS +T1201 UBERON:0000966 43018 43025 retinal +T1202 GO:0010467 43036 43046 expression +T1203 CHEBI:8984 43248 43251 SDS +T1204 GO:0042571 43291 43301 antibodies +T1205 GO:0043335 43336 43344 Unfolded +T1206 PR:000005770 43369 43374 Cox11 +T1207 PR:000005770 43427 43432 Cox11 +T1208 CHEBI:16199 43488 43492 urea +T1209 PR:000005770 43512 43517 Cox11 +T1210 CHEBI:1418 43559 43564 CHAPS +T1211 GO:0042571 43639 43647 antibody +T1212 GO:0042571 43686 43694 antibody +T1213 PR:000008608 43750 43762 Hexokinase I +T1214 PR:000008608 43771 43783 Hexokinase I +T1215 CHEBI:14314 43870 43889 glucose-6-phosphate +T1216 CHEBI:14314 43905 43924 glucose-6-phosphate +T1217 UBERON:0000955 44071 44076 brain +T1218 PR:000008608 44071 44089 brain hexokinase I +T1219 CHEBI:60004 44144 44151 mixture +T1220 CHEBI:9754 44170 44174 Tris +T1221 CHEBI:17883 44175 44178 HCl +T1222 CHEBI:6636 44196 44201 MgCl2 +T1223 CHEBI:74537 44237 44253 monothioglycerol +T1224 CHEBI:14314 44269 44288 glucose 6-phosphate +T1225 PR:000005770 44367 44372 Cox11 +T1226 PR:000008608 44394 44397 HKI +T1227 GO:0042571 44595 44605 Antibodies +T1228 NCBITaxon:9986 44608 44614 Rabbit +T1229 UBERON:0001977 44619 44623 sera +T1230 NCBITaxon:39107 44660 44666 murine +T1231 PR:000005770 44667 44672 Cox11 +T1232 PR:000005770 44788 44793 Cox11 +T1233 GO:0042571 44794 44804 antibodies +T1234 GO:0042571 44865 44873 antibody +T1235 PR:000003425 44874 44879 Hsp70 +T1236 GO:0042571 44948 44958 antibodies +T1237 SO:0000417 44989 44996 domains +T1238 PR:000013712 45000 45006 RanBP2 +T1239 GO:0042571 45062 45072 antibodies +T1240 PR:000008608 45081 45093 Hexokinase I +T1241 UBERON:0000966 45239 45245 Retina +T1242 UBERON:0000955 45370 45376 Brains +T1243 NCBITaxon:10088 45405 45409 mice +T1244 CHEBI:17992 45472 45479 sucrose +T1245 UBERON:0000955 45523 45528 brain +T1246 CL:0000540 45529 45536 neurons +T1247 CL:0000125 45541 45552 glial cells +T1248 UBERON:0000956 45576 45591 cerebral cortex +T1249 CL:0000001 45665 45678 Primary cells +T1250 CHEBI:16526 45844 45847 CO2 +T1251 GO:0042571 45910 45920 antibodies +T1252 CHEBI:52661 45964 45973 Alexa 488 +T1253 CHEBI:51248 45979 45988 Alexa 594 +T1254 MOP:0000779 45989 45999 conjugated +T1255 GO:0042571 46010 46020 antibodies +T1256 CHEBI:39442 46106 46124 fluorescent probes +T1257 NCBITaxon:10088 47655 47660 mouse +T1258 PR:000013712 47683 47689 RanBP2 +T1259 PR:000013712 47733 47739 RanBP2 +T1260 NCBITaxon:10088 47740 47744 mice +T1261 NCBITaxon:10088 47768 47773 mouse +T1262 CL:0002322 47774 47781 ES cell +T1263 PR:000013712 47850 47856 RanBP2 +T1264 SO:0000704 47897 47901 gene +T1265 SO:0000440 47907 47913 vector +T1266 NCBITaxon:10088 47987 47992 Mouse +T1267 SO:0000704 48090 48094 gene +T1268 SO:0000440 48100 48106 vector +T1269 SO:0000167 48118 48126 promoter +T1270 PR:000033987 48135 48139 lacZ +T1271 GO:0008380 48159 48165 splice +T1272 SO:0000243 48209 48237 internal ribosome entry site +T1273 GO:0005840 48218 48226 ribosome +T1274 SO:0000243 48239 48243 IRES +T1275 NCBITaxon:9606 48253 48258 human +T1276 UBERON:0001987 48259 48268 placental +T1277 PR:000003969 48259 48289 placental alkaline phosphatase +T1278 PR:000003969 48291 48295 PLAP +T1279 SO:0005853 48306 48314 cassette +T1280 PR:000013712 48320 48326 RanBP2 +T1281 CL:0002322 48334 48336 ES +T1282 UBERON:0000358 48369 48380 blastocysts +T1283 SO:0000704 48680 48687 genetic +T1284 UBERON:0002415 48776 48780 tail +T1285 SO:0001026 48781 48788 genomic +T1286 PR:000003969 48804 48808 PLAP +T1287 UBERON:0000922 48835 48842 embryos +T1288 UBERON:0000966 48847 48854 retinal +T1289 CHEBI:75055 48912 48960 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside +T1290 CHEBI:75508 48995 49031 5-bromo-4-chloro-3-indolyl phosphate +T1291 CHEBI:7586 49041 49062 nitroblue tetrazolium +T1292 CHEBI:9754 49073 49077 Tris +T1293 CHEBI:17883 49078 49081 HCl +T1294 CHEBI:26710 49099 49103 NaCl +T1295 CHEBI:6636 49110 49115 MgCl2 +T1296 GO:0010467 49200 49210 expression +T1297 CHEBI:27780 49244 49253 detergent +T1298 UBERON:0000479 49278 49285 tissues +T1299 PR:000011508 49439 49445 Nup153 +T1300 UBERON:0000479 49611 49618 tissues +T1301 CHEBI:30956 49644 49664 trichloroacetic acid +T1302 CHEBI:30956 49666 49669 TCA +T1303 CHEBI:66869 49735 49747 Tris-Acetate +T1304 GO:0008218 49827 49842 Bioluminescence +T1305 GO:0008152 50149 50158 Metabolic +T1306 NCBITaxon:10088 50187 50191 mice +T1307 GO:0007565 50228 50237 gestation +T1308 GO:0007567 50241 50246 birth +T1309 GO:0007631 50262 50265 fed +T1310 CHEBI:33290 50280 50284 chow +T1311 NCBITaxon:9989 50299 50305 Rodent +T1312 NCBITaxon:10088 50433 50438 Mouse +T1313 CHEBI:17234 50464 50471 Glucose +T1314 NCBITaxon:10088 50521 50525 mice +T1315 UBERON:0001179 50540 50552 peritoneally +T1316 CHEBI:17234 50565 50572 glucose +T1317 UBERON:0001638 50596 50602 Venous +T1318 UBERON:0000178 50603 50608 blood +T1319 CHEBI:17234 50609 50616 glucose +T1320 CHEBI:17234 50737 50744 glucose +T1321 PR:000045358 50793 50800 Insulin +T1322 CHEBI:17234 50869 50876 glucose +T1323 NCBITaxon:9606 50916 50921 human +T1324 PR:000045358 50922 50929 insulin +T1325 UBERON:0001179 50973 50985 peritoneally +T1326 NCBITaxon:10088 51000 51004 mice +T1327 CHEBI:15361 51006 51014 Pyruvate +T1328 CHEBI:17234 51083 51090 glucose +T1329 CHEBI:15361 51130 51138 pyruvate +T1330 UBERON:0001179 51199 51211 peritoneally +T1331 NCBITaxon:10088 51232 51236 mice +T1332 PR:000013712 51281 51287 RanBP2 +T1333 PR:000013712 51295 51301 RanBP2 +T1334 NCBITaxon:10088 51305 51309 mice +T1335 NCBITaxon:10088 51436 51440 mice +T1336 UBERON:0001179 51464 51476 peritoneally +T1337 CHEBI:6121 51482 51490 ketamine +T1338 UBERON:0001771 51622 51628 pupils +T1339 CHEBI:9757 51658 51669 tropicamide +T1340 CHEBI:8094 51679 51696 phenylephrine HCl +T1341 UBERON:0000970 51707 51711 eyes +T1342 CHEBI:8485 51812 51824 proparacaine +T1343 CHEBI:17883 51825 51838 hydrochloride +T1344 UBERON:0001773 51894 51900 sclera +T1345 UBERON:0001690 51983 51986 ear +T1346 GO:1990603 51988 52006 Scotopic responses +T1347 CL:0000604 52207 52210 rod +T1348 PR:000013712 52307 52313 RanBP2 +T1349 PR:000008608 52319 52322 HKI +T1350 PR:000005770 52327 52332 Cox11 +T1351 UBERON:0000479 52340 52347 Tissues +T1352 UBERON:0000479 52395 52401 tissue +T1353 UBERON:0000479 52473 52480 tissues +T1354 GO:0042571 52520 52530 antibodies +T1355 PR:000008608 52539 52542 HKI +T1356 PR:000005770 52547 52552 Cox11 +T1357 PR:000013712 52564 52570 RanBP2 +T1358 PR:000008608 52591 52594 HKI +T1359 PR:000005770 52599 52604 Cox11 +T1360 UBERON:0000479 52618 52625 tissues +T1361 PR:000013712 52777 52783 RanBP2 +T1362 NCBITaxon:10088 52784 52788 Mice +T1363 UBERON:0000966 52789 52796 Retinas +T1364 PR:000008608 52807 52810 HKI +T1365 PR:000005770 52816 52821 Cox11 +T1366 GO:0042571 52822 52832 Antibodies +T1367 UBERON:0000966 52903 52909 retina +T1368 GO:0001917 52911 52924 inner segment +T1369 CL:0000210 52956 52975 photosensory neuron +T1370 PR:000008608 53019 53022 HKI +T1371 GO:0001917 53058 53096 inner segment of photoreceptor neurons +T1372 CL:0000210 53075 53096 photoreceptor neurons +T1373 NCBITaxon:10088 53104 53108 mice +T1374 PR:000008608 53138 53141 HKI +T1375 GO:0045202 53166 53174 synaptic +T1376 PR:000008608 53323 53326 HKI +T1377 GO:0045202 53361 53369 synaptic +T1378 CL:0000604 53422 53425 rod +T1379 GO:0001750 53426 53439 outer segment +T1380 CL:0000604 53446 53449 rod +T1381 GO:0001917 53450 53463 inner segment +T1382 UBERON:0001789 53465 53468 ONL +T1383 UBERON:0001789 53470 53489 outer nuclear layer +T1384 GO:0005634 53476 53483 nuclear +T1385 UBERON:0001790 53491 53494 OPL +T1386 UBERON:0001790 53496 53517 outer plexiform layer +T1387 UBERON:0001791 53519 53522 INL +T1388 UBERON:0001791 53524 53543 inner nuclear layer +T1389 GO:0005634 53530 53537 nuclear +T1390 UBERON:0001795 53545 53548 IPL +T1391 UBERON:0001795 53550 53571 inner plexiform layer +T1392 UBERON:0001792 53573 53576 GCL +T1393 CL:0000740 53578 53591 ganglion cell +T1394 UBERON:0001792 53578 53597 ganglion cell layer +T1395 GO:0005739 53641 53653 mitochondria +T1396 CL:0000210 53686 53706 photosensory neurons +T1397 GO:0008152 53772 53781 Metabolic +T1398 PR:000013712 53795 53801 RanBP2 +T1399 NCBITaxon:10088 53805 53809 Mice +T1400 CHEBI:17234 53882 53889 glucose +T1401 PR:000013712 53913 53919 RanBP2 +T1402 PR:000013712 53927 53933 RanBP2 +T1403 NCBITaxon:10088 53936 53940 mice +T1404 SO:0000704 53966 53973 genetic +T1405 PR:000008608 54094 54097 HKI +T1406 GO:0042571 54099 54109 antibodies +T1407 PR:000008608 54118 54121 HKI +T1408 PR:000008609 54123 54127 HKII +T1409 PR:000008610 54129 54134 HKIII +T1410 PR:000008608 54212 54215 HKI +T1411 CL:0002322 54282 54284 ES +T1412 SO:0000417 54346 54352 domain +T1413 UBERON:0001017 54354 54357 CNS +T1414 UBERON:0001017 54360 54382 central nervous system +T1415 CHEBI:16856 54390 54401 glutathione +T1416 PR:000008608 54417 54420 HKI +T1417 PR:000008608 54423 54440 hexokinase type I +T1418 SO:0000417 54460 54466 domain +T1419 GO:0005643 54468 54471 NPC +T1420 GO:0005643 54474 54494 nuclear pore complex +T1421 PR:000013712 54496 54502 RanBP2 +T1422 PR:000013712 54505 54526 Ran-binding protein 2 +T1423 CHEBI:33893 55056 55064 reagents +T1424 http://purl.obolibrary.org/obo/MONDO_0001941 55307 55316 Blindness diff --git a/src/ontogpt/evaluation/craft/database/all/17069463.txt b/src/ontogpt/evaluation/craft/database/all/17069463.txt new file mode 100644 index 000000000..4ba4463df --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17069463.txt @@ -0,0 +1,267 @@ +RanBP2 Modulates Cox11 and Hexokinase I Activities and Haploinsufficiency of RanBP2 Causes Deficits in Glucose Metabolism + +Abstract + +The Ran-binding protein 2 (RanBP2) is a large multimodular and pleiotropic protein. Several molecular partners with distinct functions interacting specifically with selective modules of RanBP2 have been identified. Yet, the significance of these interactions with RanBP2 and the genetic and physiological role(s) of RanBP2 in a whole-animal model remain elusive. Here, we report the identification of two novel partners of RanBP2 and a novel physiological role of RanBP2 in a mouse model. RanBP2 associates in vitro and in vivo and colocalizes with the mitochondrial metallochaperone, Cox11, and the pacemaker of glycolysis, hexokinase type I (HKI) via its leucine-rich domain. The leucine-rich domain of RanBP2 also exhibits strong chaperone activity toward intermediate and mature folding species of Cox11 supporting a chaperone role of RanBP2 in the cytosol during Cox11 biogenesis. Cox11 partially colocalizes with HKI, thus supporting additional and distinct roles in cell function. Cox11 is a strong inhibitor of HKI, and RanBP2 suppresses the inhibitory activity of Cox11 over HKI. To probe the physiological role of RanBP2 and its role in HKI function, a mouse model harboring a genetically disrupted RanBP2 locus was generated. RanBP2−/− are embryonically lethal, and haploinsufficiency of RanBP2 in an inbred strain causes a pronounced decrease of HKI and ATP levels selectively in the central nervous system. Inbred RanBP2+/− mice also exhibit deficits in growth rates and glucose catabolism without impairment of glucose uptake and gluconeogenesis. These phenotypes are accompanied by a decrease in the electrophysiological responses of photosensory and postreceptoral neurons. Hence, RanBP2 and its partners emerge as critical modulators of neuronal HKI, glucose catabolism, energy homeostasis, and targets for metabolic, aging disorders and allied neuropathies. + +Synopsis + +The Ran-binding protein 2 (RanBP2) is a large protein with several domains. Although several protein partners were found to interact with selective domains of RanBP2, none to this date were found toward its large leucine-rich domain (LD). Cell-based experiments support several roles of RanBP2 in cell function, such as the production of functional proteins, control of protein trafficking between the nuclear and cytosol compartments, and control of multiple facets underlying cell division. Still, the genetic and physiological implications of the interactions between RanBP2 and its partners and of the function of RanBP2 in a whole-animal model remain elusive. The authors report the identification of two novel mitochondrial partners of the LD of RanBP2, Cox11 and hexokinase type I (HKI); and with multidisciplinary approaches probe the role of RanBP2 and its LD on Cox11, HKI, and functions allied to these. The authors found that RanBP2 exhibits chaperone activity toward HKI and Cox11. RanBP2 and Cox11 profoundly modulate HKI activity. Moreover, partial loss-of-function of RanBP2 in a mouse model induces deficits in growth rates and breakdown of glucose, promotes the down-regulation of HKI and ATP levels selectively in the central nervous system, and impairs visual function. These findings support a critical role of RanBP2 and its partners in metabolic processes and allied disease states. + +Introduction + +The RanBP2/Nup358 is a unique vertebrate and large scaffold protein comprised of multiple structural and functional domains [1–4]. Several roles of RanBP2 have emerged that implicate RanBP2 in nucleocytoplasmic trafficking [3,5], protein biogenesis [6,7], the formation of the mitotic spindle, assembly of the nuclear envelope [8], and the integration of the nuclear envelope breakdown with kinetochore formation and maturation during early mitotic progression [9]. The specific interaction of RanBP2 with a diverse set of partners likely reflects a pleiotropic role of RanBP2 in cell function, possibly through the integration of multiple pathways. On the other hand, the cell (tissue)-selective interaction of RanBP2 with some of its partners may also impart cell-restricted roles to RanBP2. For example, the Ran-binding domains RBDn=1-4 of RanBP2 associate with the nuclear import co-receptor, importin-β [10,11], and antibodies against RanBP2 inhibit the nuclear import pathway in HeLa cells [3]; but such a role seems dispensable in Xenopus oocytes [12]. In addition, the combination of the C-terminal domains, RBD4 and CY (of RanBP2), associates with a subset of G protein-coupled receptors, the red/green opsin, expressed in photosensory neurons and enhances opsin functional production [6,7], while the interaction of the KBD of RanBP2 with a subset of the conventional microtubule-based motor proteins, the kinesins, KIF5B and KIF5C, occurs selectively in the central nervous system (CNS) [13]. + +A diverse set of additional molecular partners, each associating specifically with a selective domain of RanBP2, are likely to impart and integrate additional roles to RanBP2. For example, the cyclophilin-like domain (CLD), the internal repeat (W1W2/IR), and zinc-finger rich (ZnF) domains of RanBP2 associate specifically with components of the 19S cap of the proteasome [14], the E2 SUMO-1-conjugating enzyme (Ubc9) [15], and the nuclear export receptor, CRM1/exportin-1 [16], respectively. RanBP2 itself was also found to exhibit SUMO1 E3 ligase activity [17], supporting a direct link between RanBP2-mediated SUMO-1 substrate modification and relocation of SUMO-1 modified cytosolic substrates (e.g., RanGAP) to the cytosolic face of the nuclear pore complex (NPC) [18]. Finally, RanBP2 was also found to localize prominently to the mitochondria-rich ellipsoid subcellular compartment of photosensory neurons [19] and to RanGTPase-restricted foci along cytoplasmic tracks [19], in addition to its localization at cytoplasmic fibrils emanating from nuclear pores [2,3,10,12,19]. + +Emerging evidence supports that the CNS-selective effects of RanBP2 may also underlie the pathogenesis of certain neuropathies. Parkin is ubiquitously expressed and interacts with the Ubc9-interacting domain of RanBP2 [20]. This promotes the ubiquitination and degradation of RanBP2 [20], possibly via the interaction of the CLD domain of RanBP2 with the 19S cap subunits of the proteasome [14]. Interestingly, the small yeast Ran-binding protein 1 (RanBP1), Yrb1p, with strong homology to the Ran-binding domains of RanBP2, is also required for cell-cycle regulated protein degradation [21]. Parkin, like RanBP2 [17], has E3-ligase activity [22–24]; and loss-of-function of parkin leads to early onset autosomal recessive juvenile Parkinsonism [25]. While the dopaminergic neuronal-restricted effects of mutations in parkin are not understood [25], parkin−/− mice exhibit mitochondrial dysfunction and energy and growth deficits, and a fraction of parkin localizes to the cytoplasmic face of the mitochondria, where together with other partners is thought to promote the degradation of selective mitochondrial substrates and to exert a neuroprotective function [26–29]. These data suggest that deficits in RanBP2 and parkin may share (patho) physiological pathways and support a multifaceted role of RanBP2. Yet clear functions of RanBP2 in animal and cell physiology remain elusive. + +Here, we report on the identification and function of two novel partners of RanBP2, Cox11 and hexokinase type I (HKI), which interact with a large and orphan domain of RanBP2, the leucine-rich domain (LD); and on the outcome of partial loss-of-function of RanBP2 on HKI, animal physiology, and glucose/energy metabolism. + +Results + +The LD of RanBP2 Interacts with Cox11 and HKI + +The LD of RanBP2 (Figure 1A) is a large and orphan domain of ~700 residues (~80 kDa), for which no molecular partners have been identified until this date. Brain and retina yeast two-hybrid libraries were screened with LD. Cox11 was identified as a partner to this domain (Figure 1B and 1C). Cox11 is a metallochaperone implicated in cytochrome c oxidase assembly [30,31]. Structure-function analysis of the interaction between mCox11, LD of RanBP2, and subdomains thereof, with quantitative yeast two-hybrid assays [32], showed optimal interaction between the intact LD and Cox11 proteins (Figure 1C). Pull-down assays of retinal extracts with glutathione S-transferase (GST)-LD precipitates a sodium dodecyl sulfate-resistant dimer isoform of Cox11 (Figure 1D, top panel), which does not bind to GST-LDZIP alone. In addition to Cox11, we also found that other mitochondrial components such as the outer membrane-associated protein, HKI [33] (Figure 1D, bottom panel) and mHsp70 (unpublished data), associated with LD of RanBP2. This association was highly specific toward the HKI isoform, because HKII, HKIII, and glucokinase did not interact with the LD of RanBP2 (unpublished data). The interaction of Cox11, HKI, and mHsp70 with RanBP2 occurred in vivo in retinal extracts, since antibodies against these and RanBP2 coimmunoprecipitated RanBP2 (Figure 1E) and HKI (Figure 1F), respectively, and these interactions were observed across different tissues (Figure S1). Since RanBP2 exhibits chaperone activity, we assessed whether the interaction between the LD of RanBP2 and Cox11 was direct and the chaperone activity of LD toward folding species of Cox11. Reconstitution binding assays were carried out between purified LD and Cox11, fully and partially denatured with GnHCl and urea, respectively, and native Cox11 (Figure 1G and 1H). Partial denatured Cox11 exhibits significantly higher and concentration-dependent binding affinity toward LD compared with the native and fully denatured Cox11 (Figure 1G). In addition, native Cox11 purified upon expression in the presence of CuSO4 (a prosthetic group tightly bound to Cox11) [31], shows significantly higher binding activity toward the LD of RanBP2, than in the absence of CuSO4 (Figure 1H). + +Figure 1 + +The LD of RanBP2 Interacts with Cox11 and HKI + +(A) Primary structure of RanBP2 and its structural/functional domains. The N-terminal LD of RanBP2 is underlined. + +(B) Sequence alignment of murine and yeast Cox11. The yeast Cox11 C- and N-terminal domains are poorly conserved. Arrow and solid line denote the predicted mitochondrial cleavage site and membrane-spanning domain. The dotted and dashed lines above the aligned sequences represent, respectively, Cox11-N and Cox11-C constructs shown in Figure 1C. + +(C) Structure-function analysis of the interaction between the LD of RanBP2 and Cox11. Optimal interaction between the LD and Cox11 occurred in the presence of constructs comprising both the complete LD and Cox11. Although removal of the cytosolic N-terminal (Cox11-C) significantly decreased the interaction with LD, the mitochondrial intermembrane domain of Cox11 (Cox11-C) together with the C-terminal half of LD (LD-C) retained most of the interaction activity. LD-N and LD-C ended and began with the leucine zipper domain of RanBP2. White and black bars denote β-galactosidase activity and growth rates in selective growth medium, respectively. Results shown represent the mean ± SD, n = 3. + +(D) GST pull-down assays with the LD of RanBP2 and its leucine zipper domain and retinal extracts. The LD, but not the leucine zipper domain of RanBP2, associate with Cox11 (top panel, lane 1) and HKI (bottom panel, lane 1). + +(E) Coimmunoprecipitation of RanBP2 with antibodies against its molecular partners shows that RanBP2 forms a complex in vivo with HKI (lanes 1 and 2), mHsp70 (lane 3), and Cox11 (lane 4). Lanes 5, 6, and 7 are control immunoprecipitation reactions with different antibodies against the RanBP2 domains, KBD, ZnF, and XAFXFG of nucleoporins. + +(F) Reciprocal coimmunoprecipitation of HKI with antibodies against RanBP2 (used and shown in (E)). + +(G) Reconstitution pull-down assays with purified LD and increasing concentrations of native (top panel), denatured (middle panel), and partially denatured (bottom panel) Cox11, respectively, in the absence and presence of denaturating agent, GnHCl and chaotropic agent, urea. Folding intermediates (lower panel) of Cox11 exhibit the highest binding activity toward the LD of RanBP2. + +(H) Similar experiments as in (G) but in the presence of native Cox11 expressed in the absence (top panel) or presence (bottom panel) of CuSO4. The mature isoform of the metallochaperone has an increased affinity toward the LD of RanBP2. LD, leucine-rich domain; LZ, leucine zipper domain; RBD1–4, Ran-binding domains 1–4; ZnF, zinc finger cluster domain; KBD, kinesin (KIF5B/KIF5C)-binding domain; CLD, cyclophilin-like domain; IR, internal repeat domain; CY, cyclophilin domain. + +Cox 11 Inhibits HKI Activity and the LD of RanBP2 Reverses the Inhibition of Cox11 over HKI + +To probe whether the interaction of Cox11 and HKI with the LD of RanBP2 modulates the enzymatic activity of HKI, we first examined the effect of increasing concentrations of Cox11 on the initial rates of HKI enzymatic activity (Figure 2A). Cox11 strongly inhibits HKI activity in a concentration-dependent fashion, and at ~15 nM of Cox11, HKI activity could not be recorded (Figure 2A). Cox11 behaves as a partial noncompetitive inhibitor of HKI by affecting the Vmax of HKI for glucose (Figure 2B). Then, we evaluated the effect of the LD of RanBP2 on the HK activity in the presence of a fixed inhibitory concentration of Cox11, saturating concentration of glucose substrate, and increasing concentrations of LD. As shown in Figure 2C, the LD domain sharply reversed the inhibitory effect of Cox11 on HKI activity in a concentration-dependent manner, but under saturating (and stochiometric) amounts of LD, the velocity of the reaction did not reach that observed for HKI activity in the absence of Cox11 (Figure 2A), suggesting the LD by itself may also have an effect on HKI activity. Indeed, a saturating concentration of LD reduced the Vmax but not the Km of HKI (Figure 2D) by ~20% under similar conditions. + +Figure 2 + +Effect of Cox11 and RanBP2 on HKI Activity + +(A) Saturation kinetics, rate versus glucose of HKI (0.24 μg) in the absence (solid circles) and presence of Cox11 (open circles, 0.25 nM; solid triangles, 7.5 nM). The activity of HKI decreases with increasing concentrations of Cox11. No measurable HKI activity was recorded in the presence of 15 nM of Cox11 (unpublished data). + +(B) Hanes-Wolf plot of (A) (1/rate versus glucose) in the absence and presence of fixed concentrations of Cox11. Linearity of reciprocal plots also supported the hyperbolic behavior of the reactions (unpublished data). Cox11 behaves as a noncompetitive inhibitor of HKI by reducing the Vmax of HKI but not its Km toward glucose. + +(C) HKI rate is plotted as a function of LD concentration at saturating glucose and fixed Cox11 (7.5 nM) concentrations. Note that increasing concentrations of the LD of RanBP2 reverse the inhibition of HKI activity by Cox11. A half-maximal effect of the LD of RanBP2 on HKI activity in the presence of 7.5 nM of Cox11 was observed at a concentration of ~0.05 nM of LD. + +(D) Rate versus glucose plot in the absence and presence of the LD of RanBP2. At a saturating concentration of the LD of RanBP2 (3.75 nM), the HKI activity was reduced by about 20%. v, rate; S, glucose. + +RanBP2 Colocalizes with HK1, Cox11, and mHsp70 in the Retina and Cultured Neurons + +In addition to its presence at the vicinity of NPCs, we have shown previously that RanBP2 is localized to and abundant in the mitochondria-rich ellipsoid subcellular compartment of photosensory (photoreceptor) neurons of the retina [19]. We extended the subcellular colocalization studies on RanBP2 and its novel partners by immunocytochemistry to determine if RanBP2, Cox11, HKI, and mHsp70 colocalize in hippocampal neurons (Figure 3A–3C), cerebral cortex neurons (Figure 3D–3F), ellipsoid (mitochondria-rich) subcellular compartments of photosensory neurons of the retina (Figure 3G–3O), and dissociated primary glia and neuron cultures from the brain (Figure 3P–3Z). Double immunostaining with antibodies against these proteins showed that they colocalize to the mitochondria (Figure 3A–3Z). + +Figure 3 + +Localization of RanBP2 and Its LD Molecular Partners + +(A–F) are thin cryosections of an area of the hipocampus (CA1 neurons) and cerebral cortex, respectively, immunostained against HKI (A and D), RanBP2 (B and E), and merged images thereof (C and F). Note that while RanBP2 and HKI are widely expressed among and colocalize to hippocampal neurons (C), HKI expression and localization with RanBP2 is restricted to a subset of cortical neurons (likely interneurons) (F). Images of the distal region of bovine retinal cryosections comprising part of the nuclear layer of photoreceptor neurons and their inner (myoid and ellipsoid) segment compartment (G–O) are immunostained against mHsp70 (G) and RanBP2 (H), mHsp70 (J) and Cox11 (K), HKI (M) and Cox11 (N), and merged images thereof (I–O). Note the prominent localization of RanBP2, mHsp70, and Cox11 at the mitochondria-rich ellipsoid compartment of photoreceptors and the colocalization of RanBP2 and Cox11 with mHsp70 (I and L), while HKI colocalization with Cox11 was limited to restricted foci (R, arrowheads). High-resolution images of dissociated primary cerebral neurons and glial cells confirmed that the colocalization of HKI and Cox11 was highly restricted (P–R), while RanBP2 extensively colocalized with HKI (S–U) and mHsp70 (V–Z). Scale bars in A–O and P–Z are 40 and 10 μm, respectively. ONL, outer nuclear layer. + +Haploinsufficiency of RanBP2 Causes Decreased Levels of HKI and Partial Mislocalization of HKI + +To determine the physiological implications of the interaction between RanBP2 and HKI (and other partners), we employed a murine embryonic stem 129Ola cell line with a targeted RanBP2 locus produced by gene-trapping to produce stable inbred (coisogenic) and mixed background lines, respectively (Figure 4A and 4B). Genotyping of 299 F2 offspring revealed a RanBP2+/+:RanBP2+/−:RanBP2−/− distribution (89:210:0) that deviated from the expected Mendelian ratio and supports that the RanBP2−/− mice were embryonically lethal. E12.5 embryos show strong expression of RanBP2 in the optic vesicle and throughout much of the embryo, which had no apparent developmental abnormalities (Figure 4C). In agreement with previous immunocytochemistry analysis [19], the RanBP2 gene is expressed across mature retinal neurons, but expression in ganglion cells of the adult retina was extremely strong (Figure 4D and 4E). + +Figure 4 + +Insertion Mutagenesis of the Murine RanBP2 Gene + +(A) Diagram of the genomic region of RanBP2 disrupted by insertion trap mutagenesis with a bicistronic reporter vector between exon 1 and 2. The bicistronic transcript produces two proteins under regulation of RanBP2. Upon splicing of RanBP2, a fusion between exon 1 and β-geo (a fusion between the β-gal and neo genes) is generated, while human placental alkaline phophatase (PLAP) is independently translated using the internal ribosome entry site. Consistent with previous studies, the expression of the former is directed to cell bodies, while expression of the latter is targeted to the axonal processes [67,68]. Transcriptional 5′ RACE analysis detects a fusion between exon 1 and β-geo. + +(B) Southern analysis of the RanBP2 locus of wild-type and heterozygous genomic DNA of tails of F1 mice digested with PpuMI (left panel) and HindIII (right panel) with probes at the 3′ (left panel) and 5′ (right panel) flanking regions of the insertion breakpoint. Q1 is a cosmid containing the RanBP2 gene up to exon 20 [4]. + +(C) Lateroventral view of a whole-mount stain of a ~12.5 dpc heterozygous embryo for PLAP and β-gal (inset picture) activities. Although PLAP was broadly expressed (e.g., somites, limbs, and CNS), the PLAP and β-Gal (inset picture) expression was particularly high in the optic vesicle (arrow). X-gal single (D) and combined staining with PLAP (E) of a retinal section of a 3-mo-old RanBP2+/− mouse. Consistent with previous immunocytochemistry studies, β-Gal activity is detected in the neuroretinal bodies and inner segment compartment of photoreceptors with conspicuously strong expression in ganglion cells. PLAP expression is found throughout the plexiform/synaptic layers and outer segment of photoreceptors (E). GC, ganglion cell; PLAP, human placental alkaline phophatase; ROS, rod outer segment; RIS, rod inner segment; ONL, outer nuclear layer; OPL, outer plexiform (synaptic) layer; INL, inner nuclear layer; IPL, inner plexiform (synaptic) layer; GC, ganglion cell layer. + +In light of the association in vivo of RanBP2 with Cox11 and HKI (Figures 1 and 3), profound in vitro modulation of HKI enzymatic activity by RanBP2 and Cox11 (Figure 2), and the critical role of HKI in catalyzing a rate-limiting step of glycolysis, we probed whether RanBP2+/− mice presented disturbances in HKI, Cox11, and energy homeostasis. Monoallelic expression of RanBP2 does not affect the number of NPCs and their distribution in hippocampal neurons (Figure 5A, unpublished data), but it led to consistent lower expression of RanBP2 by more than 50% in the CNS (brain and retina) in 129Ola (Figure 5B–5D), but not in C57BL/6J/129Ola backgrounds (unpublished data). Hence, we focused our analysis on the inbred RanBP2+/− 129Ola mouse line. Although the levels of RanBP2 were decreased in the retina, brain, and hippocampus of RanBP2+/−mice by ~50%–60% (Figure 5B and 5C), the levels of others nucleoporins, Nup153 and Nup62, and mHsp70 and Cox11, remained unchanged (Figure 5B, unpublished data). In addition, we observed a strong decrease of the levels of HKI (3- to 4-fold) (Figure 5B and 5C). This decrease was selective to the CNS, since HKI levels remained largely unaffected in the skeletal muscle, spleen, and liver (Figure 5D). Because HKI plays a key role in the production of energy intermediate substrates and HKI is virtually the sole HK isoform expressed in the CNS [33,34], we probed the impact of HKI and RanBP2 reduction in the levels of ATP. As shown in Figure 5E, there was significant and concordant reduction in levels of ATP in the CNS (brain and retina), but not in non-neuronal tissues. Finally, we also observed partial and selective delocalization of HKI, but not of Cox11, from the ellipsoid (mitochondria-rich) to the adjacent myoid subcellular compartment of rod photosensory neurons (Figure S2A–S2E). This was also accompanied by reduced HKI levels in the inner retina, in particular in the inner plexiform (synaptic) layer (Figure S2A–S2E). + +Figure 5 + +Haploinsufficiency of RanBP2 Causes a Decrease in HKI Protein and ATP Levels + +(A) Quantitative analysis of NPCs in dissociated hippocampal neurons of wild-type (+/+) and heterozygote (+/−) mice upon immunostaining with mAb414. No difference in the density of NPCs (3–4 NPC/μm2) at the nuclear envelope was found between RanBP2+/+ and RanBP2+/− mice. + +(B) Immunoblots with anti-RanBP2/Nup153/Nup62 (mAb414), −HKI, −mHsp70, and −Cox11 antibodies of retinal (top panel) and hippocampal homogenates of +/+ and +/− mice. In comparison to RanBP2+/+, RanBP2+/− mice exhibit a reduction in the expression levels of RanBP2 and HKI but not of other proteins. + +(C) Quantitative analysis of relative protein expression levels of RanBP2, Cox11, HKI, and mHsp70 in the hippocampus of RanBP2+/+ and RanBP2+/−mice. There is ~2- and 4-fold reduction of RanBP2 and HKI in heterozygote mice. + +(D) The level of HKI is reduced in the brain but not in other non-neuronal tissues tested (muscle, spleen, and liver). + +(E) The total ATP level is reduced in the CNS tissues (brain and retina) but not in non-neuronal tissues tested (e.g., spleen). + +Metabolic Disturbances Caused by Haploinsufficiency of RanBP2 + +The growth rates of inbred RanBP2+/− mice on high-fat (~10% fat) diet were significantly slower than RanBP2+/+ mice (Figure 6A). Beginning at around 4 mo of age, RanBP2+/− mice exhibit a significant slower gain in body mass than wild-type mice (Figure 6A). In addition, RanBP2+/− inbred mice presented deficits in body mass that were erased by changing the genetic background to a mixed C57BL/6J/129Ola (Figure 6B). Food consumption did not account for the body weight differences observed (Figure 6C). + +Figure 6 + +RanBP2+/− Mice on High-Fat Diet Exhibit Deficits in Growth + +(A) In comparison to wild-type mice, RanBP2+/− mice show slower growth rates beginning at 4 mo of age (arrow), and the difference in body weight between these is maintained afterward. Note that RanBP2+/− mice lack the growth spur observed in wild-type mice between 3 and 4 mo of age. + +(B) In comparison to inbred RanBP2+/−mice (129Ola genetic background), the difference in body weight between RanBP2+/+ and RanBP2+/− mice is masked upon placing these on a mixed 129Ola/C57Bl6 genetic background. + +(C) RanBP2+/+ and RanBP2+/−inbred mice exhibit similar rates of food consumption. Mice in (A), (B), and (C) were placed on a high-fat diet since birth (n = 5). + +HKI in the CNS (brain and retina) accounts virtually for all expression of HK isozymes and glucose utilization in the CNS [33,34]. Moreover, glucose is the sole reliance source of energy in the CNS under normal conditions, the CNS lacks glucose storage sources, and despite the disproportionate mass of the CNS to the rest of the body, the CNS consumes daily about 60% of the body's glucose and 25% of the total oxygen [35,36]. To determine the impact of RanBP2 haploinsufficiency on the utilization, formation, and uptake of glucose, we carried out several physiological assays. In contrast to mice placed on a normal chow diet (~5% fat; unpublished data), RanBP2+/−mice on a higher fat diet (~10% fat) performed significantly worse in the glucose tolerance test beginning at 6 mo of age (Figure 7A and 7B), thus supporting that the RanBP2+/− mice exhibited a deficit in glucose clearance. This deficit was rescued in RanBP2+/− mice of mixed C57BL/6J/129Ola background (Figure S3). Glucose clearance was not affected due to a disturbance in insulin-mediated glucose uptake (Figure 7C). Then, we probed whether RanBP2 induces impairment of gluconeogenesis, which could contribute to the pathophysiological production and clearance of glucose. To this end, the administration of the gluconeogenic substrate precursor, pyruvate (pyruvate tolerance test), showed that there was no difference in glucose production in RanBP2+/− mice (Figure 7D). Hence, partial loss-of-function of RanBP2 had no impact on the gluconeogenesis pathway. However, upon glucose production (15 min), the clearance rates of glucose were again significantly slower in RanBP2+/− than in RanBP2+/+ mice (Figure 7D), confirming an impairment in glucose breakdown. + +Figure 7 + +Metabolic Phenotypes of RanBP2+/− Inbred Mice on High-Fat Diet + +(A) 3-mo-old inbred RanBP2+/− mice (n = 5) have normal glucose clearance rates upon glucose challenge and overnight fasting. + +(B) In contrast, 6-mo-old inbred RanBP2+/− mice (n = 5) have significantly decreased glucose clearance rates upon glucose challenge and overnight fasting. + +(C) Fasted 6- to 8-mo-old RanBP2+/+ and RanBP2+/− mice have no difference in insulin-mediated glucose uptake as assayed by insulin tolerance test (n = 5). + +(D) Pyruvate tolerance test shows normal rise in glucose but decreased glucose clearance between inbred RanBP2+/+ and RanBP2+/− mice (n = 5). + +Haploinsufficiency of RanBP2 Causes Deficits in the Electrophysiological Output of Receptoral and Postreceptoral Retinal Neurons + +In light of the prominent expression of RanBP2 and HKI in retinal neurons [1,19], the vital dependence of the neuronal retina (and brain) on glucose as the main substrate source for energy production, and the determinant impact of metabolic disorders, such as diabetes, in retinal function (e.g., diabetic retinopathy) [37], we probed the impact of deficits in RanBP2, HKI, and ATP, on the electrophysiological responses of subclasses (rod and cone) photoreceptor and postreceptor retinal neurons of RanBP2+/− and in RanBP2+/+ mice. The scotopic (dark-adapted) responses mediated by the rod photoreceptor pathway at low-stimulus intensities and mixed rod and cone pathways at high-stimulus intensities were substantially reduced in RanBP2+/− mice (Figure 8A). The differences in the photopic (light-adapted) responses, initiated by cone photoreceptors, which make up 3% of the photosensory neurons in the mouse retina [38], were less obvious but still exhibited a trend toward reduced amplitudes across a range of increasing light stimulus intensities (Figure 8B). The reduction in the scotopic responses included decreases in both b-wave (Figure 8C) and a-wave (Figure 8D) amplitudes mediated by postreceptoral and receptoral neurons, respectively. Postreceptoral second-order neuron responses, represented by the b-wave, tended to be more consistently and substantially reduced than the a-waves, which directly reflect photoreceptor activity. Since second-order neuron responses depend on input from photoreceptors, this suggests that reduced b-wave amplitudes are the result of the accumulation of decreases in the light response of both photoreceptors and postreceptoral neurons. Anesthetics, particularly ketamine, can cause sustained elevation of glucose in mice, which in turn affects electroretinogram responses [39]. Thus, we were concerned that differences in electroretinogram amplitudes between RanBP2+/− and RanBP2+/+ may reflect differences in glucose level changes in response to anesthesia. However, we found no significant differences in glucose levels measured before and every 15 min during 75 min of anesthesia (n = 4–5). Glucose rose at the same rate and reached a maximum of approximately 3.3 times the pre-anesthesia level in both genotypes (unpublished data). + +Figure 8 + +Electroretinograms from 6-Mo-Old RanBP2+/−and RanBP2+/+ Inbred Mice Showing Photoreceptor and Postreceptor Neuron Electrophysiological Response Phenotypes + +(A) Scotopic (dark-adapted) responses from RanBP2+/− mice to light stimuli of increasing intensity, beginning at threshold, have reduced amplitudes compared to those observed in RanBP2+/+ mice. The three lower intensities represent responses generated in the rod photoreceptor neuronal pathway. The upper intensities are comprised of responses generated in both the rod and cone pathways. + +(B) Photopic (light-adapted, cone photoreceptor pathway) responses of RanBP2+/− mice to increasing light stimulus intensities also exhibited reduced amplitudes compared to those observed in RanBP2+/+ mice. + +(C) Average ± SE (n = 9) scotopic b-wave amplitudes from RanBP2+/− (open circles) and RanBP2+/+ (filled squares) mice representing postreceptoral neuron function. (Note: log amplitude scale.) + +(D) Average ± SE (n = 5) scotopic a-wave amplitudes, representing photoreceptor function, for RanBP2+/− and RanBP2+/+ mice in response to bright flashes. Amplitudes of responses from RanBP2+/− mice were lower over the entire range of stimulus intensities for both b- and a-waves. Asterisks represent significant differences between the groups (Student's t test, p < 0.05). Statistical significance was found across all intensities for b-wave amplitudes (2-way ANOVA, p < 0.0001), but not for the a-wave. + +Discussion + +Our findings support that RanBP2 plays a determinant role in modulating glucose and energy homeostasis. The data support that Cox1 and HKI are novel partners in vivo for the large LD of RanBP2 (Figures 1 and 2). The LD of RanBP2 exhibits chaperone activity toward folding intermediates of Cox11, and possibly, the mature HKI (Figure 1). Cox11 inhibits noncompetitively the activity of HKI with ~2–3 molecules of Cox11 (assuming formation of Cox11 dimer) required to inhibit completely the activity of a molecule of HKI (Figure 2A and 2B). Cox11 sequesters HKI by binding to HKI at a site that is distinct from the active site and effectively reduces the availability of [HKI]tot for catalysis. This is reflected by a reduction of the Vmax of HKI without significantly affecting the Km of the active site of HKI toward glucose (Figure 2A and 2B). The inhibitory property of Cox11 over HKI is suppressed by RanBP2 (Figure 2C), which by itself has a weak but significant inhibitory activity over HKI (Figure 2D). The sub-stochiometry effect of the LD of RanBP2 over the inhibition of HKI by Cox11 supports that a LD-dependent chaperonin-like mechanism underlies the suppression of Cox11-dependent inhibition of HKI by RanBP2, and that RanBP2 acts as a molecular “buffer” over HK1 and Cox11 activities. The partial loss of the RanBP2 chaperone activity in RanBP2+/− mice also leads to deficits in the sequestration of HKI in the ellipsoid compartment of photosensory neurons. This possibly underlies the mistargeting of HKI to the myoid compartment of these neurons (Figure S2). The data support that the ultimate pathophysiological outcome in HKI, caused by a reduction in RanBP2 levels and its chaperone activity, is the selective degradation of HKI as reflected by the reduced levels of HKI (and ATP) but not of other mitochondrial and NPC components (Figure 5). Through this process, it is also possible that deficits in RanBP2 cause a disturbance in the equilibrium between Cox11, HK1, and RanBP2 by leading to an increase of the inhibitory activity of Cox11 over HKI that promotes the uncoupling of the interaction of HKI from RanBP2, ultimately causing HKI degradation. Regardless, lower levels of HKI likely contribute to the decreased levels in ATP, slower growth rates, and diminished ability to metabolize glucose of RanBP2+/− mice (Figures 5–7). As discussed subsequently, the ATP deficits in the retina likely account for the reduced electrophysiological responses of retinal neurons (Figure 8). + +Various domains of RanBP2 have been previously implicated with a chaperone role in the cell. These include the enhancement of the biogenesis of red/green opsin by the combination of the RBD4-CY domains [6,7] and the stabilization by the Ran-binding domains of RanBP2 of the guanosine triphosphate-bound conformational state of RanGTPase and interaction of Ran with importin-β [11,40,41]. The data herein show that the multi-chaperone role of RanBP2 extends also to its LD in light of its ability to associate to distinct folding species of Cox11 and to prevent HKI from being degraded. This chaperone function is likely to be complemented by other partners of RanBP2 with similar and pleiotropic functions. For example, the combination of the CLD of RanBP2 with several subunits of the 19S cap of the proteasome [14], and of its neighboring internal repeat, W1W2/IR with the E3-ubiquitin ligase, parkin [20], and the E2 SUMO-1-conjugating protein, Ubc9 [15] may contribute to the down-regulation of HKI by 26S proteasome-mediated proteolysis and modulation of the molecular and subcellular partitioning of these partners. In this regard, it will be interesting to probe whether parkin also causes deficits in HKI in dopaminergic neurons, since parkin was reported to modulate RanBP2 turnover [20], and parkin loss-of-function also causes energy and growth deficits [26–29,42]. These data add a new dimension to the complexity of the regulation of the glycolytic pathway, in particular in the CNS, where glycolysis plays a major role in supplying energy and where the proteasome machinery may play a critical role in modulating components of the energy supply machinery [43]. This is further evidenced by the presence of genetic modifiers in RanBP2+/−mice on a mixed genetic background that compensates for deficits in RanBP2 and deregulation of its partners in glucose/energy homeostasis as observed in the coisogenic line. The presence of such compensatory mechanisms is also supported by the identification of a quantitative trait locus, which encompasses RanBP2, and modifies the expression of diabetes-related phenotypes [44]. + +The data herein show tissues with a decrease of RanBP2 and HKI levels mirror a reduction in the ATP levels. Since the constitutive Na+/K+ ATPase pump and the ATP-dependent conversion of glutamine to glutamate (both fundamental to maintain the electrical activity in the CNS) consumes the vast majority of the energy produced by the CNS [45–47], this likely underlies the suppression of the electrophysiological output responses of retinal neurons (Figure 8). Moreover, deficits in HKI may lead to intracellular hyperglycemia in the CNS, promote sorbitol-induced osmotic stress, and compromise further the ATPase-dependent Na+/K+ pump activity [48–50]. This may be exacerbated by a decrease of ATP, since the activity of HKI is also stimulated by ATP [51]. The cumulative effects of a reduction in ATP and intracellular hyperglycemia are known to act synergistically and modulate the electrophysiological properties of neuronal activity. Figure 9 integrates in a model these variables and implications of the data presented herein. Still, other bona fide RanBP2 partners previously identified and described in the Introduction also become strong candidates to play a role in energy homeostasis. For example, the unfolding and chaperone activity of components of the 19S cap of the proteasome may be modulated by the CLD of RanBP2 [14] and contribute to the selective HKI (and other substrates) degradation [43], while association of nuclear import receptor, importin-β with the Ran-binding domains of RanBP2 [10,11], may mediate the nuclear translocation of multifunctional substrates, such as glyceraldehyde-3-phosphate dehydrogenase. This trafficking process is selectively inhibited by the neuroprotective drug, R-(-)-deprenyl and derivates thereof, and are often employed to treat Parkinson disease [52–57]. Hence, RanBP2 and its partners emerge as key players and target genes in mediating neuropathophysiological mechanisms implicated in various genetic and environmental lesions to the CNS, as in patients with Parkinson, diabetes with insulin-resistance, and other neuropathies and neurodegenerative diseases, often linked to aging manifestations. To this effect, the RanBP2 mouse model will serve as a unique genetic tool to probe selective, multiple and novel pathways, which may have not been anticipated to be linked to metabolic processes and allied pathophysiological states. + +Figure 9 + +Model Depicting a Role of RanBP2 and Some of Its Partners in Metabolic and Neuronal Function + +RanBP2 interacts with Cox11 and HKI and the triad is in equilibrium under normal physiological conditions. RanBP2 prevents the inhibition of HKI by Cox11 and its degradation. The ultimate effect of RanBP2 on its partners is the stimulation of the glycolytic pathway and production of ATP. The glycolytic pathway is critical to fuel the constitutive Na+/K+-ATPase pump to maintain the dark current between the inner and outer segment compartments of photosensory neurons. A deficit (haploinsufficiency) in RanBP2 disturbs the equilibrium between RanBP2, HKI, and Cox11. This pathophysiological event promotes the destabilization and degradation of HKI and a decrease in ATP production required to maintain the depolarization state neurons, and, hence, a reduction in the response of receptoral and postreceptoral neurons. A reduction in ATP levels also negatively modulates HKI activity/level. Decreased levels of HKI promote intracellular hyperglycemia and activate stress kinases, which modulate negatively the Na+/K+-ATPase pump by phosphorylation. Pathophysiological pathways promoted by RanBP2 haploinsufficiency are represented by dash lines. RIS, rod inner segment; ROS, rod outer segment. + +Finally, RanBP2 appears to join other nucleoporins, such as Nup96 [58], in novel physiological functions that are vital for complex organisms and that were not anticipated from cell-based studies. The reduced expression of these proteins in whole-animal models generates phenotypes that are tissue-restricted and more importantly, do not seem to recapitulate phenotypes observed with knockdown experiments in cell-based culture assays. For example, short interfering RNA-mediated knockdown of RanBP2 [9] and members of the Nup107-Nup160 complex [59–61] have shown, respectively, to cause mitotic arrest and disruption of nuclear pore assembly and deficits in mRNA export in cell culture. Yet, haploinsufficiency of RanBP2 (this work) and Nup96 [58] predominantly produce, instead, CNS-restricted deficits in energy metabolism and alterations in the immune system linked to the down-regulation of interferon-β-regulated proteins and increased susceptibility of viral infection, respectively. While these apparent outcome disparities among experimental systems remain unclear, they could potentially result from variations in redundancies and compensatory mechanisms inherent to each experimental system. Regardless, these genetic and whole-animal models set the stage to probe novel molecular pathways in various physiological and genetic contexts, and provide also a link to pathophysiological processes underlying several human diseases. + +Materials and Methods + +Yeast two-hybrid screening and assays. + +The LD of human RanBP2 (residues 62–711) was subcloned in-frame into the GAL4 DNA-binding domain of bait vector, pBUTE (a kanamycin-resistant version of GAL4 bait vector pGBDUC1) [62] and HybriZAP pBD-GAL4 vector (Stratagene, La Jolla, California, United States). The former was used to screen ~18 million clones via mating from murine 9- to 10-d-old embryo and brain cDNA libraries at the Molecular Interaction Facility (MIF), University of Wisconsin, Madison, Wisconsin. The latter was used to screen via transformation of ~5 million clones from bovine retinal cDNA libraries [32]. The screens generated six clones. One and two in-frame clones were independently isolated from the embryonic and adult brain libraries, respectively, and the interactions were validated. The three clones encoded Cox11. Interactions between Cox11, LD, and subdomains thereof were quantified by liquid β-galactosidase (Applied Biosystems, Foster City, California, United States) and growth assays [63]. The maximum specific growth speed (μmax) was determined by calculating μmax = (ln(xt) − ln(x0))/t, where xt is the OD600 of the culture at t = t, x0 is the OD600 at t = 0 and t is the time between x0 and xt. Assays were performed with three independent clones and three samples of each clone, the results were averaged, and the standard deviations calculated. + +Site-directed and deletion mutagenesis and plasmid construction. + +Deletion mutagenesis was carried out with pairs of primers against domains of interest described in the figure legends. PCR products were subcloned into pGEX-KG [64], HybriZAP pBD-GAL4 vector (Stratagene), and pBUTE [62] vectors. GST-LDZIP alone comprised residues 447–483 of human RanBP2. + +GST pull-down and immunoprecipitation assays. + +CHAPS-solubilized retinal extracts, expression, and purification of GST-fused constructs were prepared as previously described [65]. GST pull-down assays were carried out with 0.5–2.2 μM of GST-fused proteins [65]. Co-precipitates were resolved on SDS-PAGE and analyzed by Western blot with antibodies described in the Results section. Unfolded and partially denatured Cox11 were generated by incubating recombinant and native Cox11 overnight with 5.6 and 7 M guanidine hydrochloride and urea, respectively. The Cox11 conformers were then diluted ~20-fold in CHAPS-binding buffer containing GST-LD. Immunoprecipitation assays with 5 μg of antibody and Western blots (~ 200–400 ng/ml of antibody) were performed exactly as described previously [13]. + +Hexokinase I assay. + +Hexokinase I activity was determined spectrophotometrically at 25 °C by the method of coupling the glucose-6-phosphate production via glucose-6-phosphate dehydrogenase with the change in the absorbance of NADPH at 340 nm and as described by [66]. The reaction was started by the addition of purified brain hexokinase I (0.24 μg) (gift from J. Wilson) to 1.0 ml of reaction mixture containing 0.05 M Tris-HCl (pH 8.5), 7.4 mM MgCl2, 6.6 mM ATP, 0.65 mM NADP, 11.1 mM monothioglycerol, and 1 unit of glucose 6-phosphate dehydrogenase. In the case of hexokinase activity measured in the presence of Cox11 and LD, the purified HKI was incubated with the recombinant proteins for 15 min at 4 °C before measurement of the activity. The data were fitted directly into the Michaelis-Menten equation using SIGMAPLOT (SPSS Science). + +Antibodies. + +Rabbit antisera were raised against the recombinant murine Cox11 (residues 40–275) and affinity-purified under non-denaturing conditions (Stereogene) as previously described [13]. Cox11 antibodies were used at 200 ng/ml for Western analysis. The monoclonal antibody Hsp70 was from Affinity Bioreagents (Golden, Colorado, United States) and antibodies against the KBD (JX2) and ZnF domains of RanBP2 have been described [13,16]. Polyclonal and monoclonal antibodies against Hexokinase I were provided by J. Wilson. The mAb414 was purchased from Abcam (Cambridge, Massachusetts, United States). + +Immunocytochemistry and microscopy. + +Retina dissections, radial cryosections (~6 μm), and immunohistochemistry procedures were carried out as described elsewhere [19]. Brains from 3- to 5-mo-old C57Bl/6 mice were fixed overnight in 2% paraformaldehyde, infused with 30% sucrose, and processed for cryosectioning. Primary brain neurons and glial cells were prepared from the cerebral cortex. This was macerated in the Hank's Balanced Salt solution and triturated. Primary cells were cultured in DMEM (GIBCO, San Diego, California, United States) and collagen-coated 35-mm glass bottom culture dishes (MatTek Corporation, Ashland, Maine) at 5% CO2/37 °C for ~2 d and processed for immunocytochemistry. Primary antibodies were used at concentrations ~2.5–10 μg/ml. Alexa 488- and Alexa 594-conjugated secondary antibodies (2.5 μg/ml) (Molecular Probes) were used for visualization of proteins. Crossover of fluorescent probes, background, and autofluorescence were found to be negligible. Visualization of specimens and localization of proteins were carried out by wide-field epifluorescence microscopy on Nikon (Tokyo, Japan) E600 upright and TE2000U inverted research microscopes equipped with similar Apochromat objectives. Images with the Nikon E600 were acquired with a SPOT-RT digital camera coupled to the microscope and driven by SPOT Imaging v4.0 software (Diagnostic Instruments, Sterling Heights, Michigan, United States). All images were captured at nonsaturating integration levels, 12-bit mono black/white, and then pseudo-colored. Protein colocalization analysis was carried out on the Nikon TE2000U microscope equipped with appropriate excitation and emission filter wheels, cube filters, 100 W mercury light source, Nomarski/DIC, and Plan Apochromat optics (100×, 60×, and 40× oil objectives with NA of 1.4, 40 ×LWD, and 20 ×LWD objectives and encoded motorized Z-Stage (Prior Scientific, Rockland, Maryland, United States). Images with the inverted Nikon TE2000U microscope were obtained using a CCD camera (CoolSNAP HQ; Roper Scientific, Trenton, New Jersey, United States). Images acquired under identical acquisition parameters were analyzed using Metamorph Software v6.2 (Universal Imaging, Glendale, Wisconsin, United States). Whenever applicable, serial optical Z-stacks (20–30 focal planes at 100-nm intervals) were captured and computationally processed by 3-D blind deconvolution methods with the same software. + +Generation of a mouse line with a disrupted RanBP2 locus and histological analysis of trapped RanBP2 mice. + +A feeder-independent mouse ES cell line derived from the 129P2/OlaHsd strain and harboring a disrupted RanBP2 locus by insertion mutagenesis with the gene trap vector, pGT0pfs [67,68] (generated by W. Skarnes), was obtained from the Mutant Mouse Regional Resource Center (University of California Davis, Davis, California, United States). The gene trap vector contains a promoterless neo-lacZ fusion gene with a splice acceptor site at the 5′ end followed by an internal ribosome entry site (IRES) and the human placental alkaline phosphatase (PLAP) reporter cassette. The RanBP2 129Ola ES line was injected into C57BL/6J blastocysts and four male chimeras were generated. These were bred into 129P2/OlaHsd (Harlan, Indianapolis, Indiana, United States) and C57BL/6J (Jackson Laboratory, Bar Harbor, Maine, United States) females. The two resulting and independent F1s lines, with 129P2/OlaHsd inbred and 129P2/OlaHsd/C57BL/6J mixed genetic backgrounds, were tested for germline transmission by PCR and Southern blot analysis of tail genomic DNA. β-gal and PLAP activities in whole mount embryos and retinal sections were detected, respectively, by incubation with 5-bromo-4-chloro-3-indolyl β-D-galactopyranoside and AP staining buffer (0.1 mg/ml 5-bromo-4-chloro-3-indolyl phosphate, 1 mg/ml nitroblue tetrazolium in 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl2) as described elsewhere [67,68]. + +Western blot and quantitation analysis of protein expression and ATP levels. + +Homogenates and detergent solubilized extracts of tissues and immunoblots were prepared as described elsewhere [69]. Quantitation of immunoblot bands calibrated against internal marker bands, such as mHsp70 and Nup153, was carried out with Gel Pro Analyzer (MediaCybernetics, San Diego, California, United States). For determination of ATP levels, freshly dissected and flash-frozen tissues were homogenized in 2.5% trichloroacetic acid (TCA), neutralized, and diluted to a final concentration of 0.1% with Tris-Acetate (pH 7.75). ATP measurements were carried out with the ENLITEN ATP Assay System Bioluminescence Detection Kit for ATP (Promega, Madison, Wisconsin, United States) as per the manufacturer's instruction in SpectraMax-M5 (Molecular Devices, Sunnyvale, California, United States). Protein concentration was measured by the BCA method (BioRad, Hercules, California, United States) using BSA as a standard. + +Metabolic assays. + +Two groups of male mice were kept under separate diet since gestation or birth. One group was fed with a normal chow diet (PicoLab Rodent Diet 20 −5053; LabDiet, Richmond, Indiana, United States), while the other was placed on a higher energy and fat diet (PicoLab Mouse Diet 20 −5058; LabDiet). Glucose tolerance test was performed on overnight-fasted mice injected intraperitoneally with 2 g of glucose per kg of body weight. Venous blood glucose values were determined at immediately before (0 min), and 15, 30, 60, and 120 min after the injection with an automatic glucose monitor instrument (One Touch Ultra, Lifescan). Insulin tolerance test was performed the same way as that described for the glucose tolerance test with the exception that human insulin (0.75 U/kg of body mass) was injected intraperitoneally in non-fasted mice. Pyruvate challenge test was performed the same way as that described for the glucose tolerance test with the exception that pyruvate dissolved in saline (2 g/kg of body mass) was injected intraperitoneally in overnight-fasted mice. + +Electroretinography. + +Five ~6-mo-old male RanBP2+/+ and RanBP2+/− mice in 129ola background were kept in dim cyclic light (10 lux) for 2 wk before electroretinographic responses were recorded. The mice were anesthetized intraperitoneally with ketamine (80 mg/kg) and xylazine (4 mg/kg). Body temperature was maintained with a heating pad at 37.5 ± 1 °C throughout the recording. The pupils were fully dilated with 0.5% tropicamide and 2.5% phenylephrine HCl, and both eyes were recorded simultaneously. Corneal gold-wire loops were used as the active electrodes under 0.5% proparacaine hydrochloride topical anesthesia. Gold-wire electrodes placed on the sclera near the limbus served as reference electrodes. A ground wire was attached to the ear. Scotopic responses were recorded from threshold to 9 log units of intensity above threshold, amplified, and filtered (5,000 gain, 0.1–1,000 Hz). For photopic recordings background light at 34 cd/m2 was used to suppress rod function. + +Supporting Information + +Figure S1 + +Western Blot Analysis of the Association of LD of RanBP2 with HKI and Cox11 across Tissues + +GST-JX2 (2.2 μM) was incubated with different tissue extracts (~2.0 mg) and the GST-JX2 coprecipitates from seven different tissues were analyzed by Western blot with the antibodies against HKI and Cox11. The LD of RanBP2 associated with the HKI and Cox11 in all seven tissues tested. + +(59 KB PDF) + +Click here for additional data file. + +Figure S2 + +Immunostaining of Radial Cryosections of Wild-Type (+/+) and Heterozygote (+/−) RanBP2 Mice Retinas with Anti-HKI and −Cox11 Antibodies + +A1 and B1 represent magnifications of just the distal section of the retina (inner segment subcellular compartment of the photosensory neuron layer). Note the partial delocalization of HKI to the myoid subcompartment of the inner segment of photoreceptor neurons in +/− mice (B1) and decreased levels of HKI in the inner plexiform (synaptic) layer of the retina (A and B). This is reflected by a significant increase and decrease of the mean fluorescence intensity (in arbitrary units) of HKI in the myoid and inner plexiform (synaptic) layers, respectively (E). AU, arbitrary unit; ROS, rod outer segment; RIS, rod inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer, INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer; E and M are, respectively, the ellipsoid (mitochondria-rich) and myoid compartments of photosensory neurons. + +(476 KB PDF) + +Click here for additional data file. + +Figure S3 + +Metabolic Phenotype of RanBP2+/− Mice in a Mixed Background and High-Fat Diet + +There was no difference in the glucose tolerance test between RanBP2+/+ and RanBP2+/−mice on a mixed 129Ola/C57Bl6 genetic background (C). + +(38 KB PDF) + +Click here for additional data file. + +Acknowledgements + +We thank John Wilson for purified HKI, antibodies against HKI, HKII, HKIII, and glucokinase, and support; Arthur Haas for help with kinetic analysis of HKI; and William Skarnes for helpful suggestions with the analysis of ES insertion trap lines. + +Abbreviations + +CLD - cyclophilin-like domain + +CNS - central nervous system + +GST - glutathione-S-transferase + +HKI - hexokinase type I + +LD - leucine-rich domain + +NPC - nuclear pore complex + +RanBP2 - Ran-binding protein 2 + +Footnotes + +¤ Current address: Fugent LLC, Madison, Wisconsin, United States of America + +Competing interests. The authors have declared that no competing interests exist. + +A previous version of this article appeared as an Early Online Release on September 1, 2006 (DOI: 10.1371/journal.pgen.0020177.eor). + +Author contributions. AA, RB, MG, DR, RAB, and PAF conceived and designed the experiments. AA, RB, MG, JO, DR, XL, and CBB performed the experiments. AA, RB, MG, JO, DR, RAB, PAS, and PAF analyzed the data. PAF contributed reagents/materials/analysis tools. PAF wrote the paper. + +Funding. Work supported by The National Institutes of Health EY11993/EY012665 to PAF and The National Institutes of Health 2P30-EY005722–21. PAF is the Jules and Doris Stein Research to Prevent Blindness Professor. diff --git a/src/ontogpt/evaluation/craft/database/all/17078885.ann b/src/ontogpt/evaluation/craft/database/all/17078885.ann new file mode 100644 index 000000000..3fe22e6d5 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17078885.ann @@ -0,0 +1,940 @@ +T1 PR:000000047 10 14 ALK5 +T2 UBERON:0002342 32 44 neural crest +T3 UBERON:0002342 78 90 neural crest +T4 GO:0006915 91 105 cell apoptosis +T5 UBERON:0004145 117 130 outflow tract +T6 http://purl.obolibrary.org/obo/MONDO_0021140 162 172 Congenital +T7 UBERON:0004535 173 187 cardiovascular +T8 http://purl.obolibrary.org/obo/MONDO_0004995 173 196 cardiovascular diseases +T9 GO:0007567 225 230 birth +T10 http://purl.obolibrary.org/obo/MONDO_0000839 225 238 birth defects +T11 NCBITaxon:9606 242 248 humans +T12 GO:0001755 339 348;381 383;404 416;422 427 migration ... of ... neural crest ... cells +T13 GO:0030154 350 365;381 383;422 427 differentiation ... 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UBERON:0001584 7110 7134 left subclavian arteries +T222 UBERON:0001508 7161 7172 aortic arch +T223 PR:000000047 7187 7191 Alk5 +T224 UBERON:0000922 7207 7214 embryos +T225 UBERON:0002061 7257 7271 arterial trunk +T226 UBERON:0000922 7319 7326 embryos +T227 http://purl.obolibrary.org/obo/MONDO_0009010 7347 7370 interrupted aortic arch +T228 UBERON:0001508 7359 7370 aortic arch +T229 PR:000000047 7422 7426 Alk5 +T230 UBERON:0004145 7453 7466 outflow tract +T231 UBERON:0001514 7516 7532 descending aorta +T232 UBERON:0001584 7544 7566 left subclavian artery +T233 UBERON:0005396 7572 7588 carotid arteries +T234 UBERON:0001508 7800 7811 aortic arch +T235 UBERON:0005396 7829 7845 carotid arteries +T236 UBERON:0005396 7926 7942 carotid arteries +T237 UBERON:0001534 8023 8028;8038 8057 right ... subclavian arteries +T238 UBERON:0001584 8033 8057 left subclavian arteries +T239 UBERON:0016920 8091 8109;8114 8125 descending part of ... aortic arch +T240 UBERON:0001652 8158 8162;8173 8191 left ... pulmonary arteries +T241 UBERON:0001651 8167 8191 right pulmonary arteries +T242 UBERON:0002061 8228 8242 arterial trunk +T243 PR:000000047 8257 8261 Alk5 +T244 PR:000017435 8262 8266 Wnt1 +T245 http://purl.obolibrary.org/obo/MONDO_0018072 8302 8305 PTA +T246 http://purl.obolibrary.org/obo/MONDO_0009010 8360 8383 interrupted aortic arch +T247 UBERON:0001508 8372 8383 aortic arch +T248 PR:000017435 8409 8413 Wnt1 +T249 PR:000000047 8458 8462 Alk5 +T250 UBERON:0002342 8466 8478 neural crest +T251 http://purl.obolibrary.org/obo/MONDO_0018072 8494 8523 persistent truncus arteriosus +T252 UBERON:0002061 8505 8523 truncus arteriosus +T253 CHEBI:37958 8546 8549 dye +T254 UBERON:0004145 8562 8565 OFT +T255 GO:0003151 8562 8579 OFT morphogenesis +T256 PR:000017435 8610 8614 Wnt1 +T257 http://purl.obolibrary.org/obo/MONDO_0018072 8652 8655 PTA +T258 UBERON:0002061 8667 8685 truncus arteriosus +T259 http://purl.obolibrary.org/obo/MONDO_0009010 8691 8714 interrupted aortic arch +T260 UBERON:0001508 8703 8714 aortic arch +T261 PR:000000047 8725 8729 Alk5 +T262 PR:000017435 8730 8734 Wnt1 +T263 UBERON:0002061 8814 8821 truncus +T264 UBERON:0001496 8935 8950 ascending aorta +T265 UBERON:0001496 8952 8954 Ao +T266 UBERON:0002333 8960 8975 pulmonary trunk +T267 UBERON:0002333 8977 8979 PT +T268 UBERON:0006060 9002 9013 conotruncal +T269 UBERON:0003037 9014 9020 septum +T270 PR:000000047 9025 9029 Alk5 +T271 PR:000017435 9030 9034 Wnt1 +T272 UBERON:0006060 9057 9068 conotruncal +T273 UBERON:0003037 9069 9075 septum +T274 UBERON:0001508 9140 9151 aortic arch +T275 UBERON:0005396 9187 9203 carotid arteries +T276 UBERON:0002061 9213 9220 truncus +T277 UBERON:0000947 9269 9271 Ao +T278 UBERON:0000947 9273 9278 aorta +T279 UBERON:0002333 9280 9282 PT +T280 UBERON:0002333 9284 9299 pulmonary trunk +T281 UBERON:0001534 9301 9304 RSA +T282 UBERON:0001534 9306 9329 right subclavian artery +T283 UBERON:0005396 9342 9356 carotid artery +T284 UBERON:0005396 9368 9382 carotid artery +T285 UBERON:0001584 9384 9387 LSA +T286 UBERON:0001584 9389 9411 left subclavian artery +T287 http://purl.obolibrary.org/obo/MONDO_0009010 9413 9416 IAA +T288 http://purl.obolibrary.org/obo/MONDO_0009010 9418 9441 interrupted aortic arch +T289 UBERON:0001508 9430 9441 aortic arch +T290 http://purl.obolibrary.org/obo/MONDO_0018072 9443 9446 PTA +T291 http://purl.obolibrary.org/obo/MONDO_0018072 9448 9477 persistent truncus arteriosus +T292 UBERON:0002061 9459 9477 truncus arteriosus +T293 UBERON:0004363 9507 9531 pharyngeal arch arteries +T294 UBERON:0005432 9536 9546 aortic sac +T295 PR:000000047 9550 9554 Alk5 +T296 UBERON:0004535 9579 9593 cardiovascular +T297 GO:0072358 9579 9605 cardiovascular development +T298 UBERON:0004363 9611 9615 PAAs +T299 UBERON:0001508 9708 9719 aortic arch +T300 PR:000000047 9743 9747 ALK5 +T301 UBERON:0004363 9797 9801 PAAs +T302 UBERON:0000948 9821 9828 cardiac +T303 PR:000000047 9899 9903 Alk5 +T304 PR:000017435 9904 9908 Wnt1 +T305 UBERON:0004363 9961 9965 PAAs +T306 UBERON:0003120 10081 10084;10098 10102 3rd ... PAAs +T307 UBERON:0003121 10086 10089;10098 10102 4th ... PAAs +T308 UBERON:0003123 10094 10102 6th PAAs +T309 UBERON:0010198 10118 10130 carotid duct +T310 UBERON:0005805 10136 10148 dorsal aorta +T311 UBERON:0003120 10161 10164;10173 10177 3rd ... PAAs +T312 UBERON:0003121 10169 10177 4th PAAs +T313 GO:0060033 10191 10201 regressing +T314 PR:000000047 10252 10256 Alk5 +T315 PR:000017435 10257 10261 Wnt1 +T316 UBERON:0004363 10300 10304 PAAs +T317 UBERON:0004363 10359 10363 PAAs +T318 UBERON:0010198 10417 10429 carotid duct +T319 UBERON:0010198 10531 10543 carotid duct +T320 UBERON:0005805 10640 10652 dorsal aorta +T321 UBERON:0004363 10686 10690 PAAs +T322 UBERON:0004363 10740 10744 PAAs +T323 PR:000000047 10748 10752 Alk5 +T324 PR:000017435 10753 10757 Wnt1 +T325 UBERON:0000948 10800 10807 cardiac +T326 UBERON:0004363 10851 10855 PAAs +T327 PR:000000047 10928 10932 Alk5 +T328 PR:000017435 10933 10937 Wnt1 +T329 GO:0060033 10986 10996 regressing +T330 UBERON:0010198 10997 11009 carotid duct +T331 GO:0060033 11092 11102 regression +T332 GO:0060033 11143 11153 regression +T333 UBERON:0005805 11161 11173 dorsal aorta +T334 UBERON:0003121 11186 11189;11198 11202 4th ... PAAs +T335 UBERON:0003123 11194 11202 6th PAAs +T336 UBERON:0002333 11204 11206 PT +T337 UBERON:0002333 11208 11223 pulmonary trunk +T338 UBERON:0000947 11225 11227 Ao +T339 UBERON:0000947 11229 11234 Aorta +T340 UBERON:0002061 11236 11238 TA +T341 UBERON:0002061 11240 11258 truncus arteriosus +T342 UBERON:0005432 11279 11289 aortic sac +T343 UBERON:0001529 11484 11506 brachiocephalic artery +T344 UBERON:0003121 11551 11558 4th PAA +T345 UBERON:0001508 11588 11599 aortic arch +T346 PR:000000047 11609 11613 Alk5 +T347 PR:000017435 11614 11618 Wnt1 +T348 UBERON:0005432 11645 11655 aortic sac +T349 UBERON:0002061 11697 11704 truncus +T350 UBERON:0004363 11771 11775 PAAs +T351 UBERON:0003123 11817 11825 6th PAAs +T352 UBERON:0005432 11945 11955 aortic sac +T353 UBERON:0000323 11959 11977 late stage embryos +T354 UBERON:0001529 12003 12025 brachiocephalic artery +T355 UBERON:0002061 12065 12072 truncus +T356 UBERON:0005432 12119 12129 Aortic Sac +T357 PR:000000047 12133 12137 Alk5 +T358 PR:000017435 12138 12142 Wnt1 +T359 PR:000000047 12156 12160 Alk5 +T360 PR:000017435 12161 12165 Wnt1 +T361 UBERON:0005432 12236 12246 aortic sac +T362 UBERON:0000922 12317 12324 embryos +T363 CHEBI:51686 12349 12350 H +T364 UBERON:0005432 12465 12475 aortic sac +T365 UBERON:0000948 12519 12526 Cardiac +T366 PR:000000047 12545 12549 Alk5 +T367 UBERON:0004145 12567 12580 outflow tract +T368 UBERON:0004145 12686 12699 outflow tract +T369 PR:000000047 12717 12721 Alk5 +T370 SO:0000359 12721 12725 Flox +T371 SO:0000359 12726 12730 Flox +T372 NCBITaxon:10088 12731 12735 mice +T373 NCBITaxon:10088 12778 12782 mice +T374 PR:000000047 12801 12805 Alk5 +T375 SO:0000359 12805 12809 Flox +T376 SO:0000359 12810 12814 Flox +T377 PR:000000047 12851 12855 Alk5 +T378 PR:000017435 12861 12865 Wnt1 +T379 UBERON:0000922 12891 12898 embryos +T380 UBERON:0002342 12907 12909 NC +T381 GO:0010467 12959 12969 expression +T382 UBERON:0000922 13063 13070 embryos +T383 GO:0001755 13142 13155 NCC migration +T384 UBERON:0000922 13258 13265 embryos +T385 UBERON:0004145 13335 13338 OFT +T386 PR:000000047 13383 13387 Alk5 +T387 UBERON:0004363 13419 13423 PAAs +T388 UBERON:0005432 13425 13435 aortic sac +T389 UBERON:0005967 13440 13458 conotruncal ridges +T390 PR:000000047 13550 13554 Alk5 +T391 PR:000017435 13555 13559 Wnt1 +T392 UBERON:0006562 13634 13644 pharyngeal +T393 UBERON:0004145 13649 13662 outflow tract +T394 UBERON:0000948 13690 13697 cardiac +T395 GO:0001755 13698 13711 NCC migration +T396 PR:000000047 13715 13719 Alk5 +T397 PR:000017435 13720 13724 Wnt1 +T398 UBERON:0004145 13742 13745 OFT +T399 PR:000000047 13772 13776 Alk5 +T400 PR:000017435 13777 13781 Wnt1 +T401 UBERON:0003121 13992 13995;14018 14022 4th ... PAAs +T402 UBERON:0003123 14007 14010;14018 14022 6th ... PAAs +T403 UBERON:0005432 14126 14136 Aortic sac +T404 UBERON:0006207 14141 14165 aortico-pulmonary septal +T405 http://purl.obolibrary.org/obo/MONDO_0021902 14141 14173 aortico-pulmonary septal defects +T406 PR:000000047 14177 14181 Alk5 +T407 UBERON:0000922 14197 14204 embryos +T408 UBERON:0004145 14223 14236 outflow tract +T409 UBERON:0005432 14309 14319 aortic sac +T410 UBERON:0000948 14346 14351 heart +T411 UBERON:0003104 14383 14393 mesenchyme +T412 UBERON:0002342 14411 14413 NC +T413 UBERON:0005432 14439 14449 aortic sac +T414 UBERON:0003121 14473 14476;14485 14489 4th ... PAAs +T415 UBERON:0003123 14481 14489 6th PAAs +T416 UBERON:0006207 14534 14551;14557 14563 aortico-pulmonary ... septum +T417 UBERON:0002061 14580 14587 truncal +T418 UBERON:0006207 14605 14640 aortico-pulmonary septation complex +T419 UBERON:0005432 14653 14663 aortic sac +T420 UBERON:0000922 14699 14706 embryos +T421 UBERON:0006060 14734 14745 conotruncal +T422 UBERON:0002061 14772 14779 truncus +T423 UBERON:0003983 14784 14789 conus +T424 UBERON:0002061 14878 14885 truncal +T425 UBERON:0003983 14890 14895 conal +T426 PR:000000047 14931 14935 Alk5 +T427 PR:000017435 14936 14940 Wnt1 +T428 UBERON:0004145 14957 14970 outflow tract +T429 UBERON:0006060 15036 15047 conotruncal +T430 UBERON:0005432 15143 15153 aortic sac +T431 UBERON:0005432 15241 15251 aortic sac +T432 UBERON:0002061 15256 15263 truncal +T433 UBERON:0004145 15264 15267 OFT +T434 UBERON:0006207 15371 15380 AP-septal +T435 UBERON:0003104 15381 15391 mesenchyme +T436 UBERON:0004145 15421 15424 OFT +T437 UBERON:0000947 15432 15437 aorta +T438 UBERON:0002333 15446 15461 pulmonary trunk +T439 UBERON:0000479 15524 15530 tissue +T440 UBERON:0002342 15551 15553 NC +T441 PR:000003675 15580 15585 α-SMA +T442 UBERON:0001135 15614 15627 smooth muscle +T443 PR:000000047 15641 15645 Alk5 +T444 PR:000017435 15646 15650 Wnt1 +T445 UBERON:0005432 15698 15708 aortic sac +T446 UBERON:0002061 15713 15720 truncal +T447 UBERON:0004145 15721 15724 OFT +T448 UBERON:0006207 15780 15789 AP-septum +T449 UBERON:0004145 15918 15921 OFT +T450 UBERON:0002342 15930 15932 NC +T451 UBERON:0005432 15979 15989 aortic sac +T452 UBERON:0003123 16012 16022 sixth PAAs +T453 UBERON:0002061 16031 16038 truncus +T454 PR:000003675 16059 16063 αSMA +T455 UBERON:0002342 16117 16119 NC +T456 PR:000000067 16169 16173 Alk2 +T457 http://purl.obolibrary.org/obo/MONDO_0018072 16183 16186 PTA +T458 PR:000000067 16204 16208 Alk2 +T459 PR:000017435 16209 16213 Wnt1 +T460 UBERON:0005432 16247 16257 aortic sac +T461 UBERON:0002061 16262 16269 truncal +T462 UBERON:0004145 16270 16273 OFT +T463 PR:000000047 16313 16317 Alk5 +T464 PR:000017435 16318 16322 Wnt1 +T465 PR:000000067 16348 16352 Alk2 +T466 UBERON:0004363 16381 16385 PAAs +T467 PR:000000067 16425 16429 Alk2 +T468 PR:000017435 16430 16434 Wnt1 +T469 UBERON:0003037 16480 16486 septal +T470 UBERON:0000479 16487 16493 tissue +T471 UBERON:0003121 16506 16509;16518 16522 4th ... PAAs +T472 UBERON:0003123 16514 16522 6th PAAs +T473 UBERON:0003037 16550 16556 septal +T474 UBERON:0003104 16557 16567 mesenchyme +T475 PR:000000067 16576 16580 Alk2 +T476 UBERON:0002061 16618 16625 truncal +T477 UBERON:0006207 16651 16660 AP septum +T478 UBERON:0003123 16680 16688 6th PAAs +T479 UBERON:0005432 16831 16841 aortic sac +T480 UBERON:0002061 16850 16857 truncal +T481 PR:000003675 16906 16910 αSMA +T482 PR:000000047 16962 16966 Alk5 +T483 PR:000000067 16990 16994 ALK2 +T484 UBERON:0001135 17029 17042 smooth muscle +T485 CL:0000192 17029 17047 smooth muscle cell +T486 GO:0030154 17043 17063 cell differentiation +T487 PR:000000067 17118 17122 Alk2 +T488 PR:000000047 17127 17131 Alk5 +T489 UBERON:0005432 17190 17200 aortic sac +T490 UBERON:0002061 17209 17216 truncal +T491 UBERON:0004145 17217 17220 OFT +T492 UBERON:0006207 17250 17259 AP septum +T493 UBERON:0002061 17354 17361 truncal +T494 UBERON:0004145 17362 17365 OFT +T495 PR:000000047 17385 17389 Alk5 +T496 PR:000017435 17390 17394 Wnt1 +T497 UBERON:0000922 17499 17506 embryos +T498 UBERON:0004145 17552 17555 OFT +T499 UBERON:0006060 17587 17598 conotruncal +T500 UBERON:0005432 17721 17731 aortic sac +T501 UBERON:0005432 17738 17739 s +T502 UBERON:0005432 17742 17752 aortic sac +T503 UBERON:0002061 17754 17755 t +T504 UBERON:0002061 17757 17764 truncus +T505 UBERON:0003983 17766 17767 c +T506 UBERON:0003983 17769 17774 conus +T507 PR:000000047 17801 17805 ALK5 +T508 PR:000000067 17810 17814 ALK2 +T509 GO:0065007 17815 17823 controls +T510 UBERON:0000947 17845 17852 aortico +T511 UBERON:0002048 17853 17862 pulmonary +T512 PR:000000047 17960 17964 Alk5 +T513 PR:000017435 17965 17969 Wnt1 +T514 PR:000000067 17991 17995 Alk2 +T515 PR:000017435 17996 18000 Wnt1 +T516 CHEBI:51686 18062 18063 H +T517 PR:000003675 18123 18127 αSMA +T518 UBERON:0003123 18186 18187 6 +T519 UBERON:0003123 18193 18200 6th PAA +T520 UBERON:0005432 18202 18204 AS +T521 UBERON:0005432 18206 18216 aortic sac +T522 UBERON:0002061 18218 18220 TA +T523 UBERON:0002061 18222 18240 truncus arteriosus +T524 UBERON:0000947 18242 18244 Ao +T525 UBERON:0000947 18246 18251 Aorta +T526 UBERON:0002333 18253 18255 PT +T527 UBERON:0002333 18257 18272 pulmonary trunk +T528 UBERON:0006207 18312 18321 AP septal +T529 UBERON:0003104 18322 18332 mesenchyme +T530 PR:000000047 18335 18339 Alk5 +T531 PR:000017435 18340 18344 Wnt1 +T532 GO:0006915 18375 18387;18416 18421 apoptosis of ... cells +T533 UBERON:0002342 18403 18415 neural crest +T534 PR:000000047 18443 18447 Alk5 +T535 PR:000017435 18448 18452 Wnt1 +T536 UBERON:0006207 18499 18508 AP-septal +T537 UBERON:0000479 18509 18515 tissue +T538 UBERON:0005432 18535 18545 aortic sac +T539 UBERON:0003121 18569 18572;18581 18585 4th ... PAAs +T540 UBERON:0003123 18577 18585 6th PAAs +T541 GO:0008283 18657 18670 proliferation +T542 GO:0006915 18688 18697 apoptosis +T543 CHEBI:472552 18707 18711 BrdU +T544 GO:0008283 18757 18770 proliferation +T545 PR:000000047 18791 18795 Alk5 +T546 UBERON:0000479 18899 18906 tissues +T547 UBERON:0005432 18923 18933 aortic sac +T548 UBERON:0006207 18963 18972 AP-septum +T549 PR:000033987 19010 19014 lacZ +T550 MOP:0000780 19209 19216 cleaved +T551 PR:000018762 19209 19226 cleaved caspase-3 +T552 GO:0006915 19247 19256 apoptosis +T553 CL:0000445 19284 19293;19315 19320 apoptotic ... cells +T554 UBERON:0002342 19294 19306 neural crest +T555 UBERON:0006207 19380 19389 AP septum +T556 UBERON:0005967 19409 19420 OFT cushion +T557 UBERON:0003104 19421 19431 mesenchyme +T558 GO:0006915 19459 19468 apoptosis +T559 PR:000000047 19564 19568 Alk5 +T560 PR:000017435 19569 19573 Wnt1 +T561 PR:000000047 19622 19626 Alk5 +T562 PR:000017435 19627 19631 Wnt1 +T563 UBERON:0000922 19643 19650 embryos +T564 GO:0006915 19662 19674;19686 19691 apoptosis of ... cells +T565 UBERON:0002342 19675 19677 NC +T566 UBERON:0004145 19743 19746 OFT +T567 PR:000000067 19774 19778 Alk2 +T568 GO:0006915 19820 19829 apoptosis +T569 PR:000000047 19841 19845 Alk5 +T570 PR:000017435 19846 19850 Wnt1 +T571 MOP:0000780 19880 19887 Cleaved +T572 PR:000018762 19880 19897 Cleaved Caspase-3 +T573 GO:0006915 19958 19967 apoptosis +T574 PR:000000047 19971 19975 Alk5 +T575 PR:000017435 19976 19980 Wnt1 +T576 UBERON:0005432 20010 20020 aortic sac +T577 PR:000000067 20066 20070 Alk2 +T578 PR:000017435 20071 20075 Wnt1 +T579 UBERON:0004145 20134 20137 OFT +T580 PR:000000047 20200 20204 Alk5 +T581 PR:000000067 20212 20216 Alk2 +T582 PR:000033987 20253 20257 lacZ +T583 UBERON:0000922 20266 20273 embryos +T584 CL:0000445 20292 20307 apoptotic cells +T585 UBERON:0002342 20315 20327 neural crest +T586 UBERON:0005432 20359 20361 AS +T587 UBERON:0005432 20363 20373 aortic sac +T588 CL:0000445 20403 20418 apoptotic cells +T589 UBERON:0005432 20435 20445 aortic sac +T590 PR:000000047 20489 20493 Alk5 +T591 PR:000017435 20494 20498 Wnt1 +T592 GO:0006915 20536 20545 apoptosis +T593 UBERON:0004363 20592 20596 PAAs +T594 UBERON:0005432 20605 20615 aortic sac +T595 PR:000000047 20690 20694 ALK5 +T596 GO:0006915 20771 20783;20795 20800 apoptosis of ... cells +T597 UBERON:0002342 20784 20786 NC +T598 PR:000017435 20823 20827 Wnt1 +T599 UBERON:0006562 20848 20858 Pharyngeal +T600 UBERON:0000062 20859 20865 organs +T601 PR:000000047 20885 20889 Alk5 +T602 PR:000017435 20890 20894 Wnt1 +T603 UBERON:0004145 20927 20938 cardiac OFT +T604 GO:0048513 20940 20954;20966 20972 development of ... organs +T605 UBERON:0006562 20955 20965 pharyngeal +T606 UBERON:0000062 20966 20972 organs +T607 UBERON:0001132 20984 21002 parathyroid glands +T608 UBERON:0002370 21011 21017 thymus +T609 PR:000000047 21039 21043 Alk5 +T610 PR:000017435 21044 21048 Wnt1 +T611 UBERON:0002370 21095 21101 thymus +T612 UBERON:0007124 21120 21151 third pharyngeal pouch endoderm +T613 UBERON:0008818 21203 21223 superior mediastinum +T614 UBERON:0005562 21281 21297 thymic primordia +T615 PR:000000047 21305 21309 Alk5 +T616 UBERON:0005434 21393 21404 neck region +T617 UBERON:0014387 21436 21467 neural crest-derived mesenchyme +T618 UBERON:0005562 21532 21548 thymic primordia +T619 PR:000000047 21601 21605 Alk5 +T620 UBERON:0001132 21640 21658 parathyroid glands +T621 PR:000000047 21689 21693 Alk5 +T622 PR:000017435 21694 21698 Wnt1 +T623 UBERON:0001132 21743 21755 parathyroids +T624 UBERON:0005562 21794 21810 thymic primordia +T625 UBERON:0002046 21833 21841 thyroids +T626 UBERON:0005434 21849 21860 neck region +T627 PR:000000047 21883 21887 Alk5 +T628 PR:000017435 21888 21892 Wnt1 +T629 UBERON:0001132 21910 21922 parathyroids +T630 UBERON:0005562 21952 21968 thymic primordia +T631 GO:0010467 22014 22024 expression +T632 UBERON:0001132 22028 22039 parathyroid +T633 PR:000000047 22078 22082 Alk5 +T634 UBERON:0006562 22150 22160 pharyngeal +T635 UBERON:0000062 22161 22166 organ +T636 PR:000017435 22233 22237 Wnt1 +T637 UBERON:0006562 22268 22278 Pharyngeal +T638 UBERON:0000062 22279 22285 organs +T639 PR:000000047 22305 22309 Alk5 +T640 PR:000017435 22310 22314 Wnt1 +T641 UBERON:0002370 22342 22348 thymus +T642 UBERON:0008818 22375 22395 superior mediastinum +T643 PR:000000047 22411 22415 Alk5 +T644 UBERON:0005562 22496 22512 thymic primordia +T645 UBERON:0006562 22574 22584 pharyngeal +T646 UBERON:0014387 22613 22644 neural crest derived mesenchyme +T647 UBERON:0001132 22690 22708 parathyroid glands +T648 UBERON:0002046 22743 22757 thyroid glands +T649 PR:000000047 22782 22786 Alk5 +T650 UBERON:0001132 22799 22817 parathyroid glands +T651 UBERON:0005562 22843 22859 thymic primordia +T652 GO:0010467 22910 22919 expressed +T653 UBERON:0001132 22920 22931 parathyroid +T654 CHEBI:51686 23004 23016 hematoxyllin +T655 GO:0097617 23129 23142 hybridization +T656 UBERON:0002370 23181 23182 T +T657 UBERON:0002370 23184 23190 thymus +T658 UBERON:0002046 23192 23194 Th +T659 UBERON:0002046 23196 23203 thyroid +T660 UBERON:0005562 23246 23262 thymic primordia +T661 UBERON:0001723 23296 23302 tongue +T662 PR:000017435 23347 23351 Wnt1 +T663 UBERON:0000479 23416 23422 tissue +T664 SO:0000704 23432 23436 gene +T665 UBERON:0002342 23540 23542 NC +T666 SO:0000704 23575 23579 gene +T667 UBERON:0004339 23607 23616 calvarial +T668 UBERON:0001456 23618 23624 facial +T669 UBERON:0000948 23629 23636 cardiac +T670 http://purl.obolibrary.org/obo/MONDO_0005453 23629 23644 cardiac defects +T671 UBERON:0006562 23739 23749 pharyngeal +T672 UBERON:0000948 23773 23778 heart +T673 PR:000000047 23826 23830 Alk5 +T674 PR:000000046 23850 23855 TGF-β +T675 PR:000017435 23958 23962 Wnt1 +T676 NCBITaxon:10088 23986 23990 mice +T677 PR:000000183 24004 24010 Tgf-β2 +T678 http://purl.obolibrary.org/obo/MONDO_0018072 24023 24026 PTA +T679 UBERON:0002061 24036 24054 truncus arteriosus +T680 http://purl.obolibrary.org/obo/MONDO_0009010 24060 24083 interrupted aortic arch +T681 UBERON:0001508 24072 24083 aortic arch +T682 PR:000000047 24091 24095 Alk5 +T683 PR:000017435 24096 24100 Wnt1 +T684 UBERON:0004363 24173 24177 PAAs +T685 UBERON:0004363 24232 24236 PAAs +T686 PR:000000047 24252 24256 Alk5 +T687 PR:000017435 24257 24261 Wnt1 +T688 UBERON:0005432 24312 24322 aortic sac +T689 http://purl.obolibrary.org/obo/MONDO_0018072 24361 24364 PTA +T690 UBERON:0002061 24392 24409 truncus artriosus +T691 UBERON:0002012 24423 24439 pulmonary artery +T692 UBERON:0005432 24536 24546 aortic sac +T693 UBERON:0002061 24593 24600 truncal +T694 UBERON:0003121 24658 24666 4th PAAs +T695 http://purl.obolibrary.org/obo/MONDO_0009010 24674 24689;24694 24705 interruption of aortic arch +T696 UBERON:0004363 24694 24705 aortic arch +T697 GO:0006915 24764 24776;24820 24825 apoptosis of ... cells +T698 PR:000000047 24777 24781 Alk5 +T699 UBERON:0002342 24807 24819 neural crest +T700 GO:0008219 24889 24899 cell death +T701 PR:000017435 24932 24936 Wnt1 +T702 PR:000000046 24993 24998 TGF-β +T703 GO:0007179 24993 25008 TGF-β signaling +T704 UBERON:0000948 25012 25019 cardiac +T705 PR:000000047 25059 25063 ALK5 +T706 GO:0043235 25073 25089 receptor complex +T707 PR:000000047 25091 25095 ALK5 +T708 PR:000000047 25276 25280 ALK5 +T709 GO:0032991 25297 25304 complex +T710 GO:0048180 25314 25321 Activin +T711 PR:000000071 25314 25339 Activin type IIB receptor +T712 PR:000000364 25363 25370 Smads 2 +T713 PR:000000365 25363 25368;25371 25372 Smads ... 3 +T714 PR:000000046 25407 25412 TGF-β +T715 PR:000010684 25470 25474 GDF8 +T716 PR:000007925 25476 25480 GDF9 +T717 PR:000007919 25482 25487 GDF11 +T718 PR:000007920 25492 25497 GDF15 +T719 PR:000010684 25557 25563 Gdfs 8 +T720 PR:000007925 25557 25561;25565 25566 Gdfs ... 9 +T721 PR:000007919 25557 25561;25567 25569 Gdfs ... 11 +T722 PR:000007920 25557 25561;25574 25576 Gdfs ... 15 +T723 GO:0010467 25585 25594 expressed +T724 UBERON:0000948 25613 25618 heart +T725 NCBITaxon:10088 25631 25635 mice +T726 UBERON:0000948 25682 25689 cardiac +T727 http://purl.obolibrary.org/obo/MONDO_0005453 25682 25697 cardiac defects +T728 PR:000000046 25780 25786 TGF-βs +T729 UBERON:0000948 25825 25832 cardiac +T730 GO:0003007 25825 25832;25848 25861 cardiac ... morphogenesis +T731 UBERON:0006562 25837 25847 pharyngeal +T732 GO:0006915 25888 25897 apoptosis +T733 UBERON:0005432 25918 25928 aortic sac +T734 UBERON:0006207 25940 25949 AP-septum +T735 UBERON:0003121 25979 25982;25991 25995 4th ... PAAs +T736 UBERON:0003123 25987 25995 6th PAAs +T737 GO:0006915 26036 26045 apoptosis +T738 UBERON:0003104 26082 26092 mesenchyme +T739 UBERON:0005432 26109 26119 aortic sac +T740 UBERON:0006207 26154 26163 AP septum +T741 GO:0008219 26182 26192 cell death +T742 UBERON:0006207 26206 26215 AP septal +T743 http://purl.obolibrary.org/obo/MONDO_0021902 26206 26222 AP septal defect +T744 UBERON:0002342 26254 26256 NC +T745 UBERON:0001135 26305 26318 smooth muscle +T746 CL:0000192 26305 26324 smooth muscle cells +T747 UBERON:0004145 26332 26335 OFT +T748 UBERON:0006207 26372 26381 AP septum +T749 PR:000000047 26393 26397 Alk5 +T750 PR:000017435 26398 26402 Wnt1 +T751 UBERON:0006207 26498 26507 AP septum +T752 GO:0008219 26536 26546 cell death +T753 UBERON:0006207 26600 26609 AP-septum +T754 GO:0016265 26610 26613 die +T755 UBERON:0001135 26663 26676 smooth muscle +T756 CL:0000192 26663 26682 smooth muscle cells +T757 PR:000000046 26751 26756 TGF-β +T758 GO:0007179 26751 26766 TGF-β-signaling +T759 GO:0051145 26770 26785;26794 26818 differentiation ... into smooth muscle cells +T760 UBERON:0001135 26799 26812 smooth muscle +T761 CL:0000192 26799 26818 smooth muscle cells +T762 NCBITaxon:10088 26868 26872 mice +T763 GO:0030154 26919 26939;26954 26959 differentiation into ... cells +T764 PR:000003675 26940 26944 αSMA +T765 UBERON:0006207 26967 26976 AP septum +T766 PR:000003675 27080 27084 αSMA +T767 UBERON:0004145 27092 27095 OFT +T768 PR:000000047 27142 27146 ALK5 +T769 UBERON:0001135 27167 27180 smooth muscle +T770 http://purl.obolibrary.org/obo/MONDO_0008644 27338 27354;27364 27372 velocardiofacial syndrome +T771 http://purl.obolibrary.org/obo/MONDO_0008564 27355 27372 DiGeorge syndrome +T772 http://purl.obolibrary.org/obo/MONDO_0008644 27374 27377 VCF +T773 http://purl.obolibrary.org/obo/MONDO_0008564 27378 27381 DGS +T774 http://purl.obolibrary.org/obo/MONDO_0008564 27425 27433 DiGeorge +T775 PR:000000047 27568 27572 Alk5 +T776 PR:000017435 27573 27577 Wnt1 +T777 http://purl.obolibrary.org/obo/MONDO_0008644 27627 27630 VCF +T778 http://purl.obolibrary.org/obo/MONDO_0008564 27631 27634 DGS +T779 UBERON:0002342 27674 27676 NC +T780 PR:000000047 27700 27704 Alk5 +T781 http://purl.obolibrary.org/obo/MONDO_0008644 27722 27725 VCF +T782 http://purl.obolibrary.org/obo/MONDO_0008564 27726 27729 DGS +T783 UBERON:0006562 27766 27776 pharyngeal +T784 UBERON:0000062 27777 27782 organ +T785 PR:000000047 27802 27806 Alk5 +T786 PR:000017435 27807 27811 Wnt1 +T787 UBERON:0003104 27858 27869 mesenchymal +T788 CL:0000134 27858 27874 mesenchymal cell +T789 GO:0008219 27870 27880 cell death +T790 UBERON:0006562 27888 27898 pharyngeal +T791 UBERON:0002370 27916 27922 thymus +T792 UBERON:0002046 27924 27931 thyroid +T793 UBERON:0001132 27936 27947 parathyroid +T794 GO:0016265 28046 28051 death +T795 PR:000000047 28060 28064 Alk5 +T796 SO:0000704 28137 28142 genes +T797 PR:000016143 28170 28174 Tbx1 +T798 PR:000005883 28179 28183 CrkL +T799 GO:0065007 28185 28192 control +T800 GO:0065007 28229 28239 regulating +T801 GO:0008283 28240 28253 proliferation +T802 UBERON:0009889 28261 28282 secondary heart field +T803 UBERON:0009889 28284 28287 SHF +T804 UBERON:0000925 28294 28302 endoderm +T805 UBERON:0006562 28392 28402 pharyngeal +T806 http://purl.obolibrary.org/obo/MONDO_0018072 28472 28475 PTA +T807 UBERON:0002349 28508 28518 myocardium +T808 UBERON:0009889 28528 28549 secondary heart field +T809 GO:0016477 28592 28610 migration of cells +T810 UBERON:0009889 28620 28641 secondary heart field +T811 UBERON:0004145 28649 28652 OFT +T812 UBERON:0004145 28718 28721 OFT +T813 UBERON:0001637 28755 28763 arterial +T814 UBERON:0002082 28778 28788 ventricles +T815 UBERON:0004145 28814 28817 OFT +T816 PR:000000047 28831 28835 Alk5 +T817 PR:000017435 28836 28840 Wnt1 +T818 UBERON:0002342 28906 28908 NC +T819 http://purl.obolibrary.org/obo/MONDO_0018072 28932 28935 PTA +T820 UBERON:0005432 28956 28966 aortic sac +T821 UBERON:0009889 29005 29026 secondary heart field +T822 PR:000000047 29055 29059 Alk5 +T823 UBERON:0004145 29140 29143 OFT +T824 UBERON:0002342 29147 29159 neural crest +T825 PR:000000047 29180 29184 Alk5 +T826 PR:000000067 29188 29192 Alk2 +T827 UBERON:0002342 29233 29235 NC +T828 UBERON:0009889 29263 29266 SHF +T829 UBERON:0004145 29301 29304 OFT +T830 NCBITaxon:10088 29317 29321 mice +T831 PR:000000046 29358 29363 TGF-β +T832 PR:000000034 29364 29367 BMP +T833 UBERON:0004145 29399 29402 OFT +T834 PR:000000047 29445 29449 Alk5 +T835 UBERON:0003037 29499 29505 septal +T836 UBERON:0003104 29506 29516 mesenchyme +T837 UBERON:0004145 29576 29579 OFT +T838 UBERON:0003037 29627 29633 septal +T839 UBERON:0003104 29658 29668 mesenchyme +T840 PR:000000067 29686 29690 Alk2 +T841 PR:000017435 29691 29695 Wnt1 +T842 UBERON:0003037 29719 29725 septal +T843 UBERON:0003104 29726 29736 mesenchyme +T844 GO:0001947 29778 29785 looping +T845 UBERON:0005432 29793 29803 aortic sac +T846 UBERON:0002061 29808 29815 truncal +T847 UBERON:0004145 29816 29819 OFT +T848 UBERON:0003037 29862 29868 septal +T849 UBERON:0003104 29869 29879 mesenchyme +T850 UBERON:0001135 29897 29910 smooth muscle +T851 CL:0000192 29897 29915 smooth muscle cell +T852 GO:0030154 29911 29931 cell differentiation +T853 UBERON:0004145 29955 29958 OFT +T854 PR:000000046 30017 30022 TGF-β +T855 PR:000000047 30040 30044 Alk5 +T856 SO:0000704 30046 30050 gene +T857 NCBITaxon:10088 30071 30076 mouse +T858 UBERON:0002342 30077 30089 neural crest +T859 UBERON:0002342 30091 30093 NC +T860 PR:000000047 30131 30135 ALK5 +T861 UBERON:0002342 30173 30175 NC +T862 UBERON:0006562 30271 30281 pharyngeal +T863 UBERON:0000062 30282 30288 organs +T864 UBERON:0004145 30293 30304 cardiac OFT +T865 UBERON:0000948 30310 30317 cardiac +T866 http://purl.obolibrary.org/obo/MONDO_0005453 30310 30317;30333 30340 cardiac defects +T867 UBERON:0006562 30322 30332 pharyngeal +T868 UBERON:0002342 30357 30359 NC +T869 PR:000000047 30369 30373 Alk5 +T870 PR:000000046 30456 30461 TGF-β +T871 PR:000000047 30518 30522 ALK5 +T872 PR:000000046 30555 30560 TGF-β +T873 UBERON:0000948 30576 30583 cardiac +T874 GO:0007507 30576 30583;30595 30606 cardiac ... development +T875 UBERON:0006562 30584 30594 pharyngeal +T876 GO:0060465 30584 30606 pharyngeal development +T877 PR:000000047 30618 30622 Alk5 +T878 NCBITaxon:10088 30631 30635 mice +T879 PR:000000047 30637 30641 Alk5 +T880 PR:000000067 30647 30651 Alk2 +T881 UBERON:0000922 30672 30679 embryos +T882 GO:0007618 30698 30704 mating +T883 PR:000000047 30705 30709 Alk5 +T884 PR:000000067 30714 30718 Alk2 +T885 PR:000017435 30724 30728 Wnt1 +T886 NCBITaxon:10088 30738 30742 mice +T887 PR:000000047 30775 30779 Alk5 +T888 SO:0000359 30779 30783 flox +T889 PR:000000067 30785 30789 Alk2 +T890 SO:0000359 30789 30793 flox +T891 SO:0001023 30795 30801 allele +T892 PR:000017435 30894 30898 Wnt1 +T893 NCBITaxon:10088 30903 30907 mice +T894 NCBITaxon:10088 30982 30986 mice +T895 NCBITaxon:33208 31068 31074 Animal +T896 GO:0007618 31207 31215 mataings +T897 NCBITaxon:10088 31217 31221 Mice +T898 GO:0007618 31227 31232 mated +T899 UBERON:0010148 31299 31312 vaginal plugs +T900 CHEBI:16526 31372 31375 CO2 +T901 UBERON:0000922 31381 31388 embryos +T902 UBERON:0000922 31469 31476 Embryos +T903 CHEBI:73702 31569 31572 wax +T904 CHEBI:51686 31610 31621 Hematoxylin +T905 UBERON:0000922 31687 31694 embryos +T906 CHEBI:27780 31902 31911 detergent +T907 CHEBI:75055 31949 31954 X-gal +T908 CHEBI:75958 31955 31963 solution +T909 UBERON:0000479 32035 32042 tissues +T910 PR:000003675 32099 32120 α-smooth muscle actin +T911 UBERON:0001135 32101 32114 smooth muscle +T912 MOP:0000780 32129 32136 cleaved +T913 PR:000018762 32129 32146 cleaved caspase-3 +T914 PR:000027594 32181 32183 H3 +T915 GO:0042571 32201 32211 antibodies +T916 UBERON:0000922 32320 32327 embryos +T917 CHEBI:37958 32378 32381 dye +T918 UBERON:0000922 32394 32401 Embryos +T919 CHEBI:37958 32557 32560 dye +T920 UBERON:0002082 32622 32632 ventricles +T921 CHEBI:37958 32643 32646 dye +T922 UBERON:0000922 32673 32680 Embryos +T923 CHEBI:16842 32712 32720 formalin +T924 CHEBI:41237 32761 32775 benzylbenzoate +T925 CHEBI:17987 32777 32791 benzyl alcohol +T926 GO:0010467 32803 32813 Expression +T927 UBERON:0001132 32837 32848 parathyroid +T928 GO:0010467 32857 32867 expression +T929 GO:0097617 32884 32897 hybridization +T930 SO:0000077 32905 32914 antisense +T931 NCBITaxon:10088 33056 33061 mouse +T932 PR:000000047 33151 33155 Alk5 +T933 GO:0010467 33186 33196 expression +T934 UBERON:0000922 33268 33275 embryos +T935 PR:000000067 33299 33303 Alk2 +T936 NCBITaxon:10088 33308 33312 mice +T937 PR:000017435 33494 33498 Wnt1 +T938 NCBITaxon:10088 33503 33508 mouse +T939 PR:000017435 33571 33575 Wnt1 +T940 UBERON:0000922 33580 33587 embryos diff --git a/src/ontogpt/evaluation/craft/database/all/17078885.txt b/src/ontogpt/evaluation/craft/database/all/17078885.txt new file mode 100644 index 000000000..e563f4c9a --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17078885.txt @@ -0,0 +1,133 @@ +Defective ALK5 signaling in the neural crest leads to increased postmigratory neural crest cell apoptosis and severe outflow tract defects + +Abstract + +Background + +Congenital cardiovascular diseases are the most common form of birth defects in humans. A substantial portion of these defects has been associated with inappropriate induction, migration, differentiation and patterning of pluripotent cardiac neural crest stem cells. While TGF-β-superfamily signaling has been strongly implicated in neural crest cell development, the detailed molecular signaling mechanisms in vivo are still poorly understood. + +Results + +We deleted the TGF-β type I receptor Alk5 specifically in the mouse neural crest cell lineage. Failure in signaling via ALK5 leads to severe cardiovascular and pharyngeal defects, including inappropriate remodeling of pharyngeal arch arteries, abnormal aortic sac development, failure in pharyngeal organ migration and persistent truncus arteriosus. While ALK5 is not required for neural crest cell migration, our results demonstrate that it plays an important role in the survival of post-migratory cardiac neural crest cells. + +Conclusion + +Our results demonstrate that ALK5-mediated signaling in neural crest cells plays an essential cell-autonomous role in the pharyngeal and cardiac outflow tract development. + +Background + +A considerable percentage of cardiac birth defects is caused by a failure in normal migration, differentiation or patterning of the cardiac neural crest (CNC). This subset of pluripotent neural crest stem cells forms in the dorsal aspect of the neural tube at the level of the mid-otic placode to the third somite [1]. Subsequently cardiac neural crest cells (CNCCs) delaminate, undergo a phenotypic transformation from an epithelial to mesenchymal cell type, and migrate latero-ventrally into the 3rd, 4th and 6th pharyngeal arch arteries (PAAs), where they contribute to the formation of the smooth muscle cell layer of endothelial structures derived from the aortic arch arteries [1-3]. A subset of CNCCs continues to migrate deeper into the aortic sac to form the aortico-pulmonary septum; a vital structure, which separates the pulmonary trunk from the aorta [4]. + +An indispensable role of CNCCs in the development of the cardiac outflow tract was originally demonstrated by pioneering studies of Kirby and coworkers [1], who showed that ablation of the CNC in the chick led to severe outflow tract (OFT) defects including persistent truncus arteriosus (PTA), mispatterning of the great vessels and outflow tract mal-alignments [5]. Early migratory CNCCs have been shown to retain a considerable degree of plasticity and their fate is largely controlled by instructional signals from local environments into which NCCs migrate [6]. Several recent studies have indicated that members of the TGF-β superfamily, i.e., TGF-βs and BMPs are likely candidates to provide some of these signals. Mice deficient in TGF-β2 display fourth aortic arch artery defects [7], while neural crest cell specific abrogation of TGF-β type II receptor (Tgfbr2) results in interruption of the aortic arch and PTA [8,9]. BMPs 6 and -7 are required for proper formation of the outflow tract cushions [10], while BMP type II receptor is needed for proper development of the conotruncal ridges [11]. Moreover, neural crest-specific deletion of the BMP type I receptors Alk2 and Alk3 has been shown to lead to defective aortico-pulmonary septation, among other cardiac defects [12,13]. + +TGF-β subfamily ligands signal via a receptor complex composed of two type II receptors and two type I receptors [14,15]. Ligand binding leads to phosphorylation and activation of type I receptors, which, in turn, phosphorylate and activate a specific set of downstream signaling molecules called Smads. In general terms, TGF-βs bind to the TGF-β type II receptor (TGFβRII) and TGF-β type I receptor (ALK5) activating TGF-β Smads (2 and 3), while BMPs bind to the BMP type II receptor and type I receptors ALK2, -3, or 6, activating BMP Smads (1, 5 and 8). However, it is likely that these signaling interactions are more complex in vivo, possibly allowing formation of heterotetrameric complexes composed of different type II and type I receptors [16]. In addition, recent studies have identified novel TGF-β-related ligands, which can bind to entire different combinations of receptors. For instance, growth and differentiation factors (GDFs) 8 and 9 can bind to Activin type II receptor and ALK5 to activate TGF-β Smads [17,18]. Therefore, we hypothesized that deletion of Alk5 in a specific cell lineage should reveal phenotypes which cannot be seen in comparable mutants lacking Tgfbr2. Indeed, we recently showed that neural crest cell specific Alk5 mutants display a unique spectrum of craniofacial developmental defects, e.g., cleft snout and severe mandibular hypoplasia [19]; these phenotypes were not seen in corresponding Tgfbr2 mutants [20]. To determine, whether ALK5 would also mediate unique non-redundant signaling events in cardiac neural crest cells, we focused on cardiac and pharyngeal phenotypes of mouse embryos lacking Alk5 specifically in neural crest cells. We discovered that in Alk5/Wnt1-Cre mutants, pharyngeal organs (thymus and parathyroid) fail to migrate appropriately. Moreover, the mutant embryos display severe aortic sac and pharyngeal arch artery defects, and failed aortico-pulmonary septation leading to PTA. Our data further suggest that at least some of these abnormal detected phenotypes result from a dramatic increase in apoptosis of postmigratory cardiac neural crest cells. These phenotypes differ remarkably from those seen in corresponding Tgfbr2 mutants, suggesting that ALK5 mediates a wider spectrum of signaling events than its classical binding partner TGFβRII in cardiac neural crest cells during cardiac and pharyngeal development. + +Results + +Persistent truncus arteriosus and abnormal large vessels in mice lacking Alk5 in cardiac NCCs + +To inactivate Alk5 in cardiac NCCs, mice homozygous for the floxed Alk5 allele (Alk5Flox/Flox) [21] were crossed with transgenic Wnt1-Cre mice [22], which were also heterozygous for the Alk5 knockout allele (Alk5KO) allele. The resulting mice heterozygous for the Alk5Flox and Alk5KO alleles, which also carried the Wnt1-Cre transgene, had the Alk5 gene specifically inactivated in NCCs (herein termed Alk5/Wnt1-Cre), while the littermates with remaining allelic combinations were phenotypically normal and served as controls (Alk5Flox/+, Alk5KO/+; Wnt1-Cre). When embryos were harvested during the last day of gestation, an expected number (25%) of Alk5/Wnt1-Cre mutants were recovered. However, all mutant offspring died either during the birth or during the first post-natal hours. + +To determine, if ALK5-mediated TGF-β-signaling had a role in development of the OFT and large vessels of the aortic arch, we performed casting dye experiments on E17 embryos (Fig. 1A–D). In wild-type embryos (Fig. 1A), the aorta was clearly separated from the pulmonary trunk, and the right brachiocephalic, left carotid and left subclavian arteries branched directly off the aortic arch. In contrast, Alk5/Wnt1Cre mutant embryos consistently displayed a single prominent arterial trunk (Fig. 1C–D), while corresponding Tgfbr2 mutant embryos (Fig. 1B) displayed interrupted aortic arch, as reported earlier [8]. Approximately 40% of the Alk5 mutants had a right-sided outflow tract, with the retroesophageal arch connecting to the descending aorta and to the left subclavian artery. The carotid arteries originated either from a common bud located in the ventral side of the ascending arch, or from separate adjacent sites, as verified by serial sectioning (Fig. 1M–P). The remaining mutants displayed a left-sided aortic arch, where the right carotid arteries originated from the right lateral aspect of the ascending trunk, while the left carotid arteries budded from the ventral or right ventral aspects of the trunk (Fig. 1I–L). Both right and left subclavian arteries consistently originated from the descending part of the aortic arch. Similarly, in all mutants both left and right pulmonary arteries always branched out from the common arterial trunk. To conclude, Alk5/Wnt1-Cre mutants consistently displayed PTA, which differed significantly from the characteristic interrupted aortic arch phenotype seen in Tgfbr2/Wnt1-Cre mutants [8,9]. + +Figure 1 + +Abrogation of Alk5 in neural crest cells leads to persistent truncus arteriosus type A2. A-D, Casting-dye analysis of OFT morphogenesis at E17.0. Control (A), Tgfbr2/Wnt1-Cre mutant [8] (B) demonstrating the PTA type A4 (= truncus arteriosus with interrupted aortic arch [30]) and Alk5/Wnt1-Cre mutants demonstrating the right-sided (C) and left-sided (D) arches of the truncus. E-P, Histological cross-sections on four different levels (rostral to caudal) at E17.0. In a control (E-H), the ascending aorta (Ao) and pulmonary trunk (PT) are separated by the conotruncal septum. In Alk5/Wnt1-Cre mutants (I-P) the conotruncal septum fails to form, and either left-sided (I-L) or right-sided (M-P) aortic arch can be seen. Aberrant branching of carotid arteries from the truncus has been illustrated by black arrows (J and M). Ao, aorta; PT, pulmonary trunk; RSA, right subclavian artery; RCA, right carotid artery; LCA; left carotid artery; LSA, left subclavian artery; IAA, interrupted aortic arch; PTA, persistent truncus arteriosus. + +Abnormal patterning of the pharyngeal arch arteries and aortic sac in Alk5/Wnt1Cre mutants + +During cardiovascular development, the PAAs undergo a complex set of sequential asymmetric remodeling steps resulting in the left-sided aortic arch. To determine, whether ALK5-mediated signaling was involved in remodeling of PAAs, we performed intracardiac India ink injections at different developmental stages. While at E10, Alk5/Wnt1-Cre mutants did not show obvious differences in the PAAs, abnormal remodeling became obvious in mutants a day later at E11 (Fig. 2). The controls displayed the well-formed 3rd, 4th and 6th PAAs. Moreover, the carotid duct (the dorsal aorta between the 3rd and 4th PAAs) was already regressing as demonstrated by the reduced size (Fig. 2A). In Alk5/Wnt1-Cre mutants, the 3rd and 4th pairs of PAAs were bilaterally hypoplastic, whereas the 6th pair of PAAs was notably hyperplastic (Fig. 2B). Furthermore, the carotid duct was remarkably large, when compared to controls. While the controls displayed an interruption of the carotid duct at E12 and E13 as expected (Fig. 2C), the mutants demonstrated an uncharacteristic break of the dorsal aorta between the 4th and 6th pairs of PAAs (Fig. 2D). + +Figure 2 + +Abnormal patterning of the PAAs in Alk5/Wnt1-Cre mutants. Left lateral view after intracardiac ink injections to visualize the developing PAAs at E11.0 (A,B), E12.0 (C, D) and E13.0 (E, F) in controls (A, C, E) and Alk5/Wnt1-Cre mutants (B, D, F). Arrow in A points to the regressing carotid duct. Asterisk in B depicts the corresponding structure in the mutant with no signs of regression. Asterisk in D illustrates the aberrant regression of the dorsal aorta between the 4th and 6th PAAs. PT, pulmonary trunk; Ao, Aorta; TA, truncus arteriosus. + +Around E11.5, the aortic sac normally forms a distinctive T-shaped structure, as seen in frontal sections of the control sample in Fig. 3(A,C). Subsequently, the right horn of this structure transforms into the prospective brachiocephalic artery, while the left horn together with the left 4th PAA gives rise to the definitive aortic arch [23]. In Alk5/Wnt1-Cre mutants, the T-shaped aortic sac failed to form (Fig. 3B,D). Instead, the truncus bifurcated to a left and right arm, which further branched to the PAAs, particularly to the predominant pair of 6th PAAs (Fig. 3B,D). The observed phenotype is consistent with the absence or severe hypoplasia of structures derived from the aortic sac in late stage embryos (E17), e.g., the missing brachiocephalic artery and severe shortening of the ascending truncus as shown in the Figure 1. + +Figure 3 + +Abnormal Aortic Sac in Alk5/Wnt1-Cre mutants. Alk5/Wnt1-Cre mutants (B, D) fail to form the typical T-shaped structure of the aortic sac seen in controls at E11.5. (A, C). A-B, frontal image of ink-injected embryos; C-D, frontal sections (H&E staining). Arrows in A and B point to the level of section shown in C and D (red arrows in C and D point to the aortic sac of the control and mutant, respectively). + +Cardiac NCCs deficient in Alk5 can populate the outflow tract + +Next we used the R26R lineage-tracing assay to determine whether CNCCs could appropriately populate the outflow tract region. Briefly, Alk5Flox/Flox mice were crossed with the ROSA26 Cre reporter mice, and subsequently Alk5Flox/Flox;R26R(+/+) females were crossed with Alk5KO/WT;Wnt1-Cre males. The resulting embryos had the NC-lineage permanently labeled with β-galactosidase expression, and displayed identical phenotypes to those obtained without the R26R reporter. Staining of embryos for β-galactosidase at E8-E11 did not reveal detectable differences in NCC migration between mutants and controls (data not shown). Similarly, serial transverse sectioning of whole mount embryos (E10-E12) and subsequent analysis of positively stained cells in the OFT region demonstrated that CNCCs deficient in Alk5 were capable of populating the PAAs, aortic sac and conotruncal ridges at a level comparable to that of controls (Fig 4). To conclude, the observed phenotypes in Alk5/Wnt1-Cre mutants were certainly not due to defective migration of CNCCs to the pharyngeal and outflow tract regions. + +Figure 4 + +Normal cardiac NCC migration in Alk5/Wnt1-Cre mutants. The OFT of controls (A, C, E) and Alk5/Wnt1-Cre mutants (B, D, F) display similar staining patterns when analyzed using the R26R lineage tracing assay at E11.0. A-B, whole mount staining (left lateral image); C-F, transverse sections on the level of the 4th (C, D) and 6th (E, F) PAAs. Arrows (A-F) point to the most proximal location staining positive for the β-galactosidase activity. + +Aortic sac and aortico-pulmonary septal defects in Alk5/Wnt1Cre mutant embryos + +Septation of the outflow tract lumen begins in a cranial-to-caudal direction, starting distally in the aortic sac and proceeding toward the heart [24]. Initially, the condensed mesenchyme derived from the NC forms in the base of the aortic sac between the origins of 4th and 6th PAAs. Subsequently, two prongs of the developing aortico-pulmonary (AP) septum extend into the truncal cushions and the aortico-pulmonary septation complex crosses the aortic sac cranially. In ink-injected control embryos at E11.5, a characteristic conotruncal transition separating the truncus and conus could be seen as a twisted configuration, resulting from a change in orientation of the truncal and conal cushions (Fig. 5). In contrast, in Alk5/Wnt1-Cre mutants the outflow tract appeared unusually straight, failing to demonstrate the distinct conotruncal transition (Fig. 5B,D). This assay also clearly showed a dramatic reduction in the size of the aortic sac. Histological analysis of control samples displayed the characteristic rotation of the aortic sac and truncal OFT at the level where the AP septation takes place and verified the presence of the distinctive condensed AP-septal mesenchyme, which gradually divided the OFT to the aorta and the pulmonary trunk (asterisks in Fig. 6A). R26R lineage tracing showed that this tissue is derived from the NC, while immunostaining for α-SMA showed differentiation into smooth muscle (Fig 6B). In Alk5/Wnt1-Cre mutants the characteristic rotation of the aortic sac and truncal OFT fails to take place (Fig. 6G–L), and a properly formed AP-septum was not detectable (Fig. 6G,H). R26R lineage tracing demonstrated that the defects were not due to failure of NCCs to reach the OFT region. NC-derived cells around the abnomally bifurcated aortic sac, the abnormally large sixth PAAs and the truncus demonstrated strong αSMA staining (Fig. 6H,J,L). Recently, we showed that the NC-specific mutants of the related type I receptor, Alk2, display PTA as well [12]. In Alk2/Wnt1-Cre mutants, the rotation of the aortic sac and truncal OFT failed to occur (Fig. 6M–R) as seen in Alk5/Wnt1-Cre mutants. However, in Alk2 mutants the 6th pair of the PAAs was grossly hypoplastic, and while the Alk2/Wnt1-Cre mutants displayed a noticeable amount of septal tissue between the 4th and 6th PAAs (Fig. 6M,N), the condensed septal mesenchyme lacking Alk2 failed to extend the prongs into the truncal cushions and to form the AP septum. Concurrently, the 6th PAAs were losing their patency, which may have further contributed to the failed AP septation (Fig. 6M,O,Q). While CNCCs managed to migrate to the aortic sac and the truncal cushion level (Fig. 6N,P,R), immunostaining for αSMA appeared much weaker when compared to controls and Alk5 mutants, implying that ALK2-mediated signaling is involved in smooth muscle cell differentiation as previously suggested [12]. To conclude, while both Alk2 and Alk5 mutants demonstrate a failure in both the rotation of the aortic sac and the truncal OFT, and in the formation of the AP septum, the pathogenetic mechanisms behind these defects appear remarkably different. + +Figure 5 + +The truncal OFT fails to rotate in Alk5/Wnt1-Cre mutants. Left (A, C) and right (B, D) lateral images of ink-injected control (A-B) and mutant (C-D) embryos at E11.5 demonstrate the abnormally straight OFT in mutants lacking the typical conotruncal transition (black arrow in A vs. black arrowhead in C) seen in control. Red arrowhead (C) points to the abnormally shaped aortic sac. Red "s", aortic sac; t, truncus; c, conus. + +Figure 6 + +Signaling via ALK5 and ALK2 controls different aspects of aortico-pulmonary septation. Frontal sections from distal (top row) to proximal (bottom row) of the control (A-F), Alk5/Wnt1-Cre mutant (G-L) and Alk2/Wnt1-Cre mutant (M-R) samples (E11.5). A, C, E, G, I, K, M, O, Q, H&E staining; B, D, F, H, J, L, N, P, R, double staining for αSMA (brown) and β-galactosidase (green; R26R reporter assay). 6, the 6th PAA; AS, aortic sac; TA, truncus arteriosus; Ao, Aorta; PT, pulmonary trunk; Asterisks in A, B, M and N depict the AP septal mesenchyme. + +Alk5/Wnt1-Cre mutants display increased apoptosis of post-migratory neural crest cells + +As described above, Alk5/Wnt1-Cre mutants displayed an inadequate amount of AP-septal tissue in the base of the aortic sac between the origins of 4th and 6th PAAs. To analyze whether this phenotype resulted either from defective CNCC proliferation or inappropriate apoptosis, we used BrdU and TUNEL staining, respectively. While CNCC proliferation was not affected in Alk5 mutants (data not shown), we could detect a dramatic increase in the number of TUNEL positive cells in tissues surrounding the aortic sac including the site where the AP-septum forms (Fig. 7A–C). Dual staining for lacZ and TUNEL positive cells demonstrated that these cells were postmigratory CNCCs; this phenotype was already clearly detectable at E10.5. These results were confirmed by using immunostaining for cleaved caspase-3, another marker for apoptosis (Fig. 7I,J). In the chick, apoptotic neural crest-derived cells have also been found at the sites, where the prongs of the AP septum penetrate into the OFT cushion mesenchyme [25,26]. Thus, we compared apoptosis patterns also on the more proximal level, but found no detectable differences at E11.0 between Alk5/Wnt1-Cre mutants and controls (Fig. 7D,E). Unlike in Alk5/Wnt1-Cre mutant embryos, increased apoptosis of NC-derived cells is not responsible for the observed defects in the OFT septation in corresponding Alk2 mutants (Fig. 7C,E). + +Figure 7 + +Aberrant apoptosis of NCCs in Alk5/Wnt1-Cre mutants. TUNEL (A-H) and Cleaved Caspase-3 (I, J) staining at E11.0 demonstrates a notable increase in apoptosis in Alk5/Wnt1-Cre mutants (B, H, J) on the aortic sac level when compared to controls (A, G, I) or Alk2/Wnt1-Cre mutants (C) (frontal sections), while sections on the OFT level do not demonstrate differences between controls (D) and Alk5 (E) or Alk2 (F) mutants. G,H, TUNEL staining of lacZ-stained embryos demonstrates that apoptotic cells are of neural crest origin. G, control; H, mutant. AS, aortic sac, arrows point to clusters of apoptotic cells surrounding the aortic sac. + +To conclude, our results suggest that in Alk5/Wnt1-Cre mutants a noticeable increase in apoptosis coincides with the abnormal patterning of the PAAs and the aortic sac, and with the failed AP-septation. These data support a specific role for ALK5 signaling, either directly or indirectly, in CNCC survival, since a similar apoptosis of NC-derived cells is not seen in Tgfbr2/Wnt1-Cre mutants [8,9]. + +Pharyngeal organs fail to migrate in Alk5/Wnt1-Cre mutants + +In addition to the cardiac OFT, development of pharyngeal organs, i.e., the parathyroid glands and the thymus was also abnormal in Alk5/Wnt1-Cre mutants (see Figs. 1 and 8). Normally the thymus develops from the third pharyngeal pouch endoderm and migrates caudally to its final location in the superior mediastinum as seen in controls at E14 (Fig. 8A,B). In contrast, the thymic primordia of the Alk5 mutant littermates failed to descend caudally, and were located bilaterally in the neck region, where they were surrounded by neural crest-derived mesenchyme (Fig. 8D,E). The fate determination assay demonstrated that the thymic primordia were equally populated by NCCs both in controls and Alk5 mutants (Fig. 8B,E). Likewise the parathyroid glands failed to migrate normally in Alk5/Wnt1-Cre mutants. During normal development, the parathyroids first migrate in association with the thymic primordia, until they reach the thyroids in the neck region as seen in Fig 8C. In Alk5/Wnt1-Cre mutants, the parathyroids remained associated with the thymic primordia, and despite this abnormal rostral location, expression of parathyroid hormone was indistinguishable between Alk5 mutants and controls at E14 (Fig. 8C,F). To conclude, the observed pharyngeal organ phenotypes were also in striking contrast to those seen in Tgfbr2/Wnt1-Cre mutants [8,9]. + +Figure 8 + +Pharyngeal organs fail to migrate in Alk5/Wnt1-Cre mutants. At E14.0, the thymus was not detectable in the superior mediastinum (asterisks) in Alk5 mutants (D), when compared to controls (A). Serial sectioning revealed that the thymic primordia had failed to descend and were seen bilaterally in the upper pharyngeal region (E, F) surrounded by neural crest derived mesenchyme (blue staining cells in E). In controls, the parathyroid glands were properly associated with the thyroid glands (arrows in C), while in Alk5 mutants the parathyroid glands were associated with the thymic primordia (arrows in F). However, both controls and mutants expressed parathyroid hormone (PTH) at comparable levels (blue staining in C and F). A and D, hematoxyllin and eosin staining; B and E, R26 R lineage tracing assay – counterstaining with eosin; C and F, section in situ hybridization for PTH – counterstaining with eosin. T, thymus; Th, thyroid; asterisks in D depict the absence of the thymic primordia; asterisks in E and F depict the tongue. + +Discussion + +During the last few years the Wnt1-Cre transgenic driver line has proven to be a powerful tool for tissue-specific gene deletion in NCCs [12,13,27,28]. Using this approach, several studies have independently shown that the NC-specific deletion of the Tgfbr2 gene leads to a distinct set of calvarial, facial and cardiac defects [8,9,20,29]. Interestingly, these defects appear quite different both in the craniofacial and pharyngeal regions, including the heart, when compared to the corresponding mutants of Alk5, which encodes the TGF-β type I receptor, a prototypical binding partner of TGF-βRII [19] and the present study). While Tgfbr2/Wnt1-Cre mutants as well as mice deficient in Tgf-β2 display the PTA type A4 (truncus arteriosus with interrupted aortic arch [30]), Alk5/Wnt1-Cre mutants reported here demonstrate earlier patterning defects of the PAAs, which is particularly obvious in the 3rd pair of the PAAs. Moreover, the Alk5/Wnt1-Cre mutants display an abnormal patterning of the aortic sac and defective AP septation leading to PTA, reminiscent of type A2 (= truncus artriosus with no main pulmonary artery segment present [30]). However, our results also demonstrate that significant hypoplasia of the aortic sac leads to a severe shortening of the ascending truncal arch, which masks possible defects in derivatives of the 4th PAAs, i.e., interruption of the aortic arch. These observed differences are likely due to substantial apoptosis of Alk5-deficient post-migratory neural crest cells, which is clearly detectable at E10.5, whereas similar intense cell death has not been reported in Tgfbr2/Wnt1-Cre mutants[9]. + +Our present results suggest that while TGF-β signaling in cardiac NCCs is predominantly mediated via the ALK5/TGF-βRII receptor complex, ALK5 also mediates signaling of other related ligands, which are either directly or indirectly required for appropriate NCC survival. In fact, it has been shown that, besides TGF-βRII, ALK5 can also form a complex with the Activin type IIB receptor to activate downstream Smads 2/3 [18,31]. Furthermore, a subset of TGF-β-related growth and differentiation factors (GDFs), e.g., GDF8, GDF9, GDF11 and GDF15 could induce these events [17,18,32,33]. Although relevant Gdfs 8, 9 11 and 15 are not expressed in the developing heart, nor do the mice deficient in these Gdfs display developmental cardiac defects, we cannot exclude the possibility that circulating GDFs, perhaps in concert with TGF-βs may contribute to NCC survival during cardiac and pharyngeal morphogenesis. + +We specifically studied apoptosis at the level of the aortic sac, where the AP-septum forms between the origins of 4th and 6th PAAs. Already at E10.5, we could see intense apoptosis among the postmigratory NCCs in the mesenchyme surrounding the aortic sac at the site where the prospective AP septum forms, i.e., this cell death precedes the AP septal defect seen in mutants. Although some NC-derived cells appeared to be differentiating to smooth muscle cells in the OFT (Fig. 6), we could never detect the AP septum forming in Alk5/Wnt1-Cre mutants between E10.5 and E11.5. These findings suggest that the pool of cells forming the AP septum is severely affected by the cell death. Moreover, it is likely that these cells forming the AP-septum die the before majority of them can differentiate to smooth muscle cells. + +Several in vitro studies have suggested an indispensable role for TGF-β-signaling in differentiation of NCCs into smooth muscle cells. Moreover, a recent in vivo study suggested that mice lacking Tgfbr2 in CNCCs display defective NCC differentiation into αSMA-positive cells in the AP septum [9], although this result was later disputed by another study [8]. Our immunohistochemical staining of αSMA in the OFT unequivocally demonstrated that signaling via ALK5 is not required for smooth muscle differentiation in vivo. Moreover, it has been suggested that deletion of Tgfbr2 in NCCs leads to other phenotypic features reminiscent of those seen in the velocardiofacial/DiGeorge syndrome (VCF/DGS) [9] caused by a deletion of the so called DiGeorge critical region (DGCR) on chromosome 22q11 [34,35]. Our present results suggest that although many of the observed phenotypes seen in Alk5/Wnt1-Cre mutants superficially resemble those seen in VCF/DGS, a detailed examination shows that the NC-specific abrogation of Alk5 does not lead to VCF/DGS-like phenotypes. Firstly, while the pharyngeal organ migration fails in Alk5/Wnt1-Cre mutants, perhaps as a result of increased mesenchymal cell death in the pharyngeal region, both the thymus, thyroid and parathyroid seem to develop relatively normally on the histological level in these mutants. Secondly, the NCC death seen in Alk5 mutants affects a predominantly postmigratory population of NCCs, while genes located in the DGCR, i.e., Tbx1 and CrkL, control NCC survival earlier at E8.5-E10 by regulating proliferation of the secondary heart field (SHF), and endoderm expansion, which in turn provides survival signal for NCCs allowing them to populate the pharyngeal region [36-39]. + +NCC ablation in the chick has been shown to lead to PTA and to a failure of addition of myocardium from the secondary heart field [40]. It was suggested that the defective migration of cells from the secondary heart field to the OFT in turn resulted in shortening and inappropriate rotation of the OFT, leading to mal-alignment of the arterial pole with the ventricles [41]. While the detected OFT phenotype in Alk5/Wnt1-Cre mutants shared many similarities with that seen in the chick NC ablation models, e.g., PTA and the hypoplastic aortic sac, our current results suggest that the secondary heart field is not severely affected in Alk5 mutants (data not shown). Since we could not detect appropriate rotation of the OFT in neural crest-specific mutants of Alk5 or Alk2, it appears that cells derived from the NC, as well as those from the SHF, are mutually required for proper OFT rotation in mice. However, it appears that these two TGF-β/BMP type I receptors contribute to OFT rotation through different mechanisms. In Alk5 mutants there is very little, if any, detectable septal mesenchyme present, and thus it could be argued that in these mutants OFT rotation fails due to a lack of penetration of septal prongs into the cushion mesenchyme. In contrast, in Alk2/Wnt1-Cre mutants a sizeable septal mesenchyme could be seen, still without any obvious looping of the aortic sac and truncal OFT, suggesting that the mere presence of the septal mesenchyme, without correct smooth muscle cell differentiation, is not sufficient for OFT rotation. + +Conclusion + +In this study, we have deleted the TGF-β type I receptor (Alk5) gene specifically in the mouse neural crest (NC) cell lineage. Our data suggest that ALK5 is required cell autonomously in the NC to mediate non-redundant signaling events that are essential for appropriate patterning of the pharyngeal organs and cardiac OFT. The cardiac and pharyngeal defects observed in the NC-specific Alk5 mutants differ significantly from those seen in corresponding mutants lacking the TGF-β type II receptor, suggesting that signaling mediated by ALK5 is not limited to the classical TGF-β ligands during cardiac/pharyngeal development. + +Methods + +Alk5/Wnt1Cre mice + +Alk5 (and Alk2) mutant and control embryos were generated by mating Alk5ko/+(Alk2ko/+)/Wnt1-Cre male mice with females homozygous for the Alk5flox (Alk2flox) allele and the R26R reporter[12,19]. Genotyping was performed by PCR as described earlier [21,42]. Wnt1-Cre mice were kindly provided by A. McMahon (Harvard University) and R26R reporter mice were obtained from the Jackson laboratories. All studies were carried out at the Animal Care Facility of the Saban Research Institute of Childrens Hospital Los Angeles in accordance with institutional guidelines. + +Timed mataings + +Mice were mated during the dark period of the controlled light cycle; presence of vaginal plugs was designated as day 0 hour 0. Females were euthanized by CO2, and embryos were collected in Hanks' balanced salt solution on ice. + +Histological analyses + +Embryos (E17) were fixed with 4% paraformaldehyhe for 14 hours, dehydrated and embedded in paraffin wax. Sections (7–8 um) were stained with Hematoxylin and Eosin (n≥3 for each genotype). For lineage tracing analyses, embryos were stained for β-galactosidase activity as described [43]. Briefly, the specimens (E11.0 – E11.5) were fixed in 4% paraformaldehyde for 30 minutes at room temperature, washed 3 times for 10 minutes in the detergent wash and developed for 2–12 hours in X-gal solution (n≥3 for each genotype). For immunohistochemistry, fixed sections from tissues harvested at E10.5 – E11.5 were stained with monoclonal α-smooth muscle actin (DAKO), cleaved caspase-3 (Cell Signaling) or phophohistone-H3 (Cell Signaling) antibodies. TUNEL assays were performed using the DeadEnd fluorometric TUNEL system (Promega). In each assay 3 or more embryos were analyzed for each genotype. + +Ink and casting dye injections + +Embryos (E10.0 – E13.0) were dissected and placed in ice cold PBS (n≥3 per genotype in each time point). Using a pulled glass pipette, India ink or Yellow casting dye (Connecticut Valley Biological Supply) was injected into the ventricles until ink/dye penetrated small vessels. Embryos were postfixed in 10% buffered formalin for 12 hours, dehydrated and cleared in benzylbenzoate: benzyl alcohol (2:1v/v). + +Expression analyses + +To visualize parathyroid hormone expression we used in situ hybridization and an antisense probe corresponding to nucleotides 97–534 (kindly provided by Nancy Manley) as described [44]. + +Authors' contributions + +J.W. did most of the mouse dissections and analyses, A.N. did some of the histological analyses, J.L. generated the Alk5FXFXmice, M.D. did some of the expression analyses, H.M.S. participated in design and provided the Tgfbr2 mutant embryos and V.K. generated the Alk2FXFX mice, designed and supervised the experiments and wrote the manuscript. All authors have read and approved the final manuscript. + +Acknowledgements + +We thank A. McMahon for providing the Wnt1-Cre mouse line, N. Manley for the PTH probe and B. Choudhary for Tgfbr2/Wnt1-Cre embryos. H.M.S. was supported by grants from the NIH, and V.K. by grants from the Robert E. Schneider Foundation and the NIH (HL074862 and DE013085). diff --git a/src/ontogpt/evaluation/craft/database/all/17083276.ann b/src/ontogpt/evaluation/craft/database/all/17083276.ann new file mode 100644 index 000000000..0bcf23ce3 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17083276.ann @@ -0,0 +1,1137 @@ +T1 GO:0033186 0 5 CAF-1 +T2 GO:0000792 23 38 Heterochromatin +T3 CL:0002248 55 66;77 82 Pluripotent ... Cells +T4 UBERON:0000922 67 76 Embryonic +T5 CL:0002321 67 82 Embryonic Cells +T6 NCBITaxon:40674 101 110 mammalian +T7 GO:0000785 124 133 chromatin +T8 SO:0001026 184 190 genome +T9 GO:0000785 252 261 chromatin +T10 GO:0031497 252 270 chromatin assembly +T11 GO:0031497 321 339 chromatin assembly +T12 GO:0033186 321 348 chromatin assembly factor 1 +T13 GO:0033186 350 355 CAF-1 +T14 GO:0007566 368 380 implantation +T15 NCBITaxon:10088 409 414 mouse +T16 SO:0000704 456 460 gene +T17 PR:000005402 489 498 p150CAF-1 +T18 PR:000005402 508 517 p150CAF-1 +T19 PR:000005402 603 612 p150CAF-1 +T20 UBERON:0000922 622 629 embryos +T21 GO:0005634 667 674 nuclear +T22 GO:0000792 704 719 heterochromatin +T23 UBERON:0000922 759 766 embryos +T24 GO:0000792 768 783 heterochromatin +T25 UBERON:0007232 832 840;856 862 two-cell ... stages +T26 UBERON:0000358 845 855 blastocyst +T27 PR:000005402 867 876 p150CAF-1 +T28 UBERON:0000922 898 905 embryos +T29 GO:0000792 935 950 heterochromatin +T30 GO:0000792 993 1008 heterochromatin +T31 UBERON:0007232 1012 1016;1025 1035 two- ... cell stage +T32 UBERON:0007233 1020 1035 four-cell stage +T33 UBERON:0000922 1046 1053 embryos +T34 GO:0033186 1071 1076 CAF-1 +T35 GO:0000792 1111 1126 heterochromatin +T36 GO:0007566 1137 1149 implantation +T37 UBERON:0000922 1166 1175 embryonic +T38 CL:0002322 1166 1186 embryonic stem cells +T39 PR:000005402 1201 1210 p150CAF-1 +T40 GO:0016246 1217 1233 RNA interference +T41 GO:0005721 1308 1335 pericentric heterochromatin +T42 GO:0033186 1366 1371 CAF-1 +T43 PR:000043452 1427 1434 histone +T44 CHEBI:15358 1427 1434 histone +T45 GO:0016571 1427 1446 histone methylation +T46 GO:0005721 1469 1496 pericentric heterochromatin +T47 GO:0000792 1519 1534 heterochromatin +T48 PR:000005402 1552 1561 p150CAF-1 +T49 NCBITaxon:10088 1571 1576 mouse +T50 UBERON:0000922 1577 1586 embryonic +T51 CL:0000057 1587 1598 fibroblasts +T52 GO:0033186 1668 1673 CAF-1 +T53 GO:0000792 1703 1718 heterochromatin +T54 CL:0002248 1735 1746;1757 1762 pluripotent ... cells +T55 UBERON:0000922 1747 1756 embryonic +T56 CL:0002321 1747 1762 embryonic cells +T57 GO:0000785 1803 1812 chromatin +T58 GO:0031497 1803 1821 chromatin assembly +T59 GO:0065007 1835 1846 controlling +T60 SO:0001026 1902 1908 genome +T61 UBERON:0019248 1912 1925 early embryos +T62 UBERON:0000922 1930 1939 embryonic +T63 CL:0002322 1930 1950 embryonic stem cells +T64 GO:0000785 1963 1972 Chromatin +T65 SO:0000704 1995 2002 genetic +T66 GO:0000786 2065 2076 nucleosomes +T67 PR:000043452 2114 2121 histone +T68 CHEBI:15358 2114 2121 histone +T69 GO:0000786 2195 2206 nucleosomes +T70 GO:0065007 2231 2241 regulation +T71 SO:0001026 2245 2251 genome +T72 GO:0051301 2336 2350 cell divisions +T73 GO:0000785 2381 2390 chromatin +T74 SO:0000704 2411 2418 genetic +T75 SO:0001026 2483 2489 genome +T76 GO:0006260 2490 2501 replication +T77 GO:0000785 2513 2522 chromatin +T78 GO:0031497 2513 2531 chromatin assembly +T79 GO:0000786 2560 2571 nucleosomes +T80 GO:0000786 2600 2611 nucleosomes +T81 GO:0000785 2741 2750 chromatin +T82 GO:0031497 2741 2759 chromatin assembly +T83 GO:0033186 2768 2773 CAF-1 +T84 NCBITaxon:10088 2778 2783 mouse +T85 UBERON:0000922 2784 2793 embryonic +T86 CL:0002322 2784 2804 embryonic stem cells +T87 UBERON:0019248 2809 2822 early embryos +T88 GO:0033186 2853 2858 CAF-1 +T89 SO:0001026 2902 2909 genomic +T90 GO:0033186 2957 2962 CAF-1 +T91 GO:0005634 3033 3040 nucleus +T92 GO:0000785 3112 3121 chromatin +T93 UBERON:0000922 3144 3153 embryonic +T94 CL:0002322 3144 3163 embryonic stem cell +T95 NCBITaxon:10088 3211 3216 mouse +T96 GO:0007566 3221 3233 implantation +T97 SO:0001026 3251 3257 genome +T98 UBERON:0000922 3318 3327 embryonic +T99 SO:0000704 3328 3332 gene +T100 GO:0010467 3328 3343 gene expression +T101 GO:0006306 3475 3490 DNA methylation +T102 GO:0000785 3559 3568 chromatin +T103 PR:000043452 3634 3641 histone +T104 CHEBI:15358 3634 3641 histone +T105 GO:0016570 3634 3651 histone modifying +T106 GO:0000785 3661 3670 chromatin +T107 GO:0006338 3661 3681 chromatin remodeling +T108 PR:000043452 3695 3702 histone +T109 CHEBI:15358 3695 3702 histone +T110 GO:0007566 3725 3737 implantation +T111 CL:0000034 3757 3767 stem cells +T112 UBERON:0019248 3781 3794 early embryos +T113 PR:000043452 3823 3830 histone +T114 CHEBI:15358 3823 3830 histone +T115 GO:0000785 3846 3855 chromatin +T116 GO:0006338 3846 3866 chromatin remodeling +T117 GO:0032991 3867 3876 complexes +T118 GO:0000786 3904 3914 nucleosome +T119 GO:0006334 3904 3923 nucleosome assembly +T120 GO:0065007 3978 3985 control +T121 GO:0000785 3986 3995 chromatin +T122 GO:0031497 3986 4004 chromatin assembly +T123 PR:000043452 4052 4059 histone +T124 CHEBI:15358 4052 4059 histone +T125 GO:0031497 4072 4090 chromatin assembly +T126 GO:0033186 4072 4099 chromatin assembly factor 1 +T127 GO:0033186 4101 4106 CAF-1 +T128 PR:000005402 4128 4132 p150 +T129 PR:000005403 4134 4137 p60 +T130 PR:000013776 4143 4146 p48 +T131 GO:0032991 4148 4155 complex +T132 CHEBI:15358 4171 4178 histone +T133 PR:000027594 4171 4181 histone H3 +T134 PR:000008603 4171 4178;4186 4188 histone ... H4 +T135 GO:0006260 4234 4245 replication +T136 GO:0006281 4249 4259 DNA repair +T137 GO:0033186 4282 4287 CAF-1 +T138 CHEBI:15358 4301 4308 histone +T139 PR:000027594 4301 4311 histone H3 +T140 GO:0000785 4330 4339 chromatin +T141 GO:0071897 4365 4378 DNA synthesis +T142 PR:000043452 4397 4404 histone +T143 CHEBI:15358 4397 4404 histone +T144 PR:000008570 4416 4420 HIRA +T145 PR:000008425 4459 4463 H3.3 +T146 GO:0071897 4477 4490 DNA synthesis +T147 GO:0000785 4549 4558 chromatin +T148 GO:0031497 4549 4567 chromatin assembly +T149 GO:0033186 4578 4583 CAF-1 +T150 GO:0000792 4627 4642 heterochromatin +T151 GO:0000792 4654 4669 heterochromatin +T152 PR:P05205 4654 4679 heterochromatin protein 1 +T153 PR:P05205 4681 4684 HP1 +T154 PR:000010214 4690 4694 MBD1 +T155 SO:0000417 4717 4723 domain +T156 PR:000043452 4746 4753 histone +T157 CHEBI:15358 4746 4753 histone +T158 PR:000043452 4781 4788 histone +T159 CHEBI:15358 4781 4788 histone +T160 PR:000005402 4840 4849 p150CAF-1 +T161 GO:0006260 4874 4885 replication +T162 PR:P05205 4903 4906 HP1 +T163 CHEBI:36357 4907 4916 molecules +T164 GO:0006260 4920 4931 replication +T165 SO:0000296 4920 4937 replication sites +T166 GO:0005721 4941 4968 pericentric heterochromatin +T167 GO:0051320 4985 4992 S phase +T168 GO:0033186 5044 5049 CAF-1 +T169 GO:0031507 5057 5085 formation of heterochromatin +T170 GO:0000792 5070 5085 heterochromatin +T171 PR:000043452 5182 5189 histone +T172 CHEBI:15358 5182 5189 histone +T173 NCBITaxon:40674 5324 5331 mammals +T174 CL:0002248 5339 5356 pluripotent cells +T175 UBERON:0000922 5365 5374 embryonic +T176 CL:0002322 5365 5379;5385 5390 embryonic stem ... cells +T177 CL:0002322 5381 5383;5385 5390 ES ... cells +T178 GO:0033186 5433 5438 CAF-1 +T179 NCBITaxon:10088 5452 5457 mouse +T180 SO:0000704 5473 5480 genetic +T181 CL:0002322 5497 5505 ES cells +T182 GO:0016246 5525 5541 RNA interference +T183 GO:0016246 5543 5547 RNAi +T184 GO:0033186 5563 5568 CAF-1 +T185 UBERON:0019248 5599 5604;5611 5618 early ... embryos +T186 NCBITaxon:10088 5605 5610 mouse +T187 CL:0002322 5623 5631 ES cells +T188 GO:0033186 5658 5663 CAF-1 +T189 GO:0000792 5731 5746 heterochromatin +T190 CL:0002248 5758 5769;5780 5785 pluripotent ... cells +T191 UBERON:0000922 5770 5779 embryonic +T192 CL:0002321 5770 5785 embryonic cells +T193 SO:0000704 5805 5809 gene +T194 CL:0002322 5823 5831 ES cells +T195 SO:0000147 5842 5846 exon +T196 PR:000005402 5856 5862 Chaf1a +T197 SO:0000704 5863 5867 gene +T198 PR:000005402 5883 5892 p150CAF-1 +T199 PR:000005402 5906 5912 Chaf1a +T200 NCBITaxon:10088 5916 5920 mice +T201 GO:0007567 5926 5930 born +T202 NCBITaxon:10088 6065 6069 mice +T203 PR:000005402 6104 6110 Chaf1a +T204 NCBITaxon:10088 6114 6118 mice +T205 PR:000005402 6147 6153 Chaf1a +T206 UBERON:0000922 6157 6164 embryos +T207 GO:0007566 6198 6210 implantation +T208 PR:000005402 6244 6250 Chaf1a +T209 UBERON:0000922 6254 6261 embryos +T210 UBERON:0000922 6265 6274 embryonic +T211 UBERON:0000922 6350 6357 embryos +T212 PR:000005402 6372 6378 Chaf1a +T213 NCBITaxon:10088 6382 6386 mice +T214 UBERON:0000922 6457 6464 embryos +T215 UBERON:0000922 6517 6524 embryos +T216 UBERON:0000358 6634 6645 blastocysts +T217 PR:000005402 6769 6778 p150CAF-1 +T218 UBERON:0000922 6788 6795 embryos +T219 PR:000005402 6807 6816 p150CAF-1 +T220 UBERON:0007236 6859 6875 eight-cell stage +T221 UBERON:0000922 6960 6966 embryo +T222 UBERON:0007233 6977 6992 four-cell stage +T223 PR:000005402 7013 7022 p150CAF-1 +T224 GO:0051301 7082 7096 cell divisions +T225 GO:0005634 7157 7164 nuclear +T226 GO:0000792 7181 7196 heterochromatin +T227 GO:0005634 7222 7228 nuclei +T228 PR:000005402 7232 7238 Chaf1a +T229 UBERON:0000922 7242 7249 embryos +T230 GO:0005721 7251 7278 Pericentric heterochromatin +T231 GO:0000792 7316 7331 heterochromatin +T232 GO:0051325 7361 7371 interphase +T233 GO:0005634 7372 7378 nuclei +T234 CHEBI:51217 7386 7398 fluorochrome +T235 CHEBI:51231 7399 7403 DAPI +T236 PR:000005086 7431 7435 HP1α +T237 UBERON:0000358 7461 7472 blastocysts +T238 NCBITaxon:10088 7480 7485 mouse +T239 CL:0002371 7486 7499 somatic cells +T240 GO:0005721 7501 7528 pericentric heterochromatin +T241 GO:0000785 7576 7585 chromatin +T242 GO:0010369 7604 7617 chromocenters +T243 CHEBI:51231 7648 7652 DAPI +T244 PR:000005402 7715 7721 Chaf1a +T245 UBERON:0000922 7725 7732 embryos +T246 GO:0000785 7752 7761 chromatin +T247 SO:0001026 7821 7827 genome +T248 GO:0000792 7945 7960 heterochromatin +T249 UBERON:0007232 7986 7994;8010 8016 two-cell ... stages +T250 UBERON:0000358 7999 8009 blastocyst +T251 UBERON:0007232 8021 8035 two-cell stage +T252 UBERON:0000922 8046 8053 embryos +T253 CHEBI:51231 8055 8059 DAPI +T254 GO:0005731 8137 8163 nucleolar precursor bodies +T255 GO:0000792 8177 8192 Heterochromatin +T256 CHEBI:51231 8234 8238 DAPI +T257 UBERON:0007233 8262 8271;8295 8301 four-cell ... stages +T258 UBERON:0000358 8284 8294 blastocyst +T259 PR:000005086 8331 8335 HP1α +T260 CHEBI:51231 8380 8384 DAPI +T261 GO:0000792 8421 8436 heterochromatin +T262 GO:0005634 8460 8467 nuclear +T263 GO:0000792 8484 8499 heterochromatin +T264 GO:0007566 8536 8548 implantation +T265 UBERON:0007232 8574 8582;8598 8604 two-cell ... stages +T266 UBERON:0000358 8587 8597 blastocyst +T267 PR:000005402 8609 8615 Chaf1a +T268 UBERON:0000922 8622 8629 embryos +T269 UBERON:0007236 8662 8668;8676 8686 eight- ... cell stage +T270 CHEBI:51231 8688 8692 DAPI +T271 CHEBI:51231 8749 8753 DAPI +T272 GO:0005634 8786 8793 nucleus +T273 GO:0005730 8837 8845 nucleoli +T274 GO:0034399 8857 8869;8874 8880 periphery of ... nuclei +T275 PR:000005086 8910 8914 HP1α +T276 CHEBI:51231 8955 8959 DAPI +T277 GO:0034399 8989 9006 nuclear periphery +T278 GO:0005730 9023 9031 nucleoli +T279 GO:0000792 9075 9090 heterochromatin +T280 PR:000005402 9094 9100 Chaf1a +T281 UBERON:0000922 9104 9111 embryos +T282 GO:0000792 9134 9149 heterochromatin +T283 UBERON:0007232 9172 9176;9185 9195 two- ... cell stage +T284 UBERON:0007233 9180 9195 four-cell stage +T285 UBERON:0000922 9206 9213 embryos +T286 PR:000005402 9277 9286 p150CAF-1 +T287 GO:0007566 9298 9310 implantation +T288 GO:0005720 9400 9422;9438 9444 heterochromatin within ... nuclei +T289 UBERON:0000922 9423 9432 embryonic +T290 CL:0002321 9423 9437 embryonic cell +T291 GO:0005634 9433 9444 cell nuclei +T292 GO:0033186 9499 9504 CAF-1 +T293 CL:0002322 9531 9539 ES cells +T294 UBERON:0000358 9568 9578 blastocyst +T295 UBERON:0000087 9579 9594 inner cell mass +T296 PR:000005402 9645 9651 Chaf1a +T297 UBERON:0000922 9655 9662 embryos +T298 UBERON:0000922 9715 9722 embryos +T299 GO:0016246 9744 9748 RNAi +T300 PR:000005402 9771 9780 p150CAF-1 +T301 CL:0002322 9794 9802 ES cells +T302 PR:000005402 9864 9873 p150CAF-1 +T303 GO:0016246 9874 9878 RNAi +T304 CL:0002322 9888 9896 ES cells +T305 PR:000005402 9948 9954 Chaf1a +T306 NCBITaxon:10088 9961 9965 mice +T307 CHEBI:51231 9967 9971 DAPI +T308 PR:000005086 10018 10022 HP1α +T309 CHEBI:51231 10027 10031 DAPI +T310 GO:0005730 10052 10060 nucleoli +T311 GO:0034399 10072 10084;10089 10096 periphery of ... nucleus +T312 PR:000005402 10137 10146 p150CAF-1 +T313 GO:0005634 10164 10171 nuclear +T314 GO:0000792 10188 10203 heterochromatin +T315 CL:0002322 10207 10215 ES cells +T316 GO:0007566 10246 10258 implantation +T317 UBERON:0000922 10259 10266 embryos +T318 GO:0000792 10327 10342 heterochromatin +T319 NCBITaxon:10088 10367 10372 mouse +T320 UBERON:0000922 10373 10382 embryonic +T321 CL:0000057 10383 10394 fibroblasts +T322 PR:000005402 10412 10421 p150CAF-1 +T323 GO:0016246 10435 10439 RNAi +T324 PR:000005402 10441 10450 p150CAF-1 +T325 GO:0008283 10504 10522 cell proliferation +T326 GO:0000792 10579 10594 heterochromatin +T327 PR:000005402 10635 10644 p150CAF-1 +T328 GO:0000792 10683 10698 heterochromatin +T329 GO:0033186 10768 10773 CAF-1 +T330 UBERON:0019248 10781 10793 early embryo +T331 CL:0002322 10798 10806 ES cells +T332 PR:000005402 10842 10851 p150CAF-1 +T333 CL:0002322 10855 10863 ES cells +T334 GO:0000792 10889 10904 heterochromatin +T335 GO:0005634 10905 10912 nuclear +T336 GO:0005634 10981 10988 nuclear +T337 GO:0042571 11039 11047 antibody +T338 CL:0000836 11060 11073 promyelocytic +T339 PR:000026474 11060 11082 promyelocytic leukemia +T340 PR:000026474 11084 11087 PML +T341 PR:000026474 11122 11125 PML +T342 GO:0016605 11122 11140 PML nuclear bodies +T343 GO:0005634 11174 11181 nuclear +T344 PR:000026474 11280 11283 PML +T345 GO:0016605 11280 11298 PML nuclear bodies +T346 PR:000005402 11319 11328 p150CAF-1 +T347 CL:0002322 11338 11346 ES cells +T348 GO:0005634 11381 11388 nuclear +T349 GO:0033186 11436 11441 CAF-1 +T350 GO:0033186 11509 11514 CAF-1 +T351 GO:0000785 11561 11570 chromatin +T352 CHEBI:15358 11602 11609 histone +T353 PR:000027594 11602 11612 histone H3 +T354 PR:000005086 11617 11621 HP1α +T355 GO:0033186 11641 11646 CAF-1 +T356 UBERON:0019248 11675 11688 early embryos +T357 CL:0002322 11696 11704 ES cells +T358 GO:0000792 11740 11755 heterochromatin +T359 GO:0005634 11759 11766 nuclear +T360 NCBITaxon:40674 11811 11820 mammalian +T361 GO:0033186 11844 11849 CAF-1 +T362 GO:0051320 11875 11882 S phase +T363 GO:0009294 11935 11947 transfection +T364 GO:0016246 11955 11959 RNAi +T365 SO:0000440 11960 11966 vector +T366 PR:000005402 11968 11977 p150CAF-1 +T367 GO:0033186 12030 12035 CAF-1 +T368 GO:0006260 12071 12082 replication +T369 CHEBI:472552 12137 12154 bromodeoxyuridine +T370 CHEBI:472552 12156 12160 BrdU +T371 PR:000012421 12179 12183 PCNA +T372 GO:0008283 12185 12198 proliferating +T373 PR:000012421 12185 12219 proliferating cell nuclear antigen +T374 CHEBI:59132 12212 12219 antigen +T375 GO:0006260 12306 12321 DNA replication +T376 GO:0000792 12362 12377 heterochromatin +T377 CL:0002322 12413 12421 ES cells +T378 GO:0008283 12431 12442 proliferate +T379 GO:0016265 12447 12450 die +T380 PR:000005402 12467 12476 p150CAF-1 +T381 GO:0009987 12506 12522 cellular process +T382 UBERON:0000922 12530 12537 embryos +T383 PR:000005402 12585 12594 p150CAF-1 +T384 CL:0002322 12607 12615 ES cells +T385 UBERON:0019248 12620 12633 early embryos +T386 GO:0005720 12667 12685;12690 12697 heterochromatin in ... nucleus +T387 GO:0007049 12725 12735 cell cycle +T388 GO:0000792 12799 12814 heterochromatin +T389 GO:0097617 12877 12890 hybridization +T390 NCBITaxon:10088 13002 13007 mouse +T391 CHEBI:51231 13224 13228 DAPI +T392 GO:0051325 13247 13257 interphase +T393 GO:0005634 13258 13265 nucleus +T394 PR:000005402 13381 13390 p150CAF-1 +T395 GO:0016246 13391 13395 RNAi +T396 GO:0005721 13440 13467 pericentric heterochromatin +T397 CHEBI:51231 13518 13522 DAPI +T398 GO:0034399 13781 13798 nuclear periphery +T399 CHEBI:51231 13821 13825 DAPI +T400 PR:000005402 13861 13870 p150CAF-1 +T401 GO:0005634 13959 13966 nucleus +T402 CHEBI:51231 14035 14039 DAPI +T403 GO:0097617 14060 14073 hybridization +T404 PR:000005402 14085 14094 p150CAF-1 +T405 GO:0005721 14159 14186 pericentric heterochromatin +T406 PR:000005402 14323 14332 p150CAF-1 +T407 PR:000005402 14402 14411 p150CAF-1 +T408 GO:0005721 14470 14497 pericentric heterochromatin +T409 GO:0033186 14532 14537 CAF-1 +T410 CHEBI:15358 14559 14566 histone +T411 PR:000008603 14559 14566;14576 14578 histone ... H4 +T412 GO:0071897 14595 14608 DNA synthesis +T413 GO:0000785 14666 14675 chromatin +T414 GO:0000786 14715 14726 nucleosomal +T415 PR:000005402 14864 14873 p150CAF-1 +T416 CL:0002322 14883 14891 ES cells +T417 SO:0001026 14913 14919 genome +T418 GO:0000785 14920 14929 chromatin +T419 CHEBI:15358 15034 15041 histone +T420 PR:000027594 15034 15044 histone H3 +T421 GO:0000790 15050 15069 chromatin in nuclei +T422 GO:0009294 15090 15101 transfected +T423 PR:000005402 15119 15128 p150CAF-1 +T424 GO:0016246 15129 15133 RNAi +T425 SO:0000755 15134 15149 plasmid vectors +T426 GO:0005634 15151 15157 Nuclei +T427 CHEBI:24866 15199 15203 salt +T428 CHEBI:26710 15239 15243 NaCl +T429 CHEBI:26710 15251 15255 NaCl +T430 CHEBI:15358 15290 15297 histone +T431 PR:000027594 15290 15300 histone H3 +T432 GO:0000785 15327 15336 chromatin +T433 PR:000005402 15374 15383 p150CAF-1 +T434 CL:0002322 15393 15401 ES cells +T435 GO:0005721 15478 15505 pericentric heterochromatin +T436 GO:0000786 15562 15573 nucleosomal +T437 GO:0005634 15618 15625 nuclear +T438 PR:000005086 15642 15646 HP1α +T439 PR:000005402 15670 15676 Chaf1a +T440 UBERON:0000922 15680 15687 embryos +T441 PR:000005402 15692 15701 p150CAF-1 +T442 CL:0002322 15711 15719 ES cells +T443 GO:0005721 15813 15840 pericentric heterochromatin +T444 GO:0010424 15858 15877 DNA CpG methylation +T445 PR:000005402 15966 15975 p150CAF-1 +T446 CL:0002322 16015 16022 ES cell +T447 GO:0016246 16048 16052 RNAi +T448 GO:0006306 16093 16108 DNA methylation +T449 GO:0000792 16265 16280 heterochromatin +T450 PR:000005402 16292 16301 p150CAF-1 +T451 CL:0002322 16311 16319 ES cells +T452 GO:0016246 16335 16339 RNAi +T453 SO:0000755 16340 16354 plasmid vector +T454 CL:0002322 16363 16370 ES cell +T455 PR:000005402 16404 16413 p150CAF-1 +T456 GO:0000792 16437 16452 heterochromatin +T457 GO:0006306 16497 16512 DNA methylation +T458 PR:000043452 16582 16589 histone +T459 CHEBI:15358 16582 16589 histone +T460 GO:0016570 16582 16603 histone modifications +T461 GO:0000785 16612 16621 chromatin +T462 GO:0000785 16674 16683 chromatin +T463 GO:0042571 16796 16806 antibodies +T464 PR:000005402 16817 16826 p150CAF-1 +T465 PR:000005402 16979 16988 p150CAF-1 +T466 GO:0005721 17025 17052 pericentric heterochromatin +T467 PR:000005402 17296 17305 p150CAF-1 +T468 GO:0005721 17350 17377 pericentric heterochromatin +T469 SO:0001707 17418 17425 H3K9me3 +T470 PR:000005402 17470 17479 p150CAF-1 +T471 SO:0001707 17598 17605 H3K9me3 +T472 CHEBI:51231 17642 17646 DAPI +T473 SO:0001707 17709 17716 H3K9me3 +T474 PR:000005402 17743 17752 p150CAF-1 +T475 PR:000005086 17809 17813 HP1α +T476 CHEBI:51231 18100 18104 DAPI +T477 GO:0033186 18168 18173 CAF-1 +T478 CL:0002322 18177 18185 ES cells +T479 GO:0000792 18224 18239 heterochromatin +T480 PR:000005402 18342 18351 p150CAF-1 +T481 NCBITaxon:10088 18382 18387 mouse +T482 GO:0009790 18394 18407 embryogenesis +T483 CL:0002322 18415 18423 ES cells +T484 GO:0033186 18454 18459 CAF-1 +T485 GO:0006260 18496 18511 DNA replication +T486 NCBITaxon:7742 18515 18526 vertebrates +T487 NCBITaxon:8355 18531 18545 Xenopus laevis +T488 PR:000005402 18547 18556 p150CAF-1 +T489 GO:0051301 18592 18606 cell divisions +T490 GO:0033186 18660 18665 CAF-1 +T491 NCBITaxon:40674 18682 18691 mammalian +T492 GO:0006260 18726 18741 DNA replication +T493 GO:0033186 18788 18793 CAF-1 +T494 GO:0005634 18801 18808 nuclear +T495 GO:0000792 18825 18840 heterochromatin +T496 CL:0002322 18881 18889 ES cells +T497 PR:000005402 18907 18916 p150CAF-1 +T498 CL:0002322 18926 18934 ES cells +T499 GO:0006260 18942 18957 DNA replication +T500 GO:0000786 19013 19024 nucleosomal +T501 GO:0000792 19046 19061 heterochromatin +T502 PR:000005402 19108 19117 p150CAF-1 +T503 PR:000026474 19133 19136 PML +T504 GO:0016605 19133 19143 PML bodies +T505 CL:0002322 19147 19154 ES cell +T506 GO:0005634 19150 19161 cell nuclei +T507 GO:0005634 19176 19183 nuclear +T508 PR:000005402 19235 19244 p150CAF-1 +T509 CL:0002322 19280 19287 ES cell +T510 GO:0051301 19283 19296 cell division +T511 GO:0008219 19301 19311 cell death +T512 GO:0000792 19349 19364 heterochromatin +T513 UBERON:0000922 19464 19471 embryos +T514 GO:0016265 19476 19481 death +T515 CL:0002322 19485 19493 ES cells +T516 GO:0033186 19507 19512 CAF-1 +T517 GO:0005634 19520 19527 nuclear +T518 GO:0000792 19544 19559 heterochromatin +T519 GO:0033186 19671 19676 CAF-1 +T520 NCBITaxon:9606 19725 19730 human +T521 CL:0002248 19777 19788;19799 19804 pluripotent ... cells +T522 UBERON:0000922 19789 19798 embryonic +T523 CL:0002321 19789 19804 embryonic cells +T524 UBERON:0019248 19821 19834 early embryos +T525 CL:0002322 19839 19847 ES cells +T526 SO:0001026 19855 19861 genome +T527 CL:0002371 19883 19896 somatic cells +T528 GO:0033186 19974 19979 CAF-1 +T529 GO:0000792 19983 19998 heterochromatin +T530 CL:0002322 20031 20039 ES cells +T531 GO:0000785 20061 20070 chromatin +T532 GO:0000785 20128 20137 chromatin +T533 GO:0000792 20200 20215 heterochromatin +T534 GO:0005634 20216 20223 nuclear +T535 GO:0033186 20247 20252 CAF-1 +T536 SO:0001026 20352 20358 genome +T537 CL:0002322 20401 20409 ES cells +T538 GO:0033186 20442 20447 CAF-1 +T539 GO:0033186 20486 20491 CAF-1 +T540 CL:0002248 20495 20506;20517 20522 pluripotent ... cells +T541 UBERON:0000922 20507 20516 embryonic +T542 CL:0002321 20507 20522 embryonic cells +T543 NCBITaxon:3702 20581 20601 Arabidopsis thaliana +T544 GO:0033186 20612 20617 CAF-1 +T545 GO:0033186 20663 20668 CAF-1 +T546 GO:0010369 20709 20721 chromocenter +T547 GO:0033186 20782 20787 CAF-1 +T548 GO:0000792 20808 20823 heterochromatin +T549 UBERON:0019248 20840 20845;20852 20859 early ... embryos +T550 NCBITaxon:10088 20846 20851 mouse +T551 NCBITaxon:10088 20864 20869 mouse +T552 CL:0002322 20870 20878 ES cells +T553 GO:0033186 20893 20898 CAF-1 +T554 NCBITaxon:10088 21045 21050 mouse +T555 SO:0001026 21051 21057 genome +T556 GO:0005634 21090 21097 nuclear +T557 GO:0005721 21137 21164 pericentric heterochromatin +T558 CL:0002322 21180 21188 ES cells +T559 GO:0000792 21225 21240 heterochromatin +T560 GO:0033186 21309 21314 CAF-1 +T561 GO:0033186 21346 21351 CAF-1 +T562 NCBITaxon:species 21359 21366 species +T563 CHEBI:15358 21441 21448 histone +T564 PR:000027594 21441 21451 histone H3 +T565 NCBITaxon:2759 21481 21491 eukaryotes +T566 GO:0033186 21493 21498 CAF-1 +T567 NCBITaxon:40674 21515 21522 mammals +T568 NCBITaxon:4932 21542 21566 Saccharomyces cerevisiae +T569 GO:0007114 21592 21599 budding +T570 NCBITaxon:4892 21592 21605 budding yeast +T571 SO:0001026 21606 21612 genome +T572 GO:0000792 21630 21645 heterochromatin +T573 NCBITaxon:2759 21684 21694 eukaryotes +T574 GO:0000792 21729 21744 heterochromatin +T575 GO:0036123 21753 21756;21764 21783 di- ... methylation at H3K9 +T576 GO:0034772 21753 21756;21764 21778;21796 21801 di- ... methylation at ... H4K20 +T577 GO:0036124 21761 21783 trimethylation at H3K9 +T578 GO:0070734 21761 21778;21785 21790 trimethylation at ... H3K27 +T579 GO:0034773 21761 21778;21796 21801 trimethylation at ... H4K20 +T580 GO:0035328 21826 21842 silent chromatin +T581 NCBITaxon:4932 21846 21859 S. cerevisiae +T582 NCBITaxon:40674 21864 21871 mammals +T583 GO:0036123 21873 21891 H3K9 dimethylation +T584 PR:000008425 21944 21948 H3.3 +T585 GO:0000785 22009 22018 chromatin +T586 PR:000027594 22060 22062 H3 +T587 NCBITaxon:4932 22072 22085 S. cerevisiae +T588 PR:000008425 22101 22105 H3.3 +T589 GO:0010467 22116 22125 expressed +T590 GO:0000785 22148 22157 chromatin +T591 GO:0006260 22163 22174 replication +T592 NCBITaxon:2759 22205 22215 eukaryotes +T593 GO:0000792 22269 22284 heterochromatin +T594 GO:0033186 22312 22317 CAF-1 +T595 GO:0006260 22335 22346 replication +T596 GO:0032991 22365 22372 complex +T597 GO:0005634 22412 22419 nuclear +T598 GO:0005721 22436 22463 pericentric heterochromatin +T599 UBERON:0000922 22467 22474 embryos +T600 CL:0002322 22479 22487 ES cells +T601 GO:0000785 22561 22570 chromatin +T602 GO:0005634 22598 22605 nuclear +T603 GO:0000792 22635 22650 heterochromatin +T604 UBERON:0000922 22657 22666 embryonic +T605 GO:0000792 22820 22835 heterochromatin +T606 PR:000005402 22847 22856 p150CAF-1 +T607 UBERON:0000922 22862 22869 embryos +T608 CL:0002322 22874 22882 ES cells +T609 GO:0033186 22884 22889 CAF-1 +T610 GO:0000792 22930 22945 heterochromatin +T611 GO:0006260 23056 23071 DNA replication +T612 PR:000005402 23090 23099 p150CAF-1 +T613 GO:0000792 23101 23116 heterochromatin +T614 CHEBI:36357 23147 23155 molecule +T615 GO:0033186 23278 23283 CAF-1 +T616 GO:0000792 23350 23365 heterochromatin +T617 GO:0007566 23373 23385 implantation +T618 UBERON:0000922 23386 23393 embryos +T619 GO:0005634 23426 23433 nuclear +T620 UBERON:0007232 23459 23467;23483 23489 two-cell ... stages +T621 UBERON:0000358 23472 23482 blastocyst +T622 NCBITaxon:10088 23514 23519 mouse +T623 GO:0005634 23538 23545 nuclear +T624 GO:0005720 23562 23580;23589 23595 heterochromatin in ... nuclei +T625 CL:0002322 23581 23588 ES cell +T626 GO:0005634 23584 23595 cell nuclei +T627 UBERON:0000106 23626 23640 one-cell stage +T628 GO:0005634 23661 23668 nuclear +T629 GO:0000792 23705 23720 heterochromatin +T630 GO:0005634 23748 23755 nuclear +T631 UBERON:0019248 23780 23795 early embryonic +T632 UBERON:0000922 23813 23820 embryos +T633 GO:0000792 23853 23868 heterochromatin +T634 PR:000005402 23913 23922 p150CAF-1 +T635 CL:0002322 23932 23940 ES cells +T636 PR:000005402 23988 23994 Chaf1a +T637 UBERON:0000922 23998 24005 embryos +T638 GO:0033186 24028 24033 CAF-1 +T639 GO:0000792 24067 24082 heterochromatin +T640 GO:0051301 24113 24127 cell divisions +T641 UBERON:0000922 24131 24140 embryonic +T642 GO:0005634 24171 24178 nuclear +T643 GO:0010468 24199 24225 control of gene expression +T644 SO:0000704 24210 24214 gene +T645 GO:0033186 24262 24267 CAF-1 +T646 SO:0000704 24311 24315 gene +T647 GO:0010467 24311 24326 gene expression +T648 GO:0009790 24340 24353 embryogenesis +T649 GO:0000785 24414 24423 chromatin +T650 CL:0002248 24465 24476;24487 24492 pluripotent ... cells +T651 UBERON:0000922 24477 24486 embryonic +T652 CL:0002321 24477 24492 embryonic cells +T653 GO:0006306 24536 24551 DNA methylation +T654 PR:000043452 24556 24563 histone +T655 CHEBI:15358 24556 24563 histone +T656 GO:0016570 24556 24573 histone modifying +T657 GO:0000785 24627 24636 chromatin +T658 GO:0031497 24627 24645 chromatin assembly +T659 GO:0000786 24696 24707 nucleosomes +T660 GO:0033186 24709 24714 CAF-1 +T661 GO:0000792 24762 24777 heterochromatin +T662 UBERON:0019248 24789 24802 early embryos +T663 CL:0002322 24807 24815 ES cells +T664 GO:0009790 24841 24854;24878 24885 Generation of ... embryos +T665 PR:000005402 24855 24861 Chaf1a +T666 NCBITaxon:10088 24869 24873 mice +T667 UBERON:0000922 24878 24885 embryos +T668 SO:0001026 24916 24923 genomic +T669 SO:0000357 24952 24960 flanking +T670 PR:000005402 24961 24967 Chaf1a +T671 SO:0000147 24968 24972 exon +T672 CHEBI:7507 25044 25052 neomycin +T673 http://purl.obolibrary.org/obo/MONDO_0005504 25061 25071 diphtheria +T674 CHEBI:27026 25072 25077 toxin +T675 SO:0005853 25079 25088 cassettes +T676 GO:0009294 25135 25146 transfected +T677 CL:0002322 25150 25158 ES cells +T678 CL:0002322 25205 25213 ES cells +T679 PR:000005402 25234 25240 Chaf1a +T680 PR:000005402 25260 25266 Chaf1a +T681 SO:0001023 25287 25293 allele +T682 PR:000005402 25335 25341 Chaf1a +T683 NCBITaxon:10088 25345 25349 mice +T684 CL:0002322 25374 25382 ES cells +T685 UBERON:0000358 25397 25408 blastocysts +T686 UBERON:0000922 25413 25420 embryos +T687 PR:000005402 25453 25459 Chaf1a +T688 NCBITaxon:10088 25463 25467 mice +T689 UBERON:0000922 25512 25519 Embryos +T690 CHEBI:60004 25553 25556 mix +T691 SO:0000112 25584 25591 primers +T692 SO:0001023 25654 25661 alleles +T693 SO:0000112 25805 25812 primers +T694 CHEBI:2511 25879 25886 agarose +T695 SO:0000028 25942 25944 bp +T696 SO:0000028 25967 25969 bp +T697 SO:0001023 25971 25978 alleles +T698 PR:000005402 25981 25990 P150CAF-1 +T699 GO:0016246 26004 26008 RNAi +T700 GO:0016246 26016 26020 RNAi +T701 SO:0000755 26021 26035 plasmid vector +T702 NCBITaxon:10088 26076 26081 mouse +T703 SO:0000167 26085 26093 promoter +T704 CHEBI:17939 26100 26109 puromycin +T705 SO:0000704 26120 26124 gene +T706 SO:0000440 26210 26216 vector +T707 SO:0000704 26236 26240 gene +T708 SO:0000704 26272 26277 genes +T709 CL:0002322 26281 26289 ES cells +T710 PR:000005402 26438 26447 p150CAF-1 +T711 SO:0000646 26448 26453 siRNA +T712 SO:0000646 26455 26476 short interfering RNA +T713 SO:0000155 26511 26518 plasmid +T714 SO:0000440 26532 26538 vector +T715 GO:0010467 26539 26548 expressed +T716 SO:0000646 26551 26556 siRNA +T717 GO:0006401 26574 26589 RNA degradation +T718 CL:0002322 26591 26599 ES cells +T719 GO:0009294 26605 26616 transfected +T720 SO:0000755 26649 26663 plasmid vector +T721 CHEBI:5291 26677 26684 gelatin +T722 CHEBI:17939 26751 26760 Puromycin +T723 PR:000005402 26868 26877 p150CAF-1 +T724 GO:0016246 26878 26882 RNAi +T725 SO:0000440 26883 26889 vector +T726 GO:0033186 26939 26944 CAF-1 +T727 PR:000005402 27035 27044 p150CAF-1 +T728 GO:0010467 27045 27055 expression +T729 SO:0000704 27122 27126 gene +T730 GO:0009294 27167 27178 transfected +T731 GO:0016246 27199 27203 RNAi +T732 SO:0000755 27204 27218 plasmid vector +T733 CHEBI:17939 27424 27433 puromycin +T734 CL:0002322 27454 27462 ES cells +T735 SO:0000704 27504 27508 gene +T736 CL:0002322 27525 27533 ES cells +T737 GO:0016246 27551 27555 RNAi +T738 CL:0002322 27664 27672 ES cells +T739 UBERON:0000922 27747 27754 embryos +T740 UBERON:0000922 27927 27934 embryos +T741 CHEBI:37527 27974 27978 acid +T742 UBERON:0000086 27993 28007 zona pellucida +T743 CL:0002322 28093 28101 ES cells +T744 GO:0042571 28103 28113 Antibodies +T745 PR:000005086 28119 28123 HP1α +T746 CHEBI:472552 28208 28212 BrdU +T747 PR:000012421 28255 28259 PCNA +T748 PR:000026474 28287 28290 PML +T749 GO:0042571 28387 28397 Antibodies +T750 NCBITaxon:10088 28403 28408 mouse +T751 PR:000005402 28409 28417 p150CAF1 +T752 SO:0001707 28432 28439 H3K9me3 +T753 GO:0042571 28522 28532 antibodies +T754 GO:0019835 28646 28651 lysed +T755 CHEBI:8984 28691 28694 SDS +T756 GO:0042571 28722 28732 antibodies +T757 PR:000005402 28741 28750 p150CAF-1 +T758 PR:000005086 28775 28779 HP1α +T759 CHEBI:15358 28805 28812 Histone +T760 PR:000027594 28805 28815 Histone H3 +T761 SO:0001707 28832 28839 H3K9me3 +T762 PR:000003676 28861 28868 β-actin +T763 CHEBI:15956 28941 28947 Biotin +T764 CHEBI:42098 28959 28970 Digoxigenin +T765 GO:0006412 29045 29056 translation +T766 GO:0005634 29298 29304 Nuclei +T767 GO:0000785 29366 29375 chromatin +T768 CL:0002322 29378 29386 ES cells +T769 CHEBI:9754 29439 29443 Tris +T770 CHEBI:17883 29444 29447 HCl +T771 CHEBI:17992 29464 29471 sucrose +T772 CHEBI:32588 29479 29482 KCl +T773 CHEBI:26710 29490 29494 NaCl +T774 CHEBI:6636 29501 29506 MgCl2 +T775 GO:0005634 29584 29590 Nuclei +T776 CHEBI:17992 29659 29666 sucrose +T777 CHEBI:17992 29697 29704 sucrose +T778 CHEBI:9754 29748 29752 Tris +T779 CHEBI:17883 29753 29756 HCl +T780 CHEBI:26710 29773 29777 NaCl +T781 CHEBI:17992 29786 29793 sucrose +T782 CHEBI:6636 29800 29805 MgCl2 +T783 CHEBI:3312 29812 29817 CaCl2 +T784 GO:0005634 29832 29838 nuclei +T785 CHEBI:8984 30037 30040 SDS +T786 CHEBI:15882 30123 30129 phenol +T787 CHEBI:35255 30130 30140 chloroform +T788 CHEBI:17824 30156 30167 isopropanol +T789 CHEBI:15358 30210 30217 histone +T790 PR:000027594 30210 30220 histone H3 +T791 GO:0000785 30226 30235 chromatin +T792 PR:000005402 30251 30255 p150 +T793 GO:0005634 30281 30287 nuclei +T794 CHEBI:46756 30340 30345 Hepes +T795 CHEBI:17754 30359 30367 Glycerol +T796 CHEBI:6636 30374 30379 MgCl2 +T797 CHEBI:18320 30401 30404 DTT +T798 CHEBI:8102 30413 30417 PMSF +T799 CHEBI:26710 30480 30484 NaCl +T800 CHEBI:15358 30581 30588 histone +T801 PR:000027594 30581 30591 histone H3 +T802 GO:0042571 30592 30600 antibody +T803 GO:0000785 30637 30646 chromatin +T804 GO:0000786 30671 30682 nucleosomes +T805 GO:0000785 30720 30729 chromatin +T806 GO:0042571 30780 30790 antibodies +T807 CHEBI:2511 30903 30910 agarose +T808 GO:0097617 30947 30960 Hybridization +T809 GO:0042571 31062 31072 Antibodies +T810 GO:0042571 31122 31132 Antibodies +T811 SO:0001707 31137 31144 H3K9me3 +T812 SO:0000028 31271 31273 bp +T813 PR:P23940 31280 31285 BamHI +T814 SO:0000028 31347 31349 bp +T815 PR:P43870 31356 31363 HindIII +T816 SO:0000028 31425 31427 bp +T817 PR:000005402 31614 31623 p150CAF-1 +T818 GO:0016246 31637 31641 RNAi +T819 CL:0002322 31697 31705 ES cells +T820 GO:0009294 31706 31717 transfected +T821 PR:000005402 31741 31750 p150CAF-1 +T822 PR:000005402 31752 31756 p150 +T823 GO:0016246 31758 31762 RNAi +T824 SO:0000755 31763 31778 plasmid vectors +T825 PR:000003676 31780 31787 β-actin +T826 PR:000005402 31867 31876 p150CAF-1 +T827 PR:000005086 31917 31921 HP1α +T828 CHEBI:15358 31923 31930 histone +T829 PR:000027594 31923 31933 histone H3 +T830 SO:0001707 31938 31945 H3K9me3 +T831 CL:0002322 31972 31980 ES cells +T832 GO:0009294 31981 31992 transfected +T833 PR:000005402 32016 32025 p150CAF-1 +T834 PR:000005402 32027 32031 p150 +T835 SO:0000646 32033 32038 siRNA +T836 SO:0000440 32039 32045 vector +T837 PR:000005402 32070 32079 p150CAF-1 +T838 GO:0042571 32080 32088 antibody +T839 CHEBI:51231 32101 32105 DAPI +T840 GO:0005634 32190 32197 nucleus +T841 PR:000005402 32241 32250 p150CAF-1 +T842 GO:0016246 32264 32268 RNAi +T843 GO:0033186 32316 32321 CAF-1 +T844 GO:0016246 32405 32409 RNAi +T845 SO:0000755 32410 32424 plasmid vector +T846 GO:0009294 32471 32482 transfected +T847 CL:0002322 32483 32490 ES cell +T848 SO:0000704 32560 32564 gene +T849 GO:0033186 32658 32663 CAF-1 +T850 UBERON:0000922 32673 32682 Embryonic +T851 CL:0000057 32683 32694 Fibroblasts +T852 GO:0006260 32706 32721 DNA Replication +T853 GO:0000792 32748 32763 Heterochromatin +T854 GO:0005634 32764 32771 Nuclear +T855 PR:000005402 32805 32814 p150CAF-1 +T856 CHEBI:472552 32827 32831 BrdU +T857 GO:0009294 32846 32857 transfected +T858 PR:000005402 32881 32890 p150CAF-1 +T859 GO:0016246 32891 32895 RNAi +T860 SO:0000440 32896 32903 vectors +T861 CHEBI:472552 32942 32946 BrdU +T862 PR:000005402 32986 32990 p150 +T863 CHEBI:472552 32993 32997 BrdU +T864 PR:000005402 33028 33037 p150CAF-1 +T865 CHEBI:472552 33042 33046 BrdU +T866 CHEBI:51231 33061 33065 DAPI +T867 GO:0005737 33113 33124 cytoplasmic +T868 GO:0033186 33125 33130 CAF-1 +T869 NCBITaxon:33208 33399 33405 animal +T870 PR:000026474 33460 33463 PML +T871 GO:0042571 33464 33472 antibody +T872 UBERON:0000358 33535 33545 blastocyst +T873 CHEBI:472552 33677 33681 BrdU +T874 CHEBI:472552 33684 33701 bromodeoxyuridine +T875 GO:0033186 33703 33708 CAF-1 +T876 GO:0031497 33711 33729 chromatin assembly +T877 GO:0033186 33711 33738 chromatin assembly factor 1 +T878 GO:0000785 33747 33756 chromatin +T879 UBERON:0000922 33783 33792 embryonic +T880 UBERON:0000922 33805 33814 embryonic +T881 GO:0097617 33849 33862 hybridization +T882 NCBITaxon:10088 33928 33933 mouse +T883 UBERON:0000922 33934 33943 embryonic +T884 CL:0000057 33944 33954 fibroblast +T885 PR:000012421 33956 33960 PCNA +T886 GO:0008283 33963 33976 proliferating +T887 PR:000012421 33963 33997 proliferating cell nuclear antigen +T888 CHEBI:59132 33990 33997 antigen +T889 PR:000026474 33999 34002 PML +T890 CL:0000836 34005 34018 promyelocytic +T891 PR:000026474 34005 34027 promyelocytic leukemia +T892 GO:0016246 34029 34033 RNAi +T893 GO:0016246 34036 34052 RNA interference +T894 SO:0000646 34054 34059 siRNA +T895 SO:0000646 34062 34083 short interfering RNA +T896 PR:000005402 34123 34132 p150CAF-1 +T897 GO:0000792 34196 34211 Heterochromatin +T898 PR:000005402 34244 34250 Chaf1a +T899 NCBITaxon:10088 34254 34258 mice +T900 CL:0002322 34290 34298 ES cells +T901 PR:000005402 34320 34328 p150CAF1 +T902 SO:0000417 34364 34371 domains +T903 PR:000005403 34389 34397 p60CAF-1 +T904 CHEBI:37527 34407 34413 acidic +T905 SO:0000417 34414 34420 domain +T906 NCBITaxon:39107 34453 34459 murine +T907 PR:000005402 34460 34466 Chaf1a +T908 SO:0000704 34467 34471 gene +T909 SO:0000147 34500 34505 exons +T910 SO:0000188 34510 34517 introns +T911 GO:0006413 34554 34576 translation initiation +T912 SO:0000318 34554 34581 translation initiation site +T913 PR:000005402 34602 34608 Chaf1a +T914 SO:0001644 34609 34625 targeting vector +T915 SO:0001026 34642 34649 genomic +T916 http://purl.obolibrary.org/obo/MONDO_0005504 34692 34702 diphtheria +T917 CHEBI:27026 34703 34708 toxin +T918 CHEBI:7507 34719 34727 neomycin +T919 SO:0000704 34738 34743 genes +T920 CL:0002322 34762 34770 ES cells +T921 NCBITaxon:10088 34775 34779 mice +T922 GO:0009790 34889 34902;34916 34923 production of ... embryos +T923 PR:000005402 34906 34912 Chaf1a +T924 UBERON:0000922 34916 34923 embryos +T925 SO:0000112 34989 34996 primers +T926 PR:000005402 35115 35124 p150CAF-1 +T927 PR:000005086 35137 35141 HP1α +T928 UBERON:0000922 35154 35161 embryos +T929 PR:000005402 35175 35181 Chaf1a +T930 PR:000005402 35207 35213 Chaf1a +T931 PR:000005402 35221 35227 Chaf1a +T932 UBERON:0000922 35231 35238 embryos +T933 PR:000005402 35281 35290 p150CAF-1 +T934 PR:000005402 35331 35340 p150CAF-1 +T935 GO:0010467 35341 35351 expression +T936 GO:0051320 35377 35384 S phase +T937 UBERON:0000358 35437 35447 blastocyst +T938 UBERON:0000922 35495 35501 embryo +T939 PR:000005402 35526 35535 p150CAF-1 +T940 PR:000005086 35655 35659 HP1α +T941 CHEBI:51231 35677 35681 DAPI +T942 PR:P05205 35743 35746 HP1 +T943 CHEBI:51231 35752 35756 DAPI +T944 GO:0005634 35827 35834 nucleus +T945 UBERON:0000922 35859 35865 embryo +T946 GO:0000792 35918 35933 heterochromatin +T947 CHEBI:51231 35951 35955 DAPI +T948 PR:000005086 35960 35964 HP1α +T949 UBERON:0000922 35987 35994 embryos +T950 PR:000005402 36026 36032 Chaf1a +T951 UBERON:0000922 36036 36043 embryos +T952 CHEBI:51231 36045 36049 DAPI +T953 PR:000005086 36054 36058 HP1α +T954 GO:0005634 36091 36098 nucleus +T955 PR:000005402 36102 36111 p150CAF-1 +T956 UBERON:0000922 36121 36128 embryos +T957 GO:0034399 36153 36170 nuclear periphery +T958 GO:0000792 36191 36206 heterochromatin +T959 UBERON:0000922 36264 36271 embryos +T960 UBERON:0000922 36284 36293 embryonic +T961 GO:0000792 36400 36415 Heterochromatin +T962 CHEBI:51231 36433 36437 DAPI +T963 GO:0005634 36531 36538 nucleus +T964 CHEBI:51231 36540 36544 DAPI +T965 GO:0005634 36615 36625;36632 36636 nucleus of ... cell +T966 CL:0002322 36629 36636 ES cell +T967 PR:000005402 36690 36699 p150CAF-1 +T968 CL:0002322 36703 36711 ES Cells +T969 GO:0000792 36746 36761 Heterochromatin +T970 PR:000005402 36805 36814 p150CAF-1 +T971 GO:0016246 36818 36822 RNAi +T972 CL:0002322 36826 36834 ES cells +T973 SO:0000646 36840 36845 siRNA +T974 GO:0010467 36846 36856 expression +T975 SO:0000440 36857 36863 vector +T976 CHEBI:17939 36875 36884 puromycin +T977 SO:0005853 36895 36903 cassette +T978 NCBITaxon:10088 36916 36921 mouse +T979 SO:0000167 36925 36933 promoter +T980 SO:0000646 36943 36948 siRNA +T981 CL:0002322 36968 36976 ES cells +T982 CHEBI:17939 36993 37002 puromycin +T983 GO:0009294 37035 37047 transfection +T984 GO:0000792 37063 37078 heterochromatin +T985 PR:000005402 37095 37104 p150CAF-1 +T986 CL:0002322 37114 37122 ES cells +T987 PR:000005402 37143 37152 p150CAF-1 +T988 PR:000005086 37165 37169 HP1α +T989 CL:0002322 37179 37187 ES cells +T990 GO:0009294 37188 37199 transfected +T991 PR:000005402 37224 37233 p150CAF-1 +T992 SO:0000646 37234 37239 siRNA +T993 SO:0000646 37241 37246 siRNA +T994 GO:0010467 37247 37257 expression +T995 PR:000005402 37279 37288 p150CAF-1 +T996 PR:000005086 37345 37349 HP1α +T997 CHEBI:51231 37367 37371 DAPI +T998 GO:0000792 37417 37432 Heterochromatin +T999 PR:000005402 37464 37473 p150CAF-1 +T1000 PR:000005402 37508 37517 p150CAF-1 +T1001 PR:000005086 37530 37534 HP1α +T1002 GO:0009294 37549 37560 transfected +T1003 PR:000005402 37584 37593 p150CAF-1 +T1004 SO:0000646 37594 37599 siRNA +T1005 PR:000005086 37646 37650 HP1α +T1006 CHEBI:51231 37668 37672 DAPI +T1007 PR:000026474 37699 37702 PML +T1008 GO:0016605 37699 37709 PML bodies +T1009 PR:000005402 37729 37738 p150CAF-1 +T1010 CL:0002322 37748 37756 ES cells +T1011 PR:000026474 37777 37780 PML +T1012 CL:0002322 37790 37798 ES cells +T1013 GO:0009294 37799 37810 transfected +T1014 PR:000005402 37834 37843 p150CAF-1 +T1015 SO:0000646 37844 37849 siRNA +T1016 PR:000026474 37896 37899 PML +T1017 CHEBI:51231 37917 37921 DAPI +T1018 PR:000005402 37954 37963 p150CAF-1 +T1019 GO:0000792 37986 38001 Heterochromatin +T1020 GO:0006260 38042 38057 DNA Replication +T1021 CL:0002322 38061 38069 ES Cells +T1022 CL:0002322 38075 38083 ES cells +T1023 GO:0009294 38089 38100 transfected +T1024 PR:000005402 38106 38115 p150CAF-1 +T1025 SO:0000646 38127 38132 siRNA +T1026 SO:0000440 38133 38140 vectors +T1027 CHEBI:17939 38158 38167 puromycin +T1028 CHEBI:472552 38220 38224 BrdU +T1029 CHEBI:472552 38286 38290 BrdU +T1030 PR:000005402 38343 38352 p150CAF-1 +T1031 PR:000005402 38418 38427 p150CAF-1 +T1032 CHEBI:472552 38432 38436 BrdU +T1033 PR:000012421 38494 38498 PCNA +T1034 GO:0006260 38560 38571 replication +T1035 PR:000005402 38597 38606 p150CAF-1 +T1036 CL:0002322 38616 38624 ES cells +T1037 PR:000005402 38645 38654 p150CAF-1 +T1038 PR:000005402 38692 38701 p150CAF-1 +T1039 PR:000012421 38706 38710 PCNA +T1040 GO:0007049 38776 38786 cell-cycle +T1041 PR:000005402 38811 38820 p150CAF-1 +T1042 CL:0002322 38830 38838 ES cells +T1043 GO:0022403 38897 38905;38910 38920 phase of ... cell cycle +T1044 GO:0051318 38922 38924 G1 +T1045 GO:0051320 38926 38927 S +T1046 GO:0044839 38929 38933 G2/M +T1047 CHEBI:472552 38950 38954 BrdU +T1048 GO:0009294 39133 39145 transfection +T1049 SO:0000646 39153 39158 siRNA +T1050 SO:0000440 39159 39166 vectors +T1051 PR:000005402 39192 39201 p150CAF-1 +T1052 GO:0005721 39275 39302 Pericentric Heterochromatin +T1053 GO:0051325 39394 39404 interphase +T1054 GO:0005634 39405 39414;39424 39429 nuclei of ... cells +T1055 NCBITaxon:10088 39415 39420 mouse +T1056 CL:0002322 39421 39429 ES cells +T1057 CL:0002322 39544 39552 ES cells +T1058 GO:0010467 39553 39563 expressing +T1059 SO:0000646 39579 39584 siRNA +T1060 GO:0010369 39666 39679 chromocenters +T1061 CHEBI:51231 39705 39709 DAPI +T1062 GO:0010467 39984 39994 expressing +T1063 PR:000005402 39995 40004 p150CAF-1 +T1064 SO:0000646 40005 40010 siRNA +T1065 GO:0010369 40044 40057 chromocenters +T1066 GO:0034399 40179 40196 nuclear periphery +T1067 CL:0002322 40230 40238 ES cells +T1068 GO:0005634 40307 40314 nucleus +T1069 GO:0010369 40407 40420 chromocenters +T1070 CL:0002322 40476 40484 ES cells +T1071 GO:0010467 40485 40495 expressing +T1072 PR:000005402 40496 40505 p150CAF-1 +T1073 SO:0000646 40506 40511 siRNA +T1074 PR:000005402 40513 40522 p150CAF-1 +T1075 CHEBI:51231 40626 40630 DAPI +T1076 GO:0097617 40658 40671 hybridization +T1077 GO:0010369 40799 40811 chromocenter +T1078 GO:0010369 40849 40861 chromocenter +T1079 PR:000005402 40865 40874 p150CAF-1 +T1080 GO:0000786 40906 40917 Nucleosomal +T1081 PR:000005402 40949 40958 p150CAF-1 +T1082 CL:0002322 40968 40976 ES Cells +T1083 GO:0005634 40978 40984 Nuclei +T1084 CL:0002322 41004 41012 ES cells +T1085 GO:0009294 41013 41024 transfected +T1086 PR:000005402 41041 41050 p150CAF-1 +T1087 SO:0000646 41051 41056 siRNA +T1088 SO:0000440 41057 41063 vector +T1089 GO:0005634 41065 41071 Nuclei +T1090 CHEBI:2511 41220 41227 agarose +T1091 CHEBI:4883 41255 41271 ethidium bromide +T1092 SO:0001026 41287 41294 genomic +T1093 CHEBI:25614 41332 41337 nylon +T1094 GO:0097617 41363 41373 hybridized +T1095 CHEBI:37972 41385 41388 32P +T1096 GO:0010424 41448 41467 DNA CpG Methylation +T1097 GO:0005721 41471 41498 Pericentric Heterochromatin +T1098 PR:000005402 41517 41526 p150CAF-1 +T1099 CL:0002322 41536 41544 ES Cells +T1100 GO:0009294 41580 41591 transfected +T1101 PR:000005402 41614 41623 p150CAF-1 +T1102 SO:0000646 41624 41629 siRNA +T1103 SO:0000440 41630 41636 vector +T1104 CHEBI:2511 41757 41764 agarose +T1105 GO:0005721 41893 41920 Pericentric Heterochromatin +T1106 PR:000005402 41924 41933 p150CAF-1 +T1107 PR:000005402 41954 41963 p150CAF-1 +T1108 SO:0001707 42004 42011 H3K9me3 +T1109 GO:0005721 42015 42042 pericentric heterochromatin +T1110 PR:000043452 42058 42065 histone +T1111 CHEBI:15358 42058 42065 histone +T1112 PR:000005402 42146 42155 p150CAF-1 +T1113 PR:000005402 42157 42161 p150 +T1114 SO:0000646 42163 42168 siRNA +T1115 GO:0010467 42169 42179 expressing +T1116 CL:0002322 42180 42188 ES cells +T1117 GO:0042571 42226 42234 antibody +T1118 CHEBI:2511 42267 42274 agarose +T1119 GO:0097617 42363 42376 Hybridization +T1120 GO:0097617 42481 42491 hybridized +T1121 GO:0097617 42581 42591 hybridized +T1122 PR:000005402 42687 42696 p150CAF-1 +T1123 CL:0002322 42706 42714 ES cells +T1124 SO:0001707 42868 42875 H3K9me3 +T1125 PR:000005402 42935 42944 p150CAF-1 +T1126 CL:0002322 42954 42962 ES cells +T1127 SO:0001707 42983 42990 H3K9me3 +T1128 PR:000005086 43028 43032 HP1α +T1129 PR:000005402 43054 43063 p150CAF-1 +T1130 SO:0000646 43064 43069 siRNA +T1131 GO:0010467 43070 43080 expressing +T1132 CL:0002322 43081 43089 ES cells +T1133 PR:000005086 43102 43106 HP1α +T1134 SO:0001707 43112 43119 H3K9me3 +T1135 http://purl.obolibrary.org/obo/MONDO_0004992 43859 43865 Cancer +T1136 CHEBI:33250 44001 44009 Atomique +T1137 http://purl.obolibrary.org/obo/MONDO_0004992 44069 44075 Cancer diff --git a/src/ontogpt/evaluation/craft/database/all/17083276.txt b/src/ontogpt/evaluation/craft/database/all/17083276.txt new file mode 100644 index 000000000..c815756af --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17083276.txt @@ -0,0 +1,223 @@ +CAF-1 Is Essential for Heterochromatin Organization in Pluripotent Embryonic Cells + +Abstract + +During mammalian development, chromatin dynamics and epigenetic marking are important for genome reprogramming. Recent data suggest an important role for the chromatin assembly machinery in this process. To analyze the role of chromatin assembly factor 1 (CAF-1) during pre-implantation development, we generated a mouse line carrying a targeted mutation in the gene encoding its large subunit, p150CAF-1. Loss of p150CAF-1 in homozygous mutants leads to developmental arrest at the 16-cell stage. Absence of p150CAF-1 in these embryos results in severe alterations in the nuclear organization of constitutive heterochromatin. We provide evidence that in wild-type embryos, heterochromatin domains are extensively reorganized between the two-cell and blastocyst stages. In p150CAF-1 mutant 16-cell stage embryos, the altered organization of heterochromatin displays similarities to the structure of heterochromatin in two- to four-cell stage wild-type embryos, suggesting that CAF-1 is required for the maturation of heterochromatin during preimplantation development. In embryonic stem cells, depletion of p150CAF-1 using RNA interference results in the mislocalization, loss of clustering, and decondensation of pericentric heterochromatin domains. Furthermore, loss of CAF-1 in these cells results in the alteration of epigenetic histone methylation marks at the level of pericentric heterochromatin. These alterations of heterochromatin are not found in p150CAF-1-depleted mouse embryonic fibroblasts, which are cells that are already lineage committed, suggesting that CAF-1 is specifically required for heterochromatin organization in pluripotent embryonic cells. Our findings underline the role of the chromatin assembly machinery in controlling the spatial organization and epigenetic marking of the genome in early embryos and embryonic stem cells. + +Synopsis + +Chromatin is the support of our genetic information. It is composed of numerous repeated units called nucleosomes, in which DNA wraps around a core of histone proteins. Modifications in the composition and biochemical properties of nucleosomes play major roles in the regulation of genome function. Such modifications are termed “epigenetic” when they are inherited across cell divisions and confer new information to chromatin, in addition to the genetic information provided by DNA. It is usually believed that during genome replication, the basic chromatin assembly machinery builds up “naïve” nucleosomes, and, in a subsequent step, nucleosomes are selectively modified by a series of enzymes to acquire epigenetic information. Here, the authors studied the role of a basic chromatin assembly factor (CAF-1) in mouse embryonic stem cells and early embryos. Surprisingly, they show that CAF-1 confers epigenetic information to specific genomic regions. In addition, this study revealed that CAF-1 is required for the proper spatial organization of chromosomes in the nucleus. This new knowledge may contribute to better understanding the role of chromatin in the maintenance of embryonic stem cell identity and plasticity. + +Introduction + +During mouse pre-implantation development, the genome undergoes a series of major epigenetic changes required for embryonic gene expression, the maintenance of totipotency, and the first differentiation events [1,2]. While many studies have established the importance of DNA methylation in epigenetic reprogramming, recent data point to a crucial role of chromatin in this process [3–5]. Yet we know very little about the role of histone modifying enzymes, chromatin remodeling factors, and histone chaperones during pre-implantation development, or in stem cells derived from early embryos [6]. A subset of identified histone chaperones and chromatin remodeling complexes can collaborate to promote nucleosome assembly in vitro and are therefore in a strategic position to control chromatin assembly and maturation during development [7,8]. Among histone chaperones, chromatin assembly factor 1 (CAF-1) is a three-subunit (p150, p60, and p48) complex which promotes histone H3 and H4 deposition onto newly synthesized DNA during replication or DNA repair [9,10]. Specifically, CAF-1 deposits the histone H3 variant H3.1 into chromatin, in a pathway coupled to DNA synthesis, whereas a second histone chaperone, HIRA, is involved in the deposition of the H3.3 variant in a DNA synthesis independent pathway [11,12]. In addition to this specific chromatin assembly activity, CAF-1 interacts with several proteins present in heterochromatin, including heterochromatin protein 1 (HP1) and MBD1, a methyl-CpG binding domain protein that recruits histone deacetylase and repressive histone methyltransferase activities [13–15]. Furthermore, p150CAF-1 is required to ensure a replication-specific pool of HP1 molecules at replication sites in pericentric heterochromatin during mid-late S phase [16]. Taken together, these data suggest roles for CAF-1 in the formation of heterochromatin and in the heritability of epigenetic traits. While the function of this evolutionary conserved histone chaperone has been studied extensively biochemically, we still lack information concerning its importance during early development in mammals and in pluripotent cells such as embryonic stem (ES) cells. Here, we have analyzed the importance of CAF-1 during early mouse development by genetic ablation and in ES cells by depletion using RNA interference (RNAi). We show that CAF-1 is essential for viability in early mouse embryos and ES cells. We provide evidence that CAF-1 is required for the spatial organization and epigenetic marking of heterochromatin domains in pluripotent embryonic cells. + +Results + +We used gene targeting in ES cells to delete exon 3 in the Chaf1a gene, which encodes p150CAF-1 (Figure 1A). Chaf1a+/− mice were born at a Mendelian frequency, were of normal size and weight, displayed no obvious abnormalities, and were fertile. Crossing heterozygous mice failed to generate viable newborn Chaf1a−/− mice. Furthermore, no homozygous Chaf1a−/− embryos were detected at any of the post-implantation stages. However, we could detect Chaf1a−/− embryos at embryonic day 4 (E4) using a PCR strategy (Figure 1B). They represent only 10% of E4 embryos obtained from Chaf1a+/− mice intercrosses, suggesting that more than half of the homozygous mutant embryos had degenerated before this stage. Moreover, mutant embryos contained only eight to 16 cells at the E4 stage, instead of 32 cells observed for wild-type or heterozygous blastocysts (Figure 1C and 1D). Further analysis by light microscopy using immunofluorescence (IF) allowed us to compare wild-type and p150CAF-1-depleted embryos. A loss of p150CAF-1 staining could not be detected before the eight-cell stage (unpublished data) suggesting that maternally contributed protein is present in the embryo up to the four-cell stage. Thus, depletion of p150CAF-1 protein from this stage allows a maximum of two additional cell divisions before developmental arrest. + +Strikingly, we found that the nuclear organization of heterochromatin appeared abnormal in the nuclei of Chaf1a−/− embryos. Pericentric heterochromatin, the major component of constitutive heterochromatin, can easily be visualized in interphase nuclei by the fluorochrome DAPI and by immunostaining with HP1α [16,17–19]. In wild-type blastocysts and in mouse somatic cells, pericentric heterochromatin domains cluster together and form higher-order chromatin structures called chromocenters [20], which are visualized as DAPI-dense foci (Figure 1C). These structures were not detected in Chaf1a−/− embryos (Figure 1D). Since chromatin architecture and higher-order structures are important for genome function [21], we decided to characterize this phenotype in more detail. For this purpose, we examined the status of heterochromatin organization between the two-cell and blastocyst stages. In two-cell stage wild-type embryos, DAPI staining is diffuse and fibrillar, with regions of higher density around the nucleolar precursor bodies (Figure 1E). Heterochromatin domains are progressively assembled into DAPI-dense foci between the four-cell and 32-cell blastocyst stages (Figure 1F). Localization of HP1α confirmed that the structures visualized by DAPI staining correspond to constitutive heterochromatin (Figure 1E). Thus, the nuclear organization of heterochromatin is dramatically modified during pre-implantation development, between the two-cell and blastocyst stages. In Chaf1a−/− E4 embryos, which are arrested between the eight- and 16-cell stage, DAPI-dense foci were barely detectable (Figure 1D). Instead, DAPI staining was diffuse within the nucleus, with regions of higher density around the nucleoli and at the periphery of the nuclei (Figure 1D). Localization of HP1α showed a diffuse pattern similar to the DAPI, with some enrichment at the nuclear periphery, and around the nucleoli (Figure 1D). This abnormal organization of heterochromatin in Chaf1a−/− embryos is reminiscent of the heterochromatin organization found in two- to four-cell stage wild-type embryos (compare Figure 1D and 1F). These data evidence a key role for p150CAF-1 during pre-implantation development, and reveal that this protein is required for the proper 3-D organization of heterochromatin within embryonic cell nuclei. + +Next, we wondered whether a similar requirement for CAF-1 could also be observed in ES cells, which are derived from the blastocyst inner cell mass. Given the early developmental arrest observed in Chaf1a−/− embryos, such cells could not be derived directly from null embryos, and we thus used an RNAi strategy (Figure 2A). p150CAF-1 knockdown in ES cells was quantified by Western blot analysis and IF. (Figure S1). p150CAF-1 RNAi-depleted ES cells displayed a phenotype very similar to the cells of Chaf1a−/− E4 mice. DAPI-dense foci were lost, and we observed diffuse HP1α and DAPI staining around the nucleoli and at the periphery of the nucleus (Figure 2B). This result indicates that p150CAF-1 is essential for nuclear organization of heterochromatin in ES cells in a similar way to early pre-implantation embryos. Surprisingly, we did not observe this severe alteration in heterochromatin organization in primary mouse embryonic fibroblasts (MEFs) following p150CAF-1 depletion by RNAi. p150CAF-1 depletion in MEFs resulted in a strong inhibition of cell proliferation (unpublished data), but did not alter the clustering of heterochromatin domains (Figures 2C and S2). Similarly, p150CAF-1 depletion in 3T3 cells did not affect heterochromatin organization [16]. These observations reveal a specific function for CAF-1 in the early embryo and ES cells. + +We also wondered whether loss of p150CAF-1 in ES cells specifically affects the heterochromatin nuclear subcompartment, or whether it might result in a more global loss of nuclear organization and architecture. We used a specific antibody against the promyelocytic leukemia (PML) protein to study the fate of the PML nuclear bodies, which are well-characterized subnuclear structures [22]. We could not detect any significant difference in the distribution and aspect of PML nuclear bodies between control and p150CAF-1-depleted ES cells (Figure 2D). These data show that nuclear architecture is not globally altered following CAF-1 loss-of-function. In addition, Western blot analysis revealed that CAF-1 depletion did not result in altered levels of chromatin architectural proteins such as histone H3 and HP1α (Figure S1). Thus, CAF-1 is specifically required in early embryos and in ES cells for the proper organization of the heterochromatin subnuclear compartment. + +Previous studies performed in mammalian cell lines showed that CAF-1 activity is required for S phase progression [16,23,24]. We show here that 3 d after transfection of the RNAi vector, p150CAF-1-depleted cells (identified by a complete absence of CAF-1 IF signal) appear still active for replication, as revealed by incorporation of the thymidine analog bromodeoxyuridine (BrdU) (Figure 3A), the PCNA (proliferating cell nuclear antigen) pattern (Figure 3B), and flow cytometry analysis (Figure 3C). Hence, in these cells, DNA replication persists despite the drastic changes in heterochromatin organization. However, 24 h later, ES cells cease to proliferate and die, revealing that p150CAF-1 is required for an essential cellular process, as in embryos. Altogether, our data demonstrate that loss of p150CAF-1 function in ES cells and early embryos alters first the organization of heterochromatin in the nucleus and, in a subsequent step, cell cycle and viability. + +In order to better characterize the defects in heterochromatin organization, we performed two-color DNA fluorescence in situ hybridization (FISH) experiments to reveal the spatial distribution of pericentric and centric chromosomal regions, which in mouse are mainly composed of large blocks of major and minor satellite repeats, respectively [20]. Pericentric domains from different chromosomes form clusters, which are revealed by FISH as large spots that coincide with DAPI-dense foci in the interphase nucleus (Figure 4A). At the periphery of each pericentric domain, centric regions form individual entities (Figure 4A). In p150CAF-1 RNAi-depleted cells, we observed a disruption of pericentric heterochromatin clusters that coincides with the disappearance of DAPI-dense foci (Figure 4B). Individual pericentric domains from single chromosomes are now found either isolated or aggregated in a pattern less dense than the regular clusters observed in control cells (Figure 4B). Such aggregates, which are often found at the nuclear periphery, are also revealed by DAPI staining as a typical signature of p150CAF-1 loss-of-function. Quantification of fluorescence along a line randomly drawn across the nucleus revealed lower fluorescence intensity and a broader distribution of DAPI and major satellite hybridization signals in p150CAF-1-depleted cells (Figure 4C and 4D), indicating decondensation of pericentric heterochromatin domains. In contrast, centric domains showed an intensity and shape similar to control cells, suggesting that they remain unaffected by p150CAF-1 depletion (Figure 4C and 4D). In conclusion, these results show that p150CAF-1 is required for the proper condensation and clustering of pericentric heterochromatin domains. + +Given the known role of CAF-1 in the deposition of histone H3.1 and H4 associated with DNA synthesis [11,12], we wondered whether this defect in higher-order chromatin organization could reflect an aberrant nucleosomal organization. Using DNase I and micrococcal nuclease (MNase) assays, we could not observe any significant difference between control and p150CAF-1-depleted ES cells at the level of bulk genome chromatin or at pericentric repeats (Figure 5). In a second series of experiments, we compared the association of histone H3 with chromatin in nuclei isolated from cells transfected with control and p150CAF-1 RNAi plasmid vectors. Nuclei were incubated in buffers with different salt concentrations ranging from 100 mM NaCl to 1 M NaCl. In all conditions, the amount of histone H3 remaining associated with chromatin was indistinguishable in control and p150CAF-1-depleted ES cells (unpublished data). Therefore, the loss of clustering and decondensation of pericentric heterochromatin are unlikely to be the consequence of severe defects in nucleosomal organization. + +Our findings showed that the nuclear localization of HP1α is severely altered in Chaf1a−/− embryos and p150CAF-1-depleted ES cells. We therefore examined the status of other epigenetic marks previously shown to characterize pericentric heterochromatin. We first tested DNA CpG methylation [25] at major satellite repeats and found no significant difference between control and p150CAF-1-depleted cells (Figure 6). As the LTM7 ES cell line that we used in our RNAi experiments is a female (XX) cell line, DNA methylation is globally reduced in these cells [26]. To rule out the possibility that hypomethylation of pericentric repeats might contribute to the destabilization of heterochromatin domains in p150CAF-1-depleted ES cells, we tested our RNAi plasmid vector in a XY ES cell line, and confirmed that loss of p150CAF-1 leads to disruption of heterochromatin organization independently of the degree of DNA methylation at pericentric repeats (unpublished data). We next analyzed specific histone modifications [27] in chromatin immunoprecipitation (ChIP) experiments using native chromatin [28]. We found that pericentric DNA was immunoprecipitated with a 2-fold lower efficiency with H4K20me3 [29,30] antibodies following p150CAF-1 depletion (Figure 7A and 7B). Immunoprecipitation of minor satellite repeats in the same experiment was less affected (Figure 7B), showing that loss of p150CAF-1 function mainly affects H4K20me3 at pericentric heterochromatin. Moreover, no significant difference in immunoprecipitation efficiency was detected at intracisternal A particle (IAP) elements (Figure 7B), which are noncentromeric repeated DNA elements also enriched in H4K20me3 [31]. This result shows that p150CAF-1 contributes to normal levels of H4K20me3 at pericentric heterochromatin, but not necessarily at other loci. The H3K9me3 mark [32] was also significantly reduced in p150CAF-1-depleted cells, though to a lesser extent than H4K20me3 (Figure 7A and 7B). + +In wild-type cells, most of H4K20me3 and H3K9me3 signals are present at the level of DAPI-dense domains when visualized by IF [29] (Figures 7C and 7D). H3K9me3 was severely perturbed in p150CAF-1-depleted cells, displaying a diffuse pattern similar to HP1α (Figure 7C). The H4K20me3 pattern was even more severely altered, showing both a loss of the typical dots revealed in control cells and a reduction in signal intensity (Figure 7D). Consistent with our ChIP experiments, residual H4K20me3 epitopes were detected at the level of relocated DAPI-dense material. Altogether, our results show that depletion of CAF-1 in ES cells leads to a simultaneous disruption of heterochromatin 3-D organization and an alteration in epigenetic marking. + +Discussion + +Here, we provide evidence that p150CAF-1 has a crucial function during mouse early embryogenesis and in ES cells. Previous studies showed that CAF-1 is essential for the progression of DNA replication in vertebrates. In Xenopus laevis, p150CAF-1 function is required for the rapid cell divisions that occur during early development [33]. Similarly, CAF-1 is essential in mammalian cell lines for the progression of DNA replication [16,23,24,34]. Our findings reveal a role for CAF-1 in the nuclear organization of heterochromatin domains during early development and in ES cells. We show that in p150CAF-1-depleted ES cells, while DNA replication initially persists without significant perturbation in nucleosomal organization, severe heterochromatin organization defects can be observed. Loss of p150CAF-1 did not affect PML bodies in ES cell nuclei, showing that nuclear architecture is not generally altered. Ultimately, p150CAF-1 depletion results in the arrest of ES cell division and cell death. Given the major defects observed in heterochromatin organization, it is tempting to speculate that these defects cause the developmental arrest in the embryos and death in ES cells. The role of CAF-1 in the nuclear organization of heterochromatin includes spatial localization, condensation, and clustering of pericentric domains. This important function of CAF-1 was not revealed in primary MEFs, 3T3 cells, or human cell lines, suggesting that it is specific of pluripotent embryonic cells. Alternatively, early embryos and ES cells, whose genome is more plastic than somatic cells, might represent a particularly sensitive context in which the importance of CAF-1 in heterochromatin organization is exacerbated. In ES cells, major architectural chromatin proteins are hyperdynamic and bind relatively loosely to chromatin [35]. In this cellular context, our findings show that proper heterochromatin nuclear architecture relies on CAF-1 function. In contrast, in undifferentiated cells that are already lineage committed (such as MEFs) genome architecture might be more stable than in ES cells and thus cannot be disrupted by CAF-1 depletion. This important function of CAF-1 in pluripotent embryonic cells could be partially conserved during evolution. Indeed, in Arabidopsis thaliana, in which CAF-1 is not essential for viability [36], loss of CAF-1 is associated with a mild alteration in chromocenter size [37]. This phenotype is somehow reminiscent of loss of CAF-1-mediated defects in heterochromatin organization in early mouse embryos. In mouse ES cells, we show that CAF-1 is also required for regular levels of H4K20me3 at pericentric repeats, but not at IAP elements, which are repeated sequences spread all over the mouse genome. These data suggest that proper nuclear organization and epigenetic marking of pericentric heterochromatin are coupled in ES cells. Alternatively, 3-D organization of heterochromatin and epigenetic marking might represent two independent functions of CAF-1. + +The variable requirement for CAF-1 across species might reflect the acquisition of new properties affecting the function of histone H3 variants during evolution of eukaryotes. CAF-1 is essential in mammals but dispensable in Saccharomyces cerevisiae [38]. Interestingly, the budding yeast genome does not contain heterochromatin regions equivalent to those of higher eukaryotes, and several hallmark features of heterochromatin such as di- and trimethylation at H3K9, H3K27, and H4K20 are not observed in the silent chromatin of S. cerevisiae. In mammals, H3K9 dimethylation is predominantly associated with H3.1, but not with H3.3, which carries modifications typically found in transcribed chromatin regions [39]. Outside of the centromeric H3 variant, S. cerevisiae possesses only H3.3, which is expressed and incorporated into chromatin in a replication-independent fashion in higher eukaryotes [12]. These data suggest a specific role for H3.1 in heterochromatin regions. We show here that CAF-1, which is a H3.1 replication-dependent loading complex [11], is specifically required for the nuclear organization of pericentric heterochromatin in embryos and ES cells. One possibility would be a scenario in which incorporation of H3.1 into chromatin is required for the proper nuclear organization of constitutive heterochromatin in an embryonic context. While this has to be addressed experimentally, it is worthwhile to mention an alternate possible mechanism that might explain the disruption of heterochromatin domains in p150CAF-1 null embryos and ES cells. CAF-1 could be required for the loading, into heterochromatin, of an interacting partner required for the clustering of pericentric domains. Following one or two rounds of DNA replication in the absence of p150CAF-1, heterochromatin would become deprived of this molecule, which would cause the disruption of its 3-D organization. A potential candidate is the HP1 protein, which interacts with CAF-1 [13,16]. + +In agreement with a recent report [40], our analysis of heterochromatin in pre-implantation embryos has revealed drastic changes in nuclear organization between the two-cell and blastocyst stages. Cloning experiments in mouse revealed that the nuclear organization of heterochromatin in ES cell nuclei was quickly reverted into the one-cell stage-specific form after nuclear transfer [40]. Hence, remodeling of heterochromatin 3-D organization parallels nuclear reprogramming toward an early embryonic status in cloned embryos. This drastic reorganization of heterochromatin organization is similar to that observed in p150CAF-1-depleted ES cells, and in developmentally arrested 16-cell stage Chaf1a−/− embryos. These data show that CAF-1 is involved in setting up proper heterochromatin architecture during the first cell divisions of embryonic life. Given the importance of nuclear organization in the control of gene expression [21,41], this finding suggests that CAF-1 contributes to the coordinated programs of gene expression during early embryogenesis. Our findings open new perspectives in the understanding of chromatin dynamics during early development and in pluripotent embryonic cells. While most studies focused on the role of DNA methylation and histone modifying enzymes [3], our data point to the importance of the chromatin assembly machinery. We show that in addition to assembling nucleosomes, CAF-1 provides spatial and epigenetic information to heterochromatin domains in early embryos and ES cells. + +Materials and Methods + +Generation of Chaf1a mutant mice and embryos. + +Using PCR, we amplified two genomic fragments (about 3 kb each) flanking Chaf1a exon 3. These DNA fragments were assembled by conventional cloning with the neomycin and DT (diphtheria toxin) cassettes, as described in Figure 1A. The construct was transfected in ES cells by electroporation. We identified recombinant ES cells carrying the mutant Chaf1atm1Ger (abbreviated Chaf1a− in the manuscript) allele by Southern blot (Figure 1A). We derived Chaf1a+/− mice by injecting recombined ES cells into C57BL/6N blastocysts. E4 embryos obtained from the intercross of Chaf1a+/− mice were genotyped by nested-PCR amplification. Embryos were collected in a PCR reaction mix containing oligonucleotide primers 1, 2, and 3 (Figure 1A) that amplify the wild-type and mutant alleles. After 30 cycles of amplification, 1 μl of each PCR reaction was used in a second round of PCR amplification with a new set of oligonucleotide primers. After 30 cycles of amplification, PCR reactions were run onto an agarose gel, which revealed the presence of the wild-type (150 bp) and recombinant (200 bp) alleles. + +P150CAF-1 depletion by RNAi. + +The RNAi plasmid vector that we used in this study contains the mouse H1 promoter and a puromycin selection gene (SB and MG, unpublished data). We characterized in detail the properties of this new vector using a GFP target gene and FACS: Extinction of target genes in ES cells is highly efficient in 70% of the cells, of intermediate efficiency in 15%, and inefficient in the remaining 15% of cells analysis. The sequence of p150CAF-1 siRNA (short interfering RNA) duplex [16] was cloned into this plasmid. The control vector expressed a siRNA that targets GFP RNA degradation. ES cells were transfected by electroporation with the RNA plasmid vector, seeded onto gelatin-coated slides, and cultured for 24 h in the absence of selection. Puromycin (2 μg/ml) was added to the culture medium and cells were cultured for an additional 48-h period. Using the p150CAF-1 RNAi vector, IF microscopy quantification reveals a complete CAF-1 depletion in most cells (Figure S1B). Western blot analysis (Figure S1A) reveals residual p150CAF-1 expression, as expected from the 15% of cells that do not inhibit the target gene. + +MEFs (passage 3, 90% confluence) were transfected during 4 h with the RNAi plasmid vector using Lipofectamine 2000 (Invitrogene, Carlsbad, California, United States) according to manufacturer's conditions. Cells were trypsinized, plated at 1/3 dilution, and cultured for 48 h in the presence of puromycin. + +Cell lines. + +AT-1 ES cells [42] (a gift of M. Vernet) were used for gene targeting. LTM7 ES cells were used in all RNAi experiments. We derived this cell line from (C57BL/6 × 129) F1 females bred with C3H/HeJ males. LTM7 are XX ES cells, competent for germ line transmission. Primary MEFs were derived from E13 embryos as described in [43]. + +Immunofluorescence. + +Cells were fixed for 20 min in PBS with 4% paraformaldehyde, and immunodetection was performed as previously described [44]. E4 embryos were collected and treated with tyrode acid to remove the zona pellucida, deposited onto microscope slides, and processed for immunostaining as described for ES cells. Antibodies anti-HP1α (2HP1H5, Euromedex, France), anti-H4K20me3 (Abcam, Cambridge, United Kingdom), anti-BrdU (DakoCytomation, Glostrup, Denmark), anti-PCNA (DakoCytomation), and anti-PML (Upstate Biotechnology, Lake Placid, New York, United States) were all used at 1/1000 dilution. Antibodies anti-mouse p150CAF1 [16] and anti-H3K9me3 (Upstate Biotechnology) were used at 1/250 and 1/500, respectively. All secondary antibodies were purchased from Molecular Probes (Sunnyvale, California, United States). + +Western blot analysis. + +Cells were lysed in Laemli buffer and run onto a 4%–12% SDS PAGE gradient gel. We used antibodies against p150CAF-1 ([16]; dilution 1/500), HP1α (2G9, Euromedex; 1/500), Histone H3 (Abcam; 1/500), H3K9me3 (Upstate; 1/500) and β-actin (Upstate; 1/40 000). + +DNA FISH. + +Probes were described previously [20]. Biotin-16-dUTP or Digoxigenin-11-dUTP (Roche, Basel, Switzerland) labeled probes were generated by nick translation (Roche) and FISH performed as described [20]. Image acquisition was performed with the Deltavision RT microscope (100×, 1.4 NA objective), images were deconvoluted, and fluorescence profiles measured along an arbitrary line using SoftWorx. + +Nuclei preparation, nuclease digestion, and biochemical analysis of chromatin. + +ES cells were incubated on ice for 10 min in buffer 1 (15 mM Tris-HCl [pH 7.5], 0.3 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA) with 0.15% IGEPAL (Sigma, St. Louis, Missouri, United States). Nuclei were purified by centrifugation (10,000 × g for 30 min, at 4 °C) on sucrose cushions (buffer 1 with 1.2 M sucrose) and resuspended in nuclease buffer (50 mM Tris-HCl [pH 7.5], 20 mM NaCl, 0.32 M sucrose, 4 mM MgCl2, 1 mM CaCl2). About 2.106 nuclei were incubated with increasing quantities of MNase (0.04–1.6 units) or DNase I (0.25–16 units). Digestion time at 37 °C was 10 min for MNase and 2 min for DNase I. Digestions were stopped by adding SDS to 1% and EDTA to 50 mM. DNAs were prepared by proteinase K digestion followed by phenol-chloroform extraction and isopropanol precipitation. To test the association of histone H3 with chromatin in control and p150-depleted cells, isolated nuclei were incubated on ice for 30 min in buffer 2 (50 mM Hepes [pH7.9], 20% Glycerol, 3 mM MgCl2, 0.1% IGEPAL, 0.5 mM DTT, 0.5 mM PMSF) supplemented with either 0.1 M, 0.3 M, 0.45 M, 0.7 M, or 1 M NaCl. After centrifugation at 15,000 g, pellets and supernants were analyzed by Western blot using a histone H3 antibody (Abcam). + +ChIP. + +We prepared native chromatin fragments of two to six nucleosomes in length as described [28]. 5 μg of chromatin were incubated overnight with 10 μl of commercial antibodies. After incubation with protein-G sepharose and three washes, immunoprecipitated DNA was purified, sized onto an agarose gel, and analyzed by Southern blot. Hybridization signals were quantified using an Instant Imager (PerkinElmer, Wellesley, California, United States). Antibodies specific for H4K20me3 were purchased from Abcam. Antibodies for H3K9me3 were purchased from both Abcam and Upstate Biotechnology and gave similar results. + +For Southern blot analysis, we used a 240-bp EcoRI/BamHI fragment from pSAT (major satellite probe) [45–47] and a 360-bp EcoRI/HindIII fragment from R198, corresponding to three copies of the 120-bp minor satellite repeat [49]. Probes used in FISH experiments were described in [46–48]. The probe for IAP elements was described in [50]. + +Supporting Information + +Figure S1 + +Analysis of p150CAF-1 Depletion by RNAi with Western Blot and IF + +(A) Western blot analysis of ES cells transfected with control (cont) or p150CAF-1 (p150) RNAi plasmid vectors. β-actin was used as a loading control. Amount of cells used in each lane is indicated. p150CAF-1 knockdown does not significantly affect HP1α, histone H3, or H3K9me3 levels. + +(B) IF analysis. ES cells transfected with control (cont) or p150CAF-1 (p150) siRNA vector were immunostained with p150CAF-1 antibody (green) and DAPI (blue). Scale bar = 5 μm. Fluorescence was quantified along a line drawn across the nucleus and data were plotted, revealing efficient p150CAF-1 depletion by RNAi. + +The apparent difference in the efficiency of CAF-1 knockdown revealed by Western blot and IF likely reflects the observation that the RNAi plasmid vector used in this study is efficient in 85% of the transfected ES cell population, whereas 15% of cells display no inhibition of the target gene (see Materials and Methods). + +(1.3 MB TIF) + +Click here for additional data file. + +Figure S2 + +CAF-1-Depleted Embryonic Fibroblasts Active for DNA Replication Do Not Display an Altered Heterochromatin Nuclear Architecture + +Immunodetection of p150CAF-1 (green) and BrdU (red) in MEFs transfected with control (cont) or p150CAF-1 RNAi vectors. MEFs were pulse-labeled for 1 h with BrdU prior to fixation and IF analysis. The p150 + BrdU image shows the merge between p150CAF-1 and BrdU fluorescence. DAPI staining is shown in the right-hand panel. The cytoplasmic CAF-1 IF pattern is non-specific. + +(343 KB TIF) + +Click here for additional data file. + +Acknowledgements + +We thank E. Heard and S. Khochbin for critical reading and suggestions; S. Jounier and H. Humbertclaude for cell culture; J. Mitja, S. Thessier, and J. C. Robillard for animal care; M. Vernet for AT-1 cells; K. Nacerddine for the PML antibody; N. Gilbert and T. Bestor for probes; G. Hamard for advice on blastocyst injection; P. Le Baccon for help on image analysis; and C. Mann, A. Sentenac, and E. Moustacchi for their support. + +Abbreviations + +BrdU - bromodeoxyuridine + +CAF-1 - chromatin assembly factor 1 + +ChIP - chromatin immunoprecipitation + +E4 - embryonic day 4 + +ES - embryonic stem + +FISH - fluorescence in situ hybridization + +IAP - intracisternal A particle + +IF - immunofluorescence + +MEF - mouse embryonic fibroblast + +PCNA - proliferating cell nuclear antigen + +PML - promyelocytic leukemia + +RNAi - RNA interference + +siRNA - short interfering RNA + +Figures and Tables + +Figure 1 + +Loss of p150CAF-1 Function Leads to Early Developmental Arrest and Alteration of Heterochromatin Organization + +(A) Generation of Chaf1a+/− mice by homologous recombination in ES cells. (Top) Scheme of the p150CAF1 protein indicating the interacting domains (ID) for HP1 and p60CAF-1, and the acidic domain (AD). (Middle) Structure of the murine Chaf1a gene. Blocks and lines represent exons and introns, respectively. A star indicates the translation initiation site. Below is shown the Chaf1a targeting vector, which includes genomic DNA homology regions (5 HR and 3 HR), the diphtheria toxin (DT), and neomycin selection genes. Recombined (Rec) ES cells and mice were identified by Southern blot using ScaI (S) and the indicated probe. + +(B) Breeding strategy used for the production of E4 Chaf1a−/− embryos. Genotyping was performed by PCR using the three oligonucleotide primers indicated in (A) (1, 2, and 3). An example of the result of a genotyping experiment is shown. + +(C) Immunodetection of p150CAF-1 (green) and HP1α (red) in E4 embryos derived from Chaf1a+/− intercrosses. Because Chaf1a+/+ and Chaf1a+/− embryos both stain positively for the presence of p150CAF-1, they are both designated as wild-type. p150CAF-1 expression, which indicates ongoing S phase, can be detected in most cells within the wild-type blastocyst (upper panel). The lower panel shows a 12-cell embryo labeling negatively for p150CAF-1. Only nonspecific background labeling can be observed. In each panel, the right-hand image shows the merge between the HP1α fluorescence and DAPI-stained DNA in blue. Pink color indicates the association of HP1 with DAPI-dense material. The bottom of each panel shows the magnification of a nucleus selected from the above embryo (white square). The arrowhead indicates the typical heterochromatin foci revealed by DAPI and HP1α staining in wild-type embryos. These foci are not visible in Chaf1a−/− embryos. DAPI and HP1α staining are diffuse within the nucleus of p150CAF-1-depleted embryos, with enrichment at the nuclear periphery, revealing abnormal heterochromatin organization. Scale bars represent 10 μm. + +(E) Wild-type embryos isolated at embryonic day 2 (E2, two cells), E2.5 (four cells), E3 (eight cells), E3.5 (16 cells), and E4 (32 cells) are shown. Heterochromatin was monitored by DAPI staining (upper panel) and HP1 immunolabeling (in red, lower panel). + +(F) Magnification of a nucleus (DAPI-stained) representative of each stage. The right-hand panel shows the nucleus of an ES cell. Scale bar represents 10 μm. + +Figure 2 + +Depletion of p150CAF-1 in ES Cells Results in a Severe Alteration of Heterochromatin Organization + +(A) Strategy used to deplete p150CAF-1 by RNAi in ES cells. The siRNA expression vector includes a puromycin selection cassette (Puro), the mouse H1 promoter, and the siRNA encoding sequence. ES cells were kept under puromycin selection during 48 h following transfection. + +(B) Abnormal heterochromatin organization in p150CAF-1-depleted ES cells. Immunodetection of p150CAF-1 (green) and HP1α (red) in ES cells transfected with control (cont) and p150CAF-1 siRNA. siRNA expression results in efficient p150CAF-1 depletion. The right-hand image shows the merge between HP1α fluorescence and DAPI-stained DNA in blue. Scale bar = 10 μm. + +(C) Heterochromatin organization is not altered in p150CAF-1-depleted MEFs. Immunodetection of p150CAF-1 (green) and HP1α (red) in MEFs transfected with control (cont) or p150CAF-1 siRNA. The right-hand image shows the merge between HP1α fluorescence and DAPI-stained DNA in blue. + +(D) PML bodies are not altered in p150CAF-1-depleted ES cells. Immunodetection of PML (red) in ES cells transfected with control (cont) or p150CAF-1 siRNA. The right-hand image shows the merge between PML fluorescence and DAPI-stained DNA in blue. + +Figure 3 + +p150CAF-1 Depletion and Loss of Heterochromatin Organization Are Compatible with Active DNA Replication in ES Cells + +(A) ES cells were transfected with p150CAF-1 or control siRNA vectors. After 3 d under puromycin selection, cells were pulse-labeled for 10 min with BrdU and immediately analyzed by IF. No significant difference in BrdU incorporation could be detected between control and p150CAF-1-depleted cells. The right-hand image shows the merge between the p150CAF-1 and BrdU fluorescence. Scale bar = 10 μm. + +(B) Immunodetection of PCNA (red) revealed no significant difference in the formation of replication foci between control and p150CAF-1-depleted ES cells. Immunodetection of p150CAF-1 is shown in green and the merging of p150CAF-1 and PCNA appears in yellow. + +(C) Flow cytometry analysis showed a similar cell-cycle profile for control and p150CAF-1-depleted ES cells. Results are presented as the percentage of cells in each phase of the cell cycle (G1, S, G2/M), as defined by BrdU incorporation and DNA content. Data presented are the mean of three independent experiments; error bars indicate the standard deviation. All experiments were performed 3 d after transfection of the siRNA vectors. + +Figure 4 + +Depletion of p150CAF-1 Leads to Loss of Clustering, Altered Localization, and Decondensation of Pericentric Heterochromatin Domains + +Distribution of pericentric (red) and centric (green) domains was analyzed in the interphase nuclei of mouse ES cells by DNA FISH, using major satellite (pSAT) [47] and minor satellite (pMR150) [48] DNA probes, respectively. (A) In ES cells expressing control (cont) siRNA, pericentric regions from several chromosomes associate in clusters (red). These chromocenters form foci as revealed by DAPI staining (left-hand image), while centric regions (green) remain independent entities at the periphery of these domains. The right-hand image shows the merge between the pericentric and centric FISH signals. + +(B) The organization of pericentric domains was altered in cells expressing p150CAF-1 siRNA. Instead of forming well-defined chromocenters, pericentric domains were found either isolated or associated in heterogeneous aggregates of various sizes, often at the nuclear periphery. Scale bar = 10 μm. + +(C) Control ES cells. Fluorescence was quantified along a line randomly drawn across the nucleus in the merged image and data were plotted. One can distinguish clear peaks corresponding to chromocenters (red) and the condensed minor satellites (green). + +(D) ES cells expressing p150CAF-1 siRNA. p150CAF-1 depletion led to a lower fluorescence intensity and a broader distribution of signals corresponding to DAPI (blue) and major satellite hybridization (red, plot) while the organization of the minor satellites remained unaffected. Insets in the right-hand images show a typical chromocenter in control cells (C) and a disrupted chromocenter in p150CAF-1-depleted cells (D). + +Figure 5 + +Nucleosomal Organization Is Not Altered in p150CAF-1-Depleted ES Cells + +Nuclei were prepared from ES cells transfected with control or p150CAF-1 siRNA vector. Nuclei were digested with increasing amounts of DNase I or MNase. (A) After digestion with the indicated nucleases, total DNA was prepared and run onto an agarose gel which was stained with ethidium bromide to reveal bulk genomic DNA. + +(B) The DNA was blotted onto a nylon membrane, which was then hybridized with the α-32P-labeled pSAT major satellite repeat probe [47]. + +Figure 6 + +DNA CpG Methylation at Pericentric Heterochromatin Is Not Altered in p150CAF-1-Depleted ES Cells + +Total DNA was isolated from cells transfected with a control or the p150CAF-1 siRNA vector and digested with MaeII, whose recognition sequence is present in major satellite repeats. Digested DNA was run onto an agarose gel and analyzed by Southern blotting using the pSAT major satellite probe [47]. + +Figure 7 + +Alteration of Epigenetic Marking at Pericentric Heterochromatin in p150CAF-1-Depleted Cells + +(A) p150CAF-1 depletion leads to reduced H4K20me3 and H3K9me3 at pericentric heterochromatin. Enrichment of histone marks at major satellite repeats was determined by ChIP from control (cont) and p150CAF-1 (p150) siRNA-expressing ES cells. DNA prepared from the input and the antibody-bound fraction were run onto an agarose gel and analyzed by Southern blot with the pSAT major satellite repeat probe [47]. + +(B) Hybridization signals were quantified using an Instant Imager. After autoradiography, the membrane was stripped and rehybridized with a minor satellite probe [49]. After quantification, the membrane was stripped and rehybridized with an IAP LTR probe [50]. Results are presented as the amount of DNA immunoprecipitated from p150CAF-1-depleted ES cells divided by the DNA obtained from control cells. The figure shows the mean value and standard deviation of three independent ChIP experiments. + +(C and D) H3K9me3 and H4K20me3 fluorescence patterns are severely altered in p150CAF-1-depleted ES cells. Immunodetection of H3K9me3 (C, green), H4K20me3 (D, green), and HP1α (red) in control and p150CAF-1 siRNA-expressing ES cells. Merging of HP1α with H3K9me3 (C) and H4K20me3 (D) is shown in yellow. Scale bars represent 10 μm. + +Footnotes + +¤ Current address: Unité de Génétique Fonctionnelle de la Souris, Centre National de la Recherche Scientifique URA 2578, Institut Pasteur, Paris, France + +Competing interests. The authors have declared that no competing interests exist. + +A previous version of this article appeared as an Early Online Release on September 11, 2006 (doi: 10.1371/journal.pgen.0020181.eor). + +Author contributions. MH, SB, AVP, JPQ, GA, and MG conceived and designed the experiments. MH, SB, AVP, and PH performed the experiments. MH, SB, AVP, and JPQ analyzed the data. MG wrote the paper. + +Funding. This study was funded by grants from the Association Pour la Recherche Sur le Cancer and the Fondation Pour la Recherche Medicale to MG, and grants from a Collaborative Programme Curie Institute/Commissariat à l'Energie Atomique to MG and GA. GA's team is labelized by la Ligue Contre le Cancer, and part of the Epigenome Network. AVP has been supported by the Curie Institute and the Fondation Franco Norvégienne; MH by the CEA, the Curie Institute, and the Canceropole IdF. diff --git a/src/ontogpt/evaluation/craft/database/all/17194222.ann b/src/ontogpt/evaluation/craft/database/all/17194222.ann new file mode 100644 index 000000000..fd5ddae24 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17194222.ann @@ -0,0 +1,2062 @@ +T1 SO:0000704 0 7 Genetic +T2 PR:000000164 33 37 BMP2 +T3 PR:000000165 39 43 BMP4 +T4 PR:000000168 49 53 BMP7 +T5 UBERON:0002101 57 61 Limb +T6 GO:0001501 77 91 Skeletogenesis +T7 GO:0060349 103 121 Bone morphogenetic +T8 PR:000000034 103 129 Bone morphogenetic protein +T9 PR:000000034 131 134 BMP +T10 PR:000000164 162 166 BMP2 +T11 PR:000000165 168 172 BMP4 +T12 PR:000000168 178 182 BMP7 +T13 GO:0010467 188 197 expressed +T14 UBERON:0002101 209 213 limb +T15 GO:0060173 209 225 limb development +T16 PR:000000034 227 231 BMPs +T17 UBERON:0002101 262 266 limb +T18 GO:0001501 307 321 skeletogenesis +T19 UBERON:0019248 369 384 early embryonic +T20 PR:000000164 413 417 Bmp2 +T21 PR:000000165 422 426 Bmp4 +T22 PR:000000034 465 468 BMP +T23 CHEBI:36357 469 478 molecules +T24 PR:000000034 542 545 BMP +T25 CHEBI:36357 546 555 molecules +T26 UBERON:0002101 583 587 limb +T27 GO:0060173 583 599 limb development +T28 NCBITaxon:10088 661 666 mouse +T29 PR:000000034 704 708 BMPs +T30 SO:0001023 728 735 alleles +T31 PR:000000164 751 755 Bmp2 +T32 PR:000000165 760 764 Bmp4 +T33 UBERON:0010328 802 821 limb bud mesenchyme +T34 PR:000000034 882 886 BMPs +T35 PR:000014841 934 947 Sonic Hedghog +T36 PR:000014841 949 952 SHH +T37 UBERON:0009585 1017 1040 interdigital mesenchyme +T38 UBERON:0002544 1055 1060 digit +T39 PR:000000034 1108 1111 BMP +T40 GO:0030509 1108 1121 BMP signaling +T41 GO:0051216 1151 1165 chondrogenesis +T42 GO:0051216 1182 1194 chondrogenic +T43 UBERON:0002101 1225 1230 limbs +T44 PR:000000164 1249 1253 BMP2 +T45 PR:000000165 1258 1262 BMP4 +T46 GO:0051216 1319 1331 chondrogenic +T47 PR:000000164 1389 1393 BMP2 +T48 PR:000000168 1398 1402 BMP7 +T49 PR:000000164 1406 1410 BMP2 +T50 PR:000000165 1415 1419 BMP4 +T51 PR:000000164 1464 1468 BMP2 +T52 PR:000000165 1473 1477 BMP4 +T53 GO:0001503 1512 1524 osteogenesis +T54 CHEBI:62488 1556 1575 signaling molecules +T55 GO:0060349 1583 1601 bone morphogenetic +T56 PR:000000034 1583 1610 bone morphogenetic proteins +T57 PR:000000034 1612 1616 BMPs +T58 UBERON:0002101 1699 1704 limbs +T59 UBERON:0000922 1845 1851 embryo +T60 PR:000000034 1933 1936 BMP +T61 PR:000000164 2106 2110 BMP2 +T62 PR:000000165 2115 2119 BMP4 +T63 PR:000000164 2123 2127 BMP2 +T64 PR:000000168 2132 2136 BMP7 +T65 UBERON:0002101 2171 2175 limb +T66 UBERON:0000479 2289 2296 tissues +T67 UBERON:0002101 2304 2308 limb +T68 PR:000000034 2393 2396 BMP +T69 UBERON:0007844 2450 2472 cartilaginous elements +T70 UBERON:0002101 2489 2494 limbs +T71 PR:000000164 2513 2517 BMP2 +T72 PR:000000165 2522 2526 BMP4 +T73 PR:000000034 2566 2569 BMP +T74 GO:0001503 2622 2648 development of bone tissue +T75 UBERON:0002481 2637 2648 bone tissue +T76 UBERON:0002101 2681 2686 limbs +T77 PR:000000034 2751 2754 BMP +T78 UBERON:0004755 2773 2789 skeletal tissues +T79 UBERON:0000922 2797 2803 embryo +T80 GO:0060349 2820 2838 Bone morphogenetic +T81 PR:000000034 2820 2847 Bone morphogenetic proteins +T82 PR:000000034 2849 2853 BMPs +T83 GO:0046903 2859 2867 secreted +T84 CHEBI:62488 2868 2887 signaling molecules +T85 PR:000000046 2905 2933 transforming growth factor β +T86 UBERON:0000479 3059 3065 tissue +T87 PR:000000034 3081 3084 BMP +T88 GO:0009653 3233 3246 morphogenesis +T89 CHEBI:36357 3276 3285 molecules +T90 UBERON:0002101 3350 3354 limb +T91 GO:0060173 3350 3366 limb development +T92 PR:000000034 3368 3372 BMPs +T93 GO:0001708 3458 3481 cell type specification +T94 UBERON:0000479 3514 3521 tissues +T95 UBERON:0004288 3543 3551 skeleton +T96 UBERON:0002101 3575 3579 limb +T97 GO:0060173 3575 3591 limb development +T98 PR:000000034 3601 3604 BMP +T99 GO:0030509 3601 3614 BMP signaling +T100 GO:0009948 3642 3658;3663 3681;3687 3691 establishment of ... anterior-posterior ... axis +T101 UBERON:0002101 3682 3686 limb +T102 PR:000014841 3774 3787 Sonic Hedghog +T103 GO:0007224 3780 3787;3794 3803 Hedghog ... signaling +T104 PR:000014841 3789 3792 SHH +T105 UBERON:0004347 3847 3855 limb bud +T106 PR:000014841 3982 3985 SHH +T107 PR:000000034 4104 4107 BMP +T108 PR:000000164 4116 4120 BMP2 +T109 PR:000000168 4125 4129 BMP7 +T110 GO:0010467 4140 4149 expressed +T111 PR:000014841 4175 4178 SHH +T112 UBERON:0010328 4202 4221 limb bud mesenchyme +T113 PR:000000168 4238 4242 BMP7 +T114 GO:0010467 4279 4289 expression +T115 UBERON:0010328 4306 4325 limb bud mesenchyme +T116 PR:000000164 4327 4331 BMP2 +T117 PR:000000168 4340 4344 BMP7 +T118 PR:000014841 4375 4378 SHH +T119 GO:0010467 4389 4399 expression +T120 PR:000014841 4440 4443 SHH +T121 PR:000000164 4458 4462 BMP2 +T122 PR:000000168 4467 4471 BMP7 +T123 PR:000014841 4523 4526 SHH +T124 PR:000000034 4547 4550 BMP +T125 GO:0030509 4547 4560 BMP signaling +T126 UBERON:0002101 4608 4612 limb +T127 PR:000014841 4703 4706 SHH +T128 PR:000000164 4750 4754 BMP2 +T129 PR:000000168 4759 4763 BMP7 +T130 UBERON:0002101 4821 4825 limb +T131 PR:000014841 4840 4843 SHH +T132 PR:000000164 4845 4849 Bmp2 +T133 UBERON:0000922 4857 4864 embryos +T134 GO:0016265 4865 4868 die +T135 UBERON:0002101 4895 4899 limb +T136 PR:000000168 4935 4939 Bmp7 +T137 PR:000000168 4959 4963 Bmp7 +T138 UBERON:0000922 4974 4981 embryos +T139 UBERON:0002103 4990 4998 hindlimb +T140 http://purl.obolibrary.org/obo/MONDO_0021003 4999 5010 polydactyly +T141 UBERON:0002101 5074 5079 limbs +T142 PR:000000168 5102 5106 Bmp7 +T143 NCBITaxon:10088 5116 5120 mice +T144 UBERON:0002101 5147 5151 limb +T145 PR:000000164 5230 5234 BMP2 +T146 PR:000000164 5273 5277 BMP2 +T147 PR:000000168 5282 5286 BMP7 +T148 PR:000000164 5345 5349 BMP2 +T149 PR:000000165 5351 5355 BMP4 +T150 GO:0010467 5365 5374 expressed +T151 UBERON:0004347 5388 5396 limb bud +T152 PR:000000168 5403 5407 BMP7 +T153 GO:0010467 5415 5424 expressed +T154 UBERON:0010328 5475 5494 limb bud mesenchyme +T155 PR:000014841 5548 5551 SHH +T156 GO:0010467 5576 5586 expression +T157 PR:000014841 5597 5600 SHH +T158 UBERON:0004347 5611 5620 limb buds +T159 PR:000000165 5628 5632 BMP4 +T160 PR:000014841 5689 5692 SHH +T161 CHEBI:36357 5742 5751 molecules +T162 PR:000000164 5753 5757 BMP2 +T163 PR:000000165 5759 5763 BMP4 +T164 PR:000000168 5769 5773 BMP7 +T165 UBERON:0002101 5832 5836 limb +T166 UBERON:0002544 5854 5859 digit +T167 NCBITaxon:7742 5935 5945 vertebrate +T168 UBERON:0002101 5946 5950 limb +T169 UBERON:0002544 5957 5962 digit +T170 UBERON:0003221 6027 6036 phalanges +T171 UBERON:0002470 6062 6069 autopod +T172 GO:0009952 6103 6161 establishment of anterior-posterior positioned information +T173 UBERON:0002101 6173 6177 limb +T174 PR:000014841 6181 6184 SHH +T175 PR:000000034 6192 6195 BMP +T176 GO:0030509 6192 6205 BMP signaling +T177 UBERON:0009585 6211 6234 interdigital mesenchyme +T178 UBERON:0009523 6224 6237;6242 6252 mesenchyme of ... hand plate +T179 UBERON:0006012 6365 6377 interdigital +T180 UBERON:0000479 6378 6384 tissue +T181 UBERON:0002544 6399 6404 digit +T182 PR:000000164 6422 6426 BMP2 +T183 PR:000000165 6428 6432 BMP4 +T184 PR:000000168 6438 6442 BMP7 +T185 GO:0010467 6451 6460 expressed +T186 UBERON:0009585 6468 6491 interdigital mesenchyme +T187 PR:000000034 6527 6530 BMP +T188 GO:0030509 6527 6540 BMP signaling +T189 UBERON:0000479 6549 6555 tissue +T190 PR:000000034 6594 6598 BMPs +T191 UBERON:0009585 6606 6629 interdigital mesenchyme +T192 UBERON:0002544 6669 6674 digit +T193 PR:000000168 6708 6712 BMP7 +T194 UBERON:0000922 6723 6730 embryos +T195 UBERON:0002101 6753 6758 limbs +T196 NCBITaxon:10088 6764 6768 mice +T197 PR:000000165 6805 6809 Bmp4 +T198 UBERON:0010328 6817 6836 limb bud mesenchyme +T199 UBERON:0002544 6875 6880 digit +T200 PR:000000165 6934 6938 BMP4 +T201 UBERON:0006012 6951 6963 interdigital +T202 UBERON:0002544 6977 6982 digit +T203 UBERON:0006012 7097 7109 interdigital +T204 PR:000000034 7110 7113 BMP +T205 UBERON:0002544 7154 7159 digit +T206 PR:000000164 7198 7202 BMP2 +T207 PR:000000165 7204 7208 BMP4 +T208 PR:000000168 7214 7218 BMP7 +T209 UBERON:0002101 7274 7278 limb +T210 PR:000000164 7299 7303 BMP2 +T211 PR:000000165 7305 7309 BMP4 +T212 PR:000000168 7315 7319 BMP7 +T213 GO:0016271 7361 7369;7387 7393 death of ... tissue +T214 UBERON:0006012 7374 7386 interdigital +T215 UBERON:0000479 7387 7393 tissue +T216 PR:000000034 7431 7434 BMP +T217 PR:000000034 7465 7468 BMP +T218 GO:0030509 7465 7478 BMP signaling +T219 PR:000000034 7556 7559 BMP +T220 UBERON:0002101 7600 7604 limb +T221 PR:000014841 7662 7665 SHH +T222 UBERON:0004347 7694 7702 limb bud +T223 CL:0000057 7707 7717 fibroblast +T224 GO:0090269 7707 7742 fibroblast growth factor production +T225 UBERON:0004356 7760 7783 apical ectodermal ridge +T226 UBERON:0004356 7785 7788 AER +T227 PR:000014841 7791 7794 SHH +T228 PR:000000034 7837 7840 BMP +T229 PR:000008227 7852 7859 Gremlin +T230 UBERON:0003104 7867 7877 mesenchyme +T231 PR:000000034 7911 7915 BMPs +T232 CL:0000057 7936 7946 fibroblast +T233 GO:0090269 7936 7960;7967 7977 fibroblast growth factor ... production +T234 GO:0090269 7962 7965;7967 7977 FGF ... production +T235 UBERON:0004356 7985 7988 AER +T236 UBERON:0004356 8032 8035 AER +T237 GO:0008543 8049 8062 FGF signaling +T238 PR:000014841 8086 8089 SHH +T239 UBERON:0003104 8118 8128 mesenchyme +T240 PR:000000165 8166 8170 BMP4 +T241 UBERON:0009749 8199 8214 limb mesenchyme +T242 UBERON:0004356 8247 8250 AER +T243 PR:000014841 8261 8264 SHH +T244 http://purl.obolibrary.org/obo/MONDO_0020927 8304 8325 postaxial polydactyly +T245 UBERON:0002101 8352 8356 limb +T246 GO:0060173 8352 8368 limb development +T247 PR:000000034 8370 8373 BMP +T248 GO:0001501 8426 8440 skeletogenesis +T249 PR:000000036 8538 8554 BMPR-IB receptor +T250 GO:0001502 8587 8609 cartilage condensation +T251 PR:000000035 8659 8665 BmpR1A +T252 PR:000000036 8670 8676 BmpR1B +T253 GO:0051216 8688 8700 chondrogenic +T254 NCBITaxon:10088 8724 8729 mouse +T255 UBERON:0004347 8730 8738 limb bud +T256 PR:000000021 8761 8767 noggin +T257 PR:000000034 8778 8781 BMP +T258 CHEBI:35222 8782 8791 inhibitor +T259 UBERON:0004347 8828 8837 limb buds +T260 GO:0001501 8847 8861 skeletogenesis +T261 UBERON:0003104 8863 8874 mesenchymal +T262 PR:000000034 8966 8969 BMP +T263 GO:0030509 8966 8979 BMP signaling +T264 UBERON:0003104 8997 9008 mesenchymal +T265 GO:0051216 9045 9059 chondrogenesis +T266 GO:0051216 9140 9154 chondrogenesis +T267 GO:0051216 9169 9183 chondrogenesis +T268 GO:0001501 9294 9308 skeletogenesis +T269 PR:000000034 9358 9362 BMPs +T270 GO:0008152 9434 9444 metabolism +T271 PR:000000165 9501 9505 BMP4 +T272 PR:000000168 9514 9518 BMP7 +T273 UBERON:0004288 9556 9564 skeletal +T274 PR:000000034 9677 9680 BMP +T275 GO:0030509 9677 9690 BMP signaling +T276 NCBITaxon:7742 9694 9704 vertebrate +T277 UBERON:0002101 9705 9709 limb +T278 GO:0001501 9725 9739 skeletogenesis +T279 NCBITaxon:10088 9758 9762 mice +T280 PR:000000164 9786 9790 BMP2 +T281 PR:000000165 9795 9799 BMP4 +T282 PR:000000164 9812 9816 BMP2 +T283 PR:000000168 9821 9825 BMP7 +T284 UBERON:0009749 9842 9856 limb mesechyme +T285 UBERON:0002101 9864 9868 limb +T286 GO:0060173 9864 9880 limb development +T287 PR:000000164 9888 9892 Bmp2 +T288 PR:000000165 9897 9901 Bmp4 +T289 GO:0009790 9935 9948 embryogenesis +T290 SO:0001023 9978 9985 alleles +T291 SO:0000704 10000 10005 genes +T292 SO:0001023 10023 10030 alleles +T293 UBERON:0002101 10053 10057 limb +T294 GO:0060173 10053 10069 limb development +T295 SO:0000902 10110 10119 transgene +T296 GO:0010467 10120 10129 expressed +T297 GO:0065007 10140 10147 control +T298 PR:000013316 10155 10159 Prx1 +T299 SO:0000165 10160 10168 enhancer +T300 UBERON:0002101 10183 10188 limbs +T301 PR:000000164 10202 10206 BMP2 +T302 PR:000000168 10211 10215 BMP7 +T303 PR:000000164 10239 10243 BMP2 +T304 PR:000000165 10248 10252 BMP4 +T305 PR:000014841 10402 10405 SHH +T306 UBERON:0006012 10419 10431 interdigital +T307 UBERON:0002544 10448 10453 digit +T308 PR:000000164 10468 10472 Bmp2 +T309 PR:000000165 10473 10477 Bmp4 +T310 UBERON:0002544 10525 10531 digits +T311 UBERON:0006875 10555 10565 hand plate +T312 GO:0001502 10596 10621 chondrogenic condensation +T313 UBERON:5002544 10639 10649 digit rays +T314 UBERON:0002544 10694 10699 digit +T315 PR:000000164 10760 10764 BMP2 +T316 PR:000000165 10769 10773 BMP4 +T317 PR:000000164 10781 10785 BMP2 +T318 PR:000000168 10790 10794 BMP7 +T319 GO:0051216 10811 10825 chondrogenesis +T320 GO:0001503 10827 10839 Osteogenesis +T321 PR:000000164 10956 10960 BMP2 +T322 PR:000000165 10965 10969 BMP4 +T323 GO:0001503 11002 11014 osteogenesis +T324 PR:000000034 11066 11069 BMP +T325 GO:0030509 11066 11077 BMP pathway +T326 UBERON:0004288 11085 11093 skeletal +T327 GO:0001501 11085 11105 skeletal development +T328 GO:0051216 11153 11167 chondrogenesis +T329 UBERON:0004288 11186 11194 skeletal +T330 GO:0001501 11186 11204 skeletal formation +T331 PR:000000034 11259 11262 BMP +T332 GO:0001503 11275 11285 osteogenic +T333 PR:000000034 11341 11344 BMP +T334 GO:0030509 11341 11354 BMP signaling +T335 UBERON:0002101 11376 11380 limb +T336 GO:0001501 11396 11410 skeletogenesis +T337 NCBITaxon:10088 11439 11443 mice +T338 PR:000000164 11505 11509 BMP2 +T339 PR:000000165 11511 11515 BMP4 +T340 PR:000000168 11521 11525 BMP7 +T341 PR:000000168 11527 11531 BMP7 +T342 NCBITaxon:10088 11542 11546 mice +T343 GO:0007567 11600 11605 birth +T344 PR:000000164 11616 11620 BMP2 +T345 PR:000000165 11625 11629 BMP4 +T346 UBERON:0000922 11671 11680 embryonic +T347 GO:0009790 11671 11692 embryonic development +T348 SO:0001023 11741 11747 allele +T349 PR:000000164 11751 11755 Bmp2 +T350 SO:0000346 11769 11779 loxP sites +T351 SO:0000357 11780 11788 flanking +T352 SO:0000147 11789 11793 exon +T353 SO:0001023 11823 11829 allele +T354 PR:000000165 11833 11837 Bmp4 +T355 SO:0000147 11848 11852 exon +T356 SO:0000357 11858 11865 flanked +T357 SO:0000346 11869 11879 loxP sites +T358 SO:0001023 11946 11953 alleles +T359 SO:0001023 11990 11997 alleles +T360 PR:000000164 12091 12095 Bmp2 +T361 PR:000000165 12100 12104 Bmp4 +T362 UBERON:0002101 12112 12116 limb +T363 SO:0000902 12147 12156 transgene +T364 GO:0010467 12185 12194 expressed +T365 GO:0065007 12205 12212 control +T366 PR:000013316 12220 12224 Prx1 +T367 UBERON:0002101 12225 12229 limb +T368 SO:0000165 12230 12238 enhancer +T369 SO:0000902 12250 12259 transgene +T370 GO:0010467 12260 12269 expresses +T371 UBERON:0002101 12288 12292 limb +T372 GO:0060173 12288 12304 limb development +T373 SO:0000359 12345 12351 floxed +T374 SO:0001023 12352 12359 alleles +T375 UBERON:0004347 12369 12377 limb bud +T376 PR:000013316 12413 12417 Prx1 +T377 PR:000000164 12452 12456 Bmp2 +T378 PR:000000165 12461 12465 Bmp4 +T379 SO:0001023 12466 12473 alleles +T380 UBERON:0004347 12486 12494 limb bud +T381 GO:0097617 12516 12529 hybridization +T382 PR:000000164 12531 12535 Bmp2 +T383 GO:0010467 12545 12554 expressed +T384 UBERON:0009749 12562 12577 limb mesenchyme +T385 UBERON:0000922 12581 12590 embryonic +T386 NCBITaxon:10088 12610 12615 mouse +T387 GO:0010467 12655 12665 expression +T388 SO:0000359 12691 12697 floxed +T389 PR:000000164 12698 12702 Bmp2 +T390 SO:0001023 12703 12709 allele +T391 GO:0097617 12793 12806 hybridization +T392 PR:000000164 12839 12843 Bmp2 +T393 PR:000000165 12880 12884 Bmp4 +T394 GO:0010467 12888 12897 expressed +T395 NCBITaxon:10088 12905 12910 mouse +T396 UBERON:0009749 12911 12926 limb mesenchyme +T397 GO:0010467 13011 13021 expression +T398 PR:000013316 13082 13086 Prx1 +T399 SO:0000902 13092 13101 transgene +T400 PR:000000164 13115 13119 Bmp2 +T401 PR:000000165 13124 13128 Bmp4 +T402 GO:0010467 13138 13147 expressed +T403 UBERON:0004356 13155 13158 AER +T404 PR:000013316 13166 13170 Prx1 +T405 GO:0010467 13210 13220 expression +T406 SO:0001023 13272 13279 Allelic +T407 SO:0001023 13283 13290 allelic +T408 PR:000000034 13301 13304 BMP +T409 UBERON:0002101 13315 13320 Limbs +T410 NCBITaxon:10088 13322 13326 Mice +T411 UBERON:0002101 13347 13352 limbs +T412 PR:000000164 13366 13370 BMP2 +T413 PR:000000165 13372 13376 BMP4 +T414 PR:000000168 13381 13385 BMP7 +T415 PR:000000164 13392 13396 BMP2 +T416 PR:000000165 13401 13405 BMP4 +T417 PR:000000164 13415 13419 BMP2 +T418 PR:000000168 13424 13428 BMP7 +T419 NCBITaxon:33208 13507 13514 animals +T420 UBERON:0004381 13538 13552 limb skeletons +T421 NCBITaxon:33208 13564 13571 animals +T422 PR:000000164 13573 13577 BMP2 +T423 UBERON:0002101 13588 13593 limbs +T424 UBERON:0004288 13630 13638 skeletal +T425 GO:0051216 13671 13685 chondrogenesis +T426 GO:0001503 13690 13702 osteogenesis +T427 CHEBI:16866 13728 13740 Alizarin red +T428 UBERON:0002091 13781 13802 appendicular skeleton +T429 NCBITaxon:33208 13812 13819 animals +T430 UBERON:0006849 13860 13867 scapula +T431 PR:000000164 13891 13895 BMP2 +T432 UBERON:0002101 13906 13911 limbs +T433 UBERON:0000479 13934 13940 tissue +T434 http://purl.obolibrary.org/obo/MONDO_0021002 13941 13951 syndactyly +T435 NCBITaxon:10088 13990 13994 Mice +T436 UBERON:0002101 14000 14005 limbs +T437 PR:000000165 14019 14023 BMP4 +T438 UBERON:0002101 14041 14046 limbs +T439 PR:000000168 14050 14054 Bmp7 +T440 NCBITaxon:10088 14062 14066 mice +T441 PR:000000165 14140 14144 BMP4 +T442 UBERON:0002101 14155 14160 limbs +T443 http://purl.obolibrary.org/obo/MONDO_0020927 14207 14228 postaxial polydactyly +T444 UBERON:0002544 14252 14257 digit +T445 UBERON:0004288 14289 14297 skeletal +T446 NCBITaxon:10088 14370 14374 mice +T447 PR:000000168 14409 14413 Bmp7 +T448 UBERON:0002091 14466 14487 appendicular skeletal +T449 UBERON:0004765 14479 14496 skeletal elements +T450 http://purl.obolibrary.org/obo/MONDO_0021003 14553 14564 polydactyly +T451 NCBITaxon:10088 14606 14610 mice +T452 PR:000000164 14645 14649 Bmp2 +T453 PR:000000165 14654 14658 Bmp4 +T454 UBERON:0002101 14666 14670 limb +T455 PR:000000164 14672 14676 Bmp2 +T456 PR:000000165 14681 14685 Bmp4 +T457 PR:000013316 14690 14694 Prx1 +T458 UBERON:0002101 14727 14731 limb +T459 GO:0001501 14746 14760 skeletogenesis +T460 UBERON:0004381 14792 14806 limb skeletons +T461 NCBITaxon:10088 14810 14814 mice +T462 PR:000000165 14843 14847 Bmp4 +T463 SO:0000704 14848 14852 gene +T464 PR:000000164 14873 14877 Bmp2 +T465 SO:0000704 14878 14882 gene +T466 UBERON:0002101 14922 14926 limb +T467 PR:000000164 14928 14932 Bmp2 +T468 PR:000000165 14937 14941 Bmp4 +T469 PR:000013316 14946 14950 Prx1 +T470 http://purl.obolibrary.org/obo/MONDO_0020927 15042 15063 postaxial polydactyly +T471 NCBITaxon:10088 15072 15076 mice +T472 PR:000000165 15090 15094 BMP4 +T473 NCBITaxon:10088 15143 15147 mice +T474 PR:000000164 15172 15176 Bmp2 +T475 PR:000000165 15193 15197 Bmp4 +T476 PR:000000164 15216 15220 Bmp2 +T477 PR:000000165 15225 15229 Bmp4 +T478 PR:000013316 15234 15238 Prx1 +T479 UBERON:0004288 15264 15272 skeletal +T480 UBERON:0004765 15314 15331 skeletal elements +T481 UBERON:0002544 15346 15351 digit +T482 NCBITaxon:33208 15370 15377 animals +T483 NCBITaxon:33208 15420 15427 Animals +T484 PR:000000164 15452 15456 Bmp2 +T485 PR:000000165 15461 15465 Bmp4 +T486 PR:000000164 15480 15484 Bmp2 +T487 PR:000000165 15489 15493 Bmp4 +T488 PR:000013316 15498 15502 Prx1 +T489 UBERON:0002101 15533 15538 limbs +T490 UBERON:0002472 15603 15612 stylopods +T491 UBERON:0002471 15625 15633 zeugopod +T492 UBERON:0004905 15779 15798 joint articulations +T493 UBERON:0002471 15823 15831 zeugopod +T494 UBERON:0002472 15836 15844 stylopod +T495 UBERON:0002470 15915 15923 autopods +T496 UBERON:0002470 15987 15994 autopod +T497 UBERON:0002544 16077 16083 digits +T498 UBERON:0002102 16103 16112 forelimbs +T499 NCBITaxon:33208 16122 16129 animals +T500 PR:000000164 16156 16160 BMP2 +T501 PR:000000168 16165 16169 BMP7 +T502 PR:000000164 16216 16220 BMP2 +T503 PR:000000165 16225 16229 BMP4 +T504 PR:000000164 16273 16277 BMP2 +T505 PR:000000168 16282 16286 BMP7 +T506 PR:000000164 16288 16292 Bmp2 +T507 PR:000000168 16297 16301 Bmp7 +T508 PR:000013316 16306 16310 Prx1 +T509 PR:000000164 16345 16349 BMP2 +T510 PR:000000168 16375 16379 BMP7 +T511 PR:000000164 16381 16385 Bmp2 +T512 PR:000000168 16390 16394 Bmp7 +T513 PR:000013316 16399 16403 Prx1 +T514 UBERON:0004288 16444 16452 skeletal +T515 NCBITaxon:10088 16501 16505 Mice +T516 PR:000000164 16532 16536 Bmp2 +T517 PR:000000168 16558 16562 Bmp7 +T518 PR:000000164 16564 16568 Bmp2 +T519 PR:000000168 16573 16577 Bmp7 +T520 PR:000013316 16582 16586 Prx1 +T521 UBERON:0006849 16619 16627 scapular +T522 PR:000000164 16643 16647 Bmp2 +T523 PR:000013316 16652 16656 Prx1 +T524 UBERON:0002101 16699 16704 limbs +T525 PR:000000168 16748 16752 BMP7 +T526 PR:000000164 16757 16761 BMP2 +T527 PR:000000164 16772 16776 Bmp2 +T528 PR:000000168 16781 16785 Bmp7 +T529 PR:000013316 16790 16794 Prx1 +T530 UBERON:0004300 16806 16818 last phalanx +T531 UBERON:0006050 16836 16845 digit III +T532 UBERON:0002102 16853 16861 forelimb +T533 UBERON:0002101 16873 16878 limbs +T534 UBERON:0002103 16915 16923 hindlimb +T535 UBERON:0001446 17000 17007 fibulae +T536 UBERON:0002103 17017 17026 hindlimbs +T537 UBERON:0000981 17072 17077 femur +T538 UBERON:0001465 17085 17089 knee +T539 UBERON:0006849 17147 17155 scapular +T540 PR:000000164 17172 17176 Bmp2 +T541 PR:000013316 17181 17185 Prx1 +T542 NCBITaxon:10088 17191 17195 mice +T543 UBERON:0002091 17225 17246 appendicular skeleton +T544 UBERON:0004288 17280 17288 skeletal +T545 UBERON:0002101 17327 17332 limbs +T546 NCBITaxon:33208 17342 17349 animals +T547 UBERON:0000479 17357 17363 Tissue +T548 http://purl.obolibrary.org/obo/MONDO_0021002 17364 17374 Syndactyly +T549 UBERON:0002101 17387 17392 Limbs +T550 NCBITaxon:10088 17396 17400 Mice +T551 PR:000000164 17411 17415 BMP2 +T552 PR:000000165 17420 17424 BMP4 +T553 PR:000000164 17451 17455 Bmp2 +T554 NCBITaxon:33208 17466 17473 animals +T555 UBERON:0000479 17512 17518 tissue +T556 http://purl.obolibrary.org/obo/MONDO_0021002 17519 17529 syndactyly +T557 PR:000000165 17566 17570 Bmp4 +T558 NCBITaxon:10088 17581 17585 mice +T559 http://purl.obolibrary.org/obo/MONDO_0021002 17606 17616 syndactyly +T560 PR:000000164 17690 17694 Bmp2 +T561 PR:000000165 17699 17703 Bmp4 +T562 PR:000013316 17708 17712 Prx1 +T563 NCBITaxon:10088 17718 17722 mice +T564 NCBITaxon:33208 17746 17753 animals +T565 UBERON:0002389 17759 17768;17774 17782 digits of ... forelimb +T566 UBERON:0001466 17759 17768;17806 17814 digits of ... hindlimb +T567 http://purl.obolibrary.org/obo/MONDO_0021002 17829 17839 syndactyly +T568 UBERON:0000922 17938 17947 embryonic +T569 UBERON:0002101 17948 17953 limbs +T570 UBERON:0006012 17979 17991 interdigital +T571 PR:000000164 18044 18048 Bmp2 +T572 PR:000000165 18053 18057 Bmp4 +T573 PR:000013316 18062 18066 Prx1 +T574 UBERON:0002101 18072 18077 limbs +T575 UBERON:0009551 18093 18107;18113 18118 distal tips of ... digit +T576 UBERON:0002470 18144 18151 autopod +T577 UBERON:0002544 18245 18251 digits +T578 UBERON:0006012 18302 18314 interdigital +T579 GO:0006915 18315 18324 apoptosis +T580 CHEBI:51739 18354 18369 acridine orange +T581 UBERON:0006012 18397 18409 Interdigital +T582 GO:0006915 18410 18419 apoptosis +T583 PR:000000164 18451 18455 Bmp2 +T584 PR:000000165 18460 18464 Bmp4 +T585 PR:000013316 18469 18473 Prx1 +T586 UBERON:0002101 18479 18484 limbs +T587 CHEBI:51739 18534 18549 acridine orange +T588 UBERON:0009585 18586 18609 interdigital mesenchyme +T589 UBERON:0004356 18659 18662 AER +T590 GO:0006915 18714 18723 apoptosis +T591 UBERON:0004347 18741 18749 limb bud +T592 UBERON:0004356 18764 18767 AER +T593 UBERON:0004356 18773 18776 AER +T594 UBERON:0000924 18803 18811 ectoderm +T595 UBERON:0004347 18888 18896 limb bud +T596 PR:000000164 18939 18943 Bmp2 +T597 PR:000000165 18948 18952 Bmp4 +T598 PR:000013316 18957 18961 Prx1 +T599 UBERON:0004356 18994 18997 AER +T600 PR:000000164 19028 19032 Bmp2 +T601 PR:000000165 19037 19041 Bmp4 +T602 PR:000013316 19046 19050 Prx1 +T603 NCBITaxon:33208 19056 19063 animals +T604 UBERON:0004356 19069 19072 AER +T605 UBERON:0002101 19155 19160 limbs +T606 GO:0010467 19233 19243 expression +T607 UBERON:0004356 19247 19250 AER +T608 PR:000007499 19275 19279 Fgf8 +T609 UBERON:0004356 19304 19307 AER +T610 CHEBI:51739 19339 19354 acridine orange +T611 GO:0006915 19384 19393 apoptosis +T612 PR:000000164 19499 19503 Bmp2 +T613 PR:000000165 19508 19512 Bmp4 +T614 PR:000013316 19517 19521 Prx1 +T615 UBERON:0002101 19534 19539 limbs +T616 CHEBI:51739 19561 19576 acridine orange +T617 UBERON:0004356 19605 19608 AER +T618 UBERON:0004356 19671 19674 AER +T619 GO:0006915 19791 19803;19808 19813 apoptosis in ... cells +T620 UBERON:0004356 19821 19824 AER +T621 PR:000000164 19842 19846 Bmp2 +T622 PR:000000165 19851 19855 Bmp4 +T623 PR:000013316 19860 19864 Prx1 +T624 UBERON:0000922 19877 19886 embryonic +T625 UBERON:0002101 19887 19892 limbs +T626 UBERON:0002101 19934 19938 Limb +T627 PR:000000034 19973 19976 BMP +T628 GO:0030509 19973 19986 BMP Signaling +T629 PR:000000034 19988 19991 BMP +T630 GO:0030509 19988 20001 BMP signaling +T631 UBERON:0002101 20054 20058 limb +T632 UBERON:0002101 20162 20166 limb +T633 PR:000014841 20181 20195 sonic hedgehog +T634 PR:000014841 20197 20200 Shh +T635 PR:000014841 20203 20206 SHH +T636 UBERON:0004347 20268 20276 limb bud +T637 SO:0000704 20313 20318 genes +T638 PR:000000164 20494 20498 BMP2 +T639 PR:000000168 20503 20507 BMP7 +T640 PR:000014841 20553 20556 SHH +T641 PR:000000164 20579 20583 BMP2 +T642 PR:000000168 20588 20592 BMP7 +T643 UBERON:0004347 20617 20625 limb bud +T644 GO:0010628 20673 20688;20694 20704 upregulation of ... expression +T645 PR:000000165 20689 20693 BMP4 +T646 UBERON:0002101 20731 20736 limbs +T647 UBERON:0004288 20757 20765 skeletal +T648 NCBITaxon:10088 20780 20785 mouse +T649 UBERON:0002101 20786 20791 limbs +T650 PR:000000164 20817 20821 BMP2 +T651 PR:000000168 20823 20827 BMP7 +T652 PR:000000164 20838 20842 BMP2 +T653 PR:000000168 20847 20851 BMP7 +T654 UBERON:0002544 20907 20912 digit +T655 UBERON:0004300 20949 20963 distal phalanx +T656 UBERON:0002544 20989 20995 digits +T657 PR:000000164 21019 21023 Bmp2 +T658 PR:000000168 21028 21032 Bmp7 +T659 PR:000013316 21037 21041 Prx1 +T660 UBERON:0004300 21166 21182 terminal phalanx +T661 UBERON:0009551 21196 21209;21215 21220 distal tip of ... digit +T662 PR:000000164 21335 21339 BMP2 +T663 PR:000000168 21344 21348 BMP7 +T664 PR:000014841 21388 21391 SHH +T665 UBERON:0002544 21421 21427 digits +T666 UBERON:0002544 21495 21500 digit +T667 UBERON:0004381 21517 21531 limb skeletons +T668 PR:000000164 21557 21561 BMP2 +T669 PR:000000168 21566 21570 BMP7 +T670 PR:000000034 21600 21603 BMP +T671 GO:0030509 21600 21613 BMP signaling +T672 UBERON:0009585 21661 21684 interdigital mesenchyme +T673 UBERON:0002544 21700 21706 digits +T674 UBERON:0004300 21716 21730 distal phalanx +T675 UBERON:0002389 21756 21764;21769 21778 digit of ... forelimbs +T676 NCBITaxon:33208 21788 21795 animals +T677 UBERON:0002544 21803 21809 digits +T678 UBERON:0006048 21901 21908 digit 1 +T679 UBERON:0002103 21933 21942 hindlimbs +T680 UBERON:0002544 22013 22019 digits +T681 UBERON:0004765 22120 22136 skeletal element +T682 PR:000000034 22177 22181 BMPs +T683 UBERON:0002544 22204 22209 digit +T684 PR:000000034 22303 22306 BMP +T685 GO:0030509 22303 22316 BMP signaling +T686 UBERON:0006012 22330 22344 interdigitally +T687 GO:0012501 22349 22370 programmed cell death +T688 http://purl.obolibrary.org/obo/MONDO_0021002 22393 22403 syndactyly +T689 UBERON:0002101 22432 22437 limbs +T690 PR:000000164 22457 22461 BMP2 +T691 UBERON:0000479 22509 22515 tissue +T692 http://purl.obolibrary.org/obo/MONDO_0021002 22516 22526 syndactyly +T693 UBERON:0002544 22534 22540 digits +T694 UBERON:0002101 22544 22549 limbs +T695 PR:000000164 22568 22572 BMP2 +T696 PR:000000165 22577 22581 BMP4 +T697 PR:000000034 22672 22675 BMP +T698 UBERON:0002544 22720 22726 digits +T699 UBERON:0002102 22730 22739 forelimbs +T700 PR:000000164 22758 22762 BMP2 +T701 PR:000000165 22767 22771 BMP4 +T702 UBERON:0004347 22810 22819 limb buds +T703 GO:0010467 22839 22849 expression +T704 PR:000015435 22877 22881 Sox9 +T705 UBERON:0004347 22936 22945 limb buds +T706 PR:000000164 23015 23019 Bmp2 +T707 PR:000000165 23024 23028 Bmp4 +T708 PR:000013316 23033 23037 Prx1 +T709 UBERON:0004347 23043 23052 limb buds +T710 PR:000014841 23172 23175 Shh +T711 GO:0010467 23176 23186 expression +T712 UBERON:0004356 23254 23257 AER +T713 PR:000007499 23275 23279 Fgf8 +T714 GO:0010467 23280 23290 expression +T715 PR:000014841 23341 23344 Shh +T716 GO:0010467 23345 23355 expression +T717 PR:000014841 23449 23452 Shh +T718 GO:0010467 23453 23463 expression +T719 UBERON:0004356 23526 23529 AER +T720 PR:000000034 23548 23551 BMP +T721 GO:0030509 23548 23561 BMP signaling +T722 UBERON:0005414 23626 23653 zone of polarizing activity +T723 UBERON:0005414 23655 23658 ZPA +T724 UBERON:0004356 23668 23671 AER +T725 GO:0010467 23673 23683 Expression +T726 SO:0000704 23699 23704 genes +T727 UBERON:0004356 23736 23739 AER +T728 PR:000000034 23771 23774 BMP +T729 PR:000014841 23797 23800 SHH +T730 PR:000000034 23822 23825 BMP +T731 GO:0030509 23822 23835 BMP signaling +T732 UBERON:0004356 23862 23865 AER +T733 PR:000014841 23875 23878 Shh +T734 GO:0010467 23879 23889 expression +T735 GO:0008543 23921 23934 FGF signaling +T736 UBERON:0004356 23944 23947 AER +T737 UBERON:0005414 23989 23992 ZPA +T738 PR:000014841 24004 24007 Shh +T739 GO:0010467 24008 24018 expression +T740 UBERON:0006875 24052 24062 hand plate +T741 PR:000000164 24070 24074 BMP2 +T742 PR:000000165 24076 24080 BMP4 +T743 UBERON:0004347 24091 24100 limb buds +T744 UBERON:0004288 24145 24153 skeletal +T745 UBERON:0002470 24180 24187 autopod +T746 UBERON:0002544 24251 24257 digits +T747 http://purl.obolibrary.org/obo/MONDO_0021003 24259 24270 polydactyly +T748 UBERON:0002544 24337 24343 digits +T749 UBERON:0002102 24378 24387 forelimbs +T750 UBERON:0002544 24413 24419 digits +T751 PR:000000164 24434 24438 BMP2 +T752 PR:000000165 24440 24444 BMP4 +T753 UBERON:0004347 24455 24464 limb buds +T754 GO:0001502 24486 24508 cartilage condensation +T755 UBERON:5002544 24522 24532 digit rays +T756 UBERON:0004347 24556 24565 limb buds +T757 UBERON:0002544 24595 24601 digits +T758 GO:0097617 24645 24658 hybridization +T759 PR:000015435 24689 24693 Sox9 +T760 GO:0051216 24714 24726 chondrogenic +T761 PR:000000164 24759 24763 Bmp2 +T762 PR:000000165 24768 24772 Bmp4 +T763 PR:000013316 24777 24781 Prx1 +T764 UBERON:0004347 24787 24796 limb buds +T765 PR:000015435 24807 24811 Sox9 +T766 UBERON:0001048 24825 24834 primordia +T767 UBERON:0002389 24857 24866;24871 24879 digits of ... forelimb +T768 UBERON:0002544 24932 24938 digits +T769 SO:0000704 24976 24981 Genes +T770 PR:000010687 24990 24994 Msx2 +T771 GO:0010467 25022 25031 expressed +T772 UBERON:0003859 25057 25076 forelimb mesenchyme +T773 UBERON:0002102 25108 25116 forelimb +T774 UBERON:0000479 25117 25123 tissue +T775 PR:000000164 25150 25154 Bmp2 +T776 PR:000000165 25159 25163 Bmp4 +T777 PR:000013316 25168 25172 Prx1 +T778 NCBITaxon:33208 25178 25185 animals +T779 PR:000010687 25187 25191 Msx2 +T780 GO:0010467 25204 25213 expressed +T781 GO:0051216 25225 25237 chondrogenic +T782 UBERON:0009585 25238 25261 interdigital mesenchyme +T783 GO:0010467 25318 25328 expression +T784 UBERON:0003104 25358 25368 mesenchyme +T785 GO:0010467 25375 25385 expression +T786 PR:000010687 25389 25393 Msx2 +T787 PR:000000164 25418 25422 BMP2 +T788 PR:000000165 25424 25428 BMP4 +T789 UBERON:0002102 25439 25448 forelimbs +T790 GO:0051216 25471 25485 chondrogenesis +T791 UBERON:0002389 25528 25543 forelimb digits +T792 NCBITaxon:10088 25553 25557 mice +T793 UBERON:0002544 25649 25654 digit +T794 PR:000000034 25709 25712 BMP +T795 GO:0030509 25709 25722 BMP signaling +T796 GO:0051216 25764 25778 chondrogenesis +T797 UBERON:0002102 25805 25814 forelimbs +T798 UBERON:0002103 25837 25845 hindlimb +T799 UBERON:0002544 25933 25938 digit +T800 UBERON:0005418 25965 25977 hindlimb bud +T801 GO:0010467 26013 26023 expression +T802 PR:000010687 26027 26031 Msx2 +T803 UBERON:0004288 26075 26083 Skeletal +T804 UBERON:0002101 26103 26108 Limbs +T805 PR:000000034 26143 26146 BMP +T806 GO:0030509 26143 26156 BMP Signaling +T807 PR:000000034 26158 26162 BMPs +T808 GO:0001501 26209 26221 skeletogenic +T809 PR:000000034 26298 26301 BMP +T810 CHEBI:36357 26302 26311 molecules +T811 UBERON:0004288 26355 26363 skeletal +T812 UBERON:0004288 26403 26411 skeletal +T813 SO:0001023 26457 26464 alleles +T814 UBERON:0004765 26524 26541 skeletal elements +T815 PR:000000164 26572 26576 BMP2 +T816 PR:000000165 26578 26582 BMP4 +T817 PR:000000168 26587 26591 BMP7 +T818 PR:000000164 26595 26599 BMP2 +T819 PR:000000168 26604 26608 BMP7 +T820 GO:0051216 26630 26644 chondrogenesis +T821 GO:0001503 26649 26661 osteogenesis +T822 CHEBI:16866 26751 26763 Alizarin red +T823 UBERON:0000479 26781 26787 tissue +T824 UBERON:0008883 26892 26899 osteoid +T825 GO:0031012 26919 26925 matrix +T826 UBERON:0004765 26993 27010 skeletal elements +T827 PR:000000034 27028 27031 BMP +T828 NCBITaxon:33208 27042 27049 animals +T829 CL:0000138 27083 27095 chondrocytes +T830 GO:0010467 27096 27106 expressing +T831 PR:000003266 27107 27123 type II collagen +T832 CL:0000743 27125 27150 hypertrophic chondrocytes +T833 GO:0010467 27151 27161 expressing +T834 PR:000005693 27162 27177 type X collagen +T835 CL:0000062 27194 27205 osteoblasts +T836 GO:0010467 27206 27216 expressing +T837 PR:000003263 27217 27232 type I collagen +T838 UBERON:0002101 27369 27374 limbs +T839 PR:000000164 27404 27408 BMP2 +T840 PR:000000165 27413 27417 BMP4 +T841 UBERON:0004288 27452 27460 skeletal +T842 PR:000000164 27479 27483 BMP2 +T843 PR:000000165 27488 27492 BMP4 +T844 GO:0051216 27514 27526 Chondrogenic +T845 GO:0001502 27570 27595 chondrogenic condensation +T846 UBERON:0002544 27603 27609 digits +T847 PR:000000164 27633 27637 Bmp2 +T848 PR:000000165 27642 27646 Bmp4 +T849 PR:000013316 27651 27655 Prx1 +T850 UBERON:0004347 27661 27670 limb buds +T851 GO:0097617 27682 27695 hybridization +T852 PR:000015435 27703 27707 Sox9 +T853 UBERON:0004765 27824 27841 skeletal elements +T854 PR:000000164 27894 27898 Bmp2 +T855 PR:000000165 27903 27907 Bmp4 +T856 SO:0000673 27908 27919 transcripts +T857 GO:0010467 27953 27963 expression +T858 PR:000003266 27967 27973 Col II +T859 CL:0000138 27977 27988 chondrocyte +T860 PR:000000164 28016 28020 BMP2 +T861 PR:000000165 28022 28026 BMP4 +T862 UBERON:0004381 28156 28169 limb skeleton +T863 GO:0051216 28201 28215 chondrogenesis +T864 PR:000000164 28279 28283 BMP2 +T865 PR:000000165 28285 28289 BMP4 +T866 UBERON:0004347 28300 28309 limb buds +T867 UBERON:0007844 28359 28377 cartilage elements +T868 GO:0040007 28378 28382 grow +T869 UBERON:0000976 28524 28530 humeri +T870 GO:0051216 28558 28570 chondrogenic +T871 PR:000000164 28610 28614 BMP2 +T872 PR:000000165 28616 28620 BMP4 +T873 UBERON:0000976 28631 28638 humerus +T874 PR:000005693 28773 28788 type X collagen +T875 GO:0010467 28789 28799 expression +T876 PR:000000164 28846 28850 BMP2 +T877 PR:000000165 28852 28856 BMP4 +T878 UBERON:0002101 28867 28871 limb +T879 GO:0010467 28877 28884 express +T880 PR:000015561 28920 28931 osteopontin +T881 GO:0051216 29005 29019 chondrogenesis +T882 PR:000000164 29027 29031 BMP2 +T883 PR:000000165 29033 29037 BMP4 +T884 UBERON:0002101 29048 29053 limbs +T885 GO:0001503 29104 29113 ostegenic +T886 PR:000000034 29114 29118 BMPs +T887 GO:0097617 29143 29156 hybridization +T888 PR:000000167 29161 29165 Bmp6 +T889 PR:000000168 29170 29174 Bmp7 +T890 UBERON:0002101 29184 29189 limbs +T891 GO:0010467 29228 29238 expression +T892 PR:000000167 29242 29246 Bmp6 +T893 PR:000000168 29267 29271 Bmp7 +T894 UBERON:0000479 29326 29333 tissues +T895 GO:0001503 29376 29388 Osteogenesis +T896 PR:000000164 29414 29418 BMP2 +T897 PR:000000165 29420 29424 BMP4 +T898 UBERON:0004347 29435 29444 Limb Buds +T899 CL:0000743 29508 29533 hypertrophic chondrocytes +T900 UBERON:0002222 29554 29567 perichondrium +T901 GO:0001503 29633 29645 osteogenesis +T902 PR:000000164 29674 29678 BMP2 +T903 PR:000000165 29680 29684 BMP4 +T904 UBERON:0002101 29695 29700 limbs +T905 PR:000000164 29873 29877 BMP2 +T906 PR:000000165 29882 29886 BMP4 +T907 UBERON:0000976 29970 29977 humerus +T908 GO:0031012 30017 30023 matrix +T909 GO:0010467 30024 30031 express +T910 CL:0000062 30036 30046 osteoblast +T911 PR:000003263 30054 30059 Col I +T912 UBERON:4000088 30104 30125 mineralized cartilage +T913 UBERON:0006772 30149 30169;30174 30186 hypertrophic zone of ... growth plate +T914 CL:0000092 30217 30228 osteoclasts +T915 UBERON:0002484 30307 30325 bone marrow cavity +T916 CL:0007010 30378 30399 Osteoprogenitor cells +T917 UBERON:0008883 30459 30466 osteoid +T918 UBERON:0002483 30480 30495 trabecular bone +T919 UBERON:0002484 30503 30521 bone marrow cavity +T920 UBERON:0001439 30549 30562 cortical bone +T921 GO:0007567 30566 30571 birth +T922 UBERON:0002101 30617 30622 limbs +T923 PR:000000164 30636 30640 BMP2 +T924 PR:000000165 30645 30649 BMP4 +T925 UBERON:0010363 30691 30703 endochondral +T926 GO:0001944 30731 30746 vascularization +T927 PR:000000164 30757 30761 Bmp2 +T928 PR:000000165 30766 30770 Bmp4 +T929 PR:000013316 30775 30779 Prx1 +T930 UBERON:0002102 30785 30794 forelimbs +T931 CL:0000092 30800 30811 osteoclasts +T932 UBERON:4000088 30833 30854 mineralized cartilage +T933 UBERON:0002484 30926 30944 bone marrow cavity +T934 PR:000000164 30973 30977 BMP2 +T935 PR:000000165 30982 30986 BMP4 +T936 UBERON:0002371 30997 31008 bone marrow +T937 GO:0048539 30997 31018 bone marrow formation +T938 UBERON:0002483 31023 31038 trabecular bone +T939 GO:0007567 31073 31078 birth +T940 PR:000000164 31097 31101 BMP2 +T941 PR:000000165 31106 31110 BMP4 +T942 PR:000000164 31146 31150 Bmp2 +T943 PR:000000165 31155 31159 Bmp4 +T944 PR:000013316 31164 31168 Prx1 +T945 UBERON:0001474 31174 31179 bones +T946 GO:0007567 31183 31188 birth +T947 UBERON:0000922 31216 31223 embryos +T948 PR:000000164 31322 31326 BMP2 +T949 PR:000000165 31331 31335 BMP4 +T950 PR:000000034 31381 31385 BMPs +T951 PR:000000164 31425 31429 Bmp2 +T952 PR:000000165 31434 31438 Bmp4 +T953 PR:000013316 31443 31447 Prx1 +T954 NCBITaxon:10088 31453 31457 mice +T955 NCBITaxon:10088 31527 31531 mice +T956 CL:0000743 31585 31610 hypertrophic chondrocytes +T957 CL:0000062 31644 31655 osteoblasts +T958 UBERON:0001439 31678 31691 cortical bone +T959 UBERON:0004769 31699 31716 diaphyseal region +T960 UBERON:0010357 31761 31790 secondary ossification center +T961 GO:0001503 31771 31783 ossification +T962 UBERON:0010357 31858 31887 secondary ossification center +T963 GO:0001503 31868 31880 ossification +T964 UBERON:0002371 31941 31952 bone marrow +T965 GO:0048539 31941 31962 bone marrow formation +T966 UBERON:0002483 31997 32012 trabecular bone +T967 UBERON:0000479 32035 32041 tissue +T968 PR:000000164 32132 32136 BMP2 +T969 PR:000000165 32141 32145 BMP4 +T970 GO:0007567 32166 32171 birth +T971 UBERON:0004765 32210 32227 skeletal elements +T972 UBERON:0002101 32304 32309 limbs +T973 UBERON:0008187 32366 32396 site of hypertrophic cartilage +T974 UBERON:0002484 32413 32431 bone marrow cavity +T975 UBERON:0002483 32433 32448 trabecular bone +T976 UBERON:0001439 32453 32466 cortical bone +T977 CL:0000092 32496 32507 osteoclasts +T978 UBERON:4000088 32545 32566 mineralized cartilage +T979 UBERON:0000479 32599 32605 tissue +T980 UBERON:4000088 32660 32681 mineralized cartilage +T981 UBERON:0004769 32689 32706 diaphyseal region +T982 UBERON:0000464 32734 32738 void +T983 UBERON:0000464 32827 32831 void +T984 UBERON:0000479 32862 32869 tissues +T985 GO:0045453 32944 32962 resorption of bone +T986 UBERON:0000981 33014 33019 femur +T987 NCBITaxon:33208 33030 33037 animals +T988 CL:0000062 33130 33140 osteoblast +T989 PR:000000164 33164 33168 Bmp2 +T990 PR:000000165 33173 33177 Bmp4 +T991 PR:000013316 33182 33186 Prx1 +T992 NCBITaxon:10088 33192 33196 mice +T993 UBERON:0002101 33229 33233 limb +T994 CL:0000062 33311 33321 osteoblast +T995 PR:000000164 33348 33352 Bmp2 +T996 PR:000000165 33357 33361 Bmp4 +T997 PR:000013316 33366 33370 Prx1 +T998 UBERON:0002102 33376 33385 forelimbs +T999 CL:0000062 33399 33409 osteoblast +T1000 SO:0000704 33418 33422 gene +T1001 GO:0010467 33418 33433 gene expression +T1002 NCBITaxon:10088 33461 33465 mice +T1003 CL:0000062 33467 33478 osteoblasts +T1004 UBERON:0002483 33501 33511;33525 33529 trabecular ... bone +T1005 UBERON:0001439 33516 33529 cortical bone +T1006 GO:0010467 33530 33537 express +T1007 PR:000003263 33538 33553 type I collagen +T1008 PR:000014364 33555 33560 runx2 +T1009 PR:000015445 33566 33573 osterix +T1010 SO:0000704 33574 33579 genes +T1011 GO:0007567 33609 33614 birth +T1012 PR:000000164 33659 33663 BMP2 +T1013 PR:000000165 33668 33672 BMP4 +T1014 CL:0000057 33690 33702 fibroblastic +T1015 UBERON:4000088 33755 33776 mineralized cartilage +T1016 GO:0010467 33799 33806 express +T1017 PR:000003263 33818 33833 type I collagen +T1018 PR:000014364 33838 33843 runx2 +T1019 CL:0000062 33866 33877 osteoblasts +T1020 GO:0010467 33889 33899 expression +T1021 PR:000015445 33903 33910 osterix +T1022 CL:0000057 34045 34055 fibroblast +T1023 UBERON:0004769 34083 34093 bone shaft +T1024 CL:0007010 34113 34134 osteoprogenitor cells +T1025 CL:0000062 34171 34182 osteoblasts +T1026 PR:000000164 34201 34205 BMP2 +T1027 PR:000000165 34210 34214 BMP4 +T1028 SO:0000704 34237 34244 genetic +T1029 PR:000000034 34286 34289 Bmp +T1030 SO:0000704 34290 34295 genes +T1031 UBERON:0002101 34303 34307 limb +T1032 GO:0060173 34303 34319 limb development +T1033 PR:000000164 34340 34344 BMP2 +T1034 PR:000000165 34346 34350 BMP4 +T1035 PR:000000168 34356 34360 BMP7 +T1036 UBERON:0002544 34444 34449 digit +T1037 PR:000000164 34471 34475 BMP2 +T1038 PR:000000165 34480 34484 BMP4 +T1039 UBERON:0002544 34550 34555 digit +T1040 UBERON:0007688 34556 34563 anlagen +T1041 GO:0051216 34589 34603 chondrogenesis +T1042 GO:0051216 34625 34637 chondrogenic +T1043 PR:000000164 34698 34702 BMP2 +T1044 PR:000000165 34707 34711 BMP4 +T1045 PR:000000164 34715 34719 BMP2 +T1046 PR:000000168 34724 34728 BMP7 +T1047 PR:000000164 34749 34753 BMP2 +T1048 PR:000000165 34758 34762 BMP4 +T1049 GO:0001503 34803 34815 osteogenesis +T1050 PR:000000034 34818 34821 BMP +T1051 GO:0030509 34818 34831 BMP Signaling +T1052 PR:000000034 34881 34884 BMP +T1053 GO:0030509 34881 34894 BMP signaling +T1054 UBERON:0002544 34914 34919 digit +T1055 UBERON:0005414 34972 34975 ZPA +T1056 UBERON:0006012 35012 35024 interdigital +T1057 PR:000000164 35069 35073 BMP2 +T1058 GO:0046903 35077 35085 secreted +T1059 PR:000014841 35112 35115 SHH +T1060 UBERON:0004347 35137 35145 limb bud +T1061 PR:000014841 35206 35209 SHH +T1062 UBERON:0002544 35244 35255 limb digits +T1063 PR:000000164 35282 35286 Bmp2 +T1064 UBERON:0004347 35300 35308 limb bud +T1065 GO:0060174 35300 35320 limb bud development +T1066 UBERON:0002544 35348 35353 digit +T1067 PR:000000034 35374 35378 BMPs +T1068 UBERON:0005414 35488 35491 ZPA +T1069 PR:000000168 35549 35553 BMP7 +T1070 PR:000000164 35563 35567 BMP2 +T1071 PR:000000165 35580 35584 BMP4 +T1072 PR:000014841 35617 35620 SHH +T1073 PR:000000168 35645 35649 Bmp7 +T1074 GO:0010467 35650 35660 expression +T1075 PR:000000164 35683 35687 Bmp2 +T1076 NCBITaxon:10088 35701 35706 mouse +T1077 UBERON:0004347 35707 35715 limb bud +T1078 NCBITaxon:10088 35739 35743 mice +T1079 PR:000000164 35782 35786 Bmp2 +T1080 PR:000000168 35794 35798 Bmp7 +T1081 UBERON:0002544 35837 35842 digit +T1082 UBERON:0004300 35884 35897 final phalanx +T1083 UBERON:0006050 35901 35910 digit III +T1084 UBERON:0004300 35950 35965 final phalanges +T1085 UBERON:0009551 35978 35984;35991 35996 tip of ... digit +T1086 UBERON:0003221 36047 36056 phalanges +T1087 PR:000000034 36082 36085 BMP +T1088 GO:0030509 36082 36095 BMP signaling +T1089 PR:000000034 36131 36134 BMP +T1090 GO:0030509 36131 36144 BMP signaling +T1091 UBERON:0009585 36240 36263 interdigital mesenchyme +T1092 UBERON:0002544 36304 36310 digits +T1093 PR:000000034 36336 36339 BMP +T1094 PR:000000021 36351 36357 noggin +T1095 UBERON:0006012 36365 36377 interdigital +T1096 UBERON:0000479 36378 36384 tissue +T1097 UBERON:0002544 36400 36405 digit +T1098 UBERON:0003221 36438 36447 phalanges +T1099 UBERON:0002544 36538 36543 digit +T1100 PR:000000034 36574 36577 BMP +T1101 UBERON:0006012 36593 36607 interdigitally +T1102 PR:000000021 36700 36706 noggin +T1103 UBERON:0001048 36788 36797 primordia +T1104 UBERON:0004300 36801 36817 distal phalanges +T1105 PR:000000034 36823 36826 BMP +T1106 UBERON:0006012 36890 36902 interdigital +T1107 PR:000000034 36903 36906 BMP +T1108 GO:0030509 36903 36916 BMP signaling +T1109 PR:000000034 37010 37013 BMP +T1110 GO:0030509 37010 37023 BMP signaling +T1111 GO:0051216 37070 37084 chondrogenesis +T1112 PR:000000034 37174 37177 BMP +T1113 GO:0030509 37174 37187 BMP signaling +T1114 UBERON:0002544 37217 37222 digit +T1115 PR:000000034 37280 37283 BMP +T1116 UBERON:0002544 37331 37336 digit +T1117 UBERON:0002101 37387 37392 limbs +T1118 PR:000000164 37411 37415 BMP2 +T1119 PR:000000165 37420 37424 BMP4 +T1120 PR:000000164 37450 37454 BMP2 +T1121 PR:000000168 37459 37463 BMP7 +T1122 UBERON:0006012 37493 37505 interdigital +T1123 GO:0010467 37506 37516 expression +T1124 SO:0000359 37570 37576 floxed +T1125 PR:000000034 37577 37580 BMP +T1126 SO:0001023 37581 37588 alleles +T1127 PR:000000168 37664 37668 BMP7 +T1128 UBERON:0009585 37676 37699 interdigital mesenchyme +T1129 PR:000000164 37707 37711 BMP2 +T1130 PR:000000165 37713 37717 BMP4 +T1131 UBERON:0002101 37728 37733 limbs +T1132 PR:000000034 37792 37795 BMP +T1133 GO:0030509 37792 37805 BMP signaling +T1134 UBERON:0009585 37892 37915 interdigital mesenchyme +T1135 UBERON:0002544 37932 37937 digit +T1136 PR:000000164 37966 37970 Bmp2 +T1137 PR:000000165 37975 37979 Bmp4 +T1138 PR:000013316 37984 37988 Prx1 +T1139 UBERON:0002102 37994 38003 forelimbs +T1140 UBERON:5002544 38043 38052 digit ray +T1141 PR:000000034 38106 38109 BMP +T1142 GO:0030509 38106 38119 BMP signaling +T1143 PR:000000034 38212 38215 BMP +T1144 GO:0051216 38292 38306 chondrogenesis +T1145 PR:000000164 38319 38323 BMP2 +T1146 PR:000000165 38325 38329 BMP4 +T1147 UBERON:0002101 38340 38345 limbs +T1148 SO:0001023 38524 38531 alleles +T1149 UBERON:0004347 38935 38943 limb bud +T1150 PR:000000164 38947 38951 BMP2 +T1151 PR:000000165 38953 38957 BMP4 +T1152 PR:000000168 38963 38967 BMP7 +T1153 UBERON:0004356 38984 38987 AER +T1154 PR:000000034 39006 39010 BMPs +T1155 UBERON:0004356 39018 39021 AER +T1156 PR:000000165 39073 39077 Bmp4 +T1157 PR:000013316 39082 39086 Prx1 +T1158 NCBITaxon:10088 39092 39096 mice +T1159 PR:000000165 39181 39185 Bmp4 +T1160 SO:0001023 39198 39204 allele +T1161 PR:000013316 39209 39213 Prx1 +T1162 http://purl.obolibrary.org/obo/MONDO_0020927 39281 39302 postaxial polydactyly +T1163 UBERON:0002544 39431 39437 digits +T1164 UBERON:0002544 39494 39499 digit +T1165 NCBITaxon:10088 39528 39532 mice +T1166 SO:0001023 39578 39584 allele +T1167 SO:0001023 39605 39611 allele +T1168 SO:0001023 39644 39651 alleles +T1169 NCBITaxon:10088 39659 39663 mice +T1170 PR:000000165 39696 39700 BMP4 +T1171 UBERON:0004356 39710 39713 AER +T1172 UBERON:0003104 39750 39760 mesenchyme +T1173 SO:0001023 39784 39791 alleles +T1174 UBERON:0003104 39820 39830 mesenchyme +T1175 PR:000013316 39838 39842 Prx1 +T1176 SO:0000359 39972 39978 floxed +T1177 SO:0001023 39979 39986 alleles +T1178 PR:000000165 40048 40052 BMP4 +T1179 GO:0010467 40053 40063 expression +T1180 UBERON:0004347 40144 40153 limb buds +T1181 SO:0000704 40179 40186 genetic +T1182 PR:000013316 40241 40245 Prx1 +T1183 PR:000000034 40325 40328 BMP +T1184 GO:0030509 40325 40338 BMP signaling +T1185 UBERON:0006012 40342 40354 interdigital +T1186 GO:0006915 40355 40364 apoptosis +T1187 http://purl.obolibrary.org/obo/MONDO_0021002 40400 40410 syndactyly +T1188 PR:000000164 40418 40422 BMP2 +T1189 UBERON:0002101 40433 40438 limbs +T1190 UBERON:0006022 40491 40505 3/4 interdigit +T1191 GO:0006915 40551 40560 apoptosis +T1192 PR:000000164 40588 40592 BMP2 +T1193 PR:000000165 40594 40598 BMP4 +T1194 PR:000000168 40602 40606 BMP7 +T1195 PR:000000164 40610 40614 BMP2 +T1196 PR:000000168 40619 40623 BMP7 +T1197 PR:000000034 40648 40651 BMP +T1198 GO:0030509 40648 40661 BMP signaling +T1199 UBERON:0006012 40671 40683 interdigital +T1200 GO:0008219 40684 40694 cell death +T1201 PR:000000164 40736 40740 BMP2 +T1202 PR:000000165 40745 40749 BMP4 +T1203 UBERON:0000479 40775 40781 tissue +T1204 http://purl.obolibrary.org/obo/MONDO_0021002 40782 40792 syndactyly +T1205 UBERON:0006015 40800 40815;40824 40830 webbing between ... digits +T1206 PR:000000034 40841 40844 BMP +T1207 GO:0065007 40888 40898 regulating +T1208 UBERON:0006012 40899 40911 interdigital +T1209 GO:0006915 40912 40921 apoptosis +T1210 SO:0000704 40941 40948 genetic +T1211 PR:000000034 41002 41005 BMP +T1212 GO:0030509 41002 41015 BMP Signaling +T1213 UBERON:0004288 41020 41028 Skeletal +T1214 GO:0001501 41020 41040 Skeletal Development +T1215 PR:000000034 41105 41108 BMP +T1216 GO:0030509 41105 41118 BMP signaling +T1217 GO:0001502 41147 41172 chondrogenic condensation +T1218 GO:0051216 41227 41241 chondrogenesis +T1219 PR:000000164 41347 41351 BMP2 +T1220 PR:000000165 41356 41360 BMP4 +T1221 PR:000000164 41382 41386 BMP2 +T1222 PR:000000168 41391 41395 BMP7 +T1223 GO:0010467 41407 41417 expression +T1224 PR:000000166 41428 41432 BMP5 +T1225 PR:000000167 41434 41438 BMP6 +T1226 PR:000000168 41444 41448 BMP7 +T1227 PR:000000164 41468 41472 BMP2 +T1228 PR:000000165 41477 41481 BMP4 +T1229 GO:0010467 41490 41500 expression +T1230 PR:000000165 41504 41508 BMP4 +T1231 PR:000000166 41510 41514 BMP5 +T1232 PR:000000167 41520 41524 BMP6 +T1233 PR:000000164 41544 41548 BMP2 +T1234 PR:000000168 41553 41557 BMP7 +T1235 UBERON:0003104 41584 41595 mesenchymal +T1236 CL:0000138 41613 41624 chondrocyte +T1237 UBERON:0002101 41659 41663 limb +T1238 PR:000000034 41709 41712 BMP +T1239 GO:0030509 41709 41722 BMP signaling +T1240 GO:0051216 41726 41740 chondrogenesis +T1241 NCBITaxon:10088 41774 41778 mice +T1242 PR:000000035 41789 41793 Alk3 +T1243 PR:000000035 41795 41801 BmprIa +T1244 PR:000000036 41807 41811 Alk6 +T1245 PR:000000036 41813 41818 BmpIb +T1246 PR:000000034 41863 41866 BMP +T1247 GO:0030509 41863 41886 BMP signal transduction +T1248 UBERON:0004288 41932 41940 skeletal +T1249 UBERON:0001048 41941 41951 primordial +T1250 GO:0008283 41974 41987 proliferation +T1251 GO:0006915 42004 42013 apoptosis +T1252 UBERON:0004765 42031 42048 skeletal elements +T1253 PR:000000164 42052 42056 Bmp2 +T1254 PR:000000165 42061 42065 Bmp4 +T1255 PR:000013316 42070 42074 Prx1 +T1256 UBERON:0002101 42080 42085 limbs +T1257 NCBITaxon:10088 42134 42138 mice +T1258 UBERON:0004765 42241 42258 skeletal elements +T1259 GO:0008283 42300 42313 proliferation +T1260 GO:0006915 42330 42339 apoptosis +T1261 PR:000000164 42442 42446 BMP2 +T1262 PR:000000165 42448 42452 BMP4 +T1263 PR:000000168 42458 42462 BMP7 +T1264 PR:000000035 42512 42518 BmprIa +T1265 PR:000000036 42520 42526 BmprIb +T1266 PR:000000034 42594 42598 BMPs +T1267 GO:0001503 42626 42638 osteogenesis +T1268 NCBITaxon:10088 42661 42665 mice +T1269 PR:000000164 42765 42769 Bmp2 +T1270 PR:000000165 42773 42777 Bmp4 +T1271 SO:0000704 42778 42782 gene +T1272 NCBITaxon:10088 42810 42814 mice +T1273 PR:000000164 42839 42843 BMP2 +T1274 PR:000000168 42848 42852 BMP7 +T1275 UBERON:0002101 42902 42907 limbs +T1276 PR:000000164 42911 42915 Bmp2 +T1277 PR:000000165 42920 42924 Bmp4 +T1278 PR:000013316 42929 42933 Prx1 +T1279 NCBITaxon:10088 42939 42943 mice +T1280 PR:000015445 42985 42992 osterix +T1281 NCBITaxon:10088 43002 43006 mice +T1282 CL:0000062 43047 43059 osteoblastic +T1283 CL:0000057 43094 43112 fibroblastic cells +T1284 UBERON:4000088 43125 43146 mineralized cartilage +T1285 CL:0007010 43176 43197 osteoprogenitor cells +T1286 GO:0010467 43210 43217 express +T1287 PR:000015445 43218 43225 osterix +T1288 PR:000000164 43249 43253 BMP2 +T1289 PR:000000165 43258 43262 BMP4 +T1290 PR:000000164 43286 43290 BMP2 +T1291 PR:000000165 43295 43299 BMP4 +T1292 UBERON:0010363 43307 43319 endochondral +T1293 GO:0001958 43307 43332 endochondral ossification +T1294 PR:000015445 43350 43357 osterix +T1295 SO:0000704 43358 43362 gene +T1296 GO:0010467 43358 43373 gene expression +T1297 PR:000015445 43430 43437 osterix +T1298 GO:0010467 43438 43448 expression +T1299 NCBITaxon:10088 43477 43481 mice +T1300 PR:000000164 43520 43524 Bmp2 +T1301 PR:000000165 43529 43533 Bmp4 +T1302 SO:0000704 43534 43539 genes +T1303 GO:0010467 43628 43638 expression +T1304 PR:000000034 43654 43657 BMP +T1305 SO:0000704 43658 43663 genes +T1306 PR:000000034 43748 43751 BMP +T1307 GO:0030509 43748 43761 BMP signaling +T1308 GO:0001503 43835 43847 osteogenesis +T1309 GO:0001503 43860 43870 osteogenic +T1310 PR:000000034 43871 43874 BMP +T1311 CHEBI:36357 43875 43884 molecules +T1312 PR:000000164 43897 43901 BMP2 +T1313 PR:000000165 43906 43910 BMP4 +T1314 PR:000000166 43920 43924 BMP5 +T1315 PR:000000167 43926 43930 BMP6 +T1316 PR:000000168 43936 43940 BMP7 +T1317 GO:0010467 43946 43955 expressed +T1318 UBERON:0010363 43967 43979 endochondral +T1319 PR:000000034 44017 44020 BMP +T1320 CHEBI:36357 44021 44030 molecules +T1321 PR:000000164 44074 44078 BMP2 +T1322 PR:000000165 44083 44087 BMP4 +T1323 SO:0000704 44130 44137 genetic +T1324 PR:000000034 44181 44185 BMPs +T1325 PR:000000164 44212 44216 BMP2 +T1326 PR:000000165 44218 44222 BMP4 +T1327 PR:000000166 44224 44228 BMP5 +T1328 PR:000000167 44230 44234 BMP6 +T1329 PR:000000168 44240 44244 BMP7 +T1330 PR:000000067 44274 44278 Alk2 +T1331 PR:000000035 44280 44284 Alk3 +T1332 PR:000000036 44290 44294 Alk6 +T1333 PR:000000037 44300 44317 type II receptors +T1334 PR:000000037 44319 44326 BMP RII +T1335 PR:000000070 44328 44334 ActRII +T1336 PR:000000071 44340 44347 ActRIIb +T1337 GO:0032991 44364 44371 complex +T1338 PR:000000164 44412 44416 BMP2 +T1339 PR:000000165 44418 44422 BMP4 +T1340 PR:000000166 44424 44428 BMP5 +T1341 PR:000000167 44430 44434 BMP6 +T1342 PR:000000168 44440 44444 BMP7 +T1343 PR:000000038 44491 44518 BMP receptor–specific Smads +T1344 PR:000000034 44575 44578 BMP +T1345 UBERON:0004288 44610 44618 skeletal +T1346 PR:000000034 44641 44645 BMPs +T1347 PR:000029189 44688 44691 AKT +T1348 GO:0001503 44758 44768 osteogenic +T1349 PR:000000034 44769 44773 BMPs +T1350 SO:0001023 44926 44932 allele +T1351 PR:000000164 44943 44947 Bmp2 +T1352 PR:000000165 44951 44955 Bmp4 +T1353 CL:0000062 44971 44981 osteoblast +T1354 PR:000000164 45020 45024 Bmp2 +T1355 PR:000000165 45029 45033 Bmp4 +T1356 PR:000013316 45038 45042 Prx1 +T1357 NCBITaxon:10088 45048 45052 mice +T1358 PR:000000034 45169 45172 BMP +T1359 GO:0030509 45169 45182 BMP signaling +T1360 PR:000000164 45210 45214 BMP2 +T1361 PR:000000165 45219 45223 BMP4 +T1362 PR:000000164 45296 45300 BMP2 +T1363 PR:000000165 45305 45309 BMP4 +T1364 GO:0002076 45337 45355 osteoblastogenesis +T1365 GO:0001501 45373 45387 skeletogenesis +T1366 PR:000000164 45403 45407 BMP2 +T1367 PR:000000165 45412 45416 BMP4 +T1368 GO:0002076 45438 45456 osteoblastogenesis +T1369 GO:0051216 45482 45496 chondrogenesis +T1370 PR:000000164 45547 45551 BMP2 +T1371 PR:000000165 45556 45560 BMP4 +T1372 GO:0051216 45633 45647 chondrogenesis +T1373 CHEBI:33893 45693 45701 Reagents +T1374 CHEBI:27338 45704 45710 Xylene +T1375 CHEBI:16397 45712 45721 formamide +T1376 CHEBI:36610 45727 45743 acetic anhydride +T1377 CHEBI:16866 45810 45822 Alizarin red +T1378 CHEBI:53248 45860 45880 polyvinylpyrrolidone +T1379 CHEBI:18320 45915 45929 dithiothreitol +T1380 CHEBI:10545 46022 46030 Electron +T1381 CHEBI:34674 46078 46093 Dextran sulfate +T1382 PR:000000164 46166 46170 Bmp2 +T1383 PR:000000165 46175 46179 Bmp4 +T1384 SO:0001023 46197 46203 allele +T1385 NCBITaxon:10088 46206 46210 Mice +T1386 SO:0000359 46220 46226 floxed +T1387 PR:000000164 46227 46231 Bmp2 +T1388 SO:0001023 46232 46238 allele +T1389 CHEBI:52217 46329 46344 Pharmaceuticals +T1390 NCBITaxon:10088 46355 46359 mice +T1391 SO:0000346 46361 46371 LoxP sites +T1392 SO:0000010 46409 46423 protein-coding +T1393 SO:0001215 46417 46438 coding region in exon +T1394 PR:000000164 46448 46452 Bmp2 +T1395 SO:0000704 46453 46457 gene +T1396 PR:000000165 46473 46477 Bmp4 +T1397 SO:0001023 46490 46496 allele +T1398 PR:000013316 46529 46533 Prx1 +T1399 SO:0000902 46539 46548 transgene +T1400 PR:000000164 46589 46593 Bmp2 +T1401 PR:000000165 46595 46599 Bmp4 +T1402 NCBITaxon:10088 46619 46623 mice +T1403 PR:000000164 46628 46632 Bmp2 +T1404 PR:000000168 46646 46650 Bmp7 +T1405 NCBITaxon:10088 46658 46662 mice +T1406 PR:000000164 46665 46669 Bmp2 +T1407 NCBITaxon:33208 46673 46680 animals +T1408 PR:000000165 46699 46703 Bmp4 +T1409 NCBITaxon:33208 46707 46714 animals +T1410 PR:000000164 46727 46731 Bmp2 +T1411 PR:000000165 46736 46740 Bmp4 +T1412 NCBITaxon:33208 46744 46751 animals +T1413 NCBITaxon:33208 46759 46766 animals +T1414 PR:000000164 46785 46789 Bmp2 +T1415 PR:000000165 46796 46800 Bmp4 +T1416 NCBITaxon:33208 46804 46811 animals +T1417 PR:000000164 46824 46828 Bmp2 +T1418 PR:000000165 46833 46837 Bmp4 +T1419 PR:000000164 46844 46848 Bmp2 +T1420 PR:000000165 46853 46857 Bmp4 +T1421 NCBITaxon:33208 46861 46868 animals +T1422 PR:000000164 46884 46888 Bmp2 +T1423 PR:000000165 46893 46897 Bmp4 +T1424 PR:000000164 46904 46908 Bmp2 +T1425 PR:000000165 46913 46917 Bmp4 +T1426 NCBITaxon:33208 46921 46928 animals +T1427 PR:000000164 46970 46974 Bmp2 +T1428 PR:000000165 46979 46983 Bmp4 +T1429 NCBITaxon:33208 46987 46994 animals +T1430 PR:000000164 46996 47000 Bmp2 +T1431 PR:000000165 47005 47009 Bmp4 +T1432 PR:000013316 47053 47057 Prx1 +T1433 SO:0000902 47063 47072 transgene +T1434 PR:000000164 47085 47089 Bmp2 +T1435 PR:000000165 47094 47098 Bmp4 +T1436 PR:000013316 47103 47107 Prx1 +T1437 NCBITaxon:33208 47113 47120 animals +T1438 PR:000000164 47122 47126 Bmp2 +T1439 PR:000000165 47131 47135 Bmp4 +T1440 PR:000013316 47140 47144 Prx1 +T1441 PR:000000164 47174 47178 Bmp2 +T1442 PR:000000165 47183 47187 Bmp4 +T1443 PR:000000164 47211 47215 Bmp2 +T1444 PR:000000165 47220 47224 Bmp4 +T1445 PR:000013316 47229 47233 Prx1 +T1446 NCBITaxon:33208 47239 47246 animals +T1447 PR:000000164 47248 47252 Bmp2 +T1448 PR:000000165 47257 47261 Bmp4 +T1449 NCBITaxon:10088 47265 47269 mice +T1450 SO:0000359 47279 47285 floxed +T1451 SO:0001023 47286 47293 alleles +T1452 PR:000000034 47301 47304 Bmp +T1453 SO:0000704 47305 47310 genes +T1454 PR:000013316 47319 47323 Prx1 +T1455 NCBITaxon:33208 47363 47370 animals +T1456 SO:0000112 47406 47413 primers +T1457 PR:000000164 47491 47495 Bmp2 +T1458 SO:0000112 47544 47551 primers +T1459 PR:000000165 47598 47602 Bmp4 +T1460 SO:0001026 47627 47634 genomic +T1461 UBERON:0000922 47644 47653 embryonic +T1462 UBERON:0007023 47657 47662 adult +T1463 UBERON:0002415 47663 47667 tail +T1464 PR:000000164 47673 47677 Bmp2 +T1465 SO:0000359 47683 47689 floxed +T1466 SO:0001023 47690 47696 allele +T1467 SO:0000028 47716 47718 bp +T1468 SO:0001023 47748 47754 allele +T1469 SO:0000028 47774 47776 bp +T1470 PR:000000165 47790 47794 Bmp4 +T1471 SO:0000359 47800 47806 floxed +T1472 SO:0001023 47807 47813 allele +T1473 SO:0000028 47833 47835 bp +T1474 SO:0001023 47865 47871 allele +T1475 SO:0000028 47891 47893 bp +T1476 NCBITaxon:10088 47926 47931 mouse +T1477 NCBITaxon:33208 47998 48004 Animal +T1478 PR:000000164 48081 48085 Bmp2 +T1479 PR:000000168 48110 48114 BMP7 +T1480 PR:000000164 48180 48184 Bmp2 +T1481 PR:000000165 48186 48190 Bmp4 +T1482 SO:0001023 48210 48216 allele +T1483 PR:000000168 48262 48266 Bmp7 +T1484 SO:0001023 48341 48347 allele +T1485 PR:000000168 48377 48381 Bmp7 +T1486 NCBITaxon:10088 48385 48389 mice +T1487 GO:0007567 48410 48415 birth +T1488 UBERON:0002113 48423 48429 kidney +T1489 http://purl.obolibrary.org/obo/MONDO_0001106 48423 48437 kidney failure +T1490 SO:0001023 48501 48507 allele +T1491 PR:000000164 48516 48520 Bmp2 +T1492 PR:000000168 48525 48529 Bmp7 +T1493 PR:000000164 48583 48587 Bmp2 +T1494 PR:000000168 48592 48596 Bmp7 +T1495 PR:000013316 48601 48605 Prx1 +T1496 PR:000000164 48622 48626 Bmp2 +T1497 PR:000000168 48631 48635 Bmp7 +T1498 PR:000000164 48652 48656 Bmp2 +T1499 PR:000000168 48661 48665 Bmp7 +T1500 PR:000013316 48670 48674 Prx1 +T1501 PR:000000164 48687 48691 Bmp2 +T1502 PR:000000168 48696 48700 Bmp7 +T1503 PR:000013316 48705 48709 Prx1 +T1504 PR:000000168 48795 48799 Bmp7 +T1505 SO:0000112 48835 48842 primers +T1506 PR:000000168 48844 48848 Bmp7 +T1507 PR:000000168 48892 48896 Bmp7 +T1508 PR:000000168 49027 49031 Bmp7 +T1509 PR:000000168 49043 49047 Bmp7 +T1510 SO:0000028 49086 49088 bp +T1511 SO:0000006 49089 49100 PCR product +T1512 PR:000033987 49117 49121 lacZ +T1513 PR:000000168 49179 49183 Bmp7 +T1514 SO:0000112 49219 49225 primer +T1515 SO:0005853 49256 49264 cassette +T1516 SO:0000006 49298 49309 PCR product +T1517 PR:000033987 49319 49323 lacZ +T1518 UBERON:0004288 49346 49354 Skeletal +T1519 NCBITaxon:33208 49458 49465 animals +T1520 CHEBI:16526 49484 49498 carbon dioxide +T1521 UBERON:0002075 49506 49513 viscera +T1522 UBERON:0001013 49519 49534 adipose tissues +T1523 GO:0016265 49565 49570 death +T1524 CHEBI:16236 49602 49609 ethanol +T1525 CHEBI:15347 49647 49654 acetone +T1526 UBERON:0000922 49697 49704 embryos +T1527 NCBITaxon:33208 49743 49750 animals +T1528 CHEBI:16866 49807 49819 Alizarin red +T1529 CHEBI:60004 49830 49837 mixture +T1530 CHEBI:15366 49849 49860 acetic acid +T1531 CHEBI:16236 49869 49876 ethanol +T1532 NCBITaxon:33208 49890 49897 animals +T1533 UBERON:0004288 50068 50077 skeletons +T1534 CHEBI:32035 50094 50097 KOH +T1535 CHEBI:75958 50125 50133 solution +T1536 UBERON:0004288 50162 50171 skeletons +T1537 CHEBI:17754 50199 50207 glycerol +T1538 CHEBI:51739 50227 50242 Acridine orange +T1539 CL:0000445 50256 50271 apoptotic cells +T1540 CHEBI:51739 50301 50316 acridine orange +T1541 UBERON:0000922 50362 50369 embryos +T1542 CHEBI:75958 50402 50410 solution +T1543 CHEBI:51739 50414 50429 acridine orange +T1544 UBERON:0000922 50477 50484 Embryos +T1545 GO:0097617 50589 50602 hybridization +T1546 GO:0097617 50613 50626 hybridization +T1547 UBERON:0000922 50645 50652 embryos +T1548 GO:0097617 50706 50720 hybridizations +T1549 GO:0097617 50768 50782 hybridizations +T1550 GO:0097617 50845 50858 hybridization +T1551 NCBITaxon:33208 50946 50953 animals +T1552 CHEBI:9754 50979 50983 Tris +T1553 CHEBI:53248 51020 51040 polyvinylpyrrolidone +T1554 CHEBI:16236 51115 51122 ethanol +T1555 CHEBI:27338 51142 51148 xylene +T1556 CHEBI:51686 51221 51232 hematoxylin +T1557 CHEBI:51686 51244 51245 H +T1558 GO:0097617 51316 51329 hybridization +T1559 CHEBI:42098 51335 51346 digoxigenin +T1560 GO:0097617 51411 51424 hybridization +T1561 CHEBI:27338 51550 51556 xylene +T1562 CHEBI:16236 51589 51596 ethanol +T1563 MOP:0000030 51833 51843 acetylated +T1564 CHEBI:75958 51851 51859 solution +T1565 CHEBI:17883 51876 51893 hydrochloric acid +T1566 CHEBI:28621 51901 51917 triethanol amine +T1567 CHEBI:36610 51929 51945 acetic anhydride +T1568 CHEBI:16236 52040 52047 ethanol +T1569 GO:0097617 52063 52076 Hybridization +T1570 CHEBI:75958 52120 52128 solution +T1571 CHEBI:16397 52144 52153 formamide +T1572 CHEBI:34674 52159 52174 dextran sulfate +T1573 CHEBI:75958 52190 52198 solution +T1574 CHEBI:26710 52206 52221 sodium chloride +T1575 CHEBI:9754 52230 52234 Tris +T1576 CHEBI:18320 52273 52287 dithiothreitol +T1577 CHEBI:8984 52295 52298 SDS +T1578 CHEBI:37983 52317 52320 35S +T1579 GO:0097617 52410 52423 hybridization +T1580 CHEBI:75958 52453 52461 solution +T1581 CHEBI:18320 52490 52504 dithiothreitol +T1582 CHEBI:75958 52541 52549 solution +T1583 CHEBI:16397 52565 52574 formamide +T1584 CHEBI:18320 52594 52608 dithiothreitol +T1585 CHEBI:75958 52643 52651 solution +T1586 CHEBI:9754 52695 52699 Tris +T1587 CHEBI:26710 52723 52738 sodium chloride +T1588 CHEBI:16397 52803 52812 formamide +T1589 CHEBI:18320 52832 52846 dithiothreitol +T1590 CHEBI:18320 52922 52936 dithiothreitol +T1591 CHEBI:18320 52977 52991 dithiothreitol +T1592 CHEBI:51686 53182 53183 H +T1593 CHEBI:37983 53191 53194 35S +T1594 SO:0000155 53237 53245 plasmids +T1595 PR:000003263 53255 53270 type I collagen +T1596 PR:000014364 53277 53282 runx2 +T1597 PR:000015561 53289 53300 osteopontin +T1598 PR:000015445 53311 53318 osterix +T1599 SO:0000077 53335 53344 antisense +T1600 SO:0000155 53385 53393 plasmids +T1601 GO:0051216 53502 53514 Chondrogenic +T1602 PR:000000164 53534 53538 Bmp2 +T1603 PR:000000165 53543 53547 Bmp4 +T1604 PR:000013316 53552 53556 Prx1 +T1605 NCBITaxon:33208 53562 53569 Animals +T1606 PR:000000164 53580 53584 BMP2 +T1607 PR:000000165 53586 53590 BMP4 +T1608 UBERON:0002101 53601 53606 limbs +T1609 UBERON:0002471 53626 53634 zeugopod +T1610 UBERON:0002472 53675 53683 stylopod +T1611 PR:000000164 53711 53715 Bmp2 +T1612 PR:000000165 53720 53724 Bmp4 +T1613 PR:000013316 53729 53733 Prx1 +T1614 UBERON:0000922 53739 53746 embryos +T1615 CHEBI:51686 53752 53763 Hematoxylin +T1616 UBERON:0002102 53797 53805 forelimb +T1617 CHEBI:16866 53824 53836 Alizarin red +T1618 UBERON:0004288 53845 53854 skeletons +T1619 UBERON:0002102 53869 53877 forelimb +T1620 UBERON:0002103 53888 53896 hindlimb +T1621 UBERON:0002471 53935 53943 zeugopod +T1622 UBERON:0002472 53948 53956 stylopod +T1623 PR:000000164 53960 53964 BMP2 +T1624 PR:000000165 53966 53970 BMP4 +T1625 UBERON:0002101 53981 53986 limbs +T1626 CHEBI:51739 53999 54014 Acridine orange +T1627 UBERON:0002101 54023 54028 limbs +T1628 PR:000000164 54058 54062 Bmp2 +T1629 PR:000000165 54067 54071 Bmp4 +T1630 PR:000013316 54076 54080 Prx1 +T1631 UBERON:0000922 54086 54093 embryos +T1632 CHEBI:51739 54110 54125 Acridine orange +T1633 UBERON:0002103 54134 54143 hindlimbs +T1634 PR:000000164 54173 54177 Bmp2 +T1635 PR:000000165 54182 54186 Bmp4 +T1636 PR:000013316 54191 54195 Prx1 +T1637 UBERON:0000922 54205 54212 embryos +T1638 PR:000000164 54239 54243 BMP2 +T1639 PR:000000165 54248 54252 BMP4 +T1640 UBERON:0002101 54281 54285 limb +T1641 PR:000015435 54328 54332 Sox9 +T1642 UBERON:0000922 54341 54350 embryonic +T1643 UBERON:0004347 54351 54360 limb buds +T1644 UBERON:0000922 54398 54405 embryos +T1645 UBERON:0000922 54436 54443 embryos +T1646 PR:000000164 54464 54468 Bmp2 +T1647 PR:000000165 54473 54477 Bmp4 +T1648 PR:000013316 54482 54486 Prx1 +T1649 UBERON:0000922 54492 54499 embryos +T1650 UBERON:0002102 54518 54527 Forelimbs +T1651 UBERON:0002103 54539 54548 hindlimbs +T1652 GO:0097617 54569 54583 hybridizations +T1653 PR:000003266 54589 54595 Col II +T1654 PR:000000164 54657 54661 Bmp2 +T1655 PR:000000165 54666 54670 Bmp4 +T1656 PR:000013316 54675 54679 Prx1 +T1657 UBERON:0000922 54689 54696 embryos +T1658 PR:000000167 54705 54709 Bmp6 +T1659 GO:0010467 54715 54725 expression +T1660 PR:000000164 54752 54756 Bmp2 +T1661 PR:000000165 54761 54765 Bmp4 +T1662 PR:000013316 54770 54774 Prx1 +T1663 PR:000000167 54791 54795 Bmp6 +T1664 PR:000000164 54848 54852 Bmp2 +T1665 PR:000000165 54857 54861 Bmp4 +T1666 PR:000013316 54866 54870 Prx1 +T1667 UBERON:0000922 54880 54889 embryonic +T1668 UBERON:0002102 54890 54899 forelimbs +T1669 UBERON:0004288 55024 55032 Skeletal +T1670 PR:000000164 55079 55083 BMP2 +T1671 UBERON:0000981 55131 55137 femurs +T1672 PR:000000164 55143 55147 Bmp2 +T1673 NCBITaxon:33208 55151 55158 animals +T1674 UBERON:0000981 55196 55202 femurs +T1675 NCBITaxon:33208 55216 55223 animals +T1676 GO:0097617 55224 55234 hybridized +T1677 PR:000003266 55240 55246 Col II +T1678 PR:000005693 55252 55257 Col X +T1679 PR:000003263 55267 55272 Col I +T1680 UBERON:0000981 55337 55343 femurs +T1681 PR:000013316 55358 55362 Prx1 +T1682 NCBITaxon:33208 55368 55375 animals +T1683 UBERON:0000981 55413 55419 femurs +T1684 PR:000000164 55425 55429 Bmp2 +T1685 PR:000013316 55434 55438 Prx1 +T1686 NCBITaxon:33208 55444 55451 animals +T1687 GO:0097617 55452 55462 hybridized +T1688 PR:000003266 55468 55474 Col II +T1689 PR:000005693 55480 55485 Col X +T1690 PR:000003263 55495 55500 Col I +T1691 PR:000000164 55581 55585 BMP2 +T1692 PR:000000165 55587 55591 BMP4 +T1693 UBERON:0004381 55602 55615 Limb Skeleton +T1694 CHEBI:16866 55646 55658 Alizarin red +T1695 UBERON:0001440 55667 55689 skeletons of forelimbs +T1696 UBERON:0001441 55667 55679;55700 55709 skeletons of ... hindlimbs +T1697 NCBITaxon:33208 55770 55777 animals +T1698 NCBITaxon:33208 55803 55810 animals +T1699 PR:000000164 55826 55830 Bmp2 +T1700 PR:000000165 55835 55839 Bmp4 +T1701 PR:000013316 55844 55848 Prx1 +T1702 NCBITaxon:33208 55854 55861 animals +T1703 UBERON:0002101 55867 55872 limbs +T1704 PR:000000164 55876 55880 Bmp2 +T1705 PR:000000165 55885 55889 Bmp4 +T1706 PR:000013316 55894 55898 Prx1 +T1707 NCBITaxon:33208 55908 55915 animals +T1708 NCBITaxon:33208 55967 55973 animal +T1709 UBERON:0000981 56033 56038 femur +T1710 GO:0001503 56171 56183 Osteogenesis +T1711 NCBITaxon:10088 56194 56198 Mice +T1712 SO:0001023 56221 56228 Alleles +T1713 PR:000000164 56232 56236 Bmp2 +T1714 PR:000000165 56241 56245 Bmp4 +T1715 UBERON:0000981 56291 56297 femurs +T1716 UBERON:0007023 56303 56308 adult +T1717 PR:000000164 56318 56322 Bmp2 +T1718 PR:000000165 56327 56331 Bmp4 +T1719 PR:000013316 56336 56340 Prx1 +T1720 PR:000000164 56351 56355 Bmp2 +T1721 PR:000000165 56360 56364 Bmp4 +T1722 PR:000013316 56369 56373 Prx1 +T1723 NCBITaxon:33208 56379 56386 animals +T1724 NCBITaxon:10088 56509 56514 mouse +T1725 PR:000000165 56712 56716 Bmp4 +T1726 SO:0001023 56729 56735 allele +T1727 PR:000000168 56783 56787 Bmp7 +T1728 SO:0001023 56797 56803 allele +T1729 GO:0007613 56946 56952 memory +T1730 UBERON:0004356 57140 57143 AER +T1731 UBERON:0004356 57146 57169 apical ectodermal ridge +T1732 PR:000000034 57171 57174 BMP +T1733 GO:0060349 57177 57195 bone morphogenetic +T1734 PR:000000034 57177 57203 bone morphogenetic protein +T1735 UBERON:0000922 57209 57218 embryonic +T1736 CL:0000057 57230 57240 fibroblast +T1737 PR:000014841 57256 57259 SHH +T1738 PR:000014841 57262 57276 Sonic Hedgehog +T1739 PR:000014841 57278 57281 Shh +T1740 PR:000014841 57284 57298 sonic hedgehog +T1741 UBERON:0005414 57300 57303 ZPA +T1742 UBERON:0005414 57306 57333 zone of polarizing activity +T1743 SO:0001023 57368 57375 Allelic +T1744 SO:0001023 57379 57386 allelic +T1745 PR:000000034 57397 57400 BMP +T1746 UBERON:0002101 57411 57416 Limbs +T1747 PR:000013316 57424 57428 Prx1 +T1748 PR:000000164 57457 57461 Bmp2 +T1749 PR:000000165 57466 57470 Bmp4 +T1750 SO:0001023 57483 57490 alleles +T1751 UBERON:0002101 57498 57503 limbs +T1752 PR:000000164 57505 57509 Bmp2 +T1753 PR:000000165 57524 57528 Bmp4 +T1754 GO:0097617 57564 57577 hybridization +T1755 UBERON:0002101 57585 57589 limb +T1756 PR:000000164 57609 57613 Bmp2 +T1757 PR:000013316 57618 57622 Prx1 +T1758 UBERON:0002102 57636 57645 forelimbs +T1759 NCBITaxon:10088 57657 57662 mouse +T1760 UBERON:0000922 57663 57670 embryos +T1761 UBERON:0003104 57672 57683 Mesenchymal +T1762 GO:0010467 57684 57694 expression +T1763 PR:000000164 57713 57717 Bmp2 +T1764 PR:000000164 57734 57738 Bmp2 +T1765 PR:000013316 57743 57747 Prx1 +T1766 UBERON:0000922 57753 57759 embryo +T1767 UBERON:0004356 57770 57773 AER +T1768 GO:0010467 57774 57784 expression +T1769 PR:000000164 57812 57816 Bmp2 +T1770 UBERON:0004347 57880 57888 limb bud +T1771 PR:000000165 57941 57945 Bmp4 +T1772 PR:000013316 57950 57954 Prx1 +T1773 UBERON:0002102 57968 57977 forelimbs +T1774 NCBITaxon:10088 57989 57994 mouse +T1775 UBERON:0000922 57995 58002 embryos +T1776 UBERON:0003104 58004 58015 Mesenchymal +T1777 GO:0010467 58016 58026 expression +T1778 PR:000000165 58046 58050 Bmp4 +T1779 PR:000000165 58067 58071 Bmp4 +T1780 PR:000013316 58076 58080 Prx1 +T1781 UBERON:0000922 58086 58092 embryo +T1782 UBERON:0004356 58103 58106 AER +T1783 GO:0010467 58107 58117 expression +T1784 PR:000000165 58145 58149 Bmp4 +T1785 PR:000000164 58180 58184 BMP2 +T1786 PR:000000165 58189 58193 BMP4 +T1787 UBERON:0004381 58217 58230 limb skeletal +T1788 UBERON:0004288 58258 58267 skeletons +T1789 NCBITaxon:33208 58281 58288 animals +T1790 CHEBI:16866 58318 58330 Alizarin red +T1791 UBERON:0002102 58338 58347 Forelimbs +T1792 UBERON:0002103 58355 58364 hindlimbs +T1793 PR:000000164 58397 58401 Bmp2 +T1794 PR:000013316 58406 58410 Prx1 +T1795 PR:000000165 58427 58431 Bmp4 +T1796 PR:000013316 58436 58440 Prx1 +T1797 PR:000000168 58457 58461 Bmp7 +T1798 PR:000000164 58477 58481 Bmp2 +T1799 PR:000000165 58486 58490 Bmp4 +T1800 PR:000013316 58495 58499 Prx1 +T1801 PR:000000164 58516 58520 Bmp2 +T1802 PR:000000165 58525 58529 Bmp4 +T1803 PR:000013316 58534 58538 Prx1 +T1804 PR:000000164 58555 58559 Bmp2 +T1805 PR:000000165 58564 58568 Bmp4 +T1806 PR:000013316 58573 58577 Prx1 +T1807 PR:000000164 58594 58598 Bmp2 +T1808 PR:000000168 58603 58607 Bmp7 +T1809 PR:000013316 58613 58617 Prx1 +T1810 UBERON:0006849 58671 58678 scapula +T1811 UBERON:0001446 58715 58721 fibula +T1812 UBERON:0001465 58741 58745 knee +T1813 UBERON:0003221 58793 58800 phalanx +T1814 UBERON:0006050 58804 58813 digit III +T1815 PR:000000034 58839 58842 BMP +T1816 GO:0030509 58839 58852 BMP Signaling +T1817 UBERON:0006012 58860 58872 Interdigital +T1818 http://purl.obolibrary.org/obo/MONDO_0021002 58873 58883 Syndactyly +T1819 UBERON:0002102 58891 58899 Forelimb +T1820 UBERON:0007023 58903 58908 adult +T1821 PR:000000164 58927 58931 Bmp2 +T1822 PR:000013316 58936 58940 Prx1 +T1823 NCBITaxon:10088 58946 58951 mouse +T1824 UBERON:0002103 58960 58969 hindlimbs +T1825 NCBITaxon:10088 58995 59000 mouse +T1826 PR:000000164 59013 59017 Bmp2 +T1827 PR:000000165 59022 59026 Bmp4 +T1828 PR:000013316 59031 59035 Prx1 +T1829 NCBITaxon:10088 59045 59050 mouse +T1830 UBERON:0000479 59086 59092 tissue +T1831 http://purl.obolibrary.org/obo/MONDO_0021002 59093 59103 syndactyly +T1832 PR:000000164 59107 59111 Bmp2 +T1833 PR:000013316 59116 59120 Prx1 +T1834 NCBITaxon:10088 59126 59131 mouse +T1835 PR:000000164 59158 59162 Bmp2 +T1836 PR:000000165 59167 59171 Bmp4 +T1837 PR:000013316 59176 59180 Prx1 +T1838 CHEBI:51739 59209 59224 acridine orange +T1839 UBERON:0002103 59233 59242 hindlimbs +T1840 NCBITaxon:10088 59252 59257 mouse +T1841 UBERON:0000922 59258 59265 embryos +T1842 CHEBI:51739 59267 59282 Acridine orange +T1843 CHEBI:51739 59413 59428 acridine orange +T1844 CL:0000445 59437 59452 apoptotic cells +T1845 UBERON:0009585 59460 59483 interdigital mesenchyme +T1846 UBERON:0004356 59530 59533 AER +T1847 PR:000007499 59546 59550 Fgf8 +T1848 GO:0010467 59556 59566 expression +T1849 UBERON:0002103 59574 59583 hindlimbs +T1850 PR:000000164 59611 59615 Bmp2 +T1851 PR:000000165 59620 59624 Bmp4 +T1852 PR:000013316 59629 59633 Prx1 +T1853 UBERON:0000922 59643 59650 embryos +T1854 PR:000007499 59687 59691 Fgf8 +T1855 GO:0010467 59697 59707 expression +T1856 UBERON:0002101 59742 59747 Limbs +T1857 PR:000000034 59787 59790 BMP +T1858 CHEBI:36357 59791 59800 Molecules +T1859 PR:000000165 59808 59812 Bmp4 +T1860 GO:0010467 59813 59823 expression +T1861 UBERON:0004347 59827 59836 limb buds +T1862 PR:000000164 59872 59876 Bmp2 +T1863 PR:000000168 59881 59885 Bmp7 +T1864 PR:000013316 59891 59895 Prx1 +T1865 NCBITaxon:10088 59911 59916 mouse +T1866 UBERON:0000922 59917 59924 embryos +T1867 UBERON:0002102 59936 59945 Forelimbs +T1868 UBERON:0002103 59957 59966 hindlimbs +T1869 PR:000014841 59975 59978 Shh +T1870 PR:000007499 59993 59997 Fgf8 +T1871 GO:0010467 60008 60018 expression +T1872 UBERON:0002102 60028 60037 forelimbs +T1873 UBERON:0002103 60042 60051 hindlimbs +T1874 UBERON:0000922 60087 60094 embryos +T1875 PR:000000164 60106 60110 Bmp2 +T1876 PR:000000165 60115 60119 Bmp4 +T1877 PR:000013316 60124 60128 Prx1 +T1878 UBERON:0000922 60134 60141 embryos +T1879 PR:000015435 60150 60154 Sox9 +T1880 PR:000010687 60165 60169 Msx2 +T1881 GO:0010467 60176 60186 expression +T1882 PR:000000164 60220 60224 Bmp2 +T1883 PR:000000165 60229 60233 Bmp4 +T1884 PR:000013316 60238 60242 Prx1 +T1885 NCBITaxon:10088 60248 60253 mouse +T1886 UBERON:0000922 60254 60263 embryonic +T1887 UBERON:0002102 60264 60273 forelimbs +T1888 UBERON:0002103 60288 60297 hindlimbs +T1889 PR:000014841 60319 60322 Shh +T1890 GO:0010467 60323 60333 expression +T1891 PR:000000164 60361 60365 Bmp2 +T1892 PR:000000165 60370 60374 Bmp4 +T1893 PR:000013316 60379 60383 Prx1 +T1894 UBERON:0000922 60393 60402 embryonic +T1895 UBERON:0002103 60403 60412 hindlimbs +T1896 GO:0010467 60477 60488 expressions +T1897 PR:000014841 60492 60495 Shh +T1898 PR:000007499 60500 60504 Fgf8 +T1899 GO:0051216 60531 60545 Chondrogenesis +T1900 PR:000000164 60598 60602 BMP2 +T1901 PR:000000165 60607 60611 BMP4 +T1902 UBERON:0004288 60635 60644 skeletons +T1903 UBERON:0000922 60656 60663 embryos +T1904 UBERON:0000922 60713 60719 embryo +T1905 PR:000000164 60725 60729 Bmp2 +T1906 PR:000000165 60734 60738 Bmp4 +T1907 PR:000013316 60743 60747 Prx1 +T1908 UBERON:0000922 60753 60759 embryo +T1909 UBERON:0002471 60804 60812 zeugopod +T1910 UBERON:0002472 60817 60825 stylopod +T1911 CHEBI:51686 60838 60849 Hematoxylin +T1912 UBERON:0000976 60889 60895 humeri +T1913 PR:000000164 60925 60929 Bmp2 +T1914 PR:000000165 60934 60938 Bmp4 +T1915 PR:000013316 60943 60947 Prx1 +T1916 UBERON:0000922 60957 60964 embryos +T1917 PR:000000164 61070 61074 Bmp2 +T1918 PR:000000165 61079 61083 Bmp4 +T1919 PR:000013316 61088 61092 Prx1 +T1920 UBERON:0000976 61102 61108 humeri +T1921 UBERON:0000922 61120 61127 embryos +T1922 GO:0097617 61133 61143 hybridized +T1923 CHEBI:42098 61149 61160 digoxigenin +T1924 SO:0000077 61169 61178 antisense +T1925 PR:000005693 61193 61197 ColX +T1926 UBERON:0002102 61231 61240 forelimbs +T1927 PR:000000164 61266 61270 BMP2 +T1928 PR:000000165 61272 61276 BMP4 +T1929 UBERON:0000922 61287 61294 embryos +T1930 PR:000000168 61350 61354 Bmp7 +T1931 PR:000000164 61462 61466 BMP2 +T1932 PR:000000165 61471 61475 BMP4 +T1933 GO:0001503 61477 61489 Osteogenesis +T1934 UBERON:0019248 61504 61519 Early Embryonic +T1935 GO:0009790 61510 61531 Embryonic Development +T1936 UBERON:0002102 61560 61568 forelimb +T1937 UBERON:0000922 61580 61587 embryos +T1938 UBERON:0004408 61632 61643 Distal ulna +T1939 UBERON:0000922 61659 61665 embryo +T1940 UBERON:0004408 61671 61682 distal ulna +T1941 UBERON:0004407 61671 61677;61683 61689 distal ... radius +T1942 PR:000000164 61695 61699 Bmp2 +T1943 PR:000000165 61704 61708 Bmp4 +T1944 PR:000013316 61713 61717 Prx1 +T1945 UBERON:0000922 61723 61729 embryo +T1946 UBERON:4000088 61731 61752 Mineralized cartilage +T1947 UBERON:0008883 61781 61788 osteoid +T1948 UBERON:0000976 61838 61844 Humeri +T1949 GO:0097617 61859 61869 hybridized +T1950 CHEBI:42098 61875 61886 digoxigenin +T1951 PR:000003263 61909 61913 ColI +T1952 UBERON:0000976 61940 61947 humerus +T1953 UBERON:0000922 61959 61965 embryo +T1954 PR:000000164 61976 61980 Bmp2 +T1955 PR:000000165 61985 61989 Bmp4 +T1956 PR:000013316 61994 61998 Prx1 +T1957 UBERON:0000922 62010 62016 embryo +T1958 UBERON:0002471 62046 62054 zeugopod +T1959 UBERON:0000922 62066 62073 embryos +T1960 PR:000001937 62143 62147 TRAP +T1961 UBERON:0004413 62190 62205 proximal radius +T1962 UBERON:0000922 62221 62227 embryo +T1963 UBERON:0006822 62245 62258 proximal ulna +T1964 UBERON:0004413 62245 62253;62259 62265 proximal ... radius +T1965 PR:000000164 62271 62275 Bmp2 +T1966 PR:000000165 62280 62284 Bmp4 +T1967 PR:000013316 62289 62293 Prx1 +T1968 UBERON:0000922 62299 62305 embryo +T1969 GO:0001944 62316 62331 vascularization +T1970 PR:000000164 62357 62361 BMP2 +T1971 PR:000000165 62366 62370 BMP4 +T1972 CL:0000092 62376 62387 osteoclasts +T1973 UBERON:4000088 62408 62429 mineralized cartilage +T1974 PR:000000164 62433 62437 Bmp2 +T1975 PR:000000165 62442 62446 Bmp4 +T1976 PR:000013316 62451 62455 Prx1 +T1977 NCBITaxon:10088 62461 62465 mice +T1978 UBERON:0004413 62617 62632 proximal radius +T1979 NCBITaxon:33208 62650 62656 animal +T1980 UBERON:0006822 62669 62682 proximal ulna +T1981 UBERON:0004413 62669 62677;62683 62689 proximal ... radius +T1982 PR:000000164 62699 62703 Bmp2 +T1983 PR:000000165 62708 62712 Bmp4 +T1984 PR:000013316 62717 62721 Prx1 +T1985 NCBITaxon:33208 62727 62733 animal +T1986 UBERON:0002484 62788 62806 bone marrow cavity +T1987 PR:000000164 62905 62909 BMP2 +T1988 PR:000000165 62914 62918 BMP4 +T1989 PR:000000164 62941 62945 Bmp2 +T1990 PR:000000165 62950 62954 Bmp4 +T1991 PR:000013316 62959 62963 Prx1 +T1992 NCBITaxon:10088 62969 62973 mice +T1993 UBERON:0002471 63009 63017 zeugopod +T1994 UBERON:0001490 63026 63037 elbow joint +T1995 UBERON:0001424 63104 63108 ulna +T1996 UBERON:0001423 63117 63123 radius +T1997 PR:000000164 63180 63184 BMP2 +T1998 PR:000000165 63189 63193 BMP4 +T1999 UBERON:0004406 63243 63256 distal femurs +T2000 UBERON:0000981 63273 63279 femurs +T2001 CL:0000062 63428 63444 osteoblast cells +T2002 UBERON:0001439 63467 63480 cortical bone +T2003 PR:000000164 63539 63543 Bmp2 +T2004 PR:000000165 63548 63552 Bmp4 +T2005 PR:000013316 63557 63561 Prx1 +T2006 UBERON:0000981 63567 63573 femurs +T2007 UBERON:0010357 63609 63638 secondary ossification center +T2008 GO:0001503 63619 63631 ossification +T2009 UBERON:0000479 63658 63664 tissue +T2010 UBERON:0002483 63698 63713 trabecular bone +T2011 UBERON:0002484 63715 63717 bm +T2012 UBERON:0002484 63729 63747 bone marrow cavity +T2013 PR:000000164 63763 63767 Bmp2 +T2014 PR:000000165 63772 63776 Bmp4 +T2015 PR:000013316 63781 63785 Prx1 +T2016 UBERON:0000981 63791 63797 femurs +T2017 CL:0000092 64011 64021 osteoclast +T2018 GO:0045453 64031 64046 bone resorption +T2019 UBERON:0000479 64060 64066 tissue +T2020 UBERON:0006862 64078 64092 shaft of femur +T2021 CL:0000057 64126 64136 Fibroblast +T2022 UBERON:0004769 64163 64173 bone shaft +T2023 PR:000000164 64177 64181 Bmp2 +T2024 PR:000000165 64186 64190 Bmp4 +T2025 PR:000013316 64195 64199 Prx1 +T2026 NCBITaxon:10088 64205 64210 mouse +T2027 CL:0000092 64238 64249 osteoclasts +T2028 PR:000000164 64263 64267 Bmp2 +T2029 PR:000000165 64272 64276 Bmp4 +T2030 PR:000013316 64281 64285 Prx1 +T2031 UBERON:0000981 64291 64297 femurs +T2032 UBERON:0000479 64332 64339 tissues +T2033 PR:000000164 64415 64419 Bmp2 +T2034 PR:000000165 64424 64428 Bmp4 +T2035 PR:000013316 64433 64437 Prx1 +T2036 UBERON:0001441 64447 64465 hindlimb skeletons +T2037 CHEBI:16866 64495 64507 Alizarin red +T2038 UBERON:0004412 64551 64565 proximal femur +T2039 CL:0000062 64578 64588 Osteoblast +T2040 PR:000000164 64631 64635 BMP2 +T2041 PR:000000165 64640 64644 BMP4 +T2042 UBERON:0004406 64652 64665 Distal femurs +T2043 NCBITaxon:10088 64679 64683 mice +T2044 UBERON:0004406 64707 64720 Distal femurs +T2045 PR:000000164 64726 64730 Bmp2 +T2046 PR:000000165 64735 64739 Bmp4 +T2047 PR:000013316 64744 64748 Prx1 +T2048 NCBITaxon:10088 64754 64758 mice +T2049 UBERON:0004406 64782 64795 Distal femurs +T2050 NCBITaxon:10088 64809 64813 mice +T2051 UBERON:0004406 64838 64851 Distal femurs +T2052 PR:000000164 64857 64861 Bmp2 +T2053 PR:000000165 64866 64870 Bmp4 +T2054 PR:000013316 64875 64879 Prx1 +T2055 NCBITaxon:10088 64885 64889 mice +T2056 GO:0097617 64916 64929 hybridization +T2057 CL:0000062 64939 64949 osteoblast +T2058 SO:0000704 64981 64986 genes +T2059 PR:000003263 64987 64992 Col I +T2060 PR:000014364 65011 65016 runx2 +T2061 PR:000015445 65038 65045 osterix +T2062 UBERON:0001091 65658 65664 Dental diff --git a/src/ontogpt/evaluation/craft/database/all/17194222.txt b/src/ontogpt/evaluation/craft/database/all/17194222.txt new file mode 100644 index 000000000..6aba5cce2 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17194222.txt @@ -0,0 +1,309 @@ +Genetic Analysis of the Roles of BMP2, BMP4, and BMP7 in Limb Patterning and Skeletogenesis + +Abstract + +Bone morphogenetic protein (BMP) family members, including BMP2, BMP4, and BMP7, are expressed throughout limb development. BMPs have been implicated in early limb patterning as well as in the process of skeletogenesis. However, due to complications associated with early embryonic lethality, particularly for Bmp2 and Bmp4, and with functional redundancy among BMP molecules, it has been difficult to decipher the specific roles of these BMP molecules during different stages of limb development. To circumvent these issues, we have constructed a series of mouse strains lacking one or more of these BMPs, using conditional alleles in the case of Bmp2 and Bmp4 to remove them specifically from the limb bud mesenchyme. Contrary to earlier suggestions, our results indicate that BMPs neither act as secondary signals downstream of Sonic Hedghog (SHH) in patterning the anteroposterior axis nor as signals from the interdigital mesenchyme in specifying digit identity. We do find that a threshold level of BMP signaling is required for the onset of chondrogenesis, and hence some chondrogenic condensations fail to form in limbs deficient in both BMP2 and BMP4. However, in the condensations that do form, subsequent chondrogenic differentiation proceeds normally even in the absence of BMP2 and BMP7 or BMP2 and BMP4. In contrast, we find that the loss of both BMP2 and BMP4 results in a severe impairment of osteogenesis. + +Synopsis + +A group of related signaling molecules called bone morphogenetic proteins (BMPs) are known to play important roles in the formation of the structures such as the limbs. However, because different members of this group often have similar effects on target cells and are produced in overlapping regions of the embryo and hence can be redundant with one another, removal of any single member of the BMP family may not reveal the full extent of the roles they play during development. We have therefore improved on this type of analysis by removing pairs of these factors (BMP2 and BMP4 or BMP2 and BMP7) specifically from the developing limb. Although some have speculated that these signals play an early role in organizing or “patterning” the different tissues of the limb, we find no evidence for such a role. We do find, however, that a minimal amount of BMP signal is required to form cartilage, and hence some cartilaginous elements fail to form in limbs deficient in both BMP2 and BMP4. Moreover, in the absence of these two BMP family members, there is a severe impairment in the development of bone tissue, resulting in severely deformed limbs. This study gives important new insight into the roles of these BMP signals in making skeletal tissues in the embryo. + +Introduction + +Bone morphogenetic proteins (BMPs) are secreted signaling molecules belonging to the transforming growth factor β superfamily, originally identified on the basis of their ability to induce ectopic bone formation when implanted within soft tissue in vivo [1–3]. BMP family members are now known to play an extremely diverse set of roles in a wide variety of developmental processes [4]. Even in the context of the morphogenesis of a single structure, these molecules can play a series of quite divergent roles. For example, during limb development, BMPs have been postulated to act sequentially in multiple distinct aspects of patterning, cell type specification, and differentiation of various tissues, particularly of the skeleton. + +The earliest step of limb development in which BMP signaling has been implicated is the establishment of the anterior-posterior limb axis. Differences in anterior-posterior pattern are instructed as a graded response to Sonic Hedghog (SHH) signaling emanating from the posterior margin of the limb bud [5]. It has remained controversial, however, whether this response is direct or indirect. If indeed the long-range effects of SHH are indirectly mediated by local production of secondary signals, the leading candidates have been two members of the BMP family, BMP2 and BMP7. Both are expressed in a broader domain than SHH in the early posterior limb bud mesenchyme [6,7], although BMP7 also has a second, weaker domain of expression in the anterior limb bud mesenchyme. BMP2 [8] and BMP7 [7] can be induced by ectopic SHH and their expression is greatly diminished in the absence of SHH activity [9]. BMP2 and BMP7 are thus secondary signals produced in response to SHH activity. Moreover, BMP signaling has a weak ability to posteriorly polarize the limb in ectopic grafting experiments [10], an activity enhanced by prior low-level exposure to SHH [11]. It remains unclear, however, whether BMP2 and BMP7 activity is required endogenously for anterior-posterior limb patterning by SHH. Bmp2 mutant embryos die too early to assess their limb phenotypes. A targeted deletion of Bmp7 has been made, and Bmp7-deficient embryos display hindlimb polydactyly with incomplete penetrance but otherwise phenotypically normal limbs [12,13]. Nonetheless, Bmp7 knockout mice do not show any defect in limb polarity. However, a redundant function in anterior-posterior patterning with BMP2 remains a possibility. In addition to BMP2 and BMP7, a third member of this family that is closely related to BMP2, BMP4, is also expressed in the early limb bud. Like BMP7, it is expressed in both the anterior and posterior margins of the limb bud mesenchyme [4,14]; however, it does not appear to be induced by SHH signaling, nor does its expression change in SHH-deficient limb buds. Thus, BMP4 is not a candidate for a secondary signal downstream of SHH in early patterning. However, all three of these molecules, BMP2, BMP4, and BMP7, have been suggested to act in a second distinct phase of limb patterning, when digit identities are established downstream of earlier patterning events. In the vertebrate limb, each digit can be uniquely identified based on its size, length, number of phalanges, and location within the autopod. As a consequence of the initial establishment of anterior-posterior positioned information within the limb by SHH and/or BMP signaling, the interdigital mesenchyme of the hand plate becomes specified in a graded manner. Grafting and extirpation experiments have shown that it is this polarized interdigital tissue which directs digit morphology [15]. BMP2, BMP4, and BMP7 are all expressed in the interdigital mesenchyme [4,14], and experiments inhibiting BMP signaling in this tissue suggested that differential levels of BMPs in the interdigital mesenchyme might be the relevant factor directing digit morphology [15]. As noted above, BMP7-deficient embryos produce mostly normal limbs, and mice harboring a conditional deletion of Bmp4 in the limb bud mesenchyme also do not show evidence of anterior digit transformation [16], as would have been predicted if BMP4 acted as an interdigital regulator of digit morphology. In the light of these observations, it would seem unlikely that quantitative differences in levels of interdigital BMP signal are responsible for establishing digit identity, although redundancy between BMP2, BMP4, and BMP7 in this role remains a possibility. + +A third aspect of limb patterning in which BMP2, BMP4, and BMP7 have been implicated is in the apoptotic death of the interdigital tissue. For example, ectopic application of BMP antagonists demonstrates that BMP signaling is necessary for this process, and in its absence, webbing occurs [17–19]). + +BMP activity also plays an indirect role in limb patterning as a key component of a feedback loop between SHH production in the posterior limb bud and fibroblast growth factor production in the overlying apical ectodermal ridge (AER). SHH activity leads to the upregulation of the BMP antagonist Gremlin in the mesenchyme [20,21]. This, in turn, prevents BMPs from downregulating fibroblast growth factor (FGF) production in the AER [17,22] and maintains the integrity of the AER itself [23]. FGF signaling feeds back to maintain SHH production in the posterior mesenchyme [8,24]. Thus, conditional removal of BMP4 activity from the posterior limb mesenchyme results in a persistence of the AER, expanded SHH signaling, and consequent preaxial and postaxial polydactyly [16]. + +At later stages of limb development, BMP activity is also believed to play critical roles in skeletogenesis. Activation and dominant-negative experiments in the chick have shown that signaling through the BMPR-IB receptor is necessary and sufficient for cartilage condensation in chick [25]. Likewise, conditional knockout of BmpR1A and BmpR1B blocks all chondrogenic differentiation in the mouse limb bud [26]. Similarly, when noggin, a potent BMP inhibitor, is introduced in early stage chick limb buds prior to skeletogenesis, mesenchymal condensation does not take place [27,28]. Taken together, these studies strongly implicate BMP signaling as necessary for mesenchymal condensations and the initiation of chondrogenesis. However, the phenotypes in these experiments are severe, causing either global chondrogenesis or absence of chondrogenesis, in the gain- and loss-of-function experiments, respectively. These severe defects during the early stages of skeletogenesis preclude investigation of any specific roles for BMPs in later events in cartilage differentiation, bone formation, and bone metabolism [29]. Conversely, removal of individual ligands such as BMP4 [16] or BMP7 [12,13] has not displayed defects in skeletal differentiation, presumably due to functional redundancy. + +To gain further insights into the potential roles of BMP signaling in vertebrate limb patterning and skeletogenesis, we have produced mice simultaneously lacking BMP2 and BMP4 activity or BMP2 and BMP7 activity in the limb mesechyme during limb development. Since Bmp2 and Bmp4 mutants are both lethal early in embryogenesis [30,31], we used conditional alleles of both these genes. The conditional alleles were deleted early in limb development through the action of a cre-recombinase transgene expressed under the control of the Prx1 enhancer [32]. Neither limbs deficient in BMP2 and BMP7 nor those deficient in BMP2 and BMP4 had defects suggestive of loss of anterior-posterior patterning information, arguing against these factors acting as secondary signals downstream of SHH or acting as interdigital determinants of digit identity. The Bmp2/Bmp4 double mutants did display a loss of posterior digits with a broad posterior hand plate, suggestive of a block in the chondrogenic condensation of the posterior digit rays, rather than a change in anterior-posterior digit identity. At later stages, the combined activity of neither BMP2 and BMP4 nor of BMP2 and BMP7 is required for chondrogenesis. Osteogenesis is also initiated properly in the absence of these pairs of factors. However, at later stages, the combined loss of BMP2 and BMP4 evidently becomes limiting, and osteogenesis ceases. As all prior reported manipulations of the BMP pathway during skeletal development either result in a block at the first stage of chondrogenesis or allow complete skeletal formation, this is the first demonstration of a requirement for BMP activity in osteogenic differentiation. + +Results + +To investigate the roles of BMP signaling at various stages of limb patterning and skeletogenesis, we constructed a series of mice deficient singly or in combination in the ability to produce BMP2, BMP4, and BMP7. BMP7-deficient mice (kindly provided by Dr. Liz Robertson) survive until birth. However, BMP2 and BMP4 are both required for viability early in embryonic development [30,31]. We therefore constructed a conditional allele of Bmp2, introducing loxP sites flanking exon 3. We obtained a conditional allele of Bmp4, in which exon 4 is flanked by loxP sites, from Dr. Holger Kulessa and Dr. Brigid Hogan [16]. Both of these alleles would be expected to result in null alleles following recombination (see Materials and Methods for details). To conditionally inactivate Bmp2 and Bmp4 in the limb, we used a well-characterized transgene in which cre-recombinase is expressed under the control of the Prx1 limb enhancer [32]. This transgene expresses cre very early in limb development, resulting in complete recombination of floxed alleles at early limb bud stages. We verified the ability of Prx1::cre to recombine the conditional Bmp2 and Bmp4 alleles at earliest limb bud stages using in situ hybridization. Bmp2 is first expressed in the limb mesenchyme at embryonic day (E)10.5 in the mouse (Figure 1A, asterisk). By the time its expression is first detectable, the floxed Bmp2 allele appears to be completely recombined in the limb mesenchyme, as whole mount in situ hybridization does not detect any mesenchymal Bmp2 transcription (Figure 1B). At E10.5 Bmp4 is expressed in the mouse limb mesenchyme in two stripes at the anterior and posterior margins (Figure 1C, red arrows). These expression domains are completely lost by E10.5 in the presence of the Prx1::cre transgene (Figure 1D). Bmp2 and Bmp4 are also expressed in the AER, where Prx1::cre is inactive, and these domains of expression are not affected (Figure 1A–1D, black arrows). + +An Allelic/Nonallelic Series of BMP-Deficient Limbs + +Mice were generated with limbs deficient in BMP2, BMP4, or BMP7, both BMP2 and BMP4, or both BMP2 and BMP7. To obtain an initial indication of the range of phenotypes produced in these animals, we first examined the limb skeletons of newborn animals. BMP2-deficient limbs appeared remarkably normal, both in skeletal pattern and in gross aspects of chondrogenesis and osteogenesis indicated by Alcian blue/Alizarin red staining. The one notable defect in the appendicular skeleton of these animals is a characteristic malformation of the scapula (Figure 1, red arrow). BMP2-deficient limbs also exhibit 3/4 soft-tissue syndactyly with variable penetrance (Figure 2). + +Mice with limbs deficient in BMP4 activity and the limbs of Bmp7 mutant mice have both been previously described. As reported [16], in the absence of BMP4 activity, limbs display a variable penetrance of preaxial and postaxial polydactyly, but otherwise, normal digit patterns and apparently normal skeletal differentiation (Figure 1G and 1O) take place. As previously described, mice homozygous for a null mutation in Bmp7 [12] have no defects in the formation of the normal appendicular skeletal elements (Figure 1H and 1P). We do occasionally observe preaxial polydactyly in these mutants. + +Compound heterozygous mice, with one functional copy each of Bmp2 and Bmp4 in the limb (Bmp2+/C; Bmp4+/C; Prx1::cre), show no effect on either limb patterning or skeletogenesis (unpublished data). Similarly, limb skeletons of mice in which both copies of the Bmp4 gene and one copy of the Bmp2 gene have been conditionally removed in the limb (Bmp2+/C; Bmp4C/C; Prx1::cre) are phenotypically normal other than exhibiting the variable penetrance preaxial and postaxial polydactyly seen in mice deficient in BMP4 activity alone (Figure 1J and 1R). In contrast, mice in which both copies of Bmp2 and one copy of Bmp4 had been removed (Bmp2C/C; Bmp4+/C; Prx1::cre) showed more severe skeletal defects, including significantly thinner skeletal elements. However, the digit patterns of those animals are completely normal (Figure 1I and 1Q). Animals in which both copies of Bmp2 and Bmp4 were removed (Bmp2C/C; Bmp4C/C; Prx1::cre) had extremely malformed limbs (Figure 1K and 1S). They displayed severely short and malformed stylopods; one of the zeugopod elements was almost always missing, and the remaining one was so deformed that it was difficult to identify the element correctly. Moreover, the joint articulations are defective such that zeugopod and stylopod elements are fused (see below and Figure S1A–S1C). Interestingly, the autopods are less affected than the proximal elements. Nonetheless, the autopod elements are significantly reduced in size, and strikingly the two posterior-most digits are missing in the forelimbs of these animals. + +Simultaneous removal of BMP2 and BMP7 had far less of an effect than did removal of BMP2 and BMP4 activity. Compound heterozygous removal of BMP2 and BMP7 (Bmp2+/C, Bmp7+/−; Prx1::cre) and heterozygous removal of BMP2 with complete removal of BMP7 (Bmp2+/C, Bmp7−/−; Prx1::cre) were both completely wild-type in skeletal pattern and differentiation (unpublished data). Mice homozygous for removal of Bmp2 and heterozygous for Bmp7 (Bmp2C/C, Bmp7+/−; Prx1::cre) displayed the same subtle scapular defect seen in Bmp2C/C; Prx1::cre mutants alone (unpublished data). In limbs developing in the complete absence of both BMP7 and BMP2 activity (Bmp2C/C, Bmp7−/−; Prx1::cre), the last phalanx was missing from digit III in the forelimb, and these limbs displayed the same phenotype in the hindlimb with variable penetrance (Figure 1L and 1T, black arrow). Additionally, the fibulae of these hindlimbs are malformed and do not articulate with the femur at the knee (Figure 1T, thick red arrow). They also display the same scapular defects seen in Bmp2C/C; Prx1::cre mice, and the overall size of the appendicular skeleton is slightly diminished. However, skeletal differentiation appears normal in the limbs of these animals. + +Soft Tissue Syndactyly Observed in Limbs of Mice Devoid of BMP2 and BMP4 Activity + +As noted above, Bmp2-deficient animals display a variably penetrant 3/4 soft tissue syndactyly phenotype (Figure 2B). In contrast, Bmp4-deficient mice show no evidence of syndactyly (unpublished data). However, one of the striking defects observed in the Bmp2C/C; Bmp4C/C; Prx1::cre mice is that in the newborn animals, the digits of both forelimb (unpublished data) and hindlimb show complete syndactyly (Figure 2D; compare to wild-type, Figure 2C). To determine the origin of this defect, we examined embryonic limbs at E15.5, the stage when interdigital separation is normally taking place (Figure 2E). In Bmp2C/C; Bmp4C/C; Prx1::cre limbs, only the very distal tips of each digit were separated, with the autopod adopting the shape of a notched pallet (Figure 2F). In wild-type development, the individual digits become free from one another through a process of interdigital apoptosis, observable by staining with acridine orange (Figure 2E and 2G, arrow). Interdigital apoptosis is dramatically reduced in the Bmp2C/C; Bmp4C/C; Prx1::cre limbs (Figure 2F and 2H, arrow). (Note that the strong acridine orange staining in Figure 2H is not in the interdigital mesenchyme but rather is in the remnant of the cells of the AER [red asterisk].) + +Another region of very prominent apoptosis in the wild-type limb bud is within the AER. The AER is a specialized ridge of ectoderm running along the anterior-posterior axis at the distal margin of the early limb bud. One of the phenotypes characterizing the Bmp2C/C; Bmp4C/C; Prx1::cre mutant is expansion of the AER (see below). Moreover, in the Bmp2C/C; Bmp4C/C; Prx1::cre animals, the AER is maintained as late as E15.5, long after it has started to degrade in wild-type limbs. This can be seen morphologically (unpublished data) or by staining for expression of AER-specific marker such as Fgf8 (Figure 2I and 2J). The AER displays intense staining with acridine orange, TUNEL, or other markers for apoptosis at every stage when it is present, reflecting the rapid turnover of cells in this structure [33–35]. The Bmp2C/C; Bmp4C/C; Prx1::cre mutant limbs at E15.5 show strong acridine orange staining in the remnants of AER (Figure 2H, asterisk), in domains of the distal tip where the AER remains the longest (compare staining in Figure 2F and 2J). No significant difference was observed in the extent of apoptosis in the cells of the AER in earlier stage Bmp2C/C; Bmp4C/C; Prx1::cre mutant embryonic limbs (Figure S1D–S1G). + +Patterning Defects in Limb Developing with Reduced Levels of BMP Signaling + +BMP signaling has been proposed to play several distinct roles in limb patterning. The first of these is in establishing differences along the anterior-posterior axis of the limb downstream of sonic hedgehog (Shh). SHH signaling itself extends over a long range in the developing limb bud, based on both activation of target genes [36] and protein distribution [37]. Nonetheless, some models have suggested that the patterning effect of this morphogenic signal is mediated entirely [10] or in part [11] by BMP2 and BMP7 produced as secondary signals in response to SHH. We have removed both BMP2 and BMP7 activities in the early limb bud (Figure 1), and moreover, there is no apparent upregulation of BMP4 expression in the posterior of these limbs (Figure 3A–3D). The skeletal phenotypes of mouse limbs formed in the absence of BMP2, BMP7, and both BMP2 and BMP7 are illuminating, as none of these exhibit significant digit pattern defects (Figure 1). While a distal phalanx is missing in the middle digits of the double mutants (Bmp2C/C, Bmp7−/−; Prx1::cre), this is not a phenotype consistent with a decrease or loss of a posteriorly derived polarizing activity. Indeed, the terminal phalanx forms at the distal tip of each digit by a distinct mechanism, not dependent upon anterior-posterior patterning [38]. These data strongly indicate that BMP2 and BMP7 are not required secondary signals for SHH-mediated polarization of the digits along the anterior-posterior axis. + +The lack of transformations in digit identity in the limb skeletons formed in the absence of BMP2 and BMP7 activity also argues against BMP signaling being the critical instructive signal from the interdigital mesenchyme to the forming digits. While a distal phalanx was lost from the middle digit of the forelimbs of these animals, these digits do not otherwise have a morphology consistent with their being transformed anteriorly into digit 1 (thumb). Indeed, in the hindlimbs, where the penetrance of the phenotype is variable, the mutant middle digits are otherwise indistinguishable from wild-type, other than the presence or absence of this variable skeletal element. + +In contrast to the proposed roles for BMPs in anterior-posterior digit patterning, we obtained further support for the well-accepted idea that a threshold level of BMP signaling is necessary interdigitally for programmed cell death to occur. We observed syndactyly with variable penetrance in limbs developing without BMP2 activity (Figure 2B), and nearly complete soft tissue syndactyly of all digits in limbs deficient in both BMP2 and BMP4 (compare Figure 2C and Figure 2D). + +The one dramatic patterning defect we observed in our BMP deficiency series was the loss of posterior digits in forelimbs deficient in both BMP2 and BMP4 (Figure 1K), also observed in earlier limb buds stained to show no expression of the condensation marker Sox9 (Figure 3). To understand this phenotype, we analyzed limb buds at various stages using a series of molecular markers. By E11.5, the Bmp2C/C; Bmp4C/C; Prx1::cre limb buds are noticeably broader, expanded in the posterior relative to wild-type. This correlates with an anterior expansion in Shh expression (Figure 3E and 3F, red bracket) and a concomitant expansion of the AER as visualized by Fgf8 expression (Figure 3G and 3H, red bracket). The expansion in Shh expression can first be detected at E10.5 and persists at least until E12.5, well after the time normal Shh expression disappears at E11.75 (Figure 3L and 3P). The expansion of the AER in the absence of BMP signaling may be understood on the basis of the feedback loop between the zone of polarizing activity (ZPA) and the AER. Expression of several Fgf genes [20,21] and maintenance of the AER itself [23] normally depend on BMP antagonism induced by SHH. Thus, a decrease in BMP signaling expands and maintains the AER. Because Shh expression is, reciprocally, supported by FGF signaling from the AER [8,24], this, in turn, feeds back on the ZPA, expanding Shh expression. While this explains the broader hand plate of the BMP2, BMP4–deficient limb buds, it is somewhat paradoxical in terms of the skeletal phenotype, as a broadened autopod would normally be expected to result in the formation of extra digits (polydactyly) (e.g., Figure 1G and 1O of this paper and [16]), not the loss of digits, as observed in the double mutant forelimbs. + +To examine the loss of digits more closely, BMP2, BMP4–deficient limb buds were examined during cartilage condensation phase of the digit rays at E12.5. In wild-type limb buds the condensation of all five digits can be visualized at this stage by in situ hybridization with a probe directed against Sox9, the earliest known chondrogenic marker (Figure 3I). However, in Bmp2C/C; Bmp4C/C; Prx1::cre limb buds at E12.5, Sox9 only detects primordia of the anterior three digits of the forelimb, indicating a failure in the formation of posterior digits at this earliest stage (Figure 3J). + +Genes such as Msx2 (Figure 3N) continue to be expressed throughout the posterior forelimb mesenchyme, indicating that the posterior forelimb tissue is viable at E12.5 in the Bmp2C/C; Bmp4C/C; Prx1::cre animals. Msx2 is normally expressed in the non-chondrogenic interdigital mesenchyme at this stage (Figure 3M), a continuation of an earlier expression domain throughout the distal mesenchyme. This expression of Msx2 in the posterior of the BMP2, BMP4–deficient forelimbs reflects a failure of chondrogenesis in this region. The loss of the posterior forelimb digits in these mice thus does not appear to represent a decrease in polarizing activity leading to a change in digit identity rather a consequence of the overall level of BMP signaling falling below a threshold for initiating chondrogenesis in the posterior of these forelimbs. The posterior of the hindlimb in the mutant, in contrast, appears to remain above this threshold, and more than five digit condensations form in the hindlimb bud (Figures 3K and 1S) and no ectopic expression of Msx2 is observed in the posterior (Figure 3O). + +Skeletal Differentiation in Limbs Developing with Reduced Levels of BMP Signaling + +BMPs were first discovered in the context of their skeletogenic activity. We therefore wanted to determine whether the loss of the specific BMP molecules under investigation here would perturb the skeletal differentiation pathways. Accordingly, skeletal preparations from each of the various mutant alleles were subjected to histological and molecular analyses. The skeletal elements that formed in the absence of BMP2, BMP4, or BMP7 or BMP2 and BMP7 all underwent normal chondrogenesis and osteogenesis by a series of criteria. All showed normal staining with Alcian blue (for cartilage) and Alizarin red (for mineralized tissue, see Figure 1). Most were also analyzed in histological sections stained with toluidine blue and showed osteoid formation and bone matrix deposition (compare Figure S2A and S2E; and unpublished data). The skeletal elements in these various BMP-deficient animals contained normal prehypertrophic chondrocytes expressing type II collagen, hypertrophic chondrocytes expressing type X collagen, as well as the osteoblasts expressing type I collagen, like their wild-type counterparts (Figure S2; compare Figure S2F–S2H with Figure S2B–S2D; and unpublished data). In contrast, however, limbs completely deficient in both BMP2 and BMP4 activity showed severe defects in skeletal differentiation. + +BMP2 and BMP4 Are Not Critical for Chondrogenic Differentiation + +As noted above, at E12.5, chondrogenic condensation of the digits could be visualized in Bmp2C/C; Bmp4C/C; Prx1::cre limb buds by in situ hybridization with a Sox9 probe. Similar analysis at E10.5 and E11.5 demonstrated a relatively normal process of condensation of the proximal skeletal elements (Figure S1H–S1M), despite the absence of detectable Bmp2 and Bmp4 transcripts. As shown in Figure S1N and S1O, expression of Col II, a chondrocyte marker, is observed in the BMP2, BMP4–deficient cartilage (Figure S1O) as early as E12.5, similar to a wild-type control (Figure S1N). Similarly, at E13.5, the entire limb skeleton could be visualized undergoing chondrogenesis by Alcian blue staining, albeit in a defective pattern, in the BMP2, BMP4–deficient limb buds (Figure 4A and 4B and unpublished data). + +As the cartilage elements grow, a wave of hypertrophic differentiation occurs from the center toward the distal ends (reviewed in [39–41]). Histological examination of the humeri at E13.5 showed that while chondrogenic differentiation had taken place in the BMP2, BMP4–deficient humerus, hypertrophic differentiation was delayed (Figure 4C and 4D). However, by E15.5, hypertrophy was evident in these elements as seen by type X collagen expression (Figure 4E and 4F). The hypertrophic cells in BMP2, BMP4–deficient limb also express late hypertrophic markers, such as osteopontin [42] (unpublished data). + +To examine whether the successful execution of chondrogenesis in the BMP2, BMP4–deficient limbs is a result of compensatory upregulation of other ostegenic BMPs, we carried out in situ hybridization for Bmp6 and Bmp7 in these limbs. At E13.5, we observed no increase in expression of Bmp6 (Figure S1P–S1S) or Bmp7 (compare Figure 4H with Figure 4G) mRNA in the mutant tissues compared to their wild-type counterparts + +Osteogenesis Is Initiated Normally in BMP2, BMP4–Deficient Limb Buds + +Bone formation first occurs at the interface between the late hypertrophic chondrocytes and the surrounding perichondrium, a region identified as the bone collar. We examined whether the osteogenesis program is initiated in the BMP2, BMP4–deficient limbs using several histological and molecular criteria, as described below (Figure 5). Adjacent to the hypertrophic cells, a bone collar is laid down normally in the absence of BMP2 and BMP4 (Figure 5A and 5B, red star). As can be seen in sagittal sections through an E15.5 humerus, the cells embedded in the mineralized matrix express the osteoblast marker Col I as normal (Figure 5C and 5D). By E17.5, the mineralized cartilage that makes up the late hypertrophic zone of the growth plate normally undergoes removal by osteoclasts (Figure 5G, brown-stained cells, red arrows), resulting in the formation of a bone marrow cavity (red arrow in Figure 5E and 5I, see also Figure 6). Osteoprogenitor cells from bone collar region now populate this site and deposit osteoid, forming the trabecular bone in the bone marrow cavity (Figure 6E, red arrow) and cortical bone at birth (Figure 5I, asterisk). In contrast, in E17.5 limbs deficient in BMP2 and BMP4, there is an overall delay in the normal endochondral process (Figure 5F). While vascularization occurs in Bmp2C/C; Bmp4C/C; Prx1::cre forelimbs, few osteoclasts are recruited to the mineralized cartilage (compare Figure 5G and Figure 5H, red arrows), resulting in a delay in bone marrow cavity formation in the absence of BMP2 and BMP4. In fact, bone marrow formation and trabecular bone formation have not yet started at birth in the absence of BMP2 and BMP4 (Figure 5J), and the morphology of Bmp2C/C; Bmp4C/C; Prx1::cre bones at birth is most similar to control embryos at E17.5 (Figure 5E). To differentiate between a simple delay in bone formation in the absence of BMP2 and BMP4 and the inability to form bone without these BMPs, we performed histological analyses in Bmp2C/C; Bmp4C/C; Prx1::cre mice up to 3 wks of age. As shown in Figure 6A through 6E, in the control mice, a bone collar is found adjacent to the zone of late hypertrophic chondrocytes at 1 wk (Figure 6A and 6B). Many osteoblasts line the newly formed cortical bone in the diaphyseal region (Figure 6C, blue arrow), and formation of a secondary ossification center has begun (Figure 6A, red star). At 3 wks of age, formation of the secondary ossification center is complete (Figure 6D, red star), along with robust bone marrow formation (Figure 6D, bm) and deposition of trabecular bone (Figure 6E, pale blue tissue marked by red arrow). In stark contrast, bone formation is not observed in the absence of BMP2 and BMP4 at 1 or 3 wks after birth (Figure 6F–6J). The appearance of the skeletal elements in the double mutants remains similar to E17.5 structures seen in wild-type limbs (Figure 5E), and although there is a bone collar at the site of hypertrophic cartilage (Figure 6G), no bone marrow cavity, trabecular bone, or cortical bone is present (Figure 6F). Many osteoclasts have been recruited to the remaining mineralized cartilage and are actively resorbing this tissue (red asterisks, Figure 6I), so that by 3 wks, all the mineralized cartilage in the diaphyseal region has disappeared, leaving a void where bone formation should have occurred (Figure 6J, also see Figures 6L and S3). This void is eventually invaded by soft tissues and the adjacent muscle (marked s and m, respectively, in Figure 6J). The resorption of bone at 3 wks of age is clearly visible in the proximal femur of mutant animals where a portion of that bone is missing (Figure 6L, black arrow). The inability to complete osteoblast differentiation in the Bmp2C/C; Bmp4C/C; Prx1::cre mice results in strikingly defective limb morphology at 1 wk of age (compare Figure S3G and S3H). + +To determine if any osteoblast differentiation occurs in Bmp2C/C; Bmp4C/C; Prx1::cre forelimbs, we examined osteoblast-related gene expression (Figure 7). In the control mice, osteoblasts lining the surface of trabecular and cortical bone express type I collagen, runx2, and osterix genes at both 1 wk and 3 wks after birth (Figure 7A–7D and 7I–7L). In the absence of BMP2 and BMP4, cells which are fibroblastic in appearance (Figure 6H) are found adjacent to the mineralized cartilage surfaces. These cells express amounts of type I collagen and runx2 comparable to control osteoblasts, but their expression of osterix, while noticeable at 1 wk, becomes almost undetectable at 3 wks (Figure 7E–7H and 7M–7P). These results allow us to conclude that the fibroblast-like cells resident in the bone shaft (Figure 6H) may be osteoprogenitor cells unable to differentiate into mature osteoblasts in the absence of BMP2 and BMP4. + +Discussion + +Using a genetic approach to remove activity of different Bmp genes during limb development, we have found that BMP2, BMP4, and BMP7 either individually or in combination are not required for specification of normal digit identities. However, BMP2 and BMP4 are required in concert to promote condensation of the posterior digit anlagen. We have also shown that chondrogenesis can be initiated and chondrogenic differentiation will take place even in the absence of both BMP2 and BMP4 or BMP2 and BMP7. On the other hand, BMP2 and BMP4 together are required for completion of osteogenesis. + +BMP Signaling and Pattern Formation + +It has been proposed that BMP signaling may be involved in digit patterning as a secondary signal originating in the ZPA [7,11,43] or as a later mediator of interdigital patterning information [15]. In particular, BMP2, a secreted factor that is induced by SHH [8] in the posterior limb bud, has been suggested to act as a true morphogen secondary to SHH signaling to properly pattern the limb digits. However, inactivation of Bmp2 during early limb bud development clearly does not cause any digit patterning defects. BMPs are known, in many cases, to function redundantly [44]. In considering potential secondary signals mediating ZPA activity, the most relevant family member to consider is BMP7 as, like BMP2 (and unlike BMP4), it is positively regulated by SHH signaling [7]. However, Bmp7 expression does not overlap with Bmp2 in the early mouse limb bud [45], and in any case, mice in which we conditionally knocked out Bmp2 in the Bmp7−/− background do not show any obvious digit patterning defect other than the missing final phalanx in digit III. Although still poorly understood, the final phalanges form at the tip of every digit through a process distinct from the more proximal phalanges [38]. Our data implicate BMP signaling in this process. + +At later stages, BMP signaling has been suggested to play a role in mediating the transfer of positional information from the interdigital mesenchyme to specify the identity of the adjacent digits [15]. Application of the BMP antagonist noggin to the interdigital tissue at the time of digit condensation results in loss of phalanges and a resultant pattern that can be interpreted as an anterior homeotic transformation in digit identity. However, increasing BMP concentrations interdigitally with exogenous protein does not result in the reciprocal posterior transformations, and the noggin experiments can also be interpreted simply as a block in the condensation of the primordia of distal phalanges when BMP activity is completely abolished [38]. Decreasing the level of interdigital BMP signaling, without abolishing it, provides an opportunity for differentiating between these models. If BMP signaling were only required as a permissive factor for chondrogenesis, partial reduction in the level of signaling might have no effect. However, if levels of BMP signaling were instructive in terms of digit identities, then a substantial decrease in the amount of BMP ligand present should cause transformations in digit identity. This is not what is observed, either in limbs deficient in both BMP2 and BMP4 or in those deficient in BMP2 and BMP7. It should be noted that the interdigital expression domains form several days after recombination of the floxed BMP alleles is complete. Moreover, there is no detectable compensatory upregulation of BMP7 in the interdigital mesenchyme of the BMP2, BMP4–deficient limbs (compare Figure 4G and Figure 4H). Thus, we conclude that BMP signaling is not likely to be responsible in a quantitative fashion for the instructive role of interdigital mesenchyme in establishing digit identities. However, in the Bmp2C/C; Bmp4C/C; Prx1::cre forelimbs, there is a complete loss of posterior digit ray condensations, presumably because the total level of BMP signaling falls below the threshold for initiation of condensation, as previously seen in cases where BMP antagonists have been used [27,28]. + +It is worth noting that the defects in chondrogenesis seen in the BMP2, BMP4–deficient limbs are much more severe proximally than distally. This is particularly significant because proximal elements condense and differentiate before distal ones. Thus, if the conditional alleles were partially recombined at early stages and only completely recombined at later stages, it is the distal elements, formed after greater recombination has taken place, that should be more severely affected. Since it is the proximal elements which are more severely affected, the explanation for the difference in severity must lie elsewhere. This is most likely explained by compensation in the distal limb bud by BMP2, BMP4, and BMP7 produced in the AER. + +Compensation by BMPs in the AER also likely explains a difference seen between the Bmp4C/C; Prx1::cre mice in our experiments and a recently published analysis [16], which used the identical Bmp4 conditional allele and Prx1::cre driver. The earlier report, like ours, described preaxial and postaxial polydactyly. However, they observed these phenotypes with less variability and, moreover, described multiple preaxial and postaxial ectopic digits, while we saw, at most, a single preaxial and postaxial digit. The difference is that the mice analyzed in the previous report had one null allele and one conditional allele, while ours had two conditional alleles. Their mice, therefore, lost a half-dose of BMP4 from the AER in addition to complete loss in the mesenchyme, while our conditional alleles were only recombined in the mesenchyme by the Prx1::cre driver. It is also possible that the difference in severity relates to the time required to recombine one as opposed to two floxed alleles; however, we do not favor this explanation as all detectable BMP4 expression is lost prior to any morphological differences between the mutant and wild-type limb buds. Finally, differences in genetic background could contribute to the differences as the Prx1::cre line is outbred. + +A final aspect of patterning previously associated with BMP signaling is interdigital apoptosis. Consistent with this, we see some syndactyly in the BMP2-deficient limbs. The fact that this is variable, and limited to the 3/4 interdigit, implies that the threshold for induction of apoptosis is low enough that loss of BMP2, BMP4 or BMP7 or BMP2 and BMP7 still leaves sufficient BMP signaling for most interdigital cell death to occur normally. However, loss of both BMP2 and BMP4 results in complete soft tissue syndactyly, i.e., webbing between all the digits. Although BMP activity has been previously implicated in regulating interdigital apoptosis, this is the first genetic verification of their requirement for this process. + +BMP Signaling and Skeletal Development + +As discussed above, our data indicate that threshold levels of BMP signaling are required for initiating chondrogenic condensation, consistent with prior results [26–28]. However, once chondrogenesis begins, cartilage differentiation can be sustained, albeit with some detectable delay, in the absence of BMP2 and BMP4 or in the absence of BMP2 and BMP7. Thus, the expression of either BMP5, BMP6, and BMP7 (in the absence of BMP2 and BMP4) or the expression of BMP4, BMP5, and BMP6 (in the absence of BMP2 and BMP7) is sufficient to support mesenchymal condensation and chondrocyte differentiation in the developing limb. + +In one set of previous studies implicating BMP signaling in chondrogenesis, Yoon et al. [26] showed that if mice lose both Alk3 (BmprIa) and Alk6 (BmpIb), two of the three type I receptors used in BMP signal transduction, there is a dramatic decrease in the size of skeletal primordial due to a reduction of proliferation and increase in apoptosis. The size of the skeletal elements in Bmp2C/C; Bmp4C/C; Prx1::cre limbs are also decreased compared to those of control mice, although not to the extent seen in the double receptor mutant. We do not know if reduced size of the skeletal elements in our study is similarly due to reduced proliferation and increase in apoptosis, lack of replacement by bone, or a combination of these. It is possible that a triple mutant removing BMP2, BMP4, and BMP7 activities would have a similar phenotype as the BmprIa, BmprIb double mutant, although these could also be compensated from other BMPs. We do know, however, that osteogenesis is defective in these mice, although bone formation proceeds normally in siblings retaining one functional copy of either the Bmp2 or Bmp4 gene (Figure S4), as it does in mice completely deficient in BMP2 and BMP7 (Figures 1 and S2). + +The phenotype we observe in limbs of Bmp2C/C; Bmp4C/C; Prx1::cre mice is similar to the phenotype reported for osterix knockout mice [46], which exhibited severe defects in osteoblastic differentiation. Since we observe fibroblastic cells adjacent to mineralized cartilage that have characteristics of osteoprogenitor cells but fail to express osterix in the absence of both BMP2 and BMP4, one important role of BMP2 and BMP4 during endochondral ossification may be to induce osterix gene expression in osteoprogenitors. Strikingly, however, we do observe osterix expression in the bone collar of E16.5 mice missing functional copies of both the Bmp2 and Bmp4 genes (K. Tsuji and A. Bandyopadhyay, unpublished data). This suggests that varying levels of expression from different BMP genes during preaxial and postaxial development result in fluctuations in total levels of BMP signaling, which at distinct times are above or below the threshold for supporting osteogenesis. While many osteogenic BMP molecules, apart from BMP2 and BMP4, such as BMP5, BMP6, and BMP7, are expressed during the endochondral process, our data suggest that these BMP molecules cannot compensate for the combined loss of BMP2 and BMP4 in bone formation. Recent biochemical and genetic experiments have suggested that individual BMPs have identical functions. BMP2, BMP4, BMP5, BMP6, and BMP7 can utilize the same type I (Alk2, Alk3, and Alk6) and type II receptors (BMP RII, ActRII, and ActRIIb) [47]. Once the complex between ligand and receptors is formed, BMP2, BMP4, BMP5, BMP6, and BMP7 direct the phosphorylation of the same set of BMP receptor–specific Smads (1, 5, or 8), and the signal that is transduced by each BMP appears to be identical in the skeletal target cells [48–51]. BMPs have also been shown to activate MAPK and AKT pathways and, based on current published information, each of the osteogenic BMPs appears to have the same capacity to activate these signaling pathways [52–54]. When this information is considered along with our observation that one allele of either Bmp2 or Bmp4 can rescue the osteoblast differentiation phenotype observed in Bmp2C/C; Bmp4C/C; Prx1::cre mice (see Figures 1I, 1J, 1Q, and 1R and S4), we favor the hypothesis that bone formation requires a threshold amount of BMP signaling which is not met when both BMP2 and BMP4 are completely absent. We cannot, however, exclude the possibility that BMP2 and BMP4 have distinct functions in osteoblastogenesis. Our analysis of skeletogenesis indicates that BMP2 and BMP4 are prerequisite for osteoblastogenesis while less important for chondrogenesis. These studies provide the first evidence linking BMP2 and BMP4 with bone formation in an in vivo setting where the preceding events of chondrogenesis are not compromised. + +Materials and Methods + +Reagents. + +Xylene, formamide, and acetic anhydride were purchased from Fisher Scientific (http://www.fishersci.com). Alizarin red S, toluidine blue, paraformaldehyde, polyvinylpyrrolidone, proteinase K, RNase A, tRNA, and dithiothreitol were purchased from Sigma (http://www.sigmaaldrich.com). Alcian blue 8GX was purchased from Electron Microscopy Science (http://www.emsdiasum.com). Dextran sulfate was purchased from Pharmacia (http://www.pharmacia.com). + +Generation of Bmp2 and Bmp4 conditional null allele. + +Mice carrying floxed Bmp2 allele were available through a material transfer agreement between Harvard University and Wyeth Pharmaceuticals. In these mice, LoxP sites were integrated to excise the entire protein-coding region in exon 3 of the Bmp2 gene. Generation of Bmp4 conditional allele has been described [16,55]. The Prx1::cre transgene has been described [32]. + +Generation of Bmp2, Bmp4 double conditional mice and Bmp2 conditional, Bmp7 mutant mice. + +Bmp2C/C animals were crossed with Bmp4C/C animals to generate Bmp2C/+; Bmp4C/+ animals. These animals were crossed with Bmp2C/C or Bmp4C/C animals to generate Bmp2C/C; Bmp4C/+ or Bmp2C/+; Bmp4C/C animals, respectively. Bmp2C/C; Bmp4C/+ or Bmp2C/+; Bmp4C/C animals were crossed with each other to generate Bmp2C/C; Bmp4C/C animals. Bmp2C/C; Bmp4C/C females were crossed with males bearing Prx1::cre transgene to generate Bmp2C/+; Bmp4C/+; Prx1::cre animals. Bmp2C/+; Bmp4C/+; Prx1::cre males were crossed with Bmp2C/C; Bmp4C/C females to generate Bmp2C/C; Bmp4C/C; Prx1::cre animals. Bmp2C/C; Bmp4C/C mice carrying floxed alleles of the Bmp genes without Prx1::cre are wild-type (WT). Genotyping of animals were done by PCR analyses with the primers (5′–3′) GTGTGGTCCACCGCATCAC (AHP2–9) and GGCAGACATTGTATCTCTAGG (AHP2–35) for Bmp2 and AGACTCTTTAGTGAGCATTTTCAAC (No. 79 [order of primers tried]) and AGCCCAATTTCCACAACTTC (No. 80) for Bmp4 following extraction of genomic DNA from embryonic or adult tail. For Bmp2, the floxed allele amplifies as a 545-bp product, while the wild-type allele amplifies as a 474-bp product. For Bmp4, the floxed allele amplifies as a 220-bp product, while the wild-type allele amplifies as a 180-bp product. Protocols utilized for mouse experiments were approved by the Harvard University Institutional Animal Care and Use Committee (Cliff Tabin #02735). + +The strategy for generating a Bmp2 conditional null in the BMP7−/− background is similar to the strategy used for generating the Bmp2, Bmp4 double conditional allele with three important differences. First, the Bmp7−/− (strain kindly donated by Dr. Liz Robertson [12]) is not a conditional allele but a null mutation. Second, Bmp7−/− mice do not survive past birth due to kidney failure [12] and hence cannot be maintained as homozygous for the null allele. Third, Bmp2 and Bmp7 are on the same chromosome (Chromosome 2). To obtain Bmp2C/C, Bmp7−/−; Prx1::cre we crossed Bmp2C/C, Bmp7−/+ females with Bmp2C/+, Bmp7−/+; Prx1::cre males. Bmp2C/C, Bmp7−/−; Prx1::cre progeny were obtained at a rate slightly higher than 1 in 16. For genotyping of Bmp7 locus we used the following set of primers: Bmp7_ExI_5′ (5′–3′) TCGCCTGCAGCAAGTGACCTCGGGTC; Bmp7_ExI_3′ (5′–3′) TAGGGGTAGGAGAAGCCCTGTCCGTCC and Beta-geo 5′ of 3′ (5′–3′) CTGCATACGCTTGATCCGGCTACCTGC. From a wild-type chromosome Bmp7_ExI_5′ and Bmp7_ExI_3′ amplifies an approximately 423-bp PCR product, while from the lacZ insertion mutant this pair fails to amplify any product. Bmp7_ExI_5′ and Beta-geo 5′ of 3′ (this primer is designed from the Beta-geo cassette) amplifies an approximately 1-kb PCR product from the lacZ inserted chromosome. + +Skeletal analysis. + +Our protocol is an adaptation of the procedure described [56] previously. Newborn and older animals were killed using carbon dioxide. Skin, viscera, and adipose tissues were removed within 2 h after death, and samples were fixed in 95% ethanol for 5 d. Samples were then placed in acetone to remove residual fat for 2 d. For E13.5 embryos, skin was not removed. The dehydrated animals were stained with 0.015% w/v Alcian blue and 0.005% w/v Alizarin red in a 1:19 mixture of glacial acetic acid and 70% ethanol. The younger animals were stained for 5 h at 37 °C and then overnight at room temperature, while the newborns were stained overnight at 37 °C followed by 2 d at room temperature. The stained skeletons were kept in 1% KOH, with occasional change of solution, until cleared. The cleared skeletons were transferred into 100% glycerol and photographed. + +Acridine orange staining for apoptotic cells. + +A working stock of 5 mg/ml acridine orange was diluted 1:10,000 in PBS. Dissected fresh embryos were transferred to the working solution of acridine orange and incubated for 30 min at 37 °C in the dark. Embryos were then washed twice in PBS for 5 min and viewed with a fluorescence microscope. + +Whole mount in situ hybridization. + +In situ hybridization staining on whole embryos was performed as described [57]. For section in situ hybridizations, please see below. + +Histology, section in situ hybridizations, and immunohistochemistry. + +For histology and section in situ hybridization, samples were fixed in 4% paraformaldehyde in PBS at 4 °C overnight. Newborn and older animals were then decalcified in Tris buffer containing 10% EDTA and 7.5% polyvinylpyrrolidone (pH 7.5) at 4 °C for 3 wks. Samples were then dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. 8μm sections were collected and stained with hematoxylin and eosin (H & E) or toluidine blue following standard procedure. + +Section in situ hybridization with digoxigenin labeled probes was performed as described [57]. Section in situ hybridization with radiolabeled probes was performed as described [58] with a small modification. Briefly, sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Sections were postfixed in 4% paraformaldehyde in PBS at room temperature for 15 min, digested with 10 μg/ml proteinase K at room temperature for 10 min, and again fixed in 4% paraformaldehyde at room temperature for 10 min and acetylated in the solution containing 0.2% hydrochloric acid, 0.1 M triethanol amine, and 0.25% acetic anhydride at room temperature for 10 min. Sections were then dehydrated in increasing concentrations of ethanol and air-dried. Hybridization was performed in a humidified chamber in a solution containing 50% formamide, 10% dextran sulfate, 1× Denhardt's solution, 0.6 M sodium chloride, 0.01 M Tris buffer (pH 7.5), 0.001 M EDTA, 0.05 M dithiothreitol, 0.25% SDS, 200 μg tRNA, and 35S-labeled cRNA probe at the final concentration of 5 × 106 cpm/ml at 55 °C for 16 h. After hybridization, sections were washed with a solution containing 5× SSC and 10 mM dithiothreitol at 50 °C for 30 min, incubated in a solution containing 50% formamide, 2× SSC, and 10 mM dithiothreitol at 65 °C for 30 min, treated in a solution containing 10 μg/ml RNase A in TNE [0.01 M Tris buffer (pH 7.6), 0.5 M sodium chloride, and 0.001 M EDTA] at 37 °C for 30 min and then washed with 50% formamide, 2× SSC, and 10 mM dithiothreitol at 65 °C for an additional 30 min followed by two washes with 2× SSC/10 mM dithiothreitol at 65 °C for 30 min, and 0.1× SSC/10 mM dithiothreitol at 65 °C for 30 min. + +To visualize the signal, sections were dipped into NTB-2 emulsion (Kodak, http://www.Kodak.com) and placed at 4 °C. After developing, sections were counterstained with H&E. The 35S-labeled cRNA probes were transcribed from plasmids encoding type I collagen [59], runx2 [60], osteopontin [61], and osterix [46]. Sense and antisense probes were synthesized from linearized plasmids using a Riboprobe Combination System (Promega, http://www.promega.com). + +Supporting Information + +Figure S1 + +Chondrogenic Differentiation in Bmp2C/C; Bmp4C/C; Prx1::cre Animals + +(A–C) In BMP2, BMP4–deficient limbs, there is only one zeugopod present, and it is often fused with the stylopod. All panels are from E17.5 Bmp2C/C; Bmp4C/C; Prx1::cre embryos. (A) Hematoxylin and eosin–stained section of the forelimb. Alcian blue– and Alizarin red–stained skeletons shown in (B) (forelimb) and (C) (hindlimb). Black arrow shows the fusion of the zeugopod and stylopod in BMP2, BMP4–deficient limbs. + +(D and E) Acridine orange–stained limbs from E11.5 wild-type (D) and Bmp2C/C; Bmp4C/C; Prx1::cre embryos (E). + +(F and G) Acridine orange–stained hindlimbs from E13.5 wild-type (F) and Bmp2C/C; Bmp4C/C; Prx1::cre (G) embryos. + +(H–M) In the absence of BMP2 and BMP4, condensation begins in the limb normally. Whole mount in situ staining of Sox9 mRNA in embryonic limb buds from E10.5 (H and I) and E11.5 (J–M) embryos. (H, J, and L) From wild-type embryos, (I, K, and M) from Bmp2C/C; Bmp4C/C; Prx1::cre embryos. (H, I, J, and K) Forelimbs, (L and M) hindlimbs. + +(N and O) In situ hybridizations with Col II mRNA probes of sections derived from E12.5 wild-type (N) and Bmp2C/C; Bmp4C/C; Prx1::cre (O) embryos. + +(P–S) Bmp6 mRNA expression is not increased in E13.5 Bmp2C/C; Bmp4C/C; Prx1::cre cartilage. Bmp6 in situ on sagittal sections from wild-type (Q) and Bmp2C/C; Bmp4C/C; Prx1::cre (S) embryonic forelimbs. (P and R) Bright field images of (Q) and (S), respectively. + +(23 MB TIF) + +Click here for additional data file. + +Figure S2 + +Skeletal Differentiation Occurs Normally in Absence of BMP2 + +(A) Toluidine blue–stained section of newborn femurs from Bmp2C/C animals. + +(B–D) Marker analyses. Sections of femurs from Bmp2C/C animals hybridized with Col II (B), Col X (C), and Col I (D) mRNA probes. + +(E) Toluidine blue–stained section of newborn femurs from Bmp2C/C; Prx1::cre animals. + +(F–H) Marker analyses. Sections of femurs from Bmp2C/C; Prx1::cre animals hybridized with Col II (F), Col X (G), and Col I (H) mRNA probes. + +(15 MB TIF) + +Click here for additional data file. + +Figure S3 + +BMP2, BMP4–Deficient Limb Skeleton Is Resorbed + +Alcian blue– and Alizarin red–stained skeletons of forelimbs (A–D) and hindlimbs (E–F) from 1-wk-old (A, B, E, and F) and 3-wk-old (C and D) animals. (A, C, and E) Wild-type animals, (B, D, and F) Bmp2C/C; Bmp4C/C; Prx1::cre animals. The limbs of Bmp2C/C; Bmp4C/C; Prx1::cre (H) animals are severely defective compared to a wild-type (G) animal at 1 wk of age. + +Please note that the proximal part of the femur is present at 1 wk of age but missing at 3 wks (refer to Figure 6L). + +(12 MB TIF) + +Click here for additional data file. + +Figure S4 + +Osteogenesis Occurs in Mice Lacking Three of Four Alleles of Bmp2 and Bmp4 Combined + +Toluidine blue–stained sections of femurs from adult control, Bmp2C/+; Bmp4C/C; Prx1::cre, and Bmp2C/C; Bmp4C/+; Prx1::cre animals. + +(11 MB TIF) + +Click here for additional data file. + +Acknowledgements + +We thank Dr. Andy Dudley for his help in designing mouse genotyping strategies. We also thank Jose Rivera-Feliciano and Dr. Douglas Kim for helpful discussions. We are immensely grateful to Drs. Brigid Hogan and Holger Kulessa for their kind gift of the Bmp4 conditional allele and Drs. Liz Robertson and Andy Dudley for the Bmp7 knockout allele. We would like to acknowledge the help of Dr. Ernestina Schipani and Jan Saxton for help with histology. + +This manuscript is dedicated to the memory of Dr. Holger Kulessa, whose efforts contributed to the groundwork for this study and whose dedication and intense commitment to excellence in science are greatly missed. + +Abbreviations + +AER - apical ectodermal ridge + +BMP - bone morphogenetic protein + +E - embryonic day + +FGF - fibroblast growth factor + +SHH - Sonic Hedgehog + +Shh - sonic hedgehog + +ZPA - zone of polarizing activity + +Figures and Tables + +Figure 1 + +An Allelic/Nonallelic Series of BMP-Deficient Limbs + +(A–D) Prx1::cre efficiently recombines Bmp2 and Bmp4 conditional alleles in the limbs. Bmp2 (A and B) and Bmp4 (C and D) whole mount mRNA in situ hybridization in the limb. Wild-type (A) and Bmp2C/C; Prx1::cre (B) are forelimbs from E10.5 mouse embryos. Mesenchymal expression [asterisk (A)] of Bmp2 is abolished in Bmp2C/C; Prx1::cre embryo while the AER expression [black arrow (A and B)] of Bmp2 persists. Note that pink staining in the central region of the limb bud in (B) is nonspecific background. Wild-type (C) and Bmp4C/C; Prx1::cre (D) are forelimbs from E10.5 mouse embryos. Mesenchymal expression [red arrow (C)] of Bmp4 is abolished in Bmp4C/C; Prx1::cre embryo while the AER expression [black arrow (C and D)] of Bmp4 persists. + +(E–T) Depletion of BMP2 and BMP4 together causes severe limb skeletal defects. (E–T) Whole mount skeletons from newborn animals stained with Alcian blue and Alizarin red. (E–L) Forelimbs, (M–T) hindlimbs. (E and M) Wild-type, (F and N) Bmp2C/C; Prx1::cre, (G and O) Bmp4C/C; Prx1::cre, (H and P) Bmp7 −/−, (I and Q) Bmp2C/C; Bmp4+/C; Prx1::cre, (J and R) Bmp2+/C; Bmp4C/C; Prx1::cre, (K and S) Bmp2C/C; Bmp4C/C; Prx1::cre, (L and T) Bmp2C/C, Bmp7 −/−; Prx1::cre. Thin red arrow in (F), (I), and (L), defective scapula; thick red arrow in (T), failure of fibula to articulate with knee, and thick black arrow in (L) and (T), missing phalanx in digit III. + +Figure 2 + +Depletion of BMP Signaling Causes Interdigital Syndactyly + +(A–D) Forelimb of adult wild-type (A) and Bmp2C/C; Prx1::cre mouse (B) and hindlimbs of newborn wild-type (C) mouse and newborn Bmp2C/C; Bmp4C/C; Prx1::cre (D) mouse. The black arrow in (B) shows soft tissue syndactyly in Bmp2C/C; Prx1::cre mouse. + +(E and F) Wild-type and Bmp2C/C; Bmp4C/C; Prx1::cre, respectively, showing acridine orange–stained hindlimbs of E15.5 mouse embryos. Acridine orange stain is in yellow. + +(G and H) Enlarged views of selected regions from (E) and (F), respectively. Black arrow in (G) and (H) show acridine orange–stained apoptotic cells in the interdigital mesenchyme, and asterisk in (H) shows the remnant of the AER. + +(I and J) Fgf8 mRNA expression in the hindlimbs of E13.5 wild-type (I) and Bmp2C/C; Bmp4C/C; Prx1::cre (J) embryos. The thick black arrows in (J) show Fgf8 mRNA expression. + +Figure 3 + +Patterning Defects in Limbs Deficient of Different Combinations of BMP Molecules + +(A–D) Bmp4 expression in limb buds from E11.5 wild-type (A and C) and Bmp2C/C, Bmp7 −/−; Prx1::cre (B and D) mouse embryos. (A and B) Forelimbs, (C and D) hindlimbs. + +(E–H) Shh (E and F) and Fgf8 (G and H) expression in E11.5 forelimbs and hindlimbs, respectively. (E and G) Wild-type embryos, (F and H) Bmp2C/C; Bmp4C/C; Prx1::cre embryos. + +(I–P) Sox9 (I–K) and Msx2 (M–O) expression in E12.5 wild-type (I and M) and Bmp2C/C; Bmp4C/C; Prx1::cre mouse embryonic forelimbs (J and N) and hindlimbs (K and O). (L and P) Shh expression in E12.5 wild-type (L) and Bmp2C/C; Bmp4C/C; Prx1::cre (P) embryonic hindlimbs. + +The red brackets in (F) and (H) show the broadened domains of expressions of Shh and Fgf8, respectively. + +Figure 4 + +Chondrogenesis Starts and Proceeds Normally Even in the Absence of BMP2 and BMP4 + +(A and B) Whole mount skeletons from E13.5 embryos that are stained with alcian blue. (A) Wild-type embryo, (B) Bmp2C/C; Bmp4C/C; Prx1::cre embryo. Black arrow in (B) shows the fusion of the zeugopod and stylopod. + +(C and D) Hematoxylin and eosin–stained sagittal sections of humeri from E13.5 wild-type (C) and Bmp2C/C; Bmp4C/C; Prx1::cre (D) embryos. Thick red arrow in (C) shows the hypertrophic region. + +(E and F) Sagittal sections of wild-type (E) and Bmp2C/C; Bmp4C/C; Prx1::cre (F) humeri from E15.5 embryos were hybridized with digoxigenin-labeled antisense rioprobes for ColX. + +(G and H) Sagittal sections of forelimbs from E13.5 wild-type and BMP2, BMP4–deficient embryos, respectively, stained with radioactive riboprobes for Bmp7 mRNA. (G′) and (H′) show the bright field views of (G) and (H), respectively. + +Figure 5 + +In the Absence of BMP2 and BMP4, Osteogenesis Begins During Early Embryonic Development + +(A–D) Sagittal sections of forelimb from E15.5 embryos. (A and B) Stained with toluidine blue. (A) Distal ulna from wild-type embryo, (B) distal ulna/radius from Bmp2C/C; Bmp4C/C; Prx1::cre embryo. Mineralized cartilage is shown by dark purple and osteoid is shown by light blue (red asterisk). (C and D) Humeri sections were hybridized with digoxigenin labeled riboprobe for ColI. (C) Section of wild-type humerus from E15.5 embryo, (D) from Bmp2C/C; Bmp4C/C; Prx1::cre E15.5 embryo. + +(E–H) Sagittal sections of zeugopod from E17.5 embryos. (E and F) Stained with toluidine blue while G and H are stained for TRAP (brown stain, red arrow). (E and G) E17.5 proximal radius from wild-type embryo, (F and H) E17.5 proximal ulna/radius from Bmp2C/C; Bmp4C/C; Prx1::cre embryo. Although vascularization occurs in the absence of BMP2 and BMP4, few osteoclasts are observed in the mineralized cartilage in Bmp2C/C; Bmp4C/C; Prx1::cre mice. Please note all the panels other than (G) and (H) are photographed at ×20, while (G) and (H) were photographed at ×40. + +(I and J) Sections of newborn proximal radius (I) from control animal and newborn proximal ulna/radius (J) from Bmp2C/C; Bmp4C/C; Prx1::cre animal were stained with toluidine blue. Red arrow shows the bone marrow cavity in (E) and (I). Note that bone collar (red *) forms at the right time and place in the absence of BMP2 and BMP4. Also note that since Bmp2C/C; Bmp4C/C; Prx1::cre mice occasionally have only one bone in zeugopod and the elbow joint is occasionally fused, it is difficult to distinguish between the ulna and the radius. + +Figure 6 + +Defects in Bone Formation in the Absence of BMP2 and BMP4 + +Toluidine blue staining of sagittal sections of distal femurs. + +(A–E) Control femurs at 1 wk (A) and 3 wks (D) of age. Boxed areas in (A) are enlarged in (B) and (C). Boxed area in (D) is enlarged in (E). Blue arrows in (C) point to osteoblast cells lining the surface of cortical bone. Pink arrows in (G) show similar cells to those in (C) in Bmp2C/C; Bmp4C/C; Prx1::cre femurs. Red star in (A) and (D) marks the secondary ossification center. Pale blue–stained tissue in (E), marked by red arrows, is trabecular bone. bm, marks the bone marrow cavity in (D). + +(F–J) Bmp2C/C; Bmp4C/C; Prx1::cre femurs at 1 wk (F) and 3 wks (J) of age. Boxed areas in (F) are enlarged in (G), (H), and (I). (A), (D), (F), and (J) are shown with the same magnification. Note that there are defects in bone formation but no defect in osteoclast mediated bone resorption. Mineralized tissue in the mid shaft of femur is almost resorbed at 3 wks. (H) Fibroblast-like cells present in the bone shaft of Bmp2C/C; Bmp4C/C; Prx1::cre mouse. Red star in (I) shows the osteoclasts invading the Bmp2C/C; Bmp4C/C; Prx1::cre femurs at 1 wk of age. s and m mark soft tissues and muscle in (J), respectively. + +(K and L) Three-wk-old wild-type (K) and Bmp2C/C; Bmp4C/C; Prx1::cre (L) hindlimb skeletons stained with Alcian blue and Alizarin red. The black arrow shows the missing part of proximal femur. + +Figure 7 + +Osteoblast Maturation Is Inhibited in the Absence of BMP2 and BMP4 + +(A–D) Distal femurs from control mice at 1 wk of age. + +(E–H) Distal femurs from Bmp2C/C; Bmp4C/C; Prx1::cre mice at 1 wk of age. + +(I–L) Distal femurs from control mice at 3 wks of age. + +(M–P) Distal femurs from Bmp2C/C; Bmp4C/C; Prx1::cre mice at 3 wks of age. + +In situ hybridization of early osteoblast differentiation-related marker genes Col I (B, F, J, and N), runx2 (C, G, K, and O) and osterix (D, H, L, and P). + +Footnotes + +Competing interests. The authors have declared that no competing interests exist. + +A previous version of this article appeared as an Early Online Release on November 6, 2006 (doi:10.1371/journal.pgen.0020216.eor). + +Author contributions. AB, KT, BDH, VR, and CJT conceived and designed the experiments. AB, KT, KC, and BDH performed the experiments. AB, KT, VR, and CJT analyzed the data. AB and KT wrote the paper. + +Funding. This work was supported by a grant from the National Institutes of Health (P01 DK56246 to CJT) and by funds from the Forsyth Institute and Harvard School of Dental Medicine (to VR). diff --git a/src/ontogpt/evaluation/craft/database/all/17201918.ann b/src/ontogpt/evaluation/craft/database/all/17201918.ann new file mode 100644 index 000000000..8d2ca388f --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17201918.ann @@ -0,0 +1,1513 @@ +T1 PR:000014888 0 5 SirT1 +T2 GO:0065007 6 15 modulates +T3 CHEBI:50114 20 28 estrogen +T4 PR:000045358 29 36 insulin +T5 GO:0007567 76 81 natal +T6 GO:0030879 82 110 development of mammary gland +T7 UBERON:0001911 97 110 mammary gland +T8 NCBITaxon:10088 114 118 mice +T9 CHEBI:50114 144 152 Estrogen +T10 PR:000045358 157 164 insulin +T11 UBERON:0001911 218 231 mammary gland +T12 GO:0030879 218 243 mammary gland development +T13 UBERON:0000310 248 254 breast +T14 http://purl.obolibrary.org/obo/MONDO_0007254 248 261 breast cancer +T15 PR:000014888 263 268 SirT1 +T16 GO:0065007 320 328 regulate +T17 PR:000045358 333 340 insulin +T18 NCBITaxon:1 366 375 organisms +T19 GO:0071159 441 446 NF-κB +T20 NCBITaxon:40674 451 460 mammalian +T21 PR:000014888 476 481 SirT1 +T22 GO:0065007 482 491 regulates +T23 UBERON:0001911 516 529 mammary gland +T24 GO:0030879 516 541 mammary gland development +T25 PR:000014888 614 619 SirT1 +T26 PR:000014888 645 650 SirT1 +T27 NCBITaxon:10088 661 665 mice +T28 PR:000014888 677 682 SirT1 +T29 PR:000014888 694 699 SirT1 +T30 NCBITaxon:10088 706 710 mice +T31 NCBITaxon:10088 754 758 mice +T32 PR:000014888 808 813 SirT1 +T33 SO:0000704 814 818 gene +T34 PR:000014888 820 825 SirT1 +T35 NCBITaxon:10088 856 860 mice +T36 UBERON:0001911 996 1009 mammary gland +T37 GO:0030879 996 1021 mammary gland development +T38 GO:0007565 1036 1044 pregnant +T39 NCBITaxon:10088 1045 1049 mice +T40 CHEBI:50114 1075 1083 estrogen +T41 UBERON:0006849 1168 1176 scapular +T42 PR:000014888 1216 1221 SirT1 +T43 NCBITaxon:10088 1227 1231 mice +T44 PR:000014888 1296 1301 SirT1 +T45 NCBITaxon:10088 1307 1311 mice +T46 UBERON:0000058 1330 1336 ductal +T47 GO:0035239 1330 1350 ductal morphogenesis +T48 GO:0007565 1360 1368 pregnant +T49 PR:000014888 1369 1374 SirT1 +T50 NCBITaxon:10088 1380 1384 mice +T51 GO:0007595 1396 1405 lactation +T52 CHEBI:50114 1463 1471 Estrogen +T53 UBERON:0000058 1510 1516 ductal +T54 GO:0035239 1510 1530 ductal morphogenesis +T55 CHEBI:50114 1542 1550 estrogen +T56 PR:000008947 1589 1612 IGF-1 binding protein-1 +T57 GO:0010467 1613 1623 expression +T58 PR:000014888 1627 1632 SirT1 +T59 UBERON:0000310 1638 1645 mammary +T60 UBERON:0000479 1646 1653 tissues +T61 PR:000003293 1671 1675 IκBα +T62 GO:0010467 1676 1686 expression +T63 CHEBI:50114 1724 1732 estrogen +T64 UBERON:0000058 1784 1790 ductal +T65 GO:0035239 1784 1804 ductal morphogenesis +T66 PR:000000134 1820 1824 TNFα +T67 PR:000003293 1879 1883 IκBα +T68 PR:000014888 1887 1892 SirT1 +T69 PR:000014888 1905 1910 SirT1 +T70 GO:0051716 1944 1961 cellular response +T71 PR:000014888 2006 2011 SirT1 +T72 GO:0065007 2012 2021 modulates +T73 GO:0040008 2060 2077 growth regulation +T74 UBERON:0001911 2082 2095 mammary gland +T75 GO:0030879 2082 2107 mammary gland development +T76 NCBITaxon:10088 2111 2115 mice +T77 PR:000014888 2117 2122 SirT1 +T78 GO:0065007 2136 2145 regulates +T79 GO:0010467 2150 2160 expression +T80 PR:000008947 2164 2187 IGF-1 binding protein-1 +T81 CHEBI:50114 2242 2250 estrogen +T82 UBERON:0000058 2301 2307 ductal +T83 GO:0035239 2301 2321 ductal morphogenesis +T84 PR:000014888 2377 2382 SirT1 +T85 http://purl.obolibrary.org/obo/MONDO_0004992 2420 2436 malignant growth +T86 UBERON:0003244 2440 2458 mammary epithelial +T87 CL:0002327 2440 2464 mammary epithelial cells +T88 NCBITaxon:40674 2481 2490 Mammalian +T89 PR:000014888 2491 2496 SirT1 +T90 CHEBI:13389 2520 2553 nicotinamide adenine dinucleotide +T91 PR:000043452 2564 2571 histone +T92 CHEBI:15358 2564 2571 histone +T93 PR:000014888 2592 2597 SirT1 +T94 PR:000014888 2631 2635 Sir2 +T95 PR:000014888 2689 2693 Sir2 +T96 PR:000014888 2708 2712 Sir2 +T97 GO:0016458 2729 2738 silencing +T98 GO:0007618 2781 2787 mating +T99 SO:0001789 2781 2797 mating type loci +T100 GO:0005840 2822 2831 ribosomal +T101 GO:0010467 2861 2871 expression +T102 PR:000014888 2892 2896 Sir2 +T103 NCBITaxon:1 2939 2948 organisms +T104 NCBITaxon:6231 2957 2966 nematodes +T105 UBERON:0000104 2996 3004 lifespan +T106 PR:000014888 3028 3032 Sir2 +T107 UBERON:0000104 3086 3094 lifespan +T108 PR:000014888 3164 3168 Sir2 +T109 GO:0000003 3185 3197 reproductive +T110 UBERON:0000105 3198 3206 lifespan +T111 PR:000014888 3220 3225 SirT1 +T112 GO:0065007 3226 3235 regulates +T113 GO:0000003 3240 3252 reproductive +T114 UBERON:0000105 3253 3261 lifespan +T115 UBERON:0000104 3287 3295 lifespan +T116 NCBITaxon:40674 3299 3306 mammals +T117 PR:000014888 3325 3329 Sir2 +T118 PR:000045358 3381 3388 insulin +T119 PR:000045358 3389 3396 insulin +T120 NCBITaxon:6239 3459 3481 Caenorhabditis elegans +T121 NCBITaxon:7215 3484 3495 fruit flies +T122 NCBITaxon:7215 3497 3507 Drosophila +T123 NCBITaxon:10088 3510 3514 mice +T124 NCBITaxon:9606 3520 3526 humans +T125 GO:0016020 3558 3566 membrane +T126 GO:0005737 3584 3595 cytoplasmic +T127 GO:0005634 3609 3616 nuclear +T128 GO:0010467 3663 3673 expression +T129 CHEBI:35224 3681 3689 effector +T130 SO:0000704 3690 3695 genes +T131 GO:0065007 3764 3774 regulatory +T132 PR:000014888 3926 3931 SirT1 +T133 GO:0006476 3941 3962 protein deacetylation +T134 MOP:0001030 3949 3962 deacetylation +T135 GO:0005634 4006 4013 nuclear +T136 GO:0051235 4109 4120 sequestered +T137 GO:0005737 4132 4141 cytoplasm +T138 PR:000029189 4175 4186 Akt kinases +T139 PR:000045358 4202 4209 insulin +T140 PR:000045358 4283 4290 insulin +T141 GO:0010467 4330 4340 expression +T142 CHEBI:35224 4348 4356 effector +T143 SO:0000704 4357 4362 genes +T144 CHEBI:33290 4398 4402 food +T145 NCBITaxon:1 4453 4462 organisms +T146 NCBITaxon:6239 4472 4482 C. elegans +T147 NCBITaxon:species 4510 4517 species +T148 CHEBI:26764 4519 4535 steroid hormones +T149 GO:0065007 4547 4555 regulate +T150 NCBITaxon:10088 4580 4584 mice +T151 NCBITaxon:9606 4589 4595 humans +T152 GO:0040008 4662 4668;4682 4692 growth ... regulation +T153 UBERON:0000479 4706 4713 tissues +T154 UBERON:0001911 4725 4739 mammary glands +T155 NCBITaxon:40674 4750 4759 Mammalian +T156 PR:000014888 4760 4765 SirT1 +T157 PR:000003035 4857 4860 p53 +T158 GO:0071159 4862 4867 NF-κB +T159 PR:000013059 4873 4879 PGC-1α +T160 PR:000014888 4897 4902 SirT1 +T161 GO:0033554 4932 4960 cellular responses to stress +T162 GO:0031670 4932 4953;4980 4989 cellular responses to ... nutrients +T163 CHEBI:33284 4980 4989 nutrients +T164 PR:000014888 4999 5004 SirT1 +T165 NCBITaxon:10088 5015 5019 mice +T166 UBERON:0012101 5057 5066 perinatal +T167 GO:0007567 5061 5066 natal +T168 GO:0016265 5067 5072 death +T169 UBERON:0000970 5141 5144 eye +T170 PR:000014888 5287 5292 SirT1 +T171 PR:000014888 5304 5309 SirT1 +T172 NCBITaxon:10088 5316 5320 mice +T173 PR:000014888 5357 5362 SirT1 +T174 NCBITaxon:10088 5368 5372 mice +T175 http://purl.obolibrary.org/obo/MONDO_0005047 5417 5424 sterile +T176 PR:000014888 5462 5467 SirT1 +T177 NCBITaxon:10088 5478 5482 mice +T178 PR:000014888 5531 5536 SirT1 +T179 UBERON:0001911 5567 5580 mammary gland +T180 UBERON:0000062 5592 5597 organ +T181 UBERON:0001911 5604 5617 mammary gland +T182 UBERON:0000062 5630 5635 organ +T183 GO:0007567 5662 5667 birth +T184 GO:0000003 5713 5725 reproductive +T185 UBERON:0000105 5726 5734 lifespan +T186 UBERON:0000992 5773 5780 ovarian +T187 CHEBI:50114 5781 5789 estrogen +T188 UBERON:0000058 5801 5807 ductal +T189 GO:0035239 5801 5821 ductal morphogenesis +T190 UBERON:0003244 5835 5853 mammary epithelial +T191 GO:0008283 5889 5900 proliferate +T192 CL:0000136 5924 5934 adipocytes +T193 UBERON:0003891 5939 5946 stromal +T194 CL:0000499 5939 5952 stromal cells +T195 UBERON:0012282 5960 5975 mammary fat pad +T196 UBERON:0000992 5988 5995 Ovarian +T197 CHEBI:50114 5996 6004 estrogen +T198 UBERON:0000007 6022 6031 pituitary +T199 PR:000007968 6022 6046 pituitary growth hormone +T200 UBERON:0003891 6064 6071 stromal +T201 CL:0000499 6064 6077 stromal cells +T202 UBERON:0002107 6127 6132 liver +T203 GO:0038001 6169 6178 paracrine +T204 UBERON:0000058 6201 6207 ductal +T205 GO:0035239 6201 6221 ductal morphogenesis +T206 NCBITaxon:10088 6228 6232 Mice +T207 CHEBI:50114 6245 6253 estrogen +T208 CHEBI:50114 6278 6286 estrogen +T209 PR:000007204 6278 6301 estrogen receptor alpha +T210 PR:000007204 6303 6306 ERα +T211 GO:0007567 6328 6333 natal +T212 UBERON:0000058 6334 6340 ductal +T213 GO:0035239 6334 6354 ductal morphogenesis +T214 CHEBI:26764 6388 6404 steroid hormones +T215 UBERON:0000058 6501 6507 ductal +T216 GO:0008283 6559 6572 proliferation +T217 GO:0016265 6578 6583 death +T218 GO:0046903 6587 6596 secretory +T219 UBERON:0003215 6597 6605 alveolar +T220 UBERON:0000483 6606 6616 epithelium +T221 GO:0007565 6634 6643 pregnancy +T222 UBERON:0000058 6734 6740 ductal +T223 GO:0035239 6734 6754 ductal morphogenesis +T224 PR:000014888 6765 6770 SirT1 +T225 NCBITaxon:10088 6776 6780 mice +T226 GO:0007595 6785 6794 lactation +T227 PR:000014888 6806 6811 SirT1 +T228 PR:000014888 6884 6889 SirT1 +T229 GO:0065007 6900 6910 regulatory +T230 PR:000014888 6930 6935 SirT1 +T231 GO:0065007 6936 6945 modulates +T232 CHEBI:50114 6971 6979 estrogen +T233 UBERON:0001911 7000 7013 mammary gland +T234 GO:0030879 7000 7025 mammary gland development +T235 CHEBI:50114 7031 7039 estrogen +T236 UBERON:0000992 7074 7081 ovarian +T237 CHEBI:50114 7082 7090 estrogen +T238 GO:0065007 7091 7100 regulated +T239 UBERON:0003891 7102 7109 stromal +T240 CL:0000499 7102 7114 stromal cell +T241 UBERON:0003244 7159 7177 mammary epithelial +T242 CL:0002327 7159 7183 mammary epithelial cells +T243 NCBITaxon:10088 7209 7213 Mice +T244 PR:000014888 7238 7243 SirT1 +T245 NCBITaxon:10088 7297 7301 mice +T246 PR:000014888 7351 7356 SirT1 +T247 SO:0000704 7357 7361 gene +T248 PR:000014888 7363 7368 SirT1 +T249 NCBITaxon:10088 7374 7378 mice +T250 PR:000014888 7421 7426 SirT1 +T251 NCBITaxon:10088 7432 7436 mice +T252 NCBITaxon:10088 7460 7464 mice +T253 NCBITaxon:10088 7476 7480 mice +T254 SO:0000147 7526 7530 exon +T255 PR:000014888 7540 7545 SirT1 +T256 SO:0000704 7546 7550 gene +T257 PR:000014888 7552 7557 SirT1 +T258 NCBITaxon:10088 7562 7566 mice +T259 PR:000014888 7574 7579 SirT1 +T260 NCBITaxon:10088 7585 7589 mice +T261 PR:000014888 7594 7599 SirT1 +T262 NCBITaxon:10088 7604 7608 mice +T263 PR:000014888 7665 7670 SirT1 +T264 PR:000014888 7680 7685 SirT1 +T265 NCBITaxon:10088 7691 7695 mice +T266 PR:000014888 7716 7721 SirT1 +T267 NCBITaxon:10088 7727 7731 mice +T268 PR:000014888 7736 7741 SirT1 +T269 NCBITaxon:10088 7747 7751 mice +T270 NCBITaxon:10088 7793 7797 Mice +T271 NCBITaxon:33208 7911 7917 Animal +T272 PR:000014888 8038 8043 SirT1 +T273 SO:0000704 8044 8048 gene +T274 SO:0000112 8061 8068 primers +T275 SO:0000121 8070 8072;8076 8082 5' ... primer +T276 SO:0000121 8110 8112;8116 8122 5' ... primer +T277 SO:0000132 8149 8158 3' primer +T278 NCBITaxon:39107 8185 8191 Murine +T279 UBERON:0000922 8192 8201 embryonic +T280 CL:0000057 8202 8213 fibroblasts +T281 UBERON:0000922 8244 8251 embryos +T282 UBERON:0000922 8260 8269 embryonic +T283 UBERON:0000922 8283 8292 embryonic +T284 UBERON:0001977 8346 8351 Serum +T285 PR:000045358 8352 8359 insulin +T286 CHEBI:30879 8491 8498 alcohol +T287 UBERON:0000922 8827 8836 embryonic +T288 CL:0002322 8827 8847 embryonic stem cells +T289 CHEBI:16240 8876 8893 hydrogen peroxide +T290 UBERON:0000922 8909 8918 embryonic +T291 CL:0002322 8909 8929 embryonic stem cells +T292 UBERON:0000922 9064 9073 embryonic +T293 CHEBI:16240 9096 9113 hydrogen peroxide +T294 CHEBI:16240 9190 9207 hydrogen peroxide +T295 UBERON:0000922 9223 9232 embryonic +T296 CHEBI:41688 9260 9274 crystal violet +T297 CHEBI:50114 9290 9298 Estrogen +T298 CHEBI:50114 9313 9321 Estrogen +T299 CHEBI:16469 9345 9358 17β-estradiol +T300 UBERON:0006849 9510 9518 scapular +T301 NCBITaxon:10088 9546 9550 mice +T302 UBERON:0008337 9633 9649 inguinoabdominal +T303 UBERON:0012282 9650 9666 mammary fat pads +T304 CHEBI:50913 9719 9727 fixative +T305 CHEBI:27338 9862 9868 xylene +T306 NCBITaxon:10088 10045 10049 mice +T307 CHEBI:472552 10078 10094 bromodeoxyurdine +T308 CHEBI:472552 10096 10100 BrdU +T309 UBERON:0001179 10135 10145 peritoneal +T310 UBERON:0012282 10187 10203 mammary fat pads +T311 UBERON:0000479 10222 10229 tissues +T312 CHEBI:16842 10263 10271 formalin +T313 CHEBI:75958 10272 10280 solution +T314 CHEBI:51686 10382 10383 H +T315 CHEBI:472552 10463 10467 BrdU +T316 CL:0000445 10494 10509 apoptotic cells +T317 NCBITaxon:10088 10565 10570 mouse +T318 UBERON:0001913 10571 10575 milk +T319 NCBITaxon:9986 10588 10594 rabbit +T320 NCBITaxon:10088 10600 10605 mouse +T321 UBERON:0001913 10606 10610 milk +T322 GO:0042571 10628 10636 antibody +T323 CHEBI:51247 10737 10746 Texas Red +T324 NCBITaxon:9986 10752 10758 rabbit +T325 GO:0042571 10769 10777 antibody +T326 CHEBI:51231 10801 10805 DAPI +T327 GO:0005634 10819 10825 nuclei +T328 UBERON:0000310 11089 11096 mammary +T329 UBERON:0000479 11097 11104 tissues +T330 GO:0042571 11149 11159 antibodies +T331 PR:000014888 11176 11181 SirT1 +T332 PR:000008947 11233 11256 IGF-1 binding protein-1 +T333 PR:000008947 11258 11265 IGFBP-1 +T334 PR:000003293 11328 11332 IκBα +T335 GO:0042571 11410 11420 antibodies +T336 NCBITaxon:3704 11426 11437 horseradish +T337 MOP:0000779 11449 11459 conjugated +T338 NCBITaxon:9986 11465 11471 rabbit +T339 NCBITaxon:10114 11506 11509 rat +T340 NCBITaxon:9925 11520 11524 goat +T341 PR:000014888 11585 11590 SirT1 +T342 NCBITaxon:10088 11596 11600 mice +T343 PR:000014888 11627 11632 SirT1 +T344 NCBITaxon:10088 11640 11644 mice +T345 PR:000014888 11646 11651 SirT1 +T346 NCBITaxon:10088 11657 11661 mice +T347 CHEBI:7507 11698 11706 neomycin +T348 SO:0000704 11717 11721 gene +T349 SO:0000346 11726 11739 lox sequences +T350 PR:000014888 11747 11752 SirT1 +T351 SO:0000704 11753 11757 gene +T352 SO:0000357 11758 11766 flanking +T353 SO:0000147 11767 11771 exon +T354 PR:000014888 11799 11803 Sir2 +T355 GO:0010467 11856 11866 expression +T356 PR:000014888 11870 11875 SirT1 +T357 PR:000014888 11879 11884 SirT1 +T358 NCBITaxon:10088 11890 11894 mice +T359 PR:000014888 11921 11926 SirT1 +T360 NCBITaxon:10088 11932 11936 mice +T361 NCBITaxon:10088 11989 11993 mice +T362 PR:000014888 12010 12015 SirT1 +T363 SO:0001023 12019 12025 allele +T364 PR:000014888 12035 12040 SirT1 +T365 SO:0001023 12044 12050 allele +T366 PR:000014888 12052 12057 SirT1 +T367 NCBITaxon:10088 12063 12067 mice +T368 NCBITaxon:10088 12105 12109 mice +T369 PR:000014888 12122 12127 SirT1 +T370 PR:000014888 12155 12160 SirT1 +T371 NCBITaxon:10358 12166 12169 CMV +T372 GO:0010467 12189 12199 expression +T373 NCBITaxon:10358 12207 12210 CMV +T374 SO:0000902 12215 12224 transgene +T375 SO:0000147 12251 12255 exon +T376 CL:0000586 12295 12305 germ cells +T377 PR:000014888 12324 12329 SirT1 +T378 SO:0001023 12333 12339 allele +T379 PR:000014888 12408 12413 SirT1 +T380 NCBITaxon:10088 12424 12428 mice +T381 NCBITaxon:10088 12461 12465 mice +T382 NCBITaxon:10088 12482 12486 mice +T383 PR:000014888 12548 12553 SirT1 +T384 NCBITaxon:10088 12558 12562 mice +T385 PR:000014888 12581 12586 SirT1 +T386 NCBITaxon:10088 12596 12600 mice +T387 PR:000014888 12605 12610 SirT1 +T388 NCBITaxon:10088 12622 12626 mice +T389 PR:000014888 12639 12644 SirT1 +T390 NCBITaxon:10088 12650 12654 mice +T391 PR:000014888 12686 12691 SirT1 +T392 NCBITaxon:10088 12696 12700 mice +T393 PR:000014888 12704 12709 SirT1 +T394 NCBITaxon:10088 12715 12719 mice +T395 GO:0010467 12720 12729 expressed +T396 PR:000014888 12732 12737 SirT1 +T397 SO:0001817 12764 12771 inframe +T398 SO:0000147 12784 12788 exon +T399 PR:000014888 12809 12814 SirT1 +T400 PR:000014888 12868 12873 SirT1 +T401 NCBITaxon:10088 12878 12882 mice +T402 NCBITaxon:10088 12935 12939 mice +T403 PR:000014888 12944 12949 SirT1 +T404 NCBITaxon:10088 12959 12963 mice +T405 PR:000014888 12997 13002 SirT1 +T406 NCBITaxon:10088 13008 13012 mice +T407 PR:000014888 13048 13053 SirT1 +T408 NCBITaxon:10088 13064 13068 mice +T409 PR:000014888 13091 13096 SirT1 +T410 GO:0016265 13111 13114 die +T411 GO:0007567 13129 13134 birth +T412 PR:000014888 13165 13170 SirT1 +T413 NCBITaxon:10088 13176 13180 mice +T414 GO:0065007 13307 13317 regulation +T415 GO:0006302 13319 13349 DNA double-strand break repair +T416 SO:0000985 13323 13336 double-strand +T417 PR:000014888 13393 13398 SirT1 +T418 NCBITaxon:10088 13404 13408 mice +T419 UBERON:0001977 13432 13437 serum +T420 CHEBI:35224 13485 13493 effector +T421 UBERON:0000007 13498 13507 pituitary +T422 PR:000007968 13498 13510 pituitary GH +T423 UBERON:0001977 13516 13521 serum +T424 PR:000014888 13537 13542 SirT1 +T425 NCBITaxon:10088 13548 13552 mice +T426 PR:000014888 13645 13649 Sir2 +T427 GO:0032991 13673 13680 complex +T428 NCBITaxon:10088 13693 13698 mouse +T429 PR:000014892 13699 13704 SirT6 +T430 GO:0006302 13735 13765 DNA double-strand break repair +T431 SO:0000985 13739 13752 double-strand +T432 PR:000014888 13833 13838 SirT1 +T433 UBERON:0000922 13844 13853 embryonic +T434 CL:0002322 13844 13864 embryonic stem cells +T435 CHEBI:16240 13931 13948 hydrogen peroxide +T436 PR:000017509 14034 14038 Ku70 +T437 PR:000004427 14052 14055 Atm +T438 PR:000017509 14092 14096 Ku70 +T439 NCBITaxon:10088 14107 14111 mice +T440 PR:000004427 14116 14119 Atm +T441 NCBITaxon:10088 14130 14134 mice +T442 PR:000014888 14218 14223 SirT1 +T443 NCBITaxon:10088 14229 14233 mice +T444 GO:0006281 14279 14296 DNA damage repair +T445 GO:0007595 14353 14362 Lactation +T446 PR:000014888 14393 14398 SirT1 +T447 NCBITaxon:10088 14404 14408 mice +T448 http://purl.obolibrary.org/obo/MONDO_0005047 14464 14471 sterile +T449 PR:000014888 14499 14504 SirT1 +T450 NCBITaxon:10088 14515 14519 mice +T451 PR:000014888 14536 14541 SirT1 +T452 NCBITaxon:10088 14547 14551 mice +T453 PR:000014888 14571 14576 SirT1 +T454 NCBITaxon:10088 14581 14585 mice +T455 PR:000014888 14611 14616 SirT1 +T456 UBERON:0012101 14663 14672 perinatal +T457 GO:0007567 14667 14672 natal +T458 PR:000014888 14732 14737 SirT1 +T459 NCBITaxon:10088 14742 14746 mice +T460 PR:000014888 14774 14779 SirT1 +T461 NCBITaxon:10088 14785 14789 mice +T462 GO:0007567 14807 14818 parturition +T463 PR:000014888 14820 14825 SirT1 +T464 GO:0001967 14856 14872 nursing behavior +T465 GO:0007567 14926 14931 birth +T466 GO:0016265 15031 15035 died +T467 UBERON:0001913 15088 15092 milk +T468 UBERON:0000945 15102 15110 stomachs +T469 PR:000014888 15138 15143 SirT1 +T470 GO:0007595 15171 15180 lactation +T471 UBERON:0001911 15286 15300 mammary glands +T472 PR:000014888 15323 15328 SirT1 +T473 NCBITaxon:10088 15334 15338 mice +T474 UBERON:0000058 15357 15363 ductal +T475 GO:0035239 15357 15377 ductal morphogenesis +T476 NCBITaxon:10088 15436 15440 mice +T477 UBERON:0000058 15461 15467 ductal +T478 UBERON:0000058 15521 15527 ductal +T479 GO:0035239 15521 15541 ductal morphogenesis +T480 GO:0007565 15555 15563 pregnant +T481 PR:000014888 15564 15569 SirT1 +T482 NCBITaxon:10088 15575 15579 mice +T483 GO:0007565 15596 15605 pregnancy +T484 GO:0007565 15641 15650 pregnancy +T485 UBERON:0000058 15662 15668 ductal +T486 GO:0035239 15662 15682 ductal morphogenesis +T487 GO:0007565 15704 15713 pregnancy +T488 UBERON:0001911 15730 15744 mammary glands +T489 PR:000014888 15748 15753 SirT1 +T490 NCBITaxon:10088 15766 15770 mice +T491 UBERON:0001913 15797 15801 milk +T492 GO:0007595 15797 15812 milk production +T493 UBERON:0001913 15905 15909 milk +T494 GO:0042571 15910 15918 antibody +T495 GO:0007565 15935 15944 Pregnancy +T496 UBERON:0001911 15953 15966 mammary gland +T497 GO:0030879 15953 15978 mammary gland development +T498 GO:0007595 16032 16041 lactation +T499 UBERON:0001911 16067 16081 mammary glands +T500 PR:000014888 16085 16090 SirT1 +T501 NCBITaxon:10088 16103 16107 mice +T502 GO:0007595 16111 16120 lactation +T503 UBERON:0005313 16128 16145 Terminal end buds +T504 UBERON:0005313 16147 16151 TEBs +T505 NCBITaxon:10088 16243 16247 mice +T506 UBERON:0005313 16249 16253 TEBs +T507 UBERON:0000058 16271 16277 ductal +T508 UBERON:0005313 16344 16348 TEBs +T509 UBERON:0000058 16349 16354 ducts +T510 UBERON:0012282 16373 16389 mammary fat pads +T511 UBERON:0005313 16391 16395 TEBs +T512 GO:0060033 16396 16403 regress +T513 NCBITaxon:10088 16427 16431 mice +T514 UBERON:0005313 16492 16496 TEBs +T515 PR:000014888 16526 16531 SirT1 +T516 NCBITaxon:10088 16544 16548 mice +T517 GO:0007595 16552 16561 lactation +T518 UBERON:0005313 16604 16608 TEBs +T519 UBERON:0005313 16615 16619 TEBs +T520 UBERON:0000058 16643 16649 ductal +T521 UBERON:0003215 16654 16662 alveolar +T522 PR:000014888 16710 16715 SirT1 +T523 NCBITaxon:10088 16721 16725 mice +T524 UBERON:0000058 16755 16761 ductal +T525 GO:0035295 16755 16773 ductal development +T526 UBERON:0000058 16802 16808 ductal +T527 UBERON:0000058 16840 16846 ductal +T528 UBERON:0000310 16939 16946 mammary +T529 UBERON:0000479 16947 16954 tissues +T530 GO:0007565 16968 16977 pregnancy +T531 GO:0007565 17025 17034 pregnancy +T532 UBERON:0000058 17056 17062 ductal +T533 GO:0035239 17056 17076 ductal morphogenesis +T534 PR:000014888 17087 17092 SirT1 +T535 NCBITaxon:10088 17098 17102 mice +T536 UBERON:0003215 17112 17120 alveolar +T537 GO:0009653 17121 17134 morphogenesis +T538 GO:0065007 17157 17164 control +T539 GO:0007565 17214 17223 pregnancy +T540 GO:0007595 17228 17237 lactation +T541 GO:0046903 17347 17356 secreting +T542 UBERON:0001913 17357 17361 milk +T543 GO:0007567 17368 17379 parturition +T544 NCBITaxon:10088 17395 17399 mice +T545 GO:0007595 17403 17412 lactation +T546 PR:000014888 17450 17455 SirT1 +T547 NCBITaxon:10088 17461 17465 mice +T548 UBERON:0003215 17471 17479 alveolar +T549 GO:0009653 17480 17493 morphogenesis +T550 GO:0046903 17547 17556 secretory +T551 CL:0000151 17547 17556;17577 17582 secretory ... cells +T552 UBERON:0003215 17557 17565 alveolar +T553 CL:0010003 17557 17582 alveolar epithelial cells +T554 UBERON:0000483 17566 17576 epithelial +T555 UBERON:0003215 17600 17608 alveolar +T556 UBERON:0000058 17653 17659 ductal +T557 UBERON:0003215 17726 17734 alveolar +T558 GO:0009653 17735 17748 morphogenesis +T559 PR:000014888 17752 17757 SirT1 +T560 NCBITaxon:10088 17763 17767 mice +T561 UBERON:0001913 17827 17831 milk +T562 GO:0007595 17827 17842 milk production +T563 NCBITaxon:10088 17880 17884 mice +T564 CHEBI:50114 17909 17917 estrogen +T565 UBERON:0000058 17926 17932 ductal +T566 GO:0035239 17926 17946 ductal morphogenesis +T567 CHEBI:50114 17988 17996 estrogen +T568 GO:0007565 18000 18008 pregnant +T569 NCBITaxon:10088 18009 18013 mice +T570 UBERON:0000058 18040 18046 ductal +T571 GO:0035239 18040 18060 ductal morphogenesis +T572 PR:000014888 18064 18069 SirT1 +T573 NCBITaxon:10088 18075 18079 mice +T574 CHEBI:50114 18090 18098 estrogen +T575 UBERON:0006849 18130 18138 scapular +T576 PR:000014888 18161 18166 SirT1 +T577 NCBITaxon:10088 18172 18177 mouse +T578 UBERON:0000310 18186 18193 mammary +T579 UBERON:0000479 18194 18201 tissues +T580 NCBITaxon:10088 18276 18280 mice +T581 UBERON:0000058 18291 18297 ductal +T582 GO:0007565 18367 18376 pregnancy +T583 PR:000014888 18415 18420 SirT1 +T584 NCBITaxon:10088 18426 18430 mice +T585 UBERON:0005313 18438 18442 TEBs +T586 UBERON:0000058 18447 18453 ductal +T587 UBERON:0000058 18507 18513 ductal +T588 GO:0035239 18507 18527 ductal morphogenesis +T589 NCBITaxon:10088 18550 18554 mice +T590 UBERON:0000058 18567 18573 ductal +T591 PR:000014888 18621 18626 SirT1 +T592 NCBITaxon:10088 18632 18636 mice +T593 UBERON:0000058 18655 18661 ductal +T594 GO:0035239 18655 18675 ductal morphogenesis +T595 PR:000014888 18720 18725 SirT1 +T596 NCBITaxon:10088 18738 18742 mice +T597 CHEBI:50114 18821 18829 estrogen +T598 UBERON:0000058 18860 18866 ductal +T599 GO:0035239 18860 18880 ductal morphogenesis +T600 PR:000014888 18891 18896 SirT1 +T601 NCBITaxon:10088 18902 18906 mice +T602 UBERON:0005313 18950 18954 TEBs +T603 UBERON:0000058 18972 18978 ductal +T604 UBERON:0001911 19050 19063 mammary gland +T605 GO:0030879 19050 19075 mammary gland development +T606 GO:0007565 19104 19113 pregnancy +T607 CHEBI:50114 19167 19175 estrogen +T608 CHEBI:50114 19197 19205 estrogen +T609 GO:0007565 19221 19230 pregnancy +T610 GO:0030154 19299 19317;19340 19345 differentiation of ... cells +T611 UBERON:0000483 19318 19328 epithelial +T612 UBERON:0000058 19350 19356 ductal +T613 GO:0035239 19350 19370 ductal morphogenesis +T614 PR:000014888 19381 19386 SirT1 +T615 NCBITaxon:10088 19392 19396 mice +T616 UBERON:0003244 19410 19428 mammary epithelial +T617 CL:0002327 19410 19433 mammary epithelial cell +T618 GO:0033598 19410 19447 mammary epithelial cell proliferation +T619 GO:0030154 19429 19433;19452 19467 cell ... differentiation +T620 GO:0007565 19484 19493 pregnancy +T621 UBERON:0001911 19502 19515 mammary gland +T622 GO:0030879 19502 19527 mammary gland development +T623 UBERON:0003244 19559 19577 mammary epithelial +T624 GO:0007565 19598 19606 pregnant +T625 PR:000014888 19607 19612 SirT1 +T626 NCBITaxon:10088 19618 19622 mice +T627 CHEBI:472552 19658 19662 BrdU +T628 GO:0008283 19789 19807 cell proliferation +T629 GO:0006915 19789 19793;19812 19821 cell ... apoptosis +T630 UBERON:0000310 19829 19836 mammary +T631 UBERON:0000479 19837 19844 tissues +T632 PR:000014888 19872 19877 SirT1 +T633 UBERON:0000310 19883 19890 mammary +T634 UBERON:0000479 19891 19898 tissues +T635 UBERON:0005313 19936 19940 TEBs +T636 CHEBI:472552 19946 19950 BrdU +T637 CHEBI:472552 19991 19995 BrdU +T638 UBERON:0034969 20039 20045;20058 20068 ductal ... epithelial +T639 CL:0000068 20039 20045;20058 20074 ductal ... epithelial cells +T640 UBERON:0003215 20049 20057 alveolar +T641 CL:0010003 20049 20074 alveolar epithelial cells +T642 CHEBI:472552 20110 20114 BrdU +T643 UBERON:0000310 20163 20170 mammary +T644 UBERON:0000479 20171 20178 tissues +T645 UBERON:0034969 20205 20222 ductal epithelial +T646 CL:0000068 20205 20228 ductal epithelial cells +T647 UBERON:0003215 20246 20254 alveolar +T648 CL:0010003 20246 20271 alveolar epithelial cells +T649 UBERON:0000483 20255 20265 epithelial +T650 CHEBI:472552 20277 20281 BrdU +T651 UBERON:0034969 20312 20329 ductal epithelial +T652 CL:0000068 20312 20335 ductal epithelial cells +T653 PR:000014888 20339 20344 SirT1 +T654 NCBITaxon:10088 20350 20354 mice +T655 UBERON:0000058 20372 20377 ducts +T656 UBERON:0005313 20411 20415 TEBs +T657 UBERON:0012282 20436 20451 mammary fat pad +T658 UBERON:0034969 20498 20515 ductal epithelial +T659 CL:0000068 20498 20521 ductal epithelial cells +T660 UBERON:0003215 20581 20589 alveolar +T661 UBERON:0001913 20605 20609 milk +T662 GO:0008283 20684 20697;20718 20720;20743 20748 proliferation ... of ... cells +T663 GO:0030154 20702 20720;20743 20748 differentiation of ... cells +T664 UBERON:0000483 20721 20731 epithelial +T665 GO:0000003 20817 20829 reproductive +T666 UBERON:0000105 20830 20834 life +T667 GO:0007565 20875 20884 pregnancy +T668 PR:000014888 20888 20893 SirT1 +T669 NCBITaxon:10088 20899 20903 mice +T670 GO:0006915 20906 20915 Apoptosis +T671 UBERON:0000310 20981 20988 mammary +T672 UBERON:0000479 20989 20996 tissues +T673 CL:0000445 21009 21018;21030 21035 Apoptotic ... cells +T674 UBERON:0000483 21019 21029 epithelial +T675 CL:0000066 21019 21035 epithelial cells +T676 UBERON:0034969 21070 21076;21090 21100 ductal ... epithelium +T677 UBERON:0003215 21081 21089 alveolar +T678 PR:000014888 21104 21109 SirT1 +T679 NCBITaxon:10088 21115 21119 mice +T680 GO:0007595 21123 21132 lactation +T681 CL:0000445 21179 21194 apoptotic cells +T682 NCBITaxon:10088 21245 21249 mice +T683 GO:0007595 21253 21262 lactation +T684 NCBITaxon:10088 21315 21319 mice +T685 GO:0007565 21327 21336 pregnancy +T686 GO:0007565 21353 21362 pregnancy +T687 GO:0008283 21389 21402 proliferation +T688 GO:0007565 21430 21439 pregnancy +T689 UBERON:0000058 21448 21454 ductal +T690 GO:0035295 21448 21466 ductal development +T691 PR:000014888 21470 21475 SirT1 +T692 NCBITaxon:10088 21481 21485 mice +T693 GO:0007595 21518 21527 lactation +T694 NCBITaxon:10088 21598 21602 mice +T695 GO:0007565 21610 21619 pregnancy +T696 GO:0007565 21632 21643 pregnancies +T697 PR:000014888 21700 21705 SirT1 +T698 PR:000014888 21718 21723 SirT1 +T699 GO:0007565 21749 21760 pregnancies +T700 PR:000014888 21791 21796 SirT1 +T701 GO:0007595 21839 21848 lactation +T702 PR:000014888 21869 21874 SirT1 +T703 UBERON:0000058 21905 21911 ductal +T704 GO:0035239 21905 21925 ductal morphogenesis +T705 NCBITaxon:10088 21936 21940 mice +T706 GO:0008283 21961 21974 proliferation +T707 NCBITaxon:10088 21985 21989 mice +T708 GO:0065007 21994 22003 regulated +T709 GO:0007595 22055 22064 lactation +T710 PR:000014888 22115 22120 SirT1 +T711 NCBITaxon:10088 22133 22137 mice +T712 UBERON:0000058 22151 22157 ductal +T713 GO:0035239 22151 22171 ductal morphogenesis +T714 PR:000014888 22182 22187 SirT1 +T715 NCBITaxon:10088 22193 22197 mice +T716 UBERON:0000058 22237 22243 ductal +T717 GO:0035239 22237 22257 ductal morphogenesis +T718 NCBITaxon:10088 22266 22270 mice +T719 CHEBI:50114 22283 22291 estrogen +T720 NCBITaxon:10088 22317 22321 mice +T721 PR:000007204 22402 22405 ERα +T722 CHEBI:50114 22481 22489 estrogen +T723 UBERON:0000058 22516 22522 ductal +T724 GO:0035239 22516 22536 ductal morphogenesis +T725 UBERON:0003891 22559 22566 stromal +T726 CL:0000499 22559 22571 stromal cell +T727 UBERON:0000310 22596 22603 mammary +T728 UBERON:0000479 22604 22611 tissues +T729 UBERON:0000058 22621 22627 ductal +T730 GO:0035239 22621 22641 ductal morphogenesis +T731 PR:000014888 22652 22657 SirT1 +T732 NCBITaxon:10088 22663 22667 mice +T733 GO:0007565 22762 22771 pregnancy +T734 UBERON:0001911 22780 22793 mammary gland +T735 GO:0030879 22780 22805 mammary gland development +T736 PR:000014888 22809 22814 SirT1 +T737 NCBITaxon:10088 22820 22824 mice +T738 NCBITaxon:10088 22938 22942 mice +T739 PR:000001775 22951 22967 IκB kinase alpha +T740 PR:000001775 22969 22973 IKKα +T741 GO:0071159 22988 22993 NF-κB +T742 GO:0051092 22988 23004 NF-κB activation +T743 PR:000001775 23011 23015 IKKα +T744 NCBITaxon:10088 23026 23030 mice +T745 UBERON:0000058 23046 23052 ductal +T746 GO:0035239 23046 23066 ductal morphogenesis +T747 UBERON:0000058 23110 23116 ductal +T748 GO:0007565 23145 23154 pregnancy +T749 GO:0071159 23180 23185 NF-κB +T750 PR:000005121 23196 23205 cyclin D1 +T751 GO:0010467 23206 23216 expression +T752 PR:000001775 23227 23231 IKKα +T753 NCBITaxon:10088 23242 23246 mice +T754 UBERON:0003215 23265 23273 alveolar +T755 CL:0010003 23265 23289 alveolar epithelial cell +T756 UBERON:0000483 23274 23284 epithelial +T757 GO:0050673 23274 23303 epithelial cell proliferation +T758 GO:0007595 23316 23323 lactate +T759 PR:000014888 23336 23341 SirT1 +T760 GO:0071159 23403 23408 NF-κB +T761 NCBITaxon:40674 23412 23421 mammalian +T762 PR:000014888 23440 23445 SirT1 +T763 NCBITaxon:10088 23451 23455 mice +T764 PR:000014888 23520 23525 SirT1 +T765 GO:0065007 23539 23548 regulates +T766 GO:0010467 23553 23563 expression +T767 GO:0071159 23694 23699 NF-κB +T768 GO:0065007 23700 23709 regulated +T769 SO:0000704 23710 23715 genes +T770 PR:000008947 23717 23724 IGFBP-1 +T771 PR:000003293 23729 23733 IκBα +T772 GO:0071159 23791 23796 NF-κB +T773 GO:0051092 23791 23807 NF-κB activation +T774 PR:000008947 23862 23869 IGFBP-1 +T775 PR:000003293 23874 23878 IκBα +T776 UBERON:0001013 23897 23912 adipose tissues +T777 PR:000014888 23925 23930 SirT1 +T778 NCBITaxon:10088 23936 23940 mice +T779 UBERON:0034969 23948 23965 ductal epithelial +T780 CL:0000068 23948 23971 ductal epithelial cells +T781 PR:000014888 23977 23982 SirT1 +T782 NCBITaxon:10088 23988 23992 mice +T783 GO:0007595 23996 24005 lactation +T784 GO:0010467 24040 24050 expression +T785 PR:000008947 24062 24069 IGFBP-1 +T786 PR:000003293 24074 24078 IκBα +T787 UBERON:0000310 24092 24099 mammary +T788 UBERON:0000479 24100 24107 tissues +T789 GO:0010467 24198 24208 expression +T790 PR:000008947 24212 24219 IGFBP-1 +T791 GO:0007565 24245 24254 pregnancy +T792 GO:0071159 24264 24269 NF-κB +T793 GO:0007565 24312 24321 pregnancy +T794 PR:000014888 24335 24340 SirT1 +T795 PR:000008947 24385 24392 IGFBP-1 +T796 PR:000003293 24397 24401 IκBα +T797 PR:000014888 24481 24486 SirT1 +T798 GO:0065007 24487 24496 modulated +T799 PR:000014888 24518 24523 SirT1 +T800 UBERON:0003244 24565 24583 mammary epithelial +T801 UBERON:0003215 24634 24642 alveolar +T802 CL:0010003 24634 24659 alveolar epithelial cells +T803 UBERON:0000483 24643 24653 epithelial +T804 GO:0007595 24672 24681 lactation +T805 PR:000014888 24703 24708 SirT1 +T806 GO:0042592 24744 24755 homeostasis +T807 UBERON:0000058 24775 24781 ductal +T808 GO:0035239 24775 24795 ductal morphogenesis +T809 GO:0051716 24807 24824 cellular response +T810 GO:0010467 24851 24861 expression +T811 PR:000008947 24870 24877 IGFBP-1 +T812 PR:000003293 24882 24886 IκBα +T813 UBERON:0001013 24890 24905 adipose tissues +T814 UBERON:0003244 24910 24928 mammary epithelial +T815 CL:0002327 24910 24934 mammary epithelial cells +T816 GO:0065007 24967 24977 regulation +T817 SO:0000704 24981 24985 gene +T818 GO:0010467 24981 24996 gene expression +T819 PR:000014888 25000 25005 SirT1 +T820 GO:0071159 25057 25062 NF-κB +T821 PR:000008947 25077 25084 IGFBP-1 +T822 CHEBI:35222 25097 25106 inhibitor +T823 PR:000008947 25174 25181 IGFBP-1 +T824 UBERON:0001013 25185 25200 adipose tissues +T825 PR:000014888 25204 25209 SirT1 +T826 NCBITaxon:10088 25215 25219 mice +T827 GO:0065007 25266 25275 regulated +T828 PR:000008947 25276 25283 IGFBP-1 +T829 GO:0010467 25284 25294 expression +T830 GO:0065007 25306 25315 regulated +T831 PR:000003293 25316 25320 IκBα +T832 GO:0010467 25321 25331 expression +T833 CHEBI:50114 25381 25389 estrogen +T834 UBERON:0012282 25397 25413 mammary fat pads +T835 GO:0010467 25491 25501 expression +T836 PR:000008947 25505 25512 IGFBP-1 +T837 CHEBI:50114 25546 25554 estrogen +T838 GO:0007565 25568 25576 pregnant +T839 NCBITaxon:10088 25587 25591 mice +T840 CHEBI:50114 25625 25633 estrogen +T841 UBERON:0003891 25672 25679 stromal +T842 CL:0000499 25672 25684 stromal cell +T843 PR:000008947 25726 25733 IGFBP-1 +T844 PR:000014888 25745 25750 SirT1 +T845 NCBITaxon:10088 25756 25760 mice +T846 PR:000008947 25811 25818 IGFBP-1 +T847 UBERON:0000310 25822 25829 mammary +T848 UBERON:0000479 25830 25837 tissues +T849 CHEBI:50114 25878 25886 estrogen +T850 GO:0010467 25930 25940 expression +T851 PR:000003293 25944 25948 IκBα +T852 GO:0065007 25985 25994 regulated +T853 PR:000003293 25995 25999 IκBα +T854 GO:0010467 26000 26010 expression +T855 PR:000014888 26014 26019 SirT1 +T856 PR:000000134 26069 26073 TNFα +T857 PR:000003293 26103 26107 IκBα +T858 GO:0010467 26108 26118 expression +T859 PR:000014888 26147 26152 SirT1 +T860 PR:000003293 26224 26228 IκBα +T861 GO:0010467 26229 26239 expression +T862 PR:000003293 26251 26255 IκBα +T863 PR:000000134 26279 26283 TNFα +T864 PR:000003293 26317 26321 IκBα +T865 PR:000014888 26335 26340 SirT1 +T866 PR:000014888 26445 26450 SirT1 +T867 GO:0071159 26470 26475 NF-κB +T868 PR:000014888 26598 26603 SirT1 +T869 GO:0071159 26618 26623 NF-κB +T870 GO:0051092 26618 26634 NF-κB activation +T871 GO:0071159 26644 26649 NF-κB +T872 GO:0010467 26711 26721 expression +T873 PR:000003293 26725 26729 IκBα +T874 GO:0071159 26741 26746 NF-κB +T875 GO:0051092 26741 26757 NF-κB activation +T876 SO:0001060 26774 26782 isoforms +T877 PR:000003294 26786 26789;26791 26792 IκB ... β +T878 PR:000003295 26786 26789;26797 26798 IκB ... ε +T879 GO:0071159 26819 26824 NF-κB +T880 PR:000000134 26853 26857 TNFα +T881 CHEBI:16412 26938 26955 lipopolysacharide +T882 GO:0071159 26982 26987 NF-κB +T883 GO:0051092 26982 26998 NF-κB activation +T884 GO:0071159 27012 27017 NF-κB +T885 UBERON:0001911 27046 27059 mammary gland +T886 GO:0065007 27082 27089 control +T887 PR:000001954 27093 27097 RANK +T888 PR:000001775 27112 27116 IKKα +T889 PR:000003293 27187 27191 IκBα +T890 PR:000014888 27229 27234 SirT1 +T891 GO:0065007 27273 27280 control +T892 GO:0071159 27284 27289 NF-κB +T893 PR:000001954 27434 27438 RANK +T894 PR:000001775 27453 27457 IKKα +T895 UBERON:0001911 27483 27497 mammary glands +T896 PR:000014888 27524 27529 SirT1 +T897 NCBITaxon:10088 27535 27539 mice +T898 UBERON:0000058 27570 27576 ductal +T899 GO:0035239 27570 27590 ductal morphogenesis +T900 PR:000014888 27666 27671 SirT1 +T901 PR:000001775 27700 27704 IKKα +T902 GO:0071159 27731 27736 NF-κB +T903 UBERON:0003244 27784 27802 mammary epithelial +T904 CL:0002327 27784 27807 mammary epithelial cell +T905 PR:000014888 27817 27822 SirT1 +T906 NCBITaxon:10088 27835 27839 mice +T907 PR:000014888 27902 27907 SirT1 +T908 NCBITaxon:10088 27913 27917 mice +T909 GO:0065007 27933 27943 regulatory +T910 PR:000014888 27963 27968 SirT1 +T911 GO:0065007 27969 27978 modulates +T912 CHEBI:50114 27995 28003 estrogen +T913 UBERON:0000058 28041 28047 ductal +T914 GO:0035239 28041 28061 ductal morphogenesis +T915 PR:000014888 28063 28068 SirT1 +T916 GO:0042592 28091 28102 homeostasis +T917 SO:0000704 28106 28110 gene +T918 GO:0010467 28106 28121 gene expression +T919 PR:000014888 28183 28188 SirT1 +T920 NCBITaxon:10088 28194 28198 mice +T921 UBERON:0012101 28216 28225 perinatal +T922 GO:0007567 28220 28225 natal +T923 GO:0007595 28240 28249 lactation +T924 PR:000014888 28319 28324 SirT1 +T925 GO:0007567 28337 28342 natal +T926 NCBITaxon:40674 28378 28385 mammals +T927 PR:000014888 28388 28393 SirT1 +T928 GO:0042592 28407 28418 homeostasis +T929 PR:000008947 28432 28439 IGFBP-1 +T930 PR:000014888 28449 28454 SirT1 +T931 PR:000045358 28534 28541 insulin +T932 GO:0007567 28630 28635 birth +T933 PR:000014888 28682 28687 SirT1 +T934 PR:000003273 28715 28729 IGF-1 receptor +T935 CHEBI:28874 28730 28752 phosphoinositide-3'-OH +T936 GO:0014065 28730 28759;28764 28781 phosphoinositide-3'-OH kinase ... signaling pathway +T937 PR:000029189 28760 28763 Akt +T938 GO:0043491 28760 28781 Akt signaling pathway +T939 NCBITaxon:10088 28858 28862 Mice +T940 PR:000003273 28915 28929 IGF-1 receptor +T941 SO:0000704 28930 28934 gene +T942 UBERON:0012101 28943 28952 perinatal +T943 GO:0007567 28947 28952 natal +T944 PR:000003273 29002 29016 IGF-1 receptor +T945 GO:0010467 29017 29027 expressing +T946 PR:000045358 29050 29057 insulin +T947 CHEBI:28874 29085 29107 phosphoinositide-3'-OH +T948 GO:0014065 29085 29114;29130 29137 phosphoinositide-3'-OH kinase ... cascade +T949 PR:000029189 29119 29129 Akt kinase +T950 GO:0043491 29119 29137 Akt kinase cascade +T951 CHEBI:28874 29165 29187 phosphoinositide-3'-OH +T952 GO:0014065 29165 29194;29199 29216 phosphoinositide-3'-OH kinase ... signaling pathway +T953 PR:000029189 29195 29198 Akt +T954 GO:0043491 29195 29216 Akt signaling pathway +T955 NCBITaxon:40674 29256 29263 mammals +T956 GO:0065007 29282 29290 modulate +T957 GO:0008152 29298 29308 metabolism +T958 UBERON:0000104 29313 29321 lifespan +T959 NCBITaxon:1 29331 29340 organisms +T960 NCBITaxon:40674 29346 29355 mammalian +T961 PR:000029189 29356 29366 Akt kinase +T962 PR:000002190 29393 29397 Akt1 +T963 PR:000003913 29398 29402 Akt3 +T964 NCBITaxon:10088 29447 29451 Mice +T965 PR:000002190 29469 29473 Akt1 +T966 PR:000003912 29478 29482 Akt2 +T967 SO:0000704 29483 29488 genes +T968 UBERON:0012101 29497 29506 perinatal +T969 GO:0007567 29501 29506 natal +T970 PR:000003273 29603 29617 IGF-1 receptor +T971 NCBITaxon:10088 29628 29632 mice +T972 PR:000014888 29655 29660 SirT1 +T973 NCBITaxon:10088 29666 29670 mice +T974 PR:000014888 29700 29705 SirT1 +T975 NCBITaxon:10088 29717 29721 mice +T976 UBERON:0012101 29736 29745 perinatal +T977 GO:0007567 29740 29745 natal +T978 UBERON:0012101 29850 29859 perinatal +T979 GO:0007567 29854 29859 natal +T980 NCBITaxon:10088 29933 29937 mice +T981 PR:000003273 29957 29971 IGF-1 receptor +T982 PR:000002190 29976 29980 Akt1 +T983 PR:000003912 29981 29985 Akt2 +T984 GO:0010467 30040 30050 expression +T985 CHEBI:35224 30065 30073 effector +T986 SO:0000704 30074 30079 genes +T987 GO:0007567 30097 30102 birth +T988 GO:0007567 30124 30129 birth +T989 PR:000014888 30256 30261 SirT1 +T990 NCBITaxon:10088 30267 30271 mice +T991 UBERON:0000113 30286 30295 adulthood +T992 GO:0000003 30304 30313 reproduce +T993 NCBITaxon:10088 30420 30424 mice +T994 PR:000007641 30434 30439 FoxO3 +T995 PR:000007641 30441 30447 Foxo3a +T996 PR:000007641 30448 30454 FKHRL1 +T997 NCBITaxon:10088 30466 30470 mice +T998 UBERON:0001911 30509 30523 mammary glands +T999 PR:000008947 30531 30538 IGFBP-1 +T1000 PR:000045358 30633 30640 insulin +T1001 NCBITaxon:40674 30749 30758 mammalian +T1002 PR:000007641 30766 30771 FoxO3 +T1003 SO:0000167 30797 30805 promoter +T1004 PR:000008947 30813 30820 IGFBP-1 +T1005 SO:0000704 30821 30825 gene +T1006 PR:000014888 30866 30871 SirT1 +T1007 GO:0005634 30916 30923 nuclear +T1008 PR:000007641 30924 30929 FoxO3 +T1009 GO:0010467 30950 30960 expression +T1010 PR:000008947 30964 30971 IGFBP-1 +T1011 PR:000008947 31034 31041 IGFBP-1 +T1012 PR:000014888 31105 31110 SirT1 +T1013 GO:0042592 31124 31135 homeostasis +T1014 PR:000008947 31149 31156 IGFBP-1 +T1015 GO:0042592 31199 31210 homeostasis +T1016 PR:000008947 31224 31231 IGFBP-1 +T1017 http://purl.obolibrary.org/obo/MONDO_0005047 31275 31286 infertility +T1018 NCBITaxon:10088 31321 31325 mice +T1019 PR:000008947 31330 31337 IGFBP-1 +T1020 NCBITaxon:10088 31349 31353 mice +T1021 UBERON:0001977 31370 31375 serum +T1022 CHEBI:50114 31385 31393 estrogen +T1023 NCBITaxon:10088 31420 31424 mice +T1024 PR:000007204 31467 31470 ERα +T1025 NCBITaxon:10088 31481 31485 mice +T1026 http://purl.obolibrary.org/obo/MONDO_0005047 31502 31511 infertile +T1027 PR:000014888 31562 31567 SirT1 +T1028 NCBITaxon:10088 31573 31577 mice +T1029 UBERON:0001977 31750 31755 serum +T1030 CHEBI:50114 31765 31773 estrogen +T1031 PR:000014888 31777 31782 SirT1 +T1032 NCBITaxon:10088 31795 31799 mice +T1033 CHEBI:50114 31906 31914 estrogen +T1034 PR:000007204 31924 31927 ERα +T1035 GO:0010467 31928 31938 expression +T1036 PR:000014888 32042 32047 SirT1 +T1037 GO:0065007 32061 32070 regulates +T1038 PR:000007641 32087 32092 FoxO3 +T1039 PR:000007641 32129 32134 FoxO3 +T1040 PR:000007641 32147 32152 FoxO3 +T1041 NCBITaxon:10088 32163 32167 mice +T1042 UBERON:0001305 32195 32212 ovarian follicles +T1043 UBERON:0000992 32248 32255 ovarian +T1044 UBERON:0000992 32292 32299 ovarian +T1045 UBERON:0000992 32340 32347 ovarian +T1046 http://purl.obolibrary.org/obo/MONDO_0001119 32364 32383 premature menopause +T1047 GO:0042697 32374 32383 menopause +T1048 SO:0000704 32394 32401 genetic +T1049 PR:000014888 32484 32489 SirT1 +T1050 UBERON:0001305 32535 32552 ovarian follicles +T1051 NCBITaxon:10088 32582 32586 mice +T1052 PR:000014888 32589 32594 SirT1 +T1053 GO:0065007 32595 32604 modulates +T1054 UBERON:0000058 32642 32648 ductal +T1055 GO:0035239 32642 32662 ductal morphogenesis +T1056 PR:000014888 32695 32700 SirT1 +T1057 NCBITaxon:10088 32706 32710 mice +T1058 PR:000008947 32732 32739 IGFBP-1 +T1059 UBERON:0001013 32743 32758 adipose tissues +T1060 CHEBI:50114 32809 32817 estrogen +T1061 GO:0030154 32848 32866;32897 32902 differentiation of ... cells +T1062 UBERON:0003244 32867 32885 mammary epithelial +T1063 GO:0010467 32995 33005 expression +T1064 PR:000008947 33009 33016 IGFBP-1 +T1065 UBERON:0012282 33033 33049 mammary fat pads +T1066 PR:000014888 33062 33067 SirT1 +T1067 NCBITaxon:10088 33073 33077 mice +T1068 UBERON:0000058 33116 33120 duct +T1069 GO:0007565 33155 33164 pregnancy +T1070 CHEBI:50114 33169 33177 estrogen +T1071 UBERON:0000058 33210 33216 ductal +T1072 GO:0035239 33210 33230 ductal morphogenesis +T1073 PR:000014888 33234 33239 SirT1 +T1074 NCBITaxon:10088 33245 33249 mice +T1075 CHEBI:50114 33310 33318 estrogen +T1076 NCBITaxon:10088 33419 33423 mice +T1077 UBERON:0012282 33447 33463 mammary fat pads +T1078 NCBITaxon:10088 33476 33481 mouse +T1079 PR:000014888 33533 33538 SirT1 +T1080 NCBITaxon:10088 33544 33548 mice +T1081 CHEBI:50114 33611 33619 estrogen +T1082 UBERON:0000310 33665 33672 mammary +T1083 UBERON:0000479 33673 33680 tissues +T1084 PR:000008947 33710 33717 IGFBP-1 +T1085 GO:0010467 33718 33728 expression +T1086 PR:000014888 33732 33737 SirT1 +T1087 NCBITaxon:10088 33743 33747 mice +T1088 CHEBI:50114 33754 33762 estrogen +T1089 GO:0065007 33894 33902 regulate +T1090 UBERON:0001911 33903 33916 mammary gland +T1091 GO:0030879 33903 33928 mammary gland development +T1092 UBERON:0000922 33932 33941 embryonic +T1093 GO:0007567 33947 33952 natal +T1094 GO:0000003 33958 33970 reproductive +T1095 GO:0010467 33998 34008 expression +T1096 PR:000008947 34012 34019 IGFBP-1 +T1097 UBERON:0000310 34062 34069 Mammary +T1098 GO:0030879 34062 34081 Mammary development +T1099 CHEBI:50114 34094 34102 estrogen +T1100 CHEBI:50114 34132 34140 estrogen +T1101 UBERON:0000922 34199 34208 embryonic +T1102 GO:0009790 34199 34220 embryonic development +T1103 CHEBI:50114 34297 34305 estrogen +T1104 UBERON:0000922 34333 34342 embryonic +T1105 UBERON:0003326 34353 34372 mammary mesenchymes +T1106 UBERON:0003916 34400 34408 fat pads +T1107 GO:0008283 34422 34433 proliferate +T1108 UBERON:0000058 34464 34469 ducts +T1109 CHEBI:50114 34540 34548 estrogen +T1110 UBERON:0000310 34567 34574 mammary +T1111 CL:0002451 34567 34585 mammary stem cells +T1112 UBERON:0000483 34605 34615 epithelial +T1113 GO:0030154 34689 34707;34730 34735 differentiation of ... cells +T1114 GO:0030855 34689 34704;34736 34738;34767 34783 differentiation ... to ... epithelial cells +T1115 GO:0030855 34689 34704;34736 34738;34811 34827 differentiation ... to ... epithelial cells +T1116 UBERON:0000483 34708 34718 epithelial +T1117 CHEBI:50114 34739 34747 estrogen +T1118 UBERON:0034969 34760 34777 ductal epithelial +T1119 CL:0000068 34760 34783 ductal epithelial cells +T1120 CHEBI:50114 34792 34800 estrogen +T1121 UBERON:0000483 34811 34821 epithelial +T1122 CL:0000066 34811 34827 epithelial cells +T1123 GO:0035295 34863 34877;34890 34895 development of ... ducts +T1124 UBERON:0000058 34890 34895 ducts +T1125 PR:000007204 34925 34928 ERα +T1126 NCBITaxon:10088 34938 34942 mice +T1127 UBERON:0005313 34952 34956 TEBs +T1128 GO:0060033 34966 34973 regress +T1129 GO:0007567 35003 35008 birth +T1130 UBERON:0000058 35082 35087 ducts +T1131 GO:0030154 35220 35238;35278 35283 differentiation of ... cells +T1132 GO:0030855 35220 35235;35284 35288;35308 35324 differentiation ... into ... epithelial cells +T1133 CHEBI:50114 35239 35247 estrogen +T1134 UBERON:0034969 35260 35277 ductal epithelial +T1135 CL:0000068 35260 35283 ductal epithelial cells +T1136 CHEBI:50114 35289 35297 estrogen +T1137 UBERON:0000483 35308 35318 epithelial +T1138 CL:0000066 35308 35324 epithelial cells +T1139 GO:0010467 35337 35344 express +T1140 PR:000007204 35345 35348 ERα +T1141 CHEBI:50114 35360 35368 estrogen +T1142 UBERON:0000992 35417 35424 ovarian +T1143 CHEBI:50114 35425 35433 estrogen +T1144 UBERON:0000007 35451 35460 pituitary +T1145 PR:000007968 35451 35463 pituitary GH +T1146 UBERON:0003891 35501 35508 stromal +T1147 CL:0000499 35501 35513 stromal cell +T1148 GO:0030154 35553 35571;35611 35616 differentiation of ... cells +T1149 GO:0030855 35553 35568;35617 35619;35639 35655 differentiation ... to ... epithelial cells +T1150 CHEBI:50114 35572 35580 estrogen +T1151 UBERON:0034969 35593 35610 ductal epithelial +T1152 CL:0000068 35593 35616 ductal epithelial cells +T1153 CHEBI:50114 35620 35628 estrogen +T1154 UBERON:0000483 35639 35649 epithelial +T1155 CL:0000066 35639 35655 epithelial cells +T1156 GO:0007567 35674 35679 natal +T1157 GO:0030879 35680 35708 development of mammary gland +T1158 UBERON:0001911 35695 35708 mammary gland +T1159 UBERON:0000058 35730 35736 ductal +T1160 GO:0035239 35730 35750 ductal morphogenesis +T1161 PR:000014888 35769 35774 SirT1 +T1162 NCBITaxon:10088 35780 35784 mice +T1163 PR:000008947 35856 35863 IGFBP-1 +T1164 UBERON:0000310 35867 35874 mammary +T1165 UBERON:0001013 35875 35890 adipose tissues +T1166 PR:000014888 35892 35897 SirT1 +T1167 CHEBI:50114 35949 35957 estrogen +T1168 UBERON:0034969 35978 35995 ductal epithelial +T1169 CL:0000068 35978 36000 ductal epithelial cell +T1170 GO:0050673 35985 36014 epithelial cell proliferation +T1171 GO:0030154 35996 36000;36019 36034 cell ... differentiation +T1172 GO:0065007 36061 36071 modulating +T1173 PR:000014888 36072 36077 SirT1 +T1174 UBERON:0000310 36088 36094 Breast +T1175 http://purl.obolibrary.org/obo/MONDO_0007254 36088 36101 Breast cancer +T1176 UBERON:0000104 36171 36179 Lifetime +T1177 CHEBI:50114 36192 36200 estrogen +T1178 UBERON:0000310 36228 36234 breast +T1179 http://purl.obolibrary.org/obo/MONDO_0007254 36228 36241 breast cancer +T1180 CHEBI:30879 36302 36309 alcohol +T1181 GO:0007631 36310 36321 consumption +T1182 CHEBI:50114 36410 36418 estrogen +T1183 CHEBI:50114 36548 36556 estrogen +T1184 PR:000014888 36622 36627 SirT1 +T1185 CHEBI:50114 36669 36677 estrogen +T1186 PR:000014888 36714 36719 SirT1 +T1187 UBERON:0000104 36732 36740 lifetime +T1188 CHEBI:33284 36795 36804 nutrients +T1189 UBERON:0000310 36866 36872 breast +T1190 http://purl.obolibrary.org/obo/MONDO_0007254 36866 36879 breast cancer +T1191 UBERON:0000310 37023 37029 breast +T1192 http://purl.obolibrary.org/obo/MONDO_0007254 37023 37036 breast cancer +T1193 GO:0007567 37080 37085 birth +T1194 UBERON:0001977 37106 37111 serum +T1195 CHEBI:50114 37122 37130 estrogen +T1196 PR:000014888 37333 37338 SirT1 +T1197 UBERON:0000310 37355 37361 breast +T1198 http://purl.obolibrary.org/obo/MONDO_0007254 37355 37368 breast cancer +T1199 UBERON:0000310 37466 37472 breast +T1200 http://purl.obolibrary.org/obo/MONDO_0007254 37466 37479 breast cancer +T1201 CHEBI:26195 37498 37509 polyphenols +T1202 CHEBI:36357 37527 37536 molecules +T1203 PR:000014888 37595 37600 SirT1 +T1204 CHEBI:52217 37614 37629 pharmacological +T1205 CHEBI:36357 37647 37656 compounds +T1206 NCBITaxon:9606 37660 37666 humans +T1207 PR:000014888 37693 37698 SirT1 +T1208 PR:000014888 37796 37801 SirT1 +T1209 NCBITaxon:10088 37807 37811 mice +T1210 PR:000014888 37850 37855 SirT1 +T1211 PR:000014888 37892 37897 SirT1 +T1212 GO:0042592 37918 37929 homeostasis +T1213 GO:0051716 37933 37951 cellular responses +T1214 GO:0065007 37965 37975 regulation +T1215 GO:0010467 37983 37993 expression +T1216 PR:000008947 37997 38004 IGFBP-1 +T1217 PR:000003293 38009 38013 IκBα +T1218 CHEBI:50114 38060 38068 estrogen +T1219 UBERON:0003244 38113 38131 mammary epithelial +T1220 CL:0002327 38113 38137 mammary epithelial cells +T1221 GO:0071159 38141 38146 NF-κB +T1222 GO:0051092 38141 38157 NF-κB activation +T1223 PR:000014888 38159 38164 SirT1 +T1224 UBERON:0000310 38225 38231 breast +T1225 http://purl.obolibrary.org/obo/MONDO_0007254 38225 38238 breast cancer +T1226 UBERON:0001911 38266 38279 mammary gland +T1227 GO:0030879 38266 38291 mammary gland development +T1228 PR:000014888 38340 38345 SirT1 +T1229 GO:0065007 38346 38355 modulates +T1230 CHEBI:50114 38376 38384 estrogen +T1231 GO:0065007 38405 38414 regulates +T1232 UBERON:0000058 38429 38435 ductal +T1233 GO:0035239 38429 38449 ductal morphogenesis +T1234 PR:000014888 38524 38529 SirT1 +T1235 UBERON:0000310 38533 38539 breast +T1236 http://purl.obolibrary.org/obo/MONDO_0007254 38533 38546 breast cancer +T1237 UBERON:0000062 38567 38573 organs +T1238 PR:000004427 38636 38639 Atm +T1239 http://purl.obolibrary.org/obo/MONDO_0000437 38642 38648 ataxia +T1240 CHEBI:472552 38665 38669 BrdU +T1241 CHEBI:472552 38672 38688 bromodeoxyurdine +T1242 NCBITaxon:10358 38690 38693 CMV +T1243 NCBITaxon:10358 38696 38711 cytomegalovirus +T1244 PR:000007204 38713 38716 ERα +T1245 CHEBI:50114 38719 38727 estrogen +T1246 PR:000007204 38719 38742 estrogen receptor alpha +T1247 PR:000045358 38810 38817 insulin +T1248 PR:000008947 38840 38847 IGFBP-1 +T1249 PR:000008947 38850 38896 insulin-like growth factor-1 binding protein-1 +T1250 CHEBI:51686 38898 38899 H +T1251 CHEBI:51686 38906 38917 hemotoxylin +T1252 PR:000045358 38935 38942 insulin +T1253 PR:000045358 38943 38950 insulin +T1254 PR:000003293 38983 38987 IκBα +T1255 CHEBI:35222 38990 39000 inhibitors +T1256 PR:000003293 38990 39023 inhibitors of NF-κB alpha subunit +T1257 GO:0071159 39004 39009 NF-κB +T1258 PR:000001775 39025 39029 IKKα +T1259 PR:000001775 39032 39048 IκB kinase alpha +T1260 NCBITaxon:39107 39056 39062 murine +T1261 UBERON:0000922 39063 39072 embryonic +T1262 CL:0000057 39073 39083 fibroblast +T1263 GO:0005634 39090 39097 nuclear +T1264 MOP:0000635 39123 39137 chain reaction +T1265 PR:000001954 39139 39143 RANK +T1266 PR:000001954 39146 39173 receptor activator of NF-κB +T1267 GO:0071159 39168 39173 NF-κB +T1268 GO:0016458 39181 39190 silencing +T1269 UBERON:0005313 39214 39217 TEB +T1270 UBERON:0005313 39220 39236 terminal end bud +T1271 http://purl.obolibrary.org/obo/MONDO_0005070 39244 39249 tumor +T1272 PR:000014888 39412 39417 SirT1 +T1273 NCBITaxon:10088 39425 39429 mice +T1274 PR:000014888 39575 39580 SirT1 +T1275 UBERON:0000922 39588 39597 embryonic +T1276 CL:0002322 39588 39608 embryonic stem cells +T1277 NCBITaxon:10088 39613 39617 mice +T1278 PR:000014888 39924 39929 SirT1 +T1279 NCBITaxon:10088 39935 39939 mice +T1280 PR:000014888 40032 40037 SirT1 +T1281 NCBITaxon:10088 40043 40047 mice +T1282 PR:000014888 40052 40057 SirT1 +T1283 NCBITaxon:10088 40063 40067 mice +T1284 http://purl.obolibrary.org/obo/MONDO_0004992 40160 40166 Cancer +T1285 GO:0007568 40255 40260 Aging +T1286 PR:000014888 40735 40740 SirT1 +T1287 PR:000014888 40750 40755 SirT1 +T1288 NCBITaxon:10088 40761 40765 mice +T1289 NCBITaxon:10088 40771 40776 Mouse +T1290 PR:000014888 40777 40782 SirT1 +T1291 SO:0001023 40793 40799 allele +T1292 PR:000014888 40801 40806 SirT1 +T1293 SO:0001023 40831 40837 allele +T1294 PR:000014888 40839 40844 SirT1 +T1295 SO:0001023 40862 40868 allele +T1296 PR:000014888 40870 40875 SirT1 +T1297 PR:P23940 40880 40881 B +T1298 PR:P23940 40883 40888 BamHI +T1299 PR:000014888 40919 40924 SirT1 +T1300 GO:0010467 40925 40935 expression +T1301 NCBITaxon:39107 41028 41034 murine +T1302 UBERON:0000922 41035 41044 embryonic +T1303 CL:0000057 41045 41056 fibroblasts +T1304 UBERON:0000310 41068 41075 mammary +T1305 UBERON:0000479 41076 41083 tissues +T1306 PR:000014888 41126 41131 SirT1 +T1307 NCBITaxon:10088 41137 41141 mice +T1308 NCBITaxon:10088 41167 41171 mice +T1309 NCBITaxon:10088 41187 41191 mice +T1310 UBERON:0001977 41235 41240 serum +T1311 PR:000045358 41251 41258 insulin +T1312 PR:000014888 41308 41313 SirT1 +T1313 PR:000014888 41330 41335 SirT1 +T1314 NCBITaxon:10088 41349 41353 mice +T1315 PR:000014888 41398 41403 SirT1 +T1316 PR:000014888 41410 41415 SirT1 +T1317 PR:000017509 41426 41430 Ku70 +T1318 UBERON:0000922 41434 41443 embryonic +T1319 CL:0002322 41434 41454 embryonic stem cells +T1320 PR:000014888 41523 41528 SirT1 +T1321 PR:000017509 41535 41539 Ku70 +T1322 PR:000004427 41548 41551 Atm +T1323 UBERON:0000922 41555 41564 embryonic +T1324 CL:0002322 41555 41575 embryonic stem cells +T1325 CHEBI:16240 41597 41614 hydrogen peroxide +T1326 GO:0007595 41627 41636 Lactation +T1327 PR:000014888 41648 41653 SirT1 +T1328 NCBITaxon:10088 41659 41663 mice +T1329 UBERON:0000310 41697 41704 mammary +T1330 UBERON:0000479 41705 41712 tissues +T1331 PR:000014888 41745 41750 SirT1 +T1332 NCBITaxon:10088 41756 41760 mice +T1333 UBERON:0000310 41821 41828 mammary +T1334 UBERON:0000479 41829 41836 tissues +T1335 PR:000014888 41872 41877 SirT1 +T1336 NCBITaxon:10088 41890 41894 mice +T1337 GO:0007565 41915 41924 pregnancy +T1338 GO:0007595 41934 41943 lactation +T1339 UBERON:0001913 42012 42016 milk +T1340 GO:0007595 42012 42027 milk production +T1341 UBERON:0000310 42035 42042 mammary +T1342 UBERON:0000479 42043 42050 tissues +T1343 PR:000014888 42076 42081 SirT1 +T1344 NCBITaxon:10088 42087 42091 mice +T1345 NCBITaxon:10088 42121 42126 mouse +T1346 UBERON:0001913 42127 42131 milk +T1347 GO:0005634 42153 42160 nuclear +T1348 CL:0000136 42173 42183 adipocytes +T1349 UBERON:0000483 42185 42195 epithelial +T1350 CL:0000066 42185 42201 epithelial cells +T1351 UBERON:0000310 42222 42229 mammary +T1352 UBERON:0000479 42230 42237 tissues +T1353 GO:0007565 42271 42280 Pregnancy +T1354 UBERON:0000058 42289 42295 ductal +T1355 GO:0035239 42289 42309 ductal morphogenesis +T1356 UBERON:0001911 42339 42352 mammary gland +T1357 GO:0030879 42339 42364 mammary gland development +T1358 NCBITaxon:10088 42395 42399 mice +T1359 UBERON:0005313 42405 42422 terminal end buds +T1360 UBERON:0005313 42424 42428 TEBs +T1361 UBERON:0000058 42465 42470 ducts +T1362 NCBITaxon:10088 42479 42483 mice +T1363 GO:0007565 42508 42517 pregnancy +T1364 UBERON:0001913 42559 42563 milk +T1365 GO:0007595 42559 42574 milk production +T1366 GO:0007595 42578 42587 lactation +T1367 PR:000014888 42620 42625 SirT1 +T1368 NCBITaxon:10088 42639 42643 mice +T1369 UBERON:0000058 42657 42663 ductal +T1370 GO:0035239 42657 42677 ductal morphogenesis +T1371 GO:0007565 42679 42688 Pregnancy +T1372 UBERON:0000058 42697 42703 ductal +T1373 GO:0035239 42697 42717 ductal morphogenesis +T1374 UBERON:0005313 42741 42745 TEBs +T1375 UBERON:0000058 42747 42753 ductal +T1376 PR:000014888 42807 42812 SirT1 +T1377 NCBITaxon:10088 42818 42822 mice +T1378 PR:000008947 42889 42935 insulin-like growth factor-1 binding protein-1 +T1379 PR:000008947 42937 42944 IGFBP-1 +T1380 PR:000003293 42950 42954 IκBα +T1381 UBERON:0000310 42958 42965 mammary +T1382 UBERON:0000479 42966 42973 tissues +T1383 NCBITaxon:10088 42982 42986 mice +T1384 NCBITaxon:10088 42995 42999 mice +T1385 NCBITaxon:10088 43015 43019 mice +T1386 PR:000014888 43043 43048 SirT1 +T1387 CHEBI:50114 43261 43269 Estrogen +T1388 UBERON:0000058 43294 43300 ductal +T1389 UBERON:0000310 43364 43371 mammary +T1390 UBERON:0000479 43372 43379 tissues +T1391 PR:000014888 43416 43421 SirT1 +T1392 NCBITaxon:10088 43427 43431 mice +T1393 CHEBI:50114 43447 43455 estrogen +T1394 GO:0010467 43546 43556 expression +T1395 PR:000008947 43560 43606 insulin-like growth factor-1 binding protein-1 +T1396 PR:000008947 43608 43615 IGFBP-1 +T1397 PR:000003293 43621 43625 IκBα +T1398 UBERON:0000310 43663 43670 mammary +T1399 UBERON:0000479 43671 43678 tissues +T1400 PR:000014888 43715 43720 SirT1 +T1401 NCBITaxon:10088 43726 43730 mice +T1402 CHEBI:50114 43746 43754 estrogen +T1403 UBERON:0003244 43785 43803 Mammary epithelial +T1404 CL:0002327 43785 43808 Mammary epithelial cell +T1405 GO:0033598 43785 43822 Mammary epithelial cell proliferation +T1406 GO:1990134 43793 43808;43827 43836 epithelial cell ... apoptosis +T1407 PR:000014888 43840 43845 SirT1 +T1408 NCBITaxon:10088 43851 43855 mice +T1409 GO:0007595 43859 43868 lactation +T1410 CHEBI:472552 43893 43909 bromodeoxyurdine +T1411 CHEBI:472552 43911 43915 BrdU +T1412 UBERON:0000310 43933 43940 mammary +T1413 PR:000014888 43955 43960 SirT1 +T1414 NCBITaxon:10088 43994 43998 mice +T1415 GO:0007565 44012 44021 pregnancy +T1416 GO:0007595 44032 44041 lactation +T1417 UBERON:0005313 44080 44097 terminal end buds +T1418 UBERON:0005313 44099 44103 TEBs +T1419 UBERON:0005313 44136 44139 TEB +T1420 UBERON:0000058 44141 44146 ducts +T1421 UBERON:0003215 44152 44160 alveolus +T1422 PR:000014888 44164 44169 SirT1 +T1423 NCBITaxon:10088 44175 44179 mice +T1424 UBERON:0000058 44205 44210 ducts +T1425 UBERON:0003215 44215 44223 alveolus +T1426 NCBITaxon:10088 44237 44241 mice +T1427 CL:0000445 44378 44393 apoptotic cells +T1428 CHEBI:472552 44440 44444 BrdU +T1429 CHEBI:472552 44488 44492 BrdU +T1430 UBERON:0000483 44502 44512 epithelial +T1431 CL:0000066 44502 44518 epithelial cells +T1432 UBERON:0005313 44522 44526 TEBs +T1433 UBERON:0000058 44528 44533 ducts +T1434 UBERON:0003215 44539 44546 alveoli +T1435 PR:000014888 44550 44555 SirT1 +T1436 NCBITaxon:10088 44561 44565 mice +T1437 NCBITaxon:10088 44597 44601 mice +T1438 PR:000014888 44730 44735 SirT1 +T1439 GO:0071159 44755 44760 NF-κB +T1440 PR:000000134 44786 44790 TNFα +T1441 PR:000003293 44833 44837 IκBα +T1442 GO:0010467 44838 44848 expression +T1443 PR:000014888 44852 44857 SirT1 +T1444 NCBITaxon:39107 44891 44897 murine +T1445 UBERON:0000922 44898 44907 embryonic +T1446 CL:0000057 44908 44919 fibroblasts +T1447 PR:000000134 44955 44959 TNFα +T1448 PR:000003293 45054 45058 IκBα +T1449 PR:000014888 45080 45085 SirT1 +T1450 PR:000000134 45166 45170 TNFα +T1451 PR:000003293 45219 45223 IκBα +T1452 PR:000014888 45328 45333 SirT1 +T1453 GO:0065007 45334 45343 modulates +T1454 CHEBI:50114 45344 45352 estrogen +T1455 PR:000045358 45353 45360 insulin +T1456 UBERON:0000058 45396 45402 ductal +T1457 GO:0035239 45396 45416 ductal morphogenesis +T1458 PR:000045358 45435 45442 insulin +T1459 PR:000045358 45443 45450 insulin +T1460 PR:000014888 45503 45508 SirT1 +T1461 PR:000029189 45513 45524 Akt kinases +T1462 PR:000014888 45625 45630 SirT1 +T1463 GO:0065007 45644 45653 regulates +T1464 GO:0010467 45658 45668 expression +T1465 PR:000008947 45672 45718 insulin-like growth factor-1 binding protein-1 +T1466 PR:000008947 45720 45727 IGFBP-1 +T1467 GO:0035425 45746 45755 autocrine +T1468 GO:0038001 45763 45772 paracrine +T1469 UBERON:0003244 45803 45821 Mammary epithelial +T1470 GO:0010467 45844 45851 express +T1471 PR:000003273 45852 45866 IGF-1 receptor +T1472 PR:000003273 45868 45874 IGF-1R +T1473 CHEBI:50114 45903 45911 estrogen +T1474 UBERON:0034969 45935 45952 ductal epithelial +T1475 CL:0000068 45935 45958 ductal epithelial cells +T1476 CL:0000068 45960 45963 DEC +T1477 CHEBI:50114 46008 46016 estrogen +T1478 UBERON:0003891 46029 46036 stromal +T1479 CL:0000499 46029 46041 stromal cell +T1480 CL:0000499 46043 46044 S +T1481 UBERON:0000992 46092 46099 ovarian +T1482 CHEBI:50114 46100 46108 estrogen +T1483 GO:0030855 46205 46220;46230 46232;46252 46268 differentiation ... to ... epithelial cells ... epithelial +T1484 CL:0000068 46224 46227 DEC +T1485 UBERON:0034969 46245 46262 ductal epithelial +T1486 CL:0000068 46245 46268 ductal epithelial cells +T1487 CL:0000068 46270 46273 DEC +T1488 PR:000014888 46279 46284 SirT1 +T1489 GO:0065007 46298 46307 regulates +T1490 GO:0010467 46312 46322 expression +T1491 PR:000008947 46326 46333 IGFBP-1 +T1492 UBERON:0001013 46337 46352 adipose tissues +T1493 UBERON:0000058 46439 46445 ductal +T1494 GO:0035239 46439 46459 ductal morphogenesis +T1495 PR:000014888 46470 46475 SirT1 +T1496 NCBITaxon:10088 46481 46485 mice +T1497 GO:0007565 46494 46503 pregnancy +T1498 CHEBI:50114 46517 46525 estrogen +T1499 UBERON:0000058 46551 46557 ductal +T1500 GO:0035239 46551 46571 ductal morphogenesis +T1501 PR:000014888 46582 46587 SirT1 +T1502 NCBITaxon:10088 46593 46597 mice +T1503 UBERON:0012101 46654 46663 Perinatal +T1504 GO:0007567 46658 46663 natal +T1505 GO:0007595 46690 46699 lactation +T1506 PR:000014888 46710 46715 SirT1 +T1507 NCBITaxon:10088 46721 46725 mice +T1508 PR:000014888 46749 46754 SirT1 +T1509 NCBITaxon:10088 46764 46768 mice +T1510 PR:000014888 46773 46778 SirT1 +T1511 NCBITaxon:10088 46790 46794 mice +T1512 GO:0007567 46921 46926 birth +T1513 GO:0007567 46976 46981 natal diff --git a/src/ontogpt/evaluation/craft/database/all/17201918.txt b/src/ontogpt/evaluation/craft/database/all/17201918.txt new file mode 100644 index 000000000..745104887 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17201918.txt @@ -0,0 +1,191 @@ +SirT1 modulates the estrogen–insulin-like growth factor-1 signaling for postnatal development of mammary gland in mice + +Abstract + +Introduction + +Estrogen and insulin-like growth factor-1 (IGF-1) play important roles in mammary gland development and breast cancer. SirT1 is a highly conserved protein deacetylase that can regulate the insulin/IGF-1 signaling in lower organisms, as well as a growing number of transcription factors, including NF-κB, in mammalian cells. Whether SirT1 regulates the IGF-1 signaling for mammary gland development and function, however, is not clear. In the present study, this role of SirT1 was examined by studying SirT1-deficient mice. + +Methods + +SirT1-deficient (SirT1ko/ko) mice were generated by crossing a new strain of mice harboring a conditional targeted mutation in the SirT1 gene (SirT1co/co) with CMV-Cre transgenic mice. Whole mount and histology analyses, immunofluorescence staining, immunohistochemistry, and western blotting were used to characterize mammary gland development in virgin and pregnant mice. The effect of exogenous estrogen was also examined by subcutaneous implantation of a slow-releasing pellet in the subscapular region. + +Results + +Both male and female SirT1ko/ko mice can be fertile despite the growth retardation phenotype. Virgin SirT1ko/ko mice displayed impeded ductal morphogenesis, whereas pregnant SirT1ko/ko mice manifested lactation failure due to an underdeveloped lobuloalveolar network. Estrogen implantation was sufficient to rescue ductal morphogenesis. Exogenous estrogen reversed the increased basal level of IGF-1 binding protein-1 expression in SirT1ko/ko mammary tissues, but not that of IκBα expression, suggesting that increased levels of estrogen enhanced the production of local IGF-1 and rescued ductal morphogenesis. Additionally, TNFα treatment enhanced the level of the newly synthesized IκBα in SirT1ko/ko cells. SirT1 deficiency therefore affects the cellular response to multiple extrinsic signals. + +Conclusion + +SirT1 modulates the IGF-1 signaling critical for both growth regulation and mammary gland development in mice. SirT1 deficiency deregulates the expression of IGF-1 binding protein-1 and attenuates the effect of IGF-1 signals, including estrogen-stimulated local IGF-1 signaling for the onset of ductal morphogenesis. These findings suggest that the enzymatic activity of SirT1 may influence both normal growth and malignant growth of mammary epithelial cells. + +Introduction + +Mammalian SirT1 belongs to a family of nicotinamide adenine dinucleotide-dependent histone deacetylases [1,2]. SirT1 is most closely related to yeast Sir2, the founding member of the evolutionarily conserved Sir2 family. Yeast Sir2 is required for silencing transcription at the telomeric region and mating type loci, and for suppression of ribosomal DNA recombination [3,4]. The expression of an extra copy of Sir2 in either yeast mother cells or multicell organisms such as nematodes can significantly extend the lifespan [5,6]. Inactivation of Sir2 enhances stress resistance and extends chronological lifespan of nondividing yeast cells, which is opposite to the requirement for Sir2 function in the reproductive lifespan [7]. Whether SirT1 regulates the reproductive lifespan and/or the chronological lifespan in mammals remains unknown. + +Sir2 is an integral part of an evolutionarily conserved insulin/insulin-like growth factor-1 (IGF-1) signaling (IIS) system in worms (Caenorhabditis elegans), fruit flies (Drosophila), mice, and humans [8,9]. The IIS system includes membrane-bound receptors, cytoplasmic kinases, and nuclear transcription factors. To maintain the proper expression of the effector genes for the IIS system, these conserved components form a sophisticated regulatory system, which centers on a family of forkhead transcription factors (forkhead box 'other' proteins (FoxOs)), and operates on two levels. On one level, SirT1-mediated protein deacetylation attenuates the transcriptional activity of nuclear FoxO transcription factors [10-12]. On the second level, the FoxO transcription factors can be sequestered within the cytoplasm when phosphorylated by activated Akt kinases in response to insulin and IGF-1 signals [13]. Conceivably, the IIS system senses the levels of insulin and IGF-1 and negatively regulates the expression of the effector genes. The IIS system is responsible for food storage, stress tolerance, and longevity in lower organisms, such as C. elegans [8,9,14]. In more advanced species, steroid hormones evolved to regulate the IIS system [15]. In mice and humans, the IGF-1 signaling of the IIS system mediates local effects for growth and hormonal regulation for multiple tissues, including mammary glands [16,17]. + +Mammalian SirT1 has evolved to modify the activity of a growing number of transcription factors, including p53, NF-κB, and PGC-1α, suggesting that SirT1 functions in a wide range of cellular responses to stress, inflammation, and nutrients [18-21]. SirT1-deficient mice display characteristic phenotypes of perinatal death and growth retardation as well as other diverse phenotypes, such as eye defects, with varying severity [22,23]. The underlying causal mechanism for these phenotypes, however, remains unknown. We recently generated SirT1-deficient (SirT1ko/ko) mice and found that both male and female SirT1ko/ko mice can be fertile, which is in contrast to the sterile phenotypes observed in one strain of SirT1-deficient mice [22]. This led to our study of the link between SirT1 and IGF-1 signaling using the mammary gland as a model organ. + +The mammary gland is a unique organ because it develops after birth and undergoes dynamic changes throughout the reproductive lifespan of a female. At the onset of puberty, ovarian estrogen stimulates ductal morphogenesis during which mammary epithelial progenitor cells differentiate and proliferate while interacting with adipocytes and stromal cells within mammary fat pad [17,24,25]. Ovarian estrogen, in synergy with pituitary growth hormone (GH), stimulates stromal cells to produce local IGF-1. The local IGF-1, but not liver-produced systemic IGF-1, provides a paracrine signal for commencing ductal morphogenesis [26]. Mice lacking GH, estrogen, IGF-1, GH receptor, or estrogen receptor alpha (ERα) fail to undergo postnatal ductal morphogenesis [17,24-31], indicating that both steroid hormones and IGF-1 are on the common pathway for a critical developmental checkpoint. Once the arborated ductal network is established, cycles of differentiation, proliferation, and death of secretory alveolar epithelium repeat with each pregnancy [17,24,25]. + +In the present article we report the finding and characterization of impeded ductal morphogenesis in virgin SirT1ko/ko mice and lactation failure in SirT1ko/ko mothers. The characterization of these phenotypes has identified a SirT1-dependent regulatory mechanism by which SirT1 modulates the effectiveness of the estrogen–IGF-1 signaling for mammary gland development. The estrogen–IGF-1 signaling is defined as the ovarian estrogen-regulated, stromal cell-produced local IGF-1 signal for stimulating mammary epithelial cells. + +Materials and methods + +Mice + +A previously described SirT1 targeting construct, KOII [23], was used to generate mice harboring a conditional targeted mutation in the SirT1 gene (SirT1co/co mice) (see Additional file 1). The breeding of SirT1co/co mice and CMV-Cre transgenic mice results in mice harboring a germline-transmitted deletion of exon 4 of the SirT1 gene (SirT1+/ko mice). Both SirT1co/co mice and SirT1+/co mice were used to establish breeding colonies for generating SirT1co/co and SirT1ko/ko mice, respectively. Both SirT1co/co mice and SirT1ko/ko mice were in a mixed 129SvJ/C57B6 background. Mice were housed in a special-pathogen-free facility and all procedures were approved by the University of Washington Animal Care and Use Committee. A PCR-based genotyping method was established to identify the wild-type, co, and ko loci of the SirT1 gene using three primers: 5' co primer, 5'-GGTTGACTTAGGTCTTGTCTG; 5' ko primer, 5'-AGGCGGATTTCTGAGTTCGA; 3' primer, 5'-CGTCCCTTGTAATGTTTCCC. Murine embryonic fibroblasts (MEFs) were isolated from the embryos between embryonic day 12.5 and embryonic day 14.5. The body weight was measured once a week. + +Serum insulin-like growth factor-1 + +A commercial radioimmunoassay (ALPCO Inc., Windham, NH, USA) was used. Samples (300 μl) were prepared via an alcohol extraction followed by a 2-day disequilibrium incubation at 4°C. Samples were measured in duplicate and data were calculated as the samples were counted. Six standards ranging from 10 to 500 ng/ml were used to generate the standard curve. The lower limit of detection was 10 ng. + +Survival assay + +To determine the sensitivity of embryonic stem cells after ionizing radiation or hydrogen peroxide treatment, 300 embryonic stem cells were seeded onto a 10-cm plate for 24 hours. The plates were either exposed to a 137Cs source at indicated doses or were treated with embryonic stem media containing hydrogen peroxide at indicated concentrations for 30 minutes. Seven days after irradiation or hydrogen peroxide treatment, the embryonic stem colonies stained with crystal violet were counted. + +Estrogen implantation + +Estrogen pellets in the form of 17β-estradiol, at 0.1 mg, and the 21-day releasing time were obtained from Innovative Research (Sarasota, FL, USA). One pelletwas subcutaneously implanted in the subscapular region ofindividual female mice using a sterilizedmetal trochar (Innovative Research). + +Whole mount analysis + +The inguinoabdominal mammary fat pads were spread on microscope slides, fixed in Carnoy's fixative overnight, hydrated and stained with carmine alum stain (Sigma, St. Louis, MO, USA) overnight, and were then dehydrated, treated with xylene to remove fat, and mounted with Secure Mount (Fisher Scientific, Pittsburgh, PA, USA) and cover slips. + +Histological, immunohistochemical, and immunofluorescence analyses + +The mice were given a single dose of bromodeoxyurdine (BrdU) at 100 g/kg body weight via intraperitoneal injection 2 hours before euthanasia. The mammary fat pads, as well as other tissues, were collected and fixed in 10% formalin solution (Fisher Scientific). Paraffin-embedded sections were prepared at 4 μm thickness followed by standard H & E staining for histological analysis and by immunohistochemical staining for BrdU-labeled cells (Sigma) and apoptotic cells (Promega, Madison, WI, USA). To detect the presence of mouse milk proteins, a rabbit anti-mouse milk specific protein antibody (Nordic Immunology, Tillburg, The Netherlands) was used for immunofluorescent staining, followed by Texas Red anti-rabbit secondary antibody (Molecular Probes) and DAPI staining for nuclei (Molecular Probe, Eugene, OR, USA). Microscopic analyses of all histological findings were carried out on an AxioVert 200M microscope with AxioVision 4.5 software (Carl Zeiss, München-Hallbergmoos, Germany). + +Protein analysis + +Protein extracts were prepared from mammary tissues as well as from MEFs. The following primary antibodies were used: anti-SirT1 (Upstate Biotechology, Lake Placid, NY, USA), anti-IGF-1 binding protein-1 (IGFBP-1) (R&D Systems, Minneapolis, MN, USA), and anti-actin and anti-IκBα (Santa Cruz Biotechnology, Santa Cruz, CA, USA). The corresponding secondary antibodies were horseradish peroxidase-conjugated anti-rabbit (Pierce, Rockford, IL, USA), anti-rat, and anti-goat (Santa Cruz Biotechnology). + +Results + +Growth retardation in SirT1ko/ko mice + +The conditional targeted SirT1 mutant mice (SirT1co/co mice) carry an insertion mutation of the neomycin-resistant gene and lox sequences in the SirT1 gene flanking exon 4 that encodes a conserved Sir2 motif (Figure 1a). The mutation does not affect the expression of SirT1 in SirT1co/co mice (Figure 1b). As expected, SirT1co/co mice are phenotypically indistinguishable from wild-type mice. To convert the SirT1 co allele into the SirT1 ko allele, SirT1co/co mice were crossed with CMV-Cre transgenic mice to generate SirT1 heterozygotes carrying the SirT1+/ko, CMV-Cre+ genotype. The expression of the CMV-Cre transgene catalyzes the deletion of exon 4 in most lineages of cells, including germ cells (Figure 1a). This SirT1 ko allele should be identical to the previously described Δex4 mutation [23]. SirT1+/ko, Cre+ mice were backcrossed with wild-type mice to generate the mice harboring a germline-transmitted deletion mutation (that is, SirT1+/ko mice). The breeding of SirT1+/ko male mice and SirT1+/ko female mice resulted in SirT1ko/ko mice. The cells derived from either SirT1+/ko mice or SirT1ko/ko mice expressed a SirT1 mutant protein due to the inframe deletion of exon 4 (Figure 1b). This SirT1 mutant protein may not be functional, however, since SirT1+/ko mice are phenotypically indistinguishable from wild-type mice and SirT1Δex4/Δex4 mice were phenotypically identical to SirT1 null mice [23]. + +Similar to other strains of SirT1-deficient mice, nearly two-thirds of SirT1ko/ko newborns die shortly after birth and the majority of surviving SirT1ko/ko mice manifest growth retardation (Table 1 and Figure 1c) [22,23]. Growth retardation may result from a systemic defect in hormonal regulation, DNA double-strand break repair, or other causal mechanisms. We found that SirT1ko/ko mice have reduced levels of serum IGF-1 (Figure 1d). IGF-1 often acts as a local effector for pituitary GH. The serum level of GH in SirT1ko/ko mice, however, appeared to be within the normal range (data not shown). On the other hand, yeast Sir2 and its associated Sir complex, as well as mouse SirT6, have also been implicated in DNA double-strand break repair and the maintenance of chromosome stability [32-34]. We found that SirT1ko/ko embryonic stem cells do not have increased sensitivity to either ionizing radiation or hydrogen peroxide treatment when compared with wild-type cells, or with positive control cells such as Ku70-deficient or Atm-deficient cells (Figure 1e,f). Both Ku70-deficient mice and Atm-deficient mice display growth retardation phenotypes [35,36]. The growth retardation phenotype in SirT1ko/ko mice is therefore probably not due to a defect in DNA damage repair, but rather results from a deficit in IGF-1 signaling. + +Lactation failure + +Both male and female SirT1ko/ko mice can be fertile, which is in contrast with the reported sterile phenotype in one strain of SirT1-deficient mice [22]. When male SirT1ko/ko mice impregnated female SirT1+/ko mice, the number of surviving SirT1ko/ko offspring was reduced due to the partial perinatal lethal phenotype (Table 1). In a reciprocal approach, male SirT1+/ko mice can also impregnate female SirT1ko/ko mice (Table 1). After parturition, SirT1ko/ko mothers exhibited normal nursing behavior. The pups did not survive for more than 3 days after birth, however, unless they were immediately removed and put under the care of a foster mother. All pups died of dehydration or starvation as a result of lack of milk in their stomachs. + +To determine whether the SirT1ko/ko mothers encountered a lactation defect, whole mount and histological analyses were used to characterize the morphological changes in the mammary glands. We found that virgin SirT1ko/ko mice displayed impeded ductal morphogenesis up to 9 months of age, while age-matched virgin wild-type mice displayed extensive ductal elongation and branching (Figure 2a). The absence of ductal morphogenesis persisted in pregnant SirT1ko/ko mice up to day 13 of pregnancy (Figure 2b). Despite the fact that pregnancy can induce ductal morphogenesis at the late stage of pregnancy, underdeveloped mammary glands in SirT1ko/ko female mice cause a severe deficit in milk production, as shown by histological analysis as well as the immunofluorescence analysis using an anti-milk antibody (Figure 2b,c). + +Pregnancy-induced mammary gland development + +To characterize the developmental defect leading to lactation failure, we analyzed the mammary glands of SirT1ko/ko female mice on lactation day 1. Terminal end buds (TEBs) are club-shaped transitional structures (see Figure 3a, upper panel). In wild-type female mice, TEBs form and precede ductal elongation and branching at the onset of puberty [17,24,25]. When TEBs/ducts reach the edge of mammary fat pads, TEBs regress as shown in the virgin mice in Figure 3a (upper panel). Interestingly, the transitional TEBs can be readily identified in SirT1ko/ko female mice on lactation day 1, manifesting as either newly formed TEBs or as TEBs attached to developing ductal and alveolar structures (Figure 3a, lower panel). Moreover, SirT1ko/ko mice displayed varying degrees of ductal development, ranging from a lack of any ductal structure to a fully developed ductal network, the latter of which is comparable with the morphology and cellularity of wild-type mammary tissues on day 13 of pregnancy (Figure 3a). These observations indicated that pregnancy could rescue impeded ductal morphogenesis in virgin SirT1ko/ko mice. + +Normal alveolar morphogenesis takes place under the control of additional hormones and growth factors during pregnancy and lactation [17,24,25]. These coordinated efforts are necessary to support a densely saturated lobuloalveolar system for secreting milk after parturition (see wild-type mice on lactation day 1 in Figures 2b and 3a). In some SirT1ko/ko mice, the alveolar morphogenesis was initiated, as indicated by the presence of a few secretory alveolar epithelial cells within scattered alveolar structures sprouted from a well-established ductal branching network (Figures 2c and 3a, lower panel). The extent of alveolar morphogenesis in SirT1ko/ko mice was inadequate, however, as shown by the severe deficit in milk production when compared with that in wild-type mice (Figure 2c). + +Exogenous estrogen-induced ductal morphogenesis + +To test whether the increased levels of estrogen in pregnant mice were sufficient to induce ductal morphogenesis in SirT1ko/ko mice, a single estrogen pellet was implanted in the subscapular region of each virgin SirT1ko/ko mouse and the mammary tissues were analyzed 14 and 21 days after the implantation. By day 14, wild-type mice displayed ductal side branching reminiscent of the morphological changes during early pregnancy (Figure 4a, left panel). In contrast, SirT1ko/ko mice showed TEBs and ductal elongation, which are the characteristic features of ductal morphogenesis in pubertal wild-type mice. By day 21, ductal elongation and side branching were restored in SirT1ko/ko mice and the extent of ductal morphogenesis was indistinguishable between wild-type and SirT1ko/ko female mice (Figure 4a, right panel). These observations clearly indicated that exogenous estrogen alone is sufficient to rescue ductal morphogenesis in virgin SirT1ko/ko mice. Furthermore, the presence of transitional TEBs on day 14 and of ductal side branching on day 21 suggested that the characteristic features of mammary gland development at puberty and during early pregnancy could be coupled in response to increasing levels of estrogen. Increased levels of estrogen, either during pregnancy or through implantation, were therefore sufficient to stimulate the differentiation of epithelial progenitor cells and ductal morphogenesis in virgin SirT1ko/ko mice. + +Concurrent mammary epithelial cell proliferation and differentiation + +The finding of pregnancy-induced mammary gland development prompted us to investigate how mammary epithelial progenitor cells in pregnant SirT1ko/ko mice can differentiate so rapidly. Both BrdU labeling and the TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) assay were used to measure the cell proliferation and apoptosis in the mammary tissues, respectively. We found in SirT1ko/ko mammary tissues that about one-third of the cells in TEBs were BrdU-positive as compared with the number of BrdU-positive cells in the newly differentiated ductal or alveolar epithelial cells (Figure 5a(upper panel), 5b). This BrdU-positive finding differs from that in wild-type mammary tissues, in which less than 3% of ductal epithelial cells and about 25% of alveolar epithelial cells were BrdU-positive. The newly generated ductal epithelial cells in SirT1ko/ko mice formed elongated ducts and side branches, while pushing TEBs towards the edge of mammary fat pad (Figure 3a, lower panel). The newly generated ductal epithelial cells can consequently further differentiate into a few observed alveolar cells in which milk proteins were detected (Figure 2c). These observations suggested that the proliferation and differentiation of epithelial progenitor cells, which is programmed to take place at different times of a female's reproductive life, could be concurrent at a late stage of pregnancy in SirT1ko/ko mice. + +Apoptosis is a part of the self-renewal and remodeling processes in normal mammary tissues [17,24,25]. Apoptotic epithelial cells can be readily identified in both ductal and alveolar epithelium of SirT1ko/ko mice on lactation day 1 (Figure 5a, lower panel). The levels of apoptotic cells were significantly higher than those in wild-type mice on lactation day 1, but seemed to be closer to that in wild-type mice in mid-pregnancy (e.g. day 13 of pregnancy) (Figure 5c). The rush of proliferation and differentiation during pregnancy-rescued ductal development in SirT1ko/ko mice manifested multiple features on lactation day 1 that could reassemble some characteristic features of wild-type mice in mid-pregnancy. Successive pregnancies, however, failed to improve the survival rate of either SirT1+/ko pups or SirT1ko/ko pups (three or four pregnancies in 3 months for each of three SirT1ko/ko females tested), indicating that the lactation defect persisted. A SirT1 deficiency therefore affected ductal morphogenesis in virgin mice, and lobuloalveolar proliferation in parous mice. + +Deregulated negative feedback signals + +The characterization of lactation failure has revealed two developmental defects in SirT1ko/ko female mice. The impeded ductal morphogenesis in virgin SirT1ko/ko mice bore some resemblance to the defective ductal morphogenesis seen in mice lacking GH, estrogen, or IGF-1, as well as in mice harboring mutations in the corresponding receptors, such as the GH receptor and ERα [17,24-31]. These previous studies have established the notion that GH and estrogen synergistically stimulate ductal morphogenesis, which is mediated by stromal cell-produced local IGF-1 in mammary tissues. Impeded ductal morphogenesis in virgin SirT1ko/ko mice is therefore probably due to a deficit in IGF-1 signaling. + +In addition to this early defect, pregnancy-induced mammary gland development in SirT1ko/ko mice appeared to be arrested at the beginning of lobuloalveolar development, which is reminiscent of the phenotype in mice lacking IκB kinase alpha (IKKα) activity for NF-κB activation [37]. IKKα-deficient mice exhibit normal ductal morphogenesis at the onset of puberty and exhibit normal ductal side branching during early pregnancy. Owing to the absence of NF-κB-dependent cyclin D1 expression, however, IKKα-deficient mice display defective alveolar epithelial cell proliferation and fail to lactate [37]. While SirT1 negatively regulates the transcription activity of FoxOs and NF-κB in mammalian cells [10,11,20], SirT1ko/ko mice displayed the loss-of-function phenotypes. We hypothesized that SirT1 deficiency deregulates the expression of negative feedback signals and thereby desensitizes the cells to various types of stimulation. Indeed, among the many FoxOs and NF-κB-regulated genes, IGFBP-1 and IκBα encode negative feedback signals for IGF-1 signaling and NF-κB activation, respectively. We found that the basal levels of both IGFBP-1 and IκBα were increased in adipose tissues from virgin SirT1ko/ko mice and in ductal epithelial cells from SirT1ko/ko mice on lactation day 1 (Figure 3b). Meanwhile, the expression pattern of IGFBP-1 and IκBα in wild-type mammary tissues, shown in Figure 3b, was in agreement with the findings of others [16,38,39]. Namely, the expression of IGFBP-1 was downregulated during pregnancy, whereas NF-κB activity was increased at a late stage of pregnancy. The loss of SirT1 therefore increased the basal level of both IGFBP-1 and IκBα in multilineages of cells, and potentially increased thresholds for activating SirT1-modulated signaling pathways. + +SirT1 deficiency did not affect the ability of mammary epithelial progenitor cells to differentiate into functional alveolar epithelial cells despite the lactation failure (Figure 2c). SirT1 deficiency may therefore alter the homeostasis of the signals for ductal morphogenesis and/or the cellular response to the signals. Increased expression of both IGFBP-1 and IκBα in adipose tissues and mammary epithelial cells could simply reflect a global deregulation of gene expression in SirT1ko/ko cells in which the activity of both FoxOs and NF-κB is increased. IGFBP-1 is a potent inhibitor of IGF-1 in vivo [40], suggesting that the elevated basal level of IGFBP-1 in adipose tissues in SirT1ko/ko mice could reduce the effect of IGF-1. Moreover, deregulated IGFBP-1 expression, but not deregulated IκBα expression, was reversed in response to increased levels of estrogen in the mammary fat pads after implantation (Figure 4b). The finding is relevant to the fact that the expression of IGFBP-1 is downregulated as the level of estrogen increases in pregnant wild-type mice (Figure 3b). Increased levels of estrogen therefore stimulate the production of stromal cell-derived local IGF-1, which overcomes the IGFBP-1 barrier in SirT1ko/ko mice and, ultimately, reverses the increased levels of IGFBP-1 in mammary tissues. + +It was noted that increased levels of estrogen and local IGF-1 did not interfere with the expression of IκBα (Figure 4b). To determine whether deregulated IκBα expression in SirT1ko/ko cells can be reversed, we treated MEFs with TNFα and measured the kinetics of IκBα expression. Three independent lines of SirT1ko/ko MEFs were used, which displayed varying basal levels of increased IκBα expression. Following IκBα degradation induced by TNFα, the levels of newly synthesized IκBα in all three SirT1ko/ko MEFs were always higher than that of wild-type MEFs (Figure 6). This in vitro finding implies that SirT1 deficiency affects NF-κB signaling when the IκB kinase activation is normal. + +Our current system might not be suitable for studying the effects of SirT1 deficiency on NF-κB activation in vivo. NF-κB signaling exhibits distinct biological responses because the expression of IκBα depends on NF-κB activation while the other isoforms of IκB (β and ε) are independent of NF-κB feedback [41]. For example, TNFα stimulation induces dampened oscillatory behavior after the first hour, whereas lipopolysacharide treatment leads to stable NF-κB activation [42,43]. The NF-κB signaling in the developing mammary gland is under the temporal control of RANK signaling and IKKα activation [37]. The finding of increased levels of newly synthesized IκBα within the first hour indicated that SirT1 deficiency could disrupt the temporal control of NF-κB signaling to all types of stimulation (Figure 6). It remains unknown, however, whether the reduced IGF-1 signaling would affect the efficacy of RANK signaling and IKKα activation in developing mammary glands. The fact that individual SirT1ko/ko mice manifested varying degrees of ductal morphogenesis (Figure 3a) makes it difficult to dissect the potential compound effect of SirT1 deficiency on both upstream IKKα activation and downstream NF-κB signaling. Other experimental systems, such as mammary epithelial cell-specific SirT1ko/ko female mice, may be used to address this issue. + +Discussion + +Our study of SirT1ko/ko mice has unveiled a regulatory mechanism by which SirT1 modulates the efficacy of estrogen-stimulated local IGF-1 signaling for ductal morphogenesis. SirT1 deficiency alters the homeostasis of gene expression and attenuates the efficacy of IGF-1 signaling. As a result, SirT1ko/ko mice manifest partial perinatal lethality and lactation failure phenotypes, implicating the role of evolutionarily conserved SirT1 in both postnatal survival and offspring survival of mammals. + +SirT1 balances the homeostasis of IGF-1 and IGFBP-1 in vivo + +SirT1 is an integral part of the IIS system that checks and balances the efficacy of insulin and IGF-1 signals. The IGF-1 signaling of the IIS system is required for survival after birth. As illustrated in Figure 7a, the function of SirT1 parallels the linear IGF-1/IGF-1 receptor/phosphoinositide-3'-OH kinase/Akt signaling pathway, both of which can negatively regulate the transcription activity of FoxOs. Mice harboring targeted mutations in either IGF-1 or the IGF-1 receptor gene exhibit perinatal lethality and growth retardation [44,45]. In the IGF-1 receptor-expressing cells, the binding of insulin and/or IGF-1 activates the phosphoinositide-3'-OH kinase and Akt kinase cascade for survival [8,9,13]. The phosphoinositide-3'-OH kinase/Akt signaling pathway is also highly conserved from worms to mammals, and functions to modulate energy metabolism and lifespan in lower organisms. The mammalian Akt kinase family has three members, Akt1–Akt3, which provide specificity and versatility. Mice lacking both the Akt1 and Akt2 genes display perinatal lethal and growth retardation phenotypes that are strikingly similar to that of either IGF-1 or IGF-1 receptor-deficient mice [46]. In this regard, SirT1ko/ko mice, as well as other strains of SirT1-deficienct mice, also exhibit perinatal lethal and growth retardation phenotypes (Figure 1c and Table 1) [22,23]. The consistent observation of perinatal lethal and growth retardation phenotypes from our study and studies with mice lacking IGF-1, the IGF-1 receptor, or Akt1/Akt2 suggests that upregulated FoxO activity unleashes the expression of downstream effector genes that lead to the birth-related stress. This birth-related stress may be related to the switch from a maternal level of IGF-1 to a neonatal level of IGF-1 because the surviving SirT1ko/ko mice can live into adulthood and can reproduce despite the growth retardation phenotype (Figure 1c). In this context, at least one of the FoxO-deficient mice strains, FoxO3 (Foxo3a/FKHRL1)-deficient mice, grow normally and develop functional mammary glands [47]. + +IGFBP-1 is one of six IGF-1 binding proteins, but is the only one that can be negatively regulated by insulin [48]. IGF-1 binding proteins inhibit the action of local IGF-1 in a spatial and temporal manner [40,49]. In mammalian cells, FoxO3 can directly bind to the promoter of the IGFBP-1 gene and activate the transcription [50-52]. SirT1 acts to negatively regulate the activity of nuclear FoxO3 and to decrease the expression of IGFBP-1 when the IGF-1 signal is low (Figure 7a). Increased levels of IGFBP-1 can diminish the action of IGF-1 signals in vivo. In that way, SirT1 balances the homeostasis of IGF-1 and IGFBP-1 for varying levels of IGF-1 signals. + +The homeostasis of IGF-1 and IGFBP-1 clearly affects fertility, as shown by the infertility phenotype in both IGF-1-deficient mice and IGFBP-1 transgenic mice [28,40,49]. The serum level of estrogen in IGF-1-deficient female mice appears significantly reduced, whereas in ERα-deficient mice, which are also infertile, there is a threefold increase [53]. In contrast, SirT1ko/ko mice can be fertile despite the fact that the fertility was reduced as compared with that of wild-type females (Table 1). Although our preliminary assessment indicated that the serum level of estrogen in SirT1ko/ko female mice was not overtly altered, it remains possible that a subtle irregularity or an individual variation of the estrogen level or ERα expression might be attributed to the reduced fertility. An alternative explanation for reduced fertility is that SirT1 deficiency deregulates the activity of FoxO3 and manifests an opposite effect of FoxO3 deficiency. FoxO3-deficient mice exhibit early depletion of ovarian follicles, which is reminiscent of premature ovarian failure in women [47,54]. Premature ovarian failure manifests as early cessation of ovarian function and as premature menopause caused by genetic mutations, chemo/radiation therapy, or other unknown factors. It is possible that SirT1 deficiency counterbalances normal release of ovarian follicles and reduces the fertility in mice. + +SirT1 modulates the efficacy of the IGF-1 signal for ductal morphogenesis + +We hypothesize that, in virgin SirT1ko/ko mice, increased levels of IGFBP-1 in adipose tissues effectively diminish IGF-1 signals, including the estrogen–IGF-1 signaling, and halt the differentiation of mammary epithelial progenitor cells (Figure 7b). Several results of this study provided support for this hypothesis. First, the expression of IGFBP-1 is increased in mammary fat pads from virgin SirT1ko/ko mice, in which there is no evidence of any duct (Figures 2a and 3a). Second, both pregnancy and estrogen implantation can rescue impeded ductal morphogenesis in SirT1ko/ko mice (Figures 3a and 4a), demonstrating that increased levels of estrogen can enhance the production of local IGF-1. Moreover, the varying degrees of rescue among individual mice, as well as individual mammary fat pads of the same mouse, also indicated that local IGF-1 must be halted in SirT1ko/ko mice (Figure 3a, and data not shown). Finally, increased levels of estrogen specifically activate the IGF-1 signaling in mammary tissues, as shown by the reversal of IGFBP-1 expression in SirT1ko/ko mice after estrogen implantation (Figure 4b). These results indicate that a gradient of IGF-1 signals, including both circulating and local IGF-1, may regulate mammary gland development at embryonic, postnatal, and reproductive stages, and that increased expression of IGFBP-1 decreases the efficacy of IGF-1 signals. + +Mammary development may undergo estrogen/IGF-1-independent phases and estrogen/IGF-1-dependent phases, which encompasses a late stage of embryonic development to the onset of puberty and involves maternal IGF-1, circulating IGF-1, and estrogen-stimulated local IGF-1. By embryonic day 16.5, mammary mesenchymes are connected to primitive fat pads and begin to proliferate, which results in rudimentary ducts [17,25,55,56]. We propose that this process marks the transition from estrogen/IGF-1-independent mammary stem cells to IGF-1-dependent epithelial progenitor cells, and that maternal IGF-1 is sufficient to stimulate the differentiation of epithelial progenitor cells to estrogen-independent ductal epithelial cells but not estrogen-dependent epithelial cells (Figure 7b). This explains why the development of rudimentary ducts is normal in either IGF-1 or ERα knockout mice and that TEBs form and regress immediately before and after birth, respectively [17,26,27]. During the prepubertal period, the rudimentary ducts grow isometrically under the influence of circulating IGF-1. Like maternal IGF-1, circulating IGF-1 is not sufficient to induce the differentiation of estrogen-independent ductal epithelial cells into estrogen-dependent epithelial cells, which will express ERα and become estrogen dependent (Figure 7b). At the onset of puberty, ovarian estrogen, in synergy with pituitary GH, causes a surge in the production of stromal cell-derived local IGF-1 and stimulates the differentiation of estrogen-independent ductal epithelial cells to estrogen-dependent epithelial cells, which begins postnatal development of mammary gland (Figure 7b). Impeded ductal morphogenesis results in virgin SirT1ko/ko mice because local IGF-1 is apparently not sufficient to overcome increased IGFBP-1 in mammary adipose tissues. SirT1 therefore positively regulates the efficacy of the estrogen–IGF-1 signaling for ductal epithelial cell proliferation and differentiation. + +The potential effect of modulating SirT1 activity + +Breast cancer results from the accumulation of inherited and/or somatic mutations. Lifetime exposure to estrogen is a major risk factor for breast cancer, and a number of lifestyle factors, such as diet, body fat, alcohol consumption, exercise, parity, and hormone replacement therapy, may influence a woman's exposure to estrogen. The compound personal risk to a woman is difficult to assess because the molecular link between these factors and the effect of estrogen is poorly understood. The result of this study demonstrated that SirT1 positively modulates the efficacy of the estrogen–IGF-1 signal. This suggests that if SirT1 serves as a lifetime sensor to dietary restriction and acute withdrawal of nutrients [14,57], its activity could ultimately influence the risk of breast cancer. Interestingly, an epidemiological study of Dutch famine in 1945 indicated that an exposure to famine at prepubertal age increased the risk of breast cancer, most clearly seen in women who never gave birth [58]. Moreover, the serum levels of estrogen and IGF-1 were increased among those exposed to the famine, which suggests that an epigenetic adaptation phenomenon may take place. Assessing the potential correlation between the enzymatic activity of SirT1 and the risk of breast cancer is therefore of great interest in order to identify feasible targets for chemoprevention against breast cancer. + +Several dietary polyphenols as well as small molecules have been identified as either agonists or antagonists of SirT1 [59,60]. The pharmacological effects of these compounds in humans remain elusive given that SirT1 targets multiple transcription factors and exerts pleiotropic effects. Nonetheless, our study of SirT1ko/ko mice demonstrated the potential utility of SirT1 antagonists. Specifically, reducing SirT1 activity alters the homeostasis of cellular responses, including deregulation of the expression of IGFBP-1 and IκBα, which can attenuate the stimulation from the estrogen–IGF-1 signaling and potentially desensitize mammary epithelial cells to NF-κB activation. SirT1 is therefore a candidate target for chemoprevention against breast cancer. + +Conclusion + +The study of mammary gland development is an excellent model system for unraveling how SirT1 modulates the efficacy of the estrogen–IGF-1 signaling and regulates the timing of ductal morphogenesis. These new mechanistic insights may also aid in understanding the role of SirT1 in breast cancer as well as in other organs in which local IGF-1 plays an important role. + +Abbreviations + +Atm = ataxia telangiectasia; BrdU = bromodeoxyurdine; CMV = cytomegalovirus; ERα = estrogen receptor alpha; FoxO = forkhead box 'other' protein; GH = growth hormone; IGF-1 = insulin-like growth factor-1; IGFBP-1 = insulin-like growth factor-1 binding protein-1; H & E = hemotoxylin and eosin; IIS = insulin/insulin-like growth factor-1 signaling; IκBα = inhibitors of NF-κB alpha subunit; IKKα = IκB kinase alpha; MEF = murine embryonic fibroblast; NF = nuclear factor; PCR = polymerase chain reaction; RANK = receptor activator of NF-κB; Sir = silencing information regulator; TEB = terminal end bud; TNF = tumor necrosis factor. + +Competing interests + +The authors declare that they have no competing interests. + +Authors' contributions + +HL characterized the phenotypes of the SirT1 mutant mice, participated in the design of the study, and helped to draft the manuscript. GKR and NL helped to characterize the phenotypes. CW generated the SirT1 mutant embryonic stem cells and mice. BPR helped analyze the histology data. YG conceived of the study, participated in its design and coordination, and drafted the manuscript. All authors read and approved the final manuscript. + +Supplementary Material + +Additional file 1 + +A pdf file containing the materials and methods for the generation of SirT1ko/ko mice. This supporting file documented the details of materials and methods used to generate both SirT1co/co mice and SirT1ko/ko mice. + +Click here for file + +Acknowledgements + +The study is supported by grants from the American Cancer Society (RSG-04-019-01-CNE) and the Nathan Shock Center of Excellence in the Biology of Aging at the University of Washington (to YG). The authors thank Jennifer Blasi, Thomas Gernon, Janet Leath, Jennifer Lee, Bryce Sopher, Loraine Warner, and Heather-Marie Wilson for their contributions in the early phases of the study, and Dr Fred Alt, Dr Mary Helen Barcellos-Hoff, Dr Mark Groudine, Dr Warren Ladiges, Dr George Martin, Dr Peter Rabinovitch, and Dr Jeffrey Schwartz for their support and advice. + +Figures and Tables + +Figure 1 + +Generation and characterization of SirT1co/co and SirT1ko/ko mice. (a) Mouse SirT1 wild-type allele (SirT1+), conditional targeted allele (SirT1co), and knockout allele (SirT1ko). B, BamHI. (b) Western blot analysis of SirT1 expression in wild-type (+/+), conditional targeted (co/co), heterozygote (+/ko), and knockout (ko/ko) murine embryonic fibroblasts (MEFs) and mammary tissues (MG). (c) Growth retardation in surviving SirT1ko/ko mice (open bar) using sibling mice or age-matched mice of control genotypes (filled bar). (d) The serum levels of insulin-like growth factor-1 (IGF-1) in wild-type (+/+), SirT1+/ko (+/ko), and SirT1ko/ko (ko/ko) mice. (e) The survival curves of wild-type (WT), SirT1co/co, SirT1ko/ko, and Ku70-/- embryonic stem cells after ionizing radiation. (f) The survival curve of wild-type (WT), SirT1ko/ko, Ku70-/-, and Atm-/- embryonic stem cells after treatment with hydrogen peroxide. + +Figure 2 + +Lactation failure in SirT1ko/ko mice. (a) Whole mount analysis of the mammary tissues from virgin wild-type (+/+) and SirT1ko/ko mice (ko/ko). Scale bar = 2 mm. (b) Histological analysis of the mammary tissues harvested from wild-type (+/+) and SirT1ko/ko female mice on either day 13 of pregnancy (P13) or lactation day 1 (L1). Scale bars = 100 μm. (c) Immunofluorescence staining of milk production in the mammary tissues from wild-type (+/+) and SirT1ko/ko mice on L1. Red and smear stains, mouse milk proteins; blue dots, nuclear staining of adipocytes, epithelial cells, and other cells in mammary tissues. Scale bars = 100 μm. + +Figure 3 + +Pregnancy-induced ductal morphogenesis. (a) Whole mount analysis of mammary gland development. Upper panel: wild-type (+/+) mice show terminal end buds (TEBs) at the onset of puberty, elongated ducts (virgin mice), site branching during pregnancy (P13), and lobuloalveolar structures for milk production on lactation day 1 (L1). Lower panel: virgin SirT1ko/ko (ko/ko) mice show impeded ductal morphogenesis. Pregnancy-induced ductal morphogenesis manifests transitional TEBs, ductal elongation, and side branching with variety in three SirT1ko/ko mice on L1. All scale bars = 200 μm. (b) Western blot analysis of both insulin-like growth factor-1 binding protein-1 (IGFBP-1) and IκBα in mammary tissues of male mice, virgin mice, and L1 female mice of wild type (+/+) and SirT1ko/ko (ko/ko). Actin is used as a loading control. Lower panel scores the estimated density of indicated lineages of cells in the protein extracts, which are based on the morphological analyses in (a). + +Figure 4 + +Estrogen implantation stimulates ductal elongation and site branching. (a) Whole mount analysis of the mammary tissues from the virgin wild-type (+/+) and SirT1ko/ko mice implanted with estrogen pellets (+E2) on day 14 and day 21. Scale bars = 200 μm. (b) Western blot analysis of the expression of insulin-like growth factor-1 binding protein-1 (IGFBP-1) and IκBα, with actin as a loading control, in mammary tissues from the virgin wild-type (+/+) and SirT1ko/ko mice implanted with estrogen pellets on day 21. + +Figure 5 + +Mammary epithelial cell proliferation and apoptosis in SirT1ko/ko mice on lactation day 1. (a) Upper panel: bromodeoxyurdine (BrdU) staining of the mammary sections from SirT1ko/ko (ko/ko) and wild-type (+/+) mice on day 13 of pregnancy (P13) and lactation day 1 (L1). From left to right, three terminal end buds (TEBs) at a scale of 100 μm, a single TEB, ducts, and alveolus of SirT1ko/ko mice at a scale of 20 μm, and ducts and alveolus of wild-type mice on P13 and L1 at a scale of 20 μm. Lower panel: TUNEL (Terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling) assay for apoptotic cells using sections neighbored to the sections for BrdU staining. (b) The quantitative analysis of BrdU-positive epithelial cells in TEBs, ducts, and alveoli of SirT1ko/ko mice on L1 (open bar) and wild-type mice on P13 (grey bar) and L1 (solid bar). (c) The quantitative analysis of TUNEL-positive cells in neighboring sections. + +Figure 6 + +SirT1 deficiency derails NF-κB signaling in response to TNFα stimulation. (a) Western blot analysis of IκBα expression in SirT1ko/ko (ko/ko) and wild-type (+/+) murine embryonic fibroblasts (MEFs) within the first hour after TNFα stimulation. Actin is used as a loading control. (b) The relative levels of newly synthesized IκBα in three independent SirT1ko/ko MEF lines (red circles, blue and green triangles) and wild-type MEFs after TNFα stimulation. For each line of MEFs, the unit of IκBα protein at each time point is relative to the unit 0 minutes after normalization with actin. + +Figure 7 + +SirT1 modulates estrogen–insulin-like growth factor-1 signaling for ductal morphogenesis: a model. (a) The insulin/insulin-like growth factor-1 (IGF-1) signaling system: both SirT1 and Akt kinases can negatively regulate the transcription activity of forkhead box 'other' protein (FoxO) proteins. SirT1 deficiency deregulates the expression of insulin-like growth factor-1 binding protein-1 (IGFBP-1), which may exert autocrine and/or paracrine effects to inhibit IGF-1. (b) Mammary epithelial precursor cells (EPC) express IGF-1 receptor (IGF-1R) and can differentiate into estrogen receptor (ER)-negative ductal epithelial cells (DEC-I) in response to maternal, circulating, or estrogen-stimulated, stromal cell (S)-derived local IGF-1. At the onset of puberty, ovarian estrogen, in synergy with growth hormone (GH), enhances the production of local IGF-1 and stimulates the differentiation of DEC-I to ER-positive ductal epithelial cells (DEC-II). SirT1 deficiency deregulates the expression of IGFBP-1 in adipose tissues (a), however, which attenuates the efficacy of the IGF-1 signaling and causes impeded ductal morphogenesis in virgin SirT1ko/ko mice. Either pregnancy or exogenous estrogen can overcome the impeded ductal morphogenesis in virgin SirT1ko/ko mice and can stimulate the differentiation of EPC. + +Table 1 + +Perinatal lethality, fertility, and lactation defect in SirT1ko/ko mice + +More than 10 pairs of SirT1+/ko male mice and SirT1+/ko female mice were used for breeding. Data in parentheses indicate the number of pups of this genotype found dead within 3 days after their birth. aTotal number of surviving pups at weaning (postnatal day 21). diff --git a/src/ontogpt/evaluation/craft/database/all/17206865.ann b/src/ontogpt/evaluation/craft/database/all/17206865.ann new file mode 100644 index 000000000..02a4f6c80 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17206865.ann @@ -0,0 +1,1271 @@ +T1 http://purl.obolibrary.org/obo/MONDO_0008763 11 27 Alström Syndrome +T2 PR:000003956 11 35 Alström Syndrome Protein +T3 PR:000003956 37 42 Alms1 +T4 UBERON:0002113 47 53 Kidney +T5 GO:0042384 54 66 Ciliogenesis +T6 GO:0044838 71 90 Cellular Quiescence +T7 SO:0001023 124 131 alleles +T8 PR:000003956 139 144 ALMS1 +T9 SO:0000704 145 149 gene +T10 NCBITaxon:9606 174 179 human +T11 http://purl.obolibrary.org/obo/MONDO_0008763 180 196 Alström syndrome +T12 http://purl.obolibrary.org/obo/MONDO_0008763 198 214 Alström syndrome +T13 http://purl.obolibrary.org/obo/MONDO_0000001 225 233 disorder +T14 http://purl.obolibrary.org/obo/MONDO_0011122 257 264 obesity +T15 SO:0000704 316 323 genetic +T16 http://purl.obolibrary.org/obo/MONDO_0003847 316 332 genetic diseases +T17 GO:0072372 359 373 primary cilium +T18 PR:000003956 375 380 ALMS1 +T19 GO:0005813 394 405 centrosomes +T20 GO:0036064 410 430 ciliary basal bodies +T21 PR:000003956 460 465 Alms1 +T22 PR:000003956 466 471 ALMS1 +T23 GO:0042384 488 506 formation of cilia +T24 GO:0005929 501 506 cilia +T25 PR:000003956 549 554 Alms1 +T26 NCBITaxon:10088 558 562 mice +T27 GO:0005929 578 583 cilia +T28 UBERON:0004819 587 604 kidney epithelial +T29 CL:0002518 587 610 kidney epithelial cells +T30 GO:0005929 714 720 cilium +T31 PR:000003956 772 777 Alms1 +T32 http://purl.obolibrary.org/obo/MONDO_0000001 800 807 disease +T33 SO:0001023 819 826 alleles +T34 NCBITaxon:10088 833 838 mouse +T35 http://purl.obolibrary.org/obo/MONDO_0008763 848 864 Alström syndrome +T36 PR:000003956 866 871 Alms1 +T37 GO:0010467 894 903 expressed +T38 SO:0001023 920 926 allele +T39 GO:0005929 947 952 cilia +T40 GO:0042384 947 962 cilia formation +T41 CL:0000001 966 979 primary cells +T42 NCBITaxon:10088 986 990 mice +T43 GO:0005929 1018 1023 cilia +T44 UBERON:0004134 1033 1056 kidney proximal tubules +T45 GO:0006915 1091 1100 apoptosis +T46 GO:0008283 1104 1117 proliferation +T47 UBERON:0002113 1122 1127 renal +T48 http://purl.obolibrary.org/obo/MONDO_0001106 1122 1135 renal failure +T49 http://purl.obolibrary.org/obo/MONDO_0008763 1170 1186 Alström syndrome +T50 http://purl.obolibrary.org/obo/MONDO_0000001 1219 1226 disease +T51 UBERON:0002113 1285 1290 renal +T52 http://purl.obolibrary.org/obo/MONDO_0005308 1291 1303 ciliopathies +T53 SO:0001023 1325 1332 alleles +T54 http://purl.obolibrary.org/obo/MONDO_0008763 1340 1356 Alström syndrome +T55 SO:0000704 1357 1361 gene +T56 UBERON:0002113 1381 1387 kidney +T57 GO:0042384 1388 1400 ciliogenesis +T58 SO:0001023 1434 1441 alleles +T59 UBERON:0002113 1484 1490 kidney +T60 GO:0072372 1491 1504 primary cilia +T61 http://purl.obolibrary.org/obo/MONDO_0008763 1523 1539 Alström syndrome +T62 SO:0000704 1550 1557 genetic +T63 http://purl.obolibrary.org/obo/MONDO_0003847 1550 1566 genetic disorder +T64 PR:000003956 1594 1599 ALMS1 +T65 SO:0000704 1600 1604 gene +T66 http://purl.obolibrary.org/obo/MONDO_0000001 1610 1617 disease +T67 http://purl.obolibrary.org/obo/MONDO_0001941 1638 1647 blindness +T68 GO:0008152 1663 1672 metabolic +T69 http://purl.obolibrary.org/obo/MONDO_0005066 1663 1682 metabolic disorders +T70 http://purl.obolibrary.org/obo/MONDO_0000001 1718 1726 diseases +T71 GO:0072372 1741 1755 primary cilium +T72 GO:0042995 1759 1777 cellular appendage +T73 GO:0005576 1816 1829 extracellular +T74 UBERON:0002113 1856 1862 kidney +T75 http://purl.obolibrary.org/obo/MONDO_0001106 1856 1870 kidney failure +T76 GO:0016265 1894 1899 death +T77 http://purl.obolibrary.org/obo/MONDO_0008763 1903 1919 Alström syndrome +T78 GO:0072372 2005 2018 primary cilia +T79 http://purl.obolibrary.org/obo/MONDO_0002473 2023 2045 cystic kidney diseases +T80 UBERON:0002113 2030 2036 kidney +T81 PR:000003956 2075 2080 ALMS1 +T82 GO:0005929 2109 2114 cilia +T83 UBERON:0002113 2118 2124 kidney +T84 CL:1000497 2118 2130 kidney cells +T85 SO:0001023 2139 2146 alleles +T86 SO:0000704 2154 2158 gene +T87 NCBITaxon:9606 2197 2202 human +T88 http://purl.obolibrary.org/obo/MONDO_0000001 2203 2210 disease +T89 GO:0005929 2231 2236 cilia +T90 GO:0042384 2231 2246 cilia formation +T91 NCBITaxon:33208 2270 2277 animals +T92 http://purl.obolibrary.org/obo/MONDO_0000001 2320 2327 disease +T93 SO:0001023 2328 2335 alleles +T94 NCBITaxon:10088 2358 2362 mice +T95 GO:0005929 2364 2369 cilia +T96 UBERON:0002113 2388 2394 kidney +T97 CL:1000497 2388 2400 kidney cells +T98 GO:0008283 2445 2467 cellular proliferation +T99 GO:0008219 2472 2482 cell death +T100 PR:000003956 2531 2536 ALMS1 +T101 GO:0042384 2540 2552 ciliogenesis +T102 http://purl.obolibrary.org/obo/MONDO_0008763 2578 2594 Alström syndrome +T103 GO:0005929 2622 2627 cilia +T104 http://purl.obolibrary.org/obo/MONDO_0008763 2664 2680 Alström syndrome +T105 GO:0030849 2691 2700 autosomal +T106 http://purl.obolibrary.org/obo/MONDO_0006025 2691 2719 autosomal recessive disorder +T107 http://purl.obolibrary.org/obo/MONDO_0011122 2749 2756 obesity +T108 http://purl.obolibrary.org/obo/MONDO_0005148 2758 2782 type 2 diabetes mellitus +T109 UBERON:0000966 2784 2791 retinal +T110 http://purl.obolibrary.org/obo/MONDO_0004580 2784 2804 retinal degeneration +T111 GO:0007605 2810 2817 hearing +T112 http://purl.obolibrary.org/obo/MONDO_0005365 2810 2828 hearing impairment +T113 http://purl.obolibrary.org/obo/MONDO_0000001 2851 2858 disease +T114 http://purl.obolibrary.org/obo/MONDO_0004994 2867 2881 cardiomyopathy +T115 UBERON:0002107 2883 2888 liver +T116 UBERON:0002113 2902 2908 kidney +T117 UBERON:0002113 2946 2951 Renal +T118 UBERON:0002113 2994 2999 renal +T119 http://purl.obolibrary.org/obo/MONDO_0005240 2994 3007 renal disease +T120 GO:0016265 3029 3034 death +T121 http://purl.obolibrary.org/obo/MONDO_0008763 3038 3054 Alström syndrome +T122 UBERON:0002113 3092 3099 kidneys +T123 NCBITaxon:10088 3144 3149 mouse +T124 PR:000003956 3176 3181 Alms1 +T125 GO:0072372 3192 3206 primary cilium +T126 GO:0043226 3226 3235 organelle +T127 GO:0016020 3253 3261 membrane +T128 GO:0005886 3282 3297 plasma membrane +T129 GO:0005929 3316 3321 cilia +T130 GO:0045177 3358 3364 apical +T131 GO:0009986 3365 3372;3377 3381 face of ... cell +T132 GO:0005874 3408 3419 microtubule +T133 GO:0032991 3420 3427 complex +T134 GO:0005929 3452 3457 Cilia +T135 NCBITaxon:2759 3488 3498 eukaryotic +T136 NCBITaxon:phylum 3499 3504 phyla +T137 NCBITaxon:6239 3516 3538 Caenorhabditis elegans +T138 NCBITaxon:3052 3540 3553 Chlamydomonas +T139 NCBITaxon:7742 3559 3570 vertebrates +T140 GO:0031513 3572 3594 Immotile primary cilia +T141 GO:0005874 3622 3633 microtubule +T142 GO:0005930 3669 3676 axoneme +T143 GO:0031514 3694 3706 motile cilia +T144 GO:0005874 3737 3748 microtubule +T145 GO:0072372 3817 3831 primary cilium +T146 UBERON:0001231 3904 3918 kidney tubules +T147 GO:0007224 3923 3970;3975 3983;4034 4042 transduction of extracellular signaling through ... hedgehog ... pathways +T148 GO:0016055 3923 3970;3985 3988;4034 4042 transduction of extracellular signaling through ... Wnt ... pathways +T149 GO:0048008 3923 3970;3994 4042 transduction of extracellular signaling through ... platelet-derived growth factor receptor pathways +T150 GO:0005576 3939 3952 extracellular +T151 CL:0000233 3994 4002 platelet +T152 GO:1990265 3994 4024 platelet-derived growth factor +T153 UBERON:0002113 4102 4107 renal +T154 http://purl.obolibrary.org/obo/MONDO_0001106 4102 4115 renal failure +T155 http://purl.obolibrary.org/obo/MONDO_0008763 4119 4126 Alström +T156 GO:0072372 4164 4177 primary cilia +T157 UBERON:0002113 4185 4191 kidney +T158 SO:0000704 4213 4218 genes +T159 GO:0072372 4258 4271 primary cilia +T160 UBERON:0002113 4292 4298 kidney +T161 http://purl.obolibrary.org/obo/MONDO_0005240 4292 4307 kidney diseases +T162 http://purl.obolibrary.org/obo/MONDO_0020642 4309 4334 Polycystic kidney disease +T163 UBERON:0002113 4320 4326 kidney +T164 http://purl.obolibrary.org/obo/MONDO_0020642 4336 4339 PKD +T165 UBERON:0006314 4388 4393 fluid +T166 UBERON:0002113 4440 4445 renal +T167 http://purl.obolibrary.org/obo/MONDO_0001106 4440 4453 renal failure +T168 GO:0030849 4464 4473 autosomal +T169 http://purl.obolibrary.org/obo/MONDO_0004691 4464 4486 autosomal dominant PKD +T170 http://purl.obolibrary.org/obo/MONDO_0004691 4488 4493 ADPDK +T171 PR:000012769 4505 4517 Polycystin-1 +T172 PR:000012773 4505 4515;4522 4524 Polycystin ... −2 +T173 GO:0072372 4543 4556 primary cilia +T174 GO:0005929 4579 4584 cilia +T175 UBERON:0006314 4621 4626 fluid +T176 GO:0030849 4646 4655 Autosomal +T177 http://purl.obolibrary.org/obo/MONDO_0009889 4646 4669 Autosomal recessive PKD +T178 http://purl.obolibrary.org/obo/MONDO_0009889 4671 4676 ARPKD +T179 PR:000012777 4687 4698 fibrocystin +T180 http://purl.obolibrary.org/obo/MONDO_0019005 4704 4728 nephronophthisis disease +T181 PR:000009079 4756 4764 inversin +T182 GO:0005929 4782 4789 ciliary +T183 GO:0015031 4790 4807 protein transport +T184 NCBITaxon:10088 4829 4834 mouse +T185 SO:0000704 4848 4855 genetic +T186 GO:0005929 4867 4874 ciliary +T187 http://purl.obolibrary.org/obo/MONDO_0002473 4892 4913 cystic kidney disease +T188 UBERON:0002113 4899 4905 kidney +T189 http://purl.obolibrary.org/obo/MONDO_0008763 4965 4972 Alström +T190 http://purl.obolibrary.org/obo/MONDO_0015229 4996 5008 Bardet-Biedl +T191 PR:000003956 5053 5058 ALMS1 +T192 http://purl.obolibrary.org/obo/MONDO_0000001 5071 5078 disease +T193 http://purl.obolibrary.org/obo/MONDO_0015229 5110 5113 BBS +T194 SO:0000704 5174 5179 genes +T195 http://purl.obolibrary.org/obo/MONDO_0015229 5191 5212 Bardet-Biedl syndrome +T196 http://purl.obolibrary.org/obo/MONDO_0015229 5214 5217 BBS +T197 GO:0072372 5249 5262 primary cilia +T198 PR:000003956 5279 5284 ALMS1 +T199 GO:0005813 5301 5311 centrosome +T200 GO:0036064 5316 5336 ciliary basal bodies +T201 GO:0015031 5423 5444 transport of proteins +T202 GO:0005737 5457 5466 cytoplasm +T203 GO:0005930 5475 5490 ciliary axoneme +T204 PR:000003956 5522 5527 Alms1 +T205 NCBITaxon:10088 5535 5539 mice +T206 CL:0000019 5558 5563 sperm +T207 GO:0036126 5558 5572 sperm flagella +T208 GO:0005929 5585 5592 ciliary +T209 PR:000001245 5625 5634 rhodopsin +T210 GO:0032391 5657 5696 connecting cilia of photoreceptor cells +T211 CL:0000210 5677 5696 photoreceptor cells +T212 PR:000003956 5736 5741 ALMS1 +T213 GO:0072372 5761 5774 primary cilia +T214 GO:0005929 5794 5799 cilia +T215 GO:0042384 5794 5809 cilia formation +T216 NCBITaxon:9606 5827 5832 human +T217 CL:0002551 5833 5851 dermal fibroblasts +T218 http://purl.obolibrary.org/obo/MONDO_0008763 5860 5876 Alström syndrome +T219 UBERON:0014388 5900 5933 kidney collecting duct epithelial +T220 CL:1000454 5900 5939 kidney collecting duct epithelial cells +T221 SO:0000704 5945 5949 gene +T222 PR:000003956 5955 5960 Alms1 +T223 NCBITaxon:10088 5968 5973 mouse +T224 SO:0000704 6043 6050 genetic +T225 PR:000003956 6076 6081 ALMS1 +T226 SO:0001023 6153 6159 allele +T227 PR:000003956 6163 6168 ALMS1 +T228 PR:000003956 6217 6222 ALMS1 +T229 SO:0001587 6256 6276 premature stop codon +T230 SO:0001023 6292 6299 alleles +T231 NCBITaxon:10088 6316 6320 mice +T232 http://purl.obolibrary.org/obo/MONDO_0008763 6331 6347 Alström syndrome +T233 PR:000003956 6400 6405 Alms1 +T234 PR:000003956 6440 6445 ALMS1 +T235 GO:0005929 6462 6467 cilia +T236 GO:0042384 6462 6477 cilia formation +T237 http://purl.obolibrary.org/obo/MONDO_0000001 6492 6499 disease +T238 SO:0001023 6511 6518 alleles +T239 GO:0010467 6578 6588 expression +T240 PR:000003956 6654 6659 ALMS1 +T241 GO:0005929 6674 6679 cilia +T242 GO:0042384 6674 6689 cilia formation +T243 UBERON:0002113 6728 6733 renal +T244 http://purl.obolibrary.org/obo/MONDO_0001106 6728 6741 renal failure +T245 http://purl.obolibrary.org/obo/MONDO_0008763 6745 6761 Alström syndrome +T246 GO:0005929 6796 6803 ciliary +T247 PR:000003956 6840 6845 Alms1 +T248 SO:0000646 6866 6887 Short Interfering RNA +T249 GO:0042384 6905 6917 Ciliogenesis +T250 UBERON:0002113 6921 6927 Kidney +T251 CL:1000497 6921 6933 Kidney Cells +T252 PR:000003956 6956 6961 Alms1 +T253 GO:0005929 6980 6986 cilium +T254 GO:0042384 6980 6996 cilium formation +T255 UBERON:0002113 7043 7049 kidney +T256 CL:1000497 7043 7054 kidney cell +T257 GO:0042384 7055 7067 ciliogenesis +T258 NCBITaxon:10088 7083 7088 Mouse +T259 UBERON:0004205 7089 7120 inner medullary collecting duct +T260 CL:1000617 7089 7104;7130 7135 inner medullary ... cells +T261 CL:1001225 7105 7120;7130 7135 collecting duct ... cells +T262 GO:0005929 7141 7146 cilia +T263 GO:0005929 7178 7183 cilia +T264 GO:0042995 7210 7221 protrusions +T265 GO:0009986 7231 7243 cell surface +T266 GO:0042571 7253 7261 antibody +T267 MOP:0000030 7277 7287 acetylated +T268 PR:000028799 7288 7295 tubulin +T269 SO:0000646 7327 7348 short interfering RNA +T270 SO:0000646 7350 7355 siRNA +T271 CHEBI:36357 7357 7366 molecules +T272 NCBITaxon:10088 7388 7393 mouse +T273 PR:000003956 7394 7399 Alms1 +T274 GO:0042384 7430 7448 formation of cilia +T275 GO:0005929 7443 7448 cilia +T276 GO:0005929 7464 7469 Cilia +T277 SO:0000646 7529 7534 siRNA +T278 GO:0009294 7545 7557 transfection +T279 SO:0000646 7567 7572 siRNA +T280 PR:000003956 7591 7596 Alms1 +T281 MOP:0000030 7660 7670 acetylated +T282 PR:000028799 7671 7678 tubulin +T283 GO:0005930 7806 7821 ciliary axoneme +T284 SO:0000646 7849 7854 siRNA +T285 PR:000003956 7893 7898 Alms1 +T286 SO:0000646 7951 7957 siRNAs +T287 MOP:0000030 8008 8018 acetylated +T288 PR:000028799 8019 8026 tubulin +T289 GO:0042384 8043 8055 ciliogenesis +T290 PR:000003956 8087 8092 Alms1 +T291 SO:0000646 8155 8160 siRNA +T292 PR:000003956 8194 8199 Alms1 +T293 UBERON:0001977 8226 8231 serum +T294 NCBITaxon:9986 8235 8242 rabbits +T295 SO:0000236 8298 8316 open reading frame +T296 PR:000003956 8320 8325 Alms1 +T297 UBERON:0001977 8342 8347 serum +T298 GO:0097546 8386 8399 base of cilia +T299 PR:000003956 8469 8474 ALMS1 +T300 GO:0042571 8485 8493 antibody +T301 SO:0000646 8559 8564 siRNA +T302 CHEBI:36357 8565 8574 molecules +T303 PR:000003956 8597 8602 Alms1 +T304 GO:0065007 8635 8645 Regulation +T305 GO:0005929 8649 8656 Ciliary +T306 SO:0000704 8657 8662 Genes +T307 GO:0005929 8680 8687 Ciliary +T308 NCBITaxon:3052 8735 8748 Chlamydomonas +T309 NCBITaxon:6239 8756 8766 C. elegans +T310 SO:0000704 8791 8796 genes +T311 GO:0065007 8806 8815 regulated +T312 GO:0042384 8823 8835 ciliogenesis +T313 PR:000003956 8891 8896 Alms1 +T314 PR:000003956 8944 8949 Alms1 +T315 GO:0042384 9020 9032 ciliogenesis +T316 GO:0042384 9058 9070 ciliogenesis +T317 PR:000003956 9108 9113 Alms1 +T318 GO:0005929 9175 9180 cilia +T319 GO:0005929 9227 9232 cilia +T320 SO:0000704 9241 9246 genes +T321 GO:0042384 9286 9298 ciliogenesis +T322 PR:000004667 9300 9304 Bbs4 +T323 PR:000008940 9309 9314 Ttc10 +T324 PR:000004667 9316 9320 Bbs4 +T325 PR:000004667 9322 9332 BBS gene 4 +T326 SO:0000704 9326 9330 gene +T327 GO:0005813 9384 9394 centrosome +T328 GO:0005814 9443 9453 centriolar +T329 PR:000008940 9467 9472 Ttc10 +T330 PR:000008940 9474 9479 IFT88 +T331 PR:000008940 9480 9487 polaris +T332 GO:0005929 9554 9560 cilium +T333 PR:000004667 9567 9571 Bbs4 +T334 PR:000008940 9576 9581 Ttc10 +T335 GO:0042384 9614 9626 ciliogenesis +T336 PR:000003956 9646 9652 Alms1a +T337 SO:0000646 9653 9658 siRNA +T338 PR:000003956 9727 9732 Alms1 +T339 SO:0000646 9736 9741 siRNA +T340 GO:0042384 9824 9836 ciliogenesis +T341 GO:0005929 9935 9940 cilia +T342 PR:000003956 9971 9976 Alms1 +T343 GO:0005929 10006 10011 Cilia +T344 UBERON:0004819 10015 10032 kidney epithelial +T345 CL:0002518 10015 10038 kidney epithelial cells +T346 GO:0005622 10061 10074 intracellular +T347 UBERON:0006314 10105 10110 fluid +T348 GO:0009986 10125 10137 cell surface +T349 http://purl.obolibrary.org/obo/MONDO_0020642 10188 10191 PKD +T350 GO:0005929 10213 10218 cilia +T351 SO:0000646 10255 10260 siRNA +T352 GO:0005929 10336 10341 cilia +T353 PR:000003956 10372 10378 Alms1a +T354 SO:0000646 10379 10384 siRNA +T355 PR:000003956 10510 10515 Alms1 +T356 GO:0005929 10519 10524 cilia +T357 GO:0042384 10519 10535 cilia biogenesis +T358 UBERON:0004819 10539 10556 kidney epithelial +T359 CL:0002518 10539 10562 kidney epithelial cells +T360 GO:0005929 10665 10670 cilia +T361 PR:000003956 10697 10702 Alms1 +T362 MOP:0000030 10749 10759 acetylated +T363 PR:000028799 10760 10767 tubulin +T364 GO:0005886 10775 10788 cell membrane +T365 GO:0005929 10808 10813 cilia +T366 UBERON:0006314 10839 10844 fluid +T367 PR:000003956 10885 10890 Alms1 +T368 GO:0005929 10915 10920 Cilia +T369 GO:0042384 10915 10930 Cilia Formation +T370 PR:000003956 10970 10975 Alms1 +T371 GO:0042384 10999 11011 Ciliogenesis +T372 SO:0001023 11030 11037 alleles +T373 PR:000003956 11041 11046 ALMS1 +T374 NCBITaxon:9606 11050 11055 human +T375 NCBITaxon:10088 11093 11098 mouse +T376 http://purl.obolibrary.org/obo/MONDO_0000001 11113 11120 disease +T377 SO:0001587 11137 11165 premature termination codons +T378 SO:0000147 11169 11174 exons +T379 SO:0001023 11212 11219 alleles +T380 SO:0000704 11244 11248 gene +T381 SO:0000236 11267 11285 open reading frame +T382 GO:0005929 11410 11415 cilia +T383 GO:0042384 11410 11425 cilia formation +T384 PR:000003956 11432 11437 Alms1 +T385 GO:0009294 11452 11463 transfected +T386 NCBITaxon:10088 11466 11471 mouse +T387 PR:000003956 11472 11477 Alms1 +T388 SO:0000147 11540 11545 exons +T389 SO:0000646 11599 11604 siRNA +T390 SO:0000646 11617 11622 siRNA +T391 PR:000003956 11644 11649 Alms1 +T392 SO:0000704 11650 11654 gene +T393 SO:0000673 11744 11754 transcript +T394 GO:0010467 11763 11773 expression +T395 PR:000003956 11813 11818 Alms1 +T396 GO:0009294 11833 11845 transfection +T397 SO:0000646 11858 11863 siRNA +T398 MOP:0000030 11877 11887 acetylated +T399 PR:000028799 11888 11895 tubulin +T400 GO:0009294 11917 11929 transfection +T401 PR:000003956 11937 11942 Alms1 +T402 SO:0000646 11960 11965 siRNA +T403 GO:0005929 12000 12005 cilia +T404 GO:0009294 12060 12072 transfection +T405 SO:0000333 12155 12172 boundary of exons +T406 PR:000003956 12190 12195 Alms1 +T407 SO:0000673 12252 12262 transcript +T408 GO:0009294 12275 12286 transfected +T409 GO:0009294 12309 12321 transfection +T410 SO:0000673 12377 12387 transcript +T411 PR:000003956 12444 12449 Alms1 +T412 SO:0000673 12505 12515 transcript +T413 GO:0009294 12534 12546 transfection +T414 SO:0000646 12556 12561 siRNA +T415 GO:0010467 12589 12599 expression +T416 PR:000003956 12607 12612 Alms1 +T417 SO:0001023 12679 12685 allele +T418 PR:000003956 12693 12698 Alms1 +T419 SO:0000704 12699 12703 gene +T420 GO:0042384 12723 12735 ciliogenesis +T421 GO:0010467 12750 12760 expression +T422 CL:0000001 12777 12790 primary cells +T423 NCBITaxon:10088 12798 12803 mouse +T424 CHEBI:23995 12819 12837 ethyl nitroso urea +T425 SO:0000147 12880 12884 exon +T426 PR:000003956 12895 12900 Alms1 +T427 SO:0000704 12901 12905 gene +T428 PR:000003956 12923 12928 Alms1 +T429 SO:0000704 12929 12933 gene +T430 NCBITaxon:10088 13019 13024 mouse +T431 PR:000003956 13025 13030 Alms1 +T432 SO:0001023 13067 13073 allele +T433 PR:000003956 13074 13079 Alms1 +T434 GO:0010467 13102 13112 expression +T435 PR:000003956 13116 13121 Alms1 +T436 UBERON:0000479 13153 13160 tissues +T437 PR:000003956 13189 13194 Alms1 +T438 NCBITaxon:10088 13208 13212 mice +T439 PR:000003956 13251 13256 Alms1 +T440 UBERON:0002113 13269 13275 kidney +T441 PR:000003956 13286 13291 Alms1 +T442 NCBITaxon:10088 13305 13309 mice +T443 NCBITaxon:10088 13359 13363 mice +T444 PR:000003956 13397 13402 Alms1 +T445 GO:0097546 13428 13441 base of cilia +T446 PR:000003956 13445 13450 Alms1 +T447 CL:0000001 13464 13471;13479 13484 primary ... cells +T448 UBERON:0002113 13472 13478 kidney +T449 CL:1000497 13472 13484 kidney cells +T450 SO:0001587 13530 13550 premature stop codon +T451 SO:0001023 13551 13557 allele +T452 PR:000003956 13565 13570 Alms1 +T453 SO:0000704 13571 13575 gene +T454 NCBITaxon:10088 13585 13589 mice +T455 CL:0000001 13651 13658;13682 13687 primary ... cells +T456 CL:0000057 13659 13670 fibroblasts +T457 UBERON:0002113 13675 13681 kidney +T458 CL:1000497 13675 13687 kidney cells +T459 PR:000003956 13693 13698 Alms1 +T460 NCBITaxon:10088 13712 13716 mice +T461 PR:000003956 13770 13775 Alms1 +T462 GO:0072372 13810 13823 primary cilia +T463 GO:0010467 13943 13953 expression +T464 PR:000003956 13968 13973 Alms1 +T465 SO:0000673 13974 13984 transcript +T466 GO:0005929 14009 14014 cilia +T467 GO:0042384 14009 14024 cilia formation +T468 PR:000003956 14051 14056 Alms1 +T469 NCBITaxon:10088 14070 14075 mouse +T470 PR:000003956 14104 14109 Alms1 +T471 SO:0000646 14135 14140 siRNA +T472 PR:000003956 14148 14153 Alms1 +T473 UBERON:0000922 14167 14176 embryonic +T474 CL:0000057 14177 14188 fibroblasts +T475 GO:0042384 14199 14211 ciliogenesis +T476 GO:0072372 14315 14328 Primary Cilia +T477 GO:0042592 14333 14344 Homeostasis +T478 PR:000003956 14348 14353 Alms1 +T479 NCBITaxon:10088 14361 14365 Mice +T480 PR:000003956 14401 14406 Alms1 +T481 NCBITaxon:10088 14420 14425 mouse +T482 GO:0008152 14455 14464 metabolic +T483 http://purl.obolibrary.org/obo/MONDO_0008763 14497 14513 Alström syndrome +T484 NCBITaxon:10088 14565 14570 mouse +T485 NCBITaxon:9606 14596 14601 human +T486 NCBITaxon:10088 14630 14634 mice +T487 UBERON:0001347 14884 14889;14900 14903 white ... fat +T488 CL:0000448 14884 14889;14900 14914 white ... fat adipocytes +T489 UBERON:0001348 14894 14903 brown fat +T490 CL:0000449 14894 14914 brown fat adipocytes +T491 http://purl.obolibrary.org/obo/MONDO_0004790 14919 14931;14936 14941 steatosis of liver +T492 UBERON:0002107 14936 14941 liver +T493 http://purl.obolibrary.org/obo/MONDO_0011122 14945 14950 obese +T494 NCBITaxon:10088 14958 14962 mice +T495 NCBITaxon:10088 15007 15012 mouse +T496 http://purl.obolibrary.org/obo/MONDO_0005015 15041 15049 diabetic +T497 NCBITaxon:10088 15056 15060 mice +T498 CHEBI:17234 15072 15079 glucose +T499 http://purl.obolibrary.org/obo/MONDO_0002177 15092 15108 hyperinsulinemia +T500 UBERON:0001977 15116 15121 serum +T501 CHEBI:17855 15161 15174 triglycerides +T502 CHEBI:16113 15182 15193 cholesterol +T503 CHEBI:47775 15199 15214 HDL cholesterol +T504 PR:000003956 15255 15260 Alms1 +T505 CL:0000019 15295 15300 sperm +T506 GO:0007283 15295 15310 sperm formation +T507 UBERON:0000473 15318 15324 testes +T508 PR:000001245 15339 15348 rhodopsin +T509 UBERON:0000966 15366 15372 retina +T510 PR:000003956 15430 15435 Alms1 +T511 NCBITaxon:10088 15449 15454 mouse +T512 SO:0000704 15473 15480 genetic +T513 NCBITaxon:9606 15490 15495 human +T514 http://purl.obolibrary.org/obo/MONDO_0008763 15496 15512 Alström syndrome +T515 UBERON:0002113 15584 15590 kidney +T516 GO:0005929 15591 15596 cilia +T517 UBERON:0002113 15618 15623 renal +T518 http://purl.obolibrary.org/obo/MONDO_0001106 15618 15631 renal failure +T519 NCBITaxon:9606 15635 15640 human +T520 UBERON:0002113 15656 15663 kidneys +T521 PR:000003956 15676 15681 Alms1 +T522 NCBITaxon:10088 15695 15699 mice +T523 UBERON:0000025 15727 15734 tubules +T524 UBERON:0001851 15742 15748 cortex +T525 GO:0072372 15801 15814 primary cilia +T526 UBERON:0005185 15834 15853;15858 15871 collecting ducts of ... renal medulla +T527 UBERON:0001851 15903 15909 cortex +T528 UBERON:0000025 15910 15917 tubules +T529 GO:0005929 15962 15967 cilia +T530 PR:000003956 15976 15981 Alms1 +T531 UBERON:0000025 16053 16060 tubules +T532 UBERON:0002113 16077 16083 kidney +T533 NCBITaxon:3868 16098 16118 Lotus tetragonolobus +T534 UBERON:0004134 16141 16157 proximal tubular +T535 PR:000004182 16175 16186 aquaporin-2 +T536 GO:0042571 16187 16195 antibody +T537 UBERON:0001232 16211 16227 collecting ducts +T538 PR:000003956 16267 16272 Alms1 +T539 NCBITaxon:33208 16293 16300 animals +T540 PR:000004182 16306 16317 aquaporin-2 +T541 UBERON:0001232 16326 16341 collecting duct +T542 CL:1001225 16326 16347 collecting duct cells +T543 GO:0072372 16359 16372 primary cilia +T544 UBERON:0004134 16398 16413 proximal tubule +T545 CL:1000507 16407 16419 tubule cells +T546 NCBITaxon:33208 16437 16444 animals +T547 GO:0005929 16455 16463 ciliated +T548 UBERON:0004134 16502 16518 proximal tubules +T549 PR:000003956 16522 16527 Alms1 +T550 GO:0005929 16558 16566 ciliated +T551 NCBITaxon:9606 16569 16574 Human +T552 http://purl.obolibrary.org/obo/MONDO_0020642 16575 16578 PKD +T553 UBERON:0009773 16608 16620 renal tubule +T554 GO:0008283 16663 16685 cellular proliferation +T555 GO:0006915 16663 16671;16690 16699 cellular ... apoptosis +T556 UBERON:0002113 16713 16719 kidney +T557 NCBITaxon:10088 16755 16759 mice +T558 GO:0008283 16778 16791 proliferation +T559 PR:000010425 16793 16797 Ki67 +T560 CL:0000445 16825 16840 apoptotic cells +T561 GO:0008283 16861 16874 proliferation +T562 UBERON:0002113 16897 16903 kidney +T563 GO:0008283 16931 16944 proliferation +T564 UBERON:0001225 16966 16983 cortex of kidneys +T565 PR:000003956 16989 16994 Alms1 +T566 NCBITaxon:10088 17008 17012 mice +T567 UBERON:0000025 17093 17099 tubule +T568 UBERON:0000025 17178 17185 tubules +T569 UBERON:0000483 17212 17222 epithelial +T570 CL:0000066 17212 17228 epithelial cells +T571 PR:000010425 17234 17238 Ki67 +T572 GO:0005929 17272 17277 cilia +T573 UBERON:0004819 17316 17333 kidney epithelial +T574 CL:0002518 17316 17338 kidney epithelial cell +T575 GO:1904019 17323 17348 epithelial cell apoptosis +T576 NCBITaxon:10088 17363 17367 mice +T577 PR:000010425 17377 17381 Ki67 +T578 UBERON:0000025 17500 17507 tubules +T579 UBERON:0000025 17523 17530 tubules +T580 UBERON:0002113 17610 17616 kidney +T581 http://purl.obolibrary.org/obo/MONDO_0003634 17659 17670 proteinurea +T582 UBERON:0007023 17674 17679 adult +T583 PR:000003956 17680 17685 Alms1 +T584 NCBITaxon:10088 17699 17703 mice +T585 UBERON:0002113 17729 17735 kidney +T586 UBERON:0000025 17772 17779 tubules +T587 GO:0005929 17789 17794 cilia +T588 GO:0050673 17796 17809;17824 17843 proliferation ... of epithelial cells +T589 GO:1904019 17814 17843 apoptosis of epithelial cells +T590 UBERON:0000483 17827 17837 epithelial +T591 CL:0000066 17827 17843 epithelial cells +T592 http://purl.obolibrary.org/obo/MONDO_0003634 17848 17859 proteinurea +T593 NCBITaxon:10088 17882 17886 mice +T594 PR:000003956 17934 17939 ALMS1 +T595 http://purl.obolibrary.org/obo/MONDO_0000001 17961 17968 disease +T596 SO:0000704 17969 17974 genes +T597 NCBITaxon:9606 17999 18004 human +T598 SO:0001026 18005 18011 genome +T599 PR:000003956 18196 18201 ALMS1 +T600 GO:0005813 18209 18219 centrosome +T601 GO:0036064 18224 18242 ciliary basal body +T602 GO:0042571 18346 18354 antibody +T603 PR:000003956 18420 18425 ALMS1 +T604 GO:0005929 18445 18450 cilia +T605 http://purl.obolibrary.org/obo/MONDO_0008763 18518 18534 Alström syndrome +T606 http://purl.obolibrary.org/obo/MONDO_0005308 18545 18557 ciliopathies +T607 http://purl.obolibrary.org/obo/MONDO_0015229 18570 18573 BBS +T608 NCBITaxon:10088 18589 18594 mouse +T609 http://purl.obolibrary.org/obo/MONDO_0015229 18605 18608 BBS +T610 http://purl.obolibrary.org/obo/MONDO_0008763 18613 18629 Alström syndrome +T611 CL:0000019 18635 18640 sperm +T612 GO:0036126 18635 18649 sperm flagella +T613 GO:0005929 18665 18671 cilium +T614 PR:000001245 18704 18713 rhodopsin +T615 GO:0032391 18753 18793 connecting cilium in photoreceptor cells +T616 CL:0000210 18774 18793 photoreceptor cells +T617 PR:000003956 18835 18840 Alms1 +T618 GO:0042384 18869 18887 formation of cilia +T619 GO:0005929 18882 18887 cilia +T620 GO:0005929 18908 18913 cilia +T621 NCBITaxon:9606 18931 18936 human +T622 NCBITaxon:10088 18940 18945 mouse +T623 PR:000003956 18967 18972 ALMS1 +T624 PR:000003956 18973 18978 Alms1 +T625 SO:0000704 18979 18983 gene +T626 UBERON:0002113 19014 19020 kidney +T627 GO:0042384 19021 19033 ciliogenesis +T628 PR:000003956 19067 19072 Alms1 +T629 GO:0005929 19128 19133 cilia +T630 PR:000003956 19148 19153 Alms1 +T631 MOP:0000030 19190 19200 acetylated +T632 PR:000028799 19201 19208 tubulin +T633 SO:0000646 19233 19238 siRNA +T634 CHEBI:33893 19239 19247 reagents +T635 SO:0000646 19300 19306 siRNAs +T636 GO:0010467 19343 19353 expression +T637 GO:0042384 19385 19397;19408 19413 formation of ... cilia +T638 GO:0005929 19408 19413 cilia +T639 PR:000003956 19442 19447 Alms1 +T640 SO:0000646 19448 19454 siRNAs +T641 SO:0000646 19486 19491 siRNA +T642 PR:000003956 19510 19515 Alms1 +T643 GO:0005929 19545 19552 ciliary +T644 GO:0005929 19576 19583 ciliary +T645 PR:000003956 19614 19619 Alms1 +T646 SO:0000646 19620 19625 siRNA +T647 GO:0009294 19658 19670 transfection +T648 PR:000003956 19691 19696 Alms1 +T649 SO:0000646 19733 19738 siRNA +T650 GO:0005929 19797 19802 cilia +T651 SO:0000646 19823 19828 siRNA +T652 PR:000003956 19888 19893 Alms1 +T653 GO:0042384 19916 19935 biogenesis of cilia +T654 GO:0005929 19930 19935 cilia +T655 GO:0005929 19982 19987 cilia +T656 PR:000003956 20021 20026 ALMS1 +T657 PR:000003956 20027 20032 Alms1 +T658 SO:0000646 20071 20076 siRNA +T659 PR:000003956 20140 20145 Alms1 +T660 SO:0001023 20176 20183 alleles +T661 PR:000003956 20185 20190 Alms1 +T662 NCBITaxon:10088 20210 20215 mouse +T663 NCBITaxon:10088 20275 20279 mice +T664 SO:0001587 20307 20327 premature stop codon +T665 PR:000003956 20359 20364 Alms1 +T666 GO:0036064 20405 20425 ciliary basal bodies +T667 NCBITaxon:10088 20446 20450 mice +T668 PR:000003956 20487 20492 Alms1 +T669 GO:0042571 20559 20567 antibody +T670 PR:000003956 20601 20606 Alms1 +T671 UBERON:0000006 20647 20664 pancreatic islets +T672 GO:0010467 20706 20715 expressed +T673 SO:0001023 20732 20738 allele +T674 PR:000003956 20840 20845 Alms1 +T675 GO:0042384 20861 20873 ciliogenesis +T676 CL:0000001 20877 20890 primary cells +T677 NCBITaxon:10088 20902 20906 mice +T678 GO:0005929 20932 20939 ciliary +T679 PR:000003956 20974 20979 Alms1 +T680 PR:000003956 21060 21065 Alms1 +T681 PR:000003956 21101 21106 Alms1 +T682 GO:0005929 21132 21137 cilia +T683 GO:0042384 21132 21147 cilia formation +T684 http://purl.obolibrary.org/obo/MONDO_0008763 21204 21220 Alström syndrome +T685 NCBITaxon:9606 21321 21326 human +T686 NCBITaxon:10088 21362 21367 mouse +T687 http://purl.obolibrary.org/obo/MONDO_0008763 21378 21394 Alström syndrome +T688 SO:0001023 21477 21484 alleles +T689 PR:000003956 21631 21636 ALMS1 +T690 PR:000003956 21638 21643 Alms1 +T691 SO:0000147 21687 21692 exons +T692 NCBITaxon:9606 21739 21744 human +T693 NCBITaxon:10088 21749 21754 mouse +T694 http://purl.obolibrary.org/obo/MONDO_0000001 21820 21827 disease +T695 SO:0001023 21839 21846 alleles +T696 PR:000003956 21881 21886 Alms1 +T697 SO:0001023 21887 21894 alleles +T698 NCBITaxon:33208 21973 21979 animal +T699 GO:0042073 22005 22029 intraflagellar transport +T700 GO:0042073 22031 22034 IFT +T701 GO:0072372 22126 22140 primary cilium +T702 GO:0005622 22212 22225 intracellular +T703 GO:0035556 22212 22235 intracellular signaling +T704 GO:0065007 22241 22251 regulation +T705 GO:0009653 22255 22268 morphogenetic +T706 http://purl.obolibrary.org/obo/MONDO_0015229 22290 22293 BBS +T707 http://purl.obolibrary.org/obo/MONDO_0015229 22307 22310 BBS +T708 NCBITaxon:33208 22320 22326 animal +T709 GO:0072372 22360 22373 primary cilia +T710 GO:0007608 22398 22407 olfaction +T711 UBERON:0002113 22427 22433 kidney +T712 GO:0072372 22434 22448 primary cilium +T713 UBERON:0000025 22469 22476 tubular +T714 UBERON:0000483 22477 22487 epithelium +T715 SO:0000704 22545 22552 genetic +T716 GO:0005929 22579 22584 cilia +T717 GO:0005622 22595 22608 intracellular +T718 GO:0005929 22696 22701 cilia +T719 PR:000015288 22798 22808 Smoothened +T720 CL:0000233 22817 22825 platelet +T721 GO:1990265 22817 22847 platelet-derived growth factor +T722 GO:0005929 22879 22885 cilium +T723 GO:0005929 22906 22913 ciliary +T724 PR:000003956 22993 22998 Alms1 +T725 GO:0042384 23016 23028;23047 23052 formation of ... cilia +T726 UBERON:0004819 23029 23046 kidney epithelial +T727 GO:0005929 23047 23052 cilia +T728 SO:0000704 23144 23151 genetic +T729 http://purl.obolibrary.org/obo/MONDO_0003847 23144 23161 genetic disorders +T730 GO:0072372 23165 23178 primary cilia +T731 http://purl.obolibrary.org/obo/MONDO_0020642 23201 23204 PKD +T732 http://purl.obolibrary.org/obo/MONDO_0004691 23226 23231 ADPKD +T733 http://purl.obolibrary.org/obo/MONDO_0009889 23235 23240 ARPKD +T734 http://purl.obolibrary.org/obo/MONDO_0009889 23255 23260 ARPKD +T735 http://purl.obolibrary.org/obo/MONDO_0021140 23264 23274 congenital +T736 GO:0016265 23310 23315 death +T737 http://purl.obolibrary.org/obo/MONDO_0004691 23334 23339 ADPKD +T738 UBERON:0000113 23353 23362 adulthood +T739 NCBITaxon:33208 23399 23405 Animal +T740 http://purl.obolibrary.org/obo/MONDO_0020642 23416 23419 PKD +T741 SO:0000704 23443 23450 genetic +T742 SO:0000704 23467 23472 genes +T743 UBERON:0002113 23490 23496 kidney +T744 GO:0005929 23497 23502 cilia +T745 GO:0042073 23520 23523 IFT +T746 SO:0000704 23678 23685 genetic +T747 GO:0042073 23708 23711 IFT +T748 SO:0000704 23721 23726 genes +T749 GO:0097546 23789 23796;23798 23803 base of ... cilia +T750 GO:0005929 23808 23813 cilia +T751 GO:0042073 23831 23834 IFT +T752 GO:0015031 23839 23856 protein transport +T753 GO:0005737 23873 23882 cytoplasm +T754 PR:000012769 23894 23898 PKD1 +T755 PR:000012773 23903 23907 PKD2 +T756 GO:0005929 23920 23925 cilia +T757 GO:0065007 23952 23959 control +T758 GO:0008283 23960 23978 cell proliferation +T759 UBERON:0006314 23994 23999 fluid +T760 GO:0005929 24014 24019 cilia +T761 GO:0010467 24030 24040 expression +T762 PR:000009079 24044 24052 inversin +T763 GO:0005737 24076 24087 cytoplasmic +T764 PR:000006765 24088 24091 Dsh +T765 UBERON:0001088 24162 24167 urine +T766 GO:0051301 24335 24348 cell division +T767 UBERON:0000025 24397 24403 tubule +T768 http://purl.obolibrary.org/obo/MONDO_0015229 24415 24418 BBS +T769 GO:0065007 24485 24493 regulate +T770 GO:0005929 24535 24542 ciliary +T771 GO:0015031 24543 24560 protein transport +T772 GO:0005929 24606 24611 cilia +T773 GO:0051301 24677 24690 cell division +T774 NCBITaxon:10088 24721 24726 mouse +T775 http://purl.obolibrary.org/obo/MONDO_0020642 24727 24730 PKD +T776 NCBITaxon:33208 24731 24737 animal +T777 UBERON:0001232 24808 24826 collecting tubules +T778 NCBITaxon:9606 24843 24848 human +T779 http://purl.obolibrary.org/obo/MONDO_0009889 24849 24854 ARPKD +T780 PR:000003956 24873 24878 Alms1 +T781 NCBITaxon:10088 24886 24891 mouse +T782 GO:0005929 24919 24924 cilia +T783 UBERON:0004134 24937 24953 proximal tubules +T784 UBERON:0007023 24964 24969 adult +T785 NCBITaxon:10088 24970 24974 mice +T786 UBERON:0004819 25017 25034 kidney epithelial +T787 CL:0002518 25017 25043 kidney epithelial cellular +T788 GO:0044838 25035 25054 cellular quiescence +T789 CL:0000445 25084 25093;25112 25117 apoptotic ... cells +T790 GO:0008283 25098 25111 proliferating +T791 UBERON:0000025 25138 25145 tubules +T792 GO:0005929 25161 25166 cilia +T793 UBERON:0000025 25191 25198 tubular +T794 http://purl.obolibrary.org/obo/MONDO_0003634 25219 25230 proteinurea +T795 NCBITaxon:10088 25247 25251 mice +T796 http://purl.obolibrary.org/obo/MONDO_0004691 25310 25315 ADPKD +T797 PR:000003956 25328 25333 Alms1 +T798 NCBITaxon:10088 25347 25352 mouse +T799 NCBITaxon:33208 25375 25381 animal +T800 GO:0005929 25413 25418 cilia +T801 GO:0008283 25448 25470 cellular proliferation +T802 http://purl.obolibrary.org/obo/MONDO_0002473 25493 25514 cystic kidney disease +T803 UBERON:0002113 25500 25506 kidney +T804 UBERON:0004134 25522 25538 proximal tubules +T805 http://purl.obolibrary.org/obo/MONDO_0004691 25555 25560 ADPKD +T806 SO:0001023 25636 25642 allele +T807 PR:000012769 25646 25650 PKD1 +T808 PR:000012773 25654 25658 PKD2 +T809 GO:0009294 25741 25752 transformed +T810 http://purl.obolibrary.org/obo/MONDO_0000001 25828 25835 disease +T811 GO:0010467 25883 25893 expression +T812 PR:000012769 25897 25901 PKD1 +T813 GO:0005929 25974 25981 ciliary +T814 UBERON:0002113 26001 26007 kidney +T815 GO:0005929 26073 26078 cilia +T816 GO:0006915 26085 26094 apoptosis +T817 GO:0008283 26099 26112 proliferation +T818 NCBITaxon:9606 26164 26169 human +T819 UBERON:0002113 26181 26187 kidney +T820 http://purl.obolibrary.org/obo/MONDO_0005240 26181 26196 kidney diseases +T821 SO:0000704 26202 26209 genetic +T822 http://purl.obolibrary.org/obo/MONDO_0000001 26287 26294 disease +T823 SO:0001023 26300 26306 allele +T824 PR:000003956 26310 26315 Alms1 +T825 GO:0005929 26355 26360 cilia +T826 GO:0042384 26355 26370 cilia formation +T827 NCBITaxon:10088 26390 26394 mice +T828 NCBITaxon:10088 26408 26412 mice +T829 UBERON:0000362 26420 26434 kidney medulla +T830 UBERON:0001851 26479 26485 cortex +T831 GO:0007568 26493 26498 aging +T832 UBERON:0002113 26549 26555 kidney +T833 UBERON:0000025 26629 26635 tubule +T834 UBERON:0000025 26639 26645 tubule +T835 UBERON:0000025 26658 26664 tubule +T836 GO:0005929 26702 26707 cilia +T837 GO:0008283 26726 26739 proliferation +T838 GO:0006915 26743 26752 apoptosis +T839 UBERON:0000025 26768 26774 tubule +T840 GO:0005929 26813 26818 cilia +T841 UBERON:0000483 26860 26870 epithelial +T842 CL:0000066 26860 26876 epithelial cells +T843 GO:0008283 26884 26897 proliferating +T844 GO:0006915 26904 26913 apoptosis +T845 SO:0000704 26972 26979 genetic +T846 UBERON:0000025 27008 27015 tubules +T847 UBERON:0000025 27058 27065 tubules +T848 UBERON:0000025 27224 27231 tubular +T849 http://purl.obolibrary.org/obo/MONDO_0000001 27252 27259 disease +T850 GO:0042384 27302 27314 ciliogenesis +T851 GO:0005929 27523 27528 cilia +T852 GO:0042384 27523 27538 cilia formation +T853 GO:0005929 27708 27713 cilia +T854 PR:000003956 27744 27749 Alms1 +T855 GO:0010467 27750 27760 expression +T856 GO:0016246 27764 27768 RNAi +T857 SO:0000646 27783 27788 siRNA +T858 PR:000003956 27801 27806 Alms1 +T859 GO:0016246 27826 27830 RNAi +T860 SO:0000646 27860 27865 siRNA +T861 SO:0000646 28025 28030 SiRNA +T862 GO:0016246 28088 28092 RNAi +T863 GO:0009294 28093 28105 transfection +T864 GO:0009294 28160 28171 transfected +T865 NCBITaxon:10088 28272 28277 mouse +T866 PR:000003956 28278 28283 Alms1 +T867 SO:0000440 28374 28380 vector +T868 PR:000003956 28432 28437 Alms1 +T869 SO:0000155 28443 28450 plasmid +T870 GO:0009294 28512 28523 transfected +T871 SO:0000646 28557 28562 siRNA +T872 SO:0000646 28583 28589 siRNAs +T873 PR:000003956 28607 28612 Alms1 +T874 GO:0009294 28622 28633 transfected +T875 GO:0005929 28709 28714 Cilia +T876 GO:0009294 28739 28750 transfected +T877 SO:0000646 28765 28770 siRNA +T878 GO:0009294 28771 28782 transfected +T879 SO:0000704 28813 28817 gene +T880 GO:0010467 28813 28828 gene expression +T881 GO:0009294 28868 28879 transfected +T882 SO:0000646 28921 28926 siRNA +T883 GO:0009294 28927 28938 transfected +T884 GO:0009294 28977 28988 transfected +T885 SO:0000646 28994 29000 siRNAs +T886 PR:000003956 29009 29014 Alms1 +T887 NCBITaxon:1 29512 29522 individual +T888 SO:0000704 29550 29555 genes +T889 SO:0000704 29586 29590 gene +T890 GO:0010467 29586 29601 gene expression +T891 GO:0042384 29616 29628 ciliogenesis +T892 GO:0009294 29641 29652 transfected +T893 GO:0010467 29680 29690 expression +T894 GO:0009294 29737 29748 transfected +T895 GO:0010467 29792 29802 expression +T896 GO:0010467 29932 29942 expression +T897 SO:0000704 30014 30019 genes +T898 SO:0000704 30070 30075 genes +T899 GO:0010467 30091 30101 expression +T900 GO:0009294 30121 30132 transfected +T901 GO:0009294 30196 30207 transfected +T902 SO:0000646 30244 30249 siRNA +T903 GO:0042384 30342 30354 ciliogenesis +T904 PR:000003956 30372 30377 Alms1 +T905 GO:0065007 30422 30432 regulation +T906 GO:0042384 30449 30461 ciliogenesis +T907 CHEBI:29108 30464 30468 Ca2+ +T908 GO:0040007 30499 30504 grown +T909 GO:0009294 30526 30537 transfected +T910 PR:000003956 30543 30548 Alms1 +T911 GO:0016246 30549 30553 RNAi +T912 SO:0000646 30575 30580 siRNA +T913 GO:0040007 30635 30640 grown +T914 GO:0005929 30664 30669 cilia +T915 GO:0042384 30664 30679 cilia formation +T916 CHEBI:29108 30681 30685 Ca2+ +T917 PR:000003956 31465 31470 Alms1 +T918 NCBITaxon:10088 31478 31482 mice +T919 PR:000003956 31499 31504 Alms1 +T920 NCBITaxon:10088 31518 31522 mice +T921 CHEBI:23995 31529 31547 ethyl nitroso urea +T922 NCBITaxon:10088 31580 31584 mice +T923 PR:000003956 31644 31649 Alms1 +T924 NCBITaxon:33208 31656 31663 animals +T925 SO:0000704 31687 31694 genetic +T926 UBERON:0001969 31878 31884 plasma +T927 UBERON:0000966 31911 31918 retinal +T928 UBERON:0000178 31919 31924 blood +T929 UBERON:0001977 31934 31939 Serum +T930 CHEBI:17234 31940 31947 glucose +T931 CHEBI:16113 31949 31960 cholesterol +T932 CHEBI:17855 31971 31983 triglyceride +T933 PR:000045358 32037 32044 insulin +T934 NCBITaxon:10088 32146 32150 Mice +T935 CHEBI:17992 32246 32253 sucrose +T936 CHEBI:75958 32254 32262 solution +T937 UBERON:0000479 32288 32295 Tissues +T938 UBERON:0000479 32373 32379 tissue +T939 CHEBI:51686 32408 32419 hematoxylin +T940 CHEBI:51686 32427 32428 H +T941 CHEBI:27338 32518 32524 xylene +T942 CHEBI:16236 32552 32559 ethanol +T943 GO:0042571 32569 32579 antibodies +T944 PR:000028799 32607 32614 tubulin +T945 MOP:0000030 32616 32626 acetylated +T946 PR:000028799 32627 32634 tubulin +T947 PR:000010425 32703 32707 ki67 +T948 NCBITaxon:9986 32719 32725 Rabbit +T949 PR:000004182 32739 32750 aquaporin-2 +T950 GO:0042571 32824 32834 antibodies +T951 PR:000003956 32838 32843 Alms1 +T952 NCBITaxon:9986 32857 32864 rabbits +T953 NCBITaxon:10088 32941 32946 mouse +T954 PR:000003956 32947 32952 Alms1 +T955 GO:0042571 33011 33021 Antibodies +T956 CHEBI:52661 33167 33175 Alexa488 +T957 CHEBI:51247 33180 33189 Texas Red +T958 MOP:0000779 33190 33200 conjugated +T959 GO:0042571 33211 33221 antibodies +T960 CL:0000057 33306 33317 fibroblasts +T961 UBERON:0001225 33322 33337 kidney cortical +T962 CL:0002681 33322 33337;33343 33348 kidney cortical ... cells +T963 UBERON:0000058 33338 33342 duct +T964 PR:000003956 33365 33370 Alms1 +T965 UBERON:0000922 33384 33391 embryos +T966 UBERON:0000922 33410 33419 embryonic +T967 UBERON:0000922 33490 33499 Embryonic +T968 CL:0000057 33500 33511 fibroblasts +T969 GO:0002064 33554 33567;33583 33599 generation of ... epithelial cells +T970 CL:0000001 33568 33575;33594 33599 primary ... cells +T971 UBERON:0004819 33576 33593 kidney epithelial +T972 CL:0002518 33576 33599 kidney epithelial cells +T973 NCBITaxon:10088 33601 33605 mice +T974 GO:0007567 33611 33616 natal +T975 UBERON:0001179 33650 33660 peritoneal +T976 UBERON:0001225 33688 33703 Kidney cortices +T977 UBERON:0000479 33821 33828 tissues +T978 UBERON:0004819 33998 34014 renal epithelial +T979 PR:000003956 34146 34150 Alms +T980 NCBITaxon:10088 34200 34204 mice +T981 NCBITaxon:10088 34233 34237 mice +T982 CHEBI:23995 34246 34264 ethyl nitroso urea +T983 SO:0001023 34394 34400 allele +T984 SO:0001023 34450 34456 allele +T985 NCBITaxon:10088 34651 34656 mouse +T986 SO:0001026 34657 34663 genome +T987 SO:0001248 34664 34672 assembly +T988 PR:000003956 34745 34750 Alms1 +T989 SO:0000704 34751 34755 gene +T990 NCBITaxon:10088 34766 34770 mice +T991 SO:0000147 34800 34804 exon +T992 PR:000003956 34832 34837 Alms1 +T993 SO:0001026 34868 34875 genomic +T994 PR:000003956 34886 34891 Alms1 +T995 SO:0001026 34909 34915 genome +T996 UBERON:0001977 34991 34996 Serum +T997 PR:000003956 35009 35014 Alms1 +T998 NCBITaxon:10088 35028 35032 Mice +T999 UBERON:0001969 35047 35053 Plasma +T1000 CHEBI:17234 35054 35061 glucose +T1001 CHEBI:17855 35063 35075 triglyceride +T1002 PR:000045358 35077 35084 insulin +T1003 CHEBI:16113 35100 35111 cholesterol +T1004 CHEBI:47775 35117 35132 HDL cholesterol +T1005 PR:000003956 35187 35192 Alms1 +T1006 PR:000003956 35207 35212 Alms1 +T1007 PR:000003956 35227 35232 Alms1 +T1008 NCBITaxon:10088 35236 35240 mice +T1009 http://purl.obolibrary.org/obo/MONDO_0005015 35249 35257 diabetic +T1010 GO:0005929 35343 35348 Cilia +T1011 UBERON:0001225 35356 35369 Kidney Cortex +T1012 PR:000003956 35378 35383 Alms1 +T1013 NCBITaxon:10088 35397 35401 Mice +T1014 GO:0005929 35403 35408 Cilia +T1015 UBERON:0001851 35425 35431 cortex +T1016 UBERON:0000025 35432 35439 tubules +T1017 UBERON:0000958 35459 35466 medulla +T1018 UBERON:0000025 35467 35474 tubules +T1019 GO:0005929 35507 35512 Cilia +T1020 MOP:0000030 35539 35549 acetylated +T1021 PR:000028799 35550 35557 tubulin +T1022 GO:0042571 35558 35566 antibody +T1023 SO:0000704 35631 35635 Gene +T1024 GO:0010467 35631 35646 Gene Expression +T1025 PR:000003956 35674 35679 Alms1 +T1026 SO:0000704 35681 35685 Gene +T1027 GO:0010467 35681 35696 Gene expression +T1028 SO:0000704 35710 35715 genes +T1029 GO:0009294 35763 35774 transfected +T1030 PR:000003956 35780 35785 Alms1 +T1031 SO:0000646 35786 35792 siRNAs +T1032 SO:0000646 35817 35822 siRNA +T1033 GO:0009294 35842 35853 transfected +T1034 PR:000003956 36006 36011 Alms1 +T1035 NCBITaxon:10088 36012 36017 mouse +T1036 PR:000003956 36108 36113 Alms1 +T1037 NCBITaxon:10088 36114 36119 mouse +T1038 CHEBI:29108 36421 36425 Ca2+ +T1039 http://purl.obolibrary.org/obo/MONDO_0004691 36451 36456 ADPDK +T1040 GO:0030849 36459 36468 autosomal +T1041 http://purl.obolibrary.org/obo/MONDO_0004691 36459 36503 autosomal dominant polycystic kidney disease +T1042 UBERON:0002113 36489 36495 kidney +T1043 http://purl.obolibrary.org/obo/MONDO_0009889 36505 36510 ARPKD +T1044 GO:0030849 36513 36522 autosomal +T1045 http://purl.obolibrary.org/obo/MONDO_0009889 36513 36558 autosomal recessive polycystic kidney disease +T1046 UBERON:0002113 36544 36550 kidney +T1047 http://purl.obolibrary.org/obo/MONDO_0015229 36560 36563 BBS +T1048 http://purl.obolibrary.org/obo/MONDO_0015229 36566 36587 Bardet-Biedl syndrome +T1049 GO:0042073 36589 36592 IFT +T1050 GO:0042073 36595 36619 intraflagellar transport +T1051 NCBITaxon:3868 36627 36647 Lotus tetragonolobus +T1052 NCBITaxon:10088 36669 36674 mouse +T1053 UBERON:0004205 36675 36706 inner medullary collecting duct +T1054 http://purl.obolibrary.org/obo/MONDO_0020642 36708 36711 PKD +T1055 http://purl.obolibrary.org/obo/MONDO_0020642 36714 36739 polycystic kidney disease +T1056 UBERON:0002113 36725 36731 kidney +T1057 SO:0000646 36741 36746 siRNA +T1058 SO:0000646 36749 36770 short interfering RNA +T1059 PR:000003956 36817 36822 Alms1 +T1060 GO:0010467 36823 36833 Expression +T1061 GO:0072372 36841 36855 Primary Cilium +T1062 GO:0042384 36849 36865 Cilium Formation +T1063 UBERON:0004819 36869 36886 Kidney Epithelial +T1064 CL:0002518 36869 36892 Kidney Epithelial Cells +T1065 GO:0005929 36908 36913 cilia +T1066 MOP:0000030 36943 36953 acetylated +T1067 PR:000028799 36954 36961 tubulin +T1068 GO:0009294 37012 37024 transfection +T1069 GO:0009294 37026 37038 transfection +T1070 SO:0000646 37063 37068 siRNA +T1071 GO:0009294 37073 37085 transfection +T1072 SO:0000646 37104 37110 siRNAs +T1073 PR:000003956 37128 37133 Alms1 +T1074 MOP:0000030 37173 37183 acetylated +T1075 PR:000028799 37184 37191 tubulin +T1076 GO:0005930 37200 37207 axoneme +T1077 GO:0009294 37232 37244 transfection +T1078 SO:0000646 37261 37267 siRNAs +T1079 PR:000003956 37278 37283 Alms1 +T1080 NCBITaxon:10088 37342 37347 mouse +T1081 PR:000003956 37348 37353 Alms1 +T1082 SO:0000333 37377 37395 junctions of exons +T1083 SO:0000333 37377 37389;37408 37413 junctions of ... exons +T1084 SO:0000646 37457 37463 siRNAs +T1085 PR:000003956 37496 37501 Alms1 +T1086 PR:000003956 37521 37526 Alms1 +T1087 SO:0000646 37556 37562 siRNAs +T1088 GO:0042384 37589 37601 ciliogenesis +T1089 SO:0000646 37621 37626 siRNA +T1090 PR:000003956 37657 37662 Alms1 +T1091 GO:0010467 37671 37681 expression +T1092 MOP:0000030 37683 37687 Acet +T1093 MOP:0000030 37689 37699 acetylated +T1094 PR:000003956 37739 37744 Alms1 +T1095 GO:0042384 37792 37804 Ciliogenesis +T1096 GO:0005929 37898 37903 cilia +T1097 GO:0042384 37898 37914 cilia biogenesis +T1098 GO:0005929 37938 37943 cilia +T1099 GO:0009294 37975 37987 transfection +T1100 GO:0009294 38002 38013 transfected +T1101 SO:0000646 38026 38031 siRNA +T1102 GO:0005929 38045 38050 cilia +T1103 MOP:0000030 38097 38107 acetylated +T1104 PR:000028799 38108 38115 tubulin +T1105 GO:0005929 38125 38130 cilia +T1106 GO:0005634 38151 38157 nuclei +T1107 PR:000003956 38180 38185 Alms1 +T1108 PR:000004667 38222 38226 Bbs4 +T1109 PR:000008940 38231 38236 Ttc10 +T1110 GO:0042384 38244 38256 ciliogenesis +T1111 SO:0000646 38270 38275 siRNA +T1112 SO:0000646 38288 38293 siRNA +T1113 SO:0000646 38306 38311 siRNA +T1114 GO:0042384 38382 38394 ciliogenesis +T1115 SO:0000704 38399 38404 genes +T1116 GO:0010467 38439 38449 expression +T1117 GO:0010467 38482 38492 expression +T1118 SO:0000646 38532 38537 siRNA +T1119 SO:0000646 38578 38584 siRNAs +T1120 PR:000003956 38600 38605 Alms1 +T1121 GO:0005929 38630 38635 cilia +T1122 GO:0042384 38630 38645 cilia formation +T1123 GO:0005929 38660 38665 cilia +T1124 SO:0000646 38699 38704 siRNA +T1125 CHEBI:29108 38729 38733 Ca2+ +T1126 SO:0000646 38791 38796 siRNA +T1127 GO:0005829 38839 38848 cytosolic +T1128 PR:000003956 38973 38978 Alms1 +T1129 GO:0005929 38999 39004 Cilia +T1130 GO:0042384 38999 39014 Cilia Formation +T1131 GO:0009294 39022 39034 transfection +T1132 SO:0000646 39045 39050 siRNA +T1133 PR:000003956 39075 39080 Alms1 +T1134 GO:0072372 39104 39117 primary cilia +T1135 GO:0042384 39112 39127 cilia formation +T1136 SO:0000646 39183 39188 siRNA +T1137 PR:000003956 39204 39209 Alms1 +T1138 GO:0009294 39210 39221 transfected +T1139 GO:0010467 39247 39257 expression +T1140 PR:000003956 39313 39318 Alms1 +T1141 SO:0000646 39336 39341 siRNA +T1142 PR:000003956 39380 39385 Alms1 +T1143 GO:0010467 39411 39421 expression +T1144 SO:0000646 39458 39463 siRNA +T1145 PR:000003956 39474 39479 Alms1 +T1146 SO:0000646 39497 39502 siRNA +T1147 GO:0010467 39516 39526 expression +T1148 PR:000003956 39530 39535 Alms1 +T1149 PR:000003956 39550 39555 Alms1 +T1150 SO:0001023 39569 39575 allele +T1151 PR:000003956 39603 39608 Alms1 +T1152 SO:0000704 39609 39613 gene +T1153 GO:0010467 39609 39624 gene expression +T1154 PR:000003956 39631 39636 Alms1 +T1155 NCBITaxon:10088 39650 39655 mouse +T1156 NCBITaxon:10088 39712 39717 mouse +T1157 PR:000003956 39718 39723 Alms1 +T1158 GO:0042571 39724 39732 antibody +T1159 PR:000003956 39741 39746 Alms1 +T1160 GO:0036064 39769 39787 ciliary basal body +T1161 CL:0000001 39791 39798;39806 39811 primary ... cells +T1162 UBERON:0002113 39799 39805 kidney +T1163 CL:1000497 39799 39811 kidney cells +T1164 PR:000003956 39821 39826 Alms1 +T1165 NCBITaxon:10088 39840 39845 mouse +T1166 GO:0005929 39892 39900 ciliated +T1167 CL:0000064 39892 39906 ciliated cells +T1168 GO:0097546 39933 39946 base of cilia +T1169 GO:0072372 39974 39987 primary cilia +T1170 CL:0000057 39999 40010 fibroblasts +T1171 CL:0000001 40021 40028;40036 40041 primary ... cells +T1172 UBERON:0002113 40029 40035 kidney +T1173 CL:1000497 40029 40041 kidney cells +T1174 PR:000003956 40057 40062 Alms1 +T1175 NCBITaxon:10088 40076 40081 mouse +T1176 GO:0005929 40109 40114 cilia +T1177 GO:0042384 40109 40124 cilia formation +T1178 SO:0000646 40135 40140 siRNA +T1179 PR:000003956 40149 40154 Alms1 +T1180 CL:0000057 40176 40187 fibroblasts +T1181 PR:000003956 40219 40224 Alms1 +T1182 NCBITaxon:10088 40238 40242 Mice +T1183 NCBITaxon:9606 40256 40261 Human +T1184 http://purl.obolibrary.org/obo/MONDO_0008763 40262 40278 Alström Syndrome +T1185 PR:000003956 40284 40289 Alms1 +T1186 NCBITaxon:10088 40303 40307 mice +T1187 PR:000003956 40426 40431 Alms1 +T1188 NCBITaxon:10088 40445 40449 mice +T1189 UBERON:0002107 40525 40530 liver +T1190 UBERON:0000473 40546 40552 Testis +T1191 CHEBI:51686 40553 40554 H +T1192 UBERON:0001343 40587 40607 seminiferous tubules +T1193 CL:0000019 40682 40687 sperm +T1194 GO:0036126 40682 40696 sperm flagella +T1195 UBERON:0001301 40739 40749 epididymus +T1196 PR:000003956 40753 40758 Alms1 +T1197 NCBITaxon:10088 40772 40776 mice +T1198 MOP:0000030 40783 40793 acetylated +T1199 PR:000028799 40794 40801 tubulin +T1200 CHEBI:51686 40811 40812 H +T1201 CHEBI:51686 40816 40827 hematoxylin +T1202 PR:000001245 40840 40849 Rhodopsin +T1203 UBERON:0001789 40866 40885 outer nuclear layer +T1204 GO:0005634 40872 40879 nuclear +T1205 GO:0044297 40886 40897 cell bodies +T1206 PR:000003956 40927 40932 Alms1 +T1207 NCBITaxon:33208 40946 40953 animals +T1208 UBERON:0001789 41052 41055 ONL +T1209 UBERON:0001789 41057 41076 outer nuclear layer +T1210 GO:0005634 41063 41070 nuclear +T1211 GO:0001917 41078 41080 IS +T1212 GO:0001917 41082 41095 inner segment +T1213 GO:0001750 41097 41099 OS +T1214 GO:0001750 41101 41114 outer segment +T1215 UBERON:0002113 41146 41152 Kidney +T1216 PR:000003956 41170 41175 Alms1 +T1217 NCBITaxon:10088 41183 41187 Mice +T1218 CHEBI:51686 41193 41194 H +T1219 UBERON:0002113 41205 41211 kidney +T1220 PR:000003956 41235 41240 Alms1 +T1221 NCBITaxon:10088 41254 41259 mouse +T1222 UBERON:0001851 41276 41282 cortex +T1223 UBERON:0000025 41283 41290 tubules +T1224 UBERON:0002113 41347 41353 kidney +T1225 GO:0005929 41354 41359 cilia +T1226 UBERON:0000025 41380 41387 tubules +T1227 UBERON:0001225 41395 41404;41424 41430 cortex of ... kidney +T1228 PR:000003956 41405 41410 Alms1 +T1229 GO:0005929 41440 41445 cilia +T1230 UBERON:0000958 41453 41460 medulla +T1231 CHEBI:51686 41476 41477 H +T1232 CHEBI:51686 41481 41492 hematoxylin +T1233 MOP:0000030 41500 41504 Acet +T1234 MOP:0000030 41506 41516 acetylated +T1235 UBERON:0001851 41523 41529 Cortex +T1236 GO:0005929 41530 41535 cilia +T1237 PR:000003956 41561 41566 Alms1 +T1238 UBERON:0002113 41602 41608 kidney +T1239 GO:0005634 41609 41615 nuclei +T1240 PR:000003956 41656 41661 Alms1 +T1241 GO:0005929 41763 41768 cilia +T1242 UBERON:0001225 41783 41796 kidney cortex +T1243 CL:0002584 41783 41813 kidney cortex epithelial cells +T1244 UBERON:0000483 41797 41807 epithelial +T1245 NCBITaxon:10088 41825 41829 mice +T1246 UBERON:0001225 41853 41862;41882 41888 cortex of ... kidney +T1247 PR:000003956 41863 41868 Alms1 +T1248 GO:0005929 41890 41895 cilia +T1249 UBERON:0000025 41932 41939 tubules +T1250 PR:000004182 41951 41962 aquaporin-2 +T1251 GO:0010467 41963 41973 expressing +T1252 UBERON:0000025 41974 41981 tubules +T1253 PR:000010425 42013 42017 Ki67 +T1254 GO:0008283 42027 42040 proliferating +T1255 UBERON:0000483 42041 42051 epithelial +T1256 CL:0000066 42041 42057 epithelial cells +T1257 PR:000003956 42065 42070 Alms1 +T1258 UBERON:0002113 42084 42090 kidney +T1259 UBERON:0000025 42131 42137 tubule +T1260 CL:0000445 42175 42190 apoptotic cells +T1261 PR:000003956 42194 42199 Alms1 +T1262 UBERON:0002113 42213 42220 kidneys +T1263 UBERON:0000025 42269 42275 tubule +T1264 PR:000003956 42355 42360 Alms1 +T1265 UBERON:0002113 42381 42388 kidneys +T1266 PR:000003956 42439 42444 Alms1 +T1267 NCBITaxon:10088 42458 42462 mice +T1268 UBERON:0001088 42500 42505 Urine +T1269 PR:000003956 42511 42516 Alms1 +T1270 NCBITaxon:10088 42530 42534 mice +T1271 CHEBI:33893 43129 43137 reagents diff --git a/src/ontogpt/evaluation/craft/database/all/17206865.txt b/src/ontogpt/evaluation/craft/database/all/17206865.txt new file mode 100644 index 000000000..013d46568 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17206865.txt @@ -0,0 +1,249 @@ +A Role for Alström Syndrome Protein, Alms1, in Kidney Ciliogenesis and Cellular Quiescence + +Abstract + +Premature truncation alleles in the ALMS1 gene are a frequent cause of human Alström syndrome. Alström syndrome is a rare disorder characterized by early obesity and sensory impairment, symptoms shared with other genetic diseases affecting proteins of the primary cilium. ALMS1 localizes to centrosomes and ciliary basal bodies, but truncation mutations in Alms1/ALMS1 do not preclude formation of cilia. Here, we show that in vitro knockdown of Alms1 in mice causes stunted cilia on kidney epithelial cells and prevents these cells from increasing calcium influx in response to mechanical stimuli. The stunted-cilium phenotype can be rescued with a 5′ fragment of the Alms1 cDNA, which resembles disease-associated alleles. In a mouse model of Alström syndrome, Alms1 protein can be stably expressed from the mutant allele and is required for cilia formation in primary cells. Aged mice developed specific loss of cilia from the kidney proximal tubules, which is associated with foci of apoptosis or proliferation. As renal failure is a common cause of mortality in Alström syndrome patients, we conclude that this disease should be considered as a further example of the class of renal ciliopathies: wild-type or mutant alleles of the Alström syndrome gene can support normal kidney ciliogenesis in vitro and in vivo, but mutant alleles are associated with age-dependent loss of kidney primary cilia. + +Author Summary + +Alström syndrome is a rare genetic disorder caused by mutations in the ALMS1 gene. The disease is characterized by blindness, deafness, and metabolic disorders. These symptoms are reminiscent of diseases affecting the primary cilium, a cellular appendage with a role in sensing changes to the extracellular environment. In addition, kidney failure is a frequent cause of death in Alström syndrome patients, and recent studies have suggested a causal relationship between defects in primary cilia and cystic kidney diseases. In this paper, we show that ALMS1 protein is required to form cilia in kidney cells. Mutant alleles of the gene that are similar to those seen in the human disease are able to support cilia formation in cell culture and in animals. However, a defect in the function of the disease alleles is uncovered in older mice: cilia are lost from the kidney cells, and this is associated with an increase in cellular proliferation and cell death. The data are consistent with a requirement for ALMS1 in ciliogenesis and suggest inclusion of Alström syndrome among the growing class of cilia-related pathologies. + +Introduction + +Alström syndrome is a rare autosomal recessive disorder characterized by early onset obesity, type 2 diabetes mellitus, retinal degeneration, and hearing impairment. Other aspects of the disease include cardiomyopathy, liver dysfunction, kidney dysfunction, and a delay in puberty. Renal function declines with age, and end-stage renal disease is a common cause of death in Alström syndrome patients [1]. Additionally, enlarged kidneys have been reported in a previously reported mouse strain with a mutation in Alms1 [2]. + +The primary cilium is an antenna-like organelle, surrounded by a membrane contiguous with the plasma membrane [3,4]. Typically, cilia extend several micrometers from the apical face of the cell, grounded to the cellular microtubule complex through the basal body. Cilia are conserved through several eukaryotic phyla, including Caenorhabditis elegans, Chlamydomonas, and vertebrates. Immotile primary cilia contain a scaffold of nine microtubule doublets running the length of the axoneme (9 + 0), whereas motile cilia contain an additional central microtubule pair (9 + 2 arrangement). Increasing data demonstrate roles for the primary cilium in sensory functions. These include mechanosensation of lumenal flow in kidney tubules and transduction of extracellular signaling through the hedgehog, Wnt, and platelet-derived growth factor receptor pathways [3,4]. + +Four lines of evidence suggest the hypothesis that renal failure in Alström patients is secondary to a defect in primary cilia in the kidney. First, mutations in genes that are implicated in the function of primary cilia are associated with kidney diseases. Polycystic kidney disease (PKD) is characterized by progressive development of fluid-filled cysts, ultimately leading to end-stage renal failure [5]. Both autosomal dominant PKD (ADPDK) proteins (Polycystin-1 and −2) are localized to primary cilia and are necessary for cilia-mediated signaling in response to a fluid-flow stimulus [6]. Autosomal recessive PKD (ARPKD) protein (fibrocystin) and nephronophthisis disease proteins, nephrocystin and inversin, are involved in ciliary protein transport [7,8]. Additionally, mouse strains with genetic lesions in ciliary proteins lead to cystic kidney disease [9,10]. Second, the spectrum of phenotypes seen in Alström patients is similar to Bardet-Biedl patients [11], suggesting that mutations in ALMS1 might cause disease through a similar mechanism to BBS mutations. It is now well documented that several of the 11 genes mutated in Bardet-Biedl syndrome (BBS) have roles in the function of primary cilia [12–19]. Third, ALMS1 is localized to centrosome and ciliary basal bodies in vitro [20,21], consistent with a role in the structure of the basal body or in the transport of proteins between the cytoplasm and the ciliary axoneme. Fourth, in vivo phenotypes of Alms1 mutant mice include a lack of sperm flagella, a modified ciliary structure, as well as defective rhodopsin transport through the connecting cilia of photoreceptor cells [2,22]. + +These data point to a role of ALMS1 in the function of primary cilia, but no defects in cilia formation were observed in human dermal fibroblasts from an Alström syndrome patient [21] or in the kidney collecting duct epithelial cells of a gene-trap Alms1 mutant mouse strain [2]. A possible resolution of this discrepancy comes from the genetic analysis of mutations in ALMS1 that have been found in patients. In all cases, there was at least one allele of ALMS1 that could encode an N-terminal fragment of the ALMS1 protein before the presence of a premature stop codon. Moreover, the alleles in two reported mice models of Alström syndrome would also be predicted to encode the N-terminus of Alms1 [2,22]. Thus, it is possible that ALMS1 is required for cilia formation, and that the disease-associated alleles are able to provide at least part of this activity through expression of the N-terminus of the protein. In this study, we test whether ALMS1 has a role in cilia formation or function and provide evidence that renal failure in Alström syndrome patients might be associated with ciliary dysfunction. + +Results + +Depletion of Alms1 mRNA and Protein by Short Interfering RNA Causes Defective Ciliogenesis in Kidney Cells + +To determine whether Alms1 was necessary for cilium formation and/or function, we used an in vitro model of kidney cell ciliogenesis and signaling. Mouse inner medullary collecting duct (mIMCD3) cells form cilia 5 d after confluency [23]. The cilia can be visualized as long protrusions from the cell surface using an antibody raised against acetylated tubulin (Figure 1A). We tested several short interfering RNA (siRNA) molecules designed against the mouse Alms1 sequence for their effects on formation of cilia in this model. Cilia were formed normally in the presence of a negative control siRNA. However, transfection with two siRNA sequences against Alms1, Alms1a and Alms1b, led to a markedly different phenotype. The acetylated tubulin staining on each cell manifested as a single ball of fluorescence, and very few cells showed the elongated staining typical of ciliary axoneme (Figure 1A). Both of these siRNA species also caused a decrease in the Alms1 mRNA level (Figure 1B). In contrast, two additional siRNAs (Alms1c and Alms1d) did not affect the pattern of acetylated tubulin staining in the ciliogenesis assay, nor did they affect the Alms1 mRNA level (Figure 1A and 1B). To demonstrate that the active siRNA species also decreased levels of Alms1 protein, we raised an antiserum in rabbits against the predicted N-terminal 13 amino acids of the open reading frame of Alms1. Using this antiserum, we detected positive staining at the base of cilia, consistent with previous reports on the subcellular localization of ALMS1 [21]. The antibody signal was reduced below detection in the presence of the active siRNA molecules (Figure 1C). + +Loss of Alms1 Does Not Affect Transcriptional Regulation of Ciliary Genes but Does Disrupt Ciliary Mechanosensation + +Transcriptional profiling in Chlamydomonas and in C. elegans has identified a set of genes that are regulated during ciliogenesis [24,25]. As an initial characterization of the role of Alms1 in the mIMCD3 in vitro model, we asked whether Alms1 protein was required for the transcriptional response associated with ciliogenesis. A time course of mIMCD3 ciliogenesis showed a steady rise in the level of Alms1 mRNA after confluency. This was paralleled by an increase in cilia length (Figure 2A and 2B). We chose two other cilia-related genes that have increased mRNA levels during ciliogenesis, Bbs4 and Ttc10. Bbs4 (BBS gene 4) encodes a protein localized to the basal body and centrosome that is required for targeting cargo to the pericentriolar region [13]; Ttc10 (IFT88/polaris) is required for transport of cargo from basal body to the distal cilium [26]. Bbs4 and Ttc10 mRNA were upregulated even when ciliogenesis was disrupted with Alms1a siRNA (Figure 2B). Moreover, microarray analysis showed that knockdown of Alms1 by siRNA had no effect on the general transcription program associated with confluency and ciliogenesis in the mIMCD3 cells (Figure 2C and Table S1). + +We then used this model to ask whether the stunted cilia formed in limiting amounts of Alms1 were functionally competent. Cilia on kidney epithelial cells are able to induce an intracellular calcium signal in response to fluid flow over the cell surface. Defects in this response are thought to underlie PKD [27,28]. Full-length cilia formed in the presence of a control siRNA were able to respond to flow as expected (Figure 2D). However, the stunted cilia formed in the presence of the Alms1a siRNA were unable to induce calcium flux after a flow stimulus. + +Taken together, these in vitro results suggest a critical role of Alms1 in cilia biogenesis of kidney epithelial cells, though without disruption of the transcriptional program that accompanies this process. The abnormal cilia formed after knockdown of Alms1 are characterized by a focal concentration of acetylated tubulin at the cell membrane, and these stunted cilia are unable to respond to fluid flow by calcium flux. + +A 5′ Fragment of Alms1 Is Sufficient to Rescue Cilia Formation In Vitro after Knockdown of Endogenous Alms1 and Can Support Normal Ciliogenesis In Vivo + +Reported alleles of ALMS1 in human patients, as well as in two reported mouse models of the disease, usually encode premature termination codons in exons 8, 10, and 16 [2,22,29,30]. In these alleles, the 5′ terminus of the gene encodes an intact open reading frame that might give rise to a partially functional protein. Therefore, we tested whether a truncated 5′ cDNA was able to rescue cilia formation after Alms1 knockdown. We transfected a mouse Alms1 cDNA encoding the N-terminal 1,282 amino acids, equivalent to exons 1–8, into mIMCD3 cells in the presence of the Alms1a siRNA. The Alms1a siRNA sequence matches the Alms1 gene 3′ of the cDNA fragment and so was predicted to affect only the levels of the endogenous transcript and not expression of the cDNA encoding the N-terminus of Alms1. As expected, transfection with Alms1a siRNA caused focal acetylated tubulin staining. However, cotransfection of the Alms1 cDNA with Alms1a siRNA was fully capable of rescuing the cilia phenotype (Figure 3A). To monitor the effects of cDNA transfection on knockdown of the endogenous mRNA level, we used a Taqman assay recognizing the boundary of exons 12 and 13 of the Alms1 mRNA. This assay will detect the endogenous full-length transcript but not the transfected cDNA and shows that cotransfection of the cDNA did not affect knockdown of the endogenous transcript. Conversely, using a Taqman assay recognizing the 5′ of Alms1 mRNA that detects both the endogenous and cDNA-encoded transcript, we showed that cotransfection with the siRNA Alms1a did not affect over-expression of the Alms1 5′ cDNA (Figure 3B). + +To determine whether a premature truncation allele of the Alms1 gene can support normal ciliogenesis at endogenous expression levels, we used primary cells from a mouse strain with an ethyl nitroso urea–induced, premature truncation mutation in exon 10 of the Alms1 gene (Figure S1). The Alms1 gene in this mutant strain is predicted to encode the N-terminal 2,131 amino acids of the mouse Alms1 protein, and we therefore named the allele Alms1L2131X/L2131X. Similar expression of Alms1 mRNA was observed across eight tissues in wild-type and homozygous Alms1L2131X/L2131X mice (Figure 3C). In particular, levels of Alms1 mRNA in the kidney of mutant Alms1L2131X/L2131X mice are at least as high as in the wild-type control mice. In addition, we detected mutant Alms1 protein localized at the base of cilia of Alms1L2131X/L2131X primary kidney cells (Figure 3D). Thus, despite the presence of a premature stop codon allele in the Alms1 gene of these mice, both mRNA and protein were readily detectable. By isolating primary fibroblasts and kidney cells from Alms1L2131X/L2131X mice and wild-type controls, we found that cells from the Alms1L2131X/L2131X strain showed normal primary cilia when compared to wild-type cells (Figure 3E), which is consistent with previous reports [2,21]. We then tested whether expression of the mutant Alms1 transcript was required for normal cilia formation in cells derived from the Alms1L2131X/L2131X mouse strain. Knockdown of mutant Alms1 mRNA using a 5′ specific siRNA in the Alms1L2131X/L2131X embryonic fibroblasts inhibited ciliogenesis in these cells (Figure 3F), as was seen for wild-type cells (unpublished data). + +Age-Dependent Loss of Primary Cilia and Homeostasis in Alms1 Mutant Mice + +We characterized the phenotype of Alms1L2131X/L2131X mouse strain with reference to the metabolic and sensory defects reported in Alström syndrome patients to determine whether the pathology of the mouse strain resembled that of human patients. Homozygous mutant mice increased in weight faster than wild-type controls between 7 and 10 wk of age, and body composition analysis showed that this increase was almost entirely explained by an increase in fat mass (Figure 4A). Histological analysis showed hypertrophy of white and brown fat adipocytes and steatosis of the liver in obese mutant mice (Figure 4B). Although one older male mutant mouse in this cohort of 20 became diabetic, most mice had normal glucose levels with hyperinsulinemia. Other serum abnormalities include elevated leptin, triglycerides, total cholesterol, and HDL cholesterol (Figure S2). Finally, we noted that the Alms1L2131X/L2131X strain had defective sperm formation in the testes and defective rhodopsin transport in the retina (Figure 4C and 4D). We conclude from these data that the Alms1L2131X/L2131X mouse strain is a close genetic model of human Alström syndrome [1], and we proceeded to analyze whether there were any defects in the kidney cilia that might relate to renal failure in human patients. + +The kidneys of 6-mo-old Alms1L2131X/L2131X mice contained multiple dilated tubules in the cortex (Figure 5A). Consistent with a previous report [2], primary cilia appeared normal in collecting ducts of the renal medulla. However, we noticed that some cortex tubules appeared to be almost completely denuded of cilia in aged Alms1L2131X/L2131X mutants (Figure 5A and 5B). To further characterize these tubules, we stained the kidney sections with Lotus tetragonolobus agglutinin (LTA) as a proximal tubular marker, and with aquaporin-2 antibody as a marker of collecting ducts (Figure 5C). In both the wild-type and Alms1L2131X/L2131X mutant animals, the aquaporin-2-labeled collecting duct cells have clear primary cilia as expected. LTA-labeled proximal tubule cells in the wild-type animals were also ciliated. In contrast, most of the LTA-labeled proximal tubules of Alms1L2131X/L2131X mutants were not ciliated. + +Human PKD is thought to originate from renal tubule dilatation, secondary to abnormalities in cellular proliferation and apoptosis. We examined kidney sections from wild-type and mutant mice with a marker for proliferation, Ki67, and by TUNEL staining for apoptotic cells (Figure 5D). Sparse proliferation was seen in wild-type kidney sections. Higher levels of proliferation were detected in the cortex of kidneys from Alms1L2131X/L2131X mice, and this was focused in patches, perhaps representing convolutions of the same tubule coming into the plane of sectioning. In most of the cross sections of dilated tubules, several (20%–50%) of the epithelial cells were Ki67-positive, and these cells lacked cilia. We also noted a dramatic increase in kidney epithelial cell apoptosis in the mutant mice. As with Ki67, it was striking that TUNEL-positive cells had a nonuniform distribution and seemed to be clustered within particular tubules, whereas other tubules appeared identical to wild-type controls. These changes appeared to compromise kidney function, as urinanalysis revealed a mild proteinurea in adult Alms1L2131X/L2131X mice (Figure 5E). None of the kidney phenotypes described above (dilated tubules, loss of cilia, proliferation and apoptosis of epithelial cells, or proteinurea) was seen in 2-mo-old mice (Figure S3 and unpublished data). + +Discussion + +ALMS1 is among the largest disease genes identified today in the human genome. However, amino acid sequence analysis identifies only a leucine zipper near the N-terminus, allowing limited inferences to be made about the function of this protein. Localization of ALMS1 to the centrosome and ciliary basal body has been determined using a green fluorescent protein fusion to its C-terminus, and with an N-terminal antibody, respectively [20,21]. These data are consistent with a role for ALMS1 in the function of cilia, which is supported by the overlapping clinical phenotypes between Alström syndrome and other ciliopathies, especially BBS. Additionally, mouse models of BBS and Alström syndrome lack sperm flagella and a modified cilium; and show aberrant transport of rhodopsin, pointing to defective function of the connecting cilium in photoreceptor cells. + +In this paper, we investigated whether Alms1 protein is required for the formation of cilia and, if so, whether cilia can be formed in human or mouse cells with a mutated ALMS1/Alms1 gene. We used an in vitro model of kidney ciliogenesis to demonstrate that knockdown of Alms1 mRNA caused a striking alteration in the morphology of cilia: knockdown of Alms1 caused stunted or focal staining of acetylated tubulin. The specificity of the siRNA reagents was confirmed by two pieces of evidence. First, two siRNAs that were active in decreasing mRNA expression were also active in decreasing formation of elongated cilia. Conversely, two additional Alms1 siRNAs, as well as a negative control siRNA, did not decrease Alms1 mRNA nor did they affect the ciliary phenotype. Second, the ciliary phenotype that was induced by Alms1 siRNA knockdown could be rescued by cotransfection of a 5′ fragment of Alms1 that was not targeted by the active siRNA. Together these results rule out the possibility that the cilia phenotype caused by siRNA knockdown was an off-target effect, and point to a role of Alms1 in the maintenance or biogenesis of cilia. + +Interestingly, previous work had shown that cilia were formed in cells with mutant ALMS1/Alms1. To reconcile those findings with our siRNA knockdown data, we posited a functional role for the truncated Alms1 protein encoded by the mutant alleles. Alms1 mRNA in our mutant mouse strain was present at levels similar to those in wild-type mice, despite the presence of a premature stop codon. We also found that the mutant Alms1 protein was detectable and localized to ciliary basal bodies in cells from these mice, just as was observed for wild-type Alms1. Additionally, in unpublished data, immunohistochemistry with the antibody raised against the N-terminus of Alms1 showed prominent staining in the mutant pancreatic islets, confirming that protein could be stably expressed from the mutant allele. To test whether there was a function for the mutant protein, we showed that knockdown of the mutant Alms1 mRNA inhibited ciliogenesis in primary cells from these mice. We also showed that the ciliary phenotype induced by knockdown of Alms1 could be rescued using a cDNA encoding only the N-terminal 1,282 amino acids of Alms1, confirming that the N-terminus of Alms1 is sufficient to support cilia formation. + +A spectrum of nonsense and frameshift mutations cause Alström syndrome, and all have different effects. However, no genotype/phenotype correlation has been observed among human patients [29–31]. Furthermore, two mouse models of Alström syndrome reported to date, as well as a third model reported in this manuscript, contained alleles that were predicted to encode the N-terminus of the protein, and the reported phenotypes are very consistent. Thus, we suggest that truncation of ALMS1/ Alms1, secondary to mutations in (predominantly) exons 8, 10, and 16, leads to similar phenotypes in human and mouse. In particular, we provide evidence for residual function of the disease-associated alleles. This residual function of mutant Alms1 alleles might explain a lack of the more severe developmental phenotypes described in animal models with mutations of intraflagellar transport (IFT) or kinesin motor proteins [26,32,33]. + +There is growing support for the “antenna” role of primary cilium as a key participant in sensing environmental stimuli, transduction of intracellular signaling, and regulation of morphogenetic pathways. Studies of BBS patients and BBS knockout animal models have revealed the role of primary cilia in sensing of light and olfaction [14,16,17,19]. The kidney primary cilium, extending from the tubular epithelium into the lumen, has been implicated in mechanosensation: genetic or chemical disruption of cilia inhibited intracellular calcium influx in response to laminar flow [34,35]. Recent studies have suggested that cilia constitute an essential platform where signaling processes are initiated. The Hedgehog receptor Smoothened and the platelet-derived growth factor receptor alpha localize to the cilium and, in both cases, ciliary localization is required for signaling transduction [36,37]. We show here that Alms1 is necessary for formation of kidney epithelial cilia, which in turn are known to be essential for mechanosensory signaling. + +Well-characterized genetic disorders of primary cilia include the family of PKD, inherited either as ADPKD or ARPKD traits. While ARPKD is congenital and often causes fetal or neonatal death, the onset of the ADPKD is mainly in adulthood with age-dependent progression [5]. Animal models of PKD have been developed by genetic mutation of PKD genes [38–41], loss of kidney cilia by inhibition of IFT components [10,42], or constitutive activation of the Wnt pathway [43]. + +Recent data have started to build a connection between these seemingly unrelated genetic perturbations in PKD, IFT, and Wnt genes. Localization of PKD proteins has been observed on, or at the base of, cilia and cilia are dependent on IFT for protein transport to and from the cytoplasm [44]. Both PKD1 and PKD2 function on cilia to sense lumenal flow and control cell proliferation. Specifically, fluid flow over the cilia increases expression of inversin, which in turn targets cytoplasmic Dsh, inhibiting the canonical Wnt pathway [45]. It has been proposed that urine flow terminates the canonical Wnt pathway in favor of the noncanonical Wnt pathway. The noncanonical Wnt pathway might then maintain planar cell polarity and restrict cell division in a direction parallel to the long axis of the tubule. Moreover, BBS proteins have been shown to directly interact with proteins which regulate planar cell polarity [46], as well as in ciliary protein transport [47], suggesting a model in which defects in cilia disrupt planar cell polarity signaling and lead to disorientated cell division and cyst formation. + +Existing mouse PKD animal models are characterized by the formation of early-onset cysts in the collecting tubules, reminiscent of human ARPKD. In contrast, the Alms1 mutant mouse model presented here shows cilia loss in the proximal tubules. Further, adult mice progress to a breakdown in maintenance of kidney epithelial cellular quiescence, with a dramatic increase in apoptotic and proliferating cells restricted to those tubules that have lost cilia. This is accompanied by tubular dilatation and mild proteinurea in older mutant mice. All these phenotypes are similar to clinical features of ADPKD, making the Alms1L2131X/L2131X mouse strain an interesting animal model to study the relation of cilia loss, altered signaling, and cellular proliferation in the progression of cystic kidney disease in the proximal tubules. + +Initiation of ADPKD is suggested to be dependent on a second somatic mutation in the wild-type allele of PKD1 or PKD2. Evidence for this hypothesis is controversial as somatic mutagenesis rates in nontransformed cells are likely to be too low to explain the high prevalence of bilateral disease in heterozygous carriers [48,49] and a reduced expression of PKD1 is sufficient to initiate cystogenesis [41]. In this recessive model of ciliary dysfunction in the kidney, we also observe localized expression of the cellular phenotype (cilia loss, apoptosis, or proliferation). This suggests that in this model, and perhaps in human late-onset kidney diseases, non-genetic and/or epigenetic factors are necessary for expression of the phenotype. The disease-like allele of Alms1, although functionally able to support cilia formation in vitro, in young mice and in older mice in the kidney medulla, might reveal a compromised function in the cortex during aging. We also note that the expression of the cellular kidney phenotype in our model is not random but shows a variegated pattern on a tubule-by-tubule basis. Some tubule cross sections appear to have normal cilia, and little to no proliferation or apoptosis, whereas other tubule cross sections show extensive loss of cilia, sometimes with all or nearly all of the epithelial cells either proliferating or in apoptosis. The presumed somatic event, which, in combination with a genetic predisposition, causes some tubules to appear dysfunctional while neighboring tubules have a wild-type appearance, is not known. However, more detailed characterization of the progression of the phenotype might suggest treatments that maintain tubular integrity and delay disease. + +Materials and Methods + +Cell culture and ciliogenesis assay. + +mIMCD3 cells were obtained from American Type Culture Collection (www.atcc.org) and cultured in DMEM/F12 media (Invitrogen, http://www.invitrogen.com) supplement with 10% FBS, 2.5 mM L-glutamine. For cilia formation assay, 50,000 cells were seeded on 12-mm Transwell filters (Costar). Filters were collected at day 0, day 1, day 3, day 5, and day 7 after seeding for RNA isolation and cilia immunostaining. + +Knockdown of Alms1 expression by RNAi. + +We designed siRNA specific to Alms1 by online BLOCK-iT RNAi designer (Invitrogen). These siRNA sequences are: 5′-GCTGTATGTAGTCGAATTA-3′ (Alms1a); 5′-GCCTGATTCCTTGTTTCAA-3′ (Alms1b); 5′-GCAGTAGTCTCTTCTGCTT-3′ (Alms1c); 5′-GCTTCAGCTTTGCTGAATT-3′ (Alms1d). SiRNA were synthesized by Qiagen (http://www1.qiagen.com). For RNAi transfection, mIMCD3 cells were seeded as previously described and transfected with Lipofectamine 2000 (Invitrogen) according to manufacturer's instruction. + +DNA constructs. + +The mouse Alms1 cDNA was generated from mIMCD3 mRNA. N-terminus (aa: 1–1,282) was cloned into p3XFLAG-CMV vector (Sigma, http://www.sigmaaldrich.com) to generate N-Alms1-FLAG plasmid. + +RNA isolation and labeling for array analysis. + +Cells were transfected on day 0 with either a scrambled siRNA control (one of two siRNAs directed against Alms1) or mock-transfected. Cells were seeded on day 1 and allowed to reach confluence through day 7. Cilia were formed in the mock-transfected and scrambled siRNA-transfected cells. Samples were taken for gene expression on days 0, 1, 3, 5, and 7 for the mock-transfected cells; days 0, 1, 3, and 5 for scrambled siRNA-transfected cells; and days 1, 3, and 5 for cells transfected with siRNAs against Alms1. RNA was isolated using the RNeasy Kit from Qiagen. Total RNA was examined on an Agilent Bioanalyzer (Agilent, http://www.agilent.com) and the 28S/18S ratio exceeded 2.0 for all samples. cDNA and cRNA were prepared from 4 μg total RNA according to standard Affymetrix protocols (http://www.affymetrix.com/support/technical/manual/expression_manual.affx). The in vitro transcription kit used was the GeneChip IVT labeling kit (Affymetrix). All reactions were processed at the same time by the same individual. We selected a group of 98 genes that had the highest relative gene expression change during ciliogenesis in the mock-transfected cells. The query was: (max expression level for days 0, 1, 3, 5, and 7 for the mock-transfected cells) divided by the maximum (the minimum expression level over these samples and a cutoff value of 400 arbitrary units). Four hundred units was approximately the 77th percentile of expression across all 36,000 probesets for a given sample. We selected the top 98 genes by this ratio (ratio >7.4) and then clustered the genes based on their expression levels in the mock-transfected samples. Comparison of this pattern with the samples that were transfected with either a scrambled or targeted siRNA showed no significant changes in the broad transcriptional program that was associated with ciliogenesis, suggesting that Alms1 is unlikely to play a role in transcription regulation associated with ciliogenesis. + +Ca2+ imaging assay. + +mIMCD3 cells, grown on cover glass, were transfected with Alms1 RNAi or scrambled control siRNA using Lipofectamine 2000 (Invitrogen), and cells were grown for 5 more d to induce cilia formation. Ca2+ imaging was performed as described [6]. Briefly, cells were incubated with 5 μM Fura2-AM (Molecular Probes, Invitrogen) for 30–60 min at 37 °C. Cover glasses were placed in a laminar flow perfuse chamber (Warner Instruments Corporation, http://www.warneronline.com). After 20 min equilibration, cells were perfused with media as described [6] via a local perfusion pipette. Image of Fura-2 loaded cells with excitation wavelength alternating between 340 nm and 380 nm were captured by a CCD camera. Following subtraction of background fluorescence, the ratio of fluorescence at two wavelengths was analyzed using Metafluor (Universal Imaging Corporation). All experiments have been repeated in triplicate and similar results were obtained. + +Generation and phenotypic analysis of Alms1 mutant mice. + +We identified Alms1L2131X/L2131X mice in an ethyl nitroso urea–forward genetics screen. Mutant mice were generated by successive intercrossing of heterozygote Alms1L2131X animals on a mixed C57Bl6/ NOD genetic background. Body composition was determined by quantitative magnetic resonance according to protocols supplied by the manufacturer (Echo Medical Systems, http://www.echomri.com). All plasma analytes were measured in retinal blood samples. Serum glucose, cholesterol, HDL, and triglyceride levels were determined by Olympus AU400e. Leptin and insulin levels were analyzed by ELISA (Crystal Chemical Incorporated). + +Histology and immunohistochemistry. + +Mice were anesthetized with Avertin (Sigma, 1.25%, 0.02 ml per gram body weight), perfused with 10% sucrose solution and 4% paraformaldehyde. Tissues were fixed in 4% paraformaldehyde, processed, and embedded in paraffin. 5-μm tissue sections were processed for hematoxylin-eosin (H&E) and immunostaining. For routine immunostaining, the sections were deparaffinized with xylene and rehydrated with graded ethanol. Primary antibodies used in this study were: γ-tubulin; acetylated tubulin (Sigma); β-Galactosidase (Abcam, http://www.abcam.com/index.html?); ki67 (NeoMarker Rabbit Monoclonal); aquaporin-2 (Santa Cruz Biotechnology, http://www.scbt.com). We generated polyclonal antibodies to Alms1 by injecting rabbits with the synthetic peptides MEPEDLPWPDELE, representing amino acids 1–13 of mouse Alms1. Dilutions were following the manufacturer's suggestions. Antibodies were visualized by Vectastain immunodetection kit (Vector Laboratories, Incorporated, http://www.vectorlabs.com). For indirect immunofloresence, Alexa488 and Texas Red conjugated secondary antibodies were obtained from Molecular Probes, Incorporated. + +Isolation and culturing primary fibroblasts and kidney cortical duct cells. + +Wild-type and Alms1L2131X/L2131X embryos were harvested at embryonic day 12.5 and minced by passage through a syringe with an 18 G needle. Embryonic fibroblasts were cultured in 10% FBS DMEM medium. For generation of primary kidney epithelial cells, mice (postnatal day 5) were anesthetized by intraperitoneal injection of 2.5% Avertin. Kidney cortices were dissected by handheld microtome. The slices were dissociated with DMEM containing 1 mg/ml collagenase. Digested tissues were then passed sequentially through 100-μm and 45-μm sieves, and centrifuged at 1,000 × g for 10 min at room temperature. The pellets were resuspended and cultured in renal epithelial growth medium (REGM Bullet Kit, Cambrex Bioscience, http://www.cambrex.com). + +Supporting Information + +Figure S1 + +Identification of Alms1L2131X/L2131X Strain + +(A) Genotyping data for F2 mice derived from outcross of G3 mice from an ethyl nitroso urea screen on a C57Bl/6 background to NOD, followed by intercrossing of the F1 offspring. Genotype is marked as “B” for a B6-derived allele, “H” for heterozygote, and “N” for a NOD-derived allele. Based on the phenotype and genotype data, black shading denotes the included region for the position of the mutation and gray shading denotes the excluded region. Map positions refer to public mouse genome assembly M33 (http://www.ensembl.org/Mus_musculus/index.html). + +(B) Resequencing Alms1 gene in mutant mice showing nonsense mutation at exon 10. Schematic structure of Alms1 was drawn to scale based on a genomic search of Alms1 cDNA (http://www.genome.ucsc.edu). + +(327 KB JPG) + +Click here for additional data file. + +Figure S2 + +Serum Analytes in Alms1L2131X/L2131X Mice and Controls + +Plasma glucose, triglyceride, insulin, leptin, total cholesterol, and HDL cholesterol measurements in 60- to 80-d-old and 140- to 170-d-old Alms1L2131X/L2131X, Alms1+/L2131X , and Alms1+/+ mice. Arrow, diabetic mutant male. + +(445 KB JPG) + +Click here for additional data file. + +Figure S3 + +Loss of Cilia in the Kidney Cortex of Aged Alms1L2131X/L2131X Mice + +Cilia are lost in the cortex tubules (A) but not in the medulla tubules (B) in an age-dependent manner. Cilia were stained with an anti-acetylated tubulin antibody. + +(2.0 MB JPG) + +Click here for additional data file. + +Table S1 + +Gene Expression Changes after Knockdown of Alms1 + +Gene expression levels of 98 genes with the most dramatic changes in mIMCD3 cells transfected with Alms1 siRNAs compared to a scrambled siRNA control and a mock-transfected control. + +(126 KB XLS) + +Click here for additional data file. + +Accession Numbers + +The GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession number for Alms1 mouse cDNA is AF425257 and the RefSeq (http://www.ncbi.nlm.nih.gov/RefSeq) accession number for Alms1 mouse protein is NP660258. + +Acknowledgements + +We thank Richard Cornall, Samantha Zaharevitz, Conan Liu, Hanh Garcia, David Lloyd, Satchin Panda, James Watson, John Walker, and A. Huang for help and advice throughout the course of this work. We also thank M. Bandell and T. Jegla for technical assistance in Ca2+ imaging. + +Abbreviations + +ADPDK - autosomal dominant polycystic kidney disease + +ARPKD - autosomal recessive polycystic kidney disease + +BBS - Bardet-Biedl syndrome + +IFT - intraflagellar transport + +LTA - Lotus tetragonolobus agglutinin + +mIMCD3 - mouse inner medullary collecting duct + +PKD - polycystic kidney disease + +siRNA - short interfering RNA + +Figures and Tables + +Figure 1 + +Suppression of Alms1 Expression Alters Primary Cilium Formation in Kidney Epithelial Cells + +(A) Elongated cilia, visualized with staining of acetylated tubulin (green), form normally in mIMCD3 cells after mock-transfection, transfection with a negative control siRNA, or transfection with two inactive siRNAs directed against Alms1 (Alms1c and Alms1d). Focal staining of acetylated tubulin without axoneme extension is seen after transfection with two active siRNAs targeting Alms1 (Alms1a and Alms1b). + +(B) Real-time PCR analysis with two mouse Alms1 probes recognizing the junctions of exons 1 and 2 and exons 12 and 13, respectively: Alms1a and Alms1b siRNAs both cause 70%–80% knockdown of Alms1 mRNA; no effect on Alms1 mRNA was seen with the three siRNAs that were inactive in the ciliogenesis assay. + +(C) Alms1a siRNA-treated cells lose endogenous Alms1 protein expression. Acet, acetylated. Scale bars, 10 μm. + +Figure 2 + +Loss of Alms1 Does Not Affect Transcriptional Changes during Ciliogenesis but Causes Impairment in Flow-Induced Mechanosensation + +(A) Confocal microscopic analysis of cilia biogenesis in mIMCD3 cells. Short cilia can be detected at day 3 after transfection. mIMCD3 cells transfected with Alms1a siRNA have stunted cilia at days 3 and 5. Cells were stained with anti-acetylated tubulin (yellow, cilia) and TO-PRO-3 (red, nuclei). + +(B) Suppression of Alms1 does not affect the upregulation of Bbs4 and Ttc10 during ciliogenesis. N, negative siRNA; 1a, Alms1a siRNA; 1b, Alms1b siRNA. + +(C) Heat map representation of microarray analysis of mIMCD3 during ciliogenesis. 98 genes with the most dramatic changes in expression showed approximately equivalent expression changes in the presence of a scrambled siRNA control, or in the presence of specific siRNAs that decreased Alms1 mRNA levels and blocked cilia formation. + +(D) Stunted cilia formed in the presence of Alms1a siRNA (red) lack flow-induced Ca2+ influx in mIMCD3 cells, compared with a negative control siRNA (blue). Representative data are shown for cytosolic calcium change of individual cells in response to mechanical flow. Arrow points to the start of flow. + +Figure 3 + +N-Terminal Alms1 Protein Can Support Cilia Formation + +(A) Cotransfection of Alms1a siRNA-treated cells with a 5′ Alms1 cDNA construct rescues primary cilia formation in mIMCD3 cells. + +(B) Real-time PCR analysis of Alms1a siRNA and N-terminal Alms1-transfected cells. Upper panel: over-expression of the 5′ cDNA does not affect knockdown of endogenous Alms1 mRNA with Alms1a siRNA. Lower panel: knockdown of endogenous Alms1 mRNA does not affect overexpression of the 5′ cDNA. N, negative control siRNA; cDNA, 5′ Alms1 cDNA; 1a, Alms1a siRNA. + +(C) Stable expression of Alms1 mRNA from the Alms1L2131X/L2131X allele. Real-time PCR analysis of Alms1 gene expression in an Alms1L2131X/L2131X mouse and a wild-type littermate control. + +(D) The N-terminal mouse Alms1 antibody detects Alms1 mutant protein at the ciliary basal body in primary kidney cells from the Alms1L2131X/L2131X mouse. Shown are low and high magnifications of the ciliated cells. Arrowheads point out the base of cilia. + +(E) Normal appearance of primary cilia in primary fibroblasts (MEF) and primary kidney cells (PKC) from the Alms1L2131X/L2131X mouse strain. + +(F) Inhibition of cilia formation in Alms1a siRNA-treated Alms1L2131X/L2131X primary fibroblasts. Scale bars, 10 μm. + +Figure 4 + +Alms1L2131X/L2131X Mice Recapitulate Human Alström Syndrome + +(A) Alms1L2131X/L2131X mice gain more fat mass than heterozygote or wild-type controls but equivalent lean mass. + +(B) Histological examination of Alms1L2131X/L2131X mice and wild-type littermate control. Insets show oil red O staining of frozen liver sections. + +(C) Testis H&E sections show degeneration of seminiferous tubules (arrow), which have reduced numbers of germinal cells. Reduced numbers of sperm flagella with decreased length are observed in the epididymus of Alms1L2131X/L2131X mice (anti-acetylated tubulin, green). H&E, hematoxylin-eosin. + +(D) Rhodopsin staining in the outer nuclear layer cell bodies is seen in rare cells in the Alms1L2131X/L2131X animals (arrows) but not in wild-type littermate controls. Insets illustrate higher magnification images. ONL, outer nuclear layer; IS, inner segment; OS; outer segment. Scale bars, 50 μm. + +Figure 5 + +Kidney Abnormalities in Alms1 Mutant Mice + +(A) H&E-stained kidney sections of a 6-mo-old Alms1L2131X/L2131X mouse showing dilated cortex tubules compared with an age-matched wild-type control. Lack of kidney cilia is observed in some tubules in the cortex of Alms1L2131X/L2131X kidney, whereas cilia in the medulla appear normal. H&E, hematoxylin-eosin; Acet, acetylated. + +(B) Cortex cilia count comparison between Alms1L2131X/L2131X and controls. 300–400 kidney nuclei were examined for each of six fields of Alms1L2131X/L2131X and wild-type controls. The bar chart represents the average and standard deviations of cilia count per 100 kidney cortex epithelial cells from eight mice per group. + +(C) In the cortex of Alms1L2131X/L2131X kidney, cilia are lost selectively in LTA-labeled tubules but not in aquaporin-2-expressing tubules. + +(D) Upper panel: clusters of Ki67-positive proliferating epithelial cells in the Alms1L2131X/L2131X kidney, potentially lining the same convoluted tubule. Lower panel: TUNEL staining reveals apoptotic cells in Alms1L2131X/L2131X kidneys but rarely in a wild-type control. Arrow, whole tubule cross sections were labeled by TUNEL, suggesting progression of nephropathy in Alms1L2131X/L2131X mutant kidneys. WT, wild-type. + +(E) Urinalysis of 3- to 6-mo-old Alms1L2131X/L2131X mice and age-matched littermate controls. Urine from Alms1L2131X/L2131X mice showed slight elevation of protein levels, p = 0.007. Scale bars, 50 μm. + +Footnotes + +¤ Current address: Genomics Institute of the Novartis Research Foundation, San Diego, California, United States of America + +Competing interests. RJG owns stock in Phenomix. + +A previous version of this article appeared as an Early Online Release on November 30, 2006 (doi:10.1371/journal.pgen.0030008.eor). + +Author contributions. GL, KN, CG, PM, NAH, and RG conceived and designed the experiments. GL, RV, KN, and HW performed the experiments. GL, KN, NAH, and RG analyzed the data. GL, NG, and CG contributed reagents/materials/analysis tools. GL and RG wrote the paper. + +Funding. This work was funded by Phenomix Corporation and the Genomics Institute of the Novartis Research Foundation. diff --git a/src/ontogpt/evaluation/craft/database/all/17244351.ann b/src/ontogpt/evaluation/craft/database/all/17244351.ann new file mode 100644 index 000000000..981602496 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17244351.ann @@ -0,0 +1,642 @@ +T1 SO:0001026 17 23 genome +T2 SO:0000704 79 84 genes +T3 SO:0000771 101 124 quantitative trait loci +T4 http://purl.obolibrary.org/obo/MONDO_0005578 145 154 arthritis +T5 http://purl.obolibrary.org/obo/MONDO_0000001 202 210 diseases +T6 GO:0065007 215 225 controlled +T7 SO:0000704 247 252 genes +T8 SO:0000704 344 349 genes +T9 SO:0000771 367 390 quantitative trait loci +T10 SO:0000771 392 395 QTL +T11 http://purl.obolibrary.org/obo/MONDO_0005578 417 426 arthritis +T12 SO:0001026 446 452 genome +T13 SO:0001026 503 509 genome +T14 SO:0000771 613 616 QTL +T15 SO:0000704 638 642 gene +T16 GO:0010467 638 653 gene 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http://purl.obolibrary.org/obo/MONDO_0000001 16180 16187 disease +T324 UBERON:0002405 16260 16266 immune +T325 GO:0006955 16260 16275 immune response +T326 SO:0000704 16300 16305 genes +T327 GO:0010467 16312 16322 expression +T328 SO:0000704 16403 16408 genes +T329 UBERON:0002405 16435 16441 immune +T330 GO:0006955 16435 16450 immune response +T331 GO:0031090 16452 16470 organelle membrane +T332 GO:0005576 16475 16495 extracellular region +T333 GO:0005615 16475 16488;16500 16505 extracellular ... space +T334 SO:0000704 16531 16536 genes +T335 SO:0000704 16587 16592 genes +T336 GO:0005911 16612 16634 intercellular junction +T337 SO:0000704 16658 16663 genes +T338 SO:0000704 16712 16717 genes +T339 SO:0000704 16773 16778 genes +T340 CL:0000542 16807 16817 lymphocyte +T341 GO:0046651 16807 16831 lymphocyte proliferation +T342 CL:0000084 16833 16839 T cell +T343 GO:0042110 16833 16850 T cell activation +T344 GO:0007219 16883 16903 notch signal pathway +T345 SO:0000704 16930 16935 genes +T346 SO:0000704 16990 16995 genes +T347 SO:0000704 17128 17132 gene +T348 SO:0000704 17154 17159 genes +T349 SO:0000771 17181 17184 QTL +T350 SO:0000771 17261 17264 QTL +T351 GO:0010467 17321 17330 expressed +T352 SO:0000704 17331 17336 genes +T353 http://purl.obolibrary.org/obo/MONDO_0000001 17354 17361 disease +T354 GO:0010467 17386 17395 expressed +T355 SO:0000704 17396 17401 genes +T356 SO:0000704 17407 17412 genes +T357 SO:0000704 17501 17506 genes +T358 SO:0000771 17569 17572 QTL +T359 SO:0000771 17682 17685 QTL +T360 SO:0000704 17790 17795 genes +T361 SO:0000771 17857 17860 QTL +T362 SO:0000704 17913 17918 genes +T363 SO:0000704 18013 18017 gene +T364 SO:0000704 18087 18092 genes +T365 SO:0000704 18123 18128 genes +T366 PR:000008871 18130 18136 hspa1a +T367 PR:000012101 18141 18146 Oas1a +T368 PR:000012101 18192 18197 Oas1a +T369 SO:0000704 18257 18262 genes +T370 SO:0000704 18277 18282 genes +T371 SO:0000704 18335 18340 genes +T372 PR:000008871 18349 18355 hspa1a +T373 GO:0010467 18364 18374 expression +T374 SO:0000704 18433 18438 genes +T375 GO:0010467 18459 18468 expressed +T376 PR:000008409 18501 18507 H2-Q10 +T377 PR:000003107 18509 18515 Mapk14 +T378 PR:000006153 18517 18522 Pscd1 +T379 PR:000009439 18524 18529 Kpnb1 +T380 PR:000017387 18534 18538 Wdr1 +T381 SO:0000704 18557 18562 genes +T382 PR:000008409 18564 18570 H2-Q10 +T383 GO:0010467 18597 18607 expression +T384 SO:0000704 18683 18688 genes +T385 GO:0010467 18702 18712 expression +T386 GO:0010467 18791 18801 expression +T387 SO:0000704 18966 18971 genes +T388 SO:0000704 19012 19017 genes +T389 PR:000003107 19029 19035 Mapk14 +T390 PR:000010162 19037 19045 Mapk8ip3 +T391 PR:000002091 19047 19053 Stat5a +T392 PR:000008081 19058 19063 Gna12 +T393 SO:0000771 19150 19153 QTL +T394 SO:0000771 19185 19188 QTL +T395 SO:0000771 19425 19428 QTL +T396 SO:0000704 19459 19464 genes +T397 PR:000004904 19566 19568 C5 +T398 PR:000004904 19591 19593 C5 +T399 SO:0000771 19640 19643 QTL +T400 SO:0000771 19708 19711 QTL +T401 SO:0000771 19743 19746 QTL +T402 http://purl.obolibrary.org/obo/MONDO_0005578 19792 19801 arthritis +T403 SO:0000771 19802 19805 QTL +T404 SO:0000860 19872 19880 syntenic +T405 SO:0001026 19920 19927 genomic +T406 NCBITaxon:9606 19943 19948 human +T407 SO:0001026 19949 19955 genome +T408 SO:0000771 19979 19982 QTL +T409 http://purl.obolibrary.org/obo/MONDO_0008383 19997 19999 RA +T410 SO:0000771 20037 20040 QTL +T411 SO:0001023 20051 20058 alleles +T412 http://purl.obolibrary.org/obo/MONDO_0005578 20067 20076 arthritis +T413 SO:0001023 20087 20094 alleles +T414 SO:0001023 20111 20118 alleles +T415 http://purl.obolibrary.org/obo/MONDO_0005578 20127 20136 arthritis +T416 SO:0001023 20147 20154 alleles +T417 SO:0000771 20174 20177 QTL +T418 SO:0000704 20215 20220 genes +T419 http://purl.obolibrary.org/obo/MONDO_0005578 20314 20323 arthritis +T420 SO:0000771 20332 20335 QTL +T421 SO:0001026 20414 20421 genomic +T422 SO:0000771 20441 20444 QTL +T423 GO:0065007 20445 20456 controlling +T424 GO:0042571 20463 20471 antibody +T425 PR:000003266 20482 20493 collagen II +T426 SO:0000771 20526 20529 QTL +T427 SO:0001026 20544 20551 genomic +T428 SO:0000704 20577 20581 gene +T429 GO:0065007 20601 20610 regulates +T430 GO:0065007 20627 20638 controlling +T431 GO:0042571 20649 20657 antibody +T432 PR:000003266 20668 20679 collagen II +T433 GO:0065007 20736 20744 controls +T434 CL:0000542 20745 20755 lymphocyte +T435 GO:0046651 20745 20769 lymphocyte proliferation +T436 GO:0065007 20843 20851 controls +T437 CL:0000542 20852 20862 lymphocyte +T438 SO:0000704 20915 20919 gene +T439 SO:0000771 20944 20947 QTL +T440 GO:0065007 20982 20993 controlling +T441 http://purl.obolibrary.org/obo/MONDO_0005578 20994 21003 arthritis +T442 SO:0000704 21073 21077 gene +T443 GO:0010467 21073 21088 gene expression +T444 UBERON:0004905 21119 21125 joints +T445 UBERON:0000479 21140 21146 tissue +T446 SO:0000704 21203 21207 gene +T447 GO:0010467 21203 21218 gene expression +T448 UBERON:0000029 21222 21225 LNs +T449 SO:0000704 21271 21275 gene +T450 GO:0010467 21271 21286 gene expression +T451 UBERON:0000029 21299 21302 LNs +T452 SO:0000704 21311 21322 genetically +T453 GO:0065007 21422 21431 regulated +T454 SO:0000704 21432 21437 genes +T455 SO:0000704 21515 21520 genes +T456 GO:0010467 21541 21550 expressed +T457 UBERON:0000029 21619 21622 LNs +T458 SO:0000704 21844 21849 genes +T459 SO:0000704 21880 21885 genes +T460 GO:0010467 21899 21909 expression +T461 GO:0010467 21993 22003 expression +T462 SO:0000704 22072 22077 genes +T463 CL:0000542 22094 22104 lymphocyte +T464 GO:0046651 22094 22118 lymphocyte proliferation +T465 GO:0046649 22094 22104;22123 22133 lymphocyte ... activation +T466 CL:0000542 22151 22162 lymphocytes +T467 GO:0010467 22291 22301 expression +T468 NCBITaxon:10088 22348 22352 mice +T469 SO:0000704 22394 22399 genes +T470 UBERON:0002405 22415 22421 immune +T471 GO:0006955 22415 22430 immune response +T472 GO:0042571 22553 22561 antibody +T473 GO:0016064 22553 22570 antibody response +T474 PR:000003266 22574 22585 collagen II +T475 SO:0000704 22751 22756 genes +T476 SO:0000704 22796 22801 genes +T477 SO:0000771 22836 22839 QTL +T478 SO:0000704 22859 22863 gene +T479 GO:0010467 22859 22874 gene expression +T480 SO:0001026 22905 22912 genomic +T481 SO:0000704 22937 22942 genes +T482 SO:0000771 22959 22962 QTL +T483 SO:0000704 23028 23033 genes +T484 SO:0000771 23051 23054 QTL +T485 GO:0065007 23089 23099 regulating +T486 SO:0000704 23100 23104 gene +T487 GO:0010467 23100 23115 gene expression +T488 SO:0000704 23141 23146 genes +T489 http://purl.obolibrary.org/obo/MONDO_0005578 23179 23188 arthritis +T490 PR:000003107 23190 23196 Mapk14 +T491 SO:0000704 23210 23214 gene +T492 PR:000003107 23243 23279;23287 23292 p38 mitogen-activated protein kinase ... alpha +T493 PR:000003107 23243 23246;23281 23285;23287 23292 p38 ... MAPK ... alpha +T494 CHEBI:52290 23247 23254 mitogen +T495 GO:0065007 23294 23303 regulates +T496 http://purl.obolibrary.org/obo/MONDO_0005578 23322 23331 arthritis +T497 http://purl.obolibrary.org/obo/MONDO_0005070 23361 23367 tumour +T498 PR:000001091 23388 23401 interleukin-1 +T499 CHEBI:79091 23418 23431;23436 23440 inhibitors of ... MAPK +T500 PR:000003106 23432 23440 p38 MAPK +T501 NCBITaxon:10114 23464 23468 rats +T502 PR:000003106 23479 23487 p38 MAPK +T503 http://purl.obolibrary.org/obo/MONDO_0008383 23534 23536 RA +T504 PR:000002091 23543 23549 Stat5a +T505 SO:0000704 23563 23567 gene +T506 CHEBI:36357 23596 23604 molecule +T507 PR:000002091 23656 23662 Stat5a +T508 NCBITaxon:10088 23673 23677 mice +T509 http://purl.obolibrary.org/obo/MONDO_0007179 23743 23762 autoimmune diseases +T510 UBERON:0002405 23747 23753 immune +T511 PR:000002091 23764 23770 Stat5a +T512 PR:000001004 23838 23841 CD4 +T513 PR:000001380 23842 23846 CD25 +T514 GO:0065007 23848 23858 regulatory +T515 CL:0000815 23848 23865 regulatory T cell +T516 SO:0000771 23949 23952 QTL +T517 SO:0000704 23988 23993 genes +T518 NCBITaxon:33208 24088 24095 animals +T519 SO:0001026 24156 24162 genome +T520 SO:0000771 24201 24204 QTL +T521 http://purl.obolibrary.org/obo/MONDO_0005578 24393 24402 arthritis +T522 SO:0000771 24403 24406 QTL +T523 NCBITaxon:33208 24456 24463 animals +T524 SO:0000704 24511 24515 gene +T525 GO:0010467 24511 24526 gene expression +T526 GO:0010467 24586 24595 expressed +T527 SO:0000704 24596 24601 genes +T528 GO:0010467 24633 24643 expression +T529 SO:0000704 24649 24653 gene +T530 SO:0001023 24681 24687 allele +T531 SO:0000704 24867 24872 genes +T532 SO:0000771 24890 24893 QTL +T533 SO:0000704 24942 24947 genes +T534 SO:0000771 24967 24970 QTL +T535 GO:0065007 24971 24981 regulating +T536 NCBITaxon:33208 25096 25103 animals +T537 SO:0000771 25131 25134 QTL +T538 http://purl.obolibrary.org/obo/MONDO_0005578 25209 25218 arthritis +T539 SO:0000771 25219 25222 QTL +T540 SO:0000704 25271 25276 genes +T541 NCBITaxon:10088 25288 25293 mouse +T542 NCBITaxon:9606 25298 25303 human +T543 SO:0000704 25323 25328 genes +T544 NCBITaxon:10088 25455 25459 mice +T545 SO:0000704 25491 25496 genes +T546 NCBITaxon:9606 25544 25549 human +T547 SO:0001026 25550 25556 genome +T548 http://purl.obolibrary.org/obo/MONDO_0008383 25571 25573 RA +T549 SO:0000704 25609 25614 genes +T550 http://purl.obolibrary.org/obo/MONDO_0008383 25657 25659 RA +T551 http://purl.obolibrary.org/obo/MONDO_0005578 25698 25707 arthritis +T552 http://purl.obolibrary.org/obo/MONDO_0005578 25732 25741 arthritis +T553 SO:0000704 25779 25783 Gene +T554 UBERON:0000029 25794 25796 LN +T555 UBERON:0000029 25799 25809 lymph node +T556 CHEBI:52290 25847 25854 mitogen +T557 http://purl.obolibrary.org/obo/MONDO_0005578 25915 25924 arthritis +T558 SO:0000704 25975 25979 gene +T559 SO:0000771 25981 25984 QTL +T560 SO:0000771 25987 26010 quantitative trait loci +T561 http://purl.obolibrary.org/obo/MONDO_0008383 26012 26014 RA +T562 http://purl.obolibrary.org/obo/MONDO_0008383 26017 26037 rheumatoid arthritis +T563 NCBITaxon:33208 26169 26175 animal +T564 SO:0000704 26210 26214 gene +T565 GO:0010467 26210 26225 gene expression +T566 SO:0000704 26500 26505 genes +T567 GO:0010467 26521 26530 expressed +T568 GO:0010467 26712 26721 expressed +T569 SO:0000704 26722 26727 genes +T570 SO:0000704 27004 27009 genes +T571 GO:0010467 27025 27034 expressed +T572 GO:0010467 27210 27219 expressed +T573 SO:0000704 27220 27225 genes +T574 SO:0000704 27502 27507 genes +T575 GO:0010467 27549 27558 expressed +T576 GO:0065007 27571 27580 regulated +T577 SO:0000704 27643 27648 genes +T578 NCBITaxon:33208 27767 27773 animal +T579 GO:0010467 27909 27918 expressed +T580 SO:0000704 27919 27924 genes +T581 http://purl.obolibrary.org/obo/MONDO_0005578 27950 27959 arthritis +T582 SO:0000704 28019 28023 gene +T583 GO:0010467 28019 28034 gene expression +T584 SO:0000704 28131 28136 genes +T585 GO:0010467 28162 28171 expressed +T586 SO:0000704 28237 28242 genes +T587 SO:0000704 28280 28285 genes +T588 SO:0000704 28323 28328 genes +T589 http://purl.obolibrary.org/obo/MONDO_0005578 28345 28354 arthritis +T590 SO:0000704 28373 28378 genes +T591 http://purl.obolibrary.org/obo/MONDO_0005578 28394 28403 arthritis +T592 SO:0000704 28469 28474 genes +T593 GO:0010467 28490 28499 expressed +T594 GO:0010467 28554 28563 expressed +T595 SO:0000704 28564 28569 genes +T596 http://purl.obolibrary.org/obo/MONDO_0005578 28614 28623 arthritis +T597 http://purl.obolibrary.org/obo/MONDO_0005578 28695 28704 arthritis +T598 http://purl.obolibrary.org/obo/MONDO_0005578 28722 28731 arthritis +T599 GO:0010467 28805 28814 expressed +T600 SO:0000704 28815 28820 genes +T601 SO:0000704 28876 28881 genes +T602 GO:0010467 28897 28906 expressed +T603 GO:0010467 28985 28994 expressed +T604 SO:0000704 28995 29000 genes +T605 SO:0000704 29052 29056 gene +T606 GO:0010467 29052 29067 gene expression +T607 SO:0000704 29134 29139 genes +T608 GO:0010467 29266 29276 expression +T609 SO:0000704 29288 29292 gene +T610 SO:0000704 29388 29392 gene +T611 GO:0010467 29451 29461 expression +T612 SO:0000704 29480 29484 gene +T613 SO:0000704 29559 29563 gene +T614 GO:0010467 29559 29574 gene expression +T615 GO:0010467 29599 29609 expression +T616 SO:0000704 29626 29630 gene +T617 SO:0000771 29863 29866 QTL +T618 SO:0000704 29918 29923 genes +T619 SO:0000704 29941 29945 gene +T620 GO:0010467 29941 29956 gene expression +T621 SO:0000704 30009 30014 genes +T622 SO:0000771 30043 30046 QTL +T623 SO:0000771 30113 30116 QTL +T624 SO:0000704 30175 30179 gene +T625 GO:0010467 30175 30190 gene expression +T626 http://purl.obolibrary.org/obo/MONDO_0005578 30214 30223 arthritis +T627 CHEBI:60809 30248 30256 adjuvant +T628 UBERON:0000029 30288 30290 LN +T629 UBERON:0000029 30292 30302 lymph node +T630 http://purl.obolibrary.org/obo/MONDO_0005578 30336 30345 arthritis +T631 SO:0000771 30408 30411 QTL +T632 PR:000004904 30491 30493 C5 +T633 PR:000004904 30528 30530 C5 +T634 SO:0000771 30546 30549 QTL +T635 SO:0000771 30551 30574 quantitative trait loci +T636 http://purl.obolibrary.org/obo/MONDO_0008383 30576 30578 RA +T637 http://purl.obolibrary.org/obo/MONDO_0008383 30580 30600 rheumatoid arthritis +T638 SO:0000771 30640 30643 QTL +T639 SO:0000704 30654 30659 genes +T640 http://purl.obolibrary.org/obo/MONDO_0005578 30786 30795 arthritis +T641 http://purl.obolibrary.org/obo/MONDO_0005578 30813 30822 arthritis +T642 http://purl.obolibrary.org/obo/MONDO_0005578 30907 30916 arthritis diff --git a/src/ontogpt/evaluation/craft/database/all/17244351.txt b/src/ontogpt/evaluation/craft/database/all/17244351.txt new file mode 100644 index 000000000..6e48a6efc --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17244351.txt @@ -0,0 +1,145 @@ +Combining global genome and transcriptome approaches to identify the candidate genes of small-effect quantitative trait loci in collagen-induced arthritis + +Abstract + +Quantitative traits such as complex diseases are controlled by many small-effect genes that are difficult to identify. Here we present a novel strategy to identify the candidate genes for small-effect quantitative trait loci (QTL) in collagen induced arthritis (CIA) using global genome and transcriptome approaches. First, we performed genome linkage analysis in F2 progeny of the CIA susceptible and resistant strains to search for small-effect QTL. Second, we detected gene expression patterns of both strains during CIA. The candidate genes were identified using three criteria: they are located in a genomic region linked to CIA; they are disease-specific differentially expressed during CIA; and they are strain-specific differentially expressed regarding the two parental strains. Eight small-effect QTL controlling CIA severity were identified. Of 22,000 screened genes, 117 were both strain-specific and disease-specific differentially expressed during CIA. Of these 117 genes, 21 were located inside the support intervals of the 8 small-effect QTL and thus were considered as candidate genes. + +Introduction + +Susceptibility to most complex diseases is controlled by many genes, each having a small effect on the disease. One example is rheumatoid arthritis (RA), a common complex multifactorial autoimmune disease. Several studies have been carried out to detect the genetic basis of RA, and more than 30 genomic regions have shown evidence of linkage to the disease. Most of these genomic regions did not reach a genome-wide significant threshold value of linkage, with P values between 0.05 and 0.001 [1-5]. Thus, these loci only have a small effect on RA. Small genetic contributions could also be seen from the susceptibility genes of RA identified so far, including HLA-DR4, PADI4, PTPN22 and FCRL3 [6-9]. Except for HLA-DR4, which is strongly associated with RA, all the other susceptibility genes have only a small effect on the disease. In the mouse model of RA, small genetic contributions are also often observed. For example, in a previous study, we carried out a genome screen to identify the quantitative trait loci (QTL) in collagen-induced arthritis (CIA), which is a widely used animal model of RA. Only one QTL, Cia2, was identified for the phenotype of CIA severity, but this QTL contributes to only 16% of the phenotype variations for CIA susceptibility in F2 progeny [10]. This suggests that there must be other susceptibility genes whose contributions were not big enough to reach the stringent significance threshold value of linkage analysis. + +One aim of using animal models for complex diseases is to detect the genetic basis of these diseases. With controllable environmental factors as well as the known genetic background, animal models are powerful tools to search for susceptibility genes for complex diseases, and have been intensively employed for that purpose. More than 27,000 QTL have been identified in the mouse genome since the first QTL was identified at the beginning of the 1990s [11]. By 2005, approximately 20 quantitative trait genes (QTGs) in the mouse genome had been identified [12,13]. Interestingly, most QTGs identified in animal models have the causal polymorphisms in the protein-coding region [14], which provoke protein structure changes or protein deficiency. This suggests, on the one hand, that small-effect QTL are difficult to identify with traditional strategies and, on the other hand, that the polymorphisms regulating gene expression might only slightly affect the quantitative traits, and thus are more difficult to identify. + +Microarray-based global gene expression is a powerful technique for investigating complex diseases. During disease development, genes involved in the disease are likely to be differentially regulated. Therefore, signature genes of the diseases could be identified by detecting the expression patterns of the disease-related cells/tissues and their ideal controls. In the past decade, many studies applied this technique to study both RA and its animal models [15-22]. Indeed, genes involved in arthritis show distinct expression patterns in certain tissues and pathological stages of the disease. Genes involved in immunoinflammatory responses were differentially expressed in the blood cells in RA patients [18]. Chemokines and adhesion molecules were upregulated in the joint at the initiation phase of arthritis in animal models [21,22], while genes involved in cartilage destruction and bone erosion were differentially expressed at the late phase of arthritis in animal models of RA [15,16]. Besides detecting genes involved in complex diseases, microarrays could also be used to detect the genetic polymorphisms regulating gene expression because differential expressions between two strains might be the result of a polymorphism located in regulatory elements. + +To identify the small-effect QTL of CIA as well as the potential candidate genes inside them, we investigated CIA genetically susceptible and resistant strains at both the genome and transcriptome levels. At the genome level, F2 progeny of the CIA susceptible (DBA/1) and resistant (FVB/N) strains were generated and a genome-wide linkage analysis was performed to identify small-effect QTL. At the transcriptome level, we detected the gene expression patterns of both the DBA/1 and FVB/N strains at four different phases of CIA. The potential candidate genes were identified based on three criteria: they are located within the genomic region linked to CIA; they are disease-specific differentially expressed during CIA; and they are strain-specific differentially expressed between the two parental strains during CIA. + +Materials and methods + +Animals, immunisation and assessment of arthritis + +Both DBA/1 and FVB/N mice used in this study were obtained from the Jackson Laboratory and kept in a climate-controlled environment with 12 hour light/dark cycles in the animal facility at the University of Rostock. All animal experiments were pre-approved by the State Animal Care Committee. CIA was induced in control and experimental animals according to established protocols described previously [10]. In brief, DBA/1J, FVB/N and (DBA/1J × FVB/N)F2 progeny were immunised at 8 to 12 weeks at the base of the tail with 125 μg of bovine Collagen II (Chondrex, Redmond, WA, USA) emulsified in CFA (DIFCO, Detroit, MI, USA). The clinical scoring of arthritis commenced 18 days after immunisation, and animals were monitored three times weekly for signs of CIA. Arthritis development was monitored in all four limbs using a three-score system per limb as described previously [10]. + +Eight-week old FVB/N and DBA/1J mice were used for the detection of gene expression. They were divided into four experimental groups according to the different phases of CIA, namely naive control (NC), post-immunisation (PI), onset of arthritis (OA) and chronic arthritis (CA) (Table 1). The NC group contained non-immunised mice that were sacrificed at the age of 8 weeks. The mice in the PI group were sacrificed on day 10 after immunisation. The mice in the OA group were sacrificed on day 35 after immunisation. Three FVB/N non-arthritic mice and three DBA/1 mice that showed signs of arthritis on day 33 or 34 after immunisation were sacrificed on day 35 after immunisation. The mice in the CA group were sacrificed on day 95 after immunisation. Three non-arthritic FVB/N mice and three DBA/1 mice that had developed arthritis for at least two months were sacrificed on day 95 after immunisation. + +Linkage analysis + +Detailed information on genotyping of the genome screen has been described previously [10]. In short, we genotyped 290 F2 mice using 126 informative microsatellite markers covering the genome with an average inter-marker distance of 11.5 cM for 290 F2 progeny. All linkage analyses were performed with QTX Map manager software [23]. The main clinical phenotype of CIA, arthritis severity, was taken as phenotype. To detect the small-effect QTL, the threshold value of linkage was set as P = 0.05 (Chi-square test). + +Sample preparation and microarray hybridisation + +Lymph nodes (LNs) draining the immunisation site were used for total RNA preparation. The total RNA was extracted from the tissue homogenates using a commercial kit in accordance with the provided protocol (QIGEN, Hilden, Germany). Analysis of gene expression was conducted using a U430A array (Affymetrix, Santa Clara, CA, USA) interrogating more than 22,000 genes. RNA probes were labelled in accordance with the manufacturer's instructions. Samples from individual mice were hybridised onto individual arrays. Hybridisation and washing of gene chips were done as previously described [16]. Fluorescent signals were collected by laser scan (Hewlett-Packard Gene Scanner). + +Microarray analysis + +Normalisation of the expression level was done using Affymetrix software MAS 5, which is based on global scaling of total gene expression level per microarray. The normalised expression values were imported to and analysed by dCHIP [24]. Differentially expressed genes were identified by defining the appropriate filtering criteria in the dCHIP software as: lower 90% confidence boundary of fold change between the group means exceeded twofold; the absolute difference between the two groups exceeded 100; the P value threshold of the unpaired t-test was 0.05. The false discovery rate was established with a permutation test for each pairwise comparison to estimate the proportion of false-positive genes. + +Hierarchical gene clustering was performed with dCHIP to characterise the gene expression patterns during CIA. The default clustering algorithm of genes was as follows: the distance between two genes is defined as 1 – r, where r is the Pearson correlation coefficient between the standardised expression values of the two genes across the samples used. To characterise the functional relationship between differentially expressed genes, Gene Ontology (GO) term classification incorporated in DNA-Chip Analyzer was performed. The significant level for a function cluster was set at P < 0.005, and the minimum size of a cluster was three genes. + +Results + +Small-effect QTL of CIA in (DBA/1 × FVB/N) F2 progeny + +In a previous study, we carried out a genome screen to identify QTL controlling CIA susceptibility in (DBA/1 × FVB/N) F2 progeny. For the phenotype of arthritis severity, only one QTL, Cia2, was identified, with a highly significant logarithm of the odds (LOD) score of 12 [10]. However, Cia2 contributed to only 16% of the phenotype variations, indicating that there should be some small-effect QTL whose contributions to CIA were not big enough to reach the significant threshold value of linkage. To identify these potential small-effect QTL, we reanalyzed the data using a lower threshold value of linkage (P = 0.05). We reasoned that since the main candidate gene of Cia2, complement component C5 (Hc), was proven to be essential for CIA development and because the FVB/N strain is C5 deficient [10,25], some small-effect QTL might be masked by Cia2. To exclude the masking effect of C5, we performed linkage analysis with 3 datasets, the first containing all 290 F2 progeny, the second 77 C5+/+ F2 progeny and the third 133 C5+/- F2 progeny. Eight genomic regions were linked to the phenotype of CIA severity (loci 1 to 8, Table 2), with P values varying between 0.043 and 0.003. These eight small-effect QTL were located on chromosomes 5, 6, 7, 10, 11, 16, and 17. Five loci were identified in at least two datasets. Of the eight loci, five had DBA/1 as the susceptibility allele, and three had FVB/N as the susceptibility allele. + +Lander and Botstein [26] suggested a LOD score of between 2 and 3 to ensure an overall false positive rate of 5%, which means that using a lower threshold value will prevent false negative QTL at the expense of increasing false positive QTL. Being aware of this, we examined these genomic regions to search whether they, or their syntenic genomic regions on the human genome, have been previously linked to arthritis. Four small-effect QTL overlapped with arthritis QTL on the mouse genome identified previously. Locus 1 and 2 overlap with Cia13 and Cia14, which control severity of CIA in (DBA/1 × BALB/C) F2 progeny [27]. Locus 5 located on chromosome 10 overlaps with Cia8, which was identified in (DBA/1 × B10.Q) F2 progeny [28]. Locus 6 overlaps with Pgia7, which controls susceptibility to proteoglycan-induced arthritis (PGIA) and was identified in (BALB/C × DBA/2) F2 progeny [29]. The syntenic genomic regions of five small-effect QTL on the human genome have been reported to be linked to RA. These are genomic regions 22q11 and 12p13-q24 on chromosome 22 and 12 (the counterparts of locus 2), 12p13-pter on chromosome 12 (the counterpart of locus 3), 21q22-qter and 10q22-23 on chromosome 21 and 10 (the counterparts of locus 5), 17q21-25 on chromosome 17 (the counterpart of locus 6) and 3q29-qter on chromosome 3 (the counterpart of locus 7) [2,4]. + +Strain-specific differentially expressed genes + +We detected the gene expression profiles using three mice per group, which is a small number. Being aware of the importance of data reproducibility, we determined the coefficient of variation (CV) to measure data variability. The CV for each gene on the chip and the mean CV for the entire probe set were calculated. The mean CV ranged between 18.4% and 25.8% for all experimental groups, and this relatively low CV indicated that these data could be used for further analysis (Table 1). + +To search for strain-specific differentially expressed genes, we performed comparisons of gene expression between the DBA/1 and FVB/N strains at all four phases of CIA, including NC, PI, OA and CA. For the naive mice without immunisation, 361 genes were differentially expressed between the two strains. On day 10 after immunisation, when both strains did not show any sign of the disease, 141 genes were differentially expressed. After DBA/1 mice developed CIA, 184 and 85 differentially expressed genes were identified between these two strains at the onset and chronic phases, respectively. When the lists of the differentially expressed genes at the four phases were merged and overlapping genes were excluded, 509 genes were identified (Additional file 1). Twenty-one genes consistently showed differential expression between the two strains at all phases. Besides these 21 genes, only 3 additional genes were strain-specific differentially expressed during the 3 phases after CIA induction (PI, OA and CA; Figure 1). + +Disease-specific differentially expressed genes + +To identify the disease-specific differentially expressed genes in CIA, we detected the genes that were differentially expressed in LNs during CIA in the susceptible strain. Three experimental conditions, PI, OA and CA, were compared with the NC group. On day 10 after immunisation, 102 genes were differentially expressed – most of them were upregulated (78 out of 102) – while at the onset phase of the disease, only 26 genes were differentially expressed. At the chronic phase of the disease, 184 differentially expressed genes were identified, with 156 downregulated genes. Only one gene was differentially expressed at all three phases of CIA. Besides this gene, five, one and six differentially expressed genes were shared by PI with OA, PI with CA and OA with CA, respectively (Figure 2a). Taken together, 310 disease-specific differentially expressed genes were differentially regulated during CIA in DBA/1 mice (Additional file 2). + +To further characterise the gene expression pattern during CIA, we performed hierarchical cluster analysis for these 310 genes. Six gene clusters were identified (clusters I to VI, Figure 2b), each with a distinct gene expression pattern during CIA. Cluster I contains 16 genes, representing genes that were upregulated after induction of CIA. The expression of these genes reached a peak at the onset phase of the disease and functional clustering results revealed that they are related to the immune response. Cluster II contains 12 genes whose expression was gradually upregulated and reached a peak at the chronic phase of CIA. These genes are mainly related to the immune response, organelle membrane and extracellular region and space. Cluster III contains 78 genes that were only upregulated at the PI phase. These genes are related to the intercellular junction. More than half of the genes (156 of 310) belong to cluster IV and represent genes specifically downregultaed at the chronic phase. These genes are functionally related to lymphocyte proliferation, T cell activation, protein binding as well as the notch signal pathway. Cluster V contains eight genes downregulated at the PI phase. Cluster VI contains 18 genes downregulated at the OA phase. The GO term classification showed no functional cluster that was significantly enriched in these two gene clusters. + +Candidate genes for the small-effect QTL of CIA + +To identify candidate susceptibility genes for the CIA small-effect QTL, we compared the list of strain-specific differentially expressed genes with the list of disease-specific differentially expressed genes; 117 genes were shared by both lists (Additional file 3). Figure 3 visualises positions of the 117 genes retrieved from Ensembl [30] in relation to the 8 small-effect QTL. The eight loci were located on 7 chromosomes, 5, 6, 7, 10, 11, 16 and 17. Since the confidence intervals of QTL in F2 progeny are around 20 cM [26], we used 40 Mb as the confidence intervals for all loci. Twenty-one genes were located in the confidence intervals of six of the eight QTL. We located 5, 4, 2, 1, 3 and 6 potential candidate genes within the confidence intervals of loci 1, 2, 3, 5, 6 and 8, respectively, while no candidate gene was identified for loci 4 and 7. Table 3 summarises the 21 candidate genes identified in this study. Two genes, hspa1a and Oas1a, were upregulated at the OA phase of CIA and Oas1a was also upregulated at the PI phase. Except for these two genes, all other 19 genes were downregulated at the chronic phase of CIA. All genes, except hspa1a, showed expression differences between the two strains at the NC phase. Five genes were differentially expressed at all phases of CIA, including H2-Q10, Mapk14, Pscd1, Kpnb1 and Wdr1. Among these five genes, H2-Q10 had a consistently higher expression in the DBA/1 than the FVB/N strain in all CIA phases, while the other four genes had a higher expression in the DBA/1 strain at the early stages, including NC, PI and OA, but a lower expression at the chronic phase. GO term classification analysis revealed that the functional cluster of protein kinase cascade was significantly enriched in the 21 candidate genes. This functional cluster contained four genes, including Mapk14, Mapk8ip3, Stat5a and Gna12. + +Discussion + +In this study, we attempted for the first time to identify small-effect QTL in an F2 progeny. Small-effect QTL are defined as those reaching the threshold value of P = 0.05 but that did not reach the significant threshold value suggested by Lander and Botstein [26]. Although not significant, there is evidence that most of the eight small-effect QTL likely contain susceptibility genes for CIA. First, we performed the linkage analysis in three datasets, including all 290 F progeny, 76 C5+/+ F2 progeny and 133 C5+/- F2 progeny. Five of the eight small-effect QTL were identified in at least two datasets, suggesting that these QTL are reproducible. Second, many QTL identified in the present study overlap with arthritis QTL previously identified, including loci 1, 2, 5 and 6. In addition, syntenic analysis revealed that the counterpart genomic regions on the human genome of many of these eight QTL are linked to RA. + +For five of the eight small-effect QTL the DBA/1 alleles are the arthritis-enhancing alleles, while the FVBN alleles are the arthritis-enhancing alleles in the other three QTL, indicating that some susceptibility genes could come from the resistant strain. Interestingly, loci 2 and 7 partially overlap with two arthritis-related QTL identified by us in the same F2 progeny [10]. Locus 2 was located at the same genomic region as Cia27, a QTL controlling IgG2a antibody levels to collagen II. Recently, we have refined this QTL into a 4.1 Mb genomic region and showed that a gene within this region regulates CIA severity by controlling the IgG2a antibody levels to collagen II [31]. Locus 7 on chromosome 16 overlaps with Lp1, which controls lymphocyte proliferation. Furthermore, according to our unpublished data, loci 8 on chromosome 17 controls lymphocyte adherence during development of CIA. Therefore, the gene within the small-effect QTL could affect CIA severity through controlling arthritis-related phenotypes. + +Several studies have been carried out to detect gene expression during CIA, all of which used joints as the target tissue [15,16,21,22]. This study, for the first time, detected gene expression in LNs during CIA. We present an extensive study of gene expression patterns in LNs of both genetically susceptible and resistant strains at four different phases of CIA. In both strains, differentially regulated genes were highly concentrated at the PI and CA phases, and only a small number of genes were differentially expressed in two or three phases. This indicates that biological responses in LNs were stronger in the PI and CA phases than in the OA phase, and the responses at different phases were different. When comparing the susceptible to the resistant strain, the biggest difference was found in one cluster of genes (cluster IV, Figure 2). These genes had a higher expression in DBA/1 than in FVB/N at the early phases of CIA (NC, PI and OA) and the opposite expression pattern in the CA phase. GO term classification revealed that these genes were related to lymphocyte proliferation and activation, suggesting that lymphocytes in the DBA/1 strain are more activated than those in the FVB/N strain. However, this difference is not CIA specific because the expression difference between the two strains existed in mice without immunisation. Additionally, some genes related to the immune response were upregulated in the DBA/1 strain but not in the FVB/N strain during CIA. These differences could explain why a higher antibody response to collagen II occurred in the DBA/1 strain compared to the FVB/N strain, and might partially explain the difference of the susceptibility to CIA between both strains. + +Twenty-one genes were identified as potential candidate genes for six of the eight small-effect QTL according to their gene expression patterns during CIA and their genomic locations. No candidate genes were located in QTL 4 and 7, suggesting that QTG polymorphisms of the susceptibility genes inside these two QTL might not affect the phenotype by regulating gene expression. Two of the 21 candidate genes were reported to be involved in arthritis. Mapk14, a candidate gene for locus 8 and also called p38 mitogen-activated protein kinase (MAPK) alpha, regulates the production of arthritis-essential cytokines, such as tumour necrosis factor and interleukin-1 [32]. Moreover, inhibitors of p38 MAPK could attenuate CIA in rats [33], and p38 MAPK is becoming a potential therapeutic target in RA [32]. Stat5a, a candidate gene for loci 6, is an essential molecule for lymphoid development and differentiation [34]. Stat5a-deficient mice were reported to lose tolerance, resulting in the development of autoimmune diseases. Stat5a is suggested to contribute to tolerance through maintenance of the CD4+CD25+ regulatory T cell population [35]. + +Therefore, we have presented a strategy to identify small-effect QTL and search for potential candidate genes within them. However, it is noteworthy that the low statistical threshold and small number of animals per group could lead to some false positive results. On the genome level, some of the eight small-effect QTL identified using a very low threshold value (P < 0.05) could be false positives. For example, locus 4 was identified with a low P value and does not overlap with any previously identified arthritis QTL. On the transcriptome level, the small number of animals per group and the low threshold used to detect gene expression could also result in false positives in the differentially expressed genes. Furthermore, the differential expression of a gene could result not only from allele difference between two strains, but also from other factors. Therefore, our findings should be confirmed in future studies. + +Conclusion + +We present a strategy to search candidate genes for small-effect QTL. With this strategy, we identified 21 candidate genes for 8 small-effect QTL regulating CIA susceptibility. Our future studies will be carried out using two approaches. The first is generating congenic animals for promising small-effect QTL that have relatively high P values and overlap with previously-identified arthritis QTL. The second approach is investigating candidate genes using both mouse and human studies. Candidate genes will be selected according to their function and polymorphism between the two strains. Thereafter, we will generate knock-out mice to investigate the role of the genes in CIA. For the loci whose counterparts on the human genome are linked to RA, we will investigate the candidate genes using case-control association studies in RA cohorts. + +Abbreviations + +CA = chronic arthritis; CIA = collagen-induced arthritis; CV = coefficiant of variation; GO = Gene Ontology; LN = lymph node; LOD = logarithm of the odds; MAPK = mitogen-activated protein kinase; NC = naive control; OA = onset of arthritis; PI = post-immunisation; QTG = quantitative trait gene; QTL = quantitative trait loci; RA = rheumatoid arthritis. + +Competing interests + +The authors declare that they have no competing interests. + +Authors' contributions + +XY and KB performed the animal experiments. DK and HJT performed gene expression profiling experiments. XY and DK performed the bioinformatic analysis. SI conceived and designed the experiment. XY and SI drafted the manuscript. All authors read and approved the final manuscript. + +Supplementary Material + +Additional file 1 + +Summary information on the 509 genes differentially expressed between DBA/1 and FVB/N strains. Comparison was performed between two strains at four stages of CIA, NA, PI, OA and CA. Three criteria were applied for selecting the differentially expressed genes: the lower 90% confidence bound of fold change between the group means exceeded twofold; the absolute difference between the two groups exceeded 100; the P value threshold of the unpaired t-test was 0.05 + +Click here for file + +Additional file 2 + +Summary information on the 311 genes differentially expressed in joints in the DBA/1 strain during CIA. Pairwise comparisons were performed by comparing PI, OA and CA with NA. Three criteria were applied for selecting the differentially expressed genes: the lower 90% confidence bound of fold change between the group means exceeded twofold; the absolute difference between the two groups exceeded 100; the P value threshold of the unpaired t-test was 0.05 + +Click here for file + +Additional file 3 + +Summary information on the 117 genes that were strain-specific differentially expressed and were dysregulated in the DBA/1 strain during CIA. The physical positions of the genes were retrieved from Ensembl [30] + +Click here for file + +Acknowledgements + +The authors wish to thank Ilona Klamfuss for animal care. This work was supported by a grant from the EU FP6 (MRTN-CT-2004-005693, EURO-RA). + +Figures and Tables + +Figure 1 + +Differentially expressed genes between collagen-induced arthritis (CIA) susceptible and resistant strains. Comparison of the gene expression between the DBA/1 and FVB/N strains was performed at four phases of CIA. Of the 22,000 screened genes, 509 were differentially expressed between both strains at one or more phases of CIA, including 361 genes at the naive control (NC) phase, 141 genes at post-immunisation (PI) phase, 184 genes at the onset of arthritis (OA) phase and 85 genes at the chronic arthritis (CA) phase. The Venn diagram indicates the number of overlapping genes differentially expressed at different phases of CIA. + +Figure 2 + +Differentially expressed genes in the DBA/1 strain during collagen-induced arthritis (CIA). Three experimental conditions, post-immunisation (PI), onset of arthritis (OA) and chronic arthritis (CA), were compared with naïve control (NC) to search for differentially expressed genes. (a) Venn diagram indicating the number of overlapping genes differentially expressed at three phases of CIA. (b) Hierarchical clustering of the 310 differentially expressed genes. The left panel shows the distribution of relative gene expression across the hierarchical tree structure. Rows represent individual genes; columns represent individual value of triplicate samples for each experimental group. Each cell in the matrix represents the expression level of a gene, with red and green indicating transcription levels above and below the normal values for that gene, respectively. Four sample groups are indicated above the expression matrix. Six basic gene clusters (clusters I to VI) were yielded by the analysis according to the gene expression pattern during CIA. The expression patterns of the gene cluster are graphed and the major biological activities for each cluster that were examined by functional clustering analysis are indicated on the right. + +Figure 3 + +Visualisation of all the chromosomal locations of the small-effect QTL identified in this study (blue bar) as well as 120 genes of interest from gene expression profiling (black letters). The positions of the 120 genes and the peak markers of the QTL were retrieved from Ensembl [30]. Confidence intervals of all the QTL were set as 40 Mb. + +Table 1 + +Experimental groups used for gene expression profiling + +CA, chronic arthritis; CFA; complete Freund's adjuvant; CV, coefficient of variation; LN, lymph node; NC, naive control; OA, onset of arthritis; PI, post-immunisation. + +Table 2 + +Summary of the small-effect QTL identified in this study + +aIdentified in all F2 290 progeny. bIdentified in 76 C5+/+ F2 progeny. cIdentified in 133 C5+/- F2 progeny. QTL, quantitative trait loci; RA, rheumatoid arthritis. + +Table 3 + +Summary of the small-effect QTL candidate genes + +aFold change calculated by comparing DBA/1 with FVB/N. bFold change calculated by comparing post-immunisation (PI), onset of arthritis (OA) and chronic arthritis (CA) with naive control (NC), respectively. Chr., chromosome; CIA, collagen-induced arthritis. diff --git a/src/ontogpt/evaluation/craft/database/all/17425782.ann b/src/ontogpt/evaluation/craft/database/all/17425782.ann new file mode 100644 index 000000000..eeb8c67e0 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17425782.ann @@ -0,0 +1,1321 @@ +T1 PR:000013523 0 5 Pygo1 +T2 PR:000013524 10 15 Pygo2 +T3 GO:0016055 25 38 Wnt signaling +T4 NCBITaxon:40674 42 51 mammalian +T5 UBERON:0002113 52 58 kidney +T6 GO:0001822 52 70 kidney development +T7 PR:Q9V9W8 98 105 pygopus +T8 SO:0000704 106 110 gene +T9 NCBITaxon:7215 114 124 Drosophila +T10 PR:P18824 163 172 Armadillo +T11 PR:000002198 174 183 β-catenin +T12 GO:0005667 185 213 transcription factor complex +T13 GO:0060070 217 240 canonical Wnt signaling +T14 PR:Q9V9W8 280 287 Pygopus +T15 GO:0060070 297 320 canonical Wnt signaling +T16 UBERON:0002113 324 330 kidney +T17 GO:0001822 324 342 kidney development +T18 NCBITaxon:40674 384 393 mammalian +T19 SO:0000855 394 403 orthologs +T20 PR:000013523 405 410 Pygo1 +T21 PR:000013524 415 420 Pygo2 +T22 SO:0000417 510 516 domain +T23 PR:000013524 566 571 Pygo2 +T24 GO:0016265 613 617 died +T25 GO:0007567 632 637 birth +T26 UBERON:0002113 709 715 kidney +T27 GO:0001822 709 727 kidney development +T28 http://purl.obolibrary.org/obo/MONDO_0016064 763 775 cleft palate +T29 PR:000013523 777 782 Pygo1 +T30 PR:000013523 886 891 Pygo1 +T31 PR:000013524 892 897 Pygo2 +T32 SO:0000902 979 988 transgene +T33 GO:0060070 1001 1024 canonical Wnt signaling +T34 GO:0010467 1050 1060 expression +T35 PR:000013523 1064 1069 Pygo1 +T36 PR:000013524 1073 1078 Pygo2 +T37 UBERON:0000479 1096 1102 tissue +T38 PR:000013523 1151 1156 Pygo1 +T39 PR:000013524 1161 1166 Pygo2 +T40 SO:0000704 1167 1172 genes +T41 GO:0010467 1196 1206 expression +T42 UBERON:0002113 1225 1231 kidney +T43 UBERON:0003891 1259 1266 stromal +T44 CL:0000499 1259 1271 stromal cell +T45 UBERON:0002113 1324 1331 kidneys +T46 UBERON:0000084 1363 1375 ureteric bud +T47 UBERON:0003220 1380 1402 metanephric mesenchyme +T48 GO:0001658 1425 1451;1456 1468 Branching morphogenesis of ... ureteric bud +T49 UBERON:0000084 1456 1468 ureteric bud +T50 UBERON:0002113 1582 1588 kidney +T51 UBERON:0003104 1651 1661 mesenchyme +T52 UBERON:0000084 1674 1686 ureteric bud +T53 UBERON:0001285 1688 1695 Nephron +T54 GO:0072006 1688 1705 Nephron formation +T55 GO:0010467 1771 1781 expression +T56 SO:0000704 1793 1798 genes +T57 PR:000006067 1810 1816 Cxcl13 +T58 PR:000015169 1818 1824 Slc5a2 +T59 PR:000009415 1826 1830 Klk5 +T60 PR:000014395 1832 1836 Ren2 +T61 PR:000016340 1841 1849 Timeless +T62 UBERON:0002113 1892 1898 kidney +T63 GO:0001822 1892 1910 kidney development +T64 NCBITaxon:40674 1929 1938 mammalian +T65 PR:Q9V9W8 1939 1946 Pygopus +T66 SO:0000704 1947 1952 genes +T67 GO:0001658 1977 2003;2008 2020 branching morphogenesis of ... ureteric bud +T68 UBERON:0000084 2008 2020 ureteric bud +T69 UBERON:0002113 2028 2034 kidney +T70 GO:0001822 2028 2046 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PR:Q9V9W8 5371 5378 Pygopus +T171 GO:0060070 5388 5411 canonical Wnt signaling +T172 UBERON:0000467 5459 5472 organ systems +T173 PR:000013523 5484 5489 Pygo1 +T174 PR:000013524 5494 5499 Pygo2 +T175 PR:000013523 5541 5546 Pygo1 +T176 PR:000013524 5551 5556 Pygo2 +T177 SO:0000704 5557 5562 genes +T178 SO:0000346 5576 5590 LoxP sequences +T179 SO:0000357 5594 5599 flank +T180 SO:0000417 5642 5649 domains +T181 NCBITaxon:10088 5674 5678 mice +T182 GO:0007618 5684 5689 mated +T183 NCBITaxon:10358 5706 5709 CMV +T184 NCBITaxon:10088 5714 5718 mice +T185 SO:0000346 5742 5746 LoxP +T186 SO:0001023 5791 5798 alleles +T187 SO:0000704 5902 5907 genes +T188 PR:000013523 5946 5951 Pygo1 +T189 PR:000013524 5983 5988 Pygo2 +T190 NCBITaxon:7215 6010 6020 Drosophila +T191 SO:0000417 6080 6086 domain +T192 GO:0016055 6118 6131 Wnt signaling +T193 PR:000013523 6138 6143 Pygo1 +T194 PR:000013524 6148 6153 Pygo2 +T195 PR:000013523 6173 6178 Pygo1 +T196 NCBITaxon:10088 6195 6199 mice +T197 PR:Q9V9W8 6314 6321 pygopus +T198 SO:0000704 6322 6326 gene +T199 GO:0016055 6330 6343 Wnt signaling +T200 NCBITaxon:7215 6347 6357 Drosophila +T201 GO:0010467 6376 6386 expression +T202 NCBITaxon:10088 6413 6418 mouse +T203 PR:000013523 6419 6424 Pygo1 +T204 SO:0000704 6425 6429 gene +T205 UBERON:0000955 6451 6456 brain +T206 UBERON:0002101 6458 6463 limbs +T207 UBERON:0002113 6465 6471 kidney +T208 UBERON:0002539 6476 6492 branchial arches +T209 PR:000013524 6565 6570 Pygo2 +T210 NCBITaxon:10088 6587 6591 mice +T211 GO:0007567 6604 6609 birth +T212 GO:0016265 6637 6641 died +T213 UBERON:0001555 6666 6669 gut +T214 UBERON:0000948 6671 6676 heart +T215 UBERON:0002101 6681 6686 limbs +T216 GO:0016055 6776 6789 Wnt signaling +T217 PR:000013524 6795 6800 Pygo2 +T218 UBERON:0002113 6868 6874 kidney +T219 http://purl.obolibrary.org/obo/MONDO_0016064 6924 6936 cleft palate +T220 PR:000013523 6991 6996 Pygo1 +T221 PR:000013524 7001 7006 Pygo2 +T222 NCBITaxon:10088 7007 7011 mice +T223 PR:000013524 7054 7059 Pygo2 +T224 PR:000013524 7212 7217 Pygo2 +T225 PR:000013523 7300 7305 Pygo1 +T226 PR:000013523 7358 7363 Pygo1 +T227 PR:000013524 7364 7369 Pygo2 +T228 UBERON:0000922 7377 7384 embryos +T229 PR:000013523 7396 7401 Pygo1 +T230 PR:000013524 7405 7410 Pygo2 +T231 UBERON:0000922 7414 7421 embryos +T232 PR:000013524 7447 7452 Pygo2 +T233 SO:0001023 7453 7459 allele +T234 PR:000013523 7510 7515 Pygo1 +T235 PR:000013524 7519 7524 Pygo2 +T236 UBERON:0000922 7528 7535 embryos +T237 UBERON:0000970 7555 7558 eye +T238 CHEBI:26130 7615 7624 pigmented +T239 UBERON:0000966 7625 7631 retina +T240 PR:000013523 7668 7673 Pygo1 +T241 PR:000013524 7674 7679 Pygo2 +T242 UBERON:0000922 7685 7692 embryos +T243 PR:000013524 7745 7750 Pygo2 +T244 UBERON:0000922 7792 7799 embryos +T245 PR:000013523 7805 7810 Pygo1 +T246 PR:000013524 7900 7905 Pygo2 +T247 GO:0010467 7981 7991 Expression +T248 PR:000013523 7995 8000 Pygo1 +T249 PR:000013524 8005 8010 Pygo2 +T250 UBERON:0002113 8029 8035 kidney +T251 GO:0097617 8045 8058 hybridization +T252 GO:0010467 8100 8110 expression +T253 PR:000013523 8114 8119 Pygo1 +T254 GO:0010467 8250 8260 expression +T255 PR:000013523 8277 8282 Pygo1 +T256 PR:000013524 8287 8292 Pygo2 +T257 SO:0000704 8293 8298 genes +T258 UBERON:0002113 8317 8323 kidney +T259 SO:0000704 8330 8335 genes +T260 GO:0010467 8348 8357 expressed +T261 GO:0005634 8367 8374 nuclear +T262 UBERON:0000084 8415 8427 ureteric bud +T263 UBERON:0005107 8429 8447 capping mesenchyme +T264 UBERON:0003891 8453 8460 stromal +T265 CL:0000499 8453 8466 stromal cells +T266 GO:0010467 8486 8496 expression +T267 PR:000013523 8500 8505 Pygo1 +T268 PR:000013524 8533 8538 Pygo2 +T269 UBERON:0003891 8552 8559 stromal +T270 CL:0000499 8552 8565 stromal cells +T271 GO:0005634 8583 8590 nuclear +T272 CL:1000497 8632 8640;8656 8662 cells of ... kidney +T273 UBERON:0002113 8656 8662 kidney +T274 PR:000013523 8693 8698 Pygo1 +T275 PR:000013524 8703 8708 Pygo2 +T276 GO:0010467 8709 8719 expression +T277 UBERON:0002113 8744 8750 kidney +T278 GO:0010467 8793 8803 expression +T279 PR:000013523 8820 8825 Pygo1 +T280 PR:000013524 8830 8835 Pygo2 +T281 UBERON:0001225 8852 8861;8872 8878 cortex of ... kidney +T282 PR:000013523 8885 8890 Pygo1 +T283 PR:000013524 8895 8900 Pygo2 +T284 GO:0005634 8929 8936 nucleus +T285 SO:0000704 8956 8961 genes +T286 GO:0010467 8980 8990 expression +T287 UBERON:0002113 9049 9055 kidney +T288 UBERON:0003891 9089 9096 stromal +T289 CL:0000499 9089 9101 stromal cell +T290 UBERON:0000483 9124 9134 Epithelial +T291 CL:0000066 9124 9140 Epithelial cells +T292 UBERON:0000084 9152 9164 ureteric bud +T293 PR:000001447 9208 9218 E-cadherin +T294 GO:0042571 9219 9227 antibody +T295 PR:000013523 9281 9286 Pygo1 +T296 PR:000013524 9287 9292 Pygo2 +T297 UBERON:0002113 9300 9307 kidneys +T298 PR:000013524 9337 9342 Pygo2 +T299 PR:000013523 9352 9357 Pygo1 +T300 PR:000013524 9358 9363 Pygo2 +T301 UBERON:0002113 9383 9390 kidneys +T302 GO:0001822 9427 9440 nephrogenesis +T303 UBERON:0002113 9517 9522 renal +T304 GO:0001822 9517 9534 renal development +T305 PR:000013523 9544 9549 Pygo1 +T306 PR:000013524 9550 9555 Pygo2 +T307 UBERON:0002113 9561 9568 kidneys +T308 PR:000017459 9596 9599 Wt1 +T309 PR:000005505 9610 9616 Cited1 +T310 GO:0042571 9623 9633 antibodies +T311 UBERON:0005107 9649 9679 capping metanephric mesenchyme +T312 UBERON:0006364 9691 9708 ureteric bud tips +T313 PR:000017459 9715 9718 WT1 +T314 GO:0042571 9719 9727 antibody +T315 UBERON:0004209 9740 9754 renal vesicles +T316 UBERON:0000074 9740 9745;9759 9769 renal ... glomerular +T317 UBERON:0007688 9770 9776 anlage +T318 GO:0042571 9778 9788 Antibodies +T319 PR:000001447 9792 9796 Cdh1 +T320 PR:000001447 9812 9822 E-cadherin +T321 UBERON:0000483 9853 9863 epithelial +T322 UBERON:0000084 9900 9912 ureteric bud +T323 UBERON:0001285 9925 9933 nephrons +T324 UBERON:0002113 9952 9958 kidney +T325 NCBITaxon:271171 9965 9982 Dolichos biflorus +T326 UBERON:0000084 10031 10043 ureteric bud +T327 PR:000013523 10101 10106 Pygo1 +T328 PR:000013524 10107 10112 Pygo2 +T329 UBERON:0002113 10126 10133 kidneys +T330 PR:000013523 10142 10147 Pygo1 +T331 PR:000013524 10151 10156 Pygo2 +T332 UBERON:0002113 10173 10180 kidneys +T333 UBERON:0002113 10192 10199 kidneys +T334 GO:0042571 10241 10251 antibodies +T335 PR:000005505 10262 10268 Cited1 +T336 PR:000017459 10279 10282 Wt1 +T337 GO:0010467 10289 10298 expressed +T338 UBERON:0003220 10317 10339 metanephric mesenchyme +T339 PR:000001447 10361 10365 Cdh1 +T340 UBERON:0000483 10387 10396 epithelia +T341 UBERON:0000056 10433 10441 ureteric +T342 UBERON:0006364 10532 10545 ureteric tips +T343 PR:000013523 10582 10587 Pygo1 +T344 PR:000013524 10588 10593 Pygo2 +T345 UBERON:0002113 10599 10606 kidneys +T346 UBERON:0006364 10662 10679 ureteric bud tips +T347 PR:000013523 10707 10712 Pygo1 +T348 UBERON:0003104 10766 10776 mesenchyme +T349 UBERON:0000084 10793 10805 ureteric bud +T350 PR:000017459 10885 10888 Wt1 +T351 PR:000005505 10893 10899 Cited1 +T352 UBERON:0005107 10981 10999 capping mesenchyme +T353 UBERON:0000084 11023 11036 ureteric buds +T354 UBERON:0003220 11083 11105 metanephric mesenchyme +T355 GO:0001822 11134 11147 nephrogenesis +T356 PR:000001447 11149 11153 Cdh1 +T357 UBERON:0001285 11163 11171 nephrons +T358 UBERON:0006364 11213 11230 ureteric bud tips +T359 UBERON:0000362 11259 11269;11274 11280 medulla of ... kidney +T360 GO:0001822 11320 11333 nephrogenesis +T361 UBERON:0004209 11345 11359 renal vesicles +T362 UBERON:0004198 11361 11380 comma-shaped bodies +T363 UBERON:0004199 11386 11401 S-shaped bodies +T364 PR:000013524 11450 11455 Pygo2 +T365 UBERON:0002113 11496 11503 kidneys +T366 PR:000013523 11511 11516 Pygo1 +T367 UBERON:0006364 11667 11684 ureteric bud tips +T368 PR:000013523 11693 11698 Pygo1 +T369 PR:000013524 11699 11704 Pygo2 +T370 UBERON:0002113 11710 11717 kidneys +T371 PR:000013524 11808 11813 Pygo2 +T372 SO:0001023 11814 11820 allele +T373 PR:000013523 11850 11855 Pygo1 +T374 PR:000013524 11856 11861 Pygo2 +T375 UBERON:0002113 11887 11894 kidneys +T376 UBERON:0006364 11953 11970 ureteric bud tips +T377 PR:000013524 12042 12047 Pygo2 +T378 UBERON:0006364 12088 12104 ureteric bud-tip +T379 PR:000013524 12130 12135 Pygo2 +T380 PR:000013524 12143 12148 Pygo2 +T381 UBERON:0000922 12152 12159 embryos +T382 GO:0097617 12181 12194 hybridization +T383 GO:0010467 12208 12218 expression +T384 SO:0000704 12232 12237 genes +T385 PR:000017449 12255 12260 Wnt7b +T386 GO:0010467 12264 12273 expressed +T387 PR:000017438 12294 12299 Wnt11 +T388 PR:000017453 12321 12326 Wnt9b +T389 UBERON:0012238 12334 12340;12364 12366;12381 12393 stalks ... of ... ureteric bud +T390 UBERON:0006364 12359 12366;12381 12393 tips of ... ureteric bud +T391 SO:0000704 12409 12414 genes +T392 GO:0010467 12430 12440 expression +T393 PR:000013524 12453 12458 Pygo2 +T394 PR:000013523 12466 12471 Pygo1 +T395 PR:000013524 12472 12477 Pygo2 +T396 UBERON:0002113 12492 12499 kidneys +T397 GO:0097617 12539 12552 hybridization +T398 PR:000017438 12639 12644 Wnt11 +T399 PR:000013523 12679 12684 Pygo1 +T400 PR:000013524 12685 12690 Pygo2 +T401 GO:0010467 12729 12739 Expression +T402 PR:000017449 12743 12748 Wnt7b +T403 PR:000017453 12750 12755 Wnt9b +T404 PR:000017438 12761 12766 Wnt11 +T405 PR:000013523 12776 12781 Pygo1 +T406 PR:000013524 12782 12787 Pygo2 +T407 UBERON:0002113 12802 12809 kidneys +T408 GO:0097617 12834 12848 hybridizations +T409 UBERON:0002113 12874 12881 kidneys +T410 PR:000013523 12892 12897 Pygo1 +T411 PR:000013524 12901 12906 Pygo2 +T412 UBERON:0002113 12917 12924 kidneys +T413 UBERON:0000084 12934 12946 ureteric bud +T414 PR:000017449 12974 12979 Wnt7b +T415 PR:000017453 12988 12993 Wnt9b +T416 PR:000017438 13006 13011 Wnt11 +T417 UBERON:0002113 13024 13031 kidneys +T418 GO:0010467 13046 13056 expression +T419 UBERON:0012238 13074 13088 ureteric stalk +T420 PR:000017449 13097 13102 Wnt7b +T421 PR:000017453 13107 13112 Wnt9b +T422 UBERON:0006364 13137 13150 ureteric tips +T423 PR:000017438 13166 13171 Wnt11 +T424 GO:0060070 13211 13234 canonical Wnt signaling +T425 PR:000013523 13238 13243 Pygo1 +T426 PR:000013524 13244 13249 Pygo2 +T427 NCBITaxon:10088 13257 13261 mice +T428 SO:0000902 13275 13284 transgene +T429 GO:0060070 13297 13320 canonical Wnt signaling +T430 GO:0016055 13357 13370 Wnt signaling +T431 NCBITaxon:10088 13385 13389 mice +T432 PR:000013524 13396 13401 Pygo2 +T433 PR:000013523 13409 13414 Pygo1 +T434 PR:000013524 13415 13420 Pygo2 +T435 UBERON:0000922 13439 13446 embryos +T436 GO:0060070 13468 13491 canonical Wnt signaling +T437 UBERON:0000479 13524 13530 tissue +T438 UBERON:0002329 13589 13596 somites +T439 UBERON:0001893 13654 13667 telencephalon +T440 GO:0016055 13688 13701 Wnt signaling +T441 NCBITaxon:40674 13734 13743 mammalian +T442 SO:0000704 13749 13754 genes +T443 GO:0060070 13785 13808 canonical Wnt signaling +T444 UBERON:0000467 13844 13851 systems +T445 GO:0060070 13872 13895 canonical Wnt signaling +T446 PR:000013524 13899 13904 Pygo2 +T447 PR:000013523 13909 13914 Pygo1 +T448 PR:000013524 13915 13920 Pygo2 +T449 UBERON:0000922 13928 13935 embryos +T450 UBERON:0000922 13943 13950 embryos +T451 SO:0000902 13973 13982 transgene +T452 GO:0060070 13995 14018 canonical Wnt signaling +T453 PR:000013523 14024 14029 Pygo1 +T454 PR:000013524 14033 14038 Pygo2 +T455 UBERON:0000922 14042 14049 embryos +T456 CHEBI:75055 14064 14069 X-gal +T457 UBERON:0000955 14097 14102 brain +T458 UBERON:0004117 14104 14122 pharyngeal pouches +T459 UBERON:0003051 14124 14136 otic vesicle +T460 UBERON:0004356 14138 14162 apical ectodermal ridges +T461 UBERON:0005417 14170 14174;14184 14193 fore ... limb buds +T462 UBERON:0005418 14179 14193 hind limb buds +T463 UBERON:0002329 14202 14209 somites +T464 PR:000013523 14215 14220 Pygo1 +T465 PR:000013524 14224 14229 Pygo2 +T466 UBERON:0000922 14233 14240 embryos +T467 PR:000013524 14260 14265 Pygo2 +T468 SO:0001023 14266 14273 alleles +T469 GO:0010467 14322 14332 expression +T470 UBERON:0004117 14374 14392 pharyngeal pouches +T471 UBERON:0003051 14394 14406 otic vesicle +T472 UBERON:0002329 14412 14419 somites +T473 PR:000013523 14425 14430 Pygo1 +T474 PR:000013524 14434 14439 Pygo2 +T475 UBERON:0000922 14443 14449 embryo +T476 PR:000013523 14473 14478 Pygo1 +T477 SO:0001023 14479 14486 alleles +T478 PR:000013524 14506 14511 Pygo2 +T479 SO:0001023 14512 14518 allele +T480 GO:0010467 14542 14552 expression +T481 PR:000013523 14583 14588 Pygo1 +T482 PR:000013524 14592 14597 Pygo2 +T483 UBERON:0000922 14601 14608 embryos +T484 GO:0010467 14645 14655 expression +T485 GO:0060070 14677 14700 canonical Wnt signaling +T486 UBERON:0000922 14702 14709 Embryos +T487 UBERON:0000922 14796 14803 embryos +T488 GO:0010467 15009 15019 expression +T489 UBERON:0004122 15053 15070 urogenital system +T490 PR:000013524 15119 15124 Pygo2 +T491 SO:0000704 15125 15129 gene +T492 GO:0060070 15146 15169 canonical Wnt signaling +T493 UBERON:0009201 15177 15189 nephric duct +T494 PR:000013524 15196 15201 Pygo2 +T495 PR:000013523 15211 15216 Pygo1 +T496 PR:000013524 15217 15222 Pygo2 +T497 UBERON:0000922 15241 15248 embryos +T498 GO:0010467 15279 15289 expression +T499 UBERON:0009201 15297 15309 nephric duct +T500 GO:0010467 15351 15361 expression +T501 PR:000013523 15380 15385 Pygo1 +T502 PR:000013524 15386 15391 Pygo2 +T503 UBERON:0009201 15417 15429 nephric duct +T504 UBERON:0000084 15470 15482 ureteric bud +T505 GO:0010467 15547 15557 expression +T506 PR:000013524 15584 15589 Pygo2 +T507 GO:0010467 15623 15633 expression +T508 UBERON:0000084 15637 15649 ureteric bud +T509 UBERON:0002113 15687 15693 kidney +T510 CHEBI:75055 15695 15700 X-Gal +T511 UBERON:0004122 15761 15771 urogenital +T512 UBERON:0002113 15796 15803 kidneys +T513 PR:000013523 15809 15814 Pygo1 +T514 PR:000013524 15818 15823 Pygo2 +T515 PR:000013523 15838 15843 Pygo1 +T516 PR:000013524 15847 15852 Pygo2 +T517 UBERON:0009201 15907 15919 nephric duct +T518 UBERON:0000084 15971 15983 ureteric bud +T519 PR:000013524 16005 16010 Pygo2 +T520 UBERON:0000922 16016 16022 embryo +T521 PR:000013523 16036 16041 Pygo1 +T522 PR:000013524 16045 16050 Pygo2 +T523 UBERON:0000922 16060 16066 embryo +T524 UBERON:0009201 16105 16117 nephric duct +T525 UBERON:0000084 16140 16152 ureteric bud +T526 PR:000013523 16172 16177 Pygo1 +T527 PR:000013524 16181 16185 Pygo +T528 UBERON:0000922 16189 16195 embryo +T529 GO:0010467 16211 16221 expression +T530 UBERON:0009201 16234 16246 nephric duct +T531 UBERON:0000084 16266 16278 ureteric bud +T532 CHEBI:75055 16317 16322 X-gal +T533 UBERON:0004122 16337 16347 urogenital +T534 UBERON:0000922 16368 16374 embryo +T535 SO:0000902 16395 16404 transgene +T536 PR:000013523 16478 16483 Pygo1 +T537 PR:000013524 16487 16492 Pygo2 +T538 UBERON:0000056 16535 16543 ureteric +T539 UBERON:0002113 16574 16580 kidney +T540 UBERON:0006364 16596 16609 ureteric tips +T541 UBERON:0000056 16611 16619 ureteric +T542 UBERON:0000056 16630 16636 ureter +T543 PR:000013523 16642 16647 Pygo1 +T544 PR:000013524 16651 16656 Pygo2 +T545 UBERON:0000056 16719 16727 ureteric +T546 PR:000013523 16745 16750 Pygo1 +T547 PR:000013524 16754 16759 Pygo2 +T548 GO:0010467 16778 16788 expression +T549 UBERON:0003890 16796 16816 paramesonephric duct +T550 UBERON:0000056 16839 16847 ureteric +T551 PR:000013523 16861 16866 Pygo1 +T552 PR:000013524 16870 16875 Pygo2 +T553 GO:0010467 16919 16929 expression +T554 UBERON:0003890 16937 16957 paramesonephric duct +T555 UBERON:0000056 17005 17013 ureteric +T556 GO:0010467 17019 17029 expression +T557 UBERON:0002113 17037 17043 kidney +T558 PR:000013523 17048 17053 Pygo1 +T559 PR:000013524 17057 17062 Pygo2 +T560 GO:0010467 17122 17132 expression +T561 UBERON:0000056 17136 17144 ureteric +T562 UBERON:0003890 17154 17174 paramesonephric duct +T563 PR:000013523 17182 17187 Pygo1 +T564 PR:000013524 17191 17196 Pygo2 +T565 UBERON:0003890 17235 17255 paramesonephric duct +T566 UBERON:0000056 17286 17292 ureter +T567 UBERON:0000056 17297 17305 ureteric +T568 UBERON:0002113 17336 17343 kidneys +T569 PR:000013523 17414 17419 Pygo1 +T570 PR:000013524 17423 17428 Pygo2 +T571 PR:000013523 17443 17448 Pygo1 +T572 PR:000013524 17452 17457 Pygo2 +T573 UBERON:0002113 17475 17482 kidneys +T574 UBERON:0002113 17488 17495 kidneys +T575 GO:0010467 17535 17545 expression +T576 UBERON:0000056 17562 17570 ureteric +T577 UBERON:0001851 17594 17600 cortex +T578 UBERON:0000958 17605 17612 medulla +T579 PR:000013523 17710 17715 Pygo1 +T580 PR:000013524 17719 17724 Pygo2 +T581 PR:000013523 17736 17741 Pygo1 +T582 PR:000013524 17745 17750 Pygo2 +T583 PR:000013523 17768 17773 Pygo1 +T584 PR:000013524 17777 17782 Pygo2 +T585 UBERON:0002113 17800 17807 kidneys +T586 UBERON:0000056 17846 17854 ureteric +T587 PR:000013523 17875 17880 Pygo1 +T588 PR:000013524 17884 17889 Pygo2 +T589 PR:000013523 17904 17909 Pygo1 +T590 PR:000013524 17913 17918 Pygo2 +T591 UBERON:0002113 17931 17938 kidneys +T592 PR:000013523 17956 17961 Pygo1 +T593 PR:000013524 17965 17970 Pygo2 +T594 UBERON:0002113 17982 17988 kidney +T595 PR:000013524 18106 18111 Pygo2 +T596 SO:0000704 18112 18116 gene +T597 PR:000013523 18156 18161 Pygo1 +T598 SO:0000704 18162 18166 gene +T599 GO:0060070 18184 18207 canonical Wnt signaling +T600 UBERON:0000056 18215 18223 ureteric +T601 GO:0010467 18253 18263 expression +T602 UBERON:0002113 18284 18290 kidney +T603 SO:0000902 18312 18321 transgene +T604 CHEBI:75055 18349 18354 X-gal +T605 UBERON:0002113 18406 18412 kidney +T606 PR:000013524 18441 18446 Pygo2 +T607 SO:0000704 18447 18451 gene +T608 CHEBI:75055 18466 18471 X-gal +T609 UBERON:0000056 18488 18496 ureteric +T610 PR:000013523 18528 18533 Pygo1 +T611 PR:000013524 18537 18542 Pygo2 +T612 UBERON:0002113 18552 18559 kidneys +T613 GO:0010467 18586 18596 expression +T614 GO:0060070 18643 18666 canonical Wnt signaling +T615 PR:000013523 18691 18696 Pygo1 +T616 SO:0000704 18697 18701 gene +T617 GO:0010467 18749 18759 expression +T618 PR:000013523 18807 18812 Pygo1 +T619 PR:000013524 18817 18822 Pygo2 +T620 SO:0000704 18823 18828 genes +T621 GO:0010467 18871 18881 expression +T622 PR:000013524 18895 18900 Pygo2 +T623 PR:000013523 18928 18933 Pygo1 +T624 PR:000013524 18938 18943 Pygo2 +T625 SO:0000704 18944 18949 genes +T626 GO:0010467 18990 19000 expression +T627 UBERON:0003890 19008 19023;19036 19041 paramesonephric ... ducts +T628 PR:000013524 19046 19051 Pygo2 +T629 NCBITaxon:10088 19055 19059 mice +T630 GO:0010467 19102 19112 expression +T631 CHEBI:75055 19176 19181 X-gal +T632 UBERON:0003890 19198 19219 paramesonephric ducts +T633 PR:000013523 19241 19246 Pygo1 +T634 PR:000013524 19250 19255 Pygo2 +T635 PR:000013523 19263 19268 Pygo1 +T636 PR:000013524 19272 19277 Pygo2 +T637 NCBITaxon:10088 19281 19285 mice +T638 GO:0010467 19318 19328 expression +T639 GO:0060070 19476 19499 canonical Wnt signaling +T640 UBERON:0001851 19507 19515 cortical +T641 UBERON:0000056 19516 19524 ureteric +T642 UBERON:0001224 19538 19550 renal pelvis +T643 UBERON:0002113 19569 19575 kidney +T644 UBERON:0001851 19591 19599 Cortical +T645 CHEBI:75055 19600 19605 X-gal +T646 UBERON:0000056 19631 19639 ureteric +T647 PR:000013523 19654 19659 Pygo1 +T648 PR:000013524 19663 19668 Pygo2 +T649 UBERON:0002113 19672 19678 kidney +T650 UBERON:0001225 19731 19740;19761 19767 cortex of ... kidney +T651 PR:000013523 19743 19748 Pygo1 +T652 PR:000013524 19752 19757 Pygo2 +T653 CHEBI:75055 19829 19834 X-gal +T654 UBERON:0001232 19857 19873;19891 19893;19909 19915 collecting ducts ... of ... kidney +T655 UBERON:0001224 19878 19890 renal pelvis +T656 PR:000013524 19898 19903 Pygo2 +T657 PR:000013523 19990 19995 Pygo1 +T658 PR:000013524 19999 20004 Pygo2 +T659 PR:000013523 20027 20032 Pygo1 +T660 PR:000013524 20036 20041 Pygo2 +T661 PR:000013523 20070 20075 Pygo1 +T662 PR:000013524 20079 20084 Pygo2 +T663 UBERON:0002113 20113 20120 kidneys +T664 PR:000013524 20158 20163 Pygo2 +T665 SO:0000704 20164 20168 gene +T666 GO:0060070 20172 20195 canonical Wnt signaling +T667 UBERON:0000056 20203 20211 ureteric +T668 GO:0010467 20294 20304 expression +T669 PR:000013524 20320 20325 Pygo2 +T670 PR:000013523 20335 20340 Pygo1 +T671 PR:000013524 20341 20346 Pygo2 +T672 SO:0000902 20389 20398 transgene +T673 UBERON:0002113 20440 20447 kidneys +T674 PR:000013524 20472 20477 Pygo2 +T675 SO:0000704 20478 20482 gene +T676 PR:000013523 20484 20489 Pygo1 +T677 PR:000013524 20493 20498 Pygo2 +T678 PR:000013523 20506 20511 Pygo1 +T679 PR:000013524 20515 20520 Pygo2 +T680 GO:0010467 20568 20578 expression +T681 PR:000013523 20588 20593 Pygo1 +T682 PR:000013523 20601 20606 Pygo1 +T683 PR:000013524 20610 20615 Pygo2 +T684 PR:000013523 20688 20693 Pygo1 +T685 PR:000013524 20697 20702 Pygo2 +T686 GO:0010467 20743 20753 expression +T687 PR:000013523 20790 20795 Pygo1 +T688 GO:0060070 20799 20822 canonical Wnt signaling +T689 GO:0010467 20841 20851 expression +T690 PR:000013523 20879 20884 Pygo1 +T691 PR:000013524 20888 20893 Pygo2 +T692 UBERON:0002113 20897 20903 kidney +T693 UBERON:0002113 20926 20933 kidneys +T694 UBERON:0006364 20973 20986 ureteric tips +T695 UBERON:0006364 21013 21025 ureteric tip +T696 PR:000013523 21056 21061 Pygo1 +T697 PR:000013524 21065 21070 Pygo2 +T698 UBERON:0002113 21074 21081 kidneys +T699 PR:000013523 21180 21185 Pygo1 +T700 PR:000013524 21190 21195 Pygo2 +T701 SO:0000704 21196 21201 genes +T702 GO:0060070 21205 21228 canonical Wnt signaling +T703 UBERON:0000056 21255 21263 ureteric +T704 UBERON:0002113 21276 21282 kidney +T705 GO:0010467 21337 21347 expression +T706 PR:000013523 21351 21356 Pygo1 +T707 PR:000013524 21357 21362 Pygo2 +T708 UBERON:0002113 21369 21375 kidney +T709 SO:0000902 21386 21395 Transgene +T710 PR:000013523 21458 21463 Pygo1 +T711 PR:000013524 21467 21472 Pygo2 +T712 PR:000013523 21477 21482 Pygo1 +T713 PR:000013524 21486 21491 Pygo2 +T714 PR:000013523 21499 21504 Pygo1 +T715 PR:000013524 21508 21513 Pygo2 +T716 UBERON:0002113 21517 21524 kidneys +T717 GO:0010467 21562 21572 expression +T718 PR:000013524 21581 21586 Pygo2 +T719 PR:000013523 21602 21607 Pygo1 +T720 GO:0010467 21646 21656 expression +T721 PR:000013524 21686 21691 Pygo2 +T722 GO:0010467 21719 21729 expression +T723 PR:000013523 21775 21780 Pygo1 +T724 SO:0001023 21781 21788 alleles +T725 NCBITaxon:10088 21898 21902 mice +T726 GO:0010467 21934 21944 expression +T727 UBERON:0003220 21963 21985 metanephric mesenchyme +T728 UBERON:0003220 21990 22012 metanephric mesenchyme +T729 UBERON:0004209 22041 22055 renal vesicles +T730 UBERON:0009773 22041 22046;22074 22081 renal ... tubules +T731 UBERON:0000074 22041 22046;22087 22096 renal ... glomeruli +T732 UBERON:0004199 22057 22072 S-shaped bodies +T733 GO:0060070 22180 22203 canonical Wnt signaling +T734 UBERON:0003220 22211 22233 metanephric mesenchyme +T735 GO:0060070 22303 22326 canonical Wnt signaling +T736 UBERON:0003220 22334 22356 metanephric mesenchyme +T737 UBERON:0000084 22371 22383 ureteric bud +T738 GO:0010467 22384 22394 expression +T739 PR:000017435 22398 22402 Wnt1 +T740 GO:0060070 22427 22450 canonical Wnt signaling +T741 PR:000017453 22463 22468 Wnt9b +T742 GO:0001822 22488 22501 nephrogenesis +T743 UBERON:0003220 22509 22531 metanephric mesenchyme +T744 SO:0000902 22568 22577 transgene +T745 GO:0060070 22606 22629 canonical Wnt signaling +T746 UBERON:0003220 22637 22659 metanephric mesenchyme +T747 UBERON:0003220 22706 22728 metanephric mesenchyme +T748 CHEBI:48607 22732 22748 lithium chloride +T749 CHEBI:48607 22750 22754 LiCl +T750 GO:0060070 22773 22796 canonical Wnt signaling +T751 GO:0001822 22861 22874 nephrogenesis +T752 UBERON:0002113 22878 22884 kidney +T753 UBERON:0000062 22885 22890 organ +T754 CHEBI:48607 22922 22926 LiCl +T755 UBERON:0003220 22935 22957 metanephric mesenchyme +T756 GO:0001822 22970 22983 nephrogenesis +T757 GO:0010467 23025 23035 expression +T758 SO:0000902 23075 23084 transgene +T759 GO:0060070 23116 23139 canonical Wnt signaling +T760 UBERON:0003220 23147 23169 metanephric mesenchyme +T761 UBERON:0002113 23188 23194 kidney +T762 PR:000013523 23220 23225 Pygo1 +T763 PR:000013524 23226 23231 Pygo2 +T764 UBERON:0002113 23237 23244 kidneys +T765 SO:0000704 23289 23293 gene +T766 GO:0010467 23289 23304 gene expression +T767 PR:000013523 23312 23317 Pygo1 +T768 PR:000013524 23318 23323 Pygo2 +T769 UBERON:0002113 23331 23338 kidneys +T770 SO:0000704 23392 23396 gene +T771 GO:0010467 23392 23407 gene expression +T772 SO:0000902 23437 23446 transgene +T773 GO:0065007 23456 23464 monitors +T774 SO:0000167 23485 23493 promoter +T775 GO:0016055 23497 23510 Wnt signaling +T776 GO:0010467 23546 23556 expression +T777 SO:0000704 23572 23577 genes +T778 PR:000013523 23632 23637 Pygo1 +T779 PR:000013524 23641 23646 Pygo2 +T780 UBERON:0002113 23650 23657 kidneys +T781 SO:0000704 23711 23716 genes +T782 PR:000013523 23889 23894 Pygo1 +T783 PR:000013524 23899 23904 Pygo2 +T784 UBERON:0002113 23994 24001 kidneys +T785 SO:0000704 24009 24014 genes +T786 UBERON:0002113 24052 24059 kidneys +T787 PR:000006067 24069 24075 Cxcl13 +T788 PR:000015169 24090 24096 Slc5a2 +T789 PR:000015966 24115 24122 Slco1a4 +T790 PR:000015169 24137 24143 Slc5a2 +T791 GO:0010467 24147 24156 expressed +T792 UBERON:0004134 24164 24180 proximal tubules +T793 UBERON:0007023 24188 24193 adult +T794 UBERON:0001285 24194 24201 nephron +T795 GO:0030849 24229 24238 autosomal +T796 UBERON:0002113 24249 24254 renal +T797 CHEBI:17234 24292 24299 glucose +T798 GO:0046323 24292 24306 glucose uptake +T799 UBERON:0001285 24314 24321 nephron +T800 CHEBI:25696 24332 24345 organic anion +T801 PR:000015966 24359 24366 Slco1a4 +T802 GO:0010467 24385 24394 expressed +T803 UBERON:0009773 24402 24412;24423 24429 tubules of ... kidney +T804 UBERON:0007023 24417 24422 adult +T805 PR:000013523 24467 24472 Pygo1 +T806 PR:000013524 24473 24478 Pygo2 +T807 SO:0000704 24479 24484 genes +T808 UBERON:0001285 24506 24513 nephron +T809 SO:0000704 24560 24565 Genes +T810 GO:0010467 24581 24590 expressed +T811 PR:000013523 24637 24642 Pygo1 +T812 PR:000013524 24643 24648 Pygo2 +T813 UBERON:0002113 24660 24666 kidney +T814 SO:0000704 24712 24717 genes +T815 PR:000013523 24737 24742 Pygo1 +T816 PR:000013524 24743 24748 Pygo2 +T817 UBERON:0002113 24754 24760 kidney +T818 PR:000009415 24770 24774 Klk5 +T819 PR:000009416 24787 24791 Klk6 +T820 PR:000014395 24804 24808 Ren2 +T821 PR:000016340 24825 24833 Timeless +T822 PR:000009415 24846 24850 Klk5 +T823 PR:000009416 24855 24859 Klk6 +T824 PR:000027795 24900 24907 trypsin +T825 http://purl.obolibrary.org/obo/MONDO_0004992 24969 24975 cancer +T826 UBERON:0000479 24977 24983 tissue +T827 GO:0048771 24977 24994 tissue remodeling +T828 GO:0008217 25000 25028 regulation of blood pressure +T829 UBERON:0000178 25014 25019 blood +T830 PR:000014395 25035 25039 Ren2 +T831 SO:0000853 25043 25050 homolog +T832 PR:000014394 25072 25079 Renin 1 +T833 PR:000013883 25095 25100 renin +T834 UBERON:0000178 25132 25137 blood +T835 PR:000013883 25171 25176 renin +T836 http://purl.obolibrary.org/obo/MONDO_0000839 25226 25250 congenital abnormalities +T837 UBERON:0000056 25258 25264 ureter +T838 UBERON:0004100 25269 25291 collecting-duct system +T839 PR:000016340 25298 25306 Timeless +T840 GO:0042752 25347 25378 regulation of circadian rhythms +T841 GO:0010467 25384 25393 expressed +T842 UBERON:0006364 25411 25424 ureteric tips +T843 GO:0065007 25449 25457 regulate +T844 UBERON:0000056 25458 25466 ureteric +T845 GO:0001763 25467 25490 branching morphogenesis +T846 GO:0010467 25541 25551 expression +T847 PR:000013523 25591 25596 Pygo1 +T848 PR:000013524 25600 25605 Pygo2 +T849 UBERON:0002113 25616 25623 kidneys +T850 PR:000013523 25678 25683 Pygo1 +T851 PR:000013524 25688 25693 Pygo2 +T852 SO:0000704 25809 25814 genes +T853 GO:0010467 25846 25856 expression +T854 UBERON:0002113 25886 25893 kidneys +T855 GO:0010467 25915 25925 expression +T856 PR:000005121 25929 25934 Ccnd1 +T857 PR:000005121 25944 25953 cyclin D1 +T858 PR:000017426 25964 25969 Wisp1 +T859 GO:0010467 26008 26018 expression +T860 PR:000013523 26049 26054 Pygo1 +T861 PR:000013524 26055 26060 Pygo2 +T862 UBERON:0002113 26066 26073 kidneys +T863 GO:0010467 26132 26142 expression +T864 GO:0016055 26163 26176 Wnt signaling +T865 SO:0000704 26184 26189 genes +T866 PR:000013523 26197 26202 Pygo1 +T867 PR:000013524 26203 26208 Pygo2 +T868 UBERON:0002113 26216 26223 kidneys +T869 SO:0000704 26236 26240 Gene +T870 GO:0010467 26236 26251 Gene expression +T871 GO:0016055 26270 26283 Wnt signaling +T872 PR:000013523 26305 26310 Pygo1 +T873 PR:000013524 26311 26316 Pygo2 +T874 UBERON:0002113 26322 26328 kidney +T875 PR:000013523 26395 26400 Pygo1 +T876 UBERON:0002113 26423 26430 kidneys +T877 SO:0000704 26507 26511 gene +T878 SO:0000704 26581 26586 genes +T879 GO:0010467 26596 26606 expression +T880 GO:0010467 26643 26653 expression +T881 PR:000013523 26698 26703 Pygo1 +T882 PR:000013524 26708 26713 Pygo2 +T883 GO:0010467 26773 26783 expression +T884 SO:0000704 26907 26912 genes +T885 SO:0000704 27092 27097 genes +T886 SO:0000704 27227 27231 gene +T887 GO:0010467 27227 27242 gene expression +T888 PR:000013523 27264 27269 Pygo1 +T889 PR:000013524 27270 27275 Pygo2 +T890 UBERON:0002113 27281 27288 kidneys +T891 UBERON:0002113 27319 27326 kidneys +T892 NCBITaxon:7215 27344 27354 Drosophila +T893 PR:Q9V9W8 27360 27367 Pygopus +T894 SO:0000704 27368 27372 gene +T895 GO:0060070 27394 27417 canonical Wnt signaling +T896 GO:0016055 27457 27470 Wnt signaling +T897 UBERON:0005895 27525 27528 leg +T898 UBERON:0000023 27530 27534 wing +T899 UBERON:0000970 27540 27543 eye +T900 UBERON:0000939 27544 27558 imaginal discs +T901 GO:0016055 27608 27621 Wnt signaling +T902 NCBITaxon:7215 27679 27689 Drosophila +T903 UBERON:0001002 27731 27738 cuticle +T904 UBERON:0001045 27751 27757 midgut +T905 UBERON:0001017 27772 27794 central nervous system +T906 GO:0007417 27772 27794;27808 27819 central nervous system ... development +T907 UBERON:0000948 27800 27807 cardiac +T908 GO:0007507 27800 27819 cardiac development +T909 PR:000017435 27879 27881 Wg +T910 NCBITaxon:7215 27941 27951 Drosophila +T911 NCBITaxon:10088 27987 27991 mice +T912 PR:000013523 28006 28011 Pygo1 +T913 PR:000013524 28013 28018 Pygo2 +T914 PR:000013523 28030 28035 Pygo1 +T915 PR:000013524 28036 28041 Pygo2 +T916 PR:000013524 28067 28072 Pygo2 +T917 GO:0007567 28094 28099 birth +T918 PR:000013523 28140 28145 Pygo1 +T919 PR:000013523 28266 28271 Pygo1 +T920 PR:000013524 28272 28277 Pygo2 +T921 UBERON:0000479 28306 28313 tissues +T922 GO:0010467 28375 28385 expression +T923 SO:0000704 28460 28464 gene +T924 PR:000013523 28507 28512 Pygo1 +T925 PR:000013524 28517 28522 Pygo2 +T926 SO:0000704 28523 28528 genes +T927 PR:Q9V9W8 28534 28541 Pygopus +T928 SO:0001023 28551 28558 alleles +T929 NCBITaxon:7215 28636 28646 Drosophila +T930 SO:0000417 28679 28685 domain +T931 PR:Q9V9W8 28714 28721 Pygopus +T932 PR:000017435 28734 28736 Wg +T933 SO:0000417 28756 28762 domain +T934 CHEBI:27365 28844 28853 zinc ions +T935 SO:0000417 28859 28866 domains +T936 GO:0000785 28959 28968 chromatin +T937 GO:0006338 28959 28979 chromatin remodeling +T938 NCBITaxon:7215 29021 29031 Drosophila +T939 SO:0001023 29045 29051 allele +T940 SO:0000417 29122 29128 domain +T941 GO:0016055 29162 29175 Wnt signaling +T942 GO:0009790 29193 29206 embryogenesis +T943 UBERON:0000939 29211 29224 imaginal disc +T944 GO:0007444 29211 29236 imaginal disc development +T945 PR:000013523 29246 29251 Pygo1 +T946 PR:000013524 29252 29257 Pygo2 +T947 SO:0001023 29265 29272 alleles +T948 SO:0000417 29329 29336 domains +T949 PR:000013523 29386 29391 Pygo1 +T950 SO:0000704 29392 29396 gene +T951 PR:000013524 29474 29479 Pygo2 +T952 SO:0000704 29480 29484 gene +T953 SO:0001023 29660 29667 alleles +T954 UBERON:0002113 29712 29718 kidney +T955 GO:0016055 29729 29742 Wnt signaling +T956 GO:0001822 29808 29821 nephrogenesis +T957 PR:000017453 29823 29828 Wnt9b +T958 UBERON:0000084 29844 29856 ureteric bud +T959 UBERON:0003220 29873 29895 metanephric mesenchyme +T960 GO:0001822 29907 29920 nephrogenesis +T961 PR:000017453 29940 29945 Wnt9b +T962 PR:000017444 29949 29953 Wnt4 +T963 UBERON:0003220 29980 30002 metanephric mesenchyme +T964 GO:0001822 30029 30042 nephrogenesis +T965 PR:000017438 30061 30066 Wnt11 +T966 UBERON:0006364 30086 30103 ureteric bud tips +T967 PR:000007928 30116 30120 GDNF +T968 GO:0010467 30121 30131 expression +T969 UBERON:0003220 30139 30161 metanephric mesenchyme +T970 UBERON:0002113 30219 30225 kidney +T971 GO:0001822 30219 30237 kidney development +T972 SO:0000902 30251 30260 transgene +T973 GO:0060070 30296 30319 canonical Wnt signaling +T974 UBERON:0000084 30327 30339 ureteric bud +T975 UBERON:0002113 30378 30384 kidney +T976 PR:000013524 30402 30407 Pygo2 +T977 GO:0016055 30474 30487 Wnt signaling +T978 UBERON:0006364 30538 30550 ureteric tip +T979 UBERON:0002113 30568 30574 kidney +T980 UBERON:0000056 30610 30618 ureteric +T981 GO:0060070 30658 30681 canonical Wnt signaling +T982 GO:0001658 30685 30711;30716 30728 branching morphogenesis of ... ureteric bud +T983 UBERON:0000084 30716 30728 ureteric bud +T984 GO:0016055 30774 30787 Wnt signaling +T985 UBERON:0000084 30795 30807 ureteric bud +T986 GO:0016055 30910 30923 Wnt signaling +T987 UBERON:0003220 30931 30953 metanephric mesenchyme +T988 UBERON:0003104 30969 30979 mesenchyme +T989 UBERON:0000084 30983 30995 ureteric bud +T990 PR:000013523 31012 31017 Pygo1 +T991 PR:000013524 31018 31023 Pygo2 +T992 GO:0001658 31052 31078;31083 31095 branching morphogenesis of ... ureteric bud +T993 UBERON:0000084 31083 31095 ureteric bud +T994 PR:000017438 31152 31157 Wnt11 +T995 PR:000017438 31234 31239 Wnt11 +T996 PR:Q9V9W8 31356 31363 Pygopus +T997 SO:0000704 31364 31369 genes +T998 GO:0060070 31378 31401 canonical Wnt signaling +T999 PR:000017438 31416 31421 Wnt11 +T1000 GO:0016055 31470 31483 Wnt signaling +T1001 UBERON:0000084 31493 31505 ureteric bud +T1002 UBERON:0003104 31513 31523 mesenchyme +T1003 PR:000007928 31528 31532 GDNF +T1004 GO:0035860 31528 31542 GDNF signaling +T1005 UBERON:0003104 31552 31562 mesenchyme +T1006 PR:000013523 31590 31595 Pygo1 +T1007 PR:000013524 31596 31601 Pygo2 +T1008 GO:0016055 31633 31646 Wnt signaling +T1009 UBERON:0000084 31654 31666 ureteric bud +T1010 PR:000013523 31673 31678 Pygo1 +T1011 UBERON:0003104 31739 31749 mesenchyme +T1012 UBERON:0000084 31764 31776 ureteric bud +T1013 UBERON:0003220 31803 31825 metanephric mesenchyme +T1014 UBERON:0001285 31833 31841 nephrons +T1015 PR:000017453 31890 31895 Wnt9b +T1016 UBERON:0000084 31915 31927 ureteric bud +T1017 UBERON:0003220 31935 31957 metanephric mesenchyme +T1018 GO:0001822 31983 31996 nephrogenesis +T1019 GO:0060070 32001 32024 canonical Wnt signaling +T1020 PR:Q9V9W8 32120 32127 Pygopus +T1021 NCBITaxon:7215 32145 32155 Drosophila +T1022 NCBITaxon:40674 32160 32167 mammals +T1023 NCBITaxon:7215 32172 32182 Drosophila +T1024 PR:Q9V9W8 32188 32195 Pygopus +T1025 SO:0000704 32196 32200 gene +T1026 GO:0060070 32223 32246 canonical Wnt signaling +T1027 NCBITaxon:40674 32257 32264 mammals +T1028 PR:000013523 32269 32274 Pygo1 +T1029 PR:000013524 32275 32280 Pygo2 +T1030 SO:0000704 32281 32286 genes +T1031 GO:0060070 32320 32343 canonical Wnt signaling +T1032 SO:0000902 32357 32366 transgene +T1033 GO:0060070 32379 32402 canonical Wnt signaling +T1034 PR:000013523 32453 32458 Pygo1 +T1035 PR:000013524 32459 32464 Pygo2 +T1036 UBERON:0000922 32472 32479 embryos +T1037 UBERON:0000479 32486 32492 tissue +T1038 UBERON:0002113 32553 32560 kidneys +T1039 PR:000013523 32623 32628 Pygo1 +T1040 PR:000013524 32629 32634 Pygo2 +T1041 GO:0048513 32652 32665 organogenesis +T1042 NCBITaxon:40674 32790 32799 mammalian +T1043 PR:Q9V9W8 32800 32807 Pygopus +T1044 SO:0000704 32808 32813 genes +T1045 GO:0060070 32843 32866 canonical Wnt signaling +T1046 SO:0000902 32985 32994 transgene +T1047 GO:0065007 33004 33012 monitors +T1048 GO:0010467 33017 33027 expression +T1049 SO:0000167 33061 33069 promoter +T1050 SO:0000167 33132 33141 promoters +T1051 SO:0000704 33149 33154 genes +T1052 UBERON:0002113 33182 33189 kidneys +T1053 SO:0000704 33197 33201 gene +T1054 GO:0010467 33197 33212 gene-expression +T1055 SO:0000704 33262 33267 genes +T1056 GO:0010467 33287 33297 expression +T1057 PR:Q9V9W8 33445 33452 Pygopus +T1058 SO:0000704 33453 33458 genes +T1059 NCBITaxon:40674 33462 33469 mammals +T1060 GO:0060070 33487 33510 canonical Wnt signaling +T1061 NCBITaxon:7215 33544 33554 Drosophila +T1062 SO:0000704 33560 33565 genes +T1063 GO:0010467 33571 33581 expression +T1064 UBERON:0002113 33650 33656 kidney +T1065 GO:0001822 33650 33668 kidney development +T1066 SO:0000704 33693 33698 genes +T1067 GO:0010467 33726 33736 expression +T1068 UBERON:0002113 33759 33765 kidney +T1069 GO:0060070 33798 33821 canonical Wnt signaling +T1070 NCBITaxon:40674 33855 33864 mammalian +T1071 PR:000013524 33865 33870 Pygo2 +T1072 SO:0000704 33871 33875 gene +T1073 GO:0001658 33899 33925;33930 33942 branching morphogenesis of ... ureteric bud +T1074 UBERON:0000084 33930 33942 ureteric bud +T1075 PR:000013524 34023 34028 Pygo2 +T1076 UBERON:0003220 34075 34097 metanephric mesenchyme +T1077 UBERON:0000084 34112 34125 ureteric buds +T1078 UBERON:0001285 34141 34148 nephron +T1079 GO:0072006 34141 34158 nephron formation +T1080 PR:000013523 34198 34203 Pygo1 +T1081 PR:000013524 34204 34209 Pygo2 +T1082 PR:Q9V9W8 34291 34298 Pygopus +T1083 SO:0000704 34299 34303 gene +T1084 GO:0060070 34307 34330 canonical Wnt signaling +T1085 NCBITaxon:7215 34334 34344 Drosophila +T1086 GO:0060070 34368 34391 canonical Wnt signaling +T1087 GO:0001822 34395 34408 nephrogenesis +T1088 NCBITaxon:40674 34437 34446 mammalian +T1089 PR:Q9V9W8 34447 34454 Pygopus +T1090 SO:0000704 34455 34460 genes +T1091 UBERON:0000467 34485 34492 systems +T1092 GO:0016055 34527 34540 Wnt signaling +T1093 SO:0000902 34604 34613 transgene +T1094 GO:0010467 34623 34633 expression +T1095 PR:000013523 34641 34646 Pygo1 +T1096 PR:000013524 34647 34652 Pygo2 +T1097 NCBITaxon:10088 34662 34666 mice +T1098 GO:0060070 34744 34767 canonical Wnt signaling +T1099 NCBITaxon:40674 34772 34779 mammals +T1100 PR:Q9V9W8 34816 34823 Pygopus +T1101 NCBITaxon:7215 34857 34867 Drosophila +T1102 PR:000013523 34901 34905 Pyg1 +T1103 SO:0000704 34908 34913 genes +T1104 GO:0016055 34917 34930 Wnt signaling +T1105 NCBITaxon:40674 34970 34979 mammalian +T1106 GO:0048513 34980 34993 organogenesis +T1107 PR:Q9V9W8 35013 35020 Pygopus +T1108 NCBITaxon:40674 35074 35081 mammals +T1109 SO:0000704 35089 35094 genes +T1110 PR:Q9V9W8 35143 35150 Pygopus +T1111 SO:0000704 35151 35156 genes +T1112 PR:000002198 35162 35171 β-catenin +T1113 GO:0005667 35172 35200 transcription-factor complex +T1114 GO:0005634 35279 35286 nuclear +T1115 PR:000013523 35345 35350 Pygo1 +T1116 PR:000013524 35351 35356 Pygo2 +T1117 NCBITaxon:40674 35360 35367 mammals +T1118 PR:Q9V9W8 35403 35410 Pygopus +T1119 SO:0000704 35421 35426 genes +T1120 PR:000013523 35475 35480 Pygo1 +T1121 PR:000013524 35485 35490 Pygo2 +T1122 PR:000013523 35492 35497 Pygo1 +T1123 PR:000013524 35502 35507 Pygo2 +T1124 SO:0000704 35508 35513 genes +T1125 SO:0000357 35536 35544 flanking +T1126 SO:0001215 35558 35575;35586 35590 coding regions of ... exon +T1127 SO:0000346 35621 35635 LoxP sequences +T1128 PR:P03870 35678 35681 Flp +T1129 SO:0000440 35686 35692 vector +T1130 CHEBI:7507 35736 35744 neomycin +T1131 SO:0000704 35756 35760 gene +T1132 SO:0000357 35761 35768 flanked +T1133 SO:0000350 35772 35785 FRT sequences +T1134 SO:0000061 35804 35821 restriction sites +T1135 SO:0000357 35921 35928 flanked +T1136 SO:0000346 35932 35946 LoxP sequences +T1137 SO:0001026 35978 35985 genomic +T1138 CL:0002322 36044 36051 ES cell +T1139 PR:000013523 36127 36132 Pygo1 +T1140 SO:0001026 36138 36145 genomic +T1141 SO:0001030 36178 36185 forward +T1142 SO:0001031 36216 36220 back +T1143 SO:0000147 36245 36249 exon +T1144 SO:0001030 36250 36257 forward +T1145 SO:0000147 36281 36285 exon +T1146 SO:0001031 36286 36290 back +T1147 SO:0001030 36319 36326 forward +T1148 SO:0001031 36355 36359 back +T1149 PR:000013524 36387 36392 Pygo2 +T1150 SO:0001026 36398 36405 genomic +T1151 SO:0000147 36544 36548 exon +T1152 CL:0002322 36657 36665 ES cells +T1153 CL:0002322 36690 36698 ES cells +T1154 UBERON:0000358 36716 36726 blastocyst +T1155 GO:0007618 36795 36800 mated +T1156 NCBITaxon:10088 36818 36822 mice +T1157 SO:0001023 36913 36920 alleles +T1158 PR:000013523 36929 36934 Pygo1 +T1159 PR:000013524 36939 36944 Pygo2 +T1160 GO:0007618 36963 36969 mating +T1161 SO:0000359 36983 36989 floxed +T1162 NCBITaxon:10088 36990 36994 mice +T1163 NCBITaxon:10358 37004 37007 CMV +T1164 NCBITaxon:10088 37012 37016 mice +T1165 SO:0000112 37044 37051 primers +T1166 PR:000013524 37082 37087 Pygo2 +T1167 SO:0001023 37093 37099 allele +T1168 SO:0001030 37101 37108 forward +T1169 SO:0001030 37110 37111 F +T1170 SO:0001031 37138 37145 reverse +T1171 SO:0001031 37147 37148 R +T1172 PR:000013524 37174 37179 Pygo2 +T1173 SO:0000359 37186 37192 floxed +T1174 SO:0001023 37193 37199 allele +T1175 SO:0001030 37201 37202 F +T1176 SO:0001031 37228 37229 R +T1177 PR:000013523 37256 37261 Pygo1 +T1178 SO:0001023 37267 37273 allele +T1179 SO:0001030 37275 37276 F +T1180 SO:0001031 37304 37305 R +T1181 PR:000013523 37332 37337 Pygo1 +T1182 SO:0000359 37344 37350 floxed +T1183 SO:0001023 37351 37357 allele +T1184 SO:0001030 37359 37360 F +T1185 SO:0001031 37389 37390 R +T1186 UBERON:0010148 37441 37454 vaginal plugs +T1187 UBERON:0002113 37512 37519 Kidneys +T1188 CHEBI:17790 37576 37584 methanol +T1189 CHEBI:53424 37617 37625 Tween-20 +T1190 CHEBI:53424 37631 37632 T +T1191 GO:0042571 37670 37680 antibodies +T1192 CHEBI:53424 37686 37687 T +T1193 UBERON:0000479 37720 37727 tissues +T1194 CHEBI:31624 37733 37744 fluoroscein +T1195 MOP:0000779 37745 37755 conjugated +T1196 NCBITaxon:271171 37756 37773 Dilichos biflorus +T1197 NCBITaxon:9925 37834 37838 goat +T1198 UBERON:0001977 37839 37844 serum +T1199 GO:0042571 37879 37889 antibodies +T1200 GO:0042571 37903 37913 antibodies +T1201 PR:000017459 37924 37927 WT1 +T1202 PR:000001447 37988 37998 uvomorulin +T1203 PR:000001447 38000 38010 e-cadherin +T1204 PR:000001447 38012 38016 Cdh1 +T1205 PR:000005505 38062 38068 Cited1 +T1206 GO:0042571 38138 38148 antibodies +T1207 CHEBI:52673 38154 38163 Alexa 555 +T1208 MOP:0000779 38164 38174 conjugated +T1209 NCBITaxon:9986 38180 38186 rabbit +T1210 MOP:0000779 38201 38211 conjugated +T1211 NCBITaxon:10114 38217 38220 rat +T1212 GO:0042571 38221 38231 antibodies +T1213 UBERON:0000479 38274 38281 tissues +T1214 UBERON:0002113 38577 38583 kidney +T1215 UBERON:0002113 38681 38688 kidneys +T1216 UBERON:0000922 38697 38703 embryo +T1217 UBERON:0006364 38705 38722 Ureteric bud tips +T1218 GO:0097617 38820 38833 hybridization +T1219 GO:0097617 38855 38868 hybridization +T1220 PR:000017438 38927 38932 Wnt11 +T1221 PR:000017449 38937 38942 Wnt7b +T1222 PR:000017453 38979 38984 Wnt9b +T1223 PR:000013523 39028 39033 Pygo1 +T1224 PR:000013524 39038 39043 Pygo2 +T1225 GO:0042571 39044 39052 antibody +T1226 NCBITaxon:9606 39082 39087 human +T1227 PR:000013524 39088 39093 Pygo2 +T1228 PR:Q9BRQ0 39100 39106 hPygo2 +T1229 NCBITaxon:10088 39117 39122 mouse +T1230 PR:000013523 39123 39128 Pygo1 +T1231 PR:Q9D0P5 39135 39141 mPygo1 +T1232 GO:0042571 39154 39164 antibodies +T1233 NCBITaxon:9606 39238 39243 human +T1234 PR:000013524 39244 39250 Pygo 2 +T1235 NCBITaxon:10088 39284 39289 mouse +T1236 PR:000013523 39290 39295 Pygo1 +T1237 PR:Q9BRQ0 39370 39377 hPygo 2 +T1238 PR:Q9D0P5 39382 39389 mPygo 1 +T1239 PR:Q9BRQ0 39433 39439 hPygo2 +T1240 PR:Q9D0P5 39444 39450 mPygo1 +T1241 PR:000022850 39452 39455 GST +T1242 PR:Q9BRQ0 39456 39462 hPygo2 +T1243 PR:000022850 39467 39470 GST +T1244 PR:Q9D0P5 39471 39477 mPygo1 +T1245 GO:0010467 39492 39501 expressed +T1246 NCBITaxon:2 39505 39513 bacteria +T1247 NCBITaxon:9986 39543 39550 rabbits +T1248 GO:0042571 39555 39563 antibody +T1249 NCBITaxon:9986 39626 39632 rabbit +T1250 UBERON:0001977 39637 39641 sera +T1251 PR:Q9D0P5 39650 39656 mPygo1 +T1252 PR:Q9BRQ0 39666 39672 hPygo2 +T1253 PR:000022850 39711 39714 GST +T1254 GO:0042571 39745 39755 antibodies +T1255 PR:000022850 39764 39767 GST +T1256 PR:Q9BRQ0 39778 39784 hPygo2 +T1257 PR:Q9D0P5 39794 39800 mPygo1 +T1258 UBERON:0001977 39805 39809 sera +T1259 PR:000022850 39839 39842 GST +T1260 PR:000022850 39886 39889 GST +T1261 PR:Q9BRQ0 39890 39896 hPygo2 +T1262 PR:000022850 39900 39903 GST +T1263 PR:Q9D0P5 39904 39910 mPygo1 +T1264 GO:0042571 39953 39962 antibodes +T1265 GO:0042571 40014 40022 antibody +T1266 GO:0042571 40048 40058 antibodies +T1267 PR:000013523 40091 40096 Pygo1 +T1268 UBERON:0000922 40124 40130 embryo +T1269 SO:0000902 40160 40169 transgene +T1270 GO:0060070 40188 40211 canonical Wnt signaling +T1271 CHEBI:75055 40213 40218 X-gal +T1272 UBERON:0000922 40236 40243 embryos +T1273 UBERON:0002113 40259 40266 kidneys +T1274 UBERON:0002113 40394 40400 kidney +T1275 CHEBI:75055 40425 40430 X-gal +T1276 CHEBI:75958 40440 40448 solution +T1277 SO:0000902 40468 40477 transgene +T1278 GO:0010467 40484 40494 expression +T1279 PR:000013523 40718 40723 Pygo1 +T1280 PR:000013524 40729 40734 Pygo2 +T1281 PR:000013523 40747 40752 Pygo1 +T1282 PR:000013524 40758 40763 Pygo2 +T1283 PR:000013523 40780 40785 Pygo1 +T1284 PR:000013524 40791 40796 Pygo2 +T1285 UBERON:0002113 40872 40879 kidneys +T1286 PR:000013523 40906 40911 Pygo1 +T1287 PR:000013524 40912 40917 Pygo2 +T1288 UBERON:0000922 40932 40939 embryos +T1289 GO:0097617 41192 41202 hybridized +T1290 NCBITaxon:10088 41227 41232 Mouse +T1291 GO:0010467 41235 41245 expression +T1292 SO:0000704 41314 41319 genes +T1293 GO:0010467 41324 41333 expressed +T1294 SO:0000345 41324 41347 expressed sequence tags +T1295 PR:000013523 41401 41406 Pygo1 +T1296 PR:000013524 41407 41412 Pygo2 +T1297 UBERON:0002113 41439 41446 kidneys +T1298 SO:0000704 41669 41674 Genes +T1299 SO:0000704 41826 41831 genes +T1300 PR:000013523 41926 41931 Pygo1 +T1301 PR:000013524 41932 41937 Pygo2 +T1302 UBERON:0002113 41955 41962 kidneys +T1303 UBERON:0002113 42016 42023 kidneys +T1304 SO:0000112 42302 42309 primers +T1305 PR:000003676 42383 42387 Actb +T1306 PR:000004564 42434 42441 Aldh1a7 +T1307 PR:000005720 42491 42497 Col8a1 +T1308 PR:000005958 42544 42549 Csrp1 +T1309 PR:000009415 42596 42600 Klk5 +T1310 PR:000029674 42647 42653 Picalm +T1311 PR:000013524 42699 42704 Pygo2 +T1312 PR:000014395 42751 42755 Ren2 +T1313 CHEBI:51461 42958 42968 SYBR Green +T1314 CHEBI:2511 43069 43076 agarose +T1315 SO:0000112 43098 43104 primer +T1316 PR:000003676 43220 43224 Actb +T1317 SO:0000704 43368 43373 genes +T1318 GO:0097617 43515 43529 hybridizations +T1319 GO:0042571 43595 43605 antibodies +T1320 UBERON:0000922 43833 43846 embryological +T1321 PR:000017453 43934 43939 Wnt9b diff --git a/src/ontogpt/evaluation/craft/database/all/17425782.txt b/src/ontogpt/evaluation/craft/database/all/17425782.txt new file mode 100644 index 000000000..6e5e58930 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17425782.txt @@ -0,0 +1,203 @@ +Pygo1 and Pygo2 roles in Wnt signaling in mammalian kidney development + +Abstract + +Background + +The pygopus gene of Drosophila encodes an essential component of the Armadillo (β-catenin) transcription factor complex of canonical Wnt signaling. To better understand the functions of Pygopus-mediated canonical Wnt signaling in kidney development, targeted mutations were made in the two mammalian orthologs, Pygo1 and Pygo2. + +Results + +Each mutation deleted >80% of the coding sequence, including the critical PHD domain, and almost certainly resulted in null function. Pygo2 homozygous mutants, with rare exception, died shortly after birth, with a phenotype including lens agenesis, growth retardation, altered kidney development, and in some cases exencephaly and cleft palate. Pygo1 homozygous mutants, however, were viable and fertile, with no detectable developmental defects. Double Pygo1/Pygo2 homozygous mutants showed no apparent synergy in phenotype severity. The BAT-gal transgene reporter of canonical Wnt signaling showed reduced levels of expression in Pygo1-/-/Pygo2-/- mutants, with tissue-specific variation in degree of diminution. The Pygo1 and Pygo2 genes both showed widespread expression in the developing kidney, with raised levels in the stromal cell compartment. Confocal analysis of the double mutant kidneys showed disturbance of both the ureteric bud and metanephric mesenchyme-derived compartments. Branching morphogenesis of the ureteric bud was altered, with expanded tips and reduced tip density, probably contributing to the smaller size of the mutant kidney. In addition, there was an expansion of the zone of condensed mesenchyme capping the ureteric bud. Nephron formation, however, proceeded normally. Microarray analysis showed changed expression of several genes, including Cxcl13, Slc5a2, Klk5, Ren2 and Timeless, which represent candidate Wnt targets in kidney development. + +Conclusion + +The mammalian Pygopus genes are required for normal branching morphogenesis of the ureteric bud during kidney development. Nevertheless, the relatively mild phenotype observed in the kidney, as well as other organ systems, indicates a striking evolutionary divergence of Pygopus function between mammals and Drosophila. In mammals, the Pygo1/Pygo2 genes are not absolutely required for canonical Wnt signaling in most developing systems, but rather function as quantitative transducers, or modulators, of Wnt signal intensity. + +Background + +Wnt signaling is of critical importance in several stages of kidney development. Mutual inductive interactions between the ureteric bud and metanephric mesenchyme drive nephrogenesis [1]. The ureteric bud synthesizes Wnt9b, which is essential for induction of the mesenchyme to form nephrons [2]. Wnt4 is made by the induced metanephric mesenchyme and is also required for nephrogenesis [3]. Furthermore, Wnt11, secreted by the ureteric bud tips, participates in a positive feedback loop promoting glial cell line-derived neurotrophic factor (GDNF) expression by the metanephric mesenchyme [4]. Mutations in Wnt9b or Wnt4 result in a dramatic block in nephron formation, while Wnt11 mutants show a significant reduction in nephron number. It is interesting to note that Wnt4 and Wnt11 have been shown to signal, at least in some cases, through noncanonical pathways [5-7], while there is evidence indicating that Wnt9b activates canonical Wnt signaling in the kidney [2]. + +Genetic studies in Drosophila have identified the Pygopus (Pygo) gene as a critical component of canonical Wnt signaling [8-11]. Pygo and Lgs interact with β-catenin during the formation of the canonical transcriptional complex and are required for accumulation of β-catenin in the nucleus [12]. Lgs binds the central armadillo repeats of β-catenin, while Pygo interacts with Lgs, mediating activation of Wnt targets [9,13]. The N-terminal domain of Pygo is required for Wnt transcriptional activation, while the PHD motif is required for the association of Pygo with Lgs [9,13]. Additionally, a putative nuclear localization signal (NLS) was identified within the N-terminal domain of Pygo, suggesting a possible role of nuclear importation of β-catenin [8,11]. Analyses of multiple aspects of the pygopus mutant phenotype indicate that this gene is dedicated to, and required for, canonical Wnt signaling during Drosophila development [9]. + +The mammalian genome carries two, and only two, orthologs of Drosophila Pygo, Pygo1 and Pygo2 [8,9,14]. In this report, we generated targeted mutations of Pygo1 and Pygo2 to determine their functions, with a particular interest in the contributions of these genes to canonical Wnt signaling during kidney development. The resulting double-homozygous mutant embryos showed a context-dependent reduction in canonical Wnt signaling as measured by Wnt reporter transgene expression. Development remained, however, surprisingly normal, with survival to birth and few apparent defects in most organ systems. Our phenotypic analysis focused on the kidney, which showed altered branching morphogenesis of the ureteric bud, and expansion of the zone of condensed mesenchyme surrounding the ureteric bud, yet relatively normal nephron formation, as measured by histology, confocal analysis, in situ hybridization and microarray analysis. The obvious conclusion is that in mammals, unlike Drosophila, Pygopus-mediated canonical Wnt signaling is not absolutely necessary in most developing organ systems. + +Results + +Pygo1 and Pygo2 targeted mutations + +We targeted both the Pygo1 and Pygo2 genes by inserting LoxP sequences to flank critical coding regions including the PHD domains. The resulting targeted mice were mated with transgenic CMV-Cre mice [15] to drive germline LoxP recombination, resulting in the null mutant alleles that were used for this study. PCR confirmed the deletion of the bulk of the coding sequences for both genes, including 89% of coding sequence for Pygo1 and 87% of coding sequence for Pygo2. Previous studies in Drosophila have shown that even a single missense mutation in the PHD domain can eliminate Pygo function in Wnt signaling [8]. + +Pygo1 and Pygo2 mutant phenotypes + +Pygo1 homozygous null mice were viable and fertile with no developmental defects detectable. This was surprising given the importance of the pygopus gene in Wnt signaling in Drosophila, and the reported expression during development of the mouse Pygo1 gene in, for example, the brain, limbs, kidney and branchial arches [14,16] (Yu J, Valerius MT, McMahon AP, contribution to GUDMAP, ). + +The Pygo2 homozygous null mice survived to birth, but with rare exceptions, died shortly afterwards. The gut, heart and limbs developed without detectable abnormality (data not shown) despite known requirements for Wnt signaling. The Pygo2 mutants did, however, show growth retardation, lens agenesis and a kidney phenotype with high penetrance, exencephaly, and cleft palate with incomplete penetrance. + +Double-homozygous mutant Pygo1 and Pygo2 mice had a phenotype similar to that of single Pygo2 nulls (Figure 1, Table 1). There was no significant synergism of developmental abnormalities in the double mutants. Together these results suggest that Pygo2 is required for the proper development of a limited number of structures, whereas Pygo1 is not necessary for normal development. + +Figure 1 + +Pygo1/Pygo2 mutant embryos. (A) E18.5 Pygo1-/-/Pygo2+/- embryos, with only one wild-type Pygo2 allele, appeared normal. (B, C) Double homozygous mutant Pygo1-/-/Pygo2-/- embryos were smaller, with eye defects including absent or rudimentary lens and folded pigmented retina (arrows). (C) A small percentage of Pygo1/Pygo2 null embryos also displayed exencephaly. + +Table 1 + +Phenotypes of Pygo2 wild-type, heterozyogous, and null E18.5 embryos on a Pygo1 null background. + +* Denotes a significant change (p < 0.01) of the average weight of the Pygo2 null group compared with both the Pygo wild-type and heterozygous groups. + +Expression of Pygo1 and Pygo2 in the developing kidney + +In situ hybridization has been used previously to characterize expression of Pygo1 [14,16](Yu J, Valerius MT, McMahon AP, contribution to GUDMAP, ). In this report, we used immunofluorescence to better define the expression patterns of the Pygo1 and Pygo2 genes in the developing kidney. Both genes were widely expressed, showing nuclear localization of encoded proteins in the ureteric bud, capping mesenchyme, and stromal cells (Figure 2). Raised expression of Pygo1, and to a lesser degree of Pygo2, was seen in stromal cells, and significant nuclear staining was detected in essentially all cells of the developing kidney for both proteins. + +Figure 2 + +Pygo1 and Pygo2 expression in the E18.5 developing kidney. Immunofluorescence was used to determine expression patterns of the Pygo1 and Pygo2 proteins in the cortex of the E18.5 kidney. Both Pygo1 and Pygo2 (red) were localized in the nucleus, as expected. Both genes showed widespread expression, with signal detected in all components of the developing kidney, but with elevated levels in the stromal cell compartment (arrows). Epithelial cells, primarily ureteric bud in these sections, were labeled blue using E-cadherin antibody. Original magnification × 200. + +Confocal analysis of Pygo1/Pygo2 mutant kidneys + +Histological examination of Pygo2 null and Pygo1/Pygo2 double-null mutant kidneys did not reveal any abnormalities in nephrogenesis (data not shown). Confocal analysis was therefore performed to characterize renal development in E18.5 Pygo1/Pygo2 null kidneys more precisely (Figure 3). Wt1 (red) and Cited1 (red) antibodies both stain the capping metanephric mesenchyme around the ureteric bud tips [17]. WT1 antibody also stains renal vesicles and glomerular anlage. Antibodies to Cdh1, also known as E-cadherin (blue), were used to identify epithelial structures, including the branching ureteric bud and nascent nephrons of the developing kidney [18]. Dolichos biflorus (DBA) lectin [16] was used to selectively stain ureteric bud-derived structures [19]. + +Figure 3 + +Confocal analysis of Pygo1/Pygo2 mutant E18.5 kidneys. (B, D) Pygo1-/-/Pygo2-/- mutant E18.5 kidneys and (A, C) kidneys of normal littermates were stained using antibodies to (C, D) Cited1 or (A, B) Wt1, both expressed in the condensing metanephric mesenchyme, and colored as red, Cdh1, a general marker of epithelia (blue), and DBA lectin staining the ureteric tree [16]. Confocal Z-sections were obtained every 5 μm for 75–80 μm. A reduced number of ureteric tips per area was observed in (B, D) the Pygo1/Pygo2 null kidneys compared with (A, C) control littermates. In addition, ureteric bud tips were often more dilated in Pygo1/2 null mutants compared with controls. The condensed mesenchyme surrounding the ureteric bud was also significantly expanded in the mutants. Original magnification × 200. + +Wt1 and Cited1 (red) staining revealed an increase of approximately 30% in the thickness of the capping mesenchyme surrounding the mutant ureteric buds (Figure 3, Table 2). Nevertheless, the mutant metanephric mesenchyme underwent relatively normal nephrogenesis. Cdh1-staining nephrons (blue) were identified connecting to the ureteric bud tips [16] and extending into the medulla of the kidney, as normal. Intermediate structures of nephrogenesis, including renal vesicles, comma-shaped bodies, and S-shaped bodies appeared normal. + +Table 2 + +Confocal analysis of Pygo2 wild-type, heterozygous, and null E18.5 kidneys on the Pygo1 null background. + +*Significant change (P < 0.01) between the three groups. + +SD, Standard deviation; n, number + +Confocal analysis also showed that the ureteric bud tips [16] of Pygo1/Pygo2 null kidneys were dilated and misshaped compared with those of littermates with at least one wild-type Pygo2 allele (Figure 3). In addition, the Pygo1/Pygo2 double-homozygous mutant kidneys also had a decrease of approximately 25% in the number of ureteric bud tips per area compared with littermates with at least one wild-type copy of Pygo2 (Table 2). No significant difference in ureteric bud-tip density was seen between Pygo2+/- and Pygo2+/+ embryos (P = 0.33). + +In situ hybridization + +We examined expression of three Wnt genes in Pygo mutants. Wnt7b is expressed in the stalks [20], Wnt11 in the tips [4], and Wnt9b in the stalks and weakly in the tips of the branching ureteric bud [2]. All three genes showed similar expression patterns in Pygo2+/+ and Pygo1/Pygo2 double-mutant kidneys (Figure 4). In addition, these in situ hybridization patterns confirmed the confocal microscopy results, showing a reduced number of tips (Wnt11-positive) per surface area in the Pygo1/Pygo2 double-homozygous mutants. + +Figure 4 + +Expression of Wnt7b, Wnt9b, and Wnt11 in E18.5 Pygo1/Pygo2 compound null kidneys. Whole-mount in situ of hybridizations of (A-C) E18.5 wild-type kidneys and (D-F) Pygo1-/-/Pygo2-/- mutant kidneys with the ureteric bud derivative markers: (A, D) Wnt7b, (B, E) Wnt9b, and (C, F) Wnt11. The mutant kidneys showed normal expression patterns for the ureteric stalk markers Wnt7b and Wnt9b, and reduced density of ureteric tips as measured by Wnt11. Original magnification × 32. + +Reduced canonical Wnt signaling in Pygo1/Pygo2 mutant mice + +The BAT-gal transgene reporter of canonical Wnt signaling [21] was used to examine changes in Wnt signaling in the mutant mice. Both Pygo2-/- and Pygo1/Pygo2 double-null E10.5 embryos showed a decrease in canonical Wnt signaling (Figure 5). There was, however, tissue-specific variability in the degree of reduction, with the somites, for example, showing strong reduction, while the mutant telencephalon still showed robust Wnt signaling. These results suggest that the mammalian Pygo genes are significant modulators of canonical Wnt signaling in some, but not in all developing systems. + +Figure 5 + +Reduced canonical Wnt signaling in Pygo2 and Pygo1/Pygo2 mutant embryos. E10.5 embryos, all with the BAT-gal transgene reporter of canonical Wnt signaling. (A) Pygo1+/+/Pygo2+/- embryos showed normal X-gal staining in the developing brain, pharyngeal pouches, otic vesicle, apical ectodermal ridges of the fore and hind limb buds, and in somites. (B) Pygo1+/-/Pygo2-/- embryos, with loss of both Pygo2 alleles, showed reduced but not absent BAT-gal reporter expression in many developing structures, including pharyngeal pouches, otic vesicle, and somites. (C) Pygo1-/-/Pygo2+/- embryo, with mutation of both Pygo1 alleles, but one wild-type Pygo2 allele, showed normal BAT-gal expression. (D) Double-homozygous mutant Pygo1-/-/Pygo2-/- embryos still showed some remaining BAT-gal expression, suggesting residual canonical Wnt signaling. Embryos in panels (A) and (B) were from the same litter and were processed in parallel, while embryos in (C) and (D) were from a separate litter, also processed in parallel, and were slightly more developmentally advanced. Original magnification (A, B) × 20; (C, D) × 16. + +We also examined BAT-gal reporter expression in more detail in the developing urogenital system of Pygo mutants. The results suggested that the Pygo2 gene is required for canonical Wnt signaling in the nephric duct. Both Pygo2 null and Pygo1/Pygo2 double-null E10.5 embryos showed an absence of reporter expression in the nephric duct, while control littermates showed strong expression (Figure 6A–C). In Pygo1/Pygo2 double-null mutants, the nephric duct did form, however, and give rise to the ureteric bud outgrowth, which showed reduced but not absent BAT-gal reporter expression (Figure 6A–C). + +Figure 6 + +Pygo2 is required for BAT-gal reporter expression in ureteric bud-derived structures of the developing kidney. X-Gal staining of BAT-gal transgenic (A-C) E10.5, and (E-L) E13.5 urogenital tracts, and (M-O) E18.5 kidneys. (A) Pygo1-/-/Pygo2+/+ (left) and Pygo1+/-/Pygo2-/- (right). Note the loss of reporter activity in the nephric duct (black arrowhead) and reduction of staining in the ureteric bud (white arrow) of the Pygo2 null embryo (Right). (B) Pygo1-/-/Pygo2+/- E10.5 embryo with BAT-gal reporter activity in the nephric duct (black arrowhead) and ureteric bud (white arrow). (C) Pygo1-/-/Pygo-/- embryo, with reporter expression lost in the nephric duct and reduced in the ureteric bud (white arrow). (D) Control background X-gal staining of a urogenital tract from an E13.5 embryo without the BAT-gal transgene (Non-Tg), showing absence of endogenous beta-galactosidase activity. (E) Pygo1+/-/Pygo2+/+, with BAT-gal reporter activity in the ureteric compartment of the developing kidney, including the ureteric tips, ureteric tree, and ureter. (F) Pygo1+/-/Pygo2-/-, with marked reduction of BAT-gal reporter activity in the ureteric compartment. (G) Pygo1+/-/Pygo2+/-, with reporter expression in the paramesonephric duct (white arrowhead) and ureteric tree. (H, I) Pygo1-/-/Pygo2+/-, with (H) ventral view showing reporter expression in the paramesonephric duct (white arrowhead), and (I) dorsal view showing ureteric tree expression in the kidney. (J)Pygo1+/-/Pygo2+/-, a control processed in parallel with (K) and (L), with expression in ureteric tree and paramesonephric duct. (K, L)Pygo1-/-/Pygo2-/-, reporter activity was lost in the paramesonephric duct (white arrowhead), and in the ureter and ureteric compartment of the developing kidneys (dashed circles), except for (K) a few faintly staining cells. (M, N) Pygo1+/-/Pygo2+/- (left) and Pygo1-/-/Pygo2-/- (right) E18.5 kidneys. The kidneys in (N) were bisected. BAT-gal reporter expression was seen in the ureteric tree components of the cortex and medulla of the double heterozygotes but was almost completely lost in the double-homozygous mutants. (O) Pygo1+/-/Pygo2+/+ (left), Pygo1-/-/Pygo2+/- (middle), and Pygo1-/-/Pygo2-/- (right) E18.5 kidneys. Reporter activity was present in the ureteric compartments of the Pygo1+/-/Pygo2+/+ (left) and Pygo1-/-/Pygo2+/- (middle) kidneys, but lost in the Pygo1-/-/Pygo2-/- (right) kidney. Original magnification: (A-C) × 32, (D-F) × 63, (G-I) × 40, (J-L) × 50, (M, N) × 10, and (O) × 12.5. + +At E13.5, the Pygo2 gene appeared to play a major role, and the Pygo1 gene a minor role, in canonical Wnt signaling in the ureteric tree, as measured by BAT-gal expression. A negative control kidney, without the BAT-gal transgene, showed minimal background X-gal staining (Figure 6D), whereas a BAT-gal transgenic kidney with at least one wild-type Pygo2 gene showed strong X-gal staining in the ureteric tree (Figure 6E). In contrast, Pygo1+/-/Pygo2-/- E13.5 kidneys showed very weak reporter expression (Figure 6F), suggesting a significant loss of canonical Wnt signaling. Homozygous loss of the Pygo1 gene alone, however, had a small effect on reporter expression (Figure 6G–H). Homozygous mutation of both the Pygo1 and Pygo2 genes gave a more dramatic reduction of BAT-gal expression than loss of Pygo2 alone (Figure 6F, L). + +The Pygo1 and Pygo2 genes were also required for BAT-gal reporter expression in the paramesonephric (Mullerian) ducts. In Pygo2-/- mice, there was a significant loss of reporter expression (data not shown), and double-homozygous mutants showed loss of X-gal staining in the paramesonephric ducts (Figure 6K), whereas Pygo1-/-/Pygo2+/- and Pygo1+/-/Pygo2+/- mice showed normal levels of BAT-gal expression (Figure 6G, H, J). + +BAT-gal reporter analysis of the Pygo mutants at a later developmental stage, E18.5, also identified a significant decrease in canonical Wnt signaling in the cortical ureteric branches and renal pelvis of the developing kidney (Figure 6M–O). Cortical X-gal staining was seen in the ureteric branches of a Pygo1+/-/Pygo2+/- kidney (Figure 6M, left), but was completely absent in the cortex of a Pygo1+/-/Pygo2-/- kidney (Figure 6M, right). Bisection revealed a significant loss of X-gal staining cells in the collecting ducts and renal pelvis of the Pygo2 null kidney compared with control littermates (Figure 6N). Side by side comparison of Pygo1+/+/Pygo2+/- (Figure 6O, left), Pygo1-/-/Pygo2+/- (Figure 6O, middle), and Pygo1-/-/Pygo2-/- (Figure 6O, right) E18.5 kidneys suggested a significant role for the Pygo2 gene in canonical Wnt signaling in the ureteric tree and its derivatives. + +In order to validate and quantify the BAT-gal reporter expression changes in the Pygo2 null and Pygo1/Pygo2 nulls, we performed ELISA measurements of transgene specific β-galactosidase levels in E18.5 kidneys (Figure 7). Loss of the Pygo2 gene (Pygo1+/-/Pygo2-/- and Pygo1-/-/Pygo2-/-) gave greater than 90% reduction in BAT-gal expression. Loss of Pygo1 alone (Pygo1-/-/Pygo2+/+) did not result in a significant change. Interestingly, however, the Pygo1-/-/Pygo2+/- showed only 50% of wild-type BAT-gal expression, suggesting a minor contribution by Pygo1 in canonical Wnt signaling. Although BAT-gal expression was decreased in the E18.5 Pygo1-/-/Pygo2+/- kidney, confocal analysis of kidneys with this genotype revealed no dilated ureteric tips or significant changes in ureteric tip number per area compared with Pygo1-/-/Pygo2+/+ kidneys (data not shown). Collectively, these BAT-gal reporter results suggest a significant role for the Pygo1 and Pygo2 genes in canonical Wnt signaling during development of the ureteric tree of the kidney. + +Figure 7 + +Quantitative analysis of BAT-gal reporter expression in Pygo1/Pygo2 E18.5 kidney extracts. Transgene-specific beta-galactosidase was quantified by ELISA analysis. Pygo1+/-/Pygo2+/+, Pygo1+/-/Pygo2+/- and Pygo1+/-/Pygo2+/+ kidneys all showed similar levels of BAT-gal expression. In the Pygo2 hetereozygote, Pygo1 homozygous mutants, however, reporter expression was reduced by about 50%. In Pygo2 homozygous mutants BAT-gal expression was uniformly low, with or without wild-type Pygo1 alleles. Each experimental group included a sample size of at least four. + +Interestingly, however, even in wild-type mice the BAT-gal reporter showed no expression in the developing metanephric mesenchyme, or metanephric mesenchyme-derived structures, such as renal vesicles, S-shaped bodies, tubules, and glomeruli. This has been reported previously, and was interpreted to indicate the absence of canonical Wnt signaling in the metanephric mesenchyme [21]. Results from other studies, however, argue for the presence of canonical Wnt signaling in the metanephric mesenchyme. For example, ureteric bud expression of Wnt1, thought to act through canonical Wnt signaling, can rescue Wnt9b mutants and induce nephrogenesis of the metanephric mesenchyme [2]. This suggests that the BAT-gal transgene might not accurately report canonical Wnt signaling in the metanephric mesenchyme. To address this question, we incubated E11.5 metanephric mesenchyme in lithium chloride (LiCl), which activates canonical Wnt signaling through inhibition of GSK3, and also functions as an inducer of nephrogenesis in kidney organ culture [22]. We observed that LiCl-treated metanephric mesenchyme did undergo nephrogenesis, as expected, but failed to show BAT-gal expression (data not shown), suggesting that this transgene is not an accurate reporter of canonical Wnt signaling in the metanephric mesenchyme of the developing kidney. + +Microarray analysis of Pygo1/Pygo2 null kidneys + +To further examine possible disturbance of gene expression in the Pygo1/Pygo2 mutant kidneys, we used microarrays to perform a global analysis of gene expression changes. Whereas the BAT-gal transgene reporter monitors the response of one promoter to Wnt signaling, microarrays can be used to follow expression changes of all genes, including all known Wnt targets. E18.5 wild-type and Pygo1-/-/Pygo2-/- kidneys were examined in biological triplicate. In total, 45 genes were identified as significantly changed, using a relatively low stringency screen of the data, with a p-value cutoff of <0.05, and fold change >2 (Table 3). Notably, both Pygo1 and Pygo2 were identified as downregulated (- 5.3-fold and - 3.9-fold, respectively) in the mutant kidneys. Other genes with significant decreases in mutant kidneys included Cxcl13 (- 2.3-fold), Slc5a2 (- 2.8-fold), and Slco1a4 (- 2.1-fold). Slc5a2 is expressed in the proximal tubules of the adult nephron and has been implicated in autosomal recessive renal glucosuria, characterized by loss of glucose uptake by the nephron [23]. The organic anion transporter, Slco1a4, is also strongly expressed in the tubules of the adult kidney [24]. These results suggest that the Pygo1/Pygo2 genes might play a role in nephron maturation and subsequent function. + +Table 3 + +Genes differentially expressed (p < 0.05, two-fold change or greater) in the Pygo1/Pygo2 null E18.5 kidney normalized to wild-type samples. + +Noteworthy genes upregulated in the Pygo1/Pygo2 null kidney included Klk5 (2.8-fold), Klk6 (2.7-fold), Ren2 (2.2-fold), and Timeless (2.4-fold). Klk5 and Klk6 are members of the kallikrein family of trypsin-like serine proteases. Kallikreins have diverse functions in cancer, tissue remodeling, and regulation of blood pressure [25]. Ren2, a homolog of the endopeptidase Renin 1, activates the renin-angiotensin system, increasing blood pressure [26]. Disruption of the renin-angiotensin system during development results in congenital abnormalities of the ureter and collecting-duct system [27]. Timeless, a transcription factor involved in the regulation of circadian rhythms, and expressed in the branching ureteric tips, has also been shown to regulate ureteric branching morphogenesis [28]. + +We were interested in the possible altered expression of previously known Wnt targets in the Pygo1-/-/Pygo2-/- mutant kidneys. A list of known Wnt targets was compiled from . Both Pygo1 and Pygo2 probe sets were included as references to illustrate significant down regulation. Remarkably, the known Wnt target genes showed very few differences in expression between wild-type and mutant kidneys (Figure 8). Only the expression of Ccnd1 encoding cyclin D1 [29], and Wisp1 [30] were significantly changed, with expression level changes of <1.5-fold in Pygo1/Pygo2 null kidneys. These results indicate an absence of dramatic changes in expression of previously known Wnt signaling target genes in the Pygo1/Pygo2 mutant kidneys. + +Figure 8 + +Gene expression changes of common Wnt signaling targets in the E18.5 Pygo1/Pygo2 null kidney. Microarray analysis was performed in triplicate on wild-type and Pygo1/2 compound null E18.5 kidneys. Possible Wnt targets were selected from those compiled at [39]. An initial gene list of 82 Wnt targets was created and then reduced to a total of 40 genes using an expression level restriction requiring the raw expression intensity to be >100 in at least 3 samples. Pygo1 and Pygo2 probes were included to demonstrate significant changes in expression levels. + +We used quantitative real-time PCR, with independent biological samples, to validate the microarray results. Nine genes were tested, and for seven we did observe a significant fold change in the same direction predicted by the microarray results (Table 4). It is not surprising that two of the nine genes could not be validated, considering the relatively low stringency used in screening the microarray data. + +Table 4 + +Validation of gene expression changes in the E18.5 Pygo1/Pygo2 null kidneys normalized to E18.5 wild-type kidneys. + +Discussion + +In Drosophila, the Pygopus gene is a key mediator of canonical Wnt signaling. In one study, 12 distinct measures of Wnt signaling in Pygo mutants were performed, including analysis of leg, wing, and eye imaginal discs. In 2 cases there was a significant reduction of Wnt signaling and in 10 cases a complete block [10]. A second study in Drosophila examined the effects of Pygo mutation on cuticle patterning, midgut constriction, central nervous system, and cardiac development, and concluded that "Pygo is an essential component in the Wg signal transduction pathway". [8]. + +Given these results in Drosophila, the relatively mild phenotypes of mice with targeted Pygo1, Pygo2, or double Pygo1/Pygo2 mutations were striking. Pygo2 mutants developed to birth and showed limited abnormalities, while Pygo1 homozygous mutants were normal and fertile. Furthermore, there was no detected synergism in the phenotype of the double Pygo1/Pygo2 mutant, although in several tissues the BAT-gal Wnt reporter did show more a severe reduction in expression. One possible explanation of these unexpected results is a failure of the gene targeting to eliminate functioning of the Pygo1 and Pygo2 genes. The Pygopus deletion alleles described in this report, however, are almost certainly functional nulls. In Drosophila, it has been shown that the PHD domain is absolutely necessary for Pygopus function in Wg signaling. The PHD domain is 60 amino acids with seven cysteines and a histidine, predicted to chelate two zinc ions. PHD domains are found in diverse proteins, including transcription factors, and have been implicated in chromatin remodeling and protein-protein interactions [8]. In Drosophila the pygoF107 allele, with a single missense mutation converting amino acid 802 in the PHD domain from cysteine to tyrosine, loses Wnt signaling function in both embryogenesis and imaginal disc development [8]. The Pygo1/Pygo2 mutant alleles made in this report carried deletions of the entire PHD domains, as well as most other coding sequences. For the Pygo1 gene, the coding region for 372 of 417 total amino acids was deleted, and for the Pygo2 gene, we deleted coding for 354 of 405 amino acids. It is therefore very unlikely that the relatively mild phenotypes observed were the result of residual function of the targeted alleles. + +We focused our analysis on the developing kidney, in which Wnt signaling has been shown to be of critical importance in several stages of nephrogenesis. Wnt9b is made by the ureteric bud and induces the metanephric mesenchyme to undergo nephrogenesis [2]. Downstream of Wnt9b is Wnt4 [2], which is made by the metanephric mesenchyme, and is also required for nephrogenesis [3]. In addition, Wnt11 is produced by the ureteric bud tips and induces GDNF expression in the metanephric mesenchyme [4]. + +In this report we describe a novel Wnt function in kidney development. The BAT-gal transgene reporter indicated the presence of canonical Wnt signaling in the ureteric bud and its derivatives in the developing kidney. Further, in the Pygo2 mutants this signal was lost, suggesting significant reduction of Wnt signaling. In addition, we observed a resulting decrease in ureteric tip density, reduced kidney size and altered morphology of the ureteric tree in mutants, indicating a role for canonical Wnt signaling in branching morphogenesis of the ureteric bud. While the simplest interpretation is direct Wnt signaling to the ureteric bud, it remains possible that the observed abnormalities are the result of indirect effects, with altered Wnt signaling to the metanephric mesenchyme then affecting mesenchyme to ureteric bud signaling. + +The Pygo1/Pygo2 mutant phenotype of reduced branching morphogenesis of the ureteric bud is surprisingly similar to that previously reported for Wnt11 mutants [4]. The underlying mechanisms, however, are likely to be distinct. Wnt11 is generally thought to act through a noncanonical pathway, (although exceptions have been noted [31]), whereas the Pygopus genes promote canonical Wnt signaling. Further, the Wnt11 mutants showed an altered feedback loop between Wnt signaling from the ureteric bud to the mesenchyme and GDNF signaling from the mesenchyme to the bud, whereas in the Pygo1/Pygo2 mutants, we observed disrupted Wnt signaling in the ureteric bud. + +The Pygo1/2 mutants also showed an expansion of the zone of thickened mesenchyme that caps the ureteric bud. Nevertheless, the mutant metanephric mesenchyme formed nephrons normally. This was particularly interesting, as Wnt9b signaling from the ureteric bud to the metanephric mesenchyme has been shown to induce nephrogenesis via canonical Wnt signaling [2]. + +The results in this report indicate a striking and surprising evolutionary divergence of Pygopus function between Drosophila and mammals. In Drosophila, the Pygopus gene is often required for canonical Wnt signaling, while in mammals the Pygo1/Pygo2 genes appear to play a smaller role in canonical Wnt signaling. The BAT-gal transgene reporter of canonical Wnt signaling showed reduced but generally not absent signal in Pygo1/Pygo2 mutant embryos, with tissue-specific variation in level of diminution. In addition, the kidneys were not unique in showing surprisingly normal development in Pygo1/Pygo2 mutants. Indeed, organogenesis generally proceeded without detectable abnormality, with few exceptions. These results suggest that the proteins encoded by mammalian Pygopus genes are often mere modulators of canonical Wnt signaling intensity, and not essential components. + +The microarray results further support this conclusion. Whereas the BAT-gal transgene reporter monitors the expression level of only one Wnt responsive promoter, the microarray allows the analysis of the activity levels of promoters of all genes. Interestingly, the mutant kidneys showed gene-expression profiles surprisingly similar to wild type. Some genes did, however, show expression differences, and a high percentage of these differences could be validated by real-time PCR with independent biological samples. Assuming that the Pygopus genes in mammals are dedicated to canonical Wnt signaling, as has been previously shown in Drosophila, the genes with expression differences represent candidate Wnt targets (direct or indirect) in kidney development. We would predict these genes to show greater changes in expression level in a developing kidney with a more complete removal of canonical Wnt signaling. + +Conclusion + +In conclusion, the mammalian Pygo2 gene is required for normal branching morphogenesis of the ureteric bud, with mutants showing dilated tips and reduced numbers of tips. In addition, in Pygo2 mutants there was an expansion of the zone of metanephric mesenchyme that caps the ureteric buds. Nevertheless, nephron formation proceeded remarkably normally, even in Pygo1/Pygo2 double-homozygous mutants. This was surprising considering the importance of the Pygopus gene in canonical Wnt signaling in Drosophila, and the importance of canonical Wnt signaling in nephrogenesis. The results argue that the mammalian Pygopus genes are, in most developing systems, only quantitative transducers of Wnt signaling. Previous cell culture studies [32,33] and the reduced BAT-gal transgene reporter expression in the Pygo1/Pygo2 knockout mice described in this report, do confirm an evolutionarily conserved function in canonical Wnt signaling. In mammals, however, the phenotypic effects of Pygopus mutation are much milder than in Drosophila. The degree of importance of the Pyg1/2 genes in Wnt signaling was context-dependent, but in general, mammalian organogenesis remained intact in Pygopus mutants. Perhaps the simplest explanation is that in mammals, other genes show partial functional redundancy with the two Pygopus genes. The β-catenin transcription-factor complex includes a large and growing number of proteins , some of which may share the nuclear localization and/or transcription activation functions of Pygo1/Pygo2 in mammals. The identities and roles of these Pygopus redundant genes remain to be determined. + +Methods + +Targeting of Pygo1 and Pygo2 + +Pygo1 and Pygo2 genes were each targeted by flanking the critical coding regions of the third exon containing the PHD motif with LoxP sequences. Targeting constructs were made using the Flp/Cre vector previously described [34], which carries a neomycin-resistance gene flanked by FRT sequences, and three unique restriction sites for subcloning the two blocks of homology for driving recombination, and the critical region to be flanked by LoxP sequences. For each construct, the three genomic segments required for subcloning were made by PCR from RI ES cell DNA. The resulting targeting constructs were confirmed by sequencing. + +For Pygo1, the genomic sequences used for PCR were: 5' forward, GTGAAGGAGAGATGGATAAGTATG; 5' back, TAGACCCTAACCACCTACAAG; exon forward, GGTTAGGGTCTATGTGCTGG; exon back, TCACCAAATCTCTGTTCTACAC; 3' forward, TGTGTAGAACAGAGATTTGGTG; 3' back, CAGTGAAGAAAGAGGGTCAG. For Pygo2, the genomic sequences used for PCR were: GCCTGGGTTGCTTGTCTTCTG and CCACCTTACTTGTGTGTGAGGATACATAC, CCAAGTCCCAGCATCTCTTAC and CCAGTCATACCAGCAACAAG, and exon sequences TGGGTGCTGGGAACAGAAC and CAACAACAACAGAAGACAAGC. + +Linearized constructs were electroporated into RI ES cells, and resulting targeted ES cells used for C57/Bl6 blastocyst injections according to standard protocols. Resulting chimeras were mated with Swiss Black mice, and the targeted stocks maintained on a mixed 129/Swiss Black background. + +Germline null alleles of both Pygo1 and Pygo2 were generated by mating heterozygous floxed mice with the CMV-Cre mice [15]. The sequences of the primers used for genotyping PCR were: Pygo2 null allele, forward (F) CCTGGATTCTTGTTGCTGGTATG; reverse (R) AAGGTATTTGGTGCTCCGAGGG; Pygo2 WT or floxed allele, F TGTCTTGATGACAGCGTTTAGCC, R AGATTCAGTAAGCTGAGCCTGGTG; Pygo1 null allele, F AGTTTGAAATAGCGACGAGTTTGAG, R 5'-CACTTCTGCCCCTCTCTTTGC; Pygo1 WT or floxed allele, F AAGCGTGCCTCATCTCCATCCCTAAG, R GCCCTCCCCGACGTTTATATTG. + +The noon of the day that vaginal plugs were observed was designated E0.5. + +Confocal microscopy + +Kidneys were dissected, fixed in paraformaldehyde, treated with methanol, and washed with PBS containing Tween-20 (PBS-T) prior to treatment with lectins and antibodies. PBS-T was used for incubations of the tissues with fluoroscein-conjugated Dilichos biflorus aggutinin (DBA, Vector; Burlingame, USA), and PBS-T plus 2% goat serum was used for incubations with the antibodies. The primary antibodies were anti-WT1 (c-19; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-uvomorulin (e-cadherin, Cdh1, Sigma-Aldrich St. Louis, MO, USA), and anti-Cited1 (Neomarkers-Lab Vision Corporation, Fremont, CA, USA). The secondary antibodies were Alexa 555-conjugated anti-rabbit and Alexa 633-conjugated anti-rat antibodies (Molecular Probes, Eugene, OR, USA). + +The tissues were imaged with a laser scanning microscope (Carl Zeiss, Thornwood, New York, USA) equipped with an argon (488 nm) and two HeNe lasers (543 nm and 633 nm). Optical sections approximately 2 μm thick were obtained every 5 μm to a depth of at least 65 μm. The sections began at the surface of the kidney and were on a plane tangential to it. Two Z-stack series were obtained, one from each of the two kidneys of each embryo. Ureteric bud tips identified by section tracing were counted within a defined area of the confocal image. + +In situ hybridization + +Whole-mount in situ hybridization was performed as previously described [35]. Riboprobes to Wnt11 and Wnt7b were described previously [35]. The Wnt9b riboprobe was provided by T. Carroll [2]. + +Pygo1 and Pygo2 antibody production + +To generate anti-human Pygo2 (anti-hPygo2) and anti-mouse Pygo1 (anti-mPygo1) polyclonal antibodies, we subcloned cDNA by PCR corresponding to amino-acid residues 80–327 of human Pygo 2 or amino-acid residues 76–263 of mouse Pygo1 into pGEX4T1 (Amersham Health, Piscataway, NJ, USA). The PCR fragments of hPygo 2 and mPygo 1 lack both NHD and PHD conserved regions of hPygo2 and mPygo1. GST-hPygo2 and GST-mPygo1 proteins were expressed in bacteria, purified, and injected into rabbits for antibody production by (Proteintech Group Inc., Chicago, IL, USA). The rabbit antisera of anti-mPygo1 and anti-hPygo2 were initially allowed to bind to the GST affinity matrix to remove any antibodies against GST. The anti-hPygo2 and anti-mPygo1 antisera were then separated from the GST affinity matrix and allowed to bind to the GST-hPygo2 or GST-mPygo1 affinity columns, respectively. The bound antibodes were eluted with elution buffer. To further ensure antibody specficity, the purified antibodies were extensively incubated with Pygo1/2 double-homozygous mutant embryo extract before use. + +BAT-gal transgene reporter assay of canonical Wnt signaling + +X-gal staining of both embryos and developing kidneys was performed as previously described [21]. Care was taken to reduce endogenous β-galactosidase activity within the developing kidney by increasing pH of the X-gal staining solution to 8.0. Changes in transgene β-Gal expression were quantitated using a β-Gal ELISA (Enzyme-linked immunoassay) kit (Roche, Indianapolis, IN), normalizing according to total protein. Each genotype was represented by a sample size of 4 except the non-transgenic (n = 5), Pygo1+/- ; Pygo2+/+ (n = 6), Pygo1-/- ; Pygo2+/- (n = 8), and Pygo1-/- ; Pygo2-/- (n = 6) groups. + +Microarray analysis + +Total RNA was isolated from E18.5 kidneys dissected from normal and Pygo1/Pygo2 compound null embryos using a commercial kit (Stratagene Absolutely RNA Microprep Kit; La Jolla, CA, USA). An aliquot (300 ng) of total RNA was processed and labeled using a commercial kit (TargetAmp 1-Round Aminoallyl-aRNA Ki; Epicentre, Madison, WI, USA). Labeled RNA was hybridized to microarrays (Sentrix Mouse-6 expression Beadchip; Illumina, San Diego, CA) providing coverage of over 47000 genes and expressed sequence tags as previously described [36]. Microarray analysis of Pygo1/Pygo2 null and normal wild-type kidneys was performed in biological triplicate. Raw signal intensities of each probe were obtained from data analysis software (Beadstudio ;Illumina) and imported into GeneSpring GX 7.3 (Agilent Technologies, Palo Alto, CA, USA). Genes were selected on the basis of greater than two-fold average fold change and statistical significance (p-value < 0.05). Previously described Wnt target genes were obtained from . + +Quantitative PCR validation of microarray results + +Total RNA from E18.5 Pygo1/Pygo2 null and control kidneys (both represented in duplicate and distinct from the kidneys used for microrray analysis) was purified using a commercial kit (Absolutely RNA Microprep Kit; Stratagene, La Jolla, CA USA) including DNAse1 treatment. cDNA was generated using random hexamers according to conventional protocols (Invitrogen, Carlsbad, CA, USA). The following primers were generated to include the sequence obtained from the Illumina probe: Actb (TTGCTGACAGGATGCAGAAG, ACATCTGCTGGAAGGTGGAC); Aldh1a7 (CCAGAAAGTGGTGTTTTGCT, GAGTTACAGAGAGCTTGCACCTT); Col8a1 (GCAGACAGGCATCTTCACCT, TGTGTACATCATGGGCTCGT); Csrp1 (CAGCATAGCCCAGGGTAGAG, TGGGCAAGGTAGTGAAGGTT); Klk5 (GCAGCATTACACCCGTCATA, TTGCCTCCATCCATATCTCC); Picalm (GGGAGGGAACAGAAATCCTT, GCACCGATCAACAGTGCAG); Pygo2 (TTCTGGGAACTTGTGCACTG, AACTTCCTCCAGCCCATTTT); Ren2 (TTGTGAGCTGTAGCCAGGTG, TGTGCACAGCTTGTCTCTCC) and Tia1 (TGATTGAAGGGCTACTAGAGTGGT, AGCCCCAGAGGACAATTTTT) using Primer3 software [37]. Relative quantitative PCR was performed according to the conventional SYBR Green protocol (Stratagene) using a quantitative PCR system (Mx3000p; Stratagene). Dissociation curve and agarose-gel analysis of each primer set were used to insure specificity of the amplicon. All data were normalized to an internal housekeeping control (Actb) and analyzed using the 2(-Delta Delta C(T)) method [38]. + +Authors' contributions + +SP designed and did most of the work for targeting the Pygo genes, provided oversight for the project and helped write the paper. KS did most of the experiments, including confocal analysis with LP, in situ hybridizations with HH, and helped write the paper. XL made and tested the Pygo antibodies. NS and RL provided useful suggestions and helped with the immunostaining and Bat-Gal analyses. + +Acknowledgements + +L. McClain provided essential technical assistance and we thank P. Groen and D. Witte for initial histology and embryological analyses. D. Ash helped make targeting constructs. We thank T. Carroll for providing a Wnt9b riboprobe construct. We thank H. Liang for generation of microarray data. This work was supported by grant DK61916 from the National Institutes of Health (S.S.P.). diff --git a/src/ontogpt/evaluation/craft/database/all/17447844.ann b/src/ontogpt/evaluation/craft/database/all/17447844.ann new file mode 100644 index 000000000..824ab6745 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17447844.ann @@ -0,0 +1,1699 @@ +T1 NCBITaxon:40674 2 8 Mammal +T2 PR:P23023 18 27 Doublesex +T3 SO:0000853 28 35 Homolog +T4 GO:0001739 57 70 Sex Chromatin +T5 GO:0007140 91 103 Male Meiosis +T6 GO:0007276 115 128 Gametogenesis +T7 CL:0000586 195 204 germ cell +T8 GO:0030154 200 220 cell differentiation +T9 NCBITaxon:40674 243 250 mammals +T10 GO:0000803 282 297 sex chromosomes +T11 GO:0000805 372 373 X +T12 GO:0000806 378 379 Y +T13 GO:0007126 395 402 meiotic +T14 GO:0051324 403 411 prophase +T15 GO:0031507 427 439;442 464 formation of ... heterochromatin domain +T16 GO:0000792 442 457 heterochromatin +T17 GO:0001741 470 477 XY body +T18 GO:0001741 483 490 XY body +T19 GO:0051324 510 518 prophase +T20 GO:0001739 536 549 sex chromatin +T21 GO:0007126 595 602 meiosis +T22 PR:000006549 641 646 DMRT7 +T23 NCBITaxon:40674 650 656 mammal +T24 PR:P23023 720 729 Doublesex +T25 PR:O18214 734 739 MAB-3 +T26 PR:000006549 754 759 DMRT7 +T27 GO:0001741 792 799 XY body +T28 GO:0000239 807 816 pachytene +T29 GO:0007126 826 833 meiotic +T30 GO:0051324 834 842 prophase +T31 GO:0007140 863 875 male meiosis +T32 PR:000006549 880 885 Dmrt7 +T33 GO:0007129 895 910 meiotic pairing +T34 GO:0006342 950 976 transcriptionally silenced +T35 GO:0001741 977 984 XY body +T36 GO:0000785 1002 1011 chromatin +T37 CL:0000586 1038 1048 germ cells +T38 GO:0006915 1057 1066 apoptosis +T39 GO:0000239 1074 1083 pachynema +T40 GO:0000240 1128 1137 diplonema +T41 GO:0001739 1186 1199 sex chromatin +T42 PR:000043452 1208 1215 histone +T43 CHEBI:15358 1208 1215 histone +T44 GO:0036123 1208 1224;1232 1243 histone H3K9 di- ... methylation +T45 GO:0036124 1208 1220;1229 1243 histone H3K9 ... trimethylation +T46 GO:0000792 1248 1263 heterochromatin +T47 PR:000005082 1248 1274 heterochromatin protein 1β +T48 GO:0000239 1331 1340 pachynema +T49 GO:0000240 1345 1354 diplonema +T50 PR:000006549 1385 1390 DMRT7 +T51 GO:0001741 1398 1405 XY body +T52 GO:0001739 1414 1427 sex chromatin +T53 PR:000006549 1448 1453 Dmrt7 +T54 PR:000006549 1480 1485 DMRT7 +T55 GO:0001739 1506 1519 sex chromatin +T56 GO:0016568 1510 1534 chromatin transformation +T57 GO:0000239 1555 1564 pachynema +T58 GO:0000240 1569 1578 diplonema +T59 PR:000006549 1596 1601 DMRT7 +T60 GO:0065007 1611 1618 control +T61 GO:0007126 1639 1646 meiotic +T62 GO:0000803 1647 1661 sex chromosome +T63 GO:0007126 1682 1689 meiotic +T64 GO:0001739 1690 1703 sex chromatin +T65 NCBITaxon:40674 1766 1773 mammals +T66 NCBITaxon:7742 1792 1803 vertebrates +T67 PR:000006549 1805 1810 Dmrt7 +T68 GO:0007126 1842 1849 meiosis +T69 GO:0001739 1857 1870 sex chromatin +T70 SO:0000704 1891 1896 Genes +T71 PR:P23023 1929 1938 Doublesex +T72 NCBITaxon:7215 1942 1952 Drosophila +T73 GO:0065007 1972 1979 control +T74 GO:0003006 1980 1998 sexual development +T75 NCBITaxon:33208 2020 2027 animals +T76 NCBITaxon:6231 2042 2052 roundworms +T77 NCBITaxon:40674 2056 2063 mammals +T78 PR:000006549 2115 2120 Dmrt7 +T79 SO:0000704 2121 2125 gene +T80 SO:0000704 2148 2153 genes +T81 NCBITaxon:10088 2161 2166 mouse +T82 NCBITaxon:40674 2175 2182 mammals +T83 GO:0007126 2278 2285 meiotic +T84 CL:0000015 2295 2309 male germ cell +T85 GO:0007281 2300 2321 germ cell development +T86 GO:0000805 2385 2386;2393 2404 X ... chromosomes +T87 GO:0000806 2391 2404 Y chromosomes +T88 GO:0001741 2445 2452 XY body +T89 PR:000006549 2467 2472 DMRT7 +T90 CL:0000586 2495 2505 germ cells +T91 GO:0001741 2524 2536 male XY body +T92 GO:0007126 2544 2551 meiosis +T93 GO:0001741 2609 2616 XY body +T94 PR:000043452 2708 2715 histone +T95 CHEBI:15358 2708 2715 histone +T96 GO:0000239 2737 2746 pachytene +T97 GO:0000240 2751 2760 diplotene +T98 GO:0098762 2761 2778 stages of meiosis +T99 GO:0016568 2784 2799;2809 2818 modification of ... chromatin +T100 GO:0001739 2805 2818 sex chromatin +T101 PR:000006549 2823 2828 Dmrt7 +T102 PR:000006549 2882 2887 Dmrt7 +T103 NCBITaxon:40674 2916 2923 mammals +T104 NCBITaxon:7742 2942 2953 vertebrates +T105 GO:0065007 3002 3012 regulation +T106 GO:0001739 3016 3029 sex chromatin +T107 NCBITaxon:40674 3040 3047 mammals +T108 GO:0007548 3064 3086 Sexual differentiation +T109 UBERON:0001062 3097 3107 anatomical +T110 GO:0019953 3174 3193 sexual reproduction +T111 CL:0002371 3228 3241 somatic cells +T112 GO:0019953 3297 3316 sexual reproduction +T113 CL:0000300 3334 3341 gametes +T114 CL:0000300 3354 3361 Gametes +T115 CL:0000365 3479 3485 zygote +T116 GO:0009566 3479 3495 zygote formation +T117 CL:0000300 3527 3534 gametes +T118 CL:0000300 3601 3608 gametes +T119 NCBITaxon:40674 3646 3655 Mammalian +T120 GO:0007126 3656 3663 meiosis +T121 GO:0065007 3667 3676 regulated +T122 GO:0009790 3706 3719 embryogenesis +T123 UBERON:0000113 3751 3761 adult life +T124 GO:0007126 3822 3829 meiosis +T125 CL:0000586 3879 3889 germ cells +T126 GO:0007126 3913 3920 meiosis +T127 UBERON:0000922 3928 3934 embryo +T128 GO:0007128 3953 3971 meiotic prophase I +T129 CL:0000023 3988 3995 oocytes +T130 GO:0030728 4026 4035 ovulation +T131 GO:0007137 4058 4070 metaphase II +T132 GO:0007126 4089 4096 meiosis +T133 GO:0009566 4103 4116 fertilization +T134 GO:0007140 4142 4154 male meiosis +T135 GO:0007567 4175 4182 natally +T136 GO:0007126 4246 4253 meiosis +T137 CL:0000023 4283 4289 oocyte +T138 GO:0007140 4336 4348 male meiosis +T139 CL:0000017 4374 4387 spermatocytes +T140 GO:0007126 4396 4403 meiotic +T141 GO:0007129 4441 4459 chromosome pairing +T142 GO:0007129 4461 4469 synapsis +T143 GO:0007143 4574 4592 meiosis in females +T144 NCBITaxon:9606 4599 4604 human +T145 CL:0000023 4605 4611 oocyte +T146 NCBITaxon:9606 4658 4663 human +T147 CL:0000019 4664 4669 sperm +T148 GO:0000075 4706 4717 checkpoints +T149 GO:0065007 4718 4729 controlling +T150 GO:0065007 4734 4744 monitoring +T151 GO:0007126 4759 4766 meiotic +T152 SO:0000704 4835 4842 genetic +T153 GO:0040020 4867 4885 meiotic regulatory +T154 SO:0000704 4886 4891 genes +T155 NCBITaxon:10088 4899 4904 mouse +T156 GO:0000803 5014 5029 sex chromosomes +T157 NCBITaxon:40674 5059 5066 mammals +T158 GO:0010467 5143 5153 expression +T159 SO:0000704 5168 5173 genes +T160 NCBITaxon:40674 5202 5209 mammals +T161 GO:0000805 5229 5241 X chromosome +T162 GO:0009048 5229 5254 X chromosome inactivation +T163 CL:0000015 5276 5291 male germ cells +T164 GO:0007126 5343 5350 meiosis +T165 GO:0007128 5355 5365 prophase I +T166 GO:0007129 5399 5406 synapse +T167 GO:0000805 5444 5445;5452 5462 X ... chromosome +T168 GO:0000806 5450 5462 Y chromosome +T169 GO:0007129 5452 5470 chromosome pairing +T170 NCBITaxon:9347 5584 5593;5608 5615 eutherian ... mammals +T171 NCBITaxon:9263 5598 5615 marsupial mammals +T172 GO:0000785 5685 5694 chromatin +T173 GO:0001741 5713 5720 XY body +T174 GO:0001741 5724 5732 sex body +T175 GO:0001741 5754 5761 XY body +T176 GO:0007140 5830 5842 male meiotic +T177 GO:0001741 5911 5918 XY body +T178 PR:000004803 5930 5935 BRCA1 +T179 PR:000004499 5937 5940 ATR +T180 PR:000043452 5946 5953 histone +T181 CHEBI:15358 5946 5953 histone +T182 PR:000008425 5962 5966 H3.3 +T183 PR:000043452 5981 5989 histones +T184 CHEBI:15358 5981 5989 histones +T185 PR:000027547 6012 6015 H2A +T186 PR:000027547 6020 6023 H2A +T187 PR:000008418 6044 6048 H2AX +T188 GO:0001741 6073 6080 XY body +T189 GO:0000803 6086 6101 sex chromosomes +T190 GO:0006342 6106 6132 transcriptionally silenced +T191 GO:0007126 6153 6160 meiotic +T192 GO:0000803 6161 6175 sex chromosome +T193 GO:0001741 6201 6208 XY body +T194 GO:0000239 6226 6235 pachynema +T195 SO:0000704 6262 6267 genes +T196 GO:0006342 6275 6299 transcriptionally silent +T197 GO:0007286 6305 6319 spermiogenesis +T198 GO:0016458 6346 6355 silencing +T199 GO:0000785 6393 6402 chromatin +T200 GO:0001739 6423 6436 sex chromatin +T201 GO:0007126 6455 6462 meiotic +T202 GO:0001739 6463 6476 sex chromatin +T203 GO:0007548 6508 6530 sexual differentiation +T204 NCBITaxon:1 6567 6576 organisms +T205 GO:0065007 6723 6731 regulate +T206 GO:0007548 6732 6754 sexual differentiation +T207 NCBITaxon:phylum 6783 6788 phyla +T208 SO:0000704 6819 6824 genes +T209 PR:P23023 6836 6845 doublesex +T210 PR:P23023 6847 6850 dsx +T211 NCBITaxon:7215 6855 6865 Drosophila +T212 PR:P23023 6881 6890 Doublesex +T213 PR:O18214 6891 6896 MAB-3 +T214 SO:0000417 6929 6935 domain +T215 SO:0000417 6948 6954 domain +T216 SO:0000704 6964 6969 genes +T217 GO:0065007 6989 6997 regulate +T218 GO:0007548 7017 7039 sexual differentiation +T219 NCBITaxon:6960 7043 7050 insects +T220 NCBITaxon:6231 7052 7061 nematodes +T221 NCBITaxon:40674 7067 7074 mammals +T222 PR:O18214 7085 7090 mab-3 +T223 SO:0000704 7091 7095 gene +T224 NCBITaxon:6239 7099 7121 Caenorhabditis elegans +T225 PR:P23023 7164 7167 DSX +T226 PR:P23023-1 7228 7247 male isoform of DSX +T227 SO:0001060 7233 7240 isoform +T228 SO:0000704 7305 7310 genes +T229 SO:0000417 7357 7363 domain +T230 NCBITaxon:7742 7394 7405 Vertebrates +T231 SO:0000417 7419 7425 domain +T232 SO:0000704 7426 7431 genes +T233 SO:0000704 7494 7499 genes +T234 GO:0065007 7505 7512 control +T235 GO:0007548 7513 7535 sexual differentiation +T236 NCBITaxon:40674 7537 7544 Mammals +T237 SO:0000417 7559 7565 domain +T238 SO:0000704 7566 7571 genes +T239 SO:0000704 7578 7583 genes +T240 GO:0010467 7635 7645 expression +T241 SO:0000704 7681 7686 genes +T242 PR:000006542 7688 7693 Dmrt1 +T243 GO:0010467 7698 7707 expressed +T244 UBERON:0005294 7736 7750 genital ridges +T245 UBERON:0007023 7755 7760 adult +T246 UBERON:0000473 7761 7767 testis +T247 NCBITaxon:40674 7771 7778 mammals +T248 NCBITaxon:8782 7780 7785 birds +T249 PR:000006542 7833 7838 Dmrt1 +T250 SO:0000704 7839 7843 gene +T251 GO:0000806 7861 7862 Y +T252 UBERON:0000473 7870 7876 testis +T253 SO:0000704 7889 7893 gene +T254 NCBITaxon:8088 7901 7912 Medaka fish +T255 NCBITaxon:9606 7922 7927 Human +T256 PR:000006542 7928 7933 DMRT1 +T257 GO:0030849 7945 7954 autosomal +T258 UBERON:0000473 8015 8025 testicular +T259 GO:0008584 8015 8037 testicular development +T260 NCBITaxon:10088 8086 8090 mice +T261 PR:000006542 8125 8130 Dmrt1 +T262 UBERON:0000473 8154 8160 testis +T263 CL:0000586 8192 8202 germ cells +T264 CL:0000216 8207 8220 Sertoli cells +T265 NCBITaxon:10088 8234 8238 mice +T266 PR:000006545 8249 8254 Dmrt4 +T267 CL:0000025 8264 8270 ovular +T268 SO:0000704 8303 8307 gene +T269 UBERON:0000991 8329 8336 gonadal +T270 GO:0008406 8329 8348 gonadal development +T271 SO:0000417 8412 8418 domain +T272 SO:0000704 8419 8424 genes +T273 GO:0007548 8428 8450 sexual differentiation +T274 NCBITaxon:7742 8486 8496 vertebrate +T275 SO:0000704 8502 8506 gene +T276 GO:0007548 8534 8556 sexual differentiation +T277 PR:000006543 8558 8563 Dmrt2 +T278 NCBITaxon:10088 8610 8614 mice +T279 GO:0010467 8665 8675 expression +T280 PR:000006549 8696 8701 Dmrt7 +T281 SO:0000704 8702 8706 gene +T282 NCBITaxon:10088 8714 8719 mouse +T283 PR:000006549 8721 8726 Dmrt7 +T284 GO:0010467 8730 8739 expressed +T285 UBERON:0000991 8752 8757 gonad +T286 SO:0000704 8786 8791 genes +T287 NCBITaxon:40674 8830 8837 mammals +T288 NCBITaxon:40674 8852 8861 mammalian +T289 NCBITaxon:7742 8862 8873 vertebrates +T290 PR:000006549 8896 8901 DMRT7 +T291 GO:0010467 8913 8922 expressed +T292 CL:0000586 8931 8941 germ cells +T293 GO:0001741 8978 8985 XY body +T294 CL:0000015 8989 8993;9004 9014 male ... germ cells +T295 GO:0000239 8994 9003 pachytene +T296 PR:000006549 9082 9087 Dmrt7 +T297 NCBITaxon:10088 9095 9100 mouse +T298 PR:000006549 9115 9120 Dmrt7 +T299 GO:0000239 9169 9178 pachytene +T300 GO:0007126 9188 9195 meiotic +T301 GO:0051324 9196 9204 prophase +T302 GO:0000240 9274 9283 diplonema +T303 GO:0001739 9297 9310 sex chromatin +T304 PR:000006549 9371 9376 Dmrt7 +T305 GO:0000785 9418 9427 chromatin +T306 GO:0016568 9418 9438 chromatin transition +T307 GO:0000239 9447 9456 pachynema +T308 GO:0000240 9461 9470 diplonema +T309 GO:0007126 9478 9485 meiotic +T310 GO:0051324 9486 9494 prophase +T311 GO:0010467 9506 9516 Expression +T312 PR:000006549 9520 9525 DMRT7 +T313 UBERON:0000473 9538 9544 Testis +T314 GO:0010467 9564 9574 expression +T315 GO:0007126 9605 9612 meiotic +T316 PR:000006549 9626 9631 Dmrt7 +T317 GO:0010467 9646 9656 expression +T318 PR:000006549 9660 9665 Dmrt7 +T319 UBERON:0000991 9684 9690 gonads +T320 UBERON:0000992 9727 9732 ovary +T321 PR:000006549 9734 9739 Dmrt7 +T322 GO:0007126 9811 9818 meiosis +T323 GO:0006279 9835 9858 pre-meiotic replication +T324 GO:0000239 9866 9875 pachytene +T325 PR:000006549 9895 9900 Dmrt7 +T326 GO:0010467 9901 9911 expression +T327 GO:0007126 9923 9930 meiotic +T328 UBERON:0000473 9937 9943 testis +T329 UBERON:0007023 10000 10005 adult +T330 PR:000006549 10006 10011 Dmrt7 +T331 GO:0010467 10012 10022 expression +T332 UBERON:0007023 10091 10096 adult +T333 UBERON:0000062 10097 10103 organs +T334 PR:000006549 10124 10129 Dmrt7 +T335 GO:0010467 10135 10145 expression +T336 UBERON:0000473 10153 10159 testis +T337 GO:0010467 10175 10185 expression +T338 UBERON:0000948 10189 10194 heart +T339 UBERON:0000479 10217 10223 tissue +T340 PR:000006549 10270 10275 Dmrt7 +T341 GO:0010467 10281 10291 expression +T342 GO:0007567 10303 10308 natal +T343 UBERON:0000473 10309 10315 testis +T344 GO:0008584 10309 10327 testis development +T345 GO:0010467 10348 10358 expression +T346 GO:0000239 10424 10433 pachytene +T347 GO:0007283 10477 10492 spermatogenesis +T348 GO:0010467 10522 10532 Expression +T349 PR:000006549 10536 10541 Dmrt7 +T350 PR:000006549 10583 10588 Dmrt7 +T351 UBERON:0000062 10603 10609 organs +T352 UBERON:0007023 10613 10618 adult +T353 NCBITaxon:10088 10619 10624 mouse +T354 UBERON:0000062 10641 10646 organ +T355 SO:0000112 10666 10673 primers +T356 PR:000006549 10687 10692 Dmrt7 +T357 PR:000003676 10707 10714 β-actin +T358 PR:000006549 10734 10739 Dmrt7 +T359 GO:0010467 10745 10755 expression +T360 GO:0007283 10782 10797 spermatogenesis +T361 UBERON:0000473 10819 10825 testis +T362 GO:0007567 10854 10859 birth +T363 PR:000006549 10891 10896 DMRT7 +T364 GO:0010467 10905 10915 expression +T365 UBERON:0000473 10939 10945 testis +T366 GO:0042571 10987 10995 antibody +T367 PR:000006549 10999 11004 DMRT7 +T368 CHEBI:51231 11017 11021 DAPI +T369 PR:000006549 11035 11040 DMRT7 +T370 GO:0001741 11069 11076 XY body +T371 UBERON:0000473 11078 11084 Testis +T372 GO:0042571 11126 11136 antibodies +T373 PR:000006549 11140 11145 DMRT7 +T374 PR:000015829 11156 11162 SUMO-1 +T375 PR:000015829 11172 11178 SUMO-1 +T376 GO:0001741 11199 11206 XY body +T377 GO:0000239 11295 11304 pachytene +T378 CL:0000017 11305 11318 spermatocytes +T379 GO:0001741 11324 11333 XY bodies +T380 PR:000006549 11351 11356 DMRT7 +T381 GO:0010467 11365 11375 expression +T382 GO:0042571 11393 11401 antibody +T383 GO:0042571 11453 11461 antibody +T384 SO:0000417 11512 11518 domain +T385 SO:0000417 11566 11572 domain +T386 PR:000006549 11624 11629 DMRT7 +T387 UBERON:0001977 11634 11638 sera +T388 PR:000006549 11651 11656 DMRT7 +T389 GO:0010467 11668 11677 expressed +T390 GO:0000239 11708 11717 pachytene +T391 CL:0000017 11718 11731 spermatocytes +T392 CL:0000019 11759 11764 sperm +T393 CL:0000586 11797 11806 germ cell +T394 CL:0000020 11823 11836 spermatogonia +T395 CL:0000018 11847 11857 spermatids +T396 PR:000006549 11877 11882 DMRT7 +T397 CL:0002371 11894 11907 somatic cells +T398 CL:0000216 11916 11929 Sertoli cells +T399 CL:0002481 11931 11954 peritubular myoid cells +T400 UBERON:0000025 11935 11942 tubular +T401 CL:0000178 11959 11971 Leydig cells +T402 GO:0000239 12005 12014 pachytene +T403 PR:000006549 12025 12030 DMRT7 +T404 GO:0010467 12031 12041 expression +T405 GO:0042571 12069 12077 antibody +T406 PR:000007857 12081 12086 GATA1 +T407 GO:0010467 12097 12106 expressed +T408 CL:0000216 12110 12123 Sertoli cells +T409 PR:000006549 12172 12177 DMRT7 +T410 GO:0010467 12181 12190 expressed +T411 GO:0000239 12207 12216 pachytene +T412 CL:0000017 12217 12230 spermatocytes +T413 GO:0000239 12332 12341 pachytene +T414 CL:0000017 12342 12355 spermatocytes +T415 PR:000006549 12357 12362 DMRT7 +T416 GO:0001741 12386 12393 XY body +T417 GO:0001741 12398 12406 sex body +T418 GO:0000785 12427 12436 chromatin +T419 GO:0000803 12461 12476 sex chromosomes +T420 GO:0031507 12525 12547 heterochromatinization +T421 GO:0051324 12595 12603 prophase +T422 NCBITaxon:40674 12607 12616 mammalian +T423 GO:0007140 12617 12629 male meiosis +T424 PR:000006549 12646 12651 DMRT7 +T425 GO:0010467 12660 12670 expression +T426 GO:0001741 12678 12685 XY body +T427 NCBITaxon:10088 12705 12710 mouse +T428 UBERON:0000473 12711 12717 testis +T429 PR:000006549 12731 12736 DMRT7 +T430 PR:000015829 12741 12775 small ubiquitin-related modifier 1 +T431 PR:000015829 12777 12783 SUMO-1 +T432 GO:0001741 12815 12822 XY body +T433 GO:0000239 12830 12839 pachynema +T434 PR:000006549 12849 12854 DMRT7 +T435 PR:000015829 12859 12865 SUMO-1 +T436 PR:000006549 12900 12905 DMRT7 +T437 GO:0001741 12949 12956 XY body +T438 GO:0001741 12988 12995 XY body +T439 PR:000006549 13012 13017 DMRT7 +T440 PR:000027547 13068 13071 H2A +T441 PR:000006549 13102 13107 DMRT7 +T442 GO:0001741 13147 13154 XY body +T443 UBERON:0000483 13202 13212 epithelial +T444 PR:000006549 13238 13243 DMRT7 +T445 GO:0001741 13261 13268 XY body +T446 GO:0000239 13287 13296 pachynema +T447 GO:0000239 13336 13345 pachynema +T448 GO:0000240 13365 13374 diplonema +T449 GO:0007126 13442 13449 meiotic +T450 PR:000006549 13471 13476 DMRT7 +T451 CL:0000019 13511 13516 sperm +T452 GO:0042571 13523 13531 antibody +T453 GO:0005634 13559 13566 nuclear +T454 CL:0000019 13579 13584 sperm +T455 GO:0002177 13590 13599 manchette +T456 UBERON:0000483 13634 13644 epithelial +T457 PR:000006549 13661 13666 DMRT7 +T458 GO:0001741 13684 13691 XY body +T459 CL:0000017 13695 13708 spermatocytes +T460 PR:000006549 13751 13756 Dmrt7 +T461 PR:000006549 13802 13807 Dmrt7 +T462 PR:000006549 13822 13827 Dmrt7 +T463 NCBITaxon:10088 13831 13835 mice +T464 UBERON:0000922 13862 13871 embryonic +T465 CL:0002322 13862 13876;13882 13887 embryonic stem ... cells +T466 CL:0002322 13878 13880;13882 13887 ES ... cells +T467 PR:000006549 13935 13940 Dmrt7 +T468 SO:0000704 13941 13945 gene +T469 SO:0000147 13955 13960 exons +T470 SO:0000417 13973 13979 domain +T471 SO:0000147 14012 14017 exons +T472 SO:0000417 14034 14040 domain +T473 SO:0000704 14076 14081 genes +T474 PR:O18214 14093 14098 mab-3 +T475 PR:P23023 14112 14115 dsx +T476 SO:0000359 14167 14173 floxed +T477 SO:0001023 14175 14181 allele +T478 SO:0000417 14198 14204 domain +T479 SO:0000147 14216 14221 exons +T480 PR:000006549 14225 14230 Dmrt7 +T481 SO:0000357 14235 14242 flanked +T482 SO:0000346 14289 14299 loxP sites +T483 SO:0001644 14306 14322 targeting vector +T484 CHEBI:7507 14340 14348 neomycin +T485 SO:0005853 14360 14368 cassette +T486 SO:0000357 14375 14382 flanked +T487 SO:0000350 14386 14408 Flpe recognition sites +T488 SO:0000417 14489 14495 domain +T489 GO:0006413 14504 14523 translational start +T490 SO:0000318 14504 14528 translational start site +T491 SO:0001023 14562 14568 allele +T492 CL:0002322 14612 14619 ES cell +T493 UBERON:0000358 14709 14720 blastocysts +T494 NCBITaxon:33208 14731 14738 animals +T495 SO:0001023 14785 14791 allele +T496 NCBITaxon:33208 14824 14831 animals +T497 PR:000003676 14845 14852 β-actin +T498 NCBITaxon:10088 14857 14861 mice +T499 SO:0000417 14879 14885 domain +T500 SO:0000147 14895 14900 exons +T501 PR:000006549 14917 14922 Dmrt7 +T502 SO:0001023 14924 14930 allele +T503 NCBITaxon:10088 14954 14958 mice +T504 SO:0005853 14977 14985 cassette +T505 PR:000006549 15002 15007 Dmrt7 +T506 SO:0000359 15007 15011 flox +T507 SO:0001023 15012 15018 allele +T508 PR:000006549 15020 15025 Dmrt7 +T509 NCBITaxon:10088 15029 15033 mice +T510 PR:000006549 15061 15066 Dmrt7 +T511 NCBITaxon:10088 15070 15074 mice +T512 PR:000006549 15110 15115 DMRT7 +T513 PR:000006549 15127 15132 Dmrt7 +T514 UBERON:0000473 15135 15141 testes +T515 GO:0007126 15154 15161 meiotic +T516 PR:000006549 15175 15180 Dmrt7 +T517 UBERON:0000473 15226 15232 testes +T518 PR:000006549 15333 15338 DMRT7 +T519 UBERON:0000473 15361 15367 testes +T520 PR:000006549 15370 15375 Dmrt7 +T521 GO:0048232 15392 15396;15412 15425 Male ... Gametogenesis +T522 GO:0007292 15405 15425 Female Gametogenesis +T523 PR:000006549 15439 15444 Dmrt7 +T524 GO:0040007 15597 15601 grew +T525 UBERON:0000113 15605 15614 adulthood +T526 GO:0019098 15646 15661 sexual behavior +T527 UBERON:0000992 15748 15755 ovarian +T528 PR:000006549 15838 15843 Dmrt7 +T529 http://purl.obolibrary.org/obo/MONDO_0005047 15884 15893 infertile +T530 UBERON:0000473 15902 15908 testes +T531 UBERON:0007023 15974 15979 adult +T532 UBERON:0000473 16032 16038 testis +T533 GO:0008584 16032 16050 testis development +T534 PR:000006549 16061 16066 Dmrt7 +T535 UBERON:0000473 16092 16098 testes +T536 GO:0007283 16160 16175 spermatogenesis +T537 GO:0007567 16190 16195 natal +T538 UBERON:0000473 16217 16223 testes +T539 UBERON:0000473 16263 16269 testis +T540 CL:0000020 16359 16372 spermatogonia +T541 GO:0007126 16383 16390 meiotic +T542 CL:0000586 16391 16401 germ cells +T543 UBERON:0000473 16463 16469 testes +T544 PR:000006549 16477 16482 Dmrt7 +T545 NCBITaxon:10088 16490 16494 mice +T546 GO:0040007 16505 16509 grow +T547 PR:000006549 16592 16597 Dmrt7 +T548 UBERON:0000473 16605 16611 testes +T549 CL:0000586 16626 16636 germ cells +T550 GO:0000239 16647 16656 pachynema +T551 GO:0022403 16668 16677;16683 16688 stages of ... cells +T552 CL:0000586 16678 16688 germ cells +T553 PR:000006549 16729 16734 Dmrt7 +T554 NCBITaxon:10088 16742 16746 mice +T555 GO:0007126 16768 16775 meiotic +T556 CL:0000018 16776 16786 spermatids +T557 UBERON:0001301 16796 16806 epididymal +T558 CL:0000019 16807 16818 spermatozoa +T559 CL:0000018 16862 16871 spermatid +T560 GO:0007126 16885 16892 meiotic +T561 PR:000006549 16964 16969 Dmrt7 +T562 PR:000006549 16996 17001 Dmrt7 +T563 UBERON:0000025 17009 17016 tubules +T564 CL:0000216 17061 17074 Sertoli cells +T565 CL:0000020 17079 17092 spermatogonia +T566 CL:0000656 17115 17136 primary spermatocytes +T567 UBERON:0000025 17156 17163 tubules +T568 CL:0000228 17172 17192 multinucleated cells +T569 GO:0005634 17177 17186 nucleated +T570 CL:0002242 17197 17207;17223 17229 cells with ... nuclei +T571 GO:0005634 17223 17229 nuclei +T572 CL:0000445 17250 17265 apoptotic cells +T573 UBERON:0000473 17298 17304 Testis +T574 CL:0000586 17314 17323 Germ Cell +T575 GO:0006915 17319 17333 Cell Apoptosis +T576 NCBITaxon:10088 17337 17341 Mice +T577 PR:000006549 17368 17373 Dmrt7 +T578 UBERON:0000473 17379 17385 Testes +T579 NCBITaxon:10088 17418 17423 mouse +T580 PR:000006549 17447 17452 Dmrt7 +T581 UBERON:0000473 17491 17497 testes +T582 NCBITaxon:10088 17544 17548 mice +T583 CHEBI:51686 17566 17577 hematoxylin +T584 UBERON:0000025 17706 17713 tubules +T585 CL:0000017 17735 17748 spermatocytes +T586 UBERON:0000025 17852 17859 tubules +T587 CL:0000228 17868 17887 multinucleate cells +T588 GO:0005634 17873 17881 nucleate +T589 PR:000006549 17940 17945 Dmrt7 +T590 NCBITaxon:10088 17956 17961 mouse +T591 UBERON:0000473 17962 17968 testes +T592 UBERON:0000473 17970 17976 Testes +T593 CL:0000445 18068 18083 apoptotic cells +T594 UBERON:0000473 18085 18091 Testis +T595 NCBITaxon:10088 18132 18136 mice +T596 CL:0000445 18142 18157 Apoptotic cells +T597 UBERON:0001343 18192 18212 seminiferous tubules +T598 PR:000006549 18227 18232 Dmrt7 +T599 NCBITaxon:10088 18240 18244 mice +T600 PR:000006549 18307 18312 Dmrt7 +T601 UBERON:0000473 18320 18326 testes +T602 GO:0000239 18342 18351 pachytene +T603 GO:0006915 18434 18443 apoptosis +T604 PR:000006549 18454 18459 Dmrt7 +T605 UBERON:0000473 18467 18473 testes +T606 CL:0000445 18501 18516 apoptotic cells +T607 UBERON:0000025 18569 18575 tubule +T608 GO:0005634 18613 18619 nuclei +T609 PR:000006549 18652 18657 Dmrt7 +T610 GO:0006915 18741 18750 apoptosis +T611 UBERON:0000473 18774 18780 testes +T612 CL:0000445 18819 18834 apoptotic cells +T613 UBERON:0000025 18861 18868 tubules +T614 CL:0000445 18882 18897 apoptotic cells +T615 UBERON:0001343 18933 18952 seminiferous tubule +T616 CL:0000216 18979 18992 Sertoli cells +T617 UBERON:0000473 19055 19061 testes +T618 CL:0002371 19096 19108 somatic cell +T619 GO:0006915 19104 19118 cell apoptosis +T620 PR:000006549 19195 19200 Dmrt7 +T621 GO:0007126 19219 19226 meiotic +T622 GO:0000239 19250 19259 pachynema +T623 GO:0006915 19292 19301 apoptosis +T624 GO:0000239 19327 19336 Pachytene +T625 PR:000006549 19347 19352 Dmrt7 +T626 CL:0000586 19360 19370 Germ Cells +T627 GO:0007283 19393 19406 spermatogenic +T628 PR:000006549 19422 19427 Dmrt7 +T629 CL:0000015 19431 19446 male germ cells +T630 GO:0016265 19458 19461 die +T631 GO:0042571 19471 19481 antibodies +T632 CL:0000586 19513 19522 germ cell +T633 GO:0010467 19541 19550 expressed +T634 CL:0000670 19554 19558 PGCs +T635 CL:0000020 19563 19576 spermatogonia +T636 UBERON:0007023 19600 19605 adult +T637 UBERON:0000473 19606 19612 testis +T638 UBERON:0000025 19771 19778 tubules +T639 GO:0042571 19831 19839 antibody +T640 CL:0000017 19851 19864 spermatocytes +T641 GO:0000237 19872 19881 leptotene +T642 GO:0000239 19891 19900 pachytene +T643 PR:000006549 19914 19919 Dmrt7 +T644 UBERON:0000473 19927 19933 testes +T645 UBERON:0000025 20094 20101 tubules +T646 GO:0042571 20126 20134 antibody +T647 PR:000005573 20148 20156 calmegin +T648 GO:0010467 20165 20174 expressed +T649 GO:0000239 20178 20187 pachytene +T650 CL:0000017 20188 20201 spermatocytes +T651 CL:0000018 20220 20230 spermatids +T652 UBERON:0000473 20327 20333 testes +T653 GO:0098762 20417 20430 meiotic stage +T654 CL:0000017 20437 20449 spermatocyte +T655 GO:0007129 20468 20486 chromosome-pairing +T656 PR:000015865 20510 20515 SYCP3 +T657 GO:0000795 20536 20556 synaptonemal complex +T658 PR:000006549 20583 20588 Dmrt7 +T659 GO:0000239 20608 20617 pachytene +T660 GO:0000239 20674 20683 pachynema +T661 GO:0007126 20737 20744 meiotic +T662 PR:000006549 20755 20760 Dmrt7 +T663 GO:0000239 20793 20802 pachynema +T664 GO:0007128 20870 20880 Prophase I +T665 PR:000006549 20891 20896 Dmrt7 +T666 CL:0000586 20904 20914 Germ Cells +T667 UBERON:0000473 20916 20922 Testis +T668 PR:000006549 20966 20971 Dmrt7 +T669 GO:0042571 21025 21035 antibodies +T670 GO:0007283 21048 21061 spermatogenic +T671 GO:0042571 21080 21088 antibody +T672 CL:0000020 21105 21118 spermatogonia +T673 GO:0042571 21172 21180 antibody +T674 CL:0000017 21188 21201 spermatocytes +T675 GO:0042571 21258 21266 antibody +T676 GO:0000239 21274 21283 pachytene +T677 CL:0000586 21294 21304 germ cells +T678 UBERON:0000473 21349 21355 testis +T679 GO:0007126 21379 21386 Meiotic +T680 PR:000006549 21397 21402 Dmrt7 +T681 UBERON:0000473 21410 21416 Testis +T682 GO:0098762 21443 21460;21466 21471 meiotic stages of ... cells +T683 CL:0000586 21461 21471 germ cells +T684 UBERON:0000473 21540 21546 testes +T685 GO:0000795 21575 21595 synaptonemal complex +T686 PR:000015865 21604 21609 SYCP3 +T687 GO:0007126 21687 21694 Meiotic +T688 GO:0051324 21695 21703 Prophase +T689 PR:000006549 21707 21712 Dmrt7 +T690 CL:0000586 21720 21730 Germ Cells +T691 GO:0007129 21743 21761 chromosome pairing +T692 GO:0007129 21763 21771 synapsis +T693 GO:0000239 21802 21811 pachytene +T694 GO:0006915 21823 21832 apoptosis +T695 PR:000006549 21877 21882 Dmrt7 +T696 UBERON:0000473 21890 21896 testes +T697 GO:0007129 21916 21924 synapsis +T698 GO:0042571 21934 21944 antibodies +T699 PR:000015862 21948 21953 SYCP1 +T700 GO:0000795 21957 21977 synaptonemal complex +T701 GO:0000802 21978 21996 transverse element +T702 PR:000015865 22012 22017 SYCP3 +T703 GO:0000800 22038 22051 axial element +T704 GO:0007129 22076 22084 synapsed +T705 GO:0000240 22097 22106 diplonema +T706 GO:0007130 22116 22151 Formation of synaptonemal complexes +T707 GO:0000795 22129 22151 synaptonemal complexes +T708 PR:000015862 22255 22260 SYCP1 +T709 PR:000015865 22284 22289 SYCP3 +T710 PR:000006549 22317 22322 Dmrt7 +T711 GO:0000238 22330 22338 zygotene +T712 CL:0000017 22339 22352 spermatocytes +T713 GO:0000725 22393 22413 recombination repair +T714 PR:000013672 22421 22426 RAD51 +T715 GO:0007126 22450 22457 meiotic +T716 PR:000006549 22515 22520 Dmrt7 +T717 CL:0000017 22528 22541 spermatocytes +T718 PR:000013672 22592 22597 RAD51 +T719 GO:0030849 22623 22632 autosomal +T720 GO:0000795 22633 22655 synaptonemal complexes +T721 GO:0000239 22739 22748 pachynema +T722 GO:0007129 22784 22792 synapsis +T723 GO:0000240 22800 22809 diplonema +T724 GO:0007129 22860 22879 chromosomal pairing +T725 GO:0007129 22881 22889 synapsis +T726 GO:0007129 22912 22920 synapsis +T727 GO:0007128 22928 22938 prophase I +T728 PR:000006549 22942 22947 Dmrt7 +T729 GO:0007129 22999 23007 Synapsis +T730 PR:000006549 23029 23034 Dmrt7 +T731 CL:0000586 23042 23052 Germ Cells +T732 UBERON:0000473 23058 23068 Testicular +T733 PR:000006549 23080 23085 Dmrt7 +T734 NCBITaxon:10088 23132 23136 mice +T735 GO:0042571 23166 23174 antibody +T736 PR:000015865 23178 23183 SYCP3 +T737 GO:0007126 23219 23226 meiotic +T738 GO:0051324 23227 23235 prophase +T739 PR:000015865 23245 23250 SYCP3 +T740 GO:0000239 23296 23305 pachynema +T741 GO:0030849 23311 23320 autosomes +T742 GO:0007129 23331 23339 synapsed +T743 GO:0007129 23364 23372 synapsed +T744 UBERON:0000473 23449 23459 Testicular +T745 GO:0000238 23469 23477 zygonema +T746 GO:0000239 23482 23491 pachynema +T747 GO:0042571 23516 23526 antibodies +T748 PR:000013672 23530 23535 RAD51 +T749 PR:000015865 23548 23553 SYCP3 +T750 PR:000013672 23586 23591 RAD51 +T751 CL:0000017 23638 23651 spermatocytes +T752 CL:0000216 23701 23714 Sertoli Cells +T753 CL:0000216 23716 23729 Sertoli cells +T754 CL:0000586 23744 23754 germ cells +T755 GO:0007283 23762 23777 spermatogenesis +T756 CL:0000586 23814 23823 germ cell +T757 GO:0048469 23819 23834 cell maturation +T758 PR:000006549 23868 23873 DMRT7 +T759 GO:0010467 23874 23884 expression +T760 CL:0000216 23888 23901 Sertoli cells +T761 GO:0042571 23905 23913 antibody +T762 CL:0000216 23972 23984 Sertoli cell +T763 PR:000006549 24052 24057 Dmrt7 +T764 CL:0000216 24083 24095 Sertoli cell +T765 GO:0030154 24091 24111 cell differentiation +T766 GO:0010467 24125 24135 expression +T767 CL:0000216 24143 24155 Sertoli cell +T768 PR:000007859 24164 24169 GATA4 +T769 GO:0007567 24196 24201 natal +T770 CL:0000216 24202 24215 Sertoli cells +T771 PR:000007857 24221 24226 GATA1 +T772 CL:0000216 24237 24249 Sertoli cell +T773 CHEBI:50113 24368 24376 androgen +T774 CL:0000216 24446 24459 Sertoli cells +T775 PR:000006549 24463 24468 Dmrt7 +T776 UBERON:0000473 24476 24482 testes +T777 UBERON:0000025 24505 24512 tubules +T778 PR:000007857 24513 24518 GATA1 +T779 CL:0000216 24528 24540 Sertoli cell +T780 GO:0005634 24536 24547 cell nuclei +T781 UBERON:0000025 24645 24652 tubules +T782 GO:0005634 24654 24663;24681 24686 nuclei of ... cells +T783 GO:0007126 24668 24675 meiotic +T784 CL:0000586 24676 24686 germ cells +T785 CL:0000017 24691 24704 spermatocytes +T786 CL:0000586 24753 24763 germ cells +T787 CL:0000216 24820 24832 Sertoli Cell +T788 PR:000006549 24849 24854 Dmrt7 +T789 UBERON:0000473 24862 24868 Testes +T790 UBERON:0000473 24874 24880 Testes +T791 PR:000006549 24904 24909 Dmrt7 +T792 NCBITaxon:10088 24917 24921 mice +T793 GO:0042571 24954 24962 antibody +T794 PR:000007859 24966 24971 GATA4 +T795 UBERON:0000473 24982 24988 testis +T796 GO:0042571 25011 25019 antibody +T797 PR:000007857 25023 25028 GATA1 +T798 PR:000007859 25038 25043 GATA4 +T799 PR:000007857 25048 25053 GATA1 +T800 CL:0000216 25097 25109 Sertoli cell +T801 UBERON:0000473 25137 25143 testis +T802 GO:0042571 25173 25181 antibody +T803 PR:000007857 25185 25190 GATA1 +T804 CHEBI:51231 25201 25205 DAPI +T805 CL:0000216 25219 25231 Sertoli cell +T806 GO:0005634 25227 25238 cell nuclei +T807 CL:0000216 25300 25313 Sertoli cells +T808 UBERON:0000025 25337 25344 tubules +T809 UBERON:0000473 25420 25426 Testis +T810 CL:0000216 25465 25477 Sertoli cell +T811 PR:000006549 25487 25492 Dmrt7 +T812 CL:0000216 25501 25503 SC +T813 NCBITaxon:10088 25513 25517 mice +T814 CHEBI:51686 25531 25542 hematoxylin +T815 GO:0007283 25554 25569 Spermatogenesis +T816 GO:0007286 25574 25588 spermiogenesis +T817 CL:0000216 25603 25605 SC +T818 UBERON:0000473 25614 25620 testis +T819 UBERON:0000473 25627 25633 Testis +T820 CL:0000216 25672 25674 SC +T821 NCBITaxon:10088 25683 25687 mice +T822 GO:0042571 25701 25711 antibodies +T823 PR:000007857 25715 25720 GATA1 +T824 UBERON:0001135 25731 25744 smooth muscle +T825 PR:000003675 25731 25750 smooth muscle actin +T826 UBERON:0001343 25763 25783 seminiferous tubules +T827 CL:0000216 25793 25805 Sertoli cell +T828 GO:0005634 25801 25812 cell nuclei +T829 CL:0000216 25864 25866 SC +T830 NCBITaxon:10088 25875 25879 mice +T831 CL:0000216 25895 25907 Sertoli cell +T832 PR:000006549 25924 25929 Dmrt7 +T833 UBERON:0000473 25937 25943 testes +T834 CL:0000586 25976 25985 germ cell +T835 CL:0000216 26036 26048 Sertoli cell +T836 PR:000006549 26096 26101 Dmrt7 +T837 CL:0000216 26114 26126 Sertoli cell +T838 NCBITaxon:10088 26147 26151 mice +T839 SO:0000359 26165 26171 floxed +T840 PR:000006549 26172 26177 Dmrt7 +T841 SO:0001023 26178 26184 allele +T842 PR:000006459 26190 26193 Dhh +T843 NCBITaxon:10088 26209 26213 mice +T844 PR:000006459 26224 26239 Desert hedgehog +T845 PR:000006459 26241 26244 Dhh +T846 SO:0000167 26246 26254 promoter +T847 CL:0000216 26296 26309 Sertoli cells +T848 CL:0000586 26321 26331 germ cells +T849 PR:000006549 26354 26359 Dmrt7 +T850 CL:0000216 26363 26376 Sertoli cells +T851 UBERON:0000473 26435 26445 Testicular +T852 CL:0000216 26454 26461 Sertoli +T853 CL:0000216 26472 26474 SC +T854 NCBITaxon:33208 26484 26491 animals +T855 CL:0000216 26616 26618 SC +T856 UBERON:0000473 26627 26633 testes +T857 GO:0007283 26647 26662 Spermatogenesis +T858 CL:0000019 26687 26692 sperm +T859 CL:0000216 26711 26713 SC +T860 NCBITaxon:10088 26722 26726 mice +T861 PR:000007857 26754 26759 GATA1 +T862 CL:0000216 26781 26793 Sertoli cell +T863 GO:0005634 26789 26800 cell nuclei +T864 CL:0000586 26903 26912 germ cell +T865 PR:000006549 26924 26929 Dmrt7 +T866 PR:000006549 26964 26969 Dmrt7 +T867 CL:0000216 26973 26986 Sertoli cells +T868 CL:0000216 27025 27038 Sertoli cells +T869 PR:000006549 27070 27075 Dmrt7 +T870 GO:0001741 27095 27104 XY Bodies +T871 PR:000006549 27122 27127 Dmrt7 +T872 PR:000006549 27182 27187 Dmrt7 +T873 CL:0000586 27195 27205 germ cells +T874 GO:0007126 27238 27245 meiosis +T875 GO:0000239 27269 27278 pachynema +T876 PR:000006549 27311 27316 Dmrt7 +T877 GO:0007126 27373 27380 meiotic +T878 GO:0007126 27414 27421 meiotic +T879 CL:0000586 27422 27432 germ cells +T880 GO:0001741 27466 27473 XY body +T881 GO:0007126 27512 27519 meiotic +T882 PR:000006549 27564 27569 DMRT7 +T883 GO:0030261 27584 27599;27612 27623 Condensation of ... chromosomes +T884 GO:0000805 27604 27605;27612 27623 X ... chromosomes +T885 GO:0000806 27610 27623 Y chromosomes +T886 GO:0000238 27639 27647 zygotene +T887 GO:0000239 27667 27676 pachynema +T888 GO:0001739 27734 27747 sex chromatin +T889 GO:0034399 27809 27826 nuclear periphery +T890 PR:000006549 27857 27862 DMRT7 +T891 GO:0001741 27879 27886 XY body +T892 GO:0001741 27934 27941 XY body +T893 GO:0000785 27942 27951 chromatin +T894 GO:0010467 27985 27995 expression +T895 PR:000008418 28027 28031 H2AX +T896 PR:000027547 28048 28051 H2A +T897 GO:0001741 28072 28079 XY body +T898 GO:0001741 28137 28144 XY body +T899 PR:000006549 28148 28153 DMRT7 +T900 GO:0007126 28170 28177 meiotic +T901 CL:0000586 28246 28256 germ cells +T902 PR:000006549 28260 28265 Dmrt7 +T903 UBERON:0000473 28273 28279 testes +T904 PR:000015829 28311 28317 SUMO-1 +T905 UBERON:0000473 28345 28351 testis +T906 PR:000015829 28353 28359 SUMO-1 +T907 GO:0010467 28360 28370 expression +T908 GO:0001741 28397 28404 XY body +T909 GO:0000239 28422 28431 pachytene +T910 CL:0000017 28432 28445 spermatocytes +T911 GO:0000803 28461 28475 sex chromosome +T912 GO:0030261 28465 28488 chromosome condensation +T913 GO:0007127 28521 28543 first meiotic division +T914 PR:000015829 28545 28551 SUMO-1 +T915 GO:0001741 28572 28579 XY body +T916 GO:0000805 28587 28588;28595 28606 X ... chromosomes +T917 GO:0000806 28593 28606 Y chromosomes +T918 GO:0007129 28609 28616 synapse +T919 PR:000015829 28632 28638 SUMO-1 +T920 PR:000006549 28667 28672 Dmrt7 +T921 CL:0000586 28680 28690 germ cells +T922 GO:0001741 28746 28753 XY body +T923 UBERON:0000025 28781 28788 tubules +T924 UBERON:0000119 28813 28828 layers of cells +T925 PR:000015829 28834 28840 SUMO-1 +T926 UBERON:0000119 28900 28914 layer of cells +T927 GO:0001741 28937 28944 XY body +T928 GO:0000239 29078 29087 pachytene +T929 PR:000027547 29109 29112 H2A +T930 PR:000006549 29129 29134 Dmrt7 +T931 UBERON:0000473 29142 29148 testes +T932 GO:0000239 29159 29168 pachytene +T933 PR:000027547 29173 29176 H2A +T934 GO:0001741 29200 29207 XY body +T935 GO:0000239 29216 29225 pachytene +T936 PR:000027547 29229 29232 H2A +T937 GO:0005634 29267 29274 nucleus +T938 GO:0001741 29312 29319 XY body +T939 GO:0000239 29328 29337 pachytene +T940 CL:0000017 29338 29351 spermatocytes +T941 GO:0005634 29370 29377 nuclear +T942 PR:000027547 29403 29406 H2A +T943 GO:0001741 29433 29440 XY body +T944 PR:000006549 29444 29449 Dmrt7 +T945 PR:000006549 29513 29518 Dmrt7 +T946 CL:0000586 29526 29536 germ cells +T947 GO:0001741 29554 29561 XY body +T948 GO:0000785 29595 29604 chromatin +T949 GO:0001741 29623 29630 XY Body +T950 GO:0000239 29653 29662 Pachynema +T951 PR:000006549 29666 29671 Dmrt7 +T952 NCBITaxon:10088 29679 29683 Mice +T953 UBERON:0000473 29689 29699 Testicular +T954 GO:0042571 29730 29740 antibodies +T955 PR:000015865 29760 29765 SYCP3 +T956 GO:0000239 29778 29787 pachytene +T957 GO:0001741 29824 29831 XY body +T958 PR:000006549 29854 29859 Dmrt7 +T959 UBERON:0000473 29873 29879 Testes +T960 NCBITaxon:10088 29894 29898 mice +T961 GO:0042571 29926 29934 antibody +T962 UBERON:0000473 29956 29962 Testes +T963 NCBITaxon:10088 29977 29981 mice +T964 GO:0042571 30009 30017 antibody +T965 PR:000015829 30021 30027 SUMO-1 +T966 GO:0000239 30046 30055 pachytene +T967 GO:0001741 30067 30076 XY bodies +T968 UBERON:0000473 30113 30119 testes +T969 GO:0001739 30131 30144 Sex Chromatin +T970 PR:000006549 30148 30153 Dmrt7 +T971 CL:0000586 30161 30171 Germ Cells +T972 GO:0001741 30186 30193 XY body +T973 GO:0000239 30210 30219 pachynema +T974 PR:000006549 30223 30228 Dmrt7 +T975 GO:0006342 30273 30298 transcriptional silencing +T976 PR:000006549 30368 30373 Dmrt7 +T977 GO:0000239 30385 30394 pachytene +T978 GO:0000239 30461 30470 pachytene +T979 GO:0007135 30524 30534 meiosis II +T980 GO:0007286 30539 30553 spermiogenesis +T981 GO:0000785 30589 30598 chromatin +T982 GO:0007126 30622 30629 meiotic +T983 GO:0001739 30630 30643 sex chromatin +T984 GO:0000240 30683 30692 diplonema +T985 GO:0000239 30730 30739 pachytene +T986 CL:0000586 30740 30749 germ cell +T987 GO:0008219 30745 30755 cell death +T988 PR:000006549 30759 30764 Dmrt7 +T989 GO:0000803 30836 30850 sex chromosome +T990 GO:0000239 30893 30902 pachytene +T991 GO:0001741 30903 30910 XY body +T992 GO:0097617 30996 31009 hybridization +T993 GO:0006366 31035 31066 RNA polymerase II transcription +T994 CHEBI:51231 31082 31086 DAPI +T995 GO:0001741 31110 31117 XY body +T996 UBERON:0001343 31132 31152 seminiferous tubules +T997 PR:000006549 31176 31181 Dmrt7 +T998 GO:0001741 31195 31202 XY body +T999 GO:0097617 31233 31246 hybridization +T1000 GO:0006342 31276 31301 transcriptional silencing +T1001 GO:0000239 31336 31345 pachytene +T1002 GO:0010467 31370 31380 expression +T1003 GO:0000806 31388 31389 Y +T1004 SO:0000704 31397 31401 gene +T1005 PR:000030954 31402 31406 Rbmy +T1006 GO:0000239 31445 31454 pachytene +T1007 GO:0007135 31477 31494 secondary meiosis +T1008 PR:000030954 31511 31515 Rbmy +T1009 GO:0000239 31544 31553 pachytene +T1010 PR:000006549 31563 31568 Dmrt7 +T1011 PR:000030954 31627 31631 RBMY +T1012 GO:0042571 31632 31640 antibody +T1013 GO:0000792 31671 31686 heterochromatin +T1014 PR:000005082 31671 31701 heterochromatin protein 1 beta +T1015 PR:000005082 31703 31707 HP1β +T1016 GO:0000805 31742 31743 X +T1017 GO:0000239 31762 31771 pachynema +T1018 GO:0001741 31801 31808 XY body +T1019 GO:0000240 31835 31844 diplonema +T1020 PR:000005082 31865 31869 HP1β +T1021 PR:000006549 31896 31901 DMRT7 +T1022 GO:0000239 31922 31931 pachynema +T1023 GO:0001741 31979 31986 XY body +T1024 PR:000006549 32043 32048 Dmrt7 +T1025 CL:0000586 32056 32066 germ cells +T1026 GO:0000785 32079 32088 Chromatin +T1027 PR:000006549 32106 32111 Dmrt7 +T1028 GO:0000240 32119 32128 Diplotene +T1029 CL:0000586 32129 32139 Germ Cells +T1030 GO:0000239 32179 32188 pachytene +T1031 CL:0000017 32189 32201 spermatocyte +T1032 PR:000006549 32205 32210 Dmrt7 +T1033 GO:0001741 32235 32242 XY body +T1034 GO:0006342 32294 32306;32322 32335 silencing of ... transcription +T1035 GO:0000803 32307 32321 sex chromosome +T1036 GO:0001741 32339 32346 XY body +T1037 GO:0000239 32386 32395 pachynema +T1038 PR:000005082 32437 32441 HP1β +T1039 GO:0000239 32462 32471 pachytene +T1040 CL:0000017 32472 32485 spermatocytes +T1041 PR:000005082 32487 32491 HP1β +T1042 GO:0000805 32505 32517 X chromosome +T1043 GO:0097617 32591 32604 hybridization +T1044 GO:0000240 32608 32617 diplotene +T1045 GO:0001741 32631 32638 XY body +T1046 CHEBI:51231 32649 32653 DAPI +T1047 PR:000005082 32719 32723 HP1β +T1048 GO:0000240 32727 32736 diplotene +T1049 PR:000005082 32761 32765 HP1β +T1050 GO:0001741 32777 32784 XY body +T1051 GO:0000805 32835 32847 X chromosome +T1052 PR:000027594 32883 32885 H3 +T1053 SO:0001728 32883 32891 H3-2meK9 +T1054 GO:0000240 32908 32917 diplotene +T1055 GO:0001741 32988 32995 XY body +T1056 PR:000027594 33030 33032 H3 +T1057 SO:0001707 33030 33038 H3-3meK9 +T1058 GO:0000240 33055 33064 diplotene +T1059 GO:0001741 33128 33135 XY body +T1060 GO:0030849 33156 33165 autosomes +T1061 GO:0006915 33211 33220 apoptosis +T1062 GO:0000240 33245 33254 diplotene +T1063 PR:000005082 33272 33276 HP1β +T1064 GO:0001741 33297 33304 XY body +T1065 GO:0001739 33402 33415 sex chromatin +T1066 GO:0000239 33483 33492 pachynema +T1067 PR:000006549 33531 33536 Dmrt7 +T1068 GO:0006915 33568 33577 apoptosis +T1069 GO:0000240 33587 33596 diplonema +T1070 PR:000006549 33657 33662 Dmrt7 +T1071 CL:0000017 33670 33683 spermatocytes +T1072 GO:0000239 33697 33706 pachytene +T1073 GO:0000240 33734 33743 diplonema +T1074 GO:0097617 33795 33808 hybridization +T1075 GO:0000792 33819 33834 heterochromatic +T1076 GO:0000791 33884 33895 euchromatic +T1077 GO:0001739 33949 33962 sex chromatin +T1078 GO:0016458 33991 33999 silenced +T1079 CHEBI:15358 34089 34096 histone +T1080 PR:000027594 34089 34099 histone H3 +T1081 PR:000027594 34143 34145 H3 +T1082 SO:0001728 34143 34151 H3-2meK9 +T1083 PR:000027594 34153 34155 H3 +T1084 SO:0001707 34153 34161 H3-3meK9 +T1085 PR:000005082 34180 34184 HP1β +T1086 GO:0001741 34197 34204 XY body +T1087 GO:0001739 34275 34288 sex chromatin +T1088 PR:000006549 34326 34331 Dmrt7 +T1089 GO:0000240 34332 34341 diplotene +T1090 PR:000005082 34358 34362 HP1β +T1091 GO:0000805 34383 34395 X chromosome +T1092 GO:0000239 34440 34449 pachynema +T1093 PR:000006549 34463 34468 Dmrt7 +T1094 GO:0000240 34476 34485 diplotene +T1095 PR:000005082 34525 34529 HP1β +T1096 GO:0001741 34544 34551 XY body +T1097 PR:000006549 34592 34597 Dmrt7 +T1098 GO:0000240 34605 34614 diplotene +T1099 PR:000027594 34645 34647 H3 +T1100 SO:0001728 34645 34653 H3-2meK9 +T1101 PR:000027594 34658 34660 H3 +T1102 SO:0001707 34658 34666 H3-3meK9 +T1103 GO:0001739 34682 34695 sex chromatin +T1104 PR:000006549 34721 34726 Dmrt7 +T1105 GO:0000240 34734 34743 diplotene +T1106 PR:000005082 34782 34786 HP1β +T1107 GO:0001739 34794 34807 sex chromatin +T1108 GO:0000240 34862 34871 diplonema +T1109 PR:000005082 34879 34883 HP1β +T1110 GO:0001741 34891 34898 XY body +T1111 PR:000005082 34988 34992 HP1β +T1112 GO:0007126 35024 35031 meiosis +T1113 GO:0000240 35070 35079 diplotene +T1114 GO:0001739 35103 35116 sex chromatin +T1115 GO:0030849 35135 35144 autosomal +T1116 SO:0000985 35192 35205 double-strand +T1117 MOP:0000780 35210 35216 breaks +T1118 GO:0000240 35258 35267 diplotene +T1119 GO:0006915 35309 35318 apoptosis +T1120 GO:0001739 35344 35357 sex chromatin +T1121 GO:0000240 35375 35384 diplonema +T1122 GO:0001741 35554 35561 XY body +T1123 GO:0042571 35701 35709 antibody +T1124 PR:000015865 35713 35718 SYCP3 +T1125 PR:000006549 35761 35766 Dmrt7 +T1126 GO:0000240 35774 35783 diplotene +T1127 PR:000005082 35798 35802 HP1β +T1128 GO:0001741 35823 35830 XY body +T1129 PR:000006549 35903 35908 Dmrt7 +T1130 GO:0001741 35941 35948 XY body +T1131 GO:0000239 35956 35965 pachynema +T1132 GO:0001739 36016 36029 sex chromatin +T1133 GO:0016568 36020 36040 chromatin transition +T1134 GO:0000239 36046 36055 pachynema +T1135 GO:0000240 36059 36068 diplonema +T1136 GO:0001739 36090 36103 Sex Chromatin +T1137 GO:0007129 36123 36141 Chromosome Pairing +T1138 CL:0000586 36174 36183 germ cell +T1139 CHEBI:51231 36197 36201 DAPI +T1140 PR:000015865 36208 36213 SYCP3 +T1141 PR:000005082 36224 36228 HP1β +T1142 GO:0000240 36270 36279 diplonema +T1143 GO:0001741 36297 36304 XY body +T1144 PR:000005082 36310 36314 HP1β +T1145 PR:000006549 36344 36349 Dmrt7 +T1146 CL:0000586 36357 36366 germ cell +T1147 GO:0000240 36382 36391 diplotene +T1148 GO:0001741 36431 36438 XY body +T1149 PR:000005082 36447 36451 HP1β +T1150 GO:0001741 36472 36479 XY body +T1151 SO:0000417 36574 36580 domain +T1152 PR:000006549 36589 36594 DMRT7 +T1153 CL:0000015 36611 36626 male germ cells +T1154 GO:0007126 36639 36646 meiotic +T1155 GO:0051324 36647 36655 prophase +T1156 PR:000006549 36710 36715 DMRT7 +T1157 GO:0010467 36716 36726 expression +T1158 GO:0000239 36741 36750 pachytene +T1159 CL:0000017 36751 36764 spermatocytes +T1160 GO:0001741 36814 36821 XY body +T1161 GO:0010467 36844 36854 expression +T1162 CL:0000015 36882 36897 male germ cells +T1163 GO:0000239 36908 36917 pachynema +T1164 GO:0006915 36930 36939 apoptosis +T1165 GO:0000240 36985 36994 diplonema +T1166 GO:0000785 37032 37041 chromatin +T1167 GO:0000240 37103 37112 diplonema +T1168 GO:0001739 37122 37135 sex chromatin +T1169 GO:0000239 37176 37185 pachytene +T1170 GO:0051324 37195 37203 prophase +T1171 GO:0007129 37267 37274 synapse +T1172 GO:0051598 37324 37346 pachytene surveillance +T1173 GO:0065007 37364 37371 monitor +T1174 GO:0007126 37386 37393 meiotic +T1175 GO:0006915 37485 37494 apoptosis +T1176 GO:0000239 37522 37531 pachynema +T1177 NCBITaxon:40674 37540 37547 mammals +T1178 GO:0000803 37572 37587 sex chromosomes +T1179 GO:0001741 37597 37604 XY body +T1180 GO:0007140 37651 37663 male meiosis +T1181 NCBITaxon:10088 37671 37675 mice +T1182 NCBITaxon:10088 37680 37684 mice +T1183 GO:0000803 37696 37710 sex-chromosome +T1184 GO:0030849 37714 37722 autosome +T1185 GO:0000803 37766 37780 sex chromosome +T1186 GO:0000239 37823 37832 pachynema +T1187 GO:0065007 37904 37916 surveillance +T1188 GO:0006915 37940 37949 apoptosis +T1189 PR:000006549 37953 37958 Dmrt7 +T1190 CL:0000017 37966 37979 spermatocytes +T1191 GO:0000239 38034 38043 pachytene +T1192 GO:0007129 38143 38163 chromosomal synapsis +T1193 PR:000006549 38199 38204 Dmrt7 +T1194 GO:0001741 38247 38254 XY body +T1195 PR:000006549 38283 38288 DMRT7 +T1196 GO:0001741 38332 38339 XY body +T1197 PR:000006549 38373 38378 Dmrt7 +T1198 GO:0001741 38441 38448 XY body +T1199 GO:0000785 38507 38516 chromatin +T1200 GO:0097617 38552 38565 hybridization +T1201 PR:000030954 38582 38586 RBMY +T1202 GO:0010467 38587 38597 expression +T1203 GO:0001741 38647 38654 XY body +T1204 GO:0000239 38662 38671 pachytene +T1205 PR:000006549 38672 38677 Dmrt7 +T1206 GO:0001739 38748 38761 sex chromatin +T1207 PR:000006549 38765 38770 Dmrt7 +T1208 CL:0000586 38778 38788 germ cells +T1209 GO:0000239 38812 38821 pachynema +T1210 GO:0000240 38845 38854 diplonema +T1211 PR:000027594 38882 38884 H3 +T1212 SO:0001728 38882 38890 H3-2meK9 +T1213 PR:000027594 38895 38897 H3 +T1214 SO:0001707 38895 38903 H3-3meK9 +T1215 PR:000005082 38914 38918 HP1β +T1216 GO:0001739 38934 38947 sex chromatin +T1217 GO:0000240 38968 38977 diplonema +T1218 GO:0000240 39002 39011 diplotene +T1219 PR:000006549 39071 39076 Dmrt7 +T1220 CL:0000586 39084 39094 germ cells +T1221 GO:0000239 39113 39122 pachynema +T1222 GO:0000240 39126 39135 diplonema +T1223 GO:0001739 39158 39171 sex chromatin +T1224 GO:0016568 39162 39184 chromatin modification +T1225 GO:0001739 39227 39240 sex chromatin +T1226 PR:000006549 39273 39278 DMRT7 +T1227 GO:0007126 39295 39302 meiosis +T1228 NCBITaxon:40674 39346 39353 mammals +T1229 GO:0000240 39378 39387 diplotene +T1230 GO:0001739 39417 39430 sex chromatin +T1231 GO:0000240 39515 39524 diplonema +T1232 PR:000006549 39540 39545 Dmrt7 +T1233 CL:0000586 39553 39563 germ cells +T1234 GO:0006915 39582 39591 apoptosis +T1235 GO:0001739 39629 39642 sex chromatin +T1236 GO:0006915 39679 39688 apoptosis +T1237 GO:0001739 39713 39726 sex chromatin +T1238 GO:0001739 39762 39775 sex chromatin +T1239 GO:0006915 39794 39803 apoptosis +T1240 GO:0001739 39876 39889 sex chromatin +T1241 GO:0006915 39920 39929 apoptosis +T1242 GO:0006915 39946 39955 apoptosis +T1243 GO:0001739 39969 39982 sex chromatin +T1244 PR:000006549 40022 40027 Dmrt7 +T1245 PR:000006549 40130 40135 DMRT7 +T1246 GO:0001741 40209 40216 XY body +T1247 PR:000043452 40228 40235 histone +T1248 CHEBI:15358 40228 40235 histone +T1249 PR:000043452 40258 40266 histones +T1250 CHEBI:15358 40258 40266 histones +T1251 UBERON:0000473 40270 40276 testis +T1252 PR:000043452 40286 40293 histone +T1253 CHEBI:15358 40286 40293 histone +T1254 CHEBI:32875 40294 40300 methyl +T1255 SO:0000910 40337 40343 orphan +T1256 CL:0000586 40353 40362 germ-cell +T1257 PR:000011413 40353 40377 germ-cell nuclear factor +T1258 GO:0005634 40363 40370 nuclear +T1259 GO:0000792 40475 40490 heterochromatin +T1260 PR:000006549 40526 40531 DMRT7 +T1261 GO:0001741 40549 40556 XY body +T1262 PR:000006549 40669 40674 DMRT7 +T1263 GO:0000785 40746 40755 chromatin +T1264 GO:0001741 40774 40781 XY body +T1265 GO:0000785 40817 40826 Chromatin +T1266 GO:0065007 40827 40837 regulation +T1267 SO:0000417 40871 40877 domain +T1268 SO:0000417 40913 40919 domain +T1269 GO:0000785 40944 40953 chromatin +T1270 GO:0016568 40944 40963 chromatin modifying +T1271 GO:0032991 40964 40973 complexes +T1272 PR:000006549 41059 41064 Dmrt7 +T1273 NCBITaxon:40674 41082 41091 mammalian +T1274 GO:0007126 41092 41099 meiosis +T1275 SO:0000704 41136 41140 gene +T1276 SO:0000417 41165 41171 domain +T1277 SO:0000704 41172 41177 genes +T1278 CL:0002371 41221 41234 somatic cells +T1279 SO:0000417 41249 41255 domain +T1280 SO:0000704 41256 41261 genes +T1281 PR:000006542 41263 41268 Dmrt1 +T1282 PR:000006545 41273 41278 Dmrt4 +T1283 CL:0000586 41290 41299 germ cell +T1284 GO:0007281 41290 41311 germ cell development +T1285 NCBITaxon:10088 41319 41324 mouse +T1286 PR:000006542 41326 41331 Dmrt1 +T1287 GO:0007126 41351 41358 meiotic +T1288 CL:0000015 41359 41374 male germ cells +T1289 GO:0030154 41379 41394 differentiation +T1290 CL:0000670 41398 41407 gonocytes +T1291 CL:0000020 41413 41426 spermatogonia +T1292 CL:0000216 41442 41455 Sertoli cells +T1293 GO:0010467 41471 41480 expressed +T1294 GO:0007126 41484 41491 meiotic +T1295 PR:000006542 41567 41572 DMRT1 +T1296 GO:0007126 41580 41587 meiotic +T1297 CL:0000586 41588 41598 germ cells +T1298 PR:000006549 41603 41608 DMRT7 +T1299 GO:0007126 41612 41619 meiotic +T1300 CL:0000586 41620 41630 germ cells +T1301 SO:0000417 41652 41658 domain +T1302 CL:0000015 41703 41717 male germ cell +T1303 GO:0007281 41708 41729 germ cell development +T1304 UBERON:0000992 41731 41738 Ovaries +T1305 PR:000006545 41742 41747 Dmrt4 +T1306 CL:0000025 41772 41778 ovular +T1307 CL:0000023 41820 41827 oocytes +T1308 NCBITaxon:40674 41943 41952 mammalian +T1309 SO:0000417 41956 41962 domain +T1310 SO:0000704 41963 41968 genes +T1311 UBERON:0000991 41976 41983 gonadal +T1312 GO:0008406 41976 41995 gonadal development +T1313 GO:0007546 42070 42102 sex-specific somatic development +T1314 NCBITaxon:phylum 42112 42117 phyla +T1315 PR:000006549 42132 42137 Dmrt7 +T1316 UBERON:0001987 42162 42171 placental +T1317 NCBITaxon:9347 42162 42179 placental mammals +T1318 NCBITaxon:9263 42193 42203 marsupials +T1319 NCBITaxon:9255 42210 42219 monotreme +T1320 UBERON:0007379 42221 42224 egg +T1321 NCBITaxon:9255 42221 42238 egg-laying mammal +T1322 NCBITaxon:9258 42245 42253 platypus +T1323 PR:000006549 42273 42278 Dmrt7 +T1324 SO:0000855 42279 42287 ortholog +T1325 PR:000006549 42312 42317 Dmrt7 +T1326 SO:0000855 42318 42326 ortholog +T1327 NCBITaxon:40674 42351 42360 mammalian +T1328 NCBITaxon:7742 42361 42372 vertebrates +T1329 PR:000006549 42426 42431 Dmrt7 +T1330 SO:0000704 42503 42507 gene +T1331 NCBITaxon:40674 42547 42556 mammalian +T1332 NCBITaxon:9255 42568 42578 Monotremes +T1333 GO:0000805 42589 42590;42602 42613 X ... chromosomes +T1334 GO:0000806 42600 42613 Y chromosomes +T1335 GO:0007126 42659 42666 meiosis +T1336 GO:0000803 42695 42710 sex chromosomes +T1337 NCBITaxon:40674 42724 42731 mammals +T1338 PR:000006549 42754 42759 Dmrt7 +T1339 GO:0000803 42820 42835 sex chromosomes +T1340 GO:0001739 42843 42856 sex chromatin +T1341 NCBITaxon:9255 42860 42870 monotremes +T1342 NCBITaxon:40674 42881 42888 mammals +T1343 PR:000006549 42916 42921 Dmrt7 +T1344 NCBITaxon:40674 42958 42967 mammalian +T1345 GO:0007530 42968 42985 sex determination +T1346 SO:0000704 43029 43033 gene +T1347 GO:0007129 43042 43060 chromosome pairing +T1348 GO:0007126 43086 43093 meiotic +T1349 GO:0000803 43114 43128 sex chromosome +T1350 PR:000006549 43196 43201 Dmrt7 +T1351 NCBITaxon:40674 43209 43218 mammalian +T1352 GO:0000785 43268 43277 chromatin +T1353 GO:0065007 43278 43288 regulatory +T1354 GO:0032991 43289 43298 complexes +T1355 GO:0007549 43318 43337 dosage compensation +T1356 GO:0000803 43372 43387 sex chromosomes +T1357 NCBITaxon:phylum 43399 43404 phyla +T1358 PR:000006549 43469 43474 DMRT7 +T1359 GO:0000803 43488 43503 sex chromosomes +T1360 NCBITaxon:9255 43511 43520 monotreme +T1361 GO:0007126 43521 43528 meiosis +T1362 NCBITaxon:40674 43566 43572 mammal +T1363 SO:0000417 43585 43591 domain +T1364 PR:000006549 43600 43605 DMRT7 +T1365 GO:0007126 43623 43630 meiotic +T1366 GO:0051324 43631 43639 prophase +T1367 PR:000006549 43662 43667 DMRT7 +T1368 GO:0000803 43685 43700 sex chromosomes +T1369 GO:0000792 43743 43758 heterochromatin +T1370 PR:000006549 43769 43774 Dmrt7 +T1371 GO:0016568 43819 43834;43843 43852 modification of ... chromatin +T1372 GO:0001739 43839 43852 sex chromatin +T1373 GO:0000239 43861 43870 pachytene +T1374 GO:0000240 43875 43884 diplotene +T1375 PR:000006549 43895 43900 Dmrt7 +T1376 SO:0000704 43937 43941 gene +T1377 NCBITaxon:40674 43970 43977 mammals +T1378 PR:000006549 44000 44005 DMRT7 +T1379 NCBITaxon:40674 44031 44037 mammal +T1380 GO:0007126 44077 44084 meiosis +T1381 PR:000006549 44151 44156 DMRT7 +T1382 GO:0001739 44165 44178 sex chromatin +T1383 GO:0065007 44179 44189 regulation +T1384 GO:0007140 44197 44209 male meiosis +T1385 PR:000006549 44249 44254 Dmrt7 +T1386 NCBITaxon:10088 44255 44259 mice +T1387 NCBITaxon:10088 44264 44269 mouse +T1388 PR:000006549 44270 44275 Dmrt7 +T1389 SO:0000147 44316 44320 exon +T1390 NCBITaxon:10088 44344 44349 mouse +T1391 SO:0000153 44350 44353 BAC +T1392 SO:0000167 44449 44457 promoter +T1393 PR:000006549 44506 44511 Dmrt7 +T1394 SO:0001026 44512 44519 genomic +T1395 SO:0001644 44534 44550 targeting vector +T1396 SO:0000440 44629 44635 vector +T1397 SO:0000440 44666 44672 vector +T1398 SO:0000346 44701 44710 loxP site +T1399 SO:0000188 44764 44770 intron +T1400 PR:000006549 44774 44779 Dmrt7 +T1401 SO:0000028 44790 44792 bp +T1402 SO:0000028 44802 44804 bp +T1403 SO:0000147 44819 44823 exon +T1404 SO:0000028 44920 44922 bp +T1405 SO:0000028 44930 44932 bp +T1406 PR:000006549 44943 44948 Dmrt7 +T1407 GO:0006412 44949 44962 translational +T1408 SO:0000346 44993 45002 loxP site +T1409 SO:0000061 45012 45016 site +T1410 SO:0000028 45021 45023 bp +T1411 PR:000006549 45034 45039 Dmrt7 +T1412 GO:0006412 45040 45053 translational +T1413 SO:0000440 45078 45084 vector +T1414 SO:0000147 45107 45112 exons +T1415 PR:000006549 45116 45121 Dmrt7 +T1416 SO:0000357 45126 45133 flanked +T1417 SO:0000346 45137 45147 loxP sites +T1418 SO:0000359 45149 45155 floxed +T1419 PR:000006549 45162 45167 Dmrt7 +T1420 CL:0002322 45286 45294 ES cells +T1421 CHEBI:42768 45394 45398 G418 +T1422 GO:0097617 45430 45443 hybridization +T1423 SO:0000147 45497 45501 exon +T1424 SO:0001026 45514 45521 genomic +T1425 GO:0097617 45634 45647 hybridization +T1426 GO:0097617 45669 45682 hybridization +T1427 SO:0000112 45706 45713 primers +T1428 CL:0002322 45796 45803 ES cell +T1429 SO:0000359 45826 45832 floxed +T1430 SO:0001023 45833 45839 allele +T1431 PR:000006549 45840 45845 Dmrt7 +T1432 UBERON:0000358 45876 45887 blastocysts +T1433 PR:000006549 45991 45996 Dmrt7 +T1434 PR:000006549 46001 46006 Dmrt7 +T1435 PR:000006549 46008 46013 Dmrt7 +T1436 PR:000003676 46045 46052 β-actin +T1437 NCBITaxon:10088 46068 46072 mice +T1438 SO:0000359 46087 46093 floxed +T1439 PR:000006549 46130 46135 Dmrt7 +T1440 PR:000006549 46201 46206 Dmrt7 +T1441 UBERON:0002415 46260 46264 tail +T1442 SO:0000112 46346 46353 primers +T1443 GO:0000806 46361 46373 Y chromosome +T1444 SO:0000704 46374 46378 gene +T1445 PR:000017657 46379 46382 Zfy +T1446 PR:000006549 46406 46411 Dmrt7 +T1447 SO:0001023 46412 46418 allele +T1448 PR:000006549 46419 46424 Dmrt7 +T1449 GO:0097617 46472 46481 annealing +T1450 PR:000006549 46508 46513 Dmrt7 +T1451 SO:0000359 46513 46517 flox +T1452 SO:0001023 46518 46524 allele +T1453 GO:0097617 46573 46582 annealing +T1454 PR:000006549 46617 46622 Dmrt7 +T1455 SO:0001023 46623 46629 allele +T1456 PR:000006549 46630 46635 Dmrt7 +T1457 GO:0097617 46677 46686 annealing +T1458 PR:000006549 46720 46725 Dmrt7 +T1459 GO:0010467 46726 46736 expression +T1460 SO:0000112 46774 46781 primers +T1461 GO:0097617 46802 46811 annealing +T1462 SO:0000112 46835 46842 Primers +T1463 UBERON:0000473 47486 47492 testes +T1464 CHEBI:50913 47515 47523 fixative +T1465 CHEBI:16842 47546 47554 formalin +T1466 CHEBI:16236 47611 47618 ethanol +T1467 CHEBI:73702 47652 47655 wax +T1468 CHEBI:51686 47723 47734 hematoxylin +T1469 CHEBI:16240 47853 47870 hydrogen peroxide +T1470 GO:0005634 47923 47930 nuclear +T1471 CL:0000445 47943 47958 apoptotic cells +T1472 UBERON:0000479 48073 48079 Tissue +T1473 CHEBI:53424 48164 48172 Tween 20 +T1474 CHEBI:30769 48205 48216 citric acid +T1475 UBERON:0001977 48278 48283 serum +T1476 GO:0042571 48325 48333 antibody +T1477 GO:0042571 48397 48407 antibodies +T1478 GO:0042571 48460 48470 antibodies +T1479 GO:0042571 48473 48483 Antibodies +T1480 NCBITaxon:9986 48486 48492 Rabbit +T1481 GO:0042571 48504 48514 antibodies +T1482 PR:000006549 48518 48523 DMRT7 +T1483 CHEBI:16856 48581 48592 glutathione +T1484 CHEBI:18245 48626 48627 C +T1485 PR:000006549 48656 48661 DMRT7 +T1486 GO:0042571 48663 48673 Antibodies +T1487 UBERON:0001977 48739 48744 serum +T1488 PR:000006549 48770 48775 DMRT7 +T1489 PR:000006549 48803 48808 DMRT7 +T1490 GO:0042571 48809 48817 antibody +T1491 NCBITaxon:9925 48852 48856 goat +T1492 NCBITaxon:9986 48862 48868 rabbit +T1493 GO:0042571 48879 48887 antibody +T1494 GO:0042571 48967 48977 antibodies +T1495 NCBITaxon:10114 49011 49014 rat +T1496 PR:000007857 49020 49025 GATA1 +T1497 NCBITaxon:9925 49090 49094 goat +T1498 PR:000007859 49100 49105 GATA4 +T1499 NCBITaxon:10114 49150 49153 rat +T1500 NCBITaxon:10114 49210 49213 rat +T1501 NCBITaxon:10114 49267 49270 rat +T1502 NCBITaxon:9986 49328 49334 rabbit +T1503 PR:000013672 49340 49345 RAD51 +T1504 NCBITaxon:10088 49400 49405 mouse +T1505 PR:000015829 49411 49416 GMP-1 +T1506 PR:000015829 49417 49423 SUMO-1 +T1507 NCBITaxon:9986 49472 49478 rabbit +T1508 CHEBI:32958 49484 49491 phospho +T1509 PR:000008418 49492 49496 H2AX +T1510 NCBITaxon:10088 49558 49563 mouse +T1511 CHEBI:32958 49569 49576 phospho +T1512 PR:000008418 49577 49581 H2AX +T1513 NCBITaxon:10088 49608 49613 mouse +T1514 PR:000015865 49619 49624 SYCP3 +T1515 NCBITaxon:9986 49672 49678 rabbit +T1516 PR:000005082 49684 49688 HP1β +T1517 NCBITaxon:9986 49714 49720 rabbit +T1518 PR:000027594 49726 49728 H3 +T1519 SO:0001728 49726 49734 H3-2meK9 +T1520 NCBITaxon:9986 49761 49767 rabbit +T1521 PR:000027594 49773 49775 H3 +T1522 SO:0001707 49773 49781 H3-3meK9 +T1523 NCBITaxon:9986 49808 49814 rabbit +T1524 NCBITaxon:10088 49877 49882 mouse +T1525 PR:000003675 49888 49892 αSMA +T1526 GO:0042571 49965 49975 antibodies +T1527 NCBITaxon:9925 49986 49990 goat +T1528 NCBITaxon:9986 49996 50002 rabbit +T1529 CHEBI:52661 50003 50012 Alexa 488 +T1530 NCBITaxon:9925 50014 50018 goat +T1531 NCBITaxon:9986 50024 50030 rabbit +T1532 CHEBI:51248 50031 50040 Alexa 594 +T1533 NCBITaxon:9925 50042 50046 goat +T1534 NCBITaxon:10114 50052 50055 rat +T1535 CHEBI:51248 50056 50065 Alexa 594 +T1536 NCBITaxon:9925 50071 50075 goat +T1537 NCBITaxon:10088 50081 50086 mouse +T1538 CHEBI:52661 50087 50096 Alexa 488 +T1539 NCBITaxon:9793 50131 50137 Donkey +T1540 NCBITaxon:9925 50143 50147 goat +T1541 CHEBI:37926 50148 50152 FITC +T1542 NCBITaxon:9793 50225 50231 donkey +T1543 NCBITaxon:9986 50237 50243 rabbit +T1544 CHEBI:51247 50244 50253 Texas Red +T1545 GO:0007126 50329 50336 Meiotic +T1546 GO:0007126 50379 50386 Meiotic +T1547 NCBITaxon:10088 50442 50446 mice +T1548 GO:0007126 50537 50544 meiotic +T1549 GO:0007126 50636 50643 meiotic +T1550 CL:0000017 50644 50657 spermatocytes +T1551 PR:000006549 51136 51141 Dmrt7 +T1552 GO:0007126 51149 51156 Meiotic +T1553 GO:0001741 51198 51205 XY body +T1554 PR:000015865 51207 51212 SYCP3 +T1555 GO:0042571 51213 51221 antibody +T1556 GO:0007129 51252 51260 synapsis +T1557 PR:000006549 51297 51302 Dmrt7 +T1558 PR:000006549 51316 51321 DMRT7 +T1559 GO:0001741 51356 51363 XY body +T1560 PR:000006549 51410 51415 Dmrt7 +T1561 PR:000006549 51524 51529 Dmrt7 +T1562 SO:0001644 51598 51614 targeting vector +T1563 PR:000006549 51634 51639 Dmrt7 +T1564 SO:0001023 51640 51646 allele +T1565 PR:000006549 51648 51653 Dmrt7 +T1566 SO:0001023 51681 51687 allele +T1567 SO:0001023 51702 51708 allele +T1568 SO:0000346 51718 51728 loxP sites +T1569 SO:0000357 51729 51737 flanking +T1570 GO:0006412 51742 51755 translational +T1571 SO:0000147 51787 51792 exons +T1572 SO:0000417 51812 51818 domain +T1573 CHEBI:7507 51840 51849 neomycine +T1574 SO:0005853 51861 51869 cassette +T1575 SO:0000357 51870 51877 flanked +T1576 PR:P03870 51881 51884 Flp +T1577 SO:0000350 51881 51914 Flp recombinase recognition sites +T1578 SO:0000350 51916 51925 frt sites +T1579 NCBITaxon:10088 51928 51932 Mice +T1580 PR:000006542 51954 51959 Dmrt1 +T1581 SO:0001023 51963 51969 allele +T1582 GO:0007618 51975 51980 mated +T1583 NCBITaxon:10088 51997 52001 mice +T1584 GO:0010467 52002 52012 expressing +T1585 SO:0000346 52084 52094 loxP sites +T1586 SO:0005853 52114 52122 cassette +T1587 SO:0001023 52147 52153 allele +T1588 PR:000006549 52164 52169 Dmrt7 +T1589 GO:0007618 52172 52178 Mating +T1590 PR:000006549 52182 52187 Dmrt7 +T1591 NCBITaxon:10088 52191 52195 mice +T1592 NCBITaxon:10088 52212 52216 mice +T1593 GO:0010467 52217 52227 expressing +T1594 SO:0005853 52265 52273 cassette +T1595 PR:000006549 52290 52295 Dmrt7 +T1596 SO:0000359 52295 52299 flox +T1597 SO:0001023 52300 52306 allele +T1598 CL:0002322 52354 52361 ES cell +T1599 SO:0001026 52370 52377 Genomic +T1600 CL:0002322 52388 52395 ES cell +T1601 GO:0097617 52465 52475 hybridizes +T1602 GO:0097617 52553 52563 hybridizes +T1603 UBERON:0000473 52631 52637 Testis +T1604 UBERON:0007023 52652 52657 adult +T1605 NCBITaxon:10088 52658 52662 mice +T1606 PR:000015829 52664 52670 SUMO-1 +T1607 GO:0001741 52682 52689 XY body +T1608 PR:000006549 52730 52735 Dmrt7 +T1609 PR:000006549 52761 52766 DMRT7 +T1610 PR:000006549 52812 52817 Dmrt7 +T1611 PR:000015829 52828 52834 SUMO-1 +T1612 CHEBI:50113 52971 52979 Androgen +T1613 GO:0010467 52989 52999 Expression +T1614 PR:000006549 53003 53008 Dmrt7 +T1615 CL:0000216 53016 53029 Sertoli Cells +T1616 UBERON:0000473 53031 53037 Testis +T1617 UBERON:0007023 53052 53057 adult +T1618 NCBITaxon:10088 53058 53062 mice +T1619 GO:0042571 53076 53084 antibody +T1620 CHEBI:50113 53088 53096 androgen +T1621 CHEBI:51231 53136 53140 DAPI +T1622 UBERON:0000473 53193 53199 testis +T1623 CHEBI:50113 53210 53218 androgen +T1624 GO:0010467 53239 53248 expressed +T1625 CL:0000216 53252 53264 Sertoli cell +T1626 CL:0002481 53269 53291 peritubular myoid cell +T1627 UBERON:0000025 53273 53280 tubular +T1628 UBERON:0000473 53300 53306 testis +T1629 CL:0000216 53346 53358 Sertoli cell +T1630 GO:0005634 53354 53365 cell nuclei +T1631 CHEBI:50113 53416 53424 androgen +T1632 PR:000027547 53502 53505 H2A +T1633 GO:0001741 53522 53529 XY Body +T1634 PR:000006549 53533 53538 Dmrt7 +T1635 CL:0000017 53546 53558 Spermatocyte +T1636 GO:0000239 53567 53576 pachytene +T1637 CL:0000017 53577 53590 spermatocytes +T1638 PR:000006549 53616 53621 Dmrt7 +T1639 NCBITaxon:10088 53638 53642 mice +T1640 GO:0042571 53657 53665 antibody +T1641 PR:000027547 53672 53675 H2A +T1642 CHEBI:51231 53688 53692 DAPI +T1643 GO:0001741 53717 53724 XY body +T1644 GO:0000239 53752 53761 pachytene +T1645 PR:000027547 53776 53779 H2A +T1646 GO:0001741 53802 53809 XY body +T1647 PR:000030954 53875 53879 RBMY +T1648 GO:0016458 53880 53889 Silencing +T1649 PR:000006549 53893 53898 Dmrt7 +T1650 CL:0000017 53906 53919 Spermatocytes +T1651 PR:000015865 53925 53930 SYCP3 +T1652 PR:000030954 53943 53947 RBMY +T1653 PR:000006549 53993 53998 Dmrt7 +T1654 UBERON:0000473 54006 54016 testicular +T1655 PR:000030954 54055 54059 RBMY +T1656 GO:0010467 54063 54072 expressed +T1657 GO:0000237 54090 54099 leptotene +T1658 GO:0000239 54110 54119 pachytene +T1659 CL:0000017 54120 54133 spermatocytes +T1660 PR:000030954 54135 54139 RBMY +T1661 PR:000015829 54207 54213 SUMO-1 +T1662 PR:000030954 54226 54230 RBMY +T1663 UBERON:0007023 54255 54260 adult +T1664 UBERON:0000473 54261 54267 testis +T1665 PR:000006549 54296 54301 Dmrt7 +T1666 GO:0007126 54314 54321 meiotic +T1667 GO:0010467 54352 54359 express +T1668 PR:000030954 54374 54378 RBMY +T1669 PR:000015829 54387 54393 SUMO-1 +T1670 GO:0000239 54403 54412 pachytene +T1671 GO:0010467 54426 54433 express +T1672 PR:000030954 54434 54438 RBMY +T1673 GO:0016458 54488 54497 silencing +T1674 GO:0007126 54733 54740 meiotic +T1675 GO:0042571 54832 54842 antibodies +T1676 PR:000006459 54872 54875 Dhh +T1677 NCBITaxon:10088 54880 54884 mice +T1678 NCBITaxon:10088 54923 54928 Mouse +T1679 CL:0002322 54953 54960 ES cell +T1680 PR:P23023 55254 55263 Doublesex +T1681 PR:O18214 55264 55269 MAB-3 +T1682 SO:0000417 55270 55276 domain +T1683 UBERON:0000922 55283 55292 embryonic +T1684 GO:0097617 55327 55340 hybridization +T1685 CHEBI:16856 55348 55359 glutathione +T1686 PR:000005082 55375 55379 HP1β +T1687 GO:0000792 55382 55397 heterochromatin +T1688 PR:000005082 55382 55412 heterochromatin protein 1 beta +T1689 PR:000008418 55437 55441 H2AX +T1690 GO:0007126 55450 55457 meiotic +T1691 GO:0000803 55458 55472 sex chromosome +T1692 GO:0007567 55497 55502 natal +T1693 GO:0007126 55522 55529 meiotic +T1694 GO:0001739 55530 55543 sex chromatin +T1695 PR:000015829 55581 55587 SUMO-1 +T1696 PR:000015829 55590 55624 small ubiquitin-related modifier 1 +T1697 PR:000027547 55629 55632 H2A +T1698 PR:000027547 55649 55652 H2A +T1699 CHEBI:33893 56038 56046 reagents diff --git a/src/ontogpt/evaluation/craft/database/all/17447844.txt b/src/ontogpt/evaluation/craft/database/all/17447844.txt new file mode 100644 index 000000000..6a73b1a4a --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17447844.txt @@ -0,0 +1,363 @@ +A Mammal-Specific Doublesex Homolog Associates with Male Sex Chromatin and Is Required for Male Meiosis + +Abstract + +Gametogenesis is a sexually dimorphic process requiring profound differences in germ cell differentiation between the sexes. In mammals, the presence of heteromorphic sex chromosomes in males creates additional sex-specific challenges, including incomplete X and Y pairing during meiotic prophase. This triggers formation of a heterochromatin domain, the XY body. The XY body disassembles after prophase, but specialized sex chromatin persists, with further modification, through meiosis. Here, we investigate the function of DMRT7, a mammal-specific protein related to the invertebrate sexual regulators Doublesex and MAB-3. We find that DMRT7 preferentially localizes to the XY body in the pachytene stage of meiotic prophase and is required for male meiosis. In Dmrt7 mutants, meiotic pairing and recombination appear normal, and a transcriptionally silenced XY body with appropriate chromatin marks is formed, but most germ cells undergo apoptosis during pachynema. A minority of mutant cells can progress to diplonema, but many of these escaping cells have abnormal sex chromatin lacking histone H3K9 di- and trimethylation and heterochromatin protein 1β accumulation, modifications that normally occur between pachynema and diplonema. Based on the localization of DMRT7 to the XY body and the sex chromatin defects observed in Dmrt7 mutants, we conclude that DMRT7 plays a role in the sex chromatin transformation that occurs between pachynema and diplonema. We suggest that DMRT7 may help control the transition from meiotic sex chromosome inactivation to postmeiotic sex chromatin in males. In addition, because it is found in all branches of mammals, but not in other vertebrates, Dmrt7 may shed light on evolution of meiosis and of sex chromatin. + +Author Summary + + + +Genes related to the sexual regulator Doublesex of Drosophila have been found to control sexual development in a wide variety of animals, ranging from roundworms to mammals. In this paper, we investigate the function of the Dmrt7 gene, one of seven related genes in the mouse. Female mammals are XX and males are XY, a chromosomal difference that presents specific challenges during the meiotic phase of male germ cell development. Some of these are thought to be overcome by incorporating the X and Y chromosomes into a specialized structure called the XY body. We find that DMRT7 protein is present in germ cells, localizes to the male XY body during meiosis, and is essential for male but not female fertility. The XY body normally is altered by recruitment of additional proteins and by specific modifications to histone proteins between the pachytene and diplotene stages of meiosis, but modification of the “sex chromatin” in Dmrt7 mutant cells is abnormal during this period. Because Dmrt7 is found in all branches of mammals, but not in other vertebrates, these results may indicate some commonality in regulation of sex chromatin among the mammals. + +Introduction + +Sexual differentiation generates anatomical, physiological, and behavioral dimorphisms that are essential for sexual reproduction. Many of these dimorphisms affect somatic cells, but the sexual dimorphisms that most directly mediate sexual reproduction are those of the gametes themselves. Gametes differ between the sexes in size and morphology, sometimes dramatically so, reflecting their very different roles in zygote formation. Indeed, the morphology of the gametes is what defines sex: females are the sex that produces the larger gametes and males produce the smaller ones. + +Mammalian meiosis is regulated sex-specifically starting in embryogenesis and continuing through much of adult life (reviewed in [1]). For example, the timing and synchrony of meiosis are very different in the two sexes. In females, germ cells synchronously initiate meiosis in the embryo and arrest during meiotic prophase I. After puberty, oocytes are selectively recruited for ovulation, when they proceed to metaphase II and then complete meiosis after fertilization occurs [2]. In contrast, male meiosis occurs entirely postnatally, without the arrest periods found in females. In females, each meiosis can produce a single haploid oocyte (and two extruded polar bodies), whereas each male meiosis can produce four haploid spermatocytes. + +Other meiotic processes, such as recombination and chromosome pairing (synapsis), occur in both sexes but operate somewhat differently. For example, there is a higher failure rate for meiosis in females, with human oocyte aneuploidy rates up to 25% versus about 2% in human sperm [3], and this may indicate that the checkpoints controlling and monitoring the events of meiotic progression in males are more stringent. Consistent with this idea, genetic analysis of a number of meiotic regulatory genes in the mouse has demonstrated a much stronger requirement in males than in females [1,4]. + +The existence of heteromorphic sex chromosomes, such as the XX/XY system of mammals, creates sex-specific challenges. One is the need for mechanisms to balance expression of sex-linked genes between the sexes, which in mammals is accomplished by X chromosome inactivation in females [5,6]. In male germ cells there is another sex-specific consideration during meiosis. In prophase I, when the homologous chromosomes synapse and homologous recombination occurs, X and Y chromosome pairing is limited to a region termed the pseudoautosomal region, leaving large portions of each chromosome unpaired. In eutherian and marsupial mammals, these unpaired chromosome regions are associated with a specialized chromatin domain termed the XY body or sex body. The function of the XY body is uncertain [7–11], but there is evidence that it is essential for male meiotic progression [12]. + +Several proteins are reported to localize to the XY body, including BRCA1, ATR, the histone variant H3.3, and modified histones such as ubiquitinated H2A (Ub-H2A) and phosphorylated H2AX (γH2AX) [12–15]. In the XY body, the sex chromosomes are transcriptionally silenced in a process termed meiotic sex chromosome inactivation (MSCI). The XY body disappears after pachynema; however, many sex-linked genes remain transcriptionally silent into spermiogenesis [16]. This maintenance of silencing is associated with a distinct set of chromatin marks that define a sex chromatin domain termed postmeiotic sex chromatin (PMSC) [16,17]. + +Regulators of sexual differentiation have been identified in a number of organisms, but in contrast to many other developmental processes, such as axial patterning or development of many body parts, the molecular mechanisms that regulate sexual differentiation are highly variable between phyla. A notable exception involves genes related to doublesex (dsx) of Drosophila, which share a Doublesex/MAB-3 DNA-binding motif called the DM domain [18,19]. DM domain–encoding genes have been shown to regulate various aspects of sexual differentiation in insects, nematodes, and mammals [20]. The mab-3 gene of Caenorhabditis elegans has been shown to function analogously to DSX in several respects and can be functionally replaced by the male isoform of DSX, suggesting that the similarity in the sequence of these genes may stem from conservation of an ancestral DM domain sexual regulator [18,21,22]. + +Vertebrates also have DM domain genes, and analysis to date, although limited, has shown that these genes also control sexual differentiation. Mammals have seven DM domain genes (Dmrt genes), several of which exhibit sexually dimorphic mRNA expression [23,24]. The best studied of these genes, Dmrt1, is expressed in the differentiating male genital ridges and adult testis of mammals, birds, fish, and reptiles, and a recently duplicated Dmrt1 gene functions as the Y-linked testis-determining gene in the Medaka fish [25–29]. Human DMRT1 maps to an autosomal locus, which, when hemizygous, is associated with defective testicular development and consequent XY feminization [30]. Similarly, mice homozygous for a null mutation in Dmrt1 have severe defects in testis differentiation involving both germ cells and Sertoli cells [31]. Female mice mutant in Dmrt4 have polyovular follicles, indicating that this gene also plays a role in gonadal development [32]. It appears from these studies that the involvement of DM domain genes in sexual differentiation is ancient and conserved. However, vertebrate Dmrt gene function is not limited to sexual differentiation: Dmrt2 is required in both sexes for segmentation in mice and fish [33–35]. + +Here, we have investigated the expression and function of the Dmrt7 gene in the mouse. Dmrt7 is expressed only in the gonad, and, unlike the other Dmrt genes, appears to be present exclusively in mammals and not in nonmammalian vertebrates [23,36]. We find that DMRT7 protein is expressed only in germ cells and is selectively localized to the XY body of male pachytene germ cells. To test its function, we generated a conditional null mutation of Dmrt7 in the mouse. We find that Dmrt7 is required in males for progression beyond the pachytene stage of meiotic prophase but is not required in females. In rare mutant cells that survive to diplonema, we observed sex chromatin abnormalities. Based on these observations, we suggest that Dmrt7 plays a critical role in a male-specific chromatin transition between pachynema and diplonema during meiotic prophase. + +Results + +Expression of DMRT7 Proteins in Testis + +Our previous mRNA expression analysis suggested a possible meiotic function for Dmrt7, based on the expression of Dmrt7 mRNA in the fetal gonads of the two sexes [23]. In the fetal ovary, Dmrt7 mRNA was detected primarily from E13.5 to E15.5, the time during which meiosis progresses from pre-meiotic replication to the pachytene stage [4], whereas Dmrt7 expression in the non-meiotic fetal testis was very low. Because this earlier work did not examine adult Dmrt7 expression, we first performed reverse transcriptase (RT)-PCR on mRNA from ten adult organs and detected strong Dmrt7 mRNA expression in the testis and a trace of expression in heart, but not in any other tissue tested (Figure 1A). We examined the timing of Dmrt7 mRNA expression during postnatal testis development and detected strong expression beginning at 2 wk, which roughly coincides with the onset of the pachytene stage during the first synchronous wave of spermatogenesis (Figure 1B) [37]. + +Figure 1 + +Expression of Dmrt7 mRNA and Protein + +(A) RT-PCR analysis of Dmrt7 mRNA from ten organs of adult mouse. cDNA from each organ was amplified with primers specific for Dmrt7 (top row) and β-actin (bottom row). + +(B) Dmrt7 mRNA expression during the first round of spermatogenesis. cDNAs obtained from testis at the indicated days after birth were amplified as in (A). + +(C) DMRT7 protein expression. Immunofluorescence of testis sections from 6-wk-old male stained with antibody to DMRT7 (green) and DAPI (blue). + +(D) DMRT7 subcellular localization to XY body. Testis sections from 6-wk-old male stained with antibodies to DMRT7 (red) and SUMO-1 (green). SUMO-1 is localized to the XY body. Right-most panel shows merge of other two panels. Inserts show higher magnification of pachytene spermatocytes with XY bodies. + +To investigate DMRT7 protein expression, we generated an antibody against the C-terminal portion of the protein. The antibody was raised against a unique region lacking the DM domain in order to avoid cross-reaction with other DM domain proteins. Immunofluorescent staining with purified DMRT7 antisera showed that DMRT7 protein is expressed predominantly in mid- to late-pachytene spermatocytes (Figure 1C), as well as in sperm, and is not detectable in other germ cell types including spermatogonia and round spermatids. We did not detect DMRT7 protein in somatic cells such as Sertoli cells, peritubular myoid cells, or Leydig cells. To more precisely determine the pachytene stages of DMRT7 expression, we double-stained with an antibody to GATA1, which is expressed in Sertoli cells from stages VII to IX [38]. This confirmed that DMRT7 is expressed in mid- to late-pachytene spermatocytes, starting slightly earlier than stage VII and extending through stage IX (unpublished data). + +Within pachytene spermatocytes, DMRT7 is concentrated in the XY body, or sex body, a densely staining chromatin domain that harbors the sex chromosomes. These undergo transcriptional inactivation and heterochromatinization as a result of their incomplete pairing during prophase of mammalian male meiosis [17]. To verify DMRT7 protein expression in the XY body, we double-stained mouse testis sections for DMRT7 and small ubiquitin-related modifier 1 (SUMO-1), which is concentrated in the XY body during pachynema [39,40]. DMRT7 and SUMO-1 were colocalized, confirming that DMRT7 protein is preferentially localized to the XY body (Figure 1D). We also confirmed XY body localization of DMRT7 by double staining for other markers including Ub-H2A and γH2AX (unpublished data). DMRT7 is not preferentially localized to the XY body at all stages but instead is dynamic. Based on epithelial staging, it appears that DMRT7 localizes to the XY body from mid- to late-pachynema, becomes diffusely distributed in late-pachynema, and disappears in diplonema (unpublished data). This localization was confirmed by staining of meiotic spreads (Figure S1). DMRT7 also is specifically localized in sperm, with antibody staining mainly in the perinuclear ring of the sperm head manchette. This staining coincided with the epithelial stages in which DMRT7 localizes to the XY body in spermatocytes (Figure 1C and 1D). + +Targeted Deletion of Dmrt7 + +To establish the functional requirement for Dmrt7, we generated Dmrt7−/− mice by targeted disruption in embryonic stem (ES) cells using a strategy diagrammed in Figure S2A. The Dmrt7 gene has nine exons with the DM domain encoded by the second and third exons. Because the DM domain is essential for function of other genes, including mab-3, mab-23, and dsx [18,19,41], we generated a conditionally targeted “floxed” allele in which the DM domain–containing exons of Dmrt7 are flanked by recognition sites for the Cre recombinase (loxP sites). The targeting vector also contained a neomycin resistance cassette (neo) flanked by Flpe recognition sites. The removal of these sequences by Cre-mediated recombination eliminates the DM domain and the translational start site, thus generating a putative null allele. We identified three homologously targeted ES cell clones by Southern blotting (Figure S2B) and injected cells from two clones into C57BL/6 blastocysts. Chimeric animals from both cell lines transmitted the targeted allele through the germ line. Targeted animals were bred to β-actin Cre mice to delete the DM domain–encoding exons, generating the Dmrt7− allele, or to Flpe transgenic mice to delete the neo cassette, generating the Dmrt7flox allele. Dmrt7+/− mice were interbred to generate Dmrt7−/− mice. To confirm the lack of functional DMRT7 protein in Dmrt7−/−testes, we stained meiotic spreads from Dmrt7 mutants (Figure S1) and sections from mutant testes (Figure S2C) and carried out western blot analysis (unpublished data). In each case, we detected no DMRT7 protein in the mutant testes. + +Dmrt7 Is Required for Male but Not Female Gametogenesis + +Breeding of Dmrt7 heterozygotes produced homozygous mutant progeny of both sexes at the expected frequency (63/264; 23%). Male and female homozygous mutants were viable, grew to adulthood normally, and exhibited normal sexual behavior. Female homozygotes were fertile, produced litters of normal size, and had no obvious ovarian abnormalities as judged by histological analysis (unpublished data). In contrast, Dmrt7 homozygous mutant males were completely infertile and had testes about one-third the weight of those of heterozygous or wild-type adult littermates (Figure 2). To determine when defective testis development begins in Dmrt7 mutants, we compared the testes of wild-type and mutant littermates during the first wave of spermatogenesis. Prior to postnatal day 14 (P14), mutant testes appeared histologically normal and the testis weights were similar to those of heterozygous and wild-type littermates, indicating that spermatogonia and early meiotic germ cells form normally (Figure 2B; unpublished data). Thereafter, the testes of the Dmrt7 mutant mice ceased to grow and the weight difference was significant. Microscopic examination of P21 and P42 Dmrt7 mutant testes revealed that germ cells arrest in pachynema, and later stages of germ cells are largely missing (Figure 2C and 2D). Dmrt7 mutant mice are deficient in postmeiotic spermatids and lack epididymal spermatozoa, although a few cells develop to the round spermatid stage. These meiotic defects are in agreement with a recent preliminary analysis of another Dmrt7 mutation [42]. While some Dmrt7 mutant tubules are highly vacuolated and contain primarily Sertoli cells and spermatogonia, others have abundant primary spermatocytes. In addition, some tubules contain multinucleated cells and cells with darkly stained nuclei that are typical of apoptotic cells (Figure 2D). + +Figure 2 + +Reduced Testis Size and Germ Cell Apoptosis in Mice with Targeted Deletion in Dmrt7 + +(A) Testes from a 6-wk-old wild-type (+/+) mouse and a homozygous (−/−) Dmrt7 mutant littermate. + +(B–D) Sections of testes from 14-d-old (B), 21-d-old (C), and 42-d-old mice (D) stained with hematoxylin and eosin. Wild-type is in left column and mutant in right. No significant difference is observed at 14 d (B), but by 21 d some tubules are lacking abundant spermatocytes (C, asterisk) or cells with typical apoptotic morphology are present (open arrowhead, C and D). Mutant tubules contain multinucleate cells (closed arrowhead, D). + +(E and F) TUNEL labeling of Dmrt7-deficient mouse testes. Testes from wild-type and homozygous mutant littermates were analyzed by TUNEL labeling to detect apoptotic cells. Testis sections from 21-d-old (E) and 6-wk-old mice (F). Apoptotic cells (brown) are much more abundant in seminiferous tubules of homozygous Dmrt7 mutant mice relative to wild-type. Bars in (B–F) represent 100 μm. + +Since Dmrt7 mutant testes lack most post-pachytene cells, we used TUNEL analysis to test whether the missing cells are eliminated by apoptosis. At 3 wk, Dmrt7 mutant testes contain significantly more apoptotic cells than those of wild-type controls. The percentage of tubule sections with five or more apoptotic nuclei was about three times higher in Dmrt7 mutants compared with wild-type (20% versus 7%; Figure 2E). A similar elevation of apoptosis was apparent in mutant testes at 7 wk (Figure 2F). In mutants, many apoptotic cells were in the middle of the tubules, whereas the apoptotic cells in wild-type occur mainly near the seminiferous tubule periphery. The numbers of Sertoli cells were not significantly different between wild-type and mutant testes, and we observed no difference in somatic cell apoptosis in mutants (unpublished data). From these results, we conclude that loss of Dmrt7 causes a block in meiotic progression, mainly in pachynema, leading to the elimination, by apoptosis, of the arrested cells. + +Pachytene Arrest of Dmrt7 Mutant Germ Cells + +To better define the spermatogenic stage at which Dmrt7−/− male germ cells arrest and die, we used antibodies against several stage-specific germ cell markers. TRA98 is expressed in PGCs and spermatogonia [43]. In the wild-type adult testis, strongly staining TRA98-positive cells form a layer one cell deep; however, in the mutant TRA98, strongly positive cells were abnormally organized, and some tubules had a layer several cells deep (Figure 3A). The BC7 antibody recognizes spermatocytes in the leptotene to early-pachytene stages [44]. Dmrt7 mutant testes had BC7-positive cells in approximately normal numbers, but again abnormally organized, with many positive cells in the center rather than the periphery of the tubules (Figure 3B). The TRA369 antibody recognizes a calmegin protein expressed in pachytene spermatocytes through elongated spermatids [45]. In contrast to the earlier stages, far fewer TRA369-positive cells were present in mutant testes relative to wild-type (Figure 3C). We also quantitated the number of cells at each meiotic stage using spermatocyte spreads, assaying chromosome-pairing status by staining for SYCP3, a component of the synaptonemal complex (Figure 4). We found that Dmrt7 mutants accumulate pachytene cells but have greatly reduced numbers of cells in late-pachynema and beyond. Together, these results confirm that the meiotic arrest in Dmrt7 mutants occurs primarily during pachynema and results in efficient elimination of arrested cells. + +Figure 3 + +Prophase I Arrest of Dmrt7 Mutant Germ Cells + +Testis sections from 6-wk-old wild-type (+/+) and Dmrt7 mutant (−/−) littermates stained with stage-specific antibodies specific to spermatogenic cells. + +(A) TRA98 antibody strongly stains spermatogonia, which are present in wild-type and mutant. + +(B) BC7 antibody stains spermatocytes, which are present in wild-type and mutant. + +(C) TRA369 antibody stains pachytene and later germ cells, which are severely deficient in the mutant testis. + +Figure 4 + +Profile of Meiotic Arrest in Dmrt7 Mutant Testis + +Graph of distribution of meiotic stages of germ cells from heterozygous (n = 1,970) and homozygous mutant (n = 2,084) P26 testes, spread and stained for the synaptonemal complex protein SYCP3 to permit precise staging, which was performed as described [64,65]. + +Normal Meiotic Prophase in Dmrt7 Mutant Germ Cells + +Defects in chromosome pairing, synapsis, or recombination can trigger pachytene arrest and apoptosis [46]. We therefore examined these events in Dmrt7 mutant testes. To assess homolog synapsis, we used antibodies to SYCP1, a synaptonemal complex transverse element component, and SYCP3, a component of the axial element, which remains on the desynapsed axes during diplonema [47,48]. Formation of synaptonemal complexes in the mutant was indistinguishable from that in wild-type, as indicated by the proper accumulation of SYCP1 (unpublished data) and SYCP3 (Figure 5A). Likewise, the Dmrt7 mutant zygotene spermatocytes showed normal accumulation of the early recombination repair marker RAD51, suggesting that early meiotic recombination is not significantly affected (Figure 5B). Dmrt7 mutant spermatocytes exhibited the expected decline in the presence of RAD51 foci associated with the autosomal synaptonemal complexes (Figure 5B; unpublished data) [49]. The few surviving cells that progressed beyond pachynema also underwent apparently normal desynapsis during diplonema (Figure 5A). From these results, we conclude that chromosomal pairing, synapsis, recombination, and desynapsis during prophase I in Dmrt7 mutant males are grossly normal. + +Figure 5 + +Normal Synapsis and Recombination in Dmrt7 Mutant Germ Cells + +(A) Testicular cells from Dmrt7 heterozygous (+/−) or homozygous (−/−) mutant mice were spread and stained with antibody to SYCP3 (red). The developmental stages of meiotic prophase based on SYCP3 organization are indicated in each panel. By pachynema, all autosomes are fully synapsed, and the XY bivalent is synapsed only at the pseudoautosomal region in both wild-type and mutant cells. + +(B) Testicular cells in zygonema and pachynema spread and stained with antibodies to RAD51 (green) and SYCP3 (red). Size and distribution of RAD51 foci are similar between wild-type and mutant spermatocytes. + +Abnormal Cellular Organization and the Role of Sertoli Cells + +Sertoli cells interact with germ cells during spermatogenesis and the interaction is critical for germ cell maturation [50]. Although we did not detect DMRT7 expression in Sertoli cells by antibody staining, we nevertheless considered the possibility that Sertoli cell defects might contribute to the male-specific germ line failure of Dmrt7 mutants. To characterize Sertoli cell differentiation, we examined expression of the Sertoli cell markers GATA4 (a marker of immature postnatal Sertoli cells) and GATA1 (a mature Sertoli cell marker). The levels of these proteins appeared normal relative to wild-type at P14 and P42 (Figure 6A–6C), as did the androgen receptor (Figure S3; unpublished data). However, the organization of Sertoli cells in Dmrt7 mutant testes was abnormal: in some tubules GATA1-positive Sertoli cell nuclei were displaced from their usual close apposition with the basement membrane (Figure 6C). In such tubules, nuclei of pre-meiotic germ cells and spermatocytes were packed close to the basal membrane and few germ cells were found in the adlumenal region. + +Figure 6 + +Abnormal Sertoli Cell Organization in Dmrt7 Mutant Testes + +(A) Testes from P14 wild-type and Dmrt7 mutant mice were sectioned and stained with antibody to GATA4. + +(B) P14 testis sections stained with antibody to GATA1. At P14, GATA4 and GATA1 levels are similar in wild-type and mutant Sertoli cell. + +(C) Wild-type and mutant testis sections double-stained with antibody to GATA1 (red) and DAPI (blue). Most Sertoli cell nuclei were adjacent to the basal membrane in wild-type, but mutant Sertoli cells were displaced in some tubules (arrowhead). White dotted line indicates position of basal membranes. + +(D) Testis sections from 10-wk-old wild-type and Sertoli cell-specific Dmrt7 mutant (SC-Dmrt7KO) mice stained with hematoxylin and eosin. Spermatogenesis and spermiogenesis are normal in SC-Dmrt7KO testis. + +(E) Testis sections from 10-wk-old wild-type and SC-Dmrt7KO mice stained with antibodies to GATA1 (red) and smooth muscle actin (to outline seminiferous tubules; green). Sertoli cell nuclei are positioned normally near the basal membrane in SC-Dmrt7KO mice. + +The aberrant Sertoli cell organization in Dmrt7 mutant testes raised the possibility that the germ cell phenotype might indirectly result from defects in Sertoli cell function. To test this possibility, we deleted Dmrt7 just in the Sertoli cell lineage by crossing mice carrying the floxed Dmrt7 allele with Dhh-Cre transgenic mice [51]. The Desert hedgehog (Dhh) promoter is active starting at about E12.5 in pre-Sertoli cells but not in germ cells, allowing deletion of Dmrt7 in Sertoli cells well before any likely requirement for its function [52]. Testicular size in Sertoli-targeted (SC-Dmrt7KO) animals was slightly reduced from that of wild-type, but histological analysis revealed no obvious difference between wild-type and SC-Dmrt7KO testes (Figure 6D). Spermatogenesis appeared normal, mature sperm were present, and SC-Dmrt7KO mice were fertile. In addition, GATA1 staining showed that Sertoli cell nuclei were located adjacent to the basement membrane as in wild-type (Figure 6E). These results suggest the germ cell defects of Dmrt7 mutants are not caused by lack of Dmrt7 in Sertoli cells. Rather, the abnormal organization of Sertoli cells appears to result from lack of Dmrt7 in the germ line. + +XY Bodies Form Normally in Dmrt7 Mutant Cells + +The data presented so far indicate that Dmrt7 mutant germ cells undergo apparently normal early meiosis and then arrest during pachynema due to a strict requirement for Dmrt7 in the germ line. To better understand the basis of the meiotic arrest, we more closely examined meiotic germ cells in the mutant. We focused on the XY body, which is thought to be essential for meiotic progression and is the site of preferential DMRT7 localization. Condensation of the X and Y chromosomes begins in late-zygotene cells, and, by mid-pachynema (when homologous chromosome pairs are fully aligned) the sex chromatin forms a microscopically visible spherical structure near the nuclear periphery [53]. + +We first asked whether DMRT7 is required for XY body formation by evaluating several characteristic XY body chromatin features. First, we tested γH2AX expression by immunofluorescent staining. H2AX is a variant of H2A that is crucial for XY body formation and MSCI [12]. γH2AX localized normally to the XY body of DMRT7 mutant cells in meiotic spreads (Figure 7A), and many γH2AX-positive puncta were present in germ cells of Dmrt7 mutant testes (Figure 7B). Next, we examined SUMO-1 localization in the mutant testis. SUMO-1 expression normally increases in the XY body of early- to mid-pachytene spermatocytes at the time of sex chromosome condensation. Prior to the completion of the first meiotic division, SUMO-1 disappears from the XY body as the X and Y chromosomes desynapse [40]. Punctate SUMO-1 localization was present in Dmrt7 mutant germ cells, again consistent with formation of a correctly marked XY body (Figure 7C). However, some tubules in mutants had multiple layers of cells with SUMO-1-condensed spots (Figure 7C), rather than the normal single layer of cells. This accumulation of XY body–containing cells also was apparent with γH2AX staining and is consistent with a developmental arrest of mutant cells in mid- to late-pachytene. We also examined Ub-H2A localization in Dmrt7 mutant testes. In early-pachytene, Ub-H2A is concentrated in the XY body; by mid-pachytene Ub-H2A is observed throughout the entire nucleus, but it again becomes limited to the XY body in late-pachytene spermatocytes [13]. Analysis of nuclear spreads revealed that Ub-H2A localizes normally to the XY body in Dmrt7 mutants (Figure S4). Collectively, these results indicate that Dmrt7 mutant germ cells can establish an XY body with at least some of the normal chromatin marks. + +Figure 7 + +XY Body Forms Normally during Pachynema in Dmrt7 Mutant Mice + +(A) Testicular cells spread and stained with antibodies to γH2AX (red) and SYCP3 (green). In pachytene cells, γH2AX is concentrated in the XY body both in wild-type and Dmrt7 mutant. + +(B) Testes from 6-wk-old mice sectioned and stained with antibody to γH2AX (red). + +(C) Testes from 6-wk-old mice sectioned and stained with antibody to SUMO-1 (green). Abundant pachytene cells with XY bodies are present in wild-type and mutant testes. + +Abnormal Sex Chromatin in Dmrt7 Mutant Germ Cells + +Although the XY body can form during pachynema in Dmrt7 mutants, we considered the possibility that transcriptional silencing might not be properly established. This would be consistent with the Dmrt7 phenotype: pachytene cells that escape from MSCI normally are eliminated prior to late-pachytene [17]. Recently, MSCI has been shown to continue into meiosis II and spermiogenesis, apparently mediated by a distinct chromatin compartment termed postmeiotic sex chromatin (PMSC) that is established starting in diplonema [16]. We therefore asked whether the pachytene germ cell death in Dmrt7 mutants is associated with a failure either to initiate or to maintain sex chromosome inactivation. + +First, we examined the mid-pachytene XY body. To examine XY transcriptional status, we carried out Cot-1 RNA fluorescence in situ hybridization (FISH) to detect nascent RNA polymerase II transcription, combined with DAPI staining to locate the XY body on spreads of seminiferous tubules (Figure 8A and 8B). In Dmrt7 mutants, the XY body was formed and excluded Cot-1 hybridization (Figure 8B), indicating that transcriptional silencing is established normally in mutant pachytene cells. We also examined expression of the Y-linked gene Rbmy, which normally is inactivated during pachytene and reactivated after secondary meiosis begins [54,55]. Rbmy was inactivated normally in pachytene cells of Dmrt7 mutants, based on immunofluorescent staining with an anti-RBMY antibody (Figure S5). We also examined heterochromatin protein 1 beta (HP1β), which normally localizes to the X centromere at mid-pachynema and then spreads through the XY body as it internalizes during diplonema [56]. We found that HP1β localization is normal in DMRT7 mutant cells in mid-pachynema (Figure 8C and 8D). These results suggest that XY body formation and initiation of MSCI both occur normally in Dmrt7 mutant germ cells. + +Figure 8 + +Chromatin Abnormalities in Dmrt7 Mutant Diplotene Germ Cells + +(A and B) MSCI occurs normally in mid-pachytene spermatocyte of Dmrt7 mutant. Arrows indicate XY body in all panels. Cot-1 RNA FISH (red) reveals normal silencing of sex chromosome transcription in XY body. This is consistent with wild-type mid-pachynema as described previously [16]. + +(C and D) HP1β localization in mid-pachytene spermatocytes. HP1β localizes to X chromosome centromere (arrowhead) in wild-type (C) and mutant (D). + +(E and F) Cot-1 hybridization in diplotene. Presumptive XY body, based on DAPI and γH2AX localization, is indicated by arrow in (F). + +(G and H) HP1β in diplotene. Wild-type cell (E) has HP1β throughout XY body, whereas mutant cell (H) only has localization to X chromosome centromere (arrowhead). + +(I and J) H3-2meK9 localization in diplotene cells. Mutant cell (J) lacks strong concentration of this mark to the XY body seen in wild-type (I). + +(K and L) H3-3meK9 localization in diplotene cells. Mutant cell (L) has no localization of this mark to the XY body. γH2AX localizes to autosomes in mutant cell, possibly indicating onset of apoptosis. + +(M) Example of mutant diplotene cell with normal HP1β accumulation to the XY body. + +All images except those in (M) are single Z sections. + +We next considered the possibility that sex chromatin is established normally but is not properly modified as cells exit pachynema and begin to form PMSC. Although most Dmrt7 mutant cells are eliminated by apoptosis prior to diplonema, we were able to examine epigenetic markers of PMSC in rare Dmrt7 mutant spermatocytes that escaped pachytene arrest and progressed into diplonema. First, we examined nascent transcription by Cot-1 hybridization. Although heterochromatic regions generally showed lower Cot-1 signal than euchromatic regions (Figure 8E and 8F), in some mutant cells the sex chromatin appeared to be incompletely silenced relative to wild-type (Figure 8F). We also examined three epigenetic signatures of PMSC: histone H3 dimethylated or trimethylated at lysine-9 (H3-2meK9, H3-3meK9) and spreading of HP1β through the XY body [16,57,58] (S. H. Namekawa, unpublished data). We observed defects in sex chromatin localization of all three markers in Dmrt7 diplotene cells. Although HP1β localization to the X chromosome centromere initially appeared normal at mid-pachynema, we observed Dmrt7 mutant diplotene cells that failed to show spreading of HP1β to the entire XY body (Figure 8G and 8H). Similarly, we found Dmrt7 mutant diplotene cells lacking accumulation of H3-2meK9 and H3-3meK9 marks onto the sex chromatin (Figure 8I–8L). + +Not all Dmrt7 mutant diplotene cells showed abnormal localization of HP1β to the sex chromatin (Figure 8M). In one experiment, 11/27 mutant cells in diplonema lacked HP1β on the XY body, as compared with 2/22 wild-type cells. We hypothesize that the mutant cells with normal HP1β may be those that can complete meiosis (Figures 4 and 5). Some of the mutant diplotene cells showing abnormal sex chromatin also had abnormal autosomal γH2AX staining (Figure 8L). γH2AX localizes to double-strand DNA breaks, so this staining may indicate that some diplotene mutant cells are approaching or entering apoptosis [59]. We did not observe sex chromatin defects prior to diplonema, but we cannot exclude the possibility that earlier defects exist and the affected cells are rapidly eliminated. + +In the preceding experiments, we staged cells based on XY body internalization. Because this process could be abnormal in the mutant cells, we also staged mutant cells by chromosome morphology using an antibody to SYCP3 (Figure 9). This independently identified Dmrt7 mutant diplotene cells lacking HP1β accumulation in the XY body, such as the example in Figure 9B. From these results, we conclude that Dmrt7 mutant cells establish a normal XY body in mid-pachynema, but then have multiple epigenetic defects in the sex chromatin transition from pachynema to diplonema. + +Figure 9 + +Abnormal Sex Chromatin in Cells Staged by Chromosome Pairing Status + +(A) Spread of wild-type germ cell stained with DAPI, anti-SYCP3, and anti-HP1β showing chromosome morphology typical of diplonema and internalized XY body with HP1β accumulation. + +(B) Spread of Dmrt7 mutant germ cell showing normal diplotene chromosome morphology and internalized XY body, but no HP1β accumulation in the XY body. + +XY chromosome pairs are indicated by arrow. + +Discussion + +In this study, we find that the DM domain protein DMRT7 is required for male germ cells to complete meiotic prophase but is dispensable in the female germ line. In males, DMRT7 expression is highest in pachytene spermatocytes, and the protein preferentially localizes to the XY body. Consistent with this expression, we found that most mutant male germ cells arrest in pachynema and undergo apoptosis, although a small proportion can progress to diplonema and sometimes beyond. Examination of chromatin and nascent transcription in mutant cells that progressed to diplonema revealed sex chromatin abnormalities, as discussed below. + +The pachytene stage of prophase involves tremendous chromosomal changes as the homologs align, synapse, and recombine. During this period, at least one pachytene surveillance system exists to monitor key events of meiotic progression. Cells in which any of these events is anomalous are efficiently eliminated by apoptosis [46]. Another key event of pachynema in male mammals is the packaging of the sex chromosomes into the XY body and the establishment of MSCI. Examination of male meiosis in XYY mice and mice carrying a sex-chromosome-to-autosome translocation showed that cells in which a sex chromosome escapes MSCI are eliminated prior to late-pachynema [17]. This indicates that the establishment of MSCI also is subject to surveillance. + +Since the arrest and apoptosis of Dmrt7 mutant spermatocytes could result from perturbation of any of the critical pachytene events mentioned above, we tested whether they occur abnormally in the mutant cells. We found that chromosomal synapsis and recombination appear normal in Dmrt7 mutant cells. We therefore focused on the XY body, the most prominent site of DMRT7 accumulation. First, we tested whether the XY body forms and MSCI is established in Dmrt7 mutant cells. Surprisingly, we found that these cells form an XY body with normal morphology and proper accumulation of all the chromatin marks we examined. Moreover, Cot-1 hybridization and analysis of RBMY expression demonstrated that MSCI initiates normally in the XY body of mid-pachytene Dmrt7 mutant cells. + +We did, however, observe three specific defects in the sex chromatin of Dmrt7 mutant germ cells that avoided arrest in pachynema and were able to enter diplonema. Normally cells accumulate H3-2meK9 and H3-3meK9 marks and HP1β protein on the sex chromatin as they progress to diplonema, but we observed mutant diplotene cells lacking these features. Thus, although a minority of Dmrt7 mutant germ cells can progress from pachynema to diplonema, there are defects in sex chromatin modification during the transition. A function in male sex chromatin can reconcile the findings that DMRT7 is required for meiosis, but only in males, and is present only in mammals. A proportion of mutant diplotene cells have apparently normal sex chromatin (for example, Figure 8M); these are likely to be the cells that can progress beyond diplonema. + +Because most Dmrt7 mutant germ cells are eliminated by apoptosis around the time at which we observed sex chromatin defects, a simple model is that the apoptosis is a consequence of the sex chromatin defects. The reciprocal situation (sex chromatin defects caused by apoptosis) is possible, but seems unlikely, because we observed mutant cells with sex chromatin defects but no indications of apoptosis. Alternatively, apoptosis and abnormal sex chromatin may be two independent consequences of Dmrt7 loss. This question cannot be answered definitively until we know the detailed molecular mechanism of DMRT7. + +A number of other proteins have been identified that interact with the XY body, including histone variants and modified histones, a testis-specific histone methyl transferase, chromobox proteins, an orphan receptor germ-cell nuclear factor, and recombination-related proteins [60]. A common feature of these proteins is involvement with heterochromatin and/or transcriptional repression. DMRT7 is unusual among XY body proteins in being related to highly site-specific transcriptional regulators. An attractive speculation is that DMRT7 may provide sequence specificity in recruiting other proteins, such as chromatin modifiers, to the XY body as part of the transition to PMSC. Chromatin regulation may be a common mechanism for DM domain proteins, as we find that other DM domain proteins associate with chromatin modifying complexes (M. W. Murphy, D. Zarkower, and V. J. Bardwell, unpublished data). + +The finding that Dmrt7 is essential for mammalian meiosis expands the known functions of this gene family. Invertebrate DM domain genes so far have only been found to function in somatic cells. Two other DM domain genes, Dmrt1 and Dmrt4, do affect germ cell development in the mouse. Dmrt1 is required in pre-meiotic male germ cells for differentiation of gonocytes into spermatogonia, as well as in Sertoli cells, but it is not expressed in meiotic cells [31] (S. Kim and D. Zarkower, unpublished data). The requirement for DMRT1 in pre-meiotic germ cells and DMRT7 in meiotic germ cells demonstrates that DM domain proteins act at multiple critical points of male germ cell development. Ovaries of Dmrt4 mutant females have polyovular follicles (follicles containing multiple oocytes), but it is unknown whether this reflects a defect in the germ line or the soma. It is notable that at least three mammalian DM domain genes affect gonadal development only in one sex, given the similar roles of related proteins in directing sex-specific somatic development in other phyla. + +Strikingly, Dmrt7 is present, not only in placental mammals, but also in marsupials and a monotreme (egg-laying mammal), the platypus, which has a clear Dmrt7 ortholog [36]. However, no close Dmrt7 ortholog has been reported in nonmammalian vertebrates, and our database searches did not reveal one. Thus, Dmrt7 likely arose, presumably by duplication and divergence of another Dmrt gene, shortly before or coincident with the mammalian radiation. Monotremes have five X and five Y chromosomes, which form an extended pairing chain during meiosis and appear unrelated to the sex chromosomes of the other mammals [61]. The presence of Dmrt7 in both lineages may support a common origin for either the sex chromosomes or the sex chromatin of monotremes and other mammals. A plausible model is that Dmrt7 evolved during the establishment of mammalian sex determination to help cope with ancestral differences in gene dosage, chromosome pairing, recombination, or other meiotic issues arising from sex chromosome heteromorphy. In this regard, we speculate that the recruitment of Dmrt7 during mammalian evolution may be analogous to the recruitment of chromatin regulatory complexes to achieve somatic dosage compensation during evolution of heteromorphic sex chromosomes in several phyla (reviewed in [62]). It will be of interest to determine whether DMRT7 localizes to sex chromosomes during monotreme meiosis. + +In summary, we have found that the mammal-specific DM domain protein DMRT7 is essential for meiotic prophase progression in males. DMRT7 localizes to the sex chromosomes after they are assembled into specialized heterochromatin, and many Dmrt7 mutant cells have epigenetic defects in the modification of the sex chromatin between pachytene and diplotene. Although Dmrt7 belongs to an ancient and conserved gene family, it is found only in mammals, and to our knowledge DMRT7 is the only example of a mammal-specific protein that is essential for meiosis. It will be important to determine the precise mechanism by which DMRT7 affects sex chromatin regulation during male meiosis. + +Materials and Methods + +Generation of Dmrt7 mice. + +A mouse Dmrt7 cDNA fragment containing sequences from exon 8 was used to screen a mouse BAC library from the 129/SvJ strain (Stratagene, http://www.stratagene.com), and clones containing promoter sequences were isolated and sequenced to obtain Dmrt7 genomic sequence. The targeting vector pDZ169 (diagrammed in Figure S2) was constructed by the following scheme: The vector pDZ157 was used as a backbone vector [31]. 3′ to Pgk-neo and the loxP site, we inserted, as a XmaI/XmaI DNA fragment, the third intron of Dmrt7 (from 366 bp to 2,773 bp downstream of exon 3) generated by PCR. 5′ to Pgk-neo, we inserted an EcoRI/NotI PCR fragment extending from 4,107 bp to 336 bp 5′ of the Dmrt7 translational start. Finally, we inserted a loxP site and NotI site 336 bp 5′ of the Dmrt7 translational start. In the resulting vector, the second and third exons of Dmrt7 are flanked by loxP sites (floxed). The Dmrt7-containing portions of pDZ169 were completely sequenced. + +pDZ169 was linearized with PmeI and electroporated into CJ7 ES cells (originally derived from the 129S1 strain). Three homologous recombinants were identified from 296 G418-resistant colonies by Southern hybridization by use of a DNA probe from the sequences upstream of exon 1 to screen genomic DNA digested with EcoRI. Homologous recombination was confirmed on both ends of the targeted region by Southern hybridization. Probes for Southern hybridization were made by PCR using primers DM5S10/DM5S11 (5′ probe) and DM5PR1/DM5PR2 (3′ probe), listed below. Two targeted ES cell clones containing the floxed allele Dmrt7neo were injected into C57Bl/6 blastocysts to generate chimeras. Chimeric males were bred with C57Bl/6 females to generate heterozygotes carrying Dmrt7neo. Dmrt7+/Dmrt7neo females were bred with male β-actin-Cre transgenic mice to delete the floxed sequences and generate heterozygous Dmrt7−/+ deletion mutants, which were interbred to generate homozygous Dmrt7−/− mutants. + +Genotyping and RT-PCR. + +For genotyping, tail-clip DNA was amplified for 35 cycles. Chromosomal sex was determined by PCR with primers to the Y chromosome gene Zfy (below). The wild-type Dmrt7 allele Dmrt7+ was detected by PCR with DM5S4/DM5S5, with an annealing temperature of 60 °C. The Dmrt7flox allele was detected by PCR with DM5S5F/DM7KO7R with an annealing temperature of 62 °C. The deleted Dmrt7 allele Dmrt7− was detected with DM5S3/DM7KO7R with an annealing temperature of 62 °C. RT-PCR for Dmrt7 expression analysis was as described [23] using primers SK111/SK112 with an annealing temperature of 62 °C. + +Primers. + +DM5S3: 5′-AGAGTGGATTGAATCGGATAGCTC-3′ + +DM5S4: 5′-AGGATCTTAGTGTCGAATGAATAC-3′ + +DM5S5: 5′-CCCTTATCCTCCTGCATCCAGATC-3′ + +DM5S10: 5′-CAGGCTATGGTTAGACTTGAGCAC-3′ + +DM5S11: 5′-CATCACTCGCGGACAAAGATGCAG-3′ + +DM5PR1: 5′-CTTCTGCTACAGCCACAGGTCTGG-3′ + +DM5PR2: 5′-GAATTCAACTAGTATCTGTCCC-3′ + +DM7KO7R: 5′-CGAGGATCAAGCTCAGGTCACTAGG-3′ + +DM5S5F: 5′-GATCTGGATGCAGGAGGATAAGGG-3′ + +ZFYF: 5′-CCTATTGCATGGACAGCAGTCTTATG-3′ + +ZFYR: 5′-GACTAGACATGTTCTTAACATCTGTCC-3′ + +SK111: 5′-CCCTTCTGGAAAAGAGAACATAGC-3′ + +SK112: 5′-GCTCCAGGGGCCTGTGGCTGTAGC-3′ + +β-actinF: 5′-TGCGTGACATCAAAGAGAAG-3′ + +β-actinR: 5′-GATGCCACAGGATTCCATA-3′ + +Histological analysis and TUNEL assay. + +Dissected testes were fixed in Bouin's fixative or phosphate-buffered formalin overnight at 4 °C, progressively dehydrated in a graded ethanol series, and embedded in paraffin wax. Sections (6 μm) were deparaffinized, rehydrated, and stained with hematoxylin and eosin. For TUNEL analyses, deparaffinized sections were treated with proteinase K for 15 min and quenched in 3.0% hydrogen peroxide in PBS for 5 min at room temperature. Subsequently, nuclear staining in apoptotic cells was detected using ApopTag kit (Chemicon, http://www.chemicon.com) according to the manufacturer's instructions. + +Tissue immunofluorescent staining. + +Slides with paraffin sections were washed in PBT (0.1% Tween 20 in PBS) and autoclaved in 10 mM citric acid (pH 6.0) to retrieve antigenicity. Slides were blocked in 5% serum (matched to the species of the secondary antibody) in PBS for 1 h at room temperature and incubated with primary antibodies overnight at 4 °C prior to detection with secondary antibodies. + +Antibodies. + +Rabbit polyclonal antibodies to DMRT7 were raised against a purified fusion protein containing glutathione-S-transferase (GST) fused to the C-terminal 279 amino acids of DMRT7. Antibodies to GST were removed by GST-affigel 10 chromatography and the antiserum was then purified by GST-DMRT7-affigel 15 chromatography. DMRT7 antibody was used at 1:200 dilution with a goat anti-rabbit secondary antibody (Molecular Probes, http://www.invitrogen.com) at 1:200 dilution. Other primary antibodies used for immunofluorescence were rat anti-GATA1 (1:200, Santa Cruz Biotechnology, http://www.scbt.com, sc-265), goat anti-GATA4 (1:200, Santa Cruz Biotechnology, sc-1237), rat anti-TRA98 (1:200, gift of H. Tanaka and Y. Nishimune), rat anti-BC7 (1:50, gift of H. Tanaka and Y. Nishimune), rat anti-TRA369 (1:200, gift of H. Tanaka and Y. Nishimune), rabbit anti-RAD51 (1:600 Calbiochem, http://www.calbiochem.com, PC130), mouse anti-GMP-1/SUMO-1 (1:200, Zymed, http://invitrogen.com, 33–2400), rabbit anti-phospho-H2AX (Ser139) (1:200, Upstate, http://www.millipore.com, 01–164), mouse anti-phospho-H2AX (1:200, Upstate, 05–636), mouse anti-SYCP3 (1:200, Abcam, http://www.abcam.com, ab12452), rabbit anti-HP1β (1:100, Abcam, ab10478), rabbit anti-H3-2meK9 (1:100, Upstate, 07–441), rabbit anti-H3-3meK9 (1:200, Upstate, 07–442), rabbit anti-AR (N-20) (1:200, Santa Cruz Biotechnology, sc-816), and mouse anti-αSMA clone 1A4 (1:800, Sigma, http://www.sigmaaldrich.com, A2547). Secondary antibodies used were goat anti-rabbit Alexa 488, goat anti-rabbit Alexa 594, goat anti-rat Alexa 594, and goat anti-mouse Alexa 488 (Molecular Probes) used at 1:250. Donkey anti-goat FITC (Jackson ImmunoResearch Laboratories, http://www.jacksonimmuno.com) and donkey anti-rabbit Texas Red (Jackson) were used at 1:50 according to the manufacturer's instructions. + +Meiotic chromosome spread preparations and FISH. + +Meiotic chromosome spread preparations were made from 3-wk-old mice, prepared as described by Reinholdt et al. [63]. For analysis of PMSC and Cot-1 RNA FISH, meiotic slides were prepared as previously described [16]. Slides containing chromosome spreads or meiotic spermatocytes were subjected to immunofluorescent staining or RNA FISH, as previously described [16,63]. For combined RNA FISH/immunostaining, we carried out RNA FISH first, followed by immunofluorescence. DNA FISH was performed using chromosome painting (Cambio, http://www.cambio.co.uk). Z-sections were captured by Zeiss Axioplan microscope (Zeiss, http://www.zeiss.com) and processed by Openlab (Improvision, http://www.improvision.com). + +Supporting Information + +Figure S1 + +Wild-Type and Dmrt7 Mutant Meiotic Spreads + +Arrows indicate the location of XY body. SYCP3 antibody staining (green) shows normal synapsis of homologous chromosome synapse in Dmrt7 mutant cell. DMRT7 protein (red) is localized to the XY body in wild-type (top row) and is not detected in Dmrt7 mutant (bottom row). + +(726 KB JPG) + +Click here for additional data file. + +Figure S2 + +Targeted Disruption of Dmrt7 + +(A) Diagram of targeting strategy. Homologous recombination of the targeting vector with the wild-type Dmrt7 allele (Dmrt7+) resulted in the targeted allele Dmrtneo. This allele contains loxP sites flanking the translational start and the second and third exons (containing the DM domain, gray), as well as a neomycine-resistance cassette flanked by Flp recombinase recognition sites (frt sites). Mice heterozygous for the Dmrt1neo allele were mated with transgenic mice expressing Cre recombinase, resulting in deletion of the sequence between the two loxP sites, including the neo cassette. The resulting deletion allele is called Dmrt7−. Mating of Dmrt7neo mice with transgenic mice expressing the Flpe recombinase excised the neo cassette, generating the Dmrt7flox allele. + +(B) Southern blots of targeted and wild-type ES cell clones. Genomic DNAs from ES cell clones were digested with NsiI/NotI and EcoRI. The 5′ external probe hybridizes to 14.2-kb (wild-type) and 6-kb (targeted) NsiI/NotI fragments. The 3′ probe hybridizes to 14.6-kb (wild-type) and 4.7-kb (targeted) EcoRI fragments. + +(C) Testis sections from adult mice. SUMO-1 (green) on XY body is detected in both wild-type (top) and Dmrt7 mutant (bottom) section. DMRT7 is detected in wild-type but not detected in Dmrt7 mutant in SUMO-1-positive cells. (Wild-type images are the same as in Figure 1D.) + +(3.4 MB JPG) + +Click here for additional data file. + +Figure S3 + +Normal Androgen Receptor Expression in Dmrt7 Mutant Sertoli Cells + +Testis sections from adult mice stained with antibody to androgen receptor (red) and counterstained with DAPI (blue). In both wild-type (top) and mutant (bottom) testis sections, androgen receptor protein is expressed in Sertoli cell and peritubular myoid cell. Mutant testis section shows abnormal localization of Sertoli cell nuclei, which are displaced from basement membrane. + +AR, androgen receptor. + +(2.3 MB JPG) + +Click here for additional data file. + +Figure S4 + +Ub-H2A Localization to XY Body in Dmrt7 Mutant Spermatocyte + +Spread pachytene spermatocytes from wild-type (top) and Dmrt7 mutant (bottom) mice, stained with antibody to Ub-H2A (green) and DAPI (blue). Arrows indicate XY body. Both wild-type and mutant pachytene cells have Ub-H2A properly localized to XY body. + +(722 KB JPG) + +Click here for additional data file. + +Figure S5 + +RBMY Silencing in Dmrt7 Mutant Spermatocytes + +(A) SYCP3 (green) and RBMY (red) immunostaining of spread wild-type and Dmrt7 mutant testicular cells. In wild-type and mutant cells, RBMY is expressed at low levels in leptotene stage. In pachytene spermatocytes, RBMY has fallen to background levels in both wild-type and mutant. + +(B) SUMO-1 (green) and RBMY (red) immunostaining of adult testis sections from wild-type and Dmrt7 mutant. Pre-meiotic cells near the basal membrane express high level of RBMY whereas SUMO-1-positive pachytene cells do not express RBMY, in both wild-type and mutant, indicating proper silencing. + +(5.4 MB JPG) + +Click here for additional data file. + +Acknowledgements + +We thank members of the Zarkower and Bardwell labs for many helpful discussions; Dan Camerini-Otero, John Logsdon, and Scott Hawley for insights into evolution of meiotic regulators; Hiromitsu Tanaka, Yoshitake Nishimune, and David Elliot for generously sharing antibodies; and Dies Meijer for sharing Dhh-Cre mice. We thank the University of Minnesota Mouse Genetics Laboratory for ES cell injection. Some images were obtained using the Middlebury Biology Imaging Facility, equipped in part through the National Science Foundation (Course, Curriculum, and Laboratory Improvement, 0088412). SHN is a research fellow of the Japan Society for Promotion of Science. + +Abbreviations + +DM - Doublesex/MAB-3 domain + +ES - embryonic stem + +FISH - fluorescence in situ hybridization + +GST - glutathione-S-transferase + +HP1β - heterochromatin protein 1 beta + +γH2AX - phosphorylated H2AX + +MSCI - meiotic sex chromosome inactivation + +P14 - postnatal day 14 + +PMSC - postmeiotic sex chromatin + +RT-PCR - reverse transcriptase-PCR + +SUMO-1 - small ubiquitin-related modifier 1 + +Ub-H2A - ubiquitinated H2A + +Footnotes + +Competing interests. The authors have declared that no competing interests exist. + +A previous version of this article appeared as an Early Online Release on March 7, 2007 (doi:10.1371/journal.pgen.0030062.eor). + +Author contributions. All authors conceived and designed the experiments and analyzed the data. SK, SHN, LMN, and JOW performed the experiments. JOW contributed reagents/materials/analysis tools. SK, SHN, JOW, JTL, VJB, and DZ wrote the paper. + +Funding. This work was supported by the US National Institutes of Health (GM059152) and the Minnesota Medical Foundation. LMN was supported by the Bicentennial Fund of Middlebury College. diff --git a/src/ontogpt/evaluation/craft/database/all/17465682.ann b/src/ontogpt/evaluation/craft/database/all/17465682.ann new file mode 100644 index 000000000..8f85446f8 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17465682.ann @@ -0,0 +1,1360 @@ +T1 PR:000013058 0 10 PPAR gamma +T2 GO:0065007 38 49 Controlling +T3 UBERON:0001013 50 64 Adipose Tissue +T4 CHEBI:18059 94 99 Lipid +T5 GO:0006629 94 110 Lipid Metabolism +T6 GO:0005777 122 132 Peroxisome +T7 PR:000013058 122 170 Peroxisome proliferator activated receptor gamma +T8 GO:0065007 203 212 regulated +T9 SO:0001060 213 220 isoform +T10 PR:000013058 224 229 PPARg +T11 NCBITaxon:10088 303 308 mouse +T12 PR:000045358 350 357 insulin +T13 CL:0000169 370 376 β-cell +T14 SO:0001060 442 449 isoform +T15 UBERON:0001013 485 499 adipose tissue +T16 UBERON:0002107 655 660 liver +T17 CHEBI:17855 729 744 triacylglycerol +T18 CHEBI:18059 798 803 lipid +T19 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Gene +T402 GO:0010467 14767 14782 Gene expression +T403 UBERON:0000006 14795 14801 islets +T404 NCBITaxon:10088 14819 14823 mice +T405 GO:0010467 14843 14853 expression +T406 UBERON:0001264 14857 14867 pancreatic +T407 PR:000012523 14857 14887 pancreatic duodenal homeobox-1 +T408 UBERON:0002114 14868 14876 duodenal +T409 PR:000009108 14889 14917 insulin receptor substrate 2 +T410 PR:000015064 14919 14924 Glut2 +T411 PR:000045358 14930 14937 insulin +T412 UBERON:0000006 14941 14947 islets +T413 NCBITaxon:10088 14958 14962 mice +T414 CL:0000169 15043 15050 β-cells +T415 NCBITaxon:10088 15059 15063 mice +T416 CL:0000169 15114 15120 β-cell +T417 UBERON:0001264 15190 15198 pancreas +T418 UBERON:0001264 15253 15261 pancreas +T419 PR:000045358 15336 15343 insulin +T420 UBERON:0000006 15356 15362 islets +T421 NCBITaxon:10088 15373 15377 mice +T422 CHEBI:17234 15421 15428 Glucose +T423 PR:000045358 15440 15447 Insulin +T424 NCBITaxon:10088 15466 15471 Mouse +T425 UBERON:0000006 15472 15478 Islets +T426 CHEBI:17234 15492 15499 glucose +T427 PR:000045358 15511 15518 insulin +T428 NCBITaxon:10088 15556 15560 mice +T429 UBERON:0000006 15590 15596 Islets +T430 NCBITaxon:10088 15616 15620 mice +T431 NCBITaxon:10088 15660 15664 mice +T432 UBERON:0000006 15691 15697 islets +T433 UBERON:0000006 15737 15743 islets +T434 NCBITaxon:10088 15754 15758 mice +T435 PR:000045358 15935 15942 Insulin +T436 UBERON:0000006 15954 15960 islets +T437 NCBITaxon:10088 15972 15976 mice +T438 NCBITaxon:10088 16031 16035 mice +T439 PR:000045358 16049 16056 Insulin +T440 UBERON:0000006 16076 16082 islets +T441 NCBITaxon:10088 16091 16095 mice +T442 NCBITaxon:10088 16147 16151 mice +T443 PR:000045358 16185 16192 insulin +T444 CHEBI:17234 16250 16257 glucose +T445 CHEBI:17234 16281 16288 glucose +T446 CHEBI:17234 16296 16303 glucose +T447 CHEBI:27999 16306 16317 tolbutamide +T448 NCBITaxon:10088 16357 16361 Mice +T449 NCBITaxon:10088 16380 16384 Mice +T450 NCBITaxon:10088 16408 16412 mice +T451 UBERON:0002107 16427 16434 hepatic +T452 NCBITaxon:10088 16479 16483 mice +T453 NCBITaxon:10088 16522 16527 mouse +T454 http://purl.obolibrary.org/obo/MONDO_0004790 16544 16559 hepatosteatosis +T455 NCBITaxon:10088 16575 16580 mouse +T456 GO:0010467 16618 16628 expression +T457 SO:0001060 16643 16650 isoform +T458 UBERON:0002107 16658 16663 liver +T459 NCBITaxon:10088 16673 16677 mice +T460 CHEBI:17855 16729 16733 TAGs +T461 UBERON:0002107 16741 16746 liver +T462 CHEBI:18059 16788 16793 Lipid +T463 UBERON:0001013 16805 16819 Adipose Tissue +T464 UBERON:0000006 16821 16838 Pancreatic Islets +T465 UBERON:0002107 16840 16845 Liver +T466 UBERON:0004288 16851 16859 Skeletal +T467 CHEBI:18059 17033 17039 lipids +T468 UBERON:0001013 17047 17061 adipose tissue +T469 UBERON:0000006 17063 17080 pancreatic islets +T470 UBERON:0002107 17082 17087 liver +T471 UBERON:0004288 17093 17101 skeletal +T472 NCBITaxon:10088 17126 17131 mouse +T473 UBERON:0001013 17161 17175 Adipose tissue +T474 NCBITaxon:10088 17186 17190 mice +T475 CHEBI:17855 17205 17208 TAG +T476 CHEBI:18035 17223 17226 DAG +T477 CHEBI:17761 17228 17237 ceramides +T478 CHEBI:18059 17258 17263 lipid +T479 PR:000045358 17288 17295 insulin +T480 NCBITaxon:10088 17418 17422 mice +T481 UBERON:0001013 17443 17457 adipose tissue +T482 CHEBI:17855 17484 17488 TAGs +T483 UBERON:0001013 17507 17521 adipose tissue +T484 CHEBI:18035 17570 17574 DAGs +T485 UBERON:0001347 17596 17599 WAT +T486 NCBITaxon:10088 17612 17616 mice +T487 CHEBI:18059 17702 17707 lipid +T488 UBERON:0001347 17723 17726 WAT +T489 NCBITaxon:10088 17735 17739 mice +T490 UBERON:0001347 17771 17774 WAT +T491 NCBITaxon:10088 17798 17802 mice +T492 CHEBI:17761 17845 17853 ceramide +T493 CHEBI:35366 17882 17892 fatty acid +T494 CHEBI:60479 17941 17964 lysophosphatidylcholine +T495 NCBITaxon:10088 17993 17997 mice +T496 CHEBI:17761 18070 18079 ceramides +T497 NCBITaxon:10088 18116 18120 mice +T498 CHEBI:64583 18136 18149 Sphingomyelin +T499 CHEBI:17761 18181 18189 ceramide +T500 NCBITaxon:10088 18298 18302 mice +T501 CHEBI:17855 18327 18330 TAG +T502 CHEBI:18059 18360 18365 lipid +T503 UBERON:0000006 18377 18383 islets +T504 NCBITaxon:10088 18394 18398 mice +T505 UBERON:0000006 18479 18496 pancreatic islets +T506 NCBITaxon:10088 18512 18516 mice +T507 CHEBI:18059 18531 18536 lipid +T508 NCBITaxon:10088 18612 18616 mice +T509 CHEBI:17855 18644 18648 TAGs +T510 UBERON:0000006 18667 18673 islets +T511 NCBITaxon:10088 18698 18702 mice +T512 CHEBI:16038 18777 18801 phosphatidylethanolamine +T513 CHEBI:18059 18904 18909 lipid +T514 CHEBI:17761 18939 18948 ceramides +T515 CHEBI:35366 18964 18975 fatty acids +T516 UBERON:0000006 18980 18986 islets +T517 NCBITaxon:10088 18997 19001 mice +T518 CHEBI:17855 19042 19045 TAG +T519 CHEBI:18059 19069 19074 lipid +T520 UBERON:0002107 19086 19091 liver +T521 NCBITaxon:10088 19100 19104 mice +T522 CHEBI:18059 19156 19161 lipid +T523 CHEBI:17855 19320 19324 TAGs +T524 CHEBI:18035 19329 19333 DAGs +T525 UBERON:0002107 19349 19355 livers +T526 NCBITaxon:10088 19366 19370 mice +T527 NCBITaxon:10088 19398 19402 mice +T528 UBERON:0002107 19404 19410 Livers +T529 NCBITaxon:10088 19421 19425 mice +T530 GO:0042158 19522 19531;19546 19548;19566 19578 formation ... of ... lipoproteins +T531 UBERON:0002107 19602 19608 livers +T532 CHEBI:17761 19626 19635 ceramides +T533 UBERON:0002107 19654 19660 livers +T534 CHEBI:60479 19718 19742 lysophosphatidylcholines +T535 NCBITaxon:10088 19761 19765 mice +T536 CHEBI:17855 19792 19795 TAG +T537 CHEBI:18059 19825 19830 lipid +T538 UBERON:0004288 19847 19855 skeletal +T539 CHEBI:17855 19905 19908 TAG +T540 CHEBI:18059 19932 19937 lipid +T541 UBERON:0001013 19969 19983 adipose tissue +T542 CL:0000169 19985 19991 β-cell +T543 UBERON:0002107 19997 20002 liver +T544 UBERON:0004288 20039 20047 skeletal +T545 NCBITaxon:10088 20063 20067 mice +T546 UBERON:0004288 20115 20123 skeletal +T547 UBERON:0004288 20137 20145 skeletal +T548 CHEBI:26666 20179 20201 short-chain fatty acid +T549 CHEBI:17855 20202 20206 TAGs +T550 CHEBI:17855 20264 20268 TAGs +T551 UBERON:0004288 20288 20296 skeletal +T552 NCBITaxon:10088 20312 20316 mice +T553 CHEBI:18059 20345 20351 lipids +T554 CHEBI:17761 20362 20370 ceramide +T555 CHEBI:18035 20385 20389 DAGs +T556 CHEBI:60479 20391 20415 lysophosphatidylcholines +T557 CHEBI:64583 20421 20435 sphingomyelins +T558 CHEBI:17761 20451 20460 ceramides +T559 NCBITaxon:10088 20493 20497 mice +T560 NCBITaxon:10088 20532 20536 Mice +T561 NCBITaxon:10088 20624 20629 mouse +T562 GO:0010467 20662 20672 expression +T563 GO:0008152 20676 20685 metabolic +T564 SO:0000704 20686 20691 genes +T565 SO:0000704 20761 20766 genes +T566 SO:0000704 20812 20816 Gene +T567 GO:0010467 20812 20827 Gene expression +T568 UBERON:0001347 20840 20843 WAT +T569 SO:0000704 20853 20858 genes +T570 PR:000013058 20862 20867 PPARg +T571 PR:000015066 20876 20881 Glut4 +T572 PR:000005376 20883 20890 adipsin +T573 PR:000007312 20892 20895 aP2 +T574 UBERON:0001347 20954 20957 WAT +T575 NCBITaxon:10088 20988 20992 mice +T576 NCBITaxon:10088 21011 21015 mice +T577 NCBITaxon:10088 21129 21133 mice +T578 PR:000015066 21181 21186 GLUT4 +T579 PR:000007312 21188 21191 aP2 +T580 NCBITaxon:10088 21250 21254 mice +T581 NCBITaxon:10088 21292 21296 mice +T582 NCBITaxon:10088 21311 21315 mice +T583 http://purl.obolibrary.org/obo/MONDO_0011122 21340 21345 obese +T584 PR:000045358 21350 21357 insulin +T585 GO:0010467 21373 21383 expression +T586 PR:000013058 21401 21406 PPARg +T587 UBERON:0001347 21422 21425 WAT +T588 NCBITaxon:10088 21435 21439 mice +T589 NCBITaxon:10088 21475 21479 mice +T590 GO:0010467 21549 21559 expression +T591 SO:0000704 21563 21568 genes +T592 CHEBI:18059 21581 21586 lipid +T593 GO:0006629 21581 21597 lipid metabolism +T594 GO:0010467 21611 21621 Expression +T595 CHEBI:15541 21625 21644 stearoyl-coenzyme A +T596 PR:000015921 21625 21657 stearoyl-coenzyme A desaturase 1 +T597 PR:000015921 21659 21663 Scd1 +T598 CHEBI:15889 21669 21675 sterol +T599 SO:0001861 21669 21694 sterol regulatory element +T600 PR:000028988 21669 21710 sterol regulatory element-binding protein +T601 GO:0065007 21676 21686 regulatory +T602 UBERON:0001347 21752 21755 WAT +T603 NCBITaxon:10088 21766 21770 mice +T604 NCBITaxon:10088 21789 21793 mice +T605 CHEBI:17855 21824 21828 TAGs +T606 CHEBI:18035 21843 21847 DAGs +T607 GO:0010467 21874 21884 expression +T608 CHEBI:18035 21888 21891 DAG +T609 PR:000006433 21888 21909 DAG acyltransferase 2 +T610 CHEBI:17855 21953 21956 TAG +T611 GO:0019432 21953 21966 TAG synthesis +T612 UBERON:0001347 21975 21978 WAT +T613 NCBITaxon:10088 21987 21991 mice +T614 UBERON:0001347 22006 22009 WAT +T615 NCBITaxon:10088 22021 22025 mice +T616 GO:0010467 22071 22081 expression +T617 MOP:0000619 22138 22148 hydrolysis +T618 CHEBI:18035 22152 22168 diacylglycerides +T619 UBERON:0001347 22191 22194 WAT +T620 NCBITaxon:10088 22225 22229 mice +T621 NCBITaxon:10088 22247 22251 mice +T622 NCBITaxon:10088 22297 22301 mice +T623 PR:000012942 22303 22330 Adipose triglyceride lipase +T624 CHEBI:17855 22311 22323 triglyceride +T625 NCBITaxon:10088 22402 22406 mice +T626 NCBITaxon:10088 22467 22471 mice +T627 MOP:0000568 22474 22483 Oxidative +T628 PR:000045358 22544 22551 insulin +T629 UBERON:0001013 22564 22578 Adipose tissue +T630 NCBITaxon:10088 22589 22593 mice +T631 MOP:0000568 22608 22617 oxidative +T632 NCBITaxon:10088 22651 22655 mice +T633 SO:0000704 22682 22686 gene +T634 GO:0010467 22682 22697 gene expression +T635 GO:0005576 22708 22721 extracellular +T636 PR:000015401 22708 22747 extracellular CuZn-superoxide dismutase +T637 CHEBI:18421 22727 22737 superoxide +T638 CHEBI:16856 22767 22778 glutathione +T639 CHEBI:16856 22823 22834 gluthatione +T640 CHEBI:16856 22892 22903 gluthatione +T641 CL:0000235 22941 22951 macrophage +T642 UBERON:0001013 22968 22982 adipose tissue +T643 PR:000045358 23032 23039 insulin +T644 GO:0010467 23052 23062 Expression +T645 PR:000002064 23066 23070 CD68 +T646 PR:000001813 23075 23080 F4/80 +T647 CL:0000235 23087 23097 macrophage +T648 UBERON:0001347 23128 23131 WAT +T649 NCBITaxon:10088 23155 23159 mice +T650 NCBITaxon:10088 23191 23195 mice +T651 GO:0010467 23223 23233 expression +T652 NCBITaxon:10088 23264 23268 mice +T653 NCBITaxon:10088 23284 23288 mice +T654 CL:0000235 23305 23315 macrophage +T655 PR:000045358 23373 23380 insulin +T656 NCBITaxon:10088 23404 23409 mouse +T657 NCBITaxon:10088 23432 23437 mouse +T658 SO:0000704 23440 23444 Gene +T659 GO:0010467 23440 23455 Gene expression +T660 UBERON:0002107 23468 23473 liver +T661 UBERON:0002107 23484 23491 hepatic +T662 http://purl.obolibrary.org/obo/MONDO_0004790 23484 23501 hepatic steatosis +T663 CHEBI:18059 23525 23530 lipid +T664 UBERON:0002107 23563 23570 hepatic +T665 GO:0010467 23579 23589 expression +T666 CHEBI:18059 23619 23624 lipid +T667 GO:0019915 23619 23632 lipid storage +T668 GO:0006629 23619 23624;23637 23647 lipid ... metabolism +T669 UBERON:0002107 23655 23660 liver +T670 NCBITaxon:10088 23673 23677 mice +T671 GO:0010467 23679 23689 Expression +T672 SO:0000704 23693 23698 genes +T673 CHEBI:18059 23711 23716 lipid +T674 GO:0006629 23711 23727 lipid metabolism +T675 UBERON:0002107 23731 23736 liver +T676 CHEBI:17855 23800 23804 TAGs +T677 UBERON:0002107 23812 23817 liver +T678 CHEBI:35366 23819 23829 fatty acid +T679 PR:000015921 23840 23844 Scd1 +T680 CHEBI:35366 23854 23864 fatty acid +T681 PR:000001905 23854 23876 fatty acid translocase +T682 PR:000001905 23878 23881 FAT +T683 PR:000001905 23882 23886 CD36 +T684 UBERON:0002107 23921 23927 livers +T685 NCBITaxon:10088 23943 23947 mice +T686 UBERON:0002107 23984 23989 liver +T687 NCBITaxon:10088 24000 24004 mice +T688 UBERON:0002107 24019 24024 liver +T689 NCBITaxon:10088 24036 24040 mice +T690 GO:0008610 24048 24057 lipogenic +T691 PR:000013058 24058 24063 PPARg +T692 SO:0000704 24071 24076 genes +T693 UBERON:0002107 24121 24126 liver +T694 NCBITaxon:10088 24149 24153 mice +T695 NCBITaxon:10088 24165 24169 mice +T696 GO:0010467 24210 24220 expression +T697 SO:0000704 24224 24229 genes +T698 MOP:0000568 24244 24253 oxidation +T699 PR:000013058 24261 24266 Pparg +T700 PR:000003642 24274 24277 Aox +T701 PR:000003642 24279 24283 Cpt1 +T702 PR:000017036 24289 24293 Ucp2 +T703 GO:0010467 24310 24320 expression +T704 MOP:0000568 24334 24343 oxidative +T705 SO:0000704 24344 24349 genes +T706 UBERON:0002107 24371 24376 liver +T707 NCBITaxon:10088 24385 24389 mice +T708 NCBITaxon:10088 24421 24425 mice +T709 GO:0065007 24468 24478 regulation +T710 CL:0000169 24495 24501 β-cell +T711 http://purl.obolibrary.org/obo/MONDO_0002909 24539 24553 hyperglycaemia +T712 UBERON:0002107 24585 24592 hepatic +T713 GO:0006094 24593 24608 gluconeogenesis +T714 PR:000013059 24675 24700 PPARg coactivator 1 alpha +T715 PR:000013059 24702 24710 PPARGC1a +T716 PR:000013059 24726 24731 PGC1a +T717 GO:0010467 24733 24743 expression +T718 UBERON:0002107 24756 24761 liver +T719 NCBITaxon:10088 24793 24797 mice +T720 PR:000013059 24799 24807 PPARGC1a +T721 GO:0006094 24860 24875 gluconeogenesis +T722 PR:000013059 24915 24923 PPARGC1a +T723 GO:0006094 24986 24999 gluconeogenic +T724 SO:0000704 25000 25005 genes +T725 CHEBI:18021 25006 25025 phosphoenolpyruvate +T726 PR:000012416 25006 25041 phosphoenolpyruvate carboxykinase 1 +T727 PR:000012416 25043 25049 Pepck1 +T728 CHEBI:17234 25055 25062 glucose +T729 UBERON:0002107 25091 25097 livers +T730 NCBITaxon:10088 25106 25110 mice +T731 NCBITaxon:10088 25143 25147 mice +T732 UBERON:0002107 25171 25178 hepatic +T733 GO:0006094 25179 25194 gluconeogenesis +T734 http://purl.obolibrary.org/obo/MONDO_0002909 25217 25231 hyperglycaemia +T735 NCBITaxon:10088 25249 25253 mice +T736 SO:0000704 25256 25260 Gene +T737 GO:0010467 25256 25271 Gene expression +T738 UBERON:0004288 25284 25292 skeletal +T739 NCBITaxon:10088 25308 25312 mice +T740 NCBITaxon:10088 25335 25339 mice +T741 UBERON:0004288 25340 25348 skeletal +T742 PR:000013059 25399 25407 Ppargc1a +T743 GO:0010628 25412 25428;25434 25444 up-regulation of ... expression +T744 PR:000017036 25429 25433 Ucp2 +T745 UBERON:0004288 25448 25456 skeletal +T746 NCBITaxon:10088 25474 25478 mice +T747 NCBITaxon:10088 25502 25506 mice +T748 GO:0010467 25519 25529 expression +T749 PR:000015921 25541 25545 Scd1 +T750 UBERON:0004288 25572 25580 skeletal +T751 NCBITaxon:10088 25596 25600 mice +T752 NCBITaxon:10088 25636 25640 mice +T753 SO:0000704 25664 25668 Gene +T754 GO:0010467 25729 25739 expression +T755 MOP:0000568 25743 25752 oxidative +T756 GO:0006119 25743 25768 oxidative phosphorylation +T757 GO:0044429 25773 25797 mitochondrial components +T758 CHEBI:10545 25808 25816 electron +T759 MOP:0000615 25808 25826 electron transport +T760 GO:0022900 25808 25832 electron transport chain +T761 GO:0098803 25808 25840 electron transport chain complex +T762 UBERON:0004288 25856 25864 skeletal +T763 NCBITaxon:10088 25882 25886 mice +T764 NCBITaxon:10088 25922 25926 mice +T765 http://purl.obolibrary.org/obo/MONDO_0011122 25969 25976 obesity +T766 PR:000045358 25978 25985 insulin +T767 http://purl.obolibrary.org/obo/MONDO_0005015 26002 26010 diabetes +T768 UBERON:0001013 26230 26244 adipose tissue +T769 SO:0001060 26275 26282 isoform +T770 GO:0065007 26310 26321 controlling +T771 http://purl.obolibrary.org/obo/MONDO_0011122 26322 26329 obesity +T772 GO:0065007 26383 26393 regulating +T773 UBERON:0001013 26416 26430 adipose tissue +T774 GO:0010467 26466 26475 expressed +T775 UBERON:0000479 26490 26497 tissues +T776 CHEBI:17855 26568 26571 TAG +T777 GO:0008152 26610 26619 metabolic +T778 UBERON:0007023 26637 26642 adult +T779 NCBITaxon:10088 26653 26658 mouse +T780 PR:000045358 26686 26693 insulin +T781 GO:0060612 26747 26757 adipogenic +T782 PR:000039376 26790 26796 PPARg1 +T783 NCBITaxon:10088 26829 26833 mice +T784 UBERON:0001013 26870 26884 adipose tissue +T785 PR:000045358 26917 26924 insulin +T786 PR:000013058 26955 26960 PPARg +T787 SO:0001060 26961 26968 isoform +T788 GO:0065007 26969 26978 regulated +T789 http://purl.obolibrary.org/obo/MONDO_0011122 27008 27015 obesity +T790 UBERON:0001013 27085 27099 adipose tissue +T791 GO:0008152 27153 27162 metabolic +T792 http://purl.obolibrary.org/obo/MONDO_0011122 27213 27218 obese +T793 NCBITaxon:10088 27267 27272 mouse +T794 NCBITaxon:10088 27284 27289 mouse +T795 UBERON:0001013 27343 27357 adipose tissue +T796 GO:0042755 27381 27387 eating +T797 NCBITaxon:10088 27408 27413 mouse +T798 NCBITaxon:10088 27465 27470 mouse +T799 UBERON:0001013 27514 27528 adipose tissue +T800 UBERON:0001013 27594 27608 adipose tissue +T801 GO:0008152 27640 27649 metabolic +T802 PR:000045358 27682 27689 insulin +T803 CL:0000169 27702 27708 β-cell +T804 PR:000045358 27788 27795 insulin +T805 NCBITaxon:10088 27819 27824 mouse +T806 NCBITaxon:10088 27847 27852 mouse +T807 CL:0000136 27956 27966 adipocytes +T808 CL:0000136 28016 28026 adipocytes +T809 GO:0010467 28076 28086 expression +T810 PR:000007312 28090 28093 aP2 +T811 CL:0000136 28112 28121 adipocyte +T812 NCBITaxon:10088 28157 28161 mice +T813 CL:0000136 28184 28194 adipocytes +T814 http://purl.obolibrary.org/obo/MONDO_0006573 28236 28249 lipodystrophy +T815 http://purl.obolibrary.org/obo/MONDO_0006573 28306 28320 lipodystrophic +T816 UBERON:0001013 28352 28366 adipose tissue +T817 NCBITaxon:10088 28402 28406 mice +T818 NCBITaxon:10088 28485 28489 mice +T819 NCBITaxon:10088 28511 28515 mice +T820 PR:000039376 28598 28604 PPARg1 +T821 SO:0001060 28605 28612 isoform +T822 GO:0060612 28638 28667 development of adipose tissue +T823 UBERON:0001013 28653 28667 adipose tissue +T824 NCBITaxon:10088 28710 28715 mouse +T825 UBERON:0001013 28777 28791 adipose tissue +T826 SO:0001060 28834 28841 isoform +T827 NCBITaxon:10088 28902 28906 mice +T828 NCBITaxon:39107 28922 28928 murine +T829 NCBITaxon:9606 28947 28952 human +T830 PR:000013058 28973 28978 PPARg +T831 NCBITaxon:10088 29014 29018 mice +T832 http://purl.obolibrary.org/obo/MONDO_0011122 29058 29063 obese +T833 NCBITaxon:9606 29130 29136 humans +T834 GO:0008152 29205 29218 metabolically +T835 http://purl.obolibrary.org/obo/MONDO_0005015 29231 29239 diabetic +T836 http://purl.obolibrary.org/obo/MONDO_0011122 29250 29255 obese +T837 NCBITaxon:1 29256 29267 individuals +T838 UBERON:0001013 29308 29322 adipose tissue +T839 NCBITaxon:1 29345 29356 individuals +T840 UBERON:0001013 29436 29450 adipose tissue +T841 PR:000045358 29522 29529 insulin +T842 NCBITaxon:10088 29591 29596 mouse +T843 http://purl.obolibrary.org/obo/MONDO_0011122 29610 29615 obese +T844 NCBITaxon:10088 29630 29635 mouse +T845 PR:000045358 29653 29660 insulin +T846 NCBITaxon:10088 29694 29698 mice +T847 PR:000045358 29717 29724 insulin +T848 NCBITaxon:10088 29750 29754 mice +T849 PR:000015066 29805 29810 GLUT4 +T850 UBERON:0001013 29814 29828 adipose tissue +T851 PR:000045358 30002 30009 insulin +T852 NCBITaxon:10088 30062 30066 mice +T853 PR:000045358 30080 30087 insulin +T854 http://purl.obolibrary.org/obo/MONDO_0002909 30113 30127 hyperglycaemia +T855 NCBITaxon:33208 30137 30144 animals +T856 GO:0051866 30205 30222 adaptive response +T857 GO:0051716 30214 30225;30228 30233 response of ... cells +T858 CL:0000169 30226 30233 β-cells +T859 PR:000045358 30237 30244 insulin +T860 NCBITaxon:10088 30282 30286 mice +T861 CL:0000169 30366 30372 β-cell +T862 SO:0000704 30418 30425 genetic +T863 NCBITaxon:10088 30469 30473 mice +T864 CL:0000169 30485 30491 β-cell +T865 CL:0000169 30606 30612 β-cell +T866 CL:0000169 30721 30728 β-cells +T867 PR:000045358 30741 30748 insulin +T868 UBERON:0000006 30787 30804 pancreatic islets +T869 NCBITaxon:10088 30813 30817 mice +T870 PR:000045358 30845 30852 insulin +T871 NCBITaxon:10088 30880 30884 mice +T872 CL:0000169 30921 30928 β-cells +T873 GO:0007567 30995 30999 born +T874 NCBITaxon:10088 31018 31022 mice +T875 UBERON:0000006 31050 31056 islets +T876 CL:0000169 31070 31076 β-cell +T877 NCBITaxon:10088 31099 31104 mouse +T878 http://purl.obolibrary.org/obo/MONDO_0002909 31135 31149 hyperglycaemia +T879 UBERON:0001264 31166 31176 pancreatic +T880 CL:0000169 31166 31183 pancreatic β-cell +T881 PR:000013058 31193 31198 PPARg +T882 NCBITaxon:10088 31202 31207 mouse +T883 CL:0000169 31260 31266 β-cell +T884 PR:000013058 31276 31281 PPARg +T885 NCBITaxon:10088 31285 31290 mouse +T886 GO:0010467 31296 31306 expression +T887 PR:000013058 31310 31315 PPARg +T888 CHEBI:18059 31324 31329 lipid +T889 GO:0019915 31324 31337 lipid storage +T890 UBERON:0000479 31356 31363 tissues +T891 UBERON:0001013 31382 31396 adipose tissue +T892 PR:000045358 31426 31433 insulin +T893 GO:0007631 31483 31490 feeding +T894 NCBITaxon:10088 31500 31504 mice +T895 PR:000045358 31533 31540 insulin +T896 NCBITaxon:10088 31569 31573 mice +T897 UBERON:0001264 31606 31616 pancreatic +T898 CL:0000169 31606 31624 pancreatic β-cells +T899 NCBITaxon:10088 31663 31667 mice +T900 UBERON:0000479 31700 31706 tissue +T901 SO:0000704 31716 31723 genetic +T902 UBERON:0000062 31806 31811 organ +T903 GO:0097009 31841 31859 energy homeostasis +T904 GO:0010467 31901 31911 expression +T905 SO:0001060 31922 31929 isoform +T906 CL:0000169 31933 31940 β-cells +T907 NCBITaxon:9606 31983 31989 humans +T908 SO:0001060 32071 32078 isoform +T909 PR:000045358 32139 32146 insulin +T910 http://purl.obolibrary.org/obo/MONDO_0000001 32162 32169 disease +T911 http://purl.obolibrary.org/obo/MONDO_0011122 32182 32187 obese +T912 NCBITaxon:1 32188 32199 individuals +T913 http://purl.obolibrary.org/obo/MONDO_0005015 32212 32220 diabetes +T914 UBERON:0002107 32232 32237 liver +T915 NCBITaxon:10088 32250 32255 mouse +T916 NCBITaxon:10088 32314 32318 mice +T917 http://purl.obolibrary.org/obo/MONDO_0004790 32333 32348 hepatosteatosis +T918 CHEBI:17855 32364 32376 triglyceride +T919 UBERON:0002107 32391 32396 liver +T920 NCBITaxon:10088 32415 32419 mice +T921 NCBITaxon:10088 32438 32442 mice +T922 UBERON:0001013 32466 32480 adipose tissue +T923 NCBITaxon:10088 32495 32499 mice +T924 http://purl.obolibrary.org/obo/MONDO_0004790 32509 32524 hepatosteatosis +T925 NCBITaxon:10088 32536 32540 mice +T926 SO:0001060 32568 32575 isoform +T927 CHEBI:17855 32614 32626 triglyceride +T928 UBERON:0002107 32645 32650 liver +T929 UBERON:0002107 32719 32724 liver +T930 CL:0000169 32729 32735 β-cell +T931 SO:0000704 32850 32854 gene +T932 GO:0010467 32850 32865 gene expression +T933 UBERON:0001013 32882 32896 adipose tissue +T934 UBERON:0000006 32898 32914 pancreatic islet +T935 UBERON:0002107 32916 32921 liver +T936 UBERON:0004288 32927 32935 skeletal +T937 NCBITaxon:10088 32955 32960 mouse +T938 CHEBI:18059 32966 32971 lipid +T939 UBERON:0001013 32983 32997 adipose tissue +T940 NCBITaxon:10088 33008 33012 mice +T941 CHEBI:17855 33044 33048 TAGs +T942 CHEBI:18035 33063 33067 DAGs +T943 SO:0000704 33095 33099 gene +T944 GO:0010467 33095 33110 gene expression +T945 PR:000006433 33114 33119 DGAT2 +T946 PR:000012942 33151 33178 adipose triglyceride lipase +T947 CHEBI:17855 33159 33171 triglyceride +T948 CHEBI:17855 33197 33201 TAGs +T949 UBERON:0001013 33214 33228 adipose tissue +T950 CHEBI:18059 33278 33283 lipid +T951 SO:0000704 33298 33302 gene +T952 GO:0010467 33298 33313 gene expression +T953 MOP:0000568 33346 33355 oxidative +T954 MOP:0000568 33408 33417 oxidative +T955 PR:000045358 33429 33436 insulin +T956 UBERON:0001013 33482 33496 adipose tissue +T957 CL:0000235 33500 33511 macrophages +T958 CL:0000235 33596 33606 macrophage +T959 UBERON:0001013 33627 33641 adipose tissue +T960 NCBITaxon:10088 33650 33654 mice +T961 NCBITaxon:10088 33681 33685 mice +T962 UBERON:0000006 33723 33729 islets +T963 CHEBI:18035 33774 33778 DAGs +T964 CHEBI:17761 33803 33812 ceramides +T965 CHEBI:18059 33870 33875 lipid +T966 CL:0000169 33898 33905 β-cells +T967 GO:0019432 33919 33936 formation of TAGs +T968 CHEBI:17855 33932 33936 TAGs +T969 UBERON:0002107 33976 33981 Liver +T970 UBERON:0004288 33986 33994 skeletal +T971 CHEBI:17855 34033 34036 TAG +T972 CHEBI:18059 34073 34078 lipid +T973 CHEBI:17761 34095 34104 ceramides +T974 CHEBI:60479 34109 34133 lysophosphatidylcholines +T975 NCBITaxon:10088 34142 34146 mice +T976 NCBITaxon:10088 34165 34169 mice +T977 CHEBI:18059 34176 34181 lipid +T978 GO:0065007 34242 34253 controlling +T979 GO:0008610 34262 34273 lipogenesis +T980 GO:0015908 34275 34299 transport of fatty acids +T981 CHEBI:35366 34288 34299 fatty acids +T982 MOP:0000568 34310 34319 oxidation +T983 NCBITaxon:10088 34332 34336 mice +T984 NCBITaxon:10088 34361 34365 mice +T985 PR:000013059 34380 34388 Ppargc1a +T986 GO:0006094 34399 34412 gluconeogenic +T987 SO:0000704 34413 34418 genes +T988 UBERON:0002107 34439 34444 liver +T989 NCBITaxon:10088 34453 34457 mice +T990 NCBITaxon:10088 34484 34488 mice +T991 http://purl.obolibrary.org/obo/MONDO_0002909 34546 34560 hyperglycaemia +T992 NCBITaxon:10088 34569 34573 mice +T993 CHEBI:18059 34667 34672 lipid +T994 UBERON:0000479 34693 34700 tissues +T995 UBERON:0001013 34722 34736 adipose tissue +T996 http://purl.obolibrary.org/obo/MONDO_0004790 34746 34761 hepatosteatosis +T997 NCBITaxon:10088 34774 34779 mouse +T998 NCBITaxon:10088 34802 34807 mouse +T999 CHEBI:17855 34849 34852 TAG +T1000 NCBITaxon:10088 34865 34870 mouse +T1001 CHEBI:18059 34939 34944 lipid +T1002 PR:000045358 34975 34982 insulin +T1003 UBERON:0001013 35007 35021 adipose tissue +T1004 UBERON:0000062 35041 35047 organs +T1005 NCBITaxon:1 35066 35074 organism +T1006 CHEBI:17234 35075 35082 glucose +T1007 GO:0006006 35075 35093 glucose metabolism +T1008 GO:0010467 35123 35133 expression +T1009 UBERON:0001264 35151 35159 pancreas +T1010 UBERON:0002107 35161 35166 liver +T1011 NCBITaxon:10088 35192 35197 mouse +T1012 UBERON:0000062 35271 35277 organs +T1013 CHEBI:18059 35294 35299 lipid +T1014 CHEBI:17855 35356 35360 TAGs +T1015 NCBITaxon:10088 35481 35485 mice +T1016 CHEBI:33290 35564 35568 food +T1017 GO:0007631 35564 35575 food intake +T1018 GO:0040011 35577 35586 locomotor +T1019 NCBITaxon:10088 35703 35707 mice +T1020 UBERON:0001013 35766 35780 adipose tissue +T1021 NCBITaxon:10088 35807 35811 mice +T1022 UBERON:0002107 35866 35871 liver +T1023 CL:0000169 35884 35891 β-cells +T1024 http://purl.obolibrary.org/obo/MONDO_0006573 35895 35909 lipodistrophic +T1025 NCBITaxon:10088 35910 35914 mice +T1026 NCBITaxon:10088 35925 35930 mouse +T1027 NCBITaxon:10088 36016 36020 mice +T1028 CHEBI:33284 36053 36061 nutrient +T1029 CHEBI:17855 36098 36101 TAG +T1030 UBERON:0000479 36116 36123 tissues +T1031 CHEBI:18059 36156 36161 lipid +T1032 UBERON:0000062 36179 36185 organs +T1033 UBERON:0002107 36220 36225 liver +T1034 CL:0000169 36230 36236 β-cell +T1035 NCBITaxon:10088 36258 36263 mouse +T1036 SO:0001060 36456 36463 isoform +T1037 UBERON:0001013 36477 36491 adipose tissue +T1038 GO:0008152 36537 36546 metabolic +T1039 UBERON:0001013 36591 36605 adipose tissue +T1040 UBERON:0001013 36738 36752 adipose tissue +T1041 GO:0010467 36816 36825 expressed +T1042 UBERON:0000062 36848 36854 organs +T1043 CHEBI:18059 36900 36906 lipids +T1044 PR:000045358 37040 37047 insulin +T1045 CL:0000169 37060 37066 β-cell +T1046 http://purl.obolibrary.org/obo/MONDO_0005015 37076 37084 diabetes +T1047 http://purl.obolibrary.org/obo/MONDO_0021187 37090 37105 hyperlipidaemia +T1048 NCBITaxon:9606 37138 37144 humans +T1049 NCBITaxon:1 37183 37194 individuals +T1050 http://purl.obolibrary.org/obo/MONDO_0004955 37273 37275 MS +T1051 GO:0065007 37339 37346 control +T1052 UBERON:0001013 37347 37361 adipose tissue +T1053 NCBITaxon:10088 37415 37419 mice +T1054 NCBITaxon:10088 37477 37481 Mice +T1055 SO:0000147 37515 37519 exon +T1056 NCBITaxon:10088 37621 37625 mice +T1057 NCBITaxon:10088 37660 37664 mice +T1058 NCBITaxon:10088 37767 37771 mice +T1059 SO:0000704 38020 38024 gene +T1060 NCBITaxon:33208 38080 38086 Animal +T1061 NCBITaxon:33208 38094 38101 Animals +T1062 NCBITaxon:33208 38135 38142 animals +T1063 CHEBI:33290 38225 38229 Food +T1064 CHEBI:15377 38234 38239 water +T1065 NCBITaxon:33208 38284 38290 animal +T1066 UBERON:0000178 38390 38395 Blood +T1067 UBERON:0001088 38400 38405 urine +T1068 CHEBI:33290 38420 38424 food +T1069 GO:0007631 38420 38431 food intake +T1070 NCBITaxon:10088 38465 38469 Mice +T1071 CHEBI:33290 38561 38565 chow +T1072 UBERON:0001969 38714 38720 plasma +T1073 CHEBI:17855 38760 38764 TAGs +T1074 PR:000045358 38903 38910 insulin +T1075 CHEBI:17234 39217 39224 glucose +T1076 UBERON:0000178 39228 39233 blood +T1077 UBERON:0001088 39241 39246 urine +T1078 CHEBI:33290 39251 39255 food +T1079 GO:0007631 39251 39262 food intake +T1080 CHEBI:15377 39359 39364 water +T1081 GO:0007631 39365 39371 intake +T1082 GO:0040011 39377 39386 locomotor +T1083 NCBITaxon:33208 39551 39557 Animal +T1084 CHEBI:15377 39631 39636 Water +T1085 GO:0007631 39637 39645 consumed +T1086 NCBITaxon:10088 39677 39681 Mice +T1087 CHEBI:15379 39888 39890 O2 +T1088 CHEBI:16526 39896 39910 carbon dioxide +T1089 CHEBI:16526 39912 39915 CO2 +T1090 CHEBI:15379 39969 39971 O2 +T1091 CHEBI:16526 39976 39979 CO2 +T1092 NCBITaxon:10088 39989 39993 Mice +T1093 NCBITaxon:10088 40065 40069 Mice +T1094 GO:0040011 40102 40112 Ambulatory +T1095 NCBITaxon:10088 40145 40149 mice +T1096 GO:0040011 40246 40256 ambulatory +T1097 UBERON:0001088 40369 40374 urine +T1098 UBERON:0001088 40392 40397 urine +T1099 UBERON:0001088 40507 40512 urine +T1100 CHEBI:17234 40523 40530 glucose +T1101 UBERON:0001088 40534 40539 urine +T1102 CHEBI:17234 40575 40582 glucose +T1103 CHEBI:15377 40586 40591 water +T1104 GO:0007631 40592 40598 intake +T1105 CHEBI:16646 40626 40630 carb +T1106 CHEBI:16646 40641 40645 carb +T1107 MOP:0000568 40674 40684 oxidations +T1108 CHEBI:16646 40705 40718 carbohydrates +T1109 CHEBI:17234 40722 40729 glucose +T1110 UBERON:0000006 40825 40831 islets +T1111 UBERON:0000479 40836 40843 tissues +T1112 UBERON:0000479 41191 41197 tissue +T1113 CHEBI:8984 41232 41235 SDS +T1114 CHEBI:53250 41322 41347 polyvinylidene difluoride +T1115 UBERON:0001913 41421 41425 milk +T1116 CHEBI:53424 41433 41441 Tween 20 +T1117 PR:000015066 41478 41483 GLUT4 +T1118 GO:0005576 41488 41501 extracellular +T1119 PR:000000104 41488 41527 extracellular signal-regulated kinase 1 +T1120 PR:000000103 41488 41525;41528 41529 extracellular signal-regulated kinase ... 2 +T1121 GO:0065007 41509 41518 regulated +T1122 PR:000000104 41531 41535 ERK1 +T1123 GO:0042571 41539 41549 antibodies +T1124 UBERON:0000479 41699 41705 Tissue +T1125 UBERON:0001013 41841 41855 adipose tissue +T1126 UBERON:0001264 41860 41868 pancreas +T1127 UBERON:0001013 42079 42093 adipose tissue +T1128 NCBITaxon:33208 42171 42177 animal +T1129 UBERON:0001264 42316 42324 pancreas +T1130 UBERON:0000006 42390 42407 pancreatic islets +T1131 UBERON:0001264 42414 42422 pancreas +T1132 UBERON:0002394 42444 42453 bile duct +T1133 CHEBI:75958 42471 42479 solution +T1134 UBERON:0001264 42524 42532 pancreas +T1135 UBERON:0000006 42574 42580 islets +T1136 UBERON:0000006 42616 42622 islets +T1137 CHEBI:16526 42729 42732 CO2 +T1138 UBERON:0000006 42741 42747 Islets +T1139 PR:000045358 42786 42793 insulin +T1140 PR:000045358 42832 42839 Insulin +T1141 PR:000045358 42860 42867 Insulin +T1142 UBERON:0000006 42892 42898 islets +T1143 UBERON:0000006 42905 42911 islets +T1144 CHEBI:17234 43008 43015 glucose +T1145 CHEBI:17234 43025 43032 glucose +T1146 CHEBI:17234 43045 43052 glucose +T1147 CHEBI:27999 43065 43076 tolbutamide +T1148 CHEBI:28262 43080 43084 DMSO +T1149 PR:000045358 43120 43127 insulin +T1150 PR:000045358 43129 43136 Insulin +T1151 CHEBI:16236 43170 43177 ethanol +T1152 CHEBI:15366 43178 43189 acetic acid +T1153 PR:000045358 43191 43198 Insulin +T1154 NCBITaxon:10088 43220 43225 Mouse +T1155 PR:000045358 43226 43233 Insulin +T1156 UBERON:0000006 43281 43287 Islets +T1157 NCBITaxon:10088 43313 43317 mice +T1158 CHEBI:75958 43460 43468 solution +T1159 UBERON:0000006 43499 43505 islets +T1160 PR:000045358 43524 43531 insulin +T1161 GO:0030073 43524 43539 insulin release +T1162 PR:000045358 43558 43565 insulin +T1163 NCBITaxon:10088 43604 43608 mice +T1164 CHEBI:18059 43655 43660 Lipid +T1165 UBERON:0001347 43677 43680 WAT +T1166 UBERON:0000479 43697 43703 tissue +T1167 CHEBI:26710 43747 43762 sodium chloride +T1168 CHEBI:18059 43781 43787 lipids +T1169 CHEBI:35255 43816 43826 chloroform +T1170 CHEBI:17790 43828 43836 methanol +T1171 UBERON:0002107 43896 43901 liver +T1172 UBERON:0000006 43906 43912 islets +T1173 UBERON:0002107 43936 43941 liver +T1174 UBERON:0000006 43955 43961 islets +T1175 CHEBI:60004 43987 43994 mixture +T1176 CHEBI:36357 44009 44018 compounds +T1177 CHEBI:26710 44071 44086 sodium chloride +T1178 UBERON:0002107 44092 44097 liver +T1179 CHEBI:35255 44104 44114 chloroform +T1180 CHEBI:17790 44115 44123 methanol +T1181 UBERON:0002107 44142 44147 liver +T1182 UBERON:0000006 44161 44167 islets +T1183 UBERON:0000479 44186 44192 tissue +T1184 UBERON:0002107 44260 44265 liver +T1185 UBERON:0000006 44278 44284 islets +T1186 UBERON:0002107 44309 44314 liver +T1187 UBERON:0000006 44327 44333 islets +T1188 CHEBI:60004 44464 44471 mixture +T1189 CHEBI:36357 44513 44522 compounds +T1190 CHEBI:18059 44620 44625 lipid +T1191 CHEBI:46787 44881 44888 solvent +T1192 CHEBI:15377 44933 44938 water +T1193 CHEBI:62947 44947 44952 NH4Ac +T1194 CHEBI:30751 44959 44964 HCOOH +T1195 CHEBI:38472 45024 45036 acetonitrile +T1196 CHEBI:17824 45037 45048 isopropanol +T1197 CHEBI:62947 45062 45067 NH4Ac +T1198 CHEBI:30751 45074 45079 HCOOH +T1199 CHEBI:28487 45684 45693 Reserpine +T1200 CHEBI:36357 45741 45749 compound +T1201 CHEBI:18059 45918 45923 lipid +T1202 CHEBI:18059 46238 46243 lipid +T1203 UBERON:0001013 46701 46715 Adipose Tissue +T1204 UBERON:0002107 46720 46725 Liver +T1205 SO:0000704 46726 46730 Gene +T1206 GO:0010467 46726 46741 Gene Expression +T1207 CHEBI:15377 46805 46810 Water +T1208 GO:0007631 46811 46819 Consumed +T1209 GO:0040011 46824 46833 Locomotor +T1210 PR:000015066 46906 46911 GLUT4 +T1211 GO:0010467 46920 46930 expression +T1212 SO:0000704 47001 47005 Gene +T1213 GO:0010467 47001 47016 Gene Expression +T1214 UBERON:0000006 47020 47026 Islets +T1215 SO:0000704 47090 47094 Gene +T1216 GO:0010467 47090 47105 Gene Expression +T1217 NCBITaxon:10088 47186 47191 Mouse +T1218 UBERON:0000479 47280 47286 Tissue +T1219 NCBITaxon:10088 47344 47348 mice +T1220 SO:0000704 47682 47687 genes +T1221 SO:0000704 47692 47696 gene +T1222 NCBITaxon:33208 47801 47807 Animal +T1223 UBERON:0001264 48079 48087 pancreas +T1224 NCBITaxon:10088 48266 48271 mouse +T1225 CHEBI:18035 48289 48292 DAG +T1226 CHEBI:18035 48295 48309 diacylglycerol +T1227 CHEBI:17234 48318 48325 glucose +T1228 CHEBI:51686 48339 48340 H +T1229 CHEBI:51686 48349 48361 haematoxylin +T1230 PR:000045358 48379 48386 insulin +T1231 http://purl.obolibrary.org/obo/MONDO_0004955 48452 48454 MS +T1232 GO:0008152 48457 48466 Metabolic +T1233 http://purl.obolibrary.org/obo/MONDO_0004955 48457 48475 Metabolic Syndrome +T1234 PR:000013058 48477 48482 PPARg +T1235 GO:0005777 48485 48495 peroxisome +T1236 PR:000013058 48485 48533 peroxisome proliferator activated receptor gamma +T1237 GO:0005777 48544 48554 peroxisome +T1238 PR:000013058 48544 48592 peroxisome proliferator activated receptor gamma +T1239 PR:000013059 48596 48604 PPARGC1a +T1240 PR:000013059 48607 48632 PPARg coactivator 1 alpha +T1241 PR:000015921 48634 48638 Scd1 +T1242 CHEBI:15541 48641 48660 stearoyl-coenzyme A +T1243 PR:000015921 48641 48673 stearoyl-coenzyme A desaturase 1 +T1244 CHEBI:17855 48675 48678 TAG +T1245 CHEBI:17855 48681 48696 triacylglycerol +T1246 UBERON:0001347 48698 48701 WAT +T1247 UBERON:0001347 48704 48724 white adipose tissue +T1248 NCBITaxon:10088 48811 48816 Mouse +T1249 CHEBI:33290 49049 49053 Food +T1250 GO:0007631 49049 49060 Food intake +T1251 NCBITaxon:10088 49083 49087 mice +T1252 NCBITaxon:10088 49201 49205 mice +T1253 GO:0007631 49206 49209 fed +T1254 CHEBI:33290 49210 49214 chow +T1255 NCBITaxon:10088 49220 49224 mice +T1256 CHEBI:51686 49306 49318 Haematoxylin +T1257 CHEBI:51686 49330 49331 H +T1258 UBERON:0001301 49367 49377 epididymal +T1259 UBERON:0001347 49378 49381 WAT +T1260 NCBITaxon:10088 49422 49426 mice +T1261 UBERON:0001301 49492 49502 epididymal +T1262 UBERON:0001347 49503 49506 WAT +T1263 CL:0000448 49503 49517 WAT adipocytes +T1264 NCBITaxon:10088 49569 49573 mice +T1265 PR:000045358 49603 49610 Insulin +T1266 NCBITaxon:10088 49630 49634 Mice +T1267 UBERON:0001969 49683 49689 plasma +T1268 CHEBI:17234 49690 49697 glucose +T1269 UBERON:0001969 49846 49852 Plasma +T1270 CHEBI:17234 49853 49860 glucose +T1271 NCBITaxon:10088 49922 49926 mice +T1272 CHEBI:33290 49930 49934 chow +T1273 CHEBI:51686 50115 50116 H +T1274 UBERON:0001264 50149 50157 pancreas +T1275 NCBITaxon:10088 50193 50197 mice +T1276 CL:0000169 50227 50233 β-Cell +T1277 UBERON:0002107 50247 50254 Hepatic +T1278 NCBITaxon:10088 50289 50293 Mice +T1279 CHEBI:51686 50299 50300 H +T1280 PR:000045358 50372 50379 insulin +T1281 UBERON:0001264 50396 50404 pancreas +T1282 NCBITaxon:10088 50446 50450 mice +T1283 PR:000045358 50465 50472 Insulin +T1284 UBERON:0000006 50484 50490 islets +T1285 NCBITaxon:10088 50546 50550 mice +T1286 UBERON:0000006 50608 50614 islets +T1287 PR:000045358 50621 50628 Insulin +T1288 UBERON:0000006 50644 50650 islets +T1289 NCBITaxon:10088 50705 50709 mice +T1290 CHEBI:17234 50725 50732 glucose +T1291 CHEBI:17234 50747 50754 glucose +T1292 CHEBI:27999 50763 50774 tolbutamide +T1293 UBERON:0000006 50835 50841 islets +T1294 NCBITaxon:10088 50853 50857 mice +T1295 PR:000045358 50893 50900 insulin +T1296 GO:0030073 50893 50908 insulin release +T1297 PR:000045358 50927 50934 insulin +T1298 CHEBI:51686 51010 51011 H +T1299 UBERON:0002107 51043 51048 liver +T1300 NCBITaxon:10088 51090 51094 mice +T1301 SO:0000704 51129 51133 Gene +T1302 GO:0010467 51129 51144 Gene Expression +T1303 UBERON:0001347 51162 51165 WAT +T1304 UBERON:0001347 51194 51197 WAT +T1305 NCBITaxon:10088 51239 51243 mice +T1306 UBERON:0001013 51250 51264 Adipose tissue +T1307 SO:0000704 51292 51297 genes +T1308 NCBITaxon:10088 51349 51353 mice +T1309 SO:0000704 51449 51453 Gene +T1310 GO:0010467 51449 51464 Gene Expression +T1311 UBERON:0000006 51477 51483 Islets +T1312 UBERON:0002107 51488 51493 Liver +T1313 NCBITaxon:10088 51504 51508 Mice +T1314 UBERON:0000006 51533 51539 islets +T1315 UBERON:0002107 51548 51553 liver +T1316 NCBITaxon:10088 51610 51614 mice +T1317 CHEBI:17855 51616 51618 TG +T1318 CHEBI:17855 51620 51624 TAGs +T1319 CHEBI:18035 51626 51630 DAGs +T1320 CHEBI:18035 51632 51647 diacylglycerols +T1321 CHEBI:64583 51649 51651 SM +T1322 CHEBI:64583 51653 51667 sphingomyelins +T1323 UBERON:0002107 51673 51678 Liver +T1324 SO:0000704 51679 51683 gene +T1325 GO:0010467 51679 51694 gene expression +T1326 NCBITaxon:10088 51746 51750 mice +T1327 GO:0007631 51751 51754 fed +T1328 CHEBI:33290 51755 51759 chow +T1329 GO:0019915 51905 51922 Storage of Lipids +T1330 CHEBI:18059 51916 51922 Lipids +T1331 UBERON:0001013 52011 52025 adipose tissue +T1332 CHEBI:17855 52046 52058 triglyceride +T1333 UBERON:0002107 52112 52117 liver +T1334 UBERON:0004288 52119 52127 skeletal +T1335 UBERON:0001264 52140 52148 pancreas +T1336 CHEBI:17855 52164 52167 TAG +T1337 NCBITaxon:10088 52175 52179 mice +T1338 GO:0010467 52198 52208 expression +T1339 UBERON:0002107 52212 52217 liver +T1340 CL:0000169 52231 52237 β-cell +T1341 UBERON:0000062 52292 52298 organs +T1342 CHEBI:17855 52314 52317 TAG +T1343 NCBITaxon:10088 52361 52366 mouse +T1344 UBERON:0002107 52367 52372 liver +T1345 CL:0000169 52386 52393 β-cells +T1346 CHEBI:18059 52438 52443 lipid +T1347 CHEBI:17855 52466 52469 TAG +T1348 PR:000045358 52489 52496 insulin +T1349 CL:0000169 52512 52518 β-cell +T1350 GO:0008152 52538 52547 Metabolic +T1351 GO:0007631 52562 52565 Fed +T1352 NCBITaxon:10088 52622 52626 Mice +T1353 GO:0008152 52637 52646 Metabolic +T1354 NCBITaxon:10088 52696 52700 Mice +T1355 CHEBI:33893 53063 53071 reagents +T1356 UBERON:0002107 53187 53194 Hepatic +T1357 UBERON:0001013 53199 53213 Adipose Tissue +T1358 GO:0008152 53235 53244 Metabolic +T1359 http://purl.obolibrary.org/obo/MONDO_0004955 53235 53253 Metabolic Syndrome +T1360 http://purl.obolibrary.org/obo/MONDO_0005015 53331 53339 Diabetes diff --git a/src/ontogpt/evaluation/craft/database/all/17465682.txt b/src/ontogpt/evaluation/craft/database/all/17465682.txt new file mode 100644 index 000000000..a3fad33d0 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17465682.txt @@ -0,0 +1,379 @@ +PPAR gamma 2 Prevents Lipotoxicity by Controlling Adipose Tissue Expandability and Peripheral Lipid Metabolism + +Abstract + +Peroxisome proliferator activated receptor gamma 2 (PPARg2) is the nutritionally regulated isoform of PPARg. Ablation of PPARg2 in the ob/ob background, PPARg2−/− Lepob/Lepob (POKO mouse), resulted in decreased fat mass, severe insulin resistance, β-cell failure, and dyslipidaemia. Our results indicate that the PPARg2 isoform plays an important role, mediating adipose tissue expansion in response to positive energy balance. Lipidomic analyses suggest that PPARg2 plays an important antilipotoxic role when induced ectopically in liver and muscle by facilitating deposition of fat as relatively harmless triacylglycerol species and thus preventing accumulation of reactive lipid species. Our data also indicate that PPARg2 may be required for the β-cell hypertrophic adaptive response to insulin resistance. In summary, the PPARg2 isoform prevents lipotoxicity by (a) promoting adipose tissue expansion, (b) increasing the lipid-buffering capacity of peripheral organs, and (c) facilitating the adaptive proliferative response of β-cells to insulin resistance. + +Author Summary + + + +It is known that obesity is linked to type 2 diabetes, however how obesity causes insulin resistance and diabetes is not well understood. Some extremely obese people are not diabetic, while other less obese people develop severe insulin resistance and diabetes. We believe diabetes occurs when adipose tissue becomes “full,” and fat overflows into other organs such as liver, pancreas, and muscle, causing insulin resistance and diabetes. Peroxisome proliferator activated receptor gamma (PPARg) is essential for the development of adipose tissue and control of insulin sensitivity. PPARg2 is the isoform of PPARg regulated by nutrition. Here we investigate the role of PPARg2 under conditions of excess nutrients by removing the PPARg2 isoform in genetically obese mice, the POKO mouse. We report that removing PPARg2 decreases adipose tissue's capacity to expand and prevents the mouse from making as much fat as a normal obese mouse, despite eating similarly. Our studies suggest that PPARg plays an important antitoxic role when it is induced in liver, muscle, and beta cells by facilitating deposition of fat as relatively harmless lipids and thus prevents accumulation of toxic lipid species. We also show that PPARg2 may be involved in the adaptive response of beta cells to insulin resistance. + +Introduction + +An adipocentric view of the Metabolic Syndrome (MS) considers obesity as the major factor leading to insulin resistance in peripheral metabolic tissues. However, the link between obesity and insulin resistance is complex, as indicated by the fact that some extremely obese people are glucose tolerant, while others with a mild degree of obesity develop severe insulin resistance and diabetes. This suggests that the absolute amount of fat stored may not be the most important factor determining the relationship between obesity and insulin resistance. Recent work showing the complexity of the molecular mechanisms controlling adipogenesis [1,2] suggests that adipose tissue expandability may be an important factor linking obesity, insulin resistance, and associated comorbidities. + +There are two mechanisms that have been proposed to explain how expansion of the adipose tissue stores affects insulin sensitivity. One mechanism suggests that increased adiposity induces a chronic inflammatory state characterized by increased cytokine production by adipocytes and/or from macrophages infiltrating adipose tissue. Cytokines produced by these adipocytes or macrophages may directly antagonise insulin signalling [3,4]. A second nonexclusive hypothesis is lipotoxicity. The lipotoxic hypothesis states that if the amount of fuel entering a tissue exceeds its oxidative or storage capacity, toxic metabolites that inhibit insulin action are formed [5–8]. Of particular relevance to this article, lipid metabolites, such as ceramides and diacylglycerol (DAG) or reactive oxygen species generated from hyperactive oxidative pathways, have been shown to inhibit insulin signalling and to induce apoptosis [9–11]. + +The nuclear receptor peroxisome proliferator activated receptor gamma (PPARg) is critically required for adipogenesis and insulin sensitivity [12–15]. There are two PPARg isoforms, PPARg1 and PPARg2. PPARg1 is expressed in many tissues and cell types, including white and brown adipose tissue, skeletal muscle, liver, pancreatic β-cells, macrophages, colon, bone, and placenta [16]. Under physiological conditions, expression of PPARg2, the other splice variant, is restricted to white and brown adipose tissue [16,17]. In adipose tissue PPARg is the key regulator of adipogenesis. PPARg2 is the more adipogenic PPARg isoform in vitro, it is also the isoform regulated transcriptionally by nutrition [17–20]. Although under physiological conditions expression of PPARg2 is limited to adipose tissues, we have shown that PPARg2 is ectopically induced in liver and skeletal muscle in response to overnutrition or genetic obesity [2,18]. De novo expression of PPARg2 in liver and muscle in obesity suggests that PPARg2 may have a role in insulin resistance and lipotoxicity in these tissues. Little in vivo research into the metabolic roles for the specific isoforms of PPARg has been carried out, with the studies so far focusing almost exclusively on adipose tissue [2,13,21,22]. PPARg (both isoforms) deletions have been generated in most major metabolic tissues. Liver-specific deletion of both PPARg isoforms caused an impairment in insulin sensitivity, particularly when challenged by different genetic backgrounds (lipoatrophic or leptin-deficiency) [23,24]. The effect of ablating both PPARg isoforms in muscle produced controversial results, with two groups reporting different effects on insulin sensitivity [25,26]. The role of PPARg in pancreatic β-cells is unclear, primarily due to its low expression under physiological conditions [27–29] and secondly because ablation of both PPARg isoforms in β-cells did not result in a metabolic phenotype. However PPARg may play a role in β-cell hyperplasia in response to insulin resistance, an idea supported by the fact that mice that lack PPARg in β-cells do not expand their β-cells mass in response to a high-fat diet [30]. More recently, it has been shown that heterozygous PPARg-deficient mice develop impaired insulin secretion, which is associated with increased islet triacylglycerol (TAG) content [31]. + +Here we investigate the physiological relevance of PPARg2 under conditions of positive energy balance by ablating PPARg2 in ob/ob mice. We use a new approach that integrates traditional physiological phenotyping with advanced lipidomic technology and transcriptomics. Our results indicate that in the context of positive energy balance, the absence of PPARg2 results in a major metabolic failure. Furthermore, we provide evidence that control of adipose tissue expansion by PPARg2 may be an important variable linking positive energy balance to its metabolic complications including insulin resistance, β-cell failure, and dyslipidaemia. Similarly, our lipidomic results indicate that induction of PPARg2 in nonadipose tissues should be considered as a physiological adaptation that prevents the toxic effects produced by excess nutrients. This antilipotoxic effect of PPARg2 is achieved by increasing the lipid-buffering capacity of peripheral organs and facilitating β-cell hyperplasia in response to insulin resistance. + +Results + +Ablation of PPARg2 in Ob/Ob Mice (POKO Mouse) Prevents Adipose Tissue Expansion in Response to Positive Energy Balance + +PPARg2−/− Lepob/Lepob mice with genetic ablation of the PPARg2 isoform on the obese hyperphagic ob/ob background (POKO) were generated. Matings of PPARg2+/− Lepob/Lep+ mice followed the expected Mendelian distribution (Fisher's test = 0.074 and 0.135 for males and females, respectively). PPARg1 gene expression in white adipose tissue (WAT) from five-week-old POKO mice was similar to PPARg2 KO mice and was not significantly different from wild-type (WT) mice (Figure S1). + +Figure 1A shows growth curves for male and female mice of four genotypes (WT, PPARg2 KO, ob/ob, and POKO mice) over a 12-week period. At birth, the body weight of male and female POKO mice was indistinguishable from other genotypes (unpublished data). The ob/ob mice quickly became heavier than their WT littermates, with significantly elevated body weight by four and six weeks of age in female and male mice, respectively. However, the POKO mice did not become obese, and their body weight remained close to WT and PPARg2 KO body weights mice during the 12-week study. + +POKO mice were as hyperphagic (Figure 1B) as the ob/ob mice but drank far more water compared with ob/ob littermates (81.85 ± 15.14 versus 9.05 ± 2.32 ml/70 h, p < 0.01, female POKO versus ob/ob, n = 4 at 20 wk) (Figure S2A). Dual-energy X-ray absorptiometry analysis at 20 wk (Figure 1C) confirmed that female POKO mice had slightly increased fat content (4%) compared to WT and PPARg2 KO mice, but significantly reduced fat mass compared to the 40% increase observed in ob/ob mice. At the age of 20 wk, POKO and ob/ob mice had a trend to a decreased total locomotor activity during dark and light cycles compared with the WT and PPARg2 KO mice over the 72-h period. However POKO had similar total locomotor activity compared with ob/ob mice (Figure S2B). + +At six weeks of age, female POKO mice consumed a similar amount of oxygen as ob/ob mice (vO2 = 25.06 ± 0.89 versus 23.10 ± 0.99 ml/kg bodyweight 0.75/min, p = 0.07 POKO versus ob/ob, n = 6–8) showing a lower respiratory exchange ratio (0.916 ± 0.011 versus 0.952 ± 0.007, p = 0.01, female POKO versus ob/ob) in the fed state, but similar respiratory exchange ratio in the fasted state (0.73 ± 0.014 versus 0.75 ± 0.018, p-value = 0.59 POKO versus ob/ob mice). Water intake was already significantly increased in POKO compared to ob/ob mice (13.59 ± 1.88 versus 8.15 ± 0.89 ml/d, p-value < 0.05, POKO versus ob/ob). Furthermore, levels of glucose in urine were higher in POKO mice compared with ob/ob mice (403.4 ± 49.2 versus 34.13 ± 13.5 mMol/l, POKO versus ob/ob mice, p-value = 0.001), showing an energy loss of 15.43 ± 3.06 kJ/d through urine compared with 0.70 ± 0.19 kJ/d in ob/ob mice. At this age, POKO mice showed similar locomotor activity compared with the ob/ob mice during the day, but increased locomotor activity during the night (Figure S2C). + +Histomorphometric analysis of adipose tissue from 16-wk-old male mice revealed that POKO mice had fewer small adipocytes than the ob/ob mice (Figure 1D and 1E). This analysis of adipocyte size suggests that ablation of PPARg2 in the ob/ob background impairs the potential for adipocyte recruitment. + +Early Insulin Resistance in POKO Mice Independent of Body Weight + +As expected the reduced adipose tissue expandability of the POKO mouse was associated with severe insulin resistance. Surprisingly insulin resistance developed very early in life with elevated insulin levels and blood glucose compared to ob/ob mice (Table 1). We investigated whether peripheral insulin resistance and/or a severe defect in insulin secretion may cause hyperglycaemia in the POKO mouse. No differences in plasma glucose levels were detected three to five days after birth amongst the four genotypes for both genders (unpublished data). At weaning (three weeks of age) total body weight was indistinguishable amongst the four genotypes, and blood glucose levels were similar in males and females (Figure 2A). However, by the age of four weeks, coincident with the change to a chow diet, male and female POKO mice developed severe hyperglycaemia compared to the other genotypes. Insulin plasma levels in the POKO mice at four weeks of age were increased compared to ob/ob mice (Table 1). Insulin resistance in POKO mice was confirmed by an insulin tolerance test (ITT) in four-week-old male and female mice (Figure 2B). Furthermore insulin resistance in adipose tissue was demonstrated by the extremely low levels of glucose transporter4 (GLUT4) protein in POKO adipose tissue when compared with GLUT4 levels in adipose tissue from ob/ob mice (Figure S3). Of note, insulin resistance in the POKO mice was associated with hypertriglyceridaemia as early as four-weeks of age (Table 1). + +Adult POKO Mice are Hyperglycaemic and Have Low Plasma Insulin Levels + +Given the early insulin resistance and hyperinsulinaemia in the young POKO mice, we expected to see increased insulin levels in mature POKO mice. At 16 weeks, male POKO mice exhibited severe hyperglycaemia in the fasted and fed states compared to littermate controls. Male POKO mice had inappropriately low levels of insulin (Table 2). A similar, but milder phenotype was also observed in POKO female mice (unpublished data). Of note, adult ob/ob mice compensated for their insulin resistance with increased insulin levels (Table 2). POKO mice also had hypertriglyceridaemia when compared to WT, ob/ob, or PPARg2 KO mice. + +Impaired Beta-Cell Function in the POKO Mice + +The inappropriately low insulin levels in the adult POKO mice suggested a defect in β-cells. Insulin resistance in ob/ob mice was compensated for by increasing pancreatic insulin secretion, islet number, and size (Figure 3A). However, despite being more insulin resistant than ob/ob mice, POKO mice did not increase their β-cell mass, resulting in lower plasma insulin levels than the ob/ob controls. Morphometric analysis of pancreatic sections from 16-week-old male mice confirmed that the islet-to-pancreas volume ratios were similar in the POKO, WT, and PPARg2 KO mice (0.023 ± 0.005, 0.013 ± 0.006, and 0.016 ± 0.005, respectively) and markedly increased in ob/ob mice (0.077 ± 0.017, p < 0.01 ob/ob versus POKO). Additionally, POKO mice had significantly decreased islet number and size (average area of islets POKO = 18.40 ± 2 mm2) compared to ob/ob mice (ob/ob = 61.59 ± 8 mm2). Insulin staining demonstrated that islets from POKO mice contained fewer insulin-positive cells than islets from ob/ob mice (Figure 3A). The normal cellular organization of the islet, abundant β-cells (insulin staining) in the centre of the islet and a rim of α-cells at the periphery (glucagon staining), was retained in the insulin resistant ob/ob mice but was disrupted in the islets of POKO mice (Figure 3A). Islets from POKO mice had decreased number of insulin positive β-cells when compared to islets from ob/ob mice and a scattered pattern of α-cells, which are morphological changes associated with islet remodelling in the context of β-cell failure. Gene expression analysis of islets from 16-week-old mice revealed decreased expression of pancreatic duodenal homeobox-1, insulin receptor substrate 2, Glut2, and insulin in islets from POKO mice when compared with those from WT or ob/ob (Figure S4). + +The changes seen in the β-cells of POKO mice were not the result of an inherent failure of the β-cell to develop properly as indicated by histological studies of neonatal pancreas (day 3 to day 5) (unpublished data) and four-week-old pancreas (Figure 2C), showing no morphological differences in the size, number, or insulin staining of islets from POKO mice when compared to ob/ob controls. + +Impaired Glucose-Stimulated Insulin Secretion in POKO Mouse Islets + +We measured glucose-stimulated insulin secretion in 16-week-old female POKO mice and their ob/ob littermates. Islets isolated from POKO mice were 30% smaller than those from ob/ob mice. Moreover, whereas normal islets were pure white with a smooth surface, islets from POKO mice were gray; their surface was irregular and required less time for collagenase digestion (only ten minutes instead of 30 minutes), suggesting that they were also more fragile. + +Insulin content in islets from ob/ob mice was more than 30-fold greater than in those from POKO mice (Figure 3B). Insulin secretion from the islets of POKO mice was strikingly impaired compared to those of ob/ob mice, even when expressed relative to insulin content (Figure 3C). This was observed under basal (1 mM glucose) and stimulated (16 mM glucose, 16 mM glucose + tolbutamide) release. + +Decreased Steatosis in POKO Mice Compared to Ob/Ob Mice + +As expected, the POKO mice had increased hepatic fat deposition compared to WT and PPARg2 KO mice (Table S1), but surprisingly the POKO mouse had much milder hepatosteatosis than the ob/ob mouse (Figure 3D), suggesting that ectopic expression of the PPARg2 isoform in the liver of ob/ob mice (see below), might contribute to the deposition of TAGs in the liver. + +Ablation of PPARg2 Induces a Lipotoxic Lipid Profile in Adipose Tissue, Pancreatic Islets, Liver, and Skeletal Muscle + +To investigate lipotoxicity as a potential pathogenic mechanism we used liquid chromatography/mass spectrometry (LC/MS) [32] to compare a broad spectrum of cellular lipids in the adipose tissue, pancreatic islets, liver, and skeletal muscle between the POKO mouse and controls (Protocol S1). + +Adipose tissue from POKO mice has decreased TAG but increased DAG, ceramides, and other reactive lipid species associated with insulin resistance. + +Lipidomic analysis using LC/MS identified 74 molecular species differentially present in POKO, ob/ob, and WT mice (Protocol S1). POKO adipose tissue had decreased short chain TAGs compared to ob/ob adipose tissue (Protocol S1). Conversely, the concentration of DAGs was increased in the WAT of the POKO mice compared to ob/ob littermates. There was also an increased concentration of reactive lipid species in the WAT of POKO mice compared to that of ob/ob. The WAT of both POKO and ob/ob mice (Protocol S1) had increased levels of two ceramide species (with 16:0 and 24:1 fatty acid chains, respectively) and three proinflammatory lysophosphatidylcholine species [33] compared to WT mice. Partial least squares discriminant analysis indicated these changes in ceramides were greater in the POKO than ob/ob mice (Protocol S1). Sphingomyelin (d18:1/16:0), the precursor of ceramide (d18:1/16:0) and antioxidant ethanolamine plasmalogen (36:1) [34] were markedly decreased in POKO and ob/ob mice (Figure 4A). + +Decreased TAG and accumulation of reactive lipid species in islets from POKO mice. + +Partial least-squares discriminant analysis of lipidomic profiles of isolated pancreatic islets of 16-week-old mice identified 44 lipid species accumulated at different concentrations in WT, PPARg2 KO, and POKO mice (Protocol S1). Short chain TAGs were decreased in islets from POKO and PPARg2 KO mice when compared to those from WT. This was associated with up-regulation of phosphatidylethanolamine (36:2), down-regulation of ethanolamine plasmalogen (36:2), and preferential accumulation of reactive lipid species, particularly of two ceramides (20:0 and 22:0 fatty acids) in islets from POKO mice (Figure 5A and Protocol S1). + +Decreased TAG and increased reactive lipid species in liver of POKO mice. + +Multivariate analysis of lipidomic profiles (192 lipid species) revealed large changes between the POKO, PPARg2 KO, ob/ob, and WT genotypes (Protocol S1). These included decreased levels of short and medium chain TAGs and DAGs (Figure 5B) in livers from POKO mice compared to those of ob/ob mice. Livers from POKO mice also had decrease levels of phosphatidylcholine lipid species (Protocol S1) utilised during the formation and secretion of very low density lipoproteins [35]. Conversely, POKO livers were enriched in ceramides compared to ob/ob livers, which correlated with the extent of increased levels of lysophosphatidylcholines in POKO and ob/ob mice (Protocol S1). + +Decreased TAG and accumulation of reactive lipid species in POKO skeletal muscle. + +The same lipidomic pattern of decreased TAG and increased reactive lipid species previously observed in adipose tissue, β-cell, and liver was found to a milder degree in the skeletal muscle of POKO mice (Protocol S1). Briefly, when compared to ob/ob skeletal muscle, POKO skeletal muscle showed a decrease in very short-chain fatty acid TAGs and a slight decrease in levels of medium and long chain TAGs (Protocol S1). The skeletal muscle of POKO mice also had increased reactive lipids including ceramide (d18:1/18:0), DAGs, lysophosphatidylcholines, and sphingomyelins (precursors of ceramides) when compared to that of ob/ob mice. + +Transcriptomic Analysis in POKO Mice Correlates with Lipidomic Changes + +Given the lipotoxic profiles identified in the POKO mouse, we hypothesised changes in the expression of metabolic genes directly related to PPARg2 ablation and also compensatory changes in genes associated with cellular stress (Table S4). + +Gene expression analysis in WAT. + +Target genes of PPARg such as Glut4, adipsin, aP2, and adiponectin were decreased to a larger extent in the WAT of five- and 16-week-old POKO mice than in PPARg2 KO mice (Figure S1 and Figure 4B). At five weeks of age, when differences in body fat between female WT, ob/ob, and POKO mice are only starting to become evident, levels of GLUT4, aP2, and adiponectin mRNA levels were similar in WT and ob/ob mice, yet were markedly decreased in POKO mice. As the ob/ob mice aged (16 wk) and became obese and insulin resistant, the expression pattern of these PPARg targets in the WAT of ob/ob mice became similar to that of the POKO mice. + +Results from the lipidomic analysis suggested major changes in the expression of genes involved in lipid metabolism (Figure 4B). Expression of stearoyl-coenzyme A desaturase 1 (Scd1) and sterol regulatory element-binding protein-1c (SREBP1c) were significantly lower in WAT from POKO mice compared to ob/ob mice. Furthermore, the decrease in TAGs and increased DAGs correlated with decreased expression of DAG acyltransferase 2, a key enzyme catalysing the final step in TAG synthesis, in the WAT of POKO mice compared with WAT from ob/ob mice. Again supporting the lipidomic profile, the expression of hormone-sensitive lipase, a rate-limiting enzyme for hydrolysis of diacylglycerides, was decreased in the WAT of POKO, PPARg2 KO, and ob/ob mice compared with WT mice, with the lowest levels observed in the POKO mice. Adipose triglyceride lipase levels were decreased in ob/ob and POKO compared with WT and PPARg2 KO mice, but without significant differences between ob/ob and POKO mice. + +Oxidative stress has recently been suggested as a common mechanism of insulin resistance. Adipose tissue from POKO mice had increased oxidative stress compared to that of ob/ob mice as indicated by decreased gene expression levels of extracellular CuZn-superoxide dismutase, disruption of the glutathione pathway as indicated by decreased levels of gluthatione synthase, and increased levels of peroxidase and several gluthatione transferases (Table S2). We examined macrophage infiltration of adipose tissue as a potential marker of inflammation associated insulin resistance. Expression of CD68 and F4/80, both macrophage markers, was increased in the WAT of both POKO and ob/ob mice compared with WT and PPARg2 KO mice (Figure 4B). However their expression levels were lower in the POKO mice than the ob/ob mice suggesting that macrophage infiltration was not directly related to the exacerbated insulin resistance of the POKO mouse compared to the ob/ob mouse. + +Gene expression in the POKO liver. + +Reduced hepatic steatosis accompanied by altered lipid profiles suggested that lack of hepatic ectopic expression of PPARg2 might be affecting lipid storage and metabolism in the liver of the POKO mice. Expression of genes involved in lipid metabolism in liver (Figure 5C) revealed that, proportional to the accumulation of TAGs in the liver, fatty acid synthase, Scd1, and the fatty acid translocase (FAT/CD36) were increased in ob/ob and POKO livers compared to WT mice and were significantly decreased in liver from POKO mice compared with liver from ob/ob mice. Other lipogenic PPARg target genes such as Lpl were also decreased in the POKO liver compared to the ob/ob mice. The ob/ob mice also had a compensatory increase in the expression of genes involved in β-oxidation (e.g., Pparg, Lcad, Aox, Cpt1, and Ucp2). Interestingly expression of these pro-oxidative genes was decreased in the liver of POKO mice when compared to that of ob/ob mice suggesting PPARg2 may contribute to their regulation [36]. + +Although β-cell failure could account for the severe hyperglycaemia observed in the POKO genotype, hepatic gluconeogenesis function might be affected. We observed a robust up-regulation of PPARg coactivator 1 alpha (PPARGC1a, also known as PGC1a) expression in the POKO liver compared with the WT and ob/ob mice. PPARGC1a is up-regulated in fasting and is thought to induce gluconeogenesis [37]. In parallel with the increase in PPARGC1a, microarray analysis revealed increased mRNA levels of the progluconeogenic genes phosphoenolpyruvate carboxykinase 1 (Pepck1) and glucose-6-phosphatase (G6pc) in the livers of POKO mice when compared to those of ob/ob mice (Table S2), suggesting hepatic gluconeogenesis may contribute to the hyperglycaemia observed in POKO mice. + +Gene expression analysis in skeletal muscle of POKO mice. + +In 16-week-old POKO-mice skeletal muscle we observed down-regulation of Srebp1c and Ppargc1a and up-regulation of Ucp2 expression in skeletal muscle from POKO mice compared to that of WT mice. Similarly, expression of Lpl and Scd1 was down-regulated in the skeletal muscle of POKO mice when compared with that from ob/ob mice (Figure S5; Table S2). Gene set enrichment analysis of microarray data showed decreased expression of oxidative phosphorylation and mitochondrial components including electron transport chain complex components, in skeletal muscle from POKO mice when compared with that from ob/ob mice (Table S3). + +Discussion + +The link between obesity, insulin resistance, and diabetes while epidemiologically very clear is still not properly understood at a mechanistic level. An emerging concept is that the absolute amount of fat stored may be less important than the remaining storage capacity of the adipose tissue. Here we show that the PPARg2 isoform may be an important factor controlling obesity-induced comorbidities through two mechanisms: (a) by regulating nutritionally induced adipose tissue expandability and (b) when de novo expressed in nonadipose tissues, by allowing the storage of energy in the form of relatively harmless TAG species. + +Previously we described the metabolic phenotype of the adult PPARg2 KO mouse [2], characterised by mild insulin resistance observed only in males. Given the greater adipogenic potency of PPARg2 compared with PPARg1 in vitro, we expected PPARg2 KO mice to have many more severe defects in adipose tissue than we observed, and therefore insulin sensitivity. As PPARg2 is the PPARg isoform regulated in response to nutrition and obesity [17–20], we hypothesised that PPARg2 would only become essential for adipose tissue function in the face of positive energy balance. The metabolic challenge we opted for was PPARg2 ablation in the obese (ob/ob) background (PPARg2−/− Lepob/Lepob, POKO mouse). The POKO mouse had severely decreased body-fat mass due to impaired adipose tissue expandability. Despite eating as much as an ob/ob mouse and expending a similar amount of energy, the POKO mouse was unable to store fat efficiently in its adipose tissue. This mismatch between increased energy availability and lack of adipose tissue expandability lead to a global metabolic failure characterised by severe insulin resistance, β-cell failure, and dyslipidaemia. + +The observation of reduced fat mass and increased insulin resistance in the POKO mouse compared to the ob/ob mouse strongly supports two of our hypotheses. First, we hypothesised that PPARg2 is required to recruit new adipocytes in overnutrition, but it is not required to make adipocytes during development. This is reflected by similar expression of aP2, a late marker of adipocyte differentiation, in POKO and ob/ob mice. The absence of small adipocytes was markedly different to other forms of lipodystrophy [38,39]. Additionally, and again in contrast with other lipodystrophic models that have markedly less adipose tissue than WT controls [38–40], the POKO mice had a percentage body fat that was similar (only 4% more) to WT and PPARg2 KO mice, as opposed to ob/ob mice, which had 40% fat as a proportion of body mass. This suggests that the remaining PPARg1 isoform is sufficient to support development of adipose tissue and fat deposition requirements of a lean mouse model. However, under conditions of positive energy balance, adipose tissue expandability mainly relies on the PPARg2 isoform. This idea is also suggested by the studies in heterozygous mice harbouring the murine equivalent of the human mutation (P465L) in PPARg on an ob/ob background [41]. These mice were able to accumulate fat and become obese even though showing a body mass 14% lower than ob/ob controls. In humans there is also evidence for a role for PPARg2. We have observed that metabolically healthy, nondiabetic, morbidly obese individuals have elevated levels of PPARg2 in their adipose tissue when compared to lean individuals [19]. Our second hypothesis, that the mismatch between energy availability and adipose tissue expandability is more important than fat mass itself as a predictor of insulin resistance, is also supported by our data. In fact the ob/ob mouse is much more obese than the POKO mouse but is much less insulin resistant. Furthermore, the POKO mice were already more insulin resistant than the ob/ob mice by the age of four weeks, with very low levels of GLUT4 in adipose tissue, before large differences in body weight developed, suggesting that the bioenergetic mismatch rather than the total amount of fat stored is important for the development of insulin resistance. + +Although we hypothesised that the POKO mice would become insulin resistant, the degree of hyperglycaemia in these animals was in excess of what we expected. We found that the normal adaptive response of β-cells to insulin resistance did not occur in the POKO mice as indicated by the pathological changes observed by histology and the lack of β-cell hypertrophy. Although it has been shown that genetic background can affect the ability of ob/ob mice to undergo β-cell hypertrophy [42,43], we found that the ob/ob controls on our mixed 129Sv × C57BL/6J background underwent adaptive β-cell hyperplasia and hypertrophy, suggesting that the lack of PPARg2 was responsible for the failure of the POKO β-cells to adapt to insulin resistance. Interestingly the mass of pancreatic islets in POKO mice remained similar to the noninsulin resistant WT and PPARg2 KO mice. Furthermore, these defects in POKO β-cells did not appear to be the result of a developmental defect, as new born and four-week-old mice had morphologically normal islets. + +The severe β-cell phenotype of the POKO mouse contrasts with the absence of hyperglycaemia observed in the pancreatic β-cell specific PPARg KO mouse [30]. However it should be kept in mind that in the β-cell specific PPARg KO mouse, the expression of PPARg and the lipid storage capacity of other tissues, most importantly adipose tissue, were not affected, and that insulin sensitivity was only mildly affected by high fat feeding in these mice when compared to the severe insulin resistance observed in POKO mice. Therefore the challenge to the pancreatic β-cells in this model was milder than in POKO mice. This is a clear example of how tissue-specific genetic manipulations are not always the best approach to understand the physiology of an organ in the context of the global energy homeostasis. The potential importance of the de novo expression of PPARg2 isoform in β-cells is also supported by the observation that humans harbouring the Pro12Ala mutation in PPARg2, a mutation that is located in the g2 isoform and makes PPARg2 less active, has only been associated with insulin deficiency and disease severity in obese individuals with type 2 diabetes [44]. + +The liver of the POKO mouse also displayed an unusual phenotype. We expected the POKO mice to have worse hepatosteatosis with increased triglyceride deposition in liver compared to ob/ob mice, because the POKO mice could not store fat in adipose tissue. However POKO mice had less hepatosteatosis than ob/ob mice suggesting that the PPARg2 isoform may directly contribute to facilitate triglyceride deposition in the liver. + +A common mechanistic link for the phenotypes observed in the POKO liver and β-cell was not immediately obvious. To try to determine the role of PPARg2 in these locations we performed lipidomic and gene expression analyses of the adipose tissue, pancreatic islet, liver, and skeletal muscle of the POKO mouse. The lipid pattern of adipose tissue from POKO mice was characterised by decreased TAGs and increased DAGs in parallel with decreased gene expression of DGAT2, hormone-sensitive lipase, and adipose triglyceride lipase. This decrease in TAGs in the POKO adipose tissue was associated with increased levels of reactive lipid species and a gene expression profile suggestive of increased oxidative stress [45–49]. Although it has been described that oxidative stress and insulin resistance may be related to infiltration of adipose tissue by macrophages, resulting in a chronic state of inflammation [50–52], we did not observe increased macrophage infiltration in the adipose tissue of POKO mice compared to that of ob/ob mice. + +Lipidomic analysis of POKO derived islets also showed decreased levels of triacyl and DAGs and increased levels of ceramides, suggesting that PPARg2 may contribute to increasing the lipid-buffering capacity of β-cells by promoting formation of TAGs and thus preventing lipotoxic insults. Liver and skeletal muscle lipidomics also showed reduced TAG and increased formation of reactive lipid species such as ceramides and lysophosphatidylcholines in POKO mice compared to ob/ob mice. This lipid profile was associated with impaired expression of pathways controlling de novo lipogenesis, transport of fatty acids, and beta oxidation in the POKO mice compared with the ob/ob mice. Of interest, Ppargc1a and other gluconeogenic genes were induced in the liver of POKO mice compared to that of ob/ob mice, suggesting a potential mechanism contributing to marked hyperglycaemia in POKO mice [53,54]. + +Overall, our lipidomic studies identify a remarkably similar pattern of changes in lipid species in the four tissues studied. The reduced adipose tissue mass and hepatosteatosis in the POKO mouse compared to the ob/ob mouse is explained by reduced levels of mature TAG in the POKO mouse. Similarly, ablation of PPARg2 resulted in accumulation of reactive lipid species implicated in causing insulin resistance, not only in adipose tissue, but also in other organs involved in whole-organism glucose metabolism. These results indicate that expression of PPARg2 in the pancreas, liver, and muscle of the ob/ob mouse may be performing a protective role, by increasing the capacity of these organs to buffer toxic lipid species by allowing accumulation of relatively harmless TAGs. The importance of this peripheral antilipotoxic role of PPARg2 becomes more evident if we consider that POKO and ob/ob mice are under the same degree of positive energy balance as determined by similar food intake, locomotor activity, and energy expenditure, that both models lack leptin, and that the only difference between ob/ob and POKO mice is the presence or absence of PPARg2. Given the decreased adipose tissue expandability of the POKO mice compared to ob/ob, it was anticipated that, as in the liver, muscle, or β-cells of lipodistrophic mice, the POKO mouse would accumulate more fat than the ob/ob. However, our results clearly indicate that mice lacking PPARg2, despite massive nutrient availability, are unable to deposit TAG in peripheral tissues and instead accumulate reactive lipid species in these organs. Therefore the pathologies of the liver and β-cell observed in the POKO mouse may be a result of a common lipotoxic insult facilitated by the absence of PPARg2 (Figure 6). + +In summary, in this study we provide new insights into the physiological relevance of the PPARg2 isoform and identify adipose tissue expandability as an important determinant of metabolic complications. Ablation of PPARg2 decreases adipose tissue expandability, but its pathophysiological effects only become relevant in the context of a mismatch between energy availability and adipose tissue expansion. We show that PPARg2 also plays protective role when expressed de novo in peripheral organs by increasing their capacity to buffer toxic lipids. Ablation of PPARg2 under conditions of positive energy balance determined by absence of leptin produced early development of severe insulin resistance, β-cell failure, diabetes, and hyperlipidaemia. Extrapolation of this model to humans may suggest that normal to overweight individuals with positive energy balance and inappropriately severe manifestations of the MS may have a defect in PPARg2 and/or alternative mechanisms that control adipose tissue expandability. + +Materials and Methods + +Generation of mice homozygous for PPARg2 KO and leptin deficiency (ob/ob). + +Mice heterozygous for a disruption in exon B1 of PPARg2 on a 129Sv background (PPARg2+/−) [2] were crossed with heterozygous ob/ob (Lepob/Lep+) mice on a C57Bl/6 background to obtain mice heterozygous for both the PPARg2 ablation and the leptin point mutation (PPARg2+/− Lepob/Lep+). These mice were crossed to obtain the four experimental genotypes: WT (PPARg2+/+ Lep+/Lep+), PPARg2 KO (PPARg2−/− Lep +/Lep+), ob/ob (PPARg2+/+ Lepob/Lepob), and POKO (PPARg2−/− Lepob/Lepob). Genotyping for deletion of PPARg2 and the point mutation in the ob gene was performed by PCR using standard protocols [2,55]. + +Animal care. + +Animals were housed at a density of four animals per cage in a temperature-controlled room (20–22 °C) with 12-h light/dark cycles. Food and water were available ad libitum unless noted. All animal protocols used in this study were approved by the UK Home Office and the University of Cambridge. + +Blood and urine biochemistry, food intake, and body composition analysis. + +Mice of the four experimental genotypes were placed at weaning (three weeks of age) on a normal chow diet (10% of calories derived from fat; D12450B, Research Diets, http://www.researchdiets.com). Enzymatic assay kits were used for determination of plasma FFAs (Roche, http://www.roche.com) and TAGs (Sigma-Aldrich, http://www.sigmaaldrich.com). Elisa kits were used for measurements of leptin (R & D Systems, http://www.rndsystems.com), insulin (DRG Diagnostics International Limited, http://www.drg-international.com), and adiponectin (B-Bridge International, http://www.b-bridge.com) according to manufacturers' instructions. Dual-energy X-ray absorptiometry (DEXA, Lunar Corporation, http://www.lunarcorp.com) was used to measure body composition; glucose in blood and in urine and food intake were monitored in the four experimental genotypes as previously shown [2]. + +Oxygen consumption, water intake, and locomotor activity. + +Oxygen was measured using an eight-chamber open-circuit oxygen-monitoring system attached to and sampled from the chambers of a Comprehensive Laboratory Animal Monitoring System (CLAMS; Columbus Instruments, http://www.colinst.com). Water consumed was also measured using CLAMS. Mice were housed individually in specially built Plexiglass cages maintained at 22 °C under an alternating 12:12-h light-dark cycle (light period 08:00–20:00). Sample air was sequentially passed through oxygen (O2) and carbon dioxide (CO2) sensors (Columbus Instruments) for determination of O2 and CO2 content. Mice were acclimatized to monitoring cages for 72 h before data collection. Mice were weighed before each trial. Ambulatory activity of individually housed mice was evaluated using an eight-cage rack OPTO-M3 Sensor system (Columbus Instruments). Cumulative ambulatory activity counts were recorded every 5 min throughout the light and dark cycles. + +Calculations of energy lost in urine. + +Energy lost in urine was calculated accordingly as previously shown before [56] using the following calculations: + +Energy lost in urine kJ/day = (glucose in urine [mMol/l]/1,000) × molecular weight glucose × (water intake [ml/day]/1,000) × E densitycarb; E densitycarb = energy density related to oxidations within the body for carbohydrates as glucose = 15.76 kJ/g. + +RNA preparation and real-time quantitative RT-PCR. + +Total RNA was isolated from islets and tissues samples according to the manufacturer's instructions (RNAeasy kit, Qiagen, http://www.qiagen.com) and STAT60 (Tel-Test, http://www.isotexdiagnostics.com/tel-test.html). Real-time quantitative PCR was performed using a TaqMan 7900 (Applied Biosystems, http://www.appliedbiosystems.com) according to standard protocols. + +Western blot analyses. + +The tissue samples (40 μg) were subjected to SDS-PAGE on 8% polyacrylamide gels. Proteins were then electrophoretically transferred to polyvinylidene difluoride filters. After transferring, the filters were blocked with 5% nonfat dry milk in TBS-Tween 20 followed by incubation with primary GLUT4 and extracellular signal-regulated kinase 1/2 (ERK1/2) antibodies (Promega, http://www.promega.com) overnight. The bands were quantified by scanning densitometry. + +Light microscopy and immunohistochemcal analysis. + +Tissue samples for morphological and immunohistochemcal analysis were prepared according to published protocols [2]. Morphometric analyses of adipose tissue and pancreas sections were acquired using a digital camera and microscope (Olympus BX41, http://www.olympus.com), and cell areas were measured using AnalySIS software (Soft Imaging System, http://www.soft-imaging.net). For adipose tissue, two fields from each section were analysed to obtain the mean cell-area per animal (n = 5 per genotype). The Computer Assisted Stereology Toolbox (CAST) 2.0 system from Olympus was used to perform all measurements in the pancreas according to published protocols [57]. + +Isolation and culture of pancreatic islets. + +The pancreas was injected via the bile duct with cold Hank's solution containing 0.4% (w/v) liberase (Roche). The pancreas was removed, digested for 15–30 min, and islets collected by handpicking. Isolated islets were cultured overnight in h-cell medium (SBMI 06, hcell technology, http://www.hcell.com) at 37 °C in 5% CO2 in air. Islets were used the day after isolation for insulin secretion studies or RNA extraction. + +Insulin secretion studies. + +Insulin secretion from isolated islets (five islets/well) was measured during 1-hr static incubations in Krebs—Ringer Buffer containing either 1 mM glucose, 16.7 mM glucose, or 16.7 mM glucose plus 200 μM tolbutamide in DMSO. The supernatants were assayed for insulin. Insulin content was extracted using 95:5 ethanol/acetic acid. Insulin was measured using a Mouse Insulin ELISA kit (Mercodia, http://www.mercodia.com). Islets were isolated from three mice of each genotype for these experiments. Thus, the data are the mean of three separate experiments, in which data were collected for each test solution from six samples each of five islets. For each sample, insulin release was normalised to insulin content. + +ITT. + +ITTs on four-week-old mice were performed as previously published [58]. + +Lipid profiling. + +For WAT and muscle, the tissue sample (50 mg) was homogenized with 0.15 M sodium chloride (300 μl), and the lipids were extracted with 2 ml of chloroform: methanol (2:1) and used for LC/MS as previously described [2]. + +For liver and islets, an aliquot (20 μl for liver or 10 μl for islets) of an internal standard mixture (11 reference compounds at concentration level 8–10 μg/ml), 50 μl of 0.15 M sodium chloride (for liver), and chloroform:methanol (2:1) (200 μl for liver or 90 μl for islets) was added to the tissue sample (20–30 mg). The sample was homogenized, vortexed (2 min for liver or 15 s for islets), let to stand (1 h for liver, 20 min for islets), and centrifuged at 10,000 RPM for 3 min. From the separated lower phase, an aliquot was mixed with 10 μl of a labelled standard mixture (three stable isotope-labelled reference compounds at concentration level 9–11 μg/ml), and 0.5–1.0 μl injection was used for LC/MS analysis. + +Total lipid extracts were analysed on a Waters Q-Tof Premier mass spectrometer (http://www.waters.com) combined with an Acquity Ultra Performance LC (UPLC). The column, which was kept at 50 °C, was an Acquity UPLC BEH C18 10 × 50 mm with 1.7 μm particles. The binary solvent system (flow rate 0.200 ml/min) included A, water (1% 1 M NH4Ac, 0.1% HCOOH), and B, LC/MS grade (Rathburn, http://www.rathburn.co.uk) acetonitrile/isopropanol (5:2, 1% 1 M NH4Ac, 0.1% HCOOH). The gradient started from 65% A/35% B, reached 100% B in 6 min, and remained there for the next 7 min. The total run time per sample, including a 5 min re-equilibration step, was 18 min. The temperature of the sample organizer was set at 10 °C. + +Mass spectrometry was carried out on Q-Tof Premier (Waters) run in ESI+ mode. The data were collected over the mass range of m/z 300–1,200 with scan duration of 0.2 s. The source temperature was set at 120 °C, and nitrogen was used as desolvation gas (800 l/h) at 250 °C. The voltages of the sampling cone and capillary were 39 V and 3.2 kV, respectively. Reserpine (50 μg/l) was used as the lock spray reference compound (5 μl/min; 10 s-scan frequency). + +Data processing was performed using the MZmine software [59]. Identification was performed based on an internal reference database of lipid species, or alternatively utilizing the tandem mass spectrometry. The statistical analyses were performed using Matlab (Mathworks, http://www.mathworks.com) and the Matlab library PLS Toolbox (Eigenvector Research, http://www.eigenvector.com). + +Tandem mass spectrometry was used for the identification of selected lipid species. MS/MS runs were performed by using ESI+ mode, collision energy ramp from 15–30 V, and mass range starting from m/z 150. The other conditions were as shown in the Protocol S1. + +Statistics. + +Results were expressed as mean ± standard error of mean. Statistical analysis was performed using a two-tailed unpaired t-test between appropriate pairs of groups, and significance declared if p-values were less than 0.05. + +Supporting Information + +Figure S1 + +Adipose Tissue and Liver Gene Expression + +(39 KB PPT) + +Click here for additional data file. + +Figure S2 + +Water Consumed and Locomotor Activity + +(38 KB PPT) + +Click here for additional data file. + +Figure S3 + +GLUT4 protein expression in WAT + +(65 KB PPT) + +Click here for additional data file. + +Figure S4 + +Gene Expression in Islets + +(25 KB PPT) + +Click here for additional data file. + +Figure S5 + +Gene Expression in Muscle + +(32 KB PPT) + +Click here for additional data file. + +Protocol S1 + +POKO Mouse Model Lipidomics Dataset + +(208 KB PDF) + +Click here for additional data file. + +Table S1 + +Tissue Weights of 16-Wk-Old Male POKO, Ob/Ob, PPARg2 KO, and WT mice + +(29 KB PPT) + +Click here for additional data file. + +Table S2 + +Microarray Data + +(105 KB DOC) + +Click here for additional data file. + +Table S3 + +Pathway Analysis from Microarray Data + +(112 KB XLS) + +Click here for additional data file. + +Table S4 + +Accession Numbers + +GenBank (http://www.ncbi.nlm.nih.gov/Genbank) accession numbers for the genes and gene products discussed in this paper. + +(58 KB DOC) + +Click here for additional data file. + +Acknowledgements + +Animal care and husbandry provided by J. Carter, S. Shelton, H. Wetsby, H. Williams, A. Kant, J.P. Whiting, and G. Bevan. We thank D. Lam, M. Dale, and K. Burling for their technical assistance. We also thank J. Skepper and P.M. Coan for their help with morphometry analysis in pancreas and Peter Murgatroyd for his help with oxygen consumption experiments. We acknowledge Paradigm Therapeutics (http://www.paradigm-therapeutics.co.uk) for generating the PPARg2 KO mouse. + +Abbreviations + +DAG - diacylglycerol + +GLUT - glucose transporter + +H and E - haematoxylin and eosin + +ITT - insulin tolerance test + +LC/MS - liquid chromatography/mass spectrometry + +MS - Metabolic Syndrome + +PPARg - peroxisome proliferator activated receptor gamma + +PPARg2 - peroxisome proliferator activated receptor gamma 2 + +PPARGC1a - PPARg coactivator 1 alpha + +Scd1 - stearoyl-coenzyme A desaturase 1 + +TAG - triacylglycerol + +WAT - white adipose tissue + +WT - wild type + +Figures and Tables + +Figure 1 + +Physiological Characterisation of POKO Mouse + +(A) Body weights (black circles, WT; black squares, ob/ob; white circles, PPARg2 KO; white squares, POKO) are shown for males (left) or females (right) (n = 5–12). *, p < 0.05 POKO versus ob/ob and §, p < 0.01 POKO versus WT. + +(B) Food intake from 20-wk-old female mice (n = 4) is shown. + +(C) Body composition analysis from 20-wk-old females is shown: WT, ob/ob, PPARg2 KO, and POKO mice fed chow diet mice (n = 4–7). *, p < 0.05 POKO versus WT and ###, p < 0.001 POKO versus ob/ob. + +(D) Haematoxylin and eosin (H and E)-stained sections (10×) from epididymal WAT from 16-wk-old male WT, ob/ob, and POKO mice. + +(E) Percent relative cumulative frequency analysis (PRCF) from epididymal WAT adipocytes from 16-wk-old male WT, ob/ob, PPARg2 KO, and POKO mice. (n = 4–5). + +Figure 2 + +Early Insulin Resistance in POKO Mice Independent of Body Weight + +(A) Body weight and plasma glucose levels from three, four, and five-week-old female WT, ob/ob, PPARg2 KO, and POKO. *, p < 0.05; **, p < 0.01; ***, p < 0.001 POKO versus ob/ob. + +(B) Plasma glucose levels during ITT on 4-wk-old male (left) and female (right) mice on chow diet (black triangle, WT; white triangle, PPARg2 KO; black square, ob/ob; black diamond, POKO) (n = 7). *, p < 0.05; **, p < 0.01 POKO versus ob/ob. + +(C) Morphological analysis of H and E-stained sections (10×) in pancreas from 4-wk-old males ob/ob and POKO mice (n = 5). + +Figure 3 + +Impaired β-Cell Function and Hepatic Morphological Analyis in the POKO Mice + +(A) H and E-stained sections (10×) and immunohistochemical (20×) analysis of insulin and glucagon in pancreas from 16-wk-old males WT, ob/ob, and POKO mice (n = 5). + +(B) Insulin content of islets isolated from POKO (black bars), and ob/ob (grey bars) mice. Each data point is the mean of six samples each of five islets. + +(C) Insulin secretion from islets isolated from POKO (black bars) and ob/ob (grey bars) mice in response to glucose (1, 16 mM) or glucose 16 mM + tolbutamide (200 μM). Data were collected from six samples each of five islets from three mice of each genotype. For each sample, insulin release was normalised to insulin content. *, p < 0.05; **, p < 0.01; ***, p < 0.001 POKO versus ob/ob. + +(D) H and E-stained sections (4×) in liver from 16-wk-old males WT, ob/ob, and POKO mice (n = 5). + +Figure 4 + +Lipidomic and Gene Expression Analysis of POKO WAT + +(A) Lipidomic profiling of WAT from 16-wk-old males WT, ob/ob, and POKO mice. + +(B) Adipose tissue mRNA levels from different genes from 16-wk-old male WT, PPARg2 KO, ob/ob, and POKO mice (n = 6–8). *, p < 0.05; **, p <0.01; ***, p <0.001 POKO versus ob/ob. + +Figure 5 + +Lipidomic and Gene Expression Analysis in Islets and Liver from POKO Mice + +Lipidomic profiling of islets (A) and liver (B) from 16-wk-old males WT, PPARg2 KO, ob/ob, and POKO mice. TG, TAGs; DAGs, diacylglycerols; SM, sphingomyelins. (C) Liver gene expression from 16-wk-old male WT, ob/ob, PPARg2 KO, and POKO mice fed chow (n = 6–8). *, p < 0.05; **, p, <0.01; ***, p <0.001 POKO versus ob/ob; ###, p < 0.001 POKO versus WT; ‡‡‡, p < 0.001 ob/ob versus WT. + +Figure 6 + +Storage of Lipids—Antilipotoxic Role of PPARg2 + +Antilipotoxic role of PPARg2 mediated by (a) expansion of adipose tissue and facilitation of triglyceride deposition and (b) facilitating deposition of fat in liver, skeletal muscle, and pancreas in the form of TAG. Ob/Ob mice can induce PPARg2 expression in liver, muscle, and β-cell, facilitating deposition of excess of energy in these organs in the form of TAG. Absence of inducibility of PPARg2 in POKO mouse liver, muscle, and β-cells results in increased deposition of reactive lipid species and decreased TAG, leading to marked insulin resistance and β-cell failure. + +Table 1 + +Metabolic Parameters in Fed 4-Wk-Old Male and Female POKO, Ob/Ob, PPARg2 KO, and WT Mice + +Table 2 + +Metabolic Parameters in 16-wk-Old Male POKO, Ob/Ob, and WT Mice + +Footnotes + +Competing interests. The authors have declared that no competing interests exist. + +Author contributions. GMG, SLG, FMA, and AVP conceived and designed the experiments. GMG, SLG, LY, KS, SV, MB, ML, and MO performed the experiments. GMG, SLG, LY, KS, SV, MC, RKC, MJL, TSL, and MO analysed the data. GMG, LY, KS, SV, MC, RKC, MB, and GSHY contributed reagents/materials/analysis tools. GMG and AVP wrote the paper. + +Funding. This work was supported by the European Union FP6 Hepatic and Adipose Tissue and Functions in the Metabolic Syndrome (Hepadip) integrated program (http://www.hepadip.org) (LSHM-CT-2005–018734); Diabetes UK; Medical Research Council; Wellcome Trust Integrative Physiology program; Academy of Finland (grant number 111338); and Marie Curie International Reintegration Grant from the European Community. diff --git a/src/ontogpt/evaluation/craft/database/all/17503968.ann b/src/ontogpt/evaluation/craft/database/all/17503968.ann new file mode 100644 index 000000000..b8b557cc9 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17503968.ann @@ -0,0 +1,1212 @@ +T1 GO:0008282 2 14 KATP Channel +T2 CL:0000171 40 47 α Cells +T3 GO:0065007 48 57 Regulates +T4 GO:0070091 58 74 Glucagon Release +T5 NCBITaxon:9989 85 91 Rodent +T6 NCBITaxon:9606 96 101 Human +T7 UBERON:0000006 102 122 Islets of Langerhans +T8 GO:0046903 145 153 secreted +T9 UBERON:0000006 159 175 pancreatic islet +T10 CL:0000171 159 183 pancreatic islet α cells +T11 GO:0006094 196 211 gluconeogenesis +T12 UBERON:0002107 216 221 liver +T13 CHEBI:24384 222 230 glycogen +T14 GO:0005980 222 240 glycogen breakdown +T15 GO:0065007 256 266 regulating +T16 GO:0070091 267 283 glucagon release +T17 CL:0000540 324 332 neuronal +T18 GO:0065007 333 340 control +T19 GO:0038001 342 351 paracrine +T20 GO:0065007 352 359 control +T21 CL:0000169 376 383 β cells +T22 CHEBI:17234 404 411 glucose +T23 CL:0000171 427 434 α cells +T24 CHEBI:29108 481 485 Ca2+ +T25 GO:0051716 486 498;507 512 responses of ... cells +T26 CL:0000171 499 500;507 512 α ... cells +T27 CL:0000169 505 512 β cells +T28 NCBITaxon:9989 527 533 rodent +T29 NCBITaxon:9606 538 543 human +T30 UBERON:0000006 544 550 islets +T31 CHEBI:17234 552 559 Glucose +T32 GO:0070091 585 601 glucagon release +T33 GO:0038001 617 626;640 650 paracrine ... signalling +T34 CHEBI:16865 627 631 GABA +T35 GO:0007214 627 631;640 650 GABA ... signalling +T36 CHEBI:29105 635 639 Zn2+ +T37 GO:0008282 720 736;744 751 ATP-sensitive K+ ... channel +T38 CHEBI:29103 734 736 K+ +T39 CHEBI:79085 734 735;744 758 K ... channel opener +T40 GO:0008282 738 742;744 751 KATP ... channel +T41 CHEBI:4495 759 768 diazoxide +T42 PR:000045358 793 800 insulin +T43 GO:0030073 793 808 insulin release +T44 CL:0000169 812 818 β cell +T45 GO:0051716 814 828 cell responses +T46 GO:0008282 863 875 KATP channel +T47 CHEBI:50509 863 864;868 883 K ... channel blocker +T48 CHEBI:27999 884 895 tolbutamide +T49 CHEBI:4495 913 922 diazoxide +T50 PR:000045358 970 977 insulin +T51 CL:0000171 993 995;1002 1006 α- ... cell +T52 CL:0000169 1000 1006 β-cell +T53 GO:0051716 1002 1006;1012 1021 cell ... responses +T54 CHEBI:29108 1007 1011 Ca2+ +T55 CHEBI:17234 1054 1061 glucose +T56 CHEBI:27999 1063 1074 tolbutamide +T57 CHEBI:27999 1249 1260 tolbutamide +T58 CHEBI:17234 1271 1278 glucose +T59 GO:0008282 1335 1347 KATP channel +T60 CHEBI:29101 1377 1380 Na+ +T61 CHEBI:9506 1382 1385 TTX +T62 CHEBI:29108 1398 1402 Ca2+ +T63 CHEBI:29108 1442 1446 Ca2+ +T64 CHEBI:7565 1457 1467 nifedipine +T65 CHEBI:29108 1516 1520 Ca2+ +T66 CL:0000171 1534 1540 α-cell +T67 GO:0006887 1541 1551 exocytosis +T68 GO:0016020 1584 1592 membrane +T69 NCBITaxon:9989 1605 1611 Rodent +T70 NCBITaxon:9606 1616 1621 human +T71 GO:0065007 1644 1653 regulated +T72 CL:0000171 1660 1666 α-cell +T73 GO:0008282 1667 1679 KATP channel +T74 CHEBI:17234 1726 1733 glucose +T75 GO:0006887 1766 1776 exocytosis +T76 CHEBI:24870 1820 1823 ion +T77 GO:0099610 1845 1868 action potential firing +T78 CHEBI:17234 1939 1946 glucose +T79 GO:0042593 1939 1958 glucose homeostasis +T80 CHEBI:17234 1992 1999 glucose +T81 UBERON:0002107 2009 2014 liver +T82 CL:0000171 2040 2050;2055 2086 α cells of ... pancreatic islets of Langerhans +T83 UBERON:0000006 2055 2086 pancreatic islets of Langerhans +T84 UBERON:0000178 2113 2118 blood +T85 http://purl.obolibrary.org/obo/MONDO_0005015 2164 2172 diabetes +T86 GO:0065007 2203 2213 regulation +T87 CL:0000171 2217 2223 α-cell +T88 CL:0000540 2246 2254 neuronal +T89 GO:0038001 2270 2279 paracrine +T90 UBERON:0000006 2337 2342 islet +T91 PR:000045358 2351 2358 insulin +T92 CHEBI:17234 2426 2433 glucose +T93 CL:0000171 2437 2444 α cells +T94 NCBITaxon:9989 2459 2465 rodent +T95 NCBITaxon:9606 2470 2475 human +T96 UBERON:0000006 2476 2482 islets +T97 CL:0000171 2516 2522 α-cell +T98 CHEBI:17234 2523 2530 glucose +T99 PR:000045358 2572 2579 insulin +T100 GO:0030073 2572 2589 insulin-secreting +T101 CL:0000168 2572 2589;2592 2597 insulin-secreting ... cells +T102 CL:0000169 2590 2597 β cells +T103 GO:0008282 2620 2645 ATP-dependent K+ channels +T104 CHEBI:29103 2634 2636 K+ +T105 CHEBI:17234 2665 2672 glucose +T106 GO:0016020 2700 2708 membrane +T107 GO:0051899 2700 2723 membrane depolarisation +T108 CHEBI:29101 2749 2752 Na+ +T109 CHEBI:29108 2757 2761 Ca2+ +T110 CL:0000171 2783 2789 α-cell +T111 GO:0006887 2790 2800 exocytosis +T112 UBERON:0000178 2833 2838 blood +T113 CHEBI:17234 2839 2846 glucose +T114 CL:0000171 2855 2861 α-cell +T115 CHEBI:24870 2925 2928 ion +T116 GO:0099610 2950 2973 action potential firing +T117 UBERON:0000178 3004 3009 Blood +T118 CHEBI:17234 3010 3017 glucose +T119 GO:0065007 3039 3046 control +T120 UBERON:0000006 3081 3112 pancreatic islets of Langerhans +T121 UBERON:0000006 3114 3119 Islet +T122 CL:0000169 3114 3127 Islet β cells +T123 GO:0046903 3128 3135 secrete +T124 PR:000045358 3136 3143 insulin +T125 CHEBI:17234 3149 3156 glucose +T126 CHEBI:17234 3177 3184 glucose +T127 GO:0006094 3177 3195 glucose production +T128 UBERON:0002107 3203 3208 liver +T129 CHEBI:17234 3224 3231 glucose +T130 UBERON:0000479 3252 3259 tissues +T131 GO:0065007 3261 3270 Regulated +T132 PR:000045358 3271 3278 insulin +T133 GO:0008152 3334 3343 metabolic +T134 CHEBI:29108 3380 3384 Ca2+ +T135 GO:0070509 3380 3390 Ca2+ entry +T136 GO:0006887 3396 3406 exocytosis +T137 UBERON:0000006 3412 3417 Islet +T138 CL:0000171 3412 3425 Islet α cells +T139 GO:0046903 3426 3433 secrete +T140 UBERON:0000178 3468 3473 blood +T141 CHEBI:17234 3474 3481 glucose +T142 CHEBI:17234 3500 3507 glucose +T143 GO:0070091 3524 3540 glucagon release +T144 CHEBI:17234 3587 3594 glucose +T145 GO:0006094 3587 3605 glucose production +T146 UBERON:0002107 3613 3618 liver +T147 http://purl.obolibrary.org/obo/MONDO_0005015 3623 3631 diabetes +T148 GO:0070091 3642 3658 glucagon release +T149 CHEBI:17234 3706 3713 glucose +T150 http://purl.obolibrary.org/obo/MONDO_0002909 3778 3792 hyperglycaemia +T151 http://purl.obolibrary.org/obo/MONDO_0004946 3828 3841 hypoglycaemic +T152 GO:0065007 3865 3875 regulating +T153 GO:0070091 3947 3963 Glucagon release +T154 NCBITaxon:9989 3967 3974 rodents +T155 GO:0065007 3982 3991 regulated +T156 GO:0038001 3995 4004 paracrine +T157 CHEBI:16865 4024 4043 γ-aminobutyric acid +T158 CHEBI:16865 4045 4049 GABA +T159 CHEBI:29105 4058 4062 Zn2+ +T160 PR:000045358 4072 4079 insulin +T161 CHEBI:17234 4100 4107 glucose +T162 CL:0000171 4167 4173 α-cell +T163 UBERON:0001898 4259 4271 hypothalamic +T164 GO:0065007 4272 4279 control +T165 NCBITaxon:9606 4289 4294 Human +T166 GO:0065007 4354 4361 control +T167 GO:0038001 4387 4396 paracrine +T168 GO:0065007 4410 4420 regulation +T169 CL:0000171 4424 4431 α cells +T170 NCBITaxon:9606 4513 4518 human +T171 UBERON:0000006 4519 4525 islets +T172 UBERON:0000006 4528 4533 Islet +T173 CL:0000171 4528 4541 Islet α cells +T174 GO:0010467 4542 4549 express +T175 GO:0008282 4550 4566;4574 4582 ATP-dependent K+ ... channels +T176 CHEBI:29103 4564 4566 K+ +T177 GO:0008282 4568 4572;4574 4582 KATP ... channels +T178 CHEBI:17234 4633 4640 Glucose +T179 GO:0005622 4651 4664 intracellular +T180 CL:0000171 4677 4684 α cells +T181 CHEBI:17234 4736 4743 glucose +T182 CL:0000171 4755 4761 α-cell +T183 GO:0008282 4762 4775 KATP channels +T184 PR:000003563 4802 4806 SUR1 +T185 NCBITaxon:10088 4810 4814 mice +T186 GO:0008282 4825 4838 KATP channels +T187 GO:0070091 4856 4872 glucagon release +T188 CL:0000171 4907 4908;4915 4920 α ... cells +T189 CL:0000169 4913 4920 β cells +T190 GO:0008282 4951 4964 KATP channels +T191 PR:000045358 5039 5046 insulin +T192 GO:0065007 5138 5148 regulating +T193 CHEBI:29108 5173 5177 Ca2+ +T194 GO:0070509 5173 5183 Ca2+ entry +T195 CL:0000169 5192 5199 β cells +T196 CL:0000171 5201 5208 α cells +T197 CHEBI:29101 5243 5246 Na+ +T198 GO:0070091 5277 5293 glucagon release +T199 GO:0099610 5424 5447 action potential firing +T200 CHEBI:29108 5500 5504 Ca2+ +T201 CHEBI:29108 5539 5543 Ca2+ +T202 GO:0070509 5539 5549 Ca2+ entry +T203 CL:0000171 5554 5560 α-cell +T204 CL:0000171 5596 5602 α-cell +T205 GO:0005622 5603 5616 intracellular +T206 CHEBI:29108 5617 5621 Ca2+ +T207 CHEBI:29108 5624 5628 Ca2+ +T208 GO:0005622 5629 5630 i +T209 CL:0000171 5657 5663 α-cell +T210 GO:0070091 5717 5733 glucagon release +T211 CHEBI:17234 5737 5744 glucose +T212 GO:0065007 5766 5774 regulate +T213 GO:0070091 5775 5791 glucagon release +T214 GO:0070091 5873 5889 glucagon release +T215 http://purl.obolibrary.org/obo/MONDO_0004946 5900 5913 hypoglycaemia +T216 NCBITaxon:10088 5944 5949 mouse +T217 UBERON:0000006 5950 5956 islets +T218 CL:0000169 5985 5991 β cell +T219 PR:000045358 6071 6078 insulin +T220 GO:0030073 6071 6078;6092 6099 insulin ... release +T221 GO:0070091 6083 6099 glucagon release +T222 CL:0000171 6104 6106;6113 6117 α- ... cell +T223 CL:0000169 6111 6117 β-cell +T224 GO:0051716 6113 6117;6123 6132 cell ... responses +T225 CHEBI:29108 6118 6122 Ca2+ +T226 NCBITaxon:10088 6143 6148 mouse +T227 NCBITaxon:10114 6150 6153 rat +T228 NCBITaxon:9606 6159 6164 human +T229 UBERON:0000006 6165 6182 pancreatic islets +T230 CHEBI:17234 6197 6204 glucose +T231 GO:0070091 6238 6254 glucagon release +T232 UBERON:0000006 6269 6275 islets +T233 CHEBI:29105 6299 6303 Zn2+ +T234 CHEBI:16865 6308 6312 GABA +T235 GO:0038001 6313 6322 paracrine +T236 PR:000045358 6366 6373 insulin +T237 CL:0000169 6387 6393 β-cell +T238 GO:0051716 6389 6393;6399 6408 cell ... responses +T239 CHEBI:29108 6394 6398 Ca2+ +T240 NCBITaxon:9989 6447 6453 rodent +T241 NCBITaxon:9606 6458 6463 human +T242 UBERON:0000006 6464 6470 islets +T243 CHEBI:17234 6505 6512 glucose +T244 GO:0070092 6513 6543 regulation of glucagon release +T245 GO:0032940 6536 6548;6562 6567 release from ... cells +T246 UBERON:0001264 6549 6559 pancreatic +T247 CL:0000171 6549 6567 pancreatic α cells +T248 CHEBI:17234 6579 6586 Glucose +T249 GO:0065007 6591 6599 Regulate +T250 CHEBI:16865 6651 6655 GABA +T251 CHEBI:29105 6660 6664 Zn2+ +T252 GO:0038001 6668 6677 paracrine +T253 CHEBI:16865 6742 6746 GABA +T254 CHEBI:34968 6768 6776 SR-95531 +T255 CHEBI:29105 6781 6785 Zn2+ +T256 CHEBI:29108 6801 6805 Ca2+ +T257 CHEBI:17234 6826 6833 glucose +T258 GO:0070091 6859 6875 glucagon release +T259 CHEBI:17234 6877 6884 Glucose +T260 PR:000045358 6948 6955 insulin +T261 GO:0030073 6948 6963 insulin release +T262 NCBITaxon:10088 7021 7026 mouse +T263 NCBITaxon:10114 7043 7046 rat +T264 UBERON:0000006 7047 7053 islets +T265 NCBITaxon:10088 7144 7149 mouse +T266 NCBITaxon:10114 7154 7157 rat +T267 UBERON:0000006 7158 7164 islets +T268 CHEBI:17234 7181 7188 glucose +T269 CHEBI:29108 7248 7252 Ca2+ +T270 CHEBI:34968 7309 7317 SR-95531 +T271 CHEBI:16865 7444 7448 GABA +T272 CHEBI:17234 7520 7527 glucose +T273 CHEBI:17234 7557 7564 glucose +T274 GO:0070091 7617 7633 glucagon release +T275 NCBITaxon:10088 7639 7644 mouse +T276 UBERON:0000006 7645 7651 islets +T277 UBERON:0001264 7737 7747 pancreatic +T278 CL:0000173 7737 7755 pancreatic δ cells +T279 GO:0038001 7787 7796 paracrine +T280 PR:000001669 7843 7866 somatostatin receptor 2 +T281 PR:000001669 7868 7874 SSTR-2 +T282 CHEBI:17234 7935 7942 glucose +T283 NCBITaxon:10088 7994 7999 mouse +T284 UBERON:0000006 8000 8006 islets +T285 CHEBI:16865 8022 8026 GABA +T286 CHEBI:17234 8103 8110 glucose +T287 CHEBI:17234 8237 8244 glucose +T288 CL:0000171 8317 8323 α-Cell +T289 GO:0008282 8324 8337 KATP Channels +T290 GO:0065007 8338 8346 Regulate +T291 PR:000045358 8397 8404 insulin +T292 CHEBI:52217 8422 8437 pharmacological +T293 GO:0008282 8454 8466 KATP channel +T294 GO:0008282 8511 8524 KATP channels +T295 UBERON:0000006 8535 8541 islets +T296 CHEBI:17234 8675 8682 glucose +T297 CL:0000171 8686 8692 α-cell +T298 GO:0008282 8693 8705 KATP channel +T299 CL:0000171 8779 8785 α-cell +T300 CHEBI:17234 8836 8843 glucose +T301 GO:0008282 8884 8897 KATP channels +T302 CHEBI:4495 8955 8964 diazoxide +T303 CHEBI:27999 8969 8980 tolbutamide +T304 GO:0008282 9011 9023 KATP channel +T305 CL:0000171 9036 9042 α-cell +T306 CHEBI:29108 9044 9048 Ca2+ +T307 GO:0005622 9049 9050 i +T308 GO:0008282 9108 9120 KATP channel +T309 CHEBI:4495 9131 9140 diazoxide +T310 GO:0008282 9182 9195 KATP channels +T311 CHEBI:4495 9207 9216 diazoxide +T312 NCBITaxon:10088 9275 9280 mouse +T313 NCBITaxon:10114 9297 9300 rat +T314 UBERON:0000006 9313 9319 islets +T315 GO:0070091 9336 9352 glucagon release +T316 CHEBI:4495 9392 9401 diazoxide +T317 PR:000045358 9448 9455 insulin +T318 GO:0030073 9448 9463 insulin release +T319 GO:0070091 9500 9516 glucagon release +T320 CL:0000169 9546 9552 β-cell +T321 CHEBI:4495 9596 9605 diazoxide +T322 PR:000045358 9682 9689 insulin +T323 GO:0030073 9682 9697 insulin release +T324 NCBITaxon:10088 9708 9713 mouse +T325 NCBITaxon:10114 9718 9721 rat +T326 UBERON:0000006 9722 9728 islets +T327 CHEBI:17234 9794 9801 glucose +T328 CHEBI:4495 9872 9881 diazoxide +T329 CHEBI:17234 10048 10055 glucose +T330 CHEBI:17234 10118 10125 glucose +T331 GO:0008282 10132 10145 KATP channels +T332 GO:0008282 10175 10187 KATP channel +T333 CHEBI:27999 10199 10210 tolbutamide +T334 GO:0070091 10246 10262 glucagon release +T335 CHEBI:27999 10311 10322 tolbutamide +T336 CHEBI:27999 10521 10532 tolbutamide +T337 CHEBI:17234 10557 10564 glucose +T338 GO:0070091 10613 10629 glucagon release +T339 CL:0000169 10690 10696 β-cell +T340 CHEBI:35222 10722 10731 inhibitor +T341 CL:0000171 10735 10741 α-cell +T342 GO:0032940 10737 10741;10751 10758 cell ... release +T343 GO:0070091 10742 10758 glucagon release +T344 GO:0008282 10847 10859 KATP channel +T345 CL:0000171 10906 10912 α-cell +T346 GO:0008282 10913 10926 KATP channels +T347 UBERON:0000006 10975 10981 islets +T348 NCBITaxon:10088 10998 11002 mice +T349 PR:000001980 11045 11051 Kcnj11 +T350 SO:0000704 11052 11056 gene +T351 GO:0008282 11098 11110 KATP channel +T352 CHEBI:17234 11145 11152 glucose +T353 UBERON:0000006 11208 11214 islets +T354 UBERON:0000006 11272 11278 islets +T355 CHEBI:17234 11317 11324 glucose +T356 CHEBI:52217 11340 11355 pharmacological +T357 GO:0008282 11356 11368 KATP channel +T358 CHEBI:17234 11456 11463 glucose +T359 GO:0008152 11536 11546 metabolism +T360 UBERON:0000006 11579 11585 islets +T361 CHEBI:17234 11632 11639 glucose +T362 CHEBI:17234 11673 11680 glucose +T363 CHEBI:17234 11709 11716 glucose +T364 PR:000045358 11718 11725 Insulin +T365 UBERON:0000006 11754 11760 islets +T366 CHEBI:17234 11781 11788 glucose +T367 CHEBI:17234 11825 11832 glucose +T368 PR:000045358 11844 11851 insulin +T369 GO:0070091 11910 11926 glucagon release +T370 UBERON:0000006 11947 11953 islets +T371 PR:000045358 11984 11991 insulin +T372 CL:0000169 12096 12103 β cells +T373 UBERON:0000006 12129 12135 islets +T374 CHEBI:17234 12207 12214 glucose +T375 PR:000045358 12260 12267 insulin +T376 CL:0000171 12313 12314;12321 12326 α ... cells +T377 CL:0000169 12319 12326 β cells +T378 CHEBI:29108 12354 12358 Ca2+ +T379 GO:0005622 12359 12360 i +T380 GO:0051716 12361 12373;12381 12386 responses of ... cells +T381 NCBITaxon:10088 12401 12406 mouse +T382 UBERON:0000006 12407 12413 islets +T383 CHEBI:29108 12460 12464 Ca2+ +T384 GO:0005622 12465 12466 i +T385 CHEBI:17234 12497 12504 glucose +T386 CHEBI:17234 12511 12518 Glucose +T387 CHEBI:29108 12547 12551 Ca2+ +T388 GO:0005622 12552 12553 i +T389 GO:0051716 12554 12565;12568 12573 response of ... cells +T390 CL:0000171 12566 12573 α cells +T391 UBERON:0000006 12605 12611 islets +T392 CHEBI:17234 12670 12677 glucose +T393 CHEBI:4495 12749 12758 diazoxide +T394 CHEBI:4495 12801 12810 diazoxide +T395 PR:000045358 12940 12947 insulin +T396 CL:0000169 12973 12979 β-cell +T397 GO:0051716 12975 12979;12988 12997 cell ... responses +T398 CHEBI:29108 12981 12985 Ca2+ +T399 GO:0005622 12986 12987 i +T400 CHEBI:17234 13001 13008 glucose +T401 GO:0008282 13023 13036 KATP Channels +T402 GO:0065007 13037 13045 Regulate +T403 NCBITaxon:9606 13046 13051 Human +T404 CL:0000171 13052 13058 α-Cell +T405 CHEBI:17234 13123 13130 glucose +T406 GO:0065007 13131 13140 regulated +T407 NCBITaxon:9606 13174 13179 human +T408 UBERON:0000006 13180 13186 islets +T409 PR:000045358 13206 13213 insulin +T410 UBERON:0000006 13229 13235 islets +T411 NCBITaxon:9606 13321 13326 human +T412 UBERON:0000006 13327 13333 islets +T413 CHEBI:17234 13395 13402 glucose +T414 CHEBI:27999 13434 13445 Tolbutamide +T415 GO:0070091 13465 13481 glucagon release +T416 PR:000045358 13531 13538 insulin +T417 CHEBI:17234 13584 13591 glucose +T418 CHEBI:27999 13596 13607 tolbutamide +T419 GO:0008282 13673 13686 KATP channels +T420 CHEBI:4495 13697 13706 diazoxide +T421 CHEBI:17234 13723 13730 glucose +T422 GO:0070091 13754 13770 glucagon release +T423 CHEBI:4495 13836 13845 diazoxide +T424 CHEBI:4495 13911 13920 diazoxide +T425 CHEBI:4495 13953 13962 diazoxide +T426 GO:0008282 14042 14054 KATP channel +T427 CHEBI:4495 14107 14116 diazoxide +T428 CHEBI:17234 14217 14224 glucose +T429 PR:000045358 14236 14243 insulin +T430 PR:000045358 14328 14335 insulin +T431 CHEBI:16865 14351 14355 GABA +T432 CHEBI:34968 14377 14384 SR95531 +T433 CHEBI:17234 14402 14409 glucose +T434 NCBITaxon:9606 14456 14461 human +T435 UBERON:0000006 14462 14468 islets +T436 NCBITaxon:9606 14526 14531 human +T437 CL:0000171 14532 14539 α cells +T438 NCBITaxon:9606 14548 14553 human +T439 CL:0000169 14554 14555;14562 14567 β ... cells +T440 CL:0000173 14560 14567 δ cells +T441 GO:0010467 14569 14576 express +T442 CHEBI:16865 14588 14592 GABA +T443 CHEBI:17996 14603 14606 Cl− +T444 CHEBI:29108 14697 14701 Ca2+ +T445 GO:0051716 14702 14714;14730 14735 responses of ... cells +T446 CL:0000171 14722 14723;14730 14735 α ... cells +T447 CL:0000169 14728 14735 β cells +T448 NCBITaxon:9606 14750 14755 human +T449 UBERON:0000006 14756 14762 islets +T450 CHEBI:17234 14781 14788 glucose +T451 CHEBI:29108 14800 14804 Ca2+ +T452 GO:0005622 14805 14806 i +T453 UBERON:0000006 14837 14843 islets +T454 CL:0000171 14859 14866 α cells +T455 CHEBI:4495 14926 14935 diazoxide +T456 CL:0000171 14990 14996 α-cell +T457 GO:0051716 14992 14996;15005 15014 cell ... responses +T458 CHEBI:29108 14998 15002 Ca2+ +T459 GO:0005622 15003 15004 i +T460 CHEBI:4495 15018 15027 diazoxide +T461 CHEBI:27999 15072 15083 tolbutamide +T462 GO:0008282 15108 15121 KATP channels +T463 CHEBI:4495 15184 15193 diazoxide +T464 UBERON:0000006 15290 15296 islets +T465 CL:0000169 15346 15352 β-cell +T466 GO:0051716 15348 15362 cell responses +T467 CHEBI:4495 15389 15398 diazoxide +T468 CHEBI:29108 15424 15428 Ca2+ +T469 GO:0005622 15429 15430 i +T470 GO:0051716 15431 15443;15446 15451 responses of ... cells +T471 GO:0051716 15431 15443;15458 15463 responses of ... cells +T472 CL:0000171 15444 15451 α cells +T473 CL:0000169 15456 15463 β cells +T474 PR:000045358 15520 15527 insulin +T475 GO:0030073 15520 15535 insulin release +T476 CHEBI:29101 15538 15541 Na+ +T477 GO:0065007 15551 15559 Regulate +T478 CHEBI:17234 15564 15571 Glucose +T479 CHEBI:17234 15631 15638 glucose +T480 CL:0000171 15663 15669 α-cell +T481 GO:0008282 15670 15683 KATP channels +T482 GO:0008282 15767 15779 KATP channel +T483 GO:0016020 15795 15803 membrane +T484 GO:0051899 15795 15818 membrane depolarisation +T485 CL:0000169 15865 15872 β cells +T486 CL:0000171 15874 15880 α-cell +T487 CHEBI:29101 15881 15884 Na+ +T488 GO:0016020 15935 15943 membrane +T489 CHEBI:29101 16038 16041 Na+ +T490 NCBITaxon:10088 16061 16066 mouse +T491 CL:0000171 16067 16074 α cells +T492 CL:0000171 16160 16166 α-cell +T493 CHEBI:29101 16223 16226 Na+ +T494 CHEBI:9506 16246 16258 tetrodotoxin +T495 CHEBI:9506 16260 16263 TTX +T496 CL:0000171 16302 16308 α-cell +T497 CHEBI:9506 16342 16345 TTX +T498 GO:0070091 16369 16385 glucagon release +T499 NCBITaxon:10088 16391 16396 mouse +T500 UBERON:0000006 16397 16403 islets +T501 CHEBI:17234 16445 16452 glucose +T502 CHEBI:9506 16492 16495 TTX +T503 CHEBI:17234 16507 16514 glucose +T504 CHEBI:17234 16567 16574 glucose +T505 CHEBI:9506 16599 16602 TTX +T506 CHEBI:17234 16612 16619 glucose +T507 CHEBI:17234 16675 16682 glucose +T508 CHEBI:29101 16721 16724 Na+ +T509 CHEBI:9506 16754 16757 TTX +T510 CHEBI:17234 16775 16782 glucose +T511 PR:000045358 16794 16801 insulin +T512 NCBITaxon:10088 16815 16820 mouse +T513 UBERON:0000006 16821 16827 islets +T514 CL:0000171 16901 16908 α cells +T515 CHEBI:9506 16960 16963 TTX +T516 NCBITaxon:10088 16967 16972 mouse +T517 UBERON:0000006 16973 16979 islets +T518 CL:0000171 17005 17011 α-cell +T519 GO:0051716 17007 17011;17020 17028 cell ... response +T520 CHEBI:29108 17013 17017 Ca2+ +T521 GO:0005622 17018 17019 i +T522 CHEBI:17234 17043 17050 glucose +T523 UBERON:0000006 17103 17109 islets +T524 CL:0000169 17133 17139 β-cell +T525 CHEBI:29108 17141 17145 Ca2+ +T526 GO:0005622 17146 17147 i +T527 CHEBI:29108 17176 17180 Ca2+ +T528 CHEBI:29108 17202 17206 Ca2+ +T529 CL:0000171 17251 17257 α-Cell +T530 GO:0006887 17258 17268 Exocytosis +T531 CHEBI:29101 17284 17287 Na+ +T532 CHEBI:29108 17336 17340 Ca2+ +T533 CL:0000171 17346 17353 α cells +T534 GO:0006887 17370 17383;17404 17412 exocytosis of ... vesicles +T535 GO:0031982 17404 17412 vesicles +T536 CL:0000171 17476 17482 α-cell +T537 CHEBI:29108 17483 17487 Ca2+ +T538 CHEBI:29108 17534 17538 Ca2+ +T539 CHEBI:7565 17722 17732 nifedipine +T540 CHEBI:29108 17827 17831 Ca2+ +T541 CL:0000171 17872 17878 α-cell +T542 CHEBI:29108 17879 17883 Ca2+ +T543 CHEBI:17234 17961 17968 glucose +T544 CHEBI:7565 18046 18056 nifedipine +T545 CHEBI:7565 18121 18131 nifedipine +T546 GO:0070091 18149 18165 glucagon release +T547 CHEBI:17234 18180 18187 glucose +T548 CHEBI:29108 18200 18204 Ca2+ +T549 CHEBI:38215 18200 18202;18205 18220 Ca ... channel blocker +T550 GO:0046879 18247 18257;18262 18269 release of ... hormone +T551 CHEBI:17234 18305 18312 glucose +T552 CHEBI:27999 18314 18325 tolbutamide +T553 CHEBI:9506 18331 18334 TTX +T554 CHEBI:17234 18349 18356 glucose +T555 CHEBI:17234 18472 18479 glucose +T556 CHEBI:29108 18510 18514 Ca2+ +T557 CHEBI:29108 18596 18600 Ca2+ +T558 CHEBI:29108 18628 18632 Ca2+ +T559 GO:0006887 18722 18732 exocytosis +T560 GO:0006887 18746 18756 Exocytosis +T561 GO:0046903 18880 18889 secretory +T562 GO:0030141 18880 18898 secretory granules +T563 GO:0005886 18908 18923 plasma membrane +T564 CHEBI:7565 19017 19027 nifedipine +T565 CHEBI:7565 19029 19032 nif +T566 CHEBI:29108 19124 19128 Ca2+ +T567 CHEBI:17234 19147 19154 Glucose +T568 GO:0065007 19245 19253 regulate +T569 GO:0070091 19254 19270 glucagon release +T570 GO:0016020 19286 19294 membrane +T571 GO:0051899 19286 19309 membrane depolarisation +T572 GO:0060081 19286 19294;19310 19327 membrane ... hyperpolarisation +T573 NCBITaxon:10088 19331 19336 mouse +T574 CL:0000171 19337 19344 α cells +T575 CHEBI:6073 19358 19368 isradipine +T576 CHEBI:29108 19380 19384 Ca2+ +T577 CHEBI:29108 19765 19769 Ca2+ +T578 CHEBI:29108 19904 19908 Ca2+ +T579 CHEBI:29108 20102 20106 Ca2+ +T580 CHEBI:29108 20278 20282 Ca2+ +T581 CL:0000171 20319 20326 α cells +T582 CL:0000171 20396 20402 α-cell +T583 GO:0006887 20403 20413 exocytosis +T584 CL:0000171 20744 20750 α-cell +T585 GO:0006887 20751 20761 exocytosis +T586 CL:0000171 20802 20808 α-cell +T587 GO:0006887 20809 20819 exocytosis +T588 GO:0006887 20930 20940 exocytosis +T589 GO:0006887 20981 20991 Exocytosis +T590 GO:0006887 21131 21141 exocytosis +T591 CHEBI:29108 21210 21214 Ca2+ +T592 GO:0006887 21253 21263 exocytosis +T593 CHEBI:29108 21363 21367 Ca2+ +T594 GO:0065007 21531 21540 Regulated +T595 UBERON:0001264 21565 21575 pancreatic +T596 CL:0000171 21565 21583 pancreatic α cells +T597 GO:0065007 21620 21630 regulatory +T598 http://purl.obolibrary.org/obo/MONDO_0004946 21643 21656 hypoglycaemia +T599 http://purl.obolibrary.org/obo/MONDO_0005015 21658 21675 Diabetes mellitus +T600 PR:000045358 21770 21777 insulin +T601 GO:0065007 21809 21819 regulating +T602 GO:0046879 21824 21834;21850 21857 release of ... hormone +T603 CHEBI:29108 21987 21991 Ca2+ +T604 GO:0005622 21992 21993 i +T605 GO:0051716 21994 22005;22016 22021 response of ... cells +T606 CL:0000171 22014 22024;22051 22057 α cells of ... islets +T607 NCBITaxon:9989 22034 22040 rodent +T608 NCBITaxon:9606 22045 22050 human +T609 UBERON:0000006 22051 22057 islets +T610 CHEBI:29108 22088 22092 Ca2+ +T611 CL:0000171 22145 22152 α cells +T612 GO:0065007 22179 22189 regulating +T613 GO:0070092 22201 22228 control of glucagon release +T614 SO:0000704 22250 22257 genetic +T615 NCBITaxon:10088 22258 22263 mouse +T616 PR:000003563 22282 22286 SUR1 +T617 PR:000001980 22305 22311 Kir6.2 +T618 NCBITaxon:10088 22320 22324 mice +T619 GO:0008282 22357 22370 KATP channels +T620 UBERON:0000006 22447 22452 islet +T621 GO:0008282 22453 22466 KATP channels +T622 GO:0008282 22545 22557 KATP channel +T623 UBERON:0000006 22585 22591 islets +T624 CHEBI:17234 22644 22651 glucose +T625 CHEBI:17234 22678 22685 glucose +T626 GO:0008282 22731 22743 KATP channel +T627 CHEBI:50509 22731 22732;22736 22753 K ... channel inhibitor +T628 CHEBI:27999 22754 22765 tolbutamide +T629 UBERON:0000006 22859 22865 islets +T630 GO:0008282 22892 22904 KATP channel +T631 CHEBI:4495 22915 22924 diazoxide +T632 CL:0000171 22934 22940 α-cell +T633 GO:0051716 22936 22940;22946 22955 cell ... responses +T634 CHEBI:29108 22941 22945 Ca2+ +T635 CHEBI:17234 22987 22994 glucose +T636 PR:000003563 23081 23085 SUR1 +T637 SO:0000704 23089 23096 genetic +T638 GO:0065007 23135 23145 controlled +T639 CL:0000171 23153 23159 α-cell +T640 CL:0000169 23167 23173 β-cell +T641 GO:0008282 23174 23187 KATP channels +T642 GO:0065007 23207 23215 regulate +T643 CL:0000171 23216 23222 α-cell +T644 GO:0038001 23247 23256 paracrine +T645 PR:000045358 23289 23296 insulin +T646 CHEBI:29108 23329 23333 Ca2+ +T647 GO:0051716 23334 23346;23355 23360 responses of ... cells +T648 CL:0000171 23347 23348;23355 23360 α ... cells +T649 CL:0000169 23353 23360 β cells +T650 CHEBI:17234 23493 23500 glucose +T651 GO:0038001 23529 23558 paracrine signalling pathways +T652 NCBITaxon:10114 23572 23575 rat +T653 UBERON:0000006 23576 23582 islets +T654 GO:0038001 23613 23622 paracrine +T655 GO:0070091 23648 23664 glucagon release +T656 GO:0038001 23698 23707 paracrine +T657 GO:0065007 23708 23718 regulation +T658 NCBITaxon:10088 23746 23751 mouse +T659 NCBITaxon:9606 23756 23761 human +T660 CL:0000171 23762 23769 α cells +T661 CHEBI:29105 23830 23834 Zn2+ +T662 CHEBI:16865 23836 23840 GABA +T663 GO:0038001 23871 23880 paracrine +T664 GO:0065007 23881 23888 control +T665 CHEBI:17234 23979 23986 glucose +T666 GO:0070091 24184 24200 glucagon release +T667 CHEBI:17234 24246 24253 glucose +T668 PR:000045358 24282 24289 insulin +T669 GO:0030073 24282 24297 insulin release +T670 CHEBI:17234 24307 24314 glucose +T671 CL:0000169 24380 24386 β-cell +T672 GO:0070091 24433 24449 glucagon release +T673 PR:000045358 24530 24537 insulin +T674 GO:0030073 24530 24537;24551 24558 insulin ... release +T675 GO:0070091 24542 24558 glucagon release +T676 CL:0000169 24568 24570;24577 24581 β- ... cell +T677 CL:0000171 24575 24581 α-cell +T678 GO:0051716 24577 24581;24587 24596 cell ... responses +T679 CHEBI:29108 24582 24586 Ca2+ +T680 GO:0008282 24645 24657 KATP channel +T681 CHEBI:79085 24645 24646;24650 24664 K ... channel opener +T682 CHEBI:4495 24665 24674 diazoxide +T683 PR:000045358 24729 24736 insulin +T684 GO:0030073 24729 24744 insulin release +T685 CHEBI:4495 24764 24773 diazoxide +T686 CL:0000169 24798 24804 β-cell +T687 GO:0070091 24844 24860 glucagon release +T688 UBERON:0000006 24884 24890 islets +T689 CHEBI:29101 24919 24922 Na+ +T690 CHEBI:38633 24919 24921;24923 24938 Na ... channel blocker +T691 CHEBI:9506 24939 24942 TTX +T692 CL:0000171 24994 25001 α cells +T693 GO:0065007 25037 25047 regulation +T694 CHEBI:17234 25051 25058 glucose +T695 GO:0008282 25077 25090 KATP channels +T696 CHEBI:27999 25161 25172 tolbutamide +T697 CHEBI:29108 25191 25195 Ca2+ +T698 GO:0005622 25196 25197 i +T699 CL:0000171 25218 25225 α cells +T700 CHEBI:76983 25294 25307 sulfonylureas +T701 GO:0038001 25356 25365 paracrine +T702 CL:0000171 25424 25430 α-cell +T703 GO:0008282 25496 25508 KATP channel +T704 GO:0016020 25528 25536 membrane +T705 CL:0000171 25577 25583 α cell +T706 CHEBI:4495 25772 25781 diazoxide +T707 CHEBI:27999 25786 25797 tolbutamide +T708 CHEBI:17234 25827 25834 glucose +T709 CL:0000171 25847 25853 α cell +T710 GO:0016020 25893 25901 membrane +T711 CHEBI:17234 26053 26060 glucose +T712 CL:0000171 26073 26079 α cell +T713 CHEBI:17234 26182 26189 glucose +T714 CL:0000171 26264 26270 α-cell +T715 GO:0005886 26266 26279 cell membrane +T716 CHEBI:17234 26402 26409 glucose +T717 NCBITaxon:10088 26434 26439 mouse +T718 NCBITaxon:10114 26444 26447 rat +T719 CL:0000171 26448 26455 α cells +T720 CHEBI:17234 26557 26564 glucose +T721 CHEBI:27999 26598 26609 tolbutamide +T722 GO:0070091 26632 26648 glucagon release +T723 CHEBI:17234 26678 26685 glucose +T724 GO:0002027 26778 26790 chronotropic +T725 CL:0000171 26802 26809 α cells +T726 GO:0070091 26829 26845 glucagon release +T727 CHEBI:17234 26861 26868 glucose +T728 CHEBI:17234 26947 26954 glucose +T729 NCBITaxon:9606 27071 27076 human +T730 CHEBI:52217 27099 27114 pharmacological +T731 GO:0008282 27115 27127 KATP channel +T732 CHEBI:64338 27115 27127;27151 27159 KATP channel ... agonists +T733 GO:0070092 27183 27213 regulation of glucagon release +T734 CHEBI:52217 27271 27286 pharmacological +T735 GO:0065007 27292 27302 modulation +T736 CL:0000169 27320 27327 β cells +T737 GO:0070091 27352 27368 glucagon release +T738 CL:0000169 27404 27410 β-cell +T739 GO:0008282 27445 27457 KATP channel +T740 GO:0065007 27458 27468 modulation +T741 CHEBI:29108 27527 27531 Ca2+ +T742 GO:0019722 27527 27542 Ca2+ signalling +T743 NCBITaxon:9606 27546 27551 human +T744 CL:0000171 27552 27559 α cells +T745 PR:000045358 27586 27593 insulin +T746 CL:0000171 27653 27659 α-cell +T747 GO:0008282 27660 27672 KATP channel +T748 CL:0000171 27744 27750 α-cell +T749 GO:0070091 27804 27820 glucagon release +T750 NCBITaxon:9606 27845 27850 human +T751 GO:0008282 27851 27863 KATP channel +T752 GO:0070091 27876 27892 glucagon release +T753 PR:000001980 28002 28019;28024 28036 Kir6.2 subunit of ... KATP channel +T754 GO:0008282 28024 28036 KATP channel +T755 PR:000045358 28040 28047 insulin +T756 http://purl.obolibrary.org/obo/MONDO_0005015 28078 28086 diabetic +T757 NCBITaxon:9606 28087 28092 human +T758 GO:0046879 28264 28279 hormone release +T759 http://purl.obolibrary.org/obo/MONDO_0002909 28287 28301 hyperglycaemic +T760 NCBITaxon:1 28312 28323 individuals +T761 PR:000045358 28367 28374 insulin +T762 NCBITaxon:1 28408 28419 individuals +T763 CHEBI:17234 28453 28460 glucose +T764 GO:0070091 28484 28500 glucagon release +T765 CHEBI:4495 28573 28582 diazoxide +T766 NCBITaxon:9606 28595 28600 human +T767 UBERON:0000006 28601 28607 islets +T768 GO:0008282 28654 28667 KATP channels +T769 CHEBI:4495 28678 28687 diazoxide +T770 GO:0005622 28734 28747 intracellular +T771 GO:0008282 28804 28816 KATP channel +T772 CHEBI:4495 28859 28868 diazoxide +T773 GO:0070091 28910 28926 glucagon release +T774 GO:0070091 29229 29245 glucagon release +T775 CHEBI:17234 29311 29318 glucose +T776 http://purl.obolibrary.org/obo/MONDO_0005015 29333 29341 diabetes +T777 CHEBI:17234 29372 29379 glucose +T778 NCBITaxon:1 29413 29424 individuals +T779 GO:0008282 29462 29474 KATP channel +T780 CHEBI:17234 29571 29578 glucose +T781 CL:0000171 29668 29674 α-cell +T782 GO:0008282 29675 29687 KATP channel +T783 GO:0070091 29707 29723 glucagon release +T784 GO:0008282 29774 29786 KATP channel +T785 GO:0008282 29909 29921 KATP channel +T786 GO:0031099 29942 29954 regenerative +T787 CHEBI:29101 30020 30023 Na+ +T788 CHEBI:29108 30035 30039 Ca2+ +T789 CHEBI:29101 30093 30096 Na+ +T790 CHEBI:29108 30101 30105 Ca2+ +T791 CL:0000171 30132 30139 α cells +T792 GO:0008282 30197 30210 KATP channels +T793 CHEBI:29101 30292 30295 Na+ +T794 CHEBI:29108 30312 30316 Ca2+ +T795 GO:0008282 30331 30344 KATP channels +T796 CL:0000171 30364 30370 α-cell +T797 GO:0005886 30366 30379 cell membrane +T798 CL:0000171 30477 30478;30485 30490 α ... cells +T799 CL:0000169 30483 30490 β cells +T800 CHEBI:4495 30494 30503 diazoxide +T801 GO:0008282 30559 30572 KATP channels +T802 GO:0008282 30633 30645 KATP channel +T803 NCBITaxon:10088 30690 30695 mouse +T804 CL:0000171 30696 30697;30704 30709 α ... cells +T805 CL:0000169 30702 30709 β cells +T806 CHEBI:29108 30771 30775 Ca2+ +T807 CL:0000171 30847 30854 α cells +T808 CHEBI:29101 30887 30890 Na+ +T809 CHEBI:17234 31079 31086 glucose +T810 CL:0000171 31115 31122 α cells +T811 GO:0006887 31214 31224 exocytosis +T812 CL:0000171 31399 31405 α-cell +T813 GO:0051716 31401 31405;31411 31420 cell ... responses +T814 CHEBI:29108 31406 31410 Ca2+ +T815 CHEBI:17234 31428 31435 glucose +T816 CHEBI:29101 31473 31476 Na+ +T817 CHEBI:38633 31473 31475;31477 31492 Na ... channel blocker +T818 CHEBI:9506 31493 31496 TTX +T819 CHEBI:24870 31508 31511 ion +T820 NCBITaxon:9606 31534 31539 human +T821 CL:0000171 31540 31547 α cells +T822 NCBITaxon:9606 31622 31627 human +T823 UBERON:0000006 31628 31634 islets +T824 CHEBI:4495 31662 31671 diazoxide +T825 NCBITaxon:10088 31703 31708 mouse +T826 UBERON:0000006 31709 31715 islets +T827 CHEBI:24870 31773 31776 ion +T828 CL:0000171 31798 31804 α-cell +T829 GO:0031099 31805 31817 regenerative +T830 CHEBI:17234 31848 31855 glucose +T831 NCBITaxon:9606 31901 31904 man +T832 CHEBI:29108 31961 31965 Ca2+ +T833 CL:0000171 31977 31984 α cells +T834 CHEBI:29108 32109 32113 Ca2+ +T835 CL:0000171 32135 32141 α-cell +T836 GO:0006887 32142 32152 exocytosis +T837 http://purl.obolibrary.org/obo/MONDO_0004946 32182 32195 hypoglycaemic +T838 CL:0000171 32229 32235 α-cell +T839 GO:0006887 32391 32401 exocytotic +T840 NCBITaxon:10088 32455 32459 mice +T841 UBERON:0001977 32476 32481 serum +T842 CHEBI:17234 32511 32518 glucose +T843 UBERON:0001969 32561 32567 plasma +T844 PR:000045358 32568 32575 insulin +T845 CHEBI:29108 32591 32595 Ca2+ +T846 CHEBI:29108 32618 32622 Ca2+ +T847 CHEBI:29108 32792 32796 Ca2+ +T848 GO:0070509 32792 32802 Ca2+ entry +T849 CHEBI:17489 32840 32844 cAMP +T850 CHEBI:29108 32910 32914 Ca2+ +T851 CHEBI:29103 33078 33080 K+ +T852 GO:0008282 33101 33113 KATP channel +T853 CHEBI:29108 33161 33165 Ca2+ +T854 GO:0065007 33242 33249 control +T855 GO:0008152 33322 33331 metabolic +T856 GO:0065007 33332 33339 control +T857 PR:000045358 33343 33350 insulin +T858 CHEBI:17234 33445 33452 glucose +T859 PR:000045358 33466 33473 insulin +T860 PR:000045358 33600 33607 insulin +T861 http://purl.obolibrary.org/obo/MONDO_0005015 33651 33659 diabetes +T862 http://purl.obolibrary.org/obo/MONDO_0002909 33710 33724 hyperglycaemic +T863 PR:000045358 33749 33756 insulin +T864 UBERON:0000006 33792 33797 Islet +T865 UBERON:0000006 33822 33828 Islets +T866 NCBITaxon:10088 33846 33850 mice +T867 CHEBI:17234 33928 33935 glucose +T868 CHEBI:16526 33953 33956 CO2 +T869 GO:0005622 33990 34003 intracellular +T870 CHEBI:29108 34004 34008 Ca2+ +T871 UBERON:0000006 34042 34048 islets +T872 CHEBI:29108 34069 34073 Ca2+ +T873 NCBITaxon:10088 34151 34155 mice +T874 PR:000001980 34199 34205 Kcnj11 +T875 SO:0000704 34206 34210 gene +T876 GO:0008282 34301 34314 KATP channels +T877 CL:0000171 34323 34324;34331 34336 α ... cells +T878 CL:0000169 34329 34336 β cells +T879 NCBITaxon:33208 34344 34351 animals +T880 http://purl.obolibrary.org/obo/MONDO_0005015 34368 34376 diabetic +T881 CHEBI:17234 34404 34411 glucose +T882 CHEBI:17234 34421 34428 glucose +T883 http://purl.obolibrary.org/obo/MONDO_0001076 34421 34439 glucose intolerant +T884 NCBITaxon:10088 34530 34534 mice +T885 NCBITaxon:10088 34580 34584 mice +T886 NCBITaxon:9606 34650 34655 Human +T887 UBERON:0000006 34656 34662 islets +T888 NCBITaxon:9606 34723 34728 Human +T889 UBERON:0000006 34729 34734 Islet +T890 UBERON:0000006 34815 34821 islets +T891 NCBITaxon:9606 34839 34844 Human +T892 UBERON:0000006 34845 34851 islets +T893 CHEBI:17234 34886 34893 glucose +T894 CHEBI:16526 34911 34914 CO2 +T895 GO:0005622 34974 34987 Intracellular +T896 CHEBI:29108 34988 34992 Ca2+ +T897 UBERON:0000006 35015 35021 islets +T898 CHEBI:52080 35060 35068 fura red +T899 CHEBI:17234 35086 35093 glucose +T900 UBERON:0000006 35159 35165 Islets +T901 CHEBI:26710 35398 35402 NaCl +T902 CHEBI:32588 35408 35411 KCl +T903 CHEBI:32139 35415 35421 NaHCO3 +T904 CHEBI:37585 35427 35434 NaH2PO4 +T905 CHEBI:32599 35440 35445 MgSO4 +T906 CHEBI:46756 35449 35454 HEPES +T907 CHEBI:3312 35460 35465 CaCl2 +T908 CHEBI:17234 35481 35488 glucose +T909 CHEBI:32145 35503 35507 NaOH +T910 CHEBI:51103 35737 35743 fluo-4 +T911 CHEBI:52080 35748 35756 fura red +T912 CHEBI:52080 35758 35763 FuraR +T913 GO:0005622 35801 35814 intracellular +T914 CHEBI:29108 35815 35819 Ca2+ +T915 CHEBI:51103 35992 35996 Fluo +T916 CHEBI:52080 36001 36006 FuraR +T917 UBERON:0000006 36158 36164 islets +T918 CHEBI:75958 36219 36227 solution +T919 CHEBI:29108 36233 36237 Ca2+ +T920 CHEBI:29108 36395 36399 Ca2+ +T921 GO:0046879 36705 36720 Hormone release +T922 PR:000045358 36723 36730 Insulin +T923 UBERON:0000006 36838 36844 islets +T924 CHEBI:17234 36924 36931 glucose +T925 NCBITaxon:9989 36944 36950 rodent +T926 NCBITaxon:9606 36960 36965 human +T927 CHEBI:29105 37073 37077 Zn2+ +T928 CHEBI:16865 37094 37098 GABA +T929 CHEBI:29108 37143 37147 Ca2+ +T930 CHEBI:34968 37157 37165 SR-95531 +T931 CHEBI:75958 37230 37239 solutions +T932 GO:0005576 37246 37259 extracellular +T933 CHEBI:32588 37260 37263 KCl +T934 CHEBI:26710 37279 37283 NaCl +T935 CHEBI:24870 37369 37372 ion +T936 GO:0006887 37420 37430 exocytosis +T937 CL:0000171 37564 37571 α cells +T938 CHEBI:29101 37612 37615 Na+ +T939 PR:000045358 37777 37784 insulin +T940 GO:0005576 37790 37803 extracellular +T941 CHEBI:26710 37834 37838 NaCl +T942 CHEBI:78161 37843 37849 TEA-Cl +T943 CHEBI:78161 37851 37878 tetraethylammonium chloride +T944 CHEBI:32588 37885 37888 KCl +T945 CHEBI:3312 37894 37899 CaCl2 +T946 CHEBI:6636 37905 37910 MgCl2 +T947 CHEBI:46756 37914 37919 HEPES +T948 CHEBI:32145 37933 37937 NaOH +T949 CHEBI:17234 37946 37953 glucose +T950 CHEBI:75958 38148 38156 solution +T951 CHEBI:26710 38190 38194 NaCl +T952 CHEBI:32588 38199 38202 KCl +T953 CHEBI:6636 38206 38211 MgCl2 +T954 CHEBI:46756 38219 38224 HEPES +T955 CHEBI:33988 38239 38243 CsOH +T956 GO:0006887 38246 38256 Exocytosis +T957 CL:0000171 38285 38291 α-cell +T958 CHEBI:75958 38456 38464 solution +T959 CHEBI:33988 38520 38524 CsOH +T960 CHEBI:63039 38544 38548 CsCl +T961 CHEBI:26710 38553 38557 NaCl +T962 CHEBI:6636 38561 38566 MgCl2 +T963 CHEBI:46756 38570 38575 HEPES +T964 CHEBI:32035 38590 38593 KOH +T965 CHEBI:30617 38598 38604 Mg-ATP +T966 CHEBI:29108 38648 38652 Ca2+ +T967 GO:0006887 38729 38739 exocytotic +T968 http://purl.obolibrary.org/obo/MONDO_0005015 39090 39098 Diabetes +T969 UBERON:0000006 39132 39137 Islet +T970 http://purl.obolibrary.org/obo/MONDO_0005015 39233 39241 Diabetes +T971 UBERON:0000006 39296 39301 Islet +T972 CHEBI:29108 39387 39391 Ca2+ +T973 GO:0005622 39392 39393 i +T974 GO:0005622 39396 39409 intracellular +T975 CHEBI:29108 39410 39414 Ca2+ +T976 CHEBI:52080 39416 39421 FuraR +T977 CHEBI:52080 39424 39432 fura red +T978 CHEBI:16865 39434 39438 GABA +T979 CHEBI:16865 39441 39460 γ-aminobutyric acid +T980 CHEBI:29103 39483 39485 K+ +T981 CHEBI:9506 39487 39490 TTX +T982 CHEBI:9506 39492 39504 tetrodotoxin +T983 CHEBI:29108 39531 39535 Ca2+ +T984 CHEBI:17234 39575 39582 Glucose +T985 GO:0070091 39594 39610 Glucagon Release +T986 GO:0038001 39628 39637 Paracrine +T987 CHEBI:29105 39658 39662 Zn2+ +T988 CHEBI:16865 39666 39670 GABA +T989 GO:0070091 39677 39693 Glucagon release +T990 NCBITaxon:10088 39708 39713 mouse +T991 UBERON:0000006 39714 39720 islets +T992 CHEBI:17234 39751 39758 glucose +T993 CHEBI:17234 39793 39800 Glucose +T994 GO:0070091 39836 39852 glucagon release +T995 CHEBI:29105 39873 39877 Zn2+ +T996 CHEBI:29108 39889 39893 Ca2+ +T997 CHEBI:16865 39930 39934 GABA +T998 CHEBI:34968 39947 39955 SR-95531 +T999 CHEBI:16865 39986 39990 GABA +T1000 CHEBI:17234 40027 40034 glucose +T1001 GO:0038001 40079 40088 paracrine +T1002 CHEBI:16865 40098 40102 GABA +T1003 CHEBI:17234 40122 40129 glucose +T1004 NCBITaxon:10114 40164 40167 rat +T1005 UBERON:0000006 40168 40174 islets +T1006 CHEBI:17234 40239 40246 glucose +T1007 GO:0070091 40276 40292 Glucagon Release +T1008 NCBITaxon:10088 40318 40323 Mouse +T1009 UBERON:0000006 40324 40330 Islets +T1010 GO:0065007 40334 40343 Regulated +T1011 GO:0008282 40349 40361 KATP Channel +T1012 PR:000045358 40415 40422 insulin +T1013 NCBITaxon:10088 40462 40467 mouse +T1014 UBERON:0000006 40468 40474 islets +T1015 CHEBI:17234 40501 40508 glucose +T1016 CHEBI:4495 40541 40550 diazoxide +T1017 NCBITaxon:10114 40578 40581 rat +T1018 UBERON:0000006 40582 40588 islets +T1019 CHEBI:17234 40621 40628 glucose +T1020 CHEBI:27999 40656 40667 tolbutamide +T1021 CHEBI:17234 40745 40752 glucose +T1022 CHEBI:27999 40836 40847 tolbutamide +T1023 CHEBI:17234 40866 40873 glucose +T1024 CHEBI:17234 40915 40922 glucose +T1025 CHEBI:4495 41021 41030 diazoxide +T1026 CHEBI:27999 41045 41056 tolbutamide +T1027 CHEBI:27999 41073 41084 Tolbutamide +T1028 CHEBI:17234 41089 41096 Glucose +T1029 UBERON:0000006 41169 41175 Islets +T1030 GO:0010467 41181 41188 Express +T1031 PR:000001980 41201 41207 Kir6.2 +T1032 GO:0070091 41221 41237 Glucagon release +T1033 NCBITaxon:10088 41252 41257 mouse +T1034 UBERON:0000006 41258 41264 islets +T1035 CHEBI:17234 41292 41299 glucose +T1036 CHEBI:27999 41362 41373 tolbutamide +T1037 CHEBI:17234 41382 41389 glucose +T1038 GO:0070091 41407 41423 glucagon release +T1039 UBERON:0000006 41442 41448 islets +T1040 UBERON:0000006 41481 41487 islets +T1041 PR:000045358 41524 41531 insulin +T1042 GO:0005622 41628 41641 Intracellular +T1043 CHEBI:29108 41642 41646 Ca2+ +T1044 GO:0051716 41647 41658;41668 41673 Response of ... Cells +T1045 CL:0000171 41666 41673 α Cells +T1046 UBERON:0000006 41688 41694 Islets +T1047 CHEBI:4495 41740 41749 Diazoxide +T1048 GO:0005622 41770 41783 intracellular +T1049 CHEBI:29108 41784 41788 Ca2+ +T1050 GO:0051716 41789 41803;41812 41817 responses from ... cells +T1051 CL:0000171 41804 41805;41812 41817 α ... cells +T1052 CL:0000169 41810 41817 β cells +T1053 NCBITaxon:10088 41835 41840 mouse +T1054 UBERON:0000006 41841 41846 islet +T1055 CHEBI:17234 41865 41872 glucose +T1056 CHEBI:17234 41880 41887 glucose +T1057 CHEBI:17234 41899 41906 glucose +T1058 CHEBI:4495 41917 41926 diazoxide +T1059 CHEBI:4495 41928 41932 diaz +T1060 CHEBI:29108 41974 41978 Ca2+ +T1061 GO:0051716 41979 41990;41993 41998 response of ... cells +T1062 CL:0000171 41991 41998 α cells +T1063 CHEBI:17234 42023 42030 glucose +T1064 GO:0008282 42088 42100 KATP channel +T1065 CHEBI:64338 42088 42108 KATP channel agonist +T1066 CHEBI:4495 42109 42118 diazoxide +T1067 CHEBI:17234 42158 42165 glucose +T1068 GO:0070091 42205 42221 Glucagon Release +T1069 NCBITaxon:9606 42247 42252 Human +T1070 UBERON:0000006 42253 42259 Islets +T1071 GO:0065007 42263 42272 Regulated +T1072 GO:0008282 42278 42290 KATP Channel +T1073 GO:0070091 42314 42322;42361 42368 Glucagon ... release +T1074 PR:000045358 42339 42346 insulin +T1075 GO:0030073 42339 42346;42361 42368 insulin ... release +T1076 NCBITaxon:9606 42396 42401 human +T1077 UBERON:0000006 42402 42408 islets +T1078 CHEBI:17234 42466 42473 glucose +T1079 CHEBI:27999 42484 42495 tolbutamide +T1080 PR:000045358 42527 42534 insulin +T1081 CHEBI:17234 42593 42600 glucose +T1082 CHEBI:4495 42634 42643 diazoxide +T1083 GO:0005622 42763 42776 Intracellular +T1084 CHEBI:29108 42777 42781 Ca2+ +T1085 GO:0051716 42782 42794;42797 42802 Responses of ... Cells +T1086 CL:0000171 42795 42802 α Cells +T1087 NCBITaxon:9606 42817 42822 Human +T1088 UBERON:0000006 42823 42829 Islets +T1089 GO:0065007 42834 42843 Regulated +T1090 GO:0008282 42849 42861 KATP Channel +T1091 CHEBI:29108 42887 42891 Ca2+ +T1092 NCBITaxon:9606 42918 42923 human +T1093 CL:0000171 42924 42931 α cells +T1094 UBERON:0000006 42948 42953 islet +T1095 CHEBI:17234 42974 42981 glucose +T1096 CHEBI:17234 42983 42986 glu +T1097 CHEBI:4495 43013 43022 diazoxide +T1098 CHEBI:4495 43024 43028 diaz +T1099 CHEBI:29108 43051 43055 Ca2+ +T1100 CHEBI:17234 43076 43083 glucose +T1101 CHEBI:17234 43094 43101 glucose +T1102 CHEBI:17234 43122 43129 glucose +T1103 CHEBI:4495 43142 43151 diazoxide +T1104 CHEBI:4495 43189 43198 diazoxide +T1105 CHEBI:17234 43239 43246 glucose +T1106 NCBITaxon:9606 43274 43279 human +T1107 CL:0000171 43280 43286 α-cell +T1108 GO:0051716 43282 43286;43292 43301 cell ... responses +T1109 CHEBI:29108 43287 43291 Ca2+ +T1110 CHEBI:4495 43310 43319 diazoxide +T1111 GO:0008282 43357 43369 KATP channel +T1112 CHEBI:27999 43381 43392 tolbutamide +T1113 CHEBI:4495 43452 43461 diazoxide +T1114 CHEBI:29108 43469 43473 Ca2+ +T1115 GO:0051716 43474 43485;43496 43500 response of ... cell +T1116 GO:0051716 43474 43485;43507 43511 response of ... cell +T1117 NCBITaxon:9606 43488 43493 human +T1118 CL:0000171 43494 43500 α cell +T1119 CL:0000169 43505 43511 β cell +T1120 UBERON:0000006 43528 43533 islet +T1121 CHEBI:17234 43551 43558 glucose +T1122 CHEBI:4495 43590 43599 diazoxide +T1123 CHEBI:17234 43618 43625 glucose +T1124 CHEBI:4495 43688 43697 diazoxide +T1125 CL:0000171 43701 43707 α-cell +T1126 GO:0051716 43703 43707;43713 43722 cell ... responses +T1127 CHEBI:29108 43708 43712 Ca2+ +T1128 CHEBI:17234 43783 43790 glucose +T1129 CL:0000171 43928 43934 α-Cell +T1130 GO:0051716 43930 43934;43940 43949 Cell ... Responses +T1131 CHEBI:29108 43935 43939 Ca2+ +T1132 CHEBI:29101 44003 44006 Na+ +T1133 GO:0070091 44021 44029;44068 44075 Glucagon ... release +T1134 PR:000045358 44046 44053 insulin +T1135 GO:0030073 44046 44053;44068 44075 insulin ... release +T1136 NCBITaxon:10088 44090 44095 mouse +T1137 UBERON:0000006 44096 44102 islets +T1138 CHEBI:17234 44121 44128 glucose +T1139 CHEBI:29101 44182 44185 Na+ +T1140 CHEBI:9506 44205 44208 TTX +T1141 GO:0005622 44227 44240 Intracellular +T1142 CHEBI:29108 44241 44245 Ca2+ +T1143 GO:0051716 44246 44257;44267 44272 response of ... cells +T1144 CL:0000171 44265 44272 α cells +T1145 CHEBI:17234 44283 44290 glucose +T1146 CHEBI:9506 44292 44295 TTX +T1147 CHEBI:9506 44394 44397 TTX +T1148 CHEBI:17234 44402 44409 glucose +T1149 CL:0000171 44413 44419 α-cell +T1150 GO:0051716 44415 44419;44425 44434 cell ... responses +T1151 CHEBI:29108 44420 44424 Ca2+ +T1152 CHEBI:9506 44446 44449 TTX +T1153 CHEBI:29108 44459 44463 Ca2+ +T1154 CHEBI:17234 44492 44499 glucose +T1155 GO:0065007 44683 44692 Regulated +T1156 CHEBI:29108 44703 44707 Ca2+ +T1157 GO:0070091 44722 44738 Glucagon release +T1158 CHEBI:17234 44775 44782 glucose +T1159 CHEBI:7565 44882 44892 nifedipine +T1160 GO:0006887 44907 44917 Exocytosis +T1161 CHEBI:7565 45036 45046 nifedipine +T1162 CHEBI:7565 45055 45058 nif +T1163 GO:0006887 45110 45120 exocytotic +T1164 CHEBI:7565 45178 45188 nifedipine +T1165 CHEBI:17234 45253 45260 glucose +T1166 CL:0000171 45372 45378 α-Cell +T1167 CHEBI:29108 45386 45390 Ca2+ +T1168 GO:0006887 45404 45414 Exocytosis +T1169 CHEBI:29108 45427 45431 Ca2+ +T1170 CHEBI:6073 45500 45510 isradipine +T1171 CHEBI:29108 45592 45596 Ca2+ +T1172 CHEBI:29108 45903 45907 Ca2+ +T1173 CHEBI:6073 45950 45960 isradipine +T1174 GO:0006887 46266 46276 Exocytosis +T1175 GO:0006887 46391 46401 exocytotic +T1176 GO:0006887 46417 46427 Exocytosis +T1177 GO:0006887 46570 46580 exocytotic +T1178 CL:0000171 46900 46906 α-Cell +T1179 CHEBI:17234 46959 46966 glucose +T1180 CHEBI:27999 46968 46979 tolbutamide +T1181 CHEBI:4495 46985 46994 diazoxide +T1182 CL:0000171 46998 47004 α-cell +T1183 CHEBI:29101 47011 47014 Na+ +T1184 CHEBI:29108 47027 47031 Ca2+ +T1185 PR:000045358 47095 47102 insulin +T1186 CL:0000171 47244 47250 α-cell +T1187 GO:0008282 47251 47263 KATP channel +T1188 CHEBI:29108 47305 47309 Ca2+ +T1189 CHEBI:29101 47314 47317 Na+ +T1190 CHEBI:29108 47378 47382 Ca2+ +T1191 CHEBI:29101 47387 47390 Na+ +T1192 GO:0008282 47432 47444 KATP channel +T1193 CHEBI:29108 47538 47542 Ca2+ +T1194 CHEBI:29101 47547 47550 Na+ +T1195 CHEBI:17234 47571 47578 glucose +T1196 CL:0000171 47601 47607 α-cell +T1197 GO:0008282 47608 47620 KATP channel +T1198 CHEBI:27999 47687 47698 tolbutamide +T1199 CHEBI:17234 47708 47715 glucose +T1200 GO:0008282 47761 47773 KATP channel +T1201 GO:0070091 47847 47863 glucagon release +T1202 CHEBI:4495 47896 47905 diazoxide +T1203 CHEBI:17234 47914 47921 glucose +T1204 CL:0000171 47943 47949 α-cell +T1205 GO:0008282 47950 47962 KATP channel +T1206 CHEBI:4495 48106 48115 diazoxide +T1207 CHEBI:17234 48144 48151 glucose +T1208 CHEBI:4495 48195 48204 diazoxide +T1209 GO:0008282 48215 48227 KATP channel +T1210 GO:0070091 48319 48335 glucagon release +T1211 CHEBI:33893 48640 48648 reagents +T1212 http://purl.obolibrary.org/obo/MONDO_0005015 49017 49025 Diabetes diff --git a/src/ontogpt/evaluation/craft/database/all/17503968.txt b/src/ontogpt/evaluation/craft/database/all/17503968.txt new file mode 100644 index 000000000..75deac1b7 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17503968.txt @@ -0,0 +1,265 @@ +A KATP Channel-Dependent Pathway within α Cells Regulates Glucagon Release from Both Rodent and Human Islets of Langerhans + +Abstract + +Glucagon, secreted from pancreatic islet α cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring β cells, or to an intrinsic glucose sensing by the α cells themselves. We examined hormone secretion and Ca2+ responses of α and β cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn2+ signalling was blocked, but was reversed by low concentrations (1–20 μM) of the ATP-sensitive K+ (KATP) channel opener diazoxide, which had no effect on insulin release or β cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 μM). Higher diazoxide concentrations (≥30 μM) decreased glucagon and insulin secretion, and α- and β-cell Ca2+ responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 μM) stimulated glucagon secretion, whereas high concentrations (>10 μM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na+ (TTX) and N-type Ca2+ channels (ω-conotoxin), but not L-type Ca2+ channels (nifedipine), prevented glucagon secretion. Both the N-type Ca2+ channels and α-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an α-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion. + +Author Summary + + + +Glucagon is a critical regulator of glucose homeostasis. Its major action is to mobilize glucose from the liver. Glucagon secretion from α cells of the pancreatic islets of Langerhans is suppressed by elevated blood sugar, a response that is often perturbed in diabetes. Much work has focused on the regulation of α-cell glucagon secretion by neuronal factors and by paracrine factors from neighbouring cells, including the important islet hormone insulin. In contrast, we provide evidence in support of a direct effect of glucose on α cells within intact rodent and human islets. Notably, our work implicates an α-cell glucose-sensing pathway similar to that found in insulin-secreting β cells, involving closure of ATP-dependent K+ channels in the presence of glucose. Furthermore, we find that membrane depolarisation results in inhibition of Na+ and Ca2+ channel activity and α-cell exocytosis. Thus, we propose that elevated blood glucose reduces α-cell electrical activity and glucagon secretion by inactivating the ion channels involved in action potential firing and secretion. + +Introduction + +Blood glucose levels are under the control of two hormones released from the pancreatic islets of Langerhans. Islet β cells secrete insulin when glucose is high, decreasing glucose production by the liver and increasing glucose storage in multiple tissues. Regulated insulin secretion is relatively well understood, involving the metabolic stimulation of electrical activity, Ca2+ entry, and exocytosis [1]. Islet α cells secrete glucagon in response to decreased blood glucose, whereas elevated glucose levels suppress glucagon release. Glucagon is the principal factor stimulating glucose production by the liver. In diabetes, baseline glucagon release is elevated, and glucagon secretion in the low-glucose condition is blunted [2–4]. These effects contribute to chronic hyperglycaemia and to an increased risk for acute hypoglycaemic events. + +The mechanism regulating glucagon secretion is poorly understood and remains hotly debated [5]. Glucagon release in rodents may be regulated by paracrine signals, including γ-aminobutyric acid (GABA) [6,7], Zn2+ [8], and insulin [9,10]. Conversely, glucose may suppress glucagon secretion through a direct effect on α-cell activity [11–14]. There are also studies suggesting that glucagon secretion is under hypothalamic control [15,16]. Human in vivo studies provide conflicting evidence regarding the control of glucagon secretion by paracrine or intrinsic regulation of α cells [17–30], and very little work has been done to examine this question in isolated human islets. + +Islet α cells express ATP-dependent K+ (KATP) channels [9,13,14,31,32] that can be closed by ATP [9,31]. Glucose increases intracellular free ATP in α cells [8,10], although reports vary as to the ability of glucose to inhibit α-cell KATP channels [13,31,33]. Evidence from SUR1−/− mice implicate KATP channels as regulators of glucagon release [13,34,35]. However, because both α and β cells possess molecularly identical KATP channels [36,37], it is not clear how KATP-mediated depolarisation would stimulate insulin secretion but suppress glucagon secretion. + +The answer may lie in the downstream machinery regulating electrical activity and Ca2+ entry. Unlike β cells, α cells possess a large voltage-dependent Na+ current that is essential for glucagon release [14,33]. We have previously proposed that the depolarisation-induced inactivation of this channel contributes to the cessation of action potential firing [14]. Additionally, activation of voltage-dependent Ca2+ channels (VDCCs) is essential for Ca2+ entry and α-cell function [38,39]. Accordingly, the α-cell intracellular Ca2+ ([Ca2+]i) oscillations (reflecting α-cell electrical activity) are suppressed in parallel with glucagon release by glucose [40]. Multiple VDCCs regulate glucagon release [14,41–43], although the N-type channels appear to be particularly important for glucagon release evoked by hypoglycaemia alone [33,44,45], at least in mouse islets. This is in contrast to the β cell in which the L-type VDCC functionally predominates [46]. + +We have now compared insulin and glucagon release and α- and β-cell Ca2+ responses in intact mouse, rat, and human pancreatic islets. We show that glucose retained the ability to suppress glucagon release from isolated islets during blockade of the Zn2+ and GABA paracrine pathways, and in the absence of stimulated insulin secretion or β-cell Ca2+ responses. Thus we now provide evidence in both rodent and human islets supporting the direct (intrinsic) glucose regulation of glucagon release from pancreatic α cells. + +Results + +Glucose Can Regulate Glucagon Secretion Directly + +To examine a role for GABA and Zn2+ as paracrine mediators of glucagon secretion, we examined the ability of the GABAA receptor antagonist SR-95531 and Zn2+ chelation with Ca2+-EDTA to prevent the glucose-dependent suppression of glucagon release. Glucose, at concentrations (7 and 8.3 mM) just above the threshold for insulin release (see below), suppressed glucagon secretion from isolated mouse (Figure 1A) and rat islets (Figure 1B) by 60% (n = 15, p < 0.001) and 57% (n = 10, p < 0.001), respectively. In both mouse and rat islets, the ability of glucose to inhibit glucagon secretion persisted in the presence of Ca2+-EDTA (43% and 48%, n = 10, p < 0.001, respectively) and SR-95531 (31% and 46%, n = 8 and 10, p < 0.01 and p < 0.001, respectively) (Figure 1). + +It is worth noting that in the presence of the GABAA antagonist, glucagon secretion was increased under both low- and high-glucose conditions, and furthermore, glucose was approximately 50% less effective in suppressing glucagon release from mouse islets (31% versus 60%, respectively) (Figure 1A). Additionally, somatostatin released from pancreatic δ cells is suggested to be a potential paracrine regulator of glucagon secretion. However, the somatostatin receptor 2 (SSTR-2) antagonist PRL-2903 does not interfere with the ability of glucose (at 3 and 7 mM) to inhibit glucagon secretion from mouse islets [47]. Like the GABAA receptor antagonist, however, PRL-2903 increased glucagon secretion in low-glucose conditions [47]. Thus, although the present data do not entirely rule out these pathways as modulators of glucagon secretion, glucose is clearly able to suppress glucagon secretion independently of these. + +α-Cell KATP Channels Regulate Glucagon Secretion + +We next examined glucagon and insulin secretion during pharmacological manipulation of KATP channel activity. Here we have examined the role of KATP channels in intact islets by applying an indirect, but minimally invasive, technique. It would have been difficult (if not impossible) to study the effects of glucose on α-cell KATP channel activity using the patch-clamp technique because of the smallness of the α-cell resting conductance (0.15 nS/pF in the absence of glucose, of which two thirds is attributable to KATP channels) [13]. We have instead used increasing concentrations of diazoxide and tolbutamide to “titrate” the influence of KATP channel activity on α-cell [Ca2+]i and glucagon secretion. Increasing concentrations of the KATP channel activator diazoxide demonstrated that moderate activation of KATP channels (0.3–10 μM diazoxide) relieved the suppression of glucagon secretion from both mouse (Figure 2A) and rat (Figure 2B) islets. Stimulation of glucagon release was half-maximal at approximately 1 μM diazoxide, which is well below that required to inhibit insulin release, suggesting that “re-activation” of glucagon release was not secondary to reduced β-cell secretion. Increasing the concentration of diazoxide beyond 10 μM inhibited glucagon secretion in parallel with an inhibition of insulin release from both mouse and rat islets (Figure 2A and 2B). When instead applied in the presence of 1 mM glucose (at which concentration glucagon secretion is stimulated), increasing diazoxide produced a monotonic inhibition of glucagon secretion (Figure 2C) with a half-maximal inhibitory concentration (IC50) that is much lower than what is seen under high-glucose conditions (∼2 μM versus ∼50 μM). + +In the complete absence of glucose, when KATP channels are expected to be open, the KATP channel antagonist tolbutamide also produced a biphasic effect on glucagon release. Augmentation of glucagon secretion was seen at tolbutamide concentrations of up to 1 μM (stimulation being half-maximal at 0.1 μM), whereas inhibition was observed at higher concentrations (Figure 2D). Importantly, in the presence of a maximally inhibitory tolbutamide concentration (0.5 mM), glucose was unable to produce any further inhibition of glucagon release (Figure 3A). These data are inconsistent with the idea that β-cell secretion is the primary inhibitor of α-cell glucagon release. They also suggest that glucagon secretion is maximal within a “window” of intermediate KATP channel activity. + +To further investigate the role of α-cell KATP channels as regulators of glucagon secretion, we studied islets from Kir6.2Y12X mice, which posses a Tyr12STOP mutation in the Kcnj11 gene, leading to premature termination of the KATP channel pore-forming subunit [48]. At low-glucose concentrations, glucagon secretion from the Kir6.2Y12X islets was already suppressed compared with that from wild-type islets (Figure 3B), similar to the effect of glucose stimulation or pharmacological KATP channel inhibition observed in Figures 2D and 3A. Consistent with a recent report [49], higher glucose levels stimulated glucagon secretion, perhaps due to a direct effect of metabolism on secretion [33]. In wild-type islets, glucagon secretion exhibited a nadir at 5 mM glucose, and glucagon secretion at 20 mM glucose was 40% higher than at 5 mM glucose. Insulin secretion in the Kir6.2Y12X islets was elevated at low-glucose concentrations (Figure 3C), whereas glucose-stimulated insulin secretion was blunted. It is notable that the increase in glucagon release from the Kir6.2Y12X islets was coincident with increased insulin secretion, reinforcing the view that inhibition of glucagon is not mediated by a factor released by the β cells. Furthermore, in control islets, the inhibition of glucagon secretion is maximal at a concentration of glucose (5 mM) that is without stimulatory action on insulin secretion. + +We next examined the function of α and β cells in situ by monitoring the [Ca2+]i responses of single cells within intact mouse islets. These were functionally identified by their [Ca2+]i response to 0.5, 2, and 11 mM glucose [50]. Glucose stimulation suppressed the [Ca2+]i response of α cells by 51 ± 3% (n = 63 cells in 15 islets, p < 0.001) (Figure 4A and 4B). The suppressive effect of glucose was alleviated (to 101 ± 11% of initial values) by application of 2 μM diazoxide (Figure 4A and 4B). This concentration of diazoxide was similar to that which produced maximal stimulation of glucagon secretion and much (∼15-fold) lower than necessary to inhibit insulin secretion (Figure 2B) or β-cell [Ca2+]i responses to glucose (Figure 4A). + +KATP Channels Regulate Human α-Cell Activity + +Little work has been done to examine the mechanism of glucose-regulated glucagon secretion from isolated human islets [51]. Glucagon and insulin secretion from islets isolated from healthy donors was examined. Similar to above, glucagon secretion from human islets was suppressed by 58 ± 3% (n = 9, p < 0.05) upon raising the glucose concentration from 0 to 10 mM. Tolbutamide (200 μM) inhibited glucagon release by 43 ± 14% (n = 7, p < 0.05). At the same time, insulin secretion was increased 6.4- and 4.4-fold by glucose and tolbutamide stimulation, respectively (Figure 5A). Moderate re-activation of KATP channels with 2 μM diazoxide antagonized the glucose-induced suppression of glucagon release (n = 7, p < 0.01) (Figure 5B). A 10-fold higher concentration of diazoxide had significantly less stimulatory effect (p < 0.001 versus 2 μM diazoxide), and in the presence of 200 μM diazoxide, glucagon secretion was not different from that observed in the absence of the KATP channel activator. Importantly, the lowest concentration of diazoxide (2 μM), which produced the strongest stimulation of glucagon secretion, had no inhibitory effect on glucose-stimulated insulin secretion (n = 5), whereas 20 and 200 μM produced partial or complete inhibition of insulin secretion. The GABAA receptor antagonist SR95531 had no effect on glucose-induced inhibition of glucagon secretion from human islets, and whole-cell voltage-clamp measurements indicate that human α cells, unlike human β and δ cells, express few if any GABAA receptor Cl− channels (M. Braun, R. Ramracheya, and P. Rorsman, unpublished data). + +Examination of the Ca2+ responses of single α and β cells within intact human islets demonstrated that glucose decreased [Ca2+]i by 62 ± 3% (n = 42 cells in 7 islets, p < 0.001) in α cells, and that this effect could be completely reversed by 2 μM diazoxide (Figure 6A and 6B). Furthermore, the re-activation of α-cell [Ca2+]i responses by diazoxide could be prevented by application of 100 μM tolbutamide, confirming the role of KATP channels (Figure 6C). An analysis of the dose–response relationship to diazoxide demonstrated a maximally effective concentration of 1.7 μM (Figure 6D and 6E; n = 38 cells in 9 islets), far below the concentration necessary to block β-cell responses (Figure 6D). Increases in diazoxide above 20 μM blocked the [Ca2+]i responses of α cells and β cells in parallel, consistent with the effect on glucagon and insulin release. + +Na+ Channels Regulate Low-Glucose–Stimulated Glucagon Secretion + +The above data suggest that glucose-dependent inhibition of α-cell KATP channels is involved in the suppression of glucagon secretion. It is not clear however, how KATP channel inhibition and membrane depolarisation result in suppression of secretion. Unlike in β cells, α-cell Na+ channels are active in the physiological range of membrane potentials. Previous work from our group suggested that the voltage-dependent inactivation of Na+ channels, which in mouse α cells is half-maximal at −42 mV [43], contributes to cessation of electrical activity upon α-cell depolarisation [14]. We thus used the voltage-dependent Na+ channel antagonist tetrodotoxin (TTX) to test a role for these channels in α-cell function and glucagon secretion. TTX (0.1 μg/ml) suppressed glucagon release from mouse islets by 56 ± 5% (n = 10, p < 0.001) under low-glucose conditions (Figure 7A). This effect of TTX in the low-glucose condition was similar to what we observed with high-glucose stimulation alone. With TTX present, glucose was without further inhibitory action, suggesting that glucose inhibits glucagon secretion through a Na+ channel-dependent mechanism. TTX has no effect on glucose-stimulated insulin secretion in mouse islets (Figure 7A); again suggestive of a direct rather than indirect effect on α cells. In accordance with this, application of 0.1 μg/ml TTX to mouse islets reversibly abolished the α-cell [Ca2+]i response evoked by low-glucose concentrations (Figure 7B and 7C; n = 18 cells in 4 islets), but had no effect on β-cell [Ca2+]i (unpublished data). + +N-Type Ca2+ Channels Mediate the Ca2+ Influx That Triggers Glucagon Secretion and α-Cell Exocytosis + +Downstream of Na+ channel activation, the opening of VDCCs allows Ca2+ into α cells, triggering the exocytosis of glucagon-containing vesicles. We applied whole-cell patch-clamp recordings to establish the α-cell Ca2+ channel complement. The integrated whole-cell Ca2+ current measured during 50-ms depolarisations from −70 mV to 0 mV amounted to 6.2 ± 0.8 pC (n = 15) under control conditions, 2.6 ± 0.8 pC (n = 10; p < 0.05) in the presence of 50 μM nifedipine, and 4.1 ± 0.5 pC (n = 12; p < 0.01) in the presence of 1 μM ω-conotoxin. Thus, L- and N-type Ca2+ channels account for 58% and 34% of the α-cell Ca2+ current, respectively. + +Figure 8A compares glucagon secretion at 1 and 20 mM glucose under control conditions and in the presence of 100 nM ω-conotoxin and 20 μM nifedipine, respectively. We found that whereas the L-type channel blocker nifedipine had no effect on glucagon release at 1 or 20 mM glucose, the N-type Ca2+ channel blocker ω-conotoxin inhibited the release of the hormone to a level similar to that of high glucose, tolbutamide, and TTX. Furthermore, glucose exerted no additional inhibitory action in the presence of ω-conotoxin. Thus, glucagon secretion stimulated by low-glucose levels depends principally on Ca2+ influx through N-type channels, although these only account for one third of the Ca2+ current. We confirmed this Ca2+ channel dependence by conducting high-resolution single-cell capacitance measurements of exocytosis (Figure 8B). Exocytosis elicited by 500-ms step depolarisations from −70 to 0 mV averaged approximately 150 fF (corresponding to the fusion of ∼75 secretory granules with the plasma membrane [43]) under control conditions (Ctrl). This response was not significantly affected by 50 μM nifedipine (nif), but was nearly abolished by 1 μM ω-conotoxin (ω-con; Figure 8C). + +Inactivation of N-Type Ca2+ Channels Underlie Glucose Inhibition of Glucagon Secretion + +We next examined the mechanism by which N-type channels regulate glucagon release in response to membrane depolarisation/hyperpolarisation in mouse α cells. Non–L-type (isradipine-resistant) Ca2+ currents were elicited by step depolarization to 0 mV in the absence (black) or presence (red) of 1 μM ω-conotoxin following a 200-ms conditioning pulse to −70 mV (Figure 9A) or +10 mV (Figure 9B). It is evident that the ω-conotoxin–sensitive component was abolished by the +10 mV conditioning pulse. Figure 9C summarizes the relationship between the conditioning voltage and the Ca2+ current amplitude in the absence (open squares) and presence (circles) of ω-conotoxin (upper panel). The ω-conotoxin–sensitive N-type Ca2+ current component is shown in the lower panel of Figure 9C, and underwent voltage-dependent inactivation that was half-maximal (V0.5) at −31 ± 6 mV (n = 6). A residual, ω-conotoxin–insensitive Ca2+ current also appears to undergo voltage-dependent inactivation (Figure 9C). This accounts for less than 15% of the inactivating current, and may be attributable to T-type Ca2+ channels that have been reported in α cells [14]. + +In Figure 9D, we examined the voltage-dependent activation of α-cell exocytosis using step-wise depolarisations (500 ms) from −70 mV to between −50 and 20 mV (Figure 9D). The capacitance response versus voltage relationship (Figure 9E) demonstrates a marked increase in the capacitance response between −10 and 10 mV. Thus any reduction in action potential amplitude within this range would severely attenuate α-cell exocytosis. + +The voltage-dependent inactivation of α-cell exocytosis was examined next (Figure 9F). Cells were held at conditioning potentials between −70 and −30 mV, after which exocytosis was elicited by depolarisation to 0 mV. Exocytosis stimulated from the −30 mV conditioning potential was only 25% of that elicited from −70 mV (Figure 9G). This voltage-dependent decline in exocytosis is attributable to the voltage-dependent inactivation of the N-type Ca2+ channels. The fact that inhibition of exocytosis appears to occur at more-negative voltages than that documented for the inactivation of the N-type Ca2+ channels can be attributed to the brevity of the conditioning pulses in Figure 9A–9C (200 ms), whereas in Figure 9G the holding potential was varied. + +Discussion + +Regulated glucagon secretion from pancreatic α cells is a major component of the counter-regulatory response to hypoglycaemia. Diabetes mellitus is associated with defects of glucagon secretion that exacerbate the consequences of impaired insulin secretion [2–4]. The mechanism regulating the release of this important hormone is incompletely understood and currently the source of much debate. In the present study we examined glucagon secretion and the [Ca2+]i response of in situ α cells of isolated rodent and human islets. We further characterised the Ca2+ currents and capacitance changes of single isolated α cells, to dissect the mechanism regulating downstream control of glucagon release. + +Previous work with genetic mouse models, including SUR1−/− [13,34,35] and Kir6.2−/− [16] mice, suggests an important role for KATP channels in glucagon secretion. Three pieces of evidence argue for the importance of islet KATP channels in glucagon secretion. First, introduction of the Tyr12STOP mutation into the KATP channel subunits in the Kir6.2Y12X islets results in suppression of glucagon secretion at low-glucose levels and causes loss of glucose-induced inhibition of secretion. Second, the KATP channel inhibitor tolbutamide when applied at maximally effective concentrations inhibits glucagon secretion from isolated islets. Third, a low dose of the KATP channel activator diazoxide restores α-cell Ca2+ responses and glucagon secretion in high-glucose conditions. What is not immediately clear from these arguments, and from the previous SUR1−/− genetic studies, is whether glucagon is being controlled by the α-cell or the β-cell KATP channels, as the latter may regulate α-cell function indirectly via paracrine pathways. We therefore examined insulin and glucagon secretion, and the Ca2+ responses of α and β cells in situ in parallel to determine the functional relationship between these cells. Furthermore, we measured the glucagon response to glucose during blockade of putative paracrine signalling pathways. + +Studies on rat islets support an important role for paracrine signals as regulators of glucagon release [33,52,53], whereas the case for paracrine regulation of glucagon secretion from mouse and human α cells is less clear [9,10,14,17–21,27]. Although it is clear that Zn2+, GABA, and somatostatin can exert a paracrine control of glucagon secretion under certain conditions, the data shown here firmly establish that glucose can suppress glucagon secretion independently of these pathways as demonstrated in Figure 1 (and in [47]). Furthermore, and in agreement with recently published results [47], maximal inhibition of glucagon release occurs at levels equal to or lower than 5 mM glucose, whereas the stimulation of insulin release requires glucose levels greater than 5 mM (Figure 3), suggesting that products of β-cell secretion are not required for suppression of glucagon release. + +This conclusion is further underpinned by the significant discordance between insulin and glucagon release, and the β- and α-cell Ca2+ responses, under several conditions: (1) low doses of the KATP channel opener diazoxide that stimulate glucagon secretion while not affecting insulin release; (2) high doses of diazoxide at which suppression of β-cell secretion would be expected to elevate glucagon release; (3) in the Kir6.2Y12X islets; and (4) in response to the Na+ channel blocker TTX. Therefore, our data argue that glucagon-producing α cells possess an intrinsic mechanism for regulation by glucose and that involves KATP channels. This is at variance with the data of Liu et al. [12] who report that tolbutamide has no effect on [Ca2+]i in single, isolated α cells, but in agreement with the conclusion of Ostenson et al. [54], that sulfonylureas can inhibit glucagon secretion by a direct, non-paracrine mechanism. + +On the basis of our findings, we propose that α-cell glucagon secretion occurs within a narrow window of intermediate KATP channel activity (and thus membrane potential) (Figure 10). That is, if the α cell is either too hyperpolarised (maximal KATP activity) or too depolarised (maximal KATP inhibition), then glucagon secretion is suppressed. This is supported by the biphasic effects of both diazoxide and tolbutamide. Whereas the former (in high glucose) brings the α cell in a dose-dependent manner through the membrane potential window supporting glucagon secretion in the depolarised (low KATP) to hyperpolarised (high KATP) direction (Figure 10C), the latter (in zero glucose) brings the α cell through the window in the opposite direction (from hyperpolarised to depolarised) (Figure 10B). Thus, glucose likely leads to the suppression of glucagon secretion by depolarising the α-cell membrane potential above the range that supports glucagon secretion (Figure 10A). Indeed, in some (but not all, see [55]) studies, glucose was found to depolarize mouse and rat α cells and reduce action potential amplitudes [13,52]. It is interesting that whereas low concentrations of glucose were without stimulatory effect, tolbutamide (0.1–1 μM) stimulated glucagon release beyond that observed at zero glucose. Thus, small depolarisations (as previously documented for arginine [13]) exert a positive “chronotropic” effect in α cells and thus stimulate glucagon release. The fact that glucose did not share this ability indicates that the depolarisation produced by 1 mM glucose results in sufficient inactivation of the currents to balance any increase in action potential frequency. + +Previous human studies have employed pharmacological KATP channel antagonists [23,24] or agonists [25,26] to examine the regulation of glucagon release in vivo. These were interpreted with the assumption that pharmacological KATP modulation only affects the β cells, and that any change in glucagon release was therefore secondary to altered β-cell function. Our data establish that KATP channel modulation has dramatic and direct effects on glucagon secretion and Ca2+ signalling in human α cells under conditions in which insulin secretion is unaffected. Thus, the in vivo manipulation of α-cell KATP channel activity in the above studies may well have involved direct effects on α-cell function that contributed to the observed changes in glucagon release. + +The importance of the human KATP channel pathway for glucagon release is nicely highlighted by a study investigating the effects of the common Glu23Lys (E23K) polymorphism of the Kir6.2 subunit of the KATP channel on insulin and glucagon secretion in non-diabetic human patients [29]. This variant of the channel leads to a slight decrease in the ATP sensitivity of the channel. The functional significance of this was examined by comparing hormone release during hyperglycaemic clamps in individuals carrying the polymorphism or not. Although insulin secretion in homozygous Glu23Lys individuals was not different from controls, glucose-induced suppression of glucagon release was blunted [29]. This becomes understandable in light of the effect of diazoxide on isolated human islets (Figures 4 and 5). Half-maximal activation of KATP channels occurs at diazoxide concentrations of 20–100 μM (depending on the intracellular ATP level) [56]. If the Glu23Lys polymorphism increases KATP channel activity to the same extent as 0.3–1.5 μM diazoxide, the concentration at which an effect on glucagon release is first seen, then the effect will be very difficult to detect with electrophysiology, perhaps explaining why some studies have failed to detect a functional effect of the polymorphism (reviewed in [57]). Nevertheless, such small changes can have significant biological effects, as illustrated by the glucagon release data, and may contribute to pathological states such as impaired glucose tolerance and diabetes. Thus, the reduced ability of glucose to inhibit glucagon secretion in individuals carrying the Glu23Lys variant of the KATP channel likely results from the failure of these channels to undergo complete inhibition in response to glucose. + +We have proposed that glucagon secretion is stimulated within a window of intermediate α-cell KATP channel activity, and that glucagon release is suppressed by either increases or decreases in KATP channel activity. How is this accomplished? Briefly, we suggest that this window is the result of (1) the ability of intermediate KATP channel activity to support regenerative electrical responses through the activation of voltage-dependent Na+ and N-type Ca2+ channels (grey in Figure 10); (2) the failure of the Na+ and Ca2+ channels to activate when α cells are hyperpolarised by the opening of a major fraction of KATP channels (above the grey in Figure 10); and (3) the voltage-dependent inactivation of the Na+ [14] and N-type Ca2+ channels when KATP channels are closed and the α-cell membrane potential is depolarised (below the grey in Figure 10). Thus, the differential responsiveness of α and β cells to diazoxide does not result from differential sensitivities of the KATP channels in these cells, but to the downstream responses to titrated KATP channel activity. + +One important difference between mouse α and β cells is that whereas the latter rely exclusively on voltage-gated Ca2+ channels for the upstroke of the action potentials, glucagon-producing α cells are equipped with voltage-gated Na+ channels. These channels undergo voltage-dependent inactivation at voltages more positive than −50 mV [14]. This will reduce the action potential amplitude, and indeed it is reported that glucose reduces the peak voltage in α cells from +11 mV to −1 mV [13]. This will in itself result in an approximately 35% reduction of exocytosis, which is steeply dependent on voltage between −10 and +10 mV (Figure 9E). The functional significance of this is illustrated by the observations that glucagon secretion and α-cell Ca2+ responses at low-glucose concentrations are suppressed by the Na+ channel blocker TTX. The exact ion channel complement of human α cells remains to be established. However, the fact that glucagon secretion from human islets shows the same bell-shaped diazoxide concentration dependence as in mouse islets suggests that the depolarization-induced inactivation of ion channels involved in α-cell regenerative electrical activity underlies glucose-induced suppression of glucagon secretion in man as well. + +Although L-type VDCCs mediate the majority of Ca2+ current in α cells, this work and previous studies [33,44,45] demonstrate that it is the N-type (ω-conotoxin–sensitive) VDCCs that mediate the Ca2+ influx necessary for α-cell exocytosis and glucagon secretion under hypoglycaemic conditions. We now show that the α-cell N-type VDCCs are also subject to voltage-dependent inactivation at voltages more positive than −50 mV and furthermore that this is associated with reduced exocytotic capacity. It is pertinent that N-type VDCC-deficient mice exhibit reduced serum glucagon levels and improved glucose tolerance despite a parallel reduction in plasma insulin [44]. Although Ca2+ influx through L-type Ca2+ channels does not appear to contribute much to glucagon secretion under the experimental conditions used in this study, these channels become the predominant conduit of Ca2+ entry in the presence of agents increasing cAMP [41] (unpublished data). The mechanism underlying this switch in Ca2+ channel dependence remains obscure, but may depend on the strength of depolarisation because glucagon secretion stimulated by strong depolarisation with increased K+ in combination with KATP channel block can be prevented by inhibition of L-type Ca2+ channels [47]. + +We finally point out that the model we propose here for the control of glucagon secretion shares many features with what is known about the metabolic control of insulin secretion. This in turn means that processes that interfere with the ability of, for example, glucose to stimulate insulin secretion will have the opposite effect on glucagon secretion. This would provide a simple explanation for the fact that both insulin and glucagon secretion become perturbed in diabetes and why oversecretion of glucagon exacerbates the hyperglycaemic effects of insufficient insulin secretion. + +Materials and Methods + +Islet isolation and culture. + +Islets from female NMRI mice were isolated by collagenase digestion and cultured in RPMI-1640 media (5 mM glucose) at 37 °C and 5% CO2 for 2−24 h prior to secretion or intracellular Ca2+ assays. For single-cell studies, islets were dispersed in a Ca2+-free buffer and plated in 35-mm plastic dishes. Generation of the Kir6.2Y12X mice, which possess a Tyr12STOP mutation in the Kcnj11 gene on a BALB/c background, has been described previously [48]. This results in nonfunctional KATP channels in both α and β cells. These animals are not overtly diabetic, exhibiting normal fasting glucose, but are glucose intolerant (R. Cox, unpublished data). For these experiments, age- and weight-matched wild-type C3HB mice were used as controls because the Kir6.2Y12X mice were back-crossed into this background over several generations. Human islets from four healthy donors were obtained from the Oxford DRWF Human Islet Isolation Facility. Experiments were performed in duplicate or triplicate using islets from each donor. Human islets were cultured in RPMI-1640 (10 mM glucose) at 37 °C and 5% CO2, and experiments were performed within 48 h of isolation. + +Intracellular Ca2+ measurements. + +Intact islets were loaded with fluo-4-AM (1 μM) and fura red (5 μM) in 0.5 mM glucose buffer (see below) with 0.01% pluronic acid for 30 min at 37 °C. Islets were fixed with a wide-bore holding pipette within a continuously superfused and temperature-controlled (37 °C) bath on an Axioskop 2 FS-mot microscope (Carl Zeiss, http://www.zeiss.com). The perfusion buffer contained (in mM): 140 NaCl; 3.6 KCl; 2 NaHCO3; 0.5 NaH2PO4; 0.5 MgSO4; 5 HEPES; 2.5 CaCl2; 0.5, 3, or 11 glucose; (pH 7.4 with NaOH). Laser scanning confocal microscopy was performed using an LSM 510meta system (Zeiss). Excitation was with a 488-nm argon laser, and emitted fluorescence was collected through 500–550-nm and 650–710-nm band-pass filters for the fluo-4 and fura red (FuraR) signals, respectively. Increases in intracellular Ca2+ are displayed as upward deflections. Images were acquired at 1.5-s intervals. Individual cells were selected as regions of interest, and the average ratio intensity (RI = [Fluo+1]/[FuraR+20] × 128+1) of these were analysed over time with Origin v7.0220 (OriginLab Corporation, http://www.originlab.com). Prior to experimental recordings, islets were perfused for 10 min with the appropriate control solution, and Ca2+ responses were monitored periodically during this time to ensure that responses (or lack thereof) were stable prior to beginning the experimental recording. Ca2+ responses were determined by baseline subtraction and calculation of the integrated response (i.e., the area under the curve). The slope of this response, reported here as arbitrary units (AU), was calculated for the final 60%–90% of a given treatment period to allow for equilibration of the responses. + +Hormone release. + +Insulin and glucagon secretion were measured as described elsewhere [45]. Briefly, batches of ten freshly isolated islets were pre-incubated in 1 ml of Krebs–Ringer buffer (KRB) supplemented with 1 mM glucose for 30 min (rodent) or 1 h (human) followed by 1-h incubation in 1 ml of test KRB medium supplemented as indicated. For experiments in which Zn2+ was chelated or GABAA receptors were antagonised (Figure 1), the Ca2+-EDTA and SR-95531, respectively, were present in both the pre-incubation and test solutions. When extracellular KCl was increased, NaCl was correspondingly reduced to maintain iso-osmolarity. + +Single-cell capacitance and ion current measurements. + +Whole-cell currents and exocytosis were recorded using an EPC-9 patch-clamp amplifier (HEKA Electronics, http://www.heka.com) and Pulse software (version 8.50). Single α cells were identified by their small size and Na+ current inactivation properties [58]. This method was validated by combining the electrophysiological recordings with subsequent immunostaining for glucagon and insulin. The extracellular medium contained (in mM): 118 NaCl; 20 TEA-Cl (tetraethylammonium chloride); 5.6 KCl; 2.6 CaCl2; 1.2 MgCl2; 5 HEPES (pH 7.4 with NaOH); and 5 glucose. Except for Figure 8B and 8C, in which the standard whole-cell configuration was used, the electrophysiological measurements were conducted using the perforated patch technique, and the pipette solution contained (in mM): 76 Cs2SO4; 10 NaCl; 10 KCl; 1 MgCl2; and 5 HEPES (pH 7.35 with CsOH). Exocytosis was monitored as changes in α-cell capacitance using the software-based lock-in function of the Pulse software. The standard whole-cell measurements (Figure 8B and 8C) were conducted using a pipette solution (dialyzing the cell interior) consisted of (in mM) 125 CsOH; 125 glutamate; 10 CsCl; 10 NaCl; 1 MgCl2; 5 HEPES (pH 7.15 with KOH); 3 Mg-ATP; and 25 μmol/l EGTA (measured resting free Ca2+, ∼0.2 μmol/l). Pulses were applied at low frequency (<0.05 Hz) to allow the exocytotic capacity to recover fully between the pulses. + +Statistical analysis. + +Data are presented as means and standard errors. Significance was examined by either the unpaired t-test or by multiple-comparisons analysis of variance (ANOVA) and post-test, as appropriate. + +Acknowledgements + +PEM was supported initially the European Foundation for the Study of Diabetes (EFSD)/AstraZeneca Fellowship in Islet Biology and is currently an Alberta Heritage Foundation for Medical Research Scholar, Canadian Diabetes Association Scholar, and the Canada Research Chair in Islet Biology. PR is a Wolfson Royal Society Merit Award Research Fellow. + +Abbreviations + +[Ca2+]i - intracellular Ca2+ + +FuraR - fura red + +GABA - γ-aminobutyric acid + +KATP - ATP-dependent K+, TTX, tetrodotoxin + +VDCC - voltage-dependent Ca2+ channel + +Figures and Tables + +Figure 1 + +Glucose Suppresses Glucagon Release Independently of Paracrine Signals Mediated by Zn2+ or GABA. + +(A) Glucagon release from isolated mouse islets was suppressed by 60% at 7 mM glucose compared with 1 mM (filled bars). Glucose retained its suppressive effect on glucagon release under conditions of Zn2+ chelation (Ca2+-EDTA) (open bars) and antagonism of GABAA receptors (SR-95531) (shaded bars). Antagonism of GABAA receptors increased both basal and glucose-suppressed glucagon secretion, suggesting a paracrine role for GABA independent of the glucose effect. + +(B) As in (A), but using rat islets. + +*, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with 1 mM glucose, or as indicated. + +Figure 2 + +Glucagon Release from Isolated and Intact Mouse Islets Is Regulated by a KATP Channel-Dependent Pathway + +(A) Glucagon (filled circles) and insulin (open circles) secretion measured from mouse islets in the presence of 8.3 mM glucose at increasing concentrations of diazoxide. + +(B) As in (A), but using rat islets. The glucagon responses to 1 mM glucose (filled square) and 100 μM tolbutamide (filled triangle) are indicated. + +(C) As in (A), but in the presence of 1 mM glucose and only measuring glucagon secretion. + +(D) As in (A), but examining the effect of tolbutamide in the absence of glucose. Glucagon secretion in response to 20 mM glucose is indicated by the filled square. + +*, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with zero diazoxide (A–C) or zero tolbutamide (D). + +Figure 3 + +Tolbutamide and Glucose Effects Are Non-Additive and Glucagon Response Is Altered in Kir6.2Y12X Islets That Express a Truncated Kir6.2 Subunit + +(A) Glucagon release from isolated mouse islets at 1 (open bars) and 20 mM glucose (filled bars) under control conditions and presence of 0.5 mM tolbutamide. + +(B) A glucose dose-response of glucagon release from control C3HB islets (filled circles) and Kir6.2Y12X islets (open circles). + +(C) As in (B), but insulin was measured. + +*, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with control. + +Figure 4 + +The Intracellular Ca2+ Response of Single α Cells within Intact Islets Can Be Re-Activated by Low Concentrations of Diazoxide + +(A) Representative intracellular Ca2+ responses from α and β cells within an intact mouse islet exposed to 0.5 mM glucose, 11 mM glucose, and 11 mM glucose plus 2 μM diazoxide (diaz) as indicated above the traces. + +(B) The Ca2+ response of α cells was suppressed by 11 mM glucose, and could be reactivated with low concentrations of the KATP channel agonist diazoxide. ***, p < 0.001, compared with the low-glucose condition, or as indicated. + +Figure 5 + +Glucagon Release from Isolated and Intact Human Islets Is Regulated by a KATP Channel-Dependent Pathway + +(A) Glucagon (open bars) and insulin (filled bars) release was measured from isolated human islets under control conditions and following addition of 10 mM glucose or 200 μM tolbutamide. + +(B) Glucagon (open bars) and insulin secretion (filled bars) measured in the presence of 10 mM glucose and increasing concentrations of diazoxide (0–200 μM). + +*, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with controls, unless otherwise indicated. + +Figure 6 + +Intracellular Ca2+ Responses of α Cells within Intact Human Islets Are Regulated by a KATP Channel-Dependent Mechanism + +(A) Ca2+ responses measured in two human α cells within the same islet at 0.5 mM and 11 mM glucose (glu), in the presence of 2 μM diazoxide (diaz). + +(B) Summary of the Ca2+ responses at 0.5 mM glucose, at 11 mM glucose, in the presence of glucose (11 mM) and diazoxide (2 μM), and following the removal of diazoxide, but in the continued presence of 11 mM glucose. + +(C) The re-activation of human α-cell Ca2+ responses by 2 μM diazoxide was reversed upon application of the KATP channel antagonist tolbutamide (100 μM). + +(D) The effects of increasing concentrations of diazoxide on the Ca2+ response of a human α cell and β cell within the same islet exposed to 11 mM glucose. At the end of the experiment, diazoxide was withdrawn and glucose lowered to 0.5 mM. + +(E) Dose-response curve for the effect of diazoxide on α-cell Ca2+ responses. The grey horizontal line indicates the response with 11 mM glucose alone. + +*, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with controls, unless otherwise indicated. + +Figure 7 + +Glucagon Secretion and α-Cell Ca2+ Responses Are Dependent upon the Activity of Voltage-Dependent Na+ Channels + +(A) Glucagon (open bars) and insulin (filled bars) release from isolated mouse islets at 1 mM and 20 mM glucose, under control conditions and in the presence of the Na+ channel antagonist TTX (0.1 μg/ml). + +(B) Intracellular Ca2+ response of single α cells to 0.5 mM glucose. TTX (0.1 μg/ml) was included in the perfusion medium during the indicated period. + +(C) The effects of TTX and glucose on α-cell Ca2+ responses. Note that TTX inhibits Ca2+ responses as effectively as glucose and that the action is at least partially reversible. + +*, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with controls, unless otherwise indicated. + +Figure 8 + +Glucagon Secretion Is Regulated by N-Type Ca2+ Channels + +(A) Glucagon release measured at 1 (open bars) and 20 mM glucose (filled bars) under control conditions and in the presence of 100 nM ω-conotoxin (middle) or 20 μM nifedipine (right). + +(B) Exocytosis was elicited by 500-ms depolarisations from −70 to 0 mV under control conditions (Ctrl) and in the presence of either nifedipine (50 μM; nif) or ω-conotoxin (1 μM; ω-con). + +(C) Summary of the exocytotic response under control conditions and in the presence of nifedipine and ω-conotoxin. + +*, p < 0.05; **, p < 0.01, compared with 1 mM glucose and the control capacitance response, unless indicated otherwise. + +Figure 9 + +Voltage-Dependent Inactivation of α-Cell N-Type Ca2+ Currents and Exocytosis + +(A) N-type Ca2+ currents were evaluated during blockade of the L-type channels with isradipine (2 μM). The N-type channel antagonist ω-conotoxin (1 μM; red traces) reduced the Ca2+ current elicited by a step depolarisation from −70 to 0 mV (right). + +(B) As in (A), but the pulse to 0 mV was preceded by a 200-ms conditioning depolarization to +10 mV. ω-Conotoxin was without effect on the current measured during the depolarization to 0 mV under these conditions (right). + +(C) Top: peak Ca2+ currents measured in the presence of 2 μM isradipine alone (open squares) or together with 1 μM ω-conotoxin (red circles) during a depolarization to 0 mV following 200-ms conditioning pulses to between −70 and +70 mV. Lower: inactivation of the ω-conotoxin–sensitive component. Half-maximal inactivation of the N-type current was at −31 ± 6 mV (n = 5). + +(D) Exocytosis was elicited with 500-ms depolarisations from −70 mV to between −50 and 20 mV. + +(E) The voltage dependence of the exocytotic response. + +(F) Exocytosis elicited by 500-ms depolarisations to 0 mV from holding potentials of between −70 and −30 mV. + +(G) Summary of effects of holding potential on exocytotic responses elicited by depolarisations to 0 mV. Data have been normalized to responses obtained using a holding potential of −70 mV. + +*, p < 0.05; **, p < 0.01; ***, p < 0.001, compared with ω-conotoxin (C, top) or with the initial response. + +Figure 10 + +A Model for the Suppression of Glucagon Secretion by an Intrinsic α-Cell Pathway + +Schematic representation of the effects of glucose, tolbutamide, and diazoxide on α-cell KATP, Na+, and N-type Ca2+ (VDCC) channel activities and glucagon secretion is shown. The insulin response is also shown for comparison with our experimental results (dashed lines, lower panels). The grey gradient represents a “window” of α-cell KATP channel activity that supports the activation of Ca2+ and Na+ channels. Above this window, the cell is hyperpolarized and Ca2+ and Na+ channel activation is prevented, whereas KATP channel activity below this window depolarizes the cell and causes voltage-dependent inactivation of Ca2+ and Na+ channels. + +(A) High-glucose concentration reduces α-cell KATP channel activity, reducing glucagon secretion. + +(B) Graded application of tolbutamide (in zero glucose) transiently increases glucagon secretion as KATP channel activity is reduced through, and eventually below, the window supporting glucagon release. + +(C) The graded application of diazoxide in high-glucose conditions increases α-cell KATP channel activity into, and then above the window supporting glucagon secretion. The result is a transient “re-activation” of glucagon secretion at low-diazoxide concentrations. + +(D) In low-glucose (1–2 mM) conditions, graded application of diazoxide increases KATP channel activity above the window supporting glucagon secretion, causing a monotonic inhibition of glucagon release. + +Footnotes + +Competing interests. The authors have declared that no competing interests exist. + +Author contributions. PEM, LE, and PR conceived and designed the experiments. PEM, YZDM, RR, AS, XM, and LE performed the experiments. PEM, YZDM, RR, AS, LE, and PR analyzed the data. PRVJ and RC contributed reagents/materials/analysis tools. PEM and PR wrote the paper. + +Funding. Work in the UK was supported by grants to PR from the Wellcome Trust and the European Union (Eurodia LSHM-CT-2006–518153 and BioSim LSHB-CT-2004–005137). Work conducted in Sweden was supported by the Swedish Strategic Foundation, the Göran Gustafsson Stiftelse, the Swedish Research Council, the Swedish Diabetes Association, and the Novo Nordisk Foundation (LE). diff --git a/src/ontogpt/evaluation/craft/database/all/17565376.ann b/src/ontogpt/evaluation/craft/database/all/17565376.ann new file mode 100644 index 000000000..306794043 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17565376.ann @@ -0,0 +1,1128 @@ +T1 PR:000011306 0 6 CARD15 +T2 PR:000011306 7 11 NOD2 +T3 UBERON:0001211 28 43 Peyer's Patches +T4 GO:0042592 44 55 Homeostasis +T5 NCBITaxon:10088 59 63 Mice +T6 PR:000011306 87 93 CARD15 +T7 PR:000011306 94 98 NOD2 +T8 http://purl.obolibrary.org/obo/MONDO_0005011 147 162 Crohn's Disease +T9 http://purl.obolibrary.org/obo/MONDO_0005011 164 166 CD +T10 http://purl.obolibrary.org/obo/MONDO_0013730 172 197 Graft Versus Host Disease +T11 GO:0018995 185 189 Host +T12 http://purl.obolibrary.org/obo/MONDO_0013730 199 203 GVHD +T13 http://purl.obolibrary.org/obo/MONDO_0005011 206 208 CD +T14 http://purl.obolibrary.org/obo/MONDO_0013730 213 217 GVHD +T15 UBERON:0001211 270 285 Peyer's patches +T16 UBERON:0001211 287 289 PP +T17 UBERON:0000444 304 322 lymphoid follicles +T18 UBERON:0000444 324 327 LFs +T19 NCBITaxon:10088 342 347 mouse +T20 PR:000011306 370 376 Card15 +T21 PR:000011306 377 381 Nod2 +T22 SO:0000704 423 427 gene +T23 http://purl.obolibrary.org/obo/MONDO_0005292 503 510 colitis +T24 UBERON:0001211 583 586 PPs +T25 UBERON:0000444 591 594 LFs +T26 NCBITaxon:10088 613 617 mice +T27 GO:0007567 654 659 birth +T28 UBERON:0001211 717 720 PPs +T29 UBERON:0001211 779 782 PPs +T30 NCBITaxon:10088 789 793 mice +T31 CL:0000682 838 845 M cells +T32 PR:000001004 850 853 CD4 +T33 CL:0000084 855 862 T-cells +T34 NCBITaxon:10088 867 871 mice +T35 PR:000000134 924 928 TNFα +T36 PR:000000017 930 934 IFNγ +T37 GO:0043514 936 940 IL12 +T38 PR:000001391 945 948 IL4 +T39 UBERON:0001211 1018 1020 PP +T40 UBERON:0002116 1026 1031 ileum +T41 UBERON:0002106 1040 1046 spleen +T42 NCBITaxon:10088 1053 1057 mice +T43 UBERON:0001211 1109 1111 PP +T44 NCBITaxon:2 1198 1207 bacterial +T45 NCBITaxon:10088 1235 1239 mice +T46 http://purl.obolibrary.org/obo/MONDO_0005292 1269 1276 colitis +T47 PR:000011306 1308 1314 Card15 +T48 PR:000011306 1315 1319 Nod2 +T49 GO:0048541 1351 1362;1376 1378;1383 1386 development ... of ... PPs +T50 UBERON:0001211 1383 1386 PPs +T51 UBERON:0002405 1419 1425 immune +T52 GO:0006955 1419 1434 immune response +T53 NCBITaxon:9606 1551 1556 Human +T54 PR:000011306 1557 1563 CARD15 +T55 PR:000011306 1564 1568 NOD2 +T56 http://purl.obolibrary.org/obo/MONDO_0000001 1580 1589 disorders +T57 http://purl.obolibrary.org/obo/MONDO_0005011 1591 1593 CD +T58 http://purl.obolibrary.org/obo/MONDO_0013730 1598 1602 GVHD +T59 PR:000011306 1619 1648 Caspase Recruitment Domain 15 +T60 SO:0000417 1639 1645 Domain +T61 PR:000011306 1650 1656 CARD15 +T62 PR:000011306 1672 1707 Nucleotide oligomerisation domain 2 +T63 GO:0051259 1683 1698 oligomerisation +T64 SO:0000417 1699 1705 domain +T65 PR:000011306 1709 1713 NOD2 +T66 http://purl.obolibrary.org/obo/MONDO_0005011 1740 1755 Crohn's Disease +T67 http://purl.obolibrary.org/obo/MONDO_0005011 1757 1759 CD +T68 http://purl.obolibrary.org/obo/MONDO_0013730 1774 1799 graft versus host disease +T69 GO:0018995 1787 1791 host +T70 http://purl.obolibrary.org/obo/MONDO_0013730 1801 1805 GVHD +T71 PR:000011306 1817 1821 NOD2 +T72 SO:0000704 1845 1850 genes +T73 NCBITaxon:2 1948 1957 bacterial +T74 GO:0005618 1958 1967 cell wall +T75 PR:000011306 1985 1989 NOD2 +T76 PR:000014023 2005 2009 Rick +T77 PR:000014023 2010 2014 Rip2 +T78 GO:0071159 2043 2048 NF-kB +T79 http://purl.obolibrary.org/obo/MONDO_0005011 2126 2128 CD +T80 PR:000011306 2155 2159 NOD2 +T81 http://purl.obolibrary.org/obo/MONDO_0005011 2187 2189 CD +T82 http://purl.obolibrary.org/obo/MONDO_0005011 2272 2274 CD +T83 UBERON:0002405 2303 2309 immune +T84 NCBITaxon:2 2328 2336 bacteria +T85 UBERON:0006909 2352 2361 gut lumen +T86 PR:000011306 2405 2409 NOD2 +T87 http://purl.obolibrary.org/obo/MONDO_0005011 2439 2441 CD +T88 http://purl.obolibrary.org/obo/MONDO_0005011 2558 2560 CD +T89 http://purl.obolibrary.org/obo/MONDO_0020546 2619 2629 acute GVHD +T90 UBERON:0002371 2634 2645 bone marrow +T91 UBERON:0000483 2774 2784 epithelial +T92 CL:0000066 2774 2784;2801 2806 epithelial ... cells +T93 CL:0000080 2789 2806 circulating cells +T94 http://purl.obolibrary.org/obo/MONDO_0000001 2810 2817 disease +T95 PR:000011306 2933 2937 NOD2 +T96 http://purl.obolibrary.org/obo/MONDO_0005011 2972 2974 CD +T97 http://purl.obolibrary.org/obo/MONDO_0005265 2998 3024 inflammatory bowel disease +T98 UBERON:0000160 3011 3016 bowel +T99 http://purl.obolibrary.org/obo/MONDO_0005265 3026 3029 IBD +T100 UBERON:0000344 3036 3043 mucosal +T101 http://purl.obolibrary.org/obo/MONDO_0043839 3044 3055 ulcerations +T102 GO:0007586 3063 3072 digestive +T103 UBERON:0001555 3063 3078 digestive tract +T104 http://purl.obolibrary.org/obo/MONDO_0005011 3080 3082 CD +T105 CL:0000545 3114 3122;3128 3129 T helper ... 1 +T106 GO:0042088 3114 3122;3128 3145 T helper ... 1 immune response +T107 GO:0042088 3124 3126;3128 3145 Th ... 1 immune response +T108 UBERON:0002405 3130 3136 immune +T109 UBERON:0001962 3204 3234 gut associated lymphoid tissue +T110 UBERON:0001962 3236 3240 GALT +T111 UBERON:0000444 3252 3270 lymphoid follicles +T112 UBERON:0000444 3272 3275 LFs +T113 UBERON:0000444 3278 3281 LFs +T114 UBERON:0001155 3312 3317 colon +T115 UBERON:0002108 3349 3360 small bowel +T116 UBERON:0001211 3392 3407 Peyer's patches +T117 UBERON:0001211 3409 3411 PP +T118 GO:0018995 3457 3461 host +T119 UBERON:0002405 3462 3468 immune +T120 GO:0006955 3462 3477 immune response +T121 NCBITaxon:2 3514 3522 bacteria +T122 http://purl.obolibrary.org/obo/MONDO_0005011 3524 3526 CD +T123 UBERON:0001155 3563 3568 colon +T124 UBERON:0002116 3580 3585 ileum +T125 UBERON:0000444 3597 3600 LFs +T126 http://purl.obolibrary.org/obo/MONDO_0004845 3656 3675 aphtoïd ulcerations +T127 http://purl.obolibrary.org/obo/MONDO_0005011 3720 3722 CD +T128 UBERON:0000444 3747 3750 LFs +T129 http://purl.obolibrary.org/obo/MONDO_0005011 3823 3825 CD +T130 UBERON:0001211 3830 3832 PP +T131 GO:0048541 3830 3844 PP development +T132 UBERON:0001211 3875 3878 PPs +T133 GO:0007567 3892 3897 birth +T134 UBERON:0000104 3916 3920 life +T135 http://purl.obolibrary.org/obo/MONDO_0005011 3987 3989 CD +T136 UBERON:0001211 4027 4029 PP +T137 UBERON:0002116 4071 4076 ileal +T138 UBERON:0001211 4134 4136 PP +T139 UBERON:0001962 4171 4175 GALT +T140 http://purl.obolibrary.org/obo/MONDO_0013730 4179 4183 GVHD +T141 NCBITaxon:33208 4258 4264 Animal +T142 GO:0016265 4285 4290 death +T143 UBERON:0001555 4318 4321 gut +T144 UBERON:0000344 4361 4368 mucosal +T145 http://purl.obolibrary.org/obo/MONDO_0013730 4397 4401 GVHD +T146 UBERON:0001555 4430 4433 gut +T147 http://purl.obolibrary.org/obo/MONDO_0013730 4488 4492 GVHD +T148 UBERON:0001211 4537 4539 PP +T149 NCBITaxon:10088 4550 4554 mice +T150 http://purl.obolibrary.org/obo/MONDO_0013730 4572 4576 GVHD +T151 UBERON:0001211 4608 4611 PPs +T152 http://purl.obolibrary.org/obo/MONDO_0013730 4615 4619 GVHD +T153 GO:0018995 4676 4680 host +T154 PR:000011306 4804 4808 Nod2 +T155 UBERON:0001962 4862 4866 GALT +T156 NCBITaxon:10088 4896 4901 mouse +T157 PR:000011306 4922 4928 Card15 +T158 PR:000011306 4929 4933 Nod2 +T159 PR:000011306 4968 4972 Nod2 +T160 UBERON:0001211 4991 4994 PPs +T161 PR:000011306 5023 5027 Nod2 +T162 GO:0010467 5071 5081 expression +T163 UBERON:0001211 5085 5088 PPs +T164 PR:000011306 5120 5124 Nod2 +T165 NCBITaxon:2 5165 5174 bacterial +T166 UBERON:0001211 5192 5195 PPs +T167 UBERON:0007023 5199 5204 adult +T168 NCBITaxon:10088 5205 5209 mice +T169 PR:000011306 5240 5246 Card15 +T170 PR:000011306 5247 5251 Nod2 +T171 UBERON:0001155 5275 5282 colonic +T172 CHEBI:53063 5295 5331 2,4,6-trinitrobenzene sulphonic acid +T173 http://purl.obolibrary.org/obo/MONDO_0005292 5372 5379 colitis +T174 NCBITaxon:10088 5383 5388 mouse +T175 UBERON:0000160 5413 5423 intestinal +T176 UBERON:0000160 5435 5445 intestinal +T177 NCBITaxon:10088 5479 5483 mice +T178 NCBITaxon:10088 5578 5582 mice +T179 NCBITaxon:10088 5644 5648 mice +T180 UBERON:0001211 5682 5684 PP +T181 NCBITaxon:10088 5707 5711 mice +T182 GO:0007567 5740 5745 birth +T183 UBERON:0000444 5800 5803 LFs +T184 UBERON:0002108 5808 5823 small intestine +T185 NCBITaxon:10088 5846 5850 mice +T186 NCBITaxon:10088 5862 5866 mice +T187 UBERON:0001211 5905 5908 PPs +T188 GO:0007567 5912 5917 birth +T189 NCBITaxon:10088 5948 5952 mice +T190 UBERON:0001211 6024 6026 PP +T191 UBERON:0007023 6063 6068 adult +T192 NCBITaxon:10088 6072 6076 mice +T193 UBERON:0001211 6142 6145 PPs +T194 NCBITaxon:10088 6154 6159 mouse +T195 NCBITaxon:10088 6228 6232 mice +T196 UBERON:0001211 6268 6270 PP +T197 UBERON:0002106 6326 6333 spleens +T198 NCBITaxon:10088 6347 6351 mice +T199 UBERON:0001211 6388 6390 PP +T200 NCBITaxon:10088 6410 6414 mice +T201 PR:000011306 6470 6474 NOD2 +T202 GO:0065007 6475 6484 modulates +T203 GO:0071159 6489 6498 NF-κappaB +T204 GO:0006915 6526 6535 apoptosis +T205 CL:0000445 6567 6582 apoptotic cells +T206 UBERON:0001211 6590 6593 PPs +T207 NCBITaxon:10088 6671 6675 mice +T208 PR:000002312 6694 6703 caspase 3 +T209 UBERON:0001211 6835 6838 PPs +T210 GO:0042571 6910 6920 antibodies +T211 PR:000001014 6997 7001 B220 +T212 CL:0000236 7003 7010 B-cells +T213 PR:000001018 7012 7015 CD3 +T214 CL:0000084 7017 7024 T-cells +T215 PR:000001013 7029 7034 CD11c +T216 GO:0097511 7036 7045 dendritic +T217 CL:0000451 7036 7051 dendritic cells +T218 NCBITaxon:10088 7070 7074 mice +T219 UBERON:0001211 7086 7089 PPs +T220 UBERON:0002106 7093 7100 spleens +T221 PR:000002978 7241 7246 Ly-6G +T222 CL:0000096 7248 7277 polymorphonuclear neutrophils +T223 CL:0000096 7279 7282 PMN +T224 UBERON:0001211 7299 7302 PPs +T225 CL:0000682 7331 7338 M cells +T226 UBERON:0000483 7378 7388 epithelium +T227 CL:0000096 7423 7426 PMN +T228 UBERON:0001211 7463 7466 PPs +T229 NCBITaxon:10088 7480 7484 mice +T230 CL:0000682 7513 7519 M cell +T231 NCBITaxon:10088 7558 7562 mice +T232 NCBITaxon:10088 7585 7589 mice +T233 CL:0000084 7605 7612 T-cells +T234 http://purl.obolibrary.org/obo/MONDO_0005011 7649 7651 CD +T235 PR:000001018 7677 7680 CD3 +T236 CL:0000084 7682 7689 T cells +T237 NCBITaxon:10088 7706 7710 mice +T238 PR:000001004 7736 7739 CD4 +T239 CL:0000084 7741 7747 T-cell +T240 UBERON:0001211 7781 7784 PPs +T241 PR:000025402 7812 7815 CD8 +T242 CL:0000084 7817 7824 T-cells +T243 UBERON:0001211 7873 7876 PPs +T244 NCBITaxon:10088 7885 7889 mice +T245 PR:000001018 7920 7923 CD3 +T246 PR:000001004 7924 7927 CD4 +T247 PR:000025402 7928 7931 CD8 +T248 CL:0000084 7933 7940 T-cells +T249 UBERON:0002106 8008 8015 spleens +T250 PR:000001004 8124 8127 CD4 +T251 CL:0000084 8129 8136 T-cells +T252 UBERON:0001211 8152 8155 PPs +T253 UBERON:0002106 8160 8167 spleens +T254 CL:0000898 8209 8214;8282 8289 naive ... T-cells +T255 PR:000001380 8215 8219 CD25 +T256 PR:000001016 8220 8226 CD45Rb +T257 GO:0065007 8229 8239 regulatory +T258 CL:0000815 8229 8239;8282 8289 regulatory ... T-cells +T259 PR:000001380 8240 8244 CD25 +T260 PR:000001016 8245 8251 CD45Rb +T261 GO:0007613 8257 8263 memory +T262 CL:0000813 8257 8263;8282 8289 memory ... T-cells +T263 PR:000001380 8264 8268 CD25 +T264 PR:000001016 8269 8275 CD45Rb +T265 PR:000001004 8277 8280 CD4 +T266 NCBITaxon:10088 8301 8305 mice +T267 CL:0000898 8343 8348;8377 8384 naive ... T-cells +T268 GO:0065007 8350 8360 regulatory +T269 CL:0000815 8350 8360;8377 8384 regulatory ... T-cells +T270 GO:0007613 8365 8371 memory +T271 CL:0000813 8365 8371;8377 8384 memory ... T-cells +T272 PR:000001004 8372 8375 CD4 +T273 UBERON:0001211 8388 8391 PPs +T274 UBERON:0002106 8410 8416 spleen +T275 NCBITaxon:10088 8509 8513 mice +T276 PR:000004078 8537 8546 annexin V +T277 PR:000001018 8556 8559 CD3 +T278 PR:000001018 8565 8568 CD3 +T279 PR:000001004 8569 8572 CD4 +T280 CL:0000084 8574 8581 T-cells +T281 UBERON:0001211 8597 8600 PPs +T282 PR:000001004 8645 8648 CD4 +T283 CL:0000084 8650 8657 T-cells +T284 NCBITaxon:10088 8673 8677 mice +T285 GO:0006915 8711 8720 apoptosis +T286 UBERON:0001211 8754 8756 PP +T287 UBERON:0001211 8824 8827 PPs +T288 NCBITaxon:10088 8843 8847 mice +T289 GO:0010467 8918 8928 expression +T290 PR:000001136 8932 8937 IL-1β +T291 PR:000000017 8939 8943 IFNγ +T292 PR:000000134 8945 8949 TNFα +T293 GO:0043514 8952 8957 IL-12 +T294 PR:000001391 8963 8967 IL-4 +T295 UBERON:0001211 8971 8974 PPs +T296 UBERON:0001211 8976 8978 PP +T297 UBERON:0002116 8984 8989 ileum +T298 UBERON:0002106 8994 9000 spleen +T299 PR:000000017 9056 9060 IFNγ +T300 PR:000000134 9062 9066 TNFα +T301 GO:0043514 9068 9073 IL-12 +T302 PR:000001391 9079 9083 IL-4 +T303 UBERON:0001211 9116 9119 PPs +T304 NCBITaxon:10088 9126 9130 mice +T305 NCBITaxon:10088 9205 9209 mice +T306 UBERON:0001211 9213 9215 PP +T307 UBERON:0002116 9221 9226 ileum +T308 PR:000000134 9233 9237 TNFα +T309 PR:000000017 9242 9246 IFNγ +T310 GO:0010467 9286 9295 expressed +T311 UBERON:0007023 9317 9322 adult +T312 NCBITaxon:10088 9323 9327 mice +T313 UBERON:0002106 9378 9385 spleens +T314 NCBITaxon:10088 9401 9405 mice +T315 UBERON:0001211 9484 9487 PPs +T316 UBERON:0001211 9572 9574 PP +T317 UBERON:0001211 9579 9581 PP +T318 UBERON:0002116 9587 9592 ileum +T319 UBERON:0001211 9594 9597 PPs +T320 UBERON:0001211 9602 9604 PP +T321 UBERON:0002116 9610 9615 ileum +T322 NCBITaxon:10088 9622 9626 mice +T323 GO:0010467 9799 9809 expression +T324 UBERON:0001211 9849 9851 PP +T325 NCBITaxon:10088 9858 9862 mice +T326 GO:0010467 9872 9882 expression +T327 PR:000016365 9901 9905 ZO-2 +T328 PR:000016364 9910 9914 ZO-1 +T329 PR:000002207 10002 10010 Occludin +T330 GO:0010467 10011 10021 expression +T331 PR:000011306 10056 10060 Nod2 +T332 PR:000011306 10113 10119 Card15 +T333 PR:000011306 10120 10124 Nod2 +T334 UBERON:0001555 10151 10154 gut +T335 NCBITaxon:2 10198 10206 bacteria +T336 NCBITaxon:543 10221 10239 Enterobacteriaceae +T337 NCBITaxon:286 10241 10252 Pseudomonas +T338 NCBITaxon:1279 10254 10268 Staphylococcus +T339 NCBITaxon:1301 10270 10283 Streptococcus +T340 NCBITaxon:1350 10285 10297 Enterococcus +T341 NCBITaxon:1578 10301 10314 Lactobacillus +T342 UBERON:0002116 10322 10327 ileum +T343 NCBITaxon:10088 10341 10345 mice +T344 NCBITaxon:2 10485 10494 bacterial +T345 UBERON:0001211 10511 10513 PP +T346 NCBITaxon:562 10550 10566 Escherichia coli +T347 NCBITaxon:83333 10568 10572 K-12 +T348 UBERON:0001211 10588 10590 PP +T349 NCBITaxon:10088 10597 10601 mice +T350 NCBITaxon:562 10697 10713 Escherichia coli +T351 NCBITaxon:562 10766 10782 Escherichia coli +T352 UBERON:0001211 10795 10797 PP +T353 UBERON:0000331 10798 10810 ileum mucosa +T354 UBERON:0001211 10901 10904 PPs +T355 NCBITaxon:1280 10928 10949 Staphylococcus Aureus +T356 NCBITaxon:4932 10985 11009 Saccharomyces cerevisiae +T357 NCBITaxon:10088 11043 11047 mice +T358 http://purl.obolibrary.org/obo/MONDO_0005292 11072 11079 colitis +T359 http://purl.obolibrary.org/obo/MONDO_0005292 11113 11120 colitis +T360 NCBITaxon:10088 11124 11128 mice +T361 PR:000011306 11200 11206 Card15 +T362 PR:000011306 11207 11211 Nod2 +T363 UBERON:0001155 11262 11269 colonic +T364 NCBITaxon:10088 11341 11345 mice +T365 UBERON:0000317 11422 11437 colonic mucosal +T366 NCBITaxon:10088 11468 11472 mice +T367 UBERON:0000344 11494 11501 mucosal +T368 PR:000001136 11520 11525 IL-1β +T369 PR:000000134 11527 11532 TNF-α +T370 GO:0043514 11537 11542 IL-12 +T371 PR:000011306 11613 11619 CARD15 +T372 PR:000011306 11620 11624 NOD2 +T373 http://purl.obolibrary.org/obo/MONDO_0005011 11644 11646 CD +T374 http://purl.obolibrary.org/obo/MONDO_0013730 11660 11663 GVH +T375 PR:000011306 11710 11716 CARD15 +T376 PR:000011306 11717 11721 NOD2 +T377 http://purl.obolibrary.org/obo/MONDO_0005011 11734 11736 CD +T378 http://purl.obolibrary.org/obo/MONDO_0013730 11741 11745 GVHD +T379 UBERON:0001962 11825 11829 GALT +T380 http://purl.obolibrary.org/obo/MONDO_0013730 11847 11851 GVHD +T381 http://purl.obolibrary.org/obo/MONDO_0005011 11899 11901 CD +T382 UBERON:0001211 11914 11916 PP +T383 PR:000011306 11980 11986 Card15 +T384 PR:000011306 11987 11991 Nod2 +T385 NCBITaxon:10088 12002 12007 mouse +T386 PR:000011306 12034 12040 CARD15 +T387 PR:000011306 12041 12045 NOD2 +T388 UBERON:0001211 12049 12051 PP +T389 GO:0048541 12049 12063 PP development +T390 UBERON:0007023 12095 12100 adult +T391 NCBITaxon:10088 12104 12108 mice +T392 UBERON:0001211 12130 12132 PP +T393 UBERON:0000444 12146 12149 LFs +T394 UBERON:0001555 12157 12160 gut +T395 UBERON:0001211 12162 12164 PP +T396 NCBITaxon:10088 12173 12177 mice +T397 CL:0000682 12212 12219 M cells +T398 PR:000001004 12224 12227 CD4 +T399 CL:0000084 12229 12236 T-cells +T400 GO:0010467 12250 12260 expression +T401 CL:0000545 12264 12267 Th1 +T402 CL:0000546 12272 12275 Th2 +T403 NCBITaxon:2 12365 12374 bacterial +T404 UBERON:0001211 12401 12404 PPs +T405 PR:000011306 12432 12438 Card15 +T406 PR:000011306 12439 12443 Nod2 +T407 NCBITaxon:10088 12454 12458 mice +T408 http://purl.obolibrary.org/obo/MONDO_0005292 12496 12503 colitis +T409 PR:000011306 12552 12558 Card15 +T410 PR:000011306 12559 12563 Nod2 +T411 NCBITaxon:10088 12567 12572 mouse +T412 NCBITaxon:9606 12620 12625 Human +T413 http://purl.obolibrary.org/obo/MONDO_0000001 12626 12634 diseases +T414 PR:000011306 12651 12657 CARD15 +T415 PR:000011306 12658 12662 NOD2 +T416 http://purl.obolibrary.org/obo/MONDO_0005011 12674 12676 CD +T417 http://purl.obolibrary.org/obo/MONDO_0013730 12681 12685 GVHD +T418 PR:000011306 12720 12726 Card15 +T419 PR:000011306 12727 12731 Nod2 +T420 NCBITaxon:10088 12742 12746 mice +T421 UBERON:0001211 12774 12777 PPs +T422 UBERON:0000444 12791 12794 LFs +T423 GO:0007567 12801 12806 birth +T424 UBERON:0000160 12811 12821 intestinal +T425 NCBITaxon:10088 12870 12874 mice +T426 UBERON:0000160 12925 12935 intestinal +T427 UBERON:0001211 12961 12964 PPs +T428 NCBITaxon:10088 12980 12984 mice +T429 UBERON:0000160 13048 13058 intestines +T430 UBERON:0001211 13079 13081 PP +T431 PR:000011306 13102 13108 Card15 +T432 PR:000011306 13109 13113 Nod2 +T433 GO:0065007 13114 13123 modulates +T434 UBERON:0001962 13147 13151 GALT +T435 PR:000011306 13156 13162 Card15 +T436 PR:000011306 13163 13167 Nod2 +T437 UBERON:0001744 13220 13235 lymphoïd tissue +T438 UBERON:0001555 13251 13254 gut +T439 GO:0009888 13266 13280;13294 13300 development of ... tissue +T440 UBERON:0001744 13285 13300 lymphoïd tissue +T441 UBERON:0001555 13329 13337 GI tract +T442 UBERON:0005057 13392 13405 immune organe +T443 UBERON:0002106 13411 13417 spleen +T444 UBERON:0001211 13445 13447 PP +T445 NCBITaxon:10088 13473 13477 mice +T446 GO:0007567 13481 13486 birth +T447 PR:000011306 13502 13508 Card15 +T448 PR:000011306 13509 13513 Nod2 +T449 GO:0007567 13541 13546 natal +T450 UBERON:0001962 13566 13570 GALT +T451 PR:000001317 13594 13598 IL-7 +T452 GO:0038111 13594 13609 IL-7 signalling +T453 GO:0048541 13632 13651 organogenesis of PP +T454 GO:0048568 13632 13645;13652 13658;13663 13678 organogenesis ... during ... embryonic stage +T455 UBERON:0001211 13649 13651 PP +T456 UBERON:0000922 13663 13672 embryonic +T457 UBERON:0001555 13717 13720 gut +T458 NCBITaxon:2 13731 13739 bacteria +T459 GO:0007567 13765 13770 natal +T460 GO:0002520 13771 13785;13798 13811 development of ... immune system +T461 UBERON:0004786 13786 13797 gut mucosal +T462 UBERON:0002405 13798 13811 immune system +T463 NCBITaxon:33208 13853 13860 animals +T464 NCBITaxon:33208 13872 13879 animals +T465 UBERON:0001962 13903 13907 GALT +T466 http://purl.obolibrary.org/obo/MONDO_0005292 13942 13949 colitis +T467 http://purl.obolibrary.org/obo/MONDO_0013730 13964 13968 GVHD +T468 PR:000001096 14025 14029 TLRs +T469 CHEBI:36357 14038 14047 molecules +T470 UBERON:0001962 14138 14142 GALT +T471 PR:000001096 14153 14157 TLRs +T472 UBERON:0001211 14186 14188 PP +T473 GO:0048541 14186 14200 PP development +T474 GO:0007567 14214 14219 natal +T475 NCBITaxon:10088 14229 14233 mice +T476 PR:000011306 14279 14285 Card15 +T477 PR:000011306 14286 14290 Nod2 +T478 GO:0007567 14323 14328 natal +T479 UBERON:0001962 14344 14348 GALT +T480 UBERON:0001555 14359 14362 gut +T481 UBERON:0001211 14385 14387 PP +T482 GO:0048541 14385 14399 PP development +T483 PR:000011306 14425 14431 Card15 +T484 PR:000011306 14432 14436 Nod2 +T485 UBERON:0001555 14456 14459 gut +T486 GO:0018995 14485 14489 host +T487 NCBITaxon:2 14540 14548 bacteria +T488 UBERON:0002116 14573 14578 ileum +T489 NCBITaxon:10088 14680 14684 mice +T490 PR:000011306 14701 14707 Card15 +T491 PR:000011306 14708 14712 Nod2 +T492 UBERON:0001555 14767 14770 gut +T493 NCBITaxon:33208 14881 14888 animals +T494 NCBITaxon:2 14918 14927 bacterial +T495 PR:000011306 14995 15001 Card15 +T496 PR:000011306 15002 15006 Nod2 +T497 UBERON:0001555 15022 15025 gut +T498 UBERON:0001211 15034 15037 PPs +T499 CL:0000236 15043 15049 B-cell +T500 UBERON:0010754 15064 15080 germinal centres +T501 CL:0000084 15128 15135 T cells +T502 UBERON:0001211 15157 15159 PP +T503 PR:000011306 15165 15171 Card15 +T504 PR:000011306 15172 15176 Nod2 +T505 NCBITaxon:10088 15187 15191 mice +T506 GO:0006915 15259 15268 apoptosis +T507 UBERON:0001211 15343 15345 PP +T508 CL:0000236 15354 15361 B-cells +T509 CL:0000084 15363 15370 T-cells +T510 GO:0097511 15375 15384 dendritic +T511 CL:0000451 15375 15390 dendritic cells +T512 PR:000011306 15467 15473 Card15 +T513 PR:000011306 15474 15478 Nod2 +T514 NCBITaxon:10088 15492 15496 mice +T515 UBERON:0000160 15526 15536 intestinal +T516 NCBITaxon:10088 15640 15645 mouse +T517 NCBITaxon:9606 15670 15675 Human +T518 http://purl.obolibrary.org/obo/MONDO_0005011 15676 15678 CD +T519 UBERON:0000444 15798 15801 LFs +T520 UBERON:0000483 15831 15841 epithelium +T521 CL:0000682 15852 15859 M cells +T522 NCBITaxon:2 15894 15903 bacterial +T523 CHEBI:33290 15908 15912 food +T524 CHEBI:59132 15913 15921 antigens +T525 UBERON:0006909 15931 15940 gut lumen +T526 CHEBI:59132 15944 15951 antigen +T527 GO:0019882 15944 15962 antigen presenting +T528 CL:0000145 15944 15968 antigen presenting cells +T529 UBERON:0001211 16024 16026 PP +T530 CL:0000682 16028 16035 M cells +T531 NCBITaxon:10088 16067 16071 mice +T532 UBERON:0001211 16086 16088 PP +T533 NCBITaxon:10088 16095 16099 mice +T534 PR:000001004 16133 16136 CD4 +T535 CL:0000084 16138 16145 T-cells +T536 PR:000001018 16176 16179 CD3 +T537 PR:000025402 16180 16183 CD8 +T538 PR:000001004 16184 16187 CD4 +T539 CL:0000084 16189 16196 T-cells +T540 PR:000011306 16201 16205 Nod2 +T541 GO:0065007 16210 16218 modulate +T542 GO:0006915 16223 16232 apoptosis +T543 PR:000001004 16277 16280 CD4 +T544 CL:0000084 16282 16288 T-cell +T545 GO:0006915 16315 16324 apoptosis +T546 NCBITaxon:10088 16360 16364 mice +T547 CL:0000445 16440 16449;16457 16462 apoptotic ... cells +T548 PR:000001004 16450 16453 CD4 +T549 CL:0000084 16455 16462 T-cells +T550 UBERON:0001211 16468 16470 PP +T551 NCBITaxon:10088 16501 16505 mice +T552 PR:000011306 16568 16574 Card15 +T553 PR:000011306 16575 16579 Nod2 +T554 GO:0006915 16621 16630 apoptosis +T555 UBERON:0001744 16645 16660 lymphoid tissue +T556 NCBITaxon:10088 16667 16671 mice +T557 UBERON:0002405 16694 16700 immune +T558 CL:0000738 16694 16705 immune cell +T559 CL:0000545 16770 16773 Th1 +T560 CL:0000546 16801 16804 Th2 +T561 GO:0010467 16814 16824 expression +T562 UBERON:0001211 16889 16892 PPs +T563 NCBITaxon:10088 16899 16903 mice +T564 UBERON:0002405 16940 16946 immune +T565 GO:0006955 16940 16955 immune response +T566 UBERON:0001211 17026 17028 PP +T567 UBERON:0001211 17113 17115 PP +T568 UBERON:0001211 17120 17122 PP +T569 UBERON:0002116 17128 17133 ileum +T570 NCBITaxon:10088 17138 17142 mice +T571 UBERON:0000160 17252 17262 intestinal +T572 http://purl.obolibrary.org/obo/MONDO_0005011 17288 17290 CD +T573 PR:000011306 17351 17357 CARD15 +T574 PR:000011306 17358 17362 NOD2 +T575 GO:0065007 17434 17442 modulate +T576 UBERON:0000160 17443 17453 intestinal +T577 PR:000000017 17481 17485 IFNγ +T578 PR:000000134 17487 17491 TNFα +T579 PR:000001391 17496 17500 IL-4 +T580 GO:0016020 17509 17517 membrane +T581 UBERON:0000483 17531 17541 epithelial +T582 CL:0000066 17531 17547 epithelial cells +T583 PR:000000017 17625 17629 IFNγ +T584 PR:000016364 17650 17654 ZO-1 +T585 GO:0045177 17667 17673 apical +T586 UBERON:0001211 17775 17777 PP +T587 NCBITaxon:10088 17786 17790 mice +T588 PR:000016364 17815 17819 ZO-1 +T589 PR:000016365 17824 17828 ZO-2 +T590 GO:0010467 17834 17844 expression +T591 NCBITaxon:10088 17867 17871 mice +T592 PR:000000017 17918 17922 IFNγ +T593 NCBITaxon:10088 17938 17942 mice +T594 PR:000016364 17982 17986 ZO-1 +T595 PR:000016365 17991 17995 ZO-2 +T596 GO:0010467 18001 18011 expression +T597 NCBITaxon:10088 18075 18079 mice +T598 PR:000011306 18137 18143 CARD15 +T599 PR:000011306 18144 18148 NOD2 +T600 NCBITaxon:9606 18160 18165 Human +T601 http://purl.obolibrary.org/obo/MONDO_0000001 18166 18175 disorders +T602 http://purl.obolibrary.org/obo/MONDO_0005011 18188 18190 CD +T603 PR:000000134 18260 18264 TNFα +T604 http://purl.obolibrary.org/obo/MONDO_0013730 18307 18311 GVHD +T605 GO:0042571 18341 18351 antibodies +T606 UBERON:0001555 18391 18394 gut +T607 PR:000011306 18440 18446 Card15 +T608 PR:000011306 18447 18451 Nod2 +T609 NCBITaxon:2 18455 18464 bacterial +T610 NCBITaxon:2 18480 18489 Bacterial +T611 NCBITaxon:1280 18501 18522 Staphylococcus aureus +T612 NCBITaxon:562 18527 18543 Escherichia coli +T613 NCBITaxon:4932 18565 18589 Saccharomyces cerevisiae +T614 UBERON:0001211 18610 18612 PP +T615 NCBITaxon:10088 18619 18623 mice +T616 http://purl.obolibrary.org/obo/MONDO_0005011 18652 18654 CD +T617 NCBITaxon:2 18684 18693 bacterial +T618 UBERON:0001277 18720 18741 intestinal epithelium +T619 NCBITaxon:2 18880 18888 bacteria +T620 UBERON:0001211 18911 18913 PP +T621 NCBITaxon:10088 18920 18924 mice +T622 http://purl.obolibrary.org/obo/MONDO_0005011 18982 18984 CD +T623 GO:0042571 19049 19059 antibodies +T624 NCBITaxon:4932 19063 19087 Saccharomyces cerevisiae +T625 NCBITaxon:294 19089 19112 Pseudomonas fluorescens +T626 NCBITaxon:562 19130 19146 Escherichia coli +T627 GO:0019867 19147 19161 outer membrane +T628 PR:000023451 19147 19169 outer membrane porin C +T629 PR:000022655 19180 19189 flagellin +T630 NCBITaxon:2 19210 19218 bacteria +T631 GO:0002250 19289 19314 adaptive immune responses +T632 UBERON:0002405 19298 19304 immune +T633 CHEBI:59132 19328 19336 antigens +T634 CL:0000682 19363 19370 M cells +T635 NCBITaxon:10088 19432 19436 mice +T636 CL:0000682 19455 19461 M cell +T637 GO:0030154 19457 19477 cell differentiation +T638 NCBITaxon:2 19552 19561 bacterial +T639 PR:000011306 19700 19706 CARD15 +T640 PR:000011306 19707 19711 NOD2 +T641 NCBITaxon:2 19736 19745 bacterial +T642 CHEBI:33282 19770 19783 antibacterial +T643 NCBITaxon:2 19774 19783 bacterial +T644 GO:0010467 19792 19802 expression +T645 GO:0005622 19817 19830 intracellular +T646 CHEBI:33282 19831 19843 bactericidal +T647 UBERON:0000483 19864 19874 epithelial +T648 UBERON:0002405 19875 19881 immune +T649 GO:0006952 19882 19889 defence +T650 NCBITaxon:2 19900 19909 bacterial +T651 PR:000000017 19983 19987 IFNγ +T652 UBERON:0000483 20013 20023 epithelial +T653 NCBITaxon:species 20046 20053 species +T654 UBERON:0000160 20057 20064 enteric +T655 NCBITaxon:2 20065 20073 bacteria +T656 NCBITaxon:10088 20131 20135 mice +T657 UBERON:0000062 20146 20151 organ +T658 GO:0010467 20170 20180 expression +T659 UBERON:0000317 20215 20230 colonic mucosal +T660 UBERON:0000060 20231 20238 barrier +T661 NCBITaxon:2 20261 20270 bacterial +T662 PR:000001004 20335 20338 CD4 +T663 CL:0000084 20340 20347 T-cells +T664 PR:000000017 20361 20365 IFNγ +T665 NCBITaxon:2 20403 20412 bacterial +T666 UBERON:0002405 20446 20452 immune +T667 PR:000011306 20581 20587 Card15 +T668 PR:000011306 20588 20592 Nod2 +T669 http://purl.obolibrary.org/obo/MONDO_0005292 20647 20654 colitis +T670 UBERON:0001155 20718 20725 colonic +T671 http://purl.obolibrary.org/obo/MONDO_0005292 20718 20738 colonic inflammation +T672 UBERON:0000344 20759 20766 mucosal +T673 PR:000001136 20777 20782 IL-1β +T674 PR:000000134 20784 20788 TNFα +T675 GO:0043514 20793 20798 IL-12 +T676 http://purl.obolibrary.org/obo/MONDO_0005292 20815 20822 colitis +T677 UBERON:0000317 20938 20953 colonic mucosal +T678 PR:000011306 20972 20978 Card15 +T679 PR:000011306 20979 20983 Nod2 +T680 GO:0070431 20979 20993 Nod2 signaling +T681 PR:000001153 21024 21028 TLR2 +T682 CL:0000545 21050 21053 Th1 +T683 GO:0042088 21050 21062 Th1 response +T684 CL:0000545 21097 21100 Th1 +T685 GO:0042088 21097 21100;21114 21122 Th1 ... response +T686 GO:0006954 21101 21122 inflammatory response +T687 PR:000001153 21135 21140 TLR-2 +T688 PR:000011306 21157 21163 Card15 +T689 PR:000011306 21164 21168 Nod2 +T690 NCBITaxon:10088 21179 21183 mice +T691 UBERON:0001962 21201 21205 GALT +T692 GO:0065007 21221 21229 modulate +T693 http://purl.obolibrary.org/obo/MONDO_0005292 21259 21266 colitis +T694 http://purl.obolibrary.org/obo/MONDO_0005292 21323 21330 colitis +T695 PR:000011306 21349 21355 Card15 +T696 PR:000011306 21356 21360 Nod2 +T697 NCBITaxon:10088 21364 21368 mice +T698 UBERON:0001962 21380 21384 GALT +T699 UBERON:0000160 21484 21494 intestinal +T700 NCBITaxon:2 21512 21521 bacterial +T701 UBERON:0000160 21546 21555 intestine +T702 PR:000011306 21629 21635 Card15 +T703 PR:000011306 21636 21640 Nod2 +T704 GO:0042592 21669 21680 homeostatis +T705 UBERON:0001211 21688 21691 PPs +T706 PR:000011306 21701 21707 CARD15 +T707 PR:000011306 21708 21712 NOD2 +T708 http://purl.obolibrary.org/obo/MONDO_0005011 21759 21761 CD +T709 http://purl.obolibrary.org/obo/MONDO_0013730 21762 21766 GVHD +T710 http://purl.obolibrary.org/obo/MONDO_0000001 21837 21844 disease +T711 UBERON:0001555 21948 21951 gut +T712 UBERON:0001211 21976 21978 PP +T713 UBERON:0000444 21983 21985 LF +T714 UBERON:0001962 22000 22004 GALT +T715 NCBITaxon:10088 22032 22036 mice +T716 UBERON:0001555 22069 22072 gut +T717 UBERON:0002405 22073 22079 immune +T718 GO:0006955 22073 22088 immune response +T719 NCBITaxon:2 22106 22115 bacterial +T720 NCBITaxon:9606 22187 22192 Human +T721 http://purl.obolibrary.org/obo/MONDO_0000001 22193 22201 diseases +T722 PR:000011306 22218 22224 CARD15 +T723 PR:000011306 22225 22229 NOD2 +T724 http://purl.obolibrary.org/obo/MONDO_0013730 22247 22251 GVHD +T725 http://purl.obolibrary.org/obo/MONDO_0005011 22256 22258 CD +T726 PR:000011306 22348 22354 Card15 +T727 PR:000011306 22355 22359 Nod2 +T728 NCBITaxon:10088 22363 22367 mice +T729 http://purl.obolibrary.org/obo/MONDO_0005011 22406 22408 CD +T730 http://purl.obolibrary.org/obo/MONDO_0013730 22409 22413 GVHD +T731 NCBITaxon:9606 22529 22534 Human +T732 http://purl.obolibrary.org/obo/MONDO_0000001 22535 22543 diseases +T733 SO:0000704 22589 22596 genetic +T734 http://purl.obolibrary.org/obo/MONDO_0000001 22644 22651 disease +T735 PR:000011306 22709 22715 Card15 +T736 PR:000011306 22716 22720 Nod2 +T737 NCBITaxon:10088 22724 22729 mouse +T738 http://purl.obolibrary.org/obo/MONDO_0005011 22809 22811 CD +T739 http://purl.obolibrary.org/obo/MONDO_0013730 22816 22820 GVHD +T740 NCBITaxon:33208 22845 22852 Animals +T741 PR:000011306 22854 22860 Card15 +T742 PR:000011306 22861 22865 Nod2 +T743 NCBITaxon:10088 22883 22887 mice +T744 SO:0000200 22905 22922 first coding exon +T745 SO:0000417 22979 22986 domains +T746 SO:0000318 23001 23012 start codon +T747 SO:0005853 23025 23033 cassette +T748 PR:000011306 23086 23092 Card15 +T749 PR:000011306 23093 23097 Nod2 +T750 PR:000011306 23126 23132 Card15 +T751 PR:000011306 23133 23137 Nod2 +T752 SO:0005853 23209 23217 cassette +T753 SO:0001026 23344 23351 Genomic +T754 NCBITaxon:10088 23361 23365 mice +T755 SO:0000112 23397 23404 primers +T756 SO:0000112 23483 23490 primers +T757 PR:000011306 23599 23603 Nod2 +T758 GO:0010467 23604 23614 expression +T759 SO:0000112 23688 23695 primers +T760 NCBITaxon:10088 23794 23798 mice +T761 PR:000011306 23805 23811 Card15 +T762 PR:000011306 23812 23816 Nod2 +T763 NCBITaxon:10088 23825 23829 mice +T764 NCBITaxon:10088 23922 23926 mice +T765 NCBITaxon:33208 23960 23966 animal +T766 NCBITaxon:33208 24128 24134 animal +T767 UBERON:0001211 24141 24156 Peyer's patches +T768 UBERON:0000444 24170 24187 lymphoid follicle +T769 UBERON:0002108 24208 24224 small intestines +T770 UBERON:0001211 24256 24259 PPs +T771 GO:0007567 24314 24319 birth +T772 UBERON:0001211 24326 24329 PPs +T773 UBERON:0000970 24369 24372 eye +T774 GO:0007567 24377 24382 birth +T775 UBERON:0002108 24384 24400 small intestines +T776 CHEBI:16842 24415 24423 formalin +T777 CHEBI:6872 24442 24456 methylene blue +T778 CHEBI:15366 24485 24496 acetic acid +T779 UBERON:0000444 24502 24504 LF +T780 UBERON:0002108 24513 24529 small intestines +T781 CHEBI:16842 24544 24552 formalin +T782 UBERON:0000444 24644 24647 LFs +T783 CHEBI:51686 24713 24725 haematoxylin +T784 UBERON:0001211 24763 24778 Peyer's Patches +T785 UBERON:0002106 24783 24790 spleens +T786 UBERON:0001211 24814 24817 PPs +T787 UBERON:0001211 24862 24865 PPs +T788 NCBITaxon:10088 24875 24880 mouse +T789 UBERON:0002106 25048 25054 spleen +T790 CL:0000232 25121 25133 erythrocytes +T791 GO:0019835 25134 25139 lysis +T792 CHEBI:75958 25147 25155 solution +T793 GO:0042571 25358 25368 antibodies +T794 CHEBI:37989 25473 25476 Cy5 +T795 PR:000001018 25482 25485 CD3 +T796 CHEBI:52718 25497 25500 Cy7 +T797 PR:000001018 25506 25509 CD3 +T798 CHEBI:37989 25525 25528 Cy5 +T799 PR:000001004 25534 25537 CD4 +T800 PR:000001004 25557 25560 CD4 +T801 CHEBI:37926 25568 25572 FITC +T802 PR:000025402 25578 25581 CD8 +T803 PR:000001013 25600 25605 CD11c +T804 PR:000001380 25621 25625 CD25 +T805 PR:000001014 25635 25640 CD45R +T806 PR:000001014 25641 25645 B220 +T807 CHEBI:37926 25657 25661 FITC +T808 PR:000001016 25667 25673 CD45RB +T809 PR:000004078 25690 25699 annexin V +T810 PR:000002978 25743 25748 Ly-6G +T811 CL:0000682 25783 25790 M cells +T812 UBERON:0001211 25871 25873 PP +T813 NCBITaxon:3902 25909 25924 Ulex Europeaeus +T814 GO:0042571 25925 25935 antibodies +T815 NCBITaxon:3902 25983 25997 Ulex Europeaus +T816 NCBITaxon:9986 26059 26065 rabbit +T817 CHEBI:37926 26066 26070 FITC +T818 CHEBI:64985 26071 26080 conjugate +T819 PR:000002312 26114 26123 Caspase 3 +T820 NCBITaxon:9986 26154 26160 rabbit +T821 GO:0042571 26172 26182 antibodies +T822 MOP:0000780 26186 26193 Cleaved +T823 PR:000018762 26186 26203 Cleaved Caspase-3 +T824 MOP:0000779 26307 26313 Linked +T825 UBERON:0001211 26343 26346 PPs +T826 UBERON:0002116 26348 26353 ileum +T827 UBERON:0002106 26358 26364 spleen +T828 NCBITaxon:10088 26380 26384 mice +T829 PR:000000134 26527 26531 TNFα +T830 PR:000000017 26533 26537 IFNγ +T831 PR:000001391 26539 26543 IL-4 +T832 PR:000001136 26548 26553 IL-1β +T833 UBERON:0002116 26743 26748 ileum +T834 UBERON:0001211 26765 26768 PPs +T835 UBERON:0000479 26816 26822 tissue +T836 CHEBI:75958 26874 26882 solution +T837 UBERON:0001211 26892 26894 PP +T838 UBERON:0002116 26899 26904 ileum +T839 UBERON:0000344 26974 26981 mucosal +T840 UBERON:0000042 26985 26992 serosal +T841 CHEBI:37926 27007 27011 FITC +T842 CHEBI:52071 27012 27019 dextran +T843 NCBITaxon:2 27060 27069 Bacterial +T844 CHEBI:31624 27120 27131 fluorescein +T845 MOP:0000779 27132 27142 conjugated +T846 NCBITaxon:83333 27143 27164 Escherichia coli K-12 +T847 NCBITaxon:1280 27168 27189 Staphylococcus Aureus +T848 NCBITaxon:562 27259 27275 Escherichia coli +T849 CHEBI:28077 27305 27315 rifampicin +T850 UBERON:0000344 27365 27372 mucosal +T851 NCBITaxon:4932 27384 27408 Saccharomyces cerevisiae +T852 CHEBI:31624 27459 27470 fluorescein +T853 MOP:0000779 27471 27481 conjugated +T854 NCBITaxon:4932 27482 27495 S. cerevisiae +T855 UBERON:0000344 27601 27608 mucosal +T856 GO:0001171 27631 27652 reverse transcription +T857 MOP:0000635 27664 27678 chain reaction +T858 UBERON:0001211 27711 27713 PP +T859 UBERON:0002116 27717 27722 ileum +T860 CHEBI:51461 27904 27914 SYBR Green +T861 SO:0000077 27970 27979 antisense +T862 SO:0000112 27980 27987 primers +T863 PR:000002207 28068 28076 Occludin +T864 PR:000002207 28078 28081 Occ +T865 PR:000016364 28146 28164 Zonula Occludens-1 +T866 PR:000016364 28166 28170 ZO-1 +T867 PR:000016365 28238 28256 Zonula Occludens-2 +T868 PR:000016365 28258 28262 ZO-2 +T869 GO:0010467 28397 28407 expression +T870 NCBITaxon:2 28417 28426 Bacterial +T871 UBERON:0002116 28438 28443 ileum +T872 UBERON:0002116 28456 28461 ileum +T873 UBERON:0002116 28485 28490 ileal +T874 CHEBI:15377 28534 28539 water +T875 CHEBI:53550 28577 28590 polypropylene +T876 UBERON:0002116 28606 28611 ileal +T877 NCBITaxon:9940 28754 28759 sheep +T878 UBERON:0000178 28760 28765 blood +T879 UBERON:0002116 28935 28940 ileal +T880 http://purl.obolibrary.org/obo/MONDO_0005292 28964 28971 colitis +T881 http://purl.obolibrary.org/obo/MONDO_0005292 28991 28998 colitis +T882 NCBITaxon:10088 29026 29030 mice +T883 UBERON:0001155 29048 29055 colonic +T884 CHEBI:16236 29122 29129 ethanol +T885 CHEBI:75958 29171 29179 solution +T886 UBERON:0001155 29202 29207 colon +T887 UBERON:0001245 29223 29227 anus +T888 CHEBI:53227 29243 29255 polyethylene +T889 NCBITaxon:10088 29270 29274 mice +T890 PR:000011306 29831 29835 Nod2 +T891 UBERON:0001555 29852 29855 gut +T892 UBERON:0000160 29867 29876 intestine +T893 UBERON:0001555 29937 29940 gut +T894 UBERON:0000160 29956 29965 intestine +T895 NCBITaxon:10088 29986 29990 mice +T896 NCBITaxon:10088 30037 30041 mice +T897 NCBITaxon:10088 30086 30090 mice +T898 UBERON:0002116 30166 30171 Ileal +T899 NCBITaxon:10088 30274 30278 mice +T900 NCBITaxon:2 30296 30305 bacterial +T901 NCBITaxon:10088 30349 30353 mice +T902 UBERON:0001211 30430 30433 PPs +T903 NCBITaxon:10088 30442 30446 mice +T904 PR:000001004 30471 30474 CD4 +T905 PR:000001004 30480 30483 CD4 +T906 PR:000025402 30484 30487 CD8 +T907 CL:0000084 30488 30495 T-cells +T908 PR:000001018 30518 30521 CD3 +T909 CL:0000084 30523 30530 T-cells +T910 UBERON:0001211 30546 30549 PPs +T911 UBERON:0002106 30558 30564 spleen +T912 GO:0042571 30587 30597 antibodies +T913 PR:000001018 30601 30604 CD3 +T914 PR:000001004 30606 30609 CD4 +T915 PR:000025402 30615 30618 CD8 +T916 NCBITaxon:10088 30642 30646 mice +T917 PR:000001018 30668 30671 CD3 +T918 CL:0000084 30673 30680 T-cells +T919 PR:000001018 30711 30714 CD3 +T920 PR:000001004 30715 30718 CD4 +T921 PR:000001018 30724 30727 CD3 +T922 PR:000001004 30728 30731 CD4 +T923 PR:000025402 30732 30735 CD8 +T924 CL:0000084 30737 30744 T-cells +T925 UBERON:0001211 30778 30781 PPs +T926 UBERON:0002106 30797 30803 spleen +T927 NCBITaxon:10088 30819 30823 mice +T928 NCBITaxon:10088 30859 30863 mice +T929 PR:000011306 30959 30963 Nod2 +T930 PR:000001018 30968 30971 CD3 +T931 CL:0000084 30973 30981 T -cells +T932 UBERON:0001211 30985 31000 Peyer's Patches +T933 CL:0000898 31036 31041;31065 31072 naïve ... T-cells +T934 GO:0065007 31043 31053 regulatory +T935 CL:0000815 31043 31053;31065 31072 regulatory ... T-cells +T936 GO:0007613 31058 31064 memory +T937 CL:0000813 31058 31072 memory T-cells +T938 UBERON:0001211 31076 31079 PPs +T939 UBERON:0002106 31088 31095 spleens +T940 NCBITaxon:10088 31121 31125 mice +T941 PR:000001004 31138 31141 CD4 +T942 CL:0000084 31143 31150 T-cells +T943 GO:0042571 31169 31179 antibodies +T944 PR:000001380 31183 31187 CD25 +T945 PR:000001016 31192 31198 CD45RB +T946 CL:0000445 31228 31237;31258 31263 apoptotic ... cells +T947 PR:000001018 31238 31241 CD3 +T948 PR:000001018 31247 31250 CD3 +T949 PR:000001004 31251 31254 CD4 +T950 CL:0000084 31256 31263 T-cells +T951 CL:0000445 31265 31274;31295 31300 Apoptotic ... cells +T952 PR:000001018 31275 31278 CD3 +T953 PR:000001018 31284 31287 CD3 +T954 PR:000001004 31288 31291 CD4 +T955 CL:0000084 31293 31300 T-cells +T956 GO:0042571 31343 31353 antibodies +T957 PR:000001018 31357 31360 CD3 +T958 PR:000001004 31362 31365 CD4 +T959 PR:000004078 31370 31379 annexin V +T960 PR:000001018 31401 31404 CD3 +T961 PR:000001004 31405 31408 CD4 +T962 CL:0000084 31410 31417 T-cells +T963 NCBITaxon:10088 31453 31457 mice +T964 PR:000011306 31829 31833 Nod2 +T965 GO:0007567 31842 31847 natal +T966 UBERON:0001555 31863 31866 gut +T967 UBERON:0001211 31904 31906 PP +T968 UBERON:0000160 31926 31936 intestines +T969 NCBITaxon:10088 31958 31962 mice +T970 GO:0007567 31966 31971 birth +T971 UBERON:0000444 32022 32025 LFs +T972 UBERON:0002108 32071 32086 small intestine +T973 UBERON:0001211 32137 32139 PP +T974 UBERON:0002106 32144 32150 spleen +T975 CL:0000445 32189 32204 apoptotic cells +T976 PR:000002312 32219 32228 caspase-3 +T977 NCBITaxon:10088 32290 32294 mice +T978 PR:000011306 32348 32352 Nod2 +T979 UBERON:0001211 32357 32370 Peyer's patch +T980 CL:0000084 32428 32430;32449 32454 T- ... cells +T981 CL:0000236 32432 32434;32449 32454 B- ... cells +T982 GO:0097511 32439 32448 dendritic +T983 CL:0000451 32439 32454 dendritic cells +T984 UBERON:0001211 32460 32463 PPs +T985 UBERON:0002106 32468 32475 spleens +T986 NCBITaxon:10088 32497 32501 mice +T987 CL:0000084 32514 32516;32535 32540 T- ... cells +T988 CL:0000236 32518 32520;32535 32540 B- ... cells +T989 GO:0097511 32525 32534 dendritic +T990 CL:0000451 32525 32540 dendritic cells +T991 GO:0042571 32583 32593 antibodies +T992 PR:000001018 32597 32600 CD3 +T993 PR:000001014 32602 32606 B220 +T994 PR:000001013 32611 32616 CD11c +T995 CL:0000096 32645 32674 polymorphonuclear neutrophils +T996 PR:000002978 32694 32699 Ly-6G +T997 GO:0042571 32700 32708 antibody +T998 CL:0000682 32714 32721 M cells +T999 UBERON:0000483 32765 32775 epithelium +T1000 CL:0000682 32852 32858 M-cell +T1001 UBERON:0000483 32895 32905 epithelium +T1002 NCBITaxon:10088 32941 32945 mice +T1003 PR:000011306 32978 32982 Nod2 +T1004 CL:0000084 32987 32994 T-cells +T1005 UBERON:0001211 33005 33007 PP +T1006 UBERON:0002106 33012 33018 spleen +T1007 NCBITaxon:10088 33024 33028 mice +T1008 PR:000001004 33073 33076 CD4 +T1009 PR:000025402 33079 33082 CD8 +T1010 PR:000001004 33088 33091 CD4 +T1011 PR:000025402 33092 33095 CD8 +T1012 CL:0000084 33097 33104 T-cells +T1013 UBERON:0001211 33110 33113 PPs +T1014 UBERON:0002106 33122 33128 spleen +T1015 NCBITaxon:10088 33154 33158 mice +T1016 PR:000001018 33172 33175 CD3 +T1017 CL:0000084 33177 33184 T-cells +T1018 GO:0042571 33203 33213 antibodies +T1019 PR:000001018 33217 33220 CD3 +T1020 PR:000001004 33222 33225 CD4 +T1021 PR:000025402 33231 33234 CD8 +T1022 PR:000001018 33256 33259 CD3 +T1023 CL:0000084 33261 33268 T-cells +T1024 NCBITaxon:10088 33304 33308 mice +T1025 PR:000011306 33340 33344 Nod2 +T1026 GO:0010467 33377 33387 expression +T1027 UBERON:0001211 33391 33393 PP +T1028 GO:0010467 33396 33407 Expressions +T1029 PR:000001136 33411 33416 Il-1β +T1030 PR:000000017 33418 33422 IFNγ +T1031 PR:000000134 33424 33428 TNFα +T1032 GO:0043514 33430 33435 IL-12 +T1033 PR:000001391 33437 33441 IL-4 +T1034 UBERON:0001211 33470 33473 PPs +T1035 UBERON:0002116 33475 33480 Ileum +T1036 UBERON:0002106 33485 33492 spleens +T1037 NCBITaxon:10088 33516 33520 mice +T1038 NCBITaxon:10088 33601 33605 mice +T1039 NCBITaxon:2 33677 33686 bacterial +T1040 PR:000011306 33718 33722 Nod2 +T1041 NCBITaxon:10088 33735 33739 mice +T1042 UBERON:0001211 33805 33807 PP +T1043 UBERON:0001211 33812 33814 PP +T1044 UBERON:0002116 33820 33825 ileum +T1045 CHEBI:37926 33907 33911 FITC +T1046 CHEBI:52071 33912 33919 dextran +T1047 UBERON:0001211 33930 33932 PP +T1048 UBERON:0001211 33937 33939 PP +T1049 UBERON:0002116 33945 33950 ileum +T1050 GO:0010467 33983 33993 expression +T1051 PR:000016364 34017 34021 ZO-1 +T1052 PR:000016365 34023 34027 ZO-2 +T1053 PR:000002207 34032 34035 Occ +T1054 UBERON:0001211 34042 34044 PP +T1055 NCBITaxon:2 34103 34112 Bacterial +T1056 NCBITaxon:83333 34160 34181 Escherichia Coli K-12 +T1057 NCBITaxon:562 34234 34250 Escherichia Coli +T1058 NCBITaxon:1280 34316 34337 Staphylococcus Aureus +T1059 CHEBI:31624 34378 34389 fluorescein +T1060 MOP:0000779 34390 34400 conjugated +T1061 NCBITaxon:4932 34401 34425 Saccharomyces cerevisiae +T1062 NCBITaxon:10088 34461 34465 mice +T1063 PR:000011306 34543 34547 Nod2 +T1064 http://purl.obolibrary.org/obo/MONDO_0005292 34605 34612 colitis +T1065 NCBITaxon:10088 34712 34716 mice +T1066 UBERON:0001155 34773 34780 colonic +T1067 NCBITaxon:10088 34889 34893 mice +T1068 NCBITaxon:39107 34934 34940 murine +T1069 PR:000011306 34941 34945 Nod2 +T1070 SO:0000704 34946 34950 gene +T1071 PR:000011306 35003 35009 Card15 +T1072 PR:000011306 35010 35014 Nod2 +T1073 SO:0001023 35018 35024 allele +T1074 SO:0001250 35050 35066 restriction maps +T1075 PR:000011306 35074 35080 Card15 +T1076 PR:000011306 35081 35085 Nod2 +T1077 SO:0001023 35086 35092 allele +T1078 PR:000011306 35111 35117 Card15 +T1079 PR:000011306 35118 35122 Nod2 +T1080 PR:000011306 35163 35169 Card15 +T1081 PR:000011306 35170 35174 Nod2 +T1082 SO:0001023 35175 35181 allele +T1083 CHEBI:24753 35250 35260 Hygromycin +T1084 SO:0005853 35271 35279 cassette +T1085 SO:0000147 35291 35296 Exons +T1086 SO:0000061 35327 35344 restriction sites +T1087 PR:000011306 35444 35450 Card15 +T1088 PR:000011306 35451 35455 Nod2 +T1089 SO:0000704 35497 35501 gene +T1090 SO:0001817 35513 35521 in frame +T1091 PR:000011306 35527 35533 Card15 +T1092 PR:000011306 35534 35538 Nod2 +T1093 SO:0000359 35551 35557 floxed +T1094 SO:0005853 35593 35601 cassette +T1095 SO:0000061 35631 35635 site +T1096 PR:000011306 35650 35656 Card15 +T1097 PR:000011306 35657 35661 Nod2 +T1098 SO:0000147 35662 35666 exon +T1099 SO:0000061 35682 35686 site +T1100 SO:0000147 35699 35703 exon +T1101 PR:000011306 35713 35719 Card15 +T1102 PR:000011306 35720 35724 Nod2 +T1103 SO:0000346 35742 35752 loxP sites +T1104 PR:000011306 35889 35895 Card15 +T1105 PR:000011306 35896 35900 Nod2 +T1106 PR:000011306 35929 35935 Card15 +T1107 PR:000011306 35936 35940 Nod2 +T1108 SO:0005853 36012 36020 cassette +T1109 GO:0097617 36166 36175 hybridize +T1110 SO:0000112 36239 36246 primers +T1111 PR:000011306 36278 36284 Card15 +T1112 PR:000011306 36285 36289 Nod2 +T1113 SO:0001023 36290 36297 alleles +T1114 GO:0097617 36379 36390 Hybridation +T1115 PR:000011306 36612 36616 Nod2 +T1116 NCBITaxon:10088 36627 36631 mice +T1117 SO:0001026 36640 36647 Genomic +T1118 NCBITaxon:10088 36657 36661 mice +T1119 SO:0000006 36717 36728 PCR product +T1120 SO:0000028 36736 36738 bp +T1121 GO:0010467 36745 36755 Expression +T1122 PR:000011306 36759 36763 Nod2 +T1123 UBERON:0002106 36772 36778 spleen +T1124 UBERON:0002106 36827 36833 spleen +T1125 NCBITaxon:10088 36847 36851 mice +T1126 NCBITaxon:10088 36895 36899 mice +T1127 GO:0010467 36907 36917 expression +T1128 GO:0010467 36950 36960 expression diff --git a/src/ontogpt/evaluation/craft/database/all/17565376.txt b/src/ontogpt/evaluation/craft/database/all/17565376.txt new file mode 100644 index 000000000..580b13034 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17565376.txt @@ -0,0 +1,209 @@ +CARD15/NOD2 Is Required for Peyer's Patches Homeostasis in Mice + +Abstract + +Background + +CARD15/NOD2 mutations are associated with susceptibility to Crohn's Disease (CD) and Graft Versus Host Disease (GVHD). CD and GVHD are suspected to be related with the dysfunction of Peyer's patches (PP) and isolated lymphoid follicles (LFs). Using a new mouse model invalidated for Card15/Nod2 (KO), we thus analysed the impact of the gene in these lymphoid formations together with the development of experimental colitis. + +Methodology/Principal Findings + +At weeks 4, 12 and 52, the numbers of PPs and LFs were higher in KO mice while no difference was observed at birth. At weeks 4 and 12, the size and cellular composition of PPs were analysed by flow cytometry and immunohistochemistry. PPs of KO mice were larger with an increased proportion of M cells and CD4+ T-cells. KO mice were also characterised by higher concentrations of TNFα, IFNγ, IL12 and IL4 measured by ELISA. In contrast, little differences were found in the PP-free ileum and the spleen of KO mice. By Ussing chamber experiments, we found that this PP phenotype is associated with an increased of both paracellular permeability and yeast/bacterial translocation. Finally, KO mice were more susceptible to the colitis induced by TNBS. + +Conclusions + +Card15/Nod2 deficiency induces an abnormal development and function of the PPs characterised by an exaggerated immune response and an increased permeability. These observations provide a comprehensive link between the molecular defect and the Human CARD15/NOD2 associated disorders: CD and GVHD. + +Introduction + +Caspase Recruitment Domain 15 (CARD15) also known as Nucleotide oligomerisation domain 2 (NOD2) has been associated with Crohn's Disease (CD) [1], [2] and graft versus host disease (GVHD) [3], [4]. NOD2 belongs to a family of genes involved in innate immunity [5]. It can be activated by muropeptides which are components of the bacterial cell wall. When activated, NOD2 interacts with Rick/Rip2 which in turn activates the NF-kB pathway, resulting in the production of pro-inflammatory cytokines. + +Half of CD patients have one or more NOD2 mutations [6]. Some of the CD associated mutations were found unresponsive to muropeptides [5]. By consequence, CD is usually considered as an immune deficiency toward bacteria present in the gut lumen [7]. However, the exact mechanism by which NOD2 mutations are able to induce CD lesions is still subject to debate [7]–[10]. + +Holler et al. reported that the three major mutations associated with CD (R702W, G908R and 1007fs) are also associated with severe acute GVHD and bone marrow transplantation (BMT) related mortality [3]. Mutations in both donor and recipient were found deleterious, suggesting a role of epithelial and circulating cells in disease mechanisms. Despite some differences in their conclusions, other groups recently confirmed the association between NOD2 and BMT complications [4], [11]. + +CD is a chronic relapsing inflammatory bowel disease (IBD) with mucosal ulcerations of the digestive tract. CD lesions are characterised by a T helper (Th) 1 immune response and several authors have shown that they are related with gut associated lymphoid tissue (GALT), known as lymphoid follicles (LFs). LFs are mainly encountered in the colon where they are isolated and in small bowel where they are grouped forming Peyer's patches (PP) which are known to be pivotal sites for the host immune response and for the entry of enteropathogen bacteria. CD lesions are most often localized in colon and distal ileum, where the LFs are the most abundant [12]. Fujimura et al. found that aphtoïd ulcerations (which are often considered as the earliest CD lesions) are centred by LFs [13]. In addition to this spatial relationship, a temporal link between CD and PP development has also been suggested [14]. PPs develop from birth to 10–15 years of life and then undergo involution. The age-dependent incidence curve of CD is roughly parallel to the number of PP with a delay of about 10 years. Finally, ileal lesions are uncommon in young children and seniors where PP are rare [15], [16]. + +The role of GALT in GVHD has been suspected on both experimental models and clinical observations. Animal studies showed that death after BMT was prevented by gut decontamination and that prevention of mucosal damage also prevents lethal GVHD [17]. In clinical practice, gut decontamination reduces the frequency and severity of GVHD. Finally, Murai et al. recently showed that PP deficient mice are resistant to GVHD, arguing for a crucial role of PPs in GVHD, at least in models that do not use conditioning of the host prior to adoptive transfer of the allogeneic donor cells [18], [19]. + +Considering all these elements, we hypothesized that Nod2 may play a role in the structure and function of the GALT. Consequently, we used a new mouse model deficient for Card15/Nod2 i) to evaluate the involvement of Nod2 on the number and PPs size; ii) to assess whether Nod2 modified cellular composition and cytokine expression of PPs; and iii) to determine whether Nod2 may alter paracellular permeability and bacterial translocation of PPs in adult mice. Finally, we also examined if Card15/Nod2 deficiency affects the colonic response to 2,4,6-trinitrobenzene sulphonic acid (TNBS), a classic experimental model of colitis in mouse. + +Results + +Body weight, intestinal length and intestinal weight were similar in KO and WT mice (supplementary information (SI) Table S1). Macroscopically, no inflammation was visible in KO mice according to Wallace and Ameho criteria (data not shown). KO mice exhibited an increased number of PP in comparison with WT mice at weeks 4, 12 and 52 after birth (Fig 1 A). At weeks 12 and 52, the number of isolated LFs per small intestine was also higher in KO mice than in WT mice (Fig 1 B). In contrast, the number of PPs at birth was similar between KO and WT mice (6.2±0.5 vs. 6.0±0.4; P>0.05) (Fig 1 A). + +Macroscopically, the size of PP formations appeared to be larger in adult KO mice (data not shown). To quantify this difference, the three biggest PPs of each mouse were pooled and their cells were counted. At weeks 4, 12 and 52, KO mice exhibited a higher cell number per PP (fig 1 C). In contrast, cell counts were comparable in spleens of KO and WT mice (Fig 1 D). + +Microscopic analyses of PP formations from KO mice revealed no gross abnormalities (Fig 1 E). Finally, as NOD2 modulates the NF-κappaB pathway and putatively the apoptosis, we investigated the number of apoptotic cells inside PPs. Immunohistochemistry experiments did not show differences between KO and WT mice for the number of caspase 3 positive cells (1.01±0.11 vs. 1.32±0.10; P>0.05) (Fig 1 E). + +In order to better characterise the phenotype of the cells present in PPs, we performed flow cytometry experiments using the B220, CD3 and CD11c antibodies. At week 12, no differences were seen regarding the relative proportions of B220+ B-cells, CD3+ T-cells and CD11c+ dendritic cells between KO and WT mice either for PPs or spleens (Fig 2 A–B). + +Because some cells present in a limited number may be functionally important, we also investigated the relative proportion of Ly-6G+ polymorphonuclear neutrophils (PMN) present in the PPs (Fig 2 C) and the number of M cells located inside the follicle associated epithelium (FAE) (Fig 2 D). No difference of PMN relative proportion was observed in PPs of KO and WT mice (Fig 2 C). At the opposite, M cell number was increased in the FAE of KO mice in comparison with WT mice (Fig 2 D). + +As T-cells are known to play a pivotal role in CD, we further analyzed the CD3+ T cells. At week 12, KO mice exhibited an increase of CD4+ T-cell relative proportion within their PPs, whereas the proportion of CD8+ T-cells remained constant (Fig 3 A). As a mirror image, PPs from KO mice exhibited significantly fewer CD3+CD4−CD8− T-cells (Fig 3 A). In contrast, no comparable differences were seen in the spleens (Fig 3 B). Similar data were obtained at week 4 (SI Fig S1 A–B). + +Finally, we investigated the phenotype of CD4+ T-cells present in the PPs and spleens by examining the relative proportions of naive CD25−CD45Rb+, regulatory CD25+CD45Rb− and memory CD25−CD45Rb− CD4+ T-cells. KO and WT mice had a similar relative proportion of naive, regulatory and memory CD4+ T-cells in PPs (SI Fig S2 A) and spleen (SI Fig S2 B). Flow cytometry analyses also failed to reveal a difference between KO and WT mice when investigating the annexin V positive CD3+ and CD3+CD4+ T-cells extracted from PPs (SI Fig S2 C) suggesting that the excess of CD4+ T-cells observed in KO mice does not result from a defect of apoptosis. + +Considering the differences of PP cell number and cellular composition, we further evaluated whether PPs from KO and WT mice might exhibit differences regarding their cytokine profile. Thus, the expression of IL-1β, IFNγ, TNFα , IL-12, and IL-4 in PPs, PP-free ileum and spleen were determined by ELISA, at weeks 4 and 12. Levels of IFNγ, TNFα, IL-12, and IL-4 were significantly increased in PPs of KO mice (Fig 4). In contrast, less marked differences were seen between KO and WT mice in PP-free ileum where TNFα and IFNγ were the only cytokines differentially expressed at week 4 but not in adult mice (Fig 4). Finally, no differences were observed in spleens from KO and WT mice (Fig 4). + +Cell composition and cytokine production may affect the function of PPs. We thus used Ussing chambers for determining the paracellular permeability through PP and PP-free ileum. PPs and PP-free ileum of KO mice exhibited a significant increase in paracellular permeability (Fig 5 A) while the electrical resistances were comparable (data not shown). In parallel to this change, mRNA expression levels of TJ proteins were affected in PP of KO mice. Indeed, expression of mRNAs encoding ZO-2 and ZO-1 were decreased by 45% and 36% (P<0.01 and P<0.06 respectively) (Fig 5 B). In contrast, Occludin expression was unchanged (Fig 5 B). + +Because Nod2 is involved in innate immunity we hypothesised that Card15/Nod2 deficiency may affect the gut microflora. We thus counted the numbers of bacteria classified as Enterobacteriaceae, Pseudomonas, Staphylococcus, Streptococcus, Enterococcus or Lactobacillus in the ileum of KO and WT mice. However, we did not observed differences between groups (SI Table S2). + +Ussing chamber experiments were also performed to investigate the bacterial passage through PP. The passage of a chemically killed Escherichia coli (K-12) was higher in PP of KO mice (P<0.01) (Fig 5 C). This increased translocation was also observed using living non pathogenic Escherichia coli strain J53 (Fig 5 D). In contrast, translocation of Escherichia coli across free-PP ileum mucosa was very low and not significantly increased (P>0.05, fig 5 C). The translocation through PPs of a chemically killed Staphylococcus Aureus (Wood strain without proteinA) and Saccharomyces cerevisiae was also higher in KO than in WT mice (Fig 5 E and F)). + +TNBS colitis is a well studied model of acute colitis in mice but no data are currently available in the literature on the effect of Card15/Nod2 in this experimental model. Three days after intracolonic instillation of TNBS, the body weight loss was higher in KO than in WT mice but this difference did not reach significance (Fig 6 A). Nevertheless, the colonic mucosal damage score was higher in KO mice (Fig 6 B) as well as mucosal concentrations of IL-1β, TNF-α and IL-12 (Fig 6 C). + +Discussion + +Since the discovery of an association between CARD15/NOD2 mutations and both CD [1], [2] and GVH [3], [4], the pathophysiological functions of CARD15/NOD2 involved in CD and GVHD development are poorly understood [9]. However, because an association between GALT dysfonctions and GVHD, as well as spatial and temporal links between CD lesions and PP have been suggested by several authors, we used a new model of Card15/Nod2 deficient mouse, to explore the impact of CARD15/NOD2 in PP development and function. We observed that adult KO mice exhibit an excess of PP and isolated LFs in the gut. PP from KO mice are characterized by an excess of M cells and CD4+ T-cells and a higher expression of Th1 and Th2 cytokines. These differences are associated with increased paracellular permeability and bacterial and yeast passage through PPs. Finally, we observed that Card15/Nod2 deficient mice are more susceptible to TNBS induced colitis. As a whole, the here reported phenotype of the Card15/Nod2 KO mouse is reminiscent to the observations made in the Human diseases associated with CARD15/NOD2 mutations: CD and GVHD. + +Our data demonstrate first that Card15/Nod2 deficient mice have an elevated number of PPs and isolated LFs after birth. As intestinal weight and length are similar between KO and WT mice, this finding does not seem to be secondary to an intestinal overgrowth. In addition, PPs from deficient mice are larger, as indicated by the macroscopic examination of the intestines and by the count of PP cells. As a result, Card15/Nod2 modulates the development of the GALT and Card15/Nod2 deficiency is characterised by an overgrowth of the lymphoïd tissue present in the gut. This over-development of the lymphoïd tissue seems to be specific to the GI tract as non significant change were observed in a systemic immune organe like spleen. The lack of difference in PP number between WT and KO mice at birth indicates that Card15/Nod2 plays its role during post natal development of the GALT. While lymphotoxin and IL-7 signalling are essential for the organogenesis of PP during the embryonic stage [20]–[22], it is widely believed that gut commensal bacteria are critical for the postnatal development of gut mucosal immune system, as demonstrated by studies on germ-free animals [23]. Such animals have an underdeveloped GALT and are resistant to experimental colitis and to severe GVHD [18]. Thereby, pattern recognition receptors, including TLRs and NOD molecules, can be seen as good candidates by which the resident flora stimulates the development of GALT. However, TLRs play only a limited role in PP development at early postnatal stage in mice [24]. At the opposite, our data suggest that Card15/Nod2 plays a pivotal role in the postnatal development of GALT. + +Because gut flora is important in PP development, it can be questioned if Card15/Nod2 deficiency affects gut flora composition of the host. In order to answer this question, we counted the bacteria most represented in the ileum and able to cultivate in standard conditions. We failed to demonstrate differences between KO and WT mice suggesting that Card15/Nod2 deficiency does not induce gross abnormalities of the gut microflora. However, more discrete alterations cannot be discarded and additional experiments using germ-free animals and/or molecular methods for bacterial detection are required to further explore the relationship between Card15/Nod2 and the normal gut flora. + +PPs have B-cell follicles and germinal centres surrounded by areas that contain predominantly T cells. The analysis of the PP from Card15/Nod2 deficient mice failed to reveal gross abnormalities in terms of microachitecture, apoptosis or cell composition (at least for the three main cell lineages present in PP, namely B-cells, T-cells and dendritic cells). This observation is in accordance with the previous descriptions of other Card15/Nod2 KO models of mice which failed to reveal gross intestinal abnormalities under basal conditions [7], [25]. Interestingly, the phenotype observed in the deficient mouse is reminiscent with the Human CD condition where large lymphoid aggregates with normal microarchitecture and cell composition have been reported [26]. + +LFs are covered by a specialised epithelium including M cells. These cells are able to transfer bacterial and food antigens from the gut lumen to antigen presenting cells [23]. They have thus a pivotal role in the function of PP. M cells were found more numerous in KO mice. In addition, PP of KO mice exhibited a higher proportion of CD4+ T-cells and a decreased percentage of CD3+CD8−CD4− T-cells. As Nod2 may modulate the apoptosis, we have hypothesized that this increase of CD4+ T-cell number may result from an apoptosis defect in the lymphoid cells of KO mice. However, flow cytometry analyses revealed that the relative proportion of apoptotic CD4+ T-cells from PP was similar between KO and WT mice. As a result, our experiments do not support the opinion that Card15/Nod2 deficiency is characterised by a general apoptosis defect in the lymphoid tissue of KO mice. Finally, the altered immune cell composition is concomitant with an increase of pro-inflammatory Th1 but also anti-inflammatory Th2 cytokine expression. Altogether, these results indicate that under basal condition, PPs of KO mice are characterised by an exaggerated immune response. + +Cell composition and cytokine production may affect the function of PP. We thus used Ussing chambers for determining the paracellular permeability through PP and PP-free ileum. KO mice exhibited a significant increase of paracellular permeability. This result is in accordance with the altered intestinal permeability reported in CD patients and their healthy relatives, especially in case of CARD15/NOD2 mutations [27]–[30]. Pro- and anti-inflammatory cytokines are known to modulate intestinal paracellular permeability. IFNγ, TNFα and IL-4, act on membrane receptors of epithelial cells to increase tight junction permeability [31]–[34]. For example, on T84 cells IFNγ decreased levels of ZO-1 and altered apical actin organisation, which leads to disorganisation of TJ and increased permeability [35]. Similarly, PP from KO mice exhibited a decrease of ZO-1 and ZO-2 mRNA expression in comparison with WT mice. Consequently, the excessive concentration of IFNγ observed in KO mice may down regulate the transcription of ZO-1 and ZO-2 mRNA expression and contribute to the increase paracellular permeability in KO mice. It is to note that this phenotype is reminiscent to the CARD15/NOD2 associated Human disorders. Indeed, in CD patients, increased permeability has been reported to be mediated by TNFα and to precede the clinical relapse while GVHD has been treated by anti-TNF antibodies [36], [37]. + +Because of the changes in gut permeability, we finally studied the role of Card15/Nod2 in bacterial translocation. Bacterial passage of Staphylococcus aureus and Escherichia coli and yeast ingress of Saccharomyces cerevisiae were higher through PP of KO mice. It is widely accepted that CD is related with an excessive bacterial translocation through the intestinal epithelium even if this hypothesis is not perfectly documented. The present data further support this opinion. In addition, this excess of yeast and bacteria translocation through PP of KO mice is in agreement with recent reports showing that mutated CD patients and their unaffected relatives develop more frequently antibodies to Saccharomyces cerevisiae, Pseudomonas fluorescens–related protein, Escherichia coli outer membrane porin C and CBir1 flagellin [38]. The excessive bacteria and yeast passage reported here may participate in the enhancement of adaptive immune responses to microbial antigens. + +The increased number of M cells can contribute to the high translocation rate observed in KO mice. However, because M cell differentiation is inducible by microbial challenge [39], it may also be a consequence of bacterial translocations and additional experiments are required to further dissect this complex relationship. It is also possible to consider that CARD15/NOD2 dysfunction facilitates bacterial entry through defective antibacterial peptide expression [7], impaired intracellular bactericidal capacity or reduced epithelial immune defence. Finally, bacterial translocation may also be secondary to primitive local cytokine changes. IFNγ is known to increase the epithelial adherence of selected species of enteric bacteria [40]. Ferrier et al. have shown that a chronic stress in mice drives an organ-specific cytokine expression pattern which in turn, alters the colonic mucosal barrier functions and favours bacterial translocation [31]. This effect is dependent on the presence of CD4+ T-cells and requires IFNγ production. It is thus possible that bacterial translocation is a result of the immune changes rather than its cause and additional experiments are now required to answer this question. + +Finally, we have shown that Card15/Nod2 invalidation exacerbates the severity of TNBS induced colitis, as evidenced by the increase in all parameters characterising colonic inflammation (damages scores and mucosal levels of IL-1β, TNFα and IL-12). This enhanced colitis-induced by TNBS might be explained by different mechanisms. Firstly, since microflora is required for TNBS-induced colonic mucosal damages and since Card15/Nod2 signaling has been shown to inhibit the TLR2-driven activation of Th1 response [8], one can hypothesize that the Th1 inflammatory response mediated by TLR-2 is increased in Card15/Nod2 deficient mice. Secondly, since GALT is reported to modulate the severity of TNBS-induced colitis [41], [42] it can be hypothesised that the TNBS induced colitis is exacerbated in Card15/Nod2 KO mice because of GALT overdevelopment. Finally, an alternative explanation could be that the observed defect in terms of intestinal permeability and bacterial translocation makes the intestine more susceptible to TNBS. + +Altogether, the present data demonstrate that Card15/Nod2 is required to maintain the homeostatis of the PPs. Because CARD15/NOD2 is a well demonstrated etiological factor for CD/GVHD, this conclusion is of particular importance for the understanding of disease mechanisms. Indeed, it further supports the opinion that the defect involved in the development of the gut lesions is related with PP and LF function. The GALT dysfunction observed in KO mice is associated with an excessive gut immune response and an increased bacterial translocation. These findings are consistent with our knowledge on the Human diseases associated with CARD15/NOD2 mutations namely GVHD and CD and for which similar observations have been reported. As a result, the phenotype of the Card15/Nod2 KO mice can be seen as an attenuated model of CD/GVHD. It is to note that the absence of a full phenotype is not unexpected considering the multifactorial nature of the Human diseases where exposure to several additional unknown genetic and environmental risk factors is required for disease expression. At the opposite, our work indicates that the Card15/Nod2 KO mouse is a relevant model for investigating these other risk factors associated with CD and GVHD. + +Material and Methods + +Animals + +Card15/Nod2 was disrupted in mice by replacing the first coding exon carrying the majority of the sequence encoding the CARD domains including the start codon with a EGFP cassette. (Fig. 7 A). In the first step of the strategy, the Card15/Nod2 locus was targeted with the Card15/Nod2 KO targeting fragment. In the second step, the PGKHygromycin selection cassette was removed by Cre recombinase. Targeted disruption was determined by Southern blot and long-range PCR analysis (Fig. 7 B–C). Genomic DNA from mice was amplified by PCR using the primers: 5-GTCATTTCCTGACCTCTGACC-3 and 5-AACCGCATTATTCCATGGGGC-3 to detect WT DNA and primers 5-AACCGCATTATTCCATGGGGC-3 and 5-GCCTGCTCTTTACTGAAGGCTC-3 to detect the disrupted sequence. The loss of mRNA Nod2 expression was demonstrated in splenocytes by RT-PCR (Fig. 7 D) using the following primers: (5-CTTTGAACTGTATGGGTCC-3 and 5-CTCCACTGCCTCTGCCTTA-3). As expected, no signal was observed in KO mice. + +The Card15/Nod2−/− (KO) mice used for this study were back-crossed five times with the inbred strains C57BL/6. WT and KO mice were housed and generated in the animal facility at Robert Debré Hospital, Paris, France. The absence of enteropathogens was monitored. All experiments were approved by the institutional committee for animal use. + +Peyer's patches and isolated lymphoid follicle numbers + +The entire small intestines were removed and the number of PPs was determinated by macroscopic observation except at birth where PPs were too small to be seen by the naked eye. At birth, small intestines were fixed in formalin, stained with 0.5%methylene blue and decolorized in fresh 2% acetic acid. For LF counts, small intestines were fixed in formalin and rolled up into a ‘Swiss-roll’, embedded in paraffin blocks and cut into 5 µm sections. LFs were counted in a blind fashion on two sections per blocks after haematoxylin-eosin staining. + +Cell composition of Peyer's Patches and spleens + +Cell suspensions from PPs were prepared by pressing the three largest PPs for each mouse with a 5 ml syringe piston. The preparation was then incubated with 100 U/ml collagenase D (Roche, Mannheim, Germany) for 30 minutes at 37°C in DMEM media. Cells from spleen were isolated using the same procedure with an additional step of erythrocytes lysis (Gey's-solution). After centrifugation, cells were re-suspended in DMEM media and submitted to flow cytometry analyses on a FACScalibur (Becton-Dickinson), and analyzed by Cell Quest 3.3 (Becton Dickinson). Monoclonal antibodies used to stain cell suspensions were purchased from BD Biosciences (Pharmingen Heidelberg, Germany) : PE-Cy5-anti-CD3 (17A2), PE-Cy7-anti-CD3 (145-2C11), PE-Cy5-anti-CD4 (H129.19), PE-anti-CD4 (RM4), FITC-anti-CD8 (53-6.7), PE-anti-CD11c (HL3), PE-anti-CD25, PE-anti-CD45R/B220 (RA3-6B2), FITC-anti-CD45RB (16A), APC-anti-annexin V and eBioscience (San Diego, CA) : APC-anti-Ly-6G (RB6-8C5). + +Immunohistochemistry + +M cells were counted in the FAE using a fluorescence microscope after immunostaining of PP cryostat sections (5 µm) with anti-Ulex Europeaeus antibodies (1/250, 2 h) (Sigma, France), revealed by anti-Ulex Europeaus agglutinin I (1/500, 30 min.) (Vector laboratories) and anti-rabbit FITC conjugate (1/80, 30 min.) (Sigma, France). Caspase 3 immunostaining was done using rabbit polyclonal antibodies to Cleaved Caspase-3 (Asp 175, dilution: 1/100) ( Cell Signaling Technology, Inc Ozyme, Beverly, MA, USA). + +Cytokine Enzyme-Linked Immunosorbent Assay (ELISA) + +PPs, ileum and spleen from WT and KO mice were removed, washed with cold PBS and the concentration of protein was determined using commercial kit (Biorad, Marnes la Coquette, France). TNFα, IFNγ, IL-4 and IL-1β were determined by ELISA assays (BD Biosciences) according to the manufacturer's instructions. All experimental groups were tested in duplicates. + +Ussing chamber experiments + +Biopsies from ileum with or without PPs were placed in a chamber exposing 0.196 cm2 of tissue surface to 1.5 ml of circulating oxygenated Ringer solution at 37°C. PP and ileum permeability were assessed by measuring steady-state (from 1 to 2 h) mucosal-to-serosal flux of 4 kDa FITC-dextran (Sigma, St. Quentin Fallavier, France). Bacterial translocation was studied using chemically killed fluorescein-conjugated Escherichia coli K-12 or Staphylococcus Aureus BioParticles (Molecular Probes, Leiden, the Netherlands) or a viable Escherichia coli (the J53 strain resistant to rifampicin) at a final concentration of 1.107 CFU/ml in the mucosal reservoir. Saccharomyces cerevisiae translocation was studied using chemically killed fluorescein-conjugated S. cerevisiae BioParticles (Molecular Probes, Leiden, the Netherlands) at a final concentration of 1.107 CFU/ml in the mucosal reservoir. + +Real time reverse transcription-polymerase chain reaction (RT-PCR) + +After extraction from PP of ileum by the NucleoSpin® RNA II Kit (Macherey-Nagel, Hoerdt, France), total RNA was converted to cDNA using random hexonucleotides and then used for PCR. We conducted PCR with QuantiTect SYBR Green PCR Kit (Applied, Courtaboeuf, France) using sense and antisense primers specific for: G3PDH, 5′-CACCATCTTCCAGGAGCGAG-3′ and 5′-GCCTTCTCCATGGTGGTGAA-3′; Occludin (Occ), 5′-AGCCTTCTGCTTCATCGCTTC-3′ and 5′-GTGGCAATAAACACCATGATGC-3′; Zonula Occludens-1 (ZO-1), 5′- GACTCCAGACAACATCCCGAA-3′ and 5′- AACGCTGGAAATAACCTCGTTC -3′; Zonula Occludens-2 (ZO-2), 5′-CAGCCACAATCAACGTGAATTC-3′ and 5′-CTGTCCTTCAAGCTGCCAAAC-3′. After amplification, we determined the threshold cycle (Ct) to obtain expression values. + +Bacterial content of ileum + +The entire ileum (5 cm) was removed and ileal content was collected using 3 mL of steril water (Biorad, France) administered with a polypropylene syringe. Then, ileal content was homogenized and serial dilution (50 µL) of each aliquot were plated onto 5 selective gelose (URI 4, Drigalski, Columbia ANC+5% of sheep blood, Chapman and Coccosel). Plates were incubated for 24 hours at 37°C under aerobic condition and the number of colony forming units was counted and expressed as cfu/mg of ileal content. + +TNBS induced colitis + +Under anaesthesia colitis was induced in 12 week old mice by a single intracolonic administration of 120 mg/kg TNBS (Sigma, France) dissolved in 50% ethanol. A 50 µl aliquot of the freshly prepared solution was injected into the colon, 4 cm from the anus, using a 3.5 F polyethylene catheter. The mice were weighed and killed 72 h after TNBS administration. Then, body weight, macroscopic damage score according the Wallace scores [43], and cytokines levels were assessed. + +Statistical Analyses + +Values are expressed as mean±SEM. Statistical analysis were performed using GraphPad Prism 4.00 (GraphPad Software, San Diego, CA, USA) software package for PC. Single comparisons were performed by unpaired Student's t-test. A value of P<0.05 was considered as statistically significant. All P values were two sided. + +Supporting Information + +Table S1 + +Impact of Nod2 on body weight, gut weight and intestine length. At weeks 4, 12 and 52, we investigated the body and gut weight and the intestine length of KO and WT mice. No difference was observed between KO and WT mice (P>0.05). Data represent the means±SEM of 8 mice per group. + +(0.03 MB TIF) + +Click here for additional data file. + +Table S2 + +Ileal microflora under basal condition. Under basal condition, no difference was observed between KO and WT mice (P>0.05 for each bacterial group). Data represent the means±SEM of 10 mice per group. + +(0.03 MB TIF) + +Click here for additional data file. + +Figure S1 + +PPs from KO mice exhibit higher rates of CD4+ and CD4-CD8-T-cells at week 4. At week 4, CD3+ T-cells recovered from PPs (A) and spleen (B) were stained with antibodies to CD3, CD4, and CD8 from KO (▪) and WT (□) mice. Data were gated for CD3+ T-cells. Relative proportions of both CD3+CD4+ and CD3+CD4-CD8- T-cells were significantly higher in the PPs but not in the spleen (P>0.05) of KO mice. Data represent the means±SEM of 8 mice per group. *P<0.05; **P<0.01. + +(0.08 MB TIF) + +Click here for additional data file. + +Figure S2 + +Nod2 and CD3+ T -cells in Peyer's Patches. (A and B) Relative proportions of naïve, regulatory and memory T-cells in PPs (A) and spleens (B) of KO (▪) and WT (□) mice at week 12. CD4+ T-cells were stained with antibodies to CD25 and CD45RB. (C) Relative proportions of apoptotic CD3+ and CD3+CD4+ T-cells. Apoptotic CD3+ and CD3+CD4+ T-cells were investigated by flow cytometry using antibodies to CD3, CD4 and annexin V. Data were gated for CD3+CD4+ T-cells. Data represent the means±SEM of 8 mice per group. + +(0.04 MB TIF) + +Click here for additional data file. + +Acknowledgements + +We acknowledge Veronique Mégras, Catherine Martinet, Michel Peuchmaur, Régine Paris, Pascal Blain, Jean-Baptiste Huguet, Latifa Ferkdadji, Françoise Merlin, Robert Ducroc, Christèle Madre, Sarah Cheriet and Pauline Thabuteau for their excellent assistance. + +Figures and Tables + +Figure 1 + +Nod2 and postnatal development of gut associated lymphoid formations. + +(A) PP count on the whole intestines of KO (▪) and WT (□) mice at birth and at weeks 4, 12 and 52. (B) Number of isolated LFs identified on microscopic examination of the small intestine at weeks 12 and 52. (C and D) Number of cells per PP and spleen at weeks 4, 12 and 52). (E) Number of apoptotic cells identified by caspase-3 immunostaining at week 12. Data represent the means±SEM of 8 mice per group. *P<0.05; **P<0.01, ***P<0.001. + +Figure 2 + +Nod2 and Peyer's patch cellular composition. + +(A and B) Relative proportions of T-, B- and dendritic cells from PPs and spleens of KO (▪) and WT (□) mice at week 12. T-, B- and dendritic cells were investigated by flow cytometry using antibodies to CD3, B220 and CD11c. (C) Relative proportion of polymorphonuclear neutrophils was analyzed using Ly-6G antibody. (D) M cells number inside the follicle associated with epithelium was investigated by immuno-histochemistry. Arrows indicated the presence of M-cell inside the follicle associated with epithelium. Data represent the means±SEM of 8 mice per group. **P<0.01. + +Figure 3 + +Nod2 and T-cells subset in PP and spleen from mice at 12 weeks of age. + +Relative proportion of CD4+, CD8+ and CD4−CD8− T-cells from PPs (A) and spleen (B) of KO (▪) and WT (□) mice. At week 12, CD3+ T-cells were stained with antibodies to CD3, CD4, and CD8. Data were gated for CD3+ T-cells. Data represent the means±SEM of 8 mice per group. *P<0.05. + +Figure 4 + +Nod2 invalidation increases cytokine expression in PP. + +Expressions of Il-1β, IFNγ, TNFα, IL-12, IL-4 were determined by ELISA in PPs, Ileum and spleens from KO (▪) and WT (□) mice at weeks 4 (left panel) and 12 (right panel). Data represent the means±SEM of 8 mice per group. *P<0.05; **P<0.01. + +Figure 5 + +Paracellular permeability and bacterial translocation are increased in Nod2 invalidated mice. + +Ussing-chamber and Real-time PCR experiments were performed on PP and PP-free ileum from WT (□) and KO (▪) at week 12. (A) Paracellular permeability was analysed by FITC-dextran flux from PP and PP-free ileum under basal condition. (B) mRNA expression levels of TJ proteins (ZO-1, ZO-2 and Occ) from PP were analysed by real-Time-PCR under basal condition. (C) Bacterial translocation of chemically killed fluorescent Escherichia Coli K-12. (D) Translocation of the viable non enteropathogen Escherichia Coli strain J53. (E) Translocation of a chemically killed fluorescent Staphylococcus Aureus. (F) Translocation of chemically killed fluorescein-conjugated Saccharomyces cerevisiae. Data represent the means±SEM of 8 mice per group. *P<0.05 and **P<0.01, significantly different from WT. + +Figure 6 + +Nod2 invalidation increased the suceptibility of TNBS induced colitis. + +(A) Body weight, (B) Macroscopic damage score, (C) cytokines levels were assessed in 12 week old mice. These parameters were determined three days after intracolonic administration of TNBS. Values are mean (SEM) (n = 8 in each group). *P<0.05 and **P<0.01 between WT and KO mice. + +Figure 7 + +Targeting disruption of the murine Nod2 gene by homologous recombination. + +(A) Generation of the Card15/Nod2 KO allele: targeting strategy. The restriction maps of the Card15/Nod2+allele (5′ portion), the Card15/Nod2 KO targeting fragment, and the modified Card15/Nod2 allele after homologous recombination and Cre-mediated recombination of an Hygromycin selection cassette are shown. Exons 1, 2, and 3 (black boxes) and restriction sites used for cloning and screening (X) XbaI, (A) AgeI, (P) PmlI, (S) SalI, (N) NotI are indicated. The Card15/Nod2 KO targeting fragment comprises the EGFP gene (Clontech) in frame with Card15/Nod2 ATG and the floxed PGKHygromycin (Clontech) selection cassette, introduced between the AgeI site downstream of Card15/Nod2 exon 1 and the SalI site upstream of exon 1 in the Card15/Nod2 orientation. All loxP sites are represented by open triangles. Recombination of loxP1 and loxP2 results in the loxP1+2 site. In the first step of the strategy, the Card15/Nod2 locus was targeted with the Card15/Nod2 KO targeting fragment. In the second step, the PGKHygromycin selection cassette was removed by Cre recombinase. The double-headed arrows indicate the DNA fragments resulting from digestions with different enzymes expected to hybridize with probes A, B or Hyg. Also depicted are combinations of PCR primers P1-4 that detect the different Card15/Nod2 alleles. (B) Southern blot analysis of restricted DNA resulting from digestion with XbaI.Hybridation with Probe A and B show complete integration of the targetting fragment after homologous recombination. Probe Hyg show the differents integrations of targetting fragments after homologous recombination. (C) Genotyping of Nod2-deficient mice by PCR. Genomic DNA from mice was amplified by PCR to detect the disrupted sequence (PCR product of 459 bp). (D) Expression of Nod2 mRNA in spleen. RT-PCR was performed on purified mRNA from the spleen of WT and KO mice. As expected, no signal was observed in KO mice. GAPDH expression was used as positive control of expression. + +Footnotes + +Competing Interests: The authors have declared that no competing interests exist. + +Funding: This work was supported by the Institut National de la Santé et de la Recherche Médicale, the Broad Medical Research Program, la Mairie de Paris, la Fondation pour la Recherche Médicale, BREMICI, l'association François Aupetit and la région Ile de France. diff --git a/src/ontogpt/evaluation/craft/database/all/17590087.ann b/src/ontogpt/evaluation/craft/database/all/17590087.ann new file mode 100644 index 000000000..059f6c97f --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17590087.ann @@ -0,0 +1,643 @@ +T1 PR:000009158 12 17 ITPR1 +T2 http://purl.obolibrary.org/obo/MONDO_0000437 28 34 Ataxia +T3 NCBITaxon:10088 38 42 Mice +T4 UBERON:0014643 47 62 Spinocerebellar +T5 http://purl.obolibrary.org/obo/MONDO_0011694 47 72 Spinocerebellar Ataxia 15 +T6 NCBITaxon:9606 76 82 Humans +T7 GO:0030849 115 124 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PR:000009158 5940 5945 Itpr1 +T161 GO:0010467 5946 5956 expression +T162 GO:0030849 5985 5994 autosomal +T163 http://purl.obolibrary.org/obo/MONDO_0005395 6005 6022 movement disorder +T164 NCBITaxon:9606 6058 6063 human +T165 UBERON:0001016 6064 6076 neurological +T166 http://purl.obolibrary.org/obo/MONDO_0005071 6064 6084 neurological disease +T167 NCBITaxon:9606 6119 6124 human +T168 http://purl.obolibrary.org/obo/MONDO_0000001 6125 6134 disorders +T169 SO:0005858 6177 6192 syntenic region +T170 NCBITaxon:9606 6200 6205 human +T171 SO:0001026 6206 6212 genome +T172 http://purl.obolibrary.org/obo/MONDO_0011694 6264 6269 SCA15 +T173 UBERON:0007023 6274 6279 adult +T174 GO:0030849 6286 6295 autosomal +T175 http://purl.obolibrary.org/obo/MONDO_0000437 6317 6323 ataxia +T176 PR:000009158 6383 6388 ITPR1 +T177 PR:000009158 6490 6495 Itpr1 +T178 PR:000009158 6503 6508 Itpr1 +T179 NCBITaxon:10088 6512 6516 mice +T180 http://purl.obolibrary.org/obo/MONDO_0000437 6545 6551 ataxia +T181 NCBITaxon:10088 6559 6563 mice +T182 SO:0000704 6599 6603 gene +T183 http://purl.obolibrary.org/obo/MONDO_0011694 6627 6632 SCA15 +T184 SO:0001026 6647 6654 genomic +T185 http://purl.obolibrary.org/obo/MONDO_0000001 6729 6736 disease +T186 http://purl.obolibrary.org/obo/MONDO_0011694 6795 6800 SCA15 +T187 SO:0000195 6964 6976 coding exons +T188 PR:000009158 6980 6985 ITPR1 +T189 SO:0001026 7003 7009 genome +T190 SO:0000694 7015 7018 SNP +T191 http://purl.obolibrary.org/obo/MONDO_0000001 7104 7111 disease +T192 SO:0001023 7256 7262 allele +T193 SO:0001026 7290 7296 genome +T194 SO:0000694 7302 7305 SNP +T195 SO:0001026 7380 7387 genomic +T196 PR:000009158 7427 7432 ITPR1 +T197 SO:0000704 7476 7480 gene +T198 PR:000015827 7482 7487 SUMF1 +T199 SO:0000694 7652 7655 SNP +T200 SO:0000694 7732 7736 SNPs +T201 SO:0000239 7744 7752;7761 7768 flanking ... regions +T202 SO:0000687 7812 7822;7827 7835 borders of ... deletion +T203 SO:0001026 8092 8098 genome +T204 SO:0000694 8104 8107 SNP +T205 NCBITaxon:1 8178 8189 individuals +T206 NCBITaxon:1 8238 8249 individuals +T207 UBERON:0001016 8268 8280 neurological +T208 http://purl.obolibrary.org/obo/MONDO_0005071 8268 8289 neurological disorder +T209 SO:0000704 8363 8367 gene +T210 PR:000009158 8369 8374 ITPR1 +T211 PR:000015827 8378 8383 SUMF1 +T212 NCBITaxon:1 8420 8430 individual +T213 SO:0000188 8503 8509 intron +T214 PR:000009158 8519 8524 ITPR1 +T215 SO:0000147 8550 8554 exon +T216 GO:0010467 8626 8636 expression +T217 GO:0008380 8640 8648 splicing +T218 PR:000009158 8652 8657 ITPR1 +T219 SO:0001415 8690 8704;8725 8733 breakpoints of ... deletion +T220 http://purl.obolibrary.org/obo/MONDO_0000001 8709 8716 disease +T221 SO:0000121 9025 9052 forward orientation primers +T222 SO:0000132 9113 9140 reverse orientation primers +T223 SO:0000112 9293 9300 primers +T224 SO:0000028 9394 9396 bp +T225 SO:0000028 9534 9536 bp +T226 SO:0000147 9587 9592 exons +T227 PR:000015827 9596 9601 SUMF1 +T228 SO:0000147 9630 9635 exons +T229 PR:000009158 9639 9644 ITPR1 +T230 UBERON:0001016 9763 9777 neurologically +T231 SO:0000704 9817 9824 genetic +T232 PR:000009158 9837 9842 ITPR1 +T233 http://purl.obolibrary.org/obo/MONDO_0011694 9859 9864 SCA15 +T234 http://purl.obolibrary.org/obo/MONDO_0000557 9909 9936 inherited cerebellar ataxia +T235 UBERON:0002037 9919 9929 cerebellar +T236 UBERON:0001016 10003 10012 neurology +T237 http://purl.obolibrary.org/obo/MONDO_0011694 10242 10247 SCA15 +T238 PR:000015827 10292 10297 SUMF1 +T239 PR:000009158 10306 10311 ITPR1 +T240 http://purl.obolibrary.org/obo/MONDO_0000001 10345 10352 disease +T241 SO:0001021 10465 10475 breakpoint +T242 SO:0000028 10552 10554 bp +T243 SO:0000147 10565 10570 exons +T244 PR:000015827 10578 10583 SUMF1 +T245 PR:000009158 10596 10601 ITPR1 +T246 SO:0000147 10688 10693 exons +T247 PR:000015827 10701 10706 SUMF1 +T248 SO:0000147 10711 10716 exons +T249 PR:000009158 10725 10730 ITPR1 +T250 SO:0000147 10880 10885 exons +T251 PR:000015827 10897 10902 SUMF1 +T252 UBERON:0002037 11009 11019 cerebellar +T253 http://purl.obolibrary.org/obo/MONDO_0000437 11009 11026 cerebellar ataxia +T254 PR:000015827 11050 11055 SUMF1 +T255 PR:000009158 11056 11061 ITPR1 +T256 SO:0000704 11223 11230 genetic +T257 http://purl.obolibrary.org/obo/MONDO_0000001 11244 11251 disease +T258 http://purl.obolibrary.org/obo/MONDO_0011694 11258 11263 SCA15 +T259 PR:000015827 11407 11412 SUMF1 +T260 PR:000015827 11423 11451 sulfatase modifying factor 1 +T261 http://purl.obolibrary.org/obo/MONDO_0011694 11488 11493 SCA15 +T262 PR:000015827 11518 11523 SUMF1 +T263 GO:0030849 11535 11544 autosomal +T264 http://purl.obolibrary.org/obo/MONDO_0010088 11555 11584 multiple sulfatase deficiency +T265 GO:0008152 11588 11597 metabolic +T266 http://purl.obolibrary.org/obo/MONDO_0005066 11588 11606 metabolic disorder +T267 http://purl.obolibrary.org/obo/MONDO_0000437 11705 11711 ataxia +T268 http://purl.obolibrary.org/obo/MONDO_0010088 11774 11803 multiple sulfatase deficiency +T269 PR:000009158 11829 11834 ITPR1 +T270 http://purl.obolibrary.org/obo/MONDO_0000437 11875 11881 ataxia +T271 GO:0010467 11905 11914 expressed +T272 CL:0000121 11918 11932 Purkinje cells +T273 NCBITaxon:10088 11957 11961 mice +T274 http://purl.obolibrary.org/obo/MONDO_0000437 12003 12009 ataxia +T275 CHEBI:29108 12025 12029 Ca2+ +T276 GO:0019722 12025 12039 Ca2+ signaling +T277 http://purl.obolibrary.org/obo/MONDO_0000437 12090 12096 ataxia +T278 http://purl.obolibrary.org/obo/MONDO_0007163 12109 12131 episodic ataxia type 2 +T279 http://purl.obolibrary.org/obo/MONDO_0008457 12136 12140 SCA6 +T280 PR:000009158 12215 12220 ITPR1 +T281 NCBITaxon:10376 12224 12242 Epstein-Barr virus +T282 NCBITaxon:10376 12244 12247 EBV +T283 CL:0000542 12262 12273 lymphocytes +T284 PR:000009158 12388 12393 ITPR1 +T285 PR:000009158 12472 12477 Itpr1 +T286 SO:0000417 12493 12500 domains +T287 CHEBI:25450 12516 12537 inositol triphosphate +T288 SO:0100018 12538 12552 binding domain +T289 SO:0000417 12565 12571 domain +T290 GO:0016020 12595 12603 membrane +T291 SO:0000409 12683 12695 binding site +T292 PR:000009158 12697 12702 Itpr1 +T293 CHEBI:29108 12717 12721 Ca2+ +T294 CHEBI:29108 12747 12751 Ca2+ +T295 GO:0005783 12769 12790 endoplasmic reticulum +T296 GO:0005622 12812 12825 intracellular +T297 CHEBI:33280 12833 12842 messenger +T298 PR:000009158 12876 12881 Itpr1 +T299 CL:0000121 12901 12918;12923 12933 Purkinje cells of ... cerebellum +T300 UBERON:0002037 12923 12933 cerebellum +T301 PR:000009158 12939 12944 ITPR1 +T302 http://purl.obolibrary.org/obo/MONDO_0000001 13017 13024 disease +T303 PR:000009158 13066 13071 ITPR1 +T304 http://purl.obolibrary.org/obo/MONDO_0000001 13171 13179 disorder +T305 NCBITaxon:9606 13183 13189 humans +T306 NCBITaxon:10088 13222 13226 mice +T307 http://purl.obolibrary.org/obo/MONDO_0000001 13251 13259 disorder +T308 UBERON:0000104 13306 13315 life span +T309 NCBITaxon:10088 13323 13328 mouse +T310 SO:0000318 13387 13397 start site +T311 PR:000009158 13402 13407 ITPR1 +T312 GO:0042571 13608 13616 antibody +T313 PR:000009158 13658 13663 ITPR1 +T314 http://purl.obolibrary.org/obo/MONDO_0000001 13688 13695 disease +T315 PR:000009158 13773 13778 ITPR1 +T316 http://purl.obolibrary.org/obo/MONDO_0011694 13800 13805 SCA15 +T317 http://purl.obolibrary.org/obo/MONDO_0000001 13859 13867 disorder +T318 NCBITaxon:10088 13924 13929 mouse +T319 NCBITaxon:9606 13957 13962 human +T320 http://purl.obolibrary.org/obo/MONDO_0000001 13963 13970 disease +T321 PR:000009158 14008 14013 Itpr1 +T322 NCBITaxon:33208 14020 14027 animals +T323 NCBITaxon:10088 14062 14066 mice +T324 http://purl.obolibrary.org/obo/MONDO_0011694 14122 14127 SCA15 +T325 NCBITaxon:10088 14153 14157 mice +T326 http://purl.obolibrary.org/obo/MONDO_0011694 14212 14217 SCA15 +T327 SO:0001026 14252 14258 genome +T328 SO:0000694 14264 14267 SNP +T329 SO:0001026 14319 14326 genomic +T330 http://purl.obolibrary.org/obo/MONDO_0000001 14355 14362 disease +T331 PR:000009158 14460 14465 ITPR1 +T332 http://purl.obolibrary.org/obo/MONDO_0011694 14473 14478 SCA15 +T333 PR:000009158 14510 14515 ITPR1 +T334 http://purl.obolibrary.org/obo/MONDO_0011694 14529 14534 SCA15 +T335 http://purl.obolibrary.org/obo/MONDO_0000001 14581 14588 disease +T336 PR:000009158 14779 14784 ITPR1 +T337 http://purl.obolibrary.org/obo/MONDO_0000001 14808 14815 disease +T338 SO:0000704 14833 14837 gene +T339 http://purl.obolibrary.org/obo/MONDO_0011694 14883 14888 SCA16 +T340 GO:0030849 14893 14902 autosomal +T341 http://purl.obolibrary.org/obo/MONDO_0021140 14912 14922 congenital +T342 http://purl.obolibrary.org/obo/MONDO_0000437 14938 14944 ataxia +T343 http://purl.obolibrary.org/obo/MONDO_0011694 15008 15013 SCA15 +T344 PR:000009158 15029 15034 ITPR1 +T345 SO:0000704 15040 15044 gene +T346 GO:0005622 15137 15150 intracellular +T347 GO:0035556 15137 15150;15156 15165 intracellular ... signaling +T348 CHEBI:29108 15151 15155 Ca2+ +T349 GO:0019722 15151 15165 Ca2+ signaling +T350 CL:0000121 15169 15183 Purkinje cells +T351 UBERON:0014643 15207 15222 spinocerebellar +T352 http://purl.obolibrary.org/obo/MONDO_0000437 15223 15229 ataxia +T353 SO:0001026 15255 15261 Genome +T354 NCBITaxon:10088 15278 15282 mice +T355 SO:0001026 15348 15354 genome +T356 SO:0000694 15409 15413 SNPs +T357 NCBITaxon:10088 15544 15548 mice +T358 NCBITaxon:10088 15569 15573 mice +T359 CHEBI:37958 15609 15612 dye +T360 http://purl.obolibrary.org/obo/MONDO_0000001 15884 15891 disease +T361 NCBITaxon:10088 15997 16001 mice +T362 NCBITaxon:10088 16030 16034 mice +T363 http://purl.obolibrary.org/obo/MONDO_0000001 16046 16053 disease +T364 SO:0000357 16084 16092 flanking +T365 SO:0000704 16219 16224 genes +T366 SO:0002138 16229 16250 predicted transcripts +T367 SO:0000704 16286 16293 genetic +T368 NCBITaxon:10088 16304 16308 mice +T369 NCBITaxon:10088 16351 16355 mice +T370 PR:000009158 16440 16445 Itpr1 +T371 NCBITaxon:10088 16453 16458 mouse +T372 http://purl.obolibrary.org/obo/MONDO_0000001 16469 16476 disease +T373 SO:0000147 16522 16527 exons +T374 PR:000009158 16541 16546 Itpr1 +T375 SO:0000112 16548 16554 Primer +T376 SO:0000195 16599 16611 coding exons +T377 SO:0000028 16628 16630 bp +T378 SO:0000239 16639 16647;16657 16665 flanking ... sequence +T379 SO:0000188 16648 16656 intronic +T380 PR:000009158 16669 16674 Itpr1 +T381 SO:0000147 16702 16706 exon +T382 NCBITaxon:10088 16749 16753 mice +T383 PR:000009158 16772 16777 Itpr1 +T384 NCBITaxon:10088 16838 16842 mice +T385 NCBITaxon:10088 16912 16916 mice +T386 PR:000009158 16956 16961 Itpr1 +T387 NCBITaxon:10088 16980 16985 mouse +T388 PR:000009158 17007 17012 Itpr1 +T389 PR:000009158 17026 17031 Itpr1 +T390 NCBITaxon:10088 17072 17076 mice +T391 PR:000009158 17107 17112 Itpr1 +T392 GO:0007618 17170 17176 mating +T393 GO:0007618 17207 17213 mating +T394 PR:000009158 17267 17272 Itpr1 +T395 PR:000009158 17284 17289 Itpr1 +T396 NCBITaxon:10088 17297 17301 mice +T397 PR:000009158 17316 17321 Itpr1 +T398 NCBITaxon:10088 17333 17337 mice +T399 UBERON:0000955 17487 17493 brains +T400 GO:0007567 17503 17508 natal +T401 CHEBI:9754 17574 17578 Tris +T402 CHEBI:17883 17579 17582 HCl +T403 CHEBI:26710 17591 17595 NaCl +T404 CHEBI:9750 17611 17623 Triton X-100 +T405 CHEBI:9177 17628 17647 sodium deoxycholate +T406 CHEBI:8984 17654 17657 SDS +T407 CHEBI:60004 17665 17673 cocktail +T408 MOP:0000569 17782 17790 reducing +T409 CHEBI:8984 17890 17893 SDS +T410 GO:0042571 17923 17933 antibodies +T411 PR:000009158 17937 17942 Itpr1 +T412 PR:000003676 17957 17961 Actb +T413 UBERON:0000955 18039 18045 Brains +T414 NCBITaxon:10088 18074 18078 mice +T415 CHEBI:50913 18159 18167 fixative +T416 UBERON:0000955 18169 18175 Brains +T417 CHEBI:5291 18193 18200 gelatin +T418 UBERON:0000955 18385 18391 brains +T419 UBERON:0000955 18416 18421 Brain +T420 CHEBI:75958 18499 18507 solution +T421 NCBITaxon:9925 18542 18546 goat +T422 UBERON:0001977 18547 18552 serum +T423 CHEBI:9750 18562 18574 Triton X-100 +T424 GO:0042571 18638 18648 antibodies +T425 PR:000009158 18679 18684 Itpr1 +T426 GO:0042571 18685 18693 antibody +T427 PR:000004967 18773 18778 Calb1 +T428 GO:0042571 18779 18787 antibody +T429 CHEBI:75958 18861 18869 solution +T430 GO:0042571 18982 18992 antibodies +T431 CHEBI:52673 18994 19009 Alexa Fluor 555 +T432 NCBITaxon:9925 19010 19014 goat +T433 NCBITaxon:9986 19020 19026 rabbit +T434 GO:0071735 19027 19030 IgG +T435 CHEBI:52661 19035 19050 Alexa Fluor 488 +T436 NCBITaxon:9925 19051 19055 goat +T437 NCBITaxon:10088 19061 19066 mouse +T438 GO:0071735 19067 19070 IgG +T439 UBERON:0000955 19555 19561 brains +T440 PR:000009158 19582 19587 Itpr1 +T441 GO:0042571 19588 19598 antibodies +T442 GO:0042571 19650 19658 Antibody +T443 UBERON:0000479 19730 19736 Tissue +T444 GO:0042571 19782 19792 antibodies +T445 PR:000009158 19913 19918 ITPR1 +T446 http://purl.obolibrary.org/obo/MONDO_0011694 19922 19927 SCA15 +T447 NCBITaxon:10376 19962 19965 EBV +T448 CL:0000542 19979 19990 lymphocytes +T449 SO:0000195 20025 20037 coding exons +T450 SO:0000028 20054 20056 bp +T451 SO:0000357 20060 20068 flanking +T452 SO:0000188 20069 20076 introns +T453 PR:000009158 20080 20085 ITPR1 +T454 CHEBI:37958 20125 20128 dye +T455 SO:0001026 20426 20433 genomic +T456 SO:0000112 20476 20482 Primer +T457 SO:0000704 20582 20586 Gene +T458 SO:0001026 20633 20639 Genome +T459 SO:0000694 20645 20648 SNP +T460 SO:0000694 20701 20704 SNP +T461 SO:0000694 20829 20833 SNPs +T462 SO:0001023 21040 21046 allele +T463 SO:0001026 21083 21089 genome +T464 PR:000009158 21194 21199 ITPR1 +T465 SO:0001023 21263 21269 allele +T466 NCBITaxon:1 21342 21353 individuals +T467 SO:0000357 21507 21515 flanking +T468 SO:0000112 21566 21573 primers +T469 SO:0000239 21657 21674 bordering regions +T470 SO:0000112 21676 21682 primer +T471 SO:0000112 21747 21753 primer +T472 SO:0000239 21777 21792 flanking region +T473 SO:0000239 21825 21840 flanking region +T474 SO:0000028 21887 21889 bp +T475 SO:0001026 21939 21946 genomic +T476 NCBITaxon:1 21983 21994 individuals +T477 CHEBI:37958 22026 22029 Dye +T478 SO:0000121 22092 22099;22112 22119 forward ... primers +T479 SO:0000132 22104 22119 reverse primers +T480 SO:0001026 22270 22276 genome +T481 SO:0001026 22386 22392 genome +T482 SO:0001026 22418 22425 genomic +T483 SO:0001023 22480 22486 allele +T484 SO:0001023 22501 22507 allele +T485 NCBITaxon:1 22625 22636 individuals +T486 SO:0001024 22661 22670 haplotype +T487 SO:0000357 22776 22784 flanking +T488 SO:0000121 22883 22898 forward primers +T489 SO:0000357 22929 22937 flanking +T490 SO:0000132 22978 22993 reverse primers +T491 SO:0000357 23024 23032 flanking +T492 SO:0001021 23311 23321 breakpoint +T493 SO:0000121 23369 23383 forward primer +T494 SO:0000132 23455 23469 reverse primer +T495 SO:0000028 23558 23560 bp +T496 NCBITaxon:9606 23644 23649 human +T497 SO:0001026 23650 23656 genome +T498 SO:0001415 23720 23740 deletion breakpoints +T499 SO:0000028 23796 23798 bp +T500 SO:0000006 23799 23810 PCR product +T501 SO:0001021 23822 23832 breakpoint +T502 SO:0000112 23879 23885 primer +T503 SO:0001021 24027 24037 breakpoint +T504 http://purl.obolibrary.org/obo/MONDO_0011694 24079 24084 SCA15 +T505 NCBITaxon:10376 24096 24099 EBV +T506 GO:0019835 24365 24370 lysis +T507 CHEBI:9750 24400 24412 Triton X-100 +T508 CHEBI:60004 24420 24428 cocktail +T509 GO:0019835 24475 24480 lysis +T510 MOP:0000569 24546 24554 reducing +T511 CHEBI:8984 24640 24643 SDS +T512 GO:0042571 24673 24683 antibodies +T513 PR:000009158 24687 24692 ITPR1 +T514 PR:000003676 24707 24711 ACTB +T515 NCBITaxon:10088 24840 24845 Mouse +T516 NCBITaxon:10088 24871 24875 Mice +T517 http://purl.obolibrary.org/obo/MONDO_0000001 25174 25181 disease +T518 SO:0000147 25349 25353 Exon +T519 PR:000009158 25360 25365 Itpr1 +T520 NCBITaxon:10088 25376 25380 Mice +T521 NCBITaxon:10088 25414 25419 mouse +T522 NCBITaxon:10088 25458 25463 mouse +T523 NCBITaxon:10088 25489 25493 mice +T524 SO:0000028 25516 25518 bp +T525 NCBITaxon:10088 25560 25565 mouse +T526 SO:0000028 25590 25592 bp +T527 http://purl.obolibrary.org/obo/MONDO_0011694 25778 25783 SCA15 +T528 SO:0001023 25859 25865 allele +T529 SO:0000694 26049 26052 SNP +T530 SO:0000694 26059 26062 SNP +T531 SO:0001023 26141 26147 allele +T532 SO:0001023 26205 26212 alleles +T533 SO:0000694 26246 26249 SNP +T534 SO:0000694 26283 26287 SNPs +T535 SO:0001023 26297 26303 allele +T536 SO:0000694 26351 26355 SNPs +T537 SO:0001023 26365 26371 allele +T538 SO:0001023 26440 26446 allele +T539 SO:0001026 26657 26664 genomic +T540 SO:0000006 27002 27013 PCR product +T541 SO:0000112 27030 27037 primers +T542 SO:0001026 27056 27063 genomic +T543 SO:0001415 27126 27145 deletion breakpoint +T544 SO:0000028 27171 27173 bp +T545 SO:0000357 27216 27224 flanking +T546 SO:0000028 27386 27388 bp +T547 SO:0000028 27400 27408 Basepair +T548 SO:0001026 27437 27443 genome +T549 PR:000015827 27635 27640 SUMF1 +T550 http://purl.obolibrary.org/obo/MONDO_0011694 27684 27689 SCA15 +T551 NCBITaxon:10376 27754 27757 EBV +T552 NCBITaxon:10376 27760 27778 Epstein-Barr virus +T553 UBERON:0014643 27794 27809 spinocerebellar +T554 http://purl.obolibrary.org/obo/MONDO_0000437 27810 27816 ataxia +T555 SO:0000694 27827 27830 SNP +T556 SO:0000694 27833 27863 single nucleotide polymorphism +T557 PR:000009158 27945 27950 ITPR1 +T558 NCBITaxon:10088 27969 27974 Mouse +T559 UBERON:0002037 27975 27985 Cerebellum +T560 UBERON:0002037 28017 28027 cerebellum +T561 NCBITaxon:10088 28045 28050 mouse +T562 NCBITaxon:10088 28064 28069 mouse +T563 PR:000009158 28091 28096 Itpr1 +T564 SO:0000028 28100 28102 bp +T565 NCBITaxon:10088 28129 28134 mouse +T566 SO:0000028 28157 28159 bp +T567 PR:000009158 28160 28165 Itpr1 +T568 PR:000009158 28230 28235 Itpr1 +T569 NCBITaxon:9986 28241 28247 rabbit +T570 GO:0042571 28248 28256 antibody +T571 CHEBI:52673 28267 28282 Alexa Fluor 555 +T572 PR:000004967 28329 28334 Calb1 +T573 NCBITaxon:10088 28340 28345 mouse +T574 GO:0042571 28346 28354 antibody +T575 CHEBI:52661 28365 28380 Alexa Fluor 488 +T576 PR:000009158 28434 28439 Iptr1 +T577 GO:0010467 28450 28459 expressed +T578 CL:0000121 28467 28481 Purkinje cells +T579 PR:000009158 28542 28547 Itpr1 +T580 NCBITaxon:10088 28590 28594 mice +T581 PR:000009158 28635 28640 Itpr1 +T582 UBERON:0000955 28657 28662 brain +T583 PR:000009158 28679 28684 Itpr1 +T584 PR:000009158 28696 28701 Itpr1 +T585 NCBITaxon:10088 28709 28713 mice +T586 PR:000009158 28749 28754 Itpr1 +T587 UBERON:0000955 28758 28763 brain +T588 UBERON:0000479 28764 28770 tissue +T589 PR:000009158 28776 28781 Itpr1 +T590 NCBITaxon:10088 28788 28792 mice +T591 PR:000009158 28820 28825 Itpr1 +T592 PR:000009158 28829 28834 Itpr1 +T593 NCBITaxon:10088 28842 28846 mice +T594 SO:0001023 29028 29034 allele +T595 SO:0000105 29091 29108 arm of Chromosome +T596 SO:0000694 29186 29189 SNP +T597 SO:0000694 29196 29199 SNP +T598 SO:0001023 29278 29284 allele +T599 SO:0001023 29342 29349 alleles +T600 SO:0000694 29383 29386 SNP +T601 SO:0000694 29420 29424 SNPs +T602 SO:0001023 29434 29440 allele +T603 SO:0000694 29488 29492 SNPs +T604 SO:0001023 29502 29508 allele +T605 SO:0001023 29577 29583 allele +T606 SO:0001026 29841 29848 genomic +T607 SO:0000704 29914 29919 genes +T608 PR:000009158 29947 29952 ITPR1 +T609 PR:000015827 29957 29962 SUMF1 +T610 http://purl.obolibrary.org/obo/MONDO_0011694 30011 30016 SCA15 +T611 NCBITaxon:1 30083 30094 individuals +T612 NCBITaxon:1 30121 30132 individuals +T613 http://purl.obolibrary.org/obo/MONDO_0000001 30162 30169 disease +T614 PR:000009158 30239 30244 ITPR1 +T615 PR:000009158 30279 30284 ITPR1 +T616 SO:0000112 30318 30324 primer +T617 http://purl.obolibrary.org/obo/MONDO_0011694 30431 30436 SCA15 +T618 SO:0000112 30449 30455 primer +T619 SO:0000112 30559 30565 primer +T620 SO:0000112 30820 30826 primer +T621 SO:0001415 30862 30881 deletion breakpoint +T622 SO:0000028 30948 30950 bp +T623 NCBITaxon:1 30963 30974 individuals +T624 SO:0000112 31026 31032 primer +T625 http://purl.obolibrary.org/obo/MONDO_0000001 31132 31139 disease +T626 UBERON:0001016 31163 31177 neurologically +T627 PR:000009158 31269 31274 ITPR1 +T628 NCBITaxon:10376 31293 31296 EBV +T629 PR:000009158 31383 31388 ITPR1 +T630 NCBITaxon:10376 31399 31402 EBV +T631 CL:0000542 31416 31427 lymphocytes +T632 PR:000009158 31475 31480 ITPR1 +T633 http://purl.obolibrary.org/obo/MONDO_0000001 31532 31539 disease +T634 PR:000009158 31619 31624 ITPR1 +T635 PR:000009158 31662 31667 ITPR1 +T636 GO:0042571 31789 31797 antibody +T637 PR:000003676 31806 31810 ACTB +T638 CHEBI:33893 32245 32253 reagents +T639 GO:0007568 32416 32421 Aging +T640 UBERON:0001016 32452 32464 Neurological +T641 http://purl.obolibrary.org/obo/MONDO_0005071 32452 32474 Neurological Disorders +T642 http://purl.obolibrary.org/obo/MONDO_0005098 32479 32485 Stroke +T643 NCBITaxon:9606 32563 32568 Human diff --git a/src/ontogpt/evaluation/craft/database/all/17590087.txt b/src/ontogpt/evaluation/craft/database/all/17590087.txt new file mode 100644 index 000000000..7b21ae8e2 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17590087.txt @@ -0,0 +1,173 @@ +Deletion at ITPR1 Underlies Ataxia in Mice and Spinocerebellar Ataxia 15 in Humans + +Abstract + +We observed a severe autosomal recessive movement disorder in mice used within our laboratory. We pursued a series of experiments to define the genetic lesion underlying this disorder and to identify a cognate disease in humans with mutation at the same locus. Through linkage and sequence analysis we show here that this disorder is caused by a homozygous in-frame 18-bp deletion in Itpr1 (Itpr1Δ18/Δ18), encoding inositol 1,4,5-triphosphate receptor 1. A previously reported spontaneous Itpr1 mutation in mice causes a phenotype identical to that observed here. In both models in-frame deletion within Itpr1 leads to a decrease in the normally high level of Itpr1 expression in cerebellar Purkinje cells. Spinocerebellar ataxia 15 (SCA15), a human autosomal dominant disorder, maps to the genomic region containing ITPR1; however, to date no causal mutations had been identified. Because ataxia is a prominent feature in Itpr1 mutant mice, we performed a series of experiments to test the hypothesis that mutation at ITPR1 may be the cause of SCA15. We show here that heterozygous deletion of the 5′ part of the ITPR1 gene, encompassing exons 1–10, 1–40, and 1–44 in three studied families, underlies SCA15 in humans. + +Author Summary + + + +We have identified a spontaneous in-frame deletion mutation in the gene Itpr1 that causes a recessive movement disorder in mice. In an attempt to define whether any similar disease occurs in humans we performed a literature search for diseases linked to the human chromosomal region containing ITPR1. We identified the disease spinocerebellar ataxia 15 as linked to this region. High-density genomic analysis of affected members from three families revealed that disease in these patients was caused by deletion of a large portion of the region containing ITPR1. We show here that this mutation results in a dramatic reduction in ITPR1 in cells from these patients. These data show convincingly that ITPR1 deletion underlies spinocerebellar ataxia 15 in humans. + +Introduction + +The use of forward genetics to define novel loci of interest in human disease has become increasingly viable with the implementation of large-scale mutagenesis programs. Prior to these initiatives this work was carried out in part by the investigation of spontaneous mutations that cause disorders in mouse breeding colonies. Careful observation of these serendipitous events has led to the establishment and study of many in vivo disease models [3]. + +During the generation of a knockout line of mice we noted an early movement disorder that was inherited independently of targeting vector transmission. We embarked on a series of experiments to identify the genetic lesion underlying this movement disorder and to identify a cognate disease and corresponding mutation in humans. Here we describe this effort and the discovery of deletion at the ITPR1 locus as a cause of this disorder in mice and of spinocerebellar ataxia 15 (SCA15) in humans. + +Results/Discussion + +During the generation of a line of mice with knockout of the gene Park7 we noted an early movement disorder that was inherited independently of targeting vector transmission. Our initial observations suggested the affected mice suffered from an apparently paroxysmal movement disorder, often induced by touch. The abnormal movements occurred predominantly below the cervical level, and the disorder appeared progressive. At initial examination, a human movement disorder specialist (K. G.-H.) likened the disorder to episodic intermittent ataxia or kinesiogenic paroxysmal dystonia and predicted the involvement of an ion channel mutation in the etiology. Affected mice presented at approximately postnatal day 14, and survival time without weaning was on average 4 wk after onset. + +Breeding experiments suggested that the observed disorder was inherited in an autosomal recessive manner. To map the location of the disease-causing lesion, we performed genome-wide linkage analysis using strain-specific single nucleotide polymorphisms (SNPs) at 120 loci across the mouse genome. Analysis of these data showed a single genomic region with significant linkage to disease, providing a two-point LOD score of 5.13 at marker 20.MMHAP85FLG2 on Chromosome 6qE1. The linked haplotype suggested the mutation had occurred on the 129x1/SvJ background (Figure S1). + +Literature searches revealed that among disease lines mapped to 6qE1, the spontaneous mutant opt mouse displays a strikingly similar presentation to that described here [1]. The underlying genetic lesion causing the opt phenotype is a homozygous in-frame deletion of exons 43 and 44 of the gene Itpr1 (Itpr1opt/opt), encoding inositol 1,4,5-triphosphate receptor 1 (Itpr1). Sequencing of all exons and intron–exon boundaries of Itpr1 in affected mice from the current study revealed a single mutation within Itpr1: a novel in-frame deletion of 18 bp within exon 36 (Itpr1Δ18/Δ18). To confirm the pathogenicity of this mutation we crossed heterozygous mice from the current study (Itpr1wt/Δ18) with mice heterozygous for the opt mutation (Itpr1wt/opt). This resulted in two litters of mice with a total of four affected Itpr1opt/Δ18 pups (from a total of 15) with a phenotype indistinguishable from that of the Itpr1Δ18/Δ18 and Itpr1opt/opt mice [1]. Furthermore, this phenotype was similar, although less severe, to that described in a mouse line with targeted deletion of Itpr1, where ataxia was described as a prominent feature [4]. As with the Itpr1opt/opt mice, where the deletion of exons 43 and 44 is also predicted to leave the translational reading frame unaffected, the in-frame Itpr1Δ18/Δ18 deletion mutation results in markedly decreased levels of Itpr1 in cerebellar Purkinje cells. In these two spontaneous mutants [1] and in the Itpr1-deficient mouse [4] generated by gene targeting, decreased Itpr1 expression is associated with the same autosomal recessive movement disorder (Figure 1). + +Given our interest in human neurological disease we sought to identify any cognate human disorders where linkage had been established to the syntenic region of the human genome, but where no causal mutation had been identified. SCA15, an adult-onset autosomal dominant progressive ataxia is linked to this locus [5]. Although missense mutation of ITPR1 had previously been ruled out [2] and the mode of inheritance was inconsistent with that seen in the Itpr1Δ18 and Itpr1opt mice, the phenotypic presence of ataxia in the mice led us to reexamine this candidate gene as a possible cause of SCA15. + +We obtained genomic DNA from three affected family members and one family member with unknown disease status from the kindred originally used to define and map SCA15 (family AUS1, of Australian Anglo-Celtic origin) [2]. We performed two experiments concurrently in three affected members of this family: sequence analysis of the coding exons of ITPR1 and high-density genome-wide SNP genotyping. Sequence analysis failed to show any coding alterations segregating with disease or any alterations that were inconsistent with Mendelian patterns of inheritance within the family. However, visualization of log R ratio and B allele frequency metrics from the genome-wide SNP genotyping experiments clearly showed data consistent with a heterozygous genomic deletion across the first one-third of ITPR1 and across the first half of a neighboring gene, SUMF1 (Figure 2). This deletion was apparent in all three affected family members studied and absent from the family member with unknown affection status (Figure 3). The SNP data showed a deletion of between 188 kb and 210 kb in size; examination of SNPs at the flanking unknown regions of this deletion allowed us to delimit the borders of the deletion to 7.5 kb on the telomeric side of the deletion (between rs12634249 and rs793396) and ~14.4 kb on the centromeric side of the deletion (between rs4073665 and rs17709863). In an attempt to define whether this variation was a benign polymorphism we analyzed genome-wide SNP data at this locus, produced using the same genotyping chip, from 577 individuals of European descent who were either controls or individuals with an unrelated neurological disorder. We failed to find any deletions affecting the coding sequence of either gene, ITPR1 or SUMF1; we did, however, identify a single individual with a possible heterozygous deletion approximately 6 kb in size within intron 40–41 of ITPR1, at least 5 kb away from exon 40. Given the location of this alteration it is unlikely to effect the expression or splicing of ITPR1. + +In an attempt to fine-map the breakpoints of the disease-causing deletion we performed a series of experiments designed to refine the unknown intervals at the edges between definite deleted and definite diploid sequences. These data narrowed the unknown borders to ~4 kb on the telomeric side and ~7 kb on the centromeric side. We used all possible combinations of forward orientation primers designed within the newly defined telomeric boundary and of reverse orientation primers designed within the newly defined centromeric boundary in PCR assays in an attempt to amplify across the deletion in affected family members. Using PCR primers T3F and C11R, which should be more than 200 kb apart, we were able to amplify a fragment 953 bp in size using DNA from each of the three affected family members as template. Sequencing of this fragment revealed a deletion of 201,509 bp (Figure S3), removing the first three of the nine exons of SUMF1 and the first ten of the 58 exons of ITPR1. We were unable to amplify the deletion-specific fragment in the family member of unknown affection status, or in 275 neurologically normal controls. + +To further establish genetic deletion at ITPR1 as the cause of SCA15 we analyzed two additional families with an inherited cerebellar ataxia similar to that described in the AUS1 family, ascertained through neurology clinics in London, United Kingdom. DNA extracted from probands from these two families (family H33 and family H27) were also analyzed using Illumina Infinium HumanHap550 genotyping chips. These experiments showed deletion at the SCA15 locus in all affected members assayed, from SUMF1 through ITPR1. These mutations segregated with disease in these two families (Figure S3). A strategy similar to the one outlined above enabled us to sequence over the breakpoint in family H27 but not family H33. In the former, the deletion spans 344,408 bp, removing exons 1–3 of SUMF1 and 1–44 of ITPR1; in the latter, we estimate that the deletion is 310 kb in length and that it removes exons 1–3 of SUMF1 and exons 1–40 of ITPR1. The site of mutation is of interest, particularly the fact that in each of the three families the telomeric end of the deletion is anchored between exons 3 and 4 of SUMF1; sequence searches failed to identify any repeat sequences that might explain this phenomenon. With three cerebellar ataxia families segregating a SUMF1–ITPR1 deletion, and this deletion not observed in a control population, we may reasonably conclude that the association is causal, and that the deletion is indeed the genetic basis of the disease, with SCA15 the diagnosis in the two British families as well as the original Australian family. + +It is improbable that heterozygosity for the deletion of SUMF1, encoding sulfatase modifying factor 1, of itself causes or contributes to SCA15. Homozygous mutation of SUMF1 results in autosomal recessive multiple sulfatase deficiency, a metabolic disorder characterized by hepatosplenomegaly, deafness, and developmental delay [6,7]. No co-occurrence of ataxia has been described in (heterozygous) parents of patients with multiple sulfatase deficiency. Conversely, mutation of ITPR1 is biologically plausible as a cause of ataxia: the protein is highly expressed in Purkinje cells; as we have shown here, mice with mutation at this locus present with ataxia; and perturbed Ca2+ signaling has previously been implicated in the etiology of ataxia, notably in episodic ataxia type 2 and SCA6 [8]. In further support of this conclusion, analysis of protein levels of ITPR1 in Epstein-Barr virus (EBV) immortalized lymphocytes from affected and unaffected AUS1 family members revealed that all affected members showed a dramatic decrease in ITPR1 levels when compared with the family member without the deletion (Figure 4). + +Itpr1 contains three domains, an N-terminal inositol triphosphate binding domain, a coupling domain, and a C-terminal transmembrane domain; it also contains two protein kinase A phosphorylation sites and an ATP-binding site. Itpr1 is coupled to Ca2+ channels and facilitates Ca2+ release from the endoplasmic reticulum after binding by the intracellular second messenger inositol 1,4,5-triphosphate [9]. Itpr1 is enriched in the Purkinje cells of the cerebellum [4]. ITPR1 mutations have more than one potential pathogenic mechanism. First, the disease may be a result of haploinsufficiency at ITPR1; this concept is consistent with the observation that heterozygous deletion leads to a later onset disorder in humans, whereas homozygous deletion in mice leads to an early onset disorder, able to be expressed within the much shorter life span of the mouse. Second, we cannot rule out the existence of an alternate start site for ITPR1 that may result in a product that confers a pathogenic gain of function to the protein; however, Western blot analysis of cells derived from affected AUS1 family members, which was performed using an antibody raised against the C-terminal portion of ITPR1, failed to identify any disease-specific truncated protein products. Clearly, the identification of distinct ITPR1 mutations underlying SCA15 will help elucidate the pathogenic mechanism of this disorder. + +We show here the utility of investigating spontaneous mouse mutations in understanding human disease. Currently, the small number of aged Itpr1wt/Δ18 animals precludes us from examining these mice for subtle signs and symptoms similar to those seen in SCA15 patients; however, these mice are clearly of interest to us as a potential model of SCA15. These data also demonstrate that genome-wide SNP assay can facilitate rapid detection of structural genomic mutations that may underlie disease. The data provided by these approaches provide compelling evidence that heterozygous deletion of ITPR1 causes SCA15. Clearly, sequence analysis of ITPR1 in potential SCA15 cases may provide additional insight into the disease, particularly if a stop mutation were to be identified; however, the mutational mechanism noted here means that standard sequencing approaches alone are insufficient to confidently rule out ITPR1 mutation as a cause of disease: a comprehensive gene dosage approach is also required. Given that SCA16 and autosomal dominant congenital nonprogressive ataxia have both recently been mapped to regions overlapping with the SCA15 locus [10,11], ITPR1 is a gene of importance for screening in these families. These data add weight to a role for aberrant intracellular Ca2+ signaling in Purkinje cells in the pathogenesis of spinocerebellar ataxia. + +Materials and Methods + +Genome-wide linkage in mice. + +One hundred and twenty DNA fragments were amplified across the genome, each selected to contain one or more strain-specific SNPs that would differentiate between C57BL/6J and 129x1/SvJ inbred strains [12]. Each fragment was initially amplified in 11 affected mice and nine unaffected mice; genotype calling was performed by dye-terminator sequencing of these fragments. Linkage analysis using these data was performed using mlink [13], which revealed a positive linkage at Chromosome 6qE1, on the 129x1/SvJ background (two-point LOD score 5.13 at marker 20.MMHAP85FLG2). In an attempt to narrow the disease interval we performed backcross experiments that resulted in the generation of three additional affected mice. Genotyping of all affected mice across the disease-segregating interval revealed flanking recombinants and a candidate region of ~5 Mb, between markers D6Mit37 and 44.MMHAP85FLG5 (Figure S1). This region contains 16 genes and predicted transcripts. + +Identification of the underlying genetic lesion in mice. + +Identification of similar phenotypes in mice linked to the 6qE1 interval was performed by literature searches. This revealed the Itpr1opt/opt mouse, in which disease is caused by homozygous deletion mutation of exons 43 and 44 of Itpr1. Primer pairs were designed to sequence each of the coding exons and at least 50 bp of each flanking intronic sequence of Itpr1. PCR amplification of each exon was performed using DNA from two affected mice as templates. The Itpr1Δ18/Δ18 mutation was confirmed by sequencing in all affected mice (Figure S2). + +Breeding experiments were performed between two female mice heterozygous for the current mutation (Itpr1wt/Δ18) and a male mouse heterozygous for the Itpr1opt mutation (Itpr1wt/opt). This resulted in two litters of mice with a total of four affected Itpr1opt/Δ18 pups (from a total of 15; two of seven from first mating; two of eight from the second mating) with a phenotype indistinguishable from that of the Itpr1Δ18/Δ18 and Itpr1opt/opt mice. + +Analysis of Itpr1 protein in mice. + +We performed Western blot analyses using standard techniques with ECL detection kits (Amersham, http://www.amersham.com). Briefly, dissected whole brains from postnatal day 21 littermates were homogenized in a buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, and a cocktail of protease inhibitors (Roche, http://www.roche.com). Homogenates were diluted appropriately, mixed with 4× reducing sample buffer, and loaded onto 4%–12% precast gradient gels (Novex, http://www.invitrogen.com) for SDS-PAGE and immunoblotting. The antibodies to Itpr1 (1:2,000) and Actb (1:5,000) were used as recommended by manufacturers. + +Immunohistochemistry. + +Brains were isolated from 21-d-old mice, perfused with 4% paraformaldehyde in PBS, and post-fixed overnight in the same fixative. Brains were embedded in gelatin, and 35-μm sagittal sections were cut using a sliding microtome (NeuroScience Associates, http://www.neuroscienceassociates.com). Sections from wild-type, heterozygote, and homozygous brains were placed in the MultiBrain template. Sections were washed in 1× PBS prior to 1 h of incubation in block solution containing 1× PBS with 20% normal goat serum and 0.3% Triton X-100 (pH 7.4). Sections were incubated overnight at 4 °C in primary antibodies: affinity purified polyclonal Itpr1 antibody (1:2,000, Chemicon International, http://www.chemicon.com) and monoclonal anti-Calb1 antibody (1:6,000, Sigma-Aldrich, http://www.sigmaaldrich.com) diluted in carrier solution. Following extensive washes (in 6.0 ml of PBS, three times), sections were incubated with appropriate secondary antibodies (Alexa Fluor 555 goat anti-rabbit IgG and Alexa Fluor 488 goat anti-mouse IgG [Invitrogen, http://www.invitrogen.com]) for 1 h at room temperature. Sections were washed and mounted on glass slides in a buffered medium containing Mowiol (Calbiochem, http://www.emdbiosciences.com) as described earlier [14]. Sections were imaged using a laser scanning confocal microscope (LSM 510; Zeiss, http://www.zeiss.com). Imaging parameters (pinhole, detector gain, laser power) were optimized, and were kept constant for the wild-type, heterozygous, and homozygous mutant brains. Specificity of the Itpr1 antibodies was verified by preabsorption control experiments. Antibody dilutions were incubated for 24 h at 4 °C with the immunizing peptide. Tissue sections were incubated with the preabsorbed antibodies and processed as described above. Under these conditions, no staining above autofluorescence was detected. + +Analysis of ITPR1 in SCA15 patients. + +DNA was extracted from EBV immortalized lymphocytes, derived from family members. The coding exons and at least 50 bp of flanking introns of ITPR1 were PCR amplified and sequenced using dye-terminator sequencing (BigDye version 3.1; Applied Biosystems, http://www.appliedbiosystems.com). Sequence reactions were run on an ABI3730XP automated sequencer as per the manufacturer's instructions (Applied Biosystems). This analysis was performed in all three affected family members for whom genomic DNA was available (members 6, 7, and 19). Primer sequences and conditions are available upon request. Sequence data were analyzed using Sequencher (Gene Codes Corporation, http://www.genecodes.com). Genome-wide SNP genotyping was performed using Infinium HumanHap550 SNP genotyping chips as per the manufacturer's protocol (Illumina, http://www.illumina.com). This product assays 555,352 unique SNPs. Data were collected using the Illumina BeadStation scanner and data collection software. Genotypes were produced using the genotyping module of BeadStudio (version 2.3.25; Illumina), and log R ratio and B allele frequency were visualized using the genome viewer tool within this package. In order to rule out the possibility that the observed deletion within ITPR1 was a benign copy number variant we examined log R ratio and B allele frequency metrics of HumanHap550 genotyping data at this locus from 577 individuals of Northern European descent from North America and Europe, produced by us as a part of an ongoing study. + +In an attempt to narrow the unknown intervals flanking the deletion observed in family AUS1, we designed primers for 30 PCR amplifications that would generate overlapping fragments across the two bordering regions (primer sequence and conditions available upon request). There were ten primer pairs in the telomeric flanking region and 20 pairs in the centromeric flanking region (Figure S3). On average each product was ~750 bp in size, and amplifications were performed using genomic DNA from each of the three affected individuals (family members 6, 7, and 19). Dye-terminator sequencing of each product was performed using the forward and reverse primers designed for amplification; running and analysis of each fragment was performed as described above. Amplification of a fragment from a normal diploid genome was denoted by the presence of a heterozygous polymorphism; amplification of a fragment from a region of the genome harboring a heterozygous genomic deletion was inferred when homozygosity for the major allele and the minor allele were noted among the three affected family members (i.e., this is inconsistent with Mendelian inheritance in related individuals known to share a common haplotype). + +Using the data from the experiments described above we were able to limit the size of unknown regions flanking the deletion to ~4 kb on the telomeric side and 7 kb on the centromeric side. All combinations of forward primers from the newly defined region flanking the deletion on the telomeric side with reverse primers from the newly defined region flanking the deletion on the centromeric side were used in PCR amplification reactions performed with DNA from the three affected family members and single unaffected family members. This experiment was performed in an attempt to amplify across the deleted fragment and define the exact breakpoint. A single fragment was obtained from the third forward primer from the telomeric side (T3f 5′-TGAATGCTCAATTTTCCAGC-3′) with the 11th reverse primer from the centromeric side (C11r 5′-GGGAAAATGGATAGAGGGTG-3′). The fragment, which is 953 bp in size, was sequenced as described above and compared to the current build of the human genome. A similar series of experiments was performed to identify the deletion breakpoints in families H27 and H33; we were able to amplify a 369-bp PCR product across the breakpoint found in affected members of family H27 using primer pair H27-11F 5′-GACCTCAAGAAGGCATGAATAC-3′ and H27-3R 5′-ATGGTGGCCAGGTACACAAG-3′ (Figure S4), but to date we have been unable to identify the breakpoint in family H33. + +Western blot analysis in SCA15 patients. + +EBV immortalized lymphoblasts from three affected family members who carry the deletion and one family member without the mutation were used as a readily accessible source of protein; all samples came from members of family AUS1. Protein extraction was performed using lysis buffer containing 1× TBS, 1% Triton X-100, and a cocktail of protease inhibitors (Roche) with overnight lysis at −80 °C. Homogenates were diluted appropriately, mixed with 4× reducing sample buffer, and loaded onto 4%–12% precast gradient gels (NuPAGE, Invitrogen) for SDS-PAGE and immunoblotting. The antibodies to ITPR1 (1:1,000) and ACTB (1:5,000) were used as recommended by manufacturers. + +Supporting Information + +Figure S1 + +Schematic of Genotyping Results across Mouse Chromosome 6 in Affected Mice + +Black squares are indicative of a C57BL/6J homozygous genotype; light grey squares, a 129x1/SvJ homozygous genotype; grey squares, a C57BL/6J 129x1/SvJ heterozygous genotype; white squares, undetermined genotype. The black box bounds a region of homozygous 129x1/SvJ genotypes that segregate with disease; thus, the critical region was determined to be between markers D6Mit37 and 44.MMHAP85FLG5. + +(10 MB TIF) + +Click here for additional data file. + +Figure S2 + +Sequence of Exon 36 of Itpr1 from Four Mice + +A wild-type homozygous C57BL/6J mouse (A), a wild-type homozygous 129x1/SvJ mouse (B), an affected 129x1B6 mice homozygous for the 18-bp deletion mutation (C), and an unaffected mouse heterozygous for the 18-bp deletion mutation (D). The deleted nucleotides are bounded by a green box. + +(5.1 MB TIF) + +Click here for additional data file. + +Figure S3 + +Additional Families Harboring Deletion at the SCA15 Locus + +(A) Family H33; (B) family H27. Upper panel shows log R ratio and B allele frequency metrics generated from Infinium HumanHap550 arrays for an affected family member from each family. Log R ratio is the ratio of normalized, observed R to expected R for each SNP (each SNP is a blue dot) and thus serves as a surrogate of copy number at each locus. B allele frequency is a measure of the number of times the A or B alleles are detected at each locus (each SNP is denoted by a blue dot). Thus, SNPs with a B allele frequency of one are apparent B/B homozygotes, SNPs with a B allele frequency of 0.5 are apparent A/B heterozygotes, and those with a B allele frequency of zero are apparent A/A homozygotes. These plots show a contiguous region ~310 kb long (family H33) and ~350 kb long (family H27) with decreased copy number and apparent homozygosity indicative of a genomic deletion (shaded grey). The pedigrees below show the available family members assayed for these deletions, all of whom were affected and all of whom carried a deletion at this locus. + +(6.8 MB TIF) + +Click here for additional data file. + +Figure S4 + +Deleted Regions Identified in Families AUS1 and H27 + +(Top) Family AUS1; sequence from the PCR product generated using primers T3f and C11r from genomic DNA from an affected family member. Red arrowhead denotes the deletion breakpoint; the deletion is 201,510 bp in length. + +(Bottom) Family H27; sequence flanking deleted region. Green font indicates nucleotides telomeric to the deletion; blue font indicates nucleotides centromeric to the deletion. The deletion is 344,408 bp in length. Basepair positions are based on NCBI genome build 36 reference assembly. + +(876 KB MB TIF). + +Click here for additional data file. + +Accession Numbers + +The OMIM (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=OMIM) accession number for SUMF1 is 607939. + +Acknowledgements + +We thank the SCA15 family members for participating in this study. + +Abbreviations + +EBV - Epstein-Barr virus + +SCA[number] - spinocerebellar ataxia [number] + +SNP - single nucleotide polymorphism + +Figures and Tables + +Figure 1 + +Immunohistochemistry and Western Blot Analysis of ITPR1 Protein Levels in Mouse Cerebellum + +(A–F) Immunohistochemistry of cerebellum from a wild-type mouse (A and D), a mouse heterozygous for the Itpr1 18-bp deletion (B and E), and a mouse homozygous for the 18-bp Itpr1 deletion (C and F). (A–C) Immunohistochemistry using polyclonal Itpr1 anti-rabbit antibody (1:2,000; Alexa Fluor 555); (D–F) immunohistochemistry using monoclonal Calb1 anti-mouse antibody (1:6,000; Alexa Fluor 488). Scale bars denote 100 μm. As previously described, Iptr1 is highly expressed in the Purkinje cells. Notably, there appears to be decreased immunoreactivity to Itpr1 in the heterozygous and homozygous mutant mice. + +(G) Western blot performed to examine Itpr1 levels in whole brain from wild-type, Itpr1wt/Δ18, and Itpr1Δ18/Δ18 mice; this clearly shows a reduction of Itpr1 in brain tissue from Itpr1wt/Δ18 mice and a greater reduction of Itpr1 in Itpr1Δ18/Δ18 mice. + +Figure 2 + +Metrics Derived from Analysis of DNA from Affected Family Member 7 Using Illumina Infinium HumanHap550 Genotyping Chips + +The upper and lower plots are log R ratio and B allele frequency, respectively, at an ~800-kb segment on the p arm of Chromosome 3. Log R ratio is the ratio of normalized, observed R to expected R for each SNP (each SNP is a blue dot) and thus serves as a surrogate of copy number at each locus. B allele frequency is a measure of the number of times the A or B alleles are detected at each locus (each SNP is denoted by a blue dot). Thus, SNPs with a B allele frequency of one are apparent B/B homozygotes, SNPs with a B allele frequency of 0.5 are apparent A/B heterozygotes, and those with a B allele frequency of zero are apparent A/A homozygotes. Clearly, these plots show a contiguous region ~200 kb long with decreased copy number and apparent homozygosity (bounded by a red box). As we have demonstrated previously, this is indicative of a heterozygous genomic deletion [15]. Below these plots is a schematic of the two known genes affected by this deletion, ITPR1 and SUMF1. + +Figure 3 + +Mutation Analysis in the Australian SCA15 Family + +(Top) Pedigree of kindred. Filled symbols denote affected individuals; open symbols, unaffected individuals; grey symbol denotes unknown disease status; bulls-eye symbol denotes obligate carrier. w/w, wild-type at ITPR1; w/m, heterozygous carrier of the ITPR1 deletion. + +(Middle) Schematic of primer pairs used to narrow the unknown regions between known deleted sequence and known diploid sequence at the SCA15 locus. Nine primer pairs (T1–T9) were used to amplify across the unknown region telomeric to the known deleted region; 19 primer pairs (C1–C19) were used to amplify across the unknown region centromeric to the known deleted region. All PCRs were carried out in the three affected family members. Analysis of these data narrowed the unknown region, and ultimately we were able to use primer T3f and C11r to amplify across the deletion breakpoint in the three affected family members, producing a fragment of 953 bp in affected individuals. + +(Bottom) Gel showing amplification product using primer pair T3f and C11r from affected pedigree members 6, 7, and 19; in pedigree member 23, with unknown disease affection status; in a neurologically normal control (C); and in a no template control (NC). + +Figure 4 + +Western Blot Analysis of ITPR1 Protein Levels in EBV Immortalized Lymphoblasts from AUS1 Family Members + +Western blot performed to examine ITPR1 levels in EBV immortalized lymphocytes from AUS1 affected family members carrying the ITPR1 deletion and from an AUS1 family member of unknown disease status who does not carry the deletion. Notably the samples from patients with ITPR1 deletion show a dramatic decrease in ITPR1 levels. To demonstrate equal loading, these samples were diluted one in five, and the Western blot was repeated using an antibody against ACTB. + +Footnotes + +A previous version of this article appeared as an Early Online Release on May 16, 2007 (doi:10.1371/journal.pgen.0030108.eor). + +Author contributions. EMCF, JTR, and ABS conceived and designed the experiments. JvdL, JC, MAK, LAH, SS, MRC, HCF, XL, DH, JSS, IR, and HC performed the experiments. JvdL, JC, MAK, SS, MRC, KGH, HCF, JSS, JTR, and ABS analyzed the data. HH, NWW, PG, JH, ES, RJMG, SMF, HC, and ABS contributed reagents/materials/analysis tools. RJMG, EMCF, and ABS wrote the paper. + +Funding. This research was funded in part by the intramural programs of the National Institute on Aging and the National Institute on Neurological Disorders and Stroke (NINDS), both of the National Institutes of Health, Department of Health and Human Services, United States of America. MAK was supported by a NINDS Competitive Postdoctoral Fellowship. HH is supported by the Medical Research Council, United Kingdom. + +Competing interests. The authors have declared that no competing interests exist. diff --git a/src/ontogpt/evaluation/craft/database/all/17608565.ann b/src/ontogpt/evaluation/craft/database/all/17608565.ann new file mode 100644 index 000000000..cd32821f4 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17608565.ann @@ -0,0 +1,1952 @@ +T1 PR:000013773 0 2 Rb +T2 CL:0000540 12 20 Neuronal +T3 GO:0007049 45 55 Cell-Cycle +T4 GO:0065007 68 78 Regulation +T5 PR:000028775 82 87 E2f3a +T6 http://purl.obolibrary.org/obo/MONDO_0008380 139 153 retinoblastoma +T7 PR:000013773 139 161 retinoblastoma protein +T8 PR:000013773 163 165 Rb +T9 PR:000013773 252 254 Rb +T10 GO:0007049 271 281 cell cycle +T11 GO:0070997 300 308;314 321 death of ... neurons +T12 CL:0000540 314 321 neurons +T13 GO:0051716 469 481;503 508 responses in ... cells +T14 PR:000013773 482 484 Rb +T15 UBERON:0000966 495 502 retinal +T16 CL:0009004 495 508 retinal cells +T17 GO:0006915 547 556 apoptosis +T18 PR:000006852 594 618;625 626 E2f transcription factor ... 1 +T19 PR:000006852 620 623;625 626 E2f ... 1 +T20 UBERON:0000966 697 703 retina +T21 GO:0007049 712 722 cell-cycle +T22 CL:0000561 785 799 amacrine cells +T23 CHEBI:15355 808 819 cholinergic +T24 CL:0000099 820 832 interneurons +T25 PR:000013773 925 927 Rb +T26 UBERON:0000479 1024 1030 tissue +T27 PR:000013773 1070 1072 Rb +T28 PR:000006854 1116 1120 E2f3 +T29 PR:000006854 1135 1139 E2f3 +T30 SO:0001060 1140 1148 isoforms +T31 UBERON:0000966 1191 1197 retina +T32 PR:000013773 1294 1296 Rb +T33 GO:0044838 1309 1318 quiescent +T34 SO:0001060 1393 1400 isoform +T35 PR:000013773 1431 1433 Rb +T36 PR:000028775 1471 1476 E2f3a +T37 PR:000013773 1522 1524 Rb +T38 GO:0065007 1525 1534 regulates +T39 PR:000028775 1613 1618 E2f3a +T40 UBERON:0000479 1640 1646 tissue +T41 http://purl.obolibrary.org/obo/MONDO_0008380 1688 1702 retinoblastoma +T42 PR:000013773 1688 1710 retinoblastoma protein +T43 PR:000013773 1712 1714 Rb +T44 http://purl.obolibrary.org/obo/MONDO_0005070 1730 1735 tumor +T45 GO:0016265 1768 1773 death +T46 PR:000013773 1838 1840 Rb +T47 UBERON:0000479 1895 1901 tissue +T48 PR:000013773 1970 1972 Rb +T49 GO:0016265 2044 2049 death +T50 PR:000013773 2098 2100 Rb +T51 GO:0065007 2101 2109 controls +T52 GO:0016265 2120 2125 death +T53 UBERON:0000966 2154 2160 retina +T54 PR:000006852 2171 2175 E2f1 +T55 PR:000013773 2221 2223 Rb +T56 UBERON:0000966 2234 2241 retinal +T57 CL:0000540 2242 2249 neurons +T58 GO:0016265 2262 2267 death +T59 GO:0006915 2286 2295 apoptosis +T60 CL:0000210 2380 2394 photoreceptors +T61 PR:000013773 2405 2407 Rb +T62 CL:0000561 2428 2444 amacrine neurons +T63 PR:000006852 2480 2484 E2f1 +T64 PR:000013773 2551 2553 Rb +T65 CL:0000540 2557 2565 neuronal +T66 PR:000013773 2632 2634 Rb +T67 GO:0030154 2654 2674 cell differentiation +T68 PR:000028775 2702 2707 E2f3a +T69 GO:0007049 2714 2724 cell-cycle +T70 PR:000013773 2777 2779 Rb +T71 UBERON:0000966 2915 2921 retina +T72 UBERON:0000479 2940 2946 tissue +T73 GO:0022008 2956 2968 neurogenesis +T74 UBERON:0000966 3041 3048 retinal +T75 CL:0002672 3041 3064 retinal progenitor cell +T76 GO:0008283 3060 3064;3071 3084 cell ... proliferation +T77 CL:0002672 3066 3069 RPC +T78 GO:0007067 3112 3119 mitotic +T79 UBERON:0000966 3120 3127 retinal +T80 CL:0002672 3269 3273 RPCs +T81 GO:0007049 3303 3313 cell cycle +T82 GO:0007049 3455 3465 cell cycle +T83 GO:0007067 3488 3495 mitotic +T84 UBERON:0001781 3528 3542 retinal layers +T85 CL:0000604 3544 3548 Rods +T86 CL:0000573 3553 3558 cones +T87 UBERON:0001789 3571 3590 outer nuclear layer +T88 GO:0005634 3577 3584 nuclear +T89 UBERON:0001789 3592 3595 ONL +T90 CL:0000745 3598 3608;3632 3637 horizontal ... cells +T91 CL:0000103 3610 3617;3632 3637 bipolar ... cells +T92 CL:0000561 3623 3637 amacrine cells +T93 CL:0011107 3650 3666 Müller glia cell +T94 GO:0044297 3662 3673 cell bodies +T95 UBERON:0001791 3689 3708 inner nuclear layer +T96 GO:0005634 3695 3702 nuclear +T97 UBERON:0001791 3710 3713 INL +T98 UBERON:0000045 3720 3728 ganglion +T99 CL:0000740 3720 3728;3752 3757 ganglion ... cells +T100 CL:0000561 3743 3757 amacrine cells +T101 CL:0000740 3767 3780 ganglion cell +T102 UBERON:0001792 3767 3786 ganglion cell layer +T103 UBERON:0001792 3788 3791 GCL +T104 UBERON:0001790 3810 3831 outer plexiform layer +T105 UBERON:0001790 3833 3836 OPL +T106 UBERON:0001795 3842 3863 inner plexiform layer +T107 UBERON:0001795 3865 3868 IPL +T108 GO:0045202 3876 3896 synaptic connections +T109 UBERON:0001789 3912 3915 ONL +T110 UBERON:0001791 3916 3919 INL +T111 UBERON:0001791 3924 3927 INL +T112 UBERON:0001792 3928 3931 GCL +T113 PR:000006852 3958 3962 E2f1 +T114 PR:000006853 3972 3976 E2f2 +T115 PR:000006854 3980 3984 E2f3 +T116 GO:0008219 4020 4030 Cell Death +T117 PR:000013773 4038 4040 Rb +T118 UBERON:0000966 4044 4050 Retina +T119 UBERON:0000966 4056 4063 Retinal +T120 GO:0060041 4056 4075 Retinal development +T121 UBERON:0000966 4088 4094 retina +T122 CL:0002672 4116 4120 RPCs +T123 GO:0005634 4142 4148 nuclei +T124 CL:0002672 4151 4154 RPC +T125 GO:0044297 4155 4166 cell bodies +T126 GO:0042995 4183 4192 processes +T127 GO:0007049 4222 4232 cell cycle +T128 CL:0002672 4262 4266 RPCs +T129 GO:0007067 4276 4283 mitotic +T130 GO:0005634 4312 4318 nuclei +T131 UBERON:0001792 4346 4349 GCL +T132 CL:0002672 4381 4385 RPCs +T133 CL:0000604 4431 4435 rods +T134 CL:0000604 4437 4438 r +T135 CL:0000573 4444 4449 cones +T136 CL:0000573 4451 4452 c +T137 UBERON:0001789 4461 4464 ONL +T138 CL:0000745 4466 4476;4524 4529 horizontal ... cells +T139 CL:0000745 4478 4479 h +T140 CL:0000103 4482 4489;4524 4529 bipolar ... cells +T141 CL:0000103 4491 4492 b +T142 CL:0011107 4495 4501;4524 4529 Müller ... cells +T143 CL:0011107 4503 4504 m +T144 CL:0000561 4511 4519;4524 4529 amacrine ... cells +T145 CL:0000561 4521 4522 a +T146 UBERON:0001791 4537 4540 INL +T147 UBERON:0000045 4546 4554 ganglion +T148 CL:0000740 4546 4554;4582 4587 ganglion ... cells +T149 UBERON:0000045 4556 4557 g +T150 CL:0000740 4556 4557;4582 4587 g ... cells +T151 CL:0000561 4573 4587 amacrine cells +T152 UBERON:0001792 4595 4598 GCL +T153 PR:000013773 4638 4640 Rb +T154 GO:0065007 4655 4663 regulate +T155 GO:0007049 4664 4674 cell cycle +T156 GO:0006915 4679 4688 apoptosis +T157 UBERON:0000479 4756 4762 tissue +T158 PR:000013773 4804 4806 Rb +T159 GO:0016265 4900 4905 death +T160 GO:0065007 4929 4939 regulation +T161 SO:0000704 4959 4964 genes +T162 UBERON:0000966 4996 5003 retinal +T163 GO:0005634 5066 5072 nuclei +T164 CHEBI:51231 5074 5078 DAPI +T165 GO:0051320 5095 5102 S-phase +T166 CHEBI:472552 5109 5113 BrdU +T167 GO:0006915 5127 5136 apoptosis +T168 CHEBI:472552 5206 5210 BrdU +T169 CHEBI:472552 5231 5235 BrdU +T170 UBERON:0001792 5246 5249 GCL +T171 SO:0000704 5339 5344 genes +T172 UBERON:0000966 5351 5358 retinas +T173 NCBITaxon:33208 5439 5446 animals +T174 http://purl.obolibrary.org/obo/MONDO_0008380 5627 5641 retinoblastoma +T175 PR:000013773 5627 5649 retinoblastoma protein +T176 PR:000013773 5651 5653 Rb +T177 GO:0007049 5671 5681 cell cycle +T178 UBERON:0000966 5694 5701 retinal +T179 PR:000013773 5725 5727 Rb +T180 GO:0008283 5759 5770 proliferate +T181 CL:0000604 5797 5800;5824 5829 rod ... cells +T182 UBERON:0000045 5802 5810 ganglion +T183 CL:0000740 5802 5810;5824 5829 ganglion ... cells +T184 CL:0000103 5816 5829 bipolar cells +T185 GO:0016265 5831 5834 die +T186 GO:0006915 5838 5847 apoptosis +T187 PR:000013773 5855 5857 Rb +T188 GO:0065007 5858 5866 controls +T189 GO:0007049 5871 5881 cell cycle +T190 NCBITaxon:10508 6003 6013 adenoviral +T191 GO:0065007 6017 6027 regulatory +T192 SO:0005836 6017 6036 regulatory elements +T193 GO:0007049 6075 6085 cell cycle +T194 PR:000006852 6179 6183 E2f1 +T195 PR:000006853 6185 6189 E2f2 +T196 PR:000028775 6195 6200 E2f3a +T197 CL:0000057 6245 6255 fibroblast +T198 CL:0000057 6327 6338 fibroblasts +T199 PR:000013773 6383 6385 Rb +T200 PR:000013773 6413 6415 Rb +T201 UBERON:0000922 6419 6426 embryos +T202 UBERON:0000479 6474 6481 tissues +T203 PR:000006852 6498 6502 E2f1 +T204 PR:000006853 6504 6508 E2f2 +T205 PR:000006854 6513 6517 E2f3 +T206 PR:000013773 6563 6565 Rb +T207 PR:000006855 6623 6627 E2f4 +T208 PR:000006856 6632 6636 E2f5 +T209 SO:0000704 6741 6745 gene +T210 GO:0016458 6741 6755 gene silencing +T211 GO:0044838 6759 6768 quiescent +T212 GO:0006915 6828 6837 apoptosis +T213 PR:000013773 6845 6847 Rb +T214 UBERON:0000966 6851 6857 retina +T215 PR:000006852 6883 6887 E2f1 +T216 PR:000006852 7021 7025 E2f1 +T217 PR:000006854 7029 7033 E2f3 +T218 GO:0006915 7051 7060 apoptosis +T219 UBERON:0001017 7079 7101 central nervous system +T220 UBERON:0001017 7103 7106 CNS +T221 PR:000013773 7111 7113 Rb +T222 UBERON:0000922 7117 7124 embryos +T223 UBERON:0001017 7147 7150 CNS +T224 GO:0006915 7151 7160 apoptosis +T225 UBERON:0001987 7199 7208 placental +T226 PR:000006854 7255 7259 E2f3 +T227 GO:0006915 7268 7277 apoptosis +T228 CL:0000057 7281 7292 fibroblasts +T229 PR:000006852 7328 7332 E2f1 +T230 PR:000006854 7373 7377 E2f3 +T231 GO:0006915 7394 7403 apoptosis +T232 PR:000013773 7411 7413 Rb +T233 GO:0016265 7470 7475 death +T234 PR:000013773 7488 7490 Rb +T235 UBERON:0000479 7494 7501 tissues +T236 PR:000013773 7555 7557 Rb +T237 PR:000006854 7659 7663 E2f3 +T238 SO:0001060 7664 7672 isoforms +T239 SO:0000167 7686 7695 promoters +T240 SO:0000147 7770 7775 exons +T241 PR:000028775 7782 7787 E2f3a +T242 GO:0010467 7848 7858 expression +T243 GO:0044838 7875 7884 quiescent +T244 GO:0044838 7970 7979 quiescent +T245 GO:0044838 8007 8016 quiescent +T246 CL:0000057 8017 8028 fibroblasts +T247 PR:000013773 8058 8060 Rb +T248 GO:0016458 8118 8127 silencing +T249 PR:000005279 8132 8138 Cdkn2d +T250 PR:000005279 8140 8146 p19Arf +T251 SO:0001060 8225 8233 isoforms +T252 PR:000006852 8289 8293 E2f1 +T253 PR:000006853 8298 8302 E2f2 +T254 SO:0001060 8326 8334 isoforms +T255 GO:0065007 8371 8380 regulated +T256 SO:0001060 8554 8562 isoforms +T257 PR:000013773 8627 8629 Rb +T258 CL:0000540 8644 8652 neuronal +T259 GO:0016265 8821 8826 death +T260 PR:000013773 8831 8833 Rb +T261 GO:0065007 8839 8847 regulate +T262 CL:0000540 8928 8935 neurons +T263 PR:000013773 9010 9012 Rb +T264 UBERON:0000479 9140 9146 tissue +T265 UBERON:0000966 9206 9212 retina +T266 PR:000013773 9250 9252 Rb +T267 CL:0000604 9256 9260 rods +T268 PR:000013773 9280 9282 Rb +T269 GO:0009653 9310 9323 morphogenesis +T270 UBERON:0000966 9338 9344 retina +T271 PR:000013773 9408 9410 Rb +T272 CL:0000540 9414 9420 neuron +T273 GO:0006915 9476 9485 apoptosis +T274 GO:0030154 9529 9547;9554 9559 differentiation of ... cells +T275 PR:000013773 9548 9550 Rb +T276 GO:0008283 9586 9599 proliferation +T277 GO:0016265 9604 9609 death +T278 PR:000013773 9636 9638 Rb +T279 GO:0016265 9667 9672 death +T280 PR:000006852 9687 9691 E2f1 +T281 PR:000006853 9697 9701 E2f2 +T282 PR:000006854 9705 9709 E2f3 +T283 UBERON:0000966 9749 9756 retinal +T284 CL:0000540 9757 9764 neurons +T285 CL:0000604 9776 9780 rods +T286 UBERON:0000966 9853 9859 retina +T287 PR:000013773 9919 9921 Rb +T288 PR:000013773 9964 9966 Rb +T289 PR:000006852 9967 9971 E2f1 +T290 UBERON:0000966 9992 9998 retina +T291 CHEBI:15355 10036 10047 cholinergic +T292 CL:0000561 10058 10072 amacrine cells +T293 CL:0000099 10127 10139 interneurons +T294 PR:000013773 10324 10326 Rb +T295 UBERON:0000479 10376 10382 tissue +T296 PR:000013773 10428 10430 Rb +T297 PR:000006854 10467 10471 E2f3 +T298 PR:000013773 10484 10486 Rb +T299 PR:000006854 10522 10526 E2f3 +T300 GO:0010467 10527 10537 expression +T301 PR:000006854 10558 10562 E2f3 +T302 GO:0010467 10563 10573 expression +T303 CL:0000540 10588 10595 neurons +T304 PR:000013773 10624 10626 Rb +T305 PR:000006854 10628 10632 E2f3 +T306 UBERON:0001017 10679 10682 CNS +T307 CL:0000117 10679 10690 CNS neurons +T308 PR:000013773 10747 10749 Rb +T309 GO:0022008 10762 10774 neurogenesis +T310 PR:000006854 10841 10845 E2f3 +T311 SO:0001060 10846 10853 isoform +T312 PR:000013773 10854 10856 Rb +T313 GO:0065007 10868 10875 control +T314 PR:000013773 10912 10914 Rb +T315 GO:0044838 10927 10936 quiescent +T316 CL:0000057 10937 10948 fibroblasts +T317 PR:000028775 11008 11013 E2f3a +T318 SO:0001060 11051 11058 isoform +T319 NCBITaxon:10088 11073 11078 mouse +T320 PR:000013773 11092 11094 Rb +T321 PR:000028775 11130 11135 E2f3a +T322 PR:000006852 11158 11162 E2f1 +T323 GO:0016265 11196 11201 death +T324 PR:000013773 11203 11205 Rb +T325 GO:0065007 11211 11219 regulate +T326 CL:0000540 11220 11228 neuronal +T327 CL:0000540 11267 11274 neurons +T328 PR:000028775 11302 11307 E2f3a +T329 UBERON:0000479 11313 11319 tissue +T330 PR:000013773 11364 11366 Rb +T331 GO:0065007 11367 11376 Regulates +T332 GO:0016265 11390 11395 Death +T333 PR:000006852 11404 11408 E2f1 +T334 SO:0000902 11428 11437 transgene +T335 SO:0000359 11448 11454 floxed +T336 PR:000013773 11455 11457 Rb +T337 SO:0000147 11458 11462 exon +T338 UBERON:0000922 11469 11478 embryonic +T339 UBERON:0013682 11493 11510 peripheral retina +T340 PR:000013773 11516 11518 Rb +T341 SO:0000346 11518 11522 loxP +T342 SO:0000346 11523 11527 loxP +T343 NCBITaxon:10088 11534 11538 mice +T344 PR:000006852 11570 11574 E2f1 +T345 PR:000006853 11578 11582 E2f2 +T346 SO:0000359 11624 11630 floxed +T347 PR:000006854 11631 11635 E2f3 +T348 SO:0001023 11636 11642 allele +T349 PR:000013773 11648 11650 Rb +T350 SO:0000346 11650 11654 loxP +T351 SO:0000346 11655 11659 loxP +T352 PR:000006852 11660 11664 E2f1 +T353 PR:000013773 11672 11674 Rb +T354 SO:0000346 11674 11678 loxP +T355 SO:0000346 11679 11683 loxP +T356 PR:000006852 11684 11688 E2f1 +T357 NCBITaxon:10088 11698 11702 mice +T358 PR:000013773 11724 11726 Rb +T359 SO:0000346 11726 11730 loxP +T360 SO:0000346 11731 11735 loxP +T361 PR:000006852 11736 11740 E2f1 +T362 NCBITaxon:10088 11750 11754 mice +T363 PR:000013773 11876 11878 Rb +T364 SO:0000346 11878 11882 loxP +T365 SO:0000346 11883 11887 loxP +T366 PR:000006852 11888 11892 E2f1 +T367 UBERON:0013682 11902 11919 peripheral retina +T368 PR:000013773 11927 11929 Rb +T369 PR:000006852 11930 11934 E2f1 +T370 UBERON:0000966 11957 11963 retina +T371 PR:000006853 12013 12017 E2f2 +T372 PR:000006854 12021 12025 E2f3 +T373 PR:000013773 12052 12054 Rb +T374 PR:000006854 12059 12063 E2f3 +T375 SO:0001023 12064 12071 alleles +T376 UBERON:0000966 12079 12085 retina +T377 GO:0051301 12158 12171 cell division +T378 NCBITaxon:10088 12173 12177 mice +T379 CHEBI:472552 12203 12220 bromodeoxyuridine +T380 CHEBI:472552 12222 12226 BrdU +T381 UBERON:0013682 12257 12274 peripheral retina +T382 CHEBI:472552 12288 12292 BrdU +T383 PR:000013773 12356 12358 Rb +T384 UBERON:0000966 12362 12369 retinas +T385 GO:0071897 12414 12427 DNA synthesis +T386 GO:0007567 12495 12500 natal +T387 UBERON:0000966 12524 12530 retina +T388 CHEBI:472552 12546 12550 BrdU +T389 UBERON:0000045 12552 12560 ganglion +T390 CL:0000561 12565 12573 amacrine +T391 UBERON:0000966 12629 12635 retina +T392 CHEBI:472552 12643 12647 BrdU +T393 CL:0000210 12649 12662 photoreceptor +T394 PR:000013773 12733 12735 Rb +T395 UBERON:0000966 12739 12746 retinas +T396 UBERON:0000966 12827 12833 retina +T397 GO:0051320 12895 12902 S-phase +T398 PR:000013773 13007 13009 Rb +T399 PR:000006852 13010 13014 E2f1 +T400 UBERON:0000966 13019 13025 retina +T401 PR:000006853 13086 13090 E2f2 +T402 PR:000006854 13094 13098 E2f3 +T403 GO:0007067 13154 13161 mitotic +T404 GO:0042571 13210 13220 antibodies +T405 PR:000006852 13244 13248 E2f1 +T406 PR:000006853 13258 13262 E2f2 +T407 PR:000006854 13266 13270 E2f3 +T408 PR:000006852 13326 13330 E2f1 +T409 SO:0001023 13331 13337 allele +T410 GO:0051320 13367 13374 S-phase +T411 GO:0007067 13379 13386 mitosis +T412 PR:000013773 13390 13392 Rb +T413 PR:000006852 13456 13460 E2f1 +T414 PR:000013773 13488 13490 Rb +T415 UBERON:0001017 13587 13590 CNS +T416 PR:000013773 13594 13596 Rb +T417 UBERON:0000922 13600 13607 embryos +T418 PR:000013773 13705 13707 Rb +T419 UBERON:0000966 13715 13721 retina +T420 GO:0006915 13750 13759 apoptosis +T421 CL:0000103 13778 13785;13799 13804 bipolar ... cells +T422 UBERON:0000045 13790 13798 ganglion +T423 CL:0000740 13790 13804 ganglion cells +T424 CL:0000604 13821 13825 rods +T425 PR:000013773 13860 13862 Rb +T426 CL:0000604 13866 13870 rods +T427 UBERON:0001789 13899 13902 ONL +T428 GO:0008219 13912 13920;13927 13932 death of ... cells +T429 CL:0000103 13944 13951;13965 13972 bipolar ... neurons +T430 UBERON:0000045 13956 13964 ganglion +T431 CL:0000740 13956 13972 ganglion neurons +T432 PR:000013773 14123 14125 Rb +T433 UBERON:0000045 14129 14137 ganglion +T434 CL:0000740 14129 14143 ganglion cells +T435 UBERON:0000970 14181 14186 optic +T436 PR:000006852 14239 14243 E2f1 +T437 PR:000006853 14253 14257 E2f2 +T438 PR:000006854 14261 14265 E2f3 +T439 GO:0008219 14288 14298 cell death +T440 PR:000006852 14365 14369 E2f1 +T441 UBERON:0000045 14387 14395 Ganglion +T442 CL:0000740 14387 14395;14414 14419 Ganglion ... Cells +T443 CL:0000604 14397 14400;14414 14419 Rod ... Cells +T444 CL:0000748 14406 14419 Bipolar Cells +T445 PR:000013773 14427 14429 Rb +T446 UBERON:0000966 14433 14439 Retina +T447 UBERON:0000966 14456 14463 retinal +T448 NCBITaxon:10088 14478 14482 mice +T449 GO:0005634 14536 14542 nuclei +T450 CHEBI:51231 14544 14548 DAPI +T451 UBERON:0000045 14580 14588 ganglion +T452 CL:0000740 14580 14594 ganglion cells +T453 PR:000013042 14596 14602 Pou4f2 +T454 CL:0000604 14610 14614 rods +T455 CL:0000573 14619 14624 cones +T456 PR:000014434 14626 14629 Sag +T457 CL:0000604 14631 14634 rod +T458 PR:000014434 14631 14643 rod arrestin +T459 CL:0000751 14658 14675 rod bipolar cells +T460 PR:000003058 14677 14682 Prkca +T461 PR:000004937 14695 14700 Cabp5 +T462 PR:000013042 14753 14759 Pou4f2 +T463 UBERON:0000045 14761 14769 ganglion +T464 CL:0000740 14761 14775 ganglion cells +T465 PR:000003058 14800 14805 Prkca +T466 PR:000004937 14811 14816 Cabp5 +T467 CL:0000103 14818 14831 bipolar cells +T468 UBERON:0001789 14855 14858 ONL +T469 CL:0000604 14891 14895 rods +T470 NCBITaxon:33208 14949 14956 animals +T471 UBERON:0000966 15014 15021 retinas +T472 GO:1990603 15224 15236 dark-adapted +T473 GO:1990603 15238 15246 scotopic +T474 CL:0000210 15458 15472 photoreceptors +T475 CL:0000103 15623 15631 bipolars +T476 PR:000006852 15756 15760 E2f1 +T477 GO:0010467 15778 15788 expression +T478 SO:0000704 15827 15832 genes +T479 GO:0065007 15838 15846 regulate +T480 GO:0007049 15851 15861 cell cycle +T481 GO:0006915 15866 15875 apoptosis +T482 GO:0007049 15908 15918 cell cycle +T483 PR:000013773 15969 15971 Rb +T484 UBERON:0000966 15975 15981 retina +T485 PR:000006852 16017 16021 E2f1 +T486 PR:000006853 16023 16027 E2f2 +T487 PR:000028775 16029 16034 E2f3a +T488 PR:000006858 16040 16044 E2f7 +T489 PR:000013773 16068 16070 Rb +T490 PR:000006855 16088 16092 E2f4 +T491 PR:000006856 16098 16102 E2f5 +T492 CHEBI:472552 16140 16144 BrdU +T493 PR:000006852 16200 16204 E2f1 +T494 PR:000006854 16269 16273 E2f3 +T495 PR:000013773 16341 16343 Rb +T496 PR:000006852 16344 16348 E2f1 +T497 UBERON:0000966 16353 16359 Retina +T498 PR:000006852 16369 16373 E2f1 +T499 GO:0016265 16412 16417 death +T500 PR:000013773 16425 16427 Rb +T501 UBERON:0000966 16431 16437 retina +T502 PR:000013773 16443 16445 Rb +T503 PR:000006852 16446 16450 E2f1 +T504 UBERON:0000966 16455 16461 retina +T505 PR:000013773 16512 16514 Rb +T506 GO:0065007 16515 16523 controls +T507 GO:0007049 16555 16565 cell cycle +T508 PR:000013773 16579 16581 Rb +T509 PR:000006852 16582 16586 E2f1 +T510 UBERON:0000966 16591 16597 retina +T511 PR:000014434 16607 16610 Sag +T512 PR:000014434 16613 16622 S-antigen +T513 CHEBI:59132 16615 16622 antigen +T514 CL:0000604 16623 16626 rod +T515 PR:000014434 16623 16635 rod arrestin +T516 CL:0000210 16637 16651 photoreceptors +T517 PR:000013042 16653 16659 Pou4f2 +T518 PR:000013042 16662 16667 Brn3b +T519 UBERON:0000045 16669 16677 ganglion +T520 CL:0000740 16669 16683 ganglion cells +T521 PR:000003058 16698 16703 Prkca +T522 PR:000004937 16705 16710 Cabp5 +T523 CL:0000103 16712 16727 bipolar neurons +T524 PR:000013773 16799 16801 Rb +T525 PR:000006853 16802 16806 E2f2 +T526 PR:000013773 16810 16812 Rb +T527 PR:000006854 16813 16817 E2f3 +T528 UBERON:0000966 16822 16829 retinas +T529 CL:0000540 16865 16873 neuronal +T530 PR:000010124 16882 16887 Mtap2 +T531 PR:000010124 16889 16893 MAP2 +T532 PR:000015312 16899 16905 Snap25 +T533 GO:0010467 16932 16941 expressed +T534 CL:0000103 16945 16958 bipolar cells +T535 PR:000017355 16960 16965 Chx10 +T536 PR:000013845 16967 16972 Rcvrn +T537 PR:000017354 16974 16978 Vsx1 +T538 PR:000001242 16980 16985 Tacr3 +T539 PR:000004445 16991 16997 Atp2b1 +T540 CL:0000604 17003 17021 rod photoreceptors +T541 PR:000001245 17023 17026 Rho +T542 PR:000013845 17031 17036 Rcvrn +T543 PR:000013773 17062 17064 Rb +T544 PR:000006852 17065 17069 E2f1 +T545 UBERON:0000966 17074 17080 retina +T546 CL:0000540 17103 17110 neurons +T547 UBERON:0000966 17158 17164 retina +T548 CL:0000103 17166 17178 Bipolar cell +T549 GO:0044297 17174 17185 cell bodies +T550 UBERON:0001791 17206 17209 INL +T551 GO:0042995 17243 17252 processes +T552 PR:000013773 17323 17325 Rb +T553 PR:000006852 17326 17330 E2f1 +T554 UBERON:0000966 17335 17341 retina +T555 UBERON:0001789 17366 17369 ONL +T556 CL:0000604 17407 17411 rods +T557 GO:0042995 17428 17437 processes +T558 GO:0042995 17470 17479 processes +T559 GO:0001917 17499 17504;17515 17523 inner ... segments +T560 GO:0001750 17509 17523 outer segments +T561 PR:000013773 17559 17561 Rb +T562 GO:0065007 17568 17576 regulate +T563 CL:0000210 17577 17590 photoreceptor +T564 CL:0000604 17625 17628 rod +T565 PR:000013773 17690 17692 Rb +T566 GO:0065007 17698 17706 regulate +T567 CL:0000210 17707 17720 photoreceptor +T568 PR:000006852 17763 17767 E2f1 +T569 CL:0000604 17789 17792 rod +T570 PR:000012086 17826 17830 Otx2 +T571 PR:000005904 17832 17835 Crx +T572 PR:000011432 17840 17843 Nrl +T573 PR:000006852 17878 17882 E2f1 +T574 GO:0010467 17935 17945 expression +T575 SO:0000704 17949 17954 genes +T576 GO:0065007 17960 17968 modulate +T577 GO:0008283 18022 18035 proliferation +T578 GO:0006915 18092 18101 apoptosis +T579 PR:000013773 18136 18138 Rb +T580 UBERON:0000966 18142 18149 retinal +T581 CL:0000748 18142 18157;18177 18182 retinal bipolar ... cells +T582 CL:0000740 18142 18149;18159 18167;18177 18182 retinal ... ganglion ... cells +T583 CL:0000604 18142 18149;18173 18182 retinal ... rod cells +T584 UBERON:0000045 18159 18167 ganglion +T585 PR:000006852 18200 18204 E2f1 +T586 PR:000013773 18280 18282 Rb +T587 PR:000006852 18292 18296 E2f1 +T588 CL:0002672 18318 18321 RPC +T589 CL:0002672 18352 18355 RPC +T590 GO:0008283 18356 18369 proliferation +T591 CL:0002672 18446 18449 RPC +T592 PR:000006852 18476 18480 E2f1 +T593 UBERON:0000045 18532 18540 ganglion +T594 CL:0000740 18532 18546 ganglion cells +T595 CL:0000103 18571 18584 bipolar cells +T596 UBERON:0001789 18628 18631 ONL +T597 PR:000006852 18653 18657 E2f1 +T598 PR:000013773 18665 18667 Rb +T599 PR:000006852 18668 18672 E2f1 +T600 UBERON:0000966 18677 18683 retina +T601 PR:000006852 18718 18722 E2f1 +T602 CL:0000540 18726 18733 neurons +T603 PR:000013773 18820 18822 Rb +T604 PR:000006852 18823 18827 E2f1 +T605 PR:000006852 18836 18840 E2f1 +T606 CL:0000103 18844 18857 bipolar cells +T607 GO:0016265 18954 18959 death +T608 GO:0006915 18995 19004 apoptosis +T609 GO:0007567 19029 19034 natal +T610 PR:000013773 19059 19061 Rb +T611 PR:000006852 19062 19066 E2f1 +T612 UBERON:0000966 19071 19077 retina +T613 PR:000005121 19123 19128 Ccnd1 +T614 PR:000013773 19153 19155 Rb +T615 CL:0002672 19179 19182 RPC +T616 PR:000013773 19231 19233 Rb +T617 UBERON:0000966 19237 19243 retina +T618 CL:0002672 19304 19307 RPC +T619 PR:000013773 19358 19360 Rb +T620 PR:000006852 19420 19424 E2f1 +T621 PR:000013773 19450 19452 Rb +T622 PR:000006852 19453 19457 E2f1 +T623 UBERON:0000966 19462 19468 Retina +T624 PR:000006852 19489 19493 E2f1 +T625 PR:000013773 19530 19532 Rb +T626 CL:0000540 19536 19543 neurons +T627 PR:000013773 19566 19568 Rb +T628 GO:0065007 19583 19591 regulate +T629 UBERON:0007023 19791 19796 adult +T630 PR:000006852 19809 19813 E2f1 +T631 PR:000013773 19824 19826 Rb +T632 SO:0000346 19826 19830 loxP +T633 SO:0000346 19831 19835 loxP +T634 PR:000013773 19847 19849 Rb +T635 SO:0000346 19849 19853 loxP +T636 SO:0000346 19854 19858 loxP +T637 PR:000006852 19859 19863 E2f1 +T638 NCBITaxon:10088 19867 19871 mice +T639 GO:0007602 19898 19915 visual signalling +T640 NCBITaxon:40674 19923 19932 mammalian +T641 UBERON:0000966 19933 19939 retina +T642 CL:0000210 19945 19959 photoreceptors +T643 CL:0000561 19963 19977 amacrine cells +T644 UBERON:0000045 19995 20002 gangion +T645 CL:0000740 19995 20008 gangion cells +T646 CL:0000751 20032 20035;20045 20058 rod ... bipolar cells +T647 CL:0000752 20040 20058 cone bipolar cells +T648 CL:0000210 20136 20150 photoreceptors +T649 GO:0001775 20227 20240;20252 20257 activation of ... cells +T650 CL:0000749 20241 20257 ON bipolar cells +T651 GO:1990603 20299 20311 dark-adapted +T652 GO:1990603 20313 20321 scotopic +T653 CL:0000604 20358 20361 rod +T654 PR:000013773 20396 20398 Rb +T655 UBERON:0000966 20402 20408 retina +T656 CL:0000604 20490 20493;20506 20510 rod ... cell +T657 GO:0097473 20490 20493;20511 20520 rod ... apoptosis +T658 CL:0000103 20498 20510 bipolar cell +T659 GO:0006915 20506 20520 cell apoptosis +T660 UBERON:0000966 20645 20651 retina +T661 PR:000006852 20697 20701 E2f1 +T662 UBERON:0000966 20705 20711 retina +T663 PR:000013773 20745 20747 Rb +T664 PR:000006852 20748 20752 E2f1 +T665 PR:000006852 20854 20858 E2f1 +T666 CL:0000604 20898 20901 rod +T667 PR:000013773 20916 20918 Rb +T668 UBERON:0000966 20922 20928 retina +T669 GO:0036367 20931 20944 Light-adapted +T670 GO:0036367 20946 20954 photopic +T671 CL:0000573 20994 20998 cone +T672 CL:0000573 21034 21039 Cones +T673 CL:0000210 21061 21075 photoreceptors +T674 CL:0000604 21088 21092 rods +T675 PR:000013773 21110 21112 Rb +T676 CL:0000604 21131 21135 rods +T677 PR:000013773 21161 21163 Rb +T678 UBERON:0000966 21167 21173 retina +T679 GO:0045202 21215 21223 synaptic +T680 CL:0000103 21233 21246 bipolar cells +T681 GO:0036367 21275 21292 photopic response +T682 CL:0000573 21307 21311 cone +T683 CL:0000103 21323 21330 bipolar +T684 PR:000013773 21361 21363 Rb +T685 PR:000013773 21406 21408 Rb +T686 PR:000006852 21409 21413 E2f1 +T687 UBERON:0000966 21418 21424 retina +T688 PR:000006852 21494 21498 E2f1 +T689 GO:0036367 21513 21530 photopic response +T690 PR:000006852 21534 21538 E2f1 +T691 NCBITaxon:10088 21542 21546 mice +T692 PR:000006852 21614 21618 E2f1 +T693 UBERON:0000922 21656 21665 embryonic +T694 CL:0002672 21666 21670 RPCs +T695 PR:000006852 21680 21684 E2f1 +T696 UBERON:0000966 21688 21694 retina +T697 UBERON:0000966 21751 21757 retina +T698 GO:0036367 21869 21887 photopic responses +T699 PR:000013773 21895 21897 Rb +T700 PR:000006852 21898 21902 E2f1 +T701 UBERON:0000966 21907 21913 retina +T702 CL:0000573 21955 21959 cone +T703 PR:000006852 21971 21975 E2f1 +T704 NCBITaxon:10088 21979 21983 mice +T705 CL:0000573 22027 22031 cone +T706 PR:000013773 22057 22059 Rb +T707 CL:0000561 22159 22173 amacrine cells +T708 PR:000006852 22249 22253 E2f1 +T709 PR:000006852 22304 22308 E2f1 +T710 CL:0000604 22360 22363 rod +T711 CL:0000573 22368 22372 cone +T712 PR:000013773 22396 22398 Rb +T713 UBERON:0000966 22402 22408 retina +T714 GO:0007049 22455 22465 Cell Cycle +T715 CL:0000210 22510 22523 photoreceptor +T716 CL:0000103 22528 22540 bipolar cell +T717 PR:000013773 22650 22652 Rb +T718 PR:000006852 22653 22657 E2f1 +T719 UBERON:0000966 22662 22668 retina +T720 PR:000006852 22753 22757 E2f1 +T721 PR:000013773 22766 22768 Rb +T722 PR:000006852 22769 22773 E2f1 +T723 UBERON:0000966 22778 22784 retina +T724 GO:0007049 22829 22839 cell-cycle +T725 GO:0006915 22845 22854 apoptosis +T726 PR:000004968 22916 22921 Calb2 +T727 PR:000004968 22923 22933 calretinin +T728 CL:0000561 22960 22968;22982 22986 amacrine ... cell +T729 UBERON:0000045 22973 22981 ganglion +T730 CL:0000740 22973 22986 ganglion cell +T731 GO:0044297 22982 22993 cell bodies +T732 GO:0042995 23041 23050 processes +T733 PR:000004968 23082 23087 Calb2 +T734 PR:000006852 23113 23117 E2f1 +T735 PR:000004968 23161 23166 Calb2 +T736 PR:000013773 23193 23195 Rb +T737 PR:000013773 23243 23245 Rb +T738 PR:000006852 23246 23250 E2f1 +T739 UBERON:0000966 23255 23261 retina +T740 PR:000004968 23289 23294 Calb2 +T741 GO:0044297 23296 23307 cell bodies +T742 PR:000013773 23315 23317 Rb +T743 UBERON:0001791 23321 23324 INL +T744 CL:0000561 23343 23356 amacrine cell +T745 PR:000013773 23472 23474 Rb +T746 PR:000013773 23519 23521 Rb +T747 PR:000013773 23530 23532 Rb +T748 PR:000006852 23533 23537 E2f1 +T749 UBERON:0000966 23542 23548 retina +T750 GO:0005634 23566 23572 nuclei +T751 CHEBI:51231 23574 23578 DAPI +T752 PR:000004968 23587 23592 Calb2 +T753 PR:000014968 23606 23613 Slc18a3 +T754 PR:000013773 23679 23681 Rb +T755 UBERON:0000966 23685 23691 retina +T756 GO:0005634 23709 23715 nuclei +T757 CHEBI:51231 23717 23721 DAPI +T758 PR:000014968 23739 23746 Slc18a3 +T759 PR:000003199 23763 23769 Camk2a +T760 PR:000013773 23786 23788 Rb +T761 PR:000014968 23832 23839 Slc18a3 +T762 PR:000004968 23891 23896 Calb2 +T763 GO:0044297 23898 23909 cell bodies +T764 UBERON:0001791 23917 23920 INL +T765 PR:000014968 23928 23935 Slc18a3 +T766 GO:0044297 23937 23948 cell bodies +T767 PR:000003199 23954 23960 Camk2a +T768 GO:0044297 23962 23973 cell bodies +T769 UBERON:0001791 23981 23984 INL +T770 NCBITaxon:33208 24037 24044 animals +T771 UBERON:0000966 24101 24108 retinas +T772 PR:000004968 24257 24262 Calb2 +T773 GO:0097447 24347 24361 dendritic-tree +T774 CHEBI:15355 24393 24404 cholinergic +T775 CL:0000561 24425 24441 amacrine neurons +T776 UBERON:0000966 24564 24571 retinal +T777 GO:0060041 24564 24583 retinal development +T778 UBERON:0001791 24602 24605 INL +T779 GO:0045202 24606 24613 synapse +T780 UBERON:0001792 24705 24708 GCL +T781 GO:0042995 24714 24723 processes +T782 GO:0042995 24836 24845 processes +T783 PR:000014968 24869 24876 Slc18a3 +T784 GO:0031982 24878 24887 vesicular +T785 PR:000014968 24878 24914 vesicular acetyl choline transporter +T786 CHEBI:15355 24888 24902 acetyl choline +T787 PR:000014968 24916 24921 VAChT +T788 UBERON:0013682 24979 24989;25015 25021 peripheral ... retina +T789 PR:000013773 24990 24992 Rb +T790 PR:000013773 24999 25001 Rb +T791 PR:000006852 25002 25006 E2f1 +T792 GO:0010467 25050 25059 expressed +T793 GO:0044297 25118 25129 cell bodies +T794 GO:0042995 25134 25143 processes +T795 PR:000013773 25208 25210 Rb +T796 UBERON:0000966 25214 25220 retina +T797 GO:0044297 25245 25249 soma +T798 GO:0042995 25266 25275 processes +T799 PR:000015851 25321 25325 Sv2c +T800 GO:0008021 25329 25345 synaptic vesicle +T801 PR:000000940 25374 25380 Kcnc1b +T802 PR:000000782 25385 25390 Kcnc2 +T803 GO:0010467 25411 25420 expressed +T804 GO:0044297 25428 25432 soma +T805 GO:0030425 25437 25446 dendrites +T806 UBERON:0000045 25481 25489 ganglion +T807 CL:0000740 25481 25495 ganglion cells +T808 CHEBI:16865 25502 25525 gamma-aminobutyric acid +T809 CHEBI:16865 25527 25531 GABA +T810 CHEBI:25512 25548 25564 neurotransmitter +T811 CL:0000561 25590 25604 amacrine cells +T812 CL:0000745 25632 25642;25660 25667 horizontal ... neurons +T813 CL:0000103 25652 25667 bipolar neurons +T814 PR:000004967 25678 25683 Calb1 +T815 PR:000004967 25685 25694 calbindin +T816 GO:0010467 25706 25715 expressed +T817 CL:0000561 25724 25738 amacrine cells +T818 GO:0042995 25754 25761 process +T819 PR:000013773 25862 25864 Rb +T820 PR:000017355 25905 25910 Chx10 +T821 SO:0000902 25915 25924 transgene +T822 GO:0010467 25933 25942 expressed +T823 UBERON:0000966 25974 25980 retina +T824 GO:0010467 26008 26018 expressing +T825 PR:000014968 26110 26117 Slc18a3 +T826 PR:000017355 26141 26146 Chx10 +T827 PR:000013773 26151 26153 Rb +T828 SO:0000346 26153 26157 loxP +T829 SO:0000346 26158 26162 loxP +T830 UBERON:0000966 26163 26169 retina +T831 PR:000013773 26242 26244 Rb +T832 PR:000003199 26412 26418 Camk2a +T833 UBERON:0000045 26439 26447 ganglion +T834 CL:0000740 26439 26453 ganglion cells +T835 UBERON:0000045 26472 26480 ganglion +T836 CL:0000740 26472 26486 ganglion cells +T837 PR:000013773 26509 26511 Rb +T838 UBERON:0000966 26515 26521 retina +T839 PR:000003199 26523 26529 Camk2a +T840 PR:000003199 26585 26591 Camk2a +T841 GO:0030425 26604 26613 dendrites +T842 PR:000013773 26646 26648 Rb +T843 UBERON:0000966 26652 26658 retina +T844 PR:000003199 26690 26696 Camk2a +T845 GO:0044297 26698 26702 soma +T846 PR:000013773 26725 26727 Rb +T847 UBERON:0000966 26731 26737 retina +T848 PR:000013773 26789 26791 Rb +T849 UBERON:0000966 26795 26801 retina +T850 PR:000013773 26875 26877 Rb +T851 GO:0042995 26918 26925 process +T852 GO:0065007 26960 26968 regulate +T853 GO:0010467 26973 26983 expression +T854 PR:000004968 27004 27009 Calb2 +T855 PR:000004967 27011 27016 Calb1 +T856 PR:000014968 27024 27031 Slc18a3 +T857 PR:000015851 27033 27037 Sv2c +T858 PR:000000940 27039 27045 Kcnc1b +T859 PR:000000782 27047 27052 Kcnc2 +T860 CHEBI:16865 27058 27062 GABA +T861 PR:000003199 27083 27089 Camk2a +T862 GO:0010467 27090 27100 expression +T863 GO:0044297 27152 27163 cell bodies +T864 GO:0042995 27177 27186 processes +T865 PR:000014968 27262 27269 Slc18a3 +T866 GO:0010467 27270 27280 expression +T867 PR:000014968 27327 27334 Slc18a3 +T868 GO:0042995 27346 27355 processes +T869 GO:0007567 27374 27379 natal +T870 GO:0044297 27405 27414 cell body +T871 GO:0042995 27440 27449 processes +T872 PR:000014968 27490 27497 Slc18a3 +T873 PR:000013773 27523 27525 Rb +T874 UBERON:0000966 27529 27535 retina +T875 GO:0044297 27571 27582 cell bodies +T876 PR:000013773 27610 27612 Rb +T877 GO:0042995 27616 27625 processes +T878 PR:000014968 27647 27654 Slc18a3 +T879 PR:000013773 27688 27690 Rb +T880 PR:000013773 27751 27753 Rb +T881 PR:000006854 27832 27836 E2f3 +T882 PR:000013773 27869 27871 Rb +T883 UBERON:0000966 27896 27903 retinal +T884 GO:0005634 27966 27972 nuclei +T885 CHEBI:51231 27974 27978 DAPI +T886 GO:0007067 27987 27994 mitosis +T887 PR:000014968 28019 28026 Slc18a3 +T888 GO:0044297 28050 28054 soma +T889 GO:0042995 28075 28084 processes +T890 GO:0007067 28115 28122 mitotic +T891 GO:0005634 28128 28134 nuclei +T892 PR:000013773 28138 28140 Rb +T893 PR:000013773 28145 28147 Rb +T894 PR:000006853 28148 28152 E2f2 +T895 PR:000013773 28162 28164 Rb +T896 PR:000006854 28165 28169 E2f3 +T897 UBERON:0000966 28174 28181 retinas +T898 PR:000006852 28183 28187 E2f1 +T899 GO:0007067 28213 28220 mitosis +T900 GO:0008219 28225 28235 cell death +T901 PR:000006853 28269 28273 E2f2 +T902 PR:000006854 28294 28298 E2f3 +T903 GO:0007067 28332 28339 mitosis +T904 PR:000006852 28404 28408 E2f1 +T905 PR:000006854 28413 28417 E2f3 +T906 GO:0007067 28447 28454 mitosis +T907 GO:0008219 28456 28466 cell death +T908 PR:000003199 28506 28512 Camk2a +T909 PR:000014968 28540 28547 Slc18a3 +T910 UBERON:0000966 28560 28566 retina +T911 UBERON:0000966 28587 28594 retinal +T912 GO:0005634 28621 28627 nuclei +T913 CHEBI:51231 28629 28633 DAPI +T914 PR:000014968 28642 28649 Slc18a3 +T915 PR:000009116 28663 28667 Isl1 +T916 PR:000009116 28703 28707 Isl1 +T917 PR:000014968 28709 28716 Slc18a3 +T918 UBERON:0001791 28737 28740 INL +T919 UBERON:0000966 28761 28768 retinal +T920 GO:0005634 28822 28828 nuclei +T921 CHEBI:51231 28830 28834 DAPI +T922 GO:0051301 28843 28856 cell division +T923 PR:000010425 28864 28869 Mki67 +T924 PR:000009116 28883 28887 Isl1 +T925 PR:000009116 28959 28963 Isl1 +T926 UBERON:0000966 29001 29008 retinas +T927 PR:000010425 29028 29033 Mki67 +T928 NCBITaxon:33208 29089 29096 animals +T929 UBERON:0000966 29153 29160 retinas +T930 PR:000013773 29292 29294 Rb +T931 GO:0065007 29295 29304 Regulates +T932 PR:000006854 29333 29337 E2f3 +T933 PR:000013773 29339 29341 Rb +T934 CL:0000540 29392 29400 neuronal +T935 UBERON:0004288 29416 29424 skeletal +T936 CL:0000136 29433 29443 adipocytes +T937 PR:000013773 29455 29457 Rb +T938 UBERON:0000479 29492 29498 tissue +T939 PR:000013773 29591 29593 Rb +T940 UBERON:0000966 29614 29620 retina +T941 PR:000013773 29708 29710 Rb +T942 GO:0007049 29761 29771 cell cycle +T943 GO:0008219 29761 29765;29775 29780 cell ... death +T944 GO:0065007 29851 29859 regulate +T945 SO:0000704 29881 29886 genes +T946 PR:000006853 29920 29924 E2f2 +T947 PR:000006854 29928 29932 E2f3 +T948 PR:000013773 29947 29949 Rb +T949 PR:000006852 29994 29998 E2f1 +T950 GO:0007067 30027 30034 mitosis +T951 PR:000006853 30089 30093 E2f2 +T952 PR:000006854 30168 30172 E2f3 +T953 GO:0007067 30206 30213 mitosis +T954 PR:000004968 30226 30231 Calb2 +T955 PR:000014968 30233 30240 Slc18a3 +T956 CHEBI:16865 30248 30252 GABA +T957 PR:000000940 30254 30260 Kcnc1b +T958 PR:000000782 30262 30267 Kcnc2 +T959 PR:000015851 30273 30277 Sv2c +T960 PR:000013773 30337 30339 Rb +T961 PR:000006854 30340 30344 E2f3 +T962 UBERON:0000966 30401 30407 retina +T963 GO:0045202 30447 30455 synaptic +T964 PR:000006852 30491 30495 E2f1 +T965 PR:000013773 30542 30544 Rb +T966 PR:000006852 30545 30549 E2f1 +T967 PR:000006854 30550 30554 E2f3 +T968 UBERON:0000966 30571 30577 retina +T969 CL:0000103 30585 30592;30606 30610 bipolar ... cell +T970 UBERON:0000045 30597 30605 ganglion +T971 CL:0000740 30597 30610 ganglion cell +T972 GO:0008219 30606 30616 cell death +T973 PR:000006854 30679 30683 E2f3 +T974 PR:000006854 30769 30773 E2f3 +T975 PR:000013773 30830 30832 Rb +T976 PR:000006854 30847 30851 E2f3 +T977 PR:000003199 30884 30890 Camk2a +T978 PR:000003199 30949 30955 Camk2a +T979 GO:0010467 30963 30972 expressed +T980 PR:000014968 30982 30989 Slc18a3 +T981 PR:000013773 31025 31027 Rb +T982 UBERON:0000966 31031 31037 retina +T983 PR:000013773 31071 31073 Rb +T984 PR:000006852 31074 31078 E2f1 +T985 UBERON:0000966 31083 31089 retina +T986 PR:000013773 31114 31116 Rb +T987 PR:000006854 31117 31121 E2f3 +T988 UBERON:0000966 31126 31132 retina +T989 UBERON:0000045 31192 31200 ganglion +T990 CL:0000740 31192 31206 ganglion cells +T991 PR:000003199 31239 31245 Camk2a +T992 GO:0006915 31268 31277 apoptosis +T993 PR:000009116 31376 31380 Isl1 +T994 PR:000009116 31382 31388 Islet1 +T995 GO:0010467 31406 31415 expressed +T996 UBERON:0000045 31433 31441 ganglion +T997 CL:0000740 31433 31447 ganglion cells +T998 PR:000009116 31454 31458 Isl1 +T999 UBERON:0001791 31473 31476 INL +T1000 PR:000009116 31534 31538 Isl1 +T1001 UBERON:0001791 31567 31570 INL +T1002 PR:000014968 31587 31594 Slc18a3 +T1003 PR:000009116 31613 31617 Isl1 +T1004 PR:000009116 31668 31672 Isl1 +T1005 PR:000014968 31681 31688 Slc18a3 +T1006 GO:0005634 31693 31700 nuclear +T1007 PR:000009116 31731 31735 Isl1 +T1008 PR:000010425 31737 31742 Mki67 +T1009 GO:0010467 31762 31771 expressed +T1010 PR:000014968 31785 31792 Slc18a3 +T1011 UBERON:0000966 31875 31881 retina +T1012 UBERON:0000966 31939 31945 retina +T1013 PR:000013773 31959 31961 Rb +T1014 GO:0007049 31974 31984 cell cycle +T1015 PR:000009116 32049 32053 Isl1 +T1016 CL:0000031 32074 32086 neuroblastic +T1017 UBERON:0001791 32120 32123 INL +T1018 PR:000009116 32152 32156 Isl1 +T1019 PR:000010425 32158 32163 Mki67 +T1020 PR:000013773 32192 32194 Rb +T1021 PR:000009116 32252 32256 Isl1 +T1022 PR:000013773 32309 32311 Rb +T1023 UBERON:0000966 32315 32321 retina +T1024 GO:0010467 32361 32370 expressed +T1025 PR:000013773 32437 32439 Rb +T1026 PR:000006852 32440 32444 E2f1 +T1027 UBERON:0000966 32449 32455 retina +T1028 PR:000013773 32512 32514 Rb +T1029 PR:000006854 32515 32519 E2f3 +T1030 UBERON:0000966 32524 32530 retina +T1031 PR:000004968 32629 32634 Calb2 +T1032 CL:0000561 32671 32685 amacrine cells +T1033 PR:000013773 32713 32715 Rb +T1034 PR:000006852 32725 32729 E2f1 +T1035 PR:000006854 32809 32813 E2f3 +T1036 GO:0010467 32895 32905 Expression +T1037 PR:000006854 32909 32913 E2f3 +T1038 UBERON:0001017 32943 32946 CNS +T1039 CL:0000117 32943 32954 CNS Neurons +T1040 PR:000006854 32977 32981 E2f3 +T1041 UBERON:0000966 33038 33045 retinal +T1042 CL:0000540 33046 33053 neurons +T1043 GO:0010467 33089 33099 expression +T1044 PR:000006854 33134 33138 E2f3 +T1045 NCBITaxon:10088 33222 33227 mouse +T1046 UBERON:0000479 33228 33235 tissues +T1047 PR:000006852 33284 33288 E2f1 +T1048 PR:000006853 33292 33296 E2f2 +T1049 PR:000006854 33354 33358 E2f3 +T1050 GO:0010467 33359 33369 expression +T1051 PR:000006854 33389 33393 E2f3 +T1052 CL:0002672 33410 33414 RPCs +T1053 GO:0008283 33458 33471 proliferation +T1054 PR:000006854 33533 33537 E2f3 +T1055 UBERON:0013682 33541 33558 peripheral retina +T1056 UBERON:0000966 33579 33585 retina +T1057 CL:0002672 33605 33608 RPC +T1058 PR:000006854 33644 33648 E2f3 +T1059 GO:0007067 33736 33743 mitotic +T1060 UBERON:0000966 33763 33769 retina +T1061 GO:0010467 33770 33779 expressed +T1062 PR:000006854 33780 33784 E2f3 +T1063 PR:000006854 33806 33810 E2f3 +T1064 PR:000014968 33918 33925 Slc18a3 +T1065 GO:0005737 33955 33966 cytoplasmic +T1066 PR:000006854 33967 33971 E2f3 +T1067 PR:000006854 34024 34028 E2f3 +T1068 UBERON:0013682 34032 34049 peripheral retina +T1069 PR:000006854 34059 34063 E2f3 +T1070 SO:0000346 34063 34067 loxP +T1071 SO:0000346 34068 34072 loxP +T1072 NCBITaxon:10088 34073 34077 mice +T1073 PR:000006854 34121 34125 E2f3 +T1074 PR:000014968 34146 34153 Slc18a3 +T1075 PR:000006854 34177 34181 E2f3 +T1076 GO:0044297 34205 34209 soma +T1077 GO:0030425 34214 34223 dendrites +T1078 PR:000013773 34237 34239 Rb +T1079 UBERON:0000966 34279 34285 retina +T1080 PR:000006854 34336 34340 E2f3 +T1081 UBERON:0000045 34393 34401 ganglion +T1082 CL:0000740 34393 34407 ganglion cells +T1083 CL:0011107 34412 34424 Müller cells +T1084 PR:000013773 34443 34445 Rb +T1085 GO:0042995 34462 34471 processes +T1086 UBERON:0013682 34509 34526 peripheral retina +T1087 PR:000013773 34535 34537 Rb +T1088 SO:0000346 34537 34541 loxP +T1089 SO:0000346 34542 34546 loxP +T1090 NCBITaxon:10088 34547 34551 mice +T1091 PR:000013773 34589 34591 Rb +T1092 PR:000006854 34596 34600 E2f3 +T1093 PR:000006854 34629 34633 E2f3 +T1094 GO:0010467 34701 34710 expressed +T1095 UBERON:0000966 34720 34727 retinal +T1096 CL:0000540 34728 34735 neurons +T1097 PR:000006854 34748 34752 E2f3 +T1098 PR:000013773 34757 34759 Rb +T1099 GO:0010467 34760 34770 Expression +T1100 UBERON:0000966 34824 34831 retinal +T1101 PR:000006854 34885 34889 E2f3 +T1102 CHEBI:51231 34900 34904 DAPI +T1103 PR:000006854 34958 34962 E2f3 +T1104 UBERON:0013682 34968 34978;34997 35003 peripheral ... retina +T1105 PR:000006854 35025 35029 E2f3 +T1106 PR:000006854 35056 35060 E2f3 +T1107 CL:0002672 35064 35068 RPCs +T1108 UBERON:0013682 35082 35092;35099 35106 peripheral ... retinal +T1109 CL:0000540 35107 35114 neurons +T1110 UBERON:0000966 35144 35151 retinal +T1111 PR:000013773 35205 35207 Rb +T1112 CHEBI:51231 35218 35222 DAPI +T1113 PR:000013773 35251 35253 Rb +T1114 UBERON:0013682 35269 35279;35292 35299 peripheral ... retinal +T1115 PR:000013773 35280 35282 Rb +T1116 CL:0000540 35300 35307 neurons +T1117 UBERON:0000966 35321 35328 retinal +T1118 GO:0005634 35355 35361 nuclei +T1119 CHEBI:51231 35363 35367 DAPI +T1120 PR:000006854 35376 35380 E2f3 +T1121 PR:000013773 35390 35392 Rb +T1122 PR:000014968 35414 35421 Slc18a3 +T1123 GO:0044297 35463 35467 soma +T1124 GO:0042995 35487 35496 processes +T1125 PR:000006854 35566 35570 E2f3 +T1126 CL:0000540 35613 35620 neurons +T1127 UBERON:0002616 35632 35645 brain regions +T1128 UBERON:0001876 35688 35696 amygdala +T1129 PR:000006854 35698 35702 E2f3 +T1130 CL:0000540 35732 35740 neuronal +T1131 PR:000010124 35749 35754 Mtap2 +T1132 PR:000010277 35759 35764 Mecp2 +T1133 PR:000004968 35784 35789 Calb2 +T1134 CL:0000540 35815 35822 neurons +T1135 CL:0000125 35836 35841 glial +T1136 PR:000007939 35849 35853 Gfap +T1137 UBERON:0000966 35882 35889 retinal +T1138 PR:000006854 35896 35900 E2f3 +T1139 GO:0010467 35911 35920 expressed +T1140 PR:000014968 35933 35940 Slc18a3 +T1141 CHEBI:15355 35942 35953 cholinergic +T1142 CL:0000108 35942 35961 cholinergic neurons +T1143 UBERON:0002616 35981 35991;35996 36001 regions of ... brain +T1144 UBERON:0001948 35981 35991;36006 36017 regions of ... spinal cord +T1145 CHEBI:15355 36081 36092 cholinergic +T1146 CL:0000108 36081 36092;36099 36106 cholinergic ... neurons +T1147 PR:000013773 36093 36095 Rb +T1148 UBERON:0001890 36125 36134 forebrain +T1149 PR:000013773 36146 36148 Rb +T1150 CL:0000540 36152 36159 neurons +T1151 PR:000006854 36236 36240 E2f3 +T1152 PR:000013773 36314 36316 Rb +T1153 PR:000006854 36360 36364 E2f3 +T1154 PR:000006854 36403 36407 E2f3 +T1155 SO:0001060 36408 36416 Isoforms +T1156 PR:000006854 36434 36438 E2f3 +T1157 PR:000013773 36443 36445 Rb +T1158 GO:0005634 36472 36479 nuclear +T1159 GO:0005737 36484 36495 cytoplasmic +T1160 GO:0042571 36520 36528 antibody +T1161 SO:0001060 36625 36633 isoforms +T1162 PR:000006854 36681 36685 E2f3 +T1163 SO:0001060 36686 36694 isoforms +T1164 PR:000006854 36769 36773 E2f3 +T1165 SO:0001060 36797 36805 isoforms +T1166 UBERON:0000966 36822 36828 retina +T1167 GO:0005634 36842 36849 nuclear +T1168 GO:0005737 36854 36865 cytoplasmic +T1169 PR:000006854 36953 36957 E2f3 +T1170 GO:0042571 36958 36966 antibody +T1171 PR:000028775 37019 37024 E2f3a +T1172 UBERON:0000966 37116 37123 retinal +T1173 PR:000028775 37136 37141 E2f3a +T1174 NCBITaxon:10088 37162 37166 mice +T1175 PR:000006854 37177 37181 E2f3 +T1176 SO:0000147 37182 37186 exon +T1177 GO:0010467 37199 37206 express +T1178 PR:000028775 37406 37411 E2f3a +T1179 NCBITaxon:10088 37415 37419 mice +T1180 PR:000006854 37468 37472 E2f3 +T1181 GO:0010467 37473 37483 expressing +T1182 UBERON:0000966 37500 37507 retinal +T1183 PR:000028775 37552 37557 E2f3a +T1184 PR:000028775 37674 37679 E2f3a +T1185 GO:0005634 37700 37707 nuclear +T1186 GO:0005737 37712 37723 cytoplasmic +T1187 GO:0005634 37781 37788 nuclear +T1188 PR:000028775 37837 37842 E2f3a +T1189 GO:0005634 37947 37954 nuclear +T1190 GO:0005737 37994 38003 cytoplasm +T1191 PR:000028775 38040 38045 E2f3a +T1192 PR:000028775 38096 38101 E2f3a +T1193 UBERON:0000966 38105 38111 retina +T1194 PR:000013042 38137 38143 Pou4f2 +T1195 GO:0005634 38147 38154 nuclear +T1196 GO:0010467 38176 38185 expressed +T1197 UBERON:0000045 38189 38197 ganglion +T1198 CL:0000740 38189 38203 ganglion cells +T1199 GO:0005634 38217 38224 nuclear +T1200 GO:0005737 38259 38270 cytoplasmic +T1201 PR:000014968 38297 38304 Slc18a3 +T1202 GO:0005737 38308 38319 cytoplasmic +T1203 PR:000028775 38452 38457 E2f3a +T1204 PR:000028775 38554 38559 E2f3a +T1205 GO:0065007 38580 38589 regulated +T1206 GO:0006412 38613 38626 translational +T1207 PR:000006854 38681 38685 E2f3 +T1208 SO:0001060 38686 38694 Isoforms +T1209 GO:0007049 38705 38715 Cell Cycle +T1210 UBERON:0000966 38743 38749 Retina +T1211 GO:0005634 38751 38758 Nuclear +T1212 GO:0005737 38763 38774 cytoplasmic +T1213 UBERON:0000966 38813 38820 retinal +T1214 CL:0009004 38813 38826 retinal cells +T1215 NCBITaxon:10088 38832 38836 mice +T1216 PR:000028775 38953 38958 E2f3a +T1217 NCBITaxon:10088 38962 38966 mice +T1218 PR:000028775 39016 39021 E2f3a +T1219 GO:0005737 39031 39032 C +T1220 GO:0005737 39034 39045 cytoplasmic +T1221 GO:0005634 39056 39057 N +T1222 GO:0005634 39059 39066 nuclear +T1223 PR:000028775 39092 39097 E2f3a +T1224 SO:0001060 39098 39105 Isoform +T1225 PR:000013773 39143 39145 Rb +T1226 NCBITaxon:10088 39185 39190 mouse +T1227 PR:000028775 39195 39200 E2f3a +T1228 SO:0000359 39228 39234 floxed +T1229 PR:000006854 39235 39239 E2f3 +T1230 PR:000006854 39264 39268 E2f3 +T1231 PR:000028775 39289 39294 E2f3a +T1232 NCBITaxon:10088 39298 39302 mice +T1233 PR:000006854 39316 39320 E2f3 +T1234 SO:0000147 39321 39325 exon +T1235 SO:0000188 39341 39347 intron +T1236 SO:0000112 39389 39396 primers +T1237 PR:000028775 39415 39420 E2f3a +T1238 NCBITaxon:10088 39424 39429 mouse +T1239 PR:000028775 39478 39483 E2f3a +T1240 UBERON:0000966 39506 39512 retina +T1241 SO:0000112 39531 39538 primers +T1242 UBERON:0000966 39651 39657 retina +T1243 GO:0010467 39658 39667 expresses +T1244 PR:000028775 39673 39678 E2f3a +T1245 PR:000028775 39708 39713 E2f3a +T1246 UBERON:0000966 39717 39723 retina +T1247 PR:000028775 39730 39735 E2f3a +T1248 GO:0010467 39751 39760 expresses +T1249 PR:000006854 39773 39777 E2f3 +T1250 UBERON:0000966 39781 39787 retina +T1251 PR:000028775 39806 39811 E2f3a +T1252 GO:0010467 39841 39850 expresses +T1253 SO:0000147 39876 39880 exon +T1254 SO:0000704 39922 39927 genes +T1255 UBERON:0000966 39934 39941 retinas +T1256 NCBITaxon:33208 40021 40028 animals +T1257 PR:000013773 40185 40187 Rb +T1258 PR:000028775 40199 40204 E2f3a +T1259 UBERON:0000966 40226 40233 retinal +T1260 GO:0005634 40296 40302 nuclei +T1261 CHEBI:51231 40304 40308 DAPI +T1262 GO:0000279 40317 40324 M-phase +T1263 PR:000014968 40358 40365 Slc18a3 +T1264 PR:000028775 40373 40378 E2f3a +T1265 GO:0007049 40545 40555 cell cycle +T1266 UBERON:0000966 40574 40581 retinal +T1267 GO:0060041 40574 40593 retinal development +T1268 PR:000028775 40600 40605 E2f3a +T1269 PR:000013773 40607 40609 Rb +T1270 GO:0005737 40637 40646 cytoplasm +T1271 GO:0005634 40651 40658 nucleus +T1272 PR:000013773 40697 40699 Rb +T1273 GO:0005634 40732 40739 nuclear +T1274 GO:0005737 40765 40776 cytoplasmic +T1275 PR:000013773 40777 40779 Rb +T1276 PR:000013773 40832 40834 Rb +T1277 GO:0042995 40851 40860 processes +T1278 UBERON:0000966 40924 40930 retina +T1279 PR:000006852 40937 40941 E2f1 +T1280 GO:0005634 40968 40975 nuclear +T1281 GO:0005737 40980 40991 cytoplasmic +T1282 PR:000028775 41019 41024 E2f3a +T1283 GO:0005634 41046 41053 nuclear +T1284 GO:0046983 41093 41105 dimerization +T1285 PR:000016269 41115 41120 Tfdp1 +T1286 GO:0005634 41136 41143 nuclear +T1287 SO:0001528 41136 41163 nuclear localization signal +T1288 GO:0005737 41184 41195 cytoplasmic +T1289 PR:000005274 41239 41245 Cdkn1a +T1290 PR:000003090 41250 41256 Cdkn1b +T1291 GO:0007049 41326 41336 cell cycle +T1292 GO:0005634 41432 41439 nuclear +T1293 PR:000013773 41463 41465 Rb +T1294 GO:0065007 41466 41475 Regulates +T1295 PR:000028775 41504 41509 E2f3a +T1296 PR:000006854 41525 41529 E2f3 +T1297 SO:0001060 41530 41537 isoform +T1298 PR:000013773 41566 41568 Rb +T1299 PR:000028775 41605 41610 E2f3a +T1300 NCBITaxon:10088 41614 41618 mice +T1301 PR:000028775 41706 41711 E2f3a +T1302 SO:0001023 41726 41733 alleles +T1303 PR:000028775 41801 41806 E2f3a +T1304 UBERON:0000966 41850 41856 retina +T1305 PR:000028775 41886 41891 E2f3a +T1306 PR:000028775 41903 41908 E2f3a +T1307 UBERON:0000966 41912 41918 retina +T1308 PR:000028775 41932 41937 E2f3a +T1309 PR:000028775 41960 41965 E2f3a +T1310 UBERON:0000966 41969 41976 retinal +T1311 SO:0000673 42037 42044 message +T1312 CHEBI:33695 42037 42044 message +T1313 PR:000013773 42065 42067 Rb +T1314 PR:000013773 42075 42077 Rb +T1315 PR:000028775 42078 42083 E2f3a +T1316 UBERON:0000966 42088 42094 retina +T1317 PR:000028775 42143 42148 E2f3a +T1318 PR:000013773 42277 42279 Rb +T1319 PR:000013773 42284 42286 Rb +T1320 PR:000006854 42287 42291 E2f3 +T1321 PR:000013773 42301 42303 Rb +T1322 PR:000028775 42304 42309 E2f3a +T1323 UBERON:0000966 42314 42320 retina +T1324 GO:0065007 42343 42353 regulatory +T1325 PR:000028775 42380 42385 E2f3a +T1326 GO:0007049 42398 42408 cell cycle +T1327 GO:0007049 42462 42472 cell cycle +T1328 PR:000013773 42567 42569 Rb +T1329 GO:0044838 42573 42582 quiescent +T1330 PR:000013773 42653 42655 Rb +T1331 PR:000028775 42688 42693 E2f3a +T1332 PR:000013773 42718 42720 Rb +T1333 GO:0051726 42780 42798 cell cycle control +T1334 PR:000013773 42800 42802 Rb +T1335 GO:0065007 42803 42813 regulation +T1336 PR:000028775 42817 42822 E2f3a +T1337 CL:0000540 42852 42860 neuronal +T1338 PR:000013773 42891 42893 Rb +T1339 GO:0065007 42894 42902 Controls +T1340 UBERON:0000966 42903 42910 Retinal +T1341 CL:0009004 42903 42915 Retinal Cell +T1342 GO:0051301 42911 42924 Cell Division +T1343 GO:0008219 42911 42915;42929 42934 Cell ... Death +T1344 PR:000006852 42943 42947 E2f1 +T1345 PR:000013773 42985 42987 Rb +T1346 CL:0000540 43013 43021 neuronal +T1347 GO:0007049 43022 43032 cell cycle +T1348 GO:0016265 43090 43095 death +T1349 UBERON:0001987 43153 43162 placental +T1350 PR:000013773 43234 43236 Rb +T1351 GO:0007049 43259 43269 cell cycle +T1352 CL:0000540 43286 43293 neurons +T1353 CL:0000540 43339 43346 neurons +T1354 UBERON:0000966 43368 43374 retina +T1355 PR:000013773 43411 43413 Rb +T1356 GO:0065007 43419 43428 regulates +T1357 GO:0016265 43513 43518 death +T1358 PR:000013773 43555 43557 Rb +T1359 GO:0065007 43562 43570 regulate +T1360 CL:0000540 43571 43579 neuronal +T1361 GO:0042551 43571 43590 neuronal maturation +T1362 PR:000006852 43632 43636 E2f1 +T1363 GO:0016265 43679 43684 death +T1364 PR:000013773 43692 43694 Rb +T1365 UBERON:0000966 43698 43704 retina +T1366 PR:000013773 43725 43727 Rb +T1367 PR:000006852 43728 43732 E2f1 +T1368 CL:0000540 43737 43744 neurons +T1369 CL:0000604 43802 43805 rod +T1370 CL:0000573 43811 43815 cone +T1371 CL:0000210 43874 43888 photoreceptors +T1372 CL:0000103 43892 43899;43913 43918 bipolar ... cells +T1373 CL:0000561 43904 43918 amacrine cells +T1374 GO:0016265 43933 43938 death +T1375 SO:0000704 43939 43944 genes +T1376 PR:000013773 43961 43963 Rb +T1377 PR:000006852 43987 43991 E2f1 +T1378 PR:000006853 44001 44005 E2f2 +T1379 PR:000006854 44009 44013 E2f3 +T1380 PR:000006852 44048 44052 E2f1 +T1381 GO:0065007 44062 44070 regulate +T1382 UBERON:0000966 44139 44146 retinal +T1383 CL:0009004 44139 44151 retinal cell +T1384 GO:0048469 44147 44162 cell maturation +T1385 GO:0065007 44233 44242 regulated +T1386 GO:0016265 44256 44261 death +T1387 UBERON:0000966 44301 44308 retinal +T1388 CL:0009004 44301 44314 retinal cells +T1389 CL:0000210 44326 44340 photoreceptors +T1390 PR:000013773 44424 44426 Rb +T1391 PR:000006852 44453 44457 E2f1 +T1392 GO:0016265 44509 44514 death +T1393 PR:000013773 44556 44558 Rb +T1394 NCBITaxon:11632 44591 44601 retrovirus +T1395 SO:0000440 44602 44608 vector +T1396 PR:000006852 44698 44702 E2f1 +T1397 GO:0010467 44707 44717 expression +T1398 CL:0000210 44729 44743 photoreceptors +T1399 GO:0006915 44772 44781 apoptosis +T1400 PR:000006852 44836 44840 E2f1 +T1401 GO:0006915 44928 44937 apoptosis +T1402 PR:000013773 44941 44943 Rb +T1403 GO:0065007 44970 44979 regulated +T1404 PR:000006852 44980 44984 E2f1 +T1405 UBERON:0000966 45001 45007 retina +T1406 PR:000013773 45052 45054 Rb +T1407 GO:0010467 45067 45077 expression +T1408 GO:0051301 45100 45113 cell division +T1409 GO:0006915 45127 45136 apoptosis +T1410 PR:000006852 45158 45162 E2f1 +T1411 http://purl.obolibrary.org/obo/MONDO_0008380 45248 45262 retinoblastoma +T1412 PR:000013773 45266 45269 RB1 +T1413 NCBITaxon:9606 45273 45279 humans +T1414 PR:000013773 45321 45323 Rb +T1415 CL:0000604 45327 45330 rod +T1416 CL:0000103 45331 45338 bipolar +T1417 CL:0000573 45386 45390 cone +T1418 CL:0000103 45391 45398 bipolar +T1419 PR:000006852 45416 45420 E2f1 +T1420 PR:000013773 45474 45476 Rb +T1421 PR:000006852 45477 45481 E2f1 +T1422 UBERON:0000966 45486 45492 retina +T1423 PR:000006852 45509 45513 E2f1 +T1424 UBERON:0000966 45525 45531 retina +T1425 PR:000013773 45574 45576 Rb +T1426 GO:0046549 45584 45604 development of cones +T1427 GO:0048468 45584 45598;45614 45619 development of ... cells +T1428 GO:0048468 45584 45598;45630 45635 development of ... cells +T1429 CL:0000573 45599 45604 cones +T1430 CL:0000103 45606 45619 bipolar cells +T1431 GO:0036367 45663 45671 photopic +T1432 PR:000013773 45743 45745 Rb +T1433 PR:000006852 45746 45750 E2f1 +T1434 UBERON:0000966 45755 45761 retina +T1435 PR:000013773 45764 45766 Rb +T1436 GO:0065007 45767 45775 Controls +T1437 PR:000028775 45804 45809 E2f3a +T1438 UBERON:0000966 45886 45893 retinal +T1439 CL:0000540 45894 45901 neurons +T1440 PR:000006852 45903 45907 E2f1 +T1441 PR:000013773 45945 45947 Rb +T1442 CHEBI:15355 45951 45962 cholinergic +T1443 PR:000006852 45990 45994 E2f1 +T1444 CL:0000099 46147 46159 interneurons +T1445 PR:000013773 46243 46245 Rb +T1446 GO:0065007 46246 46255 regulates +T1447 GO:0022008 46256 46268 neurogenesis +T1448 GO:0007067 46285 46292 mitosis +T1449 PR:000013773 46294 46296 Rb +T1450 CL:0000540 46350 46358 neuronal +T1451 UBERON:0004288 46374 46382 skeletal +T1452 CL:0000136 46391 46401 adipocytes +T1453 UBERON:0000479 46438 46444 tissue +T1454 PR:000013773 46528 46530 Rb +T1455 PR:000010875 46579 46584 Myod1 +T1456 PR:000013773 46727 46729 Rb +T1457 PR:000006854 46767 46771 E2f3 +T1458 PR:000013773 46795 46797 Rb +T1459 GO:0065007 46798 46807 Regulates +T1460 PR:000006852 46835 46839 E2f1 +T1461 PR:000028775 46844 46849 E2f3a +T1462 PR:000013773 46880 46882 Rb +T1463 PR:000013773 46980 46982 Rb +T1464 PR:000013773 46996 46998 Rb +T1465 CL:0002672 47025 47028 RPC +T1466 CL:0002672 47082 47086 RPCs +T1467 GO:0007067 47147 47154 mitosis +T1468 PR:000013773 47188 47190 Rb +T1469 PR:000006852 47228 47232 E2f1 +T1470 PR:000013773 47234 47236 Rb +T1471 PR:000028775 47316 47321 E2f3a +T1472 PR:000013773 47356 47358 Rb +T1473 PR:000013773 47470 47472 Rb +T1474 GO:0045595 47473 47486;47491 47506 regulation of ... differentiation +T1475 PR:000006854 47515 47519 E2f3 +T1476 GO:0065007 47551 47562 controlling +T1477 GO:0016265 47575 47580 death +T1478 PR:000006854 47582 47586 E2f3 +T1479 PR:000013773 47604 47606 Rb +T1480 GO:0008283 47652 47665 proliferation +T1481 GO:0016265 47669 47674 death +T1482 PR:000006852 47684 47688 E2f1 +T1483 GO:0008283 47716 47729 proliferation +T1484 GO:0016265 47734 47739 death +T1485 PR:000006852 47812 47816 E2f1 +T1486 PR:000006854 47825 47829 E2f3 +T1487 PR:000013773 47848 47850 Rb +T1488 PR:000006852 47887 47891 E2f1 +T1489 PR:000006854 47901 47905 E2f3 +T1490 GO:0065007 47918 47927 regulated +T1491 GO:0010467 47928 47938 expression +T1492 GO:0007049 47942 47952 cell cycle +T1493 SO:0000704 47967 47972 genes +T1494 PR:000013773 47980 47982 Rb +T1495 UBERON:0000966 47986 47992 retina +T1496 PR:000006854 47994 47998 E2f3 +T1497 GO:0010467 48002 48011 expressed +T1498 UBERON:0001017 48027 48030 CNS +T1499 CL:0000117 48027 48038 CNS neurons +T1500 GO:0007049 48071 48081 cell-cycle +T1501 PR:000013773 48105 48107 Rb +T1502 UBERON:0001890 48111 48120 forebrain +T1503 CL:0012001 48111 48128 forebrain neurons +T1504 PR:000006854 48141 48145 E2f3 +T1505 PR:000013773 48211 48213 Rb +T1506 CL:0000540 48239 48247 neuronal +T1507 PR:000013773 48312 48314 Rb +T1508 PR:000006854 48341 48345 E2f3 +T1509 SO:0001060 48346 48353 isoform +T1510 PR:000013773 48432 48434 Rb +T1511 GO:0044838 48479 48488 quiescent +T1512 SO:0001060 48597 48604 isoform +T1513 PR:000013773 48654 48656 Rb +T1514 GO:0065007 48657 48666 regulates +T1515 PR:000028775 48695 48700 E2f3a +T1516 NCBITaxon:10088 48873 48877 mice +T1517 PR:000013773 48913 48915 Rb +T1518 GO:0065007 48927 48936 regulates +T1519 PR:000006854 48980 48984 E2f3 +T1520 SO:0001060 48985 48992 isoform +T1521 PR:000028775 49004 49009 E2f3a +T1522 SO:0001060 49066 49074 isoforms +T1523 PR:000028775 49123 49128 E2f3a +T1524 SO:0001528 49188 49191 NLS +T1525 PR:000013773 49239 49241 Rb +T1526 SO:0000417 49250 49257 domains +T1527 UBERON:0000966 49330 49337 retinal +T1528 CL:0009004 49330 49343 retinal cells +T1529 PR:000028775 49345 49350 E2f3a +T1530 GO:0005634 49359 49366 nuclear +T1531 GO:0005737 49371 49382 cytoplasmic +T1532 GO:0005634 49404 49411 nuclear +T1533 PR:000028775 49458 49463 E2f3a +T1534 PR:000006852 49536 49540 E2f1 +T1535 PR:000006853 49542 49546 E2f2 +T1536 PR:000028775 49552 49557 E2f3a +T1537 PR:000005115 49564 49569 Ccna2 +T1538 GO:0051320 49645 49652 S-phase +T1539 SO:0000417 49700 49706 domain +T1540 GO:0065007 49721 49730 regulated +T1541 PR:000005115 49734 49739 Ccna2 +T1542 SO:0000417 49753 49759 domain +T1543 PR:000013773 49825 49827 Rb +T1544 PR:000013773 49934 49936 Rb +T1545 PR:000016269 49941 49946 Tfdp1 +T1546 GO:0005737 49960 49971 cytoplasmic +T1547 UBERON:0000966 49975 49982 retinal +T1548 CL:0009004 49975 49988 retinal cells +T1549 PR:000013773 50027 50029 Rb +T1550 PR:000006854 50034 50038 E2f3 +T1551 GO:0042995 50057 50066 processes +T1552 GO:0005634 50073 50080 nuclear +T1553 PR:000006856 50181 50185 E2f5 +T1554 GO:0005634 50204 50211 nucleus +T1555 GO:0005737 50215 50224 cytoplasm +T1556 PR:000006855 50232 50236 E2f4 +T1557 PR:000028775 50309 50314 E2f3a +T1558 UBERON:0000966 50332 50338 retina +T1559 PR:000013773 50397 50399 Rb +T1560 PR:000028775 50429 50434 E2f3a +T1561 PR:000028775 50512 50517 E2f3a +T1562 PR:000013773 50558 50560 Rb +T1563 GO:0007049 50645 50655 cell cycle +T1564 CL:0000540 50663 50670 neurons +T1565 UBERON:0003929 50672 50675;50685 50694 gut ... epithelia +T1566 UBERON:0019204 50680 50694 skin epithelia +T1567 CL:0011004 50708 50719 lens fibres +T1568 GO:0010467 50751 50761 expression +T1569 PR:000013773 50789 50791 Rb +T1570 UBERON:0000966 51053 51059 retina +T1571 PR:000013773 51121 51123 Rb +T1572 GO:0010467 51159 51166 express +T1573 UBERON:0000955 51211 51216 brain +T1574 PR:000013773 51218 51220 Rb +T1575 CL:0000540 51224 51231 neurons +T1576 UBERON:0003053 51254 51270 ventricular zone +T1577 PR:000016819 51285 51290 Tubb3 +T1578 PR:000016819 51292 51304 βIII-tubulin +T1579 CHEBI:472552 51335 51339 BrdU +T1580 UBERON:0003929 51353 51366 gut epithelia +T1581 CL:0000584 51383 51394 enterocytes +T1582 GO:0010467 51429 51439 expression +T1583 CHEBI:28790 51443 51452 serotonin +T1584 CHEBI:472552 51482 51486 BrdU +T1585 PR:000014968 51569 51576 Slc18a3 +T1586 GO:0051301 51853 51864;51892 51897 division of ... cells +T1587 http://purl.obolibrary.org/obo/MONDO_0008380 51970 51984 retinoblastoma +T1588 PR:000028775 52015 52020 E2f3a +T1589 PR:000028775 52051 52056 E2f3a +T1590 GO:0005737 52201 52212 cytoplasmic +T1591 PR:000028775 52214 52219 E2f3a +T1592 SO:0000704 52252 52256 gene +T1593 GO:0065007 52257 52267 regulation +T1594 GO:0065007 52308 52316 regulate +T1595 SO:0000704 52333 52338 genes +T1596 GO:0007049 52352 52362 cell cycle +T1597 PR:000003090 52383 52389 Cdkn1b +T1598 GO:0005737 52396 52407 cytoplasmic +T1599 GO:0005634 52502 52509 nucleus +T1600 GO:0005737 52514 52523 cytoplasm +T1601 GO:0022008 52531 52543 neurogenesis +T1602 PR:000028775 52613 52618 E2f3a +T1603 SO:0000704 52635 52640 genes +T1604 GO:0005737 52644 52655 cytoplasmic +T1605 CL:0000540 52685 52692 neurons +T1606 UBERON:0000966 52735 52741 retina +T1607 CL:0000561 52760 52776 amacrine neurons +T1608 PR:000013773 52804 52806 Rb +T1609 PR:000013773 52846 52848 Rb +T1610 CL:0000540 52881 52889 neuronal +T1611 PR:000013773 53024 53026 Rb +T1612 PR:000013773 53105 53107 Rb +T1613 CHEBI:75958 53218 53226 solution +T1614 GO:0000785 53307 53316 chromatin +T1615 PR:000013773 53374 53376 Rb +T1616 UBERON:0000479 53402 53408 tissue +T1617 PR:000013773 53521 53523 Rb +T1618 PR:000013773 53626 53628 Rb +T1619 PR:000013773 53782 53784 Rb +T1620 UBERON:0000479 53788 53795 tissues +T1621 NCBITaxon:10088 53893 53898 Mouse +T1622 NCBITaxon:10088 53924 53928 Mice +T1623 NCBITaxon:10088 54000 54004 mice +T1624 PR:000017355 54017 54022 Chx10 +T1625 NCBITaxon:10088 54027 54031 mice +T1626 PR:000013773 54044 54046 Rb +T1627 SO:0000346 54046 54050 loxP +T1628 SO:0000346 54051 54055 loxP +T1629 NCBITaxon:10088 54056 54060 mice +T1630 PR:000006852 54073 54077 E2f1 +T1631 NCBITaxon:10088 54081 54085 mice +T1632 PR:000006853 54087 54091 E2f2 +T1633 NCBITaxon:10088 54095 54099 mice +T1634 PR:000006854 54101 54105 E2f3 +T1635 SO:0000346 54105 54109 loxP +T1636 SO:0000346 54110 54114 loxP +T1637 NCBITaxon:10088 54115 54119 mice +T1638 PR:000028775 54125 54130 E2f3a +T1639 NCBITaxon:10088 54134 54138 mice +T1640 PR:000028775 54236 54241 E2f3a +T1641 NCBITaxon:10088 54245 54249 mice +T1642 NCBITaxon:10088 54279 54283 Mice +T1643 SO:0000112 54499 54506 primers +T1644 PR:000028775 54527 54532 E2f3a +T1645 NCBITaxon:10088 54536 54540 mice +T1646 PR:000028775 54546 54551 E2f3a +T1647 PR:000028775 54585 54590 E2f3a +T1648 PR:000028775 54629 54634 E2f3a +T1649 UBERON:0010230 54719 54727 Eyeballs +T1650 GO:0051320 54950 54957 S-phase +T1651 CHEBI:472552 54968 54972 BrdU +T1652 UBERON:0001179 55017 55029 peritoneally +T1653 CHEBI:472552 55054 55058 BrdU +T1654 CHEBI:15956 55088 55094 biotin +T1655 MOP:0000093 55088 55105 biotin-conjugated +T1656 NCBITaxon:9940 55106 55111 sheep +T1657 GO:0042571 55123 55131 antibody +T1658 GO:0042571 55216 55226 antibodies +T1659 PR:000006854 55258 55262 E2f3 +T1660 PR:000010425 55264 55269 Mki67 +T1661 PR:000013773 55275 55277 Rb +T1662 CHEBI:59132 55288 55295 antigen +T1663 CHEBI:30769 55343 55354 citric acid +T1664 CHEBI:75958 55355 55363 solution +T1665 CHEBI:60004 55528 55535 mixture +T1666 CHEBI:75958 55536 55544 solution +T1667 CHEBI:15956 55615 55621 biotin +T1668 CHEBI:35696 55641 55646 CoCl2 +T1669 CHEBI:15377 55728 55733 water +T1670 CHEBI:52661 55801 55810 Alexa 488 +T1671 CHEBI:51766 55815 55824 Alexa 568 +T1672 GO:0042571 55933 55943 antibodies +T1673 NCBITaxon:9793 55984 55990 donkey +T1674 NCBITaxon:10088 55996 56001 mouse +T1675 CHEBI:52661 56002 56011 Alexa 488 +T1676 CHEBI:51766 56015 56024 Alexa 568 +T1677 NCBITaxon:9793 56026 56032 donkey +T1678 NCBITaxon:9986 56038 56044 rabbit +T1679 CHEBI:52661 56045 56054 Alexa 488 +T1680 CHEBI:51766 56058 56067 Alexa 568 +T1681 NCBITaxon:9793 56069 56075 donkey +T1682 NCBITaxon:9925 56081 56085 goat +T1683 CHEBI:52661 56086 56095 Alexa 488 +T1684 CHEBI:51766 56099 56108 Alexa 568 +T1685 CHEBI:52661 56127 56136 Alexa 488 +T1686 CHEBI:51766 56140 56149 Alexa 568 +T1687 GO:0005634 56179 56185 Nuclei +T1688 CHEBI:51231 56212 56239 4,6-diamidino-2-phenyindole +T1689 CHEBI:51231 56241 56245 DAPI +T1690 UBERON:0000966 56566 56572 retina +T1691 UBERON:0004086 56619 56630 ventricular +T1692 UBERON:0000966 56643 56649 retina +T1693 UBERON:0001797 56695 56702 vitreal +T1694 GO:0010467 56744 56753 expressed +T1695 UBERON:0000966 56794 56800 retina +T1696 GO:0010467 56858 56865 express +T1697 GO:0051320 57090 57097 S-phase +T1698 GO:0000279 57099 57106 M-phase +T1699 CL:0000445 57112 57127 apoptotic cells +T1700 UBERON:0000970 57183 57188 optic +T1701 UBERON:0000970 57308 57313 optic +T1702 UBERON:0000970 57353 57356 eye +T1703 UBERON:0000970 57367 57371 eyes +T1704 GO:0001171 57426 57447 reverse transcription +T1705 UBERON:0013682 57497 57514 peripheral retina +T1706 CHEBI:33893 57528 57535 reagent +T1707 SO:0000112 57827 57834 primers +T1708 CHEBI:51461 58043 58053 SYBR Green +T1709 CHEBI:60004 58065 58068 Mix +T1710 CHEBI:51461 58216 58226 SYBR Green +T1711 CHEBI:60004 58238 58241 Mix +T1712 SO:0000121 58258 58265;58278 58284 forward ... primer +T1713 SO:0000132 58270 58284 reverse primer +T1714 CHEBI:15377 58315 58318 H2O +T1715 CHEBI:77182 58331 58342 Food Colour +T1716 CHEBI:15377 58432 58435 H2O +T1717 CHEBI:77182 58450 58461 Food Colour +T1718 GO:0097617 58529 58538 annealing +T1719 SO:0000112 58799 58805 primer +T1720 SO:0001026 58848 58855 genomic +T1721 PR:000008735 58949 58954 Hprt1 +T1722 NCBITaxon:10088 58985 58990 Mouse +T1723 UBERON:0000966 58991 58998 retinas +T1724 CHEBI:75958 59103 59111 solution +T1725 GO:0005634 59113 59120 Nuclear +T1726 GO:0005737 59125 59136 cytoplasmic +T1727 GO:0005634 59178 59185 Nuclear +T1728 GO:0005737 59190 59201 Cytoplasmic +T1729 CHEBI:8984 59314 59317 SDS +T1730 CHEBI:53325 59342 59356 nitrocellulose +T1731 UBERON:0001913 59402 59406 milk +T1732 GO:0042571 59448 59456 antibody +T1733 CHEBI:34683 59528 59535 Na2HPO4 +T1734 CHEBI:37585 59544 59551 NaH2PO4 +T1735 CHEBI:26710 59559 59563 NaCl +T1736 CHEBI:53424 59570 59578 Tween-20 +T1737 NCBITaxon:3704 59654 59665 horseradish +T1738 MOP:0000779 59677 59687 conjugated +T1739 GO:0042571 59688 59696 antibody +T1740 GO:0042571 59964 59974 antibodies +T1741 PR:000006852 59986 59991 E2f-1 +T1742 PR:000006854 60002 60007 E2f-3 +T1743 PR:000005274 60018 60024 Cdkn1a +T1744 PR:000005274 60026 60029 p21 +T1745 PR:000003090 60040 60046 Cdkn1b +T1746 PR:000003090 60048 60051 p27 +T1747 PR:000013042 60062 60068 Pou4f2 +T1748 PR:000013042 60070 60075 Brn3b +T1749 PR:000016269 60091 60096 Tfdp1 +T1750 PR:000016269 60098 60101 Dp1 +T1751 PR:000014968 60240 60247 Slc18a3 +T1752 PR:000014968 60249 60254 VAChT +T1753 GO:1990603 60349 60361 dark-adapted +T1754 NCBITaxon:10088 60362 60366 mice +T1755 NCBITaxon:10088 60395 60399 mice +T1756 GO:1990603 60405 60417 dark-adapted +T1757 CHEBI:6121 60475 60483 ketamine +T1758 UBERON:0001771 60552 60558 pupils +T1759 GO:1990603 60624 60636 dark-adapted +T1760 GO:1990603 60638 60646 scotopic +T1761 GO:0036367 60652 60665 light-adapted +T1762 GO:0036367 60667 60675 photopic +T1763 GO:0036367 60689 60705 Light adaptation +T1764 PR:000006852 61421 61425 E2f1 +T1765 PR:000006853 61435 61439 E2f2 +T1766 PR:000006854 61443 61447 E2f3 +T1767 GO:0008219 61478 61488 Cell Death +T1768 PR:000013773 61503 61505 Rb +T1769 UBERON:0000966 61509 61515 Retina +T1770 GO:0005634 61590 61596 nuclei +T1771 CHEBI:51231 61598 61602 DAPI +T1772 GO:0051320 61619 61626 S-phase +T1773 CHEBI:472552 61633 61637 BrdU +T1774 GO:0006915 61651 61660 apoptosis +T1775 PR:000013773 61678 61680 Rb +T1776 UBERON:0000966 61684 61691 retinas +T1777 CHEBI:472552 61693 61697 BrdU +T1778 GO:0071897 61769 61782 DNA synthesis +T1779 CL:0002672 61875 61879 RPCs +T1780 PR:000006852 61966 61970 E2f1 +T1781 GO:0006915 62000 62009 Apoptosis +T1782 UBERON:0000922 62017 62026 Embryonic +T1783 PR:000013773 62027 62029 Rb +T1784 UBERON:0000966 62033 62039 Retina +T1785 GO:0005634 62167 62173 nuclei +T1786 CHEBI:51231 62175 62179 DAPI +T1787 GO:0051320 62199 62206 S-phase +T1788 CHEBI:472552 62231 62235 BrdU +T1789 GO:0006915 62245 62254 apoptosis +T1790 PR:000013773 62290 62292 Rb +T1791 UBERON:0000966 62296 62303 retinas +T1792 CHEBI:472552 62305 62309 BrdU +T1793 UBERON:0000966 62353 62359 retina +T1794 PR:000006852 62386 62390 E2f1 +T1795 CL:0002672 62460 62464 RPCs +T1796 PR:000006852 62551 62555 E2f1 +T1797 PR:000006853 62565 62569 E2f2 +T1798 PR:000006854 62573 62577 E2f3 +T1799 GO:0007067 62595 62602 Mitosis +T1800 PR:000013773 62610 62612 Rb +T1801 UBERON:0000966 62616 62622 Retina +T1802 UBERON:0000966 62639 62646 retinal +T1803 GO:0005634 62709 62715 nuclei +T1804 CHEBI:51231 62717 62721 DAPI +T1805 GO:0000279 62733 62740 M-phase +T1806 UBERON:0000966 62961 62967 retina +T1807 PR:000006853 63124 63128 E2f2 +T1808 PR:000006854 63132 63136 E2f3 +T1809 UBERON:0000045 63153 63161 Ganglion +T1810 CL:0000740 63153 63161;63179 63183 Ganglion ... Cell +T1811 CL:0000604 63163 63166;63179 63183 Rod ... Cell +T1812 CL:0000103 63171 63183 Bipolar Cell +T1813 GO:0008219 63179 63189 Cell Death +T1814 PR:000013773 63197 63199 Rb +T1815 UBERON:0000966 63203 63209 Retina +T1816 UBERON:0000966 63226 63233 retinal +T1817 NCBITaxon:10088 63248 63252 mice +T1818 GO:0005634 63306 63312 nuclei +T1819 CHEBI:51231 63314 63318 DAPI +T1820 UBERON:0000045 63350 63358 ganglion +T1821 CL:0000740 63350 63364 ganglion cells +T1822 PR:000013042 63366 63372 Pou4f2 +T1823 CL:0000604 63380 63384 rods +T1824 CL:0000573 63389 63394 cones +T1825 PR:000014434 63396 63399 Sag +T1826 CL:0000604 63401 63404 rod +T1827 PR:000014434 63401 63413 rod arrestin +T1828 CL:0000751 63428 63445 rod bipolar cells +T1829 PR:000003058 63447 63452 Prkca +T1830 UBERON:0000045 63511 63519 ganglion +T1831 CL:0000740 63511 63519;63530 63535 ganglion ... cells +T1832 PR:000013042 63521 63527 Pou4f2 +T1833 CL:0000751 63566 63577;63587 63592 rod bipolar ... cells +T1834 PR:000003058 63579 63584 Prkca +T1835 UBERON:0001789 63616 63619 ONL +T1836 CL:0000604 63652 63656 rods +T1837 UBERON:0000966 63738 63744 retina +T1838 PR:000006852 63880 63884 E2f1 +T1839 PR:000013773 63908 63910 Rb +T1840 SO:0000346 63910 63914 loxP +T1841 SO:0000346 63915 63919 loxP +T1842 UBERON:0000966 63920 63927 Retinal +T1843 GO:0036367 63992 64005 light adapted +T1844 GO:0036367 64007 64015 photopic +T1845 PR:000013773 64225 64227 Rb +T1846 UBERON:0000966 64248 64255 retinal +T1847 GO:0005634 64314 64320 nuclei +T1848 CHEBI:51231 64322 64326 DAPI +T1849 PR:000004968 64338 64343 Calb2 +T1850 PR:000003199 64411 64417 Camk2a +T1851 PR:000014968 64436 64443 Slc18a3 +T1852 CHEBI:16865 64542 64546 GABA +T1853 CHEBI:25512 64547 64563 Neurotransmitter +T1854 PR:000013773 64571 64573 Rb +T1855 UBERON:0000966 64577 64583 Retina +T1856 PR:000017355 64605 64610 Chx10 +T1857 PR:000013773 64615 64617 Rb +T1858 SO:0000346 64617 64621 loxP +T1859 SO:0000346 64622 64626 loxP +T1860 UBERON:0000966 64627 64633 Retina +T1861 UBERON:0000966 64694 64700 retina +T1862 GO:0005634 64718 64724 nuclei +T1863 CHEBI:51231 64726 64730 DAPI +T1864 CHEBI:16865 64753 64757 GABA +T1865 PR:000014968 64768 64775 Slc18a3 +T1866 PR:000014968 64800 64807 Slc18a3 +T1867 UBERON:0000966 64830 64836 retina +T1868 CHEBI:16865 64838 64842 GABA +T1869 PR:000014968 64915 64922 Slc18a3 +T1870 PR:000013773 64961 64963 Rb +T1871 UBERON:0000966 64967 64973 retina +T1872 PR:000006854 64995 64999 E2f3 +T1873 PR:000006852 65011 65015 E2f1 +T1874 PR:000013773 65065 65067 Rb +T1875 UBERON:0013682 65077 65094 peripheral retina +T1876 CHEBI:16865 65106 65110 GABA +T1877 PR:000014968 65183 65190 Slc18a3 +T1878 PR:000017355 65214 65219 Chx10 +T1879 PR:000013773 65224 65226 Rb +T1880 SO:0000346 65226 65230 loxP +T1881 SO:0000346 65231 65235 loxP +T1882 UBERON:0000966 65236 65242 retina +T1883 PR:000013773 65284 65286 Rb +T1884 PR:000028775 65416 65421 E2f3a +T1885 SO:0001060 65422 65429 Isoform +T1886 UBERON:0000966 65448 65454 Retina +T1887 GO:0005634 65456 65463 Nuclear +T1888 GO:0005737 65468 65479 cytoplasmic +T1889 UBERON:0000966 65518 65525 retinal +T1890 CL:0009004 65518 65531 retinal cells +T1891 NCBITaxon:10088 65537 65541 mice +T1892 PR:000028775 65626 65631 E2f3a +T1893 PR:000028775 65654 65659 E2f3a +T1894 NCBITaxon:10088 65663 65667 mice +T1895 PR:000028775 65734 65739 E2f3a +T1896 GO:0005737 65749 65750 C +T1897 GO:0005737 65752 65763 cytoplasmic +T1898 GO:0005634 65774 65775 N +T1899 GO:0005634 65777 65784 nuclear +T1900 GO:0042571 65866 65876 Antibodies +T1901 PR:000013773 65900 65902 Rb +T1902 PR:000006852 65903 65907 E2f1 +T1903 SO:0000112 65996 66003 Primers +T1904 SO:0000704 66157 66162 genes +T1905 SO:0000704 66167 66171 gene +T1906 PR:000003199 66209 66215 Camk2a +T1907 PR:000006852 66247 66251 E2f1 +T1908 PR:000006853 66265 66269 E2f2 +T1909 PR:000006854 66283 66287 E2f3 +T1910 PR:000013773 66301 66303 Rb +T1911 PR:000014968 66321 66328 Slc18a3 +T1912 PR:000009116 66456 66460 Isl1 +T1913 PR:000009116 66462 66468 Islet1 +T1914 NCBITaxon:10088 66572 66576 mice +T1915 GO:0042571 66705 66715 antibodies +T1916 CHEBI:472552 66763 66767 BrdU +T1917 CHEBI:472552 66770 66787 bromodeoxyuridine +T1918 UBERON:0001017 66803 66806 CNS +T1919 UBERON:0001017 66809 66831 central nervous system +T1920 CHEBI:51231 66833 66837 DAPI +T1921 CHEBI:51231 66840 66867 4,6-diamidino-2-phenyindole +T1922 UBERON:0000922 66904 66913 embryonic +T1923 CHEBI:16865 67024 67028 GABA +T1924 CHEBI:16865 67031 67054 gamma-aminobutyric acid +T1925 UBERON:0001792 67056 67059 GCL +T1926 CL:0000740 67062 67075 ganglion cell +T1927 UBERON:0001792 67062 67081 ganglion cell layer +T1928 UBERON:0001791 67083 67086 INL +T1929 UBERON:0001791 67089 67108 inner nuclear layer +T1930 GO:0005634 67095 67102 nuclear +T1931 UBERON:0001795 67110 67113 IPL +T1932 UBERON:0001795 67116 67137 inner plexiform layer +T1933 CL:0000031 67160 67172 neuroblastic +T1934 UBERON:0001789 67180 67183 ONL +T1935 UBERON:0001789 67186 67205 outer nuclear layer +T1936 GO:0005634 67192 67199 nuclear +T1937 UBERON:0001790 67207 67210 OPL +T1938 UBERON:0001790 67213 67234 outer plexiform layer +T1939 GO:0007567 67252 67257 natal +T1940 PR:000013773 67296 67298 Rb +T1941 http://purl.obolibrary.org/obo/MONDO_0008380 67301 67315 retinoblastoma +T1942 PR:000013773 67301 67323 retinoblastoma protein +T1943 CL:0002672 67325 67328 RPC +T1944 UBERON:0000966 67331 67338 retinal +T1945 CL:0002672 67331 67354 retinal progenitor cell +T1946 UBERON:0000966 67362 67369 retinal +T1947 CL:0000561 67439 67452 amacrine cell +T1948 PR:000006854 67686 67690 E2f3 +T1949 SO:0000359 67691 67697 floxed +T1950 PR:000028775 67702 67707 E2f3a +T1951 NCBITaxon:10088 67711 67715 mice +T1952 http://purl.obolibrary.org/obo/MONDO_0001941 67933 67942 Blindness diff --git a/src/ontogpt/evaluation/craft/database/all/17608565.txt b/src/ontogpt/evaluation/craft/database/all/17608565.txt new file mode 100644 index 000000000..708670174 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17608565.txt @@ -0,0 +1,447 @@ +Rb-Mediated Neuronal Differentiation through Cell-Cycle–Independent Regulation of E2f3a + +Abstract + +It has long been known that loss of the retinoblastoma protein (Rb) perturbs neural differentiation, but the underlying mechanism has never been solved. Rb absence impairs cell cycle exit and triggers death of some neurons, so differentiation defects may well be indirect. Indeed, we show that abnormalities in both differentiation and light-evoked electrophysiological responses in Rb-deficient retinal cells are rescued when ectopic division and apoptosis are blocked specifically by deleting E2f transcription factor (E2f) 1. However, comprehensive cell-type analysis of the rescued double-null retina exposed cell-cycle–independent differentiation defects specifically in starburst amacrine cells (SACs), cholinergic interneurons critical in direction selectivity and developmentally important rhythmic bursts. Typically, Rb is thought to block division by repressing E2fs, but to promote differentiation by potentiating tissue-specific factors. Remarkably, however, Rb promotes SAC differentiation by inhibiting E2f3 activity. Two E2f3 isoforms exist, and we find both in the developing retina, although intriguingly they show distinct subcellular distribution. E2f3b is thought to mediate Rb function in quiescent cells. However, in what is to our knowledge the first work to dissect E2f isoform function in vivo we show that Rb promotes SAC differentiation through E2f3a. These data reveal a mechanism through which Rb regulates neural differentiation directly, and, unexpectedly, it involves inhibition of E2f3a, not potentiation of tissue-specific factors. + +Author Summary + + + +The retinoblastoma protein (Rb), an important tumor suppressor, blocks division and death by inhibiting the E2f transcription factor family. In contrast, Rb is thought to promote differentiation by potentiating tissue-specific transcription factors, although differentiation defects in Rb null cells could be an indirect consequence of E2f-driven division and death. Here, we resolve different mechanisms by which Rb controls division, death, and differentiation in the retina. Removing E2f1 rescues aberrant division of differentiating Rb-deficient retinal neurons, as well as death in cells prone to apoptosis, and restores both normal differentiation and function of major cell types, such as photoreceptors. However, Rb-deficient starburst amacrine neurons differentiate abnormally even when E2f1 is removed, providing an unequivocal example of a direct role for Rb in neuronal differentiation. Rather than potentiating a cell-specific factor, Rb promotes starburst cell differentiation by inhibiting another E2f, E2f3a. This cell-cycle–independent activity broadens the importance of the Rb–E2f pathway, and suggests we should reassess its role in the differentiation of other cell types. + +Introduction + +The simplicity of the retina makes it an ideal tissue to study neurogenesis. Its development proceeds through three overlapping steps starting with retinal progenitor cell (RPC) proliferation, followed by birth of post-mitotic retinal transition cells (RTCs, also referred to as precursors), and ending with terminal differentiation of seven major cell types (Figure 1A) [1]. RPCs are multipotent and exit the cell cycle to generate different RTCs at specific time periods in development [1]. This process of RTC “birth” requires coupling of differentiation and cell cycle exit. Once born, post-mitotic RTCs migrate and form different retinal layers. Rods and cones make up the outer nuclear layer (ONL); horizontal, bipolar, and amacrine cells, as well as Müller glia cell bodies, reside in the inner nuclear layer (INL); and ganglion and displaced amacrine cells form the ganglion cell layer (GCL) (Figure 1A). The outer plexiform layer (OPL) and inner plexiform layer (IPL) house synaptic connections separating the ONL/INL and INL/GCL, respectively. + +Figure 1 + +E2f1, but Not E2f2 or E2f3, Loss Rescues Ectopic Division and Cell Death in the Rb KO Retina + +(A) Retinal development. At E11 the retina is a NBL of dividing RPCs (white circle, green nuclei). RPC cell bodies oscillate along processes as they progress through the cell cycle. By P0 the NBL contains both RPCs and post-mitotic RTCs (coloured circles, red nuclei) and is separated from the GCL by the IPL. By P8 there are no RPCs, fewer RTCs, an OPL, and more differentiated rods (r) and cones (c) in the ONL; horizontal (h), bipolar (b), Müller (m), and amacrine (a) cells in the INL; and ganglion (g) and displaced amacrine cells in the GCL. Development is complete by ~P18. + +(B) Rb is thought to regulate cell cycle and apoptosis by repressing E2fs, but to promote differentiation by potentiating tissue-specific transcription factors. However, Rb loss could also perturb differentiation through the indirect effects of abnormal division or death, and/or through direct regulation of differentiation genes by E2fs. + +(C and D) Horizontal retinal sections of the indicated genotypes and ages were stained for nuclei (DAPI, blue), and (C) S-phase (anti-BrdU, red) or (D) apoptosis (TUNEL, red). Scale bars are 50 μm. + +(E–G) Quantification of (E) all BrdU+ cells, (F) ectopic BrdU+ cells in GCL at P0, and (G) total TUNEL+ cells. + +(H) Real-time RT-PCR analysis of E2fs and E2f target genes in P8 retinas of the indicated genotypes. + +Error bars represent SD of measurements from three animals, and asterisks indicate a significant difference between the WT and indicated genotypes (*, p <0.05; **; p <0.01; ANOVA and Tukey HSD test for [E–G] and Fisher test for [H]). + +The retinoblastoma protein (Rb) is critical for cell cycle exit during retinal transition cell birth. Rb knockout (KO) RTCs continue to proliferate inappropriately and some (rod, ganglion, and bipolar cells) die by apoptosis [2,3]. Rb controls the cell cycle by binding and inhibiting E2f transcription factors (E2fs) (Figure 1B), first defined as transcription factors that bind adenoviral E2 regulatory elements and subsequently shown to be critical cell cycle regulators [4,5]. E2fs bind to DNA as heterodimers with proteins of the related Tfdp family. E2f1, E2f2, and E2f3a are “activating E2fs” that are required for fibroblast division. They are strong transcriptional activators that can drive G0 fibroblasts into cycle, and are inhibited when bound to Rb [4,5]. Ectopic division in Rb KO embryos can be rescued to various extents in different tissues by knocking out E2f1, E2f2, or E2f3 [6–9], but which member(s) drive division in Rb KO RTCs is unknown. Other members of the family, such as E2f4 and E2f5, are known as “repressive E2fs” because they are weak activators and appear to be primarily involved in gene silencing in quiescent or differentiated cells. + +Activating E2fs may also promote apoptosis in the Rb KO retina (Figure 1B). Originally, E2f1 was considered the primary pro-apoptotic member of the family [10]. However, this view was reevaluated when it was shown that either E2f1 or E2f3 deletion rescues apoptosis in the developing central nervous system (CNS) of Rb KO embryos [6,11]. Subsequently, CNS apoptosis was shown to be an indirect result of placental defects and probable hypoxia [12–14]. Indeed, E2f3-induced apoptosis in fibroblasts has recently been shown to require E2f1 [15]. Thus, it is controversial whether E2f3 is required for apoptosis of any Rb KO cell type. Determining which activating E2fs promote death in distinct Rb KO tissues requires conditional rather than germ line models of Rb deletion to avoid secondary indirect effects (such as hypoxia). + +E2f family diversity is expanded by E2f3 isoforms. Alternative promoters generate two forms (a and b) that are identical except for distinct first exons [16]. E2f3a is a strong activator, and, like other activating E2fs, its expression is induced when quiescent cells are stimulated to divide [16]. E2f3b, like repressive E2fs, is present in both quiescent and dividing cells, and in quiescent fibroblasts it associates primarily with Rb, suggesting that it mediates repression [16–18]. Indeed, silencing the Cdkn2d (p19Arf) locus in unstressed cells relies on E2f3b [19]. Other E2fs may also exist in isoforms since at least two mRNA species have been detected for E2f1 and E2f2 [16]. The roles of E2f isoforms in vivo are unknown. + +E2fs are also regulated by subcellular localization. Although this feature has been best characterized for repressive E2fs [20–22], it also affects activating E2fs [23–25]. The distribution of E2f isoforms has never been assessed. + +It has been known for many years that Rb loss perturbs neuronal differentiation [26–29]. However, prior work could not exclude the possibility that differentiation defects are simply an indirect consequence of abnormal division and death. If Rb does regulate differentiation directly it is unclear whether it does so in all or a subset of neurons. Moreover, the mechanism has never been solved. In other cell types where Rb may promote differentiation directly, such as muscle and bone, it seems to do so through E2f-independent means by potentiating tissue-specific transcription factors (Figure 1B) [30–33]. In the retina, others have noted abnormally shaped Rb KO rods and have suggested Rb may directly promote their morphogenesis by activating retina-specific factors [29]. However, differentiation defects in any Rb KO neuron could be an indirect effect of ectopic division and/or apoptosis (Figure 1B). Thus, it is critical to study differentiation of Rb KO cells in the absence of ectopic proliferation and death. + +Here, we establish that Rb suppresses RTC division and death by inhibiting E2f1, not E2f2 or E2f3. When these defects were rescued, most retinal neurons, including rods, survived, differentiated, and functioned normally. Thus, unexpectedly, retina-specific differentiation factors function independently of Rb. However, comprehensive assessment of the Rb/E2f1 double-null rescued retina revealed a differentiation defect in cholinergic starburst amacrine cells (SACs). Recent breakthroughs have revealed that these interneurons are critical for direction selectivity and developmentally important rhythmic bursts [34–36]. However, their differentiation is poorly understood. Contrary to the prevailing view that Rb promotes differentiation through E2f-independent tissue-specific transcription factors, we show that Rb facilitates SAC development through E2f3. Defects in Rb null SACs correlated with specific E2f3 expression in these cells, and E2f3 expression was absent in neurons that differentiated without Rb. E2f3 is also present in a specific subset of other CNS neurons, implying that this may be a general mechanism by which Rb facilitates neurogenesis. To define the mechanism in even more detail, we determined which E2f3 isoform Rb targets to control SAC differentiation. E2f3b mediates Rb function in quiescent fibroblasts [19], yet no prior studies to our knowledge have dissected E2f3a or E2f3b functions in vivo. Using an isoform-specific null mouse we show that Rb drives SAC differentiation through E2f3a. Thus, independent of E2f1-mediated effects on division and death, Rb does regulate neuronal differentiation, but only in specific neurons and, unexpectedly, through E2f3a, not tissue-specific differentiation factors. + +Results + +Rb Regulates Division and Death through E2f1 + +We used the α-Cre transgene to delete floxed Rb exon 19 at embryonic day (E) 10 in peripheral retina [2]. RbloxP/loxP;α-Cre mice were bred with strains lacking E2f1 or E2f2 in the germ line, or a strain carrying a floxed E2f3 allele [5]. RbloxP/loxP;E2f1+/− and RbloxP/loxP;E2f1+/−;α-Cre mice were bred to produce RbloxP/loxP;E2f1−/−;α-Cre mice at a frequency of 1/8 and littermate controls at the same or higher (1/4) frequency. For simplicity we will refer to the RbloxP/loxP;E2f1−/−;α-Cre peripheral retina as the Rb/E2f1 double knockout (DKO) retina. Similar strategies were employed in the case of E2f2 or E2f3. Cre-mediated excision of Rb and E2f3 alleles in the retina was confirmed by PCR as described previously [2,5]. + +To measure ectopic cell division, mice were pulse-labelled with bromodeoxyuridine (BrdU) 2 h before sacrifice and the peripheral retina analyzed for BrdU incorporation by immunofluorescence. As reported before [2,3], Rb KO retinas exhibited both spatial and temporal ectopic DNA synthesis (Figures 1C and S1A). This is easily detected at E14, E16, and postnatal day (P) 0 in the inner retina where abnormal BrdU+ ganglion and amacrine RTCs are located, or on the outermost region of the P0 retina, where BrdU+ photoreceptor RTCs reside (Figures S1A and S2, arrows) [2]. Ectopic RTC division in Rb KO retinas is even more obvious at P8 or P18, when division is completed in wild-type (WT) retina (Figures 1C and S1A). Strikingly, the ectopically positioned S-phase cells at E14, E16, and P0 and all the abnormal division at P8 and P18 were completely suppressed in the Rb/E2f1 DKO retina (Figures 1C, 1E, 1F, S1A, and S2). In contrast, deletion of E2f2 or E2f3 had no effect at any stage of development. Analysis of mitotic cells with anti–phosphohistone 3 (PH3)–specific antibodies confirmed that loss of E2f1, but not E2f2 or E2f3, suppressed ectopic division (Figure S3). Deleting one E2f1 allele partially suppressed ectopic S-phase and mitosis in Rb KO RTCs (Figures 1C, 1E, 1F, S1A, S2, and S3), suggesting that E2f1 drives ectopic division in Rb KO RTCs in a dose-dependent fashion. These data contrast with previous findings in the lens and CNS of Rb KO embryos, where deletion of any activating E2f suppresses ectopic division to some extent [6–9]. + +Loss of Rb in the retina results in considerable RTC apoptosis, eliminating most bipolar and ganglion cells as well as many rods (Figure 2A–2D) [2,3]. The loss of Rb KO rods is evident from the thinner ONL, and the death of these cells as well as bipolar and ganglion neurons can be detected directly by double labelling for apoptotic and cell-type-specific markers [2] (M. P. and R. B., unpublished data). Loss of peripheral Rb KO ganglion cells is also evident from thinning of the optic nerve (D. C. and R. B., unpublished data). Deleting E2f1, but not E2f2 or E2f3, blocked this ectopic cell death in a dose-dependent fashion (Figures 1D, 1G, and S1B). + +Figure 2 + +E2f1 Deletion Rescues Ganglion, Rod, and Bipolar Cells in the Rb KO Retina + +(A) Horizontal retinal sections from mice of the indicated ages and genotypes were stained for nuclei (DAPI, blue) and markers that detect ganglion cells (Pou4f2, red), rods and cones (Sag [rod arrestin], green), and rod bipolar cells (Prkca, green, and Cabp5, red). Scale bars are 50 μm. + +(B) Quantification of Pou4f2+ ganglion cells. + +(C) Quantification of Prkca+ and Cabp5+ bipolar cells. + +(D) Thickness of the ONL, which represents the number of rods. + +Error bars represent SD of measurements from three animals, and asterisks indicate a significant difference between retinas of WT and the indicated genotypes, unless indicated otherwise by connecting lines (*, p < 0.05; **; p < 0.01; ANOVA and Tukey HSD test). + +(E and F) ERGs were recorded from the indicated genotypes under dark-adapted (scotopic) conditions, and (E) intensity series and (F) b-wave amplitudes as a function of the logarithm of the flash intensity were determined. (F) Further illustrates that the relative influence of the mutations on the photoreceptors (indicated by the saturated a-wave amplitude, right graph) was not substantially different from their effect on the b-wave response (dominated by the bipolars, left graph) at the same intensity of 10 cd·s/m2. + +To investigate the molecular mechanism that underlies the unique role of E2f1, we assessed the expression of known E2f targets as well as other genes that regulate the cell cycle and apoptosis. Numerous positive and negative cell cycle and apoptotic regulators were up-regulated in the Rb KO retina (Figure 1H). Among the E2f family, E2f1, E2f2, E2f3a, and E2f7 were induced following Rb loss, but E2f3b, E2f4, and E2f5 were unaffected. Consistent with the BrdU and terminal dUTP nick-end labelling (TUNEL) analyses, E2f1 deletion specifically reversed all these molecular defects, but E2f3 deletion had no effect (Figure 1H). + +Normal Differentiation in the Rb/E2f1 DKO Retina + +Because E2f1 deletion blocks abnormal division and death in the Rb KO retina, the Rb/E2f1 DKO retina provided a unique opportunity to evaluate whether Rb controls differentiation independent of cell cycle effects. The Rb/E2f1 DKO retina had many Sag+ (S-antigen/rod arrestin) photoreceptors, Pou4f2+ (Brn3b) ganglion cells, and numerous Prkca+/Cabp5+ bipolar neurons (Figure 2A–2D). In contrast, there was no such rescue of cell types in Rb/E2f2 or Rb/E2f3 DKO retinas (Figure S4). Analysis with general neuronal markers Mtap2 (MAP2) and Snap25, as well as other markers expressed in bipolar cells (Chx10, Rcvrn, Vsx1, Tacr3, and Atp2b1) and rod photoreceptors (Rho and Rcvrn) confirmed rescue of the Rb/E2f1 DKO retina (Table S1). Moreover, neurons exhibited the same complex morphology as in WT retina. Bipolar cell bodies were located in the INL and had ascending and descending processes ending in the OPL and IPL, respectively (Figure 2A). In addition, the Rb/E2f1 DKO retina had a healthy appearing ONL consisting of morphologically normal rods with descending processes ending in the OPL and ascending processes that terminated in inner and outer segments (Figure 2A). It was suggested that Rb might regulate photoreceptor differentiation, possibly through rod-specific transcription factors (Figure 1B) [29]. However, if Rb does regulate photoreceptor differentiation, it does so by inhibiting E2f1, not by potentiating rod differentiation factors, such as Otx2, Crx, or Nrl. It is impossible to tell whether E2f1 perturbs differentiation directly, by affecting the expression of genes that modulate maturation, and/or indirectly through its effects on proliferation and survival (Figure 1B). + +As with ectopic division and apoptosis (Figure 1C and 1D), the rescue of Rb KO retinal bipolar, ganglion, and rod cells was dependent on E2f1 dose (Figure 2A–2D). Separate from its role in driving ectopic division of Rb KO RTCs, E2f1 also promotes normal RPC division since in its absence RPC proliferation drops ~2-fold (D. C. and R. B., unpublished data). This modest reduction of RPC numbers in the absence of E2f1 accounts for the slight reduction in the number of ganglion cells at P0, in the number of bipolar cells at P18 or P30, and in the thickness of the ONL at P18 or P30 in the E2f1 KO and Rb/E2f1 DKO retina (Figure 2B–2D). The morphology of E2f1 KO neurons was WT (Figure 2A). Despite a slight drop in absolute cell numbers, the proportion of Rb/E2f1 DKO and E2f1 KO bipolar cells was the same as WT (data not shown). Slightly reduced cell numbers were not due to residual RTC death since we have not observed ectopic apoptosis at any embryonic or postnatal stage in the developing Rb/E2f1 DKO retina (Figures 1D, 1G, and S2). Moreover, deleting Ccnd1, which acts upstream of Rb proteins, also reduces RPC number, but does not suppress any defect in the Rb KO retina (D. C. and R. B., unpublished data). Thus, slightly reduced RPC division and dramatic rescue of severe defects in Rb KO RTCs are distinct effects stemming from the deletion of E2f1. + +Normal Function of the Rb/E2f1 DKO Retina + +The discovery that E2f1 loss rescues even the morphology of Rb KO neurons is surprising because Rb is thought to regulate differentiation primarily through E2f-independent pathways [30–33]. However, normal morphology may not equate to completely normal differentiation. Thus, we compared the electroretinograms (ERGs) of adult WT (α-Cre), E2f1−/−, α-Cre;RbloxP/loxP, and α-Cre;RbloxP/loxP;E2f1−/− mice. ERGs functionally assess visual signalling in the mammalian retina from photoreceptors to amacrine cells (but usually not gangion cells), and are dominated by rod and cone bipolar cells. Typically, an ERG signal begins with a negative deflection initiated by the photoreceptors (the a-wave), which is terminated by a large positive deflection due to the activation of ON bipolar cells (the b-wave). + +Responses to dim light in dark-adapted (scotopic) conditions specifically assess the rod system, and were defective in the Rb KO retina (Figure 2E). The substantial reduction of both a- and b-waves is consistent with rod and bipolar cell apoptosis [2]. The sensitivity of the residual response appeared unchanged, suggesting it arose from the Cre-negative portions of the retina. Responses were about the same in the WT and E2f1 KO retina, and, most importantly, also the Rb/E2f1 DKO response median lay at the lower end of the normal range for most intensities (Figure 2F). Thus, E2f1 deletion almost completely rescued the rod system in the Rb KO retina. + +Light-adapted (photopic) recordings to specifically assess the cone system yielded comparable results. Cones represent only 3% of photoreceptors and, unlike rods, develop without Rb, but they require rods for survival, and in the Rb KO retina, they have abnormal morphology and their synaptic targets, bipolar cells, are much depleted [2]. The photopic response, a product of cone and mainly bipolar activity, was much reduced by Rb loss, but was rescued considerably in the Rb/E2f1 DKO retina (Figure S5). Again, the median amplitude lay at the lower end of the E2f1 KO range. The photopic response in E2f1 KO mice was slightly reduced relative to WT (Figure S5B), possibly because E2f1 is required for maximal expansion of embryonic RPCs, and the E2f1 KO retina has, as noted earlier, slightly fewer cells than the WT retina, although cell type proportions are unaffected (D. C. and R. B., unpublished data). Thus, marginally subnormal photopic responses in the Rb/E2f1 DKO retina can be attributed to both a reduction of cone numbers in E2f1 KO mice alone, and a “genuine” slight reduction in cone function attributable to Rb loss relative to WT. This slight effect may relate to a true differentiation defect in a subset of amacrine cells discussed below. This discussion should not obscure the major outcome that E2f1 deletion recovers most of the ERG response. Thus, E2f1 deletion not only rescues morphology but also both rod and cone system function in the Rb KO retina. + +Abnormal SAC Differentiation Independent of Cell Cycle and Survival Defects + +ERGs primarily assess photoreceptor and bipolar cell function, but may miss differentiation defects in other cells. To test for subtle differences we stained the Rb/E2f1 DKO retina with 43 markers (Table S1). Thirty-two proteins displayed identical patterns in WT, E2f1 KO, and Rb/E2f1 DKO retina (Table S1). The other 11 markers revealed a cell-cycle– and apoptosis-independent differentiation defect in SACs. We first studied Calb2 (calretinin), which marks a subset of amacrine and ganglion cell bodies as well as three tracks corresponding to their processes in the IPL (Figure 3A). Normal Calb2 staining was seen in the E2f1 KO IPL (data not shown). However, only one Calb2+ track was evident in the Rb KO IPL, and this defect was not rescued in the Rb/E2f1 DKO retina (Figure 3A). We quantified Calb2+ cell bodies in the Rb KO INL (corresponding to amacrine cell staining only) and observed a reduction from P8 onwards (Figures 3C and S6). + +Figure 3 + +Differentiation Defects in Rb KO SACs + +(A) P18 horizontal sections of WT, Rb KO, and Rb/E2f1 DKO retina were stained for nuclei (DAPI, blue), Calb2 (green), and Slc18a3 (red). + +(B) Confocal images of P30 horizontal sections of WT and Rb KO retina were stained for nuclei (DAPI, blue), Chat and Slc18a3 (both red), and Camk2a (green). In the Rb KO section, the red stain is Chat only, as Slc18a3 is missing (see [A]). + +(C) Quantification of dense Calb2+ cell bodies in the INL, total Slc18a3+ cell bodies, and Camk2a+ cell bodies in the INL. Error bars represent SD of measurements from three animals, and asterisks indicate significant differences between retinas of WT and the indicated genotypes (*, p <0.05; **; p <0.01; ANOVA and Tukey HSD test). + +Scale bars are 50 μm in (A) and 20 μm in (B). + +Of the three Calb2+ tracks in the IPL, the two outer tracks are from SACs, named after their extensive dendritic-tree-like morphology [37]. SACs are cholinergic, represent ~5.2% of amacrine neurons [38], and are critical for both direction selectivity [34,35] and spontaneous rhythmic activity that occurs during normal retinal development [36]. SACs in the INL synapse in the OFF layer of the IPL that responds to decreasing light, while displaced SACs in the GCL have processes that synapse in the nearby ON layer of the IPL that responds to increasing light (reviewed in [39]). Mature SAC processes stain specifically for Slc18a3 (vesicular acetyl choline transporter, VAChT) [37], and, significantly, this marker was absent in the peripheral Rb KO or Rb/E2f1 DKO P18 retina (Figures 3A and S7B). Chat, expressed from the same locus, is also SAC specific, but marks both cell bodies and processes of mature SACs [37]. Chat was seen in fewer cells in the mature Rb KO retina, and was present in the soma but absent from processes (Figure 3B). We obtained similar results for Sv2c, a synaptic vesicle protein found in SACs [40]; Kcnc1b and Kcnc2, potassium channels expressed on SAC soma and dendrites as well as a very small number of ganglion cells [41]; gamma-aminobutyric acid (GABA), an inhibitory neurotransmitter present in about half of amacrine cells including SACs, as well as horizontal and some bipolar neurons [37]; and Calb1 (calbindin), which is expressed in many amacrine cells and labels SAC process faintly (Figure S7A and S7B; Table S1; and data not shown). Finally, we also examined the effect of Rb deletion on SAC differentiation using a Chx10-Cre transgene that is expressed in a mosaic pattern across the retina, generating patches of Cre-expressing cells [42]. Consistent with the mosaic deletion pattern, we observed markedly reduced Chat/Slc18a3 staining in the IPL of Chx10-Cre;RbloxP/loxP retina compared to WT (Figure S7C). Together, these results suggest a role for Rb in SAC differentiation. + +The above findings could indicate a defect in SAC specification, SAC survival, or the levels and/or transport of the markers described above. Camk2a marks both SACs and ganglion cells [37], but because ganglion cells are eliminated in the Rb KO retina, Camk2a is a specific SAC marker in this context. Importantly, Camk2a+ tracks and dendrites were present in both the WT and Rb KO retina (Figure 3B), and the number of Camk2a+ soma was similar in WT and Rb KO retina at P30 and beyond, although fewer cells stained in Rb KO retina at P18, suggesting a delay in its appearance (Figures 3C and S6B). Thus, Rb is not required for SAC survival or for process outgrowth, but rather it seems to regulate the expression and/or stability of Calb2, Calb1, Chat, Slc18a3, Sv2c, Kcnc1b, Kcnc2, and GABA in SACs, but leaves Camk2a expression virtually unaffected. The presence of Chat in some cell bodies but never in processes (Figure 3B) also suggests a transport defect. The developmental pattern of Slc18a3 expression also supported this notion. In mature WT SACs Slc18a3 is only in processes, but in early postnatal SACs, it is found in the cell body, and moves into emerging processes at approximately P4–P6. As noted above, Slc18a3 was absent at P18 in the Rb KO retina (Figure 3A); at P4 or P5 it was in cell bodies, yet was rarely present in Rb KO processes (Figures 4A and S6). Slc18a3 became virtually undetectable in Rb KO SACs by P8 (Figures 3C and S6C). These data suggest that Rb affects both the synthesis/stability and transport of SAC markers. + +Figure 4 + +E2f3 Loss Rescues Differentiation of Rb KO SACs + +(A) Horizontal retinal sections of the indicated ages and genotypes were stained for nuclei (DAPI, blue), mitosis marker PH3 (green), and Slc18a3 (red), which marks SAC soma at early stages and processes from ~P5 onwards. Arrows show mitotic PH3+ nuclei in Rb KO, Rb/E2f2 DKO, and Rb/E2f3 DKO retinas. E2f1 loss rescues the ectopic mitosis and cell death defects, but not the SAC defect. E2f2 loss has no effect. E2f3 loss does not rescue the ectopic mitosis and cell loss defects, but rescues the SAC defect. Inactivating E2f1 and E2f3 together rescues the ectopic mitosis, cell death, and SAC defects. + +(B) The fraction of Camk2a+ cells that are Chat + and Slc18a3+ in the P30 retina. + +(C) Horizontal P5 retinal sections were stained for nuclei (DAPI, blue), Slc18a3 (green), and Isl1 (red). Arrows show double-labelled Isl1+/Slc18a3+ cells in the inner INL. + +(D) Horizontal P0 retinal sections of the indicated genotypes were stained for nuclei (DAPI, blue), cell division marker Mki67 (green), and Isl1 (red). Arrows show double-labelled dividing SACs. + +(E) The fraction of Isl1+ cells in the inner NBL (INBL) of P0 retinas that are dividing (Mki67+). + +Error bars represent SD of measurements from three animals, and asterisks indicate significant differences between retinas of WT and the indicated genotypes (*, p <0.05; **; p <0.01; ANOVA and Tukey HSD test). Scale bars in (A), (C), and (D) are 50 μm. + +Rb Regulates SAC Differentiation through E2f3 + +Rb binds more than 100 proteins [43] and in some non-neuronal cells, such as skeletal muscle, adipocytes, and bone, Rb is thought to bind and potentiate tissue-specific transcription factors that promote differentiation [31–33]. Thus, we expected that Rb might interact with retina-specific factors to facilitate SAC differentiation. A direct role for E2f in mediating Rb-dependent differentiation defects (independent of cell cycle or death defects) has to our knowledge not been described, but because E2f can regulate some differentiation genes [44–48], we first tested whether E2f2 or E2f3 might perturb Rb KO SAC maturation. At multiple time points, E2f1 deletion suppressed ectopic mitosis (PH3+ cells), but did not reverse the SAC defect, and E2f2 deletion had no effect on either defect (Figure 4A). Remarkably, although E2f3 deletion did not reverse ectopic mitosis, it rescued Calb2, Slc18a3, Chat, GABA, Kcnc1b, Kcnc2, and Sv2c staining at multiple times (Figure 4A and data not shown). Rb/E2f3 DKO SAC tracks were slightly more disordered than in WT retina, most likely because of the absence of synaptic partner cells, which are killed by E2f1. Indeed, this minor defect was rescued in the Rb/E2f1/E2f3 triple knockout retina, where bipolar and ganglion cell death was rescued and SAC differentiation was restored (Figure 4A). E2f3 deletion alone did not affect SAC differentiation (Figure 4A); thus, it is unleashed E2f3 activity that is detrimental, and the critical role for Rb is to inhibit E2f3. + +We quantified the fraction of Camk2a+ SACs in different genotypes and found that 60% of WT P30 Camk2a+ cells expressed Chat and Slc18a3, which dropped to only 5.6% in the Rb KO retina, and remained low at 3.7% in the Rb/E2f1 DKO retina, but rose to 91% in the Rb/E2f3 DKO retina (Figure 4B). The latter fraction is higher than WT because ganglion cells, which normally make up ~40% of Camk2a+ cells, are killed by apoptosis. + +To quantify the effect of different E2fs on ectopic division specifically in SACs, we exploited Isl1 (Islet1). This marker is expressed in both SACs and ganglion cells, thus Isl1+ cells in the INL are exclusively SACs [49]. We found that 98.2% ± 1.8% of Isl1+ cells in the forming inner INL at P5 were also Slc18a3+, confirming that Isl1 is an excellent SAC marker (Figure 4C). Moreover, Isl1, unlike Slc18a3, is nuclear, which facilitates scoring of Isl1+/Mki67+ cells. It is also expressed earlier than Slc18a3, permitting analysis of SACs soon after their birth at ~E15; thus, we could study retina at P0, a time when ectopic division is high in the inner retina and prior to Rb-independent cell cycle exit associated with terminal differentiation [2]. At P0, no WT Isl1+ cells in the inner neuroblastic layer (NBL) (which is the future INL) were dividing, but 57 ± 14 Isl1+/Mki67+ cells were detected in the Rb KO inner NBL (Figure 4D). Indeed, about one-third of all Isl1+ cells in the entire inner NBL were dividing in the Rb KO retina, or ~50% in the periphery where Cre is expressed (Figure 4E and data not shown). This defect was suppressed in the Rb/E2f1 DKO retina, where we detected only 1 ± 1 dividing SAC, but not the Rb/E2f3 DKO retina, where there were 53 ± 8 dividing SACs (Figure 4D and 4E). We observed similar effects at P0 with Calb2, which marks newborn SACs and other amacrine cells (data not shown). Thus, in Rb KO SACs, E2f1 deletion suppresses ectopic division but not aberrant differentiation, whereas E2f3 deletion suppresses aberrant differentiation but not ectopic division. + +Specific Expression of E2f3 in SACs and Other Subsets of CNS Neurons + +The unique effect of E2f3 in disrupting the differentiation of SACs but not other retinal neurons might be due to cell-type-specific expression or cell-type-specific activity of E2f3. Determining between these two possibilities is not easy, as E2f immunostaining in mouse tissues is problematic. We did not solve this issue for E2f1 or E2f2, but used a modified protocol [50] to successfully track E2f3 expression (Figure 5). At P0, E2f3 was detected in RPCs, consistent with a putative role in normal proliferation (Figure 5A). The signal was specific as it was absent in the E2f3 KO peripheral retina (Figure 5A). As the retina differentiated and RPC division diminished, the number of E2f3+ cells also dropped, and by P8, when division is virtually over, only a subset of post-mitotic cells in the inner retina expressed E2f3 (Figure 5A). By P18, E2f3 was also detected in two tracks in the IPL (Figure 5A and 5B), reminiscent of SAC markers such as Chat and Slc18a3 (c.f. Figures 3 and 4). This cytoplasmic E2f3 staining was also specific, as it was absent in the E2f3 KO peripheral retina of α-Cre;E2f3loxP/loxP mice (Figure 5A). Indeed, double labelling with E2f3 (red) and Chat plus Slc18a3 (green) confirmed that E2f3 is present in both SAC soma and dendrites (Figure 5B). Rb protein was also detected in the inner retina (Figure 5A), and showed a similar distribution as E2f3 in SACs (Figure 5B), and was also present in mature ganglion cells and Müller cells as reported [51]. Rb staining in SAC processes was specific as it was absent in the peripheral retina of αCre;RbloxP/loxP mice (Figure 5A). These data suggest that Rb and E2f3 colocalize in SACs and that E2f3 triggers defects in SAC differentiation because it is specifically expressed in these retinal neurons. + +Figure 5 + +E2f3 and Rb Expression in SACs + +(A) Left panels: horizontal P0, P8, and P18 retinal sections of the indicated genotypes were stained for E2f3 (red) and DAPI (blue). The arrow indicates the junction between the E2f3 null peripheral and WT central P0 retina. Note the absence of E2f3 protein in the peripheral E2f3 KO RPCs at P0 and in peripheral inner retinal neurons at P18. Far right panel: P18 retinal sections of the indicated genotypes were stained for Rb (red) and DAPI (blue). Note the absence of Rb protein in the peripheral Rb KO inner retinal neurons. + +(B) WT P18 retinal sections were stained for nuclei (DAPI, blue), E2f3 (red) or Rb (red), and Chat plus Slc18a3 (green). Arrows indicate double-labelled soma. Note that the IPL processes are also double-labelled. + +Scale bars are 50 μm. + +We also found that E2f3 is present in a specific subset of mature neurons in various brain regions (data not shown). For example, in the P20 amygdala, E2f3 colocalized with the general neuronal markers Mtap2 and Mecp2 [52], but not with Calb2, which marks a subset of neurons, or with the glial marker Gfap (data not shown). Unlike in retinal SACs, E2f3 was not coexpressed in Chat+ or Slc18a3+ cholinergic neurons located in various regions of the brain and spinal cord (data not shown). In agreement, we could not detect defects in cholinergic Rb KO neurons in the developing forebrain, but other Rb KO neurons in this region showed differentiation defects that were rescued by deleting E2f3 [53]. Together, these results suggest that the common mechanism by which Rb promotes neural differentiation is through E2f3 inhibition. + +Distinct Localization of E2f3 Isoforms + +As noted above, E2f3 and Rb staining in SACs was both nuclear and cytoplasmic (Figure 5A and 5B). The antibody that worked in immunostaining recognizes a C-terminal region and thus, does not distinguish a/b isoforms. To our knowledge, the subcellular location of E2f3 isoforms has not been determined in any cell type. To verify the dual locations of E2f3 and to determine which isoforms were present in retina, we analyzed nuclear and cytoplasmic fractions by Western blot at different times during development. Analysis with the pan-E2f3 antibody (sc-878, Santa Cruz Biotechnology) detected a 55-kD E2f3a species and a 40-kD E2f3b polypeptide (Figure 6). To confirm that the upper species in our retinal lysates was E2f3a, we exploited novel mice that lack E2f3 exon 1a and thus express E2f3b exclusively (R. O. and G. L., unpublished data). The genotyping strategy is discussed in detail later and is outlined in Figure 7A. Western analysis confirmed that the upper band was absent in E2f3a−/− mice (Figures 6 and S8). Consistent with the drop in E2f3-expressing cells during WT retinal maturation (Figure 5A), the total amount of E2f3a was less at P18 compared to P0 (Figure 6). E2f3b was present in similar amounts at both time points. At P0 and P18, E2f3a was present in both nuclear and cytoplasmic fractions, but in marked contrast, E2f3b was exclusively nuclear at both times (Figure 6). Two closely migrating E2f3a bands were detected, more clearly evident at P18, of which the faster migrating species was dominant in nuclear and the slower species was dominant in cytoplasm (Figure 6). The identity of both as E2f3a species was confirmed by their absence in the P18 E2f3a KO retina (Figure S8). Analysis of Pou4f2, a nuclear transcription factor expressed in ganglion cells, showed that nuclear proteins had not contaminated the cytoplasmic fraction, and analysis of Slc18a3, a cytoplasmic SAC marker, confirmed that the reverse had also not occurred (Figure 6). These data show, to our knowledge for the first time, that E2f3a and E2f3b exhibit distinct patterns of subcellular distribution, and raise the possibility that E2f3a localization may be regulated by as yet unknown post-translational modifications. + +Figure 6 + +Subcellular Distribution of E2f3 Isoforms and Other Cell Cycle Proteins in the Developing Retina + +Nuclear and cytoplasmic extracts from an equivalent number of retinal cells from mice of the indicated genotypes and ages were analyzed by Western blotting to detect the indicated proteins. Lysate from E2f3a−/− mice was used as a control to confirm the location of E2f3a protein. C, cytoplasmic extracts; N, nuclear extracts. + +Figure 7 + +The E2f3a Isoform Drives the Differentiation Defect in Rb KO SACs + +(A) Schematic diagrams of the mouse WT, E2f3a−/−, and the Cre-recombined floxed E2f3 loci (indicated here as E2f3−/− for simplicity). E2f3a−/− mice lack most of E2f3 exon 1a and part of intron 1a (red dotted box). Arrows indicate PCR primers. Genotyping of an E2f3a+/− mouse is shown on the right. + +(B) RT-PCR detection of E2f3a and E2f3b mRNA in the retina. The sequences of primers are 1aF (5′-GCCTCTACACCACGCCACAAG-3′), 1bF (5′-CGGAAATGCCCTTACAGC-3′), and 4R (5′-CTCAGTCACTTCTTTGGACAG-3′). WT retina expresses both E2f3a and E2f3b mRNA. As expected, E2f3a−/− retina lacks E2f3a mRNA and still expresses E2f3b mRNA. E2f3−/− retina lacks full-length E2f3a and E2f3b mRNAs, and instead expresses a truncated mRNA lacking exon 3. + +(C) Real-time RT-PCR analysis of E2f genes in P8 retinas of the indicated genotypes. Error bars represent SD of measurements from three animals, and asterisks indicate a significant difference between WT and the indicated genotypes (*, p <0.05; **; p <0.01; ANOVA and Tukey HSD test). + +(D) Rescue of Rb KO SACs by E2f3a deletion. Horizontal retinal sections of the indicated genotypes and ages were stained for nuclei (DAPI, blue), M-phase (PH3, green), and the SAC marker Slc18a3 (red). E2f3a deletion does not suppress ectopic division, but rescues the SAC defect. Scale bars are 50 μm. + +M, molecular size marker. + +We also examined the distribution of other cell cycle regulators during retinal development. Like E2f3a, Rb was present in both the WT cytoplasm and nucleus at P0, but at P18, when the levels of Rb had increased, it was primarily nuclear (Figure 6). A very faint cytoplasmic Rb signal was evident at P18, which is consistent with Rb staining of SAC processes (Figure 5B), and with the very small proportion of SACs in the retina [38]. E2f1 was also detected in both nuclear and cytoplasmic fractions, although unlike E2f3a it was predominantly nuclear both at P0 and P18 (Figure 6). The E2f dimerization partner, Tfdp1, which lacks a nuclear localization signal [54], was primarily cytoplasmic at both P0 and P18, and the Cdk inhibitors Cdkn1a and Cdkn1b showed a similar pattern of distribution (Figure 6). Thus, among the cell cycle regulators we examined, most showed bivalent distribution, and E2f3b was unusual in its solely nuclear compartmentalization. + +Rb Regulates SAC Differentiation through E2f3a + +To test which E2f3 isoform is responsible for aberrant Rb KO SAC differentiation we exploited E2f3a−/− mice (Figure 7A). The genotyping strategy outlined in Figure 7A was used to distinguish the E2f3a, WT, and null alleles. Reverse transcriptase PCR (RT-PCR) confirmed the presence of both E2f3a and E2f3b RNA species in the developing WT retina, and the specific absence of E2f3a RNA in the E2f3a−/− retina (Figure 7B). E2f3a protein was absent in E2f3a−/− retinal lysate (Figures 6 and S8). Importantly, the levels of E2f3b message were similar in the Rb KO and Rb/E2f3a DKO retina, ruling out the possibility that any effects of E2f3a deletion we might observe were due to down-regulation of E2f3b (Figure 7C). Also, the levels of other E2fs were the same in the Rb KO, Rb/E2f3 DKO, and Rb/E2f3a DKO retina, ruling out any cross-regulatory effects (Figure 7C) [55]. E2f3a can trigger cell cycle induction, but because SAC defects are not linked to cell cycle perturbation (Figures 3A and 4), and in view of the predominant association between E2f3b and Rb in quiescent cells [16,19], we suspected that E2f3b may perturb differentiation in Rb KO SACs. Unexpectedly, however, E2f3a deletion suppressed the Rb KO SAC defect (Figure 7D). Thus, separate from its role in cell cycle control, Rb regulation of E2f3a is critical to ensure proper neuronal differentiation. + +Discussion + +Rb Controls Retinal Cell Division and Death through E2f1 + +Work in the early 1990s showed that Rb loss triggers defects in neuronal cell cycle exit, survival, and differentiation [26–28]. Much of the death is an indirect consequence of probable hypoxia linked to placental defects [12–14]. However, targeted KO and chimeric studies reveal that Rb autonomously promotes cell cycle exit in newborn neurons, and is required for survival of a subset of neurons, particularly in the retina [2,3,13,14,56–59]. However, whether Rb also regulates differentiation is obscured by potentially indirect effects of ectopic division and death. Moreover, a mechanism though which Rb may regulate neuronal maturation has not been elucidated. + +Here, deleting E2f1 specifically rescued ectopic division and death in the Rb KO retina. Importantly, major Rb/E2f1 DKO neurons differentiated normally, and ERGs revealed the rescue of rod- and cone-mediated function, implicating a regular signal flow from photoreceptors to bipolar and amacrine cells. Division and death genes were induced in Rb KO cells, and deleting E2f1, but not E2f2 or E2f3, reversed these molecular events. E2f1 may also regulate differentiation targets, but whether this contributes to defects in retinal cell maturation is impossible to separate from potentially indirect consequences of deregulated division and death. In any case, it is clear that in most retinal cells, including photoreceptors [29], transcription factors that promote differentiation function independently of Rb. + +We have also found that E2f1 deletion rescues cell-autonomous ectopic division, death, and differentiation defects in sporadic Rb KO clones generated using a Cre retrovirus vector (M. P. and R. B., unpublished data). These data are consistent with the observation that E2f1 overexpression in newborn photoreceptors drives ectopic division and apoptosis [60], and add to the growing evidence indicating that E2f1 is the major, and perhaps only, member of the three activating E2fs required to induce apoptosis in Rb KO cells [10,15]. Thus, deregulated E2f1 activity in the retina, whether resulting from the inactivation of Rb or from overexpression, promotes unscheduled cell division and triggers apoptosis in susceptible RTCs. E2f1, rather than other E2fs, may be a potential target for novel therapeutics to prevent retinoblastoma in RB1+/− humans. + +Our ERG studies revealed rescue of the Rb KO rod–bipolar system, and almost complete restoration of the cone–bipolar system following E2f1 deletion. There was a slightly lower response in the Rb/E2f1 DKO retina relative to the E2f1 KO control retina. This difference might reflect a role for Rb in the development of cones, bipolar cells, or other cells that may contribute to the photopic ERG, including potentially SACs, which do have a serious defect in the Rb/E2f1 DKO retina. + +Rb Controls SAC Differentiation through E2f3a + +Comprehensive marker analysis revealed that, in striking contrast to other retinal neurons, E2f1 deletion did not suppress defects in Rb KO cholinergic SACs. Instead, we observed E2f1-independent defects in the synthesis and transport of a large cohort of SAC proteins. These data expand insight into the development of these important interneurons, but more critically, provide to our knowledge the first unambiguous evidence that Rb regulates neurogenesis beyond terminal mitosis. Rb binds more than 100 factors [43], and in several non-neuronal cells, such as skeletal muscle, adipocytes, and bone, it binds and potentiates tissue-specific transcription factors that promote differentiation [31–33]. The idea that Rb promotes muscle differentiation by potentiating Myod1 activity was contested [61], and other mechanisms proposed [62,63], but not involving E2f repression. Strikingly, however, we discovered that Rb promotes SAC differentiation through E2f3 (Figure 8). + +Figure 8 + +Rb Regulates Distinct Processes through E2f1 and E2f3a + +Red text and arrows indicate Rb-dependent events. Black text and arrows indicate events for which there is no direct evidence of Rb involvement. Rb does not appear to temper RPC expansion and is not required for differentiation of RPCs into RTCs, but is essential to couple RTC birth to terminal mitosis, thus locking them out of cycle. Rb performs this function by inhibiting E2f1. Rb is also required for SAC differentiation, and in this case, acts by inhibiting E2f3a. There is no direct evidence that Rb is required for terminal differentiation of other cell types. Colour codes and abbreviations as in Figure 1A. + +Rb regulation of SAC differentiation through E2f3 was independent of its role in controlling division or death: E2f3 deletion rescued Rb KO SAC defects but did not suppress aberrant proliferation or death, whereas E2f1 deletion reversed abnormal proliferation and death but did not rescue SAC differentiation. Double labelling confirmed that E2f1 but not E2f3 deletion reversed Rb KO SAC division. Moreover, deleting E2f1, but not E2f3, reversed deregulated expression of cell cycle and apoptotic genes in the Rb KO retina. E2f3 is expressed in a subset of CNS neurons (this work) and drives specific cell-cycle–independent defects in Rb KO forebrain neurons [53]. Thus, E2f3 inhibition is the first, and may be the only, mechanism by which Rb participates directly in neuronal differentiation. + +To further dissect the mechanism of action of Rb in SACs we determined the E2f3 isoform it targets to promote differentiation. E2f3b was the primary candidate, since Rb and E2f3b collaborate to repress targets in quiescent cells in vitro [19]. However, in the first work to our knowledge to examine the function of any E2f protein isoform in vivo, we made the surprising observation that Rb regulates SAC differentiation through E2f3a (Figure 8). Formally, we cannot exclude the possibility that deleting E2f3b might also rescue SAC differentiation, but definitive proof will require analysis of E2f3b null mice. Nevertheless, our data prove that Rb definitely regulates SAC differentiation through the activating E2f3 isoform. + +Distinct E2f3a and E2f3b Localization + +The subcellular location of E2f isoforms has not to our knowledge been addressed before. E2f3a and E2f3b share 110 C-terminal amino acids that encode the NLS, DNA-binding, marked box, transactivation, and Rb-binding domains [16], yet they exhibit different subcellular distribution in developing retinal cells. E2f3a is both nuclear and cytoplasmic, but E2f3b is always nuclear. The unique 121- and six-residue N-termini of E2f3a and E2f3b, respectively, likely mediate this difference. This region in E2f1, E2f2, and E2f3a binds Ccna2, establishing a negative regulatory loop that deactivates E2fs in mid-late S-phase [64,65]. However, even E2f3b, which lacks this domain, binds and is regulated by Ccna2 [18], so the domain difference may not explain the unique distributions we observed. Rb family and Tfdp proteins can also determine E2f localization [20–22], and we found that a portion of both Rb and Tfdp1 proteins are cytoplasmic in retinal cells. Indeed, immunostaining revealed that Rb and E2f3 colocalize to SAC processes. + +The nuclear localization of E2f3b contrasts with that of other repressive E2fs in differentiating muscle, where E2f5 switches from the nucleus to cytoplasm, while E2f4 remains in both compartments [23]. The distinct compartmentalization of E2f3a and E2f3b in the retina suggests temporally and functionally distinct activities. Rb distribution matches that of E2f3a, consistent with its critical role in supporting SAC differentiation through E2f3a. + +Ectopic Division and Differentiation + +Rb is critical to ensure that many types of terminally differentiating cells leave the cell cycle (e.g., neurons, gut and skin epithelia, muscle, and lens fibres) (reviewed in [66]). Early overexpression studies in vitro suggested Rb might temper expansion of cycling cells, but KO studies in vivo indicate that its major role is to block division in terminally differentiating cells. In its absence, many (but clearly not all) aspects of differentiation go ahead relatively unperturbed. In the retina, differentiating transition cells are born in the absence of Rb, migrate to the correct layer, and express appropriate markers ([2] and this work). In brain, Rb KO neurons migrate away from the ventricular zone and switch on Tubb3 (βIII-tubulin), but continue to incorporate BrdU [13], and in gut epithelia, differentiated enterocytes migrate up the villi and activate expression of serotonin, yet continue to incorporate BrdU [67]. In the case of SACs, the differentiation defects we observed (e.g., loss of Slc18a3 and Chat) were not due to aberrant division, but it is possible there are other problems with these cells that are caused by ectopic division. Nevertheless, it is clear that many aspects of differentiation in multiple cell types are compatible with ectopic division. However, division of terminally differentiating cells is dangerous, since it may facilitate transformation, as is the case in retinoblastoma (reviewed in [66]). + +How Does E2f3a Perturb SAC Differentiation? + +E2f3a could disrupt SAC differentiation through its well known role as a transcriptional activator, or, in view of the discovery that it is partially cytoplasmic, E2f3a may affect processes other than gene regulation. Both scenarios are feasible since E2fs regulate differentiation genes [44–48], and cell cycle regulators, such as Cdkn1b, have cytoplasmic activities that influence differentiation [68,69]. Many transcription factors shuttle between nucleus and cytoplasm during neurogenesis (e.g., [70] and references therein). It may be difficult to identify E2f3a-specific target genes or cytoplasmic proteins in SACs since these neurons are a small proportion (<1%) of the total retina and only ~5.2% of amacrine neurons [38]. + +Do E2fs Mediate All Rb Functions? + +Others have suggested that Rb promotes differentiation in non-neuronal cells through E2f-independent means [31–33]. However, these studies did not assess whether these cell types differentiate normally if Rb is deleted along with one or more E2f family members. One study reported that Rb mutants that do not bind E2f still induce differentiation [30]. However, the binding assays were performed in solution, and we have found that several of these mutants do bind E2f, albeit weakly, on chromatin (T. Yu and R. B., unpublished data). It is possible that Rb-mediated potentiation of tissue-specific transcription factors may, at least in some cases, be a redundant activity, and that the only critical Rb function is to inhibit E2f. Our study is the first to our knowledge to assess comprehensively whether Rb KO cells can differentiate in the absence of different E2fs. In light of our findings, it will be important to reassess differentiation defects in other Rb KO tissues in the absence of individual and combined activating E2f family members. + +Materials and Methods + +Mouse strains and genotyping. + +Mice were treated according to institutional and national guidelines. α-Cre mice (P. Gruss), Chx10-Cre mice (C. Cepko), RbloxP/loxP mice (A. Berns), E2f1–/– mice, E2f2–/– mice, E2f3loxP/loxP mice, and E2f3a−/− mice were maintained on a mixed (NMRI × C57/Bl × FVB/N × 129sv) background. A detailed description of E2f3a−/− mice will be published elsewhere. Mice of different genotypes were compared within the same litter and across a minimum of three litters. We have not noted any phenotypic differences in separate litters. Genotyping was performed as before [2,5], and the primers used for genotyping E2f3a−/− mice were E2f3a KL (5′-CTCCAGACCCCCGATTATTT-3′), E2f3a KR1 (5′-TCCAGTGCACTACTCCCTCC-3′), and E2f3a KM (5′-GCTAGCAGTGCCCTTTTGTC-3′). + +Histology, immunofluorescence, and measurements. + +Eyeballs were fixed in 4% paraformaldehyde for 1 h at 4 °C, embedded in OCT (TissueTek 4583, Sakura, http://www.sakuraeu.com), frozen on dry ice, and cut into 12-μm sections on Superfrost plus slides (VWR, http://www.vwr.com). For S-phase analysis, BrdU (100 μg/g of body weight) was injected intraperitoneally 2 h prior to sacrifice. BrdU+ cells were detected using a biotin-conjugated sheep polyclonal antibody (1:500, Maine Biotechnology Services, http://www.mainebiotechnology.com). All other antibodies are described in Table S1. For E2f3, Mki67, and Rb staining, antigen retrieval was performed by boiling sections in citric acid solution for 15 min according to Ino [50], except on frozen sections. TUNEL was performed as described [13]. Briefly, sections were incubated for 1 h at 37 °C with 75 μl of mixture solution consisting of 0.5 μl of terminal deoxynucleotide transferase, 1 μl of biotin-16-dUTP, 7.5 μl of CoCl2, 15 μl of 5× terminal deoxynucleotide transferase buffer, and 51 μl of distilled water. After three washes in 4× SSC buffer, sections were incubated with Alexa 488– or Alexa 568−streptavidin (1:1,000; Molecular Probes, http://probes.invitrogen.com) for 1 h at room temperature. Primary antibodies or labelled cells were visualized using donkey anti-mouse Alexa 488 or Alexa 568, donkey anti-rabbit Alexa 488 or Alexa 568, donkey anti-goat Alexa 488 or Alexa 568, and streptavidin Alexa 488 or Alexa 568 (1:1,000; Molecular Probes). Nuclei were counter-stained with 4,6-diamidino-2-phenyindole (DAPI; Sigma, http://www.sigmaaldrich.com). Labelled cells were visualized using a Zeiss (http://www.zeiss.com) Axioplan-2 microscope with Plan Neofluar objectives and images captured with a Zeiss AxionCam camera. For double-labelled samples, confocal images were obtained with a Zeiss LSM 5.0 laser scanning microscope. + +The retina was separated into three bins by dividing the ventricular edge of the retina into equal parts and extending a line to the vitreal edge [2]. Bin 1 contains only cells that expressed Cre as progenitors; bin 3 is at central retina and contains cells derived from progenitors that did not express Cre. For cell counts or thickness measurement we used a region 0–100 μm peripheral to the boundary separating bins 1 and 2. Measurements were performed on an Axioplan-2 microscope using Axiovison software. Quantification of S-phase, M-phase, and apoptotic cells was performed on horizontal sections that included the optic nerve. Quantification of differentiated cell types was performed using horizontal sections at equal distances from the optic nerve. A minimum of three sections per eye and three eyes from different litters were counted. + +RNA extraction, reverse transcription, and PCR. + +Total RNA was isolated from dissected peripheral retina using TRIzol reagent (Invitrogen, http://www.invitrogen.com) followed by digestion with RNase-free DNase (DNA-free, Ambion, http://www.ambion.com) to remove DNA contamination. First-strand cDNA was synthesized from 0.2–0.5 μg of total RNA using the SuperScript II first-strand synthesis system (Invitrogen). PCR primers are listed in Table S2. Real-time quantitative PCR was performed using an Applied Biosystems (http://appliedbiosystems.com) PRISM 7900HT. Tests were run in duplicate on three separate biological samples with SYBR Green PCR Master Mix (Applied Biosystems) exactly as we described previously [71]. Briefly, master stocks were prepared such that each 10-μl reaction contained 5 μl of SYBR Green PCR Master Mix, 0.1 μl of each forward and reverse primer (stock 50 μM), 0.8 μl of blue H2O (0.73% Blue Food Colour; McCormick, http://www.mccormick.com), 2 μl of diluted cDNA template, and 2 μl of yellow H2O (0.73% Yellow Food Colour). PCR consisted of 40 cycles of denaturation at 95 °C for 15 s and annealing and extension at 55 °C for 30 s. An additional cycle (95 °C, 15 s, 60 °C) generated a dissociation curve to confirm a single product. The cycle quantity required to reach a threshold in the linear range was determined and compared to a standard curve for each primer set generated by five 3-fold dilutions of genomic DNA or cDNA samples of known concentration. Values obtained for test RNAs were normalized to Hprt1 mRNA levels. + +Western blots. + +Mouse retinas were homogenized by passing them through a 30-gauge BD 9 http://www.bd.com) needle 5–10 times in 1× PBS solution. Nuclear and cytoplasmic proteins were extracted using the NE-PER Nuclear and Cytoplasmic Extraction Kit (Product# 78833, Pierce Biotechnology, http://www.piercenet.com). Proteins were separated by 10% SDS-PAGE and transferred to nitrocellulose. After blocking overnight at 4 °C in 5% skim milk, membranes were incubated in the primary antibody for 2 h at room temperature. After three 10-min washes in TPBS (100 mM Na2HPO4, 100 mM NaH2PO4, 0.5 N NaCl, 0.1% Tween-20), membranes were incubated for 30 min at room temperature in the secondary horseradish peroxidase-conjugated antibody (Jackson ImmunoResearch Laboratories, http://www.jacksonimmuno.com). Blots were developed using the ECL-Plus chemiluminescent detection system (Amersham Pharmacia Biotech, http://www.pharmacia.ca), according to the manufacturer's instructions. + +The following primary antibodies were used: E2f-1 (SC-193), E2f-3 (SC-878), Cdkn1a (p21, SC-471), Cdkn1b (p27, SC-528), Pou4f2 (Brn3b, SC-6062), and Tfdp1 (Dp1, SC-610) from Santa Cruz Biotechnology (http://www.scbt.com), pRB (554136) from BD Science-Pharmingen (http://www.bdbiosciences.com), and Slc18a3 (VAChT, G448A) from Promega (http://www.promega.com). + +Electroretinography. + +ERGs were recorded from dark-adapted mice as described [72]. Briefly, mice were dark-adapted overnight and anaesthetized by subcutaneous injection of ketamine (66.7 mg/kg body weight) and xylazine (11.7 mg/kg body weight). The pupils were dilated and single-flash ERG recordings were obtained under dark-adapted (scotopic) and light-adapted (photopic) conditions. Light adaptation was accomplished with a background illumination of 30 candela (cd) per square meter starting 10 min before recording. Single white-flash stimulation ranged from 10−4 to 25 cd·s/m2, divided into ten steps of 0.5 and 1 log cd·s/m2. Ten responses were averaged with an inter-stimulus interval of either 5 s (for 10−4, 10−3, 10−2, 3 × 10−2, 10−1, and 3 × 10−1 cd·s/m2) or 17 s (for 1, 3, 10, and 25 cd·s/m2). Band-pass filter cut-off frequencies were 0.1 and 3,000 Hz. + +Statistics. + +Different genotypes were evaluated using analysis of variance (ANOVA) followed by the Tukey honestly significant difference (HSD) test or Fisher test (XLSTAT program, http://www.xlstat.com). + +Supporting Information + +Figure S1 + +Deleting E2f1, but Not E2f2 or E2f3, Rescues Ectopic Division and Cell Death in P0 and P18 Rb KO Retina + +Horizontal sections of the indicated genotypes and ages were stained for nuclei (DAPI, blue), and (A) S-phase (anti-BrdU, red) or (B) apoptosis (TUNEL, red). In Rb−/− retinas, BrdU+ cells extend beyond the normal boundaries at P0 (arrows), and ectopic DNA synthesis continues in multiple layers at later stages. Scale bar is 50 μm. The NBL is where dividing RPCs are located. + +(815 KB PDF) + +Click here for additional data file. + +Figure S2 + +Deleting E2f1 Rescues Ectopic Division and Apoptosis in the Embryonic Rb KO Retina + +Horizontal sections of the indicated genotypes and ages (E14 and E16, the period during which SACs are born) were stained for nuclei (DAPI, blue), and either S-phase (upper two panels, anti-BrdU, red) or apoptosis (lower two panels, TUNEL, red). In Rb−/− retinas, BrdU+ and TUNEL+ cells can be seen in the inner retina (arrows). Inactivation of E2f1 rescued these defects. Scale bar is 50 μm. The NBL is where dividing RPCs are located. + +(754 KB PDF) + +Click here for additional data file. + +Figure S3 + +Deleting E2f1, but Not E2f2 or E2f3, Rescues Ectopic Mitosis in the Rb KO Retina + +(A) Horizontal retinal sections of the indicated genotypes and ages were stained for nuclei (DAPI, blue) and M-phase (anti-PH3, red). Scale bar is 50 μm. + +(B) Quantification of all PH3+ cells. + +(C) Quantification of ectopic PH3+ cells. + +Error bars represent standard deviation (SD), and asterisks indicate significant difference between retina of WT and indicated genotypes (*, p <0.05; **, p <0.01; ANOVA and Tukey HSD test). + +(628 KB PDF) + +Click here for additional data file. + +Figure S4 + +Deleting E2f2 or E2f3 Does Not Rescue Ganglion, Rod, or Bipolar Cell Death in the Rb KO Retina + +(A) Horizontal retinal sections from mice of the indicated ages and genotypes were stained for nuclei (DAPI, blue) and markers that detect ganglion cells (Pou4f2, red), rods and cones (Sag [rod arrestin], green), and rod bipolar cells (Prkca, green). Scale bar is 50 μm. + +(B) Quantification of total ganglion (Pou4f2+) cells. + +(C) Quantification of total rod bipolar (Prkca+) cells. + +(D) Thickness of the ONL, which represents the number of rods. + +Error bars represent SD, and asterisks indicate significant difference between retina of WT and indicated genotypes (**, p <0.01; ANOVA and Tukey HSD test). + +(488 KB PDF) + +Click here for additional data file. + +Figure S5 + +E2f1 Deletion Rescues α-Cre;RbloxP/loxP Retinal Function + +ERGs were recorded from the indicated genotypes under light adapted (photopic) conditions. + +(A) Intensity series. + +(B) The b-wave amplitudes as a function of the logarithm of the flash intensity. + +(383 KB PDF) + +Click here for additional data file. + +Figure S6 + +Differentiation Defects in Rb KO SACs + +Horizontal retinal sections of indicated genotypes and ages were stained for nuclei (DAPI, blue) and Calb2 ([A], red; only densely stained cells were counted for Figure 3C), Camk2a ([B], green), and Slc18a3 ([C], red). Scale bars are 50 μm. + +(564 KB PDF) + +Click here for additional data file. + +Figure S7 + +GABA Neurotransmitter in the Rb KO Retina and Abnormal SACs in Chx10-Cre;RbloxP/loxP Retina + +Horizontal sections of the indicated genotypes and ages of retina were stained for nuclei (DAPI, blue), and (A and B) GABA (red) and Slc18a3 (green) or (C) Chat and Slc18a3 (red). + +(A) In P18 WT retina, GABA labelled four IPL tracks, of which the two inner tracks co-stained with Slc18a3. The latter tracks disappeared in the Rb KO retina, and were rescued by E2f3 KO but not E2f1 KO. + +(B) At the boundary of the WT (central) and Rb KO area (peripheral retina) the inner GABA+ SAC tracks can be seen disappearing towards the periphery (left). + +(C) Slc18a3 staining in the IPL of Chx10-Cre;RbloxP/loxP retina is consistent with the mosaic pattern of Rb inactivation. + +Scale bars are 50 μm. + +(633 KB PDF) + +Click here for additional data file. + +Figure S8 + +Subcellular Distribution of E2f3a Isoform in the Developing Retina + +Nuclear and cytoplasmic extracts from an equivalent number of retinal cells from mice of the indicated genotypes and ages were analyzed by Western blotting to detect the E2f3a protein. Lysates from E2f3a−/− mice of matched ages were used as a control to confirm the location of E2f3a protein. C, cytoplasmic extracts; N, nuclear extracts. + +(115 KB PDF) + +Click here for additional data file. + +Table S1 + +List of Antibodies and Marker Patterns in Rb/E2f1 DKO SACs + +(97 KB DOC) + +Click here for additional data file. + +Table S2 + +Real-Time RT-PCR Primers + +(49 KB DOC) + +Click here for additional data file. + +Accession Numbers + +The GenBank (http://www.ncbi.nlm.nih.gov/genbank) accession numbers for the major genes and gene products discussed in this paper are Camk2a (NM_009792), Chat (NM_009891), E2f1 (NM_007891), E2f2 (NM_177733), E2f3 (NM_010093), Rb (NM_009029), and Slc18a3 (NM_021712). + +Acknowledgements + +We thank K. McClellan and R. Slack for sharing unpublished data, L. Galli-Resta for suggesting Isl1 (Islet1) as an early SAC marker, J. Eubanks for advice on immunostaining, A. Berns, C. Cepko, and P. Gruss for mice, and T. Edlund, F. Haeseleer, R. Janz, S. Sugita, P. A. Hargrave, C. M. Craft, X. Zhu, R. McInnes, R. L. Chow, and J. Saari for antibodies. + +Abbreviations + +ANOVA - analysis of variance + +BrdU - bromodeoxyuridine + +cd - candela + +CNS - central nervous system + +DAPI - 4,6-diamidino-2-phenyindole + +DKO - double knockout + +E[number] - embryonic day [number] + +E2f - E2f transcription factor + +ERG - electroretinogram + +HSD - honestly significant difference + +GABA - gamma-aminobutyric acid + +GCL - ganglion cell layer + +INL - inner nuclear layer + +IPL - inner plexiform layer + +KO - knockout + +NBL - neuroblastic layer + +ONL - outer nuclear layer + +OPL - outer plexiform layer + +P[number] - postnatal day [number] + +PH3 - phosphohistone 3 + +Rb - retinoblastoma protein + +RPC - retinal progenitor cell + +RTC - retinal transition cell + +RT-PCR - reverse transcriptase PCR + +SAC - starburst amacrine cell + +SD - standard deviation + +TUNEL - terminal dUTP nick-end labelling + +WT - wild-type + +Footnotes + +Author contributions. DC and RB conceived and designed the experiments. DC performed most of the experiments. RO, PW, and GL provided the E2f3 floxed and E2f3a KO mice. DC and RB analyzed the data. MP, NT, and MWS performed the ERG analysis. DC and RB wrote the paper. + +Funding. This work was supported by grants from the Canadian Institute for Health Research and Foundation Fighting Blindness Canada to RB, and by the German Research Council (DFG Se837/4–1 and 5–1) and the European Union (IP “EVI-GenoRet” LSHG-CT-512036) to MWS. + +Competing interests. The authors have declared that no competing interests exist. diff --git a/src/ontogpt/evaluation/craft/database/all/17677002.ann b/src/ontogpt/evaluation/craft/database/all/17677002.ann new file mode 100644 index 000000000..24a390082 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17677002.ann @@ -0,0 +1,847 @@ +T1 http://purl.obolibrary.org/obo/MONDO_0005027 8 24 Seizure Disorder +T2 PR:000004825 35 42 Brunol4 +T3 NCBITaxon:10088 57 61 Mice +T4 http://purl.obolibrary.org/obo/MONDO_0005027 84 92 epilepsy +T5 NCBITaxon:9606 105 110 human +T6 http://purl.obolibrary.org/obo/MONDO_0000001 111 119 disorder +T7 SO:0000704 134 141 genetic +T8 NCBITaxon:10088 211 216 mouse +T9 NCBITaxon:10088 238 242 mice +T10 SO:0000704 299 303 gene +T11 PR:000004825 333 345 Bruno-like 4 +T12 PR:000004825 347 354 Brunol4 +T13 UBERON:0000349 365 371 limbic +T14 UBERON:0007023 482 488 adults +T15 http://purl.obolibrary.org/obo/MONDO_0005027 737 745 epilepsy +T16 PR:000004825 747 754 Brunol4 +T17 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13277 splicing +T237 NCBITaxon:10088 13313 13317 mice +T238 PR:000004825 13322 13329 Brunol4 +T239 SO:0000673 13330 13340 transcript +T240 UBERON:0007023 13533 13538 adult +T241 UBERON:0000955 13539 13544 brain +T242 SO:0000902 13625 13634 transgene +T243 GO:0010467 13642 13652 expression +T244 SO:0000704 13672 13677 genes +T245 SO:0001026 13683 13690 genomic +T246 NCBITaxon:39107 13704 13710 murine +T247 PR:000004825 13711 13718 Brunol4 +T248 SO:0000704 13730 13734 gene +T249 SO:0000704 13741 13746 Genes +T250 PR:000004825 13816 13823 Brunol4 +T251 GO:0010467 13825 13835 Expression +T252 SO:0000704 13864 13869 genes +T253 UBERON:0000955 13885 13890 brain +T254 PR:000004825 14015 14022 Brunol4 +T255 UBERON:0000955 14035 14040 brain +T256 GO:0010467 14041 14050 expressed +T257 SO:0000704 14051 14055 gene +T258 SO:0000902 14072 14081 transgene +T259 SO:0000704 14145 14149 gene +T260 SO:0001023 14164 14170 allele +T261 PR:000004825 14174 14181 Brunol4 +T262 UBERON:0007023 14258 14264 adults +T263 NCBITaxon:10088 14301 14305 mice +T264 UBERON:0000349 14316 14322 limbic +T265 SO:0000902 14498 14506 Trangene +T266 PR:000004825 14526 14533 Brunol4 +T267 NCBITaxon:10088 14546 14550 Mice +T268 SO:0001249 14556 14568 Physical map +T269 SO:0000902 14576 14585 transgene +T270 SO:0000366 14586 14601 insertion point +T271 SO:0000188 14615 14621 intron +T272 PR:000004825 14625 14632 Brunol4 +T273 SO:0000704 14633 14637 gene +T274 NCBITaxon:10088 14641 14646 mouse +T275 SO:0000902 14696 14705 transgene +T276 SO:0000902 14815 14824 transgene +T277 SO:0000902 14845 14854 transgene +T278 SO:0000902 14972 14981 transgene +T279 SO:0001026 15062 15069 genomic +T280 SO:0000366 15089 15103 insertion site +T281 SO:0000902 15141 15150 transgene +T282 SO:0000902 15196 15205 transgene +T283 SO:0000188 15214 15222 intronic +T284 PR:000004825 15237 15244 Brunol4 +T285 SO:0000902 15310 15319 transgene +T286 SO:0000366 15330 15344 insertion site +T287 SO:0001414 15360 15373;15388 15397 breakpoint of ... insertion +T288 SO:0000902 15378 15387 transgene +T289 SO:0000147 15421 15425 exon +T290 GO:0008380 15428 15434 splice +T291 SO:0001021 15461 15471 breakpoint +T292 SO:0000147 15495 15499 exon +T293 GO:0008380 15502 15508 splice +T294 SO:0000147 15528 15532 exon +T295 SO:0000188 15533 15539 intron +T296 NCBITaxon:10088 15557 15562 mouse +T297 PR:000004825 15563 15570 Brunol4 +T298 SO:0000704 15571 15575 gene +T299 SO:0000704 15605 15609 gene +T300 SO:0000236 15658 15676 open reading frame +T301 SO:0000147 15687 15691 exon +T302 SO:0000147 15706 15710 exon +T303 PR:000004825 15765 15772 BRUNOL4 +T304 SO:0000195 15809 15821 coding exons +T305 GO:0010467 15845 15855 Expression +T306 PR:000004825 15859 15866 Brunol4 +T307 NCBITaxon:10088 15891 15895 Mice +T308 PR:000004825 15909 15916 Brunol4 +T309 SO:0000673 15917 15927 transcript +T310 NCBITaxon:10088 15946 15950 mice +T311 UBERON:0000955 15960 15965 brain +T312 PR:000004825 15997 16004 Brunol4 +T313 NCBITaxon:10088 16036 16041 mouse +T314 PR:000003676 16042 16049 β-actin +T315 PR:000004825 16086 16093 Brunol4 +T316 SO:0000673 16094 16104 transcript +T317 PR:000004825 16240 16247 Brunol4 +T318 SO:0000673 16248 16258 transcript +T319 PR:000004825 16289 16296 Brunol4 +T320 UBERON:0007023 16342 16347 adult +T321 UBERON:0001851 16348 16354 cortex +T322 NCBITaxon:10088 16376 16380 mice +T323 UBERON:0002616 16444 16457 brain regions +T324 GO:0010467 16468 16478 expression +T325 PR:000004825 16482 16489 Brunol4 +T326 UBERON:0000955 16511 16516 brain +T327 UBERON:0007023 16520 16525 adult +T328 NCBITaxon:10088 16526 16530 mice +T329 SO:0000610 16555 16561 poly-A +T330 UBERON:0007023 16598 16603 adult +T331 NCBITaxon:10088 16604 16609 mouse +T332 UBERON:0000479 16610 16617 tissues +T333 GO:0097617 16635 16645 hybridized +T334 PR:000004825 16670 16677 Brunol4 +T335 PR:000003676 16682 16689 β-actin +T336 GO:0010467 16840 16850 expression +T337 PR:000004825 16862 16869 Brunol4 +T338 UBERON:0007023 16873 16879 adults +T339 UBERON:0000479 16915 16922 tissues +T340 UBERON:0000955 16929 16934 brain +T341 PR:000004825 17014 17021 BRUNOL4 +T342 UBERON:0000955 17025 17030 brain +T343 UBERON:0007023 17043 17049 adults +T344 UBERON:0000062 17098 17103 organ +T345 GO:0010467 17112 17122 expression +T346 NCBITaxon:10088 17126 17130 mice +T347 PR:000004825 17145 17152 BRUNOL4 +T348 UBERON:0000955 17165 17170 brain +T349 PR:000004825 17201 17208 Brunol4 +T350 CL:0000540 17232 17240 neuronal +T351 GO:0010467 17241 17251 expression +T352 UBERON:0000955 17267 17272 brain +T353 UBERON:0000956 17298 17313 cerebral cortex +T354 GO:0007608 17328 17337 olfactory +T355 UBERON:0002264 17328 17343 olfactory bulbs +T356 CL:0000120 17353 17365 granule cell +T357 UBERON:0002956 17353 17374;17379 17389 granule cell layer of ... cerebellum +T358 GO:0010467 17421 17431 expression +T359 UBERON:0000955 17439 17444 brain +T360 GO:0010467 17488 17498 expression +T361 CL:0000598 17519 17536 pyramidal neurons +T362 UBERON:0003882 17544 17547 CA2 +T363 UBERON:0003883 17552 17555 CA3 +T364 CL:0000598 17564 17581 Pyramidal neurons +T365 UBERON:0003881 17585 17588 CA1 +T366 UBERON:0001885 17594 17607 dentate gyrus +T367 CL:0000120 17608 17621 granule cells +T368 UBERON:0009952 17631 17655 dentate subgranular zone +T369 GO:0010467 17667 17677 expression +T370 GO:0022008 17791 17803 neurogenesis +T371 PR:000004825 17848 17855 Brunol4 +T372 GO:0010467 17856 17866 Expression +T373 UBERON:0007023 17877 17882 Adult +T374 NCBITaxon:10088 17883 17888 Mouse +T375 UBERON:0000955 17889 17894 Brain +T376 GO:0097617 17910 17923 Hybridization +T377 CHEBI:37973 17927 17930 33P +T378 SO:0000077 17939 17948 antisense +T379 GO:0097617 17996 18006 hybridized +T380 UBERON:0000955 18049 18054 brain +T381 GO:0007608 18090 18099 olfactory +T382 UBERON:0002264 18090 18104 olfactory bulb +T383 UBERON:0000956 18106 18121 cerebral cortex +T384 UBERON:0002037 18140 18150 cerebellum +T385 UBERON:0000955 18246 18251 brain +T386 UBERON:0002037 18279 18289 cerebellum +T387 CHEBI:42098 18373 18376 DIG +T388 SO:0000077 18385 18394 antisense +T389 GO:0097617 18442 18452 hybridized +T390 UBERON:0000955 18491 18496 brain +T391 UBERON:0003882 18612 18615;18624 18631 CA2 ... regions +T392 UBERON:0003883 18620 18631 CA3 regions +T393 UBERON:0002037 18698 18700 CB +T394 UBERON:0002037 18702 18712 cerebellum +T395 UBERON:0000956 18714 18716 CO +T396 UBERON:0000956 18718 18733 cerebral cortex +T397 UBERON:0002264 18752 18754 OB +T398 GO:0007608 18756 18765 olfactory +T399 UBERON:0002264 18756 18770 olfactory bulb +T400 NCBITaxon:10088 18827 18831 Mice +T401 PR:000004825 18843 18850 BRUNOL4 +T402 PR:000004825 18921 18928 BRUNOL4 +T403 GO:0045202 19050 19058 synaptic +T404 PR:000004825 19106 19113 BRUNOL4 +T405 CHEBI:36357 19121 19130 molecules +T406 GO:0065007 19141 19150 regulated +T407 CL:0000540 19176 19184 neuronal +T408 SO:0000704 19244 19249 genes +T409 GO:0010467 19265 19274 expressed +T410 NCBITaxon:10088 19315 19319 mice +T411 SO:0000673 19455 19466 transcripts +T412 SO:0000673 19501 19512 transcripts +T413 SO:0000704 19561 19566 genes +T414 SO:0000673 19695 19706 transcripts +T415 SO:0000704 19742 19747 genes +T416 PR:000004825 19771 19778 Brunol4 +T417 SO:0000704 19842 19847 genes +T418 http://purl.obolibrary.org/obo/MONDO_0000001 19922 19930 disorder +T419 CHEBI:28790 19944 19953 serotonin +T420 PR:000001168 19944 19965 serotonin receptor 2c +T421 PR:000001168 19967 19972 Htr2c +T422 PR:000001168 19975 19980 Htr2c +T423 NCBITaxon:10088 19986 19990 mice +T424 PR:000015871 20142 20153 synapsin II +T425 PR:000015871 20155 20159 Syn2 +T426 PR:000015871 20228 20232 Syn2 +T427 NCBITaxon:10088 20242 20246 mice +T428 CHEBI:44485 20271 20287 N-ethylmaleimide +T429 PR:000011443 20271 20304 N-ethylmaleimide-sensitive factor +T430 PR:000011443 20306 20309 Nsf +T431 GO:0065007 20318 20327 regulates +T432 GO:0006887 20328 20338 exocytosis +T433 GO:0045202 20342 20350 synaptic +T434 GO:0007268 20342 20363 synaptic transmission +T435 CHEBI:34018 20376 20380 AMPA +T436 PR:000015322 20430 20441 α-synuclein +T437 PR:000015322 20443 20447 Snca +T438 CL:0000540 20452 20458 neuron +T439 GO:0098793 20468 20479 presynaptic +T440 CHEBI:36357 20503 20512 molecules +T441 CL:0000598 20536 20553 pyramidal neurons +T442 PR:000004825 20579 20586 Brunol4 +T443 GO:0010467 20597 20606 expressed +T444 GO:0010467 20635 20645 expression +T445 PR:000011443 20649 20652 NSF +T446 PR:000015871 20662 20673 synapsin II +T447 UBERON:0003882 20700 20703 CA2 +T448 UBERON:0003883 20704 20707 CA3 +T449 PR:000004825 20735 20742 Brunol4 +T450 GO:0010467 20779 20789 expression +T451 SO:0000673 20802 20813 transcripts +T452 NCBITaxon:10088 20842 20846 mice +T453 GO:0010467 20867 20877 expression +T454 UBERON:0007023 20902 20907 adult +T455 UBERON:0002616 20926 20939 brain regions +T456 UBERON:0007023 21144 21149 adult +T457 NCBITaxon:10088 21173 21177 mice +T458 UBERON:0000349 21387 21393 limbic +T459 GO:0010467 21444 21454 expression +T460 SO:0000704 21467 21472 genes +T461 PR:000004825 21486 21493 Brunol4 +T462 GO:0010467 21514 21524 Expression +T463 SO:0000704 21543 21548 Genes +T464 UBERON:0000955 21558 21564 Brains +T465 GO:0097617 21637 21650 hybridization +T466 PR:000001168 21657 21662 Htr2c +T467 PR:000011443 21664 21667 Nsf +T468 PR:000015322 21669 21673 Snca +T469 PR:000015871 21675 21679 Syn2 +T470 PR:000007774 21681 21687 Gabrb3 +T471 PR:000003676 21693 21700 β-actin +T472 SO:0000610 21745 21751 poly-A +T473 UBERON:0002616 21781 21794 brain regions +T474 PR:000015871 21800 21811 synapsin II +T475 PR:000041110 21832 21844 synapsin IIa +T476 PR:000041111 21832 21840;21849 21852 synapsin ... IIb +T477 SO:0000673 21853 21864 transcripts +T478 GO:0000380 21872 21892 alternative splicing +T479 GO:0043631 21893 21908 polyadenylation +T480 PR:000041110 21951 21962 synapsin 2a +T481 PR:000041111 21951 21959;21963 21965 synapsin ... 2b +T482 UBERON:0000955 21993 21999 brains +T483 GO:0010467 22059 22069 expression +T484 PR:000007774 22073 22079 Gabrb3 +T485 PR:000007774 22099 22112;22117 22133 β3 subunit of ... GABA(A) receptor +T486 CHEBI:16865 22117 22121 GABA +T487 PR:000007774 22157 22163 Gabrb3 +T488 UBERON:0012101 22171 22180 perinatal +T489 GO:0007567 22175 22180 natal +T490 NCBITaxon:10088 22205 22209 mice +T491 GO:0010467 22267 22277 expression +T492 UBERON:0000955 22320 22326 brains +T493 UBERON:0000956 22389 22391 CO +T494 UBERON:0000956 22393 22408 cerebral cortex +T495 UBERON:0002028 22410 22412 Hb +T496 UBERON:0002028 22414 22423 hindbrain +T497 PR:000003676 22572 22579 β-actin +T498 GO:0010467 22594 22604 expression +T499 PR:000001166 22644 22649 HTR2A +T500 PR:000001168 22651 22656 HTR2C +T501 PR:000011443 22658 22661 NSF +T502 PR:000015871 22663 22674 synapsin II +T503 PR:000015322 22676 22687 α-synuclein +T504 PR:000028799 22695 22702 tubulin +T505 PR:000001166 22704 22709 HTR2A +T506 CHEBI:36357 22713 22721 molecule +T507 PR:000001168 22764 22769 HTR2C +T508 PR:000028799 22810 22817 tubulin +T509 UBERON:0007023 22881 22886 adult +T510 NCBITaxon:10088 22887 22891 mice +T511 PR:000028799 23071 23078 tubulin +T512 NCBITaxon:33208 23120 23127 animals +T513 PR:000001168 23180 23185 Htr2c +T514 PR:000004825 23262 23269 Brunol4 +T515 PR:000001168 23301 23315 Htr2c receptor +T516 SO:0000704 23316 23320 gene +T517 PR:000004825 23363 23370 Brunol4 +T518 PR:000004825 23501 23508 Brunol4 +T519 NCBITaxon:10088 23513 23517 mice +T520 PR:000001168 23631 23636 Htr2c +T521 PR:000001168 23728 23733 Htr2c +T522 GO:0010467 23743 23753 expression +T523 PR:000001168 23839 23844 Htr2c +T524 PR:000004825 23896 23903 Brunol4 +T525 NCBITaxon:10088 23908 23912 mice +T526 NCBITaxon:10088 23991 23995 mice +T527 SO:0000704 24089 24096 genetic +T528 PR:000001168 24151 24156 Htr2c +T529 PR:000004825 24193 24200 Brunol4 +T530 NCBITaxon:10088 24205 24209 mice +T531 GO:0010467 24360 24370 expression +T532 PR:000001168 24374 24379 Htr2c +T533 PR:000004825 24415 24422 Brunol4 +T534 NCBITaxon:10088 24427 24431 mice +T535 PR:000001168 24460 24465 Htr2c +T536 PR:000004825 24470 24477 Brunol4 +T537 NCBITaxon:10088 24485 24489 mice +T538 PR:000001168 24498 24503 Htr2c +T539 CHEBI:28790 24603 24612 serotonin +T540 GO:0051610 24603 24621 serotonin reuptake +T541 PR:000004825 24625 24632 Brunol4 +T542 CHEBI:5118 24653 24663 fluoxetine +T543 CHEBI:5118 24665 24671 Prozac +T544 PR:000001168 24762 24767 Htr2c +T545 PR:000004825 24804 24811 Brunol4 +T546 http://purl.obolibrary.org/obo/MONDO_0005027 24849 24865 seizure disorder +T547 PR:000015871 24982 24986 Syn2 +T548 PR:000011443 24988 24991 Nsf +T549 PR:000015322 24996 25000 Snca +T550 PR:000004825 25058 25065 Brunol4 +T551 SO:0000704 25088 25099 genetically +T552 SO:0000704 25127 25134 Genetic +T553 PR:000004825 25155 25162 Brunol4 +T554 PR:000001168 25167 25172 Htr2c +T555 UBERON:0002103 25269 25277 hindlimb +T556 PR:000001168 25404 25409 Htr2c +T557 PR:000001168 25444 25449 Htr2c +T558 PR:000001168 25496 25501 Htr2c +T559 NCBITaxon:10088 25522 25526 mice +T560 PR:000004825 25591 25598 Brunol4 +T561 UBERON:0000955 25611 25616 brain +T562 http://purl.obolibrary.org/obo/MONDO_0005027 25655 25671 seizure disorder +T563 NCBITaxon:10088 25690 25695 mouse +T564 http://purl.obolibrary.org/obo/MONDO_0000001 25723 25731 disorder +T565 PR:000004825 25765 25772 Brunol4 +T566 SO:0000704 25773 25777 gene +T567 PR:000004825 25804 25811 Brunol4 +T568 SO:0000673 25812 25822 transcript +T569 SO:0000673 25940 25950 transcript +T570 SO:0000902 26030 26039 transgene +T571 SO:0000313 26070 26077 hairpin +T572 GO:0031564 26105 26131 read-through transcription +T573 PR:000004825 26144 26151 Brunol4 +T574 NCBITaxon:10088 26161 26165 mice +T575 UBERON:0000349 26336 26342 limbic +T576 NCBITaxon:33208 26404 26410 animal +T577 http://purl.obolibrary.org/obo/MONDO_0005027 26702 26710 epilepsy +T578 PR:000004825 26769 26776 BRUNOL4 +T579 http://purl.obolibrary.org/obo/MONDO_0005027 26795 26811 seizure disorder +T580 SO:0000704 26845 26849 gene +T581 SO:0000704 26877 26884 genetic +T582 http://purl.obolibrary.org/obo/MONDO_0003847 26877 26892 genetic disease +T583 CHEBI:36357 26932 26941 molecules +T584 UBERON:0000955 27137 27142 brain +T585 SO:0000673 27184 27195 transcripts +T586 PR:000001168 27247 27252 Htr2c +T587 PR:000015871 27257 27261 Syn2 +T588 NCBITaxon:10088 27337 27341 mice +T589 PR:000011443 27366 27369 Nsf +T590 PR:000015322 27374 27378 Snca +T591 GO:0045202 27418 27426 synaptic +T592 GO:0007268 27418 27439 synaptic transmission +T593 UBERON:0007023 27486 27492 adults +T594 PR:000004825 27537 27544 Brunol4 +T595 GO:0010467 27545 27555 expression +T596 PR:000001168 27686 27691 Htr2c +T597 http://purl.obolibrary.org/obo/MONDO_0011122 27740 27747 obesity +T598 CHEBI:28790 27796 27808 serotonergic +T599 PR:000001168 27892 27897 Htr2c +T600 GO:0010467 27898 27908 expression +T601 PR:000001168 28013 28018 Htr2c +T602 NCBITaxon:10088 28024 28028 mice +T603 CHEBI:28790 28085 28097 serotonergic +T604 http://purl.obolibrary.org/obo/MONDO_0000001 28152 28160 disorder +T605 PR:000004825 28314 28321 Brunol4 +T606 CHEBI:28790 28378 28390 serotonergic +T607 GO:0051610 28425 28446 reuptake of serotonin +T608 CHEBI:28790 28437 28446 serotonin +T609 GO:0045202 28512 28520 synaptic +T610 GO:0010467 28552 28562 expression +T611 PR:000015871 28605 28609 Syn2 +T612 PR:000011443 28611 28614 Nsf +T613 PR:000015322 28619 28623 Snca +T614 SO:0000704 28692 28697 genes +T615 GO:0065007 28701 28710 regulated +T616 PR:000004825 28714 28721 Brunol4 +T617 GO:0010467 28786 28795 expressed +T618 NCBITaxon:10088 28837 28841 mice +T619 PR:000004825 28888 28895 Brunol4 +T620 GO:0010467 28932 28942 expression +T621 SO:0000673 28962 28972 transcript +T622 PR:000004825 28979 28986 BRUNOL4 +T623 GO:0065007 29005 29013 regulate +T624 GO:0000380 29014 29034 alternative splicing +T625 UBERON:0000479 29048 29055 tissues +T626 PR:000004825 29075 29082 BRUNOL4 +T627 GO:0008380 29123 29129 splice +T628 UBERON:0000955 29149 29155 brains +T629 SO:0000673 29183 29194 transcripts +T630 SO:0000704 29266 29270 gene +T631 GO:0006396 29294 29305 RNA editing +T632 GO:0006412 29322 29333 translation +T633 PR:000004825 29363 29370 Brunol4 +T634 GO:0016070 29403 29417 RNA metabolism +T635 SO:0000673 29447 29458 transcripts +T636 GO:0006412 29463 29474 translation +T637 GO:0006396 29710 29724 RNA processing +T638 PR:000004825 29783 29790 BRUNOL4 +T639 GO:0016070 29826 29840 RNA metabolism +T640 SO:0000704 29848 29853 genes +T641 http://purl.obolibrary.org/obo/MONDO_0005027 29880 29888 epilepsy +T642 CHEBI:24870 29896 29899 ion +T643 PR:000004825 29910 29917 Brunol4 +T644 CHEBI:24870 29946 29949 ion +T645 http://purl.obolibrary.org/obo/MONDO_0005027 29958 29966 epilepsy +T646 SO:0000704 29967 29972 genes +T647 NCBITaxon:9606 29981 29987 humans +T648 NCBITaxon:10088 29992 29996 mice +T649 NCBITaxon:9606 30032 30037 human +T650 PR:000004825 30038 30045 BRUNOL4 +T651 SO:0000704 30046 30050 gene +T652 NCBITaxon:9606 30069 30074 human +T653 http://purl.obolibrary.org/obo/MONDO_0005579 30147 30178 idiopathic generalized epilepsy +T654 PR:000004825 30201 30208 BRUNOL4 +T655 SO:0000704 30228 30232 gene +T656 http://purl.obolibrary.org/obo/MONDO_0005027 30243 30260 seizure disorders +T657 NCBITaxon:10088 30286 30290 Mice +T658 PR:000004825 30303 30310 Brunol4 +T659 NCBITaxon:10088 30313 30317 mice +T660 NCBITaxon:10088 30344 30348 mice +T661 GO:0010467 30440 30450 expression +T662 NCBITaxon:39107 30454 30460 murine +T663 PR:000008964 30461 30468 Ighmbp2 +T664 SO:0000568 30536 30549;30574 30582 bidirectional ... promoter +T665 CHEBI:27902 30550 30562 tetracycline +T666 PR:000008964 30618 30625 Ighmbp2 +T667 SO:0000440 30658 30664 vector +T668 SO:0000902 30723 30732 transgene +T669 GO:0045120 30766 30775 pronuclei +T670 NCBITaxon:10088 30794 30799 mouse +T671 UBERON:0000922 30800 30807 embryos +T672 GO:0007566 30833 30842 implanted +T673 NCBITaxon:10088 30863 30867 mice +T674 PR:000001168 30876 30881 Htr2c +T675 NCBITaxon:10088 30888 30892 mice +T676 NCBITaxon:10088 30907 30911 mice +T677 NCBITaxon:33208 31096 31103 animals +T678 GO:0007631 31109 31112 fed +T679 CHEBI:15377 31189 31194 water +T680 NCBITaxon:33208 31211 31217 animal +T681 NCBITaxon:33208 31297 31303 Animal +T682 NCBITaxon:33208 31355 31361 Animal +T683 NCBITaxon:10088 31423 31427 mice +T684 CHEBI:38867 31455 31465 anesthetic +T685 CHEBI:9468 31482 31492 tetracaine +T686 CHEBI:26710 31502 31506 NaCl +T687 UBERON:0000970 31528 31531 eye +T688 UBERON:0000964 31582 31589 corneal +T689 NCBITaxon:10088 31854 31858 mice +T690 UBERON:0001890 31954 31963 forebrain +T691 NCBITaxon:10088 32206 32210 mice +T692 NCBITaxon:10088 32418 32422 Mice +T693 UBERON:0013406 32530 32536 bregma +T694 UBERON:0013406 32563 32569 bregma +T695 UBERON:0003129 32592 32597 skull +T696 CHEBI:53251 32684 32690 Teflon +T697 UBERON:0002363 32777 32781 dura +T698 UBERON:0000955 32790 32795 brain +T699 UBERON:0001091 32802 32808 dental +T700 NCBITaxon:10088 32835 32839 mice +T701 CHEBI:35480 32868 32877 analgesic +T702 CHEBI:364453 32881 32890 carprofen +T703 NCBITaxon:10088 32985 32989 mice +T704 NCBITaxon:10088 33205 33209 mice +T705 CHEBI:4887 33233 33245 ethosuximide +T706 CHEBI:5118 33305 33315 fluoxetine +T707 NCBITaxon:10088 33401 33405 mice +T708 UBERON:0001179 33454 33466 peritoneally +T709 NCBITaxon:10088 33542 33546 mice +T710 UBERON:0001179 33566 33578 peritoneally +T711 SO:0000902 33733 33742 transgene +T712 SO:0000366 33743 33757 insertion site +T713 SO:0001026 33760 33767 Genomic +T714 NCBITaxon:10088 33788 33792 mice +T715 NCBITaxon:10088 33805 33809 mice +T716 SO:0000902 34023 34032 transgene +T717 SO:0001026 34033 34040 genomic +T718 SO:0000440 34181 34187 vector +T719 SO:0000440 34231 34237 vector +T720 SO:0000112 34247 34253 primer +T721 SO:0000112 34266 34272 primer +T722 SO:0000440 34395 34401 vector +T723 SO:0000440 34471 34477 vector +T724 NCBITaxon:10088 34531 34536 mouse +T725 SO:0001026 34537 34543 genome +T726 SO:0000188 34571 34577 intron +T727 PR:000004825 34587 34594 Brunol4 +T728 SO:0000704 34595 34599 gene +T729 NCBITaxon:10088 34613 34618 mouse +T730 SO:0001414 34644 34657;34672 34681 breakpoint of ... insertion +T731 SO:0000902 34662 34671 transgene +T732 SO:0000902 34719 34728 transgene +T733 SO:0000112 34738 34744 primer +T734 PR:000004825 34751 34758 Brunol4 +T735 SO:0000188 34759 34765 intron +T736 SO:0000112 34768 34774 primer +T737 SO:0001414 34821 34842 insertion breakpoints +T738 SO:0001023 34853 34859 allele +T739 SO:0000112 34865 34871 primer +T740 SO:0001023 34912 34918 allele +T741 SO:0001023 34933 34939 allele +T742 SO:0000112 34941 34948 Primers +T743 GO:0097617 35100 35109 annealing +T744 SO:0000112 35176 35183 primers +T745 SO:0000028 35198 35200 bp +T746 SO:0001023 35211 35217 allele +T747 SO:0000112 35239 35246 primers +T748 SO:0000028 35261 35263 bp +T749 SO:0001023 35271 35277 allele +T750 UBERON:0000955 35360 35365 brain +T751 UBERON:0007023 35367 35372 adult +T752 UBERON:0000955 35373 35378 brain +T753 UBERON:0002616 35393 35406 brain regions +T754 CHEBI:33893 35420 35427 reagent +T755 PR:000004825 35499 35506 Brunol4 +T756 SO:0000204 35522 35528 5′ UTR +T757 SO:0000366 35549 35563 insertion site +T758 PR:000004825 35601 35608 BRUNOL4 +T759 SO:0000673 35681 35691 transcript +T760 GO:0097617 35753 35766 Hybridization +T761 CHEBI:16397 35786 35795 formamide +T762 CHEBI:75958 35802 35810 solution +T763 NCBITaxon:10088 35941 35946 mouse +T764 PR:000003676 35947 35954 β-actin +T765 GO:0001171 36134 36155 reverse-transcription +T766 UBERON:0000956 36194 36209 cerebral cortex +T767 UBERON:0007023 36213 36218 adult +T768 GO:0001171 36428 36447 reverse transcribed +T769 NCBITaxon:11866 36453 36456 AMV +T770 CHEBI:51461 36568 36580 SYBR Green I +T771 SO:0000112 36648 36655 primers +T772 PR:000004825 36843 36850 Brunol4 +T773 SO:0000147 36851 36855 exon +T774 SO:0001030 36858 36865 forward +T775 SO:0000147 36870 36874 exon +T776 SO:0001031 36877 36884 reverse +T777 NCBITaxon:10088 37078 37082 mice +T778 CHEBI:2511 37167 37174 agarose +T779 GO:0097617 37209 37222 hybridization +T780 UBERON:0000955 37230 37235 brain +T781 NCBITaxon:10088 37275 37279 mice +T782 GO:0097617 37285 37295 hybridized +T783 CHEBI:37973 37301 37304 33P +T784 CHEBI:42098 37502 37505 DIG +T785 GO:0097617 37554 37564 hybridized +T786 CHEBI:42098 37570 37573 DIG +T787 MOP:0000779 37678 37688 conjugated +T788 CHEBI:42098 37694 37697 DIG +T789 GO:0042571 37698 37706 antibody +T790 UBERON:0000033 37900 37905 heads +T791 GO:0097617 37985 37998 hybridization +T792 SO:0000704 38026 38030 Gene +T793 SO:0000673 38328 38339 transcripts +T794 SO:0000673 38399 38410 transcripts +T795 GO:0042571 38426 38436 Antibodies +T796 GO:0042571 38472 38482 antibodies +T797 PR:000001166 38509 38514 HTR2A +T798 PR:000001168 38572 38577 HTR2C +T799 PR:000011443 38629 38632 NSF +T800 PR:000015871 38701 38711 Synapsin 2 +T801 PR:000015322 38764 38779 alpha-synuclein +T802 GO:0009293 38791 38803 Transduction +T803 PR:000028799 38848 38855 tubulin +T804 GO:0042571 38888 38898 antibodies +T805 NCBITaxon:10088 38929 38934 mouse +T806 NCBITaxon:9986 38990 38996 rabbit +T807 GO:0010467 39090 39100 expression +T808 PR:000004825 39132 39139 BRUNOL4 +T809 UBERON:0000955 39173 39179 brains +T810 PR:000001168 39185 39190 Htr2c +T811 SO:0000147 39208 39212 exon +T812 SO:0000319 39259 39269 stop codon +T813 PR:000011443 39275 39278 Nsf +T814 PR:000015871 39319 39323 Syn2 +T815 SO:0000147 39341 39345 exon +T816 SO:0000318 39393 39404 start codon +T817 GO:0043631 39458 39472 polyadenylated +T818 PR:000015871 39473 39477 Syn2 +T819 SO:0000673 39478 39489 transcripts +T820 PR:000015322 39495 39499 Snca +T821 SO:0000147 39517 39521 exon +T822 SO:0000319 39569 39579 stop codon +T823 PR:000007774 39585 39591 Gabrb3 +T824 SO:0000319 39696 39706 stop codon +T825 NCBITaxon:10088 39771 39775 mice +T826 CHEBI:33893 39979 39986 reagent +T827 GO:0042571 40095 40103 antibody +T828 MOP:0000779 40131 40141 conjugated +T829 GO:0042571 40142 40150 antibody +T830 CHEBI:53325 40230 40244 nitrocellulose +T831 GO:0042571 40385 40395 antibodies +T832 GO:0042571 40471 40481 antibodies +T833 SO:0000704 40584 40589 Genes +T834 GO:0010467 40608 40618 Expression +T835 NCBITaxon:10088 40666 40670 Mice +T836 SO:0000673 40762 40773 transcripts +T837 SO:0000673 40826 40837 transcripts +T838 SO:0000188 41066 41072 intron +T839 PR:000004825 41082 41089 Brunol4 +T840 SO:0000704 41090 41094 gene +T841 NCBITaxon:10088 41098 41103 mouse +T842 CHEBI:33893 41604 41612 reagents +T843 SO:0000704 41674 41678 Gene +T844 GO:0010467 41674 41689 Gene Expression +T845 http://purl.obolibrary.org/obo/MONDO_0005027 41964 41972 Epilepsy +T846 PR:000004825 41997 42004 Brunol4 +T847 PR:000004825 42008 42020 Bruno-like 4 diff --git a/src/ontogpt/evaluation/craft/database/all/17677002.txt b/src/ontogpt/evaluation/craft/database/all/17677002.txt new file mode 100644 index 000000000..9d62c451a --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17677002.txt @@ -0,0 +1,211 @@ +Complex Seizure Disorder Caused by Brunol4 Deficiency in Mice + +Abstract + +Idiopathic epilepsy is a common human disorder with a strong genetic component, usually exhibiting complex inheritance. We describe a new mouse mutation in C57BL/6J mice, called frequent-flyer (Ff), in which disruption of the gene encoding RNA-binding protein Bruno-like 4 (Brunol4) leads to limbic and severe tonic–clonic seizures in heterozygous mutants beginning in their third month. Younger heterozygous adults have a reduced seizure threshold. Although homozygotes do not survive well on the C57BL/6J background, on mixed backgrounds homozygotes and some heterozygotes also display spike-wave discharges, the electroencephalographic manifestation of absence epilepsy. Brunol4 is widely expressed in the brain with enrichment in the hippocampus. Gene expression profiling and subsequent analysis revealed the down-regulation of at least four RNA molecules encoding proteins known to be involved in neuroexcitability, particularly in mutant hippocampus. Genetic and phenotypic assessment suggests that Brunol4 deficiency in mice results in a complex seizure phenotype, likely due to the coordinate dysregulation of several molecules, providing a unique new animal model of epilepsy that mimics the complex genetic architecture of common disease. + +Author Summary + + + +Epilepsy is a very common brain disorder characterized by recurrent seizures, resulting from abnormal nerve cell activity in the brain. Some cases of epilepsy are caused by brain trauma, such as stroke, infection, tumor, or head injury. Others—so called “idiopathic”—do not have a clear cause. Many idiopathic epilepsies run in families, but the inheritance patterns and complex seizure types suggest that they are not due to a single defective gene but instead are caused by multiple gene defects that are inherited simultaneously in a patient. This complex inheritance makes it difficult to pinpoint the underlying defects. Here, we describe a new mutant mouse, called “frequent-flyer,” which has several different types of seizures. Although these seizures are caused by a mutation in a single gene, because this gene regulates the expression of many other genes, which, in turn, cause abnormal nerve cell activity, frequent-flyer mice provide a unique animal model of epilepsy—mimicking the complex genetic architecture of common disease. + +Introduction + +Epilepsy, defined by recurrent seizures resulting from abnormal, synchronized neuronal firing in the brain, is a very common neurological disorder. Idiopathic epilepsies do not have any antecedent disease or injury to the brain and many are suspected to have a genetic basis. The difficulty of elucidating defective genes underlying common inherited epilepsies is that they are genetically complex—being caused by multiple variants that are coinherited in affected individuals [1,2]. To date, most mutations involved in idiopathic epilepsy have been found in genes encoding ion channels or their accessory subunits with a few exceptions, for example, LGI1 [3] and EFHC1 [4] in humans, [5] and JRK/JH8 [6,7] in both humans and mice. Such exceptions are of interest in that they may lead to further understanding of epilepsy disease mechanisms beyond primary excitability defects, for example, by identification of genes that modulate the expression or function of the more proximal candidates for epilepsy—ion channels, neurotransmitter receptors, and synaptic proteins. + +Here we describe the disruption of the expression of an RNA-binding protein, BRUNOL4 (Bruno-like 4) leading to partial limbic and tonic–clonic seizures in a new mouse model of epilepsy called “frequent-flyer” (abbreviated Ff; gene symbol: Brunol4Ff). BRUNOL4 (also known as CELF4, CUG-BP, and ETR-3 like factor 4) belongs to a family of RNA-binding proteins involved in multiple aspects of RNA processing such as pre-mRNA splicing [8], mRNA editing [9], and RNA stability and translation [10]. There are six family members in both humans and mice with orthologs in nematode and fruit fly [11]. The murine and human BRUNOL4 are 99.6% identical at the amino acid level [12]. Mouse knockouts have very recently been published for Brunol1 and Brunol2, which display spermatagonial and varied developmental defects, respectively [13,14]. UNC-75, a neuron-specific ortholog in C. elegans, shares 47% identity with the human BRUNOL4 protein. UNC-75 deficiency in the nematode leads to behavioral phenotypes indicative of abnormal neurotransmission. Human BRUNOL4 can rescue the unc-75 mutant phenotype, suggesting that UNC-75 and BRUNOL4 may be involved in fine-tuning synaptic transmission through regulating RNA processing in the nervous system [15]. + +In this study we describe the seizure phenotypes of mice carrying Brunol4 disruption, and begin to explore the molecular consequences using gene expression profiling and genetic interaction tests. Our studies suggest that Brunol4 deficiency alters the expression of several molecules involved in synaptic function, which, when combined, account for the complex seizure disorder of frequent-flyer mice. + +Results + +Origin of the Mutation and Convulsive Seizure Phenotype + +The Ff mutation arose from an independent project in which a series of transgenic mouse lines was generated on the C57BL/6J (B6) strain background. One line (9/9 transgene carriers) developed frequent seizures from about three months of age, precipitated by routine handling such as cage transfer. Since the transgene construct was not expressed in all the lines and since other lines using the same construct did not have seizures, together suggested that the seizures were not caused by transgene expression per se. To distinguish between an unlinked spontaneous mutation and insertional mutagenesis, affected mice were outcrossed to normal B6 mice. In the next generation, seizures cosegregated with the presence of the transgene (25/28 carriers displayed seizures versus 0/22 non-carriers), suggesting a high-penetrance, dominant mode of inheritance for the seizure phenotype. + +Convulsive seizures ranged in severity, the mildest being muscle twitching in the face and neck, forelimb clonus, and salivation (Figure 1A). More severe seizures included rearing and falling, myoclonic jerks, and arching of the back and tail. In many cases, convulsions were followed by a very wild running–bouncing phase with occasional tonic–clonic hindlimb extension, but which, unlike the equivalent phase of some induced seizures, did not result in lethality. Hence, the allele symbol “frequent-flyer” (Ff) was assigned. The incidence of these handling-associated seizures was higher in male than in female mice. + +Figure 1 + +Phenotypes of Ff Mutant Mice + +(A) The posture of a 3-mo-old Ff/+ heterozygote mouse at the beginning of a seizure episode. Note the contraction of the muscles of the neck, ears, and forelimbs and the vertical position of the tail. + +(B) Reduced ECT of Ff/+ mice. The median response level (CC50) for minimal seizures were as follows +/+ = 9.17 mA (95% CI 8.83–9.44), Ff/+ = 7.69 mA (95% CI 7.48–7.9). Squares (□) and triangles (▵) indicate individual data points used to construct CC curves in +/+ and Ff/+ mice, respectively. + +(C) Late-onset body-weight gain in Ff/+ heterozygous mice. We monitored the body weight between the age of 12 wk and 34 wk of more than 50 male mice with similar number of mutants and controls. Single-caged mice were excluded from this study. Each mouse was weighed every other week. The body weights of mutants did not diverge significantly from those of controls until 28 wk of age and the divergence remained afterwards (t-test, assuming two-tailed distribution and two-sample equal variance, the p-values are 0.024, 0.004, 0.0011, and 0.001 from 28 wk to 34 wk). The average weight of each genotype at a fixed time-point was plotted against time to generate a growth curve using Microsoft Excel (http://www.microsoft.com/). (D) EEG recording from a Ff/Ff homozygous mouse. Shown are the six differential recordings from four subcranial electrodes, one in each quadrant corresponding to front-right (FR), front-left (FL), back-right (BR), and back-left (BL). These traces correspond to one of the longer spike-wave discharge (SWD) episodes observed. + +(E) Treatment of SWD in Ff/Ff homozygous F2 hybrid mice from crosses to FVB (white box) or 129S1 (black). Recording sessions began at least 1 h prior to ethosuximide, saline, or fluoxetine (Prozac) treatment, and the rate of SWD (minimum criteria: ≥ 0.5 s duration, amplitude ≥2× baseline) was determined and plotted (“pre”). Animals were then injected with drug, monitored for an additional hour, and SWD incidence was recorded (post). Lines between datapoints correspond to each animal pre- and post-treatment. Only the ethosuximide and fluoxetine results were significantly different comparing treatments (ETX, p = 0.009; fluoxetine, p = 0.019; saline, p = 0.804 matched pairs test). + +Although handling-associated seizures did not begin until the third month of age, by 7 wk heterozygotes had markedly reduced electroconvulsive thresholds (ECT) (Figure 1B). In addition to convulsive seizure phenotypes, heterozygotes were also slightly hyperactive, and while slightly smaller at weaning age, they had a late-onset body weight gain in Ff/+ heterozygotes (on average 10% heavier than littermate controls, Figure 1C). Despite the high frequency and the severity of seizures, Ff/+ heterozygotes do not have a reduced life span (analyzed up to 24 mo of age). The morphology of the Ff/+ brain appears normal, as evident in the proper cortical and hippocampal layering and the lack of overt gliosis (unpublished data). + +Strain Background Effects on Frequent-Flyer Phenotypes + +Ff/Ff homozygotes, however, had a much more severe phenotype; they were born alive at close to Mendelian ratios but most died during the first day. From matings between heterozygotes, only 1.1% (expect 25%) survived until 4 wk of age (Table 1). While alive, homozygotes did not display obvious signs of convulsion or respiratory stress, nor was there any obvious pathology seen in mutant brains (unpublished data). Future work will be needed to clarify the cause of perinatal lethality in Ff/Ff homozygotes. However, when we examined the F2 generation of matings between B6-Ff/+ and six different inbred mouse strains, a range of survival rates of homozygotes were observed was with the highest being 8.2% in crosses with 129S1 (Table 1), suggesting that homozygosity for B6 allele(s) makes the homozygous phenotype worse, as is the case with many neurological mutations in mice (e.g., see [16]). Interestingly, although these F2 hybrid homozygotes often lived for more than 6 mo, they were smaller than littermates and also exhibited spontaneous limbic and tonic–clonic seizures, similar in appearance to those of Ff/+ heterozygotes, except they were observed as early as 8 wk (and we suspect that lethal seizures occurred as early as 4 wk). In addition, Ff/+ heterozygotes on F1 hybrid backgrounds experienced a lower incidence of convulsive seizures later in life (unpublished data). These results show that inbred strains have polymorphisms that attenuate the frequent-flyer phenotypes. + +Table 1 + +Survival of Brunol4Ff/Ff Homozygous Mutants in F2 Generation + +Absence Epilepsy in Frequent-Flyer Mutants + +The availability of Ff/Ff homozygotes on a mixed genetic background afforded us the opportunity to determine whether they show spike-wave discharges (SWD), the electroencephalographic manifestation of absence seizures—events not observed in Ff/+ heterozygotes on the B6 background (unpublished data). Ff/Ff homozygotes tested on the F2 hybrid backgrounds (B6 × 129S1 or FVB/NJ) experienced very frequent SWD (e.g., see Figure 1D and 1E). Interestingly, heterozygotes in the FVB/NJ cross, but not in the 129S1 cross, also showed a significant rate of SWD (unpublished data). Together, these results suggest that not only do Ff/Ff homozygotes have SWD, but that the penetrance or severity is also modulated by genetic background. Although SWD in Ff/Ff homozygotes were synchronous, rhythmic, and generalized, when compared to those of other SWD-prone mice, such as stargazer or C3H/HeJ (e.g., see [17,18]), the episodes were relatively short, averaging 1.5 s in length, and the rhythmicity was more erratic than in other mutants (Figure 1D). Nevertheless, the animals remained motionless during SWD episodes, and SWD were suppressed by the anti-absence drug ethosuximide (Figure 1E, left), suggesting that they are absence seizures. + +Transgenic Insertion Mutation in Brunol4 + +To determine the identity of the gene disrupted by transgenic insertion, we cloned and sequenced a unique transgene-genomic junction fragment (see Materials and Methods) and found a 100% match to intron 1 of the Brunol4 gene on mouse Chr 18. We then evaluated the impact of transgene insertion on Brunol4 expression. The insertion expanded the 74-kb intron 1 of Brunol4 by at least 20 kb, well upstream of the exons encoding RNA binding motifs (www.ensembl.org/Mus_musculus, Figure 2). Multiple splice donor and acceptor sites were detected in the transgene, suggesting the possibility of the insertion interfering with normal Brunol4 splicing. In total RNA samples from newborn mice, no Brunol4 transcript was detected in Ff/Ff homozygotes and approximately 45% reduction was seen in heterozygotes (Figure 3A); by real-time reverse-transcriptase PCR (RT-PCR), similar reduction was observed in the adult brain of Ff/+ heterozygotes (Figure 3B). We also examined the potential impact of the transgene on the expression of the neighboring genes. The genomic region where murine Brunol4 resides is gene poor. Genes with strong annotation are at least 0.5 Mb either 5′ or 3′ away from Brunol4. Expression analysis of the neighboring genes with potential brain function did not reveal difference between Ff/Ff homozygous mutants and normal controls (unpublished data), suggesting that Brunol4 is the only brain-expressed gene affected by the transgene insertion. This is consistent with preliminary assessment of a gene-targeted null allele of Brunol4 that we made recently, which displays handling-associated seizures in older adults, resembling those of frequent-flyer mice with both limbic and wild tonic-clonic phases; younger heterozygotes also have an unusually low threshold to electroconvulsion (C. L. Mahaffey, W. N. Frankel, unpublished results). + +Figure 2 + +Trangene Insertion into the Brunol4 Locus in Ff Mice + +(A) Physical map of the transgene insertion point in the first intron of Brunol4 gene on mouse Chromosome 18. The exact number of copies of the transgene inserted is currently unknown. It appears to be more than four copies based on a Southern blot probed with a transgene-specific probe. The transgene insertion event was a simple addition at the 5′ end, but appeared complex at the 3′ end. The last intact copy of the transgene was inverted to the opposite orientation and there was an 18-kb deletion in the genomic sequence at the 3′ insertion site. Further, a 397-nt fragment from the transgene was left between the last intact copy of the transgene and the intronic sequence from Brunol4. This 397-nt fragment was in the same orientation as that of the transgene at the 5′ insertion site. The telomeric breakpoint of the transgene insertion was 28,987 nt from the exon 1 splice donor and the centromeric breakpoint was 27,056 nt from the exon 2 splice acceptor. + +(B) The exon/intron structure of the mouse Brunol4 gene. Note the orientation of the gene is reversed from (A) to facilitate viewing. The open reading frame starts in exon 1 and ends in exon 12. Three RNA recognition motifs are predicted in the BRUNOL4 peptide sequence and the respective coding exons are marked. + +Figure 3 + +Expression of Brunol4 in Mutant and Wild-Type Mice + +(A) Reduced Brunol4 transcript abundance in Ff/+ mice. Newborn brain total RNA blot was probed with Brunol4 p1 probe (upper portion) and a mouse β-actin probe (lower portion) sequentially. Brunol4 transcript was absent from Ff/Ff homozygous newborns, and reduced in abundance in littermate +/+ controls. An arrow indicates the position of the Brunol4 transcript. + +(B) Relative fold change of Brunol4 by real-time RT-PCR) shown in total RNA from adult cortex in three independent mice of each genotype. Similar results were observed from all other brain regions. + +(C) The expression of Brunol4 is restricted to the brain in adult mice. An RNA blot containing poly-A selected RNA extracted from various adult mouse tissues was sequentially hybridized with probes specific to Brunol4 and β-actin as a loading control. The position of the size markers (Ambion, http://www.ambion.com) is shown to the left side of panels. + +In order to evaluate the expression pattern of Brunol4 in adults, we examined RNA from a variety of tissues. Only brain samples showed robust signal, despite prolonged exposure time, suggesting that BRUNOL4 is brain specific in adults (Figure 3C), consistent with a recent survey of organ protein expression in mice that detected BRUNOL4 only in the brain [19]. At a higher resolution, Brunol4 showed a predominantly neuronal expression pattern in the brain—labeling was seen in the cerebral cortex, hippocampus, olfactory bulbs, and the granule cell layer of the cerebellum (Figure 4). However, strongest expression in the brain was observed in the hippocampus where high expression was detected in the pyramidal neurons of the CA2 and CA3 region. Pyramidal neurons in CA1, the dentate gyrus granule cells, and the dentate subgranular zone had weaker expression (Figure 4); the latter is interesting in light of the observation that some types of seizure activity may induce neurogenesis in this region (e.g., see [20]). + +Figure 4 + +Brunol4 Expression in Normal Adult Mouse Brain by RNA In Situ Hybridization + +A 33P labeled antisense riboprobe (A) or a sense control probe (B) was hybridized to 7-μm parasagittal paraffin sections of brain. Note the positive staining in the olfactory bulb, cerebral cortex, hippocampus, and cerebellum. Despite several attempts, persistent minor background signal was observed at the edges of the brain section, especially in the cerebellum. In order to increase the specificity and better monitor the signal development, a DIG-labeled antisense riboprobe (C) or a sense control probe (D) was hybridized to 15-μm parasagittal cryosections of brain. Representative images are shown focusing on the entire hippocampus. Note the intense labeling (blue color) in the CA2 and CA3 regions, consistent with the strong hipocampal signal seen in panel (A). + +CB, cerebellum; CO, cerebral cortex; Hi, hippocampus; OB, olfactory bulb. + +Neuroexcitability Candidates Down-Regulated in Mutant Mice + +How might BRUNOL4 deficiency result in a complex seizure phenotype? We hypothesize that BRUNOL4 is involved in the processing events of one or more mRNA-encoding proteins that are themselves more directly involved in synaptic function. Thus, in the absence or reduction of BRUNOL4, these molecules become dysregulated, leading to imbalance in neuronal excitability. We carried out microarray analysis to detect genes differentially expressed between coisogenic wild-type and mutant mice. For the primary screen, newborn Ff/Ff homozygous mutants were chosen to optimize the signal differential. Of the approximately 39,000 transcripts interrogated, changes in only 459 transcripts (corresponding to approximately 350 independent genes) were considered statistically significant in the Ff/Ff homozygotes compared with controls (Table S1). Of the 94 down-regulated transcripts (from approximately 70 independent genes), the most reduced was Brunol4 itself, with a significant decrease in Ff/Ff homozygotes. Four genes from the down-regulated list were of obvious interest for an excitability disorder. One encodes serotonin receptor 2c (Htr2c); Htr2c-null mice are known to experience frequent spontaneous seizures, at least on a mixed background, as well as a reduced seizure threshold [21]. The second encodes synapsin II (Syn2); seizures precipitated by sensory stimuli were found previously in Syn2-knockout mice [22]. The third encodes N-ethylmaleimide-sensitive factor (Nsf), which regulates exocytosis in synaptic transmission, as well as AMPA receptor trafficking [23,24]. The fourth encodes α-synuclein (Snca), a neuron-specific presynaptic protein [25]. All four molecules have been found in the pyramidal neurons in the hippocampus where Brunol4 is highly expressed [25–28]. In particular, the expression of NSF [27] and synapsin II [28] were enriched in the CA2-CA3 region, similar to that of Brunol4. + +After confirming the differential expression of all four transcripts in Ff/Ff-homozygous newborn mice (unpublished data), expression levels were assessed in adult Ff/+ heterozygous brain regions. All four RNAs had, on average, a 20%–25% reduction in the B6-Ff/+ hippocampus compared with controls (Figure 5A and 5B). Moreover, these candidates showed a significant reduction at the protein level in adult hippocampus of B6-Ff/+ mice (30%–39%; unpublished data), and on a mixed background in Ff/+ and Ff/Ff, (25%–32% Ff/+; 32%–56% Ff/Ff, Figure 5C and 5D). These region-specific decreases before the onset of seizures were consistent with the limbic-seizure phenotype and the overlapping hippocampal expression of the four genes with that of Brunol4. + +Figure 5 + +Reduced Expression of Four Candidate Genes in Ff /+ Brains + +(A) Representative images from three independent RNA blots. Sequential hybridization using Htr2c, Nsf, Snca, Syn2, Gabrb3, and β-actin probes was carried out on a blot containing poly-A RNA extracted from dissected brain regions. The synapsin II probe detected both synapsin IIa and IIb transcripts due to alternative splicing/polyadenylation [40] and there was no overt change in the synapsin 2a/2b ratio between Ff/+ and +/+ brains. In addition to the loading control actin, we examined the expression of Gabrb3, which encodes the β3 subunit of the GABA(A) receptor. Germline targeting of Gabrb3 caused perinatal lethality in homozygous mice and overt seizures in heterozygotes [41]. No significant expression difference was found between Ff/+ and +/+ brains. The position of size markers (Ambion) was shown on the left. CO, cerebral cortex; Hb, hindbrain; Hi, hippocampus. F, Ff/+; +, +/+. + +(B) Histogram summarizing results from northern blots, expressed as percent of wild type after normalization to β-actin. + +(C) Reduced expression at the protein level; Western blot for HTR2A, HTR2C, NSF, synapsin II, α-synuclein, and β-tubulin. HTR2A, a molecule with distribution and property similar to HTR2C, was used as a control in addition to β-tubulin. Representative images from experiments with three independent adult mice on a mixed background are shown. + +(D) Histogram summarizing results from (C) in Ff/+ and Ff/Ff mutants on a mixed background, expressed as % of wild type after normalization to β-tubulin, summarizing data from three independent animals. Error bars throughout are one standard deviation. + +Htr2c Deficiency Contributes to, but Is Not Sufficient for, Seizure Phenotypes in Brunol4Ff Mutants + +Null mutants of the Htr2c receptor gene have several phenotypic similarities with Brunol4Ff/+ heterozygotes, including reduced seizure threshold, hyperactivity, and late-onset weight gain [21,29]. However, the fact that Brunol4Ff/+ mice on the B6 strain background experience frequent handling-provoked convulsive seizures after 3 mo of age, whereas Htr2c null mutants do not (Y. Yang, W. Frankel, unpublished data), suggests that down-regulating Htr2c receptor expression is not sufficient to account for the handling-induced seizures. To determine whether Htr2c deficiency is sufficient to account for the ECT of Brunol4Ff/+ mice, we compared seizure threshold in wild-type, single-mutant, and double-mutant mice (Figure 6). These studies were all done on the B6 strain, to avoid the confounding effect of genetic background. Although the average seizure threshold of Htr2c null mutants was lower than that of Brunol4Ff/+ mice by approximately 2 mA, the threshold of double mutants was 1 mA lower still (p = 0.0003; |t|-test). This suggests that factors in addition to reduced expression of Htr2c contribute to seizure threshold in Brunol4Ff/+ mice. Another difference between Htr2c and Brunol4 mutant mice is that Htr2c-null mutants do not have SWD ([21]; our unpublished observations). However, we found that blocking serotonin reuptake in Brunol4Ff/Ff homozygotes by fluoxetine (Prozac) treatment lowers the SWD incidence by about 50% (Figure 1E, right)—again suggesting that Htr2c down-regulation combines with other Brunol4-downstream deficiencies to cause the seizure disorder of frequent-flyer mutants. Although further tests are required to determine whether the other causative factors are Syn2, Nsf, or Snca specifically, it is clear that the seizure phenotypes of Brunol4Ff are determined in a genetically complex manner. + +Figure 6 + +Genetic Interaction between Brunol4 and Htr2c for ECT + +Shown is a histogram of average threshold (in mA) to a minimal clonic or maximal tonic hindlimb extension seizure, determined as described in Materials and Methods, for wild-type (+/+, +/+), single mutant (Ff/+, +/+ or B6-Htr2ctm1Jul/Y), or double mutant (Ff/+; Htr2ctm1Jul/Y), littermates from crosses between B6-Htr2ctm1Jul/Y and B6-Ff/+ mice. + +Discussion + +Here, we report on the causal association between Brunol4, encoding a brain-specific RNA-binding protein, and the seizure disorder of frequent-flyer mouse mutants. The origin of the disorder is a transgenic insertion in the Brunol4 gene, resulting in very little Brunol4 transcript in homozygotes and accordingly reduced amount in heterozygotes—suggesting haploinsufficiency. We do not know why the transcript levels were very low, but one possibility is due to the inverted repeat in the transgene cluster, creating a potential hairpin structure that may prevent read-through transcription (Figure 2). Brunol4Ff mutant mice have several different kinds of seizures, depending upon genotype, age, and strain background. In heterozygotes on a B6 inbred strain background, these include recurrent limbic and tonic–clonic seizures—observed readily following routine animal handling after 3 mo of age—and a significantly lower ECT at an earlier age. Although homozygotes do not usually survive on a C57BL/6J strain background, on F2 hybrid backgrounds homozygotes (and some heterozygotes) that survived also displayed spike-wave discharges, the hallmark of absence epilepsy. + +The prospect of a defective RNA-binding protein such as BRUNOL4 causing a complex seizure disorder suggests a way in which a single gene defect can mimic a complex genetic disease, by impairing the function of multiple molecules simultaneously. This could happen either as a direct consequence of its absence, or secondarily, e.g., a cascade of effects. Microarray analysis between homozygous mutants and coisogenic control brain yielded a small number of down-regulated transcripts that were statistically significant, two of which (Htr2c and Syn2) are already known to cause seizure-related phenotypes when knocked out in mice [21,22], and two other (Nsf and Snca) have obvious functions that relate to synaptic transmission. The down-regulation of each was confirmed in adults, and was greatest in the hippocampus, where Brunol4 expression is high. Interestingly, in addition to seizure susceptibility, Ff/+ heterozygotes display two nonseizure phenotypes like those of Htr2c null mutants: mild hyperactivity and late-onset obesity [21,30,31]. This might suggest that compromised serotonergic transmission is the major factor of the frequent-flyer phenotype. However, because Htr2c expression is reduced only modestly (∼25% RNA, ∼35% protein) in Ff/+ heterozygotes, compared with complete loss in Htr2c-null mice, it seems more likely that a combination of compromised serotonergic transmission and other defects is responsible for the disorder. Further evidence for this idea was obtained in the ECT paradigm (additive phenotypic effects of double mutants), and by observing that SWD phenotype of Brunol4 homozygotes was partially mitigated by up-regulation of serotonergic transmission through blocking the reuptake of serotonin. With any of the seizure paradigms, it is plausible that reduced synaptic efficacy, e.g., due to reduced expression of the other three candidates singled out—Syn2, Nsf, or Snca—is the other contributing variable. However, we cannot ignore other genes misregulated in Brunol4 mutants, some with unknown function (Table S1), since many also expressed selectively within the hippocampus in B6 mice (Allen Brain Atlas [32]). + +We do not know why Brunol4 deficiency results in the decreased expression of these and other transcript RNAs. BRUNOL4 has been shown to regulate alternative splicing in cells and tissues without endogenous BRUNOL4 [33–35], but we did not detect aberrant splice variants in mutant brains, at least in the subset of transcripts that we analyzed (our unpublished results). Since members of the Bruno gene family are involved in RNA editing, stability, and translation, the possibility exists that Brunol4 is involved in other aspects of RNA metabolism, for example, in stabilizing transcripts for translation, a possibility that is supported, in part, by the fact that the degree of reduction at the RNA level and at the protein level was not 100% concordant in the mutant hippocampus (∼20%–25% and ∼31%–39%, respectively). However, since many RNA processing steps are believed to be coupled with transcription [36], BRUNOL4 may indeed serve multiple roles in RNA metabolism. + +Most genes known to cause idiopathic epilepsy encode ion channels. Brunol4 joins a growing list of non-ion-channel epilepsy genes in both humans and mice [3–5,7]. It is noteworthy that the human BRUNOL4 gene is in a region on human Chromosome 18 showing strong evidence for linkage with adolescent-onset idiopathic generalized epilepsy [37], suggesting that BRUNOL4 may be a candidate gene for these seizure disorders. + +Materials and Methods + +Mice. + +Origin of Brunol4Ff mice. C57BL/6J (B6) transgenic mice were generated at The Jackson Laboratory (http://www.jax.org/) using a construct where the expression of murine Ighmbp2 cDNA and enhanced green fluorescent protein (EGFP) was driven by a bidirectional tetracycline-responsive promoter. Briefly, the coding region of the Ighmbp2 cDNA was cloned in the pBI-EGFP vector (Clontech, http://www.clontech.com/). The PvuI linearized transgene (∼8.3 kb) was microinjected into pronuclei of single cell B6 mouse embryos, which were subsequently implanted into pseudopregnant mice. B6.129-Htr2ctm1Jul mice, derived from mice published in 1995 [21], were obtained from The Jackson Laboratory's Induced Mutant Resource and are now fully congenic on the B6 background after backcrossing for ten generations. All animals were fed standard National Institutes of Health diet containing 6% fat and acidified water ad libitum. All animal procedures followed Association for Assessment and Accreditation of Laboratory Animal Care guidelines and were approved by institutional Animal Care and Use Committee. + +ECT. + +As previously described [38], mice were restrained, a drop of anesthetic containing 0.5% tetracaine and 0.9% NaCl was placed onto each eye, and a preset current was applied via silver transcorneal electrodes using a electroconvulsive stimulator (Ugo Basile model 7801; http://www.ugobasile.com). The stimulator was set to produce rectangular wave pulses with the following parameters: 299 Hz, 0.2 s duration, 1.6 ms width. Sixty Ff/+ and 57 littermate +/+ male mice (ages 6–9 wk) were tested for ECT over a range of electric current settings for minimal clonic forebrain seizure and each ECT response was recorded. The data were analyzed in the computer program MiniTab (Minitab, http://www.minitab.com/) and a response curve was generated using the log-Probit procedure. To determine ECT in double mutants, male mice (ages 6–9 wk) were tested by increasing the stimulus once daily until at least a minimal clonic seizure was observed, and the average threshold determined for each genotype. + +Electroencephalogram analysis. + +Mice were anesthetized with tribromoethanol (400mg/kg i.p.) Small burr holes were drilled (1 mm anterior to the bregma and 2 mm posterior to the bregma) on both sides of the skull 2 mm lateral to the midline. Electroencephalogram (EEG) activity was measured by four Teflon-coated silver wires soldered onto a microconnector. The wires were placed between the dura and the brain and a dental cap was then applied. The mice were given a post-operative analgesic of carprofen (5 mg/kg subcutaneous) and were given a 48-h recovery period before recordings were made. The mice were recorded for a 2-h period on each of the following two days using the Grass EEG Model 12 Neurodata Acquisition System and PolyViewPro software program (Grass-Telefactor, http://www.grasstechnologies.com/). For mice that were treated with ethosuximide (200 mg/kg; Sigma-Aldrich, http://www.sigmaaldrich.com) or fluoxetine (20 mg/kg, Sigma-Aldrich), on the day following their second standard EEG recording, mice were recorded for 90 min and then injected intraperitoneally. They were then recorded for a minimum of one additional hour. The control mice were injected intraperitoneally with saline and recorded in the same manner. Matched pairs tests were done using the program JMP 6.0.3 (SAS, http://www.sas.com/). + +Identification of the transgene insertion site. + +Genomic DNA from transgenic mice and control mice was digested with BclI, SpeI, BglII, SphI, and StuI and electrophoresed and blotted onto a Nytron Plus membrane (Schleicher & Schuell, http://www.whatman.com/). The blot was probed with an EGFP probe and a unique transgene-genomic junction fragment was present in StuI-digested DNA at about 3 kb. The StuI-digested fragment of ∼3 kb was cloned into the pBluescript II-SK vector (Stratagene, http://www.stratagene.com). A vector-specific primer and an EGFP primer were used to amplify the junction fragment. Automated sequencing confirmed the presence of the EGFP cDNA as well as other vector sequence. A 652-nt fragment did not match with the pBluescript II-SK vector sequence and was used as a query to BLAST search the mouse genome. A single perfect match to intron 1 of the Brunol4 gene was found on mouse Chromosome 18. The other breakpoint of the transgene insertion was cloned by a PCR strategy using a transgene-specific primer and a Brunol4 intron 1 primer. Based on the sequence information around the insertion breakpoints in the Ff allele, a 3-primer PCR assay was designed to detect the Ff allele and wild-type allele. Primers for this assay are: s3gtf, 5′-CTCTTCATCCCTTCTGGCAAGTAG-3′; s3gtr, 5′-GTATTCAACAATTCCGTGTCGCCC-3′; and s3gtr2, 5′-CCACACAGAGACCAAGAAGATTCC-3′. At 55 °C annealing temperature, 35 cycles, standard PCR conditions, the s3gtf/s3gtr2 primers produce a 672-bp wild-type allele, and the s3gtf/s3gtr primers produce a 464-bp mutant allele. + +Northern blot analysis and quantification. + +Total RNA was prepared from newborn brain, adult brain and dissected brain regions using TRIzol reagent (Invitrogen, http://www.invitrogen.com). Two probes were generated for Brunol4, p1 containing 5′ UTR and exon1 5′ to the insertion site and p2 containing the region between BRUNOL4′s second and third RNA recognition motif. Both probes detected a single transcript on northern blots and p2 was also used for in situ analysis. Hybridization was carried out in formamide-based solution at 42 °C overnight and the blot was washed and exposed to an X-ray film at −80 °C. The same blot was stripped and reprobed with a mouse β-actin probe. Films were imaged by Fuji Luminescent Image Analyzer LAS-1000 Plus (http://fujifilmlifescienceusa.com) and subsequently quantified by Fuji Image Gauge Ver. 3.4. + +Real-time reverse-transcription PCR. + +Total RNA was prepared from the cerebral cortex of adult B6-Ff/+ and and B6-+/+ /littermates with Trizol (Invitrogen, http://www.invitrogen.com) and treated with DNase I (Promega, http://www.promega.com) under the manufacturer's suggested conditions. RNA (2 μg) was reverse transcribed with AMV reverse transcriptase (Promega). The cDNA was diluted 20-fold, and 1.5 μl was added to qPCR Mastermix Plus for SYBR Green I (Eurogentec, http://www.eurogentec.be) with pairs of the following primers: beta-actinF (5′-CATTGCTGACAGGATGCAGAA-3′) and beta-actinR (5′-GCCACCGATCCACACAGAGT-3′), Be1u (5′-TCGCAGTAGGTGAGGAAAGCGCAG-3′) and Be2d (5′-TCGCAGTAGGTGAGGAAAGCGCAG-3′), corresponding to Brunol4 exon 1 forward and exon 2 reverse, respectively. The PCR reactions were analyzed on an ABI Prism 7000 Sequence Detection System (PerkinElmer, http://www.perkinelmer.com/). The PCR amplifications from three pairs of age-matched mice were run in triplicate. Amplification of the correct size products was confirmed by agarose gel electrophoresis. + +RNA in situ hybridization. + +Thin brain sections (7 μm) from 10-wk-old B6 male mice were hybridized with 33P probes overnight at 50 °C and washed, RNase A treated, dehydrated, and air dried. Slides were dipped in liquid emulsion (Kodak, http://www.kodak.com/) and images were developed 5 d afterwards. For DIG-based in situ analysis, 15-μm cryosections were hybridized with DIG probes overnight at 65 °C and washed extensively before an overnight incubation of alkaline phosphatase–conjugated anti-DIG antibody (1:2,000, Roche, http://www.roche.com/). Staining signal was developed using BM purple (Roche) at room temperature for 12 h. + +Microarray analysis. + +Total RNA was prepared from six male newborn heads (3 Ff/Ff and 3 +/+). 10 μg of total RNA was used to generate 15 μg of cRNA for hybridization to the Affymetrix 430 v2.0 Gene Chip (Affymetrix, http://www.affymetrix.com/) according to manufacturer's recommendation. Using the R/maanova package [39], an analysis of variance (ANOVA) model was applied to the data, and F1, F2, F3, and Fs test statistics were constructed along with their permutation p-values. Changes in 459 transcripts were considered statistically significant among the 39,000 transcripts interrogated. + +Antibodies for Western blotting. + +The primary antibodies and the dilutions were: α-HTR2A, 1:500 (BD Pharmingen, http://www.bdbiosciences.com/); α-HTR2C, 1:500 (Immunostar, http://www.immunostar.com/); α-NSF, 1:1,000 (H-300, Santa Cruz Biotechnology, http://www.scbt.com/); α-Synapsin 2, 1:5,000 (Stressgen, http://www.nventacorp.com/); α-alpha-synuclein, 1:250 (BD Transduction, http://www.bdbiosciences.com/); and α-beta-tubulin, 1:1,000 (Sigma). The secondary antibodies and the dilutions were: HRP α-mouse, 1:6,000 (Zymed, http://www.invitrogen.com/) and HRP α-rabbit, 1:2,000 (PerkinElmer). + +Northern blot probes. + +The following probes were designed to detect expression differences among the putative BRUNOL4 target RNAs between Ff/+ and +/+ brains: (1) Htr2c, 437-nt probe in exon 6, the last nucleotide is 43 nt 3′ of the TAA stop codon. (2) Nsf, 470-nt probe as described in [27]. (3) Syn2, 234-nt probe in exon 1, the first nucleotide is 90 nt 3′ of the ATG start codon. This probe was able to detect the two alternatively polyadenylated Syn2 transcripts. (4) Snca, 416-nt probe in exon 6, the first nucleotide is 37 nt 3′ of the TAA stop codon. (5) Gabrb3, 412-nt probe specific to the 3′ end of the coding sequence, the last nucleotide is 14 nt 5′ of the TGA stop codon. + +Western blot. + +Hippocampi were dissected from three male Ff/+ mice (before the onset of handling-provoked seizures) and three +/+ littermates. Protein extracts were made using RIPA buffer with proteinase inhibitors (Roche) and subsequently quantified using the Bradford reagent (Bio-Rad, http://www.bio-rad.com/). Protein (50 μg) from each sample was loaded and probed with the primary antibody and a secondary peroxidase-conjugated antibody and visualized with the ECL plus kit (Amersham, http://www.amersham.com/). The nitrocellulose membrane was incubated with Restore Western blot stripping buffer (Pierce, http://www.piercenet.com/) at 37 °C for 30 min to remove all the antibodies. The membrane was washed and subsequently reprobed with a different set of antibodies. Signals were quantified using the method described above. + +Supporting Information + +Table S1 + +List of Genes with Differential Expression between Ff/Ff Homozygous and Wild-Type Newborn Mice + +Microarray analysis was carried out as described in Materials and Methods. Down-regulated transcripts (94) are listed first, followed by 365 up-regulated transcripts. + +(416 KB XLS) + +Click here for additional data file. + +Accession Numbers + +The National Center for Biotechnology Information (NCBI) Nucleotide database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=Nucleotide) accession number for intron 1 of the Brunol4 gene on mouse Chr 18 is EF639873. + +Acknowledgements + +We thank Jason Affourtit, Sandy Daigle, Barbara Beyer, Carolyne Dunbar, Jonette Gilley, Neena Haider, Sonya Kamdar, Yong Woo, and Weidong Zhang for technical assistance. We also thank Susan Ackerman, Albert Becker, Robert Burgess, and Verity Letts for comments and advice. + +Author contributions. YY and WNF conceived and designed the experiments and wrote the paper. YY, CLM, NB, and WNF performed the experiments and analyzed the data. TPM and GAC contributed reagents/materials/analysis tools. + +Funding. The Jackson Laboratory's Gene Expression and DNA Sequencing services were subsidized by an NCI core grant CA34196. This work was supported by a grant from the National Institutes of Health (NS31348 to WNF). YY was supported by a TJL Postdoctoral Fellowship and a research award from Citizens United for Research in Epilepsy (CURE). + +Abbreviations + +Brunol4, - Bruno-like 4 + +ECT - electroconvulsive threshold + +EEG - electoencephalogram + +Ff, - frequent-flyer + +RT-PCR - reverse-transcriptase PCR + +SWD - spike-wave discharge + +Footnotes + +Competing interests. The authors have declared that no competing interests exist. diff --git a/src/ontogpt/evaluation/craft/database/all/17696610.ann b/src/ontogpt/evaluation/craft/database/all/17696610.ann new file mode 100644 index 000000000..a82949bad --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17696610.ann @@ -0,0 +1,1391 @@ +T1 NCBITaxon:10088 0 5 Mouse +T2 GO:0051598 6 26 Pachytene Checkpoint +T3 PR:P38126 6 28 Pachytene Checkpoint 2 +T4 PR:000016672 30 36 Trip13 +T5 GO:0007129 65 72;95 103 Meiotic ... Synapsis +T6 NCBITaxon:40674 118 127 mammalian +T7 GO:0007126 128 135 meiosis +T8 GO:0007129 137 167 homologous chromosome synapsis +T9 NCBITaxon:2759 210 220 eukaryotes +T10 NCBITaxon:40674 222 231 mammalian +T11 GO:0000075 247 258 checkpoints +T12 GO:0065007 264 271 monitor +T13 NCBITaxon:10088 324 329 mouse +T14 SO:0000855 330 338 ortholog +T15 PR:000016672 340 346 Trip13 +T16 GO:0051598 351 371 pachytene checkpoint +T17 PR:P38126 351 373 pachytene checkpoint 2 +T18 PR:P38126 375 379 PCH2 +T19 GO:0007129 412 420 synapsis +T20 GO:0000075 421 431 checkpoint +T21 NCBITaxon:4932 435 459 Saccharomyces cerevisiae +T22 NCBITaxon:6239 464 486 Caenorhabditis elegans +T23 GO:0007126 518 525 meiosis +T24 PR:000016672 541 547 TRIP13 +T25 NCBITaxon:10088 558 562 mice +T26 CL:0000017 571 583 spermatocyte +T27 GO:0016265 584 589 death +T28 GO:0000239 593 602 pachynema +T29 CL:0000023 615 622 oocytes +T30 GO:0007567 630 635 birth +T31 CL:0000017 663 676 spermatocytes +T32 GO:0007129 677 684 synapse +T33 PR:000013672 761 766 RAD51 +T34 PR:000004759 768 771 BLM +T35 GO:0005662 777 780 RPA +T36 GO:0005712 819 828 chiasmata +T37 PR:000010442 837 841 MLH1 +T38 PR:000010443 846 850 MLH3 +T39 CHEBI:44658 856 868 okadaic acid +T40 CL:0000017 889 902 spermatocytes +T41 GO:0007132 925 936 metaphase I +T42 GO:0007129 1027 1035 synapsis +T43 SO:0000704 1036 1041 genes +T44 PR:000015553 1042 1047 Spo11 +T45 PR:000031227 1049 1053 Mei1 +T46 PR:000013858 1055 1059 Rec8 +T47 PR:000006536 1065 1069 Dmc1 +T48 PR:000016672 1091 1097 Trip13 +T49 PR:000016672 1115 1121 TRIP13 +T50 GO:0033313 1136 1154 meiotic checkpoint +T51 NCBITaxon:10088 1167 1171 mice +T52 PR:000016672 1196 1202 TRIP13 +T53 GO:0042148 1221 1236 strand invasion +T54 GO:0035825 1324 1334 crossovers +T55 NCBITaxon:40674 1430 1439 mammalian +T56 GO:0007126 1440 1447 meiosis +T57 GO:0006281 1488 1496 repaired +T58 GO:0051598 1530 1550 pachytene checkpoint +T59 NCBITaxon:10088 1563 1567 mice +T60 CL:0000019 1635 1640 sperm +T61 CL:0000025 1645 1649 eggs +T62 SO:0001026 1690 1696 genome +T63 NCBITaxon:1 1704 1714 individual +T64 GO:0007126 1757 1764 meiosis +T65 GO:0007129 1819 1826 synapse +T66 SO:0000704 1882 1889 genetic +T67 GO:0007126 1987 2009 meiotic cell divisions +T68 NCBITaxon:1 2075 2084 organisms +T69 GO:0033313 2098 2105;2107 2118 meiotic ... checkpoints +T70 GO:0065007 2125 2132 monitor +T71 GO:0007129 2145 2164 chromosome synapsis +T72 GO:0006281 2169 2189 repair of DNA damage +T73 GO:0000075 2197 2208 checkpoints +T74 CL:0000019 2279 2284 sperm +T75 CL:0000025 2288 2292 eggs +T76 NCBITaxon:10088 2329 2334 mouse +T77 PR:000016672 2335 2341 Trip13 +T78 SO:0000704 2345 2349 gene +T79 NCBITaxon:1 2366 2375 organisms +T80 GO:0033313 2396 2414 meiotic checkpoint +T81 GO:0065007 2415 2422 control +T82 GO:0000075 2476 2486 checkpoint +T83 PR:000016672 2493 2499 Trip13 +T84 GO:0007126 2565 2572 meiosis +T85 GO:0006281 2594 2603 repairing +T86 MOP:0000780 2604 2610 broken +T87 CHEBI:36357 2615 2624 molecules +T88 GO:0007129 2648 2656 synapsed +T89 NCBITaxon:33208 2671 2678 animals +T90 http://purl.obolibrary.org/obo/MONDO_0005047 2684 2691 sterile +T91 GO:0016265 2707 2712 death +T92 CL:0000023 2716 2723 oocytes +T93 CL:0000017 2728 2741 spermatocytes +T94 GO:0000075 2789 2799 checkpoint +T95 GO:0007129 2824 2832 synapsis +T96 GO:0006281 2870 2878 repaired +T97 GO:0007276 2946 2964 genesis of gametes +T98 CL:0000300 2957 2964 gametes +T99 SO:0001026 2995 3001 genome +T100 GO:0007567 3036 3041 birth +T101 http://purl.obolibrary.org/obo/MONDO_0000839 3036 3049 birth defects +T102 GO:0007127 3127 3149 first meiotic division +T103 GO:0007129 3185 3192 synapse +T104 GO:0006281 3280 3287 repairs +T105 GO:0006281 3288 3294 repair +T106 SO:0000985 3295 3308 double strand +T107 MOP:0000780 3309 3315 breaks +T108 SO:0000704 3332 3343 genetically +T109 GO:0000237 3355 3364 leptonema +T110 GO:0007129 3382 3390 synapsis +T111 GO:0007114 3394 3401 budding +T112 NCBITaxon:4892 3394 3407 budding yeast +T113 NCBITaxon:40674 3412 3419 mammals +T114 GO:0006260 3594 3609 DNA replication +T115 GO:0000725 3627 3649 recombinational repair +T116 GO:0006281 3688 3694 repair +T117 GO:0007129 3752 3781 homologous chromosome pairing +T118 GO:0006281 3791 3799 repaired +T119 MOP:0000780 3811 3817 breaks +T120 GO:0035825 3854 3867 crossing over +T121 GO:0065007 3905 3917 surveillance +T122 GO:0000075 3928 3939 checkpoints +T123 GO:0007126 3957 3964 meiotic +T124 NCBITaxon:1 4031 4040 organisms +T125 NCBITaxon:4932 4052 4065 S. cerevisiae +T126 NCBITaxon:7227 4067 4090 Drosophila melanogaster +T127 NCBITaxon:6239 4092 4102 C. elegans +T128 NCBITaxon:10088 4108 4112 mice +T129 GO:0007129 4167 4186 chromosome synapsis +T130 GO:0007126 4195 4202 meiotic +T131 GO:0000239 4217 4226 pachytene +T132 GO:0007128 4236 4254 meiotic prophase I +T133 GO:0007126 4273 4280 meiotic +T134 GO:0051598 4312 4332 pachytene checkpoint +T135 SO:0000704 4353 4360 Genetic +T136 NCBITaxon:4932 4376 4389 S. cerevisiae +T137 GO:0051598 4422 4442 pachytene checkpoint +T138 GO:0007126 4487 4494 meiosis +T139 GO:0042770 4555 4575 DNA damage signaling +T140 GO:0007067 4579 4586 mitotic +T141 NCBITaxon:3702 4601 4621 Arabidopsis thaliana +T142 GO:0051598 4648 4668 pachytene checkpoint +T143 NCBITaxon:7215 4709 4719 Drosophila +T144 GO:0051598 4726 4746 pachytene checkpoint +T145 GO:0065007 4759 4766 monitor +T146 GO:0007126 4782 4789 meiotic +T147 GO:0008152 4801 4811 metabolism +T148 NCBITaxon:4932 4815 4828 S. cerevisiae +T149 NCBITaxon:6239 4833 4843 C. elegans +T150 GO:0006302 4849 4859 DSB repair +T151 GO:0007129 4868 4887 chromosome synapsis +T152 NCBITaxon:10088 4899 4903 mice +T153 CL:0000017 4910 4923 spermatocytes +T154 CL:0000023 4928 4935 oocytes +T155 GO:0006302 4969 4979 DSB repair +T156 PR:000006536 4989 4993 Dmc1 +T157 PR:000010669 4995 4999 Msh5 +T158 PR:000004427 5005 5008 Atm +T159 GO:0000239 5040 5049 pachynema +T160 CL:0000017 5055 5068 spermatocytes +T161 GO:0006302 5096 5106 DSB repair +T162 GO:0007129 5119 5127 synapsis +T163 CL:0000023 5141 5148 oocytes +T164 GO:0007129 5205 5213 synapsis +T165 NCBITaxon:10088 5217 5221 mice +T166 SO:0000704 5250 5255 genes +T167 PR:000006536 5264 5268 Dmc1 +T168 GO:0007129 5286 5294 synapsis +T169 GO:0051598 5365 5385 pachytene checkpoint +T170 GO:0007126 5414 5421 meiotic +T171 CL:0002371 5494 5507 somatic cells +T172 GO:0051598 5536 5556 pachytene checkpoint +T173 GO:0065007 5557 5564 control +T174 NCBITaxon:40674 5583 5590 mammals +T175 PR:P38126 5650 5654 PCH2 +T176 GO:0005730 5667 5676 nucleolar +T177 NCBITaxon:4932 5735 5748 S. cerevisiae +T178 SO:0000704 5749 5756 genetic +T179 GO:0000239 5789 5798 pachytene +T180 GO:0007129 5810 5818 synaptic +T181 PR:P31111 5819 5823 zip1 +T182 GO:0051598 5897 5906;5937 5947 pachytene ... checkpoint +T183 GO:0007129 5907 5915 synapsis +T184 GO:0006302 5925 5935 DSB repair +T185 PR:P38126 5975 5979 PCH2 +T186 SO:0000855 5980 5989 orthologs +T187 NCBITaxon:1 6005 6014 organisms +T188 GO:0007129 6028 6036 synaptic +T189 GO:0007126 6037 6044 meiosis +T190 GO:0007129 6055 6063 synaptic +T191 GO:0007126 6064 6071 meiosis +T192 PR:P38126 6105 6109 Pch2 +T193 GO:0000075 6120 6130 checkpoint +T194 GO:0065007 6142 6149 monitor +T195 GO:0000795 6150 6170 synaptonemal complex +T196 GO:0000795 6172 6174 SC +T197 NCBITaxon:9606 6198 6204 humans +T198 NCBITaxon:10088 6230 6234 mice +T199 PR:000016672 6253 6259 Trip13 +T200 SO:0000855 6265 6273 ortholog +T201 PR:P38126 6277 6281 PCH2 +T202 GO:0051598 6333 6353 pachytene checkpoint +T203 GO:0000075 6400 6410 checkpoint +T204 GO:0035825 6476 6485 crossover +T205 GO:0006302 6492 6501;6510 6514 repair of ... DSBs +T206 GO:0007126 6502 6509 meiotic +T207 PR:000016672 6526 6532 Trip13 +T208 GO:0010467 6545 6554 Expressed +T209 NCBITaxon:40674 6555 6564 Mammalian +T210 SO:0000855 6565 6573 Ortholog +T211 PR:P38126 6577 6581 PCH2 +T212 NCBITaxon:40674 6627 6636 mammalian +T213 SO:0000855 6637 6645 ortholog +T214 PR:P38126 6649 6653 PCH2 +T215 PR:000016672 6655 6661 Trip13 +T216 UBERON:0002046 6663 6670 thyroid +T217 CHEBI:60311 6663 6678 thyroid hormone +T218 PR:000016672 6663 6710 thyroid hormone receptor interacting protein 13 +T219 SO:0000857 6757 6765 homology +T220 SO:0000855 6809 6818 orthologs +T221 PR:000016672 6878 6884 TRIP13 +T222 NCBITaxon:40674 6906 6915 mammalian +T223 NCBITaxon:3193 6949 6955 plants +T224 SO:0000857 6976 6990;7004 7011 evolutionarily ... related +T225 NCBITaxon:7147 7022 7027 flies +T226 PR:000016672 7126 7132 Trip13 +T227 GO:0010467 7144 7153 expressed +T228 UBERON:0005291 7170 7179;7190 7197 embryonic ... tissues +T229 UBERON:0007023 7184 7189 adult +T230 UBERON:0000473 7209 7215 testis +T231 NCBITaxon:10088 7245 7250 mouse +T232 NCBITaxon:9606 7255 7260 human +T233 SO:0000345 7261 7264 EST +T234 GO:0010467 7349 7358 expressed +T235 NCBITaxon:9606 7362 7367 human +T236 NCBITaxon:10088 7372 7377 mouse +T237 CL:0000023 7378 7385 oocytes +T238 NCBITaxon:10088 7407 7412 Mouse +T239 PR:P38126 7413 7417 PCH2 +T240 SO:0000855 7418 7426 Ortholog +T241 PR:000016672 7427 7433 TRIP13 +T242 GO:0010467 7438 7448 Expression +T243 PR:P38126 7508 7512 PCH2 +T244 PR:000016672 7513 7519 TRIP13 +T245 SO:0000855 7520 7529 orthologs +T246 NCBITaxon:2759 7867 7877 eukaryotic +T247 NCBITaxon:33511 7914 7927 deuterostomia +T248 NCBITaxon:3193 7937 7943 plants +T249 NCBITaxon:33317 7954 7965 protostomia +T250 NCBITaxon:4751 7981 7986 fungi +T251 UBERON:0000479 8053 8060 tissues +T252 UBERON:0000948 8065 8070 heart +T253 UBERON:0000955 8075 8080 brain +T254 UBERON:0002106 8085 8091 spleen +T255 UBERON:0002048 8096 8100 lung +T256 UBERON:0002107 8105 8110 liver +T257 UBERON:0004288 8115 8123 skeletal +T258 UBERON:0002113 8135 8141 kidney +T259 UBERON:0000473 8146 8152 testis +T260 UBERON:0000922 8160 8166 embryo +T261 UBERON:0000922 8176 8182 embryo +T262 UBERON:0000922 8192 8198 embryo +T263 UBERON:0000922 8212 8218 embryo +T264 SO:0000188 8225 8231 Intron +T265 SO:0000147 8232 8236 exon +T266 PR:000016672 8250 8256 TRIP13 +T267 SO:0000366 8261 8275 insertion site +T268 SO:0000704 8279 8283 gene +T269 SO:0000440 8289 8295 vector +T270 SO:0000366 8354 8368 insertion site +T271 PR:000016672 8400 8406 Trip13 +T272 SO:0000704 8421 8425 gene +T273 PR:000010303 8426 8431 Med31 +T274 UBERON:0000473 8437 8443 testis +T275 PR:000016672 8453 8459 Trip13 +T276 SO:0000112 8460 8467 primers +T277 SO:0000147 8503 8508 exons +T278 UBERON:0000473 8568 8574 testis +T279 PR:000016672 8593 8599 TRIP13 +T280 GO:0042571 8600 8608 antibody +T281 PR:000028799 8652 8659 tubulin +T282 PR:000016672 8701 8707 TRIP13 +T283 PR:000016672 8749 8755 TRIP13 +T284 UBERON:0000473 8759 8765 testes +T285 UBERON:0000473 8803 8809 testis +T286 NCBITaxon:9031 8836 8843 chicken +T287 PR:000016672 8849 8855 TRIP13 +T288 MOP:0000779 8879 8889 conjugated +T289 NCBITaxon:9031 8895 8902 chicken +T290 GO:0071735 8903 8906 IgG +T291 GO:0010467 8929 8939 Expression +T292 GO:0005634 8972 8978 nuclei +T293 CL:0000020 8989 9002 spermatogonia +T294 CL:0000020 9004 9006 Sg +T295 GO:0000237 9009 9018 leptotene +T296 CL:0000017 9019 9032 spermatocytes +T297 GO:0000239 9049 9058 pachytene +T298 CL:0000017 9059 9072 spermatocytes +T299 GO:0000239 9092 9101 pachytene +T300 CL:0000017 9102 9115 spermatocytes +T301 GO:0005634 9125 9132 nuclear +T302 UBERON:0000473 9161 9167 testis +T303 GO:0005737 9195 9206 cytoplasmic +T304 UBERON:0000483 9296 9306 epithelial +T305 UBERON:0000025 9320 9327 tubules +T306 PR:000016672 9352 9358 TRIP13 +T307 CL:0000017 9390 9403 spermatocytes +T308 PR:000015865 9447 9452 SYCP3 +T309 PR:000015865 9454 9455 S +T310 PR:000016672 9461 9467 TRIP13 +T311 PR:000016672 9469 9470 T +T312 GO:0000237 9521 9530 leptotene +T313 GO:0000237 9532 9535 Lep +T314 GO:0000238 9538 9546 zygotene +T315 GO:0000238 9548 9551 Zyg +T316 GO:0000239 9558 9567 pachytene +T317 GO:0000239 9569 9572 Pac +T318 CL:0000017 9574 9587 spermatocytes +T319 GO:0005634 9589 9596 Nuclear +T320 PR:000016672 9647 9653 Trip13 +T321 NCBITaxon:10088 9661 9665 Mice +T322 PR:000016672 9694 9700 TRIP13 +T323 NCBITaxon:40674 9704 9711 mammals +T324 NCBITaxon:10088 9726 9730 mice +T325 SO:0000704 9738 9742 gene +T326 SO:0001023 9758 9764 allele +T327 PR:000016672 9766 9772 Trip13 +T328 PR:000016672 9806 9812 Trip13 +T329 PR:000016672 9960 9966 Trip13 +T330 PR:000016672 9975 9981 Trip13 +T331 PR:000016672 9994 10000 Trip13 +T332 NCBITaxon:10088 10030 10034 mice +T333 GO:0016265 10040 10044 died +T334 PR:000016672 10156 10162 Trip13 +T335 NCBITaxon:33208 10168 10175 animals +T336 UBERON:0002415 10327 10332 tails +T337 PR:000016672 10392 10398 Trip13 +T338 NCBITaxon:10088 10406 10410 Mice +T339 UBERON:0002415 10467 10471 tail +T340 UBERON:0002415 10599 10603 tail +T341 NCBITaxon:10088 10631 10636 mouse +T342 PR:000016672 10680 10686 Trip13 +T343 UBERON:0000473 10700 10706 testes +T344 PR:000016672 10801 10807 Trip13 +T345 UBERON:0000473 10808 10814 testes +T346 UBERON:0000025 10842 10849 tubules +T347 GO:0007283 10873 10888 spermatogenesis +T348 GO:0000239 10894 10903 pachytene +T349 CL:0000017 10904 10917 spermatocytes +T350 UBERON:0000025 10933 10940 tubules +T351 UBERON:0000025 11034 11041 tubules +T352 UBERON:0000025 11048 11055 tubules +T353 GO:0000239 11064 11073 pachytene +T354 CL:0000017 11074 11086 spermatocyes +T355 PR:000016672 11179 11185 Trip13 +T356 GO:0010467 11188 11198 expression +T357 GO:0008380 11244 11251 spliced +T358 SO:0000673 11252 11263 transcripts +T359 UBERON:0000473 11267 11273 testes +T360 SO:0000704 11349 11353 gene +T361 GO:0008380 11361 11367 splice +T362 GO:0042571 11420 11428 antibody +T363 SO:0000147 11465 11469 exon +T364 UBERON:0000473 11529 11535 testes +T365 UBERON:0000473 11674 11680 testes +T366 PR:000016672 11790 11796 TRIP13 +T367 PR:000016672 11820 11826 Trip13 +T368 PR:000016672 11898 11904 Trip13 +T369 SO:0001023 11911 11917 allele +T370 CL:0000586 11961 11970 germ cell +T371 PR:000016672 11986 11992 TRIP13 +T372 GO:0010467 11996 12005 expressed +T373 GO:0010467 12030 12040 expression +T374 UBERON:0000473 12093 12099 testis +T375 PR:000016672 12132 12138 TRIP13 +T376 NCBITaxon:9031 12158 12165 chicken +T377 GO:0042571 12178 12186 antibody +T378 UBERON:0000473 12260 12266 testes +T379 CL:0000020 12279 12292 spermatogonia +T380 GO:0000237 12297 12306 leptotene +T381 CL:0000017 12307 12320 spermatocytes +T382 GO:0000238 12334 12342 Zygotene +T383 GO:0000239 12343 12352 pachytene +T384 CL:0000017 12353 12366 spermatocytes +T385 GO:0000239 12435 12444 pachytene +T386 CL:0000017 12445 12458 spermatocytes +T387 PR:000016672 12460 12466 TRIP13 +T388 GO:0005634 12482 12489 nuclear +T389 GO:0005634 12531 12537 nuclei +T390 UBERON:0001343 12548 12568 seminiferous tubules +T391 GO:0005634 12604 12611 nuclear +T392 PR:000016672 12626 12632 TRIP13 +T393 GO:0007126 12651 12658 meiotic +T394 CL:0000017 12704 12716 spermatocyte +T395 GO:0005634 12717 12723 nuclei +T396 GO:0005634 12757 12764 nuclear +T397 GO:0000795 12811 12813 SC +T398 GO:0000800 12835 12848 axial element +T399 PR:000015865 12857 12862 SYCP3 +T400 GO:0098762 12871 12887 meiotic substage +T401 PR:000016672 12901 12907 TRIP13 +T402 GO:0007126 12947 12954 meiotic +T403 GO:0005634 12955 12961 nuclei +T404 http://purl.obolibrary.org/obo/MONDO_0005047 12964 12975 Infertility +T405 GO:0007126 12983 12990 Meiotic +T406 PR:000016672 13005 13011 TRIP13 +T407 UBERON:0000991 13069 13075 gonads +T408 http://purl.obolibrary.org/obo/MONDO_0005047 13119 13126 sterile +T409 UBERON:0000992 13128 13135 Ovaries +T410 UBERON:0007023 13139 13144 adult +T411 PR:000016672 13145 13151 Trip13 +T412 CL:0000023 13254 13260 oocyte +T413 GO:0009790 13283 13296 embryogenesis +T414 GO:0007567 13313 13318 natal +T415 GO:0007567 13346 13352 partum +T416 UBERON:0000992 13353 13360 ovaries +T417 CL:0000023 13435 13442 oocytes +T418 CL:0000023 13496 13503 oocytes +T419 CL:0000023 13574 13581 oocytes +T420 GO:0000239 13587 13596 pachytene +T421 PR:000016672 13625 13631 Trip13 +T422 UBERON:0000922 13637 13646 embryonic +T423 UBERON:0000992 13647 13654 ovaries +T424 CL:0000023 13695 13702 oocytes +T425 GO:0000239 13737 13746 pachynema +T426 UBERON:0000991 13792 13798 Gonads +T427 CHEBI:51686 13808 13819 hematoxylin +T428 UBERON:0000473 13853 13859 Testes +T429 UBERON:0000992 13936 13941 ovary +T430 PR:000016672 13948 13954 Trip13 +T431 UBERON:0000992 13969 13974 ovary +T432 CL:0000023 14014 14021 oocytes +T433 PR:000016672 14028 14034 Trip13 +T434 UBERON:0000992 14055 14060 ovary +T435 CL:0000023 14078 14085 oocytes +T436 PR:000016672 14119 14125 Trip13 +T437 UBERON:0000992 14139 14144 ovary +T438 CL:0000023 14170 14177 oocytes +T439 UBERON:0000473 14246 14252 testis +T440 PR:000016672 14259 14265 Trip13 +T441 UBERON:0000473 14271 14277 testis +T442 GO:0000239 14291 14300 pachytene +T443 PR:000016672 14314 14320 Trip13 +T444 UBERON:0000473 14335 14341 testis +T445 GO:0007126 14356 14363 meiotic +T446 CL:0000018 14364 14374 spermatids +T447 PR:000015553 14390 14395 Spo11 +T448 UBERON:0000473 14399 14405 testis +T449 UBERON:0000025 14409 14415 tubule +T450 CL:0000017 14421 14434 spermatocytes +T451 GO:0000237 14438 14447 leptotene +T452 GO:0000238 14448 14456 zygotene +T453 UBERON:0000025 14487 14494 tubules +T454 CL:0000017 14510 14523 spermatocytes +T455 PR:000015553 14615 14620 Spo11 +T456 PR:000016672 14624 14630 Trip13 +T457 UBERON:0000473 14636 14642 testis +T458 UBERON:0000025 14697 14703 tubule +T459 GO:0000237 14709 14718 leptotene +T460 GO:0000238 14719 14727 zygotene +T461 CL:0000017 14728 14741 spermatocytes +T462 PR:000031227 14748 14752 Mei1 +T463 PR:000016672 14756 14762 Trip13 +T464 UBERON:0000473 14767 14773 testis +T465 PR:000031227 14837 14841 Mei1 +T466 PR:000016672 14845 14851 Trip13 +T467 UBERON:0000473 14857 14863 testis +T468 PR:000013858 14870 14874 Rec8 +T469 PR:000013858 14879 14883 Rec8 +T470 PR:000016672 14888 14894 Trip13 +T471 UBERON:0000473 14899 14905 testis +T472 PR:000013858 14911 14915 Rec8 +T473 SO:0001023 14920 14926 allele +T474 PR:000013858 15009 15013 Rec8 +T475 PR:000013858 15018 15022 Rec8 +T476 PR:000016672 15027 15033 Trip13 +T477 UBERON:0000473 15039 15045 testis +T478 PR:000006536 15052 15056 Dmc1 +T479 PR:000016672 15060 15066 Trip13 +T480 UBERON:0000473 15072 15078 testis +T481 PR:000015553 15085 15090 Spo11 +T482 PR:000016672 15094 15100 Trip13 +T483 UBERON:0000992 15114 15119 ovary +T484 PR:000015553 15183 15188 Spo11 +T485 PR:000016672 15192 15198 Trip13 +T486 UBERON:0000992 15213 15218 ovary +T487 PR:000031227 15225 15229 Mei1 +T488 PR:000016672 15233 15239 Trip13 +T489 UBERON:0000992 15253 15258 ovary +T490 PR:000031227 15322 15326 Mei1 +T491 PR:000016672 15330 15336 Trip13 +T492 UBERON:0000992 15351 15356 ovary +T493 PR:000013858 15363 15367 Rec8 +T494 PR:000013858 15372 15376 Rec8 +T495 PR:000016672 15381 15387 Trip13 +T496 UBERON:0000992 15401 15406 ovary +T497 PR:000013858 15470 15474 Rec8 +T498 PR:000013858 15479 15483 Rec8 +T499 PR:000016672 15488 15494 Trip13 +T500 UBERON:0000992 15509 15514 ovary +T501 UBERON:0000473 15549 15555 testes +T502 GO:0007126 15579 15586 meiotic +T503 UBERON:0001343 15635 15655 seminiferous tubules +T504 UBERON:0001343 15703 15723 seminiferous tubules +T505 CL:0000017 15744 15757 spermatocytes +T506 GO:0000785 15773 15782 chromatin +T507 GO:0000239 15801 15810 pachynema +T508 GO:0007283 15851 15864 spermatogenic +T509 GO:0000239 15914 15923 pachytene +T510 UBERON:0007023 16016 16021 adult +T511 UBERON:0001343 16022 16042 seminiferous tubules +T512 GO:0007126 16057 16064 meiotic +T513 CL:0000018 16065 16075 spermatids +T514 UBERON:0001301 16115 16125 epididymal +T515 CL:0000019 16126 16131 sperm +T516 GO:0007126 16147 16154 meiotic +T517 NCBITaxon:6239 16194 16204 C. elegans +T518 PR:P38126 16227 16231 Pch2 +T519 CL:0002369 16259 16264 spore +T520 GO:0030435 16259 16264;16272 16283 spore ... development +T521 CL:0000300 16265 16271 gamete +T522 GO:0007276 16265 16283 gamete development +T523 PR:000016672 16292 16298 TRIP13 +T524 GO:0007129 16327 16357 Homologous Chromosome Synapsis +T525 GO:0006281 16384 16392 repaired +T526 GO:0000239 16401 16410 Pachynema +T527 GO:0007126 16449 16456 meiotic +T528 PR:000016672 16472 16478 Trip13 +T529 CL:0000017 16484 16497 spermatocytes +T530 PR:000015865 16539 16544 SYCP3 +T531 PR:000015862 16549 16554 SYCP1 +T532 GO:0000800 16574 16579;16588 16596 axial ... elements +T533 GO:0000800 16580 16596 lateral elements +T534 GO:0000802 16601 16621 transverse filaments +T535 GO:0000795 16644 16664 synaptonemal complex +T536 GO:0000795 16666 16668 SC +T537 GO:0000239 16671 16680 Pachytene +T538 CL:0000017 16681 16693 spermatocyte +T539 GO:0005634 16694 16700 nuclei +T540 UBERON:0000473 16726 16732 testes +T541 GO:0000795 16755 16757 SC +T542 GO:0007129 16783 16806 synapsis of chromosomes +T543 PR:000015862 16834 16839 SYCP1 +T544 PR:000015865 16844 16849 SYCP3 +T545 GO:0030849 16880 16889 autosomes +T546 GO:0000805 16921 16922;16929 16940 X ... chromosomes +T547 GO:0000806 16927 16940 Y chromosomes +T548 GO:0007129 16955 16963 synapsed +T549 GO:0007567 17026 17032 partum +T550 CL:0000017 17041 17054 spermatocytes +T551 GO:0007129 17066 17074 synaptic +T552 GO:0007129 17090 17098 synapsed +T553 GO:0007567 17236 17241 natal +T554 GO:0007283 17242 17257 spermatogenesis +T555 PR:000016672 17371 17377 Trip13 +T556 CL:0000017 17383 17396 spermatocytes +T557 GO:0000239 17409 17418 pachynema +T558 GO:0000795 17433 17435 SC +T559 CL:0000023 17455 17462 oocytes +T560 GO:0007567 17490 17495 birth +T561 GO:0006281 17517 17527 DNA repair +T562 GO:0006281 17565 17573 repaired +T563 GO:0007126 17608 17615 meiotic +T564 GO:0000239 17683 17692 Pachytene +T565 CL:0000017 17693 17705 Spermatocyte +T566 GO:0042571 17784 17794 antibodies +T567 CHEBI:51217 17799 17811 fluorophores +T568 PR:000016672 17905 17911 Trip13 +T569 PR:000016672 17920 17926 Trip13 +T570 CL:0000017 18000 18013 spermatocytes +T571 GO:0000239 18029 18038 pachytene +T572 GO:0005634 18039 18046 nucleus +T573 GO:0007129 18057 18065 synapsis +T574 PR:000015862 18076 18081 SYCP1 +T575 PR:000015865 18082 18087 SYCP3 +T576 CL:0000017 18118 18131 Spermatocytes +T577 GO:0005634 18132 18139 nucleus +T578 GO:0007567 18156 18162 partum +T579 GO:0007129 18172 18180 synapsed +T580 GO:0000239 18319 18328 pachytene +T581 CL:0000017 18329 18342 spermatocytes +T582 PR:000004759 18348 18351 BLM +T583 GO:0007129 18372 18380 synapsed +T584 GO:0000239 18381 18390 pachytene +T585 PR:000013672 18422 18427 RAD51 +T586 GO:0051324 18464 18472 prophase +T587 GO:0030849 18489 18498 autosomes +T588 GO:0000239 18512 18521 pachytene +T589 GO:0005634 18522 18528 nuclei +T590 GO:0001741 18570 18577 XY body +T591 PR:000013672 18592 18597 RAD51 +T592 GO:0007129 18614 18622 synapsed +T593 PR:000008418 18657 18661 H2AX +T594 GO:0001741 18699 18706 XY body +T595 GO:0001741 18782 18789 XY body +T596 GO:0030849 18810 18819 autosomal +T597 PR:000008418 18820 18824 H2AX +T598 GO:0000239 18882 18891 pachytene +T599 CL:0000017 18892 18905 spermatocytes +T600 PR:000016548 18907 18913 TOPBP1 +T601 GO:0001741 18939 18946 XY body +T602 GO:0000803 19051 19066 sex chromosomes +T603 GO:0005662 19132 19135 RPA +T604 GO:0007129 19151 19159 synapsed +T605 PR:000010443 19225 19229 MLH3 +T606 GO:0000795 19238 19241 SCs +T607 GO:0000239 19259 19268 pachytene +T608 CL:0000017 19269 19282 spermatocytes +T609 PR:000013672 19284 19289 RAD51 +T610 PR:000013672 19354 19359 RAD51 +T611 GO:0000795 19380 19383 SCs +T612 GO:0000239 19394 19403 pachytene +T613 GO:0005634 19404 19410 nuclei +T614 PR:000010442 19438 19442 MLH1 +T615 GO:0007126 19563 19570 meiotic +T616 GO:0007126 19591 19598 meiotic +T617 GO:0007129 19678 19686 synapsis +T618 PR:000016672 19705 19711 Trip13 +T619 CL:0000017 19717 19730 spermatocytes +T620 GO:0000237 19799 19808 leptonema +T621 GO:0007126 19862 19873 meiotically +T622 PR:000013672 19893 19898 RAD51 +T623 PR:000006536 19906 19910 DMC1 +T624 GO:0005714 19926 19953 early recombination nodules +T625 GO:0005714 19955 19959 ERNs +T626 PR:000016672 19999 20005 Trip13 +T627 GO:0000238 20011 20019 zygotene +T628 CL:0000017 20020 20033 spermatocytes +T629 PR:000013672 20062 20067 RAD51 +T630 GO:0042571 20068 20076 antibody +T631 PR:000006536 20095 20099 DMC1 +T632 GO:0000725 20118 20140 recombinational repair +T633 GO:0006302 20134 20148 repair of DSBs +T634 GO:0008278 20167 20182 cohesin complex +T635 GO:0007129 20240 20248 synaptic +T636 GO:0007126 20329 20336 meiosis +T637 PR:000015706 20355 20360 STAG3 +T638 PR:000013858 20386 20390 REC8 +T639 PR:P38126 20425 20429 PCH2 +T640 PR:000016236 20544 20548 TRF2 +T641 GO:0043234 20567 20582 protein complex +T642 GO:0007129 20685 20693 synapsed +T643 GO:0007129 20713 20721 synaptic +T644 GO:0006302 20775 20785 DSB repair +T645 GO:0000239 20805 20814 pachynema +T646 CL:0000017 20838 20850 spermatocyte +T647 GO:0005634 20851 20857 nuclei +T648 GO:0042571 20863 20873 antibodies +T649 CHEBI:36357 20882 20891 molecules +T650 PR:000016672 20948 20954 Trip13 +T651 PR:000004759 20980 20983 BLM +T652 PR:000013672 21013 21018 RAD51 +T653 PR:000006536 21019 21023 DMC1 +T654 PR:000016548 21074 21080 TOPBP1 +T655 GO:0007129 21128 21136 synapsed +T656 PR:000013672 21154 21159 RAD51 +T657 PR:000006536 21160 21164 DMC1 +T658 GO:0000239 21173 21182 pachytene +T659 CL:0000017 21183 21196 spermatocytes +T660 GO:0001741 21285 21292 XY body +T661 GO:0000238 21317 21325 zygonema +T662 PR:000016548 21368 21374 TOPBP1 +T663 GO:0000077 21380 21401 DNA damage–checkpoint +T664 PR:000004427 21422 21425 ATM +T665 PR:000004499 21458 21461 ATR +T666 GO:0007129 21508 21516 synapsed +T667 GO:0007126 21528 21535 meiotic +T668 PR:000004759 21557 21560 BLM +T669 GO:0005662 21607 21610 RPA +T670 PR:000010668 21615 21619 MSH4 +T671 GO:0035825 21691 21701 crossovers +T672 GO:0035825 21703 21705 CO +T673 GO:0005662 21755 21758 RPA +T674 GO:0007129 21831 21840 synapsing +T675 GO:0007126 21841 21848 meiotic +T676 GO:0000239 21888 21897 pachynema +T677 SO:0000297 21926 21933 D-loops +T678 GO:0005662 21971 21974 RPA +T679 GO:0000239 21993 22002 pachytene +T680 GO:0006281 22069 22077 repaired +T681 GO:0000239 22137 22146 pachynema +T682 GO:0000077 22162 22183 DNA damage checkpoint +T683 GO:0007126 22241 22248 meiotic +T684 GO:0000803 22249 22263 sex chromosome +T685 GO:0007126 22288 22295 meiotic +T686 GO:0006342 22296 22308;22318 22327 silencing of ... chromatin +T687 GO:0007129 22311 22317 paired +T688 GO:0000785 22318 22327 chromatin +T689 GO:0042571 22358 22368 antibodies +T690 GO:0006302 22381 22391 DSB repair +T691 CHEBI:36357 22403 22412 molecules +T692 PR:000008418 22431 22435 H2AX +T693 PR:000004499 22489 22492 ATR +T694 PR:000004427 22498 22501 ATM +T695 GO:0007129 22546 22554 synapsed +T696 GO:0007129 22604 22612 synapsis +T697 PR:000016672 22632 22638 Trip13 +T698 GO:0006281 22661 22671 DNA repair +T699 GO:0006281 22719 22729 DNA repair +T700 GO:0006342 22742 22767 transcriptional silencing +T701 PR:000016672 22813 22819 Trip13 +T702 GO:0000239 22825 22834 pachytene +T703 CL:0000017 22835 22848 spermatocytes +T704 GO:0006281 22870 22880 DNA repair +T705 UBERON:0000473 22894 22900 testis +T706 GO:0007126 22938 22945 meiotic +T707 GO:0000240 22988 22997 diplotene +T708 GO:0005634 22998 23004 nuclei +T709 GO:0030849 23017 23026 autosomal +T710 PR:000013672 23027 23032 RAD51 +T711 PR:000006536 23033 23037 DMC1 +T712 GO:0007132 23075 23086 metaphase I +T713 PR:000016672 23137 23143 Trip13 +T714 GO:0000240 23180 23189 diplotene +T715 GO:0007132 23194 23205 metaphase I +T716 CL:0000017 23206 23219 spermatocytes +T717 PR:000016672 23273 23279 TRIP13 +T718 GO:0035825 23282 23284 CO +T719 PR:000016672 23338 23344 TRIP13 +T720 PR:000004759 23365 23368 BLM +T721 PR:000016672 23372 23378 Trip13 +T722 CL:0000017 23384 23396 spermatocyte +T723 GO:0006281 23450 23458 repaired +T724 GO:0000725 23501 23523 recombinational repair +T725 GO:0035825 23543 23559 CO recombination +T726 GO:0006298 23618 23633 mismatch repair +T727 PR:000010442 23643 23647 MLH1 +T728 PR:000010443 23652 23656 MLH3 +T729 GO:0000239 23708 23717 pachynema +T730 GO:0005712 23744 23753 chiasmata +T731 PR:000010442 23775 23779 MLH1 +T732 PR:000010443 23879 23883 MLH3 +T733 GO:0000239 23923 23932 pachytene +T734 PR:000010442 23964 23968 MLH1 +T735 PR:000016672 23994 24000 Trip13 +T736 GO:0000239 24006 24015 pachytene +T737 GO:0005634 24016 24022 nuclei +T738 GO:0006281 24034 24040 repair +T739 GO:0006302 24077 24087 DSB repair +T740 GO:0000239 24125 24134 pachytene +T741 GO:0005634 24135 24141 nuclei +T742 PR:000010442 24146 24150 MLH1 +T743 PR:000010442 24184 24188 MLH1 +T744 GO:0006281 24234 24242 repaired +T745 PR:000010442 24305 24309 MLH1 +T746 PR:000013672 24314 24319 RAD51 +T747 PR:000006536 24320 24324 DMC1 +T748 PR:000010442 24326 24330 MLH1 +T749 PR:000013672 24393 24398 RAD51 +T750 PR:000006536 24399 24403 DMC1 +T751 PR:000010442 24454 24458 MLH1 +T752 PR:000016672 24469 24475 Trip13 +T753 GO:0000239 24481 24490 pachytene +T754 CL:0000017 24491 24504 spermatocytes +T755 GO:0035825 24515 24517 CO +T756 GO:0007128 24615 24625 prophase I +T757 UBERON:0000473 24638 24648 testicular +T758 PR:000016672 24691 24697 Trip13 +T759 PR:000006536 24708 24712 Dmc1 +T760 NCBITaxon:10088 24716 24720 mice +T761 CHEBI:35222 24750 24759 inhibitor +T762 CHEBI:44658 24760 24772 okadaic acid +T763 CHEBI:44658 24774 24776 OA +T764 GO:0070194 24803 24817;24822 24824 degradation of ... SC +T765 GO:0000795 24822 24824 SC +T766 GO:0030261 24826 24849 chromosome condensation +T767 GO:0007132 24880 24891 metaphase I +T768 GO:0051323 24906 24915 metaphase +T769 PR:000006536 24978 24982 Dmc1 +T770 GO:0000793 25010 25031 condensed chromosomes +T771 PR:000016672 25052 25058 Trip13 +T772 PR:000010442 25103 25107 MLH1 +T773 PR:000016672 25118 25124 Trip13 +T774 GO:0000239 25130 25139 pachytene +T775 CL:0000017 25140 25153 spermatocytes +T776 GO:0035825 25203 25206 COs +T777 UBERON:0000473 25254 25260 testes +T778 GO:0051323 25307 25317 metaphases +T779 PR:000006536 25323 25327 Dmc1 +T780 NCBITaxon:10088 25331 25335 mice +T781 CL:0000020 25346 25359 spermatogonia +T782 CL:0000017 25365 25378 spermatocytes +T783 PR:000016672 25381 25387 TRIP13 +T784 GO:0007126 25418 25425 Meiotic +T785 GO:0007129 25468 25476 Synapsis +T786 PR:000016672 25494 25500 TRIP13 +T787 GO:0006915 25521 25530 apoptosis +T788 GO:0007129 25545 25553 synapsis +T789 NCBITaxon:6239 25560 25570 C. elegans +T790 NCBITaxon:10088 25584 25588 mice +T791 PR:000015553 25617 25622 Spo11 +T792 PR:000016672 25627 25633 Trip13 +T793 PR:000015553 25635 25640 SPO11 +T794 SO:0000704 25698 25709 genetically +T795 GO:0000237 25732 25741 leptonema +T796 NCBITaxon:1 25750 25759 organisms +T797 NCBITaxon:10088 25771 25775 mice +T798 NCBITaxon:6239 25785 25795 C. elegans +T799 PR:Q22236 25797 25803 spo-11 +T800 CL:0000300 25811 25818 gametes +T801 GO:0007129 25835 25843 synapsis +T802 PR:P38126 25860 25865 PCH-2 +T803 GO:0006915 25876 25885 apoptosis +T804 GO:0000239 25889 25898 pachynema +T805 NCBITaxon:10088 25907 25911 mice +T806 PR:000015553 25913 25918 Spo11 +T807 CL:0000017 25922 25935 spermatocytes +T808 GO:0007129 25962 25992 homologous chromosome synapsis +T809 GO:0000238 26063 26071 zygotene +T810 GO:0000239 26072 26081 pachytene +T811 CL:0000017 26106 26119 Spermatocytes +T812 PR:000016672 26123 26129 Trip13 +T813 PR:000015553 26135 26140 Spo11 +T814 UBERON:0000473 26144 26150 testes +T815 GO:0016265 26204 26209 death +T816 GO:0005819 26237 26244 spindle +T817 GO:0031577 26237 26255 spindle checkpoint +T818 GO:0005712 26273 26282 chiasmate +T819 CL:0000017 26283 26296 spermatocytes +T820 GO:0007132 26328 26339 metaphase I +T821 CL:0000017 26340 26353 spermatocytes +T822 GO:0007126 26361 26368 meiotic +T823 CL:0000018 26369 26379 spermatids +T824 UBERON:0000473 26389 26395 testes +T825 PR:000016672 26418 26424 Trip13 +T826 GO:0007129 26481 26489 synapsis +T827 PR:000016672 26493 26499 Trip13 +T828 GO:0000239 26505 26514 pachytene +T829 CL:0000017 26515 26528 spermatocytes +T830 PR:000015553 26551 26556 SPO11 +T831 GO:0000237 26573 26582 leptonema +T832 GO:0007129 26674 26693 chromosome synapsis +T833 CL:0000017 26711 26724 spermatocytes +T834 PR:000015553 26752 26757 Spo11 +T835 NCBITaxon:10088 26831 26835 mice +T836 PR:000031227 26850 26854 Mei1 +T837 NCBITaxon:7742 26858 26868 vertebrate +T838 SO:0000704 26878 26882 gene +T839 GO:0007129 26919 26938 chromosome synapsis +T840 PR:000016672 27053 27059 Trip13 +T841 GO:0042571 27143 27153 antibodies +T842 CHEBI:51217 27158 27170 fluorophores +T843 GO:0005634 27327 27333 nuclei +T844 PR:P38126 27358 27362 PCH2 +T845 GO:0000239 27378 27387 pachytene +T846 GO:0007129 27406 27414 synaptic +T847 PR:P31111 27423 27427 zip1 +T848 PR:P53061 27432 27436 zip2 +T849 NCBITaxon:10088 27451 27456 mouse +T850 PR:000015862 27457 27462 SYCP1 +T851 PR:000015862 27514 27519 Sycp1 +T852 CL:0000017 27527 27540 spermatocytes +T853 PR:000016672 27583 27589 Trip13 +T854 PR:000015862 27650 27655 Sycp1 +T855 PR:P31111 27723 27727 zip1 +T856 GO:0007129 27793 27812 chromosome synapsis +T857 GO:0007129 27893 27901 synapsis +T858 MOP:0000629 27902 27916 polymerization +T859 PR:000013858 27994 27998 Rec8 +T860 SO:0001023 27999 28005 allele +T861 PR:000013858 28007 28011 Rec8 +T862 PR:000013858 28032 28036 Rec8 +T863 GO:0007126 28040 28047 Meiotic +T864 PR:000013858 28063 28067 Rec8 +T865 CL:0000017 28075 28088 spermatocytes +T866 GO:0007129 28126 28147 interhomolog synaptic +T867 GO:0006302 28181 28191 DSB repair +T868 GO:0007129 28213 28234 interhomolog synapsis +T869 GO:0007129 28280 28287 synapse +T870 PR:000015862 28305 28310 SYCP1 +T871 PR:000013858 28329 28333 Rec8 +T872 GO:0000240 28361 28370 diplonema +T873 GO:0007132 28374 28385 metaphase I +T874 PR:000013858 28425 28429 Rec8 +T875 PR:000016672 28446 28452 Trip13 +T876 PR:000015553 28464 28469 Spo11 +T877 PR:000031227 28474 28478 Mei1 +T878 UBERON:0000473 28505 28511 testes +T879 PR:000013858 28531 28535 REC8 +T880 PR:000016672 28540 28546 TRIP13 +T881 PR:000013858 28561 28565 Rec8 +T882 GO:0007132 28609 28620 metaphase I +T883 PR:000016672 28636 28642 Trip13 +T884 NCBITaxon:10088 28648 28652 mice +T885 GO:0007129 28742 28772 homologous chromosome synapsis +T886 PR:000013858 28785 28789 Rec8 +T887 PR:000013858 28797 28801 Rec8 +T888 PR:000016672 28805 28811 Trip13 +T889 CL:0000017 28817 28830 spermatocytes +T890 PR:000013858 28859 28863 Rec8 +T891 PR:P38126 28969 28973 PCH2 +T892 GO:0007126 29020 29027 meiotic +T893 GO:0007126 29064 29071 meiosis +T894 PR:000023696 29081 29085 RecA +T895 SO:0000853 29086 29093 homolog +T896 PR:000006536 29094 29098 DMC1 +T897 NCBITaxon:10088 29140 29144 mice +T898 NCBITaxon:33208 29161 29168 animals +T899 PR:000016672 29187 29193 Trip13 +T900 PR:000006536 29198 29202 Dmc1 +T901 PR:000006536 29210 29214 Dmc1 +T902 NCBITaxon:10088 29218 29222 mice +T903 CL:0000017 29233 29246 spermatocytes +T904 GO:0007126 29255 29262 meiotic +T905 GO:0006302 29285 29295 DSB repair +T906 GO:0007129 29307 29326 chromosome synapsis +T907 GO:0007283 29333 29348 spermatogenesis +T908 PR:000006536 29352 29356 Dmc1 +T909 PR:000016672 29360 29366 Trip13 +T910 UBERON:0000473 29372 29378 testes +T911 CL:0000017 29421 29434 spermatocytes +T912 GO:0000785 29445 29454 chromatin +T913 GO:0000238 29473 29481 zygonema +T914 GO:0000239 29482 29491 pachynema +T915 PR:000006536 29552 29556 Dmc1 +T916 PR:000006536 29564 29568 Dmc1 +T917 PR:000016672 29572 29578 Trip13 +T918 GO:0007129 29611 29619 synapsis +T919 PR:000016672 29632 29638 Trip13 +T920 PR:000013672 29695 29700 RAD51 +T921 PR:000006536 29701 29705 DMC1 +T922 PR:000008418 29730 29734 H2AX +T923 PR:000006536 29774 29778 Dmc1 +T924 PR:000016672 29795 29801 Trip13 +T925 UBERON:0000992 29838 29845 ovaries +T926 PR:000006536 29860 29864 Dmc1 +T927 PR:000016672 29872 29878 Trip13 +T928 GO:0007126 29920 29927 Meiotic +T929 PR:000016672 29939 29945 Trip13 +T930 CL:0000023 29951 29958 Oocytes +T931 PR:000016672 30062 30068 Trip13 +T932 PR:000031227 30083 30087 Mei1 +T933 PR:000016672 30091 30097 Trip13 +T934 PR:000015553 30107 30112 Spo11 +T935 PR:000016672 30116 30122 Trip13 +T936 UBERON:0000992 30140 30147 ovaries +T937 PR:000031227 30186 30190 Mei1 +T938 PR:000015553 30195 30200 Spo11 +T939 PR:000015553 30238 30243 Spo11 +T940 PR:000031227 30248 30252 Mei1 +T941 PR:000016672 30270 30276 Trip13 +T942 GO:0048477 30280 30289 oogenesis +T943 PR:000006536 30311 30315 Dmc1 +T944 CL:0000023 30348 30354 oocyte +T945 PR:000016672 30363 30369 Trip13 +T946 GO:0007126 30504 30511 meiotic +T947 PR:000016672 30522 30528 Trip13 +T948 NCBITaxon:10088 30536 30540 mice +T949 GO:0006302 30562 30572 DSB repair +T950 UBERON:0000992 30587 30594 ovaries +T951 PR:000013858 30598 30602 Rec8 +T952 PR:000016672 30603 30609 Trip13 +T953 CL:0000023 30640 30647 oocytes +T954 SO:0000704 30726 30733 Genetic +T955 NCBITaxon:4932 30749 30762 S. cerevisiae +T956 GO:0051598 30790 30810 pachytene checkpoint +T957 GO:0065007 30811 30819 monitors +T958 GO:0000725 30836 30851;30856 30862 recombinational ... repair +T959 GO:0006302 30852 30862 DSB repair +T960 GO:0007129 30867 30875 synapsis +T961 GO:0000077 30925 30942 repair checkpoint +T962 PR:000013665 30946 30951 RAD17 +T963 PR:P46946 30952 30956 SAE2 +T964 GO:0007129 30978 30986 synapsis +T965 GO:0000075 30987 30997 checkpoint +T966 PR:P38126 31001 31005 PCH2 +T967 PR:P31111 31006 31010 ZIP1 +T968 SO:0000704 31041 31046 genes +T969 PR:P46946 31048 31052 SAE2 +T970 PR:P31111 31057 31061 ZIP1 +T971 NCBITaxon:40674 31080 31089 mammalian +T972 SO:0000855 31090 31099 orthologs +T973 PR:000015862 31110 31115 SYCP1 +T974 SO:0000855 31136 31144 ortholog +T975 PR:P31111 31148 31152 ZIP1 +T976 NCBITaxon:10088 31175 31180 mouse +T977 PR:000013665 31181 31186 RAD17 +T978 SO:0000855 31187 31195 ortholog +T979 PR:P06777 31197 31201 Rad1 +T980 UBERON:0000922 31221 31230 embryonic +T981 NCBITaxon:10088 31276 31281 mouse +T982 PR:P38126 31282 31286 Pch2 +T983 PR:000016672 31288 31294 Trip13 +T984 GO:0007129 31328 31336 synapsis +T985 GO:0000075 31337 31347 checkpoint +T986 NCBITaxon:6239 31351 31361 C. elegans +T987 NCBITaxon:40674 31446 31455 mammalian +T988 GO:0033313 31456 31474 meiotic checkpoint +T989 GO:0065007 31475 31482 control +T990 NCBITaxon:10088 31517 31521 mice +T991 GO:0007126 31535 31542 meiotic +T992 PR:000016672 31555 31561 TRIP13 +T993 GO:0051598 31634 31654 pachytene checkpoint +T994 GO:0065007 31655 31662 control +T995 GO:0035825 31740 31743 COs +T996 PR:000016672 31768 31774 Trip13 +T997 GO:0006302 31808 31814 repair +T998 GO:0007126 31933 31940 meiotic +T999 NCBITaxon:4932 31968 31981 S. cerevisiae +T1000 GO:0035825 31983 31985 CO +T1001 NCBITaxon:10088 32122 32126 Mice +T1002 GO:0007131 32159 32161;32173 32186 CO ... recombination +T1003 PR:000015553 32228 32233 SPO11 +T1004 MOP:0000780 32242 32248 breaks +T1005 GO:0035825 32263 32265 CO +T1006 PR:000010442 32283 32287 MLH1 +T1007 GO:0000239 32347 32356 pachynema +T1008 GO:0006302 32487 32497 DSB repair +T1009 GO:0007126 32515 32522 meiotic +T1010 PR:000013680 32542 32547 RAD54 +T1011 GO:0007126 32631 32638 meiosis +T1012 NCBITaxon:10088 32658 32662 mice +T1013 PR:000013680 32687 32692 RAD54 +T1014 CL:0000017 32703 32716 spermatocytes +T1015 PR:000013672 32749 32754 RAD51 +T1016 GO:0000239 32763 32772 pachytene +T1017 PR:000016672 32806 32812 TRIP13 +T1018 NCBITaxon:10088 32813 32817 mice +T1019 GO:0007126 32859 32866 meiotic +T1020 GO:0007114 32909 32916 budding +T1021 NCBITaxon:4892 32909 32922 budding yeast +T1022 PR:P38126 32988 32992 PCH2 +T1023 GO:0007126 33000 33007 meiotic +T1024 GO:0075317 33073 33088 ascus formation +T1025 GO:0007127 33299 33308 meiosis I +T1026 PR:000013665 33332 33337 RAD17 +T1027 PR:P46946 33338 33342 SAE2 +T1028 GO:0000075 33343 33353 checkpoint +T1029 GO:0065007 33359 33367 monitors +T1030 PR:000016672 33429 33435 TRIP13 +T1031 PR:P38126 33440 33444 Pch2 +T1032 GO:0007129 33483 33491 synapsis +T1033 PR:000016672 33502 33508 TRIP13 +T1034 CL:0000017 33519 33532 spermatocytes +T1035 PR:000015553 33580 33585 SPO11 +T1036 PR:000031227 33590 33594 MEI1 +T1037 PR:000016672 33613 33619 TRIP13 +T1038 GO:0035825 33721 33723 CO +T1039 GO:0006281 33724 33730 repair +T1040 CHEBI:44658 33782 33784 OA +T1041 PR:000016672 33848 33854 TRIP13 +T1042 PR:000016672 34008 34014 TRIP13 +T1043 GO:0032984 34031 34045;34073 34082 disassembly of ... complexes +T1044 GO:0000725 34050 34072 recombinational repair +T1045 GO:0032991 34073 34082 complexes +T1046 PR:000016672 34141 34147 Trip13 +T1047 GO:0000239 34153 34162 pachytene +T1048 PR:000016672 34185 34191 TRIP13 +T1049 SO:0000417 34216 34223 domains +T1050 PR:000022296 34251 34255 ClpA +T1051 GO:0043241 34282 34289;34305 34324 protein ... complex disassembly +T1052 GO:0032993 34293 34312 protein/DNA complex +T1053 GO:0032986 34293 34324 protein/DNA complex disassembly +T1054 GO:0007126 34391 34398 meiosis +T1055 PR:000016672 34406 34412 Trip13 +T1056 NCBITaxon:33208 34450 34457 animals +T1057 PR:000016672 34500 34506 TRIP13 +T1058 GO:0007126 34633 34640 meiosis +T1059 GO:0008219 34666 34676 cell death +T1060 PR:000016672 34680 34686 Trip13 +T1061 GO:0006302 34750 34760 DSB repair +T1062 GO:0007129 34774 34782 synapsis +T1063 CL:0000023 34833 34839 oocyte +T1064 GO:0007129 34892 34900 synapsis +T1065 CL:0000017 34914 34927 spermatocytes +T1066 UBERON:0007023 34931 34936 adult +T1067 UBERON:0000473 34937 34943 testes +T1068 GO:0007129 35030 35038 synapsis +T1069 PR:000006536 35052 35056 Dmc1 +T1070 PR:000016672 35079 35085 Trip13 +T1071 GO:0006281 35128 35136 repaired +T1072 NCBITaxon:40674 35170 35179 mammalian +T1073 GO:0051598 35180 35200 pachytene checkpoint +T1074 CL:0000023 35262 35269 oocytes +T1075 CL:0000017 35274 35287 spermatocytes +T1076 GO:0000077 35323 35333;35344 35354 DNA damage ... checkpoint +T1077 GO:0051598 35334 35354 pachytene checkpoint +T1078 GO:0007129 35380 35388 synapsis +T1079 GO:0000075 35389 35399 checkpoint +T1080 CHEBI:44658 35431 35433 OA +T1081 PR:000016672 35447 35453 Trip13 +T1082 CL:0000017 35459 35472 spermatocytes +T1083 GO:0007132 35496 35498 MI +T1084 GO:0000239 35560 35569 pachytene +T1085 CL:0000017 35570 35583 spermatocytes +T1086 CHEBI:4911 35645 35654 etoposide +T1087 GO:0000239 35714 35723 pachynema +T1088 GO:0000075 35733 35743 checkpoint +T1089 CHEBI:44658 35780 35782 OA +T1090 GO:0042148 35801 35816 strand invasion +T1091 PR:000016672 35828 35834 TRIP13 +T1092 CL:0000017 35845 35858 spermatocytes +T1093 PR:000016672 35868 35874 TRIP13 +T1094 NCBITaxon:10114 35926 35929 rat +T1095 UBERON:0002046 35930 35937 thyroid +T1096 PR:000016321 35930 35951 thyroid receptor beta +T1097 PR:000016321 35953 35957 THRB +T1098 PR:000016321 35995 35999 THRB +T1099 PR:000016672 36004 36010 TRIP13 +T1100 GO:0007126 36014 36021 meiosis +T1101 PR:000016321 36066 36070 THRB +T1102 CL:0000017 36117 36129 spermatocyte +T1103 GO:0005634 36130 36136 nuclei +T1104 GO:0001741 36162 36164;36171 36175 XY ... body +T1105 GO:0001741 36166 36169;36171 36175 sex ... body +T1106 GO:0005634 36224 36231 nuclear +T1107 GO:0000239 36252 36261 pachynema +T1108 GO:0000803 36276 36291 sex chromosomes +T1109 GO:0031507 36299 36318 heterochromatinized +T1110 GO:0006342 36323 36349 transcriptionally silenced +T1111 GO:0001741 36392 36399 XY body +T1112 CL:0000017 36431 36444 spermatocytes +T1113 GO:0001741 36482 36484 XY +T1114 GO:0031507 36485 36507 heterochromatinization +T1115 PR:000016321 36553 36557 THRB +T1116 NCBITaxon:10088 36567 36571 mice +T1117 PR:000016672 36637 36643 TRIP13 +T1118 PR:000016321 36661 36665 THRB +T1119 GO:0007126 36669 36676 meiosis +T1120 PR:P38126 36719 36723 PCH2 +T1121 SO:0000855 36724 36733 orthologs +T1122 NCBITaxon:2759 36749 36759 eukaryotic +T1123 PR:000016672 36840 36846 TRIP13 +T1124 GO:0000075 36864 36874 checkpoint +T1125 NCBITaxon:10088 36887 36891 mice +T1126 PR:000016672 36913 36919 TRIP13 +T1127 PR:P38126 36920 36924 PCH2 +T1128 NCBITaxon:3702 37014 37025 A. thaliana +T1129 GO:0033313 37052 37070 meiotic checkpoint +T1130 NCBITaxon:1 37138 37147 organisms +T1131 NCBITaxon:10088 37156 37160 mice +T1132 GO:0007114 37162 37169 budding +T1133 NCBITaxon:4892 37162 37175 budding yeast +T1134 NCBITaxon:7215 37188 37198 Drosophila +T1135 NCBITaxon:40674 37212 37221 mammalian +T1136 PR:000016672 37222 37228 TRIP13 +T1137 NCBITaxon:3701 37248 37259 Arabidopsis +T1138 PR:P38126 37260 37264 PCH2 +T1139 NCBITaxon:7147 37274 37277 fly +T1140 NCBITaxon:40674 37348 37357 mammalian +T1141 NCBITaxon:3193 37362 37367 plant +T1142 PR:P38126 37368 37372 PCH2 +T1143 PR:P38126 37499 37503 PCH2 +T1144 NCBITaxon:3193 37507 37513 plants +T1145 GO:0000075 37560 37570 checkpoint +T1146 NCBITaxon:33208 37634 37641 animals +T1147 NCBITaxon:4751 37643 37648 fungi +T1148 NCBITaxon:3193 37654 37660 plants +T1149 PR:P38126 37688 37692 PCH2 +T1150 GO:0000075 37781 37791 checkpoint +T1151 PR:P38126 37805 37809 PCH2 +T1152 GO:0000075 37855 37865 checkpoint +T1153 GO:0007114 37910 37917 budding +T1154 NCBITaxon:4892 37910 37923 budding yeast +T1155 PR:P38126 37976 37980 PCH2 +T1156 NCBITaxon:1 37990 37999 organisms +T1157 GO:0007129 38034 38042 synapsis +T1158 GO:0000075 38043 38053 checkpoint +T1159 NCBITaxon:10088 38062 38066 mice +T1160 PR:Q9SIH2 38115 38119 Dot1 +T1161 PR:Q04089 38121 38125 PCH1 +T1162 PR:000043452 38139 38146 histone +T1163 CHEBI:15358 38139 38146 histone +T1164 GO:0016458 38165 38174 silencing +T1165 GO:0000239 38203 38212 pachytene +T1166 PR:P31111 38223 38227 zip1 +T1167 PR:000006536 38232 38236 dmc1 +T1168 PR:000013680 38279 38284 RAD54 +T1169 GO:0000725 38294 38309;38314 38320 recombinational ... repair +T1170 GO:0006302 38310 38320 DSB repair +T1171 PR:Q9SIH2 38357 38361 DOT1 +T1172 PR:P38126 38379 38383 PCH2 +T1173 PR:000016672 38396 38402 TRIP13 +T1174 GO:0000075 38416 38426 checkpoint +T1175 NCBITaxon:10088 38439 38443 mice +T1176 NCBITaxon:40674 38466 38475 mammalian +T1177 PR:Q9SIH2 38476 38480 DOT1 +T1178 GO:0051598 38488 38508 pachytene checkpoint +T1179 PR:000003035 38578 38583 TRP53 +T1180 SO:0000853 38584 38591 homolog +T1181 PR:000016567 38592 38597 TRP63 +T1182 GO:0016265 38633 38638 death +T1183 CL:0000654 38657 38675 primordial oocytes +T1184 GO:0065007 38726 38736 monitoring +T1185 SO:0001026 38737 38743 genome +T1186 GO:0051598 38806 38826 pachytene checkpoint +T1187 GO:0033313 38897 38915 meiotic checkpoint +T1188 SO:0000704 38916 38921 genes +T1189 NCBITaxon:10088 38925 38929 mice +T1190 SO:0000704 38956 38961 genes +T1191 GO:0007067 38973 38980 mitotic +T1192 PR:P32641 39000 39005 RAD24 +T1193 NCBITaxon:40674 39064 39073 mammalian +T1194 GO:0051598 39074 39094 pachytene checkpoint +T1195 SO:0000855 39111 39120 orthologs +T1196 NCBITaxon:1 39147 39156 organisms +T1197 PR:000016672 39259 39265 Trip13 +T1198 PR:000016672 39273 39279 Trip13 +T1199 NCBITaxon:10088 39321 39326 Mouse +T1200 UBERON:0000479 39336 39342 Tissue +T1201 SO:0000112 39403 39410 primers +T1202 SO:0000112 39497 39504 primers +T1203 SO:0000147 39519 39524 exons +T1204 SO:0000028 39566 39568 bp +T1205 PR:000016672 39678 39684 Trip13 +T1206 NCBITaxon:10088 39695 39699 mice +T1207 NCBITaxon:10088 39706 39711 mouse +T1208 UBERON:0000922 39712 39721 embryonic +T1209 CL:0002322 39712 39731 embryonic stem cell +T1210 SO:0000704 39774 39778 gene +T1211 PR:000016672 39797 39803 Trip13 +T1212 SO:0000704 39874 39878 gene +T1213 SO:0000440 39888 39894 vector +T1214 SO:0001817 39956 39964 in-frame +T1215 SO:0000147 39987 39992 exons +T1216 SO:0000704 40008 40012 gene +T1217 CHEBI:7507 40067 40075 neomycin +T1218 SO:0000704 40104 40108 gene +T1219 SO:0001886 40133 40150 fusion transcript +T1220 SO:0000147 40162 40167 exons +T1221 PR:000016672 40175 40181 Trip13 +T1222 SO:0000366 40214 40228 insertion site +T1223 SO:0000188 40236 40242 intron +T1224 SO:0000112 40274 40280 primer +T1225 SO:0000704 40292 40296 gene +T1226 SO:0000440 40302 40308 vector +T1227 SO:0000112 40324 40330 primer +T1228 SO:0000188 40355 40361 intron +T1229 SO:0000704 40399 40403 gene +T1230 SO:0000366 40474 40488 insertion site +T1231 SO:0000028 40497 40499 bp +T1232 SO:0000188 40505 40511 intron +T1233 NCBITaxon:10088 40530 40534 mice +T1234 SO:0000112 40543 40550 primers +T1235 SO:0001023 40597 40604 alleles +T1236 PR:000016672 40608 40614 Trip13 +T1237 SO:0000112 40616 40622 primer +T1238 SO:0000112 40655 40661 primer +T1239 SO:0000112 40710 40716 primer +T1240 SO:0000112 40751 40758 Primers +T1241 SO:0000704 40825 40829 gene +T1242 SO:0000188 40856 40862 intron +T1243 SO:0000112 40866 40872 Primer +T1244 SO:0000112 40907 40914 Primers +T1245 SO:0000028 40937 40939 bp +T1246 SO:0001023 40962 40968 allele +T1247 SO:0000112 40970 40977 primers +T1248 SO:0000028 41000 41002 bp +T1249 SO:0001023 41026 41032 allele +T1250 UBERON:0007023 41281 41286 adult +T1251 UBERON:0000473 41287 41293 testes +T1252 GO:0001171 41387 41406 reverse-transcribed +T1253 PR:000016672 41479 41485 Trip13 +T1254 SO:0000010 41486 41500 protein-coding +T1255 SO:0000112 41529 41536 primers +T1256 SO:0000112 41707 41714 primers +T1257 PR:000010303 41734 41739 Med31 +T1258 NCBITaxon:9031 41995 42002 chicken +T1259 GO:0042571 42003 42013 antibodies +T1260 PR:000016672 42064 42070 TRIP13 +T1261 MOP:0000779 42093 42103 conjugated +T1262 NCBITaxon:9031 42132 42140 chickens +T1263 GO:0071760 42206 42209 IgY +T1264 UBERON:0007379 42228 42232 eggs +T1265 NCBITaxon:9031 42253 42260 Chicken +T1266 GO:0071760 42261 42264 IgY +T1267 GO:0071760 42318 42321 IgY +T1268 GO:0042571 42322 42332 antibodies +T1269 UBERON:0000473 42430 42436 testis +T1270 CHEBI:8984 42480 42483 SDS +T1271 CHEBI:53325 42524 42538 Nitrocellulose +T1272 NCBITaxon:9986 42627 42633 rabbit +T1273 NCBITaxon:9606 42639 42644 human +T1274 PR:000016672 42645 42651 TRIP13 +T1275 GO:0042571 42652 42660 antibody +T1276 CHEBI:60816 42746 42755 immunogen +T1277 SO:0000147 42829 42833 exon +T1278 NCBITaxon:9986 42904 42910 rabbit +T1279 NCBITaxon:9031 42916 42923 chicken +T1280 GO:0071735 42924 42927 IgG +T1281 NCBITaxon:3704 42928 42939 horseradish +T1282 CHEBI:64985 42951 42960 conjugate +T1283 UBERON:0000473 42996 43002 Testes +T1284 UBERON:0000992 43006 43013 ovaries +T1285 CHEBI:51686 43093 43104 hematoxylin +T1286 CHEBI:59132 43116 43123 Antigen +T1287 UBERON:0000473 43162 43168 testis +T1288 CL:0000023 43201 43207 Oocyte +T1289 CL:0000023 43290 43296 oocyte +T1290 GO:0005634 43320 43327 nucleus +T1291 CL:0000017 43397 43410 spermatocytes +T1292 CL:0000023 43415 43422 oocytes +T1293 GO:0005634 43573 43579 nuclei +T1294 GO:0098762 43594 43608 meiotic stages +T1295 PR:000015865 43680 43685 SYCP3 +T1296 PR:000015706 43689 43694 STAG3 +T1297 PR:000013672 43821 43826 RAD51 +T1298 GO:0005662 43830 43833 RPA +T1299 PR:000010442 44008 44012 MLH1 +T1300 PR:000010443 44016 44020 MLH3 +T1301 PR:000013672 44026 44031 RAD51 +T1302 PR:000015865 44047 44052 SYCP3 +T1303 PR:000015706 44056 44061 STAG3 +T1304 GO:0005634 44107 44113 Nuclei +T1305 PR:000010442 44157 44161 MLH1 +T1306 GO:0042571 44236 44246 antibodies +T1307 NCBITaxon:10088 44283 44288 mouse +T1308 PR:000015865 44294 44298 SCP3 +T1309 NCBITaxon:9986 44337 44343 rabbit +T1310 PR:000015862 44349 44354 SYCP1 +T1311 NCBITaxon:9986 44395 44401 rabbit +T1312 PR:000013858 44407 44411 REC8 +T1313 NCBITaxon:9986 44445 44451 rabbit +T1314 PR:000013672 44457 44462 RAD51 +T1315 GO:0042571 44487 44495 antibody +T1316 PR:000013672 44512 44517 RAD51 +T1317 PR:000006536 44522 44526 DMC1 +T1318 NCBITaxon:9986 44592 44598 rabbit +T1319 NCBITaxon:9986 44667 44673 rabbit +T1320 PR:000015706 44679 44684 STAG3 +T1321 NCBITaxon:9986 44723 44729 rabbit +T1322 PR:000010443 44735 44739 MLH3 +T1323 NCBITaxon:10088 44771 44776 mouse +T1324 NCBITaxon:9606 44782 44787 human +T1325 PR:000010442 44788 44792 MLH1 +T1326 NCBITaxon:9986 44847 44853 rabbit +T1327 PR:000016548 44859 44865 TopBP1 +T1328 NCBITaxon:10088 44901 44906 mouse +T1329 CHEBI:15358 44922 44929 histone +T1330 PR:000027547 44922 44933 histone H2A +T1331 NCBITaxon:9986 44966 44972 rabbit +T1332 PR:000016236 44978 44982 TRF2 +T1333 NCBITaxon:9986 45021 45027 rabbit +T1334 PR:000004759 45033 45036 BLM +T1335 GO:0042571 45082 45092 antibodies +T1336 MOP:0000779 45093 45103 conjugated +T1337 CHEBI:52661 45116 45131 Alexa Fluor 488 +T1338 CHEBI:51248 45116 45127;45135 45138 Alexa Fluor ... 594 +T1339 GO:0007132 45296 45307 Metaphase I +T1340 CL:0000017 45308 45320 spermatocyte +T1341 CHEBI:44658 45333 45335 OA +T1342 GO:0051323 45348 45357 Metaphase +T1343 CL:0000017 45364 45377 spermatocytes +T1344 PR:000016672 45392 45398 Trip13 +T1345 NCBITaxon:10088 45443 45447 mice +T1346 CHEBI:44658 45522 45524 OA +T1347 CHEBI:44658 45563 45565 OA +T1348 CHEBI:16526 45661 45664 CO2 +T1349 CHEBI:51231 45725 45729 DAPI +T1350 GO:0051323 45743 45752 metaphase +T1351 GO:0005634 45753 45759 nuclei +T1352 PR:000016672 45802 45808 TRIP13 +T1353 SO:0000855 45809 45818 orthologs +T1354 SO:0000704 45893 45897 gene +T1355 SO:0000855 45935 45944 orthologs +T1356 SO:0000417 46118 46124 domain +T1357 SO:0000730 46315 46319 gaps +T1358 NCBITaxon:10088 46504 46509 Mouse +T1359 PR:000016672 46510 46516 TRIP13 +T1360 PR:P38126 46525 46529 PCH2 +T1361 SO:0000855 46530 46539 Orthologs +T1362 GO:0042571 46663 46673 Antibodies +T1363 CHEBI:51217 46678 46690 Fluorophores +T1364 PR:000016672 46755 46761 Trip13 +T1365 CL:0000017 46769 46782 Spermatocytes +T1366 GO:0000239 46804 46813 Pachynema +T1367 GO:0006281 46819 46827 Repaired +T1368 GO:0007132 46855 46866 Metaphase I +T1369 PR:000016672 46941 46947 TRIP13 +T1370 PR:P38126 47293 47297 PCH2 +T1371 GO:0042571 47325 47335 antibodies +T1372 PR:000015553 47350 47355 Spo11 +T1373 UBERON:0000922 47357 47366 embryonic +T1374 CL:0002322 47357 47377 embryonic stem cells +T1375 NCBITaxon:10088 47417 47421 mice +T1376 GO:0007129 47504 47512 synaptic +T1377 MOP:0000629 47513 47527 polymerization +T1378 GO:0035825 47570 47572 CO +T1379 GO:0035825 47575 47584 crossover +T1380 SO:0000985 47592 47605 double strand +T1381 MOP:0000780 47606 47611 break +T1382 GO:0007126 47620 47627 meiotic +T1383 GO:0000803 47628 47642 sex chromosome +T1384 GO:0007126 47664 47671 meiotic +T1385 GO:0006342 47672 47684;47694 47703 silencing of ... chromatin +T1386 GO:0000785 47694 47703 chromatin +T1387 GO:0035825 47714 47723 crossover +T1388 CHEBI:44658 47725 47727 OA +T1389 CHEBI:44658 47730 47742 okadaic acid +T1390 GO:0000795 47780 47782 SC +T1391 GO:0000795 47785 47805 synaptonemal complex diff --git a/src/ontogpt/evaluation/craft/database/all/17696610.txt b/src/ontogpt/evaluation/craft/database/all/17696610.txt new file mode 100644 index 000000000..20f7bdcf1 --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/17696610.txt @@ -0,0 +1,309 @@ +Mouse Pachytene Checkpoint 2 (Trip13) Is Required for Completing Meiotic Recombination but Not Synapsis + +Abstract + +In mammalian meiosis, homologous chromosome synapsis is coupled with recombination. As in most eukaryotes, mammalian meiocytes have checkpoints that monitor the fidelity of these processes. We report that the mouse ortholog (Trip13) of pachytene checkpoint 2 (PCH2), an essential component of the synapsis checkpoint in Saccharomyces cerevisiae and Caenorhabditis elegans, is required for completion of meiosis in both sexes. TRIP13-deficient mice exhibit spermatocyte death in pachynema and loss of oocytes around birth. The chromosomes of mutant spermatocytes synapse fully, yet retain several markers of recombination intermediates, including RAD51, BLM, and RPA. These chromosomes also exhibited the chiasmata markers MLH1 and MLH3, and okadaic acid treatment of mutant spermatocytes caused progression to metaphase I with bivalent chromosomes. Double mutant analysis demonstrated that the recombination and synapsis genes Spo11, Mei1, Rec8, and Dmc1 are all epistatic to Trip13, suggesting that TRIP13 does not have meiotic checkpoint function in mice. Our data indicate that TRIP13 is required after strand invasion for completing a subset of recombination events, but possibly not those destined to be crossovers. To our knowledge, this is the first model to separate recombination defects from asynapsis in mammalian meiosis, and provides the first evidence that unrepaired DNA damage alone can trigger the pachytene checkpoint response in mice. + +Author Summary + + + +It is critical that the chromosomes carried by sperm and eggs contain faithful representations of the genome of the individual that produced them. During the process of meiosis, the maternal and paternal copies of each chromosome “synapse” with each other (become tightly associated), exchange genetic material via the process of recombination, then separate into daughter cells in the first of two meiotic cell divisions. The intricate chromosome behavior is subject to errors, so most organisms have evolved meiotic “checkpoints” that monitor fidelity of chromosome synapsis and repair of DNA damage. These checkpoints cause defective cells to self destruct rather than generate defective sperm or eggs. We studied the effects of deleting mouse Trip13, a gene that in distant organisms plays a key role in meiotic checkpoint control. These experiments revealed that instead of having a checkpoint role, Trip13 is required for one of the two major classes of recombination in meiosis that is required for repairing broken DNA molecules. The chromosomes still synapsed normally, but animals were sterile due to massive death of oocytes and spermatocytes. These results indicate that, in addition to a checkpoint that responds to failed synapsis, one exists to specifically detect unrepaired DNA damage that is due to failed recombination. + +Introduction + +The genesis of gametes containing an intact, haploid genome is critical for the prevention of birth defects, and is highly dependent upon the fidelity of chromosome dynamics before the first meiotic division. Homologous chromosomes must pair, synapse, undergo recombination, and segregate properly to opposite poles. Recombination, which repairs repair double strand breaks (DSBs) that are genetically induced in leptonema, is coupled with synapsis in budding yeast and mammals. While our knowledge of the assembly and nature of recombination machinery is extensive, little is known about the disassembly of recombination intermediates, recruitment of DNA replication machinery during recombinational repair, and how the choice between different repair pathways is made. + +Defects in recombination can preclude homologous chromosome pairing, leave unrepaired chromosome breaks, and cause aneuploidy by abrogating crossing over. To avoid such deleterious outcomes, surveillance systems (“checkpoints”) exist to sense meiotic errors and eliminate cells containing unresolved defects. In many organisms, including S. cerevisiae, Drosophila melanogaster, C. elegans, and mice [1–4], meiocytes with defects in recombination and/or chromosome synapsis trigger meiotic arrest in the pachytene stage of meiotic prophase I. This response to meiotic defects is referred to as the “pachytene checkpoint” (reviewed in [5]). Genetic experiments in S. cerevisiae have identified elements of the pachytene checkpoint machinery (reviewed in [5]). In addition to meiosis-specific proteins, these include factors that play roles in DNA damage signaling in mitotic cells [6–10]. Arabidopsis thaliana does not appear to have a pachytene checkpoint akin to that in yeast [11], nor do male Drosophila. + +The pachytene checkpoint is known to monitor two aspects of meiotic chromosome metabolism in S. cerevisiae and C. elegans: (1) DSB repair and (2) chromosome synapsis [2,12]. In mice, both spermatocytes and oocytes harboring mutations that disrupt DSB repair (such as Dmc1, Msh5, and Atm) are efficiently eliminated in pachynema, but spermatocytes are much more sensitive to DSB repair–independent synapsis defects than oocytes [13–15]. However, because recombination is required for synapsis in mice (mutations in recombination genes such as Dmc1 cause extensive asynapsis [16]), it has remained formally uncertain whether there is a distinct pachytene checkpoint that responds to defects in meiotic recombination, and if so, whether it would be identical to that used in somatic cells. The mechanisms of putative pachytene checkpoint control remain unknown in mammals, since no mutations have been identified that abolish it. + +PCH2, encoding a nucleolar-localized AAA-ATPase that was originally identified in an S. cerevisiae genetic screen for mutants that relieve pachytene arrest of asynaptic zip1 mutants [8], was recently determined to be an essential component of the pachytene synapsis (but not DSB repair) checkpoint in yeast and worms [2,12]. PCH2 orthologs are present in organisms that undergo synaptic meiosis, but not asynaptic meiosis, prompting the suggestion that a Pch2-dependent checkpoint evolved to monitor synaptonemal complex (SC) defects from yeast to humans [12]. Here, we generated mice deficient for the Trip13, the ortholog of PCH2, and evaluated whether it also plays a role in the pachytene checkpoint. Surprisingly, while we found no evidence for checkpoint function, we did uncover a potential role for this protein in noncrossover (NCO) repair of meiotic DSBs. + +Results + +Trip13 Is a Widely Expressed Mammalian Ortholog of PCH2 with Unusual Phylogenetic Relationships + +The mammalian ortholog of PCH2, Trip13 (thyroid hormone receptor interacting protein 13), encodes a protein with extensive amino acid homology in regions alignable to the yeast and worm orthologs (Figure S1) [12]). Interestingly, phylogenetic analysis of TRIP13/Pch2p shows that the mammalian protein clusters more closely to plants than it does to the evolutionarily more closely related worms and flies (Figure 1A; see Discussion). Semi-quantitative reverse-transcriptase PCR (RT-PCR) analysis showed Trip13 mRNA to be expressed in a variety of embryonic and adult tissues, including testis (Figure 1B), consistent with mouse and human EST data summarized in Unigene (http://www.ncbi.nlm.nih.gov/UniGene). It is also highly expressed in human and mouse oocytes [17]. + +Figure 1 + +The Mouse PCH2 Ortholog TRIP13 and Expression in Wild Type and Mutant + +(A) Phylogenetic tree of presumed PCH2/TRIP13 orthologs. The database sequence accession number of each protein is presented in Table S1. Numbers shown are bootstrap values (see Materials and Methods). The clustering was the same regardless of whether the whole entire AA sequence or trimmed sequences (where regions showing little conservation were removed) were used for the analysis. Major eukaryotic groups are indicated in color, with deuterostomia in blue, plants in green, protostomia in purple, and fungi in maroon. + +(B) Amplification products of cDNA from the following tissues: 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, testis; 9, E7 embryo; 10, E11 embryo; 11, E15 embryo; and 12, E17 embryo. + +(C) Intron–exon structure of TRIP13 and insertion site of gene-trap vector. See Materials and Methods for details on how the precise insertion site was identified. + +(D) RT-PCR of Trip13 and a control gene Med31 from testis RNA. The Trip13 primers are situated in the first and last exons (see Materials and Methods). + +(E) Western blot analysis of testis protein with anti-TRIP13 antibody. The blot was later probed with anti-alpha tubulin actin as a loading control. The expected TRIP13 protein is ∼48 KDa. + +(F) Localization of TRIP13 in testes. Wild-type (top) and mutant (bottom) testis sections were probed with chicken anti-TRIP13, and detected with HRP-conjugated anti-chicken IgG (brown/red staining). Expression in WT was most prominent in the nuclei of Type B spermatogonia (Sg), leptotene spermatocytes (LS), and early pachytene spermatocytes (PS), but not late pachytene spermatocytes (LP). No nuclear staining was seen in mutant testis sections, although reddish cytoplasmic background is present. Identification of cell types was judged in part by estimating the epithelial stage of the tubules as described [67]. + +(G) TRIP13 localization in surface-spread spermatocytes. Preparations were immunolabeled with anti-SYCP3 (S) and TRIP13 (T). Both individual and merged images are shown for leptotene (Lep), zygotene (Zyg), and pachytene (Pac) spermatocytes. Nuclear staining was absent in the mutant. + +Generation of Trip13 Mutant Mice + +To explore the function of TRIP13 in mammals, we generated mice with a gene trap-disrupted allele, Trip13RRB047 (Figure 1C; abbreviated as Trip13Gt). Heterozygotes were normal in all respects, but homozygotes were present at ∼2/3 the expected ratio from intercrosses between heterozygotes (91 Trip13+/+, 183 Trip13Gt/+, and 61 Trip13Gt/Gt). Since >90% of prewean mice that died were mutant homozygotes, this discrepancy is apparently due to a partially penetrant lethality. Most surviving Trip13Gt/Gt animals were grossly normal. However, homozygotes that were semi-congenic (N4) on the C57BL/6J strain were often markedly smaller and/or had kinked or shorter tails (Figure 2A and 2B). + +Figure 2 + +Developmental Phenotypes of Trip13 Mutant Mice + +(A) Shown are 21-d-old littermates. Note the shortened tail in the mutant, but overall similar body size. + +(B) Shown are 23-d-old littermates. The mutant is smaller in this case, but the tail is not as truncated as the mouse in (A). + +(C) Wild-type (WT) and homozygous Trip13 mutant (MUT) testes. + +(D) and (E) are cross sections through 17.5-d-old heterozygous (“WT”) and homozygous mutant Trip13 testes, respectively. Whereas the tubules in WT show coordinated spermatogenesis with pachytene spermatocytes present in all tubules (proximal to the lumen), developmental progression in the mutant is not synchronized between tubules. Some tubules have no pachytene spermatocyes (asterisks), while in others, development is somewhat disorganized (#). + +RT-PCR analysis of Trip13Gt expression (Figure 1D) revealed a low level of normally spliced transcripts in testes of homozygotes that is presumably a consequence of incomplete usage of the gene trap's splice acceptor. Western blot analysis, using a polyclonal antibody raised against a peptide encoded by exon 3, revealed multiple species in wild-type and heterozygous testes, one of which corresponds to the expected size of 48 kDa (Figure 1E). This and three other species were undetectable in homozygous mutant testes, but a reduced amount of an intense ∼38 kDa smaller band was present. It is not clear if this corresponds to TRIP13. The greatly decreased Trip13 mRNA and predicted correct-length protein in mutants indicate that the Trip13RRB047 allele is severely hypomorphic. + +To determine the germ cell types in which TRIP13 is expressed, and to assess possible expression in the mutant by means other than Western analysis, testis sections were immunolabeled for TRIP13 using a polyclonal chicken antipeptide antibody (see Materials and Methods). The most intensely labeled cells in control testes were Type B spermatogonia and leptotene spermatocytes (Figure 1F). Zygotene/pachytene spermatocytes stained less strongly, and there was no detectable staining in late pachytene spermatocytes. TRIP13 appeared to be nuclear localized. There was no such staining of nuclei in mutant seminiferous tubules (Figure 1F). To further assess the nuclear localization, TRIP13 was used to probe meiotic chromosomes prepared by surface spreading of spermatocyte nuclei. In wild type, there was diffuse nuclear staining, and no evidence of concentration on SC cores (marked by the axial element protein SYCP3) at any meiotic substage (Figure 1G). TRIP13 signal was noticeably absent in mutant meiotic nuclei. + +Infertility Due to Meiotic Disruption in TRIP13-Deficient Meiocytes + +Homozygotes of both sexes had small gonads (Figure 2C; see below) and were invariably sterile. Ovaries of adult Trip13Gt/Gt females were severely dysmorphic and had few or no follicles (Figure 3A and 3B). The majority of oocyte loss occurred in late embryogenesis or early in postnatal development, since 2 d postpartum ovaries were markedly smaller than those of control littermates, and were lacking oocytes or newly forming follicles (Figure 3C and 3D). Thus, oocytes failed to progress to the dictyate (resting) phase. Since we observed oocytes with pachytene stage chromosomes in 17.5 d Trip13Gt/Gt embryonic ovaries (unpublished data), this indicates that oocytes were eliminated somewhere between pachynema and dictyate. + +Figure 3 + +Histology of Mutant Gonads + +All are hematoxylin/eosin-stained paraffin sections. Testes are from 6-wk-old males, except as indicated below. + +(A) Wild-type 25-d-old ovary. + +(B) Trip13Gt/Gt 25-d-old ovary, showing dysgenesis from an absence of oocytes. + +(C) Trip13Gt/+ 2-d-old control ovary. Arrows point to oocytes in newly forming follicles. + +(D) Trip13Gt/Gt 2-d-old ovary, dysgenic due to lack of oocytes. Magnification is the same as its littermate in “C.” + +(E) Wild-type testis. + +(F) Trip13Gt/Gt testis with uniform pachytene arrest. + +(G) Trip13Gt/Gt 3-mo-old testis with some postmeiotic spermatids (arrows). + +(H) Spo11−/- testis. A tubule with spermatocytes at leptotene/zygotene transition is labeled ZP, and tubules with apoptotic spermatocytes are marked with an asterisk. The specimen was taken from a littermate of that in (I). + +(I) Spo11−/− Trip13Gt/Gt testis. Labeling is the same as in (H). The inset contains a tubule with leptotene-zygotene spermatocytes. + +(J) Mei1−/− Trip13Gt/+ testis. The specimen was taken from a littermate of that in (K). + +(K) Mei1−/− Trip13Gt/Gt testis. + +(L) Rec8Mei8/Rec8Mei8 Trip13Gt/+ testis. The Rec8Mei8 allele was described [39]. The specimen was taken from a littermate of that in (M). + +(M) Rec8Mei8/Rec8Mei8 Trip13Gt/Gt testis. + +(N) Dmc1−/− Trip13Gt/Gt testis. + +(O) Spo11−/− Trip13Gt/+ 25-d-old ovary. The specimen was taken from a littermate of that in (P). + +(P) Spo11−/− Trip13Gt/Gt 25-d-old ovary. + +(Q) Mei1−/− Trip13Gt/+ 25-d-old ovary. The specimen was taken from a littermate of that in (R). + +(R) Mei1−/− Trip13Gt/Gt 25-d-old ovary. + +(S) Rec8Mei8/Rec8Mei8 Trip13Gt/+ 25-d-old ovary. The specimen was taken from a littermate of that in (T). + +(T) Rec8Mei8/Rec8Mei8 Trip13Gt/Gt 25-d-old ovary. + +Histological sections of mutant testes revealed a lack of postmeiotic cell types that are characteristic of wild-type seminiferous tubules (Figure 3E). The most developmentally advanced seminiferous tubules contained adluminal spermatocytes with condensed chromatin characteristic of pachynema (Figure 3F). The absence of coordinated spermatogenic progression beyond this stage is indicative of a pachytene arrest. This was revealed more clearly by chromosome analysis (see below). Some sections of adult seminiferous tubules contained postmeiotic spermatids (Figure 3G), although we saw no motile epididymal sperm. These drastic meiotic defects stand in contrast to yeast and C. elegans, in which deletion of Pch2 alone has minor effects on spore/gamete development [2,8]. + +TRIP13-Deficient Meiocytes Undergo Homologous Chromosome Synapsis Despite the Presence of Unrepaired DSBs in Pachynema + +To better characterize the degree of meiotic progression in Trip13Gt/Gt spermatocytes, we immunostained chromosome spreads for SYCP3 and SYCP1, components of the axial/lateral elements and transverse filaments, respectively, of the synaptonemal complex (SC). Pachytene spermatocyte nuclei from postpubertal mutant testes could assemble normal SC cores and exhibited full synapsis of chromosomes as judged by colabeling of SYCP1 and SYCP3 along the full lengths of all autosomes (Figure 4A). Additionally, the X and Y chromosomes were normally synapsed at their pseudoautosomal region. More prepubertal (17.5 d postpartum) mutant spermatocytes contained asynaptic or terminally asynapsed chromosomes than age-matched controls (62.5% versus 25%, respectively; Figure 4B). We attribute this to a delay in the first wave of postnatal spermatogenesis (Figure 2D and 2E), likely related to systemic developmental retardation (Figure 2A and 2B). Nevertheless, since Trip13Gt/Gt spermatocytes progress to pachynema with no gross SC abnormalities, and oocytes were eliminated soon after birth (a characteristic of DNA repair mutants [13]), this suggested that unrepaired DSBs are responsible for eventual meiotic arrest and elimination. + +Figure 4 + +Immunohistochemical Analysis of Pachytene Spermatocyte Chromosomes + +Surface-spread chromosomes were immunolabeled with the indicated antibodies and fluorophores. As indicated in the upper right of each panel, cells were from wild type (WT, either +/+ or Trip13Gt/+) or Trip13Gt/Gt (Mut). There were no differences seen between heterozygotes and +/+ spermatocytes. + +(A) A mutant pachytene nucleus with full synapsis. Areas of SYCP1/SYCP3 colabeling are yellow. + +(B–E) Spermatocytes nucleus from 17.5 d postpartum mutant. Asynapsed chromosomes or regions of chromosomes are indicated by white and yellow arrows, respectively. Unlike the normal distribution in wild-type pachytene spermatocytes (C), BLM foci are present on synapsed pachytene chromosomes in the mutant (D). RAD51 foci, which are abundant earlier in prophase, disappear from autosomes in wild-type pachytene nuclei (E) and the bulk of staining is over the XY body (arrow). + +(F) RAD51 persists on the synapsed mutant chromosomes (arrows). + +(G) H2AX phosphorylation is restricted to the XY body in WT. + +(H) In addition to a large area of γH2AX staining (arrow) over the XY body, there is extensive autosomal H2AX phosphorylation (arrows). + +(I, J) Note that in wild-type pachytene spermatocytes, TOPBP1 is present only over the XY body (yellow arrow). In the mutant (J), an arrow denotes one area of intensive staining that may be over the sex chromosomes, but many other chromosome cores are positively stained. + +(K, L) RPA persists along synapsed cores in the mutant, not WT. + +(M, N) Arrows indicate examples of MLH3 foci on SCs. + +(O) In WT late pachytene spermatocytes, RAD51 is present only at background levels. + +(P) As in (F), extensive RAD51 staining delineates SCs in mutant pachytene nuclei (indicated by white arcs). MLH1 foci colocalize with these tracts (arrows) at the typical 1–2 foci per chromosome as in (M). + +To elucidate the cause of meiotic arrest, we analyzed meiotic chromosomes with a variety of markers that are diagnostic of recombination and synapsis. Recombination in Trip13Gt/Gt spermatocytes appeared to initiate normally as judged by the presence of γH2AX in leptonema (Figure S2A and S2B), which reflects the presence of meiotically induced DSBs [18]. RAD51 and/or DMC1, components of early recombination nodules (ERNs), was also present as abundant foci in Trip13Gt/Gt zygotene spermatocytes (unpublished data; the anti-RAD51 antibody cross-reacts with DMC1), indicating that recombinational repair of DSBs is initiated. The cohesin complex, which is essential for completion and/or maintenance of synaptic associations, appeared to assemble normally as judged by immunolabeling for the meiosis-specific cohesins STAG3 (Figure S2C and S2D) and REC8 (unpublished data). Because yeast PCH2 localizes to telomeres in a Sir3p-dependent manner, we tested for possible telomere defects by immunolabeling for TRF2, a component of a protein complex that plays an essential role in telomere protection [19]. It was localized to telomeres of both fully synapsed and telomerically asynaptic mutant chromosomes (Figure S2E and S2F). + +Defects in DSB repair became apparent in pachynema upon probing of mutant spermatocyte nuclei with antibodies against molecules involved in various stages of recombination. In >99% of Trip13Gt/Gt chromosome spreads, BLM helicase (Figure 4C and 4D), RAD51/DMC1 (Figure 4E and 4F), γH2AX (Figure 4G and 4H), and TOPBP1 (Figure 4I and 4J) all persisted abnormally on synapsed chromosomes. For RAD51/DMC1, mutant pachytene spermatocytes contained 138 ± 6 foci (compared to 11 ± 3 foci in wild type, most of which were on the XY body), down from 218 ± 13 in zygonema (compared to 220 ± 13 foci in wild type). TOPBP1 is a DNA damage–checkpoint protein involved in ATM protein–dependent activation of ATR protein [20,21]. It binds sites of DSBs and unsynapsed regions of meiotic chromosomes [22,23]. BLM has been reported to colocalize with markers (RPA and MSH4) of recombination at sites distinct from those that become resolved as crossovers (CO) [24]. We therefore assessed the distribution of RPA, the ssDNA binding protein, which is normally present at focal sites of synapsing meiotic chromosomes before disappearing in mid-pachynema [25]. It is thought to bind D-loops of recombination intermediates [26]. RPA also persisted on pachytene mutant chromosomes (Figure 4K and 4L). These data indicate that unrepaired DSBs, or unresolved recombination intermediates, remain in pachynema and activate a DNA damage checkpoint system. + +It should be noted that chromosomes affected by meiotic sex chromosome inactivation (MSCI) and meiotic silencing of unpaired chromatin (MSUC) are heavily stained by antibodies for several DSB repair-associated molecules, including γH2AX. H2AX phosphorylation due to MSCI and MSUC is conducted by ATR, not ATM [27–29]. Since mutant chromosomes are fully synapsed, and MSUC is known to occur only as a result of asynapsis, the decoration of Trip13Gt/Gt chromosomes with DNA repair markers is probably attributable to incomplete DNA repair rather than transcriptional silencing. + +Consistent with the presence of rare (<1%) Trip13Gt/Gt pachytene spermatocytes devoid of persistent DNA repair markers, and testis histology showing some degree of postmeiotic progression (Figure 3G), we observed both diplotene nuclei that lacked autosomal RAD51/DMC1 and γH2AX (Figure S3A–S3D), and also metaphase I spreads with 20 bivalents (Figure S3E–S3F). Since Trip13Gt may not be a complete null, these diplotene and metaphase I spermatocytes might arise by virtue of having sufficient wild-type TRIP13. + +CO-Associated Markers Appear Normally in the Absence of TRIP13 + +The persistence of BLM on Trip13Gt/Gt spermatocyte chromosomes suggests that at least a subset of the unrepaired DSBs correspond to sites of defective NCO recombinational repair. To assess whether CO recombination occurs in the mutant, we examined the distribution of the mismatch repair proteins MLH1 and MLH3, which are normally detectable as foci in mid-late pachynema and mark the locations of chiasmata [30,31]. Remarkably, MLH1/3 foci were formed; we observed 1–2 foci/chromosome as in wild type and at typical overall levels (MLH3 = 23 ± 2, N = 10; [30,32]) on mid-late pachytene chromosomes (Figure 4M and 4N; MLH1 not shown). Since <1% of Trip13Gt/Gt pachytene nuclei had normal repair (as judged by absence of persistent DSB repair markers; see above), but most of the pachytene nuclei had MLH1/3 foci, it was unlikely that the MLH1/3 foci formed only on chromosomes with fully repaired DSBs. To test this directly, we conducted double staining for MLH1 and RAD51/DMC1. MLH1 foci were present on chromosomes that also contained numerous RAD51/DMC1 foci (Figure 4O and 4P). + +To assess whether these MLH1/3 foci in Trip13Gt/Gt pachytene spermatocytes represent CO events completed to a point where they could maintain interhomolog attachments though the end of prophase I, we treated testicular cells from 17.5–20.5-d-old control (+/+), Trip13Gt/Gt, and Dmc1−/− mice with the protein phosphatase inhibitor okadaic acid (OA), a chemical that induces degradation of the SC, chromosome condensation, and premature progression to metaphase I [33]. Fifteen metaphase spreads were identified for each genotype. Whereas all of the Dmc1−/− spreads had ∼35 or more condensed chromosomes, all of the +/+ and Trip13Gt/Gt spreads had 20–25, suggesting that the MLH1/3 foci in Trip13Gt/Gt pachytene spermatocytes represent sites of completed, or near-completed, COs. Because the preparations were made from whole testes, it is possible that the univalent-containing metaphases from Dmc1−/− mice were from spermatogonia, not spermatocytes. + +TRIP13 Deficiency Does Not Alleviate Meiotic Arrest Phenotypes of Mutants Defective in Synapsis + +To determine if TRIP13 deficiency prevents apoptosis triggered by asynapsis as in C. elegans, we analyzed mice that were doubly mutant for Spo11 and Trip13. SPO11 is a transesterase that is essential for the creation of genetically programmed DSB during leptonema of many organisms, including mice [18]. In C. elegans, spo-11 mutant gametes have extensive asynapsis, which triggers PCH-2 dependent apoptosis in pachynema [2]. In mice, Spo11−/− spermatocytes are severely defective in homologous chromosome synapsis [34,35], and arrest with chromosomes in a state characteristic of the zygotene/pachytene transition (Figure 3H). Spermatocytes in Trip13Gt/Gt Spo11−/− testes progressed maximally to that point before undergoing death (Figs 3I), well before the spindle checkpoint that eliminates achiasmate spermatocytes [36]. There was no evidence of metaphase I spermatocytes or postmeiotic spermatids in these testes, unlike those seen in Trip13 single mutants (Figure 3G). In contrast to the complete synapsis in Trip13Gt/Gt pachytene spermatocytes (Figure 5A), in which SPO11 is available in leptonema to initiate (via DSB induction, Figure S2A and S2B) a recombination-driven homolog search, chromosome synapsis in doubly mutant spermatocytes was highly disrupted as in Spo11 single mutants (Figure 5B and 5C). Identical studies were performed with mice deficient for Mei1, a vertebrate-specific gene also required for DSB formation and chromosome synapsis [37], with similar results (Figure 3J and 3K; immunocytology not shown). + +Figure 5 + +Immunocytological Analysis of Trip13 Compound Mutants + +Surface-spread chromosomes were immunolabeled with the indicated antibodies and fluorophores. Genotypes are indicated, as are those panels in which dual staining patterns are merged. Note that (H) and (I) are at lower magnification to show multiple nuclei. + +In yeast, deletion of PCH2 alleviates the pachytene arrest caused by asynaptic mutants zip1 and zip2 [8]. Although mouse SYCP1 might be a functional equivalent of Zip1p, because Sycp1 mutant spermatocytes arrest at approximately the same point as Trip13 mutants, there would be no opportunity to observe bypass of Sycp1−/−. Since Zip2p is present at sites of axial associations, even in zip1 mutants, it has been suggested that Zip2p promotes initiation of chromosome synapsis [38]. These observations raise the possibility that in yeast, Pch2p responds to synapsis polymerization rather than initiation. To test this, we performed epistasis analysis with a Rec8 allele (Rec8Mei8, abbreviated as Rec8−). Meiotic chromosomes of Rec8 mutant spermatocytes undergo apparent homolog pairing and interhomolog synaptic initiation, but are defective in DSB repair and fail to maintain interhomolog synapsis [39,40]. Rather, sister chromatids appear to synapse and are bound by SYCP1 along their axes. Rec8 mutants do not progress to diplonema or metaphase I. Double mutant analysis indicated that Rec8 is epistatic to Trip13. As in the Spo11 and Mei1 experiments, histology of testes deficient for both REC8 and TRIP13 resembled the Rec8 mutant, with no evidence of progression to metaphase I that occurs in Trip13Gt/Gt mice (Figure 3L and 3M). Immunocytological analysis of spread chromosomes showed a failure of homologous chromosome synapsis in both the Rec8−/− and Rec8−/− Trip13Gt/Gt spermatocytes, as previously reported for Rec8 mutants (Figure 5D and 5E) [39,40]. + +Although subsequent reports indicate otherwise [10,12], deletion of PCH2 in yeast was originally reported to alleviate meiotic arrest caused by deficiency for the meiosis-specific RecA homolog DMC1 [8]. To investigate this relationship in mice, we constructed animals doubly mutant for Trip13 and Dmc1. As in Dmc1−/− mice, in which spermatocytes undergo meiotic arrest from defective DSB repair and failed chromosome synapsis [16], spermatogenesis in Dmc1−/− Trip13Gt/Gt testes was uniformly arrested at the point where spermatocytes contained chromatin characteristic of zygonema/pachynema (Figure 3N). Immunocytological analysis indicated that both Dmc1−/− and Dmc1−/− Trip13Gt/Gt chromosomes had extensive asynapsis compared to Trip13Gt single mutants (Figure 5F–5H), and all had persistent RAD51/DMC1 foci and phosphorylated H2AX (γH2AX; Figure 5I–5L), confirming that Dmc1 is epistatic to Trip13. Doubly mutant females had residual ovaries, phenocopying Dmc1−/− and Trip13Gt/Gt single mutants (unpublished data). + +Meiotic Defects in Trip13Gt/Gt Oocytes Are DSB-Dependent + +Epistasis analysis of females was insightful with respect to the cause of arrest in Trip13 mutants. Both Mei1−/−/Trip13Gt/Gt and Spo11−/−/Trip13Gt/Gt females had ovaries with numerous follicles, identical to Mei1 and Spo11 single mutants (Figure 3O–3R). Thus, Spo11 and Mei1 are epistatic to Trip13 in oogenesis, just as they are to Dmc1 [13,41]. This demonstrates that oocyte loss in Trip13Gt/Gt females is dependent on DSB formation. In conjunction with the immunohistochemical data, these data provide strong evidence that meiotic arrest in Trip13 mutant mice is due to defects in DSB repair. As expected, ovaries of Rec8 Trip13 double mutants were devoid of oocytes as were those from either single mutant (Figure 3B, 3S, and 3T). + +Discussion + +Genetic experiments in S. cerevisiae provided evidence that the pachytene checkpoint monitors and responds to recombinational DSB repair and synapsis independently. Wu and Burgess concluded that the repair checkpoint is RAD17-SAE2 dependent, while the synapsis checkpoint is PCH2-ZIP1 dependent [12]. Of these four genes, SAE2 and ZIP1 do not have clear mammalian orthologs (although SYCP1 may be a functional ortholog of ZIP1), and mutation of the mouse RAD17 ortholog, Rad1, presumably causes embryonic lethality [42]. Thus, mutational analysis of mouse Pch2 (Trip13), which is also critical for the synapsis checkpoint in C. elegans [2], was the best remaining option to evaluate potential functional conservation in mammalian meiotic checkpoint control. + +Our results demonstrate that in mice, the primary meiotic function of TRIP13 is in recombination itself. We found no evidence that it is involved in pachytene checkpoint control. Our data suggest that while recombination events destined to be resolved as COs can proceed normally in Trip13 mutants, DSBs that enter the NCO repair pathway are incompletely resolved or processed inefficiently. This hypothesis is compatible with current knowledge of meiotic recombination pathways. In S. cerevisiae, CO and NCO pathways are distinct [43]; they have different recombination intermediates, and are dependent upon different proteins [44,45]. Mice also appear to have independent CO versus NCO recombination pathways [46]. As in yeast, both require SPO11-induced breaks, but only the CO pathway requires MLH1. Both types of recombinant products are formed by mid-late pachynema. Another possibility is that the recombination defects are a result of defective intersister recombination. However, this type of DSB repair is suppressed in meiotic cells. Ablation of RAD54, which mediates intersister recombination in yeast, does not significantly disrupt meiosis in either yeast or mice [47,48]. Interestingly, RAD54-deficient spermatocytes display abnormal persistence of RAD51 foci on pachytene chromosomes, similar to those in TRIP13 mice, but there are no deleterious effects on meiotic progression or fertility [49]. + +Data from budding yeast also indicate that Pch2p functions in recombination. Deletion of PCH2 delays meiotic progression by ∼2 h in SK1 yeast, and causes a minor decrease in ascus formation [50]. DSBs persist >2 h longer in pch2Δ yeast than in wild type, and hyperresection of DSBs in dmc1Δ pch2Δ double mutants is lower than in dmc1Δ cells [10]. Additionally, it was reported that pch2Δ yeast had a meiosis I delay dependent on the RAD17–SAE2 checkpoint that monitors recombination intermediates [12]. However, the exact role of TRIP13 (or Pch2) in recombination is unclear. Because synapsis occurs in TRIP13-deficient spermatocytes and is dependent on DSB formation (activity of SPO11 and MEI1), we suggest that TRIP13 functions after homology recognition and strand exchange, and that recombination events entering the CO repair pathway are either completed or nearly so (because OA treated resulted in bivalent chromosomes). One possibility for TRIP13′s role in recombination is that it is directly involved in a step specific to resolution of NCO recombination intermediates. Another possibility is that TRIP13 is required for disassembly of NCO recombinational repair complexes [51] containing those proteins that persist abnormally on Trip13Gt/Gt pachytene chromosomes. Notably, TRIP13 has two putative ATPase domains, a signature of AAA-ATPase ClpA/B chaperones that perform protein or protein/DNA complex disassembly [52]. These potential recombination roles might not be limited to meiosis, since Trip13 is widely transcribed and the mutant animals exhibited developmental defects. Finally, TRIP13 might play an indirect role, such as providing a “licensing” signal for the resolution of NCO intermediates and completion of meiosis. + +Regarding the cause of cell death in Trip13 mutants, our data indicate that this is triggered by defective DSB repair rather than asynapsis. We base this conclusion on two observations: (1) oocyte elimination is dependent upon DSB formation and (2) synapsis is normal in spermatocytes of adult testes. Indeed, this mutant is unique in that recombination defects occur in the absence of asynapsis (e.g., as in Dmc1 knockouts). Thus, the Trip13 mutant provides the first evidence that unrepaired DNA damage alone can trigger the mammalian pachytene checkpoint response. Furthermore, our results allow us to conclude that oocytes and spermatocytes share a similar, if not identical, DNA damage pachytene checkpoint that is decoupled from a synapsis checkpoint. + +Interestingly, we found that OA treatment of Trip13Gt/Gt spermatocytes could propel them into MI, despite a report that the same did not occur when wild-type pachytene spermatocytes were treated with the DSB-inducing agents gamma radiation or etoposide [53]. It is possible that the nascent induction of DSBs in pachynema evokes a checkpoint response that cannot be bypassed by OA, whereas the post-strand invasion lesions in TRIP13-deficient spermatocytes do not. + +TRIP13 was originally discovered to be an interactor with rat thyroid receptor beta (THRB; [54]), but the relationship between THRB and TRIP13 in meiosis is unknown. Interestingly, we observed that THRB is distributed diffusely throughout wild-type spermatocyte nuclei but is excluded from the XY (sex) body (unpublished observations), a compartmentalized nuclear domain beginning in pachynema, in which the sex chromosomes become heterochromatinized and transcriptionally silenced in the process of MSCI [55]. However, the XY body appeared intact in most mutant spermatocytes upon probing with several markers of XY heterochromatinization (unpublished observations). Considering that THRB knockout mice are viable and fertile [56], the functional relationship between TRIP13 and its receptor THRB in meiosis is unclear. + +Given the high similarity of PCH2 orthologs throughout the eukaryotic world, one or more essential functions of this protein must be conserved. Since TRIP13 does not exhibit checkpoint function in mice, we surmise that the TRIP13/PCH2 ancestral protein had a function in recombination that persists to the present. Notably, A. thaliana does not appear to have a meiotic checkpoint activity that eliminates mutant meiocytes in a manner analogous to organisms such as mice, budding yeast, and female Drosophila [11,57], and mammalian TRIP13 is more similar to Arabidopsis PCH2 than the fly or worm proteins (Figures 1A and S1). The unusual relatedness between mammalian and plant PCH2 may therefore be attributable to both the presence of a common conserved function (namely recombination, although the role of PCH2 in plants has yet to be determined), and the absence of checkpoint function. Nevertheless, the evolutionary relationships between animals, fungi, and plants (which are discordant with PCH2 sequence phylogeny) do not allow parsimonious models addressing the points in time that checkpoint functions in PCH2 were gained or lost. It is possible that its checkpoint function evolved independently in worms and budding yeast. The picture will become clearer as the function of PCH2 in other organisms is elucidated. + +The nature of the synapsis checkpoint in male mice remains unidentified. One possible candidate is Dot1 (PCH1 in yeast), a histone methyltransferase silencing factor that is required for pachytene arrest of zip1 and dmc1 mutants in yeast [58], and for preventing RAD54-mediated recombinational DSB repair between sister chromatids. However, DOT1 acts upstream of PCH2. Given that TRIP13 doesn't have checkpoint function in mice, a potential role for mammalian DOT1 in the pachytene checkpoint is dubious but awaits investigation. Recently, it was shown that the TRP53 homolog TRP63 is required for DNA damage–induced death of dictyate-stage primordial oocytes, leading to the suggestion that it is involved in monitoring genome integrity [59]. However, this activity occurs subsequent to a pachytene checkpoint. As alluded to earlier, a complicating problem for studying potential meiotic checkpoint genes in mice is that as in yeast, such genes often have mitotic functions (such as RAD24 [7]), and their ablation can cause lethality [42]. Unless mammalian pachytene checkpoint components have orthologs with similar functions in organisms such as yeast, their identities are likely to remain elusive. + +Materials and Methods + +PCR analysis of Trip13 cDNA. + +Trip13 was amplified from samples of Clontech's Mouse Multiple Tissue cDNA Panel I (http://www.clontech.com), using the following primers: 5′-GCACCATTGCACTTCACATC-3′ (TRP3-6F) and 5′-TGACCATCAGACTGTCGAGC-3′ (TRP3-6R). These primers correspond to exons 3 and 6, respectively, and amplify a 330-bp cDNA product. The cDNAs in this panel are equalized to allow quantitative analysis by RT-PCR. + +Generation of Trip13-deficient mice. + +The mouse embryonic stem cell line RRB047 (strain 129/Ola) containing a gene trap insertion in Trip13 was obtained from BayGenomics (http://www.baygenomics.ucsf.edu/). The gene-trapping vector used to create this line, pGT1lxf, was designed to create an in-frame fusion between the 5′ exons of the trapped gene and a reporter, βgeo (a fusion of β-galactosidase and neomycin phosphotransferase II). The gene-trapped locus creates a fusion transcript containing exons 1–3 of Trip13 and βgeo. To identify the exact insertion site within intron 3, PCR was performed using one primer within the gene trap vector, and the other primer at various positions in intron 3 pointing towards the 3′ end of the gene. Product from a productive reaction was sequenced, revealing that the insertion site was 445 bp into intron 3. + +Genotyping of mice. + +Three primers were used to distinguish wild-type and mutant alleles of Trip13: primer 1, 5′-CGTCGCTCCATTGCTTTGTGC-3′; primer 2, 5′-AGTAGTGGTACACTGTATTTTTGCTTTCATTGA-3′; and primer 3, 5′-GTAGATCCCGGCGCTCTTACCAA-3′. Primers 1 and 2 are located upstream and downstream, respectively, of the gene trap insertion within the intron 3. Primer 3 corresponds to pGTlxf sequence. Primers 1 and 2 amplify a 700-bp band from a wild-type allele; primers 1 and 3 amplify a 540-bp fragment from a mutant allele. Separate reactions were used to assay the presence or absence of each amplicon from a DNA sample. The cycling conditions were: 94 °C 2 min; 35 cycles of 94 °C 30 s, 57 °C 45 s, and 72°C 50 s; and 72 °C 2 min. + +RT-PCR. + +Total RNA was isolated from adult testes with the RNeasy Mini Kit (Qiagen, http://www.qiagen.com), and 4.0 μg was oligo dT–primed and reverse-transcribed with Superscript II (Stratagene, http://www.stratagene.com). The entire Trip13 protein-coding sequence was amplified with primers 5′-ATGGACGAGGCGGTG-3′ and 5′-TCAAACATAAGCTGAAAGTT-3′. The cycling conditions were: 94 °C 2 min; 94 °C 30s, 55 °C 45 s, and 72 °C 80 s for 35 cycles; and 72 °C 2 min. The primers for amplifying the Med31 coding sequence as control were : 5′-ATGGCCGCGGCCGTCGCTATGG-3′ and 5′-TCATTTCCCTGCTGTGTTATTCTGCTGCTGCTGC-3′. The cycling conditions were: 94 °C 2 min; 94 °C 30 s, 55 °C 30 s, and 72 °C 35 s for 35 cycles; and 72 °C 2 min. + +Development and purification of chicken antibodies. + +A peptide corresponding to amino acids 25–40 of TRIP13, VLQRSGSTAKKEDIK, was conjugated to KLH and used to immunize chickens (done by Sigma Genosys, http://www.sigmaaldrich.com). Polyclonal IgY was isolated from eggs with the Eggcellent Chicken IgY Purification kit (Pierce, http://www.piercenet.com). IgY antibodies were then affinity purified using the immunizing synthetic peptide. + +Western blotting. + +50 μg of testis extract in RIPA buffer was separated by 8% SDS-PAGE and electrotransferred onto a Pure Nitrocellulose membrane (Bio-Rad, http://www.biorad.com). The membrane was incubated with a polyclonal rabbit anti-human TRIP13 antibody (18-003-42687; Genway, http://www.genwaybio.com). According to the manufacturer, the immunogen was a synthetic peptide embedded in sequence we deduced to correspond to exon 3. Binding was detected by chemiluminescence ECL kit (Pierce) using a rabbit anti-chicken IgG horseradish peroxidase conjugate (Pierce). + +Histological analyses. + +Testes or ovaries were fixed in Bouin's, embedded in paraffin, sectioned at 6 μm, and stained by hematoxylin and eosin. Antigen retrieval for immunohistochemistry of testis sections was as described [60]. Oocyte and follicle numbers were counted as described [61]. Only follicles containing an oocyte with a clearly visible nucleus were scored. + +Immunocytochemistry. + +Immunolabeling of surface-spread spermatocytes and oocytes was performed as described [39,62]. To reach conclusions on the pattern of staining for various proteins, 30 (unless otherwise indicated) well-spread nuclei of particular meiotic stages were first identified under the fluorescent microscope on the basis of SYCP3 or STAG3 staining, then imaged at both appropriate wavelengths to determine the pattern of second proteins with focal patterns such as RAD51 or RPA. Unless otherwise indicated, the panels shown in the figures were the exclusive or predominant patterns seen. The exception for this approach was in the case of staining for MLH1 or MLH3 plus RAD51 (in which case SYCP3 or STAG3 was not available to find chromosome cores). Nuclei in this situation were identified first by MLH1/3 foci clustering, then imaged for both fluorescent wavelengths. + +Primary antibodies used in this study were as follows: mouse anti-SCP3 (1:500; Abcam, http://www.abcam.com); rabbit anti-SYCP1 (1:1,000; a gift from C. Heyting) [63]; rabbit anti-REC8 (1:100; a gift from C. Heyting); rabbit anti-RAD51 (1:250, this polyclonal antibody recognizes both RAD51 and DMC1; Oncogene Research Products, http://www.merckbiosciences.co.uk); rabbit anti-γH2AX (1:500; Upstate Biotechnology, http://www.upstate.com/); rabbit anti-STAG3 (1:1,000; a gift from R. Jessberger); rabbit anti-MLH3 (1:400; a gift from P. Cohen); mouse-anti-human MLH1 (1:50; BD Biosciences, http://www.bdbiosciences.com); rabbit-anti-TopBP1 (1:100; a gift from J. Chen) [22]; mouse-anti-ubiquityl-histone H2A (1:200; Upstate Biotechnology); rabbit-anti-TRF2 (1:500; a gift from T. de Lange); and rabbit-anti-BLM (1:50; a gift from R. Freire). All secondary antibodies conjugated with either Alexa Fluor 488 or 594 (Molecular Probes, http://probes.invitrogen.com/) were used at a dilution of 1:1,000. All images were taken with a 100× objective lens under immersion oil. + +Metaphase I spermatocyte spreads and OA treatment. + +Metaphase fixed spermatocytes from 8-mo-old Trip13RRB047 homozygotes, using 23-d-old wild-type mice as control, were prepared and stained with Giemsa as described [64]. + +For OA treatment, cells were exposed to 5 μM OA (Calbiochem, http://www.emdbiosciences.com) for 6 h at 32 °C in a humidified environment of 5% CO2 before spreading [65]. These preparations were stained with DAPI to visualize metaphase nuclei and chromosomes. + +Phylogenetic analyses. + +TRIP13 orthologs were identified by BLASTP searches of Genbank and other sources providing gene models such as Ensembl. The selected orthologs can be found in Table S1. Amino acid alignments were done with Clustal W, using the default settings with and without removing the regions outside of the AAA-ATPase central domain. The trees were constructed by using the neighbor-joining method with Poisson correction. The reliability of internal branches was assessed by using 500 bootstrap replicates, and sites with gaps were ignored in this analysis. Neighbor-joining searches were conducted by using the computer program MEGA3 [66]. + +Supporting Information + +Figure S1 + +Depiction of Conserved Regions of Mouse TRIP13 and Its PCH2 Orthologs + +(75 KB PDF) + +Click here for additional data file. + +Figure S2 + +Surface-Spread Chromosomes Immunolabeled with the Indicated Antibodies and Fluorophores + +(947 KB PDF) + +Click here for additional data file. + +Figure S3 + +Trip13 Mutant Spermatocytes That Progress Beyond Pachynema Have Repaired DSBs and Form Bivalents at Metaphase I + +(1.5 MB PDF) + +Click here for additional data file. + +Table S1 + +Sources of TRIP13 Amino Acid Sequences Used to Construct the Phylogenetic Tree in Figure 1 + +(22 KB PDF) + +Click here for additional data file. + +Acknowledgements + +We thank P. Burgoyne, P. Cohen, E. Alani, and P. Moens for helpful discussions, R. Viswanatha for helpful comments on the manuscript, W. Pawlowski and T. Pawlowska for input on the evolutionary tree of PCH2, R. Freire and J. Chen for antibodies, M. Jasin for Spo11− embryonic stem cells, and R. Munroe for generating chimeric mice. We also thank an anonymous reviewer for pointing out that Pch2p might be sensing synaptic polymerization in yeast, not initiation. + +Abbreviations + +CO - crossover + +DSB - double strand break + +MSCI - meiotic sex chromosome inactivation + +MSUC - meiotic silencing of unpaired chromatin + +NCO - noncrossover + +OA - okadaic acid + +RT-PCR - reverse-transcriptase PCR + +SC - synaptonemal complex + +Footnotes + +A previous version of this article appeared as an Early Online Release on June 21, 2007 (doi:10.1371/journal.pgen.0030130.eor). + +Author contributions. XL and JCS conceived and designed the experiments and analyzed the data. XL performed the experiments. JCS wrote the paper. + +Funding. This work was supported by NIH grant GM45415 to JS. + +Competing interests. The authors have declared that no competing interests exist. diff --git a/src/ontogpt/evaluation/craft/database/all/annotation.conf b/src/ontogpt/evaluation/craft/database/all/annotation.conf new file mode 100644 index 000000000..50cafa64c --- /dev/null +++ b/src/ontogpt/evaluation/craft/database/all/annotation.conf @@ -0,0 +1,4840 @@ +[entities] +NCBITaxon:kingdom +NCBITaxon:phylum +NCBITaxon:species +NCBITaxon:subspecies +PR:O01367 +PR:O14782 +PR:O15066 +PR:O18214 +PR:O35066 +PR:O54908 +PR:O55165 +PR:O75330 +PR:O75410-1 +PR:O95359-1 +PR:O95359-4 +PR:P00441 +PR:P00573 +PR:P03870 +PR:P03882 +PR:P03962 +PR:P04386 +PR:P04483 +PR:P04692 +PR:P04925 +PR:P05084 +PR:P05187 +PR:P05205 +PR:P06002 +PR:P06492 +PR:P06633 +PR:P06777 +PR:P09088 +PR:P09493 +PR:P09615 +PR:P10040 +PR:P10041 +PR:P10083 +PR:P10084 +PR:P12757-1 +PR:P13104 +PR:P17536 +PR:P18168 +PR:P18824 +PR:P20154 +PR:P21440 +PR:P22082 +PR:P22810 +PR:P23023 +PR:P23023-1 +PR:P23940 +PR:P26659 +PR:P26675 +PR:P28741 +PR:P29673 +PR:P31111 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+UBERON:0011002 +UBERON:0011078 +UBERON:0011137 +UBERON:0011270 +UBERON:0011768 +UBERON:0011817 +UBERON:0011905 +UBERON:0011919 +UBERON:0011997 +UBERON:0012101 +UBERON:0012170 +UBERON:0012198 +UBERON:0012238 +UBERON:0012246 +UBERON:0012274 +UBERON:0012282 +UBERON:0012283 +UBERON:0012427 +UBERON:0012429 +UBERON:0012652 +UBERON:0013141 +UBERON:0013147 +UBERON:0013235 +UBERON:0013406 +UBERON:0013623 +UBERON:0013636 +UBERON:0013682 +UBERON:0013691 +UBERON:0013768 +UBERON:0014374 +UBERON:0014387 +UBERON:0014388 +UBERON:0014399 +UBERON:0014540 +UBERON:0014548 +UBERON:0014626 +UBERON:0014643 +UBERON:0014907 +UBERON:0014930 +UBERON:0015143 +UBERON:0015178 +UBERON:0016405 +UBERON:0016459 +UBERON:0016467 +UBERON:0016525 +UBERON:0016530 +UBERON:0016538 +UBERON:0016884 +UBERON:0016920 +UBERON:0017648 +UBERON:0018260 +UBERON:0018264 +UBERON:0018545 +UBERON:0018707 +UBERON:0019204 +UBERON:0019248 +UBERON:0019252 +UBERON:0024914 +UBERON:0025534 +UBERON:0028194 +UBERON:0034705 +UBERON:0034969 +UBERON:1000019 +UBERON:2000083 +UBERON:2000084 +UBERON:2000164 +UBERON:2000280 +UBERON:2000468 +UBERON:2000532 +UBERON:2000732 +UBERON:2001463 +UBERON:2001977 +UBERON:2001995 +UBERON:2002283 +UBERON:2002284 +UBERON:2005411 +UBERON:3011048 +UBERON:4000088 +UBERON:4100005 +UBERON:4200011 +UBERON:4200132 +UBERON:4200215 +UBERON:4200230 +UBERON:5002544 +[attributes] +[relations] + From 3c67d4d16f8c9332ef6f373e082b7472bc896d7a Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Wed, 29 Nov 2023 17:24:52 -0500 Subject: [PATCH 12/24] Add EvalCRAFTConcepts to eval resolver --- src/ontogpt/evaluation/resolver.py | 5 ++++- 1 file changed, 4 insertions(+), 1 deletion(-) diff --git a/src/ontogpt/evaluation/resolver.py b/src/ontogpt/evaluation/resolver.py index 023035394..6f1cdaa17 100644 --- a/src/ontogpt/evaluation/resolver.py +++ b/src/ontogpt/evaluation/resolver.py @@ -3,12 +3,15 @@ from class_resolver import ClassResolver +from ontogpt.evaluation.craft.eval_craft_ner import EvalCRAFTConcepts from ontogpt.evaluation.ctd.eval_ctd import EvalCTD from ontogpt.evaluation.ctd.eval_ctd_ner import EvalCTDNER from ontogpt.evaluation.maxo.eval_maxo import EvalMAXO from ontogpt.evaluation.evaluation_engine import SPIRESEvaluationEngine -resolver = ClassResolver([EvalCTD, EvalCTDNER, EvalMAXO], base=SPIRESEvaluationEngine) +resolver = ClassResolver( + [EvalCRAFTConcepts, EvalCTD, EvalCTDNER, EvalMAXO], base=SPIRESEvaluationEngine +) def create_evaluator( From 1e636829aebb0f939ed1056cc7dc345d300387d0 Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Wed, 29 Nov 2023 17:37:06 -0500 Subject: [PATCH 13/24] Simple annotation loading --- .../evaluation/craft/eval_craft_ner.py | 36 ++++++++++++------- 1 file changed, 23 insertions(+), 13 deletions(-) diff --git a/src/ontogpt/evaluation/craft/eval_craft_ner.py b/src/ontogpt/evaluation/craft/eval_craft_ner.py index fb62b3526..9441cef2b 100644 --- a/src/ontogpt/evaluation/craft/eval_craft_ner.py +++ b/src/ontogpt/evaluation/craft/eval_craft_ner.py @@ -35,7 +35,6 @@ """ -import gzip import logging from collections import defaultdict from dataclasses import dataclass @@ -56,7 +55,7 @@ ) THIS_DIR = Path(__file__).parent -DATABASE_DIR = Path(__file__).parent / "database" +DATABASE_DIR = Path(__file__).parent / "database" / "all" # These are the entity types involved in this dataset. TARGET_TYPES = [ @@ -93,11 +92,12 @@ class PredictionNER(BaseModel): # results stores all the TP, FP, FN, and TP entities # with entity name from TARGET_TYPES as key - results = Dict[Dict[List[Tuple]]] - for target in TARGET_TYPES: - results[target] = {} - for result_type in RESULT_TYPES: - results[target[result_type]] = [] + # results is a dict of dict of lists of tuples + results: Dict[str, Dict[str, List[Tuple[str, str]]]] = {} + # for target in TARGET_TYPES: + # results[target] = {} + # for result_type in RESULT_TYPES: + # results[target[result_type]] = [] scores: Optional[Dict[str, SimilarityScore]] = None predicted_object: Optional[TextWithEntity] = None @@ -134,12 +134,23 @@ def __post_init__(self): def load_test_cases(self) -> Iterable[Document]: return self.load_cases(DATABASE_DIR) + # Load cases from txt and ann files def load_cases(self, path: Path) -> Iterable[Document]: logger.info(f"Loading {path}") - # Retrieve corpus if not present - # Preprocess corpus # Load documents + # Remove extra empty lines from input text + for docfilepath in path.glob("*.txt"): + logger.info(f"Loading text doc {docfilepath}") + with open(docfilepath, "r") as docfile: + doctext = docfile.read().replace('\n\n', '\n') + annfilepath = docfilepath.with_suffix('.ann') + logger.info(f"Loading corresponding annotation file {annfilepath}") + with open(annfilepath, "r") as annfile: + annotations = annfile.readlines() + print(doctext) + print(len(annotations)) + # Validate documents # with gzip.open(str(path), "rb") as f: @@ -203,17 +214,16 @@ def load_cases(self, path: Path) -> Iterable[Document]: # ) - # def eval(self) -> EvaluationObjectSetNER: - # """Evaluate the ability to extract chemical and disease named entities.""" + def eval(self) -> EvaluationObjectSetNER: + """Evaluate the ability to extract and ground entities in CRAFT corpus.""" - # # TODO: need other labelers # labeler = get_adapter("sqlite:obo:mesh") # if self.num_tests and isinstance(self.num_tests, int): # num_test = self.num_tests # else: # num_test = 1 # ke = self.extractor - # docs = list(self.load_test_cases()) + docs = list(self.load_test_cases()) # shuffle(docs) # eos = EvaluationObjectSetNER( # test=docs[:num_test], From 79597f4fdf5e67e025810c3b53b0844dac5d392a Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Wed, 29 Nov 2023 17:51:48 -0500 Subject: [PATCH 14/24] More document parsing --- .../evaluation/craft/eval_craft_ner.py | 25 ++++++++++++------- 1 file changed, 16 insertions(+), 9 deletions(-) diff --git a/src/ontogpt/evaluation/craft/eval_craft_ner.py b/src/ontogpt/evaluation/craft/eval_craft_ner.py index 9441cef2b..51deaeafb 100644 --- a/src/ontogpt/evaluation/craft/eval_craft_ner.py +++ b/src/ontogpt/evaluation/craft/eval_craft_ner.py @@ -57,6 +57,8 @@ THIS_DIR = Path(__file__).parent DATABASE_DIR = Path(__file__).parent / "database" / "all" +MONDO_PURL_PREFIX = "http://purl.obolibrary.org/obo/MONDO_" + # These are the entity types involved in this dataset. TARGET_TYPES = [ "AnatomicalElement", @@ -138,8 +140,12 @@ def load_test_cases(self) -> Iterable[Document]: def load_cases(self, path: Path) -> Iterable[Document]: logger.info(f"Loading {path}") + entities_by_text = defaultdict(list) + # Load documents # Remove extra empty lines from input text + # Parse annotations to identifier only + # MONDO ids are full PURLs for some reason, so fix those too for docfilepath in path.glob("*.txt"): logger.info(f"Loading text doc {docfilepath}") with open(docfilepath, "r") as docfile: @@ -148,18 +154,19 @@ def load_cases(self, path: Path) -> Iterable[Document]: logger.info(f"Loading corresponding annotation file {annfilepath}") with open(annfilepath, "r") as annfile: annotations = annfile.readlines() - print(doctext) - print(len(annotations)) + + doc = {} + these_annotations = [] + for annotation in annotations: + uri = (annotation.split())[1] + if uri.startswith(MONDO_PURL_PREFIX): + uri = uri.replace(MONDO_PURL_PREFIX, 'MONDO:') + if uri not in these_annotations: + these_annotations.append(uri) + # Validate documents - # with gzip.open(str(path), "rb") as f: - # collection = biocxml.load(f) - # chemicals_by_text = defaultdict(list) - # diseases_by_text = defaultdict(list) - # for document in collection.documents: - # these_annotations = [] - # doc = {} # for p in document.passages: # doc[p.infons["type"]] = p.text # for a in p.annotations: From 3a52bfa5eeecb48fea920077682b197cb3f388fa Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Thu, 30 Nov 2023 15:47:04 -0500 Subject: [PATCH 15/24] Add Cell class to schema --- src/ontogpt/templates/craft_concept.py | 8 ++++++++ src/ontogpt/templates/craft_concept.yaml | 19 +++++++++++++++++++ 2 files changed, 27 insertions(+) diff --git a/src/ontogpt/templates/craft_concept.py b/src/ontogpt/templates/craft_concept.py index 42076e3f7..417b3eeea 100644 --- a/src/ontogpt/templates/craft_concept.py +++ b/src/ontogpt/templates/craft_concept.py @@ -82,6 +82,12 @@ class BiologicalProcess(NamedEntity): label: Optional[str] = Field(None, description="""The label (name) of the named thing""") +class Cell(NamedEntity): + + id: str = Field(..., description="""A unique identifier for the named entity""") + label: Optional[str] = Field(None, description="""The label (name) of the named thing""") + + class CellularComponent(NamedEntity): id: str = Field(..., description="""A unique identifier for the named entity""") @@ -169,6 +175,7 @@ class Document(TextWithEntity): """ anatomicalelements: Optional[List[str]] = Field(default_factory=list, description="""One or more parts of the body or anatomy.""") biologicalprocesses: Optional[List[str]] = Field(default_factory=list, description="""One or more biological processes, as defined by the Gene Ontology.""") + celltypes: Optional[List[str]] = Field(default_factory=list, description="""One or more cell types or cell lines.""") cellularcomponents: Optional[List[str]] = Field(default_factory=list, description="""One or more cellular components, as defined by the Gene Ontology.""") chemicals: Optional[List[str]] = Field(default_factory=list, description="""One or more chemical substances, drugs, or small molecules.""") diseases: Optional[List[str]] = Field(default_factory=list, description="""One or more diseases or conditions.""") @@ -210,6 +217,7 @@ class AnnotatorResult(ConfiguredBaseModel): NamedEntity.model_rebuild() AnatomicalElement.model_rebuild() BiologicalProcess.model_rebuild() +Cell.model_rebuild() CellularComponent.model_rebuild() Chemical.model_rebuild() Disease.model_rebuild() diff --git a/src/ontogpt/templates/craft_concept.yaml b/src/ontogpt/templates/craft_concept.yaml index a419e0240..f9f5d9324 100644 --- a/src/ontogpt/templates/craft_concept.yaml +++ b/src/ontogpt/templates/craft_concept.yaml @@ -62,6 +62,15 @@ classes: medial surface of mandible; ribosomal subunit export from nucleus; pole cell development biologicalprocesses: + celltypes: + range: Cell + multivalued: true + description: One or more cell types or cell lines. + annotations: + prompt: >- + A semi-colon separated list of cell types or cell lines, for + example: type I AECs; mesenchymal cell; neuron; + smooth muscle cells; photoreceptors cellularcomponents: range: CellularComponent multivalued: true @@ -168,6 +177,16 @@ classes: values_from: - GOBiologicalProcessType + Cell: + is_a: NamedEntity + annotations: + annotators: "sqlite:obo:cl" + prompt.examples: >- + type I AECs, mesenchymal cell, neuron, smooth muscle cells, + photoreceptors + id_prefixes: + - CL + CellularComponent: is_a: NamedEntity annotations: From 759aeb2d262fe05f4e1ee61901526c1f0684afac Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Thu, 30 Nov 2023 15:54:51 -0500 Subject: [PATCH 16/24] Don't consider SNOMED valid prefix for Taxon --- src/ontogpt/templates/craft_concept.yaml | 1 - 1 file changed, 1 deletion(-) diff --git a/src/ontogpt/templates/craft_concept.yaml b/src/ontogpt/templates/craft_concept.yaml index f9f5d9324..29747d9d3 100644 --- a/src/ontogpt/templates/craft_concept.yaml +++ b/src/ontogpt/templates/craft_concept.yaml @@ -265,7 +265,6 @@ classes: is_a: NamedEntity id_prefixes: - NCBITaxon - - SNOMEDCT annotations: annotators: "sqlite:obo:ncbitaxon, bioportal:SNOMEDCT" prompt.examples: >- From 8198486fed0f5919a267677aec0214c6ab9fd87f Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Thu, 30 Nov 2023 16:11:18 -0500 Subject: [PATCH 17/24] Validate and sort entities in test docs --- .../evaluation/craft/eval_craft_ner.py | 142 +++++++++--------- 1 file changed, 71 insertions(+), 71 deletions(-) diff --git a/src/ontogpt/evaluation/craft/eval_craft_ner.py b/src/ontogpt/evaluation/craft/eval_craft_ner.py index 51deaeafb..c901e2ec9 100644 --- a/src/ontogpt/evaluation/craft/eval_craft_ner.py +++ b/src/ontogpt/evaluation/craft/eval_craft_ner.py @@ -51,6 +51,8 @@ from ontogpt.evaluation.evaluation_engine import SimilarityScore, SPIRESEvaluationEngine from ontogpt.templates.craft_concept import ( Document, + NamedEntity, + Publication, TextWithEntity, ) @@ -59,20 +61,23 @@ MONDO_PURL_PREFIX = "http://purl.obolibrary.org/obo/MONDO_" -# These are the entity types involved in this dataset. -TARGET_TYPES = [ - "AnatomicalElement", - "BiologicalProcess", - "CellType", - "CellularComponent", - "Chemical", - "Disease", - "MolecularFunction", - "MolecularProcess", - "Protein", - "SequenceFeature", - "Taxon", -] +# These are the entity types involved in this dataset, +# along with their corresponding prefixes. +# GO terms need to be checked during sorting +# so they go to the correct subset. +TARGET_TYPES = { + "AnatomicalElement": "UBERON", + "BiologicalProcess": "GO", + "CellType": "CL", + "CellularComponent": "GO", + "Chemical": "CHEBI", + "Disease": "MONDO", + "MolecularFunction": "GO", + "MolecularProcess": "MOP", + "Protein": "PR", + "SequenceFeature": "SO", + "Taxon": "NCBITaxon", +} RESULT_TYPES = [ "true_positives", @@ -149,13 +154,15 @@ def load_cases(self, path: Path) -> Iterable[Document]: for docfilepath in path.glob("*.txt"): logger.info(f"Loading text doc {docfilepath}") with open(docfilepath, "r") as docfile: + title = docfile.readline().rstrip() doctext = docfile.read().replace('\n\n', '\n') + logger.debug(f"Title: {title}\nDocument Text (truncated): {doctext[0:100]}...({len(doctext[100:])} chars)") annfilepath = docfilepath.with_suffix('.ann') logger.info(f"Loading corresponding annotation file {annfilepath}") with open(annfilepath, "r") as annfile: annotations = annfile.readlines() - doc = {} + # Get annotations and validate as CURIEs these_annotations = [] for annotation in annotations: uri = (annotation.split())[1] @@ -163,62 +170,55 @@ def load_cases(self, path: Path) -> Iterable[Document]: uri = uri.replace(MONDO_PURL_PREFIX, 'MONDO:') if uri not in these_annotations: these_annotations.append(uri) - - - # Validate documents - - # for p in document.passages: - # doc[p.infons["type"]] = p.text - # for a in p.annotations: - # if a.infons["type"] in TARGET_TYPES: - # these_annotations.append(a) - # title = doc["title"] - # abstract = doc["abstract"] - # logger.debug(f"Title: {title} Abstract: {abstract}") - # for a in these_annotations: - # i = a.infons - # if i["type"] == "Chemical": - # e = Chemical.model_validate( - # { - # "id": f"{self.subject_prefix}:{i[self.subject_prefix]}", - # } - # ) - # chemicals_by_text[(title, abstract)].append(e.id) - # elif i["type"] == "Disease": - # e = Disease.model_validate( - # { - # "id": f"{self.subject_prefix}:{i[self.subject_prefix]}", - # } - # ) - # diseases_by_text[(title, abstract)].append(e.id) - - # all_entities_by_text = chemicals_by_text | diseases_by_text - - # i = 0 - # for (title, abstract), _entities in all_entities_by_text.items(): - # i = i + 1 - # pub = Publication.model_validate( - # { - # "id": str(i), - # "title": title, - # "abstract": abstract, - # } - # ) - # chemical_entities = chemicals_by_text[(title, abstract)] - # disease_entities = diseases_by_text[(title, abstract)] - # logger.debug( - # f"Chemicals: {len(chemical_entities)} for Title: {title} Abstract: {abstract}" - # ) - # logger.debug( - # f"Diseases: {len(disease_entities)} for Title: {title} Abstract: {abstract}" - # ) - # yield ChemicalToDiseaseDocument.model_validate( - # { - # "publication": pub, - # "chemicals": chemical_entities, - # "diseases": disease_entities, - # } - # ) + e = NamedEntity.model_validate( + { + "id": uri, + } + ) + entities_by_text[(title, doctext)].append(e.id) + + i = 0 + for (title, doctext), _entities in entities_by_text.items(): + i = i + 1 + pub = Publication.model_validate( + { + "id": str(i), + "title": title, + "abstract": doctext, # Yes, it's the full text, but this is the slot name + } + ) + all_entities = entities_by_text[(title, doctext)] + + # Do entity sorting + # Results should be unique - no duplicates + # TODO: Need to check on GO terms too + logger.info(f"Sorting {len(all_entities)} redundant entities for Title: {title}") + entities_by_type = {key: [] for key in TARGET_TYPES.keys()} + for entity in all_entities: + prefix = (entity.split(":"))[0] + for entity_type in TARGET_TYPES: + if prefix == TARGET_TYPES[entity_type]: + if entity not in entities_by_type[entity_type]: + entities_by_type[entity_type].append(entity) + break + for entity_type in entities_by_type: + logger.debug(f"{entity_type}: {len(entities_by_type[entity_type])}") + yield Document.model_validate( + { + "publication": pub, + "anatomicalelements": entities_by_type["AnatomicalElement"], + "biologicalprocesses": entities_by_type["BiologicalProcess"], + "celltypes": entities_by_type["CellType"], + "cellularcomponents": entities_by_type["CellularComponent"], + "chemicals": entities_by_type["Chemical"], + "diseases": entities_by_type["Disease"], + "molecularfunctions": entities_by_type["MolecularFunction"], + "molecularprocesses": entities_by_type["MolecularProcess"], + "proteins": entities_by_type["Protein"], + "sequencefeatures": entities_by_type["SequenceFeature"], + "taxa": entities_by_type["Taxon"] + } + ) def eval(self) -> EvaluationObjectSetNER: From 07bc3ff6faa2cd981dd98e1ec3c04c9579a9ba5f Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Fri, 1 Dec 2023 16:14:32 -0500 Subject: [PATCH 18/24] Check GO subtypes and deprecated status --- .../evaluation/craft/eval_craft_ner.py | 76 +++++++++++++------ 1 file changed, 51 insertions(+), 25 deletions(-) diff --git a/src/ontogpt/evaluation/craft/eval_craft_ner.py b/src/ontogpt/evaluation/craft/eval_craft_ner.py index c901e2ec9..c04287d24 100644 --- a/src/ontogpt/evaluation/craft/eval_craft_ner.py +++ b/src/ontogpt/evaluation/craft/eval_craft_ner.py @@ -110,6 +110,7 @@ class PredictionNER(BaseModel): predicted_object: Optional[TextWithEntity] = None named_entities: Optional[List[Any]] = None + class EvaluationObjectSetNER(BaseModel): """A result of performing named entity recognition.""" @@ -128,15 +129,12 @@ class EvaluationObjectSetNER(BaseModel): @dataclass class EvalCRAFTConcepts(SPIRESEvaluationEngine): - # TODO: nope, not these subject_prefix = "MESH" object_prefix = "MESH" def __post_init__(self): - self.extractor = SPIRESEngine( - template="craft_concept.Document", model=self.model - ) + self.extractor = SPIRESEngine(template="craft_concept.Document", model=self.model) def load_test_cases(self) -> Iterable[Document]: return self.load_cases(DATABASE_DIR) @@ -155,52 +153,80 @@ def load_cases(self, path: Path) -> Iterable[Document]: logger.info(f"Loading text doc {docfilepath}") with open(docfilepath, "r") as docfile: title = docfile.readline().rstrip() - doctext = docfile.read().replace('\n\n', '\n') - logger.debug(f"Title: {title}\nDocument Text (truncated): {doctext[0:100]}...({len(doctext[100:])} chars)") - annfilepath = docfilepath.with_suffix('.ann') + doctext = docfile.read().replace("\n\n", "\n") + logger.debug( + f"Title: {title}\nDocument Text (truncated): {doctext[0:100]}...({len(doctext[100:])} chars)" + ) + annfilepath = docfilepath.with_suffix(".ann") logger.info(f"Loading corresponding annotation file {annfilepath}") with open(annfilepath, "r") as annfile: annotations = annfile.readlines() - + # Get annotations and validate as CURIEs these_annotations = [] for annotation in annotations: uri = (annotation.split())[1] if uri.startswith(MONDO_PURL_PREFIX): - uri = uri.replace(MONDO_PURL_PREFIX, 'MONDO:') + uri = uri.replace(MONDO_PURL_PREFIX, "MONDO:") if uri not in these_annotations: these_annotations.append(uri) e = NamedEntity.model_validate( { "id": uri, } - ) + ) entities_by_text[(title, doctext)].append(e.id) i = 0 + go_map = {} + goad = get_adapter("sqlite:obo:go") for (title, doctext), _entities in entities_by_text.items(): i = i + 1 pub = Publication.model_validate( { "id": str(i), "title": title, - "abstract": doctext, # Yes, it's the full text, but this is the slot name + "abstract": doctext, # Yes, it's the full text, but this is the slot name } ) all_entities = entities_by_text[(title, doctext)] # Do entity sorting # Results should be unique - no duplicates - # TODO: Need to check on GO terms too + # Check on GO terms too logger.info(f"Sorting {len(all_entities)} redundant entities for Title: {title}") entities_by_type = {key: [] for key in TARGET_TYPES.keys()} for entity in all_entities: prefix = (entity.split(":"))[0] - for entity_type in TARGET_TYPES: - if prefix == TARGET_TYPES[entity_type]: - if entity not in entities_by_type[entity_type]: - entities_by_type[entity_type].append(entity) - break + # Check if this is a GO CURIE first and find substype + # We store these subtypes too + if prefix == "GO": + if entity in go_map: + go_type = go_map[entity] + else: + try: + go_meta = goad.entity_metadata_map(entity) + go_type = go_meta["oio:hasOBONamespace"][0] + except KeyError: + go_dep = go_meta["owl:deprecated"][0] + if go_dep: + logger.warning(f"{entity} is deprecated. Ignoring...") + else: + logger.warning(f"{entity} has something wrong with it. Ignoring...") + go_map[entity] = go_type + if go_type == "biological_process": + entities_by_type["BiologicalProcess"].append(entity) + elif go_type == "molecular_process": + entities_by_type["MolecularProcess"].append(entity) + elif go_type == "cellular_component": + entities_by_type["CellularComponent"].append(entity) + continue + else: + for entity_type in TARGET_TYPES: + if prefix == TARGET_TYPES[entity_type]: + if entity not in entities_by_type[entity_type]: + entities_by_type[entity_type].append(entity) + break for entity_type in entities_by_type: logger.debug(f"{entity_type}: {len(entities_by_type[entity_type])}") yield Document.model_validate( @@ -216,21 +242,21 @@ def load_cases(self, path: Path) -> Iterable[Document]: "molecularprocesses": entities_by_type["MolecularProcess"], "proteins": entities_by_type["Protein"], "sequencefeatures": entities_by_type["SequenceFeature"], - "taxa": entities_by_type["Taxon"] + "taxa": entities_by_type["Taxon"], } ) - def eval(self) -> EvaluationObjectSetNER: """Evaluate the ability to extract and ground entities in CRAFT corpus.""" - # labeler = get_adapter("sqlite:obo:mesh") - # if self.num_tests and isinstance(self.num_tests, int): - # num_test = self.num_tests - # else: - # num_test = 1 - # ke = self.extractor + # labeler = get_adapter("sqlite:obo:mesh") + # if self.num_tests and isinstance(self.num_tests, int): + # num_test = self.num_tests + # else: + # num_test = 1 + # ke = self.extractor docs = list(self.load_test_cases()) + # shuffle(docs) # eos = EvaluationObjectSetNER( # test=docs[:num_test], From 6992c176297cb5b8ffce09b03d90a793b2ccba40 Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Fri, 1 Dec 2023 16:17:17 -0500 Subject: [PATCH 19/24] Debug output --- src/ontogpt/evaluation/craft/eval_craft_ner.py | 1 + 1 file changed, 1 insertion(+) diff --git a/src/ontogpt/evaluation/craft/eval_craft_ner.py b/src/ontogpt/evaluation/craft/eval_craft_ner.py index c04287d24..7c11f24b2 100644 --- a/src/ontogpt/evaluation/craft/eval_craft_ner.py +++ b/src/ontogpt/evaluation/craft/eval_craft_ner.py @@ -182,6 +182,7 @@ def load_cases(self, path: Path) -> Iterable[Document]: goad = get_adapter("sqlite:obo:go") for (title, doctext), _entities in entities_by_text.items(): i = i + 1 + logging.debug(f"Loading document {i}") pub = Publication.model_validate( { "id": str(i), From 98ba4a782f5b6eaa345eb44ec994a3639bb9cc64 Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Fri, 1 Dec 2023 16:21:49 -0500 Subject: [PATCH 20/24] Parse molecular_function properly --- src/ontogpt/evaluation/craft/eval_craft_ner.py | 4 ++-- 1 file changed, 2 insertions(+), 2 deletions(-) diff --git a/src/ontogpt/evaluation/craft/eval_craft_ner.py b/src/ontogpt/evaluation/craft/eval_craft_ner.py index 7c11f24b2..71795e9ae 100644 --- a/src/ontogpt/evaluation/craft/eval_craft_ner.py +++ b/src/ontogpt/evaluation/craft/eval_craft_ner.py @@ -217,8 +217,8 @@ def load_cases(self, path: Path) -> Iterable[Document]: go_map[entity] = go_type if go_type == "biological_process": entities_by_type["BiologicalProcess"].append(entity) - elif go_type == "molecular_process": - entities_by_type["MolecularProcess"].append(entity) + elif go_type == "molecular_function": + entities_by_type["MolecularFunction"].append(entity) elif go_type == "cellular_component": entities_by_type["CellularComponent"].append(entity) continue From f3fb5f2a6a255fd859290d98558bebdb63883347 Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Fri, 1 Dec 2023 16:56:48 -0500 Subject: [PATCH 21/24] Expanding eval --- .../evaluation/craft/eval_craft_ner.py | 253 ++++++++++-------- 1 file changed, 135 insertions(+), 118 deletions(-) diff --git a/src/ontogpt/evaluation/craft/eval_craft_ner.py b/src/ontogpt/evaluation/craft/eval_craft_ner.py index 71795e9ae..44695313b 100644 --- a/src/ontogpt/evaluation/craft/eval_craft_ner.py +++ b/src/ontogpt/evaluation/craft/eval_craft_ner.py @@ -79,6 +79,21 @@ "Taxon": "NCBITaxon", } +TARGET_ATTR_TYPES = [ + "publication", + "anatomicalelements", + "biologicalprocesses", + "celltypes", + "cellularcomponents", + "chemicals", + "diseases", + "molecularfunctions", + "molecularprocesses", + "proteins", + "sequencefeatures", + "taxa", +] + RESULT_TYPES = [ "true_positives", "num_true_positives", @@ -111,6 +126,8 @@ class PredictionNER(BaseModel): named_entities: Optional[List[Any]] = None +# TODO: expand to cover all entity types +# and adapt to the PredictionNER results form defined above class EvaluationObjectSetNER(BaseModel): """A result of performing named entity recognition.""" @@ -250,123 +267,123 @@ def load_cases(self, path: Path) -> Iterable[Document]: def eval(self) -> EvaluationObjectSetNER: """Evaluate the ability to extract and ground entities in CRAFT corpus.""" - # labeler = get_adapter("sqlite:obo:mesh") - # if self.num_tests and isinstance(self.num_tests, int): - # num_test = self.num_tests - # else: - # num_test = 1 - # ke = self.extractor + if self.num_tests and isinstance(self.num_tests, int): + num_test = self.num_tests + else: + num_test = 1 + ke = self.extractor + docs = list(self.load_test_cases()) - # shuffle(docs) - # eos = EvaluationObjectSetNER( - # test=docs[:num_test], - # training=[], - # predictions=[], - # ) - # n = 1 - # for doc in eos.test: - # logger.info(f"Iteration {n} of {num_test}") - # n += 1 - # logger.info(doc) - # text = f"Title: {doc.publication.title} Abstract: {doc.publication.abstract}" - # pub = Publication.model_validate( - # { - # "id": str(doc.publication.id), - # "title": doc.publication.title, - # "abstract": doc.publication.abstract, - # } - # ) - # predicted_obj = Document.model_validate( - # { - # "publication": pub, - # "chemicals": [], - # "diseases": [], - # } - # ) - # named_entities: List[str] = [] # This stores the NEs for the whole document - # ke.named_entities = [] # This stores the NEs the extractor knows about - - # if self.chunking: - # text_list = chunk_text(text) - # else: - # text_list = iter([text]) - - # for chunked_text in text_list: - # extraction = ke.extract_from_text(chunked_text) - # if extraction.extracted_object is not None: - # logger.info( - # f"{len(extraction.extracted_object.chemicals)}\ - # chemical entities from window: {chunked_text}" - # ) - # logger.info( - # f"{len(extraction.extracted_object.diseases)}\ - # disease entities from window: {chunked_text}" - # ) - # if not predicted_obj and extraction.extracted_object is not None: - # predicted_obj = extraction.extracted_object - # else: - # if predicted_obj is not None and extraction.extracted_object is not None: - # predicted_obj.chemicals.extend(extraction.extracted_object.chemicals) - # predicted_obj.diseases.extend(extraction.extracted_object.diseases) - # logger.info( - # f"{len(predicted_obj.chemicals)} chemical entities, after concatenation" - # ) - # logger.info( - # f"{len(predicted_obj.diseases)} disease entities, after concatenation" - # ) - # logger.debug(f"concatenated chemical entities: {predicted_obj.chemicals}") - # logger.debug(f"concatenated disease entities: {predicted_obj.diseases}") - # if extraction.named_entities is not None: - # for entity in extraction.named_entities: - # if entity not in named_entities: - # named_entities.append(entity) - - # def included(t: str): - # if t.startswith("MESH:"): - # return t - - # predicted_obj.chemicals = [t for t in predicted_obj.chemicals if included(t)] - # predicted_obj.diseases = [t for t in predicted_obj.diseases if included(t)] - - # logger.info(f"{len(predicted_obj.chemicals)} filtered chemical entities (MESH only)") - # logger.info(f"{len(predicted_obj.diseases)} filtered disease entities (MESH only)") - # pred = PredictionNER( - # predicted_object=predicted_obj, test_object=doc, named_entities=named_entities - # ) - # named_entities.clear() - # logger.info("PRED") - # logger.info(yaml.dump(data=pred.model_dump())) - # logger.info("Calc scores") - # pred.calculate_scores(labelers=[labeler]) - # logger.info(yaml.dump(data=pred.model_dump())) - # eos.predictions.append(pred) - # self.calc_stats(eos) - # return eos - - # def calc_stats(self, eos: EvaluationObjectSetNER): - # num_true_positives_ce = sum(p.num_true_positives_ce for p in eos.predictions) - # num_false_positives_ce = sum(p.num_false_positives_ce for p in eos.predictions) - # num_false_negatives_ce = sum(p.num_false_negatives_ce for p in eos.predictions) - # if num_true_positives_ce + num_false_positives_ce == 0: - # logger.warning("No true positives or false positives for chemical entities.") - # return - # eos.precision_ce = num_true_positives_ce / (num_true_positives_ce + num_false_positives_ce) - # eos.recall_ce = num_true_positives_ce / (num_true_positives_ce + num_false_negatives_ce) - # if eos.precision_ce + eos.recall_ce == 0: - # logger.warning("No precision or recall for chemical entities.") - # return - # eos.f1_ce = 2 * (eos.precision_ce * eos.recall_ce) / (eos.precision_ce + eos.recall_ce) - - # num_true_positives_de = sum(p.num_true_positives_de for p in eos.predictions) - # num_false_positives_de = sum(p.num_false_positives_de for p in eos.predictions) - # num_false_negatives_de = sum(p.num_false_negatives_de for p in eos.predictions) - # if num_true_positives_de + num_false_positives_de == 0: - # logger.warning("No true positives or false positives for disease entities.") - # return - # eos.precision_de = num_true_positives_de / (num_true_positives_de + num_false_positives_de) - # eos.recall_de = num_true_positives_de / (num_true_positives_de + num_false_negatives_de) - # if eos.precision_de + eos.recall_de == 0: - # logger.warning("No precision or recall for disease entities.") - # return - # eos.f1_de = 2 * (eos.precision_de * eos.recall_de) / (eos.precision_de + eos.recall_de) + shuffle(docs) + eos = EvaluationObjectSetNER( + test=docs[:num_test], + training=[], + predictions=[], + ) + n = 1 + for doc in eos.test: + logger.info(f"Iteration {n} of {num_test}") + n += 1 + logger.info(doc.publication.title) + text = f"{doc.publication.title}\n{doc.publication.abstract}" + pub = Publication.model_validate( + { + "id": str(doc.publication.id), + "title": doc.publication.title, + "abstract": doc.publication.abstract, + } + ) + predicted_obj = Document.model_validate( + { + "publication": pub, + "anatomicalelements": [], + "biologicalprocesses": [], + "celltypes": [], + "cellularcomponents": [], + "chemicals": [], + "diseases": [], + "molecularfunctions": [], + "molecularprocesses": [], + "proteins": [], + "sequencefeatures": [], + "taxa": [], + } + ) + named_entities: List[str] = [] # This stores the NEs for the whole document + ke.named_entities = [] # This stores the NEs the extractor knows about + + if self.chunking: + text_list = chunk_text(text) + else: + text_list = iter([text]) + + for chunked_text in text_list: + extraction = ke.extract_from_text(chunked_text) + if extraction.extracted_object is not None: + logger.info("Found entities in active window.") + if not predicted_obj and extraction.extracted_object is not None: + predicted_obj = extraction.extracted_object + else: + if predicted_obj is not None and extraction.extracted_object is not None: + for entity_type in TARGET_ATTR_TYPES: + pred_entity_list_attr = getattr(predicted_obj, entity_type) + extraction_list_attr = getattr(extraction.extracted_object, entity_type) + new_entity_list = pred_entity_list_attr.extend(extraction_list_attr) + setattr(predicted_obj, entity_type, new_entity_list) + logger.info(f"{len(new_entity_list)} {entity_type} entities, after concatenation") + if extraction.named_entities is not None: + for entity in extraction.named_entities: + if entity not in named_entities: + named_entities.append(entity) + + # TODO: update based on known prefixes + def included(t: str): + if t.startswith("MESH:"): + return t + + # TODO: repeat for all entities, as above + predicted_obj.chemicals = [t for t in predicted_obj.chemicals if included(t)] + + # TODO: repeat for all entities, as above + logger.info(f"{len(predicted_obj.chemicals)} filtered entities") + pred = PredictionNER( + predicted_object=predicted_obj, test_object=doc, named_entities=named_entities + ) + named_entities.clear() + logger.info("PRED") + logger.info(yaml.dump(data=pred.model_dump())) + logger.info("Calc scores") + pred.calculate_scores(labelers=[labeler]) + logger.info(yaml.dump(data=pred.model_dump())) + eos.predictions.append(pred) + self.calc_stats(eos) + return eos + + # TODO: adapt to aggregated form + def calc_stats(self, eos: EvaluationObjectSetNER): + num_true_positives_ce = sum(p.num_true_positives_ce for p in eos.predictions) + num_false_positives_ce = sum(p.num_false_positives_ce for p in eos.predictions) + num_false_negatives_ce = sum(p.num_false_negatives_ce for p in eos.predictions) + if num_true_positives_ce + num_false_positives_ce == 0: + logger.warning("No true positives or false positives for chemical entities.") + return + eos.precision_ce = num_true_positives_ce / (num_true_positives_ce + num_false_positives_ce) + eos.recall_ce = num_true_positives_ce / (num_true_positives_ce + num_false_negatives_ce) + if eos.precision_ce + eos.recall_ce == 0: + logger.warning("No precision or recall for chemical entities.") + return + eos.f1_ce = 2 * (eos.precision_ce * eos.recall_ce) / (eos.precision_ce + eos.recall_ce) + + num_true_positives_de = sum(p.num_true_positives_de for p in eos.predictions) + num_false_positives_de = sum(p.num_false_positives_de for p in eos.predictions) + num_false_negatives_de = sum(p.num_false_negatives_de for p in eos.predictions) + if num_true_positives_de + num_false_positives_de == 0: + logger.warning("No true positives or false positives for disease entities.") + return + eos.precision_de = num_true_positives_de / (num_true_positives_de + num_false_positives_de) + eos.recall_de = num_true_positives_de / (num_true_positives_de + num_false_negatives_de) + if eos.precision_de + eos.recall_de == 0: + logger.warning("No precision or recall for disease entities.") + return + eos.f1_de = 2 * (eos.precision_de * eos.recall_de) / (eos.precision_de + eos.recall_de) From c27cb1adc363038b8caa515fccf565d2e8fac80a Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Tue, 12 Dec 2023 15:37:58 -0500 Subject: [PATCH 22/24] More documentation for eval --- src/ontogpt/evaluation/craft/eval_craft_ner.py | 11 ++++++++--- 1 file changed, 8 insertions(+), 3 deletions(-) diff --git a/src/ontogpt/evaluation/craft/eval_craft_ner.py b/src/ontogpt/evaluation/craft/eval_craft_ner.py index 44695313b..aaa19ec14 100644 --- a/src/ontogpt/evaluation/craft/eval_craft_ner.py +++ b/src/ontogpt/evaluation/craft/eval_craft_ner.py @@ -4,12 +4,12 @@ This is an NER evaluation, or specifically concept recognition. -It uses a specific template (craft_concepts) +It uses a specific template (craft_concepts). -The corpus is retrieved from: +The corpus is from: https://github.com/UCDenver-ccp/CRAFT -This evaluation use the v5.0.2 release. +This evaluation uses the v5.0.2 release. Funk et al. 2014 BMC Bioinformatics (https://doi.org/10.1186/1471-2105-15-59) @@ -32,6 +32,11 @@ Molecular Process Ontology (MOP) UBERON Ontology +Note: documents in this test corpus are full-texts +and may not fit into the context window of all +models. Please consider models with context window +of 12K tokens or greater. + """ From 8315cc00243626353e4e9523e7c7d339f3b3eb7f Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Tue, 12 Dec 2023 15:47:52 -0500 Subject: [PATCH 23/24] Filter each entity type in predicted_obj --- .../evaluation/craft/eval_craft_ner.py | 48 ++++++++++++++----- 1 file changed, 37 insertions(+), 11 deletions(-) diff --git a/src/ontogpt/evaluation/craft/eval_craft_ner.py b/src/ontogpt/evaluation/craft/eval_craft_ner.py index aaa19ec14..b0060bf62 100644 --- a/src/ontogpt/evaluation/craft/eval_craft_ner.py +++ b/src/ontogpt/evaluation/craft/eval_craft_ner.py @@ -336,22 +336,48 @@ def eval(self) -> EvaluationObjectSetNER: extraction_list_attr = getattr(extraction.extracted_object, entity_type) new_entity_list = pred_entity_list_attr.extend(extraction_list_attr) setattr(predicted_obj, entity_type, new_entity_list) - logger.info(f"{len(new_entity_list)} {entity_type} entities, after concatenation") + logger.info( + f"{len(new_entity_list)} {entity_type} entities, after concatenation" + ) if extraction.named_entities is not None: for entity in extraction.named_entities: if entity not in named_entities: named_entities.append(entity) - # TODO: update based on known prefixes - def included(t: str): - if t.startswith("MESH:"): - return t + predicted_obj.anatomicalelements = [ + t for t in predicted_obj.anatomicalelements if t.startswith("UBERON") + ] + predicted_obj.biologicalprocesses = [ + t for t in predicted_obj.anatomicalelements if t.startswith("GO") + ] + predicted_obj.celltypes = [ + t for t in predicted_obj.anatomicalelements if t.startswith("CL") + ] + predicted_obj.cellularcomponents = [ + t for t in predicted_obj.anatomicalelements if t.startswith("GO") + ] + predicted_obj.chemicals = [ + t for t in predicted_obj.anatomicalelements if t.startswith("CHEBI") + ] + predicted_obj.diseases = [ + t for t in predicted_obj.anatomicalelements if t.startswith("MONDO") + ] + predicted_obj.molecularfunctions = [ + t for t in predicted_obj.anatomicalelements if t.startswith("GO") + ] + predicted_obj.molecularprocesses = [ + t for t in predicted_obj.anatomicalelements if t.startswith("MOP") + ] + predicted_obj.proteins = [ + t for t in predicted_obj.anatomicalelements if t.startswith("PR") + ] + predicted_obj.sequencefeatures = [ + t for t in predicted_obj.anatomicalelements if t.startswith("SO") + ] + predicted_obj.taxa = [ + t for t in predicted_obj.anatomicalelements if t.startswith("NCBITaxon") + ] - # TODO: repeat for all entities, as above - predicted_obj.chemicals = [t for t in predicted_obj.chemicals if included(t)] - - # TODO: repeat for all entities, as above - logger.info(f"{len(predicted_obj.chemicals)} filtered entities") pred = PredictionNER( predicted_object=predicted_obj, test_object=doc, named_entities=named_entities ) @@ -365,7 +391,7 @@ def included(t: str): self.calc_stats(eos) return eos - # TODO: adapt to aggregated form + # TODO: adapt to aggregated form def calc_stats(self, eos: EvaluationObjectSetNER): num_true_positives_ce = sum(p.num_true_positives_ce for p in eos.predictions) num_false_positives_ce = sum(p.num_false_positives_ce for p in eos.predictions) From fa4759200f3bb4f7f054053ea9e0f1c1d64a02ab Mon Sep 17 00:00:00 2001 From: caufieldjh Date: Tue, 12 Dec 2023 16:04:41 -0500 Subject: [PATCH 24/24] Expanding --- .../evaluation/craft/eval_craft_ner.py | 69 +++++++++++++++---- 1 file changed, 54 insertions(+), 15 deletions(-) diff --git a/src/ontogpt/evaluation/craft/eval_craft_ner.py b/src/ontogpt/evaluation/craft/eval_craft_ner.py index b0060bf62..9bfddb6fb 100644 --- a/src/ontogpt/evaluation/craft/eval_craft_ner.py +++ b/src/ontogpt/evaluation/craft/eval_craft_ner.py @@ -121,28 +121,67 @@ class PredictionNER(BaseModel): # with entity name from TARGET_TYPES as key # results is a dict of dict of lists of tuples results: Dict[str, Dict[str, List[Tuple[str, str]]]] = {} - # for target in TARGET_TYPES: - # results[target] = {} - # for result_type in RESULT_TYPES: - # results[target[result_type]] = [] + for target in TARGET_ATTR_TYPES: + results[target] = {} + for result_type in RESULT_TYPES: + results[target[result_type]] = [] scores: Optional[Dict[str, SimilarityScore]] = None predicted_object: Optional[TextWithEntity] = None named_entities: Optional[List[Any]] = None + def calculate_scores(self): + self.scores = {} + + def all_objects(dm: Optional[TextWithEntity]): + if dm is not None: + return list(set(dm.chemicals + dm.diseases)) + else: + return list() + + def chem_entities(dm: TextWithEntity) -> Set: + if dm.chemicals is not None: + return set(((entity)) for entity in dm.chemicals) + else: + return set() + + def disease_entities(dm: TextWithEntity) -> Set: + if dm.diseases is not None: + return set(((entity)) for entity in dm.diseases) + else: + return set() + + self.scores["similarity"] = SimilarityScore.from_set( + all_objects(self.test_object), + all_objects(self.predicted_object), + ) + + if self.predicted_object is not None and self.test_object is not None: + pred_ce = chem_entities(self.predicted_object) + test_ce = chem_entities(self.test_object) + pred_de = disease_entities(self.predicted_object) + test_de = disease_entities(self.test_object) + + self.true_positives_ce = list(pred_ce.intersection(test_ce)) + self.false_positives_ce = list(pred_ce.difference(test_ce)) + self.false_negatives_ce = list(test_ce.difference(pred_ce)) + self.num_false_negatives_ce = len(self.false_negatives_ce) + self.num_false_positives_ce = len(self.false_positives_ce) + self.num_true_positives_ce = len(self.true_positives_ce) + + self.true_positives_de = list(pred_de.intersection(test_de)) + self.false_positives_de = list(pred_de.difference(test_de)) + self.false_negatives_de = list(test_de.difference(pred_de)) + self.num_false_negatives_de = len(self.false_negatives_de) + self.num_false_positives_de = len(self.false_positives_de) + self.num_true_positives_de = len(self.true_positives_de) -# TODO: expand to cover all entity types -# and adapt to the PredictionNER results form defined above class EvaluationObjectSetNER(BaseModel): """A result of performing named entity recognition.""" - precision_ce: float = 0 - recall_ce: float = 0 - f1_ce: float = 0 - - precision_de: float = 0 - recall_de: float = 0 - f1_de: float = 0 + precisions: List[float] = [0] + recalls: List[float] = [0] + f1s: List[float] = [0] training: Optional[List[TextWithEntity]] = None predictions: Optional[List[PredictionNER]] = None @@ -385,14 +424,14 @@ def eval(self) -> EvaluationObjectSetNER: logger.info("PRED") logger.info(yaml.dump(data=pred.model_dump())) logger.info("Calc scores") - pred.calculate_scores(labelers=[labeler]) + pred.calculate_scores() logger.info(yaml.dump(data=pred.model_dump())) eos.predictions.append(pred) self.calc_stats(eos) return eos - # TODO: adapt to aggregated form def calc_stats(self, eos: EvaluationObjectSetNER): + #for attr_type in TARGET_ATTR_TYPES: num_true_positives_ce = sum(p.num_true_positives_ce for p in eos.predictions) num_false_positives_ce = sum(p.num_false_positives_ce for p in eos.predictions) num_false_negatives_ce = sum(p.num_false_negatives_ce for p in eos.predictions)